key: cord-348547-wmvqvbqi authors: desmons, aurore; terrade, caroline; boulagnon, camille; giusti, delphine; nguyen, yohan; andreoletti, laurent; fornes, paul; digeon, beatrice; leveque, nicolas title: post-mortem diagnosis, of cytomegalovirus and varicella zoster virus co-infection by combined histology and tissue molecular biology, in a sudden unexplained infant death date: 2013-10-31 journal: journal of clinical virology doi: 10.1016/j.jcv.2013.08.007 sha: doc_id: 348547 cord_uid: wmvqvbqi abstract background an autopsy case of a two-month-old male infant who suddenly and unexpectedly died during his sleep, eight days after the onset of benign varicella. objectives to describe post-mortem combined histological and tissue molecular biological techniques for the diagnosis of cytomegalovirus and varicella zoster virus co-infection as a cause of death. study design real-time quantitative pcr and rt-pcr assays for herpesviruses, respiratory viruses, adenovirus, enterovirus and parvovirus b19 were performed on multi-organ frozen samples and paraffin-embedded tissues in combination with histology. results cytomegalovirus and varicella zoster virus were detected by molecular biology with highest viral loads detected in the lungs (4.6×107 and 1.9×105 genome copies per million of cells, respectively). pulmonary extensive necrotizing inflammation and immunohistochemistry correlated to virological data. virological molecular biology was negative on paraffin-embedded tissues. conclusions this case shows that thorough quantitative virological investigations on frozen tissues must be performed in combination with histology and immunohistochemistry for the determination of the cause of a sudden unexplained infant death. previous studies have demonstrated the major role of viral diseases in the pathogenesis of sudden unexpected infant death (suid) [1] [2] [3] [4] . we report on a case of a two-month-old male infant who suddenly and unexpectedly died during his sleep, without premonitory symptoms. post-mortem virological quantitative molecular analyses performed on frozen tissue samples revealed cytomegalovirus (cmv) and varicella zoster virus (vzv), mainly in the lungs. these results correlated to histological findings. virological molecular biology was negative on paraffin-embedded tissues. this case shows that quantitative virological investigations on multi-organ frozen samples must be performed in suid in combination with histology and immunohistochemistry. without this diagnostic approach, viral infections can be underestimated as a cause of death in infants. a two-month-old male infant was found dead in his cot. according to national diagnostic protocol in suid, an autopsy was performed in our institution. his mother had positive vzv and cmv serologies with antibodies titers of, respectively, 1800 international units (iu) and 560 arbitrary units (au) per liter (enzygnost ® assay, siemens, erlangen, germany) associated with high anti-cmv igg avidity during pregnancy, suggestive of a maternal primary infection before pregnancy. the infant was prematurely delivered at thirty-six weeks. the newborn was hospitalized during the first sixteen days after delivery in a neonatal intensive care unit, because of a severe hypotrophy (weight: 1725 g) associated with thrombopeniarelated diffuse petechiae (58,000/mm 3 platelets). platelet count increased spontaneously, up to 148,000/mm 3 , with no explanation. no perinatal investigations of cmv congenital infection were performed. the newborn weighted 2080 g when he was discharged. at the age of one month and 21 days, he had varicella contracted from his brother who had been diagnosed with chickenpox 15 days earlier. the newborn's infection was mild, consisting of three successive skin rashes, with no other manifestations. no antiviral treatment was given. eight days after the onset of the varicella, he was found dead on the back in his cot. in this context of a suid, an autopsy was performed according to national protocol. his weight was 3725 g. disseminated skin necrotic lesions were present. the organs were found normal. no cause of death was found. viral (herpes simplex virus type 1 and type 2 (hsv-1 and -2), cmv, epstein-barr virus (ebv), vzv, human herpes virus type 6 (hhv-6) and enterovirus) and bacterial tests were negative in the cerebrospinal fluid (csf). interferon alpha was negative in the csf. viral cultures performed from skin lesions, throat and anal swabs, as well as from tracheobronchial aspiration were negative. hemocultures, throat and fecal cultures were negative for main bacterial pathogens. histology showed extensive necrotizing inflammation in the lungs and moderate mononuclear inflammation in the kidneys ( fig. 1a and b ). other organs were otherwise normal. immunohistochemistry on paraffin blocks using monoclonal cmv antibody (argene biomerieux, verniolle, france) revealed a few cmv cell inclusions in the lungs whereas numerous cmv inclusions were observed in the kidneys ( fig. 1c and d) . heart, lung, kidney, liver, small intestine and colonic samples were collected for virological analyses and stored at −80 • c. these samples were analyzed with real-time pcr and rt-pcr assays for herpesviruses (hsv-1, hsv-2, vzv, cmv, ebv and hhv-6), respiratory viruses (influenza a&b viruses, parainfluenza 1 to 4 viruses, respiratory syncytial virus (rsv), rhinovirus, human bocavirus (hbov), human coronavirus nl63, oc43, hku1, 229e and the human metapneumovirus), adenovirus, enterovirus and parvovirus b19 (hsv1 hsv2 vzv r-gene ® , respiratory multi well system r-gene ® , argene biomerieux, verniolle, france) [5] [6] [7] [8] [9] [10] . cmv and vzv genomes were detected in all tissue samples (fig. 2) . the highest cmv and vzv loads were found in the lungs, 4.6 × 10 7 and 1.9 × 10 5 genome copies per million of cells, respectively (fig. 2) . in contrast, cmv and vzv dna were not detected in paraffin-embedded tissues. several studies have demonstrated frequent detection of viruses in post-mortem specimens suggesting their involvement in the pathogenesis of suid [1] [2] [3] [4] . the main viruses reported were respiratory viruses, such as rsv, adenovirus, influenza and parainfluenza viruses, responsible for respiratory tract inflammation, apnoea and hypoxemia, which can exceptionally lead to death [2,3,11.-18] . other viruses involved in common childhood diseases such as hsv, ebv, cmv, hhv-6, parvovirus b19 and enterovirus have also been detected in lungs, heart and csf of deceased infants, but their involvment in the death remained sometimes uncertain [2, 3, [19] [20] [21] [22] [23] [24] [25] . vzv has been reported in only one case of suid involving a healthy 17-month-old boy who unexpectedly died 3 days after onset of benign varicella. enzyme-immunoassay with monoclonal antibodies to vzv revealed disseminated infection involving the skin, the lungs, the liver, the spleen and the gastrointestinal tract [26] . to our knowledge, the present case is the first description of a cmv and vzv co-infection in suid. in this case report, the infant's clinical presentation and circumstances of the death were consistent with a suid syndrome. the term suid syndrome refers to those suid cases, in which no cause of death is found despite a complete autopsy, histology of all organs and thorough biological investigations. the varicella diagnosed clinically a few days prior to the death was considered mild with no alarming symptoms. however, pulmonary necrotizing inflammatory lesions, immunohistochemistry, and molecular detection of cmv and vzv in multi-organs samples demonstrated the role of the co-infection in the death. varicella zoster virus is responsible for a common childhood disease, which is benign in most cases. the morbidity rate is estimated at 4% and the mortality at 1 per 100,000 children, mainly due to viral or bacterial pulmonary superinfections [27, 28] . several risk factors to develop a severe form of varicella were evidenced in the present case. the main risk factor was prematurity. premature newborns tend to be at higher risk to develop a severe infection because of their lymphocyte-related immune system immaturity. moreover, some studies have shown a link between the pregnancy term and the newborn's antibodies titers against vzv in an immune mother [29, 30] . specific igg antibodies are mainly transmitted to the fetus during the third trimester of the pregnancy leading to lower titer in a premature newborn. furthermore, the antibodies decrease is faster in the first months following the birth of a premature than in an infant born on due term. it is noteworthy that in our case, the severe infection occurred despite maternal protecting antibodies against vzv. the protection level might have not been sufficient because of prematurity. another risk factor could be the virulence of the strain, itself. vzv strains that had lost an immunodominant b-cell epitope on the glycoprotein e ectodomain have been reported to have exhibited enhanced cell-to-cell spread in cell culture as well as in the scid-hu mouse [31, 32] . in the present case, the sequencing of the glycoprotein e coding gene did not reveal any particular virulence regarding the strain isolated (not shown; accession number: kf056799). the cmv co-infection should also be considered as a risk factor to develop a severe and disseminated varicella. cmv is well known for its immunosuppressive properties through many mechanisms including homologue il-10 production [24, 33] . even if the kinetics of viral infections is impossible to prove because of the lack of samples previous to the death, cmv infection should have occurred first, before the infection due to vzv, either during the pregnancy or at the birth by reinfection or reactivation of the mother or during the two months of the infant's life, leading to cmv-related immunosuppression therefore promoted the multivisceral spread of the vzv. as far as diagnostic methods are concerned, molecular tools were used in several post-mortem protocols to diagnose viral infections in suid and revealed more viruses than reported with less sensitive methods such as immunofluorescence assay or cell culture [2] . published studies also showed that qualitative pcr was not sufficient to determine the role of viruses in suid [2, 3, 25, 34] . in our case, the use for the first time of virus quantitation in combination with histology permitted the assessment of the role of the viruses in the pathogenesis of suid. moreover, molecular tests were positive on frozen tissues, but not on paraffin-embedded tissues. this could be explained by modification of nucleic acids caused by formalin fixation. indeed, this process can cause significant protein-nucleic acid crosslinks as well as fragmentation of nucleic acids reducing the quantity and size of nucleic acids suitable for amplification [35] . moreover, nucleic acids extract from paraffin-embedded tissues often contains remnants of substances that can inhibit the amplification reaction. these results showed that, without quantitative virology on frozen tissues, viral infection can be underestimated as a cause of death in suid. the high viral loads assessed in the lungs associated with necrotizing inflammatory lesions suggest subclinical hypoxia as the possible mechanism of the death [14] . despite the presence of virus in other organs, histology was normal raising the question of the pathogenic role of viruses in this condition. in conclusion, this first reported case of cmv and vzv coinfection as a cause of suid demonstrates that quantitative molecular biology on frozen tissues is mandatory for the diagnosis in combination with histology. none. none of the authors of the present manuscript have a commercial or other association that might pose a conflict of interest (e.g., pharmaceutical stock ownership, consultancy). the present study was conducted by the university hospital of reims (champagne-ardenne, france) and was approved by the hospital's ethics committee. contribution of bacteriology and virology in sudden unexpected death in infancy virological investigations in sudden unexpected death in infancy (sudi) virological analysis in the diagnosis of sudden children death: a medico-legal approach microbiology in sudden infant death syndrome (sids) and other childhood deaths identification and characterization of persistent human erythrovirus infection in blood donor samples assessment of automated dna extraction coupled with real-time pcr for measuring epstein-barr virus load in whole blood, peripheral mononuclear cells and plasma real-time pcr for rapid diagnosis of entero-and rhinovirus infections using lightcycler pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr development of a real-time polymerase chain reaction assay for the diagnosis of human herpesvirus-6 infection and application to bone marrow transplant patients development of a real-time pcr procedure including an internal control for the measurement of hcmv viral load sudden death in a toddler with laryngotracheitis caused by human parainfluenza virus-1 sudden death in toddlers with viral meningitis, massive cerebral edema, and neurogenic pulmonary edema and hemorrhage: report of two cases sudden death from human parainfluenza virus 2 viruses and sudden infant death detection of rna viruses in sudden infant death (sid) detection of respiratory syncytial virus using immunohistochemistry detection and significance of adenoviruses in cases of sudden infant death role of respiratory viral infection in sids: detection of viral nucleic acid by in situ hybridization detection of human herpesvirus-6, epstein-barr virus and cytomegalovirus in formalin-fixed tissues from sudden infant death: a study with quantitative real-time pcr cytomegalovirus-induced pneumonia and myocarditis in three cases of suspected sudden infant death syndrome (sids): diagnosis by immunohistochemical techniques and molecularpathologic methods pcr-based diagnosis of enterovirus and parvovirus b19 in paraffin-embedded heart tissue of children with suspected sudden infant death syndrome coxsackie b3 myocarditis in 4 cases of suspected sudden infant death syndrome: diagnosis by immunohistochemical and molecular-pathologic investigations cmv-dna detection in parenchymatous organs in cases of sudden infant death syndrome cytomegalic inclusion disease of the salivary glands in sudden infant death syndrome enterovirus in sudden unexpected deaths in infants varicella-zoster virus replication site in internal organs of an otherwise healthy child with varicella and sudden death paediatric varicella hospitalisations in france: a nationwide survey neonatal antibody titers against varicella-zoster virus in relation to gestational age, birth weight, and maternal titer transplacental transport of igg antibodies to preterm infants: a review of the literature varicellazoster virus ge escape mutant vzv-msp exhibits an accelerated cell-to-cell spread phenotype in both infected cell cultures abd scid-hu mice antigenic variation of varicella zoster virus fc receptor ge: loss of major b cell epitope in the ectodomain potent immunosuppressive activities of cytomegalovirus-encoded interleukin-10 role of virus-induced myocardial affections in sudden infant death syndrome: a prospective postmortem study morphological quality and nucleic acid preservation in cytopathology key: cord-293886-gbv1ipmn authors: cunha, burke a.; pherez, francisco; walls, nicole title: severe cytomegalovirus (cmv) community-acquired pneumonia (cap) in a nonimmunocompromised host date: 2008-10-01 journal: heart lung doi: 10.1016/j.hrtlng.2008.05.008 sha: doc_id: 293886 cord_uid: gbv1ipmn background: community-acquired pneumonia (cap) in an immunocompetent host may be severe because of a variety or combination of host and microbial factors. in patients with severe cardiopulmonary dysfunction, even relatively avirulent pathogens, that is, mycoplasma pneumoniae, moraxella catarrhalis, may compromise borderline cardiac/heart function and present clinically as severe cap. alternately, patients with streptococcus pneumoniae and impaired humoral immunity/splenic dysfunction may present as severe cap. with the exception of legionnaire's disease, influenza, and adenovirus, pathogen virulence is not a key determinant of cap severity. methods: diagnostically, patients with severe cap may be approached based on the pattern of infiltrates on chest x-ray together with the severity of hypoxemia (ie, increased a-a gradient: >35). results: we present the case of an immunocompetent adult who presented with severe cap during peak influenza season. direct fluorescent antibody testing of his respiratory secretions was negative for influenza, adenovirus, and other respiratory viruses. diagnostic bronchoscopy was negative for bacterial and fungal pathogens. the only clues to the cause of his severe cap was the presence of relative lymphopenia, atypical lymphocytosis and elevated serum transaminases. after influenza and adenovirus were ruled out, cytomegalovirus (cmv) cap was considered. the diagnosis of cmv cap was made serologically by demonstrating highly elevated igm cmv titers. because the diagnosis was made during the patient's recovery late in hospitalization, he did not receive cmv antiviral therapy. conclusion: this case should remind clinicians that influenza and adenovirus are diagnostic considerations in patients presenting with severe cap with diffuse bilateral interstitial infiltrates accompanied by severe hypoxemia in normal hosts. if influenza and adenovirus are ruled out, then cmv cap, although rare, should be considered, particularly when viral cap is accompanied by relative lymphopenia, atypical lymphocytosis and increased serum transaminases. c ytomegalovirus (cmv) is a dna virus in the family herpesviridae. cmv transmission from person to person requires body fluid contact. cmv initially penetrates host epithelial cells and produces cell enlargement, thus the term "cytomegalovirus." after initial infection by the immune system suppresses the virus and cmv remains latent unless reactivated by impaired cell-mediated immunity (cmi). [1] [2] [3] there are many clinical manifestations of cmv infection that may be divided into acute primary infection and reactivation. acute infection occurs in neonates via vertical transmission from the mother. in immunocompetent adults, acute infection is manifested by an infectious mononucleosis-like syndrome that is benign and self-limited. in immunocompro-mised hosts (ie, organ transplant recipients), patients with impaired cmi, and patients with human immunodeficiency virus (hiv), severe disease is often seen. 4, 5 acute cmv or reactivation of latent infection can affect almost any organ. however, the most common manifestations are retinitis, pneumonia, encephalitis, hepatitis, and colitis. severe infection is rare in immunocompetent hosts. [1] [2] [3] 6, 7 we present a case of severe cmv community-acquired pneumonia (cap) in an immunocompetent host. a 64-year-old man was admitted with shortness of breath. he had a 2-day history of a "flu-like illness" characterized by fever, myalgias, and progressive breathlessness. the patient had seen his primary physician, who prescribed oseltamivir and moxifloxacin, but the shortness of breath progressed and he went to the hospital for further evaluation and treatment. on admission, the patient's temperature was 103°f with a simultaneous pulse of 107 beats/min. he appeared to be in mild to moderate respiratory distress. he had a negative medical history, was a normal host, and had no risk factors for hiv. his physical examination results were unremarkable except for bilateral diffuse rales. admission laboratory tests revealed a white blood cell count of 35 k/mm 3 (polymorphonuclear neutrophils ϭ 83%, lymphocytes ϭ 14%, atypical lymphocytes ϭ 5%, and monocytes ϭ 3%), a platelet count of 581 k/mm 3 (n ϭ 160-392 k/mm 3 ), an erythrocyte sedimentation rate of 82 mm/h (n ϭ 1-20 mm/h), a c-reactive protein level of 11.3 mg/l (n ϭ ͻ3 mg/l), a blood urea nitrogen level of 24 mg/dl (n ϭ 8-21 mg/dl), and a creatinine level of 0.9 mg/dl (n ϭ 0.6-1.2 mg/dl). serum transaminases were mildly elevated: serum glutamate oxaloacetate transaminase ϭ 171 iu/l (n ϭ 13-39 iu/l), serum glutamate pyruvate transaminase ϭ 135 iu/l (n ϭ 4-36 iu/l), and alkaline phosphatase ϭ 181 iu/l (n ϭ 25-100 iu/l). respiratory fluorescent antibody panel for influenza a and b virus, respiratory syncytial virus, adenovirus, parainfluenza virus, and metapneumovirus was negative. his initial chest x-ray showed bilateral interstitial markings that rapidly progressed over 24 hours (figs 1 and 2) . diagnostic bronchoscopy was unrevealing, and bronchoalveolar lavage (bal) was negative for pneumocystis (carinii) jiroveci pneumonia. bacterial and fungal cultures were negative. bal cytology was negative for viral inclusions. during the patient's hospitalization, he became progressively hypoxemic and required intubation and mechanical ventilation. his a-a gradient on room air was 194. mycoplasma pneumoniae, c. pneumoniae, herpes simplex virus (hsv) 1 and 2, and q fever titers were negative. legionella titers and urinary legionella antigen testing were also negative. atypical lymphocytosis persisted and peaked at 13%. he slowly improved over 14 days, and antibiotics were discontinued after 20 days. his oxygen requirements gradually decreased, and he was eventually extubated. influenza a titers were 1:8 (n ϭ ͻ1:8), and influenza b titers were 1:16 (n ϭ 1:8). cold agglutinins were repeatedly negative. cmv polymerase chain reaction (pcr) was negative. his initial immunoglobulin igm cmv titer was 1.87 (n ͻ 1.1 isr). ten cap may be caused by a variety of typical and atypical bacterial pathogens, as well as some viruses. the severity of cap depends on host factors (ie, cardiopulmonary function), immune system status (ie, splenic function), and, in some cases, the virulence of the pathogen. patients with preexisting severe cardiac or pulmonary disease, if pneumonia develops, may present with severe cap. superimposed cap stresses already compromised cardiopulmonary function and may manifest clinically as severe cap. even relatively avirulent pathogens (eg, m. pneumoniae, m. catarrhalis) may manifest as severe cap in patients with impaired cardiopulmonary function. with cap caused by encapsulated bacteria (eg, streptococcus pneumoniae, haemophilus influenzae), splenic function is the primary determinant of cap severity. any of the above factors alone or in combination in immunocompetent hosts may present clinically as severe cap. nonetheless, some pathogens are inherently highly virulent and often present as severe cap. all other things being equal, cap is likely to be severe in patients with legionnaire's disease, influenza, systemic acute respiratory distress syndrome (sars), hanta pulmonary virus syndrome (hps), or adenovirus. [8] [9] [10] cmv pneumonia occurs almost exclusively in patients with impaired cmi/impaired t-lymphocyte function (eg, organ transplant recipients and those with hiv) or who are receiving immunomodulating/ immunosuppressive agents. [1] [2] [3] [4] [5] 8 cmv cap results from the reactivation of a previous/latent dormant cmv infection. although clinically similar, hsv-i pneumonia, an often overlooked but important cause of nosocomial pneumonia, does not present as cap. 8, [11] [12] [13] [14] in the compromised host with impaired cmi, cmv cap pneumonia presents early (ͻ48 hours) with fever and profound/prolonged hypoxemia with minimal or no pulmonary infiltrates. pulmonary infiltrates may appear later (ͼ48 hours) with cmv cap. nonspecific laboratory abnormalities often associated with cmv cap include leukopenia, relative lymphopenia, and thrombocytopenia. 8, 15, 16 atypical lymphocytes may or may not be present in the peripheral smear. cmv infection regularly involves the liver, manifested in cmv cap by mild elevations of serum transaminases. [1] [2] [3] in patients with hiv, cmv is an "innocent bystander" copathogen in approximately 75% of cases with pneumocystis (carinii) jiroveci pneumonia (pcp). in patients with pcp, the underlying cmv does not need to be treated. in patients with hiv with pcp, in contrast, pcp resolves without specifically treating the cmv component. however, in organ transplant recipients, cmv cap is treated with specific anti-cmv antiviral therapy. 8, 17 because cmv cap is rare in immunocompetent adults, the natural history of cmv cap is not well understood. cap cmv is clinically indistinguishable from other viral pneumonias. as with cmv, other viral pneumonias may be accompanied by leukopenia, relative lymphopenia, or thrombocytopenia. viral pneumonias typically present with minimal or few infiltrates on chest x-ray and are accompanied by severe hypoxemia with a high a-a gradient (ie, ͼ35). 8, 18 the diagnosis of cmv cap may be made serologically or pathologically. the serologic diagnosis of cmv depends on demonstrating an elevated cmv igm titer. cmv pcr is often negative, but if positive it is also diagnostic. pathologically, cmv inclusion bodies may be seen in transbronchial/open lung biopsy specimens pathognomonic for cmv, that is, cytoplasmic inclusions, cellular gigantism, and "kidney bean-shaped" intranuclear inclusions. bal is usually negative and therefore unhelpful in diagnosing cmv cap. in contrast, in hsv-1 nosocomial pneumonia, cytopathic examination of bronchial epithelial cells is diagnostic for hsv-1. therefore, in the absence of viral inclusion bodies in transbronchial, percutaneous, or open lung biopsy specimens, the clinical diagnosis of cmv cap is based on the clinical presentation of viral pneumonia plus increased igm cmv titers or a positive cmv pcr. 3, 8, 19 in the case presented, an immunocompetent adult presented with severe cap requiring intensive care and ventilatory support. [20] [21] [22] [23] [24] his hypoxemia was out of proportion to auscultation and chest x-ray findings. minimal bilateral, symmetrical, interstitial chest x-ray infiltrates on the chest x-ray with an increased a-a gradient (ͼ35) and minimal infiltrates suggested a process causing an oxygen diffusion defect (ie, viral pneumonia). 8, 22, 24 because he was admitted during influenza season, the initial working diagnosis was viral influenza. however, rapid influenza testing and respiratory viral fluorescent antibody panel were negative for influenza and other respiratory viruses. viral influenza and adenoviral titers were also negative. because he was an immunocompetent host, cmv was not an initial diagnostic consideration. 5, 6 the possibility of cmv was later considered in view of some of his otherwise unexplained laboratory findings, for example, atypical lymphocytosis (5%-13%) and increased serum transaminases (serum glutamate oxaloacetate transaminase ϭ 103-331 iu/l, n ϭ 13-39 iu/l; serum glutamate oxaloacetate transaminase ϭ 164-441 iu/l, n ϭ 4-36 iu/l). diagnostic bronchoscopy was performed, and cytology was negative for hsv-1 and cmv. viral cultures of bronchial washings were negative for cmv. because the diagnosis was confirmed after the patient's recovery, which was slow and prolonged, he was not treated for cmv cap. the clinical approach to a patient with severe cap depends on the assessment of the cardiopulmonary and splenic function, distribution of the pulmonary infiltrates on chest x-ray or chest computed tomography scan, and degree of hypoxemia/ magnitude of the a-a gradient. 8, [22] [23] [24] patients with severe cap may be approached clinically as having either focal or nonfocal infiltrates. those with focal chest x-ray infiltrates without a large a-a gradient (ͻ35) have severe cap usually as the result of impaired cardiopulmonary/ splenic function. the pathogens causing severe cap with focal chest x-ray infiltrates without a large diffusion defect (ie, a-a gradient ͻ 35) are the typical bacterial cap pathogens: s. pneumoniae, h. influenzae, m. catarrhalis, m. pneumoniae, and c. pneumoniae. in contrast, in patients presenting with severe cap with focal chest x-ray infiltrates and a large a-a gradient (ͼ35), legionnaire's disease or, less commonly, adenoviral cap is the primary diagnostic consideration. if a patient presents with severe cap and no or minimal infiltrates, when accompanied by a large a-a gradient (ͼ35), then an interstitial infectious process should be considered. interstitial pathogens causing pneumonia with an oxygen diffusion defect are respiratory viruses: influenza, respiratory syncytial virus, systemic acute respiratory distress syndrome and hanta pulmonary virus syndrome. 8 pcp is found virtually only in individuals with hiv and immunosuppressed patients (ie, transplants), steroids (table i) . 8, 22 in an "apparently normal" host, the diagnosis of otherwise unexplained pcp should prompt hiv testing. 8 influenza a is the prototypical cause of severe viral cap. patients with severe influenza a have a severe oxygen diffusion defect and die of profound hypoxemia without bacterial suprainfections. a minority of patients with influenza have superimposed s. aureus cap. influenza with superimposed bacterial pneumonia is easily recognized in patients because there are focal infiltrates on chest x-ray. later in viral influenza (ie, ͼ48 hours), there may be bilateral patchy chest x-ray infiltrates which are not nonsegmental or lobar infiltrates, and indicate a superimposed bacterial pneumonia. 8, 18 the clinical presentation of respiratory viral pathogens presenting as severe cap is difficult to differentiate clinically. many clinical and laboratory features overlap. common differential diagnostic problems include differentiating viral influenza from adenoviral cap. 8, 18, [22] [23] [24] ordinarily, cmv cap in immunocompetent hosts is not a diagnostic consideration. in the case presented, a viral influenza was suspected because the case occurred during the peak of the influenza season. adenoviral cap also occurs during the late winter and early spring months and was also a likely diagnostic possibility. because the patient did not have leukopenia, relative lymphopenia, or thrombocytopenia, cmv was considered as a possible pathogen only after influenza and adenoviral cap were ruled out. although it is true that influenza and adenovirus pneumonia may be associated with mildly increased serum transaminases, tests for these pathogens were negative, prompting testing for an unusual diagnosis (ie, cmv). the patient's igm cmv titer was highly elevated (5.65 isr; n ͻ 1.1 isr), which confirmed the diagnosis in this case (table ii) . 8, 18 we believe the mildly elevated influenza b titers were the result of cmv cross-reactivity. 8, 25, 26 severe cmv infections are rare in immunocompetent hosts. in the largest review published to date, 34 cases of cmv infection ranging from isolated encephalitis to disseminated disease with multiple organ involvement were described. 7 in this case series, cmv cap was the sole manifestation of cmv infections in only 1 case (1/34). cmv cap was present in 9 of 34 cases. in 5 of 9 cmv cap cases, lung infection was accompanied by hepatic involvement, an important clue to the diagnosis. the diagnosis of cmv infection was confirmed serologically in 23 of 34 patients. the single patient with isolated cmv cap without liver involvement survived without anti-cmv therapy, as was the case in the patient presented. 7 in severe cap with normal/near-normal chest x-rays and profound hypoxemia accompanied by leukopenia, relative lymphopenia, thrombocytopenia, or elevated serum transaminases, the primary diagnostic considerations should be viral influenza and adenovirus. the diagnosis of influenza should be doubted without leukopenia and relative lymphopenia, which are key features of influenza with both diagnostic and prognostic significance. the diagnosis of severe influenza or adenovirus cap should be questioned in the absence of leukopenia with relative lymphopenia, and alternate diagnoses should be considered. however, if viral influenza and adenovirus are ruled out in patients with otherwise unexplained atypical lymphocytosis and increased serum transaminases, clinicians should consider the possibility of cmv in immunocompetent adults presenting with severe cap. cytomegalovirus biology and infection philadelphia: lippincott-raven publishers cmv pneumonia after human marrow transplantation cytomegalovirus pneumonia in adult nontransplantation patients with cancer: review of 20 cases occurring from 1964 through 1990 cytomegalovirus infection in the normal host severe cytomegalovirus infection in immunocompetent patients community acquired pneumonia severe legionella pneumonia: rapid diagnosis with winthrop-university hospital's weighted point score system (modified) acute respiratory disease associated with adenovirus serotype 14 -four states herpes simplex-1 (hsv-1) pneumonia herpes simplex virus type 1 (hsv-1) pneumonia presenting as failure to wean herpes simplex virus (hsv) pneumonia in a heart transplant: diagnosis and therapy a cluster of nosocomial hsv-1 pneumonia in an intensive care unit cytomegalovirus pneumonia. semin community-acquired pneumonia due to cytomegalovirus, herpes simplex 1 virus, and human herpesvirus-6. in: marrie tj, ed. community-acquired pneumonia cmv in the lungs of patients with aids: respiratory pathogen or passenger? the clinical diagnosis of severe viral influenza a the diagnosis of cmv pneumonitis in lung and heart/ lung transplant patients by pcr compared with traditional laboratory criteria severe community-acquired pneumonia severe community-acquired pneumonia pulmonary infections in the compromised host severe community acquired pneumonia: determinants of severity and approach to therapy severe community acquired pneumonia in the critical care unit diagnostic significance of elevated cold agglutinin titers in infectious disease mycoplasma pneumoniae pneumonia: diagnostic significance of highly elevated cold agglutinin titers key: cord-011197-bmigh2rs authors: yener, nazik; üdürgücü, muhammed title: airway pressure release ventilation as a rescue therapy in pediatric acute respiratory distress syndrome date: 2020-03-03 journal: indian j pediatr doi: 10.1007/s12098-020-03235-w sha: doc_id: 11197 cord_uid: bmigh2rs objectives: to describe experience with airway pressure release ventilation (aprv) in children with severe acute respiratory distress syndrome (ards) refractory to conventional low tidal volume ventilation. methods: this retrospective observational study was performed in an 11-bed, level 3 pediatric intensive care unit. evaluation was made of 30 pediatric patients receiving airway pressure release ventilation as rescue therapy for severe ards. results: patients were switched to aprv on an average 3.2 ± 2.6 d following intubation. when changed from conventional mechanical ventilation (cmv) to aprv, there was an expected increase in the spo(2)/fio(2) ratio (165.1 ± 13.6 vs. 131.7 ± 10.2; p = 0.035). mean peak inspiratory pressure was significantly lower during aprv (25.4 ± 1.26 vs. 29.8 ± 0.60, p < 0.001) compared to cmv prior to aprv but mean airway pressure (p(aw)) was significantly higher during aprv (19.1 ± 0.9 vs. 15.3 ± 1.3, p < 0.001). hospital mortality in this study group was 16.6%. conclusions: the results of this study support the hypothesis that aprv may offer potential clinical advantages for ventilatory management and may be considered as an alternative rescue mechanical ventilation mode in pediatric ards patients refractory to conventional ventilation. acute respiratory distress syndrome (ards) is the most severe form of acute respiratory failure, characterised by severe diffuse inflammation and hypoxemia that poses a significant threat to patients of all age groups. currently, lung-protective ventilation strategies, including open lung and low-tidal-volume, are among the major ards mechanical ventilation strategies to prevent ventilator-induced lung injury for both adults and children [1] . despite advancements in our understanding of lung-protective low-tidal-volume ventilation, the mortality associated with pediatric ards remains high and has changed little in the last 20 years (22-40%) [2, 3] . no consensus has been reached on the optimal mode of ventilation for pediatric ards patients refractory to conventional mechanical ventilation (cmv) using low tidal volume combined with sufficient positive end expiratory pressure (peep). over the past 3 decades, such patients have commonly transitioned from cmv to high frequency oscillation ventilation (hfov) for refractory hypoxemia or to limit cyclic high peak pressures [1] . unfortunately, there is a lack of relevant hfov research on pediatric populations as only one small cohort randomized controlled trial (rct) has been conducted [4] . approximately 20 y ago, airway pressure release ventilation (aprv) was introduced as a partial ventilatory mode for mechanical ventilation in clinical practice. however, until recently, it was not widely used as a rescue mode for the difficult-to-oxygenate patient with ards. as the name suggests, aprv mode has been described as a continuous positive airway pressure with a brief intermittent release phase allowing the release of only partial lung volume. in adult patients with ards, compared with other conventional ventilatory modes, aprv may improve oxygenation due to increased recruitment of lung volumes, length of stay in the intensive care unit and ventilator-free days [5, 6] . until 2018, pediatric research on aprv was restricted to case reports, case series and prospective crossover studies [7] . a recent study by lalgudi ganesan et al. [8] was the first randomised control trial (rct) of aprv as a primary ventilation strategy in children with ards. the trial was terminated early after 50% enrolment when the analysis demonstrated a trend toward higher mortality in the aprv group compared with the conventional low-tidal volume ventilation. however, there is a lack of data investigating the safety and efficacy of aprv as a rescue therapy in pediatric ards. the aim of this study was to describe authors' experience with aprv in children with severe ards refractory to conventional low tidal volume ventilation. this retrospective observational study was conducted in the 11-bed pediatric intensive care unit of ondokuz mayıs university hospital, a tertiary level hospital in turkey. approval for the study was granted by the university ethics committee. the study included patients aged between 1 mo and 18 y, receiving aprv ventilation during index admission to the picu and who fulfilled the diagnostic criteria of ards, according to the pallic definition and were refractory to conventional low tidal volume ventilation. determination of failure of cmv and the decision to employ alternative modes was left to the discretion of the attending intensive care physician. despite the lack of a standardized ventilator protocol, the authors' institutional practice for ards is to initiate cmv with a minimum of 5 cm h 2 o of positive end-expiratory pressure (peep), low tidal volume (6-8 ml/kg predicted body weight) and to attempt to wean fio 2 to ≤0.60. inability to wean fio 2 prompts elevation level of peep (10-15 cm h 2 o) with the goal of maintaining plateau airway pressure at no more than 30 cmh 2 o. prone positioning is a part of the authors' clinical practice if it is not possible to wean fio 2 to ≤0.60 during conventional low tidal volume ventilation. the prone position and the other non-ventilatory co-interventions (inhaled nitric oxide or extracorporeal membrane oxygenation) for ards were not used during aprv. persistently elevated plateau pressures (≥ 30 cm h 2 o), or oxygenation difficulties (inability to wean fio 2 ≤ 0.60 despite increasing peep) prompted consideration of changing the mode of ventilation. as there is no hfov ventilator in authors' clinic, aprv was used as a rescue mode in children with severe ards when cmv does not achieve a specific target level of oxygenation as previously described. patients receiving aprv for <8 h, or for an indication other than ards were excluded. all patients were ventilated with a servo-i (maquet, germany) ventilator and aprv parameters were initially adjusted by the attending intensive care physician. the authors' instutitional practice is personalized-setting aprv (p-aprv) where the low pressure (p low) is set at zero to facilitate rapid emptying and the brief expiratory time (t low) prevents full deflation of the lung end-expiratory pressure from reaching zero [9] . data were retrieved from the medical records of eligible patients including the diagnosis on admission, demographic data, co-morbidities, date and time of intubation, mode of ventilation prior to initiation of aprvand date and time of aprv initiation, length of mechanical ventilation, length of stay in picu and survival to hospital discharge. a record was made of ventilator settings, the ph and venous co 2 values, sedation and vasopressor use before and after aprv (at 3 h) and evidence of pneumomediastinum or pneumothorax at any time on aprv. the oxygen saturation to fraction of inspired oxygen ratio (spo 2 /fio 2 ) was calculated before and after aprv, and the oxygen saturation index [osi = mean airway pressure (p aw ) × fio 2 × 100 ÷ spo 2 ] was calculated prior to aprv. radiographic requirements for the diagnosis of ards were derived from the attending radiologist's final chest x-ray report. data obtained in the study were analysed statistically using ibm spss vn 23 (spss inc., chicago, il, usa). data were expressed as mean ± standard deviation and as the median and interquartile ranges (iqr), or percentages. conformity of quantitative data to normal distribution was assessed with the shapiro-wilk test and the paired samples t-test was used in the comparisons of normally distributed variables. a value of p < 0.05 was accepted as statistically significant. of the patients ventilated with aprv between october 2015 and april 2019, 33 remained on aprv for more than 8 h and 30 patients met the study inclusion criteria. three patients were on aprv ventilation for <8 h, did not tolerate it and were placed back on cmv; of these, mortality was seen in 2 with a diagnosis of drowning, and 1 with sepsis survived. of the remaining 33 patients who were on aprv ventilation for >8 h, 3 patients were on aprv for non-ards reasons (2 with cardiogenic pulmonary edema, 1 with pulmonary hemorrhage) and were excluded. the median age of the patients enroled in the study was 28 mo (iqr 14.75-42.5), 83% were younger than 5 y old and 20% were infants. the demographic data and outcomes are presented in table 1 . following a diagnosis of ards, all 30 patients were ventilated with conventional low tidal volume and high peep ventilation with the synchronized intermittent mandatory ventilation mode with pressure support (simv+ps). ventilation measurements before and after transition to aprvare presented in table 2 . mean maximum peep was 13 ± 1.3 (range 11-15) during cmv. all patients had severe ards as per the pallic criteria, with mean osi 13.4 (12.5-18.5) prior to aprv. patients were switched to aprv on average 3.2 ± 2.6 d (range: 1-11 d) following intubation. during the observational period, all patients required sedation (including, benzodiazepines and narcotics). when changed from cmv to aprv, the sedation requirements increased in 4 patients, decreased in 5 and remained unchanged in all the others. at 3 h after patients were switched to aprv there was an expected increase in the spo 2 / fio 2 ratio (165.1 ± 13.6 vs. 131.7 ± 10.2, p = 0.035). mean peak pressure was significantly lower during aprv (25.4 ± 1.26 vs. 29.8 ± 0.60, p < 0.001) compared to cvm prior to aprv but p aw was significantly higher during aprv (at 3 h) (19.11 ± 0.97 vs. 15.32 ± 1.3, p < 0.001. mean mandatory breaths in aprv were 12.8 ± 1.0 (10-15). when changed from cmv to aprv, 53% of the patients received vasopressor. vasopressor requirement and the ph and venous co 2 values were not significantly different before and after aprv. renal replacement therapy was given to six patients in the study group. patients were switched to simv+ ps as a weaning mode. after a weaning period of 4.9 ± 1.6 d, all patients were extubated with non-invasive ventilation (niv). mortality was seen in 5 patients during aprv. the primary cause of death was worsening hypoxemia in 4 patients and multi organ dysfunction in 1. in 4 of these 5 patients, there was a co-morbidity, and in 1, the cause of death was sepsis. all the patients with mortality were aged <5 y old and 3 were < 2 y old. of the 25 patients who survived, 23 (80%) were spontaneously breathing at 4 d after extubation. two patients could not tolerate weaning from niv and were discharged home with niv. in 1 patient with a history of reactive airway diseases, bilateral pneumothorax developed, which required bilateral chest tubes during aprv. these events occurred 3 d post-aprv initiation and there was no associated early mortality. in-hospital mortality for the cohort was 16.6% (5/30). aprv is an inverse ratio pressure control mode of mechanical ventilation that was first described and introduced into clinic practice more than two decades ago [10] . although different mode names are used, aprv is now available in almost all critical care ventilators. the main findings of this study were that in a cohort of pediatric patients with severe ards, when cmv did not achieve a specific target level of oxygenation, initiation of aprv was able to significantly and sustainably improve oxygenation. inverse ratio ventilation (irv), hfov and aprv are non-conventional modes of ventilation considered as a rescue treatment for patients with moderate to severe ards who are refractory to cmv [11, 12] . all these nonconventional modes increase oxygenation by increasing the average p aw [13] . in the current study, when transfer was made from cmv to aprv mode, the average p aw increased significantly. as the inspiration duration is relatively much longer than the expirium duration in both irv and aprv, the duration at high pressure in the respiratory cycle is prolonged, causing elevation in the p aw values. it is thought that high p aw values increase oxygenation by opening the lungs [14] . in the current study, there was a statistically significant decrease in peak airway pressure when cmv was switched to aprv. this decrease is thought to be protective against lung damage associated with the ventilator. in literature, there is no consensus on how the initial settings should be determined for aprv [15] . in accordance with the findings of the current study, it could be considered appropriate to start with a lower p high than pip in cmv when determining the initial settings in the transfer to aprv from cmv as a rescue mode in children. the number of mandatory breaths in aprv is set much lower compared to cmv, but the patient can breathe spontaneously at each point of the ventilation cycle. a large part of the spontaneous respiration occurs in the longer inspirium duration. although spontaneous respiration has a positive effect on the protection of diaphragm functions, there is insufficient information about the effect on lung dynamics and vili formation of spontaneous breathing occurring in the long p high duration during aprv [16] . in the current study, the mean number of mandatory breaths during aprv was determined as 12.8 ± 1.0. although the results of a low number of breaths have not been investigated for direct aprv, previous animal experimental studies have shown that a high number of mandatory breaths during cmv, especially with high tv, led to additional damage in the lungs [17, 18] . to increase patient compliance during both hfov and irv, heavy sedation and muscle blockage are often required [13] . that the use of intense sedation and analgesia is not required in adults during aprv has been emphasised as an advantage in literature. in two different prospective studies of adults with trauma and cardiac surgery, controlled mechanical ventilation was compared with aprv and it was concluded that there was a reduced requirement for sedation and analgesia in aprv [19, 20] . in the current study, there was no significant difference in the use of sedation and analgesia in the transition from cmv to aprv. nevertheless, in the current study, there was no requirement for the use of muscle blockage in any patient during both cmv and aprv. as the benefits of aprv are based on the spontaneous breathing component, the advantage of this method may be lost on patients who need heavy sedation or neuromuscular paralysis with lack of spontaneous breathing. possible contraindications to aprv include patients with obstructive lung disease (asthma or chronic obstructive pulmonary disease) who require prolonged exhalation time and conditions that may worsen with the elevation of the mean airway pressure, such as unmanaged increases of intracranial pressure and large bronchopleural fistulas. in addition, the limited research and experience with this form of ventilation can be a potential problem in certain facilities (neuromuscular disease) [13, 21] . no consensus has been reached in literature on the subject of how weaning can be achieved in patients applied with aprv. while some authors have reduced p high until it is cpap mode, it is recommended that weaning is applied by increasing t high [22, 23] . in the current study, first p high was reduced before starting weaning and when a sufficient drop was achieved, transition was made when tolerated to simv+ ps (peep = 5 cmh 2 o) mode, as this mode is well known by authors' team. following ventilation with simv+ps mode for mean 4.9 ± 1.8 d after aprv, all the surviving patients were extubated with niv. to minimise failure of extubation, prophylactic niv was applied immediately on extubation. of the 25 patients of the current series who survived, 23 were able to be discharged with spontaneous breathing. in the other 2 patients, pulmonary damage developed, so niv was set and they were discharged home with niv. all the current study patients were applied with aprv in rescue mode, and all had been previously ventilated with lung-protective cmv (at mean 13 peep) for mean 3.2 d. therefore, the ventilator-related lung damage that developed was not considered to be related to aprv alone. with the exception of 1 patient with a history of reactive airway disease who developed pneumothorax, no significant complications were observed. although the number of cases in this study is low, aprv can be considered a safe ventilation method for children. a trend toward mortality benefit has only been shown in one small retrospective study as 25 of 58 patients: 31% in the aprv group, compared to 59% in the simv group (p < 0.05) [24] . however, lalgudi ganesan et al. [8] recently conducted the first rct of aprv in pediatric ards cases. the study had to be terminated after 50% enrolment (52 children) as analysis showed higher mortality rates in the aprv group. it was concluded that the use of aprvas a primary ventilation strategy in children with ards resulted in higher mortality rates compared with conventional low tidal volume ventilation. in an rct by yehya et al., [9] of 60 immunocompromised and ards pediatric patients refractory to conventional ventilation, the mortality rate was 63% and did not differ between patients transitioned to aprv and hfov. hospital mortality in the current study group (16.6%) was below the range of recently published mortality rates (22-40%) in clinical trials of pediatric ards [2, 3] . there are several limitations to this study. first, as this was a retrospective observational study, conducted with the intent of describing the outcomes of patients with severe ards refractory to cmv, who were then treated with aprv, there was no direct comparison with other conventional ventilation methods. second, the sample size was small so it may not have been of sufficient power to detect improvements in oxygenation before and after transition to aprv. third, the determination of failure of cmv and initiation of aprv were based on the decision of the intensive care physician rather than a unit guideline. in addition, as a sedation scale was not used, there could not be any evaluation of whether or not there was a difference in the requirement for sedation and analgesia. in conclusion, the results of this study demonstrated that in a cohort of pediatric patients with severe hypoxemic respiratory failure when cmv did not achieve a specific target level of oxygenation, initiation of aprv is associated with a significant and sustained improvement in oxygenation. these results support the hypothesis that aprv may offer potential clinical advantages for ventilatory management and may be considered as an alternative rescue mechanical ventilation mode in pediatric ards patients refractory to conventional ventilation. as aprv is commonly available on intensive care ventilators when hfov is not always accessible, this mode can be considered a relatively simple modality that can be implemented easily. pediatric acute lung injury consensus conference group. pediatric acute respiratory distress syndrome: definition, incidence, and epidemiology: proceedings from the pediatric acute lung injury consensus conference pediatric acute respiratory distress syndrome prospective, randomized comparison of highfrequency oscillatory ventilation and conventional mechanical ventilation in pediatric respiratory failure combined effects of prone positioning and airway pressure release ventilation on gas exchange in patients with acute lung injury airway pressure release ventilation as a primary ventilatory mode in acute respiratory distress syndrome airway pressure release ventilation in children airway pressure release ventilation in pediatric acute respiratory distress syndrome: a randomized controlled trial high frequency oscillation and airway pressure release ventilation in pediatric respiratory failure airway pressure release ventilation current role of high frequency oscillatory ventilation and airway pressure release ventilation in acute lung injury and acute respiratory distress syndrome severe hypoxemic respiratory failure: part 1: ventilatory strategies airway pressure release ventilation: what do we know? mean airway pressure: physiologic determinantsand clinical importance: part 2: clinical implications does airway pressure release ventilation offer important new advantages in mechanical ventilator support? respir care should airway pressure release ventilation be the primary mode in ards? respir care airwayv pressure release ventilation prevents ventilator-induced lung injury in normal lungs effects of respiratory rate on ventilator-induced lung injury at a constant paco2 in a mouse model of normal lung the influence of controlled mandatory ventilation (cmv), intermittent mandatory ventilation (imv) and biphasic intermittent positive airway pressure (bipap) on duration of intubation and consumption of analgesics and sedatives. a prospective analysis in 596 patients following adult cardiac surgery long-term effects of spontaneous breathing during ventilatory support in patients with acute lung injury airway pressure release ventilation: an alternative mode of mechanical ventilation in acute respiratory distress syndrome other approaches to open-lung ventilation: airway pressure release ventilation airway pressure release ventilation: theory and practice practical use of airway pressure release ventilation for severe ards -a preliminary report in comparison with a conventional ventilatory support publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contribution ny: study design, literature search and manuscript preparation; mu: data collection, analysis of data and review of manuscript. ny is the guarantor fort his paper. conflict of interest none. key: cord-276343-sb3vd7fq authors: humar, atul; doucette, karen; kumar, deepali; pang, xiao-li; lien, dale; jackson, kathy; preiksaitis, jutta title: assessment of adenovirus infection in adult lung transplant recipients using molecular surveillance date: 2006-12-31 journal: the journal of heart and lung transplantation doi: 10.1016/j.healun.2006.09.015 sha: doc_id: 276343 cord_uid: sb3vd7fq background little is known about adenovirus infections in adult lung transplant recipients. because the virus can establish latency, re-activation may be relatively common after transplantation. methods we assessed adenovirus infection in 80 adult lung transplant recipients. adenovirus polymerase chain reaction (real-time pcr assay; limit of detection ∼25 copies/ml plasma) was done on plasma samples collected at regular intervals until 1 year post-transplant. results adenovirus dna was detected in 18 of 80 patients (22.5%) and in 19 of 595 (3.4%) plasma samples up to 12 months post-transplant. median time to detection of viremia was 134 days post-transplant (range 1 to 370 days). median viral load was 180 copies/ml plasma (range 50 to 360 copies/ml). symptoms were evaluated at the time of adenovirus detection: 14 of 18 (78%) patients were asymptomatic; 4 of 18 (22%) patients had otherwise unexplained febrile/flu-like illness that resolved spontaneously. adenovirus was not found to be a trigger for acute rejection. no detrimental effect on pulmonary function was seen immediately after adenovirus infection. conclusions adenovirus viremia is common in adult lung transplant recipients. in contrast to findings on adenoviral pneumonitis in lung transplant recipients, isolated episodes of low-level viremia are self-limited and do not trigger acute rejection or a decline in pulmonary function. adenoviruses are double-stranded dna viruses. over 50 serotypes are known to cause human infections, including respiratory syndromes, gastrointestinal disease and febrile syndromes in immunocompetent hosts. 1,2 after infection, lifelong immunity appears to be serotypespecific and therefore infections with other serotypes may occur. in addition, the virus can establish latency in numerous tissues, including tonsils, adenoids, intestine and urinary tract. 2 therefore, transplant patients may be at risk of adenovirus infection either from new acquisition or from re-activation of latent virus. in transplant patients, the spectrum of clinical sequelae due adenovirus infections seems to be broad, and may also differ in pediatric and adult populations. [3] [4] [5] in children, acquisition of new viruses may be more common, whereas in adults re-activation of endogenous latent virus may be more likely to occur. severe life-threatening disseminated disease is well described in both adult and pediatric hematopoietic stem cell recipients and solidorgan recipients. [5] [6] [7] [8] overall, lung transplant recipients with adenovirus infection appear to be at high risk for severe necrotizing pneumonitis, resulting in significant graft dysfunction and associated with high mortality. 9, 10 infection of the allograft by adenovirus has also been associated with acute and chronic rejection and graft dysfunction in studies of lung and heart transplant recipients. 11, 12 however, recent data have suggested that adenovirus re-activation and viremia may be relatively common in transplant recipients and, in most cases, may not be associated with adverse clinical sequelae, especially if there is no evidence of allograft infection. 13 we undertook the present study to assess the incidence, timing and clinical sequelae of adenovirus viremia in lung transplant recipients. patients in this study were enrolled as part of a clinical trial comparing valganciclovir prophylaxis with ganci-clovir (intravenous and oral) as cytomegalovirus (cmv) prophylaxis in d ϩ /r ϫ and r ϩ lung transplant recipients. the design of the previous study with regard to inclusion and exclusion criteria has been described in detail elsewhere. 14 patients were enrolled at two canadian transplant centers and were transplanted between 1999 and 2002. the institutional review board at each center approved the study and informed consent was obtained from the patients. the original study evaluated cmv prophylaxis and, as part of this study, patients had received oral ganciclovir, intravenous ganciclovir or oral valganciclovir up to 3 months post-transplant. all patients in both groups had plasma samples saved for further testing at regular intervals until 1-year posttransplant. initially, this was done usually every 2 weeks and then at monthly intervals. all plasma samples underwent adenovirus testing using a quantitative realtime polymerase chain reaction (pcr) assay (see later) and cmv viral load testing by quantitative pcr (roche cobas amplicor assay, roche diagnostic systems, branchburg, nj). the results of viral load testing (both adenovirus and cmv) were not available to the treating physician. no pre-transplant samples were available for testing as part of this study. in the first year, protocol bronchoscopies were performed at only one of the two participating centers. these took place at weeks 2 and 6 post-transplant and then at months 3, 6, 9 and 12 post-transplant. routine microbiologic testing was performed as well as a transbronchial biopsy to assess for rejection. two sets of primers and probes were designed from the highly conserved hexon gene of adenovirus type 2 and 4 using primerexpress software (abi) designed for the detection of all serotypes. 15 dna was extracted from 200 l of plasma using a qiagen dna mini-kit according to the manufacturer's protocol (qiagen) and eluted from the column with 50 l of elution buffer. to quantify adenovirus in plasma, an external standard curve was established with a 10-fold series of dilution from one copy to 1.0eϩ06 copies using a 375-bp dna fragment produced from type 2 adenovirus. real-time taqman pcr was conducted in a closed-tube sequence detection system (model 7000, abi-prism, applied biosystems). briefly, the pcr was performed in a 25-l volume containing 12.5 l of universal dna master mix (applied biosystems), 10 l dna, 400 nmol/liter each primer and 200 nmol/liter of each probe. after initial incubation at 50°c for 2 minutes to activate uracil-nglycosylase and then at 95°c for 10 minutes for denaturing, pcr amplification was performed with two-step thermal cycles of 94°c for 20 seconds and 60°c for 1 minute by 45 cycles after re-heating at 95°c for 5 minutes. amplification data were collected and analyzed by computer (sequence detection system, version 1.0, applied biosystems). the dynamic range of detection of the pcr was 25 to 2.5eϩ07 copies/ml plasma in normal healthy controls. adenovirus dna is not detectable from plasma samples when using this assay. in patients with detectable adenovirus viremia by pcr, clinical disease was attributed to adenovirus if detectable viremia was accompanied by compatible symptoms (including febrile illnesses, respiratory, gastrointestinal disease or evidence of infection of the allograft) in the absence of another defined etiology. in addition, all complications and microbiologic data during the first year post-transplant were assessed to determine whether adenovirus infection was diagnosed clinically outside the study evaluation. acute rejection (grade ն2) was diagnosed on the basis of a transbronchial biopsy that demonstrated characteristic perivascular lymphocytic infiltrates using criteria defined by the international society for heart and lung transplantation (ishlt). 16 in patients in whom a biopsy could not be performed, a clinical diagnosis of rejection was permitted (i.e., a deterioration in lung function with no other identifiable etiology that responded to high-dose corticosteroid therapy). effects on pulmonary function in patients with adenovirus infection were determined based on measurement of forced expiratory volume in 1 second (fev 1 ). an fev 1 decline of ͼ15% within 3 months of adenovirus infection was considered potentially significant and occurring within a time period in which virally triggered declines in graft function may be playing a role. cmv disease was defined according to standard criteria. 17 informed consent was obtained from all patients for participation in the study. all clinical research was conducted according to institutional guidelines for human experimentation. a total of 80 lung transplant recipients were analyzed (63 double-lung and 17 single-lung transplant recipients) (table 1). a total of 595 plasma samples were tested (mean number of samples per patient ϭ 7.4, range 4 to 12). mean age was 49.6 years (range 19 to 68), which included 27 women and 53 men. pre-transplant underlying lung disease included cystic fibrosis (n ϭ 18), emphysema/ chronic obstructive pulmonary disease (copd; n ϭ 28), idiopathic pulmonary fibrosis (n ϭ 21), ␣ 1 -anti-trypsin deficiency (n ϭ 5) or "other" (n ϭ 8). maintenance immunosuppression was cyclosporine/prednisone/azathioprine (n ϭ 43), cyclosporine/prednisone/mycophenolate (n ϭ 29) or other (n ϭ 8). a total of 23 of 80 (28.8%) patients received induction anti-lymphocyte globulin. cmv prophylaxis consisted of intravenous ganciclovir for 2 weeks (all patients), followed by 12 weeks of oral ganciclovir (n ϭ 30), intravenous ganciclovir (n ϭ 10) or oral valganciclovir (n ϭ 40). adenovirus dna was detected in 18 of 80 patients (22.5%) in the first 12 months post-transplant. of the total 595 samples tested from these patients, 19 (3.4%) were positive. quantitative viral load data are shown in figure 1 . the median viral load was 180 copies/ml (range 50 to 410 copies/ml). viremia was detected on one occasion in 17 of the 18 patients and on two occasions in 1 patient. timing of viremia post-transplant is shown in figure 2 , and was relatively evenly distributed throughout the first year post-transplant. median time to detection of first viremia was 134 days posttransplant (range 1 to 370 days). donors were not specifically tested for adenovirus. donor chart review for the cases in which virus was detected early in the recipient did not reveal any clinical or radiologic evidence of active infection in the donor. symptoms were reviewed at the time of viremia. of the 18 patients with viremia, 14 (78%) had no symptoms at the time of viremia. four of 18 patients (22%) had symptoms. all symptoms consisted of a febrile, flu-like illness. no patients had clinical or radiologic evidence of pneumonitis at the time of viremia, and adenovirus pneumonitis was not documented in any of the other study patients during the study period. in the 4 patients with viremia and symptoms, no other etiology of symptoms was found (all were negative for cmv pp65 antigenemia). in all cases, symptoms resolved spontaneously without any specific therapy and without any change in immunosuppression. adenovirus viral load was compared in symptomatic vs asymptomatic patients. median viral load was 185 copies/ml in patients with symptoms (range 86 to 346) vs 180 copies/ml in asymptomatic patients (range 50 to 410; p ϭ not significant [ns] ). underlying disease in these 4 patients was cystic fibrosis (n ϭ 2), idiopathic pulmonary fibrosis (n ϭ 1) and emphysema (n ϭ 1). immunosuppression consisted of cyclosporine, prednisone and azathioprine in 3 of the 4 patients. the low viral loads observed in the present study are in contrast to the high viral loads we observed in the clinical setting among transplant patients with documented adenoviral pneumonitis. in the previous respiratory virus season, 2 patients (1 kidney transplant recipient and 1 lung transplant recipient, neither part of this study) were diagnosed with adenovirus pneumonitis. viral loads in these patients were 6.5 ϫ 10 5 and 9.5 ϫ 10 5 copies/ml plasma, respectively. risk factors for viremia were also analyzed ( (table 1) . no specific epidemiologic links were observed between patients with viremia, and no evidence of seasonality was apparent. adenovirus infection was not found to be a trigger for acute rejection (clinically treated or biopsy proven). the overall incidence of one or more acute rejection episodes in patients with adenovirus viremia was 3 of 18 (16.7%) vs 34 of 62 (54.8%) in non-viremic patients (p ϭ 0.01). in only 1 case did an episode of acute rejection develop within the 3 months after viremia, whereas, in 2 cases, the rejection episode occurred before viremia. in all 18 patients with viremia, pulmonary function tests (fev 1 ) remained stable in the immediate 3 months after detection of viremia. no interaction with the development of cmv viremia or symptomatic cmv disease was observed. cmv viremia occurred in 8 of 18 (44.4%) patients with adenovirus vs 27 of 62 (43.5%) patients without adenovirus (p ϭ ns). symptomatic cmv disease occurred in 4 of 18 (22.2%) patients with adenovirus vs 11 of 62 (17.7%) patients without adenovirus (p ϭ ns). in patients with both adenovirus and cmv viremia (n ϭ 8), cmv preceded adenovirus in 5 patients and occurred after adenovirus in 3 patients. mean peak cmv viral load in patients with adenovirus was 8,630 copies/ml (range undetectable to 56,000 copies/ml) vs 8,645 copies/ml (range undetectable to 81,900 copies/ ml) in patients without adenovirus (p ϭ ns). cmv prophylaxis did not influence detection of adenovirus viremia. the number of patients who were positive while on anti-cmv prophylaxis was 8 of 18 (45%), similar to the rate of positivity after discontinuation of prophylaxis. we have shown that adenovirus viremia is very common in lung transplant recipients, and was detected in 18 of 80 (22.5%) of the patients during the first year post-transplant. the majority of infections were not associated with any symptoms. in 4 of 18 (22%) patients, symptoms likely attributable to adenovirus were present and consisted of a febrile, flu-like illness. in all patients with symptoms, resolution occurred without use of anti-viral therapy or reduction of immunosuppression. viral loads were generally low in these patients (range 86 to 346 copies/ml), and viremia was not sustained. however, this did represent true replicative infection rather than detection of latent virus, because only cell-free samples (i.e., plasma) were used for viral detection. adenovirus viremia was not associated with any evidence of indirect effects on graft function or presence of acute rejection. our results are in contrast to previous reports of adenovirus infection in lung transplant recipients. for example, in a study of pediatric lung and heart-lung recipients, specimens (swabs, bronchoalveolar lavage fluid) were prospectively tested for respiratory viruses by culture, pcr and antigen detection. 12 in 16 patients adenovirus was identified in the transplanted lungs of 8 patients. of these patients, 2 appeared to have donor-transmitted early fulminant adenovirus pneumonitis. overall, adenovirus infection of the transplanted lung was significantly associated with graft loss, obliterative bronchiolitis (ob) and death due to respiratory failure. 12 in another study, biopsy and autopsy specimens were analyzed for adenovirus by immunohistochemistry and in situ hybridization in 308 lung transplant recipients. 9 adenovirus pneumonitis was identified in 4 patients (1.3%). all cases had severe necrotizing pneumonia and were fatal. three of the 4 cases were in pediatric transplant recipients and all cases occurred within 45 days of transplant, suggesting either donor transmission or early acquisition/re-activation in the recipient. 9 late fatal adenovirus pneumonitis occurring several years after the lung transplant has also been described. 10 the pcr assay we used had a sensitivity of 25 copies/ml. this would be expected to be significantly more sensitive than the culture or direct antibody staining methods commonly used for respiratory specimens. also, we assessed blood samples as opposed to respiratory specimens. both of these factors may have partially accounted for the higher rate of infection observed compared with other studies. respiratory viral infections in adult lung transplant recipients have long been suspected as potential triggers for allograft rejection and the development of obliterative bronchiolitis or its clinical equivalent, bronchiolitis obliterans syndrome (bos). taking into account the limitations of recent studies (most with small, retrospective sample sizes), the literature supports an association between respiratory virus infection and bos. 18 for example, khalifah et al 19 retrospectively reviewed 259 lung transplant recipients (respiratory samples for cultures and antigen detection), and found 21 respiratory viral infections (including 2 cases of adenovirus respiratory tract infection); these patients were at increased risk for bos and death. kumar et al 20 prospectively studied 100 lung transplant patients, including 50 patients with respiratory virus infection and 50 controls. for respiratory specimens, molecular detection methods (pcr) were used in addition to standard viral techniques. adenovirus was not identified in any of the patients, but the most common etiologies of viral infection were rhinovirus, coronavirus, respiratory syncytial virus (rsv) and influenza. viral infection was associated with acute rejection and a significant decline in fev 1 over time, consistent with the development of bos. there is evidence from other organ recipients that adenovirus infection of the allograft may be associated with indirect effects. shirali et al 11 performed viral pcr on endomyocardial biopsies from pediatric heart transplant recipients and found that adenovirus was the most commonly detected virus found in 24 of 149 (16.1%) patients. viral detection was associated with an increased risk of coronary vasculopathy and graft loss. overall, the data on adenovirus infection in lung transplant recipients suggest that infections are severe, especially in pediatric patients, and are associated with necrotizing pneumonia and result in a decline in pulmonary function. however, our data suggest that, in adults, infections are very common and often asymptomatic or associated with mild self-limited symptoms. this discrepancy may be due to several factors. first, we assessed viremia rather than infection within the allograft. it may be that the two sites of infection behave very differently in terms of symptomatology and clinical consequences. second, used active molecular surveillance techniques, which are more likely to pick up sub-clinical cases, as compared with symptom-based diagnostic evaluation. third, we did not assess pediatric patients, who may be more likely to have newly acquired adenovirus in the absence of pre-existing immunity. therefore, it should be emphasized that, although no significant clinical effects of low-level viremia were observed, patients with higher viral loads may well have more severe disease or an association with rejection. it is possible that most cases of adenovirus we detected may have been due to re-activation of a previously latent virus. however, the possibility of newly acquired adenovirus due to exogenous exposure cannot be excluded. our results are consistent with recently published molecular surveillance data in other organ transplant recipients. 13 in a study of 263 adult kidney (and kidneypancreas), liver and heart transplant recipients, who had testing of blood samples at regular intervals, the incidence of detectable viremia was 7.2%. in 58% of these patients viremia was asymptomatic, whereas in the remaining patients viremia was associated with self-limited respiratory or gastrointestinal symptoms. in that study, the investigators did not detect an association with subsequent allograft rejection. 13 limitations of our study include the small sample size and the fixed testing intervals. for example, testing may not have necessarily been carried out at all time-points at which patients may have had compatible symptoms (e.g., respiratory or gastrointestinal symptoms). it is therefore possible that the incidence of infection may have been even higher. no additional cases of adenovirus were clinically diagnosed other than the patients who were positive by our surveillance; however, specific pcr testing for adenovirus was not routinely performed for clinical purposes during the study period. finally, this study was considered a sub-study, part of a cmv prophylaxis study, and so adenovirus surveillance was not the primary objective. in conclusion, adenovirus viremia is very common in adult lung transplant recipients. such infections may be different from those in which the allograft is primarily involved. viremic episodes were either asymptomatic or associated with only self-limited febrile syndromes despite no change in immunosuppression or specific anti-viral therapy. no association with acute rejection episodes or with a decline in lung function was observed after infection. at present, routine pcr surveillance after lung transplantation would not be indicated based on the data presented, although further studies are required. the impact of adenovirus infection on the immunocompromised host principles and practice of infectious diseases adenovirus, parvovirus b19, papilloma virus, and polyomaviruses after hemopoietic stem cell or solid organ transplantation adenovirus infection in pediatric liver and intestinal transplant recipients: utility of dna detection by pcr adenovirus infection in adult orthotopic liver transplant recipients: incidence and clinical significance adenovirus infections in adult recipients of blood and marrow transplants fatal disseminated adenoviral infection in a renal transplant patient adenovirus: an increasingly important pathogen in paediatric bone marrow transplant patients adenovirus pneumonia in lung transplant recipients late fatal adenovirus pneumonitis in a lung transplant recipient association of viral genome with graft loss in children after cardiac transplantation adenovirus infection in the lung results in graft failure after lung transplantation a surveillance study of adenovirus infection in adult solid organ transplant recipients a trial of valganciclovir prophylaxis for cytomegalovirus prevention in lung transplant recipients enhanced identification of viral and atypical bacterial pathogens in lower respiratory tract samples with nucleic acid amplification tests revision of the 1990 working formulation for the classification of pulmonary allograft rejection: lung rejection study group american society of transplantation recommendations for screening, monitoring and reporting of infectious complications in immunosuppression trials in recipients of organ transplantation infectious etiology of bronchiolitis obliterans: the respiratory viruses connection-myth or reality? respiratory viral infections are a distinct risk for bronchiolitis obliterans syndrome and death clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant the authors thank jayne fenton and kate toogood for assistance with data collection and sample preparation. key: cord-354374-rtgjjglc authors: c.g. pollok, richard; j.g. farthing, michael title: enteric viruses in hiv-related diarrhoea date: 2000-12-01 journal: mol med today doi: 10.1016/s1357-4310(00)01816-5 sha: doc_id: 354374 cord_uid: rtgjjglc hiv-related diarrhoea is an important cause of morbidity and mortality in hiv infection. cytomegalovirus is a well-established cause of diarrhoea, but the role of other enteric viruses is less clear and will be discussed here. the clinical manifestations, disease mechanisms, diagnostic techniques and current treatments for the management of these infections are reviewed. cmv infection of the gi tract, in patients with aids have diminished greatly. following the initiation of haart, aids patients with cmv can successfully discontinue anticmv treatment without reactivation of the disease and with a parallel reduction in cmv viraemia. the pathogenic role of cmv infection is well established. although retinitis is the most common manifestation of infection, before the introduction of combination antiretroviral therapy, gi infection of the oesophagus, stomach, small bowel and colon occur in 5-15% of patients during the course of hiv infection. the incidence of cmv retinitis has declined following the introduction of haart, with a concomitant increase in survival 5 . this is also presumably true of gi cmv, although published data are limited. although cmv can infect any part of the gi tract, the most common site of infection is the colon 6 , and the most common manifestation of cmv colitis is chronic or intermittent diarrhoea in association with abdominal pain. the disease is also associated with mild or severe rectal bleeding or abdominal pain in the absence of diarrhoea and, in addition, fever is common at presentation. pain can precede the development of toxic megacolon, and intestinal perforation is rare but clearly life threatening. colonic cmv infection can occur in association with infection elsewhere in the gi tract including the oesophagus, which usually results in dysphagia and odynophagia, and the pancreatico-biliary tree, which results from aids-related cholangiopathy or pancreatitis and manifests as pain in the upper abdomen. gi infection can also herald cmv retinitis and careful retinal assessment is, therefore, essential in this group. the mechanisms by which cmv induces gi disease are poorly understood. cmv infection of the mucosa is associated with a marked local inflammatory response, which might have a role in inducing mucosal ulceration. wilcox and colleagues found that mrna levels of the proinflammatory cytokine tumour necrosis factor ␣ (tnf-␣) were elevated in oesophageal mucosa of patients with cmv oesophagitis, but returned to normal following treatment of cmv (ref. 7) . similarly, sharpstone et al. noted increased levels of tnf-␣ in faecal samples from patients with gi cmv (ref. 8) . the enteric ischaemia that results from cmvassociated vasculitis might conceivably play a role in gi cmv (ref. 9) . although this hypothesis remains unexplored in cmv enteritis, a similar mechanism has been invoked to explain the aetio-pathogenesis of the opportunistic viral enteritis is a potentially important gastrointestinal (gi) manifestation of hiv-related disease. however, with the exception of cytomegalovirus (cmv) and human herpes simplex virus (hsv), which are established aetiological agents of disease in the gi tract in patients with hiv, the role of other enteric viruses remains controversial. between 44% and 82% of hiv patients with chronic diarrhoea have a pathogen that is readily identifiable using a well-established diagnostic protocol that includes stool culture, microscopy and histological examination of biopsies obtained by endoscopy of the upper and lower gi tract [1] [2] [3] [4] . for example, using such a protocol, 82% of 155 cases of persistent diarrhoea were related to infection with identifiable bacteria, parasite or cmv, with cmv alone accounting for 18% of these cases 4 . the additional techniques of stool electron microscopy and specific immunological staining implicated other enteric viruses in a further 11% of cases. the role of these other viruses in so-called 'pathogen-negative' diarrhoea remains uncertain. the clinical importance of hiv enteropathy is probably limited. several viruses, including astrovirus, picobirnavirus, small round structured virus (srsv) and rotavirus, are implicated in hiv-related diarrhoea. in addition, adenovirus is associated with persistent diarrhoea in patients with characteristic adenovirus colitis. the evidence for a pathological role for these viruses will be discussed ( table 1) . it is well established that cmv can infect the gi tract and cause diarrhoea and methods of diagnosis and current treatments are reviewed. the spectrum of disease morbidity and mortality among hiv patients has altered dramatically since the widespread introduction of highly active antiretroviral therapy (haart). opportunistic infections, including a spectrum of both upper and lower gi manifestations of cmv infection have been described and a definitive diagnosis of cmv enterocolitis requires intestinal biopsy. in a prospective study, wilcox and colleagues evaluated 55 patients with hiv by sigmoidoscopy and colonoscopy. chronic diarrhoea and abdominal pain occurred in 80% and 50% of patients respectively and 9% presented with lower gi bleeding with a previous history of diarrhoea 12 . endoscopically, appearances were heterogeneous. three main categories were identified: (1) colitis associated with ulceration (39%); (2) ulceration alone (38%); or (3) colitis alone (20%). subepithelial haemorrhage was common in all groups. a total of 31 patients underwent complete endoscopy to the caecum. of these, four (9%) had disease that was proximal to the splenic flexure without distal involvement that was therefore inaccessible by flexible sigmoidoscopy. this contrasts with early reports suggesting higher rates of right-sided colitis 13 . the same group has assessed the varied endoscopic appearances of cmv infection of the oesphagus 14 . multiple ulcers, located in the middle or lower oesophagus, were identified in 58% of patients. these were usually less than 1 cm in diameter and superficial. the heterogeneous manifestation of cmv disease in both the colon and oesophagus makes biopsy essential for accurate diagnosis. a histologically based diagnosis of cmv enterocolitis depends largely on identification of characteristic cytomegalovirus inclusion bodies in samples usually obtained from the base of cmv ulcers. in addition, specific immunoperoxidase staining for cmv is also useful in identifying gi disease 15 ; positive staining is more likely from the edge of ulcers and some authors claim greater sensitivity compared to conventional histology, although it is unclear from these reports how rigorously inclusions bodies have been excluded 16, 17 . the value of a viral culture of intestinal biopsies is limited as this is a time-consuming procedure. furthermore, cmv viraemia can occur in the absence of mucosal disease, which can lead to a false-positive diagnosis if biopsies are contaminated with blood. although using in situ hybridization to assess gi cmv disease is both sensitive and specific, it probably offers little additional benefit over conventional histology 18 . using the pcr for the detection of cmv might also be more sensitive than using standard histological techniques; however, evidence suggests that this technique lacks specificity, with 28% of normal gi biopsies positive by pcr. furthermore, as with in situ hybridization, there is the risk of pcr giving a false-positive result in patients with viraemia. the sensitivity of conventional histology, viral culture, pcr and immunohistochemistry are summarized in table 2 (ref. 19 ). cotte and colleagues monitored cmv dna levels during treatment and levels broadly reflected clinical response 20 . newer techniques are now available to monitor the natural history and progression of cmv disease in the post-haart era. salmon-ceron and colleagues have compared three blood markers of cmv, levels of pp65 antigenaemia in viral culture, plasma levels of cmv dna and levels of late cmv mrna, in the assessment of cmv-disease progression in patients on haart. in a multivariate analysis, plasma cmv dna, raised pp65 antigenaemia or a cd4 count of 75 cells l ϫ1 were identified as independent risk factors in the development of cmv disease 21 . others have compared these techniques with retinal assessment in the evaluation of cmv retinitis. the digene hybrid capture cmv dna system was 85% as sensitive as retinal assessment, which is similar to the more cumbersome pp65 antigenaemia assay (80% as sensitive) 22 . table 3 glossary cholangiopathy -pathology relating to the biliary tree. crohn's disease -chronic inflammatory condition of the gastrointestinal tract, the cause of which remains unknown. enteric virus -virus with a tissue tropism for the intestinal tract. odynophagia -pain on swallowing. oesophagitis -inflammation of the oesophagus. pancreatitis -inflammation of the pancreas. retinitis -inflammation of the retina that can lead to visual impairment or blindness. toxic megacolon -pathological dilatation of colon associated with certain infections and inflammatory conditions of the bowel. viraemia -blood-borne carriage of virus. treatment of cmv enterocolitis requires parenteral therapy with either ganciclovir (5 mg kg ϫ1 twice daily) or foscarnet (90 mg kg ϫ1 twice daily), both of which can cause severe side effects. although these agents improve both endoscopic and symptomatic markers of cmv disease, the survival benefit is uncertain and needs to be re-evaluated following the introduction of haart (ref. 23) . ganciclovir can cause severe bone marrow depression with resultant anaemia and neutopaenia, and foscarnet can cause severe renal impairment (although a concomitant infusion of normal saline largely diminishes the risk of renal damage). in an open label, randomized study comparing a two-week course of foscarnet with ganciclovir, at the doses stated above, there was no significant difference in response of gi cmv disease between the two therapies 24 . approximately 75% of patients had good clinical and endoscopic responses with disappearance of inclusion bodies as determined histologically. relapse occurred within ten weeks in at least 50% of patients and survival without haart was ϸ20 weeks. surprisingly, blanshard and colleagues found that maintenance therapy did not increase the time taken for relapse of gi disease, although numbers were small and allocation of maintenance therapy was not randomized. the use of oral ganciclovir in maintenance therapy for retinitis is effective but has not been specifically evaluated for gi disease 25 . the efficacy of treatment with oral ganciclovir for primary prophylaxis of cmv disease is disputed and concerns about the development of viral resistance have been raised 26, 27 . it is hoped that newer agents, such as cidofovir, famiclovir, valganciclovir and formivirsen, will have a role in the management of cmv disease 28 . encouragingly, the advent of haart has led to a marked reduction in mortality and morbidity of hiv patients 29 . haart can result in remission of previously persistent opportunistic infections, including cmv infection 5 . however, the development of viral resistance and difficulties with compliance could lead to breakthrough hiv viraemia and the re-emergence or reacquisition of opportunistic gi infections. unfortunately, combination antiretroviral therapy is costly and, consequently, unavailable to the vast majority of hiv patients worldwide. patients on haart with stable cmv retinitis and cd4 counts of 150 cells l ϫ1 were able to successfully discontinue anticmv maintenance therapy without relapse of retinitis or development of extraocular disease over a mean follow-up period of 16 months 30 . importantly, immune reactivation retinitis occurred in 90% of patients started on haart before discontinuation of anticmv treatment, with substantial visual loss in the minority of patients. macdonald and colleagues report similar findings, although the follow-up period was considerably shorter, and they do not comment on immune reactivation retinitis 31 . others find that haart causes a significant and progressive decline in cmv viraemia in the absence of specific anticmv treatment 32 . it is intriguing to speculate about the possible occurrence of immune reactivation enteritis in patients with gi cmv; this has not been described to date. many questions remain about the host immune response to opportunistic infections following immune reconstitution with haart. what are the specific immune mechanisms leading to disease resolution following haart? does the t-cell repertoire expand following reconstitution to recognize 'new' cmv antigens? when can secondary prophylaxis be discontinued in patients that respond to haart? what are the immune consequences of retroviral rebound when haart fails? how can cmv indices be monitored to predict relapse in patients on haart? adenovirus is reported to cause infection in other immunosuppressed groups, including individuals with primary immunodeficiency and bone-marrow-transplant patients 33 . in hiv patients, dionisio and colleagues report increasing stool carriage of subgenus f type 40 adenovirus with increasing immunosuppression 34 . janoff and colleagues first described adenovirus colitis 35 . electron microscopy or culture of colonic biopsies from 67 hiv patients investigated for diarrhoea identified adenovirus in five patients. colonoscopy revealed mild inflammatory change in two of these patients. focal necrosis and amphophilic nuclear inclusions within degenerating epithelial cells were shown using light microscopy, and electron microscopy revealed characteristic hexagonal adenovirus particles within the inclusions (fig. 1) . maddox et al. have confirmed these characteristic features and found that specific immunostaining for adenovirus is both sensitive and specific for the identification of adenovirus inclusions 36 . the pathogenic role of adenovirus remains unclear as this group of patients is frequently co-infected with other known pathogens. thomas and co-workers report that adenovirus colitis is significantly more likely to be associated with chronic diarrhoea 37 , while schmidt and colleagues were able to detect adenovirus only in aids patients who are severely immunosuppressed 38 . although in the severely immunosuppressed group, adenovirus was detected more frequently in patients with diarrhoea than without (10% vs 3.3%), both positive and negative correlations between adenovirus and diarrhoea have been reported by other groups (table 1) [37] [38] [39] [40] [41] [42] [43] . most studies indicate a strong association between infection with adenovirus and co-infection with other pathogens, in particular cmv. it is difficult, therefore, to ascribe a pathogenic role to this virus with any certainty [37] [38] [39] . infection of enterocytes by hiv is well documented and is implicated as the cause of so-called 'hiv enteropathy', in which morphological and functional abnormalities of the gut are described in the absence of any other detectable pathogen 44 . the clinical importance of hiv enteropathy is probably limited, certainly 'pathogen-negative' diarrhoea is comparatively short lived and associated with a good prognosis 45 . several other enteric viruses are associated with hiv-related diarrhoea. grohman and colleagues examined stool specimens from patients with or without diarrhoea 39 . electron microscopy, polyacrylamide-gel electrophoresis and enzyme immunoassay were used to examine samples for rotavirus, adenovirus, calcivirus and picobirnavirus. paired sera were also analysed for antibodies to norwalk and picobirnavirus. overall, virus was detected in 35% of patients with diarrhoea and 12% without diarrhoea. astrovirus, picobirnavirus, calcivirus (including srsv) and adenovirus were identified significantly more often in patients with diarrhoea. unfortunately, co-infection with other known pathogens was not evaluated systematically and no information regarding the relative distribution of acute and chronic diarrhoea was provided. schmidt and colleagues detected virus in 17% of stool samples from 256 hiv-infected patients 38 . adenovirus and coronavirus were detected more frequently in patients with diarrhoea than without (10% vs 3.3% and 15% vs 6.6% respectively) and both were associated with severe immunosuppression. thea et al. found no association between enteric virus shedding and diarrhoea in a study performed in zaire 40 . overall, this group identified enteric virus, including rotavirus, srsv, coronavirus and adenovirus in 17% of samples analysed. they noted a trend towards increased shedding with greater immunosuppression, a finding in common with cunningham and colleagues 43 . an association of rotavirus with prolonged diarrhoea in hiv patients, as detected by enzyme immunoassay was also reported, although other groups do not support this finding 41 . data from selected studies that have evaluated enteric virus carriage in hiv patients are summarized in table 3 . in summary, data regarding the association of non-cmv enteric virus infection and diarrhoea is conflicting. the best evidence relates to adenovirus infection although its pathogenic role is far from certain. we speculate that many of these viruses cause only self-limiting acute diarrhoea or, as in the case of adenovirus, act as a cofactor in cmv infection. little is known about therapeutic options for these putative pathogens, although ribavarin might be effective in the treatment of adenovirus (r.c.g. pollok, unpublished). the impact of haart on non-cmv enteric virus infection has not yet been evaluated and warrants study. new developments in the diagnosis and treatment of cmv are being established. assays for cmv dna and the cmv pp65 antigen assay offer the prospect of cmv surveillance before the development of end-organ disease, which will allow the tailored introduction of prophylaxis. oral ganciclovir and valganciclovir (which, of the two, has greater bioavailability) are potentially useful prophylactic agents. second generation cmv treatments are becoming available and are being evaluated, largely in transplant patients. the advent of haart has dramatically reduced the occurrence of all opportunistic infections, including cmv. 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count remain risk factors for cytomegalovirus disease in patients receiving highly active antiretroviral therapy comparison of three assays for cytomegalovirus detection in aids patients at risk for retinitis ganciclovir treatment of cytomegalovirus colitis in aids: a randomized, double-blind, placebo-controlled multicenter study treatment of aids-associated gastrointestinal cytomegalovirus infection with foscarnet and ganciclovir: a randomized comparison oral ganciclovir for cytomegalovirus retinitis in patients with aids: results of two randomized studies response of asymptomatic cytomegalovirus viraemia to oral ganciclovir 3 g/day or 6 g/day in hiv-infected patients a randomized, placebo-controlled trial of the safety and efficacy of oral ganciclovir for prophylaxis of cytomegalovirus disease in hiv-infected individuals. terry beirn community programs for recent advances in the therapy and prevention of cmv infections revision to the british hiv association guidelines for antiretroviral treatment of hiv seropositive individuals. bhiva guidelines writing committee discontinuation of anticytomegalovirus therapy in patients with hiv infection and cytomegalovirus retinitis lack of reactivation of cytomegalovirus (cmv) retinitis after stopping cmv maintenance therapy in aids patients with sustained elevations in cd4 t cells in response to highly active antiretroviral therapy decrease of cytomegalovirus replication in human immunodeficiency virus infected-patients after treatment with highly active antiretroviral therapy adenoviruses in the immunocompromised host chronic intestinal infection due to subgenus f type 40 adenovirus in a patient with adenovirus colitis in the acquired immunodeficiency syndrome adenovirus infection of the large bowel in hiv positive patients enteric viral infections as a cause of diarrhoea in the acquired immunodeficiency syndrome stool viruses, coinfections, and diarrhea in hiv-infected patients. berlin diarrhea/wasting syndrome study group enteric viruses and diarrhea in hiv-infected patients prevalence of enteric viruses among hospital patients with aids in kinshasa rotavirus antigen detection in patients with hiv infection and diarrhea prevalence of acute enteric viral pathogens in acquired immunodeficiency syndrome patients with diarrhea gastrointestinal viral infections in homosexual men who were symptomatic and seropositive for human immunodeficiency virus natural history and prognosis of diarrhoea of unknown cause in patients with acquired immunodeficiency syndrome (aids) key: cord-315304-pge45105 authors: kotton, c.n.; kuehnert, m.j.; fishman, j.a. title: organ transplantation, risks date: 2015-03-06 journal: reference module in biomedical sciences doi: 10.1016/b978-0-12-801238-3.02629-5 sha: doc_id: 315304 cord_uid: pge45105 viruses are among the most common causes of opportunistic infection after solid organ and hematopoietic stem cell transplantation (sot and hsct). viral infection is associated with both direct (invasive disease) and indirect (immune modulation) effects affecting susceptibility to other infections and promoting allograft rejection. the transplantation recipient is susceptible to a broad array of viral pathogens. some may be transmitted with the allograft as donor-derived infections, while others result from exposure, either soon after the transplant or from more distant exposures when infection is latent and reactivates in the setting of immune suppression. simultaneous infections with multiple viral or viral and nonviral pathogens are common. the risk for viral infection is a function of the intensity of exposure and virulence of the specific virus, the intensity of immune suppression used to prevent graft rejection or graft-versus-host disease, underlying immune deficits, and factors affecting host susceptibility. studies of viral latency, reactivation, and of the cellular effects of viral infection will provide clues for future strategies in prevention and treatment of viral infections. this article covers specific issues relating to viral infection in sot and hsct; additional details regarding these viral infections are also found elsewhere in this text. transaminases, coagulopathy, graft dysfunction, and either fever or leukocytosis within several weeks after transplantation. given the rarity of such infection diagnosed in normal hosts, there may be a predilection for this in transplant recipients. transplant-transmitted infection is rare and might be difficult to recognize, but physicians should consider the possibility, particularly when unexplained neurologic complications occur. these investigations underscore the challenge in detecting and diagnosing infections that occur in recipients of organs or tissues from a common donor. the potential for disease transmission from donor source may not be considered in recipient evaluation. in these investigations, the ability to connect illnesses to a common organ donor was facilitated by the fact that multiple recipients were hospitalized at the same facility. as organ and tissue transplantation becomes more common, the potential risks of disease transmission may also increase. because of improved diagnostic assays, donor-transmitted infections are increasingly recognized, although often times, the impact of infection is not well understood. the advent of polymerase chain reaction (pcr) and other genetic material-based tests have allowed for detection of active viremia, in contrast to serologic tests, which reflect past acquisition of infection. this is important not only for recipient diagnosis, but also for recognition of donor infection retrospectively; donor diagnosis is only possible if appropriate specimens are stored postmortem. recognition of viral infection transmitted through transplantation is increasing, likely both due to increased recognition of unexpected symptoms consistent with transmission, and due to increasingly sophisticated diagnostic testing in both the recipient and the donor. investigation of potential donor-transmitted infection requires rapid communication among physicians in transplant centers, organ procurement organizations, and public health authorities. an immediate system for tracking and disseminating pertinent patient data to evaluate donor-derived infection and associated adverse event outcomes is needed. until such a system can be established, clinicians should report unexpected outcomes or unexplained illness in transplant recipients to their local organ and tissue procurement organizations. given the frequency of latent viral infection, notably among herpesviruses, reactivation of latent infection provides a major source of infection after transplantation. the specific virus, the tissue infected, stimuli for activation, and the nature of the host immune response impact the nature of viral latency. some viruses are metabolically inactive when latent, while others continue to replicate at low levels that may be determined by the effectiveness of the host's immune response. multiple factors contribute to viral reactivation after transplantation, including graft rejection and therapy, immune suppression (especially reduction of t-cell mediated, cytotoxic immunity), inflammation, and tissue injury. numerous cellular pathways are involved in the control of viral replication and are activated after transplantation, such as nuclear factor kb, ikb, and jak-stat (the janus family of protein tyrosine kinases (jaks) and signal transducers and activators of transcription (stat) proteins). antirejection therapy can also result in a significant release of pro-inflammatory cytokines which may increase viral replication. in general, reactivation of viruses, especially late after transplantation, should suggest new immune defects (e.g., cancer) or relative over-immunosuppression. latency and reactivation has been best studied in the herpesviruses, which establish lifelong, latent infection after primary infection. in general, latency is considered to be the absence of viral replication, with viral genomes present in the cell without replication or spread. studies of other viruses, such as friend virus in mice, suggest that protective antiviral immunity is an active process mediated by 'leaky' (low-level) viral replication. the existence of true latency, as opposed to low-level replication, remains controversial. herpesviruses make 'latency' proteins that both control viral persistence within the target cell and influence other cellular processes. the latent state is characterized by low levels or the absence of detectable viral antigens, minimal transcription of productive or lytic cycle genes, and expression of the latency-associated viral transcripts. viral latency may be occasionally interrupted, leading to reactivation and spread of infectious virus with or without recurrent disease. ebv establishes latency in b lymphocytes in association with expression of a limited set of viral genes. immune control of hsv infection and replication occurs at the level of skin or mucosa during initial or recurrent infection and in the dorsal root ganglion, where latency and reactivation are controlled by immune mechanisms mediated by interferons, myeloid and plasmacytoid dendritic cells, cd4(+) and cd8(+) t cells, and other cytokines. despite similarities, the molecular details and mechanisms of latency and reactivation vary considerably among the herpesviruses. mechanisms responsible for maintenance of latency are unclear. reactivation of cmv has been extensively studied. cmv viral genomes can be found in cd14+ monocytes and cd34+ progenitor cells, although the primary reservoir for latent cmv and the mechanisms by which latency is maintained are unknown. allogeneic immune responses and fever (via tumor necrosis factor-a (tnf-a)) have been shown in vitro to increase both cmv promoter activity and viral replication. immune suppression is not essential for the reactivation of latent cmv, but serves to perpetuate such infections once activated. subclinical activation of cmv is common and increasing diagnosed by sensitive molecular assays. for other viruses such as bk polyomavirus, specific types of tissue damage such as warm ischemia and reperfusion injury may precipitate viral activation; they have been linked to an inflammatory state in grafts (via activation of tnf-a, nuclear factor kappa b (nf-kb), neutrophil infiltration, and nitric oxide synthesis), tubular-cell injury, and enhanced expression of cell-surface molecules, all of which may contribute to viral activation. thus, immune injury, inflammatory cytokines, and ischemia-reperfusion injury stimulate viral replication and change expression of virus-specific cell-surface receptors. the hosts' direct pathway antiviral cellular immune response within allografts is less effective due to mismatched major histocompatibility antigens between the organ donor and host with dependence on indirect pathways of antigen presentation. these factors may render the allograft more susceptible to viral infection. common reactivation infections after transplantation include cmv, hbv, hcv, hiv, hsv-1 and hsv-2, hpv, and vzv (as zoster). other less clinically common viral infections related to reactivation include the polyoma viruses bk and jc, human herpes virus 6 (hhv-6), human herpes virus 7 (hhv-7), and hhv-8. reactivation of one virus may lead to reactivation of others; multiple studies have shown that infection with hhv-6 and/or hhv-7 are risk factors for cmv disease and cmv infection may trigger hhv-6 and hhv-7 reactivation. while some reactivation infections routinely cause significant clinical disease, such as cmv, hsv, and vzv, others may cause more variable illness. hhv-6, for example, reactivates with immunosuppression, with clinically significant infection after hsct. by contrast, the role of both hhv-6 and hhv-7 in sot recipients is less well defined; while reactivation is common, clinical disease is generally not evident. based on epidemiologic exposures, new infections from the environment are commonly acquired after transplantation. the respiratory viruses are the most common new infections after transplantation, including rsv, influenza, parainfluenza, and adenovirus. newer respiratory pathogens (metapneumovirus and sars coronavirus) also cause major infections in immunocompromised hosts. gastrointestinal viruses such as rotavirus or norwalk virus are common and can cause significant diarrhea and dehydration; diarrheal syndromes may alter absorbance and metabolism of calcineurin inhibitors (e.g., cyclosporine and tacrolimus), with unexpectedly elevated levels of tacrolimus. nonimmune patients can acquire primary ebv, cmv, vzv, parvovirus b19, and other infections in the post-transplantation period. in the absence of previous immunity and with the attenuation of immunity due to the immunosuppressive regimen, new infections are often more severe and prolonged than in the general population. for example, parvovirus b19 infection is often more persistent and relapsing in transplantation patients, occasionally complicated by the unusual findings of hepatitis, myocarditis, pneumonitis, glomerulopathy, arthritis, or transplantation graft dysfunction. the effects of viral infection are conceptualized as 'direct' and 'indirect' (see table 2 ). this classification serves to separate the tissueinvasive viral infection (cellular and tissue injury) from effects mediated by inflammatory responses (e.g., cytokines) or by alterations in host immune responses. syndromes such as fever and neutropenia (e.g., with cmv infection) or invasive disease resulting in pneumonia, enteritis, meningitis, and encephalitis are considered direct effects. indirect effects of viral infections are generally thought to be immunomodulatory responses to viral infections mediated by cytokines, chemokines, and/or growth factors. the impact of these effects is diverse and includes systemic immune suppression predisposing to other opportunistic infections (notably with cmv or hcv infections). in addition, viral infection may alter the expression of cell-surface antigens (e.g., major histocompatibility antigens) provoking graft rejection and/or cause disregulated cellular proliferation (contributing to atherogenesis in cardiac allografts, obliterative bronchiolitis in lung transplantation, or to oncogenesis). increased viral replication and persistence may contribute to allograft injury (fibrosis) or chronic rejection. infection with one virus may stimulate replication of other viruses in a form of viral 'cross talk'. as was noted above, infection with hhv-6 and/or hhv-7 serve as risk factors for cmv disease and vice versa. the direct and indirect effects of hhv-6 reactivation can be significant: hhv-6 infection is associated with high levels of il-6 and tnf-a, and in pediatric renal or bone marrow transplantation, hhv-6 reactivation is strongly associated with acute rejection. co-infection with hcv and cmv predicts an accelerated course for hepatitis. co-infection with cmv and ebv increases the risk for post-transplantation lymphoproliferative disorders (ptld) by 12-20-fold. a more theoretical concern is that t-cell responses against viral infections are thought to produce cross-reacting immune responses against graft antigens, possibly via 'alternative recognition' within the t-cell receptor. this cross-reactivity is termed 'heterologous immunity' and may provoke abrogation of graft tolerance. the hhv family has eight members affecting humans, all of which can cause significant disease in transplantation recipients. the risk of many of these infections is reduced by the use of valganciclovir or acyclovir after transplantation. hsv-1 and à 2 (hhv-1 and à2) usually cause oral and genital ulceration, although it may occur in more unexpected areas as well. recurrent disease can be problematic for some patients and warrants consideration of secondary prophylaxis with antiviral therapy. vzv (hhv-3) is common with an incidence of herpes zoster among 869 patients after sot of 8.6% (liver 5.7%, renal 7.4%, lung 15.1%, and heart 16. . ptld constitutes a spectrum of disease, which is often responsive to reduced immunosuppression in previously immune hosts. ebv-seronegative individuals with primary infection after transplantation are at increased risk for ebv-mediated ptld. the clinical presentation of cmv (hhv-5) can range from a 'cmv syndrome' including fever, malaise, leukopenia, to a 'flu-like' illness with myalgias and fatigue, to a more significant end-organ disease with pneumonitis, colitis, encephalitis, hepatitis, or chorioretinitis. cmv is the single most important pathogen in transplantation recipients due to direct and indirect effects (see above). hhv-6 commonly reactivates after transplantation, especially after hsct where it is associated with hepatitis, pneumonitis, cmv reactivation, bone marrow suppression, and encephalitis. hhv6 causes less symptomatic clinical infection after sot, although the indirect effects of reactivation have not been studied. hhv-7 commonly reactivates after transplantation. the clinical symptoms caused by hhv-7 are uncertain, although neurological symptoms seem to be significant, especially in children. hhv-8 causes kaposi's sarcoma and is seen in sot recipients at a rate 500-1000 higher than the general population, with a prevalence of 0.5-5% depending on the patient's (and donor's) country of origin. hepatitis b and c are among the most common indications for liver transplantation, and can complicate other transplantations as well. hepatitis c is currently the most common indication for liver transplantation, accounting for 40-45% of cases in recent times. recurrent post-transplantation hepatitis c infection poses a conundrum between treating the hepatitis c and reducing immunosuppression without precipitating rejection. given the risk of precipitating graft dysfunction, hepatitis c treatment with interferon and ribavirin was often deferred in extra-hepatic transplant recipients. novel therapies for hepatitis c should reduce the risk of treatment complications after transplant. for hepatitis b, the goal is complete viral suppression before and after transplantation, using antiviral agents and sometimes hepatitis b immunoglobulin. respiratory viruses are the most common community-acquired infections in transplantation recipients. given the increased rates of pneumonia and bacterial and fungal superinfection, prevention (vaccination, avoidance of sick individuals) is essential. diagnosis of respiratory viruses within a few hours via enzyme-linked immunosorbent assay (elisa) or immunofluorescent staining is available in many medical centers. viral cultures are time consuming and expensive. respiratory syncytial virus and parainfluenza are the most common community-acquired respiratory viruses, followed by influenza and adenovirus. antiviral medications (rimantidine, amantidine, or oseltamivir) may prevent or reduce the severity of illness. the use of ribavirin or rsv table 2 direct and indirect effects of cmv infection fever and neutropenia syndrome (leukopenia, fever, myalgia, fatigue, thrombocytopenia, hepatitis, nephritis) increases risk of ptld gastrointestinal invasion with colitis, gastritis, ulcers, bleeding, or perforation increases risk of cardiovascular events hepatitis increases risk of immunosenescence, mortality pancreatitis chorioretinitis increases risk of new-onset diabetes mellitus after transplantation immune globulin in adults to prevent rsv infection is not well studied. ribavirin is sometimes used for documented rsv infections of the lower respiratory tract in transplant recipients. metapneumovirus and severe acute respiratory syndrome-associated coronavirus (sars-cov) are emerging pathogens in transplantation patients. the clinical spectrum of disease from metapneumovirus ranges from symptomatic (even fatal) to asymptomatic cases. some groups have suggested a possible correlation with graft rejection in lung transplant recipients. diagnosis is often made using molecular assays; the full impact of this infection in transplantation is yet to be realized. sars, caused by a zoonotic coronaviruses, is a highly contagious and rapidly progressive form of viral pneumonia, which spread from asia to many parts of the world in early 2003. a number of transplantation patients were infected, some of whom died. the impact of sars and resulting infection control issues was significant for both active organ transplantation (i.e., concerns about transmitting donorderived infections) as well as routine follow-up care for transplantation patients, some of who deferred healthcare visits. gastrointestinal viruses such as rotavirus or norwalk virus may cause significant diarrhea and dehydration; prolonged infections for months have been seen in transplant recipients. enteroviral infections in the summer months in the northern hemispheres are common and can have a more complicated and prolonged course in renal transplantation recipients. bk virus is associated with a range of clinical syndromes in immunocompromised hosts: viruria and viremia, ureteral ulceration and stenosis, and hemorrhagic cystitis. the majority of patients with bk virus infections are asymptomatic. infection by jc polyomavirus has been observed in renal allograft recipients as both nephropathy (in association with bk virus or alone) and/or progressive multifocal encephalopathy (pml). jcv establishes renal latency but receptors are present in multiple tissues including the brain. infection of the central nervous system generally presents with focal neurologic deficits or seizures and may progress to death following extensive demyelination. human papilloma virus (hpv) infections can cause significant disease in renal transplantation recipients, including oral, skin, genital, and rectal lesions ranging from warts and dysplasia to malignancy (especially squamous cell carcinoma). the recent arrival of a vaccine for genital hpv infections may help reduce these infections. in transplantation recipients, parvovirus b19 infection can cause erythropoietin-resistant anemia, pancytopenia, myocarditis, or pneumonitis. direct renal involvement with glomerulopathy and allograft dysfunction has been reported in renal transplantation recipients. clinical and virologic responses to treatment with intravenous immunoglobulin are usually excellent. several zoonotic viruses have caused major illness and death in the transplantation setting, including wnv, rabies, and lymphocytic choriomeningitis virus (lcmv). all have been recently reported as donor-derived infections related to sot, with clinically subtle infections in the donor and often deadly infections in the recipients. wnv is more morbid after sot; the risk of meningoencephalitis in a sot patient infected with wnv has been estimated to be 40%, compared with <1% in normal hosts. donors in endemic areas should be screened for wnv, as the prevalence can be high. aside from donor-derived infections, rabies and lcmv have not been reported. although hiv-infected patients are living longer and dying less often from complications related to acquired immunodeficiency syndrome (aids), they are experiencing significant morbidity and mortality related to end-stage liver and renal disease. preliminary studies suggest that both patient and graft survival are similar in hiv-negative and hiv-positive kidney and liver transplantation recipients. however, hcv infection appears to be accelerated even in controlled hiv infection. ongoing multicenter trials of transplantation in hiv infections are continuing. drug interactions between the immunosuppressive regimen and antiretroviral drugs necessitate careful monitoring. rapid and sensitive molecular biology-based assays for many of the common viruses after transplantation have replaced, for the most part, serologic testing and in vitro cultures for the diagnosis of infection. serologic assays are generally less sensitive in transplant patients, as humoral immune responses may be delayed or absent. in one series, 29% of patients with parvovirus b19 infection as shown by pcr assay had a negative igm assay. quantitative molecular tests allow the optimization and individualization of antiviral therapies for prevention and treatment of infection. this advance is most significant in the management of cmv, ebv, hepatitis b, and hepatitis c viruses, where quantitative assays (such as viral loads or antigenemia tests) guide antiviral therapy. nonquantitative (i.e., qualitative) assays are less useful in management as they do not assess responses to therapy and cannot differentiate primary infection, from reactivation or reinfection. for example, chromosomal integration of latent hhv-6 dna (which happens in 1-3% of immunocompetent subjects) leads to high levels of viral dna, whether or not the infection is active; one group concludes that any diagnosis of hhv-6 encephalitis should not be made without first excluding chromosomal hhv-6 integration by measuring dna load in csf, serum and/or whole blood. latent infections due to ebv and cmv may be qualitatively positive by pcr, confirming the need for quantitative assays. blood tests may not always accurately reflect the level of end-organ diseases; thus, it may be useful to test specific affected tissues as well the blood. in this regard, histologic evaluation of tissues using pathogen-specific immunohistochemistry may augment systemic assays. patients with cmv colitis, for example, may have negative molecular or antigenemia blood assays for cmv. in addition, patients may shed cmv in secretions without true infection, limiting the diagnostic capacity of a positive culture. bk polyomavirus may be detectable in the urine (either by cytology, looking for the classic decoy cells, or by pcr) before it is detectable in the blood, providing a window of opportunity for reducing immunosuppression possibly in advance of invasive disease. adenovirus may be detectable in local infections, such as cystitis, with negative blood assays. the treatment of viral infections in the renal transplantation recipient includes: the reduction of immunosuppression, antiviral therapy, diagnosis and treatment of co-infections (such as cmv, ebv, hhv-6, or à7), and use of adjunctive therapies such as immunoglobulins or colony stimulating factors. the overall level of immunosuppression has a major impact on both the risk of reactivation of latent infection and the ability to clear such an infection. reducing the immunosuppressive regimen during active viral infection can be a major contribution toward clearing infection, although it presents a risk of graft rejection. as protective cytotoxic immunity to viruses is generally t-cell (cd8 +) mediated, an initial reduction of antimetabolites (if neutropenic) and calcineurin inhibitors merits consideration. in contrast, a reduction of the steroid dose during the acute phase of a febrile illness may cause acute adrenal insufficiency. when available, antiviral therapies (such as acyclovir, ganciclovir, ribavirin, lamivudine, and oseltamivir) are often essential. the toxicity of some agents (such as cidofovir, foscarnet, and ribavirin) may complicate management, notably in the face of reduced renal function in patients receiving calcineurin inhibitors as part of the immunosuppressive regimen. the duration of therapy is often longer in transplant patients than in normal hosts, and often reflects the ability to reduce the overall level of immunosuppression, that is. less immunosuppression may result in a shorter treatment time. the increased information provided by molecular diagnostics may allow for more directed treatment regimens. antiviral therapy is often used for prophylaxis in the post-transplantation period, especially for individuals at risk for primary infection. in general, acyclovir and related agents are used to prevent hsv and vzv and ganciclovir or valganciclovir to prevent cmv as well as hsv and vzv. the relative advantages of universal prophylaxis (i.e., the use of antiviral medication in all susceptible patients for a period after transplantation) or monitoring with preemptive (early) therapy remain to be established. meta-analyses suggest that universal prophylaxis with antiviral medications in sot recipients reduces cmv disease and cmv-associated opportunistic infections, graft rejection, and mortality; their use is recommended for high risk individuals (cmv-positive recipients and in cmv-negative recipients of cmv-positive organs). some studies indicate that universal prophylaxis and preemptive therapy are effective in reducing the incidence of cmv disease. in the hsct setting, where the risks of bone marrow toxicity from valganciclovir are higher, many programs choose to use preemptive monitoring and therapy for cmv. various adjunctive therapies have been helpful in treating and preventing viral infections. immunoglobulins (i.e., intravenous immune globulins (ivig), cmv, and hbv hyper-immune globulins (both prepared from plasma preselected for high titer antibodies to cmv and hbv, respectively), as well as monoclonal antibodies such as those for rsv) have been helpful in preventing and treating viral infections, likely due to both direct and indirect immunomodulatory properties. a significant percent of patients have post-transplantation hypogammaglobulinemia, which has been linked to increased mortality and may benefit from globulin repletion. this may be most apparent in the setting of active infections, as well as prophylaxis. immunostimulatory agents, such as interferon-alpha used to treat hepatitis c, can be helpful at treating the viral infection but may perturb the relationship between the graft and the host, precipitating rejection. reversal of neutropenia can be done using colony stimulating factors such as g-csf, which is generally well tolerated in sold organ transplantation patients. vaccines should be given to patients as early as possible in the course of organ failure and well in advance of transplantation to optimize immune responses. in general, a response to vaccination is more likely to occur when given pre-transplantation rather than after transplantation. nonlive viral vaccines such as hepatitis b, hepatitis a, injectable influenza, rabies, hpv, injectable polio may be given both pre-and post-transplantation (see table 3 for classifications). live viral vaccines such as attenuated influenza for protection against varicella in nonimmune subjects, as well as zostavax, for protection against zoster), yellow fever, oral polio, and vaccinia (smallpox) should generally be avoided after sot and in patients who are chronically immunosuppressed (such as those with gvhd); some may be given after hsct. consideration of future travel or trips home should also be considered in the pre-transplantation period a prospective survey of human herpesvirus-6 primary infection in solid organ transplant recipients ast infectious diseases community of practice (2013) vaccination in solid organ transplantation parvovirus b19 infection after transplantation: a review of 98 cases transmission of lymphocytic choriomeningitis virus by organ transplantation infection in solid-organ transplant recipients prospective study of polyomavirus type bk replication and nephropathy in renal-transplant recipients zoonoses in solid-organ and hematopoietic stem cell transplant recipients cmv: prevention, diagnosis and therapy viral infection in the renal transplant recipient prevention of infection in adult travelers after solid organ transplantation updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation a seroprevalence study of west nile virus infection in solid organ transplant recipients idsa clinical practice guideline for vaccination of the immunocompromised host notes from the field: a cluster of lymphocytic choriomeningitis virus infections transmitted through organ transplantation -iowa hhv-6 dna level in csf due to primary infection differs from that in chromosomal viral integration and has implications for the diagnosis of encephalitis key: cord-350807-qdq96723 authors: reckziegel, maria; weber-osel, claudia; egerer, renate; gruhn, bernd; kubek, florian; walther, mario; wilhelm, stefanie; zell, roland; krumbholz, andi title: viruses and atypical bacteria in the respiratory tract of immunocompromised and immunocompetent patients with airway infection date: 2020-05-27 journal: eur j clin microbiol infect dis doi: 10.1007/s10096-020-03878-9 sha: doc_id: 350807 cord_uid: qdq96723 respiratory tract infections (rti) can take a serious course under immunosuppression. data on the impact of the underlying pathogens are still controversial. samples from the upper (n = 322) and lower rt (n = 169) were collected from 136 children and 355 adults; 225 among them have been immunocompromised patients. exclusion criteria were presence of relevant cultivable microorganisms, c-reactive protein > 20 mg/dl, or procalcitonin > 2.0 ng/ml. samples were tested by pcr for the presence of herpesviruses (hsv-1/-2; vzv; cmv; hhv6; ebv), adenoviruses, bocaviruses, entero-/rhinoviruses (hrv), parechoviruses, coronaviruses, influenza viruses (iv), parainfluenza viruses as well as for pneumoviruses (hmpv and rsv), and atypical bacteria (mycoplasma pneumoniae, m.p.; chlamydia pneumoniae, c.p.). viral/bacterial genome equivalents were detected in more than two-thirds of specimens. under immunosuppression, herpesviruses (ebv 30.9%/14.6%, p < 0.001; cmv 19.6%/7.9%, p < 0.001; hsv-1: 14.2%/7.1%, p = 0.012) were frequently observed, mainly through their reactivation in adults. immunocompromised adults tended to present a higher rsv prevalence (6.4%/2.4%, p = 0.078). immunocompetent patients were more frequently tested positive for iv (15.0%/5.8%, p = 0.001) and m.p. (6.4%/0.4%, p < 0.001), probably biased due to the influenza pandemic of 2009 and an m.p. epidemic in 2011. about 41.8% of samples were positive for a single pathogen, and among them ebv (19.9%) was most prevalent followed by hrv (18.2%) and iv (16.6%). hsv-2 and c.p. were not found. marked seasonal effects were observed for hrv, iv, and rsv. differences in pathogen prevalence were demonstrated between immunocompetent and immunocompromised patients. the exact contribution of some herpesviruses to the development of rti remains unclear. electronic supplementary material: the online version of this article (10.1007/s10096-020-03878-9) contains supplementary material, which is available to authorized users. infections of the upper respiratory tract (urti) are among the most frequent infections worldwide. these are mainly caused by rna viruses. among them, influenza viruses (iv), pneumoviruses (respiratory syncytial virus, rsv; human metapneumovirus, hmpv), but also parainfluenza viruses (piv), coronaviruses (cov), and rhinoviruses (hrv) are considered by world health organization a global health burden [1] . serious rti through respiratory viruses are frequently observed under immunosuppression, for example, in solid organ transplant recipients [2] . the current breakthroughs of immunomodulating therapies in medicine contribute to the continuous increase of patients being under iatrogenic immunosuppression and being at risk for pulmonary infections [3] . in general, suppression of t cell function is associated with a higher susceptibility for infection or reactivation of various viruses [4, 5] . impairment of th1cell activity but also of humoral immunity, both, facilitates the development of viral rti. in immunocompromised patients, higher morbidity and sometimes also mortality rates through infections, for example, with adenoviruses (adv), iv, piv, rsv, hmpv, but also of secondary complications like bacterial pneumonia have been observed [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] . furthermore, particularly transplant patients are at risk for reactivation of diverse herpesviruses (herpes simplex virus-1/-2, hsv-1/-2; varicella zoster virus, vzv; cytomegalovirus, cmv; human herpesvirus 6, hhv-6; epstein-barr virus, ebv) [12, 15, [17] [18] [19] [20] . multiple viral infections/reactivations can occur [21] as well as indirect interactions of viruses with bacteria [22] [23] [24] . these aspects may challenge interpretation of diagnostic findings. the frequency of viral infections/reactivations is also influenced by factors like the underlying disease, therapeutic regimes, as well as the type of transplant and hla mismatches [12, 19] . thus, fast and efficient diagnostic methods that cover a broad spectrum of viruses, bacteria, but also fungi and parasites are in urgent need to deal with the aforementioned challenges. the availability of such methods is of particular importance in stem cell transplant recipients where clinical symptoms of rti are variable or may be mimicked by graft-versus-host disease [25, 26] . early diagnosis enables limited antiviral interventions [16, 27] and may prevent further cross-transmission [28] . however, detection of viral genome equivalents does not necessarily mean a causative role of this virus, and particularly immunocompromised patients can shed viruses over a prolonged time period [13, 29, 30] . furthermore, there is increasing evidence of the existence of a respiratory virome which is defined by the presence of common viral pathogens, rare viruses, and viruses of unknown pathogenicity [31] . thus, the exact contribution of a single virus to the development of rti is still controversial. we tried to consider most of these aspects by performing an observational study addressing the spectrum and impact of respiratory viruses but also of herpesviruses and the atypical bacteria mycoplasma pneumoniae (m.p.) and chlamydia pneumoniae (c.p.) in patients with respiratory symptoms. for this, an underlying infection through relevant cultivable microorganisms was largely ruled out. data were analyzed with respect to patients' immune status and age. this study included 322 samples from the upper (nasal or throat swabs and washings), and 169 samples from the lower (broncheoalveolar/tracheal washings, induced sputum) respiratory tract (urt/lrt) collected over a period of 40 months beginning in september 2009 to december 2012 from 266 healthy and 225 immunocompromised patients with symptoms of a rti (i.e., common cold, cough with/without sputum, dyspnea, and fever). this setting included samples from patients with respiratory symptoms under neutropenia or lungtransplant recipients with a recently observed decrease in forced expiratory volume in one second (fev1). a compromised immune status was defined (i) for solid organ or stem cell transplant recipients under iatrogenic immunosuppression, (ii) in patients with autoimmune disorders under immunosuppressing therapy but also (iii) in cancer patients under chemotherapy/radiation, and (iv) in patients with primary or secondary causes of immunodeficiency including hiv infection. samples found to contain relevant cultivable bacteria (streptococcus pneumoniae, staphylococcus aureus, haemophilus influenzae, various enterobacterales and nonfermenting gram-negative bacteria, mycobacterium tuberculosis) and fungi were excluded. furthermore, specimens obtained from patients with a present bacteremia/ sepsis were excluded as well as samples from patients with rti through pneumocystis jirovecii or legionella pneumophila. the oropharyngeal and tracheopulmonal flora was considered if data were available from routine diagnostics. in addition, samples obtained from the lrt of patients with more than 100,000 colonies/ml of oropharyngeal or tracheopulmonal flora, or more than 10,000 colonies/ml of enterococcus spp. or candida spp., were also excluded since such high concentrations may represent an infection rather than colonization. in addition, the presence of procalcitonin (pct) > 2 ng/ml and/or of c-reactive protein (crp) > 20 mg/ dl in serum leads to exclusion of the rt sample. the remaining samples were obtained from 99 immunocompetent (median age 1.0 years; 56 males/43 females) and 37 immunocompromised (median age 4.6 years; 18 males/19 females) children and adolescents as well as 167 immunocompetent (median age 46.2 years; 73 males/94 females) and 188 (median age: 57.2 years; 101 males/87 females) immunocompromised adults. among the immunocompromised cohort, most samples were obtained from patients with hemato-oncological malignancies (28.4%), followed by samples from patients after organ (24.9%) and stem cell (16.4%) transplantation or with autoimmune disorders (13.8%). about 9.8% of samples were included from patients with other conditions of immunosuppression or from solid tumor patients (6.7%). specimens were immediately deep frozen (− 80°c) until nucleic acid extraction. the extraction was done manually with the qiaamp minelute virus spin kit or automatically with the ez1 virus mini kit (both qiagen, hilden, germany). the nucleic acids were stored at − 20°c and used for synthesis of copy dna (cdna) applying the revertaid h minus first strand cdna synthesis kit (thermofisher scientific, langen, germany) with random hexamers. integrity of cdna was demonstrated by amplification of ß-actin or glyceraldehyde 3-phosphate dehydrogenase (gapdh) dna [32, 33] with dreamtaq dna polymerase (thermofisher scientific). presence of herpesviral dna (hsv-1/-2, vzv, cmv, and hhv-6) was demonstrated by applying conventional pcr [34] [35] [36] [37] [38] [39] [40] [41] together with the hotstartaq dna polymerase and qsolution (qiagen). quantitative detection of ebv dna in samples from the lrt was done in accordance with krumbholz et al. (2006) [42] , but sybr-green (quantitect sybr green pcr kit; qiagen) was used instead of hybridization probes. these diagnostic pcrs were continuously approved by successful participation in the external quality assurance service (eqas) program of instand e.v. (düsseldorf, germany). for detection of diverse respiratory viruses, all cdnas were tested with the seeplex rv5 ace (covers iv-a; iv-b; rsv-a/-b; adv; piv1-3; bocavirus, bov; hmpv; hrv-a/ b; cov 229e/nl63/oc43/hku1) and rv12 (covers the same spectrum as rv5, but is not able to detect bov) ace detection kits. the seeplex rv15 ace detection kit (includes also piv-4, hrv-c, and enterovirus detection but is not able to detect cov hku1) was used from november 2011, since the distribution of rv5 and rv12 versions was abandoned (all kits seegene, eschborn, germany). all three multiplex assays have been established in the laboratory using defined eqas samples from instand e.v. before testing of study samples. the rv5 kit is a screening kit and neither allows discrimination between adv, piv, and bov nor between hmpv, hrv, and cov. to overcome this problem, amplicons were purified after agarose gel-electrophoresis applying the qiaquick gel extraction kit (qiagen). purified dna was ligated into the pdrive cloning vector included in the qiagen pcr cloning kit, and used for transformation of competent escherichia coli cells. then, colonies were screened for inserts by pcr applying the dreamtaq dna polymerase and oligonucleotides specific for pdrive. amplicons with inserts were purified and sequenced using the dtcs quick start master mix. sequence analysis was done on a beckman ceq 8000 genetic analyzer (all beckman coulter, krefeld, germany). for detection of human parechovirus (hpev) genome equivalents, a semiquantitative real-time pcr was established using cdna and oligonucleotides [43] together with the quantitect sybr green pcr kit (qiagen) on a lightcycler 1.5 (roche, mannheim, germany). positive controls for hpev-pcr were kindly provided by dr. corinna pietsch and prof. dr. uwe gerd liebert (institute of virology, university of leipzig, germany). since enteroviruses were not covered by rv5 and rv12 assays, nearly all samples were screened by a nested pcr protocol detecting a conserved sequence of the 5′nontranslated region (5′-ntr) [44] . then, rough-typing was done by sequence analysis of purified pcr products. detection of hrv was performed by nested amplification of the vp4/2-encoding region [45] . parallel testing for m.p. and c.p. was done by applying the diagenode mycoplasma pneumoniae and chlamydophila pneumoniae kit (r-diamcpn, diagenode s. a., liège, belgium) on an abi7500 real-time pcr system (thermofisher scientific). sequence data were analyzed using mega 6.0 [46] . all other data were analyzed applying the two-sided fisher's exact test implemented in ibm® spss® statistics 20. a p value < 0.05 was considered statistically significant. this study included 491 samples from immunocompromised or immunocompetent patients with symptoms of rti collected over a period of 40 months. all samples tested positive for the presence of gapdh and/or ß-actin. thus, quality of sampling and nucleic acid extraction was demonstrated (data not shown). among the overall study population, genome equivalents of ebv were most frequently detected (22.3%, 84/377), followed by hhv-6 (20.3%, 32/158), hrv (14.1%, 69/ 491), cmv (13.2%, 65/491), rsv (11.2%, 55/491), and iv (10.8%, 53/491). genome equivalents of hsv-2 and c.p. were generally not detected (table 1) . with respect to patients' immune status, dna of ebv (30.9% vs. 14.6%), cmv (19.6% vs. 7.9%), and hsv-1 (14.2% vs. 7.1%) was significantly more prevalent in immunocompromised patients while genome equivalents of iv (5.8% vs. 15.0%) or m.p. (0.4% vs. 6.4%) were more frequently observed in their immunocompetent counterpart. the higher prevalence of cmv and ebv was only observed (supplementary fig. 1 ). in 26.3% (129/491) of samples found to be pathogen-positive, multiple agents were detected. among them were samples with two (20.4%), three (5.1%), four (0.6%), or even five (0.2%) different viruses/bacteria (supplementary fig. 1 ). the combination of two herpesviruses (hhv-6/ebv 13.2%, 5/38; cmv/ebv 9.6%, 8/83; cmv/hsv-1 8.0%, 8/100) but also of ebv and iv (6.0%, 5/83) or m.p. (6.0%, 5/83) as well as of rsv and hrv (5.0%, 5/100) was frequently observed. bocaviral dna was found together with other viruses/m.p. supplementary fig. 1 ). significant seasonal effects were recorded in the immunocompetent group for hrv with a high prevalence in autumn and for iv with an increased prevalence in winter. seasonal effect was significant in both patient groups for rsv with increased prevalence in winter and spring (supplementary table 2 ). interestingly, slight seasonality was also observed for hhv-6 in the immunocompetent group. in addition, some interannual variation was found for m.p. and iv (data not shown), which was most likely associated to the 2009influenza pandemic and an m.p. epidemic in 2011, respectively. in this monocentric study, genome equivalents of viruses and m.p. were frequently detected in immunocompromised (66.7%) and immunocompetent (69.2%) patients with respiratory symptoms (table 1) . since a contribution of relevant cultivable microorganisms to patient symptoms was largely excluded, a causative role of the pathogens detected in this study has to be considered. previously, a comparable approach was used to identify viral causes of severe rti in children [47] . the stringent exclusion criteria may account for the low number of patient samples included in this study and may have neglected possible additive or synergistic effects between bacteria, fungi, and viruses. in particular, we found a high prevalence of herpesviruses in immunocompromised adults with respiratory infections. nearly one-third of them was tested positive for ebv and every fourth patient presented cmv in his respiratory tract. in children, herpesviral dna was rarely detected which reflects the generally increasing infestation rate observed over life-time [48] [49] [50] [51] [52] and indicates viral reactivation as a major cause for pathogen detection. the higher prevalence of ebv, cmv, and hsv-1 in the airways of adults was associated with the state of immunosuppression. this is in line with the fact that herpesviral reactivation is facilitated by the impaired immune system [53] . it is still controversial whether this reactivation contributes to respiratory pathology or just represents an indicator of excessive immunosuppression. for cmv, however, there is no doubt that this betaherpesvirus is responsible for lrti in immunocompromised patients [17, 20] . cmv pneumonia is considered as likely when viral dna has been detected in bal of symptomatic patients [17] . thus, in our study, a remarkable proportion of immunocompromised adults revealed signs of suspected cmv pneumonia (table 1 ) and may benefit from antiviral prophylaxis or therapy. while hsv-1 dna was slightly more prevalent in the urt of immunocompromised patients, we found nearly comparable detection rates in the lrt of both patients groups (table 1 ). this is in line with a previous study [54] . interestingly, the same authors found that higher hsv-1 concentrations were associated with a poor patient outcome [54] . as for cmv, definitive diagnosis of hsv-1 pneumonitis depends on the presence of viral antigen within the lrt tissues [17] . in contrast to a recent report [55] , we found two-times higher ebv dna prevalence in immunocompromised patients compared to their immunocompetent counterparts (table 1) . previously, ebv dna was frequently detected in patients with pneumonia, respiratory insufficiency, and other bronchopneumopathies, but its presence was not associated with increased 28-day mortality [55] . in addition, the same authors reported no difference in ebv concentration between immunocompromised and immunocompetent patients [55] , which is in contrast to our findings (fig. 1, supplementary table 1 ). nevertheless, the contribution of ebv to the development of respiratory symptoms is still controversially discussed in the literature and remains unclear so far [55] [56] [57] . there is, however, some evidence that ebv reactivation-like that of other herpesviruses-may trigger fig. 1 comparison of ebv-dna copies/ml in respiratory specimens from immunocompromised and immunocompetent patients. data are presented in a logarithmic scale. the median ebv concentration is significantly higher in immunocompromised patients (p = 0.030, mann-whitney u test) inflammation which is associated to transplant rejection or interstitial lung disease [20, [58] [59] [60] . hhv-6 dna was found at similar high frequencies of ca. 20% in both patient collectives. interpretation of our results, however, is limited since our pcr protocol may have also detected chromosomally integrated viral dna [61] and cannot differentiate between hhv-6a and hhv-6b. the latter variant is more commonly implicated in human disease [62] . moreover, hhv-6 was frequently observed in combination with other herpesviruses ( table 2) as also seen by others [62] . thus, the contribution of hhv-6 to rti remains unclear. other herpesviruses (vzv, hsv-2) were found to be negligible in this study (table 1) which is in line with the literature [17, 20, 54, 63] . most of our results were obtained by end-point pcr. the consideration of viral concentration-as it is exemplarily shown here for ebv-may be useful in order to better unravel the contribution of herpesviruses to the development of lung pathology [20] . the observed frequencies of respiratory viruses were comparable to data from the german laboratory network (https://clinical-virology.net/en/charts/chart/ctype/ count/network/resp/section/viruses) and to another study from germany [64] . genome equivalents of rsv and hrv were prevalent in children while hrv and iv were frequent in adults. interestingly, immunocompromised adults tended to have a higher prevalence of rsv (table 1) . this supports previous data on the contribution of rsv to morbidity and mortality in this patient group [65] . there were various examples of single detections, which are probably indicative for infection, but also of co-presence of two or more pathogens ( table 2 , supplementary fig. 1 ). bocaviral dna, for instance, was frequently found in combination together with further viral genomes as also reported by others [66] . in children of 2 years and younger, however, this parvovirus was detected solely. previously, isolated bov infection was shown to be a likely cause of severe acute rti in children [67] . in a german study, bov dna was demonstrated in 10.3% of nasal swabs obtained from children with respiratory symptoms [68] . this prevalence is largely comparable to our results. same authors indicated a mean age of 1.8 years table 2 detection of multiple pathogens in the respiratory tract of the overall study population (a) as well as of immunocompromised (b) and immunocompetent (c) patients. the gray boxes indicate frequent co-infections. note that due to multiple detection (i.e., more than two pathogens), the sum of the frequencies given in these boxes may be higher the total frequency given in the black box. see also suppl. figure 1 a) overall study populaɵon adv bov cov ev hmpv hpev hrv iv-a iv-b piv rsv-a rsv-b cmv ebv hhv-6 hsv-1 for bov detection. in 39.1%, bocaviral dna was detected together with other pathogens [68] . interestingly, analysis of the ev 5′-ntr sequences gave some evidence for the presence of ev-d68 in the airways of three adults and one toddler. ev-d68 infection is associated with the development of acute flaccid myelitis and severe respiratory illness [69] . parechoviral rna was found only in a 2-year-old immunocompetent child with ia-v infection. human parechoviruses can cause mild gastrointestinal and respiratory disease but also sepsis-like illness and meningitis in infants [70] . the general low prevalence of hpev in this study is in line with a previous report [64] . the possible etiology of rti was not clarified in 45.9% of immunocompromised children (table 1) . under these conditions, application of broad diagnostic technologies like nextgeneration sequencing could be useful in identification of the underlying pathogen [71] . moreover, cmv detection rate in samples from the urt of immunocompetent children was surprisingly high (table 1) . seasonal effects were evident for several respiratory viruses (supplementary table 2 ). slight seasonality was also observed for hhv-6 in immunocompetent patients. interpretation of this finding, however, is unclear. the high prevalence of m.p. in 2011 may be explained by an epidemic observed in germany [72] . in line with this, the 2009 pandemic caused by a/h1n1pdm09 may account for a further study bias. dna of c.p. was generally not detected in our study. this is in line with the low prevalence of 0.2% reported recently [73] , but also with data from the respiratory viruses network (https://clinical-virology.net/en/charts/chart/ctype/count/ network/resp/section/bacteria). it is hypothesized that the prevalence of c.p. was overestimated in previous reports, most likely due to 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authors would like to thank all patients and their families for study support. furthermore, the great contribution of attended physicians is kindly acknowledged. the authors thank fabian cundano maltez, rosemarie carius, and martina müller for their excellent technical service. in addition, we are grateful for the continuous support given authors' contributions ak and rz conceived the study, helped with interpretation of data, and wrote the manuscript together with mw who performed statistical analyses. mr and cwo tested all respiratory tract samples by pcr and analyzed available patient data in frame of their m.d. theses. testing of nucleic acids for the presence of ev, hrv, and hpev was partially done by fk and sw as part of their bachelor theses. bg and re continuously supported the collection and analysis of samples. all authors read and approved the final version of this manuscript. the study was approved by the ethics committee of the jena university hospital (2612-07/09). the authors declare that they have no conflict of interest. key: cord-323691-5s5almd2 authors: mishin, vasiliy p; cominelli, fabio; yamshchikov, vladimir f title: a ‘minimal’ approach in design of flavivirus infectious dna date: 2001-12-04 journal: virus research doi: 10.1016/s0168-1702(01)00371-9 sha: doc_id: 323691 cord_uid: 5s5almd2 abstract the ‘infectious dna’ approach, which is based on in vivo transcription of (+)rna virus genome cdna cassettes from eukaryotic promoters in transfected cells, became a popular alternative to the classical scheme in the infectious clone methodology. its use, however, is often limited by the instability of plasmids due to a transcriptional activity of eukaryotic promoters in escherichia coli resulting in synthesis of products toxic for the bacterial host. using a highly unstable representative infectious clone of japanese encephalitis (je) flavivirus, we tested a new approach in design of such problematic ‘infectious dna’ constructs, which is based on minimizing unwanted transcription in the bacterial host. a plasmid containing full genome size je cdna under control of the minimal cytomegalovirus (cmv) promoter can be propagated in e. coli with growth and stability characteristics similar to that of constructs controlled by the t7 promoter. transfection of this plasmid into susceptible cells leads to the establishment of a productive infectious cycle. reinsertion of the cmv enhancer at the 3′-end of the je cassette substantially increased the specific infectivity without affecting the stability and growth characteristics of the construct. this approach can be useful when stabilization of infectious clones by modification of a viral cdna cassette is not the feasible or suitable alternative. modern (+)rna virus studies increasingly rely on the infectious clone methodology (reviewed, boyer and haenni, 1994) , which allows targeted manipulation of viral genomes. in the classical approach, (+ )rna viruses are recov-ered from cells transfected with synthetic rna made by in vitro transcription of infectious clone cdna templates (campbell and pletnev, 2000; gritsun and gould, 1995; kapoor et al., 1995; polo et al., 1997; rice et al., 1989; sumiyoshi et al., 1992) . in a layered dna/rna approach, also known as 'infectious dna' (herweijer et al., 1995) , (+ )rna viruses are recovered directly after transfection of plasmids carrying viral genome cdna into susceptible cells. it was first reported for poliovirus (racaniello and balti-more, 1981) , and has been widely used for plant viruses (reviewed, boyer and haenni, 1994) . recently, it was adapted for alphaviruses (reviewed, schlesinger and dubensky, 1999) . substantial difficulties, however, were encountered in design of flavivirus 'infectious dna', requiring either modification of the viral genome cassette (yamshchikov et al., 2001) to prevent unwanted expression of viral genome segments encoding toxic for escherichia coli products, or deletion of the structural protein region (varnavski et al., 2000) . substantial stabilization of viral genome cassettes by blocking unwanted expression was also reported for a number of plant viruses (johansen, 1996; lopez-moya and garcia, 2000; olsen and johansen, 2001) . in this approach, deleterious effects of spurious transcription from eukaryotic promoters in bacterial cells are eliminated at the translational level by insertion of intron(s) into viral cdna cassettes. there may be situations, however, when such modification of a viral cassette is not feasible or is not acceptable. for this reason we sought to investigate if the stability of constructs containing an unmodified virus genome cassette can be improved by preventing its deleterious expression at the transcriptional level, i.e. by minimizing spurious transcription from eukaryotic promoters in e. coli. as a model, we have used a highly unstable infectious clone of japanese encephalitis flavivirus (je) (sumiyoshi et al., 1992; yamshchikov et al., 2001) . je is a member of the fla6i6irus genus of the family fla6i6iridae, which also includes yellow fever (yf), west nile, tick borne encephalitis, dengue, and hepatitis c viruses (kuno et al., 1998) . the flavivirus virion contains singlestranded rna of positive polarity, which is approximately 11 kb in length and encodes three structural and seven nonstructural proteins (rice, 1996) . viral rna is capped at its 5%-end, but has no poly(a) tract at the 3%-end. both virion rna and in vitro synthesized capped rna are infectious when transfected into susceptible cells (rice et al., 1989) . in this report we demonstrate that spurious transcription in e. coli can be substantially reduced by utilizing a minimal eukaryotic promoter (davis and huang, 1988) to control transcription of the viral cassette. as an enhancement of this 'minimal approach', a substantial increase in the specific infectivity of 'infectious dna' was achieved by placing the enhancer at the 3%-end of the viral genome cassette. while this methodology does not resolve completely the instability problem, it may be considered as an alternative approach when for any reason the viral genome cassette cannot be modified. dna manipulations were performed using standard procedures (sambrook et al., 1989) with commercially available enzymes according to manufacturer's protocols. e. coli strain hb101 was used for cloning and maintenance of recombinant constructs. sequence analysis of plasmids and of recovered viruses was performed on abi 310 genetic analyzer (perkin-elemer) using manufacturer's kits and protocols. luciferase encoding plasmids (fig. 1a) were derived from pbr322. plasmids pcmv5%utrje/ luc and pcmvmp5%utrje/luc include the first 69 nucleotides of je 5%-utr followed by the cmv promoter or minimal cmv promoter, respectively. plasmids pcmvmp/luc-3%enhd and pcmvmp/luc-3%enhr were designed by blunt-end cloning of a bgl ii-pvu i cmv enhancer fragment (from pcineo; promega) into bam hi site at the 3%-end of the luciferase gene in pcmvmp/luc. these plasmids contain the cmv enhancer in the opposite orientations. plasmids used in the design of the final construct (fig. 2) were described earlier (yamshchikov et al., 2001) . digestion of pcmvje5%(943) at the pvu i (situated between the enhancer and promoter regions in the cmv promoter/enhancer fragment) and mlu i sites yielded a cmv minimal promoter-je 5%-end conjoint fragment, which replaced the sal i-mlu i fragment in pt7je5% resulting in a pcmvm-pje5% construct. an eag i-bam hi cmvmp-je 5% half genome fragment from this plasmid was inserted into pje3%(bghtt) (yamshchikov et al., 2001) digested with the same enzymes. the resulting plasmid pcmvmpje(bghtt) contained full-length je genome cdna under transcriptional control of the cmv minimal promoter and the bgh transcriptional terminator. plasmids pcmvmpje(bghtt)enhd and pcmvmpje(-bghtt)enhr were derived from pcmvmpje(-bghtt) by blunt-end ligation of the bgl ii-pvu i enhancer fragment into the ssp i site (fig. 2 ). luciferase activity in transfected eukaryotic cells was measured using the dual-luciferase reporter assay kit (promega) according to manufacturer's recommendations. plasmid prlnull (promega) was used for normalization of expression by admixing to luciferase-containing plasmids in proportion 1:10. bhk cells (bredenbeek et al., 1993) were transfected with plasmid dna using superfect (qiagen) according to the manufacturer. transfection mix containing 0.275 mg plasmid dna per well was placed into wells of 24 well plate in triplicate per each time interval. cells were lysed with 75 ml of the lysis buffer (promega) at 6, 12 and 24 h post transfection and luciferase activities were quantified as recommended by the manufacturer. to determine of the cmv promoter activity in bacteria, overnight cultures of e. coli dh10b cells (gibco-brl) transformed with plasmids were diluted 1:50 with fresh lb and grown 6 and 12 h. cells from 1 ml culture were resuspended in 100 ml buffer i (100 mm tris-hcl, ph 8.0; 100 mm nacl; 2 mm edta) and lysozyme was added to 0.5 mg/ml. after incubation for 1 h at 0°c, cells were lysed by adding 100 ml of 2× lysis buffer (promega). after centrifugation at 14,000 rpm at 4°c for 10 min, 5 ml of the clear lysate was used for luciferase assay. normalization of luciferase activity was done to amounts of plasmid dna isolated from 1 ml of culture. isolated plasmid dna was treated with atp-dependent dnase, rnase a and proteinase k before measuring plasmid concentration using dyna quant fluorimeter (hoefer) according to the manufacturer's protocol. chambers et al. (1990) . an asterisk denotes the silent mutation, which appeared during cloning (see text for details). upper letters indicate relative positions of endonuclease restriction sites used in assembly and analysis of designed constructs: m, mlu i; b, bam hi; s, sal i; p, pvu i; e, eag i; a, asp 718 (kpni); ss, ssp i; t7, t7 promoter, enh, cmv enhancer, mp, cmv minimal promoter, bghtt, bovine growth hormone transcriptional terminator. the scheme is not drawn exactly to the scale. (b) sequence at junctions of cmv promoter, 5%-and 3%-ends of the je genome, and bghtt. bhk and vero (atcc crl 1586) cells were maintained at 37°c, in a humidified atmosphere containing 5% co 2 in dulbecco's modified eagle's medium (dmem) supplemented with 5% fetal calf serum (fcs; hyclone) and 1× antibiotic -antimycotic (gibco-brl). wild-type virus jaoars982 (sumiyoshi et al., 1987) and attenuated vaccine strain sa14-14-2 (nitayaphan et al., 1990) were grown in vero cells. virus growth characteristics were determined either after infection of 4×10 5 bhk cells in 6-well plates with 10 3 pfu of virus (moi= 0.0025) or after transfection with 1 mg of infectious plasmid. media of the infected or transfected cells were sampled at various times and assayed for virus titers using bhk cells. for evaluation of the specific infectivity of dna, bhk cells were transfected with serial 10-fold dilutions of plasmid dna in the presence of 1 mg carrier dna (pbr322). in either case, after 1 h for virus adsorption or 3 h for dna transfection, cells were washed two times with dulbecco's pbs (dpbs) and overlaid with dmem supplemented with 1% methylcellulose (sigma) and 2% fcs (hyclone). cells were incubated for 3-4 days and stained with 1% crystal violet in 70% methyl alcohol to visualize foci of cytopathology (plaques). all plaque assays were performed in duplicate, and median virus concentration (log pfu/ml) was plotted as function of time. all procedures involving infectious material were performed under the biological safety level 3 containment. bhk cells seeded on 12 mm glass coverslips were transfected with dna-superfect complexes (0.25 mg of plasmid per 2× 10 5 cells). at daily intervals, cells were fixed in cold methanol-acetic acid (95:5), washed with dpbs containing 1% fcs (dpbs/fcs) and reacted with je (atcc vr 1259af) mouse hyperimmune ascites fluid (at 1:1000 dilution in dpbs/fcs). a goat anti-mouse igg-fluorescein conjugate (gibco-brl) was used as the secondary antibody at 1:200 dilution in dpbs/fcs and cells were observed under fluorescent microscope equipped with a video imaging system. total cellular rna from infected and transfected cells was isolated using the rneasy mini kit (qiagen) with dnase treatment as recommended by the manufacturer. removal of residual dna was monitored by pcr of rna samples using the same primer sets as for rt-pcr assays. analysis of the 5%-end of je-specific rna in transfected and infected cells was performed by primer extension assays (sambrook et al., 1989) using amv reverse transcriptase (panvera) and [ 32 p]-labeled primer je100r (5%-gtcatggt-tatcttccgttct-3%), complementary to pos. 80-100 of the je genome. primer extension products were analyzed on 6% paag containing 7 m urea. to determine the 5%-end of a primary cmv transcript we used first choice rlm-race kit (ambion) according to manufacturer's recommendation. total rna from cell transfected with pcmv5%utr-je/luc was analyzed. primers luc22r (5%-ttatgtttttggcgtcttc-3%) and luc87r (5%-gccttatgcagttgctctc-3%) complementary to pos. 4-22 and 69-87 of the luciferase gene were used for rt, amplification and sequencing reaction. the 3%-end of je specific rna was determined by using an adapted race-pat assay (salles et al., 1999) . briefly, total rna polyadenylated in the presence of polya polymerase (gibco-brl) was reverse transcribed from a dt15-anchor reverse primer (salles et al., 1999) following by pcr amplification using the same reverse primer and a specific primer corresponding to pos. 10 468-10 485 of the je genome. products of rt-pcr were purified using 1% agarose gel and sequenced. earlier, it has been demonstrated (johansen, 1996; lopez-moya and garcia, 2000; olsen and johansen, 2001; yamshchikov et al., 2001 ) that interruption of a viral open reading frame either by frameshift mutations or by insertion of introns leads to substantial stabilization of plasmids containing viral genome cassettes under control of eukaryotic promoters. this suggests that spurious transcription from eukaryotic promoters in e. coli (antonucci et al., 1989; davis and huang, 1988) may substantially contribute to the observed instability of such constructs. as an alternative to the mentioned above modification of viral genome cassettes, we sought to explore if a decrease of such spurious transcription can eliminate the destabilizing effect of eukaryotic promoters on 'infectious dna' constructs. the powerful and versatile cmv promoter, often used to drive expression of engineered constructs in a variety of mammalian cells (foecking and hofstetter, 1986) , consists of a transcriptionally active minimal promoter region of approximately 100 bp immediately adjacent to the rna transcription start, and of an about 600 bp upstream enhancer element, which displays only a minute transcriptional activity in mammalian cells (thomsen et al., 1984) . to determine if the enhancer has any effect on the promoter activity in e. coli, we designed plasmids pbr/luc (no promoter), pcmv/luc (full promoter), pcmvmp/luc (no enhancer), and a pair of plasmids pcmvmp/luc-enhd and pcmvmp/luc-enhr containing the enhancer relocated to the end of the reporter gene (fig. 1a) . comparative evaluation of luciferase production in e. coli transformed with the above plasmids, shown in fig. 1b , indicated that deletion of the enhancer leads to a 5-6-fold reduction of luciferase gene expression. in contrast to position-independent function of enhancers in mammalian cells (jonsson et al., 1994) , and certain activators in prokaryotic cells (molina-lopez and santero, 1999) , the cmv enhancer relocated downstream from the reporter gene had no dis-cernible effect on production of luciferase in e. coli. we also compared expression of the luciferase gene in bhk cells transfected with the designed plasmids. as indicated in fig. 1c , luciferase production from the plasmid containing the minimal promoter was 7-10-fold less than from the full promoter construct. in contrast to bacterial expression described above, relocation of the enhancer to the 3%-end of the luciferase gene restored 40-50% of the full cmv promoter activity. position-dependent modulation of the enhancer activity has been observed (jonsson et al., 1994) . flavivirus genome rna has a short 5%-untranslated region (5%-utr), which forms a highly conserved secondary structure (brinton and dispoto, 1988) . a positive effect of flavivirus 5%-utr on the translation efficiency of synthetic rna in cell-free systems has been reported earlier (preugschat et al., 1990; ruiz-linares et al., 1989) . since an enhancing effect of flavivirus 5%-utr on translation would be beneficial in the devised 'minimal' approach, we were interested to determine if such enhancing effect can be observed with je 5%-utr in vivo. to address this question, we designed plasmids pcmv5%utr-je/luc and pcmvmp5%utr-je/luc (fig. 1a) , in which the first 69 nucleotides of je 5%-utr have been placed between the luciferase reporter gene and the cmv or minimal cmv promoters, respectively. the effect of 5%-utr was accessed by measuring luciferase activity in lysates of bhk cells transiently transfected with these plasmids and with plasmids pcmv/luc and pcmvmp/luc (fig. 1a) . surprisingly, luciferase production from pcmv5%utr-je/luc was about 15-20% less than from pcmv/luc, and the presence of je 5%-utr had no discernible effect on amounts of luciferase produced from the construct containing the minimal cmv promoter (fig. 1c) . since for the purposes of the present study we were interested primarily in the activity of the minimal promoter, we did not investigate this question in more detail. based on the evidence described above, we attempted to assemble an 'infectious dna' of japanese encephalitis flavivirus under control of the minimal cmv promoter. sequence analysis of one isolate of the pcmvje5%(943) plasmid described earlier (yamshchikov et al., 2001 ) revealed a silent substitution at pos. 551 of the je cdna fragment resulting in formation of an additional site for restriction endonuclease sau3a i (gatt gatc). this mutation has created a convenient marker allowing to distinguish between genomes of the parent and recovered viruses. in contrast to the similar construct controlled by the full cmv promoter (yamshchikov et al., 2001) , a pcmvmpje5% plasmid containing 5%-half (1-5576 nt) of the je genome cdna controlled by the minimal promoter appeared stable with growth characteristics in e. coli hb101 resembling those of pt7je5% (sumiyoshi et al., 1992; yamshchikov et al., 2001) . preservation of the open reading frame in the je fragment was confirmed by its complete sequencing. similarly, pcmvmpje(-bghtt) containing full-length je genome cdna and the bgh transcriptional terminator (fig. 2) had the same growth characteristics in e.coli hb101 as the half je genome-containing plasmid pcmvmpje5%. to evaluate the stability of the final construct during propagation in e.coli hb101, three independent isolates of pcmvmpje(bghtt) were subjected to two transformation-plasmid isolation cycles. plasmid transformation usually resulted in formation of small uniform colonies, with large colonies observed with a frequency of less than 10 − 3 . plasmid dna isolated from small colonies was infectious and had the similar specific infectivity in bhk cells. plasmids isolated from the large colonies were not infectious and had substantial rearrangements in the viral cdna cassette. finally, we have reinserted the enhancer fragment into pcmvmpje(bghtt) downstream from the bghtt fragment. in agreement with the evidence described above, resulting plasmids pcmvmpje(bghtt)enhd and pcmvmpje(-bghtt)enhr had the same stability and growth characteristics as the parent plasmid. after transfection of bhk with the full-length pcmvmpje(bghtt) construct, individually stained cells were observed on the 1st day after transfection and positive cells progressively increased in number from the 1st to the 4th day after transfection. by day 4 all cells became je-positive, indicating formation of the virus and the spread of infection. similar result was obtained for the plasmids pcmvmpje(bghtt)enhd and pcmvm-pje(bghtt)enhr. the specific infectivity of plasmid pcmvmpje(bghtt) appeared dependent on a transfection agent and transfection protocol used, and was in the range of 10 2 -10 3.5 pfu/mg. the presence of the cmv enhancer downstream from bghtt led to an increase in the specific infectivity to 1-5 × 10 5 pfu/mg, which became comparable to that of the full cmv-controlled intron-containing construct pcmvp/ eje(i356i2217)bghtt reported earlier (yamshchikov et al., 2001) cytopathic effects resembling those resulting from infection of bhk cells with the parent strain jaoars982 were reproducibly observed 3-4 days after exposure of bhk monolayers to dnase treated media from cells transfected with pcmvm-pje(bghtt), indicating the presence of a dnaseinsensitive infectious agent. the time course of virus accumulation in the media was determined at 12-h intervals after transfection of bhk cells with 1 mg of the plasmid pcmvmpje(bghtt). virus was undetectable in the media during 24 h after transfection of bhk cells (fig. 3) . the virus recovered from dna had the same growth characteristics in bhk cells as the parent jaoars982 virus, with titers reaching 10 6.0 -6.5 pfu/ml, and producing plaques on monolayers of bhk cells similar by the size and morphology to plaques produced by the parent virus (fig. 4) . plaques produced by either virus (turbid) were distinct from plaques produced by attenuated virus sa14-14-2 (fig. 4) , which produced clear plaques. to verify that virus was recovered from the recombinant dna, we examined the presence of the t c mutation at genome pos. 551, which is characteristic for the pcmvmpje(bghtt) construct. total rna isolated from cells, infected with either the parent virus jaoars982 or with the virus recovered from cells transfected with pcmvm-pje(bghtt), was reverse transcribed using a reverse primer complementary to pos. 2306-2330 of the je genome, followed by pcr amplification of a 780 bp fragment with a pair of primers corresponding to pos. 1-25 and 759-780 of the je genome and digestion with sau3a i. the additional sau3a i site formed as result of the above mutation was present only in the fragment amplified from rna of the recovered virus, but not in the fragment originating from parent virus rna (results not shown). this result confirmed that the recovered virus has originated from the plasmid pcmvmpje(bghtt); this conclusion was confirmed also by complete sequence fig. 4 . plaque morphology of parent virus jaoars982, the virus recovered from infectious dna, and attenuated virus sa14-14-2. after infection, bhk cells were overlaid with 1% methylcellulose. plaques were visualized with crystal violet after 4 days. fig. 5 . primer extension analysis of je specific rna with the 5%-[ 32 p]-labeled primer. the sequence ladder gatc was prepared with the same primer and pcmvmpje(bghtt). the corresponding sequence is given on the left with the je genome-specific sequence shown in capital letters. lane 1, uncapped rna transcript synthesized from pt7je5%; lane 2, extension products synthesized from jaoar982-specific rna; lane 3, extension products synthesized from recovered virusspecific rna. sky, 1999 sky, ), coronavirus (almazan et al., 2000 , as well as plant viruses (reviewed, boyer and haenni, 1994; johansen, 1996; lopez-moya and garcia, 2000) . design of flavivirus 'infectious dna', however, has proven to be a more challenging task, perhaps due to the well known instability of plasmids carrying flavivirus genome cdna, which is only exacerbated by the transcriptional activity of eukaryotic promoters in e. coli (antonucci et al., 1989; davis and huang, 1988) . earlier we have reported design of flavivirus 'infectious dna' stabilized by modification of the viral genome cdna cassette (yamshchikov et al., 2001) . our attempts to assemble full je genome cdna under control of the cmv promoter were unsuccessful; a half genome cassette containing je structural proteins was also extremely unstable. similar instability problems were encountered with je constructs driven by the rous sarcoma virus (rsv) long terminal repeat. this may have resulted from expression of viral genome segments encoding hydrophobic protein domains, which are characteristic for membrane-embedded structural proteins of enveloped viruses and are toxic for e. coli (lopez-moya and garcia, 2000) . a stable kunjin replicon construct, which did not include genes of the structural proteins, has been reported (varnavski et al., 2000) . the intron-based approach has been successfully used also for stabilization of 'infectious dna' for a number of 'difficult' viruses (johansen, 1996; lopez-moya and garcia, 2000; olsen and johansen, 2001) . however, since modification of viral genome cassettes may not always be desirable or feasible, we have explored other options allowing use of unmodified viral genome cassettes. because transcription from eukaryotic promoters in e. coli (antonucci et al., 1989; davis and huang, 1988) appears to be a major destabilization factor, we have attempted to reduce its deleterious effects by manipulating the promoter to minimize its activity in bacteria. in experiments with luciferase reporter constructs, the minimal cmv promoter was 5 -6 times less active than the full cmv promoter both in e. coli and in bhk cells. we expected that while in eukaryotic cells ensuing replication of viral rna will compensate analysis of je-specific rna isolated from cells infected with the recovered virus. the 5%-end of the recovered virus genome was analyzed by primer extension of rna isolated from cells infected with either the parent virus jaoars982 or with the virus recovered from dna. synthetic control rna with the 5%-end authentic to je genomic rna was prepared by in vitro transcription of pt7je5% (sumiyoshi et al., 1992; yamshchikov et al., 2001) with t7 polymerase. as shown in fig. 5 , extension products for both viruses demonstrated the same electrophoretic mobility (compare lanes 2 and 3), and both appeared one nucleotide longer than the extension product obtained from the uncapped synthetic transcript (lane 1). this likely resulted from reverse transcription of the 5%-cap structure, which is present in viral genomic rna. we did not investigate this in more detail. both viral genomes had identical 3%-ends (data not shown). eukaryotic promoters have been successfully used for the design of 'infectious dna' of several animal (+ )rna viruses, such as poliovirus (semler et al., 1984) , fmdv (beard et al., 1999) , alphaviruses (reviewed, schlesinger and duben-for the decrease in the transcription rate, substantial reduction in the spurious transcription from the promoter fragment will be beneficial for stabilization of the plasmid during propagation in e. coli. indeed, replacement of the t7 promoter in pt7je/kpn infectious clone (yamshchikov et al., 2001) with the minimal cmv promoter did not result in destabilization of the plasmid or in change of its growth characteristics. this suggests that in e. coli a substantial part of the spurious transcriptional activity was associated with the enhancer, since its exclusion enabled us to obtain a construct reasonably stable in e. coli with the frequency of stabilizing mutations less than 10 − 3 . using indirect immunofluorescence, we detected synthesis of virus-specific proteins in cells transfected with the full genome constructs; however we did not detect virus-specific proteins when cells were transfected with the half-genome construct pcmvmpje5%. this is probably indicative of a low transport rate through nuclear membrane of non-polyadenylated rna or/and a low transcription rate from the minimal promoter in vivo, which may also explain the substantially lower specific infectivity of the dna construct driven by such promoter. certain regulatory elements of the eukaryotic genes, such as enhancers, can influence gene expression in position-and orientation-independent manner (jonsson et al., 1994) . we reasoned that such capability would be beneficial in our system, since we could increase transcription rate in mammalian cells without affecting the stability of constructs in e. coli. indeed, plasmids containing the luciferase reporter gene under control of the minimal cmv promoter and the enhancer inserted at the end of the gene directed increased production of luciferase in bhk cells, i.e. only 40-50% of that directed by the full promoter, and expression levels did not depend on the enhancer orientation. in contrast, no effect on luciferase production was observed in e. coli transformed with these plasmids. similarly, insertion of the cmv enhancer into pcmvmpje(bghtt) after the polyadenylation signal resulted in a 10-100-fold increase in the specific infectivity as compared to the parent plasmid. in our hands, the specific infectivity of the designed dna construct reached 5× 10 5 pfu/mg and became comparable to the specific infectivity of the intron-stabilized plasmid (10 6 pfu/mg; yamshchikov et al., 2001 ). yet, no substantial difference in the stability or in the growth characteristics of the resulting plasmid has been observed in e. coli. in this study we have demonstrated an alternative 'minimal' approach useful in design of problematic 'infectious dna' constructs of (+ )rna viruses, which is based on manipulation of eukaryotic promoter with the goal to reduce its spurious transcription in e. coli. while this approach cannot resolve instability problems associated with the presence in viral genome cdna prokaryotic promoter-like elements (johansen, 1996; lopez-moya and garcia, 2000; olsen and johansen, 2001) , it simplifies design of 'infectious dna' constructs, in which spurious transcription from an eukaryotic promoter fragment does serve as the major destabilizing factor. together with the intron-based stabilization approach, it provides a certain flexibility and a choice in the design strategy. the availability of more convenient flavivirus 'infectious dna' should facilitate advances in studies on mechanisms of flavivirus replication and in design of novel flavivirus vaccines. eukaryotic promoters drive gene expression in escherichia coli development of dna vaccines for foot-and-mouth disease, evaluation of vaccines encoding replicating and non-replicating nucleic acids in swine infectious transcripts and cdna clones of rna viruses sindbis virus expression vectors: packaging of rna replicons by using defective helper rnas sequence and secondary structure analysis of the 5%-terminal region of flavivirus genome rna infectious cdna clones of langat tick-borne flavivirus that differ from their parent in peripheral neurovirulence flavivirus genome organization, expression, and replication transfer and expression of plasmids containing human cytomegalovirus immediateearly gene 1 promoter -enhancer sequences in eukaryotic and prokaryotic cells powerful and versatile enhancer -promoter unit for mammalian expression vectors infectious transcripts of tick-borne encephalitis virus, generated in days by rt-pcr a plasmidbased self-amplifying sindbis virus vector. hum intron insertion facilitates amplification of cloned virus cdna in escherichia coli while biological activity is reestablished after transcription in vivo an enhancer in the first intron of the human purine nucleoside phosphorylase-encoding gene synthesis and characterization of an infectious dengue virus type-2 rna genome phylogeny of the genus flavivirus construction of a stable and highly infectious intron-containing cdna clone of plum pox potyvirus and its use to infect plants by particle bombardment an artificial enhancer with multiple response elements stimulates prokaryotic transcriptional activation medicated by various regulatory proteins nucleotide sequence of the virulent sa-14 strain of japanese encephalitis virus and its attenuated vaccine derivative, sa-14-14-2 nucleotide sequence and infectious cdna clone of the l1 isolate of pea seed-borne mosaic potyvirus infectious rna transcripts from full-length dengue virus type 2 cdna clones made in yeast in vitro processing of dengue virus type 2 nonstructural proteins ns2a, ns2b, and ns3 cloned poliovirus complementary dna is infectious in mammalian cells flaviviridae: the viruses and their replication transcription of infectious yellow fever rna from fulllength cdna templates produced by in vitro ligation modulations of the in vitro translational efficiencies of yellow fever virus mrnas: interactions between coding and noncoding regions assaying the polyadenylation state of mrnas molecular cloning: a laboratory manual alphavirus vectors for gene expression and vaccines production of infectious poliovirus from cloned cdna is dramatically increased by sv40 transcription and replication signals infectious japanese encephalitis virus rna can be synthesized from in vitro-ligated cdna templates complete nucleotide sequence of the japanese encephalitis virus genome rna promoter-regulatory region of the major immediate early gene of human cytomegalovirus stable high-level expression of heterologous genes in vitro and in vivo by noncytopathic dna-based kunjin virus replicon vectors a new strategy in design of +rna virus infectious clones enabling their stable propagation in e. coli we thank dr s. schlesinger for bhk-21 cells, dr d. trent for pm343 and ph756 plasmids, and jennifer ascañ o for help in manuscript preparation. this study was supported by research award v22/181/128 to v.f.y. from the world health organization. almazan, f., gonzalez, j.m., penzes, z., izeta, a., calvo, e., plana-duran, j., enjuanes, l., 2000. from the cover: engineering the largest rna virus genome as an infectious bacterial artificial chromosome. proc. natl. acad. sci. usa 97, 5516 -5521. key: cord-279638-jr1mbh7s authors: calore, elisabetta; marson, piero; pillon, marta; tumino, manuela; tison, tiziana; mainardi, chiara; de silvestro, giustina; rossin, sara; franceschetto, genny; carraro, elisa; pescarin, matilde; varotto, stefania; destro, roberta; gazzola, maria vittoria; basso, giuseppe; messina, chiara title: treatment of acute graft-versus-host disease in childhood with extracorporeal photochemotherapy/photopheresis: the padova experience date: 2015-07-14 journal: biol blood marrow transplant doi: 10.1016/j.bbmt.2015.07.007 sha: doc_id: 279638 cord_uid: jr1mbh7s acute graft-versus-host disease (agvhd) is the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. systemic steroid treatment represents the first-line therapy for agvhd and is associated with a response rate of 30% to 60%. steroid-resistant patients have a poor prognosis with high transplantation-related mortality (trm). several second-line therapies have been proposed for the management of unresponsive agvhd, without proven beneficial effects on patients' outcome or overall long-term survival. for these reasons, extracorporeal photochemotherapy/photopheresis (ecp), a cell-based approach to control gvhd that spares generalized immunosuppression, seems to be promising. in this study, we report the outcome of 72 consecutive pediatric patients treated with ecp between 1997 and 2013 for agvhd. among them, 21 patients had steroid-resistant agvhd, 42 had steroid-dependent agvhd, and 9 did not receive steroid as first-line therapy because of clinical contraindications. a complete response was obtained in 72% of patients, a partial response was observed in 11%, and there was no response in 17% of patients. at day +180, trm was 4% in the whole cohort; trm was 3% and 20% among responders and nonresponders to ecp, respectively (p < .0001). the 5-year overall survival was 71%, showing a difference between responders and nonresponders of 78% and 30%, respectively (p = .0004). the 5-year time to progression of primary disease was 81%, without any significant difference between the 2 groups. moreover, the 5-year progression-free survival of primary disease was 72%, with a significant difference (p = .0007) between responders (79%) and nonresponders (30%) to ecp. in conclusion, this study demonstrates that ecp is highly effective in agvhd without a negative impact on primary disease. allogeneic (allo) hematopoietic stem cell transplantation (hsct) is increasingly used as a therapeutic approach for hematological and nonhematological diseases. in the last decade, improvements in infection monitoring and prophylaxis, immunosuppressive (is) strategies, high-resolution hla typing, and supportive care measures led to better outcomes after this procedure. despite these advancements, acute graft-versus-host disease (agvhd) remains the major cause of morbidity and mortality after allo-hsct [1, 2] . to standardize diagnosis and management of agvhd, a british guideline was published by a joint working group of the british committee for standards in haematology and the british society for bone marrow transplantation. this document included recommendations for diagnosis and management of agvhd as well as primary treatment options for patients with steroid-refractory (sr) disease [3] . standard management of agvhd included steroids at different doses depending on agvhd grade. if no improvement of agvhd after 7 days was noted or progression within 72 hours were observed, then the addition of secondline agents should be considered. second-line options are mycophenolate mofetil (mmf), anti-tnf antibodies, mammalian target of rapamycin inhibitors, il-2 receptor antibodies, and extracorporeal photochemotherapy/photopheresis (ecp) [3] . unfortunately, despite multiple clinical trials, no single agent improving overall survival (os) for patients who failed steroid treatment has been identified [4, 5] . moreover, the current survival at years in patients who respond to steroids is about 36% versus 17% in nonresponders (nr) [6] and it has been shown that transplantation-related mortality (trm) is higher in steroidresistant patients [7, 8] . in our study, we focused on ecp, one of the most promising treatments for agvhd. briefly, ecp consists of 3 procedures: collection of peripheral leukocytes cells, irradiation of cells by ultraviolet a light in presence of 8methoxypsoralen , and reinfusion of treated cells in the patient. the underlying mechanism of action of ecp in gvhd is not completely understood [9, 10] . within 24 hours, this process induces apoptosis of all treated cells (including t cells) and subsequent phagocytosis by antigen-presenting cells; as a result, this might regulate immune homoeostasis, modulate the cytokine production, and induce tolerance of antigen-presenting cells [10] [11] [12] [13] . ecp has been demonstrated to be effective in treating both steroid-resistant and steroid-dependent patients with agvhd [14] [15] [16] [17] . in the pediatric setting in particular, the reported response rate ranges from 50% to 100%, according to the organs involved. in agvhd steroid-resistant patients, 5year os is significantly increased in complete responders to ecp, 69% compared with 12% for nr [18] . in 2013, the italian society of haemapheresis and cell manipulation and the italian group of bone marrow transplantation elaborated best practice recommendations for ecp, which reflected the common clinical practice in most italian transplantation centers where ecp is performed with a total of 4500 procedures per year [19] . despite this large use of ecp, most of the published reports deal with retrospective data that are difficult to compare, as patients' selection criteria, treatment schedules, patients' monitoring, and patients' assessment protocols differ among institutions. moreover, no randomized studies have been conducted in patients with agvhd. here, we report our single-center experience on ecp treatment in 72 pediatric patients with agvhd. the response to ecp, trm, os, progression-free survival (pfs) of primary disease, and time to progression (ttp) of primary disease of patients treated with ecp were analyzed. from january 1997 to june 2013, 72 consecutive pediatric patients (44 males, 28 females) affected by agvhd were treated with ecp at the hsct unit of university hospital of padova. fifteen of these 72 patients have been previously reported [20] . the clinical characteristics of patients are shown in table 1 . the median age at ecp was 8.3 years (range, .9 to 20.3 years) and the median body weight was 25 kg (range, 7 to 98 kg). fifteen children weighted less than 15 kg. the last follow-up was fixed on june 2014. in detail, 21 patients were treated with ecp for agvhd refractory to steroids, which was defined as a progression or no improvement in agvhd after at least 3 days or 7 days on methylprednisolone (mp) ! 2 mg/kg body weight, respectively (sr group); 21 patients for steroid-dependant agvhd, defined as a flare-up of agvhd during the tapering of mp (sd group); and 30 children with agvhd who required a reduction of pharmacological is or contraindications to is therapy for viral reactivations, systemic mycoses, or intolerable side effects (group with infectious complications [ic]). in particular, 9 of 30 patients in the ic group (ic-a group) underwent ecp without steroids as a first-line therapy because of contraindications: 1 for tcr ab and cd19edepleted haploidentical transplantation with probable invasive pulmonary aspergillosis (ipa) and adenovirus (adv), 1 for proven ipa, 1 for concomitant proved ipa and cytomegalovirus (cmv) reactivation, 1 for probable ipa and cmv and bk virus (bkv) reactivations, 1 for proven ipa and multiple viral reactivations, including adv, cmv, and epstein-barr virus (ebv), 2 for cmv reactivation, 1 for cmv and ebv reactivations, and 1 for cmv, ebv, adv, and bkv reactivations. the other 21 patients (ic-b group) had sd agvhd and cyclosporin a (csa)erelated renal insufficiency in only 1 patient; sd agvhd and concomitant infections in the remaining 20 patients: cmv reactivations, 2; cmv and ebv reactivations, 4; cmv and bkv reactivations, 2; cmv, adv, and bkv reactivations, 1; ebv reactivations, 4; ebv and adv reactivations, 1; ebv reactivations and probable aspergillosis, 1; hepatitis b virus, 1; hepatitis b virus and ebv reactivations, 1; cmv, ebv, and bkv reactivations, 1; adv, human herpesvirus-6, bkv, and coronavirus, 1; hepatitis c virus, cmv, and proven ipa, n ¼ 1. in our practice, surveillance for viral and fungal infections in blood is routinely performed during the first 100 days after hsct in all patients and includes ebv-dna, cmv-dna, adv-dna, human herpesvirus-6 dna, bkv-dna, jc-dna, and galactomannan antigen search. this schedule is performed once each week in allo-hsct recipients from hla-identical sibling and twice each week in allo-hsct recipients from unrelated or haploidentical donors. monitoring viral infections in urine comprises cmv-dna, bkv-dna, jcv-dna in a weekly search. blood, urine, and stool cultures; nasal and throat swabs; and nasopharyngeal aspirates were weekly performed. search for other viruses or microbiological agents was performed upon clinical symptoms. viral reactivations were detected by pcr positivity for ebv-dna (cut-off: 1000 copies/ml), cmv-dna (cut-off: 1000 copies/ ml), and adv-dna in qualitative test. clinical systemic fungal infections were defined proved or probable according to european organization for research and treatment of cancer criteria [21] . the cut-off for the galactomannan index was set at .5 (enzyme immuno assay (e.i.a.) method). the algorithm for agvhd treatment used in our center is shown in figure 1 . csa was administered for 6 and 12 months in children who received hsct from an hla-identical sibling or unrelated donor, respectively. in unrelated hsct, short-term methotrexate and rabbit antithymocyte globulin (atg) were given. in unrelated cord blood hsct, prophylaxis included csa and atg. in haploidentical setting, ex vivo elimination of ab t cells and cd19 b cells was done and atg was administered before the cells were infused; no other is therapy was given after hsct. informed consent was obtained from patients' parents, as well as from the patients themselves when possible, and the use of ecp was approved by the ethical committee of the hospital of padova. the clinical organ involvement was graded and then combined to obtain an overall grade, according to glucksberg criteria for agvhd [22] . histological confirmation was obtained whenever clinically indicated to confirm gvhd diagnosis. eligibility criteria for ecp treatment were as follows: children with sr agvhd (n ¼ 21); children with sd agvhd (n ¼ 21); patients with agvhd in whom is therapy was contraindicated or who required a rapid decrease of is therapy for increasing ebv viral load, cmv reactivation in 2 subsequent samples, systemic fungal infections, intolerable side effects (n ¼ 30). all children must present in complete hematological remission and full donor chimerism; white blood cell (wbc) count > 1 â 10 9 /l, and no concomitant treatment with either atg or monoclonal antibodies. ecp was performed using 2 different techniques: "in-line" treatment (uvar photopheresis instrument, therakos, exton, pa) was used in 19 of 72 patients and the "off-line" technique (cobe spectra, bct terumo, lakewood, co) was used in 53 of 72 children. technical descriptions have already been published [19] . the "off-line" technique was introduced in 2003 to treat lowweight children. for patients weighing less than 15 kg, priming of the leukapheresis circuit with irradiated and leuko-reduced red blood cells (regardless of baseline hemoglobin level) was performed, as recommended in the italian society of haemapheresis and cell manipulation-italian group of bone marrow transplantation indications [19, 23] . pre-ecp hemoglobin levels were maintained between 10 g/dl and 12 g/dl. the cell product was treated with 8-mop and diluted to a final concentration of 20 mg/100 ml to 34 mg/ 100 ml, according to specific procedures (in-line technique, 34 mg/100 ml; off-line technique, 20 mg/100 ml). in all patients, a 7 to 9 french hickman double-lumen central catheter was systematically used for the procedure. to provide adequate flow rates, ie, 1 to 2 ml/kg/minute, anticoagulation with urokinase 10,000 u for 2 hours as lock-therapy was performed on the day of the procedure. the leukapheresis product contained a median of wbc of 19.4 â 10 3 /ul (range, 10.7 to 70.1 â 10 3 ), a median of mononuclear cells (mnc) of 80.5% (range, 50% to 90%). the median number of wbc reinfused to the patients was 2970 â 10 6 (range, 1150 to 10,420 â 10 6 ), whereas the median number of mnc reinfused to the patients was 2794 â 10 6 (range, 782.3 to 9805.4 â 10 6 ). patients were treated with ecp twice each week for the first month, every 2 weeks during the second and third months, and then monthly for at least 3 more months, for a total of 22 procedures. progressive tapering and discontinuation of ecp were decided upon evaluation of individual response. any concomitant is therapy was initially maintained, then modified or discontinued according to the clinical response. criteria for defining response to ecp were previously reported [20] . all patients enrolled for ecp before day þ100 were included in this group and response to ecp was evaluated at day þ28, day þ56, and at the end of ecp treatment. complete response (cr) was defined as the resolution of all signs of agvhd and partial response (pr) as at least a 50% improvement in the clinical signs. in the latter case, given the complexity of assessing response, we defined pr for each organ as follows: for the skin, at least a 50% reduction in the body surface area affected; for the gi tract and liver a 50% reduction in the volume of diarrhea or value of bilirubin. any worsening of organ involvement, as well as the appearance of new signs or symptoms of gvhd, was defined as progressive disease (pd). patients with stable or pd were considered nr. seventy-two consecutive patients with agvhd received ecp at a median time of 46 days (range, 13 to 91) after hsct and 22 days (range, 4 to 81) from the diagnosis of agvhd. sixty-four patients had skin involvement (grade iv, n ¼ 13; grade iii, n ¼ 20; grade ii, n ¼ 21; grade i, n ¼ 10). fifty-five patients had gastrointestinal (gi) agvhd (grade iv, n ¼ 8; grade iii, n ¼ 2; grade ii, n ¼ 18; grade i, n ¼ 27). twelve patients had liver involvement (grade iii, n ¼ 3; grade ii, n ¼ 4; grade i, n ¼ 5). regarding the number of organs involved: in 17 patients skin was affected and 7 presented gi involvement, whereas 36 patients had combined skin and gi agvhd, 1 patient had combined gi and liver agvhd, and 11 patients had combined skin, gi, and liver agvhd. ic-a group is those with infectious complications and no steroid before ecp; the ic-b group is those with infectious complications and steroid before ecp. * hla match considered 6/6. the overall clinical grading of agvhd was as follows: grade i, n ¼ 8; grade ii, n ¼ 29; grade iii, n ¼ 17; and grade iv, n ¼ 18; details of different grades in the patients' subgroups can be found in table 1 clinical evaluation of the patients was conducted at every ecp procedure. sixty-three of 72 patients with agvhd grades i to iv received 2 mg/kg/ day of mp as first-line therapy. the median dose of steroid at the beginning of ecp was 2 mg/kg/day. in detail, the is therapies before ecp were csa, n ¼ 9; csa plus steroid (2 mg/kg), n ¼ 42; tacrolimus plus steroid (2 mg/kg), n ¼ 12; and csa or tacrolimus plus mmf plus steroid, n ¼ 9. ecp was used as first-line therapy in 8 of 72 patients, as second line therapy in 43 of 72 patients (among them, 1 haploidentical hsct was treated only with csa), as third-line in 15 of 72 patients, and in 6 of 72 patients as fourth-line therapy. patients' characteristics were compared using the chi-squared or fisher's exact test (as appropriate) in case of discrete variables, or the mann-whitney test in case of continuous variables. trm was calculated from the date of hsct to day þ180 and to the last follow-up, considering as event any nonrelapse cause of death. os was calculated from the date of hsct to the date of death from any cause, or to the last follow-up. pfs was calculated from the date of hsct to the date of relapse of underlying primary disease or death for any cause or to the last follow-up. ttp was calculated from the date of hsct to the date of relapse of primary disease or to the last follow up. cumulative incidences (ci) of relapse of underlying disease were estimated in the competing risk model, considering death from any cause or cgvhd as the competing events. survival analysis was performed using kaplan-meier method with 95% confidence interval. standard error (se) for each survival and incidence rate is given. differences between groups were compared using the log-rank test and the gray's test. all reported p values were 2-sided, and statistical significance was set at a ¼ .05 (sas institute, cary, nc; release 8.2) [24] . response to ecp treatment, evaluated according to the overall grading of agvhd and to the organ involvement at day þ28, day þ56, and at the end of ecp, is summarized in table 2 . at the end of treatment with ecp, 52 of 72 (72%) patients had a cr, 8 of 72 (11%) had a pr, and 12 of 72 (17%) were nr. among the 52 patients showing a cr, 7 patients had agvhd grade i, 22 patients had grade ii, 12 had grade iii, and 11 had grade iv. in particular, the cr rate for patients with agvhd grades i and ii and grades iii and iv were 78% and 66%, respectively (p ¼ .70), whereas the pr rate for patients with agvhd grades i and ii and grades ii to iv were 5% and 17%, respectively (p ¼ .80). no significant statistical difference in cr rate was observed according to the subgroups analyzed (sr, 67%; sd, 81%; ic groups, 70%) (p ¼ .91). at ecp discontinuation, cr of agvhd manifestations of skin, gut, and liver was observed in 78%, 76%, and 84% of patients, respectively. maximal response to ecp was observed after 8 weeks of treatment (16 procedures). as a result of ecp, at the end of treatment, it was possible to discontinue is therapy in 12 patients (17%) and reduce it in 44 patients (61%), of them 32 who received allo-hsct from an unrelated donor. regarding the steroid tapering, in 63 patients treated with 2 mg/kg/day before ecp, the steroid dose was reduced by 80% after 1 month of ecp treatment, 84% after 2 months, and 88% after 3 months of ecp treatment. the median lansky/karnofsky performance score improved from 70% before ecp to 100% after completing the treatment. no association was found between responders and nr to ecp and the major clinical risk factors affecting agvhd (table 3) . cgvhd twenty-three of 72 patients (32%) presented clinic manifestations of cgvhd (table 4 ). in detail, 19 patients (26%) had progressive cgvhd (11 nr and 8 pr to ecp) and 4 patients (5%) had quiescent cgvhd onset after a median of 6 months (range, 5 days to 16 months) from the end of ecp. overall grading of cgvhd, based on the national institutes of health consensus [25] , was mild for 6 patients, moderate for 10 patients, and severe for 7 patients. among the patients with progressive cgvhd, ecp was used with other is therapies in 4 of 19 patients, obtaining cr in only 1 of them. overall, 10 of 19 patients were alive at the last follow-up: 9 of 10 had no cgvhd and discontinued is therapy, whereas only 1 patient presenting with cgvhd was still in treatment. all the patients with quiescent cgvhd were alive at the last follow-up: 2 patients were free from cgvhd and without is therapy and the other 2 patients had cgvhd and were still on treatment with is therapy plus ecp. no association was found between responders and nr to ecp and the onset of quiescent cgvhd. at day þ180, the overall trm was 4% (se, 1%). trm was 3% (se, 2%) and 20% (se, 13%) for responders and nr to ecp, respectively (p < .0001). at last follow-up, the overall trm was 11% (se, 4%), whereas trm stratified between responders and nr was 3% (se, 2%) and 58% (se, 20%), respectively (p < .0001) (figure 2a,b) . the 5-year os was 71% (se, 5%) with a statistically significant difference between responders and nr (78%; se, 5% versus 30%; se, 14%, respectively; p ¼ .0004) ( figure 3a,b) . the 5-year pfs of primary disease for all the group was 72% (se, 5%), with a significant difference (p ¼ .0007) between responders (79%; se, 5%) and nr (30%; se, 14%) ( figure 4a,b) . overall, the 5-year ttp of primary disease was 81% (se, 5%), without any significant difference between the 2 groups (responders: 82%; se 5% versus nr: 78%; se, 14%; p ¼ .65) ( figure 5a,b) . we compared patients' survival rates on ecp treatment used as first, second, or third/fourth-line therapy. no difference was observed at 5-years between responders and nr in term of os (p ¼ .56), pfs (p ¼ .55), and ttp (p ¼ .62). the overall 5-year ci of relapse of the underlying disease was 20% (se, 5%); in particular, it was 21% (se, 6%) and 20% (se, 9%) for responders and nr to ecp, respectively ( figure 6a,b) . overall, at the last follow-up (median time from hsct of 5 years; range, .18 to 17.6 years), 51 patients were alive (71%); 48 of them (94%) were without gvhd and without any is therapy. twenty-one patients (29%) died: 14 from relapse of primary disease and 7 from nrm. among this last group, 1 patient with agvhd died at day þ65 from hsct because of sepsis; 5 patients with cgvhd died from cmv pneumonia (1 case), acute hepatitis from hcv infection (1 case), encephalopathy (1 case), and multiorgan failure (2 cases); and 1 patient died from cmv pneumonia at day þ135 from hsct, without evidence of cgvhd. side effects observed during ecp were generally mild and more frequent in low-weight children. ecp caused mild hypotension in 10 patients associated with abdominal pain in all cases (16 episodes out of 1382 apheresis sessions). these adverse effects did warrant suspending the procedure. a transient reduction in hemoglobin, platelet, and/or wbc count during the ecp treatment, likely independent from the post-transplantation course and putatively ecp-related, was observed in 26, 20, and 25 patients, respectively. one patient with grade iv agvhd on high-dose steroid therapy (5 mg/kg/ day) experienced acute gi bleeding after the second course of ecp: gi endoscopy showed multiple ulcers in the stomach. a girl with pre-existing cardiac impairment showed acute heart failure for fluid overload after the procedure that quickly responded to adequate therapy. one girl, after 10 ecp procedures, had anaphylaxis (cough, vomiting, abdominal pain, hypotension, and palpebral edema) a few minutes after the end of 8-mop irradiated bag infusion. she responded to antihistamine and steroid therapy, but ecp treatment was then stopped. the aim of this retrospective study was to analyze the role of ecp for the treatment of agvhd. the efficacy of ecp is well established for treatment of cgvhd [26, 27] , whereas in agvhd, no prospective randomized studies have been published. however, the use of ecp is suggested as second-line therapy, together with mammalian target of rapamycin inhibitors, mmf, il-2 receptor antibodies, and anti-tnf antibodies [3] . the largest phase 2 prospective study exploring feasibility and efficacy of ecp in treatment of agvhd in adults, involving 59 patients, was performed by greinix et al. [14] and reported a cr rates of 82% for skin and 61% for liver and gi agvhd. so far, data on 207 pediatric patients treated with ecp for agvhd have been reported, showing an overall cr rate ranging from 32% to 73% and a survival rate ranging from 44% to 85% [18, 20, [28] [29] [30] [31] [32] [33] [34] . to date, our is the largest pediatric case series treated in a single center. in our sample size, we found a higher overall response rate to ecp compared with a multicenter retrospective study of the italian association for pediatric hematology/oncology (aieop) (72% versus 54%) [18] . we attempted to determine the factors that may have influenced our observed higher response. in the last 15 years, many changes have been introduced in hsct, such as highresolution hla typing, new agents in the conditioning regimen, more use of atg, monoclonal antibodies, and new antifungal drugs. it is difficult to determine which modification may have influenced the outcome. we could also hypothesize that specific expertise in pediatric hsct and earlier treatment with ecp (22 days in our series versus 30 days in aeiop study) may have improved the overall outcome. further studies are needed to address this topic. no association was found between responders and nr setting to ecp and major risk factors for agvhd. in addition, in our series, no difference was found according to the grade of gvhd (grade i and ii, 78%; versus grade iii and iv, 66%; p ¼ .70) and to the subgroups of patients analyzed (sr, 67%; sd, 81%; ic, 70%; p ¼ .91). our results showed better response rate than those reported in literature for advanced stages of disease, where higher grades and poorer response to is therapy correlate with a worst outcome [6] . nevertheless, higher cr rates were observed in grade ii gvhd, suggesting that an early start of ecp sessions may be beneficial, even if in our study the timing to start ecp (<22 versus > 22 days) did not influence the response. in our group, ecp seemed to be effective in all the involved organs. as previously reported, our results support that ecp is a steroid-sparing treatment; in fact, the steroid dose was reduced by 80%, 84%, and 88% after 1, 2, and 3 months, respectively from the onset of ecp. we performed ecp as front-line therapy in 8 patients with fungal infection and viral reactivation and agvhd, with complete response in 7 of them. to our knowledge, this is the first report of ecp as first-line treatment. is therapy was either discontinued or reduced in 78% of responding patients. it is well known from the literature that is therapy increases the risk of infectious complications and relapse of underlying disease after allo-hsct [1, 2, 18, 20, 28, 35] . in children, who may be particularly vulnerable to the consequence of gvhd itself and prolonged treatment with is agents, the use of ecp is particularly appealing. the efficacy of ecp in controlling gvhd did not affect the preservation of graft-versus-leukemia effect; in fact, the low incidence of relapse of underlying disease was recorded by us and others [17, 18, 20, 28] . many concerns has been raised related to the technical aspects of apheresis in the pediatric setting. in children with low body weight, the caregivers should carefully monitor patients for signs and symptoms of hypovolemia. in our experience, ecp was well tolerated, with few and mild adverse effects, the most frequent of which were mild hypotension, abdominal pain, and headache. curiously, these symptoms were recorded more often during ecp compared with other apheretic procedures [19, 34] . the majority of side effects were observed in the earliest period in which ecp was performed in our center. all these observations support the idea that there has been a learning process for the management of technical elements and side effects. in our experience, ecp was feasible even in 15 very young children with low body weight (<15 kg). technically, we performed priming of the circuit with irradiated and leukodepleted red blood cells (regardless of baseline hemoglobin level). some authors recently reported that saline infusion or albumin boluses may be an alternative priming approach in patients with body weight ranging from 19 to 39 kg [36] . however, it should be proven that this approach could be safely transferred to population weighting less than 15 kg. currently used ecp techniques include the "off-line" and the "in-line" devices [19] . in our center, both techniques were used in different time periods with no difference in response rate observed. the number of wbc collected and mnc reinfused did not affect clinical outcome. notably, all patients underwent the procedure with the bilumen central venous line already in place (hickman-broviac bard access systems, salt lake city, ut, usa), which is different from the majority of reports, in which a larger central venous line (for instance, quinton) is placed. it would be interesting to extend our experience to determine if urokinase anticoagulation allows the proper flow rate of pre-existing central venous line. further, because of the experience of the staff in completing the procedure, no patient required sedation. the ci of cgvhd in pediatric population ranged from 6% to 65% according to the source of stem cells (hla-identical sibling cord blood versus matched unrelated donor peripheral blood) [37, 38] , whereas in the aieop experience, the ci of cgvhd was reported to be 27% [39] . in our small series, the incidence of cgvhd was 32%. the majority of our children presented progressive cgvhd (26%) and few had quiescent cgvhd (5%). for this reason, it is hard to determine if patients previously treated with ecp for agvhd could benefit from retreatment. our data are consistent with literature and the results encourage us in exploiting this promising approach for agvhd. in conclusion, a standardized approach to ecp treatment is needed for pediatric patients. from this perspective, sharing single-center experience is 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there are no conflicts of interest to report. key: cord-347761-wgodcsav authors: cant, andrew; cole, theresa title: infections in the immunocompromised date: 2009-10-24 journal: hot topics in infection and immunity in children vi doi: 10.1007/978-1-4419-0981-7_1 sha: doc_id: 347761 cord_uid: wgodcsav infections in the immunocompromised differ significantly from those in the immunocompetent. they can be more serious, more often life threatening, more difficult to diagnose and are caused by more unusual organisms. children can be immunocompromised for a variety of reasons and the numbers, worldwide, are growing. cryptosporidium and aspergillus species. b lymphocyte defects can result in infections by bacteria such as streptococcus pneumoniae, haemophilus influenzae and staphylococcus aureus, as well as echo virus and protozoa such as giardia, whereas phagocytic defects will result in infections by staphylococcus, pseudomonas and aspergillus species. when considering infections in those undergoing hsct, it is also important to note that there is a recognized sequence of risk for different infections at different times after hsct, which equates with different aspects of the immune system compromise at these times (fig. 1) . within the first 30 days, when the patient is neutropenic, there is a significant risk of infection by both gram-negative and grampositive bacteria, along with herpes-simplex virus. between 30 and 90 days after transplant, when t-cell immunity is still limited, there is a rise in the numbers of fungal and cmv infections. later infections are more commonly caused by varicella zoster virus (vzv) or s. pneumoniae. whatever the cause of the immunocompromise, possible infection requires a different approach to investigation and management from those in an immunocompetent child. although an understanding of the type of immunocompromise is helpful to predict the likely organism, infection in the immunocompromised also needs to be considered by the system affected. infections can occur in any system of the body, but the respiratory system and gastrointestinal tract are especially vulnerable, as they have large surface areas and their barrier defences are, of necessity, compromised by the need to transport oxygen and nutrients, respectively. disseminated viral and fungal infections are another important risk, whilst central venous catheter (cvc) infections also constitute a frequent problem in the immunocompromised. each of these will be discussed in turn. the respiratory tract can be exposed to a wide variety of different organisms. pneumocystis jiroveci pneumonia (pcp), cmv and aspergillus are particularly important and well recognized sources of infection in the immunocompromised host; however, other significant pathogens have more recently been identified. these include respiratory syncytial virus (rsv); influenza; parainfluenza; adenovirus; picornaviruses; measles; human metapneumovirus; cocavirus; coronaviruses nl63, and hku1 and polyomaviruses wu and k1. pneumonitis and bronchiolitis are the most common presentations of respiratory infection, but lobar pneumonia may also occur. a defective immune/inflammatory response means that patients may have few respiratory symptoms, so there should be a low threshold for investigation. in one study where broncho-alveolar lavage (bal) was performed in 69 children with immunodeficiency pre-hsct, pathogens were isolated in 26 of these, six of whom were asymptomatic. pcp and bacteria were the most commonly identified organisms, followed by parainfluenza virus, cmv, rsv, influenza b and human herpes virus-6 (hhv6) (slatter et al., 2007) . accurate diagnosis depends on collecting the right samples and using appropriate diagnostic techniques. these include throat swabs, nasopharyngeal aspirates (npa), bal fluid and even lung biopsy, as deemed appropriate. samples must be sent to look specifically for bacteria, fungi and viruses. some respiratory pathogens will not be isolated from the upper respiratory tract; for example, pcp will not be identified on npa, whilst other organisms found on npa may not be found in the lower respiratory tract. this highlights the importance of bal as a diagnostic procedure. lung biopsy may be particularly important in the diagnosis of fungal infection, especially when there is a negative bal in patients with persistent signs, symptoms or chest x-ray changes. diagnosis may require culture of organisms (bacteria, mycobacteria or viruses), immunofluorescence (viruses), polymerase chain reaction (bacteria, viruses and fungi) or antigen testing (e.g. galactomannan for aspergillus). serological testing is often ineffective, as immunodeficient children may not mount an antibody response or may be receiving intravenous immunoglobulin (ivig), which will make results impossible to interpret. it is important to know what tests are available in your local laboratory. discussion with the local microbiologist or virologist is essential to ensure the right samples are sent for appropriate investigations, so as not to miss a serious infection. high resolution computerized tomography (hrct) of the chest is more sensitive than chest x-ray, aiming to classify a disease as interstitial, airway or involving airspace, which may aid diagnosis. pcp has historically been associated with hiv but is also a significant cause of morbidity in other groups of immunocompromised patients, particularly those with haematological malignancies, brain tumours requiring prolonged courses of steroids, prolonged neutropaenia or lymphopaenia, and those undergoing hsct. therefore pcp prophylaxis is important, as recommended by a recent cochrane review (green et al., 2007) . this treatment is generally in the form of cotrimoxazole given three times per week. in children that cannot tolerate cotrimoxazole, either dapsone or aerosolized pentamidine can be used. p. jiroveci infection commonly presents with tachypnoea, non-productive cough and fever, but the severity can vary. there is usually a sub-acute diffuse pneumonitis and chest x-ray changes can be subtle. these often take the form of bilateral diffuse interstitial changes, although lobar, miliary or nodular changes can be seen. hrct may show ground glass attenuation, consolidation, nodules, thickening of interlobular septa and thin walled cysts. mortality ranges between 5 and 40%, if treated, but can reach nearly 100% if left untreated. identification of pcp can be difficult. definitive diagnosis depends on identifying the organism in respiratory tract secretions or lung tissue, usually from tracheal secretions, bronchial secretions or from lung biopsy. more recently, pcr technology has been developed for identifying pcp from secretions. in a review of children diagnosed with severe combined immune deficiency (scid) treated at a supra-regional center, 10 out of 50 were identified as having pcp. one was diagnosed on bal prior to transfer to the supra-regional center, one was diagnosed on nasopharyngeal secretions and bal, seven were diagnosed on bal alone, and in one diagnosis was not made until lung biopsy was performed (berrington et al., 2000) . recommended first line pcp treatment is high dose cotrimoxazole. this can, however cause a number of adverse effects, for example, neutropenia, anaemia, renal dysfunction, rash, vomiting and diarrhea. those that cannot tolerate cotrimoxazole or those that have not improved after 5-7 days of treatment should be changed to a different agent. choices include pentamidine, atovaquone, clindamycin/primaquine or dapsone, but experience with these agents in children is limited. corticosteroids should be given as an adjunctive therapy in moderate and severe pcp. a number of studies have shown a reduction in acute respiratory failure, decreased need for ventilation and decreased mortality (sleasman et al., 1993; bye et al., 1994; mclaughlin et al., 1995) . a recent cochrane review supports the use of corticosteroids in hiv-infected patients with pcp, especially in those with substantial hypoxaemia (briel et al., 2006) . a wide variety of respiratory viruses will also cause significant morbidity and mortality in the immunocompromised. measles is an important respiratory pathogen in the immunocompromised host and it must be remembered that the typical rash may not develop. mortality can be high, especially amongst patients with leukaemia and those undergoing hsct. a prospective multi-center review of patients undergoing hsct found direct rsv-associated mortality to be 17.4%, and mortality directly attributable to influenza a to be 15.3% (ljungman et al., 2001) . respiratory viruses often present with non-specific symptoms but progress to a significant lower respiratory tract infection. chest x-ray will often show a pneumonitis picture with diffuse interstitial changes. hrct may show peri-bronchial thickening and ground glass attenuation without consolidation in a lobular distribution. diagnosis requires identification of the organism from respiratory secretions. this may be possible on nasopharyngeal secretions or throat swab but may require more invasive testing, such as bronchoscopy and bal. laboratory techniques include immunofluorescence, pcr and viral culture. treatment is mainly supportive, but specific treatment options are evolving, making rapid and accurate diagnosis increasingly important. appropriate isolation and infection-control measures are essential to prevent transmission between immunocompromised patients, as these viruses can be easily spread. one uk study in a hsct unit identified 10 cases of rsv over one winter season, and eight of the nine rsv strains that could be tested by molecular methods were found to be identical (taylor et al., 2001) . specific treatments for rsv infection include ribavirin and rsv monoclonal antibody (palivizumab). ribavirin can be given orally, intravenously or via inhalation; however, the aerosolized route has been used most frequently for rsv infection. historically, pooled hyperimmune rsv immunoglobulin has been proposed as an additional treatment, but this has been superseded by the anti-rsv monoclonal antibody, palivizumab. combinations of inhaled ribavirin and intravenous palivizumab have shown encouraging results. palivizumab has been shown to be safe and well tolerated in patients undergoing hsct, with a suggestion of better outcome (improved survival) when compared to ribavirin alone (boeckh et al., 2001; chavez-bueno et al., 2007) . there are two groups of drugs available for the treatment of influenza -namely the adamantanes (effective against influenza a, e.g. rimantadine) and the neuraminidase inhibitors (effective against both influenza a & b, e.g. oseltamivir). in recent years, there has developed increasing resistance to adamantanes. the neuraminidase inhibitors have been shown to reduce the duration of illness by one day when given to an immunocompetent host within 48 h of onset of symptoms. although there are few data on the benefit of treating influenza in an immunocompromised patient with a neuraminidase inhibitor, their use appears sensible and safe. there is, thus far, no specific treatment available for rhinovirus, coronavirus or human metapneumovirus. ribavirin has been proposed as a treatment for parainfluenza virus infection but evidence, so far, of benefit is disappointing. although there is little clinical data on the use of ribavirin for measles pneumonitis, it does have in vitro activity and therefore, due to the high level of mortality with this condition, should be considered. a review of respiratory viral infection in children with primary immune deficiencies in a hsct unit found 22 of 73 patients admitted for hsct had respiratory viral infection. of these, 11 had paramyxoviruses (rsv or parainfluenza i-iv), and were treated with aerosolized ribavirin and ivig. five of these patients also received nebulized immunoglobulin and corticosteroid. three of these five survived, compared to two out of the six who did not receive nebulized treatment. it was concluded that the nebulized treatment was well tolerated and could be a useful adjunctive therapy (crooks et al., 2000) . in children who have undergone hsct, infection and inflammation can become inextricably interwoven to generate pneumonitis. in this case, in addition to the need for anti-infective agents, immunomodulation will be required through agents such as steroids, ivig and anti-tumour necrosis factor monoclonal antibodies. the gastrointestinal tract is also exposed to a wide variety of organisms and viruses which are of particular concern in the immunocompromised child, notably enteroviruses, adenovirus, rotavirus, caliciviruses, but also protozoa, mainly cryptosporidium and giardia. presentation is most commonly with diarrhea and vomiting, which may protracted. cryptosporidium can also be responsible for ascending cholangitis and liver disease. in some cases, identification of the causative organism can be difficult. culture may be required to identify some viruses. pcr can be useful, for example, for adenovirus and is more sensitive than microscopy alone in detecting cryptosporidium. prevention of transmission between immunocompromised patients is essential. there must be strict adherence to infection-control policies to prevent hospital wards from becoming sources of infection. one study looking at the extent of gastroenteric virus contamination in a pediatric-primary immunodeficiency ward and a general pediatric ward found viruses on 17 and 19% of environmental swabs, respectively. interestingly, these were contaminating objects used by parents rather than stafffor example the parents' room television, the parents' toilet tap and the microwave used by parents on the pediatric-primary immunodeficiency ward (gallimore et al., 2008) . this highlights the importance of ensuring that parents and visitors, as well as staff, comply with hand washing and infection control measures. rotavirus infection, which is usually relatively mild and self-limiting in the immunocompetent, can lead to persistent vomiting and diarrhea and, if untreated, severe malnutrition, in the immunocompromised. it can be identified in stool by using enzyme immunoassay and may also be identified on electron microscopy. there is no specific treatment. fluid and electrolyte management is important. orally administered immunoglobulin has been used in some cases. caliciviruses, namely noroviruses and sapoviruses can also cause significant problems in the immunocompromised. symptomatic infection and virus shedding can be prolonged; for example, one case report of a child undergoing hsct for cartilage hair hypoplasia demonstrated norovirus shedding for 156 days following transplant, during the period of immune reconstitution. the child was symptomatic throughout this time (gallimore et al., 2004) . again, there is no specific treatment but meticulous management of fluids, electrolytes and nutritional support is essential, allowing time for immune reconstitution and consequent viral clearance. adenovirus will be discussed in more detail in the section on disseminated infection in the immunocompromised. cryptosporidium species are oocyst-forming protozoa that cause watery diarrhea which can result in severe dehydration and even death, if not treated. disease is normally confined to the gastrointestinal tract, but there is a risk of biliary tree, pulmonary or even disseminated disease in the immunocompromised. infection may be diagnosed on identification of oocysts by microscopy. enzyme immunoassays have also been used and pcr, too, can be helpful. treatment of cryptosporidium infection can be difficult and a number of agents have been proposed, including nitazoxanide, paromomycin, rifabutin and the macrolides. evidence is limited but a recent review has indicated that nitazoxanide may reduce parasite load and therefore be useful (abubakar et al., 2007) . in the authors' experience, azithromycin and nitazoxanide are safer options in post-hsct patients, as paromomycin has been associated with significant hearing loss, particularly when given with ciclosporin. supportive care remains essential. in those with hiv, anti-retroviral therapy, with its associated improvement in cd4 count, can result in improvement in the cryptosporidium infection. giardia intestinalis is a flagellate protozoan that exists in trophozoite or cyst forms. the cysts are the infective form. children with humoral immunodeficiencies are particularly at risk of chronic symptomatic infection, with foul-smelling stool, abdominal distension and anorexia. cysts may be identified on stool microscopy or by using immunofluorescent antibody testing. treatment is with metronidazole, tinidazole or nitazoxanide. it may be necessary to use combination therapy in the immunocompromised if they have failed to respond to single-agent treatment. disseminated viral infection in the immunocompromised is of particular concern. the most significant culprits are adenovirus and members of the human herpes virus: cmv, ebv, hhv6, hsv and vzv. these can affect the lungs, gastrointestinal tract and brain, resulting in a variety of symptoms. reactivation of latent herpes viral infection is more common than primary infection after sot or hsct. investigation using pcr techniques allows early diagnosis and quantification of viral load, and is now possible for adenovirus, cmv, ebv and hhv6. prophylaxis to prevent cmv and hsv reactivation is used for children undergoing hsct and many sots. surveillance in high-risk patients enables pre-emptive treatment to be given before damaging disease occurs. treatment will depend on the causative virus. adenovirus is usually responsible for relatively minor upper respiratory tract or gastrointestinal infection but can result in life-threatening pneumonia, meningitis, encephalitis and disseminated disease in the immunocompromised. those most at risk are patients who receive allogeneic bone marrow transplant, those with active graft versus host disease and those who receive total body irradiation. there are a number of different species of adenovirus, and these are divided into serotypes, some of which are primarily associated with the respiratory tract, while others have a predilection for the gastrointestinal tract. young children are particularly vulnerable, as they often carry adenovirus in their gastrointestinal tract, predisposing them to reactivation and dissemination when they become immunocompromised. in view of this, screening can be important in the immunocompromised and adenovirus is usually identified in urine, stool, or sometimes respiratory secretions prior to being identified in blood. a study of 132 patients undergoing hsct were screened for adenovirus in stool, urine, on throat swab and in peripheral blood during the post transplant period. 27% had a positive adenoviral pcr on at least one screening test, but this was not associated with clinical signs unless it was detected in peripheral blood and, even then, there was a median delay of 3 weeks from first detection of adenovirus until the patient demonstrated clinical signs. in one study, mortality was as high as 82% in those with adenovirus detected on peripheral blood. this highlights the importance of early recognition and consideration of pre-emptive use of antivirals (lion et al., 2003) . successful treatment of adenovirus infection has so far been limited. the most widely used agents are cidofovir or ribavirin, which may be given together with ivig. although cidofovir has potent nephrotoxic effects, these can be greatly reduced by the concurrent use of intravenous hyperhydration and probenecid. cidofovir has been shown to be more effective in adenovirus and is now considered the best first-line treatment. data on the clinical effectiveness of ribavirin in adenoviral infections are more conflicting. in vitro data suggest that ribavirin alone has activity against subgenus c serotypes. in a post-hsct patient with adenoviral infection, immune suppression should be reduced as much as possible, as t-cell immune reconstitution is very important for viral elimination. cmv infection is often asymptomatic in the immunocompetent; however, in the immunocompromised it can lead to pneumonia, colitis and retinitis. cmv persists in a latent form after primary infection and can result in reactivation in someone who later becomes immunosuppressed -for example, when undergoing hsct. cmv can be identified from respiratory secretions, urine and blood. as with adenovirus, pcr screening may be useful in identifying the virus before a child becomes symptomatic, especially in cases where reactivation is likely with immunosuppression. treatment is usually with intravenous ganciclovir, with foscarnet or cidofovir as second-line treatment. oral valganciclovir is very well absorbed and is also now an option for treatment. foscarnet has also been used in cases of children undergoing hsct to avoid the myelosuppressive effects of ganciclovir. ivig should be used alongside antiviral therapy. there has been one case report of ganciclovir-and foscarnet-resistant cmv being successfully treated with artesunate (shapira et al., 2008) . there is also interest in the new antiviral agent maribavir for resistant cmv. ebv is associated with lymphoproliferative disorders in the immunocompromised. replication of ebv in b cells is usually inhibited by natural killer cells, antibodydependent cell cytotoxicity and t-cell cytotoxic responses. therefore, children with cellular immune deficiencies are at risk of uncontrolled lymphoproliferation. those at particular risk are children who are transplant recipients, both sot or hsct, and those with hiv. ebv can be detected in blood by pcr and viral load can be monitored. alongside monitoring of the virus, it is important to monitor for signs of lymphoproliferation, both clinically and biochemically. biopsy of suspicious lesions is often needed to make a diagnosis. ebv infection requires treatment if it causes b lymphoproliferation or posttransplant lymphoproliferative disease (ptld). this may take the form of the anti-cd20 monoclonal antibody rituximab, chemotherapy or radiotherapy. decreasing immunosuppression whenever possible in a post-transplant patient is very important. more recently there have been encouraging results from work with cytotoxic t-cell therapy in ptld. this involves the infusion of ebv-specific cytotoxic t lymphocytes (ctls) generated from ebv sero-positive blood donors. in one recent multi-center study, 33 patients who had failed conventional therapy were recruited and monitored for response: 14 patients achieved complete remission while three showed a partial response (haque et al., 2007) . primary hhv6 infection in the immunocompetent host leads to the typical clinical picture of roseola or a non-specific febrile illness. the virus remains latent after primary infection and therefore, similar to cmv, can reactivate in immunocompromised states. the importance of hhv6 as a pathogen in the immunocompromised is probably underestimated, and many labs do not screen for infection; thus, many infections may not be recognized. hhv6 can cause fever, rash, hepatitis, pneumonia and encephalitis, as well as bone marrow suppression. hhv6 also appears to have synergistic effects and interactions with other infectious agents, such as cmv, adenovirus and fungi. it can be identified and quantified on blood samples by pcr. treatment, where necessary, is with intravenous ganciclovir or foscarnet. primary varicella infection results in chickenpox, a common and generally selflimiting childhood illness. in the immunocompromised, there is a significant risk of both primary or reactivated disease becoming disseminated. this is particularly associated with t lymphocyte defects. vzv is the second most common cause of viral pneumonitis in children with aids. it should be remembered that fatal vzv infection has been reported in cases where the only immunosuppressant medication has been corticosteroids at a dose of 1 mg/kg/day of prednisolone for 2 weeks. the virus can be identified from vesicular fluid. treatment is usually in the form of intravenous aciclovir, but, oral valaciclovir is a useful alternative in older children. an important area to consider in relation to vzv infection is that of postexposure prophylaxis. although long-term prophylaxis for vzv is not usually recommended, post-exposure prophylaxis in non-immune immunocompromised children is important. two options are available. the most widely used is varicella zoster immunoglobulin (vzig). however, due to a shortage of vzig a few years ago, oral aciclovir was reconsidered and has been shown to be effective. it must be remembered, however, that aciclovir has low bioavailability when given orally and requires multiple daily dosing. it may be more appropriate to consider the oral pro-drug valaciclovir, which has been shown to be effective and well tolerated (nadal et al., 2002) . further work to clarify the best prophylactic and pre-emptive treatment regimens is needed. fungal infections must be considered in specific circumstances; for example, in those who are neutropenic (where risk increases exponentially with duration of neutropenia), those on steroids and those with graft versus host disease. candida and aspergillus are of particular interest in children who have undergone hsct. symptoms that should raise the suspicion of fungal infection are persistent fevers unresponsive to antibiotics, skin nodules, chest pain and radiological evidence of infection crossing tissue planes. candida is most commonly associated with cvc infection but can also cause disseminated disease. aspergillus infection can have an insidious onset, frequently affecting the respiratory tract but then spreading to involve other areas such as the spine and intracranial cavity. investigation and diagnosis remain difficult and may require antigen testing, pcr, cross-sectional imaging and biopsy of suspicious lesions/areas. persistent mucocutaneous candidiasis is seen in patients with defects in t-cell function and may be a presenting feature for hiv infection or primary immune deficiency. disseminated infection can involve almost any organ or any anatomical site and can be rapidly fatal. it is a particular concern in patients with cvc, especially those receiving multiple infusions and/or parenteral nutrition. there are a number of different candida species that can result in disseminated infection. candia albicans is the most common but c. parapsilosis, c. glabrata, c. tropicalis and c. krusei are increasingly common (fig. 2) . diagnosis may be difficult, as blood cultures are not always positive. however, identification can be made by microscopy of biopsy specimens. suspicious lesions, which are often found in organs such as the liver, kidney, spleen and brain, are best identified by cross-section imaging. pcr techniques have been developed, as well as detection of antigen from the fungal cell wall (mannan). however, these techniques are not as yet wholly reliable. there are a number of agents available for treatment, including amphotericin b, caspofugin or an azole, such as voriconazole. prolonged treatment is usually required and if there is a cvc invasive aspergillus infection in the immunocompromised usually involves lungs, sinuses, brain or skin and commonly crosses tissue planes. less commonly, it can cause endocarditis, osteomyelitis, meningitis and infection around the eye or orbit. it can cause angio-invasion, resulting in thrombosis and, occasionally, erosion of the blood vessel wall, often with catastrophic hemorrhage as a consequence. there are a number of aspergillus species that cause invasive disease. most commonly it is due to aspergillus fumigatus, but a. flavus, a. terreus, a. nidulans and a. niger are also responsible for invasive infection. diagnosis can be challenging. crosssectional imaging is very important in identifying suspicious lesions. aspergillus is infrequently identified from blood and is most commonly indicated from biopsy specimens. galactomannan, a complex sugar molecule found in the cell wall of the aspergillus species, may also be identified from blood and can be useful in aiding diagnosis. treatment is usually with amphotericin b, voriconazole or caspofungin and requires a prolonged course. surgical excision of fungal lesions may be required, especially if there are significant areas of necrotic tissue into which antifungal agents will not penetrate effectively. there is also an important association between aspergillus infection and building work on a hospital site. one study in an italian hematology unit found three cases of proven aspergillosis in patients with acute leukemia that coincided with renovation work on the hospital site and high levels of a. fumigatus in the corridors (pini et al., 2008) . this highlights the importance for high-risk patients (e.g. after hsct) of sterile isolation in cubicles maintained at positive pressure with highly purified air. extra attention must be paid to reducing exposure of immunocompromised patients when there is building work on any hospital site. many immunocompromised children will have indwelling cvc for treatment, be this an external broviac or hickman line, or an internal portacath. although very beneficial they, unfortunately, provide a site for infection. catheter-related blood stream infections can be serious and in some cases life-threatening. clinical features of catheter-related blood stream infection can be very non-specific. diagnosis is often made on identification of organisms from blood culture along with lack of focal infective symptoms/signs. organisms causing cvc infection are often those that would be non-virulent normal flora in an immunocompetent host; for example, coagulase negative staphylococci, enterococci and viridans streptococci. however, mycobacterial cvc infections also occur (hawkins et al., 2008) , as do candida cvc infections. prevention has to be the priority. lines should be inserted under strict aseptic technique and, once in place, access should be by fully trained staff using aseptic technique. local policies should be followed for accessing and flushing cvcs. historically, cvcs were often removed when infection was identified; however, many patients were left in the difficult situation of poor venous access and in need of a further general anaesthetic to replace the line. many catheter-related blood stream infections can be treated with antibiotics, without requiring cvc removal. if there is clinical suspicion of catheter-related blood stream infection, antibiotics for both coagulase negative staphylococcus and gram negative organisms should be introduced. once organisms are identified from blood culture, antibiotics can be tailored appropriately. antibiotic "locks" can be used alongside systemic antibiotics to reduce colonization within the cvc. antibiotic "locking" involves instilling 1-2 ml of concentrated antibiotic solution in to the cvc and leaving it for a pre-determined time before removal. antibiotics used in studies to treat cvc colonization have included vancomycin, amikacin and minocycline. there is also limited evidence on the use of amphotericin locks. studies have attempted to look at whether using locks alone or in combination with systemic antibiotics has benefits. the results are variable and, at this stage it must be concluded that locks are a useful adjunct to systemic treatment. there is not enough evidence to suggest they can be used alone in immunocompromised children with cvcs (berrington and gould 2001) . in an attempt to present cvc infection, antibiotic-impregnated cvcs have also been developed. a recent systematic review found significant reductions in catheterrelated blood stream infections in heparin-coated or antibiotic-impregnated cvcs, when compared to standard cvcs, as well as those coated with chlorhexidine, silver sulphadiazine, or silver-impregnated. there were, however, some concerns about the development of antibiotic resistance and further study is required before recommendations can be made about the most appropriate cvc to be used (gilbert and harden, 2008) . it must be remembered that catheter-related blood stream infection can be life threatening and there should be a low threshold for removal of the cvc if there are signs of clinical deterioration on treatment or if blood cultures drawn from cvcs are repeatedly positive, despite ongoing appropriate antibiotic treatment. there is increased mortality associated with delayed catheter removal in s. aureus and fungal infections, and so removal must be considered urgent if these organs are isolated. the benefits of removing the cvc if gram-negative organisms are identified is slightly more difficult to assess due to scarcity of data; however, it is likely that immediate removal does contribute to increased survival. in all infections the risk/benefit ratio of removing or retaining cvcs should be carefully considered. in children receiving treatment for malignancy, febrile neutropenia is a significant cause of morbidity and mortality. over time, outcome has improved dramatically but it still remains a frequent reason for hospitalization. it has been shown that (schmipff et al., 1971) ; hence, empiric antibiotics have become a standard part of treatment for children and adults with febrile neutropenia. fever with neutropenia in any immunocompromised child should be acted on promptly. however, exactly how this is defined and what is appropriate management varies widely. this was highlighted by a recent review of febrile neutropenia management in the united kingdom children's cancer study group centers (phillips et al., 2007) . there was wide variation in the definition of fever (from persistent temperature higher than 37.5 â�¢ c to a single reading of 39 â�¢ c) and neutropenia (absolute neutrophil count <1 ã� 10 9 , < 0.75 ã� 10 9 or < 0.5 ã� 10 9 ). empirical antibiotic regimes also varied greatly, including aminoglycosides plus a second agent (piperacillin based, cephalosporin or carbapenem), carbapenem alone or, in two cases, cefuroxime plus flucloxacillin and ciprofloxacin plus ceftazidime. timing of the anti-fungal therapy was even more variable, in terms of when to start and the duration of empirical treatment. some of this variation can be explained by variations in organisms isolated and antibiotic sensitivity from unit to unit, but this does not seem to account for all the differences in practice. therefore, although local findings should influence presenting patterns, further work is required to devise a framework within which local policies that target specific patient populations and microbiological flora are implemented. a specimen protocol is shown in fig. 3 infections in immunocompromised children offer a variety of challenges in both diagnosis and management. organisms that result in mild, self-limiting illness in an immunocompetent host can have catastrophic effects on an immunocompromised child. signs and symptoms are often less specific and finding a causative organism can be more difficult. it is important to have a low threshold for thinking about infections and looking for them. negative tests should not be taken to be reassuring if there is clinical suspicion and it may be necessary to look further and more closely. it is important to develop a good relationship with local microbiology and virology laboratories to aid this process. once an infection is identified, it must be acted upon quickly as delay may be disastrous. treatment of any infection in an immunocompromised child is likely to be more intense and prolonged than in a child with a fully functioning immune system. it is also important to consider prophylaxis for specific patient groups in specific situations (e.g. post hsct) and each unit should have defined policies and guidelines to follow for these patients. in summary, when dealing with an immunocompromised child, for whatever reason, when there is suspicion about infection, think early, look carefully and treat now! treatment of crypotspodiiosis in immunocompromised individuals: systematic review and meta-analysis infectious complications in children with cancer and children with human immunodeficiency virus infection. clinical approach to infection in the compromised host adenovirus. mandell, douglas and bennet's principles & practice of infectious disease second line salvage treatment of aids-associated pneumocystis jiroveci pneumonia: a case series and systematic review use of antibiotic locks to treat colonized central venous catheters unsuspected pneumocystis carinii pneumonia at presentation of severe primary immunodeficiency antiviral drugs for cytomegalovirus 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foscarnet contamination of the hospital environment with gastroenteric viruses: comparison of two pediatric wards over a winter season oral valganciclovir leads to higher exposure to ganciclovir than intravenous ganciclovir in patients following allogeneoic stem cell transplantation chronic excretion of a norovirus in a child with cartilage hair hypoplasia(chh) respiratory viruses other than influenza virus: impact and therapeutic advances identification of a novel polyomavirus from patients with acute respiratory tract infections prophylaxis for pneumocystis pneumonia (pcp) in non-hiv immunocompromised patients. cochrane database syst rev effectiveness of impregnated central venous catheters for catheter related blood stream infection: a systematic review allogeneic cytotoxic t cell therapy for ebv positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial catheter related bloodstream infections caused by rapidly growing nontuberculous mycobacteria: a case series including rare species diagnosis, prevention and management of catheter related bloodstream infection during long term parenteral nutrition investigation of blood cultures (for organisms other than mycobacterium species) viral respiratory tract infections in transplant patients antiviral therapy for adenovirus infections molecular monitoring of adenovirus in peripheral blood after allogeneic bone marrow transplantation permits early diagnosis of disseminated disease respiratory virus infections after stem cell transplantation: a prospective study from the infectious diseases working party of the european group for blood and marrow transplantation effect of corticosteroids on survival of children with acquired immune deficiency syndrome and pneumocystis carinii-related respiratory failure in investigation of the steady-state pharmacokinetics of oral valaciclovir in immunocompromised children epidemiology of invasive candidiasis: a persistent public health problem variation in policies for the management of febrile neutropaenia in united kingdom children's cancer study group centres invasive pulmonary aspergillosis in neutropenic patients and the influence of hospital renovation human bocavirus: passenger of pathogen in acute respiratory tract infections? empiric therapy with carbenicillin and gentamicin for febrile patients with cancer and granulocytopenia management of pneumocystis jiroveci pneumonia in children receiving chemotherapy artesunate as a potent antiviral agent in a patient with late drug-resistant cytomegalovirus infection after hematopoietic stem cell transplantation value of bronchoalveolar lavage before haematopoietic stem cell transplantation for primary immunodeficiency or autoimmune diseases corticosteroids improve survival of children with aids and pneumocystis carinii pneumonia respiratory virus infections in the immunocompromised host molecular epidemiology of outbreak of respiratory syncytial virus within bone marrow transplant unit infections in recipients of blood and marrow transplantation. mandell, douglas and bennet's principles & practice of infectious disease adenovirus: an increasingly important pathogen in paediatric bone marrow transplant patients beta-herpesviruses in febrile children with cancer key: cord-291960-1is0rv6c authors: piñana, josé luis; giménez, estela; gómez, maría dolores; pérez, ariadna; gonzález, eva maría; vinuesa, víctor; hernández-boluda, juan carlos; montoro, juan; salavert, miguel; tormo, mar; amat, paula; moles, paula; carretero, carlos; balaguer-roselló, aitana; sanz, jaime; sanz, guillermo; solano, carlos; navarro, david title: pulmonary cytomegalovirus (cmv) dna shedding in allogeneic hematopoietic stem cell transplant recipients: implications for the diagnosis of cmv pneumonia date: 2019-02-21 journal: j infect doi: 10.1016/j.jinf.2019.02.009 sha: doc_id: 291960 cord_uid: 1is0rv6c objectives: to date no definitive cut-off value for cytomegalovirus (cmv) dna load in bronchoalveolar lavage (bal) fluid specimens has been established to discriminate between cmv pneumonia and pulmonary cmv dna shedding in allogeneic hematopoietic stem cell transplant (allo-hsct) recipients. methods: the current retrospective study is aimed at assessing the range of cmv dna loads quantified in bal fluid specimens from allo-hsct patients with pneumonia in which different microorganisms were causally involved. results: a total of 144 bal specimens from 123 patients were included. cmv dna was detected in 56 out of 144 bal fluid specimens and the median cmv dna load from patients in whom cmv pneumonia was unlikely or could be tentatively ruled out was 1210 (31–68, 920) iu/ml. the frequency of cmv dna detection and median cmv dna loads were comparable, irrespective of the attributable cause of pneumonia. detection of cmv dna loads in bal fluid specimens >500 iu/ml was independently associated with pneumonia-attributable mortality. conclusions: the current study highlights the difficulty in establishing universal cmv dna load thresholds in bal fluid specimens for distinguishing between cmv pneumonia and pulmonary cmv dna shedding, and suggests that the presence of cmv dna in bal fluid specimens beyond a certain level may have a deleterious impact on patient outcome. the implementation of effective prevention strategies has significantly reduced the incidence of cytomegalovirus (cmv) pneumonia after allogeneic hematopoietic stem transplantation (allo-hsct) 1 ; nevertheless, this clinical entity persists as a major clinical problem owing to poor survival, despite timely initiation of targeted antiviral therapy. 1, 2 currently, diagnosis of cmv pneumonia still remains a challenge. proven cmv pneumonia can only be diagnosed using virological, histopathological or immunochemistry methods on biopsied lung tissue 1 ; this specimen type, however, is rarely obtained due to potential life-threatening complications. traditionally, culture-based bronchoalveolar lavage (bal) testing has been the mainstay for suggesting cmv involvement ex vivo , 3 although detection of viable cmv in bal fluids only points to a probable causality. 4 may be present in the absence of probable or proven disease (pulmonary cmv shedding). [5] [6] [7] [8] recent studies suggested that quantitation of cmv dna in bal fluids may permit discrimination between these two conditions 9 -11 ; specifically, a cut-off cmv dna level > 500 iu/ml was proposed to serve that purpose, this displaying a positive predictive value of roughly 50% for probable cmv pneumonia using current prevalence rates. 10 nevertheless, validation of diagnostic viral load threshold across centers using different real-time pcr assays for cmv dna quantitation and procedures for bal obtention seems of paramount relevance. this task is hampered by the very low incidence of cmv pneumonia nowadays ( < 2%) 3, 12 and the difficulty in establishing an incontrovertible diagnosis, even when lung tissue specimens are available for testing. 13 -16 exploratory studies gathering information on the range of cmv dna loads measured in bal specimens from allo-hsct patients with pneumonia in whom cmv causality is unlikely or can be reasonably ruled out may provide useful information and are thus warranted. here, we report on our experience on this matter. the study cohort consisted of 123 consecutive patients who received an allo-hsct at hospital clínico universitario-hcu-( n = 61) or at hospital universitario politécnico "la fe" -hlf-( n = 62) and underwent diagnostic bronchoscopy. clinical and microbiological data of patients and bal fluid specimens ( n = 144; hcu, n = 80; hlf, n = 64) submitted to the respective microbiology service between may 2012 and may 2017, respectively, were retrospectively reviewed. as per protocol, quantitative cmv pcr testing has been routinely performed on bal specimens at both centers since 2012. all patients had clinical and radiography signs of pneumonia at the time of sampling. bronchoscopy was performed using standard procedures according to international consensus guidelines. 17 the first bal fluid specimen per pneumonia event was taken into consideration for analysis purposes. this study was approved by the hospital clínico, fundación incliva ethics committee and informed consent was obtained from all patients. a preemptive antiviral therapy approach was used at hcu to prevent cmv end-organ disease. 18 cmv dna in plasma was quantified using the realti me cmv pcr assay (abbott molecular, des plaines, il, usa), which exhibits a limit of detection of approximately 31 iu/ml. 19 patients were preemptively treated with oral valganciclovir, i.v. ganciclovir or i.v. foscarnet upon detection of cmv dna levels exceeding 1500 iu/ml or a cmv dna doubling time ≤ 2 days, as previously reported. 18, 20 surveillance for cmv dna detection and quantitation was conducted at least once a week within the first 100 days after allo-hsct and this was extended beyond day 100 in patients at risk for recurrent episodes of cmv dnaemia. 21 in turn, a universal prophylaxis strategy was deployed at hlf. 22 briefly, hla-matched related allo-hsct recipients were given oral valganciclovir (900 mg/day, three times a week) through day 90 after transplantation. unrelated allo-hsct recipients were treated with oral valganciclovir (900 mg/day) through day 180 after transplantation. at this center, plasma cmv dna load was quantified using the cmv r-gene® (biomerieux, l'etoile, paris, france), 23 which displays a limit of detection of 150 iu/ml, prior dna extraction using the virus/pathogens mini kit (qiagen, valencia, ca, usa) on the qiasymphony dsp platform (qiagen). plasma cmv dna monitoring was performed once a week within the first 100 days, fortnightly from day 100 to day 180, and every 2-4 weeks thereafter through day 365 after transplantation. detection of any level of cmv dna in plasma prompted the administration of antiviral therapy with (val)ganciclovir or foscarnet at the doses specified above. anti-cmv therapy was given at the physician discretion when patients with cmv dna detected in bal specimens had concurrent cmv dnaemia with cmv dna levels below the threshold for preemptive antiviral therapy. plasma specimens obtained within 72 h of bal sampling were available from all patients for cmv dna quantitation. all bal fluid specimens underwent cmv pcr testing within 24-48 h. upon reception. samples were kept at 4 °c until processed. bal fluid specimens obtained from different locations (when available) were collected in sterile containers, pooled and vortexed for 30 s after the addition of sterile glass beads. two-hundred μl of each undiluted bal fluid samples were subjected to nucleic acid extraction using the m20 0 0 sp system (abbott diagnostics) or the qiaamp dna blood mini kit (qiagen) on either the qia symphony or the ez-1 platforms (qiagen). cmv dna quantitation was performed using the realti me cmv pcr assay (abbott molecular) at hcu or the cmv r-gene® assay (biomerieux) at hlf. commutability of the cmv who international standard 24 for cmv dna quantitation in bal specimens was assessed. the cmv who standard was reconstituted in 1 ml of deionized water as recommended by the manufacturer. 24 a pool of bal specimens free of cmv dna, as determined by the abbott pcr assay was made and spiked with the who international standard with a predefined cmv dna concentration to achieve nominal values of 10 2 10 3 , 10 4 and 10 5 iu/ml. the reference materials were assayed in triplicate in a single run. cmv dna levels measured in bal fluid specimens closely matched those expected (the mean standard deviation for both pcr assays was < 0.2 log 10 iu/ml for each cmv who standard concentration tested). gram stain, fungal stain and acid-fast bacilli stain were routinely performed. cytology examination (bal fluid cytospins) was performed systematically in patients attended at hcu, and nonroutinely at hlf. in all, bal fluid cytospin data were available from 83 patients. in addition, bal fluid specimens were examined for the presence of respiratory viruses (rvs) using either the luminex xtag rvp fast assay (luminex molecular diagnostics, austin, tx,usa) at hcu, which allows detection of 19 rvs, or the clart® pneumovir assay (genomica, coslada, spain)-at both centers-that makes it possible to detect simultaneously 17 rvs, as previously reported. 25 quantitative cultures of bal specimens for bacterial isolation were performed on conventional media as recommended 26 ; bacterial loads > 10 4 cfu/ml were deemed to be clinically relevant. 26 bal specimens were cultured on bcye-alpha agar, bd (becston dickinson) mgit® (mycobacteria growth indicator tube)/lowenstein-jensen agar slants and sabouraud agar for recovery of legionella pneumophila, mycobacterium spp., and fungal organisms, respectively. the platelia tm aspergillus ag kit (bio-rad, hercules, ca, usa) was used for quantitation of aspergillus spp. galactomannan in bal fluid and serum specimens. calcofluor white, blue toluidine or direct immunofluorescence staining procedures were used for detection of pneumocystis jiroveci. 26 lung biopsy tissue specimens were not obtained. likewise, neither shell vial and conventional viral cultures for cmv detection or recovery nor direct fluorescent antibody testing for cmv detection were performed. [39] . acyclovir/valacyclovir prophylaxis against herpes simplex and varicella zoster viruses was given to all patients as previously described. 18,20 -22 all patients received standard antibacterial and antifungal prophylaxis. 18,20 -22 definitions cmv dnaemia and pulmonary cmv dna shedding were defined as the detection of cmv dna (at any level) in one or more plasma or bal fluid specimens, respectively. proven cmv pneumonia categorization required histopathological evidence (i.e., viral inclusions and immunohistochemical staining) in biopsy (autopsy) lung tissue. cmv pneumonia diagnosis was reasonably excluded if cmv-induced cytopathogenetic effect was not observed in post-mortem (autopsy) lung tissue (data were available from 23 patients) or bal fluid cytospins (data available from 83 patients), there was a lack of chest x ray and computed tomography (ct) evidence consistent with cmv pneumonia (ie. reticulonodular infiltrates, the presence of bilateral ground-glass opacities, air-space opacities, small centrilobular nodules < 1 cm, and absence of larger nodules -see 27 , for review -) and alternative diagnoses that could account for the signs and symptoms, particularly when the presence of other significant bacterial, virus or fungal pathogens was demonstrated. the clinical and radiographic response to targeted antimicrobial therapy (for bacterial, fungal and non-cmv viral infections) was also considered to be against the involvement of cmv. the probable cmv pneumonia category, according to recent criteria 4 was not considered herein, since culture-based bal fluid testing was not performed and no diagnostic cmv dna cut-off level has been definitely established. diagnosis of proven, probable, and possible invasive fungal infection and pneumocystis jiroveci pneumonia was achieved following consensus criteria. 28, 29 acute graft versus host disease (agvhd) was diagnosed and graded according to standard criteria. 30 frequencies were compared using the χ 2 test (fisher exact test) for categorical variables. differences between medians were compared using the mann-whitney u test (for two independent variables) or the kruskal-wallis test (for more than two independent variables). cumulative incidence plots of mortality from pneumonia were generated with the graphpad prism software (la jolla, ca, usa) and the curves were compared by using the gehan-wilcoxon test. cox proportional hazards models were used to evaluate unadjusted and adjusted hazard ratios (ahrs) for mortality attributable to pneumonia, as previously reported. 21 for multivariate analyses, only variables with parameter estimates showing a p value ≤ 0.10 in the univariate analyses were included. two-sided exact p values are reported and p values ≤ 0.05 were considered statistically significant. the data were analyzed with the spss (version 20.0) statistical package. table 1 shows relevant demographic and clinical characteristics of the 123 patients in the cohort. bal fluid samples were obtained at a median of 172.5 days after allo-hsct (range 3 days to five years). specific details on the microbiological yield of bal fluid specimens are shown in table 2 . proven cmv pneumonia was diagnosed in 2 patients (1.6%) on the basis of histopathological and immunohistochemistry findings in lung autopsy tissue. in the remaining 142 episodes, pneumonia was attributable to bacteria in 18 cases (12.5%), and to one or more viruses in 37 (25.7%). there were 22 invasive aspergillosis infection cases (13.3%), these being categorized as possible ( n = 11), probable ( n = 7), or proven ( n = 4). mixed infections were documented in 42 patients (29.2%). the individual pathogenetic contribution of each detected or cultured microbial agent to pneumonia was not attempted to be settled. twenty-three pneumonia cases (15.9%) were deemed not to have an infectious cause on the basis of clinical and radiographic data and the lack of detection of any potential pathogenic microorganism (other than cmv) in bal fluid specimens. cmv dna was detected in 56 out of 144 bal fluid specimens (38.9%): 49 from patients with pneumonia episodes in which potential respiratory pathogens (one or more) were either cultured or detected, and 7 from cases deemed not to have an infectious origin. in these latter cases, the involvement of cmv as the causative agent was reasonably ruled out on clinical and radiographic grounds. cmv dna was present in bal fluid specimens from the two patients with proven cmv pneumonia; no other microorganisms were concurrently detected/cultured in these two specimens. the frequency of cmv dna detection was comparable ( p = 0.40), regardless of the established (or presumed) etiology of pneumonia ( table 3 ). in 41 episodes, cmv dna bal detection occurred in the face of an ongoing episode of cmv dnaemia (including two episodes that occurred in the setting of autopsy-proven cmv pneumonitis), and isolately in the remaining 15 episodes. clinical and laboratory characteristics of patients ( n = 33) in whom cmv dna was co-detected in bal and plasma specimens in the absence (presumed) of cmv pneumonitis ( n = 39 episodes) merit special attention, as cmv lung disease usually occurs concomitantly with cmv dnaemia. 3, 4 the data are shown in supplementary table 1 . cmv pneumonitis was deemed to be unlikely in these patients owing to one or more of the following: (i) lack of typical findings in cts (in all episodes); (ii) negative bal cytospin results (in 25 episodes); (iii) negative lung histopathology at autopsy (in table 2 pneumonia attributable etiology and microbiological findings in bronchoalveolar lavage fluid specimens. no. (%) c according to [28] . d according to [4] . e including idiopathic pneumonia syndrome, bronchiolitis obliterans and cryptogenic organizing pneumonia among other causes. 7 out of 16 patients who died); (iv) survival of patients who did not undergo specific anti-cmv treatment courses with appropriate doses for cmv pneumonitis (23 episodes); of note, no patient in this series was treated with anti-cmv drugs for cmv pneumonitis as recommended 3 ; (v) documentation of the presence of bacteria, viruses (other than cmv) or fungal pathogens known to cause pneumonia (in 25 episodes); (vi) clinical response (survival) to targeted anti-bacterial, anti-viral (influenza virus) or anti-fungal therapy (in 17 episodes). nevertheless, in particular, there were four episodes in which the causative involvement of cmv raised doubts (second episode in patient 31, and episodes in patients 35, 37 and 51-supplementary table 1. despite the fact that ct scans were not suggestive of cmv pneumonitis, no alternative microbial etiology was documented and all these patients died (pneumonia was the attributable cause of death). in addition, cmv dna was detected at high levels in both bal (ranging from 1382 to 40,048 iu/ml) and plasma (ranging from 3510 to 54,540 iu/ml) specimens. one of these patients (patient 51) had negative bal cytospin results. in turn, cmv dnaemia was documented in 33 cases in the absence of cmv dna detection in bal fluid samples. recipient cmv seropositivity and treatment with corticosteroids were associated with detection of cmv dna in bal fluid specimens in univariate analyses (supplementary table 2 ). overall, the median cmv dna load in bal fluid specimens from patients categorized as not having cmv pneumonia was 1210 iu/ml (range, 31-68,920 iu/ml). this magnitude was greater ( p = 0.001) in specimens analyzed at hlf (median, 1938 iu/ml; range, 180-68,920 iu/ml) than in those being tested at hcu (median, 345 iu/ml; range, 21-11,263 iu/ml) (supplementary fig. 1) . the cmv dna load was > 500 iu/ml in 32 pneumonia episodes and < 500 iu/ml in the remaining 22 ( table 4 ). the cmv dna load in bal fluid in the two proven cmv pneumonia cases was 1453 and 12,998 iu/ml. a trend towards higher cmv dna loads in bal fluid specimens was observed ( p = 0.09) in episodes in which cmv dnaemia was detected concurrently (1382 iu/ml; range, 31-68,920 iu/ml vs. 289 iu/ml; range, 66-12,839 iu/ml in its absence). cmv dna loads in bal fluid specimens were comparable ( p = 0.62), irrespective of the etiology (attributable) of pneumonia ( fig. 1 , and supplementary table 3) . overall, there was a trend towards an inverse correlation between the level of immunosuppression, as inferred by the immunodeficiency index (isi) -see footnotes in table 1 -, and the cmv dna load quantified in bal specimens (rho, −0.15; p = 0.08). overall, 72 patients (50%) were under anti-cmv therapy at the time of bal sampling (prophylaxis, n = 14; preemptive therapy, n = 58). among those with cmv dna detectable in bal fluids, 37 (66%) were receiving anti-cmv therapy (preemptive therapy, n = 35, and antiviral prophylaxis, n = 2). the cmv dna load in bal was significantly higher in patients who were under antiviral therapy (median, 1585 iu/ml; range, 31-68,920 iu/ml vs. median, 345 iu/ml; range, 39-7159 iu/ml; p = 0.04 in non-treated patients). two out of the 19 patients with cmv dna detected in bal fluids and no concurrent cmv dnaemia were treated with i.v. ganciclovir. forty-six patients (37%) died within 60 days after bal sampling, including one patient with proven cmv pneumonia. the cause of death was deemed to be related to the pneumonia episode in 40 patients (87%). the cumulative incidence of pneumoniaattributable mortality was comparable across the different etiological categories ( fig. 2 ) . we investigated whether the presence of cmv dna in bal fluid specimens was associated with increased mortality, and found this not to be the case. nevertheless, roc curve analysis enabled to determine a cut-off cmv dna level in bal fluid identifying those patients with increased mortality; this threshold was 500 iu/ml (sensitivity: 84.2%; 95% ci 62.4-94.5%; specificity, 53.1%; 95% ci, 36.4 −69.1%) (supplementary fig. 2 ). adjusted cox models including in addition to this variable a number of others that could have had an impact on pneumoniaattributable mortality identified the detection of a cmv dna load in bal fluid specimens at levels > 500 iu/ml, treatment with corticosteroids (at any dose) and lymphocyte counts < 0.7 × 10 9 /l, the [30] . b the cut-off was established by roc analysis (not shown). cumulative incidence of mortality attributable to pneumonia complications by day 60 after bronchoalveolar lavage fluid testing according to the etiology. cumulative incidence plots were generated with the graphpad prism software (la jolla, ca, usa) and the curves were compared by using the gehan-wilcoxon test (the p value is shown). latter two at the time of bal sampling, as the parameters that were independently associated with pneumonia-attributable mortality ( table 5 ) . moreover, as shown in fig. 3 , these factors appeared to exert a synergistic impact on mortality. the definitive abandonment of traditional culture-based procedures for cmv testing in most laboratories and the non-availability of lung biopsy specimens for histopathological and immunohistochemistry examination make the diagnosis of proven or probable cmv pneumonia 4 elusive nowadays. moreover, no cut-off value for cmv dna load in bal fluid specimens discriminating between cmv pneumonia and pulmonary cmv dna shedding in allo-hsct recipients has been established at the present time. 4 the assessment of the performance of quantitative cmv dna pcr testing for the diagnosis of cmv pneumonia faces several difficulties including: (i) the low incidence of this clinical event, (ii) the lack of a normalized procedure for cmv dna pcr testing on bal fluids, (iii) the non-negligible possibility of miscategorization of pneumonia cases as either being causally linked or unrelated to cmv when using bal fluid specimens, or even lung tissue material, for cmv diagnosis, 8 (iv) the persistence of a large variability of cmv dna loads provided by different real-time pcr assays, 31 -33 despite their calibration to the who international standard for cmv dna 24 and, as already mentioned, (v) the relegation of virological procedures for cmv detection in clinical specimens. inevitably, all these drawbacks were encountered in this study, so that we aimed not to establish a diagnostic cut-off, but rather to assess the range of cmv dna loads quantifiable in bal fluid specimens from patients in whom the causal involvement of cmv was highly unlikely or could be reasonably discarded; this, having in mind that we surely incurred cmv pneumonia infradiagnosis. nevertheless, we perceived this limitation as not being an insoslayable obstacle to our purpose fig. 3 . cumulative incidence of mortality attributable to pneumonia complications by day 60 after bronchoalveolar lavage fluid testing according to the presence of one or more factors associated with mortality in multivariate cox models (cmv dna load in bal fluid 500 iu/ml, treatment with corticosteroids and total lymphocyte counts < 0.7 × 10 9 /l). cumulative incidence plots were generated with the graph-pad prism software (la jolla, ca, usa) and the curves were compared by using the gehan-wilcoxon test (the p values for comparisons are shown. as: (i) cmv is unlikely to be the etiological agent of more than 10% of pneumonia cases among allo-hsct recipients who undergo routine bronchoscopy, 10 and (ii) survival of patients with cmv pneumonia who do not undergo specific anti-cmv treatment with appropriate doses of (val)ganciclovir or foscarnet is highly unlikely, provided the high rate of cmv pneumonia-associated mortality. 4 we retrospectively reviewed clinical and microbiological data from patients who underwent routine quantitative cmv pcr testing on bal fluid specimens at two transplant centers in our city. we deliberately chose not to include archived bal specimens in our series because of the lack of data on the impact of cryopreservation on cmv dna load quantitation in this sample type. several findings arose from the present study. first, we confirmed previous observations 8 -10 indicating that detection of cmv dna in bal fluid specimens using highly-sensitive pcr assays is a very common finding in allo-hsct patients with pneumonia, irrespective of the definitive etiological diagnosis. in our series, cmv dna was detected in more than one third of bal fluid samples, the frequency of detection varying between 30% in patients seemingly with not having an infectious pneumonia and 45% in patients with mixed infections. in our series, recipient cmv seropositivity and treatment with corticosteroids were associated with detection of cmv in bal fluid specimens. of interest, the two patients with proven cmv pneumonia had cmv dna detectable in bal fluid. second, we found a wide range of cmv dna loads measured in bal fluid specimens from patients with pneumonia in whom cmv causality was unlikely or reasonably ruled out attending to clinical (including therapeutic response to nonanti-cmv drugs), radiographic, lung autopsy histopathology or bal fluid cytology (in some patients) and microbiological criteria. interestingly, median cmv dna loads were comparable irrespective of the nature and the number of co-detected microorganisms (at both participating centers), and were overall higher in the presence of concurrent dnaemia. as for the latter observation, in agreement with boeckh et al., 10 we found this not to be due to pulmonary hemorrhage (not shown). overall, cmv dna loads in bal fluid specimens processed at hlf were of greater magnitude than those analyzed at hcu. since cmv dna loads produced by the argene pcr assay are slightly lower than those measured by the abbott pcr assay (not shown), differences in the net state of immunosuppression of patients at the time of bal sampling across centers, as reflected by the immunodeficiency score index (higher for hlf patients), may account for this observation. in fact, a trend towards an inverse correlation was found between the isi score and the cmv dna load quantified in bal specimens. in a recent study, 10 a cmv dna load cut-off of 500 iu/ml in bal fluid was found to have a positive predictive value of ∼50% for the presence of probable cmv pneumonia (considering a prevalence of this event of 10% among patients at risk and undergoing bal testing). tan et al., 8 in contrast, found cmv dna levels in bal fluid samples to have a limited value to distinguish between cmv and non-cmv pneumonia cases. it is pertinent to mention here that the above threshold is between one and two log 10 lower than those tentatively proposed for diagnosing cmv pneumonia in lung transplant recipients. 34 -38 interestingly, control patients with non-cmv pneumonia in the boeckh study 10 showed a median cmv dna load of 0 log 10 iu/ml (iqr, 0-1.6 iu/ml), with cmv dna levels between 100 and 500 iu/ml in roughly 64% of cases and > 500 iu/ml in 36% of them. here, the opposite was true, with nearly 60% of bal fluid samples from patients with non-proven cmv pneumonia having cmv viral loads > 500 iu/ml, of which 75% had > 1000 iu/ml. it is worth highlighting that these figures were comparable at both participating centers, despite the above-referred differences in cmv dna loads provided by pcr assays used at each center. to gauge the potential relevance of these data, it must be taken into consideration that in nearly 70% of pneumonia episodes in our cohort, bal sampling was performed while patients were under anti-cmv therapy ( > 3 days), whereas in the aforementioned study, 10 only 24% of patients with cmv pneumonia and 35% of patients with non-cmv pneumonia had been treated with antivirals for at least two days. despite this fact, higher cmv dna loads in bal fluid specimens were quantified in the current study, likely reflecting major differences regarding the dnaemia cut-off triggering the inception of antiviral therapy between these studies (much higher in the current cohort). in fact, in this series, the median cmv dna load in bal was significantly higher in patients who were under antiviral therapy than in non-treated patients. as stated above, the limited number of proven cmv pneumonia cases in our series precluded any attempt to establish a diagnostic cmv dna load cutoff; nevertheless, in light of the data presented herein, a threshold value of 500 iu/ml is unlikely to be discriminative between cmv pneumonia and pulmonary cmv dna shedding in our setting. in this sense, we fully support the idea that the magnitude of such a diagnostic cut-off is likely to vary depending upon the patient's characteristics, the bal procedure and processing, the assay used for cmv dna quantitation and the severity of cmv pneumonia at the time of sampling. 4 our data should be interpreted with caution as the exclusion of cmv as the probable causative agent can be judged as dubious in some cases provided that no virological methods were used to investigate the presence of cmv in bal fluid specimens. in particular, there were 4 episodes occurring in 4 patients who died, in whom cmv dna was detected at high levels in both bal and plasma specimens ( > 10 0 0 iu/ml) and no alternative microbial etiology was documented. nevertheless, the conclusions drawn on the basis of the overall dataset stood when cases in which little or no doubt existed on the lack of involvement of cmv (i.e. bacterial pneumonia, tuberculosis.) were analyzed separately (representative examples are shown in supplementary table 4) . cmv is a highly pro-inflammatory and immunosuppressive virus; as such it may act as a synergistic co-pathogen in the absence of documented cmv-induced cytopathogenicity, and may have a relevant impact on patient outcome. 3 in this sense, we found that the presence of cmv dna in bal fluid specimens at levels > 500 iu/ml, in addition to receipt of corticosteroids and low lymphocyte counts at the time of bal sampling, was associated with increased pneumonia-attributable mortality in cox multivariate models. the relative scarce number of pneumonia cases in which bal specimens had cmv dna loads > 500 iu/ml did not allow to investigate whether this apparent effect exhibited a dose-response pattern. again, this finding must be interpreted cautiously, as in order to avoid overfitting, cox models were not adjusted to a number of factors that may have had an impact on mortality (i.e., adequacy of antimicrobial treatment, severity of pneumonia, among others). the limited size of the current cohort also precluded any meaningful statistical analysis evaluating the impact of cmv dna load in bal at each center, separately. further studies are urgently needed to validate this observation, since this subset of patients may benefit from short courses of anti-cmv therapy. 10 limitations of the current study, in addition to the ones highlighted above, are its retrospective nature, the potential biased selection of patients requiring bronchoscopy for the etiological diagnosis of pneumonia, the lack of normalization of cmv dna loads in bal fluids to cellular dna content (although this was reported to be expendable in a previous study 10 ) and the use of different dna extraction platforms and real-time pcr assays for cmv dna quantitation. nevertheless, this study has several strengths, including the inclusion of consecutive specimens, the use of fresh bal fluid specimens and highly-sensitive real-time pcr assays for cmv dna load quantitation, and the performance of a comprehensive and systematic evaluation of specimens for the presence of rvs pathogens using molecular assays. in summary, our study highlights the difficulty in establishing universal cmv dna load thresholds in bal fluid specimens for distinguishing between cmv pneumonia and pulmonary cmv dna shedding. despite this, the potential impact of the presence of cmv in bal fluid specimens on pneumonia-attributable mortality observed herein merits to be further investigated. to this end, only large multicenter prospective studies using consensus protocols for cmv dna pcr bal testing and conventional culture-based virological testing may shed light on this issue. reduced mortality of cytomegalovirus pneumonia after hematopoietic cell transplantation due to antiviral therapy and changes in transplantation practices cytomegalovirus pneumonia in hematopoietic stem cell recipients an update on the management and prevention of cytomegalovirus infection following allogeneic hematopoietic stem cell transplantation definitions of cytomegalovirus infection and disease in transplant patients for use in clinical trials open lung biopsy diagnosis of diffuse pulmonary infiltrates after marrow transplantation a randomized, controlled trial of prophylactic ganciclovir for cytomegalovirus pulmonary infection in recipients of allogeneic bone marrow transplants; the city of hope-stanford-syntex cmv study group preemptive ganciclovir administration based solely on asymptomatic pulmonary cytomegalovirus infection in allogeneic bone marrow transplant recipients: long-term follow-up molecular and culture-based bronchoalveolar lavage fluid testing for the diagnosis of cytomegalovirus pneumonitis cmv viral load in bronchoalveolar lavage for diagnosis of pneumonia in allogeneic hematopoietic stem cell transplantation cytomegalovirus (cmv) dna quantitation in bronchoalveolar lavage fluid from hematopoietic stem cell transplant recipients with cmv pneumonia cytomegalovirus (cmv) dna quantification in bronchoalveolar lavage fluid of immunocompromised patients with cmv pneumonia cytomegalovirus infection and disease after reduced intensity conditioning allogeneic stem cell transplantation: single-centre experience rapid diagnosis of invasive cytomegalovirus infection by examination of tissue specimens in centrifugation culture open lung biopsy diagnosis of diffuse pulmonary infiltrates after marrow transplantation rapid diagnosis of cytomegalovirus pneumonia in marrow transplant recipients by bronchoalveolar lavage using the polymerase chain reaction, virus culture, and the direct immunostaining of alveolar cells pulmonary cytomegalovirus infection in immunocompromised patients report of ers task force: guidelines for measurement of acellular components and standardization of bal re-emptive antiviral therapy for active cmv infection in adult allo-sct patients guided by plasma cmv dnaemia quantitation using a realtime pcr assay: clinical experience at a single center comparison of the new abbott real time cmv assay and the abbott cmv pcr kit for the quantitation of plasma cytomegalovirus dnaemia preemptive antiviral therapy for cmv infection in allogeneic stem cell transplant recipients guided by the viral doubling time in the blood impact of cytomegalovirus dnaemia on overall and non-relapse mortality in allogeneic stem cell transplant recipients incidence, risk factors, and outcome of cytomegalovirus infection and disease in patients receiving prophylaxis with oral valganciclovir or intravenous ganciclovir after umbilical cord blood transplantation comparative evaluation of the qiasymphony rgq system with the easymag/r-gene combination for the quantitation of cytomegalovirus dna load in whole blood the collaborative study group. collaborative study to evaluate the proposed 1st who international standard for human cytomegalovirus (hcmv) for nucleic acid amplification (nat)-based assays comparison of the performance of 2 commercial multiplex pcr platforms for detection of respiratory viruses in upper and lower tract respiratory specimens a guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2013 recommendations by the infectious diseases society of america (idsa) and the american society for microbiology (asm) diagnostic evaluation of pulmonary abnormalities in patients with hematologic malignancies and hematopoietic cell transplantation revised definitions of invasive fungal disease from the european organization for research and treatment of cancer/invasive fungal infections cooperative group and the national institute of allergy and infectious diseases mycoses study group (eortc/msg) consensus group ecil guidelines for the diagnosis of pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients clinical manifestations of graft-versus-hostmdisease in human recipients of marrow from hl-a-matched sibling donors are we there yet? impact of the first international standard for cytomegalovirus dna on the harmonization of results reported on plasma samples spanish society for infectious diseases and clinical microbiology quality control study group would kinetic analyses of plasma cytomegalovirus dna load help to reach consensus criteria for triggering the initiation of preemptive antiviral therapy in transplant recipients progress in quantitative viral load testing: variability and impact of the who quantitative international standards human cytomegalovirus load in plasma and bronchoalveolar lavage fluid: a longitudinal study of lung transplant recipients clinical utility of cytomegalovirus viral load in bronchoalveolar lavage in lung transplant recipients relationship between cytomegalovirus dna load in epithelial lining fluid and plasma of lung transplant recipients and analysis of coinfection with epstein-barr virus and human herpesvirus 6 in the lung compartment preemptive therapy for systemic and pulmonary human cytomegalovirus infection in lung transplant recipients cytomegalovirus viral load in bronchoalveolar lavage to diagnose lung transplant associated cmv pneumonia immunodeficiency scoring index to predict poor outcomes in hematopoietic cell transplant recipients with rsv infections this research was supported by a grant ( 12/1992 ) from fis ( fondo de investigaciones sanitarias, ministerio de sanidad y consumo, spain).estela giménez holds a río hortega research contract from the carlos iii health institute (ref. cm16/0 020 0 ). none. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.02.009 . key: cord-282618-tjvjlyn9 authors: luke, j m; vincent, j m; du, s x; gerdemann, u; leen, a m; whalen, r g; hodgson, c p; williams, j a title: improved antibiotic-free plasmid vector design by incorporation of transient expression enhancers date: 2010-11-25 journal: gene ther doi: 10.1038/gt.2010.149 sha: doc_id: 282618 cord_uid: tjvjlyn9 methods to improve plasmid-mediated transgene expression are needed for gene medicine and gene vaccination applications. to maintain a low risk of insertional mutagenesis-mediated gene activation, expression-augmenting sequences would ideally function to improve transgene expression from transiently transfected intact plasmid, but not from spurious genomically integrated vectors. we report herein the development of potent minimal, antibiotic-free, high-manufacturing-yield mammalian expression vectors incorporating rationally designed additive combinations of expression enhancers. the sv40 72 bp enhancer incorporated upstream of the cytomegalovirus (cmv) enhancer selectively improved extrachromosomal transgene expression. the human t-lymphotropic virus type i (htlv-i) r region, incorporated downstream of the cmv promoter, dramatically increased mrna translation efficiency, but not overall mrna levels, after transient transfection. a similar mrna translation efficiency increase was observed with plasmid vectors incorporating and expressing the protein kinase r-inhibiting adenoviral viral associated (va)1 rna. strikingly, htlv-i r and va1 did not increase transgene expression or mrna translation efficiency from plasmid dna after genomic integration. the vector platform, when combined with electroporation delivery, further increased transgene expression and improved hiv-1 gp120 dna vaccine-induced neutralizing antibody titers in rabbits. these antibiotic-free vectors incorporating transient expression enhancers are safer, more potent alternatives to improve transgene expression for dna therapy or vaccination. supplementary information: the online version of this article (doi:10.1038/gt.2010.149) contains supplementary material, which is available to authorized users. gene therapy using transiently expressed non-integrative plasmid vectors is the ideal solution for many public health applications. for example, no current therapy for diabetic foot ulcers directly improves the biology of wound healing as would gene therapy using the hypoxia-inducible factor 1a (hif-1a) master regulator of wound healing genes. 1 a topically delivered gene-based drug is ideal for such growth factor replacement therapies, as plasmid has a far longer duration of expression compared with rapidly degraded recombinant peptides. extensive research to improve plasmid delivery has resulted in advanced methods such as electroporation (ep)-facilitated uptake, which dramatically improves gene transfer compared with passive dna uptake. plasmid-directed gene expression is now reaching the efficacy barrier that, to date, has prevented commercialization of gene-based plasmids for human application. 2 although plasmid delivery has been the focus of gene transfer research, complementary vector design innovations may also be applied to improve transgene expression. we previously reported the development of potent minimalized antibiotic-free (af) mammalian expression vectors. 3 af vectors are derivatives of the pdnavac-cultra expression vectors, 4 which incorporate and express a 150 bp rna-out antisense rna that represses expression of a host strain chromosome-encoded counter-selectable marker (sacb-encoded levansucrase) that is toxic in the presence of sucrose (figure 1a ). these sucrose selectable vectors combine af selection with highly productive fermentation manufacturing (41 g l à1 plasmid dna yields) and improved transgene expression levels compared with existing vectors. 3, [5] [6] [7] [8] these plasmids were designed to remove non-essential extraneous sequences ( figure 1 ) and are minimalized fda (food and drug administration)-compliant 9 vectors for gene therapy or genetic immunization applications. these af vectors are also compliant with european regulatory agency recommendations to eliminate antibiotic resistance markers from plasmid therapy vectors. 10 although individual expression-augmenting sequences have been identified, they have not been used combinatorially to improve vector performance. we report herein the incorporation of rationally designed additive combinations of expression enhancers into optimized minimalistic af vectors to effect improved transgene expression. the resultant high-production-yield, minimal, af mammalian expression vectors incorporate novel vector backbone functionalities that further improve plasmid-directed transgene expression after transient transfection (transient expression enhancers; tee platform: figures 1b and c) . the viral human t-lymphotropic virus type i (htlv-i) r, adenoviral viral associated (va) rnai (va1) and sv40 enhancers used in this study were derived from non-coding regions of the respective viruses and did not have significant sequence homology to the human genome. these studies demonstrate that dramatic increases in vector-directed transgene expression can be obtained through innovations in vector design. to reduce chances in chromosomal integration, sequences added to a plasmid to increase transgene expression should contain no significant homology to the human genome. this may be determined by blast search, specifying to search for short, nearly exact matches against the human genome. 5, 11 regions encoding antigenic peptides should also not be present in vector backbones. these include cryptic open reading frames (orfs) in bacterial or eukaryotic sequences that may be expressed in eukaryotic cells to generate unwanted and potentially detrimental cytotoxic t-cell 12-14 or humoral responses. the removal of spacer and junk sequences and the use of rna-based selectable markers to eliminate the kanamycin-resistant (kanr) orf reduce this risk. the ntc8385, ntc8485 (figures 1b and c) and ntc8685 vectors (ntc8485 incorporating the boundary element deletion; figure 1c ) described herein are minimalized vectors that do not contain extraneous spacer or junk sequences. these vectors incorporate a short 140 bp rna-based selection marker rather than an antibiotic resistance marker (figure 1a ). this resulted in much higher vector potency through elimination of approximately 2 kb of dna compared with the gwiz (genlantis, san diego, ca, usa) vector, which includes the kanr gene and associated extraneous dna including in a plasmid-free cell, levansucrase was expressed from a chromosomally integrated sacb gene, leading to cell death in the presence of sucrose. right: rna-out from the plasmid repressed translation of the sacb gene, achieving plasmid selection; (b) ntc8385 egfp af vector with rna-out selectable marker and htlv-i r transient expression enhancer; (c) ntc8485 egfp af vector, with transient expression enhancers htlv-i r, va1 and sv40 enhancer. optimization of the sv40-cmv boundary (be deletion) resulted in a vector, ntc8685, with further improved expression; (d) gwiz egfp kanr vector with locations of non-essential spacer and junk dna (tn903 inverted repeat, polyc, polyg and ampr promoter), annotated. the basepairs 1-245 puc19 region functioned to maintain an optimal junction between the cmv promoter and the prokaryotic backbone. this sequence was retained in the equivalent location (in the ntc8385 vector-up) and was replaced by the sv40 enhancer in the ntc8485 and ntc8685 vectors. in these vectors, the 1-245 bp puc19 region was moved and added as an extension to the puc origin to add back a leading strand primosomal assembly site (pas-bh) present in pbr322. this site was deleted when the puc vector was created by imprecise deletion of the repressor of primer (rop) gene. 6 ntc8485 and ntc8685 pas-bh vectors had higher plasmid copy number and manufacturing yields than did ntc8385 or gwiz. 6 the transposon tn903 inverted repeat, the ampicillin resistance marker promoter and polyc and polyg tailing site cloning footprints ( figure 1d ). 3¢ untranslated region (utr): 3¢ utr orfs are translated many dna vaccine vectors contain a single copy of the human or woodchuck hepatitis b virus posttranscriptional regulatory element (pre or wpre) immediately downstream of the stop codon before the transcriptional terminator. this element is commonly used in retroviral and lentiviral vectors as an alternative to the lentiviral rev-response element to direct export of unspliced full-length viral genomic mrna. pre elements are included in dna vaccines to increase spliced mrna nuclear export and transgene expression. however, hepatitis genomes are highly compacted such that this enhancer also encodes a 178 amino-acid fragment of the hepatitis c virus polymerase gene. 15 the pre, by design, is within the eukaryotic expression cassette; this type of design is known to generate t cells reactive to major histocompatibility complex-i epitopes in a cryptic orf. 13 such antigenic regions might significantly alter immune responses in individuals with previous exposure and memory t cells reactive to hepatitis. to assess this risk, we quantified the translation of a coding region inserted, in different reading frames, downstream of the transgene stop codon in a cytomegalovirus (cmv) promoter expression vector. a neomycin resistance gene (neor) without an upstream kozak sequence was cloned downstream of an enhanced green fluorescent protein (egfp) transgene in different configurations similar to that used with pres. quantifiable neor translation products were present in all tested configurations, as was biologically active neor protein after plasmid transfection into both hek293 and cho cell lines (supplementary table s1 ). these results demonstrated that expression enhancers should ideally be non-coding regions and that cryptic orfs present downstream of the transgene within the exported eukaryotic mrna can be translated, even when the orf is not in the same reading frame as the upstream transgene. although a number of 5¢ utr sequences have been identified that can be used to increase transgene expression (for example, see 16 ) , the htlv-i r element is very potent. when cloned downstream of the transcription start site, htlv-i r improved expression through a variety of promoters, including sv40 17 and cmv, 18 and improved transgene expression in non-human primates. 18 cmv-htlv-i r promoter plasmid vectors were found to be safe for human vaccination in nih vaccine research center-sponsored clinical trials of hiv, ebola and severe acute respiratory syndrome (sars) dna vaccines. the cmv-htlv-i r promoter has htlv-i r incorporated as part of exon 1 and intron 1 downstream of the cmv promoter ( figure 1b ). this configuration improved dna vaccine expression and immunogenicity. 3, 18 incorporation of htlv-i r into ntc8385 (figure 1b ) dramatically increased transgene expression in hek293 total cellular extracts ( figure 2a) and fluorescence-activated cell sorting (facs) sorted cells (figure 2b ) after transient transfection. this was due to increased mrna translation efficiency (that is, htlv-i r increased the amount of egfp transgene produced per pg cytoplasmic egfp mrna; figure 2c ), but not due to increased overall cytoplasmic mrna levels. by contrast, no effect on integrated expression (either total mean fluorescence or fluorescence per integrated transgene copy) was observed in hek293 ( figure 2d ) or cho-k1 (supplementary figure s1 ) cells. consistent with this, the cytoplasmic mrna translational efficiency improvement observed with htlv-i r from transiently transfected plasmid did not occur with cytoplasmic rna produced from genomically integrated plasmid ( figure 2c) . interestingly, the overall amount of egfp transgene produced per pg of cytoplasmic egfp mrna was dramatically higher than that from genomically integrated plasmid compared with transiently transfected plasmid for both cmv and cmv-htlv-i r promoter plasmids ( figure 2c ). the in vivo effect of the htlv-i r region on plasmid vaccination was determined in rabbits. immunization of rabbits with a cmv-htlv-i r promoter hiv-1 gp120 dna vaccine vector combined with ep delivery induced up to ½ log higher titers of hiv-1 virusneutralizing antibodies (ic 50 ) than a comparator cmv promoter vector ( figure 3 ). improved ic 50 titers were observed with both neutralization-sensitive (nl4-3 and sf162) and -resistant (6535 and jrcsf) viruses. this is likely attributable to a higher gp120 expression with the cmv-htlv-i r plasmid. these results demonstrate that this modified 5¢ utr vector design improved vector performance in vivo when combined with an optimal dna delivery platform (ep). activation of pkr inhibits mrna translation through eukaryotic initiation factor-2a phosphorylation. 19, 20 pkr is activated either by its ligand, double-stranded rna or by the pkr-associated activator protein (pact, and in murine cells by the rax ortholog) in response to diverse non-nucleic acid-based cell stresses including endoplasmic reticulum stress (the unfolded protein response). 21 cell stress is induced by certain plasmid deliveries (for example, liposomal or calcium phosphate formulations) and/or by expression of aggregation-prone proteins (for example, hydrophobic membrane-domaincontaining proteins, polyepitope synthetic proteins). therefore, inclusion of a pkr inhibitor in the vector backbone may improve plasmidmediated expression under stress conditions. the adenovirus serotype 5 va rnai (va1) and the epstein-barr virus eber1 rnas are non-coding rna pol iii-expressed small rna-based pkr inhibitors. va rnai was included in the padvantage vector (promega, madison, wi, usa) plasmid backbone to increase target gene expression of co-transfected plasmids. 22 for human vaccination, va rnai is preferable to eber1, as eber1 binds multiple cellular proteins and has documented cell growth-stimulating properties. these adverse properties have not been attributed to va1, and, as a natural adenovirus gene, va1 is present in multiple adenoviral vectors evaluated in human clinical trials. cmv-htlv-i r promoter plasmids encoding va1 (for example, ntc8385-va1-egfp; figure 1b ) had increased transgene expression compared with the parent cmv-htlv-i r promoter vector after transient expression of human hek293 cells (figure 4b ). cmv-htlv-i r-and cmv promoter-directed transgene expression levels were not affected by addition of rna pol iii inhibitor to the transfection (rna pol iii independent; figure 4a ). in contrast, as expected, va1-mediated increased transgene expression was rna pol iii dependent, as it was inhibited at all tested rna pol iii inhibitor concentrations ( figure 4b ). va1 increased transiently transfected mrna translation efficiency (egfp transgene produced per pg of cytoplasmic egfp mrna; supplementary figure s2 ), but had no effect on translation efficiency after forced integration in hek293 cells (egfp protein produced per integrated transgene; figure 4d ). va1-and htlv-i r-directed transient expression enhancement was also observed in human a549 and murine nih-3t3 and l929 cell lines (supplementary figure s3) . these results demonstrated that both htlv-i r and va1 increased translation of transiently expressed mrnas and that their effects were not redundant. the variability in the activity of these elements between these cell lines probably reflected differences in levels of cellular cofactors required for va1 and htlv-i r activity. va1-mediated pkr inhibition may account for increased transgene expression. alternatively, pkr-independent stimulation of gene expression may be through va1 inhibition of adenosine deaminase acting on rna 23 or through rna interference. 24, 25 this was determined by testing the effects of function-specific point mutations in the vector-encoded va1 gene (figure 4c ) on egfp expression. va1-pkr3 incorporated the l3 mutation. this is a 2 bp change in the central domain that has been shown to be inactive for pkr inhibition, 26 yet is transcribed at high level in cell culture and maintains the va rnailike rna secondary structure necessary for rna interference inhibition. va1-pkr5 incorporated a 1 bp change (pm91) in the central domain that is strongly reduced for pkr inhibition, 27 but retained adenosine deaminase acting on rna-inhibiting activity 23 and the va rnai-like rna secondary structure required for rna interference inhibition. the results (figure 4c ) suggest that pkr inhibition, and not adenosine deaminase acting on rna or rna interference inhibition, was critical for va1-mediated transgene expression enhancement, as this effect was lost with both pkr3 and pkr5 point mutants. nuclear membrane transit: sv40 enhancer transient expression enhancer sequences that, when incorporated into a vector backbone, improve plasmid nuclear localization will also enhance non-viral gene expression, as dna transport to the nucleus is a limiting factor in non-dividing cells (reviewed in lam and dean, 28 and wagstaff and jans 29 ). such sequences would be true transient expression enhancers, as they function to enhance plasmid nuclear entry, not mrna production or translation, which could also affect chromosomal gene expression. mechanisms for sequence-specific plasmid dna nuclear import are being delineated, 30 and a number of dna transcription factor binding sites have been identified that increase plasmid nuclear import when added to a plasmid vector backbone. 28 several of these were incorporated into the ntc8385-va1 vector backbone and evaluated for improved expression. sv40 enhancer. inclusion of the sv40 72 bp enhancer upstream or downstream of the expression cassette in a dna vaccine vector backbone increased in vivo expression up to 20-fold using ep delivery in muscle tissue. 31, 32 this enhancement was not observed in vitro. 31 the ntc8485 vector (figure 1c ) contains the sv40 enhancer (two copies of the 72 and the 21 bp repeats) (figure 5a ). this enhancer region, as part of the sv40 promoter, has not caused safety concerns in human clinical trials of sv40 promoter-containing vectors. ntc8485 had improved manufacturing yields because of improved copy number mediated by the sv40 enhancer and the pas-bh replication origin. 6 this configuration of the sv40 enhancer with the cmv promoter was also shown to improve transgene expression after ep-mediated delivery to murine muscle. 33 as expected, in vitro expression of ntc8485 was similar to the ntc8385 vector (figure 5b) . treatment with tumor necrosis factor-a, which induces nuclear localization of nuclear factor-kb (nf-kb), thereby increasing transgene expression by activation of multiple nf-kb sites in the cmv promoter, resulted in a higher expression than ntc8385 (figure 5b) , perhaps through activation of the two nf-kb sites in the sv40 enhancer (figure 5a ). addition of three nf-kb binding sites to ntc8485 (ntc8485-kb) further improved expression after tumor necrosis factor-a activation (figure 5b) . ntc8485 contains the at-rich unique region upstream of the cmv enhancer. the unique region contains a boundary domain 34 and several binding sites for cellular repressor proteins pdx1 35 and cdp (figure 5a ). 36 these sequences may interfere with sv40 enhancer interactions with the cmv promoter. consistent with this, deletion of the unique region between the sv40 and cmv enhancer junction (to create ntc8685) increased transgene expression in dividing hek293 cells compared with the ntc8485 parent vector (figures 5b and c) . as the sv40 enhancer did not generally improve expression in vitro, 31 the unique region deletion in ntc8685 may allow sv40 enhancermediated activation of the cmv promoter. this optimized configuration had higher expression than ntc8485-kb, with and without tumor necrosis factor-a, and approximately 10-fold higher expression than the kanr pvax1 (invitrogen, carlsbad, ca, usa) cmv promoter vector (figure 5b) . the sv40 enhancer nf-kb sites are substrates for the e coli dcm methylase that methylates the internal cytosine residues in the recognition sequence 5¢-cc*agg-3¢ or 5¢-cc*tgg-3¢ to 5-methyl-cytosine (5mc). methylation of these sites may reduce nf-kb binding and sv40 enhancer-mediated activation of the cmv promoter in the optimized ntc8685 vector. consistent with this, ntc8685 plasmid produced in a dcmà host strain had much higher expression than dcm+plasmid (figure 5b) . expression levels of ntc8385, ntc8485 and ntc8685 after in vitro ep were determined in human dendritic cells (dc) versus a cmv promoter comparator (supplementary figure s4 ). all three ntc cmv-htlv-i r promoter vectors had improved expression compared with a cmv promoter vector. in this system, in which isolated dividing cells are subjected to in vitro ep, a strong effect of the ntc8485 and ntc8685 encoding va1 rna or sv40 enhancer was not observed, although ntc8685 was the highest expressing vector in all three donors. this would indicate that cell stress was not induced by these in vitro conditions, and cell division eliminated the need for sv40-mediated nuclear entry (as reported in li et al. 31 ). other nuclear import binding proteins. nf-kb and nm23-h2 nuclear shuttle protein binding sites increase plasmid nuclear localization when added to base vectors. 37, 38 the sp1 protein is speculated to be a chromatin modulator that functions to maintain expressioncompetent euchromatin through association with histone acetyl transferases. 39 the sp1 and nm23-h2 sites also form a g-quadruplex (g4) structure. this is a local single-stranded dna region, with a single-stranded three-loop structure formed by interactions with four 3-guanine motifs and three linking loops. g4 structures exclude nucleosomes and are recognized by a variety of dna binding enzymes 40 that may function as nuclear shuttle proteins. binding sites for these proteins were integrated into the ntc8685 backbone. ntc8685 expression in dividing or non-dividing (aphidicolin treated) a549 was not improved by inclusion of dna binding sequences for nuclear localizing proteins nf-kb (kb), nm23-h2 (g4) or sp1 (figures 5e and f) . a549 is known to express nm23-h2. 40 however, expression of ntc8385, ntc8485 and ntc8685 base vectors in aphidicolin-treated non-dividing a549 (figure 5f ) and non-dividing hek293 (figure 5d ) cells was much higher than that of the gwiz comparator. all these vectors were produced in dcmà hosts; hence, differences in expression levels could not be attributed to effects of plasmid dcm methylation. this implies that nuclear localization was optimal in these ntc backbones, and that inclusion of additional shuttle protein binding sites was redundant to existing nuclear localization signals present in these vectors. cell growth was inhibited with 20 mm of pol iii inhibitor, which accounts for the observed reduced fu at this concentration; (b) pol iii transcription was required for va1 expression enhancement effect. the assay was in the same format as in (a), except for the fact that pol iii inhibitor-treated cells were transfected with egfp plasmids containing the cmv-htlv-i r promoter with or without va1; (c) inhibition of pkr, not of adenosine deaminase acting on rna (adar) or rna interference (rnai), was required for va1 expression enhancement effect. hek293 cells were transfected with ntc7485-egfp (same backbone as ntc8485, except for kanr instead of rna-out) and ntc7485-egfp-modified to contain: va1 (pkr+rnai+adar+inhibitor); va1-pkr3 (pkràrnai+adar?inhibitor), in which va1 incorporated the l3 point mutation, 26 (the designation 'adar?' was used, as the effect of l3 on adar inhibition has not been established); or va1-pkr5 (pkràrnai+adar+inhibitor) in which va1 incorporated a 1 bp change (pm91) in the central domain. 27 (d) va1 did not increase transgene expression after genomic integration. postintegration expression of (1) kanr gwiz (cmv) compared with (2) af gwiz derivative containing the cmv-htlv-i r promoter (instead of cmv) and the va1 gene (cmv/af-htlv-i-va1). cho cells were transfected with egfp-ires-neo transgene versions of both plasmids using lipofectamine ltx in quadruplicate (using four different ratios of plasmid to ltx). integrants were selected with geneticin for 10 days. results presented are after nine generations post selection (g9). total mean ± s.d. fluorescence of egfp extracts from four g9 integrated cell lines per vector (fu) and fu per integrated transgene copy number per genome (fu/copy) are shown. integrated copy number was determined using rt-pcr quantification of the egfp transgene in isolated quantified genomic dna. we report the development of regulatory-agency-compliant minimal af dna vaccine/gene therapy plasmid vectors that combined 41 g l à1 fermentation yields 41 matrix-attachment site vectors, tee sequences did not increase the frequency of genome integration (williams et al. manuscript in preparation) . a vector family, incorporating various tee combinations, is now available, allowing researchers to select the optimal combination for their application. tee vectors could also be adapted to improve expression in specific cell types, through swapping of an alternative tissue-specific cellular promoter for the ubiquitous cmv promoter. three transient expression-augmenting sequences were identified and incorporated into minimal af vectors in order to increase the in vivo expression of encoded transgenes. the htlv-i r region, inserted downstream of the cmv promoter, dramatically increased transgene expression by enhancing mrna translation efficiency. a similar and additive mrna translation efficiency increase was observed with plasmid vectors further incorporating and expressing the protein kinase r (pkr)-inhibiting adenoviral va rnai (va1). finally, the sv40 enhancer, cloned upstream of the cmv enhancer, increased transgene expression in non-dividing cells. importantly, these vectors did not increase transgene expression from plasmid dna after forced genomic integration. this phenomenon of translation stimulation restriction to extrachromosomal vector-derived mrnas has been previously observed with va1, 42 and was attributed to localized activation of pkr leading to translation inhibition through eukaryotic initiation factor-2a phosphorylation. 42, 43 the similar cis-acting effect of htlv-i r to enhance transient but not integrated plasmid mrna translation is mechanistically distinct, because htlv-i r and va1 effects were additive. a critical difference between transient and genomically produced rna is that transient mrna is inefficiently translated (figure 2c ). this may be due to aberrant mrna splicing, differential cytoplasmic mrna localization, altered stability of ribosome bound-rna or differences in ribosome loading/composition/stability with transient compared with genomic source mrna. the fragment of htlv-i r incorporated into the 5¢ utr may alleviate these transient rna-restricted effects. incorporation of dna binding sites for nuclear shuttling proteins nf-kb and nm23-h2 into the ntc8685 vector backbone did not result in improved nuclear localization. previous studies with these factors were with less-optimal vectors. 37, 38, 44 nuclear localization capacity may be maximized with the extensively optimized minimalized ntc8685 vector. the vector platform described herein, when combined with advanced delivery methods such as ep, further increased gene expression. this led to improved hiv-1 gp120 neutralizing antibody titers in rabbits after ep-mediated delivery of an htlv-i r tee vector. the reduced size compared with alternative vectors may be critical to improving gene transfer with large transgenes such as dystrophin, as large vectors have reduced transfection efficiency with ep delivery. 45 tee vectors therefore have application to generally improve dna vaccination or gene therapy. these improvements may be critical to enable future gene medicine licensure for public health applications. vectors were constructed using standard molecular biology methods. 46 plasmids ntc8385, ntc8485 and ntc8685 contain a 140 bp draiii-kpni rna-based sucrose selectable marker (rna-out). ntc8485 and ntc8685 incorporate the high copy number pas-bh-sv40 backbone from the kanr vector ntc7485. 6 ntc8485-kb and ntc8685-kb contain three copies of an optimized nf-kb binding site (underlined 44 ) inserted into the noti site (bold) (figure 1c ). ntc8685-kb 5¢-gcggccgggactttccagctggg gactttccagctggggactttccgcggccgc-3¢. ntc8685-2x sp1 contains two copies of an optimized sp1 transcription factor binding site (underlined 47 ) inserted between the noti and nhei sites (bold) of the vector:2x sp1: 5¢-gcggccgctagacggggcgggggctcgacggggcgggggctagc-3¢. ntc8685-g4 contains one copy of the c-myc nm23-h2 transcription factor binding site (underlined 38 ) inserted between the noti and nhei sites (bolded) of the vector: g4: 5¢-gcggccgctaggggagggtggggagggtgggg aaggtggggagctagc-3¢. kanr plasmids were grown in either escherichia coli strain coli dh5a (f-f80dlaczdm15 d(laczya -argf) u169 reca1 enda1 hsdr17 (rkà, mk+) phoa supe44 l-thi-1 gyra96 rela1) or dcmà version ntc48107 dh5a dcm, whereas af plasmids were grown in either dcm+ ntc4862 (dh5a attl::p5/6 6/6-rna-in-sacb, cm r ) or dcmà ntc48165 (dh5a dcm att l ::p 5/6 6/6 -rna-in-sacb, cm r ). for cell culture and immunization testing, low endotoxin (o100 eu mg à1 ) plasmid dna was purified using nucleobond ax 2000 or ax 10 000 columns (macherey nagel, düren, germany). where indicated, plasmid dna was linearized by restriction enzyme digestion before transfection. linearized plasmid dna was purified by phenol/chloroform extraction and ethanol precipitation before being resuspended in te buffer (10 mm tris, 1 mm edta, ph 8.0) for transfection. adherent hek293 (human embryonic kidney), a549 (human lung carcinoma), nih3t3 (murine embryonic fibroblast), l929 (murine areolar fibroblast) and cho-k1 (chinese hamster ovary) cell lines were obtained from the american type culture collection (manassas, va, usa). cell lines were propagated in dulbecco's modified eagle's medium/f12 containing 10% fetal bovine serum and split (0.25% trypsin-edta) using invitrogen (carlsbad, ca, usa) reagents and conventional methodologies. for transfections, cells were plated on either 6-or 24-well tissue culture dishes. supercoiled or linear plasmids were transfected into cell lines using lipofectamine 2000 (or lipofectamine ltx for cho-k1) following the manufacturer's instructions (invitrogen) and analyzed for duration and variability of expression in integrated (egfp-ires-neo transgene) and transient (egfp transgene) expression systems as indicated. plasmid integrants were selected using media containing 500 mg ml à1 geneticin (invitrogen) over 10 days. total cellular lysates for egfp determination were prepared by resuspending cells in cell lysis buffer (bd biosciences pharmingen, san diego, ca, usa), lysing cells by incubating for 30 min at 37 1c, followed by a freeze-thaw cycle at à80 1c. lysed cells were clarified by centrifugation and the supernatants assayed for egfp by flx800 microplate fluorescence reader (bio-tek, winooski, vt, usa). single cell suspensions for facs flow cytometry were prepared by dissociating cells with trypsin treatment, and washing with media to remove trypsin and then phosphate-buffered saline for facs analysis using the bd (franklin lakes, nj, usa) facscalibur dual-laser cytometer. transfection of non-dividing cells was as described in monkonge et al. 38 briefly, hek293 or a549 cells were pretreated overnight with 5 mg ml à1 aphidicolin (sigma, st louis, mo, usa), which inhibits cell division and synchronizes cells to early s phase. transfection of egfp plasmids and outgrowth were then performed in the presence of aphidicolin. cell outgrowths were incubated with or without 25 ng ml à1 tumor necrosis factor-a (invitrogen) for 3 h to induce nf-kb nuclear shuttling. fresh medium was then added and total cell extracts were prepared for egfp fluorescence determination 24 h (a549) or 48 h (hek293) post transfection. genomic dna was isolated from integrated cell lines using the dneasy blood and tissue kit (qiagen sciences, germantown, md, usa) and total dna was quantified using the flâ800 microplate fluorescence reader (bio-tek) by determining picogreen (invitrogen) fluorescence of samples versus a linearized vector standard curve. rt-pcr to quantify vector copies in genomic dna was performed used a taqman egfp transgene 6fam-acagccacaac tct-mgbnfq probe and flanking primers (5¢-gggcacaagctggagtaca ac-3¢; 5¢-tctgcttgtcggccatgata-3¢) in a taqman gene expression assay using applied biosystems (foster city, ca, usa) taqman reagents and the step one real time pcr system (applied biosystems, carlsbad, ca, usa). linearized vector was used for the rt-pcr standard curve. cytoplasmic rna was isolated from transfected hek293 cells using the protein and rna isolation system (paris kit, ambion, austin tx, usa) and quantified by a 260 . samples were dnase treated (dna-free dnase; ambion) before reverse transcriptase rt-pcr using the agpath-id one step rt-pcr kit (ambion) and the egfp transgene specific probe. dnase treatment was confirmed by the lack of detectable signal in control rt-pcr reactions run without the initial 48 1c reverse transcriptase step using heat-treated 25â pcr buffer. heat treatment inactivates heat-labile reverse transcriptase but not taq dna polymerase. assay linearity was verified using sample dilutions. the hiv-1 gp120 envelope glycoprotein gene from the jrcsf strain was cloned into the pmamp and af ntc8382 backbones. the pmamp plasmid has been described previously 48 and contains the cmv promoter and intron 1 for gene expression and the bovine growth hormone polyadenylation site. ntc8382 is the ntc8385 vector modified to secrete transgene using a tissue plasminogen activator secretion leader. 4 both vectors express the identical tissue plasminogen activator-gp120 fusion protein. each plasmid was injected into rabbits (six per group) on days 0, 28 and 56, followed by ep with the medpulser electroporation system (inovio biomedical, san diego, ca, usa). all rabbits were bled on days 42 and 70. on day 84, the rabbits received a booster injection of alumadjuvanted gp120 protein. rabbits were bled on day 98 and the study was terminated. sera were prepared from the day 70 and 98 blood samples for measurement of the levels of hiv-1 neutralization activity. neutralization was tested on two neutralization-sensitive viruses (sf162 and nl4-3) and two resistant viruses (6535 and jrcsf). rabbit ep and virus neutralization assays were as described. 48 this animal study was conducted at aldevron (fargo, nd, usa) and was approved by aldevron's institutional review board. dcs from three independent donors were generated as previously described 49 and nucleofected 24 h after maturation using the amaxa dc nucleofection kit (amaxa, koeln, germany). a total of 0.5-1â10 6 dcs were prepared as per manufacturer's instructions, nucleofected with 2 mg of plasmid dna and subsequently resuspended in complete rpmi media (10% fetal bovine serum, 2 mmol l à1 glutamax and 1 mmol l à1 sodium pyruvate (sigma)), supplemented with the cytokine maturation cocktail containing 10 ng ml à1 interleukin-1b, 10 ng ml à1 tumor necrosis factor-a, 100 ng ml à1 interleukin-6 (r&d systems, minneapolis, mn, usa), 1 mg ml à1 prostaglandin e2 (sigma), 800 u ml à1 granulocyte macrophage colony-stimulating factor (sargramostim leukine; immunex, seattle, wa, usa) and 1000 u ml à1 interleukin-4 (r&d systems). nucleofection efficiency and egfp transgene expression were assessed 18 h after nucleofection using the bd facscalibur flow cytometer. the data were analyzed using cell quest software (bd). electroporation for the delivery of dna-based vaccines and immunotherapeutics: current clinical developments improved antibiotic-free dna vaccine vectors utilizing a novel rna based plasmid selection system pdnavaccultra vector family: high throughput intracellular targeting dna vaccine plasmids plasmid dna vector design; impact on efficacy, safety and upstream production generic plasmid dna production platform incorporating low metabolic burden seed-stock and fed-batch fermentation processes low metabolic burden plasmid production plasmid dna production combining antibiotic-free selection, inducible high yield fermentation, and novel autolytic purification guidance for industry: considerations for plasmid dna vaccines for infectious disease indications non-clinical studies required before first clinical use of gene therapy medicinal products detection of integration of plasmid dna into host genomic dna following intramuscular injection and electroporation cryptic open reading frames in plasmid vector backbone sequences can provide highly immunogenic cytotoxic t-lymphocyte epitopes translation from cryptic reading frames of dna vaccines generates an extended repertoire of immunogenic, mhc class i-restricted epitopes robust, vaccine-induced cd8+ t lymphocyte response against an out-of-frame epitope the genome of hepatitis b virus contains a second enhancer: cooperation of two elements within this enhancer is required for its function evaluating post-transcriptional regulatory elements for enhancing transient gene expression levels in cho k1 and hek293 sr alpha promoter: an efficient and versatile mammalian cdna expression system composed of the simian virus 40 early promoter and the r-u5 segment of human t-cell leukemia virus type 1 long terminal repeat a human t-cell leukemia virus type 1 regulatory element enhances the immunogenicity of human immunodeficiency virus type 1 dna vaccines in mice and nonhuman primates pkr; a sentinel kinase for cellular stress impact of protein kinase pkr in cell biology: from antiviral to antiproliferative action the double-strand rna-dependent protein kinase pkr plays a significant role in a sustained er stress-induced apoptosis increased gene expression in mammalian cell lines using padvantage dna as a cotransfectant adenovirus vai rna antagonizes the rna-editing activity of the adar adenosine deaminase suppression of rna interference by adenovirus virus-associated rna adenovirus va1 noncoding rna can inhibit small interfering rna and microrna biogenesis secondary and tertiary structure in the central domain of adenovirus type 2 va rna i effect of single-base substitutions in the central domain of virus-associated rna i on its function progress and prospects: nuclear import of nonviral vectors nucleocytoplasmic transport of dna: enhancing non-viral gene transfer identification of protein cofactors necessary for sequence-specific plasmid dna nuclear import muscle-specific enhancement of gene expression by incorporation of sv40 enhancer in the expression plasmid electroporation in combination with a plasmid vector containing sv40 enhancer elements results in increased and persistent gene expression in mouse muscle vectors and methods for genetic immunization identification of a boundary domain adjacent to the potent human cytomegalovirus enhancer that represses transcription from the divergent ul127 promoter pdx1, a cellular homeoprotein, binds to and regulates the activity of human cytomegalovirus immediate early promoter cellular homeoproteins, satb1 and cdp, bind to the unique region between the human cytomegalovirus ul127 and major immediate-early genes a regulated nfkb-assisted import of plasmid dna into mammalian cell nuclei identification and functional characterization of cytoplasmic determinants of plasmid dna nuclear import taf1 histone acetyltransferase activity in sp1 activation of the cyclin d1 promoter metastases suppressor nm23-h2 interaction with g-quadruplex dna within c-myc promoter nuclease hypersensitive element induces c-myc expression critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust rna pol ii and pol iii-mediated eukaryotic expression with high bacterial production yields translational control mediated by eukaryotic initiation factor-2 is restricted to specific mrnas in transfected cells the antiviral enzymes pkr and rnase l suppress gene expression from viral and non-viral based vectors nuclear delivery of nfb-assisted dna/polymer complexes: plasmid dna quantification by confocal laser scanning microscopy and evidence of nuclear polyplexes by fret imaging factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles vectors and methods for genetic immunization two synthetic sp1-binding sites functionally substitute for the 21-base-pair repeat region to activate simian virus 40 growth in cv-1 cells effect of trimerization motifs on quaternary structure, antigenicity, and immunogenicity of a noncleavable hiv-1 gp120 envelope glycoprotein nucleofection of dcs to generate multivirus-specific t cells for prevention or treatment of viral infections in the immunocompromised host we thank marni england-hill (aldevron) and jennifer bath (concordia college) for oversight of the rabbit study at aldevron, and danielle shea (university of nebraska, lincoln) for performing flow cytometry. we also thank kim hanson (nature technology) for purifying the plasmid dna used in this study and sheryl anderson (nature technology) for linear vector preparation. this paper described work supported by nih grants r44 gm072141-03 and r43 gm080768-01 to jaw. aml is supported by a specialized centers for cell-based therapy grant nih-nhlbi 1 u54 hl081007 and an amy strelzer manasevit scholar award. ug is supported by an asbmt young investigator award and a leukemia and lymphoma society special fellow in clinical research award. key: cord-016255-kkko1xne authors: van der meer, j.t.m.; nouwen, j.l. title: 14 intravasale infecties en sepsis date: 2011 journal: microbiologie en infectieziekten doi: 10.1007/978-90-313-7944-6_14 sha: doc_id: 16255 cord_uid: kkko1xne infecties in het hart en de bloedbaan worden intravasale of endovasculaire infecties genoemd. de circulatie van bloed door het hart is essentieel voor de aanvoer van zuurstof en voedingstoffen naar weefsel en organen en voor de afvoer van afvalstoffen. in dit hoofdstuk worden infecties besproken die zich vooral afspelen in lymfoïde weefsels en meerdere orgaansystemen kunnen aantasten. cellen van het immuunsysteem spelen een belangrijke rol in de pathogenese van deze vaak chronische infecties. dit uit zich onder andere in gegeneraliseerde lymfadenopathie (lymfekliervergroting), een gemeenschappelijk klinisch kenmerk van deze ziekten. in dit hoofdstuk worden de pathofysiologische processen die aan deze lymfekliervergroting ten grondslag liggen kort samengevat en worden de belangrijkste infectieuze ziektebeelden met lymfadenopathie en hun verwekkers besproken. de lymfeklier verzamelt eiwitrijke vloeistof (lymfe) uit de extracellulaire ruimte van perifere weefsels. lymfe bevat antigenen, micro-organismen en fagocytaire cellen en vloeit continu via afferente lymfevaten naar de regionale lymfeklieren. vanuit de subcapsulaire ruimte van de lymfeklier bereikt de lymfe via weefselspletende sinussen -de hilus, waar zij de lymfeklier via de efferente lymfvaten verlaat. na passage van een aantal lymfeklierstations komt de lymfe uiteindelijk via de ductus thoracicus in de v. cava superior. tijdens de passage door de lymfeklier komt de lymfe in contact met een zeer groot aantal fagocyterende cellen die zich op een skelet van reticulinevezels hebben vastgezet. door fagocytose wordt de lymfe vrijwel volledig gefilterd van antigenen en micro-organismen. in de lymfeklier komen t-en b-lymfocyten in contact met antigene fragmenten die worden gepresenteerd door dendritische cellen. de lymfeklier fungeert dus behalve als mechanisch filter ook als kraamkamer van het specifieke immuunsysteem, waar door intensief contact tussen antigeenpresenterende cellen en lymfocyten een antigeenspecifieke cellulaire (t-cel) en humorale (b-cel) respons wordt gegenereerd. bij infecties kan zowel het niet-specifieke als het specifieke immunologische proces aanleiding geven tot lymfekliervergroting (lymfadenopathie). bij acute bacteriële infecties (zoals door staphylococcus aureus of streptococcus pyogenes) wordt de vergroting van de lymfeklier veroorzaakt door een toevloed van macrofagen en granulocyten, die in de lymfeklier doorgedrongen bacteriën afkapselen en fagocyteren. de vergroting is pijnlijk en gaat ook klinisch gepaard met andere ontstekingsverschijnselen (lymfadenitis), waarbij ook ontsteking van de afferente lymfvaten (lymfangitis) kan optreden. in de lymfeklier kan abcedering optreden. bij chronische virale infecties wordt daarentegen de lymfadenopathie veroorzaakt door een sterke toename van het aantal geactiveerde lymfocyten en immunoblasten. lymfeklieren bij chronische virale infecties zijn niet of nauwelijks pijnlijk. aspecifieke ontsteking en immuunactivatie kunnen ook naast elkaar voorkomen: in subacuut verlopende lymfadenopathieën, zoals veroorzaakt door bartonella henselae en chlamydia trachomatis, kunnen door histiocyten omringde microabcesjes optreden. lymfadenopathie door mycobacterium tuberculosis wordt gekenmerkt door het optreden van granulomen: haarden van epitheloïde cellen in palissadestand met veelkernige reuscellen en in het midden verkazende necrose. een overzicht van de belangrijkste ziektebeelden waarbij lymfadenopathie optreedt, met hun verwekkers en belangrijkste differentiële criteria, wordt gegeven in tabel 13.1. naast deze infectieuze oorzaken bestaan er tal van niet-infectieuze ziektebeelden die gepaard gaan met lymfadenopathie. hieronder vallen zowel systeemziekten (zoals bepaalde auto-immuunziekten en sarcoïdose) als maligne aandoeningen (primaire (non-)hodgkinlymfomen en secundaire lymfekliermetastasen). ten slotte kan lymfadenopathie optreden als reactie op bepaalde farmaca of corpora aliena (bijv. siliconen). mononucleosis infectiosa is een ziektebeeld met als belangrijkste klinische kenmerken moeheid, koorts, zwelling van (vooral de cervicale) lymfeklieren en een meer of minder uitgesproken faryngitis. de ziekte verloopt in de meeste gevallen subacuut en komt vooral voor bij adolescenten. in verreweg de meeste gevallen kan een recente infectie met epstein-barr-virus (ebv) worden aangetoond. ook kan het worden veroorzaakt door een recente cytomegalovirusinfectie (cmv-infectie) (5-10%), een toxoplasmose ( 1%), of een (primaire) hivinfectie. ebv en cmv behoren tot de humane herpesviridae (zie hoofdstuk 1). omdat lymfadenopathie tijdens de primaire infectie van ebv, cmv en hiv zo'n prominente rol kan spelen, zullen deze virussen in dit hoofdstuk uitvoeriger worden besproken. epstein-barr-virus (ebv, officiële taxonomische benaming: humaan herpesvirus 4, hhv-4) werd in 1961 ontdekt door epstein, achong en barr. zij zagen het virus met behulp van een elektronenmicroscoop in cellen die gekweekt waren uit een tumor die veel voorkwam bij kinderen in afrika (burkitt-lymfoom). vervolgens bleek dat de meeste volwassenen antistoffen hadden tegen ebv, passend bij een vroegere infectie. de toevallige observatie van de onderzoekers gertrud en werner henle dat zich antistoffen tegen ebv vormden bij een laboratoriummedewerker die mononucleosis infectiosa doormaakte, leidde tot de identificatie van ebv als een van de verwekkers van dit ziektebeeld. speeksel bevat wisselende hoeveelheden van dit virus, geproduceerd in de tonsilcrypten. primaire infectie via speekselcontact leidt tot productie van virus in de initieel geïnfecteerde epitheelcellen, waarop vervolgens de aanwezige b-cellen worden geïnfecteerd. infectie van deze b-cellen leidt tot een transformatie, wat in het laboratorium kan worden aangetoond met spontane uitgroei van lymfoblastoïde cellijnen. de infectie van b-cellen leidt tot een uitgesproken t-celrespons, waardoor in het bloedbeeld van patiënten een bont beeld van afwijkend gevormde (atypische) lymfocyten (geactiveerde t-lymfoblasten) wordt gezien. het kenmerkende symptomencomplex van mononucleosis infectiosa wordt niet rechtsreeks door virusreplicatie veroorzaakt, maar is het resultaat van de sterke t-celrespons (lymfadenopathie) en de daarmee gepaard gaande cytokine-en interleukineproductie, wat zich uit door extreme moeheid en koorts. deze t-celrespons is verantwoordelijk voor de onderdrukking van de door ebv geïnduceerde b-celproliferatie in vivo. ten gevolge van de virusspecifieke t-celrespons kan ebv na de primaire infectie alleen in latent geïnfecteerde b-geheugencellen voortbestaan. bij asymptomatische dragers is ongeveer 1 op de 100.000 b-geheugencellen geïnfecteerd. in deze cellen is weliswaar het ebvgenoom in de kern aanwezig, maar wordt slechts een beperkt aantal genen (10 van de ongeveer 100) tot expressie gebracht. hierdoor worden slechts weinig virale peptiden op het celoppervlak in de context van moleculen van hla-klasse i gepresenteerd, waardoor deze cellen niet door cytotoxische cd8+-t-cellen kunnen worden herkend. reactivatie van virusreplicatie treedt mogelijk op als gevolg van stimulatie van de b-geheugencel door herkenning van zijn specifieke antigeen. als gevolg van deze reactivatie kan bij 5-10% van de dragers uit speeksel virus worden geïsoleerd. het vermogen van ebv om b-celproliferatie te induceren kan leiden tot het ontstaan van b-celtumoren. aanvankelijk oligoklonale, in latere stadia vaak monoklonale b-cellymfomen kunnen worden gezien bij patiënten met een verstoorde t-celfunctie, zoals hiv-geïnfecteerde personen en ontvangers van orgaantransplantaten (post-transplant lymphoproliferative disorder, ptld). verder is aangetoond dat ebv-infectie een cofactor is in de pathogenese van burkitt-lymfoom en nasofarynxcarcinoom (frequent in china) en bij agressievere vormen van de ziekte van hodgkin. ebv komt wereldwijd voor. er is geen verschil in prevalentie tussen de geslachten of tussen etnische groepen. de leeftijd waarop infectie optreedt, wordt vooral bepaald door sociaal-economische factoren. in niet-westerse landen treedt infectie vaak al op de vroege kinderleeftijd op vanwege intensieve contacten; bij 5-jarigen ligt de seroprevalentie al boven de 50%, op volwassen leeftijd is uiteindelijk wereldwijd 90-95% geïnfecteerd. in zeldzame gevallen kan ebv-infectie aanleiding geven tot neurologische complicaties, zoals myelitis transversa, nervusfacialisparese, neuritis optica of cerebellitis. de soms heftig verlopende faryngitis kan naast slikstoornissen aanleiding geven tot pseudomeningisme door een verhoogde tonus van de cervicale musculatuur. respiratoire obstructie door zwellingen in hypofarynx en larynx komt incidenteel ook voor en kan ernstig zijn. karakteristiek is de rash die kan optreden tijdens ebvinfectie na empirische therapie met amoxicilline. een belangrijke hematologische complicatie is een hemolytische anemie op auto-immuunbasis. er zijn geen aanwijzingen voor betrokkenheid van ebv bij het chronisch vermoeidheidssyndroom. de diagnose ebv-infectie wordt gesteld op grond van serologisch onderzoek. waarschijnlijk ten gevolge van de massale polyklonale activatie van b-cellen kunnen tijdens de primaire ebv-infectie autoantistoffen en heterofiele antistoffen ontstaan (dat zijn meestal igm-antistoffen, gericht tegen erytrocytaire antigenen bij andere species). deze heterofiele antilichamen zijn dus niet gericht tegen ebv maar zijn een direct gevolg van polyklonale b-celstimulatie. de paul-bunnell-test, waarbij deze heterofiele antistoffen worden aangetoond in serum, is een specifieke surrogaattest voor ebv-infectie. deze test is positief bij 90% van de volwassenen met een primaire infectie, maar veel minder gevoelig bij jonge kinderen met een acute ebv-infectie. heterofiele antistoffen zijn vooral aantoonbaar in de eerste drie tot zes maanden na het begin van symptomen. voor screening, ook in de huisartsenpraktijk, zijn sneltests beschikbaar. specifieke ebv-serodiagnostiek kan ook worden verricht. hiermee kunnen igm-en igg-antistoffen worden aangetoond tegen het virale capsideantigeen (vca), een aantal vroege antigenen (early antigens, ea) en antigeen uit de latente cyclus van het virus (epstein-barr virus-associated nuclear antigen, ebna). de kinetiek van de verschillende antistofresponsen is weergegeven in figuur 13.2. aan de hand van het antistofpatroon kan een inschatting worden gemaakt van het moment van ebvinfectie. anti-vca-igg-antistoffen ontstaan tijdens de acute fase en blijven ook na herstel aantoonbaar. hoge titers anti-ea-antistoffen wijzen op een recente infectie. reconvalescentie gaat gepaard met het dalen van de anti-ea-titers en stijgende titers van anti-ebna-antistoffen. de kweek van ebv wordt niet gebruikt in de routinediagnostiek. de ontwikkeling van kwantitatieve moleculaire (pcr) technieken maakt het mogelijk om de hoeveelheid ebv in plasma te kwantificeren. bij patiënten die immuunsuppressieve therapie ontvangen vanwege een transplantatie wordt de hoeveelheid ebv gevolgd om ebv-gemedieerde posttransplantatie-lymfoproliferatieve ziekte vroegtijdig op te sporen en te voorkomen. de behandeling van een acute ebv-infectie is voornamelijk ondersteunend. aciclovir remt ebv-replicatie in vitro, maar heeft klinisch geen effect. dit past bij de beschreven vooral immunopathologische basis van het ziektebeeld. incidenteel kunnen corticosteroïden aangewezen zijn bij dreigende luchtwegobstructie of hemolytische anemie. een vaccin is nog niet voorhanden. bij ebv-positieve lymfoproliferatieve ziektebeelden speelt therapie gericht tegen b-cellen (anti-cd20 monoklonale antistoffen) een belangrijke rol. humaan cytomegalovirus (cmv, humaan herpesvirus 5) dankt zijn naam aan de reuzen-(megalo)cellen, die werden aangetroffen in organen van zuigelingen met een letaal verlopende congenitale infectie. humaan cytomegalovirus kent wereldwijd een groot aantal stammen met ongeveer 95% homologie op sequentieniveau; bij een groot aantal diersoorten (primaten, knaagdieren enz.) komen eigen cmv-soorten voor. besmetting met cmv treedt frequent prenataal of perinataal op (tijdens passage door het baringskanaal of door lactatie), of door contact met besmet speeksel of urine. monocyten kunnen tijdens de primaire infectie worden geïnfecteerd en produceren cmv na hun differentiatie tot macrofagen. op deze wijze dragen zij vermoedelijk bij tot de hematogene verspreiding van het virus. cmv infecteert onder andere epitheelcellen van nieren (proximale tubuli) en speekselkliercellen (ductusepitheel), endotheel en fibroblasten. het virus kan de placenta passeren en een intra-uteriene infectie veroorzaken (zie hoofdstuk 15). bij het onder controle krijgen van de primaire infectie spelen waarschijnlijk zowel specifieke cytotoxische t-lymfocyten als neutraliserende antistoffen een rol. de humorale en cellulaire respons leiden tot partiële immuniteit tegen andere cmv-stammen. in de latente fase kan cmv-dna worden aangetoond in monocyten en hemopoëtische stamcellen, maar niet in granulocyten, de cellen waarin het virus juist gedetecteerd wordt tijdens viremische episoden. actieve replicatie van cmv in de monocyten-/macrofagenreeks is waarschijnlijk beperkt tot gedefinieerde fasen in de differentiatie. de beperkte replicatie in de tussenliggende fasen draagt mogelijk bij tot de persistentie van de infectie. mogelijk geldt hetzelfde voor andere celtypen. bij immunocompetente dragers treedt episodisch een asymptomatische reactivatie op, waarbij het virus in urine en speeksel wordt uitgescheiden. lymfadenopathieën en hiv epidemiologie prenatale, intra-uteriene infecties kunnen ernstig verlopen (congenitale cmv, zie hoofdstuk 15), maar peri-en postnatale cmv-infecties verlopen vrijwel altijd asymptomatisch. overdracht vindt vooral plaats via speeksel, maar ook via moedermelk, urine, feces, bloed en sperma. tot 10% van de pasgeborenen wordt besmet met cmv tijdens de baring of via lactatie, maar intensief contact met leeftijdgenootjes die virus uitscheiden (crèches) zorgt voor een snelle toename van het aantal geïnfecteerden met de leeftijd. een tweede snelle stijging van de seroprevalentie wordt gezien bij jonge volwassenen. ook dan verlopen de meeste (90%) van de primo-infecties asymptomatisch. afhankelijk van de hygiënische omstandigheden varieert de seroprevalentie wereldwijd op volwassen leeftijd tussen 50 en 100%. ongeveer 10% van de primo-infecties op de volwassen leeftijd gaat gepaard met symptomen. deze treden op na een incubatietijd van gemiddeld zes weken, vergelijkbaar met ebv. ongeveer 5-10% van de klinische beelden van mononucleosis infectiosa wordt veroorzaakt door cmv. cmv-mononucleosis treedt doorgaans op wat latere leeftijd op dan ebv-mononucleosis. meestal blijven de symptomen beperkt tot twee à drie weken koorts. faryngitis en cervicale lymfadenopathie zijn minder gebruikelijk dan bij primaire ebv-infectie. het perifere bloedbeeld vertoont meestal een lymfocytose met atypische lymfocyten, en de leverfuncties kunnen gestoord zijn. volledig herstel treedt op na ongeveer zes weken. de paul-bunnell-reactie is kenmerkend negatief. zeldzame complicaties zijn hepatitis, pneumonie, aseptische meningitis en het syndroom van guillain-barré. ditzelfde ziektebeeld van koorts, leukopenie, atypische lymfocytose en splenomegalie kan optreden bij een cmv-seronegatieve ontvanger drie tot zes weken na bloedtransfusie met vers cmv-seropositief bloed. de kans op cmv-transmissie via bloedtransfusie (geschat op ongeveer 2,5% per unit getransfundeerd bloed) kan worden gereduceerd door gebruik van bloed van seronegatieve donoren, leukocytenarm bloed of bevroren bloed-of bloedproducten. ernstige ziektebeelden veroorzaakt door cmv treden vooral op bij intra-uteriene infectie (zie hoofdstuk 15) en patiënten met afweerstoornissen (zie hoofdstuk 17). bij orgaantransplantaties is cmv-ziekte een van de meest voorkomende complicaties. cmv-ziekte kan het gevolg zijn van reactivatie van het virus van de (seropositieve) ontvanger of infectie vanuit het donororgaan of door gedoneerd bloed. vanwege het beperkte aanbod van donororganen is het niet altijd mogelijk naast een goede hla-match ook de cmv-serostatus van donor en ontvanger op elkaar af te stemmen. cmv-infectie bij transplantatiepatiënten kan gepaard gaan met langdurige koorts, trombo-en/of leukopenie en gestoorde leverenzymen (hepatitis), spierpijn en ge-wrichtsklachten. daarnaast kunnen ernstige gastro-intestinale infecties optreden (oesofagitis, gastritis, colitis), die gepaard kunnen gaan met perforatie. cmvpneumonitis is vooral een levensbedreigende ziekte bij patiënten met een allogene beenmergtransplantatie. diverse vormen van cmv-ziekte kunnen optreden bij aidspatiënten, vooral bij een sterk gestoorde immuniteit (cd4+-cellen < 50/mm 3 ). naast pneumonitis, encefalitis en gastro-intestinale infecties is vooral retinitis een beruchte complicatie. de laatste manifestatie kan zich bilateraal voordoen en leidt bijna altijd tot blindheid, tenzij antivirale therapie wordt gegeven. incidenteel doet zich een ernstige cmv-colitis voor bij jonge volwassenen met een op het oog normale of hooguit licht gestoorde immuniteit (zoals bij zwangeren). diagnostiek de diagnose primaire cmv-infectie bij immuuncompetente gastheren wordt meestal gesteld aan de hand van het antistofpatroon. igm-antistoffen (in de vroege fase) en igg-antistoffen (levenslang) zijn aantoonbaar. een reactivatie van de cmv-infectie kan gepaard gaan met de hernieuwde vorming van igm-antistoffen maar dit is weinig betrouwbaar. een foutpositieve cmv-igm-test kan soms optreden bij een primaire ebv-infectie. het betreft hier antistoffen gegenereerd als gevolg van de tijdens de ebv-infectie optredende polyklonale b-celactivatie, of mogelijk antistoffen die gemeenschappelijke antigene determinanten van ebv en cmv herkennen. heterofiele antistoffen komen bij cmv-infectie echter niet voor. naast serologisch onderzoek bestaat de mogelijkheid om cmv als virus aan te tonen, door middel van kweek, door antigeendetectie of tegenwoordig vooral door cmv-dna-detectie. dit is vooral van groot belang bij de infecties van immuungecompromitteerde gastheren. cmv is in die gevallen als typisch systemische infectie aantoonbaar op vele plaatsen, zoals in de keel, in urine, in bronchiaal spoelsel en in leukocyten. ook bij congenitaal geïnfecteerde kinderen vindt soms nog jarenlang sterke cmv-uitscheiding in de urine plaats. positieve kweekresultaten wijzen niet noodzakelijkerwijs op een symptomatische cmv-infectie. asymptomatische uitscheiding van cmv komt regelmatig voor, in het bijzonder bij patiënten met een verminderde afweer. een goede indruk van de relevantie van een cmv-infectie is in die gevallen te verkrijgen door het meten van de hoeveelheid viraal dna in bloed of plasma. de drie middelen met klinisch bewezen werkzaamheid, ganciclovir, foscarnet en cidofovir, worden gekenmerkt door een hoge frequentie van toxische neveneffecten. ganciclovir is nauw verwant aan aciclovir, maar blijkt een veel effectievere remmer van de cmv-replicatie. dit is het meest toegepaste middel tegen cmv-infectie; het is ook oraal toepasbaar door middel van de prodrug valganciclovir. door het ter beschikking komen van snelle moleculaire tests voor de kwantitatieve bepaling van de hoeveelheid cmv in plasma wordt bij transplantatiepatiënten therapie vaak toegepast op geleide van de hoeveelheid cmv in het bloed. met deze strategie blijkt ernstige cmv-ziekte bij deze patiënten grotendeels te kunnen worden voorkomen (zie hoofdstuk 17). onderzoek naar preventie van cmv-ziekte door vaccinatie is gaande. immunisatie van ontvangers van niertransplantaten acht weken voor transplantatie met een verzwakte cmv-stam (towne) leidde niet tot een verminderde cmv-uitscheiding na transplantatie. de incidentie van cmv-ziekte was wel lager ten opzichte van de placebogroep en ook verliep de ziekte in die gevallen minder ernstig. naast onderzoek met dit type vaccin is ook onderzoek gaande met vaccins bestaande uit gezuiverd of recombinant gb, het voornaamste envelopglycoproteïne van cmv-virus. ook het voorkómen van schade door congenitale cmv-infectie is een belangrijk doel van vaccinatie. naar schatting 1% van de mononucleosis infectiosa-achtige ziektebeelden wordt veroorzaakt door toxoplasma gondii, een intracellulair protozoön. in hoofdstuk 18 zal nader op deze infectie worden ingegaan. casus 13.2 een 45-jarige vrouw wordt opgenomen in het ziekenhuis vanwege toenemende kortademigheid. tien jaar eerder werkte zij als reisleidster ruim acht maanden in tanzania, kort waarop zij een griepachtig beeld met lymfadenopathie ontwikkelde, dat toen werd geduid als mononucleosis infectiosa. twee jaar voor presentatie begon ze langzamerhand steeds meer last te krijgen van seborroïsch eczeem, genitale ulcera en eenmaal een ernstige gordelroos in het gelaat. de laatste maanden is ze 8 kg afgevallen. bij onderzoek blijkt er sprake van een verlaagde zuurstofspanning door een dubbelzijdige, interstitiële pneumonie. bij een longspoeling wordt pneumocystis in de alveolaire macrofagen herkend en wordt een hiv-antistoftest ingezet. deze blijkt positief. haar cd4-aantal blijkt 30/mm 3 . in 1981 werd in de verenigde staten een epidemie van longontstekingen met pneumocystis jirovecii beschreven, vaak gepaard gaand met een zeldzame vorm van huidkanker, het kaposi-sarcoom. al snel werd duidelijk dat een ernstige immuunstoornis aan deze verschijnselen ten grondslag lag en dat de slachtoffers allen in korte tijd overleden. aanvankelijk waren de slachtoffers jonge homoseksuele mannen, maar al snel werden vergelijkbare casus waargenomen bij heteroseksuele mensen uit haïti en bij ontvangers van bloedtransfusies. de aandoening werd aids genoemd (acquired immune deficiency syndrome) en gezien de epidemiologie vermoedde men al snel een infectieuze oorzaak. in 1983 isoleerden de latere nobelprijswinnaars (2008) barré-sinoussi en montagnier als eersten uit de lymfeklier van een franse aidspatiënt een nieuw retrovirus, dat uiteindelijk humaan immunodeficiëntievirus type 1 (hiv-1) werd genoemd. in 1986 werd een verwant virus, hiv-2, aangetoond bij aidspatiënten in west-afrika. inmiddels is duidelijk geworden dat beide virussen voorkomen bij primaten in afrika (als vormen van simian immunodeficiency virus, siv). men heeft aannemelijk kunnen maken dat de voorlopers van beide humane virussen in de jaren dertig (hiv-1) en veertig (hiv-2) van de vorige eeuw vanuit primaten bij de mens geïntroduceerd zijn op het afrikaanse continent. het begin van de epidemie in afrika is niet herkend, wel heeft men met terugwerkende kracht een geval van aids kunnen vaststellen bij een zeeman uit manchester in 1959, die vermoedelijk in afrika was geïnfecteerd. het virusdeeltje heeft een sferische vorm en is ongeveer 100 nm groot (figuur 13.3a). het virus heeft een envelop die bestaat uit de virale glycoproteïnen gp120 en gp41, en bestanddelen die afkomstig zijn van de gastheercelmembraan. deze envelop omgeeft de capside, met als voornaamste bestanddeel het virale p24-eiwit. binnen de capside bevinden zich twee identieke enkelstrengs rna-moleculen (het virale genoom) en een aantal moleculen van de enzymen reverse transcriptase, integrase en protease. het genoom is relatief klein (9 kb) en bevat negen genen, die in totaal coderen voor vijftien verschillende eiwitten. het genoom wordt aan weerszijden geflankeerd door een long terminal repeat (ltr), een niet-coderend deel van het genoom betrokken bij de regulatie van de virale expressie (figuur 13.3b). de genproducten zijn onder te verdelen in: 1 structurele proteïnen (o.a. de gag-genproducten (p24 en p17) en envelopglycoproteïnen (gp41 en gp120); 2 door het virus gecodeerde enzymen die nodig zijn voor de virale replicatiecyclus (reverse transcriptase, integrase en protease); 3 eiwitten die betrokken zijn bij de regulatie van de genexpressie (o.a. tat, rev en nef). hiv infecteert humane gastheercellen die op hun buitenmembraan een cd4+-receptormolecuul bezitten (figuur 13.4). deze cd4+-receptoren zijn uiteraard aanwezig op cd4+-positieve t-lymfocyten, maar in mindere mate ook op macrofagen en dendritische cellen. hiv gebruikt dit cd4+-molecuul als eerste receptor om de gastheercel te kunnen binden. door binding van het virale gp120 aan dit cd4+-molecuul ontstaat in de virusenvelop een conformatieverandering, waardoor het virus zich kan binden aan een tweede (co)receptor. deze tweede receptor komt uit de familie van de chemokinereceptoren, waarvan er twee belangrijk zijn voor hiv: ccr-5 en cxcr-4. de ccr-5-receptor bevindt zich vooral op macrofagen, dendritische cellen en cd4+-positieve t-lymfocyten, de cxcr-4-receptor komt vooral tot expressie op geactiveerde cd4+-positieve t-lymfocyten. op basis van hun coreceptorgebruik kan hiv worden onderverdeeld in varianten die vooral de ccr-5receptor gebruiken (r5-virussen, ook wel macrofagotrope virussen) en varianten die vooral de cxcr-4-receptor gebruiken (x4-virussen, ook wel lymfocytotrope virussen). er zijn ook varianten die beide receptoren kunnen gebruiken (duotrope virussen). het overgrote deel van de patiënten wordt geïnfecteerd met r5-trope virussen. gedurende de infectie kunnen virussen muteren met als gevolg dat bij ruim 50% van de geïnfecteerde individuen in de loop van de infectie virussen ontstaan die ook de cxcr-4-coreceptor kunnen gebruiken. na binding aan de coreceptor treedt een tweede conformatieverandering op en kan het envelopeiwit gp41 binden aan de gastheercelmembraan. binding van de virus-en celmembraan leidt tot fusie van beide membranen, waarop de nucleocapside het cytoplasma van de cel kan binnengaan. vervolgens worden met behulp van het enzym reverse transcriptase de twee enkelstrengs virale rna-kopieën omgezet in proviraal dubbelstrengs dna (figuur 13.4). dit dna migreert naar de kern en integreert in het gastheercel-dna met behulp van het reeds aanwezige virale integrase. het geïntegreerde dna noemen we een provirus. in feite is er nu sprake van een irreversibele ('ongeneeslijke') situatie: zo lang de geïnfecteerde gastheercel(populatie) overleeft, zal ook het geïntegreerde hiv-dna overleven. wanneer een provirus bevattende cd4+-cel geactiveerd wordt, wordt de transcriptiefactor nfkb geproduceerd. deze bindt aan de promotor van het provirus, waarop transcriptie wordt gestart. de virale eiwitten tat en rev die daarbij gemaakt worden, zorgen vervolgens voor een efficiënter verlopende transcriptie, waarop virale mrna's en de precursors van de structurele (glyco)proteïnen worden geproduceerd. de virale glycoproteïnen groeperen zich bij de celmembraan. het virale protease klieft het gag-precursoreiwit, waarop de eindproducten worden geproduceerd, onder meer de virale capside-eiwitten. samen met het genomisch rna assembleren deze eiwitten aan de binnenkant van de celmembraan tot nieuwe virionen, die zich door lokale uitstulping en afsnoering van de celmembraan (budding) buiten de cel begeven (zie figuur 13.4). men schat dat er bij een hiv-1-geïnfecteerde patiënt gemiddeld 10 8 -10 10 virusdeeltjes per dag worden geproduceerd, waarbij dagelijks 10 8 -10 9 cd4+-cellen worden geïnfecteerd. doordat het reverse transcriptase een slordig enzym is (1:10 4 nucleotiden wordt foutief gekopieerd), treden er mutaties op die leiden tot virusvarianten. men kan daardoor niet spreken van één virus maar van een viruspopulatie. afhankelijk van suppressieve factoren, zoals de gastheerimmuniteit tegen hiv of de aanwezigheid van medicatie, zullen vooral die virussen expanderen, die door hun mutatie(s) aan deze suppressieve druk kunnen ontsnappen (survival of the fittest). het kenmerk van een hiv-infectie is een geleidelijke afname van het aantal cd4+-positieve lymfocyten in het perifere bloed (figuur 13.5), waardoor uiteindelijk een functionele immuundeficiëntie ontstaat. aanvankelijk nam men aan dat de cd4+-daling een direct gevolg was van de celdood door virusreplicatie, dan wel door toegenomen geprogrammeerde celdood (apoptose), of door immuungemedieerde destructie. tegenwoordig neemt men aan dat de hiv-infectie een hyperactivatie van het gehele cd4+-compartiment veroorzaakt, waardoor de cd4+-cellen veel korter overleven en uiteindelijk onvoldoende kunnen worden aangevuld. aangezien de cd4+-lymfocyt een centrale rol speelt in de regulering van de immuunrespons, leidt het verlies van deze celpopulatie uiteindelijk tot deficiënties in die immuunrespons. vooral de cellulaire immuniteit is sterk afhankelijk van de aansturende rol van de cd4+-cel en is daarom als eerste gestoord. de cellulaire immuniteit controleert onder meer micro-organismen die in of op het lichaam latent aanwezig zijn, zoals schimmels, mycobacteria en herpesvirussen. wanneer de cellulaire immuniteit verminderd functioneert, kunnen deze micro-organismen tot ziekteverschijnselen leiden. men spreekt dan van opportunistische infecties (zie ook hoofdstuk 17). de humorale immuniteit ontwikkelt zich vooral in de lymfadenopathieën en hiv eerste levensjaren. de cd4+-cel speelt in die fase wel een belangrijke rol in deze ontwikkeling, maar als het humorale immuunrepertoire eenmaal gevormd is, wordt de rol van de cd4+-cel hierin minder belangrijk. een volwassen persoon met een laag cd4+-aantal kan gekapselde micro-organismen, zoals pneumokokken en stafylokokken, die vooral afhankelijk zijn van een humorale immuunrespons, daarom nog redelijk effectief bestrijden. jonge kinderen met hiv hebben daar duidelijk meer moeite mee door een nog onvoldoende gerijpt humoraal repertoire. vaccinatie, waarbij humorale immuniteit moet worden opgebouwd onder invloed van cd4+-cellen, is om diezelfde reden minder effectief bij hiv-geïnfecteerde patiënten met een laag cd4+-aantal. een hiv-infectie onderscheidt zich in het begin niet van andere virusinfecties waartegen het lichaam zowel een humorale als een cellulaire immuunrespons opbouwt. nadat het virus in de eerste weken vrij spel heeft gehad, waarbij grote hoeveelheden virus in het bloed kunnen worden aangetroffen (figuur 13.5), zorgen tegen hiv gerichte neutraliserende antistoffen en cytotoxische lymfocyten (ctl) voor controle over het virus, waarop de hoeveelheid celvrij virus in het plasma daalt. afhankelijk van de kracht van deze gastheerrespons en de mogelijkheid van het virus om hieraan te ontsnappen, ontstaat na een aantal weken een soort balans (set-point) waarbij hiv en het immuunsysteem elkaar in evenwicht houden. doordat het virus echter continu muteert, ont-staan virusvarianten die aan deze immuuncontrole ontsnappen, waarop een nieuwe immuunrespons tegen deze varianten de balans weer moet proberen te herstellen. doordat de daarvoor benodigde cd4+-cellen echter langzaam verdwijnen (en daardoor ook de ctlrespons tegen hiv vermindert), raakt de balans steeds meer verstoord en zal het virus deze strijd uiteindelijk winnen: de hoeveelheid virus in het plasma stijgt weer. in het algemeen geldt dat stijging van de hoeveelheid in het plasma (meer dan 100.000 viruskopieën per ml) gepaard gaat met een snellere cd4+-daling en dus een snellere ziekteprogressie. door de mutaties in de envelop kan het virus ook zijn tropisme veranderen: naast macrofagotrope virussen (r5) ontstaan duotrope (r5/x4) virussen, met als gevolg dat het virus in meer cellen kan repliceren. hierdoor stijgt de hoeveelheid virus snel en zal het aantal cd4+-cellen in korte tijd sterker dalen, wat gepaard gaat met snelle ziekteprogressie. het beloop van de hiv-infectie kent grote interindividuele verschillen: de duur van de asymptomatische eerste fase kan variëren van twee tot meer dan vijftien jaar. de genetische achtergrond, zoals de hla-typering van de geïnfecteerde persoon en ook andere genetische polymorfismen binnen het immuunsysteem, draagt daar belangrijk aan bij. zo zijn er individuen die een homozygote deletie hebben voor de ccr-5-receptor, waardoor infectie met r5-virussen niet kan plaatsvinden. bij personen met de heterozygote ccr-5-deletie kan een r5-virus zich minder gemakkelijk verspreiden en verloopt de ziekteprogressie trager. de kliniek rondom hiv-infectie is grofweg in drie fasen te verdelen: de acute hiv-infectie in de eerste drie maanden, daarna een relatief asymptomatisch verlopende fase, die twee tot meer dan vijftien jaar kan duren, en tot slot een symptomatische fase, die snel progressief kan verlopen en eindigt in aids. nadat iemand met hiv geïnfecteerd is geraakt, ontstaan in de acute fase klinische symptomen bij 80-90% van de personen, waarbij 50-60% medische hulp zoekt. de symptomen beginnen meestal twee tot vier weken na de infectie en lijken sterk op een griepbeeld of op mononucleosis infectiosa, dat eerder werd besproken bij de acute ebv-infectie. koorts treedt vaak op (> 90%), gepaard gaand met faryngitis (75%), lymfadenopathie (50-75%), spier-en gewrichtspijn (50-90%) en moeheid (80-90%). regelmatig worden bovenstaande verschijnselen begeleid door een uitgebreide maculopapulaire huiduitslag (30-70%), gewichtsverlies (50-70%), hoofdpijn (40%), darmklachten met diarree en/of braken (30-50%) en ulcera in mond, rectum en genitalia. bij bloedonderzoek ziet men vaak atypische lymfocytose (> 80%), leuko-en trombopenie (35-45%) en milde hepatitis (20%). meestal houden de klachten en afwijkingen niet langer dan twee weken aan. in deze fase van de acute hiv-infectie komt de immuunrespons tegen hiv op gang: er ontstaan antistoffen tegen alle hiv-eiwitten (seroconversie) en er ontstaat ook een sterke ctl-respons. initieel is er veel virus in het plasma aanwezig (hiv-rna viral load) en zullen de hiv-tests (die berusten op het aantonen van anti-hiv-antistoftests) nog negatief zijn; men spreekt van de window-fase. seroconversie treedt bij de meeste patiënten binnen acht tot tien weken op, zodat deze window-fase slechts enkele weken duurt. tijdens de acute hiv-infectie daalt het aantal cd4+-cellen in het bloed, wat zich meestal grotendeels weer herstelt. de daling kan soms zo diep zijn, dat ernstige opportunistische infecties kunnen optreden. de grootste daling van cd4+-cellen in de acute fase vindt echter plaats in het darmgeassocieerde lymfoïde weefsel (galt, gut associated lympoïd tissue). in dit compartiment vindt nauwelijks herstel van het aantal cd4+-cellen plaats. met het op gang komen van een krachtige immuunrespons daalt de hoeveelheid hiv-rna in het plasma en herstelt het aantal cd4+-cellen in het perifere bloed zich weer redelijk (figuur 13.5). na de acute fase volgt de al genoemde asymptomatische fase, waarbij virus en gastheer elkaar in evenwicht lijken te houden rond een soort set-point. algemene klachten zoals moeheid, nachtzweten en lymfadenopathie worden in wisselende mate gezien, evenals huidklachten zoals folliculitis en seborroïsch eczeem. bepaalde opportunistische infecties zoals herpes zoster en herpes simplex treden vaker op, evenals sinusitis en pneumokokkenpneumonieën, maar zo lang het aantal cd4+-lymfocyten hoger is dan 200/mm 3 zijn deze beelden mild en van voorbijgaande aard. zodra het cd4+-aantal duidelijk onder deze grens komt, worden de opportunistische infecties ernstiger. infecties door candida albicans van mondholte (stomatitis) en slokdarm (oesofagitis), longontstekingen met pneumocystis jirovecii (vroeger pneumocystis carinii genoemd) en diarree met giardia lamblia, cryptosporidium, isospora en microsporidia nemen in frequentie toe, naarmate het cd4+-gehalte lager wordt. bij cd4+-waarden onder de 100/mm 3 zorgen toxoplasmose (cerebraal abces), cryptococcus neoformans (meningitis, maar ook gedissemineerd), cmv (colitis en retinitis) en atypische mycobacteria (bacteriëmie, darmwand en beenmerg) voor ernstig verlopende en dodelijke infecties. los van deze cd4+-grenzen worden als belangrijke opportunistische problemen gewone tuberculose maar ook kwaadaardige aandoeningen als non-hodgkinlymfomen en kaposisarcomen gezien. wanneer een hiv-geïnfecteerde zich presenteert met een van deze opportunistische problemen, spreekt men per definitie van aids. onbehandeld is de gemiddelde overleving van een aidspatiënt meestal niet langer dan 18 maanden, afhankelijk van de ernst van de ziekte waarop de diagnose aids wordt gesteld. de belangrijkste aids-indicatordiagnosen zijn: -pneumocystis jirovecii-pneumonie (voorheen: pneumocystis jirovecii); -oesofageale candidiasis; -cerebrale toxoplasmose; -cryptosporidiose met > 1 maand diarree; -extrapulmonaire cryptokokkose; -cmv-ziekte van tractus digestivus, retinitis, pneumonitis, encefalitis; -hsv mucocutane ulcera > 1 maand, pneumonitis, oesofagitis; -pulmonale of gedissemineerde tuberculose; -gedissemineerde infectie met mycobacterium avium intracellulare of m. kansasii; hiv-wasting: > 10% gewichtsverlies en diarree, of langdurige febris e.c.i. en chronische malaise; -hiv-geassocieerde dementie; -kaposi-sarcoom; -non-hodgkin-of b-cellymfoom. sinds de eerste hiv-patiënten werden beschreven, is er al direct veel ontwikkeling geweest in het zoeken naar behandelingen. in eerste instantie bestond dit uit het steeds beter behandelen en voorkomen van opportunistische infecties. zo werd al snel duidelijk dat een van de beruchtste opportunistische infecties, de pneumocystis jirovecii-pneumonie (zie ook hoofdstuk 17) kon worden voorkomen als patiënten een profylaxe met co-trimoxa-zol ontvingen zodra hun aantal cd4+-cellen lager werd dan 200/mm 3 . vanaf 1987 werd het mogelijk de hiv-infectie zelf te behandelen met een middel dat reverse transcriptase remde: azidothymidine (azt). dit middel is een gemodificeerd nucleoside (thymidine), dat wanneer het in de dna-keten wordt ingebouwd verdere dna-verlenging (en dus uiteindelijk virusreplicatie) onmogelijk maakt. in eerste trials met dit middel was een duidelijk klinische verbetering aantoonbaar, maar het overlevingsvoordeel bleek van korte duur. nieuwe, vergelijkbare medicijnen, alle nrti's (nucleoside-analogue reverse transcriptase inhibitors) genoemd, kwamen op de markt, maar ook zij bleken in monotherapie geen aanwinst. duotherapie was effectiever, maar de echte doorbraak kwam pas met het gebruik van tripelcombinatietherapie. al snel werd het arsenaal medicijnen uitgebreid met een groep medicijnen die reverse transcriptase op een andere manier remmen (nnrti's, non-nucleoside reverse transcriptase inhibitors), maar ook met nieuwe nrti's en proteaseremmers. ook werd toen duidelijk waarom monoen duo-nrti-therapie niet werkten: deze middelen konden de virusreplicatie niet geheel remmen, omdat een of twee mutaties in het enzym reverse transcriptase al tot antivirale resistentie konden leiden. virussen met een tot twee mutaties waren al aanwezig in de viruspopulatie van onbehandelde mensen, met als gevolg dat deze mutanten werden uitgeselecteerd en konden repliceren. tripelcombinatietherapie (een proteaseremmer of een nnrti gecombineerd met twee nrti's) bleek wel tot volledige onderdrukking van de virale replicatie te leiden. hiervoor wordt het acroniem haart gebruikt (highly active anti-retroviral therapy). in enkele weken tijd kan de hoeveelheid hiv-rna in plasma tot onmeetbaar lage waarden worden teruggebracht, gevolgd door een snelle stijging van het aantal cd4+-cellen in het perifere bloed, deels als gevolg van reallocatie van deze cellen vanuit de lymfeklier, deels als gevolg van remming van virusgeïnduceerde celdood. met het stijgen van het cd4+-celaantal verdwijnen de ziekteverschijnselen snel en blijken de cd4+-waarden vervolgens geleidelijk te kunnen terugkeren tot relatief normale waarden, mits de virussuppressie voortdurend wordt gehandhaafd. belangrijk hierbij is dat haart dagelijks en goed geslikt wordt. met de eerstegeneratie-tripeltherapieën viel dit niet mee, aangezien zij bestonden uit grote aantallen pillen die meerdere keren per dag genomen moesten worden en bovendien frequent gepaard gingen met bijwerkingen als diarree, bloedarmoede en neuropathie. desalniettemin daalde in korte tijd de sterfte aan aids. ook bleek de virusoverdracht van moeder naar kind te kunnen worden geblokkeerd, door de hiv-positieve moeder tijdens het laatste deel van de zwangerschap een combinatiebehandeling te geven. na 2003 werden nog nieuwe klassen hiv-remmers geïntroduceerd: een remmer van de fusie van gp41 met de celmembraan (2003), een middel dat bindt aan ccr5-coreceptor (2008 ccr5-coreceptor ( ) en een integraseremmer (2008 , zodat het totale aantal geregistreerde antiretrovirale middelen in 2011 al ruim twintig telt. een bespreking van de aangrijpingspunten van antivirale therapie is te vinden in hoofdstuk 1 (paragraaf 1.7.2). meerdere combinaties van vrijwel steeds drie middelen kunnen op die manier worden samengesteld, waarbij ook resistente virussen goed kunnen worden geremd. behalve dit betere antivirale effect, maakten de nieuwere medicijnen combinaties mogelijk met een aanmerkelijk minder aantal pillen en ook met veel minder bijwerkingen. vooral deze laatste twee eigenschappen maakten dat patiënten daadwerkelijk hun medicatie goed konden blijven innemen. dankzij de haart lijkt de levensverwachting van hiv-geïnfecteerde personen inmiddels vergelijkbaar aan die van niet hiv-geïnfecteerden. langetermijnbijwerkingen, bijkomende ziekten (zoals chronische hepatitis b en c), maar ook ouderdomsverschijnselen spelen daarom een steeds grotere rol in de hiv-zorg anno 2011. met haart wordt gestart als het risico op opportunistische infecties begint te stijgen. aanvankelijk stelde men die grens onder een cd4+-aantal van 300/mm 3 , mede in overweging nemend dat de eerste combinaties ook veel bijwerkingen hadden. met de nieuwere combinaties is een vroegere start mogelijk en schuift de startgrens daarom op naar een hoger cd4+-getal (400/mm 3 anno 2010). haart is kostbaar en het bovenbeschreven succes kon aanvankelijk vooral worden bereikt in de rijkere landen. na 2000 is haart echter met vereende krachten ook geïntroduceerd in landen met de grootste patiëntenconcentraties. eind 2009 ontvingen zo ruim 5 miljoen patiënten over de hele wereld haart, weliswaar tegen een geschat totaal patiëntenaantal van 33 miljoen (2008). de bovenstaande beschrijving van het beloop en de behandeling van hiv-infectie betreft vooral hiv-1. wereldwijd is bij ongeveer 5% van de hiv-geïnfecteerde patiënten echter sprake van infectie met hiv-2. de prevalentie van dit virus is het hoogst in west-afrika, waar dit virus vermoedelijk rond 1940 door een meerkattensoort (zwarte mangabey) bij de mens werd geïntroduceerd. het ziektebeeld van hiv-2 verschilt niet van hiv-1, maar het beloop van een hiv-2-infectie is doorgaans veel milder en verloopt in een trager tempo. de hoeveelheid hiv-2-rna in plasma is veel lager dan bij hiv-1. dit draagt er waarschijnlijk toe bij, dat hiv-2 minder gemakkelijk kan worden overgedragen via seksueel contact of van moeder op kind. de diagnostiek van hiv-1 en hiv-2 verschilt enigszins: in de standaard hiv-serologie (elisa) zullen hiv-1-en hiv-2-infecties niet van elkaar onderscheiden kunnen worden. dit is pas mogelijk bij de zogeheten bevestigingstest (western-blot), waarbij specifieke antistoffen te-gen hiv-2-eiwitten apart worden gemeten. de pcr-test, die gebruikt wordt voor het meten van hiv-1-rna in plasma, detecteert hiv-2-rna niet. hierop dient men bedacht te zijn omdat een specifieke hiv-2-rna-test moet worden aangevraagd. ook therapeutisch zijn er verschillen tussen hiv-1 en hiv-2: non-nucleoside reverse transcriptase inhibitors (nnrti's) en fusieremmers zijn niet werkzaam tegen hiv-2 vanwege een andere structuur van het reverse transcriptase en gp41. het aantal behandelingsmogelijkheden tegen hiv-2 is daardoor beperkter. hiv kan worden overgedragen via seksueel contact, bloed-bloedcontact (zoals transfusie van bloedproducten en het gebruik van verontreinigde naalden) en door verticale transmissie (van moeder op kind). het risico op seksuele overdracht wordt vergroot wanneer er sprake is van slijmvliesbeschadigingen, zoals bij geslachtsziekten of bij agressieve c.q. traumatische seks (verkrachting), maar is ook afhankelijk van de seksuele techniek. zo is de transmissiekans groter bij anale seks ten opzichte van vaginale seks en ook zal de ontvangende partner eerder een infectie oplopen (tabel 13.2). de kans op overdracht wordt mede bepaald door de hoeveelheid aanwezig infectieus virus. bij een hoge virale load in het plasma, zoals vooral het geval is tijdens de acute hiv-infectie, is de transmissiekans veel groter dan bij een lage virale load. effectieve haart reduceert het transmissierisico tot bijna nihil. transmissie via bloed-bloedcontact verloopt efficiënter dan seksuele overdracht, maar is afhankelijk van het inoculum en ook weer van de hoeveelheid aanwezig virus. mondiaal gaat het hierbij vooral om hergebruik van injectienaalden bij intraveneus drugsgebruik, maar er zijn ook diverse voorbeelden van hergebruik van naalden en infuussystemen in medische setting (libië, china, roemenië), dan wel infusie van met hiv besmette bloedproducten. ook in nederland zijn vóór 1985 tientallen hemofiliepatiënten op deze manier met hiv geïnfecteerd, doordat er nog geen hiv-test beschikbaar was. het risico op verticale transmissie van hiv van moeder naar kind is 15-25% wanneer geen borstvoeding wordt geven, maar loopt op tot 40% als dat laatste wel gebeurt. de meeste kinderen raken geïnfecteerd in de laatste fase van de zwangerschap, vooral tijdens de partus. ook hier speelt de hoogte van de virale load weer een belangrijke rol. door alle zwangere vrouwen vroeg op hiv te testen en ten minste in het derde trimester van de zwangerschap met haart te behandelen (zie verder), is in de westerse wereld het risico op verticale transmissie tot minder dan 0,1% teruggedrongen. de incidentie van hiv-1 in de verschillende risicogroepen is sterk afhankelijk van de geografische lokalisatie en transmissieroute. in centraal-afrika, waar het virus eind jaren zeventig al wijdverbreid was, is heteroseksuele overdracht het frequentst en wordt het virus veel aangetroffen bij jonge mannen en vooral vrouwen. in sommige afrikaanse landen blijkt meer dan 30% van de zwangere vrouwen geïnfecteerd. ook infectie van pasgeborenen door verticale transmissie komt in afrika veel voor. in de verenigde staten, europa en australië komt een hiv-1-infectie vooral voor bij mannen die seks hebben met mannen (msm), ook al neemt het aantal heteroseksuele overdrachten geleidelijk toe. in oost-europa en veel aziatische landen, waaronder china en indonesië, is een snelle uitbreiding van de epidemie gaande, vooral als gevolg van intraveneus drugsgebruik. eind 2008 bedroeg het geschatte aantal in leven zijnde hiv-geïnfecteerden wereldwijd ongeveer 33 miljoen, waarvan het overgrote deel woonachtig was in afrika bezuiden de sahara. in nederland was eind 2010 het aantal in leven zijnde hiv-geïnfecteerden in de landelijke registratie 14.000, maar wordt het werkelijke aantal geschat op ruim 24.000. de belangrijkste transmissieroute bij de nederlandse patiënten is msm-contact (57,2 %), gevolgd door heteroseksueel contact (32%), (ex-)intraveneus drugsgebruik (3,2 %) en bloedproducten (1,5%). hoewel er direct na de ontdekking van hiv optimisme ontstond over het ontwikkelen van een beschermend vaccin, is hier tot op heden nog steeds geen zicht op. kandidaatvaccins bleken wel in staat tot het opwekken van hiv-specifieke immuniteit, maar dit bleek geenszins beschermend. toen uit een veelbelovende studie in 2008 bleek dat er wellicht meer hiv-infecties voorkwamen in de groep met het beoogde vaccin dan in de placebogroep, werden de verwachtingen nog verder getemperd, tabel 13.2 gemiddeld risico op hiv-1-transmissie bij eenmalig contact met een hiv-1-positieve, onbehandelde bron. al bleek er bij een intensieve gecombineerde vaccinatietrial in 2009 voor het eerst sprake van een zwak beschermend effect. goede voorlichting en vermijding van risicogedrag (veilige seks, schone naalden voor intraveneuze drugsgebruikers) blijven daarom vooralsnog de belangrijkste wapens in de strijd tegen de verspreiding van de hivepidemie. seksuele transmissie kan vooral voorkomen worden door goed en consequent gebruik van mannencondooms en ook mannenbesnijdenis blijkt de kans op transmissie te verlagen. geen van deze methoden biedt echter honderd procent bescherming. nieuwe methoden, zoals virucide vaginale crèmes en vrouwencondooms, bleken nog niet effectief. effectieve haart verlaagt ook het risico op seksuele overdracht, al maakt het daarmee condoomgebruik niet overbodig. transmissie via bloed-bloedcontact kan vermeden worden door goede screening van bloedproducten op hiv en het verstrekken van schone naalden aan drugsverslaafden. verticale transmissie kan worden voorkomen door het toedienen van antiretrovirale middelen aan de zwangere vrouw, waarbij in westerse landen ook de pasgeborene nog vier weken antiretrovirale therapie krijgt toegediend als een soort postexpositieprofylaxe (pep). in westerse landen worden daardoor weinig hiv-positieve kinderen meer geboren. in derdewereldlanden is een dergelijke benadering vaak niet mogelijk, maar lukt het dikwijls toch de transmissie terug te dringen door kort voor de bevalling een eenmalige dosis nnrti (nevirapine) toe te dienen. het aantal hiv-geïnfecteerde kinderen loopt hierdoor wel terug, maar de keerzijde van deze korte monotherapie zijn resistentieontwikkeling bij de moeders en de toch hiv-positief geboren kinderen. de resistentieprevalentie tegen nevirapine kan daarbij oplopen tot 60%. de genoemde postexpositieprofylaxe kan ook worden gegeven aan personen die door een incident (prikaccident, onbeschermd seksueel contact) aan hiv zijn geëxposeerd en mogelijk geïnfecteerd. hoewel hiervoor vooral dierexperimenteel bewijs bestaat, lijkt pep effectief als het zo snel mogelijk na het incident wordt toegediend. kernpunten -infecties met ebv, cmv, hiv-1 en t. gondii behoren tot de frequentste oorzaken van gegeneraliseerde lymfadenopathie. -een primaire infectie met ebv of cmv verloopt vaak asymptomatisch (kinderleeftijd) maar leidt op oudere leeftijd vaak tot een klinisch beeld met faryngitis, koorts, lymfadenopathie en atypische lymfocytose (mononucleosis infectiosa). -mononucleosis infectiosa kan ook gezien worden bij primaire infecties met toxoplasma gondii en hiv. -ebv en cmv zijn humane herpesvirussen die persisteren in latente vorm. vooral bij immuungecompromitteerde patiënten kunnen herpesvirussen ernstige systemische infecties of lymfoproliferatieve syndromen (ebv) veroorzaken. -een acute of doorgemaakte infectie met ebv of cmv is aan te tonen met serologisch onderzoek. moleculaire diagnostiek kan worden gebruikt om de hoeveelheid virusreplicatie te monitoren. -hiv is een retrovirus dat via seksueel of bloed-bloedcontact wordt overgedragen en een persisterende infectie veroorzaakt. ook perinatale transmissie komt voor. na de primo-infectie ontstaat een klinische latentieperiode die kan variëren van maanden tot jaren. -hiv-infectie leidt op den duur tot cd4+-t-celdepletie en een ernstige cellulaire immunodeficiëntie met als gevolg levensbedreigende opportunistische infecties en maligniteiten. -hiv-infectie kan worden aangetoond met serologisch onderzoek. de activiteit van de infectie kan worden vervolgd door seriële bepaling van het aantal cd4+-cellen en de hoeveelheid hiv in plasma. -hiv-infectie kan worden behandeld met een combinatie van antiretrovirale middelen (haart). haart leidt tot volledige virusonderdrukking met daarbij uiteindelijk goed herstel van de cellulaire immuniteit. -met behulp van levenslange antiretrovirale therapie kunnen patiënten een normaal leven leiden, met waarschijnlijk een bijna normale levensverwachting. hiv immunopathogenesis and strategies for intervention infectious diseases 3rd infectious mononucleosis principles and practice of infectious diseases clinical virology key: cord-290976-dhwlr2ui authors: lednicky, john a; waltzek, thomas b; mcgeehan, elizabeth; loeb, julia c; hamilton, sara b; luetke, maya c title: isolation and genetic characterization of human coronavirus nl63 in primary human renal proximal tubular epithelial cells obtained from a commercial supplier, and confirmation of its replication in two different types of human primary kidney cells date: 2013-06-27 journal: virol j doi: 10.1186/1743-422x-10-213 sha: doc_id: 290976 cord_uid: dhwlr2ui background: cryopreserved primary human renal proximal tubule epithelial cells (rptec) were obtained from a commercial supplier for studies of simian virus 40 (sv40). within twelve hrs after cell cultures were initiated, cytoplasmic vacuoles appeared in many of the rptec. the rptec henceforth deteriorated rapidly. since sv40 induces the formation of cytoplasmic vacuoles, this batch of rptec was rejected for the sv40 study. nevertheless, we sought the likely cause(s) of the deterioration of the rptec as part of our technology development efforts. methods: adventitious viruses in the rptec were isolated and/or detected and identified by isolation in various indicator cell lines, observation of cytopathology, an immunoflurorescence assay, electron microscopy, pcr, and sequencing. results: cytomegalovirus (cmv) was detected in some rptec by cytology, an immunofluorescence assay, and pcr. human herpesvirus 6b was detected by pcr of dna extracted from the rptec, but was not isolated. human coronavirus nl63 was isolated and identified by rt-pcr and sequencing, and its replication in a fresh batch of rptec and another type of primary human kidney cells was confirmed. conclusions: at least 3 different adventitious viruses were present in the batch of contaminated rptec. whereas we are unable to determine whether the original rptec were pre-infected prior to their separation from other kidney cells, or had gotten contaminated with hcov-nl63 from an ill laboratory worker during their preparation for commercial sale, our findings are a reminder that human-derived biologicals should always be considered as potential sources of infectious agents. importantly, hcov-nl63 replicates to high titers in some primary human kidney cells. cell lines and primary cells obtained from commercial suppliers or through inter-laboratory transfer can contain adventitious (i.e., contaminating) viruses. this happens primarily because cytopathic effects (cpe) are not always apparent in virus-infected cell cultures, and consequently, the cells are unwittingly sold or transferred between laboratories [1] . the adventitious viruses that are encountered in cell cultures often stem from bovine serum that is used to supplement cell growth media, and include: bovine viral diarrhea virus (bvdv) [1] [2] [3] [4] [5] [6] , bovine polyomavirus [1, 7, 8] , bovine parvovirus [1, [9] [10] [11] (j. lednicky, unpublished) , and bovine herpes viruses [1, [12] [13] [14] [15] . unintentional contamination of cultured cells by these serum-derived viruses has obvious consequences not only with regard to data generation, but also because it exerts a toll on time wasted in the performance of laboratory work, and the costs thereof. other common sources of contaminating viruses are: (a) laboratory workers, and (b) animal-sourced enzymes (such as porcine trypsin) and (c) other biologicals that are used for cell culture [1] . examples of viruses that stem from porcine trypsin that have recently been found as contaminants of many cell lines including those used for vaccine production are torque teno sus virus (ttsuv), a member of the family anelloviridae, and porcine circoviruses 1 and 2 (pcv1 and pcv2) [1, [16] [17] [18] [19] [20] . anelloviruses and circoviruses are relatively small viruses with single-stranded, circular dna genomes that replicate within the nuclei of infected cells. cpe due to the presence of anelloviruses have not been well described at present. finally, primary cells can contain endogenous retroviruses and other viruses. for example, primary monkey kidney cells, which are used for the detection of paramyxoviruses and picornaviruses in many american diagnostic microbiology laboratories, can contain endogenous simian viruses that are either latent in the kidneys, or cause persistent but inapparent kidney infections in their hosts [21] . the work described in this manuscript resulted from a previous study of sv40 transcription in primate cells (j. lednicky, unpublished) . sv40 is a polyomavirus that was once referred to as "vacuolating agent" or "simian vacuolating virus 40" because commonly studied sv40 strains induce the formation of cytoplasmic vacuoles late during infection of most permissive primate cells [22] . a batch of primary human rptec that had been obtained for our previous transcription study of well-known vacuolating strains of sv40 proved unsuitable, as about 60% of the cells exhibited cytoplasmic vacuolation within 12 hours after they were seeded in flasks. necrosis and apoptosis were also evident in some of the attached cells. due to vacuolation and obvious cell deterioration, the rptec were rejected for our sv40 study. nevertheless, as we often work with primary cells and continuously refine our research methodologies, we sought to determine a likely root cause(s) of the deterioration of the rptec to (a) advance our understanding of primary cell culture technology, and (b) explore whether proper biosafety practices were being observed. for example, might the rptec be contaminated with a significant pathogen best suited for work in biosafety level-3 or −4 laboratories? we first tested whether vacuolation of the rptec stemmed from faulty media preparation. for example, vacuoles can form in madin darby canine kidney (mdck) cells due to: (a) shortage of l-glutamine in the cell growth medium, (b) inappropriate addition of antifungal agents to the medium, (c) improper co 2 environment for the sodium bicarbonate concentration of the medium, (d) nutrient depletion of the medium, and (e) mycoplasma contamination [23] . faulty media formulation was ruled out as the root cause of the failure of this batch of rptec to thrive. instead, based on the progressive formation of cpe, the results of our initial diagnostic tests, and our cumulative experience with cell culture [1] , we predicted that adventitious agents were causing the rapid demise of our rptec cultures. dna extracted from the rptec tested negative by pcr for mycoplasma species, and polyomaviruses sv40 and bk virus (bkv), suggesting none of these was causing vacuolation and/or cell deterioration. however, a single cause of the rptec deterioration was unlikely, as we detected 3 different human viruses in the rptec: human cytomegalovirus (cmv), human coronavirus nl63 (hcov-nl63), and human herpesvirus 6b (hhv-6b). cmv, also known as human herpesvirus-5 (hhv-5), (subfamily betaherpesvirinae), is a double-stranded dna virus that establishes lifelong persistence; it can remain latent in different human tissues and is known to infect renal tubular epithelial cells. a majority of humans are seropositive for cmv [24, 25] . whereas cmv infections are typically asymptomatic in healthy humans, the virus can reactivate and cause disease in immunosuppressed patients, including those undergoing kidney transplantation. indeed, cmv antigens and dna are found in renal epithelial cells in kidneys of trauma victims examined during autopsy as well as in biopsies of renal allografts, indicating that these cells can harbor cmv in both healthy persons and allograft recipients [26, 27] . hcov-nl63 is a single-stranded positive-sense rna virus of the genus alphacoronavirus (family coronaviridae, order nidovirales). first identified in 2003 from a child with bronchiolitis in the netherlands, it is now recognized that hcov-nl63 can cause upper and lower respiratory tract infections in humans, primarily in infants and the elderly [28] [29] [30] [31] [32] [33] . wild-type hcov-nl63 is difficult or impossible to isolate from clinical specimens in continuous cell lines [34] , although the prototype hcov-nl63 strain was propagated in llc-mk2 cells [33] and in primary, differentiated human bronchial-tracheal respiratory epithelial cells cultured at the air-liquid interface [35] . there are at least three different hcov-nl63 genotypes (a, b, and c) [34] . hhv-6b is a double stranded dna virus (subfamily betaherpesvirinae, genus roseolovirus) that infects up to 100% of humans and is the causative agent of exanthem subitum, which is also known as roseola infantum or sixth disease [36] . after the primary infection, hhv-6b generally persists in latent form in t-lymphocytes and other cells. hhv-6b reactivation is common in transplant recipients, which can cause several clinical manifestations such as encephalitis, bone marrow suppression and pneumonitis [37] . the work presented herein serves as a reminder that biologicals (such as calf serum and cultured cells) can be contaminated with adventitious agents. the focus of this article is on the detection and isolation of hcov-nl63, which to our knowledge, heretofore has not been reported in a natural infection of human kidney cells, or tested in vitro in primary human rptec. within 12 hrs after cryopreserved rptec were thawed and seeded in cell culture flasks, we observed that about 60% of the attached cells were vacuolated. since vacuolation may have been a sign of cytotoxicity due to residual cryopreservative, the rptec basal growth medium [basal growth medium (bgm)], which had been supplied with the cells, was changed. we noted by phase-contrast microscopy that prominent intranuclear inclusions surrounded by a clear halo ("owl-eyes") were present in enlarged nuclei in some of the rptec, and that the same cells were enlarged relative to a majority of the others. these findings were considered pathognomonic for cytomegalovirus (cmv) [38] (table 1) . vacuoles were still present 24 hrs post-seeding of the rptec (and after the rgm change at 12 hrs) ( figure 1a ), but there were no signs of contamination by extracellular bacteria or fungi. the ph at 37°c of fresh bgm was approximately 7.36 (within normal range), and ammonia was not detected using a salicylate-based method (data not shown). these findings suggested neither incorrect ph nor presence of ammonia in bgm were causing vacuolation of the rptec. moreover, cv-1, llc-mk2, and vero cells, which are cell lines derived from monkey kidneys, did not get vacuolated after 24 hrs incubation with bgm. thus, no evidence of cytotoxicity due to bgm was uncovered. by 36 hrs post-seed, vacuoles were still present in rptec in bgm that had been boosted with additional l-glutamine, suggesting glutamine deficiency was not an issue. a bioactive agent release assay indicated something in the spent bgm of the 24 hr rptec cultures induced enlargement and/or vacuolation of wi-38 ( figure 1b and c), llc-mk2 (figures 2a and b) , vero e6 cells ( figure 2c ), and cv-1 and hek-293 cells (not shown) within 12 hrs. cell enlargement, rounding, and vacuolation were more notable in wi-38 cells than other cells (table 1 ). these observations suggested the rptec were releasing either a biomolecule(s) or virus(es) that adversely affected some of the cell lines. immunofluorescence assay (ifa) and pcr for cmv some rptec from 48 hr cultures were positive for cmv by ifa (their nuclei were fluorescent), and dna extracted and purified from the cells also tested positive for cmv by pcr (data not shown). however, the extracted dna was pcr negative for human herpes virus (hhv)-1 and hhv-2, and polyomaviruses sv40 and bkv ( table 1) . cpe consisting of cell swelling/rounding and/or vacuoles also occurred at 34°and 37°c in wi-38, cv-1, llc-mk2, and vero cells inoculated with spent media from 5-day old rptec cultures. as for the bioagent release assay, morphological aberrations were most notable in wi-38 cells. trypsin did not enhance cpe in llc-mk2, mdck, mdck-london, mv1 lu, or vero cells. the wi-38 cells (but not the other cells) died 2 days afterwards. however, starting day 5 post-inoculation (p.i.), occasional syncytia with 8 or more nuclei were noted in llc-mk2, cv-1, hek-293, mv1 lu, and vero cells, and smaller syncytia (with up to 8 nuclei) in mdck and mdck-london cells (table 1) . thereafter, cpe were most pronounced in llc-mk2 cells and in hek293 cells. in llc-mk2 cells, most of the early cpe consisted of vacuolation and the formation of foci of detached rounded cells, many forming elongated oblong clumps of rounded cells above the monolayer (referred to as "striations" in ref. [50] ). at later times, cytolysis of syncytia occurred. vacuolation in llc-mk2 cells appeared more pronounced at 37°than 34°c, and conversely, syncytia appeared larger at 34°than 37°c ( figures 3a-d) . rounding followed by eventual detachment from the growing surface occurred in infected hek-293 cells (not shown). in mdck cells, vacuoles were also more pronounced at 37°than 34°c. due to cell vacuolation and deterioration, electron micrographs of five day old rptec cultures were difficult to interpret. at low magnification, vacuoles and cell deterioration were obvious ( figure 4a ). occasional viral particles consistent in appearance and size with cmv at different stages of maturation were observed at higher magnifications (data not shown). in addition to nuclear inclusions, homogenous electron-opaque, dense cytoplasmic bodies were present. however, unlike the irregular-shaped cytoplasmic bodies we usually observe in cmv-infected cell cultures (j. lednicky, unpublished observations), these were distinctly circular, as described by smith and de harven for cmv in infected cells [39] ( figure 4b ). additionally, we also noted many virus-like particles (vlp) that were morphologically different from cmv; these vlp were present as free particles, within vacuoles, and in transport vesicles. a majority of the virus-like particles were spherical, and they collectively ranged from about 80 to 120 nm in diameter, and some seemed to have surface projections ( figure 4c ). somewhat different results were obtained when the indicator cells of table 1 were inoculated with freeze-thawed rptec lysate from 7-day old cultures instead of spent media from 5-day old rptec cultures. in contrast to previous findings, cpe were not observed in wi-38 cells at early times onto 30 days p.i. however, cpe were seen in llc-mk2 cells starting 4 days p.i., and in other cells at later times ( table 1) . as before, vacuolation was more pronounced at 37°than 34°c. since syncytia were observed, we focused pcr efforts on the detection of the viruses that we considered the most likely candidates: coronaviruses, human paramyxoviruses, and reoviruses (hhv-1 and −2 were already ruled out, section 3, above). we did not test for retroviruses, acknowledging that exogenous or endogenous retroviruses may have been causing syncytia in the cells. extracted nucleic acids were tested by pcr or rt-pcr using assays designed to detect known human coronaviruses [33, [40] [41] [42] , paramyxoviruses [43] [44] [45] , and reoviruses [46] . rt-pcr and sequencing showed one of the viruses in the cv-1, hek-293, llc-mk2, mdck, mdck-london, mv1 lu, and vero e6 cells was coronavirus hcov-nl63. an example of rt-pcr reactions performed with 2 primer sets specific for hcov-nl63 is shown in figure 5 . proof that hcov-nl63 was replicating in the llc-mk2 cells was obtained by electron microscopy ( figure 6a -e). characteristic features of hcov-nl63 replication in llc-mk2 cells [35, 47] were detected, such as the formation of double membrane and laminar structures, and inclusion bodies ( figure 6a ). packets of granular nucleocapsid material were also evident in infected cells (6b). virus particles at various stages of maturation were present in the cytoplasm (6c) and in the rer outside the nuclei (6d). free virus particles 80 -100 nm in diameter were present in spent media (6e). a counterstain was not used to easily visualize the viral spikes ("crown") surrounding the viruses in figure 6e . the complete consensus genomic sequence of hcov-nl63 was obtained for virus in llc-mk2 cells that had been incubated at 34°c. the virus, designated hcov-nl63 strain rptec/2004/1, has a genomic length of 27,553 bp, and the complete sequence has been deposited in genbank (accession no. jx504050). a dataset was prepared containing complete or nearly complete hcov-nl63 genomes in genbank. to construct a phylogram, the for more comprehensive analyses. pcr tests for herpesviruses that were not included in our previous assays (for hhv-3,-4,-6,-7, and −8) were performed on dna extracted from rptec. a 151-bp amplicon was generated using nested primers for hhv-6 [48] . identity was confirmed by sequencing (data not shown). biotypes of plaque-purified hcov-nl63/rptec/2004 compared to hcov-nl63/amsterdam-1 since it was likely that multiple viruses contributed to the observations described in table 1 , an attempt was made to plaque purify hcov-nl63 in caco-2 cells [49] at 37°c [29, 49] and in llc-mk2 cells at 32°c (32°to 34°c are considered optimal temperatures for the in-vitro cultivation of hcov-nl63 [33] [34] [35] 47, 50, 51] ). whereas hcov-nl63 replicates more effectively in caco-2 cells than llc-mk2 cells [49] , that information was not available and therefore caco-2 cells were not used in our initial studies (table 1) , which were performed in 2004. nine days p.i., llc-mk2 cells were stained with neutral red, individual plaques picked, and subjected to 1 more round of plaque purification [50] . similarly, foci of cpe were identified under an unstained agarose overlay in caco-2 cells 5 days p.i., picked, and subjected to 2 more rounds of plaque purification [49] . plaque-purified stocks resulting from llc-mk2 figure 5 rt-pcr detection of hcov-nl63 in llc-mk2 cells. lane m, 100 bp mw markers (new england biolabs); lane 1, hcov-nl63specific pcr product (314 bp) amplified by pcr primers n5-pcr1 and n3-pcr1 [33] ; lane 2, hcov-nl63-specific pcr product (237 bp) amplified by pcr primers repsz-1 and sz-3 [33] ; lane 3, non-infected llc-mk2 control tested using pcr primers n5-pcr1 and n3-pcr1; lane 4, non-infected llc-mk2 control tested using pcr primers repsz-1 and sz-3. table 1 were infected at a moi of 0.1 pfu/cell. hcov-nl63/rptec/ 2004 pp a -f) formed the same cpe described in table 1 that were observed for freeze-thawed rptec, though formation of cpe was delayed by at least 1 day. a few examples are depicted in figure 8a cells. cpe were least obvious in mdck and mv1 lu cells. with the exception of hek-293 cells, which were only tested at 37°c (below 35°c, these cells do not adhere well to the growing surface of a flask), cpe were first detected at 33°c. from spent media harvested from 7-day old cultures, viral titers were obtained for hcov-nl63/rptec/2004 pp isolates a -f and hcov-nl63-amsterdam-1 using plaque assays in caco-2 cells [49] . for each cell line that was tested (as listed in table 1 ), the viral titer was similar for each virus. representative results, obtained for hcov-nl63 /rptec/2004 pp isolate a ( figure 9a ), indicate the highest titer (3.2 × 10 5 pfu/ml) was attained when the virus was propagated in llc-mk2 cells. using a moi of 0.1 pfu/cell, we tested progeny virus production by hcov-nl63/rptec/2004 pp a and d in llc-mk2 cells. the virus yields over a 9-day infection period were determined by plaque assays in caco-2 cells. similar results were obtained for the 2 viruses; the results for rptec/2004 pp d are shown in figure 9b . newly acquired (in 2013) primary rptec, hre, and hrce cells did not release a detectable bioagent (data not shown). what may have been "owl's eye" nuclei were observed rarely only in hre cells. both hcov-nl63 /rptec/2004 and hcov-nl63/amsterdam-1 caused rapid formation of cpe in rptec ( figure 10 ) and hre cells (figure 11 ) infected at a moi of 0.1 with plaque purified hcov-nl63/rptec/2004 pp a or hcov-nl63/amsterdam-1. we noted that the rptec were not vacuolated when sub-confluent ( figure 10a ) yet became vacuolated once confluent ( figure 10b ), but otherwise stayed viable when re-fed every 2 days with rebm. extensive cpe consisting of rounding of the cells and cytolysis occurred by 3 dpi in rptec ( figure 10c -e) and 4 dpi in hre cells ( figure 11b -c). when 1 ml of spent rebm was obtained from rptec or hre cells 3 days after they had been infected with hcov-nl63 rptec/2004 pp a or hcov-nl63/amsterdam-1, and inoculated onto llc-mk2 cells in t25 flasks, cpe were extensive 3 days later ( figure 10f and figure 11d ). in contrast, 1 ml of spent media from non-infected (negative control) rptec and hre cells had no effect on llc-mk2 cells (data not shown). the presence of hcov-nl63 in the spent media of rptec and hre that had been inoculated with the viruses, and in the indicator llc-mk2 that had been inoculated with spent media from the virus-infected cells, was confirmed by rt-pcr (data not shown). in contrast, cpe were sparse in hrce cells 7 dpi with either hcov-nl63/rptec/ 2004 pp a or hcov-nl63/amsterdam-1 (data not shown). both hcov-nl63/rptec/2004 and hcov-nl63/ amsterdam-1 formed relatively high viral titers by 4 dpi in rptec and hre cells, but not in hrce cells ( figure 12 ). the viral titers exceeded those formed in llc-mk2 cells by about 2 orders of magnitudes (ie, by 2 logs). in contrast, viral titers for both virus strains remained low (10 3 pfu/ml) in hrce cells by 9 dpi (data not shown). the presence of cmv in the original batch of viruscontaminated rptec was not a surprise to us, as we have isolated cmv from frozen (−80°c) simian kidneys and from primary simian kidney cells (lednicky, unpublished) . we learned from the supplier that the donor of the virus-contaminated rptec of this study was seropositive for cmv. however, our batch of viruscontaminated rptec was not checked for the presence of cmv by the supplier (personal communication). as precedence for the presence of cmv in human kidney cells in vivo, it is known that reactivation of cmv in renal tubule epithelial cells can complicate kidney transplantation, leading to poor long-term graft function [52] . the apparent complete inactivation of cmv by the freeze-thaw procedure we used was unexpected, as the process does not always completely inactivate cmv [53] , but was nevertheless fortuitous, leading to observations resulting in the detection of hcov-nl63. then again, it may have inactivated other viruses in the rptec. to our knowledge, ours is the first description of hcov-nl63 in primary rptec. overall, our observations of hcov-nl63 growth in various cell lines appear consistent with literature reports. growth of the virus in llc-mk2 and vero cells is well known [29, 33, 54] . the ability of the virus to form cpe in mdck was previously described [54] . the lack of hcov-nl63 growth in human fibroblasts has been reported [54] . in particular, mrc-5 cells, did not support the replication of hcov-nl63 [54] , and those cells are used interchangeably with wi-38 cells in american diagnostic virology laboratories for the isolation of respiratory viruses and cmv (both cell lines are derived from human fetal lung cells). thus, it is not surprising that hcov-nl63 does not replicate in wi-38 cells. growth of hcov-nl63 at 37°c has been reported and should not be a surprise [29, 49] . that hcov-nl63 might induce vacuolation is not a surprise, as that is a common property of coronaviruses. it will be interesting to see if interaction with ganglioside gm1 is related to the vacuolation process, as reported for sv40 [22] . hcov-nl63 replicated in hek-293 cells, as does sars-cov [55, 56] . both sars-cov and hcov-nl63 can use angiotensin-converting enzyme 2 (ace2) as a viral receptor [57] , and ace2 is expressed in kidneys [58] , and may be reasons hcov-nl63 was present in our batch of rptec and could infect hek-293 cells. replication of sars-cov in mv1 lu cells was previously reported [59] , so perhaps it is not surprising that hcov-nl63 does as well, if the viruses share receptor specificity, and mv1 lu cells contain the cellular machinery necessary for the replication of these viruses. however, the origin of hek-293 is unclear, as the cells express neurofilament (nf) subunits nf-h, nf-l, nf-m, alphainternexin, and other proteins found in neurons [60] . thus, hek-293 may be of neuronal origin, and it will be interesting in the future to discern which neural and kidney cells support the replication of hcov-nl63. it is not clear why rapid cell swelling rounding, and vacuolation, followed by cell death, occurred in wi-38 cells. our current hypothesis is that cmv was latent in the kidney cells of the donor of the rptec, and that the virus was reactivated during the initial harvest of cells from the donor's kidney. we surmise that within our batch of rptec, that many of the cells had been inadvertently frozen when they were at an early stage of cmv infection. it is likely that the cells produced a large yield of cmv when they were brought out of cryopreservation, and that the high-titer cmv infected the permissive wi-38 at a high moi, and this resulted in rapid killing of those cells. since we were unprepared for such analyses, a quantitative enumeration of infectious cmv particles was not performed. we also suspect that cmv from the rptec had infected vero, llc-mk2, and cv-1 cells, but the infection was abortive [38] , unlike the situation in wi-38 cells, which are permissive for that virus. finding that the hcov-nl63 is similar to viruses from 2004 and 2005 is perhaps not surprising, as the rptec of this report were prepared from a donor and purchased (by us) that same year. to our knowledge, hcov-nl63 has not been reported in natural infections of human kidneys. the ability of hcov-nl63 to replicate to high titers in primary rptec and hre cells suggests that at least some human kidney cells are fully permissive for the virus. however, we are unable to resolve whether (a) the original batch of contaminated rptec were infected (naturally) with the virus prior to harvest, or (b) a worker with a respiratory infection accidentally contaminated the rptec during their initial preparation, or (c) the rptec were contaminated in our laboratory. we are unable to resolve the issue whether the cells were contaminated during preparation for many reasons, foremost being the company that sold the cells was merged with a different entity. it is unlikely that the rptec were infected in our laboratory, as we did not have hcov-nl63 in our laboratory in 2004, and acquired hcov-nl63 /amsterdam-1 only recently (sept. 2012) so that we could compare the biotype of hcov-nl63/rptec with that of amsterdam-1. moreover, our laboratory policy dictates that workers refrain from cell culture work when they have a respiratory tract infection. it is plausible (but we lack proof) that hcov-nl63 may have been latent in the donor's kidneys, a possibility consistent with the known biology of various coronaviruses that establish long-term but sub-clinical infections. noteworthy, sars-cov, which shares the same ace2 receptor as hcov-nl63, has been associated with kidney disease [61] [62] [63] [64] . sars-cov causes a systemic infection with viral shedding not only in respiratory secretions, but also in stool and urine [63, 65, 66] . perhaps hcov-nl63 is capable of causing systemic infections as well, though the severity is much less than that of sars-cov. a parallel to this notion is the finding that hcov-nl63 replicates to high titers in caco-2 cells [49] , which are derived from a human colon carcinoma. in april of 2012, a new coronavirus capable of causing severe acute respiratory infections of humans emerged in jordan. the same coronavirus was isolated in the summer of 2012 from a patient with acute pneumonia and renal failure in saudi arabia [67, 68] . the new virus has been fully sequenced, classified as a group c β-coronavirus [69] [70] [71] , and termed middle east respiratory syndrome coronavirus (mers-cov) by the coronavirus study group of the international committee on taxonomy of viruses (announced in j. virology on may 15, 2013) . genetically, mers-cov is closely related to sars-cov, and is another example of a coronavirus associated with respiratory disease that can also infect kidney cells. the donor of the rptec of our study did not have kidney disease (otherwise, the cells would not have been harvested and sold for research purposes), suggesting a persistent, sub-clinical infection of the kidneys by hcov-nl63 is more likely. to what extent, if any, hhv-6b may have somehow modulated the growth of the other viruses in the rptec is unclear. noteworthy, hhv-6b has also been reported in association with renal epithelial cells and kidney transplant rejection [72] . lastly, whereas the virus-like particles of figure 4c appear similar to those in an electron micrograph of sars-cov in kidney tissue [63] , we have no formal proof that they are in fact hcov-nl63 and may be another virus we did not identify in our work. taken together, our findings are a reminder that human-derived biologicals should always be considered as potential sources of infectious agents. moreover, our findings raise the possibility of kidney involvement during the course of infection with hcov-nl63. cryopreserved primary human rptec were obtained from a commercial source in the usa. bgm, supplements, and growth factors [fetal bovine serum, insulin, transferrin, triiodothyonine (t3), human recombinant epidermal growth factor, hydrocortisone, epinephrine, gentamicin sulfate, and amphotericin-b] were concurrently obtained as a kit from the rptec supplier. the rptec were first seeded onto four t25 flasks and manipulated following instructions included with the kit. glutamine, which was later substituted with 2 mm l-alanyl-l-glutamine (glutamax™, invitrogen corp.). both dmem and emem were supplemented with antibiotics [psn; 50 μg/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml neomycin (invitrogen corp.)], and 10% (v/v) low igg, heat-inactivated gamma-irradiated fetal bovine serum (hyclone, logan, ut). additionally, sodium pyruvate (invitrogen corp.) and non-essential amino acids (hyclone) were added to emem., with the exception: emem formulated with calf serum (hyclone) instead of fbs was used for nih/3 t3 cells. before seed stocks were prepared, the cell lines were propagated in growth media with plasmocin (invivogen, san diego, ca) for 2 weeks to reduce the chances of mycoplasma contamination. next, the cell lines were incubated for a minimum of 2 weeks in the absence of antibiotics to determine whether fast-growing microbial contaminants were present or abnormal morphological changes would occur (associated with intracellular mycoplasma). following 2-3 weeks of propagation without antibiotics, the plasmocin-treated cell lines and rptec cells were tested by pcr for the presence of mycoplasma dna using a takara pcr mycoplasma detection kit (fisher scientific, pittsburgh, pa) [1] . the cells tested negative for mycoplasma. an independent laboratory (at the university of florida) confirmed that the stock of llc-mk2 cells that was used for the isolation of hcov-nl63 in this manuscript was negative for human respiratory viruses including human coronaviruses 229e, hku1, oc43, and nl63 using a genmark multiplex respiratory pcr esensor xt-8 respiratory viral panel (esensor rvp; genmark diagnostics, inc., carlsbad, ca). fresh l-glutamine was added to bgm in a 24 hr rptec culture and the cells observed every six hrs for one day to assess the effect on cell morphology, vacuolation, and viability. complete, freshly prepared bgm was substituted for dmem in subconfluent cultures of cv-1, llc-mk2, mdck, vero, and wi-38 cells, and the cells incubated at 37°c and observed every 12 hours over 3 days for morphological changes or cell death as evidence of cytotoxicity. to find out whether the rptec were releasing a bioactive agent, spent bgm from a 24 hr rptec culture was equally subdivided and added to subconfluent cv-1, hek-293, llc-mk2, vero e6, and wi-38 cells in t-25 flasks. these particular cell lines were chosen on the assumption that a virus growing in rptec would preferentially infect primate over non-primate cells. after inoculation, the cells were incubated at 37°c (the same temperature used for rptec) and observed for morphological aberrations over 48 hrs. a standard cytospin procedure was used to deposit rptec from a 48 hr culture onto a glass slide. ifa was performed using a commercial kit with a primary antibody directed against a cmv immediate early protein, and a secondary antibody that was labeled with fluorescein isothiocyanate (light diagnostics™ cmv ifa kit, millipore, billerica, ma). the bgm of a five day rptec culture was replaced with fresh ice-cold cacodylate-buffered 4% gluteraldehyde (ph 7.2). after 2 hrs at room temperature, the fixed cells were scraped free using a cell scraper, and pelleted by centrifugation at 8,000 x g for 10 minutes. the fixative was removed, and the cell pellet resuspended with cold fixative to a final volume of 500 μl, then stored overnight at 4°c. the fixed cells were post-fixed with osmium tetroxide, stained with uranyl acetate, embedded in spurr's embedding medium, then thin-sectioned. the thin sections were stained with uranyl acetate and lead citrate and transmission electron microscopy performed using a hitachi h-600. five days after being seeded, about 50% of the rptec had completely deteriorated, whereupon spent bgm media was added to 2 groups of subconfluent a549, bhk-21, cv-1, hek-293, llc-mk2, mdck, mdck-london, mv1 lu, nih/3 t3, vero e6, and wi-38 cells in complete growth media, and to 2 groups of llc-mk2 and mdck and mv1 lu cells in serum-free media containing l-1-tosylamide-2-phenylethyl chloromethyl ketone (tpck)-treated trypsin. the tpck-trypsin was at a final concentration of 2 μg/ml (mdck and mdck-london cells) or 0.2 μg/ml (llc-mk2 and mv1 lu). for each group, 1 set was incubated at 37°c, the other at 34°c (incubation at 2 different temperatures is standard in our laboratory, as many of the respiratory viruses we work with preferentially replicate at temperatures lower than 37°c). tpck-trypsin in serum-free media was used to facilitate the isolation of influenza and other viruses that require protease cleavage of some viral component for infectivity. after inoculation, the cells were re-fed every 3 days with 3% serum media or serum-free media with trypsin for long-term (up to 30 day) observations. at day 7 post-seed, only about 10% of the rptec remained attached to the flask, a majority of which were vacuolated and showed other signs of cpe. to facilitate the isolation of viruses other than cmv, the cells were scraped free and transferred along with the spent bgm into a sterile 50 ml polypropylene centrifuge tube, and frozen at −20°c for one week (this step reduces the number of viable cmv virions by a factor of many logs, since cmv loses viability when stored at −20°c) [38] ; [j. lednicky, unpublished] . next, the frozen tube of scraped rptec was freeze-thawed three times, alternating between freezing at −20°c for 12 hrs and a 30 minute thaw at room temperature, as an additional measure to further reduce the number of viable cmv particles. after the third thaw, an aliquot was tested using the cells and methods of section 2.5 above, and the remainder frozen at −80°c for retrospective analyses. intracellular dna was purified from a 48 hr rptec culture using a qiaamp dna mini kit (qiagen, valencia, ca) and tested by pcr for cmv, hhv-1 and −2, and polyomaviruses sv40 and bkv. total rna was purified from a freeze-thawed seven-day old rptec culture supernatant using a qiaamp viral rna kit (qiagen). the primers and conditions that were used for pcrbased detection of viruses were based on published literature and will be provided upon request. since syncytia were formed by the second virus (not cmv) that we were attempting to identify, pcr efforts were focused on human herpes, paramyxo (measles, mumps, metapneumovirus, parainfluenza viruses 1-5, respiratory syncytial virus), and coronaviruses. rt-pcr for rna virus screens was performed with omniscript reverse transcriptase (qiagen) followed by pcr with hotshot taq (new england biolabs, ipswich, ma) 68°c. hcov-nl63 was first detected using a pancoronavirus rt-pcr assay for the viral polymerase gene with primer pair cor-fw and cor-rv [42] , followed by sequencing of the 251 bp amplicon. that was accomplished using cor-rv for cdna synthesis (with reverse transcription performed for 1 hr at 37°c), and pcr performed as: initial denaturation step: 94°c (1.5 min); 30 cycles of 94°c (20 sec), 48°c (30 sec), 68°c (30 sec); terminal extension step at 68°c (3.5 min); 4°c ∞. for confirmation, primer pairs n5-pcr1 and n3-pcr1 [42] and repsz-1, and repsz-3 [33] were used with pcr parameters similar to those for cor-fw and cor-rv, and the resulting amplicons sequenced. n5-pcr1 and n3-pcr1 amplify a 314 bp amplicon from the hcov-nl63 nucleocapsid region. n3-pcr1 was used to generate cdna, and pcr performed at an annealing temperature of 46°c. following cdna synthesis primed with repsz-rt [33] , primer pair repsz-1, and repsz-3 amplify a 237 bp amplicon from the hcov-nl63 orf1b region at a pcr annealing temperature of 46°c. electron microscopy of llc-mk2 cells infected with hcov-nl63 from rptec llc-mk2 cells that were rt-pcr positive for hcov-nl63 were trypsinized to detach them from the growing surface of a t75 flask, pelleted, and the pellet resuspended in icecold 4% paramormaldehyde, 2% gluteraldehyde, in 0.1 m sodium cacodylate, ph 7.2. they were subsequently analyzed as described above. targeted hcov-nl63/rptec/2004 sequences were rt-pcr-amplified from purified rna using a genome walking strategy. briefly, overlapping primers described by h. geng et al. (genbank jx524171) and others [33, 42] were used to obtain the viral sequence. accuscript high fidelity reverse transcriptase (agilent technologies, inc., santa clara, ca) was used for first-strand cdna synthesis in the presence of superase-in rnase inhibitor (ambion). pcr was performed using phusion polymerase (new england biolabs) with denaturation steps performed at 98°c. the 3′ and 5′ ends of hcov-nl63 /rptec/2004 were determined from vrna using a race (rapid amplification of cdna ends) kit (rlm race, ambion, austin, tx) following the manufacturer's instructions. sequences were analyzed using an applied biosystem 3130 dna analyzer by using bigdye terminator (v. 3.1) chemistry and the same primers used for amplifications. the genomic sequence for isolate nl63/rptec/2004/1 was combined with other representative nl63 genomic sequences [34] available in genbank (ncbi.nlm.nih.gov/ genbank/index.html) to build the final dataset. full genome alignments were performed using mafft 5.8 [73] followed by minor manual adjustments in clustalw [74] . the e-ins-i alignment strategy was used with the following parameters: scoring matrix (blosum62), gap open penalty (1.53), and offset value (0). the aligned dataset was imported into jmodeltest version 0.1.1 [75] and the akaike information criterion (aic) was used to select a best-fit model of evolution for phylogenetic analysis. phylogenetic trees were constructed using mrbayes 3.1.2 [76] . the markov chain was run for a maximum of 10 million generations, with a stopping rule implemented so that the analysis would halt when the average deviation of the split frequencies was < 0.01. four independent analyses were conducted, each with 1 cold and 3 heated chains with the default heating parameter (temperature = 0.2). every 1000 generations were sampled and the first 25% of mcmc samples discarded as burn-in. hcov-nl63/amsterdam-1 hcov-nl63/amsterdam-1 was obtained from the biodefense and emerging infections research resources repository (bei resources, manassas, va). plaque assays were performed following the procedures outlines in references 39 and 50. the art of animal cell culture for virus isolation methods for detection and frequency of contamination of fetal calf serum with bovine viral diarrhea virus and antibodies against bovine viral diarrhea virus bovine viral diarrhea disease associated with a contaminated vaccine demonstration and genotyping of pestivirus rna from mammalian cell lines bovine viral diarrhea virus contamination of nutrient serum, cell cultures and viral vaccines identification of pestiviruses contaminating cell lines and fetal calf sera bovine polyomavirus, a frequent contaminant of calf serum bovine polyomavirus, a frequent contaminant of calf sera a virus discovery method incorporating dnase treatment and its application to the identification of two bovine parvovirus species identification of novel porcine and bovine parvoviruses closely related to human parvovirus 4 the association of calf serum with the contamination of bhk21 clone 13 suspension cells by a parvovirus serologically related to the minute virus of mice (mvm) replication of bovine herpesvirus type 4 in human cells in vitro bovine herpesvirus type 4: a special herpesvirus (review article) an adventitious viral contaminant in commercially supplied a549 cells: identification of infectious bovine rhinotracheitis virus and its impact on diagnosis of infection in clinical specimens growth characteristics of bovine herpesvirus 1 (infectious bovine rhinotracheitis) in human diploid cell strain wi-38 infection studies on human cell lines with porcine circovirus type 1 and porcine circovirus type 2 investigations of porcine circovirus type 1 (pcv1) in vaccine-related and other cell lines evaluation of the human host range of bovine and porcine viruses that may contaminate bovine serum and porcine trypsin used in the manufacture of biological products torque teno sus virus (ttsuv) in cell cultures and trypsin presence of antibodies reacting with porcine circovirus in sera of humans, mice, and cattle comparative analysis of the full-length genome sequence of a clinical isolate of human parainfluenza virus 4b mutations in the gm1 binding site of simian virus 40 vp1 alter receptor usage and cell tropism atcc w ccl-34) have developed vacuoles. what is wrong? seroprevalence of cytomegalovirus infection in the united states detection of human cytomegalovirus dna, rna, and antibody in normal donor blood the histopathologic identification of cmv infected cells in biopsies of human renal allografts. an evaluation of 100 transplant biopsies by in situ hybridization widespread presence of cytomegalovirus dna in tissues of healthy trauma victims understanding human coronavirus hcov-nl63 osterhaus ad: a previously undescribed coronavirus associated with respiratory disease in humans epidemiology and clinical presentations of human coronavirus nl63 infections in hong kong children the novel human coronaviruses nl63 and hku1 human coronavirus nl63, a new respiratory virus identification of a new human coronavirus genomic analysis of 16 colorado human nl63 coronaviruses identifies a new genotype, high sequence diversity in the n-terminal domain of the spike gene, and evidence of recombination human airway epithelial cell culture to identify new respiratory viruses: coronavirus nl63 as a model identification of human herpesvirus-6 as a causal agent for exanthem subitum human herpesvirus 6 infection in hematopoietic stem cell transplant patients human cytomegalovirus herpes simplex virus and human cytomegalovirus replication in wi-38 cells. iii. cytochemical localization of lysosomal enzymes in infected cells generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-pcr and nonfluorescent low-density microarray identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel pancoronavirus rt-pcr assay: frequent detection of human coronavirus nl63 in children hospitalized with respiratory tract infections in belgium fatal hemorrhagic pneumonia concomitant with chlamydia pneumoniae and parainfluenza virus 4 infection sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses a family-wide rt-pcr assay for detection of paramyxoviruses and application to a large-scale surveillance study novel human reovirus isolated from children with acute necrotizing encephalopathy. emerg infect dis morphogenesis of coronavirus hcov-nl63 in cell culture: a transmission electron microscopic study human herpesvirus-6 infection in children. a prospective study of complications and reactivation plaque assay for human coronavirus nl63 using human colon carcinoma cells systematic assembly of a full-length infectious clone of human coronavirus nl63 replication-dependent downregulation of cellular angiotensin-converting enzyme 2 protein expression by human coronavirus nl63 extensive human cytomegalovirus (hcmv) genomic dna in the renal tubular epithelium early after renal transplantation: relationship with hcmv dnaemia and long-term graft function freeze-thawing of breast milk does not prevent cytomegalovirus transmission to a preterm infant identification of cell lines permissive for human coronavirus nl63 sars-associated coronavirus replication in cell lines inhibition of cytokine gene expression and induction of chemokine genes in non-lymphatic cells infected with sars coronavirus human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry angiotensin-converting enzyme 2-a new cardiac regulator exogenous ace2 expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication preferential transformation of human neuronal cells by human adenoviruses and the origin of hek 293 cells acute renal impairment in coronavirus-associated severe acute respiratory syndrome fatal severe acute respiratory syndrome is associated with multiorgan involvement by coronavirus multiple organ infection and the pathogenesis of sars tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases viral loads in clinical specimens and sars manifestations evaluation of reverse transcription-pcr assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction isolation of a novel coronavirus from a man with pneumonia in saudi arabia is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans genetic relatedness of the novel human group c betacoronavirus to tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5. emerg microb infect human herpesvirus 6 infection in renal transplantation mafft version 5: improvement in accuracy of multiple sequence alignment clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice posada d: jmodeltest: phylogenetic model averaging mrbayes: bayesian inference of phylogenetic trees isolation and genetic characterization of human coronavirus nl63 in primary human renal proximal tubular epithelial cells obtained from a commercial supplier, and confirmation of its replication in two different types of human primary kidney cells part of this work, including electron microscopy, was performed when the corresponding author was at the dept of pathology at the loyola university medical center (lumc), maywood, illinois. at lumc, linda fox provided excellent assistance with electron microscopy. some of this work was performed in partial fulfillment of internship requirements for em and mcl. the authors thank dr. gary heil for mdck-london cells. electron microscopy at the university of florida was performed by karen kelley. this work was financed by intramural funds made available to j. lednicky. the authors declare that they have no competing interests.authors' contributions jal conceived of the work, participated in all procedures, interpreted data; tbw performed phylogenetic analyses, interpreted data, and both jal and tbw wrote the manuscript, em, jcl, sbh, and mcl performed cell culture and virology work, and photographed cells; em assisted with dna and rna extractions, and ifa. jcl helped format the manuscript. all authors read and approved the final manuscript. key: cord-280374-yj0r4rwt authors: jain, richa; gupta, kirti; bhatia, anmol; bansal, arun; bansal, deepak title: hepatic sinusoidal-obstruction syndrome and busulfan-induced lung injury in a post-autologous stem cell transplant recipient date: 2018-01-04 journal: indian pediatr doi: 10.1007/s13312-017-1172-5 sha: doc_id: 280374 cord_uid: yj0r4rwt veno-occlusive disease of the liver is mostly encountered as a complication of hematopoietic stem cell transplantation with myeloablative regimens with an incidence estimated to be 13.7%. it is clinically characterized by tender hepatomegaly, jaundice, weight gain and ascites. strong clinical suspicion and an early recognition of clinical signs are essential to establish the diagnosis and institute effective regimen. another complication of cytotoxic drugs given for cancers, is development of busulfan-induced lung injury. a strong index of suspicion is needed for its diagnosis, especially in setting where opportunistic fungal and viral infections manifest similarly. we illustrate the clinical and autopsy finings in a 2½-year-old boy who received autologous stem-cell transplantation following resection of stage iv neuroblastoma. he subsequently developed both hepatic veno-occlusive disease and busulfan-induced lung injury. the autopsy findings are remarkable for their rarity. volume 54 __ september 15, 2017 jain, et al. hepatic sinusoidal-obstruction syndrome increased efforts, hypoxia (saturation on room air: 90%; increasing to 95% on 40% fio2). tachycardia was present (heart rate 132/minute); however, circulatory parameters were normal (bp 98/54 mm hg, normal capillary refill time and pulse pressure, warm extremities). pallor was present, with no evidence of skin or mucosal bleeding. systemic examination was consistent with pneumonitis as the patient had tachypnea, bilateral equal air entry, and presence of coarse crepitations in bilateral lung fields. abdominal examination showed dark brown pigmentation over abdomen with no tenderness, guarding or rigidity. hepatomegaly was present with liver palpable 3 cm below costal margin (span 8 cm). spleen was not palpable. there was no free fluid. cardiac and neurological examinations were essentially normal. the index case was managed as a case of pneumonitis post auto-sct. respiratory support was initially provided with nasal prongs-continuous positive airway pressure. intravenous antibiotics were started cefoperazone-sulbactam, amikacin and azithromycin. due to rapidly progressive respiratory distress, child was transferred to pediatric intensive care unit where mechanical ventilation was provided. there was single episode of fever on the day of admission (38.7 o c) with a subsequent afebrile period throughout the hospital stay. progressive respiratory distress worsened into acute respiratory distress syndrome (ards). ventilation strategy was modified accordingly. on day 63, there was development of hypotensive shock, initially responding to fluid boluses. on day 65, the shock necessitated inotropic support (dopamine, adrenaline, noradrenaline and pre-terminally, and vasopressin). there was development of left sided pneumothorax followed by cardiac arrest on the same day. the patient could be revived and pneumothorax was drained. multi-organ dysfunction developed with acute kidney injury (onset day 63), requiring peritoneal dialysis. significant transaminitis with elevated bilirubin levels was documented. antibiotics were changed to vancomycin and meropenam on day 63; azithromycin was continued. intravenous co-trimoxazole and gancyclovir were added in therapeutic, renal modified doses. platelet concentrates were transfused to maintain a platelet count above 20 × 10 6 /mm 3 . there was development of refractory shock on day 67. the child suffered another cardiac arrest on day 68, and could not be revived. the fig. 1a-d) . with these chest radiographs, possible etiologies considered were infective, including fungal pneumonia and pcj pneumonia, cmv disease and miliary tuberculosis (tb). non-infective etiologies under consideration included pulmonary graft-versus host disease (gvhd) and pulmonary veno-occlusive disease (vod). this is a case of neuroblastoma, stage iv, day 68 post auto-sct, presenting with fever, pneumonia, hypoxia, and investigations showing polymorphic leucocytosis with deranged liver function tests (lft). in a posttransplant patient, complications can be divided according to the duration subsequent to transplant. in the initial 30 days, there is presence of neutropenia; between 30-100 days is the early post-engraftment phase and beyond 100 days is late post-engraftment phase. the index case developed symptoms in the early post engraftment phase. common complications seen in early post-engraftment phase can be divided into infective and non-infective. infective etiologies include cmv which can explain both pneumonia and hepatitis. it is a common pathogen causing disease 3 weeks post sct. india is an endemic country for cmv. in the index case, eta demonstrated polymerase chain reaction (pcr) positivity volume 54 __ september 15, 2017 jain, et al. hepatic sinusoidal-obstruction syndrome for cmv along with radiological findings which were supportive of the diagnosis. however, the child deteriorated despite administration of gancyclovir from day 2 onwards, which is unusual. typically blood pcr is positive in such cases, though not mandatory for diagnosis of cmv pneumonia. fungal infections are the next possibility, supported by the presence of candida in eta on two occasions. presence of normal neutrophil count and a normal serum galactomannan are odd points. galactomannan <0.5 has shown a good negative predictive value for aspergillus infection [2] . other viral infections that are important in post-sct scenario include respiratory syncytial virus (rsv), para-influenza virus, influenza, metapneumovirus, and coronavirus. multiorgan failure and lymphopenia is common in these patients. patients with rsv often require ventilation. associated co-infection with fungus, especially aspergillus can be seen. in the absence of investigations directed towards the myriad respiratory viruses, it is difficult to rule in or rule out these infections. tuberculosis should be considered in an immunocompromised patient in an endemic country; however, the rapid onset of disease, absence of a contact and negative evaluation make it unlikely. in our case other bacterial infections typically seen in an immunocompromised child are also unlikely in view of sterile cultures, complete absence of fever and normal creactive protein (crp).though this clinical presentation can be caused by infection with pcj, it is an uncommon infection. other atypical infections like nocardia and cryptococcus are rarer still. the non-infective etiologies causing respiratory symptoms in a post-transplant setting can be pulmonary gvhd, idiopathic pneumonia syndrome (ips), bronchiolitis obliterans syndrome (bos), cryptogenic organising pneumonia (cop) and sos. ips is a very common disease in this situation, but is typically seen post allo-sct and hepatitis is not an associated feature. on-going hepatic sos is unlikely as there was no weight gain or tender hepatomegaly. gvhd and bos are also typically diseases seen in allo-sct setting. pulmonary sos is very rare and normal echo findings negate this possibility. the clinical presentation is consistent with cop, though it is more common in females undergoing allogenic transplant. the final diagnosis is neuroblastoma stage iv, day + 68 post auto-sct (bu-mel) with pneumonitis, ards and multi-organ failure; likely etiology being fungal pneumonia or cmv pneumonia and hepatitis secondary to ischemia with underlying sos. pediatric hemato-oncologist 1: ips occurs post sct day+60 to 80. this child had typical bilateral basilar infiltrates and hypoxia. moreover, ips has a relationship with use of busulfan and pre-existing sos. presence of cmv positivity in eta is of questionable significance as it is a common organism. histopathological evidence from lung biopsy is essential to prove cmv pneumonia. liver dysfunction in the form of transaminitis was likely due to shock and ischemia. pediatric hemato-oncologist 2: bacterial and fungal infections cannot be excluded despite absence of fever, several sterile cultures and continued normal values of crp, though less likely. however, both cmv and pcj infections are possible with normal crp. absence of adventitious lung sounds at initial presentation, along with presence of hypoxia may be a pointer towards pcj pneumonia. immunocompromised state, lymphopenia and the fact that the child was not on pcj prophylaxis are important here. moreover, cmv is ubiquitous in our volume 54 __ september 15, 2017 jain, et al. hepatic sinusoidal-obstruction syndrome pediatric population, and in pediatric oncology patients, we have seen a near 100% seropositivity. reactivation of cmv can occur at any point of time in these patients. important non-infective possibilities are ips and cryptogenic pneumonia. pulmonary sos is quite unlikely given the normal echo findings. adult hematologist: immune reconstitution posttransplant takes typically 6 to 12 months. this child was immunocompromised. adenovirus infection can be considered. it can be rarely seen in association with hepato-pulmonary syndrome. the excised tumor on histology was categorized as differentiating neuroblastoma ( fig. 2a-c) . autopsy revealed normal serous cavities. liver weighed 290 g and revealed irregular areas of sinusoidal congestion, confluent at places with necrosis of adjoining parenchyma involving both right and left lobe (fig. 2d) . no thrombi were identified in right and left hepatic vein or inferior vena cava. microscopically, areas of centrizonal congestion were identified (fig. 2e) . furthermore, the dominant pathology was seen in the central vein and terminal hepatic venule (thv). there was varying degree of obliterative changes with subendothelial fibrosis and laying down of reticulin fibres and collection of extracellular matrix in subintimal zone. at places, the thv was completely obliterated with wipe out of centrizonal hepatocytes while the periportal hepatocytes were preserved (web fig. 1a-e) . in other regions, extravasated rbcs and areas of hemorrhage were noted in centri-zonal regions. besides acute obliterative changes, subacute changes in form of deposition of collagen around the hepatic venule and collection of hemosiderin laden macrophages were also noted. loss of hepatic parenchyma resulted in approximation of central veins structures (web fig. 1a) . both lungs weighed 245g with dull pleura. microscopy revealed features of busulfan-induced lung injury with marked prominence of type ii pneumocytes; many of them demonstrated nuclear atypia and hyperchromasia. marked thickening of interstitium with fibrosis was also noted (web fig. 2a-b) . other regions showed patchy acute bronchopneumonia and alveolar haemorrhages. features of pulmonary arteriopathy were also noted with prominence of intra-acinar arterioles. there were no features of vod in the pulmonary veins. an occasional focus of septic emboli with candida infiltration into parenchyma was noted. no cmv inclusions were noted in lungs. pcr carried out on lung tissue for adenovirus, rsv and metapneumovirus were negative. acute ulcers with candida infiltration were noted in stomach and small intestine. candida had disseminated to heart causing mural endocarditis, myocardial abscess and tiny (2-4 mm) vegetations on left atrial wall (webfig. 2 c-f). both tricuspid and mitral valves were normal. dissemination with formation of fungal abscesses were also detected in psoas muscle and omental fat. subsequent to septic emboli, infarcts were detected in right kidney (upper pole) with thrombi within the branches of renal vessels and spleen. right kidney also revealed features of acute tubular necrosis in noninfracted regions. no residual tumor was detected in lymph nodes, thymus and bone marrow. the autopsy diagnosis is concluded as follows: in a known case-of neuroblastoma, undifferentiated (adrenal) post-autologous stem-cell transplant: • features of busulfan-induced lung injury with organizing bronchopneumonia and pulmonary arterial hypertension; • veno-occlusive disease in liver. • fungal (candida) ulcers in git with extensive dissemination to heart (mural endocarditis and myocardial abscess), lungs, skeletal muscle and omental fat producing embolic infarcts in right kidney and spleen. • no residual disease in bone marrow. hepatic sinusoidal obstruction syndrome (sos) is an obliterative venulitis of thv which occurs as a result of cytoreductive therapy prior to hematopoietic stem cell transplantation (hsct), ingestion of pyrrolizidine alkaloids, or radiation therapy [3] [4] [5] [6] . the primary pathogenetic event is the endothelial injury of sinusoids and small hepatic veins. following which, there is deposition of fibrin-related aggregates and oedema in the subendothelial zone [3] . accumulation of these aggregates and entrapment of fluid and cellular debris progressively occlude the hepatic venous flow and leads to post-sinusoidal intrahepatic hypertension. this is accompanied by necrosis of perivenular hepatocytes. histologically, acute, sub-acute and chronic forms of sos have been described depending upon collagenization and fibrosis of terminal hepatic venule. incidence of sos varies from 0-70%, as it depends on the conditioning regimen used as well as upon the patient's risk factors [4] [5] [6] . sos occurs more often after allo-than after auto-hsct (8 v/s 3%, respectively), suggesting a role of immune reactions in this disorder [7] . few independent studies have documented increase in circulating levels of plasminogen activator inhibitor-1 (pai-1), a molecule released by the endothelial cells, in patients developing sos [8, 9] . increased pai-1 levels might be of clinical utility in challenging clinical situations in patients with hyperbilirubinemia occurring after hsct. it forms one of the therapeutic targets for defibrotide, which reduces circulating pai-1 levels along with other actions. other endothelial markers, like intercellular adhesion molecule-1 (icam-1), e-selectin, von willebrand factor (vwf), and thrombomodulin may also be helpful in early identification of patients at risk of sos who may benefit from early introduction to therapies [10] . diagnosis of sos is based on constellation of signs and symptoms and serum bilirubin levels. hepatic sos is clinically characterized by jaundice caused mainly by conjugated hyperbilirubinemia, tender hepatomegaly, fluid accumulation manifested as rapid weight gain and ascites [4] . most commonly used diagnostic criteria for sos includes the seattle criteria [11] , the modified seattle criteria [1] , and the baltimore criteria [12] . because of its high incidence and mortality, prophylaxis for hepatic sos is widely practiced, using different regimens in different centres. when hepatic sos is established, specific therapy is usually given in addition to general supportive care, especially in moderate or severe cases. hepatic sos is a formidable challenge both for patients undergoing stem cell transplantation and for their physicians. the second pathology in this child which significantly contributed to his downhill course was busulfan induced lung injury. intriguingly, in the present clinical setting, busulfan induced lung injury remains an diagnosis of exclusion, particularly with respect to considering usual and atypical infections. its clinical presentation includes a spectrum ranging from acute, rapidly progressive respiratory distress to chronic, interstitial lung disease with insidious onset [13, 14] . the pathophysiology of drug-induced lung injury is not fully understood but direct toxicity of the drug to parenchymal cells, cell-mediated immune reactions and release of cytokines are believed to contribute to the lung injury. the pathologic findings consist of mainly diffuse interstitial pneumonitis, organizing alveolitis and cellular atypia within type ii pneumocytes. the injury pattern with busulfan is diffuse alveolar damage (dad) either in acute exudative phase with alveolar and interstitial oedema and hyaline membranes; or late reparative phase, which is characterized by proliferation of type ii pneumocytes and interstitial fibrosis [15] . marked atypia of the type ii pneumocytes is a morphological clue in favour of busulfan induced lung injury in contrast to organizing bacterial pneumonia. moreover, pcr for cmv is helpful in excluding viral pneumonia. the prevalence of drug-induced pulmonary toxicity is increasing, and more than 100 drugs are now known to cause lung injury. because this lung injury can be progressive and fatal, early recognition is important. the diagnosis of pulmonary drug toxicity should be considered in any patient with a history of drug therapy who presents with new or progressive respiratory complaints. the superadded fungal ulcer which developed preterminally with extensive dissemination to heart causing mural endocarditis and myocardial abscess eventually led to the demise of the child. hepatic sos contributes considerably to transplantation-related morbidity and mortality. recognition of this disease in the post-transplantation setting remains a challenge in the absence of specific diagnostic features as many other more common conditions can mimic it. a high index of suspicion is needed to identify patients with sos. while hepatic sos and busulfan induced lung injury are commonly reported as isolated findings following autologous sct, the coexistence of these are extremely rare and have not been documented in the literature thus far. the present case adds observational data to the existing literature and highlights the importance of keeping high index of suspicion for these two entities in patients following hsct, and early institution of effective therapy. veno-occlusive disease of the liver and multiorgan failure after bone marrow transplantation: a cohort study of 355 patients serum galactomannan assay for the diagnosis of invasive aspergillosis in children with haematological malignancies sinusoidal obstruction syndrome (hepatic veno-occlusivedisease) hepatic veno-occlusive disease (sinusoidal obstruction syndrome) after hematopoietic stem cell transplantation sinusoidal obstruction syndrome vascular disorders of the liver. american association for the study liver diseases incidence and outcome of hepatic venoocclusive disease after blood or marrow transplantation: a prospective cohort study of the european group for blood and marrow transplantation. european group for blood and marrow transplantation chronic leukemia working party the relevance of plasminogen activator inhibitor 1 (pai-1) as a marker for the diagnosis of hepatic veno-occlusive disease in patients after bone marrow transplantation endothelial dysfunction after bone marrow transplantation: increase of soluble thrombomodulin and pai-1 in patients with multiple transplant-related complications prediction of veno-occlusive disease using biomarkers of endothelial injury venocclusive disease of the liver after bone marrow transplantation: diagnosis, incidence, and predisposing factors venoocclusive disease of the liver following bone marrow transplantation busulphan lung in childhood lung function 5 yrs after allogeneic bone marrow transplantation conditioned with busulphan and cyclophosphamide interstitial pneumopathies caused by busulfan. histologic, developmental and bronchoalveolar lavage analysis of 3 cases key: cord-340228-mvqoyror authors: al-herz, waleed; essa, sahar title: spectrum of viral infections among primary immunodeficient children: report from a national registry date: 2019-05-29 journal: front immunol doi: 10.3389/fimmu.2019.01231 sha: doc_id: 340228 cord_uid: mvqoyror objective: to present the frequency and spectrum of viral infections in primary immunodeficient children. methods: the data was obtained from the kuwait national primary immunodeficiency disorders (pids) registry during the period of 2004-2018. results: a total of 274 pid children were registered in knpidr during the study period with predominance of immunodeficiencies affecting cellular and humoral immunity, followed by combined immunodeficiencies with associated syndromic features and diseases of immune dysregulation. overall infectious complications affected 82.4% of the patients, and viral infections affected 31.7% of the registered patients. forty-five patients (16.4%) developed viral infections caused by at least 2 organisms, among those 20 patients were affected by three or more viral infections. there was a statistically significant association between viral infections and pid category. however, there was no statistically significant association between viral infections and gender or the patients' onset age. there was a total of 170 viral infections during the study period and the causes of these infections were predominated by cmv (22.2%), adenovirus (11.7%), ebv (11.1%), and enteroviruses (7.4%). cmv and parainfluenza infections were more common in the group of immunodeficiencies affecting cellular and humoral immunity while ebv and human papilloma virus (hpv) were more common in the immune dysregulation group and combined immunodeficiencies with associated syndromic features, respectively. the most common presentation was viremia (28.8%) followed by pneumonia (28.2%) and skin infections (17.6%). the most common causes of viremia were cmv followed by adenovirus and ebv, while the most common organisms causing pneumonia were cmv followed by rhinovirus and parainfluenza. there were 80 deaths among the registered patients, 10% were caused by viral infections. conclusions: viral infections are common in pids and result into a wide-range of clinical manifestations causing significant morbidity and mortality. primary immunodeficiency disorders (pids) are monogenic defects affecting the innate and/or adaptive immune systems (1) . patients are at increased risk of a wide range of manifestations including autoimmunity, immune dysregulation, and malignancies, but infectious complications are the commonest (2) (3) (4) . historically, pid patients used to die before recognition because of infections due to the lack of effective measures to either prevent or treat them. advances in public health and the discovery of antimicrobial agents made the diagnosis of pids possible (5) . although therapeutic interventions like intravenous immunoglobulins and hematopoietic stem cell transplants have helped to decrease morbidity and mortality, physicians caring for pid patients frequently struggle with treating infections which are usually recurrent or chronic, severe and are frequently caused by opportunistic organisms. among these microbes are viruses that in pid patients can be challenging with an increased risk of mortality and may predispose to malignancies (6) (7) (8) . pids may also lead to reduced clearance and prolonged shedding of certain viruses like rhinovirus and poliovirus (9, 10) . while most pids predispose to a wide spectrum of viral infections, certain diseases enhance vulnerability to specific viral infections (11) . whereas, the risk of viral infections in pids is well-established and was recently reviewed (12, 13) , we are not aware of any report that characterizes such infections in a large cohort of patients with different types of pids. in this report, we present the frequency and spectrum of viral infections in primary immunodeficient children from kuwait between january 2004 and december 2018. the data was obtained from the kuwait national primary immunodeficiency disorders registry (knpidr) which was approved by the research and ethics committee of the ministry of health in kuwait and the kuwait university health sciences center ethical committee in accordance with the declaration of helsinki. the patients were followed prospectively and classified according to the international union of immunological societies, primary immunodeficiency diseases committee report on inborn errors of immunity (2017) (1). secondary immunodeficiencies (drug induced, hiv induced, and immunodeficiency associated with metabolic disorders... etc.), were ruled out by obtaining a detailed history and by performing appropriate testing when these disorders were suspected. the clinical diagnosis was based on the standard of care depending on the patient's signs and symptoms supported by laboratory and/or radiologic findings. for example, patients who were diagnosed with pneumonia presented with respiratory signs and symptoms associated with radiologic findings. the diagnosis of herpes simplex virus (hsv) keratitis and stomatitis, warts caused by human papilloma virus (hpv), varicella-zoster virus (vzv) and molluscum contagiosum infections was based on clinical evaluation. in-house polymerase chain reaction (pcr) was used initially to test for cytomegalovirus (cmv), epstein-barr virus (ebv), herpesvirus 6 (hhv-6), adenovirus, respiratory and gastrointestinal viruses. this was replaced later by commercial kits as indicated below. for patients with gastrointestinal manifestations stool or colonic samples were collected. deep nasopharyngeal aspirate or bronchoalveolar lavage samples were collected from patients with respiratory manifestations using sterile nylon flocked swab or by bronchoscopy and placed in viral transport medium. serum and cerebrospinal fluid samples were collected for viral infections as required. all samples were labeled and transported the earliest to virology laboratory, faculty of medicine, kuwait university for day-to-day routine screening for viral infections and storage. viral nucleic acid from samples was extracted using an automated magna pure lc 2.0 system (roche diagnostic systems, branchburg, nj). hsv (2) keratitis (2) hpv (2) warts (2) cmv (8) viremia (4), pneumonia (6) adenovirus (3) viremia (2), pneumonia (1) enterovirus (3) hemophagocytosis (1), viremia (1), pneumonia (1) norovirus (1) immunodeficiency with centromeric instability and facial anomalies (2) sapovirus (2) enteritis (2) adenovirus (2) enteritis (1), viremia (1) predominantly antibody deficiencies (n = 6) btk deficiency (1) enterovirus (1) meningo-encephalitis µ heavy chain deficiency (1) enterovirus (1) (1) vzv (1) chickenpox lyst deficiency (1) ebv (1) hemophagocytosis cmv (2) colitis (1), viremia (2) ebv (2) stool samples were homogenized. prior to extraction, a 200 mg aliquot was suspended in 1 ml of nuclease-free water or 1 ml of stool transport and recovery buffer (roche diagnostics, meylan, france). then the suspension was immediately clarified by centrifugation at 11,000 g for 5 min. according to the manufacturer's instructions rt-pcrs was performed on 10 µl of nucleic acid by using lightcycler 480 rt-pcr thermocycler (roche, meylan, france). cmv, ebv, enterovirus, hadv, and hhv-6 detection were performed on serum, stool/colonic, respiratory or csf samples by lightmix r kits (tib molbiol, berlin, germany). amplifications were performed according to the manufacturer's instructions on lightcycler 480 rt-pcr thermocycler (roche, meylan, france). the presence of poliovirus rna in clinical samples was confirmed by one-step reverse transcription-pcr, followed by a direct sequencing of pcr products, as described previously (14) . data were processed using ibm spss, version 25 (ibm corporation, armonk, ny, usa 2017). pearson's chi-square test was used to assess the association between two categorical variables. the non-parametric mann-whitney u-test was applied to assess whether the patients' ages at onset of a symptom of pid have a significant effect on the risk of viral infection. the effect of age at onset was assessed both as quantitative and qualitative variables after dividing them into groups (0-5, 6-11, 12-24, 25-48, > 48 months). the p ≤ 0.05 was used as the cut-off level for statistical significance. a total of 274 pid children (142 males and 132 females) were registered in knpidr during the study period. the distribution of these patients according to pid categories is: immunodeficiencies affecting cellular and humoral immunity, 97 patients (35.4%); combined immunodeficiencies with associated syndromic features, 67 patients (24.5%); predominantly antibody deficiencies, 34 patients (12.4%); diseases of immune dysregulation, 47 patients (17.2%); congenital defects of phagocyte number or function, 17 patients (6.2%); autoinflammatory disorders, 1 patient (0.3%); and complement deficiencies, 11 patients (4%). no patients with defects in innate immunity were registered. seventy-one patients were treated with hematopoietic stem cell transplant (hsct) and 141 received intravenous immunoglobulins. it is important to mention that of the reported viral infections occurred prior to hsct in patients who received such treatment. overall infectious complications affected 226 patients (82.4%), and viral infections affected 87 patients (31.7% of the registered patients). forty-five patients (16.4%) developed viral infections caused by at least 2 organisms, mostly in the category of immunodeficiencies affecting cellular and humoral immunity (31 patients). among those, 20 patients were affected by three or more viral infections. there was a statistically significant association between viral infections and pid category after excluding patients who belong to congenital defects of phagocyte number or function, autoinflammatory disorders and complement deficiencies due to low numbers (p < 0.001) ( table 1) . however, there was no statistically significant association between viral infections and gender (p = 0.488), or the patients' onset age when assessed both as quantitative and qualitative variables (p-values 0.23 and 0.655, respectively). there was a total of 170 viral infections during the study period, 33% were detected at the time of pid diagnosis while 67% were documented after establishing the diagnosis. the causes of these infections were: cmv (22.2%); adenovirus (11.7%); ebv (11.1%); enteroviruses (7.4%), hsv and hpv (6.8% each); vzv and rhinovirus (6.2% each); molluscum contagiosum (5.5%) (figure 1) ; norovirus and parainfluenza virus (3% each); h1n1 virus (1.85%); rotavirus, rsv, sapovirus, hhv-6 and corona virus (1.2% each). a patient with severe combined immunodeficiency presented with myocarditis caused by poliovirus type 2. two patients (1 with severe combined immunodeficiency and 1 with mhc ii deficiency) had prolonged excretion of poliovirus type 1 in the stool. the details of the viral infections are presented in table 2 . figure 2 shows the number of patients affected by different viruses according to pid categories. the most prominent findings are that cmv and parainfluenza infections are more common in the group of immunodeficiencies affecting cellular and humoral immunity while ebv and hpv are more common in the immune dysregulation group and combined immunodeficiencies with associated syndromic features, respectively. the most common presentation was viremia (28.8%) followed by pneumonia (28.2%) and skin infections (17.6%) ( table 3) . the most common causes of viremia were cmv followed by adenovirus and ebv, while the most common organisms causing pneumonia were cmv followed by rhinovirus and parainfluenza ( table 3) . in the current study, we present the characteristics of viral infections in a large cohort of pid children who were followed prospectively over a period of 15 years. viral infections affected more than 1/3 of the registered patients, many of whom were affected by more than 1 virus. the patients were affected by a range of viral organisms but cmv, adenovirus and ebv were the culprits in almost half of the cases. the high frequency of cmv infections (>20%) in our cohort can be explained by the fact that most of the cases are affected by combined immunodeficiencies. patients with such defects are extremely susceptible to progressive infection with cmv (12) . our finding that cmv and parainfluenza infections are more common in the group of immunodeficiencies affecting cellular and humoral immunity has been documented previously (7, 15) . the observation that ebv is more common in the immune dysregulation group specifically triggering hlh is also welldocumented (8, 12) . we have found that patients with dock8 deficiency are particularly predisposed to mucocutaneous viral infections like molluscum contagiosum and hsv infections. this is probably since dock8 is an important regulator of the actin cytoskeleton that is critical for cell migration through collagendense tissue, hence playing an important antiviral immunity in the skin (16) . the presented cohort of patients are characterized by the high frequency of combined immunodeficiencies which are more severe with a higher predisposition to viral infections compared to other pid categories. another prominent feature of our cohort is that none of the registered patients suffer from adenovirus (12) ebv (10) enteroviruses (6) hhv-6 (2) pneumonia 28.2 cmv (18) rhinovirus (9) parainfluenza (5) adenovirus (4) h1n1 (3) coronavirus (2) rsv (2) ebv (2) enterovirus (1) skin infections 17.6 hpv (11) vzv (10) molluscum contagiosum (9) gastrointestinal infections 9.4 norovirus (5) enteroviruses (4) adenovirus (2) cmv (2) sapovirus (2) rotavirus ( increased susceptibility to specific viral infections. examples of such diseases are tlr3, trif, or unc93b1 deficiencies which predispose to hsv-1 encephalitis and epidermodysplasia verruciformis or cxcr4 deficiencies which predispose to hpv. it is important to stress that physicians should be aware of pids and consider them in patients with severe or recurrent viral infections. importantly, they should be aware that many pids result in poor antibody response, hence serologic testing should be avoided while testing a patient for infectious complications and antigenic detection method should be used instead. health care providers should also be aware of the recommendations for live viral vaccines in immunodeficient patients and their close contacts (17) . live vaccines, such as the chicken pox, measles, mumps, rubella (mmr), rotavirus, yellow fever, oral polio, and the influenza nasal spray should be avoided in certain types of pids. furthermore, any infants born into a family with a suspicious history of pid should avoid all live viral and bacterial vaccines until pids is ruled out. historically, oral polio vaccine (opv) was the only form used in the vaccination schedule in kuwait. since 2008 the first dose of opv given at the age of 2 months was replaced with the inactivated formulation. fortunately, only 1 patient from our cohort who was diagnosed with rag1 deficiency developed opv related complication (i.e., myocarditis). two more patients with cid had prolonged excretion of poliovirus type 1 in the stool. unfortunately, stool surveillance program of pid patients for vaccine derived polio virus is not available in the country. the present study has some limitations since we did not determine the true burden of viral infections in pids. this could be established by documenting the number of admissions to the intensive care unit and the type of care provided like mechanical ventilation and the use of inotropes during these admissions. other important variables that can be considered are the number of viral infection reactivations, the number of admissions to the hospital, the length of stay, and duration of using antiviral treatments. however, an important strength of the study is that the patients were followed prospectively by the same clinical immunologist. another important strength of the study is that most patients were diagnosed at the molecular level. this may help in determining the genotype-phenotype correlation. yet, collaborative efforts will be needed to collect a bigger number of patients. viral infections in pids should be treated aggressively with appropriate antiviral medications and definitive treatments like hsct when possible since failure to eradicate viral pathogens creates an inflammatory environment that promotes cell survival and proliferation and may predispose to malignancy (18) . innovative treatments like virus-specific t cells should be explored to improve clinical outcomes for this group of patients (19) . all datasets generated for this study are included in the manuscript and/or the supplementary files. the data was obtained from the kuwait national primary immunodeficiency disorders registry (knpidr) which was approved by the research and ethics committee of the ministry of health in kuwait and the kuwait university health sciences center ethical committee in accordance with the declaration of helsinki. wa-h: development of the research concept and goals, design of methodology, data collection and analysis, writing the initial manuscript draft, approval of the submitted manuscript, and agreement to be accountable for the content of the work. se: contributed to the research idea, writing of the manuscript and approval of the submitted manuscript and agreement to be accountable for the content of the work. knpidr was funded by kuwait foundation for the advancement of sciences. international union of immunological societies: 2017 primary immunodeficiency diseases committee report on inborn errors of immunity primary immunodeficiency disorders in iran: update and new insights from the third report of the national registry primary immunodeficiency disorders in kuwait: first report from kuwait national primary immunodeficiency registry primary immunodeficiency diseases in oman: 10-year experience in a tertiary care hospital history of primary immunodeficiency diseases survival and predictors of death among primary immunodeficient patients: a registry-based study patients with primary immunodeficiencies in pediatric intensive care unit: outcomes and mortality-related risk factors primary immunodeficiency diseases associated with increased susceptibility to viral infections and malignancies patients with primary immunodeficiencies are a reservoir of poliovirus and a risk to polio eradication virus shedding after human rhinovirus infection in children, adults and patients with hypogammaglobulinaemia human tlrs and il-1rs in host defense: natural insights from evolutionary, epidemiological, and clinical genetics recurrent and sustained viral infections in primary immunodeficiencies severe viral infections and primary immunodeficiencies echovirus type 9 is an important cause of viral encephalitis among infants and young children in kuwait respiratory virus infection in immunocompromised patients dock8 regulates lymphocyte shape integrity for skin antiviral immunity recommendations for live viral and bacterial vaccines in immunodeficient patients and their close contacts inflammation as a tumor promoter in cancer induction virus-specific t cells: current and future use in primary immunodeficiency disorders the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2019 al-herz and essa. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-310217-p9nqcz5d authors: nikolina, basic-jukic title: can hyperimmune anti-cmv globuline substitute for convalescent plasma for treatment of covid-19? date: 2020-05-31 journal: med hypotheses doi: 10.1016/j.mehy.2020.109903 sha: doc_id: 310217 cord_uid: p9nqcz5d information on treatment of covid-19 infection in renal transplant recipients is scarce, especially in symptomatic patients and patients with recent major clinical events. this group of patients suffers from different opportunistic infections which may coexist with covid-19. currently available expert opinions suggest reduction of immunosuppression therapy for renal transplant recipients with symptomatic covid-19 infection with either antiviral drugs, hydroxychloroquine and/or azithromycin. inspired by our experience in treatment of cmv pneumonia and literature data on the potential benefit of convalescent plasma for treatment of different viral diseases we suggest use of the hyperimmune anti-cmv gamma globulins in addition to other available therapies. besides the immunosuppression reduction which is supposed to be beneficial, immunoglobulins with their immunomodulatory effects and possible antiviral role, may increase a possibility for favorable outcome. since december 2019, the coronavirus covid-19 pandemic has affected almost 2,5 million people worldwide with more than 170.000 proven deaths by april 21th 2020 (1). renal transplant recipients are at increased risk for development of infection due to their immunocompromised state, but may also have more severe forms of the disease and an increased mortality risk due to numerous comorbidities. information on treatment of covid-19 infection in renal transplant recipients is scarce, especially in symptomatic patients and patients with recent major clinical events. current epidemiologic situation with the covid-19 pandemic present a great challenge for transplant physitians. lack of experience and well known fact that even in the simplest cases "one size does not fit all", we should more than ever focus on the individual approach to each patient. currently available expert opinions suggest reduction of immunosuppression therapy for renal transplant recipients with symptomatic covid-19 infection. however, a huge gap in knowledge exists for patients with additional problems besides the covid-19 infection. inspired by our experience in treatment of cmv pneumonia and literature data on the potential benefit of convalescent plasma for treatment of different viral diseases we suggest use of the hyperimmune anti-cmv gamma globulins in addition to other available therapies. besides the immunosuppression reduction which is supposed to be beneficial, immunoglobulins with their immunomodulatory effects and possible antiviral role, may increase a possibility for favorable outcome. hyperimmune anti-cmv immunoglobulin is a cmv-specific polyclonal immunoglobulin preparation that binds to cmv surface antigens neutralizing the potential of cmv from entering host cells. additionally, it presents the cmv particle for phagocytosis by binding to the cmv surface. finally, the preparation has immunomodulatory actions which may be beneficial. we decided to use hyperimmune anti-cmv globulins while the preparation contains immunoglobulins directed against the multiple viral pathogens (ebv, measles, parvovirus b19…) (2), and thus may imitate (at least partially) the convalescent plasma. convalescent plasma therapy, has been used in treatment of numerous infectious diseases including sars and mers pandemic (3) . based on the theory that it may neutralize viremia in patients with sars-cov-2 infection, one dose of 200 ml of convalescent plasma derived from recently recovered donors, was transfused to the patients along with the supportive care and antiviral drugs. the treatment was well tolerated, resulted with clinical and laboratory improvement, but with varying degrees of absorption of lung lesions (4, 5) . in conclusion, we suggest the use of hyperimmune anti-cmv immunoglobulins for treatment of covid-19 especially when occur as coinfection with cmv instead of the convalescent plasma which may be unavailable for majority of patient. the author declares no conflict of interests literature internal report. data on file the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis convalescent plasma as a potential therapy for covid-19 effectiveness of convalescent plasma therapy in severe covid-19 patients key: cord-348130-t9tysvr8 authors: cho, sung-yeon; lee, hyeon-jeong; lee, dong-gun title: infectious complications after hematopoietic stem cell transplantation: current status and future perspectives in korea date: 2018-02-27 journal: korean j intern med doi: 10.3904/kjim.2018.036 sha: doc_id: 348130 cord_uid: t9tysvr8 hematopoietic stem cell transplantation (hsct) is a treatment for hematologic malignancies, immune deficiencies, or genetic diseases, ect. recently, the number of hscts performed in korea has increased and the outcomes have improved. however, infectious complications account for most of the morbidity and mortality after hsct. post-hsct infectious complications are usually classified according to the time after hsct: pre-engraftment, immediate post-engraftment, and late post-engraftment period. in addition, the types and risk factors of infectious complications differ according to the stem cell source, donor type, conditioning intensity, region, prophylaxis strategy, and comorbidities, such as graft-versushost disease and invasive fungal infection. in this review, we summarize infectious complications after hsct, focusing on the korean perspectives. hematopoietic stem cell transplantation (hsct), known previously as bone marrow transplantation, is performed to treat two broad categories of diseases. the first category consists of functional failure of bone marrow or marrow-derived cells, including aplastic anemia, myelodysplastic syndrome, immunodeficiency syndromes (severe combined immune deficiency or chronic granulomatous disease), genetic diseases (mucopolysaccharidosis, glycogen storage diseases, etc.), or hemoglobinopathies (thalassemia, sickle cell anemia, etc.). in such cases, hsct is applied to replace the de-fective, non-functional bone marrow tissues. diseases in the second category-which includes hematologic malignancies such as acute or chronic leukemia, multiple myeloma, lymphomas, and myeloproliferative neoplasms-are more common indications for hsct, the performance of which aims (1) to restore the myelosuppressive or myeloablative effect of cytotoxic treatment (such as intensive chemotherapy and/or total body irradiation [tbi]) to eliminate malignant cells, and (2) to induce a graft-versus-leukemic effect by providing anti-neoplastic immune cells expressing tumor-specific or -associated antigens [1, 2] . until the 1970s, hscts were simply classified as autologous or allogeneic according to their donor status, and survival rates were very low. since the introduction of the human leukocyte antigen (hla) concept, the incidence of graft failure and/or graft-versus-host disease (gvhd) has decreased. in recent years, allogeneic hsct has involved various sources of hematopoietic stem cells (bone marrow, peripheral stem cell, cord blood, and mesenchymal cells), donors (sibling, unrelated, haploidentical), and conditioning regimens (standard [myeloablative], reduced intensity). supportive therapies such as transfusion, colony stimulating factors, and antimicrobial agents have also been developed. although various early diagnosis and therapeutic techniques have been developed to improve transplant performance, infectious diseases still affect the prognosis of hsct recipients [2] [3] [4] . in korea, hsct was first performed in 1983 [5] , and the number of transplants has rapidly increased over the past 30 years. in addition to being susceptible to infectious diseases due to neutropenia immediately after hsct, delayed immune recovery is evident over a long period of time after engraftment, depending on the type of hsct, immunosuppressants, and acute or chronic gvhd. because the prevalent infectious diseases vary geographically and over time, state-of-the-art knowledge is needed [6] [7] [8] . in this manuscript, infectious complications after hsct, particularly those common in korea, are reviewed, as well as the epidemiology, diagnosis, preventive or therapeutic strategies, and novel drugs for improving outcomes of patients. the post-hsct period is usually divided into the (1) pre-engraftment period (day 0 to days , (2) immediate post-engraftment period (engraftment to day 100), and (3) late post-engraftment period (days 100 to 365). in general, the neutrophil count recovers 2 to 3 weeks after hsct. however, the functional recovery of various cell types is also important. immunological recovery takes 3 to 6 months for nk cells, 6 to 12 months for b-cells and cd8 t-cells, and 1 to 2 years for cd4 t-cells [9] . the use of t-cell-depleting agents (anti-thymocyte globulin [atg] , alemtuzumab, etc.) to reduce the risk of gvhd and graft failure could improve the outcomes of transplantation but may delay immune recovery [10] . recipients of t-cell-depleted transplantation may be vulnerable to viral infections-such as cytomegalovirus (cmv), epstein-barr virus (ebv), or adenovirus-and adoptive immunotherapy may be applied [11] . during the pre-engraftment period, the risk of opportunistic infection varies depending on the type of anticancer drugs used, conditioning intensity (myeloablative or reduced intensity), and presence of acute gvhd. autologous hsct leads to more rapid recovery of immune function than allogeneic hsct. in the post-engraftment period, the immune system is reconstituted and recovered in autologous hsct recipients. however, allogeneic hsct recipients undergoing long-term immunosuppressive therapy for chronic gvhd remain at risk of infection. table 1 shows the risk factors for, and infectious diseases commonly encountered after, hsct. as shown in table 1 , the infectious diseases that occur before the engraftment are similar to those that develop during the neutropenic phase after chemotherapy [2, 12] . neutrophils are important in innate immunity against microorganisms. a decrease in the number of neutrophils increases the susceptibility to infection. in addition, patients with neutropenia have a reduced number of leukocytes. therefore, inflammatory findings, which are common in patients with normal leukocyte counts, are not often seen, with the exception of fever, which is difficult to diagnose, so the appropriate time to start treatment may be missed [13] . the main sources of bacterial infections in the pre-engraftment phase are the normal gastrointestinal flora and indwelling vascular catheters. gram-negative bacilli (gnb) are common pathogens in the former, while gram-positive cocci (gpc) are more common in the latter [14] . in general, microbiologically defined infections (mdis) account for only 30% to 40% of cases of neutropenic fever [15] . the distribution of causative organisms in febrile neutropenic patients varies geographically. in the united states and europe, gpc accounted for 60% to 70% of mdi until the early 1990s, while the proportion of the korean journal of internal medicine vol. 33, no. 2, march 2018 gnb increased after the mid-2000s. in a multicenter epidemiological survey, the proportions of gram-positive and -negative cases were 55% (range, 30% to 85%) and 45% (range, 15% to 70%), respectively [16] [17] [18] . enterobacteriaceae was the most common causative organism, accounting for 30% (range, 8% to 56%). the second most common organism was coagulase-negative staphylococci (24% [range, 7% to 51%]). in korean studies, gnb were also more common until the mid-2000s, which is consistent with other reports from the asia-pacific region [19] . intestinal bacteria such as escherichia coli, klebsiella pneumoniae, and pseudomonas aeruginosa are frequently reported gnb, and enterococcus, streptococcus, and staphylococcus species are common among gpc [6] [7] [8] [20] [21] [22] . because the epidemiology of bacterial pathogens and the resistance profiles varies, early empirical antimicrobial agents must be selected according to the distributions and susceptibilities of the frequently detected bacterial taxa in that center [13, 15, 19] . in the late post-engraftment phase after autolo-gous hsct, the immune system is reconstituted and restored, which is consistent with the time at which chronic gvhd typically develops after allogeneic hsct. therefore, autologous transplant recipients rarely experience opportunistic infections at this time, but allogeneic hsct recipients (who take chronic immunosuppressive drugs for several months) are at risk of infection. in this period, infectious diseases may continue to develop. in addition, the immune deficiency resulting from chronic gvhd is associated with infections by encapsulated bacteria such as streptococcus pneumoniae, haemophilus influenzae, and neisseria meningitidis [2, 12] . deficiencies in immunoglobulin g2 (igg2) and igg4 have been reported in patients with chronic and severe gvhd, and are associated with severe pneumonia, meningitis, and sepsis due to s. pneumoniae [23] . early diagnosis and treatment are essential, and vaccination should be emphasized in this high-risk group of patients. immunization after hsct will be described in a separate section of this review. over the past decade, the frequency of detection of resistant bacteria has increased worldwide, including in patients with neutropenic fever and hematologic malignancies [24] . among extended-spectrum β-lactamase (esbl)-producing enterobacteriaceae, k. pneumoniae comprises around two-thirds, and e. coli about one-thirds (range, 11% to 69%), depending on the region [18, [25] [26] [27] . in the late 2000s, 26% of e. coli and k. pneumoniae bacteremia isolates from neutropenic patients were es-bl-producing strains in a single-center study [26] . the incidence of infections caused by carbapenem-resistant enterobacteriaceae (cre), along with esbl-producing enterobacteriaceae, is increasing in hematologic malignancy patients [18, [28] [29] [30] [31] . k. pneumoniae, e. coli, p. aeruginosa, and acinetobacter baumannii are clinically important strains associated with the acquisition of carbapenem resistance. a study of prior colonization by cre as a risk factor for cre bloodstream infections found that 45% of neutropenic patients with carbapenem-resistant k. pneumoniae rectal colonization experienced bloodstream infection by an identical strain [30] . there is no multicenter study of cre in hsct recipients. however, a single-center, retrospective cohort study performed in korea reported that the incidence of carbapenem-resistant a. baumannii bacteremia was 0.53 cases per 10,000 patient-days; post-engraftment infection by this organism can be fatal [31] . the rate of methicillin-resistant coagulase-negative staphylococci is reportedly higher than 50% in most centers, while the incidence rate of staphylococcus aureus in hsct patients is low with a high methicillin-resistance rate of median 56% (range, 18% to 100%) [18] . in the late 2000s, coagulase-negative staphylococci were reported to have a methicillin resistance rate of more than 90% and s. aureus a rate of more than 60% [22] . the incidence of enterococcus bloodstream infections was 1.76 cases per 1,000 patient-days, with vancomycin-resistant enterococcus (vre) accounting for 20.6% [22, 32] . most vre bloodstream infections are caused by e. faecium and are associated with long-term hospitalization and an underlying medical condition, but the resistance itself has not been associated with mortality [32] . the epidemiology of drug-resistant pathogens is important for establishing strategies for prophylaxis and initial empirical antimicrobial therapy. because domes-tic data are limited to single-institution and retrospective studies, multicenter nationwide studies of the epidemiology of bacterial infections in korea are required. prophylaxis plays an important role during the pre-engraftment phase ( table 2 ). oral fluoroquinolone prophylaxis can reduce febrile neutropenic episodes and related mortality in allogeneic hsct recipients [33, 34] . according to the guidelines from the infectious diseases society of america and infectious diseases working party of the german society of hematology and oncology, ciprofloxacin and levofloxacin are equally effective, but levofloxacin may be more effective in terms of broader coverage of viridans streptococci when oral mucositis is present [15, 35] . although fluoroquinolone prophylaxis has led to an increase in the frequency of drug-resistant (including quinolones) pathogens, the benefit reportedly outweighs the risk [33] . fluoroquinolone prophylaxis decreased the rate of identification of p. aeruginosa [22] . however, fluoroquinolone prophylaxis is reportedly correlated with resistance development and clostridium difficile-associated diarrhea [36] [37] [38] [39] [40] [41] . moreover, fluoroquinolone prophylaxis may reduce the rate of bloodstream infections, but not the overall mortality rate [42] . therefore, the benefit of routine fluoroquinolone prophylaxis should be weighed against its toxicity and effect on the local epidemiology. in neutropenic patients during the pre-engraftment phase, infections can progress rapidly. in addition, as it is difficult to distinguish between bacterial infection and non-infectious fever in the early stages, empirical antimicrobial therapy is recommended immediately in all patients with febrile neutropenic episodes. except for the presence of definite infection foci, the broad-spectrum β-lactam antibiotics ceftazidime, cefepime, piperacillin/tazobactam, and carbapenem are recommended as they have activity against gram-positive and -negative bacteria, including p. aeruginosa [13, 15, 43, 44] . step-down therapy can be considered after an initial regimen comprising two or more antimicrobial agents, depending on the local epidemiology and resistance profiles. the de-escalation approach is generally recommended when high incidence of resistant pathogens in neuthe korean journal of internal medicine vol. 33, no. 2, march 2018 tropenic fever, or high colonization rates of multiple drug resistance bacteria, or if the patient has a history of infection with drug-resistant pathogens [13, 24, 44] . however, the data on cut-offs for resistance are insufficient to formulate such a strategy. in addition, no randomized controlled study has compared the therapeutic efficacy of the escalation and de-escalation approaches in hsct recipients. selection of the appropriate strategy should take into account the patient's condition and the prevalence of resistant organisms in the center. aspergillus species are the most common causes of invasive fungal infections (ifis) in patients with hematologic diseases, followed by candida spp. and rare fungi [45] . the causative fungi are not different after hsct [46, 47] . the characteristics of, and risk factors for, fungal infections differ according to the phase after transplantation. during the pre-engraftment period, neutropenia and mucosal damage are risk factors for invasive candidiasis [4] . antifungal prophylaxis regimens used during and/or after hsct are listed in table 3 [13, 48] . recent advances in the use of prophylactic antifungal agents during hsct have led to changes in the epidemiology of candidiasis [49] . the korean epidemiologic data show that the incidence of candida albicans infection is markedly reduced by antifungal prophylaxis [22] . nonetheless, the incidence of c. albicans has been decreased by the increasing prophylactic use of fluconazole or echinocandins, and attention should be paid to the selection of antifungal agents for empirical or targeted therapy for invasive candidiasis [50, 51] . post-engraftment, the risk of candida infection decreases with the recovery of neutropenia and mucosal skin loss, but the risk of aspergillosis remains [52] . invasive aspergillosis (ia) that develops during the neutropenic period typically involves angioinvasion due to host immunosuppression. in contrast, ia that occurs in non-neutropenic patients, usually due to long-term use of steroids or immunosuppressive agents for gvhd, is more likely to be caused by local inflammation (i.e., airway invasiveness) than by vascular invasion [53] . in the seifem b-2004 study, the most frequent etiological agents of ifi were aspergillus (70.1%) and candida (24.8%) species. the ifi-attributable mortality rate was 65.3% (72.4% for allogeneic hsct recipients and 34.7% for autologous hsct recipients) [46] . in a prospective observational study performed in the asia-pacific region, table 2 examples of therapy antimicrobial prophylaxis intermediate autologus hsct anticipated neutropenia less than 7-10 days bacteria: consider fluoroquinolone prophylaxis during neutropenia a fungus: consider prophylaxis during neutropenia and for anticipated mucositis, consider pcp prophylaxis virus: during neutropenia or longer depending on risks high allogenenic hsct including unrelated or family mismatched donor anticipated neutropenia more than 10 days prolonged neutropenia secondary neutropenia after engraftment status of malignancy not in remission ghvd with significant steroids treatment (> 20 mg/day of prednisolone equivalents) use of secondary immunosuppressive agents due to refractory gvhd (e.g., tnf-α inhibitor) bacteria: consider fluoroquinolone a fungus: consider prophylaxis during neutropenia, consider pcp prophylaxis virus: during neutropenia or longer depending on risks hsct, hematopoietic stem cell transplantation; pcp, pneumocystis jirovecii pneumonia; gvhd, graft-versus-host disease; tnf-α, tumor necrosis factor-α. a recent data concern the correlation with fluoroquinolone prophylaxis and development of resistance or clostridium difficile associated diarrhea. ia accounted for 65.9% of ifi, followed by 26.7% of invasive candidiasis. interestingly, aspergillus spp. was not the major fungal pathogen in centers in thailand and vietnam, probably because galactomannan testing was unavailable. the heterogeneity of diagnostic, prophylactic, and therapeutic approaches for ifi necessitates local epidemiological data [54] . the incidence of ifi is higher in allogeneic than in autologous hsct, and the established risk factors include chronic gvhd and steroid use [3] . according to a recent multicenter study performed in korea (risk study), the cumulative incidence of ifi after transplantation is 15 [47] . therefore, active anti-mold prophylaxis should be considered in patients with those risk factors. currently, voriconazole is available in korea for secondary prophylaxis of ia after hsct, and po-saconazole for patients on significant immunosuppressive agents for gvhd. the diagnosis of ifi after hsct is not markedly different from that before engraftment. however, the computed tomography (ct) findings of invasive pulmonary aspergillosis in patients with hematologic diseases may differ depending on the time of neutropenia and the time of neutrophil recovery after hsct, suggesting that the diagnostic criteria may need to be redefined [55] . to diagnose ifis, radiologic examinations such as chest x-ray and ct, and microbiological studies including fungal culture, and galactomannan and β-d-glucan assays should be performed periodically. for accurate diagnosis, bronchoscopy and bronchoalveolar lavage (bal) fluid examination, lung biopsy, and culture are required. voriconazole is the drug of choice for treating ia. voriconazole therapeutic drug monitoring can be used in many centers, and the target trough level is 1 to 5.5 mg/l. the cyp2c19 polymorphism affects the pharmacokinetics of voriconazole. among korean hematologic patients, 28% are extensive metabolizers (ems), 48% are heterozygous ems, and 14% are poor metabolizers. while subtherapeutic initial trough levels were common in ems, there was no significant relationship between cyp2c19 genotype and the clinical outcomes of ia or the toxicity of voriconazole [56] . isavuconazole is non-inferior to voriconazole for the primary treatment of suspected invasive mold disease, with fewer drug-related adverse events [57, 58] . salvage therapy includes liposomal amphotericin b, caspofungin, posaconazole, and itraconazole, or a combination of them [58] . treatment duration is at least 6 to 12 weeks, which should be individualized according to the changes in radiologic, microbiologic, and immunologic parameters. ifis can occur during treatment with anti-mold agents. patients with gvhd under long-term immunosuppressive therapy should be on antifungal agents to prevent ifis (table 3 ). in particular, there is a risk of ifis other than aspergillosis, such as mucormycosis or other rare molds [48, 59, 60] . a retrospective single-center study reported that the prevalence and incidence of voriconazole-breakthrough ifis were 2.25% and 0.22 cases per year, respectively. the overall mortality rate was 44.4% [61] . the possible causes of breakthrough ifi during voriconazole treatment were persistent immunodeficiency, neutropenia, low voriconazole concentration, or poor vascular supply (i.e., abscess or necrotic tissue) [61] . in voriconazole-refractory ia, clinicians should consider the following: misdiagnosis or coinfection with another mold, inadequate blood voriconazole concentration, inadequate tissue drug concentration, immune reconstitution inflammatory syndrome, or infection with voriconazole-resistant aspergillus [62] . proven/probable ia patients reportedly have a low culture-positive rate (17.7%) [63] . to determine the azole-resistance rate of aspergillus clinical isolates in korea, culture-positive cases should undergo susceptibility testing. the major symptoms of pneumocystis jirovecii pneumonia (pcp) are fever, difficulty breathing, and dry cough rather than purulent sputum. typical radiologic findings are bilateral diffuse infiltrates originating from the periphery of both lungs, but may be normal at the beginning. pcp can present as segmental consolidation in the upper lobe, or subtle ground glass opacities and pneumothorax. it usually occurs within 6 months after transplantation but also after 6 months of immunosup-pressive therapy for chronic gvhd [3, 4] . the known risk factors for pcp in hematologic patients are acute lymphoblastic leukemia, allogeneic hsct recipients, alemtuzumab, fludarabine, cyclophosphamide, rituximab, and steroid use (> 20 mg/day prednisone for 4 weeks) [64] . if pcp is clinically suspected, ct, bronchoscopy, and bal should be performed as early as possible. in a retrospective study of bronchial washings or bal fluid from non-human immunodeficiency virus (hiv)-infected patients suspected of respiratory infection, among the 169 polymerase chain reaction (pcr)-positive patients, 63.3% were classified as pcp and 46.7% as non-pcp. the majority of patients (84%) in the non-pcp group recovered without treatment for pcp [65] . in two recent meta-analyses, the sensitivity and specificity of quantitative pcr (qpcr) assays were superior to those of non-qpcr assays [66, 67] . the serum β-d-glucan test result has a high negative-predictive value but is not useful for the follow-up of pcp [68] . the prognosis is worse in the presence of concomitant infectious diseases, particularly cmv, aspergillus, and so on. steroids can be used as an adjunctive therapy in patients with hypoxia (pao 2 ≤ 70 mmhg or pao 2 -pao 2 ≥ 35 mmhg). the incidence of pcp has been significantly reduced by trimethoprim/sulfamethoxazole prophylaxis in highrisk patients. high-risk patients who do not take prophylaxis or who show poor compliance with the drug regimen are the main populations at risk of pcp. in a systematic review and meta-analysis of non-hiv immunocompromised hosts (patients with acute leukemia and recipients of hsct and solid organ transplant), the incidence of pcp was reduced by 91% (relative risk [rr], 0.09) in trimethoprim/sulfamethoxazole prophylaxis group compared with placebo [69, 70] . in addition, pcp-related mortality was significantly reduced by 83% (rr, 0.17). however, trimethoprim/sulfamethoxazole prophylaxis did not markedly reduce all-cause mortality in a hematology population [70] . mortality rates remain very high in hematology patients (30% to 59%), particularly in hsct recipients (48% to 70%), compared with 17% to 30% in patients with hiv infection [71] . nevertheless, given the more severe course of pcp and the higher pcp-related mortality rates, trimethoprim/sulfamethoxazole prophylaxis can likely save lives in other www.kjim.org https://doi.org/10.3904/kjim.2018.036 immunocompromised groups as well. pcp should be prevented for at least 6 months or until the immunosuppressant is discontinued in allogeneic hsct recipients [13, 48, 71] . although the optimum duration of pcp prophylaxis is controversial, it is suggested to be continued for a period of time after the immunosuppressant is discontinued. in the setting of corticosteroid-containing regimens, prophylaxis should be continued while steroids are being weaned and/or for 6 weeks after their cessation [72] . with some chemotherapy regimens (i.e., alemtuzumab) consideration should be given to extended pcp prophylaxis for up to 12 months because of the high rate of late-onset pcp [73] . table 4 summarizes the methods of preventing pcp after hsct. cmv infection can occur after hsct and can be fatal. cmv can be reactivated or the patient can be reinfected after transplantation. the spectrum of cmv infection is extensive, from cmv reactivation without organ involvement (presenting mainly as asymptomatic antigen-emia or dnaemia) to cmv disease such as esophagitis, gastritis, colitis, hepatitis, pneumonia, retinitis, and encephalitis. in addition to direct organ involvement, cmv reactivation can exert indirect effects-such as graft failure or immunosuppression-that may result in the development of concurrent bacterial and/or fungal infections [74] . cmv disease, especially cmv pneumonia or encephalitis, can be fatal despite aggressive anti-cmv therapy [75, 76] . a 10-year retrospective single-center study performed in korea reported that cmv disease occurred in 2.9% of allogeneic hsct recipients and 0.5% of autologous hsct recipients. pneumonia (38.6%), retinitis (36.4%), and enteritis (15.9%) frequently developed in patients with cmv infection. the average time of onset of cmv disease was 90 days after transplantation, ranging from 12 to 936 days [77] . another study in the same institution reported that cmv dnaemia developed in about 51% of hsct recipients [78] . cmv reactivation can be associated with a higher non-relapse mortality rate (rr, 1.61 to 1.95) [79] . in another report, 70% of hsct recipients had experienced any level of cmv antigenemia, of whom 41% had received ganciclovir therapy for significant cmv reactivation and 4% had cmv diseases. however, this incidence could be underestimated because disease prevalence was not evaluated in all patients undergoing pre-emptive therapy [80] . because the diagnosis of cmv disease is based on the pathology findings, the diagnosis might be limited depending on the patient's condition. particularly for the diagnosis of cmv pneumonia, obtaining lung tissue from critically ill patients by transbronchial lung biopsy is problematic. one study aimed to measure the cmv level in bronchial washing fluid and suggest a cut-off for pneumonia diagnosis. cmv dnaemia of > 18,900 copies/ml (137,970 iu/ml) may be associated with cmv pneumonia in post-hsct patients, as determined by the receiver operating characteristics curve [81] . however, further data are needed because it is difficult to distinguish whether the viral dna in alveolar hemorrhage reflects viremia rather than lung tissue involvement, or whether cmv is detected as a bystander in bronchial washing fluid. management of cmv is categorized into prevention, pre-emptive treatment, and definitive treatment. pre-emptive therapy is anti-cmv treatment even in the absence of clinical symptoms in cases with cmv infection (reinfection or reactivation). most transplantation centers introduce pre-emptive therapy rather than routine universal prevention because of insurance coverage, cost-benefit ratio, and adverse drug reactions. studies of monitoring strategies and early detection have resulted in the use of cmv pp65 antigenemia testing and real-time qpcr for the surveillance and identification of patients suitable for pre-emptive therapy [82, 83] . however, in hsct, the relationship between cmv viral load and cmv disease is different from that in solid organ transplantation. cmv gastrointestinal disease can develop without preceding cmv antigenemia or dnaemia, while > 75,000 copies/ml of cmv dnaemia is reportedly associated with an increased risk of cmv retinitis after hsct [78, 84] . however, the correlations were moderate, and antigenemia or dnaemia does not necessarily precede or accompany cmv disease. therefore, it is important to identify the at-risk groups and clinical features of the various cmv diseases to facilitate early diagnosis and treatment. a recent prospective double-blind trial evaluated letermovir prophylaxis for preventing cmv in hsct recipients. a total of 565 patients underwent randomization and received letermovir or placebo through week 14 after transplantation. the incidence of clinically significant cmv infection was lower in the letermovir group than in the placebo group by week 24 after transplantation (122 of 325 patients [37.5%] vs. 103 of 170 patients [60.6%], p < 0.001). letermovir prophylaxis resulted in a significantly lower risk of clinically significant cmv infection than placebo. adverse events with letermovir were mainly of low grade [85] . as a result, the u.s. food and drug administration has approved letermovir for the prevention of cmv infection and diseases in adult cmv-seropositive patients undergoing allogeneic hsct. in korea, which has a high cmv-seropositive rate (95% to 100%) in adults, most donors and recipients are seropositive (d+/r+) and the frequency of cmv infection after transplantation is up to 50% [76] . in addition, owing to active cmv monitoring and pre-emptive therapy, there has been a decrease in the incidence of cmv infection immediately after transplantation. however, late cmv infection (> 3 months after transplantation) is increasing, especially if cmv-specific t-cell function has not been restored due to chronic gvhd. therefore, immune monitoring is required in patients with high-risk of cmv disease. cmv-specific immune recovery has been studied in kidney transplantation (kt) and hsct recipients [86] [87] [88] [89] [90] [91] . cmv-specific cytotoxic t lymphocytes (ctls) play an important role in the reconstitution of cmv-specific immunity in immunocompromised patients. the methods used to evaluate cmv-specific immunity include tetramer assay, intracellular cytokine analysis by flow cytometry, measurement of interferon-γ secretion by cmv-specific cd8 t-cells, and enzyme-linked immunospot assay using ie-1 and pp65 peptide pools [92] . quantification of cmv-specific t-cell immunity after hsct facilitates the identification of patients at risk of cmv-related complications [87, 88] . currently available anti-cmv agents in korea are ganciclovir, valganciclovir, foscarnet, and cidofovir. since february 2018, the use of valganciclovir in hsct patients with cmv infection has increased due to changes in the reimbursement rules. regarding adverse events, attention should be paid to bone marrow suppression, and in patients with renal insufficiency. gvhd or the use of www.kjim.org https://doi.org/10.3904/kjim.2018.036 monoclonal antibodies (i.e., alemtuzumab) can increase the incidence of cmv infections. such infections may not be distinguishable from gvhd, or may coexist with gvhd, and may not respond to antiviral drugs if diagnosis is delayed [93] . if there is persistent infection despite anti-cmv therapy, cmv refractoriness can be considered if a > 1 log 10 decrease in cmv dna level in blood or plasma is not achieved after ≥ 2 weeks of treatment. in cmv-refractory cases, resistance should also be suspected. although cmv resistance remains uncommon in hsct recipients from hla-matched donors (0% to 7.9%), in high-risk patients, the incidence of cmv resistance is up to 14.5% [94] [95] [96] . the gold standard of cmv resistance testing is an increase in the ic 50 (half maximal inhibitory concentration) value by plaque reduction assay. however, cmv resistance can be defined when one or more genetic mutations associated with ganciclovir, valganciclovir, foscarnet, and cidofocir are identified with clinical refractoriness. therefore, in recent years, mutations in ul97 and ul54 can be tested. predisposing factors for cmv resistance include prolonged use of anti-cmv agents, recurrent cmv infections, inadequate antiviral absorption, subtherapeutic antiviral level, haploidentical transplantation, or t-cell depletion [97] . if resistance is confirmed, a drug to which resistance has not been identified, or combination of drugs, may be used, but treatment is currently limited. clinician's experience is important in such complicated cases. herpes zoster is caused by the reactivation of virus latent in the posterior ganglia after primary infection with varicella zoster virus (vzv). in korea, vzv is transmitted by natural infection in most cases and its seroprevalence in adults is > 90%; previous vzv infection is a prerequisite for herpes zoster [98] [99] [100] . the clinical features of herpes zoster include abnormal sensation or pain through skin segments 2 to 3 days before skin lesions develop, erythematous spots with irritation, and rapid formation of blisters. the blisters burst and form ulcers, which scar and become dry. pain and postherpetic neuralgia are major problems. in most cases, one or two dermatomes are involved unilaterally, but dissemination to several other dermatomes or systemically can occur and is associated with visceral or central nervous system involvement [101] . treatment consists of antiviral administration and adjuvant therapy to reduce the acute pain and postherpetic neuralgia. it is not clear whether antiviral treatment is effective 72 hours after the onset of rash. however, immunocompromised patients with persistent or new vesicular rash, eye involvement, and/or neurologic complications should receive antiviral treatment even > 72 hours after the onset [1] . the risk of varicella is highest in the first 24 months after hcst, or during immunosuppressive therapy for gvhd [101] . in a korean transplantation center, 34.3% of patients who underwent allogeneic hsct in 2004 to 2005 experienced herpes zoster. the cumulative incidence was 22% at 1 year post-transplant, and 30.8%, 38.6%, and 41.2% at 2, 3, and 4 years (fig. 1) . chest dermatomes were most commonly involved (43.8%), and 11.4% of cases were disseminated. there was no herpes-zoster-related mortality. a live attenuated zoster vaccine is available but cannot be used for hsct recipients. acyclovir prophylaxis can reduce the incidence of herpes simplex virus (hsv) and vzv infections not only before engraftment but also in the long term until the immunosuppressant is stopped [102] . it is recommended that acyclovir be maintained for at least 1 year after allogeneic hsct and for 6 to 12 months after autologous hsct [3, 48] . antiviral prophy ebv reactivation can occur 3 to 6 months after transplantation, typically in patients with chronic gvhd. however, progression to disease is relatively rare. fever and neutropenia may occur as a result of ebv symptoms, which are similar to those of infectious mononucleosis. most ebv reactivations are subclinical and require no therapy [101] . in addition, aplastic anemia, oral hairy leukoplakia, and post-transplant lymphoproliferative disease (ptld) can occur. ptld occurs less frequently after hsct than after transplantation of other solid organs. ebv-related ptld occurs in cases of unrelated donor transplantation, t-cell-depleted transplantation, gvhd, and use of an anti-lymphocyte antibody to prevent gvhd [104] . the diagnosis of ebv-associated ptld can be established by tissue biopsy for histopathology and detection of ebv [105] . among other herpesviruses, human herpes virus 6 (hhv6) may be responsible for meningitis and hemorrhagic cystitis [106] . hemorrhagic cystitis in hsct recipients can be classified according to the onset time (before vs. after engraftment), and cases occurring within 7 days after transplantation are usually non-infectious. non-infectious causes include radiotherapy and chemotherapy (e.g., cyclophosphamide, ifosfamide, busulfan, and etoposide), while post-engraftment hemorrhagic cystitis can be caused by virus. viral reactivation can be accompanied by bladder urothelial damage due to the conditioning regimen. viral hemorrhagic cystitis can be caused by polyomaviruses (bk and jc viruses), adenovirus, cmv, hsv, and hhv6 [107, 108] . of these viruses, bk virus (bkv) is the most common; 80% to 90% of adults are bkv seropositive [109] [110] [111] . bkv-associated hemorrhagic cystitis is predominant in allogeneic hsct patients, and bkv-associated nephropathy in kt patients. why bkv reactivation manifests as these two major forms in kt and hsct patients is unclear [112] . the diagnostic criteria defined by the european conference on infections in leukemia (ecil)-6 comprise the following triad: viral replication (urine bkv > 10 7 copies/ml), symptoms of cystitis, and hematuria of grade ii or higher. a blood bkv dna level of > 10 3-4 copies/ml is reportedly associated with significant viruria. however, a negative plasma viral load does not rule out bkv-associated hemorrhagic cystitis [113] [114] [115] . the risk factors for hemorrhagic cystitis are myeloablative conditioning regimen, atg, and gvhd. the incidence is higher in allogeneic than in autologous hsct patients, and in adults than in pediatric cases. treatment for virus-associated hemorrhagic cystitis should be initiated by minimizing immunosuppressants. cidofovir might be effective for adenovirus, cmv, and bkv, and can be considered if there is ≥ grade iii hematuria (gross hematuria with blood clots) and no response to conservative treatment [113] . in eight retrospective and two prospective studies, the use of cidofovir with or without probenecid (either 3 to 5 mg/kg with probenecid or 0.5 to 1.5 mg/kg without probenecid) was investigated. a reduction in the urine and blood bkv load reportedly occurs in 38% to 62% and 67% to 84% of patients, respectively [116] [117] [118] [119] [120] [121] [122] . a retrospective study did not recommend levofloxacin because of insufficient data and the low level of evidence [113] . in addition, retrospective data are available for vidarabine, leflunomide, hyaluronic acid, and mesenchymal cells, but these are not recommended by the guidelines [113] . brincidofovir markedly reduces bkv replication in vitro [123] . in cases of persistent hematuria, renal function can be impaired by blood clots and related post-renal obstructions. conservative care such as intravenous hydration and removal of obstructions is also important. tients. crv infections can also occur during neutropenia and show significant morbidity and mortality. the epidemiology of crv infection in hsct patients is likely to reflect that in the community, with seasonal variations. influenza and respiratory syncytial virus (rsv) infections usually occur in winter, parainfluenza virus (piv) infections in summer, and rhinovirus throughout the year. in hsct recipients, crv infection is not limited to urtis and is more likely to progress to lrtis [124, 125] . respiratory virus multiplex pcr enables rapid diagnosis of crv infections in clinical practice [126] . there are little data on crv infection in hsct patients in korea. in a 4-year retrospective study in the authors' hsct center from 2007 to 2011, 67 of 1,038 hsct patients (6.5%) had 71 cases of crv-lrti. rsv (43.6%) was the most common pathogen of crv-lrti, followed by piv (26.8%), influenza virus (19.7%), and rhinovirus (9.9%) [127] . the overall mortality rate at day 30 after crv-lrti was 32.8%, and high-dose steroid usage (> 1 mg/kg/day), severe immunodeficiency, and lymphopenia (absolute lymphocyte count < 200 cells/mm 3 ) were significantly associated with mortality [127] . these findings are similar to the prevalence, mortality rate, and mortality-related risk factors of crv-lrti in europe and the united states. the average mortality rate of rsv-lrti is 32% (range, 0% to 70%) in international studies, and the major risk factors for progression to lrti are lymphopenia, old age, mismatched/unrelated donor, and neutropenia [128, 129] . in cases of piv-lrti, the overall mortality rate is 10% to 30%, and high-level corticosteroid exposure, neutropenia, lymphopenia, and early onset after hsct are the major risk factors for lrti [129] [130] [131] . the rate of progression of influenza to lrti is 25% to 28% and the overall mortality rate is 25% to 58% [132] [133] [134] . the risk factors for progression to influenza-lrti are early onset after hsct, lymphopenia, old age, neutropenia, and delayed antiviral administration [132] [133] [134] . in cases of rhinovirus, most infections are asymptomatic, less than 10% of patients progress to definite pneumonia, and the mortality rate is < 10% [135, 136] . in addition, human metapneumovirus, adenovirus, coronavirus, and bocavirus can cause urtis and lrtis in hsct patients [129, 137] . management of crv infection in hsct patients can be classified into four categories: prevention (infection control), selective therapy for urti, antiviral therapy, and supportive care. currently, a commercialized vaccine is available only for influenza, and so preventive strategies to stop the spread of crv infections-such as hand hygiene, wearing gloves and mask, and isolation of symptomatic patients-have been emphasized [129, 134, 137, 138] . it is recommended that healthcare workers and family members of patients be vaccinated against influenza, as well as undergo post-exposure prophylaxis (in the korea influenza guidelines, oseltamivir can be administered prophylactically for 10 days if an immunocompromised patient comes into contact with a patient with confirmed influenza or influenza-like illness) [134, [137] [138] [139] . because some crv-urtis do not progress to lrti and therapeutic agents are limited, it is important to identify crv-urti patients at increased risk of lrti [129, 137, 140] . treatment of rsv-urti with aerosolized ribavirin significantly reduces the frequency of progression to lrti (25% vs. 47%) [128] . in addition, the following immunodeficiency scoring index has been proposed: neutropenia (< 500 cells/mm 3 , 3 points), lymphopenia (< 200 cells/mm 3 , 3 points), age ≥ 40 years (2 points), myeloablative conditioning (1 point), gvhd (acute/chronic, 1 point), corticosteroids (1 point), and pre-engraftment or within 30 days of transplant (1 point). a score of > 7 is associated with a significantly increased risk of progression to lrti, suggesting the necessity of criteria for identifying high-risk groups [141] . palivizumab, a humanized anti-rsv monoclonal antibody, does not reduce the progression to lrti in hsct patients and is currently recommended only for prophylactic usage in high-risk children [142] . the risk factors for progression to lrti have been discussed for piv, human metapneumovirus, and rhinovirus urtis, but treatment is not recommended because of a lack of evidence [129, 137, 140] . oseltamivir, zanamivir, and peramivir can be used to treat influenza urtis and lrtis and should be administered within 48 hours of symptom onset [134, 139] . delayed administration is also beneficial, so these agents should be administered even after 48 hours [134, 139] . although there is no definitive evidence, most guidelines recommend that antiviral treatment be extended the korean journal of internal medicine vol. 33, no. 2, march 2018 beyond the usual duration in cases of severe influenza lrti [134, 139, 143] . aerosolized ribavirin and intravenous immunoglobulin (ivig) combination treatment significantly reduces rsv-lrti-related mortality and is thus recommended by most guidelines [129, 137, 140, 141] . although evidence is lacking, if aerosolized ribavirin cannot be used, oral ribavirin or intravenous ribavirin may be considered [129, 137, 140, 141] . a recent double-blind, placebo-controlled study of gs-5806 (presatovir), an oral rsv-entry inhibitor, involved 54 rsv-infected healthy adult volunteers who received rsv challenge intranasally. gs-5806 reduced the viral load and the severity of clinical disease [143] . proven piv-lrti should be treated with aerosolized ribavirin and ivig in combination [129, 137, 140] . unfortunately, aerosolized ribavirin is costly and available only through the korea orphan & essential drug center in korea, so its use is limited. no antiviral is available to treat crv, rhinovirus, human metapneumovirus, and bocavirus lrtis, so supportive care is the mainstay. hsct patients with crv infection are often susceptible to other pathogens, and so work-up for co-existent pathogens is required [129, 137, 140] . s. aureus and s. pneumoniae infections are commonly followed by influenza infection. therefore, patients with influenza lrtis should be administered antibiotics to which these bacteria are susceptible [144] . as it is not possible to distinguish these bacteria from other co-pathogens (i.e., pcp, cmv, or fungi, etc.) using only imaging tests and clinical symptoms, examinations such as bronchoscopy are required [129, 137, 140] . tuberculosis (tb) is an important opportunistic infection among hsct recipients, in whom its incidence is 2-to 40-fold higher than that in the general population; however, this is lower than in solid-organ-transplant patients [145] [146] [147] . hsct recipients are immunosuppressed as a result of their hematologic disease, chemotherapy, radiotherapy, immunosuppressive agents, and gvhd [146] . several risk factors for the development of tb after hsct have been reported, including acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, busulfan, cyclophosphamide, tbi, corticosteroid therapy, hla-mismatched transplant, gvhd, or a history of tb infection [148] . of hsct patients who develop tb, 80% and 20% had undergone allogeneic and autologous transplant, respectively. a single-center study performed in korea reported that 2.5% of allogeneic hsct recipients were diagnosed with tb after hsct. the median time to development of tb was 386 days after transplantation (range, 49 to 886). the standardized incidence ratio of tb, compared with that in the general population, was 9.10 (95% ci, 5.59 to 14.79). extensive chronic gvhd was associated with the development of tb (p = 0.003) (fig. 2) [146] . another center in korea reported that hsct patients with tbi-based conditioning are likely to have tb disease [149] . extrapulmonary tb comprises 42% of post-hsct tb cases, which is higher than that in the general population (15% to 20%) [146] . owing to the risk of reactivation or new infection, prophylaxis is recommended for hsct recipients with a positive tb-specific interferon-γ release assay result [3] . isoniazid is well tolerated in the post-hsct period. however, concurrent itraconazole is not recommended due to drug interactions, and the impact of voriconazole or posaconazole is unclear. isoniazid should be continued for at least 9 months until immunosuppression is reduced. rifampin may undergo drug-drug interactions with immunosuppressants, which makes this option not practical. to determine the optimum timing and duration of isoniazide prophylaxis in hsct patients, further studies are needed. post-hsct vaccination is recommended regardless of the type of transplantation and source of stem cells. guidelines recommend that both allogeneic and autologous hsct recipients should be immunized as scheduled even during immunosuppressive therapy for chronic gvhd [150, 151] . however, if > 0.5 mg/kg prednisolone is administered for gvhd, it may be temporarily delayed to increase the immune response until the dosage of immunosuppressants become decreased. the recommended schedules in korea are presented in table 5 [152] . hsct is performed to treat various hematologic malignancies and other disorders. although survival rates have improved due to developments in transplantation techniques, infectious complications remain major causes of morbidity and mortality after hsct. the severity of infectious complications varies according to the phase post-transplant, type of transplantation, gvhd, and the degree of immunosuppression or reconstitution. further studies are required to improve the treatment of infectious diseases, and the outcomes of hsct patients are expected to improve in future. no potential conflict of interest relevant to this article was reported. hsct, hematopoietic stem cell transplantation. a following the three doses of 13-valent pneumococcal conjugate vaccine (pcv), a dose of 23-valent polysaccharide pneumococcal vaccine may be given to broaden the covered spectrum. in hsct recipients with chronic graft-versus-host disease (gvhd) who are likely to respond poorly to polysaccharide vaccine, a fourth pcv should be considered at intervals of more than 6 months. b dtap (diphtheria-tetanus-reduced acellular pertussis vaccine) is preferred over tetanus toxoid-reduced diphtheria toxoid-reduced acellular pertussis vaccine (tdap). if only tdap is available, it can be used. c re-immunization with td (tetanus toxoid-reduced diphtheria toxoid vaccine) or tdap at least every 10 years. d indicated ≥ 24 months after hsct (if no gvhd or ongoing immunosuppression and patient is seronegative for each vaccine). common infectious diseases in hematopoietic stem cell transplant recipients hematopoietic stem cell transplantation: an overview of infection risks and epidemiology guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective infections in patients with hematologic neoplasms and hematopoietic stem cell transplantation: neutropenia, humoral, and splenic defects the activity of hematopoietic stem cell transplantation in korea infectious complications and outcomes after allogeneic hematopoietic stem cell transplantation in korea current trends of infectious complications following hematopoietic stem cell transplantation in a 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prophylaxis in haematological cancer patients with neutropenia: ecil critical appraisal of previous guidelines evidence-based approach to treatment of febrile neutropenia in hematologic malignancies. hematology european guidelines for empirical antibacterial therapy for febrile neutropenic patients in the era of growing resistance: summary of the epidemiology and treatment outcome of invasive fungal infections in patients with hematological malignancies fungal infections in recipients of hematopoietic stem cell transplants: results of the seifem b-2004 study. sorveglianza epidemiologica infezioni fungine nelle emopatie maligne epidemiology and risk factors for invasive fungal diseases among allogeneic hematopoietic stem cell transplant recipients in korea: results of national comprehensive cancer network. national comprehensive cancer network clinical practice guidelines in oncology: prevention and treatment of cancer-related infections pa): nccn, c2018 fungal infections in leukemia 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the respiratory virus multiplex reverse transcription-polymerase chain reaction test for adult patients at a korean tertiary care center lower respiratory tract diseases caused by common respiratory viruses among stem cell transplantation recipients: a single center experience in korea management of rsv infections in adult recipients of hematopoietic stem cell transplantation ecil-4): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus the characteristics and outcomes of parainfluenza virus infections in 200 patients with leukemia or recipients of hematopoietic stem cell transplantation parainfluenza virus infections after hematopoietic stem cell transplantation: risk factors, response to antiviral therapy, and effect on transplant outcome outcome of pandemic h1n1 infections in hematopoietic stem cell transplant recipients risk factors for pneumonia in immunocompromised patients with influenza 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oncology immunodeficiency scoring index to predict poor outcomes in hematopoietic cell transplant recipients with rsv infections palivizumab treatment of respiratory syncytial virus infection after allogeneic hematopoietic stem cell transplantation oral gs-5806 activity in a respiratory syncytial virus challenge study severe influenza treatment guideline the risk of tuberculosis in transplant candidates and recipients: a tbnet consensus statement the demanding attention of tuberculosis in allogeneic hematopoietic stem cell transplantation recipients: high incidence compared with general population long-term risk of tuberculosis in haematopoietic stem cell transplant recipients: a 10-year nationwide study infections caused by mycobacterium tuberculosis in recipients of hematopoietic stem cell transplantation tuberculosis in hematopoietic stem cell transplant recipients in korea vaccinations in patients with hematological malignancies a comprehensive review of immunization practices in solid organ transplant and hematopoietic stem cell transplant recipients vaccination of hematopoietic stem cell transplantation recipients: perspective in korea key: cord-342000-h4jo2bir authors: aggarwal, ashim; pyle, joseph; hamilton, john; bhat, geetha title: early cardiac allograft vasculopathy: are the viruses to blame? date: 2012-05-31 journal: case rep med doi: 10.1155/2012/734074 sha: doc_id: 342000 cord_uid: h4jo2bir this paper describes a case of early (7 months after transplant) cardiac allograft vasculopathy. this-43-year-old (cmv positive, ebv negative) female patient underwent an orthotopic heart transplant with a (cmv negative, ebv positive) donor heart. she had a history of herpes zoster infection and postherpetic neuralgia in the past. the patient's panel reactive antibodies had been almost undetectable on routine surveillance testing, and her surveillance endomyocardial biopsies apart from a few episodes of mild-to-moderate acute cellular rejection (treated adequately with steroids) never showed any evidence of humoral rejection. the postoperative course was complicated by multiple admissions for upper respiratory symptoms, and the patient tested positive for entero, rhino, and coronaviruses serologies. during her last admission (seven months postoperatively) the patient developed mild left ventricular dysfunction with an ejection fraction of 40%. the patient's endomyocardial biopsy done at that time revealed concentric intimal proliferation and inflammation resulting in near-total luminal occlusion in the epicardial and the intramyocardial coronary vessels, suggestive of graft vasculopathy with no evidence of rejection, and the patient had a fatal ventricular arrhythmia. cardiac transplantation is a well-defined therapy for endstage heart failure. following transplantation, median survival is 10 years rising to 13 years for those who survive the first year [1] . the leading causes of death in the first year following transplantation include infection, rejection, and graft failure. cardiac allograft vasculopathy (cav) is the second leading cause of death after 1 year following transplantation, second only to malignancy [1] . we present a case of early (7 months after transplant) cardiac allograft vasculopathy (cav) in a cytomegalovirus (cmv) positive patient with a history of herpes zoster infection and multiple other viral infections in the postoperative course possibly contributing to graft vasculopathy. a 43-year-old caucasian female with a history of nonischemic dilated cardiomyopathy with left ventricular ejection fraction (lvef) of 10-15% status following thoratec heart-mate ii left ventricular assist device (lvad) (implanted 2 years prior as a bridge to transplant) was transferred to our tertiary care facility for management of unresolving pseudomonas driveline infection. the patient secondary to persistent pseudomonas bacteremia despite adequate treatment with intravenous antibiotics underwent lvad removal with reimplantation with another vad. the patient also underwent an aicd lead extraction with generator change secondary to questionable vegetation on the defibrillator lead on transesophageal echocardiogram. the patient did well following that and remained home for 4 months while awaiting a cardiac transplant. her past history was significant for hypertension, dyslipidemia, recurrent pulmonary embolism, history of herpes zoster infection with postherpetic neuralgia, and intracerebral hemorrhage. four months later the patient was electively admitted for transplant evaluation. her panel reactive antibody (pra) levels were found to be low at 4% as measured by flow cytometry using hla class i luminex-coated beads. the patient (cmv positive) finally underwent a cmv negative, epstein-barr virus (ebv) positive orthotopic heart transplant without the need for desensitization. the patient's immediate postoperative course was complicated by multiple failed attempts at extubation secondary to fluid overload that required tracheostomy and acute kidney injury requiring temporary hemodialysis (with complete eventual recovery of renal function). the patient after 4 weeks, on routine surveillance endomyocardial biopsy (emb), was found to have ishlt grade 2r acute cellular rejection which was successfully treated with intravenous pulsed steroids and mycophenolate mofetil. the patient was eventually discharged home 2 weeks later and was followed as an outpatient. three months subsequent to transplant the patient started to develop signs and symptoms of upper respiratory tract infections manifesting as unremitting cough. the patient admitted was found to have viral infection with positive serologies for entero, rhino, and coronaviruses, and the emb was negative for rejection. the patient was managed conservatively without any antiviral treatment except prophylactic ganciclovir for cmv prophylaxis and discharged home. the patient did present again with similar respiratory symptoms a month later at which time it was decided to treat the patient with a course of oseltamivir (tamiflu) for a clinical suspicion of influenza. the patient was discharged only to be readmitted 2 months later (6 months after transplant) for symptoms of exertional dyspnea, nausea, and abdominal pain. the patient was found to have low cardiac index (1.59 l/min/m 2 ) and elevated right sided pressures on right heart catheterization while the emb remained negative for cellular or humoral rejection. an echocardiogram at the time revealed a mildly depressed left ventricular ejection fraction at 40% with mild right ventricular dysfunction. the patient's panel reactive antibodies were undetectable. table 1 lists the trends in the available viral titres and other laboratory data (glucose and lipids). the patient was treated with intravenous methylprednisolone and plasmapheresis to treat for possible graft dysfunction. the next day the patient had a sudden cardiorespiratory arrest and died despite prolonged attempts at resuscitation. a postmortem analysis revealed microscopic changes of concentric intimal proliferation and inflammation resulting in near-total luminal occlusion in the epicardial and the intramyocardial coronary vessels, suggestive of graft vasculopathy (figures 1(a)-1(c) ). there was no evidence of rejection seen. cardiac transplantation is the definitive treatment for eligible patients with end-stage heart failure. cav and graft failure are the leading cause of death in patients who survive the first year after transplant. despite a small decrease in the cumulative incidence documented recently, the incidence of cav after transplant remains significant: 8% at 1 year, 20% at 3 years, 30% at 5 years, and more than 50% at 10 years [1] . cav is a risk factor for long-term mortality, but the diagnosis of cav also carries a short-term mortality riskapproximately 10% of patients die in the 12 months after the diagnosis cav [1] . the diagnosis of cav is challenging because of deinnervation as well as the concentric and diffuse nature of the disease. classical symptoms of angina are often missing, and patients tend to present with heart failure [2] . there are multiple purported etiologies for graft coronary artery disease including both immunologic and nonimmunologic factors. immunologic factors include human leukocyte antigen mismatching, cytokine production, and activation of the cellular immune system. nonimmunologic factors include diabetes, hypertension, and hyperlipidemia [3, 4] . there is also accumulating data suggesting that infections play a role in atherosclerosis and in cav. cmv is one of the pathogens that has been most convincingly implicated in the pathophysiology of cav [5] . viral infections including those due to cmv have been associated with accelerated cav [6, 7] . positive pretransplantation cmv serology has been shown to be a risk factor for cav in children [8, 9] . cardiovascular risk has also been associated with seropositivity for chlamydia pneumonia, helicobacter pylori, cmv, and other herpes viruses. however, more recent studies in larger patient cohorts have demonstrated only modest associations [10, 11] . given the modest and variable risk that has been linked to individual pathogens, zhu and colleagues have proposed the sum of the relevant infectious agents, defined as the "total pathogen burden," as the important risk factor to be considered. exposure to a panel of five pathogens was found to be predictive of angiographic disease [12] and cardiovascular events [13] . similar results were reported in a study showing that exposure to increasing number of pathogens was associated with increased risk [14] . an increasing "pathogen burden" defined as the aggregate number of positive serologies (mainly driven by burden of herpesviridae) was significantly predictive of the long-term prognosis in a doseresponse manner. these observations have led the investigators to propose the "herpes burden" (aggregate seropositivity to cmv, herpes simplex-1 and -2, and ebv) as a more effective predictor of cardiovascular risk [14] . our patient had a cmv positive status who received an ebv positive donor heart along with a history of documented herpes zoster infection in the past. whether the multiple serologically positive viral infections (entero, rhino, and coronaviruses) amongst the many other nonimmune factors augmented the existent viral burden thereby triggering a selfperpetuating immune cascade, inducing inflammation and intimal proliferation, is a concept that can only be hypothesized and arduous to prove. kumar [15] . this study adds to the increasing evidence highlighting a possible immunogenic potential of respiratory viruses in solid organ recipients manifesting as acute or chronic rejection. treatment of established cav has been disappointing. the options have included adjusting the immunosuppressive regimen, percutaneous or surgical revascularization or retransplantation. because cav has such an important influence on morbidity and mortality, it is evident that if further improvement in graft and patient survival is to be made, attention must focus on reducing the risk of cav. there is proven benefit from lipid lowering therapy, mammalian target of rapamycin (mtor) inhibitors (everolimus and sirolimus), and possibly diltiazem (calcium channel blocker) [4] . our case is an addition to a list of very limited number of cases of early allograft vasculopathy (within the first year after transplant) that aims to highlight its poor clinical outcome and survival compared to the form that develops after the first year. the lack of data on the patients viral titres is an important limitation of this case. allograft coronary disease remains one of the greatest obstacles to long-term recipient survival after cardiac transplantation. this process has been extensively studied, yet remains poorly understood. although it typically becomes clinically significant years after transplantation, it can also occur early and result in recipient death as early as 6 months following transplant. why a given patient develops an aggressive form of the disease early after transplantation and another patient may live ten or even twenty years without evidence of clinically significant disease is unknown. the authors of this paper declare that they have no conflict of interests. furthermore, no commercial organization took part in its preparation or funding. the registry of the international society for heart and lung transplantation: twenty-seventh official adult heart transplant report-2010 cardiac allograft vasculopathy: advances in diagnosis cardiac allograft vasculopathy: pathology, prevention and treatment cardiac allograft vasculopathy: recent developments the role of viruses in cardiac allograft vasculopathy viral infection induces de novo lesions of coronary allograft vasculopathy through a natural killer cell-dependent pathway prophylaxis versus preemptive anti-cytomegalovirus approach for prevention of allograft vasculopathy in heart transplant recipients positive pretransplantation cytomegalovirus serology is a risk factor for cardiac allograft vasculopathy in children does cytomegalovirus serology impact outcome after pediatric heart transplantation? coronary heart disease, helicobacter pylori, dental disease, chlamydia pneumoniae, and cytomegalovirus: metaanalyses of prospective studies chlamydia pneumoniae igg titres and coronary heart disease: prospective study and meta-analysis effects of total pathogen burden on coronary artery disease risk and creactive protein levels prospective study of pathogen burden and risk of myocardial infarction or death impact of viral and bacterial infectious burden on long-term prognosis in patients with coronary artery disease a prospective molecular surveillance study evaluating the clinical impact of community-acquired respiratory viruses in lung transplant recipients key: cord-335692-5uxtua9o authors: kilic, a.; elliott, s.; hester, l.; palavecino, e. title: evaluation of the performance of diasorin molecular pneumocystis jirovecii-cmv multiplex real-time pcr assay from bronchoalveolar lavage samples date: 2020-01-31 journal: j mycol med doi: 10.1016/j.mycmed.2020.100936 sha: doc_id: 335692 cord_uid: 5uxtua9o the aim of this study was to evaluate the performance of the diasorin molecular pj-cmv multiplex real-time pcr (pj-cmv pcr) assay (diasorin molecular llc, usa) in bronchoalveolar lavage (bal) samples compared to direct immunofluorescence assay (ifa) for the detection of pneumocystis jirovecii and assess cmv and p. jirovecii co-infection rate in immunosuppressed patients with suspected pneumonia. a total of 125 bal samples from immunosuppressed patients submitted for pjp-ifa were tested. surplus samples were saved and further tested by using the pj-cmv pcr assay. among the 125 samples, p. jirovecii was detected in 31.2% (39/125) and in 40% (50/125) of the specimens using ifa and pj-cmv pcr respectively. eleven of the pj-cmv pcr positive samples were negative by direct ifa for p. jirovecii. all samples positive by direct ifa were also positive by pj-cmv pcr. using the direct ifa as a gold standard, the pj-cmv pcr sensitivity, specificity, positive predictive value and negative predictive value for detection of p. jirovecii were 100%, 87.2%, 78% and 100%, respectively. however, after reviewing the clinical diagnosis, the specificity and ppv increased to 100%. of the 50 p. jirovecii samples positive by pj-cmv pcr, 18 (36%) were also positive for cmv by the pj-cmv pcr. the co-infection rate was found to be 37.5% (6/16) and 35.2% (12/34) in hiv infected and non-hiv infected patients. this study indicated that the diasorin molecular pj-cmv multiplex real-time pcr assay has higher sensitivity than direct ifa for detection of p. jirovecii and provides rapid detection of pj and cmv infection in bal samples. the aim of this study was to evaluate the performance of the diasorin molecular pj-cmv multiplex realtime pcr (pj-cmv pcr) assay (diasorin molecular llc, usa) in bronchoalveolar lavage (bal) samples compared to direct immunofluorescence assay (ifa) for the detection of pneumocystis jirovecii and assess cmv and p. jirovecii co-infection rate in immunosuppressed patients with suspected pneumonia. a total of 125 bal samples from immunosuppressed patients submitted for pjp-ifa were tested. surplus samples were saved and further tested by using the pj-cmv pcr assay. among the 125 samples, p. jirovecii was detected in 31.2% (39/125) and in 40% (50/125) of the specimens using ifa and pj-cmv pcr respectively. eleven of the pj-cmv pcr positive samples were negative by direct ifa for p. jirovecii. all samples positive by direct ifa were also positive by pj-cmv pcr. using the direct ifa as a gold standard, the pj-cmv pcr sensitivity, specificity, positive predictive value and negative predictive value for detection of p. jirovecii were 100%, 87.2%, 78% and 100%, respectively. however, after reviewing the clinical diagnosis, the specificity and ppv increased to 100%. of the 50 p. jirovecii samples positive by pj-cmv pcr, 18 (36%) were also positive for cmv by the pj-cmv pcr. the co-infection rate was found to be 37.5% (6/16) and 35.2% (12/34) in hiv infected and non-hiv infected patients. this study indicated that the diasorin molecular pj-cmv multiplex real-time pcr assay has higher sensitivity than direct ifa for detection of p. jirovecii and provides rapid detection of pj and cmv infection in bal samples. c 2020 elsevier masson sas. all rights reserved. clinical suspicion of pjp were tested for p. jirovecii by direct ifa using the monofluo p. jirovecii ifa test kit (monofluo tm bio-rad, laboratories, usa) according to the manufacturer's instructions. according to the clinical data reviewed, all patients evaluated were considered immunosuppressed and bal samples were submitted for testing because patients met clinical and radiographic criteria for possible pjp and other infections had been ruled out. the non-hiv infected patients included patients with hemato. data collection and analysis were performed with liaison 1 mdx studio software. the following cycling conditions were used: 1 cycle at 978c for 120 s followed by 40 cycles of 978c for 10 s with a ramp speed of 28c/s, and 608c for 30 s with a ramp speed of 28c/s with capture mode on. the test time of the pj-cmv pcr assay from sample processing to final result is approximately 60 minutes. extraction and amplification controls were used to detect pcr failure and/or inhibition. positive and negative control samples were included in each run. both cmv and pj positive molecular controls were purchased from exact diagnostics (exact diagnostics, fort worth, tx). standard procedures were used to obtain the analytical sensitivity of the test for the detection of p. jirovecii and cmv. briefly, commercially available controls (cmv ad 169-zeptometrix, buffalo, ny, and p. jirovecii pcpp100-exact diagnostics, fort worth, tx) were chosen for the limit of detection (lod) determination. serial dilutions were prepared in a pooled bal negative matrix ranging from 5000 copies/ml to 500 copies/ml for cmv and from 2500 copies/ml to 1000 copies/ml for p. jirovecii. lod was defined as the minimum concentration with a detection rate of at least 95% by probit analyses. for the analytical specificity assessment, the manufacturer tested a panel of 31 lod was determined to be 2,063 ae 46 copies/ml or 103 copies/ rxn for cmv and 1,826 ae 44 copies/ml or 91 copies/rxn for p. jiroveci in the current study. the assay yielded negative results for the panel of microorganisms other than the target organisms and thus no crossreactivity was demonstrated. the results for the clinical samples showed that p. jirovecii was detected in 31.2% (39/125) and 40% (50/ 125) of the specimens using ifa and pj-cmv pcr, respectively. when we compared the results obtained from hiv-infected and non-hiv infected patients, 16 (45.7%) of the 35 hiv infected and 34 (37.7%) of 90 non-hiv infected patients were positive for p. jirovecii by pj-cmv pcr. eleven of the samples positive by pj-cmv pcr were negative by ifa. all 39 samples positive by ifa were also positive by pcr. there were no samples that were negative by pj-cmv pcr and positive by ifa. the ct for detection of pj (number of cycles needed for a positive result) obtained with the pj-cmv pcr was higher than 25 cycles (in 7 samples higher than 30) in the 11 samples negative by ifa suggesting that the pj organism load was lower in the pj-cmv pcr positive and ifa negative samples. using ifa as a gold standard, the pj-cmv pcr sensitivity, specificity, positive predictive value and negative predictive value for p. jirovecii were 100%, 87.2%, 78% and 100%, respectively (table 1) . of the 50 p. jirovecii positive samples, 18 (36%) were also positive for cmv by the pj-cmv pcr. the co-infection rate was found to be 37.5% (6/16) and 35.2% (12/34) in hiv infected and non-hiv infected patients with immunosuppressed conditions, respectively. there are several methods for detection of p. jirovecii from respiratory clinical samples including cytologic examination and ifa. direct ifa test has been widely used and is still in first line for pjp diagnostic in association with pathogen dna detection. these tests detect both the cystic and trophic forms of p. jirovecii. although the direct ifa tests are more rapid to perform and easier to interpret than cytochemical stains, they might have false-negative results, especially in specimens from non-hiv infected patients since these tend to have fewer organisms present in the specimens [8, 9] . these tests also depend upon the skills and experience of the observer in terms of familiarity with the different morphologies of the organisms [10] . an overall 55.5% sensitivity and 98.6% specificity of direct ifa tests has been demonstrated in a meta-analysis article evaluating seven prospective studies [11] . molecular techniques such as real-time pcr have been used in the diagnostic evaluation of pjp, which have better sensitivity than traditional cytologic stains and ifa [3, 4] . in the present study, we evaluated the performance of the pj-cmv pcr assay on stored bal samples against ifa for diagnosis of pjp. to our knowledge, this is the first evaluation of the pj-cmv pcr assay using reagents from diasorin molecular. the pj-cmv pcr showed a higher sensitivity and specificity at diagnosing pjp in both hiv-infected and non-hiv infected immunocompromised patients compared to the direct ifa test. the higher sensitivity observed in this study is consistent with the results of previous studies on the detection of p. jirovecii [2, 12, 13] . a bivariate metaanalysis study by lu et al. presented the diagnostic accuracy of p. jirovecii pcr techniques, with sensitivity ranging from 96% to 100% and specificity ranging from 87% to 93% [12] . in another review, reported sensitivities ranged from 82% to 100% and specificities ranged between 83% and 100% for pjp diagnosis using pcr assays [13] . a bivariate meta-analysis and systemic review by table 1 comparison of the p. jirovecii by direct immunofluorescence (ifa) and the pj-cmv multiplex real-time pcr assay results (n = 125). negative total [2] . previous studies that have compared the performance of realtime pcr to ifa have also reported that a small number of samples were positive by real-time pcr but negative by ifa testing due to the higher sensitivity of the pcr [12, 14, 15] . in this study, eleven samples found positive by pcr were negative by ifa. all 11 of these with potentially false positive results compared to ifa had clinically proven pjp with clinical symptoms. these specimens had an overall higher cycle threshold value compared to samples positive by both methods, which became positive at less than 25 cycles. the specificity increased from 87.2 to 100% and the ppv increased from 78 to 100% once these eleven results were considered true-positive according to patients' clinical diagnostic criteria. one of the advantages of the pj-cmv pcr assay is that it is designed as a closed system to reduce the potential carry-over contamination from run-to-run with amplicons causing falsepositive results. clinical samples are not open simultaneously during any step in the process. pj-cmv pcr assay can be also used on other respiratory fluids like sputum or bronchial fluid. cmv infection has immunomodulatory actions and aggravates the state of immunosuppression. it might lead to higher risk of opportunistic infections with other pathogens such as pj and significantly influence on the outcome of the pjp infections [16, 17] . however, some studies have reported no significant effect of cmv co-infection on the outcome of pjp [5, 7, 18] . although the clinical significance of concomitant cmv infection with pjp is poorly understood, some investigators advise that cmv should be monitored closely in pjp infected patients [5] . the pj-cmv pcr assay detects both of these pathogens in a single sample, eliminating the need for a separate assay for cmv pcr testing in respiratory specimens. previous studies have reported that 23.4-61.5% of pjp patients also exhibit co-infection with cmv [7, 17, 19, 20] . the percentage of cmv co-infection of this study was found to be 36% of patients, which is consistent with previous studies. there was no significant difference between the hiv infected and non-hiv infected patients in terms of the cmv co-infection. there are a few limitations to this study. our initial study question restricted the scope of this study and the data that we had access to. in particular, we compared the pj-cmv pcr assay against direct ifa, the test routinely used at our laboratory, but not against another molecular test or against a quantitative test that could provide a specific organism load. in addition, although all samples analyzed were from patients meeting clinical criteria of pjp, we do not have further clinical data on the patients. in summary, our findings demonstrate that the pj-cmv multiplex real-time pcr assay showed higher sensitivity compared with direct ifa for detection of p. jirovecii in bal samples and might be easily adapted for use in the clinical microbiology laboratories allowing the simultaneous detection of pj and cmv in a single sample. the authors declare that they have no competing interest. development and validation of a pneumocystis jirovecii real-time polymerase chain reaction assay for diagnosis of pneumocystis pneumonia evaluation of pcr in bronchoalveolar lavage fluid for diagnosis of pneumocystis jirovecii pneumonia: a bivariate meta-analysis and systematic review detection of pneumocystis jirovecii by quantitative real-time pcr in oral rinses from pneumocystis pneumonia asymptomatic human immunodeficiency virus patients comparison of a commercial real-time pcr assay realcycler(r) pjir kit, progenie molecular, to an in-house real-time pcr assay for the diagnosis of pneumocystis jirovecii infections a pneumocystis jirovecii pneumonia outbreak in a single kidney-transplant center: role of cytomegalovirus co-infection cytomegalovirus in the bronchoalveolar lavage fluid of patients with aids outcomes of non-hiv-infected patients with pneumocystis pneumonia and concomitant pulmonary cytomegalovirus infection diagnosis and management of pneumocystis pneumonia in resource-poor settings pneumocystis jirovecii pneumonia in human immunodeficiency virus infection prevalence of pneumocystis jirovecii among immunocompromised patients in hospitals of tehran city metaanalysis of diagnostic procedures for pneumocystis carinii pneumonia in hiv-1-infected patients pcr diagnosis of pneumocystis pneumonia: a bivariate meta-analysis pneumocystis jirovecii pneumonia in non-hivinfected patients: new risks and diagnostic tools prospective clinical evaluation of respiratory samples from subjects at risk for pneumocystis jirovecii infection by use of a commercial realtime pcr assay pneumocystis jirovecii testing by real-time polymerase chain reaction and direct examination among immunocompetent and immunosuppressed patient groups and correlation to disease specificity assessment of cytomegalovirus and cell-mediated immunity for predicting outcomes in non-hiv-infected patients with pneumocystis jirovecii pneumonia medicine (baltimore human herpes virus co-infection is associated with mortality in hiv-negative patients with pneumocystis jirovecii pneumonia prognostic markers of short-term mortality in aids-associated pneumocystis carinii pneumonia the association between cytomegalovirus co-infection with pneumocystis pneumonia and mortality in immunocompromised non-hiv patients pneumocystis jirovecii pneumonia in tropical and low and middle income countries: a systematic review and meta-regression the diasorin pj and cmv pcr reagents used in this study were provided by diasorin molecular llc. key: cord-296402-rd5clf8h authors: josé castón, juan; miguel cisneros, josé; torre-cisneros, julián title: efectos de la infección viral en el paciente trasplantado date: 2007-10-31 journal: enfermedades infecciosas y microbiología clínica doi: 10.1157/13109990 sha: doc_id: 296402 cord_uid: rd5clf8h las infecciones virales continúan siendo una importante causa de morbimortalidad en los pacientes trasplantados. en estos pacientes, el riesgo de infección viral depende de varios factores como el tipo de trasplante, la intensidad de la inmunosupresión y la susceptibilidad del receptor. además de efectos directos, la infección viral puede causar efectos indirectos derivados del efecto inmunomodulador de algunos virus como citomegalovirus o herpesvirus 6. entre estos efectos se encuentran un mayor riesgo de replicación de otros virus, de rechazo, de otras infecciones oportunistas y de otras entidades específicas en cada tipo de trasplante. los tests moleculares cuantitativos han reemplazado en la mayoría de las ocasiones a los métodos serológicos y al cultivo para el diagnóstico de estas infecciones, especialmente en el caso de citomegalovirus, virus de epstein-barr, y los virus de la hepatitis b y c. sin embargo, estos avances diagnósticos no se han acompañado del desarrollo de antivirales específicos o de vacunas eficaces, por lo que las medidas de prevención continúan siendo fundamentales en estos pacientes. viral infection remains an important cause of morbidity and mortality in transplant recipients. the risk of viral infection in these patients depends on several factors, such as the type of organ transplanted, the intensity of immunosuppression, and the recipient's susceptibility. in additional to direct effects, viral infection cause indirect effects, including greater risk of replication of other viruses, graft rejection, opportunistic infections and other specific entities for each type of transplant. these indirect effects result from the immunomodulatory activity of some viruses, such as cytomegalovirus and human herpes virus-6. for the most part, quantitative molecular tests have replaced serologic testing and in vitro culture for diagnosing infection. this approach is particularly prominent for cytomegalovirus, epstein-barr virus, hepatitis b virus, and hepatitis c virus. despite these diagnostic advances, the development of specific antiviral agents and effective antiviral vaccines is limited. thus, prophylactic strategies are still essential in transplant recipients. las infecciones por virus continúan siendo una importante fuente de morbilidad y mortalidad en los pacientes trasplantados, tanto de órgano sólido (tos) como de progenitores hematopoyéticos (tph). la adquisición de estas infecciones puede producirse de forma exógena, a través de los órganos del donante (citomegalovirus, virus de epstein-barr), mediante transfusión de hemoderivados o bien por exposición en la comunidad (influenza, adenovirus). además de esta vía exógena, la reactivación endógena de virus latentes, fundamentalmente herpesvirus, es un problema frecuente en los pacientes trasplantados y puede conducir a la aparición tanto de efectos directos como indirectos sobre el órgano trasplantado. esta clasificación permite distinguir los efectos producidos por la invasión viral directa que acarrean daño celular y tisular (p. ej., neumonitis por citomegalovirus), de los efectos inmunomoduladores mediados por la respuesta inflamatoria o por las alteraciones en el sistema inmune del huésped. el riesgo de infección viral tras el trasplante está condicionado por la suma de muchos factores entre los que se incluyen la intensidad, virulencia y mecanismos de la exposición viral, la intensidad y tipo del régimen de inmunosupresión y la presencia o ausencia de inmunidad antiviral preexistente. a pesar de esta variabilidad, podemos distinguir un patrón general en la cronología de estas infecciones tras el trasplante 1 (fig. 1 ). de este modo, los pacientes infectados por virus de herpes simple (vhs) pueden presentar una reactivación precoz durante los primeros dos meses postrasplante. igualmente, las infecciones adquiridas a través del injerto, como las producidas por virus de la hepatitis b (vhb) y c (vhc) y virus de la inmunodeficiencia humana (vih) pueden aparecer en el primer mes tras la cirugía. respecto al citomegalovirus (cmv), tanto la reactivación como la infección adquirida en período peritrasplante, suelen producirse entre el primero y el cuarto mes postrasplante, aunque en la actualidad se describen cada las infecciones virales continúan siendo una importante causa de morbimortalidad en los pacientes trasplantados. en estos pacientes, el riesgo de infección viral depende de varios factores como el tipo de trasplante, la intensidad de la inmunosupresión y la susceptibilidad del receptor. además de efectos directos, la infección viral puede causar efectos indirectos derivados del efecto inmunomodulador de algunos virus como citomegalovirus o herpesvirus 6. entre estos efectos se encuentran un mayor riesgo de replicación de otros virus, de rechazo, de otras infecciones oportunistas y de otras entidades específicas en cada tipo de trasplante. los tests moleculares cuantitativos han reemplazado en la mayoría de las ocasiones a los métodos serológicos y al cultivo para el diagnóstico de estas infecciones, especialmente en el caso de citomegalovirus, virus de epstein-barr, y los virus de la hepatitis b y c. sin embargo, estos avances diagnósticos no se han acompañado del desarrollo de antivirales específicos o de vacunas eficaces, por lo que las medidas de prevención continúan siendo fundamentales en estos pacientes. palabras clave: infección viral. trasplante. citomegalovirus. hepatitis. enfermedad linfoproliferativa postrasplante. viral infection remains an important cause of morbidity and mortality in transplant recipients. the risk of viral infection in these patients depends on several factors, such as the type of organ transplanted, the intensity of immunosuppression, and the recipient's susceptibility. in additional to direct effects, viral infection cause indirect effects, including greater risk of replication of other viruses, graft rejection, opportunistic infections and other specific entities for each type of transplant. these indirect effects result from the immunomodulatory activity of some viruses, such as cytomegalovirus and human herpes virus-6. for the most part, quantitative molecular tests have replaced serologic testing and in vitro culture for diagnosing infection. this approach is particularly prominent for cytomegalovirus, epstein-barr virus, hepatitis b virus, and vez con más frecuencia episodios de enfermedad por cmv más allá de este período (enfermedad tardía por cmv). ello se ha relacionado fundamentalmente con las pautas de profilaxis universal en el caso de tos o con la aparición de enfermedad injerto contra huésped (eich) crónica en receptores de tph. otros virus latentes como el virus de epstein-barr (veb) y el virus herpes-zoster (vhz) se presentan mayoritariamente entre el segundo y el sexto mes postrasplante. en el caso del poliomavirus bk, se ha observado una distribución bimodal en la presentación de los casos; el 50% de ellos aparece durante las primeras 8 semanas postrasplante y el resto en períodos posteriores que pueden abarcar desde meses hasta años tras el trasplante. finalmente, las infecciones por virus adquiridos en la comunidad como influenza, virus sincitial respiratorio (vsr) y adenovirus pueden aparecer en cualquier período. la infección por cmv es la principal causa de morbilidad y mortalidad de origen viral en los pacientes trasplan-tados. este virus se encuentra distribuido ampliamente en la población general con tasas de seroprevalencia que oscilan entre el 60 y el 90%. después de la infección primaria, el cmv permanece latente, y puede encontrarse su genoma en múltiples tipos celulares como los monocitos cd14+ y las células progenitoras cd34+, aunque su reservorio y los mecanismos por los cuales permanece latente son mal conocidos. el cmv puede producir tanto efectos directos como indirectos. entre los efectos directos se incluyen el síndrome viral (fiebre acompañada de neutropenia, trombopenia o alteración de transaminasas) y la enfermedad invasora por cmv, cuyo riesgo se incrementa de forma exponencial con el aumento de la carga viral 2,3 . entre los órganos más frecuentemente afectados se encuentran el pulmón, el hígado, el tracto gastrointestinal y la retina, en los cuales se produce una respuesta inflamatoria responsable del daño tisular. para el diagnóstico de certeza de enfermedad por cmv se requiere la presencia de un cuadro clinicoanalítico compatible junto con la demostración de lesiones histológicas en una biopsia (inclusiones intracelulares en "ojo de lechuza") y/o cultivo positivo para cmv ( fig. 2 ). en el caso de la neumonía, se acepta la detección del virus en el lavado broncoalveolar 4 . los efectos indirectos, a diferencia de la enfermedad invasora, son independientes del grado de viremia, y resultan de la interacción del cmv con la respuesta inmune del huésped 5 . estos efectos pueden estar, al menos en parte, relacionados con la presencia durante largos períodos de tiempo de baja concentración de replicación viral, lo que facilitaría una disfunción de los linfocitos cd4+ y macrófagos inducida directamente por el cmv e indirectamente a través de la producción de citocinas supresoras. entre los numerosos efectos indirectos del cmv descritos hasta el momento se encuentran un aumento en el riesgo de rechazo y disfunción del injerto, ateroesclerosis acelerada en el trasplante cardíaco, bronquiolitis obliterante (bos) en el trasplante pulmonar, infecciones oportunistas, neoplasias, síndrome de guillain-barré y diabetes mellitus postrasplante 2,6 ( fig. 3 ). la asociación entre infección por cmv y rechazo se ha evidenciado en estudios aleatorizados con fármacos frente a cmv en receptores de trasplante renal. en uno de ellos se observó que receptores seronegativos que habían recibido injertos procedentes de donantes seropositivos (d+/r-) presentaron una reducción del 50% en la incidencia de rechazo cuando se empleó profilaxis frente a cmv respecto a placebo 7 . en otros estudios realizados en trasplante renal se ha evidenciado una mayor supervivencia y una menor tasa de rechazo a los 3 años del trasplante en aquéllos sometidos a profilaxis 8 . en el caso del trasplante hepático, la profilaxis frente a cmv se ha relacionado con una mayor supervivencia del injerto, una menor tasa de rechazo y una disminución de la mortalidad de los pacientes 9 . este aumento de la supervivencia se ha observado igualmente en el análisis retrospectivo de una cohorte de trasplantados renales y cardíacos en situación d+/r-sometidos a profilaxis frente a cmv 8 . además del rechazo, la disfunción del injerto y la disminución de la supervivencia, la enfermedad cardiovascular ha sido relacionada con la infección por cmv en pacientes trasplantados. en un análisis post hoc de un ensayo que incluyó a pacientes aleatorizados a recibir ganciclovir o placebo durante los primeros 28 días postrasplante, los pacientes que recibieron profilaxis con ganciclovir y gammaglobulina presentaron menor riesgo de arteriopatía coronaria que los del grupo placebo 10 . por otro lado, el efecto inmunosupresor mediado por cmv, puede traducirse en un aumento en la incidencia de infecciones oportunistas así como de neoplasias. en este sentido se ha considerado la infección por cmv un factor de riesgo de infecciones bacterianas y fúngicas (pneumocystis jiroveci, aspergillus spp. y candida spp.), así como de enfermedad linfoproliferativa postrasplante asociada al virus de epstein-barr 11 . en la actualidad desconocemos el impacto de las diferentes pautas de profilaxis frente a cmv sobre la aparición de efectos indirectos 12 . entre estas pautas se incluyen la terapia anticipada (vigilancia y administración de antivirales cuando se detectan concentraciones significativas de viremia) y la profilaxis universal (administración de antivirales a todos los pacientes). en trasplantados hepáticos, la terapia anticipada se ha asociado con tasas de infección oportunista, rechazo y supervivencia similares a las de los pacientes que no han recibido ninguna profilaxis antiviral 13 . igualmente, no se han encontrado diferencias entre las tasas de rechazo y disfunción renal al comparar pacientes trasplantados renales sometidos a terapia anticipada frente a aquéllos en quienes se ha empleado tratamiento diferido de la enfermedad 14 . la incapacidad de la terapia anticipada para impedir la presencia de baja replicación viral podría justificar estos resultados. por el contrario y desde un punto de vista teórico, las pautas de profilaxis universal al impedir cualquier grado de replicación del cmv podrían disminuir el riesgo de aparición de efectos indirectos. sin embargo, estos beneficios podrían contrarrestarse por un mayor riesgo de aparición de resistencias y por una mayor probabilidad de aparición de enfermedad tardía por cmv tras la retirada de dicha profilaxis 15 . en la actualidad, la aparición de enfermedad por cmv más allá de los primeros 6 meses postrasplante es un hecho cada vez más frecuente, el cual se encuentra relacionado con la supervivencia a largo plazo de estos pacientes 16 . en un ensayo realizado con pacientes sometidos a trasplante hepático que realizaron profilaxis con ganciclovir oral, la mediana del comienzo de aparición de enfermedad por cmv fue de 10 meses 17 . igualmente, en otro ensayo que comparó valganciclovir con ganciclovir oral en castón profilaxis durante 90 días en receptores de tos (hepático, cardíaco, renal y páncreas y riñón) en situación d+/r-, se observó cómo en ambos grupos todos los casos de enfermedad por cmv aparecieron después de los primeros 6 meses postrasplante 18 . la aparición tardía de enfermedad por cmv probablemente sea debida a una alteración en la reconstitución inmune dependiente de los linfocitos t específicos frente al virus. de hecho, se ha comprobado que la disminución en la inmunidad celular específica frente a cmv es un factor de riesgo de enfermedad posterior por este virus 19 . la profilaxis antiviral se ha postulado como un factor fundamental en el desarrollo de alteraciones de la reconstitución inmune responsables de la enfermedad tardía por cmv, tanto en receptores de tos como de tph. en el caso del tph, los pacientes que reciben profilaxis con ganciclovir durante los primeros 100 días postrasplante presentan una disfunción en la respuesta específica frente a cmv por parte de los linfocitos cd4+ y de los cd8+, cuando se les compara con pacientes que no reciben dicha profilaxis 19 . de la misma forma, en receptores de trasplante hepático en tratamiento durante 6 semanas con ganciclovir oral, se ha comprobado una mayor supresión de la respuesta inmune inducida por linfocitos t-helper específicos en comparación con pacientes que no habían recibido ganciclovir 20 . estos resultados podrían explicarse porque en los pacientes que reciben ganciclovir existe una disminución en la proliferación de células t, así como una menor velocidad de expansión de precursores de células t específicas frente a cmv. la enfermedad tardía por cmv ha sido igualmente asociada con los regímenes no mieloablativos de tph. aunque estos pacientes presentan una baja incidencia de enfermedad por cmv en los primeros 100 días postrasplante cuando se comparan con los que reciben regímenes mieloablativos, las tasas de enfermedad por cmv al año son similares en ambos grupos, y es más tardío el comienzo de los síntomas en receptores de tph no mieloablativo 21 . este hecho podría deberse al efecto protector de las células t residuales procedentes del receptor en el postrasplante inmediato y a un comienzo tardío de la eich en estos pacientes. la aparición cada vez más frecuente de complicaciones tardías relacionadas con el cmv ha motivado que en la actualidad la profilaxis antiviral esté siendo sometida a evaluación. aunque se desconoce cuál será la mejor estrategia para evitar estas complicaciones, parece razonable pensar que entre las posibles alternativas se encuentren el desarrollar estrategias que faciliten la reconstitución inmune de los pacientes d+/r-en los primeros meses postrasplante, la prolongación de la profilaxis antiviral más allá de los primeros 3 meses postrasplante en pacientes de alto riesgo y el empleo de terapia anticipada basada en la antigenemia pp65 22 o en la detección de adn viral mediante reacción en cadena de la polimerasa (pcr). en estos casos sería deseable desarrollar marcadores objetivos de riesgo de padecer enfermedad tardía (virológicos o inmunológicos), para extender la profilaxis únicamente en estos pacientes. para el tratamiento de la enfermedad establecida por cmv ganciclovir continúa siendo el fármaco de elección tanto en tos como en tph. la buena biodisponibilidad de valganciclovir, un l-valyl éster de ganciclovir ha posibilitado que sea empleado por algunos clínicos como una alternativa a ganciclovir, aunque su eficacia deberá ser demostrada en ensayos bien diseñados. la resistencia a ganciclovir puede deberse a mutaciones en los genes ul97, ul54 o ambos. como pautas alternativas se han propuesto el foscarnet, cidofovir y la leflunomida. además, otros fármacos como benzimidazol, adefovir, lobucavir y maribavir se encuentran actualmente en estudio, aunque no disponemos de datos clínicos. en pacientes sometidos a tph que presentan neumonitis por cmv se recomienda, además del tratamiento antiviral, el empleo de gammaglobulina inespecífica. no existe evidencia de que la inmunoglobulina hiperinmune específica frente a cmv sea más eficaz que la inespecífica. en receptores de tos no existen datos concluyentes sobre el beneficio de ninguna de estas inmunoglobulinas. la enfermedad linfoproliferativa postrasplante (elpt) engloba a un grupo heterogéneo de trastornos linfoproliferativos que comprenden desde un síndrome mononucleósico inducido por el veb hasta proliferaciones monomórficas muy agresivas, las cuales pueden ser indistinguibles de formas graves de linfoma 23 (tabla 1). esta proliferación linfoide aparece como consecuencia de las pautas de inmunosupresión que son instauradas tras el trasplante, las cuales condicionan un descenso en la función de células t específicas frente al veb. esto desencadena una proliferación incontrolada de células b infectadas por dicho virus. la elpt, sin embargo, no se asocia exclusivamente con la infección por el veb, sino que cada vez más frecuentemente aparecen formas de elpt negativas a veb, las cuales tienden a desarrollarse en períodos más tardíos tras el trasplante y presentan mayor mortalidad. de hecho, para el diagnóstico de elpt no se requiere la presencia de veb en células tumorales 24 . el período de mayor riesgo de aparición de elpt es el primer año postrasplante. en receptores de tos la mediana del tiempo de comienzo de elpt es de 6 meses, mientras que en el caso del tph es de 2 meses tras el trasplante, presentándose generalmente con formas más extensas y agresivas de la enfermedad 23 . la incidencia de elpt varía según el tipo de trasplante, y son el trasplante pulmonar y el de intestino delgado los que presentan mayor incidencia (5-32%) y el trasplante renal el de menor riesgo, con una incidencia inferior al 1%. estas diferencias pueden deberse a la mayor intensidad de la inmunosupresión empleada en estos tipos de trasplante, la cual es uno de los factores de riesgo más importantes para el desarrollo de elpt. en este sentido, se ha evidenciado que la inducción y el tratamiento del rechazo con okt3 y globulina antitimocítica se relacionan con un mayor riesgo de elpt 25 . en la actualidad no existen datos concluyentes sobre la asociación particular entre algunos de los fármacos inmunosupresores con el desarrollo de elpt. aunque existe discusión a este respecto sobre los efectos del tacrolimus en comparación con ciclosporina a, el fármaco más reciente micofenolato mofetil no se ha asociado con mayor riesgo de elpt 25 . el efecto de los inhibidores de la serina-treonina cinasa de mamíferos, sirolimus y evorolimus no se ha aclarado aún. sin embargo, podríamos pensar que dado su efecto inhibidor sobre las celulas derivadas de elpt en modelos animales, su empleo no se asocie a mayor riesgo de elpt 26 . en general, podemos concluir que probablemente el grado de inmunosupresión global más que cada fármaco inmunosupresor en particular, interviene de forma más importante en el desarrollo de elpt. además de con la inmunosupresión, la elpt se ha relacionado con el estado serológico del receptor frente al donante respecto al veb. de esta forma, los receptores seronegativos que reciben órganos de donantes seropositivos presentan entre 10 y 50 veces mayor riesgo de elpt como consecuencia del desarrollo de infección primaria por veb 27 . esto justifica además la mayor incidencia de elpt en la población pediátrica en períodos precoces tras el trasplante. el mayor riesgo de elpt en receptores seronegativos que reciben injertos de donantes seropositivos frente a veb ha posibilitado que se haya sugerido la inmunización pretrasplante frente al virus como una de las estrategias de prevención de la enfermedad. sin embargo, y aunque se encuentra en desarrollo, en la actualidad no se dispone de una vacuna frente al veb 28 . por otro lado, existen datos contradictorios sobre la utilidad de la profilaxis frente a cmv para la prevención de elpt. además, se ha observado que el cmv es un factor de riesgo independiente de elpt en pacientes seronegativos con respecto al veb, que reciben injertos de donantes seropositivos. la correcta profilaxis frente a cmv en esta población es crucial para evitar la elpt asociada al veb 29 . la localización donde puede desarrollarse la elpt parece estar en relación con el tiempo transcurrido desde la realización del trasplante. en receptores de trasplante pulmonar, más del 50% de todas las elpt durante el primer año se desarrollan sobre el injerto, mientras que esta localización se afecta solamente en el 15% de las ocasiones tras este período 30 . este hecho se ha comprobado igualmente en el caso del trasplante renal 30 . además de la implicación del injerto, otras localizaciones extralinfoides pueden verse afectadas por la elpt; predomina fundamentalmente el tracto gastrointestinal. se ha especulado que la continua exposición antigénica que tiene lugar en esta localización podría desencadenar una respuesta inflamatoria local que sería la responsable de la aparición de elpt. otras localizaciones frecuentemente afectadas por la elpt son los senos paranasales, el sistema nervioso central, los ganglios linfáticos y la piel. para el diagnóstico precoz de elpt se requiere un alto grado de sospecha clínica, debido a que generalmente no existen formas clínicas específicas de presentación. esto es especialmente importante cuando se produce afectación del injerto por elpt. los receptores de trasplante renal con elpt del injerto se presentan por lo general con insuficiencia renal, hidronefrosis o fiebre. en estos casos, la ultrasonografía puede revelar la presencia de adenopatías. si se tiene en cuenta que el aparato gastrointestinal es una localización frecuente de elpt, la aparición de síntomas como diarrea o sangrado digestivo deben hacernos pensar en la posibilidad de elpt como causante del cuadro. debido a la inespecificidad de los síntomas, resulta necesario el empleo de métodos diagnósticos que permitan establecer un diagnóstico de certeza de la forma más precoz posible. entre estos métodos se encuentra la detección del adn de veb mediante técnicas de pcr, entre las cuales se considera de elección la de pcr cuantitativa en tabla 1. clasificación actual de la oms de la enfermedad linfoproliferativa postrasplante elpt zarse en cada paciente, y puede estar limitado por el riesgo de rechazo o cuando el injerto es imprescindible para la supervivencia del paciente. esta estrategia presenta diferentes tasas de respuesta, que oscilan entre el 0 y el 89%, en función de factores pronósticos tales como valores elevados de láctico deshidrogenasa, la afectación multiorgánica de elpt y la presencia de disfunción orgánica en el momento del diagnóstico 34 . la quimioterapia citotóxica convencional puede ser empleada en pacientes con elpt que no presentan respuesta o cuando resulta imposible reducir el grado de inmunosupresión. varios regímenes de quimioterapia como el chop (ciclofosfamida, adriamicina, vincristina y prednisona) han sido empleados en estos pacientes con buenos resultados, aunque la alta mortalidad asociada a las complicaciones del tratamiento limita su empleo en numerosas ocasiones. en cuanto al valor de los fármacos antivirales como aciclovir o ganciclovir en el tratamiento de la elpt existe una experiencia limitada que incluye un pequeño número de estudios no aleatorizados con diferentes poblaciones de riesgo y dosificaciones desiguales 33 . finalmente, los anticuerpos monoclonales constituyen una opción atractiva para el tratamiento de elpt debido a su baja capacidad inmunosupresora, su actividad frente a células linfocitarias y su potencial para activar las células del sistema inmune. entre este grupo, rituximab, un anticuerpo monoclonal quimérico anti-cd20 ha demostrado los mejores resultados. choquet et al 35 realizaron un ensayo prospectivo y multicéntrico de rituximab en pacientes con elpt en el que se incluyeron pacientes pediátricos y adultos tratados con dosis estándar de 375 mg/m 2 semanalmente durante 4 semanas. el 68% de los pacientes presentó respuesta mantenida al año de seguimiento con una baja incidencia de efectos adversos. hasta hace poco tiempo, los pacientes con elpt sin respuesta a la disminución del tratamiento inmunosupresor eran sometidos a tratamiento con quimioterapia citotóxica convencional. en la actualidad no existen estudios que hayan comparado de forma prospectiva y aleatorizada las pautas de quimioterapia frente a rituximab. sin embargo, en un estudio retrospectivo que incluyó a 35 pacientes, las tasas de supervivencia y curación fueron similares en ambos grupos, aunque con una menor toxicidad y mortalidad relacionada con el tratamiento en el grupo que recibió rituximab 36 . estos resultados han posibilitado que algunos autores sugieran que rituximab debería ser considerado antes que la quimioterapia como segunda línea de tratamiento en los casos de elpt cd20 positivos, reservando la quimioterapia para los pacientes en los que no pueda usarse o que no respondan a rituximab 33 . otros herpesvirus: herpes-6, herpes-7, herpes-8, herpes simple-1, herpes simple-2 y virus varicela zóster virus herpes 6 (vhh-6) y virus herpes 7 (vhh-7) vhh-6 y vhh-7 son beta-herpesvirus linfotropos que generalmente causan infección en los primeros años de vida. la infección primaria casi siempre es asintomática o se manifiesta únicamente como una enfermedad febril de la infancia, posteriormente permanece en estado latente al igual que ocurre con otros herpesvirus. en adultos la seroprevalencia llega hasta el 90%, lo que condiciona que la mayoría de infecciones en pacientes trasplantados se producen por reactivación del virus latente. en el trasplante de órgano sólido las tasas de infección por vhh-6 oscilan entre el 31 y el 55% 37 , y existen dos variantes distintas del virus, vhh-6a y vhh-6b; esta última es la responsable de la mayoría de infecciones en pacientes trasplantados. en el tph, la tasa de infección documentada llega hasta el 40%. en el caso del vhh-7 las tasas de infección oscilan entre el 0 y el 46%, aunque la información disponible es menor que para el vhh-6. en receptores de tos, la mayoría de infecciones por vhh-6 ocurren entre las dos y cuatro semanas postrasplante, y se han asociado con el empleo de anticuerpos monoclonales frente a cd3 (okt3) o globulina antitimocítica, y con los regímenes que contienen sirolimus y anticuerpos frente al receptor il-12 como terapia de inducción 38 . en el caso del tph, el mayor nivel de replicación se alcanza dentro de las primeras 4 semanas postrasplante, siendo el tph alogénico, la presencia de enfermedad hematológica avanzada, el tratamiento con esteroides y la presencia de antígeno mayor de histocompatibilidad (hla) no idéntico entre donante y receptor los principales factores de riesgo. al igual que ocurre con el cmv, las manifestaciones clínicas del vhh-6 y vhh-7 pueden producirse por la acción directa del virus o bien a través de sus efectos inmunomoduladores. las infecciones sintomáticas parecen ser más frecuentes en receptores de tph que en pacientes sometidos a tos, y pueden estar relacionadas con el tipo de trasplante y la intensidad de la inmunosupresión. en receptores de tos, la primoinfección suele ser asintomática 39 y la manifestación clínica más frecuente es la aparición de un síndrome febril inespecífico. en este sentido, se ha especulado que los cuadros febriles atribuidos al cmv en pacientes trasplantados pudieran estar relacionados con infecciones concomitantes por vhh-6 o vhh-7, más que con infección únicamente por cmv. de hecho, se ha comunicado que el 89% de los receptores de trasplante hepático con infección por cmv presentan infección concomitante por vhh-6 variedad b o vhh-7 40 . por otro lado, el vhh-6 puede producir afectación del sistema nervioso central tanto en pacientes sometidos a tos como a tph. esta afectación puede traducirse en encefalitis, alteración del estado mental y convulsiones, y son poco frecuentes los síntomas de focalidad. además de estos síntomas se han descrito otras manifestaciones asociadas al vhh-6 como hepatitis, enfermedad gastrointestinal y mielosupresión, generalmente en forma de leucopenia o trombopenia y que aparecen tanto en tos como en tph. entre los efectos indirectos asociados al vhh-6 en tos se han comunicado un mayor riesgo de desarrollo de enfermedad por cmv y de infección fúngica invasora en trasplantados hepáticos. en estos pacientes la infección por vhh-6 se ha asociado además a formas más agresivas de recurrencia de la hepatopatía por virus de la hepatitis c y con disfunción del injerto 41, 42 . otros efectos indirectos descritos son un mayor riesgo de enfermedad por veb, virus varicela-zóster y micobacterias. en pacientes sometidos a tph, existen datos conflictivos respecto a la influencia del vhh-6 en el riesgo de rechazo así como de eich. en general, de los datos disponibles podemos deducir que en el caso del tos la afectación producida por el vhh-6 se manifiesta fundamentalmente a través de los efectos indirectos, y son menos frecuentes los casos de enfermedad invasora. sin embargo, en los pacientes sometidos a tph, las secuelas clínicas directas parecen ser predominantes, aunque los efectos indirectos especialmente sobre la eich podrían contribuir a un aumento de la mortalidad de estos pacientes. en cuanto al diagnóstico, al igual que para otros herpesvirus la serología no resulta de utilidad en el postrasplante ya que carece de la suficiente sensibilidad en el momento de la infección aguda. las técnicas de pcr son las que aportan mayor sensibilidad para detectar estos virus, aunque presentan la limitación de no diferenciar entre infección latente y activa, lo que puede evitarse mediante el empleo de técnicas cuantitativas. en la actualidad no existe suficiente evidencia para recomendar la monitorización sistemática de vhh-6 y vhh-7 en pacientes asintomáticos. carecemos de la suficiente evidencia para recomendar profilaxis universal o terapia anticipada frente a vhh-6 y vhh-7. existen datos in vitro que han evidenciado actividad de ganciclovir, foscarnet y cidofovir frente a vhh-6 43 . estos datos se han confirmado in vivo por la disminución de reactivaciones de vhh-6 en pacientes sometidos a tratamiento con ganciclovir o foscarnet. ambos fármacos se han empleado con éxito en el tratamiento de meningoencefalitis por vhh-6 tras el trasplante. sin embargo, la tasa de curación de este tratamiento que podemos extraer de los casos publicados llega únicamente al 60% 37 . tanto vhh-6 como vhh-7 presentan baja sensibilidad frente a aciclovir. la elección del fármaco antiviral deberá basarse fundamentalmente en el perfil de seguridad del mismo. aunque ganciclovir presenta riesgo de nefrotoxicidad, parece razonable evitar la administración de foscarnet en pacientes con riesgo elevado o que presentan insuficiencia renal. por el contrario, en aquellos casos en los que existan citopenias hematológias, foscarnet puede ser una alternativa debido a que no posee los efectos mielosupresores de ganciclovir. el vhh-8 es un gamma-herpesvirus conocido por ser el agente etiológico del sarcoma de kaposi (sk), linfoma de cavidades y la enfermedad de castleman 44, 45 . a diferencia de otros herpesvirus, la infección por este virus no es ubicua, presenta tasas de seroprevalencia que oscilan dependiendo de las regiones geográficas entre el 0 y el 5% en norteamérica, el 5 y el 20% en el área mediterránea y más del 50% en regiones de áfrica. el riesgo de reactivación y de infección primaria después del trasplante dependerá, por tanto, de la seroprevalencia del virus en la región, lo que justifica las diferentes tasas de incidencia de sk que han sido comunicadas (0,5% en norteamérica y norte de europa, y 5,3% en arabia saudí) 46 . la seropositividad previa al trasplante, así como la infección primaria adquirida a través del donante, incrementan el riesgo de sk. además, la intensidad de la inmunosupresión y el empleo de productos antilinfocitarios pueden desempeñar un papel importante en la patogenicidad del vhh-8 47 . la realización de técnicas de pcr constituye una opción atractiva para monitorizar la carga viral del vhh-8, sobre todo si tenemos en cuenta que las concentraciones de vhh-8 en linfocitos de sangre periférica se han asociado con el desarrollo de sk 48 . el empleo de cribado serológico del donante y receptor puede resultar útil sobre todo en poblaciones de alto riesgo. sin embargo, la utilidad de la detección rutinaria de la carga viral en el seguimiento de estos pacientes deberá demostrarse en futuras investigaciones. aunque existen estudios in vitro que han demostrado actividad de ganciclovir, foscarnet y cidofovir frente a vhh-8, el papel de estos antivirales en estrategias de profilaxis o terapia anticipada resulta incierto. se desconoce si las estrategias empleadas para la prevención del cmv ejercen un efecto frente a la infección por vhh-8. en cuanto a la prevención, resulta complicado realizar recomendaciones específicas. en receptores seropositivos frente a vhh-8 o que reciben un órgano de un donante seropositivo, la monitorización de la carga viral podría resultar de utilidad. en pacientes con sk, la reducción de la inmunosupresión puede llevar a la regresión del tumor y constituye la primera línea de tratamiento 46 . en pacientes que no responden a esta medida puede emplearse radiación o quimioterapia. el empleo de fármacos antivirales en pacientes trasplantados con sk u otras manifestaciones de infección por vhh-8 requiere futuras investigaciones. el virus herpes simple (vhs)-1 y vhs2, y el virus varicela zóster (vvz) pertenecen a la familia de los alfaherpesvirus. las tasas de seroprevalencia para vhs varían entre las diferentes poblaciones. oscila entre el 60 y el 80% para el vhs-1, con cifras algo inferiores para vhs-2. la incidencia de enfermedad por vhs en pacientes que no reciben profilaxis oscila entre el 25 y el 35%, y el período de máximo riesgo son las primeras 4 semanas postrasplante 49 . en los pacientes trasplantados la mayoría de las infecciones por vhs son originadas por reactivación de una infección latente, aunque se han descrito infecciones primarias transmitidas desde el injerto. la presentación más frecuente es la gingivoestomatitis causada por vhs-1 o la enfermedad genital o perianal originada por vhs-2. además de estas manifestaciones el vhs puede ocasionar enfermedad invasora y producir esofagitis, hepatitis y neumonitis, que afectan fundamentalmente a pacientes trasplantados de pulmón o de pulmón y corazón 50 . en cuanto al vvz, la seroprevalencia se sitúa en torno al 75% y al igual que para otros herpesvirus, en la mayoría de los casos la enfermedad en pacientes trasplantados se origina a partir de la reactivación de una infección latente. la incidencia de herpes zóster se sitúa entre el 2 y el 10% en trasplante renal y hepático y en el 20% para el trasplante cardíaco 49, 51, 52 . en el caso del tph la incidencia es mayor y puede llegar hasta el 50%. en estos pacientes la afectación puede ser metamérica o generalizada y aparece la mayoría de los casos después de los primeros 3 meses postrasplante. al igual que para vhs, además de la afectación superficial el vvz puede producir complicaciones viscerales como neumonitis, hepatitis y encefalitis. el estado serológico del receptor condiciona el riesgo de infección después del trasplante. los pacientes seropositi-vos para vhs o vvz previamente al trasplante presentan mayor riesgo de reactivación posterior. los pacientes seronegativos pueden adquirir la infección a partir de la exposición en la comunidad. en receptores de tph, la eich aguda y crónica, los receptores hla no idénticos y el trasplante autólogo por linfoma se han descrito como factores de riesgo de reactivación clínica de vvz 38 . además, la eich aguda y el desarrollo de varicela por infección primaria se han asociado a mayor riesgo de enfermedad diseminada en estos pacientes. en el caso del tos la intensidad de la inmunosupresión empleada condiciona el riesgo de reactivación viral. en la mayoría de los casos las manifestaciones clínicas típicas de la enfermedad por vhs y vvz son la principal herramienta para el diagnóstico. los métodos de laboratorio se reservan para los casos de presentación atípica o afectación visceral. entre estos métodos, el cultivo viral obtenido de muestras de la lesión es comúnmente empleado, así como las técnicas de pcr que pueden emplearse para detectar el genoma viral tanto en muestras de tejido como de fluidos. en cuanto a la profilaxis, aciclovir y valaciclovir han mostrado ser efectivos para prevenir la enfermedad por vhs y vvz 53 . además, los regímenes empleados en la profilaxis frente a cmv son efectivos para la prevención de vsh y probablemente también para vvz 7 . en candidatos a trasplante que son seronegativos frente a vvz, la vacunación con virus atenuados estaría indicada para prevenir la infección primaria postrasplante. en estos pacientes se aconseja un intervalo de entre cuatro y seis semanas entre la vacunación y la realización del trasplante al ser una vacuna de virus atenuados. además, y debido a su alta contagiosidad, en estos pacientes resulta recomendable la vacunación de los potenciales contactos seronegativos. la administración de la vacuna se desaconseja una vez que se ha realizado el trasplante al ser una vacuna de virus atenuados. finalmente, los pacientes seronegativos frente a vvz expuestos a un caso de varicela, zóster o exantema tras vacunación deben recibir profilaxis postexposición mediante la administración de gammaglobulina específica tan pronto como sea posible (dentro de las 92 h siguientes) 54 . pasado este período la administración de antivirales como profilaxis postexposición debe ser considerada. en pacientes seropositivos la profilaxis con aciclovir no se recomienda de forma sistemática dada la buena respuesta al tratamiento y el riesgo potencial de desarrollo de resistencias asociado a la profilaxis. para el tratamiento de la enfermedad por vhs o vvz, el fármaco de elección es aciclovir administrado por vía intravenosa. la administración de aciclovir oral, famciclovir o valaciclovir puede considerarse en casos de afectación orolabial por vhs o de zóster monometamérico. en los pacientes trasplantados la resistencia a aciclovir es un hecho infrecuente y el tratamiento con foscarnet es la alternativa en estos casos. el vhb se encuentra distribuido ampliamente en todas las zonas del mundo; existen aproximadamente unos 500 millones de portadores crónicos y una incidencia de in-fección de 30.000-50.000 casos al año, de los cuales el 10 y el 15% desarrollarán hepatitis crónica. la prevalencia de vhb entre los pacientes incluidos en programa de hemodiálisis y trasplantados renales se encuentra entre el 0,1 y el 1,4%. en estos pacientes, la positividad del antígeno de superficie del virus de la hepatitis b (hbs-ag) implica un peor pronóstico que los pacientes negativos de hbs-ag, especialmente cuando se acompaña de coinfección por virus de la hepatitis c 55 . en trasplantados cardíacos, la prevalencia de infección por vhb se sitúa en el 3-10%. la mayoría de estos pacientes permanecen asintomáticos durante largos períodos aunque hasta un 50% desarrollará cirrosis a los 7-10 años del trasplante. en el caso de los trasplantados hepáticos, a pesar de que el empleo de inmunoglobulina específica frente al vhb (ighb) junto con lamivudina ha reducido las tasas de recurrencia hasta menos del 10%, la recidiva de la infección postrasplante continúa limitando el pronóstico especialmente en aquellos con replicación activa en el momento de ser trasplantados 55 . entre los principales factores de riesgo de adquisición de novo de vhb tras el trasplante se encuentran el estado serológico donante/receptor, la intensidad de la inmunosupresión, el empleo de hemoderivados y la ausencia de vacunación previa al trasplante 56 . el trasplante de un órgano positivo de hbs-ag a un receptor seronegativo supone un alto riesgo de transmisión del virus 57 . en los casos de donantes negativos de hbs-ag y positivos de hbc-ac existe un alto riesgo de transmisión a receptores de trasplante hepático a menos que se administre una pauta intensiva de profilaxis. sin embargo, el riesgo de transmisión a receptores de trasplante no hepático seronegativos es escaso 58 . por otro lado, los candidatos positivos de hbs-ag que presentan adn viral o hbe-ag positivo tienen una mayor mortalidad de origen hepático que aquellos hbs-ag positivos sin adn viral o hbe-ag. para la prevención de la infección por vhb se aconseja la vacunación de forma precoz de los candidatos negativos de hbs-ag, realizando una monitorización anual de los títulos de anticuerpos con administración de una dosis de recuerdo si el título desciende por debajo de 10 mu/ml 59 . además de la vacunación debe realizarse un cribaje riguroso de los hemoderivados así como la monitorización de parámetros serológicos y virales en todos los pacientes en programa de hemodiálisis. para prevenir o al menos retrasar la reinfección por vhb tras el trasplante hepático se recomienda el empleo combinado de ighb y lamivudina. en trasplantados no hepáticos puede emplearse lamivudina en casos de hepatitis activa y alta carga viral, teniendo en cuenta que el tratamiento con interferón alfa es menos eficaz en este contexto y que ha sido asociado con un mayor riesgo de rechazo. en algunos casos de resistencia a lamivudina, la continuación de este tratamiento se ha mostrado eficaz en algunas ocasiones. además, otro fármaco como adefovir ha sido satisfactoriamente empleado en infecciones por vhb resistentes a lamivudina, aunque debido a la posibilidad de toxicidad renal su empleo puede estar limitado en pacientes con disfunción renal asociada a los inhibidores de la calcineurina. en la actualidad se estima que existen 150 millones de pacientes infectados por el vhc en todo el mundo. de los pacientes infectados, el 85% desarrolla una infección crónica mientras que el 10-30% evolucionan a cirrosis a largo plazo. los factores de riesgo universales para la adquisición de la infección son la transfusión de hemoderivados y el uso de drogas por vía parenteral. además, el vhc puede transmitirse a través del injerto con un riesgo que varía en función del órgano trasplantado. en pacientes en diálisis la incidencia de infección por vhc de novo ha disminuido mediante la aplicación de medidas de control y el cribaje de productos sanguíneos, hasta situarse actualmente en el 0,7-3% 60 . una vez que se realiza el trasplante renal, la infección por vhc se asocia con mayor morbilidad y mortalidad que los pacientes trasplantados vhc negativos. además, los receptores de trasplante renal infectados por vhc presentan mayor riesgo de glomerulonefritis membranoproliferativa, membranosa, vasculitis leucocitoclástica y crioglobulinemia 61 . es importante destacar que en estos pacientes no existe correlación entre el grado de elevación de transaminasas, la carga viral y el genotipo del vhc con la gravedad de las lesiones histológicas. la enfermedad hepática avanzada, particularmente la cirrosis, en pacientes con insuficiencia renal crónica, se considera una contraindicación para el trasplante renal aislado, por lo que se debe considerar la biopsia renal para los candidatos sin evidencia clínica de cirrosis. en estos casos se recomienda evaluar la realización de trasplante doble de riñón e hígado. una vez que se realiza el trasplante renal, la disfunción en la respuesta de los linfocitos t puede originar una menor progresión de la enfermedad, lo que podría explicar la mayor supervivencia de los pacientes trasplantados infectados por vhc en comparación con los pacientes vhc positivos en hemodiálisis 62 . ésta es la razón por la que se recomienda aceptar a los pacientes con insuficiencia renal terminal para la realización de trasplante. en receptores de trasplante cardíaco negativos de vhc el empleo de donantes seropositivos implica un significativo riesgo de transmisión del vhc, aunque no parece existir gran influencia sobre el injerto y la supervivencia del paciente. respecto al trasplante hepático, la hepatopatía crónica por vhc constituye su principal indicación a pesar de que la tasa de reinfección sobre el injerto es virtualmente universal, y en el 50-80% se desarrollará hepatitis del injerto. de hecho, la supervivencia tanto del órgano trasplantado como del paciente es inferior a la de los trasplantados hepáticos negativos de vhc, dependiendo del grado de replicación viral previa al trasplante. además de la intensidad de la replicación, el tipo y la intensidad de la inmunosupresión presentan una influencia sobre la progresión de la hepatopatía por vhc postrasplante, con una mayor incidencia de fibrosis del injerto y cirrosis con pautas de doble o triple terapia que la inmunosupresión con un único fármaco. en algunos estudios se ha evidenciado que el trasplante hepático desde un donante positivo de vhc a un receptor seropositivo parece ser una estrategia segura 63 . sin embargo, y dado que el genotipo 1 presenta menor respuesta al tratamiento antiviral y requiere una mayor duración de éste, se recomienda evitar el trasplante de órganos de un donante con genotipo 1 a un receptor seropositivo pero con un genotipo más favorable como el 2 o 3. el diagnóstico de infección crónica y activa se realiza detectando el arn del virus en sangre. el interferón alfa en combinación con ribavirina es el tratamiento más eficaz para la hepatopatía crónica por vhc. sin embargo, en trasplantados renales el empleo de interferón se ha asociado a rechazo, pérdida del injerto y a un rebote de la carga viral a los valores anteriores al tratamiento, por lo que se desaconseja su uso en estos pacientes. la eficacia a largo plazo de la monoterapia con ribavirina en esta población es desconocida. en contraste con los trasplantados renales, el tratamiento con interferón en monoterapia o combinado con ribavirina se ha recomendado en receptores de trasplante hepático positivos de vhc. la respuesta virológica completa se presenta hasta en el 50% de los pacientes, aunque sólo una parte de ellos presentará respuesta viral sostenida después de la retirada del tratamiento. la escasez de los datos disponibles sobre tratamiento del vhc en trasplantados cardíacos, pulmonares o de pulmón y corazón no permite realizar recomendaciones generales en el momento actual. el virus bk pertenece a la familia de los poliomavirus y se encuentra ampliamente distribuido con tasas de seroprevalencia del 80%. en el huésped inmunocomprometido la mayoría de casos de infecciones por virus bk suceden como consecuencia de la reactivación del virus latente, que se encuentra fundamentalmente en las células epiteliales del túbulo renal. en esta población, hasta el 90% de las infecciones son asintomáticas aunque pueden aparecer una gran variedad de manifestaciones como viruria y viremia, ulceración y estenosis uretral, cistitis hemorrágica y nefritis intersticial 64 . la mayoría de casos de infecciones por virus bk se producen en pacientes sometidos a trasplante renal. en estos pacientes la nefritis tubulointersticial (nefropatía bk) constituye la expresión más frecuente de enfermedad por este virus 65 . esta entidad se manifiesta típicamente como una elevación en la concentración de creatinina plasmática como consecuencia de una progresiva pérdida de función del injerto, por lo que puede resultar difícil su distinción del rechazo o de toxicidad farmacológica. aunque los factores de riesgo no están bien definidos, el estado donante/receptor d+/r-y d+/r+, la intensidad de la inmunosupresión, el empleo de algunos inmunosupresores específicos (tacrolimus y ácido micofenólico) y la presencia de rechazo se han asociado a mayor riesgo de nefropatía bk 38 . para el diagnóstico definitivo de enfermedad por virus bk se requiere la realización de biopsia renal. en estadios iniciales de la enfermedad, pueden observarse inclusiones virales junto con un pequeño grado de inflamación. las fases tardías se caracterizan por un infiltrado de células mononucleares con invasión focal del uroepitelio y el túbulo renal. las células infectadas muestran grandes núcleos hipercromáticos junto con inclusiones virales intra-nucleares. este hallazgo es importante ya que para confirmar el diagnóstico resulta necesaria la demostración del virus en el interior de la célula infectada, lo que permite distinguir a la nefropatía bk del rechazo. además del examen anatomopatológico pueden emplearse otros métodos no invasores, como el estudio citológico de la orina que permite la visualización de las células decoy (células epiteliales con grandes núcleos e inclusiones basófilas). la presencia de estas células es sugestiva de viruria, y presentan una sensibilidad de aproximadamente el 100% para el diagnóstico de infección por virus bk, aunque con un valor predictivo positivo del 30% 66 . esto puede ser debido a que las células decoy pueden aparecer en la infección por otros virus como el jc, cmv o adenovirus, o bien porque la presencia de viruria presenta escasa correlación con la nefritis por virus bk. la escasa especificidad del estudio citológico ha motivado el empleo de técnicas moleculares en sangre y orina para el diagnóstico de esta infección. de esta forma se ha evidenciado cómo la viremia plasmática de bk se correlaciona con la presencia de nefropatía por este virus y puede emplearse para monitorizar la respuesta al tratamiento 66 . para el tratamiento de la nefropatía bk la medida más importante consiste en la disminución de la inmunosupresión. sin embargo, en algunas ocasiones la aplicación de esta estrategia se encuentra limitada por el rechazo de forma concomitante con la nefritis viral. esto ha motivado el empleo de fármacos antivirales como vidarabina, cidofovir y leflunomida, de los cuales disponemos de escasos datos sobre su eficacia 67 . parvovirus b-19 es un virus adn cuya seroprevalencia en adultos oscila entre el 60% y el 90%. la infección primaria por este virus ocurre más frecuentemente en la infancia y se manifiesta clásicamente como eritema infeccioso, aunque en la mayoría de ocasiones se presenta de forma asintomática. en adultos, la infección primaria por parvovirus b-19 puede manifestarse como artritis y en los casos de adquisición durante el embarazo se ha asociado con el desarrollo de hydrops fetalis. en pacientes trasplantados la incidencia de infección por parvovirus b-19 es baja, a pesar de que el virus persiste en los tejidos de los individuos seropositivos, y que, por tanto, podría transmitirse potencialmente a través del injerto o de productos sanguíneos trasfundidos al receptor 68 . en pacientes sometidos a tos la infección por parvovirus b-19 se manifiesta típicamente por el desarrollo de aplasia pura de serie roja con un escaso o ausente recuente reticulocitario. ello es debido a que parvovirus b-19 infecta a los precursores eritroides mediante la unión al receptor conocido como antígeno p. además, parvovirus b-19 se ha asociado con otras manifestaciones como hepatitis, neumonitis, miocarditis y disfunción del injerto. en receptores de tph, la anemia asociada a parvovirus b-19 es un hecho infrecuente probablemente por el efecto profiláctico de la inmunoglobulina intravenosa que es administrada en muchos de estos pacientes. para el diagnóstico de infección por parvovirus b-19 se han empleado la serología, el examen histológico de la mé-dula ósea y la detección del adn viral mediante pcr. la serología presenta la importante limitación de su escasa sensibilidad en pacientes inmunosuprimidos, por lo que la presencia de igm negativa frente a parvovirus b-19 no descarta el diagnóstico de infección. en estos pacientes el diagnóstico puede realizarse mediante técnicas de pcr en sangre periférica. en los casos en los que exista alta sospecha de enfermedad por parvovirus b-19 en los que el resultado de la pcr haya sido negativo, el diagnóstico puede confirmarse mediante estudio histológico de la médula ósea 68 . para el tratamiento de la enfermedad por parvovirus b-19 no existen en la actualidad fármacos antivirales disponibles, por lo que se aconseja si es posible la reducción en la inmunosupresión. el hecho de que la viremia desaparezca con la generación de anticuerpos, ha llevado a la inclusión de la inmunoglobulina intravenosa en el tratamiento de la enfermedad por parvovirus b-19 69 . la inmunoglobulina intravenosa contiene inmunoglobulinas específicas frente a parvovirus b-19 y resulta útil en el tratamiento de la anemia asociada a la replicación viral. sin embargo, en la actualidad se desconocen tanto las dosis como la duración óptima del tratamiento con inmunoglobulina y se observan recaídas tras el cese de su administración. para el diagnóstico precoz de estas recaídas puede ser recomendable la monitorización de las concentraciones de hemoglobina una vez suspendido el tratamiento y considerar la realización de pcr en aquellos pacientes que desarrollen anemia. en aquellos pacientes en quienes se confirme el diagnóstico, el retratamiento con inmunoglobulina intravenosa puede ser empleado. los virus respiratorios causan frecuentemente enfermedad que afecta al tracto respiratorio tanto en pacientes inmunosuprimidos como inmunocompetentes, y afectan sobre todo a la población infantil. entre estos virus se incluyen el virus respiratorio sincitial (vrs), parainfluenza (vp), influenza (vi), adenovirus, rhinovirus, coronavirus y enterovirus. el vrs es un virus arn causante de epidemias anuales por todo el mundo. aunque los síntomas de la infección por vrs son generalmente leves en niños y adultos, se han descrito casos de enfermedad invasora fundamentalmente en receptores de tph relacionados con la adquisición de la infección por debajo del primer año de edad, la enfermedad pulmonar subyacente y el grado de inmunosupresión 70 . el diagnóstico definitivo se realiza mediante el cultivo del virus en determinadas líneas celulares, aunque el coste y el tiempo requeridos para su crecimiento han posibilitado la aparición de otras alternativas como la detección antigénica y los métodos de shell vial. para estas infecciones se recomienda el tratamiento de soporte en los casos de afectación del tracto respiratorio superior en ausencia de factores de riesgo. en los casos de afectación del tracto respiratorio inferior o de presencia de factores de riesgo se recomienda el empleo de aerosoles de ribavirina en combinación con inmunoglobulina intravenosa o palivizumab, un anticuerpo monoclonal específico frente a vrs 38 . el vi se considera uno de los virus causantes de morbimortalidad más importantes a lo largo de todo el mundo. en la actualidad la información prospectiva sobre la incidencia de este virus en la población trasplantada es escasa. sin embargo, en estudios retrospectivos se ha evidenciado el papel del virus en la aparición de enfermedad grave en pacientes pediátricos. en trasplantados adultos la gravedad de la enfermedad por vi se ha relacionado con la intensidad de la inmunosupresión, el tipo de trasplante y la variación estacional en su virulencia 38 . el diagnóstico de certeza de infección por vi se obtiene por el aislamiento del virus en el cultivo. además, otros métodos como el cultivo shell vial, la detección antigénica y la detección de actividad neuraminidasa son relativamente sensibles y específicos aunque no siempre se encuentran disponibles. el tratamiento precoz de la infección por influenza a con amantadina o los inhibidores de neuraminidasa zanamivir y oseltamivir se ha mostrado efectivo en adultos sanos. únicamente estos últimos presentan eficacia frente a influenza b. el tratamiento de vi con zanamivir inhalado u oseltamivir oral parece reducir el riesgo de complicaciones respiratorias aunque ninguno de estos fármacos ha demostrado eficacia en la prevención de complicaciones graves del vi. a pesar de la ausencia de datos en pacientes trasplantados, algunos autores recomiendan el uso de un antiviral para el tratamiento de los pacientes trasplantados con infecciones por vi. la vacunación de los pacientes de alto riesgo parece la medida más eficaz frente al vi. esta vacuna debe administrarse además a los contactos cercanos y a los trabajadores sanitarios 38 . la respuesta a la vacuna en los pacientes trasplantados no es, sin embargo, tan intensa como la que aparece en la población general. las infecciones por los tipos 1 y 2 del vp se producen generalmente en invierno, a diferencia del tipo 3, cuyas infecciones se distribuyen a lo largo de todo el año. el vp puede producir enfermedad grave en pacientes pediátricos y en receptores de trasplante pulmonar de cualquier edad. en estos últimos, la infección por vp se ha asociado con la presencia de rechazo agudo y bronquiolitis obliterante. las manifestaciones clínicas de esta infección incluyen desde un cuadro catarral de vías altas hasta insuficiencia respiratoria grave. al igual que para el vrs y el vi, el cultivo del virus y la detección antigénica mediante inmunofluorescencia constituyen las principales herramientas para el diagnóstico. a ellas se ha sumado recientemente la detección del arn viral. aunque no se dispone de tratamiento antiviral específico frente a vp, se ha propuesto el empleo de ribavirina en aerosol en pacientes de alto riesgo con enfermedad grave asociada a este virus. sin embargo, en el análisis de esta estrategia procedente de estudios no aleatorizados en receptores de tph se ha evidenciado escasa respuesta clínica. las medidas de control de la transmisión nosocomial representan la mejor actitud profiláctica ya que no existen pautas específicas de profilaxis frente a vp. los pacientes trasplantados pueden adquirir el virus west nile (vwn) a través del donante, mediante transfusión o a través de exposición en la comunidad. en es-tos pacientes, el riesgo de desarrollo de meningoencefalitis tras la exposición es mayor que en individuos inmunocompetentes. en áreas endémicas, el cribado de los hemoderivados, la monitorización de los donantes y las medidas de precaución de los receptores frente a la adquisición comunitaria del virus constituyen las principales estrategias preventivas. la reducción de la inmunosupresión representa la principal estrategia en los receptores con enfermedad por vwn 71 . financiado por el ministerio de sanidad y consumo, instituto de salud carlos iii, red española de investigación en patología infecciosa (reipi rd06/0008). los artículos publicados en la sección "formación médica continuada" forman parte de grupos temáticos específicos (antibiograma, antimicrobianos, etc.). una vez finalizada la publicación de cada tema, se irán presentando al sistema español de acreditación de la formación médica continuada (seaformec) para la obtención de créditos. una vez concedida la acreditación, esta se anunciará oportunamente en la revista y se abrirá un período de inscripción gratuito para los socios de la seimc y suscriptores de la revista, al cabo del cual se iniciará la evaluación, durante un mes, que se realizará a través de la web de ediciones doyma. a) el citomegalovirus puede causar enfermedad después del cuarto mes postrasplante. b) la reactivación del virus de epstein-barr se presenta mayoritariamente entre el segundo y el sexto mes postrasplante. c) no está influida por la intensidad de la inmunosupresión. d) las infecciones adquiridas a través del injerto, como la hepatitis b, pueden aparecer en el primer mes tras la cirugía. e) la enfermedad por virus respiratorios puede producirse en cualquier momento tras el trasplante. a) comparte gran parte de su genoma con el herpesvirus 6. b) se ha asociado con rechazo en pacientes con trasplante renal. c) las pautas de terapia anticipada se han asociado con la aparición de infecciones tardías. d) se ha asociado con mayor riesgo de infecciones fúngicas. e) puede reactivar la replicación de otros herpesvirus. 3. respecto a la enfermedad linfoproliferativa postrasplante señale la respuesta falsa:. a) se asocia al virus de epstein-barr. b) suele aparecer durante el primer año postrasplante. c) existen una gran variedad de formas clínicas. d) la detección del virus de epstein-barr es imprescindible para el diagnóstico. e) la reducción de la inmunosupresión es fundamental para su tratamiento. a) en receptores de trasplante de órgano sólido la primoinfección suele ser asintomática. b) no tiene propiedades inmunomoduladoras. c) los efectos indirectos son excepcionales en trasplantados de órgano sólido. d) se recomienda la monitorización sistemática del vhh-6 en los pacientes trasplantados. e) aciclovir es eficaz frente a vhh-6. a) en pacientes trasplantados seronegativos se considera indicada la vacunación. b) en pacientes seropositivos no se recomienda la profilaxis con aciclovir. c) la incidencia de herpes zóster es superior en tph. d) los pacientes seronegativos expuestos a un paciente con exantema por vvz deben recibir profilaxis postexposición. e) aciclovir constituye el tratamiento de elección. a) el trasplante de un órgano de donante positivo de hbs-ag a un receptor seronegativo supone alto riesgo de transmisión del virus. b) la coinfección por virus de la hepatitis c implica peor pronóstico en el trasplante renal. c) se considera indicada la vacunación pretrasplante de los candidatos seronegativos. d) en casos de resistencia a lamivudina, la continuación del tratamiento se ha mostrado eficaz en algunos casos. e) todas las anteriores son correctas. a) la tasa de reinfección sobre el injerto es prácticamente del 100%. b) el genotipo 1 presenta mayor respuesta al tratamiento antiviral. c) el interferón alfa se asocia a rechazo en trasplantados renales. d) la supervivencia es mayor en trasplantados renales que en pacientes en hemodiálisis. e) la serología negativa no descarta enfermedad activa. a) la presencia de rechazo se ha asociado a nefropatía por bk. b) existe buena asociación entre viruria y nefropatía por bk. c) existe buena asociación entre viremia y nefropatía por bk. d) las células decoy pueden aparecer en la infección por adenovirus. e) en la mayoría de ocasiones se distingue fácilmente del rechazo. a) su incidencia en pacientes trasplantados es alta. b) la anemia por parvovirus b-19 es frecuente en receptores de tph. c) la presencia de igm negativa descarta el diagnóstico. d) la manifestación más típica de la infección en la infancia es la artritis. e) todas las respuestas anteriores son falsas. a) se producen fundamentalmente en la infancia. b) los aerosoles de ribavirina pueden emplearse en el tratamiento de la infección por vrs. c) la vacunación de los pacientes de alto riesgo es la medida de prevención más eficaz frente a la infección por virus influenza. d) no se dispone de tratamiento antiviral específico frente al virus parainfluenza. e) todas las anteriores son correctas. a review of critical periods for opportunistic infection in the new transplantation era cytomegalovirus in transplantation: challenging the status quo pneumonia after heart 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prevent cytomegalovirus disease and early death in recipients of solid-organ transplants: a systematic review of randomised controlled trials preemptive oral ganciclovir can reduce the risk of cytomegalovirus disease in liver transplant recipients indirect outcomes associated with cytomegalovirus (opportunistic infections, hepatitis c virus sequelae, and mortality) in liver-transplant recipients with the use of preemptive therapy for 13 years pre-emptive therapy of cmvpp65 antigen positive renal transplant recipients with oral ganciclovir: a randomized, comparative study antiviral drugs can inhibit lymphocyte apoptosis induced by cytomegalovirus antigens efectos de la infección viral en el paciente trasplantado impact of cytomegalovirus in organ transplant recipients in the era of antiviral prophylaxis emergence of ganciclovir-resistant cytomegalovirus disease among solid organ transplant recipients efficacy and safety of valganciclovir vs. oral ganciclovir for prevention of cytomegalovirus disease in solid organ transplant recipients recovery of hla restricted cytomegalovirus-specific t-cell responses after allogeneic bone marrow transplant: correlation with cmv disease and effect of ganciclovir prophylaxis delayed occurrence of cytomegalovirus disease in organ transplant recipients receiving antiviral prophylaxis are we winning the battle only to lose the war? incidence and outcome of cytomegalovirus infections following nonmyeloablative compared with myeloablative allogeneic stem cell transplantation, a matched control study quantitative pp65 antigenemia in the diagnosis of cytomegalovirus disease: prospective assessment in a cohort of solid organ transplant recipients presentation and early detection of post-transplant lymphoproliferative disorder after solid organ transplantation differences between early and late posttransplant lymphoproliferative disorders in solid organ transplant patients: are they two different diseases? lymphomas after solid organ transplantation: a collaborative transplant study report immunosuppressive tor kinase inhibitor everolimus (rad) suppresses growth of cells derived from posttransplant lymphoproliferative disorder at allograft-protecting doses epstein-barr virus seronegativity is a risk factor for late-onset posttransplant lymphoroliferative disorder in adult renal allograft recipients therapeutic lmp1 polyepitope vaccine for ebv-associated hodgkin disease and nasopharyngeal carcinoma posttransplant lymphoproliferative disease in primary epstein-barr virus infection after liver transplantation: the role of cytomegalovirus disease early onset post-transplant lymphoproliferative disease is associated with allograft localization role of epstein-barr virus dna load monitoring in prevention and early detection of post-transplant lymphoproliferative disease epstein-barr virus (ebv) load and interleukin-10 in ebv-positive and ebv-negative post-transplant lymphoproliferative disorders management of 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kidney and liver transplantation effects of hepatitis c infection and renal transplantation on survival in end-stage renal disease viral hepatitis in the liver transplant recipient viral infection in the renal transplant recipient polyomavirus infection of renal allograft recipients: from latent infection to manifest disease prospective study of polyomavirus type bk replication and nephropathy in renal-transplant recipients quantitative viral load monitoring and cidofovir therapy for the management of bk virus-associated nephropathy in children and adults parvovirus b19 infection after transplantation: a review of 98 cases pure red-cell aplasia of 10 years'duration due to persistent parvovirus b19 infection and its cure with immunoglobulin therapy respiratory virus infections after stem cell transplantation: a prospective study from the infectious diseases working party of the european group for blood and marrow transplantation emerging viral infections in transplant recipients infection in organ transplant recipients efectos de la infección viral en el paciente trasplantado key: cord-307016-4hdsb5oq authors: allen, upton; green, michael title: prevention and treatment of infectious complications after solid organ transplantation in children date: 2010-04-30 journal: pediatric clinics of north america doi: 10.1016/j.pcl.2010.01.005 sha: doc_id: 307016 cord_uid: 4hdsb5oq effective prevention, diagnosis, and treatment of infectious diseases after transplantation are key factors contributing to the success of organ transplantation. most transplant patients experience different kinds of infections during the first year after transplantation. children are at particular risk of developing some types of infections by virtue of lack of immunity although they may be at risk for other types due the effect of immunosuppressive regimens necessary to prevent rejection. direct consequences of infections result in syndromes such as mononucleosis, pneumonia, gastroenteritis, hepatitis, among other entities. indirect consequences are mediated through cytokines, chemokines, and growth factors elaborated by the transplant recipient in response to microbial replication and invasion, which contribute to the net state of immunosuppression among other effects. this review summarizes the major infections that occur after pediatric organ transplantation, highlighting the current treatment and prevention strategies, based on the available data and/or consensus. upton allen, mbbs, msc, frcpc a,b,c, *, michael green, md, mph d,e organ transplantation is the most practical means of rehabilitating patients with a variety of forms of end organ dysfunction. this procedure is arguably the outstanding clinical biomedical accomplishment of the last 3 decades. potent immunosuppressive drugs have dramatically reduced the incidence of rejection of transplanted organs, but have also increased the susceptibility of patients to opportunistic infections. 1 thus, the success of organ transplantation is dependent in part on effective prevention, diagnosis, and treatment of infectious diseases after transplantation. to this end, emphasis is increasingly being placed on prevention. most transplant patients will have evidence of microbial invasion in the first year after transplant. the effects of this microbial invasion are diverse, resulting in direct and indirect consequences. the direct consequences result in a variety of clinical infectious disease syndromes such as mononucleosis, pneumonia, gastroenteritis, hepatitis, among other entities. the indirect consequences are mediated through cytokines, chemokines, and growth factors elaborated by the transplant recipient in response to microbial replication and invasion, which contribute to the net state of immunosuppression, the pathogenesis of acute and chronic allograft injury, and in some cases, the development of lymphoproliferative or malignant disorders. the risk of infection in the solid organ transplant patient is largely determined by the interaction of 3 factors: technical/anatomic factors that involve the transplant procedure itself, and the perioperative aspects of care such as the management of vascular access, drains, and the endotracheal tube; environmental exposures (box 1); and the patient's net state of immunosuppression (box 2). in the case of technical/anatomic mishaps, the best way to prevent infection is to correct the anatomic abnormality under coverage of appropriate antimicrobial therapy as antimicrobial treatment alone will not eliminate the risk of developing recurrent infections related to the uncorrected problem. as a consequence, the transplant recipient remains at high risk of subsequent infections with an increased risk of developing antimicrobial resistance until successful correction of the underlying abnormality. 1, 2 when one is considering therapy in the transplant patient, the concept of the therapeutic prescription package is useful. this package has 2 major components: an immunosuppressive component to prevent and treat rejection and an antimicrobial component to make it safe. thus, the nature of the antimicrobial program being administered must be closely linked to the nature and intensity of the immunosuppressive program required and the resulting net state of immunosuppression. 1, 2 there are 3 modes in which antimicrobial agents can be administered to the transplant recipient: a therapeutic mode, in which antimicrobial agents are administered in the treatment of established clinical infection; a prophylactic mode, in which antimicrobial agents are administered to an entire population before an event to prevent the occurrence of an infection important enough to justify this intervention: and a preemptive mode, in which antimicrobial agents are administered to a subpopulation noted to be at particular risk of clinically important infection based on clinical, epidemiologic, or laboratory markers. this review focus on preventive strategies (prophylactic and preemptive) and on the diagnosis and management of established infection. infection in the posttransplant period has a stereotyped temporal pattern, a timetable. although some clinical syndromes, such as pneumonia, can occur at any time point after transplant, the causes may be very different at different time points. fig. 1 delineates the timetable for the onset of infections after organ transplantation in the absence of effective preventative strategies. when preventative antimicrobial therapy fails to completely protect the patient, a common clinical effect is to extend the time period in which the infectious complication will likely appear. for example, in the case of cytomegalovirus (cmv) infection, in the absence of prophylaxis cmvinduced clinical disease is most common 1 to 3 months after transplantation. when prophylaxis is used, but fails, it is common for the disease to occur 4 to 8 months after transplantation (depending on the nature and duration of the prophylaxis and the immunosuppressive regimen). 2, 3 like all patients, the transplant recipient is at risk of acquiring infections in the health care and community settings. such infections are not necessarily transplant specific. fig. 1 is a graphical representation of the timing of infections during the posttransplant period. 2 in general, 3 time periods are recognized, each with differing forms of infection: [1] [2] [3] in the first month, there are 3 major causes of infection: (1) infection that was present in the recipient before transplant, with its effects now increased as a result of surgery, anesthesia, and immunosuppressive therapy; (2) infection conveyed with a contaminated allograft; and (3) the same bacterial and candidal infections of the wound, lungs, drainage catheters, and vascular access devices that are seen in nonimmunosuppressed patients undergoing comparable surgery. most (more than 95%) of the infections occurring in the first month after transplant fall into this last category; the main factor determining the incidence of such infections is the technical aspects of surgery as well as specific aspects of perioperative and postoperative care. this second time period is when the effect of immune suppression is most notable on the risk of infection. during this period, 2 major classes of infection predominate. the first of these is attributable to a group of viral pathogens that are associated with latent and/or chronic infections. examples include cmv, epstein-barr virus (ebv), human herpes virus 6 (hhv-6), and the hepatitis viruses (b and c); all of which may cause disease through acquisition of primary infection (typically from the donor) or secondary infection within the recipient under the pressure of immune suppression (secondary infection includes reactivation of latent pathogens and reinfection with a new strain). the second set of pathogens observed in this time period cause socalled opportunistic infections and include organisms such as listeria monocytogenes, aspergillus fumigatus, and pneumocystis jiroveci. development of infection with these opportunistic pathogens is attributable to the combination of sustained immunosuppression, which is often combined with the immunomodulating effects of viral infection creating a net state of immunosuppression great enough that these opportunistic infections can occur without an especially intensive environmental exposure. information describing infections occurring in children more than 6 months after transplant is limited because transplant recipients commonly return to their homes, which are often far from their transplant centers. accordingly, details regarding infectious complications occurring in this time period may be biased to include more significant infections resulting in hospitalization. despite this limitation, experience supports dividing individuals with infections during this last time period into 2 main categories: (1) most patients with a good result from transplantation (maintenance immunosuppression, good allograft function) are at greatest risk from typical community-acquired infections (such as influenza, parainfluenza, and respiratory syncytial virus); (2) a smaller group of patients with poorer outcomes from transplantation (excessive acute and chronic immunosuppression, poor allograft function, and, often, chronic viral infection). these patients remain at high risk for recurrent infections related to uncorrected mechanical problems as well as opportunistic infections attributable to organisms like pneumocystis jiroveci, listeria monocytogenes, cryptococcus neoformans, and nocardia asteroides. the time line of infections after transplantation outlines the wide spectrum of infections that occur after transplantation. among these infections, the major burden is represented by bacteria, candida species, cmv, ebv, adenovirus, varicella zoster virus, and community-acquired respiratory viruses. in addition, certain infections represent challenges for specific organ groups (eg, bk virus infection in renal transplant recipients and toxoplasma infection in heart/heart-lung transplant recipients). selected aspects of these infections are summarized later. as indicated earlier, bacterial infections are most commonly seen during the early posttransplant period. however, bacterial infections can occur at any time after transplantation. risk factors include the presence of indwelling catheter devices, including endotracheal tubes, foley catheters and central venous catheters. in this regard, hospital-acquired gram-negative organisms, coagulase-negative staphylococci and staphylococcus aureus are often encountered. the nature of these infections and the specific pathogens involved vary according to the organ transplanted, sites of infection, the microbiologic flora of the institution, and the pretransplant status of the patient. in general, the most common site of bacterial infection is at or near the site of transplantation. urinary tract infection, notably pyelonephritis, has been recognized as the most common infectious complication among renal transplant recipients. 4 infections after organ transplantation liver transplant recipients, the most frequent site of bacterial infection is within the intraabdominal space, often accompanied by bacteremia. 5, 6 intraabdominal and wound infections are also commonly seen in intestinal transplant recipients. bacteremia, which can be partly explained by disruption of the mucosal barrier associated with harvest injury or rejection, is commonly seen. 7, 8 infection of the lower respiratory tract (including pneumonia and lung abscess) is the most common site of infection reported in most, but not all, series of pediatric heart transplant recipients. 9-12 mediastinitis is another important infection after thoracic transplantation, particularly if reexploration of the chest is required. pathogens associated with mediastinitis include s aureus and gram-negative enteric bacilli. children undergoing lung transplantation because of cystic fibrosis experience a high rate of infectious complications as they often have preexisting colonization with resistant organisms, including pseudomonas species, burkholderia species and other bacterial pathogens. [13] [14] [15] [16] given the importance and difficulty in treating these often resistant organisms, transplant centers usually recommend a thorough microbiologic evaluation of heart-lung or lung transplant candidates before transplantation. the transplant patient is also at risk of developing infection as a result of community-acquired bacterial pathogens, the most important of which is streptococcus pneumoniae (pneumococcus). transplant recipients are known to be at increased risk of pneumococcal sepsis. 17 among these patients, heart recipients who have been transplanted at a young age seem to be at an increased risk compared with other pediatric organ recipients. 17 the frequency of fungal infections varies according to the type of organ transplanted. [18] [19] [20] for example, invasive fungal infections are uncommon after renal transplantation. for these patients, the most frequently encountered entity is candida urinary tract infection. similarly for liver, heart, and intestinal transplant recipients, the major fungal infections are also caused by candida species. for all of these patients, invasive aspergillosis and other mycoses occur uncommonly. the consequences of invasive aspergillosis and other noncandidal mycoses associated with invasive infections are frequently devastating. lung transplant recipients are unique in that they experience proportionately more infections with aspergillus species compared with other organ recipients. these infections are often seen in children undergoing transplantation as treatment of cystic fibrosis and reflect infection with aspergillus that was present in the recipient before transplantation. however, aspergillus is also frequently recovered from the lungs of transplant recipients with obliterative bronchiolitis (chronic rejection of the lung) regardless of the cause of their original lung disease leading to transplantation. 21 cmv cmv infection and disease remain important causes of mortality and morbidity among pediatric organ transplant recipients. 22 data on the precise burden in pediatric organ transplant recipients are limited, however, by wide differences in data collection and reporting. in addition, nonuniform approaches to the laboratory diagnosis and definition of cmv disease applied in retrospective studies affects the ability to interpret available data. in 5 centers in the united states, 10% to 20% of liver transplant patients experienced cmv disease within 2 years after transplantation. 23 a review of first-time pediatric lung transplant patients indicated that among at-risk subjects, the incidence of cmv viremia was 29% to 32%, whereas the incidence of cmv pneumonitis was 20% during the first year after transplantation. 24, 25 cmv disease is often associated with fever and hematologic abnormalities, including leucopenia, atypical lymphocytosis, and thrombocytopenia. visceral sites affected may include the gastrointestinal tract, lungs, and liver. central nervous system involvement, including chorioretinitis, is rare in organ transplant recipients. the diagnosis of cmv infection and disease in organ transplant recipients can be affected by the variable lack of sensitivity and/or specificity of different diagnostic tests. serology has no role in the diagnosis of active cmv disease after transplantation as it does not differentiate between prior infection and active disease. the interpretation of serologic results is further confused by the potential presence of passive antibody from blood products provided during or after the transplant procedure. in addition, the altered immune responses after transplantation might impair the patient's ability to mount predictable humoral responses. viral culture of blood for cmv has limited clinical usefulness for diagnosis of disease caused by poor sensitivity. there is no role for cmv urine culture in the diagnosis of disease caused by poor specificity. 26 a positive culture from bronchoalveolar lavage specimens may not correlate with disease. 27, 28 the presence of a positive measurement of cmv load in the peripheral blood (measured by either nucleic acid amplification techniques (nat) or pp65 antigenemia assay) in a patient with a compatible cmv clinical syndrome is strongly suggestive of cmv disease. however, the cmv load may be positive before the onset or in the absence of clinical disease and may be seen in the presence of disease from other causes. further, the cmv load in the peripheral blood may be negative in some patients with tissue invasive disease, especially cmv involving the gastrointestinal tract. given the variable usefulness of these tests, histopathologic examination of involved organs is essential to confirm the presence of cmv when the diagnosis of invasive cmv disease is being considered. intravenous ganciclovir (10 mg/kg/d, given twice daily) remains the preferred drug for the treatment of cmv disease in pediatric transplant recipients. reduction of immunosuppression is desirable unless concurrent evidence of rejection precludes this. ganciclovir therapy is sometimes accompanied by cmv hyperimmune globulin therapy in some centers. typically, a clinical response to treatments is expected in 5 to 7 days after treatment has been initiated. foscarnet and cidofovir may be considered in the setting of ganciclovir resistance. the optimal length of treatment should be determined by monitoring viral loads weekly. 22 treatment is typically continued until 2 consecutive negative samples are obtained. in cases of serious disease and in tissue invasive disease without viremia, longer treatment periods with clinical monitoring of the specific disease manifestation are recommended. data are emerging on the use of valganciclovir in the prevention and treatment of cmv infection/disease among adult transplant recipients. 29, 30 considerably less data are available for children. 31 a summary of the approach to prophylaxis is outlined in table 1 , including the roles of ganciclovir with or without immune globulin and suggestions on duration of their use, where indicated. although the most feared ebv-associated disease after transplantation is posttransplant lymphoproliferative disorder (ptld), patients may experience a broad range of clinical symptoms that do not meet the definitions of ptld. these might include the manifestations of infectious mononucleosis (fever, malaise, exudative pharyngitis, lymphadenopathy, hepatosplenomegaly, and atypical lymphocytosis), specific organ diseases such as hepatitis, pneumonitis, gastrointestinal symptoms, and hematological manifestations such as leucopenia, thrombocytopenia, hemolytic anemia, and haemophagocytosis. 32 ebv-associated leiomyosarcoma has also been described. 33 ebv disease is seen most frequently in patients experiencing primary ebv infections following transplantation. rates of ebv disease and ptld vary according to the organ transplanted with recipients of intestines and lungs being at the highest risk and those receiving liver, kidney, and heart at lower risk. as for cmv disease, serology is not useful for diagnosis in the posttransplant period. the presence of increased ebv viral load in the peripheral blood as determined by quantitative polymerase chain reaction (pcr) is widely accepted as an assay to predict or indicate the likely presence of ptld. however, these assays are limited in specificity and may remain persistently elevated in asymptomatic patients. the definitive diagnosis of ebv diseases, including ptld requires histopathologic examination of biopsy material. the use of ebv-specific assays (eg, ebv encoded rna [eber] staining) enhances the sensitivity and specificity of histologic examination in these patients. the approach to the treatment of ebv disease and ptld remains somewhat controversial. reduction of immune suppression is widely accepted as critical in the management of patients with these complications. the role of the antiviral agents acyclovir and ganciclovir are unproven, although many transplant clinicians use them in the treatment of ebv infection. 34, 35 treatment approaches are often modified from regimens used to treat cmv disease. currently, when antiviral agents are used to treat ebv, the agent of choice is ganciclovir, as in vitro it is 10 times more active against ebv compared with acyclovir. the controversy on the use of these agents for ebv/ptld arises because although these agents can suppress ebv lytic infection, infections after organ transplantation they seem to be of limited value in treatment nonlytic ebv proliferation, which is believed to be the dominant component of ebv-related ptld. increasing evidence (albeit anecdotal) supports the use of the anti-cd20 monoclonal rituximab in the treatment of ebv disease and ptld. however, the optimal timing and treatment strategy for this agent remain to be defined. additional alternative strategies such as the use of chemotherapy require collaborative input from oncologists familiar with the management of ebv-related disease in organ transplant recipients. the prevention of posttransplant ebv diseases, including ptld remains controversial. antiviral regimens have been modeled from the cmv scenario. to date, preemptive reduction in immunosuppression in the setting of increasing viral load may have the most supportive data and is increasingly being used (see table 1 ). adenovirus infection may be acquired by exogenous means or endogenously as a result of reactivation of latent infection. the clinical spectrum of infection and disease in pediatric transplant recipients is variable. 36 there are more than 51 serotypes that generally show some fidelity as this relates to the types of organs affected and the resultant syndromes. 37 among liver transplant patients, disease manifestations include self-limited fever, gastroenteritis, cystitis, hepatitis, and pneumonitis. these manifestations may occur in other transplant recipients, depending on the level of immunosuppression. adenovirus dna can be detected in the peripheral blood using qualitative or quantitative pcr techniques. in the appropriate clinical setting, the presence of adenovirus dna in the blood provides presumptive evidence of infection, with examination of tissue by histopathology providing more definitive evidence of infection. the management of adenovirus infection poses challenges because of limited effective treatment options. cidofovir is currently accepted as the drug of choice. however, this conclusion is primarily based on a retrospective review of historical experience and the agent is not approved for this indication by the us food and drug administration or similar agencies. nonetheless, ongoing experience continues to support a role for the treatment of adenoviral infections with this agent. before the advent of cidofovir, intravenous ribavirin was used with anecdotal reports of successes and failures. 38 although the major burden of bk virus infection is among adult renal transplant patients, the role of this virus in pediatric organ transplantation is becoming more clearly defined. most infections are as a result of reactivation in adults. primary infection may occur, notably among pediatric transplant recipients. the major clinical manifestation in the renal transplant recipient is tubulointerstitial nephritis. renal biopsy is required for definitive diagnosis. noninvasive testing modalities include screening of blood and urine for bk dna using pcr. 39 there is no firm consensus on the preferred approach to the management of bk nephropathy. early detection is a desired goal. to that end, quantitative pcr monitoring for bk dna is performed in some centers. this often provides opportunities to modulate immunosuppression. in situations where antiviral therapy is used, the agent most often used is cidofovir, for which there are reports of success. 40 however, at present no consensus exists supporting the therapeutic efficacy of this agent. varicella zoster virus (vzv) is a major threat to pediatric transplant patients and many individuals enter transplantation without immunity to this virus. 41 immunosuppressed individuals are at risk of severe outcomes from vzv infection. visceral involvement may accompany severe infection and clinicians should be reminded that disseminated disease can rarely occur in the absence of typical cutaneous vesicles. 42 pretransplant vaccination has been shown to provide sustained humoral immunity for at least 2 years after transplantation. 43 it is strongly recommended that transplant candidates be vaccinated before transplantation. given that this is a live vaccine, the minimum interval between vaccination and transplantation is recommended to be 4 to 6 weeks. although some centers have selectively considered the use of vzv vaccine in susceptible children after transplantation, this approach cannot be recommended at this time because of the lack of safety data, given the known risk of live vaccines in immunosuppressed individuals. families of transplant patients should be educated to be alert to potential exposures in settings such as schools and should report them promptly to health care providers to allow for postexposure prophylaxis. varicella-susceptible transplant recipients should receive varicella zoster immune globulin within 96 hours after a varicella exposure. 44 if this window has passed or if varicella zoster immune globulin is not available, there is the option for the use of postexposure chemoprophylaxis with acyclovir (80 mg/kg/d, given 4 times daily for 7 days; maximum dose 800 mg, 4 times daily) starting at day 7 to 10 after exposure. 44 in the absence of profound immunosuppression, no prophylaxis is usually necessary for exposed organ recipients who are immune to vzv as a result of prior infection or vaccination before transplantation. treatment of the transplant patient with vzv infection is usually initiated with intravenous acyclovir until there is evidence of clinical improvement (fever abates, no new lesions, lesions starting to crust, no visceral disease). outpatient treatment with oral acyclovir or valacyclovir has been used in children with mild infection, low levels of immunosuppression, and when there are no concerns regarding the adequacy of follow-up. famciclovir and valacyclovir are approved for use in adults. famciclovir is the prodrug of penciclovir, which has an extended half-life in infected cells. valayclovir is the prodrug of acyclovir and produces fourfold greater serum levels than those produced by acyclovir. pediatric formulations are current not available. most children who have undergone organ transplantation experience communityacquired viral infections and have no significant problems. however, it is well recognized that children who are significantly immunocompromised can have severe disease caused by these viruses, including respiratory syncytial virus infection, parainfluenza, and influenza viruses. 45, 46 for pediatric transplant recipients, the likelihood of more severe outcomes is greater during the early months after transplantation or during periods of peak immunosuppression. in 2009, the advent of a pandemic strain of influenza a (pandemic h1n1 2009) has been cause for concern. 47, 48 in general, the principles that govern the prevention and treatment of pandemic h1n1 in pediatric transplant patients are similar to those for seasonal influenza. transplant patients are among those who are known to be at increased risk of severe outcomes from pandemic h1n1. they are candidates for treatment with oseltamivir or zanamivir (where appropriate) if they have acute respiratory illness that is suspected or confirmed to be caused by h1n1. 49 like other immunocompromised patients, they are at an increased risk of having prolonged shedding of virus and the harboring of drug-resistant strains of influenza a, including pandemic h1n1. pediatric transplant patients are candidates for vaccination against this virus (as they are for seasonal influenza a) if they are greater than 6 months of age. most infections after organ transplantation experts currently delay vaccination until after the first months following organ transplantation. 49 pneumocystis pneumonia (pcp) is a recognized threat in the posttransplant period. 50, 51 the risk is greatest during the first 6 to 12 months after transplantation, with the time of onset being usually after the first month. trimethoprim-sulfamethoxazole remains the prophylactic agent of choice. 52 this agent is also preferred for initiation of therapy in individuals who develop pcp. although the optimal duration of pcp prophylaxis remains unclear, most experts provide pcp prophylaxis for a minimum of 6 to 12 months, with some recommending indefinite use, especially for solid organ transplant recipients requiring more prolonged periods of higher levels of immunosuppression. intravenous pentamidine is an alternative for treatment of pcp for patients who are intolerant of trimethoprim-sulfamethoxazole or whose disease has not responded to this agent after 5 to 7 days. 52 however, pentamidine is associated with a relatively high incidence of adverse events, including pancreatitis, renal dysfunction, hypoglycemia, and hyperglycemia. atovaquone may be used to treat milder forms of pcp among adults; however, pediatric data are limited. alternatives to trimethoprim-sulfamethoxazole for prophylaxis include oral atovaquone or dapsone. aerosolized pentamidine is recommended if children cannot tolerate these oral agents. another alternative is intravenous pentamidine, albeit at the risk of greater toxicity. 52 toxoplasma gondii infection is of greatest concern among heart transplant patients, but infection can occur in other categories of transplant recipients, including kidney and liver recipients. 53, 54 toxoplasma organisms can remain encysted within muscle tissue, such as cardiac muscle. thus, infection is acquired as a result of the reactivation of cysts that remain dormant in the donor hearts of toxoplasma seronegative children. clinical manifestations can occur as early as 2 weeks after transplantation. manifestations include pneumonia, fever syndrome, myocarditis, chorioretinitis, and central nervous system disease. current prophylaxis includes pyrimethamine/sulfadiazine for d1rã� patients. trimethoprim-sulfamethoxazole is typically used in r1 patients. however, some experts also recommend trimethoprim-sulfamethoxazole for d1rã� patients. the duration of prophylaxis is usually 6 months. infection with this parasitic worm is of relevance to individuals who previously acquired infection following a period of residence in endemic regions. 55, 56 donorassociated transmission of stronygloides has also occurred. asymptomatic immunocompromised persons, including transplant recipients are at risk of strongyloides hyperinfection, which results from dissemination of larvae via the systemic circulation, resulting in abdominal pain, diffuse pulmonary infiltrates, and septicemia or meningitis from enteric gram-negative bacilli. serologic screening is recommended for individuals from endemic regions ( table 2 ). ivermectin treatment is indicated for screenpositive individuals. tuberculosis (tb) is always a concern for immunocompromised hosts. 57-60 incidence rates are low in most transplant centers in the developed world, but outcomes of tb can be devastating in organ transplant recipients. before transplantation a careful history for tb exposure or infection, mantoux test screening, and a chest radiograph can assists in establishing the diagnosis of latent tuberculosis infection. the interferon-gamma release assays are currently being evaluated to define their role in settings where the tb skin test has poor utility. 61, 62 the use of antituberculous agents in transplant patients poses challenges because of the interaction between isoniazid and rifampin with immunosuppressive medications. however, this should not be seen as a contraindication to the use of antituberculous agents, which have to be used when warranted by the clinical situation. the pretransplant phase is arguably the most important phase of transplantation. a detailed history and physical examination are necessary to identify conditions that influence the risk or management of infections after transplantation. this assessment allows for the identification of preexisting conditions that require treatment or prophylaxis in the period before or after transplantation. table 2 summarizes screening tests that should be performed in the pretransplant period. immunizations represent an important strategy for preventing infections in the transplant patient. [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] wherever possible, vaccines should be administered in the pretransplant period to improve the chances of optimal immunologic take. a guideline on vaccinations for the transplant candidate/patient has recently been published. 73 in some situations, accelerated vaccination schedules may be used for selected vaccines. given differences in childhood vaccination schedules in different jurisdictions, clinicians should acquaint themselves with the appropriate schedules and the circumstances under which accelerated schedules could be used. when using vaccines after transplantation, one needs to be concerned about safety as well as efficacy. in general, all live virus vaccines should be avoided in the transplant recipient. the oral polio, yellow fever, and oral typhoid vaccines are live and are contraindicated in immunosuppressed patients. the live attenuated intranasal influenza vaccine is also contraindicated. measles, mumps, and rubella vaccines are somewhat contraindicated and their use should be limited to outbreak scenarios. the varicella vaccine is also somewhat contraindicated and is not approved for use in transplant patents. although limited published data support the potential use of this vaccine in transplant recipients, 72 most experts continue to advise against this practice. in the cases of the nonlive vaccines, the major concern is not safety, but efficacy. thus, in general, it is advisable to give nonlive vaccines at times when the level of immunosuppression would allow for immunogenicity. table 3 summarizes the vaccines that are indicated and contraindicated in transplant recipients. given the relative burden and importance of invasive pneumococcal disease in pediatric transplant recipients, the importance of pneumococcal vaccination should not be underestimated. 17, 63, 69 donor organ screening the organ donor is a frequent source of exposure to pathogens in the organ transplant recipient. accordingly, screening of the donor organ is a crucial aspect of the preventive strategies aimed at minimizing adverse outcomes from infections in the posttransplant period. despite a long-standing recognition of the importance of donor-derived infections, increased concern about this problem has emerged because of recent donor-related transmission of human immunodeficiency virus (hiv). this case, as well as concerns about the lack of sensitivity of serologic testing and the relatively long time period until seroconversion against hiv, hepatitis b virus (hbv), and hepatitis c virus (hcv), have led to interest in the use of nat-based testing for the pathogens hiv, hcv, and hbv. although arguments exist for and against the use of nat testing, a final international consensus addressing if and when to use these tests is only beginning to emerge. 74 decisions relating to the use of such tests must consider not only the reliability of this technology but also the feasibility of universal implementation of these testing procedures for all procurement organizations. recent cases of donor-associated transmission of lymphocytic choriomeningitis virus and west nile virus have also raised questions on whether the panel of routine tests performed on potential donors should be expanded to include these and other potential donorderived pathogens. to date, a consensus has not been reached on whether or not screening against these pathogens should be routinely included in donor testing panels. it is to be hoped that the implementation of working groups and committees focusing on the problem of donor-derived infections in north america and europe will lead to improved data to better inform subsequent recommendations regarding donor testing. current requirements for screening of nonliving donors are shown in table 4 . at present, no specific requirements have been implemented for screening allen & green of live donors. in general, testing strategies that are in place for deceased donors are applied to the use of these organs. the importance of documenting the presence of potential donor-transmissible pathogens is imperative not only to inform decision making regarding the use of potential donor organs but also because results of donor testing can inform specific preventative strategies even when donor-associated exposure to pathogens is unavoidable. various prophylaxis regimens are used in the posttransplant period. although there are common basic principles, the specific regimens vary across centers and by the type of organ transplanted. for most patients, the major targets of prophylaxis are infections after organ transplantation bacterial pathogens, herpes group viruses, and fungal pathogens, including pneumocystis. perioperative antibiotics are typically used for 48 to 72 hours to provide prophylaxis against surgical contamination. the burden of cmv infection in transplant patients is such that it represents the major focus of prevention in the posttransplant period, when intravenous ganciclovir is usually used with or without cmv hyperimmune globulin in selected patient groups. table 1 summarizes various pathogens and the regimens that are often used for prevention of infection in the posttransplant period. in the evaluation of the febrile transplant patient, clinicians should consider if the child's fever is related to common childhood infections or infections that are unique to the immunosuppressed transplant recipient. to this end, the timing of infections after transplantation (see fig. 1 ) provides guidance regarding the most likely pathogens. for example, as discussed earlier, the most likely causes of infection within the first month after transplantation are often bacterial or candidal and are largely similar to what is seen in nonimmunosuppressed patients who have undergone comparable surgery. the nature of the evaluation will depend on the clinical status of the patient and whether or not a source of infection has been identified. examination abnormal, focus of infection defined. admission to hospital may be indicated depending on the clinical status of the patient and the site of the infection. the diagnostic evaluation varies, but should include a minimum of a complete blood count and differential, blood, and urine cultures. additional investigations depend on the clinical assessment and the timing of presentation after transplantation. examination normal, no focus of infection defined. patients who are clinically unwell typically require admission for evaluation and treatment. the diagnostic evaluation should consider the likely differential diagnoses. consultation with infectious diseases is recommended. patients who are well may not necessarily require admission. however, this depends on several factors, including the adequacy of follow-up, the degree of immune suppression and the suspected diagnoses. the diagnostic evaluation should include a minimum of a complete blood count and differential, blood, and 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implications for management american society of transplantation infectious diseases community of practice. mycobacterium tuberculosis interferon-gamma release assays improve the diagnosis of tuberculosis and nontuberculous mycobacterial disease in children in a country with a low incidence of tuberculosis performance of an interferon-gamma release assay for diagnosing latent tuberculosis infection in children seven-valent pneumococcal conjugate vaccine in pediatric solid organ transplant recipients: a prospective study of safety and immunogenicity a prospective, comparative study of the immune response to inactivated influenza vaccine in pediatric liver transplant recipients and their healthy siblings double-dose accelerated hepatitis b vaccine in patients with end-stage liver disease failure of hepatitis b immunization in liver transplant recipients: results of a prospective trial immunogenicity and safety of hepatitis a vaccine in liver and renal transplant recipients immunity to poliomyelitis, diphtheria and tetanus in pediatric patients before and after renal or liver transplantation safety and immunogenicity of the american academy of pediatrics-recommended sequential pneumococcal conjugate and polysaccharide vaccine schedule in pediatric solid organ transplant recipients report from the advisory committee on immunization practices (acip): decision not to recommend routine vaccination of all children aged 2-10 years with quadrivalent meningococcal conjugate vaccine (mcv4) pretransplant varicella vaccination is cost-effective in pediatric renal transplantation ast infectious diseases community of practice. guidelines for vaccination of solid organ transplant candidates and recipients safety and immunogenicity of varicella-zoster virus vaccine in pediatric liver and intestine transplant recipients special report. nucleic acid testing (nat) of organ donors: is the ''best'' test the right test? key: cord-354325-r73datur authors: berger, mitchell; shankar, vidya; vafai, abbas title: therapeutic applications of monoclonal antibodies date: 2002-07-31 journal: the american journal of the medical sciences doi: 10.1097/00000441-200207000-00004 sha: doc_id: 354325 cord_uid: r73datur abstract researchers have sought therapeutic applications for monoclonal antibodies since their development in 1975. however, murine-derived monoclonal antibodies may cause an immunogenic response in human patients, reducing their therapeutic efficacy. chimeric and humanized antibodies have been developed that are less likely to provoke an immune reaction in human patients than are murine-derived antibodies. antibody fragments, bispecific antibodies, and antibodies produced through the use of phage display systems and genetically modified plants and animals may aid researchers in developing new uses for monoclonal antibodies in the treatment of disease. monoclonal antibodies may have a number of promising potential therapeutic applications in the treatment of asthma, autoimmune diseases, cancer, poisoning, septicemia, substance abuse, viral infections, and other diseases. i n 1975, kohler and milstein revolutionized the field of immunology by developing monoclonal antibodies (mabs). since that time, many mabs have been developed for use in diagnostic procedures and in immunotherapy. ever since it was observed that the therapeutic use of heterologous mabs elicited immunogenic responses in humans, significant research efforts have been devoted toward creating chimeric and humanized antibodies for use in human patients. major achievements have been in the production of mabs in transgenic plants and animals. the use of phage display libraries has created customized antibodies with defined affinity and specificity. this review describes how rodent, chimeric, and humanized antibodies have each been used, with varying degrees of success to treat cancer, septicemia, autoimmune disorders, and infectious diseases. we also describe here recent applications of antibody engineering, such as the use of bispecific antibodies and antibody fragments in immunotherapy. von behring and kitasato discovered in 1890 that the serum of vaccinated persons contained certain substances, which they termed "antibodies." in 1895, they treated diphtheria with an antiserum raised against the toxin. on the basis of research on tetanus toxin and trypanosome parasites, ehrlich proposed in 1900 the "side-chain theory" of antibody formation, which hypothesized that physiologically active substances, including toxins, attach to cell surface receptors that are produced in response to toxin-cell interactions and then ejected from the cells into the bloodstream, leading to circulating antibodies. 1, 2 not until the 1950s, however, did scientists' understanding of antibodies become sufficient to lay the foundation for the development of mabs. jerne 3 postulated in 1955 a theory of natural selection for antibody formation. animals vaccinated with an antigen were expected to produce several distinct antibodies against several epitopes of the antigen. frank macfarlane burnet subsequently refined and expanded jerne's theory. 4 burnet's "clonal selection theory," as it is generally known, postulates that cells specific for synthesizing 1 type of antibody are spontaneously generated due to random somatic mutations during the maturation of the immune system and that these cells proliferate when exposed to an antigen. at about the same time, porter isolated fragment antigen binding (fab) and fragment crystalline (fc) from proteolytically cleaved rabbit ␥-globulin. 5 until the 1960s, antibody-producing cells were difficult to maintain in culture, because they died after a few days. in addition, only polyclonal antibodies could be obtained. in 1964, littlefield 6 developed a way to isolate hybrid cells from 2 parent cell lines using the hypoxanthine-aminopterin-thymi-dine (hat) selection media. in 1975, khler and milstein 7 provided the most outstanding proof of the clonal selection theory from results of heterokaryons-cell hybrids formed by the fusion of normal and malignant cells. twenty-five years after kohler and milstein produced the first monoclonal antibodies, dramatic progress has been made in using antibodies for diagnostic purposes, but the uses of mabs to treat disease have-until recently-remained somewhat limited. 8, 9 however, as this review indicates, the potential of mabs to aid in the treatment of a wide range of diseases is now beginning to be realized. antibodies are y-shaped proteins composed of peptides called heavy and light chains, the ends of which vary from antibody to antibody. each combination of heavy and light chains binds to a particular antigenic site. these glycoprotein chains are folded into domains of about 110 amino acids that become twisted into the immunoglobulin (ig) fold and are stabilized by disulfide bonds. structurally, each ig molecule consists of 2 50-kd heavy chains and 2 25-kd light chains, linked by disulfide bonds. human immunoglobulins are divided into 5 classes or isotypes based on the amino acid composition of their heavy chains: ␣, ␦, ⑀, ␥, and , denoted iga, igd, ige, igg, and igm, respectively. there are 2 kinds of light chains, (k) and (l), which are common to all 5 classes. four subclasses of igg (igg1, igg2, igg3, and igg4) and 2 subclasses of iga (iga1 and iga2) exist, each with a distinct function. secretory iga exists in dimeric form held together by a j chain and is associated with a secretory component that helps it pass through the cell membrane. 10, 11 each chain has a constant domain to bind host effector molecule and a variable domain to bind to the target antigen. light chains have 1 variable (v l ) and 1 constant domain (c l ), whereas heavy chains have 1 variable (v h ) and either 3 (␣, ␦, and ␥ chains) or 4 constant domains (⑀ and heavy chains) depending upon the isotype class. each variable domain contains 3 regions known as "hypervariable loops," also known as complementarity-determining regions (cdrs), that identify the antigen. the other amino acids in the variable (fv) domain are known as framework residues and act as a scaffold to support the loops. the v l and the v h , the c h 1 and the c l , and the 2 c h 3 domains are paired; the 2 c h 2 domains have carbohydrate side chains attached to them and are not paired. the folded constant domains may be homologous among different species, allowing hybrid domains (eg, mouse-human) to be produced. the variable domain confers specificity and affinity. the cdr amino acid sequences are extremely variable and play a large role in interac-tion with the targeted antigen. the chain type, region, and distance from the amino terminus characterize the domains. thus, c h 2 domain refers to the second constant domain of the heavy chain. upon digestion with papain, the antibody molecule is cleaved on the amino-terminal side of the disulfide bridges into 2 identical fabs and an fc fragment, whereas pepsin cleaves the antibody on the carboxy-terminal side of the disulfide bridges into 1 f(ab') 2 fragment containing both the arms of the antibody, and many small pieces of the fc fragment. currently, the following are available: whole antibodies, enzymatically produced 50-kd fab fragments, engineered 25-kd single-chain fv (scfv) antibodies consisting of the v h and v l connected by a flexible peptide linker, diabodies (noncovalent dimers of scfv), minibodies (scfv-c h 3 dimers), and heavy chain iggs found in species of the camelidae family, which are devoid of light chains and are referred to as v hh . the first mab described by kohler and milstein was created by the fusion of murine myeloma cells with murine-antibody-secreting lymphocytes. 7, 8 myeloma cells are immortalized b lymphocytes capable of secreting homogeneous antibodies. the immortal myeloma cell lacks the enzyme hypoxanthine guanosine phosphoribosyl transferase (hgprt) and is sensitive to the hat media. however, a hybrid cell known as a hybridoma, generated by the fusion of myeloma cell and an antibody-producing b cell, can survive in the hat media. the spleen b lymphocytes contribute the hgprt gene to the hybrid cell and, hence, unfused myeloma cells and spleen cells die in the hat media. the conventional method of generating mabs is the hybridoma technology in which spleen cells from immunized mice are fused with murine myeloma cells. whereas the myeloma cell imparts immortality to the hybridoma allowing cells to be cultivated indefinitely, the immune spleen b-cell confers antigen specificity. because each hybridoma is derived from a single cell, the cells within a hybridoma cell line are identical and make the same antibody molecule with same antigen-binding site and isotype, hence it is called a mab. among several excellent reviews that detail mab production with hybridomas is a recent review by dean and shepherd. 12 initial attempts to bypass the mouse to make human mabs involved fusion of human immune spleen lymphocytes with nonsecreting human myeloma partners to obtain hybrid cells that continually secrete a specific antibody. however, poor fusion of human myelomas, unsatisfactory performance of the hybrid cells, and the difficulty in accessing immune lymphocytes have prevented success. although heteromyelomas-which are fusions of hu-man and mouse myelomas-work better, these hybrids are usually unstable. attempts to use mouse myeloma cells to create hybrids and derive human mabs led to the loss of human chromosomes and the inability to make human igs. 13 unfortunately, in vitro immunization is limited by its inability to produce a secondary response and by the absence of the affinity maturation process that occurs in vivo. 13 affinity maturation process is a complex phenomenon, a consequence of intense bcell proliferation, somatic hypermutation of ig variable domain genes, and selection for b cells with high-affinity antigen binding, all occurring in the specialized microenvironment of the germinal center within the lymphoid tissue. thus, the search for an ideal fusion partner for generating human mabs has been difficult, which is why the epstein-barr virus (ebv) technique for immortalization of b lymphocytes is preferred. but immortalized b cells do not always replicate exactly the in vivo antibody response, because the tissues from which these cells were selected and the manipulations to which they are subjected to in the laboratory may alter the antibody specificity. 14, 15 protocols for the preparation of ebv virus, b cells, and cell fusion have been described elsewhere. 14, 16, 17 methods used for large-scale production of mabs may include the generation of ascites tumors in mice or in vitro mammalian cell culture fermentation by using bioreactors and continuous perfusion culture systems. 18, 19 the key issues in scale-up productions are the growth media, fermenter size, fermentation time, and purification procedures. 19 purification or downstream processing is accomplished by chromatography, fragmentation, conjugation with chelating agents, ultrafiltration, and controlled precipitation. 18 a more recent technique for producing antibodylike molecules uses what is known as the phage display library. it involves the construction of v h and v l gene libraries and their expression on the surface of a filamentous bacteriophage. developed in the 1990s, the phage display method requires repeated "panning" or screening of different antibodies based on their affinity for a specific antigen. antibody genes are linked to bacteriophage coat protein genes and the bacteriophages with the fusion genes are used to infect bacteria to create the phage display library. the resulting bacteriophages express the fusion proteins and display them on their surface, and the phage display library comprises recombinant phages, each displaying a different antigenbinding site on its surface. the phage expressing an antigen-binding domain specific for a particular antigen can be detected and isolated by binding to the surface coated with that antigen. libraries of v h and v l genes may be generated from nonimmunized donors, immunized donors who have an immune response against a particular antigen or from a synthetic library consisting of antibody fragments. 20, 21 one promising way to increase antibody yield or develop new antibodies may be by using genetically altered animals and plants. abgenix, a company in fremont, ca, has developed the transgenic "xeno-mouse," in which the mouse antibody-producing genes have been inactivated and functionally replaced by approximately 90% of the human ig gene loci in germline configuration, coding for the heavy and light chains. 22, 23 upon immunization with any specific human or nonhuman antigens, the "xeno-mouse" generates mabs, which are fully human igs, with high affinities and antigen-binding specificities. "xenomouse" strains producing specifically igg1, igg2, or igg4 isotypes have also been created to generate panels of diverse and highly specific mabs. kirin brewery company, japan, has developed another transgenic mouse known as the "trans-chromo" mouse. the endogenous igh and igg loci of the "trans-chromo" mouse were inactivated, but it harbors 2 individual human chromosome fragments, derived from human chromosomes 2 and 14, that contain whole human ig light-and heavy-chain loci, respectively. 24 these mice are capable of producing every subtype of fully human ig, including iga and igm. in these transgenic mouse models, human antibodies with high affinity to an immunized antigen are naturally selected by the murine immune system via an affinity maturation process, and thereby show increased diversity of the mabs. transgenic mice may be a suitable alternative to chimeric or humanized antibody production or the use of phage display systems to create less immunogenic or novel antibodies. 25 for instance, transgenic mice that express mabs to coronavirus in their milk have been developed. 26, 27 because ruminant animals, such as cows, goats, and sheep, produce relatively large amounts of milk, genetically-modified members of these species could also be used to produce large quantities of therapeutic proteins, including mabs. 28 plants may be a potential source of recombinant proteins, including mabs. 29 -31 plant virus vectors, such as the tobacco mosaic virus, may be used to make mabs. transgenic tobacco plants may also be used for large-scale production of recombinant iga, which is used in passive mucosal immunotherapy. 30, 31 this mab could be added to toothpaste to effectively protect against bacteria that cause tooth decay. 31, 32 recombinant antigens obtained from plants may also have therapeutic applications. for instance, attempts to immunize mice with escherichia coli heat labile enterotoxin b produced in transgenic tobacco and potato plants have proved promising. 33 hepatitis b viral surface protein produced in transgenic tobacco plants has been shown to be immunogenic in mice. 32 the genes coding for murine malignant b-cell specific markers are inserted into tobacco mosaic virus to cultivate an immunogenic protein in tobacco plants that may eventually be used in developing a vaccine against non-hodgkin lymphoma. 34, 35 other immunotherapeutic proteins under development include norwalk virus capsid proteins produced in tobacco and potatoes, cholera toxin and ct-b produced in potatoes, hepatitis b antigen produced in tobacco, anti-human igg used to detect nonagglutinating antibodies produced in alfalfa, and humanized anti-herpes simplex virus (hsv)-2 grown in soybeans. 36 -39 the food and drug administration (fda) considers antibodies to be "biopharmaceuticals"; as such, mab applications are regulated by the agency's center for biologics evaluation and research (cber) and the center for drug evaluation and research. 40, 41 the fda has created a "points to consider" document advising manufacturers of factors to consider in the production and testing of mabs intended for human use and identified information that should appear in investigational new drug or biologics license applications. 40 the "points to consider" document serves to "indicate the agency's current thinking on mab products for human use." 40 the agency's recommendations are aimed at protecting human health because viruses and cellular dna from antibody-producing cells with malignant phenotypes may be integrated into host cells after transformation. accordingly, among the points to consider are several steps-such as taking care to ensure purity of immunoconjugates and demonstrating the ability of any purification scheme to remove adventitious agents-designed to prevent contamination of the final product by human pathogens. 40 manufacturers must also adhere to animal care standards and detail steps to prevent contamination of cell culture. the points to consider document includes a list of normal human tissues used in cross-reactivity testing, tests for murine viruses, and organs to be considered in dosimetry estimates. the fda recognizes that because of species differences, animal models expressing the antigen of interest or cross-reactive epitopes are not always available. the agency has also published a guidance entitled s6 preclinical safety evaluation of biotechnology derived pharmaceuticals based on international conference of harmonization technical requirements. 41 before beginning phase 1 clinical studiesconducted to assess the safety of the drug and its mechanism of action-the fda recommends that researchers conduct in vivo and in vitro testing of the mab to assess cross-reactivity with human tissues or non-target-tissue binding. preclinical safety testing aims to evaluate the immunogenicity, crossreactivity, stability and effector functions of mabs. the metabolism, carcinogenicity, and genotoxicity of the product should also be evaluated. 41 guidance has also been published for mabs used as reagents in drug manufacturing. 42 the guidance emphasizes biological safety, performance characteristics of the reagent, and potential presence of residual amounts of the reagent in the final product. the fda general principles for testing and manufacturing are broadly applicable to many classes of antibodies, including those produced by phage display systems or transgenic plants and animals. depending on the expression system being used, other fda guidance documents, such as cber's points to produce biologicals should also be referenced. [43] [44] [45] humanizing monoclonal antibodies rodent mabs with excellent affinities and specificities have been generated using conventional hybridoma technology, but their use in clinical medicine is limited due to the immune responses they elicit in humans. for instance, the human antimouse antibody (hama) response can compromise the clinical effectiveness of murine mabs. although hama responses are directed against the murine constant regions, which represent the major antigenic features of the mouse ig, significant responses are also directed toward the murine variable regions. as a result, patients may mount an immune response against the injected murine antibodies, leading to allergic or immune complex hypersensitivities, rapid clearance of the antibody, and reduced clinical efficacy. 13,46 -51 initially, morrison and colleagues 48,52 introduced chimeric mabs in 1984, which showed several advantages over unmodified rodent antibodies. generally, chimeras combine the human constant regions with the intact rodent variable regions, replicating the rodent antibody variable regions by pcr and then cloning them into eukaryotic expression vectors containing human constant regions. 49 ideally, this allows better interaction with human effector cells and the complement system. because the fc region has little influence on the structure of the fv region, the chimeric constructs' affinity and specificity are virtually unchanged, and both rodent and chimeric antibodies cause apoptosis at a similar rate and intensity against target cells in vitro. 50, 51, 55 although chimeric antibodies have helped solve some of the problems associated with the use of rodent mabs, they still show significant immunogenicity in humans; because of their approximately 30% mouse sequence, they cause an human-antichimeric antibody response. humanized antibodies containing only the cdrs of the rodent variable region grafted onto the human variable region framework have been introduced to overcome these deficiencies. 53 the early work on recombinant, chimeric, and rodent/human antibodies happened during the mid 1980s and, by the late 1980s, greg winters and his colleagues demonstrated that a functional human-like antibody could be created by grafting the antigen-binding cdrs from variable domains of rodent antibodies onto human variable domains. numerous humanized antibodies have now been designed and constructed, and many are currently being evaluated in clinical trials. 13, 14, 39, 47, 54 efficient procedures for constructing humanized antibodies have been developed. 13, 14, 47, 55 the first step is to clone and sequence the complementary dnas (cdnas), coding for the variable domains of the mouse antibody to be humanized. the mouse hybridoma cell line is grown in an appropriate culture medium, and cells are harvested for rna isolation. polymerase chain reaction (pcr) primers that hybridize to the 5' ends of the mouse leader sequences and to the 5' ends of the mouse constant regions are designed for cloning light chain variable regions and heavy chain variable regions. cdna is synthesized from total rna, followed by pcr amplification with light and heavy chain specific primers. positive bacterial colonies containing mouse variable regions are then screened. construction of a chimeric antibody involves modifying the cloned mouse leader-variable regions at the 5'-and 3'-ends, using pcr primers to create restriction enzyme sites for convenient insertion into expression vectors, to incorporate sequences for efficient eukaryotic translation, and to incorporate splice-donor sites for rna splicing of the variable and constant regions. the adapted mouse light and heavy chain leader-variable regions are inserted into vectors containing, for example, human cytomegalovirus enhancer and promoter for transcription, a human light or heavy chain constant region, a neomycin gene for selection of transformed cells, and the simian virus 40 origin of replication in cos cells. these vectors are designed to express chimeric or reshaped human light and heavy chains in mammalian cells. the design and construction of an engineered human antibody require an analysis of the primary amino acid sequences of the mouse variable regions to identify the residues most critical in forming the antigen-binding site. a structural model of the mouse variable region is built on the basis of homology to known antibody variable regions. the framework regions (frs) of the new variable regions are modeled on frs from structurally similar immunoglobulin variable regions. the design process involves selecting human light and heavy chain variable regions that will serve as templates for the construction of a reshaped human antibody. the mouse cdrs are then joined to the frs from selected human variable regions. the primary amino acid sequences are then carefully analyzed to ascertain whether they would recreate an antigen-binding site that mimics the original mouse antibody. within the frs, the amino acid differences between the mouse and the human sequences are examined, and the relative importance of each amino acid in the formation of antigen-binding site is evaluated. minimum changes in the frs are desirable and should closely match the sequences from natural human antibodies. any potential glycosylation site in the frs of either mouse or human sequence needs to be identified and its influence on antigen binding considered. the dna sequences coding for the reshaped human variable regions, either made synthetically or based on an existing sequence that is very similar to the newly designed reshaped human variable region, are modified by pcr with specially designed oligonucleotide primers. the human variable regions together with their leader sequences are then cloned into a mammalian expression vector that already contains human constant regions. each human variable region is linked to the desired constant region via an intron. preliminary expression and analysis of the reshaped human antibodies are done by cotransfection of mammalian cell-expression vectors, 1 coding for human light chain and 1 coding for human heavy chain. the vectors will replicate in the cos cells and transiently express and secrete reshaped human antibodies. the concentration of the antibody produced can be analyzed by using an enzyme-linked immunosorbent assay. specific changes in the amino acids of the framework region may also be required to preserve the orientation and structure of the rodent cdr required for binding. computer modeling using databases containing human variable genes will identify sequences homologous to the rodent v regions. 13, 47 a computer model of the rodent fv can identify the non-cdr residues that interact with the cdr sequences, and choices can be made regarding which residues need to be included in the variable region. antibodies humanized in this way may have binding affinities up to one-third greater than the corresponding murine antibodies. 49 allergenicity is also significantly reduced. about 20 to 40% of patients exhibit a hama reaction to murine antibodies, whereas only about 7% have a similar reaction to humanized antibodies. 47,53,56 -58 humanization of mabs still has several practical difficulties. first, a detailed knowledge of the antibody structure and function is required. second, methods for efficient construction of humanized mabs are limited. third, unpredictable immunogenicity may result when a new amino acid sequence is introduced to balance affinity retention. fourth, the antibody repertoire is limited to the animal in which the progenitor mab originated. fifth, the rodent mab producing hybridoma must be isolated and thoroughly characterized. however, obstacles to humanization are gradually being surmounted. karpas et al 15 reported creating a hat-sensitive and ouabain-resistant human myeloma cell line that can fuse with human lymphoblast cells. a hat-sensitive subline of the myeloma cells that secreted only light chains was fused with ebv-transformed white blood cells that produced igg mabs to hiv-1 gp41. eventually, a clone was isolated that was polyethylene glycol-resistant and would not revert when placed in hat medium. standard polyethylene glycol fusion protocol was followed to fuse the myeloma cells with both ebvtransformed white cells and fresh white cells obtained from the peripheral blood of adults and tonsil cells from 2 children. the authors reported promising rates of hybridoma formation, stable ig production, and high yield of secreted antibodies compared with antibody-producing cell lines from mouse myeloma cells. the authors have now developed 40 hybridomas that have been secreting cells for more than 5 months. a human myeloma cell line may eventually prove useful in creating mabs against certain autoimmune diseases and cancers. this technique may also make it easier for other researchers to generate human mabs for use in therapy. antibodies may act directly when binding to a target molecule by inducing apoptosis, inhibiting cell growth, mimicking or blocking a ligand, or interfering with a key function. 53, 59 in addition, antibodies may modulate or potentiate drugs or other therapies. the antibody may itself act as an effector-as in antibody-dependent cellular cytotoxicity (adcc) or antibody-dependent complement-mediated cascade-or it may involve effector elements such as cytotoxins, enzymes, radioactive isotopes, signals for other parts of the immune system, and/or cytotoxic drugs. 49,50,60 -62 adcc occurs if fc regions on the antibodies are recognized by receptors present on cytotoxic cells, such as natural killer cells, macrophages, granulocytes, and monocytes. complement-mediated cytotoxicity ensues if the antibody binding prompts a complement cascade to occur. active immunotherapy can be accomplished if the antigen can provoke a long-lasting t-cell response, which may be achieved by administering whole cancer cell extracts or by using small antigenic peptides isolated from tumors in experimental patients or animals. 58 for instance, mabs mimicking breast cancer-specific antigens elicit anti-idiotype antibodies; this is another way of creating active immunity, which can lead to a humoral auto antibody-like immune response. 58 the use of bispecific molecules that recognize antigens on both the target cell and effector cell can increase adcc. the most effective mechanisms are blockade of a crucial ligand or growth factor or adcc in which tumor cells are killed by fc receptorbearing cytotoxic effectors. cell killing by adcc is proportional to the amount of antibody bound to a cell, whereas the blockade of an essential growth factor may not show effects until most of its receptor is saturated. a higher antigen expression by the target cell will increase antibody binding and subsequent adcc. but high receptor expression will also make it difficult to prevent the binding of a cytokine or ligand at minimum threshold. 53, 59, 63 with a bispecific antibody, the specificity of mab is combined with the cytotoxicity of immune effector cells, for instance, to neutralize a tumor. 9, 45, 63, 64 bispecific antibodies link the tumor cell directly to the killer cell via cytotoxic trigger molecules, such as t-cell receptors or fc receptors, leading to lysis and/or phagocytosis by the effector cells. although bispecific antibodies can enrich effectors at the tumor site and activate tumor bound effector cells by enabling cross-linking between effector and target cells, they can cause system-wide immune activation because of t-cell receptor cross-linking. bispecific antibodies can also mediate cellular cytotoxicity via various effector cells, including phagocytes, natural killer cells, and t-lymphocytes. another way to use the binding properties of antibodies is by conjugating antibodies to cytotoxic drugs, radioisotopes, or toxins. 49, 58, 63, 65, 66 techniques for conjugation have been described in several recent reviews and articles. 49, 58, 67, 70 mabs can be conjugated to chemotherapeutic drugs such as doxorubicin, mitomycin, and methotrexate. chemotherapeutic agents constitute cytotoxic or cytostatic drugs that can be conjugated to antibodies; thus far, however, these drugs have shown poor specificity for target cells and frequently lead to toxicity. 55, 62, 65 often, antibodies may lose reactivity upon conjugation with such agents. immunotoxins used in cancer therapy are conjugated antibodies that combine plant (ricin, gelonin, saponin, abrin, pokeweed antiviral protein) or bacterial (diphtheria toxin and pseudomonas toxin a) toxins with antibody specificity. 52, 59, 62, 63, 65, 66, 68, 69 toxins may inhibit protein synthesis even at picomolar concentrations. natural toxins, such as diphtheria toxin, can act at low concentrations, enter cells, and disrupt key cellular processes. 69 enzymes may also be conjugated to antibodies in antibody-directed enzyme-prodrug therapy. 55, 61, 62, 66, 71 an enzyme that can convert a prodrug to an active form may be conjugated to an antibody and targeted to a desired location using the antibody-binding region. they may more effectively localize tumors than chemical conjugates, and a prodrug is converted to an active drug by the enzyme at the target cells. another therapeutic technique relies on using antibody fragments, which may be produced by the traditional method (proteolytic digestion with papain) or with newer methods that may reduce damage to the binding sites. 66, 71 some newer methods attempt to construct mimetics that combine multiple cdrs from several antibodies into single molecules with molecular weights substantially lower than single chain fragments. because of their smaller size, antibody fragments may be able to penetrate tissues and tumors more readily and be less immunogenic than whole mabs. however, although antibody fragments may have better penetration than whole antibodies, their shorter half-lives may compromise clinical usefulness. 68 heteropolymerized antibodies have recently been developed by chemically linking a mouse igg mab, specific to a complement receptor site (cr1) on the human or nonhuman primate erythrocyte, to a second mouse igg mab that is specific to the targeted antigen. 72 the heteropolymer technique is based on immune adherence, in which antibody-antigen immune complexes bind to a complement receptor on the red blood cell, facilitating the phagocytosis of these complexes. 72 once introduced into the patient, the heteropolymers will bind to both the red blood cells and the targeted antigen. the antigen-antibody heteropolymer complexes are then transported to the liver and spleen, where the complexes are destroyed by macrophages, whereas the red blood cells return to the circulation unharmed. [72] [73] [74] [75] a wide variety of conditions, of both autoimmune and foreign origin, may potentially be treated with this method, including hiv infection, systemic lupus erythematosus, marburg virus infection, and myasthenia gravis. 76 -78 although mouse antibodies are currently being used with this technique, chimeric mousehuman antibodies could also be constructed. 72 it may also be possible to use the heteropolymers as "sentinels" by injecting them before antigen exposure. in 1 recent study, multiple infusions of heteropolymers provided nonimmunized monkeys with protection against an antigen (⌽174) for as long as 2 weeks. 79 when milstein and kohler announced their isolation of a mab in 1975, many thought mabs would provide an effective cancer treatment. however, early clinical trials were disappointing. 80 -81 producing mabs to tumor antigens is a complex process. first, proteins peculiar to human cancer cells are identified; then, mice are injected with human tumor cells (antigen) to stimulate an immune response. after about 30 days, the mouse lymphocytes are removed and fused with myeloma cells to isolate and reproduce hybrid cells specific to a predetermined antigen. rodent antibodies alone have been used in several trials, but the clinical effects are often minimal because of poor activation of human effector or complement cells by the fc region of the murine antibody and the accompanying hama responses. 55, 57 substituting the rodent fc region with a human fc fragment may help overcome these effects. 81 with the discovery of proto-oncogenes, secondgeneration trials in the 1990s turned to more specific tumor antigens that could be potential targets. 58 in 1994, mab 17-1a, an antibody to epithelial cell surface antigen expressed on human colorectal carcinomas, was approved for the identification of adenocarcinomas; mab17-1a may reduce the mortality and occurrence rate of colorectal cancer. 82, 83 in 1997, rituximab, a mouse-human chimeric anti-cd20 antibody, was approved for the treatment of non-hodgkin b cell lymphoma. 53, 83 rituximab binds to cd20 antigen on b cells and b cell tumors and then elicits a natural immune response that can kill malignant cells. recent animal trials have shown the potential of mab therapy in cancer. 52 increases in epidermal growth factor (egf) receptor expression have been found on many human cancers, and a fully human anti-egf receptor igg2 mab has been shown to inhibit human cancer growth in vitro and in vivo. 22 other efforts are focusing on antibody to her2 antigen in breast cancer. patients with cancer often mount an immunologic response to tumor-associated antigens with increases in cytotoxic lymphocytes and antibodies. 14 several tumor-reactive antibodies have been cloned against melanomas, colon carcinomas, ovarian, breast, and lung tumors. often, these antibodies cross-react with other malignant tissues or cell lines. 14 recently, trastuzumab (herceptin), a humanized mab that binds directly to the c-erb2 protein (her2), has been approved by the fda for the treatment of breast cancer. 52,84 -89 herceptin can be used to treat metastatic breast cancer in patients whose tumors overexpress the her2 protein (about 30% of breast cancer patients) and may be used in conjunction with other therapies such as paclitaxel (taxol). 89 antibody was developed by humanizing murine mab 4d5 by inserting antigen-binding regions of mab 4d5 into the framework of a consensus human igg1, resulting in rhumab-her2 or trastuzumab. 85 cancer cells have antigens that are specific to the tumor (tumor-specific antigens) or are present in greater concentration than normal (tumor-associated antigens). antibodies may eliminate target cells by complement action or through adcc. the variable region of the antibody recognizes and attaches to a specific antigen and the constant region, then joins with an effector cell capable of killing the targeted cancer cell. when the antigen and antibody bind, precipitation and agglutination may isolate the complex. anti-idiotype mabs that mimic-tumor associated antigens also may be used in cancer therapy. 82 the idiotype network hypothesis, formulated by lindemann and jerne, suggests that because each mature b cell secretes an antibody with unique antigenbinding specificity in the variable domain (referred to as the idiotype), anti-idiotype mabs can be generated and used as surrogate antigens or vaccines for immunization against the tumor. 82, 90 a murine anti-idiotype mab, aca125, which mimics the tumor-associated antigen ca125, was recently found to induce a specific anti-anti-idiotypic immune response in 28 of 42 patients with platinum-pretreated recurrent ovarian cancer enrolled in a phase i/ii clinical trial. 90,91 a positive immune response was associated with statistically significant (p ͻ 0.0001) prolonged survival times (19.9 ϯ 3.1 months in patients shown to have an anti-antiidiotypic response compared with 5.3 ϯ 4.3 months in patients who were anti-anti-idiotypic negative). 90, 91 in addition, peripheral blood lymphocyte mediated lysis of ca-125 expressing tumor cells increased in 9 of 18 patients after vaccination with the mab. 91 despite the use of a murine antibody, both this study and a previous phase i study involving patients with ovarian cancer found minimal side effects. 90, 91 promising early results have also been demonstrated in patients with advanced colorectal carcinoma who received a murine anti-idiotype mab that mimics an epitope of carcinoembryonic antigen (ceavac) 82, 90 and in patients with malignant melanoma who received an anti-idiotype mab (trigem) that mimics disialoganglioside gd2 92 despite promising developments, however, there are still several obstacles to effective cancer therapy with mabs. problems with the tumor, the mab, or conjugate characteristics all continue to challenge researchers. 52, 63, 84 thus far, chemotherapeutic mab therapies have faced obstacles due to the poor ability of agents to affect tumor cells preferentially over healthy cells, the intrinsic insensitivity of many tumors to these drugs, and the rapid development of resistance in tumor cells. 65, 66, 93, 94 mabs often decrease the size of tumors, but rarely lead to complete remission of solid tumors. 59 at the cellular level, mabs at low doses ideally should bind with excellent affinities. because no antigens have been identified that are expressed exclusively on tumor cells, cross-reactivity of mabs must be examined with histochemical tests on tissue sections or in animal models. careful testing must also be done to ensure that the effector molecules such as drugs, toxins, or isotopes in the conjugates do not inadvertently target healthy cells. although humanized antibodies have greater affinity than murine antibodies, this potential has yet to be translated into improved clinical outcomes against cancer. 52 however, recent success using the xenomouse technology to develop a fully human igg2 mab specific to epidermal growth factor receptor indicates the potential for future advances in this area. 22 recombinant immunotoxins, fusion proteins containing the fv of a mab and a bacterial toxin, are under development for cancer therapy. because immunotoxins exploit only the variable region-binding function of the mab, in theory just a fragment of the binding region, such as the bivalent f(ab')2 fragment, monovalent fab, scfv, or disulfide-stabilized fv fragments may be used as opposed to the larger and probably more immunogenic whole antibody. disulfide-stabilized fragments may be more stable or have higher affinity for the antigen than scfv, in which the linker may interfere with binding or fail to stabilize the fragment. 66 recombinant immunotoxins derived from pseudomonas enterotoxin have been shown to be active against lymphomas, solid tumors, and leukemias. 66 because binding specificity and affinity are the key elements, it is not known which form of the immunoconjugate is usually more effective in treating human tumors; some animal tests suggest that the whole antibody, with its longer half-life and bivalent binding, is more effective than the antibody fragments with decreased binding affinity. bispecific antibodies and single chain fvs are being developed that may be more effective because they clear faster from nontumor tissues and more deeply penetrate tumors than whole antibodies. 49, 64, 70 bispecific antibodies may be used to target tumor vascular endothelial cells, thereby limiting the tumor's blood supply and possibly inhibiting its spread. 70 advances in radiolabeling have allowed immunoconjugates to be delivered to cells and showed promise in clinical trials. 14, 63, 80, 95, 96 radioimmunotherapy uses a radiolabeled mab to deliver radioactive isotopes to targeted cells. radioisotopes such as iodine-131 and yttrium-90, which are ␤ emitters, can cause damage not only to the bound cell but also to cells adjacent to tumor cells that antibodies may not be able to reach within the tumors. lack of knowledge about the appropriate dose, biodistribution, and shedding of antigen hinders use of radioisotopes. radiolabeled mabs may also affect normal cells, depending on the extent to which reticuloendothelial cells expressing fc receptors bind to the constant regions of intact antibody molecules. using antibody fragments or constructs may modify this nonspecific uptake. 63, 68, 82, 93 radiotherapy exerts most of its effect by emitting a low dose, which exponentially decreases continuous radiation, but the antibody per se may have a cytotoxic effect. the duration of radiotherapy is determined by the half-lives of the antibody and the isotope used. the success of radioimmunotherapy is affected by the specificity, affinity, dose, and immunoreactivity of the antibody, heterogeneity of antigen expression, diffusion rate, tumor volume, blood supply and tumor location, dose rate effects, and variability in dose deposition. the choice of radionuclide, selection of the chelate used to link the mab to the radionuclide, and the mab selected are critical in development of radiolabeled mabs. 80 suitable radio nuclides include yttrium-90, iodine-131, and copper-67. iodine-131 was the first radioisotope to be used in treatment of hodgkin disease and other lymphomas. 68 yttrium-90 is potentially useful for lymphoma therapy because it decays with ␤, but not ␥, emissions that may kill other tumor cells in a cross-fire effect. 68 the energy released by yttrium is 5 times higher than that of iodine-131, which degrades rapidly after uptake into a tumor cell, causing toxicity. 80 in addition to their uses in radiotherapy, radiolabeled mabs can also be used to diagnose cancer. 18, 56, 57, 82, 94 radioactive isotopes linked with mabs may help localize tumors in a form of diagnostic imaging called immunoscintigraphy. for instance, oncoscint, a mab coupled to indium-111, may be used to detect an antigen (tag-72) found on colorectal adenocarcinomas. 56 mabs may potentially be used to suppress the immune system after transplant or to induce tolerance to transplanted organs or tissues. thus far, however, only antibodies to cd3 and cd25 are licensed for clinical use. 97 okt3, a murine igg2a antibody to human cd3, and antibodies to cd25 (il-2 receptor) have been used to reduce allograft rejection. 97 graft-versus-host disease (gvhd) is a complication of allogeneic stem cell transplant that occurs despite histocompatibility testing and use of cyclosporine and its analogs. 98 gvhd is a frequent cause of illness or death in allogeneic transplant patients and occurs when alloselective donor t cells recognize and interact with major and minor histocompatibility antiigens in the host, leading to cytokine release. mab therapy against gvhd is most effective when it is administered after a bone marrow transplant but before gvhd development by targeting t-cells before their activation. 99 for instance, 1 target is the interleukin-2 receptor ␣-chain (il-2r␣ tac protein or cd25), whose expression is a crucial step in the activation of alloreactive t cells. 97, 100 recently, it was reported that daclizumab, a humanized anti-il-2a antibody, was an effective complement to dual immunosuppression therapy in renal transplant patients. 101 because only activated cells express il-2, antibodies to this cytokine might inhibit t cells during allograft rejection and prevent generation of cytotoxic t cells. the development of hama response in patients and the decreased effectiveness of murine mab relative to human mab have thus far compromised the effectiveness of this approach. however, humanized anti-tac mabs, with better human effector functions, may survive longer in vivo and may prove less immunogenic than its murine counterpart. short-term cd4 antibody therapy may also contribute to long-term acceptance of skin and islet allografts, even after gaining immunocompetence. 102 more generally, the ability of mabs to induce tolerance to transplanted tissues and self-antigens may hold great therapeutic potential. 97, 102 mabs to cd4 and cd8 have been studied, although thus far these have not resulted in clinical success. 97 depletion or blockade of t cells by antibodies may facilitate tolerance by preventing t cells from attacking the graft, and recent t cell depletion studies in primates have proven promising. 97 mab treatments for other complications after transplants are also under study, including potential treatment of posttransplant lymphoproliferative disorder with the anti-cd20 mab rituximab and the use of anti-lfa-1 mab (odulimomab) to protect against ischemia-reperfusion injury after kidney transplants. [103] [104] [105] daclizumab, a humanized antibody that targets the anti il-2 receptor, may reduce the risk of acute rejection of a renal transplant and also lower cytomegalovirus infection rates among transplant recipients. 106 the inflammatory bowel disorder known as crohn disease has also been treated with mab therapy. 107 a chimeric igg1k antibody, infliximab (remicade), acts by binding to soluble and transmembrane tumor necrosis factor ␣ (tnf␣), preventing it from binding to its receptors on activated macrophages. the anti-tnf␣ antibody may provide relief to patients with moderately to severely active crohn's disease. in addition, the fda recently approved infliximab for the treatment of rheumatoid arthritis in combination with methotrexate. 108 -111 high levels of ige may cause bronchial hyperresponsiveness, a risk factor for asthma. [112] [113] [114] im-therapeutic applications of monoclonal antibodies mune responses mediated by ige are important in the pathogenesis of allergic asthma. in 1 recent study with subjects reporting moderate to severe allergic asthma, twice-weekly injections of recombinant humanized anti-ige antibody (rhumab-e25), which forms complexes with free ige and blocks its interaction with mast cells and basophils led to a fall in serum ige levels and slightly decreased asthma symptom scores relative to the placebo group. 112 patients receiving anti-ige were able to reduce reliance on corticosteroids. although the study must be interpreted cautiously, it is nonetheless a promising step in finding more effective asthma therapies. 112, 114, 116 approximately 400,000 cases of sepsis in the us and about 25,000 cases in the uk are reported each year. 116, 117 the mortality rate could be as high as 40 to 70%, depending on the population studied. 117, 118 mabs have been targeted to tnf␣ and tnf␣ receptors, which are key elements of the inflammatory response. unfortunately, this may inhibit many cytokines, which can impair the patients' ability to fight infection and increase the risk of secondary sepsis; also inhibiting a few cytokines may not be sufficient. although mabs targeted toward components of the host immune system, such as individual receptors or mediator cells, help in the treatment of septic shock, targeting the bacterial endotoxin or lipid a of gram-negative bacteria with mabs may be more efficacious. lipopolysaccharide endotoxin, a component of the outer cell membrane of gramnegative bacteria, consisting of a highly variable o-linked polysaccharide chain, an r core region, and lipid a, which in turn is composed of a glucosamine disaccharide backbone substituted with amide-and ester-linked long-chain fatty acids, is believed to be important in many cases of septic shock and septicemia, and can trigger an inflammatory cascade that can cause serious injury and even death. 119, 120 lipid a is linked by the core region to the o-linked side chain, and variations in the o-linked side chains result in an enormous diversity among gram-negative bacteria, making adoptive immunotherapy against the o-linked antigenic determinants difficult. 114 directing antibodies to the core region or lipid a, which tend to be more conserved, would allow for therapy against a diverse array of gram-negative bacteria. favorable results with polyclonal j5 antibody have spurred attempts to develop a mab. 116, 117 a murine igm mab, e5 (xoma, berkeley, ca) binds to an epitope on lipid a of e coli j5. a human igm, ha-1a (centoxin) is derived from a heterohybridoma fused from spleen cells of a patient vaccinated with e coli j5 before splenectomy. 116, 117, 119, 121 both the antibodies have thus far shown mixed success in patients with sepsis and neither is currently cleared by the fda for therapeutic use. because patients with bacteremia often have endotoxemia, ha-1a, an igm antibody to endotoxin may be effective against endotoxin in the bloodstream as well. unfortunately, the high cost of antibody ($3500/dose in europe) combined with the uncertainty that the sepsis is caused by gram-negative bacteria has hindered the widespread use of ha-1a. 112, 115, 117 although it is less efficacious, the only other option may be to target tnf␣, but 1 recent study reported a high (54.1%) rate of adverse reactions in patients receiving an anti-tnf␣ antibody. 118 the antibody was no more effective than the placebo given to control subjects. however, research on a mab for the treatment of septicemia continues. cytomegalovirus (cmv) causes serious illness affecting the immunocompromised, such as patients with aids and those undergoing organ transplants. infection rates may be up to 75% in those negative for cmv who receive kidneys from seropositive patients. 122 cmv infection can result in retinitis and gastroenteritis in hiv-infected patients and may also cause chronic intrauterine infection. 122, 123 up to 40,000 cases of congenital cmv infection are reported each year; mental retardation and hearing loss may occur in about 25% of these cases. 122 currently, there is no vaccine against cmv. ganciclovir, foscarnet, and (s)-1-[3-hydroxy-(2 phosphonylmethoxy)propyl]cytosine are some of the potential treatments for cmv infection. 122 another mode of treatment is via administration of anti-cmv hyperimmunoglobulin, derived from pooled sera of cmvseropositive persons. passive immunization has been shown to reduce the severity of cmv and prevent mother-to-infant transmissions. 122 moreover, antibodies may be able to clear the virus from infected tissues, a function previously thought to be exclusive to cytotoxic t lymphocytes. many physicians use a combination of antiviral agents and immunoglobulins in patients at risk for cmv infection. mabs may also decrease the amount of antiviral agents required for treatment. mabs against murine cmv polypeptides have been shown to be protective in animal models. 124 although mabs may act synergistically with foscarnet or ganciclovir in vitro, it is unclear whether this advantage could be extrapolated to in vivo conditions. experiments in which murine cmv is used as a model system suggest that high concentrations of antibody, along with ganciclovir or (s)-1-[3-hydroxy-(2 phosphonylmethoxy)propyl]cytosine, synergistically inhibit the growth of murine cmv in cell culture, whereas lower antibody concentrations produce only an additive effect. however, antibody ad-ministered along with ganciclovir in severe combined immunodeficient mice also produced only an additive effect. 123 the extent to which a mab can protect the host from cmv infection cannot reliably be predicted from its immunoreactivity, neutralizing titers in vitro, or from its antigen-binding data, because the mechanism of action of a mab in vitro and in vivo may be entirely different. 124 if an antibody is used along with drugs, not only is the treatment efficacy improved but also the side effects of a high drug concentration may be avoided. for instance, 25 mg of ganciclovir/kg of body weight, along with an antibody, was almost as effective as 50 mg/kg ganciclovir administered alone in cmv-infected severe combined immunodeficient mice. 124 the msl109 antibody, a human igg1 directed against human cmv glycoprotein, is marginally synergistic in inhibiting cmv in vitro when conjugated to ganciclovir or foscarnet. in a recent trial involving 209 aids patients with active cmv retinitis, the rate of retinitis progression was similar in the group receiving the antibody (60 mg, intravenous) for 2 weeks and the placebo control group. 125 surprisingly, mortality increased in the msl-109 group, and the poor performance of msl-109 in this trial may have been due to the inability of the antibody to neutralize the established cmv infection in aids patients or its failure to cross the blood-ocular barrier in sufficient quantities. liver transplant patients are also at risk of cmv infection and may require treatment with the murine mab okt3, an anti-t cell antibody that is directed to cd3 antigen, along with ganciclovir to mitigate transplant rejection. 126 human mabs neutralizing cmv were tested for their safety and pharmacokinetics over a period of 3 to 73 days in bone marrow transplant recipients and were found to be safe and efficacious, as evidenced by a steady increase in the neutralizing activity. 127 also, a human anti-cmv mab designated c23 that was purified from hybridomas generated by the fusion of human lymphocytes with mouse myeloma cells has virus neutralization titers about 1000-fold higher than those produced after conventional human ␥ globulin used in humans. 128 a humanized mab has been developed that binds to the 86-kd glycoprotein, gpul75 (gh), of cmv, recognizes a variety of virus strains, and neutralizes clinical isolates of cmv; it could be used as a potential agent for the prevention or treatment of cmv infections in humans. 129 respiratory syncytial virus (rsv) causes serious lower respiratory tract disease requiring considerable supportive care, administration of humidified oxygen, and respiratory assistance. the fda approved the use of a humanized mab against rsv, which afflicts mostly infants and children younger than 24 months of age. 130, 131 known as palivizumab, the antibody (produced by synagis and medimmune, inc.) treatment showed a 55% reduction in rsv infection in hospitalized patients. 130, 131 the american academy of pediatrics' committee on infectious diseases has published recommendations on the use of this antibody in children at risk for rsv infection. 130, 131 mabs have also been used in the therapy of hsv infections. the incidence of neonatal herpes is 30 to 50% in babies born vaginally to mothers with primary infection but only 1 to 3% in babies born to mothers with recurrent infections. 132 this difference is attributed to the passive transfer of protective antibodies to the fetus. antibodies to hsv-1 may also confer partial protection against type 2 strains. based on this observation, the potential of mab therapy against herpes has been studied for more than a decade. experiments in mice, for instance, indicate that microgram quantities of antibodies may promote healing of corneal opacity and blepharitis caused by herpes simplex. 133 however, attempts to confer immunity in humans and other higher animals by passive immunization alone have been disappointing, because antibodies to hsv are restricted to only a few specific epitopes. 133 among the 11 glycoproteins expressed by the herpes virus on its surface, 5 glycoproteins (gb, gd, gh, gk, and gl) are thought to be crucial to infection. the initial step in hsv infection seems to be the binding of gc to heparan sulfate proteoglycans followed by the binding of gb, and the binding of gb and gc is stabilized by gd. gh is involved in initiation of viral fusion and other glycoproteins may help in expanding the fusion process. evidence that neutralizing antibodies to gh, gd, and gb prevent membrane fusion but not viral attachment is consistent with this model. 133 most human antibodies are directed against gd and gb epitopes. mabs can target at least 7 different antigenic sites on hsv glycoprotein d in the murine model. 133, 134 glycoprotein d is necessary for virus replication in tissue culture. yamamoto et al 135 reported the antibody-dependent cellular cytotoxicity effect of hs1, a neutralizing mab to glycoprotein b of hsv, in athymic nude mice inoculated with hsv intracutaneously. when hs1 is administered, the skin lesions healed in 50% of the mice given the antibody. also, latent infection in the ganglia was prevented in mice that survived, as evidenced by the failure to detect hsv upon co-cultivation with vero cells. administration of hs1 after development of zosteriform lesions (5 to 9 days after infection) reduced the virus in the ganglia and prolonged survival time; however, the disease was not totally avoided and eventually the mice succumbed to the disease. the antibody therapy could prevent or decrease the severity of herpetic keratitis, iritis, and blepharitis after corneal infection by hsv. 136, 137 thus, mabs could potentially be used to prevent congenital herpes and sexual transmission of the herpes virus. 47, 133, 138, 139 currently, no treatments exist for the hemorrhagic fever caused by the filovirus ebola. recently, a team of researchers at the us army's medical research institute identified protective antibodies directed against epitopes on ebola virus membraneanchored glycoprotein. some of the antibodies protected mice as long as 2 days after exposure; doses were equivalent to acceptable (3 to 5 mg/kg) human levels. antibody specificity is important, because some mabs studied bound to ebola zaire but not to the ivory coast or sudan serotypes. much research remains to be done before the potential of antibodies in the treatment of ebola will become realized. 140 toxins are poisonous proteinaceous substances. antibodies were first clinically used in the 1970s for protection against the toxin digitalis. 48, 102 today, antidigitalis immunotoxicotherapy is a standard therapy. digoxin is produced from sheep immunized with digoxin-serum albumin conjugate. igg antibody specific for digoxin is purified and cleaved into fabs, because the smaller molecular mass of the fragments allows faster renal clearance and more rapid action than the whole antibody. the fab fragments bind to circulating digitalis molecules and generate a complex that is unable to bind to receptors. adverse effects are rare, and the immunotherapy is generally effective against digoxin and digitoxin within 1 hour. 120, 141 however, because polyclonal antibodies may be difficult to produce and may cause hypersensitive reactions, researchers have attempted to develop humanized mabs against digoxin and other toxins. 141 for example, 1 group has used a transgenic mouse to produce hybridomasecreted human mabs against digoxin. 141 more recently, the effect of goat anti-colchicine igg antibodies has been studied in mice exposed to a lethal intraperitoneal dose of anti-inflammatory colchicines, and preliminary studies in a 25-yearold woman with colchicine poisoning showed promise. 102, 141 attempts have also been made to use drug-specific goat antibody fragments to treat anti-tricyclic antidepressant (tca) overdose. 120, 141 antidepressant overdose is the most common cause of intentional drug overdose in the us. however, lethal doses of tricyclics are 10-to 100-fold higher than those of colchicine, digoxin, and snake venom, which require higher antibody doses (up to several g/kg) to reverse the toxicity. 120, 142 high-affinity tricyclic antidepressant-specific mabs have been shown to reverse the cardiovascular toxicity of antidepressant desipramine in rats and prolong their survival, and tricyclic antidepressant-specific fab fragments, along with sodium bicarbonate, a standard treatment for tricyclic overdose, also minimizes the required dose of antibody. 142 the use of smaller antibody fragments, such as single chain fv fragments, half the size of a fab, may also be a promising approach because the single chain fragment retains affinity and has shorter half-life in the body. monoclonal antibodies may also help to alleviate poisoning due to environmental contamination. hexachlorobiphenyl, paraquat, atrazine, 3,5,6-trichloro-2-pyridinol, the chief degradation byproduct of the insecticides chloropyrifos, triclopyr, and chloroprifosmethyl, and other chemicals may soon be detected and remediated with the help of antibodies. 139, [143] [144] [145] antibodies may also be used in the treatment of poisoning due to paraquat, hexachlorobiphenyl, domoic acid [amnesic shellfish poisoning], and other contaminants that are otherwise difficult to remove from the body or surrounding environment. [145] [146] [147] [148] the primary target of many abused drugs is the central nervous system (cns), and immunotherapy against such chemicals must be able to penetrate the cns. 149 pcp or phencyclidine (angel dust) is a type of arylcyclohexamine that affects multiple sites in the brain. 150, 151 it is linked to violent psychotic episodes that are similar to schizophrenia. 149 treatment is difficult because there is no known antagonist identified, pcp has a high volume of distribution, and about 95% of it is cleared after being metabolized. 151 recently, mabs have been described that could bind to cocaine or pcp and act like sponges in the bloodstream to prevent them from reaching the brain. 152 it has been shown that a single dose of antibody reduced pcp effects for up to 2 weeks in animals, which might be equivalent to a several months in humans. high-affinity antibody 6b5 fragments against pcp function better in that the fragments with bound pcp are more quickly cleared from the body than the whole antibodybound pcp. 153 the crystal structure of the antibody fragment complexed with pcp has been studied in detail. 154 antibody fragments are also preferable to whole antibodies in the treatment of pcp addiction because of their lower antigenicity and improved pharmacokinetics. 149, 151 anti-idiotype antibodies could potentially be produced against the drug-mab binding site and mimic drug structural features, although these antibodies do not cross the bloodbrain barrier. 151 by increasing protein binding in the vascular compartment and lowering the drug's volume of distribution, the antibody acts like a pharmacokinetic antagonist. 150 evidence that the antibody can also reverse effects of other potent arylcyclohexylamine drugs suggests that antibody medications can be used to treat different classes of drugs. 150, 151 an anti-pcp igg has been produced from a hybridoma cell line using both ascites and bioreactor methods, and the pharmacological and immune specificity of the antibody has been confirmed by administering the anti-pcp fragment to rats in three different studies without producing observable effects in behavior. 155 active immunotherapy against methamphetamine addiction is also being contemplated. 156 antibody therapy for cocaine addiction is also a possibility, especially with the development of catalytic antibodies having enzyme-like characteristics that can be used to inactivate drugs. 151, [157] [158] [159] antibodies with esterase activity have been successfully mimicked, and because cocaine is believed to be metabolized by in vivo esterases, such antibodies would be useful in the treatment of cocaine addiction. 151 catalytic antibodies probably would not be as useful as high-affinity anti-cocaine antibodies, but could be used in medical emergencies or as part of a withdrawal therapy. 151 potential antibody applications against cocaine addiction have been tested in rats with a vaccine approach by (1) attaching cocaine molecules to a carrier protein for immunizing rats to produce antibodies against cocaine and (2) humanizing anticocaine antibodies derived from rats and producing them in bacteria. 152, 160 a catalytic mab (15a10) has recently been reported to attenuate cocaine's cardiovascular effects in mice. 155, 159 conclusion about 200 years have elapsed since edward jenner vaccinated a young child against smallpox. since that time, the field of immunology has evolved at a rapid pace and has yielded many critical developments. although vaccination has thus far proven to be the most cost-effective method of preventing diseases worldwide, the development of mabs that use the specificity of immunological responses is one of the most successful applications of immunology to date. chimeric and humanized antibodies have reduced the risk of allergenicity from exposure to nonself antibodies and heightened the clinical effectiveness of mab treatments. developments in radiology and pharmacology have allowed radiolabeled and immunoconjugated antibodies to be produced. antibody fragments, heteropolymers, and bispecific antibodies are now available in addition to whole mabs. these promising developments may soon allow mabs to be used to treat afflictions as varied as substance abuse, cancer, asthma, viral infection, septicemia, and poisoning. as heddy zola observed in 1995, "the therapeutic application of mabs, whilst still limited in scope, promises to break its substantial shackles and realize the potential forecast by its proponents." 46 on immunity: with special reference to cell life ehrlich's passion: the origins of his receptor immunology the natural selection theory of antibody formation a modification of jerne's theory of antibody production using the concept of clonal selection the hydrolysis of rabbit gamma globulin and antibodies with crystalline papain selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants continuous cultures of fused cells secreting 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antibodies in plants exploring transgenic plants as a new vaccine source oral immunization with a recombinant bacterial antigen produced in transgenic plants tobacco can be good for you. the new york times rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain fv epitopes in tobacco plants production of antibodies in transgenic plants a humanized monoclonal antibody produced in transgenic plants for immunoprotection of the vagina against genital herpes production of a diagnostic monoclonal antibody in perennial alfalfa plants using monoclonal antibodies to prevent mucosal transmission of epidemic infectious diseases points to consider in the manufacture and testing of monoclonal antibody products for human use guidance for industry. s6 preclinical safety evaluation of biotechnology-derived pharmaceuticals center for biologics evaluation and research and center for drug evaluation and research, us food & drug administration points to consider in the production and testing of therapeutic products for human use derived from transgenic animals points to consider in the production and testing of new drugs and biologicals produced by recombinant dna technology points to consider in the characterization of cell lines used to produce biologicals. center for biologics evaluation and research, us food & drug administration monoclonal antibodies: the second generation human antibodies by design genetically engineered antibody molecules engineering antibodies for therapy recombinant antibodies: alternative strategies for developing and manipulating murine-derived monoclonal antibodies monoclonal antibody-based therapy for solid tumors: an overview monoclonal antibodies: innovations in diagnosis and therapy unconjugated monoclonal antibody therapy of lymphoma monoclonal antibodies: the second generation. herndon (va): bios scientific preparation of recombinant antibodies from immune rodent spleens and the design of their humanization 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cocaine. i. monoclonal antibody 15a10 and the reinforcing effects of cocaine in rats evaluation of anti-cocaine antibodies and a cocaine vaccine in a rat selfadministration model we thank carolyn m. black and the national center for infectious diseases editorial office for the critical review of this manuscript. key: cord-304066-rirbdhz3 authors: reddehase, matthias j. title: adverse immunological imprinting by cytomegalovirus sensitizing for allergic airway disease date: 2019-05-10 journal: med microbiol immunol doi: 10.1007/s00430-019-00610-z sha: doc_id: 304066 cord_uid: rirbdhz3 cytomegalovirus (cmv) infection has a profound impact on the host’s immune system. immunological imprinting by cmv is not restricted to immunity against cmv itself, but can affect immunity against other viral or non-viral infectious agents and also immunopathological responses. one category is heterologous immunity based on molecular mimicry, where antigen recognition receptors specific for a cmv antigen with broad avidity distribution also bind with some avidity to unrelated antigens and exert effector functions against target structures other than those linked to cmv. another category is induction of cytokines by cmv infection that inhibit or drive immune responses to bystander antigens unrelated to cmv, and a third category is the activation of antigen-presenting cells by cmv from which unrelated antigens profit as “stowaways”. a striking example of the “stowaway” category, actually one that is of medical importance, has been published recently and will be discussed here for the more general reader. specifically, in a murine model, cmv airway infection and inhaled environmental antigen of poor intrinsic allergenic potential were found to sensitize for allergic airway disease (aad) only when combined. as to the mechanism, viral activation of cd11b(+) conventional dendritic cells (cd11b(+) cdc) that localize to airway mucosa facilitates uptake and processing of inhaled antigen. thus, cmv serves as a “door opener” for otherwise harmless environmental antigens that have no intrinsic property to activate dc. antigen-laden cd11b(+) cdc migrate selectively to the airway draining lymph nodes, where they prime type-2 cd4(+) t helper (th-2) cells. upon airway re-exposure to the inhaled antigen, th-2 cells secrete interleukins (il-4, il-5, il-9, and il-25) known to induce goblet cell metaplasia, the lead histopathological manifestation of aad that is characterized by thickening of airway epithelia and increased numbers of mucus-producing goblet cells, resulting in enhanced mucus secretion and airflow obstruction. human cytomegalovirus (hcmv) is not a textbook example of a respiratory viral infection. nonetheless, hcmv infection has a respiratory phase that usually is not diagnosed because of its mild and unspecific symptoms in the immunocompetent host. so, it is subsumed under the popular term "common cold" that is used for symptoms of respiratory virus infections for which there is no medical indication to identify the cause and for which symptomatic treatment is sufficient, for instance infections with rhinoviruses, common coronaviruses, and parainfluenza viruses. historically, cmvs were named "salivary gland viruses" ( [1, 2] , reviewed in [3, 4] ), as they replicate in acinar glandular epithelial cells of the salivary glands, from where they are released into the salivary duct [5, 6] . a frequent route of host-to-host transmission for primary infection is thus through saliva of a virus-shedding contact person, for instance in mother-to-child transmission. transmission probability is increased whenever very young children crowd together, such as in multi-child families and in daycare centers. as an example of real life epidemiology for lectures on medicine, toy sharing, that is the mouthing of toys that other children have mouthed, has been identified as a way by which hcmv is transmitted between peers (reviewed in [7] , see also the contribution by adler and reddehase in this issue of mmim [8] ). with the saliva, virus reaches upper airway mucosas, where it replicates persistently for a prolonged period, and from there it also reaches lower airway mucosas in bronchi, bronchioli, and the lungs, as shown long ago and revisited more recently in murine models of intranasal infection using murine cmv (mcmv) [9, 10] . this topic is specifically addressed and more comprehensively referenced by zhang and colleagues in this issue of mmim [11] . children who undergo a primary airway infection after transmission of hcmv are unavoidably at the same time exposed to a universe of inhaled environmental antigens, few of which are classical allergens, whereas by far most have lowto-no intrinsic allergenic potential, as otherwise all of us would suffer from allergy. clearly, allergic airway disease (aad) elicited by inhaled environmental antigens is not the rule. the development of aad requires a "sensitization phase", which, in immunological terminology, means "priming" of allergenspecific t cells, usually type-2 cd4 + t helper (th-2) cells, as well as re-exposure to the allergen for a recall response. in their effector phase, restimulated allergen-specific th-2 cells secrete cytokines that induce goblet cell (gc) metaplasia, such as the interleukins il-4, il-5, il-9, and il-25 [12] [13] [14] . remodeling of the airway mucosa by transdifferentiation of ciliated cells and clara/club cells to mucus-producing gc, rather than by proliferation of gc [15] , associated with thickening of airway epithelia and chronic mucus hypersecretion and consequent airflow obstruction, is the lead histopathological manifestation of aad. mouse models, provided they are reasonably designed to meet a clinical correlate, have proven predictive value for human disease [16] . as airway exposure to environmental antigens at the time of primary airway infection after hcmv transmission is a realistic scenario of medical interest, recent work modeled this scenario in the mouse with the aim to investigate a possible virus-allergen interplay [17] . in the model (fig. 1, top and [17] ), immunocompetent c57bl/6 mice were simultaneously exposed via the airways to mcmv and to purified ovalbumin (ova). ova was chosen to represent a protein antigen that has low allergenic potential on its own and fails to sensitize for aad. for tracking the fate of this low-potency allergen, ova was tagged with a red fluorescent dye. on three consecutive days (days 14, 15, and 16) after sensitization, mice were re-exposed to ova aerosol for a recall response, and airway histopathology was evaluated 2 days after the last challenge exposure (fig. 1, histological images) . mcmv airway infection alone did not trigger aad, nor did the combination of ova-sensitization and ova re-exposure. combined sensitization by ova and mcmv, however, followed by challenge exposures to ova, caused aad that is characterized by a massive increase in the number of mucus-producing gc. this histopathology is referred to in the literature as gc hyperplasia or metaplasia, depending on whether one focuses on the increase in gc numbers [12] or on the remodeling of the mucosa by transdifferentiation of ciliated cells and clara/club cells to gc [15] , respectively. it should be noted that aad was not induced when either ova sensitization or ova reexposure were missing. these controls verified the ovaspecificity of aad. depletion of cd4 + t cells shortly before the first ova challenge prevented aad, whereas depletion of cd8 + t cells did not. so, the effector cells in aad are ova-specific cd4 + t cells. based on the data by reuter et al. [17] , fig. 2 provides a graphical summary that illustrates the mechanism by which mcmv imprints the immune system for aad immunopathology. upon co-exposure of airway mucosa to inhaled antigen/low potency allergen, represented by ova in the specific case, and mcmv, the virus activates mhc-ii + cd11c + dendritic cells (dc) that localize to the mucosa, specifically dc of the cd11b + subset of conventional b220 low ly6c low dc, briefly cd11b + cdc. their activation is indicated by upregulation of cd28 ligands cd80 and cd86, and is associated with enhanced uptake of ova. the ova-laden cd11b + cdc then migrate selectively to the airway draining lymph nodes, where they present processed ova peptides by mhc-ii molecules to naïve ova peptide-specific th-2 cells for priming. upon re-exposure to inhaled ova, restimulated ova peptidespecific th-2 cells secrete effector cytokines il-4, il-5, il-9, and il-25, all known to be involved in the induction of gc metaplasia. in essence, the work by reuter and colleagues [17] has shown that the key event in sensitization for aad is the efficient uptake and processing of an inhaled antigen by migratory dc that localize to the airway mucosa as sentinels. classical allergens and most pathogens have the intrinsic property to activate dc, whereas inhaled environmental protein antigens do not, and are thus ignored by the sentinels. this is a "healthy ignorance", as otherwise all environmental antigens would cause allergy. activation of dc by cmvs is beneficial for priming a protective antiviral immune response, though it can result in allergic disease when used by otherwise harmless "stowaways" for entering dc. the findings suggests that by this "door opener" function, cmv airway infection may broaden the spectrum of potential allergens. current experimental protocols did not lead to full-blown asthma. future work will address the question if repetitive allergen exposure will eventually lead to clinical asthma. another interesting aspect for future research is the finding that aad in the here discussed model was not accompanied by eosinophilia [17] , unlike in established aad/asthma mouse models and in most clinical forms of asthma. so, is it a bad model? interestingly, there exists a clinical correlate, namely an asthma phenotype in humans known as non-eosinophilic asthma (nea). nea accounts for particularly severe cases of asthma, and its most relevant clinical trait is its poor response to asthma treatment with corticosteroids [18] . testing if non-eosinophilic aad, reviewed here and described in greater detail in [17] , qualifies as a model, or can be developed into a model, for clinical nea is therefore of top priority. uptake & processing fig. 2 graphical abstract of the mechanism that leads to aad/gc metaplasia. for detailed explanation, see the body of the text. the waved symbol represents peptide(s) derived by antigen processing. cdc conventional dendritic cell. the graphics concerning the airway mucosa was inspired by artwork presented in [12] propagation of salivary gland virus of the mouse in tissue cultures propagation in tissue cultures of a cytopathogenic virus from human salivary gland virus (sgv) disease the history of cytomegalovirus and its diseases margaret gladys smith, mother of cytomegalovirus: 60th anniversary of cytomegalovirus isolation mouse cytomegalovirus. necrosis of infected and morphologically normal submaxillary gland acinar cells during termination of chronic infection site-restricted persistent cytomegalovirus infection after selective long-term depletion of cd 4+ t lymphocytes the epidemiology and public health impact of congenital cytomegalovirus infection pediatric roots of cytomegalovirus recurrence and memory inflation in the elderly interstitial pneumonia and subclinical infection after intranasal inoculation of murine cytomegalovirus murine cytomegalovirus (cmv) infection via the intranasal route offers a robust model of immunity upon mucosal cmv infection persistent viral replication and the development of t-cell responses after intranasal infection by mcmv airway goblet cell hyperplasia in asthma: hypersecretory and anti-inflammatory? the cytokine network in asthma and chronic obstructive pulmonary disease importance of cytokines in murine allergic airway disease and human asthma the airway epithelium in asthma mouse model of cytomegalovirus disease and immunotherapy in the immunocompromised host: predictions for medical translation that survived the "test of time coincident airway exposure to low-potency allergen and cytomegalovirus sensitizes for allergic airway disease by viral activation of migratory dendritic cells non-eosinophilic asthma: current perspectives publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations this article is part of the special issue on immunological imprinting during chronic viral infection.acknowledgements mjr is supported by the deutsche forschungsgemeinschaft sfb 1292, individual project tp11. conflict of interest the author declares that he has no conflict of interest. key: cord-311505-akcc9oms authors: geisen, will r.; berger, justin; schwartz, chelsea; reddy, abhimanyu; rai, balaj; peck, joshua; wadih, george title: cytomegalovirus enterocolitis secondary to experimental covid-19 therapy date: 2020-09-22 journal: idcases doi: 10.1016/j.idcr.2020.e00962 sha: doc_id: 311505 cord_uid: akcc9oms the novel coronavirus-2019 (covid-19) has caused a global pandemic of historical proportions, infecting millions of people worldwide. due to its high mortality rate and a paucity of clinical data, experimental therapies have been utilized with uncertain success and, unfortunately, poor outcomes. we describe a gentleman who was treated with experimental therapies and subsequently developed cytomegalovirus colitis and hypovolemic shock. additionally, this case validates colonoscopy as a mode to rule out concurrent infectious etiologies causing diarrhea in covid-19-positive patients. the novel coronavirus-2019 (covid-19) has caused a global pandemic of historical proportions, infecting millions of people worldwide. due to its high mortality rate and a paucity of clinical data, experimental therapies have been utilized with uncertain success and, unfortunately, poor outcomes. we describe a gentleman who was treated with experimental therapies and subsequently developed cytomegalovirus colitis and hypovolemic shock. additionally, this case validates colonoscopy as a mode to rule out concurrent infectious etiologies causing diarrhea in covid-19-positive patients. keywords: covid-19; immunocompromised; colitis; endoscopy introduction: the novel 2019 coronavirus (covid-19), a form of severe acute respiratory syndrome (sars-cov-2), has caused a pandemic of historical proportions. as of this publication, 16.7 million people have been diagnosed worldwide with 660,792 deaths reported. approximately 10-15% of these patients develop respiratory distress requiring hospitalization and, often, icu admission (1, 2) . due to its worldwide distribution, a paucity of clinical trial data, and a high mortality rate, the covid pandemic has led to widespread implementation of experimental therapies with varying levels of success and, in some instances, poor outcomes (3, 4, 5) . we present a patient who was treated with experimental therapies and subsequently developed severe gastrointestinal pathology that was diagnosed by colonoscopy. case report: a 68-year-old caucasian male with medical history of hypertension and glaucoma presented with nausea, emesis, and fevers of 1-week duration. in the emergency department he became increasingly hypoxic and required 4 liters of supplemental oxygen, warranting chest radiography. chest x-ray demonstrated bilateral opacities concerning for atypical pneumonia and patient was started on cefepime, vancomycin, and azithromycin. respiratory polymerase chain reaction (pcr) was positive for streptococcus and staphylococcus species and nasopharyngeal swab for sars-cov-2 was obtained prior to transfer to the intensive care unit (icu). the patient developed acute respiratory distress syndrome (ards) requiring intubation, mechanical ventilation, and extracorporeal membrane oxygenation (ecmo). computed tomography (ct) of the chest/abdomen/pelvis showed extensive ground glass opacities, pelvic ascites, and no intracolonic abnormalities. six days after initial presentation, coronavirus pcr resulted positive. while awaiting confirmatory testing for sars-cov-2, the patient received five doses of hydroxychloroquine per experimental protocols. on day seven of admission, the patient received one dose of tocilizumab, an interleukin-6 (il-6) inhibitor approved for giant cell arteritis. in addition, the patient received a ten-day course of remdesivir, an anti-viral agent with noted efficacy against rna viruses. in the icu, the patient developed high-output diarrhea (2-4 liters daily) and hypovolemic shock. clostridioides difficile toxin was negative on three separate occasions during his admission. gi panel j o u r n a l p r e -p r o o f polymerase chain reaction (pcr) for bacterial, viral, fungal, and protozoan causes of infectious diarrhea were also found to be negative. gastroenterology was consulted and added octreotide, cholestyramine, atropine/diphenoxylate, and probiotics without significant improvement. ct of the abdomen and pelvis showed extensive bowel wall thickening of the colon and distal ileum with rectal sparing. etiologies of infectious colitis, including cytomegalovirus (cmv), were entertained but initial dna pcr were negative. two weeks later, as the patient continued to have high output stools, cmv igg and igm were tested and demonstrated elevated igg (1.50) and undetectable igm. cmv quantitative pcr was highly elevated (55,937 iu/ml) indicating viremia and the patient was started on intravenous ganciclovir. colonoscopy yielded multiple raised plaques within the terminal ileum and pan-colonic ulcerations that were biopsied and clipped (figure 1a, b) . the obtained pathology showed focal glandular atypia, pseudostratification of enlarged nuclei, and inclusion bodies with positive immunohistochemical staining for cmv (figure 2a, b) . the management of sars-cov-2 has included the use of many experimental therapies, including hydroxychloroquine, tocilizumab, and remdesivir. several of these agents, specifically those approved for rheumatologic disorders, result in an immunocompromised state. hydroxychloroquine has been shown to inhibit toll-like receptor pathways, thus dampening antigen presenting cells from activating inflammatory processes (6) . il-6 is heavily involved in b-cell maturation into antibody producing cells and in vivo data from patients taking tocilizumab has noted a significant decrease the production of iga/igg (7) . these reductions in immunocompetency, especially iga/igg which are heavily involved in the protection of mucosal barriers, make the gastrointestinal tract vulnerable to opportunistic infections. while anecdotal studies have led to the widespread use of experimental therapies (3, 4) , more comprehensive studies analysis have shown that these agents may cause increased mortality (5). additionally, studies have shown a significant alteration of gut microbiome and increased rates of c. difficile in covid-19 patients, most presumably from immunomodulating therapies (6, 8) . it is difficult to extrapolate the effect that covid-19 will have on the field of gastroenterology, but one can assume that a rise in prevalence of infectious diarrhea is likely. this case exemplifies the notion that unproven therapies can lead to severe disease, including cmv colitis. of note, the presence of gastrointestinal symptoms, specifically diarrhea, have been widely reported in patients with covid-19 (9, 10) . a survey of 2,506 patients with sars-cov-2 infection found that 5.8 % (145/2506) experienced diarrhea as a presenting symptom (9) . because the virus attaches to and enters cells through angiotensin-converting enzyme 2 (ace2), which has significantly higher expression in the small intestine than the lungs, it is believed that the gastrointestinal tract may play a key role in the infectivity of covid-19 (10) . some speculate that viral invasion through the intestinal mucosa and the ensuing inflammation cause diarrhea in these patients (9, 10) . however, while diarrhea may be a common symptom in early covid-19 disease, this case illustrates that there is a role for colonoscopy to exclude concurrent infection in these patients. colonic biopsy of the of inflamed lesions with hematoxylin and eosin (h&e) staining and immunohistochemistry is highly specific (92-99%) for cmv and necessary to definitively diagnose cmv colitis (11) . with immunomodulating therapies being used in experimental protocols and clinical trials for sars-cov-2 j o u r n a l p r e -p r o o f infection, colonoscopy is vital to diagnose gastrointestinal opportunistic infections that are associated with these treatment modalities clinical characteristics of coronavirus disease 2019 in china a novel coronavirus from patients with pneumonia in china tocilizumab for the treatment of severe covid-19 hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial observational study of hydroxychloroquine in hospitalized patients with covid-19 clostridioides difficile in covid-19 patients in vivo effects of the anti-interleukin-6 receptor inhibitor tocilizumab on the b cell compartment alterations in gut microbiota of patients with covid-19 during time of hospitalization novel coronavirus infection: gastrointestinal manifestations pooled prevalence of diarrhea among covid-19 toward an algorithm for the diagnosis and management of cmv in patients with colitis the occurrence of diarrhea in covid-19 patients mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology excessive anxiety in ibd patients is unnecessary for covid-19 dear editor-in-chief,we are submitting the manuscript cytomegalovirus enterocolitis secondary to experimental covid-19 therapy for consideration of publication as a case report. the report describes a patient with sars-cov-2 pneumonia who was treated with experimental immunomodulating therapies and, subsequently, developed cytomegalovirus (cmv) colitis. our manuscript stresses the potential dangers of experimental therapies in lieu of clinical trial data. it also emphasizes the role of colonoscopy in the evaluation of patients with infectious diarrhea secondary to covid-19 and its management.thank you in advance for your consideration of this manuscript. key: cord-193356-hqbstgg7 authors: widrich, michael; schafl, bernhard; ramsauer, hubert; pavlovi'c, milena; gruber, lukas; holzleitner, markus; brandstetter, johannes; sandve, geir kjetil; greiff, victor; hochreiter, sepp; klambauer, gunter title: modern hopfield networks and attention for immune repertoire classification date: 2020-07-16 journal: nan doi: nan sha: doc_id: 193356 cord_uid: hqbstgg7 a central mechanism in machine learning is to identify, store, and recognize patterns. how to learn, access, and retrieve such patterns is crucial in hopfield networks and the more recent transformer architectures. we show that the attention mechanism of transformer architectures is actually the update rule of modern hopfield networks that can store exponentially many patterns. we exploit this high storage capacity of modern hopfield networks to solve a challenging multiple instance learning (mil) problem in computational biology: immune repertoire classification. accurate and interpretable machine learning methods solving this problem could pave the way towards new vaccines and therapies, which is currently a very relevant research topic intensified by the covid-19 crisis. immune repertoire classification based on the vast number of immunosequences of an individual is a mil problem with an unprecedentedly massive number of instances, two orders of magnitude larger than currently considered problems, and with an extremely low witness rate. in this work, we present our novel method deeprc that integrates transformer-like attention, or equivalently modern hopfield networks, into deep learning architectures for massive mil such as immune repertoire classification. we demonstrate that deeprc outperforms all other methods with respect to predictive performance on large-scale experiments, including simulated and real-world virus infection data, and enables the extraction of sequence motifs that are connected to a given disease class. source code and datasets: https://github.com/ml-jku/deeprc transformer architectures (vaswani et al., 2017) and their attention mechanisms are currently used in many applications, such as natural language processing (nlp), imaging, and also in multiple instance learning (mil) problems . in mil, a set or bag of objects is labelled rather than objects themselves as in standard supervised learning tasks (dietterich et al., 1997) . examples for mil problems are medical images, in which each sub-region of the image represents an instance, video a pooling function f is used to obtain a repertoire-representation z for the input object. finally, an output network o predicts the class labelŷ. b) deeprc uses stacked 1d convolutions for a parameterized function h due to their computational efficiency. potentially, millions of sequences have to be processed for each input object. in principle, also recurrent neural networks (rnns), such as lstms (hochreiter et al., 2007) , or transformer networks (vaswani et al., 2017) may be used but are currently computationally too costly. c) attention-pooling is used to obtain a repertoire-representation z for each input object, where deeprc uses weighted averages of sequence-representations. the weights are determined by an update rule of modern hopfield networks that allows to retrieve exponentially many patterns. classification, in which each frame is an instance, text classification, where words or sentences are instances of a text, point sets, where each point is an instance of a 3d object, and remote sensing data, where each sensor is an instance (carbonneau et al., 2018; uriot, 2019) . attention-based mil has been successfully used for image data, for example to identify tiny objects in large images (ilse et al., 2018; pawlowski et al., 2019; tomita et al., 2019; kimeswenger et al., 2019) and transformer-like attention mechanisms for sets of points and images . however, in mil problems considered by machine learning methods up to now, the number of instances per bag is in the range of hundreds or few thousands (carbonneau et al., 2018; lee et al., 2019 ) (see also tab. a2). at the same time the witness rate (wr), the rate of discriminating instances per bag, is already considered low at 1% − 5%. we will tackle the problem of immune repertoire classification with hundreds of thousands of instances per bag without instance-level labels and with extremely low witness rates down to 0.01% using an attention mechanism. we show that the attention mechanism of transformers is the update rule of modern hopfield networks (krotov & hopfield, 2016 demircigil et al., 2017) that are generalized to continuous states in contrast to classical hopfield networks (hopfield, 1982) . a detailed derivation and analysis of modern hopfield networks is given in our companion paper (ramsauer et al., 2020) . these novel continuous state hopfield networks allow to store and retrieve exponentially (in the dimension of the space) many patterns (see next section). thus, modern hopfield networks with their update rule, which are used as an attention mechanism in the transformer, enable immune repertoire classification in computational biology. immune repertoire classification, i.e. classifying the immune status based on the immune repertoire sequences, is essentially a text-book example for a multiple instance learning problem (dietterich et al., 1997; maron & lozano-pérez, 1998; wang et al., 2018) . briefly, the immune repertoire of an individual consists of an immensely large bag of immune receptors, represented as amino acid sequences. usually, the presence of only a small fraction of particular receptors determines the immune status with respect to a particular disease (christophersen et al., 2014; emerson et al., 2017) . this is because the immune system has already acquired a resistance if one or few particular immune receptors that can bind to the disease agent are present. therefore, classification of immune repertoires bears a high difficulty since each immune repertoire can contain millions of sequences as instances with only a few indicating the class. further properties of the data that complicate the problem are: (a) the overlap of immune repertoires of different individuals is low (in most cases, maximally low single-digit percentage values) (greiff et al., 2017; elhanati et al., 2018) , (b) multiple different sequences can bind to the same pathogen (wucherpfennig et al., 2007) , and (c) only subsequences within the sequences determine whether binding to a pathogen is possible (dash et al., 2017; glanville et al., 2017; akbar et al., 2019; springer et al., 2020; fischer et al., 2019) . in summary, immune repertoire classification can be formulated as multiple instance learning with an extremely low witness rate and large numbers of instances, which represents a challenge for currently available machine learning methods. furthermore, the methods should ideally be interpretable, since the extraction of class-associated sequence motifs is desired to gain crucial biological insights. the acquisition of human immune repertoires has been enabled by immunosequencing technology (georgiou et al., 2014; brown et al., 2019) which allows to obtain the immune receptor sequences and immune repertoires of individuals. each individual is uniquely characterized by their immune repertoire, which is acquired and changed during life. this repertoire may be influenced by all diseases that an individual is exposed to during their lives and hence contains highly valuable information about those diseases and the individual's immune status. immune receptors enable the immune system to specifically recognize disease agents or pathogens. each immune encounter is recorded as an immune event into immune memory by preserving and amplifying immune receptors in the repertoire used to fight a given disease. this is, for example, the working principle of vaccination. each human has about 10 7 -10 8 unique immune receptors with low overlap across individuals and sampled from a potential diversity of > 10 14 receptors (mora & walczak, 2019) . the ability to sequence and analyze human immune receptors at large scale has led to fundamental and mechanistic insights into the adaptive immune system and has also opened the opportunity for the development of novel diagnostics and therapy approaches (georgiou et al., 2014; brown et al., 2019) . immunosequencing data have been analyzed with computational methods for a variety of different tasks (greiff et al., 2015; shugay et al., 2015; miho et al., 2018; yaari & kleinstein, 2015; wardemann & busse, 2017) . a large part of the available machine learning methods for immune receptor data has been focusing on the individual immune receptors in a repertoire, with the aim to, for example, predict the antigen or antigen portion (epitope) to which these sequences bind or to predict sharing of receptors across individuals (gielis et al., 2019; springer et al., 2020; jurtz et al., 2018; moris et al., 2019; fischer et al., 2019; greiff et al., 2017; sidhom et al., 2019; elhanati et al., 2018) . recently, jurtz et al. (2018) used 1d convolutional neural networks (cnns) to predict antigen binding of t-cell receptor (tcr) sequences (specifically, binding of tcr sequences to peptide-mhc complexes) and demonstrated that motifs can be extracted from these models. similarly, konishi et al. (2019) use cnns, gradient boosting, and other machine learning techniques on b-cell receptor (bcr) sequences to distinguish tumor tissue from normal tissue. however, the methods presented so far predict a particular class, the epitope, based on a single input sequence. immune repertoire classification has been considered as a mil problem in the following publications. a deep learning framework called deeptcr (sidhom et al., 2019) implements several deep learning approaches for immunosequencing data. the computational framework, inter alia, allows for attention-based mil repertoire classifiers and implements a basic form of attention-based averaging. ostmeyer et al. (2019) already suggested a mil method for immune repertoire classification. this method considers 4-mers, fixed sub-sequences of length 4, as instances of an input object and trained a logistic regression model with these 4-mers as input. the predictions of the logistic regression model for each 4-mer were max-pooled to obtain one prediction per input object. this approach is characterized by (a) the rigidity of the k-mer features as compared to convolutional kernels (alipanahi et al., 2015; zhou & troyanskaya, 2015; zeng et al., 2016) , (b) the max-pooling operation, which constrains the network to learn from a single, top-ranked k-mer for each iteration over the input object, and (c) the pooling of prediction scores rather than representations (wang et al., 2018) . our experiments also support that these choices in the design of the method can lead to constraints on the predictive performance (see table 1 ). our proposed method, deeprc, also uses a mil approach but considers sequences rather than k-mers as instances within an input object and a transformer-like attention mechanism. deeprc sets out to avoid the above-mentioned constraints of current methods by (a) applying transformer-like attention-pooling instead of max-pooling and learning a classifier on the repertoire rather than on the sequence-representation, (b) pooling learned representations rather than predictions, and (c) using less rigid feature extractors, such as 1d convolutions or lstms. in this work, we contribute the following: we demonstrate that continuous generalizations of binary modern hopfield-networks (krotov & hopfield, 2016 demircigil et al., 2017) have an update rule that is known as the attention mechanisms in the transformer. we show that these modern hopfield networks have exponential storage capacity, which allows them to extract patterns among a large set of instances (next section). based on this result, we propose deeprc, a novel deep mil method based on modern hopfield networks for large bags of complex sequences, as they occur in immune repertoire classification (section "deep repertoire classification). we evaluate the predictive performance of deeprc and other machine learning approaches for the classification of immune repertoires in a large comparative study (section "experimental results") exponential storage capacity of continuous state modern hopfield networks with transformer attention as update rule in this section, we show that modern hopfield networks have exponential storage capacity, which will later allow us to approach massive multiple-instance learning problems, such as immune repertoire classification. see our companion paper (ramsauer et al., 2020) for a detailed derivation and analysis of modern hopfield networks. we assume patterns x 1 , . . . , x n ∈ r d that are stacked as columns to the matrix x = (x 1 , . . . , x n ) and a query pattern ξ that also represents the current state. the largest norm of a pattern is m = max i x i . the separation ∆ i of a pattern x i is defined as its minimal dot product difference to any of the other patterns: we consider a modern hopfield network with current state ξ and the energy function for energy e and state ξ, the update rule is proven to converge globally to stationary points of the energy e, which are local minima or saddle points (see (ramsauer et al., 2020) , appendix, theorem a2 ). surprisingly, the update rule eq. (1) is also the formula of the well-known transformer attention mechanism. to see this more clearly, we simultaneously update several queries ξ i . furthermore the queries ξ i and the patterns x i are linear mappings of vectors y i into the space r d . for matrix notation, we set x i = w t k y i , ξ i = w t q y i and multiply the result of our update rule with w v . using y = (y 1 , . . . , y n ) t , we define the matrices and the patterns are now mapped to the hopfield space with dimension d = d k . we set β = 1/ √ d k and change softmax to a row vector. the update rule eq. (1) multiplied by w v performed for all queries simultaneously becomes in row vector notation: this formula is the transformer attention. if the patterns x i are well separated, the iterate eq. (1) converges to a fixed point close to a pattern to which the initial ξ is similar. if the patterns are not well separated the iterate eq.(1) converges to a fixed point close to the arithmetic mean of the patterns. if some patterns are similar to each other but well separated from all other vectors, then a metastable state between the similar patterns exists. iterates that start near a metastable state converge to this metastable state. for details see ramsauer et al. (2020) , appendix, sect. a2. typically, the update converges after one update step (see ramsauer et al. (2020) , appendix, theorem a8) and has an exponentially small retrieval error (see ramsauer et al. (2020) , appendix, theorem a9). our main concern for application to immune repertoire classification is the number of patterns that can be stored and retrieved by the modern hopfield network, equivalently to the transformer attention head. the storage capacity of an attention mechanism is critical for massive mil problems. we first define what we mean by storing and retrieving patterns from the modern hopfield network. definition 1 (pattern stored and retrieved). we assume that around every pattern x i a sphere s i is given. we say x i is stored if there is a single fixed point x * i ∈ s i to which all points ξ ∈ s i converge, for randomly chosen patterns, the number of patterns that can be stored is exponential in the dimension d of the space of the patterns (x i ∈ r d ). theorem 1. we assume a failure probability 0 < p 1 and randomly chosen patterns on the sphere with radius m = k √ d − 1. we define a := 2 d−1 (1 + ln(2 β k 2 p (d − 1))), b := 2 k 2 β 5 , and c = b w0(exp(a + ln(b)) , where w 0 is the upper branch of the lambert w function and ensure then with probability 1 − p, the number of random patterns that can be stored is examples are c ≥ 3.1546 for β = 1, k = 3, d = 20 and p = 0.001 (a + ln(b) > 1.27) and c ≥ 1.3718 for β = 1 k = 1, d = 75, and p = 0.001 (a + ln(b) < −0.94). see ramsauer et al. (2020) , appendix, theorem a5 for a proof. we have established that a modern hopfield network or a transformer attention mechanism can store and retrieve exponentially many patterns. this allows us to approach mil with massive numbers of instances from which we have to retrieve a few with an attention mechanism. deep repertoire classification problem setting and notation. we consider a mil problem, in which an input object x is a bag of n instances x = {s 1 , . . . , s n }. the instances do not have dependencies nor orderings between them and n can be different for every object. we assume that each instance s i is associated with a label y i ∈ {0, 1}, assuming a binary classification task, to which we do not have access. we only have access to a label y = max i y i for an input object or bag. note that this poses a credit assignment problem, since the sequences that are responsible for the label y have to be identified and that the relation between instance-label and bag-label can be more complex (foulds & frank, 2010) . a modelŷ = g(x) should be (a) invariant to permutations of the instances and (b) able to cope with the fact that n varies across input objects (ilse et al., 2018) , which is a problem also posed by point sets (qi et al., 2017) . two principled approaches exist. the first approach is to learn an instance-level scoring function h : s → [0, 1], which is then pooled across instances with a pooling function f , for example by average-pooling or max-pooling (see below). the second approach is to construct an instance representation z i of each instance by h : s → r dv and then encode the bag, or the input object, by pooling these instance representations (wang et al., 2018) via a function f . an output function o : r dv → [0, 1] subsequently classifies the bag. the second approach, the pooling of representations rather than scoring functions, is currently best performing (wang et al., 2018) . in the problem at hand, the input object x is the immune repertoire of an individual that consists of a large set of immune receptor sequences (t-cell receptors or antibodies). immune receptors are primarily represented as sequences s i from a space s i ∈ s. these sequences act as the instances in the mil problem. although immune repertoire classification can readily be formulated as a mil problem, it is yet unclear how well machine learning methods solve the above-described problem with a large number of instances n 10, 000 and with instances s i being complex sequences. next we describe currently used pooling functions for mil problems. pooling functions for mil problems. different pooling functions equip a model g with the property to be invariant to permutations of instances and with the ability to process different numbers of instances. typically, a neural network h θ with parameters θ is trained to obtain a function that maps each instance onto a representation: z i = h θ (s i ) and then a pooling function z = f ({z 1 , . . . , z n }) supplies a representation z of the input object x = {s 1 , . . . , s n }. the following pooling functions are typically used: average-pooling: where e m is the standard basis vector for dimension m and attention-pooling: z = n i=1 a i z i , where a i are non-negative (a i ≥ 0), sum to one ( n i=1 a i = 1), and are determined by an attention mechanism. these pooling functions are invariant to permutations of {1, . . . , n } and are differentiable. therefore, they are suited as building blocks for deep learning architectures. we employ attention-pooling in our deeprc model as detailed in the following. modern hopfield networks viewed as transformer-like attention mechanisms. the modern hopfield networks, as introduced above,have a storage capacity that is exponential in the dimension of the vector space and converge after just one update (see (ramsauer et al., 2020) , appendix).additionally, the update rule of modern hopfield networks is known as key-value attention mechanism, which has been highly successful through the transformer (vaswani et al., 2017) and bert (devlin et al., 2019) models in natural language processing. therefore using modern hopfield networks with the key-value-attention mechanism as update rule is the natural choice for our task. in particular, modern hopfield networks are theoretically justified for storing and retrieving the large number of vectors (sequence patterns) that appear in the immune repertoire classification task. instead of using the terminology of modern hopfield networks, we explain our deeprc architecture in terms of key-value-attention (the update rule of the modern hopfield network), since it is well known in the deep learning community. the attention mechanism assumes a space of dimension d k in which keys and queries are compared. a set of n key vectors are combined to the matrix k. a set of d q query vectors are combined to the matrix q. similarities between queries and keys are computed by inner products, therefore queries can search for similar keys that are stored. another set of n value vectors are combined to the matrix v . the output of the attention mechanism is a weighted average of the value vectors for each query q. the i-th vector v i is weighted by the similarity between the i-th key k i and the query q. the similarity is given by the softmax of the inner products of the query q with the keys k i . all queries are calculated in parallel via matrix operations. consequently, the attention function att(q, k, v ; β) maps queries q, keys k, and values v to d v -dimensional outputs: att(q, k, v ; β) = softmax(βqk t )v (see also eq. (2)). while this attention mechanism has originally been developed for sequence tasks (vaswani et al., 2017) , it can be readily transferred to sets ye et al., 2018) . this type of attention mechanism will be employed in deeprc. the deeprc method. we propose a novel method deep repertoire classification (deeprc) for immune repertoire classification with attention-based deep massive multiple instance learning and compare it against other machine learning approaches. for deeprc, we consider immune repertoires as input objects, which are represented as bags of instances. in a bag, each instance is an immune receptor sequence and each bag can contain a large number of sequences. note that we will use z i to denote the sequence-representation of the i-th sequence and z to denote the repertoire-representation. at the core, deeprc consists of a transformer-like attention mechanism that extracts the most important information from each repertoire. we first give an overview of the attention mechanism and then provide details on each of the sub-networks h 1 , h 2 , and o of deeprc. attention mechanism in deeprc. this mechanism is based on the three matrices k (the keys), q (the queries), and v (the values) together with a parameter β. values. deeprc uses a 1d convolutional network h 1 (lecun et al., 1998; hu et al., 2014; kelley et al., 2016) that supplies a sequence-representation z i = h 1 (s i ), which acts as the values v = z = (z 1 , . . . , z n ) in the attention mechanism (see figure 2 ). keys. a second neural network h 2 , which shares its first layers with h 1 , is used to obtain keys k ∈ r n ×d k for each sequence in the repertoire. this network uses 2 self-normalizing layers (klambauer et al., 2017) with 32 units per layer (see figure 2 ). query. we use a fixed d k -dimensional query vector ξ which is learned via backpropagation. for more attention heads, each head has a fixed query vector. with the quantities introduced above, the transformer attention mechanism (eq. (2)) of deeprc is implemented as follows: where z ∈ r n ×dv are the sequence-representations stacked row-wise, k are the keys, and z is the repertoire-representation and at the same time a weighted mean of sequence-representations z i . the attention mechanism can readily be extended to multiple queries, however, computational demand could constrain this depending on the application and dataset. theorem 1 demonstrates that this mechanism is able to retrieve a single pattern out of several hundreds of thousands. attention-pooling and interpretability. each input object, i.e. repertoire, consists of a large number n of sequences, which are reduced to a single fixed-size feature vector of length d v representing the whole input object by an attention-pooling function. to this end, a transformer-like attention mechanism adapted to sets is realized in deeprc which supplies a i -the importance of the sequence s i . this importance value is an interpretable quantity, which is highly desired for the immunological problem at hand. thus, deeprc allows for two forms of interpretability methods. (a) a trained deeprc model can compute attention weights a i , which directly indicate the importance of a sequence. (b) deeprc furthermore allows for the usage of contribution analysis methods, such as integrated gradients (ig) (sundararajan et al., 2017) or layer-wise relevance propagation (montavon et al., 2018; arras et al., 2019) . see sect. a8 for details. classification layer and network parameters. the repertoire-representation z is then used as input for a fully-connected output networkŷ = o(z) that predicts the immune status, where we found it sufficient to train single-layer networks. in the simplest case, deeprc predicts a single target, the class label y, e.g. the immune status of an immune repertoire, using one output value. however, since deeprc is an end-to-end deep learning model, multiple targets may be predicted simultaneously in classification or regression settings or a mix of both. this allows for the introduction of additional information into the system via auxiliary targets such as age, sex, or other metadata. table 1 with sub-networks h 1 , h 2 , and o. d l indicates the sequence length. network parameters, training, and inference. deeprc is trained using standard gradient descent methods to minimize a cross-entropy loss. the network parameters are θ 1 , θ 2 , θ o for the sub-networks h 1 , h 2 , and o, respectively, and additionally ξ. in more detail, we train deeprc using adam (kingma & ba, 2014) with a batch size of 4 and dropout of input sequences. implementation. to reduce computational time, the attention network first computes the attention weights a i for each sequence s i in a repertoire. subsequently, the top 10% of sequences with the highest a i per repertoire are used to compute the weight updates and prediction. furthermore, computation of z i is performed in 16-bit, others in 32-bit precision to ensure numerical stability in the softmax. see sect. a2 for details. in this section, we report and analyze the predictive power of deeprc and the compared methods on several immunosequencing datasets. the roc-auc is used as the main metric for the predictive power. methods compared. we compared previous methods for immune repertoire classification, (ostmeyer et al., 2019) ("log. mil (kmer)", "log. mil (tcrb)") and a burden test (emerson et al., 2017) , as well as the baseline methods logistic regression ("log. regr."), k-nearest neighbour ("knn"), and support vector machines ("svm") with kernels designed for sets, such as the jaccard kernel ("j") and the minmax ("mm") kernel (ralaivola et al., 2005) . for the simulated data, we also added baseline methods that search for the implanted motif either in binary or continuous fashion ("known motif b.", "known motif c.") assuming that this motif was known (for details, see sect. a4). datasets. we aimed at constructing immune repertoire classification scenarios with varying degree of difficulties and realism in order to compare and analyze the suggested machine learning methods. to this end, we either use simulated or experimentally-observed immune receptor sequences and we implant signals, specifically, sequence motifs or sets thereof weber et al., 2020) , at different frequencies into sequences of repertoires of the positive class. these frequencies represent the witness rates and range from 0.01% to 10%. overall, we compiled four categories of datasets: (a) simulated immunosequencing data with implanted signals, (b) lstm-generated immunosequencing data with implanted signals, (c) real-world immunosequencing data with implanted signals, and (d) real-world immunosequencing data with known immune status, the cmv dataset (emerson et al., 2017) . the average number of instances per bag, which is the number of sequences per immune repertoire, is ≈300,000 except for category (c), in which we consider the scenario of low-coverage data with only 10,000 sequences per repertoire. the number of repertoires per dataset ranges from 785 to 5,000. in total, all datasets comprise ≈30 billion sequences or instances. this represents the largest comparative study on immune repertoire classification (see sect. a3). hyperparameter selection. we used a nested 5-fold cross validation (cv) procedure to estimate the performance of each of the methods. all methods could adjust their most important hyperparameters on a validation set in the inner loop of the procedure. see sect. a5 for details. table 1 : results in terms of auc of the competing methods on all datasets. the reported errors are standard deviations across 5 cross-validation (cv) folds (except for the column "simulated"). real-world cmv: average performance over 5 cv folds on the cmv dataset (emerson et al., 2017) . real-world data with implanted signals: average performance over 5 cv folds for each of the four datasets. a signal was implanted with a frequency (=witness rate) of 1% or 0.1%. either a single motif ("om") or multiple motifs ("mm") were implanted. lstm-generated data: average performance over 5 cv folds for each of the 5 datasets. in each dataset, a signal was implanted with a frequency of 10%, 1%, 0.5%, 0.1%, or 0.05%, respectively. simulated: here we report the mean over 18 simulated datasets with implanted signals and varying difficulties (see tab. a9 for details). the error reported is the standard deviation of the auc values across the 18 datasets. results. in each of the four categories, "real-world data", "real-world data with implanted signals", "lstm-generated data", and "simulated immunosequencing data", deeprc outperforms all competing methods with respect to average auc. across categories, the runner-up methods are either the svm for mil problems with minmax kernel or the burden test (see table 1 and sect. a6). results on simulated immunosequencing data. in this setting the complexity of the implanted signal is in focus and varies throughout 18 simulated datasets (see sect. a3). some datasets are challenging for the methods because the implanted motif is hidden by noise and others because only a small fraction of sequences carries the motif, and hence have a low witness rate. these difficulties become evident by the method called "known motif binary", which assumes the implanted motif is known. the performance of this method ranges from a perfect auc of 1.000 in several datasets to an auc of 0.532 in dataset '17' (see sect. a6). deeprc outperforms all other methods with an average auc of 0.846 ± 0.223, followed by the svm with minmax kernel with an average auc of 0.827 ± 0.210 (see sect. a6). the predictive performance of all methods suffers if the signal occurs only in an extremely small fraction of sequences. in datasets, in which only 0.01% of the sequences carry the motif, all auc values are below 0.550. results on lstm-generated data. on the lstm-generated data, in which we implanted noisy motifs with frequencies of 10%, 1%, 0.5%, 0.1%, and 0.05%, deeprc yields almost perfect predictive performance with an average auc of 1.000 ± 0.001 (see sect. a6 and a7). the second best method, svm with minmax kernel, has a similar predictive performance to deeprc on all datasets but the other competing methods have a lower predictive performance on datasets with low frequency of the signal (0.05%). results on real-world data with implanted motifs. in this dataset category, we used real immunosequences and implanted single or multiple noisy motifs. again, deeprc outperforms all other methods with an average auc of 0.980 ± 0.029, with the second best method being the burden test with an average auc of 0.883 ± 0.170. notably, all methods except for deeprc have difficulties with noisy motifs at a frequency of 0.1% (see tab. a11) . results on real-world data. on the real-world dataset, in which the immune status of persons affected by the cytomegalovirus has to be predicted, the competing methods yield predictive aucs between 0.515 and 0.825 (see table 1 ). we note that this dataset is not the exact dataset that was used in emerson et al. (2017) . it differs in pre-processing and also comprises a different set of samples and a smaller training set due to the nested 5-fold cross-validation procedure, which leads to a more challenging dataset. the best performing method is deeprc with an auc of 0.831 ± 0.002, followed by the svm with minmax kernel (auc 0.825 ± 0.022) and the burden test with an auc of 0.699 ± 0.041. the top-ranked sequences by deeprc significantly correspond to those detected by emerson et al. (2017) , which we tested by a mann-whitney u-test with the null hypothesis that the attention values of the sequences detected by emerson et al. (2017) would be equal to the attention values of the remaining sequences (p-value of 1.3 · 10 −93 ). the sequence attention values are displayed in tab. a14. we have demonstrated how modern hopfield networks and attention mechanisms enable successful classification of the immune status of immune repertoires. for this task, methods have to identify the discriminating sequences amongst a large set of sequences in an immune repertoire. specifically, even motifs within those sequences have to be identified. we have shown that deeprc, a modern hopfield network and an attention mechanism with a fixed query, can solve this difficult task despite the massive number of instances. deeprc furthermore outperforms the compared methods across a range of different experimental conditions. impact on machine learning and related scientific fields. we envision that with (a) the increasing availability of large immunosequencing datasets (kovaltsuk et al., 2018; corrie et al., 2018; christley et al., 2018; zhang et al., 2020; rosenfeld et al., 2018; shugay et al., 2018) , (b) further fine-tuning of ground-truth benchmarking immune receptor datasets (weber et al., 2020; olson et al., 2019; marcou et al., 2018) , (c) accounting for repertoire-impacting factors such as age, sex, ethnicity, and environment (potential confounding factors), and (d) increased gpu memory and increased computing power, it will be possible to identify discriminating immune receptor motifs for many diseases, potentially even for the current sars-cov-2 (covid-19) pandemic minervina et al., 2020; galson et al., 2020) . such results would greatly benefit ongoing research on antibody and tcr-driven immunotherapies and immunodiagnostics as well as rational vaccine design (brown et al., 2019) . in the course of this development, the experimental verification and interpretation of machine-learningidentified motifs could receive additional focus, as for most of the sequences within a repertoire the corresponding antigen is unknown. nevertheless, recent technological breakthroughs in highthroughput antigen-labeled immunosequencing are beginning to generate large-scale antigen-labeled single-immune-receptor-sequence data thus resolving this longstanding problem (setliff et al., 2019) . from a machine learning perspective, the successful application of deeprc on immune repertoires with their large number of instances per bag might encourage the application of modern hopfield networks and attention mechanisms on new, previously unsolved or unconsidered, datasets and problems. impact on society. if the approach proves itself successful, it could lead to faster testing of individuals for their immune status w.r.t. a range of diseases based on blood samples. this might motivate changes in the pipeline of diagnostics and tracking of diseases, e.g. automated testing of the immune status in regular intervals. it would furthermore make the collection and screening of blood samples for larger databases more attractive. in consequence, the improved testing of immune statuses might identify individuals that do not have a working immune response towards certain diseases to government or insurance companies, which could then push for targeted immunisation of the individual. similarly to compulsory vaccination, such testing for the immune status could be made compulsory by governments, possibly violating privacy or personal self-determination in exchange for increased over-all health of a population. ultimately, if the approach proves itself successful, the insights gained from the screening of individuals that have successfully developed resistances against specific diseases could lead to faster targeted immunisation, once a certain number of individuals with resistances can be found. this might strongly decrease the harm done by e.g. pandemics and lead to a change in the societal perception of such diseases. consequences of failures of the method. as common with methods in machine learning, potential danger lies in the possibility that users rely too much on our new approach and use it without reflecting on the outcomes. however, the full pipeline in which our method would be used includes wet lab tests after its application, to verify and investigate the results, gain insights, and possibly derive treatments. failures of the proposed method would lead to unsuccessful wet lab validation and negative wet lab tests. since the proposed algorithm does not directly suggest treatment or therapy, human beings are not directly at risk of being treated with a harmful therapy. substantial wet lab and in-vitro testing and would indicate wrong decisions by the system. leveraging of biases in the data and potential discrimination. as for almost all machine learning methods, confounding factors, such as age or sex, could be used for classification. this, might lead to biases in predictions or uneven predictive performance across subgroups. as a result, failures in the wet lab would occur (see paragraph above). moreover, insights into the relevance of the confounding factors could be gained, leading to possible therapies or counter-measures concerning said factors. furthermore, the amount of data available with respec to relevant confounding factors could lead to better or worse performance of our method. e.g. a dataset consisting mostly of data from individuals within a specific age group might yield better performance for that age group, possibly resulting in better or exclusive treatment methods for that specific group. here again, the application of deeprc would be followed by in-vitro testing and development of a treatment, where all target groups for the treatment have to be considered accordingly. all datasets and code is available at https://github.com/ml-jku/deeprc. the cmv dataset is publicly available at https://clients.adaptivebiotech.com/pub/emerson-2017-natgen. in section a2 we provide details on the architecture of deeprc, in section a3 we present details on the datasets, in section a4 we explain the methods that we compared, in section a5 we elaborate on the hyperparameters and their selection process. then, in section a6 we present detailed results for each dataset category in tabular form, in section a7 we provide information on the lstm model that was used to generate antibody sequences, in section a8 we show how deeprc can be interpreted, in section a9 we show the correspondence of previously identified tcr sequences for cmv immune status with attention values by deeprc, and finally we present variations and an ablation study of deeprc in section a10. input layer. for the input layer of the cnn, the characters in the input sequence, i.e. the amino acids (aas), are encoded in a one-hot vector of length 20. to also provide information about the position of an aa in the sequence, we add 3 additional input features with values in range [0, 1] to encode the position of an aa relative to the sequence. these 3 positional features encode whether the aa is located at the beginning, the center, or the end of the sequence, respectively, as shown in figure a1 . we concatenate these 3 positional features with the one-hot vector of aas, which results in a feature vector of size 23 per sequence position. each repertoire, now represented as a bag of feature vectors, is then normalized to unit variance. since the cytomegalovirus dataset (cmv dataset) provides sequences with an associated abundance value per sequence, which is the number of occurrences of a sequence in a repertoire, we incorporate this information into the input of deeprc. to this end, the one-hot aa features of a sequence are multiplied by a scaling factor of log(c a ) before normalization, where c a is the abundance of a sequence. we feed the sequences with 23 features per position into the cnn. sequences of different lengths were zero-padded to the maximum sequence length per batch at the sequence ends. 1d cnn for motif recognition. in the following, we describe how deeprc identifies patterns in the individual sequences and reduces each sequence in the input object to a fixed-size feature vector. deeprc employs 1d convolution layers to extract patterns, where trainable weight kernels are convolved over the sequence positions. in principle, also recurrent neural networks (rnns) or transformer networks could be used instead of 1d cnns, however, (a) the computational complexity of the network must be low to be able to process millions of sequences for a single update. additionally, (b) the learned network should be able to provide insights in the recognized patterns in form of motifs. both properties (a) and (b) are fulfilled by 1d convolution operations that are used by deeprc. we use one 1d cnn layer (hu et al., 2014) with selu activation functions (klambauer et al., 2017) to identify the relevant patterns in the input sequences with a computationally light-weight operation. the larger the kernel size, the more surrounding sequence positions are taken into account, which influences the length of the motifs that can be extracted. we therefore adjust the kernel size during hyperparameter search. in prior works (ostmeyer et al., 2019) , a k-mer size of 4 yielded good predictive performance, which could indicate that a kernel size in the range of 4 may be a proficient choice. for d v trainable kernels, this produces a feature vector of length d v at each sequence position. subsequently, global max-pooling over all sequence positions of a sequence reduces the sequence-representations z i to vectors of the fixed length d v . given the challenging size of the input data per repertoire, the computation of the cnn activations and weight updates is performed using 16-bit floating point values. a list of hyperparameters evaluated for deeprc is given in table a3 . a comparison of rnn-based and cnn-based sequence embedding for motif recognition in a smaller experimental setting is given in sec. a10. regularization. we apply random and attention-based subsampling of repertoire sequences to reduce over-fitting and decrease computational effort. during training, each repertoire is subsampled to 10, 000 input sequences, which are randomly drawn from the respective repertoire. this can also be interpreted as random drop-out (hinton et al., 2012) on the input sequences or attention weights. during training and evaluation, the attention weights computed by the attention network are furthermore used to rank the input sequences. based on this ranking, the repertoire is reduced to the 10% of sequences with the highest attention weights. these top 10% of sequences are then used to compute the weight updates and the prediction for the repertoire. additionally, one might employ further regularization techniques, which we only partly investigated further in a smaller experimental setting in sec. a10 due to high computational demands. such regularization techniques include l1 and l2 weight decay, noise in the form of random aa permutations in the input sequences, noise on the attention weights, or random shuffling of sequences between repertoires that belong to the negative class. the last regularization technique assumes that the sequences in positive-class repertoires carry a signal, such as an aa motif corresponding to an immune response, whereas the sequences in negative-class repertoires do not. hence, the sequences can be shuffled randomly between negative class repertoires without obscuring the signal in the positive class repertoires. hyperparameters. for the hyperparameter search of deeprc for the category "simulated immunosequencing data", we only conducted a full hyperparameter search on the more difficult datasets with motif implantation probabilities below 1%, as described in table a3 . this process was repeated for all 5 folds of the 5-fold cross-validation (cv) and the average score on the 5 test sets constitutes the final score of a method. table a3 provides an overview of the hyperparameter search, which was conducted as a grid search for each of the datasets in a nested 5-fold cv procedure, as described in section a4. computation time and optimization. we took measures on the implementation level to address the high computational demands, especially gpu memory consumption, in order to make the large number of experiments feasible. we train the deeprc model with a small batch size of 4 samples and perform computation of inference and updates of the 1d cnn using 16-bit floating point values. the rest of the network is trained using 32-bit floating point values. the adam parameter for numerical stability was therefore increased from the default value of = 10 −8 to = 10 −4 . training was performed on various gpu types, mainly nvidia rtx 2080 ti. computation times were highly dependent on the number of sequences in the repertoires and the number and sizes of cnn kernels. a single update on an nvidia rtx 2080 ti gpu took approximately 0.0129 to 0.0135 seconds, while requiring approximately 8 to 11 gb gpu memory. taking these optimizations and gpus with larger memory (≥ 16 gb) into account, it is already possible to train deeprc, possibly with multi-head attention and a larger network architecture, on larger datasets (see sec. a10). our network implementation is based on pytorch 1.3.1 (paszke et al., 2019) . incorporation of additional inputs and metadata. additional metadata in the form of sequencelevel or repertoire-level features could be incorporated into the input via concatenation with the feature vectors that result from taking the maximum of the 1d cnn outputs w.r.t. the sequence positions. this has the benefit that the attention mechanism and output network can utilize the sequence-level or repertoire-level features for their predictions. sparse metadata or metadata that is only available during training could be used as auxiliary targets to incorporate the information via gradients into the deeprc model. limitations. the current methods are mostly limited by computational complexity, since both hyperparameter and model selection is computationally demanding. for hyperparameter selection, a large number of hyperparameter settings have to be evaluated. for model selection, a single repertoire requires the propagation of many thousands of sequences through a neural network and keeping those quantities in gpu memory in order to perform the attention mechanism and weight update. thus, increased gpu memory would significantly boost our approach. increased computational power would also allow for more advanced architectures and attention mechanisms, which may further improve predictive performance. another limiting factor is over-fitting of the model due to the currently relatively small number of samples (bags) in real-world immunosequencing datasets in comparison to the large number of instances per bag and features per instance. we aimed at constructing immune repertoire classification scenarios with varying degree of realism and difficulties in order to compare and analyze the suggested machine learning methods. to this end, we either use simulated or experimentally-observed immune receptor sequences and we implant signals, which are sequence motifs weber et al., 2020) , into sequences of repertoires of the positive class. it has been shown previously that interaction of immune receptors with antigens occur via short sequence stretches . thus, implantation of short motif sequences simulating an immune signal is biologically meaningful. our benchmarking study comprises four different categories of datasets: (a) simulated immunosequencing data with implanted signals (where the signal is defined as sets of motifs), (b) lstm-generated immunosequencing data with implanted signals, (c) real-world immunosequencing data with implanted signals, and (d) real-world immunosequencing data. each of the first three categories consists of multiple datasets with varying difficulty depending on the type of the implanted signal and the ratio of sequences with the implanted signal. the ratio of sequences with the implanted signal, where each sequence carries at most 1 implanted signal, corresponds to the witness rate (wr). we consider binary classification tasks to simulate the immune status of healthy and diseased individuals. we randomly generate immune repertoires with varying numbers of sequences, where we implant sequence motifs in the repertoires of the diseased individuals, i.e. the positive class. the sequences of a repertoire are also randomly generated by different procedures (detailed below). each sequence is composed of 20 different characters, corresponding to amino acids, and has an average length of 14.5 aas. in the first category, we aim at investigating the impact of the signal frequency, i.e. the wr, and the signal complexity on the performance of the different methods. to this end, we created 18 datasets, whereas each dataset contains a large number of repertoires with a large number of random aa sequences per repertoire. we then implanted signals in the aa sequences of the positive class repertoires, where the 18 datasets differ in frequency and complexity of the implanted signals. in detail, the aas were sampled randomly independent of their respective position in the sequence, while the frequencies of aas, distribution of sequence lengths, and distribution of the number of sequences per repertoire, i.e. the number of instances per bag, are following the respective distributions observed in the real-world cmv dataset (emerson et al., 2017) . for this, we first sampled the number of sequences for a repertoire from a gaussian n (µ = 316k, σ = 132k) distribution and rounded to the nearest positive integer. we re-sampled if the size was below 5k. we then generated random sequences of aas with a length of n (µ = 14.5, σ = 1.8), again rounded to the nearest positive integers. each simulated repertoire was then randomly assigned to either the positive or negative class, with 2, 500 repertoires per class. in the repertoires assigned to the positive class, we implanted motifs with an average length of 4 aas, following the results of the experimental analysis of antigenbinding motifs in antibodies and t-cell receptor sequences by . we varied the characteristics of the implanted motifs for each of the 18 datasets with respect to the following parameters: (a) ρ, the probability of a motif being implanted in a sequence of a positive repertoire, i.e. the average ratio of sequences containing the motif, which is the witness rate. in this way, we generated 18 different datasets of variable difficulty containing in total roughly 28.7 billion sequences. see table a1 for an overview of the properties of the implanted motifs in the 18 datasets. in the second dataset category, we investigate the impact of the signal frequency and complexity in combination with more plausible immune receptor sequences by taking into account the positional aa distributions and other sequence properties. to this end, we trained an lstm (hochreiter & schmidhuber, 1997 ) in a standard next character prediction (graves, 2013) setting to create aa sequences with properties similar to experimentally observed immune receptor sequences. in the first step, the lstm model was trained on all immuno-sequences in the cmv dataset (emerson et al., 2017) that contain valid information about sequence abundance and have a known cmv label. such an lstm model is able to capture various properties of the sequences, including positiondependent probability distributions and combinations, relationships, and order of aas. we then used the trained lstm model to generate 1, 000 repertoires in an autoregressive fashion, starting with a start sequence that was randomly sampled from the trained-on dataset. based on a visual inspection of the frequencies of 4-mers (see section a7), the similarity of lstm generated sequences and real sequences was deemed sufficient for the purpose of generating the aa sequences for the datasets in this category. further details on lstm training and repertoire generation are given in section a7. after generation, each repertoire was assigned to either the positive or negative class, with 500 repertoires per class. we implanted motifs of length 4 with varying properties in the center of the sequences of the positive class to obtain 5 different datasets. each sequence in the positive repertoires has a probability ρ to carry the motif, which was varied throughout 5 datasets and corresponds to the wr (see table a1 ). each position in the motif has a probability of 0.9 to be implanted and consequently a probability of 0.1 that the original aa in the sequence remains, which can be seen as noise on the motif. in the third category, we implanted signals into experimentally obtained immuno-sequences, where we considered 4 dataset variations. each dataset consists of 750 repertoires for each of the two classes, where each repertoire consists of 10k sequences. in this way, we aim to simulate datasets with a low sequencing coverage, which means that only relatively few sequences per repertoire are available. the sequences were randomly sampled from healthy (cmv negative) individuals from the cmv dataset (see below paragraph for explanation). two signal types were considered: (a) one signal with one motif. the aa motif ldr was implanted in a certain fraction of sequences. the pattern is randomly altered at one of the three positions with probabilities 0.2, 0.6, and 0.2, respectively. (b) one signal with multiple motifs. one of the three possible motifs ldr, cas, and gl-n was table a1 : properties of simulated repertoires, variations of motifs, and motif frequencies, i.e. the witness rate, for the datasets in categories "simulated immunosequencing data", "lstm-generated data", and "real-world data with implanted signals". noise types for * are explained in paragraph "real-world data with implanted signals". implanted with equal probability. again, the motifs were randomly altered before implantation. the aa motif ldr changed as described above. the aa motif cas was altered at the second position with probability 0.6 and with probability 0.3 at the first position. the pattern gl-n, wheredenotes a gap location, is randomly altered at the first position with probability 0.6 and the gap has a length of 0, 1, or 2 aas with equal probability. additionally, the datasets differ in the values for ρ, the average ratio of sequences carrying a signal, which were chosen as 1% or 0.1%. the motifs were implanted at positions 107, 109, and 114 according to the imgt numbering scheme for immune receptor sequences (lefranc et al., 2003) with probabilities 0.3, 0.35 and 0.2, respectively. with the remaining 0.15 chance, the motif is implanted at any other sequence position. this means that the motif occurrence in the simulated sequences is biased towards the middle of the sequence. we used a real-world dataset of 785 repertoires, each of which containing between 4, 371 to 973, 081 (avg. 299, 319) tcr sequences with a length of 1 to 27 (avg. 14.5) aas, originally collected and provided by emerson et al. (2017) . 340 out of 785 repertoires were labelled as positive for cytomegalovirus (cmv) serostatus, which we consider as the positive class, 420 repertoires with negative cmv serostatus, considered as negative class, and 25 repertoires with unknown status. we changed the number of sequence counts per repertoire from −1 to 1 for 3 sequences. furthermore, we exclude a total of 99 repertoires with unknown cmv status or unknown information about the sequence abundance within a repertoire, reducing the dataset for our analysis to 686 repertoires, 312 of which with positive and 374 with negative cmv status. we give a non-exhaustive overview of previously considered mil datasets and problems in table a2 . to our knowledge the datasets considered in this work pose the most challenging mil problems with respect to the number of instances per bag (column 5). table a2 : mil datasets with their numbers of bags and numbers of instances. "total number of instances" refers to the total number of instances in the dataset. the simulated and real-world immunosequencing datasets considered in this work contain a by orders of magnitudes larger number of instances per bag than mil datasets that were considered by machine learning methods up to now. we evaluate and compare the performance of deeprc against a set of machine learning methods that serve as baseline, were suggested, or can readily be adapted to immune repertoire classification. in this section, we describe these compared methods. this method serves as an estimate for the achievable classification performance using prior knowledge about which motif was implanted. note that this does not necessarily lead to perfect predictive performance since motifs are implanted with a certain amount of noise and could also be present in the negative class by chance. the known motif method counts how often the known implanted motif occurs per sequence for each repertoire and uses this count to rank the repertoires. from this ranking, the area under the receiver operator curve (auc) is computed as performance measure. probabilistic aa changes in the known motif are not considered for this count, with the exception of gap positions. we consider two versions of this method: (a) known motif binary: counts the occurrence of the known motif in a sequence and (b) known motif continuous: counts the maximum number of overlapping aas between the known motif and all sequence positions, which corresponds to a convolution operation with a binary kernel followed by max-pooling. since the implanted signal is not known in the experimentally obtained cmv dataset, this method cannot be applied to this dataset. the support vector machine (svm) approach uses a fixed mapping from a bag of sequences to the corresponding k-mer counts. the function h kmer maps each sequence s i to a vector representing the occurrence of k-mers in the sequence. to avoid confusion with the sequence-representation obtained from the cnn layers of deeprc, we denote u i = h kmer (s i ), which is analogous to z i . specifically, where #{p m ∈ s i } denotes how often the k-mer pattern p m occurs in sequence s i . afterwards, average-pooling is applied to obtain u = 1/n n i=1 u i , the k-mer representation of the input object x. for two input objects x (n) and x (l) with representations u (n) and u (l) , respectively, we implement the minmax kernel (ralaivola et al., 2005) as follows: where u (n) m is the m-th element of the vector u (n) . the jaccard kernel (levandowsky & winter, 1971 ) is identical to the minmax kernel except that it operates on binary u (n) . we used a standard c-svm, as introduced by cortes & vapnik (1995) . the corresponding hyperparameter c is optimized by random search. the settings of the full hyperparameter search as well as the respective value ranges are given in table a4a . the same k-mer representation of a repertoire, as introduced above for the svm baseline, is used for the k-nearest neighbor (knn) approach. as this method clusters samples according to distances between them, the previous kernel definitions cannot be applied directly. it is therefore necessary to transform the minmax as well as the jaccard kernel from similarities to distances by constructing the following (levandowsky & winter, 1971) : d jaccard (u (n) , u (l) ) = 1 − k jaccard (u (n) , u (l) ). (a2) the amount of neighbors is treated as the hyperparameter and optimized by an exhaustive grid search. the settings of the full hyperparameter search as well as the respective value ranges are given in table a5 . we implemented logistic regression on the k-mer representation u of an immune repertoire. the model is trained by gradient descent using the adam optimizer (kingma & ba, 2014) . the learning rate is treated as the hyperparameter and optimized by grid search. furthermore, we explored two regularization settings using combinations of l1 and l2 weight decay. the settings of the full hyperparameter search as well as the respective value ranges are given in table a6 . we implemented a burden test (emerson et al., 2017; li & leal, 2008; wu et al., 2011) in a machine learning setting. the burden test first identifies sequences or k-mers that are associated with the individual's class, i.e., immune status, and then calculates a burden score per individual. concretely, for each k-mer or sequence, the phi coefficient of the contingency table for absence or presence and positive or negative immune status is calculated. then, j k-mers or sequences with the highest phi coefficients are selected as the set of associated k-mers or sequences. j is a hyperparameter that is selected on a validation set. additionally, we consider the type of input features, sequences or k-mers, as a hyperparameter. for inference, a burden score per individual is calculated as the sum of associated k-mers or sequences it carries. this score is used as raw prediction and to rank the individuals. hence, we have extended the burden test by emerson et al. (2017) to k-mers and to adaptive thresholds that are adjusted on a validation set. the logistic multiple instance learning (mil) approach for immune repertoire classification (ostmeyer et al., 2019) applies a logistic regression model to each k-mer representation in a bag. the resulting scores are then summarized by max-pooling to obtain a prediction for the bag. each amino acid of each k-mer is represented by 5 features, the so-called atchley factors (atchley et al., 2005) . as k-mers of length 4 are used, this gives a total of 4 × 5 = 20 features. one additional feature per 4-mer is added, which represents the relative frequency of this 4-mer with respect to its containing bag, resulting in 21 features per 4-mer. two options for the relative frequency feature exist, which are (a) whether the frequency of the 4-mer ("4mer") or (b) the frequency of the sequence in which the 4-mer appeared ("tcrβ") is used. we optimized the learning rate, batch size, and early stopping parameter on the validation set. the settings of the full hyperparameter search as well as the respective value ranges are given in table a8 . for all competing methods a hyperparameter search was performed, for which we split each of the 5 training sets into an inner training set and inner validation set. the models were trained on the inner training set and evaluated on the inner validation set. the model with the highest auc score on the inner validation set is then used to calculate the score on the respective test set. here we report the hyperparameter sets and search strategy that is used for all methods. deeprc. the set of hyperparameters of deeprc is shown in table a3 . these hyperparameter combinations are adjusted via a grid search procedure. table a3 : deeprc hyperparameter search space. every 5 · 10 3 updates, the current model was evaluated against the validation fold. the early stopping hyperparameter was determined by selecting the model with the best loss on the validation fold after 10 5 updates. * : experiments for {64; 128; 256} kernels were omitted for datasets with motif implantation probabilities ≥ 1% in the category "simulated immunosequencing data". known motif. this method does not have hyperparameters and has been applied to all datasets except for the cmv dataset. the corresponding hyperparameter c of the svm is optimized by randomly drawing 10 3 values in the range of [−6; 6] according to a uniform distribution. these values act as the exponents of a power of 10 and are applied for each of the two kernel types (see table a4a ). knn. the amount of neighbors is treated as the hyperparameter and optimized by grid search operating in the discrete range of [1; max{n, 10 3 }] with a step size of 1. the corresponding tight upper bound is automatically defined by the total amount of samples n ∈ n >0 in the training set, capped at 10 3 (see table a5 ). number of neighbors {1; max{n, 10 3 }} type of kernel {minmax; jaccard} table a5 : settings used in the hyperparameter search of the knn baseline approach. the number of trials (per type of kernel) is automatically defined by the total amount of samples n ∈ n >0 in the training set, capped at 10 3 . logistic regression. the hyperparameter optimization strategy that was used was grid search across hyperparameters given in table a6. learning rate 10 −{2;3;4} batch size 4 max. updates 10 5 coefficient β 1 (adam) 0.9 coefficient β 2 (adam) 0.999 weight decay weightings {(l1 = 10 −7 , l2 = 10 −3 ); (l1 = 10 −7 , l2 = 10 −5 )} table a6 : settings used in the hyperparameter search of the logistic regression baseline approach. burden test. the burden test selects two hyperparameters: the number of features in the burden set and the type of features, see table a7 . number of features in burden set {50, 100, 150, 250} type of features {4mer; sequence} table a7 : settings used in the hyperparameter search of the burden test approach. logistic mil. for this method, we adjusted the learning rate as well as the batch size as hyperparameters by randomly drawing 25 different hyperparameter combinations from a uniform distribution. the corresponding range of the learning rate is [−4.5; −1.5], which acts as the exponent of a power of 10. the batch size lies within the range of [1; 32]. for each hyperparameter combination, a model is optimized by gradient descent using adam, whereas the early stopping parameter is adjusted according to the corresponding validation set (see table a8 ). learning rate 10 {−4.5;−1.5} batch size {1; 32} relative abundance term {4mer; tcrβ} number of trials 25 max. epochs 10 2 coefficient β 1 (adam) 0.9 coefficient β 2 (adam) 0.999 table a8 : settings used in the hyperparameter search of the logistic mil baseline approach. the number of trials (per type of relative abundance) defines the quantity of combinations of random values of the learning rate as well as the batch size. in this section, we report the detailed results on all four categories of datasets (a) simulated immunosequencing data (table a9 ) (b) lstm-generated data (table a10) , (c) real-world data with implanted signals (table a11) , and (d) real-world data on the cmv dataset (table a12) , as discussed in the main paper. ± 0.000 ± 0.000 ± 0.271 ± 0.000 ± 0.000 ± 0.218 ± 0.000 ± 0.000 ± 0.029 ± 0.000 ± 0.001 ± 0.017 ± 0.001 ± 0.002 ± 0.023 ± 0.001 ± 0.048 ± 0.013 ± 0.223 svm (minmax) 1.000 1.000 0.764 1.000 1.000 0.603 1.000 0.998 0.539 1.000 0.994 0.529 1.000 0.741 0.513 1.000 0.706 0.503 0.827 ± 0.000 ± 0.000 ± 0.016 ± 0.000 ± 0.000 ± 0.021 ± 0.000 ± 0.002 ± 0.024 ± 0.000 ± 0.004 ± 0.016 ± 0.000 ± 0.024 ± 0.006 ± 0.000 ± 0.013 ± 0.013 ± 0.013 ± 0.013 ± 0.014 ± 0.011 ± 0.009 ± 0.007 ± 0.008 ± 0.011 ± 0.012 ± 0.012 ± 0.007 ± 0.014 ± 0.017 ± 0.010 ± 0.020 ± 0.012 ± 0.016 ± 0.016 ± 0.074 known motif b. 1.000 1.000 0.973 1.000 1.000 0.865 1.000 1.000 0.700 1.000 0.989 0.609 1.000 0.946 0.570 1.000 0.834 0.532 0.890 ± 0.000 ± 0.000 ± 0.004 ± 0.000 ± 0.000 ± 0.004 ± 0.000 ± 0.000 ± 0.020 ± 0.000 ± 0.002 ± 0.017 ± 0.000 ± 0.010 ± 0.024 ± 0.000 ± 0.016 ± 0.020 ± 0.001 ± 0.014 ± 0.020 ± 0.001 ± 0.013 ± 0.017 ± 0.001 ± 0.012 ± 0.012 ± 0.001 ± 0.018 ± 0.018 ± 0.002 ± 0.010 ± 0.009 ± 0.002 ± 0.012 ± 0.013 ± 0.202 table a9 : auc estimates based on 5-fold cv for all 18 datasets in category "simulated immunosequencing data". the reported errors are standard deviations across the 5 cross-validation folds except for the last column "avg.", in which they show standard deviations across datasets. wildcard characters in motifs are indicated by z, characters with 50% probability of being removed by d . table a10 : auc estimates based on 5-fold cv for all 5 datasets in category "lstm-generated data". the reported errors are standard deviations across the 5 cross-validation folds except for the last column "avg.", in which they show standard deviations across datasets. characters affected by noise, as described in a3, paragraph "lstm-generated data", are indicated by r . table a12 : results on the cmv dataset (real-world data) in terms of auc, f1 score, balanced accuracy, and accuracy. for f1 score, balanced accuracy, and accuracy, all methods use their default thresholds. each entry shows mean and standard deviation across 5 cross-validation folds. we trained a conventional next-character lstm model (graves, 2013) based on the implementation in https://github.com/spro/practical-pytorch (access date 1st of may, 2020) using pytorch 1.3.1 (paszke et al., 2019) . for this, we applied an lstm model with 100 lstm blocks in 2 layers, which was trained for 5, 000 epochs using the adam optimizer (kingma & ba, 2014) with learning rate 0.01, an input batch size of 100 character chunks, and a character chunk length of 200. as input we used the immuno-sequences in the cdr3 column of the cmv dataset, where we repeated sequences according to their counts in the repertoires, as specified in the templates column of the cmv dataset. we excluded repertoires with unknown cmv status and unknown sequence abundance from training. after training, we generated 1, 000 repertoires using a temperature value of 0.8. the number of sequences per repertoire was sampled from a gaussian n (µ = 285k, σ = 156k) distribution, where the whole repertoire was generated by the lstm at once. that is, the lstm can base the generation of the individual aa sequences in a repertoire, including the aas and the lengths of the sequences, on the generated repertoire. a random immuno-sequence from the trained-on repertoires was used as initialization for the generation process. this immuno-sequence was not included in the generated repertoire. finally, we randomly assigned 500 of the generated repertoires to the positive (diseased) and 500 to the negative (healthy) class. we then implanted motifs in the positive class repertoires as described in section a3.2. as illustrated in the comparison of histograms given in fig. a2 , the generated immuno-sequences exhibit a very similar distribution of 4-mers and aas compared to the original cmv dataset. real-world data deeprc allows for two forms of interpretability methods. (a) due to its attention-based design, a trained model can be used to compute the attention weights of a sequence, which directly indicates its importance. (b) deeprc furthermore allows for the usage of contribution analysis methods, such as integrated gradients (ig) (sundararajan et al., 2017) or layer-wise relevance propagation (montavon et al., 2018; arras et al., 2019; montavon et al., 2019; preuer et al., 2019) . we apply ig to identify the input patterns that are relevant for the classification. to identify aa patterns with high contributions in the input sequences, we apply ig to the aas in the input sequences. additionally, we apply ig to the kernels of the 1d cnn, which allows us to identify aa motifs with high contributions. in detail, we compute the ig contributions for the aas and positional features in the kernels for every repertoire in the validation and test set, so as to exclude potential artifacts caused by over-fitting. averaging the ig values over these repertoires then results in concise aa motifs. we include qualitative visual analyses of the ig method on different datasets below. here, we provide examples for the interpretation of trained deeprc models using integrated gradients (ig) (sundararajan et al., 2017) as contribution analysis method. the following illustrations were created using 50 ig steps, which we found sufficient to achieve stable ig results. a visual analysis of deeprc models on the simulated datasets, as illustrated in tab. a13 and fig. a3 , shows that the implanted motifs can be successfully extracted from the trained model and are straightforward to interpret. in the real-world cmv dataset, deeprc finds complex patterns with high variability in the center regions of the immuno-sequences, as illustrated in figure a4 . real-world data with implanted signals extracted motif implanted motif(s) g r s r a r f r l r d r r r {l r d r r r ; c r a r s; g r l-n} motif freq. ρ 0.05% 0.1% 0.1% table a13 : visualization of motifs extracted from trained deeprc models for datasets from categories "simulated immunosequencing data", "lstm-generated data", and "real-world data with implanted signals". motif extraction was performed using integrated gradients on the 1d cnn kernels over the validation set and test set repertoires of one cv fold. wildcard characters are indicated by z, random noise on characters by r , characters with 50% probability of being removed by d , and gap locations of random lengths of {0; 1; 2} by -. larger characters in the extracted motifs indicate higher contribution, with blue indicating positive contribution and red indicating negative contribution towards the prediction of the diseased class. contributions to positional encoding are indicated by < (beginning of sequence), ∧ (center of sequence), and > (end of sequence). only kernels with relatively high contributions are shown, i.e. with contributions roughly greater than the average contribution of all kernels. b) c) figure a3 : integrated gradients applied to input sequences of positive class repertoires. three sequences with the highest contributions to the prediction of their respective repertoires are shown. a) input sequence taken from "simulated immunosequencing data" with implanted motif sz d z d n and motif implantation probability 0.1%. the deeprc model reacts to the s and n at the 5 th and 8 th sequence position, thereby identifying the implanted motif in this sequence. b) and c) input sequence taken from "real-world data with implanted signals" with implanted motifs {l r d r r r ; c r a r s; g r l-n} and motif implantation probability 0.1%. the deeprc model reacts to the fully implanted motif cas (b) and to the partly implanted motif aas c and a at the 5 th and 7 th sequence position (c), thereby identifying the implanted motif in the sequences. wildcard characters in implanted motifs are indicated by z, characters with 50% probability of being removed by d , and gap locations of random lengths of {0; 1; 2} by -. larger characters in the sequences indicate higher contribution, with blue indicating positive contribution and red indicating negative contribution towards the prediction of the diseased class. figure a4 : visualization of the contributions of characters within a sequence via ig. each sequence was selected from a different repertoire and showed the highest contribution in its repertoire. the model was trained on cmv dataset, using a kernel size of 9, 32 kernels and 137 repertoires for early stopping. larger characters in the extracted motifs indicate higher contribution, with blue indicating positive contribution and red indicating negative contribution towards the prediction of the disease class. table a14 : tcrβ sequences that had been discovered by emerson et al. (2017) with their associated attention values by deeprc. these sequences have significantly (p-value 1.3e-93) higher attention values than other sequences. the column "quantile" provides the quantile values of the empiricial distribution of attention values across all sequences in the dataset. in this section we investigate the impact of different variations of deeprc on the performance on the cmv dataset. we consider both a cnn-based sequence embedding, as used in the main paper, and an lstm-based sequence embedding. in both cases we vary the number of attention heads and the β parameter for the softmax function the attention mechanism (see eq. 2 in main paper). for the cnn-based sequence embedding we also vary the number of cnn kernels and the kernel sizes used in the 1d cnn. for the lstm-based sequence embedding we use one one-directional lstm layer, of which the output values at the last sequence position (without padding) are taken as embedding of the sequence. here we vary the number of lstm blocks in the lstm layer. to counter over-fitting due to the increased complexity of these deeprc variations, we added a l2 weight penalty to the training loss. the factor with which the l2 weight penalty contributes to the training loss is varied over 3 orders of magnitudes, where suitable value ranges were manually determined on one of the training folds beforehand. to reduce the computational effort, we do not consider all numbers of kernels that were considered in the main paper. furthermore, we only compute the auc scores on 3 of the 5 cross-validation folds. the hyperparameters, which were used in a grid search procedure, are listed in tab. a15 for the cnn-based sequence embedding and tab. a16 for the lstm-based sequence embedding. results. we show performance in terms of auc score with single hyperparameters set to fixed values so as to investigate their influence in tab. a18 for the cnn-based sequence embedding and tab. a17 for the lstm-based sequence embedding. we note that due to restricted computational resources this study was conducted with fewer different numbers of cnn kernels, with the auc estimated from only 3 of the 5 cross-validation folds, which leads to a slight decrease of performance in comparison to the full hyperparameter search and cross-validation procedure used in the main paper. as can be seen in tab. a18 and a17, the lstm-based sequence embedding generalizes slightly better than the cnn-based sequence embedding. table a17 : impact of hyperparameters on deeprc with lstm for sequence encoding. mean ("mean") and standard deviation ("std") for the area under the roc curve over the first 3 folds of a 5-fold nested cross-validation for different sub-sets of hyperparameters ("sub-set") are shown. the following sub-sets were considered: "full": full grid search over hyperparameters; "beta=*": grid search over hyperparameters with reduction to specific value * of beta value of attention softmax; "heads=*": grid search over hyperparameters with reduction to specific number * of attention heads; "lstms=*": grid search over hyperparameters with reduction to specific number * of lstm blocks for sequence embedding. table a18 : impact of hyperparameters on deeprc with 1d cnn for sequence encoding. mean ("mean") and standard deviation ("std") for the area under the roc curve over the first 3 folds of a 5-fold nested cross-validation for different sub-sets of hyperparameters ("sub-set") are shown. the following sub-sets were considered: "full": full grid search over hyperparameters; "beta=*": grid search over hyperparameters with reduction to specific value * of beta value of attention softmax; "heads=*": grid search over hyperparameters with reduction to specific number * of attention heads; "ksize=*": grid search over hyperparameters with reduction to specific kernel size * of 1d cnn kernels for sequence embedding; "kernels=*": grid search over hyperparameters with reduction to specific number * of 1d cnn 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ellis unit linz, the lit ai lab and the institute for machine learning are supported by the land oberösterreich, lit grants deeptoxgen ( in the following, the appendix to the paper "modern hopfield networks and attention for immune key: cord-288945-c9ow1q5c authors: spengler, ulrich title: liver disease associated with non-hepatitis viruses date: 2019-11-01 journal: encyclopedia of gastroenterology doi: 10.1016/b978-0-12-801238-3.65782-3 sha: doc_id: 288945 cord_uid: c9ow1q5c hepatitis is commonly associated with certain viruses labeled as “hepatitis” viruses. however, many other viral infections can also affect the liver ranging from mild asymptomatic elevations of aminotransferases to fulminant hepatic failure. this article will provide a brief overview on a variety of different viral infections that may be associated with significant liver pathology at least under certain conditions, for example, immunosuppression. this overview discusses key virological features, clinical presentation of associated liver disease and provides some information on diagnosis and an outline of treatment options. thus, the overview can provide first orientation when infectious hepatitis is encountered in a patient that cannot be explained by the usual hepatitis viruses. the liver is a highly perfused organ and the first filter for blood coming from the intestines. thus, the liver is particular prone to become involved in blood-borne infections. apart from the so-called hepatitis viruses, many other viruses, primarily targeting other extrahepatic tissues, also lead to liver damage and hepatitis. damage can range from asymptomatic elevations of aminotransferases to fulminant hepatic failure depending on the virus and the host's immune response. when the immune system controls infection poorly, direct infection of hepatocytes and liver necrosis may occur. this situation applies to patients under severe immunosuppression or infections with particularly virulent agents such as the viruses that cause hemorrhagic fevers. alternatively, liver cells may become victims of collateral damage without direct infection when cytolytic cd8 þ t effector lymphocytes are expanded outside the liver and then recruited via liver-resident macrophages such as kupffer cells presenting viral antigens (polakos et al., 2006; schumann et al., 2000) . this type of liver damage is particularly associated with respiratory viruses such as influenza virus. however, since many viral infections expand cd8þ t lymphocytes, this by-stander mechanism may affect the liver more often (murali-krishna et al., 1998) . here, we provide a brief overview over viral infections primarily not designated as hepatitis viruses which may lead to liver disease and hepatic complications. this overview will largely focus on the hepatic aspects of viral infections. thus, readers interested in more detailed information should consult a comprehensive textbook in microbiology or clinical infectious diseases. approximately 8% of travelers to the developing world require medical care during or after travel, and fever is the underlying problem in 28% of them (wilson et al., 2007) . physicians evaluating returned travelers frequently suspect rare or exotic diagnoses. although exotic diseases, in particular viral hemorrhagic fevers are a severe threat in certain regions of the world, liver disease due to exotic infections such as ebola virus, rift valley fever or lassa fever have been reported only sporadically but do not represent a frequent health problem in returning travelers. viral hemorrhagic fevers share some epidemiological and clinical features and cause rather similar liver pathology. most viruses are transmitted via arthropod vectors. the viruses cause small vessel damage in multiple organs resulting in overt hemorrhage. the spectrum of diseases and their geographical distribution are listed in table 1 . much attention has been paid to abnormal liver function and altered hepatic pathology. nevertheless, clinically significant liver disease or death from liver failure are rare complications except in yellow fever. dengue fever is among the top three etiological agents, accounting for approximately 6% of febrile illnesses in the traveler (wilson et al., 2007) . of note, although malaria is the leading cause of systemic febrile illness worldwide, apart from sub-saharan africa and central america travelers returning from tropical or sub-tropical regions had dengue fever more frequently than malaria. chikungunya fever is an emerging novel virus infection recently expanding in asia and africa, which also causes fever, myalgia, arthralgia and skin rash in increasing numbers of patients. table 1 viral hemorrhagic fevers affecting the liver the dengue virus complex comprises four antigenically related but distinct flaviviruses termed dengue virus serotypes 1 through 4 (den-1 to . dengue viruses are transmitted by aedes aegypti mosquitos in epidemic and endemic outbreaks and cause acute infections. three to six days after a mosquito bite the virus spreads via the blood-stream, and among the various organs it can be isolated frequently from liver samples (rosen and khin, 1989) . dengue virus infection usually causes a flu-like illness with a rash-dengue fever (fig. 1 ). hepatomegaly and elevated serum aminotransferases, which are usually mild, are common in dengue virus infections (wahid et al., 2000) . clinically more severe diseases, for example, dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) can follow from secondary infection with dengue virus of different serotype. in 2009, the world health organization issued new guidelines and introduced a revised classification scheme consisting of the following categories: dengue without warning signs, dengue with warning signs, and severe dengue (world health organization, 2009 ). however, there are no reliable warning signs for severe dengue. in dhf there are widespread petechial hemorrhages together with multiple organ damage; in dss, which mostly affects children below the age of 15, there is extensive capillary leakage and severe fluid depletion leading to hypovolemic shock. if untreated, mortality approaches 50%. in fatal cases of dhf the liver is enlarged, pale from steatosis and shows multifocal hemorrhages. laboratory diagnosis of dengue virus infection is done by detection of viral components, for example, pcr in serum or indirectly by serology. the sensitivity of each approach depends on the duration and course of the patient's illness when the patient presents for evaluation. currently vaccines against dengue virus are being developed but have not become licensed, yet. there is no direct antiviral therapy available against the dengue viruses. thus, treatment is supportive, requiring meticulous fluid management and intensive care in dss. details for treatment are summarized in recent who guidelines (world health organization, 2012). chikungunya is an arthropod-borne toga virus initially endemic to west africa, which next spread to the indian ocean islands and southeast asia (charrel et al., 2007) . chikungunya fever was originally considered a disease of tropical and subtropical regions, until an outbreak in northern italy was recorded in 2007 (rezza et al., 2007) . since then chikungunya infections have become exported to other western countries, the americas and caribbean region via international travel. of note, dengue and zika viruses are transmitted by the same mosquito vectors as chikungunya. thus, the viruses can co-circulate in the same geographic region, and coinfections have been documented. transmission of chikungunya via blood products has been reported in france, where a nurse was infected by exposure to blood from an infected patient. chikungunya, might also be transmitted inadvertently by organ transplantation since viremia can exceed high levels prior to onset of symptoms. the infection begins abruptly with high fever, symmetric polyarthralgia and macular or maculopapular skin rash (taubitz et al., 2007) . pruritus and bullous skin lesions have also been described. previously chikungunya fever has been considered a self-limited disease. however, severe complications also comprising acute viral hepatitis and deaths have been reported in the recent outbreaks; particularly in elderly patients (> 65 years) and people with pre-existing chronic medical problems. some patients have persisting symptoms for a variable length of time after the acute illness. manifestations include arthritis/arthralgia, edematous polyarthritis of fingers and toes, morning pain and stiffness, and severe tendosynovitis. clinically chikungunya fever must be differentiated from dengue fever, which shares many symptoms and features. the diagnosis of chikungunya virus infection should be suspected in a patient with acute onset of fever and polyarthralgia who had possible exposure by travel to or residency in an epidemiological risk area. the diagnosis of chikungunya is established by detection of chikungunya viral rna via real-time reverse-transcription polymerase chain reaction (rt-pcr) or chikungunya virus serology (pan-american health organisation, 2017). chikungunya igm antibodies become detectable by elisa 5 days after the onset of symptoms. for certain regions simultaneous testing for dengue and zika virus infection is recommended. there exists no specific antiviral therapy for acute chikungunya virus infection. treatment consists of supportive care and includes rest, fluid replacement, and eventually the use of nonsteroidal antiinflammatory drugs (nsaids) or acetaminophen to relieve pain and fever. in patients suspected to have dengue virus coinfection nsaids should be avoided before the patient becomes afebrile for >2 days, in order to avoid bleeding complications associated with severe dengue virus infection. chikungunya infection cannot be prevented by vaccination. hanta viruses are examples of emerging viruses, which belong to the genus hantavirus within the bunyaviridae family. they are negative-sense single-stranded rna viruses, primarily harbored in rodents and shed in rodent urine, saliva and feces. the virus is inhaled by man as aerosols from dried rodent excreta, or in unusual circumstances, transmitted by bites (nichol et al., 2000) . hantaviruses exist in multiple serotypes worldwide, which differ in their virulence. some are considered apathogenic, while certain isolates can produce two distinct severe syndromes in humans: the hanta virus cardiopulmonary syndrome, mostly due to isolates in the americas; and the hemorrhagic fever with renal syndrome caused by isolates (seoul virus, dobrava virus, puumala virus, hantaan virus) in europe and asia (plyusnin et al., 2001) . in some instances, patients with hanta virus hemorrhagic fever suffered from severe acute hepatitis, whereas renal damage was rather mild (wong et al., 1987; chan et al., 1987; lledó et al., 2003) . hanta virus has also been incriminated as a potential cause of cryptogenic hepatitis in southwestern china. however, this hypothesis has not been confirmed by antibody studies in japan. hanta viruses, however, may still play some role, because hanta virus infection has been observed to trigger autoimmune liver disease (yotsuyanagi et al., 1998) . thus, this mechanism may contribute to community-acquired hepatitis (martin et al., 2008) . nevertheless, the precise role of hanta virus infections for human liver disease still awaits clarification. by the time symptoms are evident, patients uniformly have antiviral igm antibodies and most have igg antibodies. diagnostic assays to detect hanta virus antibodies include enzyme-linked immunosorbent assay (elisa), immunoblot test (sia), western blot, indirect immunofluorescence (ifa), complement fixation, and hemagglutinin inhibition as well as neutralization assays. hanta virus strains associated with hemorrhagic fever and hepatorenal syndrome are sensitive to ribavirin in vitro. a prospective, randomized, double-blind, placebo-controlled trial in the people's republic of china reported a sevenfold decrease in mortality among ribavirin-treated patients with serologically confirmed hanta virus disease (huggins et al., 1991) . however, ribavirin appeared to be less effective in hanta virus cardiopulmonary syndrome. thus, it has not yet been approved, but nevertheless may be tried as rescue treatment in emergency situations (sidwell and smee, 2003) . yellow fever is a member of the flaviviridae family and constitutes a single-stranded plus strand rna virus. it comprises a single conserved serotype and seven major genotypes reflecting distinct regions in west africa, central-east africa and south america (vasconcelos et al., 2004; barnett, 2007) . yellow fever virus is transmitted by a variety of different aedes vectors and causes endemic and epidemic outbreaks in africa and south america. yellow fever can be prevented by vaccination, and thus, has become rare in travelers. the spectrum of yellow fever virus infection ranges from subclinical infection to a life-threatening disease with fever, jaundice, renal failure and hemorrhage. usually yellow fever initially presents as an acute, flu-like illness of sudden onset with fever, myalgia and headache, which cannot be distinguished easily from other acute infections such as severe malaria, leptospirosis, fulminant viral hepatitis or dengue hemorrhagic fever. between 48 and 72 h after onset, aminotransferases start to rise in blood heralding the development of jaundice. the degree of liver abnormalities at this stage predicts the severity of liver disease during the course of the illness later on. next, a period of apparent remission lasting up to 48 h may follow the initial infection. patients with abortive infection recover at this stage. about 15% of patients will enter the third stage of intoxication characterized by the return of fever, prostration and organ dysfunction. patients suffer from nausea, vomiting, or epigastric pain, and develop jaundice and oliguria. bleeding can occur from the mouth, nose, eyes or stomach. serum aspartate transferase (ast) levels usually exceed those of alanine transferase (alt). the outcome of yellow fever infection is determined during the second week after onset, when many patients either rapidly recover, while between 20% and 50% of the patients, who have progressed to the stage of intoxication, will ultimately die from circulatory shock. convalescence may be associated with fatigue over several weeks, and occasionally jaundice and elevated aminotransferases may persist for months. the diagnosis of yellow fever is confirmed by demonstration of specific igm by elisa, by pcr or by isolating the virus from the blood. polymerase chain reaction (pcr) testing in blood and urine can detect the virus in early stages of the disease. in later stages, testing to identify antibodies is recommended. liver biopsies should be avoided due to a high risk of bleeding complications. there is currently no specific anti-viral drug to treat yellow fever but specific care to manage dehydration, liver and kidney failure as well as fever improves outcomes. ribavirin inhibits yellow fever virus in vitro. however, the extremely high concentrations of the drug needed cannot be achieved in vivo. recently there have been attempts to treat liver failure resulting from yellow fever infection by high urgency liver transplantation (song et al., 2019) . however, outcomes after liver transplantation were mixed and the few survivors had frequent postoperative bacterial and cytomegalovirus infections. a highly active attenuated live-vaccine is available, which induces seroconversion rates >95% and provides a high level of protection. due to potential risks associated with a live virus vaccine children below the age of 9 months, pregnant women and immunosuppressed individuals should not receive the vaccine, nor should subjects be vaccinated who are allergic to egg proteins. infrequently two serious vaccine-related complications may occur: a form of encephalitis termed yellow fever-associated neurotropic disease and a syndrome resembling natural infection designated as yellow fever vaccine-associated viscerotropic disease. adenoviruses have a worldwide distribution and cause febrile diseases. over 50 serotypes can be distinguished which are further subdivided into the six subgoups a through f. typical syndromes comprise conjunctivitis, upper respiratory tract infections such as pharyngitis and coryza, pneumonia and otitis media. in young children an acute diarrheal illness is caused by subgroup f type 40 and 41 adenoviruses. adenoviruses can persist in human tissue over prolonged periods (garnett et al., 2009) , and can cause a variety of clinical syndromes in immunocompromised individuals including severe hepatitis (kojaoghlanian et al., 2003) . in particular, transmission of latent adenovirus with the donated organ is a risk factor for adenoviral hepatitis in pediatric liver transplantation (michaels et al., 1992) . adenoviral hepatitis occurs in congenital and acquired immunodeficiency syndromes and resembles severe necrotizing hepatitis associated with herpes simplex virus infection. patients develop extensive areas of liver cell necrosis (fig. 2) , massively elevated aminotransferases, and a severe coagulopathy (south et al., 1982; janner et al., 1990; krilov et al., 1990) . ultimately, outcomes may be fatal. liver biopsy specimens may reveal typical intranuclear inclusion bodies in necrotic areas of the liver, but biopsies carry an extremely high bleeding risk. viral isolation and pcr techniques help to identify the causative viral strain. to date a proven therapy or vaccine to prevent adenoviral hepatitis does not exist, but ribavirin may be helpful in selected cases (wulffraat et al., 1995) . liver damage in fatal influenza has been considered immune-mediated, because high cytokine levels were detected (murali-krishna et al., 1998; peiris et al., 2004) . moreover, volunteers infected experimentally with intranasal influenza a/kawasaki/86 (h1n1) transiently developed elevated aminotransferases (polakos et al., 2006) . since the rise in liver enzymes occurred after pyrexia had settled, it was concluded that the host's immune responses rather than viral infection caused damage to the liver. immune mediated liver damage may also be the cause for elevated aminotransferases in other viral respiratory infections such as respiratory syncytial virus (peiris et al., 2004) . however, cardiovascular failure and hepatic ischemia must be considered in as alternative factors in patients with severe respiratory infections ( fig. 3) (eisenhut et al., 2004) . influenza viruses represent three genera in the orthomyxoviridae family. generally influenza a viruses is associated with more severe disease in humans than influenza viruses b and c. influenza a is further subdivided with respect to genetic variation in its hemagglutinin (h) and neuraminidase (n) genes. influenza viruses commonly cause a self-limited acute respiratory infection with fever, rhinorrhea, sore throat and occasionally gastrointestinal symptoms. therefore, aminotransferases are not monitored routinely. in the 2004 h1n5 influenza outbreak, however, about 60% of patients with pneumonia had deranged liver function tests with gastrointestinal symptoms such as vomiting, abdominal pain and diarrhea on initial presentation (yuen and wong, 2005) . although molecular evidence for viral liver disease was not found, autopsy revealed hepatic centro-lobular necrosis in some cases . in influenza virus infection the clinical presentation is dominated by fever and respiratory symptoms. the diagnosis be established by detecting viral antigen or antibodies, but nowadays the gold standard has become detection of viral rna in throat washings by pcr. influenza virus infection can be treated and prevented by neuraminidase inhibitors such as oseltamivir and zanamivir, which have been recommended especially for treatment in influenza h1n5 infection by the who in 2010 (schünemann et al., 2007) . however, their benefit is limited due to the appearance of resistant isolates in recent outbreaks. a preventive vaccine is adapted annually to the circulating strains. severe acute respiratory syndrome (sars) is caused by a novel coronavirus (sars-coronavirus, sars-cov), which caused outbreaks of severe infections of the lung and gastrointestinal tract in the far east and canada (ksiazek et al., 2003; drosten et al., 2003; lee et al., 2003; poutanen et al., 2003) . there was also other organ involvement. middle east respiratory syndrome coronavirus (mers-cov) first appeared in the arabian peninsula and meanwhile has occasionally been observed also in few travelers returning from risk areas. apart from viral pneumonia other internal organs may become affected including hepatitis (alsaad et al., 2018) , particularly when mers-cov hits patients with concomitant diseases, for example, diabetes mellitus. sars-cov and mers-cov have been detected in masked palm civets, dogs and cats as well as camel and thus represent zoonotic infections in man. approximately 25% of patients with sars had elevated liver enzymes at the onset of infection, and further 45% of patients with normal liver enzymes at initial presentation developed elevated aminotransferases later on, so that overall up to 70% of patients showed elevated liver enzymes during their illness (booth et al., 2003; choi et al., 2003; wong et al., 2003; chan et al., 2005) . jaundice was observed in <10% of cases. in most patients aminotransferases started to rise toward the end of the first week and peaked at the end of the second week. with resolution of sars aminotransferases normalized spontaneously in the majority of patients. severe liver damage (alt >5 times the upper limit of normal) was observed more frequently in male patients, and those with significant other comorbidities or elevated serum creatinine levels (chan et al., 2005) . diagnosis of sars-cov and mers-cov infection is suspected in persons with typical symptoms who had contact to risk areas. the diagnosis is confirmed in certified specialized microbiology labs by pcr from respiratory fluids. hygienic prevention measures have to be respected when handling samples and caring for patients with suspected coronavirus pneumonia. during sars outbreaks both ribavirin and kaletra (baby dose ritonavir/lopinavir) were tested as experimental therapy but showed limited success. herpesviruses form a large family of dna viruses, which comprises eight members that can cause disease in man (table 2 ). herpes simplex virus (hsv), varizella zoster virus (vzv), epstein-barr virus (ebv), cytomegalovirus (cmv) and human herpesvirus type 6 or 7 (hhv6, hhv7) can directly affect the liver and are infections in the human population usually acquired during childhood or adolescence. hhv8 can be transmitted sexually and presumeably also vertically from mother to child but has a more limited prevalence. in severely immunosuppressed patients hhv8 can cause kaposi sarcoma and body cavity lymphoma. herpesviruses persist life-long and can reactivate liver disease in immunosuppressed patients later on. primary herpes simplex infection produces characteristic oral (hsv-1) or genital (hsv-2) vesicular lesions. symptoms can be severe with fever and malaise but primary infections are frequently asymptomatic. fulminant hepatitis is a complication both of hsv-1 and hsv-2 infection (pinna et al., 2002) . organ transplantation and treatment for hematological malignancies are the most frequent underlying predispositions (johnson et al., 1992) . further individuals at risk include neonates, patients on steroids, hivinfected patients, and patients with cancer or myelodysplastic syndromes (pinna et al., 2002; johnson et al., 1992; kusne et al., 1991; zimmerli et al., 1988; frederick et al., 2002) . rarely fatal hsv-hepatitis has also been reported in immunocompetent adults (goodman et al., 1986) . varicella zoster virus (herpesvirus type 3) causes chicken pox; and shingles when latent infection is reactivated. after primary infection there is replication of varicella zoster virus in the epithelia of gut, respiratory tract, liver and endocrine glands. secondary viraemia then leads to infection of the skin and causes the typical rash. liver disease is rare and limited to patients with severe immunodeficiency. hsv-related hepatitis has a high (>80%) mortality and resembles septic endotoxic shock; jaundice is not always present (kusne et al., 1991) . patients suffer from fever, anorexia, nausea, vomiting, abdominal pain, leucopenia, and coagulopathy. typical oral or genital vesicular lesions may be present in only about 30% of patients (pinna et al., 2002) . some patients have disseminated further extrahepatic involvement, for example, lung, lymphnodes, spleen, and adrenal glands. in severe varicella zoster virus infection hepatic lesions are similar to herpes simplex hepatitis. varicella zoster virus has also been reported to trigger severe autoimmune type hepatitis (al-hoamoudi, 2009). the diagnosis of hsv-related hepatitis must be rapidly established. serologic assays are of little use. herpes simplex and varicella zoster viruses are detected preferentially by the polymerase chain reaction (finström et al., 2009) , or occasionally by viral isolation or immunofluorescence staining. prompt systemic treatment with acyclovir reduces hsv-associated morbidity and serious complications in hiv-infected patients. antiviral acyclovir prophylaxis has markedly reduced hsv re-activation after organ transplantation (seale et al., 1985; pettersson et al., 1985) . acyclovir resistance occurs in about 5% of immunocompromised patients and is negligible (<0.5%) in immunocompetent subjects (tyring et al., 2002) . valacyclovir is a prodrug of acyclovir, and famciclovir a prodrug of penciclovir, which have similar antiviral mechanisms as acyclovir. thus hsv isolates resistant to acyclovir are also resistant to these drugs (levin et al., 2004) . cidofovir and foscarnet are alternative choices to treat acyclovir-resistant hsv but are less well tolerated (safrin et al., 1991) . treatment and prophylaxis of varicella zoster hepatitis is similar to herpes simplex viruses because acyclovir also inhibits replication of varicella zoster virus. in immunocompetent hosts cytomegalovirus (cmv) infection may be rather asymptomatic but occasionally causes transient minimally symptomatic acute disease. congenital cytomegalovirus infection occurs in <2% of newborns and is encountered in (kylat et al., 2006) . when newborns or immunocompromised patients, for example, hiv infection, cancer, solid organ or bone marrow transplantation become cmv-infected, they may develop serious disease. cmv-related liver disease represents the commonest cause of viral hepatitis after organ transplantation. infection may result from re-activation of endogenous virus under immunosuppression, infection from the transplanted organ or blood transfusion of a cmv-positive donor. in liver transplantation, most cmv disease occurs at 1-4 months after transplantation. cmv infection is also a potential factor triggering acute and chronic rejection. vice versa, rejection therapy with corticosteroid boluses may induce endogenous cmv reactivation. although cmv re-activation is a frequent complication also in hiv-positive patients with advanced immunodeficiency (cd4 counts <200/ml), involvement of the liver seems to be rather rare (palmer et al., 1987) , but cmv occasionally causes bile-duct necrosis and a so-called hiv-cholangiopathy, a sclerosing cholangitis encountered in patients with terminal hiv-immunodeficiency (bonacini, 1992) . in about 10% of immunocompetent subjects primary cytomegalovirus infection produces an infectious mononucleosis-like syndrome, which is associated with elevated aminotransferases and a mild hepatitis (fig. 4) . liver histology may show focal hepatocyte and bile duct damage with lymphocytic infiltration into the sinusoids and occasionally epithelioid granulomas without necrosis, while cmv inclusion bodies or cmv immunostaining are only rarely seen (snover and horwitz, 1984) . fetal cmv infection has also been associated with obstructive biliary disease and neonatal hepatitis with giant cell transformation, cholestasis and viral inclusion bodies (finegold and carpenter, 1982) . in liver biopsies hepatocytes are swollen and may contain basophilic granules in the cytoplasm. a typical intranuclear amphophilic inclusion body can be present, resembling an "owl's eye" (fig. 3c ). both nuclear and cytoplasmic inclusions are full of virions (desmet, 1983) . however, in posttransplantation cmv hepatitis cytomegalovirus inclusion bodies are scanty. instead small foci of necrosis and inflammation (microabscesses) may be present (fig. 3b) . de novo appearance of cmv igm antibodies or a fourfold rise in igg antibodies herald cmv infection in immunocompetent individuals. however, serology is unreliable in immunocompromised patients and is replaced by quantitative molecular dna amplification assays (humar et al., 1999; caliendo et al., 2003) . meanwhile most transplant centers perform cmv surveillance by weekly quantitative determination of cmv dna. in most transplant units organ recipients at high risk for acquiring cmv disease receive immune prophylaxis with cmvhyperimmune antibodies and antiviral drugs (paya, 2001) . however, cmv infection and disease may still develop. cmv hepatitis should be treated promptly in patients with immunodeficiency. intravenous ganciclovir or oral valganciclovir over 3 weeks is the treatment of choice. drugs must be continued in reduced doses as chemoprophylaxis, if prolonged immunosuppression is anticipated (crumpacker, 1996) . fortunately, ganciclovir resistance is still rare (martin et al., 2008) , so that the more toxic alternatives cidofovir and foscarnet are rarely needed. epstein-barr virus (ebv) is shedded in oral secretions, and most primary ebv infections occur in adolescents. ebv accounts for 90% acute infectious mononucleosis syndromes. it persists life-long in a latent state, which results from a dynamic interplay between viral evasion strategies and the host's immune responses. while-unlike other herpesviruses-ebv reactivation-associated liver disease is not a prominent feature of persistent ebv infection, this herpesvirus is a potent cause for various malignancies such b-and t cell lymphomas, hodgkin lymphoma, and nasopharyngeal carcinoma. ebv has also been associated with an aggressive lymphoproliferative disease after liver transplantation. ebv intrauterine infection may lead to diverse congenital anomalies also comprising biliary atresia (goldberg et al., 1981) . however, only few pregnant women are susceptible, thus intrauterine ebv infection is rare. in infants and young children primary infection is frequently asymptomatic, while in adults it results in the infectious mononucleosis syndrome. patients develop malaise, headache, low-grade fever, before the more specific symptoms such as pharyngitis/ tonsillitis, swelling of cervical lymphnodes and moderate to high-grade fever occur. nausea, vomiting, and anorexia are frequent findings. a mild clinical hepatitis accompanies infectious mononucleosis in approximately 90% of patients (fig. 5) . splenomegaly is found in about half of patients, but hepatomegaly and jaundice are infrequent findings. patients show peripheral blood lymphocytosis with characteristic large abnormal lymphocytes in their blood smears. the vast majority of patients recover over 2-4 weeks, but fatigue may persist over several months after infection. ebv does not infect hepatocytes but lymphoid tissue. thus, liver damage is due to immune-mediated pathology and-when exceptionally done-biopsy specimens show diffuse lymphocytic infiltrates in the sinusoids but only occasionally focal apoptotic hepatocytes (fig. 3a ) (purtilo and sakamoto, 1981) . moreover, ebv-related immune activation can lead to several complications: patients with x-linked lymphoproliferative disease (xlp) caused by a mutation in the sh2d1a gene on the x chromosome are particularly vulnerable to the epstein-barr virus and may suffer fatal infections with extensive liver necrosis (seemayer et al., 1995) . in patients with severe immunodeficiency lymphomatoid granulomatosis is a further unusual complication of epstein-barr virus infection leading to granuloma formation in multiple organs including the liver, which may require interferon-alpha antiviral therapy (wilson et al., 1996) . in patients with hiv infection an ebv-associated lymphoproliferative disorder with hepatic infiltration of immunoblasts (beissner et al., 1987) , and a hemophagocytic syndrome have been reported (albrecht et al., 1997) . epstein-barr virus is also the major causative agent for the so-called posttransplant lymphoproliferative disease (ptld), which after organ transplantation may result in lymphocytic infiltration of the liver and other organs ranging from benign polyclonal b cell proliferation to malignant b cell lymphoma (hanto, 1995) . ptld occurs more commonly in children than in adults, depending on the degree of immunosuppression. it is primarily a complication in ebv-negative organ recipients, who develop primary ebv infection under immunosuppression owing to a graft from an ebv-positive donor. finally, ebv is a potent risk factor to develop lymphoma in later life even in patients without overt immunosuppression (fig. 6) . the clinical suspicion of epstein-barr virus infection is confirmed by detection of heterophilic or ebv-specific antibodies in infectious mononucleosis and quantitative polymerase chain reaction assays in patients with lymphoproliferative disorders (bruu et al., 2000; weinberger et al., 2004) . liver biopsy is not recommended for routine diagnostics. treatment of epstein-barr virus infection is primarily supportive. corticosteroid therapy can ameliorate symptoms. however, this option should be only considered in individuals with immune-mediated life-threatening complications, for example, imminent liver failure. because of theoretical concerns to suppress the immune system in an infection with a potentially oncogenic virus, corticosteroids are not recommended in general. acyclovir inhibits the ebv dna polymerase, and antiviral therapy with this drug has shortened virus shedding but failed to demonstrate a convincing clinical benefit even in severe acute epstein-barr virus infection (torre and tambini, 1999) . acyclovir antiviral therapy is not effective against latent ebv-infection. thus, reduction in immunosuppression, anticancer chemotherapy, and b-cell depleting antibodies are needed to treat ebv-related lymphoproliferative disorders. human herpesviruses types 6 (hhv6) and 7 (hhv7) hhv-6 exists in two variants, hhv6-a and hhv-6b, which infect t cells and various other cells types expressing the cd46 receptor (santoro et al., 1999) . although genetically clearly distinct from hhv-6, hhv-7 is another b-herpesvirus that shares many features with hhv-6. primary infection with either virus commonly occurs at young age and can lead to a febrile illness known as exanthema subitum or roseola infantum (leach, 2000) . pityriasis rosea reflects primary infection with hhv-7. hvv-6 also integrates into the host's genome and is transmitted via the germline. hhv6 and hhv-7 can reactivate each other (tanaka-taya et al., 2000) as well as cytomegalovirus leading to symptomatic cmv-disease in liver transplantation (humar et al., 2000) . the full spectrum of diseases caused by chronic hhv-6 and -7 infection is still unclear, but these viruses are putatively involved in a variety of different syndromes such as encephalitis, multiple sclerosis, pneumonitis, an infectious mononucleosis-like syndrome, postinfectious drug hypersensitivity as well as lymphoproliferative disorders and systemic disease in immunocompromised patients (stoeckle, 2000) . hhv-6 can cause severe and fatal hepatitis in neonates, children and adults (mendel et al., 1995; chevret et al., 2008; härmä et al., 2003) . hepatitis due to infection and reactivation of hhv-6 and hhv-7 can also complicate organ transplantation (dockrell and paya, 2001; härmä et al., 2006; ohashi et al., 2008) . in addition, hhv-6 has been associated with autoimmunity and giant-cell hepatitis and giant-cell transformation of bile duct cells (potenza et al., 2008) . hhv6-igm antibodies develop within a week after infection but are an unreliable marker, because false-positive test results in about 5% of healthy controls. beyond that serology does not distinguish between hhv-6a and hhv-6b variants and may cross-react with hhv7. the preferred method to diagnose hhv-6 and -7 infection is by quantitative dna amplification assays (deback et al., 2008) . detecting high viral loads in liver specimens or hhv-6 viremia is associated with approximately twofold increased mortality after liver transplantation (pischke et al., 2012) . in immunocompetent patients hhv-6 and -7 cause a benign self-limited infection, which does not require specific antiviral treatment. unlike hhv-6b both hhv-6a and hhv-7 are relatively resistant against ganciclovir, while foscarnet acts against all three viruses (yoshida et al., 1998; de clercq et al., 2001) . cidofovir may be a therapeutic alternative, but some resistant hhv-6 isolates have been identified (bonnafous et al., 2008) . human herpesvirus 8 is a g-herpesvirus, which has potential for malignant transformation. although primary hhv-8 infection can cause rash and fever in children and immunocompromised individuals, the onset of hhv-8-related diseases usually occurs several years after hhv-8 acquisition: kaposi sarcoma, body cavity lymphoma, and multicentric castleman's disease are the typical presentations of hhv-8 infection but bone marrow aplasia and multiple myeloma has also been described in association with hhv-8 infection (lee and henderson, 2001) . in autopsy studies kaposi sarcoma involved the liver in approximately 20% of patients with aids and was usually part of a widespread cutaneous and visceral disease. due to highly active antiretroviral combination therapy, kaposi sarcoma has become a rare complication of hiv infection. however, fulminant hepatic kaposi sarcoma may occur after organ transplantation (cahoon et al., 2018, fig. 7) . macroscopically there are dark-red tumors on the skin, the liver capsule and the parenchyma. under the microscope the typical lesion is a mesh of spindle-cell-like tumor cells and dilated thin-walled vessels (glasgow et al., 1985) . hepatosplenomegaly, fever, and weight loss are typical features of multicentric castleman's disease, a pre-malignant proliferation of b-lymphocytes (fig. 8a ). lymphocytes in multicentric castleman's disease and kaposi sarcoma seem to cooperate with each other, and thus the two hhv-8-related lesions occasionally occur within the same lymphnode (naresh et al., 2008) . ascites and pleural effusions are the hallmark of hhv-8-related body cavity lymphoma possibly giving rise to a false initial diagnosis of decompensated liver cirrhosis. however, abundant lymphoma cells in the aspirated fluids provide a pivotal diagnostic hint (fig. 8b ). hhv-8 can also cause solid organ lymphoma involving the liver (cesarman and knowles, 1999) . the diagnosis of hhv-8 associated malignancies is established from biopsies via their characteristic histopathological features. the virus itself is detected by various antibody assays or polymerase chain reaction (pcr) assays (chiereghin et al., 2017) . reconstitution of immune function is the primary goal for treatment of hhv-8 associated diseases. this can be achieved by highly active antiretroviral therapy in hiv infection and alternatively by antiproliferate m-tor (mammalian target of rapamycin) inhibitors for immune suppression in transplantation (barozzi et al., 2009) . immune stimulation with imiquimod and interferon-alpha (babel et al., 2008; van der ende et al., 2007) has been attempted. oncological therapy with liposomal anthracyclines or paclitaxel in kaposi sarcoma (di trolio et al., 2006; stebbing et al., 2003) , or rituximab in the case of castleman's disease and lymphoma are further potent treatment options (bower et al., 2007) . ganciclovir, cidofovir, foscarnet, adefovir and lobucavir but not acyclovir can block hhv-8 replication in vitro, and in a controlled crossover trial valganciclovir reduced oropharyngeal shedding of hhv-8 by 80% (casper et al., 2008) . this overview addresses the currently most relevant viral infections involving the liver. however, it provides only an outline and is in far not exhaustive. in rare instances, hepatitis occurred in the context of infections with enteroviruses (sun and smith, 1966) , measles (khatib et al., 1993) and rubella viruses (mclellan and gleiner, 1982) as well as parvovirus b19 (yoto et al., 1996; hayakawa et al., 2007) . the reader will find details of these rare infections in microbiology textbooks. beyond that, international travel and global warming are likely to introduce new exotic infections, which must be considered in the differential diagnosis of severe hepatitis. this problem is illustrated by the recent autochthonous crimean-congo hemorrhagic fever (cchf) virus infections in spain (negredo et al., 2017) , which did not occur in this country before. thus, hepatologists must be constantly prepared to face new challenges. epstein-barr-virus-associated hemophagocytic syndrome. a cause of fever of unknown origin in hiv infection severe autoimmune hepatitis triggered by varicella zoster infection histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection-clinicopathological and ultrastructural study development of kaposi's sarcoma under sirolimus-based immunosuppression and successful treatment with imiquimod yellow fever: epidemiology and prevention indirect antitumor effects of mammalian target of rapamycin inhibitors against kaposi sarcoma in transplant patients fatal case of epstein-barr virus-associated lymphoproliferative disorder associated with a human immunodeficiency virus infection hepatobiliary complications in patients with human immunodeficiency virus infection clinical features and short term outcome of 144 patients with sars in the greater toronto area rare tumor entities associated with persistent hhv8 infection. (a) castleman's disease in a lymphnode of hiv-positive patient who developed malaise, fever and cutaneous kaposi sarcoma. castleman's disease is a hhv8-associated pre-malignant lymphoproliferative disorder, which can progress to b-cell lymphoma. 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asymptomatic hiv infection histology slides were kindly provided by prof. dr. hans-peter fischer, department of pathology, rheinische friedrich-wilhelms university of bonn, germany. key: cord-266218-r6xg9zts authors: law, arjun datt; salas, maria queralt; lam, wilson; michelis, fotios v.; thyagu, santhosh; kim, dennis (dong hwan); lipton, jeffrey howard; kumar, rajat; messner, hans; viswabandya, auro title: reduced-intensity conditioning and dual t lymphocyte suppression with antithymocyte globulin and post-transplant cyclophosphamide as graft-versus-host disease prophylaxis in haploidentical hematopoietic stem cell transplants for hematological malignancies date: 2018-08-07 journal: biol blood marrow transplant doi: 10.1016/j.bbmt.2018.07.008 sha: doc_id: 266218 cord_uid: r6xg9zts haploidentical hematopoietic stem cell transplantation (haplohsct) with conditioning regimens using post-transplant cyclophosphamide (ptcy) for peripheral blood stem cell (pbsc) grafts is limited by comparably higher rates of acute and chronic graft-versus-host disease (gvhd). antithymocyte globulin (atg) may mitigate this risk. we evaluated haplohsct after reduced-intensity conditioning (ric) with atg, ptcy, and cyclosporine to prevent rejection and gvhd. fifty adults underwent haplohsct from august 2016 to february 2018. ric included fludarabine (30 mg/m(2)/day on days –5 to –2), busulfan (3.2 mg/m(2)/day on days –3 and –2), and total body irradiation (200 cgy) on day –1. unmanipulated pbscs were infused on day 0. gvhd prophylaxis included atg (4.5 mg/kg over days –3 to –1), ptcy (50 mg/kg/day on days +3 and +4), and cyclosporine from day +5. median age was 56 years (range, 22 to 70 years); 25 (73.5%) patients were in first complete remission (cr1), 5 (14.7%) were in second complete remission (cr2), and 8 (23.5%) had active disease. median time to neutrophil engraftment was 16 days (range, 8 to 43 days). at day +100, the cumulative incidence of acute gvhd of any grade, and grades iii to iv was 38.3% and 5.2%, respectively. mild chronic gvhd was seen in 15.5%. cytomegalovirus (cmv) reactivation occurred in 37 (74%) cases and cmv disease occurred in 4 (11.5%) cases. epstein-barr virus (ebv) reactivation occurred in 21 (61.8%) patients. the incidence of histologically confirmed post-transplantation lymphoproliferative disorder (ptld) was 5.8%. four patients received rituximab. there were no cmv, ebv, or ptld-related deaths. six-month and 1-year overall survival (os), cumulative incidence of relapse (cir), and nonrelapse mortality (nrm) were 73.9%, 10.2%, and 19.4%, respectively, and 48.1%, 16% and 38.2%, respectively. infection was the most common cause of death (18%). unmanipulated haploidentical pbsc transplantation following ric with atg, ptcy, and cyclosporine as a gvhd prevention strategy results in low rates of acute and chronic gvhd. allogeneic hematopoietic stem cell transplantation (hsct) is a potentially curative therapy for patients with high-risk or advanced hematological disorders [1] . an hlamatched sibling donor is considered the first choice. approximately 70% of patients do not have a suitably matched sibling donor (msd) available for transplantation. alternatives such as matched unrelated donors (muds) can be identified for only 50% to 60% of patients, with the donor search and procurement process requiring a median of 4 months [2] . for patients who do not have suitable msds or muds, alternative stem cell sources include unrelated single or double umbilical cord blood transplants, hla-mismatched unrelated donors (mmuds), and hla haploidentical family members. despite the expansion of mud registries and cord blood banks, donor availability can often be uncertain particularly for under-represented ethnic minorities, which may be subject to prolonged and unproductive registry searches [3] . the use of haploidentical donors reduces this uncertainty to a large degree, as almost all patients have an immediately available related donor with whom they share a single hla haplotype. this not only facilitates a reduced time to transplantation, but also provides a more reliable source of donor there remains considerable variation between centers in conditioning regimens, graft sources and choice of graft-versus-host disease (gvhd) prophylaxis to achieve lower nonrelapse mortality (nrm) and optimum long-term outcomes in haploidentical hsct (haplohsct). the combination of a nonmyeloablative conditioning regimen with antithymocyte globulin (atg) and post-transplant high-dose cyclophosphamide (ptcy) has only been described in the setting of haplohsct for sickle cell disease [4] . in our institution, we established a reduced-intensity conditioning (ric) regimen consisting of fludarabine (flu), reduced-dose busulfan (bu), and total body irradiation (tbi) (total dose 200 cgy) combined with atg and followed by ptcy and cyclosporine for the prevention of graft rejection and gvhd for haplohsct recipients. we previously reported the use of this protocol in mud transplants wherein lower rates of severe acute gvhd were demonstrated compared with nonàatg-based regimens [5] . in the present study, we report the use of this protocol in 50 consecutive patients who underwent haplohsct at our center. from august 2016 to february 2018, 50 adult patients with hematological malignancies underwent haplohsct at the princess margaret cancer centre, toronto, canada. all patients were 18 years old with no upper age limit, had a karnofsky performance score (kps) >60%, and did not have active/ untreated infection or human immunodeficiency virus seropositivity. criteria for pretransplant organ function included left ventricular ejection fraction 45% without significant preexisting cardiac disease, pulmonary function testing demonstrating forced expiratory volume >50% predicted and diffusing capacity of carbon monoxide >50, normal/stable kidney function on biochemistry, and liver functions tests showing total bilirubin <2.5 times normal with transaminases <3 times the upper limit of normal. informed consent was obtained from all patients. the use of this protocol was approved by the institutional board of ethics. all patients with positive donor specific antibodies were excluded. high-resolution molecular typing for hla class i (a, b, c) and class ii (dr, dq) was performed for recipients and donors when patients were referred to the allogeneic blood and marrow transplantation program. haploidentical related donors were selected in all cases where msd or 9/10 mud could not be identified. peripheral blood stem cells (pbsc) were collected after granulocyte colony-stimulating factor (g-csf) mobilization. a minimum dose of 6 £ 10 8 cd34 + cells/kg recipient body weight was requested in all cases. cd3 + cell enumeration is not routinely performed in our institute, as unmanipulated stem cells are used in all cases. all donors provided informed consent. patients received conditioning therapy with fludarabine 30 mg/m 2 /day i. v. on days -5 to à2, busulfan 3.2 mg/m 2 /day i.v. days à3 and à2, and 200 cgy of tbi on day à1. rabbit atg (thymoglobulin; genzyme-sanofi, lyon, france) was administered in doses of .5 mg/kg on day à3, 2 mg/kg on day à2, and 2 mg/kg on day à1 (total 4.5 mg/kg). t cellàreplete pbsc grafts were infused on day 0. all patients received ptcy (cyclophosphamide 50 mg/kg/day i.v.) on days +3 and +4, with the first dose starting 72 hours after the start of allograft infusion. four doses of mesna were administered on days +3 and +4 at a total daily dose of 80% of the cyclophosphamide dose. cyclosporine was initiated at a dose of 2.5 mg/kg i.v. on day +5 and adjusted to achieve a therapeutic level of 200 to 400 mg/l ( figure 1 ). all patients received daily filgrastim 300 mg subcutaneously, starting on day +7 until neutrophils were >1.5 £ 10 9 /l. ursodeoxycholic acid 500 mg oral twice daily with meals (starting on the first day of conditioning) was used for prevention of sinusoidal obstruction syndrome (sos) in all patients. prophylactic antimicrobial therapy included posaconazole 300 mg daily, acyclovir 400 mg twice daily, ciprofloxacin 500 mg twice daily, and inhaled pentamidine 300 mg monthly. postengraftment, cytomegalovirus (cmv) titer was monitored twice per week and epstein-barr virus (ebv) was monitored weekly. cyclosporine tapering started around days +45 to 60 for all patients without gvhd. the schematic for the conditioning protocol and post-transplant immunosuppressive regimen is shown in figure 1 . neutrophil engraftment was defined as the first of 3 consecutive days with an absolute neutrophil count .5 £ 10 9 /l and platelet recovery was defined as a sustained and transfusion-independent platelet count >20 £ 10 9 /l (first of 3 days) without transfusion times 7 days. donor total chimerism was assessed in whole blood by pcr for variable number tandem repeat polymorphisms at days +30 and +60 in all patients. all patients underwent bone marrow examination at day +60 for marrow cellularity and disease monitoring. clinical features, post-transplant outcome, and side effects were collected through retrospective chart review. last follow-up was updated in february 2018. the study was conducted in accordance with the declaration of helsinki, and was reviewed and approved by the ethics committee at the princess margaret cancer centre, toronto, canada. patient and disease characteristics were reported using descriptive statistics (count and percentage). the time to event was calculated from the date of transplant to the date of event or last date of patients known to be alive. the main outcome variables of interest included overall survival (os), relapse-free survival (rfs), and nonrelapse mortality (nrm). the kaplan-meier method was used to estimate os and rfs. the cumulative incidences of acute and chronic gvhd were calculated accounting for death and relapse as competing risks. the incidence rate of acute gvhd was estimated at day +100 and chronic gvhd at day +180. nrm was estimated using the cumulative incidence method accounting for relapse as a competing risk (fine-and-gray analysis). survival rates were calculated 6 months and 1 year after hap-lohsct. analyses were performed using spss version 16.0 for windows (spss inc, chicago, il), and ezr [6] . table 1 shows baseline patient characteristics. table 2 shows donor characteristics, graft parameters, and gvhd characteristics. median follow-up for survivors was 168 days (range, 22 to 536 days), or 5 months (range, 0 to 17 months). nearly 80% of donor-recipient pairs were mismatched at 5 hla loci. sixteen patients and donors were cmv mismatched. while cryopreserved grafts were used in the majority, 10 patients received fresh product. the hematopoietic cell transplantation comorbidity index score was 3 in almost 40% cases. median time to neutrophil engraftment was 17 days (range, 8 to 43 days). median time to platelet engraftment was 22 days (range, 7 to 217 days). engraftment syndrome (characterized by fever, fluid retention/weight gain, hypotension, and hypoxemia in the absence of documented infection, and temporally correlated with count recovery) occurred in 3 (6%) patients, and all cases were successfully treated with steroids. ninetyfour percent of patients achieved donor chimerism >95% at day +30. four (8%) patients failed to engraft by day +30. one patient developed secondary graft failure 3 months post-transplant. this included 1 patient with primary myelofibrosis, 1 with myelodysplastic syndrome, and 2 with acute myelogenous leukemia. median of 9.94 £ 10 6 cd34 + /kg recipient body weight (range, 8.2 to 11.7 £ 10 6 cd34 + /kg recipient body weight) were infused. no donor-specific antibodies were documented. all patients had developed cmv reactivation, and 1 patient with secondary graft failure had biopsy-proven cmv colitis. ebv reactivation was documented in 2 cases, and 1 developed asymptomatic bk viruria. median length of hospitalization was 32 days (range, 13 to 83 days). post-transplant clinical course, organ toxicity, and infections are summarized in supplementary tables 1 and 2 . three (6%) cases developed low-grade cytokine release syndrome following stem cell infusion. this occurred on day 0 in all cases and resolved after cyclophosphamide administration in all cases by day +4. mild mucositis was seen in all patients, while 9 (18%) patients developed grade 3 to 4 mucositis requiring short-term total parenteral nutrition. mild-to-moderate sos was documented in 7 (17.4%) patients, with grade 3 sos in 1 case. all cases resolved with fluid restriction and diuretics. ten patients developed features of fluid overload, and 3 patients developed cardiogenic pulmonary edema secondary to fluid retention and hypoalbuminemia during the first 30 days during the post-transplant course. all cases responded to aggressive diuresis and fluid optimization, and no patient needed intensive care support. no ischemic events occurred. three patients developed pericardial complications (2 cases with pericarditis and 1 myopericarditis) during follow-up. all effusions were mild, resolved spontaneously, and did not show features of cardiac tamponade. diagnostic or therapeutic pericardiocentesis was not performed. one patient demonstrated an appreciable decline in left ventricular ejection fraction on follow-up echocardiography at day+60 but remained asymptomatic. one patient with a history of anthracycline-associated cardiomyopathy with induction chemotherapy sustained a fatal ventricular arrhythmia while undergoing treatment for cmv reactivation and gvhd. transient elevation of renal parameters without oliguria was seen in 13 (26%) patients. creatinine returned to baseline in all cases with conservative management. neutropenic fever occurred in 42 (84%) of the patients with positive blood cultures in nineteen cases (38%). no proven invasive fungal infections were encountered during hospitalization for the transplant. laboratory evidence of cmv reactivation by peripheral blood pcr was observed in 37 (74%) patients, with a median time to reactivation of 26 days (range, 13 to 61 days) post-transplant. cmv was also identified in bronchoalveolar lavage specimens in 3 cases and on intestinal biopsy in 1 case. all patients received intravenous ganciclovir or valgancyclovir until viremia clearance. two patients required foscarnet therapy due to persistence of cmv viremia despite appropriate doses of ganciclovir. twenty-seven (64%) patients developed ebv reactivation after transplantation with a median time of 56 days (range, 20 to 233 days). four patients (8%) developed biopsy proven post-transplant lymphoproliferative disease (ptld). ptld was managed by tapering immunosuppression and weekly rituximab for a maximum of 4 doses. rates of acute and chronic gvhd were 44% and 10%, respectively. acute gvhd was graded according to the keystone criteria and chronic gvhd was graded in accordance with the national institutes of health chronic graft-versus-host disease consensus [7, 8] . acute gvhd of any grade was identified in 20 (44%) patients; however, grade iii to iv acute gvhd occurred in only 2 cases (4%). the cumulative incidence of acute gvhd of any grade, grade ii to iv, and grade iii to iv at day +100 was 38.3%, 20.3%, and 5.2%, respectively ( table 2) . skin (36%) was the most commonly affected site, followed by liver (14%) and gut (10%). first-line therapy of clinically significant acute gvhd consisted of topical or systemic steroids based on site and severity. the cumulative incidence of chronic gvhd at 3 months was 15.5%. no systemic immunosuppression was required for patients with chronic gvhd. no patients developed moderate or severe chronic gvhd (table 3) . nineteen (38%) patients died during follow-up. table 4 lists the causes of death among transplanted patients. five (10%) patients relapsed during the follow-up period with a median time to relapse of 4 months (range, 1 to 9 months). six-month os, rfs, cir, and nrm were 73.9%, 57%, 10.2%, and 19.4%, respectively. no significant differences were found in os (p = .411) and progression-free survival (p = .563) between patients in cr1 versus cr2 versus active disease. one-year os, rfs, cir, and nrm were 48.1%, 35.7%, 16%, and 38.2%, respectively ( table 2 ). causes of death were relapse in 4 (8%) cases, graft failure in 2 (4%) patients, infection in 9 (18%) patients, multiorgan failure in 2 (4%) cases, acute gvhd in 1 case, and cardiac arrhythmia in 1 case (figures 2à4). haplohsct was initially described in the late 1950s. this was historically associated with high rates of graft rejection, severe acute gvhd, and worse outcomes when compared with msd transplants [9] . an important milestone in haplohsct was the use of t cellàreplete grafts in combination with posttransplantation high-dose cyclophosphamide [10] . in the setting of haplohsct, ptcy decreases the risk of gvhd and graft rejection by targeting and inducing cell apoptosis of alloreactive t cells rapidly proliferating early after transplant, sparing regulatory t cells and preserving nondividing hematopoietic stem and progenitor cells [10à13]. ptcy may be associated with high rates of severe acute gvhd if used alone, with 4 of 5 patients receiving single-agent ptcy succumbing to fatal, steroid-refractory gvhd in 1 single-center report [14] . therefore, post-transplant immunosuppression commonly includes additional agents such as calcineurin inhibitors, sirolimus, and/or mycophenolate mofetil [13, 14] . haplohsct using unmanipulated t cellàreplete pbsc grafts with ptcy, calcineurin inhibitors, and mycophenolate mofetil as gvhd prophylaxis have been associated with increased rates of acute and chronic gvhd and have shown commensurate outcomes with acceptable nrm when compared with other donor sources [12, 15, 16] . atg has also been extensively studied as a method to prevent graft rejection, acute gvhd, and chronic gvhd in msd and mud transplants [17à19]. however, its effectiveness in modulating severe acute and chronic gvhd is offset by a higher rate of infectious complications, including cmv and ebv reactivation [20à22] . ptcy induces selective destruction of proliferating alloanti-genàstimulated t cells. this effect is invaluable for haplohsct where there is an intense t cell proliferation due to major hla mismatch. donor memory t cells are relatively protected from ptcy and provide donor immunity in addition to immune reconstitution post-transplant [13] . reduced-intensity conditioning with fludarabine, reduceddose busulfan, and tbi (200 cgy) is a low-toxicity regimen that induces sufficient immunosuppression to permit donor hematopoietic cells to consistently engraft despite the hlahaplotype barrier. the feasibility of ric in haplohsct in adult patients with hematological malignancies is well described [23à25] . the combination of fludarabine and busulfan with ptcy may potentiate the antineoplastic activity of the conditioning regimen which may lead to resulting in further disease reduction. data in haplohsct using ric and pbsc as the stem cell source is limited. additionally, centers using pbsc in haploidentical transplants have shown rates of extensive chronic gvhd as high as 38% [16] . the combination of atg and low dose ptcy was found to increase regulatory t cell reconstitution in an animal model leading to lower rates of acute gvhd in a murine experiment [26] . our own experience with this combination in mud transplants led to its use in the haplohct setting [5] . we had also encountered a high incidence of severe acute and chronic gvhd with ric for msd and mud transplants [27] . this led to a concerted effort to design a conditioning regimen that would reduce both acute and chronic gvhd. ric with low-dose busulfan and fludarabine and atg + cyclosporineàbased gvhd prophylaxis showed outcomes comparable with mud with lower rates of cgvhd [18] . the inclusion of atg in conditioning regimens results in faster achievement of donor chimerism, especially when using alternative donors [17, 18] . however, the use of atg is also associated with delayed immune reconstitution and increased infective complications, particularly viral reactivations with cmv or ebv [19à21] . due to the higher degree of immunosuppression, hap-lohsct recipients have an increased risk of infectious complications, particularly viral infections including cmv reactivation. high incidence of cmv reactivation was noted in our study; however cmv-related mortality was very low. t cellàreplete haplohsct recipients have a lower incidence of infections than t cellàdepleted haplohsct patients do because of improved immune reconstitution; the incidence of cmv reactivation has been reported to be 60% [12] . the best approach to prevent cmv reactivation in the setting of hap-lohsct is not well established. the use of novel agents such as letermovir as prophylaxis may be potentially useful avenues of research [28] . ebv-related ptld after allogeneic hsct is associated with mortality rates as high as 50% to 90% [29, 30] . based on european data, the incidence of ptld after allogeneic hsct was 3.2%, varying from 1.2% in muds to 2.8% in mismatched related donors (including haploidentical and mismatched related donors). in the context of atg-based conditioning, the incidence of ptld has been reported to be 9.7% [31] . the risk of developing ebv-ptld is predominantly related to the degree of t cell depletion or impairment, as ebv-infected b cells are held in check by cytotoxic t cell lymphocytes. this loss of this mechanism leads to uncontrolled b cell proliferation and the development of ptld [32] . although the incidence of ebv reactivation and ptld in our patients was high, all patients responded well to rituximab monotherapy and no ptldrelated deaths occurred. the observed high rate of ebv reactivation and ptld may be attributed to t cell impairment between donor and recipient secondary to hla mismatch, the use of dual t cell depletion with ptcy and atg, and the high rate of cmv reactivation. patients were monitored closely with weekly pcr-based testing for ebv. this may have led to a higher rate of detection of ebv reactivation. ebv seromismatch has been described as a risk factor for ptld development, and the selection of an ebv-matched donor may affect rates of reactivation in a manner similar to cmv [31] . there are no clear guidelines for the monitoring and preemptive treatment of ebv reactivation or ptld in haplohsct. our study's major limitation is the short duration of followup. the focus of this report is to demonstrate the applicability of this novel conditioning regimen in a broad selection of patients. while a longer follow-up would certainly be more informative for outcomes such as chronic gvhd and longerterm complications, the lower incidence of acute gvhd grade ii to iv was encouraging. the incidence of nrm was of considerable concern and has been attributed to infective complications, particularly after viral infections. we have implemented a reduction in the atg dose in an attempt to mitigate the infection risk. finally, the proportion of older (35% over 65 years of age) patients may explain lower tolerance to infectious and other post-transplant complications. future modifications to this protocol may also include more aggressive antiviral prophylaxis and therapy. to summarize, our experience indicates that t cellàreplete haplohsct after ric with low-dose busulfan, fludarabine, tbi (200 cgy) combined with atg, ptcy, and cyclosporine is a feasible and effective transplant regimen. this approach produced consistent donor cell engraftment with low rates of acute gvhd; however, this was tempered by high rates of viral reactivation and consequently, nrm. haplohsct may be used when a suitable hlamatched donor is not available or when allogeneic hsct is needed urgently. prospective studies are needed to compare the outcomes of different conditioning regimens in haplohsct and the outcome of allogeneic hsct using alternative graft sources. financial disclosure: the authors have nothing to disclose. conflict of interest statement: there are no conflicts of interest to report. authorship statement: a.l. and q.s. collected and analyzed the data and contributed equally to the writing of this manuscript. w.l., s.t., j.l., f.m., r.k., d.k., h.m., and a.v. provided valuable input into the study design, analysis, and interpretation and reviewed the manuscript. supplementary data related to this article can be found online at doi:10.1016/j.bbmt.2018.07.008. hematopoietic stem-cell transplantation hla match likelihoods for hematopoietic stem-cell grafts in the u.s. registry on modeling human leukocyte antigen-identical sibling match probability for allogeneic hematopoietic cell transplantation: estimating the need for an unrelated donor source hla-haploidentical bone marrow transplantation with posttransplant cyclophosphamide expands the donor pool for patients with sickle cell disease reduction of severe acute graft-versus-host disease using a combination of pre transplant anti-thymocyte globulin and post-transplant cyclophosphamide in matched unrelated donor transplantation investigation of the freely available easy-to-use software 'ezr' for medical statistics consensus conference on acute gvhd grading measuring therapeutic response in chronic graft-versus-host disease: national institutes of health consensus development project on criteria for clinical trials in chronic graft-versushost disease: iv. response criteria working group report clonable t lymphocytes in t cell-depleted bone marrow transplants correlate with development of graft-v-host disease hla-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide durable engraftment of major histocompatibility complex-incompatible cells after nonmyeloablative conditioning with fludarabine, low-dose total body irradiation, and posttransplantation cyclophosphamide improved early outcomes using a t cell replete graft compared with t cell depleted haploidentical hematopoietic stem cell transplantation post-transplant high-dose cyclophosphamide for the prevention of graft-versus-host disease single-agent high-dose cyclophosphamide for graft-versus-host disease prophylaxis in human leukocyte antigenàmatched reduced-intensity peripheral blood stem cell transplantation results in an unacceptably high rate of severe acute graft-versushost disease alternative donor transplantation after reduced intensity conditioning: results of parallel phase 2 trials using partially hla-mismatched related bone marrow or unrelated double umbilical cord blood grafts haploidentical transplantation using t cell replete peripheral blood stem cells and myeloablative conditioning in patients with high-risk hematologic malignancies who lack conventional donors is well tolerated and produces excellent relapse-free survival: results of a prospective phase ii trial role of thymoglobulin in matched sibling allogeneic hematopoietic stem cell transplantation with busulfan and fludarabine conditioning in myeloid malignancies reduced-intensity conditioning with busulfan, fludarabine, and antithymocyte globulin for hematopoietic cell transplantation from unrelated or haploidentical family donors in patients with acute myeloid leukemia in remission antilymphocyte globulin for prevention of chronic graft-versus-host disease impact of donor and recipient cytomegalovirus serostatus on outcomes of antithymocyte globulin-conditioned hematopoietic cell transplantation optimal dose of rabbit thymoglobulin in conditioning regimens for unmanipulated, haploidentical, hematopoietic stem cell transplantation: long-term outcomes of a prospective randomized trial epstein-barr virus-related post-transplantation lymphoproliferative disorder after unmanipulated human leukocyte antigen haploidentical hematopoietic stem cell transplantation: incidence, risk factors, treatment, and clinical outcomes haploidentical transplant with posttransplant cyclophosphamide vs matched unrelated donor transplant for acute myeloid leukemia haploidentical transplantation for older patients with acute myeloid leukemia and myelodysplastic syndrome reduced-intensity transplantation for lymphomas using haploidentical related donors vs hlamatched unrelated donors low-dose post-transplant cyclophosphamide can mitigate gvhd and enhance the g-csf/atg induced gvhd protective activity and improve haploidentical transplant outcomes myeloablative versus reduced-intensity conditioning in patients with myeloid malignancies: a propensity score-matched analysis letermovir prophylaxis for cytomegalovirus in hematopoietic-cell transplantation strategies to prevent ebv reactivation and posttransplant lymphoproliferative disorders (ptld) after allogeneic stem cell transplantation in high-risk patients response to rituximab-based therapy and risk factor analysis in epstein barr virus-related lymphoproliferative disorder after hematopoietic stem cell transplant in children and adults: a study from the infectious diseases working party of the european group for blood and marrow transplantation risk factors for post-transplant lymphoproliferative disorder after thymoglobulin-conditioned hematopoietic cell transplantation post-transplantation lymphoproliferative disorders in adults key: cord-347064-ljd121no authors: josé, ricardo j.; brown, jeremy s. title: opportunistic bacterial, viral and fungal infections of the lung date: 2016-05-05 journal: medicine (abingdon) doi: 10.1016/j.mpmed.2016.03.015 sha: doc_id: 347064 cord_uid: ljd121no opportunistic infections are a major cause of morbidity and mortality in severely immunocompromised patients, such as those receiving chemotherapy or biological therapies, patients with haematological malignancy, aplastic anaemia or hiv infection, and recipients of solid-organ or stem cell transplants. the type and degree of the immune defect dictate the profile of potential opportunistic pathogens; t-cell-mediated defects increase the risk of viral (cytomegalovirus, respiratory viruses) and pneumocystis jirovecii infections, whereas neutrophil defects are associated with bacterial pneumonia and invasive aspergillosis. however, patients often have combinations of immune defects, and a wide range of other opportunistic infections can cause pneumonia. importantly, conventional non-opportunistic pathogens are frequently encountered in immunocompromised hosts and should not be overlooked. the radiological pattern of disease (best assessed by computed tomography) and speed of onset help to identify the likely pathogen(s); radiological imaging can subsequently be supported by targeted investigation including the early use of bronchoscopy in selected patients. rapid and expert clinical assessment can identify the most likely pathogens, which can then be treated aggressively, providing the best opportunity for a positive clinical outcome. opportunistic infections occur when a loss of established innate or adaptive immune responses allows an organism that is normally weakly virulent to cause infection. the type and degree of immune defect dictate which potential opportunistic pathogens are likely (table 1) . opportunistic lung infections are a major cause of morbidity and mortality for patients immunocompromised because of hiv infection, haematological malignancy, aplastic anaemia or chemotherapy treatment, or who are recipients of solid-organ or stem cell transplants, and also can complicate treatment with the new biological therapies for inflammatory conditions. immunocompromised patients also have an increased risk of infections caused by more conventional pathogens, which should be considered in the differential diagnosis. expert clinical assessment with early diagnosis and aggressive treatment are required for a positive outcome. computed tomography (ct) is more sensitive than plain chest radiography for defining the predominant pattern(s) of lung involvement. combined with knowledge of the patient's immune status (loss of t-cell or antibody-mediated immunity, or defects in neutrophil-mediated immunity), this can often identify the most likely pathogens. this article provides a concise overview of the most common opportunistic lung infections. conventional bacterial pathogens although the risk of opportunistic infection is high in immunocompromised patients, most pneumonias are related to the more key points c knowledge of the immune defect helps to narrow down the potential pathogens c computed tomography of the chest is better than radiographs at defining the radiological pattern of disease in immunocompromised hosts c in selected patients, early bronchoscopy increases the yield of microbiological identification of a potential pathogen c prolonged high-dose glucocorticoids (>20 mg/day for >21 days) predispose to pneumocystis jirovecii pneumonia (pjp) c biological agents are associated with specific immune defects that increase the risk of opportunistic lung infections (e.g. tumour necrosis factor-a inhibitors and risk of mycobacterial disease, endemic fungi and legionella pneumophila; anti-cd20 drugs and mycobacterial disease, cytomegalovirus pneumonitis and pjp) c due to the increase in azole resistance of aspergillus fumigatus, combination of an azole with an echinocandin antifungal agent is recommended in immunocompromised hosts with severe invasive pulmonary aspergillosis conventional bacterial pathogens. these are particularly common after a viral illness. they usually present similarly to pneumonia in immunocompetent individuals 1 with fever, respiratory symptoms, focal consolidation and rapid rises in inflammatory markers. the major risk factors are neutropenia, antibody deficiencies and high-dose corticosteroids. the organisms involved are more diverse than those seen in conventional pneumonia and are more likely to be resistant to first-line antibiotics. they include both gram-positive (streptococcus pneumoniae, staphylococcus aureus) and gram-negative (e.g. pseudomonas aeruginosa, proteus species, escherichia coli, other enteric pathogens) organisms. reactivation of latent tuberculosis occurs in patients with t-cell immune defects. mycobacterium tuberculosis cultures and polymerase chain reaction (pcr) should be carried out on respiratory samples from immunocompromised individuals with pulmonary infiltrates who were born or live in countries with a high prevalence of tuberculosis. non-tuberculous mycobacterial infections can present similarly. mycobacterial infections are usually readily diagnosed by microscopy and culture or by biopsy of affected lung tissue. nocardiosis is an uncommon gram-positive bacterial infection with a high mortality when disseminated. there are over 80 nocardia species, but human disease is usually caused by the nocardia asteroides complex. nocardia is found in soil, decaying vegetable matter and stagnant water. inhalation is the most common route of entry so pneumonia is the most common infection. the main risk factors are defects in t-cell-mediated immunity (e.g. after transplantation), prolonged glucocorticoid therapy, malignancy, graft-versus-host disease (gvhd), diabetes mellitus, chronic granulomatous disease and alveolar proteinosis. nocardia pneumonia usually develops over weeks with cough, haemoptysis, weight loss, fever and night sweats but can be more acute. common radiological features are patches of dense consolidation or macronodules, frequently pleurally based. cavitation and pleural effusions are common. these appearances can be mistaken for metastases, mycobacterial infection or invasive aspergillosis. local spread to the pericardium and mediastinum, and haematogenous spread to brain, joints and soft tissue, occur in about half of patients. a rapid diagnosis can be made by identifying the characteristic beaded, branching gram-positive and weakly acid-fast filaments on microscopy of tissue or respiratory samples. blood and sputum cultures can be positive but require prolonged aerobic culture. susceptibility to antibiotics varies among the nocardia species, and treatment with two or three intravenous antibiotics may initially be necessary in immunocompromised individuals. trimethoprimesulphamethoxazole is firstline therapy, with carbapenems, amikacin, third-generation cephalosporins, tetracyclines or amoxicillineclavulanate as alternatives. the duration of treatment is prolonged (up to 12 months) in immunocompromised patients and with central nervous system (cns) disease. lower respiratory tract infections with the respiratory viruses (respiratory syncytial virus, parainfluenza, influenza, adenovirus, metapneumovirus, coronavirus, rhinovirus) are relatively common in immunocompromised patients with defects in t-cellmediated immunity. respiratory viruses usually cause bronchiolitis that presents with coryzal symptoms, cough, fever and dyspnoea. in a minority of patients, auscultation of the lungs reveals characteristic squeaks or wheeze. the chest radiograph is often normal or non-specific. ct classically demonstrates 'tree-in-bud' changes suggestive of small airways inflammation or diffuse ground-glass opacities. the diagnosis is confirmed rapidly using nasopharyngeal aspirate samples for viral antigen immunofluorescence or pcr for viral nucleic acids, the latter being favoured in immunocompromised hosts. if nasopharyngeal aspirate findings are negative, immunofluorescence or pcr of bronchoalveolar lavage fluid (balf) has a higher sensitivity. in the absence of pneumonia, mortality from respiratory virus infection is relatively low, although infection can persist for several weeks. treatment is supportive, but in immunocompromised hosts specific antiviral treatment is recommended ( table 2 ). in cases of severe infection, combination with intravenous immunoglobulin should be considered. viral infection, particularly influenza (including h1n1) has effects on lung host defences and predisposes to secondary bacterial infection, 1, 2 which in immunocompromised hosts (particularly after chronic glucocorticoid use, chemotherapy for cancer and haemopoietic stem cell transplantation (hsct)) can lead to more severe illness. clinically, this is suspected when there is relapse of fever, increased oxygen requirements and c-reactive protein concentration, and radiographic evidence of more dense consolidation or infiltrates. antibiotic treatment for secondary bacterial infection should cover the organisms most commonly encountered after influenza, including strep. pneumoniae, staph. aureus and haemophilus influenzae. novel viruses that have recently emerged, such as middle east respiratory syndrome coronavirus and avian influenza a strain h7n9, are potential rare causes of severe pneumonias in immunocompromised patients. 1 the herpesvirus cmv is an important cause of lung infection in patients with impaired t-cell-mediated immunity, such as transplant recipients. cmv infection is defined as active cmv replication irrespective of symptoms or signs, while cmv disease is infection associated with evidence of organ-specific disease. cmv infection in immunocompromised patients is usually caused by reactivation of latent cmv acquired in early life, but can also be primary infection in previously uninfected individuals, in whom it is often more severe. pneumonitis is an important complication and commonly presents with an insidious onset of fever, malaise, cough and dyspnoea with hypoxia. classic features on ct are symmetrical peribronchovascular and alveolar infiltrates predominantly affecting the lower lobes, but asymmetrical changes, consolidation and effusions are not uncommon. in suspected cmv infection/disease, cmv replication can easily be identified and the viral load determined by pcr or cmv pp65 antigen testing of blood or balf. cmv infection is also identified by culture of urine, throat and balf specimens. evidence of cmv reactivation does not always mean that concurrent lung disease is caused by cmv, and conversely cmv viraemia can occasionally be absent in patients with cmv pneumonitis. cmv pneumonitis is more likely with high-level viraemia, especially if the viral load increased rapidly. cmv pneumonitis can be confirmed by finding inclusion bodies in balf cells or transbronchial or video-assisted thoracic surgery (vats) biopsy samples. first-line treatment of cmv pneumonitis is intravenous ganciclovir or oral valganciclovir (unlicensed indication). secondline treatments include foscarnet (unlicensed indication), cidofovir (unlicensed indication) and maribavir. cmv immunoglobulin can be used as an adjunct to therapy in immunocompromised individuals. treatment efficacy is monitored by measuring blood cmv viral load, with treatment usually continued for at least 2 weeks after resolution of viraemia. other herpesviruses, such as herpes simplex virus (hsv), varicella zoster (vzv) (both associated with the characteristic rash) and human herpesvirus (hhv) 6, are rare causes of diffuse pneumonitis similar to cmv in the immunocompromised host. firstline treatment of hsv and vzv is with aciclovir, but valaciclovir, famciclovir (licensed for hzv, unlicensed for hsv), cidofovir (licensed for hsv) and foscarnet (licensed for hsv, unlicensed indication for hzv) can also be used. no drug has been specifically approved for the treatment of hhv-6, but ganciclovir and foscarnet are recommended by experts for the treatment of severe hhv-6 infection. 3 treatment options for fungal pneumonias are listed in table 3 . pneumocystis jirovecii (formerly p. carinii) pneumocystis jirovecii pneumonia (pjp) is the most common aids-defining illness (cd4 counts <200 cells/mm 3 ) but is also important in non-hiv immunocompromised patients with defects in t-cell-mediated immunity or who are taking prolonged high-dose systemic glucocorticoids. clinical presentation is classically with slowly increasing dyspnoea, dry cough and hypoxaemia, with few physical or radiological findings, but can be more rapidly progressive fulminant disease. exercise-induced oxygen desaturation is a sensitive clinical marker. chest radiographs may show diffuse, bilateral, interstitial infiltrates but can be normal, whereas high-resolution ct is much more sensitive and often shows extensive ground-glass opacities with an apical distribution and peripheral sparing. pneumatocoeles are not uncommon, and chronic infection can lead to bizarre-looking cystic changes. p. jirovecii cannot be cultured, and diagnosis requires identification of the organism in induced sputum or balf by microscopy with giemsa and grocott staining. immunofluorescence and pcr techniques increase the diagnostic yield, but false-positive pcr can occur because of lung colonization by p. jirovecii. p. jirovecii can be found in balf for 48e72 hours after starting empirical treatment. first-line treatment is high-dose trimethoprim esulphamethoxazole for 21 days, with adjunctive corticosteroids for severe hypoxaemia (po 2 <8 kpa) ( table 3) . second-line therapies include clindamycin plus primaquine, pentamidine, atovaquone, or trimethoprim plus dapsone. prophylaxis with trimethoprimesulphamethoxazole or nebulized pentamidine is recommended in patients with hiv infection (cd4 count <200 cells/mm 3 ), transplant recipients (solid-organ and hsct) and patients receiving prolonged high-dose glucocorticoids (>20 mg/ day for 21 days or longer). mortality is approximately 10%. aspergillus species are ubiquitous and are continuously inhaled but usually establish infection only when there are major defects in phagocyte function, such as severe and prolonged neutropenia (e.g. after hsct or aplastic anaemia), in patients taking highdose glucocorticoids, or with haematological malignancy or chronic granulomatous disease. chronic gvhd is also a significant risk factor and, rarely, patients with chronic lung disease or milder forms of immunosuppression develop semi-invasive forms of aspergillosis. the most common infective species is aspergillus fumigatus. the respiratory tract (including the sinuses) is most often affected, although blood-borne spread to internal organs (especially the cns) and skin can occur. the classic presenting triad in invasive pulmonary aspergillosis (ipa) is fever, chest pain and haemoptysis, although fever alone or various respiratory symptoms can occur. aspergillus has a predilection for growing into blood vessels, potentially causing fatal massive haemorrhage. chest radiographs show patchy infiltrates or nodules that can cavitate. ct features include macronodules (single or multiple, with or without cavitation) or patchy consolidation. nodules can show the 'halo' (surrounding ground-glass infiltrates caused by haemorrhage) or 'air-crescent' (cavitation around a fungal ball) signs. when the patient's immune function recovers, fungal balls can form in a walled-off cavity created by the invasive phase of the disease. other manifestations of invasive aspergillus infections affecting the lung include: aspergillus tracheobronchitis, in which infection is restricted to the tracheobronchial tree, causing a relentless cough. ct may show focal bronchial wall thickening and 'tree-in-bud' changes. bronchoscopy is diagnostic, identifying highly inflamed mucosa with necrotic white slough that is positive for aspergillus on culture and histology. chronic necrotizing pulmonary aspergillosis (cnpa) or chronic cavitary pulmonary aspergillosis (ccpa), which are more indolent forms of invasive aspergillosis associated with mild immunosuppression or chronic lung disease. these present with a long history of cough and frequently with marked systemic symptoms. there is also a slowly progressive patch of consolidation with or without cavitation (cnpa) or an expanding dry upper lobe cavity with a thickened wall (ccpa). diagnosis of ipa is suggested by detection of galactomannan (a relatively specific cell wall component) or b-d-glucan (a cell wall component of many fungi including aspergillus and pneumocystis) antigen in blood or balf. false-positives galactomannan antigen results occur with concomitant treatment with b-lactam antibiotics. definitive diagnosis of ipa is made by positive culture for aspergillus and histopathological demonstration of tissue invasion on ct-guided or vats biopsy specimens. histology is highly sensitive, showing dichotomous (45 ) branching of septated hyphae on gomori methenamine silver or periodic acideschiff staining. however, histology specimens are often unavailable, and culture is relatively insensitive, so diagnosis is frequently made on clinical grounds (suggestive ct appearances, high-risk patient, with or without a positive galactomannan test). aspergillus antibodies have no role in the diagnosis of ipa but are positive in ccpa and sometimes in cnpa. there has been a worldwide increase in a. fumigatus resistance to azoles, 4 and treatment with a combination of an azole and an echinocandin antifungal agent may be necessary in immunocompromised hosts with severe ipa. non-aspergillus filamentous fungi filamentous fungi, including fusarium, zygomycetes, scedosporium and penicillium, can cause invasive pulmonary infections in immunocompromised patients with a clinical presentation similar to ipa. diagnosis is made by culture from respiratory samples or lung biopsy, and is important as some species are resistant to conventional antifungal agents. galactomannan and b-d-glucan cell wall antigen tests are negative in zygomycetes infections. mortality is high. direct pulmonary invasion by candida species is rare even in immunocompromised patients, despite frequent isolation from sputum. pulmonary infection usually occurs in neutropenic patients as haematogenous spread from infected indwelling vascular catheters or infections related to transplant surgery. lung nodules are often peripheral and sometimes very large. candida albicans is most commonly identified, but a range of non-albicans candida (e.g. candida parapsilosis, candida tropicalis, candida glabrata, candida krusei) can cause disease. a novel culture-independent test allows for the rapid detection of candida in blood, with a particularly high negative predictive value e positive results require confirmation by culture. 5 infected indwelling lines should be removed. cryptococcus neoformans pneumonia almost always affects only immunocompromised patients and can present with dyspnoea, cough and fever. hiv/aids (cd4 count <200 cells/mm 3 ) is the most common risk factor but cryptococcal pneumonia also occurs in other defects of t-cell-mediated immunity (especially after solid organ transplantation). radiological features include diffuse interstitial infiltrates, focal consolidation, discrete nodules and hilar lymphadenopathy. diagnosis is by microscopic identification (indian ink stain) or culture from respiratory tract samples. the lung is the port of entry for disseminated infection (usually cns), and neurological symptoms should prompt a lumbar puncture and cerebrospinal fluid culture. endemic fungi are found in specific geographical areas and cause primary infection by inhalation or inoculation of contaminated material (e.g. bat faeces). reactivation of latent infection can occur in immunocompromised patients, especially with defects in t-cell-mediated immunity, so a history of travel or residence in a high-risk area can be relevant. common endemic fungi causing pulmonary infections include histoplasma capsulatum, coccidioides (candida immitis, candida posadasii), blastomyces dermatitidis and sporothrix schenckii. presentation varies by pathogen but tends to mimic tuberculosis, with cavitating pneumonias, pulmonary nodules, enlarged mediastinal and hilar lymph nodes, or a miliary pattern. systemic dissemination is not uncommon in immunocompromised patients. diagnosis requires identification of the fungus in respiratory samples or biopsy material, including bone marrow aspirates. culture can take 6 weeks. hist. capsulatum can be rapidly detected with an antigen detection assay but this can cross-react with other endemic fungi. serology identifies patients with previous exposure for most fungi but is not reliable in immunocompromised patients. mortality is high without timely appropriate treatment. community-acquired pneumonia viral pneumonia prevention and treatment of cancer-related infections genomic context of azole resistance mutations in aspergillus fumigatus determined using whole-genome sequencing t2mr and t2candida: novel technology for the rapid diagnosis of candidemia and invasive candidiasis key: cord-259194-9zllvfqb authors: cupples, sandra a. title: transplant infectious disease: implications for critical care nurses date: 2011-11-02 journal: crit care nurs clin north am doi: 10.1016/j.ccell.2011.08.001 sha: doc_id: 259194 cord_uid: 9zllvfqb infection is an important issue for critical care nurses as they care for patients throughout all phases of the transplant continuum: potential organ donors, transplant candidates, and transplant recipients. this article has reviewed salient issues relative to infections in each of these patient populations, including patients with vads, and has highlighted key points pertaining to bacterial, viral, and fungal infections. while they are on the transplant waiting list. urinary tract infections are common in kidney and pancreas transplant candidates. kidney transplant candidates are also at risk for infections in the native kidneys and occult abscesses. liver transplant candidates may have intra-abdominal infections or aspiration pneumonia. pneumonia is also common in the heart and lung candidate populations. hospitalized candidates are at risk for catheter-or device-related infections, such as those associated with dialysis access devices or ventricular assist devices (vads). as of august 2011, there were more than 3100 candidates on the heart transplant waiting list in the united states. 2 to date, only 949 heart transplant procedures have been performed in the united states in 2011. thus the demand for donor hearts far exceeds the supply. 3 vads were developed to augment circulation in patients with end-stage heart disease. these devices have been approved by the food and drug administration for three purposes: to bridge patients to heart transplantation, to bridge patients to recovery of their native myocardial function, and to provide permanent support for patients who are not deemed to be suitable heart transplant candidates ("life-time" or "destination" therapy). 4 vads can support the right or left ventricle or both. they stabilize the patient's condition by increasing cardiac output, improving perfusion to vital organs, and restoring mobility. 5, 6 these devices are typically implanted through a median sternotomy incision and placed in a pre-peritoneal or intra-abdominal pocket. the major components of a vad are inflow and outflow cannulae, unidirectional valves, a polyurethane chamber (for pulsatile devices), and a pump or rotor. the device is connected to an external power source through a driveline that exits through the abdominal wall (fig. 1) . 7, 8 vads contain biomaterials and, unfortunately, none of these materials are biologically inert. 9 therefore, events that occur at the host-implant interface can trigger aberrant immune activation. when the patient's blood comes in contact with the foreign vad surface, t cells can become activated and initiate a defective proliferative response and subsequent activation-induced cell death. as a result, the patient's immune system is impaired and the patient may be more susceptible to infection. 4, 5, 7, 9 infection is a common complication of vad therapy. 6 vad-related infections may delay or prevent transplantation altogether and they are a major cause of morbidity and mortality in lifetime therapy patients. 10 -12 the most recent international society for heart and lung/mechanical circulatory support device registry data indicate that infection occurred in 32.5% of the 655 vad patients enrolled in this database and that patients with vad infections had a 7.9% mortality rate. 13 device-related infection rates reported in the literature have ranged between 13% and 80%. 5 potential infection sites include the surgical site or any component of the vad (driveline, device pocket, or pump membrane). driveline infections are the most common; however, more than half of all infections involve several device sites simultaneously. 7 vad infections may remain localized or become systemic. if the infection spreads to multiple sites, serious complications such as bloodstream infections, bacteremia, sepsis, and endocarditis can ensue. 7 device-, patient-, and mechanical-related factors can contribute to vad infections. device-related factors include the exposure of percutaneous drivelines to pathogens and the vad cavities and pockets that can harbor pathogens. these microorganisms can cause blood flow through the pump to become turbulent; this in turn enables the pathogens to adhere to the surface of the device. 4 patient-related risk factors for infection include older age, poor nutritional status, indwelling catheters, prolonged intubation, postoperative bleeding, blood transfusions, multiorgan dysfunction, comorbidities such as diabetes mellitus and obesity, prolonged hospitalization before vad implantation, and surgical reexploration. 4, 6, 7 mechanical trauma to the driveline exit site is frequently associated with late-onset (ͼ30 days after implantation) infections. driveline trauma occurs when, for example, the controller or battery pack is dropped or when the driveline is snared on an object. these accidents result in shearing or torsion injuries that can lead to infection. 12 device-related infections can occur at any time; however, the majority develop between 2 weeks and 2 months of implantation. 7 gram-positive pathogens, particularly staphylococcus species, cause most infections. 14 these organisms are able to form a protective biofilm that blunts the host immune response and enables them to attach to and grow on inanimate surfaces. 8 fungal and gram-negative bacilli, such as pseudomonas aeruginosa and the enterobacter and klebsiella species, are other causative agents; these particular pathogens are associated with poorer outcomes. 4, 7 the administration of broad-spectrum antibiotics often leads to the development of fungal infections. 7 the clinical manifestations of vad-related infections are varied. presentation may be subtle or acute. if a device-related infection is suspected, the patient must have a thorough evaluation that includes a comprehensive physical examination and extensive work-up including blood cultures with gram stains. if possible, cultures should be obtained before initiation of antimicrobial therapy. 7 other sources of infection, such as pneumonia, urinary tract or catheter-related infections, must be investigated appropriately. additional diagnostic tests are site specific. for example, ultrasound is used to evaluate suspected pump pocket infections; transesophageal echocardiograms are useful in the setting of vad-related endocarditis. table 1 lists the typical signs and symptoms of devicerelated infections and potential treatment options. the evidence regarding the impact of device-related infection and posttransplant outcomes is mixed. some studies have indicated that these infections do not reduce 1-year 15 or overall 7, 16 survival. other studies have found that serious device-related infections can persist into the posttransplant period 7, 17 and are associated with decreased early 11 and long-term 17 posttransplant survival. although assist devices are often associated with infection, the benefits of this life-saving therapy are thought to outweigh the infection risk. 7 the major clinical implications for pre-and postoperative nursing care are listed in table 2 . infections can be transmitted via the allograft itself. 18 a donor-derived disease transmission is defined as "any disease present in the organ donor that is or has the potential to be transmitted to at least one of the recipients." 19(p234) donor-derived infectious diseases are rare. unexpected transmissions, that is, those that were either unrecognized in the donor or for which the donor was not screened, occur in fewer than 1% of all solid organ transplantation procedures. 19 although rare, these infections cause significant morbidity and mortality. factors that promote infection in potential organ donors include the use of medical devices and the treatment of patients in certain units that have high rates of bacterial contamination. 20 it is important to note that treatment of donor infections itself can further increase the potential donor's risk of iatrogenic infection, for example, via the insertion of intravascular catheters for antimicrobial therapy, the administration of immunomodulating medications such as corticosteroids, and prolonged hospitalization. 21 for a number of reasons, infections in potential organ donors may be difficult to diagnose: • the donor may not have the clinical manifestations of infection due to insufficient numbers or virulence of pathogens. • hemorrhage or aggressive fluid resuscitation may dilute both organisms and serologic infection markers such that they are undetectable by conventional laboratory tests. • the donor may not mount a fever response because brain death causes loss of temperature control and poikilothermia (a phenomenon whereby body temperature decreases to that of the environment). • the donor's white blood cell count may be already elevated due to trauma, tissue inflammation, or medications such as corticosteroids. 21 as a consequence, the diagnosis of infection may rely on culture and urinalysis reports, polymerase chain reaction (pcr) and nucleic acid testing results, characteristics of sputum, and changes in chest radiographs and computed tomography (ct) scans. 21 potential organ donors undergo a rigorous infectious disease evaluation. organ procurement and transplantation network (optn) policies mandate that potential donors must be screened for the following pathogens: human immunodeficiency 22 potential heart donors are screened for toxoplasmosis. many donors are also screened for nosocomial infections such as methicillinresistant staphylcoccus aureus or vancomycin-resistant enterocci. because infection can be transmitted via transfusions, serologic testing is typically performed both before and after a potential donor receives blood products. in addition, family members are questioned about the potential donor's infection risk, including prior infection exposure, history, and immunizations; travel to endemic areas; and risky behaviors such as intravenous drug abuse. table 3 displays donor organ acceptance and exclusion criteria based on the results of infectious disease screening. the acceptance of organs from donors with known infections with or exposure to hiv, hepatitis, or other viruses remains controversial. 21 given that the number of transplant candidates on the waiting list far exceeds the number of available organs, strategies to expand the donor pool include accepting donors with certain infections, higher-risk serological profiles, and social histories suggestive of prior exposure to bloodborne infections as well as donors who may be more at risk for transmitting infections (eg, older donors and donors with long icu stays). 20 informed consent effective treatment of bacterial infections in potential organ donors can result in successful transplantation. 21 box 1 displays important principles of antibiotic selection and administration for potential organ donors. the organ procurement organization's (opo's) coordinator has major responsibilities regarding the prevention and treatment of infections and reporting known infections to transplant centers that could potentially receive organs from infected donors. infections that must be reported to the transplant center are listed in box 2. moreover, all antimicrobial agents that are given to the potential donor must be documented and reported to each transplant center that receives an organ from that donor. 21 when a transplant center is informed that one of its organ recipients is confirmed positive for or has died from a potential donor-derived transmissible disease, that center must notify, within one working day, the opo that procured that organ. the opo must then notify the optn. these reports are forwarded to unos and uploaded to the disease transmission advisory committee's (dtac's) secure website. 1. select a bactericidal antibiotic over a bacteriostatic antibiotic. 2. use medication that will most directly target the identified bacteria to prevent the removal of harmless bacteria, the promotion of selective overgrowth of fungi, resistant organisms, or abnormal bacterial strains (eg, clostridium difficile), and the development of gene mutations and highly resistant organisms. 3. substitute directed antibiotic for broad-spectrum agent once sensitivities are available. 9. consider antibiotics specifically effective against anaerobic bacteria in the setting of facial injuries, pulmonary aspiration, or contaminated wounds from an injury scene. 10. administer antibiotics intravenously to maximize bioavailability. 11. adjust antibiotic doses in the setting of renal failure, hepatic failure, and older donor age. dtac data indicate that, between 2005 and 2007, there were 80 donors with reported possible donor-derived infectious disease transmission, 30 recipients with confirmed (proven, probable, or possible) donor-derived infections, and 14 recipient deaths attributed to donor-derived infections. these deaths were due to hepatitis c, tuberculosis, hiv, chagas disease, bacteremias, candidemia, strongyloides, and lymphocytic choriomeningitis virus. 18 there are three major factors that determine a transplant recipient's risk of infection. these include the patient's epidemiological exposure, either in the box 2 infections that must be reported to the transplant center 35 known conditions that may be transmitted by the donor organ must be communicated to the transplant center. these may include, but are not limited to, the following: hospital or in the community; the patient's current antimicrobial regimen, if any; and the patient's net state of immunosuppression, which is defined as "the combined effect of all of the factors that determine the patient's susceptibility to infection." 24(p138), 25 the net state of immunosuppression includes the patient's current immunosuppressive regimen (number and strength of antirejection agents), as well as any of the following concurrent factors: infection with an immunomodulating virus (eg, cmv or ebv); metabolic or autoimmune disorders (eg, diabetes mellitus); neutropenia or lymphopenia; disruption of mucocutaneous barriers; and surgical sequelae (eg, fluid collections). 25 approximately 80% of all transplant recipients have at least one significant infection during the first posttransplant year. 26 the three major groups of posttransplant pathogens are bacteria, viruses, and fungi (fig. 2) . bacterial infections are the most common, 26 followed by viral and fungal infections. bacterial infections frequently occur at the transplant site. bacterial pneumonias are common among all types of solid organ transplant recipients. nosocomial pathogens of particular concern include clostridium difficile, vancomycin-resistant enterococcus, methicillin-resistant staphylococcus aureus, and extended-spectrum ␤-lactamase gram-negative bacilli. common organ-specific bacterial infections and associated risk factors are listed in table 4 . most posttransplant viral infections are caused by two groups of pathogens: the herpes viruses (cmv, ebv, hsv 1 and 2, and varicella zoster) and the hepatitis viruses. viral infections are particularly deleterious because they have both direct and indirect effects. the direct effect is the clinical syndrome caused by the virus itself, such as cmv pneumonia or hepatitis. indirect effects include potential injury to the allograft, rejection, oncogenesis, and the virus's ability to alter the net state of immunosuppression, thereby increasing the patient's susceptibility to other opportunistic infections. the herpes viruses are characterized by latency. this means that once the virus is present, the patient will harbor the viral genome for life. immunosuppression, particularly augmented immunotherapy in the setting of rejection, can trigger replication of latent herpes viruses. 24 cytomegalovirus is the most important pathogen that affects transplant recipients. there is a bidirectional relationship between cmv and rejection. cmv can trigger rejection and the inflammatory effects of rejection and rejection therapy can increase cmv viral replication. the allograft is more likely to be affected by a cmv infection than a native organ. thus, liver transplant recipients with cmv infections are prone to develop vanishing bile duct syndrome, heart transplants recipients are at risk for coronary artery vasculopathy, lung transplant recipients are at risk for bronchiolitis obliterans, and so forth. the most common types of cmv disease are hepatitis, pneumonitis, and gastroenteritis. with regard to cmv serostatus, the risk of developing a posttransplant cmv infection is highest in cmv-seronegative recipients who receive an allograft from a cmv-seropositive donor and lowest in cmv-seronegative recipients who receive an allograft from a cmv-seronegative donor. recipients who receive potent antirejection therapy such as antithymocyte globulin are also at increased risk for developing a cmv infection. a concurrent critical illness can lead to the reactivation of a latent cmv infection; this is thought to be associated with proinflammatory cytokines and subsequent downregulation of the immune system. agents used to prevent or treat cmv infection include ganciclovir, valganciclovir, acyclovir, and cmv immune globulin. foscarnet is often used to treat ganciclovirresistant organisms. because cmv can be transmitted through blood transfusions, cmv-seronegative transplant candidates and recipients should receive cmv-negative, leukocyte-poor, or filtered blood products. 1, 24 given that most adults are ebv-seropositive, most posttransplant ebv infections in adults are reactivated from latent pretransplant infections. however, ebv-seronegative recipients can acquire an ebv infection through blood transfusions or community exposure. the incidence of posttransplant ebv infections is highest in multiorgan and intestinal transplant recipients followed, in decreasing order, by kidney-pancreas, lung, heart, liver, and kidney recipients. intravenous ganciclovir has been used as preemptive therapy for patients at high risk for ebv infections, for example, patients receiving antilymphocyte antibody therapy for rejection. the clinical sequelae of ebv infection range from a relatively mild mononucleosis-like syndrome to posttransplant lymphoproliferative disease (ptld). treatment options for mononucleosis include acyclovir. ptld is a set of syndromes that ranges from a benign, self-limiting polyclonal proliferation of b cells to an aggressive, malignant, monoclonal lymphoma. risk factors for ptld include pretransplant ebv-negative serostatus, primary ebv infection, high ebv viral load, cmv serostatus mismatch (recipient is cmv negative and donor is cmv positive), cmv disease, potent rejection treatment, and type of allograft. the incidence of ptld is highest in intestinal transplant recipients. treatment options for ptld range from antiviral agents (acyclovir, ganciclovir) and decreased immunosuppression for the benign polyclonal form to chemotherapy, radiation, resection, and decreased immunosuppression for malignant monoclonal lymphoma. although invasive fungal infections have the lowest incidence of all infections, they are associated with the highest morbidity and mortality rates. 27 risk factors for fungal infections include the use of high-dose corticosteroids and broad-spectrum antibiotics, rejection that requires increased immunosuppression, allograft dysfunction, and a simultaneous infection with an immunomodulating virus such as cmv. 24 two genera, aspergillus and candida, cause the vast majority of posttransplant fungal infections. together, these two pathogens account for more than 80% of invasive fungal infections. these infections typically present during the first month posttransplant, 27 but they can occur at any time. the most common fungal infection that involves the respiratory tract is invasive aspergillosis, which may affect approximately 30% of solid organ transplant recipients. 28 other portals of entry include the gastrointestinal tract and the skin. the risk of disseminated candidiasis is highest in neutropenic patients with central venous catheters who have received broadspectrum antibiotics and who have had prolonged icu stays. 29 liver transplant recipients are at highest risk for invasive candidiasis, followed, in decreasing order, by pancreas, lung, heart-lung, kidney, and heart transplant recipients. 29 pediatric transplant recipients are often at higher risk for posttransplant infections for a number of reasons, including: • lack of immunity to common pathogens such as cmv and ebv • incomplete immunizations • increased technical difficulty and prolonged transplant operative time due to pretransplant palliative surgeries • inability to close the abdomen or chest due to placement of a large allograft into a small child • social behavior of children in densely populated day care and school settings. 30 acute mediastinitis can develop after the implantation of mechanical circulatory assist devices or after heart, lung, and heart-lung transplantation. the risk of posttransplant mediastinitis is higher if the patient had a mechanical circulatory assist device or a total artificial heart as a bridge to transplantation. there are preoperative, intraoperative, and postoperative risk factors for mediastinitis. examples of preoperative risk factors include diabetes mellitus, prior sternotomy, renal failure requiring dialysis, prolonged hospitalization before the transplant surgery, and obesity. the risk of developing mediastinitis is more than double in patients with a body mass index greater than 30. intraoperative risk factors include blood transfusions and prolonged cardiopulmonary bypass, aortic cross-clamp, and operative times. examples of postoperative risk factors include surgical reexploration, prolonged icu stay, prolonged mechanical ventilation (ͼ24 -48 hours), having a tracheostomy, cardiopulmonary resuscitation, poor perioperative and postoperative glucose control, and low posttransplant cardiac output. 31 the major etiologic pathogens associated with mediastinitis include, in decreasing order, gram-positive cocci (staphylococcus aureus, staphylococcus epidermidis, enterococcus spp., streptococcus spp.), gram-negative bacilli (escherichia coli, enterobacter spp., klebsiella spp., proteus spp., other enterobacteriaceae, and pseudomonas spp.), and fungi (candida albicans). 31 the initial clinical manifestations of mediastinitis may be subtle: mild chest pain, and edema or erythema along the sternal incision. 24 the most common presenting symptom is fever; it may be associated with localized infection, erythema, cellulitis, purulent drainage, pleuritic-like pain, and sternal instability. diagnostic studies include ct scans, cultures, and laboratory tests. laboratory findings include elevations in the white blood cell count, c reactive protein, and procalcitonin. the latter test is particularly useful in distinguishing between rejection and infection. once mediastinitis is diagnosed, treatment should be initiated promptly. therapeutic options include surgical drainage/débridement, wound irrigation, tailored parenteral antimicrobial agents, and nutritional support. 31 in transplant recipients, central nervous system (cns) infections are among the most deleterious because they can be difficult to diagnose and treat. diagnosis is often challenging because presenting symptoms, such as mental status changes, seizures, focal neurologic deficits, and headache, may be blunted by immunosuppressive therapy. moreover, the neurotoxic effects of antibiotics, antiviral agents, and immunosuppressants themselves may make diagnosis even more complicated. 32, 33 the first step in diagnosing a suspected cns infection is a neuroimaging study to establish the presence, location, potential etiology, and characteristics of any lesion(s). magnetic resonance imaging studies of the brain or spinal cord or both are typically preferred to ct scans. neuroimaging studies are useful in determining if the infection is focal or nonfocal and if it involves the meninges. other diagnostic tests include cerebrospinal fluid analyses, electroencephalograms, viral polymerase chain reaction tests, cultures, and serologic tests. brain biopsies are rarely done, except in the setting of posttransplant lymphoproliferative disease and brain abscesses. the posttransplant interval, patient-specific risk factors, and the timing and evolution of clinical manifestations also help to inform the diagnosis. 32 cns infections may be caused by fungi, viruses, or bacteria. fungal infections carry the highest mortality rate-90% or higher-of all pathogens. 32 most brain abscesses are associated with aspergillus. these abscesses tend to occur early in the posttransplant period, particularly in recipients who have multiple risk factors for infection such as surgical reexploration, dependence on mechanical ventilation or dialysis, or retransplantation. 33 unlike meningitis in immunocompetent patients, posttransplant meningitis is typically caused by fungi. in this setting, the patient often develops a systemic infection that subsequently spreads to the cns. viral cns infections may be associated with the reactivation of a latent virus, such as jc virus-induced progressive multifocal leukoencephalopathy, or they may be caused by a new exposure to a pathogen such as west nile virus. other pathogens commonly associated with posttransplant encephalitis include the herpes simplex virus, varicella zoster virus, ebv, and cmv. bacterial cns infections are more frequently caused by listeria and nocardia rather than more common bacterial pathogens. 32 due to the severity of cns infections in transplant recipients, an infectious disease consult, coupled with prompt diagnosis and treatment, is imperative. empiric, broad-spectrum antimicrobial agents are typically administered until the causative organism is identified. 32 immunosuppressive agents blunt the inflammatory response to infection; however, in most cases, transplant recipients with infections will have an increase in temperature. some infections, however, tend to occur in the absence of fever. these include pneumocystis pneumonia, focal fungal lung infections, and cryptococcal meningitis. 29 patients with a persistent fever greater than 38°c or acute pulmonary infiltrates or both are typically hospitalized for an infection workup. fevers of unknown origin are most commonly associated with cmv or ebv viral syndromes. it is important to note that fevers in transplant recipients may also be caused by drug reactions (particularly antilymphocyte therapy), pulmonary emboli, deep vein thrombosis, and rejection. rejection-induced fever typically occurs in lung transplant recipients. it occurs less commonly in kidney and liver transplant recipients and rarely in heart recipients. 24, 29 given that other human beings are the most frequent source of infection in the patient's environment, it is essential that nurses prevent the nosocomial transmission of respiratory viruses and the transmission of organisms through contaminated hands or inanimate objects. 29 in addition, it is important for critical care nurses to: • follow standard precautions • use aseptic techniques with vascular and urinary catheters • ensure that ventilator circuits, catheters, and dressings are changed per protocol • inspect all percutaneous catheter sites for signs of infection • maintain closed systems for urinary and suction catheters • keep the head of the bed elevated to decrease the risk of aspiration. • restrict access to patients by visitors and staff with colds or other contagious illnesses • avoid transporting transplant recipients through areas of hospital construction • promptly recognize and report the clinical manifestations of infections • obtain and report diagnostic test results in a timely manner • administer and document antimicrobial agents in a timely manner • assess and report the vaccination status of transplant candidates and recipients • know the cmv, ebv, and other pertinent serostatuses of the donor and recipient. 21, 29 infection is an important issue for critical care nurses as they care for patients throughout all phases of the transplant continuum: potential organ donors, transplant candidates, and transplant recipients. this article has reviewed salient issues relative to infections in each of these patient populations, including patients with vads, and has highlighted key points pertaining to bacterial, viral, and fungal infections. approach to the immunocompromised host with infection in the intensive care unit organ procurement and transplantation network organ procurement and transplantation network. transplants by donor type complications in patients with ventricular assist devices nonvalvular cardiovascular device-related infections ventricular assist devices in the adult ventricular assist device-related infections device-related infections: a review immunobiologic consequences of assist devices for the randomized evaluation of mechanical assistance for the treatment of congestive heart failure (rematch) study group. long-term mechanical left ventricular assistance for end-stage heart failure left ventricular assist device-related infection: treatment and outcome late-onset driveline infections: the achilles' heel of prolonged left ventricular assist device support mechanical circulatory support device database of the international society for heart and lung transplantation: third annual report -2005 infection in ventricular assist devices: prevention and treatment effect of left ventricular assist device infection on post-transplant outcomes infections during left ventricular assist device support do not affect posttransplant outcomes lvad bloodstream infections: therapeutic rationale for transplantation after lvad infection donor-derived disease transmission events in the united states: data reviewed by the optn/unos disease transmission advisory committee the epidemiology and prevention of donor-derived infections donor infection and transmission to recipient of a solid allograft bacterial infection during adult donor care available at: http:// optn.transplant.hrsa.gov/policiesandbylaws2 the ast infection disease community of practice. screening of donor and recipient prior to organ transplantation transplant complications: infectious diseases infection in solid organ transplant recipients bacterial infections in the early period after liver transplantation: etiological agents and their susceptibility clinical aspects of invasive candidiasis in solid organ transplant recipients new therapies for fungal pneumonia risk factors and approaches to infections in transplant recipients intestine transplantation mandell, douglas and bennett's principles and practice of infectious diseases neurologic manifestations of transplant complications infections of the central nervous system in transplant recipients drive-line exit site infection in a patient with axial flow pump support: successful management using vacuum-assisted therapy lung and heart-lung transplantation liver transplantation liver transplantation pancreas and kidney-pancreas transplantation pancreas and simultaneous pancreas-kidney transplantation key: cord-257114-pxmflm2c authors: burguete, sergio r.; maselli, diego j.; fernandez, juan f.; levine, stephanie m. title: lung transplant infection date: 2012-12-26 journal: respirology doi: 10.1111/j.1440-1843.2012.02196.x sha: doc_id: 257114 cord_uid: pxmflm2c lung transplantation has become an accepted therapeutic procedure for the treatment of end‐stage pulmonary parenchymal and vascular disease. despite improved survival rates over the decades, lung transplant recipients have lower survival rates than other solid organ transplant recipients. the morbidity and mortality following lung transplantation is largely due to infection‐ and rejection‐related complications. this article will review the common infections that develop in the lung transplant recipient, including the general risk factors for infection in this population, and the most frequent bacterial, viral, fungal and other less frequent opportunistic infections. the epidemiology, diagnosis, prophylaxis, treatment and outcomes for the different microbial pathogens will be reviewed. the effects of infection on lung transplant rejection will also be discussed. significant progress has been made since the first human lung transplant (lt) in 1963, and although survival after transplantation was initially plagued by issues of rejection, the advent of immunosuppression ushered in a new era in transplantation science and made long-term survival a possibility. with this success came the dilemma of post-transplant infectious complications, which, to this day, remain a significant contributor to overall morbidity and mortality in the lung transplant recipient (ltr). of all solid organ transplants, lungs are the most prone to infection, and this is likely due to several factors unique to the lung allograft. apart from constant exposure to the outside environment, the lungs are exposed to the colonized native airway and have been stripped of their usual mechanisms of defence including the cough reflex, bronchial circulation and lymphatic drainage. these factors, coupled with the induction of an immunosuppressed state collaborate to produce an environment that is ripe for the development of infection. apart from direct injury, infection leads to several complications that may then have an effect on overall survival including the development of both acute and chronic rejection with eventual graft failure. the immune modulating effects of some pathogens, such as cytomegalovirus (cmv), can also augment the risk of developing other infections further leading to increased morbidity. 1 a thorough and comprehensive screening and management approach must be undertaken to optimize the survival of these patients and minimize the risk of infectious complications. we present a review of the major infectious complications following lt as well as recent recommendations for the evaluation and management of these entities. the respiratory tract is the most common area of infection after lt, and bacterial pneumonia is the most common infectious complication. cmv is the second most common complication, and its occurrence is much higher than in other solid organ recipients. 2 it appears that the critical period for infections after lt is within the first 90 days. in a recent epidemiological study in which 51 ltr were followed for a mean of 38.2 months, 75% of infectious episodes occurred within the first year after transplantation, and nearly half (42%) occurred within the first 3 months. 3 bacterial disease accounted for the largest proportion of infections (48%) followed by viral, fungal and mycobacterial disease (35%, 13% and 4%, respectively). in the early post-lt period (days to 1 month), nosocomial organisms account for the majority of infections. following this period and for the next several months, at a time when immunosuppression is at the highest level, opportunistic organisms such as cmv and fungi account for the majority of infections. in the late post-transplant period, community-acquired bacterial and viral infections develop, although infection with health care-associated organisms remains common (fig. 1) . it is within the first year that infection makes the biggest impact on mortality. according to the registry of the international society for heart and lung transplantation, infection is listed as the leading cause of mortality, accounting for 31% of deaths within the first year after transplant. 5 thereafter, infection is a close second to bronchiolitis obliterans syndrome (bos) as a cause of death. recently, it has been increasingly recognized that infection may both predispose the airways to the development of bos and increase the mortality of those with bos, thus still contributing significantly to this mortality. 6 the lungs are unique organs in that they are constantly exposed to antigens from both the environment (inhaled antigens) and the bloodstream (blood-borne antigens). the upper airways and pulmonary tissue have defence mechanisms composed of physical barriers and cellular components. physical barriers include hairs in the nasal cavity, mucus secretions, cilia and turbulent airflow generated by the nasal cavity that prevent pathogens from reaching the lower airways. despite these barriers, pathogens may still reach and infect the pulmonary tissue. there are several risk factors that make ltr more vulnerable to infection (table 1) . immediately postsurgery, ltr may have disruption of normal physical barriers and are at risk of aspiration and infection (e.g. use of nasogastric and endotracheal tubes). 7, 8 there are also other important changes that happen postsurgery. first, during the surgical procedure of lt, there is a complete disruption of the bronchial circulation, and this may cause a loss of epithelium integrity, ciliary function and mucus production. 9 these effects are transient because of the development of collateral circulation but remain at risk of infection during the initial stages. [9] [10] [11] second, denervation of the allograft may suppress the cough reflex and promote bronchial hyperresponsiveness. 2 third, the lymphatic drainage of the allograft is also severed promoting stasis and oedema in the bronchial tissues impairing normal healing. 2 fourth, stenosis or necrosis may occur at the site of the bronchial anastomosis, which may in turn facilitate colonization and invasion by opportunistic pathogens and decrease the clearance of secretions beyond the anastomosis. 12 at the cellular level, the ltr is vulnerable to infection due to the immunosuppression regimen used to prevent rejection affecting multiple inflammatory cellular lines and cytokines. the regimen consists of induction agents (medications used immediately post-transplant) and maintenance agents for prolonged use. because immunosuppression is needed indefinitely, ltr has a life-long increased risk for opportunistic pathogens to proliferate and cause significant complications. the maintenance immunosuppression regimen consists typically of a calcineurin inhibitor, an antimetabolite and corticosteroids. 13, 14 the calcineurin inhibitors used in lt are cyclosporine a and tacrolimus. cyclosporine a binds to cyclophylin preventing the activation of the nuclear factor of activated t-lymphocytes (t cells) by calcineurin. tacrolimus binds to fk-binding protein 12 inhibiting calcineurin and preventing the activation of the nuclear factor of activated t cells. 13, 15 by reducing the activation of nuclear factor of activated t cells, both drugs reduce the production of interleukin-2 limiting the clonal expansion of activated t cells (fig. 2) . 16 azathioprine and mycophenolate mofetil (mmf) are the commonly used antimetobolites after lt. azathioprine, a derivative of 6-mercaptopurine, inhibits both ribonucleic acid and deoxyribonucleic acid production, reducing the proliferation of both t cells and b-lymphocytes. mmf is a prodrug of mycophenolic acid, an inhibitor of the inosine monophosphate dehydrogenase (fig. 2 ). this enzyme is responsible for the synthesis of guanine nucleotides, which both t cells and b-lymphocytes are critically dependent of. 17 other maintenance agents that have been used less frequently to maintain immunosuppression include sirolimus and everolimus. sirolimus binds to the fk-binding protein 12 and through the mammalian target of rapamycin pathway prevents the synthesis of deoxyribonucleic acid and proteins by t cells (fig. 2) . 18 through an independent mechanism, sirolimus also affects b-lymphocytes and decreases cytokine and antigen production. 19 everolimus reduces the mammalian target of rapamycin kinase activity, inhibiting the downstream pathways of proliferation and activation of t cells. 20 finally, through the alteration of gene transcription factors, corticosteroids can exert a wide variety of immunosuppressive effects: interruption of antigen presentation, changes in the production of cytokines and alteration in the proliferative responses of various cell lines. 21 the use of induction agents after lt varies among centres. these agents include okt3, antithymocyte globulin (atg), alemtuzumab and basiliximab. okt3 is a murine monoclonal antibody that inactivates the t cell receptor-cd3 complex preventing the activation of circulating t cells with a partial sparing of t regulatory cells. atg is a polyclonal antibody directed against lymphocytes. it depletes circulating lymphocytes through complement-mediated lysis and destruction by the reticuloendothelial system after opsonization. 13 basiliximab is a chimeric monoclonal antibody that targets the a subunit of the interleukin-2 receptor inhibiting the differentiation and proliferation of t cells. 22, 23 alemtuzumab is a murine monoclonal antibody that targets cd52. this receptor is present in macrophages, monocytes, b-lymphocytes and t cells among other inflammatory cells. the binding of cd52 causes complementmediated cytolysis and activation of pathways leading to apoptosis. 13 the use of okt3 is now significantly limited due to an increase risk of infection. [24] [25] [26] [27] for this reason, most centres have elected to use atg, basiliximab or alemtuzumab, in combination with corticosteroids for induction of immunosuppression after lt. 28 evaluation of large series of solid organ recipients has shown that this combination prevents graft rejection and improves survival. 29 atg does not increase the rate of infections in transplant recipients and has been associated with a survival benefit. 30, 31 basiliximab compared with atg does not increase the risk of infection and was safer than okt3 in heart and ltr. 22, 23, 26, 32 alemtuzumab was recently shown to improve survival compared with atg. 33 despite these positive outcomes, the immunosuppression is more profound during induction, and patients should be monitored closely for infection during this period. despite the removal of both lungs during bilateral procedures, residual colonization and/or infection can remain in the thoracic cavity, the bloodstream, the upper airways or the sinuses. those patients with cystic fibrosis (cf) present the highest risk for recipient-harboured infection due to the frequent colonization and infection with multiresistant microorganisms including bacteria (gram-negative rods and gram-positive cocci) and fungi. resistant gramnegative organisms pose perhaps the greatest risk, and some studies suggest an association between pretransplant colonizing organisms from patients with suppurative lung disease and pneumonias following lt. 34 the majority of recent data suggests that patients colonized with multi-drug-resistant pseudomonas appear to have acceptable outcomes, including survival following lt, and should not be excluded on that criterion alone. 35, 36 in contrast, a former subspecies of pseudomonas, now subspeciated as burkholderia cenocepacia due to its unique resistance patterns, can pose significant problems in transplant recipients. there have now been at least nine distinct genotypic variants (genomovars) identified in the burkholderia cenocepacia complex. 37 colonization with burkholderia cenocepacia complex (genomovar 3) can result in significant morbidity and mortality post-transplant and should be considered a strong relative contraindication to lt, 38, 39 although isolated reports of successful outcomes have been reported. 40 in one study of 75 patients, 38 there was a significant difference in 1-year-survival between those patients not infected (92%) and those colonized with a non-burkholderia cenocepacia strain (89%) compared with those colonized with burkholderia cenocepacia (29%). similar results of variable survival rates based on burkholderia cenocepacia species have been found in other studies. 37, 39 because of these overwhelming data, the majority of transplant centres will not transplant colonized or infected patients with this organism. when evaluating the potential lt donor, routine screening is done to prevent transmission of donorharboured infection to the recipient. 41 donor screening includes routine serology for viral infection including cmv, epstein-barr virus, varicella-zoster, hepatitis b and c, and human immunodeficiency virus, among others. in addition, the potential donor lungs are evaluated radiographically and bronchoscopically. despite these measures, infection may still occur. to potentially pre-empt the development of donortransmitted infection at the time of the transplant procedure, a culture swab or wash, or a portion of the donor bronchus is sent for culture. in contrast with some older studies, 42, 43 more recent data suggest that recovery of an organism from the donor lung, a schematic overview of the mechanisms of action of medications used for immunosuppression. il-2 is required for the activation of the mtor pathway and progression of the t cell cycle. both csa and tacrolimus reduce the activation of nfat, which in turn results in a decreased production of il-2. basiliximab is a monoclonal antibody that inhibits the il-2 receptor. sirolimus and everolimus inhibit the mtor pathway through inhibition of specific enzymes. alemtuzumab targets protein cd52 causing t cell dysfunction. both mmf and aza disrupt key elements of the deoxyribonucleic acid synthesis affecting the progression of the t cell cycle. aza, azathioprine; csa, cyclosporine a; fkbp12, fk-binding protein 12; impdh,inosine-50-monophosphate dehydrogenase; il-2, interleukin-2; il-2r, il-2 receptor; mmf, mycophenolate mofetil; mtor, mammalian target of rapamycin; nfat, nuclear factor of activated t-lymphocytes. including a positive gram stain, or subsequent growth in culture does not always translate into infection and/or poor outcomes in the recipient. 34, 44, 45 in one study of 80 ltr, the investigators noted that organisms were grown from 57% or 89% of donors for a total number of isolates of 149. 44 of these, most isolates were staphylococci or streptococci. post-transplant pneumonias were found in 41% of recipients in this study; however, pseudomonas, and not gram-positive organisms, was the most prevalent causative organism. the results of this study and others 45 suggest that the presence of organisms in the donor does not necessarily predict post-transplant pneumonia, and perhaps this donor criterion should be re-evaluated. despite these suggestions and because empirical bacterial prophylaxis was used in the majority of these studies, the general practice is to routinely initiate prophylactic, broad-spectrum antibiotics (regimens are discussed later) and then narrow the antibiotic therapy based on donor isolates. 41 any patient with suppurative lung disease, such as cf or bronchiectasis, being considered for lt will receive a bilateral procedure with attempts at avoiding infection from a remaining native lung. however, in those diagnoses where a single lt may be performed, such as chronic obstructive pulmonary disease or interstitial lung disease, the native lung may harbour infectious organisms that can infect the new graft, particularly when the patient is subjected to immunosuppression. alternatively, the native lung can develop severe infection leading to sepsis and further compromise. although attempts at avoiding this risk are undertaken by routine pretransplant screening, examples of infection that can be harboured in the native lung include bacteria, fungi (perhaps contained in a mycetoma) or non-tuberculous mycobacteria (ntm). 46 as part of the initial pretransplant evaluation, all potential transplant recipients should undergo careful screening for infection. although there may be some variation between transplant centres, routine screening includes serological measurement for cmv, epstein-barr virus, varicella-zoster, hepatitis b and c, and human immunodeficiency virus, and screening for latent tuberculous infection. the results obtained from this screening are used to assess the patient's overall candidacy for lt (e.g. human immunodeficiency virus is generally an exclusion) and also to stratify the patient for screening and prophylaxis in the post-lt period (e.g. cmv and epstein-barr virus). recommendations for recipient and donor presolid organ transplant screening are published from the american society of transplantation. 41 pneumonias comprise the most common cause of infection following lt, and bacterial pathogens remain the most common cause of all pneumonias. 34, 47 in a multicentre, prospective study from spain, with a median follow-up of 180 days, 85 episodes of pneumonia were documented in 236 ltr for an incidence of 72 episodes/100 lt years. 47 of these, bacteria were the most common pathogen accounting for 82.7% of the pneumonias. bacterial pneumonia is most common in the early post-transplant period (1-30 days) usually due to infection with health care-associated and nosocomial organisms (fig. 1 ). in the spanish study, 40 of 85 of pneumonias (44%) occurred in the first 30 days following transplant. nearly 3/4 of all bacterial pneumonias (72%) were due to gram-negative organisms-most commonly pseudomonas (incidence 118.6 episodes per 1000 ltr/year). staphylococcus aureus and acinetobacter infections were the second most common bacterial isolates (each with an incidence of 67.8 episodes/1000 ltr/year). the median time to development of gram-negative pneumonia was 31 days with a range of 3-394 days. grampositive cocci-related pneumonias also occurred in the early post-transplant period at a median of 35.5 days (range 2-486 days) post-transplant. other bacterial isolates from this and other studies span the spectrum of health care-acquired infectious organisms. similarly, p. aeruginosa was found to be the most common isolate accounting for 33.3%, staphylococcus aureus comprised 26.8%, and aspergillus 16%. 34 pneumonia is also seen in the late post-transplant period. throughout the lifespan of the ltr, ongoing contact with hospital settings, both outpatient and inpatient, and frequent antibiotic exposure commonly result in infections with health careassociated, often resistant, pathogens. communityacquired pneumonias can also develop in the late post-transplant period. 48 in a single-centre study, 14 out of 220 ltr (6.4%) developed invasive pneumococcal infection (pneumonia and/or sepsis) at a median of 1.3 years after transplantation (incidence rate: 22.7 cases per 1000 person-years). routine vaccination for pneumococcus with the pneumococcal polysaccharide vaccine is recommended both before and every 5 years following lt. 41 in general, the approach to suspected pneumonia at any time period post-transplant includes sputum, blood cultures and often bronchoscopy with bron-choalveolar lavage (bal), sterile brush and sometimes biopsy. the role of new biomarkers such as procalcitonin for diagnosis or follow-up has not been well established in the ltr. due to the high incidence of early post-transplant pneumonia, whether derived from the recipient, donor or nosocomially acquired, broad-spectrum postoperative prophylaxis is routinely used. prophylaxis in the post-transplant period varies by centre but typically includes a third generation cephalosporin and vancomycin and is then tailored to the results of donor and recipient cultures, or as clinically indicated for 7-10 days. prophylactic antibiotic treatment should be extended to 14 days for known pretransplant recipient colonization. for specific prophylactic regimens for viral and fungal pathogens, see later. treatment of bacterial pneumonia includes standard regimens as outlined by the american thoracic society and infectious disease society of america treatment for health care-acquired pneumonia. 49 in the setting of known prior colonization or infection, initial antibiotic selection may be based on prior culture and sensitivity results. typical antibiotics used should include coverage for gram-negative (including pseudomonas) and gram-positive (including staphylococcus aureus) pathogens. in general, 8-14 days of therapy is recommended. in the case of resistant organisms, inhaled aminoglycosides may also be added to the treatment regimen. pneumonia has significant impact on overall posttransplant survival and the eventual complication of chronic rejection. in the spanish study, attributable 1-year survival was reduced in those patients developing pneumonia of any aetiology (29.5% mortality) versus those without pneumonia (14% mortality), although bacterial pneumonia alone was not separated out in this analysis. these authors also found that the probability of survival during the first year of follow-up was significantly higher in the multivariate analysis in lt recipients who did not have a pneumonia episode compared with those that had at least one episode of pneumonia. 47 in the bonde et al. study, pneumonia was found to be an independent predictor of overall mortality. 44 viral infection after lt is common and classified into disease caused by cmv or caused by other community-acquired respiratory viruses (carv). a recent study showed that a viral pathogen was responsible for 25 of 71 infectious episodes in a cohort of ltr, with cmv accounting for 68% of those cases. additionally, the majority of cmv episodes occurred within the first 3 months following lt, while the majority of the later infections were due to influenza and occurred after 1 year (fig. 1) . 3 among the opportunistic infections following lt, cmv is the most prevalent and most important despite significant advances in both diagnosis and management. as well as contributing directly to both morbidity and mortality, mounting evidence suggests a relationship between cmv pneumonitis and chronic rejection in the form of bos and decreased survival despite treatment. 50 cmv seropositivity can range from 30% to 97% in the general population, and after infection, the patient will harbour the virus for life. of all solid organ transplants, ltr has the highest risk of developing cmv disease. 51 the incidence of cmv infection has been reported to range from 30% to 86% in post-ltr, with a mortality of 2-12%. 52 this increased incidence is thought to be due partly to the high viral load of cmv transmitted in the lymphatics of the lung compared with other solid organs, as well as the high level of immunosuppression required for lung allograft. the most important risk factor for the development of cmv infection is the donor-positive/recipientnegative serostatus of a transplant patient, as these patients will lack immunity to cmv. the lowest risk occurs in donor-negative/recipient-negative patients. 51 other important risk factors include type and intensity of both induction and maintenance immunosuppression, concurrent infections, rejection and host factors such as age or comorbities. 51, 52 there is almost a symbiotic relationship between rejection and cmv infection. both of these individual processes induce a cytokine cascade that in essence promotes the development of the other. tumour necrosis factor-alpha, a key signal in the reactivation of cmv from latency, is released during allograft rejection, thereby facilitating the onset of viral replication and subsequent infection. conversely, infection of the vascular endothelium and smooth muscle by cmv leads to an upregulation of adhesion molecules promoting an increase in the quantity of inflammatory cells in the graft and subsequent development of rejection. additionally, molecular mimicry and the production of anti-endothelial antibodies with cmv may also play a role in the development of rejection. 52 cmv serology of both donor and recipient must be checked prior to transplant. 53 there is an important distinction between cmv infection and disease. infection is defined as 'cmv replication regardless of symptoms', while disease is defined as 'evidence of cmv infection with attributable symptoms', such as 'a viral syndrome with fever and/or malaise, leukopenia, thrombocytopenia or as tissue invasive disease'. 51, 54 recent technologies have effected a shift in the diagnosis of cmv infection and disease. the previous method of diagnosis, pp65 antigen detection, has been replaced by quantitative nucleic acid-based amplification testing via polymerase chain reaction (pcr) for the recognition of viraemia by most centres, with 85% of institutions using this method for monitoring and diagnosis. 55 there are no universally accepted viral load cut-offs for positive and negative results, and that reported values may be dissimilar between different laboratories. despite this, current guidelines on the management of cmv in solid organ transplant patients do not clearly favour one test over the other and cite both as acceptable options for diagnosis. additionally, viral culture of blood or urine has a limited role for diagnosis and is not routinely recommended. 53 most recently, tests for cell-mediated immunity against cmv have shown promise for predicting risk of developing disease. lisboa and colleagues demonstrated that cell-mediated immunity to cmv, as shown by a cd8+ t cell response assay, was associated with decreased risk of developing disease in patients with detectable low-level viraemia. twenty four of 26 patients (92.3%) with a positive interferon-gamma release assay were able to clear their viraemia without disease compared with 5 of 11 (45.5%) in patients with a negative cell-mediated immunity at onset (p = 0.004). 56 in a similar study, the same group was able to show that a negative assay was associated with a higher chance of developing late-onset cmv after prophylaxis. in their study, cmv disease occurred in 2/38 (5.3%) patients with a detectable interferongamma response versus 16/70 (22.9%) patients with a negative response (p = 0.038). 57 there are two accepted approaches to the prevention of disease from cmv, universal prophylaxis and preemptive therapy, and although there are no randomized trials comparing one strategy versus the other in ltr, most centres favour the former or may sometimes employ both. 55 the first, universal prophylaxis, involves administration of antivirals to all transplant patients deemed to be at high risk by serostatus. the second, pre-emptive therapy, is comprised of monitoring at-risk patients for viral replication and administering antivirals at a predetermined level of replication in the hopes of treating patients prior to the onset of disease. a cochrane review comparing prophylaxis in different groups of solid organ transplant patients with antivirals versus placebo or no treatment showed a significant reduction in disease (relative risk 0.42), infection (relative risk 0.61), mortality from cmv disease (relative risk 0.26) and allcause mortality (relative risk 0.63). interestingly, the review also found a decrease in the risk of developing herpes-simplex virus, varicella-zoster virus and bacterial infections. 58 prophylaxis may not only be beneficial in decreasing direct morbidity and mortality from cmv disease but may also have secondary effects by decreasing the morbidity and mortality of both acute and chronic rejection. the cochrane review mentioned earlier failed to show a difference in acute rejection episodes, but other small studies have shown statistically significant differences in ltr specifically and it is generally believed that prevention of cmv decreases the risk for acute rejection. [58] [59] [60] the data for bos are more encouraging. a recent study by chmiel and colleagues was able to show a 23% absolute risk reduction of developing bos in a group of ltr on cmv prophylaxis as compared with a historical cohort that was not prophylaxed and a 35% absolute risk reduction compared with data in the literature (p = 0.002). 1 most centres provide prophylaxis for a period of 3-6 months after transplantation; however, the optimal duration of prophylaxis has not been well established and is currently under debate. 55 the guidelines recommend a minimum of 6 months for ltr. 53 recent data suggest that this window of prophylaxis should possibly be extended, especially for donor-positive/ recipient-negative patients. palmer and colleagues report the first randomized, placebo-controlled trial showing a decrease in the risk of cmv disease with extended prophylaxis. in this study, 136 ltr who completed 3 months of valganciclovir prophylaxis were randomized to an additional 9 months of valganciclovir versus placebo. the risk of cmv disease was reduced (32% vs 4%; p < 0.001) in the extendedcourse group versus the short-course group. there were also statistically significant reductions in cmv infection (64% vs 10%; p < 0.001) and disease severity as measured by viral load with extended treatment. acute rejection episodes, opportunistic infections, adverse events and cmv ul97 ganciclovir-resistance mutations were similar between both groups. 61 the international consensus guidelines list valganciclovir and ganciclovir (oral or intravenous (iv)) as the antivirals of choice for the prevention of cmv disease and state that cmv immunoglobulin may also be used in combination with these two, but there are limited data to support its use. 53 although foscarnet was commonly used in the past for cmv disease, the significant risk of nephrotoxicity with concomitant calcineurin-inhibitor use has made it fall out of favour for the relatively safer agents ganciclovir and valganciclovir. 55 and, although the recommendation for treatment of severe disease is still iv ganciclovir, the results of the valcyte in cmv disease treatment of solid organ recipients trial have made valganciclovir a viable choice in the treatment of less severe cmv. 53 the in cmv disease treatment of solid organ recipients trial randomized 321 solid organ transplant recipients with non-life-threatening cmv disease to either oral valganciclovir or iv ganciclovir. valganciclovir demonstrated non-inferiority in regard to clinical resolution of disease as well as eradication of viraemia in both the intent-to-treat and the per-protocol arms of the study. 62 the current guidelines recommend oral valganciclovir at twice-daily dosing or iv ganciclovir for the treatment of nonsevere cmv disease. as there are no efficacy data for valganciclovir in severe or life-threatening disease, iv ganciclovir is still the 'gold standard' for those patients. in both groups, serial monitoring of viraemia should occur optimally at 1-week intervals, and treatment should be continued for a minimum of 2 weeks and until viral eradication has been documented with two consecutive tests. the use of secondary prophylaxis is generally recommended for 1-3 months after treatment of disease. 53 infection with a carv is common after lt, and with the development of new diagnostic techniques, the incidence quoted in older literature is likely underestimated. a study of ltr undergoing serial surveillance and diagnostic bal over a 3-year period showed that a respiratory virus was isolated in 51.6% of patients on at least one bal sample. rhinovirus was the most common pathogen isolated, followed by parainfluenza, coronavirus, influenza, metapneumovirus and respiratory syncytial virus (rsv). 63, 64 carv is being increasingly recognized as contributors to significant morbidity in immunocompromised hosts and can cause severe and life-threatening pneumonitis. additionally, there appears to be evidence that infection with these organisms can also lead to a decrease in graft survival. a retrospective cohort study of 259 ltr followed over 5 years showed a significantly increased risk of developing bos or death from bos in the group that was diagnosed with a carv infection. 65 given the paucity of effective antiviral treatment for most of these viruses, early diagnosis is essential for both treatment and to minimize spread among other immunocompromised patients. with the exception of influenza and rsv, for which treatments exist, supportive care and a reduction in immunosuppression remain the cornerstones of care for the treatment of carv. a complete listing of all the viruses that commonly affect ltr would be beyond the scope of this article so we will focus on those that have the most clinical bearing, namely influenza, rsv, human metapneumovirus and parainfluenza. as it typically does not cause respiratory tract disease, we will not discuss epstein-barr virus, except to mention its known association with post-transplant lymphoproliferative disorder after lt. infection of normal hosts with influenza most commonly causes a self-limited disease with upper respiratory symptoms, myalgias and fever; however, infection in ltr appears to be associated with increased risk of lower respiratory tract involvement by either a primary viral or a concomitant bacterial superinfection. this was illustrated in a small series of ltr admitted for influenza where all appeared to have pulmonary parenchymal involvement on imaging and by bal as well as in another series by vilchez and colleagues, where 7 of 15 patients with influenza were found to have pulmonary infiltrates, 5 of which were attributed to a primary viral pneumonia after bal. 66, 67 novel h1n1 influenza appears to have similar clinical features, although there appears to be an increased rate of gastrointestinal symptoms such as nausea and diarrhoea; which may be prominent. 68 due to the increased severity of disease, all ltr and their household contacts should receive annual influenza vaccination for prevention of disease. 69 diagnosis is essential, and efforts should be made to establish the type, as specific therapy will depend on resistance patterns. 69 diagnosis of seasonal influenza is made by rapid antigen detection of nasopharyngeal swabs, but this method appears to be unsatisfactory for detection of novel h1n1 and molecular real-time pcr methods are currently approved for use when swine flu is suspected. 70 in addition to supportive care and isolation, treatment involves the use of the antiviral agents amantadine and rimantidine for susceptible influenza a strains, and zanamavir and oseltamivir for both influenza a and b strains. due to the variation in circulating strains from year to year, it is important to stay abreast of the current recommendations from the centers for disease control and prevention 71 for appropriate treatment. 72 in addition, given the prolonged viral shedding, the typical treatment course of 5 days may be insufficient in ltr, and prolonged therapy may be required. some experts advocate treating influenza even if symptom onset is greater than 48 h and treating until viral replication ceases. 73 treatment of novel h1n1 is limited by the resistance of the strain to the m2 inhibitors: amandatine and rimantidine. as such, current guidelines recommend treatment with oseltamivir or perhaps even zanamavir if resistance is suspected to this agent. iv or higher dose therapy is recommended for critically ill patients, and immunosuppression should be decreased. 63, 64 rsv by the age of 2, virtually, all children have been infected with rsv, although reinfection can occur throughout life, and early acquisition after transplant or with augmented immunosuppression is a risk factor for severe disease. 72 as with influenza, infection can vary from a self-limited upper respiratory illness to severe pneumonia and occurs through inhalation of infectious droplets and contact with fomites, making isolation precautions paramount for prevention. there are currently no available vaccines for rsv and no recommended therapies for prevention. due to a lack of data for effective antiviral treatment, the only universally accepted recommendations for therapy are supportive care and a reduction of immunosuppression. 72 ribavirin, which has shown in vitro activity against rsv, is approved for treatment of lower tract disease by showing benefit in stem cell recipients. 74, 75 there are otherwise no controlled studies showing efficacy with the use of inhaled ribavirin in transplant patients. despite this, inhaled ribavirin remains the most commonly used treatment for rsv with one report showing a multidrug regimen of ribavirin, steroids, rsv-iv immunoglobulin and palivizumab to be safe, effective and associated with stability of lung function. 76 two small case series have shown promise for parenteral and oral ribavirin in ltr. 77, 78 an optimal treatment strategy for disease due to rsv is yet to be determined, and further studies are needed to better delineate effective agents that can safely be used in the lt setting. like rsv, human metapneumovirus and parainfluenza are members of the paramyxovirus family and present similarly to rsv. although typically they are milder than rsv, they have been shown to cause severe disease and have also been associated with both acute rejection and bos. 67, 79, 80 real-time pcr is the diagnostic modality of choice, and a diagnosis should be pursued, as clinical features alone are not specific enough to distinguish between the carv. supportive care remains the mainstay of treatment although inhaled ribavirin appears to be increasingly used for the treatment of these pathogens in patients with lower respiratory tract involvement despite a lack of controlled trials. furthermore, some experts also consider the use of iv immunoglobulin with significant disease for both parainfluenza and human metapneumovirus. 72, 80 fungal infections are a common complication after lt with an estimated incidence of 15-35% and an overall mortality of 80%. 81 complications at the site of the anastomosis (i.e. stenosis or necrosis) create the ideal environment for these infections to thrive. other risk factors include the immunomodulatory effect of coexistent infections (i.e. viral) and neutropenia. [82] [83] [84] as previously mentioned, transmission of infection from donor to host after lt can occur, or the native lung may serve as a reservoir of fungal organisms during single lt. 85 this is particularly important in chronic obstructive pulmonary disease patients in whom the lung surfaces are irregular and may have colonized bullae. 84 pretransplant fungal colonization is common, especially in patients with cf and chronic obstructive pulmonary disease, and it has been associated with post-transplant fungal infection and bos, 86 although not all colonized patients develop active/invasive infection. 83 the most common fungal pathogens in ltr are candida and aspergillus species, while zygomycetes, scedosporium, fusarium, cryptococcus species, histoplasmosis and coccidiomycosis occur less commonly. in general, these infections are more prevalent during the first few months after transplantation and, in some cases such as with cryptococcus species, histoplasmosis or coccidiomyocosis, can present as a reac-tivation of a latent infection. fungal infections can manifest as invasive disease with a reported 1-year cumulative incidence of 8.6% in ltr. 87 similarly, disseminated disease, post-transplant empyema, and airway and anastomotic infection have been reported. aspergillus species are the most common cause of invasive fungal infection after lt with an incidence of 32%. 84 more than half the cases occur within the first six months following lt, 84 (fig. 1 ) and more often involve ltr than other solid organ recipients. 88 several species have been described as pathogenic: aspergillus terreus, aspergillus flavus, aspergillus fumigatus and aspergillus niger. among these species, aspergillus fumigatus remains the most common cause of invasive disease. 89 the majority of aspergillus isolates in sputum or bal represent colonization (23%), and only a fraction of these will develop invasive disease (<10%), which carries a high mortality. 69, 90, 91 in ltr, the risk of invasive pulmonary aspergillosis rises with airway colonization by aspergillus species. 84, 89, 92 colonization is found in up to 50% of patients with cf. despite higher colonization compared with other populations, these patients have lower risk of invasive aspergillosis, but a higher risk for aspergillus tracheobronchitis. 93 in addition to colonization, airway ischaemia and bos have also been implicated as risk factors for invasive aspergillosis. 84, 89, 92 disseminated disease has been reported with an incidence of 22%, occurring as reactivation from an occult focus and/or as a new post-transplant infection. 84 other less common manifestations, such as mediastinal masses, skin, softtissue, sinus, orbit, central nervous system, sternal wound and chest wall infections, have also been described. 89, 91 diagnosis there are limited data on the role of minimally invasive tests such galactomannan, pcr and 1,3-b-dglucan assay for the diagnosis of invasive aspergillosis in ltr. 94,95 1,3-b-d-glucan, a cell component of all fungi, has been used in the diagnosis of multiple invasive fungal infections, but unfortunately, the role in ltr has limitations. 96 diagnosis of invasive aspergillosis may require aggressive procedures (i.e. biopsy) to verify tissue involvement; however, this is not always possible, and often, the diagnosis is reached on evaluation of computed tomography chest findings and fungal staining/culture from bronchoscopy (i.e. bal). the radiological findings of invasive aspergillosis include consolidations, nodules, cavitary lesions and mass-like opacities, often with a 'halo sign'. 84 in cases where the diagnosis is not possible with a less invasive approach, a biopsy with fungal stain/culture and histopathology may be required. once the diagnosis of invasive pulmonary aspergillosis is made, computed tomography or magnetic resonance of the central nervous system is suggested to rule out disseminated disease. over the years, the use of antifungal prophylaxis has decreased the overall risk of aspergillosis. despite this, the risk of late infection after discontinuation of prophylaxis or even while using it is still present. 97 the treatment of pretransplant colonization has not been shown to reduce the incidence of post-transplant aspergillosis, but invasive disease in the pretransplant setting should be treated. 90 recent data has shown the superiority of voriconazole compared with amphotericin b deoxycholate in patients with invasive pulmonary aspergillosis, but solid organ transplant patients were poorly represented in the study. 98 a major concern with the use of voriconazole in ltr is the interaction with most of the immunosuppressants used in this population. tacrolimus, sirolimus and cyclosporine can potentially increase the serum concentrations of voriconazole. for this reason, close monitoring of drug levels is needed. other options for the treatment of invasive aspergillosis are posaconazole and itraconazole, but their roles as first-line agents are not well established. the echinocandins (caspofungin, micafungin and anidulafungin) have shown some in vitro activity against aspergillus species, but their utility as firstline antifungals for this infection has not been studied either. the evidence for combined therapy with two or more agents as initial therapy is limited and not recommended. despite several alternatives, voriconazole remains the standard therapy for invasive aspergillosis along with reduction of immunosuppression. 99 voriconazole levels should be monitored carefully, especially in cf patients where serum concentrations can be variable. 99, 100 in general, target trough levels should range between 1 and 5 mg/ml. duration is typically recommended for a minimum of 12 months and depends on clinical and radiographical improvement. finally, surgical resection might be indicated when there is progression of disease despite optimal antifungal therapy, life-threatening haemoptysis, sinus infection or lesions in the proximity of great vessels, pericardium or in the brain. 82 severe candidal infections can appear within weeks to months after transplant, especially in the presence of heavier donor or recipient colonization. 91 typically candida infections occur within the first 30 days after lt and appear to be the second most common cause of invasive fungal infection in ltr. 69 candidaemia usually occurs during the first 4 weeks and is often related to the intensive care unit stay and the surgical procedure; however, parenchymal lung infection is rare. 101 mortality for invasive candidal infections, excluding anastomotic infections, has been estimated at more than 50%. 102 cultures are essential for the diagnosis of candidal infection in ltr. identification of species and susceptibilities need to be obtained as intrinsic resistance and dose-dependent susceptibility has been reported in different candida species. 103 other methods such as b-d-glucan have not reached significant accuracy for clinical use, 104 while others such as pcr are still experimental. candida species are commonly found in the oropharynx and can potentially colonize the airway. their presence in respiratory secretions may make it difficult to differentiate between invasive infection and colonization. invasive lung infection with candida is very infrequent even in the lt recipient colonized with candida. 97 clinical suspicion, culture results and direct bronchoscopic findings should guide any decision for treatment of candidal infections. echinocandins and liposomal amphotericin b are the first-line agents for empirical therapy of suspected candidal infection. 69 this is especially true in ltr who are at risk of developing severe candidal disease. fluconazole has been put forward as an empirical agent as well but is frequently reserved for patients with mild-to-moderate disease, nonneutropenic and at low risk for candida glabrata and candida krusei, for which it has less activity. empirical therapy should then be adjusted based on susceptibilities. for candida albicans infections, fluconazole and echinocandins have been effective, but in widespread disease, amphotericin b might be considered. finally, the duration of therapy varies among patients and with the degree and severity of infection. in candidaemia, treatment can extend up to 2 weeks but may be even longer in cases of more invasive disease. 69 histoplasmosis, coccidioidomycosis and rarely, blastomycosis are endemic mycoses that can potentially cause infection in transplant recipients. when present in this population, pulmonary and disseminated disease can occur with a high mortality. 105 these are especially important in endemic areas of the united states such as the midwest for histoplasmosis and the southwest for coccidiomycosis. 106 histoplasmosis can present in the early or late posttransplant period as a consequence of reactivation of a latent infection, new exposure or donor-derived infection. 106 the diagnosis can be delayed, but in ltr, urinary antigen appears to be a better diagnostic tool than the fungal antibody serologies. 106 the presence of fever without a clear source should raise clinical suspicion for disseminated histoplasmosis in any transplant patient, especially when pancytopenia and absence of pulmonary manifestations are present. in patients whose explanted lung is found to have histoplasmosis, antifungal prophylaxis after transplant seems effective at preventing reactivation of this infection. 106 there is no clear consensus about the duration of prophylaxis, and 18 months has been reported to be effective. 106 coccidioidomycosis is typically acquired when patients are exposed to the desert soil of the southwestern united states and northern mexico. the most common mechanism of infection in lt recipients is reactivation, but donor-derived transmission has also been reported. 107 patients in whom there is evidence of prior coccidioidomycosis, either radiographically or serologically, may require lifelong antifungal prophylaxis after transplant. 91 cryptococcus infections can present in solid organ transplant recipients as a pulmonary or extrapulmonary process. 108 the incidence of cryptococcus infection in ltr has been estimated around 2% and has been commonly associated with exposure to pigeons and other birds. 90 interestingly, ltr may be less likely to have a positive cryptococcal antigen test in the setting of isolated pulmonary cryptococcosis. 38, 108 an immunosuppressive regimen containing a calcineurin inhibitor has been associated with decreased mortality possibly due to synergistic effects between calcineurin inhibitors and antifungal agents use to treat cryptococcus. 109 however, a recent study has reported the occurrence of an immune reconstitution syndrome-like illness in some transplant patients after the initiation of antifungal therapy for cryptococcal infection. 110 zygomycotic infections appear to be escalating in frequency in immunosuppressed patients, and this trend has been partially attributed to the increasing use of voriconazole for therapy and prophylaxis. 111 this infection is characterized by vascular invasion of affected tissues with subsequent infarction and necrosis. in ltr, it can manifest as bronchial anastomotic or parenchymal infection with a mortality of 87% in the latter. 112, 113 its management includes the combination of surgical debridement and antifungal agents. in the united states, 80% of transplant centres use antifungal prophylaxis, 114 and approximately 81% perform pretransplant surveillance for fungal colonization. 115 despite this, there is still no general consensus regarding the most appropriate prophylactic strategy in the peritransplant window. although there are no randomized trials evaluating their efficacy, several antifungal agents have been used for prophylaxis in ltr. for universal prophylaxis, voriconazole, itraconazole and amphotericin b are commonly used, while targeted prophylaxis with fluconazole (candida), voriconazole and itraconazole (aspergillus) are used based on the results of surveillance bronchoscopy. 114 in general, the choice for antifungal prophylaxis depends, in part, on the presence of specific risk factors such as colonization with aspergillus, presence of airway stents or ischaemia, single lung transplantation, cmv infection, hypogammaglobulinaemia or treatment of acute rejection. 69 despite a lack of controlled trials, several studies suggest potential prevention of invasive aspergillosis with the use of either compound of amphotericin b. 116, 117 inhaled amphotericin b has lower systemic toxicity, better delivery to the site of fungal exposure and a lower likelihood of resistance when compared with systemic antifungal therapy. 116, 118, 119 the data regarding voriconazole for prophylaxis in ltr is promising, especially given the excellent bioavailability, broad antifungal coverage and good drug levels achieved in lung tissue. 120, 121 unfortunately, the numerous drug interactions with some of the immunosuppressants, and its potential adverse effects may preclude its use as a first-line prophylactic agent. itraconazole has clinical effectiveness similar to the combination of voriconazole and inhaled amphotericin b and may have lower hepatotoxicity when compared with voriconazole. 114 duration of antifungal prophylaxis varies from centre to centre. the use of voriconazole or itraconazole for 3-6 months with or without amphotericin b has been shown to decrease the incidence of aspergillus infection after transplantation. 88 the use of inhaled amphotericin b is typically for 2 weeks or is discontinued at the moment of discharge. in cases where pretransplant fungal colonization is present, patients may be treated for several weeks before lt and continued for up to 3 months after transplantation. because ltr is at high risk for fungal infections, antifungal prophylaxis should be started in most patients after lt with careful consideration of sideeffects and interactions to improve outcomes and be guided by cultures from donor, graft and recipient. mycobacterial infection after lt is rare. previously, most of these infections were secondary to mycobacterium tuberculosis. 122 more recently, data have shown an increase in the incidence of ntm, particularly mycobacterium abscessus, ranging between 3% and 9%. 123, 124 chalermskulrat et al., reported higher isolation of ntm in end-stage cf patients undergoing pre-lt evaluation (19.7%) than in post-lt cf patients (13.7%). 124 colonization, especially when m. abscessus was isolated, was associated with an increased risk for invasive mycobacterial infection in cf patients. 124 over the last 10 years, multiple cases of m. abcessus in lt recipients have been reported with pleuropulmonary and disseminated disease. [125] [126] [127] in addition, there is an increase in both mortality and disseminated disease associated with m. abcessus in solid organ transplant recipients. 128 on the other hand, m. avium complex and other ntm infections are less common, and their impact on morbidity and mortality is less severe compared with m. abcessus. 129 if during the pretransplant evaluation, the clinical presentation and radiographical findings are suggestive of ntm infection, diagnostic testing and therapy should be considered before transplantation. in the cf population, the presence of ntm should not preclude lt, but careful monitoring for recurrence after transplant should be performed. 124 the diagnostic criteria of the american thoracic society and infectious disease society of america apply to pre-and post-ltr (symptoms, radiological findings and microbiology). 130 similarly, the antimicrobial therapy recommended in the ntm guidelines is applicable to ltr. 130 therapy for mycobacterial infection in the immunosuppressed patient can be problematic particularly due to drug interactions and increased toxicity. nevertheless, these infections can be controlled, and some patients achieve an appropriate response and cure. anastomotic tracheobronchitis is a unique form of pulmonary infection 131 that usually develops in the first 6 weeks to 3 months following lt. during the transplant procedure, the bronchial circulation is not reanastomosed, and thus, the bronchial anastomosis must receive collateral blood flow from the pulmonary circulation, is subject to ischaemia and may be susceptible to infection. this diagnosis is easily confirmed with bronchoscopic examination revealing purulence, ulcerations, pseudomembranes, necrotic material, dehiscence and sometimes narrowing at the site of the anastomoses, and histological and culture results. the organisms most commonly causing tracheobronchitis in this setting are bacteria-(pseudomonas, staphylococcus) and fungi aspergillus (an incidence of 32% and 20%, respectively) and candida. 84, 132, 133 treatment includes appropriate antibacterial and/or antifungal antimicrobials. the treatment of airway anastomotic infections with fungi is with a combination of both systemic and sometimes inhaled antifungal agents. 134, 135 for aspergillosis, the combination of voriconazole and nebulized amphotericin b along with reduction of immunosuppression has been advocated. 99, 134 duration of therapy for tracheobronchitis is usually determined by resolution under bronchoscopic surveillance. late sequelae may include stenosis and or stricture requiring intervention with balloon dilation or occasionally endobronchial stent placement. a study demonstrated a decrease in 5-year survival in single ltr who developed bronchial anastomosis fungal infections. 132 other types of bacterial infection described in ltr include those of the pleural space, blood stream and wounds, with organisms often isolated in the nosocomial setting, and clostridium difficile. pneumocystis jiroveci pneumonia (pjp) occurs exclusively in immunosuppressed states. the risk of infection is higher during the first 6 months after lt due to the degree of immunosuppression during this period. 136 cmv infection is also an independent risk factor for pjp. 137 despite this, pjp remains a rare complication after lt. 138 the low rate of infection is due to the use of prophylaxis with trimethoprimsulfamethoxazole as a first-line agent, and dapsone, pentamidine and atovaquone as alternatives. 139, 140 trimethoprim-sulfamethoxazole has been shown to have better tolerance, potentially treat a wider range of infections, and has fewer side-effects. 139 there is controversy regarding the duration of prophylaxis after transplant. a study revealed that the rate of pjp did not decline after 1 year of transplantation, suggesting that prophylaxis should be continued beyond this period. 141 ltr should receive at least 6 months of prophylaxis post-transplant, and if tolerated, adequately, it should be continued indefinitely. in those patients in whom prophylaxis has been discontinued, it should be resumed if the patient develops acute or chronic rejection requiring augmented immunosuppression. the standard therapy for pjp is trimethoprim-sulfamethoxazole in combination with corticosteroids. as previously noted, mmf is used frequently as part of the immunosuppression regimen after lt. interestingly, this medication has shown antimicrobial properties against several pathogens including pneumocysitis spp. 142, 143 in three comparative studies, none of a total of 1152 transplant patients who received mmf developed pjp compared with an infection rate of 1.8% in a similar group that did not receive mmf. [144] [145] [146] the mechanism for these effects remains unknown, but it is likely that mmf may benefit ltr by two different mechanisms. in lt, nocardia remains an important pathogen with a frequency of 0.6-2.1% and a directly attributable mortality of up to 30%. 147 it is important to note that some of these patients (60-100%) were on treatment with prophylactic trimethoprim-sulfamethoxazole, a medication to which nocardia is classically susceptible to, underscoring the resistance of some strains to prophylaxis therapy. 147 the treatment for nocardia is trimethoprim-sulfamethoxazole, but resistance has been documented and other alternatives have been used successfully: imipenem, amikacin, third generation cephalosporins, minocycline, moxifloxacin, linezolid and dapsone. 148 despite the relatively low frequency of nocardia in lt, because of the high risk of mortality and the ability to mimic other infections, clinicians must have awareness of this pathogen to improve an early diagnosis to initiate appropriate therapy. chronic rejection following lt is manifested pathologically by bronchiolitis obliterans and clinically by worsening obstructive dysfunction on pulmonary function, the bos. bos is the rate-limiting factor in long-term survival following lt, and up to 50% of ltr will develop bos. 5, 149 the aetiology remains unclear, although acute rejection is one of the identified risk factors. emerging evidence continues to point towards infectious aetiologies as important factors in the pathogenesis of bos. several different viral, bacterial and fungal pathogens have been implicated in this process. 150, 151 these findings are critical regarding the understanding the mechanisms of rejection and possible therapies to prevent it. cmv was the first pathogen linked to the development of bos. cmv pneumonitis is associated not only with bos but also with decreased survival despite treatment. 50 furthermore, there has been an absolute risk reduction in the development of bos with the use of cmv prophylaxis, supporting the evidence that this virus may play an important role in the pathogenesis of rejection. 1 carv infections, including rsv, human metapneumovirus and parainfluenza virus, were also identified as a significant risk factor for developing bos. 65, 67, 79, 80 bacterial colonization and infection may be a contributing risk factor to the development of bos. [152] [153] [154] [155] because macrolides are felt to slow the progression of bos, it has been postulated that this response is due to the potential treatment of a chronic infection with mycoplasma pneumoniae or chlamydia pneumoniae, 154, 156 although macrolide immunomodulation also plays an important role. it has been shown that a positive serology and pcr testing for chlamydia pneumoniae on bal samples increases the rate of bos and early mortality. 157, 158 supporting this theory further, a study recently demonstrated that macrolides can prevent the development of bos. 153 fungal pathogens have been also associated with the development of bos. 159 fungal pneumonitis and aspergillus colonization have been identified as independent risk factors for bos and mortality related to rejection. 151, 159, 160 moreover, the combination of lateonset aspergillosis and chronic allograft dysfunction was a risk factor for poorer survival. 132 despite several advances in surgical technique, immunosuppression and prophylaxis, infection continues to remain an important cause of death and disease in the ltr. although there are non-modifiable factors that are innate to the patient or to the nature of the procedure, there are several modifiable factors that can be recognized and changed so as to optimize the patient's chances for survival and further extend life. prompt recognition and treatment of these factors is paramount for appropriate management. prophylaxis strategies continue to evolve and show promise for several of the infectious agents. avoidance of these infectious complications may not only lead to a decrease in the direct consequences of infection but also to a reduction in the subsequent causes of ultimate graft failure including both acute and chronic rejection. antimicrobial resistance is a growing problem, and although newer antimicrobials will likely be of benefit, especially against viral and fungal pathogens, prevention of these diseases remains the best approach. careful consideration and further research are needed regarding the mechanisms by which infection and subsequent inflammation alters the immunoregulatory machinery of the host and subsequently leads to the development failure of the allograft. factors that are important in evaluating an infectious episode include time after transplant, immunosuppression, cmv serostatus, prophylaxis regimen and treatment for acute rejection. 3 given that outcomes appear to be improved with early recognition and treatment of disease, all practitioners must always maintain a high index of suspicion caring for these patients. ganciclovir/valganciclovir prophylaxis decreases cytomegalovirus-related events and bronchiolitis obliterans syndrome after lung transplantation epidemiology and management of infections after lung transplantation early and late infections in lung transplantation patients the registry of the international society for heart and lung transplantation: twenty-eighth adult lung and heart-lung transplant report-2011 bronchiolitis obliterans syndrome development in lung transplantation patients risk factors for icu-acquired pneumonia effect of nasogastric tubes on the nose and maxillary sinus pulmonary infection defense after lung transplantation: does airway ischemia play a role? airway epithelium of transplanted lungs with and without direct bronchial artery revascularization bronchial transsection and reanastomosis in pigs with and without bronchial arterial circulation anastomotic airway complications after lung transplantation immunosuppression for lung transplantation current trends in immunosuppression for lung transplantation conventional and novel approaches to immunosuppression use of cyclosporine in lung transplantation clinical pharmacokinetics and pharmacodynamics of mycophenolate in solid organ transplant recipients rapamycin blocks cell cycle progression of activated t cells prior to events characteristic of the middle to late g1 phase of the cycle rapamycin in transplantation: a review of the evidence clinical pharmacokinetics of everolimus corticosteroid effects on cell signalling basiliximab in lung transplantation: preliminary experience basiliximab versus rabbit anti-thymocyte globulin for induction therapy in patients after heart transplantation risk/benefit ratio of perioperative okt3 in cardiac transplantation risk factors for early, cumulative, and fatal infections after heart transplantation: a multiinstitutional study a randomized multicenter comparison of basiliximab and muromonab (okt3) in heart transplantation: simcor study induction therapy in lung transplantation: a prospective, controlled clinical trial comparing okt3, anti-thymocyte globulin, and daclizumab anonymous organ procurement and transplantation network (optn), scientific registry of transplant recipients (srtr) annual data report. department of health and human services, health resources and services administration, healthcare systems bureau, division of transplantation induction immunosuppression improves long-term graft and patient outcome in organ transplantation: an analysis of united network for organ sharing registry data rabbit antithymocyte globulin as induction immunotherapy in pediatric heart transplantation the impact of induction on survival after lung transplantation: an analysis of the international society for heart and lung transplantation registry basiliximab as an alternative to antithymocyte globulin for early immunosuppression in lung transplantation five-year outcomes with alemtuzumab induction after lung transplantation bacterial and fungal pneumonias after lung transplantation the impact of panresistant bacterial pathogens on survival after lung transplantation in cystic fibrosis: results from a single large referral centre survival of lung transplant patients with cystic fibrosis harboring panresistant bacteria other than burkholderia cepacia, compared with patients harboring sensitive bacteria impact of burkholderia infection on lung transplantation in cystic fibrosis survival after lung transplantation of cystic fibrosis patients infected with burkholderia cepacia complex clinical outcome following lung transplantation in patients with cystic fibrosis colonised with burkholderia cepacia complex: results from two french centres survival of burkholderia cepacia sepsis following lung transplantation in recipients with cystic fibrosis disease community of practice. screening of donor and recipient prior to solid organ transplantation bacterial colonization of the donor lower airways is a predictor of poor outcome in lung transplantation a review of lung transplant donor acceptability criteria impact of donor lung organisms on post-lung transplant pneumonia a positive donor gram stain does not predict outcome following lung transplantation native lung complications in single-lung transplant recipients and the role of pneumonectomy pneumonia after lung transplantation in the resitra cohort: a multicenter prospective study invasive pneumococcal infections in adult lung transplant recipients infectious diseases society of america. guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcareassociated pneumonia cytomegalovirus pneumonitis is a risk for bronchiolitis obliterans syndrome in lung transplantation cytomegalovirus in solid organ transplant recipients cytomegalovirus and lung transplantation international consensus guidelines on the management of cytomegalovirus in solid organ transplantation working group on infectious disease monitoring. american society of transplantation recommendations for screening, monitoring and reporting of infectious complications in immunosuppression trials in recipients of organ transplantation an international survey of cytomegalovirus management practices in lung transplantation clinical utility of cytomegalovirus cell-mediated immunity in transplant recipients with cytomegalovirus viremia cell-mediated immunity to predict cytomegalovirus disease in high-risk solid organ transplant recipients antiviral medications for preventing cytomegalovirus disease in solid organ transplant recipients cytomegalovirus prevention in high-risk lung transplant recipients: comparison of 3-vs 12-month valganciclovir therapy update and review: state-of-the-art management of cytomegalovirus infection and disease following thoracic organ transplantation extended valganciclovir prophylaxis to prevent cytomegalovirus after lung transplantation: a randomized, controlled trial oral valganciclovir is noninferior to intravenous ganciclovir for the treatment of cytomegalovirus disease in solid organ transplant recipients a prospective molecular surveillance study evaluating the clinical impact of community-acquired respiratory viruses in lung transplant recipients guidance on novel influenza a/h1n1 in solid organ transplant recipients respiratory viral infections are a distinct risk for bronchiolitis obliterans syndrome and death influenza pneumonia in lung transplant recipients: clinical features and association with bronchiolitis obliterans syndrome influenza and parainfluenza respiratory viral infection requiring admission in adult lung transplant recipients the novel 2009 h1n1 influenza virus pandemic: unique considerations for programs in cardiothoracic transplantation common infections in the lung transplant recipient rapid-test sensitivity for novel swine-origin influenza a (h1n1) virus in humans pneumonia and influenza death rates-united states, 1979-1994 diseases community of practice. rna respiratory viral infections in solid organ transplant recipients respiratory viral infections in transplant recipients community respiratory virus infections in immunocompromised patients: hematopoietic stem cell and solid organ transplant recipients, and individuals with human immunodeficiency virus infection ribavirin therapy in bone marrow transplant 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donor-to-host transmission of bacterial and fungal infections in lung transplantation assessment of infection risks prior to lung transplantation invasive fungal infections among organ transplant recipients: results of the transplant-associated infection surveillance network (tran-snet) the incidence of invasive aspergillosis among solid organ transplant recipients and implications for prophylaxis in lung transplants aspergillosis in lung transplantation: incidence, risk factors, and prophylactic strategies infections after lung transplantation antifungal prophylaxis in lung transplantation aspergillus infections in transplant recipients aspergillus infection in lung transplant recipients with cystic fibrosis: risk factors and outcomes comparison to other types of transplant recipients aspergillus galactomannan antigen in the bronchoalveolar lavage fluid for the diagnosis of invasive aspergillosis in lung transplant recipients molecular detection and species-specific identification of medically important aspergillus species by real-time pcr in experimental invasive pulmonary aspergillosis the (1,3){beta}-dglucan test as an aid to early diagnosis of invasive fungal infections following lung transplantation fungi and molds following lung transplantation voriconazole versus amphotericin b for primary therapy of invasive aspergillosis treatment of aspergillosis: clinical practice guidelines of the infectious diseases society of america voriconazole pharmacokinetic variability in cystic fibrosis lung transplant patients significance of blood stream infection after lung transplantation: analysis in 176 consecutive patients fungal infections after lung transplantation diseases community of practice. candida in solid organ transplant recipients approach to the diagnosis of invasive aspergillosis and candidiasis histoplasmosis in solid organ transplant recipients: 10 years of experience at a large transplant center in an endemic area posttransplantation disseminated coccidioidomycosis acquired from donor lungs cryptococcosis in solid organ transplant recipients calcineurin inhibitor agents interact synergistically with antifungal agents in vitro against cryptococcus neoformans isolates: correlation with outcome in solid organ transplant recipients with cryptococcosis an immune reconstitution syndrome-like illness associated with cryptococcus neoformans infection in organ transplant recipients emerging invasive zygomycosis in a tertiary care center: epidemiology and associated risk factors fungal infections in lung transplant recipients mucormycosis of the bronchial anastomosis: a case of successful medical treatment and historic review antifungal prophylaxis with voriconazole or itraconazole in lung transplant recipients: hepatotoxicity and effectiveness a survey of antifungal management in lung transplantation nebulized liposomal amphotericin b prophylaxis for aspergillus infection in lung transplantation: pharmacokinetics and safety feasibility, tolerability, and outcomes of nebulized liposomal amphotericin b for aspergillus infection prevention in lung transplantation safety of aerosolized amphotericin b lipid complex in lung transplant recipients invasive fungal infections in lung transplantation: role of aerosolised amphotericin b voriconazole prophylaxis in lung transplant recipients intrapulmonary penetration of voriconazole in patients receiving an oral prophylactic regimen tuberculosis in transplanted lungs the spectrum of mycobacterial infection after lung transplantation non-tuberculous mycobacteria in end stage cystic fibrosis: implications for lung transplantation mycobacterium abscessus chest wall and pulmonary infection in a cystic fibrosis lung transplant recipient lung transplantation in patients with cystic fibrosis and mycobacterium abscessus infection mycobacterium abscessus infections in lung transplant recipients: the international experience mycobacterium abscessus infection in solid organ transplant 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impact of invasive fungal diseases on survival after lung transplantation antimicrobial prophylaxis regimens following transplantation pneumocystis pneumonia should prophylaxis for pneumocystis carinii pneumonia in solid organ transplant recipients ever be discontinued? mycophenolate mofetil: effects on cellular immune subsets, infectious complications, and antimicrobial activity novel anti-pneumocystis carinii effects of the immunosuppressant mycophenolate mofetil in contrast to provocative effects of tacrolimus, sirolimus, and dexamethasone rs-61443 (mycophenolate mofetil). a multicenter study for refractory kidney transplant rejection anonymous mycophenolate mofetil for the treatment of refractory, acute, cellular renal transplant rejection the tricontinental mycophenolate mofetil renal transplantation study group. anonymous a blinded, randomized clinical trial of mycophenolate mofetil for the prevention of acute rejection in cadaveric renal transplantation nocardia infection in lung transplant recipients pulmonary nocardiosis: risk factors, clinical features, diagnosis and prognosis report of the ishlt working group on primary lung graft dysfunction part ii: definition. a consensus statement of the international society for heart and lung transplantation bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria effect of etiology and timing of respiratory tract infections on development of bronchiolitis obliterans syndrome pseudomonas aeruginosa colonization of the allograft after lung transplantation and the risk of bronchiolitis obliterans syndrome a randomised controlled trial of azithromycin to prevent chronic rejection after lung transplantation long-term azithromycin therapy for bronchiolitis obliterans syndrome: divide and conquer? impact of graft colonization with gram-negative bacteria after lung transplantation on the development of bronchiolitis obliterans syndrome in recipients with cystic fibrosis azithromycin therapy for patients with bronchiolitis obliterans syndrome after lung transplantation chlamydia pneumoniae infection after lung transplantation chlamydia pneumoniae serology in donors and recipients and the risk of bronchiolitis obliterans syndrome after lung transplantation aspergillus colonization of the lung allograft is a risk factor for bronchiolitis obliterans syndrome infections in lung allograft recipients: ganciclovir era key: cord-018943-5zf0eya3 authors: michels, guido; ruhparwar, arjang; welte, tobias; gottlieb, jens; teschner, sven; burst, volker; mertens, jessica; stippel, dirk; herter-sprie, grit; von bergwelt-baildon, michael; theurich, sebastian; vehreschild, janne; scheid, christoph; chemnitz, jens; kochanek, matthias title: transplantationsmedizin in der intensivmedizin date: 2011-09-22 journal: repetitorium internistische intensivmedizin doi: 10.1007/978-3-642-16841-3_24 sha: doc_id: 18943 cord_uid: 5zf0eya3 die postoperative intensivmedizinische behandlung von patienten nach herztransplantation basiert häufig auf erfahrung und deren weitergabe zwischen den transplantationszentren (hohe variabilität). ▬ die 5-jahres-überlebensrate nach herztransplantation beträgt ca. komplikationen nach herztransplantation (⊡ tab. 24.4) pumpversagen (»low-cardiac-output«-syndrom) ▬ die (r-/d+)-situation macht eine prophylaktische therapie mit ganciclovir/valciclovir erforderlich (tts-leitlinie level ii/iii). ▬ anti-cmv-medikamente werden nur präemptiv gegeben, wenn es anhalt für eine replikation (cmv-pp65 oder pcr positiv) gibt. ▬ auch diese strategie reduziert das risiko von cmv-reaktivierung und infektion. cmv-negative blutprodukte anfordern! wenn nicht möglich, kann auch cmv-unbekannt transfundiert werden ▬ bei ab0-inkompatibilität zwischen spender und empfänger gilt die transfusionstabelle (⊡ tab. 24.27) für blutprodukte ab tag 0 (blutbank über szt informieren!) new classification of chronic gvhd: added clarity from the consensus diagnoses diagnosis and treatment of transplantation-associated thrombotic microangiopathy: real progress or are we still waiting? the international society of heart and lung transplantation guidelines for the care of heart transplant recipients therapeutic drug monitoring of mycophenolic acid after lung transplantation--is it clinically relevant antifungal prophylaxis in liver transplant recipients infection in organ-transplant recipients accf/aha/ acp/hfsa/ishlt 2010 clinical competence statement on management of patients with advanced heart failure and cardiac transplant: a report of the accf/aha/acp task force on clinical competence and training consensusrecommendations for sirolimus in liver transplantation update on lung transplantation canadian cardiovascular society consensus conference update on cardiac transplantation 2008: executive summary focused update incorporated into the acc/aha 2005 guidelines for the diagnosis and management of heart failure in adults: a report of the american college of cardiology foundation/american heart association task force on practice guidelines: developed in collaboration with the international society for heart and lung transplantation international consensus guidelines on the management of cytomegalovirus in solid organ transplantation current concepts on cytomegalovirus infection after liver transplantation the survival benefit of liver transplantation evaluation of the lung allocation score in highly urgent and urgent lung transplant candidates in eurotransplant alternatives to heart transplantation key: cord-272835-6nx4f8ss authors: paulsen, grant c.; danziger-isakov, lara title: respiratory viral infections in solid organ and hematopoietic stem cell transplantation date: 2017-12-31 journal: clinics in chest medicine doi: 10.1016/j.ccm.2017.07.012 sha: doc_id: 272835 cord_uid: 6nx4f8ss respiratory viruses are common in solid organ and hematopoietic stem cell transplant recipients and a recognized cause of significant morbidity and mortality. epidemiology, risk factors, and attributable mortality in both populations are reviewed. in addition, virus-specific prevention and treatment options, including emerging investigational therapies, are discussed for respiratory syncytial virus, influenza, adenovirus, parainfluenza, and other respiratory viruses. common respiratory viral infections (rvis) are an important cause of morbidity and mortality following solid organ transplant (sot) and hematopoietic stem cell transplant (hsct). 1,2 rvis are typically caused by respiratory syncytial virus (rsv), influenza, parainfluenza, rhinovirus, adenovirus, and human metapneumovirus (hmpv). there is also increasing recognition of human coronavirus and human bocavirus in these populations. in addition, in sot and hsct patients, respiratory infections can be caused by viruses less commonly associated with the respiratory tract, such as cytomegalovirus (cmv), human herpesviruses (herpes simplex virus [hsv] 1, hsv2), and varicella zoster virus (vzv). this article focuses on the epidemiology, outcomes, and specific prevention and treatment options for rvis in sot and hsct patients. rvis are a well-recognized cause of morbidity and mortality following sot, especially within the thoracic transplant population. recent prospective surveillance of 98 lung transplant recipients in spain found an overall rate of respiratory viruses, asymptomatic and symptomatic, of 0.76 per patient-year and a significantly higher rate of 2.1 rvis per patient-year in symptomatic patients. 3 nasopharyngeal swabs collected from asymptomatic patients were positive 11.5% of the time compared with 55.4% positive in symptomatic patients. the most frequently detected rvis in symptomatic patients were picornaviruses, such as rhinovirus and enterovirus, at 43%, followed by coronavirus (16.7%) and influenza (16.7%). symptomatic rvi detection progressed to lower respiratory tract infection (lrti) in 40% of patients. 3 a prospective swiss study reported similar results with an rvi incidence of 0.83 per patient-year, detection of respiratory viruses in 14% of those screened and 34% of symptomatic patients, and rhinovirus/enterovirus as the most common rvi. 4 human bocavirus, identified in 2005, has been reported to cause upper respiratory infection (uri), fevers, wheezing, lrti, and diarrhea in normal hosts, and plays an unclear role in sot recipients. 5 parainfluenza, rsv, hmpv, and influenza were the most frequently found viruses in lrti and were associated with higher rates of hospitalization. analyses of rvi in lung transplant recipients have reported a rate of infection from 1.4% to 60%, with detection 5 times more frequent if symptoms were present (table 1) . 6,7 reported risks for rvi in lung transplant patients include increased calcineurin inhibitor levels, age less than 15 years, and underlying cause for transplant other than cystic fibrosis. 4, 8 as noted for sot patients, rvis in hsct recipients have been well-characterized causes of significant morbidity and mortality. reported incidences of rvi in hsct recipients vary between 4% in earlier reports using antigen detection and culture 9 and 20% to 40% using polymerase chain reaction (pcr) testing (see table 1 ). 2,10-12 bocavirus, as mentioned earlier, also plays an unclear role in the hsct population, with 1 report of disseminated bocavirus in a pediatric hsct recipient. 13 risk factors for progression to lrti include age greater than 65 years, lymphopenia, neutropenia, alternative/nongenoidentical sibling donor, and chronic graft-versus-host disease (gvhd). 10,14 several publications have attempted to delineate morbidity and mortality following rvi in thoracic transplant recipients, specifically with respect to acute rejection and chronic lung allograft dysfunction (clad)/bronchiolitis obliterans syndrome (bos). in the study from spain mentioned earlier, lrti was associated with significant change in lung function (forced expiratory volume in 1 second [fev 1 ]) at 1 and 3 months following infection and nested case-control analysis reported a significant association between rvi within 3 months and acute rejection (hazard ratio [hr], 6.54; confidence interval [ci], 1.47-29.08; p 5 .01). 3 alternatively, the swiss study with similar incidence rates found no such association. 4 clad compromises long-term survival following lung transplant and although an association with previous viral infection has been explored in published literature, a definitive link remains unclear. pooled incidence rates for clad in the meta-analysis mentioned earlier for virus-positive cases were 18% (9 out of 50 cases) compared with 11.6% (37 out of 319) in virusnegative cases, but because of limited number of overall events a link could not be confirmed. 6 however, there are published epidemiologic links between rvi and clad as well as data on biologically plausible mechanisms underlying a causal relationship. 5,15-17 in addition, a recent large retrospective cohort (n 5 250) of lung transplant recipients found an independent association between rvi and development of clad within 3 the next months (hr, 4.8; ci, 1.9-11.6; p<.01). 18 paulsen & danziger-isakov published attributable mortality caused by lrti in hsct from a respiratory virus varies, up to 28% to 30% in some reports, most commonly involving influenza, rsv, adenovirus, and hmpv. 10,19 important risk factors for mortality in the hsct population include lymphopenia, corticosteroids (>1 mg/kg), and viral or bacterial coinfection. 11 bos following allogeneic stem cell transplant is also associated with significant morbidity and mortality. the most recognized risk factor for bos following hsct is gvhd, but there is some evidence that rvis may play a role as well. 20,21 the array of diagnostic tools for rvis in immunocompromised patients has greatly increased over the last few years, providing increased sensitivity as well as decreased processing times. 22 in general, testing for rvis has moved away from viral antigen and culture methods and now relies heavily on molecular methods. real-time quantitative and qualitative reverse transcription pcr testing is a well-established method for identifying viral infection and is now incorporated into many of the guidelines in use. 23 multiplex platforms that test for multiple viruses simultaneously from a single sample are now common. as the sensitivity of molecular diagnostic tools has improved, the issues of asymptomatic viral shedding and sampling methods have become more relevant than ever. as noted earlier, asymptomatic viral shedding is seen frequently, including reports of asymptomatic rsv shedding in hsct recipients for 35 to 80 days and persistent rhinovirus detection for 8 to 15 months in lung transplant recipients. 3,18,24,25 the ability of current molecular testing methods to detect virus in samples with few viral copies combined with prolonged viral shedding in sot and hsct recipients can create a challenge for clinicians trying to determine whether a positive molecular test indicates a true pathogen. 26 the source or sampling method used is an important consideration. respiratory samples are routinely obtained from aspirates/washes or swabs from the nasopharynx (np) or oropharynx (op) as well as more invasive collection methods such as bronchoalveolar lavage (bal). however, the choice of upper respiratory sample collection methodology for optimal viral recovery is uncertain. in children hospitalized because of respiratory infections, sensitivity of rvi detection with np aspirates of 86% to 100% have been reported versus nasal swab sensitivity of 67% to 95%. 27 a similar comparison of np swabs with or without op swabs in immunocompetent children found a higher sensitivity of np swabs for rvi of 91% to 100%, which was greater than or equal to op swabs (83%-98%), with combined testing increasing detection by 2% to 9%, depending on the virus. 28 investigation in adults reported higher sensitivity with np washes (85%) than np swabs (73%) or op swabs (54%), and noted that maximal sensitivity was achieved through a combination of all 3 methods. 29 rvi prevention is a key component to minimize infections and subsequent complications. interventions can be patient specific, such as antiviral prophylaxis following transplant and immunization both before and following transplant. for the most common rvis, immunization is only available for influenza, as discussed later. vaccines for many of the other rvis are still under development and not commercially available. in addition, interventions at the level of the health care system decrease the incidence of rvi, including the appropriate use of respiratory or contact precautions, screening of visitors, and immunization of health care staff. 30,31 these interventions are included in the general us centers for disease control and prevention (cdc) recommendations to reduce health care-associated pneumonia. 32 prevention of rvi in hsct recipients is also addressed in the joint cdc, infectious diseases society of american (idsa), and american society for blood and marrow transplantation guidelines as well (http://asheducationbook.hematologylibrary. org/content/2001/1/392.long). 33 other interventions, such as universal mask use for all health care staff and visitors on an hsct unit, strict isolation of all patients, mandatory hand washing, and visitor restriction for children less than 12 years of age, have been shown to significantly reduce the incidence of rvis and are used at some centers. [34] [35] [36] virus-specific outcomes, prevention, and treatment respiratory syncytial virus epidemiology, risk, and attributable mortality rsv has long been recognized as a concerning pathogen in immunocompromised hosts, with increased mortality if the infection involves the lower respiratory tract. 19 in the solid organ population, lung transplant recipients are at increased risk for rsv-related mortality and morbidity compared with the other organs. 37,38 incidence of rsv in lung transplant recipients is variable and accounts for roughly 6% to 12% of rvi infections. 4 risk factors for morbidity and mortality are not clearly defined, with reports of young age (<2 years), recent transplant, preexisting lung disorder, recent rejection, and multivisceral transplant as risk factors. 39, 40 rsv infection following hsct depends on several factors, including patient age and type of transplant. incidence of rsv infection following hsct has been reported at 7% to 9% for allogeneic and 1.5% for autologous, with pediatric patients at the greatest risk of rsv. 41,42 several risk factors have been reported for rsv as well as progression to lrti or severe infection. in hsct, risk factors for rsv include less than 1 month posttransplant (preengraftment), both younger (<2 years) and older (>60 years) age, gvhd, relapsed disease, and smoking. 40,43 risks for presenting with lrti or progression to lrti include lack of rsv-directed therapy, high-dose total body irradiation, respiratory coinfection, absolute neutrophil count less than 500 cells/mm 3 , and an absolute lymphocyte count (alc) less than 100 to 200 cells/mm 3 . 42,44 in contrast, an alc greater than 1000 cells/mm 3 was protective against progression to lrti. attributable mortality in rsv infection has been reported in several publications; with mortality approaching 80% in untreated hsct patients and decreased to 6% to 25% with prompt supportive care and/or treatment. 40 more recent assessment of adult hospitalized patients reported 5% to 16% mortality in hsct and 10% to 13% in sot recipients in the setting of ribavirin treatment. 39,45,46 age greater than 60 years and lymphopenia were risk factors for mortality and compared with nontransplant patients. a recent report in pediatric hsct recipients also showed better outcomes than historical reports, with only 19% progression to lrti and no mortality, with a primary treatment modality of intravenous immunoglobulin (ivig) supplementation. 42 although transplant patients are likely at increased risk for morbidity and mortality, especially those with lrti, pediatric patients presenting in clinic with upper respiratory tract infection (urti) symptoms have also been managed with good outcomes in the outpatient setting. 47 therefore, although rsv infection in sot and hsct populations is associated with increased morbidity and mortality, precise determination of its impact remains elusive, but overall outcomes seem to be improving over time. in addition to the general preventive measures reviewed earlier, the only us food and drug administration (fda)-approved agent for the prevention of severe rsv infection is palivizumab. palivizumab is a humanized monoclonal antibody targeting the f glycoprotein of rsv, and is approved for prevention of rsv in high-risk patients less than 2 years of age. recommendations were published in 2014 by the american academy of pediatrics for palivizumab prophylaxis for infants and children less than 24 months of age with specific predisposing conditions. 48 with respect to immunocompromised children, no specific recommendations were made, aside from considering prophylaxis in profoundly immunocompromised patients younger than 24 months of age. a more recent multidisciplinary consensus conference in italy, referencing the lack of adequate clinical trials and statistical power, recommended against palivizumab for children with primary or acquired immunodeficiencies. 49 in practice, the use of palivizumab for rsv prophylaxis in immunocompromised patients varies widely. approximately 50% of surveyed pediatric sot centers used palivizumab for prophylaxis, in both candidates and recipients, with 93% of those targeting infants 0 to 12 months old and 79% extending use to 0 to 24 months. 50 only 10% provided palivizumab for patients 2 to 4 years old and 7% gave palivizumab for patients more than 4 years of age. this variability in practice is likely caused by the limited data on the efficacy of palivizumab prophylaxis in immunocompromised patients. in a retrospective pediatric hsct cohort from memorial sloan kettering cancer center (n 5 275), nearly half of the high-risk patients received intravenous (iv) palivizumab. in the palivizumab treatment group, 30% developed rsv compared with only 4% of those who did not receive prophylaxis. 51 similarly, an approach limiting palivizumab only to those hsct pediatric patients younger than 12 months with either a chronic oxygen requirement or severe combined immunodeficiency, pretransplant to 100 days posttransplant, found no increase in the incidence of rsv or patient outcomes compared with historical controls with wider use of palivizumab. 52 if time allows, delaying hsct has also been reported to be an effective strategy to prevent serious rsv infection in hsct candidates with rsv urti before conditioning. 53,54 one reported strategy delayed transplant until symptom resolution and negative repeat rsv testing, resulting in a significant reduction in rsv pneumonia following transplant and improved mortality compared with those patients whose transplants were not delayed. 53 careful thought must be given to the underlying disease process, risk of progression, as well as type of transplant before making the decision to delay hsct. several management options for rsv have been considered and reported in the literature, and although there are reports of improved outcomes, no placebo-controlled trial has clearly delineated the indication for and efficacy of treatment. the only randomized controlled trial accrued 14 hsct patients over 5 years and reported that aerosolized ribavirin decreased rsv viral load compared with supportive care but did not significantly improve outcomes. 55 ribavirin is a broadspectrum nucleoside analogue with activity against dna and rna viruses. reported toxicities of inhaled ribavirin include bronchospasm, cough, nausea, rash, and decreased pulmonary function. iv ribavirin adverse effects include hemolysis, hyperbilirubinemia, and leukopenia, whereas oral ribavirin can cause anemia and rash. 56 at present, aerosolized ribavirin remains the only fdaapproved drug for the treatment of severe rsv infection, and it is only approved for use in children. 57 immunomodulators that have also been investigated include ivig and anti-rsv monoclonal antibody (palivizumab). because of the lack of clear evidence of efficacy, wide variation in management of rsv exists. 58 recommendations and guidelines have been published for hsct and sot patients, and although based on the best available data, they are not strong recommendations in many cases. for example, the sot recommendations for rsv lrti are for consideration of aerosolized ribavirin in combination with rsv ivig or palivizumab. 59 because of the increased mortality in hsct patients, recommendations for treatment within this population are stronger. based on published reports as well as selfreported treatment strategies in surveys from sot centers, lung and heart-lung recipients are often treated for urti or lrti with rsv; lrtis may be treated in non-lung transplant sot recipients, although this is inconsistent. 58 retrospective and prospective studies report improved outcomes in symptomatic lung transplant patients treated with iv ribavirin plus corticosteroids 60 ; oral ribavirin plus corticosteroids 61 ; oral or iv ribavirin 62,63 ; and inhaled ribavirin plus corticosteroids, ivig, and palivizumab, 64 highlighting the lack of consensus on treatment strategies in this population. recommendations for hsct patients from the infectious diseases working party of the german society for haematology and medical oncology recommend ivig in general and ribavirin in particular for rsv in patients with cancer, largely based on data in hsct recipients. 65 guidelines from the united kingdom recommend inhaled ribavirin and ivig for allogeneic hsct recipients with either lrti or urti and risk factors for progression to lrti; they also suggest oral ribavirin if the inhaled form is not available. 66 outside of guidelines and recommendations, several prospective and retrospective studies have been published on the treatment of rsv in hsct patients, and, despite the available literature, there is no commonly accepted approach. reports of improved outcomes in the treatment of rsv in hsct patients can be found for inhaled, oral, or iv ribavirin, 46,67,68 as well as combinations of ribavirin with ivig, palivizumab, and/or rsv-specific ivig 56 ; most support treatment at the urti stage, before progression to lrti. a systematic review of the available retrospective studies in 2011 reported that any form of ribavirin, alone or in combination with an immunomodulatory agent, was effective in preventing progression of urti to lrti, with a trend toward better outcomes with inhaled ribavirin plus an immunomodulator. 56 although negative studies are potentially less likely to achieve publication, there are data available suggesting that adjunctive corticosteroid use and palivizumab alone do not improve outcomes. 69, 70 complicating the assessment of rsv treatments in sot and hsct patients are recent reports of good outcomes with minimal intervention, such as the 54 immunocompromised pediatric patients diagnosed with symptomatic rsv without any mortality despite only 8 (15%) receiving directed therapy. 47 another analysis of 32 pediatric hsct recipients with rsv reported no attributable mortality with no ribavirin therapy; all patients were managed either with supportive care alone or immunoglobulin therapy. 42 although it is unclear which, if any, rsv-specific treatment is the most effective intervention, there are potentially effective investigational drugs being developed. treatment with aerosolized aln-rsv01 (alnylam pharmaceuticals, cambridge, ma), a small interfering rna that targets the rsv nucleocapsid messenger rna, has shown some early promise in potentially preventing bos in lung transplant recipients with rsv. 71 another agent, ri-001 (adma biologics, inc, ramsey, nj) contains standardized levels of high-titer anti-rsv neutralizing antibody. in a report of compassionate use in 15 patients with rsv lrti already receiving treatment with ribavirin, ri-001 was well tolerated and showed at least a 4-fold increase in geometric mean titer of rsv antibodies. 72 ri-002 (adma biologics, inc, ramsey, nj) is a new immunoglobulin formulation that was developed using plasma collected from individuals tested to have high-titer anti-rsv antibodies, and in early trials showed a significant increase in anti-rsv neutralizing antibodies when administered to primary immunodeficient patients. 73 in addition, there are several other small molecule therapies in various stages of development, including early clinical trials. 74 one such molecule, gs-5806, an oral rsv entry inhibitor, was reported to have significantly lower viral load, total mucus weight, and total symptom score versus placebo in a healthy adult challenge. 75 phase 2b trials with the same novel small molecule in lung transplant recipients and bone marrow transplant (bmt) patients have completed enrollment, with results pending (nct02534350, nct02254421). epidemiology, risk, and attributable mortality the impact of influenza infection in sot patients can be particularly severe, especially in lung transplant recipients. rates of severe influenza in lung transplant patients have been reported in 16% to 20% of those infected, with an attributable mortality of 4% to 8%; even higher mortalities of 21% caused by the 2009 h1n1 infection in lung transplant recipients with preexisting bos grade 3 were reproted. [76] [77] [78] a report of australian lung transplant recipients with h1n1 influenza noted a 16% fev 1 decline at presentation and 39% of patients had prolonged allograft dysfunction. 78 reports of the most severe infections are largely based on outcomes following the 2009 h1n1 pandemic; however, a recent retrospective cohort study of brazilian renal transplant recipients with influenza a between 2009 and 2014 reported a 14% incidence of both intensive care unit (icu) admission and mortality, which is higher than expected. 79 similar to solid organ recipients, influenza infection in hsct patients causes significant morbidity and mortality. progression from urti to lrti varies widely depending on the report, but has been found in 23% to 30% of adult hsct patients or patients with hematologic malignancy, with overall mortality of 4% to 12%. 14,80,81 risk factors for either presentation with lrti or progression to lrti include an alc less than 100 to 200 cells/ml, lack of influenza-directed therapy, increased creatinine level, and delay in seeking care. there is a significant survival benefit if treated with influenza-directed therapy, with 9% mortality in treated patients versus 27% mortality in untreated patients. 14, 80, 81 prevention vaccination remains the primary focus and most strongly recommended method of influenza prevention. 82 seasonal influenza vaccines cover either 3 or 4 strains of influenza based on antigenic characterization of the previous year's circulating strains. in general, quadrivalent vaccines cover an additional influenza b strain compared with the trivalent vaccines without interfering with vaccine response. 83 influenza vaccines are available in inactivated (intramuscular or intradermal administration) and live, attenuated (intranasal) formulations. the live, attenuated vaccine is not recommended for immunocompromised recipients, and the inactivated vaccine is preferred for household contacts. 82, 84 overall influenza vaccine response in sot and hsct patients remains variable based on the population. the optimal timing of vaccination following sot has not been precisely determined, with guidelines from the american society of transplantation infectious diseases community of practice and the idsa recommending vaccination between 2 and 6 months after transplant. 85, 86 reports of immunogenic response to influenza vaccine in sot show a wide variation depending on the organ transplanted and the year assessed, with historical ranges between 15% and 93% protection. 82 seroprotective responses have been reported between 19% and 43% in lung transplant recipients, [87] [88] [89] 19% and 55% in adult renal transplant recipients, 90 and greater than 75% in pediatric liver recipients. 91 various adjuvants and dosing strategies have been evaluated in an effort to increase immunogenicity, with variable results. high-dose influenza vaccine contains 4 times the antigen dose of the standard influenza vaccine, and in early trials has been reported to increase the percentage of pediatric sot patients who achieved an increase in protective titers. 92 there are several likely explanations behind the reported variability in response, including the organ transplanted, duration of time from transplant, and degree of immunosuppression. the impact of immunosuppression on immunogenicity shows mycophenolate mofetil as most consistently associated with a decrease in response, whereas sirolimus has been associated with an increase in vaccine responsiveness. 87, 93 because of concerns about the potential development of human leukocyte antigen alloantibodies following the adjuvanted pandemic h1n1 influenza vaccine, evaluations of acute rejection following influenza immunization have been conducted. [94] [95] [96] a subsequent postauthorization safety study following the 2009/2010 ph1n1 vaccine found no increased risk of acute rejection associated with vaccination. 97 further, seasonal influenza immunization from 2006 to 2009 also found no increased risk of acute rejection. 98 similar to sot patients, influenza immunization is recommended for hsct recipients and is less effective compared with healthy controls. again, response rates to influenza in hsct vary based on time from transplant, influenza season/year studied, and degree of immunosuppression. rates of seroconversion of 30% to 40% are typical. 99, 100 in addition, there are encouraging reports of increased immunogenicity with high-dose inactivated influenza vaccine compared with standard dose. 101 although inactivated influenza vaccination is strongly recommended for sot and hsct recipients, other approaches have been suggested to prevent infection. in addition to use for treatment of influenza, oseltamivir and zanamivir are also approved for influenza chemoprophylaxis. a prospective study, predominantly in adult sot recipients, found that daily oseltamivir for 12 weeks during seasonal influenza circulation significantly reduced the incidence of laboratory-confirmed influenza as determined by reverse transcription pcr. 102 oseltamivir, given for 7 to 10 days, has also been reported to be effective in prevention of influenza infection in a hematology/hsct inpatient unit during a nosocomial h1n1 outbreak. 103 treatment early treatment with antiviral drugs has been shown to improve outcomes and reduce hospital admissions and mechanical ventilation use. 77, 80, 81, 104 the mainstay of treatment of influenza are the neuraminidase inhibitors (nais), oseltamivir, peramivir, and zanamivir. the influenza a m2 protein inhibitors amantadine and rimantadine are active against influenza a only, but are no longer recommended because of significant resistance in circulating influenza strains. 76 literature on the treatment of influenza in sot and hsct are reliably reproducible with maximum benefit seen the earlier in the course that virus-specific therapy is initiated. 81 during the 2009 pandemic h1n1 season, sot recipients treated with antiviral agents within 48 hours of symptom onset were significantly less likely (8% vs 22%) to require icu admission. 77 although treatment within 24 to 48 hours is optimal, benefit has been shown even with delayed treatment. 80 most experts therefore endorse influenza-specific antiviral treatment of sot and hsct patients with influenza at any point in their illness. oseltamivir is an oral nai indicated for the treatment of influenza a and b in adults and children more than 2 weeks of age. recommended duration of treatment is 5 days in immunocompetent children and adults. in general, treatment recommendations and practice in sot patients with influenza are for 5 days of oseltamivir as well. 105, 106 although 5 days of oseltamivir is the typical treatment duration, there are reports of treatment for 10 days or longer in patients with persistent symptoms. 107 a clinical trial also investigated conventional-dose versus doubledose oseltamivir for 10 days in immunocompromised patients, with no results yet released (clinicaltrials.gov, nct00545532). zanamivir is an inhaled nai approved for treatment of influenza a and b in adults and children 7 years of age and older. zanamivir is used less frequently than oral oseltamivir, likely because of the delivery route and rare reports of inhaled zanamivir failure. 108 peramivir is active against influenza a and b and currently is the only iv nai approved for clinical use in patients aged 18 years and older. iv formulations of both zanamivir and oseltamivir are under investigation in clinical trials. 109, 110 there is generally less experience with peramivir compared with oral oseltamivir, but published reports of clinical effectiveness and reduction in viral load are encouraging. peramivir is a viable treatment option, especially in those patients in whom oral or inhaled antivirals are not the optimal route. 111, 112 the pandemic 2009 h1n1 influenza strain was also notable for an increased frequency of nai resistance in up to 14% of strains. 113 however, nai resistance is currently uncommon, with an overall incidence of 0.5% to 1.9% of isolates, but does remain an area of growing concern. 114 the most frequent neuraminidase mutation is the h275y substitution, which results in highlevel oseltamivir resistance, reduced peramivir susceptibility, and generally preserved zanamivir activity. 110, 114 options for oseltamivir-resistant influenza are limited. inhaled zanamivir may have activity in many, but not all, cases. peramivir has been reported to be effective in patients with oseltamivir-resistant influenza and has shown encouraging results in preventing lethality in mouse models of nai-resistant h5n1/avian influenza models. 115,116 das181 (ansun biopharma, san diego, ca) is a recombinant fusion protein that removes the sialic acid receptor for influenza binding and entry into the cell, potentially inhibiting influenza and parainfluenza infection. das181 has shown promising in vitro results of activity against oseltamivir-resistant influenza strains, and additional testing versus several nai-resistant strains is ongoing. 117, 118 aside from advances in supportive care, no specific adjunctive therapies are routinely recommended. corticosteroids have been shown to decrease the need for mechanical ventilation and progression to lrti but at the cost of prolonged viral shedding. 80 therefore, although corticosteroids are not routinely recommended, if corticosteroids are indicated for another reason, such as rejection or gvhd, worsening infection with influenza is not clear reason to withhold steroids. epidemiology, risk, and attributable mortality adenovirus is a double-stranded dna virus made up of 52 immunologically distinct types, with serotypes 1 and 2 most commonly associated with pneumonia. 119 in nonimmunocompromised patients, adenovirus typically causes self-limited disease, such as uri, conjunctivitis, and/or gastroenteritis. adenovirus infection in immunocompromised patients can range from asymptomatic viremia to significant localized or disseminated disease. in contrast with many of the other community-acquired respiratory viruses, adenoviral infection can occur from primary acquisition or through reactivation of virus. in 1 study, viral reactivation was linked to the timing of immune reconstitution and cd41 t-cell counts. 120 depending on patient factors, clinical disease in immunocompromised patients can include pneumonia, hepatitis, colitis, hemorrhagic cystitis, and encephalitis. 5 in a 1993 to 2006 retrospective cohort of rvis in pediatric sot, hsct, and oncology patients, adenoviral infection was associated with the greatest length of stay, and was the only specific virus that increased the risk of morbidity and mortality related to rvi (odds ratio, 3.7; ci, 1.1-12.6; p 5 .03). 1 adenoviral infections have been reported in renal, liver, small bowel, lung, and heart transplant recipients, and although most are asymptomatic, they can be associated with severe disease. [121] [122] [123] [124] [125] [126] although adenoviral infection in hsct is more likely to cause severe disease than in sot, there are reports of graft infection and rejection with adenovirus in essentially all sot populations. 123, 127, 128 infection in lung recipients is common (up to 22% in 1 series) and there are multiple reports of severe infection, including graft loss, progression to bos, and death. 125, [129] [130] [131] [132] infection in hsct recipients can be severe and associated with significant mortality. in 14 adult hsct recipients with adenovirus viremia, most of whom were treated with antivirals, almost 50% developed invasive adenoviral disease and 23% died of the infection. 133 adenoviral lrti and disseminated disease generally carry the greatest mortality in pediatric and hsct patients (up to 80% in some reports). [134] [135] [136] treatment treatment options for adenoviral infection or disease are limited because there are currently no approved antiviral agents for treatment. reports of recovery with reduced immune suppression alone make the need for therapy and optimal timing for intervention uncertain. 124 as for most viral infections in sot and hsct patients, immunosuppression reduction is recommended if possible. cidofovir, approved for treatment of cmv retinitis in patients with acquired immunodeficiency syndrome, has been the most common antiviral used for treatment. cidofovir is a nucleotide analogue that inhibits viral dna polymerase with broad antiviral activity against dna viruses, such as herpesviruses and adenovirus. although cidofovir has in vitro activity against adenovirus and is generally accepted as the standard of care, cidofovir treatment efficacy is controversial. 135, 137, 138 dosing of cidofovir is generally 5 mg/kg iv once every 7 days, or 1 mg/kg iv 3 times per week, often in conjunction with probenecid and hydration. 129 cidofovir nephrotoxicity, which is not dose dependent, is the most common reason for discontinuation of therapy. 137 alternative therapies, including ribavirin and combination cidofovir plus ivig, have been reported with limited data, and these are not routinely recommended for use. 119, 122, 139 investigational adenovirus therapies because of new antiviral development and alternative treatment modalities, there may be additional options in the future. brincidofovir is an orally bioavailable lipid conjugate of cidofovir that has potent in vitro activity against adenovirus, and has shown promising results for both treatment of serious invasive adenovirus infections and asymptomatic viremia. 140, 141 recognizing the vital role of t-cell immunity in control of viral infections and the loss of this immunity during hsct and sot, the use of adoptive t-cell immunity is promising as well. adoptive t-cell immunity uses donor virus-specific t-cells to treat infection, and has been reported to be safe and effective when performed early in the course of the infection. 134, 142 adoptive t-cell transfer has generally been limited to a few centers and predominantly in hsct recipients, because of both the time and the expertise needed for cell preparation, but more recent methods may allow shorter generation time and more access to this therapy. 143, 144 parainfluenza epidemiology, risk factors, and attributable mortality parainfluenza virus (piv) is a single-stranded, enveloped rna virus with 4 distinct serotypes (types 1-4). serotypes vary in seasonality and disease, with piv3 associated with pneumonia and bronchiolitis, and year-round activity that peaks in spring and summer. piv1 and piv2 are commons causes of pediatric laryngotracheobronchitis (croup) and typically peak in fall and winter. 5 piv4 infection is rarely associated with disease. a retrospective analysis of rvi in pediatric immunocompromised patients reported that 26% of rvis in sot patients were caused by piv. parainfluenza infections presented more commonly as urti with or without lrti symptoms, and less frequently as lrti alone. 1 although there are previous reports of significant mortality in sot recipients caused by piv (up to 15% in 1 series 145 ), more recent publications in lung and other organ recipients report decreased mortality. 8, 146 retrospective reports place the incidence of piv infection following hsct between 2% and 7%, with greater incidence in children than in adults. [147] [148] [149] lrti after hsct is associated with high morbidity and mortality. risk factors for lrti caused by piv are lymphopenia (<300 cells/mm 3 ), neutropenia (<500 cells/ml), apache (acute physiology and chronic health evaluation) ii score greater than 15, myeloablative conditioning, high-dose corticosteroids for gvhd, and coinfection. 104, 150 reported risk factors for mortality in hsct include lrti, early infection, mismatched related donor, apache ii score greater than 15, new oxygen requirement at diagnosis, low monocyte counts (<100 cell/ml), and high-dose steroid use (>2 mg/kg/d). 149, 151, 152 in a series of 28 hsct patients with piv lrti, mechanical ventilation was necessary in 29% and attributable mortality was 46%. 150 other recent publications report 17% to 37% mortality in hsct recipients with probable or proven parainfluenza lrti. 149, 151, 152 treatment there are no currently fda-approved antiviral treatments for parainfluenza disease. treatment is supportive and includes reduction in immunosuppression. ribavirin and/or ivig have been used, off label, in parainfluenza infections with variable results and no definitive evidence of efficacy. 147, [149] [150] [151] [152] [153] [154] [155] investigational parainfluenza virus therapies das181, as discussed previously for nai-resistant influenza, can potentially inhibit piv binding to respiratory epithelial cells. das181 is an inhaled treatment typically administered via a dry powder inhaler for 5 to 10 days, and has been used under compassionate use and clinical trial protocols in hsct and sot recipients, including 2 lung transplant patients. in published reports, das181 has shown encouraging results, including reduction in piv quantitative viral load and overall outcomes. [156] [157] [158] [159] in addition, there are intriguing studies examining the impact of cholesterol reducing agents such as gemfibrozil and lovastatin on disrupting viral assembly in piv, rsv, and influenza. 160 reports of parainfluenza-specific t-cell generation from healthy donors may also ultimately lead to effective adoptive t-cell therapy for piv. 161 epidemiology, risk factors, and attributable mortality human metapneumovirus (hmpv) is an rna virus identified in 2001, in the same paramyxovirus family as rsv and parainfluenza, that typically causes a self-limited urti in immunocompetent persons. hmpv is found worldwide and occurs predominantly in the late winter and spring months, often following the rsv season. 162 as with many of the other community-acquired rvis in sot and hsct patients, progression from the upper respiratory tract to the lower respiratory tract is associated with increased morbidity and mortality. hmpv has been a well-documented cause of symptomatic and asymptomatic infection in lung transplant recipients. in a retrospective population of 49 symptomatic adult lung transplant recipients, 25% were hmpv positive via nasopharyngeal aspirate or bal. 163 as with most rvis, up to 20% can be asymptomatic, whereas others develop severe pneumonia or acute graft dysfunction. rhinorrhea, cough, and sputum production are the most frequently reported symptoms, with mortality caused by acute respiratory distress syndrome and graft rejection also reported. 4,164 identification of replicating hmpv in respiratory samples has been seen with simultaneous biopsy-proven graft rejection, suggesting a potential association between hmpv and acute rejection. 163 infection in hsct patients is variable as well, with recent reviews reporting an overall incidence of 5% to 7%. 165, 166 the same analysis found that lrti occurred in 34% of hmpv infections in hsct recipients, with a mortality of 6%, greatest in those with lrti. mortalities caused by hmpv infection have been reported to be as high as 39%. 167 progression from urti to lrti is seen in up to 60% of hsct recipients and has been associated with steroid use (>1 mg/kg), low lymphocyte count (<300 cells/mm 3 ), and onset of infection less than 30 days from hsct. 168 there are no currently approved antivirals for the prevention or treatment of hmpv. as with rsv, ribavirin has reported in vitro activity against hmpv. 169 the efficacy of ribavirin for hmpv infection has not been reliably shown. there are reports in lung transplant recipients of oral ribavirin resulting in quicker return to baseline and decreased incidence of subsequent bos, and case reports of survival with iv and inhaled ribavirin. 170, 171 however, most reports are from small single-centered studies and do not include a control population. in addition, other studies have not been able identify a similar beneficial effect of ribavirin for hmpv treatment. 167, 172 investigational human metapneumovirus therapies adoptive t-cell transfer has not yet been achieved, but hmpv-specific t cells have been generated, with further work ongoing. 173 there are also reports in vitro and in mouse models of a human monoclonal antibody for prophylactic and therapeutic hmpv infections. 174 cytomegalovirus epidemiology, risk factors, and attributable mortality cytomegalovirus (cmv) is a b-herpesvirus well recognized as a significant pathogen in immunocompromised patients. cmv has been reported to affect from 12% to 80% of heart and lung transplant recipients and 50% of hsct patients. [175] [176] [177] although cmv can cause a wide array of infections, from asymptomatic to tissue-invasive disease, cmv pneumonitis/pneumonia is of particular concern for thoracic transplant recipients. the diagnosis of proven cmv pneumonitis is based on compatible clinical signs and/or symptoms and documented cmv in lung tissue. traditionally, tissue-invasive cmv is based on histopathologic or immunohistochemical (ihc) findings consistent with tissue invasion on biopsy. 178, 179 cmv cultures or quantitative nucleic acid amplification testing of tissue samples are difficult to interpret because a positive finding could indicate tissue-invasive disease, shedding in the setting of active viremia, or both. 178 recent updates to the definition of cmv pneumonia now include proven, probable, and possible cmv pneumonia. proven disease still relies on identification of viral antigens or inclusion bodies via immunohistochemistry in biopsy material, but probable cmv pneumonia is defined as compatible symptoms plus cmv detection via viral isolation, culture, or quantitation of cmv dna in bal fluid. 180 there is no definitive cutoff for cmv dna load in the setting of cmv pneumonia; however, some reports suggest greater than 500 to 5500 iu/ml as a possible cutoff. 180, 181 possible cmv pneumonia has been suggested based on positive quantitative pcr performed on biopsy tissue. risk factors for cmv disease have been reported as advanced age and reduced-dose valganciclovir. 179 delayed-onset cmv pneumonitis (>100 days posttransplant), donor with positive cmv serology, asymptomatic cmv infection, and cmv disease at any time have been associated with increased mortality in lung transplant recipients. [182] [183] [184] cmv in hsct patients can also cause a wide array of clinical manifestations, with cmv pneumonia being the most serious, resulting in a mortality of approximately 30%. 185, 186 cmv reactivation alone is associated with lower overall mortality following hsct. 187 incidence of cmv pneumonia is unclear, largely because of the difficulties with definitive diagnosis, but an autopsy study of 999 patients with cancer and hsct reported an incidence of cmv pneumonia of 3%. 176 risk factors associated with cmvattributable mortality include female sex, lymphopenia, and mechanical ventilation at onset. 185, 188 diagnostic classification of cmv pneumonia/ pneumonitis is the same as discussed earlier for sot. iv ganciclovir (gcv) and oral valganciclovir, with or without cmv immunoglobulin (cmvig), are the agents recommended for prophylaxis of cmv infection in sot recipients. 178 the most common strategies used are universal prophylaxis versus preemptive therapy, with consensus recommendations favoring universal prophylaxis for highrisk heart and lung recipients. risk stratification in organ transplant recipients relies heavily on donor and recipient cmv serologic results before transplant. donors who are cmv immunoglobulin (ig) g positive (d1) paired with recipients who are igg negative (rã�) are considered the highest risk group. recipients who are cmv dã�/rã� are generally considered low risk, although community-acquired infection can occur posttransplant, and r1 patients are variably classified as intermediate-risk or high-risk, often center dependent. recommended duration of prophylaxis varies based on organ system and risk stratification. for d1/rã� heart transplant recipients, the recommended minimum duration of cmv prophylaxis is between 3 and 6 months. for d1/rã� lung transplant recipients the minimum recommended duration is between 6 and 12 months, with some advocates for longer, even indefinite, prophylaxis. centers using indefinite prophylaxis for lung transplant recipients report low incidence of cmv infection, which must be balanced against reports of association between gcv-resistant cmv and prolonged cmv prophylaxis, with an incidence of 10% to 50% gcv-resistant infection in some cases. 177, 189, 190 for r1 recipients the minimum duration in lung is 6 months, and 3 months in heart recipients. in dã�/rã� populations the routine use of cmv prophylaxis is not generally recommended. 178 there are several published reports of potential benefit to the addition or sole use of cmvig for prophylaxis, but, because of the limited data to support routine use, the addition of cmvig is not routinely recommended. 178, 191 the most recent consensus guidelines note that some experts add cmvig for intermediate and higher-risk recipients, but there are no randomized studies indicating that cmvig is any better than gcv or valganciclovir alone. as opposed to sot recipients, hsct patients with positive cmv serology (r1) before transplant are at higher risk for reactivation and non-relapserelated mortality. 192 preventive strategies following hsct are similar and generally consist of either universal prophylaxis for at least 100 days following hsct or a preemptive approach, with the latter as the more commonly reported practice. 192, 193 there are also reports that suggest immunosuppression choice alters cmv infection risk. lower incidences of cmv infection are reported in patients treated with regimens including a mammalian target of rapamycin (mtor) inhibitor. [194] [195] [196] [197] although these results are not consistently reproducible, some experts recommend considering the use of mtor inhibitors in the presence of clinically relevant, recurrent, or gcv-resistant cmv infection. [198] [199] [200] further, cmv vaccination is under evaluation with several candidate vaccines assessed in clinical trials, including live attenuated; recombinant glycoprotein b (gb); dna plasmid; and virus-like particle systems. 201 investigations in immunocompromised patients have reported on the safety of a cmv dna vaccine candidate (asp0113) and a chimeric peptide vaccine (pf03512676) in hsct, with some early evidence of potential clinical benefit as well. 202, 203 similarly, a phase 2 trial of an adjuvanted gb vaccine versus placebo in pretransplant liver and kidney patients showed reduced cmv viremia and days of gcv therapy following transplant, with the greatest effect seen in cmv seronegative patients. 204 therefore, although the current cmv vaccine candidates are not yet ready for clinical use, the available data plus the number of clinical trials either planned or ongoing in immunocompromised patients are very encouraging. iv gcv and oral valganciclovir are the most commonly used treatments for cmv infection or disease. 178, 205 foscarnet and cidofovir are active agents as well, but are generally reserved for gcv-resistant infection or those hsct recipients with concern for potential bone marrow suppression caused by of gcv, such as during the preengraftment period. 178, 185 cmvig has been reported to be effective in several publications and may have a role in certain settings. 188, 206 cmvig is often used for cmv pneumonia in hsct recipients, although a recent analysis failed to find significant improvement with adjunctive ivig or cmvig administration in that population. 185 the consensus guidelines on cmv treatment in sot recipients recommend consideration of adjunctive ivig or cmvig for recurrent cmv disease in thoracic organ recipients as adjunctive therapy in cases of hypogammaglobulinemia. 178 investigational cytomegalovirus therapies investigational drugs and therapies for cmv prevention and treatment are currently being developed and/or tested in clinical trials. letermovir interacts with ul56, a component of viral dna cleavage and packaging, and has been reported to be effective in reducing cmv infection in hsct recipients and reducing viral load in sot patients. 207, 208 maribavir is a competitive inhibitor of ul97 that failed to show noninferiority of cmv prevention in liver transplant patients and hsct recipients, but is currently under investigation for use in cmv infections that are refractory to gcv, valganciclovir, foscarnet, or cidofovir (clinicaltrials.gov, nct02931539). 209, 210 brincidofovir, referenced earlier for adenovirus, has been shown to reduce cmv events compared with placebo in hsct recipients. 211 in addition, adoptive t-cell therapy for cmv disease has been reported to be effective in a lung transplant recipient and as well as in several hsct recipients. [212] [213] [214] herpes simplex virus and varicella zoster virus hsv1 and hsv2 are a-herpesviruses and common causes of infection in immunocompetent and immunocompromised persons. in immunocompromised patients, hsv can cause a wide variety of clinical infection from asymptomatic oropharyngeal shedding to mucocutaneous and disseminated disease, including pneumonitis. hsv pneumonitis is uncommon in the era of antiviral prophylaxis, but thoracic transplant and hsct recipients are reportedly at greatest risk. [215] [216] [217] similar to cmv pneumonitis, the recommended diagnostic test for hsv pneumonitis is tissue histopathology with ihc for hsv. positive bal pcr testing for hsv may represent contamination or oropharyngeal shedding. 218 iv acyclovir is the treatment of choice for severe, disseminated hsv, including pneumonitis. it is recommended to continue iv acyclovir until resolution, or 14 days, at which time oral medication may be given. 218 vzv, another herpesvirus, can also cause significant disease in sot and hsct recipients. the most common manifestations of vzv are primary varicella, or chickenpox, in susceptible seronegative patients or herpes zoster, or shingles, in those with previous vzv infection or vaccination. 219 visceral dissemination is more common in hsct and less common in sot. disseminated disease includes infection of lung tissue resulting in vzv pneumonia with significant morbidity and mortality. 220, 221 risk of dissemination is increased in acute and chronic gvhd, as well as increased immunosuppresion. 219, 220 iv acyclovir is also the recommended treatment of disseminated or invasive vzv for at least 7 days, and potentially longer in patients with extensive involvement. 219 rvis are common in sot and hsct recipients and cause a broad array of infections, ranging from asymptomatic to significant virus-associated mortality. although there are some potential new and/or novel therapeutic options under evaluation, current treatment options remain limited in immunocompromised patients and generally consist of supportive care; reduction of immunosuppression; and, if available, antiviral medications. preventive measures with infection control and appropriate immunization remain vital. effective use of oral ribavirin for respiratory syncytial viral infections in allogeneic haematopoietic stem cell transplant recipients successful systemic high-dose ribavirin treatment of respiratory syncytial virus-induced infections occurring pre-engraftment in 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disease and resistance to antiviral therapy transfer of minimally manipulated cmv-specific t cells from stem cell or third-party donors to treat cmv infection after allo-hsct multicenter study of banked third-party virus-specific t cells to treat severe viral infections after hematopoietic stem cell transplantation herpes simplex virus infection in heart-lung transplant recipients herpes simplex virus (hsv) pneumonia in a heart transplant: diagnosis and therapy acyclovirresistant herpes simplex virus pneumonia postunrelated stem cell transplantation: a word of caution herpes simplex virus in solid organ transplantation varicella zoster virus in solid organ transplantation varicella-zoster virus: pathogenesis, immunity, and clinical management in hematopoietic cell transplant recipients pneumonia due to varicella-zoster virus reinfection in a renal transplant recipient key: cord-017782-dtveihrj authors: fong, i. w. title: litigations for hiv related complications date: 2010-11-30 journal: medico-legal issues in infectious diseases doi: 10.1007/978-1-4419-8053-3_13 sha: doc_id: 17782 cord_uid: dtveihrj in 1992, a 27-year-old male with same sex exposure requested human immuno-deficiency virus (hiv) testing anonymously at a walk-in clinic. he was advised that the test (hiv serology) was positive and he requested a repeat test (anonymously) 1 month later, which was also reported as being positive. about 2 years later, he was assessed by a general practitioner for symptoms of depression and continued medical care. at that time, investigations revealed a cd4 t-cell count of about 700 cells/ul. sometime in 1996 a repeat blood test revealed a cd4 cell count just <500 cells/ul. no consultation to an infectious diseases specialist or hiv clinic was made. the gp(general practitioner) then initiated a regimen consisting of didanosine, lamivudine, and saquinavir for hiv infection. at that time, testing for hiv viral load was not generally available to the medical community, but became procurable in 1997. initially, the patient tolerated the regimen well and over the next 3 years his cd4 cell count was maintained above 600–700 cells/ul and the hiv viral load remained undetectable (<50 copies). however, the patient started to show morphologic changes of moderate facial and peripheral lipoatrophy, developed mild sensory peripheral neuropathy, and increased liver enzymes attributable to fatty liver, and elevations of the fasting serum glucose. in the summer of 2000, although the cd4 cell count remained stable, the hiv viral load was reported as being over 7,000 copies/ul. at this time, the patient was referred to a university hospital hiv clinic. load of >7,000 copies/ul came to the conclusion that there was either a mix-up in the blood specimen samples or error in the labeling or reporting. efforts to verify or clarify the initial hiv serology were unsuccessful as no permanent records were kept for anonymous hiv serology results. the patient (plaintiff ) initiated litigation against the gp (defendant) for medical malpractice. specific charges were: (1) the gp should have repeated the hiv serology to confirm that the plaintiff was hiv infected, (2) the defendant was negligent in starting treatment for hiv infection without proof of disease, (3) the physician lacked knowledge of hiv infection and should have referred the patient to a specialist or hiv clinic, (4) treatment of toxic medications were given for several years without any clear indication, and (5) the gp did not adequately inform the patient on the pros and cons of therapy, nor explain the potential toxicities and side-effects. financial compensation by the plaintiff was sought for psychological suffering over the years with the false impression that he was hiv infected, and physical suffering from the side effects of the medications and the need to take unnecessary large amounts of pills for several years. the side effects had affected his social life and left a permanent physical stigma, and also adversely affected his performance at work (due to absenteeism from adverse events). the latter had resulted in his inability to perform at a high level and thus retarded his progress in his career path. all these effects have indirectly affected his earning ability over 3-4 years, and also future earning capacity. the present aids pandemic is caused by the hiv-1 strain and hiv-2 is predominantly found in west and central africa but is rare in developed nations. seroconversion after exposure usually occurs within 2 weeks to 3 months, but occasionally may take 12 months or longer. 1 delayed or protracted time for seroconversion may be seen especially in immunosuppressed subjects. 2 usually by 6 months after exposure, seroconversion should occur in 95% or more of cases. 3 a period of viremia and antigenemia without detectable antibodies occurs within 4-6 weeks of initial hiv infection. at this phase, high levels of plasma p24 antigen or viral rna can be detected, and the viremia and antigenemia decline to very low levels coinciding with seroconversion. detection of antibodies to hiv remains the most cost-effective and commonly used method to prove hiv infection. enzyme-linked immunoassay (elisa) is the most commonly used assay to test for hiv-1 and hiv-2 because of its low cost, standardized procedure, reliability, and rapid turnaround. 1 for experienced laboratories under optimal conditions (commonly licensed kits) the sensitivity and specificity of the elisa are both 99%. 1 false negative reactions can occur in infected persons early in the course before seroconversion and in immunosuppressed patients. false positive elisa results can occur for various reasons, including human error, variability in the test kits, hemodialysis, auto-immune disease, multiple myeloma, hemophilia, alcohol hepatitis, positive rapid plasma regain (rpr) test, and for unknown reasons (idiopathic). 1 the elisa uses hiv antigen to bind igg hiv antibodies in the test sample. the western blot test (wb) is the most commonly used confirmatory test for the presence of hiv specific antibodies. compared to elisa, the wb is more expensive, time-consuming and requires more technical expertise to interpret. false negative wb can also occur in the very early phase of hiv infection before development of antibodies. false positive reactions can occur in auto-immune disorders, polyclonal gammopathies, hyperbilirubinemia, subjects with human leukocyte antigen (hla) antibodies, and healthy individuals. in low risk populations, the chance of false-positive reaction of elisa and wb combined is extremely low -1 in 135,000. 4 the probability of another test being false positive in the same person tested at another time for both tests would be 1:135,000 â 135,000 or 1 in 18 billion chance. although the polymerase chain reaction (pcr) can be used to detect the hiv genome before antibody production, the pcr is highly prone to contamination with nucleic acids, which causes many false-positive reactions and therefore has not been recommended for diagnostic purposes. these pcr tests are thus mainly used for serial measurements of plasma hiv-1 rna for quantitation over the range of 75-500,000 rna copies/ml to monitor progress and response to therapy. in high risk populations, detection of hiv-dna by pcr has been found to have falsepositive rates of 2-3.4%. 5, 6 data from bayer on the versant hiv-1 rna 3.0 assay (bdna) found that all 22 of 912 false-positive samples quantitated were 1,000 copies/ml or less (personal communication with dr. r. ziermann from bayer). the main issue in this case is related to acceptance of a patient's history of a serious disease (from a test performed elsewhere) without verifying the results. although physicians commonly accept the history of a patient's underlying illness as valid, treatment for a disorder with potentially toxic agents should always require verification of the diagnosis. it could therefore be argued that the gp was remiss in instituting a cocktail of medications without having a confirmed copy of the test result for hiv infection. this is particularly damaging for an asymptomatic subject with no history of opportunistic infection or clinical evidence of aids complication. moreover, a cd4 count cannot be used as a surrogate marker for the diagnosis of hiv infection. although the cd4 + t lymphocyte quantitative count is a very useful and standard test to monitor patients for progression of hiv disease or response to therapy, it can be low in many conditions. the normal cd4 + t lymphocyte count usually averages 8.0-10.5 â 10 8 cells/l (800-1,050/mm 3 ), but the range of normality (2 standard deviations of the mean) is quite wide (500-1,400 cells/mm 3 ). 7 about 80% of the normal blood lymphocytes are t lymphocytes and nearly twothirds of blood t lymphocytes are cd4 + (helper) lymphocytes and most patients with lymphocytopenia have reduction in absolute number of cd4 + t lymphocytes. 8 there are many conditions that can be associated with lymphocytopenia and lower than normal cd4 + lymphocyte count. although hiv infection is the most common viral infection associated with cd4 + lymphocytopenia, other viral infections can transiently decrease cd4 + cell counts (including measles, corona viruses and others). 8 the list of conditions associated with cd4 + lymphocytopenia (besides viral infections) include bacterial and fungal sepsis (including tuberculosis), major surgery, recent trauma or hemorrhage, malignancy, glucocorticoid use, cytotoxic chemotherapy, radiotherapy, auto-immune diseases, nutritional deficiencies, organ transplantation, acquired common variable immunodeficiency, and idiopathic cd4 + lymphocytopenia. 8 furthermore, it is common to observe biologic variations in the absolute cd4 + cell count even in hiv infected subjects without any other factors. a healthy adult may have, at some time, transient decrease in cd4 + cell count below 500. whether the plaintiff's cd4 + cell count decline was due to viral upper respiratory tract infection or other causes was not clear. in hiv-infected patients, the t lymphocytes decline by 4% per year for every log 10 hiv rna copies/ml in the plasma. 7 currently, the optimal time to start antiretroviral therapy (art) for asymptomatic hiv patients is not clear. there is consensus that patients with aids complications or symptomatic disease should be started on art. there is still controversy as to the optimal time to initiate art in asymptomatic patients. some guidelines recommend considering starting art below 350 cells/mm 3 and others recently <500 cells/mm 3 , but there are no randomized controlled trials to provide a clear answer. how can we resolve the issue of two hiv serology tests taken at separate times in the same subject being false positive? there are several possibilities, none of which can be proven in or out of court. it is possible, that since blood samples taken in the clinic were labeled with a code number to provide anonymity, that the samples were mislabeled and originated from a truly hiv infected subject. however, the chance of that occurring twice in a row would be extremely low or unlucky. it is also possible that the plaintiff suffered from a mental disorder or delusion (such as munchausen's syndrome) and imagined that he had a positive hiv serology. there was no indication of a psychiatric disorder from the gps office records. rare false claims of a medical disease (including hiv infection) may be encountered under unusual conditions where the person can expect some form of material gain, i.e., financial, improved living conditions, sympathetic reduction in sentences for criminal offenses, etc. none of these appeared evident from review of the records. feigned hiv infection has been reported in malingering patients [9] [10] [11] and in young women with psychosocial disorders with history of prolonged sexual, physical and emotional abuse. 12 a retrospective study from an hiv clinic in a municipal hospital identified seven patients with fictitious hiv infections, six of whom had a history of illicit narcotic abuse. 13 a survey of ten other local hospitals found that known cases of alleged (fictitious) hiv infection occurred at eight of the hospitals but only one of the ten hospitals routinely documented hiv infections before initiating care. 13 in a specialist hiv unit in central london over a 5-year period, 12 patients (1.7% of admissions) with feigned hiv/aids were identified. 14 a young man, aged 36 years, presented to a new family physician (fp) in 1994 with symptoms of 15 lb weight loss, chronic diarrhea for 3 weeks and night sweats. he was found to be unwell, with evidence of significant weight loss from wasting, oral thrush, and oral hairy leukoplakia. an hiv serology performed was positive (screen and confirmatory), and his cd4 + lymphocyte count was 80 cells/mm 3 . he was started on antiretrovirals and prophylaxis for pneumocystic pneumonia. the patient was a practicing homosexual with multiple partners (none of whom were known hiv infected) and he used condoms sometimes, but inconsistently for sexual encounters. he had no known past medical illness and claimed to have previously tested negative for hiv infection in 1988. he claimed to have requested an hiv test in 1990 but there was no record of this in his previous fp records. an hiv serology was performed in the summer of 1993 (which was positive), but the patient was never informed of the results and apparently was lost to follow-up. the records subsequently in 2000 indicated that the young man was attending an hiv clinic regularly with no opportunistic infection and was clinically stable on a combination of art with a stable cd4 + lymphocyte count of 160 cells/mm 3 and undetectable hiv (<50 copies). the patient in 2000 initiated lawsuit against his original fp for medical malpractice. the charges against the physician were that he failed to perform an hiv test in 1990 despite the plaintiff's request and he was negligent in failing to notify the plaintiff of the result of the hiv serology in 1993. these acts of negligence by the defendant resulted in delay in the diagnosis and treatment, thus allowing his hiv infection to progress to aids. furthermore, failure on the part of the defendant had resulted in missed opportunities to start earlier treatment, and the delay in initiation of art resulted in a decrease in his expected life span and affected the quality of his life. the defendant countered that there was no record of the plaintiff ever requesting an hiv test in 1990. furthermore, the plaintiff never kept the appointment after the positive hiv serology in 1993 to be notified of the result. moreover, since the plaintiff was subsequently lost to follow-up, he never had a chance to counsel him on hiv disease or institute treatment. following acute hiv infection, about 50-70% of subjects develop clinical symptoms of variable severity, from mild flu-like illness to aseptic meningitis. 15 there is also evidence that severity and duration of clinical acute illness of a primary infection is of prognostic importance. the risk of developing aids within 3 years of seroconversion in subjects who were asymptomatic or had mild illness was only 10% versus 78% (eight times greater) in those with seroconversion illness of at least 14 days. 16 peak viral replication soon after infection occurs in 2-4 weeks and levels of virus can exceed >10 7 copies/ml in plasma. this is associated with a dramatic drop in circulating cd4 + lymphocytes, then a slowing of t-cell loss, and rebound by 9-12 weeks. this rebound of cd4 + cells corresponds to a decline in viral load, which reaches a steady state (set point), which is variable by 9-12 weeks. clinical progression of hiv disease has been tied to a set point level with lower levels associated with better prognosis. 17 at 1 year after seroconversion, patients demonstrate a fall of about 349 cd4 + cells/mm 3 (mean baseline 999 cells/mm 3 ), followed by a more gradual decline in cd4 + cells in the later period of infection. 18 there is usually a variable period of 8-10 years span before patients develop aids (about 50%). the typical hiv-infected person shows a progressive decline of cd4 + lymphocytes (50-100 cells/year) over time. long-term non-progression or elite controllers represent <5% of hiv-infected subjects who maintain relatively normal cd4 + cell count and very low or immeasurable viral load for 8 years to decades without therapy. 19 this is a heterogenous group of elite controllers whose benign course may result from robust immune responses against hiv, or defective poorly replicative virus secondary to deletion of the nef gene. 20, 21 there is evidence that host factors that influence the course of hiv disease are correlated to polymorphisms dominating the hla region, with class i polymorphism dominating the hla associations. 22 there is also evidence from studies in the united states and europe that hla-b57 and hla b-27 are strongly associated with long term survival or non-progression. 22 not all long-term non-progressors are "elite or viremic controllers" (patients with undetectable hiv or plasma hiv rna levels of 50-2,000 copies/ml). these patients with cd4 + cell counts of >500 cells/mm 3 for at least 10 years most often had hiv rna levels of >2,000 copies/ml, but had significantly lower hiv rna than subjects with typical progression. thus plasma set point hiv rna levels explain <50% of the variability in rates of clinical progression. 24 the chemokine receptor 5 (ccr5) protein serves as a co-receptor on cd4 + lymphocytes for certain strains of hiv-1. homozygosity for a 32-base pair deletion allele (ccr5d32) protects against hiv infection (1% of caucasians) and heterozygosity (individuals with one allele) show a decreased progression to aids. 25 there is also evidence that co-infection with gb virus (gbv-c), a flavivirus not known to cause disease (in subjects with gbv-c viremia), have slower progression and slower decrease in cd4 + cell counts than those without gbv-c infection. 23 although the reasons for this protective effect are unclear, there is evidence that gbv-c inhibits hiv replication in peripheral blood mononuclear cells in vitro, and since gbv-c infects cd4 + cells, this may compete with the hiv for target cells for infection. 23 a minority of patients with hiv infection can rapidly progress to aids within 1-3 years of their infection. this may be related to host genetic factors and age at the time of infection and other extrinsic conditions. older age at the time of infection (>25 years) has been associated with faster progression of the disease in hemophiliacs and older homosexuals. 23 concomitant co-infection with cytomegalovirus (cmv) has been associated with rapid progression to aids in hemophiliacs and others. 26, 27 co-infection with htlv-i may increase the risk for development of aids while htlv-2 can delay the progression of disease. 23, 28, 29 active tuberculosis can also enhance hiv replication and cause rapid progression to aids. 30 however, although treatment of active tuberculosis for 6 months is associated with increased cd4 + cell count, it does not markedly affect the hiv viral loads. 31 the role of hepatitis c-virus (hcv) co-infection on the progression of hiv disease has been conflicting, with some studies showing more rapid progression, but others have found no effect on the development of aids. 23 the clade or strain of hiv-1 may play a role in the course of the disease. clade d of hiv-1 is associated with faster progression to death in africa than clade-a and b. 32 women in senegal infected with c, d or g hiv clade were eight times more likely to develop aids than those infected with clade a subtype (the predominant sub-type). 33 infection with multiple strains of hiv-1 (more common in women) have also been associated with faster disease progression. 34 socio-economic factors such as poverty, homelessness, drug and alcohol abuse, and black race play indirect roles in the prognosis and disease progression of hiv infection, primarily through lower access to medical management, delay in instituting antiretrovirals and poorer compliance with medications. although a previous study found that alcohol and psychoactive drugs did not accelerate hiv disease, 35 there is in vitro evidence that alcohol, cocaine and narcotics can impair the immune response to hiv-1 and allow enhanced replication in peripheral blood mononuclear cells. 23 a case report of rapid progression to aids within a year of infection has also been attributed to alcoholism. 36 it would appear that the plaintiff became infected with hiv sometime between 1988 (reported hiv-negative) and 1993 (first noted hiv-positive). however, by 1994 he had progressed to symptomatic aids. thus, his course was more rapid than usual hiv infected individuals were, and especially as no other conditions or factors were recognized that could accelerate his course of disease. however, the failure of the fp to notify the patient of his hiv-positive status before recognition of his condition was only 1 year. would an earlier diagnosis by 1 year and assuming institution of art then, affected the outcome as to lifespan and quality of life? appropriate treatment a year earlier with art would likely have aborted or ameliorated his symptomatic disease, of weight loss, diarrhea, and malaise. however, it is less clear whether his expected lifespan would be any greater. if we assume that over the preceding year his cd4 + cell count probably declined by 50-100 cells/mm 3 , then even at that time he would have already progressed to aids (cd4 + cell count <200 cells/mm 3 ). there is reasonable good cumulated evidence that starting therapy when the cd4 + cell count is very low (<200 cells/ mm 3 ) is associated with less chance of immune reconstitution and greater risk of opportunistic complications than those started on art when the cd4 + cell count was >200 cells/mm 3 . the optimum cd4 + cell count for initiating art has not been well established although recent large observational cohort studies (retrospective and prospective ) and suggests better outcomes for hiv infected patients receiving earlier art with cd4 + cell count !350 cells/mm 3 or >500 cells/mm 3 ; the data however is flawed and controversial. 37, 38 lack of randomization in these studies could result in significant biases as motivated, health-conscious individuals would likely do better that those less motivated. it is not clear in these studies as to the cause of excess mortality in those not accepting treatment. for instance, it would be expected and predictable to find excess mortality (from any disease) in marginalized people (homeless, alcoholics, drug abusers), who are less likely to start art, which may be unrelated to hiv complications, such as suicides, homicide, accidents, drug overdose, liver failure or other diseases more prevalent in these groups (diabetes mellitus, cardiovascular disease, chronic lung disease and cancer). the defendant denied the plaintiffs claim that earlier hiv test (in 1990) was requested. it could be argued, however, that the fp should have been doing regular hiv serology in an individual that belongs to a high-risk group (with the patient's consent). the center for disease control and prevention (cdc) estimate that nearly 50% of men who have sex with men (msm) with hiv infection are unaware of their status. the cdc national behavior surveillance system of high-risk venue-based recruitment found 25% of msm tested to be infected with hiv, and nearly 50% of the hiv infected individuals were unaware of their infection. 39 in new york city, the hiv incidence rate among msm was 2.3%, with 52% of those infected being unaware of their hiv seropositivity. 39 it is estimated that 21% of hiv-infected people in the us who are unaware of their infection may account for up to 52% of new infections. cdc hiv testing guidelines recommend annual testing for high-risk populations (including msm), and since 2006 have recommended universal opt-out hiv screening in all health care settings. 40 thus, the plaintiff could argue that the defendant fell below the standard of care by not recommending and performing annual hiv tests. furthermore, if he were found to be hiv seropositive earlier, (by 1990 or before) with careful monitoring and institution of art before his cd4 + cell count fell <350 cells/mm 3 , his quality of life and life expectancy would be greater. 41 what is the effect of life expectancy with late treatment initiation for hiv disease? in a recent study using a state-transition model of hiv disease, the projected life expectancy of hiv uninfected and hiv infected persons with similar risk profiles were compared. 41 those with hiv infection lost 11.92 years of life if they received care concordant with guidelines and late treatment initiation resulted in 2.60 additional years of life lost (greatest for hispanics [3.90 years]). 41 an infectious disease (id) specialist/internist was consulted to assess a 41-year-old male with mild pancytopenia and a past history of bilateral pneumonia the year before. the patient had a history of multiple sexual contacts with prostitutes 5 years prior and had refused hiv testing the year before when he developed pneumonia (which was suspected to be pneumocystic pneumonia [pcp]). at this office visit, he agreed to an hiv test and a cd4 + cell count. the patient was called for a return appointment to discuss the results of the test a month later, but this appointment was cancelled by the patient for personal reasons. the blood test revealed the patient was hiv seropositive with a very low cd4 + cell count of 5 cells/mm 3 , but the results were not given over the phone or by mail. thus, the subject remained unaware of his hiv status and severe immune deficiency. about 3 months later, the patient attended an optician for blurred vision and he was referred to a hospital er for an ophthalmologist consultation. he was briefly assessed by the attending er physician, but due to the long waiting period pending full eye assessment, he left prematurely. the patient arranged an appointment with the id specialist in the er of the suburban community hospital. the subject was told of his hiv status and a brief retinal examination (without pupillary dilatation) by the id physician revealed no abnormality. an appointment was arranged for another office visit to the id specialist to discuss hiv therapy in 2 weeks. one week later the subject returned to the er with respiratory symptoms and poor vision. he was admitted as possible pcp under the care of the id physician, but no eye examination was performed. a week after his admission to hospital, a neurologist who was consulted found very poor vision with light perception only in the right eye and finger counting on the left eye. fundoscopy revealed bilateral chorioretinitis and ophthalmology consultation was requested, but treatment of cmv retinitis was only instituted 2 days later. his course was complicated by retinal detachment secondary to cmv retinitis with almost complete blindness in the right eye and severe visual impairment of the left eye -legally blind. malpractice litigation was brought by the patient against the id physician and the admitting er physician of the hospital. the charges against the id consultant were: (1) failure to notify the plaintiff of his hiv status and seriousness of his condition, (2) failure to do a proper eye examination or refer him to an ophthalmologist when he was first seen in the er a week before his admission, (3) failure to do a fundoscopy or arrange urgent ophthalmology consultation on admission to the hospital, (4) delay in starting appropriate treatment for cmv retinitis even after the neurologist findings were consistent with the diagnosis. the claim filed against the er physician was for neglect in performing an eye examination, despite the patient's symptoms of poor vision and failure to require an urgent ophthalmology consultation. that prompt recognition of cmv retinitis and immediate institution of antiviral therapy could have resulted in better visual result. failure of the id physician to inform the plaintiff of the seriousness of his condition, even by phone, could have resulted in prevention of visual loss and admission to hospital if treatment with art and pcp prophylaxis were started 3 months before his hospital admission. the defendant (id specialist) countered that it was the plaintiff who canceled the follow-up appointment for counseling on his condition, and it was neither his policy nor the recommended standard to discuss these issues on the phone. therefore, failure to initiate earlier art before the aids complications was due to the fault of the plaintiff. furthermore, his eye examination performed at the first er visit revealed no abnormalities. visual complaints in hiv infected persons can be unrelated (as in normal people) or related directly to complications of aids or indirectly due to medications. ocular manifestations are common in people with aids, and before the advent of highly active art (haart), the majority of patients with aids developed some ocular involvement at some time. 42 the most frequent ocular abnormality was usually silent or asymptomatic and occurred in nearly 50% of aids patients before the era of haart -hiv microangiopathy, consisting of cotton wool exudates, and less frequently hemorrhages. 42 occasionally hiv retinopathy could present with visual impairment from larger branch vein or central retinal vein occlusion. the most dreaded ocular complications of aids were from opportunistic ocular infections (cmv retinitis, herpes zoster (vzv) retinitis, or herpes zoster ophthalmicus, toxoplasma retinitis and ocular syphilis), or neoplasm (kaposi sarcoma of the lids and conjunctivae, and orbital or intraorbital lymphoma). 42 cmv retinitis is the most frequent sight threatening ocular complication of aids, occurring in the late stages when the cd4 + lymphocyte counts <50 cells/ml. in the pre-haart era cmv retinitis occurred in 30% of patients with aids, and the number of new cases has dramatically fallen since widespread use of haart by 55-95% (average 80%). 42 the incidence of cmv retinitis among patients with cd4 + cell count <100 cells/ml was 10% per year and for many patients with cd4 + cell count <50 cells/ml, it was 20% per year. symptoms of cmv retinitis include floaters, flashing lights, loss of visual field, or visual loss. in the early stages with small peripheral retinal lesions patients can be asymptomatic and 13-15% of persons with cd4 + cell count 50 cells/ml have asymptomatic cmv retinitis. 43 lesions adjacent to the optic nerve or fovea (posterior pole of the retina or macula) are immediately vision threatening. the retina has been divided into three zones for clinical assessment of risk to vision. zone 1 lies within 1,500 mm from the edge of the optic nerve, zone 2 extends from the edge of zone 1 to the equator of the eye, and zone 3 extends from the equator to the pars plana (pigmented posterior zone of the ciliary body). see fig. 13 .1 for the schematic diagram of the zones of the retina. lesions of zone 1 are immediately sight-threatening and require urgent treatment, whereas lesions of zones 2-3 may be observed for short periods of time without risk of loss of visual acuity. 42 the mean time for progression of peripheral lesions without treatment was found to be 22 days (enlargement to uninvolved retina by !750 mm in width). 44 the complications of untreated or delayed treatment of cmv retinitis include impaired vision to blindness, secondary to progressive retinitis with hemorrhages, scarring and retinal detachment. in the pre-haart era, retinal detachment in cmv retinitis occurred in 25% at 6 months and in 50-60% at 1 year. 42 the diagnosis of cmv retinitis can be made reliably by an experienced ophthalmologist by dilated direct or indirect ophthalmoscopy. examination of the fundus through an undilated pupil is inadequate to diagnose or exclude cmv retinitis as only 10% of the retina can be evaluated. 42 the aim of treatment with anti-cmv drugs (ganciclovir intravenously or oral valganciclovir) is to arrest progression of the disease, prevent further spread, and preserve vision. treatment with anti-cmv agents does not eradicate the virus but delays progression and relapse, until immunity can be restored by haart. anti-cmv therapy, half the dose after induction for 3 weeks, can be discontinued once the cd4 + cell count is >100-150 cells/ml for 6 months. some experts advocate intravitreal injection or ganciclovir implant in addition to systemic therapy for zone 1 cmv retinitis to avoid loss of vision. 42 for persons recently discovered to be hiv-seropositive, it is ideal to give the results in person confidentially, and at the same time counsel the patient on the disease. however, there are several options available if the individual were reluctant to return for follow-up appointment (or cancels the appointment). the results could be forwarded to the fp and notify him or her of the patient's cancelation plus need for counseling, close monitoring, and for initiating art or pcp prophylaxis depending on the cd4 + cell count. if the subject has no fp, then the person can be notified of the results by letter or phone, or through the public health department. although some physicians are reluctant to discuss confidential issues on the phone, there is no edict against this practice. however, confidentiality needs to be maintained and the identification of the person on the phone should be verified. this has become a common practice with financial institutions and even lawyers who discuss medicolegal cases with medical experts on the phone. since the defendant knew the plaintiff had advanced hiv disease (aids) as indicated by the very low cd4 + cell count, it was mandatory that the patient be made aware of the seriousness of his condition as soon as possible by one of the above mechanisms. his failure to impart this information to the plaintiff directly or indirectly could be considered negligence by the court. failure of the defendant to perform a dilated ophthalmoscopy or arrange for urgent ophthalmology consultation when the plaintiff initially presented with impaired vision also falls below the standard of practice in an hiv infected individual with a cd4 + cell count of <50 cells/ ml. the physician ought to have known that cmv retinitis was a main concern, and could be sight threatening and that examination by un-dilated fundoscopy would be insensitive and inaccurate. based on the evidence presented, it could be argued by the plaintiff's lawyer that had the patient been notified earlier of the seriousness of his condition and accepted treatment with haart 3 months before his hospital admission, it is likely that he would have had a better quality of life and preservation of his vision. although counsel for the defendant may counter that the plaintiff should be responsible for his own health (as he canceled the follow-up appointment), there were several avenues available to the defendant to ensure that the patient became aware of his serious illness, and he failed to utilize any of them. whether or not a court may consider these failures as human errors from oversight in a busy medical practice and not medical malpractice would be difficult to predict. review of testing for human immunodeficiency virus low sensitivity of elisa testing in early hiv infection duration of human immunodeficiency virus infection before detection of antibody measurement of the false positive rate in a screening program for human immunodeficiency virus infection detection of human immunodeficiency virus dna using polymerase chain reaction in a well-characterized group of homosexual and bisexual men concordance of polymerase chain reaction with human immunodeficiency virus antibody detection immunology of hiv infection william's hematology, 7 th edition fraudulent aids factitious aids the baron has aids: a case of fictitious human immunodeficiency virus infection and review factitious hiv syndrome in young women factitious hiv infection: the importance of documenting infection feigned hiv infection/aids: malingering and munchausen's syndrome primary human immunodeficiency virus type i infection: review of pathogenesis and early treatment intervention in humans and animal retrovirus infection clinical course of primary hiv infection prognosis in hiv infection predicted by the quantity of virus in plasma cd4 + lymphocyte cell enumeration for prediction of clinical course of human immunodeficiency virus disease immunopathogenic mechanisms of hiv infection immunologic and virologic status after 14 to 18 years of infection with an attenuated strain of hiv-1 brief report: absence of intact nef sequence in a long-term survivor with non-progressive hiv-1 infection consistent associations of class i and ii and transporter gene products with progression of human immunodeficiency virus type i infection in homosexual men overall features of hiv pathogenesis prognosis for long term survival natural control of hiv-1 replication and long-term non-progression: overlapping but distinct phenotypes genetic restriction of hiv-1 infection and progression to aids by a deletion allele of the ckr5 structural gene cytomegalovirus infection and progression towards aids in hemophiliacs with human immunodeficiency virus infection cytomegalovirus seroconversion as a cofactor for progression to aids progression to aids in homosexual men coinfected with hiv-1 and htlv-1 in trinidad htlv-i/ii seropositivity and death from aids among hiv-i seropositive intravenous drug users influence of tuberculosis on human immunodeficiency virus (hiv-i): enhanced cytokine expression and elevated b2-microglobulin in hiv-i associated tuberculosis human immunodeficiency virus-i rna levels and cd4 lymphocyte counts during treatment for active tuberculosis in south african patients different rates of disease progression of hiv type i infection in tanzania based on infecting subtype human immunodeficiency virus type i subtypes differ in disease progression infection with multiple human immunodeficiency virus type i variant is associated with faster disease progression no evidence for a role of alcohol or other psychoactive drugs in accelerating immunodeficiency in hiv-i positive individuals alcoholism and rapid progression to aids after seroconversion effect of early versus deferred antiretroviral therapy for hiv on survival timing of initiation of antiretroviral therapy in aids-free hiv-i infected patients: a collaborative analysis of 18 hiv cohort studies hiv prevalence, unrecognized infection and hiv testing among men who have sex with men -five us cities sexually transmitted diseases treatment guidelines early versus standard antiretroviral therapy for hiv-infected adults in haiti cytomegalovirus retinitis and low cd4 + t-lymphocyte counts intravenous cidofovir for peripheral cytomegalovirus retinitis in patients with aids what lessons can we learn from these cases? l all patients with self-reported hiv-seropositive status should be verified by repeating the test. l physicians should pay more careful attention to patients' symptoms and complaints and act with reasonable promptness. l deal with patients' complaints as you would want to be done to yourself or relatives. key: cord-017030-tzuyo6tx authors: henao-martínez, andrés f.; montoya, josé g. title: infections in heart, lung, and heart-lung transplantation date: 2018-12-08 journal: principles and practice of transplant infectious diseases doi: 10.1007/978-1-4939-9034-4_2 sha: doc_id: 17030 cord_uid: tzuyo6tx half a century has passed since the first orthotopic heart transplant took place. surgical innovations allowed for heart, lung, and heart-lung transplantation to save lives of patients with incurable chronic cardiopulmonary conditions. the complexity of the surgical interventions, chronic host health conditions, and antirejection immunosuppressive medications makes infectious complications common. infections have remained one of the main barriers for successful transplantation and a source of significant morbidity and mortality. recognition of infections and its management in this setting require outstanding clinical skills since transplant recipients may not exhibit classic signs or symptoms of disease, and laboratory work has some pitfalls. the prevention, identification, and management of infectious diseases complications in this population are a priority to undertake to improve the medical outcomes of transplantation. herein, we reviewed the historical aspects, epidemiology, and prophylaxis of infections in heart, lung, and heart-lung transplantation. we also discuss the most prevalent organisms affecting the host and the organ systems involved. louder! louder!" dr. john gibbon jr. used for the first time in 1953 a heart-lung respirator to keep a patient alive while performing heart surgery. dr. norman shumway at stanford developed and perfected the first surgical technique leading to heart transplantation surgery. after dr. christian barnard's first orthotopic heart transplant in december 1967, and dr. shumway first heart transplant in the united states in january 1968, heart transplantation became a standard therapeutic option for life-threatening congestive failure and started to be performed in the hundreds over the next following years at different centers. heart transplant surgery faced complications due in part to rejection and infection. however, the development of more selective immunosuppressive therapy and improvements in prevention, detection, and treatment of infections allowed for heart transplant surgery to increase rapidly worldwide. four thousand and ninety six heart (3529 adults) transplants were reported to the international society of heart and lung transplant registry (ishl) in 2011 [1] . the landscape of infection affecting heart transplant patients has been shaped by different factors: (a) implementation of more selective calcineurin-based immunosuppressive protocols, (b) lessened immunosuppressive induction regimens, (c) the institution of antimicrobial prophylaxis resulting in a significant decrease or delay in the emergence of major infections episodes including p. jirovecii (pcp), nocardia spp., listeria spp., toxoplasma gondii, cytomegalovirus, toxoplasmosis, cytomegalovirus (cmv), herpes simplex virus (cmv), varicella zoster virus (vzv), and invasive fungal infections, (d) introduction of novel diagnostic technology facilitating earlier recognition and treatment of infections, (e) expansion in the criteria to select donors and recipients to include various scenarios dealing with hbv, hcv, and hiv infections [2] , and (f) shift toward predominantly grampositive bacterial infections and multiresistant bacteria in recent years [3] [4] [5] . a stanford team lead by dr. bruce reitz performed a lung transplantation as a combined heart-lung transplant procedure in 1981 [6] . shortly after, thoracic surgeons optimized the single-and double-lung transplant procedures. improvement of surgical techniques, especially bronchial anastomosis and evolution of flush perfusion lung preservation, decreased the perioperative bronchial complications substantially. similarly to heart transplantation, improvements in immunosuppressive regimens, antimicrobial prophylaxis, and graft preservation led to enhancement in survival among lung transplant recipients. in contrast to cardiac, lung transplantation has faced the challenge of infections unique to the transplant of this organ. mold infections of the anastomotic site, host versus graft disease, and serious infections with mycobacterium abscessus, chlamydia spp., bronchiolitis, and burkholderia cepacia complex are among infectious complications rarely observed in other transplant patients [7] . transplantation of thoracic organs has improved the quality of life and prevented the death of thousands of individuals worldwide. graft survival and life expectancy have been markedly improved in these patients due to the introduction of more optimal immunosuppression, antimicrobial prophylaxis, and diagnostic technology allowing the earlier diagnosis and treatment of infection and rejection. finally, further control of infection is likely to result from implementation of new approaches to assess the net state of immunosuppression in these patients. infection was recognized as a major threat to thoracic transplantation from the early inception days [8] . there are several factors predisposing thoracic transplant recipients to infections: (a) factors present before transplantation: age, presence of comorbidities (e.g., chronic kidney disease, diabetes mellitus, cancer, etc.), nutrition status, latent infections, colonization with healthcare-associated organisms, and occult community-acquired infections; (b) factors during the surgery: duration of the transplant procedure, graft injury including ischemic time, colonization or latent infection of the graft, surgical instrumentation (e.g., mechanical ventilation, invasive devices such as catheters, drains, foley catheters, etc.), icu stay, and need for re-interventions; and (c) factors present after transplant: degree of immunosuppression, cmv infection, and rejections ( a total of 4096 heart transplants were performed in 2011. heart transplant recipients have an average age of 54 years and are predominantly man (76%). they have a significant history of smoking (46%) and hypertension (45%) and have cardiomyopathy (54%) followed by coronary artery disease (37%) as the leading causes of transplant [1] . the historical (pediatric and adult transplants between 1982 and 2011) 1-year, 5-year, and 10-year survival rates are 81%, 69%, and 50%, respectively. overall median survival is 11 years, but it increases up to 13 years for those surviving the first year after transplantation. although not associated with increased posttransplant mortality, infections before transplant can affect up to 25% of heart transplant candidates. being bronchitis and soft tissue infections, the more commonly present [9] . despite no major changes in the distribution of causes of death since 1994, infections remained a predominant factor of mortality during the first 3 years after transplant. it contributes with up to almost 20% of causes of death [3] . the global incidence of infections in heart transplant ranges between 30% and 60% and the associated mortality between 4% and 15% [10] . the incidence of infection measured as major infectious episodes per patient has steadily declined from 2.83 in the early 1970s to 0.81 in the early 2000s [3, 8, 11] . the most frequent type of infection is bacterial (44%), followed by viral (42%), fungal including pneumocystis jirovecii (14%), and protozoa (0.6%). unfavorable functional outcomes are observed in patients who developed infections in the first year of transplant, mainly associated with bloodstream, cmv, and lung infections [12] . pulmonary and central nervous system (cns) infections are independent predictors of mortality among heart transplant recipients. reactivation of latent parasitic infections residing in extra-cardiac tissues in the host or transmitted in the transplanted heart is an important consideration. the classic example is the reactivation of trypanosoma cruzi. chagas disease is a vectorborne illness transmitted by triatomine bugs, and it is endemic in latin america. the ethnicity or origin of either the donor or the recipient from these regions should raise the concern for possible reactivation. chagas reactivation was documented in 38.8% of cases in a cohort of brazilian heart transplant recipients, where chagas cardiomyopathy was the second most common indication for transplant (34.9%) [13] . chagas can also reactivate from the transplanted heart procured from a seropositive donor and transplanted into a seronegative recipient. although with a substantial decreased on its prevalence in the most recent eras, toxoplasmosis is another important consideration in this setting. similarly to chagas, toxoplasma gondii-also with a predilection to invade the myocardium-can be transmitted by reactivation of quiescent cysts in the recipient or the transplanted heart [14] . by 2011, 3640 adults received lung transplantation, the highest reported number of procedures up to that date, driven mainly by the increase of double-lung transplants. doublelung transplant is indicated for septic lung diseases (e.g., cystic fibrosis). around 66% of recipients were aged 45-65 years old. the most frequent indications for transplant were copd (34%), followed by interstitial lung disease (ild) (24%), bronchiectasis associated with cystic fibrosis (cf) (17%), and α1at deficiency-related copd (6%) [15] . the overall (from 1994 to 2011) 1-year, 5-year, and 10-year survival rates among lung recipients are 79%, 53%, and 31%, respectively. overall median survival is 5.6 years. lung transplants from cmv seronegative donors have better survival rates than from cmv seropositive donor. thirty-day mortality was led by graft failure (24.7%) and non-cmv infections (19.6%). during the remainder of the year, non-cmv infections were the leading cause of death (35.6%). infection is still prominent as the cause of death following the first year of transplant after bronchiolitis obliterans syndrome (bos)/chronic lung rejection or graft failure [15] . other infections complications historically present among the ten primary causes of death within the first year include sepsis, pneumonia, and fungal infections [16] . high lung allocation score (las) at the time of transplantation is associated with a lower 1-year survival and higher rates of infections among lung transplant recipients [17] . sixty-three adult heart-lung transplantations were reported to the ishl registry in 2011. sixty-six percent of recipients were in the group range from 18 to 49 years old. sixty-three percent of the indications were for congenital heart disease and idiopathic pulmonary arterial hypertension. heartlung transplant for cf was higher in europe and other centers compared to north american. when compared to lung only transplants, short-term survival was worse, but long-term survival was better for the heart-lung transplant recipients. their 1-year, 5-year, and 10-year survival rates were 63%, 44%, and 31%, respectively. the median survival was 3.3 years and 10 years for those surviving the first year. similarly, they have graft failure (27%), technical complications (21.9%), and non-cmv infections (17.8%) as leading causes of death during the first 30 days posttransplant. non-cmv infections (35.1%) were the top cause of death after 1 month and within 1 year of transplant. after the first year, bos/late graft failure and non-cmv infections were the predominant causes of death [15] . among other risk factors for mortality in lung transplantation are cystic fibrosis, nosocomial infections, and mechanical ventilation before transplant [18] . infections in lung transplant recipients are predominantly bacterial (48%), viral (35%), fungal (13%), and mycobacterial (4%) [19] . in 60%, the infection site is pulmonary. risk factors for infection vary by the type of organism. mechanical ventilation (mv) for >5 days immediately following transplant surgery and isolation of staphylococcus aureus (sa) from airway cultures in the recipient were considered risk factors for invasive sa infections in a retrospective study of patients with lung and heart-lung transplants [20] . likewise, risk factors for the development of healthcare-associated infections with gram-negative organisms, aspergillus, legionella, and mrsa (methicillin-resistant staphylococcus aureus), include prolonging mv, renal failure, use of atg (antithymocyte globulin), and recurrent rejections episodes [21] . additionally, α-1-antitrypsin deficiency and repeat transplantation are also risk factors for nosocomial infections. mycobacterium tuberculosis transmission from lung donors with latent infection has been documented in highly endemic areas [22] . colonization with mdr organisms (pseudomonas aeruginosa, burkholderia, acinetobacter, nontuberculous mycobacteria (ntm), and scedosporium) before transplantespecially important in cf patients-can predict the development of challenging infections to treat after transplant [23] . patients should undergo a comprehensive evaluation of potential infectious complications associated with transplantation. a detailed medical history including previous vaccinations, history of past infections, exposures (geographical, occupational, animal, etc.), travel, and foreign-born status among others should be obtained. clinicians shuold perform routine serologies for the detection of pathogen-specific igg for cmv, hsv, ebv (vca), vzv, hepatitis b (hbsag, hbsab, hbcab), hiv, hepatitis c, and syphilis. toxoplasma igg should also be performed in heart and heart-lung transplant candidates. additionally, we recommend to obtain ua, urine culture, cxr, and tuberculin skin test (tst), or a quantiferon assay. in lung and heart-lung transplant candidates, sputum should be cultured for bacterial, fungal, and afb studies. some centers advocate the screening of patients for colonization with mdr (multidrug resistant) bacteria such as mrsa and vre (vancomycin resistant enterococci), which it may have an impact on the type of antibacterial prophylaxis used preoperatively or the empirical antibiotics should sepsis develop in the immediate postoperative period. in potential lung recipients, previous respiratory colonization with mdr pseudomonas, especially in cf patients, should not exclude them from transplant [24] . on the other hand, if colonization with b. cenocepacia (genomovar iii) in cf is present transplant is relatively contraindicated [25, 26] . checking for endemic fungi such as coccidioides immitis or for the parasites trypanosoma cruzi, strongyloides stercoralis, and leishmania spp. is indicated in the presence of the appropriate risk factors [27] [28] [29] [30] [31] . histoplasma capsulatum has reactivated during immunosuppressive therapy [32] . infections after solid organ transplantation (sot) are rare and attributable to transmission from the donor [33] . furthermore, latent histoplasmosis can be present with negative serologies and treatment after transplant carries a good outcome. therefore the role of screening for histoplasmosis is of questionable significance [34] . the type of evaluation may change if the donor is alive or deceased depending on the available time to collect the samples. similarly to recipients, donors should undertake a comprehensive assessment including a complete history, assessment of risk factors, exposures, immunizations, and previous or current infections. donors should be screened for hiv, hepatitis b/c, syphilis, and tuberculosis. furthermore, we recommend to obtain serologies for cmv, ebv, hsv, vzv, and toxoplasma gondii, and for htlv-1/ htlv-2 in endemic areas. in high-risk donors, the use of nucleic acid amplification tests (naat) for hbv, hcv, and hiv should be considered. additionally, blood cultures to document an occult bacteremia are recommended. in lung transplant donors, we recommend obtaining respiratory cultures through bronchoscopy to detect colonizing organisms and target them to prevent invasive infections in the donor. culturing the media of the allograft during acquisition or processing have been advocated to reduce the risk of mycotic aneurysms among kidney transplant recipients, which may apply to other sot [35] . screening of donors for endemic mycosis is not well established. on the other hand, heart transplant donors should be screened for chagas if the donor was born in latin america [29] . finally, it is important to highlight the increase recognition of emerging, unusual viral infections such as west nile virus, lymphocytic choriomeningitis virus, rabies, and different human coronaviruses [34, 36] . testing for those organisms should be done based on individual assessments. immunization should be optimized before transplantation since the recipient will have better chances to mount an adequate immune response [37] . the advisory committee on immunization practices (acip) [38] and the guidelines for immunizations in solid organ transplantation [39] recommend inactivated influenza vaccine annually. tetanus, diphtheria, and acellular pertussis (tdap) should be administered to all adults who have not previously received tdap or have an unknown status. varicella vaccination with two doses in patients without evidence of immunity or a single dose of zoster vaccination, inactivated polio vaccine, hepatitis a/b, hpv (three series through 26 years of age), and meningococcal and pneumococcal vaccines should be administered [38] . it is remarkably important to vaccinate all household members as well. bcg and rabies vaccines can be considered under some extenuating or exposure-related indications. see table 2 .3. education of the patient and the family members is a cornerstone to establishing effective preventive measures. emphasis should be enforced about hand hygiene and food handling. additionally, potential sources of bacteria, fungi (e.g., aspergillus), and toxoplasmosis such as plants and flowers, cleaning pet's litter or cages, eating uncooked meat, acquiring new pets, construction areas, farming, barnyard activities, and smoking marihuana should be avoided. if those recreational or occupational exposures are unavoidable; appropriate gear, such gloves, must be worn. education about possible community exposures is also important. close contacts with persons with fevers or rash potentially infected with vzv, herpes zoster, or influenza should be circumvented as well. patients should cook all meals thoroughly, wash all fruits and vegetables, and shun all unpasteurized products. safe sex practices are recommended. if any foreign travel is planned, seeking evaluation in a specialized travel clinic is advisable. guidelines for the management of surgical antimicrobial prophylaxis list cefazolin (2 g, 3 g for patients with weight >120 kg every 4 h) as the recommended regimen for heart, lung, and heartlung transplantation surgery. clindamycin (900 mg every 6 h) or vancomycin (15 mg/kg) can be substituted as alternative agents in beta-lactam allergic patients [40, 41] . this recommendation can be adjusted individually, based on local hospital surveillance data or previous knowledge of colonizing organisms (e.g., addition of aztreonam, gentamicin, or a single-quinolone dose). however, the widespread use of quinolones may increase the resurgence of antimicrobial resistance. the antibiotic should be administered within 60 min before surgical incision (within 120 min for vancomycin or quinolones) and to be continued for 24-48 h in heart transplants and 48-72 h and no longer than 7 days in lung and heart-lung transplant recipients. recommendation to continue antibacterial prophylaxis until chest and mediastinal tubes are removed lacks sufficient evidence. redosing will depend on the procedure duration and associated blood loss. the recipient does not need treatment if a localized infection was present in the donor, except during meningitis where concomitant bacteremia often coexist. in meningitis and bacteremia, it is prudent to treat the recipient for 2-4 weeks [34] . indications for antifungal prophylaxis in heart transplant recipients are not clear. a systemic review showed no benefit of antifungal therapy to prevent invasive fungal infections in transplants recipients other than liver [42] . although a prospective cohort of heart transplant recipients showed targeted prophylaxis-an echinocandin for a median of 30 days with the presence of at least one risk factor for invasive aspergillosis (ia) (reoperation, cytomegalovirus disease, posttransplantation hemodialysis, and another patient with ia in the program 2 months before or after the procedure)-was highly effective and safe in preventing ia episodes [43] , no consensus exists for universal antifungal prophylaxis in heart transplant recipients. most centers have adopted antifungal prophylaxis including inhaled amphotericin b, oral itraconazole, or iv targeted echinocandin prophylaxis. in lung and lung-heart transplant recipients, fungal prophylaxis should be considered, especially if pretransplantation respiratory cultures either from the donor lung or recipient airways shows aspergillus or candida. one approach is to use inhaled amphotericin b (50 or 100 mg in extubated or intubated patients, respectively) daily until 4 days after transplant and then weekly until hospital discharge in patients with no known colonization [44, 45] . if a mold has been isolated, voriconazole is recommended up to 4 months after transplant. although evidence and efficacy need to be confirmed, combination antifungal prophylaxis therapies is used at some centers [46] . pneumocystis jiroveci prophylaxis is done with trimethoprimsulfamethoxazole (tmp-smx) for 6 months, up to 1 year. some centers extend the pjp prophylaxis to lifelong. tmp-smx also confers protection against toxoplasma, nocardia, and listeria species infections. alternatively, dapsone, inhaled pentamidine, or atovaquone can be used in patients with a history of sulfa allergy. tmp-smx is recommended at many centers for lifelong in toxoplasmosis seronegative recipients of seropositive cardiac donors (toxoplasma d+/r−) [11] . cmv prevention is recommended to all d+/r− and r+ patients. there are two common strategies for cmv prevention: antiviral prophylaxis and preemptive therapy. both approaches possess similar success rate and their advantages and disadvantages [47] . guidelines recommend valganciclovir or intravenous ganciclovir as the preferred antivirals. oral ganciclovir is an option in heart transplant patients, although it possesses a low oral bioavailability and therefore the theoretical risk of increased resistance. often, cmv immune globulin is used as an adjunctive agent. in heart recipients, prophylaxis is recommended for 3-6 months in d+/r− and 3 months in r+. in lung and heart-lung recipients, the duration of prophylaxis is 12 months and 6-12 months in d+/r− and r+ recipients, respectively [48] . in d−/r− patients, otherwise not receiving cmv active agents, antiviral prophylaxis against other herpes viruses, such as hsv and vzv, should be considered. use of oral cmx001 (oral liposomal formulation of cidofovir) in hematopoietic-cell transplants reduced cmv-related events and may have a potential role in preventing cmv in other transplant settings [49] . refer to table 2 .4 for a list of prophylaxis recommendations. this period is characterized more commonly for nosocomial, bacterial infections. thus, the bacterial organisms present are often mdr (e.g., vre, mrsa). in heart transplant recipients, skin and soft tissue infections (ssti), surgical site infection, and mediastinitis are of concern during this period. likewise, lung and lung-heart transplant recipients may develop infections related to previous respiratory colonization (pseudomonas, aspergillus). other significant infections include aspiration pneumonitis, healthcare-and ventilatorassociated pneumonia, catheter-related bloodstream infections (crbsi), nosocomial utis, and clostridium difficile colitis. donor-derived infections during this period can be present and will include hsv, lymphocytic choriomeningitis virus (lcmv), rhabdovirus (rabies), west nile virus (wnv), and hiv. toxoplasma gondii and trypanosoma cruzi are also serious donor-derived infections in heart transplant recipients that can develop within the first 6 months posttransplantation [50] . during this period, reactivation of latent infections usually occurs. hence, bacterial infections such as those caused by nocardia asteroides, listeria monocytogenes, and mycobacteria tuberculosis typically occur. additionally, fungal infections by aspergillus spp., cryptococcus neoformans, and p. jiroveci and parasitic by toxoplasma gondii, leishmania spp., strongyloides, and trypanosoma cruzi can also be seen. viral infections present during this period include herpesviruses (hsv, vzv, cmv, and ebv) and adenovirus. development of infections after 6 months are predominantly community-acquired pneumonia and urinary tract infections. other diseases include aspergillus and mucor species, nocardia, rhodococcus, and late viral infections including cmv, hepatitis b and c, jc polyomavirus infection, posttransplant lymphoproliferative disorder (ptld), hsv encephalitis, and viral community-acquired infections (e.g., coronavirus, west nile virus, influenza). it is important to recognize transplant recipients as a patient population with increased susceptibility to infections and the antibiotic should be administered within 60 min before surgical incision (within 120 min for vancomycin or quinolones) and to be continued for 24-48 h in heart transplants and 48-72 h and no longer than 7 days in lung and heart-lung transplant recipients b doses of valganciclovir, ganciclovir, and other antibiotics may require adjustment for renal function have a low threshold to perform diagnostic workup in the presence of any concerning signs or symptoms. infections monitoring is also done in a structured way when preemptive therapy for cmv is in place (as opposed to universal prophylaxis). protocols vary by the transplant center but, usually, implies a weekly cmv pcr or pp65 ag monitoring [51] . likewise, monitoring of cell-mediated immunity (cmi) using a quantiferon-cmv assay may be useful predicting late-onset cmv disease once cmv prophylaxis has been stopped [52] . cmi also have been monitored for ebv using an enzyme-linked immunospot assay [53] . immunoglobulin g (igg), c3, igg2 levels, and nk cell counts have been proposed as an attempt to identify the risk of infection in heart transplant recipients within the first year [54] . significant drug-drug interactions exist among antimicrobial and immunosuppressive agents. patient medication list should be reviewed carefully. ctp3a4 strong inducers such as nafcillin reduce tacrolimus serum concentrations. in contrast, azoles such as fluconazole can result in increased levels of tacrolimus or cyclosporine. for voriconazole, the dose of tacrolimus needs to be reduced by two-thirds [55] and the cyclosporine dose by 50% [56] . rifamycins can have an opposite drug-drug interaction by decreasing the concentrations of prednisone, cyclosporine, tacrolimus, sirolimus, and mycophenolate mofetil (mmf) [57, 58] . likewise, tacrolimus administration along with quinolones may cause qt prolongation [59] . in heart transplant patients, bacterial infections have similar clinical manifestations commonly observed in other patient populations. however, clinical signs may be subtle or absent (e.g., afebrile). they are the most frequent type of infections in this setting, reaching up to 50% of all infections [3] . the most common are pulmonary infections followed by bacteremias, mediastinal, and skin infections. staphylococcus aureus-predominantly methicillin-resistant-can cause ssti, ventilator-associated pneumonia, mediastinitis, crbsi, other forms of bacteremia, and osteomyelitis. in contrast, coagulase-negative staphylococcus is more commonly associated with crbsi. among gram-negative bacteria, pseudomonas aeruginosa is common, usually of pulmonary origin. escherichia coli is the primary causal organism of utis. extended-spectrum β-lactamase (esbl)producing klebsiella pneumoniae, escherichia coli, klebsiella oxytoca, and citrobacter freundii are also found in 2.2% of heart transplant recipients [60] . nocardia species are well recognized as an opportunistic pathogen in this setting. although relatively rare in heart transplant recipients (frequency <1%), nocardia is only second in frequency in heart transplant after lung transplant recipients [61] [62] [63] . pertinent-independent risk factors associated with the development of this infection in sot include high-dose steroids, history of cmv disease, and high levels of calcineurin inhibitors [62] . with the almost universal prophylaxis with tmp-smx, nocardia infection is less common and often present late, usually after 1 year posttransplant [63] . when they occurred, they affect the lung predominantly, which is the port of entry for disseminated infections and cns invasion. also, it can cause skin nodules and abscesses. listeria monocytogenes can also be seen in heart transplant recipients and can count for a significant proportion of the bacterial meningitis cases in this setting [64] . additionally, myocarditis and myocardial abscesses with this organism have also been documented [65] . mycobacterium tuberculosis and nontuberculous mycobacteria (ntm), although, documented to occur in heart transplantation, are rare in the united states [66, 67] . however, it is important to recognize that the development of tuberculosis (tb) can be more prevalent in some endemic regions and often present with extrapulmonary involvement [68, 69] . legionellosis and rhodococcus equi with mainly pulmonary manifestations (pneumonia, pulmonary infiltrates, or cavitation) are another significant infections among heart transplant recipients [70] . fungal infections excluding pcp represent around 4.0% of all the infections. from them, invasive mold infections (imi) are a significant contribution to morbidity and mortality among heart transplant recipients. the incidence in this population can reach 10 per 1000 person-years, and its associated mortality is approximately 17% [71] . aspergillus represents up to 65% of all imi. its median time of onset is about 46 days, although late presentation (>90 days) has been more recently recognized associated with receipt of sirolimus in conjunction with tacrolimus for refractory rejection or cardiac allograft vasculopathy [72] . the most common clinical presentation for aspergillosis includes fever, cough, and single or multiple pulmonary nodules [73] . extrapulmonary manifestations include spondylodiscitis, infective endocarditis, mediastinitis, endophthalmitis, and brain and cutaneous abscesses [74] [75] [76] [77] [78] . dissemination tends to affect the cns in a good proportion of the cases. mucormycosis is the second most frequent mold affecting heart transplant recipients. mucor, along with other non-aspergillus molds (e.g., scedosporium, ochroconis gallopava), are associated with disseminated infections, cns involvement, and poorer outcomes [79, 80] . pneumocystis jiroveci (pcp)-although with a marked reduction in inci-dence with the introduction of universal prophylaxis-is still a significant pathogen and cases may occur late after heart transplant. cryptococcosis, although infrequent among sot patients, has its higher incidence in heart transplant recipients [81] . usually, its manifestations present late and affect the lungs and the cns predominantly. histoplasmosis and coccidioidomycosis occurred typically in the first year after transplant. antigenuria was the most sensitive diagnostic test in sot for histoplasmosis [82] . finally, candida infections are an important cause of morbidity and mortality as well. rate of colonization is higher than in the general population [83] . candida most commonly causes an oral mucosa infection. although there has been a decline of invasive infections over time, these do occur and typically in the form of bloodstream infections secondary to catheter-related infections, tracheobronchitis, or disseminated disease [84] . additionally, other confined end-organ injuries such as endophthalmitis and esophagitis can also be seen. cmv infection is of critical importance among sot. in heart transplant recipients, cmv has been inconsistently associated with cardiac allograft vasculopathy [85] . furthermore, cmv leads to upregulation of pro-inflammatory cytokines, increase procoagulant response, left ventricular dysfunction, allograft rejection, and an increase of opportunistic infections [86] . the greatest risk for developing cmv disease is cmv-negative recipients of cmvpositive organs (d+/r−), followed by d+/r+ and d−/r+. a clinical report estimated that the rate of infections in heart transplant ranges between 9% and 35%, and disease is present in around 25% of patients [87] . the clinical manifestations are not unique to heart transplant recipients and include a cmv syndrome (fevers, myalgias, arthralgias, malaise, leukopenia, and thrombocytopenia). cmvassociated end-organ injury in this setting includes most frequently pneumonitis and gastrointestinal disease [10] . other manifestations comprise myelosuppression, hepatitis, and pancreatitis. in contrast to the high frequency observed in aids patients, chorioretinitis in heart transplant patients is relatively rare [87] . guidelines on cmv diagnosis and managements are discussed in more detail in chap. 55 and also have been published elsewhere [88] . other herpes viruses are of important consideration as well. ebv-associated t-cell ptlds are more frequent in heart transplant recipients (0.4%) than in other sot patients [89] . ptld is a significant contributor to morbidity and mortality in the pediatric heart transplant population [90] . human t-lymphotropic virus type i (htlv1), human herpes virus (hhv)-6, hhv-7, and hhv-8 might play a role in ebv(−) t-cell ptlds as well. herpes viruses can manifest, as in other hosts, as mucocutaneous lesions for hsv, herpes zoster for vzv, infectious mononucleosis in the case of ebv, kaposi sarcoma for hhv-8, and encephalitis for hhv-6/7. hepatitis, colitis, pneumonitis, and gastrointestinal disease have also been attributed to dissemination with certain herpes viruses. herpes viruses can present with disseminated skin lesions (with or without vesicle formation) and fever of unknown origin. adenovirus has been associated with rejection, ventricular dysfunction, coronary vasculopathy, and the need for retransplantation. the current standard treatment for adenovirus is cidofovir, but outcomes are not optimal [91] . chronic hepatitis without an identifiable cause should prompt testing for hepatitis e virus (hev). chronic hev infection leads to the rapid development of fibrosis. hev testing should be done with rna pcr due to a delay in the antibody response. we recommend decreased immunosuppression and ribavirin therapy for 3 months [92, 93] . other less common manifestation that should be considered under the correct epidemiologic risk factors include htlv-1/ htlv-2-associated myelopathy, rabies, lymphocytic choriomeningitis virus, subacute measles encephalitis, mumps (associated parotitis, orchitis, vestibular neuritis, and allograft involvement), dengue virus, orf virus, human coronavirus, and influenza [36] . cardiac transplant itself is one the predictors for development of toxoplasmosis [94] . other associated risk factors include negative serum status before transplant, diagnosis of cytomegalovirus (cmv) infection, and high-dose prednisone. toxoplasmosis can be transmitted by the donor heart (d+/r−, especially during the first 3 months) or can reactivate from the recipient (>3 months). most of the infections developed during the first 6 months posttransplant and are predominantly primary infections. about 22% of infected patients had a disseminated infection carrying an estimated 17% mortality. toxoplasmosis can manifest otherwise with myocarditis, encephalitis, pneumonitis, or chorioretinitis. diagnosis requires identification of tissue cysts surrounded by an abnormal inflammatory response, detection of toxoplasma dna in body fluids by pcr, or positive toxoplasma-specific immunohistochemistry in affected organs. posttransplant serological tests are not helpful for diagnosis and may be misleading since results may change or not regardless of the presence of toxoplasmosis [95] . the preferred treatment regimen is a combination of pyrimethamine with sulfadiazine [96] . advanced chagasic cardiomyopathy is a primary indication for heart transplantation in some centers [13] . trypanosoma cruzi, the causal organism of chagas disease, can be transmitted up to 75% of the time from infected heart donors (d+/r−) [97] . additionally, chagas disease can reactivate from the donor once immunosuppression is in place (r+). the reactivation rate can range between 22% and 90% in recipients with chronic chagasic cardiomyopathy undergoing heart transplant [98] [99] [100] . additional risk factors for reactivation include rejection episodes, neoplasms, and use of mmf [98] . the mean onset of symptoms is approximately 112 days [101] . once manifested, chagas can present with nonspecific symptoms such as fever, malaise, anorexia, hepatosplenomegaly, and lymphadenopathy. myocarditis, pericarditis, and encephalitis are also seen. reactivation can mimic rejection and exhibits congestive heart failure, av block and skin manifestations such as nodules and panniculitis. increased eosinophil count and anemia can be indirect indicators of reactivation [102] . diagnosis is made with the visualization of circulating trypomastigotes in peripheral blood. additionally, blood and tissue pcr can be used. tissue amastigotes can be seen in biopsy h&e preparations (fig. 2.1) . finally, serologies are a crucial aspect in the diagnosis especially if seroconversion have been documented. in asymptomatic individuals, when the diagnosis of chagas has been established in the donor, monitoring should be instituted with weekly blood t. cruzi pcr and microscopy [29] . preferred antitrypanosomal therapy consists on benznidazole. nifurtimox is an alternative treatment option. posaconazole has anti-parasitic activity but carries high failure rates [103, 104] . gi disease with isospora (cystoisospora) belli, cryptosporidium, cyclospora, and microsporidia has been reported to affect sot recipients. microsporidiosis can manifest with disseminated disease: fever, keratoconjunctivitis, cns involvement, cholangitis, cough, and thoracic/ abdominal pain [94] . other rare parasitic infections affecting heart transplants include leishmaniasis, strongyloidiasis, and free-living amoebas [94, 105] . the rate of surgical site infections (ssi)-sternal wound infections-in patients receiving antimicrobial prophylaxis ranged from 5.8% to 8.8% following heart transplant procedures [41] . heart transplantation itself is an independent risk factor for ssis. other risk factors include age, prophylaxis with ciprofloxacin alone, positive wire cultures, female gender, previous left ventricular assist device (vad) placement, bmi >30 kg/m 2 , previous cardiac procedures, and inotropic support for hemodynamic instability [41, 106] . similarly to other hosts, staphylococcus species are the predominant organism causing sstis. mrsa can reach up to 21% of the cases. gram-positive organisms: vre (e. faecalis), coagulase-negative staphylococci, and other enterococcus species are other etiologic agents. candida and selected gram negatives such as enterobacteriaceae, p. aeruginosa, and stenotrophomonas maltophilia can cause ssis as well [107] . sternal osteomyelitis often complicates deep ssi. additionally, sternal wound infections by ntm and fungi such as aspergillus and scedosporium have been documented [108, 109] . herpes zoster is also an important consideration and source of morbidity. herpes zoster (hz) is found as a complication in 19-22% of the patients with a median time of presentation ranging from 0.73 to 2.10 years [64, 110] . close to half may develop postherpetic neuralgia. multi-dermatome involvement, zoster ophthalmicus, and meningoencephalitis are also described. exposure to mmf is an independent risk factor. conversely, cmv prophylaxis reduces the risk for hz. bloodstream infections (bsis) are a risk factor for mortality among heart transplant recipients. likewise, sot recipient status is an independent risk factor for developing bacteremia [111] . in heart transplant recipients; the rate of bsi ranged between 16% and 24%. the median onset is about 51-191 days, and the sources are in order of frequency: lower respiratory tract, urinary tract, and crbsi. gram-negative bacteria were more commonly isolated. they are in order of appearance e. coli, p. aeruginosa, and k. pneumoniae. more common grampositive bacteria were s. aureus, s. epidermidis, e. faecalis, and l. monocytogenes. directly attributable mortality is 12.2%. among the identifiable independent risk factors to develop bsi are hemodialysis, prolonged intensive care unit stay, and viral infections [112, 113] . infective endocarditis (ie) is seen more frequently among heart transplant recipients than in the general population. with ie occurred, it most commonly involves the mitral and tricuspid valves and staphylococcus aureus and aspergillus are the main etiologic organisms. the main predisposing factors in this setting are believed to be the frequent use of vascular indwelling catheters and the frequency of endomyocardial biopsies [114] . staphylococcus aureus bacteremia in heart transplant recipients ranges from 10% to 38% [11, 115] . the sources of sa bacteremia in sot are crbsi (30%), pneumonia (24%), wound (14%), endocarditis (10%), intra-abdominal infections (9%), bone and joint (7%), cardiac devices (3%), uti (1%), and ssti (1%) [115] . immediately following heart transplant and during the 1st month, patients are more susceptible to develop pneumonia, most of which are healthcare or ventilator associated and therefore caused by nosocomial organisms such as mrsa, pseudomonas aeruginosa, and other gram negatives including acinetobacter and esbl-enterobacteriaceas. pneumonia is one the major contributors to mortality in the early postoperative period. pneumonia-related mortality approaches 15% [116] . after the 1st month, interstitial pneumonia and pneumonitis can develop, and the differential includes herpesviruses (hsv, cmv, vzv) and respiratory syncytial virus (rsv), toxoplasma gondii and pneumocystis jiroveci. pulmonary nodules with or without cavitation can be caused by fungi such as coccidioidomycosis, aspergillosis, mucormycosis, cryptococcosis; bacterial including actinomycosis, tuberculosis, atypical mycobacterial infections, nocardia, rhodococcus equi, and gramnegative bacilli; and noninfectious causes like pulmonary infarction or lymphoproliferative disorders [117, 118] . pulmonary nodules are seen in about 10% of the patients, and the median detection time is about 66 days. the associated symptoms are fever and cough. the most frequent etiology is aspergillus followed by nocardia, and rhodococcus. cmv is an exceedingly rare cause of pulmonary nodules. the diagnostic approach with the higher yield is transthoracic fine needle aspiration followed by bronchoalveolar lavage and transtracheal aspiration [118] . communityacquired pneumonia caused by streptococcus pneumonia, legionella spp., mycoplasma, and influenza is another source of morbidity [10] . mediastinitis is a common complication in this setting. in patients receiving antimicrobial prophylaxis, mediastinitis develops in 3-7% of the patients [107, 119] . a ct scan is usually necessary to determine the extension of the infection. mrsa staphylococcus epidermidis, gram-negative bacteria, and aspergillus fumigatus are frequently found as the causal organisms [120] . antimicrobial therapy should be accompanied by aggressive surgical debridement [121] . there are not distinctive abdominal-pelvic complications among heart transplant recipients. clostridium difficile is a common hospital-related cause of diarrhea associated with the use of antimicrobials. other etiology for diarrhea second-ary to acute gastroenteritis can present in a protracted way in this setting. listeria infection can present as a febrile gastroenteritis illness as well. nontyphoid salmonella infection has been described to complicate the early postoperative period in a center in taiwan [122] . acute cholecystitis can affect heart transplant recipients advocating to have a low threshold to use ultrasound as a screening method [123] . acute pancreatitis with abscess formation has also been described [124] . as pointed above, hepatitis e can present with persistently abnormal liver tests. although less frequent than in kidney transplant recipients, urinary tract infections are an important cause of morbidity. utis are predisposed by foley catheters. the organisms most commonly involved are gram-negative bacteria, enterococcus, and candida. polyomavirus nephropathy by bk virus has been described in heart transplant recipients and might be a contributor to chronic kidney disease [125] . the need for urgent transplantation and multiple transfusions are independently associated with infectious, neurologic complications. its overall mortality can reach 12% [64] . donor-derived meningoencephalitides affecting heart transplant recipients usually manifest within the first 30 days. these infections include west nile virus, arenaviruses (e.g., lcmv), and rabies. wnv can manifest with a guillain-barré-like axonopathy with cerebrospinal fluid (csf) pleocytosis. in addition to meningitis or encephalitis, ataxia, myelitis, optic neuritis, polyradiculitis, and seizures can also be observed [126] . wnv can be also acquired by the recipient in the community or through blood transfusions and present at a later time [127] . other infectious forms of meningitis and encephalitis that can present after the 1st month include listeriosis, streptococcus pneumoniae, trypanosoma cruzi, toxoplasma, hhv-6, and disseminated herpes virus infections (cmv, vzv, hsv, and ebv) [128] [129] [130] . the absence of appropriate primary prophylaxis or monitoring increases their risk. aspergillus causes the majority of brain abscess. additionally toxoplasma, tuberculosis, listeria spp., cryptococcus neoformans, scedosporium spp., and nocardia can also be causative agents [129] . concomitant pulmonary involvement is common, particularly for those whose portal of entry is the respiratory tract. progressive multifocal leukoencephalopathy (pml), a demyelinating disease caused by the reactivation of jc virus, has a usual median onset of 27 months. it carries a marked high case fatality rate and a median survival of 6.4 months in sot [131] . the use of rituximab as an antirejection treatment seems to confer an increased risk for pml [132] . htlv-1-associated myelopathy (ham) has been described as well in sot. bacterial infections are the most common type of infections among lung and lung-heart transplant recipients. the anatomic site most frequently affected is the respiratory tract, usually manifested with pneumonia, sinusitis, or tracheobronchitis. previous colonization, healthcare associated, and procedures related are the primary sources. for patients with cystic fibrosis (cf), knowledge of previous colonization results may provide some diagnostic and therapeutic advantages. pseudomonas aeruginosa is a predominant colonizing pathogen in cf. however, acinetobacter baumannii, burkholderia species, stenotrophomonas maltophilia, achromobacter xylosoxidans, ntm, pandorea, and ralstonia are also observed [23] . furthermore, pathogens that are known to cause nosocomial pneumonia during the 1st month include staphylococcus aureus, pseudomonas aeruginosa, other gram negatives (klebsiella pneumoniae, enterobacter cloacae, serratia marcescens, escherichia coli, acinetobacter species), and anaerobes. gram-positive bacteria are a common source of infections making up to 40% of them [133] . the most common sites affected were the respiratory tract, followed by bacteremia, skin, wound, and catheter related. the pathogens more frequently identified are staphylococcus species (77%), enterococcus species (12%), streptococcus species (6%), pneumococcus (4%), and eubacterium lentum (1%). staphylococcus aureus infection can develop up to 20% of lung recipients. sa commonly causes pneumonia, followed by tracheobronchitis, bacteremia, intrathoracic infections, and sstis [20] . streptococcus pneumoniae is community acquired and present with pneumonia, usually after 6 months posttransplant. pseudomonas aeruginosa has high rates of colonization (up to 40%) and disease (30%) [134] . other significant bacterial infections that may present after the 1st month are mycobacterium tuberculosis, ntm, nocardia, rhodococcus, and legionella. isolation of ntm in lung transplant recipients without evidence of disease is not associated with increased mortality [135] . nocardiosis can occur in about 2% of the lung transplant recipients. the median time of onset ranges from 14.3 to 34.1 months [136, 137] . nocardia asteroides, n. farcinica, n. nova, and n. brasiliensis have been reported. n. farcinica appears to carry worse outcomes. this infection can present as a breakthrough in the presence of trimethoprim-sulfamethoxazole for p. jiroveci prophylaxis, although the isolates may remain susceptible. mortality has been reported to range between 18% and 40%. the native lung is more frequently affected in single-lung transplant recipients. nodules are the more prevalent radio-graphic finding. extrapulmonary involvement affecting the skin and brain can be seen. hypogammaglobulinemia and neutropenia seem to confer additional risk factors for nocardiosis in this setting [137] . fungal infections are frequent complications in lung and lung-heart transplant. they present in about 15-35% and carry an overall mortality close to 60% [138] . aspergillus and candida are the most frequent causative agents. other important fungi include cryptococcus spp., mucormycosis, endemic fungi (histoplasma, coccidioides, and blastomyces spp.), scedosporium spp., fusarium spp., and dematiaceous molds. candida infections are prominent during the 1st month after transplantation. it can be one of the most common causes of bsi in this setting [139] . although colonization of the upper airways and gastrointestinal tract is common, candida additionally can cause mucocutaneous disease, tracheobronchitis, anastomosis site infections, crbsi, and disseminated disease. aspergillus spp. lead as the cause of invasive fungal infections. its attack rate of infection is almost ten times compared to that in other sot patients (estimated incidence of 6% among lung transplant recipients) [140, 141] . a. fumigatus is the most common species, but a. terreus, a. flavus, and a. niger have been described as well. the main predisposing risk factors in this setting are intense immunosuppression, previous colonization with aspergillus spp., airway ischemia, and bos. single-lung transplant possesses the greatest risk to developing an invasive aspergillus infection carrying a higher mortality than double-lung and heart-lung transplant recipients. single-lung recipients are usually older and more likely to have copd as the indication for transplantation [140] . aspergillus infections can present as tracheobronchitis, pneumonia, or disseminated disease. extrapulmonary involvement includes sinusitis, cns or orbits infections, and vertebral osteomyelitis. aids in the diagnosis can include surveillance bronchoscopies (bronchoalveolar lavage stain and culture; biopsy), chest ct and serum/bal galactomannan, beta-d-glucan, and pcr. the presence of pulmonary nodular lesions in invasive infections can carry better outcomes [142] . voriconazole is the treatment of choice. it is important to note that immune reconstitution inflammatory syndrome (iris) can develop at a median of 56 days in 7% of treated lung transplant recipients [143] . in aspergillus tracheobronchitis, nebulized amphotericin b and debridement of the bronchial anastomosis are important adjuvant measures to systemic antifungal therapy [144, 145] . pneumocystis jirovecii pneumonia manifests from 1 to 6 months. its incidence has been reduced dramatically with universal tmp/smx prophylaxis. cryptococcosis with a rate of 2% in lung transplant recipients presents with pulmonary involvement, but dissemination with meningitis can occur. furthermore, cryptococcus skin manifestations like cellulitis and cryptococcus-associated iris have been documented [146, 147] . viral infections are a common cause of morbidity among lung transplant recipients. the most common viruses are (1) cmv among the herpes viruses and (2) community-acquired respiratory viruses. as in other sot recipients, the higher risk to develop cmv infection is among d+/r−, followed by d+/r+, d−/r+, and d−/r−. this last scenario carries less than 5% of risk [48, 148] . lung transplant recipients possess higher risk for cmv than other sot with an estimated incidence of 30-86% [87] . the lung is considered a primary reservoir for cmv latency, and abundant lymphocytic tissue surrounds the transplanted organ. additionally, the use of antilymphocyte antibodies to treat rejection or for immunosuppression and other herpesviruses infections are additional risk factors for cmv disease [149] . interferon (ifn)-γ (+874t/t) polymorphism increases ifn levels and may be a predisposition for cmv disease [150] . cmv is significantly associated with bos, which reduces survival after the first year posttransplant [151] . cmv disease is most commonly manifested by pneumonitis or viral syndrome and less frequently with gastrointestinal disease. among lung transplant recipients, ganciclovir-resistant cmv carries an increased morbidity and mortality [152] . infections with community-acquired respiratory viruses ranged from 7.7% to 64%. these infections are associated with increased risk to develop pneumonia, graft dysfunction manifested by lung function loss, bos, high calcineurin inhibitor blood levels, and increase mortality [153] [154] [155] . these viruses include influenza, parainfluenza, respiratory syncytial virus (rsv), coronaviruses, human rhinovirus, adenovirus, human metapneumoviruses, and bocaviruses. the hospitalization rates are higher for influenza and parainfluenza (50% and 17%, respectively) [154] . symptoms are usually nonspecific. diagnosis often requires detection of viral nucleoprotein antigens in nasopharyngeal swabs or bronchoalveolar lavage (bal) by enzyme immunoassay or fluorescent antibody or the amplification of nucleic acid by pcr. ribavirin may possess activity against paramyxoviruses (rsv, metapneumovirus, and parainfluenza). ribavirin is administered inhaled, orally, or intravenously. oseltamivir or zanamivir is the treatment choice of influenza a or b [156] . adamantanes (amantadine and rimantadine) are not active against influenza b, and there is a marked increase resistance among influenza a strains [156] . similarly to other sot recipients, dna viruses like non-cmv herpesviruses (hsv-1,-2), vzv, hhv-6,-7,-8, and ebv are a source of significant morbidity including but not limited to cmv-negative viral syndrome, rash, pneumonitis, hepatitis, and encephalitis [157] . lastly, polyomavirus such as bk virus (bkv), jc virus (jcv), and simian virus 40 (sv40)-although fre-quently encountered in lung transplant recipients with an unclear causality-may cause worsening renal function or survival [158] . ptld is also a well-recognized complication. a trend toward late ptld presentation (>1 year) has been documented where b symptoms are more predominant as well as extra-graft involvement [159] . as other immunosuppressive states, certain parasitic infections can complicate lung and heart-lung transplants recipients. it is critical to elicit a detailed history and geographic risk factors to determine the risk of acquisition and the potential etiologic agent. toxoplasmosis can result from primary infection or reactivation of previous latent infections. toxoplasmosis can develop in patients with negative epidemiological history for cat ownership or consumption of undercooked meat. in patients with primary toxoplasmosis, nonspecific symptoms such as fever, lymphadenopathy, or organ injury may be present. reactivation can cause encephalitis with or without space-occupying brain lesions, seizures, chorioretinitis, fever of unknown origin, pneumonitis, myocarditis, and rash. although cases of the lung fluke, paragonimus westermani have not been reported in lung transplantation, it can be a potential threat in endemic areas where this organism is endemic. other parasites that can target the lung in immunosuppressive states include echinococcus, schistosoma, and strongyloides stercoralis [160] . strongyloidiasis can present as hyperinfection syndrome [161] . leishmania, although infrequently seen, has been reported among lung and lung-heart recipients [30] . free-living amoebas can affect this population as well. amoebic granulomatous dermatitis and disseminated infection presenting with ulcerative skin lesions, respiratory failure, and seizures have been described in lung transplant recipients [162, 163] . finally, alimentary protozoa, including cryptosporidium, which present with diarrhea and may elevate tacrolimus levels [164] , and microsporidia, which present with unusual manifestations like myositis or granulomatous interstitial nephritis, affects lung transplant recipients [165, 166] . the overall rate of ssis is about 13% with a significant proportion of infections being organ or space occupying (72%), deep incisional (17%), and superficial (10%) [18, 41] . independent risk factors to develop ssi are diabetes, female donor, prolonged ischemic time, and the number of red blood cells transfusion during the perioperative period [167] . ssis are associated with a 35% mortality within the first year of transplantation. the most common organisms found to cause ssi or mediastinitis are p. aeruginosa, candida species, s. aureus (including mrsa), enterococcus, coagulasenegative staphylococci, burkholderia cepacia, e. coli, proteus mirabilis, serratia marcescens, acinetobacter baumannii, enterobacter cloacae, and klebsiella species. there is a correlation in up to 33% of the patients' ssi causative organisms with previous pathogens colonizing recipients' native lungs at the time of the transplant [167] . the median onset is 25 days after lung transplant [167] . although rare, ntm can cause ssi infections among lung transplant recipients. the most frequently encountered are mycobacterium avium complex followed by mycobacterium abscessus and mycobacterium gordonae. ntm ssi infections can be complicated by progressive disseminated disease or requirement of lifelong suppressive therapy [135] . other organisms such as mycoplasma hominis and lactobacillus spp. have also been described. deep infections can affect up to 5% of the patients. sternal osteomyelitis can reach up to 6% of these deep infections. causative organisms for sternal osteomyelitis include pseudomonas aeruginosa, serratia marcescens, and scedosporium. non-sternal osteomyelitis affecting the calcaneus bone has complicated a disseminated infection with aspergillus fumigatus [168] . bloodstream infections (bsis) occur with an estimated rate of 25% among lung transplant recipients. a major proportion of bsis occur in the early posttransplant period. bsis infections are significantly associated with worse survival [139, 169] . the most common organisms encountered are staphylococcus aureus, pseudomonas aeruginosa, and candida [139] . pseudomonas aeruginosa bsi-predominantly present during the transplant hospitalization period and more commonly affecting cf patients-is followed in frequency by burkholderia cepacia and candida albicans. conversely, staphylococcus aureus was the predominant organism after transplantation discharge. in an estimated 70% of bsi, the source was pulmonary, followed in frequency by crbsi, gastrointestinal infection, peritonitis, and uti. a pulmonary source of bacteremia in sot often develops into septic shock [170] . although unusual, cases of aspergillus fumigatus endocarditis have been described following lung transplantation [171] . often patients had cf as the underlying lung disease and a median of 8 ± 6 months presentation. this complication carries a high mortality and often requires a combination of antifungal therapy with valvular replacement surgery. infectious complications related to the chest cavity include mediastinitis, cardiac (pericarditis and myocarditis), lung parenchyma infections (nodular infiltrates, cavitation, or pneumonia), bronchial anastomosis infections, and pleural space infections (bronchopleural fistula and empyema). empyema followed by mediastinitis and pericarditis, in addition to surgical wound infections and sternal osteomyelitis, is the most frequent deep ssi complications affecting the chest cavity. empyema presents in around of 3.6% of cases. it occurs during the first 6 months after transplantation (median 46 ± 39 days) carrying an estimated mortality of 28.6% [172] . most common organisms found are staphylococcus spp., e. coli, enterobacter spp., klebsiella spp., mycoplasma hominis, vre, and candida. furthermore, mycobacterium abscessus was isolated as a rare causative agent of empyema as well [173] . the degree of immunosuppression, reduced renal function, previous sternotomy, and re-exploration due to bleeding are listed as potential risk factors for mediastinitis [119] . there is an increased prevalence of mediastinitis caused by gram negatives and fungi among lung transplant recipients. causative organisms for mediastinitis are similar to ssi and are listed above. infectious pericarditis can be present up to 6% of the patients (isolated organisms include mssa, mycoplasma hominis, and scedosporium prolificans) [167, 174, 175] . due to their high fatal rate, fungal bronchial anastomotic infections are critical to recognize. pneumonia is believed to affect around 21% of lung recipients and 40% of heart-lung recipients. nosocomial organisms cause early pneumonia as in other posttransplant settings. the donor's lung seems to be the primary source for pneumonic infections, although the recipients' upper airways or sinuses are also potential sources. preoperative colonization with gram-negative rods and colonized infected donor bronchus or perfusate are recognized risk factors for pneumonia. likewise, pretransplantation colonizing microorganisms from suppurative lung disease are associated with pneumonia development posttransplant [176] . the most common causal organisms are pseudomonas aeruginosa, staphylococcus aureus, and aspergillus spp. other pathogens include bacteria such as b. cepacia, enterobacter species, s. maltophilia, klebsiella species, s. epidermidis, and e. coli, and fungi such as fusarium spp., cryptococcus neoformans, and paracoccidioides brasiliensis [176] . after the 1st month, pneumonia can present as local infiltrates, diffuse interstitial infiltrates, and nodules with or without cavitation. this type of presentation may aid in the possible causative microorganism. the list of potential pathogens is extensive and includes in addition to the already mentioned nocardia, chlamydia pneumonia, legionella, tb, ntm, pneumocystis jirovecii, rhodococcus, herpesviruses (cmv, hsv, and vzv), respiratory viruses, endemic fungi (e.g., histoplasmosis), mucormycosis, and scedosporium spp. [177] [178] [179] . similarly to other sot, common infectious complications affecting the gastrointestinal or genitourinary tract include clostridium difficile colitis and utis. intra-abdominal com-plication carries an overall increase mortality [180] . frequent gi symptoms presenting posttransplant are diarrhea which can affect almost 30% of lung transplant recipients and abdominal pain. abdominal pain should prompt further investigation for potential intra-abdominal causes. in the pediatric population, the possibility of ptld should be investigated since it carries a high mortality [181] . other described infectious intra-abdominal complications include digestive perforation (seen in 6%) [182] , retroperitoneal abscesses, cholecystitis, perianal abscesses, esophagitis, pancreatitis, pancreatic abscesses, hepatitis, diverticulitis, appendicitis, cmv colitis, megacolon, and colon rupture [180, 183, 184] . in developing countries, persistently abnormal liver enzymes should prompt testing for hev. hev rna should be used for screening. oral ribavirin seems to be safe and effective in this setting [185] . cns symptoms developing during the 1st month following lung or heart-lung transplantation should trigger the concern for donor-derived viral infections. lcmv often is accompanied by csf normal to low glucose, marked elevated protein, and mild pleocytosis [36] . although with unclear benefit, ribavirin has been used. donor-transmitted rabies is an uncommon but neurologic devastating complication that occurs within the first 30 days of transplant. lung transplantation has been described as a potential causal mechanism [186] . other organisms known to cause meningitis in lung transplant recipients are cryptococcus, tuberculosis, wnv, and herpesviruses [187, 188] . diagnosis of wnv in this setting requires nuclear acid amplification due to the unreliability of serologic testing. scedosporium apiospermum infections often cause dissemination including cns abscesses in addition to pulmonary involvement among lung transplant recipients [189] . it is important to differentiate from other molds, since amphotericin b is ineffective against scedosporium spp. in severe cases or refractory disease without an appropriate surgical debridement, the addition of terbinafine to voriconazole may prove to be useful [190] . other recognized organisms causing occupying brain lesions are fusarium, nocardia, aspergillus, toxoplasmosis, cryptococcus neoformans, listeria, and cladophialophora bantiana [191] [192] [193] . pml, a late manifestation, can be associated with intensified immunosuppression or rituximab. cidofovir followed by mirtazapine can be considered as a form of therapy for pml. infections in heart, lung, and heart-lung transplant recipients are a complex, dynamic, and evolving process. many factors such as demographics, timing, type of transplant, anatomy, and microbiology, among others, interplay in the development of these fatal complications. pertinent recognition and treatment of these infections improve transplantation outcomes. the registry of the 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management challenge randomized trial of posaconazole and benznidazole for chronic chagas' disease leishmaniasis in a heart transplant patient incisional surgical infection in heart transplantation incidence, treatment strategies and outcome of deep sternal wound infection after orthotopic heart transplantation scedosporium apiospermum pneumonia and sternal wound infection in a heart transplant recipient aspergillosis with aspergillus osteomyelitis and diskitis after heart transplantation: surgical and medical management incidence and risk factors for herpes zoster following heart transplantation population-based study of the epidemiology and the risk factors for pseudomonas aeruginosa bloodstream infection bloodstream infections among heart transplant recipients bloodstream infection in heart transplant recipients: 12-year experience at a university hospital in taiwan infective endocarditis following orthotopic heart transplantation: 10 cases and a review of the literature staphylococcus aureus bacteremia in solid organ transplant recipients: evidence for improved survival when compared with nontransplant patients pulmonary complications in heart transplant recipients pulmonary nocardiosis in a heart transplant patient: case report and review of the literature lung nodular lesions in heart transplant recipients mediastinitis in heart and lung transplantation: 15 years experience bacterial mediastinitis after heart transplantation: clinical presentation, risk factors and treatment surgical treatment of mediastinitis after cardiac transplantation nontyphoid salmonella infection in heart transplant recipients cholelithiasis in heart transplant patients survival following rupture of a pancreatic abscess in a heart transplant recipient polyomavirus nephropathy in native kidneys of nonrenal transplant recipients west nile virus infection after cardiac transplantation community-acquired west nile virus infection in solid-organ transplant recipients trypanosoma 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single institution experience parasitic lung infections detection and treatment of strongyloides hyperinfection syndrome following lung transplantation a case of successful treatment of cutaneous acanthamoeba infection in a lung transplant recipient disseminated acanthamoebiasis after lung transplantation cryptosporidium enteritis in solid organ transplant recipients: multicenter retrospective evaluation of 10 cases reveals an association with elevated tacrolimus concentrations myositis due to the microsporidian anncaliia (brachiola) algerae in a lung transplant recipient key diagnostic features of granulomatous interstitial nephritis due to encephalitozoon cuniculi in a lung transplant recipient epidemiology and outcomes of deep surgical site infections following lung transplantation aspergillus fumigatus empyema, arthritis, and calcaneal osteomyelitis in a lung transplant patient successfully treated with posaconazole epidemiology of bloodstream infections in the first year after pediatric lung transplantation bacteremia and septic shock after solid-organ transplantation aspergillus endocarditis in lung transplant recipients: case report and literature review empyema complicating successful lung transplantation mycobacterium abscessus empyema in a lung transplant recipient scedosporium prolificans pericarditis and mycotic aortic aneurysm in a lung transplant recipient receiving voriconazole prophylaxis mycoplasma hominis pericarditis in a lung transplant recipient: review of the literature about an uncommon but important cardiothoracic pathogen bacterial and fungal pneumonias after lung transplantation mold infections in lung transplant recipients nosocomial legionellosis in three heart-lung transplant patients: case reports and environmental observations chlamydia pneumoniae infection after lung transplantation intraabdominal complications after lung transplantation abdominal involvement in pediatric heart and lung transplant recipients with posttransplant lymphoproliferative disease increases the risk of mortality prevalence and management of gastrointestinal complications in lung transplant patients: mitos study group the spectrum of colonic complications in a lung transplant population gastrointestinal complications in heart and in heart-lung transplant patients chronic hepatitis e infection in lung transplant recipients management and outcomes after multiple corneal and solid organ transplantations from a donor infected with rabies virus tuberculous meningitis in a lung transplanted patient impact of rituximab-associated b-cell defects on west nile virus meningoencephalitis in solid organ transplant recipients scedosporium apiospermum (pseudoallescheria boydii) infection in lung transplant recipients combination antifungal therapy in the treatment of scedosporium apiospermum central nervous system infections cladophialophora bantiana brain abscess in a solid-organ transplant recipient: case report and review of the literature disseminated fusarium infection with brain abscesses in a lung transplant recipient central nervous system infections in heart and heart-lung transplant recipients no funding agencies had any role in the preparation, review, or approval of this paper. the views expressed in this paper are those of the authors and do not necessarily represent the views of the university of colorado denver or stanford university. no conflict of interests was reported by andrés f. henao-martínez and josé g. montoya. key: cord-006393-jcj9nqfu authors: tutschka, peter j. title: the use of immunoglobulin in bone marrow transplantation date: 1990 journal: j clin immunol doi: 10.1007/bf00918696 sha: doc_id: 6393 cord_uid: jcj9nqfu the role of bone marrow transplantation is to restore lymphohematopoietic function of a recipient whose marrow has been destroyed, either by disease or by the preparative therapy employed in an attempt to eradicate the patient's lymphohematopoietic malignancy. the restoration of lymphohematopoietic function through the donor graft occurs in stages, requires several months, and is often not completed until 1 to 2 years after transplantation. these sequential steps of immuno-reconstitution are associated with a number of definable and predictable immune deficiencies and seem to be responsible for the pattern of complications that emerges after transplantation. most of these complications are either the result of, or associated with, infections that also occur in an almost predictable pattern. in the various phases of immune deficiency following sequentially after transplantation, the humoral immune system is greatly affected, thus raising the possibility that passively administered antibodies in the form of immune globulin therapy might be beneficial in all phases of the marrow transplant procedure. this paper attempts to summarize the use of immune globulin preparations in clinical bone marrow transplantation, showing the rationale for and some of the results of therapeutic immune globulin administration. intravenous immune globulin (iv1g) is now widely used in bone marrow transplantation, despite the fact that critical randomized, controlled studies necessary to permit utilization on a scientific basis have largely not been done yet. this paper attempts to give an overview of the use of immune globulins and the rationale for their use in clinical marrow transplantation. prior to bone marrow transplantation the recipient's marrow must be completely eradicated. the hematopoietic system and the immune system are 88s then rebuilt, almost in their entirety. this rebuilding of the lymphohematopoietic system does not occur immediately; instead, it goes through several separate, sequential phases which are associated with an almost predictable pattern of complications, especially infectious complications. in the preengraftment phase, pancytopenia is the most important event. this is followed by an early postengraftment phase, characterized immunobiologically by acute graft-vs-host disease (gvhd) and severe combined immunodeficiency. the late postengraftment phase is characterized by chronic gvhd and immunodeficiency but of a more humoral type. almost predictably we first see bacterial infections followed by nonbacterial, viral infections, particularly interstitial pneumonias. later, there are some well-defined infections with encapsulated organisms, the latter sometimes even very late after bone marrow transplantation. in the preengraftment phase there is complete aplasia with absolute neutropenia and a severe lymphopenia. immunoglobulin levels decrease to less than 50% of normal about 3 weeks after bone marrow transplantation. the patient's defenses have also been weakened by severe impairment of the physical barrier functions due to the very aggressive preparative therapy necessary for bone marrow transplantation. this gives rise to infections derived primarily from the bacterial microflora reservoirs of the patient, such as gram-negative bacteria from the gastrointestinal tract and grampositive bacteria from the integument such as staphylococcus epidermidis from the skin. in recent experience gram-positive bacteria have replaced gram-negative bacteria as the dominant sources of infection. in 15 to 20% of patients in the preengraftment phase bacteremia is present, usually associated with sepsis and circulatory collapse. there are several theoretical ways to prevent those early infections. first, one could try to develop less toxic preparative regimens so that the barrier function was less impaired. the preparative regimen that we developed in our center is one in which total-body irradiation (tbi) is replaced by a seemingly less toxic agent such as busulfan (1). such a regimen permits a very short period of aplasia (for only 5 to 7 days), which markedly reduces the danger to the patient. second, one could try to reduce the patient's own microflora reservoirs by providing a gnotobiotic, highly restricted environment. it has been shown that patients with aplastic anemia who were placed in such an environment had a significantly increased probability of survival following bone marrow transplantation (2) . third, one could try to bolster the humoral immunity by passively administering antibodies against the relevant microorganisms. commercially available immune globulin preparations do indeed contain antibodies against a wide range of pathogens and toxins, in fact against virtually all the pathogenic organisms that are important for infections in the transplant setting. the activation of the complement system by the administration of immune globulins will lead to increased phagocytosis and an increased granulocytochemotaxis. immune globulins may also prevent bacterial attachment on the mucosal surfaces because the receptor sites will be occupied by the infused antibodies. furthermore, some reasonably wellcontrolled studies have shown that passive immune globulin therapy is effective for burn patients as well as for trauma patients (3, 4) . in our own program we have tried to utilize all the accepted measures for infectious prophylaxis. we studied 110 consecutive transplant patients for whom we substituted busulfan in place of tbi. we used selective body decontamination in an ultraclean environment and administered ivig at a dose of 500 mg/kg every 2 weeks for a total of 4 months. with this regimen the incidence of bacteremia was only 2.7% and almost half of the patients developed no fever during aplasia. unfortunately, this study was not a controlled one and the important question if ivig given systemically wilt reduce infectious complications during aplasia remains unanswered. several placebo-controlled trials are under way in a number of transplant centers to resolve this matter. from the viewpoint of the immunobiologist, phase ii, the phase early after marrow engraftment, is the more important period. a number of critical immune events take place in this second phase. by this time host b-cell immunity has disappeared completely but donor b-cell immunity has not yet been reestablished. adoptive t-cell donor immunity (due to mature cells that were transferred with the bone marrow) has disappeared as well. the function of the helper t cells is markedly impaired, either because the stem cells have not yet developed into the appropriate mature t cells or because the patients are receiving immunosuppressive agents. the cytotoxic effector function is also impaired in these patients. in addition to these difficulties, the donor immune system must reorient itself in a different mhc-restriction setting. furthermore, nonspecific suppressor cells will appear 30 to 50 days after transplantation, resulting in a global impairment of the immune defense system. acute gvhd might be present, markedly worsening the already present immune defect in the second phase of transplantation. an oversimplified definition of gvhd describes it as an immune attack by donor t lymphocytes on certain host target cells in the gastrointestinal tract, the liver, and particularly, the skin. the resultant cutaneous disease is characterized by discoloration and desquamation followed by severe erythema together with epidermal lysis in which the entire integument is affected. a number of infectious complications are associated with acute gvhd, especially those due to resistant gram-negative bacteria, but also candida infections and gram-positive infections caused by corynebacteria. interstitial pneumonia, a very important infection which is often associated with acute gvhd, is discussed shortly. there is an intricate relationship between gvhd and infections, germ-free rodents which are transplanted in a germ-free environment will not develop gvhd even if they receive a mismatched transplant. conversely, intentional contamination of these chimeric animals will cause gvhd (5). patients in germ-free environments have a reduced incidence of severe gvhd. furthermore, cutaneous gvhd will develop in areas which have tissue damage, especially if this damage is caused by a virus. finally, viral infections are known to trigger gvhd in rats (6) . could ivig have a role in preventing gvhd? animal studies, including some from our laboratory, indicate that when antibody against escherichia coli is given systemically to rats, it will reduce gvhd in matched bone marrow transplants. this suggests that ivig might have some influence on gvhd. it is possible that ivig could act as an immune modulator. an fcdependent blockade of reticuloendothelial structures by ivig has been described and studies in animal models have shown that immune globulins can induce antiidiotypic regulation. also on the basis of animal models, we know that suppressorcell function can be restored with the use of ivig. since specific suppressor cells are important in counteracting gvhd, this is another instance in which ivig may play a role, at least in theory. the most important effect of ivig, however, might be an indirect one by which prevention of infections will, in turn, avert the trigger for gvhd. although at the present time gvhd is no longer a major problem in most transplant programs, gvhdassociated viral infections are still of great importance. the two major viral infections in phase 1i express themselves either as interstitial pneumonia or as a hemorrhagic gastroenteritis not unlike the necrotizing enterocolitis in the pediatric population which is described elsewhere in this issue. the interstitial infiltrate of the early pneumonia quickly spreads over both lungs, often resulting in a complete whitening ~f the lungs which is accompanied by severe adult respiratory disease syndrome (ards). in at least half the cases, cytomegalovirus (cmv) is associated with ards. the incidence of this disease varies between 15 and 40%, with cmv being responsible for about half of the cases. when cmv is the agent, mortality can range from 85 to as much as 100%. there are several risk factors for the development of interstitial pneumonia such as tbi (if the lung dose exceeds 600 rads), acute gvhd, increased age of the patient, cmv seropositivity of the patient before the transplant, transfusion of blood products from cmv-positive donors, inability to generate an antiviral cytotoxicity (which is very common in the postengraftment phase), and inability to mount a humoral antibody response. the latter defect is quite important and suggests a potential rote for passive therapy with antibodies. historically bone marrow transplant recipients who have high titers of complement-fixing antibodies to cmv prior to transplant have had a better prognosis for overcoming such a severe viral pneumonia. in the murine model, it could be shown that passive immunization can modify cmv infection. several studies have been carried out to see if passive immunization with antibodies against cmv would prevent interstitial pneumonia. investigators from ucla, minnesota, and sloan-kettering all showed, in reasonably well-controlled studies, that the incidence of cmv interstitial pneumonia was reduced in patients given high-titered antibody preparations compared to that in the control group. this finding triggered numerous other studies, mostly uncontrolled, which can be summarized as follows. ivig is effective in preventing cmv infections in cmv-seronegative recipients who receive cmvseronegative grafts. it is likely to be ineffective in preventing the development of interstitial pneumonia in seronegative recipients who receive seropositive grafts. furthermore, it is likely to be ineffective in cmv-seronegative recipients who receive cmv-seropositive blood products; however, the latter statement is not fully substantiated and a number of studies are in progress to delineate that point. it is likely that immunoglobulin is ineffective in cmv-seropositive recipients who receive either cmv-seropositive or cmv-seronegative blood products. we conducted our own trial with ivig in which 119 patients were enrolled (7) . slightly more than half of the patients were cmv seronegative. the median observation time was 29 months. all the patients received cmv-negative blood products and were given 500 mg/kg of ivig every 2 weeks for eight administrations. there were no cmv infections and there was no cmv pneumonia in the first group of 57 patients, who were cmv seronegative and had received a graft from a cmvseronegative donor. in the next group of cmvseronegative recipients, who were given a graft from a cmv-seropositive donor, 3 of 12 patients developed viremia or viruria but none of them developed pneumonia. the low incidence of cmv interstitial pneumonia in this group indicated that ivig might be effective in the cmv-seronegative patient even when receiving a seropositive donor graft. unfortunately, ivig was largely ineffective in seropositive patients. of the 50 seropositive recipients, 33 (66%) developed a positive cmv culture and 8 developed cmv pneumonia, which was fatal in 6 cases. when all groups are combined the overall effect of ivig appears impressive: a 13% incidence of cmv infection and a 7% incidence of cmv pneumonia, suggesting that ivig was able to reduce the incidence of cmv pneumonia, perhaps even in cmv-seropositive patients. we tried to obtain more definitive information about the role of ivig in seropositive recipients. we were unwilling to do a randomized, placebocontrolled study since we had ethical problems with not treating all our patients with ivig. we speculated that if we were to give ivig to seropositive patients in higher doses and more frequently, we might prevent not only cmv excretion (such as viremia or viruria) but also the development of cmv infection. for these reasons we limited our study to cmv-seropositive recipients and stratified them between two ivig schedules, giving either 500 mg/kg every 2 weeks for 4 months or 500 mg/kg every week for 4 months. if patients became excretors of the virus, they received a dose of 500 mg/kg daily for 10 days. there were 24 patients in the standard group and 22 in the high-dose group. we observed no difference in the number of patients who became excretors--15 in the standard-dose group and 17 in the high-dose group. there was no difference in the number of patients that developed systemic disease or in the number of fatalities observed. thus, a higher dose of antibody does not appear to be more beneficial than a lower dose in preventing cmv disease. new strategies need to be developed to overcome the severe problem presented by the cmv-seropositive patient. the treatment of established cmv pneumonia is not very effective. we have learned that ara-a, acyclovir, and tft are not effective in the therapy of this disease, although acyclovir might be effective as a preventive agent. ivig alone is as effective as 9(l,3-dihydroxy-2-proxymethyl) guanine (dhpg) alone in treating established cmv pneumonia, but surprisingly, the combination of ivig and dhpg is quite effective, and in a number of studies it has reduced the mortality rate from 85 to 50-60% (8, 9) . hemorrhagic gastroenteritis is the other major viral complication in the early postengraftment phase (10) . the mortality is relatively high (29%) even in the absence of acute gvhd. a number of viruses (such as rotavirus) are associated with hemorrhagic gastroenteritis, but recently cmv is becoming prevalent. about half of the cases of hemorrhagic gastroenteritis in our own patient population are now due to cmv; the remainder are split between rotavirus and adenovirus. effective measures to prevent this syndrome might include the following: strict isolation, effective prevention of gvhd (since gvhd might well set the stage for hemorrhagic gastroenteritis), and possibly therapy with orally administered immune globulin. there might be a rationale for such a therapy if we consider that the gastrointestinal tract is severely injured after transplantation. the preparative therapy as well as gvhd have caused severe mucosal injury. the peyer's patches and other gutassociated lymphoid tissues have been destroyed and must be rebuilt. in the lamina propria iga-and igm-producing plasma cells have disappeared. most importantly the degradation of igg when administered orally is much impaired in such a devastated gut. it has been shown that igg antibody survives passage through the gastrointestinal tract and emerges as an intact molecule. this led us to consider administering ivig orally to patients at risk for hemorrhagic gastroenteritis. we conducted a pilot trial in which 30 patients were given ivig orally at a dose of 50 mg/kg for 4 weeks. prior to administration of ivig we could not demonstrate any igg in the stool. after oral administration of ivig, 20 of 30 patients had considerable amounts of igg, up to 400 mg/dl, in the stool. eighteen patients had greater than 20 mg/dl. only 1 of the 30 patients developed a positive adenovirus culture and 12 patients remained afebrile (11) . we have just completed a double-blind, randomized study comparing orally administered ivig to placebo. hopefully, the analysis of the study will show that the concept of oral administration of immune globulin has some merit. the immunobiology of phase iii, the late postengraftment phase, is characterized by a continuous impairment of the t helper-cell function, a continuous defect in humoral immunity, and, in particular, the development of chronic gvhd. a number of infections are seen in this posttransplant phase, especially sinopulmonary infections with encapsulated organisms such as streptococcus pneumoniae. initially we thought that the hyposplenism associated with chronic gvhd was responsible for these infections (12) . however, we considered that there might also be an association with an iggsubclass deficiency and we conducted a small study. igg subclasses were determined in 28 patients. one group of 14 patients showed a marked igg2-subclass deficiency with a very high infection rate. twelve of these 14 patients developed infections with gram-positive organisms. another group of 14 patients with no igg2-subclass deficiency had a very low infection rate, with only 2 of 14 being infected. studying these patients further we could show that the patients continued to show subclass deficiency even after they resolved the infections. however, it is not only igg2 but also igg4 subclass which was markedly reduced (13). to prevent these late infections one could maintain coverage against gram-positive organisms beyond day i00 and should maintain gram-positive coverage for an even longer period in patients who demonstrate chronic gvhd. most importantly, one should maintain ivig supplementation beyond day 120 for the first year. in our transplant center we continue ivig supplementation as long as the subclass deficiency persists, not unlike a patient with an inborn iggsubclass deficiency syndrome. utilizing this strategy one can virtually eliminate late gram-positive infections. since there are a number of sequential events in the immunobiology after transplantation which correlate with infectious patterns, it should be possible to develop rational strategies to counteract the various immune defects. the prophylactic and therapeutic use of ivig might have a central role because it is likely to affect all phases of bone marrow transplantation. we know that immune globulins have a beneficial effect in both the prevention and the treatment of viral interstitial pneumonia. it is likely that immune globulin will also have a beneficial effect in the prevention of viral enteritis and in counteracting late subclass deficiency. there may also be some modulating influence on the entire immunobiology of bone marrow transplantation. bone marrow transplantation for leukemia following a new busuifan and cyclophosphamide regimen graft versus host disease and survival in patients with aplastic anemia treated by marrow grafts from hla-identical siblings: beneficial effect of a protective environment clinical use of intravenous immunoglobulins antibody therapy in gram-negative bacterial disease the role of microflora in development of graft versus host disease graftvs-host disease and sialodacryoadenitis viral infection in bone marrow transplanted rats cytomegalovirus (cmv) infection in bone marrow transplant recipients: use of intravenous gammaglobulin as prophylactic and therapeutic agents successful treatment of serious cytomegalovirus disease with 9(1,3-dihydroxy-2-propoxymethyl) guanine in bone marrow transplant (bmt) patients the diagnostic, prophylactic and therapeutic uses of monoclonal antibodies to human cytomegalovirus infectious gastroenteritis in bone marrow transplant recipients oral administration of igg in marrow transplant recipients functional asplenia in patients with chronic graftversus-host disease: concise communication igg subclass deficiency and pneumococcal pneumonia following allogeneic marrow transplantation dr. gulati: what has been your experience with autologous transplants? do you need to do all this with patients who are undergoing autologous transplants?dr. tutschka: i do not think so. the incidence of interstitial pneumonia as well as hemorrhagic gastroenteritis is much reduced in autologous transplants-perhaps a quarter of that seen in allogeneic transplants. furthermore, the immune system recovers much more readily after autologous bone marrow transplantation. on the other hand, autologous transplantation patients might provide an excellent target population in which to study more nonspecific effects, such as the reduction of infections during aplasia, because we do not have the compounding variables of gvhd, immune modulation phases, and so on. we are currently not using ivig to the same extent in autologous transplants as we do in auogeneic transplants but i think it is a good study population. key: cord-103297-4stnx8dw authors: widrich, michael; schäfl, bernhard; pavlović, milena; ramsauer, hubert; gruber, lukas; holzleitner, markus; brandstetter, johannes; sandve, geir kjetil; greiff, victor; hochreiter, sepp; klambauer, günter title: modern hopfield networks and attention for immune repertoire classification date: 2020-08-17 journal: biorxiv doi: 10.1101/2020.04.12.038158 sha: doc_id: 103297 cord_uid: 4stnx8dw a central mechanism in machine learning is to identify, store, and recognize patterns. how to learn, access, and retrieve such patterns is crucial in hopfield networks and the more recent transformer architectures. we show that the attention mechanism of transformer architectures is actually the update rule of modern hop-field networks that can store exponentially many patterns. we exploit this high storage capacity of modern hopfield networks to solve a challenging multiple instance learning (mil) problem in computational biology: immune repertoire classification. accurate and interpretable machine learning methods solving this problem could pave the way towards new vaccines and therapies, which is currently a very relevant research topic intensified by the covid-19 crisis. immune repertoire classification based on the vast number of immunosequences of an individual is a mil problem with an unprecedentedly massive number of instances, two orders of magnitude larger than currently considered problems, and with an extremely low witness rate. in this work, we present our novel method deeprc that integrates transformer-like attention, or equivalently modern hopfield networks, into deep learning architectures for massive mil such as immune repertoire classification. we demonstrate that deeprc outperforms all other methods with respect to predictive performance on large-scale experiments, including simulated and real-world virus infection data, and enables the extraction of sequence motifs that are connected to a given disease class. source code and datasets: https://github.com/ml-jku/deeprc transformer architectures (vaswani et al., 2017) and their attention mechanisms are currently used in many applications, such as natural language processing (nlp), imaging, and also in multiple instance learning (mil) problems . in mil, a set or bag of objects is labelled rather than objects themselves as in standard supervised learning tasks (dietterich et al., 1997) . examples for mil problems are medical images, in which each sub-region of the image represents an instance, video a pooling function f is used to obtain a repertoire-representation z for the input object. finally, an output network o predicts the class labelŷ. b) deeprc uses stacked 1d convolutions for a parameterized function h due to their computational efficiency. potentially, millions of sequences have to be processed for each input object. in principle, also recurrent neural networks (rnns), such as lstms (hochreiter et al., 2007) , or transformer networks (vaswani et al., 2017) may be used but are currently computationally too costly. c) attention-pooling is used to obtain a repertoire-representation z for each input object, where deeprc uses weighted averages of sequence-representations. the weights are determined by an update rule of modern hopfield networks that allows to retrieve exponentially many patterns. classification, in which each frame is an instance, text classification, where words or sentences are instances of a text, point sets, where each point is an instance of a 3d object, and remote sensing data, where each sensor is an instance (carbonneau et al., 2018; uriot, 2019) . attention-based mil has been successfully used for image data, for example to identify tiny objects in large images (ilse et al., 2018; pawlowski et al., 2019; tomita et al., 2019; kimeswenger et al., 2019) and transformer-like attention mechanisms for sets of points and images . however, in mil problems considered by machine learning methods up to now, the number of instances per bag is in the range of hundreds or few thousands (carbonneau et al., 2018; lee et al., 2019 ) (see also tab. a2). at the same time the witness rate (wr), the rate of discriminating instances per bag, is already considered low at 1% − 5%. we will tackle the problem of immune repertoire classification with hundreds of thousands of instances per bag without instance-level labels and with extremely low witness rates down to 0.01% using an attention mechanism. we show that the attention mechanism of transformers is the update rule of modern hopfield networks (krotov & hopfield, 2016 demircigil et al., 2017) that are generalized to continuous states in contrast to classical hopfield networks (hopfield, 1982) . a detailed derivation and analysis of modern hopfield networks is given in our companion paper (ramsauer et al., 2020) . these novel continuous state hopfield networks allow to store and retrieve exponentially (in the dimension of the space) many patterns (see next section). thus, modern hopfield networks with their update rule, which are used as an attention mechanism in the transformer, enable immune repertoire classification in computational biology. immune repertoire classification, i.e. classifying the immune status based on the immune repertoire sequences, is essentially a text-book example for a multiple instance learning problem (dietterich et al., 1997; maron & lozano-pérez, 1998; wang et al., 2018) . briefly, the immune repertoire of an individual consists of an immensely large bag of immune receptors, represented as amino acid sequences. usually, the presence of only a small fraction of particular receptors determines the immune status with respect to a particular disease (christophersen et al., 2014; emerson et al., 2017) . this is because the immune system has already acquired a resistance if one or few particular immune receptors that can bind to the disease agent are present. therefore, classification of immune repertoires bears a high difficulty since each immune repertoire can contain millions of sequences as instances with only a few indicating the class. further properties of the data that complicate the problem are: (a) the overlap of immune repertoires of different individuals is low (in most cases, maximally low single-digit percentage values) (greiff et al., 2017; elhanati et al., 2018) , (b) multiple different sequences can bind to the same pathogen (wucherpfennig et al., 2007) , and (c) only subsequences within the sequences determine whether binding to a pathogen is possible (dash et al., 2017; glanville et al., 2017; akbar et al., 2019; springer et al., 2020; fischer et al., 2019) . in summary, immune repertoire classification can be formulated as multiple instance learning with an extremely low witness rate and large numbers of instances, which represents a challenge for currently available machine learning methods. furthermore, the methods should ideally be interpretable, since the extraction of class-associated sequence motifs is desired to gain crucial biological insights. the acquisition of human immune repertoires has been enabled by immunosequencing technology (georgiou et al., 2014; brown et al., 2019) which allows to obtain the immune receptor sequences and immune repertoires of individuals. each individual is uniquely characterized by their immune repertoire, which is acquired and changed during life. this repertoire may be influenced by all diseases that an individual is exposed to during their lives and hence contains highly valuable information about those diseases and the individual's immune status. immune receptors enable the immune system to specifically recognize disease agents or pathogens. each immune encounter is recorded as an immune event into immune memory by preserving and amplifying immune receptors in the repertoire used to fight a given disease. this is, for example, the working principle of vaccination. each human has about 10 7 -10 8 unique immune receptors with low overlap across individuals and sampled from a potential diversity of > 10 14 receptors (mora & walczak, 2019) . the ability to sequence and analyze human immune receptors at large scale has led to fundamental and mechanistic insights into the adaptive immune system and has also opened the opportunity for the development of novel diagnostics and therapy approaches (georgiou et al., 2014; brown et al., 2019) . immunosequencing data have been analyzed with computational methods for a variety of different tasks (greiff et al., 2015; shugay et al., 2015; miho et al., 2018; yaari & kleinstein, 2015; wardemann & busse, 2017) . a large part of the available machine learning methods for immune receptor data has been focusing on the individual immune receptors in a repertoire, with the aim to, for example, predict the antigen or antigen portion (epitope) to which these sequences bind or to predict sharing of receptors across individuals (gielis et al., 2019; springer et al., 2020; jurtz et al., 2018; moris et al., 2019; fischer et al., 2019; greiff et al., 2017; sidhom et al., 2019; elhanati et al., 2018) . recently, jurtz et al. (2018) used 1d convolutional neural networks (cnns) to predict antigen binding of t-cell receptor (tcr) sequences (specifically, binding of tcr sequences to peptide-mhc complexes) and demonstrated that motifs can be extracted from these models. similarly, konishi et al. (2019) use cnns, gradient boosting, and other machine learning techniques on b-cell receptor (bcr) sequences to distinguish tumor tissue from normal tissue. however, the methods presented so far predict a particular class, the epitope, based on a single input sequence. immune repertoire classification has been considered as a mil problem in the following publications. a deep learning framework called deeptcr (sidhom et al., 2019) implements several deep learning approaches for immunosequencing data. the computational framework, inter alia, allows for attention-based mil repertoire classifiers and implements a basic form of attention-based averaging. ostmeyer et al. (2019) already suggested a mil method for immune repertoire classification. this method considers 4-mers, fixed sub-sequences of length 4, as instances of an input object and trained a logistic regression model with these 4-mers as input. the predictions of the logistic regression model for each 4-mer were max-pooled to obtain one prediction per input object. this approach is characterized by (a) the rigidity of the k-mer features as compared to convolutional kernels (alipanahi et al., 2015; zhou & troyanskaya, 2015; zeng et al., 2016) , (b) the max-pooling operation, which constrains the network to learn from a single, top-ranked k-mer for each iteration over the input object, and (c) the pooling of prediction scores rather than representations (wang et al., 2018) . our experiments also support that these choices in the design of the method can lead to constraints on the predictive performance (see table 1 ). our proposed method, deeprc, also uses a mil approach but considers sequences rather than k-mers as instances within an input object and a transformer-like attention mechanism. deeprc sets out to avoid the above-mentioned constraints of current methods by (a) applying transformer-like attention-pooling instead of max-pooling and learning a classifier on the repertoire rather than on the sequence-representation, (b) pooling learned representations rather than predictions, and (c) using less rigid feature extractors, such as 1d convolutions or lstms. in this work, we contribute the following: we demonstrate that continuous generalizations of binary modern hopfield-networks (krotov & hopfield, 2016 demircigil et al., 2017) have an update rule that is known as the attention mechanisms in the transformer. we show that these modern hopfield networks have exponential storage capacity, which allows them to extract patterns among a large set of instances (next section). based on this result, we propose deeprc, a novel deep mil method based on modern hopfield networks for large bags of complex sequences, as they occur in immune repertoire classification (section "deep repertoire classification). we evaluate the predictive performance of deeprc and other machine learning approaches for the classification of immune repertoires in a large comparative study (section "experimental results") exponential storage capacity of continuous state modern hopfield networks with transformer attention as update rule in this section, we show that modern hopfield networks have exponential storage capacity, which will later allow us to approach massive multiple-instance learning problems, such as immune repertoire classification. see our companion paper (ramsauer et al., 2020) for a detailed derivation and analysis of modern hopfield networks. we assume patterns x 1 , . . . , x n ∈ r d that are stacked as columns to the matrix x = (x 1 , . . . , x n ) and a query pattern ξ that also represents the current state. the largest norm of a pattern is m = max i x i . the separation ∆ i of a pattern x i is defined as its minimal dot product difference to any of the other patterns: we consider a modern hopfield network with current state ξ and the energy function for energy e and state ξ, the update rule is proven to converge globally to stationary points of the energy e, which are local minima or saddle points (see (ramsauer et al., 2020) , appendix, theorem a2 ). surprisingly, the update rule eq. (1) is also the formula of the well-known transformer attention mechanism. to see this more clearly, we simultaneously update several queries ξ i . furthermore the queries ξ i and the patterns x i are linear mappings of vectors y i into the space r d . for matrix notation, we set x i = w t k y i , ξ i = w t q y i and multiply the result of our update rule with w v . using y = (y 1 , . . . , y n ) t , we define the matrices and the patterns are now mapped to the hopfield space with dimension d = d k . we set β = 1/ √ d k and change softmax to a row vector. the update rule eq. (1) multiplied by w v performed for all queries simultaneously becomes in row vector notation: this formula is the transformer attention. if the patterns x i are well separated, the iterate eq. (1) converges to a fixed point close to a pattern to which the initial ξ is similar. if the patterns are not well separated the iterate eq.(1) converges to a fixed point close to the arithmetic mean of the patterns. if some patterns are similar to each other but well separated from all other vectors, then a metastable state between the similar patterns exists. iterates that start near a metastable state converge to this metastable state. for details see ramsauer et al. (2020) , appendix, sect. a2. typically, the update converges after one update step (see ramsauer et al. (2020) , appendix, theorem a8) and has an exponentially small retrieval error (see ramsauer et al. (2020) , appendix, theorem a9). our main concern for application to immune repertoire classification is the number of patterns that can be stored and retrieved by the modern hopfield network, equivalently to the transformer attention head. the storage capacity of an attention mechanism is critical for massive mil problems. we first define what we mean by storing and retrieving patterns from the modern hopfield network. definition 1 (pattern stored and retrieved). we assume that around every pattern x i a sphere s i is given. we say x i is stored if there is a single fixed point x * i ∈ s i to which all points ξ ∈ s i converge, for randomly chosen patterns, the number of patterns that can be stored is exponential in the dimension d of the space of the patterns (x i ∈ r d ). theorem 1. we assume a failure probability 0 < p 1 and randomly chosen patterns on the sphere with radius m = k √ d − 1. we define a := 2 d−1 (1 + ln(2 β k 2 p (d − 1))), b := 2 k 2 β 5 , and c = b w0(exp(a + ln(b)) , where w 0 is the upper branch of the lambert w function and ensure then with probability 1 − p, the number of random patterns that can be stored is examples are c ≥ 3.1546 for β = 1, k = 3, d = 20 and p = 0.001 (a + ln(b) > 1.27) and c ≥ 1.3718 for β = 1 k = 1, d = 75, and p = 0.001 (a + ln(b) < −0.94). see ramsauer et al. (2020) , appendix, theorem a5 for a proof. we have established that a modern hopfield network or a transformer attention mechanism can store and retrieve exponentially many patterns. this allows us to approach mil with massive numbers of instances from which we have to retrieve a few with an attention mechanism. deep repertoire classification problem setting and notation. we consider a mil problem, in which an input object x is a bag of n instances x = {s 1 , . . . , s n }. the instances do not have dependencies nor orderings between them and n can be different for every object. we assume that each instance s i is associated with a label y i ∈ {0, 1}, assuming a binary classification task, to which we do not have access. we only have access to a label y = max i y i for an input object or bag. note that this poses a credit assignment problem, since the sequences that are responsible for the label y have to be identified and that the relation between instance-label and bag-label can be more complex (foulds & frank, 2010) . a modelŷ = g(x) should be (a) invariant to permutations of the instances and (b) able to cope with the fact that n varies across input objects (ilse et al., 2018) , which is a problem also posed by point sets (qi et al., 2017) . two principled approaches exist. the first approach is to learn an instance-level scoring function h : s → [0, 1], which is then pooled across instances with a pooling function f , for example by average-pooling or max-pooling (see below). the second approach is to construct an instance representation z i of each instance by h : s → r dv and then encode the bag, or the input object, by pooling these instance representations (wang et al., 2018) via a function f . an output function o : r dv → [0, 1] subsequently classifies the bag. the second approach, the pooling of representations rather than scoring functions, is currently best performing (wang et al., 2018) . in the problem at hand, the input object x is the immune repertoire of an individual that consists of a large set of immune receptor sequences (t-cell receptors or antibodies). immune receptors are primarily represented as sequences s i from a space s i ∈ s. these sequences act as the instances in the mil problem. although immune repertoire classification can readily be formulated as a mil problem, it is yet unclear how well machine learning methods solve the above-described problem with a large number of instances n 10, 000 and with instances s i being complex sequences. next we describe currently used pooling functions for mil problems. pooling functions for mil problems. different pooling functions equip a model g with the property to be invariant to permutations of instances and with the ability to process different numbers of instances. typically, a neural network h θ with parameters θ is trained to obtain a function that maps each instance onto a representation: z i = h θ (s i ) and then a pooling function z = f ({z 1 , . . . , z n }) supplies a representation z of the input object x = {s 1 , . . . , s n }. the following pooling functions are typically used: average-pooling: where e m is the standard basis vector for dimension m and attention-pooling: z = n i=1 a i z i , where a i are non-negative (a i ≥ 0), sum to one ( n i=1 a i = 1), and are determined by an attention mechanism. these pooling functions are invariant to permutations of {1, . . . , n } and are differentiable. therefore, they are suited as building blocks for deep learning architectures. we employ attention-pooling in our deeprc model as detailed in the following. modern hopfield networks viewed as transformer-like attention mechanisms. the modern hopfield networks, as introduced above,have a storage capacity that is exponential in the dimension of the vector space and converge after just one update (see (ramsauer et al., 2020) , appendix).additionally, the update rule of modern hopfield networks is known as key-value attention mechanism, which has been highly successful through the transformer (vaswani et al., 2017) and bert (devlin et al., 2019) models in natural language processing. therefore using modern hopfield networks with the key-value-attention mechanism as update rule is the natural choice for our task. in particular, modern hopfield networks are theoretically justified for storing and retrieving the large number of vectors (sequence patterns) that appear in the immune repertoire classification task. instead of using the terminology of modern hopfield networks, we explain our deeprc architecture in terms of key-value-attention (the update rule of the modern hopfield network), since it is well known in the deep learning community. the attention mechanism assumes a space of dimension d k in which keys and queries are compared. a set of n key vectors are combined to the matrix k. a set of d q query vectors are combined to the matrix q. similarities between queries and keys are computed by inner products, therefore queries can search for similar keys that are stored. another set of n value vectors are combined to the matrix v . the output of the attention mechanism is a weighted average of the value vectors for each query q. the i-th vector v i is weighted by the similarity between the i-th key k i and the query q. the similarity is given by the softmax of the inner products of the query q with the keys k i . all queries are calculated in parallel via matrix operations. consequently, the attention function att(q, k, v ; β) maps queries q, keys k, and values v to d v -dimensional outputs: att(q, k, v ; β) = softmax(βqk t )v (see also eq. (2)). while this attention mechanism has originally been developed for sequence tasks (vaswani et al., 2017) , it can be readily transferred to sets ye et al., 2018) . this type of attention mechanism will be employed in deeprc. the deeprc method. we propose a novel method deep repertoire classification (deeprc) for immune repertoire classification with attention-based deep massive multiple instance learning and compare it against other machine learning approaches. for deeprc, we consider immune repertoires as input objects, which are represented as bags of instances. in a bag, each instance is an immune receptor sequence and each bag can contain a large number of sequences. note that we will use z i to denote the sequence-representation of the i-th sequence and z to denote the repertoire-representation. at the core, deeprc consists of a transformer-like attention mechanism that extracts the most important information from each repertoire. we first give an overview of the attention mechanism and then provide details on each of the sub-networks h 1 , h 2 , and o of deeprc. attention mechanism in deeprc. this mechanism is based on the three matrices k (the keys), q (the queries), and v (the values) together with a parameter β. values. deeprc uses a 1d convolutional network h 1 (lecun et al., 1998; hu et al., 2014; kelley et al., 2016) that supplies a sequence-representation z i = h 1 (s i ), which acts as the values v = z = (z 1 , . . . , z n ) in the attention mechanism (see figure 2 ). keys. a second neural network h 2 , which shares its first layers with h 1 , is used to obtain keys k ∈ r n ×d k for each sequence in the repertoire. this network uses 2 self-normalizing layers (klambauer et al., 2017) with 32 units per layer (see figure 2 ). query. we use a fixed d k -dimensional query vector ξ which is learned via backpropagation. for more attention heads, each head has a fixed query vector. with the quantities introduced above, the transformer attention mechanism (eq. (2)) of deeprc is implemented as follows: where z ∈ r n ×dv are the sequence-representations stacked row-wise, k are the keys, and z is the repertoire-representation and at the same time a weighted mean of sequence-representations z i . the attention mechanism can readily be extended to multiple queries, however, computational demand could constrain this depending on the application and dataset. theorem 1 demonstrates that this mechanism is able to retrieve a single pattern out of several hundreds of thousands. attention-pooling and interpretability. each input object, i.e. repertoire, consists of a large number n of sequences, which are reduced to a single fixed-size feature vector of length d v representing the whole input object by an attention-pooling function. to this end, a transformer-like attention mechanism adapted to sets is realized in deeprc which supplies a i -the importance of the sequence s i . this importance value is an interpretable quantity, which is highly desired for the immunological problem at hand. thus, deeprc allows for two forms of interpretability methods. (a) a trained deeprc model can compute attention weights a i , which directly indicate the importance of a sequence. (b) deeprc furthermore allows for the usage of contribution analysis methods, such as integrated gradients (ig) (sundararajan et al., 2017) or layer-wise relevance propagation (montavon et al., 2018; arras et al., 2019) . see sect. a8 for details. classification layer and network parameters. the repertoire-representation z is then used as input for a fully-connected output networkŷ = o(z) that predicts the immune status, where we found it sufficient to train single-layer networks. in the simplest case, deeprc predicts a single target, the class label y, e.g. the immune status of an immune repertoire, using one output value. however, since deeprc is an end-to-end deep learning model, multiple targets may be predicted simultaneously in classification or regression settings or a mix of both. this allows for the introduction of additional information into the system via auxiliary targets such as age, sex, or other metadata. table 1 with sub-networks h 1 , h 2 , and o. d l indicates the sequence length. network parameters, training, and inference. deeprc is trained using standard gradient descent methods to minimize a cross-entropy loss. the network parameters are θ 1 , θ 2 , θ o for the sub-networks h 1 , h 2 , and o, respectively, and additionally ξ. in more detail, we train deeprc using adam (kingma & ba, 2014) with a batch size of 4 and dropout of input sequences. implementation. to reduce computational time, the attention network first computes the attention weights a i for each sequence s i in a repertoire. subsequently, the top 10% of sequences with the highest a i per repertoire are used to compute the weight updates and prediction. furthermore, computation of z i is performed in 16-bit, others in 32-bit precision to ensure numerical stability in the softmax. see sect. a2 for details. in this section, we report and analyze the predictive power of deeprc and the compared methods on several immunosequencing datasets. the roc-auc is used as the main metric for the predictive power. methods compared. we compared previous methods for immune repertoire classification, (ostmeyer et al., 2019) ("log. mil (kmer)", "log. mil (tcrb)") and a burden test (emerson et al., 2017) , as well as the baseline methods logistic regression ("log. regr."), k-nearest neighbour ("knn"), and support vector machines ("svm") with kernels designed for sets, such as the jaccard kernel ("j") and the minmax ("mm") kernel (ralaivola et al., 2005) . for the simulated data, we also added baseline methods that search for the implanted motif either in binary or continuous fashion ("known motif b.", "known motif c.") assuming that this motif was known (for details, see sect. a4). datasets. we aimed at constructing immune repertoire classification scenarios with varying degree of difficulties and realism in order to compare and analyze the suggested machine learning methods. to this end, we either use simulated or experimentally-observed immune receptor sequences and we implant signals, specifically, sequence motifs or sets thereof weber et al., 2020) , at different frequencies into sequences of repertoires of the positive class. these frequencies represent the witness rates and range from 0.01% to 10%. overall, we compiled four categories of datasets: (a) simulated immunosequencing data with implanted signals, (b) lstm-generated immunosequencing data with implanted signals, (c) real-world immunosequencing data with implanted signals, and (d) real-world immunosequencing data with known immune status, the cmv dataset (emerson et al., 2017) . the average number of instances per bag, which is the number of sequences per immune repertoire, is ≈300,000 except for category (c), in which we consider the scenario of low-coverage data with only 10,000 sequences per repertoire. the number of repertoires per dataset ranges from 785 to 5,000. in total, all datasets comprise ≈30 billion sequences or instances. this represents the largest comparative study on immune repertoire classification (see sect. a3). hyperparameter selection. we used a nested 5-fold cross validation (cv) procedure to estimate the performance of each of the methods. all methods could adjust their most important hyperparameters on a validation set in the inner loop of the procedure. see sect. a5 for details. table 1 : results in terms of auc of the competing methods on all datasets. the reported errors are standard deviations across 5 cross-validation (cv) folds (except for the column "simulated"). real-world cmv: average performance over 5 cv folds on the cmv dataset (emerson et al., 2017) . real-world data with implanted signals: average performance over 5 cv folds for each of the four datasets. a signal was implanted with a frequency (=witness rate) of 1% or 0.1%. either a single motif ("om") or multiple motifs ("mm") were implanted. lstm-generated data: average performance over 5 cv folds for each of the 5 datasets. in each dataset, a signal was implanted with a frequency of 10%, 1%, 0.5%, 0.1%, or 0.05%, respectively. simulated: here we report the mean over 18 simulated datasets with implanted signals and varying difficulties (see tab. a9 for details). the error reported is the standard deviation of the auc values across the 18 datasets. results. in each of the four categories, "real-world data", "real-world data with implanted signals", "lstm-generated data", and "simulated immunosequencing data", deeprc outperforms all competing methods with respect to average auc. across categories, the runner-up methods are either the svm for mil problems with minmax kernel or the burden test (see table 1 and sect. a6). results on simulated immunosequencing data. in this setting the complexity of the implanted signal is in focus and varies throughout 18 simulated datasets (see sect. a3). some datasets are challenging for the methods because the implanted motif is hidden by noise and others because only a small fraction of sequences carries the motif, and hence have a low witness rate. these difficulties become evident by the method called "known motif binary", which assumes the implanted motif is known. the performance of this method ranges from a perfect auc of 1.000 in several datasets to an auc of 0.532 in dataset '17' (see sect. a6). deeprc outperforms all other methods with an average auc of 0.846 ± 0.223, followed by the svm with minmax kernel with an average auc of 0.827 ± 0.210 (see sect. a6). the predictive performance of all methods suffers if the signal occurs only in an extremely small fraction of sequences. in datasets, in which only 0.01% of the sequences carry the motif, all auc values are below 0.550. results on lstm-generated data. on the lstm-generated data, in which we implanted noisy motifs with frequencies of 10%, 1%, 0.5%, 0.1%, and 0.05%, deeprc yields almost perfect predictive performance with an average auc of 1.000 ± 0.001 (see sect. a6 and a7). the second best method, svm with minmax kernel, has a similar predictive performance to deeprc on all datasets but the other competing methods have a lower predictive performance on datasets with low frequency of the signal (0.05%). results on real-world data with implanted motifs. in this dataset category, we used real immunosequences and implanted single or multiple noisy motifs. again, deeprc outperforms all other methods with an average auc of 0.980 ± 0.029, with the second best method being the burden test with an average auc of 0.883 ± 0.170. notably, all methods except for deeprc have difficulties with noisy motifs at a frequency of 0.1% (see tab. a11) . results on real-world data. on the real-world dataset, in which the immune status of persons affected by the cytomegalovirus has to be predicted, the competing methods yield predictive aucs between 0.515 and 0.825 (see table 1 ). we note that this dataset is not the exact dataset that was used in emerson et al. (2017) . it differs in pre-processing and also comprises a different set of samples and a smaller training set due to the nested 5-fold cross-validation procedure, which leads to a more challenging dataset. the best performing method is deeprc with an auc of 0.831 ± 0.002, followed by the svm with minmax kernel (auc 0.825 ± 0.022) and the burden test with an auc of 0.699 ± 0.041. the top-ranked sequences by deeprc significantly correspond to those detected by emerson et al. (2017) , which we tested by a mann-whitney u-test with the null hypothesis that the attention values of the sequences detected by emerson et al. (2017) would be equal to the attention values of the remaining sequences (p-value of 1.3 · 10 −93 ). the sequence attention values are displayed in tab. a14. we have demonstrated how modern hopfield networks and attention mechanisms enable successful classification of the immune status of immune repertoires. for this task, methods have to identify the discriminating sequences amongst a large set of sequences in an immune repertoire. specifically, even motifs within those sequences have to be identified. we have shown that deeprc, a modern hopfield network and an attention mechanism with a fixed query, can solve this difficult task despite the massive number of instances. deeprc furthermore outperforms the compared methods across a range of different experimental conditions. impact on machine learning and related scientific fields. we envision that with (a) the increasing availability of large immunosequencing datasets (kovaltsuk et al., 2018; corrie et al., 2018; christley et al., 2018; zhang et al., 2020; rosenfeld et al., 2018; shugay et al., 2018) , (b) further fine-tuning of ground-truth benchmarking immune receptor datasets (weber et al., 2020; olson et al., 2019; marcou et al., 2018) , (c) accounting for repertoire-impacting factors such as age, sex, ethnicity, and environment (potential confounding factors), and (d) increased gpu memory and increased computing power, it will be possible to identify discriminating immune receptor motifs for many diseases, potentially even for the current sars-cov-2 (covid-19) pandemic minervina et al., 2020; galson et al., 2020) . such results would greatly benefit ongoing research on antibody and tcr-driven immunotherapies and immunodiagnostics as well as rational vaccine design (brown et al., 2019) . in the course of this development, the experimental verification and interpretation of machine-learningidentified motifs could receive additional focus, as for most of the sequences within a repertoire the corresponding antigen is unknown. nevertheless, recent technological breakthroughs in highthroughput antigen-labeled immunosequencing are beginning to generate large-scale antigen-labeled single-immune-receptor-sequence data thus resolving this longstanding problem (setliff et al., 2019) . from a machine learning perspective, the successful application of deeprc on immune repertoires with their large number of instances per bag might encourage the application of modern hopfield networks and attention mechanisms on new, previously unsolved or unconsidered, datasets and problems. impact on society. if the approach proves itself successful, it could lead to faster testing of individuals for their immune status w.r.t. a range of diseases based on blood samples. this might motivate changes in the pipeline of diagnostics and tracking of diseases, e.g. automated testing of the immune status in regular intervals. it would furthermore make the collection and screening of blood samples for larger databases more attractive. in consequence, the improved testing of immune statuses might identify individuals that do not have a working immune response towards certain diseases to government or insurance companies, which could then push for targeted immunisation of the individual. similarly to compulsory vaccination, such testing for the immune status could be made compulsory by governments, possibly violating privacy or personal self-determination in exchange for increased over-all health of a population. ultimately, if the approach proves itself successful, the insights gained from the screening of individuals that have successfully developed resistances against specific diseases could lead to faster targeted immunisation, once a certain number of individuals with resistances can be found. this might strongly decrease the harm done by e.g. pandemics and lead to a change in the societal perception of such diseases. consequences of failures of the method. as common with methods in machine learning, potential danger lies in the possibility that users rely too much on our new approach and use it without reflecting on the outcomes. however, the full pipeline in which our method would be used includes wet lab tests after its application, to verify and investigate the results, gain insights, and possibly derive treatments. failures of the proposed method would lead to unsuccessful wet lab validation and negative wet lab tests. since the proposed algorithm does not directly suggest treatment or therapy, human beings are not directly at risk of being treated with a harmful therapy. substantial wet lab and in-vitro testing and would indicate wrong decisions by the system. leveraging of biases in the data and potential discrimination. as for almost all machine learning methods, confounding factors, such as age or sex, could be used for classification. this, might lead to biases in predictions or uneven predictive performance across subgroups. as a result, failures in the wet lab would occur (see paragraph above). moreover, insights into the relevance of the confounding factors could be gained, leading to possible therapies or counter-measures concerning said factors. furthermore, the amount of data available with respec to relevant confounding factors could lead to better or worse performance of our method. e.g. a dataset consisting mostly of data from individuals within a specific age group might yield better performance for that age group, possibly resulting in better or exclusive treatment methods for that specific group. here again, the application of deeprc would be followed by in-vitro testing and development of a treatment, where all target groups for the treatment have to be considered accordingly. all datasets and code is available at https://github.com/ml-jku/deeprc. the cmv dataset is publicly available at https://clients.adaptivebiotech.com/pub/emerson-2017-natgen. in section a2 we provide details on the architecture of deeprc, in section a3 we present details on the datasets, in section a4 we explain the methods that we compared, in section a5 we elaborate on the hyperparameters and their selection process. then, in section a6 we present detailed results for each dataset category in tabular form, in section a7 we provide information on the lstm model that was used to generate antibody sequences, in section a8 we show how deeprc can be interpreted, in section a9 we show the correspondence of previously identified tcr sequences for cmv immune status with attention values by deeprc, and finally we present variations and an ablation study of deeprc in section a10. input layer. for the input layer of the cnn, the characters in the input sequence, i.e. the amino acids (aas), are encoded in a one-hot vector of length 20. to also provide information about the position of an aa in the sequence, we add 3 additional input features with values in range [0, 1] to encode the position of an aa relative to the sequence. these 3 positional features encode whether the aa is located at the beginning, the center, or the end of the sequence, respectively, as shown in figure a1 . we concatenate these 3 positional features with the one-hot vector of aas, which results in a feature vector of size 23 per sequence position. each repertoire, now represented as a bag of feature vectors, is then normalized to unit variance. since the cytomegalovirus dataset (cmv dataset) provides sequences with an associated abundance value per sequence, which is the number of occurrences of a sequence in a repertoire, we incorporate this information into the input of deeprc. to this end, the one-hot aa features of a sequence are multiplied by a scaling factor of log(c a ) before normalization, where c a is the abundance of a sequence. we feed the sequences with 23 features per position into the cnn. sequences of different lengths were zero-padded to the maximum sequence length per batch at the sequence ends. 1d cnn for motif recognition. in the following, we describe how deeprc identifies patterns in the individual sequences and reduces each sequence in the input object to a fixed-size feature vector. deeprc employs 1d convolution layers to extract patterns, where trainable weight kernels are convolved over the sequence positions. in principle, also recurrent neural networks (rnns) or transformer networks could be used instead of 1d cnns, however, (a) the computational complexity of the network must be low to be able to process millions of sequences for a single update. additionally, (b) the learned network should be able to provide insights in the recognized patterns in form of motifs. both properties (a) and (b) are fulfilled by 1d convolution operations that are used by deeprc. we use one 1d cnn layer (hu et al., 2014) with selu activation functions (klambauer et al., 2017) to identify the relevant patterns in the input sequences with a computationally light-weight operation. the larger the kernel size, the more surrounding sequence positions are taken into account, which influences the length of the motifs that can be extracted. we therefore adjust the kernel size during hyperparameter search. in prior works (ostmeyer et al., 2019) , a k-mer size of 4 yielded good predictive performance, which could indicate that a kernel size in the range of 4 may be a proficient choice. for d v trainable kernels, this produces a feature vector of length d v at each sequence position. subsequently, global max-pooling over all sequence positions of a sequence reduces the sequence-representations z i to vectors of the fixed length d v . given the challenging size of the input data per repertoire, the computation of the cnn activations and weight updates is performed using 16-bit floating point values. a list of hyperparameters evaluated for deeprc is given in table a3 . a comparison of rnn-based and cnn-based sequence embedding for motif recognition in a smaller experimental setting is given in sec. a10. regularization. we apply random and attention-based subsampling of repertoire sequences to reduce over-fitting and decrease computational effort. during training, each repertoire is subsampled to 10, 000 input sequences, which are randomly drawn from the respective repertoire. this can also be interpreted as random drop-out (hinton et al., 2012) on the input sequences or attention weights. during training and evaluation, the attention weights computed by the attention network are furthermore used to rank the input sequences. based on this ranking, the repertoire is reduced to the 10% of sequences with the highest attention weights. these top 10% of sequences are then used to compute the weight updates and the prediction for the repertoire. additionally, one might employ further regularization techniques, which we only partly investigated further in a smaller experimental setting in sec. a10 due to high computational demands. such regularization techniques include l1 and l2 weight decay, noise in the form of random aa permutations in the input sequences, noise on the attention weights, or random shuffling of sequences between repertoires that belong to the negative class. the last regularization technique assumes that the sequences in positive-class repertoires carry a signal, such as an aa motif corresponding to an immune response, whereas the sequences in negative-class repertoires do not. hence, the sequences can be shuffled randomly between negative class repertoires without obscuring the signal in the positive class repertoires. hyperparameters. for the hyperparameter search of deeprc for the category "simulated immunosequencing data", we only conducted a full hyperparameter search on the more difficult datasets with motif implantation probabilities below 1%, as described in table a3 . this process was repeated for all 5 folds of the 5-fold cross-validation (cv) and the average score on the 5 test sets constitutes the final score of a method. table a3 provides an overview of the hyperparameter search, which was conducted as a grid search for each of the datasets in a nested 5-fold cv procedure, as described in section a4. computation time and optimization. we took measures on the implementation level to address the high computational demands, especially gpu memory consumption, in order to make the large number of experiments feasible. we train the deeprc model with a small batch size of 4 samples and perform computation of inference and updates of the 1d cnn using 16-bit floating point values. the rest of the network is trained using 32-bit floating point values. the adam parameter for numerical stability was therefore increased from the default value of = 10 −8 to = 10 −4 . training was performed on various gpu types, mainly nvidia rtx 2080 ti. computation times were highly dependent on the number of sequences in the repertoires and the number and sizes of cnn kernels. a single update on an nvidia rtx 2080 ti gpu took approximately 0.0129 to 0.0135 seconds, while requiring approximately 8 to 11 gb gpu memory. taking these optimizations and gpus with larger memory (≥ 16 gb) into account, it is already possible to train deeprc, possibly with multi-head attention and a larger network architecture, on larger datasets (see sec. a10). our network implementation is based on pytorch 1.3.1 (paszke et al., 2019) . incorporation of additional inputs and metadata. additional metadata in the form of sequencelevel or repertoire-level features could be incorporated into the input via concatenation with the feature vectors that result from taking the maximum of the 1d cnn outputs w.r.t. the sequence positions. this has the benefit that the attention mechanism and output network can utilize the sequence-level or repertoire-level features for their predictions. sparse metadata or metadata that is only available during training could be used as auxiliary targets to incorporate the information via gradients into the deeprc model. limitations. the current methods are mostly limited by computational complexity, since both hyperparameter and model selection is computationally demanding. for hyperparameter selection, a large number of hyperparameter settings have to be evaluated. for model selection, a single repertoire requires the propagation of many thousands of sequences through a neural network and keeping those quantities in gpu memory in order to perform the attention mechanism and weight update. thus, increased gpu memory would significantly boost our approach. increased computational power would also allow for more advanced architectures and attention mechanisms, which may further improve predictive performance. another limiting factor is over-fitting of the model due to the currently relatively small number of samples (bags) in real-world immunosequencing datasets in comparison to the large number of instances per bag and features per instance. we aimed at constructing immune repertoire classification scenarios with varying degree of realism and difficulties in order to compare and analyze the suggested machine learning methods. to this end, we either use simulated or experimentally-observed immune receptor sequences and we implant signals, which are sequence motifs weber et al., 2020) , into sequences of repertoires of the positive class. it has been shown previously that interaction of immune receptors with antigens occur via short sequence stretches . thus, implantation of short motif sequences simulating an immune signal is biologically meaningful. our benchmarking study comprises four different categories of datasets: (a) simulated immunosequencing data with implanted signals (where the signal is defined as sets of motifs), (b) lstm-generated immunosequencing data with implanted signals, (c) real-world immunosequencing data with implanted signals, and (d) real-world immunosequencing data. each of the first three categories consists of multiple datasets with varying difficulty depending on the type of the implanted signal and the ratio of sequences with the implanted signal. the ratio of sequences with the implanted signal, where each sequence carries at most 1 implanted signal, corresponds to the witness rate (wr). we consider binary classification tasks to simulate the immune status of healthy and diseased individuals. we randomly generate immune repertoires with varying numbers of sequences, where we implant sequence motifs in the repertoires of the diseased individuals, i.e. the positive class. the sequences of a repertoire are also randomly generated by different procedures (detailed below). each sequence is composed of 20 different characters, corresponding to amino acids, and has an average length of 14.5 aas. in the first category, we aim at investigating the impact of the signal frequency, i.e. the wr, and the signal complexity on the performance of the different methods. to this end, we created 18 datasets, whereas each dataset contains a large number of repertoires with a large number of random aa sequences per repertoire. we then implanted signals in the aa sequences of the positive class repertoires, where the 18 datasets differ in frequency and complexity of the implanted signals. in detail, the aas were sampled randomly independent of their respective position in the sequence, while the frequencies of aas, distribution of sequence lengths, and distribution of the number of sequences per repertoire, i.e. the number of instances per bag, are following the respective distributions observed in the real-world cmv dataset (emerson et al., 2017) . for this, we first sampled the number of sequences for a repertoire from a gaussian n (µ = 316k, σ = 132k) distribution and rounded to the nearest positive integer. we re-sampled if the size was below 5k. we then generated random sequences of aas with a length of n (µ = 14.5, σ = 1.8), again rounded to the nearest positive integers. each simulated repertoire was then randomly assigned to either the positive or negative class, with 2, 500 repertoires per class. in the repertoires assigned to the positive class, we implanted motifs with an average length of 4 aas, following the results of the experimental analysis of antigenbinding motifs in antibodies and t-cell receptor sequences by . we varied the characteristics of the implanted motifs for each of the 18 datasets with respect to the following parameters: (a) ρ, the probability of a motif being implanted in a sequence of a positive repertoire, i.e. the average ratio of sequences containing the motif, which is the witness rate. in this way, we generated 18 different datasets of variable difficulty containing in total roughly 28.7 billion sequences. see table a1 for an overview of the properties of the implanted motifs in the 18 datasets. in the second dataset category, we investigate the impact of the signal frequency and complexity in combination with more plausible immune receptor sequences by taking into account the positional aa distributions and other sequence properties. to this end, we trained an lstm (hochreiter & schmidhuber, 1997 ) in a standard next character prediction (graves, 2013) setting to create aa sequences with properties similar to experimentally observed immune receptor sequences. in the first step, the lstm model was trained on all immuno-sequences in the cmv dataset (emerson et al., 2017) that contain valid information about sequence abundance and have a known cmv label. such an lstm model is able to capture various properties of the sequences, including positiondependent probability distributions and combinations, relationships, and order of aas. we then used the trained lstm model to generate 1, 000 repertoires in an autoregressive fashion, starting with a start sequence that was randomly sampled from the trained-on dataset. based on a visual inspection of the frequencies of 4-mers (see section a7), the similarity of lstm generated sequences and real sequences was deemed sufficient for the purpose of generating the aa sequences for the datasets in this category. further details on lstm training and repertoire generation are given in section a7. after generation, each repertoire was assigned to either the positive or negative class, with 500 repertoires per class. we implanted motifs of length 4 with varying properties in the center of the sequences of the positive class to obtain 5 different datasets. each sequence in the positive repertoires has a probability ρ to carry the motif, which was varied throughout 5 datasets and corresponds to the wr (see table a1 ). each position in the motif has a probability of 0.9 to be implanted and consequently a probability of 0.1 that the original aa in the sequence remains, which can be seen as noise on the motif. in the third category, we implanted signals into experimentally obtained immuno-sequences, where we considered 4 dataset variations. each dataset consists of 750 repertoires for each of the two classes, where each repertoire consists of 10k sequences. in this way, we aim to simulate datasets with a low sequencing coverage, which means that only relatively few sequences per repertoire are available. the sequences were randomly sampled from healthy (cmv negative) individuals from the cmv dataset (see below paragraph for explanation). two signal types were considered: (a) one signal with one motif. the aa motif ldr was implanted in a certain fraction of sequences. the pattern is randomly altered at one of the three positions with probabilities 0.2, 0.6, and 0.2, respectively. (b) one signal with multiple motifs. one of the three possible motifs ldr, cas, and gl-n was table a1 : properties of simulated repertoires, variations of motifs, and motif frequencies, i.e. the witness rate, for the datasets in categories "simulated immunosequencing data", "lstm-generated data", and "real-world data with implanted signals". noise types for * are explained in paragraph "real-world data with implanted signals". implanted with equal probability. again, the motifs were randomly altered before implantation. the aa motif ldr changed as described above. the aa motif cas was altered at the second position with probability 0.6 and with probability 0.3 at the first position. the pattern gl-n, wheredenotes a gap location, is randomly altered at the first position with probability 0.6 and the gap has a length of 0, 1, or 2 aas with equal probability. additionally, the datasets differ in the values for ρ, the average ratio of sequences carrying a signal, which were chosen as 1% or 0.1%. the motifs were implanted at positions 107, 109, and 114 according to the imgt numbering scheme for immune receptor sequences (lefranc et al., 2003) with probabilities 0.3, 0.35 and 0.2, respectively. with the remaining 0.15 chance, the motif is implanted at any other sequence position. this means that the motif occurrence in the simulated sequences is biased towards the middle of the sequence. we used a real-world dataset of 785 repertoires, each of which containing between 4, 371 to 973, 081 (avg. 299, 319) tcr sequences with a length of 1 to 27 (avg. 14.5) aas, originally collected and provided by emerson et al. (2017) . 340 out of 785 repertoires were labelled as positive for cytomegalovirus (cmv) serostatus, which we consider as the positive class, 420 repertoires with negative cmv serostatus, considered as negative class, and 25 repertoires with unknown status. we changed the number of sequence counts per repertoire from −1 to 1 for 3 sequences. furthermore, we exclude a total of 99 repertoires with unknown cmv status or unknown information about the sequence abundance within a repertoire, reducing the dataset for our analysis to 686 repertoires, 312 of which with positive and 374 with negative cmv status. we give a non-exhaustive overview of previously considered mil datasets and problems in table a2 . to our knowledge the datasets considered in this work pose the most challenging mil problems with respect to the number of instances per bag (column 5). table a2 : mil datasets with their numbers of bags and numbers of instances. "total number of instances" refers to the total number of instances in the dataset. the simulated and real-world immunosequencing datasets considered in this work contain a by orders of magnitudes larger number of instances per bag than mil datasets that were considered by machine learning methods up to now. we evaluate and compare the performance of deeprc against a set of machine learning methods that serve as baseline, were suggested, or can readily be adapted to immune repertoire classification. in this section, we describe these compared methods. this method serves as an estimate for the achievable classification performance using prior knowledge about which motif was implanted. note that this does not necessarily lead to perfect predictive performance since motifs are implanted with a certain amount of noise and could also be present in the negative class by chance. the known motif method counts how often the known implanted motif occurs per sequence for each repertoire and uses this count to rank the repertoires. from this ranking, the area under the receiver operator curve (auc) is computed as performance measure. probabilistic aa changes in the known motif are not considered for this count, with the exception of gap positions. we consider two versions of this method: (a) known motif binary: counts the occurrence of the known motif in a sequence and (b) known motif continuous: counts the maximum number of overlapping aas between the known motif and all sequence positions, which corresponds to a convolution operation with a binary kernel followed by max-pooling. since the implanted signal is not known in the experimentally obtained cmv dataset, this method cannot be applied to this dataset. the support vector machine (svm) approach uses a fixed mapping from a bag of sequences to the corresponding k-mer counts. the function h kmer maps each sequence s i to a vector representing the occurrence of k-mers in the sequence. to avoid confusion with the sequence-representation obtained from the cnn layers of deeprc, we denote u i = h kmer (s i ), which is analogous to z i . specifically, where #{p m ∈ s i } denotes how often the k-mer pattern p m occurs in sequence s i . afterwards, average-pooling is applied to obtain u = 1/n n i=1 u i , the k-mer representation of the input object x. for two input objects x (n) and x (l) with representations u (n) and u (l) , respectively, we implement the minmax kernel (ralaivola et al., 2005) as follows: where u (n) m is the m-th element of the vector u (n) . the jaccard kernel (levandowsky & winter, 1971 ) is identical to the minmax kernel except that it operates on binary u (n) . we used a standard c-svm, as introduced by cortes & vapnik (1995) . the corresponding hyperparameter c is optimized by random search. the settings of the full hyperparameter search as well as the respective value ranges are given in table a4a . the same k-mer representation of a repertoire, as introduced above for the svm baseline, is used for the k-nearest neighbor (knn) approach. as this method clusters samples according to distances between them, the previous kernel definitions cannot be applied directly. it is therefore necessary to transform the minmax as well as the jaccard kernel from similarities to distances by constructing the following (levandowsky & winter, 1971) : d jaccard (u (n) , u (l) ) = 1 − k jaccard (u (n) , u (l) ). (a2) the amount of neighbors is treated as the hyperparameter and optimized by an exhaustive grid search. the settings of the full hyperparameter search as well as the respective value ranges are given in table a5 . we implemented logistic regression on the k-mer representation u of an immune repertoire. the model is trained by gradient descent using the adam optimizer (kingma & ba, 2014) . the learning rate is treated as the hyperparameter and optimized by grid search. furthermore, we explored two regularization settings using combinations of l1 and l2 weight decay. the settings of the full hyperparameter search as well as the respective value ranges are given in table a6 . we implemented a burden test (emerson et al., 2017; li & leal, 2008; wu et al., 2011) in a machine learning setting. the burden test first identifies sequences or k-mers that are associated with the individual's class, i.e., immune status, and then calculates a burden score per individual. concretely, for each k-mer or sequence, the phi coefficient of the contingency table for absence or presence and positive or negative immune status is calculated. then, j k-mers or sequences with the highest phi coefficients are selected as the set of associated k-mers or sequences. j is a hyperparameter that is selected on a validation set. additionally, we consider the type of input features, sequences or k-mers, as a hyperparameter. for inference, a burden score per individual is calculated as the sum of associated k-mers or sequences it carries. this score is used as raw prediction and to rank the individuals. hence, we have extended the burden test by emerson et al. (2017) to k-mers and to adaptive thresholds that are adjusted on a validation set. the logistic multiple instance learning (mil) approach for immune repertoire classification (ostmeyer et al., 2019) applies a logistic regression model to each k-mer representation in a bag. the resulting scores are then summarized by max-pooling to obtain a prediction for the bag. each amino acid of each k-mer is represented by 5 features, the so-called atchley factors (atchley et al., 2005) . as k-mers of length 4 are used, this gives a total of 4 × 5 = 20 features. one additional feature per 4-mer is added, which represents the relative frequency of this 4-mer with respect to its containing bag, resulting in 21 features per 4-mer. two options for the relative frequency feature exist, which are (a) whether the frequency of the 4-mer ("4mer") or (b) the frequency of the sequence in which the 4-mer appeared ("tcrβ") is used. we optimized the learning rate, batch size, and early stopping parameter on the validation set. the settings of the full hyperparameter search as well as the respective value ranges are given in table a8 . for all competing methods a hyperparameter search was performed, for which we split each of the 5 training sets into an inner training set and inner validation set. the models were trained on the inner training set and evaluated on the inner validation set. the model with the highest auc score on the inner validation set is then used to calculate the score on the respective test set. here we report the hyperparameter sets and search strategy that is used for all methods. deeprc. the set of hyperparameters of deeprc is shown in table a3 . these hyperparameter combinations are adjusted via a grid search procedure. table a3 : deeprc hyperparameter search space. every 5 · 10 3 updates, the current model was evaluated against the validation fold. the early stopping hyperparameter was determined by selecting the model with the best loss on the validation fold after 10 5 updates. * : experiments for {64; 128; 256} kernels were omitted for datasets with motif implantation probabilities ≥ 1% in the category "simulated immunosequencing data". known motif. this method does not have hyperparameters and has been applied to all datasets except for the cmv dataset. the corresponding hyperparameter c of the svm is optimized by randomly drawing 10 3 values in the range of [−6; 6] according to a uniform distribution. these values act as the exponents of a power of 10 and are applied for each of the two kernel types (see table a4a ). knn. the amount of neighbors is treated as the hyperparameter and optimized by grid search operating in the discrete range of [1; max{n, 10 3 }] with a step size of 1. the corresponding tight upper bound is automatically defined by the total amount of samples n ∈ n >0 in the training set, capped at 10 3 (see table a5 ). number of neighbors {1; max{n, 10 3 }} type of kernel {minmax; jaccard} table a5 : settings used in the hyperparameter search of the knn baseline approach. the number of trials (per type of kernel) is automatically defined by the total amount of samples n ∈ n >0 in the training set, capped at 10 3 . logistic regression. the hyperparameter optimization strategy that was used was grid search across hyperparameters given in table a6. learning rate 10 −{2;3;4} batch size 4 max. updates 10 5 coefficient β 1 (adam) 0.9 coefficient β 2 (adam) 0.999 weight decay weightings {(l1 = 10 −7 , l2 = 10 −3 ); (l1 = 10 −7 , l2 = 10 −5 )} table a6 : settings used in the hyperparameter search of the logistic regression baseline approach. burden test. the burden test selects two hyperparameters: the number of features in the burden set and the type of features, see table a7 . number of features in burden set {50, 100, 150, 250} type of features {4mer; sequence} table a7 : settings used in the hyperparameter search of the burden test approach. logistic mil. for this method, we adjusted the learning rate as well as the batch size as hyperparameters by randomly drawing 25 different hyperparameter combinations from a uniform distribution. the corresponding range of the learning rate is [−4.5; −1.5], which acts as the exponent of a power of 10. the batch size lies within the range of [1; 32]. for each hyperparameter combination, a model is optimized by gradient descent using adam, whereas the early stopping parameter is adjusted according to the corresponding validation set (see table a8 ). learning rate 10 {−4.5;−1.5} batch size {1; 32} relative abundance term {4mer; tcrβ} number of trials 25 max. epochs 10 2 coefficient β 1 (adam) 0.9 coefficient β 2 (adam) 0.999 table a8 : settings used in the hyperparameter search of the logistic mil baseline approach. the number of trials (per type of relative abundance) defines the quantity of combinations of random values of the learning rate as well as the batch size. in this section, we report the detailed results on all four categories of datasets (a) simulated immunosequencing data (table a9 ) (b) lstm-generated data (table a10) , (c) real-world data with implanted signals (table a11) , and (d) real-world data on the cmv dataset (table a12) , as discussed in the main paper. ± 0.000 ± 0.000 ± 0.271 ± 0.000 ± 0.000 ± 0.218 ± 0.000 ± 0.000 ± 0.029 ± 0.000 ± 0.001 ± 0.017 ± 0.001 ± 0.002 ± 0.023 ± 0.001 ± 0.048 ± 0.013 ± 0.223 svm (minmax) 1.000 1.000 0.764 1.000 1.000 0.603 1.000 0.998 0.539 1.000 0.994 0.529 1.000 0.741 0.513 1.000 0.706 0.503 0.827 ± 0.000 ± 0.000 ± 0.016 ± 0.000 ± 0.000 ± 0.021 ± 0.000 ± 0.002 ± 0.024 ± 0.000 ± 0.004 ± 0.016 ± 0.000 ± 0.024 ± 0.006 ± 0.000 ± 0.013 ± 0.013 ± 0.013 ± 0.013 ± 0.014 ± 0.011 ± 0.009 ± 0.007 ± 0.008 ± 0.011 ± 0.012 ± 0.012 ± 0.007 ± 0.014 ± 0.017 ± 0.010 ± 0.020 ± 0.012 ± 0.016 ± 0.016 ± 0.074 known motif b. 1.000 1.000 0.973 1.000 1.000 0.865 1.000 1.000 0.700 1.000 0.989 0.609 1.000 0.946 0.570 1.000 0.834 0.532 0.890 ± 0.000 ± 0.000 ± 0.004 ± 0.000 ± 0.000 ± 0.004 ± 0.000 ± 0.000 ± 0.020 ± 0.000 ± 0.002 ± 0.017 ± 0.000 ± 0.010 ± 0.024 ± 0.000 ± 0.016 ± 0.020 ± 0.001 ± 0.014 ± 0.020 ± 0.001 ± 0.013 ± 0.017 ± 0.001 ± 0.012 ± 0.012 ± 0.001 ± 0.018 ± 0.018 ± 0.002 ± 0.010 ± 0.009 ± 0.002 ± 0.012 ± 0.013 ± 0.202 table a9 : auc estimates based on 5-fold cv for all 18 datasets in category "simulated immunosequencing data". the reported errors are standard deviations across the 5 cross-validation folds except for the last column "avg.", in which they show standard deviations across datasets. wildcard characters in motifs are indicated by z, characters with 50% probability of being removed by d . table a10 : auc estimates based on 5-fold cv for all 5 datasets in category "lstm-generated data". the reported errors are standard deviations across the 5 cross-validation folds except for the last column "avg.", in which they show standard deviations across datasets. characters affected by noise, as described in a3, paragraph "lstm-generated data", are indicated by r . table a12 : results on the cmv dataset (real-world data) in terms of auc, f1 score, balanced accuracy, and accuracy. for f1 score, balanced accuracy, and accuracy, all methods use their default thresholds. each entry shows mean and standard deviation across 5 cross-validation folds. we trained a conventional next-character lstm model (graves, 2013) based on the implementation in https://github.com/spro/practical-pytorch (access date 1st of may, 2020) using pytorch 1.3.1 (paszke et al., 2019) . for this, we applied an lstm model with 100 lstm blocks in 2 layers, which was trained for 5, 000 epochs using the adam optimizer (kingma & ba, 2014) with learning rate 0.01, an input batch size of 100 character chunks, and a character chunk length of 200. as input we used the immuno-sequences in the cdr3 column of the cmv dataset, where we repeated sequences according to their counts in the repertoires, as specified in the templates column of the cmv dataset. we excluded repertoires with unknown cmv status and unknown sequence abundance from training. after training, we generated 1, 000 repertoires using a temperature value of 0.8. the number of sequences per repertoire was sampled from a gaussian n (µ = 285k, σ = 156k) distribution, where the whole repertoire was generated by the lstm at once. that is, the lstm can base the generation of the individual aa sequences in a repertoire, including the aas and the lengths of the sequences, on the generated repertoire. a random immuno-sequence from the trained-on repertoires was used as initialization for the generation process. this immuno-sequence was not included in the generated repertoire. finally, we randomly assigned 500 of the generated repertoires to the positive (diseased) and 500 to the negative (healthy) class. we then implanted motifs in the positive class repertoires as described in section a3.2. as illustrated in the comparison of histograms given in fig. a2 , the generated immuno-sequences exhibit a very similar distribution of 4-mers and aas compared to the original cmv dataset. real-world data deeprc allows for two forms of interpretability methods. (a) due to its attention-based design, a trained model can be used to compute the attention weights of a sequence, which directly indicates its importance. (b) deeprc furthermore allows for the usage of contribution analysis methods, such as integrated gradients (ig) (sundararajan et al., 2017) or layer-wise relevance propagation (montavon et al., 2018; arras et al., 2019; montavon et al., 2019; preuer et al., 2019) . we apply ig to identify the input patterns that are relevant for the classification. to identify aa patterns with high contributions in the input sequences, we apply ig to the aas in the input sequences. additionally, we apply ig to the kernels of the 1d cnn, which allows us to identify aa motifs with high contributions. in detail, we compute the ig contributions for the aas and positional features in the kernels for every repertoire in the validation and test set, so as to exclude potential artifacts caused by over-fitting. averaging the ig values over these repertoires then results in concise aa motifs. we include qualitative visual analyses of the ig method on different datasets below. here, we provide examples for the interpretation of trained deeprc models using integrated gradients (ig) (sundararajan et al., 2017) as contribution analysis method. the following illustrations were created using 50 ig steps, which we found sufficient to achieve stable ig results. a visual analysis of deeprc models on the simulated datasets, as illustrated in tab. a13 and fig. a3 , shows that the implanted motifs can be successfully extracted from the trained model and are straightforward to interpret. in the real-world cmv dataset, deeprc finds complex patterns with high variability in the center regions of the immuno-sequences, as illustrated in figure a4 . real-world data with implanted signals extracted motif implanted motif(s) g r s r a r f r l r d r r r {l r d r r r ; c r a r s; g r l-n} motif freq. ρ 0.05% 0.1% 0.1% table a13 : visualization of motifs extracted from trained deeprc models for datasets from categories "simulated immunosequencing data", "lstm-generated data", and "real-world data with implanted signals". motif extraction was performed using integrated gradients on the 1d cnn kernels over the validation set and test set repertoires of one cv fold. wildcard characters are indicated by z, random noise on characters by r , characters with 50% probability of being removed by d , and gap locations of random lengths of {0; 1; 2} by -. larger characters in the extracted motifs indicate higher contribution, with blue indicating positive contribution and red indicating negative contribution towards the prediction of the diseased class. contributions to positional encoding are indicated by < (beginning of sequence), ∧ (center of sequence), and > (end of sequence). only kernels with relatively high contributions are shown, i.e. with contributions roughly greater than the average contribution of all kernels. b) c) figure a3 : integrated gradients applied to input sequences of positive class repertoires. three sequences with the highest contributions to the prediction of their respective repertoires are shown. a) input sequence taken from "simulated immunosequencing data" with implanted motif sz d z d n and motif implantation probability 0.1%. the deeprc model reacts to the s and n at the 5 th and 8 th sequence position, thereby identifying the implanted motif in this sequence. b) and c) input sequence taken from "real-world data with implanted signals" with implanted motifs {l r d r r r ; c r a r s; g r l-n} and motif implantation probability 0.1%. the deeprc model reacts to the fully implanted motif cas (b) and to the partly implanted motif aas c and a at the 5 th and 7 th sequence position (c), thereby identifying the implanted motif in the sequences. wildcard characters in implanted motifs are indicated by z, characters with 50% probability of being removed by d , and gap locations of random lengths of {0; 1; 2} by -. larger characters in the sequences indicate higher contribution, with blue indicating positive contribution and red indicating negative contribution towards the prediction of the diseased class. figure a4 : visualization of the contributions of characters within a sequence via ig. each sequence was selected from a different repertoire and showed the highest contribution in its repertoire. the model was trained on cmv dataset, using a kernel size of 9, 32 kernels and 137 repertoires for early stopping. larger characters in the extracted motifs indicate higher contribution, with blue indicating positive contribution and red indicating negative contribution towards the prediction of the disease class. table a14 : tcrβ sequences that had been discovered by emerson et al. (2017) with their associated attention values by deeprc. these sequences have significantly (p-value 1.3e-93) higher attention values than other sequences. the column "quantile" provides the quantile values of the empiricial distribution of attention values across all sequences in the dataset. in this section we investigate the impact of different variations of deeprc on the performance on the cmv dataset. we consider both a cnn-based sequence embedding, as used in the main paper, and an lstm-based sequence embedding. in both cases we vary the number of attention heads and the β parameter for the softmax function the attention mechanism (see eq. 2 in main paper). for the cnn-based sequence embedding we also vary the number of cnn kernels and the kernel sizes used in the 1d cnn. for the lstm-based sequence embedding we use one one-directional lstm layer, of which the output values at the last sequence position (without padding) are taken as embedding of the sequence. here we vary the number of lstm blocks in the lstm layer. to counter over-fitting due to the increased complexity of these deeprc variations, we added a l2 weight penalty to the training loss. the factor with which the l2 weight penalty contributes to the training loss is varied over 3 orders of magnitudes, where suitable value ranges were manually determined on one of the training folds beforehand. to reduce the computational effort, we do not consider all numbers of kernels that were considered in the main paper. furthermore, we only compute the auc scores on 3 of the 5 cross-validation folds. the hyperparameters, which were used in a grid search procedure, are listed in tab. a15 for the cnn-based sequence embedding and tab. a16 for the lstm-based sequence embedding. results. we show performance in terms of auc score with single hyperparameters set to fixed values so as to investigate their influence in tab. a18 for the cnn-based sequence embedding and tab. a17 for the lstm-based sequence embedding. we note that due to restricted computational resources this study was conducted with fewer different numbers of cnn kernels, with the auc estimated from only 3 of the 5 cross-validation folds, which leads to a slight decrease of performance in comparison to the full hyperparameter search and cross-validation procedure used in the main paper. as can be seen in tab. a18 and a17, the lstm-based sequence embedding generalizes slightly better than the cnn-based sequence embedding. table a17 : impact of hyperparameters on deeprc with lstm for sequence encoding. mean ("mean") and standard deviation ("std") for the area under the roc curve over the first 3 folds of a 5-fold nested cross-validation for different sub-sets of hyperparameters ("sub-set") are shown. the following sub-sets were considered: "full": full grid search over hyperparameters; "beta=*": grid search over hyperparameters with reduction to specific value * of beta value of attention softmax; "heads=*": grid search over hyperparameters with reduction to specific number * of attention heads; "lstms=*": grid search over hyperparameters with reduction to specific number * of lstm blocks for sequence embedding. table a18 : impact of hyperparameters on deeprc with 1d cnn for sequence encoding. mean ("mean") and standard deviation ("std") for the area under the roc curve over the first 3 folds of a 5-fold nested cross-validation for different sub-sets of hyperparameters ("sub-set") are shown. the following sub-sets were considered: "full": full grid search over hyperparameters; "beta=*": grid search over hyperparameters with reduction to specific value * of beta value of attention softmax; "heads=*": grid search over hyperparameters with reduction to specific number * of attention heads; "ksize=*": grid search over hyperparameters with reduction to specific kernel size * of 1d cnn kernels for sequence embedding; "kernels=*": grid search over hyperparameters with reduction to specific number * of 1d cnn kernels for sequence embedding. a compact vocabulary of paratope-epitope interactions enables predictability of antibody-antigen binding predicting the sequence specificities of dna-and rna-binding proteins by deep learning explaining and interpreting lstms solving the protein sequence metric problem rank-loss support instance machines for miml instance annotation augmenting adaptive immunity: progress and challenges in the quantitative engineering and analysis of adaptive immune receptor repertoires multiple instance learning: a survey of problem characteristics and applications vdjserver: a cloud-based analysis portal and data commons for immune repertoire sequences and rearrangements tetramer-visualized gluten-specific cd4+ t cells in blood as a potential diagnostic marker for coeliac disease without oral gluten challenge ireceptor: a platform for querying and analyzing antibody/b-cell and t-cell receptor repertoire data across federated repositories support-vector networks quantifiable predictive features define epitope-specific t cell receptor repertoires on a model of associative memory with huge storage capacity bert: pre-training of deep bidirectional transformers for language understanding solving the multiple instance problem with axis-parallel rectangles predicting the spectrum of tcr repertoire sharing with a data-driven model of recombination immunosequencing identifies signatures of cytomegalovirus exposure history and hla-mediated effects on the t cell repertoire predicting antigen-specificity of single t-cells based on tcr cdr3 regions. biorxiv a review of multi-instance learning assumptions deep sequencing of b cell receptor repertoires from covid-19 evaluation and benchmark for biological image segmentation the promise and challenge of high-throughput sequencing of the antibody repertoire tcrex: detection of enriched t cell epitope specificity in full t cell receptor sequence repertoires. biorxiv identifying specificity groups in the t cell receptor repertoire generating sequences with recurrent neural networks. arxiv a bioinformatic framework for immune repertoire diversity profiling enables detection of immunological status learning the high-dimensional immunogenomic features that predict public and private antibody repertoires improving neural networks by preventing co-adaptation of feature detectors long short-term memory fast model-based protein homology detection without alignment neural networks and physical systems with emergent collective computational abilities convolutional neural network architectures for matching natural language sentences attention-based deep multiple instance learning nettcr: sequence-based prediction of tcr binding to peptide-mhc complexes using convolutional neural networks basset: learning the regulatory code of the accessible genome with deep convolutional neural networks detecting cutaneous basal cell carcinomas in ultra-high resolution and weakly labelled histopathological images self-normalizing neural networks capturing the differences between humoral immunity in the normal and tumor environments from repertoire-seq of b-cell receptors using supervised machine learning observed antibody space: a resource for data mining next-generation sequencing of antibody repertoires dense associative memory for pattern recognition dense associative memory is robust to adversarial inputs gradient-based learning applied to document recognition set transformer: a framework for attention-based permutation-invariant neural networks imgt unique numbering for immunoglobulin and t cell receptor variable domains and ig superfamily v-like domains distance between sets methods for detecting associations with rare variants for common diseases: application to analysis of sequence data the extended cohnkanade dataset (ck+): a complete dataset for action unit and emotion-specified expression high-throughput immune repertoire analysis with igor a framework for multiple-instance learning computational strategies for dissecting the high-dimensional complexity of adaptive immune repertoires longitudinal high-throughput tcr repertoire profiling reveals the dynamics of t cell memory formation after mild covid-19 infection. biorxiv methods for interpreting and understanding deep neural networks layer-wise relevance propagation: an overview how many different clonotypes do immune repertoires contain? current opinion in systems biology treating biomolecular interaction as an image classification problem -a case study on t-cell receptorepitope recognition prediction. biorxiv sumrep: a summary statistic framework for immune receptor repertoire comparison and model validation biophysicochemical motifs in t-cell receptor sequences distinguish repertoires from tumor-infiltrating lymphocyte and adjacent healthy tissue pytorch: an imperative style, high-performance deep learning library needles in haystacks: on classifying tiny objects in large images interpretable deep learning in drug discovery pointnet: deep learning on point sets for 3d classification and segmentation graph kernels for chemical informatics cov-abdab: the coronavirus antibody database. biorxiv immunedb, a novel tool for the analysis, storage, and dissemination of immune repertoire sequencing data a $$k$$-nearest neighbor based algorithm for multi-instance multi-label active learning machine learning in automated text categorization high-throughput mapping of b cell receptor sequences to antigen specificity vdjtools: unifying post-analysis of t cell receptor repertoires vdjdb: a curated database of t-cell receptor sequences with known antigen specificity deeptcr: a deep learning framework for understanding t-cell receptor sequence signatures within complex t-cell repertoires prediction of specific tcr-peptide binding from large dictionaries of tcr-peptide pairs. biorxiv axiomatic attribution for deep networks attention-based deep neural networks for detection of cancerous and precancerous esophagus tissue on histopathological slides learning with sets in multiple instance regression applied to remote sensing attention is all you need revisiting multiple instance neural networks novel approaches to analyze immunoglobulin repertoires immunesim: tunable multi-feature simulation of b-and t-cell receptor repertoires for immunoinformatics benchmarking genome-wide protein function prediction through multiinstance multi-label learning rare-variant association testing for sequencing data with the sequence kernel association test polyspecificity of t cell and b cell receptor recognition practical guidelines for b-cell receptor repertoire sequencing analysis learning embedding adaptation for few-shot learning convolutional neural network architectures for predicting dna-protein binding pird: pan immune repertoire database multi-instance multi-label learning with application to scene classification predicting effects of noncoding variants with deep learning-based sequence model the ellis unit linz, the lit ai lab and the institute for machine learning are supported by the land oberösterreich, lit grants deeptoxgen ( in the following, the appendix to the paper "modern hopfield networks and attention for immune key: cord-021977-yu0hrg6h authors: pham, phuong-thu t.; danovitch, gabriel m.; pham, phuong-chi t. title: medical management of the kidney transplant recipient: infections and malignant neoplasms date: 2010-12-27 journal: comprehensive clinical nephrology doi: 10.1016/b978-0-323-05876-6.00101-5 sha: doc_id: 21977 cord_uid: yu0hrg6h nan despite prophylactic therapy against common bacterial, viral, and opportunistic pathogens in the perioperative and postoperative period, infections are the second most common cause of death after cardiovascular disease (cvd) in renal transplant recipients. according to the u.s. renal data system (usrds), infections occurred at a rate of 45 per 100 patient-years during the first 3 years after transplantation. 1 the most common infections are bacterial, followed by viral and fungal. parasitic infections are rare. notably, cytomegalovirus (cmv) and herpes simplex virus (hsv) infection rates have decreased since the mid-1990s as a result of effective antiviral prophylaxis; hepatitis b virus (hbv) and hepatitis c virus (hcv) infection rates increased during the same period for unclear reasons. both the type and occurrence of infections in the immunocompromised transplant recipient follow a "timetable pattern" (fig. 101.1 ). 2 although rare, both blood-borne and kidney infections have been transmitted during donation. these include viral infections (e.g., hcv, hbv, human immunodeficiency virus (hiv), cmv, and bk, among others), parasitic infections (malaria, babesia), and bacterial infections (from undiagnosed bacteremia or renal infections). most infections in the first month are due to common bacteria and candida acquired in the hospital setting. except for hsv, other viral infections are uncommon during this period. similar to those that follow any major surgical procedure, most bacterial infections during this period involve wounds, catheters, and drainage sites. aspiration pneumonia and urinary tract infections (utis) are common. infections specific to renal transplant recipients include perinephric fluid collections due to lymphoceles, wound hematomas, or urine leaks; indwelling urinary stents; and utis secondary to urinary tract abnormalities, such as ureteral stricture, vesicoureteral reflux, or neurogenic bladder. most utis are caused by common gram-negative bacteria (escherichia coli, enterobacteriaceae, and pseudomonas) and gram-positive bacteria (enterococcus). preventive measures for utis include early urethral catheter removal and antibiotic prophylaxis. trimethoprim-sulfamethoxazole or ciprofloxacin prophylaxis during the first 3 months after transplantation effectively reduces the frequency of utis to less than 10% and essentially eliminates urosepsis unless anatomic or functional derangement of the urinary tract is present. infections with multidrug-resistant microorganisms have recently emerged as an important cause of morbidity and mortality in organ transplantation. hence, in some centers, the routine use of antibiotic prophylaxis is no longer recommended. although strict aseptic surgical techniques and perioperative use of firstgeneration cephalosporins reduce the incidence of wound infections, infections are still observed, especially in subjects with comorbid conditions such as diabetes mellitus (dm) and obesity. antibiotic-associated clostridium difficile infection (particularly cephalosporins, ciprofloxacin, and amoxicillin-clavulanate) has become a serious epidemiologic problem worldwide. judicious use of antibiotic prophylactic therapy may decrease the incidence of iatrogenic c. difficile infections. whereas most infections during the first month are due to routine bacterial infections, nosocomial outbreaks have also been reported for rarer infections, such as legionella from contaminated hospital water supplies. during months 1 to 6, opportunistic infections secondary to immunosuppression are most common. viral infections, such as cmv, hsv, varicella-zoster virus (vzv), epstein-barr virus (ebv), hbv, and hcv, may occur from exogenous infection or reactivation of latent disease due to the immunosuppressed state. repeated courses of antibiotics and corticosteroid therapy increase the risk of fungal infections, whereas viral infections may not only result from the immunosuppression but may themselves further impair immunity to increase the risk for additional opportunistic infections. opportunistic infections may occur with pneumocystis jiroveci (previously pneumocystis carinii), aspergillus species, listeria monocytogenes, nocardia species, and toxoplasma gondii. trimethoprim-sulfamethoxazole prophylaxis (see early post-transplantation period, outbreaks of donor-transmitted viral infections, such as lymphocytic choriomeningitis and west nile virus, have been reported. lymphocytic choriomeningitis occurs within the first 4 weeks after transplantation and is associated with a greater than 90% mortality rate. 3 in the late posttransplantation period, infections with community-acquired viral pathogens, including vaccine-preventable diseases such as mumps and measles, have reemerged. there is currently no effective antiviral therapy against either infection, and adherence to current guidelines for vaccinations in solid organ transplantation is recommended (discussed later). other emerging or reemerging viral infections include adenovirus, human herpesvirus 6, metapneumovirus, parainfluenza, and respiratory syncytial virus. interestingly, only rare cases due to severe acute respiratory syndrome (sars) coronavirus have been reported. the following sections discuss selected infections in renal transplant recipients. suggested prophylactic therapy is shown in figure 101 cmv infection may be a primary infection in a seronegative recipient (donor seropositive, recipient seronegative), reactivation of endogenous latent virus (donor seropositive or seronegative, recipient seropositive), or superinfection with a new virus strain in a seropositive recipient (donor seropositive, recipient seropositive). primary cmv infection is usually more severe than reactive infection or superinfection. cmv infection occurs primarily after the first month of transplantation and continues to be a significant cause of morbidity in the first 6 months after organ transplantation through both direct and indirect effects. cmv infection may be asymptomatic, presenting as a mononucleosis-like syndrome or influenza-like illness with fever and leukopenia or thrombocytopenia, or a severe systemic disease. hepatitis, esophagitis, gastroenteritis with colonic ulceration, pneumonia, chorioretinitis (associated with retinal hemorrhage), and even otitis 4 may occur. in enterically drained pancreas transplantation, cmv has been reported to cause bleeding ulcer from the duodenal segment. clinical manifestations usually after 6 months, the infection risk can be categorized on the basis of the patient's status. the first category consists of the majority of transplant recipients (70% to 80%), who have satisfactory or good allograft function, relatively low doses of immunosuppression medication, and no history of chronic viral infection. the risk of infection in these patients is similar to that of the general population, with community-acquired respiratory viruses constituting the major infective agents. opportunistic infections are unusual unless environmental exposure has occurred. the second group (approximately 10% of patients) consists of those with chronic viral infection that may include hbv, hcv, cmv, ebv, bk virus, or papillomavirus. in the setting of immunosuppression, such viral infections may lead to the development of progressive liver disease or cirrhosis (hbv, hcv), bk nephropathy, post-transplantation lymphoproliferative disease (ebv), or squamous cell carcinoma (papillomavirus). the third group (approximately 10% of patients) consists of those who experience multiple episodes of rejection requiring repeated exposure to heavy immunosuppression. these patients are the most likely to develop chronic viral infections and superinfection with opportunistic organisms. causative opportunistic pathogens include p. jiroveci, l. monocytogenes, nocardia asteroides, and cryptococcus neoformans and geographically restricted mycoses (coccidioidomycosis, histoplasmosis, blastomycosis, and paracoccidioidomycosis). in these high-risk candidates, lifelong prophylactic therapy with trimethoprim-sulfamethoxazole (80 mg/ 400 mg daily) has been advocated. lifelong antifungal prophylaxis should also be considered and environmental exposure minimized (primarily avoidance of pigeons and areas of active building construction). several uncommon viral infections have recently been reported in both the early and late post-transplantation periods. 3 in the figure 101 .1 timetable of infections.* *geographically focused infections will need to be considered in certain cases, such as malaria, leishmaniasis, trypanosomiasis, and strongyloidiasis. 1 sources of infections specific to recipients of renal transplant: perinephric fluid collections (e.g., lymphoceles, wound hematomas, urine leaks), indwelling urinary stents, or anatomic or functional genitourinary tract abnormalities (e.g., ureteral stricture, vesicoureteric reflux, neurogenic bladder blood, such as cmv dna polymerase chain reaction (pcr) or pp65 antigenemia during surveillance studies. the former assay is highly specific and sensitive for the detection of cmv viremia. the latter is a semiquantitative fluorescent assay in which circulating neutrophils are stained for nonspecific uptake of cmv early antigen (pp65). various prophylactic and preemptive protocols have been developed. oral acyclovir provides effective cmv prophylaxis solely in recipients of seronegative donor organs. oral or intravenous ganciclovir or oral valganciclovir provides superior prophylactic or preemptive therapy against primary cmv infection or cmv reactivation. prophylactic or preemptive therapy should be based on the intensity of immunosuppression (i.e., during antilymphocyte antibody therapy) and the seropositive status of the donor, the recipient, or both. seronegative individuals who receive organs from latently infected seropositive donors are at greatest risk for primary infection and severe cmv disease. a suggested cmv prophylaxis protocol is shown in figure 101 clinical cmv disease is treated with intravenous ganciclovir (5 mg/kg twice daily for 3 weeks, dose adjusted for renal dysfunction) with reduction of immunosuppression, such as withholding of mmf. treatment is continued until clearance of viremia as assessed by pcr or antigenemia. anecdotal reports have suggested that calcineurin inhibitor (cni) to sirolimus switch in conjunction with ganciclovir therapy may be beneficial in patients with apparent ganciclovir-resistant cmv. 8 in patients with gastrointestinal cmv infection, the use of these assays is unreliable, and repeated endoscopy should be considered to assess response to therapy. in patients who have primary infection and respond slowly to therapy, the addition of cmv hyperimmune globulin (150 mg/kg per dose given intravenously every 3 to 4 weeks for 3 months) may be of benefit. 5 in patients with tissue invasive disease, intravenous ganciclovir is recommended with conversion to oral therapy when there is evidence of a good response, followed by a 3-month course of oral ganciclovir or valganciclovir prophylaxis. 5 whereas oral valganciclovir provides good bioavailability and may be effective in mild cmv disease, it is not recommended for the treatment of occur 1 to 4 months after transplantation except for chorioretinitis, which occurs later in the transplant course. 5 quantitative cmv assays of serum in patients with invasive colitis and gastritis or neurologic disease including chorioretinitis are often negative. diagnosis in such cases may require invasive testing and biopsies. cmv infection is associated with immune modulation and dysregulation of helper/suppressor t cells and may be a risk factor for chronic allograft rejection, secondary infection with opportunistic agents (such as p. jiroveci, candida, and aspergillus), reactivation of human herpesvirus hhv-6 and hhv-7, and the development of post-transplantation lymphoproliferative disease. cmv infection is also associated with acceleration of hcv infection and the development of new-onset dm after transplantation. 6 donor and recipient seropositive status and the use of blood products from a cmv-seropositive donor are well-established risk factors for cmv infection. other factors associated with an increased risk of cmv infection include the use of antilymphocyte antibodies, prolonged or repeated course of antilymphocyte preparations, comorbid illnesses, neutropenia, and acute rejection episodes. mycophenolate mofetil (mmf) has been reported to increase the risk for cmv viremia and cmv disease in some studies, especially in patients receiving more than 3 g/day. although the cause-effect of allograft rejection and cmv infection remains conjectural, several studies suggest that one may increase the risk for the other, possibly owing to the release of inflammatory cytokines. prevention of cmv infection, for example, results in a lower incidence of graft rejection. 7 prophylactic therapy begins in the immediate postoperative period. preemptive therapy involves treatment of those who are found to seroconvert by quantitative laboratory assays of the † check glucose-6-phosphate dehydrogenase deficiency before initiation of therapy. ‡ in order of efficacy. § fluconazole is recommended for recipients of combined kidney-pancreas or combined kidney-liver transplants. consider reinstitution of prophylactic therapy for 3 months after acute rejection episodes requiring intensification of immunosuppression. cmv, cytomegalovirus. trimethoprim-sulfamethoxazole (tmp-its routine use reduces or eliminates the incidence of smz)* (80/400 mg) one tablet daily pneumocystis jiroveci, listeria monocytogenes, × 3 months nocardia asteroides, and toxoplasma gondii in renal transplant recipients, tmp-smz reduces the incidence of urinary tract infection from 30%-80% to <5%-10% monthly intravenous or aerosolized replaces tmp-smz for patients with sulfa allergies pentamidine > dapsone † > or atovaquone ‡ nystatin 100,000 units/ml, 4 ml after for fungal prophylaxis meals and before bedtime or fluconazole § 200 mg one tablet daily close monitoring of cyclosporine or tacrolimus levels × 2 months when starting and stopping antifungal agents acyclovir/valganciclovir/ganciclovir for cmv prophylaxis, see figure 101 .3 bk virus is a ubiquitous human virus with a peak incidence of primary infection in children 2 to 5 years of age and a seroprevalence rate of more than 60% to 90% among the adult population worldwide. after primary infection, bk virus preferentially establishes latency within the genitourinary tract and frequently is reactivated in the setting of immunosuppression. in renal transplant recipients, bk virus is associated with a range of clinical syndromes including asymptomatic viruria with or without viremia, ureteral stenosis and obstruction, interstitial nephritis, and bk allograft nephropathy. during the last decade, bk nephropathy has emerged as an important cause of allograft dysfunction after renal transplantation. most series report that 30% to 40% of renal transplant recipients develop bk viruria, 10% to 20% develop bk viremia, and 2% to 5% develop bk nephropathy. the highest prevalence of bk viruria and viremia occurs at 2 to 3 months and 3 to 6 months, respectively. the risk for development of bk viremia increases when urine viral load is greater than 10 4 copies/ml, whereas bk nephropathy is unusual in the absence of bk viremia. bk nephropathy commonly presents with an asymptomatic rise in serum creatinine during the first posttransplantation year. however, bk nephropathy may occur as early as the first week to as late as 6 years after transplantation. diagnosis is made by allograft biopsy, which demonstrates bk viral inclusions in renal tubular cell nuclei and occasionally in glomerular parietal epithelium (fig. 101.4a ). there are variable degrees of interstitial mononuclear inflammation (fig. 101 .4b), often with plasma cells, degenerative changes in tubules, and focal tubulitis, which may mimic acute rejection. bk nephropathy often is associated with very focal and sharply demarcated areas of tubulointerstitial inflammation, corresponding to foci of viral infection. immunohistochemistry (fig. 101.4c ), in situ hybridization, or electron microscopy is required to confirm the diagnosis. bk infection and acute rejection may occur simultaneously, and distinguishing between bk nephropathy and acute rejection or the presence of both can be a diagnostic challenge. in late bk nephropathy, few characteristic intranuclear inclusions are seen, and the histologic changes may be indistinguishable from chronic rejection. a histologic classification system for bk nephropathy based on the degree of active inflammation, acute tubular injury, and tubulointerstitial scarring may have prognostic significance. 10 urine cytology for decoy cells and quantitative determinations of viruria and of viral load in blood have been proposed as surrogate markers for the diagnosis of bk nephropathy. treatment strategies include reduction in immunosuppression that involves reduction or discontinuation of mmf and azathioprine with judicious reduction in cni therapy or other immunosuppressive regimen. switching from tacrolimus to cyclosporine or to sirolimus (rapamycin) has resulted in resolution of bk nephropathy and viremia or viruria in anecdotal case reports. switching from cni to sirolimus may have the added benefit of avoiding the long-term nephrotoxic effect of cni therapy. although no approved antiviral drug is available, adjunctive therapy with leflunomide, cidofovir, quinolones, or intravenous immune globulin (ivig) may be beneficial, especially in patients with progressive allograft dysfunction. quinolones are preferred by some centers because of low cost and ease of administration; leflunomide is used by others because of its potential simultaneous antiviral and immunosuppressive properties. cidofovir is highly concentrated in urine and renal tissue, and the use of low-dose cidofovir in bk nephropathy has been reported to be devoid of nephrotoxicity or serious adverse events. anecdotal established cmv disease, and intravenous ganciclovir is required. cidofovir and foscarnet are alternative therapeutic agents, but in view of their nephrotoxicity and potential synergistic nephrotoxicity with cnis, they are reserved for use when ganciclovirresistant strains are clinically suspected. candida infections are common in transplant recipients; candida albicans and candida tropicalis account for 90% of the infections. dm, high-dose corticosteroids, and broad-spectrum antibacterial therapy predispose patients to mucocutaneous candidal infections such as oral candidiasis, intertriginous candidal infections, esophagitis, vaginitis, and uti. skin infections are treated with nystatin and topical clotrimazole; candidal utis are treated with fluconazole or voriconazole or more rarely with liposomal amphotericin or caspofungin for fluconazole-resistant species (see chapter 53). whenever possible, foreign objects such as bladder catheters, surgical drains (e.g., percutaneous nephrostomy tube), and urinary stents should be promptly removed. the ideal management of asymptomatic candiduria in immunocompromised patients remains uncertain (see chapter 53), but a short course (7 to 10 days) of fluconazole is generally recommended. systemic antifungal therapy is indicated in the presence of any positive blood culture for candida species. 1 if cmv status is unknown, give intravenous dhpg until cmv status is determined. 2 dose adjustment for renal function is necessary. dhpg, 9-(1,3-dihydroxy-2-propoxymethyl) guanine. 3 although low-dose valganciclovir, 450 mg daily, has been shown to be effective, the canadian society of transplantation consensus workshop on cmv management recommends dosing valganciclovir at 900 mg daily for cmv+ recipients of a cmv+ organ (kidney, liver, pancreas, heart). (from reference 9.) prophylaxis protocol 1 acyclovir 400 mg daily (or valganciclovir 450 mg daily) × 3 months cmv dna every 2 weeks × 3 months during antibody treatment, dhpg 2 5.0 mg/kg iv everyday, then following antibody treatment/valganciclovir 900 mg po everyday × 6 months if no antibody treatment: valganciclovir 900 mg everyday for 6 months cmv dna every 2 weeks x 3 months during antibody treatment, dhpg 5.0 mg/kg iv everyday, then following antibody treatment, valganciclovir 900 mg po everyday × 6 months if no antibody treatment: acyclovir 400 mg daily (or valganciclovir 450 mg daily) × 3 months cmv dna every 2 weeks × 3 months during antibody treatment, dhpg 5.0 mg/kg iv everyday, then following antibody treatment, valganciclovir 900 mg po everyday × 6 months if no antibody treatment: acyclovir 400 mg daily (or valganciclovir 450 mg daily) 3 × 3 months cmv dna every 2 weeks × 3 months reports have suggested that ivig may be effective in treating corticosteroid-resistant rejection, 11 and its use may be beneficial in patients with concomitant rejection and bk nephropathy or in those with histopathologic changes that are indistinguishable from those of rejection. despite treatment, 30% to more than 60% of patients with established bk nephropathy developed progressive decline in renal function with graft loss. early diagnosis and intervention may improve prognosis. intensive monitoring of urine and serum for bk by pcr during the first year with preemptive reduction of immunosuppressive therapy may lead to the resolution of viremia and prevent bk nephropathy. in the absence of active viral replication, patients with graft loss due to bk nephropathy can safely undergo retransplantation. active surveillance for bk virus reactivation after transplantation is recommended. suggested guidelines for post-transplantation screening and monitoring for bk replication are shown in figure 101 .5. tuberculosis (tb) infection in the renal transplant recipient varies according to the prevalence in the general population (e.g., the incidence of tb in transplant recipients has been reported to occur in 0% to 1.3% in the united states, compared with 11% in south africa and 11% to 14% in india and pakistan). 12 most tb infection in the transplant recipient results from reactivation of dormant lesions in the setting of immunosuppressive therapy. hence, all renal transplant candidates should have a ppd skin test (tuberculin skin test) placed before transplantation. a positive skin test response or a prior history of tb mandates further evaluation to rule out active disease. isoniazid prophylaxis for a total of 9 months is recommended for those who have a positive skin test response. of interest, most of the patients who develop tb after transplantation had negative ppd skin test results before transplantation. 13 some centers recommend isoniazid prophylactic therapy in selected ppd-negative patients with (1) a history of inadequately treated tb, (2) radiographic evidence of granulomatous disease and no history of adequate treatment, (3) an organ from a ppd-positive donor, or (4) close and prolonged contact with a case of active tb. 13 in patients with a known history of adequately treated tb infection, we advocate the use of isoniazid prophylaxis for the first 9 months after transplantation and during intensification of immunosuppression. others, however, have suggested that isoniazid prophylaxis is not indicated for those patients whose tb had been properly treated. 12 clinical, radiologic, or culture evidence of active tb infection is a contraindication to transplantation. enzyme-linked immunospot (elispot), which detects t cells specific for mycobacterium tuberculosis antigens, is unaffected by bacille calmette-guérin (bcg) vaccination and has become a major advance in tb screening. in some centers, the tuberculin skin test has been replaced by the elispot assays (t-spot.tb assay). a rare but important cause of infection in transplant patients, particularly those from endemic areas such as southeast asia, is strongyloides. in the presence of immunosuppression, a "hyperinfection" syndrome may be observed with parasitic pneumonia (fig. 101.6 ) and gastrointestinal involvement. post-transplantation gastrointestinal complications are common and can arise from a variety of causes. only selected complications are discussed; for a comprehensive review of nystatin "swish and swallow" during the first month after transplantation is recommended. in high-risk candidates, including liver or pancreas transplant recipients and those receiving antilymphocyte antibody therapy, fluconazole prophylactic therapy (3 to 6 months) is warranted. clostridium difficile infection may be asymptomatic or present with diarrhea, intestinal obstruction, or even fulminant pseudomembranous colitis with toxic megacolon and perforation. c. difficile colitis is reported in 3.5% to 16% of transplant recipients. 15 risk factors include young (<5 years) or advanced age, female gender, use of monoclonal antibodies to treat acute rejection episodes, and intra-abdominal graft placement. among transplant recipients receiving antimicrobial therapy, c. difficileassociated diarrhea develops in approximately 50% of patients. 15 in mild cases of c. difficile infection, oral metronidazole is as effective as oral vancomycin and is the preferred first-line treatment. treatment failure, however, requires treatment with oral vancomycin. in severely ill patients with gastrointestinal dysmotility or ileus, in which oral agents may not reach the colonic mucosa, metronidazole should be administered intravenously. severe colonic disease refractory to medical treatment may necessitate colectomy. helicobacter pylori infection is associated with a wide range of gastrointestinal complications including chronic gastritis, duodenal and gastric ulcers, mucosa-associated lymphoid tissue (malt) lymphoma, and gastric carcinoma, both in the general population and in recipients of solid organ transplants. treatment includes a triple-drug regimen consisting of two antibiotics and an acid-suppressive agent such as an h 2 blocker or a proton pump inhibitor. the first-line h. pylori regimen as recommended by the american college of gastroenterology is shown in figure 101 .7. in recipients of orthotopic heart transplants, triple-drug therapy resulted in a lower eradication rate compared with the general population, suggesting that immunosuppression may hinder the clearance of h. pylori. unexplained dyspeptic or reflux symptoms should be investigated further with endoscopy and biopsy to exclude malignant transformation. h. pylori is now recognized as a risk factor for malt lymphoma, which may occur in kidney, liver, and heart transplant recipients. in renal transplant recipients infected with h. pylori, malt lymphoma may be less aggressive than other lymphomas, and the disorder may be cured by eradication of h. pylori. post-transplantation colonic complications, such as diverticulitis and colonic perforation, may be life-threatening and difficult to diagnose because symptoms may be masked by immunosuppressive therapy, particularly in the early postoperative period. diverticulitis complicated by perforation, abscess formation, phlegmon, or fistula has been reported to occur in 1.1% of renal transplant recipients 16 and may be increased in patients with polycystic kidney disease (pkd). early post-transplantation colonic perforations are largely due to high-dose corticosteroids, diverticulitis, cmv colitis, and intestinal ischemia; perforations occurring late or years after transplantation are commonly due to diverticulosis or malignant disease. abdominal symptoms may be absent because of the effects of immunosuppression and may only be suggested by the post-transplantation gastrointestinal complications, readers are referred to chapter 83 and references 13 and 14. mmf commonly causes gastrointestinal side effects, including nausea, vomiting, dyspepsia, anorexia, flatulence, and diarrhea. dose reduction, transient discontinuation of the drug, or dividing the dose into three or four times a day often ameliorates or resolves the symptoms. switching to the enteric-coated formulation of mmf may improve gastrointestinal tolerability in some patients but has not been consistently shown to be better than the original formulation. a large randomized double-blind study using patient-reported outcomes to assess the impact of gastrointestinal symptoms on patients' health-related quality of life and symptom burden is currently under way. sirolimus may cause oral mucocutaneous lesions that can be confused with hsv or cmv infection but are culture negative. drug-related oral ulcers usually resolve after discontinuation of the offending agent. sirolimus, tacrolimus, and cyclosporine have also been suggested to cause diarrhea in some patients. post-transplantation infections of the gastrointestinal tract may be viral, fungal, or bacterial in etiology. viral infections are most commonly caused by cmv and hsv; c. albicans and c. tropicalis are common opportunistic fungal infections. leukoplakia and post-transplantation lymphoproliferative disorder (ptld) may develop in patients with ebv infection (ptld is discussed in a later section). commonly encountered bacterial pathogens include clostridium difficile and helicobacter pylori. cmv can affect any segment of the gastrointestinal tract. patients may present with dysphagia, odynophagia, nausea, vomiting, gastroparesis, abdominal pain, diarrhea, or gastrointestinal bleeding. leukopenia and elevated transaminases are common. persistent or unexplained symptoms of nausea, vomiting, or diarrhea, particularly in the early post-transplantation period or during intensification of immunosuppression, warrant further investigation with upper or lower endoscopies and biopsies. hsv infection results primarily from reactivation of endogenous latent virus, causing clinical infection within the first 1 to 2 months after transplantation. patients commonly present with oral mucocutaneous lesions or gingivostomatitis with or without odynophagia and dysphagia. hsv esophagitis has been noted to occur in patients receiving high-dose corticosteroids and antilymphocyte preparations for acute rejection. limited oral mucocutaneous lesions are treated with oral acyclovir; extensive infections require intravenous acyclovir or ganciclovir. rare cases of hsv hepatitis have been reported. 14 the routine use of acyclovir prophylaxis in the early post-transplantation period is recommended. candida stomatitis and esophagitis are common during the first 6 months after transplantation and are increased in subjects with leukopenia or with severe immunosuppression, diabetes, or concomitant infections. bleeding or perforation with formation of tracheoesophageal fistulas has been reported. prophylactic oral recipients of organ transplants are at increased risk for development of neoplasms compared with the general population. similar to post-transplantation infectious complications, the time to occurrence of different types of malignant neoplasms after transplantation appears to follow a timetable pattern. the israel penn international transplant tumor registry data on the time of appearance of different neoplasms after solid organ transplantation are shown in figure 101 .9. ptld generally occurs early after transplantation; skin cancers occur with increasing frequency with time. the intensity and duration of immunosuppression as well as the ability of these agents to promote replication of various oncogenic viruses are important risk factors. the associations between human papillomaviruses and cervical and vulvar carcinoma, ebv and ptld, hbv and hcv and hepatocellular carcinoma, and hhv-8 and kaposi's sarcoma are well established. figure 101 .10 provides a summary presence of tachypnea and tachycardia. mortality after colonic perforation is high. management includes prompt exterioration of the perforated colon, early and broad-spectrum antimicrobial therapy, and minimization of immunosuppressive therapy. although uncommon, the presence of abdominal pain and gastrointestinal bleeding with unexplained fevers or weight loss should raise the suspicion for gastrointestinal tb. the characteristic endoscopic findings include circular ulcers, small diverticula, and sessile polyps. the presence of caseating granulomas or acid-fast bacilli, or both, confirms the diagnosis. all potential renal transplant candidates should receive immunization for hepatitis b, pneumococcus, and other standard immunizations appropriate for age. up-to-date recommendations for routine adult immunizations are available through the centers for disease control and prevention website (www.cdc.gov/nip/rec/ adult-schedule.pdf). immunizations should ideally be administered at least 4 to 6 weeks before transplantation to achieve optimal immune response and to minimize the possibility of live vaccinederived infection in the post-transplantation period. household members, close contacts, and health care workers should also be fully immunized. live virus or live organism vaccines should be avoided after transplantation. these include measles-mumps-rubella (mmr), live oral poliovirus (which is also contraindicated for household contacts), smallpox (vaccinia), varicella, yellow fever, adenovirus, live oral typhoid (ty21a), bcg, and intranasal influenza vaccine. in addition, exposure to persons who have chickenpox or herpes zoster should be avoided until the lesions have crusted over and no new lesions are appearing. vaccinations using inactivated or killed microorganisms, components, and recombinant moieties are safe for transplant recipients. these include hepatitis a and hepatitis b, intramuscular influenza a and b, pneumococcal, haemophilus influenzae b, inactivated polio virus vaccine, diphtheria-pertussis-tetanus (dpt), and neisseria meningitidis. in general, vaccination should be avoided in the first 6 months after transplantation because of the potential for stimulating the immune response, with a higher chance of graft dysfunction and rejection. in addition, vaccinations within the first 6 months after transplantation are often ineffective because of heavy immunosuppression. for prevention of infection in adult travelers after solid organ transplantation, readers are referred to reference 17. recommended vaccinations before and after transplantation are listed in figure 101 .8. no * standard-dose ppi twice daily (or esomeprazole once daily) + clarithromycin 500 mg twice daily + amoxicillin 1000 mg twice daily for 10-14 days yes † standard-dose ppi twice daily + clarithromycin 500 mg twice daily + metronidazole 500 mg twice daily for 10-14 days yes bismuth subsalicylate 525 mg orally 4 times daily + metronidazole 250 mg orally 4 times daily + tetracycline 500 mg orally four times daily + ranitidine 150 mg orally twice daily (or standard-dose ppi once daily to twice daily) for 10-14 days measles-mumps-rubella x renal cell carcinomas (27%), and breast carcinomas (23%). 21 in an analysis of registry data involving 90 patients with a history of pretransplantation prostate adenocarcinoma (77 renal, 10 heart, and 3 liver transplant recipients), prostate cancer recurrences were found to relate to the stage of disease at initial diagnosis. 22 tumor recurrence rates were 14%, 16%, and 33% for stage i, stage ii, and stage iii diseases, respectively. hence, a longer waiting time may be necessary for more advanced disease. suggested guidelines for tumor-free waiting periods for common pretransplantation malignant neoplasms are shown in figure 101 .12. ptld is the most common post-transplantation malignant neoplasm in children; in adults, it is the second most common malignant neoplasm after skin cancer. ptld has been reported to occur in 1% to 5% of renal transplant recipients. the majority of ptld is non-hodgkin's lymphoma of b-cell origin, and more than 80% to 90% are linked to ebv infection. based on the world health organization classification, ptld can be divided into three distinct morphologic groups: (1) diffuse b-cell hyperplasia, (2) polymorphic ptld (usually monoclonal), and (3) monomorphic ptld that includes high-grade invasive lymphoma of b-or t-lymphocyte centroblasts. diffuse b-cell hyperplasia is usually seen in children and young adults and commonly occurs within the first year after transplantation. polymorphic ptld represents the most common type of ptld in both children and adults and may occur at any time after transplantation. in contrast, monomorphic b-cell ptld is often seen several years after transplantation and may resemble non-hodgkin's lymphoma in the general population. in a retrospective analysis of registry data for 402 recipients of kidney transplants, ptld occurred at a median of 18 months (range, 1 to 310 months) after transplantation. ptld may present with constitutional symptoms such as fevers, night sweats, and weight loss or localized symptoms of the respiratory tract (infection or mass, including tonsillar or even gingival involvement), gastrointestinal tract (diarrhea, pain, perforation, bleeding, mass), or central nervous system (cns) (headache, seizure, confusion). in contrast to lymphomas in the general population, in which lymph nodes are almost always involved, lymph node involvement is absent in more than 80% of patients with ptld. risk factors for ptld include primary ebv infection, younger age, antecedent history of cmv disease, and use of antilymphocyte antibody (e.g., antithymocyte globulin, okt3). a history of pretransplantation malignant disease and fewer hla matches are associated with an increased risk of ptld. cyclosporine and tacrolimus may enhance the development of ebv-associated ptld by directly promoting the survival of ebv-infected b cells, presumably through the inhibition of ebv-transformed cells from apoptosis. 23 reduction or discontinuation of immunosuppressive therapy, particularly antilymphocyte antibody, cyclosporine, tacrolimus, or mmf, is recommended as first-line treatment; prednisone is increased to 10 to 15 mg daily to prevent allograft rejection. sirolimus has a strong antiproliferative effect on ptld-derived b-cell lines, 24 but whether sirolimus may limit b-cell lymphoma growth while simultaneously providing immunosuppression to prevent graft rejection awaits studies. acyclovir or ganciclovir therapy and reduction in immunosuppression are beneficial and may be curative in benign polyclonal b-cell proliferation. the of the incidence of cancers related to infections in transplant recipients. 18 an analysis of the usrds database 19 documented that the cancer rates for most common cancers, such as colon, lung, prostate, stomach, esophagus, pancreas, ovary, and breast, are nearly twofold higher after kidney transplantation compared with the general population. although registry studies have limitations, all transplant recipients should adhere to standard cancer surveillance appropriate for age (fig. 101.11 ). 20 in patients with a history of pre-transplantation malignant neoplasms, close monitoring for recurrences in the post-transplantation period is mandatory. the highest recurrence rates have been observed with multiple myeloma (67%), non-melanoma skin cancers (53%), bladder carcinomas (29%), sarcomas (29%), symptomatic of 375 mg/m 2 ) in patients with ptld (in conjunction with reduction in immunosuppression) have shown promising results. complete remission rates of 30% to 60% have been reported. 24 although the response rates appear to vary substantially among patients and centers, rituximab in conjunction with reduction in immunosuppression is evolving as the treatment of choice for cd20 + ptld. the role of cytokine-based therapy, such as interferon alfa and anti-il-6, remains poorly defined 25 ; increased risk of allograft rejection is seen with anti-il-6 treatment. sirolimus, an immunosuppressant with antiproliferative properties, has been demonstrated to prevent proliferation of b-cell (but not t-cell) ptld-derived tumor cell lines in vitro and in vivo. 26 limited data from nine european transplant centers have shown tumor regression in 15 of 19 patients with ptld who underwent minimization or withdrawal of cnis and sirolimus conversion. 27 factors that adversely affect survival include multiple-versus single-site involvement, increasing age, b-cell predominance, use of antilymphocyte globulin or antithymocyte globulin and okt3, and "early" versus "late" onset (within 6 to 12 months versus more than 12 months). in recipients of renal transplants with ptld restricted to the allograft alone, transplant nephrectomy may improve survival. role of antiviral therapy in b-cell monoclonal malignant transformation is less well defined; 50% to 90% mortality has been reported despite antiviral therapy. surgical resection with or without adjunctive local irradiation has been suggested for localized disease. local irradiation has been advocated as the treatment of choice for ptld involving the central nervous system. in lesions not amenable to surgery or more aggressive monoclonal types of ptld, chemotherapy has been used with favorable results compared with reduction in immunosuppression alone. the most frequently used regimens are chop (cyclophosphamide, doxorubicin [adriamycin], vincristine, and prednisone) and vapec-b (doxorubicin, etoposide, cyclophosphamide, methotrexate, bleomycin, and vincristine). other reported promising novel therapies include promace-cytabom (prednisone orally, doxorubicin, cyclophosphamide, etoposide-cytarabine, bleomycin, vincristine [oncovin], methotrexate). 25 adverse effects of chemotherapy include high mortality rates from sepsis and treatment-related toxicities. rituximab, a chimeric monoclonal antibody with murine variable regions targeting the cd20 antigen and human igg1-κ constant regions, has antitumor activity against cd20-expressing b-cell lymphomas. early experiences with rituximab (two to six weekly doses figure 101 .11 preventive care recommendations for cancer surveillance in renal transplant recipients. 1 as recommended by the american transplant society and the european best practice guidelines on renal transplantation. 2 the american college of preventive medicine recommends regular screening for high-risk individuals but none for low-risk individuals. 3 neoplasms. it has been proposed by experts in the field that immunosuppression dose reduction or withdrawal may permit recovery of the immune system and control the progression of life-threatening malignant neoplasms. the former allows intact immune surveillance against malignant cells. nonetheless, this approach is not without its attendant risk of graft rejection and graft loss. furthermore, little is known as to how much and to what extent immunosuppression reduction or withdrawal might alter the natural history of established post-transplantation malignant neoplasms. in our opinion, cni to sirolimus switch or cni minimization in conjunction with sirolimus may be a viable therapeutic option (the antitumoral effect of sirolimus is discussed later). in patients with metastatic cancer, manipulation of immunosuppression is probably futile, and the risk of rejection and graft loss necessitating a return to dialysis is likely to outweigh the benefit. studies suggest that immunosuppressive agents have different effects on cancer risk after transplantation. the carcinogenic effects of okt3, antithymocyte globulin, cyclosporine, tacrolimus, and azathioprine have been well documented. in contrast to azathioprine, mmf has been shown to have antiproliferative effects and has been suggested to protect against posttransplantation malignant neoplasms. 30, 31 analysis of more than 17,000 adult patients with preexisting dm indicated a significantly higher incidence of malignant transformation in azathioprine-treated than in mmf-treated patients (3.7% versus 2.2%; p < .01). 31 however, whether mmf is protective of posttransplantation malignant neoplasia remains speculative. both preclinical and clinical studies have demonstrated that mtor inhibitors such as sirolimus and everolimus have antiproliferative and antitumor effects. early studies in renal transplant recipients demonstrated a lower incidence of skin cancer with sirolimus-based therapy without cyclosporine or sirolimus maintenance therapy after early cyclosporine withdrawal compared with those who remained on cyclosporine and sirolimus combination therapy. it has been suggested that the protective effect of sirolimus against skin cancer is due to its inhibition of several ultraviolet light-induced mechanisms involved in skin carcinogenesis. the 5-year malignancy data of the rapamune maintenance regimen trial demonstrated a lower incidence of both skin and non-skin cancers at 5 years after transplantation in recipients receiving sirolimus-based therapy and cyclosporine withdrawal at month 3 compared with those receiving sirolimus and cyclosporine combination therapy. 29 sirolimus therapy has also been reported to result in successful clinical and histologic remission of kaposi's sarcoma in renal transplant recipients. 32 although sirolimus appears to provide satisfactory outcomes in certain cancers after transplantation, its use in the management of malignant disease after solid organ transplantation remains to be defined and should be tailored to each individual patient. there is no consensus on the management of immunosuppressive therapy in patients with post-transplantation malignant rates of first infection following kidney transplantation in the united states infection in organ transplant recipients emerging virus in transplantation: there is more to infection after transplant than cmv and ebv sun-exposed areas and 7 times higher in non-sunexposed areas. the use of sirolimus, an inhibitor of mammalian target of rapamycin (mtor)-induced signaling, may delay the onset or reduce the incidence of post-transplantation skin and non-skin malignant neoplasms (discussed under management of post-transplantation malignant neoplasms) risk factors for skin cancer include light skin color, intensity of sun exposure (ultraviolet light exposure), genetic factors, and duration of follow-up after transplantation. in addition, immunosuppression in combination with enhanced sunlight exposure may induce malignant changes in papilloma cancer screening in renal transplant recipients: what is the evidence? evaluation of transplant candidates with pre-existing malignancies prostate cancer prior to solid organ transplantation: the israel penn international transplant tumor registry experience effect of cyclosporine and tacrolimus on the growth of esptein-barr virus-transformed b-cell lines rapamycin inhibits the interleukin 10 transduction pathway and the growth of epstein-barr virus b cell lymphomas prospective study of sequential reduction in immunosuppression, interferon alpha-2, and chemotherapy for posttransplant lymphoproliferative disorder the immunosuppressive macrolide rad inhibits growth of human epstein-barr-virus-transformed b lymphocytes in vitro and in vivo: a potential approach to prevention and treatment of posttransplant lymphoproliferative disorders post-transplant lymphoproliferative disorder-the potential of proliferation signal inhibitors immunosuppressants and skin cancers in transplant patients: focus on rapamycin sirolimus therapy after early cyclosporine withdrawal reduces the risk for cancer in adult renal transplantation the changing pattern of posttransplant malignancies post-transplant de novo malignancies in renal transplant recipients: the past and the present cancers after renal transplantation infection in renal transplant recipient new onset diabetes mellitus after transplantation valacyclovir for the prevention of cytomegalovirus disease after renal transplantation. international valacyclovir cytomegalovirus prophylaxis transplantation study group the use of sirolimus in ganciclovir-resistant cytomegalovirus infections in renal transplant recipients canadian society of transplantation consensus workshop on cytomegalovirus management in solid organ transplantation final report histological patterns of polyomavirus nephropathy: correlation with graft outcome and viral load reversal of steroid-and anti-lymphocyte antibody-resistant rejection using intravenous immunoglobulin (ivig) in renal transplant recipients tuberculosis and renal transplantation mycobacterium tuberculosis gastrointestinal complications of transplant immunosuppression gastrointestinal complications in renal transplant recipients herpes simplex virus hepatitis after renal transplantation prevention of infection in adults travelers after solid organ transplantation incidence of cancers in people with hiv/aids compared with immunosuppressed transplant recipients: a meta-analysis cancer after kidney transplantation in the united states key: cord-005225-7uuilki4 authors: paduch, darius a. title: viral lower urinary tract infections date: 2008-03-26 journal: curr urol rep doi: 10.1007/s11934-007-0080-y sha: doc_id: 5225 cord_uid: 7uuilki4 lower urinary tract infections (utis) are common among the general population and are most often caused by bacterial pathogens. viruses are an uncommon cause of utis in an immunocompetent host; however, viruses are increasingly recognized as the cause of lower uti, especially hemorrhagic cystitis, among immunocompromised patients. bk virus, adenovirus, and cytomegalovirus are predominant pathogens involved in hemorrhagic cystitis after stem cell and solid organ transplantation, and their early diagnosis and treatment may prevent significant morbidity of hemorrhagic cystitis. the diagnosis of viral lower uti is based on molecular techniques, and real-time polymerase chain reaction is often the method of choice because it allows for quantification of viral load. cidofovir is becoming a drug of choice in viral utis because it is active against the most common viral pathogens. this review discusses the epidemiology, pitfalls in diagnosis, and current treatment of viral utis. lower urinary tract infections (utis) are commonly seen in urologic practice, and most urologists are familiar with typical bacterial pathogens and current treatment paradigms; however, viral pathogens, which can cause lower utis, are less known. viral infections of the lower urinary tract are usually seen in immunocompromised patients, especially in solid organ and stem cell transplantation recipients, and are the most common cause of hemorrhagic cystitis in this group of patients [1••,2,3••] anatomically lower utis can be divided into cystitis, prostatitis, seminal vesiculitis, and urethritis; however, bladder and ureter are most commonly affected by viral lower tract utis. one of the main differences between bacterial and viral pathogens affecting the lower urinary tract is that no bacteria should be found in the urine of healthy people, especially in men. the same can not be assumed with viruses because some of them (eg, bk virus [bkv]) can be found in healthy, asymptomatic, immunocompetent patients. therefore, viral infection is defined as a presence of an identifiable viral organism with inflammatory symptoms. the symptoms of lower uti include hematuria, genital or lower abdominal pain, urgency, frequency (secondary to inflammatory response and irritation of the bladder wall), pyuria, and hematospermia. in rare instances of prostatic abscess, the obstructive voiding symptom and urinary retention can be found. the presentation, as well as the natural history of lower utis, depends on existing anatomic abnormalities and (more importantly) on the immune status of the host. with the emergence of new medications and protocols to treat systemic diseases (such as leukemia, lymphoma, chronic renal insufficiency, and rheumatologic disease) bone marrow transplantation, chemotherapy, immune modulators, and immunosuppressants are commonly used and add to the number of immunocompromised patients. the immunologic status of the patient dramatically changes one's ability to fight the infection and alters clinical course. mortality among immunocompetent patients with lower uti is extremely low; however, viral uti with high viral load can be associated with high mortality in immunocompromised patients because of associated viremia and multiorgan viral infections and failure. antivirals themselves have significant side effects, and their use may induce organ rejection [4] . thus, lower urinary tract symptoms in an immunocompromised patient should be diagnosed and treated promptly. this review brings the reader up-to-date with typical viral pathogens that can cause lower uti and provides information on clinical management. the most common presenting symptom of viral uti is hemorrhagic cystitis. recently, hemorrhagic cystitis was considered a complication of chemotherapy, especially with alkylating drugs such as busulfan. however, with improved methods of viral detections, it was found that viral infections are a common cause of hemorrhagic cystitis. in a prospective study of more than 100 children who underwent bone marrow transplantation, hemorrhagic cystitis occurred in 25.5%, and viral cause was identified in more than 95% of children with hemorrhagic cystitis. polyoma bkv was detected in the urine of 21 patients (80.8%), adenovirus (adv) was detected in four (14.4%), and jc virus was detected in one patient (3.8%). the highdose chemotherapy conditioning was the best predictor of developing viral hemorrhagic cystitis [5••] . male sex and unrelated or mismatched donor are additional risk factors for hemorrhagic cystitis [4] . viral hemorrhagic cystitis can also occur after renal transplantation, but its prevalence is lower than in bone marrow transplantation recipients, which may be attributed to lower risk of bkv reactivation rate after renal transplantation. this is thought to be secondary to less pronounced suppression of cellular immune response, which is needed to prevent rejection in solid organ transplantation [6] . bkv and adv are the most common viral pathogens isolated in hemorrhagic cystitis after renal transplantation. bkv can cause interstitial nephritis, ureteral stenosis, and hemorrhagic cystitis and is almost always treated with antivirals [7] . adenoviral hemorrhagic cystitis is usually self-limiting, and treatment depends on clinical picture. detection of adv in urine in patients with hemorrhagic cystitis is pathognomonic of adenoviral cystitis [5••,8,9] . lower abdominal pain, dysuria, frequency, urgency, and lack of high-grade fever are common symptoms of lower uti among the immunocompetent population, regardless of type of pathogen (table 1 ). the symptomatology is altered by the immune status of the host and gross hematuria; fever and malaise are seen more commonly in immunocompromised patients with lower uti. the majority of utis are caused by bacteria with escherichia coli and enterococcus [10] . thus, bacteriologic cultures have to be obtained in every patient; however, in patients not improving clinically (despite antibiotic treatment) or in patients who are at high risk of viral uti (ie, bone marrow transplantation recipients, patients undergoing treatment with a multidrug regimen for leukemia or lymphoma), the diagnosis of viral uti has to be strongly considered. early diagnosis may prevent rejection. diagnosis of viral uti is more challenging because viruses are small organisms, and they can not be visualized with even the best optical microscope. the culture of viruses may take up to 14 to 28 days, and often it is too late to treat a patient with disseminated multiorgan viral infections at that time. thus, molecular and immunofluorescence techniques are used more commonly. the reliability of diagnosis depends on adequate technique, obtaining and transporting the specimen as well as technique of detection. clinicians should be familiar with commonly used methods of virus detection: culture, direct immunofluorescence of organism, serologic-and antigen-based assays, and genomic amplification (quantitative or qualitative). viruses are too small to be detected by direct light microscopy after staining the specimen. viruses live in the host cells, and the presence of some viruses (cytomegalovirus [cmv], bkv) may be suspected by characteristic changes on urine cytology [11] . otherwise, a virus has to be grown in culture, and the type of virus is determined based on characteristic cytopathologic changes of cell culture inoculated with the specimen. this diagnostic method is cumbersome and prone to false-negative results. not all viruses can be cultured. adv, cmv, enteroviruses, herpes simplex virus (hsv), influenza, mumps, parainfluenza, respiratory syncytial virus, and varicella-zoster virus can be cultured, but this technology is not applicable for detection of coxsackie a viruses, hepatitis viruses, arbovirus, parvovirus, human papillomavirus, reovirus, measles virus, and gastrointestinal viruses. to increase detection, 1 ml of body fluids or tissues have to be placed in a special transportation medium (eg, m4) as soon as possible and placed at 4°c until they reach the laboratory. medium can not be frozen and most laboratories will not accept specimen obtained on a bacterial transportation swab. because each of the viruses requires specific cell line, media, and method of detection, it is especially important to provide adequate clinical information and to be specific about the type of virus to be detected. unlike bacterial specimens, one can not order a general "viral culture." because of the cost and time required for diagnosis using culture, most laboratories have shifted to other techniques, which can be generally divided into methods detecting the presence of pathogen (antigen), such as direct immunofluorescence and enzyme-linked immunosorbent assay (elisa); methods detecting genetic material (dna or rna), which is a specific finger print of the virus; and methods detecting antibodies in the serum or central nervous system, such as elisa or competitive elisa. each of these techniques differs by their cost, specificity, and sensitivity. in direct immunofluorescence, the pathogen is detected in the spun body fluid (or cells). the antibody against the antigen specific for virus (eg, hsv or bkv) is used and then detected under fluorescent microscope by secondary antibodies coupled with fluorochrome [12] . the method is relatively simple and fast; however, it is not a quantitative method and can not be used for viruses with rapidly changing antigens. this test is most commonly used to detect hsv by scraping from the genital ulcers, but it can also be performed on urine specimen [13, 14] . for collection, one would use a dacron swab to collect the cells from the ulcer and then smear the cells on two clean slides with the patient's name and medical record number. after the cells have air-dried the test can be transported to the virology laboratory. elisa and competitive elisa are used to detect viral antigens and antibodies against the viruses in the specimen. the antibodies against the antigen are immobilized on the styrene plate, and secondary antibodies are then applied to detect the antigen. the plates are washed to remove excess antibody and chemiluminescence, or colorimetric detection reagents are added. the darker the color in the well, the more antigens (viral particles) present in the specimen. in competitive elisa, the tracer antigen competes with the specimen antigen for a set number of binding sites. in this assay, the lighter color after developing reaction means that there is a higher concentration of virus in the patient's sample. elisa is also used in the detection of antibodies. in this assay, the antibody is an antigen. elisa is a relatively simple and fast technique that is often used in automatic assays; however, small changes in volumes of sandwich antibodies or developer will affect the results (pipetting error). in automatic instrumentation, the chemicals are dispensed from prefilled containers, and "carry-over" contamina-tion between different runs of assay can easily occur. this is a known problem; hence, if the results do not match the clinical picture, one should contact the laboratory and inquire about recent quality-control problems or controls. because the amount of primary antibody is fixed per well, the concentrations of antigens or tracer can not be too high because they will occupy all of the binding sites by chance, and the assay results can be outside of the linear standard curve. given that each assay uses a different (and often) proprietary amount of antigen and antibodies as well as tracers, the results between different assays may differ, and it is preferable to use the same assay from a single supplier. serologic methods detect changes in titers of antibody against the known pathogen. because developments of antibodies against a virus take time and use of immunosuppressants may modulate immune response, lack or presence of antibodies may not exclude or confirm current infection. this is especially true in viruses, such as hsv, cmv, and bkv, with a high prevalence (17%, > 60%, and > 90%, respectively) among an immunocompetent population in the united states [15, 16] . hsv and bkv are especially important in urology. to find out if a patient was ever exposed to hsv, one can measure immunoglobulin g against hsv levels, which should be elevated. during acute or recent infection, the immunoglobulin m titer allows for differentiation of new viral infection versus history of exposure in the past. immunoglobulin m increases within 2 weeks of exposure. the results for antibodies are reported as titers, and increasing titers make it more likely that the patient has current infection. because of some nonspecific binding of a patient's antibodies to the antigen, good quality laboratories stratify their titers into negative, undetermined, and positive. undetermined titers can be a result of nonspecific binding or of current infection when the host has not yet produced enough antibodies to be detected. if levels of antibodies are within an undetermined level, the assay should be repeated in 2 to 4 weeks, or direct method of detection should be used. because of problems with detection of antibodies and poor correlation between the viral load and the elisa, as well as the fact that many viruses are present in immunocompetent hosts, the current state-of-the-art detection techniques are based on molecular techniques [17] . these techniques are based on polymerase chain reaction (pcr), which allows for specific and fast amplification of a small region of viral genome. because the genome of most of the clinically important viruses is known, it is relatively easily to amplify viral dna or rna and to detect amplicon by gel electrophoresis, chromatography, or real-time pcr. real-time pcr allows for relative or absolute quantification of viral load. the results are reported as number of virions per ml or number of units per ml of specimen. commercially available bkv tests are able to detect 500 copies of bkv in 1 ml of urine. although pcr is a sensitive diagnostic method and even a minute amount of virus can be detected, its sensitivity is also one of its drawbacks. the genome of some viruses is not stable, and one needs to choose the most stable "conserved" sequence that can identify the virus of interest with high specificity. even a single change in the nucleotide sequence can affect the binding of primers, and if the annealing temperature is high, the presence of a virus may not be detected (false-negative result). the viruses also occur in multiple genotypes (example hpv or adv); thus, often one assay is not able to detect all genotypes. because the pcr reaction produces millions of copies of viral dna "amplicon," it is easy to obtain falsepositive results from airborne amplicon contamination. therefore, in many laboratories, the preparatory and analytic areas are physically separated, and high-performance flow hoods are used when amplified samples are handled. the real-time pcr, which eliminates transferring of amplified product to the gel, avoids many of the contamination problems and has become a method of choice for molecular detection of clinically important viruses. realtime pcr and quantification of viral load has significant prognostic value in predicted clinical outcomes [18] . classification of clinically important viruses is somehow difficult because viruses with quite different biochemical and genomic properties may cause similar diseases. generally, viruses are divided based on type of nuclei acid that they are made from (dna or rna), and subsequently, they are divided into groups based on replication properties such as single-or double-strand. the most commonly used classification is called the baltimore classification (coined for its creator, american biologist and nobel laureate, dr. david baltimore), and it divides viruses into seven groups. clinically, viruses are often group-based on clinical picture, though viruses with different biochemical properties can lead to a similar pathology ( table 2 ). basic knowledge of viral classification is useful because the majority of antiviral drugs are active against viruses with similar biochemical and molecular properties (table 3) . the human polyomavirus (bkv) is a subgroup of papovavirus and is a common and normally nonpathogenic virus, with approximately 97% of the adult population having antibodies against it [19] . bkv has a significant homology to a neurotropic virus, causing progressive multifocal leukoencephalopathy, or jcv. bkv was first identified in the urine of a renal transplantation recipient with the initials b.k. (by dr. sylvia d. gardner in 1971). the bkv virus has a urotheliotropic nature and can be identified in the collecting system epithelium and transitional epithelium. the typical cytopathologic changes of bkv are often found in a healthy person, and it is believed to represent transient shedding of virions in urine, most likely secondary to stress or decreased immune response. the patient's cytopathologic changes resolve within 3 months, and there seems to be no clinically significant sequelae of bkv-positive cytology in immunocompetent people [20] . high-grade transitional cell carcinoma can be difficult to distinguish from bkv on cytology, and follow-up cytology may be considered in patients with risk factors for transitional cell carcinoma [21] . up to 0.3% of the healthy population and 3% of pregnant women will have bkv-positive urine cytology [22] . the interest in bkv paralleled development in potent immunosuppressants and the discovery of an association between reactivation of bkv in patients with renal transplantation and progressive nephropathy and allograft loss-polyomavirus-induced nephropathy [23] . the bkv is commonly found in the urine of patients with hemorrhagic cystitis and ureteral strictures, especially after bone marrow and solid organ transplantations [6, 24] . fifteen of 90 patients developed late-onset bkv hemorrhagic cystitis 16 to 95 days after bone marrow transplantation in the study by giraud et al. [3••] . most of these patients (10 of 15; 67%) developed hemorrhagic cystitis within 30 days after transplantation. the hemorrhagic cystitis was severe in more than 80% of the patients. reduced conditioning decreases risk of bkv-related hemorrhagic cystitis [3••] . bkv infection should be suspected in a patient with immune deficiencies (table 1 ) who presents with hemorrhagic cystitis, microscopic hematuria, hydronephrosis, and an increase in creatine. most bkv infections occur within 1 to 6 months after transplantation ( table 4 ). the clinical diagnosis needs to be confirmed by detecting the virus in the urine or blood. recipients of renal transplantation may also have associated graft dysfunction, and renal biopsy may show typical interstitial nephritis with characteristic changes in tubules. the presence of bkv in renal parenchyma may be detected by commercially available antibodies. because of the high prevalence of positive antibodies in the serum and lack of a reliable viral cell culture, the diagnosis of bkv lower utis requires molecular techniques, such as quantitative real-time pcr, which allows for the detection of bkv and an estimation of the number of viral copies per ml of urine or blood [25] . bkv dna is rarely detected in the urine of a healthy individual. urine cytology can be indicative of bkv infection by identifying so-called "decoy" cells; however, sensitivity of decoy cells in diagnosis of bkv infection in hemorrhagic cystitis is low [26] . real-time pcr tests and quantification of viral copies seem to have prognostic value and can be used to monitor response to therapy. bkv is detected in 87% of patients with hemorrhagic cystitis after bone marrow transplantation, and the prevalence of bkv is statistically higher than in patients without hemorrhagic cystitis [3••] . bkv is more prevalent than adv in patients with hemorrhagic cystitis [5••,27] . urine should be sent for bkv and adv detection by pcr for every patient with hemorrhagic cystitis who is immunocompromised. until recently, the treatment of the bkv infection and its urologic complications focused on supportive measures (hydration, correction of coagulopathy, bladder irrigation), reduction in immunosuppression, and leflunomide with an overall poor response [28] . acyclovir, ganciclovir, brivudine, ribavirin, foscarnet, and cytarabine have poor antiviral activity against bkv in in vitro studies [29] . over the past few years, cidofovir, administered intravenous (iv) or intravesical, proved to be effective therapy against bkv with a relatively low rate of side effects [5••,30,31] . in the case of hemorrhagic cystitis, hydration, correction of coagulopathy, and bladder irrigation may be followed by cidofovir intravesical instillation, 5 mg/kg in 60 ml of saline instillation for 1 hour once a week [30] . cidofovir can also be given iv at a similar dose; however, bladder instillation may avoid nephrotoxic complications of the drug. cidofovir is active against the cmv and adv, though it is less active against bkv, and by itself, it can cause nephropathy. thus, intravesical instillation may be a better option for hemorrhagic cystitis, especially because the adv plays a role in some of the patients with hemorrhagic cystitis, and cidofovir may be active against both organisms [1••,32,33••,34] . the response to treatment is measured by quantitative real-time pcr and resolution of hematuria. although bkv is (without a question) the dominant viral cause of cystitis, only 50% of patients with bkv viruria will develop hemorrhagic cystitis, and prophylactic treatment with cidofovir may not be necessary. however, preemptive management-starting therapy as soon as the patient develops cystitis-may be beneficial, taking into account the high morbidity associated with hemorrhagic cystitis after bone marrow transplantation. one study showed that ciprofloxacin prophylaxis decreased the viral load of bkv as compared with cephalosporin. the mechanism of this finding is unclear [35] . it is important to remember that bkv is associated with an increased risk of bladder cancer, and follow-up cytologic studies (once hematuria resolves) may be indicated [36] . it is unknown if bkv is a cause or one of the modulators that increase the chance of neoplastic transformation. adv are double-strand dna viruses with at least 51 serologic subtypes. adv are known to cause upper respiratory, gastrointestinal, and conjunctival infections in healthy people and children; however, their pathogenicity is altered by the immunologic status of the host, and in immunocompromised patients, adv can affect many other systems [32] . normally, the adv causes asymptomatic infection of lymphoepithelial tissues, but in the immunocompromised patient, they can reactivate the latent infection or cause de novo infection. the adenoviral infections are more common in stem cell transplantation and solid organ transplantations. adv can be detected in 10% of urine samples after transplantation, and over 12 months of follow-up, adenoviral utis occurred in 9% of patients [37] . children, recipients of allogeneic versus autogeneic stem cell graft, and patients with graft versus host disease are much more prone to adenoviral diseases, which is a reflection of a more pronounced immunosuppression used in the above conditions. immunocompetent patients have limited disease that hardly ever leads to serious mortality or morbidity. adenoviral cystitis can present as gross and microscopic hematuria in up to 20% of patients [38] . history of solid organ or bone marrow transplantation and use of immunosuppressants aids in diagnosis because adv cystitis occurs almost exclusively in immunocompromised patients [8] . most cases of adv-related hemorrhagic cystitis occur within 12 months of transplantation [8] . hemorrhagic cystitis is most commonly caused by immunotype 11, and presence of adv in urine is almost exclusively seen in hemorrhagic cystitis [39] . the adenoviral infection can often coexist with aspergillosis and cmv in immunocompromised patients, and broad cultures should be obtained. adv are never detected in healthy patients. presence of adv in the urine of an immunocompromised patient is always associated with cystitis, but only 50% of bkv viruria will present clinically as cystitis. cystitis is the most common clinical presentation of adv infection of the genitourinary tract. infection with adv is defined as presence of virus in culture, presence of viral antigen by immunofluorescence, or presence of adv dna by pcr, irrespective of symptoms. the diagnosis using molecular techniques is faster because the culture can take up to 21 days. adenoviral disease refers to presence of virus and symptoms of invasive disease. in stem cell transplantation patients, adv can cause hepatitis, hemorrhagic colitis, hemorrhagic cystitis, or pneumonitis and often leads to disseminated disease and death [40] . adenoviral infections are associated with significant mortality and morbidity, and some advocate preemptive treatment and a high level of suspicion in immunocompromised patients, especially if adv can be detected in the blood by pcr [40, 41] . unfortunately, until recently there was no single antiviral drug that would be potent and devoid of drug toxicity. cidofovir is becoming an optional treatment of adenoviral infections and should be considered a first-choice antiviral to treat adv cystitis. lower dose (1 mg/kg three times per week for 3 weeks) is used in renal transplantation patients because of concern of nephrotoxicity of cidofovir, but this regimen fails to prevent hsv or cmv infections, and a higher dose (5 mg/kg once a week, iv or intravesical) may be a better choice [33, 42, 43] . it is possible that the nephrotoxicity of cidofovir is a result of often reduced immunosuppression and rejection and not the result of cidofovir itself, and a higher dose with continued immunosuppression may result in less long-term complications. ribavirin, which has relatively poor activity against adv, has been more successful in treating human leukocyte antigen-matched bone marrow recipients. ribavirin is less successful in decreasing mortality in children [44] [45] [46] . ganciclovir is mostly used for prevention of cmv infection; however, it has been used in the treatment of hemorrhagic cystitis in transplantation patients [47] . vidarabine (10 mg/kg/day for 5 days) has been successfully used in the treatment of hemorrhagic cystitis and may be a viable alternative to cidofovir, but it is less active in generalized adv infections [45, [48] [49] [50] . vidarabine and its metabolite achieve a high concentration in urine, which may explain its success in treatment of adv hemorrhagic cystitis [48] . cmv infection is common, and more than 60% of adults are seropositive. cmv belongs to a large group of herpes viruses that are of limited pathologic significance in immunocompetent patients. cmv reactivation or new infection is common in transplantation patients, and it can cause significant mortality and morbidity; hence cmv prophylaxis is commonly employed in patients after transplantation. cmv is a relatively rare cause of lower utis; however, circumferential evidence supports the association between cmv and hemorrhagic cystitis [51] . reports of resolution of hematuria after treatment with iv ganciclovir have added further evidence linking cmv and hemorrhagic cystitis [51] . although rare, cmv cystitis can also occur in immunocompetent patients [52] . hemorrhagic cystitis has been clearly associated with reactivation of cmv [53, 54] . cmv is also believed to cause ureteritis and ureteral stenosis [55] . diagnosis cmv can be detected by seroconversion in a previously negative host and increase in immunoglobulin m and immunoglobulin g antibodies titers [56••] , but a high prevalence of latent cmv infection makes serologic diagnosis difficult, and detection of cmv antigen (pp65), rna, or dna is commonly used. recently the pp65 antigenemia and real-time pcr have been compared with each other, and real-time pcr seems to correlate better with the clinical picture [57] . most patients receive prophylaxis with ganciclovir after solid organ transplantation; however, in active diseases, other drugs, such as foscarnet, 60 mg/kg iv twice daily for 14 days, can be used because they are active against cmv [58] . cidofovir is active against both bkv and cmv and has been used if both viruses are detected [59] . for this reason, cidofovir may become a drug of choice in patients who present with hemorrhagic cystitis after solid organ or bone marrow transplantation. hsv type-2 is a common cause of genital ulcers in immunocompetent hosts, but bladder involvement is rare. hsv cystitis can occur in immunocompetent patients who have some other predisposing factors, such as diabetes mellitus or rheumatologic disorders [60, 61] . hemorrhagic cystitis can be a sign of disseminated hsv infection in immunocompromised patients [62] . diagnosis is relatively easy because hsv can be detected by serology, direct immunofluorescence, or cell culture. treatment of hsv infection depends on the host's age and immune and serologic status. neonates who are born of mothers with primary hsv infection are at high risk of transmission and central nervous system complications, and they are treated with iv acyclovir for 14 days. acyclovir or valacyclovir is commonly used in symptomatic subjects with primary infection for 10 to 14 days using oral medications. suppression is recommended in patients with recurrent flare-ups or in discordant couples to decrease risk of infection [63•] . other viruses that are important in urology are the human papillomavirus, poxvirus causing molluscum contagiosum, and hiv; however, they do not (per se) cause lower utis and thus are outside the scope of this review. viral infections of the genitourinary tract are associated with significant morbidity and suffering, including increased mortality in immunocompromised patients. as urologists we need to include the most recent developments in genitourinary virology in our practice because knowledge of viral biology and clinical pathology may prevent viral transmission (hsv and human papillomavirus), and early management of viral cystitis may decrease mortality related to disseminated viral infections of the lower urinary tract in selected patients. particular interest, published recently, have been highlighted as: • of importance •• of major importance cidofovir for the treatment of adenoviral infection in pediatric hematopoietic stem cell transplant patients this study address the indications, outcomes, and possible side effects of cidofovir use in adenoviral infections. given that there are few potent drugs against the adenovirus, this manuscript is very important because it proves that cidofovir is clinically useful as a first-line of therapy for bkv and adenovirus polyoma virus-induced hemorrhagic cystitis in renal transplantation patient with polyoma virus nephropathy the incidence of hemorrhagic cystitis and bk-viruria in allogeneic hematopoietic stem cell recipients according to intensity of the conditioning regimen this is an important study evaluating risk factors for hemorrhagic cystitis in patients after bone marrow transplantation hemorrhagic cystitis after allogeneic bone marrow transplantation in children: clinical characteristics and outcome incidence, clinical outcome, and management of virus-induced hemorrhagic cystitis in children and adolescents after allogeneic hematopoietic cell transplantation this is one of very few studies following the children who underwent bone marrow transplantation in the prospective way. this manuscript is critical because it specifically focuses on hemorrhagic cystitis post-transplantation polyomavirus infections bk virus in solid organ transplant recipients: an emerging syndrome hemorrhagic adenovirus cystitis after renal transplantation acute hemorrhagic cystitis caused by adenovirus following renal transplantation: review of the literature infectious complications and antibiotic use in renal transplant recipients during a 1-year follow-up periodic assessment of urine and serum by cytology and molecular biology as a diagnostic tool for bk virus nephropathy in renal transplant patients rapid detection and identification of jc virus and bk virus in human urine by using immunofluorescence microscopy asymptomatic herpes simplex virus type 2 (hsv-2) infection among pregnant women in turkey comparison of the detection of herpes simplex virus in direct clinical specimens with herpes simplex virus-specific dna probes and monoclonal antibodies trends in herpes simplex virus type 1 and type 2 seroprevalence in the united states seroepidemiology of the human polyomaviruses detection of herpes simplex virus type 1, herpes simplex virus type 2 and varicella-zoster virus in skin lesions: comparison of real-time pcr, nested pcr and virus isolation polymerase chain reaction detection of bk virus and monitoring of bk nephropathy in renal transplant recipients at the university hospital la fe serological diagnosis of human polyomavirus infection boon me, van keep jp, kok lp: polyomavirus infection versus high-grade bladder carcinoma: the importance of cytologic and comparative morphometric studies of plastic-embedded voided urine sediments a prospective study of human polyomavirus infection in pregnancy the natural history, risk factors and outcomes of polyomavirus bk-associated nephropathy after renal transplantation cmv and bkv ureteritis: which prognosis for the renal graft? quantitative real-time pcr assay for detection of human polyomavirus infection urine cytology findings of polyomavirus infections detection of bk virus and adenovirus in the urine from children after allogeneic stem cell transplantation urological manifestations of bk polyomavirus in renal transplant recipients activities of various compounds against murine and primate polyomaviruses cidofovir bladder instillation for the treatment of bk hemorrhagic cystitis after allogeneic stem cell transplantation cidofovir treatment of human polyomavirus-associated acute haemorrhagic cystitis adenovirus infections in transplant recipients intravesical instillation of cidofovir in the treatment of hemorrhagic cystitis caused by adenovirus type 11 in a bone marrow transplant recipient authors have shown that the intravesical instillation of cidofovir can be used successfully to treat hemorrhagic cystitis. this form of treatment probably decreases the risk of nephrotoxicity ciprofloxacin decreased polyoma bk virus load in patients who underwent allogeneic hematopoietic stem cell transplantation bladder carcinoma in a transplant recipient: evidence to implicate the bk human polyomavirus as a causal transforming agent adenoviral infection after allogeneic stem cell transplantation (sct): report on 130 patients from a single sct unit involved in a prospective multi center surveillance study adenovirus associated haematuria hemorrhagic cystitis associated with urinary excretion of adenovirus type 11 following allogeneic bone marrow transplantation adenovirus infections following allogeneic stem cell transplantation: incidence and outcome in relation to graft manipulation, immunosuppression, and immune recovery hemorrhagic cystitis after conditioning for bone marrow transplantation and its prophylaxis cidofovir for treating adenoviral hemorrhagic cystitis in hematopoietic stem cell transplant recipients early diagnosis of adenovirus infection and treatment with cidofovir after bone marrow transplantation in children intravenous ribavirin treatment for severe adenovirus disease in immunocompromised children successful ribavirin therapy for severe adenovirus hemorrhagic cystitis after allogeneic marrow transplant from close hla donors rather than distant donors adenovirus-associated haemorrhagic cystitis after bone marrow transplantation successfully treated with intravenous ribavirin treatment of adenovirus-associated haemorrhagic cystitis with ganciclovir therapeutic basis of vidarabine on adenovirus-induced haemorrhagic cystitis vidarabine therapy for virus-associated cystitis after allogeneic bone marrow transplantation successful vidarabine therapy for adenovirus type 11-associated acute hemorrhagic cystitis after allogeneic bone marrow transplantation cytomegalovirus -induced hemorrhagic cystitis following bone marrow transplantation cytomegalovirus-induced haemorrhagic cystitis in a patient with neurogenic bladder high incidence of adeno-and polyomavirus-induced hemorrhagic cystitis in bone marrow allotransplantation for hematological malignancy following t cell depletion and cyclosporine cmv-induced hemorrhagic cystitis as a complication of peripheral blood stem cell transplantation: case report cytomegalovirus ureteritis as a cause of renal failure in a child infected with the human immunodeficiency virus meta-analysis: the efficacy of strategies to prevent organ disease by cytomegalovirus in solid organ transplant recipients although prevention of cmv infection is commonly done, this is one of very few studies that compares different methods of prevention assessment of cmv load in solid organ transplant recipients by pp65 antigenemia and real-time quantitative dna pcr assay: correlation with pp67 rna detection cmv reactivation induced bk virus-associated late onset hemorrhagic cystitis after peripheral blood stem cell transplantation treatment of bk virusassociated hemorrhagic cystitis and simultaneous cmv reactivation with cidofovir hemorrhagic cystitis associated with herpes simplex virus hemorrhagic cystitis with herpes simplex virus type 2 in the bladder mucosa hemorrhagic cystitis due to herpes simplex virus as a marker of disseminated herpes infection hsv shedding although not so commonly seen in lower utis, this is an important update regarding the biology and transmission of hsv key: cord-005794-3u4iu41r authors: berner, michel e.; hanquinet, sylviane; rimensberger, peter c. title: high frequency oscillatory ventilation for respiratory failure due to rsv bronchiolitis date: 2008-05-24 journal: intensive care med doi: 10.1007/s00134-008-1151-3 sha: doc_id: 5794 cord_uid: 3u4iu41r objective: to describe the time course of high frequency oscillatory ventilation (hfov) in respiratory syncytial virus (rsv) bronchiolitis. design: retrospective charts review. setting: a tertiary paediatric intensive care unit. patients and participants: infants with respiratory failure due to rsv infection. intervention: hfov. measurements and results: pattern of lung disease, ventilatory settings, blood gases, infant’s vital parameters, sedation and analgesia during the periods of conventional mechanical ventilation (cmv, 6 infants), after initiation of hfov (hfovi, 9 infants), in the middle of its course (hfovm), at the end (hfove) and after extubation (post-extub) were compared. all infants showed a predominant overexpanded lung pattern. mean airway pressure was raised from a mean (sd) 12.5 (2.0) during cmv to 18.9 (2.7) cmh(2)o during hfovi (p < 0.05), then decreased to 11.1(1.3) at hfove (p < 0.05). mean fio(2) was reduced from 0.68 (0.18) (cmv) to 0.59 (0.14) (hfovi) then to 0.29 (0.06) (p < 0.05) at hfove and mean peak to peak pressure from 44.9 (12.4) cmh(2)o (hfovi) to 21.1 (7.7) p < 0.05 (hfove) while mean (sd) paco(2) showed a trend to decrease from 72 (22) (cmv) to 47 (8) mmhg (hfvoe) and mean infants respiratory rate a trend to increase from 20 (11) (hfovi) to 34 (14) (hfove) breaths/min. with usual doses of sedatives and opiates, no infant was paralysed and all were extubated to cpap or supplemental oxygen after a mean of 120 h. conclusion: rsv induced respiratory failure with hypercapnia can be managed with hfov using high mean airway pressure and large pressure swings while preserving spontaneous breathing. (0.18) (cmv) to 0.59 (0.14) (hfovi) then to 0.29 (0.06) (p \ 0.05) at hfove and mean peak to peak pressure from 44.9 (12.4) cmh 2 o (hfovi) to 21.1 (7.7) p \ 0.05 (hfove) while mean (sd) paco 2 showed a trend to decrease from 72 (22) (cmv) to 47 (8) mmhg (hfvoe) and mean infants respiratory rate a trend to increase from 20 (11) (hfovi) to 34 (14) (hfove) breaths/min. with usual doses of sedatives and opiates, no infant was paralysed and all were extubated to cpap or supplemental oxygen after a mean of 120 h. conclusion: rsv induced respiratory failure with hypercapnia can be managed with despite the use of non invasive respiratory support such as cpap [1, 2] , helium-oxygen mixture [3] , alone or in combination [4] , the proportion of infants with rsv infection requiring conventional mechanical ventilation (cmv) is still reported to vary around 25% in those requiring intensive care admission [1, 5] . adequate cmv in obstructive lung disease represents a challenge. high tidal volumes and minute ventilation are required implying high peak pressures, risk of air leaks, and an almost universal use of muscular paralysis [6] . the use of high frequency oscillatory ventilation (hfov) has been described in a few cases [7-10] as a rescue intervention to improve severe hypoxemic respiratory failure while co 2 elimination was yet achieved. in this report, we aim to describe the time course of hfov in rsv proven infection with predominantly hypercapnic respiratory failure as an effective alternative to conventional mechanical respiratory support. charts of all infants with confirmed rsv bronchiolitis treated with high frequency oscillatory ventilation between 01.01.1998 and 01.01.2007 were reviewed. those with a known underlying condition susceptible to modify the nature or the course of the lung disease such as broncho-pulmonary dysplasia, pulmonary congenital malformation or non operated congenital heart disease were excluded. the obstructive pattern of the lung disease for 6 infants initially on cmv, was determined according to the relation between mean airway pressure and the alveolo-arterial oxygen pressure difference (aado 2 ) described by tasker et al. [11] and lung expansion was assessed for each infant by counting the total number of visible ribs (on both hemithoraces) above the diaphragm on the initial chest x-ray as described by greenough et al. [12] , 16 ribs being the cut-off value for the presence of lung overexpansion. during the course of mechanical ventilation, no pharmacological treatment such as corticosteroids, b2 or a-1adrenegic agonists were administered. bacterial coinfection was suspected on clinical deterioration and antibiotics given if gram staining of tracheal secretions, blood leucocytes count, differential and serum crp were considered abnormal. cmv was delivered with a babylog 8000 (dräger, lubeck, germany) in a pressure controlled mode and hfov through an electromagnetically driven membrane oscillator (3100 a sensor medics critical care, yorba linda, ca, usa). frequency was started between 8-12 hz with an i:e ratio of 0.33 and mean airway (mean aw ) pressure 2 cmh 2 o above the last value set during conventional ventilation. it was raised by steps of 2 cmh 2 o aiming at an arterial saturation above 88% until a reduction in fio 2 could be initiated. additional recruitment manoeuvres were not attempted. peak to peak pressure amplitude was started at a value where chest movements could be visually detected and stepwise increased to reach and maintain the ph above 7.25 irrespective of the paco 2 . infants received midazolam or chloral hydrate for sedation and morphine sulphate for analgesia. muscular paralysis was avoided as long as not specifically required. ventilatory settings (fio 2 , mean aw pressure, peak to peak amplitude, ventilatory frequency, i:e ratio), blood gases and vital signs (infants respiratory rate, mean arterial blood pressure and heart rate) were extracted during conventional mechanical ventilation (period cmv), a mean of 3 h (±40 min) after the switch to hfov (period hfovi), mid course (period hfovm), just prior stopping hfov (period hfove) and after extubation either on cpap and/or supplemental oxygen therapy (post-extub). kruskall-wallis non parametric analysis of variance was performed to compare periods of respiratory support for ventilatory variables, gas exchanges and vital signs using a prism 4.0 package (graphpad software). p \ 0.05 was considered significant. a total of 13 infants with rsv proven lung infection were treated with hfov during the study period. four cases were excluded from analysis (two with congenital pulmonary malformation, two with unrepaired congenital cardiac disease). three infants, transferred after intubation from another unit or hospital were treated with hfov as first intention. six infants, intubated in our icu, were put on hfov after a median (25-75%) period of cmv of 3.5 (1-32.5) h. the relevant demographic data, initial lung pattern and clinical course are described in table 1 . all infants attested of lung overexpansion on chest x-ray [12] while none fulfilled the criteria for an ards type of rsv infection [11] . bacterial co-infection was diagnosed in five infants (56%). before initiating hfov in the six cases under cmv, mean (sd) ph was 7.27 (0.15) units and paco 2 72 (22) mmhg with a mean peak inspiratory pressure of 26 (5) cmh 2 o and a peep of 6.5 (1) cmh 2 o. changes in ventilatory settings, blood gases, infants own respiratory rates, heart rates and mean arterial pressures, doses of medication given during the various periods of ventilation are summarized in table 2 . no infant was paralyzed. hemodynamically, two infants were treated with dopamine. one infant had developed a bilateral pneumothorax under cmv which resolved with drainage and hfov. five infants were extubated to cpap, the other to simple supplemental oxygen and all survived. although mechanical ventilation at high frequencies is usually considered to be contraindicated in lung disease with increased airway resistance, the successful use of rescue hfov in a few infants with bronchiolitis to correct severe hypoxemia [7-9] or in a case of status asthmaticus [14] has yet been published. our report documents that hfov can be used as an elective alternative to cmv to support spontaneous respiration and improve gas exchanges in infants with predominantly obstructive and hypercapnic respiratory failure from small airway disease. indeed, despite bacterial co-infection in half of them, either by scoring the chest x-ray [12] or plotting the individual alveolo-arterial oxygen difference against mean aw pressure [11] , all infants showed a predominantly overexpanded lung pattern [15] . in our view, the use of hfov in bronchiolitis demonstrates several advantages over cmv. it obviates the need for infant ventilator synchrony and allows muscular paralysis to be avoided. paco 2 can be controlled by adjusting peak to peak pressure while the infant continues actively to contribute to expiration under usual sedation. we consistently observed that the infant's respiratory rate slowly increased as pressure amplitudes were decreased (see fig. 1 ). a bilateral pneumothorax observed under cmv resolved during hfov and no new air-leak occurred but the number of infants is too small to draw conclusions. hemodynamically, a few infants did require adrenergic support but renal function was well preserved as suggested by the physiological metabolic compensation of the respiratory acidosis that occurred over time. the mean duration of hfov was either similar [11, 16] or slightly shorter to that reported in infants with obstructive bronchiolitis using cmv [6, 15] and all patients could be extubated directly to cpap or supplemental oxygen. the major limitation of this report is its retrospective and descriptive nature. hfov settings were set on pragmatic grounds because the actual knowledge on mechanisms governing pressure and flow transmission when peripheral airway resistance is high, is scarce [17]. cmv conventional mechanical ventilation, hfovi initial period of high frequency oscillatory ventilation, hfovm middle period during high frequency oscillatory ventilation, hfove end period of high frequency oscillatory ventilation. *p \ 0.05 cmv vs hfovi. **p \ 0.05 hfovi vs hfove in this view, three settings deserve comments: the large peak to peak pressure, the low ventilatory frequency and the high mean aw pressure compared to hfov in idiopathic respiratory distress syndrome [18] . such settings seem at first poorly adapted to the ventilation of obstructive lung disease. however, with hfov, the transmission of pressures and volumes delivered by the ventilator is exquisitely sensitive to the mechanical properties of the respiratory system and the endotracheal tube [19] . thus, the requirement of high peak to peak pressure can be explained by the dampening that occur through the small endotracheal tube and down the highly resistive airway of these infants, reducing pressure swings and effective tidal volume [19, 20] . a low frequency can thus be advocated to maintain an adequate tidal volume once high amplitude has been set. the high mean aw pressure required at the airway opening is a hallmark of all cases reported with hfov in small airway disease [9, 10, 13] and represents the most intriguing setting. it is partly attributed to the pressure drop occurring through the flow dependant resistance of the endotracheal tube which is accentuated when using an i:e of 0.33 generating higher inspiratory than expiratory flows [19] . more importantly, this high mean aw pressure is considered necessary to generate a sufficient pressure to open and stent the collapsed small airway, allowing pressure swings to be transmitted peripherally and alveolar ventilation to resume in obstructed lung units. in this concept termed the ''open airway'' concept, mean aw pressure becomes a major determinant of co 2 elimination [9, 10, 13, 14] . the optimal pressure required to open the peripheral airway without causing alveolar overdistention and adverse cardiovascular effects must lie in a narrow range. it still requires an experimental set-up to be approximated scientifically [20] . in conclusion, hfov seems a valuable tool to support spontaneous respiration and gas exchange in infants with respiratory failure due to the common obstructive lung pattern of rsv infection. determination of optimal ventilatory settings remains a difficult clinical task due to the complexity of the interaction between mean aw pressure, frequency, pressure amplitude and mechanical characteristics of the respiratory system. effects of nasal continuous positive airway pressure ventilation in infants with severe acute bronchiolitis randomized controlled trial of nasal continuous positive airway pressure (cpap) in bronchiolitis high-frequency oscillatory ventilation (hfov) facilitates co 2 elimination in small airway disease: the open airway concept time course of severe respiratory syncytial virus infection in mechanically ventilated infants a simple chest radiograph score to predict lung disease in prematurely born infants high-frequency oscillatory ventilation in children: a single-center experience of 53 cases status asthmaticus treated by highfrequency oscillatory ventilation acute respiratory distress syndrome caused by respiratory syncytial virus bronchiolitis treated with mechanical ventilation: prognosis factors and outcome in a series of 135 children dependence of intrapulmonary pressure amplitudes on respiratory mechanics during high-frequency oscillatory ventilation in preterm lambs the provo multicenter early-frequency oscillatory ventilation trial: improved pulmonary and clinical outcome in respiratory distress syndrome high-frequency oscillatory ventilation: mechanisms of gas exchange and lung mechanics attenuation of pressure swings along the endotracheal tube is indicative of optimal distending pressure during high-frequency oscillatory ventilation in a model of acute lung injury key: cord-274012-56i4sikj authors: gavaldà, joan; román, antonio title: infección en el trasplante de pulmón date: 2007-11-30 journal: enfermedades infecciosas y microbiología clínica doi: 10.1157/13112940 sha: doc_id: 274012 cord_uid: 56i4sikj actualmente, el trasplante de pulmón se considera un tratamiento válido para un buen número de pacientes con insuficiencia respiratoria grave. de todas formas, las complicaciones son muy frecuentes y pueden llevar a fracaso del injerto a medio y largo plazo y menor supervivencia. de acuerdo con el registro de la international society for heart and lung transplantation, las tasas de supervivencia al primer, segundo y quinto año fueron, en 2006, del 74, 65 y 47%, respectivamente. el principal obstáculo para el éxito a largo plazo del trasplante de pulmón es el rechazo crónico, caracterizado histológicamente como bronquiolitis obliterante, que acontece en cerca de dos terceras partes de los pacientes. uno de los factores más importantes para el desarrollo de bronquiolitis obliterante, además del número de rechazos agudos, es la infección y la enfermedad por citomegalovirus (cmv). recientemente, se ha destacado el papel de la infección por diferentes virus respiratorios como factores de riesgo para el desarrollo de rechazo crónico en receptores de un trasplante de pulmón. las complicaciones infecciosas son una causa frecuente de morbimortalidad en este tipo de pacientes, y la causa de muerte de cerca de la mitad de ellos. la infección bacteriana es la complicación más frecuente de un receptor de un trasplante de pulmón. del total, el 35-66% son bacterianas y el 50-85% de los pacientes presentan como mínimo un episodio de infección bacteriana. la segunda causa más frecuente de infección, después de la bacteriana, es la infección por cmv. a pesar de utilizar diferentes estrategias de prevención, la incidencia sigue siendo elevada, y se sitúa alrededor del 7% el primer año postrasplante. es el único tipo de trasplante de órgano sólido en el cual la etiología más frecuente de la infección fúngica es aspergillus spp., a diferencia del resto, en que típicamente se deben a candida spp. la incidencia de aspergilosis invasora se cifra en alrededor del 4%. lung transplantation is now considered an established therapeutic option for patients with severe respiratory failure. nevertheless, complications are frequent and can lead to intermediateor long-term graft dysfunction and decreased survival. according to the registry of the international society for heart and lung transplantation, survival rates in these patients at one, two, and five years are 74%, 65%, and 47%, respectively. the main obstacle to long-term success of lung transplantation, however, is chronic rejection, which is characterized histologically as bronchiolitis obliterans and occurs in up to two-thirds of patients. one of the most important risk factors for the development of bronchiolitis obliterans, in addition to the number of previous acute rejection episodes and the incidence of persistent rejection, is cytomegalovirus infection and disease. moreover, recent evidence has indicated a role for respiratory viruses as risk factors for the development of chronic rejection in lung transplant recipients. infectious complications are a frequent cause of morbidity and mortality in these patients and are the cause of death in nearly half of them. bacterial infection is the most frequent infectious complication in lung transplant patients. among the total of infections, 35%-66% are bacterial and 50%-85% of patients present at least one episode. cmv is the second most frequent cause of infectious complications following lung transplantation. despite the use of various preventive strategies, the risk of developing cmv disease in lung transplant recipients is over 5% during the first year. this is the only type of solid organ transplant in which the etiology of fungal infection is characteristically aspergillus spp., in contrast to others in which infection by candida spp. is most common. the incidence of invasive aspergillosis is about 4%. la era moderna del trasplante se inicia con la introducción de la ciclosporina como inmunosupresor. después de la fase de aprendizaje, muchos hospitales han acumulado práctica y experiencia, con lo que se mejoran las tasas de supervivencia. actualmente, el trasplante de pulmón se considera un tratamiento válido para un buen número de pacientes con insuficiencia respiratoria grave [1] [2] [3] [4] [5] [6] . actualmente, el trasplante de pulmón se considera un tratamiento válido para un buen número de pacientes con insuficiencia respiratoria grave. de todas formas, las complicaciones son muy frecuentes y pueden llevar a fracaso del injerto a medio y largo plazo y menor supervivencia. de acuerdo con el registro de la international society for heart and lung transplantation, las tasas de supervivencia al primer, segundo y quinto año fueron, en 2006, del 74, 65 y 47%, respectivamente. el principal obstáculo para el éxito a largo plazo del trasplante de pulmón es el rechazo crónico, caracterizado histológicamente como bronquiolitis obliterante, que acontece en cerca de dos terceras partes de los pacientes. uno de los factores más importantes para el desarrollo de bronquiolitis obliterante, además del número de rechazos agudos, es la infección y la enfermedad por citomegalovirus (cmv). recientemente, se ha destacado el papel de la infección por diferentes virus respiratorios como factores de riesgo para el desarrollo de rechazo crónico en receptores de un trasplante de pulmón. las complicaciones infecciosas son una causa frecuente de morbimortalidad en este tipo de pacientes, y la causa de muerte de cerca de la mitad de ellos. la infección bacteriana es la complicación más frecuente de un receptor de un trasplante de pulmón. del total, el 35-66% son bacterianas y el 50-85% de los pacientes presentan como mínimo un episodio de infección bacteriana. la segunda causa más frecuente de infección, después de la bacteriana, es la infección por cmv. a pesar de utilizar diferentes estrategias de prevención, la incidencia sigue siendo elevada, y se sitúa alrededor del 7% el primer año postrasplante. es el único tipo de trasplante de órgano sólido en el cual la etiología más frecuente de la infección fúngica es aspergillus spp., a diferencia del resto, en que típicamente se deben a candida spp. la incidencia de aspergilosis invasora se cifra en alrededor del 4%. de todas formas, las complicaciones son muy frecuentes y pueden llevar a fracaso del injerto a medio y largo plazo y menor supervivencia. de acuerdo con el registro de la international society for heart and lung transplantation, las tasas de supervivencia al primer, segundo y quinto año fueron, en el año 2006, del 74, 65 y 47%, respectivamente 6 . el trasplante de pulmón puede ser unilateral o bilateral. el unilateral se utiliza fundamentalmente para las neumopatías no sépticas, mientras que el bilateral se reserva para las sépticas, tales como la fibrosis quística y las bronquiectasias. las infecciones y el rechazo agudo son las principales complicaciones en el período peritrasplante. el mayor obstáculo para el éxito a largo plazo del trasplante de pulmón es el rechazo crónico caracterizado histológicamente como bronquiolitis obliterante, que acontece en cerca de dos terceras partes de los pacientes [7] [8] [9] [10] . uno de los factores más importantes para el desarrollo de bronquiolitis obliterante, además del número de rechazos agudos [7] [8] [9] , es la infección y la enfermedad por cmv 10, 11 . recientemente, se ha destacado el papel de la infección por diferentes virus respiratorios como uno de los factores de riesgo para el desarrollo de rechazo crónico en receptores de un trasplante de pulmón [12] [13] [14] [15] [16] [17] . las complicaciones infecciosas son una causa frecuente de morbimortalidad en este tipo de pacientes, y suponen la causa de muerte de cerca de la mitad de ellos 6 . en este capítulo nos centraremos en la epidemiología, la clínica, el diagnóstico, el tratamiento y la prevención de las infecciones bacterianas, víricas y fúngicas de los receptores de un trasplante de pulmón. asimismo, comentaremos aspectos específicos relacionados con la infección en el donante y en el pulmón residual. uno de los principales problemas con que nos encontramos es el que supone la ausencia de estudios clínicos aleatorizados que nos ayuden a resolver de forma definitiva muchas de las cuestiones relacionadas con la infección en los receptores de un trasplante de pulmón. el riesgo de infección en un receptor de un trasplante de pulmón está determinado por la interrelación de diferentes grupos de factores, como son los vinculados con el receptor, los del acto quirúrgico, los propios del microorganismo infectante y aquellos propios del estado de inmunosupresión (tabla 1). la situación clínica del paciente, sobre todo si tiene obesidad mórbida o malnutrición, insuficiencia renal, diabetes o está ventilado mecánicamente, influye en el riesgo de presentar una infección postrasplante. los receptores de edad avanzada tienen mayor riesgo de tener una infección después del trasplante. asimismo, aquellos pacientes que antes del trasplante han sido tratados con corticoides o antimicrobianos pueden presentar mayor incidencia de infecciones y éstas ser producidas por microorganismos multirresistentes. la ausencia de inmunidad específica frente a cmv, herpes simple tipo 1, virus de la varicelazóster o virus de epstein-barr (veb) implica que el receptor esté en riesgo de adquirir estas infecciones cuando el donante tenga la infección latente por dichos virus. estas infecciones primarias producen enfermedad con mayor frecuencia y suele ser grave 18 . las localizaciones más frecuentes de infección en el postrasplante inmediato son el pulmón y la cavidad torácica extrapulmonar, pues después del trasplante, la integridad de la pleura visceral no se recupera y el mediastino no existe debido a la comunicación entre los espacios pleurales 18 . en el trasplante de pulmón hay una serie de factores predisponentes específicos para la neumonía bacteriana. la extracción dejándolo isquémico durante horas y reimplantándolo sin restablecer su drenaje linfático ni inervación afecta a sus mecanismos de defensa 18 . la mucosa de la vía aérea se daña y se paraliza el mecanismo de aclaramiento mucociliar. las anastomosis también dificultan el drenaje de las secreciones respiratorias. la denervación gavaldà j elimina o disminuye el reflejo tusígeno, con lo que las secreciones se acumulan en los bronquios segmentarios. la interrupción del drenaje linfático altera la llegada de células efectoras del sistema inmune al injerto [19] [20] [21] . además, este último tiene un microambiente de antígeno linfocitario de histocompatibilidad hla incompatible entre los macrófagos alveolares del huésped y los linfocitos alveolares del donante 22 . inóculos muy pequeños de microorganismos en el injerto pueden producir neumonía después del trasplante 18 . finalmente, el pulmón es un órgano en contacto constante con el exterior y, por ello, con microorganismos aéreos ubicuos. la condición más importante que predispone a la infección postrasplante es el rechazo crónico del injerto o bronquiolitis obliterante. en estos pacientes existe un alto grado de fibrosis y bronquiectasias. por otra parte la causa más frecuente de muerte en los pacientes con bronquiolitis obliterante es la infección 18 . por lo general, los receptores de un trasplante de pulmón reciben triple terapia inmunosupresora con ciclosporina o tacrolimus, más esteroides y, en la actualidad, micofenolato. algunos grupos emplean terapia de inducción y, en algunas ocasiones, se utiliza rapamicina o sirulimus como rescate. no hay ningún estudio que haya podido determinar una mayor o menor incidencia de infección con las diferentes pautas de inmunosupresión. la única excepción es el riesgo incrementado de enfermedad por cmv con la utilización de anticuerpos antilinfocitarios. la infección por virus inmunomoduladores, como el cmv, inmunodeprime y es un factor de riesgo para la aparición de infecciones oportunistas 10, 18 . la infección bacteriana es la complicación más frecuente de un receptor de un trasplante de pulmón. la frecuencia es difícil de conocer, ya que las series son antiguas y se remontan al inicio de los programas de trasplante. los pacientes tienen más de un episodio de infección (0,8-1,2 episodios por paciente; 1,5-2,5 episodios por paciente infectado), y la más frecuente es la respiratoria 18 . iniciándose por una colonización persistente, el receptor de un trasplante de pulmón puede presentar todos los tipos de infección respiratoria: traqueobronquitis y neumonía. las infecciones en el postrasplante inmediato están en relación con: a) una colonización pretrasplante del donante (infección del injerto) o del receptor (fibrosis quística); b) complicaciones relacionadas con el acto quirúrgico, y c) la estancia prolongada en el hospital de un paciente sometido a una cirugía mayor (intubación, cateterización, infección nosocomial). las infecciones en el postrasplante tardío se hallan en relación con el grado de bronquiolitis obliterante que presente el paciente y con la repercusión en forma de bronquiectasias y fibrosis en sus injertos. los pacientes sin bronquiolitis obliterante tendrán infecciones parecidas a la población general, aunque su presentación y su evolución pueden ser diferentes debido al tratamiento inmunosupresor. la etiología de la infección bacteriana en los tres primeros meses dependerá de la ecología del centro donde se realiza el trasplante. en este punto merece un comentario el estudio realizado en resitra (red de estudio de infección en el trasplante) que evaluó 85 episodios de neumonía en 236 trasplantados pulmonares (con una incidencia de 72 episodios por 100 pacientes/año) en un período actual (de los años 2003 a 2005). la neumonía bacteriana (82,7%) fue más frecuente que la fúngica (14%) y la vírica (10,4%). la neumonía bacteriana fue causada por bacilos gramnegativos en 34 casos (59,9%) (pseudomonas aeruginosa en 14 y acinetobacter baumannii en 8) y por cocos grampositivos en 8 casos (14%) (en todos el agente causal fue staphylococcus aureus). es de destacar la ausencia de neumonía por legionella pneumophila probablemente por efecto de la profilaxis con cotrimoxazol. el diagnóstico no difiere del de otro tipo de pacientes, aunque de forma característica se ha de realizar la identificación y el antibiograma de todos los aislados procedentes de las muestras respiratorias. otro factor muy importante es determinar si la anastomosis ofrece signos de isquemia. esto implicaría riesgo de infección, dehiscencia de la sutura y la necesidad de nebulización de antibióticos para tratar una colonización o infección. la mediastinitis es una rara complicación en los receptores de un trasplante de pulmón. más que mediastinitis hemos de hablar de toraquitis, puesto que el mediastino, como ya hemos comentado anteriormente, no existe como tal. es una complicación muy grave que requiere drenajes quirúrgicos. la infección por mycobacterium tuberculosis ha sido publicada de forma ocasional y puede ser debida a reactivación, a enfermedad oculta en el pulmón residual o a transmisión por el injerto [23] [24] [25] . en nuestro programa de trasplante fue diagnosticada en 10 de los 186 receptores (5,4%) que fueron trasplantados de agosto de 1990 a mayo de 2001. la media de tiempo postrasplante fue de 115 días (intervalos entre 1 y 381 días). en el 40% de los casos, el diagnóstico se realizó en los pulmones explantados. a pesar de la inmunosupresión, se observó una respuesta adecuada sin efectos adversos relacionados con la terapia en la mayoría de los casos 26 . las infecciones por nocardia spp. son raras pero están bien descritas 27, 28 . los pacientes con episodios frecuentes de rechazo agudo e insuficiencia renal presentaron mayor riesgo. una revisión retrospectiva de 540 receptores de pulmón o corazón-pulmón identificó 10 pacientes con infección por nocardia (1,9%), que se diagnosticó tras un período de 13 ± 14,5 meses (media ± desviación estándar [de]) después del trasplante. todos los pacientes tuvieron enfermedad pulmonar sin evidencia de enfermedad extrapulmonar. no contribuyó directamente a la muerte de ningún paciente y había coinfección con otros microorganismos en seis de ellos 27 . aunque los datos de incidencia son parecidos a los de la serie de la universidad de pittsburgh, estos autores describen una mortalidad relacionada del 30% (3 de 10 pacientes). cos, sino también en la experiencia en el cuidado de estos pacientes. cuando se aísla un microorganismo en las secreciones respiratorias se inicia tratamiento antibiótico, aun estando el paciente asintomático, añadiendo un aminoglucósido o colistina nebulizada en caso de que la anastomosis esté isquémica. las únicas situaciones en que no está indicado el tratamiento son la colonización por estreptococos del grupo viridans o staphylococcus epidermidis. consideramos que la neumonía por p. aeruginosa ha de tratarse con la combinación de un betalactámico y un aminoglucósido. la traqueobronquitis por este microorganismo también es tratada con biterapia, pero en este caso utilizamos tobramicina nebulizada en dosis de 100 mg/12 h. las indicaciones de colistina o aminoglucósidos nebulizados en nuestro hospital son la detección de microorganismos multirresistentes (acinetobacter baumannii, pseudomonas spp., stenotrophomonas maltophilia y s. aureus resistente a cloxacilina [sarc]), la traqueobronquitis recidivante en pacientes con fibrosis quística (p. aeruginosa, s. maltophilia) y la traqueobronquitis si la sutura está isquémica. del segundo al sexto mes sólo tratamos aquellos episodios de infección, mientras que los episodios de colonización son tratados si se evidencia el mismo microorganismo en dos muestras respiratorias separadas por una semana. el tratamiento de la tuberculosis tiene unas características peculiares en el trasplante de órgano sólido. la rifampicina no debería ser utilizada por su efecto estimulador del metabolismo del citocromo p-450, lo que hace muy complicado mantener las concentraciones plasmáticas de ciclosporina o tacrolimus, y esto puede suponer un rechazo persistente y pérdida del injerto 29 . nuestra recomendación es isoniacida, etambutol, piracinamida y ofloxacino los primeros tres meses; posteriormente se retira la piracinamida y el resto se mantiene entre 18 y 24 meses. ninguno de los pacientes de nuestro programa que recibieron esta pauta falleció en relación con la tuberculosis. todos los candidatos con prueba de intradermorreacción de mantoux positiva o anérgicos han de tomar 12 meses de profilaxis con isoniacida, después de descartar una infección tuberculosa activa. ha de realizarse un estudio microbiológico e histológico de los pulmones extraídos para descartar tuberculosis activa. el tratamiento de la neumonía por legionella pneumophila también es complicado, ya que los macrólidos inhiben el metabolismo del citocromo p-450, y dado que la eficacia de levofloxacina es similar, nosotros utilizamos este fármaco en una dosis de 500 mg/12 h para el tratamiento de esta infección. de entre los trasplantados de órgano sólido, el de pulmón es el que tiene una mayor incidencia de infección fúngica invasora. es el único tipo en que la etiología más frecuente es aspergillus spp., a diferencia del resto, en que típicamente este tipo de infecciones son debidas a candida spp. la infección por aspergillus spp. en el receptor de un trasplante de pulmón se manifiesta en forma de una simple colonización hasta una enfermedad invasora. una forma muy característica de estos pacientes es la traqueobronquitis, que puede ser simple, ulcerativa, seudomembranosa o nodular. como sucede en la infección bacteriana, es di-fícil conocer la incidencia real de la infección fúngica invasora en estos pacientes, ya que las series son muy antiguas y en la mayoría de ellas se mezclan pacientes con colonización, traqueobronquitis y enfermedad invasora. en la re-sitra, que siguió en nuestro país de forma prospectiva una cohorte de 155 trasplantados de pulmón desde 2003 hasta 2005, la incidencia de enfermedad invasora con profilaxis fue del 3,9%. es difícil conocer la incidencia sin profilaxis aunque puede variar entre el 13 y el 26% [30] [31] [32] . la mortalidad de las formas invasoras es muy elevada; se cifra entre el 70 y el 100% [30] [31] [32] . la incidencia de infección fúngica invasora y de traqueobronquitis ulcerativa en 104 trasplantados de pulmón de nuestro centro que utilizaron anfotericina b liposomal nebulizada como profilaxis fue del 2% para ambas manifestaciones de la aspergilosis. los factores de riesgo que se han demostrado para la aspergilosis invasora son la colonización previa por aspergillus spp., la neumonitis por cmv, la isquemia de las vías aéreas, la colonización durante los primeros seis meses postrasplante y el trasplante unipulmonar [30] [31] [32] . no se ha podido demostrar una relación con un aumento de la inmunosupresión, aunque obviamente no se puede descartar. los pacientes con bronquiolitis obliterante también tienen un riesgo aumentado. no se considera contraindicación para indicar un trasplante el aislamiento de ninguna especie de hongo pretrasplante, pero es obligado un trasplante bipulmonar y la realización de un escáner torácico para descartar la posibilidad de adherencias de micetomas a la pared torácica. la traqueobronquitis es una forma característica de aspergilosis en los receptores de un trasplante de pulmón y es casi exclusiva de estos pacientes 33 . esta enfermedad puede ir desde una simple bronquitis hasta una forma nodular, seudomembranosa o ulcerativa. frecuentemente, se afecta la anastomosis y en los casos más graves puede llevar a dehiscencia de la sutura, hemorragia o enfermedad diseminada. cuando se aísla aspergillus spp. de las secreciones respiratorias en los primeros 6-9 meses después del trasplante, siempre se realiza una broncoscopia a fin de descartar una traqueobonquitis ulcerativa o seudomembranosa. la aspergilosis invasora es parecida a la que acontece en otros tipos de trasplante de órgano sólido. la única forma diferente es la del pulmón nativo o residual de los receptores de un trasplante unipulmonar. puede aparecer inmediatamente después del trasplante porque no se haya detectado en la evaluación, o puede ser una infección de novo [30] [31] [32] . es muy compleja de diagnosticar, ya que el pulmón residual está desestructurado y es muy difícil ver cambios. hay que sospecharla en trasplantados unipulmonares que no responden al tratamiento de una supuesta traqueobronquitis aspergilar. responden muy mal al tratamiento, y la única posibilidad de curación, si es posible, sería la neumectomía. la mayoría de infecciones por candida spp. acontecen en los primeros dos meses postrasplante, están relacionadas con un ingreso prolongado en unidad de cuidados intensivos (uci) y las más frecuentes son las candidemias 18 . la incidencia de neumonía por pneumocystis jirovecii en las primeras series sin profilaxis era superior al 80% 18 . dado que los pacientes son tratados toda la vida con esteroides, nuestro grupo recomienda mantener la profilaxis con cotrimoxazol. la tabla 2 resume las estrategias terapéuticas frente a la infección aspergilar en el trasplante de pulmón. para intentar disminuir la elevada mortalidad relacionada con la aspergilosis invasora, tenemos que apoyarnos en tres puntos: un diagnóstico lo más precoz posible, un tratamiento antifúngico en dosis plenas y la posibilidad de disminuir la inmunosupresión, o bien intentar aumentar la respuesta inmunológica del huésped. el diagnóstico precoz y el inicio inmediato del tratamiento deberían reducir la mortalidad relacionada con esta infección. el problema reside en que, en la actualidad, no están a nuestra disposición técnicas que permitan esta aproximación. aunque el hallazgo de aspergillus spp. en cultivo es una indicación para el inicio del tratamiento, en algunos pacientes el primer cultivo positivo se obtiene en la necropsia, y en los receptores de un trasplante de pulmón puede ser simplemente una colonización. la utilización de técnicas como la reacción en cadena de la polimerasa o la detección de antígeno han demostrado su eficacia en el paciente neutropénico, pero su valor en el receptor de un órgano sólido no está establecido. en el caso de la aspergilosis, el escáner torácico es un método diagnóstico muy válido, incluso antes del inicio de los síntomas. la tomografía computarizada (tc) helicoidal o la de alta resolución pueden aumentar la sensibilidad de la tc clásica. los hallazgos de la tc torácica in-cluyen, de forma inicial, el signo del halo (un área de baja atenuación alrededor de una lesión nodular debida al edema o sangrado que rodea un área isquémica) y, tardíamente, una zona de aire creciente alrededor de un nódulo pulmonar causado por la contracción de un tejido infártico. las alteraciones de la tc preceden, por lo general, a las alteraciones de la radiografía de tórax, por lo que la tc torácica debería ser considerada en todo paciente con sospecha de infección. la presencia de signos sugestivos de aspergilosis invasora nos obliga a aplicar técnicas diagnósticas invasivas para intentar su diagnóstico y, probablemente, plantearnos el inicio del tratamiento en espera de resultados. la duración óptima del tratamiento es desconocida y depende de la extensión de la enfermedad, la respuesta al tratamiento y el estado inmunitario del paciente. una aproximación razonable sería continuar el tratamiento con el fin de tratar microfocos después de que los signos clínicos y radiológicos hayan desaparecido, los cultivos sean negativos y el estado inmunitario sea lo más correcto posible. la duración del tratamiento debería estar guiada por la respuesta clínica, más que por una dosis total arbitraria. la respuesta final de estos pacientes al tratamiento antifúngico está relacionada con factores del huésped, como pueden ser la disminución de la inmunosupresión y la recuperación de la función del injerto. una secuencia lógica de tratamiento sería la utilización primegavaldà j los pacientes colonizados han de ser tratados con anfotericina b nebulizada o itraconazol de forma anticipada para prevenir el desarrollo de enfermedad invasora. el tratamiento de la traqueobronquitis con anfotericina b liposomal o itraconazol o voriconazol asociado o no a anfotericina b nebulizada es eficaz en la mayoría de las ocasiones, y se utilizará uno u otro tratamiento en función del tipo de traqueobronquitis y de si está o no afectada la sutura 18 . en caso de dehiscencia se requiere la resección quirúrgica y la colocación de un stent, aunque los resultados no son excesivamente positivos. la cirugía ha de indicarse en los casos de hemoptisis, cuando sea masiva o secundaria a una lesión que se localice cerca de los grandes vasos, en la enfermedad sinusal, en la progresión de una lesión pulmonar cavitada y única a pesar del tratamiento antifúngico apropiado y en la infiltración del pericardio, grandes vasos, hueso o del tejido subcutáneo torácico mientras se está recibiendo tratamiento. como ya hemos indicado, hay algún caso descrito de curación de una aspergilosis invasora del pulmón nativo mediante la práctica de una neumectomía. probablemente, la única posibilidad de curación de la aspergilosis cerebral es la resección quirúrgica amplia 18 . es importante la reducción de la inmunosupresión como coadyuvante al tratamiento antifúngico pero sin poner en riesgo la viabilidad del injerto. el trasplante de pulmón es el tipo de trasplante de órgano sólido en el que se ha de tener más en cuenta la in-fección en el donante. en un estudio realizado en el hospital vall d'hebron 34 se diagnosticó una infección en 103 de los 197 donantes de un trasplante de pulmón (52%). los tipos de infección del donante fueron contaminación del líquido de preservación en 30 ocasiones (29,1%), colonización del injerto en 65 (63,1%) y bacteriemia en 8 (8%). es de resaltar que la etiología de la colonización del injerto fue s. aureus en 26 ocasiones (40%) y p. aeruginosa en 11 (17%). esto obliga a tener en cuenta estos dos microorganismos en la profilaxis inicial del trasplante de pulmón. los pacientes que llevaban ventilados más de 48 h tenían una incidencia de infección mayor que los que llevaban ventilados menos de 48 h. se transmitió la infección a 15 receptores (7,6%). excluyendo cinco casos en los cuales era imposible realizar una profilaxis efectiva (aspergillus fumigatus, s. maltophilia y sarc), se produjo un fallo en la profilaxis en 11 de 197 trasplantes (5,6%). estos resultados son parecidos a otros publicados en la literatura médica, pero con casuística mucho menor. estos datos nos indican que es imprescindible realizar cultivos de lavado bronquioalveolar, broncoaspirado, líquido de preservación y hemocultivos para adecuar la profilaxis y tratar una posible infección en el receptor. se han de realizar tinciones de gram, plata o calcofluor y ziehl-nielsen, además de cultivos específicos para bacterias, hongos y micobacterias. los resultados tienen que estar disponibles lo antes posible para adecuar la pauta antibiótica. el hallazgo de secreciones con una tinción de gram que sea positiva no es contraindicación para aceptar un pulmón para trasplante. la existencia de neumonía, aspiración de jugo gástrico, crecimiento de hongos filamentosos o abundantes secreciones después de la aspiración mediante el fibrobroncoscopio sí se consideran contraindicaciones para el trasplante por la mayoría de grupos. en el caso del trasplante unipulmonar es importante efectuar un análisis patológico exhaustivo para descartar infecciones que hubieran pasado inadvertidas en la evaluación del donante. la infección crónica del tracto respiratorio antes del trasplante distingue a los pacientes con fibrosis quística del resto de pacientes sometidos a un trasplante de pulmón por otras causas y, con ello, la preocupación por un incremento en el riesgo de infección postrasplante. diferentes estudios 35, 36 han demostrado que este riesgo es similar. de todas formas, se han demostrado las mismas cepas (p. ej., p. aeruginosa) mediante electroforesis de campo pulsátil o análisis de adn que infectaban antes del trasplante, probablemente debido a contaminación durante el acto quirúrgico o por la sinusitis crónica de estos pacientes. aunque ciertos grupos recomiendan el drenaje pretrasplante o postrasplante de los senos paranasales, su eficacia no ha podido ser demostrada. hasta hace poco, algunos programas de trasplante rechazaban posibles receptores portadores de patógenos respiratorios como p. aeruginosa multirresistente u otros bacilos gramnegativos multirresistentes como burkholderia cepacia, s. maltophilia, o alcaligenes xylosoxidans, pero datos recientes han demostrado que la supervivencia postrasplante es similar a la de los pacientes que no son portadores de estos microorganismos 18 . en ningún programa de trasplante del estado español existe contraindicación por el hecho de que un paciente sea portador de un determinado microorganismo. la profilaxis antibiótica en los receptores de un trasplante de pulmón ha de ser de amplio espectro y cubrir fundamentalmente p. aeruginosa y s. aureus por las consideraciones que ya hemos expuesto anteriormente, además de las propias de la cirugía mayor del trasplante, excepto en los receptores de una neumopatía séptica (fibrosis quística o bronquiectasias), en los que se realizará una profilaxis dirigida en función de los cultivos pretrasplante. para la profilaxis inicial, nuestro grupo utiliza amoxicilina-ácido clavulánico 2 g más ceftacidima 2 g cada 3 h durante la cirugía y después de cada 8 h. para los pacientes con neumopatía séptica, adecuamos el cultivo previo y añadimos una penicilina antiestafilocócica más tobramicina instilada o nebulizada empezando en el momento en que el paciente llega a la uci. la duración de la profilaxis dependerá de los cultivos del donante y del receptor. si los cultivos son negativos, se retiran al tercero o quinto día y si son positivos, se adecuan a ellos y se mantienen durante 15 días o hasta que sean negativos. el primer punto que ha de tenerse en cuenta es que la duración del riesgo es muy prolongada, como mínimo un año, y es mayor si el paciente sufre bronquiolitis obliterante. esto hace que la administración parenteral sea inviable. por tanto, en este momento existen varias alternativas, como los triazoles, en concreto itraconazol, voriconazol y posaconazol, y la anfotericina b nebulizada. el grupo de pittsburg presentó recientemente su experiencia con voriconazol como profilaxis en 65 pacientes con una incidencia del 1,5% de aspergilosis invasora, comparándola al 23% de un grupo al que se le administró itraconazol, con o sin anfotericina b nebulizada en el momento del primer cultivo positivo. el problema del grupo de voriconazol fue que presentó hepatotoxicidad cerca del 40% de los pacientes 37 . el papel de posaconazol no ha sido estudiado pero los resultados en pacientes con leucemia o trasplante de médula ósea hacen que puedan ser prometedores en el receptor de un trasplante de pulmón 38 . las concentraciones en la vía aérea con 24 mg de anfotericina b liposomal son adecuadas para la protección frente a la infección por aspergillus spp. durante un período de 15 días. esta conclusión se basa en un estudio farmacocinético efectuado por nuestro grupo y todavía no publicado. en este estudio, las concentraciones medias de 12 muestras broncoscópicas a los 14 días después de la inhalación de 24 mg de anfotericina b liposomal fue de 4,13 g/ml. además, se realizó un ensayo clínico en que se incluyó un total de 104 pacientes, en el cual se diagnosticaron dos episodios infección fúngica invasora (1,9%). a raíz de este ensayo clínico, en nuestro centro se sigue la siguiente pauta de profilaxis antifúngica en los receptores de un trasplante de pulmón: 25 mg de anfotericina b liposomal nebulizada: a) los dos primeros meses o hasta que la sutura esté completamente curada, tres veces por semana; b) del segundo al sexto mes, una vez por semana, y c) a partir del sexto mes, una vez cada 15 días. en caso de que se produzca tos o broncoespasmo, se controla con salbutamol. en los casos de intolerancia a la nebulización o detección de especies resistentes a anfotericina b se administra voriconazol. aunque puede administrarse itraconazol, ya que su espectro de acción cubre aspergillus spp., su mala tolerancia digestiva incapacita su empleo en un buen número de pacientes. la segunda causa más frecuente de infección, después de la bacteriana, es la infección por cmv. la incidencia sin profilaxis según la literatura médica ronda el 50%, una incidencia mucho más alta que en otro tipo de trasplantes. este porcentaje puede acercarse al 75% de pacientes seronegativos que reciben un órgano seropositivo [39] [40] [41] . como veremos más adelante, a pesar de utilizar diferentes estrategias de prevención, la incidencia continúa siendo todavía elevada, y se sitúa alrededor del 7% el primer año postrasplante. los factores de riesgo relacionados con la enfermedad por cmv son parecidos a otros tipos de trasplantes. así, se han incriminado la utilización de anticuerpos antilinfocitarios, el receptor seronegativo para cmv que recibe un pulmón de un donante seropositivo, la replicación del virus herpes 6 y una carga viral de cmv elevada estando el paciente asintomático 18 . con la utilización de la profilaxis, la forma de enfermedad más frecuente es el síndrome viral. la neumonitis por cmv tiene un comienzo insidioso y se manifiesta de forma inicial como un síndrome constitucional y fiebre. posteriormente, puede presentar taquipnea y disnea. la hipoxemia está siempre presente. en la exploración física sólo se hallará taquipnea. ante un deterioro de la función respiratoria durante el tratamiento de una neumonitis por cmv se ha de descartar primero rechazo agudo y superinfección por bacilos gramnegativos u hongos antes que mala evolución de la neumonitis por cmv. las manifestaciones radiológicas son diversas, y lo más frecuente son infiltrados intersticiales y/o alveolares bilaterales en ambas bases. los episodios más graves acontecen en los receptores seronegativos de donantes seropositivos (d+/r-). característicamente, en las pruebas de función respiratoria las de difusión pueden ser las únicas que se alteren 18 . el diagnóstico de la infección y de la enfermedad por cmv está establecido internacionalmente a partir de un artículo de ljungman, griffiths y paya 42 y, posteriormente, a partir de un documento de consenso del grupo de estudio de la infección en el trasplante de la seimc 43 . para el tratamiento de la enfermedad por cmv se utiliza ganciclovir o foscarnet asociado o no a gammaglobulina hiperinmune. el tratamiento es, esencialmente, el mismo que en otros tipos de trasplante de órgano sólido. la utilización de gammaglubulina es controvertida y, en todo caso, su evidencia científica, si la tiene, viene derivada a partir de los estudios en trasplantados de médula ósea. algunos autores recomiendan su uso en pacientes con neumonitis, afectación gastrointestinal, leucopenia grave y recaídas. la infección por cepas resistentes a ganciclovir es un problema emergente en la población trasplantada. un estudio publicado en 2002 describió una incidencia cerca-na al 10% de infección por cepas de cmv resistentes a ganciclovir, definidas por mutación del gen ul97, que era más frecuente en la población d+/r44 . de acuerdo con nuestro protocolo, cuando diagnosticamos una enfermedad por cmv iniciamos ganciclovir i.v. en dosis de 5 mg/kg/12 h. por lo general, no se disminuye las dosis de tacrolimus o corticoides excepto en los casos de neumonitis. la azatioprina o el micofenonelato se retiran. en los pacientes con neumonitis se asocia gammaglobulina hiperinmune en dosis de 200 mg/kg cada 48 h durante la primera semana de tratamiento. se monitoriza la carga viral y si se observa un incremento significativo al final de la primera semana ha de sospecharse una infección por una cepa resistente a ganciclovir. un incremento de la antigenemia al segundo o tercer día de tratamiento no es un hecho infrecuente. cuando se utiliza ganciclovir se recomienda controlar, al menos semanalmente, el número de neutrófilos, que ha de mantenerse por encima de 1.000/l. cuando los neutrófilos descienden entre 500-1.000/l se puede intentar mantener el ganciclovir, administrando factor estimulante de colonias de granulocitos (g-csf), y cambiarlo sólo si la cifra de neutrófilos no se incrementa o se estabiliza en 24 h. puede estar indicado el tratamiento con foscarnet, controlando convenientemente la función renal cuando se tenga que suspender el ganciclovir por toxicidad, o en los casos de sospecha fundada de resistencia a ganciclovir. la duración del tratamiento ha de ser de 15 días, excepto para los casos de neumonitis, en que se prolonga hasta tres semanas. por lo general, la antigenemia cuantitativa es negativa al final del tratamiento, pero la cuantificación del adn puede ser positiva, por lo que no serviría para la monitorización. cuando el paciente tiene una infección asintomática se inicia tratamiento anticipado (véase más adelante). una alternativa al tratamiento con ganciclovir endovenoso es valganciclovir, cuya excelente farmacocinética, con una biodisponibilad por vía oral que le permite conseguir concentraciones séricas similares a 5 mg/kg i.v. tras una dosis de 900 mg, permitiría el tratamiento ambulatorio. nosotros nos planteamos utilizar valganciclovir en el tratamiento anticipado de la infección asintomática y en el caso del síndrome viral, siempre que el estado clínico del paciente lo permita y se haya descartado una neumonitis subclínica. el pronóstico del receptor de un trasplante de pulmón con neumonitis es excelente y la mortalidad relacionada, prácticamente nula [39] [40] [41] . de todas formas, puede darse cerca de un 15-20% de recaídas. este hecho es más frecuente en receptores seronegativos y si la antigenemia persiste positiva al final del tratamiento. además de producir la enfermedad, el cmv puede producir una serie de efectos indirectos que tienen relación con situaciones de inmunomodulación. por un lado, inmudeprime al paciente y lo hace más susceptible a infecciones oportunistas y, por otro, le hace incrementar el riesgo de presentar un rechazo crónico del injerto 18 . linda sharples et al 45 realizaron una revisión excelente sobre los factores de riesgo de bronquiolitis obliterante en los receptores de un trasplante de pulmón. utilizando el parámetro más objetivo, como puede ser la neumonitis, existen ocho estudios en siete centros con 543 pacientes que relacionan la neumonitis por cmv con la bronquiolitis obliterante. por otra parte, estudios de tres centros con 154 casos en 302 tras-plantados no hallan esta correlación. tres estudios parecen describir una caída en la incidencia de bronquiolitis obliterante después del inicio de la profilaxis con ganciclovir 45 . aunque falta conocer la respuesta definitiva a esta pregunta y, sobre todo, saber si la profilaxis con ganciclovir puede disminuir la incidencia de bronquiolitis obliterante, sí parece que hay una asociación entre cmv y rechazo crónico en el trasplante de pulmón. recientemente, se ha descrito una correlación entre la infección ocasionada por diferentes virus respiratorios y el desarrollo posterior de bronquiolitis obliterante, es decir, de rechazo crónico del injerto [12] [13] [14] [15] [16] [17] . la incidencia de neumonitis por virus herpes simple tipo 1 se describió al inicio de los programas de trasplante de pulmón cuando no se utilizaba profilaxis con ganciclovir y su incidencia se cifró entre el 5 y el 10% 18, 46 . se asociaba frecuentemente a neumonía bacteriana o neumonitis por cmv. es una entidad grave que causa infiltrados alveolares bilaterales e insuficiencia respiratoria. la mortalidad se cifraba próxima al 20%. la incidencia de enfermedad linfoproliferativa tras el trasplante 47 , relacionada con el veb, en receptores de un injerto pulmonar varía según las series entre el 1,6 y el 20%, y es de 2 a 6 veces superior a otros tipos de trasplante de órgano sólido. por otra parte, aparece de forma más precoz y tiene un curso más agresivo. estas diferencias en la incidencia pueden ser debidas a distintas pautas de inmunosupresión, al número de receptores seronegativos para veb y al porcentaje de pacientes pediátricos de las diferentes series. el riesgo es mayor en los receptores seronegativos para el veb que reciben un injerto de un donante seropositivo. aunque no de una forma concluyente, hay indicios que demuestran que otros virus de la familia de los herpesviridae podrían incrementar la actividad del veb 18 . existen pocos estudios en la literatura médica que evalúen la incidencia de los virus diferentes al herpes que puedan causar infecciones respiratorias, y la mayoría de ellos están enfocados en buscar la relación entre ellos y el rechazo crónico del injerto. kumar et al 48 , de un total de 50 infecciones respiratorias, diagnosticaron 33 (66%) cuya etiología fue: rinovirus 9; coronavirus 8; virus respiratorio sincitial (vrs) 6; virus de la influenza a 5; virus de la parainfluenza 4, y metapneumovirus 1. la clínica inicial de todos los pacientes fue la de una infección del tracto respiratorio superior. cuatro (8%) casos evolucionaron a neumonía. milstone et al 49 estudiaron la frecuencia y las complicaciones de las infecciones respiratorias virales de 50 trasplantados pulmonares durante una temporada invernal. en 32 pacientes (64%) se produjeron 49 episodios sintomáticos. la incidencia de neumonitis por adenovirus es muy baja; se cifra en alrededor del 1% de la población adulta trasplantada de pulmón 18 . aparece en los primeros tres meses después del trasplante y tiene un curso muy agresivo, con necesidad de ventilación mecánica y una elevada mortalidad. por contra, la incidencia en la población infantil es más elevada y el curso clínico también es muy agresivo. es común el desarrollo de bronquiolitis obliterante en los supervivientes. la infección por virus de la familia de los paramixoviridae no es infrecuente, pero hay muy poca información en la literatura médica. wendt et al 50 informan de 19 episo-dios en 18 pacientes de un total de 84 receptores de un trasplante de pulmón, lo que da una incidencia del 22%. la infección por vrs tiene predominio estacional (enerojunio) y la producida por virus de la parainfluenza anual. dos pacientes presentaron neumonía y necesitaron ventilación mecánica. se administró ribavirina en aerosol en 14 ocasiones sin efectos adversos. un paciente falleció y dos presentaron una reducción permanente de la función respiratoria. en la literatura no hay referencias a la epidemiología y la clínica de la infección por vrs en trasplantados de pulmón. existen dos estudios 51,52 que evalúan la eficacia y seguridad de la ribavirina en aerosol en infecciones por vrs en trasplante de pulmón con resultados excelentes 50, 51 . la incidencia de la infección por el virus de la parainfluenza es muy variable, entre el 10 y el 66%. la mayoría de episodios aparece después de un año del trasplante. todos los pacientes inician el cuadro con una clínica de vías respiratorias altas, y un porcentaje no despreciable se complica con clínica de tracto respiratorio inferior y un 16% con neumonía. aun así, el pronóstico es bueno. si se realiza biopsia transbronquial por alteración de las pruebas funcionales respiratorias es frecuente hallar rechazo agudo concomitante. existen estudios que han demostrado que hasta el 32% de los pacientes han desarrollado bronquiolitis obliterante, con una media de seis meses (intervalo 1-14) después de la infección por este virus. la incidencia de infección por rinovirus es menor al 5%, en la mayoría de los casos es asintomática o cursa con sintomatología de vías respiratorias superiores. también se ha descrito su asociación con bronquiolitis obliterante 18 . hay dos estrategias para la prevención de la infección por cmv en el trasplante de órgano sólido y en el de pulmón en particular: la profilaxis y el tratamiento anticipado. la profilaxis supone administrar un antiviral con o sin gammaglobulina hiperinmune a todos los pacientes trasplantados. el tratamiento anticipado significa administrar antivirales a aquellos pacientes que están en riesgo porque tienen una carga viral significativa o se les ha administrado un determinado tratamiento, como pueden ser los anticuerpos antilinfocitarios. en el trasplante de pulmón han de utilizarse las dos estrategias de prevención en combinación. es difícil realizar recomendaciones en este tipo de trasplante, puesto que la mayoría de estudios son de cohorte y no existen estudios aleatorizados. en general, la profilaxis con ganciclovir intravenoso retrasa la aparición de la enfermedad por cmv pero no la evita, y se observa enfermedad por cmv tardía (hasta 2 años), incluso tras profilaxis mantenidas durante los primeros seis meses postrasplante 53, 54 . la excelente biodisponibilidad de valganciclovir permite obviar la necesidad de utilizar la vía intravenosa durante períodos largos de tiempo, manteniendo concentraciones plasmáticas elevadas después de su administración oral. zamora et al 55 determinaron la seguridad y eficacia de valganciclovir en receptores de un trasplante de pulmón. un total de 90 receptores de un trasplante de pulmón que sobrevivían más de 30 días recibieron gammaglobulina anti-cmv asociada a ganciclovir intravenoso y, cuando se iniciaba la ingesta oral, valganciclovir 450 mg/12 h hasta completar 180, 270 o 365 días, respectivamente, como profilaxis. posteriormente, los pacientes fueron controlados y tratados al diagnosticarse una infección significativa. los resultados se compararon con un grupo histórico (140 pacientes) que recibieron dosis alta de aciclovir después de ganciclovir intravenoso más gammaglobulina anti-cmv. la enfermedad por cmv fue significativamente menor en el grupo con valganciclovir (2,2% frente a 20%; p < 0,01). no se observaron diferencias al prolongar más de 180 días la duración de la profilaxis. nuestro grupo, en colaboración con el grupo de trasplante pulmonar de la separ, realizó un estudio similar. un grupo prospectivo de 76 pacientes recibió ganciclovir i.v. 10 mg/kg/día hasta que toleraban la ingesta oral y después valganciclovir 900 mg/ 24 h hasta completar 120 días. este grupo se comparó con un grupo de 83 controles históricos que recibieron ganciclovir endovenoso durante 21 días seguido de ganciclovir oral en dosis de 1 g/8 h hasta completar 120 días. asimismo, después de la profilaxis se realizaba tratamiento anticipado de todos los episodios de infección por cmv significativa. la incidencia de enfermedad por cmv en el grupo de valganciclovir fue del 7,9%, algo superior a la del estudio de zamora (2,2%), y sin diferencias significativas con el grupo control histórico (16,1%) . en ambos estudios, valganciclovir fue bien tolerado, con una tasa de neutropenia similar a la reportada para ganciclovir. en cuanto a utilizar el tratamiento anticipado como única estrategia de prevención en el trasplante de pulmón, el gran problema radica en que en dicho trasplante el seguimiento ha de realizarse durante un tiempo muy prolongado y algunos pacientes no siguen los controles, de manera que el riesgo para el desarrollo de neumonitis puede ser elevado. por todo ello desaconsejaríamos esta estrategia como la única que aplicar en el trasplante de pulmón. según los datos existentes y nuestra experiencia personal, consideramos que la profilaxis universal seguida del tratamiento anticipado es la mejor estrategia para la prevención de la enfermedad por cmv en el trasplantado de pulmón. recomendamos ganciclovir intravenoso en dosis de 5 mg/kg/12 h, hasta que se tolere la vía oral y después, un cambio a valganciclovir en dosis de 450/12 h, hasta el tercer mes después del trasplante. en los pacientes de alto riesgo (d+/r-) debe administrarse gammaglobulina intravenosa, siempre en asociación con ganciclovir. a partir de este momento iniciamos monitorización con antigenemia y tratamiento anticipado con ganciclovir intravenoso (5 mg/kg/12 h) o valganciclovir (900 mg/12 h) en caso de infección clínica significativa. la monitorización debería ser semanal al menos hasta el sexto mes, y posteriormente, en cada visita médica hasta el segundo año postrasplante. el tratamiento anticipado se inicia en las siguientes situaciones: 1) ante cualquier evidencia de replicación en pacientes de alto riesgo (d+/r-), y 2) en los casos de receptor seropositivo para cmv en circunstancias de antigenemia elevada (variable en función de la técnica y del laboratorio), de aumento progresivo de sus valores o de positividad asociada a tratamiento de rechazo. el futuro de la infección por cmv como la de otros herpesviridae como el veb pasa por la vacunación de los receptores seronegativos y, sobre todo, por la utilización de inmunoterapia adaptativa. algunos grupos europeos han reportado su experiencia en trasplante de órgano sólido con resultados muy esperanzadores 56 . los artículos publicados en la sección "formación médica continuada" forman parte de grupos temáticos específicos (antibiograma, antimicrobianos, etc.). una vez finalizada la publicación de cada tema, se irán presentando al sistema español de acreditación de la formación médica continuada (seaformec) para la obtención de créditos. una vez concedida la acreditación, ésta se anunciará oportunamente en la revista y se abrirá un período de inscripción gratuito para los socios de la seimc y suscriptores de la revista, al cabo del cual se iniciará la evaluación, durante un mes, que se realizará a través de la web de ediciones doyma. a) la lesión de preservación del injerto favorece la infección. b) la enfermedad por citomegalovirus es la infección más frecuente. c) es obligatorio realizar un drenaje quirúrgico de los senos paranasales antes del trasplante en los pacientes afectados de una fibrosis quística. d) la infección por virus respiratorios favorece la progresión a rechazo crónico. e) las respuestas a y b son ciertas. 2. en el trasplante de pulmón, es falso que: a) la infección más frecuente del donante es la colonización del injerto. b) la detección de pseudomonas aeruginosa multirresistente contraindicaría el trasplante en un paciente con fibrosis quística. c) no hay ningún estudio que haya podido determinar una mayor o menor incidencia de infección con las diferentes pautas de inmunosupresión. d) los microorganismos que colonizan con más frecuencia el injerto y que hay que considerar en toda profilaxis antibacteriana son p. aeruginosa y staphylococcus aureus. e) la causa más frecuente de muerte en los pacientes con bronquiolitis obliterante es la infección. 3. con relación a la epidemiología de la infección bacteriana, es falso que: a) la infección más frecuente sea respiratoria. b) el aclaramiento mucociliar es un mecanismo de defensa importante en estos pacientes. c) la etiología más frecuente de las neumonías sea por por bacilos gramnegativos. d) la incidencia de infección por legionella pneumophilla sea muy baja. e) la bronquiolitis obliterante es un factor de riesgo de infeccion bacteriana. a) el tratamiento de la tuberculosis debería ser isoniacida, etambutol, piracinamida y ofloxacino los primeros tres meses; posteriormente se retira la piracinamida y el resto se mantiene entre 18 y 24 meses. b) en caso de que la sutura esté isquémica y se aísle un microorganismo significativo debe asociarse al tratamiento colistina o tobramicina nebulizada. c) deban tratarse las colonizaciones por p. aeruginosa que acontecen dentro de los tres primeros meses postrasplante. d) los macrólidos son los fármacos de elección de la neumonía atípica. e) los aminoglucósidos sistémicos deben evitarse por su riesgo de nefrotoxicidad sinérgica con los inmunosupresores anticalcineurínicos. a) debemos diferenciar los receptores portadores de una neumopatía séptica (bronquiectasias, fibrosis quística) de los no séptica. b) la pauta inicial de los portadores de una neumopatía séptica debe adecuarse a los cultivos previos. c) inicialmente, debe tenerse en cuenta la posible infección del donante cubriendo las especies más frecuentes, es decir, s. aureus y p. aeuruginosa. d) la duración de la profilaxis en los receptores de una neuropatía no séptica será de 3-5 días si los cultivos del donante son negativos. e) todas las respuestas son ciertas. 6. respecto a la infección fúngica a los portadores de un trasplante de pulmón, es falso que: a) el galactomanano no es útil en el diagnóstico de una aspergilosis invasora en estos pacientes. b) la traqueobronquitis ulcerativa es una forma característica de enfermedad. c) la incidencia de aspergilosis invasora con profilaxis se cifra en alrededor del 4%. d) recientemente se ha demostrado que el posaconazol puede ser el fármaco de elección para la prevención de la infección fúngica invasora en estos pacientes. e) el único tipo en la etiología más frecuente es aspergillus spp., a diferencia del resto, en que típicamente este tipo de infecciones son debidas a candida spp. 7. en el tratamiento de la aspergilosis invasora, es falso que: a) la presencia de signos sugestivos de aspergilosis invasora en la tomografía computarizada helicoidal de tórax nos obliga a realizar técnicas diagnósticas invasivas para intentar su diagnóstico y, probablemente, plantearnos el inicio del tratamiento en espera de resultados, incluso en ausencia de clínica. b) una secuencia lógica de tratamiento sería aplicar primero una terapia intravenosa con voriconazol o anfotericina b liposomal hasta la desaparición de los signos radiológicos, o como mínimo hasta detener la progresión de la enfermedad, y, posteriormente, continuar con voriconazol o itraconazol oral de 12 a 18 meses. c) un estudio reciente demuestra que el tratamiento de elección de la aspergilosis invasora es la combinación de voriconazol y caspofungina. d) la traqueobronquitis ulcerativa puede ser tratada incrementando el intervalo de dosificación de la anfotericina b liposomal nebulizada a tres veces por semana. e) el fracaso terapéutico puede ser tratado con posaconazol. a) actualmente el factor de riesgo más importante para que un trasplantado tenga una enfermedad por cmv es que sea cmv negativo y reciba un pulmón de un donante cmv positivo. b) la incidencia al primer año postrasplante es menor del 10%. c) debamos tratar con ganciclovir siempre que se evidencia replicación. d) la forma de enfermedad más frecuente es el síndrome viral. e) la alteración más común en las pruebas de función respiratoria en el contexto de una neumonitis es una alteración de la difusión. 9. respecto a la profilaxis de la enfermedad por cmv, es falso que: a) la mejor estrategia de prevención es el tratamiento anticipado. b) ganciclovir i.v. es la mejor estrategia de prevención, hasta que el paciente tolere la ingesta oral, seguir con valganciclovir hasta el día 120 y posteriormente, tratamiento anticipado. c) en los receptores seronegativos que reciben un órgano de un donante seropositivo realizaremos tratamiento anticipado ante cualquier evidencia de replicación. d) el tratamiento anticipado nos obliga a hacer monitorización mediante antigenemia cuantitativa en cada visita médica hasta un año postrasplante. e) en caso de intolerancia a ganciclovir i.v. se puede administrar valganciclovir por sonda nasogástrica. 10. en el tratamiento de la enfermedad por cmv, es falso que: a) el tratamiento de elección sea ganciclovir. b) el incremento de la antigenemia los primeros días pueda ser normal. c) siempre deba administrarse gammaglobulina. d) el incremento de la antigenemia al final de la primera semana deba hacernos sospechar una infección por una cepa resistente aganciclovir. e) foscarnet puede ser una alternativa terapéutica para el tratamiento de la infección por cepas resistentes a ganciclovir. lung transplantation at the university of pittsburgh: 1982 to 1994 acute and chronic rejection after lung transplantation improved results with lung transplantation for cystic fibrosis update in transplantation lung transplantation: current status and challenges the registry of the international society for heart and lung transplantation bronchiolitis obliterans following lung transplantation acute and chronic rejection after lung transplantation incidence of obliterative bronchiolitis after heart-lung transplantation in children analysis of time-dependent risks for infection, rejection, and death after pulmonary transplantation prevention of cytomegalovirus infection-enhanced experimental obliterative bronchiolitis by antiviral prophylaxis or immunosuppression in rat tracheal allografts respiratory viral infections aggravate airway damage caused by chronic rejection in rat lung allografts chronic rhinoviral infection in lung transplant recipients clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant respiratory viral infections are a distinct risk for bronchiolitis obliterans syndrome and death respiratory viruses and chronic rejection in lung transplant recipients infectious etiology of bronchiolitis obliterans: the respiratory viruses connection -myth or reality? risks and epidemiology of infections after lung or heart-lung transplantation ciliary beat frequency and structure of recipient and donor epithelia following lung transplantation mucociliary function after lung transplantation impairment of bronchial mucociliary clearance in long-term survivors of heart/lung and double-lung transplantation. the paris-sud lung transplant group hla phenotype of lung lavage cells following heart-lung transplantation tuberculosis in transplanted lungs mycobacterium tuberculosis in lung transplant recipients transmission of mycobacterium tuberculosis to recipients of single lung transplants from the same donor tuberculosis in lung transplant recipients nocardia infection in heartlung transplant recipients at alfred hospital nocardia infection in lung transplant recipients clinical presentation and outcome of tuberculosis in kidney, liver, and heart transplant recipients in spain aspergillus airway colonization and invasive disease after lung transplantation saprophytic fungal infections and lung transplantation -revisited aspergillus infection in single and double lung transplant recipients ulcerative tracheobronchitis after lung transplantation. a new form of invasive aspergillosis donorto-host transmission of bacterial and fungal infections in lung transplantation infectious complications of lung transplantation. impact of cystic fibrosis recent advances in cystic fibrosis voriconazole prophylaxis in lung transplant recipients posaconazole or fluconazole for prophylaxis in severe graft-versus-host disease cytomegalovirus infection and survival in lung transplant recipients cytomegalovirus infection and pneumonitis. impact after isolated lung transplantation impact of ganciclovir prophylaxis on heart-lung and lung transplant recipients definitions of cytomegalovirus infection and disease in transplant recipients consensus document from gesitra-seimc on the prevention and treatment of cytomegalovirus infection in transplanted patients high incidence of ganciclovir-resistant cytomegalovirus infection among lung transplant recipients receiving preemptive therapy risk factors for bronchiolitis obliterans: a systematic review of recent publications incidence and significance of noncytomegalovirus viral respiratory infection after adult lung transplantation lung retransplantation after posttransplantation lymphoproliferative disorder (ptld): a single-center experience and review of literature of ptld in lung transplant recipients clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant a single-season prospective study of respiratory viral infections in lung transplant recipients paramyxovirus infection in lung transplant recipients intravenous ribavirin is a safe and cost-effective treatment for respiratory syncytial virus infection after lung transplantation clinical features and outcomes of paramyxoviral infection in lung transplant recipients treated with ribavirin impact of ganciclovir prophylaxis on heart-lung and lung transplant recipients randomized trial of daily versus three-times-weekly prophylactic ganciclovir after lung and heart-lung transplantation following universal prophylaxis with intravenous ganciclovir and cytomegalovirus immune globulin, valganciclovir is safe and effective for prevention of cmv infection following lung transplantation cmv-specific immunotherapy infección en el trasplante de pulmón key: cord-016998-6n662amh authors: nan title: nierentransplantation date: 2007 journal: praxis der nephrologie doi: 10.1007/978-3-540-48556-8_13 sha: doc_id: 16998 cord_uid: 6n662amh die nierentransplantation ist die effektivste behandlungsmethode der chronischen terminalen niereninsuffizienz. seit den 1960er jahren entwickelte sie sich zu einer standardtherapie. wichtige voraussetzungen waren die entdeckung des hla-systems, die entwicklung der immunsuppressiva sowie die technische perfektionierung des organerhaltes außerhalb eines lebenden körpers. die 5-jahres-überlebensrate für allotransplantate beträgt etwa 65%, diejenige von lebendspenden 79%. immunsuppression -293 13 von klasse-1-hla-genen kodierte antigene sind auf allen kernhaltigen zellen vorhanden, von klasse-2-hla-genen kodierte antigene hauptsächlich auf b-lymphozyten und monozyten, also zellen des abwehrsystems. klasse-i-und klasse-ii-antigene bestehen aus zwei ketten, die als α und β-kette bezeichnet werden und ihre letztliche konfiguration durch dimerisierung bilden. die klasse-i-antigene enthalten eine polymorphe antigenspezifische kette (»heavy chain«) und kommen auf der zelloberfläche immer mit einem β2-mikroglobulinmolekül (»light chain«) assoziiert vor. durch kristallisation konnte die struktur der klasse-i-moleküle des hla-komplexes sichtbar gemacht werden. auf der moleküloberfläche findet sich eine 2×1×1 nm große grube, in welcher fremdantigene von 8-9 aminosäuren größe gebunden werden können. bei den klasse-ii-antigenen bilden immer eine α-(»heavy chain«) und β-kette (»light chain«) zusammen eine antigenbindende tasche. ▬ das langzeitüberleben eines transplantates hängt u. a. vom ausmaß der hla-übereinstimmung ab. die auslösung einer frühen abstoßung hängt mehr von patientenspezifischen faktoren und der immunsuppression ab. eine analyse aus den usa von 1994 fand eine halbwertszeit von 24 jahren für das transplantatüberleben bei hlaidentischer lebendspende (zwillinge), bei leichennierentransplantation von 20 jahren bei 6facher hla-übereinstimmung, von 9 jahren bei zufallsmatching. primär glomeruläre erkrankungen schwächen die prognostische aussagekraft des hla-matches aufgrund der möglichen rezidive im transplantat. hla-a-, hla-b-und hla-dr-region findet man eine transplantathalbwertszeit von 20 jahren und ein 10-jahres-transplantatüberleben von 65-70%. sechs identische hla-antigene führen zu dem besten 1-jahres-(88%) und auch langzeittransplantatüberleben. ein mismatch in der hla-a-, hla-b-oder hla-dr-region ist mit einer transplantathalbwertszeit von 10 jahren und einem 10-jahres-transplantatüberleben von 40-50% verbunden. stimmen mehrere hla-antigene nicht überein, liegt die transplantathalbwertszeit bei 7-9 jahren und das 10-jahres-überleben bei 30-35%. diese erkenntnisse sind der verdienst großer datenbanken, die überregional, z. t. sogar weltweit daten gesammelt und ausgewertet haben. problematisch bei der datenauswertung ist die weiterentwicklung sowohl der testsysteme als auch die einführung neuer immunsuppressiva. wichtige transplantationsorganisationen: ▬ cts -collaborative transplant study, prof. opelz, heidelberg ▬ eurotransplant, leiden ▬ united kingdom transplant service ▬ unos -united network for organ sharing, nordamerika ▬ seopf -american southeast organ procurement foundation toleranz gegenüber nichteigenen hla-antigenen erwirbt man vermutlich pränatal und in der stillzeit. untersuchungen an transplantatempfängern, die vor der transplantation z. b. über bluttransfusionen mit spenderantigenen konfrontiert wurden, deuten auf die möglichkeit einer induktion von immuntoleranz hin. die bildung von hla-antikörpern wird durch schwangerschaft, geburt und vorherige transplantationen stark, durch bluttransfusionen in geringerem ausmaß induziert. eine erfolgreiche transplantation ist nahezu ausgeschlossen, wenn beim empfänger zytotoxische antikörper gegen folgende antigene vorhanden sind: ▬ blutgruppenantigene (ab0-unverträglichkeit) des spenders ▬ klasse-i-oder -ii-hla-antigene des spenders ▬ endotheliale oder monozytäre antigene des spenders ein positiver t-zell-crossmatch (s. unten) z. b. führt zu einer hyperakuten abstoßung. komplementsystem und gerinnungskaskade werden aktiviert, eine anaphylaktische reaktion tritt ein und polymorphkernige leukozyten und mononukleäre zellen wandern in das transplantat ein. dies führt innerhalb von minuten bis stunden zu einer fibrinoiden nekrose der blutgefäße des transplantates und ischämischer nekrose des nierenparenchyms. in japan wurde aufgrund extremen organmangels blutgruppeninkompatibel transplantiert. durch immunadsorption in kombination mit b-zell-antikörpern konnte im vergleich mit gematchten patienten ein gleiches langzeitüberleben des transplantates erreicht werden. ähnliche ergebnisse wurden bei der transplantation 13.1 · transplantationsimmunologie von organen mit der gering immunogenen blutgruppe a 2 in empfänger der blutgruppen 0 und b erreicht. t-und b-zell-crossmatch. beim t-zell-crossmatch und b-zell-crossmatch wird die stimulation der b-oder t-lymphozyten durch empfängerserum getestet. bei der gemischten lymphozytenkultur (»mixed lymphocyte culture«, mlc) wird die stimulation von empfänger und spenderlymphozyten getestet, die miteinander inkubiert werden. stimulation kann anhand der anzahl von blasten oder des einbaus radioaktiver nukleinsäuren getestet werden. »panel reactive antibodies«, pra. bei diesem test wird das serum des empfängers auf das vorhandensein zytotoxischer antikörper gegen eine große zahl von häufigen (»populären«) antigenen getestet. hohe sensibilisierung zeigt sich in reaktivität gegen einen hohen prozentsatz der angebotenen antigene und wird als prozent-panel-reaktivität ausgedrückt. patienten mit hoher panel-reaktivität haben eine geringere transplantationschance, da sie häufig im crossmatch positiv sind. in einem amerikanischen zentrum z. b. war die wartezeit bis zur transplantation für patienten mit einer panel-reaktivität über 50% 5-mal so lange als bei einer reaktivität unter 10%. die 1-und 2-jahres-überlebensraten des transplantates waren ebenfalls geringer. auch »alte« tests mit hoher reaktivität (>6 monate vor transplantation) verschlechtern das transplantatüberleben, auch wenn das ergebnis unmittelbar vor transplantation besser war. ein großes problem besteht in der interpretation von positiven antikörpertests, denn nicht alle zytotoxischen antikörper des empfängers gegen spenderantigene sind für den transplantationserfolg von bedeutung. die bei der grunderkrankung lupus erythematodes vorhandenen multiplen autoantikörper können die testergebnisse verfälschen. positiver b-zell-crossmatch bei negativem t-zell-crossmatch führt in der regel nur bei anti-körpern gegen klasse-i-hla-antigene zu einer häufung frühen transplantatversagens. positiver b-zell-crossmatch führt zu einer 5% geringeren 2-jahres-überlebensrate bei ersttransplantation und 10% geringeren bei retransplantation. flow-cytometry-test. ein positiver flow-cytometry-test ist sowohl bei erst-als auch bei retransplantation ein negativer prognostischer faktor. dieser sehr empfindliche test wird auch bei vorhandensein, niedrig titriger, nicht komplementaktivierender, inkompatibler hla-antikörper positiv. viele zentren führen unmittelbar vor der transplantation einen crossmatch-test zwischen spender und empfänger durch, um eine hyperakute abstoßung auszuschließen. dieser dauert aber ca. 4 h und verlängert damit die kalte ischämiezeit. bei patienten mit 0% panel-reaktivität kann dieser letzte test vermutlich unterlassen werden. der wachsende anteil von zweit-und drittnierentransplantierten und die damit höhere zahl von hochimmunisierten patienten gewinnt zunehmend an bedeutung. eurotransplant hat den hochimmunisierten transplantatempfänger folgendermaßen definiert: »panel reactive antibodies« (pra) >85%; d. h., dass hla-antikörper im patientenserum mit >85% unselektierter patienten im crossmatch reagieren. das vorhandensein donor-spezifischer hla-antikörper (dsa) resultiert aus einer früheren exposition gegenüber fremden hla-antigenen durch bluttransfusionen, vorausgegangenen berücksichtigt wird die summe der mismatches bzw. der übereinstimmenden hla-antigene. dies wird in einer punktzahl ausgedrückt und hat eine gewichtung von 40%. sie bezeichnet die wahrscheinlichkeit, ein in den hla-merkmalen weitgehend übereinstimmendes daten des ucla-registers (»ucla tissue typing laboratories«) zeigen eine deutlich schlechtere langzeitprognose für nierentransplantationen bei diabetikern. nach 5 jahren werden patientenüberlebensraten von 45-75% beschrieben! diese liegen jedoch deutlich über der 5-jahres-überlebensrate von diabetischen dialysepatienten von 0-35%. im usrds (»united states renal data system«) wurden 7200 transplantierte diabetiker mit 15000 diabetischen dialysepatienten auf der warteliste verglichen: das mortalitätsrisiko nach 18 monaten war bei den dialysepatienten signifikant höher. diese sehr hohe mortalität wird zu einem großen teil durch extrarenale gefäßerkrankungen verursacht. vermutlich ist die elimination von age-proteinen (advanced glycation endproducts, kap. 9) durch das transplantat mit ausschlaggebend für die bessere gefäßsituation der transplantierten diabetiker. in der vorbereitungsphase der nierentransplantation ist eine invasive abklärung der koronarien mittels koronarangiographie unumgänglich. wenn dabei die notwendigkeit einer acvb-operation zu tage tritt, aber nicht zugemutet werden kann, ist eine transplantation vermutlich ebenfalls ein nicht zumutbarer eingriff. die u. a. aus neurologischen gründen gehäuften harnwegsinfekte der diabetiker zwingen zu einer gründlichen urologischen abklärung, oft sind langzeitprophylaxen mit antibiose indiziert. proteinurie und langsamer funktionsverlust können die entwicklung einer diabetischen nephropathie im transplantat anzeigen. auslöser der diabetischen nephropathie im transplantat ist ebenfalls die blutzuckerentgleisung. beim jüngeren patienten ohne ausschlusskriterien sollte eine 13.3 · vorbereitung der transplantation kombinierte pankreas-nieren-transplantation angestrebt werden ( kap. 9). die transplantation von organen nicht verwandter lebendspender sowie die akzeptanz alter spender sind versuche, die bestehende lücke zwischen organangebot und nachfrage zu füllen. gründe für das schlechte ansehen der nicht-verwandten lebendspende sind ungelöste ethische probleme: ▬ wo liegt die wahre motivation der spende? (psychologische evaluierung und betreuung sicher sinnvoll) ▬ wenig akzeptable morbidität und mortalität von spender und empfänger ▬ schlechtes transplantatüberleben während die anzahl der amerikanischen transplantationszentren, die nicht-verwandte lebendspenden akzeptieren ständig zunimmt, ist die nicht-verwandte lebendspende in den europäischen ländern unüblich. auch bei nicht verwandten lebendspendern ist die ab0-kompatibilität ausgangstest für alle weiteren abklärungsuntersuchungen, die denen der lebendspende durch verwandte entsprechen. das 1-jahres-überleben in einigen studien lag bei 92-95%, nach 2 jahren funktionierten in einer studie noch 83% der transplantate. somit liegt die rate funktionierender transplantate nach 1 jahr näher bei derjenigen verwandter lebendspender, als bei derjenigen von leichennierentransplantationen. dies ist leicht mit den kürzeren ischämiezeiten und planbaren operationsumständen zu begründen. nieren von ehepartnern hatten in einer studie eine halbwertsüberlebenszeit von 12 jahren. im jahr 2005 wurden in deutschland 165 pankreastransplantationen an 23 kliniken durchgeführt, davon 144 in kombination mit einer niere (»simultanous pancreas-kidney transplantation«, spk). die zahl der kombinierten pankreas-(nieren)-transplantationen und auch die zahl der neuanmeldungen zur transplantation nahm leicht ab. die patientenüberlebensrate und das überleben der niere entsprechen derjenigen der alleinigen nierentransplantation. patienten über 45 jahre haben ein doppelt so hohes risiko des transplantatverlustes und ein 3faches mortalitätsrisiko. ihnen ist eine alleinige nierentransplantation anzuraten. besteht das angebot einer hla-identischen oder zumindest sehr gut passenden niere, so sollte die einzeltransplantation der organe (»pancreas after kidney«, pak) in erwägung gezogen werden. viele zentren fordern eine koronarographie vor aufnahme auf die warteliste, da koronare komplikationen die mortalität der potentiellen transplantatempfänger vervierfacht. blindheit, hochdruck, periphere bypässe, amputationen sowie zerebrovaskuläre komplikationen haben keinen einfluss auf das transplantatüberleben. unter einer induktionstherapie versteht man die einleitende, meist bereits präoperativ beginnende immunsuppression. prinzipiell unterscheidet man protokolle mit antikörpern gegen t-zellen in kombination mit niedrig dosierten konventionellen immunsuppressiva von protokollen mit hochdosierten konventionellen immunsuppressiva (ohne antikörper). als antikörper finden einsatz: ▬ antilymphozytenserum (atg = antithymozytenglobulin = anti-t-lymphozytenglobulin) ▬ basiliximab, daclizumab: ursprünglich in der maus gezüchtete, humanisierte il-2-rezeptorantikörper eine akute abstoßung ist oft schwer abgrenzbar von einer verzögerten funktionsaufnahme. manche zentren befürworten dann die gabe von atg, um eine okkulte abstoßung nicht untherapiert zu lassen. die transplantatbiopsie ist zur diagnosesicherung erforderlich. es gibt derzeit keine konsensusempfehlungen für die induktionstherapie. die derzeit wichtigsten medikamente zur erhaltungsimmunsuppression nach nierentransplantation sind steroide, azathioprin, mycophenolat, ciclosporin und tacrolimus sowie sirolimus. in den ersten 3 monaten ist das risiko einer akuten abstoßung am höchsten. man setzt deswegen in dieser zeitspanne höhere dosen der immunsuppressiva ein. langfristig werden jedoch möglichst niedrige dosierungen angestrebt, da malignomund infektionsrisiko mit der gesamtdosis der immunsuppression korrelieren. die dosis der immunsuppression wird außerdem höher angesetzt bei: ▬ vorhandener sensibilisierung ▬ retransplantation (höhere dosen als bei ersttransplantation) ▬ hoher anzahl von abstoßungen bei der ersttransplantation ▬ geringer hla-übereinstimmung auch bei völliger hla-übereinstimmung wird mit steroiden und azathioprin oder ciclosporin weiterbehandelt. ein komplettes absetzen kann nicht empfohlen werden, da es auch spät noch zu akuten abstoßungen oder beschleunigter chronischer abstoßung kommen kann. ganz selten kann die entwicklung einer spenderspezifischen toleranz beobachtet werden. glukokortikoide hemmen die aktivierung von t-lymphozyten. sie beeinflussen die zellulären immunreaktionen über eine synthesehemmung von zytokinen (interleukin 1, interleukin 2). die antikörperbildung wird nur bei der gabe hoher dosen beeinflusst. außerdem lagern sich hochdosierte steroide in die zellmembran ein und verändern die struktur und damit funktion der oberflächenproteine durch störung der membranintegrität. die hohe steroiddosis der induktionstherapie wird in abhängigkeit vom verlauf langsam reduziert. ab etwa 0,5 g/kg kg prednisolonäquivalente können transplantierte aus der stationären behandlung entlassen werden. die dosis sollte wenn möglich auf unter 10 mg/24 h bzw. 0,1 mg/kg kg reduziert werden, um langzeitnebenwirkungen der steroide wie cushing-syndrom, osteoporose, aseptische knochennekrosen, muskelatrophie und steroiddiabetes zu vermeiden. erfreuliche nebenresultate der steroidreduktion sind blutdruckabfall, absinken des gesamtcholesterols, erleichterung der diabeteseinstellung und stabilisierung der knochensituation. ein frühes völliges absetzen der steroide ist mit einer deutlichen zunahme akuter abstoßungen, spätes absetzen mit einer reduktion der nierenfunktion und zunahme der proteinurie verbunden. gibt man die steroiddosis jeden 2. tag (sog. »alternate day regimen«), wird die störung der hormonellen feedbackmechanismen der nebenniere bzw. der hypothalamus-hypophysen-nebennierenachse reduziert und die gefahr eines cushing-syndroms sinkt. fieber, schwäche, myalgien, arthralgien und gewichtsverlust können zeichen einer subtilen nebenniereninsuffizienz sein, die mit einem falschnormalen acth-test einhergeht. azathioprin (imurek) führt als antimetabolit der purinbiosynthese zur suppression zellulärer immunreaktionen hauptsächlich der t-lymphozyten. die erhaltungsdosis liegt bei 1,5-2,5 g/kg kg. hohe initialdosen können die inzidenz akuter abstoßungen reduzieren. schwerste nebenwirkung ist die leukopenie. bei leukozytenzahlen unter 3000/mm 3 muss eine behandlungspause unter fortlaufender blutbildkontrolle erfolgen. sinken die leukozytenzahlen unter 1000/mm 3 , muss eine stationäre aufnahme, unter 700/mm 3 eine isolation erfolgen. bei weiter sinkenden leukozytenzahlen sollte granulozytenstimulierender wachstumsfaktor (g-csf=neupogen) verabreicht werden. beim wiedereinsetzen wählt man eine niedrigere dosis. eine häufige nebenwirkung ist die hepatotoxizität, die sich klinisch in oberbauchbeschwerden äußert, welche von einer enzymatischen cholestase und transaminasenanstieg begleitet sein können. allopurinol erhöht den plasmaspiegel von azathioprin über eine hemmung der xanthinoxidase und darf deswegen nicht gleichzeitig verabreicht werden. bei schwerer gicht kann durch umsetzen auf mycophenolat die gabe von allopurinol ermöglicht werden. neoplasmen der haut sind bei nierentransplantierten unter azathioprin vermutlich ebenfalls gehäuft. direkte sonneneinstrahlung muss gemieden bzw. sonnencreme mit hohem lichtschutzfaktor verwendet werden. azathioprin ist heute wegen der besseren wirksamkeit und geringeren myelosuppression weitgehend durch mmf ersetzt worden. mycophenolat-mofetil (mmf, cellcept) blockiert die purinbiosynthese über eine inhibition der inosinmonophosphat-dehydrogenase. es wird als ersatz von azathioprin und zur »rescue«-therapie bei okt 3 resistenten abstoßungskrisen eingesetzt. es ist nicht nephrotoxisch und weniger knochenmarksupprimierend als azathioprin. häufig sind gastrointestinale nebenwirkungen mit diarrhö und gastritis. unter tripletherapie mit steroiden und ciclosporin a (oder tacrolimus) treten akute abstoßungen wesentlich seltener auf als unter zweifachtherapie mit steroiden und ciclosporin a (oder tacrolimus) alleine. bei dem vergleich von 2 und 3 g mycophenolat/tag vs. azathioprin (beide gruppen mit steroiden und ciclosporin a) waren nach 6 monaten transplantatverluste und abstoßungen unter mycophenolat seltener, antilymphozytenglobulin (alg) musste seltener eingesetzt werden und die 1-jahres-transplantatüberlebensrate war tendenziell höher. diese ergebnisse bestätigten sich nach 3 jahren. trotz der höheren therapiekosten war mycophenolat durch die selteneren abstoßungsbehandlungen kostengünstiger als azathioprin. der im tierexperiment gefundene günstige effekt auf chronische abstoßung konnte beim menschen noch nicht nachvollzogen werden. unter azathioprin stabile patienten werden im allgemeinen nicht auf mycophenolat umgesetzt. es ist bisher unklar, ob das absetzen von steroiden unter ciclosporin und mycophenolat möglich ist. ciclosporin ist ein lipophiles peptidantibiotikum, welches von dem pilz tolypodadium inflatum gebildet wird. es hemmt die zelluläre immunantwort über eine bindung an intrazelluläre cyclophylline. dies führt zu einer synthesestörung von interleukin-2 und anderen zytokinen. seit den frühen 1980er jahren hat ciclosporin seinen festen platz in der erhaltungsimmunsuppression. die kombination von ciclosporin a mit steroiden und azathioprin bezeichnet man als »tripletherapie«. die meisten nierentransplantierten patienten erhalten derzeit diese tripletherapie oder ciclosporin a mit entweder steroiden oder azathioprin/mycophenolat. gelegentlich wird ciclosporin auch als einziges immunsuppressivum eingesetzt. auch spätes absetzen von ciclosporin führt gehäuft zu akuten abstoßungen und dadurch schlechterem transplantatüberleben. die inzidenz chronischer abstoßung wird allerdings von ciclosporin nicht vermindert. viele medikamente beeinflussen den abbau von ciclosporin a und können so den plasmaspiegel verändern. der talspiegel sollte in der erhaltungsphase zwischen 50 und 150 ng/ml liegen, der vollblutspiegel zwischen 150 und 300 ng/ml. niedrigere dosen werden bei stabiler transplantatfunktion toleriert. manche patienten sind sehr ciclosporinempfindlich und kommen mit spiegeln um 35 ng/ml gut zurecht. es senkt die rate von zytomegalievirus-infektionen und ermöglicht über eine reduktion der ciclosporin-dosis auch die minimierung der nephrotoxischen effekte von ciclosporin. als teil einer tripeltherapie, zusammen mit ciclosporin-mikroemulsion (sandimmun optoral) und steroiden, hat es sich als ähnlich effektiv erwiesen wie mycophenolatmofetil, das in den letzten jahren am häufigsten in einer ciclosporin-basierten tripeltherapie eingesetzt wurde. die therapie mit in der maus gezüchteten antikörpern führt beim menschen zu durch zytokinausschüttung von t-zellen bedingten nebenwirkungen (fieber, schüttelfrost, übelkeit, erbrechen, diarrhö, hypotonie, thoraxschmerzen, dyspnoe). die gentechnische herstellung von hybridantikörpern aus der antigenbindenden region des murinen antikörpers und dem grundgerüst menschlichen iggs konnte sowohl die immunogenität der antikörper vermindern als auch die halbwertszeit der antikörper verlängern. sie werden zur induktionstherapie und bei der akuten abstoßung eingesetzt. die nebenwirkungen sind geringer als bei okt 3. der erste zugelassene monoklonale antikörper war muromonab-cd3 (okt 3). okt 3 ist ein sehr potentes immunsuppressivum, wird heute aber selten verwendet, da die moderneren antikörper und neuere immunsuppresiva okt 3 mit seinen z. t. heftigen nebenwirkungen weitgehend verdrängt haben. die monoklonalen rekombinanten antikörper, die bisher in der transplantationsmedizin eingesetzt werden, binden an die α-kette des il-2-rezeptors und verhindern die bindung von il-2 an die aktivierte t-zelle. derzeit sind basiliximab (simulect) und daclizumab (zenapax) erhältlich. die prohylaktische gabe als induktionstherapie reduziert die rate akuter abstoßungen, die 1-jahres-funk-tionsrate unterscheidet sich jedoch nicht zur induktionstherapie ohne antikörper. vorwiegender einsatz bei immunologischen risikopatienten. tagesdosen von 8-10 mg/kg kg ciclosporin-mikroemulsion zusammen mit 2-maliger gabe von 1 g mycophenolat und niedrig dosierten steroiden ist die derzeit am häufigsten eingesetzte induktionstherapie. als erhaltungstherapie bieten sich 3-5 mg/kg kg ciclosporin-mikroemulsion, mycophenolat cyclophosphamid und mercaptopurin sind im tierexperiment teratogen und sollten beim menschen vermieden werden. methotrexat ist sicher embryotoxisch, führt zum abort und wird als substanz zur beendigung ektoper schwangerschaften getestet. für mycophenolat gibt es keine anwendungsuntersuchungen in der schwangerschaft. gleiches gilt für den antikörper okt 3. die federal drug administration (fda) stuft beide substanzen bezüglich ihrer teratogenität als sogenannte c-klasse ein (⊡ tab. 13.2). dies bedeutet, dass adäquate studien fehlen und risiken nicht ausgeschlossen werden können, eine vitale indikation die gabe aber rechtfertigen kann. der zeitpunkt des auftretens einer transplantatfunktionsstörung ist oft charakteristisch für die auslösende ursache. polyomaviren sind eine klasse von dns-viren aus der familie der papovaviridae, die zu opportunistischen infektionen führen können. die durchseuchung ist hoch, es kommt jedoch nur selten zum krankheitsausbruch. das zu den polyomaviren zählende jakob-creutzfeld-virus z. b. ist wahrscheinlich die ursache der progressiven multifokalen leukenzephalopathie. bei knochenmarktransplantierten wurde eine assoziation mit dem auftreten einer hämorrhagischen zystitis, bei nierentransplantierten mit dem auftreten einer ureterstenose beobachtet. mittels zytologie, urinkulturen und elektronenmikroskopie konnten polyomaviren im urin von 10-45% der patienten nach nierentransplantation nachgewiesen werden. es kann vermutlich sowohl im rahmen der immunsuppression reaktiviert als auch mit dem transplantat übertragen werden. tritt etwa 11 monate nach transplantation eine akute verschlechterung der transplantatfunktion mit den zeichen einer interstitiellen nephritis auf, so bildet die polyomavirusinfektion eine wichtige differentialdiagnose. in der bisher größten untersuchung an 22 patienten konnte das transplantat bei den 8 patienten, deren immunsuppression reduziert wurde, erhalten werden, während 8 von 12 patienten, die unter der annahme einer abstoßung verstärkt immunsuppressiv therapiert wurden, ihr transplantat verloren. mit steigender viruslast ist das virus zuerst im urin, dann im plasma und schließlich in der niere nachweisbar. die häufigsten ursachen einer langsam progredienten verschlechterung der transplantatfunktion sind: ▬ chronische abstoßung ▬ ciclosporintoxizität ▬ hypertensive nephrosklerose als folge mangelhafter blutdruckkontrolle ▬ obstruktion der ableitenden harnwege ▬ rezidiv der grunderkrankung oder neue nierenerkrankung unter transplantatabstoßung versteht man die immunologischen abwehrreaktionen des empfängers gegen das transplantat. diese wird durch vorgebildete, zytotoxische antikörper gegen klasse-i-antigene oder durch blutgruppeninkompatibilität hervorgerufen. diese form der abstoßung ist durch den ausschluss einer vorbestehenden sensibilisierung des empfängers gegen spenderantigene selten geworden. die aktivierung von komplement-und die gerinnungskaskade führt innerhalb von minuten nach öffnung der gefäßklemmen zur mikroembolisa-13.6 · transplantatabstoßung tion hauptsächlich mittlerer und kleiner gefäße und nekrose des transplantats (⊡ abb. 13.1). eine therapie ist nicht möglich. unter einer akuten abstoßung versteht man eine akute funktionsverschlechterung des transplantates, die mit charakteristischen, histologischen veränderungen einhergeht. sie tritt bei 30% der leichennieren-ersttransplantationen, 27% der lebendspenden und 37% der zweittransplantationen auf. mehr als die hälfte der transplantierten erleidet mindestens eine akute abstoßung. tritt diese in den ersten beiden monaten nach tpl auf, so besteht ein höheres risiko für eine chronische abstoßung. histologisch unterscheidet man zwischen zellulärer und vaskulärer abstoßung. zeichen zellulärer abstoßung sind interstitielle infiltration mit mononukleären, zellen, sowie ruptur der tubulären basalmembran. neutrophile im interstitium deuten dagegen eher auf eine infektion hin. zeichen der antikörpervermittelten, humoralen abstoßung (früher als »vaskulär« bezeichnet) sind endothelschwellung, fibrinoide nekrosen der arteriolen, fibrinthromben in den glomerulären kapillarschlingen und in schweren fällen eine nierenrindennekrose. glomeruläre beteiligung ist ein schlechtes prognostisches zeichen. sind gleichzeitig interstitielle, mononukleäre infiltrate vorhanden, so spricht man von einer gemischten abstoßung. mit hilfe der »banff-97-klassifizierung« können akute abstoßungen standardisiert eingestuft werden. dies ist wichtig, um therapieschemata vergleichen zu können (⊡ tab. 13.3). die erfüllung der »borderline«-kriterien wird nicht als abstoßung gewertet. beweisend für eine abstoßung ist bisher allein die histologische analyse. ein idealer abstoßungsmarker in form eines spezifisch und sensitiv auf abstoßung reagierenden messwerts im blut oder noch besser im urin steht derzeit nicht zur verfügung. geforscht wird nach molekularbiologischen nachweismethoden einer erhöhten expression abstoßungsspezifischer zytokine (z. b. il-7, il-10, il-15, fas ligand, perforin und granzym b). eine akute abstoßung tritt bei 10-35% der nierentransplantierten auf. zur behandlung stehen die steroidstoßtherapie, atg (s. unten) und deren kombination zur verfügung. bei hochgradigem klinischen verdacht sollte bereits vor erhalt des biopsieergebnisses zumindest mit der steroidtherapie begonnen werden. die steroidstoßtherapie besteht aus der i.v.-verabreichung von 3-5 mg/kg kg methylprednisolon über 3-5 tage mit konsekutivem raschem ausschleichen bis zur ursprünglichen erhaltungsdosis. bei bisher eher niedrigem spiegel kann auch die ciclosporindosis angehoben werden. nebenwirkungen der steroide sind erhöhte infektanfälligkeit, blutzuckerentgleisungen, hypertonie, ulkuserkrankung und steroidpsychosen. eine prophylaktische, antimykotische therapie für den gastrointestinaltrakt in form von dünndarmlöslichen amphotericin-b-dragees und -emulsion (ampho-moronal für mund und speiseröhre), sowie eine ulkusprophylaxe mit einem h 2 -blocker wird empfohlen. wenn nach 5-7 tagen kein abfall des serumkreatinins sowie steigerung der urinausscheidung eingetreten ist, spricht man von steroidresistenter abstoßung. antilymphozytenserum wird durch die immunisierung von kaninchen oder pferden mit menschlichen lymphozyten aus dem thymus (deswegen atg = antithymozytenglobulin) oder aus b-zellkulturen gewonnen. eine typische dosierung wäre 10-15 mg/kg kg/24 h. nach einigen tagen bis einer woche kommt es in 75-100% zu einer rückkehr des kreatinins zum ausgangswert. nachteilig ist die serienabhängig unterschiedliche wirkstärke, die aufwendige produktion, die fehlende spezifität sowie die notwendigkeit eines zentralen zuganges zur applikation. um die infektionsgefahr nicht zu groß werden zu lassen, wird die dosis von ciclosporin, tacrolimus, azathioprin und mycophenolat während atg-therapie reduziert. gleichzeitig werden prophylaktisch antibiotika, virostatika und antimykotika gegeben. bei der infusion von atg treten als zeichen der reaktion auf fremdeiweiß fieber und schüttelfrost auf, selten kommt es auch zu anaphylaktischen reaktionen. viele zentren versuchen diesen begleitreaktionen durch gabe eines »cocktails« von steroiden, antihistaminika und antipyretika vorzubeugen. okt 3 wird wegen der starken nebenwirkungen kaum noch oder nur noch als reservemedikament eingesetzt. bei irreversiblem funktionsverlust des transplantates muss die immunsuppression abgesetzt werden. die infektionsgefahr aufgrund von medikamenten und zunehmender urämie potenziert sich. mit zunehmender niereninsuffizienz addiert sich auch die neurotoxizität von urämie und ciclosporin. beim absetzen der immunsuppression kann trotz terminalen transplantatversagens eine abstoßung auftreten, die zu einer nephrektomie zwingen kann. dies ist besonders, wenn ein transplantatversagen innerhalb des 1. jahres eintritt, der fall. ein häufig gewähltes vorgehen ist das sofortige absetzen von ciclosporin, tacrolimus, azathioprin und mycophenolat gefolgt von einem ausschleichen der steroiddosis. kommt es wiederholt zu abstoßungskrisen und nimmt das transplantat an größe zu, besteht die gefahr der transplantatruptur. nach vorübergehender restitution einer höheren steroiddosis muss das transplantat dann entfernt werden. vier fünftel aller nierentransplantierten erleiden mindestens eine infektion im 1. jahr nach transplantation. das spektrum der auslöser von infektionen ist bei immunsupprimierten patienten um die opportunistischen keime erweitert. je stärker die immunsuppression, umso geringer die abstoim 1. monat nach transplantation kommen die typischen, auch bei nicht transplantierten auftretenden, postoperativen infektionen gehäuft vor. dazu gehören wundinfektionen durch bakterien oder pilze, pneumonien, katheterinfektionen etc. erstaunlicherweise sind trotz der in dieser zeit maximalen immunsuppression die opportunistischen infektionen ( pneumocystis jiroveci, nocardiose, listeriose) selten. zwischen 4 wochen und 6 monaten nach transplantation kommen infektionen mit immunmodulierenden viren -insbesondere cmv -besonders häufig vor. diese können den boden für eine weitere infektion bereiten. in dieses intervall fallen auch infektionen durch hsv, hzv, hepatitisviren, mykobakterium tuberculosis und ebv. ebv kann die entwicklung lymphoproliferativer erkrankungen induzieren. infektionserkrankungen, die später als 6 monate nach transplantation auftreten, entsprechen bezüglich der erreger weitgehend den die allgemeine bevölkerung betreffenden infektionen. es gibt jedoch auch chronische virusinfekte, die dann erst klinisch manifest werden (aids, chronische hepatitis, lymphoproliferative erkrankungen nach ebv, chorioretinitis durch cmv). die meisten zentren verabreichen bereits bei der transplantation eine perioperative antibiotikaprophylaxe mit breitem wirkspektrum sowie eine cmv-prophylaxe mit gancyclovir. trimethoprim-sulfamethoxazol wird zur prophylaxe von harnwegsinfekten und speziell zu pneumocystisprophylaxe eingesetzt. bei rezidivierenden harnwegsinfekten, anomalien der ableitenden harnwege oder neurogener blasenentleerungsstörung wird es als dauerantibiose verabreicht. alternativ können gyrasehemmer (z. b. ciprofloxazin, ofloxazin) eingesetzt werden. das zytomegalievirus ist eines der vier herpesviren. die infektionsrate der bevölkerung mit cmv steigt mit dem alter. die transfusion von blut ist eine potentielle infektquelle, der gebrauch von leukozytenfiltern kann die virustransmission deutlich verringern. mehr als zwei drittel aller spender und empfänger von organtransplantaten haben antikörper gegen cmv als zeichen einer durchgemachten, wenn auch klinisch vielleicht nicht manifesten infektion. man unterscheidet eine cmv-infektion von einer cmv-erkrankung. von infektion spricht man bei: ▬ serokonversion von igg zu igm ▬ bei einem 4fachen anstieg des igg titers ▬ beim nachweis von cmv-antigen in infizierten zellen ▬ bei isolation des virus aus kulturen klinik cmv-erkrankung bedeutet das auftreten von klinischen symptomen wie fieber, leukopenie und organmanifestationen (z. b. pneumonitis, hepatitis, pankreatitis, kolitis, meningoenzephalitis). eine symptomatische cmv-infektion tritt meist zwischen 1 und 4 monaten nach transplantation auf, die cmv-chorioretinitis gelegentlich auch später. kardinalsymptom ist eine leukopenie, die bei protokollen mit azathioprin zu einer dosisreduktion oder umsetzen auf mycophenolat zwingt. die häufigste klinische manifestation ist ein der mononukleose ähnliches bild mit fieber, myalgien, arthralgien, schwäche, leukopenie, milder lymphozytose und gelegentlich leichtem transaminasenanstieg. unter cmv-induzierter transplantatglomerulopathie versteht man ein klinisches bild mit verschlechterter transplantatperfusion und akuter tubulusnekrose. dieses syndrom ist weder in seiner klinischen signifikanz, noch in seiner abgrenzung von einer durch eine cmv-infektion getriggerten abstoßung, noch in seiner häufigkeit gesichert. die behandlung richtet sich insgesamt nach der schwere der erkrankung. okt 3 muss abgesetzt, azathioprin reduziert und ggf. durch mycophenolat ersetzt werden. die i.v.-gabe von gancyclovir wird an die nierenfunktion angepasst. man behandelt normalerweise über 3 wochen und verabreicht ggf ergänzend hyperimmunglobulin (s. unten). eine gleichzeitige infektion mit opportunistischen keimen (pneumocystis jiroveci, nocardiose, listeriose) muss ausgeschlossen werden. eine cmv-prophylaxe ist insbesondere in folgenden situationen wichtig: ▬ bei der transplantation von organen cmvpositiver spender auf cmv-negative empfänger zur vermeidung einer neuinfektion ▬ bei cmv-positiven empfängern zur vermeidung einer reaktivierung ▬ bei cmv-positiven empfängern und cmvpositiven spendern zur vermeidung einer infektion mit einem anderen virussubtyp die meisten zentren geben derzeit gancyclovir als prophylaxe. da die bioverfügbarkeit oralen gancyclovirs nicht besonders gut ist, wird primär die i.v.-applikation gewählt, eventuell gefolgt von einer oralen therapie. acyclovir vermindert zwar auch die erkrankungsrate, aber im unterschied zu gancyclovir ist die infektionsrate unverändert. valacyclovir, eine weiterentwicklung von acyclovir, hat interessanterweise im unterschied zu letzterem gute prophylaktische wirkung gegen cmv und eine bessere bioverfügbarkeit als gancyclovir. eine cmv-prophylxe mit cmv hyperimmunglobulin (cytoglobin, cytotect biotest) existiert bereits seit den 1980er jahren. eine infektion wird zwar nicht verhindert, aber die rate an cmv-erkrankungen, an parasitären oder pilzinduzierten superinfektionen sowie an leukopenie wird signifikant reduziert. veränderungen der leberwerte findet man bei 7-24% von organempfängern. leberversagen ist die todesursache bei 8-28% nierentransplantierter patienten. etwa die hälfte der lebererkrankungen bei nierentransplantierten wird durch hcv ver-13.7 · infektionen bei nierentransplantierten ursacht, der rest durch hbv, cmv, ebv, medikamentennebenwirkung (azathioprin, ciclosporin a), alkohol oder hämosiderose. empfänger hcv-positiver organe entwickeln 4-mal so häufig eine hepatitis. die auswirkung auf die überlebensrate sowie die transplantatüberlebensrate ist jedoch umstritten. britische und amerikanische untersuchungen zeigten, dass die nierentransplantation auch beim anti-hcv-positiven patienten der dialysebehandlung überlegen ist. bei der transplantation zwischen hcv-positivem spender und empfänger können gleiche oder unterschiedliche genotypen des hcv-virus vorliegen. nach aktueller datenlage hat dies keine auswirkung auf die schwere einer hepatitis. hcv-positive empfänger entwickeln signifikant häufiger eine hepatitis, meist eine chronisch aktive hepatitis. selten kommt es auch zu einer rasch progredienten, mit cholestase und schwerer fibrose einhergehenden sog. »fibrosierenden cholestatischen hepatitis«. endgültige sicherheit über den hcv-status des spenders gibt nur der rna-test, auch die elisa-systeme der 2. generation haben noch eine geringe rate falsch-positiver ergebnisse. eine leberbiopsie zur bestimmung des ausmaßes der hepatitis ist sehr gut geeignet, die qualität der prognostischen aussage bezüglich des verlaufes nach transplantation bei anti-hcv-positiven patienten zu verbessern. für die prognose bei normaler leberhistologie fehlen studien. bei ausgeprägten leberparenchymschäden, die bei dialysepatienten auch bei fast normalen transaminasen vorkommen können, sollte die entscheidung zur transplantation mit vorsicht gewählt werden. nierengesunde patienten mit hepatitis c können mit interferon-α und bei einer kreatininclearance über 50 ml/min zusätzlich mit ribavirin behandelt werden. da die gabe von zytokinen abstoßungen triggert, ist eine nierentransplantation eine kontraindikation für interferon-α. derzeit wird die auswirkung einer interferon-α-therapie bei dialysepatienten mit hepatitis c vor nierentransplan-tation auf das auftreten eines rezidiv der erkrankung nach transplantation untersucht. ribavirin kann bei ausreichender transplantatnierenfunktion die viruslast mindern. proteinurie beim nierentransplantierten mit hcv-infektion kann auf eine hcv-assoziierte erkrankung des transplantates hinweisen. rezidiv der grunderkrankung 13.8.1 primäre nierenerkrankungen die rezidivrate der iga-glomerulonephritis liegt bei etwa 50%, wobei lebendspenden häufiger rezidive erfahren (bis zu 83%). nicht jede histologische läsion führt zu klinischen symptomen. der verlauf ist ähnlich den iga-glomerulonephritiden in nativen nieren langsam, die transplantatverlustrate durch das rezidiv ist gering (etwa 75% 5-jahres-transplantatüberleben). lebendspenden werden daher als ethisch akzeptabel erachtet. ciclosporin a hat keinen einfluss auf häufigkeit, schwere und verlauf der rezidive. insgesamt ist das transplantatüberleben (leichenniere) von patienten mit iga-glomerulonephritis vermutlich besser als bei anderen grunderkrankungen. hier liegt die rezidivrate bei etwa 20%. es existieren jedoch abgrenzungsprobleme zu den sekundären glomerulosklerosen. patienten unter 20 jahren mit rasch progredienter niereninsuffizienz durch fsgs, haben im transplantat eine rezidivrate von fast 50% und sind oft nach etwa 3 jahren erneut dialysepflichtig. das rezidiv präsentiert sich oft mit nephrotischer proteinurie. die patienten erleiden häufig akute abstoßungen und ein anv in der ersten woche nach transplantation. ein rezidiv im ersttransplantat erhöht das risiko eines rezidivs in folgetransplantaten. geschwindigkeit und kontinuität der rezidive lässt einen im serum zirkulierenden pathogenen faktor vermuten. temporäre verbesserungen der proteinurie nach behandlung mit proteinadsorptionssäulen (plasmaseparation) unterstützen diese these. aufgrund der hohen wahrscheinlichkeit eines rezidivs sollte bei der aggressiven form der fsgs und bei patienten mit fsgs-rezidiv im ersttransplantat keine lebendspende durchgeführt werden. eine membranoproliferative glomerulonephritis rezidiviert häufig im transplantat. bei typ i in 20-30%, bei typ ii in 50-100%. für den seltenen typ iii liegen nur einzelfallstudien mit rezidiven vor. klinisch zeigt sich eine proteinurie und bei typ i auch häufig hämaturie. rezidive bei typ i führen in 30-40%, bei typ ii in 10-20% (mindestens) zum transplantatverlust. eine effektive therapie ist nicht bekannt. eine hepatitis-c-assoziierte mpgn kann ebenfalls im transplantat auftreten. die membranöse glomerulonephritis rezidiviert eher selten (3-5%). betroffen sind hauptsächlich patienten mit rasch progredientem verlauf der primären erkrankung. die rezidive treten nach etwa 10 monaten auf und führen bei 30-50% zum transplantatverlust. häufiger als das rezidiv ist eine mit chronischer abstoßung assoziierte »de-novo«-membranöse glomerulonephritis, die 18-21 monate nach tpl auftritt und klinisch als proteinurie auffällt. die antikörperproduktion bei diesem krankheitsbild ist normalerweise selbstlimitierend. nach dem 13.8 · rezidiv der grunderkrankung verschwinden der antikörpertiter (meist 1 jahr) ist die rezidivrate verschwindend gering. allerdings findet man in bis zu 50% der transplantierten organe lineare igg-ablagerungen an der glomerulären basalmembran. bei nach dem verschwinden der antikörpertransplantierten patienten mit rezidiv findet man hämaturie und proteinurie, der transplantatverlust ist jedoch gering. patienten mit alport-syndrom können nach nierentransplantation eine de-novo-anti-gbm-glomerulonephritis entwickeln. in ihren eigennieren ist nämlich das goodpasture-antigen nicht nachweisbar, so dass eine »gesunde« spenderniere sozusagen ein goodpasture-mismatch mit sich bringt, gegen welches dann vom empfänger antikörper produziert werden. hus-rezidive im transplantat sind für 20-50% der patienten beschrieben. unklar ist jedoch, wie hoch der anteil sekundärer hämolytisch-urämischer syndrome durch vaskuläre abstoßung oder ciclosporintherapie ist. risikofaktoren für ein rezidiv sind infektiöse diarrhö, primäres hus vom autosomal-rezessiven typ, höheres alter, lebendspende, gabe von ciclosporin oder tacrolimus und kurzes intervall zwischen primärem hus und transplantation. therapeutisch kann sowohl atg als auch ciclosporin (mit vorsicht) eingesetzt werden. plasmapherese wurden ebenfalls erfolgreich eingesetzt. beim auftreten eines hus unter ciclosporin sollte dieses reduziert werden. vermutlich gilt gleiches für die extrem selten vorkommende ttp. ein hämolytisch-urämisches syndrom im transplantat bei anderer grunderkrankung ist meist mit ciclosporintoxizität, akuter vaskulärer abstoßung oder hiv assoziiert. prophylaktisch kann niedrig dosierte acetylsalicylsäure oder dipyridamol verabreicht werden. bisher sind nur etwa 80 fälle von nierentransplantation bei sklerodermieerkrankung beschrieben. die transplantatüberlebenszeit ist kürzer als bei anderen grunderkrankungen, nach 5 jahren beträgt sie z. b. nur 47%. die rezidivrate ist aufgrund der ähnlichkeit der renalen sklerodermieläsionen mit den veränderungen bei vaskulärer abstoßung schwer einzuschätzen. sie wird in der literatur mit 20% angegeben und hat meist einen aggressiven verlauf. eine nierentransplantation führt nur selten zu einer reaktivierung der erkrankung. zeichen der lupusnephritis nur in 2-9%. diese rekurrieren in bis zu 20%. anca-titer sagen das risiko der rekurrenz nicht voraus. typische histologische veränderungen der diabetischen nephropathie im transplantat sind die verdickung der basalmembran und eine verbreiterung des mesangiums, die klassischen nodulären läsionen der glomeruli fehlen meist. nur knapp 2% der transplantatverluste sind auf die diabetische nephropathie zurückzuführen, da sie sich sehr langsam entwickelt. ihr wiederauftreten wird durch die kombinierte pankreas-nieren-transplantation verhindert. die meisten chirurgischen und auch urologischen probleme treten relativ früh nach transplantation, oft noch während der stationären nachbehandlung auf. eine erfolgreiche transplantation kann viele der durch die terminale niereninsuffizienz hervorgerufenen knochenstoffwechselstörungen beheben. die plasmaspiegel von phosphat, ap, β 2 -mikroglo-bulin und parathormon fallen, die kalzifikationen nehmen ab. aluminiumosteopathie, hyperparathyreoidismus, β 2 -ablagerungen und diabetische knochenstörungen können jedoch persistieren. die persistenz eines hyperparathyreoidismus ist häufig. sie beruht meist auf einer hyperplasie der nebenschilddrüsen, die jahre bis zur rückbildung benötigen kann. nur selten ist sie zeichen eines adenoms. die meist etwa 10 tage nach tpl auftretende hyperkalzämie beruht auf der resorption von gewebekalzifikationen und der normalisierten vitamin-d-produktion (und gelegentlich auch auf dem albuminanstieg). in extremfällen kann eine kalziphylaxie auftreten. falls die korrigierte kalziumkonzentration nach einem längeren zeitraum (max. 1 jahr) noch über 3,1 mmol/l liegt und die ipth-werte nicht adäquat absinken, muss eine parathyreoidektomie erwogen werden. die rückbildung der aluminiumosteopathie nach transplantation ist auch durch dauertherapie mit desferal nicht zu erreichen. hypophosphatämie nach transplantation wird durch hyperparathyreoidismus oder durch tubulären renalen phosphatverlust verursacht. folgen sind muskelschwäche und osteomalazie. die progression der β 2 -amyloidose wird durch erfolgreiche transplantation unterbrochen, da jetzt wieder ausreichend β 2 -mikroglobulin renal eliminiert wird. alte knochenzysten bleiben jedoch bestehen. osteopenie und osteonekrosen sind die beiden wichtigsten knochenkomplikationen nach transplantation. ursächlich sind fortbestehende störungen des kalziumstoffwechsels und die auswirkungen der immunsuppressiva -insbesondere der steroide -auf den mineralstoffwechsel. zwar konnte durch ciclosporin die steroiddosis gesenkt werden, die längere lebensdauer führt jedoch zu einer höheren gesamtdosis an steroiden. die inzidenz osteopenisch bedingter frakturen bei nierentransplantierten beträgt bis zu 22%. der verlust der knochenmasse ist kurz nach transplantation am höchsten. nach etwa 1,5 jahren liegt die knochendichte von 60% der patienten unterhalb der frakturschwelle. mit hilfe der dexa (dual energy x-ray absorptionsosteometrie) können verlaufskontrollen der knochendichte durchgeführt werden. lymphoproliferative erkrankungen treten nach organtransplantation 30-bis 50-mal häufiger auf, ihre inzidenz beträgt 1%. ihre verläufe und verteilung unterscheiden sich von denen der normalbevölkerung. zum beispiel sind normalerweise ca. zwei drittel der lymphome non-hodgkin-lymphome, bei transplantierten dagegen sind es 93%. extranodales auftreten, beteiligung des zentralen nervensystems und infiltration des transplantats sind häufig. ebv-induzierte b-zellproliferation spielt eine wichtige ursächliche rolle. insgesamt kommen drei formen der ebv-assoziierten lymphoproliferativen erkrankungen bei transplantierten vor: ▬ in 55% eine gutartige polyklonale b-zell-lymphoproliferation mit mononukleoseartigem krankheitsbild und normalen zytogenetischen parametern ohne hinweis auf maligne transformation. ▬ in etwa 30% liegt das gleiche mononukleoseartige bild auch mit polyklonalen b-zellproliferationen, allerdings mit zeichen früher maligner zellentartung vor. ▬ am seltensten ist ein ausschließlich extranodales wachstum solider tumoren von monoklonaler b-zellherkunft mit malignen charakteristika in der zytogenese. es gibt jedoch auch lymphome ohne ebv-assoziation, d. h. eine negative ebv-serologie gibt keine sicherheit. eine nach transplantation erworbene ebv-infektion stellt aufgrund der fehlenden immunität ein größeres risiko dar als eine vorbestehende infektion. weitere risiken für eine lymphoproliferative erkrankung sind therapiezyklen mit okt 3 oder ein sog. cmv-mismatch (spender positiv, empfänger negativ). alle drei risikofaktoren zusammen -fehlende ebv-immunität, cmv-mismatch, okt 3-gabe -erhöhen das risiko einer lymphoproliferativen erkrankung um den faktor 654. ergebnisse der nierentransplantation 13.11.1 transplantatüberleben unter kurzeitüberleben wird üblicherweise die 1-jahres-transplantatüberlebensrate verstanden. diese ist seit einführung von ciclosporin a, also in etwa den letzten 25 jahren deutlich gestiegen. dies gilt allerdings hauptsächlich für die nicht vorsensibilisierten patienten. die ergebnisse für stark hlavorsensibilisierte patienten haben sich leider nicht wesentlich verbessert. im schnitt lag die 1-jahres-transplantatüberlebensrate ende der 1970er jahre bei etwa 55%, in der ersten hälfte der 1980er jahre bei etwa 75% und in der zweiten hälfte bei fast 90%. dabei hatten 16-bis 35-jährige transplantationskandidaten besonders gute resultate. das gleiche gilt für eine kurze kalte ischämiezeit und gute initiale transplantatfunktion. die 1-bis 5-jahres-transplantatüberlebensraten für lebendspenden und postmortale organspenden sind in ⊡ abb. 13.4 dargestellt. die aktuellen transplantationsstatistiken werden im jeweiligen jahresbericht der dso (deutsche stiftung organtransplantation) veröffentlicht. die transplantationshalbwertszeit ist die zeit, innerhalb derer die hälfte der zu einem bestimmten zeitpunkt verpflanzter organe nicht mehr funktioniert. sie wird erst ab dem 1. jahr nach transplantation berechnet. dass sie in den letzten beiden jahrzehnten nicht wesentlich gestiegen ist, bedeutet, dass der effekt von ciclosporin auf das kurzzeitüberleben von nierentransplantaten nicht auf das langzeitergebnis übertragbar ist. letzteres wird negativ beeinflusst durch: ▬ hypertensive nierenschädigung ▬ chronische ciclosporintoxizität ▬ rezidiv der grunderkrankung ▬ hyperfiltration eventuell positiv wirken dagegen vermutlich ace-hemmer. management von patienten auf der transplantationsliste unter besonderer beachtung immunologischer aspekte, mitteilungen de deutschen arbeitsgemeinschaft für klinische nephrologie,xxxv long-term pancreas allograft outcome in simultaneous pancreas-kidney transplantation: a comparison of enteric and bladder drainage mycophenolate mofetil, together with ciclosporin a, prevents anti-okt 3 antibody response in kidney transplant recipients immunosuppression in organ transplantation evaluation of pathologic criteria for acute renal allograft rejection: reproducibility, sensitivity, and clinical correlation the humoral immune response towards hla class ii determinants in renal transplantation development of sang-35: a ciclosporine formulation bioequivalent to neoral infection in organ-transplant recipients glomerular diseaseduring hcv infection in renal transplantation clinical practice guidelines: prevention of cytomegalovirus disease after renal transplantation immunosuppressive effects and safety of a sirolimus/ciclosporine combination regimen for renal transplantation the impact of an acute rejection episode on long-term renal allograft survival (t1/2) analysis of hla-dr matching in dna-typed cadaver kidney transplants prospective evaluation of pretransplant blood transfusions in cadaver kidney recipients plasma exchange and tacrolimus-mycophenolate rescue for acute humoral rejection in kidney transplantation a preliminary report of diltiazem and ketoconazole. their ciclosporine-sparing effect and impact on transplant outcome epstein-barr virusinduced posttransplant lymphoproliferative disorders task force and the mayo clinic organized international consensus development meeting transplantation tolerance -the search continues köhler a für die deutsche stiftung organtransplantation (2000) organspende und transplantation in deutschland international standardization of criteria for the histologic diagnosis of renal allograft rejection: the banff working classification of kidney transplant pathology advantage of antithymocyte globulin induction in sensitized kidney recipients: a randomized prospective study comparing induction with and without antithymocyte globulin maintenance pharmacological immunosuppressive strategies in renal transplantation reduced kidney transplant rejection rate and pharmacoeconomic advantage of mycophenolate mofetil das ausmaß der immunsuppression spielt ebenfalls eine rolle. die entwicklung der lymphoproliferativen erkrankung findet hauptsächlich im ersten jahr statt, denn zu diesem zeitpunkt ist die totale immunsuppression am höchsten.die diagnose einer ebv-assoziierten lymphoproliferativen erkrankungen setzt höchste aufmerksamkeit und misstrauen allen auffälligkeiten gegenüber voraus. sie wird gestellt, wenn aus dem tumor entnommenes gewebe lymphoid ist und folgende bedingungen erfüllt, die in einer konsensuskonferenz (paya et al. 1999 ) festgelegt wurden: ▬ ebv-infektion in vielen zellen ▬ mono-oder oligoklonale zellpopulationen ▬ zerstörung der normalen gewebestruktur durch den lymphoproliferativen prozessdurch eine perioperative gabe von gancyclovir sowie reduktion der tacrolimusdosis konnte bei hochrisikopatienten mit ebv-mismatch die inzidenz der ebv-assoziierten lymphoproliferativen erkrankungen signifikant gesenkt werden. angeregt durch retrospektive analysen wird derzeit die prophylaktische gabe von gancyclovir parallel zu okt 3 getestet. neue entwicklungen umfassen die gabe von b-zell-antikörpern, ein in pilotstudien bereits erfolgreich eingesetztes therapieprinzip. die immuntherapie mit lymphokinaktivierten, autologen killerzellen ist noch umstritten. die infusion von spenderleukozyten unter der rationale des vorhandenseins von gegen ebv sensibilisierten zytotoxischen t-zellen in den leukozytenseparationen hatte dagegen bei 3 von 5 knochenmarktransplantierten einen 10-bis 16-monatigen teilremissionserfolg.bezüglich der photochemotherapie gibt es noch keine empfehlungen, lediglich positive erfahrungen bei lungentransplantationen. es werden photosensibilisierende substanzen verabreicht, die sich bevorzugt in malignen stoffwechselaktiven lymphozyten anreichern. die leukozyten werden dann nach leukapherese extrakorporal bestrahlt, maligne lymphozyten sterben bevorzugt ab.bei lymphoproliferativen erkrankungen nach transplantation kann von einer 25-35% überlebensrate ausgegangen werden. t-zell-lymphome haben eine sehr schlechte prognose. key: cord-283826-lgyc3sro authors: stiehm, e. richard; orange, jordan s.; ballow, mark; lehman, heather title: therapeutic use of immunoglobulins date: 2010-11-05 journal: adv pediatr doi: 10.1016/j.yapd.2010.08.005 sha: doc_id: 283826 cord_uid: lgyc3sro nan medical science and thereby placed in the hands of the physician a victorious weapon against illness and death.' ' since then antibodies in multiple forms (animal and human serums, immune globulins and monoclonal antibodies) have been developed, primarily for prevention of infectious diseases, and less commonly for their treatment. these antibodies are presented in table 1 . this section reviews their uses, with an emphasis on their value in the treatment of human infections, as summarized in table 2 . antibody works by several mechanisms. it can neutralize viruses and bacterial toxins, lyse bacteria with the aid of complement, prevent the spread of microbes to adjacent cells or along nerve roots, coat bacteria for opsonization by phagocytes, block microbial attachment by saturating microbial receptors, and facilitate lysis of infected cells by binding them to cytotoxic cells with an fc receptor. antibody is particularly valuable in bacterial diseases associated with toxin production because much of the tissue damage results from action of the toxin; these can be neutralized rapidly by antibody before antibiotics kill the bacterium. anthrax (bacillus anthracis) anthrax is a rare but serious infection, predominantly of ruminant animals, caused by an aerobic gram-positive rod [1] . humans are infected through the skin (cutaneous anthrax), by ingestion (gastrointestinal anthrax), or by inhalation of anthrax spores (inhalational anthrax) [1] . the last often results from prolonged exposure to animal hides or carcasses or infected soil, and rarely by deliberate spore exposure in the bioterrorism setting. after inhalation the spores are ingested by alveolar macrophages and transported to regional nodes, where the spores germinate and release potent exotoxins. these toxins damage cell membranes, increase capillary permeability, cause pulmonary damage, and lead to shock and cardiovascular collapse. a vaccine is available for individuals at high risk for exposure and for the military. before the antibiotic era and as early as 1903, anthrax antitoxin (usually equine) was used in therapy [2] . an antitoxin is of value in a bioterrorism attack, both before and after exposure. the us government is collecting plasma from immunized donors to develop a human high-titer igiv [3] . a human monoclonal antibody is being tested in animals and humans [4] . diphtheria (corynebacterium diphtheriae). many of the adverse effects of diphtheria result from the action of its potent toxin on the heart, central nervous system, and other organs [5] . thus the prompt use of antitoxin is indicated, in addition to antibiotics [6] . the dose used depends on the localization and severity of infection, ranging from 20,000 units for mild infection of short duration to 120,000 units for severe illness with neck edema. the equine antitoxin is given intravenously, so must be preceded by skin testing for hypersensitivity and possible desensitization. the antitoxin is available through the us centers for disease control (cdc). a smaller dose of antitoxin can be used in asymptomatic, exposed, susceptible individuals. before the availability of diphtheria vaccine, antitoxins were given to health care workers caring for patients with diphtheria [7] . tetanus (clostridium tetani). equine antitoxin for the treatment of tetanus was initiated by von behring in the 1890s for toxin neutralization. extensive studies have been carried out to determine the optimal dose of antitoxin and the possible benefit of intrathecal antitoxin, particularly in tetanus neonatorum, a common problem in developing countries [8] . since the 1960s a human tetanus immune globulin (tig) has been available, but in some areas of the world equine antitoxin is still used. tig is given to unimmunized or incompletely immunized patients who sustain contaminated or deep puncture wounds [8] . the recommended dose of tig is 250 iu, along with initiation of active immunization. if tig is unavailable, human ivig can also be used; it contains variable titers of tetanus antitoxin but a minimal dose of 200-400 mg/kg is suggested for tetanus prophylaxis [8, 9] . clostridium difficile gastroenteritis. clostridium difficile infection of the gastrointestinal tract is usually associated with antibiotic-associated diarrhea, often with pseudomembranous colitis and sometimes toxic megacolon [10] toxic strains of clostridium difficile release 2 distinct toxins, both of which have potent cytotoxic and inflammatory properties [11] . infection generally leads to an antibody response to the toxin, and most individuals older than 2 years have such antibodies. high levels of these antibodies acquired after colonization may result in the asymptomatic carrier state [12] . some patients with symptomatic infection, many of whom are immunodeficient or immunosuppressed, develop antibiotic-resistant diarrhea; many have low or absent igg antibodies to toxin a. such patients may respond to ivig given 300 to 500 mg/kg every 1 to 3 weeks [13] . such therapy increases antitoxin levels, controls the diarrhea, and prevents relapses [14, 15] . controlled trials have not been performed. botulism (clostridium botulinum). botulism is a severe paralytic poisoning resulting for the ingestion or absorption of neurotoxin or spores of clostridium botulinum. several variants are recognized: food poisoning from ingestion of contaminated canned food, wound botulism from a contaminated soft-tissue infection, inhalational botulism among individuals working with the toxin or in a bioterrorist event, infantile botulism (see next section), and adult-type infant botulism in adults with preexisting gastrointestinal disease [16] [17] [18] . in the last 2 types, ingested spores multiply in the gastrointestinal tract to elaborate toxin; the absorbed toxin results in a paralytic disorder. a few cases of botulism have been associated with use of botulism toxin for cosmetic use [19, 20] . an heptavalent fab fragment equine antitoxin (hbat) to types a, b, c, d, e, f and g is available in the united states through the cdc [21, 22] . sensitivity testing must be conducted before their use. antitoxin to all 3 types is given unless the toxin type is known. additional doses may be needed in severe wound botulism. antitoxin can also by used prophylactically in individuals known to have ingested contaminated food. it is not used for infantile botulism. infantile botulism (clostridium botulinum). this severe paralytic disorder of infants results from the ingestion of clostridium botulinum spores in baby formulae or food, resulting in slow onset of constipation, abdominal bloating, poor feeding, and respiratory paralysis [22] . such infants must be hospitalized for prolonged periods for tube feeding and respiratory support, often for as long as 6 to 9 months. human iv botulism immune globulin is available for treatment of infantile botulism [23] . despite its high cost ($50,000 per vial) it is cost-effective because of the shortened hospital stay needed. gas gangrene (clostridium perfringens). there is no antitoxin for gas gangrene. respiratory infections with streptoccocci, streptococcus pneumonia, haemophilus influenzae, and neisseria meningitides are reduced in immunodeficient patients receiving immunoglobulin therapy. these patients include young infants with poor antibody responses to polysaccharide antigens, patients infected with the human immunodeficiency virus (hiv), and patients with primary antibody immunodeficiencies. before antibiotics, immune serum or animal serum was used as therapy for severe bacterial infection [24, 25] . other studies suggest that a large dose of ivig decreases the frequency of otitis in patients with recurrent otitis and normal immunity [26] . thus regular use of ivig in antibody-deficient patients in doses of 400 to 600 mg/kg every 3 to 4 weeks or an equivalent amount given subcutaneously decreases the frequency and severity of otitis and other respiratory tract infections [27, 28] . circulating antibody may play a role in the prevention and treatment of invasive group a streptococcal infection [29] . newborns with transplacental antibody and patients on ivig rarely develop streptococcal illnesses. equine antitoxin was used with some success in the treatment of erysipelas and scarlet fever in the 1920s and 1930s [30] . a preventive vaccine against the streptococcal m protein has been contemplated but is not yet unavailable. treatment with ivig, in addition to antibiotics, is probably beneficial [25, 31] . streptococcal pyrogenic exotoxins types a, b, and c and mitogenic factor elaborated by certain strains of streptococci may be responsible for these complications. these exotoxins are potent superantigens that activate certain t lymphocytes directly, leading to synthesis and/or release of multiple cytokines with resultant shock, fever, and organ failure. ivig contains neutralizing antibodies to these antigens of varying titers from batch to batch [32] . despite this variability ivig is recommended, in addition to antibiotics, in the management of these infections, not only to neutralize pyrogenic toxins but to dampen cytokine storm and release [33] . controlled trials are unavailable but case reports and large series compared with historical controls are encouraging [34] . large doses of ivig are recommended (eg, 1-2 g/kg over several days). staphylococcal infections are ubiquitous and of varying severity, ranging from superficial skin infections to deep-seated cellulitis, osteomyelitis, and overwhelming shock [35, 36] . these severe infections occur when the organism is resistant to antibiotics or is a strain associated with toxin production. one well-recognized syndrome is toxic shock associated with tampon use in menstruating women [36] . this syndrome results from release of the toxic shock syndrome toxin-1, a potent superantigen that initiates the release of multiple cytokines and a clinical picture of rapidly progressive fever, shock, and organ failure. most authorities recommend a high dose of ivig to neutralize the toxin and dampen cytokine storm [35, 37] . a second situation in which ivig may be of value is in neonatal staphylococcal infection, usually coagulase-negative staphylococcus epidermidis. this is the most common cause of sepsis in premature infants and is aggravated in part by the use of catheters and central lines [38, 39] . one controlled study indicated that ivig was of value in decreasing the incidence of this infection [40] . other studies were not confirmatory, possibly because of differences in titer for the protective antibodies [39] . immunoglobulin is also used in the treatment of antibiotic-resistant staphylococcal infection. older studies from waisbren [41] and current studies from russia suggest clinical benefit [42] . animal studies support such a combined approach [43] . newborns, particularly premature newborns with birth weight less than 2000 g are potential candidates for immunoglobulin therapy in view of the frequency and severity of infections. all newborns have low levels of igm and iga, and, if premature, a deficiency of transplacental maternal igg, the deficiency of which is proportional to the degree of immaturity [44] . premature infants also have defects in antibody synthesis, complement levels, opsonic activity, neutrophil mobilization and killing, and cellular immune responses [44] . accordingly several studies sought to determine the value of igiv in the prevention or early treatment of infection in premature infants. these studies differ in terms of entry criteria, immunoglobulin dose and duration, and end points (eg, type and severity of infection, survival). meta-analyses of prospective, randomized, placebo-controlled prevention studies suggest a slight reduction (3%) in the frequency of sepsis but no difference in mortality, length of nursery stay, or other complications of prematurity [45] [46] [47] . by contrast meta-analysis of 6 controlled studies for the treatment of proven sepsis, involving 262 premature infants, showed that igiv therapy reduced mortality from 20% to 11%, a significant difference [48] . there was a suggestive benefit for infants with suspected sepsis also. infants with neutropenia may particularly benefit. because a common cause of neonatal sepsis is staphylococcus epidermidis, a hyperimmune staphylococcal ivig may be of particular benefit in the prevention of neonatal sepsis. two recent studies of igiv from either immunized donors (altastaph) [49] or selected donors with high titers to a fibrinogen-binding protein (veronate) [50] did not show a significantly decreased incidence of infection. studies of monoclonal antibodies to staphylococcal antigens are in progress. thus the 1990 national institutes of health consensus statement that igiv should not be given routinely to infants of low birth weight but that it may be of value in selected premature newborns with proven or suspected infection remains valid [51] . patients undergoing severe stress associated with trauma, extensive surgery, or intensive care have profound exposure to and susceptibility to infection, usually as a result of enteric gram-negative infections [52, 53] . monoclonal antibodies, igm-enriched igiv, and regular igiv have been studied in these situations with inconclusive results [5] . laupland and colleagues [54] reviewed 14 randomized trials of igiv and found suggestive benefit in terms of length of stay in the intensive care unit (icu) and mortality. similar studies in pediatric patients in the icu have not been performed. despite the lack of controlled trials, igiv is often used in critically ill patients, particularly neutropenic patients, because of possible benefit and rare side effects. although many viral diseases are prevented by immunoglobulin, just a few are amenable to antibody therapy, as presented in table 2 . this section focuses on some viral diseases in which antibodies can be used in therapy. although smallpox (variola) has been eradicated from the world since 1977, immunization with live vaccinia virus (cowpox virus) is still used by the military and by certain laboratory personnel working with vaccinia [5] . further, smallpox is a potential bioterrorism weapon so a supply of vaccinia immune globulin (vig) is being stockpiled by the us government for complications of smallpox vaccine and for a response to biological warfare. kempe [55] used immune globulin from vaccinated individuals (vig) to prevent the spread in a 1953 outbreak of smallpox in madras, india. he also showed that vig could be used to treat the not infrequent complications of smallpox vaccine including vaccinia eczematum, generalized vaccinia, autoinoculation, and prevention of spread to high-risk individuals exposed to a recently vaccinated individual. vig, both for iv and intramuscular (im) use, is prepared from vaccinated donors and is commercially available. the usual dose is 100 mg/kg [56] . parvovirus is a dna virus that causes fifth disease (slapped cheek syndrome, a common exanthem of childhood that usually provides lifelong immunity to subsequent exposure [57] ). parvovirus infects erythroid progenitors (its receptor is the common red cell p antigen) to cause red cell aplasia in patients with congenital or acquired immunodeficiencies including hiv, immunosuppressed organ transplant recipients, and patients with sickle cell disease [57] [58] [59] . igiv contains neutralizing antibody to parvovirus such that prolonged highdose therapy can eradicate the infection. parvovirus infection during pregnancy can also cause fetal hydrops [60] . arthritis and chronic fatigue syndrome are uncommon manifestations of chronic parvovirus infections [60, 61] . the ivig dose needed to eradicate parvovirus in not established but is large (1-2 g/kg) and should be repeated until the virus is eradicated as indicated by serum polymerase chain reaction analysis [62, 63] . antibodies to cytomegalovirus (cmv) either in the form of hyperimmune iv cmv immune globulin (cmvig-cytogam) or regular igiv have been used for more than a decade to prevent cmv infection in recipients of bone marrow and solid organ transplant [64] . cmvig is prepared from donors with high anti-cmv titers but regular igiv also contains cmv antibodies at lower titers. testing of donor and recipient for cmv infection, the use of cmv antibodynegative blood donors, and the use of antiviral drugs have greatly reduced the indications for cmv antibody [65] . cmvig is still used in heart and heartlung transplants (along with antivirals) if either the donor or the recipient is cmv-seropositive [66] . cmvig is also of suggestive benefit in severe cmv pneumonitis along with antiviral treatment [67] . cmvig may also be of value for in utero cmv infection; infusions of cmvig were given intraperitoneally at 28 and 29 weeks to a cmv-infected fetus, with possible benefit [68] . nigro and colleagues [69] gave 31 pregnant women with primary cmv infection cmvig during pregnancy; some women received additional cmvig into the amniotic sac or umbilical cord. only one woman gave birth to an infant with cmv infection compared with cmv infection in 7 of 14 infants of control women who did not receive antibody therapy. these data are encouraging but are not from well-controlled studies. thus the use of cmvig in recipients of organ transplant, severe cmv infections, or in utero cmv infections is unproved but of suggestive therapeutic benefit. transplacental maternal antibody has a proven preventive effect in herpes simplex virus (hsv) infection in the newborn period: mothers with a reactivated herpex infection (ie, preexisting infection) during delivery are 10-fold less likely to transmit hsv to their newborn infants during vaginal delivery than are mothers with primary hsv infection acquired during late pregnancy [70] . masci and colleagues [71] used ivig to prevent recurrent genital hsv infection with suggestive benefit. the value of hsv monoclonal antibody or ivig is being evaluated for treatment of disseminated neonatal disease. epstein-barr virus (ebv) antibodies are present in variable titers in ivig, particularly in cmvig, because donors with high titers of cmv often have high titers of ebv. a few patients with posttransplant ebv-induced lymphoproliferative syndrome or hepatitis have been treated successfully with a combination of igiv or cmvig, antiviral therapy and interferon-a [72] [73] [74] . similar results have been achieved in ebv infection in x-linked lymphoproliferative syndrome: such patients have a hereditary predisposition to overwhelming ebv infection [75] . varicella-zoster immune globulin (vzig), available since 1978, is prepared from plasma with high titers to vz virus [76] . the commercial product vari-zig is used for the prevention or modification of susceptible high-risk immunodeficient or immunosuppressed children exposed to chickenpox or shingles. it is also used in susceptible women during late pregnancy, newborn infants whose mother develops chickenpox perinatally, and exposed premature infants of less than 28 weeks' gestation. it is not of benefit in established chickenpox or zoster infection [77] . encephalomyelitis. before poliovirus vaccine was introduced, immunoglobulin was used in the prevention of poliomyelitis [78] . immunodeficient individuals are susceptible to chronic enteroviral encephalitis, usually echovirus or coxsackievirus or less commonly, attenuated poliovirus vaccine strains [79] [80] [81] [82] . regular doses of igiv given to antibody-deficient patients have markedly reduced the frequency of enterovirus encephalitis in these patients. attenuated poliovirus has been replaced in many countries by inactivated (salk) vaccine. high-dose ivig (sufficient to increase the serum igg levels to 1000 mg/ml) has been used successfully in immunodeficient patients with enteroviral encephalomyelitis [80] [81] [82] [83] . some patients have been given intrathecal infusions [80, 81] . not all ivig-treated patients are cured: some may have viral strains for which the ivig has no neutralizing antibody. for these instances typing of the cerebrospinal fluid and treatment with selective ivig units with antibodies to the infecting serotype may be necessary. antiviral therapy with pleconoril has also been used [83] . neonatal enteroviral infection. severe and sometimes fatal disseminated enterovirus infection can develop in neonates [84] [85] [86] . high-dose ivig has been used in such infants with suggested benefit in decreasing the severity of the illness [84] . maternal plasma may also be used in the likelihood that the mother has antibody to the organism involved [85] . ivig has also been used to prevent spread to unaffected infants in a nursery [86] . unless the titer in the ivig is known, large doses are recommended. an increasingly important use of hyperimmune hepatitis b immune globulin (hbig) is to prevent hepatitis b recurrence in hepatitis b-seropositive recipients of liver transplant, many of whom are transplanted because of complications of hepatitis b [87, 88] . hepatitis b reoccurs in half of the patients in 3 years [89] . such recurrences can be reduced significantly by giving large doses of hbig for a prolonged period beginning at the time of transplantation and continuing indefinitely after transplantation [89] . antiviral agents such as lamivudine are also given simultaneously. the dose of hbig after transplantation is varied so as to maintain a continuous serum anti-hbs titer. hepatitis b vaccine can also be given to induce active immunity. the 2 types of hbig available include the 16% igim used for prophylaxis in newborns of hepatic b-positive mothers and for unimmunized exposed susceptibles and a 5% hbig for iv use in liver transplantation. the use of the latter adds a considerable cost to liver transplantation. the university of california at los angeles medical center spends $500,000 per year on hbig, nearly all for the liver transplant program. a hyperimmune hepatitis c immune globulin for hepatitis c liver transplantation is also under study. monoclonal antibodies to hepatitis b and c are under development. west nile fever. west nile fever, caused by the west nile virus, is common in many tropical regions where culex mosquitoes are endemic. it has spread to europe and the united states, and can also be transmitted by infected blood and organ transplantation. several case reports and animal studies suggest that ivig prepared from seropositive donors modifies the severity and mortality [90, 91] . ebola. ebola virus, a filivirus, causes severe and often fatal hemorrhagic fever in tropical africa. there is no effective antiviral agent. goat hyperimmune serum protected guinea pigs from experimental infection if given within 72 hours of exposure. this product was used for emergency prophylaxis in 4 patients exposed by a laboratory accident. only one developed mild infection [92] . equine serum has protected monkeys against low-dose virus challenge but not high-dose virus challenge [93] . blood from convalescing patients has also been used with promising results [94] . other animal antisera have been developed, as have monoclonal antibodies. tick-borne encephalitis. tick-borne encephalitis caused by a flavivirus is endemic in central europe. a vaccine is available as is a hyperimmune immune globulin. a combination has been also used [95, 96] . argentine hemorrhagic fever. argentine hemorrhagic fever caused by the junin virus has a high mortality from vascular or neurologic complications. maiztegui and colleagues [97] found that immune plasma given before the ninth day of illness reduced mortality to 1% among 91 patients given immune plasma compared with 16.5% mortality among 97 patient given normal plasma. severe acute respiratory distress syndrome. convalescent plasma and ivig have been used in the treatment of severe acute respiratory distress syndrome caused by a corona virus. studies were inconclusive [98] . antibody is a time-honored way to prevent viral infection after exposure, and has a crucial role in the treatment of bacterial diseases associated with toxin production. it is also of value in prevention of certain viral infections as well as in the treatment of parvovius, enterovirus infection, and certain regional viral infections. polyclonal immunoglobulin, now used in scores of diverse disorders [99] [100] [101] , was first used in the prevention of infectious diseases. in 1952, ogden bruton [102] reported a child with agammaglobulinemia and initiated the first use of repeat injections of immunoglobulin as replacement therapy. in his report c-globulin fractionated from human plasma was administered subcutaneously to an 8-year-old boy who had no known c-globulin in a serum protein electorophoresis. this child had multiple infections, including 19 episodes of septicemia, which were ameliorated by chronic treatment with the immunoglobulin. this experience represented the dawn of immunoglobulin therapy for primary immunodeficiency and defined its use in a disease for which no therapeutic alterative was available. since then, the study of primary immunodeficiency has expanded markedly. there are now more than 140 distinct diagnoses, most of which have defects of humoral immunity [103] . approximately 1 in 2000 people are living with a primary immunodeficiency in the united states, of whom greater than 50% have an antibody deficiency potentially requiring immunoglobulin replacement therapy [104] . other primary immunodeficiency registries confirm that greater than 50% have an antibody deficiency [105] [106] [107] [108] . treatment with immunoglobulin remains the best therapeutic option for most of these patients. characteristics of antibody immunodeficiencies appropriate for replacement therapy are presented in table 3 . the clearest indications for immunoglobulin therapy are those associated with an absence of b cells (category i). these patients are unable to make antibodies or immunoglobulin i. examples include agammaglobulinemia and certain types of severe combined immunodeficiency. several gene defects may be responsible for these illnesses [109] , but all need immunoglobulin replacement therapy. the next category (ii) of patients needing immunoglobulin are those who have b cells but cannot make igg and generate specific igg antibodies. because igg represents the major defense of humoral immunity against infection, these patients also require immunoglobulin replacement therapy. this diagnostic category includes the hyper igm syndrome (higm) and common variable immunodeficiency (cvid). higm is caused by several specific gene mutations [110] , but most cvid cases have no identifiable genetic lesions [111] . diagnosis can be made by either identifying a specific gene mutation, or by defining the quantitative and qualitative deficit of igg [112] . as in patients in category i, continuous and uninterrupted replacement therapy with immunoglobulin is warranted. if the diagnosis is confirmed molecularly, immunoglobulin therapy must be continued. in a few cases, it may be clinically appropriate to stop immunoglobulin therapy once during a lifetime to determine if the defect is fixed [1] . this strategy should not be repeated if the single trial indicates a persistent deficit. if a trial off immunoglobulin therapy is considered, this should be performed in late spring or summer, when respiratory infections are less prevalent. a third diagnostic category (iii) of antibody deficiencies is those associated with qualitative defects in humoral immunity [113] . these patients have b cells and produce normal quantities of igg but the quality of igg is diminished. these individuals are unable to respond appropriately to specific antigenic challenges such as vaccinations or infections. this category includes those with specific antibody deficiency with normal immunoglobulins [113] and certain patients with nemo (nf-kappa;-b essential modulator) deficiency [114, 115] . diagnosis is made after documentation of an ineffective vaccination response, a failed humoral response to an infection, or a specific molecular/ genetic diagnosis linked to this category [112] . a fourth category (iv) includes patients with lower than expected levels of igg but who are able to mount effective antibody responses. this category forms a subset of individuals referred to as having ''isolated hypogammaglobulinemia'' when only the igg level is low. although hypogammaglobulinemia can be a component of many immunologic defects, in isolated hypogammaglobulinemia antibody quality is adequate, with normal responses to vaccination or infection. because the normal age-specific ranges of igg define the lower limit at the 2.5th percentile, one of 40 individuals has low levels of igg. the question becomes, when there is no deficit of antibody quality, is isolated hypogammaglobulinemia clinically a problem? it is also important to discern when hypogammaglobulinemia represents a primary versus a secondary problem with increased loss of igg. examples of the latter include draining chylothorax [116] or intestinal lymphangiectasia [117] . in these individuals, the hypogammaglobulinemia is less likely to cause a problem because antibody synthesis is intact and often accelerated. in patients with primary hypogammaglobulinemia, the level of igg that is associated with a definitive risk for infection is not defined, especially when antibody quality is intact [112] . some insurance companies recommend replacement therapy for patients who have an igg level less than 400 mg/dl and a history of recurrent infection. although that situation may be reasonable, questions still exist about how to manage the patient recognized as having primary hypogammaglobulinemia with low igg levels (ie, <150) but no history of infection. diagnostic examples include transient hypogammaglobulinemia of infancy (thi) [118] [119] [120] or otherwise unexplained primary hypogammaglobulinemia [121] . the former diagnosis is established in retrospect, as the igg level normalizes with age. thus, in select cases of thi immunoglobulin replacement may be considered as a temporizing measure. however, primary hypogammaglobulinemia remains a difficult diagnostic and therapeutic dilemma. other patients (a fifth diagnostic category [v]) have a deficiency of one of the 3 major igg subclasses, igg1, igg2, or igg3. igg4 deficiency is common and should not be considered an abnormality [99] . although a deficiency of one of the major igg subclasses indicates some immunologic deviation, most of these patients have a normal total igg level, intact responses to specific antigens, and are not candidates for immunoglobulin replacement therapy. those with impaired antibody specificity do not fall in to this category, but into the third category. however, even without impairment in antibody quality, immunoglobulin replacement in some patients in a deficiency subclass does reduce the incidence of infections [122, 123] . nevertheless, most insurers in the united states have additional criteria for justifying therapy in patients with igg in deficiency subclasses. a final diagnostic category is patients with recurrent infection who do not have hypogammaglobulinemia subclass deficiency or deficits of antibody quality. thus, they have infectious susceptibility without evidence of identifiable immune abnormality. the infectious burden in these individuals can be high and most certainly has an explanation, so nonhumoral diagnoses should be aggressively sought. there are also patients an explanation of whose infectious susceptibility presently evades clinical science. immunoglobulin replacement therapy has been considered in these individuals under certain circumstances. immunoglobulin preparations for antibody immunodeficiencies although bruton [102] gave immunoglobulin to his patient by the subcutaneous (sc) route, subsequent patients until 1970 received immunoglobulin by weekly im injections [124] . this strategy was necessary because the immunoglobulin preparations were not purified to the degree required for iv administration. in the early 1970s, immunoglobulin preparations with low quantities of immunoglobulin aggregates were developed for iv administration. ivig and igiv have numerous advantages, including achieving high peak and trough igg levels and convenient monthly dosing regimens. although limited studies have compared ivig with imig, the iv route has become the preferred route of immunoglobulin administration worldwide [125] . seven ivig preparations are currently approved by the us food and drug administration (fda) for replacement therapy in primary immunodeficiency (table 4 ). each has been studied in a licensing trial in patients with primary immunodeficiency and found to be safe and effective. the primary end point in most of these clinical trials has been the prevention of serious bacterial infection compared with the expected frequency of such infections before diagnosis [126] . the rate of infection can be surprisingly high, as shown by bruton's [102] first patient mentioned earlier. the early diagnosis and treatment of primary immunodeficiency with immunoglobulin products has reduced morbidity and mortality and considerable savings of health care expenditures [127, 128] . all ivig products are purified from human plasma pools under strict manufacturing guidelines. although each manufacturer has its own process there are more similarities than differences in the various methods. all processes remove non-igg impurities and igg aggregates and add stabilizers to prevent in vitro aggregate formation. despite these efforts, adverse reactions during ivig administration are not uncommon [129] . all immunoglobulin manufacturers have robust measures to screen donors and to inactivate blood-borne pathogens; the safety of immunoglobulin preparations in the last decade has been superb [130] . there are subtle differences among different ivig products from different companies; several companies have more than one product on the market [131] . this situation can lead to confusion about which ivig to administer to which patient. in general, most ivig products are tolerated by most patients. the characteristics of the individual ivig preparations, as outlined in table 2 , may help in selecting the best product for each patient. they differ as to concentration, stabilizers, sugar content, iga content, sodium content, and osmality. the volume of individual vials, storage requirements, need to reconstitute a lyophilized product before use, local availability, and price are also variable. many patients may tolerate one product more effectively than another. thus, when a patient tolerates a particular immunoglobulin product it is advisable to continue with that product whenever possible [99] . three other preparations of immunoglobulin are approved by the us fda (table 5) . one is approved for im administration and two for sc administration. few patients receive their immunoglobulin by the im route. the sc administration of immunoglobulin resurfaced in 1980 in the united states [132] . subcutaneous immunoglobulin (scig) is usually given in the abdominal wall or thigh with a thin bored needle and an infusion pump, delivered over several hours. although its initial use in the united states was limited, the sc route gained popularity in europe; extensive clinical experience indicated that it was equivalent to ivig therapy [133, 134] . a crossover trial with ivig and a us fda licensing trial showed that scig was equivalent to ivig in preventing infection in primary immunodeficiency [135, 136] . scig has advantages and disadvantages compared with ivig therapy (many related to patient preferences) and these have been reviewed extensively [137] [138] [139] . one advantage of scig over ivig is the markedly decreased incidence of systemic reactions [134, 135] . another is eliminating the need for iv access or indwelling iv access devices. the most serious disadvantage is the need for more frequent administration (at least weekly) to administer sufficient immunoglobulin [140] . another disadvantage is less frequent physician encounters because most scig infusions are given at home by caretakers or home infusion companies. the dose and frequency of immunoglobulin therapy is a complex topic and draws on both evidence-and experience-based sources. these recommendations are presented in a several reviews and consensus statements [99, 112, 138, 139, 141] . the recommendations include starting doses of 400 to 600 mg/kg/mo. after several months this dose can be altered depending on the trough level and the clinical response. patients vary as to their requirement to maintain reasonable resistance to infection [141, 142] . scig is typically used after the patient has been on ivig for several months. the weekly scig dose is usually one-fourth of the previous monthly ivig dose. some immunoglobulin-naive patients are started on immunoglobulin therapy with scig so the number of initial doses may need to be increased. the amount of scig given at a single site for an adult is usually 20 ml of the 16% solution (ie, 3.2 g). more than one site can be used simultaneously to deliver the target dose. this procedure has been facilitated by the availability of special tubing, needle sets, catheters, and pumps. infusion site reactions are not uncommon but are rarely severe [136] . ivig is usually administered monthly and scig is usually administered weekly, but other schedules are often used. these schedules include shorter or longer intervals between infusions of ivig to achieve a satisfactory clinical response. scig can be given biweekly, or divided into more frequent injections, even small daily doses. the latter is generally self-administered at home, well tolerated, and preferred by some patients because of the small daily dose needed [143, 144] . trough levels of immunoglobulin achieved must be considered. several studies have correlated resistance to infection with specific igg trough levels. targeting a specific trough level may be feasible for patients with agammaglobulinemia who have a profound deficiency of igg [126] but more difficult for other antibody deficiencies [145] [146] [147] . in agammaglobulinemia, a trough level of 500 mg/dl is a minimally acceptable level and 800 mg/dl a more desirable trough level [99, 126] . these recommendations may not be appropriate in other disorders in which baseline igg levels and antibody titers are variable; in these cases the clinical response must be considered. polyclonal immunoglobulin is essential therapy for the primary antibody immunodeficiency diseases. the different disorders in which immunoglobulin therapy are used are reviewed. several immunoglobulin products are available for their treatment; they have similar therapeutic properties but there are individual differences among the available products. immunoglobulin can be given either intravenously (ivig) or subcutaneously (scig). dosage, frequency of infusions, achieved trough levels, and advantages and disadvantages of ivig and scig are discussed. the early 1980s witnessed an increase in the use of ivig as an immunomodulator for inflammatory and autoimmune disorders. more than 70% of the ivig prescribed is for patients with autoimmune and inflammatory diseases, despite the fact that ivig is approved for just a handful of indications (box 1). in the late 1990s, this situation led to an ivig shortage, compromising those patients who depend on igg replacement therapy to correct their underlying antibody deficiency. in 2006, the american academy of allergy, asthma and immunology's committee on primary immunodeficiency evaluated the use of ivig for multiple disorders. the strength of the evidence for a beneficial effect and the basis for this recommendation were classified (box 2). this section reviews the use of ivig for the autoimmune and inflammatory conditions in this report (box 3), in the context of a review of the mechanisms of action of ivig in these conditions. the multiple effects of ivig on the innate and adaptive immune system are illustrated in fig. 1 . historical note: ivig in immune thrombocytopenic purpura the first use of ivig for an autoimmune process was in children with immune thrombocytopenic purpura (itp). imbach and colleagues [148] observed that antibody-deficient patients receiving ivig who also had itp had a marked increase in platelet count after ivig infusions. subsequently, these investigators examined the therapeutic effects of ivig in children with a primary diagnosis of itp; they used high-dose ivig (400 mg/kg) for 4 consecutive days. the investigators reported a dramatic increase in platelet count within hours of the administration of ivig. in some patients, the increase in platelet count was sustained; in others, repeat ivig treatments were necessary. box 3 presents the indications for ivig in autoimmune cytopenias as well its likely benefit. several hypotheses have been proposed to explain the rapid increase in platelet count (or other antibody-coated cells) after ivig administration. the most accepted hypothesis is that high-dose ivig induces an fc receptor blockade of reticuloendothelial cells in the liver and spleen, preventing them from removing antibody-sensitized cells. debre and colleagues [149] provided evidence for this hypothesis when they infused fcc fragments in children with itp, and showed an increase in platelet count after the infusion. the fc receptor blockade theory may account for the rapid increase in platelet count after the ivig infusion, but not for the longterm benefits of ivig. thus additional mechanisms have been sought. one such mechanism, supported by animal studies, is that ivig stimulates inhibitory fccriib receptors found on a variety of cell types including b cells that in turn inhibit antibody and immune function [150] . samuelsson and colleagues [151] showed in a mouse model of itp that ivig suppresses or inhibits antiplatelet antibody production through this fccriib receptor. subsequently ravetch and colleagues [152, 153] identified distinct motifs in the ivig that have a propensity to engage and activate the fccriib inhibitor receptor that inhibits antibody synthesis. these distinct properties were attributed to the carbohydrate moiety in the ivig molecule, representing about 5% of the total igg molecule. more than 30 different covalently attached carbohydrate glycans in the igg molecule have been identified. glycosylation of the igg is essential for binding to all fcc receptors. the important glycan moiety in the igg molecule is attached to the asparagine (asn 297 ) in the second domain of the constant region of the igg molecule. using a k/bxn serum-induced arthritis model in mice, kaneko [152] showed that igg at 1 g/kg inhibited the inflammatory arthritic process. deglycosylated or neuraminidase-treated ivigs were unable to inhibit this inflammation. kaneko then showed that ivig enriched for the sialylated glycan moiety had comparable inhibitory effects on the inflammatory process at one-tenth of the dosage used with intact ivig. this investigator showed that this inhibitory activity resided in the igg fc fragment, and was dependent on fccriib expression on effector macrophages. anthony and colleagues [154] have engineered a recombinant/sialylated human igg1 fc protein that had the same immune modulating activity as native ivig. these investigators showed that the action of sialylated fc in the rheumatoid arthritis mouse model is mediated through the interaction of sialylated fc with the sign-r1 receptor on macrophages [154] . the investigators propose that the interaction between sialylated fc and sign-r1 produces an antiinflammatory state that upregulates inhibitory fccriib receptors on effector cells, making these cells more resistant to triggering by immune complexes. they suggest that dc-sign, the human homolog of sign-r1, has a comparable role for the antiinflammatory effects of igg fc fragments. another mechanism proposed by yu and lennon [155] suggested that the administration of high-dose ivig augments the catabolism of endogenous serum igg. igg catabolism occurs through a process by which the igg molecule binds to a specialized fc receptor found on endothelial cells (eg, fcrn), which protects the igg molecule from normal catabolism and its removal from the plasma. this process accounts for the long serum igg half-life (21 days). high-dose ivig saturates the fcrn receptor, resulting in the accelerated catabolism of autoantibodies [156] [157] . hansen and balthasar [157] have supporting data in a rat model of immune thrombocytopenia using monoclonal antibodies. the uses of ivig in several autoimmune inflammatory neuropathies are presented in box 3. the fda has recently approved the use of ivig in chronic inflammatory demyelinating polyneuropathy. this table also shows the evidence-based efficacy of ivig in rheumatic disorders. aside from the mechanisms involving the fccriib inhibitory receptor and the accelerated catabolism of autoimmune antibodies through the fcrn receptor, it has also been proposed that the administration of ivig can regulate autoreactive b cells by restoring the idiotypic-antiidiotypic network. other autoimmune diseases may be associated with a deficiency of these antiidiotypic antibodies, which are believed to regulate the production and activity of these autoantibodies (box 4). kazatchkine and colleagues [158] showed that f(ab 9 ) 2 fragments prepared from ivig could bind to several autoantibodies (eg, antifactor viii, antithyroglobulin, anti-dna, antiintrinsic factor, neutrophil cytoplasmic antigens), and thus lead to increased catabolism of these autoimmune antibodies and prevent them from inducing tissue injury [159] . these investigators postulated that ivig may work, at least in part, in certain autoimmune diseases by neutralizing the functional activity of various autoantibodies or inhibiting their binding to their respective autoantigens [160] . another mechanism by which ivig may benefit autoimmune disease is by preventing the uptake of complement on target tissues. berger and colleagues [161] showed that high concentrations of igg inhibit the uptake of c3 on antibodysensitized erythrocytes. thus, any inflammatory or autoimmune process that involves a c3b-or c4b-dependent process could be modulated by ivig therapy. this situation is best exemplified in patients with dermatomyositis in whom the disease is mediated by activation of c3 and deposition of the membrane attack complex on the endomysial capillaries [162] . treatment with ivig inhibits complement-induced inflammation by decreasing complement deposition on the endomysial capillaries of muscle tissues [163, 164] . this mechanism of ivig is relevant not only in dermatomyositis but also in guillain-barrã© syndrome and myasthenia gravis [165, 166] . as shown in box 3, ivig is used in many other inflammatory diseases. however, the evidence-based data for several of these diseases are not so strong as some of the autoimmune disorders discussed earlier. nevertheless, one inflammatory disease in which ivig may be beneficial is toxic epidermal necrolysis or stevens-johnson syndrome. patients with toxic epidermal necrolysis have high levels of serum-soluble fas ligand that bind to fas receptors on keratinocytes to induce apoptosis (cell death). viard and colleagues [167] showed that the anti-fas antibodies in ivig block the interaction of fas ligand with fas receptors on the keratinocytes, preventing destruction of the epithelium. ivig contains antibodies to several cell-surface molecules [168] including antibodies to a 10-peptide sequence containing the (arg-gly-asp) motif that is expressed on cell surfaces and matrix proteins that are part of the integrin adhesion system. ivig inhibits the adhesion of b cells to fibronectin and inhibits platelet aggregation [169] . turhan and colleagues [170] and chang and colleagues [171] investigated the effect of ivig on a mouse model of sickle cell acute vasoocclusive crisis, in which the adhesion of sickled red blood cells to leukocytes causes the vasoocclusive disease. in this model, high-dose ivig given after the onset of a crisis resulted in improved blood flow and prolonged survival. these investigators showed that ivig reverses acute vasoocclusive crisis in sickle cell mice by inhibiting neutrophil adhesion to the capillary endothelial cells. the various mechanisms of the antiinflammatory and immunomodulatory properties of ivig are reviewed. the first use of ivig was in the treatment of immune thrombocytopenia, presumably because of fc receptor blockade. other mechanisms are reviewed as well as the evidence for the value of ivig in multiple disorders. ivig may have yet undiscovered immunomodulating properties on both the innate and adaptive immune systems. future advances will include a better understanding of its mechanisms of action and modification of the igg molecule to enhance its immunomodulating properties. idiopathic thrombocytopenic purpura (ia-a) might provide benefit autoimmune neutropenia autoimmune hemolytic anemia (iii-d) posttransfusion purpura (iii-d) inflammatory neuropathies definitely beneficial: guillain-barrã© syndrome (ia-a) chronic inflammatory demyelinating polyneuropathy multifocal motor neuropathy (ia-a) probably beneficial: myasthenia gravis (ib-iia-b) eaton myasthenic syndrome (ib-a) igm antimyelin-associated glycoprotein paraprotein-associated peripheral neuropathy (ib-a) stiff man syndrome (ib-a) might provide benefit: relapsing-remitting multiple sclerosis (ia-a) intractable childhood seizures rasmussen syndrome (iib-b) acute disseminated encephalomyelitis (iii-c) lumbosacral or brachial plexitis human t-lymphotropic virus-1-associated myelopathy (iii-c) postinfectious cerebellar ataxia acute idiopathic dysautonomia (iii-d) unlikely to be beneficial: demyelinating neuropathy associated with monoclonal igm (ib-a) amyotrophic lateral sclerosis (iii-c) poems syndrome paraneoplastic neuropathies (iii-c) rheumatologic and organ-specific autoimmune diseases definitely beneficial: graves ophthalmopathy (ib-a) probably beneficial: autoimmune uveitis (iia-b) might provide benefit: severe rheumatoid arthritis autoimmune diabetes mellitus (iib-b) vasculitides and antineutrophil antibody syndromes systemic lupus erythematosus (iii-d) unlikely to be beneficial: antiphospholipid antibody syndrome toxic epidermal necrolysis/ stevens-johnson syndrome (iia-b) might provide benefit: steroid-dependent asthma (ib-a) prevention of acute humoral rejection in renal transplants (ib-a) treatment of acute humoral rejection in renal transplants pediatric autoimmune neuropsychiatric disorder associated with streptococcus (pandas) (iib-b) subset of women (repeat second-trimester loss) with spontaneous recurrent abortions unlikely to be beneficial: nonsteroid-dependent asthma (ib-a) prevention of chronic gvhd after bone marrow transplantation (ib-a) chronic fatigue syndrome atopic dermatitis (iia-b) use of intravenous immunoglobulin in human disease: a review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology principles and practice of infectious diseases studies on anthrax: clinical report of ten human cases hhs to buy 20,000 courses of anthrax antitoxin. available at: cidrap.umn raxibacumab or the treatment of inhalational anthrax philadelphia: saunders/elsevier report of the committee on infectious diseases history of the american pediatric society 1887-1965 report of the committee on infectious diseases anti-tetanus toxoid antibodies in intravenous gamma globulin: an alternative to tetanus immune globulin treatment of clostridium difficile infection clostridium difficile-associated diarrhea and colitis: clinical manifestations, diagnosis, and treatment asymptomatic carriage of clostridium difficile and serum levels of igg antibody against toxin intravenous immunoglobulin for the treatment of severe, refractory, and recurrent clostridium difficile diarrhea treatment with intravenously administered gamma globulin of chronic relapsing colitis induced by clostridium difficile toxin intravenous immunoglobulin therapy for severe clostridium difficile colitis botulinum toxin as a biological weapon: medical and public health management syndrome of infant botulism handbook for epidemiologists, clinicians and laboratory workers botulism in 4 adults following cosmetic injections with an unlicensed, highly concentrated botulinum preparation severe botulism after focal injection of botulinum toxin investigational heptavalent botulinum antitoxin (hbat) to replace licensed botulinum antitoxin ab and investigational botulinum antitoxin e botulism and infant botulism human botulism immune globulin for the treatment of infant botulism treatment of haemophilus influenzae infection and of meningococcic and pneumococcic meningitis return to the past: the case for antibody-based therapies in infectious diseases respiratory syncytial virus-enriched globulin for the prevention of acute otitis media in high risk children prophylactic intravenous immunoglobulin in hivinfected children with cd4 ã¾ counts of 0.20 ã� 10 9 /l or more: effect on viral, opportunistic, and bacterial infections national institute of child health and human development (nichhd) intravenous immunoglobulin study group. intravenous immune globulin for the prevention of bacterial infections in children with symptomatic human immunodeficiency virus infection passive antibody therapies: progress and continuing challenges antitoxin versus no antitoxin in scarlet fever adjunctive treatment of streptococcal toxic shock syndrome using intravenous immunoglobulin: case report and review different preparations of intravenous immunoglobulin vary in their efficacy to neutralize streptococcal superantigens: implications for treatment of streptococcal toxic shock syndrome clinical usefulness of intravenous human immunoglobulins in invasive group a streptococcal infections: case report and review intravenous immunoglobulin for streptococcal toxic shock syndrome-a comparative observational study report of the committee on infectious diseases vaginal tampon model for toxic shock syndrome philadelphia: saunders/elsevier opsonic antibodies to staphylococcus epidermidis: in vitro and in vivo studies using human intravenous immune globulin the role of intravenous immunoglobulin for the prevention and treatment of neonatal sepsis intravenous immune globulin for the prevention of nosocomial infection in low-birth-weight neonates the treatment of bacterial infections with the combination of antibiotics and gamma globulin immunotherapy against antibiotic-resistant bacteria: the russian experience with an antistaphyloccocal hyperimmune plasma and immunoglobulin synergism between human gamma globulin and chloramphenicol in the treatment of experimental bacterial infections the physiologic immunodeficiency of immaturity meta-analyses of the effectiveness of intravenous immune globulin for prevention and treatment of neonatal sepsis administration of intravenous immunoglobulins for prophylaxis or treatment of infection in preterm infants: meta-analysis intravenous immunoglobulin for preventing infection in preterm and/ or low birth-weight infants (cochrane review). the cochrane library intravenous immunoglobulin for suspected or subsequently proven infection in neonates a blinded, randomized, multicenter study of an intravenous staphylococcus aureus immune globulin multicenter study to assess safety and efficacy of inh-a21, a donor-selected human staphylococcal immunoglobulin, for prevention of nosocomial infections in very low birth weight infants nih consensus development conference: diseases, doses, recommendations for intravenous immunoglobulin. hlb newsletter polyvalent immunoglobulins for prophylaxis of bacterial infections in patients following multiple trauma the secondary immunodeficiencies polyclonal intravenous immunoglobulin for the treatment of severe sepsis and septic shock in critically ill adults: a systematic review and meta-analysis studies on smallpox and complications of smallpox vaccination smallpox (variola) doling r, editors. mandell, douglas and bennett's principles and practice of infectious diseases parvovirus b19 infection: aplastic crisis, erythema infectiosum and idiopathic thrombocytopenic purpura parvovirus infection and its treatment report of the committee on infectious diseases persistent parvovirus-associated chronic fatigue treated with high dose intravenous immunoglobulin persistent b19 parvovirus infection in patients infected with human immunodeficiency virus type 1 (hiv-1): a treatable cause of anemia with aids chronic pure red cell aplasia caused by parvovirus b19 in aids: use of intravenous immunoglobulin: a report of eight patients cytomegalovirus infection in kidney transplantation: prophylaxis and management clinical practice guidelines: prevention of cytomegalovirus disease after renal transplantation comparison of combined prophylaxis of cytomegalovirus hyperimmune globulin plus ganciclovir versus cytomegalovirus hyperimmune globulin alone in high-risk heart transplant recipients immunotherapy of cmv infections intraperitoneal administration of cytomegalovirus hyperimmunoglobulin to the cytomegalovirus-infected fetus passive immunization during pregnancy for congenital cytomegalovirus infection report of the committee on infectious diseases intravenous immunoglobulins suppress the recurrences of genital herpes simplex virus: a clinical and immunological study successful treatment of epstein-barr virus infection with ganciclovir and cytomegalovirus hyperimmune globulin following kidney transplantation treatment with ganciclovir and ig for acute epstein-barr virus infection after allogeneic bone marrow transplantation the effect of intravenous immunoglobulin and interferonalpha on epstein-barr virus-induced lymphoproliferative disorder in a liver transplant recipient immune-mediated hematologic and oncologic disorders, including epstein-barr virus infection passive immunization against varicella zoster infections varicella-zoster infections experimental studies on passive immunization against poliomyelitis: i. protection with human gamma globulin against intramuscular inoculation and combined passive and active immunization chronic enteroviral meningoencephalitis in agammaglobulinemic patients intraventricular gamma-globulin for the management of enterovirus encephalitis successful treatment of echovirus meningoencephalitis in sex-linked agammaglobulinaemia by intrathecal and intravenous injection of high titer gammaglobulin chronic enteroviral meningoencephalitis, in agammaglobulinemia: case report and literature review enteroviral meningitis: natural history and outcome of pleconaril therapy neonatal enterovirus infection: virology, serology, and effects of intravenous immune globulin investigation of treatment failure in neonatal echovirus 7 infection use of normal immunoglobulin in an echovirus 11 outbreak in a special-care baby unit prophylaxis in liver transplant recipients using a fixed dosing schedule of hepatitis b immunoglobulin liver transplantation in hbsag-positive hbv-dnanegative cirrhotics: immunoprophylaxis and long term outcome hbv-infection in liver transplantation in hbsag positive patients: experience with long-term immunoprophylaxis hyperimmune gammaglobulin for the treatment of west nile virus encephalitis using high titer west nile intravenous immunoglobulin from selected israeli donors for treatment of west nile virus infection preparation and use of hyperimmune serum for prophylaxis and therapy of ebola virus infections evaluation of immune globulin and recombinant interferon-a2b for treatment of experimental ebola virus infections treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients tick-borne encephalitis vaccination against tick-borne encephalitis (tbe): influence of simultaneous application of tbe immunoglobulin on seroconversion and rate of adverse events efficacy of immune plasma in treatment of argentine haemorrhagic fever and association between treatment and a late neurological syndrome sars: systematic review of treatment effects use of intravenous immunoglobulin in human disease: a review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology intravenous immunoglobulin utilization in the canadian atlantic provinces: a report of the atlantic collaborative intravenous immune globulin utilization working group prescribing intravenous immunoglobulin: summary of department of health guidelines primary immunodeficiency diseases: an update from the international union of immunological societies primary immunodeficiency diseases classification committee population prevalence of diagnosed primary immunodeficiency diseases in the united states primary immunodeficiency diseases in latin america: the second report of the lagid registry primary immunodeficiency diseases in australia and new zealand primary immunodeficiency syndromes in italy: a report of the national register in children and adults primary immunodeficiency diseases in norway primary b cell immunodeficiencies: comparisons and contrasts defects of class-switch recombination common variable immunodeficiency: an update on etiology and management practice parameter for the diagnosis and management of primary immunodeficiency natural history of selective antibody deficiency to bacterial polysaccharide antigens in children hypomorphic nuclear factor-kappab essential modulator mutation database and reconstitution system identifies phenotypic and immunologic diversity human nuclear factor kappa b essential modulator mutation can result in immunodeficiency without ectodermal dysplasia acute chylothorax in children: selective retention of memory t cells and natural killer cells intestinal lymphangiectasia: a protein-losing enteropathy with hypogammaglobulinemia, lymphocytopenia and impaired homograft rejection the outcome of patients with hypogammaglobulinemia in infancy and early childhood impaired specific antibody response and increased b-cell population in transient hypogammaglobulinemia of infancy infants presenting with recurrent infections and low immunoglobulins: characteristics and analysis of normalization therapy for patients with recurrent infections and low serum igg3 levels efficacy of intravenous gammaglobulin for immunoglobulin g subclass and/or antibody deficiency in adults the gamma globulins. iv. therapeutic uses of gamma globulin efficacy of intravenous immunoglobulin in primary humoral immunodeficiency disease early and prolonged intravenous immunoglobulin replacement therapy in childhood agammaglobulinemia: a retrospective survey of 31 patients impact of a physician education and patient awareness campaign on the diagnosis and management of primary immunodeficiencies pharmacoeconomics of immunoglobulins in primary immunodeficiency adverse reactions and pathogen safety of intravenous immunoglobulin european surveillance of immunoglobulin safety-results of initial survey of 1243 patients with primary immunodeficiencies in 16 countries differences between igiv products: impact on clinical outcome immunoglobulin replacement therapy by slow subcutaneous infusion subcutaneous immunoglobulin replacement in patients with primary antibody deficiencies: safety and costs home treatment of hypogammaglobulinaemia with subcutaneous gammaglobulin by rapid infusion the comparison of the efficacy and safety of intravenous versus subcutaneous immunoglobulin replacement therapy safety and efficacy of self-administered subcutaneous immunoglobulin in patients with primary immunodeficiency diseases subcutaneous immunoglobulin replacement in primary immunodeficiencies subcutaneous administration of igg subcutaneous immunoglobulin replacement therapy for primary antibody deficiency: advancements into the 21st century pharmacokinetics of immunoglobulin administered via intravenous of subcutaneous routes individualizing the dose of intravenous immune serum globulin for therapy of patients with primary humoral immunodeficiency biologic igg level in primary immunodeficiency disease: the igg level that protects against recurrent infection replacement igg therapy and self-therapy at home improve the health-related quality of life in patients with primary antibody deficiencies quality of life and health-care resource utilization among children with primary immunodeficiency receiving home treatment with subcutaneous human immunoglobulin the effect of two different dosages of intravenous immunoglobulin on the incidence of recurrent infections in patients with primary hypogammaglobulinemia. a randomized, double-blind, multicenter crossover trial comparison of the efficacy of igiv-c, 10% (caprylate/chromatography) and igiv-sd, 10% as replacement therapy in primary immune deficiency. a randomized double-blind trial efficacy of intravenous immunoglobulin in the prevention of pneumonia in patients with common variable immunodeficiency high dose intravenous gammaglobulin for idiopathic thrombocytopenic purpura in childhood infusion of fc gamma fragments for treatment of children with acute immune thrombocytopenic purpura fcg receptor as regulators of immune responses anti-inflammatory activity of ivig mediated through the inhibitory fc receptor anti-inflammatory activity of immunoglobulin g resulting from fc sialylation recapitulation of ivig anti-inflammatory activity with a recombinant igg fc identification of a receptor required for the anti-inflammatory activity of ivig mechanism of intravenous immune globulin therapy in antibody-mediated autoimmune diseases accelerated autoantibody clearance by intravenous immunoglobulin therapy: studies in experimental models to determine the magnitude and time course of the effect effects of intravenous immunoglobulin on platelet count and antiplatelet antibody disposition in a rat model of immune thrombycytopenia v region-mediated selection of autoreactive repertoires by intravenous immunoglobulin (ivig) the concept of anti-idiotypic regulation of selected autoimmune diseases by intravenous immunoglobulin anti-idiotypic suppression of autoantibodies to factor viii (antihaemophilic factor) by high-dose intravenous gammaglobulin intravenous and standard immune serum globulin preparations interfere with uptake of 125i-c3 onto sensitized erythrocytes and inhibit hemolytic complement activity modulation of complement-mediated immune damage by intravenous immune globulin intravenous immune globulin for dermatomyositis controlled studies with high-dose intravenous immunoglobulin in the treatment of dermatomyositis, inclusion body myositis, and polymyositis increased in vitro uptake of the complement c3b in the serum of patients with guillain-barrã© syndrome, myasthenia gravis and dermatomyositis intravenous immune globulin therapy for neurologic diseases inhibition of toxic epidermal necrolysis by blockade of cd95 with human intravenous immunoglobulin intravenous immunoglobulin: an update on the clinical use and mechanisms of action inhibition of cell adhesion by antibodies to arg-gly-asp (rgd) in normal immunoglobulin for therapeutic use intravenous immune globulin prevents venular vasoocclusion in sickle cell mice by inhibiting leukocyte adhesion and the interactions between sickle erythrocytes and adherent leukocytes intravenous immunoglobulins reverse acute vaso-occlusive crises in sickle cell mice through rapid inhibition of neutrophil adhesion key: cord-018545-fk17n2bx authors: dorofaeff, tavey; mohseni-bod, hadi; cox, peter n. title: infections in the picu date: 2012 journal: textbook of clinical pediatrics doi: 10.1007/978-3-642-02202-9_268 sha: doc_id: 18545 cord_uid: fk17n2bx nan effective control of infections starts at the community level, outside the hospital. there are a number of important initiatives that, although simple and not necessarily intensive care related, have the greatest impact on the outcomes of infections. these include provision of adequate and age appropriate foods, breast feeding, drinkable water, provision of mosquito nets and shelter, avoidance of overcrowding, sanitization, and prevention of disease by vaccination. these are basic needs and requirements of mankind as a basis of good health. they are attainable in the largest cities or the most remote areas. infections are one of the commonest causes of mortality in the pediatric intensive care unit (picu), with a mortality of up to 50%, depending on the origin of the infection. infections in the intensive care unit can be divided into those that occur outside the hospital (community acquired) and those that occur within the walls of the hospital and beyond 48 h of admission (nosocomial). preventive measures give the most benefit, both outside and inside the hospital. good hand washing, good respiratory care practice, and judicious use of antibiotics are examples of effective interventions that reduce the rate of nosocomial infections. sepsis comprises up to 25% of admissions to a typical pediatric intensive care unit. shock and management of septic shock are discussed elsewhere in this text. however basic principles of management are the same and are not, and should not be, limited to the intensive care unit. treatment should commence as soon as the recognition of any septic process is underway be it in the field, the clinics, emergency departments, or on the wards. in the community, on the hospital wards, and in the picu, timely identification of illness and access to skilled healthcare personnel are crucial steps limiting the development of organ dysfunction and failure. early identification means early resuscitation and early treatment. this may be hours or in some cases days prior to the admission to the picu. this early recognition and intervention gives the patient the greatest chance of surviving a significant infection. crucial to the management of any serious infection in the intensive care unit and elsewhere is the early use of appropriate antibiotics. early identification of most bacteria is almost universally by the way of a gram stain. this can be performed in any microbiology lab, in the field, or a clinic that is suitably equipped. though references such as the ''red book'' (american academy of pediatrics) give invaluable information on the appropriate antimicrobial therapy for a given microbe or infectious syndrome, there is no substitute for a well informed, up-to-date infectious diseases physician or microbiologist. they are able to provide information on local isolates, patterns of sensitivity, and best management practices for a large variety of infections. the majority of the bacteria listed below will be referenced elsewhere; they are highlighted to reflect their frequency of identification in the picu. additionally, the immunocompromised host will be at risk from a number of opportunistic infections that will be discussed later in this chapter. these bacteria have a tendency for multiple antibiotic resistances. there are a number of other medically significant bacteria that only periodically present in the pediatric intensive care unit. these bacteria are prevalent in some regions and not elsewhere. they will not be discussed further in this chapter. fungi (of importance in the picu) classification • yeasts (e.g., candida or cryptococcus sp.) • molds -filamentous fungi (e.g., aspergillus sp. and trichophyton sp.) • dimorphic fungi -yeasts in tissue but grow in vitro as molds (e.g., histoplasmosis) candida. c. albicans has the highest incidence in the critical care environment followed by c. parapsilosis, c. tropicalis, c. glabrata, and c. krusei. localized and systemic infections in neonates and immunocompromised children. any organ can be involved: mucous membranes, larynx, esophagus, brain, eyes, lungs, heart, kidneys, liver, and spleen. aspergillus. a. fumigatus is the most common species in invasive aspergillosis, followed by a. flavus, and a. nigra. localized and systemic infection in immunocompromised children (particularly post stem cell transplant and in children with aml); in the skin, subcutaneous tissues, nasopharynx, lungs, brain, and virtually any other organ. endemic mycosis (histoplasmosis, blastomycosis). pneumonitis, hepatosplenomegaly, fever in children with t-cell dysfunction and in those with hiv infection. coccidioides immitis. pneumonia, meningitis in children with t-cell dysfunction and in those with hiv infection. cryptococcus neoformans. pneumonia, meningitis in children with t-cell dysfunction and in those with hiv infection. human herpes viruses 1. herpes simplex virus (hsv, types 1 and 2) (a) systemic infection in the neonate with shock and coagulopathy and severe liver failure (b) encephalitis, hepatitis (c) local (mouth, esophagus, larynx, lungs, heart, liver, kidneys, cns) or systemic disease in organ and stem cell transplant and immunocompromised patients 2. cytomegalovirus (cmv) (a) congenital infection in the neonate with systemic involvement (b) localized (liver, lungs, heart, kidneys, gi, cns, eyes) or systemic infection in solid organ and stem cell transplant as well as immunocompromised patients 3. epstein-barr virus (ebv) (a) infectious mononucleosis (b) burkitt's lymphoma, x-linked lymphoproliferative disorder (xl-lpd), post transplant lymphoproliferative disorder (ptld) (c) localized (liver, heart, lungs, kidneys, gi, cns) it is likely that viral infections have been underestimated both in their frequency and the degree of morbidity they cause. indisputably, in the modern day, hiv (human immunodeficiency virus) is one of the most significant viral pathogens worldwide, particularly in africa and developing nations that do not have the available resources to prevent spread among the community and more importantly maternal infection of the newborn. this leads to a range of morbidities as discussed in the immunocompromised section of this chapter. viral infections are mostly diagnosed clinically on the basis of history and physical examination as well as the regional prevalence of viral diseases. there are a number of ways to test for the presence of a particular virus from either patient blood or other body fluids. these are: tissue culture, serology and seroconversion, immunoflouresence, and pcr (polymerase chain reaction). only two of these are of any use to the intensivist: pcr and immunoflouresence. the turnaround time for viral culture and serum serology is inefficient in the critical care context. respiratory viruses (respiratory syncytial virus, influenzae, adenovirus, parainfluenzae, and human metapneumovirus) are the main contributors to viral disease in the picu, as they are in the general pediatric population. in the picu the majority of patients that develop significant degrees of illness are those who have significant comorbidities. conditions such as ex-prematurity, chronic lung disease, neuromuscular diseases, and congenital heart disease are probably the most common of these. herpes virus family, particularly herpes simplex virus (hsv), is the next important contributor to the burden of viral disease. all members of this family (hsv, ebv, cmv, and vzv) can cause serious infections in the neonate and in immunocompromised children. all services that treat the acutely unwell child (and adult) are at risk of being overwhelmed in an epidemic. national and regional planning needs to be undertaken prior to the advent of any serious infection where ever possible. (examples are sars -severe acute respiratory syndrome, or h1n1 ''swine flu.'') this is encapsulated in the worldwide pandemic planning taking lessons from the sars epidemic and the last major (in terms of mortality) influenza epidemic, the ''spanish flu'' that was prevalent from 1918 to 1920. at a hospital or an organizational level the concept of ''surge strategy'' is used. this is an organization based contingency plan to deal with large numbers of patients admitted simultaneously (i.e., mass trauma casualties or epidemics). in the case of influenza (or sars) this is relevant to the intensive care in that there is a finite capacity of any unit to provide mechanical ventilation. in addition to this, the institution is responsible for the protection of health care workers who are at high risk to contract an infectious illness and become a patient themselves. this would further increase the burden of illness and has the potential to limit available human resources. the prevalence of hiv in the general population and, in particular, in children in many developing countries poses significant stress on limited resources. hopefully, with more effective preventive programs to control vertical transmission of the infection and with availability of affordable anti-hiv medications, the quality of care for hiv-infected children will improve and the need for intensive care will diminish. regional or local experience is crucial in the management of many infections. dengue fever and viral hemorrhagic fevers, which are of more global importance, will be reviewed in more detail. dengue infections, caused by the four antigenically distinct dengue virus serotypes (den1, den2, den3, den4) of the family flaviviridae, are the most important arbovirus diseases. dengue is the most widely distributed mosquito-borne viral infection of humans, affecting an estimated 100 million people worldwide annually. dengue hemorrhagic fever usually occurs in children, with peaks in incidence at 7 months of age (with dengue-immune mothers), and at 3-5 years of age (during a second infection with a new serotype). it is spread throughout the tropical and subtropical zones between 30 n and 40 s where environmental conditions are optimal for viral transmission by aedes mosquitoes, principally aedes aegypti. the disease is endemic in se asia, the pacific, west africa, the caribbean, and the americas. global warming, by increasing the range of aedes mosquito, has the potential to lead to more widespread disease. who has classified the severity of dengue infection on the basis of a combination of clinical and laboratory findings (presence of hypotension and shock, tourniquet test, lowest platelet count, plasma leakage represented by high hematocrit level) in to: • dengue fever • dengue hemorrhagic fever • dengue shock syndrome (dss) viral hemorrhagic fever is a loosely defined category that includes infections from a host of viruses leading to similar clinical syndromes and sharing a similar severity of illness. otherwise, these viruses are different from each other with regard to their reservoir hosts, geographic distribution, and taxonomy. risk factors for exposure also vary among these infections and hence the control methods are geared to specific infections and their causative agents and intermediate hosts. in endemic areas diagnosis is by and large clinical and is confirmed by serological tests and viral pcr or culture. there are vaccines developed for some of these viruses. as a group, the treatment for these infections in the picu is mainly supportive and includes measures to: • optimize hemodynamic state and treat shock • monitor and control brain edema and intracranial hypertension • support ventilation and gas exchange with noninvasive or invasive ventilation • treat coagulopathic state if symptomatic • provide renal replacement therapy if needed; monitor and optimize glucose and electrolyte levels exhaustive discussion of this topic is beyond the current chapter, so the most important ones are briefly mentioned here. yellow fever is endemic in tropical africa between 15 n to 10 s and in parts of central and south america between 10 n and 40 s. in the life-cycle of this virus, in different parts of the world, mosquitoes (a. aegypti, haemagogus, and sabethes), monkeys, and people are involved; however, epidemic mosquito-borne human-to-human transmission can occur. after an incubation period of 3-10 days, fever, headache, malaise, nausea and vomiting, and musculoskeletal pain occur suddenly. initially, the clinical signs may include conjunctivitis, flushing of the skin, and relative bradycardia. in about 10% of cases the illness deteriorates with development of shock, systemic toxicity, gi bleeding, renal dysfunction, liver failure and jaundice, encephalopathy, and systemic bleeding. this latter picture is associated with a high mortality rate (30-50%). differential diagnosis includes other viral hemorrhagic fevers, viral hepatitis, leptospirosis, malaria, typhus, typhoid fever, brucellosis, rickettsial disease, and some intoxications. therapy is supportive and these patients may need intensive care admission for hepatic, renal and circulatory failure. who has recommended routine childhood vaccination in endemic areas (for children >4 months of age). vector control is important in highly populated areas to reduce the risk of epidemic transmission. lassa fever causes as many as 300,000 cases and 5,000 deaths each year in west africa and is a leading cause of maternal and fetal deaths. the virus is carried by mastomys huberti and mastomys erythroleucus, the rodent reservoirs whose infectious excretions are the source of human infections in west africa. in adults and children, early illness includes fever, malaise, headache, and musculoskeletal pain. these nonspecific symptoms progress over 4-5 days to include pharyngitis, cough, chest pain, diarrhea, and vomiting. in endemic areas, a purulent pharyngitis, with conjunctivitis, head and neck edema, and mucosal bleeding are highly specific signs of lassa fever. in severe cases, the illness may be complicated by hypovolemic shock, encephalopathy, respiratory distress caused by laryngeal edema, pleural effusions, or pneumonitis. liver failure, systemic and gi/gu bleeding, and myocarditis can occur. mortality is between 15% and 30%. there are anecdotal reports of the use of intravenous ribavirin in critically ill children with lassa fever but treatment is mainly supportive. lassa fever has been transmitted from person to person during hospitalization. universal exposure precautions should be observed as well as contact and droplet precautions. cchf is caused by a nairovirus (family bunyaviridae), and is transmitted by hyalomma ticks and by contact with infectious body fluids. the geographical distribution of the hyalomma ticks covers africa, the middle east and mediterranean areas, eastern russia, and west asia. the incubation period is from 2 to 9 days. illness onset is abrupt and nonspecific, with fever, chills, rigors, intense headache, and generalized muscle pain. onset of bleeding in the skin, mucous membranes, and the gi tract usually occurs after 3-6 days of illness. hepatitis, liver failure, circulatory failure, shock, and ards can ensue with mortality in up to 30% of cases. treatment is mainly supportive. the virus is sensitive in vitro to ribavirin, and this agent has been used in management of cchf with variable success (who). the value of immune plasma from recovered patients for therapeutic purposes has not been demonstrated, although it has been employed on several occasions (who). patients with suspected or confirmed cchf should be cared for by staff using added droplet and contact precautions. hfrs is caused by old world hantaviruses (family bunyaviridae). the reservoirs are small rodents, and humans are infected percutaneously or by direct exposure. clinical illness has an abrupt onset with fever, severe musculoskeletal pain, renal failure, systemic and gi bleeding, circulatory failure, and shock. this form of the disease is more common in asia and eastern europe. in hantavirus pulmonary syndrome (hps) (mainly seen in the americas), within 12-24 h of onset of symptoms, most patients develop some degree of hemodynamic instability and pulmonary edema accompanied by hypoxemia to full blown ards. petechiae of the head and neck are common but overt hemorrhagic symptoms are not. treatment is supportive and in those who survive, recovery is usually rapid. when given early in the course of illness, intravenous ribavirin has improved survival rate in hfrs but not in hps. steroids reduce the severity of the symptoms but do not increase the survival rate. malaria is singled out here because it is the most significant parasitic disease in humans with an estimated 500 million infections annually that result in 1-3 million deaths. the majority of these deaths are in children younger than 5 years of age and most are in africa. in developed countries malaria is the most common cause of febrile illness with no localizing signs in travelers returning from developing countries. the most important aspects of severe malaria are reviewed, which, for the most part, is caused by plasmodium falciparum. indicators of severe and complicated falciparum malaria and prognostic signs (world health organization 2000) cerebral malaria unrousable coma (gcs < 11/15), with peripheral p. falciparum parasitemia after exclusion of other causes of encephalopathy severe anemia hgb < 5 g/dl in the presence of parasitemia >10,000 per ml impaired consciousness of any degree, prostration, jaundice, intractable vomiting, parasitemia >2% in nonimmune individuals. levels of parasitemia should be interpreted in the light of immunity. patients with complicated malaria should be managed as severe malaria, i.e., with parenteral antimalarials even though they do not necessarily meet the criteria of severe disease. for details of management, review > chap. 101, ''malaria''. the world health organization defines cerebral malaria as unrousable coma in the presence of p. falciparum parasitemia when other causes of encephalopathy have been excluded. the precise etiology of cerebral malaria is not certain. most likely it is caused by sequestration of infected erythrocytes. this condition has a high mortality that likely results from brain micro vascular ischemia, infarction, and secondary cerebral edema. cerebral malaria is a medical emergency that requires: 1. supportive care: (a) continuous monitoring of vital signs. for a detailed discussion on sepsis, and the diagnosis and management of shock, please review the appropriate chapters (> chap. 61, ''bacterial sepsis and shock''). toxic shock and necrotizing fasciitis are two particular sepsis syndromes that require a special reference. toxic shock syndrome (tss) is caused by two bacteria: staphylococcus and streptococcus. s. aureus is a gram-positive coccus that is grouped in clusters. it is responsible for a number of infections ranging from skin sepsis, pneumonia, and joint infections to endocarditis. phage transformed staphylococcus produces a toxin that initiates a syndrome known as toxic shock syndrome (tss). this came to light in the 1980s with the ''menstrual shock'' syndrome. a non menstrual form was also identified. this was associated with staphylococcus sepsis at surgical sites, skin or joint infections, and with staphylococcal pneumonia. this syndrome is said to be ''superantigen'' mediated. the toxin proteins produced by the staphylococcus are able to ''cross-link'' the t-cell receptor without being processed by an antigen presenting cell (apc). this leads to an uncontrolled cascade of cytokines and immune system up regulation. at the level of the capillary this leads to inflammation and increasing permeability with secondary organ dysfunction (renal impairment, cardiac, pulmonary, and liver dysfunction). clinically this is manifested by skin erythema, tachycardia, hypotension, hypoxia and other critical organ dysfunction. initially this is subtle but rapidly develops into multi organ dysfunction. see the table below for the criterion upon which a diagnosis of staphylococcal toxic shock is made. treatment consists of recognition of the process, draining any collections of pus, and debridement, if that is appropriate. at the same time initiation of large volume fluid resuscitation, inotropic support and support of failing lungs with oxygen and ventilation if needed. antistaphylococcal antibiotics should be administered (this includes an antibiotic to cover for methicillin resistant staphylococcus). clindamycin being an anti-ribosomal antibiotic (50s bacterial ribosome) has a theoretical advantage in reducing the amount of toxin produced prior to antibiotic induced death of the bacterium. intravenous immunoglobulin (ivig) is a treatment for severe toxic shock that is progressing to multi-systems dysfunction. it has a proven efficacy in toxic shock in reducing the mortality of severe disease. this is thought to be via two general mechanisms. the first is by binding directly to the toxin. the second is by its immuno-mediatory properties. major criteria (all required) 1. fever !38.8 c 2. hypotension (orthostatic or shock) 3. rash (erythematous early and desquamative later) minor criteria (any three required) 1. gastrointestinal: vomiting or diarrhea 2. muscular: severe myalgia or cpk !2x upper limit of normal 3. mucous membranes: vaginal, oropharyngeal, or conjunctival hyperemia 4. renal: urea or creatinine !2x upper limit of normal, or urinalysis with >5 wbc per high-power field 5. hepatic: total bilirubin, ast or alt !2x upper limit of normal 6. blood: platelet count <100,000/ml 7. cns: disorientation or change in level of consciousness without focality, noted when fever and hypotension are absent streptococcal toxic shock is a syndrome that is analogous to staphyloccal toxic shock syndrome in that it is a superantigen mediated toxin related dysfunction of the immune system. group a beta-hemolytic streptococcus is most commonly associated with streptococcal toxic shock syndrome. clinical presentation is very similar to staphylococcal toxic shock. see table below. treatment consists of appropriate antibiotics. clindamycin is used for antimicrobial and antitoxin producing properties as previously mentioned. ivig here too has a role in reducing the mortality of severe disease. intensive care therapy consists of fluid resuscitation (large volume) and support of organ dysfunction (inotropes, ventilation, renal replacement therapy). hypotension or shock, plus any two of the following: 1. scarlet fever rash 2. abnormal liver function tests 3. renal insufficiency 4. disseminated intravascular coagulopathy (dic) 5. acute respiratory distress syndrome (ards) 6. soft tissue necrosis definite: preceding requirements + isolation of group a streptococcus from a normally sterile body site probable: preceding requirements + isolation of group a streptococcus from a non sterile body site necrotizing soft tissue infections are aggressive soft tissue infections that cause extensive necrosis, and include necrotizing cellulitis, fasciitis, and myonecrosis. the following clinical findings may be present: • erythema or discolored skin also the following systemic signs may be present: • local pain and tenderness out of proportion to physical findings • pain or tenderness that extends past the margin of apparent affected skin area necrotizing fasciitis is a surgical emergency. it is caused by a number of organisms: group a beta-hemolytic streptococci and other streptococci, staphylococcus, clostridium, pseudomonas, klebsiella, serratia, neisseria, escherichia, morganella, proteus, shigella, vibrio, salmonella, pasturella, enterobacter, corynebacterium, cryptococcus, fusobacterium, peptococcus, eikenella, bacteroides. the most common causative agent is group a streptococcus. pathologically it is characterized by micro angiopathic thrombosis and necrosis along superficial and deep fascial planes. the illness is associated with a breach of the integument. this can be by superficial infection, surgery or trauma. non steroidal antiinflammatory drugs are implicated in the pathogenesis. in children there is an association with varicella (chickenpox) infection. clinically the lesions appear either pale or have violaceous discoloration, often edematous, and there may be crepitus from gas forming bacteria. pain and tenderness in excess of that expected is a feature. the concern for the intensivist is the physiological decompensation that can lead to rapid cardiovascular collapse. broad-spectrum (and appropriate) antibiotics are indicated, and mechanical ventilation and cardiovascular support may be needed. urgent and wide surgical debridment of the affected areas is indicated. in cases of streptococcal necrotizing fasciitis there may be additional benefit from human immunoglobulin (ivig) therapy. though this has not been subjected to clinical trials, given the high mortality rate of necrotizing fasciitis and the biologically plausible consideration that ivig could neutralize the effects of streptococcal superantigens, its use can be justified. other treatments that have been used are: • vacuum-assisted wound closure (particularly in patients who have had large wound debridement) • hyperbaric oxygen (anecdotal evidence) the child, especially the infant, presenting with upper airway obstruction (uao) demands immediate attention. acute inflammation of the upper airway is of greater importance in small children because of the smaller diameter of the airway, hence the greater degree of obstruction from a similar amount of inflammation (resistance changes inversely to the fourth power of the radius of the airway). the following signs and symptoms are particularly worrisome: • inspiratory and expiratory stridor • active expiration (use of the rectus abdominis muscle when exhaling) • apnea or irregular breathing • increasing tachycardia (if no intervention is done tachycardia may be followed by decreasing heart rate which is usually a pre-arrest sign) • hypoxemia (late sign) • change in neurological status (becoming increasingly inconsolable and restless, or a child who ''stops fighting'' and becomes fatigued and hypotonic) there are many scoring systems for severity of the uao in children. the following is one suggested by downes et al. in 1980 . of note there is no mention of the neurological status in this scoring system. level of alertness and consolability of a small child are very important indicators of the severity of the uao. immediate management of acute severe stridor outside the picu, independent of underlying cause: • keep the child and the parent as calm as possible. do not separate the child from parent. • give the parent an oxygen mask to hold near the child's face. • call for help urgently from someone with expertise in airway management (usually an anesthesiologist). • give nebulized epinephrine (im epinephrine if airway obstruction is due to anaphylactic reaction). (skip nebulized epinephrine if you suspect epiglottitis.) • do not send the child to the radiology department for a lateral x-ray of the neck. • do not administer any sedative medications to the patient. • do not do attempt to draw blood for investigations. • place ecg monitoring leads and pulse oximetry probe without disturbing the child. • do not attempt to place an iv line (obviously you would place an iv/io access if the child has already had a respiratory or cardiac arrest). • the airway expert will decide to take the child to the or for intubation, or transfer to the picu. in children, there are many causes of acute uao, including infections (viral, bacterial) such as infectious mononucleosis, croup, epiglottitis, tracheitis, peritonsillar abscess, retropharyngeal abscess, diphtheria. noninfectious causes include foreign body, severe allergic reactions, acute angioneurotic edema, airway burn, trauma, and post-extubation in the picu. there are many causes of chronic/recurrent uao. in the history there may be chronic/recurrent symptoms. these patients may become symptomatic acutely (often with a viral respiratory infection) mimicking acquired acute upper airway obstruction. examples are: choanal atresia, laryngotracheomalacia, vascular ring, laryngeal web, subglottic stenosis, subglottic haemangioma, vocal cord palsy, recurrent angioneurotic edema. in this section the infectious causes of uao are addressed to. the more common infectious etiologies that may present with severe uao in children are: • croup or viral laryngotracheobronchitis • bacterial tracheitis • epiglottitis viral croup is the most common form of uao in children 6 months to 6 years of age (mostly 6 months to 2 years) and is more common in the autumn and early winter. the site of obstruction is the subglottic area. obstruction is caused by inflammation and edema. the most common viral etiology is parainfluenza, but influenza, enterovirus (coxsakie and echovirus), rsv, adenovirus, paramixovirus, rhinovirus, and hsv can cause a similar clinical picture. human metapneumovirus has been implicated in a few reports. in immunocompromised children, candida sp. can cause a similar presentation. there is a prodrome of mild fever and uri symptoms for 1-2 days before the onset of stridor. the stridor is characteristically harsh, dry, high pitched, and inspiratory. a ''barking'' or ''seal-like'' cough is prominent and usually worse at night. these children do have a voice, though hoarse, and they do not have trismus, dyphagia, or significant drooling. children with stridor at rest should be admitted for observation, while those with severe uao should be admitted to a picu. up to 15% of children with croup require hospitalization. usually no investigations are needed. administration of steroids (oral route is as good as intramuscular) in the emergency room has decreased the rate of hospitalization. hospitalized children with croup should receive a short course of oral or intravenous steroids (an example of a regimen is: dexamethasone 0.6 mg/kg iv/ po as an initial dose followed by 0.15 mg/kg q 6 h iv/po). inhaled nebulized epinephrine 1:1,000 solution (0.5 ml/kg, up to 5 mg) reduces the severity of obstruction and stridor. this can be repeated as required. the child must be observed for at least 2 h after a dose of nebulized epinephrine as the effects are transient. the decision on when to intubate a child with croup is a clinical one. if, despite maximum medical treatment there is not a clinical improvement or perhaps deterioration, a decision to intubate should be made or at least considered. a gentle and smooth intubation, using a tube one size smaller than usual for the age of the child, should be performed by a skilled and experienced practitioner. these children are at risk of accidental extubation and need proper securing of the ett, skilled nursing care, and adequate sedation only once the airway has been secured. most clinicians extubate the child 2-6 days later, when an audible air leak has developed around the ett and fever has settled. epiglottitis, or acute bacterial supraglottitis, is a bacterial infection of the laryngeal inlet, and is usually caused by h. influenzae type b (hib). with ''classical'' hib epiglottitis, the peak age of involvement is 2-3 years of age. since the introduction of the hib vaccine, the incidence of this disease has fallen dramatically, but the vaccine does not offer 100% protection. also, other organisms like s. aureus, s. pneumoniae, group a + b streptococcus, and n. meningitidis have been implicated as causative agents. the incidence of these latter organisms is higher in adolescents and older children. noninfectious causes of epiglottitis have been described in the following conditions: kawasaki's disease, stevens-johnson syndrome, airway burn, caustic ingestion, post-radiotherapy, angioneurotic edema, trauma (including trauma from intubation), leukemia, and lymphohistiocytosis. granulomatous states can cause a more chronic picture (sarcoidosis, tb, or wegener's granulomatosis). as fewer and fewer physicians have seen even one case of epiglottitis, it is important to have a high index of suspicion in any febrile child with uao. the following signs are highly suspicious of epiglottitis: • usually there are no prodromal signs and symptoms. • a few hours of high fever and tachypnea. • pain with swallowing, hence drooling is common. • reluctance to speak. • the child looks ill, with circumoral pallor, and a ''toxic'' appearance. • there is minimal or no coughing. • stridor is low-pitched and muffled, more like a snore. • child prefers to sit forward in the tripod position with mouth open and is reluctant to move his head or neck. if you have suspicion (on clinical grounds) that a child may have epiglottitis: • do not make the child lie down. • do not separate the child from parent. • do not examine the throat. • do not place an iv cannula. • do not order a lateral x-ray of the neck. • do not order any blood work. • do not transport a child with epiglottitis between hospitals unintubated. • the child should be accompanied by an expert in difficult airway management to the operating room for examination under anesthesia and securing airway if needed. the technique for induction of anesthesia is beyond the scope of this chapter. generally the inhalational method is performed in the sitting position (position of comfort for the child), and once the child loses consciousness intravenous access is secured and the rest of the monitoring is applied. laryngoscopy and intubation is only attempted after adequate depth of anesthesia has been obtained. blood cultures and a swab from the inflamed epiglottis should be sent and a 3 rd generation cephalosporin should be given once an iv is in place. when back in picu, accidental extubation can have disastrous consequences. skilled taping of the ett, nursing care, and adequate anaelgesia/sedation cannot be over emphasized. usually after 12-48 h of intravenous antibiotics the patient can be safely extubated, once the fever has subsided and presence of a leak is documented and the child is able to swallow (the child is not drooling). some practitioners prefer to reevaluate by direct laryngoscopy with the patient deeply sedated or anesthetized. if the causative organism is proved to be hib, in families with siblings under 4 years of age or families with an immunocompromised child, prophylaxis with rifampicin should be provided. bacterial tracheitis is characterized by profuse purulent secretions or sometimes by pseudomembrane formation in the tracheal lumen. the median age of the patient is 5 years. s. aureus is the most common etiology, though other gram-positive and less commonly gram-negative microorganisms might be causative. in immunocompromised children candida and aspergillus can cause tracheitis. these children usually have a high fever, they look toxic, and the stridor characteristically is high pitched and composed of both inspiratory and expiratory components. cough is usually prominent and they may have dysphonia or aphonia. drooling can be seen with bacterial tracheitis. these patients are at risk of airway obstruction. they require appropriate antimicrobial therapy, observation, and intubation by experts if warranted. community-acquired pneumonia (as opposed to nosocomial or hospital acquired pneumonia) is a common pediatric diagnosis that leads to admission to hospital for intravenous antibiotics and supportive respiratory therapy. pneumonia means inflammation of the lung parenchyma caused by infection and the diagnosis is made clinically in a febrile child with respiratory signs and symptoms who has evidence of consolidation on cxr. blood cultures frequently fail to reveal the infecting organism in pneumonia. tracheal aspirate, or more reliably, bronchoalveolar lavage (bal) and on occasions lung biopsy are required. children with immunodeficiency or malignancy undergoing therapy are a common example of where bal and/or lung biopsy may be necessary. • mycobacterium tuberculosis s. pneumoniae and s. aureus are the most important bacterial pathogens in children with pneumonia who need intensive care admission. as a general rule, truly focal disease, confined to a single lobe, is more likely to be due to bacteria. an ill child with unilateral pleural effusion most likely has s. pneumoniae or s. aureus pneumonia. viral • rsv • influenza a, b, c • parainfluenza routes to acquire infection: • inhalation of infected particles (most common) • aspiration • hematogenous invasion of the lower respiratory tract with viruses and bacteria leads to inflammatory changes characterized by migration of neutrophils into the alveoli. together with alveolar macrophages they provoke the production of inflammatory exudates and cellular debris that lead to consolidation of the lung parenchyma. the surrounding areas can be affected by atelectasis. • spo 2 <90% in high concentrations of inspired oxygen (>60% fio 2 ) • excessive work of breathing which may lead to exhaustion • shock and hemodynamic instability • change in neurological status (agitation or alteration of the level of consciousness) reasons of failure to respond to treatment on the pediatric ward or as outpatient: • development of an empyema or less commonly a lung abscess • underlying lung disease such as: bronchopulmonary dysplasia (bpd, in ex-premies), cystic fibrosis, inhaled foreign body, tracheobronchomalacia or post tracheal surgery, or infected congenital lung cyst • diagnosed or undiagnosed immunodeficiency states (primary, hiv, leukemia) • children with neuromuscular diseases, weakness, or spasticity such as muscular dystrophies, myasthenia, spinal muscular atrophy, or cerebral palsy • inappropriate antibiotics, inappropriately low dose or resistant bacteria • non bacterial pneumonia (viral pneumonia or alternative pathogen such as tuberculosis) once the culture results (bal, blood culture, sputum culture) and sensitivities are known the therapy should be tailored to the antibiotic sensitivities of the causative organism(s). • empyema. more commonly seen with s. pneumoniae and s. aureus pneumonia. generally a chest drain is needed. the use of fibrinolytics and surgery are areas for debate and local advice from thoracic or general surgeons and physicians from the respiratory and infectious diseases services should be sought. bronchiolitis is a seasonal viral infection of the lower respiratory tract that mainly affects infants. the usual cause is respiratory syncytial virus (rsv), although influenza, parainfluenza, adenovirus, and human metapneumovirus can cause a similar syndrome. in young infants, chlamydia and b. pertussis can cause respiratory illness with a more prolonged course that initially may resemble bronchiolitis. 20-25% of infants with bronchiolitis may have a secondary bacterial infection. infants with bronchiolitis have fever, cough, difficulty in feeding, and, on occasion, audible wheezing. on examination bronchiolitis is a syndrome characterized by respiratory distress, hyperinflation of the chest, and wheezes with fine inspiratory crackles heard on auscultation. apnea may occur even before onset of clinically significant respiratory distress, especially in ex-premature infections in the picu and very young infants. though not common, neonates may present with hypothermia and a sepsis like syndrome. the following groups are at increased risk of severe infection: • ex-premature infants and neonates • infants with congenital heart disease • infants with immune deficiency • infants with neuromuscular disease the virus causes direct damage to the respiratory epithelium with resultant inflammation, increased secretions, small airway obstruction. areas of hyperinflation and atelectasis exist simultaneously throughout the lung. this leads to ventilation and perfusion (v/q) mismatch and hypoxia. hyperinflation flattens the diaphragm and makes breathing less efficient. should they require respiratory support, many of these infants can be managed with noninvasive continuous positive airway pressure (cpap) at 4-8 cm h 2 o. this reduces the work of breathing and improves oxygenation. suction to maintain patency of airways is of crucial importance. if the saturations remain low and/or the infant continues to have frequent apneas despite providing noninvasive ventilatory support intubation of the trachea is indicated. • intubation is usually required for several days. • inadequate humidification and inadequate tracheal suctioning cause endotracheal tube blockage or lung atelectasis followed by increasing pressure and fio 2 requirements. • as a general rule, the best ventilatory mode is one that assists spontaneous respiratory efforts; keep the child's own respiratory and coughing efforts by providing enough comfort (sedation) and pressure support. • peep or cpap (initially at 4-6 cm h 2 o) may reduce the work of breathing. • apply enough peak inspiratory pressure (pip) to achieve visible chest excursions and if higher pressures (>30 cm h 2 o) are needed, let the paco 2 gradually rise to 75-80 mmhg with arterial ph > 7.2 (permissive hypercapnia). particular issues in infants with congenital heart disease and bronchiolitis: • infants with left to right shunts have more frequent viral and bacterial respiratory infections, and have higher morbidity and more prolonged course with bronchiolitis. • infants with palliated single ventricle physiology and those with limited cardiac output (for example severe valvar aortic stenosis) have high morbidity and mortality with bronchiolitis. • bronchiolitis and other viral respiratory infections in infants with congenital heart disease lead to operative delays and increasing complications post cardiac bypass surgery (e.g., pulmonary hypertension). in any infant with rsv bronchiolitis and congenital heart disease awaiting surgery, it is suggested to wait for 2-3 weeks before proceeding with bypass and surgery. the american academy of pediatrics has specific recommendations on prophylactic monthly injection of rsv monoclonal antibody in ''at risk'' infants during the cold season. however, the use of this approach has only been shown to aid a small number of patients. this section reviews: • myocarditis • infective endocarditis • infectious pericarditis • wound infection after cardiac surgery myocarditis is an inflammatory disease of the heart muscle characterized in its active phase by cellular infiltrates and myocardial necrosis. however myocarditis can have cellular infiltrates with little or no myonecrosis. most cases of myocarditis are thought to have a viral etiology; however, viruses are infrequently isolated. the most common viral causes include the enterovirus family particularly coxsackievirus b and adenovirus. other viral causes are influenza, cmv, hsv, parvovirus, rubella, varicella, mumps, hiv, and ebv. myocarditis has a number of other non viral etiologies, some infective and some not. they include bacteria, rickettsia, fungi, protozoa, pharmaceuticals, toxins, and connective tissue/autoimmune disorders. typically viral myocarditis begins as a systemic viral illness with flu-like symptoms. as the virus infects the myocytes the immune system is up regulated and cd4 t-helper cells and cd8 cytotoxic t cells are stimulated along with proinflammatory cytokines. persistence of the viral rna and production of no by the myocytes have been linked to myocardial tissue damage. myocarditis can present in a number of ways: • out of hospital cardiac arrest/sudden death • cardiogenic shock (may mimic sepsis) • congestive heart failure (increasing dyspnea, lethargy) • dysrhythymias -bradycardia, tachycardia whilst sepsis and hypovolemic shock are more common than cardiogenic shock from myocarditis, it should always be in the differential. sometimes acute ''decompensation'' of these children is heralded by abdominal distension and vomiting. teenagers may complain of a feeling of ''impending doom'' or severe chest discomfort. clinically, signs of tachycardia/tachypnea, gallop rhythm, hyperdynamic precordium, and displaced apex are often present. hepatomegaly and in older children elevated jugular venous pressure (jvp) are usually present. crackles on auscultation are often present in the chests of older children. chest x-ray (cxr) may show an enlarged heart, pulmonary venous congestion, alveolar edema, kerley b lines, and in some cases pleural effusions. in acute myocarditis the heart may often look normal in size on the cxr. a 12-lead ecg is useful to assess underlying rhythm, assess for ischemia and for the subtle ecg changes that are sometimes evident with myocarditis; st-t changes, reduced qrs voltage, widened qrs. echocardiography is absolutely necessary to assess structure and function of the heart and to assess for a pericardial effusion. involvement of appropriate specialists is important -cardiologists, intensivists, and cardiothoracic surgeons work cooperatively to manage and stabilize these patients. treatment of myocarditis is largely supportive. immunomodulation using steroids, intravenous immunoglobulin, and immunosuppressive agents is controversial. identifying any modifiable contributors, i.e., toxins and drugs, is of crucial importance. supportive therapy for heart failure associated with myocarditis ranges from diuretics and afterload reduction, addition of inotropic support to placing the patient on mechanical circulatory support. dopamine and dobutamine increase contractility but also heart rate and myocardial oxygen consumption. milrinone is an intravenous phosphodiesterase inhibitor that improves contractility and at the same time, reduces the afterload. enoximone is an oral phosphodiesterase inhibitor that is available in europe, but not in north america. levosimendan is a calcium sensitizer and improves contractility. it has limited availability world wide. positive intrathoracic pressure, given noninvasively via a face mask (cpap), reduces lv afterload and may improve cardiac output in the setting of lv dysfunction. failed medical therapy or deteriorating function will usually indicate the need for extracorporeal support and ultimately heart transplantation. mechanical support (ecls, ''berlin'' heart) is frequently used as a ''bridge'' to recovery or transplant. for a complete review of endocarditis review the cardiology chapter in this book. the major reasons a child with endocarditis may need admission to picu are: infections in the picu 1. congestive heart failure due to worsening valvar regurgitation 2. congestive heart failure with abrupt onset due to valve apparatus rupture/perforation, or dehiscence of a prosthetic valve 3. systemic to pulmonary artery shunt obstruction 4. arrhythmia 5. renal failure 6. embolic events to (a) brain (b) heart (c) lungs (d) bowel (e) extremities for a complete review of pericarditis and tamponade, please review the cardiology chapter in this book. acute inflammation of the pericardium in a previously healthy child has usually been assumed to be viral. in most cases a causative agent is not detected (hence the term ''idiopathic'' pericarditis). an upper respiratory infection usually precedes the onset of symptoms by 10-14 days. the reported viral pathogens include coxsackievirus, adenovirus, rsv, varicella, hepatitis b, hiv, and post influenza vaccine. primary infectious pericarditis that may need picu care is usually purulent bacterial pericarditis. these patients are generally toxic looking. the infection in the pericardium rarely occurs in the absence of infection elsewhere (hematogenous spread). in comparison with the viral (idiopathic) pericarditis, the incidence of tamponade and hemodynamic instability is much higher with purulent pericarditis. s. aureus is the most common cause of purulent pericarditis. other bacteria include h. influenzae, n. meningitides, and s. pneumoniae. in developing countries, tubeculous pericarditis is a common cause of chronic constrictive pericarditis. therapy depends on the hemodynamic status of the patient. a toxic-looking child with physiological signs and symptoms of tamponade should be transferred urgently to the catheterization laboratory or to the intensive care unit for percutaneous drainage of the pericardial collection. sometimes the pus in pericardium is so thick or organized (esp. with h. influenzae) that percutaneous drainage may not be sufficient and the child will need open surgical drainage. with tamponade physiology, administration of fluid boluses can temporarily increase the intracardiac ''filling'' and stabilize the patient until the definitive treatment (percutaneous or surgical drainage) is performed. broad-spectrum intravenous antibiotics with good antistaphylocccal coverage should be commenced promptly if purulent pericarditis is suspected. surgical wound infections after cardiac surgery can be categorized as superficial (cellulitis) or deep (mediastinitis). the patient usually presents a few days after the procedure, but may occur up to 2 months after the initial operation. the important signs are erythema and induration at the surgical incision. the child may be irritable and have a mild fever. there may be a leukocytosis with ''left shift'' and elevated inflammatory markers such as c-reactive protein (crp) or erythrocyte sedimentation rate (esr). in addition to the wound erythema with or without purulent discharge there may be signs of sternal instability with ''crepitus'' on direct pressure over the sternum. diagnosis nevertheless is a clinical one and relies on a high index of suspicion. the risk factors are: neonates, long cardiopulmonary bypass time, delayed sternal closure, and reexploration of the chest for postoperative bleeding. the most common organisms associated with sternal wound infections are s. aureus, s. epidermidis, enterococcus species, and candida species. antibiotic treatment should begin as soon as a sternal wound infection is suspected and a wound swab has been sent. blood cultures should be sent from both peripheral and central venous sites whenever possible. the initial antibiotic regimen should consist of broad-spectrum gram-positive (anti-staphylococcal) coverage, with the addition of gram-negative coverage if the patient is septic or mediastinitis is suspected. if there is not a rapid improvement or the patient deteriorates the sternal wound may need to be surgically explored and debrided. antibiotics should be given for 10 days to 2 weeks for cellulitis and for 4-6 weeks for deep wound infections. meningitis is an inflammation of the leptomeninges of the brain. for a review of ''aseptic'' meningitis which also includes viral causes of meningitis, please look at the neurology and infectious disease chapters in this textbook. suffice to mention that patients with ''aseptic'' meningitis are usually not as sick as those with purulent meningitis, and the csf abnormalities are not as prominent. patients with bacterial meningitis have a number of reasons for requiring intensive care. the most common clinical scenarios are coma and seizures. the local inflammatory response to bacteria multiplying in the csf involves polymorphonuclear leukocytes, the endothelium, complement, and cytokines. this results in an alteration in the cerebral blood flow and venous drainage, vascular inflammation, and obstruction to csf flow and reabsorption. the infection within the meninges may extend to the surrounding brain parenchyma. the commonest bacteria are s. pneumoniae, n. meningitidis, and h. infuenzae. in the neonatal period, the likely causative organisms are different: group b streptococcus, l. monocytogenes, and gram-negative bacilli are the commonest. this profile will be modified depending on the local vaccination policy, socioeconomic status of the children in the area, and local/regional epidemics of disease. none the less the intensive care management is similar: 1. broad-spectrum cns penetrating antibiotics with narrowing of spectrum of antibiotic cover once results of cultures of the blood and csf are known. antibiotics with high csf/brain tissue penetrance must always be used. in areas with high incidence of s pneumonia penicillin resistance (including the united states), empiric therapy for community-onset bacterial meningitis is both vancomycin and a 3rd generation cephalosporin. acute complications of bacterial meningitis are: • hyponatremia (serum sodium <135 micromole/l): this is usually due to the syndrome of inappropriate secretion of antidiuretic hormone (siadh). there is hyponatremia and low serum osmolarity without signs of hypovolemia. hyponatremia can cause convulsions. cerebral salt wasting is a much rarer condition that gives hyponatremia with signs of volume contraction. siadh is treated by free water restriction and cerebral salt wasting is treated with sodium (either iv or po) supplementation. in a hyponatremic child with convulsions give 3% nacl 3-5 ml/kg intravenously. it is important to note that the change of serum sodium (and hence serum osmolarity) is of more importance in some cases than the absolute serum sodium. a sudden drop in serum sodium (greater than 0.5-1.0 micromole/h) should be treated with hypertonic saline. • seizures: convulsions that occur early in the course of purulent meningitis are usually generalized and have less prognostic significance than those occurring later. etiology of convulsions in meningitis can be any of the following: brain edema, diffuse ischemia, hyponatremia, subdural collection, sinus venous thrombosis, or focal infarction. • subdural effusion: this complication is seen more commonly in neonates and infants. a good practice is to measure the head circumference daily in any infant with meningitis. the diagnosis is made or confirmed by neuroimaging. if the effusion is large, or if it is associated with focal signs, convulsions, or signs of increased intracranial pressure, a neurosurgical consultation is necessary. • obstructive hydrocephalus: obstructive hydrocephalus occurs when the pus (often with high protein content) in the ventricles blocks the outflow of csf. this situation occurs more frequently in small infants and neonates. similar to meningitis, seizures, focal or generalized signs, and coma, are common presentations. the list of differential diagnosis is long and includes: aseptic meningitis, post infectious encephalitis and noninfectious encephalopathies (metabolic, vascular, demyelinating disease, tumor). the most frequent causes of acute encephalitis include: enteroviruses, hsv, vzv, ebv, adenovirus, influenza virus, and m. pneumoniae. mumps, measles, and rubella infections are rarely seen in developed countries. in many parts of the world arboviruses are major causes of endemic encephalitides. tuberculosis is always high on the list of differential diagnosis in developing countries. diagnosis: csf can be completely normal, but usually contains >10 leukocytes/mm 3 (mainly lymphocytes), mildly increased protein level and mildly reduced to normal glucose level. in children with hsv encephalitis, the csf may contain red blood cells. csf in addition to cell count, chemistry and culture should be sent for pcr for viral agents. csf pcr can be helpful in a number of the infectious encephalopathies. for example: m. pneumoniae, mycobacterium, cmv, ebv, vzv; where the organism may not be cultured from the csf. brain imaging (ct or mri) can be helpful in the diagnosis. in hsv encephalitis there may be focal edema and enhancement seen in the temporal area. this is relatively specific for hsv infection of the brain. the electroencephalogram may show focal periodic epileptiform activity in frontal and temporal parts of the brain. this is common in hsv disease. diffuse slow waves generalized over the cerebral cortex may also be seen. this may either represent encephalitis or be secondary to sedatives and anticonvulsants used in the picu. the primary use of eeg is in the management of seizures. finally, when the etiologic diagnosis is not clear, or the patient is deteriorating despite treatment, brain biopsy may be performed. treatment is largely supportive. as for meningitis this consists of appropriate antimicrobial (antiviral) therapy. there should be no delay in starting acyclovir if hsv is suspected or considered. the dose is 30 mg/kg/day for 10 days. when m. pneumoniae is suspected antibiotics with good penetration into the brain (ciprofloxacin, or azithromycin) should be used. additional management includes airway protection for coma and seizures. medical and surgical therapies for management of intracranial hypertension, as discussed above should be adhered to. adem is an acute or sub acute inflammatory demyelinating disease of the cns (brain and spinal cord). in contrast to multiple sclerosis it is a monophasic illness. adem is considered a parainfectious disease and the precipitants include infection with upper respiratory tract viruses, influenza, group a streptococcus, ebv, vzv, measles, mumps, rubella, and mycoplasma. clinical presentation may involve fevers, seizures and a constellation of neurological phenomena. commonly these are coma, focal neurological deficits or alterations in personality and behavior. often a recent ''viral infection'' is present in the history. the lumbar puncture is done to exclude infections. csf may have normal white cell count or mild pleocytosis (mainly lymphocytes), and mild to significant protein elevation. the cultures and pcr should be negative. neuroimaging is the main diagnostic tool for adem mri is the modality of choice, as the ct is normal in 40% of cases. the typical mri findings are multiple disseminated asymmetrical hyperintense lesions on t2wi and flair in the white matter and basal ganglia. the cerebrum is more involved than the cerebellum. treatment: (1) continue antibiotics and antivirals until final csf cultures and pcr results are confirmed to be negative. (2) once infection is ruled out methylprednisolone ''pulse'' dose at 30 mg/kg/day (maximum 1 g) for 3 days followed by oral prednisolone 2 mg/kg/day for 2 weeks. this is followed by a 4 weeks weaning regimen. (3) plasma exchange or ivig for relapsed or refractory adem. gbs is an immune-mediated polyneuropathy that is usually preceded by a viral or bacterial infection of the respiratory or gi tract 1-3 weeks prior to presentation. gbs is the most common cause of acute paralysis in developed countries and is characterized by progressive, ascending, symmetric motor weakness and loss of reflexes. the sensory symptoms (extremity pain, paresthesia) and autonomic irregularities (tachycardia, bradycardia, hypertension, hypotension, arrhythmia) can be prominent. there are usually no sensory deficits in physical examination. infections known to precede the onset of paralysis are: cmv, ebv, vzv, campylobacter jejuni, mycobacterium tuberculosis, hiv, and m. pneumoniae. the pathogenesis involves an immune response against the infectious agent and has components that cross-react with those of the peripheral nervous system. diagnosis is clinical but is aided by csf and electrophysiology testing. the csf shows a high protein content and low/normal white cell count. this may be missed if the lumbar puncture is done early in the first week of the illness. electrophysiology will show decreased conduction velocity in the peripheral nerves. treatment: ivig at 2 g/kg/day for 2-5 days or plasma exchange. (two courses for mild gbs and four to five courses for severe illness.) corticosteroids have not demonstrated effectiveness in gbs and are not recommended. indications for picu admission: • for respiratory support • for plasma exchange • autonomic instability (hypo-or hypertension, arrhythmia) botulism is a toxin mediated disease caused by c. botulinum. symptoms start a few hours and up to 6 days after exposure to the toxin. the cranial nerves are involved initially with difficulty swallowing, abnormal speech (abnormal cry in infants) and eye movements (ptosis). other symptoms may include nausea, vomiting, constipation, and abdominal distension. as the illness progresses it causes paralysis of the extremities and respiratory muscles to various degrees. in infants the disease can be mild with hypotonia and constipation as the main findings. also in infants there is sometimes a history of ingestion of honey before the onset of symptoms (honey may contain the spores of c. botulinum). diagnosis is made from a combination of clinical findings and electromyography. stool for botulinum toxin or serum serology is confirmatory but takes time. treatment is with antitoxin to remove circulating toxin but this will not affect the toxin already present at the neuromuscular junction. specific botulinum immunoglobulin is not readily available world wide. the cost is prohibitive for a lot of countries and the cost-benefit analysis is only favorable for those patients requiring mechanical ventilation. penicillin and metronidazole are given to eradicate the source of toxin production. aminoglycosides and steroids should not be given as they may worsen the neurosmuscular transmission defect and increase muscular weakness. if the source of the c. botulinum is a wound (i.e., wound botulism) then it will need surgical debridement. indications for admission to picu: • respiratory support • autonomic instability children are increasingly surviving diseases that until recently were considered untreatable. there are more potent, intense chemotherapy regimens being used and increasingly there are more patients who undergo solid organ and stem cell transplants. these treatments and interventions particularly with immunosuppressants, though frequently successful, leave patients at considerable risk for severe infections. neutropenia is defined as the absolute neutrophil count (anc) [anc = pmn + band count] <1,000/mm 3 , and is generally associated with cancer and its treatment. the risk of infection is particularly high with: • rapid drop in anc • anc < 100/mm 3 (profound neutropenia) • prolonged neutropenia fever in neutropenic patients is defined as an oral/ tympanic membrane temperature >38 c in two repeated measurements over a 4 h period or one measurement above 38.5 c. the portals of entry of infectious agents are usually: the oral mucosa, the gut, the upper/lower respiratory tract, and central vascular lines. the most common organisms are gram-positive cocci (s. aureus, s. epidermidis, and strep. viridans), gram-negative bacilli (e. coli, k. pneumoniae, p. aeroginosa), and fungi (candida, aspergillus). recently the spectrum of pathogens has begun to change, with the emergence of more gram-negative and fungal infections. this is likely due to an increase in resistant pathogens in the face of the use of very broadspectrum antibiotics, intensity of therapy (high-dose chemotherapy and stem cell transplant) and prolonged neutropenia. the single most important risk factor for fungal infection is the duration of neutropenia. it is standard of practice to start antibiotics for a child with anc < 500 who is febrile. initial empiric therapy for febrile neutropenia consists of a b-lactam antibiotic and an aminoglycoside, plus a glycopeptide if a coagulase negative staph or enterococcus is suspected or isolated, if the child is in shock, has an endoprosthesis or a vascular tunnel infection. if patient has a history of arabinoside-c administration and has severe mucositis strep viridans infection is highly suspected and vancomycin should be added. if perianal infections in the picu tenderness is present add anaerobic coverage (metronidazole or clindamycin). after 4-5 days of fever and neutropenia adding an antifungal is the usual practice of most oncologists. infants with a severe combined or t-cell immune deficiency usually present early in the first few months. defects in cell-mediated immunity can result from congenital disorders such as digeorge syndrome, severe combined immunodeficiency disease (scid) and wiskott-aldrich syndrome. they can be secondary to lymphomas, immunosuppressive medications or chronic illness. acute viral infections such as measles and pertussis are also known to decrease a patient's cellmediated immunity. typical infections are pneumocystis jiroveci pneumonia (formerly carinii), cmv pneumonitis, rsv pneumonitis, disseminated enteroviral infection, and invasive fungal infection. patients are highly susceptible to infections with intracellular organisms such as salmonella, listeria, mycobacteria, herpes family viruses (cmv, ebv, and hsv), as well as fungi and protozoa. in older children and those with secondary immunodeficiencies, these infections tend to be reactivated disease. children with chronic mucocutaneous candidiasis and chronic granulomatous disease typically present early in life with recurrent candida and staphylococcal infections. neonates with adhesion molecule deficiency usually present with delayed separation of the umbilical cord stump, increased polymorphonuclear count, and increased incidence of bacterial infections. children with primary humoral immune deficiency usually present between 6 months and 5 years of age. the onset is consistent with the time when the level of placentally transferred maternal antibodies (igg) has declined. these defects as well as complement deficiency and asplenia are more commonly associated with infections by encapsulated microorganism such as h. influenza, n. meningitides, and s. pneumoniae. although patients with these conditions are mainly susceptible to bacterial infections involving the upper and lower respiratory tract, they can have protracted diarrhea with giardia or echovirus. defects in the late complement component (the ''attack'' component, c5-9) are prone to recurrent neisseria infections. children with early complement defects usually have autoimmune and rheumatologic manifestations. fulminant meningococcemia is also associated with properdin deficiency (alternative complement pathway). hsct is now an established treatment for a host of immunologic, metabolic, hematological, and neoplastic disorders. the ''stem cells'' may be obtained from the patient (autologous). alternatively from an hlacompatible related or unrelated donor (allogeneic). there is little risk of acute or chronic graft versus host disease (gvhd) with autologous hsct. currently there are three sources for stem cells: bone marrow, peripheral blood, and umbilical cord blood. generally with the umbilical cord stem cell transplant the speed of engraftment is lower, but so is the risk of gvhd. with peripheral blood stem cell transplant engraftment occurs faster but the risk of gvhd is also higher. with bone marrow stem cell transplant the speed of engraftment and the risk of gvhd is somewhere between the other two sources. with unrelated umbilical cord blood and t-cell depleted bone marrow or blood stem cell transplant, the risk of graft failure is higher. there is a higher risk of infection with occurrence of gvhd, and with graft failure. the conditioning regimens used to prepare the patients generally consists of high-dose chemotherapy with or without regional or total body irradiation. post transplant, there is a period of pancytopenia and though the neutrophil count usually normalizes after 3-4 weeks it is not unusual for these patients to need red cell or platelet transfusions for much longer. the risk of infection is influenced by rapidity of myeloid recovery and the rate of lymphoid reconstitution. the speed of restoration of adequate immune function is highly variable. the stem cell source, hla compatibility, purging or t-cell depletion of the graft prior to transplant, and severity of gvhd are important factors. in the early post-transplant period (first 100 days), transplant centers employ prophylactic measures to reduce the risk of infection. these measures vary between different centers, and include: • prophylactic antibiotics (for pcp, candida, hsv, cmv) • administration of ivig • environmental precautions (isolation and barrier nursing) in the early post-transplant period, patients are most susceptible to infections caused by both gram-negative and gram-positive organisms and by fungi. there is a higher risk of cmv pneumonitis in patients with gvhd (largely due to the need for immunosuppressive treatments). cmv negative recipients who receive transplant from a cmv positive donor are at highest risk for cmv pneumonitis. children who have undergone hsct and are admitted to picu have a particularly poor prognosis. pneumonia, mechanical ventilation, and the need for renal replacement therapy are especially poor prognostic factors. those with septic shock and line sepsis have the best prognosis. in addition to bacteria, the most commonly isolated organisms are cmv, rsv, adenovirus, candida, aspergillus, and pcp. in a recent report from great ormond street hospital in london, uk, only 56% of these patients survived to discharge. similar to children who have received hsct, children receiving solid organ transplants are prone to infections before and after the transplant. the main differences are: 1. patients after solid organ transplants are usually less immunosuppressed than hsct patients and are not at risk of immune reconstitution syndrome and its associated inflammatory and infectious complications. however they are at risk of surgery-related complications and postsurgical infection issues (wound infection, bacteremia, atelectasis/pneumonia, urinary tract infection). 2. infection of the transplanted organ due to latent or colonizing organisms present in either the donor or recipient can lead to invasive widespread disease in the immune suppressed post-transplant recipient. an example of this is the child with cystic fibrosis colonized with pseudomonas species; those colonized with b. cepacia are particularly at risk of developing resistant infection post lung or combined heart/lung transplants. 3. children receiving solid organ transplants are at risk of reactivation of latent infections, such as cmv. but, unlike hsct patients who most commonly present with cmv pneumonitis in recipients of solid organ transplants the cmv disease depends on the sites where the virus is latent and on the organ that has been transplanted. lung, heart/lung, and liver transplant patients are most vulnerable to systemic disease. cmv infection can precipitate rejection and increase vulnerability to other infections such as fungal infections. 4. ebv-associated post transplant lymphoproliferative disorder (ptld) is a potentially fatal complication of solid organ transplant. the risk of ptld is higher in children who were ebv seronegative prior to the sot. ebvrelated infection in sot patients may present in several different ways: asymptomatic or nonspecific viral syndrome, mononucleosis syndrome, and ptld. the latter can have a spectrum from fever, lymphadenopathy and diarrhea to full blown lymphoma. tissue biopsy is necessary to establish the diagnosis of ptld. the mainstay of therapy consists of decreasing immunosuppression. chemotherapy and biological treatments (such as anti-cd20 monoclonal antibodies) have been used. the most common reason children with hiv/aids are admitted to the picu is respiratory distress. septic shock and cns involvement (encephalopathy, encephalitis, and meningitis) are other common conditions leading to admission. in addition to the bacteria, viruses and mycoplasma that cause infections of the lower respiratory system in the non-hiv patient, there are a number of other opportunistic infections and inflammatory conditions to consider in the hiv-infected patient. these are commonly: • pneumocystis jiroveci • cmv pneumonitis • tuberculosis • fungal infections • lymphoid interstitial pneumonitis (lip) • immune reconstitution inflammatory syndrome (iris) most infants with respiratory failure will not have a previous diagnosis of hiv infection when they present. in non-endemic areas, especially during the colder season, such infants would be diagnosed and treated initially as cases of bronchiolitis. the possibility of an underlying immune deficiency and or aids should be considered in any infant who responds poorly to treatment or who has risk factors. these include failure to thrive, history of recurrent chest infections, hepatosplenomegaly, adenopathty, severe persistent oral thrush, or abnormal neurological signs. in sub saharan africa, tb and other bacterial pneumonias were common in both hiv-infected and uninfected children who presented with respiratory failure. however pneumocystis and cmv pneumonitis occurred almost exclusively in infants who were hiv-infected. the majority of cases of p. jiroveci pneumonia present in the first 6 months of life. usually these children are quite hypoxemic. high fever is uncommon compared with bacterial pneumonia. there is a diffuse, bilateral air space or interstitial involvement on the chest x-ray. occasionally there is a ''ground glass'' appearance. in an hiv positive patient with bilateral diffuse parenchymal or interstitial infiltrates on cxr, the development of pneumothorax is suggestive of p. jiroveci infection. diagnosis is by bronchoalveolar lavage (bal). even if a child develops pneumocystis while on prophylactic therapy, high-dose intravenous trimethoprim/sulfamethoxazole should be started. prophylaxis may have failed because of poor compliance. if one suspects drug resistance then other agents should be used (pentamidine, dapsone). methylprednisolone at 2-4 mg/kg/day divided in four doses should be administered in moderate to severe cases (practically all children with pneumocystis who are admitted to picu) for 5 days and then tapered. untreated, it is universally fatal. with proper therapy the mortality is less than 10%. the risk factors for mortality are the severity of respiratory failure and the severity of immunosuppression. lip in children with aids is associated with increased risk of lower respiratory tract infections including bronchiectatsis. this condition can produce severe ventilation/perfusion mismatch and hypoxemia but may also be asymptomatic. the cxr shows diffuse infiltrates and hilar lymphadenopathy persisting for >2 months despite antibiotic therapy. usually cxr changes are worse than clinical symptoms. lip can be related to ebv infection or to an exaggerated immunological response to inhaled or circulating antigens or both. steroids have been used in the treatment of symptomatic lip. coexistence of hiv and tb accelerates the course of both of these infections. the risk of miliary and extra pulmonary tb is higher in children with aids and the course is more likely to be severe and rapid. a child with hiv infection is five to ten times more at risk of active tb. in countries with high prevalence of tuberculosis, the who suggests bcg vaccination of all neonates at birth but not in any child/infant with clinical aids. for treatment of tb, please refer to the chapter in this book. mac can produce a systemic illness in children infected with hiv that is characterized by fever, chronic diarrhea, abdominal pain, malabsorption, lymphadenopathy, and obstructive jaundice. mac usually would not present with significant lung disease in these children. iris occurring weeks after the initiation of specific anti-hiv treatment may on occasion be severe enough to cause respiratory failure warranting admission to the icu, though it can also indicate latent or incipient mycobacterial disease. this (iris) is a diagnosis of exclusion and requires a bal and possibly a transbronchial biopsy. management is usually with corticosteroids. other than sepsis and respiratory failure, the other conditions that may bring the child with aids to the intensive care unit are (to name the more common ones): • cns infections (bacterial, mycobacterium, fungi, cryptococcus, viruses, and rarely cns toxoplasmosis), acute hiv encephalopathy • hiv-related cardiomyopathy • severe diarrhea and shock due to cryptosporidium or other microorganisms • liver failure due to infections or drugs • complications of antiviral medications such as acute pancreatitis, acute liver failure, stevens-johnson syndrome an important aspect of care for these children in the picu for the staff is risk of exposure to body fluids and of needle stick injury. universal exposure precautions should be strictly adhered to. it is imperative that all staff be aware of the guidelines and procedures after exposure to biological fluids in their institutions and seek advice from the occupational health department immediately if exposed. as increasing numbers of immunocompromised children with fungal and viral infections are admitted to the pediatric intensive care units, common antifungal and antiviral medications that are used against these infections are briefly reviewed. for a complete review of these topics, the reader is referred to chapters on individual fungal and viral infections in this book. increasingly systemic fungal infections have become more significant in morbidity and mortality of immunocompromised patients in intensive care units. factors that have been associated with this increase are: • use of more potent and broad-spectrum antibacterial agents • prolonged and severe neutropenia • prolonged and severe immune dysfunction (primary or secondary) • having central venous lines and invasive devices • total parenteral nutrition (tpn) the most common fungal pathogens causing systemic illness in critically ill children are candida and aspergillus species. in recent years there has been an increasing importance of uncommon fungal pathogens such as non-albicans candida species, fusarium species, trichosporon species, and dematiaceous fungi. in an immunocompromised patient with a positive fungal culture from a central venous line, current guidelines strongly advocate removal of the line. traditionally with invasive candidiasis, amphotericin b (amb) has been the first-line drug to use. however, intravenous flucanozole and itraconazole could be considered. in non-neutropenic patients positive for c. albicans (but not other candida species) fluconazole is as effective as amb. in empirical treatment of prolonged febrile neutropenic patients (>4-5 days) amb should be started. clinical trials have shown that liposomal preparations of amphotericin b (l-amb) have similar, but not better, efficacy compared with conventional amb preparations. some authors recommend a liposomal preparation of amphotericin as a preferred first-line treatment. unfortunately, high cost can be prohibitive in many parts of the world. in general, the l-amb agents cause less fever, rigors, nausea, and vomiting. they are also less nephrotoxic. voriconazole, a second generation triazole, can be used for empirical treatment of febrile neutropenic patients in place of l-amb. in patients with invasive aspergillosis it has been shown that initial therapy with voriconazole leads to a better response and improved survival with fewer side effects. echinocandins such as caspofungin have been used in combination with amb or voriconazole in more resistant cases of invasive aspergillosis with persisting fevers. micafungin and anidulafungin are two other agents in this family. antifiungals (old and new) have a large potential for side effects and drug-drug interactions. clinicians need to be aware of the specific profiles of the drugs they use from this antimicrobial family. below are the common agents likely to be used in the intensive care unit. hiv drugs have not been discussed. please refer to the chapter on hiv for more detailed discussion of these agents. there are two main groups of antiviral agents: various types of nosocomial infections in pediatric intensive care units, blood stream infections had the highest incidence, followed by lower respiratory infections and urinary tract infections. a basic mandate of medicine is ''primum non nocere'' -first do no harm. while it is inevitable that some patients may acquire nosocomial infections, these infections cause significant morbitiy and mortality. the overall mortality attributable to the various nosocomial infections within the picu has been estimated to be between 10% and 15% and infections acquired in the picu are associated with an increased risk of death, with a relative risk of 3.4. it is widely appreciated that these infections can be minimized by a number of simple interventions, most important of which is hand washing; ''clean hands save lives.'' in a review of the related literature between 1990 and 2002, it was shown that between 11% and 48% of nosocomial infections could have been prevented. blood stream infections are common. not surprisingly they are most common in those patients who are the most debilitated, receiving mechanical ventilation and have central venous lines in situ for longest time, urinary catheters and other artificial surfaces. the spectrum of infections also has a predictable frequency. gram +ve infections are the most common bacteremias (whether or not associated with a central line) followed by gram àve and then fungi. typically fungi are found in those patients on tpn or long term, broad-spectrum antibiotics and immuosuppressed. central venous lines (cvl) are used to provide secure intravenous access for administration of medications such as vasopressors and inotropes, to monitor pressures, blood oxygen saturations, and for intravenous nutrition. in a survey of picu's in the united states the rate of cvl infection was 7.6 per 1,000 catheter-days. in neonates the corresponding figure was 11.3 per 1,000 catheter-days. in europe the cvl infections occurred at a rate of 10.9 infections per 1,000 catheter-days. measures taken at time of insertion of the cvl significantly reduce the incidence of infection. strict aseptic technique (gowns/gloves/mask and wide sterile field), use of chlorhexidine (as opposed to povodine/iodine), and minimal trauma (use of ultrasound and experienced operators) are all very important factors at the time of insertion. chlorhexidine disks topically placed upon the skin at the insertion site and antibiotic impregnated lines are used by some units but not proven to be of value. cuffed and tunneled lines such as hickman lines, port-a-cath lines and picc (peripherally inserted central catheter) have a significantly lower rate of infections than standard central lines that are inserted in the intensive care. where long term therapy >1 week is required consideration should be undertaken to the insertion of one of these types of lines. site of insertion is important. the femoral vein is easy to cannulate with fewest insertion complications. however, it is more likely to become infected and thrombosed. it is good as a temporary line but early consideration should be given to removing and/or repositioning access. to prevent central line infection minimization of the ''opening'' of the line on a daily basis is important. asepsis on line ports prior to use (with alcohol or chlorhexidene) is critically important. ultimately to reduce infection rates lines should be kept for the briefest time possible. difficulty of insertion and type of ongoing therapy come into this cost-benefit analysis. when there is a suspicion that a central venous line has become infected then blood cultures should be drawn from the line and from a peripheral puncture. broad-spectrum antibiotics that cover the bacteria above (vancomycin and gentamicin for example) should be commenced. the line should be removed if at all possible. an attempt to sterilize the line may be made in the circumstances where the line is ''precious'' and not easily replaced. this can involve alternate infusion of antibiotics through all lumens and the use of antibiotic ''locks.'' this is defined as a respiratory infection that occurs 48 h post admission for mechanical ventilation. the respiratory infection is defined by: fever/hypothermia, crackles on physical exam, new respiratory infiltrates on cxr, deteriorating ventilatory status (tachypnea), cough, deteriorating gas exchange, elevated or depressed white cell counts. this may or may not be in the presence of bacterial isolates from a sterile respiratory sample (i.e., bal). there are age specific criteria for the diagnosis of vap. the cdc has produced a document that lists the specific criteria. this can be found at: http://www.cdc.gov/ncidod/hip/nnis/members/pneumonia/final/pneumocriteriav1.pdf. the incidence of vap in the picu is 6-11.6 per 1,000 ventilator-days. the diagnosis of vap is challenging and controversial. there are a number of simple interventions to reduce the incidence of vap. they are: 1. elevate head of the bed to 30 2. ventilator tubing: (a) dependant positioning of ventilator tubing to avoid aspiration (b) removal of excess condensate (c) limit frequency of tubing change unless required 3. suctioning: (a) limiting amount of saline lavage when suctioning (b) sterile technique (gloves and sterile catheter) + (gowns/masks and eye wear for protection of staff) 4. mouth care: frequent mouth cares with chlorhexidene-based wash 5. feeding: (a) early institution of feeding (b) avoidance of gastric over distension (c) limiting use of antacid therapy to high risk patients (i.e., burns, head injury) 6. avoid/limit antibiotic therapy to minimize chance of colonization with antibiotic resistant flora urinary tract infection is directly proportional to the length of time that a foley catheter is in place. frequently, patients are on antibiotics that will suppress urinary infections. however, virtually all intensive care patients with urinary catheters will acquire urinary sepsis if their stay is prolonged. more than 90% of hospital acquired uti's occur in catheterized children. the best intervention (as with central lines) is early removal of the catheter. when strict measurement of urinary output is not needed and the likelihood of urinary retention (due to illness or drugs) is not an issue then catheters should be removed. intermittent catheterization can be considered as an intervention to avoid a permanent foley catheter where retention is an issue in a longer term patient. if an icu patient develops fever or unexplained sepsis then it is mandatory that a urine specimen be sent for microscopy and culture. this is especially important in the patient with a catheter. if urinary sepsis is proven then consideration for catheter removal should be given. broad-spectrum antibiotics that cover the spectrum of bacteria listed above should be commenced. antibiotics should be specifically weighted to cover the gramnegative bacteria as these are most common. surgical site infections are a less frequent infection but none the less important source of infectious morbidity. if a wound is ''dirty'' or contaminated such as a traumatic soiled wound or contaminated peritoneum from perforated appendicitis then broad-spectrum antibiotics should be commenced in high dose. at the same time, appropriate surgical management should be undertaken to deal with the contaminated wound. the surgical team will usually offer guidance on this issue. if a wound is ''clean,'' for example a surgical incision, the surgical team will generally have a preference for antibiotic prophylaxis. commonly a second generation cephalosporin will be used. this should be given at time of the operation and for a defined and limited time thereafter. prolonged prophylaxis has been shown not to prevent inevitable wound infections and promotes emergence of multiple antibiotic resistances. for wounds that become infected in the intensive care unit, swabs should be taken of any discharge. surgical review should be initiated and the wound dressed (with frequent changes). appropriate antibiotics should commence. opening of the wound and drainage/debridement of infected tissue is the responsibility of the managing surgical team. all intensive care units should have the ability to isolate for airborne and body fluid infectious organisms. simple hand washing is very important (before and after examining patients or attending the bed side). where this is not infections in the picu practical an alcohol-based hand gel can be used. from simple hand washing a graduated appropriate degree of isolation and infection control processes should be undertaken, i.e., gowns, gloves, respiratory protection -masks with increasing filtering ability to full respirators. negative pressure rooms (with antechambers) are usually reserved for respiratory isolation for the protection of staff and other patients. positive pressure rooms are for protective isolation of the patient who is immunocompromised. negative pressure isolation and strict barrier isolation is reserved for highly infectious pathogens. sars and ebola virus are examples where this may be necessary. all intensive care staff should strictly adhere to hand washing practices (with a chlorhexidine based product). unfortunately this is not the case and medical staff are often the worst offenders in this regard. active and repeated awareness campaigns should be carried out to reinforce this basic but very important healthcare related activity. severe infectious processes are common reasons for admission to the pediatric intensive care unit. children in the picu are at risk for developing severe infections. increasingly children with a dysfunctional immune system survive their primary illnesses and are admitted to the picu with severe infections. secondary immune deficiency is common in the course of prolonged critical illness. rapid sampling of body fluids and commencement of broad-spectrum antibiotic cover is of the utmost importance. it is shown that even 5 min of delay in starting appropriate antibiotics has been associated with increased mortality. if there are reasons to believe there is an anatomical source of infection (collection of pus, infected central venous line, infected prosthesis etc.) often the antibiotics would not achieve their effects until the source of infection is dealt with effectively (surgical evacuation/ removal, drainage). optimizing the hemodynamic status of the patient (oxygen delivery, addressing preload, after load, and contractility) should start from the moment one considers the diagnosis of sepsis or severe life threatening infection. diagnostic and therapeutic interventions all go hand in hand and start in parallel from the initial encounter with the patient. it is vital that every unit has an updated knowledge of the prevalence and sensitivity of the micro organisms prevalent in their community and in the hospital. colleagues in clinical microbiology or infectious disease departments are invaluable members of any picu team in dealing with these issues. prophylactic measures such as effective hand washing, observing strict sterility while placing central venous lines, measures to reduce incidence of the vap, discontinuing the invasive lines and catheters when not indicated anymore, and adherence to universal exposure precautions should be implemented and monitored and audited regularly. they save more lives and money than much more expensive interventions. effective antibiotic stewardship, tailoring the antibiotic coverage when sensitivities of the causative organisms are known, and discontinuing 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pediatric shock in the emergency department cdc guidelines on the diagnosis of ventilator associated pneumonia toxic shock syndrome in children epidemiology, pathogenesis, and management manson's tropical disease british thoracic society guidelines for the management of pleural infection prognostic factors in pediatric cancer patients admitted to the pediatric intensive care unit epidemiology and outcome of necrotizing fasciitis in children: an active surveillance study of the canadian paediatric surveillance program acute bronchiolitis and croup ventilator-associated pneumonia in neonatal and pediatric intensive care unit patients risk factors for healthcare-associated infection in a pediatric intensive care unit a national point-prevalence survey of pediatric intensive care unit-acquired infections in the united states; pediatric prevention network concept of operations for triage of mechanical ventilation in an epidemic outcome of children requiring admission to an intensive care unit after bone marrow transplantation infections of the airway the global neonatal and pediatric sepsis initiative infections in the intensive care unit urgences et soins intinsifs paediatriques severe malaria: lessons learned from the management of critical illness in children sepsis and septic shock: a global overview pediatric critical care surge capacity critical care outcomes in the hematologic transplant recipient clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 update by the infectious diseases society of america management of severe dengue in children infectious diseases in the pediatric intensive care unit rogers' textbook of pediatric intensive care red book: 2009 report of the committee on infectious diseases nosocomial infections in pediatric patients: a european, multicenter prospective study; european study group hand washing in the intensive care unit: a big measure with modest effects evidence behind the who guidelines: hospital care for children: what treatments are effective for the management of shock in severe dengue? epiglottitis and croup a prospective study of ventilator-associated pneumonia in children predictors of mortality in patients undergoing autologous hematopoietic cell transplantation admitted to the intensive care unit london world health organisation (2005) pocket book of hospital care for children: guidelines for the management of common illness with limited resources. who, geneva zar hj, apolles p, argent a et al (2001) the etiology and outcome of pneumonia in human immunodeficiency virus-infected children admitted to intensive care in a developing country infections in the picu key: cord-023729-dipjubn7 authors: serlin, michael h.; dieterich, douglas title: gastrointestinal disorders in hiv date: 2009-05-15 journal: global hiv/aids medicine doi: 10.1016/b978-1-4160-2882-6.50027-7 sha: doc_id: 23729 cord_uid: dipjubn7 nan gastrointestinal disease in human immunodeficiency virus (hiv) spans the entire gi tract from the mouth to the rectum. the spectrum of gastrointestinal symptoms in hiv ranges from odynophagia and dysphagia, to nausea and vomiting, to abdominal pain and fi nally diarrhea and tenesmus. as with normal hosts, gastrointestinal disorders are very common in hiv patients, whether it be from opportunistic infections secondary to the patient's immunosuppressed status, medication induced, or through other etiologies. almost all hiv and aids patients have some gastrointestinal complaints throughout the course of their illness. with the dramatic changes in hiv care because of highly active antiretroviral therapy (art) in the mid-1990s, the incidence of opportunistic infections are decreasing, and as a result, the clinical picture of gastrointestinal illnesses in hiv is changing. the evaluation of the hiv patient with gastrointestinal complaints requires a thorough history and physical exam, in addition to selected studies, in order to diagnose the correct disease and treat accordingly. patients with hiv and aids typically can have upper gastrointestinal symptoms, which can range from dysphagia, or diffi culty swallowing, to odynophagia, or the feeling of pain upon swallowing. at least one-third of patients with hiv before the art era had esophageal complaints, 1 and the incidence increased with the progression of the disease. most of the symptoms in these patients are secondary to opportunistic infections caused by the patient's immunosuppressed state, and related to the degree of immunosuppression. the most common etiologies of esophageal pathology and esophagitis in aids patients (table 23 .1) are candida species, herpes simplex virus (hsv), and cytomegalovirus (cmv). 2 in addition, there is also an entity of idiopathic esophageal ulcers (ieu) that is also seen in hiv patients, which may be immunologically mediated, 3 or caused by hiv itself. 4 other etiologies of esophageal complaints include malignancy (especially lymphoma and kaposi's sarcoma) and other noninfectious causes. however, with the introduction of protease inhibitors (pis) in 1996 and art, and the decreased incidence of aids, more esophageal complaints in hiv these days are related to common etiologies like gastroesophageal refl ux disease (gerd) than opportunistic infections. 5 in addition to the most common symptoms of dysphagia and odynophagia, other symptoms can also suggest esophageal disease in hiv patients, like chest pain, nausea, vomiting, anorexia and weight loss. the symptoms can be acute or have a more chronic, progressive course. in addition, dysphagia is often associated with candidal esophagitis, whereas odynophagia is generally symptomatic of esophageal ulcerative disease. patients can have both dysphagia and odynophagia, and because they also may have more than one illness concurrently, it is imperative to pursue a thorough investigation as to the etiology of esophageal complaints in hiv patients. the evaluation of hiv patients with esophageal symptoms does not defi nitively need to include endoscopy with biopsy, but this is the gold standard, as it allows the physician to visualize the esophageal lumen, and to biopsy affected sites ( fig. 23.1) . the history and physical exam is obviously important, as it may lead to a discovery of gerd, or pill-induced esophagitis. in addition, patients with disseminated cmv (e.g. cmv retinitis) with esophageal symptoms (especially odynophagia) may respond to cmv antiviral therapy in the absence of diagnostic endoscopy and biopsy. the most common sign on physical exam that relates to esophageal complaints is oral thrush, which can be suggestive of esophageal candidiasis in patients with esophageal complaints. in these patients, especially those with only dysphagia (or dysphagia and odynophagia, but not those with solely odynophagia) it may be benefi cial to document the response from an empiric trial of oral fl uconazole, as opposed to endoscopy. 6 if there is a symptomatic response to the fl uconazole, then it can be presumed the patient had candidal esophagitis and proceed accordingly. in addition to history and physical exam, there are other ways to evaluate esophageal complaints. barium esophagography is relatively insensitive and non-specifi c and should not be used for diagnostic purposes, but the most characteristic fi nding in candidal esophagitis is diffuse mucosal irregularity resulting in a 'shaggy' appearance mimicking diffuse ulceration. 7 cmv and ieu may appear as well circumscribed ulcers that may be shallow or deep, and are indistinguishable on barium swallow. 8 of course, radiography can determine a neoplastic origin to dysphagia in patients with malignancies. another form of evaluation is brush cytology, where a cytology brush is passed through a nasogastric tube and obtains tissue for viral cultures and immunohistochemistry. unfortunately, this leads to large sampling error, because it is done blindly without visualization of the lumen, and misses diagnoses (as patients may have two infectious processes, and ieu cannot be diagnosed). viral culture and cytologic brushings add little in the evaluation of aids patients with esophagitis over endoscopy with biopsy. 9 endoscopy with biopsy generally yields a viral or fungal diagnosis based on culture and hematoxylin and eosin staining (which will exclude a viral etiology); only after several biopsy samples do not show any etiology of the ulcerations can a tentative, exclusionary diagnosis of ieu be made. candidal esophagitis is the most common cause of esophagitis in hiv patients, especially in patients with complaints of dysphagia, or odynophagia and dysphagia. 10 the fungal isolate is generally candida albicans, though other species of candida can also affect the esophagus. it should be suspected in patients with a cd4+ lymphocyte count of <100 cells/mm 3 (though can occur at any cd4+ count), and esophageal symptoms with or without thrush, which can be absent in 30% of patients. 11 in patients with hiv and new onset symptoms, an empiric trial of standard dosed fl uconazole is an effective strategy, as 82% of patients in a prospective study responded. if there is no response, endoscopy can be pursued. 6 in patients that fail empiric antifungal therapy, the most common etiology (in 77% of the patients) is ulcerative esophagitis as opposed to persistent candidiasis. 12 fluconazole is the drug of choice for candidiasis, with a loading dose of 200 mg orally followed by 100 mg daily from 10-14 days. clotrimazole troches are also successful, as topical treatment of esophageal candidiasis, 13 but nystatin is not. 14 itraconazole and ketoconazole are effi cacious systemic therapies, but not as effective as fl uconazole. 15, 16 amphotericin b is also a helpful therapy, but is generally used only in azole-resistant patients because of the toxicity of the medication. low-dose amphotericin (0.3-0.5 mg/kg per day for 7-14 days) is usually adequate. caspofungin can also be used in candidal esophagitis, and is felt to be as effi cacious as fl uconazole and well tolerated. 17 however, it is only available in intravenous form and is more expensive, but may be the drug of choice for azoleresistant mucosal candidiasis because of the relative lack of toxicity compared with amphotericin b. primary prophylaxis of candidal disease is not recommended because of the non-life-threatening nature of the disease, the effectiveness of acute therapy, and the risk of antifungal resistance. while candida is the most common esophageal pathogen, it can also occur in addition to ulcerative esophagitis in hiv patients. cmv esophagitis is the most common etiology of odynophagia in hiv patients and therefore esophageal ulcerations, up to 45% of patients in one prospective study. 18 fever and substernal chest pain can be reported in addition to odynophagia, and thrush can be concomitant, but dysphagia is very uncommon. the diagnosis of cmv is best made by endoscopy and biopsy, with the pathology showing viral cytopathic effect in the gastrointestinal mucosa via intranuclear inclusions. immunohistochemistry is also helpful for confi rmation, as viral cultures are less sensitive and specifi c. 19 cmv is the most common viral pathogen in the esophagus in hiv patients, and esophagitis is the most common extraocular manifestation of cmv. 20 it can appear as a diffuse esophagitis, single or multiple ulcers, or giant ulcers involving the whole esophagus, and generally occurs when the cd4+ lymphocyte count is <50. it may be discovered only after treatment for candida, as they may exist concurrently in 20% of patients. 21 the incidence of cmv esophagitis has declined dramatically in the art era. treatment for cmv esophagitis involves a wide array of antiviral medications, namely ganciclovir, valganciclovir, foscarnet, and cidofovir. ganciclovir was the fi rst agent used to combat cmv and has the most data behind it; the response rate is about 70-80%. it is given intravenously in a 2-4 week induction period, 5-10 mg/kg per day, but has dose limiting side-effects, mainly bone marrow suppression (and resultant neutropenia and thrombocytopenia). oral ganciclovir can also be used as maintenance therapy, but the data is limited. intravenous foscarnet (90 mg/kg b.i.d., 2-3 weeks) is seen as equally effective to intravenous ganciclovir in the treatment of cmv esophagitis, but comes with a nephrotoxic side-effect profi le; however, randomized studies have shown equal effi cacy and safety. 22 foscarnet is generally used in cases of clinical failure after ganciclovir induction. 23 oral valganciclovir (900 mg/ day) has not been tested in cmv esophagitis, but does have 60% of the bioavailability of intravenous ganciclovir. cidofovir also can treat cmv, but is not typically used because of nephrotoxicity. hsv esophagitis is a relatively uncommon cause of esophageal ulceration in hiv patients compared with cmv and ieu. the endoscopic appearance is described as being well circumscribed and having a 'volcano' like appearance, distinguishing them from the ulcers seen in cmv infection, which tend to be linear or longitudinal and are deeper. 24 treatment is with oral acyclovir (15-30 mg/kg per day), though if patients have diffi culty with swallowing, intravenous acyclovir can be used. valacyclovir and famci-clovir can also be used because of their effi cacy and convenient dosing schedules. foscarnet intravenously (40 mg/kg b.i.d.) is used in cases of acyclovir resistance. 25 secondary prophylaxis with valacyclovir (500-1000 mg/day) is recommended in patients with frequent relapses. idiopathic esophageal ulcers (ieu) are a diagnosis of exclusion if none of the pathologic, fungal, or viral studies return a diagnosis. the treatment of idiopathic esophageal ulcers is done with oral corticosteroids, generally starting at 40 mg of oral prednisone daily. 26 if the patient cannot take medications orally, then the corticosteroids can be given intravenously. in addition, thalidomide can also be used for ieu if corticosteroids are not effi cacious. 27 in addition to the infections and ulcerations discussed above, there are other esophageal issues in hiv, namely motility and neuropathic issues. there is a form of hiv neuropathy that could lead to gastrointestinal complaints, like gastroparesis. these would be treated symptomatically, with prokinetic agents like metoclopramide. additionally, in patients with both symptomatic esophageal complaints, as well as those that are asymptomatic, there are fi ndings of esophageal motility abnormalities. this is probably because of the neurotropic nature of hiv leading to autonomic dysfunction in the gastrointestinal neurologic plexus. 2 the problems associated with the stomach in hiv patients are similar to the stomach problems of non-hiv infected individuals. opportunistic infections that affect hiv patients typically do not affect the stomach. symptoms and presentation are often related to abdominal pain, nausea or vomiting. the most common manifestations of gastric illness in hiv patients is like the general population, namely gerd, peptic ulcer disease (pud) and gastritis. care needs to be used when prescribing histamine-2 (h 2 ) blockers or proton-pump inhibitors (ppis), however, as these medications can interact with art, especially ppis. work-up is the same as with the general population, and endoscopy with biopsy is the gold standard for diagnosis. helicobacter pylori, a common cause of peptic ulcer disease in non-hiv positive patients, seems to have a decreased incidence in hiv patients compared with the general population; cmv may actually be the leading cause of pud in hiv patients. 28 as far as actual hiv-related gastric pathology, gastric lymphoma, kaposi's sarcoma, and some of the opportunistic organisms (e.g. cmv, tuberculosis, toxoplasmosis, and cryptococcosis) can be seen. in addition, dyspepsia, nausea and vomiting can all be related to the side-effects of various antiretroviral medications. one of the most common complaints of hiv patients is diarrhea. the reasons for diarrhea are multifold, but most commonly relate to opportunistic infection and antiretroviral medications. a more in depth coverage of diarrheal illness in hiv patients is discussed later in ch. 65: etiology and management of diarrhea in hiv-infected patients and impact on antiretroviral therapy, but an overview will follow here. as with esophageal disease, the incidence of opportunistic infections has decreased in the art era, though the incidence of chronic diarrhea has remained steady even in the art era. 29 the evaluation of diarrhea includes a thorough history and physical, as much can be determined from just eliciting the patient's history of symptoms. if the diarrhea occurs with upper abdominal cramps, or bloating, this suggests an upper intestinal source, or enteritis. bloody diarrhea, tenesmus, and lower abdominal cramping imply colonic involvement. in addition, patients with a history of receptive anal intercourse have a higher involvement of colitis and sexually transmitted pathogens (e.g. gonorrhea, hsv, etc.) in the anorectal area. homosexual or bisexual men have a 3-fold higher incidence of diarrhea than patients in other risk groups. obviously, travel and diet history can also be important in the histories of hiv patients with diarrhea. as with other aspects of hiv, opportunistic infections tend to be more common in the setting of lower cd4+ lymphocyte counts (and therefore greater immunosuppression). the diagnostic studies involved in the work-up of diarrhea in the hiv patient are similar to those in the general population. in addition to history and physical exam (exam specifi cally adds little to diagnosis, other than evidence of malnutrition), the fi rst route of investigation is often the stool examination. the tests to order include bacterial culture, clostridium diffi cile toxin assay, as well as looking for ova and parasites, especially isospora, cryptosporidium, and microsporidia. these generally need to be specifi ed as potential pathogens when the sample is sent to microbiology when looking at ova and parasites. in addition, a gram stain or methylene blue stain for fecal leukocytes can also be helpful, as evidence of fecal leukocytes may point towards a picture of colitis. another form of non-invasive testing is blood cultures and serologies, which can be helpful for the diagnosis of systemic opportunistic infections like mycobacterium avium intracellulare (mai) or viral etiologies like cmv. especially in the patients who have diarrhea and fever, mai may be a possibility. however, with all of these etiologies, a diagnosis may be treated, and symptoms may still persist as secondary infections may also be present. for colitis, a barium enema or abdominal radiography may detail the presence of a toxic megacolon, a complication of clostridium diffi cile colitis. in addition to the non-invasive studies listed above, in the absence of a diagnosis, the workup for diarrhea should include some invasive studies as well. the fi rst step is generally a fl exible sigmoidoscopy, which can give visualization of the colon (to diagnose or rule out pseudomembranous colitis, among other etiologies) and provide tissue for biopsy. abnormal tissue should be biopsied, but if the mucosa is normal then random tissue can be sent. the fl exible sigmoidoscopy is better than the alternatives as it does not require sedation. if small intestinal etiology is suspected, an egd (going past the second portion of the duodenum) can help determine the cause of the diarrhea through biopsies of the small bowel, sent not only for pathology, but also electron microscopy and culture analysis. if the fl exible sigmoidoscopy is nondiagnostic, and there is still no diagnosis from other studies, a colonoscopy can be performed so more biopsies can be done to rule out opportunistic infections, especially in the ileum. this is rarely done, however, and in general, the absence of defi nitive diagnosis leads to treatment and evaluation (as will be discussed later). the symptoms associated with enteritis are typically associated with diarrhea and prolonged malabsorption leading to malnutrition. this is generally because of opportunistic infections, and, similar to other pathologies in hiv patients, the incidence of enteritis has decreased in the highly active antiretroviral therapy (art) era. the main symptoms of enteritis are copious voluminous diarrhea (>2 l/day) with dehydration and malabsorption, as opposed to colitis, which is a bloody, painful diarrhea. the work-up of enteritis is detailed above, but specifi cally should include stool studies, and, if non-diagnostic, esophagogastroduodenoscopy (egd) with biopsy. the etiologies of enteritis in hiv patients are multifold, and include bacterial, viral, infections of the small intestine (enteritis) fungal, and parasitic pathogens. these can be diagnosed by the stool studies detailed earlier. parasites may be the most common etiology for enteritis, especially in those patients not on art and greatly immunosuppressed. parasites are typically diagnosed through stool analysis for ova and parasites, including direct fl uorescent antibody (dfa), enzyme-linked immunosorbent assay (elisa) or polymerase chain reaction (pcr). light microscopy can be used, though pcr can be diagnostic at much lower levels of parasitic infection. cryptosporidium parvum is the most commonly identifi able pathogen in aids related persistent diarrhea, especially in patients with cd4+ lymphocyte counts <200. it is typically treated with paromomycin 1500-3000 mg every 6-8 h orally, or azithromycin 900-1200 mg four times a day, though albendazole 400 mg twice a day has also shown to be effective. nitazoxanide (1 g twice daily for at least 2 weeks) can also be used in the treatment of cryptosporidiosis, but a cure is generally not possible if the cd4+ lymphocyte count is <50. however, a study with children showed no benefi t to nitazoxanide at all in hiv+ children (just hiv seronegative ones). 30 hyperimmune bovine colostrums can also be used, but typically not to cure the parasitic infection. 31 microsporidiosis is the next most commonly identifi able refractory diarrhea in hiv patients. this can be treated with albendazole as well (400-1600 mg every 12 h orally) but most cases are poorly responsive to treatment and require indefi nite therapy. isospora belli is rare in the usa, but is more common in developing countries. trimethoprim-sulfamethoxazole, one double strength tablet every 6 h for 10 days is the treatment of choice, but pyrimethamine 50-75 mg four times daily (with folinic acid 5 mg orally four times daily) is acceptable for patients with allergies to sulfa medications. 32 lastly, giardia lamblia and amoebic dysentery can also occur in hiv patients, at the same incidence as the general population, and can be treated with metronidazole 750 mg thrice daily for 5-10 days, or tinidazole 2 g orally once for giardiasis, 3 days for amebic dysentery. 30 for strongyloidosis, thiabendazole 25 mg/kg twice daily orally is the drug of choice. albendazole seems to be active against all of the parasitic organisms associated with diarrhea in hiv patients, and could be the fi rst line of therapy when parasitic infection is suspected, pending microbiological study. with all of the aforementioned parasites, treatment cannot only be targeted at the pathogen, but also at the diarrheal symptoms, with the use of somatostatin analogs like octreotide to try to reduce the amount of diarrhea. in addition, since the para-sites are both more common and more chronic at lower cd4+ lymphocyte counts, art, which reduces the degree of immunosuppression, can also be curative. opioids such as tincture of opium or codeine can provide symptomatic relief in cases of severe diarrhea through its constipating actions, as well as pain relief. 33 bulking agents, lactose-free diets, and antidiarrheal medications like diphenoxylate with atropine or loperamide are also benefi cial in treating diarrheal symptoms. viral infection in hiv patients can cause diarrhea, typically through colitis, but also rarely through an enteritis. cmv, in addition to causing esophagitis, can also affect the gi tract through diarrheal illness, and runs the spectrum from asymptomatic carriage to severe diarrheal illness including appendicitis, bleeding and perforation. it typically occurs in the setting of severe immunosuppression with a cd4+ lymphocyte count <100. the diagnosis of cmv enterocolitis is best made through demonstrating a viral cytopathic effect in tissue specimens, but viral stool cultures can also signify disease (though are less sensitive). the treatment for cmv enterocolitis is mainly ganciclovir and foscarnet, as described earlier in the esophagitis section. valganciclovir may not achieve adequate bioavailability because of the enterocolitis. other viruses can affect the hiv patient gastrointestinal tract, including rotavirus, adenovirus, norwalk virus, or unusually, picornaviruses, and coronaviruses. these tend to be less common than the other pathologies previously described, and are diffi cult to diagnose as well. adenovirus can cause a hemorrhagic colitis; acute diarrhea is seen in patients with only adenovirus in stools, but patients with adenovirus on biopsy specimen generally have a chronic diarrhea. 34 hsv can cause diarrhea via systemic infection in end-stage hiv patients, or can cause a colitis and proctitis through hiv mucosal lesions. hsv can be treated with acyclovir, valacyclovir, famciclovir, or, if acyclovir resistant, foscarnet (as previously described in the esophagitis section). in addition, hiv itself may be a cause of hiv enteropathy and a diarrheal pathogen, and may be identifi ed in gut tissue in up to 40% of patients, but this is controversial. idiopathic aids enteropathy, on the other hand, is the term used for a chronic diarrhea in an aids patient that is without identifi able pathogen or diagnosis (despite intensive investigation). mucosal hyperproliferation is noted on biopsy. for these etiologies, in addition to art to increase cd4+ lymphocyte count and reduce immunosuppression, should be treated symptomatically with bulking agents, antidiarrheals, and opioids. the combination of antidiarrheal therapy with art has been shown to be more benefi cial than antidiarrheal therapy alone in hiv patients with chronic diarrhea. 35 other agents that can cause enteritis include mycobacterium avium intracellulare (mai) and pneumocystis jiroveci (pcp). small intestinal disease is the most common site of gastrointestinal luminal involvement by mai. 36 it is often seen with diffuse small bowel infi ltration (mimicking whipple's disease) and causes severe malabsorption in patients with cd4+ lymphocyte counts of <50. if a malabsorptive diarrhea occurs with fever and night sweats, in addition to weight loss, mai must be considered, and blood cultures or a bone marrow biopsy may be diagnostic for disseminated mai infection. diagnosing mai enteritis is more diffi cult, however, as a positive stool is not diagnostic for gastrointestinal disease (though can suggest subsequent disseminated disease). 37 an endoscopic biopsy and acidfast staining can show acid-fast bacilli and give an ideal diagnosis. treatment is with a multitude of options, using combinations of clarithromycin 500 mg twice daily, ethambutol 800-1200 mg orally daily, azithromycin 600 mg daily, rifampin 600 mg daily, rifabutin 300 mg daily, amikacin 15 mg/kg three times weekly, and ciprofl oxacin 750 mg twice daily. these can reduce, but not eradicate, mai. luminal tuberculosis can also occur as an example of extrapulmonary involvement, but is rare; in contrast to mai, it can generally be treated to cure with antituberculous therapy. pneumocystis jiroveci (pcp) can also be seen as the cause of diarrhea in hiv patients, but is very uncommon, especially in the setting of pcp prophylaxis for aids patients; treatment is with antipneumocystis therapy, 38 generally with trimethoprim-sulfamethoxazole. bacterial infections in hiv patients typically cause a picture of colitis instead of enteritis, with bloody diarrhea and tenesmus. the most common bacterial pathogens seen in hiv patients include salmonellae, shigella, e. coli, campylobacter jejuni, and clostridium diffi cile. salmonellosis is 100 times more common in hiv patients than in immunocompetent hosts, 39 and recurrent salmonella bacteremia establishes the diagnosis of aids in an hiv patient. the diagnosis is straightforward, as salmonella can normally be cultured in stool specimens in addition to blood culture results. salmonella gastroenteritis can present with either watery diarrhea or dysentery (mucopurulent diarrhea) with or without fever, abdominal pain, or nausea and vomiting. though the diarrhea may be self-limited, the treatment is generally ciprofl oxacin 500 mg orally twice a day, for 2-4 weeks, for eradication. shigella has a similar presentation to salmonellosis, with a similar wide spectrum of presentation of illness. it does come with a high rate of severe complications, including anemia, hypoglycemia, sepsis, hemolytic uremic syndrome, disseminated intravascular coagulopathy and renal failure, with which mortality obviously increases. it also can be treated with ciprofl oxacin 500-750 mg twice a day orally for 5-7 days, and is diagnosed by stool culture as well. campylobacter jejuni generally presents as a watery diarrhea, and its incidence is probably decreased due to widespread pcp prophylaxis with trimethoprimsulfamethoxazole. it is typically harder to culture from stool, and may be diagnosed by endoscopic biopsy. antimicrobial therapy is not essential, though erythromycin 250-500 mg orally four times daily, or ciprofl oxacin 500 mg orally twice a day for 5-7 days may reduce the duration of the illness. e. coli may be seen (in any of several strains), and, like the other enteric bacterial diarrheal illnesses, can be treated with a fl uoroquinolone like ciprofl oxacin. as illustrated, in cases of suspected bacterial diarrhea, ciprofl oxacin would cover most enteric pathogens and would be the empiric drug of choice. clostridium diffi cile is seen in hiv patients not only in the presence of antibiotic therapy, but also in the absence of recent antibiotic therapy. the most common antibiotics that cause c. diffi cile are clindamycin, ampicillin, cephalosporins and aminoglycosides. the clinical presentations and response to therapy are not different in hiv patients than in patients without hiv. diagnosis is made by detecting c. diffi cile toxin in stool assay, and treatment is with metronidazole 250-500 mg orally every 6-8 h for 10-14 days, or tinidazole, in addition to stopping the offending initial antibiotic therapy. in resistant cases, oral vancomycin 250 mg every 6 h can be used, as can rifaximin 200 mg three times daily, though it is not as effective as vancomycin. in cases of suspected c. diffi cile without diagnosis via stool toxin assay, a fl exible sigmoidoscopy can look for pseudomembranous colitis, which can be diagnostic for c. diffi cile. fungal etiologies of diarrhea in hiv patients are relatively rare, but can occur in patients with immunocompromised states and low cd4+ lymphocyte counts. gastrointestinal histoplasmosis appears to be the most commonly described fungal etiology of diarrhea in hiv patients, and typically occurs in the colitis setting of a systemic infection. diagnosis is made by fungal culture and smear of tissue or blood, 40 and treatment is with amphotericin b 0.5-1 mg/kg per day intravenously initially, with maintenance therapy with itraconazole 200 mg orally daily. coccidiomycosis and cryptococcosis are also rare, and occur in the presence of systemic infections as well. in addition, as candidal infections are the most common opportunistic infections of hiv patients, a dehydrating diarrhea can also occur as a manifestation of the infection. obviously, not all causes of diarrhea in hiv patients are secondary to opportunistic infections. several noninfectious etiologies of diarrhea in hiv patients can occur as well, including the most common, drug-induced diarrhea. the most common drugs that cause diarrhea in hiv patients are nucleoside reverse transcriptase inhibitors (nrtis) and protease inhibitors (pis). with protease inhibitors, diarrhea is the most common side-effect reported with nelfi navir and saquinavir, most commonly occurs at the initiation of treatment, and is a cause of cessation of therapy and lack of adherence to treatment. 41 newer agents like lopinavir-ritonavir seem to cause less diarrhea than older pis like nelfi navir. 42 treatment is generally guided towards treatment of symptoms, namely with bulking agents or antidiarrheals like loperamide. other causes of diarrhea in hiv patients include infl ammatory bowel disease, including crohn's disease and ulcerative colitis, neither of which have increased incidence in hiv patients. treatment is tailored according to the disease process itself, though an active disease process may decrease cd4+ lymphocyte count; this may be reversed by colectomy. 43 in addition, aids related illnesses that are noninfectious but can also cause diarrhea and gastrointestinal issues include lymphoma and kaposi's sarcoma, both of which are diagnosed by biopsy. infi ltration of the mucosal tract by the neoplasm can lead to diarrhea and weight loss. anorectal disease is a big component of hiv gastrointestinal care, especially among homosexual males. interestingly, the incidence of anorectal pathology in hiv patients has not been affected by art. 44 many anorectal pathologies are seen in hiv patients, including anal fi stulas and fi ssures, perirectal abscesses, ulcerations and proctitis (box 23.1). in addition, anal neoplasms as a result of human papillomavirus (hpv) and other etiologies can also occur. anorectal carcinoma has an increased incidence in both hiv patients and among homosexual males, and is the fourth most common malignancy seen in hiv; 45 hiv+ homosexual men have twice the incidence than hiv negative homosexual men. anal squamous cell carcinoma is frequently associated with squamous intraepithelial neoplasia and hpv, much like cervical carcinoma, and can be detected by anorectal cytology, similar to papanicolaou smears. 46 the gold standard for diagnosis of anorectal neoplastic disease is still anoscopy with biopsy, though anorectal cytology can be useful as a screening test. in addition to anal carcinoma, other anorectal symptoms are common in the hiv population, especially among homosexual males. anal condyloma is the most common hiv related anal pathology, and is associated with hpv infection; treatment options include a variety of surgical options, including cryotherapy. the four most common infectious causes of proctitis in men who have sex with men are gonorrhea, herpes simplex, chlamydia and syphilis. 47 gonorrhea and chlamydia are typically treated together with ceftriaxone 125 mg i.m. once and azithromycin 1 g orally once; fl uoroquinolones, oral cephalosporins and doxycycline (100 mg orally twice a day for 7 days) can also be used. primary syphilis is treated with benzathine penicillin g 2.4 million units i.m. once (or doxycycline 100 mg twice daily for 2 weeks if penicillin allergic). hsv infections, as described earlier with esophagitis and colitis, can also cause perianal and rectal ulcerations, with associated symptoms of tenesmus, pain, and bleeding. treatment is with antiherpetic medications like acyclovir, valacyclovir, or famciclovir, as explained earlier, with foscarnet in acyclovir-resistant cases. other etiologies of proctitis include lymphogranuloma venereum, as well as other causes of colitis detailed above; clinical overlap can also happen in hiv patients. in addition to a thorough physical examination, all patients with anorectal symptoms should have anoscopy and sigmoidoscopy with mucosal biopsy to look for fi ssures, perirectal abscesses, and fi stulas in addition to searching for opportunistic infections, with microbiological studies sent for viral, fungal and bacterial cultures. oesophageal symptoms, their causes, treatment and prognosis in patients with aids esophageal motility disorders in hiv patients odynophagia from aphthous ulcers of the pharynx and esophagus in aids chronic idiopathic esophageal ulceration in aids: characterization and treatment with corticosteroids declining prevalence of opportunistic gastrointestinal disease in the era of combination antiretroviral therapy fluconazole compared with endoscopy for human immunodefi ciency virus-infected patients with esophageal symptoms aids therapy prospective endoscopic characterization of cytomegalovirus esophagitis in patients with aids prospective comparison of brush cytology, viral culture, and histology for the diagnosis of ulcerative esophagitis in aids natural history of hivassociated esophageal disease in the era of protease inhibitor therapy prospective evaluation of oropharyngeal fi ndings in human immunodefi ciency virusinfected patients with esophageal ulcer etiology of esophageal disease in human immunodefi ciency virusinfected patients who fail antifungal therapy comparison of oral fl uconazole and clotrimazole troches as treatment of oral candidiasis in patients infected with human immunodefi ciency virus oropharyngeal candidiasis in patients with aids: randomized comparison of fl uconazole versus nystatin oral suspensions fluconazole versus itraconazole for candida esophagitis in aids fluconazole compared with ketoconazole for the treatment of candida esophagitis in aids: a randomized trial a randomized double-blind study of caspofungin versus fl uconazole for the treatment of esophageal candidiasis esophageal ulceration in human immunodefi ciency virus infection: causes, response to therapy, and long-term outcome frequency of positive tests for cytomegalovirus in aids patients: endoscopic lesions compared with normal mucosa cytomegalovirus infection in patients with hiv infection cytomegalovirus and candida esophagitis in patients with aids treatment of cytomegalovirus esophagitis in patients with acquired immune defi ciency syndrome: a randomized controlled study of foscarnet versus ganciclovir foscarnet treatment of cytomegalovirus gastrointestinal infections in acquired immunodefi ciency syndrome patients who have failed ganciclovir induction herpes esophagitis: clinical syndrome, endoscopic appearance, and diagnosis in 23 patients successful foscarnet therapy for acyclovir-resistant mucocutaneous infection with herpes simplex virus in a recipient of allogeneic bmt a pilot study of oral corticosteroid therapy for idiopathic esophageal ulcerations associated with human immunodefi ciency virus infection thalidomide for the treatment of esophageal aphthous ulcers in patients with human immunodefi ciency virus infection. national institute of allergy and infectious disease aids clinical trials group low prevalence of helicobacter pylori but high prevalence of cytomegalovirusassociated peptic ulcer disease in aids patients: comparative study of symptomatic subjects evaluated by endoscopy and cd4 counts the changing etiology of chronic diarrhea in hiv-infected patients with cd4 cell counts less than 200 cells/mm 3 effect of nitazoxanide on morbidity and mortality in zambian children with cryptosporidiosis: a randomised controlled trial management of protozoal diarrhoea in hiv disease management of diarrhea in hiv-infected patients palliative care and aids: 2 -gastrointestinal symptoms enteric viral infections as a cause of diarrhoea in the acquired immunodefi ciency syndrome impact of protease inhibitors on the outcome of human immunodefi ciency virus-infected patients with chronic diarrhea atypical mycobacterial infection of the gastrointestinal tract in aids patients mycobacterium avium complex in the respiratory or gastrointestinal tract and the risk of m. avium complex bacteremia in patients with human immunodefi ciency virus infection pneumocystis colitis in a patient with the acquired immunodefi ciency syndrome risk factors for primary bacteremia and endovascular infection in patients without acquired immunodefi ciency syndrome who have nontyphoid salmonellosis disseminated histoplasmosis in the acquired immune defi ciency syndrome: clinical fi ndings, diagnosis, and treatment, and review of the literature management of protease inhibitorassociated diarrhea differences in rates of diarrhea in patients with human immunodefi ciency virus receiving lopinavir-ritonavir or nelfi navir infl ammatory bowel disease in individuals seropositive for the human immunodefi ciency virus anorectal pathology in hiv/aids-infected patients has not been impacted by highly active antiretroviral therapy cancer incidence in a population with a high prevalence of infection with human immunodefi ciency virus type 1 anorectal cytology as a screening tool for anal squamous lesions: cytologic, anoscopic, and histologic correlation etiology of clinical proctitis among men who have sex with other men key: cord-004986-en7taikk authors: nagy, nathalie; remmelink, myriam; van vooren, j. p.; salmon, isabelle title: infections gastro-intestinales chez le patient immunocompromis date: 2002 journal: acta endoscopica doi: 10.1007/bf03016656 sha: doc_id: 4986 cord_uid: en7taikk the gastrointestinal tract is frequently involved in immunocompromised hosts. the most common digestive manifestations are dysphagia, odynophagia and diarrhea. these diseases are more frequent in patients with acquired immunodeficiency virus (aids). these gi diseases are of several categories: hiv related inflammatory conditions (hiv related enteropathy, idiopathic esophageal ulceration), infections due to germs also commonly present in immunocompetent patients (salmonellosis, shigellosis,…), opportunistic infections (cmv, mucormycosis,cryptosporidium, mycobacterium, isospora belli,…). the prevalence, pathogenesis, clinical manifestation, gross pathological findings and microscopic features are discussed for each entity. muqueuse, l'absence quasi complete de lymphocytes cd4+ au niveau intra-6pith61ial et de mani~re concomitante une diminution des lymphocytes cdll+ intra-6pith61iaux du gr~le et du rectum. le rapport t4/t8 de la lamina propria est identique h celui observ6 dans le sang p6riph6rique. le nombre et la fonction rdduits des cd4 rdsultent en une diminution du nombre de plasmocytes ig a s~crdtant au sein de la muqueuse. ii existe dgalement une rdduction de la s~cr~tion acide gastrique, probablement lide gt la presence d'anticorps anti cellules paridtales, ainsi qu'une diminution de la motilitd intestinale rdsultant d'une ddnervation autonomique. la combinaison de tous ces facteurs permet une colonisation aisle de l'intestin grgle par une flore anadrobie, source de diarrhdes pr~cddant trds souvent des infections opportunistes [3] . dans les pays industrialis6s, 75 % des patients hiv ou sida d6veloppent des diarrh6es persistantes ; ce chiffre atteint 100 % dans les pays en voie de d6veloppement. dans 80 % des cas, ces diarrhdes sont imputables ~ des ent6ropathog~nes dont la coccioidose est la plus fr6quente [1] . une malabsorption, des anomalies de la muqueuse gr~le en l'absence d'agent pathog6ne a pour la premiere fois 6t6 d6crite en 1984. le virus hiv a 6t6 d6tect6 dans la muqueuse intestihale, suscitant l'hypothbse qu'il soit lui-m6me responsable, du moins en partie, des 16sions observdes. n6anmoins, cette hypoth6se n'a pas 6t6 formellement confirm6e et certains auteurs sceptiques sugg6rent que ces diarrh6es seraient dues ?a des agents opportunistes non d6tectables par les techniques usuelles ou inconnus. enfin, les facteurs alimentaires tels que le r6gime pauvre en prot6ines, le d6ficit en acide folique ou en vitamine a peuvent 6galement affecter l'int6grit6 de l'6pithflium intestinal. approximativement 30 ?a 50 % des patients hiv pr6senteront durant le d6cours de la maladie une atteinte cesophagienne. l'agent pathog6ne le plus fr6quemment isol6 est le candida albicans survenant dans plus ou moins 50 70 % des cas, suivi des infections herp6tiques et cmv, estimdes h 15-35 % [4, 5] . n6anmoins, lorsque toutes les causes 6tiologiques causant des ulc6rations oesophagiennes ont 6t6 61imi-n6es, 40 a 50 % des patients hiv pr6sentent des ulc6rations idiopathiques. au sein du tissu de granulation, la prot6ine hivp24core a 6t6 d6tect6e, sugg6rant une participation active dans le processus d'ulc6ration en l'absence d'autre agent pathog6ne. ces ulc6rations surviennent le plus souvent lorsque le taux de cd4 est bas. les sympt6mes en sont gdn6ralement de l'odynophagie, l'aspect endoscopique mimant les ulc~res her-p6tiques et ~ cmv. ces ulcbres sont souvent difficile h gu6rir ; une instillation intral6sionnelle de corticoides de m6me que le thalidomide semblent donner de bons r6sultats. dans les proctocolites li6es au virus hiv, les marqueurs de l'inflammation sont non sp6cifiques, tels que l'augmentation du nombre de granules lysosomiaux au sein des lymphocytes intra-6pith61iaux, la pr6sence de cellules 6pith61iales apoptotiques, la pr6sence de structures r6ticulaires au sein des cellules endoth61iales, des lymphocytes ou des monocytes. le degr6 d'inflammation semble corr61er avec le taux d'antig~ne p24 d6cel6 dans la muqueuse ainsi qu'avec l'importance des sympt6mes cliniques, sug-g6rant un rfle 6tiologique du virus hiv [1] . l'ent6rocolite neutrop6nique est une condition inflammatoire non li6e au virus hiv ou aux infections opportunistes, 6galement appel6e typhlite. celle-ci est caract6ris6e par une inflammation aigue affectant essentiellement le caecum, l'appendice et parfois l'il6on terminal. d'abord d6crite chez l'enfant leuc6mique et s6v~rement neutrop6nique, l'affection se rencontre actuellement chez les patients souffrant d'h6mopathies malignes ou de cancers. la neutrop6nie associ6e aux effets de la chimioth6rapie permettent aux baet6ries intraluminales d'envahir et d'endommager la muqueuse. une septic6mie, le plus souvent causee par des bacilles gram n6gatifs, survient chez 79 % des patients. au niveau histologique, des h6morragies, un ~ed~me marqu6, une inflammation focale pauvre en cellules inflammatoires sont observ6s. parfois, une zone ndcrotique r6sultant d'un ulc6re bien ddlimit6, provoque une perforation. focalement, des colonies bact6riennes intramurales et des kystes gazeux sousmuqueux peuvent 6tre vus. les infections opportunistes representent la majo-rit6 des infections rencontrees chez l'hete immunocompromis, la plupart d'entre elles se rencontrant au niveau du tractus gastro-intestinal. le spectre de celles-ci s'est rapidement elargi. lors d'etudes anterieures (1990) , 43 % des causes d'enteropathies associees ~ des diarrhees chroniques 6taient inconnues. une etude 4 ans plus tard n'en revelait plus que 19 %, r6sultant de l'identification de nouveaux patho-g~nes [1] . les affections opportunistes varient egalement en fonction du sexe, du groupe h risque, de la localisation et de la periode etudiee. par exemple, la candidose eesophagienne, atteinte gastro-intestinale la plus frequente chez le patient hiv aux etats-unis et en europe, affecte plus particulierement les femmes. de m~me, les infections ~ cmv ou les cryptosporidioses surviennent plus frequemment chez tes homosexuels que chez les patients sida ayant contracte la maladie par drogues intraveineuses. l'isosporiase affectant 30 % des patients sida haitiens est pratiquement absente aux usa. lorsque les traitements sont efficaces et que la survie des patients augmente, l'epidemiologie des infections opportunistes se modifie. par exemple, les pneumonies resultant du pneumocystis carinii ont largement diminue depuis 1987, alors que les infections herpetiques et ~ cmv ont particuli~rement progress6 surtout chez les hommes homosexuels. dans 44 h 68 % des patients sida presentant une enteropathie due ~un ou plusieurs agents pathogenes concomitant, des symptemes gastro-intestinaux sont retrouves. le diagnostic d'infections opportunistes est en general base sur une combinaison de culture de selles, examen direct des selles ~ la recherche d'ceufs ou de larves, et d'une biopsie endoscopique. les symptemes gastro-intestinaux sont plus frdquents chez les patients africains que chez les europeens et les nord americains. les deux plaintes majeures sont l'odynophagie et les diarrhees. l'atteinte ~esophagienne survient chez 30 ~ 40 % des patients ; l'incidence des diarrhees est plus elevee atteignant 90 % particulierement chez les patients sevbrement immunocompromis. les infections virales se rencontrent chez tous les groupes de patients immunocompromis. l'infection cmv est le pathog~ne gastro-intestinal le plus frequent. l'infection herpetique semble 6tre plus frequente chez le patient hiv que chez les autres patients immunodeprimes. dans une importante etude prospective r6alisee sur 100 patients hiv pr6sentant une cesophagite her-petique, le virus hsv n'a 6te identifi6 que darts 5 % des cas alors que la prevalence du virus cmv atteignait 50 % [4] . les infections herpetiques chez l'hete immunocompromis semblent representer une reactivation d'une infection latente contractee plus tet dans la vie, et son incidence augmente lorsque l'immunodepression gagne du terrain. des anticorps igg specifiques diriges eontre l'anti-g~ne herpetique ont 6te documentes chez plus de 80 % des hommes homosexuels. chez le patient hiv, la reactivation du virus hsv survient lorsque le taux de cd4 diminue au-dessous de 50/ram 3. l'infection herpetique infecte essentiellement la muqueuse malpighienne ; d~s lors la muqueuse peri-anale et l'cesophage sont les muqueuses les plus frequemment tou-chees. l'oesophagite herpetique peut parfois, dans des formes severes, se compliquer d'une necrose transmurale associee ~ une fistule tracheo-~esophagienne. les une atteinte gastrique s'accompagne en g6n6ral d'une atteinte diss6min6e dans le tube digestif. les colites ~ cmv prddominent dans le caecum et le c6lon droit ; la muqueuse peut paraitre normale mais en g6n6ral on y observe de profondes ulcdrations. dans certaines atteintes s6vhres, une n6crose de la paroi avec perforation peut s'observer comme dans les m6gac61ons toxiques. l'aspect histologique des cellules infectdes par le virus est caract6ristique ; on retrouve au sein du cytoplasme ou du noyau des inclusions virales ; les cellules semblent ballonis6es. les inclusions nucl6aires sont acidophiles et fr6quemment entour6es d'un halo ; les inclusions cytoplasmiques sont souvent multiples, granulaires et basophiles (fig. 1) . le cmv infecte plusieurs types cellulaires, essentiellement les cellules endoth61iales, mais aussi les cellules musculaires lisses, les fibroblastes, et les cellules ganglionnaires. les cellules glandulaires sont nettement moins infect~es que les cellules malpighiennes. le diagnostic est r6alis6 par endoscopie et biopsies [6] . les immunomarquages et l'hybridation in situ sont des m6thodes plus sensibles que i'he ; la pcr repr6sente la technique diagnostique la plus sensible. le r6sultat du traitement par gancyclovir peut ~tre monitor6 par un grading histologique sur les sp6cimens biopsiques [4] . certains auteurs ont d6velopp6 un syst~me de gradation simple ddfini en trois grades selon le hombre de cellules infectdes visualis6es : grade i = de 1 ~ 4 cellules infect6es, grade ii = de 5 ~ 9, grade lii= plus de 10. les patients sida prdsentent plus fr6quemment des sympt6mes li6s/t ces organismes, ainsi que des infections prolong6es par rapport fi la population g6n6rale. la salmonellose r6sultant d'une infection ~ partir de salmonella typhimurium est 20 fois plus frequente chez les patients sida. la shigellose est souvent isolde des coprocultures chez ces patients sida; dans cette population, l'infection est potentiellement fatale. les infections ~ campylobacter ont 6t6 identifi6es dans approximativement 11% des coprocultures des patients sida, qu'ils souffrent ou non de diarrh6es ; ces patients, pr6sentant une incidence d'infection, sont 39 fois plus importants que dans la population g6n6rale. les femmes sont plus fr6quemment infect6es que les hommes. le clostridium difficile affecte la plupart des types de patients immunocompromis et est li6 essentiellement h la prise d'antibiotiques et aux hospitalisations prolong6es ou r6p6t6es [6] . la tuberculose gastro-intestinale reste rare malgr6 une atteinte pulmonaire fr6quente atteignant 60 70 % des patients sida ; celle-ci ne survient que dans 3 ~ 5 % des cas, chiffre comparable fi ceux retrouv6s dans la population gdn6rale (2 %). le tractus gastro-intestinal semble ~tre la porte d'entr6e de la mycobact6rie et son atteinte est deux fois plus fr6quente que la forme pulmonaire. bien que tousles segments du tractus digestif puissent ~tre entrepris, c'est la portion intestinale qui est le site primaire le plus fr6quent, le foie et la rate 6rant les sites les plus fr6quents de dissdmination [6] . le diagnostic est r6alis6 ~i partir de coprocultures ou de biopsies. au niveau histologique, la lamina propria de la muqueuse intestinale est diffus6ment infiltrde d'histiocytes, comblant les villosit6s et s6parant les cryptes. lorsque cette infiltration est massive, celle-ci est aisdment reconnuc aux colorations de routine d'he ; cependant, la coloration de ziehl facilite le diagnostic. cette coloration permet en effet de visua-liser les nombreux baciues au sein des macrophages mais aussi en extra-cellulaire. le diagnostic diff6rentiel se pose avec la maladie de whipple au sein de laquelle les macrophages sont fortement color6s par le pas et n6gatifs pour la coloration de ziehl. une culture sanguine positive 6tablit le diagnostic de mac diss6min6e, mais pas celui d'une infection active au niveau digestif. une coproculture positive sugg~re mais ne prouve pas une infection digestive. les cultures sanguines peuvent ~tre n6gatives n6cessitant des cultures r6p6-t6es, des biopsies de moelle osseuse ou des traitements empiriques. r6cemment, un nouveau type de mycobact6rie a 6merg6, il s'agit de la mycobactorie genovense. mycobactdrie gordonae est 6galement une cause rare d'atteinte digestive. la spiroch6tose survient relativement frdquemment chez les mgles homosexuels ; l'affection touche 6galement environ 5 % de la population gdndrale, et 30 % des homosexuels ne pr6sentent pas de tableau d'immunod6ficience. la spiroch6tose ne s'accompagne d'aucune 16sion macroscopiquement ou endoscopiquement visible. au niveau histologique, les spirochbtes adh6rent h la surface de la muqueuse ot~ ils apparaissent comme un tapis chevelu bleut6. ils se r6v~lent aux colorations de pas et de warthin-starry [7] . une majorit6 d'6tudes indique que la pr6valence des infections ~ hp est plus faible chez les patients sida que clans la population g6n6rale pour des cohortes appari6es pour le sexe, l'fige et les symp-t6mes. la pr6valence est certainement plus faible lorsque le taux de cd4 est inf6rieur ?a 200. ceci sugg~re un r61e des cd4 et une fonction immune ndcessaire aux infections a hp et aux ulc~res peptiques r6sultant de l'hp. cependant, lorsque les patients sida sont infect6s, la maladie peut 6tre particuli~rement virulente et la charge d'organismes, importante [8] . les candidoses gastro-intestinales surviennent dans une grande cat6gorie des patients immunocompromis mais affecte particuli~rement les patients sida. la candidose est l'affection gastro-intestinale la plus fr6quente chez le patient sida (31%). l'~esophage est l'organe cible. une co-infection avec le virus cmv est frdquente. chez les patients neutrop6niques, la candidose intestinale est une importante source de diss6mination h6matog6ne. le candida albicans est l'espbce la plus fr6quemment isolde. chez les patients sida, la flore orale dif-f~re de celle de la population g6n6rale par une diver-sit6 appauvrie; la pathogen~se de la candidose oesophagienne d6bute done par un remplacement de la flore orale par une flore moins complexe progressant vers l'oropharynx lorsque le nombre de cd4 diminue sous 179/mm 3, r6sultant en une infection eesophagienne invasive lorsque le taux de cd4 est > 129/ram 3 [4] . le diagnostic final se fait g partir de l'identification des filaments myc61iens et des spores h partir de sp6cimens biopsiques ou des brossages cesophagiens. au niveau histologique, la candidose invasive implique la pr6sence de filaments myc61iens p6ndtrant au sein d'une muqueuse intacte, alors que la colonisation se caract6rise par la pr6sence de filaments et de spores au niveau de zones ulcdr6es ou n6crotiques. les filaments peuvent ~tre ddtectds aux colorations de routine d'he mais sont mieux identifiables aux colorations de pas ou de grocott. une histoplasmose colique a 6galement 6t6 d6crite en association avec le syndrome de job. l'identification de l'agent pathog~ne sur mat6riel biopsique est primordiale 6tant donn6 que les mises en culture peuvent prendre plusieurs semaines [1] . la cryptoccocose digestive a 6t6 identifi6e dans 33 % des autopsies or) celle-ci se prdsentait sous sa forme diss6min6e. on la rencontre chez les patients sida mais aussi chez les patients souffrant d'h6mopathies malignes ou b6n6ficiant de corticoth6rapie au long court [1] . au niveau digestif, ce sont l'eesophage et le c61on qui sont le plus souvent affect6s. l'aspergillus infecte rarement le tractus digestif, l'cesophage reste le site pr6f6rentiel. l'aspergillose invasive se rencontre surtout chez les patients sdv~rement immunod6primds (fig. 2) la mucormycose est une affection rare et souvent fatale rencontr6e chez les h6tes immunocompromis. les facteurs de risque principaux incluent le diabbte, l'acidoc6tose, les neutrop6nies s6v6res, les leuc6mies et les traitements immunosuppresseurs. seuls quelques cas ont 6t6 rapport6s chez les patients sida. le tractus gastro-intestinal est rarement affect6, repr6sentant seulement 7 % de tousles cas [7] . l'infection initiale du tractus digestif r6sulte probablement de l'ingestion de spores. 66 % des infections digestives surviennent au niveau gastrique ; les perforations sont fr6quentes. le c61on (21%), le grole (4 %) et l'cesophage (2 %) peuvent 6galement 6tre infect6s. la caraet6ristique histologique principale est l'invasion locale des vaisseaux sanguins par les filaments, induisant des vasculites aigu~s, la formation de thrombi et de n6crose isch6mique du tissu adjacent. les filaments sont peu sept6s, branch6s de manibre irr6gulibre ~ 90 ~ peu color6s par i'he. la plupart des filament sont 6pais et atteignent 10 ~ 15 microns de diam6tre. certains filaments pr6sentent 6galement un aspect torsad6. les diarrh6es sont le sympt6me gastro-intestinal le plus frdquemment rencontr6 chez les patients sida, affectant 50 h 90 % des individus pr6sentant un taux de cd4 rdduit. les protozoaires sont actuellement reconnus comme les principaux agents pathog6nes des diar-rh6es infectieuses, les deux organismes les plus fr6quemment identifi6s 6tant le cryptosporidium et le comme les virus et les mycoses, c'est principalemerit l'intestin gr~le qui est affect6 en premier. ii existe certaines variations g6ographiques, surtout chez les patients sida, l'ispora belli est par exemple frdquemment rencontr6 ~ haiti, alors que le cryptosporidium est le pathog6ne le plus souvent responsable de diarrh6es h washington. une pneumocystose extra-pulmonaire a 6t6 identi-fi6e chez 2,5 % des patients sida/~ new york, avec une atteinte digestive de 22 %. l'affection digestive r~sulte en g~n6ral soit d'une diss6mination h partir des ganglions ou de la voie h6matog~ne suivant une infection pulmonaire soit par r6activation d'une infection gastrique latente. approximativement 50 % des patients prdsentant une forme extra-pulmonaire ont bdn6fici6 d'un traitement ~ la pentamidine, r6duisant le risque de la forme pulmonaire mais ne pr6venant pas la forme diss6min6e. la forme diss6min6e est typiquement un 6v6nement survenant tardivement dans le ddcours de la maladie, lorsque le taux de cd4 est infdrieur 50/mm 3. l'atteinte par pneumocystis a tout d'abord 6t6 d6crite au niveau ~esophagien et duod6nal chez un patient vivant en 1987 ; depuis lors, des infections gastriques, coliques et gr61es ont 6galement 6t6 identi-fi6es. au niveau histologique, il existe de nombreux organismes et macrophages spumeux dans la lamina propria [1] . le cryptosporidium est devenu l'agent pathog~ne le plus fr6quemment responsable des diarrh6es ren-contr6es chez les patients sida. identifi6 en 1976 comme agent responsable d'ent6rites chez l'humain, l'affection reste limit6e chez les h6tes immunocom-p6tents mais peut ~tre s6vbre chez les sujets immunocompromis. la cryptosporidiose survient chez 3 /a 11% des patients sida, quel que soit le groupe ~ risque mais est plus fr6quente chez les homosexuels. l'infection se rencontre surtout chez les hiv pr6sentant des diarrh6es chroniques. lorsque le taux de cd4 est inf6rieur ~ 50/mm 3, l'infection est souvent dramatique. d'autres maladies li6es au sida pr6-c~dent le d6veloppement de la cryptosporidiose dans 85 % des cas [1] . l'organisme infecte le plus souvent le j6junum, mais se rencontre 6galement ailleurs dans le tube digestif ou darts les voies biliaires. le c61on est le deuxi~me site le plus fr6quemment infect6, suivi de l'estomac et de l'~esophage. le diagnostic repose sur l'identification de l'organisme soit dans les aspirations duod6nales, soit sur mat6riel biopsique. les protozoaires sont clairement visualis6s aux colorations de routine d'he, mais sont mieux observ6s 5 la coloration de giemsa. ils se caract6risent par des organismes sph6riques, basophiles, mesurant entre 2 et 4 microns de diambtre et sont attach6s aux microvillosit6s formant la bordure des cellules 6pith61iales (fig. 3) . le sommet ainsi que les bords lat6raux des villosit6s comptent le plus grand nombre d'organismes au nlveau de la muqueuse gr~le; alors qu'au niveau colique, les cryptes et l'6pith61ium de surface sont infect6s de mani~re 6gale. la variabilit6 de taille des organismes correspond aux diff6rentes ~tapes du cycle de vie [4, 6] . le diagnostic peut 6galement 6tre 6tabli par identification des oocystes dans les selles, en utilisant une coloration de ziehl ou une immunofluorescence. les formes tissulaires sont elles ndgatives pour la coloration de ziehl ou les colorations du mucus. les microsporidium sont un groupe h6t6rog~ne de protozoaires, pr6sentant un cycle obligatoirement intra-cellulaires. identifi6s pour la premibre fois chez l'humain en 1985, ils peuvent se rencontrer dans le tractus digestif et les voies biliaires, la v6sicule biliaire et l'arbre respiratoire. l'incidence des diarrh6es ~ microsporidium identitides chez les patients sida varie de 1,7 a 39 %, mais m0me les patients asymptomatiques peuvent ~tre porteurs de l'organisme dans l'intestin gr~le. les ces agents pathog~nes sont difficiles ~ identifier en microscopie optique, la microscopie 61ectronique est souvent n6cessaire. ils peuvent ~tre mis en 6vidence dans les aspirations j6junales et les selles en utilisant une coloration acid-fast afin de d6tecter les oocytes, qui sont autofluorescents bleu lorsqu'ils sont examin6s en microscopie, en 6pifluorescence ultra-violette. la pr6sentation clinique est similaire a celle obser-v6e dans les infections dues au cryptosporidium parvum [9] . entomoeba histolytica est consid6r6 comme un agent commensal non pathog~ne chez les patients homosexuels. l'infection peut survenir lors d'ingestion de kystes par la voie oro-faecale. les patients hiv ne semblent pas pr4senter de susceptibilit6 particulibre ~ amoebae. cependant une colonisation m6me par des agents non pathog6nes peut 8tre responsable d'affections s6vhres chez les patients immunocompromis [6] . giardia lamblia peut occasionnellement provoquer une diarrh6e chez les patients sida. comme c'est le cas pour les entomoeba, le patient hiv ne pr6sente pas de susceptibilit6 particuli6re. la microscopie optique d6tecte largement les giardia, et moins fr6quemment les trophozoites, et constitue la m6thode diagnostique de choix. bien avant les 6pid6mies de sida, le toxoplasme 6tait reconnu comme protozoaire responsable d'infections opportunistes chez les patients immunocompromis. l'atteinte digestive r6sulte en g6n6ral d'une toxoplasmose diss6min6e et s'observe darts 20 % des autopsies. chez les patients sida, l'atteinte digestive peut otre g4n6ralis6e ou n'affecter qu'un seul segment, et peut exceptionnellement 8tre diagnostiqu6e du vivant du patient [7] . c'est un protozoaire obligatoirement intra-cellulaire, qui peut se d6velopper darts n'importe quel type cellulaire, ~ l'exception des globules rouges. certaines 6tudes rapportent des strongyloidoses diss6min6es chez des patients immunocompromis. le d6nominateur commun semble 4tre une corticothdrapie. on ne remarque pas d'incidence plus 61ev6e chez les patients sida. la leishmaniose visc6rale est une maladie s6vhre, acquise en g6n6ral au niveau du bassin m6diterran6en, se ddveloppant le plus souvent chez les h6tes immunocompromis lors de voyages dans les zones end6miques. l'infection s'aequihre par morsure de la femelle de la mouche des sables, transfusions sanguines, infections g6nitales et rapports sexuels. les infections visc6rales sont major6es chez les patients hiv et les atteintes digestives sont fr6quentes; certaines 6tudes rapportent 100% d'atteinte gastro-intestinale [7] . le diagnostic est r6alis6 par identification des amastigotes de la leishmania, dans les frottis ou les sp6cimens biopsiques. les biopsies j6junales pr6sentent des alt6rations villositaires telles que des villosit6s massu6es, dues h l'infiltration massive de la lamina propria par des histiocytes gorg6s de leishmania. les ent6rocytes bordant les cryptes et les villositds ne comprennent pas d'organisme. ceux-ci apparaissent bleut6s h la coloration de giemsa. chez les patients immuno-comp6tents, des granulomes peuvent 8tre observ6s dans la lamina propria ; ils sont en g6ndral absents ou peu form6s chez les immunocompromis. dans une 6tude, le blastocystis homminis 6tait le troisihme organisme le plus fr6quemment identifi6 aprss le cryptosporidium et le cmv chez les patients hiv. ils se rencontrent toujours lorsque l'immunosuppression est sdv6re dans un contexte de diarrh6es chroniques. les biopsies en gdndral apparaissent normales; lorsqu'elles sont anormales, elles exhibent des alt6rations non sp6cifiques. although gastrointestinal infections occur in all groups of immunocompromised patients, the frequency is highest in patients with acquired immunodeficiency syndrome (aids). the reduced number and the reduced function of the cd4+ cells affect the terminal differentiation of ig a-bearing to ig a-secreting b cells, resulting in a decreased number of mucosal iga-bearing plasma cells. there is also a reduction in gastric acid production, probably related to parietal cell antibodies and a decrease in intestinal motility resulting from autonomic denervation. the combination of all these factors promotes colonisation of the small bowel by anaerobic bacteria, which commonly causes diarrhea preceding opportunistic infections [3] . 100 % in some developing countries. at least 80 % of cases of chronic diarrhoea can be attributed to specific enteropathogens among which coccidial parasites are the most frequent [1] . malabsorption and mucosal abnormalities of the small bowel in the absence of detectable pathogens were first described in 1984. hiv has been detected within the intestinal mucosa and it has been proposed that the virus itself might be responsible, at least in part, for the enteropathy. however, no direct evidence of this theory has been produced so far and sceptics favour the suggestion that diarrhoea is actually due to opportunistic infection which has failed to be detected or to unrecognised pathogens. approximately 30-50 % of patients with hiv will have oesophageal disease some time during their illness. candida albicans is the most frequently isolated pathogen, accounting for 50-70 % of cases, followed by cmv and hsv in that order, accounting for 15 to 35 % [4, 5] . however, after all known aetiologies of hiv-related oesophageal ulceration are excluded, 40 to 50 % of patients have idiopathic oesophageal ulceration. hiv p24 core protein has been detected in the granulation tissue. these findings suggest that hiv is capable of producing ulcers in the absence of other pathogens. these ulcers occur mostly when the patients have a low cd4 count. these ulcers can be very difficult to treat, intralesional injection of steroids has shown some therapeutic potential as well as thalidomide. in hiv-related proctocolitis non specific markers of inflammation are seen such as an increased number of lysosomal granules in intraepithelial lymphocytes, focal crypt epithelial cell apoptosis, and tubuloreticular structures in endothelial cells, lymphocytes and monocytes. the degree of inflammation appears to correlate with the mucosal level of p24 antigen and clinical symptoms, suggesting an aetiologic role of hiv [11. first described in children with leukaemia and severe neutropenia, this condition is now known in immunocompromised patients suffering from lymphoma, anaplastic anaemia and cancer. the combined effects of neutropenia and chemotherapy allows luminal bacteria to invade and injure the bowel wall. septicaemia, mostly caused by gram-negative bacilli, occurs in more than 79 % of patients. histologically, haemorrhage, marked edema, and patchy inflammation with a paucity of inflammatory cells are seen. sometimes necrosis results in well demarcated ulcers that may perforate. focally, intramural bacterial colonies and submucosal gas cysts may be seen. in a large prospective study of 100 hiv-infected patients with ulcerative oesophagitis, hsv was identified in only 5 % of cases, whereas the prevalence of cmv was almost 50 % [4] . [1, 4] . pneumocystis carinii. cmv infections typically occur when immunodeficiency is severe (cd4+ < lo0/mm 3) and also represents reactivation of a latent virus conversely, persistent cmv infection itself may promote the decline of immune function. the infection may involve any part of the gi tract, and the risk of involvement of a given segment varies with the type of immunosuppression. in patients with aids, the colon is mostly affected; and in the upper gi tract, oesophageal disease is more common. in immunocompromised patients without aids, the lower and upper gi tract is equally affected and gastroduodenal disease is more common than oesophageal. in the oesophagus cmv preferentially affects the distal segment. gastric involvement is usually associated with involvement elsewhere in the gi tract. in cmv colitis', predominantly seen in the caecum and the right colon, the mucosa may appear normal but usually deep ulcerations are seen. severe cases may manifest as necrotising colitis or toxic megacolon with perforation. nuclear inclusion are acidophilic and often surrounded by a halo, cytoplasmic inclusion bodies are multiple, granular and often basophilic (fig. 1) . the diagnosis is made by endoscopy and biopsy [6] . immunohistochemistry and in situ hybridisation are more sensitive than he; pcr represents the most sensitive technique. results of treatment with ganciclovir can be monitored by means of histological grading of cmv in biopsy specimen. some autors developed a simple grading system that defines grade i as one to four, grade h as five to nine, and grade iii as ten or more cmv-infected cells per biopsy specimen [4] . clostridium difficile affects individuals with variable types of immunosuppression and related to the prevalence of antibiotic use and frequent hospitalisation [6] . these infections include mycobacterium tuberculosis, mycobacterium avium complex or intracellulare and other atypical mycobacteria. tuberculosis, despite extrapulmonary tuberculosis being common in aids patients (occurring in 60 to 70 %), rarely involves the gi tract (3 % to 5 %), and not significantly more frequently than in general population (2 %). oesophageal, small bowel and colonic tuberculosis have been described. oesophageal involvement may develop after mediastinal lymph node tuberculosis and can result in a tracheo-oesophageal fistula. [1, 4] . the gi tract appears to be the most common portal entry and gi involvement is twice as common as the respiratory tract. although segments of the gi tract may be implicated, the intestine is the most common primary site and liver and spleen are the most common sites for dissemination [6] . [7] . the majority of studies indicate that the prevalence of hp in aids patients is significantly lower than in age, sex, and symptom-matched hiv patients. the prevalence is certainly lower when the cd4 count is lesser than 200. this suggests a role of cd4 cell and immune function in sustaining hp infections and hp-related peptic ulcer disease. nevertheless, when aids patients become infected, the d&ease may exhibit particularly aggressive lesions and large number of organisms [8] . candidiasis is the most frequent aids-defining gi disease (31%). the oesophagus is the prime target organ. co-infection with cmv is frequent. in neutropenic patients, gi candidiasis is an important source of haematogenous dissemination. candida albicans is the species most commonly isolated. in aids patients the oral flora differs from that of the general population by a low level of genetic diversity; the pathogenesis of oesophageal candidiasis begins thus with the replacement of the normal oral flora with a less complex flora and progresses to oropharyngeal candidiasis as the number of cd4+ decreases below 179/mm 3 and results in invasive oesophageal disease when the cd4+ are < 129/mm 3 [4] . the definitive diagnosis rests on the identification of typical yeast forms in endoscopic mucosal biopsies or oesophageal brushings. histopathologically, invasive candidiasis implies hypheal penetration into intact mucosa as opposed to colonisation, which implies the presence of yeasts on an intact mucosa surface or in necrotic tissue. candida can be seen on he stains but is better visualised by a pas stain or grocott methenamine silver (gms). other species than c. albicans have been found in aids patients in 7 % to 8 % including c. parapsilosis, c. krusei, c. tropicalis and torulopsis glabrata. disseminated histoplasmosis develops in approximately 5 % of patients with aids in the midwest of the usa where histoplasmosis is endemic. colonic histoplasmosis has also been described in association with job's syndrome. recognition of the organism in biopsy specimen is crucial because culture may take several weeks [1] . cryptococcal gi disease has been identified at autopsy in 33 % of patients with disseminated cryptoccoccosis, including patients with aids and haematological malignancies as well as those receiving corticosteroid therapy. the oesophagus and colon are mostly involved [1] . aspergillosis rarely affects the gi tract, but the target site is the oesophagus. invasive aspergillosis affects severely debilitated patients (fig. 2) [7] . [1] . the organism infects the jejunum most heavily, but it can be found throughout the gi tract including the biliary tract. the colon is the second more common location, followed by the stomach and finally the oesophagus. the diagnosis is made by identification of the organism in either duodenal aspirates, stool, or tissue samples. the protozoan can be clearly observed on he stain, but is best seen with giemsa stain as rows or clusters of basophilic spherical structures 2 to 4 mm in diameter attached to the microvillous border of the epithelial cells (fig. 3) . tips and lateral aspects of villi show the greatest number of organisms in the small bowel whereas in the colon crypt and surface epithelial involvement appears equal. the variation of the size of the organisms corresponds to different stages of the life cycle [4, 6] . the diagnosis can also be made by identification of the oocysts in stools using acid-fast or irnmunofluorescent stains. however, tissue forms do not stain with acid-fast stains, and are mucous stain negative. microsporidia are a heterogeneous group of obligate intracellular spore-forming (coccidian) protozoa. first described in the human in 1985, they can be found in the gi tract, in the biliary tract, the gallbladder and in the respiratory tract. the reported incidence of microsporidium in aids patients with diarrhoea ranges between 1.7 % and 39 %, but even patients without symptoms may harbour the organism in the small bowel. microsporidiosis resulting from infection with enterocytozoon bieneusi affects exclusively the small intestine and the enterocytes, while septata intestinalis, first described in 1992, may disseminate and infect other organs like kidney and gallbladder parasites are then localised in macrophages, fibroblasts and endothelial cells. although electron microscopy of small bowel biopsies is considered to be the best method, the examination of a biopsy specimen stained with he, giemsa or a modified trichrome method, has sensitivities of 77 %-83 % and specificities approaching 100 % [1, 9] . diagnosis can also be made by identification of microsporidial spores in stools and duodenal aspirates. microsporidium is mostly identified in the jejunum as an intracellular parasite seen in the villous enterocytes from just above the mouths of the crypts to the tips of the villi where they are more numerous. parasites are 4 to 5 mm in diameter, supranuclear, either paler or darker than the surrounding cytoplasm, and contain prominent clefts. spores are less frequent than the parasite, but are supranuclear, clustered, dark, and refractile. the cytoplasm of the enterocyte becomes progressively vacuolated, and the nucleus is hyperchromatic. taking into account that albendazole is highly effective for e. intestinalis but largely ineffective for e. bienusi, it is important to make a specific diagnosis [6] . enteritis resulting from isospora belli has been observed in a number of settings of immunosuppres-sion, including aids, alpha-chain disease, lymphoblastic leukaemia and t-cell lymphoma. isospora is common in haitian and african patients with aids (15 % to 60 %), but is rare in the usa (3 % to 7 %). the symptoms are similar to those of cryptosporidiosis, but eosinophilia may be present. the diagnosis is made by identification of the oocysts in stool specimens using acid-fast stain, or by biopsy; the diagnosis is of importance because specific therapy is available. isospora can be recognised on he stains of small bowel specimens, the intracellular protozoon is larger than microsporidium and measures between 10 and 30pro in diameter the common merozoite is bananashaped and found at all levels of the enterocyte cytoplasm. although poorly stained and pale, the central nucleus, large nucleolus, perinuclear halo, and location within a parasitophorous vacuole give it a characteristic appearance. the infection produces mucosal atrophy and tissue eosinophilia. cyclospora [1, 4] . these pathogens are difficult to appreciate on optical microscopy, although electron microscopy is often diagnostic. they can be identified in jejunal aspirates and in stool specimens using acid-fast stain to detect oocysts which are autofluorescent blue when examined with ultraviolet epifluorescence microscopy. the clinical presentation is quite similar to that of cryptosporidium parvum [9] . entamoeba histolytica is often a non-pathogenic commensal in homosexual men. infection can occur by ingesting cysts through oral-anal contact. hiv patients do not appear to have an increased susceptibility to amoebae. as expected, colonisation, even with non-pathogenic strains, may cause significant disease in immunocompromised patients [6] . giardia lamblia can occasionally cause diarrhoea in aids patients. as in the case of amoebae, hiv patients do not appear to have an increased susceptibility to giardia. light microscopic detection of giardia cysts and, less frequently, trophozoites, continues to be the mainstay of diagnosis. even before the aids epidemic toxoplasmosis was well known as a protozoal opportunistic infection of a variety of immunocompromised patients. disseminated toxoplasmosis resulting in gi involvement, was observed at autopsy in 20 % of cases. aids-related gi toxoplasmosis may involve the entire gi tract or only one segment and may exceptionally be diagnosed antemortem by endoscopic biopsy [7] . it is an obligatory intracellular protozoan, that lives in any type of cells with the exception of red blood cells. trophozoites appear round to slightly oval and 2 to 4 pm in diameter in he-stained section. a central or eccentric haematoxylinophylic dot, representing the nucleus surrounded by pale cytoplasm, is seen in trophozoites. pas stains confirm the presence of glycogen-containing bradyzoites and true cysts. some studies report the cases of disseminated strongyloidiasis in a variety of clinical settings of immunosuppression. the common denominator appears to be corticosteroid therapy. visceral leishmaniasis is a severe disease, usually acquired (mediterranean countries) and usually developing in immunocompromised patients, by travel to an endemic region. the infection is acquired through bites of infected female sand flies, blood transfusion, genital infections and sexual contact. visceral infection is increasingly reported in hiv patients, and gi tact involvement is very frequent, some studies reporting an involvement of lo0 % of cases [7] . in one study blastocystis hominis was the third most common organism isolated after cryptosporidiosis and cmv in hlv patients. it is always recovered from severely immunosuppressed patients with chronic diarrhea. biopsies often appear normal; when abnormal, they only exhibit mild non specific abnormalities. gastrointestinal pathology, an atlas and text gastrointestinal disease in the immunocompromised patient. human pathol -review article : the therapy of gastrointestinal infections associated with the acquired immunodeficiency syndrome -aids and the gut recently recognised microbial enteropathies and hiv infection -gastrointestinal manifestations of aids in children c --idiopathic esophageal ulceration in acquired immunodeficieney syndrome : successful treatment with misoprostol and viscous lidocain -lower helicobacter priori infection and peptic ulcer disease prevalence in patients with aids and suppressed cd4 counts e --aids and the gastrointestinal tract. postgraduate medicine key: cord-004643-uu4uipfy authors: hasan, mohammad rubayet; sundaram, manu somasundaram; sundararaju, sathyavathi; tsui, kin-ming; karim, mohammed yousuf; roscoe, diane; imam, omar; janahi, mohammad a.; thomas, eva; dobson, simon; tan, rusung; tang, patrick; lopez, andres perez title: unusual accumulation of a wide array of antimicrobial resistance mechanisms in a patient with cytomegalovirus-associated hemophagocytic lymphohistiocytosis: a case report date: 2020-03-20 journal: bmc infect dis doi: 10.1186/s12879-020-04966-z sha: doc_id: 4643 cord_uid: uu4uipfy background: infections with multidrug-resistant organisms (mdro) pose a serious threat to patients with dysregulated immunity such as in hemophagocytic lymphohistiocytosis (hlh), but such infections have rarely been comprehensively characterized. here, we present a fatal case of hlh secondary to cytomegalovirus (cmv) infection complicated by both anti-viral drug resistance and sepsis from multiple mdros including pandrug-resistant superbug bacteria. case presentation: a previously healthy, six-year-old boy presented with a 45-day history of fever prior to a diagnosis of hemophagocytic lymphohistiocytosis and hemorrhagic colitis, both associated with cmv. on hospital admission, the patient was found to be colonized with multiple, multidrug-resistant (mdr) bacteria including vancomycin-resistant enterococci (vre) and carbapenamase-producing organisms (cpo). he eventually developed respiratory, urine and bloodstream infections with highly drug-resistant, including pandrug-resistant bacteria, which could not be controlled by antibiotic treatment. antiviral therapy also failed to contain his cmv infection and the patient succumbed to overwhelming bacterial and viral infection. whole genome sequencing (wgs) of the mdr bacteria and metagenomic analysis of his blood sample revealed an unusual accumulation of a wide range of antimicrobial resistance mechanisms in a single patient, including antiviral resistance to ganciclovir, and resistance mechanisms to all currently available antibiotics. conclusions: the case highlights both the risk of acquiring mdr superbugs and the severity of these infections in hlh patients. antimicrobial resistance (amr) is one of the most important public health challenges of current times as the burden of infectious diseases with multidrug-resistant organisms (mdro) is increasing at an alarming rate. globally, approximately 500,000 people die each year due to drug-resistant infections and, if not controlled, these deaths are predicted to exceed 10 million by 2050 [1] . particularly vulnerable are patients with immune deficiency or dysregulation whose inability to fight infections leads to increased risk of disseminated infection and greater dependency on antimicrobial therapy [2] . hemophagocytic lymphohistiocytosis (hlh) is a potentially life-threatening condition characterized by overactivation of lymphocytes and macrophages that results in dysregulation of inflammatory responses [3] . while these factors likely put hlh patients at high risk of infection with mdros, no such reports has been published to date and the clinical course of such infections in these patients remains unknown. here we report severe infections with multiple mdros in a patient with cytomegalovirus (cmv)-associated hlh. we also show an unusual accumulation of a wide-array of antiviral and antibiotic resistance mechanisms in a single patient based on data generated by shotgun metagenomic sequencing of blood from the patient and whole genome sequencing (wgs) of mdros isolated from the patient. a previously healthy 6-year-old boy presented at our hospital in august 2018 with a 45-day history of intermittent, high-grade fever without a clear source, accompanied by loss of appetite, weight loss, painful tongue ulcers, diffuse abdominal pain and intermittent left calf muscle pain. he was a resident of qatar who had just returned from a family holiday in india, where he was hospitalized twice due to fever of unknown origin (fuo) and received intravenous (iv) ceftriaxone and amikacin without any improvement. on examination, he looked moderately ill and pale, and was found to have mouth ulcers and splenomegaly. initial laboratory investigations revealed neutropenia, thrombocytopenia, normocytic normochromic anemia with high ferritin, elevated liver enzymes and c-reactive protein (crp). on admission, surveillance cultures for mdros were positive for vancomycin-resistant enterococcus faecium (vre) and carbapenemase-producing escherichia coli (ndm1) and klebsiella pneumoniae (oxa-48) (fig. 1) . he was also found to be positive for cytomegalovirus (cmv) igm with a viral load of 27,946 iu/ml. bone marrow examination performed twice showed hypocellularity with myeloid preponderance and no morphologic evidence of malignancy. immunological workup revealed severe reduction in cd19+ b-cells and cd16 + cd56+ nk cells. genetic testing (invitae, san franscico, usa) with a primary immunodeficiency (pid) panel comprised of 207 genes revealed three variants of uncertain significance. his liver enzymes were elevated. liver needle biopsy and electron microscopy revealed mild steatosis with steatohepatitis. patchy sinusoidal dilation with hepatocellular plate atrophy was noted. wilson's disease was excluded by genetic testing. all bacterial, mycobacterial and fungal cultures of bone marrow were negative. extensive infectious disease workup during the course of his hospitalization were negative except that the respiratory pathogen pcr panel was positive for adenovirus and rhinovirus and that vre and candida spp. were isolated from his urine culture. antiviral therapy for cmv viremia was started with iv ganciclovir for 3 weeks. cmv viral load dropped to 6167 iu/ml. patient showed symptomatic improvement and was discharged with oral valganciclovir for another 3 weeks. four months post discharge, he developed fever and a productive cough. he had several visits to the emergency department, and infectious disease and hematology outpatient clinics, and was briefly hospitalized again for fever, recurrent respiratory and candida infections, recurrent thrombocytopenia and suspected immunodeficiency. after discharge, he continued to have intermittent fever, and his parents took him to india for further evaluation. he was diagnosed with hemophagocytic lymphohistiocytosis (hlh) secondary to cmv infection, and started treatment with dexamethasone and cyclosporine in february 2019. in order to manage the cmv viremia, he was also treated with multiple antivirals including cidofovir and foscarnet. the child was re-admitted to sidra medicine after 1 month of therapy for hlh with a one-week history of fever, hematochezia and fatigue. on the same day, he developed massive rectal bleeding with blood clots and required resuscitation and was transferred to the pediatric intensive care unit (picu). the rectal bleeding and hemodynamic instability required blood and platelet transfusions and urgent colonscopy, which showed erosion and signs of colitis. cmv-induced hemorrhagic colitis was suspected, with the possibility of transmural infiltration of the bowel with hemophagocytic cells. he was intubated on day two. on picu day three, the patient became hypotensive during interventional radiology and required resuscitation. blood culture was positive with multidrug-resistant e. coli. prolonged antiviral therapy and the lack of response led to a suspicion of cmv ganciclovir resistance, so foscarnet was started. he was extubated 5 days after intubation but on picu day 16, he was reintubated after developing pulmonary hemorrhage. the chest x-ray showed worsening pulmonary hemorrhage and he developed catecholamineresistant shock that required initation of steroids. also, he continued to have bleeding per rectum requiring continuous support with blood products. a ct angiogram showed bleeding from the terminal ileum and colitis in the large intestine. he was put on total parenteral nutrition. surgical intervention for the gastrointestinal bleeding was considered too risky. thus, he was conservatively managed with octreotide and esomeprazole infusion and blood product transfusions. he also developed acute kidney injury likely from the combination of nephrotoxic medications, hematuria and infection, and continued to be anuric, requiring continuous renal replacement therapy. he also developed microangiopathic hemolytic anemia requiring 5 cycles of plasmapheresis. for hlh, his treatment was escalated to increasing doses of dexamethasone, intravenous immunoglobulin (ivig) and cyclosporine. microbiological findings during this time were remarkable for multiple cultures positive for mdros. the first was a positive blood culture on picu day 5 with an extensively drug resistant, carbapenemase-producing e. coli, which prompted the addition of colistin to his antimicrobial treatment (fig. 1 ). this was followed by multiple positive blood cultures with elizabethkingia meningoseptica, e. faecium and pandrug-resistant k. pneumoniae, and a positive urine culture with vre. whole genome sequencing (wgs) was performed on several multi-dug resistant bacteria isolated from the patient at various time points showing accumulation of resistance mechanisms to almost all classes of antibiotics used to treat these bacteria ( table 1 , supplemental methods). shotgun metagenomic sequencing performed on serum on day 6, revealed an a594v mutation in the ul97 gene of cmv (fig. 2 , supplemental methods), which is known to confer van vancomycin *bacterial whole genome sequencing (wgs) was performed as described in the supplemental methods and data were analyzed as described previously [4] resistance to ganciclovir. as a result, ganciclovir was discontinued leaving foscarnet as his anti-viral treatment. iv levofloxacin and vancomycin was added to cover e. meningoseptica and vre, along with prophylactic trimethoprim/sulfamethoxazole and antifungal treatment. his cmv titres continued to be high in spite of antiviral therapy. over the course of the last few days of picu, the patient continued to deteriorate clinically: pulmonary bleeding persisted with intermittent respiratory acidosis despite maximal ventilator support. he continued to be hypotensive despite inotropic support, remained anuric and continued to have gastrointestinal (gi) bleeds. the patient passed away on picu day 20 from multiorgan failure associated with sepsis with highly drug-resistant bacteria. hlh is characterized by dysregulation of the immune system whereby hematopoietic cells are phagocytosed by activated macrophages and lymphocytes [7] . primary or familial hlh may be inherited in an autosomal recessive manner and are grouped into 5 types based on the affected gene. type 1 hlh is caused by a defect in chromosome 9, while types 2, 3, 4 and 5 are known to be caused by mutations in familial hlh (fhlh) genes prf1, unc13d, stx11 and stxbp2 genes, respectively [7] . at the initial presentation to our hospital in june 18, 2018 with cmv viremia, the patient did not meet criteria for hlh. this diagnosis was made when abroad in india, and treatment started there. subsequent review identified that the patient developed 8/8 hlh-2004 criteria [8] . in our patient, the history of recurrent infection, the cmv viremia, the clinical diagnosis of hlh and marked reduction in b-cell and nk-cell numbers could suggest an underlying primary immunodeficiency (pid) disorder. however, a sample sent to a referral laboratory to test for pid genes including above hlhassociated genes did not identify significant mutations in known pid genes, suggesting that hlh in our patient was cmv-associated. hlh in an immunocompetent patient secondary to cmv infection is extremely rare but has been reported in the literature [9] . 2 genotype and antiviral resistance profiles of the cytomegalovirus strain. nucleic acid extract from patient serum was subjected to ngs library preparation using nextera xt kit (illumina, usa) and sequencing was performed on a miseq (illumina). paired sequence reads were mapped to ul97 and ul54 sequences (gene id 3077517 and genbank accession abv71585.1, respectively) to obtain corresponding gene sequences from the patient's cmv strain. the sequences were then analyzed by using an online mutational resistance analyzer (mra) available from the university of ulm, https://www.informatik.uni-ulm.de/ni/mitarbeiter/hkestler/mra/app/index.php?plugin=form [5] . ganciclovir resistance was confirmed by the presence of the a594v mutation in ul97 [6] what was unique in our patient compared to other reported cmv-associated hlh cases was the overwhelming infection with mdros. on hospital admission, the patient was found to be colonized with multiple mdros including vre, and carbapenamase-producing enterobacteriaceae, which may have been acquired during his previous hospital course in india. wgs revealed that the vre isolate harbored the vanhax gene cluster that encodes vana, providing high level resistance to vancomycin and teicoplanin. apart from vancomycin resistance, the isolate also possessed genes encoding resistance to macrolides, lincosamides, tetracyclines and aminoglycosides (table 1) . phenotypically, the isolate was also resistant to linezolid but we were unable to identify the genetic determinant. the e. coli and k. pneumoniae isolates harbored genes encoding a wide array of antimicrobial resistance mechanisms affecting the vast majority of antibiotic classes, including ctx-m-15 and ndm β-lactamases, the most common extendedspectrum β-lactamases and carbapenemases, respectively, found in enterobacterales in india [10, 11] . notably, the e. coli isolate had 5 different modifying enzymes conferring resistance to aminoglycosides, which has rarely been reported [12] . there were also several narrow host range plasmids belonging to the incf family (replicons fia, fib and fii) in the e. coli and k. pneumoniae strains (data not shown) [10] . since these plasmids can simultaneously harbor most of the genes detected and can be transferred both within the same species and between both species, it is presumed that they played a role in the acquisition and subsequent exchange of resistance determinants among these isolates. consequently, our patient developed respiratory, urine and bloodstream infections with highly drug-resistant, including pandrug-resistant, bacteria, which could not be controlled by antibiotic treatment. although the patient initially responded to the antiviral drug ganciclovir as reflected by a drop in cmv viral load (fig. 1) , he later became non-responsive to antiviral treatment. due to the severity of the cmv infection despite ganciclovir treatment, real-time metagenomic analysis was performed, revealing ganciclovir resistance and allowing for tailoring of the antiviral therapy. although the ul97 mutation detected in this case is well described in the literarture for its association with ganciclovir resistance, the reduced absorption of the drug because of cmvassiciated enterocolitis may have contributed to the development of resistance as well [13] . by wgs, the vre isolate from his urine and the e. coli and k. pneumoniae isolates from his blood culture were found to be of different sequence types compared to the isolates the patient was previously found to be colonized with, but the later clinical isolates harbored both the resistance mechanisms found in the colonizing strains as well as newly acquired resistance mechanisms. in particular, the pandrug-resistant k. pneumoniae isolated from the patient's blood had ndm β-lactamases in addition to oxa-48 β-lactamases. phenotypically, the isolate was resistant to all currently used antibiotics including the last resort antibiotic, colistin. we were unable to detect the mcr gene that is commonly known to encode for colistin resistance and the genetic determinants of colistin resistance remained unknown in this study. while the exchange of resistance determinants via horizontal gene transfer is a possibility, the invasive superbug infections in our patient may have been facilitated by his travel history as well as his cmv-associated enteropathy, which eventually culminated with untreatable sepsis, multiorgan failure and the untimely death. our study has a few limitations. although the patient was tested for a panel of genes for pid, wgs was not performed to look for other genetic causes of hlh or pid, outside of the commonly known mutations. also, the clinical course of our patient in india was not well documented and details regarding antibiotic and antiviral treatment of the patient are unknown. however, it appears most likely that the patient acquired mdros while seeking medical treatment in india, where mdro infection is frequently associated with hospitalization [14] [15] [16] . therefore, the risks of mdro infections associated with medical tourism to regions with high rates of antibiotic resistance should be discussed with patients, who are at higher risk for complications from these organisms. it is important that the travel and other relevant history be elicited from patients so that appropriate screening measures for mdros can be implemented. strict infection control measures are necessary to reduce nosocomial transmission of mdros, especially in centers caring for immunocompromised or critically ill patients. in our institution, we perform risk-based screening for mdros, implement appropriate isolation measures to prevent spread to other patients and health care workers, and perform wgs to monitor for nosocomial transmission. this case depicts the dire circumstances associated with severe infection with mdr superbugs in a particularly vulnerable patient, and underscores the need for urgent measures to prevent the development of antibiotic resistnace through appropriate use of antimicrobials and to prevent the spread of mdros through surveillance and implementation of appropriate infection control measures. supplementary information accompanies this paper at https://doi.org/10. 1186/s12879-020-04966-z. additional file 1. supplemental methods. review on antimicrobial resistance: tackling drug-resistant infections globally pulmonary infections in immunocompromised patients an overview of hemophagocytic lymphohistiocytosis draft genome sequences of two streptococcus pneumoniae strains causing invasive infections in children in qatar extended pairwise local alignment of wild card dna/rna sequences using dynamic programming characterization of multiple cytomegalovirus drug resistance mutations detected in a hematopoietic stem cell transplant recipient by recombinant phenotyping treatment of hemophagocytic lymphohistiocytosis with hlh-94 immunochemotherapy and bone marrow transplantation acute cytomegalovirus (cmv) infection associated with hemophagocytic lymphohistiocytosis (hlh) in an immunocompetent host meeting all eight hlh 2004 diagnostic criteria the role of epidemic resistance plasmids and international high-risk clones in the spread of multidrug-resistant enterobacteriaceae prevalence and clonality of extended-spectrum betalactamases in asia emergence of five kinds of aminoglycoside-modifying enzyme genes simultaneously in a strain of multidrug-resistant escherichia coli in china cytomegalovirus viremia and resistance patterns in immunocompromised children: an 11-year experience antimicrobial resistance: the next big pandemic carbapenemase-producing klebsiella pneumoniae, a key pathogen set for global nosocomial dominance high reported rates of antimicrobial resistance in indian neonatal and pediatric blood stream infections publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions mrh designed the study, analyzed data and drafted the manuscript; mss collected clinical data and prepared the case report; ss collected sequence data and revised the manuscript; kmt analyzed sequence data and revised the manuscript; myk, dr, oi, maj, et, sd, rt, pt and apl interpreted data and substantially revised the manuscript. all authors read and approved the final version of the manuscript. bacterial whole genome sequencing (wgs) and metagenomic sequencing were performed as part of other research projects supported by sidra medicine, qatar (grant no. sirf_200026 to m.r.h. and sirf_200040 to a.p.l.). bacterial whole genome sequence (wgs) data and metagenomics data are available under bioprojects prjna564977 and prjna576033 in ddbj/ena/ genbank with the accession numbers srx6852761, srx6852760, srx6852759, srx6852758, srx6852757, srx6852756, srx6852755 and srr10236626, respectively.ethics approval and consent to participate not applicable the parents of the patient provided written informed consents to publish their child's personal or clinical details along with any identifying images. the authors declare that they have no competing interests.received: 11 november 2019 accepted: 11 march 2020 key: cord-267269-05mezubh authors: plazolles, n.; humbert, j.‐m.; vachot, l.; verrier, b.; hocke, c.; halary, f. title: pivotal advance: the promotion of soluble dc‐sign release by inflammatory signals and its enhancement of cytomegalovirus‐mediated cis‐infection of myeloid dendritic cells date: 2010-10-12 journal: j leukoc biol doi: 10.1189/jlb.0710386 sha: doc_id: 267269 cord_uid: 05mezubh dc‐sign is a member of the c‐type lectin family. mainly expressed by myeloid dcs, it is involved in the capture and internalization of pathogens, including human cmv. several transcripts have been identified, some of which code for putative soluble proteins. however, little is known about the regulation and the functional properties of such putative sdc‐sign variants. to better understand how sdc‐sign could be involved in cmv infection, we set out to characterize biochemical and functional properties of rdc‐sign as well as naturally occurring sdc‐sign. we first developed a specific, quantitative elisa and then used it to detect the presence sdc‐sign in in vitro‐generated dc culture supernatants as cell‐free secreted tetramers. next, in correlation with their inflammatory status, we demonstrated the presence of sdc‐sign in several human body fluids, including serum, joint fluids, and bals. cmv infection of human tissues was also shown to promote sdc‐sign release. based on the analysis of the cytokine/chemokine content of sdc‐sign culture supernatants, we identified ifn‐γ and cxcl8/il‐8 as inducers of sdc‐sign production by modc. finally, we demonstrated that sdc‐sign was able to interact with cmv gb under native conditions, leading to a significant increase in modc cmv infection. overall, our results confirm that sdc‐sign, like its well‐known, counterpart mdc‐sign, may play a pivotal role in cmv‐mediated pathogenesis. dc-sign is a tm type ii protein, which belongs to a family of calcium-dependent lectins diversely used by human apcs, such as tissue-residing myeloid dcs, alveolar and ln macrophages, and endothelial cells from liver sinusoids [1] [2] [3] [4] . dc-sign contains a crd that is highly conserved in lectins and a neck region consisting of ig-like domain repetitions, which mediate a ph-dependent oligomerization of dc-sign monomers and thus, increase affinity with its ligands [5] [6] [7] [8] . during monocyte differentiation toward modcs, dc-sign expression has also been reported to be induced by il-4 and to be negatively regulated by ifns, tgf-␤, and dexamethasone [9] . dc-sign is a receptor for self-glycoproteins, such as icam-3/2 [10, 11] , and is also able to recognize high mannose-containing structures and fucosylated lewis blood ags (le x/y/b/a ) [7, 12] , expressed by several pathogenic microorganisms including viruses, bacteria, yeasts, and parasites (for review, see ref. [13] ). interactions between dc-sign and pathogenic-derived sugar moieties have been shown to play a prominent role in vivo in favoring the capture and internalization of microbes [14] , with or without the help of other ag-capture receptors, such as the macrophage mannose receptor (cd206), dec-205 (cd205) [15] , or the mac-rophage galactose-type c-type lectin [16] . in contrast to other c-type lectins, several transcripts have been described for dc-sign, most likely originating from alternative splicing and potentially leading to the expression of sdc-sign proteins [17] . as described already for the dc-sign homologue, dc-signr (or cd209 ligand) [18] , such cdnas have been reported by others to encode nonsecreted sdc-sign molecules [19] . in addition, the same study showed that sdc-sign displayed no functional activity in terms of icam-3-dependent cosignaling compared with mdc-sign. some years ago, we demonstrated that cmv, like hiv, is able to bind to dcs via dc-sign and enter them more easily than the free viral particles [20] . consequently, the dc-sign-mediated increase in virus entry resulted in a strong cmv infection of modc. the study in question shed new light on the molecular interactions that favor dc infection by cmv and especially the pivotal role played by dc-sign as a docking and internalizing receptor. cmv is a widespread herpesvirus that infects 40 -100% of the populations worldwide. it induces lifelong viral persistence and may cause severe disease in immunocompromised individuals, such as those with hiv or transplant patients. the major cmv host entry sites are myeloid dc-containing peripheral tissues, especially oropharyngeal and genital mucosa. in the present study, we investigated expression regulation and functional properties of naturally occurring as well as rsdc-sign variants. we demonstrated that such variants can promote cmv infection of modcs, as already shown for their counterparts, mdc-sign. these data suggest that sdc-sign variants may play a crucial role in the cmv-mediated pathogenesis. after signing an informed consent, peripheral or cord blood and bal samples, as well as vaginal mucosa explants, resulting from a partial or complete hysterectomy (vaginal tumor resection or prolapsus), were collected and used in our experiments, in accordance with protocols approved by the local ethical committee. bal samples were isolated from hospitalizedinfected (cmv primary infection, influenza, coronavirus, pneumococcus, or hemofilus influenza) or polytrauma patients. bal were classified as "inflammatory" or "noninflammatory" according to the crp serum concentration (respectively, [crp] serum ͼ5 mg/ml, and [crp] serum ͻ5 mg/ml). two endotheliotropic human cmv strains, tb40/e and vhl/e, were kindly provided by dr. christian sinzger (institute for medical virology and epidemiology, university of tubingen, tubingen, germany) [21] . tbgfp was a kind gift of dr. martin messerle (department of virology, hannover medical school, hannover, germany). all viruses were propagated, purified, and titered as described [22] . for gel filtration analyses, the rcrd of dc-sign was kindly provided by dr. franck fieschi (institute for structural biology, grenoble, france). this molecule was produced in escherichia coli. anti-dc-sign antibodies were obtained from dr. bernard verrier (fre2736 cnrs-biomérieux, lyon, france; mab clone 3e1a8, purified and biotinylated) from the french agency for aids research program (mab clone 1b10; ac14.1 [20] ) or purchased from bd biosciences (san jose, ca, usa; mab clone dcn46) or santa cruz biotechnology (santa cruz, ca, usa; pab h-200). mouse mab against specific human surface ags were used in a direct immunostaining assay: pe-cyanin5-conjugated anti-cd1a (clone bl6), pe-conjugated anti-cd80/83/86 (clones mab104, hb15a, and ha5.2b7), and conjugated isotypic control mab (beckman coulter, fullerton, ca, usa); and pe-conjugated anti-hla dr (clone l243) and fitc-conjugated anti-dc-sign/cd209 (#dcn46; bd biosciences). neutralizing polyclonal antibodies directed against human cxcl-8/il-8, cxcl-10/ip-10, ifn-␥, or il-6 were purchased from r&d systems (minneapolis, mn, usa). hek 293t clones were cultured in 8% fcs dmem supplemented with 2 mm glutamine. hek cells were used for transient recombinant protein expression assays. dcs were generated in vitro from adult blood monocytes according to a modified version of the protocol described previously by sallusto and lanzavecchia [23] . briefly, cd14 ϩ monocytes were highly enriched from pbmcs of healthy donors by a negative magnetic depletion using hapten-conjugated cd2, cd3, cd19, cd56, cd66b, and glycophorin a antibodies (stemsep™ human monocyte enrichment kit, stem cell technologies, vancouver, canada). routinely, two enrichment steps resulted in ͼ98% pure cd14 ϩ cells. purified cd14 ϩ monocytes were cultured in six-well plates (nunc, thermo scientific, rochester, ny, usa) for 6 days with rpmi-1640 medium, supplemented with 8% fcs (biowhittaker, cambrex, charles city, ia, usa) and 20 ng/ml human ril-4 and 100 ng/ml human rgm-csf (peprotech ec ltd., london, uk). every 2 days, one-half of the medium was replaced by fresh il-4/gm-csf-supplemented medium. at day 5, virtually all cells displayed the typical phenotype cd1a ϩ , cd14 -, hla-dr -, cd80 low , cd86 low , cd83 -, dc-sign ϩ of immature modcs, as assessed by flow cytometry. alternatively, dcs were generated from cd34 ϩ cbps, as described already elsewhere [17] . immunomagnetically purified cbps were cultured in medium supplemented with 20 ng/ml stem cell factor and 50 ng/ml gm-csf (r&d systems). tnf-␣ (10 ng/ml) was added on day 7, and on day 11 of culture, il-4 (10 ng/ml) was added to induce dc-sign synthesis for 3 additional days. total rna was isolated from modc using the trizol™ reagent isolation method according to the manufacturer's instructions (invitrogen corp., carlsbad, ca, usa). total rna was then reverse-transcribed with the m-mlv rt and polydt oligonucleotide (promega, madison, wi, usa). dc-sign-encoding cdnas were amplified by pcr using a high-fidelity taq polymerase (roche diagnostics, meylan, france) with the following oligonucleotides: forward 5ј-aagaattcgactacaaggatgacgatga-caagggaatgagtgactccaaggaa-3ј, allowing to insert a flag tagencoding sequence, and reverse 5ј-tattatgcatctacgcag-gaggggaattctt -3ј. pcr products were directly cloned at the ecori sites of the pet21a prokaryotic expression vector (novagen, vwr international s.a.s, fontenay sous bois, france) or alternatively, of the pcdna3.1 plasmid (invitrogen corp.). tm-missing cdna were screened using two internal primer pairs to amplify, respectively, all and tm-containing cdna: forward/reverse dc-sign_sm/dc-sign_as (tmϭ59.8°c, 5ј-ctc-caaggaaccaagactgc-3ј/tmϭ59.8°c, 5ј-ttgttgggctctcctct-gtt-3ј) and forward/reverse dc-sign_ss/dc-sign_as (tmϭ55.3°c, 5ј-aactcctctccttcacgc-3ј). retained, tm-missing cdnas were then sequenced by the dideoxynucleotide termination method (genomexpress, meylan, france) and submitted to a basic local alignment search tool request (http://blast.ncbi.nlm.nih.gov/blast.cgi). for clarity reasons, our constructions consisting of the sdc-sign1a type i isoform encoding cdna cloned into pet21a and pcdna3.1 vectors and were renamed pet21-flag-sdcsign1at1 and pcdna3.1-flag-sdcsign1at1, respectively. the sdc-sign1a type iii-encoding sequence was also cloned into the pet21a plasmid and produced similarly as described below for sdcsign1at1. for protein production, pet21a-flag-sdcsign1at1 and pet21a-flag-sdcsign1at3 recombinant plasmids were transferred to rosetta™ (de3) plyss-competent cells (novagen, a merck company, germany). this modified bacterial strain is usually used to synthesize native recombinant eukaryotic protein, even in the absence of a signal peptide. the recombinant protein expression was induced for 6 -12 h by the addition of 1 mm iptg to a growing culture at 30°c, according to the manufacturer's instructions, and in the presence of 34 g/ml chloramphenicol (c0378, sigma-aldrich, st. louis, mo, usa). iptg-induced cells were harvested and lysed directly with the bacterial-protein extraction reagents (thermo scientific). both sdc-sign isoforms were then purified by a two-step affinity chromatography consisting of one passage on mannan-conjugated agarose (elution was done with 50 g/ml mannan in tbs), followed by a second passage on a m2 affinity resin-loaded column at low pressure (sigma-aldrich). then, rsdc-sign elution was obtained with a 100-g/ml flag peptide solution (in tbs), according to sigma-aldrich protocols. after the second round of purification, each eluted fraction was concentrated onto centricon-3 filtration devices (millipore, bedford, ma, usa) to allow the contaminating flag peptide to be removed. each treated fraction was then analyzed by western blot and silver nitrate staining to estimate purity (90 -95% estimated purity was usually achieved). positive fractions were then pooled and stored in glycerol (50% v/v) at -80°c until used. sdc-sign concentration was measured by elisa for every flag-sdc-sign batch production. when required, samples were submitted to gel filtration chromatography to allow mw determination of native multimer or monomer proteins. briefly, 0.5-2 ml dc-sign-containing samples were loaded onto a hiprep 26/60 sephacryl s-200 hr column (ge healthcare, waukesha, wi, usa) and separated according to their mw. fractions (1 ml) were collected and analyzed by western blot or elisa to document the presence or absence of dc-sign. mean elution volumes were then determined for each tested sample, thus enabling the calculation of dc-sign mw using mw standards (bio-rad france, marnes-la-coquette, france). a specific sdc-sign elisa was developed in our laboratory. a polyclonal anti-dc-sign antibody (clone h-200, santa cruz biotechnology) was coated onto presaturated reactibind 96-well plates, according to the manufacturer's instructions (pierce biotechnology, rockford, il, usa). after washing plates, samples (sera, bal, synovial fluid, cell culture supernatants, etc.) were diluted in tbs or left undiluted, mixed with an equal volume of tbst containing the biotin-conjugated 3e1 anti-dc-sign mab (0.2 g/ ml), and incubated further for 1 h at room temperature. a final incubation step with a hrp-conjugated neutravidin™ solution (pierce biotechnology) allowed for the detection of sdc-sign with tetramethylbenzidine, a specific chromogenic hrp substrate. absorbance was read at 450 nm and 570 nm (background substraction) on a labsystems multiskan microplate reader (labsystem multiskan ms, finland) and analyzed with the biolise™ software (version 2.0, labtech international, uk). quantitative sdc-sign concentration determination was achieved when ods were compared with a standard curve, obtained with flag-sdc-sign1at1 protein, produced in e. coli. validation tests of our homemade elisa sdc-sign are presented in table 1 . exosomes were prepared from the supernatant of 6-day-old il-4/gm-csfdifferentiated modcs cultured in complete medium using a simplified version of a protocol reported previously by raposo et al. [24] . three successive centrifugations at 300 g (5 min), 1200 g (20 min), and 10,000 g (30 min) were performed to eliminate cells and debris. supernatants were ultracentrifuged at 100,000 g for 2 h onto a d20/30% sucrose gradient-density cushion (dϭ1.217). the exosome-enriched pellet was resuspended in pbs and kept frozen for further experiments, and clarified supernatant was concentrated with an amicon centrifugal filter device (10 kda cut-off, millipore). total protein contents of supernatants and exosome pellets were quantified using the bca assay (pierce biotechnology). cytokines and chemokines were quantified into cell/tissue culture supernatants or biological fluids by the cba flex set (bd biosciences). when needed, cervical or vaginal epithelium biopsies were infected with tb40/e cmv strain (6ϫ10 5 percentages of gfp ϩ cells directly reflected the infectious rate, as assessed already in our former work [20] . total proteins were obtained from concentrated cell culture supernatants or cell lysates. when required, hek 293t cells were transiently transfected with the pcdna3.1-flag-sdcsign1at1 eukaryotic expression vector using the exgen 500 transfection reagent according to the manufacturer's instructions (euromedex, france). after 3 days, cells were harvested, washed three times with pbs, and resuspended into a lysis buffer (50 mm tris, phϭ7.5, 150 mm nacl, 0.5% triton x-100), supplemented with a protease inhibitor cocktail (p8340; sigma-aldrich) and cleared from nuclei by centrifugation at 10,000 g. for each kind of experiment, indicated total protein amounts were loaded and separated onto 10% acrylamide sds-page gels before being transferred to nitrocellulose membranes. saturated membranes (2 h, room temperature in 0.1% tbst, 5% nonfat milk) were then hybridized with specific primary antibodies against dc-sign (3e1a8 or 1b10 clones) or actin (rabbit polyclonal antibody; sigma-aldrich) and secondary hrp-conjugated goat anti-mouse or rabbit mab (amersham pharmacia biotech, sweden). proteins of interest were finally detected using an ecl detection kit (pierce biotechnology). alternatively, protein lysates were diluted in tbs and spotted onto appropriate nitrocellulose membranes using a minifold-i dot-blot system (schleicher & schuell, germany). then, membranes were saturated and hybridized as described in the previous paragraph. human cervical or vaginal mucosa explants were collected by prorfessor c. hocke (department of gynecological surgery, general hospital of bordeaux, france), in accordance with international ethical rules. explants were extracted as circular pieces of ϳ1 cm 2 and submitted or not to cmv infection (vhl/e; 6ϫ10 5 pfu/cm 2 ). oct (sakura finetek usa, inc., torrance, ca, usa)-embedded frozen tissue sections were air-dried for 30 min, washed in pbs (ph 7.4), and fixed/permeabilized in 1:1 vol:vol cold acetone/methanol at 4°c for 10 min. the fixed sections were saturated using pbs containing 0.5% bsa and 10% normal goat serum (sigma-aldrich) at room temperature for 1 h. subsequently, the sections were incubated with the anti-dc-sign dcn46 mab or the anti-ie/e cmv ag antibody (argene sa, varilhes, france), followed by incubation with an alexa 488-conjugated goat anti-mouse mab (molecular probes, invitrogen corp.). concomitantly, nuclei were stained with dapi. slides were then washed twice in pbs for 5 min and rinsed in distilled water before being air-dried. stained sections were mounted with dako fluorescent mounting medium (dako, carpinteria, ca, usa) and analyzed on a sp5 confocal microscope (leica microsystems, germany). statistics were generated with the graphpad prism 5.0 software (graphpad software inc., la jolla, ca, usa). unpaired sample comparisons were performed using a mann-whitney nonparametric rank test. a previous study by mummidi and colleagues [17] reported the cloning of cdna, potentially coding for sdc-sign. to determine whether corresponding proteins exist, we first isolated dc-sign-encoding cdnas from monocyte-or cd34 ϩ cord blood cell-derived dcs by pcr. the primer used in this pcr allowed for the insertion of a flag tagencoding sequence in the 5ј position (fig. 1a) . only 10 selected cdna clones were submitted to a second round of pcr to retain the sole tm region lacking cdnas, thought to probably code for sdc-sign (fig. 1b) . four of the 10 clones were not amplified by the tm-specific internal pcr (clones 2, 3, 6, and 7; fig. 1a and b). these cdna clones lacking the tm exon were sequenced and blasted against known cdna libraries. then, we confirmed that three out of these four isolated cdnas were completely identical to a sequence reported previously by mummidi et al. [17] (i.e., sdc-sign1a type i isoform-encoding sequence; genbank accession number nm_001144896) and that the last one encoded a spliced variant, the sdc-sign1a type iii (gen-bank accession number ay042227). we then subcloned sdc-sign1a type i-and iii-encoding cdnas (called thereafter flag-sdc-sign1at1 and t3) into prokaryotic and eukaryotic expression plasmids as required. an elisa was produced simultaneously to measure sdc-sign isoform concentrations in cell culture supernatants or in any other biological fluids. the flag-sdc-sign1at1 protein of prokaryotic origin was used to obtain a standard curve and set up our homemade elisa (fig. 1c ). although our sdc-sign elisa was moderately sensitive with a typical concentration range of 100 -0.1 ng/ml, we were able to validate its specificity. in fact, by means of selective ip experiments on sdc-sign standard solutions, we could demonstrate that 1b10 mab or h-200 pab (the coating pab), directed against dc-sign, abrogated sdc-sign detection, whereas an irrelevant mab had no effect, i.e., flag-sdc-sign1at1 was still detected by elisa (table 1) . interestingly, a homotypic elisa, i.e., the same antibody (clone 3e1) used as the primary (capture) and secondary antibody, led to low signal detection. this observation evoked the possibility that the flag-sdc-sign1at1 protein was expressed as multimers. this issue is addressed at a later stage in this paper. previous studies have reported that tm-lacking encoding dc-sign sequences are transcribed as soluble cytoplasmic pro-teins but not secreted by the sdc-sign-expressing transfectant cells or immature modcs [19] . it was our hypothesis that the lack of secreted proteins in culture supernatants may be a result of a secretion defect or the lack of a signal peptide [25] . of note, certain proteins, such as il-1␣, fgf2, or galectin-1/3, are secreted, despite being devoid of a signal peptide [26 -29] . as a result, we surmised that the same could be true for sdc-sign. thus, using our quantitative and specific elisa, we observed the presence of potential sdc-sign isoforms in 10ϫ concentrated cell culture supernatants of mdc-sign-expressing dcs. in vitro dcs were generated from adult monocytes or cd34 ϩ cbps, according to already well-known protocols [17, 23, 30] . somewhat surprisingly, increasing quantities of sdc-sign were found in both dc-derived culture supernatants in relation to postdifferentiation time ( fig. 2a and b) . sdc-sign appearance in culture supernatants was correlated with the addition of ril-4. this observation was consistent with previously published observations showing that upon differentiation, il-4 was required to induce mdc-sign expression [9] . to know whether sdc-sign and mdc-sign expressions could be differentially regulated during differentiation, modcs were generated with several cytokine cocktails (il-4/gm-csf, ifn-␣/gm-csf, and il-13/gm-csf) or il-4/il-13 alone. all combinations, except the one using ifn-␣, were able to induce sdc-sign and mdc-sign to the same extent, as assessed by western blot (fig. 2c and d) . taken together, these results provided strong evidence that sdc-sign may be secreted by in vitro-generated dcs and that sdc-sign and mdc-sign were regulated in a similar manner in the course of dc differentiation. we next tried to purify sdc-sign from modc culture supernatants to definitely prove that this molecule is derived from an alternative splicing event, as expected in previous studies [17] . however, we failed to purify sufficient amounts of highpurity sdc-sign for n-terminal sequencing. to circumvent these difficulties, we alternatively treated modcs with a broadspectrum inhibitor of mmps (i.e., a disintegrin and metalloprotease/tace family), called marimastat (british biotech, uk), which was supposed to have no effect on sdc-sign release. differentiating dcs were incubated with increasing doses of marimastat. at day 6, mdc-sign expression was analyzed by flow cytometry, and sdc-sign was quantified in culture supernatants by elisa (fig. 3a , respectively, black and open bars). despite the use of high concentrations of marimastat (10 m), no modification of sdc-sign versus mdc-sign expression patterns could be observed, thus weighing in favor of a sliced, sequence-derived product and not a shedding of mdc-sign by mmps. like many cell types, dcs are able to secrete 60 -80 nm membrane vesicles, called exosomes. to know whether sdc-sign isoforms could be secreted as exosome-borne proteins, we separated the exosome-enriched fraction from cleared il-4/gm-csf-derived modc culture supernatant, according to the modified protocol published by raposo et al. [24] , and titered sdc-sign in all samples by elisa, i.e., medium alone, nonultracentrifuged supernatant, ultracentrifuged supernatant, and exosome-enriched pellet. the results are described in fig. 3b . sdc-sign was retained almost completely in the supernatant cleared from exosomes by ultracentrifugation, as it was shown to be absent from the exosome-enriched fraction (p). we concluded that sdc-sign was not expressed as exosomeassociated proteins. several groups have provided strong evidence that mdc-sign or its homologue dc-signr is expressed as homotetramers to increase affinity for their ligands [5, 7, 31] . tetramerization is dependent on the neck length consisting of various ig-like domain repeats [5, 32] and also on the extracellular ph [8] . the question we posed ourselves was whether this was the case for sdc-sign using gel filtration. here again, we were unable to purify sufficient sdc-sign from modc cultures to perform our analysis. as a result, we produced rflag-sdc-sign1at1 and t3 in e. coli and separated them according to their apparent mw by gel filtration. for each sample, fractions were collected and analyzed by elisa. the results are shown in fig. 3c . sdc-sign quantities were plotted against elution volumes, and elution volume of the maximum sdc-sign quantities for each sample was determined on the plot. column precalibration allowed us to calculate each apparent mw meas of the most abundant sdc-sign isoform found in each sample. thus, the flag-sdc-sign1at1-associated peak was eluted with 52 ml, corresponding to an apparent mw of 175 kd. an approximate, mw theor of 46 kd was calculated on specialized websites (mw theor ; http://www.expasy.org/) for flag-sdc-sign1at1. as mw meas was about four times larger than mw theor (ratioϭ3.8), we concluded that flag-sdc-sign1at1 could be expressed as tetramers in nondenaturing and nonreducing conditions. applying the same procedure for the flag-sdc-sign1at3, it was estimated that unlike sdc-sign1at1, sdc-sign1at3 could probably be found in culture medium as a dimer (mw meas ϭ81 kd; mw theor ϭ32 kd; ratioϭ2.5). purified crd (ϳ19 kd) was used as a control here, as it was unable to multimerize. peak-associated fractions of the flag-sdc-sign1at1 gel filtration run were pooled and concentrated ten-to 15-fold onto centrifugal filter units (10 kda cutoff) and finally analyzed by western blotting in denaturing and reducing conditions. the results are shown in fig. 3d . in this setting, we only observed flag-sdc-sign1at1 as a 40-to 45-kda protein, suggesting that flag-sdc-sign1at1 is expressed as a homotetramer in native conditions. all of our data indicate that sdc-sign proteins are secreted in a mmp-independent manner as exosome-free homotetramers, at least for fulllength sdc-sign variants. we demonstrated above that sdc-sign is secreted by human blood-borne, precursor-derived dc-sign ϩ dcs in vitro. thus, we assumed that it could be secreted by dcs in vivo. our study first analyzed human sera from healthy donors by western blot (nϭ21; fig. 4a ). much to our surprise, sdc-sign was detected in 19 of the 21 sera with a heterogeneity of expression between individuals. following this, we tested a broader panel of human serum from healthy donors (nϭ62) by elisa. consistently with the western blot analysis, almost all sera contained detectable amounts of sdc-sign ranging from 0 to ͼ150 ng/ml with a mean value of 65.36 ng/ml (minϭ0 ng/ml; maxϭ154 ng/ml; medianϭ72 ng/ml; 25th percentileϭ32.41 ng/ml; 75th percentileϭ95.75 ng/ml; fig. 4b ). the serum sdc-sign concentrations were comparable with the lower amounts of mbl variants, which are found in human serum in a broad range of concentrations, depending on their genotype [33] . further gel-filtration experiments demonstrated that serum sdc-sign was more probably expressed as tetramers, which was consistent with our previous results obtained with rflag-sdc-sign1at1 (supplemental fig. 1 ). mdc-sign has previously been associated with th2 responses, at least in vitro [34, 35] . however, certain experimental findings came to our attention, showing that dc-sign may be up-regulated in inflammatory diseases such as crohn's disease [36, 37] . we hypothesized that sdc-sign may be found in inflammatory body fluids. for this purpose, bals were harvested from patients suffering from diverse lung diseases, including primary cmv infections, and tested for the presence of sdc-sign. when segregated according to the inflammatory status of samples (based on diagnoses, i.e., viral infections, etc.), the quantity of sdc-sign was clearly higher within inflammatory (meanϭ24.4 ng/ml) compared with noninflammatory bal (meanϭ1.37 ng/ml; pϭ0.0005; fig. 4c ). to confirm these observations, we compared sera and joint fluids from ra versus osteoarthritissuffering patients. joint fluids from ra patients were indeed prototypical inflammatory fluids, whereas joint fluids from osteoarthritis were considered as mechanical fluids marked by the absence of inflammatory cytokines and chemokines (supplemental fig. 2 ). as shown in fig. 4d (left panel) , there was a highly significant difference between sdc-sign amounts in ra versus control joint fluids (pϭ0.006). when analyzing the related sdc-sign concentrations in the sera of the same patients (fig. 4d, right panel; pϭ0.02) , a similar difference could be observed, thus promoting the conclusion that sdc-sign expression was closely correlated to the inflammatory status of these human biological fluids (fig. 4e) . taken together, these results largely confirmed the fact that sdc-sign is produced in distinct biological fluids, and its expression ap-pears to be up-regulated by inflammatory disorders, as exemplified here in the case of ra. on the basis of previous results of this study showing that higher amounts of sdc-sign are generally found in inflammatory, virally infected bal (fig. 4c) , we next endeavored to ascertain whether such inflammation was dependent on viral infection. we therefore infected freshly isolated mucosal explants of human origin (vagina or cervix biopsies) with a high viral load and further incubated them for 7 days before freezing. frozen tissue sections were stained to reveal nuclear ie/e cmv ag in infected cells or dc-sign. as shown in fig. 5a , dc-sign ϩ cells were located exclusively in the lamina propria (fig. 5a, d and e) of freshly isolated explants (fig. 5a, a) or of 7-day noninfected explants (fig. 5a, b) , whereas after a 7-day infection period, dc-sign ϩ cells were distributed equally among the mucosal epithelium and the lamina propria (fig. 5a, f) , as shown by a sharp nuclear staining of ie/e cmv agpositive cells (fig. 5a, c) . in the course of these experiments, explant culture supernatants were collected and analyzed by elisa to quantify secreted dc-sign. the results are described in fig. 5b . upon infection, sdc-sign release was ϳ2.5 times higher in explants when compared with the basal sdc-sign release after 7 days in noninfected explant supernatants, as well as in freshly isolated explant supernatants. we then confirmed that cmv infection contributed to the establishment of an inflammatory environment by showing that typical cytokines (il-6 and ifn-␥) or chemokines (cxcl-8/il-8 and cxcl-10/ip-10) were overproduced in explant supernatants upon infection (fig. 5c ). lps stimulation for 24 h was used as a positive control of cytokine/chemokine release. together, these results clearly demonstrate that sdc-sign expression could be up-regulated by a viral infection of freshly isolated peripheral tissues. we next sought to identify proinflammatory cytokines or chemokines that might be responsible for the induction of sdc-sign up-regulation. focusing on molecules overexpressed in explant culture supernatants upon cmv infection or lps activation, experiments were performed using fully differentiated immature modc, subcultured with various doses of human rcxcl-8/il-8, rcxcl-10/ip-10, rifn-␥, and ril-6, alone or in combination. to minimize the effect of exogenous il-4, which is necessary to differentiate modc but also able to induce sdc-sign, potentially leading to misleading conclusions, cells were first starved of il-4. in that setting, sdc-sign release was up-regulated significantly by ifn-␥ and to a lesser extent, by cxcl-8/il-8 in a dosedependent manner when compared with the spontaneous level of sdc-sign secretion by il-4-starved cells (i.e., "no il-4" experimental conditions; respectively, pϭ0.01 and pϭ0.001), whereas no significant effect could be shown after adding exogenous cxcl-1/ip-10 or il-6 ( fig. 6) . it should be noted that cxcl-8/il-8 and ifn-␥ act in an additive but not synergistic manner to induce sdc-sign production by il-4-starved immature modcs. the stimulation level in that case is equivalent to the "il-4 alone" experimental condition. il-6 unresponsiveness is most likely a result of the absence of a functional receptor on the cell surface, as no significant staining for gp130, i.e., the signaltransducing chain of il-6r, could be observed by flow cytometry (data not shown). in accordance with our previous data, we hereby demonstrated that sdc-sign is re-in-duced by exogenous ifn-␥ and/or cxcl-8/il-8 in fully differentiated immature modc cultures. we demonstrated above that sdc-sign secretion is promoted by an inflammatory setting and even upon cmv infection of human tissue explants. thus, it remained to be determined whether sdc-sign has a protective or facilitating effect on cmv infection. first, we tested the functionality of flag-sdc-sign1at1 through its ability to interact directly with the cmv envelope gb, which we identified previously as a ligand for mdc-sign [20] . lysates of transiently transfected hek cells were spotted on a nitrocellulose membrane and incubated further with flag-sdc-sign1at1 or specific mab as a detection control to document interactions between gb and sdc-sign under native conditions (fig. 7a) . when incubated with flag-sdc-sign1at1, gb ϩ cell lysates could be revealed by a hrp-conjugated anti-flag antibody, whereas the anti-flag alone did not provide any signal. flag-sdc-sign1at1 ϩ hek cell lysates were used as positive controls for dc-sign and flag detection. an additional actin detection was used as a loading sample control. these results suggested that the flag-sdc-sign1at1 was functional. however, the functional role of sdc-sign during the cmv infection remained unclear. on the basis of the mbl-mediated inhibition of hiv susceptibility reported in the literature, we first hypothesized that sdc-sign might neutralize the cmv infection of modc. in this setting, we were unable to block modc infection, even using higher concentrations of flag-sdc-sign1at1 (data not shown). in addition, as mdc-sign expression was reported to be responsible for modc cmv cis-infection, we assumed that flag-sdc-sign1at1 may function as a promoter of the infection. for this purpose, modc were infected with tbgfp cmv (moiϭ1) for 24 -48 h in the presence of decreasing amounts of flag-sdc-sign1at1 (from 400 to 12.5 ng/ml). the gfp ϩ modc, indicating the percentage of early infected cells, was estimated by flow cytometry. infected modc frequencies were approximately twice as high (33.1ϯ6%) as the control infection (14.8ϯ7.2%) when incubated with 50 ng/ml flag-sdc-sign1at1-supplemented culture medium (pϭ0.01; fig. 7b ). a 100-ng/ml concentration was not sufficient to mediate a significant infection enhancement when compared with control infection. surprisingly, it was observed that flag-sdc-sign1at1 concentrations higher than 100 ng/ml and lower than 50 ng/ml did not modify the infectious rate of modc. to confirm that the deleterious effect that we could observe was specific to sdc-sign, we conducted similar experiments but only using an optimal amount of flag-sdc-sign1at1 (50 ng/ml), which was first captured or not onto a mannan-conjugated resin (fig. 7c, upper panels) . here again, the addition of flag-sdc-sign1at1 doubled the gfp ϩ modc percentage (32.5% vs. 15.7%). this enhanced infection was readily a result of flag-sdc-sign, as it was abrogated by a preincubation step of the recombinant lectin on mannan-conjugated agarose. as a negative control, we also performed the same assay with fsf, which we reported previously as being prone to cmv infection in a dc-sign-independent manner ( [20] ; fig. 7c lower panels). all of these results led us to conclude that flag-sdc-sign1at1 is functional and is able to facilitate cmv infection of modcs. we previously reported the crucial role of mdc-sign as a docking and internalizing receptor for cmv on modc [20] . simultaneously, other groups reported the existence of potentially sdc-sign variants at the cdna level, generated by alternative splicing of the tm-encoding exon [17] . soluble but nonsecreted and nonfunctional dc-sign proteins were described by others [19] . to better delineate the role of an extended dc-sign repertoire on cmv infection pathogenesis, the present study appraised the biochemical properties, the regulation, and the role of recombinant as well as naturally occurring sdc-sign variants in the cmv cis-infection of modc. martinez et al. [19] had previously shown that sdc-sign was produced by transfected cells. they reported that sdc-sign failed to be secreted in the cell culture supernatant but instead, was retained in the cytoplasm. strangely, they sought to test its ability to promote the transmission of cosignals to t cells in comparison with mdc-sign but to no avail. in the present study, we developed and used a specific and quantitative elisa to demonstrate the secretion of sdc-sign in concentrated modc culture supernatants. the fact that martinez et al. [19] failed to detect sdc-sign in culture supernatant may be a result of the lack of sensitivity of their own elisa and the absence of a culture supernatant concentration step before measuring sdc-sign. what is more, in keeping with the work of mummidi et al. [17] , we cloned cdnas lacking the tm-encoding exon and coding for putative sdc-sign from distinct, in vitro-generated dc populations. in our study, a broad-spectrum inhibitor of mmps was used to avoid a possible shedding of mdc-sign at the plasma membrane. nevertheless, modc and tissue-residing dcs expressed two or three other membrane-associated mmps that might not be targeted by mmp inhibitors [38] . however, mmp inhibition did not result in a difference of sdc-sign and mdc-sign expression by modcs. these results all confirmed previous observations reporting sdc-sign expression as a soluble, full-length molecule. another crucial issue was to check whether sdc-sign could be expressed as an exosomeassociated protein, as suggested in a recent report [39] . briefly, exosomes derived from dc-sign ϩ murine bone marrow-derived dcs were shown to express dc-sign on the basis of cytometric analyses. on the other hand, as a result of very slight dc-sign staining of exosomes, these results were not convincing and had to be interpreted with caution. in this case, based on the measurement of sdc-sign concentrations with our elisa, we could undoubtedly argue in favor of the expression of sdc-sign as exosome-free molecules. the final proof of the secretion of a full-length sdc-sign would be to sequence the naturally occurring sdc-sign protein, but we failed to purify sufficient amounts of it to obtain any kind of irrefutable proof. feinberg et al. [5] previously demonstrated that mdc-sign was expressed as tetramers at the plasma membrane of transfected cell lines. the mdc-sign tetramerization was dependent on the presence of at least five ig-like domain repeats in the neck region. another study also provided sound evidence that immature modcs express mdc-sign tetramers, which are supposed to physically interact with the coreceptor cd4 [40] . here, by gel filtration analyses under native conditions (i.e., neutral ph and isotonic buffer), we proved that not only recombinant but also blood-borne sdc-sign consisted of tetramers, thus confirming results published previously by others [5, 8] . we next demonstrated that il-4 and il-13 were potent inducers of sdc-sign and mdc-sign. these results were in line with those published by relloso and colleagues [9] in a previous report. in addition, we did not test ifn-␥ during monocyte differentiation in dcs, but we tested ifn-␣, which was sufficient to prevent dc-sign expression when added at the very beginning of modc differentiation. in contrast, it had no effect when added after 2 days postdifferentiation (data not shown), strongly suggesting that dc-sign expression was irreversible. this idea was borne out by a former study reporting the il-4-dependent and irreversible up-regulation of pu.1, a specific transcription factor of the myeloid lineage, mainly responsible for the induction of dc-sign expression in modcs and monocyte-derived macrophages during their in vitro differentiation [41, 42] . in this study, besides il-4 or il-13, we used fully differentiated immature modcs as a cellular model to document what inflammatory soluble factors could be involved in the sdc-sign secretion that we detected in several human body fluids. surprisingly, ifn-␣ and most probably, type i ifns, in general (although this remains to be experimentally ascertained), were shown to impair dc-sign during cell differentiation, whereas ifn-␥ was identified as a good inducer of sdc-sign by fully differentiated immature modc. cxcl-8/il-8 was also demonstrated to promote sdc-sign expression but to a lesser extent. when added together as exogenous cytokines on il-4-starved modcs, we observed additive effects on sdc-sign secretion, suggesting that they may be both required to induce optimal sdc-sign secretion, albeit weakly. even at higher doses of ifn-␥ and/or cxcl-8/il-8, we were unable to attain the sdc-sign secretion level obtained with il-4 or il-13 in vitro. to explain why, in such a case, ifn-␥ induced and did not repress sdc-sign secretion by modcs, one could argue once again that fully differentiated immature modcs have a distinct transcription program coordinated by distinct transcription factors (i.e., pu.1, etc.) compared with monocytes during their differentiation. in genital human mucosa, after 7 days of ex vivo cmv infection, we noted a significant increase of sdc-sign secretion by residing dcs. the analysis of the cytokine content of infected biopsy supernatants confirmed the presence of ifn-␥ and cxcl-8/il-8 in sufficient amounts to imagine their direct involvement in the induction of sdc-sign. in these experiments, no difference of mdc-sign expression by dermal dcs could be observed by immunostaining. based on our own results, we expected that sdc-sign induction could be correlated with the same up-regulation of membrane isoforms. however, it is our belief that the explanation for this discrepancy can be found in a recent study reported by liu et al. [18] , indicating that the vast majority of dc-signr, a closely related dc-sign gene product found in hiv mucosal inoculation sites, i.e., anal and genital mucosa, was encoded by tm lacking spliced cdna sequences [17] . these data pointed to a differential expression of sdc-sign versus mdc-signr isoforms in virally infected peripheral tissues. despite being unable to document any differential expression of dc-sign in vitro, we cannot rule out this possibility in vivo. to better understand if this were the case, discriminating antibodies are currently being generated in our laboratory. interestingly, former studies provided strong evidence that ra synovia contained high levels of cxcl-8/il-8, which may be a result of viral infection caused by cmv [43] . here, these molecules were detected in high amounts in infected bal or ra joint fluids, suggesting that they could also be sdc-sign inducers in vivo. moreover, cxcl-8/il-8 was up-regulated in the female genital tract in the course of coinfection with cmv and hiv-1 [44] . this observation substantially supported our sdc-sign induction data in genital mucosa explants. cxcl-8/il-8 was already known as an efficient neutrophil and macrophage chemoattractant, and a link had been established between high serum concentrations and the cmv disease occurrence in patients after liver allotransplantation [45] . the analysis of the sdc-sign content in sera from healthy volunteers showed a high heterogeneity of concentrations. on the basis of previous reports in the literature, we endeavored to forge a link between sdc-sign concentrations and a th2 cytokine environment, marked by high amounts of il-4/13 in sera and bal from atopic patients. unfortunately, we were unable to show significant correlation (data not shown). in contrast, we demonstrated that high amounts of sdc-sign could be correlated to high amounts of cytokines and chemokines, usually overexpressed upon inflammation in diverse body fluids, such as serum, bal, or joint fluids, from patients suffering from autoimmune, inflammatory, or infectious diseases. these results match the data reporting an increased number of mdc-sign-expressing macrophages in bal from mycobacterium tuberculosis-infected patients [3] , as well as in ra synovia [4] . several studies have already reported a link between high frequencies of mdc-sign ϩ -infiltrating cells and the proinflammatory environment found in crohn's disease lesions [37] . one can easily imagine that these mdc-sign ϩ cells may also be able to secrete sdc-sign upon inflammation and/or infection. thus, on the basis of these unexpected results, we suggest that dc-sign, whether sdc-sign or mdc-sign, may be considered as a new marker of tissue inflammation. our final investigation focused on sdc-sign functions. several studies have already documented the fact that rsdc-sign was able to neutralize hiv and dengue virus infection of transfectants or modc at similar mean concentrations (i.e., from 0.5 to 50 g/ml [46, 47] ). although navarro-sanchez et al. [47] reported convincing data, they used truncated rsdc-sign, only consisting of the dc-sign crd domain, which was shown to be expressed as monomers in solution by others [5] . crd monomers had a limited affinity for their ligands in comparison with tetramers [40] . here, we provided strong evidence that flag-sdc-sign1at1 was capable of interacting with the cmv gb, already known to be a ligand of mdc-sign [20] . using our tetrameric rsdc-sign, we were unable to inhibit cmv infection of fsf, even when sizeable sdc-sign amounts were used (up to 17 m; data not shown). we then hypothesized that sdc-sign could function as an enhancer of cmv infection. as expected, low flag-sdc-sign1at1 concentrations (varying from 10 to 100 ng/ml) allowed a significant increase in the percentage of ie/e cmv ag ϩ immature modc from three distinct blood donors, whereas sdc-sign concentrations below and above this range had no potentializing effect. to explain this bell-curve effect, we proposed a hypothetical model at low but constant viral input. according to this model, at high sdc-sign concentrations, free virions are rapidly complexed, leading to an impaired binding and internalization of cmv into modc. between 100 and 10 ng/ml, sdc-sign may serve as an opsonin with cmv virions with an optimal stoichiometry leading to the capture and internalization of a maximum number of sdc-sign-immobilized virions. when using very low sdc-sign concentrations (below 10 ng/ ml), the majority of virus is free, and the infection efficacy decreases to levels of infection without sdc-sign, as we postulated that sdc-sign-opsonized cmv was captured more efficiently by modc than free virions. this suggests the existence of one or several yet-unknown opsonic receptors for sdc-sign, which may not be able to interact with previously reported, truncated rsdc-sign [5, 46] . a novel study is under progress in our lab to fulfil the identification of such a receptor. however, based on the literature, we discuss putative candidates. first, icam-2 and -3, but not icam-1, are ligands for mdc-sign [10, 11] . however, only icam-1 is expressed by modc [48] . although we cannot rule out, so far, the possibility for sdc-sign to interact with icam-1, the involvement of icam-2/3 in the sdc-sign-mediated infection enhancement in modc is likely to be inconceivable. second, mdc-sign complexes have the propensity to form multimers (di-, tri-, and tetramers, depending on the neck length [7] ). as a consequence, one can imagine that sdc-sign, whether bound to cmv virions or not, may be able to dock on mdc-sign to form aggregates with pre-existing lectin homodimers or trimers. new experiments are needed to confirm this hypothesis. we finally considered a last potential candidate receptor enabling the sdc-sign-mediated cmv infection enhancement, the cd11b molecule, also known as the ␣m integrin, a macrophage antigen-1 or complement receptor 3 component when associated with the ␣2 integrin. cd11b is highly expressed by immature modc [49] . interestingly, it has been involved recently in the uptake of mannosylated liposomes by macrophages in cooperation with a murine homologue of mdc-sign [50] . furthermore, cd11b has also been described to function as a facilitating agent for hiv opsonization by immature modc in a mdc-sign-dependent manner [49] . it is thus highly tempting to speculate on the fact that sdc-sign-opsonized cmv particles may interact directly with cd11b, leading to their internalization and thus, promoting modc infection. further experiments are obviously needed to confirm this hypothesis. in summary, our work has strengthened the notion that dc-sign, whether sdc-sign or mdc-sign, should be considered as an inflammatory rather than a regulatory marker. our results also raised the question of the existence of a cell surface receptor for sdc-sign-opsonized cmv virions on modc. as such, our findings shed new light on the diversity of the dc-sign repertoire and extend our knowledge of the use of mdc-sign as well as sdc-sign by cmv to divert the human innate immune response to its own benefit. f.h. designed the research and was responsible for the project and manuscript preparation; and p.n., l.v., and b.v. participated in the data analyses. all authors reviewed the paper and had access to raw data. dc-sign, a dendritic cell-specific hiv-1-binding protein that enhances trans-infection of t cells expression of dc-sign and dc-signr on human sinusoidal endothelium: a role for capturing hepatitis c virus particles dc-sign induction in alveolar macrophages defines privileged target host cells for mycobacteria in patients with tuberculosis expression of the dendritic cell-associated c-type lectin dc-sign by inflammatory matrix metalloproteinase-producing macrophages in rheumatoid arthritis synovium and interaction with intercellular adhesion molecule 3-positive t cells. arthritis rheum extended neck regions stabilize tetramers of the receptors dc-sign and dc-signr binding-site geometry and flexibility in dc-sign demonstrated with surface force measurements a novel mechanism of carbohydrate recognition by the c-type lectins dc-sign and dc-signr. subunit organization and binding to multivalent ligands dc-sign neck domain is a ph-sensor controlling oligomerization: saxs and hydrodynamic studies of extracellular domain dc-sign (cd209) expression is il-4 dependent and is negatively regulated by ifn, tgf-␤, and anti-inflammatory agents dc-sign-icam-2 interaction mediates dendritic cell trafficking identification of dc-sign, a novel dendritic cell-specific icam-3 receptor that supports primary immune responses cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cellspecific icam-3-grabbing nonintegrin on dendritic cells pathogen recognition by dc-sign shapes adaptive immunity the c-type lectin dc-sign (cd209) is an antigen-uptake receptor for candida albicans on dendritic cells how c-type lectins detect pathogens the macrophage c-type lectin specific for galactose/n-acetylgalactosamine is an endocytic receptor expressed on monocyte-derived immature dendritic cells extensive repertoire of membrane-bound and soluble dendritic cell-specific icam-3-grabbing nonintegrin 1 (dc-sign1) and dc-sign2 isoforms. inter-individual variation in expression of dc-sign transcripts most dc-signr transcripts at mucosal hiv transmission sites are alternatively spliced isoforms dc-sign, but not sdc-sign, can modulate il-2 production from pmaand anti-cd3-stimulated primary human cd4 t cells human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements human cytomegalovirus impairs dendritic cell function: a novel mechanism of human cytomegalovirus immune escape efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/ macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor ␣ b lymphocytes secrete antigenpresenting vesicles sequence and expression of a membrane-associated c-type lectin that exhibits cd4-independent binding of human immunodeficiency virus envelope glycoprotein gp120 determinants in the n-terminal domains of galectin-3 for secretion by a novel pathway circumventing the endoplasmic reticulum-golgi complex a novel secretory pathway for interleukin-1 ␤, a protein lacking a signal sequence unconventional secretion of fibroblast growth factor 2 and galectin-1 does not require shedding of plasma membrane-derived vesicles regulation of expression and secretion of galectin-3 in human monocyte-derived dendritic cells cd34ϩ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to gm-csfϩtnf ␣ the structure of dc-signr with a portion of its repeat domain lends insights to modeling of the receptor tetramer structural requirements for multimerization of the pathogen receptor dendritic cell-specific icam3-grabbing non-integrin (cd209) on the cell surface interplay between promoter and structural gene variants control basal serum level of mannan-binding protein helicobacter pylori modulates the t helper cell 1/t helper cell 2 balance through phase-variable interaction between lipopolysaccharide and dc-sign dc-sign association with the th2 environment of lepromatous lesions: cause or effect? unique cd14 intestinal macrophages contribute to the pathogenesis of crohn disease via il-23/ifn-␥ axis increased expression of dc-signϩil-12ϩil-18ϩ and cd83ϩil-12-il-18 -dendritic cell populations in the colonic mucosa of patients with crohn's disease expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases define the migratory characteristics of human monocyte-derived dendritic cells mature dendritic cells pulsed with exosomes stimulate efficient cytotoxic t-lymphocyte responses and antitumour immunity proteomic analysis of dc-sign on dendritic cells detects tetramers required for ligand binding but no association with cd4 the transcription factor pu.1 controls dendritic cell development and flt3 cytokine receptor expression in a dose-dependent manner pu.1 regulates the tissue-specific expression of dendritic cell-specific intercellular adhesion molecule (icam)-3-grabbing nonintegrin enhancement human cytomegalovirus replication in a human lung fibroblast cell line by interleukin-8 hiv type 1 and cytomegalovirus coinfection in the female genital tract interleukin-8 serum concentrations after liver transplantation dc-sign-mediated internalization of hiv is required for transenhancement of t cell infection dendritic-cellspecific icam3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses immature monocyte-derived dendritic cells are productively infected with herpes simplex virus type 1 opsonization of hiv with complement enhances infection of dendritic cells and viral transfer to cd4 t cells in a cr3 and dc-sign-dependent manner cooperation of specific icam-3 grabbing nonintegrinrelated 1 (signr1) and complement receptor type 3 (cr3) in the uptake of oligomannose-coated liposomes by macrophages key words: opsonin ⅐ c-type lectin ⅐ inflammation this work was supported by an institutional grant from in-serm avenir (national french health institute), the aquitaine, and the pays de loire regions (j-m.h.'s salary). we thank olivier neyrolles for his critical reading of the manuscript, the etablissement français du sang d'aquitaine for blood samples, and the bordeaux imaging center (bic; confocal imaging). all authors certify that they have no conflicts of interest or competing financial interests. key: cord-022472-q2qtl26d authors: fishman, jay a.; ramos, emilio title: infection in renal transplant recipients date: 2009-05-15 journal: chronic kidney disease, dialysis, & transplantation doi: 10.1016/b978-1-4160-0158-4.50041-0 sha: doc_id: 22472 cord_uid: q2qtl26d nan successful management of infections in the immunocompromised renal transplant recipient is complicated by a variety of factors. 1 these include increased susceptibility to a broad spectrum of infectious pathogens and the difficulty in making a diagnosis of infection in the face of diminished signs and symptoms of infection, an array of noninfectious etiologies of fever (e.g., graft rejection, drug toxicity), and the possibility that multiple processes are present simultaneously. further, because immunocompromised patients tolerate invasive and established infection poorly with high morbidity and mortality, the urgency for an early and specific diagnosis to guide antimicrobial therapy is increased. given the primacy of t-lymphocyte dysfunction in transplantation, viral infections in particular are increased and contribute to graft dysfunction, systemic illness, graft rejection, and enhancing the risk for other opportunistic infections (e.g., pneumocystis and aspergillus species) and for virally-mediated cancers. the risk of infection in the renal transplant recipient is determined by the interaction of two factors: 1. the epidemiologic exposures of the patient, including those unrecognized by the patient or distant in time ( the prevention and treatment of infection is central to the optimal management of transplant recipients, given the adverse impact of infections on quality of life. consideration of the epidemiology of infection allows the clinician to establish a differential diagnosis for a given "infectious" presentation and to design the optimal preventive strategy for each patient. donor and recipient screening are critical components to the post-transplant health maintenance of the patient (table 37-3) . of these, consideration should be given to empiric therapy for purified protein derivative (ppd) positive patients, for strongyloides stercoralis in patients from endemic regions, and for patients known to have received organs from donors with acute bacterial and fungal infections. specific antiviral strategies stratified according to individual risk should be considered for all kidney recipients. exposures of importance can be divided into four overlapping categories: donor-or recipient-derived infections, and community-or nosocomial-acquired exposures. infections that are derived from the donor tissues and activated in the recipient are among the most important exposures in transplantation. some of these are latent while others are the result of bad timing-active infection transmitted at the time of transplantation. all of the known types of infections have been recognized in transplant recipients. the activation of these infections may reflect the intensity of immune suppression or result from the allogeneic response (graft rejection), which activates latent viral pathogens. three types of infection merit special attention. first, in donors who are bacteremic or fungemic at the time of donation, these infections-staphylococci, pneumococcus, candida species, salmonella, e. coli-tend to "stick" to anastamotic sites (vascular, urinary) and may produce leaks or mycotic aneurysms. second, viral infections, including cytomegalovirus (cmv) and epstein-barr virus (ebv), are associated with particular syndromes and morbidity in the immunocompromised population (discussed later in text). the greatest risk of such infections is in recipients who are seronegative (immunologically naïve) and receive infected grafts from seropositive donors (latent viral infection). third, late, latent infections, including tuberculosis, may activate many years after the initial exposure. disseminated mycobacterial infection is often difficult to treat once established due largely to interactions between the antimicrobial agents used to treat infection (e.g., rifampin, streptomycin, isoniazid) and the agents used in immune suppressive therapy. given the risk of transmission of infection from the organ donor to the recipient, certain infections should be considered relative contraindications to organ donation. given that renal transplantation is, in general, elective surgery, it is reasonable to avoid donation from individuals with unexplained fever, rash, or infectious syndromes. some of the common criteria for exclusion of organ donors are listed in table 37 -4. infections in this category are generally latent infections activated in the setting of immune suppression. it is necessary to obtain a careful history of travel and exposures to guide preventive strategies and empiric therapies. notable among these infections are tuberculosis, strongyloidiasis, viral infections (herpes simplex and varicella zoster or shingles), histoplasmosis, coccidioidomycosis, hepatitis b or c, and human immunodeficiency virus (hiv). vaccination status should be evaluated (tetanus, hepatitis b, childhood vaccines, influenza, pneumococcal vaccine). dietary habits should also be considered, including the use of well water (cryptosporidia), uncooked meats (salmonella, listeria), and unpasteurized dairy products (listeria). common exposures in the community are often related to contaminated food and water ingestion, exposure to infected children or coworkers, or exposures due to hobbies (gardening), travel, or work. respiratory virus infection due to influenza, respiratory syncytial virus, and adenoviruses and more atypical pathogens (herpes simplex virus, herpes zoster virus) carries the risk for viral pneumonia but increased risk for bacterial superinfection. community (social or transfusion-associated) exposure to cmv and ebv may produce severe primary infection in the nonimmune host. recent and remote exposures to endemic, geographically restricted systemic mycoses (blastomyces dermatitidis, coccidioides immitis, and histoplasma capsulatum) and mycobacterium tuberculosis can result in localized pulmonary, systemic, or metastatic infection. asymptomatic strongyloides stercoralis infection may activate more than 30 years after initial exposure due to the effects of immunosuppressive therapy. such reactivation can result in either a diarrheal illness and parasite migration with hyperinfestation syndrome (characterized by hemorrhagic enterocolitis, hemorrhagic pneumonia, or both) or disseminated infection with accompanying (usually) gramnegative bacteremia or meningitis. gastroenteritis due to salmonella species, campylobacter jejuni, and a variety of enteric viruses can result in persistent infection, more severe and prolonged diarrheal disease as well as an increased risk of bloodstream invasion and metastatic infection. nosocomial infections are of increasing importance because organisms with significant antimicrobial resistance predominate in many centers. these include vancomycin, linezolid and quinupristin/dalfopristin-resistant enterococci, methicillinresistant staphylococci, and fluconazole-resistant candida species. a single case of nosocomial aspergillus infection in a compromised host should be seen as an indication of the failure of infection control practices. antimicrobial abuse has resulted in increased rates of c. difficile colitis. outbreaks of infections due to legionella species have been associated with hospital plumbing and contaminated water supplies or ventilation systems. each nosocomial infection should be investigated to ascertain the source and prevent subsequent infections. nosocomial spread of p. jiroveci between immunocompromised patients has also been suggested by a variety of case series. respiratory viral infections may be acquired from medical staff and should be considered among the causes of fever and respiratory decompensation among hospitalized or institutionalized, immunocompromised individuals. the net state of immunosuppression is a measure of all of the factors contributing to the patient's risk for infection (table 37 -2). among these are: 1. the specific immunosuppressive therapy, including dose, duration, and sequence of agents. 2. technical problems from the transplant procedure, resulting in leaks (blood, lymph, urine) and fluid collections, devitalized tissue, poor wound healing, and surgical drainage catheters for prolonged periods. 3. prolonged airway intubation 4. prolonged use of broad-spectrum antibiotics 5. renal and/or hepatic dysfunction 6. prolonged use of vascular access or dialysis catheters presence of infection with one of the immunomodulating viruses, including cmv, ebv, hepatitis b (hbv) or c (hcv), or hiv. specific immunosuppressive agents are associated with increased risk for certain infections (table 37-5) . combinations of these agents may enhance this risk or cause toxicity (e.g., nephrotoxicity) and may further enhance risk. as immunosuppressive regimens have become more standardized, the specific infections that occur most often will vary in a predictable pattern depending on the time elapsed infection i in r renal t transplant r recipients 683 (figure 37-1 ). this is a reflection of the changing risk factors (surgery/hospitalization, immune suppression, acute and chronic rejection, emergence of latent infections, and exposures to novel community infections. 1 the pattern of infections will be changed with alterations in the immunosuppressive regimen (pulse dose steroids or intensification for graft rejection), intercurrent viral infection, neutropenia (drug toxicity), graft dysfunction, or significant epidemiologic exposures (travel or food). the time line reflects three overlapping periods of risk for infection: (1) the perioperative period to approximately 4 weeks after transplantation; (2) the period 1 to 6 months after transplantation (depending on the rapidity of taper of immune suppression and the type and dosing of antilymphocyte "induction" that may persist); and (3) the period beyond the first year after transplantation. these periods reflect the changing major risk factors associated with infection: (1) surgery and technical complications; (2) intensive immune suppression with viral activation; and (3) community-acquired exposures with the return of normal activities. the time line may be used in a variety of ways: (1) to establish a differential diagnosis for the transplant patient suspected of having infection; (2) as a clue to the presence of an excessive environmental hazard for the individual, either within the hospital or in the community; and (3) as a guide to the design of preventive antimicrobial strategies. infections occurring outside the usual period or of unusual severity suggest either excessive epidemiologic hazard or excessive immunosuppression. the prevention of infection must be linked to the risk for infection at various times after transplantation. routine preventive strategies from the massachusetts general hospital are outlined in table 37 -6. it should be noted that such strategies serve only to delay the onset of infection in the face of epidemiologic pressure. the use of antibiotic prophylaxis, vaccines, and behavioral modifications (e.g., routine hand washing or advice against digging in gardens without masks) may only result in a "shift to the transplantation 684 (1) a combination of atovaquone 1500 mg po with meals once daily plus levofloxacin (or equivalent fluoroquinolone without anti-anaerobic spectrum) 250 mg once daily; (2) pentamidine (300 mg iv or inhaled q 3-4 weeks); and (3) dapsone (100 mg po qd to biweekly) +/− pyrimethamine. each of these agents has toxicities that must be considered, including hemolysis in g6pd-deficient hosts with dapsone. none of these alternative programs offer the same broad protection of tmp-smx. continued right" of the infection time line, unless the intensity of immune suppression is reduced or immunity develops. during the first month after transplantation, three types of infection occur. the first type of infection is that present in the recipient prior to transplantation, was inadequately treated, and now has emerged in the setting of surgery, anesthesia, and immunosuppression. pre-transplantation pneumonia and vascular access infections are common examples of this type of infection. colonization of the recipient with resistant organisms is also common (e.g., mrsa). the first rule of successful transplant infectious disease is the eradication of all infection possible prior to transplantation. the second type of early infection was present in the donor before transplantation. this is often a nosocomial-acquired organism (resistant gram-negative bacilli and s. aureus or candida species) due to (1) systemic infection in the donor (e.g., line infection) or (2) contamination during the organ procurement process. the end result is a high risk of infection of vascular suture lines with resultant mycotic aneurysm. uncommonly, infections have been transmitted from donor to recipient, including tuberculosis or fungal (e.g., histoplasmosis) infection that may emerge earlier in the time line than would be predicted (i.e., in the first month). the third type and the most common source of infections in this period are related to the complex surgical procedure of transplantation. these include surgical wound infections, pneumonia (aspiration), bacteremia due to vascular access or surgical drainage catheters, urinary tract infections, or infections of fluid collections-leaks of vascular or urinary transplantation 686 table 3 37-6 renal transplantation antimicrobial protocols at the massachusetts general hospital, boston, massachusetts-cont'd prophylaxis is achieved with 50% of the therapeutic dose of ganciclovir or valganciclovir (corrected for renal function). in some patients, intravenous immune globulin (ivig or hyperimmune globulin) is used as an adjunctive therapy for prophylaxis. certain subgroups merit routine prophylaxis. these include: • solid organ transplant recipients who are naïve (seronegative) and receive an organ from a seropositive donor (d+/r−) • solid organ transplant recipients who are seropositive (r+) and receive antilymphocyte antibodies or other intensive immune suppression (e.g., for graft rejection) symptoms, fever/neutropenia mo (or valacyclovir 500 bid or acyclovir 400 tid) use of cmv-negative or leukocyte-filtered blood status unknown with als intravenous ganciclovir 5mg/kg iv for first dose and qd (corrected for renal function) until sero-status determined. neutropenia: the dose of antiviral and antibacterial therapies are not, in general, reduced for neutropenia. consider other options first! + als: antilymphocyte antibodies include any of the lytic, lymphocyte-depleting antisera *note: not fda approved at these doses prevention of mucocutaneous infection can be accomplished with oral clotrimazole (may increase cya levels) or nystatin 2 to 3 times per day at times of steroid therapy or in the face of antibacterial therapy. fluconazole, at a dose of 200-400 mg/day for 10-14 days is utilized in the treatment of prophylaxis failures. routine prophylaxis with fluconazole is used for pancreas transplants. anastamoses or of lymphoceles. these are nosocomial infections and, as such, are due to the same bacteria and candida infections observed in nonimmunosuppressed patients undergoing comparable surgery. however, given the immune suppression, the signs of infection may be subtle and the severity or duration may be greater. the technical skill of the surgeons and meticulous postoperative care (i.e., wound care, endotracheal tubes, vascular access devices, and drainage catheters) are the determinants of risk for these infections. also among the common infections is c. difficile colitis. limited perioperative antibiotic prophylaxis (i.e., from a single dose to 24 hours of an antibiotic such as cefazolin) is usually adequate with additional coverage only for known risk factors (e.g., prior colonization with mrsa). for pancreas transplantation, perioperative prophylaxis against yeasts with fluconazole is used in addition, bearing in mind the interactions between azole antifungal agents and calcineurin inhibitors and sirolimus (levels may be increased significantly). notable by their absence in the 1st month after transplantation are opportunistic infections, even though the daily doses of immunosuppressive drugs are at their highest during this time. the implications of this observation are important: the net state of immunosuppression is not great enough to support the occurrence of opportunistic infections unless an exposure has been excessive; this observation suggests that it is not the daily dose of immunosuppressive drugs that is of importance but rather the sustained administration of these drugs, the "area under the curve," in determining the net state of immunosuppression. thus, the occurrence of a single case of opportunistic infection in this period should trigger an epidemiologic investigation for an environmental hazard. infection in the transplant recipient 1 to 6 months after transplantation has one of three causes: 1. lingering infection from the peri-surgical period, including relapsed c. difficile colitis, inadequately treated pneumonia, or infection related to a technical problem (e.g., urine leak, lymphocele, hematoma). fluid collections require drainage. 2. viral infections, including cmv, hsv, shingles (vzv), human herpesvirus 6 or 7, ebv, relapsed hepatitis (hbv, hcv), and hiv. this group of viruses is unique: lifelong infection; tissue-associated (often transmitted with the allograft from seropositive donors); immunomodulating-systemically immune suppressive and, potentially, predisposing to graft rejection. it is also notable that the herpesviruses are prominent due to the attenuated ability of t cells to control these infections. among the other viral pathogens of this period must be included bk polyomavirus in association with allograft dysfunction and community-acquired respiratory viruses (adenovirus, influenza, parainfluenza, respiratory syncytial virus, metapneumovirus). the suppression of antibody production (e.g., using tacrolimus and mycophenylate mofetil or with lymphopenia) may predispose to other infections. 3. opportunistic infection due to p. jiroveci, listeria monocytogenes, t. gondii, nocardia species, aspergillus species, and other agents. in this period, the stage is also set for the emergence of a subgroup of patients, the "chronic ne'er-do-wells"-individuals who require higher than average immune suppression to maintain graft function or who have prolonged untreated viral infections and other opportunistic infections, predicting long-term susceptibility to many other infections (third phase, discussed later). such individuals may merit prolonged (lifelong) prophylaxis (antibacterial and/or antiviral) to prevent life-threatening infection. the specific opportunistic infections that occur, reflect the specific immunosuppressive regimen used and the presence or absence of immunomodulating viral infection. viral pathogens (and rejection) are responsible for the majority of febrile episodes that occur in this period. during this period, anti-cmv strategies and trimethoprim-sulfamethoxazole prophylaxis are effective in decreasing the risk of infection. trimethoprim-sulfamethoxazole prophylaxis eliminates p. jiroveci pneumonia (pcp) and reduces the incidence of urinary tract infection and urosepsis, l. monocytogenes meningitis, nocardia species infection, and toxoplasma gondii. transplant recipients who are more than 6 months past the procedure can be divided into three groups in terms of infection risk. the first group consists of the majority of transplant recipients (70%-80%) who had a technically good procedure with satisfactory allograft function, reduced and maintenance immunosuppression, and absence of chronic viral infection. these patients resemble the general community in terms of infection risk, with community-acquired respiratory viruses constituting their major risk. occasionally, such patients will develop primary cmv infection (socially acquired) or infections related to underlying diseases (e.g., skin infections in diabetes). the second group (~10% of patients) suffers chronic viral infection, which, in the absence of effective therapy, will lead inexorably to one of three results: • end organ damage (e.g., bk polyomavirus nephropathy, cryoglobulinemia, or cirrhosis from hcv-hbv being relatively well managed at present) • malignancy (post-transplantation lymphoproliferative disease [ptld] due to ebv, skin, or anogenital cancer due to papilloma viruses) • acquired immunodeficiency syndrome (hiv/aids) the third group of patients (~10% of all recipients) has less than satisfactory allograft function and requires excessive amounts of immunosuppressive therapy for recurrent graft rejection. this may be associated with chronic viral infection. this is the subgroup of transplant recipients, often termed the "chronic ne'er-do-wells," who are at highest risk for opportunistic infection with such pathogens as p. jiroveci, l. monocytogenes, n. asteroides, and cryptococcus neoformans. it is our practice to give these patients lifetime maintenance trimethoprim-sulfamethoxazole prophylaxis and to consider the use of fluconazole prophylaxis. also, this group is susceptible to organisms more often associated with immune dysfunction of aids (bartonella, rhodococcus, cryptosporidium, and microsporidium species) and invasive fungal pathogens (aspergillus, zygomycetes, and the dematiaceae, or pigmented, molds). minimal signs or symptoms merit careful evaluation in this group of "high-risk" individuals. guidelines for pre-transplant screening have been the subject of several recent publications including a consensus conference of the immunocompromised host society (ichs), the american society for transplantation (ast) clinical practice guidelines on the evaluation of renal transplant candidates, and the astp clinical practice guidelines on the evaluation of living renal transplant donors. [2] [3] [4] [5] [6] [7] [8] [9] the transplant donor the critical feature of screening for deceased donors is time limitation. a useful organ must be procured and implanted before some microbiologic assessments have been completed. thus, major infections must be excluded and appropriate cultures and stored samples obtained for future reference. as a result, bacteremia or fungemia may not be detected until after the transplant has occurred. such infections have not generally resulted in transmission of infection as long as the infection has been adequately treated, both in terms of use of antimicrobial agents to which the organism is susceptible and time. in recipients of tissues from 95 bacteremic donors, a mean of 3.8 days of effective therapy post-transplantation appeared adequate to prevent transmission; longer courses of therapy in the recipient are preferred, targeting known potential pathogens from the donor. 10 bacterial meningitis must also be treated with antibiotics that penetrate the csf before procurement. similarly, due to the limited time for testing, certain acute infections (cmv, ebv, hiv, hbv, or hcv) may be undetected in the period prior to antibody formation, and viral dna detection is preferred. as a result, the donor's clinical, social, and medical histories are essential to reducing the risk of such infections. however, in the presence of known infection, such infections must be treated prior to procurement, if possible. major exclusion criteria are outlined in table 37 -4. the differences in screening of the living donor and the cadaver donor are largely based on the different time frames during which this screening takes place. the living donor procedure should be considered elective-and, thus, evaluation completed and infections treated prior to such procedures. an interim history must be taken at the time of surgery to assess the presence of new infections since the initial donor evaluation. intercurrent infections (flu-like illness, headache, confusion, myalgia, cough) might be the harbinger of important infection (west nile virus, sars, rabies, trypanosoma cruzi). live donors undergo a battery of serologic tests (table 37-3) as well as ppd skin test and, if indicated, chest radiograph. the testing must be individualized based on unique risk factors (e.g., travel). of particular importance to the renal transplant recipient is the exclusion of urinary tract infection. whether focal infections in the donor outside the procured organs merit therapy remains unresolved. mycobacterium tuberculosis. this bacterium from the donor represented approximately 4% of reported post-transplant tb cases in a review of 511 patients by singh and colleagues. 11 active disease should be excluded in ppd positive donors, including chest radiograph, sputum cultures, and chest ct, if the chest radiograph is abnormal. urine afb cultures may be useful in the ppd-positive kidney donor. isoniazid prophylaxis of the recipient should be considered for untreated, ppdpositive donors. 12 factors mitigating towards prophylaxis include donor from endemic region, use of high-dose steroid regimen, or high-risk social environment. chagas' disease (t. cruzi). this parasitic disease has been transmitted by transplantation in endemic areas and recently in the united states. schistosomiasis and infection by strongyloides stercoralis are generally recipient-derived problems. epstein-barr virus. the risk for post-transplant lymphoproliferative disease (ptld) is greatest in the ebv seronegative recipient of an ebv seropositive allograft (i.e., d+/r−). this is most common in pediatric transplant recipients and in adults coinfected with cmv or on higher levels of immune suppression. monitoring should be considered for at-risk individuals using a quantitative, molecular assay (e.g., pcr) for ebv. 13, 14 ebv is also a cofactor for other lymphoid malignancies. varicella screening should be used to identify seronegative individuals (no history of chicken pox or shingles) for vaccination prior to transplantation. hsv screening is performed by most centers despite the use of antiviral prophylaxis during the post-transplant period. vzv serologic status is particularly important in children who may be exposed at school (for antiviral or varicella immune globulin prophylaxis) and in adults with atypical presentations of infection (pneumonia or gi disease). other herpesviruses may reactivate with hhv-6 and hhv-7 serving as cofactors for cmv and fungal infections and in endemic regions, kaposi's sarcoma-associated herpesvirus (hhv-8/kshv) causing malignancies. hepatitis b virus (hbv). hbsag and hbv core antibody (hbcab) are used for screening purposes with hbsab positivity indicating either vaccination or prior infection. hbcab-igm positivity suggests active hbv infection, whereas igg positivity suggests a more remote or persistent infection. the hbsag negative, hbcab-igg positive donor may have viral dna in the liver but may be appropriate as a donor for hbvinfected renal recipients. quantitative assays for hbv should be obtained to guide further therapy. the presence of hbsag negative, hbcab-igg positive assays may be a false-positive or reflect true, latent hbv infection. hepatitis c virus (hcv) infection will generally progress more rapidly with immune suppression and with cmv coinfection. hcv seropositive renal transplant candidates are more likely to develop cirrhosis and complications of liver failure. there is no good therapy for hcv infection; management is by quantitative molecular viral assays. hiv-infected donors have not been utilized. the progression of disease is rapid and outweighs the benefits of transplantation. donors may be excluded based on historic evidence of "high-risk" behavior for hiv infection. western blot testing and molecular assays (pcr) should be obtained prior to the use of tissues from any hivseropositive donor. human t-lymphotropic virus i (htlv-i) is endemic in the caribbean and parts of asia (japan) and can progress to htlv-i-associated myelopathy/tropical spastic paraparesis (ham/tsp) or to adult t cell leukemia/lymphoma (atl). htlv-ii is similar to htlv-i serologically but is less clearly associated with disease. use of organs from such donors is generally avoided. 15, 16 west nile virus (wnv) is a flavivirus associated with viral syndromes and meningoencephalitis and may be transmitted by blood transfusion and organ transplantation. 17, 18 routine screening of donors is not advocated other than in areas with endemic infection of the blood supply. donors with unexplained changes in mental status or recent viral illness with neurologic signs should be avoided. sars (severe acute respiratory syndrome) is a recently described coronavirus, thought to be associated with exposure to civets or other animals common to the diet of certain regions of china. tissue persistence is prolonged and infection of transplant recipients appears to be severe and often symptomatic. organ procurement should exclude patients with recent acute illnesses meeting sars criteria. the pre-transplant period is useful for a thorough travel, animal, and environmental and exposure history; updating immunizations; and counseling of the recipient regarding travel, food, and other infection risks. ongoing infection must be eradicated prior to transplantation. two forms of infection pose a special risk: 1. bloodstream infection: this is related to vascular access, including that for dialysis and pneumonia, which puts the patient at high risk for subsequent lung infection with nosocomial organisms. infected ascites or peritoneal dialysis fluid must also be cleared prior to surgery. urinary tract infection (uti) must be eliminated prior to transplantation with antibiotics with or without nephrectomy. similarly, skin disease that threatens the integrity of this primary defense against infection should be corrected before transplantation, even if doing so requires the initiation of immunosuppression prior to transplantation (e.g., the initiation of immune suppression to treat psoriasis or eczema). finally, the history of more than one episode of diverticulitis should initiate an evaluation to determine whether sigmoid colectomy should be carried out prior to transplantation. 2. tuberculosis: both the incidence of active disease and the occurrence of disseminated infection due to m. tuberculosis are far higher in the transplant recipient than in the general population. active tuberculous disease must be eradicated prior to transplantation. the major antituberculous drugs are potentially hepatotoxic, and significant drug interactions are common between the anti-tb agents and the agents of immune suppression. in patients with active infection, from endemic regions or with high risk exposures, tb therapy should be initiated in all ppd positive individuals prior to transplantation. some judgment may be used as to the optimal timing of treatment in individuals without evidence of active or pleuropulmonary disease. greater risk may include: • previously active tuberculosis or significant signs of old tuberculosis on chest radiograph • recent tuberculin reaction conversion • known exposure to active disease • protein-calorie malnutrition, cirrhosis, or other immune deficiency • living in a shelter or other group housing aids for those benefiting from haart, aids has been converted from a progressively fatal disease to a chronic infection controlled by complex regimens of antiviral agents. haart has been associated with reduced viral loads, improved cd4 lymphocyte counts, and reduced susceptibility to opportunistic infections. in the pre-haart era, organ transplantation was generally associated with a rapid progression of aids. as a result, hiv-infected individuals have been excluded at most transplantation centers. however, prolonged disease-free survival with haart has lead to a reconsideration of this policy. renal transplantation in hiv has been associated with good outcomes in individuals with controlled hiv infection and in the absence of hcv co-infection. 19 management requires some sophistication regarding both the immune suppressive agents and the various haart regimens. the spectrum of infection in the immunocompromised host is quite broad. given the toxicity of antimicrobial agents and the need for rapid interruption of infection, early, specific diagnosis is essential in this population. advances in diagnostic modalities (ct or mri scanning, molecular microbiologic techniques) may greatly assist in this process. however, the need for invasive diagnostic tools cannot be overemphasized. given the diminished immune responses of the host and the frequency of multiple simultaneous processes, invasive diagnosis is often the only method for optimal care. the initial therapy will, by necessity, be broad with a rapid narrowing of the antimicrobial spectrum as data become available. the first choice of therapy is to reduce the intensity of immune suppression. the risk of such an approach is that of graft rejection. the selection of the specific reduction may depend upon the organisms isolated. similarly, reversal of some immune deficits (neutropenia, hypogammaglobulinemia) may be possible with adjunctive therapies (colony stimulating factors or igg). co-infection with virus (cmv) is common and merits additional therapy. cmv is the single most important pathogen in transplant recipients, having a variety of direct and indirect effects. 1, 27 the direct effects include: • fever and neutropenia syndrome with features of infectious mononucleosis, including hepatitis, nephritis, leukopenia, and/or thrombocytopenia • pneumonia • gastrointestinal invasion with colitis, esophagitis, gastritis, ulcers, bleeding, or perforation • hepatitis, pancreatitis, chorioretinitis with the exception of chorioretinitis, the direct clinical manifestations of cmv infection usually occur 1 to 4 months after transplantation; chorioretinitis usually does not begin until later in the transplant course. although cmv is the most common cause of clinical infectious disease syndromes, its "indirect effects" are often more important. cmv infection produces a profound suppression of a variety of host defenses, predisposing to secondary invasion by such pathogens as p. jiroveci, candida and aspergillus species, and some bacterial infections. cmv also contributes to the risk for graft rejection, ptld, hhv6, and hhv7 infections. the mechanisms for this effect are complex, including altered t-cell subsets and mhc synthesis, and the elaboration of an array of pro-inflammatory cytokines, chemokines, and growth factors. transmission of cmv in the transplant recipient occurs in one of three patterns: primary infection, reactivation infection, and superinfection. 1 virus may be reactivated in the setting of an allograft from a seropositive donor transplanted into a seropositive recipient (d+r+). control of cmv infection is via mhc-restricted, virusspecific, cytotoxic t lymphocyte response (cd8+ cells) controlled by cd4+ lymphocytes. seroconversion is a marker for the development of host immunity. thus, the major effector for activation of virus is the nature of the immunosuppressive therapy being administered. the lytic antilymphocyte antibodies, both polyclonal and monoclonal, are direct activators of viral infection (mimicking the alloimmune response) and also provoke the elaboration of tnf and the other pro-inflammatory cytokines that enhance viral replication. cyclosporine, tacrolimus, sirolimus, and prednisone (other than pulse doses) have limited ability to reactivate latent cmv while azathioprine, mycophenolate, and cyclophosphamide are moderately potent in terms of promoting viral reactivation. these agents perpetuate infection once established. allograft rejection is a major stimulus for cmv activation and vice versa. thus, the cmv infection has been linked to a diminished outcome of renal and other allografts. as a result, reinke and colleagues 27 showed that 17 of 21 patients for whom biopsy revealed evidence of "late acute rejection" demonstrated a response to antiviral therapy. further, lowance and colleagues 28 demonstrated that the prevention of cmv infection also resulted in a lower incidence of graft rejection. clinical management of cmv, both prevention and treatment, is of great importance for the transplant recipient. it is based on a clear understanding of the causes of cmv activation and the variety of diagnostic techniques available. cmv cultures are generally too slow and insensitive for clinical utility. further, a positive cmv culture (or shell vial culture) derived from respiratory secretions or urine is of little diagnostic value-many patients secrete cmv in the absence of invasive disease. serologic tests are useful prior to transplantation to predict risk but are of little value after transplantation in defining clinical disease (this statement includes measurements of anti-cmv immunoglobulin m [igm] levels). should a patient seroconvert to cmv, this is evidence that the patient has been exposed to cmv and has developed some degree of immunity. however, seroconversion in transplantation is generally delayed and, thus, not useful for clinical diagnosis. the demonstration of cmv inclusions in tissues in the setting of a compatible clinical presentation is the "gold standard" for diagnosis. quantitation of the intensity of cmv infection has been linked to the risk for infection in transplant recipients. [29] [30] [31] [32] [33] two types of quantitative assays have been developed: the molecular assays and the antigen detection assays. the antigenemia assay is a semiquantitative fluorescent assay in which circulating neutrophils are stained for cmv early antigen (pp65), which is taken up nonspecifically as a measure of the total viral burden in the body. the molecular assays (direct dna pcr, hybrid capture, amplification assays) are highly specific and sensitive for the detection of viremia. most commonly used assays include plasma-based pcr testing and the whole-blood hybrid capture assay, noting that whole blood and plasma-based assays cannot be directly compared. the highest viral loads are often associated with tissue-invasive disease with the lowest in asymptomatic cmv infection. viral loads in the cmv syndrome are variable. either assay can be used in management. the advent of quantitative assays for the diagnosis and management of cmv infection has allowed noninvasive diagnosis in many patients with two important exceptions: 1. neurologic disease, including chorioretinitis 2. gastrointestinal disease, including invasive colitis and gastritis. in these syndromes, the cmv assays are often negative and invasive (biopsy) diagnosis may be needed. the central role of assays is illustrated by the approach to prevention and treatment of cmv (table 37-6). the schedule for screening is linked to the risk for infection. thus, in the high risk patient (d+/r− or r+ with antilymphocyte globulin) after the completion of prophylaxis, monthly screening is performed to assure the absence of infection for 3 to 6 months. in the patient being treated for cmv infection, the assays provide an end point (zero positivity) for therapy and the initiation of prophylaxis. prevention of cmv infection must be individualized for immunosuppressive regimens and the patient. two strategies are commonly used for cmv prevention: (1) universal prophylaxis and (2) preemptive therapy. universal prophylaxis involves giving antiviral therapy to all "at-risk" patients beginning at or immediately post-transplant for a defined time period. in preemptive therapy, quantitative assays are used to monitor patients at predefined intervals to detect early disease. positive assays result in therapy. preemptive therapy incurs extra costs for monitoring and coordination of outpatient care while reducing the cost of drugs and the inherent toxicities. prophylaxis has the possible advantage of preventing not only cmv infection during the period of greatest risk, but also diminishing infections due to hhv6, hhv7, and ebv. further, the indirect effects of cmv (i.e., graft rejection, opportunistic infection) may also be reduced by routine prophylaxis. in practice neither strategy is perfect. both breakthrough disease and ganciclovir resistance have been observed in both approaches. given the risk for invasive infection, patients at risk for primary infection (cmv d+/r−) are generally given prophylaxis for 3 to 6 months after transplantation. we utilize 6 months of prophylaxis in patients receiving lytic antilymphocyte antibodies. other groups are candidates for preemptive therapy if an appropriate monitoring system is in place and patient compliance is good. the standard of care for treating cmv disease is 2 to 3 weeks of intravenous ganciclovir (5 mg/kg twice daily, with dosage adjustments for renal dysfunction). in patients slow to respond to therapy and who are seronegative, the addition of 3 months of cmv hyperimmune globulin in seronegative individuals (150 mg/kg/dose iv) may be useful. relapse does occur, primarily in those not treated beyond the achievement of a negative quantitative assay. therefore, we treat intravenously until viremia has been cleared and following it with prophylaxis with 2 to 4 months of oral ganciclovir (1 g two or three times daily) or valganciclovir (based on creatinine clearance). this approach has resulted in rare symptomatic relapses and appears to prevent the emergence of antiviral resistance. a number of issues remain. first, the role of oral valganciclovir in treatment has not been well studied. this agent provides good bioavailability but is not approved for this indication. further, some relapses occur in gi disease because the assays used to follow disease are not reliable in this setting. thus, repeat endoscopy should be considered to assure the clearance of infection. the optimum dosing of valganciclovir for prophylaxis in renal transplant recipients is also unclear. many centers use 450 mg/day po (given reduced creatinine clearance) although the fda approved dosing 900 mg/day. it is worth measuring the creatinine clearance to ensure appropriate dosing. alternative therapies are available in intravenous form only. these include foscarnet and cidofovir. foscarnet has been used extensively for therapy of cmv in aids patients. it is active against most ganciclovir-resistant strains of cmv, although we prefer combination therapy (ganciclovir and foscarnet) for such individuals, given the toxicities of each agent and the antiviral synergy demonstrated. cidofovir has been used in renal transplant recipients, often with nephrotoxicity. both foscarnet and cidofovir may exhibit synergistic nephrotoxicity with calcineurin inhibitors. a newer class of agents (leflunamide) has been approved for immune suppression and treatment of rheumatologic diseases but also appears to have useful activity against cmv (and possibly bk polyomavirus). ebv is a ubiquitous herpesvirus (the majority of adults are infected) that has b-lymphocytes as a primary target for infection. in immunosuppressed transplant recipients, primary ebv infection (and relapses in the absence of antiviral immunity) causes a mononucleosis-type syndrome, generally presenting as a lymphocytosis (b-cells) with or without lymphadenopathy or pharyngitis. meningitis, hepatitis, and pancreatitis may also be observed. remitting-relapsing ebv infection is common in children and may reflect the interplay between evolving antiviral immunity and immune suppression. this syndrome should suggest relative over-immune suppression. ebv also plays a central role in the pathogenesis of posttransplant lymphoproliferative disorder or ptld. [34] [35] [36] [37] the most clearly defined risk factor for ptld is primary ebv infection that increases the risk for ptld by 10-to 76-fold. ptld may occur, however, in the absence of ebv infection or in seropositive patients. post-transplant non-hodgkin's lymphoma (nhl) is a common complication of solid organ transplantation. lymphomas comprise up to 15% of tumors among adult transplant recipients (51% in children) with mortality of 40% to 60%. many deaths are associated with allograft failure after withdrawal of immune suppression during treatment of malignancy. compared with the general population, ptld has increased extranodal involvement, poor response to conventional therapies, and poor outcomes. the spectrum of disease ranges from benign polyclonal, b-cell infectious mononucleosis-like disease to malignant, monoclonal lymphoma. 38 the majority is of b-cell origin, although t-cell, nk-cell and null cell tumors are described. it should be noted that ebv-negative ptld has been described and that t-cell ptld has been demonstrated in allografts, confused with graft rejection or other viral infection. ptld late (more than 1-2 years) after transplantation is more often ebv-negative in adults. the clinical presentations of ebv-associated ptld vary: 1. unexplained fever (fever of unknown origin) 2. a mononucleosis-type syndrome, with fever, malaise, with or without pharyngitis or tonsillitis (often diagnosed incidentally in tonsillectomy specimens); often no lymphadenopathy is observed. 3. gastrointestinal bleeding, obstruction, perforation 4. abdominal mass lesions 5. infiltrative disease of the allograft 6. hepatocellular or pancreatic dysfunction 7. central nervous system disease diagnosis serologic testing is not useful for the diagnosis of acute ebv infection or ptld in transplantation. thus, quantitative ebv viral load testing is required for the diagnosis and management of ptld. [39] [40] [41] [42] serial assays are more useful in an individual patient than specific viral load measurements. these assays are not standardized and cannot be directly compared between centers. there are some data to suggest that assays using unfractionated whole blood are preferable to plasma samples for ebv viral load surveillance. clinical management depends on the stage of disease. in the polyclonal form, particularly in children, reestablishment of immune function may suffice to cause ptld to regress. at this stage, it is possible that antiviral therapy might have some utility given the viremia and role of ebv as an immune suppressive agent. with the progression of disease to extra-nodal and monoclonal malignant forms, reduction in immune suppression may be useful, but alternate therapies are often required. in renal transplantation, the failure to regress with significant reductions in immune suppression may suggest the need to sacrifice the allograft for patient survival. combinations of anti-b-cell therapy (anti-cd20 rituximab), chemotherapy (chop), and/or adoptive immunotherapy with stimulated t cells have been utilized. [43] [44] [45] [46] polyomaviruses polyomaviruses have been identified in transplant recipients in association with nephropathy and ureteral obstruction (bk virus) and in association with demyelinating disease of the brain (jc virus) similar to that in aids. polyomaviruses are small nonenveloped viruses with covalently closed, circular, double-stranded dna genomes. adult levels of seroprevalence are 65% to 90%. bk virus appears to achieve latency in renal tubular epithelial cells. jc virus has also been isolated from renal tissues but appears to have preferred tropism for neural tissues. reactivation occurs with immune deficiency and suppression and tissue injury (e.g., ischemia-reperfusion). bk virus is associated with a range of clinical syndromes in immunocompromised hosts: viruria and viremia, ureteral ulceration and stenosis, and hemorrhagic cystitis. [47] [48] [49] [50] [51] [52] [53] [54] active infection of renal allografts has been associated with progressive loss of graft function (bk nephropathy) in some individuals. this may be referred to as polyomavirus-associated nephropathy or pvan. bk nephropathy is rarely recognized in recipients of nonrenal organs. the clinical presentation of disease is usually as sterile pyuria, reflecting shedding of infected tubular and ureteric epithelial cells. these cells contain sheets of virus and are detected by urine cytology as "decoy cells." in most cases, such cells are not detected and the patient presents with diminished renal allograft function or with ureteric stenosis and obstruction. in such patients, the etiologies of decreased renal function must be carefully evaluated (e.g., mechanical obstruction, drug toxicity, pyelonephritis, rejection, thrombosis, recurrent disease), and choices must be made between increasing immune suppression to treat suspected graft rejection and reducing immune suppression to allow the immune system to control infection. patients with bk nephropathy treated with increased immune suppression have a high incidence of graft loss. reduced immune suppression may stabilize renal allograft function but risks graft rejection. polyoma-associated nephropathy manifested by characteristic histologic features and renal dysfunction is found in about 1% to 8% of renal transplant patients. risk factors for nephropathy are poorly defined. nickeleit and colleagues 51, 52 found that cellular rejection occurred more commonly in patients with bk nephropathy than in controls. other studies have implicated high dose immunosuppression (particularly tacrolimus and mycophenolate mofetil), pulse dose steroids, severe ischemia-reperfusion injury, exposure to antilymphocyte antibody therapy, increased number of hla mismatches between donor and recipient, cadaver renal transplants, and presence and degree of viremia in the pathogenesis of disease. the role of specific immunosuppressive agents has not been confirmed. the use of urine cytology to detect the presence of infected decoy cells in the urine has approximately 100% sensitivity for bk virus infection but a low (29%) predictive value. 53, 54 it is, therefore, a useful screening tool but cannot establish a firm diagnosis. the use of molecular techniques to screen blood or urine has also been advocated but is more useful in management of established cases (viral clearance with therapy) than in specific diagnosis. [55] [56] [57] [58] [59] [60] hirsch and colleagues 53 showed that patients with bk nephropathy have a plasma viral load statistically significantly higher (>7700 bk virus copies per ml of plasma, p<.001, 50% positive predictive value, 100% negative predictive value) when compared to patients without such disease. 53 given the presence of viremia in renal allograft recipients, it is critical to reduce immune suppression when possible. however, the possible coexistence of rejection with bk infection makes renal biopsy essential for the management of such patients. renal biopsies will demonstrate cytopathic changes in renal epithelial cells without cellular infiltration with the gradual evolution of cellular infiltration consistent with the diagnosis of interstitial nephritis. fibrosis is often prominent occasionally with calcification. immunostaining for cross reacting sv40 virus demonstrates patchy staining of viral particles within tubular cells. there is no accepted treatment for pvan other than a marked reduction in the intensity of immune suppression. it is possible to monitor the response to such maneuvers using urine cytology (decoy cells) and viral load measures in blood and/or urine. the greatest incidence of bk nephropathy is at centers with the most intensive immune suppressive regimens. thus, it is unclear whether reduction of calcineurin inhibitors or antimetabolites should be considered first. given the toxicity of calcineurin inhibitors for tubular cells and the role of injury in the activation of bk virus, as well as the need for anti-bk t-cell activity, we have generally reduced these agents first. other centers have selected reduction of the antimetabolite first. regardless of the approach, renal function, drug levels, and viral loads must be monitored carefully. some centers advocate the use of cidofovir for bk nephropathy in low doses (0.25-1 mg/kg every 2 weeks). [61] [62] [63] [64] significant renal toxicity may be observed with this agent, especially in combination with the calcineurin inhibitors. retransplantation has been achieved in such patients with failed allografts, possibly as a reflection of immunity developing subsequent to reduction in immune suppression. 65 infection of the central nervous system by jc polyomavirus has been observed uncommonly in renal allograft recipients as progressive multifocal encephalopathy. this infection generally presents with focal neurologic deficits or seizures and may progress to death following extensive demyelination. pml may be confused with calcineurin neurotoxicity; both may respond to a reduction in drug levels. it is thought that these are distinct entities, but further studies are underway. in addition to the endemic mycoses, transplant recipients are at risk for opportunistic infection with a variety of fungal agents, the most important of which are candida species, aspergillus species, and c. neoformans. the most common fungal pathogen in these patients is candida, with c. albicans and c. tropicalis accounting for 90% of the infections and c. glabrata for most of the rest. mucocutaneous candidal infection (e.g., oral thrush, esophageal infection, cutaneous infection at intertriginous sites, candidal vaginitis) occurs particularly when candidal overgrowth is promoted by the presence of high levels of glucose and glycogen in tissues and fluids (e.g., with poorly controlled diabetes, high-dose steroid therapy) and by broad-spectrum antibacterial therapy). these infections are usually treatable through correction of the underlying meta-bolic abnormality and topical therapy with clotrimazole or nystatin. more difficult to manage is candidal infection occurring in association with the presence of foreign bodies that violate the mucocutaneous surfaces of the body (e.g., vascular access catheters, surgical drains, and bladder catheters). optimal management of these infections requires removal of the foreign body and systemic antifungal therapy with either fluconazole or amphotericin. a special problem in renal transplant recipients is candiduria, even if the patient is asymptomatic. particularly in individuals with poor bladder function, obstructing fungal balls can develop at the ureteropelvic junction, resulting in obstructive uropathy, ascending pyelonephritis, and the possibility of systemic dissemination. a single positive culture result for candida species from a blood specimen necessitates systemic antifungal therapy, because this finding carries a risk of visceral invasion of more than 50% in this population. fluconazole (400-600 mg/day, with adjustment for renal dysfunction), because of its better safety profile, is usually used as initial therapy, unless the patient is critically ill or a fluconazole-resistant species (e.g., c. glabrata or c. krusei) is present. in these instances, therapy is with caspofungin or amphotericin b, usually in a lipid preparation. flucytosine may be useful as an adjunctive therapy in resistant infections but must be guided by drug levels and attention to hematopoietic toxicity. invasive aspergillosis is a medical emergency in the transplant recipient, with the portal of entry being the lungs and sinuses in more than 90% of patients and the skin in most of those remaining. two species, a. fumigatus and a. flavum, account for most of these infections, although amphotericin-resistant isolates (a. terreus) are occasionally recognized. the pathologic hallmark of invasive aspergillosis is blood vessel invasion, which accounts for the three clinical characteristics of this infection: tissue infarction, hemorrhage, systemic dissemination with metastatic invasion. early in the course of transplantation, central nervous system involment with fungal infection is most often due to aspergillus species; more than 1 year after transplantation, other fungi (zygomycetes, dematiaceous fungi) are increasingly prominent. the drug of choice for this infection is probably voriconazole, noting the intense interactions between this agent and the calcineurin inhibitors and sirolimus. liposomal amphotericin is a reasonable alternative, and combination therapies are under study. of note, surgical debridement is often essential for the successful clearance of such invasive infections. central nervous system (cns) infection in the transplant recipient is an important differential for the clinician. the spectrum of causative organisms is broad and must be considered in terms of the timeline for infection in this population. many infections are metastatic to the cns, often from the lungs. thus, a "metastatic workup" is a component of evaluation of cns lesions, including those due to aspergillus, cryptococcus, nocardia, or strongyloides stercoralis. viral infections include cytomegalovirus (nodular angiitis), herpes simplex meningoencephalitis, jc virus (pml), and varicella zoster virus. common bacterial infections include listeria monocytogenes, mycobacteria, nocardia, and occasionally salmonella species. brain abscess and epidural abscess may be observed with methicillin-resistant staphylococcus, penicillin resistant pneumococcus and quinolone-resistant streptococci problematic. metastatic fungi include aspergillus and cryptococcus but also spread from sinuses (mucoraceae), skin (dematiaceae), and bloodstream (histoplasma and pseudoallescheria/scedosporium, fusarium species). parasites include toxoplasma gondii and strongyloides. given the spectrum of etiologies, precise diagnosis is essential. in particular, empiric therapy must "cover" listeria (ampicillin), cryptococcus (fluconazole or amphotericin), and herpes simplex virus (acyclovir) while awaiting data from lumbar puncture, blood cultures, and radiographic studies. included in the differential diagnosis are noninfectious etiologies, including calcineurin inhibitor toxicity and lymphoma, as well as metastatic cancer. biopsy is often needed for a firm diagnosis. cryptococcal infection is rarely seen in the transplant recipient until more than 6 months after transplantation. in the relatively intact transplant recipient, the most common presentation of cryptococcal infection is that of an asymptomatic pulmonary nodule, often with active organisms present. in the "chronic ne'er-do-well" patient, pneumonia and meningitis are common with skin involvement at sites of tissue injury (catheters) also being observed. cryptococcosis should be suspected in transplant recipients present with unexplained headaches (especially when accompanied by fevers), decreased state of consciousness, failure to thrive, or unexplained focal skin disease (which requires biopsy for culture and pathologic evaluation) more than 6 months after transplantation. diagnosis is often achieved by serum cryptococcal antigen detection, but all such patients should have lumbar puncture for cell counts and cryptococcal antigen studies. initial treatment is probably best with amphotericin and 5-flucytosine followed by high dose fluconazole until the cryptococcal antigen is cleared from blood and cerebrospinal fluid. scarring and hydrocephalus may be observed. the spectrum of potential pathogens of the lungs in transplantation is too broad for this discussion. however, some general concepts are worth mentioning. as for all infections in transplantation, invasive diagnostic techniques are often necessary in these hosts. the depressed inflammatory response of the immunocompromised transplant patient may greatly modify or delay the appearance of a pulmonary lesion on radiograph. focal or multifocal consolidation of acute onset will quite likely be caused by bacterial infection. similar multifocal lesions with subacute to chronic progression are more likely secondary to fungi, tuberculosis, or nocardial infections. large nodules are usually a sign of fungal or nocardial infection, particularly if they are subacute to chronic in onset. subacute disease with diffuse abnormalities, either of the peri-bronchovascular type or miliary micronodules, are usually caused by viruses (especially cmv) or pneumocystis jiroveci. 66, 67 additional clues can be found by examining pulmonary lesions for cavitation; cavitation suggests such necrotizing infections as those caused by fungi (aspergillus or mucoraceae), nocardia, staphylococcus, certain gram-negative bacilli, most commonly with klebsiella pneumoniae and pseudomonas aeruginosa. [68] [69] [70] ct of the chest is useful when the chest radiograph is negative or when the radiographic findings are subtle or nonspecific. ct is also essential to the definition of the extent of the disease process, the possibility of multiple simultaneous processes (superinfection), and to the selection of the optimal invasive technique to achieve microbiologic diagnosis. the risk of infection with pneumocystis is greatest in the first 6 months after transplantation and during periods of increased immune suppression. 1, 66, 67 the natural reservoir of infection remains unknown. aerosol transmission of infection has been demonstrated by a number of investigators in animal models, and clusters of infections have developed in clinical settings, including between hiv-infected persons and renal transplant recipients. activation of latent infection remains a significant factor in the incidence of disease in immunocompromised hosts. in the solid organ transplant recipient, chronic immune suppression that includes corticosteroids is most often associated with pneumocystosis. bolus corticosteroids, cyclosporine, or co-infection with cmv may also contribute to the risk for pneumocystis pneumonia. in patients not receiving trimethoprim-sulfamethoxazole (or alternative drugs) as prophylaxis, most transplant centers report an incidence of pneumocystis jiroveci pneumonia of approximately 10% in the first 6 months post-transplant. there is a continued risk of infection in three overlapping groups of transplant recipients: (1) those who require higher than normal levels of immune suppression for prolonged periods of time due to poor allograft function or chronic rejection; (2) those with chronic cytomegalovirus infection; and (3) those undergoing treatments that increase the level of immune deficiency, such as cancer chemotherapy or neutropenia due to drug toxicity. the expected mortality due to pneumocystis pneumonia is increased in patients on cyclosporine when compared to other immunocompromised hosts. the hallmark of infection due to p. jiroveci is the presence of marked hypoxemia, dyspnea, and cough with a paucity of physical or radiologic findings. in the transplant recipient, pneumocystis pneumonia is generally acute to subacute in development. atypical pneumocystis infection (radiographically or clinically) may be seen in patients who have coexisting pulmonary infections or who develop disease while receiving prophylaxis with second choice agents (e.g., pentamidine or atovaquone). patients outside the usual period of greatest risk for pcp may present with indolent disease confused with heart failure. in such patients, diagnosis often has to be made by invasive procedures. the role of sirolimus therapy in the clinical presentation is unknown. a number of patients have been identified with interstitial pneumonitis while receiving sirolimus; it is not known whether this syndrome is directly attributable to sirolimus or reflects concomitant infection. the characteristic hypoxemia of pneumocystis pneumonia produces a broad alveolar-arterial po 2 gradient. the level of serum lactic dehydrogenase (ldh) is elevated in most patients with pneumocystis pneumonia (>300 international units [iu]/ml). however, many other diffuse pulmonary processes also raise serum ldh levels. like many of the "atypical" pneumonias (pulmonary infection without sputum production), no diagnostic pattern exists for pneumocystis pneumonia on routine chest radiograph. the chest radiograph may be entirely normal or develop the classical pattern of perihilar and interstitial "ground glass" infiltrates. microabscesses, nodules, small effusions, lymphadenopathy, asymmetry, and linear bands are common. chest computerized tomography (ct-scans) will be more sensitive to the diffuse interstitial and nodular pattern than routine radiographs. the clinical and radiologic manifestations of p. jiroveci pneumonia are virtually identical to those of cmv. indeed, the clinical challenge is to determine whether both pathogens are present. significant extrapulmonary disease is uncommon in the transplant recipient. identification of p. jiroveci as a specific etiologic agent of pneumonia in an immunocompromised patient should lead to successful treatment. a distinction should be made between the diagnosis of pneumocystis infection in aids and in non-aids patients. the burden of organisms in infected aids patients is generally greater than that of other immunocompromised hosts and noninvasive diagnosis (sputum induction) more often achieved. in general, noninvasive testing should be attempted to make the initial diagnosis, but invasive techniques should be used when clinically feasible. the diagnosis of p. jiroveci infection has been improved by the use of induced sputum samples and of immunofluorescent monoclonal antibodies to detect the organism in clinical specimens. these antibodies bind both cysts and trophozoites. the cyst wall can be displayed by a variety of staining techniques; of these, the gomori's methenamine-silver nitrate method (which stains organisms brown or black) is most reliable, even though it is susceptible to artifacts. sporozoites and trophozoites are stained by polychrome stains, particularly the giemsa stain. early therapy, preferably with trimethoprim-sulfamethoxazole (tmp-smz) is preferred; few renal transplant patients will tolerate full-dose tmp-smz for prolonged periods of time. this reflects both the elevation of creatinine due to trimethoprim (competing for secretion in the kidney) and the toxicity of sulfa agents for the renal allograft. hydration and the gradual initiation of therapy may help. alternate therapies are less desirable but have been used with success, including: intravenous pentamidine, atovaquone, clindamycin with primaquine or pyrimethamine, and trimetrexate. although a reduction in the intensity of immune suppression is generally considered a part of anti-infective therapy in transplantation, the use of short courses of adjunctive steroids with a gradual taper is sometimes used in transplant recipients (as in aids patients) with severe respiratory distress associated with pcp. the importance of preventing pneumocystis infection cannot be overemphasized. low dose trimethoprim-sulfamethoxazole is well tolerated and should be used in the absence of concrete data demonstrating true allergy. alternative prophylactic strategies including dapsone, atovaquone, inhaled or intravenous pentamidine, are less effective than trimethoprim-sulfamethoxazole but useful in the patient with significant allergy to sulfa drugs. tmp-smx is the most effective agent for prevention of infection due to p. jiroveci. the advantages of tmp-smx include increased efficacy, lower cost, the availability of oral preparations, and possible protection against other organisms, including toxoplasma gondii, isospora belli, cyclospora cayetanensis, nocardia asteroides, and common urinary, respiratory, and gastrointestinal bacterial pathogens. it should be noted that alternative agents lack this spectrum of activity. due to concerns about the efficacy of vaccines following transplantation, patients should complete vaccinations at least 4 weeks beforehand to allow time for an optimal immune response and resolution of subclinical infection from live vaccines. vaccinations should include pneumococcal vaccine (if not vaccinated in last 3-5 years), documentation of tetanus and mmr (measles, mumps, rubella) and polio status, as well as vaccines for hepatitis b and varicella zoster (if no history of chickenpox or shingles) (see also . after transplant, influenza vaccination should be performed yearly or as per local guidelines. recommended schedules and doses for routine vaccinations can be obtained from the united states centers for disease control and prevention (cdc) at www.immunize.org or the cdc immunization information hotline, (800) 232-2522. infection in organ-transplant recipients 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transplants-tubular necrosis, mhc-class ii expression and rejection in a puzzling game polyomavirus infection of renal allograft recipients: from latent infection to manifest disease prospective study of polyomavirus type bk replication and nephropathy in renaltransplant recipients bk virus nephropathy? polyomavirus adding insult to injury morphological spectrum of polyoma virus disease in renal allografts: diagnostic accuracy of urine cytology prospective study of the human polyomaviruses bk and jc and cytomegalovirus in renal transplant recipients clinical course of polyoma virus nephropathy in 67 renal transplant patients bk virus infection in a kidney allograft diagnosed by needle biopsy polyomavirus reactivation in native kidneys of pancreas alone allograft recipients bk virus nephropathy diagnosis and treatment: experience at the university of maryland renal transplant program the use of vidarabine in the treatment of human polyomavirus associated acute haemorrhagic cystitis activities of various compounds against murine and primate polyomaviruses clinical pharmacokinetics of cidofovir in human immunodeficiency virus-infected patients quantitative viral load monitoring and cidofovir therapy for the management of bk virusassociated nephropathy in children and adults successful retransplantation following renal allograft loss to polyoma virus interstitial nephritis prevention of infection caused by pneumocystis carinii in transplant recipients prevention of infection due to pneumocystis carinii nocardiosis in transplant recipients recurrent nocardiosis in a renal transplant recipient central venous catheter-associated nocardia bacteremia: an unusual manifestation of nocardiosis key: cord-018393-5jlqn7wq authors: finke, ernst-jürgen; tomaso, herbert; frangoulidis, dimitrios title: bioterrorismus, infektiologische aspekte date: 2011-12-14 journal: lexikon der infektionskrankheiten des menschen doi: 10.1007/978-3-642-17158-1_3 sha: doc_id: 18393 cord_uid: 5jlqn7wq infektionskrankheiten sind ständige begleiter und gefürchtete geißeln der menschheit. pest und pocken versetzen als todbringende seuchen die menschen nicht erst seit dem altertum in schrecken (lat.: terror). archaische ängste und vor allem eine hohe medienaufmerksamkeit sorgen immer wieder für panik und irrationale reaktionen: im indischen surat setzte im herbst 1994 während eines ungewöhnlichen pestausbruchs eine massenflucht ein, nachdem die presse den verdacht auf lungenpest und terroristische anschläge verbreitet hatte. über 800.000 menschen, darunter auch zahlreiche ärzte und pflegekräfte, verließen daraufhin ihre arbeitsplätze und wohnorte. allein die drastischen flugund handelsbeschränkungen brachten indien einen ökonomischen schaden von etwa 3 milliarden us $. . als bioterrorismus wird gewöhnlich eine drohung mit oder der einsatz von biologischen waffen, biologischen kampfstoffen und vergleichbaren biologischen agenzien durch staaten, gruppen oder einzelpersonen aus politischen, militärischen, religiösen, ökonomischen oder anderen beweggründen bezeichnet. angedrohte oder realisierte bioterroristische anschläge verfolgen oft die absicht, physische und psychische schäden auszulösen und bevölkerungsgruppen in angst und panik zu versetzen und zu demoralisieren. damit wird bezweckt, eine gesellschaft wirtschaftlich zu schwächen, politisch zu destabilisieren oder sogar zu paralysieren. aber allein schon blinde alarme ("weißes pulver") durch trittbrettfahrer und gezielte desinformation ("pockenpandemie", "bioterror") können ähnlich wirken und eine massenhysterie auslösen. mit einem massenanfall an geschädigten wäre insbesondere zu rechnen, wenn b-kampfmittel aus geheimen staatlichen b-waffenarsenalen genutzt und extrem hohe dosen "professioneller" biologischer kampfstoffe ausgebracht würden. experten gehen davon aus, dass gegenwärtig eine derartige biologische bedrohung sehr unwahrscheinlich ist. dafür sprechen auch verschiedene historische analysen von ereignissen, bei denen biologische agenzien gegen personen eingesetzt wurden. so kam es im zeitraum von 1900 bis 2001 weltweit lediglich zu 77 biologischen ereignissen, wobei in den letzten 100 jahren in den usa weniger als 10 menschen durch biologische anschläge getötet wurden [24] . biologische kampfstoffe sind zu nicht friedlichen zwecken produzierte vermehrungsfähige organismen und daraus gewonnene physiologisch aktive stoffe, die tod oder krankheit bei mensch, tier und pflanze verursachen können. über 30 arten und typen von krankheitserregern und toxinen (7 tab. 1) gelten nach ansicht internationaler expertengremien und der us-amerikanischen centers for disease control and prevention (cdc) als potenzielle b-kampfstoffe [9, 34, 36, 38] . sie werden häufig auch als sogenannte "dual threat", "critical", "select" oder "biologische agenzien" bezeichnet. einige der in tab. 1 aufgeführten agenzien sind aus bisher offengelegten b-waffen-programmen bekannt, wurden schon einmal waffenfähig gemacht oder bei terroristischen bzw. kriminellen aktionen eingesetzt. es handelt sich dabei um besonders wirksame mikrobielle, pflanzliche und tierische toxine sowie hoch virulente stämme verschiedener natürlich vorkommender human-und tierpathogener bakterien-, viren-und pilzarten. einige sind aufgrund ihrer dauerformen (z. b. sporen von b. anthracis) besonders umweltresistent und bleiben dadurch in aerosolform in der luft für mehrere stunden infektiös. die mehrzahl der als biologische agenzien infrage kommenden mikroorganismen sind zoonoseerreger, von denen einige beim menschen gefährliche, d. h. lebensbedrohliche und hoch ansteckende, infektionskrankheiten verursachen können (7 tab. 1, 2 und 3) . sie treten in deutschland normalerweise nicht oder sehr selten auf. infektionen mit den meisten dieser krankheitserreger verlaufen überwiegend klinisch manifest. unbehandelt weisen sie eine schlechte prognose und relativ hohe letalität auf. abgesehen von pocken, lungenpest und bestimmten viralen hämorrhagischen fiebern ist eine übertragung von mensch zu mensch selten. angesichts der fehlenden oder geringen inzidenz derartiger "exotischer" krankheiten kann eine valide diagnostik nur in wenigen hoch spezialisierten referenz-, konsiliar-und expertenlaboratorien der schutzstufen 3 oder 4 gewährleistet werden. wirksame mittel zur kausalen therapie und zur immun-sowie chemoprophylaxe sind entweder nicht oder nur begrenzt verfügbar. so könnten z. b. bestimmte, in deutschland nicht zugelassene, impfstoffe gegen pest, anthrax, tularämie oder pocken erst bei eintritt eines biologischen schadensereignisses freigegeben werden. daher würde ein angriff mit erregern gefährlicher infektionskrankheiten aufgrund des hohen epidemischen potenzials und der unzureichenden immunität einer ungeschützt exponierten bevölkerung schwer alle bisher bekannt gewordenen biologischen drohungen und anschläge mit terroristischem oder kriminellem hintergrund zeigen: es ist nahezu unmöglich, rechtzeitig festzustellen, wer biologische kampfstoffe besitzt oder anzuwenden gedenkt. und selbst wenn man unmittelbar vor einem wahrscheinlichen biologischen anschlag stünde, ist nicht exakt vorauszusagen, von wem, gegen wen, wann und wo welches b-agens in welcher menge, wie oft und auf welche weise zum einsatz käme. aus infektiologischer und epidemiologischer sicht sind daher folgende aspekte beachtenswert: biologische anschläge werden höchstwahrscheinlich unangekündigt und verdeckt ausgeführt. bei einer simultanen infizierung größerer personengruppen müssen nicht alle exponierten in gleichem maße geschädigt werden. je nachdem, in welcher menge, wann und wie oft biologische agenzien eingesetzt werden, kann eine unterschiedlich große zahl von menschen entweder gleichzeitig oder zeitlich versetzt an demselben oder an unterschiedlichen orten betroffen sein. einmal als aerosol oder durch kontamination von lebensmitteln und trinkwasser ausgebracht, wären selbst extrem hohe erregerkonzentrationen mit menschlichen sinnen nicht erfassbar. dabei würde eine nicht gewarnte und folglich ungeschützte zielpopulation äußerst wirksam infiziert. sofern "professionelle" b-kampfstoffe oder nicht endemische erreger als b-agenzien ausgebracht würden, träfen sie auf eine voll empfängliche population, in der dann ein erheblicher anteil der exponierten erkranken dürfte. nach ablauf der minimalen inkubationszeit würden sich eine oder mehrere primäre explosivepidemien mit variierender intensität entwickeln. im falle übertragbarer krankheiten können in abhängigkeit von der kontaktrate und empfänglichkeit der exponierten weitere epidemiewellen folgen. bei erregern gefährlicher infektionskrankheiten dürften nicht nur infektionen durch inhalation von b-agenzien fulminant verlaufen, sondern auch nach oraler aufnahme von massiv kontaminierten lebensund genussmitteln oder trinkwasser. explosive aus-bruchsgeschehen wären aber ebenso nach einer exposition gegen einheimische krankheitserreger, z. b. salmonellen, shigellen oder noroviren, zu erwarten, gegen die zumeist keine belastbare immunität bestünde. so haben anhänger der rajneesh-sekte in the dallas (us-staat ohio) im jahre 1984 unbemerkt salatbüfetts einiger restaurants mit salmonella typhimurium kontaminiert, um eine gemeindewahl zu beeinflussen. dadurch kam es zu mehreren salmonellose-ausbrüchen mit insgesamt 751 erkrankten, von denen 45 stationär behandelt werden mussten. im jahre 1995 "beimpfte" ein mitarbeiter eines medizinischen zentrums in texas (usa) gebäck mit shigella dysenteriae typ 2 und verursachte damit eine shiga-ruhr bei zwölf seiner kollegen. für ihre straftaten nutzten die täter übrigens bakterienstämme aus den laboratorien, in denen sie beschäftigt waren. fazit: aufgrund der vielzahl potenzieller b-agenzien und ausbringungsmöglichkeiten einerseits und der heterogenität (alter, geschlecht, immunität, disposition) der gefährdeten population andererseits ist mit einem außerordentlich großen spektrum biologischer szenarien und krankheitsformen und -verläufen zu rechnen (7 tab. 2) . daher können mögliche szenarien nicht vorausgesagt und das schadensausmaß eines biologischen anschlags nur schwer abgeschätzt werden. hinzu kommt, dass ohne hinweise auf einen unmittelbar bevorstehenden oder erfolgten angriff biologische agenzien in der umwelt, wenn überhaupt, erst sehr spät festzustellen sind. aufgrund der biologischen und physikalischen alterung vegetativer bakterienformen und viren bestünden chancen für einen erfolgreichen erregernachweis unmittelbar nach einem anschlag und auch noch nach einigen wochen bei einsatz umweltresistenter agenzien, z. b. anthraxsporen oder coxiella burnetii. ein biologischer wirkungsherd (= potenziell verseuchte räume, objekte oder gebiete einschließlich flora, personen, tierbeständen, technik) lässt sich daher anfangs kaum exakt lokalisieren und der umfang der kontamination nur mit einem enormen technischen und zeitlichen aufwand bestimmen. somit besteht die gefahr, dass äußerlich kontaminierte oder schon infizierte exponierte den wirkungsherd ohne vorherige dekontamination verlassen. dabei könnten sie b-kampfstoffe oder biologische agenzien mit ihrer kleidung oder über genutzte verkehrsmittel weiter verbreiten und sekundär infektionen an anderen orten auslösen. deshalb gilt es, frühzeitig einen "reinen" absperrbereich (grüne zone) für die behandlung und betreuung der betroffenen, einen übergangsbereich (gelbe zone) zur dekontamination und zum ausschleusen und einen "unreinen" gefahren-bzw. kontaminationsbereich (rote zone) festzulegen [4, 11] . "eine schwere krankheit lässt sich anfangs leicht heilen, aber schwer erkennen. wenn sie sich jedoch verstärkt, kann man sie leicht erkennen, aber nur schwer heilen." (nicolo macchiavelli, 1449 -1527 es ist wenig wahrscheinlich, dass biologische anschläge rechtzeitig als solche erkannt werden, sofern kein automatisches monitoring mit einem zuverlässigen echtzeit-nachweis von b-agenzien existiert. in der praxis wird man erst durch einen ungewöhnlichen krankheitsausbruch bzw. ein außergewöhnliches seuchengeschehen in der bevölkerung oder auch in tierbeständen aufmerksam werden (7 tab. 5) . ein ausbruch stellt ein gehäuftes auftreten von zwei und mehr erkrankungs-oder todesfällen in engem epidemiologischem zusammenhang dar. nicht selten bilden sie die anfangsphase von epidemien. ungewöhnlich sind ausbrüche dann, wenn sie in ihren ökologischen, epidemiologischen, infektiologischen und mikrobiologischen merkmalen von der "norm" abweichen. als "norm" gilt dabei das typische auftreten einer infektionskrankheit entsprechend dem erwartungsgemäßen saisonalen, geo-und demografischen verteilungsmuster und dem bekannten klinischen erscheinungsbild. verdacht auf den einsatz von b-agenzien bzw. einen ungewöhnlichen krankheitsausbruch besteht, wenn der erreger nicht endemisch ist, die krankheit wie im fall der pocken als ausgerottet gilt oder keine exposition in einem endemiegebiet im ausland oder bei labortätigkeiten stattgefunden haben kann. auffällig sind auch atypische, bei natürlicher infektion unübliche, krankheitsverläufe, z. b. inhalationsmilzbrand und primäre pestpneumonie, oder auffällig hohe manifestations-und letalitätsraten. für eine nicht natürliche ursache können auch extrem kurze inkubationszeiten und der nachweis "exotischer" erreger oder einer sonst nicht beobachteten multiresistenz bei einer endemischen erregerart sprechen. wichtige kriterien für die aufklärung der ursachen können zusätzlich nachrichtendienstliche oder kriminalpolizeiliche hinweise auf eine biologische bedrohung bilden. die erkennung eines nicht natürlich verursachten krankheitsgeschehens würde zusätzlich erschwert, wenn man bisher unbekannte bzw. gentechnisch manipulierte agenzien oder endemisch vorkommende erreger ausbrächte. im letzteren falle würden, analog zu den beiden o. a. anschlägen mit salmonellen und shigellen, "natürliche" seuchengeschehen vorgetäuscht. deshalb sind eine kontinuierliche epidemiologische überwachung von infektionskrankheiten und konsequente einhaltung der meldepflicht nach § § 6 und 7 infektionsschutzgesetz (ifsg) sowie der übermittlung gemäß § 12 abs. 1 des ifsg sowie bei zoonosen gemäß der tierseuchenerregerverordnung notwendig [40, 41] . auf eu-ebene bestehen zwei frühwarnsysteme, das "early warning"-system für infektionskrankheiten und das bichat-system (= biological, chemical and atomic threats). darüber werden alle mitgliedsstaaten per e-mail unverzüglich bei besonderen ereignissen informiert [2, 6] . hinweise auf das vorkommen und die ausbreitung ungewöhnlicher krankheitsausbrüche bieten neben den melde-und frühwarnsystemen auch nationale und internationale programme, websites des robert koch-instituts und der who, syndromorientierte und laborsentinels sowie netzwerke zur überwachung (surveillance) ausgewählter infektionskrankheiten, z. b. influenza, invasive pneumokokken-infektionen oder gastroenteritiden [39] . damit lassen sich relativ zeitnah untypische anstiege in der morbidität oder mortalität, abweichungen in der normalen demografischen, geografischen und saisonalen verbreitung verfolgen. außerdem können auch lokale oder regionale häufungen (cluster) und geno-oder phänotypische besonderheiten endemischer, neu auftretender oder absichtlich freigesetzter erreger erfasst werden das setzt aber voraus, dass ärzte, tierärzte und konsultierte laboratorien entsprechend eingebunden, sensibilisiert und geschult sind, um derartige besondere ereignisse wahrzunehmen und frühzeitig zu alarmieren. allerdings zeigen die bisherigen erfahrungen, dass außergewöhnliche seuchengeschehen nicht immer als biologische anschläge erkannt werden. so wurde die nicht natürliche genese der salmonellen-enteritisepidemie 1984 in den usa nur durch die selbstanzeige eines mitglieds der rajneesh-sekte, und zwar erst ein jahr später, offenkundig. nicht immer ist damit zu rechnen, dass sich infektionen mit einem bestimmten b-agens in allen alters-, berufs-oder geschlechtsgruppen gleichermaßen klinisch manifestieren bzw. gleich schwer verlaufen. geschädigte dürften anfangs eine relativ unspezifische, zumeist influenza-ähnliche symptomatik oder syndrome üblicher endemischer infektionskrankheiten zeigen. das erschwert die rechtzeitige erkennung eines b-angriffs erheblich. hinzu kommt, dass krankheitsfälle bei der gegenwärtig hohen mobilität (ausnahme: zielgruppen in geschlossenen einrichtungen) an unterschiedlichen orten, zeitlich versetzt, vereinzelt oder gruppiert auftreten können. die schnelligkeit, mit der dann die richtige diagnose gestellt und eine geeignete therapie sowie präventive maßnahmen eingeleitet werden, beeinflusst das weitere schicksal der primär erkrankten und der noch gesunden exponierten sowie kontaktpersonen. dies gilt insbesondere, wenn es sich um eine gefährliche infektionskrankheit oder ein ereignis von möglicher internationaler tragweite für die öffentliche gesundheit, gemäß § 12 abs. (7 tab. 4) . nach einer mutmaßlichen aerogenen exposition sollten auch von ansteckungsverdächtigen (= noch "gesunde" b-exponierte und kontaktpersonen zu krankheitsverdächtigen und potenziell kontaminierten personen) baldmöglichst venenblut und nasen-/rachenabstriche zum nachweis biologischer agenzien gewonnen werden [4, 5, 11] . alle probenmaterialien sind unter einhaltung der gesetzlichen vorschriften zu verpacken und der untersuchungseinrichtung umgehend per kurier zuzustellen. der transport ist dem empfangenden labor rechtzeitig telefonisch anzukündigen. die mikrobiologische labordiagnostik kann mehrere tage erfordern. untersuchungsergebnisse würden jedoch bei fulminanten krankheitsverläufen zu spät vorliegen. sofern verfügbar, lässt sich zwar eine vorläufige diagnose mit modernen molekularbiologischen und immunologischen schnellmethoden stellen. allerdings ist die schnelldiagnostik nur für ein begrenztes spektrum von b-agenzien verfügbar und nicht routinemäßig etabliert, sondern auf wenige spezialisierte referenzlaboratorien beschränkt [4, 5] . diese sollten daher frühzeitig in das management biologischer schadensereignisse eingebunden werden. sofern es keinerlei drohungen oder hinweise bezüglich eines bevorstehenden oder stattgefundenen biologischen anschlags gibt, wird es bei einzelnen erkrankungsfällen oder einem krankheitsausbruch am anfang kaum einen verdacht auf einen b-anschlag und demzufolge auch keine schutzvorkehrungen geben. somit wird gerade das mit der sichtung, dekontamination, medizinischen notversorgung und dem transport von krankheitsverdächtigen betraute medizinische personal als kontaktpersonen besonders infektionsgefährdet sein. unter diesen "first respondern" kann es daher ebenfalls zu erkrankungen kommen, sofern sie "ungeschützt" eingesetzt würden. für sie sollten geeignete persönliche schutzausrüstungen (sog. infektionsschutz-sets) und gegebenenfalls mittel zur prä-oder postexpositionellen prophylaxe (pep) bereitgestellt werden [4, 27] . prinzipiell sind nach einem biologischen anschlag alle ansteckungsverdächtigen aktiv zu ermitteln, zu erfassen und zu sichten. bei bedarf sollten sie dekontaminiert werden und, sofern verfügbar, eine pep und medizinische beratung erhalten [14, 32] . eine behelfsmäßige (not-) dekontamination (wechsel der kleidung, duschen) dieses personenkreises sowie der krankheitsverdächtigen kann entweder sofort im biologischen wirkungsherd oder im verlaufe von 24 stunden auch noch im rahmen einer ambulanten oder stationären quarantäne bzw. isolierung erfolgen [11, 27] . krankheitsverdächtige (= b-geschädigte), die innerhalb von 24 stunden nach einer vermuteten b-exposition in stationären behandlungseinrichtungen eintreffen, sind ebenfalls vor der aufnahmen zu dekontaminieren. für einen massenanfall an b-geschädigten sollten gesonderte dekontaminationsbereiche im rahmen der krankenhausalarmplanung vorgesehen werden [4, 11] . eine sichtung ist sinnvoll, wenn man das schadenausmaß nicht voraussehen kann und bei begrenzten ressourcen möglichst vielen opfern helfen muss [11, 26, 29] . dabei sind vorrangig diejenigen patienten zu identifizieren, welche einer sofortigen isolierung und/ oder dringenden intensivbehandlung mit beatmung bedürfen. die sichtungskriterien sind, der lage angepasst, vom leitenden notarzt festzulegen. hier sollten auch kinderärzte eingebunden werden, da sich sowohl die pathophysiologie als auch die klinik und behandlung entsprechender krankheiten bei kindern wesentlich von der bei erwachsenen unterscheiden. frühzeitig ist eine falldefinition zu erstellen, um ersthelfern und ärzten das erkennen der jeweils vorliegenden krankheit und die differenzialdiagnostik zu erleichtern [41] . da unter umständen verschiedene b-agenzien gleichzeitig oder zeitlich versetzt und auf unterschiedliche weise (aerogen, alimentär) ausgebracht werden könnten, muss die sichtung auch auf verschiedene syndrome ausgerichtet sein (7 tab. 2) . die prozentualen anteile der erkrankten in den einzelnen schweregradgruppen hängen primär von der art und menge des b-agens, dem expositionsgrad und der empfänglichkeit sowie disposition der betroffenen ab. ansteckungsverdächtige, einschließlich ungeschütztes medizinisches personal, sind bis zum ausschluss einer gefährlichen infektionskrankheit möglichst außerhalb des biologischen wirkungsherdes unter quarantäne abzusondern und täglich medizinisch zu überwachen (mindestens zweimal täglich temperaturmessung und inspektion). bei erkrankung müssen sie als ansteckungsfähig betrachtet und sofort isoliert werden. alle krankheitsverdächtigen sollten im zuge der sichtung je nach schwere und syndrom in kohorten zusammengefasst werden [4, 11, 26] . solange der verdacht auf eine gefährliche infektionskrankheit besteht, sind sie räumlich isoliert unterzubringen und zu transportieren sowie unter barrierebedingungen zu behandeln (7 tab. 3) . sobald die diagnose feststeht, richtet sich die weitere unterbringung der patienten im krankenhaus primär nach ihrer ansteckungsfähigkeit und dem schweregrad der erkrankung (7 tab. 3 [4, 14, 15] . eine kalkulierte prä-oder post-expositionelle prophylaxe (pep) durch breitbandantibiotika und deren kombinationen sind bei einigen b-agenzien möglich und sollten für alle ansteckungsverdächtigen sowie bei kontaktpersonen zu krankheitsverdächtigen grundsätzlich vorgesehen werden [5, 11, 17, 34] . dabei müssen u. a. alter, bestimmte grundleiden, erworbene immunsuppression oder schwangerschaft, ähnlich wie im falle von pocken oder influenzapandemien, bei der auswahl der chemoprophylaxe und -therapie oder bei impfungen, berücksichtigt werden. stets sollte das risiko, an der krankheit zu sterben, gegen mögliche nebenwirkungen der arzneimittel sorgfältig abgewogen werden. zugelassene impfungen gibt es in der regel nicht (7 tab. 3) . manche impfstoffe sind zwar als sogenannte "investigational new drugs" in den usa erhältlich und z. synonym(e) "100-tage-husten". circa 10 tage . länger dauernder husten, anfallsartiger husten mit würgen oder erbrechen, inspiratorischer stridor, apnoe bei säuglingen, paroxysmale hustenattacken. die krankheit verläuft beim ungeimpften in drei phasen: die erreger haften und vermehren sich ausschließlich an den schleimhäuten der atemwege. immunantwort nach infektionskrankheit ca. 10 jahre. infektionen durch adenoviren, respiratory-syncytial virus, rhinoviren und andere erreger akuter respiratorischer infektionen sind bei jugendlichen und erwachsenen häufig von keuchhusten nicht zu unterscheiden. nasopharyngealabstrich. [4, 5] . weitere detailbeschreibungen zur taxonomie, siehe schlüsselliteratur [7] . kürzlich entdeckung eines neuen vogelvirus, genannt aviäres bornavirus (abv) [6] . könnte wegen der unterschiede zu bdv als subspezies eingestuft werden. [3] . die entdeckung, dass bdv genomanteile in das wirtserbgut eingebaut werden [5] , unterstreicht die risiken mentaler störungen bei persistent infizierten menschen [4] . humanes bdv ist erstmalig aus peripheren weißen blutzellen isoliert worden, d. h. es hat zielzellen außerhalb des gehirns [3] . virale rna konnte durch rt-pcr in hirnautopsie-proben verstorbener mit psychiatrischen vorerkrankungen amplifiziert [7] und virusantigene im liquor cerebrospinalis als zumindest transiente virusaktivität im gehirn (nur bei patienten mit major depression) nachgewiesen werden [3, 8] . antikörper sind im serum/plasma nachweisbar, allerdings (methodenabhängig) nicht zu jedem zeitpunkt der persistenten infektion. dies ist erklärbar mit der bildung von immunkomplexen, die im blut zirkulieren und als folge von antigenschüben entstehen. die antikörper sind bei mensch und tier vor allem gegen das n-und p-protein gerichtet und haben keine schutzwirkung [3] . neutralisierende antikörper (bisher nur bei tieren nachgewiesen) sind erheblich seltener [8] . sie erkennen vor allem das g-protein, teilweise allerdings auch das m-protein. die zelluläre immunantwort ist bisher nur gründlich im experimentellen tiermodell (ratte) untersucht [7] . immunpathologische ereignisse treten gegenüber den ätiopathogenetisch bedeutsamen balancestörungen im neurotransmitter-netzwerk in den hintergrund oder spielen zumindest keine initiale rolle bei verhaltensänderungen. erkrankungen des peripheren und zentralen nervensystems mit negativem bdv-blutbefund, z. b. enzephalitiden viraler genese sowie die frühstadien von nvcjd und möglicherweise auch die frühsymptomatik der alzheimer-krankheit; außerdem die borreliose-infektion (neuroborreliose), bei der ein heterogenes symptomenbild angenommen wird, das sich wenig mit der ausprägung einer (bdv-spezifischen) dysfunktion im limbischen system deckt. citratblutproben (ca.10 ml) sind optimal geeignet, vor allem im plasma, aber auch in leukozyten, infektionsparameter zu überwachen. beim tier kommt post mortem gehirnmaterial des limbischen systems hinzu. die labor-diagnostik der bdv-infektion wurde über jahrzehnte von der fluoreszenz-antikörper technik beherrscht. ein negativer befund schließt aber eine infektion nicht aus [3] . neu entdeckte laborparameter erlauben eine sichere diagnose [3] . benötigt werden eine oder mehrere citratblut-proben (10 ml), möglichst während akuter krankheitsepisoden entnommen, aus denen plasma und weiße blutzellen getrennt gewonnen werden. im plasma werden mit elisa-techniken, die auf spezifischen epitop-definierten monoklonalen antikörpern basieren [1, s30-31] zirkulierende bdv-spezifische immunkomplexe (cics) sowie virale proteine (plasma-antigen) und ggf. antikörper (3 teste) gemessen. bdv-cics sind die am häufigsten nachweisbaren infektionsmarker und eignen sich optimal für suchtests [3] . in den blutzellen können zeitweise ebenfalls virusproteine (intrazelluläres antigen) sowie virusnukleinsäure (mit nested rt-pcr) gefunden werden [2] . bei schweren psychiatrischen erkrankungen, aber auch bei normalen blut-(spender)proben mit hohen antigenwerten bei gleichzeitiger cic-präsenz kann der bdv-spezifische nukleinsäurenachweis direkt aus plasma (serum) gelingen [3] . die diagnostik der humanen bdv-infektion (die tier infektion einschließend) wird gegenwärtig nur von wenigen forschungslaboratorien im in-und ausland (italien, tschechoslowakei, ungarn, iran, china), eine aussagekräftige serologie unter einschluss von antigen und cics [3] , in deutschland nur von dem unten erwähnten referenzlabor angeboten. b. duttonii, b. hispanica, b. crocidurae, b. persica, b. caucasica, b. latyschewii, b. hermsii, b. turicatae, b. parkeri, b. mazzottii, b. graingeri, b. venezuelensis, b. burgdorferi sensu lato, b. burgdorferi sensu stricto, b. garinii, b. afzelii, b. spielmanii, b. lonestari, b. anserina filarienfieber. einige jahre (absterben der adulten würmer, mittlere lebenszeit 5 jahre). lymphknotenschwellung, fieber, schmerzen im befallenen bereich (meist arm oder bein, einseitig). akut auftretendes fieber > 38 °c, verbunden mit schmerzen und überwärmung bevorzugt im bereich der vergrößerten lymphknoten, rötung der lymphbahnen. insbesondere der femorale lymphknoten kann dabei abszedieren und nach außen durchbrechen. typisch sind bleibende große narben. die fieberattacken können mehrmals pro jahr auftreten und dauern mehrere tage, meist aber nicht länger als eine woche. bei brugia-infektionen verweilen die adulten im gegensatz zur lymphatischen filariose durch wuchereria bancrofti (7 wuchereria) nicht in der genitalregion, entsprechend beschränken sich lymphadenitis und lymphangitis auf die region der extremitäten (gelegentlich sind die mammae betroffen). die symptome entstehen durch das (natürliche oder durch medikamente induzierte) absterben von adulten würmern. der lokal in den lymphbahnen auftretende entzündungsreiz führt zur entzündung der lymphgefäße (lymphangitis) wie auch der zugehörigen lymphknoten (lymphadenitis). das absterben der würmer führt zu einer freisetzung sowohl von "klassischen" wurmantigenen, welche eine antikörper-abhängige zelluläre zytotoxizitätsantwort (adcc) mit beteiligung von eosinophilen und makrophagen induzieren, als auch von antigenen der wolbachia-endobakterien, die eine typische sofortreaktion (toll-like-rezeptor-vermittelt) gegen bakterien, ähnlich einer sepsis, hervorrufen. wichtige mediatoren sind hier tnf, il-6, il-1, und il-8. okkulte filariose, tropisches eosinophiles asthma, weingartner-syndrom. monate bis jahre nach infektion. asthmoide bronchitis. keine. hervorgerufen durch bck. pneumonie, exazerbationen, hämoptysen, sepsis, respiratorische insuffizienz. die symptome entsprechen einer chronischen pneu-monie mit rezidivierenden exazerbationen und verschlechterung der gesamtprognose. selten tritt das sog. "cepacia-syndrom" auf, das mit nekrotisierender pneumonie mit septischem verlauf, rapider verschlechterung der lungenfunktion und hoher mortalität einhergeht. cf-patienten mit einer b.-cenocepacia-infektion zeigen nach lungentransplantation eine signifikant schlechtere prognose. pneumonie durch andere cf-erreger, v. a. p. aeruginosa. hervorgerufen durch bck, b. gladioli (pneumonie, harnwegsinfekt, kathetersepsis, peritonitis) bzw. r. pickettii (meningitis, endokarditis, osteomyelitis, kathetersepsis). die klinische symptomatik manifestiert sich in abhängigkeit der lokalisation. infektionen durch andere nosokomiale erreger. hervorgerufen durch b. pseudomallei. gebogene, spiral-oder s-förmige gramnegative stäbchen, 0,2-0,9 μm dick, 0,5-5 μm lang, von älteren kulturen oder nach sauerstoffexposition auch kokkoid. aufgrund ihrer schlankheit sind sie in der gram-färbung häufig nur schwer zu erkennen. charakteristisch ist eine gute beweglichkeit (oft "windradartig"), welche vor allem bei phasenkontrastbetrachtung oder in der dunkelfeld-mikroskopie deutlich erkennbar wird. die beweglichkeit wird durch je eine uni-oder bipolare geißel vermittelt, welche aber auch fehlen kann. keine wirksame immunreaktion nach infektion. ausschlüsse von erkrankungen bakterieller, parasitärer oder viraler genese sind bei oberflächlichen candidosen notwendig, da die krankheitsbilder wenig spezifisch sind. die art der prädisposition oder grundkrankheit kann richtungsweisend sein. synonym(e) tiefe candidose, tertiäre peritonitis. unbekannt, da oft endogene infektion. peritonitis. peritonitis. meist tertiäre peritonitis nach darmoperation, perforation oder bei capd via besiedelte peritonealkatheter. keine wirksame immunreaktion nach infektion. andere, peritonitis verursachende erreger. unbekannt, da meist endogene infektion. fieber, uncharakteristisches krankheitsbild. sepsis mit möglicher absiedelung in auge (endophthalmitis, chorioretinitis), hirn (basale meningitis, meningitis mit intraparenchymalen abszessen, enzephalitis), knochen (osteomyelitis), leber (chronische entzündung mit multiplen abszessen), nieren (interstitielle nephritis), herz (endokarditis mit besiedelung der herzklappen, perikarditis). besiedelung von plastikimplantaten (katheter, herzklappen etc.) mit gefahr der dissemination. nach einschwemmung der pilze in die blutbahn absiedelung in allen organen möglich, insbesondere aber in nieren, gehirn, myokard, auge, milz und leber. im gewebe entstehen multiple mikroabszesse. granulozyten sind wichtige abwehrzellen, mitentscheidend für prävention der systemischen dissemination. mononukleäre phagozyten müssen aktiviert werden (ifnγ -hauptsächlich aus cd4+, aber auch cd8+ und nk-zellen), um phagozytierte candida-zellen abtöten zu können. reduktion der cd4+ zellen mit auftreten von soorösophagitis korreliert. antikörperproduktion bei immunkompetenten menschen vorhanden, unterscheidung zwischen schleimhautbesiedelung, infektion und dissemination kaum möglich, protektion durch antikörper fraglich. andere opportunistische infektionen. schlankes fusiformes oder fadenförmiges gramnegatives stäbchen, teilweise gebogen oder coccoid, gleitende (taumelnde) beweglichkeit. die komplette genomsequenz von c. canimorsus ist in genbank unter der accession-nr. cp001632.1 hinterlegt. langsames wachstum auf blut-oder kochblut-agar in anaerober oder mikroaerophiler atmosphäre (5-10 % co 2 ). nach 2-4 tagen kleine, flache, raue, teilweise gelblich pigmentierte kolonien, in den agar eingesunken, mit unregelmäßigem rand und schwärm zonen oder mit glattem rand und glatter oberfläche. c. ochraceae, c. gingivalis, c. sputigena sind mit parodontitis assoziiert. parodontitis. 7 parodontitis (aggregatibacter). 7 parodontitis (aggregatibacter). pathophysiologie 7 parodontitis (aggregatibacter). keine daten verfügbar. parodontitis durch andere erreger. blutkultur bei endokarditis, sepsis, osteomyelitis und anderen systemischen infektionen. liquor cerebrospi-nalis und blutkultur bei meningitis. gewebeprobe, punktat bzw. abstrich bei lokalen infektionen. mikroskopie: direkter nachweis des erregers im grampräparat. kultur stellt das routineverfahren im mikrobiologischen labor dar. capnocytophaga wächst auf blut-oder kochblut-agar, nicht aber auf mcconkey-agar. oxidase-und katalasereaktion variabel, indolnegativ, schwache fermentation von kohlenhydraten. molekularbiologische methoden zum nachweis und zur identifizierung der bakteriellen dna (nukleinsäureamplifikation, sequenzierung) finden in besonderen fällen anwendung. nachweis aus primär sterilen untersuchungsmaterialien wie blut, herzklappe, abszesspunktat spricht für die kausale rolle des erregers, während der nachweis aus mit normalflora besiedelten proben meist eine kolonisation anzeigt. in vitro häufig empfindlich gegen breitspektrum-cephalosporine, carbapeneme, fluorchinolone, chloramphenicol, erythromycin, clindamycin. teilweise resistent gegen cotrimoxazol und aminoglykoside, i. d. r. resistent gegen metronidazol und aztreonam. der erreger kommt weltweit vor. einige capnocytophaga arten gehören der physiologischen rachenflora des menschen an. c. canimorsus und c. cynodegmi besiedeln den rachenraum von hunden und katzen. immunsupprimierte, v. a. patienten mit granulozytopenie oder asplenie. ein erhöhtes risiko für endokarditis besteht bei vorschädigung der herzklappen, trägern künstlicher und biologischer herzklappen und conduits, nach shuntanlage sowie nach durchgemachter bakterieller endokarditis. die infektion ist i. d. r. endogen bei den spezies, die zur physiologischen standortflora des menschen gehören. andere arten können durch hund-bzw. katzenbiss oder speichel übertragen werden. bei vorgeschädigten herzklappen wird eine antibioti-sche endokarditisprophylaxe bei chirurgischen bzw. zahnärztlichen eingriffen empfohlen. keine daten verfügbar. keine. keine. cardiobacterium hominis, c. valvarum. genus cardiobacterium gehört der familie cardiobacteriaceae an. c. hominis gehört der hacek-gruppe an. schlankes, gramnegatives stäbchen mit grampositiv erscheinenden polkappen, pleomorph, einzeln, in paaren, ketten oder rosetten gelagert. unbeweglich. die sequenz des 16s rrna-gens ist in genbank unter der accession-nr. m35014 verfügbar. cardiobacterium wächst unter mikroaerophilen (5-10 % co 2 ) oder anaeroben bedingungen auf blutbzw. kochblutagar. nach 2-4 tagen kleine konvexe, runde kolonien, opaleszierend ohne oder mit leichter β-hämolyse, die später flach und trocken werden, netzartig konfluieren und in den agar einsinken. keine daten verfügbar. c. hominis ist meist mit endokarditis assoziiert, seltener mit meningitis oder anderen eitrigen infektionen. der erreger wurde ferner aus dentalen plaques und bei parodontitis isoliert. keine daten verfügbar. keine daten verfügbar. fieber und neu aufgetretenes herzgeräusch bei endokarditis. weitere symptome der endokarditis können eine splenomegalie, petechien, hämaturie und andere zeichen der embolisation sowie eine anämie sein. cardiobacterium ist bestandteil der schleimhautflora des menschen und kann nach hämatogener verbreitung infektionen der herzklappen, meningen, etc. verursachen. keine daten verfügbar. endokarditis durch andere erreger. blutkultur bei endokarditis und sepsis. liquor cerebrospinalis und blutkultur bei meningitis. gewebeprobe, punktat bzw. abstrich bei lokalen infektionen. mikroskopie: direkter nachweis des erregers im gram-präparat. kultur stellt das routineverfahren im mikrobiologischen labor dar. cardiobacterium wächst auf blut-oder kochblut-, nicht aber auf mc-conkey-agar und ist i. d. r. katalasenegativ und oxidasepositiv. indol wird produziert (teilweise nur schwach), glucose und andere kohlenhydrate werden fermentiert. molekularbiologische methoden zum nachweis und zur identifizierung der bakteriellen dna (nukleinsäureamplifikation, sequenzierung) finden in besonderen fällen anwendung. nachweis aus primär sterilen untersuchungsmaterialien wie blut, herzklappe, abszesspunktat spricht für die kausale rolle des erregers, während der nachweis aus mit normalflora besiedelten proben meist eine kolonisation anzeigt. normalerweise besteht gute in-vitro-empfindlichkeit gegen viele antibiotika. zur empirischen therapie der endokarditis werden cephalosporine der 3. generation (ceftriaxon, cefotaxim), z. t. in kombination mit aminoglykosiden empfohlen. resistenz β-laktamase produzierende stämme wurden beschrieben. der erreger kommt vermutlich weltweit vor. bestandteil der physiologischen standortflora des oberen respirationstrakts des menschen. ein erhöhtes risiko für endokarditis besteht bei vorschädigung der herzklappen, trägern künstlicher und biologischer herzklappen und conduits, nach shuntanlage sowie nach durchgemachter bakterieller endokarditis. die infektionen entstehen i. d. r. endogen, d. h. sie gehen von der körpereigenen normalflora aus. bei vorgeschädigten herzklappen wird eine antibiotische endokarditisprophylaxe bei chirurgischen bzw. zahnärztlichen eingriffen empfohlen. keine daten verfügbar. keine. keine. schlüsselliteratur han rueckert, 1996 ). an das 5´-terminale uracil der rna ist das kleine hydrophobe protein vpg (virus protein genome linked, 2,4 kda) kovalent gebunden. in der 5´-ntr (emcv 833 nukleotide und tmev 1064 nukleotide) befindet sich mit einer ausgeprägten sekundärstruktur der initiationsort der translation (internal ribosome entry site = ires) und bei emcv eine poly-cytosin-region (poly-c-tract; 80-250 cytosine). die 3´-ntr ist in unterschiedlicher länge polyadenyliert. während der proteinbiosynthese wird der kodierende bereich der polycistronischen mrna in ein polyprotein übersetzt, das im vergleich zu den enteroviren am n-terminus zusätzlich ein leader-protein aufweist. die region p1 enthält die kapsidproteine vp0 (vorläufer von vp4 und vp2), vp3 und vp1. die regionen p2 und p3 enthalten funktionelle proteine (u. a. 2a = protease, 3b = vpg, 3c = protease, 3d = rna-polymerase). die prozessierung der proteine wird durch 3 proteasen bewirkt. protease 2a (pfeil; nur zusammen mit 2b proteolytisch aktiv) setzt ein vorläuferprotein l-p1-2a frei. die protease 3c spaltet das vorläuferprotein an den l-p1 und p1-2a schnittstellen und setzt das vorläuferprotein p1 für die kapsidproteine frei. die protease 3c übernimmt auch die meisten übrigen proteolytischen spaltungen vor dem zusammenbau des virus (assembly). im viruskapsid wird nach aufnahme der viralen rna das vorläuferprotein vp0 in die kapsidproteine vp2 und vp4 gespalten, wobei für enteroviren eine beteiligung der rna postuliert wird. die enteroviren (poliovirus, coxsackieviren, echoviren und enteroviren 68-71) und die humanen rhinoviren haben eine gleiche genomorganisation, besitzen jedoch keine leader (l)-sequenz und können in der länge der kodierenden und nichtkodierenden bereiche der jeweiligen rnas voneinander abweichen (7 polioviren) . die akute virusvermehrung findet in den epithelzellen des darms statt. es wird davon ausgegangen, dass analog zur poliovirus-infektion (7 polioviren) das virus über eine virämie zum erfolgsorgan (z. b. zns) transportiert wird. der reproduktionsmechanismus von cardioviren ist erst ansatzweise aufgeklärt. da cardioviren in ihren strukturellen und funktionellen eigenschaften teilweise den enteroviren gleichen, ist für cardioviren eine vermehrungsstrategie wie bei enteroviren anzunehmen (7 polioviren keine. gurkenkernbandwurm. in der regel asymptomatischer verlauf, selten gastrointestinale symptome, perianaler juckreiz. synonym(e) larva migrans. unbekannt. abhängig von der lokalisation, schmerzhafte hautund bindehautschwellungen. abhängig von der lokalisation, entzündlicher tumor. der mensch ist fehlzwischenwirt. die larve wandert durch diverse organe des körpers und kann bis zu 30 cm lang werden. untersuchungsmaterial d. caninum: stuhlprobe; spirometra (sparganum): biopsiematerial. stuhlmikroskopie bzw. histologie. die proglottiden des gurkenkernbandwurms lassen sich leicht von anderen bandwürmern unterscheiden, da sie in der mitte breiter sind als an den schmalen enden, was ihnen das aussehen von gurkenkernen verleiht. spirometra: die spargana können bis zu 36 cm lang werden. einmalbehandlung mit praziquantel oder niclosamid. verbreitung d. caninum: weltweite verbreitung, seltene infektion des menschen. sparganose kommt in ost-und südostasien, selten in afrika, mittelamerika und südeuropa vor. katzen und wild lebende carnivore. kinder. akzidentieller verzehr von infizierten flöhen, unsauberes trinkwasser, verzehr von schlangen-oder froschfleich. infektion auch durch auflegen von rohem schlangen-oder froschfleisch auf wunden oder auge (tradionelle medizin in asien). regelmäßige entwurmung der hauskatzen und gele-gentliche behandlung mit insektiziden, um die ektoparasiten abzutöten. eine meldepflicht besteht nicht. referenzzentren / expertenlaboratorien 5 nationale referenzzentren gibt es nicht. als fachlich qualifiziert anzusehen sind sämtliche parasitolo gischen und tropenmedizinischen institutionen. chikungunya-virus wurde 1952 in tansania während eines ausbruchs mit fieber und gelenkschmerzen isoliert und beschrieben. der name "chikungunya" stammt aus der sprache der einheimischen bevölkerung (suaheli: "das was sich aufkrümmt", nach den durch die gelenkschmerzen bedingten körper-und gliedmaßenkrümmungen der patienten). chikungunya-fieber eine der wichtigsten arbovirus-erkrankungen und rangiert unter den drei wichtigsten sogenannten "emerging infections". in den großen epidemien der letzten jahre konnten bei älteren patienten und patienten mit grunderkrankungen vereinzelt tödliche verläufe beobachtet werden. atypische verläufe (enzephalitis, hepatitis) konnten keinen spezifischen risikogruppen zugeordnet werden. kleinkinder zeigen häufiger atypische verlaufsformen mit fieber und exanthem ohne die typischen gelenksbeschwerden. chikungunya-virus wird in der natur überwiegend durch stechmücken der gattung aedes übertragen. am sylvatischen zyklus sind unterschiedliche stechmücken-arten (u. a. aedes furcifer-taylori, aedes luteocephalus, aedes dalzieli u. a.), am urbanen zyklus sind hauptsächlich aedes aegypti und aedes albopictus beteiligt. bisher sind keine impfstoffe verfügbar. einzige möglichkeit der prävention ist damit die individuelle stechmücken-expositionsprophylaxe. ausbrüche mit chikungunya-fieber können im persönlichen umfeld durch entfernen aller für stechmücken-brutplätze geeigneten gegenstände eingedämmt werden. in einer ausbruchssituation können großflächige ausbringungen von insektiziden (larvizide, adultizide) eine epidemie kurzfristig eindämmen, jedoch erfahrungsgemäß nicht stoppen. der erregernachweis und der serologische nachweis einer akuten chikungunya-virusinfektion sind namentlich meldepflichtig gemäß § 7 ifsg unter der rubrik "virale hämorrhagische fieber -sonstige formen". da reinfektionen möglich und auch häufig sind, hinterlässt die primärinfektion offenbar keine protektive immunität. möglicherweise spielt eine überschießende immunantwort, die vor allem gegen das innerhalb der chlamydiaceae stark konservierte "heat shock protein 60 (chsp60)" gerichtet ist, eine pathogenetisch entscheidende rolle. zumindest bei affen führte die wiederholte experimentelle inokulation der konjunktiven mit chsp60 zu einem dem trachom sehr ähnlichen krankheitsbild. da chsp60 homologien zu humanen "heat shock" proteinen zeigt, wird auch ein autoimmungeschehen diskutiert. in den frühstadien der erkrankung (einschluss-) konjunktivitis durch die c.-trachomatis-serotypen d-k sowie durch viren. die infektionen werden durch befall der entsprechenden schleimhautepithelzellen mit c. trachomatis der serovare d-k ausgelöst. der großteil der infektionen verläuft insbesondere bei frauen oligosymptomatisch bzw. gänzlich asymptomatisch. als folge unerkannter infektionen können chronisch verläufe mit irreversiblen folgeschäden entstehen. die pathophysiologischen details sind dabei nicht vollständig aufgeklärt. offenbar führt jedoch die aufsteigende chlamydieninfektion der adnexe zu chronisch inflammatorischen prozessen mit fibrotischem umbau des gewebes und tubaler okklusion. persistente chlamydienformen mit modifizierter gen-und antigen-expression könnten dabei eine wichtige rolle spielen. die natürliche infektion verleiht nur eine partielle immunität. reinfektionen, insbesondere durch unterschiedliche serovare sind möglich. tierexperimentelle daten weisen daraufhin, dass die th-1 vermittelte zelluläre immunantwort kritisch für die erregererradikation ist. hohe titer c.-trachomatis-spezifischer antikörper korrelieren nicht mit der beseitigung der infektion sondern im gegenteil mit schweren folgeerkrankungen oder invasiven infektionen wie pelveoperitonitis, tubarinfertilität oder dem lgv. dabei werden auch antikörper gegen virulenz-assoziierte proteine induziert [9] . bei umschriebenen lokalen infektionen wie der urethritis oder konjunktivitis kommt es häufig zu keiner serologisch messbaren humoralen immunantwort. gonorrhoe sowie andere erreger der nicht-gonorrhoischen urethritis wie ureaplasma urealyticum, trichomonas vaginalis, mycoplasma genitalium und herpes simplex-virus. lgv. genitalulkus mit schmerzhafter lymphadenopathie, proktitis. im stadium i der infektion zeigt sich an der genitalschleimhaut oder angrenzenden haut eine primärläsi-on in form einer kleinen papel oder eines schmerzlosen herpetiformen ulkus. dabei kommt es häufig zur abheilung des ulcus ohne narbenbildung. in abhängigkeit der sexualpraktiken kann sich die primärläsion auch als proktitis oder pharyngitis klinisch manifestieren. im stadium ii unilaterale, manchmal beidseitige, in der regel inguinale schmerzhafte lymphadenopathie. im fortgeschrittenen verlauf einschmelzung der lymphknoten mit sogenannter bubobildung (fluktuierende lymphknoten) und fistelbildung. im stadium iii lymphabflussstörungen mit elephantiasis. die lokale symptomatik wird häufig von allgemeinsymptomen wie fieber, abgeschlagenheit und krankheitsgefühl begleitet. das lymphogranuloma venereum wird durch die c.trachomatis-serovare l1-l3 verursacht. initial werden die epithelzellen des genitaltraktes befallen. die eintrittspforte stellen kleinere hautabschilferungen oder verletzungen dar. die erreger gelangen in die drainierenden lymphknoten, wo sie in monozyten und lymphozyten replizieren und zur abszess-(bubo-)bildung führen. die rupturierung eingeschmolzener lymphknoten kann zur fistelbildung führen. bei unbehandelten entwickelt sich eine chronisch granulomatöse entzündung mit fibrotischem umbau und verlegung der lymphabflussgefäße. es kommt insbesondere ab dem zweiten erkrankungsstadium zu einer messbaren humoralen immunantwort. andere ulzerierende erkrankungen des genitaltraktes wie herpes genitales, syphilis, ulcus molle und granuloma inguinale. sinusitis, pharyngitis, bronchitis und pneumonie. twar-pneumonie. die exakte inkubationszeit ist unbekannt, vermutlich 1-3 wochen. typische leitsymptome für c.-pneumoniae-infektionen fehlen. für c.-pneumoniae-infektionen ist ein biphasischer verlauf beschrieben. unklar ist jedoch, wie häufig sich aus initial milden infektionen der oberen luftwege eine bronchitis oder eine pneumonie entwickelt und was die auslösenden faktoren dafür sind. eine ätiologische rolle von c. pneumoniae in der atherogenese wird seit 1988 diskutiert, als saikku und mitarbeiter aufgrund serologischer daten eine assoziation zwischen c. pneumoniae und der koronaren herzkrankheit sowie des akuten myokardinfarktes postulierten [24] . in den folgestudien wurden die erreger in atherosklerotischen läsionen von patienten mittels kultur, pcr, immunhistochemie und transmissions-elektronenmikroskopie detektiert. dabei wurde diskutiert, ob der erreger bzw. einzelne bestandteile (antigene, nukleinsäuren) durch das monozyten-makrophagensystem aus dem respirationstrakt in die gefäßwand gelangen. in-vitro-studien weisen darauf hin, dass die präsenz hochimmunogener chlamydien-antigene wie heat shock-protein 60 sowie das chlamydiale lps eine entscheidende rolle bei der induktion proinflammatorischer prozesse in der gefäßwand spielen könnten. die diskrepanz der publizierten daten, einschließlich der tierexperimentellen befunde sowie das scheitern groß angelegter therapiestudien [4] hat dazu geführt, dass die ätiologische relevanz des erregers bei der arteriosklerose zunehmend kritisch diskutiert wird [13] . die c.-pneumoniae-infektion induziert eine spezifische humorale und zelluläre immunantwort, die jedoch offenbar nicht vollständig vor reinfektionen schützt. frühestens 2 wochen nach primärinfektion finden sich spezifische igm-antikörper, frühestens 4 wochen nach primärinfektion spezifische igg-antikörper. tierexperimentelle daten weisen darauf hin, dass eine effektive th1-vermittelte immunantwort un-ter beteiligung von cd8-positiven zellen entscheidend für die erregerradikation ist. in frage kommen vor allem andere erreger atypischer pneumonien und des oberen respirationstraktes insbesondere respiratorische viren sowie mycoplasma pneumoniae, c. psittaci und legionella pneumophila. synonym(e) papageienkrankheit, ornithose. der begriff "ornithose" ist eigentlich die korrekte krankheitsbezeichnung, denn dadurch wird deutlich, dass nicht nur papageien, sondern viele unterschiedliche vogelarten als erregerreservoir in frage kommen. variabel, zwischen 1 und 4 wochen. initial grippeähnliche symptomatik die typischerweise in eine atypische pneumonie mündet. oft wird die infektion initial verkannt, da keine gezielte anamnese (vogelkontakt) erhoben wurde oder die infektion als systemische allgemeininfektion imponiert. hohes fieber, gliederschmerzen und starke kopfschmerzen finden sich in verlaufsformen mit und ohne respiratorische beteiligung. unproduktiver husten (im erkrankungsverlauf oft spät), dyspnoe sowie infitrate im röntgen-thorax weisen auf eine pneumonie hin, die auch einen karnifizierenden verlauf nehmen kann [7] . in der präantibiotischen ära verlief die psittakose oft tödlich. durchfall, übelkeit und erbrechen können den verdacht anfangs auf eine gastroenteritis und bei ikterus und hepatomegalie auf eine hepatitis lenken. eine kardiale beteiligung kann sich als perikarditis, myokarditis und kulturnegative endokarditis manifestieren. bei zns-beteiligung relativ häufig meningoenzephalitische symptomatik mit somnolenz und verwirrung. die nicht selten auftretenden exantheme können vielgestaltig sein, typischerweise kommt es zur ausbildung eines roseolenähnlichen makulo-papulären erythems. die psittakose oder ornithose wird durch c. psittaci (aviäre serovare ) verursacht. unklar ist, welche determinanten auf wirts-und erregerseite für den variablen klinischen verlauf verantwortlich sind. in-vitro-studien sowie die rasche systemische ausbreitung deuten darauf hin, dass c. psittaci sich auch in monozyten vermehren kann. bisher kaum charakterisiert. bei klinisch manifester c ornithose in der regel ausgeprägte humorale immunantwort gegen das chlamydien-lipopolysaccharid und weitere bisher schlecht charakterisierte antigene. bei atypischer pneumonie kommen vor allen dingen respiratorische viren, coxiella burnetii, legionella pneumophila, mycoplasma pneumoniae und c. pneumoniae differenzialdiagnostisch in frage. bei systemischem verlauf ohne lungenbeteiligung sind differenzialdiagnostisch die ursachen des fiebers unklarer genese zu bedenken. keine. unklar. spontanabort, unklares fieber und sepsis in der schwangerschaft. die infektion mit c. abortus in der schwangerschaft kann zunächst zu einer influenzaähnlichen symptomatik führen. schwere und lebensbedrohliche verläufe sind beschrieben worden, die mit einem multiorganversagen (niere, leber) und disseminierter intravasaler gerinnung einhergehen [29] . die erkrankung wird durch c. abortus verursacht, dem erreger einer insbesondere bei schafen und ziegen weit verbreiteten zoonose, die bei diesen tieren zum abort führen kann. offenbar besitzt c. abortus einen tropismus zu den plazentaepithelien. falls schwangere frauen z. b. in der landwirtschaft gegenüber c. abortus infizierten nutztierbeständen (v. a. schafen) exponiert sind, insbesondere aber wenn sie kontakt mit abortmaterial von infizierten tierbeständen, kann es aufgrund einer schweren plazentitis zum verlust des feten kommen. infektionen bei männern wurden bisher noch nicht beschrieben. es wird vermutet, dass eine th2-vermittelte zelluläre antwort bei der interferon-γ herunterreguliert ist, möglicherweise zur raschen ausbreitung des erregers beiträgt. differenzialdiagnose q-fieber. da es sich bei chlamydien um obligat intrazellulär re-plizierende bakterien handelt, ist bei der entnahme der untersuchungsproben grundsätzlich auf die gewinnung möglichst zellreicher materialien zu achten. in abhängigkeit der im untersuchenden labor eingesetzten verfahren zum direktnachweis der erreger ist die verwendung spezieller abstrichbestecke und transportmedien erforderlich. sofern der zellkulturelle nachweis angestrebt wird, muss das untersuchungsmaterial in einem speziellen chlamydien-transportmedium möglichst unverzüglich nach entnahme gekühlt ins labor gebracht werden. für den serologischen nachweis wird eine serummonovette entnommen. kommerziell verfügbare nukleinsäureamplifikationsverfahren erlauben den nachweis von urogenitalen c. trachomatis-infektionen mit hoher zuverlässigkeit. diese nachweisverfahren können jedoch noch bis zu 3 wochen nach abgeschlossener therapie positiv bleiben. bei der interpretation serologischer verfahren ist auf speziesspezifität des testes zu achten. hohe c.trachomatis-spezifische antikörper finden sich bei aufsteigenden c.-trachomatis-infektionen des kleinen beckens und beim lymphogranuloma venereum. der direkte nachweis von c. pneumoniae mittels kultur oder pcr weist auf eine aktive behandlungsbedürftige infektion hin. allerdings ist zu berücksichtigen, dass selbst in speziallaboratorien mit einer beträchlichen variabilität insbesondere der pcr-befunde zu rechnen ist. ein igm-titer von 1 ≥ 16 und/oder ein igg-titer von 1 ≥ 512 gelten neben einem 4fachen titeranstieg in gepaarten serumproben als diagnostisch hinweisend auf eine aktive infektion. allerdings sollte ein igg-titer von 1 ≥ 512 in einer einzelnen serumprobe mit vorsicht interpretiert werden, da erhöhte igg-titer u. u. mehrere jahre auch ohne klinisch-apparente symptomatik persistieren können. igg-titer zwischen 1:16 und 1:256 weisen auf eine zurückliegende infektion hin. der diagnostische nutzen von spezifischen iga-antikörpern ist unklar [5] . es gibt ganz offensichtlich einen mangel an standardisierten und evaluierten kommerziell verfügbaren nachweisverfahren, was die beurteilung der klinischen relevanz von c. pneumoniae erheblich erschwert [15] . die verfügbaren serologischen nachweisverfahren erlauben keinen nachweis speziesspezifischer antikörper gegen c. psittaci und c. abortus [6] . daher ist zu bedenken, dass ein erhöhter kbr-titer auch durch eine infektion mit einer anderen chlamydienspezies zustande kommen kann. beim nachweis hoch positiver (häufig mit anderen chlamydienspezies kreuzreagierender) antikörper ist bei entsprechender klinischer symptomatik die gezielte erhebung der anamnese hinsichtlich einer möglichen exposition des patienten gegenüber den natürlichen wirten diagnostisch wegweisend. gramnegative peritrich begeißelte stäbchenbakterien. genom vollständig sequenziert, siehe auch www.ncbi. nlm.nih.gov. fakultativ anaerob, wachstum innerhalb von 24 stunden auf nährmedien. nicht bekannt. nicht bekannt. der jeweiligen erkrankung entsprechend. der jeweiligen erkrankung entsprechend. fakultativ pathogener opportunistischer erreger, infektion bei standortwechsel. nicht bekannt. abgrenzung von anderen krankheitsauslösenden erregern. durch einschwemmen von citrobacter sp. in die blutbahn kann es zur sepsis und extrem selten zur endokarditis kommen. nicht bekannt. nicht bekannt. fieber. fieber. mehrfach-resistente stämme werden beobachtet. die therapie entsprechend dem antibiogramm wird empfohlen. wirksam sind häufig ureidopenicilline, cefotaxim, cefmenoxim, ceftriaxon, carbapeneme, chinolone und aminoglykoside. nicht bekannt. eine epidemische ausbreitung von citrobacter sp. im rahmen von nosokomialen infektionen wurde bisher nicht beobachtet. angehörige des genus citrobacter finden sich in faezes von menschen und tieren, in wasser, abwasser und abfall. säuglinge, immunsupprimierte, karzinom-und transplantationspatienten. citrobacter spp. werden durch direkten kontakt oder auch indirekt über gegenstände oder lebensmittel übertragen. prävention / impfstoffe 7 fakultativ pathogene e. coli (escherichia coli). nicht erforderlich. § 23 ifsg abs. 1: multiresistenz ist zu dokumentieren. heidi schütt-gerowitt synonym(e) gasbranderreger, welch-fraenkel-"bazillus" (c. perfringens = c. welchii), prarauschbrand-"bazillus" (= c. septicum). clostridium perfringens, c. septicum, c. histolyticum, c. novyi sowie einige andere clostridien-arten. nekrotisierende fasziitis, streptokokken-fasziitis, infektionen mit anderen gasbildenden bakterien. darmbrand, pig-bel, nekrotisierende enterokolitis. 1-2 wochen nach dem ernährungsfehler (7 unten). bauchschmerzen, erbrechen, blutiger durchfall. unter der einwirkung des von c. perfringens gebildeten β-toxins kommt es zu nekrosen im jejunum. jedoch ist nicht der erreger allein die ursache für die erkrankung: das krankheitsbild wird bzw. wurde vor allem bei kindern mit fehlernährung (verzehr von großen mengen fleisch) beobachtet. andere durchfallerreger. gastroenteritis. grampositive kurze stäbchen mit subterminaler spore. die für den menschen pathogenen toxintypen sind chromosomal kodiert, während andere (tierpathogene) typen phagenkodiert sind. c. botulinum vermehrt sich nur in einer streng anaeroben atmosphäre, z. b. in luftdicht verpackten lebensmitteln. der pathogenitätsfaktor von c. botulinum ist ein exotoxin (neurotoxin), von dem es acht verschiedene typen gibt. die typen a, b, e und f sind für den menschen pathogen. botulismus: lebensmittelbedingt, wundbotulismus, säuglingsbotulismus, botulismus "ungeklärter ursache" synonym(e) wurstvergiftung. nach oraler oder aerogener aufnahme des toxins treten die symptome nach 8 stunden bis einigen tagen auf, bei den übrigen formen lässt sich keine inkubationszeit angeben. schlaffe lähmungen, insbesondere doppelbilder sehen. beim nicht eruierbar, meist abhängig von der antibiotikagabe bzw. der gabe anderer medikamente (sieherisikopatienten) blutig-schleimige durchfälle mit fieber und krampfartigen bauchschmerzen. die cdad tritt meist einige tage -eventuell aber auch wochen -nach dem beginn einer antibiotischen oder antineoplastischen therapie auf. symptome sind leichte bis schwere z. t. blutig-schleimige durchfälle mit fieber und krampfartigen bauchschmerzen. die diagnose der schwersten verlaufsform, der pseudomembranösen kolitis, wird aufgrund des endoskopischen bildes gestellt, wobei eine ödematös veränderte darmschleimhaut mit charakteristischen gelblichweißen plaques und pseudomembranen zu sehen ist. als weitere komplikation kann ein toxisches megakolon auftreten und es kann zu einer darmperforation kommen. c. difficile kann in geringer zahl im darm des menschen vorkommen und aufgrund seiner resistenzeigenschaften selektioniert werden, wenn durch eine antibiotische therapie die übrige darmflora zerstört wird. im prinzip können alle antibiotika sowie auch einige andere medikamente dieses krankheitsbild auslösen, am häufigsten wird es aber bei bzw. nach der gabe von ampicillin, amoxicillin, clindamycin und cephalosporinen beobachtet. in seltenen fällen kann auch die stagnation des darminhaltes (z. b. postope-rativ) die ursache für die vermehrung von c. difficile sein. nicht bekannt. differenzialdiagnostisch kommen die enteropathogenen erreger in betracht. stuhlproben, die möglichst innerhalb von 2 stunden zum labor gelangen sollten. das meist verwendete diagnostische verfahren ist der nachweis der toxine a und b mittels enzymimmunoassay. wegen der bedeutung des toxins b sollten tests, die allein das toxin a nachweisen, nicht verwendet werden. außerdem kann c. difficile auf spezialkulturmedien selektiv angezüchtet werden. dann muss im zweiten schritt der nachgewiesene stamm auf sein toxinbildungsvermögen untersucht werden. ein alleiniger kultureller nachweis von c. difficile lässt keine aussage über die ätiologische bedeutung zu. die kulturelle untersuchung ist aber notwendig, um aussagen über die besiedelungshäufigkeit machen zu können. ferner existiert ein schnelltest, der auf dem nachweis der glutamatdehydrogenase beruht. dieser test hat einen hohen negativen prädiktiven wert, aber seine spezifität ist so gering, dass im positiven fall immer weitere tests erforderlich sind. außerdem kann auch eine pcr zum nachweis von c. difficile durchgeführt werden. in schweren fällen soll immer ein kultureller nachweis erfolgen, um ggf. eine stammtypisierung durchführen zu können. die befunde müssen immer in verbindung mit der klinischen symptomatik gesehen werden, da c. difficile und seine toxine auch bei klinisch gesunden menschen vorhanden sein können. eine therapieüberwachung mittels der laboruntersuchungen hat keinen aussagewert. eventuell kann allein das absetzen einer noch laufenden antibiotischen therapie die symptome stoppen; bei schwer verlaufenden fällen muss metronidazol oder ggf. vancomycin oral gegeben werden. die resistenzeigenschaften von c. difficile sind die ursache für seine selektionierung und somit für die erkrankung überhaupt. resistenzen gegen die zur the-rapie eingesetzten antibiotika metronidazol und vancomycin sind bisher nicht beobachtet worden. verbreitung c. difficile kommt in geringer zahl im darm von menschen und tieren vor. 3-15 % der erwachsenen, jedoch 30-50 % der säuglinge sind mit c. difficile besiedelt. die sporen von c. difficile sind auch im boden und in gewässern weltweit verbreitet. wirtsbereich / reservoir 7 verbreitung. vor allem ältere patienten und kinder, die eine antibiotische oder antineoplastische therapie bekommen, sowie seltener patienten, bei denen es postoperativ oder aus anderen gründen zu einer stagnation des darminhaltes gekommen ist. auch für patienten, die mit protonenpumenhemmern, h2-rezeptor-antagonisten oder nicht steroidalen antiphlogistika behandelt werden, besteht ein erhöhtes risiko einer cdad. es gibt jedoch auch fälle, bei denen eine cdad bei jungen menschen ohne risikofaktoren auftritt. die untersuchung der hierfür verantwortlichen clostridien-stämme führte zu der entdeckung der toxin-überproduzenten (z. b. ribotyp 027). in der regel handelt es sich um ein endogenes geschehen, jedoch kann eine nosokomiale übertragung im sinne einer cross infection erfolgen, z. b. durch die hände des pflegepersonals. ausbrüche auf stationen kommen immer wieder vor, so dass c. difficile jetzt als einer der wichtigsten verursacher nosokomial erworbener diarrhoen anzusehen ist. dazu trägt bei, dass die sporen von c. difficile sehr resistent gegen umwelteinflüsse und auch gegen desinfektionsmittel sind. um dieser schweren erkrankung vorzubeugen, ist es wichtig, dass antibiotika nur unter strenger indikationsstellung gegeben werden. eine impfung gibt es nicht. patienten, bei denen c.-difficile-toxine nachgewiesen sind, müssen isoliert werden, um eine weitere übertragung zu vermeiden, da bei ihnen c. difficile in großer zahl im darm vorhanden ist. heidi schütt-gerowitt erreger synonym(e) wundstarrkrampf-erreger. gegen das tetanustoxin bzw. das zur impfung verwendete tetanus-toxoid entsteht eine antitoxische humorale immunität. differenzialdiagnostisch ist an eine strychninvergiftung zu denken. serum, wundabstrich. tetanus ist vor allem eine klinische diagnose. durch laboruntersuchungen kann man ihn weder beweisen noch ausschließen. prinzipiell kann ein toxinnachweis im tierversuch (maus) durchgeführt werden, er bleibt aber meist erfolglos. die kulturelle anzüchtung des erregers ist mit den üblichen methoden der anaerobier-diagnostik möglich, sie hat aber praktisch keine bedeutung. aufgrund der typischen symptomatik. wichtig ist eine sorgfältige wund"toilette". auch wenn bereits klinischer verdacht auf tetanus besteht, muss die eintrittspforte exzidiert werden, um eine weitere vermehrung und die dabei erfolgende toxinbildung zu unterbinden. die gabe von antibiotika hat keinen einfluss auf den krankheitsverlauf. wenn klinische symptome vorliegen, die für tetanus sprechen, sind in der regel intensivmedizinische maßnahmen erforderlich. wenn der impfstatus unklar ist oder die letzte impfung mehr als 10 jahre zurückliegt, muss auch bei kleinen verletzungen eine passive immunisierung mit tetanus-antitoxin i. m. und gleichzeitig die aktive immunisierung durchgeführt werden ("simultanimpfung"). die antitoxingabe muss so früh wie möglich erfolgen, da die antikörper nur das noch nicht gebundene toxin neutralisieren können. wenn die letzte tetanusimpfung zwischen 5 und 10 jahre zurückliegt, muss bei einer verletzung eine aktive impfung gegeben werden. keine maßnahmen sind erforderlich, wenn die letzte tetanusimpfung weniger als 5 jahre zurückliegt. die sporen von c. tetani haben eine hohe umweltresistenz. verbreitung c. tetani kommt im darm von tieren, selten auch des menschen vor und ist weltweit im erdboden und im staub verbreitet. die erkrankung ist in deutschland wegen des guten impfstatus der bevölkerung selten, in den entwicklungsländern asiens und afrikas kommt sie jedoch immer noch häufig vor. in diesen ländern sterben jährlich auch heute noch tausende neugeborene an nabelschnur-tetanus. darm von tieren, staub. risikogruppen sind alle nicht oder unvollständig geimpften bzw. nicht adäquat "aufgefrischten" menschen. die sporen von c. tetani dringen über wunden -auch bagatellverletzungen -in den körper ein. durch tierbisse kann die vegetative form übertragen werden. die wichtigste präventionsmaßnahme ist die aktive immunisierung. sie soll nach der grundimmunisierung, die in der regel bereits im ersten lebensjahr erfolgt, alle 10 jahre aufgefrischt werden. bei adäquat durchgeführter impfung sind andere präventionsmaßnahmen nicht notwendig. die aktive immunisierung erfolgt mit dem tetanus-toxoid. nicht erforderlich. keine meldepflicht nach ifsg. unbekannt. trockene und wüstenartige gebiete im südwesten der usa, mittel-und südamerika. mensch, wild-und nutztiere in endemiegebieten; seeottern. landarbeiter, archäologen und touristen in endemiegebieten, immunsupprimierte. die infektiösen arthrokonidien werden mit der stauboder sandhaltigen luft übertragen, insbesondere bei sandstürmen. sehr selten erfolgt die übertragung durch mikrotraumata direkt in das gewebe. keine übertragung von mensch zu mensch. eine spezifische prophylaxe ist nicht möglich. expositionsprophylaxe für immunsupprimierte. nationale surveillance-programme erfassen infektionen in endemiegebieten. keine. lokale ausbildung eines granuloms, das viele eosinophile leukozyten enthält. basidiobolomykose, zygomykose, sarkom. schleimhautbiopsie der nase oder subkutane gewebebiopsie. im gegensatz zur basidiobolomykose sind überwiegend gesunde männliche erwachsene betroffen. es sind keine prädisponierenden grunderkrankungen oder beruflichen risikofaktoren bekannt. es wird angenommen, dass inhalierte sporen von conidiobolus spp. die traumatisch veränderte nasenschleimhaut penetrieren. keine daten verfügbar. die erkrankung ist nicht von mensch zu mensch übertragbar. ausbrüche kommen nicht vor. nach dem infektionsschutzgesetz besteht für die conidiobolomykose in deutschland keine meldepflicht. es besteht kein lang andauernder immunologischer schutz vor coronaviren nach infektion. durch coronavirus nl 63. new-haven-coronavirus. ähnlich wie bei hmpv 3-6 tage, zumeist zunächst mit beteiligung der oberen atemwege. keine. keine. nach einer inkubationszeit von 2-5 tagen erkranken ca. 20 % der nicht immunen infizierten personen (maximale variabilität: 1-10 tage). tonsillitis oder pharyngitis mit pseudomembranen. coxsackieviren werden wie die anderen enteroviren hauptsächlich fäkal-oral übertragen (7 polioviren das zur infektion führende verbreitungsstadium des parasiten ist die oozyste. dieses sphärische dauerstadium hat einen durchmesser von etwa 8 bis 10 μm und ist von einer gegen umwelteinflüsse widerstandsfähigen, derben hülle umschlossen. die oozysten enthalten zwei sporozysten mit je zwei sporozoiten, deren größe etwa 9 x 1,5 μm beträgt. die oozysten sind desinfektionsmittel-unempfindlich. der monoxene entwicklungszyklus gleicht dem von cryptosporidium parvum. die entwicklung findet nach oraler aufnahme der oozysten und freisetzung der sporozoiten intrazellulär im dünndarm statt. die genauen abläufe beim menschen sind jedoch noch nicht vollständig aufgeklärt. menschen in jedem alter sind gefährdet und die infektionen sind weltweit verbreitet. hohe pathogenität besteht bei immunkompromittierten oder geschwächten personen. die virulenz ist beträchtlich und der von kryptosporidien vergleichbar. cyclosporidiose, aids-related diarrhea, traveller's disease. die inkubationszeit beträgt 2 bis 7 tage (in ausnahmefällen bis zu 18 tagen). drei-bis viermal täglich wässrige durchfälle, übelkeit, anorexie, appetitlosigkeit, gewichtsverlust, magenkrämpfe, abdominale schmerzen, blähungen, muskelschmerzen, müdigkeit, schwäche, fieber und schüttelfrost, elektrolytverlust. histopathologisch werden mäßige bis massive erytheme bei infektionen mit c. cayetanensis im distalen duodenum beobachtet. weiterhin treten reaktive hyperämie mit vaskulärer dilatation, zottenkapillarkongestion, asexuelle und sexuelle stadien enthaltende parasitophore vakuolen im darmepithel, kryptenhyperplasie sowie partielle villusatrophie auf. in elektronenmikroskopischen aufnahmen sind intrazelluläre sporozoitenähnliche partikel sichtbar. eine neuinfektion ist trotz durchlaufener krankheit möglich. aus patientenseren wurden cyclospora-spezifische antikörper isoliert, jedoch sind die vorgänge der immunantwort auf cyclospora noch nicht vollständig geklärt und ob sich eine immunität entwickelt, ist fraglich. differenzialdiagnostisch ist die cyclosporiasis von anderen erkrankungen durch die parasiten isospora, microspora, giardia lamblia und cryptosporidium spp. zu unterscheiden. enteritiden durch bakterien und viren sind abzugrenzen. die oozysten von c. cayetanensis sind deutlich größer (faktor 2) als die der kryptosporidien (5-6 μm) und kleiner als die von isospora belli (20-33 x 10-19 μm). außerdem enthalten die oozysten von isospora acht sporozoiten. die infektiösen stadien der mikrosporidien sind einzellige kleine sporen, die einen polfaden aufweisen. darstellung der oozysten mittels mikroskopischer untersuchung bei 400facher vergrößerung nach säurefester färbung (z. b. modifizierte ziehl-neelsen-färbung). in saf-oder formalin-fixierten stuhlproben sind die oozysten leicht zu übersehen. cyclospora-oozysten leuchten unter uv-licht blau (autofluoreszenz). wegen der unregelmäßigen ausscheidung der oozysten ist eine mehrfachuntersuchung des stuhls notwendig. sensitivität und spezifität der pcr liegen bislang unter denen des goldstandards (mikroskopische untersuchung von stuhlproben). mehrfache stuhluntersuchungen sind unerlässlich. bereits der positive nachweis von wenigen oozysten stellt eine indikation zur therapie dar. eine routinemäßige stuhluntersuchung beinhaltet nicht obligatorisch den nachweis von cyclospora-oozysten, es sei denn, dies wird ausdrücklich angefordert. bei den meisten ärzten ist allerdings die cyclosporiasis unbekannt. immunsupprimierte patienten (hiv/aids, nach transplantation oder chemotherapie) und kleinkinder. die diagnosestellung ist bei erstgenanntem personenkreis von besonderer bedeutung, da mit einem schwereren, verlängerten und möglicherweise extraintestinalen krankheitsverlauf gerechnet werden muss. zudem treten häufig rezidive auf. bei immunkompetenten ist die erkrankung in der regel selbstlimitierend. die übertragung findet durch die orale aufnahme der oozysten in kontaminierter nahrung (wasser oder gemüse) statt. ein impfstoff existiert derzeit nicht. reisende sollten die präventionsmaßnahmen für sauberes trinkwasser berücksichtigen. keine. die cmv-infektion zeichnet einen breiten zelltropismus aus, der epithel-, endothel-, glatte muskelzellen, fibroblasten und myeolmonozytäre zellen (monozyten, makrophagen, granulozyten, cd34 + -vorläuferzellen, megakaryozyten) einschließt. in vitro vermehrt sich hcmv in fibroblastenkulturen besonders effizient. hierbei tritt ein zytopathischer effekt (cpe) und plaquebildung auf. der replikationszyklus beansprucht mindestens 3 tage. dabei durchläuft hcmv wie alle herpesviren drei replikationsphasen ("immediate early", "early" und "late"). bei der primärinfektion des patienten kann keine präzise inkubationszeit angegeben werden. das auftreten von klinischen symptomen und der beginn der virusausscheidung können zwischen wenigen wochen und monaten liegen. wie alle herpesviren kann hcmv in vivo eine latente, nicht produktive infektionsform mit episomaler lokalisation der viralen dna etablieren, aus der die produktive virusvermehrung reaktiviert werden kann. das intakte immunsystem kann die cmv-replikation beenden, das virus jedoch nicht aus dem organismus eliminieren. als latenzort von cmv werden unterschiedliche zelltypen diskutiert, u. a. monozytäre vorläufer-und endothelzellen. exogene oder endokrine faktoren, die zur reaktivierung der cmv-replikation führen, sind bisher schlecht definiert (z. b. stress). immundefizienz begünstigt das ausmaß der reaktivierten infektion. diese führt zur virusausscheidung über körpersekrete. vermehrt findet man episoden der virusausscheidung während der schwangerschaft und stillperiode. für das verständnis der cmv-infektion ist eine unterscheidung der cmv-erkrankung mit vielfältigen klinischen manifestationsmöglichkeiten einerseits von der cmv-infektion mit virusausscheidung bei fehlender symptomatik andererseits notwendig. ausmaß und verlauf der aktiven cmv-infektion sind in hohem maße vom immunstatus des patienten bestimmt. dabei kommt der zellulären immunität die entscheidende rolle zu. antikörper haben eine unterstützende funktion. primär asymptomatische infektionen können bei ungenügender immunologischer kontrolle im weiteren verlauf zu allgemeinen und organspezifischen symptomen führen. hierbei findet sich eine starke virusreplikation und virämie. in produktiv infizierten zellen wirkt die cmv-infektion lytisch. histologisch resultieren veränderungen einzelner zellen (z. b. "eulenaugenzellen") und von zellverbänden (z. b. endothelschäden, ulzera der gastrointestinalen mucosa, retinits etc.). multiple genetische funktionen befähigen das virus, sich der immunkontrolle partiell zu entziehen. so ist das virus in der lage, die mhc-vermittelte antigenpräsentation zu kontrollieren und damit der t-zell-kontrolle entgegenzuwirken. ebenso attenuiert cmv die erkennung infizierter zellen durch natürliche killerzellen (nk). schließlich unterbricht das virus zahlreiche intrazelluläre signalkaskaden und so die wirkung von interferonen und antiviralen zytokinen. die virale immunevasion kann durch spezifische funktionen des immunsystems kompensiert werden. dies setzt aber eine intakte zelluläre immunität voraus. die antigenität ist stabil. sub-und serotypen werden nicht unterschieden. mithilfe von molekulargenetischen analysen lassen sich cmv-isolate genotypisch unterscheiden. die klinische bedeutung der genetischen variabilität ist bislang unklar. lymphadenopathie, hepatosplenomegalie, fieber, lymphozytose. die cmv-primärinfektion verläuft in der mehrzahl der fälle asymptomatisch. auch bei immunkompetenten personen können jedoch uncharakteristische symptome auftreten: fieber, lymphknotenschwellungen, hepatosplenomegalie, lymphozytose. ca. 20 % aller fälle mit infektiöser mononukleose sind durch cmv verursacht. in seltenen fällen kann die cmv-primärinfektion durch komplikationen wie ikterus, myokarditis, interstitielle pneumonie, thrombozytopenie oder ein guillain-barré-syndrom in erscheinung treten. vermutlich sind eine ausgeprägte aktivierung des immunsystems und die lyse virusinfizierter zellen für das fieber, die lymphadenopathie und die gewebsentzündung verantwortlich. es finden sich aktivierte t-lymphozyten, cmv-spezifisches igm und iga sowie die igg-serokonversion. infektiöse mononukleose durch ebv, maligne erkrankungen mit b-symptomatik. cmv-infektion der lunge. die symptome entwickeln sich in abhängigkeit von der immundefizienz unterschiedlich schnell, in der regel innerhalb weniger tage. dyspnoe, röntgenologisch interstitielle pneumonie. husten, fieber, tachypnoe. gefährdet sind insbesondere patienten nach knochenmark-oder stammzelltransplantation. es besteht eine massive virusreplikation und inflammation in der lunge mit nachfolgender hypoxie. diskutiert wird ein pathophysiologischer zusammenhang mit der graft-versus-host-disease. die antivirale immunantwort in der lunge ist ungenügend. interstitielle pneumonien anderer ursache. cmv-netzhautentzündung. die symptome entwickeln sich in abhängigkeit von der immundefizienz und netzhautlokalisation innerhalb von tagen bis wochen. visusverlust, sehstörungen, erblindung. funduskopisch imponieren weiße exsudative und schließlich nekrotische netzhautbezirke (cotton wool spots). eine papillitis ist ebenfalls möglich. dazu können perivaskuläre hämorrhagien treten. die cmv-infektion des auges ist schmerzlos und kann bei peripherer netzhautlokalisation längere zeit unbemerkt bleiben. die cmv-replikation in der retina führt zu entzündung, schwellung und einblutung. ohne frühzeitige antivirale behandlung tritt innerhalb von monaten die erblindung ein. die cmv-retinits tritt am häufigsten bei hiv-patienten mit fortgeschrittenem cd4-t-zellverlust (< 100 zellen/μl) ein. risiko, ausmaß und prognose hängen vom immunstatus ab. retinitis bei toxoplasmose, hsv, vzv, bartonellose, candidose. synonym(e) cmv-infektion des gastrointestinaltraktes. die symptome entwickeln sich in abhängigkeit von der immundefizienz unterschiedlich schnell, in der regel innerhalb weniger tage. diarrhoe, schmerzen, schluckbeschwerden. bei colitis meist wässrige, gelegentlich auch blutige durchfälle und fieber. möglich sind cmv-infektionen im rektum und proktitis. bei ösophagitis schmerzhafte schluckbeschwerden. wird in der regel nur bei immundefizienten patienten beobachtet. die cmv-replikation in der epithelschicht von speiseröhre bzw. darm führt zu zunächst flachen defekten der mucosa, aus denen sich tiefere, meist scharfbegrenzte ulzerationen mit pseudomembranen entwickeln. die zelluläre immunantwort in speiseröhre bzw. darm ist ungenügend. in der regel sind die patienten virämisch. enterokolitis durch cryptosporidien, mikrosporidien, mykobakterien, adenovirus. angeborene cmv-infektion. in abhängigkeit vom schweregrad zeigen sich die schäden bereits in utero. entwickeln sich ausschließlich schäden des zns, zeigen sich diese erst im laufe des ersten lebensjahres. innenohrschwerhörigkeit, hepatosplenomegalie, blutbildveränderungen. hepatosplenomegalie, intra-oder extrahepatische gallengangsatresie, chorioretinitis, mikrozephalie, enzephalitis (mit oder ohne periventrikuläre verkalkungen), hydrozephalus, sprachstörungen, krampfanfälle, retinitis, thrombozytopenie, anämie und selten auch kardiovaskuläre defekte. oligosymptomatische formen mit passagerer viszeraler symptomatik sind häufiger als das vollbild. fetopathien sind häufiger als embryopathien, das risiko einer schwerwiegenden infektion ist in der ersten schwangerschaftshälfte erhöht. die spätschäden sind in form von geistigen und körperlichen entwicklungsrückständen, intelligenzdefiziten, taubheit und sprachstörungen erheblich. die diaplazentare transmission von der schwangeren auf den embryo/fetus kann bei primärer wie reaktivierter cmv-infektion der schwangeren erfolgen. allerdings sind die klinischen folgen für das kind bei einer primärinfektion sehr viel schwerwiegender. die organschäden (z. b. innenohr, leber, retina, knochenmark) werden durch die zytopathogenen effekte des virus und eine mangelnde immunkontrolle der virusreplikation erklärt. infolge der immunologischen unreife des feten ist die zelluläre immunantwort ungenügend. cmv-igm-antikörper sind nur in einem teil der fälle nachweisbar. kongenitale und perinatale infektionen anderer ursa-che (z. b. toxoplasmose, röteln), gendefekte, stoffwechselerkrankungen. serum, blut-leukozyten, plasma, trachealsekret, urin, liquor, stuhl, fruchtwasser, nabelschnurblut, augenkammerwasser, gewebebiopsien. der direkte virusnachweis erfolgt am schnellsten und empfindlichsten durch pcr. quantifizierende nachweisverfahren eignen sich auch zur therapiesteuerung. die virusanzucht gelingt mittels humaner fibroblastenkulturen. der antigenämietest wird mit monoklonalen antikörpern gegen das intranukleäre pp65-antigen durchgeführt. indirekter virusnachweis durch den nachweis cmvspezifischer igg-bzw. igm-antikörper mittels elisa oder ift. mithilfe der aviditätsbestimmung cmvspezifischer igg-antikörper kann der infektionszeitpunkt frischer infektionen näher eingegrenzt werden. western blot und neutralisationstest werden nicht routinemäßig durchgeführt. bei verdacht auf resistenzentwicklung gegen ganciclovir und foscarnet genotypische analyse (sequenzierung) der gene ul97 bzw. ul54. positive cmv-dna-befunde belegen eine aktive infektion, der cmv-dna-nachweis im blut bzw. in blutzellen eine virämie. der nachweis von igm-antikörpern deutet auf eine primäre oder reaktivierte infektion. er ist bei immundefizienten patienten und neugeborenen häufig falsch negativ. ganciclovir, foscarnet und cidofovir werden bei organ-oder lebensbedrohlichen infektionen intravenös verabreicht, ihre grundsätzliche wirksamkeit ist in studien belegt. valganciclovir wird oral eingesetzt. die auswahl der eingesetzten medikamente wird durch die teilweise erhebliche toxizität der mittel bestimmt. die wirksamkeit von cmv-hyperimmun globulin ist bei spezifischen indikationen nachgewiesen. resistenzbildung gegen die genannten medikamente tritt gelegentlich auf. verbreitung cmv ist weltweit verbreitet. mensch. die durchseuchung in deutschland beträgt bei erwachsenen in abhängigkeit von der sozialen herkunft im durchschnitt 50 %. in asien, afrika und südamerika ist die durchseuchung deutlich höher. bei spezifischen bevölkerungs-und patientengruppen wurde eine durchseuchung von > 90 % ermittelt (z. b. aids-patienten, homosexuelle männer). das virus wird bei engen zwischenmenschlichen kontakten über speichel, genitalsekrete und muttermilch übertragen. es bestehen infektionsgipfel im kleinkindesalter und in der adoleszenz. iatrogene übertragungswege sind die organtransplantation und die bluttransfusion, sofern die weißen blutzellen nicht entfernt werden. in deutschland wird etwa 1 % der neugeborenen mit einer kongenitalen cmv-infektion geboren. 10-15 % dieser kinder entwickeln eine dauerhafte schädigung. wirksame impfstoffe sind nicht verfügbar. die immunprophylaxe kongenitaler cmv-erkrankungen durch cmv-hyperimmunglobulinpräparate in der schwangerschaft scheint wirksam, muss aber durch weitere klinische studien bestätigt werden. bei immundefizienten transplantationspatienten hat sich eine befristete chemoprophylaxe mit valganciclovir oder aciclovir bewährt. seronegative schwangere mit haushaltskontakten zu kleinkindern, die cmv-ausscheider sind, können mithilfe spezifischer hygienemaßnahmen eine wirksame reduktion des infektionsrisikos erreichen. frühgeborene cmv-seropositiver mütter sollten virusinaktivierte muttermilch erhalten. besteht nicht. der befall mit apathogenen amöben wird nicht therapiert. über resistenzen gibt es demzufolge keine berichte. darmamöben kommen weltweit vor. ihre häufigkeit korreliert mit den lebensmittel-und wasserhygienischen bedingungen. während die meisten apathogenen darmamöben im menschlichen intestinaltrakt vorkommen, ist e. gingivalis im mund zu finden. e. moshkovskii wurde auch aus limnischen sedimenten und klärschlamm isoliert. von e. gingivalis ist bekannt, dass auch primaten, hunde und katzen befallen werden können. iodamoeba bütschlii wurde ebenfalls bei anderen primaten sowie bei schweinen gefunden. e. histolytica/dispar konnte bei hunden, schweinen sowie affen nachgewiesen werden. aufgrund der oft schlechteren hygienischen verhältnisse besteht insbesondere bei reisen in entwicklungsländer oder länder mit niedrigem hygienischen standard eine erhöhtes infektionsrisiko. die infektionsfähigen zysten werden in der regel fäkal-oral übertragen. fliegende und kriechende arthropoden können die zysten taktil (mechanisch) verbreiten (z. b. anheftung an den tarsen). praktisch alle in diesem kapitel behandelten amöbenzysten werden durch die aufnahme kontaminierten wassers oder den verzehr kontaminierter lebensmittel übertragen. die prävention besteht in der persönlichen hygiene zur wirksamen unterbrechung der infektkette. insbesondere die strikte einhaltung der maßnahmen zur nahrungshygiene bietet in ländern mit niedrigem hygienestandard einen gewissen schutz. es besteht keine meldepflicht. referenzzentren / expertenlaboratorien 5 als fachlich qualifizierte ansprechstellen gelten prakt isch d alle parasitologischen und tropenmedizi nischen institutionen. andere infektiöse und nicht infektiöse ursachen einer chronisch intermittierenden diarrhoe. zum nachweis intestinaler flagellaten ist nativer (unfixierter) oder fixierter stuhl in geeigneten transport-. weltweit. bei den meisten darmflagellaten ist der mensch das einzige bekannte reservoir. d. fragilis wurde auch bei affen gefunden, chilomastix mesnili bei affen und schweinen. bei reisen in entwicklungsländer oder in länder mit niedrigen hygienischen standards besteht ein erhöhtes infektionsrisiko. immunkompromittierte menschen sind prädisponiert. die prävention von darmflagellaten-infektionen besteht in der persönlichen hygiene zur wirksamen unterbrechung der infektkette. insbesondere die strikte einhaltung der maßnahmen zur nahrungshygiene bietet in ländern mit niedrigem hygienestandard einen gewissen schutz. zur eliminierung sowie zur prävention der trichomons-tenax-besiedlung genügt eine verbesserung der mundhygiene. es besteht keine meldepflicht. referenzzentren / expertenlaboratorien 5 als fachlich qualifizierte ansprechstellen gelten alle parasitologischen und tropenmedizinischen institutionen. der ausdruck "dengue" entstand aus dem versuch, die phrase "ki denga pepo" (kommt aus dem swahili und bedeutet so viel wie "krampfähnliche anfälle verursacht durch einen bösen geist") in die spanische sprache zu integrieren. ausbrüche von dengue-fieber wurden erstmals 1779 in batavia (heute jakarta) und kairo registriert. danach traten epidemien in intervallen von 10-30 jahren in tropischen und subtropischen gebieten auf. erst 1944 konnte albert sabin dengueviren von soldaten isolieren, die in kalkutta, auf neuguinea und auf hawaii erkrankt waren. dengueviren gleichen morphologisch dem gelbfiebervirus. die sequenzierung einer region des hüllprotein-gens hat gezeigt, dass die serotypen denv 1-4 weiter in genotypen unterteilt werden können. die genome von denv 1-4 sind ähnlich wie dasjenige des gelbfiebervirus organisiert. genbank-nr.: m23027 (denv 1), m19197 (denv 2), a34774 (denv 3), m14931 (denv 4). nach der übertragung durch einen infizierten moskito vermehrt sich das virus in den regionalen lymphknoten und verbreitet sich über lymphe und blut. als weitere replikationsorte werden endothelzellen und zellen im knochenmark diskutiert. das virus konnte auch aus leber, lunge, nieren, lymphknoten, gehirn und gastrointestinaltrakt isoliert werden. die vermehrung im retikuloendothelialen system und der haut führt schließlich zur virämie. außerdem infizieren dengueviren dendritische zellen des immunsystems, welche die angeborene und adaptive immunantwort überbrücken. alle 4 serotypen verursachen identische krankheitsbilder. die meisten infektionen mit dengueviren verlaufen jedoch asymptomatisch. synonym(e) "breakbone fever". die inkubationszeit beträgt 4-7 tage (in einzelfällen bis zu 14 tage). das klassische dengue-fieber beginnt mit plötzlichem fieberanstieg, schweren kopfschmerzen (typischerweise frontal lokalisiert), schwindel und erbrechen. charakteristischerweise treten muskelschmerzen, knochenschmerzen ("breakbone fever") und arthralgien auf. besonders das rückgrat ist davon betroffen. vor der rekonvaleszenzphase tritt in 50 % der fälle ein diffus verteiltes, makulopapuläres exanthem auf. es dauert 2 bis 4 tage und kann von pruritus und schälen der haut begleitet sein. trotz der schweren klinischen symptomatik und zum teil langwieriger rekonvaleszenz ist die prognose günstig. dem immunsystem, wird eine wichtige rolle bei der entstehung von symptomen zugeschrieben (immunpathogenese). nach infektion mit einem serotyp gibt es nur kurzzeitig eine gruppenspezifische immunität, langfristig bleibt nur ein typenspezifischer immunschutz. eine sichere klinische diagnose der apparenten infektionen mit dengueviren ist oft nicht möglich. das klassische dengue-fieber unterscheidet sich kaum von anderen erkrankungen, die ebenfalls mit plötzlichem fieber einhergehen. allerdings sollten schwere muskel-und knochenschmerzen an dengueviren denken lassen. infektionen mit anderen arboviren (z. b. west-nil-virus, rift-valley-fieber-virus und sandmückenfiebervirus) sowie malaria und leptospirose sind auszuschließen. inkubationszeit dhf/dss beginnt 4-7 tage nach auftreten der ersten krankheitsphase. die erste krankheitsphase verläuft ähnlich wie beim klassischen dengue-fieber. danach kommt es zu einer kurzen remission. bauchschmerzen, erbrechen, unruhe, bewusstseinsstörungen oder ein plötzlicher wechsel von fieber zur hypothermie können den be-ginn des dhf ankündigen. schließlich tritt eine plötzliche verschlechterung auf, die durch blutdruckabfall und kreislaufkollaps gekennzeichnet ist. in haut und schleimhäuten zeigen sich petechien (thrombozytenzahl < 10 5 /mm 3 ). gastrointestinale blutungen und hämorrhagische pneumonien können folgen. bei sehr ungünstigem verlauf des dhf kommt es zum dss mit meist letalem ausgang. das auftreten von dhf wird u. a. durch zwei wirtsfaktoren begünstig: alter < 15 jahre und frühere infektion mit dengueviren. über 90 % der patienten mit dhf hatten bereits eine frühere denguevirus-infektion mit einem anderen serotyp. bei etwa 1-2 % aller sekundärinfektionen tritt ein dhf auf. die erkrankten personen sind in der regel nicht älter als 15 jahre. beim dhf und seiner schwersten komplikation, dem dss, spielt das so genannte "immunoenhancement" eine rolle. dabei binden nichtneutralisierende antikörper, die sich nach einer früheren infektion mit einem anderen serotyp gebildet haben, an dengueviren, ohne diese unschädlich zu machen. die so entstandenen immunkomplexe binden schließlich über den fc-teil der igg-antikörper an fc-rezeptoren (insbesondere an fcγri wegen dessen hoher avidität) auf monozyten/makrophagen und werden dann in das zellinnere aufgenommen. über diesen mechanismus können dengueviren leichter in mononukleäre zellen eindringen. daraus resultiert im infizierten organismus eine größere anzahl von infizierten zellen, die eine größere menge an viruspartikel produzieren. daher ist die zirkulation von mehreren verschiedenen serotypen in einem endemiegebiet eine wesentliche voraussetzung für das auftreten des dhf/dss. das dhf ist kaum von anderen ursachen des hämorrhagischen fiebers abgrenzbar. thrombozytopenie und die zeichen einer leichten verbrauchskoagulopathie deuten in richtung dengueviren. serum, plasma. wichtigste nachweismethode ist die rt-pcr. im amplifizierten material kann dann mit hilfe der sequenzierung der subtyp bestimmt werden. die virusisolierung ist schwierig. zu diesem zweck werden am besten moskito-zelllinien inokuliert. etwa 2-3 tage später kann das virus nach anfärbung mit spezifischen monoklonalen antikörpern in der immunfluoreszenz identifiziert werden. zum nachweis von virusspezifi-schen antikörpern werden kbr, hht und nt eingesetzt. elisa-verfahren zum nachweis von virusspezifischen igm-antikörpern sind erhältlich, werden aber erst 4-5 tage nach beginn der symptome positiv. bei der interpretation der serologie sind mögliche kreuzreaktionen mit anderen flaviviren (u. a. auch bei impfung gegen gelbfieber oder japanische enzephalitis) zu berücksichtigen. igm-antikörper können bis zu 3 monaten nach infektion persistieren. die reiseanamnese ist sehr wichtig, da aufgrund der inkubationszeit eine denguevirus-infektion unwahrscheinlich wird, wenn die symptome mehr als 2 wochen nach verlassen eines endemiegebiets für dengueviren auftreten. fieber, welches länger als 10 tage persistiert, ist in der regel nicht auf eine denguevirus-infektion zurückzuführen. die einzigen vertebraten, die als wirte für dengueviren fungieren, sind der mensch und mehrere arten von asiatischen und afrikanischen primaten. babymäuse können nur sehr schwer infiziert werden. dengueviren werden hauptsächlich durch den stich des moskitos aedes aegypti auf den menschen übertragen. dieser ist bei tage aktiv und hält sich in der nähe von menschlichen behausungen auf. andere, für die infektion von menschen wichtige vektoren sindbesonders in asien und ozeanien -aedes albopictus, aedes polynesiensis und aedes scutellaris. als infektionsquelle für moskitos fungieren menschen in der virämischen phase. nach der aufnahme von infiziertem blut muss sich das virus noch im moskito 1-2 wochen vermehren, bis der vektor die infektion an andere menschen weitergeben kann. ähnlich wie beim gelbfiebervirus existieren im wesentlichen zwei verschiedene übertragungszyklen: ein silvatischer und ein urbaner. im unterschied zum gelbfiebervirus ist jedoch bei den dengueviren der urbane zyklus für epidemien und endemien am wichtigsten. eine vakzine ist zum jetzigen zeitpunkt noch nicht verfügbar. daher ist das vermeiden von moskitostichen (adäquate kleidung, gebrauch von moskito-repellentien und moskitonetzen) hier besonders wichtig. gegenwärtig ist der vielversprechendste impfstoffkandidat eine rekombinante lebendvakzine, die auf dem erfolgreichen 17d-impfstoff gegen gelbfieber basiert und immunogene proteine der dengueviren enthält. sie wird zurzeit klinisch getestet. die einzige mögliche kontrolle besteht gegenwärtig in der elimination von aedes-brutstätten in der nähe von menschlichen siedlungen. endemische länder sollten ein labor-gestütztes überwachungssystem aufbauen, welches eine zumindest grobe vorhersage der epidemiologischen situation liefert. nach § 6 des infektionsschutzgesetzes (ifsg) ist vom feststellenden arzt bei krankheitsverdacht, erkrankung sowie tod an virusbedingtem hämorrhagischen fieber der patient namentlich dem gesundheitsamt zu melden (unverzüglich, spätestens innerhalb von 24 stunden). außerdem ist nach § 7 jeder direkte oder indirekte (serologische) nachweis von dengueviren durch das labor dann namentlich zu melden, wenn er auf eine akute infektion hinweist. diese meldungen werden entsprechend § 11 über die zuständigen landesbehörden an das robert-koch-institut übermittelt. zusätzlich ist das auftreten einer erkrankung auch nach § 12 ifsg übermittlungspflichtig. dies bedeutet, dass -zusätzlich zum übermittlungsweg nach § 6 und 7 -bestätigte hämorrhagische verläufe von denguevirus-infektionen vom gesundheitsamt aus unverzüglich an die zuständige oberste gesundheitsbehörde und von dort unverzüglich an das robert koch-institut zu übermitteln sind, welches dann die informationen an die who weitergibt. weiterführende informationen zum ifsg und zur falldefinition für gesundheitsämter sind auf der unten aufgeführten web-adresse des robert-koch-instituts zu finden. differenzialdiagnostisch sind eine opisthorchose sowie gallenwegerkrankungen anderer genese zu bedenken. stuhl. die labordiagnose stützt sich auf den mikroskopischen nachweis der mit dem stuhl ausgeschiedenen dunkelbraunen, ovalen und gedeckelten eier von 38-45 × 22-30 μm größe. der positive befund aufgrund einer einzigen stuhluntersuchung ist noch nicht beweisend für einen tatsächlichen befall mit d. dendriticum (ausschluss eines pseudoparasitismus bei darmpassage durch verzehr befallener schafleber). eine gezielte therapie ist unbekannt; möglicherweise kommen praziquantel, albendazol oder triclabendazol in frage. die dicrocoeliose ist als zoonose weltweit, vor allem aber in eurasien und nordafrika verbreitet. in europa sind vor allem die kalkhaltigen mittelgebirge und das alpenvorland betroffen. lanzettegel sind in erster linie parasiten der wiederkäuer und anderer herbivorer säugetiere. der mensch stellt nur einen akzidentiellen endwirt dar. die dicrocoeliose wird nur selten beim menschen beobachtet. gehäuft tritt sie bei hirten und feldarbeitern auf, einzelfälle bei hiv-infizierten und bei einem patienten mit morbus crohn unter immunsuppressiver therapie sind in der literatur beschrieben. eine übertragung auf den menschen kann akzidentiell durch die orale aufnahme infizierter ameisen zustande kommen, die sich an grashalmen und anderen pflanzen befinden. eine dicrocoelium-infektion ist allein dadurch vermeidbar, dass keine an gräsern oder vegetabilien befindlichen ameisen verschluckt werden. eine meldepflicht nach dem infektionsschutzgesetz besteht nicht. referenzzentren, expertenlaboratorien 5 offizielle referenzzentren existieren nicht; als fachlich qualifiziert anzusehen sind parasitologische und tropenmedizinische institutionen. es besteht keine meldepflicht. referenzzentren / expertenlaboratorien qualifiziert anzusehen sind sämtliche para sitologischen und tropenmedizinischen insti tu tionen. 1-7, 9, 11-21, 24-27 und 29-33) aseptische 9, 11, [13] [14] [15] [16] [17] [18] [19] [20] [21] 25, 27, 30, 31 paralyse (selten) 4, 6, 9, 11, 30 wahrscheinlich 1, 7, 13, 14, 16, 18, 31 enzephalitis, ataxie oder guillain-barré syndrom 2, 6, 9, 19 wahrscheinlich 3, 4, 7, 11, 14, 18 exantheme 2, 4, 6, 9, 11, 16, 18 wahrscheinlich 1, 3, 5, 7, 12, 14, 19, 20 respirationstraktinfekte (u. a. sommergrippe) 4, 9, 11, 20 zum routinenachweis von echoviren und parechoviren wird wie bei allen anderen enteroviren rachenabstrich und stuhl verwendet (7 polioviren und coxsackieviren) . in abhängigkeit der organmanifestation eignen sich zusätzlich konjunktival-, rektal-und andere abstriche, rachenspülwasser, nasensekret, urin, liquor, biopsie-oder autopsiematerialien von herz und/oder gehirn. eine aktive immunisierung gegen echoviren und parechoviren ist nicht verfügbar. nosokomiale echovirus-und parechovirus-infektionen können von klinischem personal durch vernachlässigung der üblichen hygiene übertragen werden. wegen der fulminanten verläufe sind infektionen mit echoviren wie auch mit coxsackieviren auf neugeborenenstationen besonders gefürchtet. wesentliche präventionsmaßnahme ist die fachgerechte windelentsorgung und kittelwechsel. gegebenenfalls ist eine räumliche trennung der infizierten patienten vorzunehmen. die aktuellen maßnahmen zur hygienischen händedesinfektion sind unbedingt einzuhalten. es besteht keine meldepflicht. gramnegative stäbchenbakterien, beweglich durch peritriche begeißelung. genom vollständig sequenziert, siehe auch www.ncbi. nlm.nih.gov. fakultativ anaerob, wachstum innerhalb von 24 stunden. endotoxin. nicht bekannt. dem krankheitsbild entsprechend. dem krankheitsbild entsprechend. fakultativ pathogener opportunistischer erreger, infektion bei standortwechsel. opsonisierung durch antikörper, phagozytose. ausschluss anderer infektionserreger. nicht bekannt. fieber. fieber. erregernachweis ausschlaggebend. fluorochinolon, ampicillin, cefalosporine, aminoglykoside. weltweit, gastroenteritis in tropischen und subtropischen klimazonen, häufig einhergehend mit dem nachweis von entamoeba histolytica. nachweis in faeces vom menschen, haustieren, ratten, fröschen, vögeln, fischen. immunsupprimierte und abwehrgeschwächte patienten, reisende in warme klimazonen. endogen, schmierinfektion. siehe obligat pathogene e.-coli-stämme und fakultativ pathogene e.-coli-stämme. 7 escherichia coli. nicht erforderlich. aufgrund der an schildzecken gebundenen übertragungsweise sind ausbrüche nicht zu erwarten. es besteht keine meldepflicht. brouqui p et al (2004) kleine, regelmäßige, gramnegative stäbchen. manche isolate zeigen eine gleitende (taumelnde) beweglichkeit, während andere unbeweglich sind. genom die komplette sequenz des 16s rrna-gens steht in genbank unter der accession-nr. m22512 zur verfügung. anzucht in mikroaerophiler atmosphäre (5-10 % co 2 ) auf blut-oder kochblutagar. nach 2-3 tagen sehr kleine (1 mm), weißlich-grau oder gelblich gefärbte kolonien, die in den nährboden eindringen (korrodieren), nach dem wegwischen einen abdruck hinterlassen und von einem saum umgeben sind. daneben gibt es nicht korrodierende stämme. gewebeprobe, punktat bzw. abstrich bei lokalen infektionen. blutkultur bei endokarditis, sepsis, osteomyelitis und anderen systemischen infektionen. liquor cerebrospinalis und blutkultur bei meningitis. mikroskopie: direkter nachweis des erregers im grampräparat. kultur stellt das routineverfahren im mikrobiologischen labor dar. e. corrodens wächst auf blut-oder kochblut-, nicht aber auf mcconkey-agar und ist oxidase-positiv und katalase-negativ. kohlenhydrate werden nicht fermentativ abgebaut. e. corrodens kommt häufig in mischkulturen mit fakultativ oder obligat anaeroben erregern vor. nachweis der bakteriellen dna mittels nukleinsäureamplifikation findet in besonderen fällen anwendung. nachweis aus primär sterilen untersuchungsmaterialien wie blut, herzklappe, abszesspunktat spricht für die kausale rolle des erregers, während der nachweis aus mit normalflora besiedelten proben meist eine kolonisation anzeigt. therapeutische maßnahmen e. corrodens ist in vitro empfindlich gegen penicillin, aminopicilline, acylureidopenicilline, cephalosporine der 2. und 3. generation, carbapeneme, fluorochinolone und tetrazykline. die in-vivo-wirksamkeit von aminopenicillinen, auch in kombination mit β-laktamaseinhibitoren, ist jedoch umstritten. resistenz besteht häufig gegen cephalosporine der 1. gerneration, erythromycin, clindamycin und metronidazol. der erreger kommt vermutlich weltweit vor. wirtsbereich / reservoir e. corrodens ist physiologischer bestandteil der rachenflora und kommt vermutlich auch auf anderen schleimhäuten des menschen vor. personen mit menschenbissverletzungen, z. b. nach faustschlag (clenched fist injury). der erreger kann durch bissverletzung übertragen werden. endogene infektionen sind auch möglich. keine daten verfügbar. keine daten verfügbar. keine. web-adressen schlüsselliteratur paul k, patel ss (2001) zeitweise eingestellt werden. mit der entdeckung des (mittlerweile in einigen ländern verbotenen) ddt (dichlor-diphenyl-trichlor-methylmethan) 1939 wurde die bekämpfung von ektoparasiten revolutioniert. immer mehr ektoparasiten wurden in den letzten jahrzehnten als überträger von z. t. unbekannten oder wieder aufkommenden ("emerging" und "reemerging") infektionserregern erkannt. die morphologie ist sehr unterschiedlich, da unter die ektoparasiten sowohl spinnentiere (zecken und milben) als auch insekten (insecta, hexapoda) gestellt werden. zecken sind bis zu 2 cm große, dorsoventral abgeplattete ektoparasiten (familienzuordnung abhängig von dem vorhandensein oder fehlen eines rückenschilds: schildzecken bzw. lederzecken/weichzecken), die am ersten beinpaar ein spezielles sinnesorgan, das haller'sche organ, zum aufspüren von wirten besitzen. der körper des weibchens schwillt beim blutsaugen enorm an. die larven der zecken und milben besitzen sechs beine, die nymphen und adulten tiere acht. flöhe sind 2-4 mm große, meist sprunggewaltige, lateral abgeplattete, flügellose insekten mit arttypischen beborstungen. stechmücken sind zarte, langbeinige, fliegende nematocera mit langen stechborsten, deren genital-und fühlermorphologie auskunft über geschlecht und artzugehörigkeit gibt. sie besitzen ein flügelpaar, halteren und fadenförmige antennen. bettwanzen sind bis max. 1 cm große, dorsoventral abgeflachte, rotbraune, behaarte insekten, deren rudimentäre vorderflügel schuppenförmig sind und denen hinterflügel fehlen. raubwanzen sind mit bis zu 3 cm deutlich größer (7 trypanosoma cruzi). der entwicklungszyklus ist temperaturabhängig. die vermehrung erfolgt durch kopulation von männchen und weibchen. die dorsoventral abgeplatteten zecken machen ebenso wie die läuse, milben und wanzen eine hemimetabole entwicklung durch. stechmücken und flöhe durchlaufen eine holometabole entwicklung. aus dem ei schlüpft eine larve, die sich zur nymphe weiterentwickelt und nach häutungen zur imago heranreift. zur eireifung benötigen die ektoparasiten eine blutmahlzeit. die reaktion der menschlichen haut auf die speichelinjektion der ektoparasiten ist wahrscheinlich auf die wirkung von eiweißfraktionen, die als antikoagulans, haemolysin oder als nervengift fungieren, zurückzuführen. bei zecken kann es durch neurotoxisch wirkende substanzen im zeckenspeichel zur zeckenparalyse kommen, die sogar letal ausgehen kann. durch toxisch wirkende speicheldrüsensekrete können auch hämorrhagien und ödeme an den einstichstellen oder systemische reaktionen wie fieber, lähmungen oder krämpfe ausgelöst werden. darüber hinaus führt die allergische reaktion auf die in die stichwunde abgegebenen antigene zu klinischen manifestationen. insekten-, zecken-, wanzen-, floh-, milbenstich synonym(e) arthropodenstich, stich von blutsaugern, bei trombiculiden: trombidiose. rötung, schmerzen, juckreiz, v. a. an der einstichstelle. innerhalb weniger minuten bildet sich um die (arthropoden-) stichstelle ein unterschiedlich weit ausgedehntes erythem (hautrötung) aus, das eine zentrale quaddel aufweisen kann und mehr oder weniger stark juckt (kutane sofortreaktion ige, typ 1). nach einiger zeit (kutane spätreaktion, t-zell-typ, meist innerhalb 24 stunden) entsteht daraus vielfach eine papel (knötchen), die ebenfalls juckt und lange erhalten bleiben kann. selten treten vesikel oder pusteln auf. symptome wie juckreiz, schmerzen, überempfindlichkeitsreaktionen, entzündungen etc. können durch den stich von dipteren verursacht werden. schmerzen und juckreiz treten nicht beim stich selbst, sondern erst später auf. insbesondere bei lederzecken (z. b. taubenzecke) ist eine deutliche punktförmige hämorrhagie (blutaustritt) an der einstichstelle beschrieben. die durch zeckenbefall direkt hervorgerufenen symptome, wie z. b. ödeme oder fieber und krämpfe, werden hauptsächlich durch das toxisch wirkende speicheldrüsensekret ausgelöst. zeckenparalysen beginnen mit symptomen wie schwindel, kopfschmerzen, erbrechen und allgemeiner muskelschwäche, bis hin zur aufsteigenden lähmung, sprachstörungen und tod durch lähmung der atemmuskulatur. raubwanzen stechen oft ins gesicht ("kissing bug") aufgrund der systemischen reaktion auf ihre stiche sind todesfälle beschrieben. bei längerer exposition gegenüber bettwanzen können asthma, schockzustände und beeinträchtigung des sehvermögens folgen. charakteristisch für flohstiche ist, dass sie perlschnurartig in reihe angeordnet sind (probestiche). zudem tritt hierbei zunächst eine punktförmige hämorrhagie mit hellrotem hof direkt am floheinstich (roseola pilicosa) auf, der nach einiger zeit einem blauroten kleinen fleck (purpura pulicosa) weicht. bei jedem erneuten einstich eines flohs jucken auch die früheren stichstellen (repetieren). bei milbenstichen kann es (durch den als allergen wirkenden speichel) zur sogenannten gebüschkrätze (scrub itch) sowie zur bildung einer schwarzen kruste (tache noir) kommen. die trombi-diose tritt vorwiegend im herbst auf, die auftretenden entzündungen gehen mit heftigem juckreiz einher. es handelt sich bei den hautreaktionen auf stiche von ektoparasiten weniger um reaktionen auf die mechanische verletzung als auf komponenten des in den stichkanal injizierten speichels. dementsprechend fallen die reaktionen je nach ektoparasit bzw. auch je nach wirt (mensch) unterschiedlich aus (bis hin zum anaphylaktischen schock). sekundärschäden können auftreten, wenn durch kratzen an den juckenden einstichstellen hautläsionen und damit eintrittspforten für erreger (sekundärinfektionen) geschaffen werden. die sensibilisierung gegen die in die stichwunde abgegebenen antigene kann heftige allergische reaktionen bewirken, die sich z. b. in fieber, kopfschmerzen, lymphadenitis etc. äußern. bei bettwanzen reagiert das immunsystem ebenfalls auf das fremdeiweiß des wanzenspeichels. bei längerem befall kann eine immunisierung erfolgen. bei flöhen spricht man von allergischer dermatitis. igg-und ige-antikörperreaktionen gegen antigene von ektoparasiten sind vielfach beschrieben. abzugrenzen sind die stiche der beschriebenen arthropoden von denen anderer arthropoden (z. b. läuse, fliegen, sandmücken, kriebelmücken, krätzemilben). auch dermatitiden (z. b. durch bakterien oder pilze) anderer genese bei mensch und tier sind von stichreaktionen zu unterscheiden. eine mechanische übertragung von krankheitserregern ist durch die mundwerkzeuge der arthropoden möglich. dadurch möglicherweise ausgelöste lokale infektionen sind von den reinen stichreaktionen abzugrenzen. die krankheitsbilder nach arthropodenstichen werden in der regel rein klinisch diagnostiziert. der zusammenhang zu einem arthropodenstich ergibt sich aus der noch sichtbaren einstichstelle oder der anamnestischen angabe des patienten. bei feststellbarem befall durch arthropoden (z. b. zecken, läuse, milben) ist die artdiagnose anzustreben. vor allem die adulten tiere, oft auch die eier und larven, eignen sich zur sicheren bestimmung. hierzu sind diese an den bevorzugten stellen des körpers, ggf. auch des haustieres oder der umgebung zu suchen. mikroskopische artbestimmung: die gefundenen eier, larven, nymphen oder adulten tiere werden unter dem mikroskop anhand spezieller morphologi-scher merkmale beurteilt, die der einschlägigen bestimmungsliteratur zu entnehmen sind. makroskopisch kann die anordnung der abgelegten eier im bruthabitat bei stechmücken zur artdifferenzierung hilfreich sein. adulte flöhe werden z. b. beim sprung erkannt, die larven erscheinen als weiße pünktchen. eine speziesdifferenzierung ist essenziell, um geeignete, der biologie der jeweiligen ektoparasiten folgende, bekämpfungs-oder prophylaxemaßnahmen durchführen zu können. eine gefährdungsanalyse ist vor allem auch anhand der durch die ektoparasiten übertragenen endemischen erreger in dem jeweiligen gebiet vorzunehmen. zur behandlung von mückenstich-reaktionen helfen eiswürfel oder kühle kompressen. zur linderung des juckreizes und zur verhinderung von sekundärinfektionen sollte eine desinfektion der stichstellen erfolgen (alkohol, lokal!). lokale und orale antiallergika (antihistaminika, in schweren fällen auch kortikosteroide) können bei allergischen reaktionen topisch (gel oder creme) oder systemisch appliziert werden. die bekämpfung der ektoparasiten sollte von erfahrenen fachleuten mithilfe von insektiziden oder akariziden durchgeführt werden. mannigfaltige resistenzen der genannten ektoparasiten gegen insektizide sind bekannt. so werden beispielsweise resistenzen gegen pyrethroide (z. b. permethrin und alphacypermethrin) bei bettwanzen beschrieben. saugende und stechende arthropoden findet man weltweit in den jeweiligen arttypischen habitaten bzw. klimazonen. insbesondere die stechmücken sind auf biotope angewiesen, die ihnen geeignete brutmöglichkeiten bieten, z. b. die umgebung stehender oder sehr langsam fließender gewässer oder auch temporäre wasseransammlungen, neotrombicula autumnalis und verwandte arten kommen vor allem in mitteleuropa vor. ctenocephalides felis ist die in mitteleuropa häufigste flohart (> 80%). die hier behandelten arten besitzen im gegensatz zu den läusen (7 läuse) keine wirtsspezifität und können sowohl an tieren als am menschen blut saugen. sie finden ihren wirt mittels chemischer und optischer signale. ein risiko besteht für alle, die sich in den habitaten der ektoparasiten während deren aktivitätszeiten ungeschützt aufhalten (meist in der dämmerung, nachts). befinden sich die ektoparasiten in der häuslichen umgebung, sind praktisch alle bewohner gefährdet. meist sind es bei den ektoparasiten die weiblichen tiere, die blut saugen, damit ihre eier heranreifen können. die in diesem kapitel behandelten arthropoden sind potenzielle überträger (engl: vectors) von krankheitserregern (z. b. endoparasiten, 7 tab. 1). oft fungieren sie jedoch nicht nur als reine überträger sondern zugleich auch als endwirte, wie z. b. anopheles sp. für die plasmodien. die übertragung von krankheitserregern erfolgt z. b. über die stechendsaugenden mundwerkzeuge, entweder durch regurgitation der erreger aus dem darmbereich oder durch ihr einwandern in die speicheldrüsen und ihre abgabe in die stichwunde mit dem speichelsekret. die chagas-krankheit wird durch das einkratzen des raubwanzenkots in die stichstelle übertragen (siehe kapitel trypanosoma cruzi). die ektoparasiten selbst, bzw. deren stadien, können anthropogen weiterverbreitet werden, wie es von aedes-larven in gebrauchten autoreifen oder ähnlichen wassergefüllten "behältnissen" beschrieben ist. moskitonetze, die z. b. mit permethrin imprägniert werden, und korrekt getragene, helle kleidung etc. bieten einen gewissen schutz gegen die blutsaugenden ektoparasiten (persönliche schutzmaßnahmen). auf die haut aufgetragene repellents, wie z. b. deet-oder icaridin-haltige mittel, stellen einen schutz gegen zahlreiche blutsaugende arthropoden dar. gute wirksamkeit wird auch oft von neu entwickelten formulierungen mit sekundären pflanzenstoffen berichtet. die vermeidung bzw. beseitigung von brutstätten (auch kleinste wasseransammlungen) stellt eine flankierende maßnahme im rahmen der habitatveränderung dar. haustiere sind z. b. zur vermeidung einer flohplage ebenfalls in die maßnahmen mit einzubeziehen. die beachtung der bevorzugten stechzeiten (und von präferierten orten) hilft bei der vermeidung von stichen durch ektoparasiten ebenfalls. der einsatz von räucherspiralen, verdampfern, sprays etc. muss sehr vorsichtig erfolgen, eine intensive nutzung in geschlossenen räumen vermieden werden. gaze am fenster schützt vor dem eindringen vieler ektoparasiten. aufklärung und verhaltensänderungen-bzw. anpassungen sowie die meidung von befallenen gebieten (expositionsprophylaxe) sind weitere eckpfeiler einer stichvermeidung. menschen, die allergisch auf stiche von ektoparasiten reagieren, sollten stets ein antihistaminikum griffbereit haben. die pathophysiologie der endokarditis wird bestimmt durch hämodynamische veränderungen bedingt durch die destruktion der herzklappen oder durch eine systemische inflammationsreaktion im sinne einer sepsis. diese sind ursache der im rahmen der ie auftretenden organschäden, sofern sie nicht direkt durch septische embolisation verursacht werden. zur erreger-und resistenzgerechten einleitung einer antimikrobiellen therapie ist der blutkulturelle nachweis des auslösenden pathogens essentiell. [3] , die heute in modifizierter form vorliegen [4] , als hilfreich erwiesen. sie können jedoch in keinem fall eine klinische beurteilung ersetzen. grundsätzlich sollte die therapie der ie unter stationären bedingungen mittels parenteraler gabe bakterizider antibiotika erfolgen. bei infektionen durch penicillin-empfindliche streptokokken mit unkompliziertem verlauf ist im anschluss an eine 14-tägige stationäre initialbehandlung eine ambulante fortführung der therapie möglich. bei akuten erkrankungsformen und bei hämodynamischer instabilität, wird nach akquisition von blutkulturen umgehend eine kalkulierte antimikrobielle therapie eingeleitet. bei klinisch stabilen patienten kann oft das ergebnis der mikrobiologischen diagnostik abgewartet werden. bezüglich der spezifischen erreger-und resistenzgerechten therapie sei auf die aktuellen leitlinien verwiesen [2] . frühzeitig sind herzchirurgen hinzuzuziehen, um im falle nicht-kontrollierbarer infektionen eine rasche chirurgische sanierung zu ermöglichen. nach neurologischen ereignissen sollte es bei bestehender indikation zu keiner verzögerung der operation kommen, sofern die neurologische prognose gut ist. nach intrakraniellen blutungen jedoch sollte der operationszeit-gnaformen" bezeichnet werden. die reifen zysten haben einen durchmesser von 10-16 μm und besitzen immer vier kerne mit einem zentralen karyosom. zysten von entamoeba histolytica und den kommensalisch im darm des menschen vorkommenden arten entamoeba dispar und entamoeba moshkovskii lassen sich morphologisch nicht unterscheiden. das genom von entamoeba histolytica ist weitgehend aufgeklärt (http://pathema.jcvi.org/cgi-bin/entamoeba/pathemahomepage.cgi). nach dem aktuellen kenntnisstand umfasst es etwa 24 megabasen, die auf 14 chromosomen verteilt sind. bisher wurden knapp 10.000 offene leserahmen identifiziert. nur etwa ein viertel aller protein-kodierenden gene ist durch introns unterbrochen und nur 6 % der gene besitzen mehr als ein intron. die vermehrung von entamoeba histolytica findet im dickdarm statt. nach oraler aufnahme infektionstüchtiger zysten, die im gegensatz zu den trophozoiten über monate an der außenwelt stabil bleiben können und resistent gegenüber dem sauren millieu des magens sind, entwickeln sich im dünndarm die einkernigen, teilungsfähigen trophozoiten, die vor allem den oberen dickdarm besiedeln. im distalen kolon kommt es zur erneuten enzystierung mit zwei anschließenden kernteilungen. eine infizierte person kann bis zu 500 millionen zysten pro tag ausscheiden. die pathogenität von entamoeba histolytica beruht primär auf der fähigkeit des parasiten, wirtsgewebe und zellen zu lysieren. hierbei spielen oberflächenrezeptoren, cysteinproteinasen und porenbildende peptide der amöben eine entscheidende rolle. in abhängigkeit von der stärke der expression dieser moleküle, kann die virulenz einzelner amöbenisolate variieren. antigenvariabilität konnte bisher bei entamoeba histolytica nicht nachgewiesen werden. allerdings verlaufen die meisten infektionen mit entamoeba histolytica asymptomatisch (nicht invasive amöbiasis). nur in etwa 10 % der fälle kommt es zur invasion in das gewebe (invasive amöbiasis), wobei sowohl der darm (intestinale amöbiasis) als auch andere organe (extraintestinale amöbiasis), vornehmlich die leber, betroffen sind (amöbenleberabszess). synonym(e) amöbenruhr, amöben-colitis, amöben-dysenterie, amöbom. sehr variabel, zwischen wenigen tagen und mehreren monaten. blutig-schleimige diarrhoe. enteritis oder kolitis von variablem schweregrad, diarrhoe, fieber, tenesmen, druckdolenter oberbauch. für die pathologie der intestinalen amöbiasis sind die amöben direkt verantwortlich. durch eine kontaktabhängige lyse von darmgewebe verursacht entamoeba histolytica tiefe ulzerationen der darmschleimhaut. amöbom = sehr seltene, durch entamoeba histolytica hervorgerufene tumorartige granulomatöse entzündungsreaktion des dickdarms. in mehr als 90 % aller fälle mit invasiver amöbiasis finden sich zum zeitpunkt der klinischen symptomatik signifikante serum-antikörpertiter gegen entamoeba-histolytica-antigen. andere infektiöse darmerkrankungen, insbesondere durch shigellen und invasive escherichia coli, sowie nicht infektöse darmerkrankungen wie colitis ulcerosa oder morbus crohn. amöbenleberabszess. sehr variabel, im mittel 3-5 monate, in einzelfällen mehrere jahre. fieber, schmerzen im rechten oberbauch. hepatomegalie, übelkeit, erbrechen, schmerzen in der rechten schulter (abhängig von der lokalisation des abszesses), ikterus, diarrhoe (nur in 10 % der fälle!). nach invasion in die darmschleimhaut können die amöben hämatogen in andere organe gestreut werden und dort ausgedehnte abszesse induzieren. diese finden sich in erster linie in der leber. amöbenleberabszesse führen häufig zu transsudaten der pleura und zu atelektasen der lunge, können aber gelegentlich auch je nach lokalisation in die lunge oder das perikard rupturieren. primäre amöbenabszesse au-ßerhalb der leber, wie etwa in der lunge oder dem gehirn, sind eine rarität. amöbenleberabszesse führen regelmäßig zur bildung von serum-antikörpern gegen entamoeba-histolytica-antigene. sie sind bei über 90 % der patienten bereits zu beginn der klinischen symptomatik nachweisbar. bakterielle leberabszesse, echinokokkuszyste, primäre leberzyste, einschmelzender tumor. die diagnostik der invasiven amöbiasis richtet sich nach dem manifestationsort der erkrankung: 5 bei der intestinalen amöbiasis stehen der rektoskopische oder koloskopische nachweis entsprechender schleimhautveränderungen sowie der direktnachweis des erregers im vordergrund. letzteres geschieht durch stuhluntersuchungen oder den histologischen nachweis von amöben im biopsiematerial. 5 bei der extraintestinalen amöbiasis bedient man sich vor allem bildgebender verfahren wie sonographie und computertomographie zum nachweis entsprechender organmanifestationen und strukturdefekte. gleichzeitig ist der serologische nachweis spezifischer antikörper gegen entamoeba histolytica ein wichtiges, oftmals richtungweisendes diagnostisches hilfsmittel. gramnegative stäbchenbakterien, beweglich durch peritriche begeißelung. kapselbildung fakultativ. genom vollständig sequenziert, siehe auch www.ncbi. nlm.nih.gov. fakultativ anaerob, wachstum innerhalb von 24 stunden. endotoxin. ca. 10 % aller nosokomialen infektionen, postoperative wundinfektionen nach abdomineller und vaginaler hysterektomie, meningitis, gastroenteritis, harnwegsinfektionen keine. nicht bekannt. dem jeweiligen krankheitsgeschehen entsprechend. dem jeweiligen krankheitsgeschehen entsprechend. fakultativ pathogene opportunistische infektionserreger, infektion bei standortwechsel. opsonisierung durch antikörper, phagozytose. ausschluss anderer infektionserreger. durch einschwemmung von enterobacter spp. in die blutbahn kann es zur sepsis kommen keine. nicht bekannt. fieber. fieber. bei enterobacter-cloacae-isolaten aus tropischen ländern wurde eine enterotoxin-ähnliche aktivität nachgewiesen, daher selten auch durchfallerreger keine. nicht bekannt. durchfall. durchfall. ausschluss anderer infektionserreger. wundabstrich, urin, faeces, blutkultur, liquor. hauptwirt von e. vermicularis ist der mensch; daneben wurden gelegentlich menschenaffen infiziert gefunden. befallen sind in erster linie kinder oder behinderte. verhaltensbedingt (vermutlich aber auch aufgrund von immunreaktionen) ist die infestation im erwachsenenalter in mitteleuropa selten. die übertragung erfolgt auf oralem wege über die eier, die direkt über schmierinfektion (eier unter den fingernägeln! finger-after-mund-weg) oder indirekt über aufgewirbelten staub (aufschütteln der bettwäsche) aufgenommen werden. synonym(e) enterococcus-infektion. zu den inkubationszeiten der verschiedenen enterokokken-infektionen gibt es keine dezidierten daten; nach eindringen in sterile oder prädisponierte anatomische kompartimente ist aber von kurzen inkubationszeiten von wenigen tagen auszugehen. harnwegsinfektionen, endokarditiden, weichteilinfektionen, intraabdominelle infektionen, postoperative wundinfektionen, gefäßkatheter-infektionen, abszesse, dekubitalulzera sowie verschiedene andere infektionen. keine. selten kommt es zu persistierenden oder reaktivierten ebv-infektionen, die zu dysfunktionen verschiedenster organe führen können. so kann es im verlauf einer persistierenden ebv-infektion zur knochenmarksaplasie, pneumonitis, rekurrierenden febrilen episoden, dysgammaglobulinämien, hepatitis oder neurologischen abnormalitäten kommen. patienten mit x-linked lymphoproliferative disease, sind nicht in der lage, ebv-infektionen zu kontrollieren. serum. igm und igg-antikörper gegen viruskapsid-antigen (vca), "early-antigen" (ea) oder "epstein-barr nuclear antigen" (ebna) können im immunfluoreszenztest oder elisa nachgewiesen werden. frische ebv-infektionen zeichnen sich meist durch die anwesenheit von igm-und igg-antikörpern gegen vca, serokonversion gegen ea und die abwesenheit von anti-ebna-antikörpern aus, die meist erst im späteren verlauf der infektion auftreten. das blutbild weist erhöhte lymphozytenzahlen auf, mikroskopisch können monozytoide lymphozyten (pfeifferzellen) nachgewiesen werden. der bei erwachsenen gebräuchliche paul-bunnel-test (nachweis heterophiler antikörper) gibt bei erwachsenen in 80-85 % der fälle ein positives resultat, ist aber bei kindern in 50 % der fälle negativ. frische ebv-infektionen zeichnen sich meist durch die anwesenheit von igm-und igg-antikörpern gegen vca, serokonversion gegen ea und die abwesenheit von anti-ebna-antikörpern aus, die meist erst im späteren verlauf der infektion auftreten. es sind keine kausalen therapieoptionen bekannt. bei chronisch aktiven ebv-infektionen wurden behandlungsversuche mit acyclovir, ganciclovir, ifn, il-2 und vidarabin beschrieben. keine daten verfügbar. weltweit. ebv ist durch einen starken lymphotropismus gekennzeichnet und infiziert vorwiegend b-lymphozyten. die infektion erfolgt wahrscheinlich über den c3d-rezeptor. außer seinem natürlichen wirt, dem menschen, können auch affenzellen infiziert werden. es sind keine risikogruppen bekannt. menschen können in jedem lebensalter erkranken, wobei der infektionsgipfel im kindes-bzw. jugendalter liegt. keine prävalenz für bestimmte bevölkerungsgruppen. die übertragung erfolgt über tröpfcheninfektion, setzt aber einen sehr engen kontakt voraus (z. b. küssen). auch durch bluttransfusionen ist das virus übertragbar. kein impfstoff erhältlich. keine daten verfügbar. keine. unbewegliches, schlankes, grampositives, zuweilen gramlabiles stäbchen mit ketten-und fadenbildung. das genom ist in teilbereichen sequenziert. weitere informationen unter http://www.genedb.org. langsames wachstum auf nährmedien. über virulenzfaktoren ist nichts bekannt. die inkubationszeit beträgt 1-4 tage. an der eintrittspforte der haut juckende schmerzende schwellung. risikogruppen sind tierärzte sowie arbeiter in der fleisch und fisch verarbeitenden industrie. infektionen können allerdings auch im häuslichen milieu beim umgang mit tierischen produkten auftreten. die übertragung der krankheit erfolgt meistens über kleine verletzungen an der haut und im kontakt mit infizierten tieren, bzw. ihrem fleisch. präventionsmaßnahmen bestehen in der vermeidung des kontakts mit infizierten tieren, bzw. deren produkten. impfschutz ist nicht gegeben. wegen des vorkommens als seltene einzelerkrankung ist ein ausbruchsmanagement nicht erforderlich. es besteht keine meldepflicht nach ifsg. grampositive kleine stäbchen, keine sporenbildung. das genom der eubakterien ist mittels pcr nachweisbar, 16s rrna-vergleiche führten zu der oben angesprochenen taxonomischen neuordnung. die bakterien der gattung eubacterium vermehren sich nur unter streng anaeroben bedingungen. nicht bekannt. abszesse: nierenabszess, bartholinischer abszess, intraabdomineller abszess, infektionsprozesse der orofazialregion, parodontalerkrankungen, wundinfektionen, intrauterine infektionen bei liegendem intrauterinpessar nicht bekannt, endogene infektion. abszedierende prozesse. unspezifische, abszedierende prozesse und wundinfektionen. eubakterien kommen in der regel in den genannten prozessen nicht in reinkultur vor, sondern in verbindung mit virulenteren anaeroben oder fakultativen bakterien. nicht bekannt. infektionen durch andere erreger. durch abszesspunktion gewonnener eiter, tiefe wundabstriche. kulturelle anzüchtung unter anaeroben bedingungen mit nachfolgender identifizierung mittels biochemischer reaktionen oder gensequenzierung. wie bei jedem nachweis von bakterien der normalen körperflora ist eine ätiologische bedeutung nachgewiesener eubacterium spp. nur bei adäquat entnommenem untersuchungsmaterial (ohne kontamination mit der flora der angrenzenden besiedelten körperbereiche) anzunehmen. ubiquitär. eubacterium spp. sind bestandteil der normalen körperflora des menschen (darmflora, mundflora: vor allem e. lentum, urogenitaltrakt: e. nodatum) sowie von tieren. frauen mit liegendem intrauterinpessar. nicht bekannt. keine. nicht erforderlich. keine. es findet sich eine hyperplasie des gallengangepithels. die abgabe von prolin durch die adulten würmer (stimulieren die kollagenproduktion) führt zu fibrosen. die durch den leberegel hervorgerufene geringgradige immunantwort führt weder zur abtötung des parasiten, noch schützt sie vor reinfektion. leberstörungen anderer genese (z. b. schistosomiasis, zirrhosen anderer genese). zum nachweis der charakteristischen eier ist nativer (unfixierter) oder fixierter stuhl in geeigneten transportgefäßen bzw. speziellen transportsystemen einzusenden. der einachweis gelingt evtl. auch im gallensaft oder im sekret des zwölffingerdarms. mikroskopische stuhl-diagnostik nach anreicherungsverfahren. die infektiösen stadien von f. hepatica werden beim verzehr der als "zwischenwirte" dienenden wasserpflanzen aufgenommen. werden die beschriebenen wasserpflanzen nicht oder nur im abgekochten zustand verzehrt, kommt es nicht zu einer infektion. die bekämpfung der fasciolose muss bei der behandlung der herbivoren säugetiere ansetzen, um eine infektion der schnecken zu verhindern. maßnahmen zur schneckenbekämpfung sind problematisch. es besteht keine meldepflicht. referenzzentren / expertenlaboratorien 5 als fachlich qualifizierte ansprechstellen gelten alle parasitologischen und tropenmedizinischen institutionen. marburgvirales hämorrhagisches fieber. wie 7 ebolavirus-infektion (erkrankung 1). wie 7 ebolavirus-infektion (erkrankung 1). wie 7 ebolavirus-infektion (erkrankung 1). wie 7 ebolavirus-infektion (erkrankung 1). wie 7 ebolavirus-infektion (erkrankung 1). wie 7 ebolavirus-infektion (erkrankung 1). wegen der hohen pathogenität dieser viren müssen beim umgang mit infektiösem material besondere sicherheitsvorkehrungen beachtet werden. bei personen, die mit dem sle-v infiziert wurden, lassen sich wie bei anderen flavivirusinfektionen igmund igg-antikörper nachweisen. es ist anzunehmen, dass auch eine t-zell-antwort generiert wird. einzelheiten zur immunantwort bei infektionen des menschen mit mve-v, pow-v und roc-v sind nicht bekannt. viele andere viren, die enzephalitis hervorrufen, müssen in betracht gezogen werden. einige der durch moskitos übertragenen seltenen flaviviren -wie z. b. wsl-v -rufen hauptsächlich fieber, arthralgien und exantheme hervor. die inkubationszeit von wsl-v beträgt 2-4 tage. die meisten infektionen mit wsl-v bleiben symptomlos oder verlaufen sehr mild, so dass sie diagnostisch nicht weiter abgeklärt werden. es kann sich jedoch eine grippeähnliche symptomatik entwickeln mit plötzlichem fieber sowie schweren kopf-, glieder-und muskelschmerzen. lichtphobie und hyperästhesie der haut sind weitere symptome. nicht selten wird ein exanthem beobachtet. das fieber verschwindet bereits nach 2-3 tagen während die muskelschmerzen länger anhalten können. berichte über tödliche infektionen liegen nicht vor. antikörper können mit dem hämagglutinationshemmtest nachgewiesen werden. andere viren, die eine grippeähnliche symptomatik hervorrufen können. seltene humanpathogene flaviviren, die hämorrhagisches fieber verursachen, sind kfd-v und ohf-v. beide werden durch zecken übertragen (7 tab. 1) . die inkubationszeit für kfd-v und ohf-v beträgt ca. 3-8 tage. keine meldepflicht nach ifsg. keine bekannt. innerhalb eines fsme-v-subtyps variiert die aminosäuresequenz nicht mehr als bis zu 2,2 %. dagegen ist diese variabilität zwischen den subtypen naturgemäß größer (5,6 %) . in einigen studien wird dem fernöstlichen subtyp eine größere virulenz im menschen zugeschrieben als den anderen. tierexperimente unterstützen diese annahme. außerdem gibt es hinweise, dass der sibirische subtyp einen chronisch-progressiven krankheitsverlauf verursachen kann, während dies für den europäischen subtyp nicht beobachtet wird. zentraleuropäische zeckenenzephalitis (infektion mit dem europäischen fsme-v-subtyp); russische frühjahrs-sommer-enzephalitis (infektion mit dem fernöstlichen fsme-v-subtyp). die inkubationszeit beträgt 7-14 tage, in einzelfällen bis zu 28 tage. das klinische erscheinungsbild von fsme ist nicht charakteristisch, sodass letztlich die ergebnisse des virologischen labors für die diagnosestellung entscheidend sind. die meisten infektionen mit dem europäischen fs-me-v subtyp verlaufen inapparent. nur bei 5-30 % der infektionen treten grippeähnliche symptome auf mit fieber (< 38 °c), kopfschmerzen, schwindelgefühl und erbrechen ("sommergrippe"). diese 1. phase der krankheit dauert 4-6 tage. danach lassen die beschwerden für ungefähr 2-3 tage nach (in einzelfällen bis zu 21 tage). in der 2. phase der erkrankung, die nur 20-30 % der patienten erleiden, stellen sich hohes fieber (bis > 40 °c), starkes krankheitsgefühl und meningitis-symptome ein. es können zeichen der enzephalitis hinzu kommen (meningoenzephalitis, in ca. 30-40 % der fälle mit einer 2. phase). vor allen dingen bei älteren patienten kann sich zusätzlich eine myelitis oder radikulitis entwickeln (gefahr der bulbärparalyse und phrenikusparese). in diesen schweren fällen (ca. 10 % der fälle mit einer 2. phase) beträgt die letalität ungefähr 1-2 % und die gefahr von bleibenden schäden besteht. extrapyramidale und zerebelläre symptome können oft noch monate nach rekonvaleszenz persistieren. gewöhnlich kommt es aber selbst bei schweren verläufen zur völligen heilung ohne bleibende neurologische ausfälle. in 10-20 % der schwereren verläufe muss jedoch mit bleibenden psychomotorischen defekten gerechnet werden. paraplegien, tetraplegien und paresen der schultergürtelund kopfmuskulatur sind als prognostisch ungünstige zeichen zu werten. insgesamt betrachtet, sind die krankheitsbilder paresen und bleibenden schäden bei erwachsenen ausgeprägter als bei kindern. durch den fernöstlichen fsme-v-subtyp hervorgerufene erkrankungen beginnen weniger akut. die letalität (5-40 %) ist jedoch wesentlich höher als bei beim europäischen subtyp. einen weiteren unterschied stellt die tatsache dar, dass hier infizierte kinder schwerer erkranken als erwachsene. auch die rate der bleibenden neurologischen schäden ist mit 30-60 % höher. der sibirische subtyp des fsme-v verursacht krankheitsverläufe (letalität von 1-3 %), die weniger schwer sind als beim fernöstlichen aber schwerer als beim europäischen subtyp verlaufen. ähnlich wie bei dem fernöstlichen subtyp werden chronische infektionsverläufe beobachtet. der erste fieberanstieg ist auf die virämie zurückzuführen, während der zweite fiebergipfel dann auftritt, wenn das stadium der zns-infektion erreicht ist. das gehirn verändert sich ödematös. vereinzelt treten umschriebene blutungen auf. die beobachtete lähmung der oberen extremitäten lässt sich mit der hohen empfindlichkeit der anterioren motorischen rückenmarkszellen im halswirbelbereich für virus-assoziierte zytopathogene mechanismen erklären. gegen das glykoprotein der hülle werden hämagglutinationshemmende und neutralisierende antikörper gebildet. in der regel werden zunächst igm-antikörper produziert und erst danach igg-antikörper. auch eine zelluläre immunantwort wird generiert. serum, plasma, liquor. die diagnose wird in der regel aufgrund des nachweises von virusspezifischen igm-und igg-antikörpern im serum und ggf. im liquor (bei ca. 50 %) durch das elisa-verfahren gestellt. meist sind schon in der ersten krankheitsphase virusspezifische igm-antikörper im serum nachweisbar. bei zunächst negativem test und weiterbestehendem klinischen verdacht empfiehlt sich die testwiederholung nach etwa 1 woche. in der zweiten krankheitsphase werden dann in der regel sowohl igm-als auch igg-antikörper gefunden. andere serologische testverfahren wie kbr und hht spielen keine rolle mehr. die virusisolierung aus dem blut von infizierten und der nachweis mittels rt-pcr gelingen nur in der virämischen phase und sind für die routinediagnostik daher meist bedeutungslos (bei arztkonsultation ist die virämische phase in der regel schon vorbei). in der zweiten erkrankungsphase kann eventuell im liquor mittels rt-pcr virale nukleinsäure gefunden werden. berücksichtigt werden müssen mögliche kreuzreaktionen durch antikörper gegen andere flaviviren. diese können durch neutralisations-testverfahren ausgeschlossen werden, die allerdings nur speziallaboratorien durchführen. bei der befundinterpretation muss auch daran gedacht werden, dass fsme-impfungen zu lange zeit nachweisbaren spiegeln von fsme-vspezifischen igm-antikörpern führen können. bei infektionen mit dem epstein-barr-virus nach fsme-impfung können fsme-v-reaktive igm-antikörper auftreten, die nicht auf eine fsme zurückzuführen sind. es kommen nur supportive maßnahmen (z. b. strenge bettruhe) in betracht, da eine spezifische antivirale therapie nicht zur verfügung steht. weltweit treten mehr als 10.000 krankheitsfälle jährlich auf. besonders russland ist betroffen. damit ist das fsme-v nach dem japanische enzephalitis-virus der zweithäufigste krankheitserreger unter den neurotropen flaviviren. das verbreitungsgebiet des europäischen subtyps erstreckt sich bis zum ural im osten, über finnland und schweden im norden, nach deutschland und frankreich im westen bis herunter nach italien, griechenland und dem ehemaligen jugoslawien im süden. im unterschied zu den erregern der lyme-borreliose (borrelia burgdorferi) wird das fsme-v nur in bestimmten risikogebieten gefunden. die topographische darstellung der fsme-risikogebiete in deutschland (angabe auf landkreisebene) beruht größtenteils auf einer speziellen fallsammlung von fsme-erkrankungen und wird ständig aktualisiert (robert-koch-institut in berlin in enger kooperation mit den betroffenen bundesländern). naturherde mit hohem risiko befinden sich überwiegend in baden-württemberg und bayern. solche mit niedrigem risiko liegen in hessen, rheinland-pfalz und thüringen (7 abb. 1 bieten sollte lange kleidung getragen werden. diese sollte hell sein, damit herumwandernde zecken besser erkannt werden können. repellentien wie beispielsweise autan haben eine zeitliche begrenzte wirkung (2 stunden) gegen zecken. nach risikoaufenthalten sollte der körper nach zecken abgesucht werden (bevorzugte lokalisationen: unter den armen, im nacken, am haaransatz des kopfes und generell an dünnen, gut durchbluteten hautpartien). zecken, die sich mit ihrem halteapparat bereits in der haut verankert haben, sollten möglichst rasch entfernt werden. mit einer zeckenzange oder pinzette wird die zecke möglichst hautnah gefasst und vorsichtig -nach leichtem hin-und herhebeln -herausgezogen. danach sollte die einstichstelle desinfiziert werden. die verwendung von öl, wachs oder nagellack, um die zecke zu ersticken, sind obsolet (gefahr der vermehrten erregerausschüttung). die wunde sollte sofort nach entfernung der zecke desinfiziert werden. die fsme-impfung ist eine indikationsimpfung für risikogruppen (s. o.). zurzeit sind vier impfstoffe verfügbar. neben zwei russischen impfstoffen, die den fernöstlichen subtyp enthalten, gibt es zwei vakzine, welche in europa zugelassen sind und auf dem europäischen subtyp basieren (fsme-immun von baxter; encepur von novartis). die impfung mit dem europäischen subtyp schützt sehr wahrscheinlich auch vor infektionen mit dem fernöstlichen subtyp. für die impfung wird ein komplettes, durch formalin inaktiviertes virus verwendet, welches in kulturen von embryonalen hühnerfibroblasten angezüchtet und anschließend gereinigt wurde. als adjuvans enthält der impfstoff aluminiumhydroxid. drei impfungen sind für einen vollständigen impfzyklus notwendig, wobei die effizienz des impfschutzes bei 95 % bis 99 % liegt. auffrischimpfungen sollten alle 3 bis 5 jahre durchgeführt werden. früher beobachtete nebenwirkungen (kopfschmerzen, fieber, abgeschlagenheit) treten heute kaum mehr auf, da die vakzine nun durch einen ultrazentrifugationsschritt hochaufgereinigt wird. an einer weiteren verbesserung der impfstoffe wird gearbeitet. die durchimpfungsrate der österreichischen bevölkerung ist die höchste in ganz europa und beträgt etwa 86 %. dadurch konnte die die zahl der fs-me-bedingten hospitalisierungen um 90 % gesenkt werden. die postexpositionelle gabe von fsme-spezifischem hyperimmunglobulin (passive impfung) wird "generell nicht empfohlen" (stiko) und von herstellerseite in deutschland nicht mehr angeboten. berichte über schwere krankheitsverläufe nach zu später postexpositioneller passiver immunisierung liegen vor. der zugrunde liegende mechanismus ist möglicherweise eine verstärkung der infektion durch die verabreichten antikörper (immunoenhancement). wenn fsme in regionen beobachtet wird, die bislang nicht als risikogebiet eingestuft wurden, dann müssen weiterführende epidemiologische untersuchungen eingeleitet werden. darüber hinaus sollten die ärzte dieser regionen ausführlich über fsme informiert und weitergebildet werden. nach § 7 abs. 1 nr. 14 infektionsschutzgesetzes (ifsg) muss das labor dem gesundheitsamt jeden direkten oder indirekten (serologischen) nachweis von fsme-v durch das labor dann namentlich melden, wenn er auf eine akute infektion hinweist. darüber hinaus stellt das gesundheitsamt gemäß § 25 abs. die taxonomie auf spezies-ebene wurde in den letzten jahren aufgrund molekulargenetischer untersuchungen der its-rdna, der 28s-rdna, der mitochondrialen dna und der sequenz des beta-tubulin-gens neu definiert. danach werden mehr als 20 spezies unterschieden. bei f. oxysporum und f. solani wurden mehrere varianten beschrieben, sodass beide arten als spezies-komplex anzusehen sind. in der ehemaligen udssr starben 1944-1947 mehr als 100.000 menschen an einer nahrungsmittelvergiftung durch verschimmeltes getreide. die erkrankung war eine mykotoxikose verursacht durch toxine von f. sporotrichoides und f. poae. der erste fall einer disseminierten invasiven fusariose wurde 1973 von cho et al. bei einem kind mit einer akuten lymphatischen leukämie beschrieben. histologisch finden sich bei invasiver fusariose im wirtsgewebe radiär wachsende, hyaline, septierte, sich spitzwinklig (30-50°) verzweigende myzelien einheitlichen kalibers (3-4 μm) . diese sind anfärbbar mit der grocott-gomorri-versilberung oder dem perjodsäure-schiff-reagenz (pas). ohne immunhistologie sind die hyphen nicht von aspergillus und scedosporium (pseudallescheria) unterscheidbar. in der kultur wachsen nach 3-5 tagen bei 37 °c auf sabouraud-glukose-agar weißliche kolonien mit reichlich luftmyzel. 5 fusobakterien besitzen eine reihe von adhäsinen, die es ihnen erlauben, mit anderen bakterienarten zu interagieren bzw. zu aggregieren. diese adhäsine ermöglichen zudem die adhäsion an schleimhautepithelzellen, aber auch an künstlichen oberflächen (zahnersatzmaterialien) und stellen auf diese weise einen wichtigen faktor bei der plaqueformation dar. fusospirochätose. unspezifisch. die angina plaut-vincent stellt eine mischinfektion aus spirochäten (treponema vincentii) und fusobacterium nucleatum dar. sie verursacht meist kein fieber und nur geringfügige störungen des allgemeinbefindens und äußert sich lediglich in meist einseitigen schluckbeschwerden. trotzdem finden sich typischerweise erheblich geschwürig und nekrotisch veränderten gaumenmandeln, die meist fibrinöse übelriechende grau-grünliche beläge aufweisen. schwellungen der submandibularen lymphknoten können oft beobachtet werde. unklar. eine bleibende immunität nach infektion entsteht nicht. streptokokkenangina, lues. synonym(e) nekrobazillose. unspezifisch. in folge einer in der regel durch f. necrophorum hervorgerufenen pharyngotonsillitis kommt es zu einer thrombophlebitis der vena jugularis interna. metastatische absiedelungen aus diesem fokus können alle organe betreffen und schnell zu schweren krankheitserscheinungen bis hin zum septischen schock führen. unklar. eine bleibende immunität nach infektion entsteht nicht. streptokokkenangina. wangenbrand. unspezifisch. die noma ist eine in der regel durch fusobakterien hervorgerufene infektion der wangenschleimhaut, die im weiteren verlauf zu einer zerstörung von weichgewebe und knöchernen anteilen des gesichts führt. später kommt es zu septischen absiedlungen, die mit einer hohen letalität behaftet sind. unklar. eine bleibende immunität nach infektion entsteht nicht. staphylokokken-und streptokokkeninfektionen, aktinomykose. gemeinsam ist neben dem vorkommen von geradkettigen, gesättigten und einfach-ungesättigten, langkettigen zellulären fettsäuren der aufbau der peptidoglykanschicht sowie der glutamatstoffwechsel (glutamatdehydrogenase positiv). differenzierende biochemische charakteristika sind u. a. der indolabbau (nur f. mortiferum und f. russii sind negativ), wachstum in gegenwart von 20 % galle und äskulinhydrolyse (nur f. mortiferum ist positiv), nitratreduktion (f. ulcerans ist positiv) sowie die hippurathydrolyse (f. periodonticum ist positiv). der nachweis von fusobacterium spp. ist bei entsprechenden klinischen infektzeichen als relevant zu bewerten. neben der chirurgischen abszessbehandlung muss immer eine antibiotikatherapie erfolgen. fusobakterien sind empfindlich gegen eine vielzahl von antibiotika wie penicilline, cephalosporine, peneme, tigecyclin, β-laktam/β-laktamaseinhibitor-kombinationen, metronidazol, clindamycin, linezolid und chloramphenicol. von der angina plaut-vincent sind häufiger jugendliche betroffen. die noma tritt bei mangelernährten immunsupprimierten kindern in entwicklungsländern auf. meist endogene infektionen. keine. nicht relevant. keine. die bakterielle vaginose betrifft hauptsächlich jüngere, sexuell aktive frauen oder frauen mit häufig wechselnden partnern. eine erhöhte prävalenz der bv findet sich auch bei homosexuellen frauen. ein erhöhtes risiko besteht auch für frauen mit iup (intrauterinpessar). transmission / vektoren g. vaginalis kann durch geschlechtsverkehr übertragen werden, kann jedoch auch bei frauen ohne sexuelle kontakte nachgewiesen werden. eine übertragung z. b. in schwimmbädern oder über toilettensitze ist nicht möglich. erhalt des physiologischen vaginalmilieus, vermeidung unnötiger irritationen (antibiotika, scheidenspülungen, vaginalcremes etc.). im allgemeinen ist die antibiotische behandlung der erkrankten ausreichend, eine partnerbehandlung wird nicht zwingend empfohlen. weder die erkrankung noch der erreger sind im infektionsschutzgesetz aufgeführt. referenzzentren / expertenlaboratorien 5 nationale referenzzentren sowie konsiliarlaboratorien in deutschland sind nicht bekannt. g werden können. auch kausale assoziationen mit anderen, nichthepatischen erkrankungen gelten als nicht gesichert, so dass gbv-c als ein für den menschen nicht pathogenes virus angesehen werden kann. koinfektionen von patienten sowohl mit gbv-c als auch viralen hepatitis-erregern (hbv, hcv) oder hiv sind durch den ähnlichen parenteralen übertragungsweg erklärbar. eine reihe von studien hat gezeigt, dass bei hiv-infizierten patienten mit einer gbv-c-koinfektion der krankheitsverlauf bis zum stadium aids deutlich langsamer erfolgt und mit einer längeren überlebenszeit verbunden ist. dieser protektive effekt ist nicht in allen studien bestätigt worden, wobei möglicherweise unterschiedliche gbv-c-stämme einen unterschiedlichen einfluss auf den verlauf der hiv-koinfektion haben. bisher liegen nur daten über humorale und zelluläre immunantworten gegen das e2-hüllprotein vor. das erscheinen von anti-e2-antikörpern korreliert in der regel mit dem verschwinden der virämie und mit einer durchgemachten infektion. das vorliegen von anti-e2-antikörpern vor orthotroper lebertransplantation schützt vor einer gbv-c-de-novo-infektion, was für eine protektive rolle der anti-e2-antikörper spricht. im gegensatz zur situation beim hcv ist der e2-genabschnitt hoch konserviert. bezüglich der zellulären immunantwort weisen patienten mit ausgeheilter oder florider gbv-c-infektion im proliferationsassay keine unterschiede auf. entfällt. eine infektion mit gbv-c kann mittels rt-pcr für gbv-c-rna aus serum oder plasma nachgewiesen werden. antikörper gegen das oberflächenglykoprotein e2 (anti-e2) sind kurz vor oder nach verschwinden der virämie mittels elisa nachweisbar, das heißt, ihr auftreten korreliert in der regel mit dem verschwinden der virämie. es gibt jedoch einzelne fälle von rna-elimination ohne erscheinen nachweisbarer e2-antikörper. eine indikation zur medikamentösen behandlung einer gbv-c infektion liegt nicht vor. allerdings wurden im rahmen der interferon-therapie von chronischer hepatitis c bei patienten mit gbv-c koinfektion ähnliche erfolgsraten für die elimination von hcv und gbv-c gefunden. resistenz gbv-c kann mit üblichen viruziden maßnahmen inaktiviert werden. verbreitung gbv-c ist weltweit verbreitet. die gbv-c-rna-prävalenz bei blutspen dern in europa und nordamerika liegt zwischen 1 und 4 %, in risikogruppen (s.o.) und in der bevölkerung anderer kontinente kann sie deutlich höher sein. anti-e2-antikörper liegen bei bis zu 16 % der europäischen blutspender vor, was häufiger als der rna-nachweis ist und für die hohe ausheilrate der gbv-c infektion spricht. gbv-c ist bisher nur beim menschen nachgewiesen worden. experimentell sind affen infizierbar. gbv-a und gbv-b sind affen-spezifische viren (u. a. tamarine). drogenabhängige, empfänger von blutprodukten, hämodialysepatienten, transplantatempfänger, personen mit häufig wechselndem und ungeschütztem geschlechtsverkehr. gbv-c kann parenteral über blut und blutprodukte, "needle sharing", direkten kontakt oder auch perinatal von der mutter auf das kind übertragen werden. insgesamt ähnelt die übertragungsweise sehr stark der für hiv. im vergleich zu hcv spielt die vertikale übertragung eine größere rolle. gbv-c ist auch in samenflüssigkeit und im speichel nachgewiesen worden. bei gbv-c-positiven (und hcv-negativen) blutspendern wurden als risikofaktoren für eine gbv-c-infektion sexualkontakte und vorangegangene medizinische eingriffe ermittelt. angesichts der fehlenden klinischen relevanz von gbv-c sind besondere maßnahmen zur infektionsvermeidung nicht vorgesehen (wie z. b. screening von blutprodukten auf gbv-c rna oder anti-e2-antikörper). darüber hinaus gelten die üblichen ver haltens regeln zur verhütung parenteral übertragbarer krankheiten (einmalspritzen, handschuhe, desinfektion und sterilisation, kondome). aufgrund der bisher nicht erwiesenen pathogenität von gbv-c sind keine strategien zur krankheitsvorbeugung entwickelt und implementiert worden. wahrscheinlich greifen aber die allgemeinen strategien zur verhütung anderer parenteral übertragbarer viruserkrankungen (7 hcv-infektion). zwar ist die akute non-a-e-hepatitis nach § 6 des ifsg meldepflichtig, jedoch wird diese offensichtlich nicht durch gbv-c ausgelöst. es sind weder der nachweis von gbv-c rna noch von anti-e2 zu melden. giardiasis synonym(e) giardiasis, lambliasis. malabsorptionssyndrom. es kommt zu abdominalen schmerzen, abgeschlagenheit, übelkeit, erbrechen, flatulenz sowie intermittierenden, dünnbreiigen diarrhöen, malabsorption, unterernährung bei kindern und immundefizienten personen. g. lamblia verursacht lokale darmepithelschäden, entzündungen der propria und störungen des gallenflusses. die entwicklung einer immunität ist fraglich. einen schutz vor reinfektionen gibt es offensichtlich nicht. diarrhoen anderer genese (amöbenbefall, diarrhöen durch bakterien, viren). nicht fixierter stuhl oder duodenalsaft. der nachweis der trophozoiten erfolgt im frischpräparat und im gefärbten ausstrich (heidenhain-, trichrom-oder lawless-färbung), am besten aus frischem stuhl. ist die untersuchung von durchfallstühlen nicht innerhalb einer stunde und von geformten stühlen nicht am selben tag gewährleistet, sollte der stuhl sofort nach dem absetzen fixiert werden (pva-, mif-oder saf-lösung), da die trophozoiten rasch desintegrieren können. zysten lassen sich im gegensatz zu trophozoiten auch in stuhlproben nachweisen, die nicht frisch gewonnen oder fixiert wurden. zudem können sie unter erhaltung ihrer morphologie effektiv angereichert werden (mif-oder saf-anreicherung). die ausscheidung von zysten variiert jedoch häufig sehr und kann bei akuter giardiasis mit den flüssigen durchfällen völlig fehlen. zum ausschluss einer giardiasis sollten daher mehrere (mindestens drei) stuhlproben von verschiedenen tagen auf zysten und/oder trophozoiten untersucht werden. gelegentlich lässt sich die infektion nur durch den nachweis von trophozoiten in endoskopischen oder per sonde gewonnenen aspiraten bzw. bei abstrichen (sofortige untersuchung oder fixierung wie oben) oder biopsaten (nach giemsa gefärbte tupfpräparate und histologie) aus dem duodenum sichern. immunologische nachweisverfahren haben eine geringe aussagekraft. molekularbiologische nachweisverfahren (dna-nachweis aus stuhl oder duodenalmaterial oder anderen geweben) sind für den nachweis von giardia-infektionen besonders bei subklinischen, asymptomatischen oder chronischen verlaufsformen sehr nützlich. das auftreten von zysten oder trophozoiten ist eine therapieindikation. sexuell übertragbare krankheit mit weichen, meist schmerzhaften ulzerationen im genitalbereich mit inguinaler lymphadenitis. weicher schanker (engl. chancroid). 3-14 tage, in der regel 3-5 tage. schmerzhafte ulzeration im genitalbereich mit inguinaler lymphadenitis. leitsymptome, weiche papel mit erythematösem hof. nicht bekannt. vorübergehender schutz aufgrund der zellgebundenen immunität (verzögerter hypersensitivtiätsreaktion vom typ iv), hinterlässt jedoch keine bleibende immunität. treponema pallidum (syphillis), herpes-simplex-virus (hsv) typ 1 und 2, calymmatobacterium granulomatis (granuloma inguinale). abstrich, punktat. eine inkubationszeit lässt sich nicht präzise definieren, da das entstehen von krankheitserscheinungen von der zahl der -in der regel akkumulativ -eingedrungenen bzw. aufgenommenen larven und der dauer der infektion abhängt. pneumonie, uncharakteristische gastrointestinale symptomatik, eisenmangel-anämie. die infektion erfolgt in erster linie durch aktives eindringen der larven in die intakte haut, aber auch orale infektionen durch aufnahme filariformer larven sind möglich. die prävention besteht generell in der hygienischen entsorgung menschlicher fäkalien in gruben bzw. in dem verbot einer verwendung als dünger. individuell ist barfußgehen und sitzen auf nacktem boden in endemiegebieten zu vermeiden. ein impfstoff gegen hakenwürmer ist derzeit nicht verfügbar. eine impfstoffentwicklung auf der basis rekombinanter antigene wird versucht. wegen des indirekten übertragungsweges sind ausbruchsgeschehen nicht möglich. nach dem infektionsschutzgesetz (ifsg) vom 1. die häufigkeit von h.-pylori-stämmen mit resistenzen gegen clarithromycin und metronidazol hat in den letzten jahren zugenommen und macht die therapie zunehmend problematisch. alternative therapieformen bei versagen der ersttherapie (so genannte rescue-therapieformen) sind die quadrupeltherapieschemata. andere therapieschemata sollten auf der basis von publizierten effektivitätsdaten und nach resistenztestung eingesetzt werden. weltweit. mensch. ein reservoir in der unbelebten umwelt ist nicht bekannt. die epidemiologie der h.-pylori-infektion ist bisher nur unvollständig untersucht und es gibt viele offene fragen. hierzu gehören die fragen nach dem vorherrschenden transmissionsweg (7 oben), nach möglichen epidemiologisch bedeutsamen umweltreservoirs oder nach der entstehung der stammheterogenität innerhalb der spezies h. pylori, um nur einige zu nennen. alles deutet jedoch darauf hin, dass sowohl in industrienationen als auch in entwicklungsländern der größte teil der infektionen in der kindheit erworben wird und dass prophylaktische strategien bei der transmission im kindesalter angreifen müssen. verbesserung der allgemeinen sozioökonomischen bedingungen und der hygiene reduziert die prävalenz der h.-pylori-infektion. da nicht genau bekannt ist, wie die transmission erfolgt, gibt es zurzeit keine spezifischen empfehlungen zur expositionsprophylaxe. impfungen gegen h. pylori befinden sich in der entwicklung. es besteht keine meldepflicht. christoph springfeld synonym(e) veraltet: equine morbillivirus. isolierung infizierter pferde. nein. differenzierung von anderen virushepatitiden erfordert jedoch die bestimmung der hepatitis-b-antigene und -antikörper. in 7 tab. 2 ist das zeitliche auftreten der hbv marker in verschiedenen phasen der hepatitis-b-virusinfektion dargestellt. bei verdacht auf eine hepatitis-b-infektion sollte als erster schritt hbsag (nachweis ab ca. 100 pg/ml) und anti-hbc-igg aus dem serum bestimmt werden. die höhe der anti-hbc-igm antikörper geben einen hinweis auf das stadium der erkrankung (werte > 500 pei einheiten sprechen für akute hepatitis). der nachweis von hbeag korreliert in der regel mit einer hohen virämie (≥ 10 6 genomäquivalente/ml). ein akutselbstlimitierter verlauf ist in der regel durch den abfall des hbsag innerhalb von sechs wochen um mehr als 50 prozent mit verlust von hbeag und hbv-dna gekennzeichnet. anti-hbe verschwindet in der regel einige jahre nach der akuten-selbstlimitierten hepatitis. bei isoliert anti-hbc-positiven patienten, insbesondere bei koinfektionen mit hiv, ist die bestimmung der hbv-dna indiziert. gegenwärtig sind in europa für die therapie der chronischen hepatitis b interferon-α (ifn-α) und pegyliertes ifn-α zugelassen, daneben drei nukleosidanaloga (lamivudin, telbivudin, entecavir) und zwei nukleotidanaloga (adefovir dipivoxil, tenofovir diso proxil). eine therapieindikation besteht in der regel, wenn patient hbv-dna-positiv ist und zwei der drei folgenden kriterien erfüllt sind: 5 virusreplikation von ≥ 10 4 viruskopien/ml, 5 fortgeschrittene fibrose, zirrhose, 5 erhöhte transaminasen. therapieziele sind das verschwinden von hbv-dna und (wenn vorhanden) hbeag sowie anhaltende normalisierung der transaminasen. die immunstimulatorische therapie mit ifn-α über 6 monate oder pegyliertem interferon über 1 jahr ist bei bis zu 40 % der patienten erfolgreich, bei ca. 10 % verschwindet später auch hbsag, wobei die ansprechrate vom genotyp abhängt (a > b > c > d). nukleosid-und nukleotidanaloga hemmen die virusreplikation direkt und effektiv und sind sehr gut verträglich. sie werden erfolgreich bei der therapie chronischer hbv campylobacter jejuni and 1. related species hrsg.) manual of clinical microbiology die gattungen streptobacillus, campylo-3. bacter, arcobacter und helicobacter campylobacter und arcobacter spp mikrobiologische diagnostik. 2.aufl., georg thieme verlag stuttgart theiler's viruses the viruses and 2. their replication picornaviridae. the viruses and 3. their replication encyclopedia of virology picornaviridae. in: fauquet cm et 5. al (eds) virus taxonomy, classification and nomenclature of viruses, eighth report of the international committee on taxonomy of viruses diagnostic procedures for viral, rickettsial and chlamydial infections, 5 th edn picornaviren -klinik, 7. diagnostik und prävention medizinische mikrobiologie und infektiologie. 6. aufl epidemiological impact and 2. disease burden of congenital cytomegalovirus infection in europe immunomodulation by cytomegalo-3. viruses: manipulative strategies beyond evasion fields virology, 5 th edn. lippincott williams & williams cytomegaloviruses. molecular 5. biology and immunology medizinische mikrobiologie und infektiologie. 6. auflage principles and practice of infectious diseases konsiliarlabor für ehrlichien epidemiologie, diagnos-1. tik und tropenmedizin in klinik 3. und praxis, 4. aufl. thieme, stuttgart stanley sl medizinische mikrobiologie und infektiologie. 4. auflage mikroskopische diag nos tik: bakteriologie, mykologie, virologie, para sitologie diagnostik und therapie der parasitosen des menschen. 2. aufl picornaviridae. in: fauquet cm et 4. al. (eds) virus taxonomy, classification and nomenclature of viruses, eighth report of the international committee on taxonomy of viruses enteroviruses: polio-5. viruses, coxsackieviruses, echoviruses and enteroviruses 68-71 picornaviren -klinik, 6. diagnostik und prävention institution: institut für virologie haus 47 universitätskliniken, homburg/saar 66421 homburg/saar; ansprechpartner: herr prof all the virology on the www epstein-barr virus infection the epstein barr virus epstein-barr virus epstein-barr-virus: 40 4. years on ch-8028 zürich web-adressen hrsg) (1995) prin-3. ciples and practice of infectious diseases, 4 th edn. churchill livingstone epidemic profile of shiga-toxin-2. producing-escherichia coli o104:h4 outbreak in germany -preliminary report infektionen des darmes: miq 9 zentrum für infektionsmedizin principles 2. and practice of infectious disease manual of clinical micro biology 8 th edn fungi causing eumycotic mycetoma atlas of 2. clinical fungi, 2 nd edn mycetoma: a review flaviviruses. in: zuckerman 3 principles and practice of clinical virology principles and practices of infectious diseases clini-1. cal manifestations of tularemia in japan -analysis of 1355 cases observed between (ed) manual of clinical microbiology frühsommer-meningoenzephalitis-virus und russisches frühjahrs-sommer-enzephalitis-virus referenzzentren / expertenlaboratorien bun desforschungsinstitut für tiergesundheit, standort jena, naumburger str. 96a, 07743 jena, tel. +49 deutsche vereinigung zur bekämpfung der virus krankheiten e who world health organization (aktuelles über infektionskrankheiten tick-borne encephalitis virus -a review of an emerging zoonosis principles and practice of clinical virology institut für medizinische mikrobiologie phone: +31-30-2122600, fax +31-30-2512097, e-mail: info@cbs.knaw.nl web-adressen atlas of 1. clinical fungi, 2nd edn hyalohyphomyzeten. in: neu-2. meister b, geiss hk, braun rw, kimmig p (hrsg.) mikrobiologische diagnostik, 2. aufl. georg thieme verlag infections 3. caused by fusarium species aspergillus, fusarium and 4. other opportunistic moniliaceous fungi liebigstr. 21, 04103 leipzig taxonomy, biol-1. ogy, and periodontal aspects of fusobacterium nucleatum (hrsg) the prokaryotes, 2. aufl intrageneric relationships of members of the genus fusobacterium as determined by reverse transcriptase sequencing of small-subunit rrna lemierre's syndrome: still an important clinical entity philadelphia: lippincott williams and wilkins flaviviruses. in: zuckerman 2 principles and practice of clinical virology mosquito-borne diseases 1886 von j. e. weeks gezüchtet, bis 1950 als koch-weeks-bazillen bezeichnet infections due to haemophilus spe-1. cies other than h. influenzae diagnostic tests for chancroid chancroid and haemophilus 5. ducreyi: an update medizinische mikrobiologie und infektiologie. 4. auflage offizielle referenzzentren existieren nicht; als fachlich qualifiziert anzusehen sind sämtliche parasitologischen und tropenmedizinischen institutionen clinical parasitolo-1. gy. 9 th edn miq, qualitätsstandards in der mikrobiologisch-infektiologischen diagnostik. 4, parasitosen. gustav fischer hookworm disease: 6. current status and new directions. taylor & francis, london h onen. die natürliche infektion hinterlässt eine homologe (serotypspezifische) immunität helmut ruska haus antikörpernachweis bei hantavirusinfektion einschließlich serologischer bestätigungstests genomnachweis durch rt-pcr für hantaviren allgemein (genusspezifisch) sowie für die einzelnen virustypen (speziesspezifisch) voraussetzungen zur virusanzucht vorhanden web-adressen characterization and outcome following puumala virus infection: a retrospective analysis of 75 cases a global 2. perspective on hantavirus ecology, epidemiology, and disease zunehmende verbrei-3. tung der nephropathia epidemica in deutschland von der maus zum men-4. schen: gefahren durch hantaviren hantavirus-induced immunity in rodent reservoirs and humans network "rodent-borne pathogens" in germany: longitudinal studies on the geographical distribution and prevalence of hantavirus infections lehnert h, wer-1. dan k (hrsg) nephrologie und hochdruck. innere medizin. essentials, 4. auflage urinary tract infections and antibiotic 2. resistance hummers-pradier e (2010) the diagnosis of urinary tract infection: a systematic review medikamentöse therapie 4. von harnwegsinfekten helico-1. bacter pylori infection: a clinical overview pathogenese, dia-2. gnostik und therapie der helicobacter pylori-infektion bundesgesundheitsbl gesundheitsforsch gesundheitsschutz current concepts in the ma helicobacter pylori evo-4. lution and phenotypic diversification in a changing host strauß-allee 11, 93042 regensburg merkblatt/ratgeber hbv und mitteilungen der ständigen impfkommission prophylaxis, diagnosis and therapy of hepatitis-b-virus-(hbv-) infection: upgrade of the guideline hepatitis b virus infection 2. -natural history and clinical consequences hepatitis b virus and hepa mandell, douglas and bennett's principles and practice of infectious diseases natural history of chronic hepatitis 4. b virus infection hepatitis-b-vi-5. rus (hepadnaviridae) fields virology 5th edition ne, virusähnliche partikel oder rekombinante virale vektoren gehören hepatitis-c-virus -grundla-1. gen american gastro-2. enterological association technical review on the management of hepatitis c hepatitis c virus hepati-4. tis-c-virus -klinik und therapie hepatitis-c-virus -diag-5. nostik und prävention principles and practice of infectious diseases 6th edition expert opinion on the treatment of patients with chronic hepatitis c hepatitis d (delta) 1. virus hepatitis delta virus epidemiology, patho-3. genesis and management of hepatitis d: update and challenges ahead hepatitis-e-virus all the virology on the www hepatitis e: an over-1. view and recent advances in clinical and laboratory research safety and effica-3. cy of a recombinant hepatitis e vaccine hepatitis e virus characterization of a prototype strain 5. of hepatitis e virus dann können 2. aphasische symptome, hemiparese, kurze psychotische episoden, krampfanfälle (komplex-fokal beginnend mit sekundärer generalisation) sowie quantitative bewusstseinsstörungen bis hin zum koma auftreten konsiliarlaboratorium für herpes-simplex-virus (hsv) und institut für virologie und antivirale therapie, nordhäuserstr. 78, 99089 erfurt ansprechpartner: prof. dr. p. wutzler, prof. dr. a. sauerbrei all the virology on the www herpes simplex infections of the nervous system pathogenicity of human 2. herpesviruses due to specific pathogenicity genes, frontiers of virology 3 herpes-simplex-virus das risiko einer hantavirus-infektion kann nur durch vermeidung der exposition gegenüber den reservoirwirten gemindert werden. nahrungsmittel sollten für nager unzugänglich aufbewahrt werden. nagetier-in die bekämpfung der ektoparasiten (im rahmen des "integrated pest management") ist ein eckpfeiler bei der eindämmung der durch sie übertragenen krankheiten. bekämpfungsmaßnahmen sind, falls aus epidemiologischen gründen erforderlich, nur nach einer artbestimmung gezielt durchzuführen (schädlingsmonitoring mit befallsanalyse). bei flöhen ist insbesondere die larvenbekämpfung (mittel zur häutungsverhinderung, wachstumsregulatoren) an den lagerstätten der haustiere wichtig. die bekämpfung von zecken-und milbenplagen ist recht schwierig und wird ebenfalls mit insektiziden (z. b. malathion) durchgeführt. bettwanzen und raubwanzen werden mit insektiziden (z. b. pyrethroide) bekämpft. bei der stechmückenbekämpfung wird zwischen der larvenbekämpfung und der bekämpfung der adulten tiere unterschieden. während die larven im wasser z. b. mit bti (bacillus thuringiensis var. israelensis) bekämpft werden können, müssen gegen die adulten tiere insektizide wie z. b. pyrethroide eingesetzt werden. residualanwendungen von insektiziden durch das besprühen der innenwände von behausungen werden in endemiegebieten ebenfalls erfolgreich angewandt. der einsatz von natürlichen feinden (prädatoren, parasiten) ist im einzellfall nützlich (z. b. einführung von gambusien gegen anopheleslarven). zur entfernung der stationär saugenden zecken werden zeckenzangen, zeckenkarten o. ä. angeboten. die vernebelung oder ulv (ultra low volume)-ausbringung von insektiziden sind fachkundigem personal zu überlassen. auch die konsequente und fachgerechte bekämpfung der natürlichen reservoire (z. b. nager) stellt eine maßnahme zur eindämmung von ausbrüchen dar. eine meldepflicht nach dem infektionsschutzgesetz kann sich für spezielle, durch ektoparasiten übertragene erkrankungen ergeben 7 jeweilige krankheitserreger. referenzzentren / expertenlaboratorien 5 als fachlich qualifizierte ansprechstellen gelten praktisch alle parasitologischen und tropenmedizinische institutionen. die inzidenz der ie liegt bei etwa 3-10 episoden/100.000 personenjahre [1, 2] unbekannt. keratitis, onychomykose, hautläsionen. am auge finden sich keratitis und endophthalmitis;an der haut kutane und subkutane infektionen bei verbrennungswunden oder hautulzera, an den nägeln eine onychomykose. bei immunkompetenten entstehen nach traumatischer inokulation lokalisierte pilzherde. keine daten verfügbar. helga meisel, wolfgang jilg, detlev h. krüger synonym(e) gbv-c, hepatitis-g-virus, hgv. taxonomie gbv-c wurde (bisher ohne zuordnung zu einem virusgenus) als eigene spezies in die familie flaviviridae eingeordnet. innerhalb der flaviviren ist gbv-c eng mit dem hepatitis-c-virus (hcv) verwandt und weist auf aminosäureebene eine 28 %ige homologie zum hcv auf. der nachweis der milben im untersuchungsmaterial erfolgt mikroskopisch. im nativmaterial zeigen sich die milben recht beweglich. bei der demodex-blepharitis gelingt der milbennachweis auch mit der spaltlampe. besondere risikogruppen sind nicht bekannt. durch den engen kontakt zwischen mutter und kind erfolgt die übertragung wahrscheinlich vielfach schon im frühen kindesalter. da haarbalgmilben sehr beweglich sind, von einem follikel zum andern wandern können und auch frisch geschlüpfte larven schnell neue haarfollikel befallen, ist die übertragung aber auch in späterem alter möglich. wirksame maßnahmen zur verhütung des milbenbefalls sind nicht bekannt. eine meldepflicht nach dem infektionsschutzgesetz besteht nicht. jansen t, plewig g (1996) demodex-milben und ihre be-1.deutung für gesichtsdermatosen. münch med wochenschr 138:483-487 hans-günther sonntag koch-weeks-bazillen. nicht bekannt. eitrige konjunktivitis. eitrige konjunktivitis, hohes fieber bei bpf. nicht näher bekannt, mögliche/r von h. influenzae abweichende/r virulenzfaktor/en. konjunktivitis jeglicher genese (bakteriell, viral, durch pilze, allergisch u. a.). abstrich, bei verdacht auf bpf blut. mikroskopie: schlankes, nicht bekapseltes gramnegatives stäbchen. kultur: anspruchsvoll, wächst auf kochblutagar. biochemische differenzierung: nach wuchsfaktoren und biochemischen kriterien. keimnachweis und keimcharakterisierung. augentropfen oder -salben mit chloramphenicol, rifampicin, sulfonamiden oder chinolonen (norfloxacin, ciprofloxacin). sehr empfindlich gegen umwelteinflüsse. konjunktivitis v. a. in warmen ländern (nordafrika, südstaaten der usa), brazilian purpuric fever (v.a. in sao paulo, brasilien). mensch, nachweis auch in stechmücken gelungen. kinder. übertragung durch kontaktinfektion. unterbrechung durch kontaktinhibition. keine meldepflicht nach ifsg. schlüsselliteratur albritton wl (1982) nicht genau bekannt, häufig nur wenige tage. epiglottitis, tracheobronchitis, meningitis. epiglottitis, sinusitis, tracheobronchitis, meningitis, bronchopulmonale infektion, septische arthritis, weichteilinfektionen. kapselsubstanz (polyribitolphosphat) des typs b als wichtiger virulenzfaktor fördert die invasion und blockiert die phagozytose. immunität (gegen typ b) nach impfung von unbekannter dauer, gute schutzwirkung bei kindern. invasive erkrankungen können einer meningokokken-meningitis/sepsis ähneln, ansonsten je nach betroffenem organsystem. alle materialien von infizierten organsystemen. klassische therapie mit aminopenicillinen (ampicillin, amoxycillin). schwere infektionen wegen des risikos der ampicillinresistenz parenteral mit cefotaxim und analogen. leichtere infektionen mit oralcephalosporinen oder aminopenicillin mit betalaktamase-inhibitor. ampicillinresistenz durch plasmid kodierte betalaktamase, in mittel-und nordeuropa 10 %, usa, spanien, italien bis 50 %; dabei oft multiresistenz (chloramphenicol, tetrazykline, cotrimoxazol). häufungen in kinderheimen und krankenhäusern, epidemien nicht bekannt. nur beim menschen, vorwiegend nasopharynx, seltener mundhöhle, genitalschleimhaut (h. influenzae biotyp iv). keimträger häufig unter kindern und erwachsenen. sehr empfindlich gegen umwelteinflüsse. kleinkinder bis zum 2. lebensjahr, patienten mit virusinfektionen der atemwege und defekten der mukoziliären clearance. übertragung durch kontakt-und tröpfcheninfektion, begünstigt durch enge lebensverhältnisse. hohes übertragungsrisiko bei kindern. aktive impfung mit konjugatimpfstoff, empfohlen für kinder bis zum 6. lebensjahr und bei funktioneller oder anatomischer asplenie, gute schutzwirkung, deutlicher rückgang der infektionen durch den typ b. chemoprophylaxe bei meningitis oder epiglottitis für kontaktpersonen mit rifampicin oral (über 4 tage). ausbrüche nicht bekannt, bei auftreten der erkrankung chemoprophylaxe bei kontaktpersonen. namentlich nach § 7 abs.1 ifsg nur für den direkten nachweis aus liquor oder blut (durch das labor). hafnia ist die alte bezeichnung für den namen kopenhagen. gramnegative stäbchenbakterien, beweglich durch peritriche begeißelung. das genom von h. alvei ist vollständig sequenziert. siehe auch www.ncbi.nlm.nih.gov. wachstum innerhalb von 24 stunden. nicht bekannt. postoperative wundheilungsstörungen, pneumonien, abszesse, harnwegsinfektionen. entsprechend dem jeweiligen krankheitsbild. entsprechend dem jeweiligen krankheitsbild. differenzialdiagnose ausschluss anderer infektionserreger. durch übertritt in die blutbahn kann es zur sepsis kommen. nicht bekannt. fieber. fieber. differenzialdiagnose ausschluss anderer infektionserreger. kulturelle anzüchtung: s. fakultativ pathogene e. coli 7 escherichia coli. serologische differenzierung: es wurden 68 o-und 64 h-antigentypen nachgewiesen. phagentypisierung: speziallaboratorien. blut, eiter, sputum, urin. befund / interpretation erregernachweis ausschlaggebend. häufig multiresistenz. durch hafnia alvei bedingte erkrankungen sind selten und werden allenfalls im hospital bei krankenhauserworbenen infektionen nachgewiesen. hafnia alvei kommt im darm von menschen, tieren und vögeln vor, aber auch in wasser, abwasser, mist sowie im erdreich risikogruppen sind immunsupprimierte und abwehrgeschwächte patienten. mit hoher wahrscheinlichkeit schmierinfektion. das hfrs wird in erster linie symptomatisch behandelt. bei schweren hfrs-fällen erwies sich die frühzeitige antivirale chemotherapie mit ribavirin als erfolgreich, für das hantavirus-lungensyndrom fehlt bisher der nachweis der wirksamkeit. hantaviren sind in vitro gegenüber ribavirin empfindlich. andere wirksame virostatika sind nicht bekannt. hantaviren sind weltweit verbreitet. generell werden die in der alten welt vorkommenden viren, von den in der neuen welt endemischen unterschieden (7abb. 1). zielorgane sind urethra, blase, ureter und niere. bei der immunabwehr spielen lokale immunmechanismen der mukosa, die bildung von sekretorischem immunglobulin a und die anwesenheit von tamm-horsfall-protein (thp) eine rolle. thp bindet uropathogene erreger und bewirkt so die ausscheidung mit dem urin. bei kindern, älteren frauen und diabetikern ist die thp-ausscheidung vermindert. ein wichtiger faktor in der pathogenese rezidivierender harnweginfekte ist die abwesenheit von toll-like-rezeptoren (tlr). toll-like-rezeptoren sind teil des innaten immunsystems. diese sowohl auf epithelialen als auch nicht-epithelialen zellen lokalisierte faktoren erkennen pathogene und aktivieren das innate immunsystem sowie inflammatorische prozesse. insbesondere der tlr-4 wird mit harnwegsinfektionen in verbindung gebracht. experimentelle untersuchungen deuten daraufhin, dass dysregulierte trl bzw. die defizienz von trl für chronisch rezidivierende verläufe von harnwegsinfektionen verantwortlich sind. an dieser induktion ist eine gruppe von 29 genen beteiligt, die auf einer so genannten pathogenitätsinsel (cag-pathogenitätsinsel) lokalisiert sind. die gene erlauben h. pylori wahrscheinlich, einen so genannten typ-iv-sekretionsapparat zu bilden, mit dem die bakterien das caga-protein in die magenschleimhautzellen "injizieren". caga wird nach der injektion von zellulären kinasen phosphoryliert und löst multiple signaltransduktionsvorgänge aus, die zur malignen transformation der zellen beitragen könnten. beim kontakt von cag-positiven stämmen mit epithelzellen können auch peptidoglykanbruchstücke in die zelle eingeschleust werden, die das angeborene immunsystem über den musterkennungsrezeptor nod1 aktivieren. alle mit h. pylori infizierten personen entwickeln eine entzündliche reaktion der magenschleimhaut, die in der regel im magenantrum besonders ausgeprägt ist (chronische typ-b-gastritis). auf dem boden der durch die h.-pylori-infektion ausgelösten gastritis (die selbst entweder asymptomatisch sein oder auch zu uncharakteristischen oberbauchbeschwerden führen kann) können verschiedene folgekrankheiten entstehen. die h.-pylori-gastritis heilt in der regel nicht spontan aus, nur im hohen alter kann es infolge einer schleimhautatrophie zur spontanen elimination der erreger kommen. anämie, dysphagie, erbrechen, gewichtsverlust. das zwölffingerdarmgeschwür kommt praktisch ausschließlich bei patienten vor, die mit h. pylori infiziert sind. die eradikation der h.-pylori-infektion verhindert ulkusrückfälle mit großer sicherheit. 7 chronische gastritis, schmerzen, blutiges erbrechen. die h.-pylori-infektion ist ein wichtiger risikofaktor für die entstehung des magenadenokarzinoms. das karzinomrisiko ist umso größer, je früher die infektion erworben wurde. die magenschleimhaut ist bei gesunden personen praktisch frei von lymphatischem gewebe. die h.-pylori-infektion führt häufig zur bildung von lymphfollikeln in der submukosa (sekundäres malt). sie ist daher die voraussetzung für die entstehung von malignen non-hodgkin-lymphomen des magens. ein zusammenhang zwischen h.-pylori-infektionen und idiopathischer thrombozytischer purpura, eisenmangel-anämie und asthma wie auch anderen atopischen erkrankungen wird diskutiert immunantwort im verlauf der h.-pylori-infektion werden antikörper gegen h.-pylori-antigene gebildet, die sich zwar für die serologische diagnostik eignen, aber nicht zu einer protektiven immunität führen. die reaktion der magenschleimhaut auf die infektion ist die ausbildung einer so genannten chronisch aktiven gastritis, die durch infiltration mit neutrophilen granulozyten und lymphozyten charakterisiert ist. eine zentrale rolle in der steuerung der entzündungsreaktion spielt interleukin-8. im infiltrat herrschen t-lymphozyten vom th1-typ vor. eine ausführliche darstellung der immunpathogenese der h. pylori-infektion findet sich in der angegebenen literatur. die umfangreichen differenzialdiagnosen der h.-pylori-assoziierten magenerkrankungen (z. b. medikamentös induzierte ulzera) fallen in den bereich der gastroenterologie und können im rahmen dieses lexikons nicht dargestellt werden. biopsien von magen-oder duodenalschleimhaut, serum, stuhl. atemtest. bei infizierten pferden wurde das virus vor allem im endothel der lungengefäße, aber auch in anderen organen nachgewiesen. die pathophysiologie der menschlichen erkrankung, insbesondere die ursache der verzögert aufgetretenen enzephalitis, ist nicht bekannt. im serum infizierter menschen und tiere können antikörper gegen das virus nachgewiesen werden. eine hendra-virus-infektion sollte nach derzeitigem stand des wissens nur bei patienten in erwägung ge-zogen werden, die kontakt mit infizierten pferden hatten. differentialdiagnostisch kommen andere erreger von atemwegsinfektionen und enzephalitiden in betracht. blut, serum, liquor, abstriche. derzeit sind keine kommerziellen tests zur diagnose einer hendra-virus-infektion erhältlich. bei begründetem verdacht sollte kontakt z. b. zum bernhard-nocht-institut aufgenommen werden, um die diagnostik sowie das notwendige untersuchungsmaterial zu besprechen. die anzucht des virus sollte nur in hochsicherheitslabors (s4) durchgeführt werden. die untersuchungsergebnisse sollten mit den zuständigen ärzten im referenzzentrum und dem gesundheitsamt besprochen werden. ribavirin ist in vitro wirksam, so dass ein therapieversuch mangels alternativen gerechtfertigt erscheint. monoklonale antikörper zur postexpositionsprophylaxe werden entwickelt. keine bekannt. alle 13 hendra-virus-ausbrüche fanden in australien (bundesstaaten queensland und new south wales) statt. als natürlicher wirt des hendra-virus gelten flughunde der gattung pteropus, die nach infektion jedoch keine krankheitssymptome zeigen. alle infizierten menschen hatten kontakt zu erkrankten pferden. der genaue übertragungsweg von flughunden auf pferde und von pferden auf menschen ist nicht bekannt. es gibt keinen anhalt für eine übertragung von mensch zu mensch oder von flughunden auf den menschen. eine replikation des hav konnte bisher nur in der leber nachgewiesen werden. die destruktion der infizierten zellen erfolgt über zytotoxische t-zellen. anti-hav-igm ist bei fast allen patienten mit beginn der symptomatik nachweisbar. kurz darauf finden sich anti-hav-igg antikörper, die lebenslang persistieren. die hepatitis-a-virusinfektion ist von anderen hepatitiden viraler oder nicht viraler ätiologie mit einer serologischen labordiagnostik abzugrenzen. die routinediagnostik einer akuten hav-infektion erfolgt über den nachweis spezifischer antikörper im serum. in speziellen fällen kann der virusnachweis im stuhl oder blut angezeigt sein. die diagnose einer akuten hepatitis-a-virusinfektion erfolgt über den nachweis von anti-hav-igm im serum. der nachweis der hav-rna über rt-pcr im stuhl oder blut kann insbesondere bei unklaren protrahierten verläufen zur diagnose-sicherung herangezogen werden. zur feststellung der immunität wird ein gesamtantikörpernachweis gegen hav (anti-hav) durchgeführt. der nachweis von anti-hav-igm ist zeichen einer akuten oder in der regel kürzlich abgelaufenen hav-infektion. falsch positives anti-hav-igm kommt vor, daher muss ein positives ergebnis grundsätzlich durch eine gesamtantikörperbestimmung gegen hav (anti-hav) ergänzt werden. bei protrahierten verläufen findet sich anti-hav-igm teilweise über monate bis jahre. in diesen fällen kann häufig der nachweis der hav-rna im stuhl oder blut die diagnose sichern. immunität gegen hav wird ab einer anti-hav konzentration von 10 u/l angenommen. eine hav-spezifische therapie steht nicht zur verfügung. die epidemiologie der hepatitis a unterliegt einer kontinuierlichen veränderung. obgleich das hav weltweit verbreitet ist, zeigen sich extreme unterschiede in der seroprävalenz von land zu land. neben dem menschen sind nur wenige nicht-humane primaten infizierbar. in den westlichen industriestaaten ist das risiko einer hav-infektion niedrig. einem erhöhten erkrankungsrisiko unterliegen aber auch dort bestimmte berufsgruppen, wie z. b. personal in kindertagesstätten und kliniken oder arbeiter in kanal-und kläranlagen. ein hohes risiko einer hav-infektion haben personen, die aus einem gebiet mit niedriger hav-inzidenz in ein endemiegebiet reisen. die transmission des hav findet fast ausschließlich über den fäkal-oralen weg, insbesondere über kontaminiertes wasser, kontaminierte nahrungsmittel und schmierinfektionen statt. übertragungen des hav via bluttransfusion und kontaminierte blutprodukte sind beschrieben, aber selten. zur prävention einer hav-infektion stehen gut verträgliche inaktivierte impfstoffe zur verfügung, die zu einem sicheren schutz führen. als post-expositionelle maßnahme werden gegebenenfalls immunglobuline verabreicht. bei infektionsgefährdeten personen im umfeld eines ausbruchs werden impfungen mit einem impfstoff, der ein 2-dosen-schema zulässt, empfohlen. bei individuell besonders gefährdeten personen kann eine passive immunisierung mit immunglobulin erfolgen. eine neuere untersuchung zeigt, dass zur unterbrechung der infektkette auch die familienangehörigen von geimpften kontaktpersonen aktiv immunisiert werden sollten. der aktuelle stand der empfohlenen präventiv-und bekämpfungsmethoden ist im merkblatt "hepatitis-a-erkennung und verhütung" des robert koch-instituts zusammengefasst. zur primärprävention wird eine impfung mit rekombinantem, heterolog exprimierten hbsag, das an aluminiumhydroxid adsorbiert ist, eingesetzt. seit 1995 empfiehlt die ständige impfkommission (stiko) in deutschland die generelle aktive hepatitis-b-immunisierung von säuglingen, kindern und jugendlichen sowie von risikogruppen. die grundimmunisierung erfolgt in der regel durch dreifache gabe der vakzine in dem zeitintervall von einem monat zwischen den ersten beiden und sechs bis zwölf monaten zwischen der zweiten und dritten impfung. der anti-hbs-titer ein bis zwei monate nach erfolgter grundimmunisierung gestattet aussagen über den impferfolg. liegt er über 100 ie/l, ist ein langzeitschutz für zu erwarten. die genaue schutzdauer ist nicht bekannt, sie dürfte aber über 15 jahren liegen. bei menschen mit hohem infektionsrisiko sollte aus sicherheitsgründen nach 10 jahren eine auffrischimpfung durchgeführt werden. menschen mit immundefekten, aber auch fünf bis 10 % der gesunden bevölkerung sprechen auf die impfung nicht oder schlecht an (non-responder, anti-hbs nach 3. impfung 10 ie/l, und hypo-respondern, anti-hbs nach 3. impfung 10-100 ie/l). ihnen wird eine erneute impfung mit kontrolle empfohlen. zur verhinderung von neuinfektionen sind das blutspender-screening auf hbsag und anti-hbc, das hbsag-screening von schwangeren, die kontinuierliche arbeitsmedizinische überwachung von medizinischem personal (hbv-serostatus, impfung) inklusive deren unterweisung in adäquater arbeitsweise (z. b. schutzhandschuhe, einmalspritzen, desinfektion von medizinischen geräten), die information der bevölkerung und insbesondere der risikopersonen (kondombenutzung, gefahren des "needle sharings") wichtig. helga meisel, wolfgang jilg, detlev h. krüger erregerspezies aufgrund von ähnlichkeiten mit den flavi-und pestiviren in genomorganisation, replikationsmechanismus und prozessierung des polyproteins wurde hcv der familie flaviviridae zugeordnet. im unterschied zu den humanen flaviviren (z. b. gelbfiebervirus, fs-me-virus) und den animalen pestiviren wird hcv nicht durch arthropoden übertragen und führt im hohen maße zu chronischen verläufen. aus diesem grund wurde hcv 1999 als ein eigenständiges genus (hepaciviren) in die familie flaviviridae eingeordnet. interessanterweise gibt es auch eine ferne genetische verwandtschaft von hcv zu pflanzenpathogenen erregern (potyviren, carmoviren). nachdem klar wurde, dass mindestens ein erreger von infektiösen non-a-non-b-hepatitiden existieren müsste, wurden aus dem plasma eines infizierten schimpansen cdna-expressionsbanken hergestellt. ihre testung mit dem serum eines patienten mit einer chronischen non-a-non-b-hepatitis führte 1989 zum auffinden des ersten hcv-spezifischen klons 5-1-1, der wiederum zur identifizierung eines größeren genabschnitts diente und mit hilfe spezieller molekularer "walking"-techniken schließlich zur definition des kompletten virusgenoms führte. die histologischen befunde bei der akuten und chronischen hepatitis c unterscheiden sich nicht von denen bei anderen virushepatitiden: bei akuten hepatitiden treten hepatozelluläre nekrosen mit entzündungszellen (makrophagen, lymphozyten) im leberparenchym und in der portalen region auf. die schwere chronische hepatitis c ist histologisch durch inflammatorische destruktion und progressive fibrose gekennzeichnet. verschiedene scoring-systeme erfassen sowohl die aktivität als auch das stadium einer chronischen hepatitis. bei der chronischen hepatitis c bilden sich typischerweise lymphoplasmazelluläre infiltrate in den portalfeldern. daneben treten wie bei anderen chronischen virushepatitiden auch nekrosen (mottenfraßnekrosen, intralobuläre nekrosen), leberparenchymverfettung und fibrose auf. bei der hcv-infektion werden antikörper gegen struktur-und nichtstrukturproteine gebildet. obwohl jene gegen die hypervariable region 1 (hvr1) von e2 neutralisierend wirken, scheint ihr auftreten nur im frühen stadium der infektion zu einem limitierten verlauf beizutragen. neben der angeborenen immunität (nk-zellen, ifn) ist während der akuten hcv-infektion eine starke multispezifische immunantwort von cd4 + -und cd8 + -t-zellen für die erfolgreiche viruselimination entscheidend. bei chronisch infizierten patienten wurden abgeschwächte t-zell-antworten beobachtet. hcv-spezifische cd4 + -t-zellen können im gegensatz zu den antikörpern und cd8 + -t-zellen noch jahrzehnte nach akut limitierter hcv-infektion nachgewiesen werden und stellen somit einen marker für eine durchgemachte, bereits seronegative hepatitis c dar. nach infektionsschutzgesetz (ifsg § § 6 und 7) müssen akute hepatitis-c-infektionen oder der nachweis einer hcv-infektion, soweit nicht schon eine chronische hcv-infektion bekannt ist, gemeldet werden. in einigen bundesländern ist darüber hinaus jeder anti-hcv-träger nach länderverordnung meldepflichtig. igg-und igm-antikörper werden gegen antigene determinanten aller drei orfs gebildet. andere virale hepatitiden. serum. die diagnostik der hev-infektion beruht auf dem nachweis von spezifischen antikörpern der igm-und igg-klasse. dieser test wird routinemäßig von den meisten labors angeboten. darüber hinaus steht für bestimmte fragestellungen auch der nachweis viraler hev-rna mittels nukleinamplifikationstest zur verfügung. igm-und igg-antikörper gegen hev können im elisa untersucht werden. igm anti-hev kann 1-4 wochen nach der infektion nachgewiesen werden. etwa 3 monate nach beginn der erkrankung sind die igm-antikörper nicht mehr nachweisbar. auch ein ansteigender igg-titer ist beweisend für die floride hev infektion. es gibt keine spezifische therapie der hev-infektion. bisherige seroepidemiologische studien zeigten, dass der erreger in südostasien, indien, dem nahen osten, zentralasien und mittel-und südamerika vorkommt. aufgrund des vorkommens des erregers bei verschiedenen tierspezies, wie schweinen, affen, ratten sowie mäusen, geht man davon aus, dass es sich bei der infektion um eine zoonose handelt. bei schwangeren kann es zu fulminanten verläufen kommen. hev wird fäkal-oral übertragen und ist vorwiegend mit kontaminiertem trinkwasser assoziiert. ein sexueller übertragungsweg erscheint ebenso wahrscheinlich, da eine infektionshäufung im jungen erwachsenenalter beobachtet wird. ein impfstoff wurde in einer phase-ii-studie erprobt [3] und wird wahrscheinlich bald zur verfügung stehen. hygienemaßnahmen wie bei anderen viralen hepatitiden, die fäkal-oral übertragen werden. nach dem infektionsschutzgesetz (ifsg) § 6 besteht beim krankheitsverdacht, der erkrankung sowie beim tod an einer akuten hepatitis auch bei hepatitis e eine namentliche meldepflicht. darüber hinaus besteht nach ifsg § 7 beim nachweis einer akuten infektion mit hepatitis-e-virus durch das labor eine namentliche meldepflicht. 7 hepatitis e virus (hev) herpes-simplex-virus 1, herpes-simplex-virus 2 der genus simplexvirus ist der familie herpesviridae und der unterfamilie der alphaherpesvirinae zugeordnet. anhand von dna-homologien, serologischer typisierung und klinischer symptomatik unterscheidet man zwei serotypen: das humane herpesvirus 1 (herpes-simplex-virus 1, hsv-1) und das humane herpesvirus 2 (herpes-simplex-virus 2, hsv-2). herpes bedeutet "kriechen, kribbeln, schleichen" und wurde von hippokrates für bestimmte hautkrankheiten verwendet. morton (1694) gibt eine genaue beschreibung des krankheitsbildes "herpes febrilis". gegen ende des 19. jahrhunderts wird die terminologie herpesvirus hominis (simplex) eingeführt. generalisierter herpes des neugeborenen. die inkubationsperiode beträgt beim herpes neonatorum 9-11 tage. die manifestation beginnt am 9. bis 11. lebenstag mit einer lokalen infektion der haut, der mundschleimhaut und der augen, mukokutanen bläschen, keratokonjunktivitis oder choriorenitis. anschließend erfolgt das stadium des herpes generalisatus mit dem befall der inneren organe und einem sepsisähnlichen bild. die herpes-sepsis bei neugeborenen ist überwiegend die folge einer infektion im geburtskanal. die häufigkeit des subklinischen verlaufs ist unbekannt. das herpes neonatorum verläuft mit der häufigkeit von 1 auf 2.000-5.000 geburten unbehandelt meistens letal. die letalität beträgt ohne behandlung 80 %. entwicklung einer enzephalitis meist am 9./10. lebenstag bei hämatogener genese, bei retrogradem axonalem virustransport am 16./17. lebenstag mit den symptomen von fokalen oder generalisierten krampfanfällen, tremor, unruhe oder lethargie. andere infektionen, intrakranielle blutung. meningoencephalitis herpetica. stunden bis tage. kopfschmerze, fieber, bewusstseinsstörung. oft ist eine aktivierung von cd3-und cd8-positiven t-lymphozyten sowie cd68-positiven makrophagen sichtbar, die auch nach abklingen der akuten infektion oft noch nachweisbar sind. enzephalitis durch andere viren (z. b. vzv, cmv) bzw. bakterien (z. b. tbk, mykoplasmen), hirnabszess, hirntumor, durchblutungsstörungen, alkoholentzugssyndrom. inkubationszeit unbekannt, meist reaktivierung. rötung, fremdkörpergefühl, "sand im auge", lichtscheu und sehverschlechterung. die hsv-infektion der kornea und der bindehaut führt zu läsionen vor allem auf dem epithel der hornhaut. erwachsene sind am häufigsten betroffen. wenn neugeborene betroffen sind, dann meistens nur an einem auge. schwere serös-eitrige konjunktivitis. trübung und oberflächliche ulzerationen der hornhaut. eventuelles auftreten von herpesbläschen im bereich der augenlider. rasche ohthalmologische behandlung. choriorenitis bei generalisation. zoster ophthalmicus. eccema herpeticatum, pustulosis acuta varioliformis juliusberg. es kann sich um eine erstinfektion oder um eine reaktivierung handeln. monomorphe vesikuloerosive eruption auf ekzematöser haut bei teils schweren allgemeinsymptomen, fieber und lymphknotenschwellung. charakterisiert durch eine schwere generalisierte infektion meistens auf dem boden einer bestehenden hauterkrankung (meistens atopisches ekzem) mit flüssigkeitsverlust sowie der gefahr einer bakteriellen superinfektion und möglicher sepsis bei virämien mit nachfolgendem multiorganversagen. die übertragung erfolgt mittels kontakt-oder schmierinfektion, die virusausbreitung im patienten kann per continuitatem, lymphogen oder hämatogen erfolgen. es entstehen verdickte krusten auf der haut mit ekzem-effloreszenzen, die sich diffus und rasch ausdehnen können. akute varizelleninfektion des neurodermitikers, eczema vaccinatum, varioliforme pyodermie. müdigkeit, abgeschlagenheit, appetitlosigkeit, druckgefühl im rechten oberbauch, erbrechen, fieber, ikterus. ein sehr seltenes krankheitsbild mit meistens tödlicher folge. infektiöse (z. b. hepatitis a, b, c) und nichtinfektiöse hepatitiden (z. b. autoimmun, toxisch). als untersuchungsmaterial dienen bläschenflüssigkeit, liquor, tränenflüssigkeit, bronchiallavage, rachenspülwasser. neugeborene von müttern mit primärem oder rekurrierendem herpes genitalis, personen mit ekzemen, immunsupprimierte oder immungeschwächte perso-nen, z. b. unter zytostatischer therapie, mit infekten, mit aids. das virus wird durch direkten kontakt über speichel, urin, stuhl und andere körperflüssigkeiten übertragen. hsv-2 meist über sexuellen kontakt oder unter der geburt im geburtskanal. es besteht ein dringender bedarf zur entwicklung einer effizienten prophylaxe gegen die herpes-simplex-virus-infektion. prophylaxe des herpes neonatorum: bei rekurrierendem herpes genitalis der schwangeren regelmäßiger versuch des virusnachweises. falls negativ, natürliche geburt; falls in den letzten wochen der schwangerschaft positiv, schnittentbindung erwägen. bei sichtbaren erscheinungen stets sectio innerhalb von 24 h nach blasensprung. passive immunisierung des kindes wirkungslos: herpes neonatorum tritt auch dann auf, wenn antikörper der mutter passiv übertragen wurden. aciclovir beim kind bei den ersten anzeichen einer herpesinfektion verabreichen. pflegepersonal mit rezidivierender herpes-simplex-infektion nicht auf neugeborenenstationen beschäftigen. enzephalitis ist meldepflichtig. key: cord-022752-bdve1ydv authors: knuf, markus title: infektiologie date: 2019-08-09 journal: lehrbuch kinderund jugendmedizin doi: 10.1016/b978-3-437-21661-9.00010-8 sha: doc_id: 22752 cord_uid: bdve1ydv nan hier sind die symptome fieber, enanthem der mundschleimhaut mit bläschen, vor allem im bereich der zunge, des harten gaumens, der gingiva und der wangenschleimhaut sowie ein exanthem mit bläschenbildung an den handinnenflächen, fußsohlen und am gesäß zu nennen. die hand-fuß-mund-krankheit wird durch enteroviren der gruppe a verursacht. hierzu gehören coxsackie-a-viren, das humane enterovirus 71 und andere serotypen. zu den primär durch eine blickdiagnose zu diagnostizierenden infektionskrankheiten gehören auch die windpocken (varizellen) (› abb. 10.5) mit kurz vor exanthemausbruch bestehendem fieber sowie kopf-und gliederschmerzen. die inkubationszeit reicht von 10-21 (meistens 14-17) tagen. es bilden sich dann im bereich des rumpfes und gesichts, typischerweise aber auch des behaarten kopfes juckende rote flecken, aus denen dann knötchen und blasen entstehen. das nebeneinander von papeln, krusten und pusteln ist typisch (sog. heubnersche sternenkarte) für das windpocken-exanthem. auch die schleimhäute im bereich des mundes (hier v. a. der gaumen), der nase und der augen können betroffen sein. bei den windpocken ist zu beachten, dass schwerwiegende komplikationen wie eine optikusneuritis, zerebellitis oder enzephalitis letztendlich auch bei mildem exanthem zur vorstellung in der praxis oder klinik führen können. als "folgeerkrankung" der windpocken und weitere blickdiagnose ist herpes zoster (gürtelrose, kopfrose) zu nennen. nach einer latenzphase kommt es zu einer reaktivierung der varizella-zoster-viren (vzv), die nach erstinfektion in den spinalganglien verblie-ben sind. bei etwa 90 % der patienten bestehen prodromalsymptome mit schmerzen, dysästhesien, parästhesien, intermittierenden oder brennenden missempfindungen. die symptome treten typischerweise 2-5 tage vor auftreten der hauterscheinungen auf. dann kommt es zu einem (meist) unilateralen erythem, das typischerweise ein bis drei dermatome umfasst, mit makulopapulösen effloreszenzen sowie gruppierten bläschen (oft erst 1 tag später), aus denen vzv nachweisbar sind. nach 2-4 wochen ist das krankheitsbild hinsichtlich der hauterscheinungen ausgeheilt. langfristig können jedoch schmerzen und dysästhe sien persistieren. sonderformen sind zoster ophthalmicus (7-17 %, steigende inzidenz im alter, 50 % mit komplikationen) (› abb. 10.6), zoster oticus (ramsay-hunt-syndrom) sowie zoster generalisatus. weitere typische blickdiagnosen sind das erythema infectiosum (ringelröteln), › abb. 10.7 gibt das typische exanthem mit girlandenartigen veränderungen wieder. der erreger der ringelröteln ist das parvovirus b19. bei scharlach bestehen ein feinfleckiges exanthem mit aussparung der mundpartie ("periorale blässe") sowie eine tonsillopharyngitis, ein enanthem und eine himbeerzunge (› abb. 10.8). in der rekonvaleszenz kommt es zu einer schuppung, besonders an den händen. herz, nieren und nervensystem können beteiligt sein. scharlach ist von der unkomplizierten tonsillopharyngitis abzugrenzen (› abb. 10.9). beide krankheitsbilder werden durch β-hämolysierende streptokokken der gruppe a (gas) hervorgerufen. im vordergrund stehen bei infektionskrankheiten im kindesund jugendalter fieber und atemwegsinfektionen. fieber › tab. 10.1 gibt einen überblick über häufige und seltene ursachen für fieber bei kindern (und jugendlichen). überwiegend sind viren für atemwegsinfektionen im kindesalter verantwortlich. die unterscheidung kann gut klinisch vorgenommen werden. › tab. 10.2 gibt typische kriterien für die unterscheidung viraler von bakteriellen atemwegsinfektionen wieder. ein weiteres typisches klinisches symptom sind petechien (und sugillationen) (› abb. 10.10). petechien können praktisch bei allen viruserkrankungen passager und in milder form auftreten. daneben sind bluterkrankungen • vor der initiierung einer antiinfektiven therapie (z. b. antibiotika, antivirale medikamente, antimykotika) sollte möglichst immer eine spezifische infektionskrankheit diagnostiziert werden! • die diagnose einer infektionskrankheit kann klinisch und / oder mittels einer infektiologischen (mikrobiologie / virologie) labordiagnostik erfolgen! • der einsatz von antiinfektiva wird nicht ausschließlich durch laborwerte indiziert! • antiinfektiva sind keine antipyretika! • in der pädiatrie wird die mehrzahl der infektionskrankheiten durch viren hervorgerufen! die klinische diagnose einer infektionskrankheit besteht aus drei elementen: 1. erreger 2. erfolgsorgan 3. expositionsanamnese / grundkrankheit (disposition) bei kenntnis von zwei der drei genannten faktoren kann auf den dritten geschlossen und eine kalkulierte antiinfektive therapie begonnen werden. • material nicht formalinfixieren oder einfrieren. • material aus der pathologie ist häufig bakteriell kontaminiert (keine strenge sterilität bei verarbeitung). • punktate nicht in blutkulturflaschen einsenden. • nativurin in urinmonovette, keine sog. "uricults". begleitinformation für das labor: • art des untersuchungsmaterials und möglichst genaue lokalisation • klinische symptome • verdachtsdiagnose • fragestellung • ggf. reiseanamnese, immunstatus • wichtig: therapie mit antiinfektiva, ggf. welche eine mikrobiologische diagnostik gelingt dann besonders gut, wenn vor einsatz eines antiinfektivums eine blutkultur oder materialprobe gewonnen wird und diese mit kurzer transportzeit im labor ankommt. bezüglich des direkten mikrobiologischen erregernachweises ist immer der zu erwartende erreger bei der auswahl des entnahme-und kulturmediums zu berücksichtigen. im zweifelsfall ist mit dem mikrobiologischen labor rücksprache zu halten, welches entnahmemedium (z. b. bei v. a. pertussis: abstrich mit flexiblem kalziumalginat-oder dacron-tupfer vom hinteren nasopharynx, nicht rachen!) geeignet ist, den erreger auch tatsächlich zu identifizieren. es stehen verschiedene nährmedien zur verfügung. man unterscheidet selektivnährmedien (chemische zusätze, antibiotika, balance zwischen selektivität und zu starker unterdrückung des wachstums der zielkeime) von differenzialnährmedien / chromogenagar (orientierende identifizierung der bakterien), universalnährmedien (keine wachstumshemmer), optimalnährmedien (für sehr anspruchsvolle bakterien) von spezialnährmedien (zusätze für spezielle spezies-identifizierung) und selektive differenzialnährmedien (kombination von selektion und differenzierung). zu berücksichtigen ist, dass chlamydien, mykoplasmen, borrelien oder treponema pallidum (syphilis) nicht kultivierbare bakterien sind. hierfür sind alternative diagnostische verfahren (pcr, serologie) zu wählen. hier sind die polymerase-kettenreaktion (pcr) und die multiplex-pcr fest etablierte verfahren. die vorteile einer pcr sind meist eine höhere sensitivität sowie der nachweis von schwer anzüchtbaren bakterien. nachteilig ist, dass sich keine informationen über resistenzen (ausnahme multiresistente tbc, mrsa, vre, manche carbapenemasen) gewinnen lassen. eine begleitende kultur ist daher trotzdem notwendig. weitere molekularbiologische verfahren sind: pulsfeld-gelelektrophorese (pfge; genetische verwandtschaft von isolaten), sequenzierung (typisierungsmethode in der virologie: z. b. enteroviren), genomsequenzierung. serologische methoden weisen einen erreger indirekt nach: es wird nicht der krankheitserreger an sich nachgewiesen, sondern die gegen den krankheitserreger (vorsicht: kreuzreaktionen!) gerichteten antikörper. es wird also indirekt eine infektion diagnostiziert. wichtig bei der interpretation von serologischen ergebnissen sind die infektionsanamnese und der klinische kontext! viele infektionen weisen eine lang anhaltende immunantwort und damit im serum nachweisbare antikörper auf. es ist essenziell, eine frische von einer abgelaufenen infektion zu unterscheiden. folgende methoden stehen zur verfügung: • nachweis eines igg-titeranstiegs (2-bis) 4-facher anstieg nach 2-4 wochen, serumpaare sollten immer angestrebt werden (keine igg-einzelbestimmungen!) • nachweis von immunglobulin m (igm) • aviditätsbestimmung der antikörper (spezialuntersuchung) eine frische infektion kann durch eine igm-einzelbestimmung diagnostiziert werden. igm-nachweise gelingen etwa 1 woche nach beginn der inkubationszeit. da in der pädiatrie bei einigen sog. "kinderkrankheiten" (z. b. varizellen) lange inkubationszeiten bestehen, gelingt der igm-nachweis oft schon zu krankheitsbeginn. in der regel persistieren igm-antikörper 4-8 wochen. bei immunsupprimierten patienten können sie aber monate-bis jahrelang persistieren. ferner kann es durch oligoklonale stimulation zu einer langzeitpersistenz kommen (borrelien, ebv, vzv u. a.), die damit eine akute infektion vortäuscht. daneben sind kreuzreaktionen zwischen einzelnen erregern bekannt (viren der herpesgruppe, zwischen mumps-und parainfluenzavirus) folgende serologische verfahren stehen zur verfügung: • neutralisationstest (hohe spezifität) schnellteste sind antigennachweise gegen oft virale erreger und beruhen auf immunchromatografischen, meist auf enzym-immunoassays (eia). beispiele: rsv-, influenza-schnelltest. grundsätzlich sind bei der infektiologischen diagnostik und insbesondere bei der verwendung von schnelltestverfahren testtheoretische grundlagen zu berücksichtigen. › abb. 10.12 gibt eine vierfeldertafel mit angabe der sensitivität und spezifität wieder. wichtig für den klinischen alltag sind nicht so sehr die sensitivität und spezifität eines testverfahrens (qualitätsmerkmale des testverfahrens), sondern vor allem der positive bzw. negative vorhersagewert. ein positiver oder negativer vorsagewert hängt vom gewählten test, vor allem aber von der erkrankungshäufigkeit ab. es ist daher besonders wichtig, eine geeignete "klientel" für die anwendung des testverfahrens auszuwählen. als beispiel sei die simulation eines rsv-schnelltests genannt. rsv-schnellteste weisen eine sensitivität von 93-98 % sowie eine spezifität von 92-97 % auf. auf basis der sensitivität (› abb. 10.13) lässt sich der positive vorhersagewert ermitteln, wenn die häufigkeit der erkrankung bekannt ist. bei einer auswahl aus mit hoher wahrscheinlichkeit an rsv-erkrankten säuglingen (90 %) unter annahme einer sensitivität von 95 % und einer spezifität von 95 % liegt der positive vorhersagewert bei 99,4 %, der negative vorhersagewert allerdings nur bei 67,9 %. hieraus folgt, dass ein negatives testergebnis nicht zweifelsohne als "negativ" im klinischen kontext gewertet werden kann. in umgekehrter weise ist die anwendung eines simulierten rsv-schnelltests in einer population mit wenigen rsv-infektionen nicht geeignet, einen hohen positiven vorhersagewert zu ermitteln. problematisch sind influenza-schnellteste, da diese häufig über eine schlechte sensitivität verfügen. in einer metaanalyse lag die sensitivität bei 73 %, die spezifität bei 99 %. ferner ist der test (test-kit) an sich ("das produkt") zu berücksichtigen. › abb. 10.14 ergibt ergebnisse einer ringversuch-analyse wieder. mit zwei verschiedenen proben (1 : 10 und 1 : 20 verdünnt) wurden in abhängigkeit vom verwendeten test höchst unterschiedliche ergebnisse ermittelt. dies ist bei der interpretation von inkongruenten befundlagen zu berücksichtigen. neben typischen klinischen zeichen ("blickdiagnosen") führen fieber und zeichen einer systemischen inflammation (sirs, entzündungszeichen -biomarker) zur diagnose. beispielhaft hierfür ist das toxic-shock-syndrom (tss) (› abb. 10.15). hier stehen hohes fieber und dann nach kurzem verlauf zeichen eines septischen schocks im vordergrund. neben laborparametern wie blutbild und differenzialblutbild, crp u. a. werden zur diagnose einer pneumonie radiologische verfahren eingesetzt. zweifelsohne kann eine röntgenuntersuchung des thorax (a.-p.) hilfreich sein. vorsichtig ist geboten, da eine erregerdiagnose mit der radiologischen diagnostik trotz "typischer befunde" nicht zuverlässig zu führen ist. für den klinischen alltag (pädiatrische "community-acquired pneumonia" [pcap]) ist die definition einer pneumonie, bestehend aus der trias "fieber -husten -dyspnoe", sehr hilfreich. eine röntgenaufnahme des thorax ist sinnvoll bei patienten mit chronischen erkrankungen (neuromuskuläre erkrankungen, zystische fibrose u. a.), soll aber nicht regelhaft bei atemwegsinfektionen im kindesalter eingesetzt werden. bezüglich der biomarker ("entzündungszeichen") ist zu sagen, dass diese eine unterschiedliche kinetik aufweisen (› abb. 10.16) und zum zeitpunkt einer etwaigen diagnostik bereits wieder herunterreguliert worden sind. hier bietet sich eine verlaufskontrolle nach 24-48 h (crp) an. gemeinhin werden die biomarker zur differenzierung zwischen viralen und bakteriellen infektionen benutzt. hierfür liegt eine reihe von untersuchungen vor. der sensitivitätsbereich ist höchst variabel und reicht von 17 bis 100 %. hieraus folgt, dass auch bei virusinfektionen (adenoviren!) erhöhte konzentrationen von biomarkern im blut gemessen werden können. es konnte bislang kein biomarker ermittelt werden, der klar zwischen bakterieller und viraler infektion differenziert (und damit auskunft über die notwendigkeit einer antibiotischen therapie gibt). aktuell wird die kombination verschiedener biomarker (z. b. "tumour necrosis factor-rela-ted apoptosis-inducing ligand" [trail] , interferon-γ induzierendes protein 10 [ip10], crp) zur besseren differenzierung zwischen viraler und bakterieller infektion wissenschaftlich evaluiert. für die diagnose einer infektionskrankheit sind immer die anamnese, das klinische bild, die mikrobiologische befundlage sowie hilfsweise der einsatz von biomarkern geeignet. daneben gilt es die epidemiologie von erregern zu berücksichtigen (z. b. "influenzawelle"). im herbst und winter ist die kenntnis über den rsv-bzw. influenzastatus sehr hilfreich, um eine diagnose zu stellen. entsprechende netzwerke publizieren daten hierzu. ein beispiel für eine veränderte epidemiologie ist der keuchhusten. in der vergangenheit waren vor allen dingen kleinkinder mit "typischem keuchhusten" betroffen. nach der flächendeckenden einführung der pertussis-impfung hat sich das hauptmanifestationsalter verschoben; nunmehr sind vermehrt jugendliche und erwachsene betroffen, die jedoch nicht charakteristisch an pertussis erkranken ("atypischer keuchhusten" -chronische bronchitis). aus diesem grund wird die diagnose häufig nicht gestellt, sodass jugendliche bzw. erwachsene dann noch nicht immunisierte neugeborene und junge säuglinge infizieren. daher ist insbesondere bei jungen säuglin-abb. 10.14 ringversuch virusimmunologie röteln (341) [w1054] gen mit anfallsartigem husten und erbrechen, aber auch mit atempausen, grau-blassem hautkolorit und reduziertem trinkverhalten immer auch an pertussis zu denken. pertussis stellt insbesondere bei säuglingen eine lebensbedrohliche erkrankung dar. hier kann es zu einer hyperleukozytose mit leukozytenzahlen > 100 / nl kommen. die hyperleukozytose wird vermutlich durch pertussistoxin (pt) ausgelöst. der diagnostik von keuchhusten kommt daher eine besondere bedeutung zu. die pertussisdiagnostik besteht aus kulturellen verfahren, pcr und serologie. zu beginn der erkrankung (husten) sollte die diagnose kulturell oder mittels pcr gesichert werden. nach 2-4 wochen kann eine serologie (keine allgemeine pertussisserologie! antipertussis-igm bzw. -igg) angewendet werden. bei säuglingen und kleinkindern ist eine serologie gegen anti-pt nicht hilfreich und sollte daher nicht zum einsatz kommen. • bei säuglingen und kleinkindern wird die diagnose "pertussis" kulturell oder mittels pcr gesichert. eine serologie ist nicht sinnvoll. • eine allgemeine "pertussisserologie" existiert nicht. es erfolgt der antikörpernachweis gegen pt. infektionskrankheiten können als epidemie, endemie und pandemie auftreten. eine epidemie ist definiert als das auftreten von mehr krankheitsfällen, spezifischen gesundheitsbezogenen verhaltensweisen oder anderen gesundheitsbezogenen ereignissen in einer bevölkerung oder einer bestimmten bevölkerungsgruppe, als üblicherweise zu erwarten wäre. die sog. hintergrundaktivität ist also für die falldefinition "epidemie" von großer bedeutung. die hintergrundaktivität lässt sich nur bestimmen, wenn eine kontinuierliche erfassung von krankheitsfällen erfolgt. epidemien und ausbrüche sind in der regel örtlich und zeitlich eingrenzbar. im infektionsschutzgesetz (ifsg) ist "ausbruch" definiert als "auftreten von zwei oder mehr gleichartigen erkrankungen, bei denen ein epidemischer zusammenhang wahrscheinlich ist oder vermutet wird" ( § 6 [2b] und [6b] ifsg). eine endemie bezeichnet das konstante auftreten einer bestimmten erkrankung oder eines erregers innerhalb eines geografisch definierten gebiets oder einer definierten bevölkerungsgruppe. zuweilen gibt die endemie die prävalenz einer erkrankung in einem bestimmten gebiet oder einer definierten bevölkerungsgruppe wieder. im gegensatz zu einer epidemie ist eine pandemie ein gehäuftes auftreten von infektionskrankheiten, das weltweit oder über ein weites gebiet mit überschreiten internationaler grenzen vorkommt. ein wesentliches instrument im ausbruchsmanagement von infektionen mit pandemievorsorge ist die krankheitsüberwachung (surveillance). eine systematische und kontinuierliche datenerfassung, die analyse und interpretation der daten sowie die zugänglichkeit für akteure im gesundheitswesen ist dabei essenziell. basis für die erfassung von infektionskrankheiten in deutschland ist das infektionsschutzgesetz (ifsg). ausbrüche lassen sich mithilfe von 10 .1 allgemeine infektiologie interventions-, querschnitt-, kohorten-oder fall-kontroll-studien analysieren. bei randomisierten, kontrollierten interventionsstudien ("randomized controlled trials", rcts) werden probanden aus einer population nach einem zufallsverfahren einer interventionsgruppe oder einer kontrollgruppe zugeteilt. rcts stellen in der epidemiologie den höchsten standard dar. querschnittstudien ("crosssectional studies") untersuchen einen möglichen zusammenhang zwischen einer infektionskrankheit und bestimmten variablen in einer bevölkerungsgruppe zu einem definierten zeitpunkt. hiermit lässt sich die prävalenz einer bestimmten variable bestimmen. kohortenstudien ("cohort studies") sind untersuchungen, die eine definierte gruppe von personen über eine bestimmte zeitspanne beobachten. fall-kontroll-studien ("case control studies") vergleichen probanden, die an einer bestimmten infektionskrankheit leiden, mit einer geeigneten kontrollgruppe, in der die infektionskrankheit nicht vorkommt. mittels statistischer methoden lässt sich das relative risiko (rr) (verhältnis der erkrankungsrate unter exponierten studienteilnehmern im vergleich zur inzidenz dieser rate unter nicht exponierten studienteilnehmern) und das chancenverhältnis ("odds ratio" -or; quotient der "odds" einer exposition bei fällen geteilt durch die "odds" der exposition bei kontrollen) ermitteln. voraussetzungen für eine infektionskrankheit sind: • erreger • übertragungsvorgang • empfänglicher wirtsorganismus die infektiosität eines erregers ist maßgeblich für die "chance", einen potenziellen wirt zu infizieren. als kontagiosität beschreibt man die häufigkeit der infektion. die pathogenität ist ein maß für das "krankmachende potenzial" des erregers. als virulenz wird das ausmaß der pathogenität beschrieben. die infektionsdosis bezeichnet die zahl der notwendigen erreger, die zu einer infektion führen. ein weiterer wichtiger faktor in diesem zusammenhang ist das reservoir (wirt). als infektionsquelle gilt jener teil des reservoirs, der zum ausgangspunkt einer neuen infektion wird. bei der beschreibung der übertragungsvorgänge gilt es die direkte von einer diaplazentaren (vertikalen) übertragung sowie von einer indirekten übertragung über vehikel (lebensmittel, wasser, ärztliche instrumente, fremdkörper u. ä.) oder einen vektor (z. b. insekten, nagetiere) zu unterscheiden. daneben ist auch eine infektion indirekt durch die luft (z. b. varizellen, tuberkulose u. a.) denkbar. die empfänglichkeit eines wirtsorganismus hängt von der konstitution des immunsystems (primärer oder sekundärer immundefekt?) sowie der angeborenen bzw. spezifischen immunität ab. die vergleichsweise hohe inzidenz von infektionen durch bekapselte erreger (s. pneumoniae, n. meningitidis, früher: haemophilus influenzae typ b) bei säuglingen und kleinkindern wird u. a. durch spezifische defekte (mutationen) in der signaltransduktionskaskade der angeborenen immunität (z. b. irak-4, myd88 u. v. m.) erklärt. im weiteren verlauf des lebens können diese dann durch redundante mechanismen der adaptiven immunität kompensiert werden. eine spezifische immunität kann durch frühere durchgemachte infektionen mit demselben erreger oder aktiv durch impfung bzw. passiv durch spezifische immunglobuline erreicht werden. zur bekämpfung von pandemien werden nationale erregerspezifische pandemiepläne veröffentlicht. pandemiepläne sind umfangreiche wissenschaftliche ausarbeitungen, die der vorbereitung auf eine mögliche pandemie einer bestimmten infektionskrankheit (z. b. influenza) dienen. pandemieplanungsaktivitäten finden sowohl auf internationaler ebene (world health organization, who) oder im europäischen zentrum für krankheitsüberwachung und prävention (european center for disease prevention and control, ecdc) als auch auf nationaler (robert koch-institut, rki) und regionaler (landesbehörden) ebene statt. beispielhaft sei die influenzapandemieplanung genannt, die bereits 2005 in einen nationalen pandemieplan mündete und weiter aktualisiert wird. der influenzapandemieplan besteht aus einem teil 1 (strukturen und maßnahmen) und einem teil 2 (wissenschaftliche grundlagen). so werden wesentliche daten zur epidemiologie der influenza, zu den virologischen grundlagen und zum diagnostischen nachweis, surveillance-konzepte und studien, eine risikoeinschätzung während einer pandemie und das klinische bild der influenza dargestellt. daneben sind ausführungen zu nichtpharmakologischen maßnahmen, impfstoffkonzepten und pandemierelevanten arzneimitteln und fachliche grundlagen der kommunikation teil der pandemieplanung. unter www.rki.de ist die jeweils aktuelle pandemieplanung einsehbar. eine nosokomiale infektion ist nach § 8 abs. 8 ifsg "eine infektion mit lokalen oder systemischen infektionszeichen als reaktion auf das vorhandensein von erregern oder ihrer toxine, die im zeitlichen zusammenhang mit einer stationären oder einer ambulanten medizinischen maßnahme steht, soweit die infektion nicht bereits vorher bestand". wesentlich für die definition einer nosokomialen infektion ist der zeitliche zusammenhang, weniger ein kausaler zusammenhang in bezug auf eine medizinische maßnahme. bestimmte maßnahmen können dazu führen (intensivtherapie, immunsuppressive behandlung u. a.), dass bei patienten das risiko für infektionen durch erreger steigt, mit denen sie bereits zuvor besiedelt waren. hierbei handelt es sich um sog. endogene infektionen. die inkubationszeit ist geeignet zur abgrenzung einer nosokomialen von einer ambulant erworbenen infektion. beispielhaft sei die rsv-infektion genannt. wenn ein patient ab dem 5. tag seines stationären aufenthalts symptomatisch wird, so ist von einer nosokomialen infektion auszugehen. nosokomiale infektionen können sich auch nach der entlassung manifestieren. die kommission für krankenhaushygiene und infektionsprävention (krinko) beim rki hat empfehlungen zur prävention nosokomialer infektionen sowie zu betrieblich-organisatorischen und baulich-funktionalen maßnahmen der hygiene in krankenhäusern und anderen medizinischen einrichtungen ( § 23 abs. 1 ifsg) formuliert. zu den maßnahmen gehören: • identifikation patientenbezogener risikofaktoren für nosokomiale infektionen: risikofaktoren, die vom patienten ausgehen, sind z. b. angeborene oder erworbene immundefekte, eine immunsuppressive therapie, eine gestörte barrierefunktion von haut und schleimhäuten (z. b. nach operationen), bei frühgeborenen mit einem geburtsgewicht < 1.500 g, nach verbrennungen und verbrühungen. der einsatz von fremdkörpern (katheter, gastrostomien, shunts, invasive beatmung u. a.) erhöht das risiko für eine nosokomiale infektion ebenso wie der langfristige einsatz von antiinfektiva. • personelle voraussetzungen: hygienefachpersonal soll in ausreichender zahl in einer klinik zur verfügung stehen und regelmäßig auf den stationen präsent sein. das hygienefachpersonal arbeitet eng mit dem krankenhaushygieniker, der mikrobiologie sowie der virologie zusammen. eine zeitnahe kommunikation von mikrobiologischen bzw. virologischen befunden, ggf. ein gezieltes kolonisationsscreening zur erkennung einer besiedelung mit bestimmten multiresistenten erregern, sowie die enge zusammenarbeit mit "antibiotic stewardship teams" (abs) und abs-beauftragten ärzten stellen wesentliche elemente zur verhinderung nosokomialer infektionen dar. • strukturell-organisatorische voraussetzungen: hierzu gehören ein abteilungsspezifischer hygieneplan, hygienedatenblätter, standardarbeitsanweisungen (sops) zu hygienerelevanten arbeitsabläufen, festgelegte konzepte für die einarbeitung, schulung und das praktische training neuer mitarbeiter in bezug auf die standards der infektionsprävention, die verfügbarkeit von spendern zur händedesinfektion, die bereitstellung erforderlicher schutzkleidung und viele weitere maßnahmen. • baulich-funktionelle voraussetzungen zur isolierung etwaiger kontagiöser patienten müssen in ausreichender zahl vorhanden und entsprechend ausgestattet sein. • für patienten mit einem besonderem risiko (onkologie, zystische fibrose u. a.) muss sichergestellt sein, dass es nicht zu einer erregerexposition aus dem trinkwasser kommt (z. b. legionellen, pseudomonas aeruginosa, atypische mykobakterien u. a.). • maßnahmen in der basishygiene: maßnahmen der basishygiene (früher: standardhygiene) werden bei jedem kontakt zum patienten und seiner umgebung durchgeführt, damit eine übertragung von infektionserregern auf patienten oder personal verhindert wird. zu den basismaßnahmen gehören insbesondere händedesinfektion, der gezielte einsatz von viruziden händedesinfektionsmitteln sowie der gebrauch der persönlichen schutzausrüstung beim umgang mit blut, körperflüssigkeiten, exkreten und sekreten; maßnahmen der kontaktisolierung, aseptisches vorgehen bei allen invasiven maßnahmen und bei der erhaltungspflege von kathetern, drainagen usw., aseptisches vorgehen bei der rekonstitution von arzneimitteln und der zubereitung von mischinfusionen sowie die sichere injektionsund infusionspraxis. die maßnahmen der basishygiene sollten mit den hygienefachkräften für die jeweiligen bereiche detailliert ausgearbeitet und besprochen werden. • persönliche schutzausrichtung: hierzu gehört ein mund-nasen-schutz (mns), die ggf. erregerabhängig als partikelfiltrierende halbmasken (ffb) angewendet werden sollten. weitere ausrüstungsgegenstände sind medizinische einmalhandschuhe sowie einmalkittel. es muss betont werden, dass alle maßnahmen der individuellen sorgfältigen händedesinfektion nachzuordnen sind und diese nicht durch eine scheinsicherheit konterkarieren dürfen. die überwachung von nosokomialen infektionen beinhaltet die erfassung von ereignissen nach einheitlichen definitionen mit standardisierten methoden sowie die analyse der ergebnisse mittels größeninzidenzdichte (ni pro 1.000 patiententage) oder inzidenzrate (ni pro 1.000 anwendungstage, z. b. von zentralen gefäßkathetern). die ergebnisse müssen dem behandlungsteam rückgemeldet werden. kliniken für kinder und jugendliche sollen über eine besucherregelung verfügen, die einerseits die medizinischen und psychosozialen gründe für die anwesenheit von vertrauenspersonen im krankenhaus gewährleistet, andererseits aber patienten, angehörige und mitarbeiter schützt bzw. dafür sorgt, dass bestimmte infektionen wie z. b. varizellen, masern, rsv oder influenza nicht in die klinik eingetragen werden. krankheitserreger wie bakterien oder viren können durch direkten oder indirekten kontakt, parenteral sowie durch tröpfchen (< 2 m) oder aerosole (> 2 m) übertragen werden. › tab infektionskrankheiten können durch eine aktive oder passive immunisierung verhindert werden. weitere effekte sind die abschwächung bzw. verhinderung von komplikationen bei infektionskrankheiten (z. b. varizellen-impfung), die verhinderung von folgekrankheiten wie hepatozelluläres karzinom oder gebärmutterhalskrebs (hepatitis-b-, hpv-impfung), einsatz als riegelungsimpfung (z. b. meningokokken-impfungen) oder die induktion einer herdenimmunität (impfung gegen bekapselte erreger, rotavirus-impfung). schutzimpfungen werden von der ständigen impfkommission (stiko) (www.stiko.de) am rki beraten und jährlich publiziert (epidemiologisches bulletin nr. 34 des jeweiligen jahres). die öffentliche empfehlung von schutzimpfungen erfolgt auf der grundlage von § 20 ifsg. neben den empfohlenen standardimpfungen sind auch individuelle impfindikationen denkbar. zur verfügung stehen lebendimpfstoffe (enthalten vermehrungsfähige, attenuierte erreger) und sog. totimpfstoffe (inaktivierte impfstoffe, die komplette, abgetötete mikroorganismen, gereinigte oder rekombinant hergestellte antigene strukturen oder abgewandelte virulenzfaktoren wie z. b. toxoide beinhalten). neben den eigentlichen impfantigenen enthalten die impfstoffe meist weitere substanzen wie lösungsmittel, adjuvanzien, stabilisatoren, konservierungsmittel und u. u. auch spuren von antibiotika. die genaue zusammensetzung der impfstoffe ist den fachinformationen zu entnehmen. quecksilberverbindungen (thiomersal), die früher als stabilisatoren von inaktivierten impfstoffen dienten, werden heute für allgemein empfohlene impfungen nicht mehr verwendet. man unterscheidet grundsätzlich oral, nasal oder parenteral anwendbare impfstoffe. zur injektionstechnik hat die stiko empfehlungen veröffentlicht (schmerzreduktion). die impfstelle sollte vor der injektion mit einer alkoholischen lösung (70 %) desinfiziert werden und wieder trocknen. vor der injektion des impfstoffs sollte nicht aspiriert werden. im säuglingsalter ist der m. vastus lateralis (anterolateraler oberschenkel) die impfstelle, die mit dem geringsten risiko einer verletzung von nerven und gefäßen assoziiert ist. alternativ bietet sich der m. deltoideus an. aktive immunisierungen in den glutealbereich werden wegen der gefahr einer verletzung des n. ischiadicus und der geringen antikörperantwort (z. b. nach hepatitis-b-impfung) nicht mehr empfohlen. um möglichst wenige injektionen zu verabreichen, sollten kombinationsimpfstoffe verwendet werden. › tab. 10.5 gibt den aktuellen impfkalender der stiko (2018 / 2019) wieder. in bestimmten situationen können simultanimpfungen (aktivpassiv) sinnvoll sein. hierzu gehört die postnatale hepatitis-b-impfung bei neugeborenen von hbsag-positiven müttern sowie die simultane tetanusimpfung nach verletzung bei unzureichendem impfschutz. passive immunisierungen spielen, abgesehen von der rsv-immunisierung bei extremen frühgeborenen, kaum noch eine rolle. jede impfung sollte exakt dokumentiert werden. bei personen ohne immundefizienz wird nach der durchführung einer empfohlenen standardimpfung keine antikörperbestimmung empfohlen. impflücken sollten bei jeder sich bietenden gelegenheit durch nachimpfungen geschlossen werden. es gilt die regel: "jede impfung zählt." auch bei längeren intervallen zwischen einzelnen impfungen muss eine grundimmunisierung nicht neu begonnen werden. die stiko hat hierzu instruktive empfehlungen publiziert. bei den impfnebenwirkungen werden lokale von systemischen reaktionen unterschieden. zu den häufigsten systemischen reaktionen gehören subfebrile temperaturen oder fieber. impfnebenwirkungen lassen sich kategorisieren: • lokale und allgemeine reaktionen: diese ereignisse (schmerz an der einstichstelle, rötung und schwellung im bereich der einstichstelle, fieber u. a.) werden generell als ausdruck der normalen auseinandersetzung des organismus mit dem impfstoff verstanden. kenntnisse über art und häufigkeit der reaktion resultieren aus klinischen studien im zusammenhang mit der zulassung eines neuen impfstoffs oder aus klinischen beobachtungen nach der markteinführung. • komplikationen: der übergang von "üblichen" reaktionen zu komplikationen ist fließend. komplikationen sind klar der impfung bzw. den impfungen zuzuordnen. es besteht ein gesicherter ursächlicher oder ein überwiegend als wahrscheinlich anzusehender zusammenhang nach impfungen in bezug auf eine impfkomplikation. beispiele sind eine postvakzinale anaphylaxie oder eine neuritis nach tetanus-impfung. das risiko "komplikation" haftet der impfung in solchen fällen spezifisch an. insbesondere die masern-impfung wird in der öffentlichkeit häufig als komplikationsträchtig diskutiert. • krankheiten, krankheitserscheinungen in (noch) ungeklärtem oder nicht vollständig geklärtem ("assoziation") ursächlichem zusammenhang mit der impfung: hierbei handelt es sich um ereignisse nach impfungen mit begrenzten studienergebnissen. es werden krankheiten oder krankheitserscheinungen im zeitlichen zusammenhang mit einer impfung dargestellt, bei denen jedoch weder klare evidenz für noch gegen einen ursächlichen zusammenhang vorliegt. hierzu gehört z. b. das auftreten des kawasaki-syndroms nach impfung. die auswertung von klinischen studien legt einen (statistisch nichtsignifikanten) zusammenhang zwischen der impfung mit einer pentavalenten rotavirus-impfung (rv5) und einem erhöhten risiko für eine kawasaki-erkrankung nahe. • hypothesen und unbewiesene behauptungen: hierbei handelt es sich um ereignisse, die einen ursächlichen zusammenhang zwischen einer bestimmten impfung und einer bestimmten krankheit annehmen. neben einzelnen veröffentlichungen, die einen zusammenhang beobachten, liegen zur thematik qualifizierte studien vor, die keine evidenz für einen ursächlichen zusammenhang der postulierten krankheit mit der impfung finden konnten. besonderes beispiel ist der vielfach vorgetragene etwaige zusammenhang zwischen der mmr-impfung und autismus und morbus crohn oder zwischen hib-impfung und diabetes mellitus typ 1 sowie hepatitis b und multipler sklerose bzw. optikusneuritis. alle postulate konnten in mehreren wissenschaftlich hochwertigen studien widerlegt werden und sollten nicht mehr zur diskussion stehen. impfnebenwirkungen und komplikationen kommen vor. dennoch können alle heute zugelassenen impfstoffe als "sicher" angesehen werden. bei suszeptiblen individuen (genetische disposition) oder einer bislang unerkannten erkrankung des immunsystems kann es zu einer triggerung einer bislang nicht bekannten krankheit (z. b. dravet-syndrom nach mmr[v]-impfung) oder zu schwerwiegenden komplikationen kommen. wichtig ist, dass unerwünschte ereignisse in zeitlichem zusammenhang mit einer impfung möglichst dezidiert und lückenlos dokumentiert werden, um einen etwaigen kausalzusammenhang zu sichern oder das auftreten eines zufälligen postvakzinalen unerwünschten ereignisses (koinzidenz) zu dokumentieren. die dakj hat eine stellungnahme zum vorgehen beim auftreten ungewöhnlicher neurologischer symptome in zeitlichem zusammenhang mit impfungen im kindes-und jugendalter abgegeben (› abb. 10.17). jede über das übliche ausmaß einer impfreaktion hinausgehende impfreaktion muss nach § 6 abs. 1 nr. 3 ifsg an das gesundheitsamt gemeldet werden. in der diskussion sind sog. unspezifische impfeffekte, die auf populationsbezogene studien zurückgehen und impfungen einen (positiven wie negativen) einfluss auf die "allgemeine" morbidität bzw. mortalität von bevölkerungsgruppen zuschreiben. immundefekte können angeboren oder erworben sein. angeborene immundefekte sind deutlich seltener als erworbene. patienten mit einer immundefizienz können abhängig von der vorliegenden funktionsstörung oft auf impfungen nicht adäquat reagieren und werden durch lebendimpfstoffe u. u. sogar gefährdet. solche verläufe sind beschrieben. impfungen mit lebendimpfstoffen sind deshalb bei den meisten patienten mit angeborener immundefizienz standardimpfung für mädchen und jungen im alter von 9-14 jahren mit 2 impfstoffdosen im abstand von mindestens 5 monaten; beginnend > 14 jahren oder bei impfabstand von < 5 monaten zwischen 1. und 2. dosis ist eine 3. dosis erforderlich (fachinformation beachten). 5 td-auffrischimpfung alle 10 jahre. nächste fällige td-impfung einmalig als tdap-bzw. als tdap-ipv-impfung. 6 einmalige mmr-impfung für alle nach 1970 geborenen personen ≥ 18 jahre mit unklarem impfstatus, ohne impfung oder mit nur 1 impfung in der kindheit. 7 impfung mit dem 23-valenten polysaccharid-impfstoff. 8 standardimpfung für alle personen ab 60 jahren mit einem adjuvantierten herpes-zoster-subunit-(hz / su-)totimpfstoff zur verhinderung von herpes zoster und postherpetischer neuralgie, 2 impfstoffdosen im abstand von mindestens 2 und maximal 6 monaten. (agammaglobulinämie, t-zell-defekte, kombinierte immundefekte) kontraindiziert. ein selektiver iga-mangel, igg-subklassen-mangel sowie phagozytosedefekte, komplementdefekte und asplenie stellen dagegen keine kontraindikation für impfungen dar, sondern sind sogar indiziert. totimpfstoffe können im prinzip bei allen formen der immundefizienz angewendet werden. offen ist häufig die frage der impfeffektivität. bei patienten mit angeborenen oder erworbenen immundefekten ist eine überprüfung des impferfolgs mittels antikörperbestimmung angezeigt. zu beachten ist, dass es oftmals keine definierten "schutztiter" gibt, sondern lediglich die auseinandersetzung des immunsystems mit der impfung dadurch dokumentiert wird. ist eine immunsuppression absehbar (einleitung einer therapie), so sollte möglichst vor der immunsuppression der impfstatus komplettiert werden. dies gilt auch für eine geplante splenektomie. bezüglich der anwendung von kortikosteroiden gelten folgende grenzdosen: • zuvor gesunde kinder, die mit hohen dosen (> 2 mg / kg kg / tag bei einem kg von ≤ 10 kg bzw. > 20 mg / tag prednison-äquivalent bei einem kg von > 10 kg) systemisch wirkender kortikosteroide über längere zeit (> 2 wochen) behandelt werden, dürfen nicht mit lebendimpfstoffen geimpft werden. dies ist frühestens 1 monat nach beendigung der therapie möglich. • inaktivierte impfstoffe können hingegen verabreicht werden. sie sind möglicherweise von eingeschränkter wirksamkeit. • dieses gilt auch für die impfung nach infektionskrankheiten mit einer suppression des immunsystems (z. b. ebv, adenoviren, masern). • zuvor gesunde patienten, die eine kurzzeittherapie (< 2 wochen) oder eine therapie mit niedrigen bis mittleren dosen (< 20 mg bzw. < 2 mg / kg kg prednison) erhalten, oder solche mit topischer (z. b. intraartikulärer oder inhalations-) therapie gelten nicht als immunsupprimiert und können alle impfungen regulär erhalten. die anwendung von anderen immunsuppressiv wirkenden medikamenten (z. b. methotrexat, azathioprin) sowie die verabreichung von biologika sollte in bezug auf impfungen immer mit dem behandelnden zentrum besprochen werden. patienten mit asymptomatischer hiv-infektion sollten alle standardimpfungen, inkl. lebendimpfungen, erhalten. bezüglich der lebendimpfung liegt bei einer schweren immundefizienz (cd4-zahl < 200 / µl bei alter ≥ 5 jahre bzw. < 15 % bei alter < 5 jahre) eine kontraindikation für die masern-, mumps-, röteln-, varizellenimpfung vor. ein operativer eingriff bzw. die damit verbundene narkose können das immunsystem u. u. beeinflussen. die art der verwendeten narkosetechnik sowie die chirurgische intervention (z. b. mit kardiopulmonalem bypass) an sich und deren dauer bestimmen das ausmaß von • im säuglingsalter (grundimmunisierung) sollte, wenn möglich, ein größerer eingriff und nicht die impfung verschoben werden. notfalleingriffe können immer erfolgen und sollen unter berücksichtigung der aktuellen zu erwartenden impfreaktionen mit entsprechend angepassten anästhesieverfahren und postoperativer überwachung durchgeführt werden. • nach operativen eingriffen sind keine bestimmten zeitabstände einzuhalten. impfungen können erfolgen, sobald der patient in einem stabilen allgemeinzustand ist. • impfungen aus vitaler indikation (z. b. tetanus, tollwut, hepatitis-b-impfung) können jederzeit verabreicht werden. nach größeren operationen oder schwersten verletzungen (u. a. immunsuppression nach transplantation, kardiopulmonaler bypass, polytrauma) ist ein individualisiertes vorgehen anzustreben und ggf. der impferfolg zu überprüfen. bezüglich der infektiologischen versorgung von flüchtlingen im kindes-und diagnostik, therapie die diagnose wird klinisch und sonografisch gestellt. die therapie richtet sich nach der schwere der erkrankung. in der symptomatischen behandlung (u. a. ruhigstellung, hochlagerung, kühlung) wird die therapie mit nichtsteroidalen analgetika / antiphlogistika favorisiert. bei begründetem verdacht auf eine bakterielle infektion ist eine antiinfektive therapie angezeigt. vor allem e. coli sollte im wirkspektrum eingeschlossen sein. daneben ist an gonokokken und enterokokken zu denken. neben defekten des angeborenen oder adaptiven immunsystems sind erworbene immundefekte (hiv) sowie eine immunsuppressive therapie (z. b. in der hämato-onkologie und der rheumatologie) prädisponierend für infektionen. infektionen bei defekten des angeborenen immunsystems sind defektspezifisch ("typisch"). rezidivierende respiratorische infektionen, schleimhautulzerationen, eine protrahiert verlaufende omphalitis, granulome und organabszesse, infektionen durch nichttuberkulöse mykobakterien (ntm) sowie virusinfektionen, invasive pyogene infektionen, osteomyelitiden oder lymphadenitiden, chronische mukokutane und invasive candidosen, eine enzephalitis sowie meningitis müssen an das vorliegen eines defekts der angeborenen immunität denken lassen. patienten mit intensivzytostatisch behandelten neoplasien (aml-induktionstherapie, therapie eines leukämierezidivs), nach allogener stammzelltransplantation bis zur erholung der granulozytenzahl und patienten mit einer schweren gvhd und augmentierter immunsuppression erhalten eine chemoprophylaxe im hinblick auf pilzinfektionen. im mittelpunkt stehen hier fluconazol, itraconazol, voriconazol, posaconazol, micafungin oder liposomales amphotericin b. zur vzv-prophylaxe gehört die expositionsprophylaxe (isolierung, aktive varizellenimpfung, wenn möglich) sowie die postex-positionsprophylaxe mit passiver immunprophylaxe und chemoprophylaxe (aciclovir). bei patienten mit allogener stammzelltransplantation ist eine etwaige cmv-reaktivierung zu bedenken. zur cmv-prophylaxe gehört die auswahl von leukozytendepletierten (und bestrahlten?) blutprodukten zur transfusion. als präventive therapie ist ganciclovir empfohlen. die "gram-färbung" ist eine von dem dänischen arzt hans christian gram (1853 gram ( -1938 in berlin entwickelte methode zur differenzierung von bakterien. bakterien unterscheiden sich hinsichtlich der zellhülle, die eine unterscheidung mittels der unten beschriebenen färbung zulässt. grampositive bakterien besitzen eine dicke hülle aus peptidoglykan, gramnegative bakterien verfügen über eine dünne mureinschicht, der sich außen eine weitere schicht die äußere membran, anschließt. bei der gram-färbung werden bakterien mit einem basischen farbstoff (kristallviolett) angefärbt. es folgt eine nachbehandlung mit jodkalium-jodid-komplex. der dabei entstehende farbkomplex ist wasserunlöslich, in ethanol jedoch löslich und kann aus gramnegativen bakterien mit ethanol extrahiert werden. wegen der dickeren mureinschicht bei grampositiven bakterien verbleibt der farbstoff im bakterium. • zu den grampositiven bakterien gehören actinomyces, streptomyces sowie streptokokken, enterokokken, staphylokokken, listerien, bacillus, clostridium und lactobacillus. • beispiele für gramnegative bakterien sind enterobakterien (escherichia coli u. a.), salmonella, shigella, klebsiella, proteus, enterobacter sowie die gattungen pseudomonas, legionella, neisseria, rickettsia und die art pasteurella multocida. multiresistente erreger (mre) können sowohl grampositiv als auch gramnegativ sein. ein bakterium gilt als multiresistent, wenn es gegen zwei oder mehr antibiotikaklassen resistent ist, die in der empirischen standardtherapie gegen diese erregerspezies eingesetzt werden. in der klinischen praxis lassen sich folgende mre unterscheiden: • vancomycin-bzw. glykopeptid-resistente enterokokken (vre) differenzialdiagnostisch sind immer auch nichtinfektiöse ursachen einer enzephalopathie abzugrenzen, z. b. intoxikationen, reye-syndrom, zns-tumoren, blutungen, vaskulitiden, metabolische erkrankungen oder auch das weite feld der autoimmunenzephalitiden (z. b. nmda-rezeptor-ak-vermittelte enzephalitis). interessanterweise scheint die häufigkeit immunologisch vermittelter enzephalitiden die der erregerbedingten offenbar zu übersteigen. weitere wichtige diagnostische maßnahmen sind laboruntersuchungen (serologie) sowie liquoranalysen (schrankenstörung, zellzahl, proteingehalt, pcr-untersuchungen auf neurotrope viren). auch das eeg sowie ein mrt gehören zum diagnostischen spektrum. eine hirnbiopsie ist selten erforderlich, kann aber mitunter zur klärung einer nichtinfektiösen enzephalitis (rasmussen-enzephalitis) beitragen. klinisch relevante enzephalitiden sind: herpes-simplex-enzephalitis, masern-enzephalitis, enzephalitis durch flaviviren sowie japan-enzephalitis und west-nil-virus-fieber bzw. enzephalitis. insbesondere die herpes-enzephalitis kommt bei neugeborenen vor. im gegensatz zur herpes-enzephalitis des älteren kindes wird diese meist durch hsv 2 verursacht. neugeborene von müttern mit primärinfektion eines herpes genitalis am ende der schwangerschaft sollen aufgrund der hohen manifestationsrate per sectio caesarea entbunden und prophylaktisch für 10 tage mit aciclovir in einer dosis von 60 mg / kg / kg in 3 ed behandelt werden. prognose die prognose einer enzephalitis hängt von der grundkrankheit sowie dem erreger ab. oftmals wird keine restitutio ad integrum erreicht, und es sind rehabilitative maßnahmen notwendig. aus pragmatischen gründen empfiehlt es sich, bei der bakteriellen meningitis drei altersgruppen zu unterscheiden: • neugeborene • säuglinge nach der 5. bis 6. lebenswoche • kinder jenseits des 1. lebensjahrs epidemiologie in europa wird die jahresinzidenz der meningitis auf 0,5-4 erkrankungen je 100.000 einwohner angegeben. der häufigkeitsgipfel befindet sich in den ersten beiden lebensjahren. bei neugeborenen stellen frühgeburtlichkeit und niedriges geburtsgewicht die hauptrisikofaktoren dar. eine bakterielle meningitis im neugeborenenalter kann im rahmen einer early-onset-sepsis oder als late-onset-sepsis entstehen. therapie eine analgetische und fiebersenkende therapie, z. b. ibuprofen (bis 30 mg / kg kg / tag in 3 ed) oder paracetamol (bis zu 40-60 mg / kg kg / tag in 4 ed), ist bei patienten mit einer aom indiziert. bei verlegter nasenatmung können 0,9-prozentige nacl-lösungen intranasal oder α-adrenerge abschwellende nasentropfen (bis maximal 7 tage) appliziert werden. schmerzstillende ohrentropfen, die lokalanästhetika enthalten, haben nur einen kurzfristigen effekt. zuvor muss eine trommelfellperforation ausgeschlossen werden. die spontane heilungsrate der aom ist hoch und beträgt 70-90 % innerhalb von 2-7 tagen. die wirksamkeit einer antiinfektiven therapie in bezug auf eine verkürzung der symptome fieber und schmerzen ist relativ gering. da der übermäßige antibiotikaverbrauch zur resistenzentwicklung beiträgt und antibiotika nebenwirkungen verursachen, ist eine möglichst zielgerechte therapie für patienten mit aom, die wirklich davon profitieren, anzustreben. kinder unter 2 jahren mit klinisch eindeutiger, vor allem bilateraler aom und / oder otorrhö profitieren von einer antibiotischen therapie. ab einem alter von 2 jahren kann bei nicht schwer kranken patienten mit einer aom zunächst eine symptomatische therapie erfolgen, sofern eine klinische kontrolle innerhalb von 48 h gewährleistet ist. › tab. 10.17 gibt kriterien zur antiinfektiven therapie bzw. beobachtung der aom bei kindern ohne risikofaktoren wieder. eine otitis externa ist eine entzündung des äußeren gehörgangs aufgrund infektiöser, allergischer und dermatologischer ursachen. häufig ist eine bakterielle infektion nachweisbar. die wichtigsten bakterien sind pseudomonas aeruginosa, staphylococcus aureus und kns sowie streptokokken der gruppe a, enterobacteriaceae und pilze. die schwere otitis externa ist oft nicht leicht zu behandeln. es empfiehlt sich die zusammenarbeit mit einem hno-arzt. bei der mastoiditis liegt eine akute entzündung der schleimhaut der lufthaltigen zellen im proc. mastoideus des schläfenbeins vor. epidemiologie die inzidenz der akuten mastoiditis beträgt 1-4 je 100.000 einwohner. die patienten sind meist zwischen 7 monaten und 3 jahren alt. kinder im alter unter 2 jahren weisen die höchste inzidenz auf. klinisches bild, diagnose das klinische bild ist gekennzeichnet durch ohrenschmerzen, fieber und reduzierten allgemeinzustand. zusätzlich finden sich retroaurikulär eine rötung, schmerzhaftigkeit, eine teilweise fluktuierende schwellung sowie eine abstehende ohrmuschel. bei mehr als 75 % der patienten besteht gleichzeitig eine akute otitis media. bei bis zu 30 % kommt es zu extra-oder intrakraniellen komplikationen wie abszessbildung oder hirnnervenlähmungen. die diagnose wird primär klinisch und in enger zusammenarbeit mit einem hno-arzt gestellt. therapie therapeutisch ist eine chirurgische intervention erforderlich. bei einer mastoiditis besteht immer eine indikation zur i. v. antiinfektiven therapie. bei patienten ohne risikofaktoren ist ampicillin / sulbactam (150-200 mg / kg kg / tag in 3 ed) die therapie der wahl. alternativ kann mit einer kombination aus einem cephalosporin der gruppe 3 (cefotaxim oder ceftriaxon) und clindamycin (40 mg / kg kg / tag) behandelt werden. die rhinitis wird ausschließlich durch viren, meist durch rs-, parainfluenza-, influenza-, rhino-, corona-, boca-und adenoviren hervorgerufen. sehr selten liegt primär eine bakterielle infektion vor (z. b. konnatale syphilis). etwa 5 % der kinder entwickeln sekundär eine bakterielle infektion. die behandlung ist symptomatisch; es können schleimhautabschwellende sprays eingesetzt werden. bei der akuten sinusitis handelt es sich um eine nasennebenhöhlenentzündung (rhinosinusitis), die durch die entzündung mit schwellung und starker sekretion der nasenschleimhaut hervorgerufen wird. neben verlegung, sekretstau und ausfluss in die vorderen und hinteren nasenwege treten fieber und je nach alter abgeschlagenheit und lokale schmerzen oder kopfschmerzen auf. die im kindesalter häufigen viralen rhinosinusitiden führen nach schätzungen in weniger als 10 % der fälle als komplikation zu einer bakteriellen sinusitis. die akute sinusitis ist beim kind im unterschied zur akuten otitis nicht sehr häufig und eher eine krankheit des erwachsenenalters. neben viralen atemwegsinfektionen sind allergien die wichtigsten prädisponierenden faktoren. sekretabfluss über die hinteren nasenwege und die rachenhinterwand kann zu anhaltendem husten führen. die lokale entzündung und der sekretstau gehen je nach betroffener nebenhöhle mit zahn-, gesichts-und augenschmerzen einher. weitere symptome sind mundgeruch und bauchschmerzen. komplikationen der akuten sinusitis sind selten. wegen der anatomischen nähe ist immer auch an eine ophthalmologische komplikation zu denken. die diagnose basiert vorwiegend auf anamnese und klinischer untersuchung. in der bildgebung hat sich das mrt durchgesetzt. das ct bleibt ausnahmefällen vorbehalten. eine akute sinusitis sollte antiinfektiv behandelt werden, wenn folgende kriterien erfüllt sind: • persistierende zeichen der rhinosinusitis über 10 tage ohne besserung • auftreten schwerer symptome wie hohes fieber, eitriges nasensekret oder gesichtsschmerzen • zunahme der symptome oder biphasischer verlauf mit neu auftretendem fieber oder kopfschmerzen mittel der wahl ist amoxicillin (50 mg / kg kg / tag in 2-3 ed) für 10 tage p. o. bei nichtansprechen stehen amoxicillin plus clavulansäure oder ceftriaxon zur verfügung. bei penicillinunverträglichkeit kann clarithromycin eine alternative darstellen. bei einer tonsillopharyngitis handelt es sich um eine entzündung des waldeyerschen rachenrings. als symptom treten vorzugsweise halsschmerzen auf. bei säuglingen und kleinkindern können unspezifische allgemeinsymptome wie nahrungsverweigerung oder fieber darauf hindeuten. aufgrund der weißen bis gelblichen tonsillenbeläge lassen sich morphologisch die angina follicularis (stippchenförmige beläge) und die angina lacunaris (konfluierende beläge) unterscheiden. erreger und epidemiologie als erreger kommen viren und bakterien infrage. › tab. 10.18 gibt das mögliche erregerspektrum, symptome und differenzialdiagnosen der tonsillopharyngitis wieder. zu 70-95 % handelt es sich bei der akuten tonsillopharyngitis um eine virusinfektion. der häufigste bakterielle erreger sind bei immunkompetenten kindern (zu 20-30 %) und erwachsenen (5-15 %) gruppe-a-streptokokken (gas). die übertragung der erreger der infektiösen tonsillopharyngitis erfolgt insbesondere durch engen kontakt in sozialgemeinschaften wie familien, kindergärten, heimen und kasernen. typischerweise tritt die gas-tonsillopharyngitis von november bis mai auf, während die tonsillopharyngitis durch enteroviren eher im sommer und im herbst vorkommt. eine gas-tonsillopharyngitis bei kindern vor dem 3. lebensjahr ist äußerst selten, jedoch können diese kinder gas-träger sein. problematisch ist, dass eine vielzahl von erregern den pharynx kolonisiert. hierzu gehören gas, streptococcus pneumomiae, staphylococcus aureus, haemophilus influenzae, corynebacterium spp. und actinomyces spp. weiterhin finden sich regelmäßig neisserien, moraxella catarrhalis u. a. für die diagnose einer gas-tonsillopharyngitis wird neben typischen klinischen symptomen der mcisaac-score verwendet. › abb. 10.18 gibt das vorgehen bei v. a. akute tonsillopharyngitis / tonsillitis wieder. zu den differenzialdiagnosen der akuten tonsillopharyngitis gehören prinzipiell erkrankungen durch corynebacterium diphtheriae und hib, wenngleich beide historisch und nach einführung der impfung in deutschland praktisch nicht mehr vorkommen. differenzialdiagnostisch ist an einen peritonsillarabszess (unilaterale tonsillenschwellung, starke schmerzen, schluckstörung, kloßige sprache), eine epiglottitis (speichelfluss / schluckstörung, hohes fieber, inspiratorischer stridor, kloßige sprache) sowie eine ebv-infektion (weiß-graue tonsillenbeläge, hepatosplenomegalie) zu denken. therapie da eine bakterielle tonsillopharyngitis durch andere erreger außer gas hundertfach seltener ist als eine virale ätiologie, ist der nachweis von gas für die initiierung einer antiinfektiven therapie sinnvoll. die behandlung kann zunächst nur aus einer schmerztherapie bestehen. mittel der wahl zur behandlung der gas-tonsillopharyngitis ist nach wie vor penicillin g. bei nachweis β-hämolysierender streptokokken ist grundsätzlich die indikation zu einer antiinfektiven behandlung gegeben, die sich im wesentlichen aus verkürzter infektiosität sowie einer gering verkürzten symptomdauer begründet. für die hno-chirurgische therapie liegt eine interdisziplinär verfasste awmf-s2k-leitline vor. "therapie entzündlicher erkrankungen der gaumenmandeln-tonsillitis": awmf.org/uploads/tx_szleitlinien/017-024l_s2k_tonsillitis_gaumenman-deln_2015-08_01.pdf https://else4.de/m6o7g › tab. 10.19 gibt die indikation für die empfehlung zur tonsillektomie bzw. tonsillotomie in anlehnung an die awmf-leitlinie wieder. in der mundhöhle können verschiedene anatomische strukturen bzw. gewebe von infektionen betroffen sein. am häufigsten sind odontogene infektionen, die oftmals von einem geschädigten zahn ausgehen. da die normale mundflora aus vielen verschiedenen bakterienspezies besteht, wobei sowohl grampositive erreger (grüne streptokokken, β-hämolysierende streptokokken) als auch anaerobier und actinomyzeten besonders häufig vorkommen, kann sich bei prädisposition leicht eine infektion entwickeln. therapie eine sofortige stationäre versorgung ist bei v. a. epiglottitis angezeigt. als antiinfektive therapie stehen cephalosporine der gruppe 3 (cefotaxim, ceftriaxon oder amoxicillin / clavulansäure oder ampicillin / sulbactam) zur verfügung. der begriff krupp wurde in der vergangenheit für die diphtherie verwendet. bei der akuten stenosierenden laryngotracheitis handelt es sich nicht um einen "pseudo-krupp" (ursache unbekannt), sondern um eine infektionskrankheit, also einen "infekt-krupp". therapie das krankheitsbild kann mit antiobstruktiven medikamenten behandelt werden. bei versagen der antiobstruktiven therapie ist auf eine fremdkörperaspiration hin zu untersuchen. der nutzen der sauerstofftherapie ist umstritten. die systemische steroidtherapie ist bei der schweren form als therapieversuch generell angezeigt. die gabe von sauerstoff bei hypoxämie ist umstritten. die wirksamkeit von sekretolytika und atemluftbefeuchtung ist nicht erwiesen. bei obstruktiver bronchitis und bronchiolitis kann die inhalative gabe eines kurz wirkenden β 2 -mimetikums, bei säuglingen zusammen mit ipratropiumbromid, versucht werden. auf die behandlung von rsv-infektionen wird in › kap. 10.3.56 eingegangen. die häufigste form der lungenentzündung im kindesalter ist die ambulant erworbene pneumonie ("pediatric community-acquired pneumonia" = pcap), die in erster linie klinisch diagnostiziert wird. hierzu gehören respiratorische symptome wie husten und atemnot, thorakale symptome sowie fieber, tachykardie, nahrungsverweigerung, dehydratation, bauchschmerzen, inaktivität und vigilanzveränderungen. die pneumonie ist eine häufige erkrankung im kindesalter. bei unter 5-bzw. 1-jährigen kindern ist die inzidenz mit 28-150 pneumonien pro 10.000 kindern pro jahr hoch. klinisch wird die nicht schwere pcap von der schweren pcap mit dehydratation, stark reduziertem allgemeinzustand, nahrungsverweigerung, somnolenz und bewusstlosigkeit unterschieden. hinweise für eine bakterielle pneumonie sind hohes fieber, stark reduzierter allgemeinzustand, hypoxämie und initial fehlender husten. die wichtigsten erreger der pneumonie im kindesalter sind nach alter und risikofaktoren gegliedert. › tab. 10.21 nennt die häufigsten erreger verschiedener pneumonieformen im kindesalter. pneumokokken verursachen lobärpneumonien. komplikationen einer pneumonie können parapneumonische pleuraergüsse oder pleuraempyeme sein. eine basale pneumonie kann mit ausgeprägten bauchschmerzen einhergehen, die eine appendizitis oder ein anderes intraabdominales geschehen vortäuschen können. hier ist die beobachtung der atmung in phasen, in denen das kind nicht schmerzgeplagt ist (auch nach gabe eines analgetikums), für die diagnosestellung hilfreich. kinder mit einer mykoplasmenpneumonie zeigen oft einen trockenen reizhusten und dyspnoe sowie häufig einen unauffälligen auskultationsbefund. bei 1-4 monate alten säuglingen mit tachypnoe und einem pertussiformen husten ohne fieber ist an eine chlamydia-trachomatis-pneumonie zu denken, insbesondere, wenn gleichzeitig eine meist eitrige einseitige konjunktivitis vorliegt. feinblasige, inspiratorische rasselgeräusche weisen eine hohe spezifität für die prädiktion einer pneumonie auf. diagnose die pcap wird primär klinisch diagnostiziert. röntgenaufnahmen des thorax sind routinemäßig nicht erforderlich und sollten nur bei schwerer erkrankung, bei v. a. pleuraerguss, atelektase, tbc, tumor und lungenödem sowie bei ausbleibender besserung / entfieberung durchgeführt werden. anhand radiologischer veränderungen allein kann nicht zuverlässig zwischen viraler und bakterieller pneumonie unterschieden werden. bei patienten mit einer nicht schweren pcap sollte keine routinemäßige blutentnahme erfolgen, da anhand der parameter auch nicht zuverlässig zwischen viraler und bakterieller pneumonie unterschieden werden kann. ein erregernachweis sollte bei patienten mit schwerer pcap, therapieresistenz oder komplikationen mittels nachweis bakterieller erreger aus blutkultur, induziertem sputum (schulalter, patienten mit cf) oder pleuraerguss angestrebt werden. bei ausgeprägten pleuraergüssen ist eine diagnostische (identifikation des erregers und resistenzbestimmung), notfalls auch eine therapeutische punktion des pleuraraums sinnvoll. eine erregergewinnung mittels bronchoskopie und bronchoalveolärer lavage (bal) ist nur bei patienten mit schwer verlaufender, therapierefraktärer pneumonie bzw. immundefizienz angezeigt. in der jüngeren vergangenheit wird vermehrt die pcr-(multiplex-pcr-)diagnostik eingesetzt, um eine vielzahl von bakteriellen und viralen pneumonieerregern zu erfassen. akut sind die ergebnisse allerdings oft nicht verfügbar. die serologische diagnostik aus einzelproben kann bei mycoplasma pneumoniae bzw. chlamydia pneumoniae versucht werden. serologische verlaufsuntersuchungen mit bestimmung von antikörpern in serenpaaren (initiale und in der rekonvaleszenz) spielen in der praxis kaum eine rolle. die candidiasis der haut (soordermatitis) ist ein häufiges krankheitsbild bei kindern, die eine windel tragen. candida-infektionen der schleimhäute und inneren organe sind opportunistische infektionen bei immunsupprimierten patienten oder patienten mit anderen risikofaktoren. übertragung candida spp. können direkt mit einer kontaktoder schmierinfektion übertragen werden. sie verfügen über eine äußerst geringe kontagiosität. hygienische basismaßnahmen sind daher in der regel ausreichend. infektionen von der mutter auf das neugeborene unter der geburt oder postnatal sind denkbar. weitere übertragungsmöglichkeiten sind geschlechtsverkehr, von patient zu patient oder von pflegepersonal / ärzten zu patient. indirekte übertragungen sind als schmierinfektion über händekontakt, stuhlhaltige windeln, pflegeutensilien und einrichtungsgegenstände möglich. nahrungsmittel können eine orale infektion auslösen. nosokomiale candida-infektionen werden über katheter, infusionslösungen oder medizinische geräte vermittelt. grundsätzlich ist immer auch eine endogene infektion, ausgehend von der besiedelung der schleimhäute, möglich. therapie eine schleimhaut-candidiasis kann mit miconazol (säuglinge: 100 mg in 4 ed, mundgel), nystatin (säuglinge: < 1.500 g 300.000 e in 3 ed, > 1.500 g 450.000 e in 3 ed als suspension), amphotericin b (säuglinge: < 1.500 g 0,8 ml [80 mg] in 4 ed, > 1.500 g 1,6 ml [160 mg] in 4 ed als suspension) sowie fluconazol (säuglinge: 6 mg / kg kg in 1 ed p. o.) behandelt werden. jenseits der neugeborenenperiode kommen bei oberflächlichen candida-infektionen (z. b. der oropharyngealen candidiasis [mundsoor]) topisches nystatin, clotrimazol oder fluconazol zum einsatz. die ösophageale candidiasis wird mit fluconazol behandelt. die therapie der vulvovaginalen candidose besteht aus topisch antimykotisch wirksamen azolen bzw. miconazol / clotrimazol oder fluconazol / itraconazol. risikofaktoren risikofaktoren für invasive candida-infektionen sind: prophylaxe zur prophylaxe invasiver candida-infektionen gehören ein sorgfältiges hygieneregime, die behandlung des vaginalen hefebefalls am ende der schwangerschaft, die klinische überwachung mykosegefährdeter patienten mit dem ziel der frühdiagnostik und -therapie invasiver candida-mykosen. hier sind insbesondere extrem frühgeborene kinder zu nennen und solche, die mit einer immunsuppressiven therapie, breitband-antibiotika und intensivmedizinischen maßnahmen behandelt werden. fadenpilze der gattung aspergillus können verschiedene krankheitszustände auslösen. hierzu gehören die allergische bronchopulmonale aspergillose (abpa) sowie die chronische pulmonale aspergillose. unter den verschiedenen formen der invasiven aspergillose ist die invasive pulmonale aspergillose am häufigsten. absiedelungen, insbesondere in das zns, kommen nicht selten vor. epidemiologie die wichtigsten klinischen risikofaktoren für invasive aspergillus-infektionen sind eine prolongierte und profunde granulozytopenie (< 500 neutrophile granulozyten / µl über ≥ 10 tage) sowie funktionelle defekte von granulozyten und makrophagen (septische bzw. chronische granulomatose, glukokortikoidtherapie, gvhd). patienten mit aml bzw. leukämierezidiven weisen ein hohes risiko für invasive aspergillosen auf. klinisches bild das klinische bild der invasiven aspergillose besteht aus fieber, respiratorischen und infarktartigen symptomen, bei beteiligung des zns sind fokale oder diffuse neurologische ausfälle zu registrieren. die radiologischen befunde sind oftmals unspezifisch und nicht immer wegweisend. diagnose die wichtigste diagnostische maßnahme ist, bei risikopatienten an eine aspergillus-infektion zu denken. pulmonale bzw. zentralnervöse befunde können mittels mrt identifiziert werden. herdförmige läsionen erscheinen charakteristisch mit umgebender milchglasartiger verdichtung ("halo sign"). einschmelzungen sind charakteristisch. aspergillus-arten sind in blutkulturen schwer nachweisbar. der nachweis von galactomannan (einem membranassoziierten glykosid) im serum, in der bal bzw. im liquor sowie verfahren der nukleinsäureapplikation von aspergillus spp. an punktions-und biopsiematerial kann die diagnose erleichtern. galactomannan-bestimmungen weisen eine hohe variabilität hinsichtlich der sensitivität und spezifität sowie der vorhersagewerte auf, weswegen die befunde nur im klinischen kontext interpretiert werden können (vortestwahrscheinlichkeit). therapie initialtherapie der 1. wahl: voriconazol (16 mg / kg kg / tag in 2 ed, tag 1: 18 mg / kg kg in 2 ed für die altersgruppe 2-14 jahre; 8 mg / kg kg in 2 ed, tag 1: 12 mg / kg kg in 3 ed ab 15 jahre und für 12-bis 14-jährige mit einem kg von > 50 kg) oder die i. v. gabe von liposomalem amphotericin b (3 mg / kg kg / tag in 1 ed). zur zweitlinientherapie stehen je nach vorbehandlung liposomales amphotericin b, amphotericin-b-lipidkomplex, voriconazol, caspofungin, posaconazol und itraconazol zur verfügung. bei einer zns-aspergillose gilt wegen der guten gewebegängigkeit voriconazol als mittel der wahl. frühgeborene, neugeborene und kinder in den ersten beiden lebensjahren werden aufgrund der weitgehend fehlenden daten zur therapie mit liposomalem amphotericin b (3 mg / kg kg / tag in 1 ed) oder amphotericin-b-lipidkomplex (5 mg / kg kg / tag in 1 ed) behandelt. bei dringender indikation kann die therapie mit caspofungin auch bei früh-und neugeborenen erwogen werden (25 mg / m 2 kof / tag in 1 ed). nach klinischer stabilisierung kann eine orale weiterbehandlung mit voriconazol erfolgen. prophylaxe die tragende säule der prophylaxe invasiver aspergillosen ist die expositionsprophylaxe gegenüber aerogenen konidien. aus diesem grund sind spezielle raumlufttechnische vorkehrungen zu treffen. bei hochrisikopatienten in der hämato-onkologie kann eine präventive therapie mit itraconazol, zu den stichverletzungen gehören sicher auch nadelstichverletzungen (berufliches umfeld). das infektionsrisiko nach einer kanülenverletzung für hepatitis b (hbv) liegt bei 10-40 %, für hepatitis c (hcv) bei 3-6 % sowie für hiv bei 0,1-0,3 %. zu den maßnahmen im verletzungsfall gehören: bluten lassen (ausdrücken, aber nicht die wunde quetschen), 5-10 min desinfektion. danach sollte eine unmittelbare vorstellung beim betriebsarzt erfolgen und kontakt zur infektiologischen bzw. mikrobiologischen abteilung aufgenommen werden. entscheidung zur postexpositionsprophylaxe (pep) und einleitung eines d-arzt-verfahrens. bei messerstichverletzungen ist meistens von einer infektion mit s. aureus, seltener mit gas auszugehen. das infektionsrisiko ist abhängig vom ort der verletzung (umgebungsflora) sowie vom verwendeten messer. neben den hautständigen erregern (staphylokokken, streptokokken) kommt somit eine vielzahl von pathogenen infrage. es bedarf einer individuellen evaluation des infektionsrisikos. verbrennungen werden, abgesehen von chirurgischen maßnahmen wie escharotomie oder fasziotomie, internistisch oder intensivmedizinisch (nach ausmaß) versorgt. es gilt nekrotische zu den knochen-und gelenkinfektionen gehören die osteomyelitis, (meistens) eine infektion des knochens, die von bakterien und seltener von pilzen oder anderen mikroorganismen hervorgerufen wird, sowie die septische arthritis. die inzidenz der hämatogenen osteomyelitis und septischen arthritis wird in europa mit etwa 5-10 fällen je 100.000 kinder pro jahr angegeben. zuletzt war eine abnehmende tendenz zu erkennen. bei den häufigen nachweisen von koagulase-negativen staphylokokken kann es sich auch um kontaminationen handeln. etwa 2 % der neugeboreneninfektionen sind durch anaerobier, besonders bacteroides fragilis bedingt. häufigkeit und epidemiologie man unterscheidet die frühe ("early onset", eos) neugeboreneninfektion (inzidenz bei reifgeborenen ca. 0,8 je 1.000 für kulturgesicherte infektionen, frühgeborene: inzidenz ca. 10 je 1.000 für kulturgesicherte sepsis bei vlbw-frühgeborenen). die inzidenz für eine klinische, nicht kulturgesicherte neugeborenensepsis liegt je nach definition um den faktor 10-30 höher. früh-und neugeboreneninfektionen durch gbs beginnen in der regel schon intrauterin oder unmittelbar nach der geburt. von einer "late-onset"-infektion (los) spricht man, wenn diese später als im alter von 72 h bzw. 7 tagen (bei gbs) auftritt. weitere symptome sind neurologischer natur, z. b. lethargie, hypotonie, hyperexzitabilität oder allgemeine symptome wie trinkschwäche, nahrungsunverträglichkeit. bei frühgeborenen mit bakteriellen infektionen können ähnliche symptome auftreten, sie sind jedoch oftmals weniger ausgeprägt als bei reifgeborenen. eine nosokomiale sepsis ist definiert als sepsis, die sich später als 72 h nach klinikaufnahme entwickelt. an einer early-onset-sepsis (eos) erkranken in den usa 0,5-1,2 / 1.000 lebendgeborene (kinder ≥ 34 ssw), an einer late-onset-sepsis (los) ca. 6,2 je 1.000 lebendgeborene (frankreich). bei einer eos entstammen die infektionserreger meist der mütterlichen vaginalflora. bei los sind gefäßkatheter, beatmungstuben oder andere medizinische maßnahmen entscheidende eintrittspforten, sodass insbesondere bei frühgeborenen bevorzugt kns, s. aureus oder enterobakterien auftreten. diagnostik zu bakteriellen infektionen bei neugeborenen wurde kürzlich eine s2k-leitlinie erarbeitet, auf die an dieser stelle verwiesen wird: "bakterielle infektionen bei neugeborenen": awmf.org/leitlinien/detail/ ll/024-008.html https://else4.de/l9p8v zur infektionsdiagnostik bei v. a. neugeboreneninfektion / -sepsis werden neben der blutkulturdiagnostik folgende parameter empfohlen: blutbild und differenzialblutbild (i / t-quotient), crp, il-6 (ggf. il-8 augeninfektionen können sich als konjunktivitis (viral und bakteriell) als keratitis, blepharitis sowie infektionen des tränensystems und als orbitale oder intraokuläre infektionen manifestieren. die neugeborenenkonjunktivitis ist definiert als eine konjunktivitis, die im 1. monat auftritt. erreger als erreger kommen n. gonorrhoeae, chlamydia trachomatis, mischinfektionen sowie s. aureus, e. coli, pneumokokken, gbs, h. influenzae u. a. infrage. in industrienationen wird bei 0,5-1 % der geburten mit einer neugeborenenkonjunktivitis gerechnet. klinisch liegt meist eine perakute beidseitige konjunktivitis mit reichlich purulentem sekret und massivem lidödem vor. diagnose die diagnose wird klinisch und mittels abstrich gestellt. therapie therapeutisch werden gonokokken mit ceftriaxon (25-50 mg / kg kg / tag i. v. in 1 ed) oder mit cefotaxim (50-100 mg / kg kg / tag i. v. in 2-3 ed für 1 tag), chlamydien mit erythromycin (30-50 mg / kg kg / tag p. o. in 2-3 ed) für 2 wochen behandelt. die credé-prophylaxe (1 % silbernitrat oder silberacetatlösung, einmalig nach geburt) wird nicht mehr empfohlen. eine einmalige direkt postnatale gabe einer antibiotischen augensalbe (erythromycin 0,5 % oder tetrazyklin 1 %) stellt eine gute prophylaxe gegen gonokokken, nicht aber gegen chlamydien dar. alternativ kann 2,5 % jodlösung verwendet werden. hierbei handelt es sich um eine ein-oder beidseitige bindehautentzündung durch bakterien. läsionen durch hsv im genitoanalbereich entfallen zu 80 % auf hsv2 und zu 20 % auf hsv1. die hsv-infektion kann durch sexuellen kontakt und unter der geburt auf das neugeborene übertragen werden. bei genitalen hsv-infektionen sollte immer eine systemische therapie erfolgen (aciclovir). die syphilis wird durch treponema pallidum hervorgerufen. therapie die antiinfektive behandlung erfolgt erregerspezifisch. bei männern kann eine infektion mit trichomonas vaginalis symptome einer urethritis, epididymitis oder prostatitis verursachen, bei frauen vaginalen ausfluss, der diffus, übelriechend oder gelbgrün sein kann. viele patienten (70-85 %) haben minimale oder gar keine symptome, und unbehandelte asymptomatische infektionen können sogar jahre persistieren. humane papillomaviren (hpv) werden entsprechend ihrem onkogenen potenzial in die hpv-typen "low risk" (hpv6 und hpv11), ursächlich für die bildung von condylomata acuminata (feigwarzen), sowie "high risk" (hpv16 und hpv18 u. v. m.) dazu gehören die trichinellose sowie die cholera (3 fälle: indien 2, philippinen 1). fleckfieberfälle wurden zuletzt nicht gemeldet. bei den 2015 gemeldeten 43 erkrankungen an läuserückfallfieber handelt es sich um asylsuchende vom horn von afrika. im winter 2013 / 2014 kam es zu einem anhaltenden chikungunya-fieberausbruch auf verschiedenen inseln der karibik (martinique, san martin, guadeloupe). der ausbruch breitete sich in ganz mittelund südamerika aus. in deutschland wurden 110 fälle gemeldet. keine meldungen in deutschland. 2015 wurden 722 dengue-fieber-erkrankungen übermittelt. dies ist die zweithöchste fallzahl seit einführung des ifsg. die häufigsten nennungen kommen nach reisen aus thailand (29 %), gefolgt von indonesien (16 %) und brasilien (7 %). es wurden erstmalig einzelne fälle berichtet. die 2015 gemeldeten 16 fälle einer kutanen leishmaniose traten nach reisen in folgende länder auf: syrien 6, tunesien 4, mallorca 2, israel 2, brasilien 1, peru 1. zoonosen sind infektionskrankheiten, die von bakterien, parasiten, pilzen, prionen oder viren verursacht und wechselseitig zwischen tieren und menschen übertragen werden können (mensch → tier = anthropozoonose; tier → mensch = zooanthropose • bakterien: wichtige bakterielle zoonosen sind: trichophytien, mikrosporidien. bei den parasitären zoonosen werden einzeller, würmer und arthropoden differenziert. die wichtigsten seien an dieser stelle genannt. amöbenruhr (amöbiasis) ist eine infektion des darms durch das protozoon entamoeba histolytica. nach who-schätzungen erkranken pro jahr bis zu 50 mio. menschen an einer invasiven amöbiasis. die amöbiasis ist in den meisten tropischen und subtropischen regionen der erde endemisch. sie ist nicht auf die tropen beschränkt, sondern findet sich überall dort, wo aufgrund niedriger hygienestandards eine fäkal-orale übertragung möglich ist. in deutschland ist die amöbiasis vor allem eine erkrankung von reiserückkehrern. der mensch ist der einzig relevante wirt für entamoeba histolytica. entamoeba histolytica verlaufen asymptomatisch. in 10-20 % der fälle kommt es zu einer invasion des parasiten in das gewebe mit dem klinischen bild einer amöbenkolitis (amöbenruhr). die inkubationszeit ist sehr variabel und kann bis zu mehrere jahre betragen. typische symptome sind bauchschmerzen und blutige diarrhöen. komplikationen der verlauf kann durch einen amöbenleberabszess, darmulzerationen bzw. -perforationen mit konsekutiver peritonitis kompliziert sein. ferner wurden abszedierungen an pleura und in der lunge sowie im herzbeutel beschrieben. diagnose die diagnose der amöbenruhr erfolgt durch den erregernachweis, wobei die mikroskopie in der regel unzureichend ist. nur der nachweis von 20-60 µm großen trophozoiten von erreger campylobacter gehören zu einer rna-superfamilie von gramnegativen bakterien, zu der auch helicobacter und arcobacter gehören. charakteristisch für diese bakterien ist, dass sie die schleimhäute des magen-darm-trakts oder die reproduktionsorgane besiedeln. durch ihre spiralform und begeißelung sind sie auch in schleimhäuten mobil. es wurden 26 spezies, 2 provisorische spezies und 9 subspezies identifiziert, von denen campylobacter (c.) jejuni, c. coli und c. fetus die wichtigsten humanpathogenen spezies sind. einige stämme produzieren toxine. schon eine sehr geringe ingestionsdosis kann eine infektion auslösen. campylobacter, besonders c. jejuni, gehören weltweit zu den häufigsten verursachern der akuten bakteriellen enteritis. es ist eine steigende inzidenz zu beobachten. die häufigkeitsverteilung zeigt zwei spitzen: 1. kinder < 2-5 jahre 2. junge erwachsene die inzidenz kann sehr unterschiedlich ausfallen (14-1.500 je 100.000 gesamtpopulation / jahr). eine infektion mit campylobacter ist für etwa jeden 5. fall einer reisediarrhö verantwortlich. komplikationen komplikationen sind selten und kommen in der regel nur bei immunsupprimierten patienten vor. bei gesunden können cholezystitis, peritonitis, meningitis, vaskulitis, erythema nodosum, perikarditis und myokarditis sowie septische arthritis und weichteilinfektionen den verlauf komplizieren. selten kann ein guillain-barré-syndrom auftreten. die induktion eines aborts durch c. fetus muss als komplikation bezeichnet werden. die diagnose wird durch den nachweis in der stuhlkultur geführt. zur erhöhung der sensitivität können eine speziesspezifische pcr oder 16s-rrna eingesetzt werden. therapie neben der symptomatischen behandlung (rehydratations-, flüssigkeits-und elektrolyterhalt) wird eine antiinfektive therapie nur bei schwerem, lang anhaltendem verlauf (> 1 woche) durchgeführt. eine behandlungsindikation stellt die erkrankung von jungen säuglingen oder kindern mit beeinträchtigter immunabwehr dar. makrolide sind die antiinfektiva der 1. wahl. azithromycin wird meist über 3 tage in einer dosis von 10 mg / kg kg / tag mit 1 ed verabreicht, wobei auch die einzelgabe von 30 mg / kg kg / tag als sehr effektiv gilt. bei einer sepsis werden meropenem, gentamicin, clindamycin und cephalosporine der gruppe 3 eingesetzt. prävention eine impfung steht nicht zur verfügung. die wichtigsten präventiven maßnahmen sind konsequente küchenhygiene und sorgfalt bei der speisenzubereitung. erkrankte personen werden isoliert. es besteht meldepflicht (ifsg). pilze lassen sich systematisch einteilen in dermatophyten, hefepilze und schimmelpilze. hinsichtlich der klinischen manifestation spricht man von lokalen und oberflächlichen mykosen sowie systemischen mykosen. dermatophyten, hefepilze und schimmelpilze werden auch als "dhs-system" zusammengefasst. nahezu drei viertel aller pilzinfektionen werden durch dermatophyten (epidermophyton, microsporum, trichophyten) hervorgerufen. etwa 20 % entfallen auf hefepilze. hier sind insbesondere candida, cryptococcus und pityrosporum zu nennen. bei den schimmelpilzen (ca. 5 % der infektionen) sind vor allem aspergillus und mucor wichtige vertreter. pilzinfektionen kommen in deutschland durch folgende spezies vor: aspergillus spp., candida spp., kryptokokken, mucor spp., pseudallerischia bzw. scedosporium. am häufigsten kommen candida-infektionen der schleimhäute, überwiegend durch c. albicans verursacht, vor. unter den erregern invasiver infektionen ist c. albicans mit 50-70 % der häufigste erreger, gefolgt von c. parapsilosis, c. glabrata und c. tropicalis. wesentlich für die abwehr von haut-und schleimhautinfektionen sind mechanismen der erworbenen zellulären immunität. candida-infektionen werden vorzugsweise durch phagozytose abgewehrt. störungen der erworbenen zellulären immunität sowie der granulozytenfunktion und eine "unreife" prädisponieren für candida-infektionen im allgemeinen. eine spezifische inkubationszeit kann nicht angegeben werden. man unterscheidet die candidiasis der haut von candida-infektionen der schleimhäute und inneren organe. haut ist eine typische infektionskrankheit des säuglingsalters. in späteren lebensabschnitten deutet eine solche infektion auf prädisponierende faktoren, wie z. b. diabetes mellitus oder immundefekte, hin. neben dem windelsoor, der konnatalen kutanen candidiasis sowie der candidiasis des genitale kann sehr selten eine chronische mukokutane candidiasis (cmc) im rahmen von autoimmunerkrankungen vorkommen. diagnose die diagnose der haut-candidiasis wird klinisch gestellt. therapie es haben sich miconazol-zinkoxid-kombinationspasten bzw. nystatin-zinkpasten als effektiv erwiesen. bei den candida-infektionen der schleimhäute und inneren organe unterscheidet man die oropharyngeale candidiasis sowie eine candidiasis des magens und des darms von einer candida-sepsis, die überwiegend als nosokomiale infektion bei frühgeborenen < 1.000 g, bei patienten mit hämatologischen neoplasien und intensivmedizinischen erkrankungen auftritt und eine hohe letalität aufweist. im prinzip kann eine candidiasis jedes organ betreffen und als endokarditis, infektion von larynx und bronchien, osteomyelitis sowie infektionen der nieren und ableitenden harnwege, endophthalmitis und zns-infektionen auftreten. invasive candida-infektionen mit hämatologischer grunderkrankung sind mit folgenden risikofaktoren assoziiert: protrahierte granulozytope nie, zvk, therapie mit kortikosteroiden und ggf. biologika sowie mukositis und gabe von breitspektrum-antiinfektiva. diagnose die diagnose bei schleimhautinfektionen bzw. invasiver candida-infektion wird neben den typischen klinischen symptomen durch eine pilzkultur gestellt. aspergillus spp. sind ubiquitär verbreitet. sie wachsen bevorzugt im erdreich, in kompost und anderem organischem abfall. in wohnbereichen befinden sie sich häufig an feuchten wänden, auf topfpflanzen oder hydrokulturen sowie im biomüll. im krankenhaus sind vor allem klimaanlagen, baustellen, wasserhähne und duschköpfe mögliche habitate von aspergillus. eine ansteckung von mensch zu mensch ist nicht bekannt. die übliche eintrittspforte der < 5 µm großen konidien ("sporen") ist der respirationstrakt (inkl. der nasennebenhöhlen). häufigkeit die wichtigsten klinischen risikofaktoren für eine invasive aspergillus-infektion sind eine prolongierte granulozytopenie (< 500 neutrophile granulozyten / µl über ≥ 10 tage) sowie funktionelle defekte von granulozyten und makrophagen. neben der invasiven aspergillose werden unter dem begriff chronische pulmonale aspergillose verschiedene erkrankungen zusammengefasst, die bei nicht abwehrgeschwächten patienten auftreten können. klinische formen sind die chronische kavitäre pulmonale aspergillose, herdförmige noduläre manifestationen und singuläre aspergillome. in europa erworbene kryptokokkosen werden meist durch die hefe cryptococcus (c.) neoformans verursacht, seltener durch c. gattii. von c. neoformans liegen mehrere serotypen vor. die hefe ist weltweit verbreitet und kommt hauptsächlich in vogelfäkalien (tauben, papageienarten), in kontaminierter erde oder staub vor. die übertragung erfolgt durch inhalation der hitze-und austrocknungsresistenten erreger. auch verletzungsmykosen sind beschrieben worden. die inkubationsdauer nimmt bis zu mehrere wochen ein. klinische symptome das klinische bild tritt überwiegend bei immunsupprimierten patienten mit t-zell-defekten auf. weniger häufig werden kryptokokkosen bei personen mit organtransplantation oder malignen tumoren unter chemotherapie bzw. langzeit-steroidmedikation diagnostiziert. im primärstadium ist die infektion häufig inapparent. mit der hämatogenen disseminierung (sekundärstadium) können alle parenchymatösen organe erreicht werden. diagnostik es stehen kulturelle (mikroskopischer direktnachweis bekapselter hefen im tusche-präparat aus liquorsediment, urin, biopsat u. a.), serologische (antigennachweis im serum und liquor) sowie histologische nachweismethoden zur verfügung. therapie bei einer disseminierten und zerebralen kryptokokkose wird eine kombinationstherapie mit amphotericin b (ggf. als liposomale formulierung) und 5-flucytosin empfohlen. die therapie wird bis zur sterilisierung des liquors durchgeführt. sodann erfolgt eine konsolidierungstherapie mit fluconazol für mindestens 8 wochen. kann amphotericin b nicht verordnet werden, so bietet sich die kombination von fluconazol mit 5-flucytosin an. bei personen mit immunsuppression muss eine langfristige rezidivprophylaxe diskutiert werden. die lungenkryptokokkose braucht bei intakter immunabwehr und milder klinik nicht zwingend behandelt zu werden. prävention eine spezifische prävention ist nicht möglich. immunsupprimierten patienten wird empfohlen, streuquellen zu meiden. aktinomykosen werden durch actinomyces-arten (a. israelii, a. gerencseriae) hervorgerufen. bislang wurden 25 verschiedene actinomyces spp. identifiziert. viele actinomyces-arten sind bestandteil der schleimhautflora. eine differenzierung zwischen infektion und kolonisation ist daher keineswegs einfach. alle aktinomykosen, mit ausnahme der nach menschenbiss oder faustschlagverletzungen entstandenen kutanen formen, sind endogene infektionskrankheiten, die nicht ansteckend und sporadisch weltweit verbreitet sind. risikofaktoren sind immundefizienz, diabetes mellitus, immunsuppression und alkoholismus. chlamydien sind obligat intrazelluläre bakterien, die extrazellulär als infektiöse, aber metabolisch inaktive elementarkörperchen vorkommen können. intrazellulär spricht man von aktiven retikularkörperchen. aus retikularkörperchen entstehen inaktive elementarkörperchen, die unter zerstörung der wirtszelle freigesetzt werden. die früher übliche einteilung der familie chlamydiacae in die gattungen chlamydia und chlamydophila wurde aufgegeben. klinisch bedeutsam sind c. trachomatis, c. suis, c. psittaci, c. abortus sowie c. pneumoniae u. a. im folgenden werden chlamydia-trachomatis-, chlamydia-pneumoniae-sowie chlamydia-abortus-infektionen besprochen. chlamydia-trachomatis-infektionen führen zu einem trachom, einer konjunktivitis oder zu respiratorischen bzw. urogenitalen infektionen sowie dem lymphogranuloma venereum. erreger erreger sind c. trachomatis der serogruppen b und d -k. bei sexuell aktiven jugendlichen wurden urogenitale infektionen mit c. trachomatis bis zu 13 % im weiblichen urogenitaltrakt nachgewiesen. bei personen > 30 jahren sinkt die prävalenz unter 2 %. infektionen durch c. trachomatis gehören zu den häufigsten sexuell übertragbaren infektionskrankheiten. bei präpubertären kindern können genitale chlamydien-infektionen auf sexuellen missbrauch hinweisen. das klinische bild entspricht dem einer urethritis mit dysurie und ausfluss. diagnose die diagnose wird durch den erregernachweis aus zellhaltigen abstrichen vom urogenitaltrakt, ggf. einschließlich laparoskopischer oder aus der urethra gewonnenem sekret gestellt. serologischen verfahren kommt keine bedeutung zu. therapie bei kindern ab 8 jahren doxycyclin für 7-10 tage p. o., azithromycin über 3 tage bzw. alternativ ofloxacin oder levofloxacin. prävention verwendung von kondomen. urogenitale infektionen durch c. trachomatis sind meldepflichtig (ifsg). hierbei handelt es sich um eine infektion mit einer indolenten, vesikulären, papulösen oder ulzerösen primärläsion im genitalbereich, die nach einigen tagen ohne narbenbildung spontan abheilt. nach einem symptomfreien intervall von 1-8 wochen kommt es zu einer schmerzhaften schwellung, u. a. der leistenlymphknoten, mit rötlich livider verfärbung der darüber liegenden haut und möglicher abszedierung sowie allgemeinem krankheitsgefühl und fieber. erreger die infektionskrankheit wird durch c. trachomatis der serogruppen l1 -l3 (am häufigsten l2) hervorgerufen. der erreger wird sexuell übertragen. endemische regionen sind asien, afrika, südamerika und teile der karibik. in europa werden lokale ausbrüche vor allem bei hiv-positiven männern beobachtet. die inkubationszeit beträgt 2-12 tage. diagnose die diagnose wird durch den erregernachweis aus zellhaltigem abstrich vom primärulkus aus der urethra oder dem rektum, eiter oder punktionsmaterial befallener lymphknoten gestellt. therapie doxycyclin, erythromycin. prävention verwendung von kondomen. es besteht meldepflicht (ifsg). chlamydia pneumoniae ist weltweit verbreitet. um die pubertät herum lässt sich ein steiler anstieg spezifischer antikörper mittels seroepidemiologischer untersuchungen feststellen. bei erwachsenen werden mit zunehmendem alter seroprävalenzraten von > 80 % erreicht. klinische symptome der erreger verursacht im kindesalter bis zu 11 % der ambulant erworbenen pneumonien. reinfektionen können während des gesamten lebens auftreten. die inkubationszeit beträgt 1-4 wochen. klinisch lassen sich infektionen der oberen atemwege (sinusitis, pharyngitis, otitis media) sowie der unteren atemwege (bronchitis, pneumonie) beobachten. seltene extrapulmonale manifestationen umfassen meningoenzephalitis, reaktive arthritis, myokarditis, guillain-barré-syndrom. diagnose die diagnose wird typischerweise durch den erregernachweis aus den unteren atemwegen (bal) oder anderen sekreten gestellt. trotz erregernachweis können antikörper fehlen. therapie doxycyclin, erythromycin, roxithromycin. meldepflichtig sind gehäuft auftretende infektionen (ifsg). erreger chlamydia-psittaci-infektionen gehören zu den ornithosen. die übertragung der erreger erfolgt mittels getrocknetem kot oder über sekrete (staub) oft asymptomatisch infizierter vögel. die erkrankung ist in deutschland selten. die inkubationszeit beträgt 5-14 tage (bis zu 40 tage). krankheitsbild mit hohem fieber, starken muskel-und kopfschmerzen sowie meningismuszeichen nach kürzlichem kontakt mit vögeln. bei vielen patienten entwickelt sich dann eine interstitielle pneumonie mit trockenem, anhaltendem, nicht produktivem husten. diagnose antikörper sollten mittels mikroimmunfluoreszenztest oder einem immunoblot gesucht werden, der antigene der drei humanpathogenen chlamydien enthält. der erregernachweis wird mit molekularbiologischem verfahren aus respiratorischen proben geführt. therapie siehe chlamydia pneumoniae. es besteht eine meldepflicht bei erkrankungen oder tod (ifsg). erreger nach kontakt schwangerer frauen mit infizierten lammenden mutterschafen oder kühen kann es zu einer schweren fieberhaften erkrankung mit plazentitis bis hin zum abort kommen. die seltene zoonose ist potenziell letal und wird durch chlamydia abortus hervorgerufen. die diagnose erfolgt mittels pcr. • latente cmv-infektion (persistenz im gewebe, nicht im blut) • aktive cmv-infektion (symptome, "cmv-erkrankung") • asymptomatische cmv-infektion cmv ist die häufigste ursache einer konnatalen infektion. 0,2-0,5 % der neugeborenen werden pränatal infiziert. eine cmv-erstinfektion tritt bei ca. 0,5 % aller schwangerschaften auf. in 30-50 % der fälle kommt es dabei auch zur infektion des feten. bei schwangeren kann es zu einer reaktivierung einer latenten cmv-infektion oder zu einer "neuen" (re-)infektion durch einen anderen cmv-stamm kommen. es ist somit möglich, dass es auch bei einer zu beginn der schwangerschaft cmv-igg-"positiven" schwangeren zu einer erneuten infektion kommen kann. die hohe inzidenz latenter infektionen führt dazu, dass fetale infektionen durch reaktivierung ähnlich häufig sind wie durch primärinfektionen. die inkubationszeit einer cmv-infektion über infektiöse körpersekrete beträgt 4-8 wochen. nach einer bluttransfusion liegt die inkubationszeit bei 3-12 wochen, nach organtransplantationen kommt es nach 4-8 wochen zur infektion. stillen kann nach ca. 48 tagen inkubationszeit zur infektion führen. eine fetale cmv-infektion kann zu einem hydrops fetalis oder abort führen. in abhängigkeit vom gestationsalter lassen sich charakteristische, bleibende schäden ausmachen. hierbei ist insbesondere das zns betroffen. im ersten trimenon kann es zu einer zns-atrophie mit lissenzephalie, im zweiten trimenon mit schizenzephalie, balkenmangel, periventrikulären verkalkungen und polymikrogyrie, im dritten trimenon mit okzipitoparietal betonter leukenzephalopathie, subkortikalen zysten, ventrikulären adhäsionen und thalamustriatalen vaskulopathien kommen. andererseits bieten nur ca. 25 % aller feten mit cmv-infektion sonografische veränderungen des zns. postnatal weisen ca. 10 % aller neugeborenen mit intrauteriner cmv-infektion bei geburt symptome auf. klinische symptome sind hepatosplenomegalie, transaminasenerhöhung, blutbildungsstörungen, pneumonie, kardiomyopathie, chorioretinitis und enzephalitis sowie intrauterine wachstumsretardierung. die letalität der symptomatischen konnatalen cmv-infektionen beträgt 10-15 %. bis zu 50 % der kinder weisen neurologische defizite bzw. eine progrediente innenohrschwerhörigkeit auf, die sich bei normalem hörscreening in der neonatalzeit auch erst im weiteren verlauf entwickeln kann. auch bei kindern mit einer bei geburt asymptomatischen cmv-infektion (ca. 90 %) kann sich in 5-15 % der fälle eine innenohrschwerhörigkeit ausbilden. es wird vermutet, dass bis zu 20 % der behandlungsbedürftigen hörstörungen auf cmv-infektionen zurückgehen. diagnose eine aktive cmv-infektion wird durch den direkten virusnachweis aus blut, liquor, urin, speichel-und rachensekret, amnionflüssigkeit oder muttermilch diagnostiziert. eine konnatale cmv-infektion wird durch den nachweis des virus im urin oder speichel in der 1. bis 3. lebenswoche diagnostiziert. ein pcr-nachweis im blut ist möglich, aber nicht zuverlässig. ab der 3. woche nach der geburt kann die diagnose einer konnatalen cmv-infektion nur noch retrospektiv durch den cmv-dna-nachweis in getrocknetem blut aus der stoffwechsel-screeningkarte erfolgen. therapie im vordergrund der behandlung einer konnatalen cmv-infektion steht die vermeidung einer innenohrschwerhörigkeit. die behandlung mit valganciclovir wird bei symptomatischer konnataler cmv-infektion empfohlen (32 mg / kg kg / tag p. o. in 2 ed). aus klinischen studien konnten leichte vorteile für eine 6-monatige (gegen 6-wöchentliche) behandlung zur verhinderung von langzeitkomplikationen (hörstörung, neurologische entwicklung) ermittelt werden. alternativ kann initial für 1-2 wochen ganciclovir (12 mg / kg kg / tag i. v. in 2 ed) verabreicht werden. beide substanzen sind für neugeborene nicht zugelassen. die behandlung erfolgt off-label. bei immunsupprimierten patienten mit symptomatischer cmv-infektion (meningoenzephalitis, pneumonie, hepatitis, ösophagitis, kolitis) ist eine therapie mit ganciclovir oder valganciclovir indiziert. prävention expositionsprophylaxe: das risiko einer cmv-konversion in der schwangerschaft beträgt in deutschland ca. 0,5 %. wichtigste ansteckungsquelle sind urin und speichel von asymptomatisch erkrankten kindern. während eine laktogene cmv-infektion bei reifen und fast reifen neugeborenen fast immer asymptomatisch verläuft, kann es bei sehr unreifen frühgeborenen (< 32 ssw) zu einer symptomatischen infektion kommen. das pasteurisieren einer etwaigen cmv-haltigen muttermilch zur verabreichung an sehr unreife frühgeborene wird kontrovers beurteilt. bei den tiefen dermatophytosen (tinea profunda) unterscheidet man entzündliche tiefe dermatophytosen von nicht entzündlichen tiefen dermatophytosen. bei der häufigen tinea pedis (prävalenz > 10 %) kommt es zur interdigitalen mazeration und / oder diffusen plantaren schuppung, die nur selten mit vesikeln einhergeht. häufig ist bei der tinea pedis der nagel befallen (tinea unguium oder bronchomykose). diagnose einsatz der wood-lampe bei 365 nm: innerhalb der befallenen läsion zeigt sich eine hellgrüne bzw. schwachgrüne fluoreszenz. daneben kommen die mikroskopie sowie der kulturelle nachweis der dermatophyten zum einsatz. therapie man unterscheidet die topische von der systemischen therapie. topisch kommen metronidazolderivate wie clotrimazol, econazol, isoconazol, bifonazol, sertaconazol und oxiconazol zum einsatz. systemisch ist griseofulvin (20 mg / kg kg / tag für 8-12 wochen) mittel der wahl. eine meldepflicht besteht nicht. erreger und pathogenese die diphtherie wird durch corynebacterium (c.) diphtheriae hervorgerufen. es lassen sich vier biotypen unterscheiden: gravis, mitis, belfanti und intermedius. der pathogenetische faktor ist die bildung des diphtherietoxins (exotoxin), das nach integrieren eines bakteriophagen mit dem diphtherietoxin-gen dtx in der bakterienzelle gebildet wird. neben c. diphtheriae können c. ulcerans (rachendiphtherie, hautdiphtherie) und c. pseudotuberculosis (beide kommen bei haus-und nutztieren vor) diphtherietoxin produzieren und diphtherieähnliche symptome verursachen. c.-diphtheriae-stämme ohne toxinbildung sind apathogen. erregerreservoir für c. diphtheriae ist der mensch. diphtheriefälle werden weltweit beobachtet. in westlichen industrieländern ist die zahl der erkrankungen erheblich zurückgegangen. dies ist nicht allein durch die impfung zu erklären. in deutschland werden importierte erkrankungsfälle beobachtet. infektionsquelle sind erkrankte und keimträger. die übertragung erfolgt aerogen bei "face-to-face"-kontakt oder durch tröpfcheninfektion. das übertragungsrisiko durch erkrankte ist höher als durch gesunde keimträger. die inkubationszeit beträgt 2-5 tage, gelegentlich länger. klinische symptome häufigste manifestationsform der diphtherie ist die rachendiphtherie mit entzündung in der tonsillopharyngealregion. auch der kehlkopf, die nase oder der tracheobronchialbaum können betroffen sein. es bestehen hohes fieber, halsschmerzen und schluckbeschwerden. später kommen stridor sowie gaumensegellähmung und lymphknotenschwellungen hinzu. die letalität der diphtherie liegt bei 5-10 %; unter ungünstigen verhältnissen kann sie bei säuglingen und kleinkindern sowie erwachsenen auf 25-40 % steigen. todesursache ist meistens eine atemwegsob struktion oder herzversagen infolge einer myokarditis. diagnose wegweisend ist der klinische befund. der erregernachweis (pcr) sollte aus abstrichen der verdächtigen läsion angestrebt werden. erkrankte personen erhalten sofort antitoxin zur toxinneutralisation kombiniert mit einer antiinfektiven therapie (penicillin, erythromycin) über 14 tage. prävention es steht eine impfprävention zur verfügung. die diphtherie-impfung ist als standardimpfung für alle säuglinge, kinder, jugendlichen und erwachsenen in deutschland von der stiko empfohlen. darüber hinaus bestehen indikationen für zugereiste personen. krankheitsverdacht, erkrankung und tod sind meldepflichtig. primärinfektion mit ebv meist asymptomatisch oder unspezifisch. ältere kinder, jugendliche und erwachsene bieten das bild der "akuten infektiösen mononukleose" (aim; synonym: "pfeiffersches drüsenfieber") mit hohem fieber, exsudativer angina tonsillaris, pharyngitis, lymphadenopathie, abgeschlagenheit und splenomegalie. komplikationen können alle organsysteme betreffen, insbesondere aber das immunsystem sowie das hämatopoetische system. bei kindern mit angeborenem oder erworbenem immundefekt (z. b. hiv-infektion, therapie mit immunsuppressiva) kann eine ebv-primärinfektion oder ebv-reaktivierung zu schweren, häufig letalen lymphoproliferativen krankheitsbildern ("lymphoproliferative disease", lpd) bis hin zu malignen b-zell-lymphomen führen. bei ebv-assoziierten komplikationen nach transplantation von soliden organen oder hämatopoetischen stammzellen spricht man von einer "posttransplant lymphoproliferative disorder" (ptld). es sind verschiedene malignome (burkitt-, hodgkin-, t-/ nk-zell-lymphome, nasopharynx-, magenkarzinome sowie leiomyosarkome) mit ebv assoziiert. weltweit ist das ebv für ca. 2 % aller krebserkrankungen verantwortlich. diagnose das klinische bild der aim ist typisch und lässt sich meistens ohne weitere laboruntersuchungen diagnostizieren. das blutbild weist vermehrt atypische, reaktive lymphozyten auf. › tab. 10.36 fasst typische antikörpermuster für verschiedene infektionsstadien zusammen. therapie eine antivirale therapie existiert nicht. im wesentlichen wird symptomatisch behandelt. enteroviren inkl. coxsackie-, echo-und polioviren sind kleine, hüllenlose rna-viren innerhalb der familie picorna viridae. zur gruppe der coxsackie-a-viren zählen 22, zur gruppe der coxsackie-b-viren 6 und zur gruppe der echoviren 28 serotypen. humane enteroviren werden genotypisch in die gruppen a, b, c und d eingeteilt. diagnose und therapie die diagnosestellung erfolgt mittels nukleinsäureamplifikation, virusisolierung oder in ausnahmefällen mittels serologie. eine spezifische therapie steht nicht zur verfügung. prävention impfstoffe gegen enteroviren befinden sich in der vorbereitung. keine frühere ebv-infektion ----frische ebv-primärinfektion / aim + + + chronische aktive ebv-infektion + / -+++ +++ -/ + vca: "virus capsid antigen"; ea: "early antigen"; ebna1: "ebv nuclear antigen 1"; aim = akute infektiöse mononukleose haemophilus-influenzae-infektionen werden durch ein gramnegatives, oft kokkoides, unbewegliches und sporenloses stäbchen aus der familie der pasteurellaceae hervorgerufen. es sind bekapselte und unbekapselte stämme bekannt. man unterscheidet 6 serotypen (a -f) und 8 biotypen (i -viii). ein großteil der invasiven infektionen wurde vor einführung der impfung durch hib hervorgerufen. unbekapselte stämme verursachen häufig atemwegsinfektionen wie otitis media, sinusitis, konjunktivitis, bronchopneumonie sowie exazerbation einer chronischen bronchitis. influenzae kommt weltweit und ausschließlich beim menschen vor. unbekapselte stämme gehören zur normalflora des nasen-rachen-raums. invasive infektionen kommen besonders bei säuglingen und kleinkindern vor. die inzidenz betrug vor einführung der impfung ca. 23 je 100.000 und jahr bei kindern unter 16 jahren. inzwischen ist sie auf unter 0,5 je 100.000 und jahr gesunken. für hib beträgt die inzidenz < 0,05 pro 100.000 und jahr. der häufigkeitsgipfel der hib-meningitis liegt in den ersten beiden lebensjahren, bei der epiglottitis durch hib dagegen im 3. bis 4. lebensjahr. nach einführung der hib-impfung sind beide krankheitsbilder nahezu verschwunden. die übertragung erfolgt mittels tröpfcheninfektion oder durch direkten kontakt von mensch zu mensch. diagnose ein kultureller nachweis ist bei allen kindern mit v. a. eine invasive infektion anzustreben. therapie cefotaxim, ceftriaxon, cephalosporine der gruppe 2, amoxicillin plus clavulansäure. prävention eine chemoprophylaxe mit rifampicin bei indexpatienten mit einer invasiven hib-infektion ist sinnvoll, wenn nicht mit ceftriaxon oder cefotaxim behandelt wurde. nach einem kontakt zu einem patienten mit invasiver hib-infektion wird von der die hepatitis c wird durch das hepatitis-c-virus (hcv), ein rna-virus aus der flaviviren-gruppe, hervorgerufen. von diesem virus existieren mindestens 18 genotypen. in deutschland wurden 2016 über 4.000 hepatitis-c-fälle beim rki gemeldet. etwa 0,4 % der einwohner in deutschland sind anti-hcv-positiv. übertragung und inkubationszeit das hcv wird überwiegend durch i. v. drogengebrauch und sexualkontakte übertragen, seltener durch dialyse und hausarztkontakte. eine vertikale übertragung kommt bei etwa 1-6 % der kinder hcv-rna-positiver mütter vor und ist bei kindern inzwischen der häufigste übertragungsweg. die inkubationszeigt beträgt 8 (2-26) wochen. klinische symptome das klinische bild der hepatitis c ist oftmals symptomarm und unspezifisch. häufig lässt sich die hepatitis c kaum von einer akuten hepatitis a oder b unterscheiden. diagnose die diagnose erfolgt über den nachweis von anti-hcv-antikörpern. therapie die hepatitis c kann erfolgreich behandelt werden. zur behandlung der hepatitis c stehen polymeraseinhibitoren (nukleotidisch, nichtnukleotidisch) sowie ns3 / 4a-protease-inhibitoren und ns5a-inhibitoren zur verfügung. prävention es existiert keine spezifische immunprophylaxe. die akute hepatitis-d-infektion ist meldepflichtig (ifsg). die hepatitis e wird durch ein rna-virus aus der gruppe der caliciviren hervorgerufen. vier genotypen sind bekannt. herpes-simplex-1-und -2-viren (hsv1, hsv2) gehören zur gruppe der humanpathogenen herpesviren. infektionen mit hsv1 betreffen meist die haut und schleimhaut, hsv2-assoziierte infektionen betreffen die genitalregion. eine strikte trennung ist nicht möglich. übertragung und inkubationszeit hsv kommen ubiquitär vor. die übertragung erfolgt von mensch zu mensch, insbesondere durch engen körperkontakt (geburt, geschlechtsverkehr) mit symptomatischen oder asymptomatischen personen. eine neonatale hsv-transmission erfolgt selten pränatal (ca. 5 %), meist unter der geburt (85 %). in seltenen fällen (10 %) ist auch eine postnatale ansteckung durch infektiöse kontaktpersonen möglich. das erregerreservoir stellen infizierte personen dar. patienten mit primärer infektion (gingivostomatitis, herpes genitalis) können das virus mehrere wochen ausscheiden. hsv2-infektionen treten abhängig von der sexuellen aktivität auf. die inkubationszeit schwankt zwischen 2 und 12 tagen (median 3-6 tage). bei neugeborenen fällt die inkubationszeit deutlich länger aus (3 wochen). die inzidenz der neonatalen hsv-infektionen wird auf 1 : 3.000 bis 1 : 20.000 aller lebendgeborenen geschätzt. das risiko einer hsv-infektion bei vaginaler entbindung beträgt für das neugeborene 30-60 % (primärinfektion der mutter) und sinkt auf unter 5 % bei rekurrierender hsv-infektion der mutter. prävention schwangere und gebärende frauen sollten bzgl. einer etwaigen hsv-infektion befragt werden. schwangere mit einer genitalen hsv-primärinfektion sollten mit aciclovir über 10 tage behandelt werden. bei frauen mit rezidivierendem herpes genitalis in der spätschwangerschaft senkt eine aciclovir-suppressionstherapie die häufigkeit von hsv-rezidiven zum zeitpunkt der geburt und wahrscheinlich die hsv-2-bedingte sectiorate. bei einer floriden genitalen hsv-infektion der schwangeren nach der 36. ssw, vor allem bei einem primären herpes genitalis, soll per sectio entbunden werden. mütter, die hsv ausscheiden und an einer aktiven kutanen (herpes labialis), nicht aber genitalen hsv-infektion erkrankt sind, sollten möglichst isoliert werden. der kontakt des neugeborenen zu infektiösen hauteffloreszenzen (mutter, vater, personal) muss durch abdeckung (mundschutz), basishygiene (händedesinfektion) und geeignetes verhalten (kein küssen des kindes) verhindert werden. beim stillen des neugeborenen ist auf effloreszenzen an der brust zu achten. in einzelfällen kann bei exponierten kindern bis zum erhalt der hsv-diagnostik eine präsymptomatische aciclovir-therapie erwogen werden. eine impfung gegen hsv steht nicht zur verfügung. alle 3 monate sollten die viruslast, die cd4-zahl und weitere laborparameter (monitoring von uaw) kontrolliert werden. da im säuglingsalter ein hohes risiko besteht, aids-definierende symptome, besonders eine hiv-enzephalopathie bzw. hiv-hepatopathie zu entwickeln, ist es konsens, alle kinder im säuglingsalter unabhängig von der viruslast und unabhängig von der cd4-zahl zu behandeln. für ältere kinder kann dann eine indikation zur behandlung nach zellzahl und viruslast gefunden werden. impfungen hinsichtlich der impfprävention bei hiv-infektionen wird auf die empfehlungen der stiko verwiesen. totimpfungen können unabhängig vom immunstatus ohne risiko eingesetzt werden. die verabreichung einer lebendimpfung (masern, mumps, röteln, windpocken) wird für alle asymptomatischen hiv-positiven kinder empfohlen, wenn keine schwere immunsuppression vorliegt (grenzwert an cd4+ zellen je µl: säuglinge 750, 1-5 jahre: 500, > 5 jahre: 200). die hiv-infektion ist nicht meldepflichtig. es existieren lediglich eine anonyme labormeldepflicht und bei aids eine freiwillige anmeldung beim aids-fallregister des rki. papillomaviren gehören zur gruppe der papovaviren und sind nicht umhüllte viren mit einer zirkulären doppelstrang-dna unter einem kapsid aus 72 kapsomeren. persistenz ohne klinische apparenz. eine virämie findet fast nie statt. die humanpathogenen papillomaviren (hpv) umfassen über 100 genotypen. hpv-infektionen bei kindern sind häufig. 10-20 % der schulkinder weisen kutane warzen auf. die inkubationszeit bei kutanen warzen beträgt 6 monate bis 2 jahre. larynxpapillome sind selten (inzidenz 0,1-2,8 auf 100.000) und treten vorzugsweise bei säuglingen und kleinkindern auf. juvenile larynxpapillome weisen eine starke assoziation zu genitalen hpv-infektionen der mutter auf (hpv6 und hpv11). genitale hpv-infektionen gehören zu den häufigsten sti und betreffen vorwiegend adoleszente und junge erwachsene. sie werden durch schmierinfektionen übertragen. verschiedene hpv-typen führen zu anogenitalen warzen. die inkubationszeit beträgt 4 wochen bis mehrere monate. klinische symptome gemeine plantare warzen (verrucae vulgaris, verrucae plantaris) werden vorzugsweise durch die hpv-typen 1, 2, 3, 4 und 7 hervorgerufen. sie treten insbesondere im bereich von bradytrophen arealen auf. filiforme warzen finden sich an augenlidern, lippen und nase. plane juvenile warzen (verrucae planae juveniles) werden durch die hpv-typen 3, 10, 28 und 41 hervorgerufen und finden sich als flache, hautfarbene oder hellbräunliche papeln insbesondere von gesicht und handrücken. die spontanregressionsrate kutaner oder anogenitaler warzen im kindesalter liegt innerhalb von 2 jahren bei über 60 %. infektionen mit bestimmten hpv-typen (16, 18, 31, 45 u. a.) sind von großer bedeutung für die entstehung von zervix-, vulva-, penis-und analkarzinomen. diagnose die diagnose von warzen wird klinisch gestellt. hpv lassen sich nicht anzüchten. eine analyse der viralen dna aus der läsion mittels pcr ist möglich. bei karzinomverdacht ist eine invasive diagnose (biopsie) angezeigt. therapie therapeutisch kann das von warzen befallene gewebe chirurgisch angegangen werden, oder es kommt eine antiproliferative, z. t. immunmodulatorische therapie zum einsatz. larynxpapillome werden gelasert. prävention es stehen immunogene hpv-vakzine (9-valent und 2-valent) zur verfügung. durch umfangreiche klinische studien konnte gezeigt werden, dass beide impfstoffe sehr gut gegen malignomvorstufen wirken, die durch die im impfstoff enthaltenen hpv-typen hervorgerufen wurden. hieraus wird abgeleitet, dass ein sehr guter schutz gegen sich später entwickelnde karzinome angenommen werden darf. der 9-valente impfstoff schützt darüber hinaus auch vor genital-und analwarzen. es besteht keine meldepflicht (ifsg). influenzaviren gehören zur familie der orthomyxoviren. sie lassen sich in drei typen (a, b, c) unterteilen, influenza-a-und -b-viren sind für menschliche infektionen von größerer bedeutung als die influenza-c-viren; so rufen sie die interpandemische saisonale influenza hervor. die viren bestehen aus einem segmentierten genom mit 8 einsträngigen dna-molekülen, die für insgesamt 11 proteine codieren. influenza-a-viren werden nach ihrem oberflächenglykoprotein, hämagglutinin und neuraminidase-subtypen unterteilt (z. b. a / h1n1; a / h3n2). hämagglutinin bewirkt die zelluläre adhäsion, wohingegen neuraminidase für die zelluläre hämagglutinin und neuraminidase der influenza-a-und -b-viren spielen bei der virusreplikation und der bildung protektiver antikörper eine zentrale rolle. durch fehler bei der viralen rna-replikation entstehen virusvarianten (antigen-drift). diese verursachen die jährlichen influenzaausbrüche. von antigen-shift spricht man, wenn es zu einer raschen, deutlichen veränderung der antigeneigenschaften des virus mit entstehung eines völlig neuen subtyps des influenza-a-virus kommt. ursache ist ein austausch der gensegmente für hämagglutinin und / oder neuraminidase. antigen-shifts sind voraussetzungen für influenza-pandemien. von influenza-b-viren gibt es keine subtypen, es zirkulieren jedoch zwei genetisch unterschiedliche linien (b / victoria-linie, b / yamagata-linie). alle 16 unterschiedlichen hämagglutinine und 9 neuraminidasen finden sich nur bei wildlebenden wasservögeln. beim menschen traten bisher nur die subtypen a / h1n1, a / h2n2 und a / h3n2 auf. influenza-b-viren kommen nur beim menschen vor. influenza-infektionen sind weltweit verbreitet. jährlich kommt es während der herbst-und wintermonate zu grippeausbrüchen, die in ihrer stärke erheblich variieren. seit vielen jahren zirkulieren weltweit zwei influenza-a-subtypen (a / h1n1 und a / h3n2) sowie die zwei influenza-b-viruslinien. etwa 25-30 % der grippeerkrankungen werden durch die influenza-b-viren verursacht. die saisonale grippe betrifft gewöhnlich 5-15 % der bevölkerung, wobei die inzidenz im kindesalter zwischen 20 und 35 % liegt. übertragung, inkubationszeit und klinische symptome kinder weisen häufig schwere respiratorische symptome auf. im frühen kindesalter sind für die hohe krankheitsbelastung neben rsv-und rhinovirus-infektionen vor allem die jährlichen influenzaausbrüche verantwortlich. seroprävalenzstudien in deutschland und den niederlanden ergaben, dass 75-99 % der kinder bis zum 6. lebensjahr bereits eine influenza-a-infektion durchgemacht haben. die morbiditätsraten bei kindern unter 5 jahren liegen zwischen 9 und 30 %. die übertragung der influenzaviren erfolgt durch tröpfchen (> 5 µm), insbesondere beim husten oder niesen. eine aerogene übertragung (atmen, sprechen) oder durch direkten kontakt (händeschütteln) ist ebenfalls möglich. die inkubationszeit ist kurz und beträgt durchschnittlich 2 (1-4) tage. die dauer der infektiosität beträgt 4-5 tage ab dem auftreten der ersten symptome. › tab. 10.44 gibt die wichtigsten klinischen symptome der influenza im vergleich zum banalen, sog. grippalen infekt wieder. zu den komplikationen gehören u. a. enzephalopathien, fieberkrämpfe, pneumonien sowie tracheobronchitiden, sinusitiden und mittelohrentzündungen. todesfälle sind auch im kindesalter bekannt. die influenza-assoziierten hospitalisationsraten sind insbesondere im säuglings-und kleinkindesalter erhöht. diagnose die diagnose kann klinisch oder durch direkten nachweis viraler antigene mittels immunfluoreszenz, elisa oder sog. schnelltests aus klinischen materialien des oberen und unteren respirationstrakts gestellt werden. die nachweismethode der wahl ist der typenspezifische nachweis mittels pcr aus nasen-rachen-sekret bzw. -abstrich. therapie die neuraminidasehemmer oseltamivir und zanamivir blockieren die aktivität der viralen neuraminidase und damit die freisetzung neu gebildeter viren. sie wirken gegen influenza-aund -b-viren. oseltamivir und zanamivir sind für kinder ab 1 bzw. ab 5 jahren zugelassen. eindeutige belege für eine vermeidung oder verkürzung schwerer krankheitsverläufe sowie eine reduktion der letalität liegen bei kindern nicht vor. prävention eine jährlich anzuwendende impfung gegen influenza ist verfügbar. als besonderheit ist hervorzuheben, dass bei der influenza altersspezifisch wirksame influenza-impfstoffe unterschiedlicher konzeption und komposition angewendet werden sollten. aufgrund der schlechten vorhersehbarkeit von zirkulierenden b-stämmen werden aktuell quadrivalente influenza-impfstoffe empfohlen. es wird auf die aktuellen empfehlungen der stiko verwiesen. bartonellen sind gramnegative bakterien. bartonella henselae ist der erreger der häufigen katzenkratzkrankheit, die weltweit auftritt. übertragung und inkubationszeit bartonella henselae wird überwiegend bei biss-und kratzwunden durch symptomlose katzen, seltener durch hunde übertragen. etwa 13 % der hauskatzen in deutschland weisen eine bakteriämie für den nachweis von bartonella henselae auf. die katzenkratzkrankheit tritt überwiegend im herbst und winter auf. eine übertragung von mensch zu mensch ist nicht dokumentiert. die inkubationszeit ist unterschiedlich, wahrscheinlich 3-10 tage. klinische symptome nach auftreten einer hautläsion und weiteren 15-50 tagen wird eine lymphadenitis beobachtet. das klinische bild der katzenkratzkrankheit ist eine überwiegend einseitige lymphadenitis. überwiegend sind die axillären bzw. supraklavikulären oder zervikalen lymphknoten betroffen. es besteht eine selbstheilungsrate der lymphadenitis innerhalb von 2-4 monaten. bei 60-90 % der betroffenen patienten entwickelt sich an der eintrittspforte des erregers nach 3-10 tagen ein kleines bläschen oder eine pustel, die rasch in eine papel übergeht und verkrustet. weitere symptome der katzenkratzkrankheit sind fieber, kopfschmerzen, gliederschmerzen, appetitlosigkeit und übelkeit. bei weniger als 10 % der patienten tritt ein sog. okuloglanduläres syndrom (parinaud-syndrom) auf. es besteht eine nichteitrige konjunktivitis mit prä-und supraaurikulärer lymphadenitis. eintrittspforte ist wahrscheinlich die bindehaut. auch neurologische manifestationen wie enzephalitis, neuroretinitis oder polyneuritis sind in sehr seltenen fällen zu beobachten. als komplikationen sind endokarditiden, myokarditiden oder osteomyelitiden zu nennen. intermittierende fieberschübe sowie rezidivierende, heftige bauchschmerzen können auf den befall von bauchorganen hinweisen. diagnose die diagnose der katzenkratzkrankheit wird aufgrund der anamnese und des klinischen verlaufs vermutet. serologisch steht ein immunfluoreszenztest mit in zellkulturen produziertem antigen zur verfügung. bei einem igg-titer von > 1 : 200 im immunfluoreszenztest (ift) kann die diagnose angenommen werden. der nachweis von igm (≥ 1 : 20) kann zur klärung beitragen, gelingt bei der katzenkratzkrankheit aber nicht immer. elisa-verfahren sind nicht verfügbar. auch eine biopsie kann im zweifelsfall zur diagnose führen. therapie in den meisten fällen ist weder eine chirurgische intervention noch eine antiinfektive therapie angezeigt. bei sehr schweren fällen kann mit azithromycin behandelt werden. prävention immunsupprimierte patienten sollten den kontakt zu katzen und hunden meiden. kingella kingae ist ein unbewegliches, plumpes, gramnegatives, in paaren und kurzen ketten angeordnetes bakterium. einer systemischen infektion geht die besiedelung des oropharynx voraus. kingella kingae lässt sich vor allem bei kleinkindern mit einer prävalenz von bis zu 10 % in der oropharyngealflora nachweisen. diagnose bildgebend wird eine knochen-und gelenkinfektion diagnostiziert. der erregernachweis lässt sich mittels nadelpunktion (biopsie, gelenkflüssigkeit) sichern. therapie kingella kingae ist gut empfindlich gegen penicillin, betalaktam-antibiotika (z. b. penicilline und cephalosporine). die prävention die pertussis-impfung gehört zu den standardimpfungen für alle altersgruppen. diesbezüglich wird auf die empfehlungen der stiko verwiesen. hervorzuheben ist, dass weder die pertussis-infektion noch eine pertussis-impfung zu einer lang anhaltenden immunität führen. eine bindehautentzündung zeichnet sich durch eine meist leicht schmerzhafte, oft follikuläre rötung der bindehaut aus. sie geht mit vermehrtem tränenfluss und verklebten lidern einher. eine infektiöse ursache muss ggf. von einer anderen genese (allergisch, autoimmun, sekundär bei blepharitis, fremdkörper oder andere traumen) unterschieden werden. infektiöse erreger für eine konjunktivitis sind neisseria gonorrhoeae (neugeborenenkonjunktivitis), chlamydia trachomatis der serotypen d -k (neugeborenenkonjunktivitis), mischinfektionen mit chlamydien und gonokokken (neugeborenenkonjunktivitis), andere bakterien wie staphylococcus aureus, e. coli, pneumokokken, β-hämolysierende streptokokken, haemophilus influenzae, neisserien und moraxella catarrhalis. zur physiologischen bindehautflora gehören kns, staphylococcus aureus, corynebacterium spp. und α-hämolysierende streptokokken. als virale erreger sind vor allem hsv und adenoviren zu nennen. seltener lässt sich eine hämorrhagische keratokonjunktivitis durch bestimmte enteroviren beobachten. weitere informationen: › kap. 10.2.20. die kopflaus (pediculosis capitis) ist 2-4 mm lang und befällt sowohl kinder als auch erwachsene. besonders häufig sind kinder im grundschulalter sowie mädchen betroffen. getrennt von ihrem obligaten wirt überleben kopfläuse nur 24-36 h. wesentliche schädigungen oder gesundheitsfolgen gehen von der kopflaus in der regel nicht aus. die prävalenz in der gesamtbevölkerung ist gering. in kjge kommt es jedoch regelmäßig zu größeren infestationen. übertragung, klinische symptome und diagnose die übertragung erfolgt ausschließlich von mensch zu mensch bei körperkontakt (kjge, sozialverhalten, "selfies" u. a.). andere übertragungswege (spielzeug) stellen bei der kopflaus eine sehr seltene möglichkeit dar. kopfläuse befallen vor allem das capillitium mit bevorzugung der retroaurikulären region. selten sind läuse auch im bereich der bart-und schamhaare anzutreffen. ein kopflausbefall ist für betroffene meistens symptomlos. wenn symptome auftreten, dann ist juckreiz zu nennen. gelegentlich sind urtikarielle, juckende, hochrote papeln zu beobachten. die haare sind häufig verklebt und verfilzt. differenzialdiagnostisch ist an ein kopfekzem, impetigo contagiosa und tinea capitis zu denken. das leitsymptom der pedikulose ist ein erheblicher pruritus. neben kopfläusen sind auch nissen, die knospenartig an den haaren kleben und sich im gegensatz zu kopfschuppen nicht vom haar abstreichen lassen, anzutreffen. die diagnose wird klinisch gestellt. therapie national und international variieren die angaben zur therapie. die vorschläge reichen vom alleinigen auskämmen bis hin zur anwendung von "läusemitteln" und detaillierten anweisungen für reinigungs-, putz-oder waschmaßnahmen von räumen, polstermöbeln, betten, kleidung und spielzeug. auch kombinationen der maßnahmen werden empfohlen. rki und dgpi empfehlen die abtötung von läusen und ihren eiern. auch kontaktpersonen sollen behandelt werden. in deutschland ist permethrin mittel der wahl. es wird auf das feuchte haar aufgetragen und nach etwa 30-bis 45-minütiger einwirkzeit mit wasser ausgewaschen. nach 8-10 tagen sollte eine zweite behandlung erfolgen (rki-empfehlung). zur desinfektion von matratzen, bettwäsche etc. eignet sich allethrin. pyrethrumextrakte sind schwächer wirksam als permethrin. malathion ist in deutschland nicht im handel. eine orale therapie mit ivermectin hat sich in der behandlung von kopfläusen als wirksam erwiesen. der einsatz von ivermectin bei pediculosis capitis erfolgt "off-label". daneben wird dimeticon eingesetzt. es sind präparate mit sehr unterschiedlicher zusammensetzung auf dem markt. die wirksamkeit hängt wesentlich von den physikalischen eigenschaften der eingesetzten dimeticone ab. zusammenfassend ist festzustellen, dass der evidenzlevel für die therapie von kopfläusen relativ gering ist. prävention nach ifsg schließt der festgestellte kopflausbefall eine betreuung oder eine tätigkeit in einer gemeinschaftseinrichtherapie die behandlung ist symptomatisch (rehydratation, isotonie). sollte eine kausale therapie notwendig sein (immunsuppression), so ist nitazoxanid bei gesunden kindern ab 12 monaten bis ≤ 11 jahren zugelassen. therapieversuche ohne eindeutige wirksamkeit wurden auch mit paromomycin und azithromycin unternommen. prävention neben der aufrechterhaltung der immunfunktion (reduktion der iatrogenen immunsuppression) stehen die aufklärung über ansteckungswege und eine gute basishygiene (händewaschen) im vordergrund. der nachweis von kryptosporidien ist meldepflichtig (ifsg). erreger fetale und neonatale infektionen können selten postnatal (meist nosokomial) auftreten. die infektion erfolgt nach der geburt in einer kontaminierten geburtswanne oder einem inkubator. die letalität ist hoch (bis zu 60 %). das pontiac-fieber ist ein grippeähnliches krankheitsbild mit fieber, kopfschmerzen, myalgien, arthralgien, übelkeit, schwindel und husten. es sind vor allem menschen ohne grundkrankheit betroffen. diagnose die diagnose der legionellose wird durch den direktnachweis des erregers (pcr, kultur) aus rachenabstrichen, sputum, trachealsekret oder bal gestellt. daneben ist der nachweis von legionella pneumophila serogruppe 1-antigen im urin am 1. bis 3. erkrankungstag mittels elisa oder immunchromatografie (schlechte sensitivität, gute spezifität) möglich. therapie therapie der wahl im kindesalter ist clarithromycin (15 mg / kg kg / tag i. v. in 2 ed) oder azithromycin (10 mg / kg kg / tag in 1 ed i. v. für 5 tage). unkomplizierte formen werden 5-10 tage, schwere verläufe 21 tage lang behandelt. kontrollierte prospektive studien zur antiinfektiven therapie einer legionellose im kindesalter fehlen allerdings. prävention in krankenhäusern werden warmwassersysteme mit stagnierendem wasser sowie entsprechende medizinische geräte (inkubatoren) mikrobiologisch untersucht. um das ansteckungsrisiko zu minimieren, soll bei immunsupprimierten patienten in risikobereichen zur mund-und gesichtspflege, zur anfeuchtung der atemluft, zur verneblung oder herstellung von medikamenten kein trinkwasser verwendet werden. es besteht eine meldepflicht (ifsg). die leishmaniose wird von geißeln (flagellen) tragenden protozoen der gattung leishmania hervorgerufen, die zur familie der trypanosomatidae gehören. die viszerale leishmaniose wird insbesondere von erregern des sog. leishmania-donovani-komplexes (leishmania donovani, leishmania infantum) verursacht. die kutane leishmaniose in europa, im arabischen raum sowie in afrika wird durch leishmania major, leishmania tropica und leishmania aethiopica hervorgerufen. in mittel-und südamerika sind leishmaniamexicana-komplexe sowie die subgattung viannea als erreger zu nennen. leishmaniosen sind zoonosen und anthroponosen. das reservoir sind säugetiere, vor allem hunde und nager, in einigen regionen vorwiegend menschen. übertragung der mensch wird durch einen stich der weiblichen schmetterlingsmücke der gattung phlebotomus und lutzomyia infiziert. kontaminierte blutkonserven und infusionsnadeln sowie organtransplantationen können in seltenen fällen zur übertragung führen. die leishmaniose kommt ist mehr als 80 tropischen und subtropischen ländern vor. exakte prävalenzdaten liegen nicht vor. leishmaniose ist eine benigne, selbstlimitierende erkrankung, die in weiten teilen südeuropas, asien, afrika sowie mittelund südamerika bei kindern und erwachsenen vorkommt. infolge eines infizierenden stichs entwickelt sich über viele wochen eine juckende papulöse hauteffloreszenz, die auf einen durchmesser bis 2 cm anwachsen kann. zentral entwickelt sich ein ulkus mit erhabenem randwall, das nach 3-18 monaten abheilt und eine hypo-oder hyperpigmentierte narbe zurücklässt. • mukokutane leishmaniose (espundia): bei der mukokutanen form, die überwiegend in süd-und mittelamerika vorkommt, disseminieren leishmanien meist bei nicht oder unzureichend behandelten primären hautläsionen in die schleimhäute. bis es zur abheilung kommt, vergehen monate bis jahre. eine häufige folge sind gewebsulzerationen im bereich der nasenschleimhaut, die zu einer septumperforation führen können. auch eine verschleppung in nasenpharynx, auf larynx und trachea kann vorkommen. diagnose die typischen laborbefunde bei einer viszeralen leishmaniose sind eine beschleunigte blutsenkungsgeschwindigkeit, eine panzytopenie sowie eine markante hypergammaglobulinämie als folge einer polyklonalen b-zell-aktivierung. die diagnose der leishmaniose kann durch eine kombination von verschiedenen methoden (parasitologisch, molekularbiologisch und serologisch) gesichert werden. parasiten können kultiviert oder alternativ mittels pcr nachgewiesen werden. bei immunkompetenten patienten kann eine elisa-technik oder immunfloreszenz zum einsatz kommen. differenzialdiagnostisch sind bei der viszeralen leishmaniosis mit fieber und hepatosplenomegalie und ggf. lymphadenopathie vor allem leukämien und lymphome zu berücksichtigen. bei der kutanen leishmaniose muss eine pyodermie anderer ursache abgegrenzt werden. therapie zuletzt wurde zur therapie der viszeralen leishmaniose als mittel der 1. wahl bei kindern und erwachsenen liposomales amphotericin b (3 mg / kg kg an 4 aufeinanderfolgenden tagen sowie am 10. tag) angegeben. erreger aus mittel-und südamerika erfordern eine dosierung von 3-4 mg pro kg kg über 10 tage. eine neue leitlinie zur diagnostik und therapie der viszeralen leishmaniose befindet sich in vorbereitung. die kutane leishmaniose kann bei immunsupprimierten patienten oder bei ausgeprägten läsionen in problematischen regionen medikamentös therapiert werden. zu den maßnahmen gehören die lokale applikation von paromomycin mit methylbenzethoniumchlorid sowie peri-und intraläsionale injektionen von antimon. prävention als vorbeugende maßnahme beim aufenthalt in regionen, in denen die leishmaniose vorkommt, gilt vor allem der schutz vor kontakt mit phlebotomen. dazu gehören das auftragen von repellenzien wie deet oder icaridin sowie das das tragen langer hosen und langärmeliger oberbekleidung. listerien sind grampositive stäbchenbakterien. sie lassen sich im erdboden, im wasser und auf pflanzen detektieren. bei 1-3 % der gesunden sind listerien im stuhl nachweisbar. wichtigster vertreter ist listeria monocytogenes. es sind mehrere serovare bekannt. listerien verursachen infektionen bei verschiedenen tierspezies. sie verfügen über virulenzfaktoren, die das eindringen und überleben innerhalb von wirtszellen ermöglichen. die inzidenz der listeriose beträgt in mittel-und westeuropa 1-4 bis 10 fälle je 1 mio. einwohner. in deutschland werden pro jahr ca. 300 fälle gemeldet. es zeigt im typischen fall eine zentrale abblassung und livide verfärbung. bei kindern lässt sich das erythema migrans häufig im kopf-und halsbereich beobachten. das borrelien-lymphozytom tritt wesentlich seltener als das erythema migrans auf. es handelt sich um eine erkrankung der haut mit bevorzugung der ohren (ohrmuschel, ohrläppchen) und der mamillen. es imponieren eine livide rötung und derbe infiltration. die acrodermatitis chronica atrophicans zählt zu den späten manifestationen der lyme-borreliose, die monate bis jahre nach der infektion auftritt. die lymphozytäre meningitis ohne oder mit hirnnervenausfällen, am häufigsten mit akuter peripherer fazialisparese, ist mit einem anteil von > 80 % der erkrankungsfälle die häufigste klinische manifestation der neuroborreliose im kindesalter. die lyme-borreliose ist die häufigste ursache der akuten peripheren fazialisparese im kindesalter. die bilaterale fazialisparese geht praktisch immer mit einer borrelien-ätiologie einher. in den sommer-und herbstmonaten ist jeder 2. erkrankungsfall einer akuten peripheren fazialisparese im kindesalter auf eine infektion mit borrelia burgdorferi zurückzuführen. eine beteiligung anderer hirnnerven (n. oculomotorius, n. trochlearis, n. abducens, n. vestibularis) ist möglich. im unterschied zu anderen, viralen meningitiden zeichnet sich die borrelienmeningitis durch eine einige tage längere krankheitsgeschichte mit allgemeinbeeinträchtigung, kopfschmerzen und kaum ausgeprägten meningealen reizzeichen aus. bei erwachsenen tritt besonders häufig die lymphozytäre meningoradikuloneneuritis (bannwarth-syndrom) auf. die späte neuroborreliose ist bei kindern selten und geht mit länger bestehenden beschwerden wie kopfschmerzen, pseudotumor cerebri, fokaler enzephalitis, zerebellärer ataxie, querschnittsmyelitis oder guillain-barré-syndrom einher. frühe gelenkmanifestationen der lyme-borreliose umfassen arthralgien wechselnder lokalisationen. die klassische lyme-arthritis ist eine späte manifestation, die nach einer inkubationszeit von monaten bis jahren nach einem zeckenstich auftritt. sie imponiert als akute mon-oder oligoarthritis und manifestiert sich vor allem an den großen gelenken. die arthritis sistiert nach 1-2 wochen, tritt aber nach einem symptomfreien intervall von wochen und monaten an gleicher stelle wieder auf. weitere klinische manifestationen sind: lyme-karditis sowie konjunktivitis, chorioretinitis, keratitis, uveitis intermedia, iridozyklitis und optikusneuritis. diagnose die diagnose der lyme-borreliose wird stadienabhängig gestellt (› tab. 10.46). das erythema migrans ist pathognomonisch, und die diagnose wird klinisch ermittelt. frühe manifestationen einer borreliose werden durch anamnestische daten, klinische befunde sowie den spezifischen nachweis von antikörpern gegen borrelia burgdorferi diagnostiziert. die serologische routinediagnostik zur lyme-borreliose stützt sich auf den nachweis spezifischer igm-und / oder igg-antikörper gegen borrelia burgdorferi im blut und bei v. a. neuroborreliose auch im liquor. standard ist ein enzymimmunoassay (elisa als suchtest). bei spätmanifestation lassen sich in nahezu 100 % der fälle spezifische antikörper nachweisen. als bestätigungstest, insbesondere zur überprüfung der spezifität, wird ein immunoblot durchgeführt. es sind kreuzreaktionen mit anderen spirochäten (leptospirose, syphilis, rückfallfieber) sowie falsch positive igm-befunde bei herpesvirus-infektionen (ebv, vzv, cmv) und beim vorliegen von rheumafaktoren möglich. da spezifische antikörper lange persistieren, ist die serologie zur verlaufs-und therapiekontrolle obsolet. da die symptome einer neuroborreliose vielfältig und unspezifisch sind, erfordert die diagnose "neuroborreliose" den nachweis einer lymphozytären liquorpleozytose oder einer spezifischen authochtonen antikörpersynthese im zns. im kindesalter liegen einige besonderheiten vor: die lymphozytäre liquorpleozytose findet sich bei > 90 % der frühen neuroborreliosen. die diagnose "neuroborreliose" kann als gesichert gelten, wenn neben der liquorpleozytose spezifische igm-antikörper gegen borrelia burgdorferi und evtl. auch igg-antikörper nachweisbar sind. der erregernachweis (pcr oder kultur) im liquor ist durchschnittlich nur bei maximal 30 % der fälle möglich. der nachweis des b-zellen anziehenden chemokins cxcl13 im liquor ist neueren untersuchungen zufolge für die diagnose der frühen neuroborreliose geeignet. andere verfahren, insbesondere der lymphozytentransformationstest, werden nicht empfohlen. 10 .3 spezifische erreger und infektionskrankheiten nach zecken abgesucht werden. eine prophylaktische antiinfektive therapie ist nicht indiziert. ein impfstoff steht nicht zur verfügung. malaria wird durch einzeller der gattung plasmodium hervorgerufen. erreger und übertragung die übertragung erfolgt durch den stich der dämmerungs-und nachtaktiven weiblichen anopheles-mücke. es sind über 100 plasmodienarten bekannt, von denen fünf als humanpathogen gelten: • plasmodium (p.) falciparum (malaria tropica) • p. vivax und p. ovale (malaria tertiana) • p. malariae (malaria quartana) • p. knowlesi (in südostasien bei affen, auch ursache von malaria bei menschen: malaria knowlesi) bei p. falciparum, p. vivax und p. ovale ist der mensch neben den stechmücken das einzige reservoir. der mensch fungiert als zwischenwirt und ermöglicht die ungeschlechtliche vermehrung der parasiten; im endwirt, der anopheles-mücke, erfolgt die geschlechtliche vermehrung. mücken benötigen blut nur für die eiproduktion (weibliche mücke). nach dem stich erfolgt die invasion der hämoparasiten in hepatozyten und eine ungeschlechtliche vermehrung. abhängig von der plasmodienart kommt es nach frühestens 5 tagen, ggf. auch nach monaten zur freisetzung von merozoiten in den blutkreislauf. sodann befallen die parasiten erythrozyten und transformieren zu trophozoiten, die sich teilen und mehrkernige schizonten bilden, die in merozoiten zerfallen. die freigesetzten merozoiten befallen neue erythrozyten und setzen den zyklus fort. die malaria tropica und die knowlesi-malaria können als tropenmedizinischer notfall bezeichnet werden. neben fieber kommen bewusstseinsstörungen (somnolenz, koma), zerebrale krampfanfälle, pulmonale störungen sowie nierenversagen, hypoglykämien u. a. symptome vor. malaria tertiana und malaria quartana gehen selten mit schweren verläufen einher. eine malaria tropica in der schwangerschaft stellt eine lebensbedrohliche situation für mutter und kind dar. es ist eine diaplazentare infektion durch maternal-fetale transfusion während der geburt möglich. man spricht von einer konnatalen malaria. die neugeborenen sind zunächst gesund und fallen 4-12 wochen später durch uncharakteristische symptome wie trinkschwäche, gedeihstörung und anämie auf. zusätzlich kann eine hepatosplenomegalie bestehen und fieber auftreten. die malaria muss als kompliziert angesehen werden und sollte bei vorliegen der folgenden symptome als lebensbedrohliche krankheit eingestuft werden: bewusstseinstrübung, zerebraler krampfanfall, respiratorische insuffizienz, hypoglykämie, azidose, hyperkaliämie, schock, spontanblutungen sowie zeichen einer relevanten dehydratation. eine malaria tropica kann durch eine schwere anämie, niereninsuffizienz, hämoglobinurie, transaminasenerhöhung, ikterus und hyperparasitämie kompliziert werden. diagnose die diagnostik ist im zusammenhang mit den anamnestischen daten zu sehen. ein tropenaufenthalt und vorstellung mit fieber sollten immer anlass sein, an eine malaria zu denken. entscheidend ist der mikroskopische nachweis von plasmodien im dünnen, panoptisch gefärbten blutausstrich oder im parallel angefertigten dicken blutausstrich ("dicker tropfen"). bei anhaltendem verdacht ist eine wiederholung nach 12-24 h angezeigt. die bestimmung von antikörpern gegen plasmodien ist zur akutdiagnostik nicht geeignet. neben der erregerdiagnostik sollten ein blutbild angefertigt sowie leber-und nierenparameter erhoben werden. typische laborzeichen einer malaria sind eine thrombozytopenie und hyperbilirubinämie. therapie die therapie der malaria richtet sich nach der erregerart und dem schwergrad der erkrankung. malaria tertiana und quartana können ambulant behandelt werden, malaria tropica und knowlesi-malaria sind stets stationär zu behandeln. eine malaria durch p. falciparum und eine unkomplizierte knowlesi-malaria sowie die malaria tertiana werden mit einer oralen artemisinin-kombinationstherapie (act) behandelt. alternativ kann atovaquon / proguanil eingesetzt werden. chloroquin wird nur noch zur behandlung der malaria quartana eingesetzt. bei der malaria tertiana muss im anschluss an die elimination der erythrozytären parasiten eine nachbehandlung mit primaquin angeschlossen werden, um hypnozoiten in der leber abzutöten und rezidiven vorzubeugen. die komplizierte malaria durch p. falciparum und die knowlesi-malaria werden i. v. mit artesunat oder einer kombination aus chinin und doxycyclin bzw. clindamycin i. v. behandelt. prävention die entscheidung zur durchführung einer malariaprophylaxe muss anhand von reiseziel, reisezeit, reisedauer und reisestil sowie unter berücksichtigung individueller gegenanzeigen getroffen werden. die prävention einer malaria basiert auf dem schutz vor mückenstichen (expositionsprophylaxe) und der einnahme von malaria-medikamenten (chemoprophylaxe). beide maßnahmen ergänzen sich, bieten jedoch keinen absolut sicheren schutz vor einer malaria. da anopheles-mücken dämmerungs-und nachtaktiv sind, gilt es, unter imprägnierten moskitonetzen zu schlafen und insbesondere am abend lange kleidung zu tragen und repellents anzuwenden. die art und form der chemoprophylaxe sollte nach tropenmedizinischer beratung erfolgen. das masernvirus ist ein virus mit einsträngiger rna. es gibt nur einen serotyp, aber mehr als 20 genotypen. das masernvirus gehört zur familie der paramyxoviren. masern sind hochkontagiös mit einem manifestationsindex von nahezu 100 %! einziges reservoir ist der mensch. übertragung und inkubationszeit die übertragung erfolgt als tröpfcheninfektion, in sehr seltenen fällen auch durch luftzug über größere entfernungen. die infizierten sind 3-5 tage vor bis 4 tage nach exanthemausbruch infektiös, wobei die infektiosität im prodromalstadium am höchsten ist. nach erkrankung besteht eine lebenslange immunität. in vielen ländern konnten die masern durch konsequente umsetzung von impfprogrammen eliminiert werden. in deutschland treten jährlich viele hundert bis wenige tausend fälle auf, da die impfquote von weniger als 95 % nicht ausreicht, um die masern zu eliminieren. die inkubationszeit beträgt 8-12 tage. klinische symptome masern sind klinisch durch einen zweiphasigen verlauf charakterisiert. im prodromalstadium treten fieber und katarrhalische erscheinungen wie konjunktivitis, schnupfen, halsschmerzen, heiserkeit und trockener husten auf. die trias "fieber, bronchitis / pneumonie und konjunktivitis" muss an masern denken lassen. pathognomonisch sind koplik-flecken (kalkweiße stippchen auf hochroter, etwas granulierter schleimhaut, bevorzugt an der wangenschleimhaut). es entwickelt sich ein konfluierendes, makulopapulöses exanthem, das 3-4 tage nach dem prodromalstadium mit hohem fieberanstieg einhergeht. es besteht eine generalisierte lymphadenopathie. mitigierte masern treten bei jungen säuglingen auf, die noch maternale antikörper besitzen. bei immunsupprimierten patienten können masern vom klassischen krankheitsverlauf abweichen. insbesondere bei patienten mit einem t-zell-defekt entwickelt sich eine riesenzellpneumonie, die in der regel zum tod führt. bei diesen patienten lässt sich auch eine spezifische enzephalitisform, die "measles inclusion body encephalitis" (mibe) beobachten, die auf einer direkten virusinvasion beruht. hiervon abzugrenzen ist die subakute sklerosierende panenzephalitis (sspe), die sich bereits nach einer latenz von 5 wochen bis zu 6 monaten klinisch manifestiert und insbesondere bei säuglingen und kleinkindern auftritt, die zuvor an masern erkrankt waren. zu den komplikationen der masern gehören: masernkrupp, bronchiolitis, masernpemphigoid und akute masernenzephalitis, die bevorzugt am 3. und 9. tag nach exanthembeginn auftritt und in einer häufigkeit von 1 : 500 bis 1 : 2.000 zu beobachten ist. zu den weiteren neurologischen symptomen gehörten zerebrale krampfanfälle, neurologische herdsymptome mit hirnnervenparesen und hemiplegie sowie gelegentlich auch myelitische symptome. die masernenzephalitis hat eine letalität von 10-20 % und eine defektheilungsrate von 20-30 %. die o. g. sspe, eine persistierende infektion des zns, die sich bei immunologisch gesunden typischerweise erst nach einer latenz von 5-10 jahren manifestiert, verläuft in drei stadien (verhaltensauffälligkeiten und nachlassen intellektueller leistung, myoklonien und zerebrale anfälle, dezerebrationsstatus). die sspe führt innerhalb von 3-5 jahren nach krankheitsbeginn zum tod. die häufigkeit in den ersten 5 lebensjahren wird mit 1 : 700 bis 1 : 1.300 masernfälle angegeben. der erreger von meningokokken-infektionen ist neisseria (n.) meningitidis. diese neisserien sind unbewegliche, sporenlose, gramnegative diplokokken. sie wachsen aerob und sind kapnophil. pathogene varianten besitzen eine polysaccharidkapsel der serogruppen a, b, c, w, x oder y. das erregerreservoir stellt der mensch dar. in europa sind etwa 10 % der einwohner asymptomatische träger von meningokokken im nasen-rachen-raum. meningokokken-infektionen treten besonders bei säuglingen und kleinkindern mit einem erkrankungsgipfel um den 6. lebensmonat auf. ein zweiter inzidenzgipfel tritt im jugendalter auf. insgesamt sind invasive meningokokken-infektionen jedoch sehr selten (0,5 je 100.000 einwohner). streptococcus (s.) pneumoniae ist ein bekapseltes grampositives bakterium, von dem mindestens 94 verschiedene serotypen identifiziert wurden. pneumokokken können den menschlichen nasopharynx passager oder permanent besiedeln und sind potenziell pathogen. die besiedelung ist altersabhängig und hängt von der beeinträchtigung der wirtsabwehr bzw. einer potenziellen schleimhautschädigung infolge einer virusinfektion oder durch tabakrauchexposition ab. vor einführung der generellen impfempfehlung waren invasive pneumokokken-infektionen am häufigsten innerhalb der ersten 2 lebensjahre anzutreffen. nach einführung der konjugierten impfung kam es zu einer deutlichen absenkung von invasiven pneumokokken-erkrankungen. ein sog. replacement-phänomen führt zu einer zunahme der nicht im impfstoff enthaltenden serotypen. die epidemiologie der pneumokokken-infektion ist "im fluss". diagnose idealerweise wird bei einer invasiven pneumokokken-infektion ein erregernachweis im blut oder liquor angestrebt. abstriche sind nicht sinnvoll, da nicht sicher zwischen kolonisierung und verursachung der erkrankung unterschieden werden kann. therapie therapie der wahl ist bei einer lokal begrenzten pneumokokken-infektion amoxicillin. bei gesicherter pneumokokken-empfindlichkeit kann auch penicillin eingesetzt werden. bei invasiven infektionen werden neben penicillin g und alternativ ampicillin auch cephalosporine eingesetzt. seit den 1970er-jahren sind vermehrt penicillin-resistente pneumokokken-stämme nachgewiesen worden. prävention zur prophylaxe der pneumokokken-infektion stehen pneumokokken-konjugat-und kapselpolysaccharid-impfstoffe zur verfügung. bezüglich des einsatzes wird auf die aktuellen stiko-empfehlungen verwiesen. isolationsmaßnahmen werden nicht empfohlen. krankheit oder tod durch pneumokokken-infektionen sind in deutschland nicht meldepflichtig. als prionen werden proteine bezeichnet, die sowohl in physiologischen als auch pathogenen strukturen vorliegen können. die bezeichnung "prionen" ist abgeleitet von "protein" und "infektion". diagnose die diagnose wird häufig durch antigennachweise mittels elisa-schnelltest ermittelt. die sensitivität ist relativ niedrig. pcr-nachweismethoden, vor allem in multiplex-tests integriert, setzen sich mehr und mehr durch. kulturelle und serologische verfahren haben praktisch keine bedeutung. therapie die behandlung ist symptomatisch. prävention rsv-positive neugeborene und säuglinge sowie kleinkinder sollten im krankenhaus kohortiert werden. basishygienemaßnahmen sind von größter bedeutung. seit einigen jahren kann palivizumab zur prophylaxe von rsv-infektionen zur passiven immunisierung eingesetzt werden. empfohlen ist die passive immunisierung für risiko-frühgeborene, kinder mit einer hämodynamisch relevanten herzerkrankung sowie solche mit syndromalen oder neurologischen grundkrankheiten. eine aktive immunisierung steht nicht zur verfügung. impfstoffe zur aktiven immunisierung und medikamente (nukleosidanaloga) zur behandlung der rsv-infektion sind in der vorbereitung. eine meldepflicht besteht nicht. rhinoviren sind kleine und unbehüllte viren aus der familie der picornaviridae. man unterscheidet mindestens drei untergruppen (a -c). die infektionsinzidenz beträgt in den ersten lebensjahren 1-2 pro jahr und nimmt auf 1-4 pro jahr bei senioren ab. unter streptokokken-infektionen werden gemeinhin infektionen durch β-hämolysierende streptokokken der gruppe a, c und g verstanden. β-hämolysierende streptokokken der gruppe c und g können asymptomatisch kolonisieren oder krankheitsbilder verursachen, die streptokokken der gruppe a (gas) sehr ähnlich sind. in deutschland bedeutsam sind vor allem gas-infektionen. streptokokken werden sehr häufig (mehr als 25 % der kinder im nasen-rachen-raum) ohne zeichen einer entzündung detektiert (v. a.im winter). β-hämolysierende streptokokken sind grampositive, in kurzen ketten angeordnete kokken. anhand des lancefield-antigens können streptokokken der gruppe a, c und g serologisch unterschieden werden. als wichtiger virulenzfaktor gilt das m-protein. nach einer streptokokken-infektion wird lediglich eine m-typ-spezifische immunität erlangt. weitere wichtige virulenzfaktoren von β-hämolysierenden streptokokken sind streptolysin s und o sowie die pyrogenen exotoxine spe-a, -b, -c und -d. therapie es sollte zwischen gas-trägertum, viraler und gas-tonsillopharyngitis unterschieden werden. die indikation zur antiinfektiven therapie der gas-tonsillopharyngitis kann nicht als absolut betrachtet werden. bei scharlach bzw. gesicherter gas-tonsillopharyngitis wird im regelfall eine antiinfektive therapie empfohlen. therapie der wahl ist penicillin (penicillin v: 50.000-100.000 ie / kg kg / tag, 4 ed. tagesmaximaldosis für kinder: 2 mio. ie, für erwachsene: 3 mio. ie in 2-3 ed). neben penicillin v kann auch phenoxymethylpenicillin-benzathin mit einer deutlich längeren halbwertszeit angewendet werden (50.000 ie / kg kg / tag, 2 ed). auch amoxicillin ist alternativ denkbar. die therapiedauer beträgt 7 tage. bei penicillin-allergie gelten oralcephalosporine als mittel der wahl. makrolide führen wegen der auch in deutschland erkennbaren resistenzrate nicht immer zu einer zuverlässigen heilung. prävention 24 h nach einleitung einer antiinfektiven therapie sind patienten mit tonsillopharyngitis und scharlach nicht mehr infektiös. die prophylaxe bei patienten nach arf kann mit einem oralen penicillin (400.000 ie / tag, 2 ed) oder mit benzylpenicillin-benzathin (1,2 mio. ie i. m., alle 4 wochen) erfolgen. die prophylaxe nach arf ist besonders für patienten nach rheumatischer karditis bedeutsam, da sie eine hohe resistenzrate aufweisen. die gesamtdauer der arf-prophylaxe sollte minimal 5 jahre dauern, bei einem arf-rezidiv lebenslang. es besteht keine meldepflicht. invasive streptokokken-infektionen können als nekrotisierende fasziitis, sepsis oder streptokokkentoxin-schocksyndrom (früher toxischer scharlach) auftreten. das akute rheumatische fieber gehört zu den immunvermittelten streptokokken-folgekrankheiten und tritt 1-4 wochen nach einer akuten gas-infektion auf. diagnosekriterien, diagnostik und therapie › kap. 15.4.2. die akute post-streptokokken-glomerulonephritis tritt vorwiegend 1-4 wochen nach einer gas-tonsillopharyngitis mit "nephrophilen" gas-stämmen auf. zur klinischen symptomatik gehören fieber, makrohämaturie, oligurie, periorbitale ödeme, hypertonie, kopfschmerzen und sehstörungen sowie proteinurie, erythrozyten-und leukozytenzylinder im urin und eine c3-hypokomplementämie. weitere erreger der syphilis ist treponema pallidum. man unterscheidet die erworbene von der konnatalen syphilis. in deutschland lag die inzidenz für syphilis zuletzt bei 8,5 je 100.000 einwohner mit steigender tendenz. die übertragung erfolgt durch sexuellen kontakt mit einer partnerin oder einem partner mit syphilitischen läsionen. bei kindern und adoleszenten mit erworbener syphilis muss an sexuellen missbrauch gedacht werden, wobei nur wenige daten zu einer erworbenen syphilis bei kindern und jugendlichen in deutschland vorliegen. die inkubationszeit der erworbenen syphilis beträgt 10-90 tage. das klinische bild beginnt mit einer schmerzlosen schleimhautulzeration ("schanker") und regionalen lymphknotenschwellungen (primärstadium). danach kommt es bei einem teil der infizierten zu einem chronischen verlauf mit einer grippalen symptomatik und lymphadenopathie, exanthem und ggf. haarausfall (sekundärstadium). das tertiärstadium mit multipler organbeteiligung (aorta, haut, zns, augen) ist selten. die diagnostik wird weiter unten besprochen. antiinfektiv ist penicillin g das mittel der wahl. alternativ kommen doxycyclin, ceftriaxon und ggf. azithromycin bei der frühsyphilis in betracht. die infektion der schwangeren erfolgt ausschließlich über sexuelle kontakte. die transplazentare infektion des feten kann in jedem syphilisstadium der schwangeren erfolgen. infiziert sich die mutter während der schwangerschaft, beträgt die übertragungsrate ohne behandlung 50 %. neben der transplazentaren infektion ist auch eine infektion des kindes bei passage der geburtswege möglich (perinatale infektion); daneben kommt eine übertragung durch stillen in betracht, wenn sich syphilitische läsionen an der brust befinden. in deutschland ist es vor allem durch die systematische orale immunisierung der heimischen fuchspopulation gelungen, die tollwut zurückzudrängen. deutschland gilt als frei von klassischer tollwut. die fledermaustollwut ist hiervon jedoch ausgenommen. inkubationszeit die inkubationszeit der tollwutinfektion ist sehr variabel: sie schwankt von wenigen tagen bis zu jahren. erreger der erreger der varizellen ist das varizella-zoster-virus (vzv), ein doppelsträngiges dna-virus, das zu den herpesviren gehört. das vzv persistiert nach abklingen der windpocken in den dorsalen spinal-und hirnnervenganglien sowie in den enterischen und autonomen ganglien. eine reaktivierung der latenten infektion führt zu einem herpes zoster (gürtelrose). eine intrauterine vzv-infektion kann zu einem "fetalen varizellensyndrom", neonatalen varizellen und / oder herpes zoster im 1. lebensjahr führen. varizellen sind weltweit verbreitet. in regionen ohne varizellenimpfung liegt der häufigkeitsgipfel im kleinkindesalter. in der vorimpfära waren im alter von 14-17 jahren etwa 95 % der kinder und jugendlichen bereits an varizellen erkrankt. der herpes zoster tritt in den meisten fällen erst nach dem 5. lebensjahrzehnt auf. die inzidenz liegt bei den 75-bis 90-jährigen bei 14 je 1.000. auch kinder < 10 jahren können betroffen sein (1-2 je 1.000 personen). das erregerreservoir für vzv ist der mensch. übertragung und inkubationszeit das virus wird durch speichel und konjunktivalflüssigkeit übertragen. die infektion erfolgt durch infektiöse tröpfchen und direkten kontakt mit varizelleneffloreszenzen, seltener durch kontakt mit zostereffloreszenzen. im gegensatz zum herpes zoster scheiden patienten mit varizellen 1-2 tage vor ausbruch des exanthems vzv aus. die aerogene übertragung ("windpocken") von vzv ist möglich, aber eher selten. begünstigend wirkt der relativ intensive kontakt in familien oder kjge. die inkubationszeit beträgt 14-16 tage, sie kann auf bis zu 8-10 tage verkürzt bzw. zu 21 tage verlängert sein. nach gabe von vzv-immunglobulin verlängert sich die inkubationszeit auf bis zu 28 tage. als taeniasis wird der intestinale befall des menschen mit adultwürmern der zestodengattung taenia (t.) bezeichnet. drei verschiedene humanpathogene arten kommen vor: rinderbandwurm (t. saginata), ostasiatischer finnenbandwurm (t. asiatica) und schweinebandwurm (t. solium). als zystizerkose wird der befall des menschen als zwischenwirt mit dem finnen-bzw. larvenstadium (cysticercus cellulosae von t. solium) bezeichnet. selten sind infektionen des menschen als zwischenwirt durch andere taenia-arten beschrieben worden. t. saginata besteht aus einem kopf mit vier saugnäpfen, einem kurzen halsteil und der 6-10 m lang werdenden gliederkette aus 1.000-2.000 gliedern. t. solium wird selten viel länger als 3-4 m und ist ähnlich gebaut. der mensch dient als endwirt der im dünndarm lebenden adulten bandwürmer; er scheidet dann mit dem stuhl die reifen proglottiden (glieder) mit einer großen anzahl infektionstüchtiger larven aus, die ihre eier enthalten. die taeniasis ist weit verbreitet. bedingt durch fleischbeschau und korrekte fleischlagerung geht die prävalenz von t. saginata in europa zurück. übertragung und inkubationszeit eine mensch-zu-mensch-übertragung ist nicht bekannt. die übertragung erfolgt durch fäkal kontaminierte nahrungsmittel. die präpatenzzeit der taeniasis bis zum beginn der ausscheidung von bandwurmgliedern bzw. eiern beträgt 8-12 wochen. inkubationszeit für die zystizerkose ist extrem variabel. klinisches bild das klinische bild ist oligosymptomatisch. zu erwähnen ist die neurozystizerkose mit parenchymatösen zysten im gehirn. bei multiplen zysten mit entzündlicher reaktion kann es zur schwerwiegenden zystizerkose-enzephalitis kommen. wenn der liquorraum durch die zysten verlegt wird, kann eine shunt-versorgung notwendig sein. diagnostik und therapie die diagnose der taeniasis basiert auf der entdeckung der bandwurmglieder im stuhl und deren untersuchung. daneben spielen bildgebende verfahren eine rolle. therapiert wird mit praziquantel (1 ed 10 mg / kg kg). alternativ steht niclosamid oder mebendazol zur verfügung. die zystizerkose wird mit albendazol behandelt. die echinokkokose besteht aus zwei unterschiedlichen erkrankungen: • die zystische echinokokkose wird durch den hundebandwurm (echinococcus granulosus) hervorgerufen; der mensch fungiert als fehlwirt. die zystische echinokokkose ist weit verbreitet. es kommt zur zystenbildung in leber, lunge und anderen organen. alle altersgruppen sind betroffen. die diagnose wird durch detektion der zysten gestellt. die serologie dient allenfalls der bestätigung des bildgebenden verdachts. die zystische echinokokkose wird zystenstadienspezifisch (nach einer sonografischen einteilung) therapiert. mittel der wahl ist albendazol. stadienabhängig kommen chirurgische verfahren hinzu. • die alveoläre echinokokkose wird durch den fuchsbandwurm hervorgerufen. der mensch fungiert ebenfalls als fehlwirt. die alveoläre echinokokkose kommt vor allem in europa vor. aufgrund der langen inkubationszeit (5-15 jahre) sind erkrankungen bei kindern und jugendlichen selten. auch hier steht die bildgebende diagnostik im vordergrund. serologisch wird, wie bei der zystischen echinokokkose, zunächst ein sensitiver antikörpersuchtest durchgeführt und im falle der positivität durch einen immunoblot oder elisa verifiziert. zum zeitpunkt der diagnosestellung sind viele patienten nicht mehr kurativ operierbar. die resektion wird von einer 2-jährigen albendazol-therapie begleitet. übertragung und inkubationszeit trichinella spiralis und andere trichinella spp. gehören zum stamm der nematoden. die erkrankung wird durch die aufnahme von trichinenlarven mit rohem und ungekochtem fleisch von schweinen und wildtieren ausgelöst. die larven werden freigesetzt und besiedeln anschließend den dünndarm. nach penetration durch das epithel einer zottenbasis entwickeln sich geschlechtsreife entwicklungsstadien. die weiblichen adultwürmer beginnen 5-7 tage post infectionem mit der larvenproduktion. einige larven verbleiben im stuhl, die mehrzahl jedoch migriert via mukosa über lymph-und blutgefäße in die quergestreifte skelettmuskulatur, wo sie von einer kollagenkapsel umgeben persistieren. man geht von etwa 10.000 erkrankungen pro jahr in der nördlichen hemisphäre aus. diagnose und therapie bei bestehendem krankheitsverdacht ist die anamnese hinsichtlich der essgewohnheiten zu durchleuchten. die trias lidödeme, muskelschmerzen und fieber ist typisch für die trichinellose. serologische tests (elisa, immunoblot) werden 2-6 wochen nach einer infektion positiv. klinisch können 1-7 tage nach der ingestion der larven durchfälle auftreten. die symptomatik ist mit übelkeit und erbrechen unspezifisch. das intervall zwischen infektion und beginnender symptomatik in der migrationsphase beträgt meist 1-3 wochen. intermittierendes fieber sowie eine "b-symptomatik" können auf eine trichinellose hinweisen. therapeutisch steht albendazol zur verfügung. fleisch sollte stets gekocht oder durchgebraten gegessen werden. es besteht meldepflicht (ifsg). yersinien gehören zur familie der enterobacteriaceae und sind gramnegative stäbchen. yersinia (y.) enterocolitica ruft eine akute enteritis mit stärkstem befall des terminalen ileums hervor und y. pseudotuberculosis die mesenteriale lymphadenitis. die höchste inzidenz findet sich im 2. lebensjahr. übertragung und inkubationszeit die yersiniose wird typischerweise durch die ingestion kontaminierter nahrung hervorgerufen. yersinien können wahrscheinlich auch über eine vielzahl von säugern übertragen werden. der langfristige einsatz von ppi begünstigt die empfänglichkeit für eine yersinien-infektion. eine fäkal-orale übertragung von mensch zu mensch ist möglich. auch ausbrüche in familien und kjge wurden beobachtet. die ausscheidung der yersinien mit dem stuhl kann wochen nach ende des durchfalls fortbestehen (6 wochen). eine übertragung über kontaminierte blutkonserven ist möglich. die inkubationszeit beträgt 1-14 tage, bei y. pseudotuberculosis bis zu 20 tage. klinische symptome klinisch kommt es zur akuten enteritis und mesenterialen lymphadenitis und komplizierend zu einer reaktiven arthritis. die mesenteriale lymphadenitis ist zuweilen schwer von einer akuten appendizitis zu unterscheiden. die atypischen bzw. oligosymptomatischen yersiniosen gehen mit schmerzen im rechten unterbauch, invagination, pharyngitis, fieber unbekannter ursache oder kawasaki-syndrom-ähnlichen symptomen einher. bei diffusen bauchschmerzen, lymphknotenschwellungen und v. a. eine ced sollte eben auch an eine yersiniose gedacht werden. sehr selten kommt es zu intestinalen blutungen oder zur perforation des ileums. abszedierungen sind möglich. eine wichtige komplikation ist die reaktive arthritis, besonders bei hla-b27-positiven kindern ab dem 7. lebensjahr. diagnose die diagnose erfolgt bei der akuten enteritis durch anzucht des erregers, meist y. enterocolitica aus dem stuhl. aber auch andere abstriche (rachen) können zur diagnose führen. neben der erregeranzucht kann der antikörpernachweis die diagnose sichern. bei der reaktiven arthritis finden sich in gelenken und bakteriellen produkten keine vermehrungsfähigen erreger. therapie die meisten infektionen verlaufen selbstlimitierend. die therapie ist in der regel symptomatisch. eine antiinfektive behandlung ist bei septischen krankheitsbildern und infektionen jenseits des gastrointestinaltrakts indiziert. als medikamente stehen cotrimoxazol, cephalosporine der gruppe 3 und aminoglykoside zur verfügung. prävention die prophylaxe erfolgt durch vermeidung der kontamination von nahrungsmitteln. isolierung und sorgfältige händedesinfektion sind angezeigt. es besteht meldepflicht (ifsg). isoxazolylpenicilline isoxazolylpenicilline sind penicillinasefeste penicilline und haben gegenüber penicillin-g-resistenten staphylokokken eine über 20-bis 50-fach stärkere wirkung. allerdings ist die wirkung gegenüber penicillin-g-empfindlichen bakterien um das 10-bis 100-fache schwächer als die wirkung von penicillin. • di-und flucloxacillin stehen in oraler und parenteraler form zur verfügung. • oxacillin wird aufgrund seiner geringen oralen bioverfügbarkeit von nur 30 % parenteral angewendet. bei längerer hoch dosierter anwendung sind kontrollen des blutbildes (granulozytopenie) und der leberwerte erforderlich. • gruppe 5: ceftobiprol und ceftarolin sind parenteral verfügbar und wirksam gegenüber gramnegativen bakterien, ähnlich wie die cephalosporine der gruppe 3, jedoch zusätzlich aktiv gegenüber mrsa, penicillinresistenten pneumokokken und e. faecalis. die cephalosporine der gruppen 1-5 haben eine unterschiedlich ausgeprägte gute betalaktamasestabilität. die basis-cephalosporine der gruppe 1 und 2 haben eine gute staphylokokkenaktivität. gegen methicillin-resistente staphylokokken (mrsa) sind alle cephalosporine, mit ausnahme der bisher bei kindern nicht zugelassenen cephalosporine der gruppe 5, unwirksam. die empfindlichkeit gegen gramnegative erreger ist stark variabel und nimmt grundsätzlich von gruppe 1 zu 4 zu. cephalosporine sind, mit ausnahme der gruppe 5, inaktiv gegen enterokokken, listerien und bordetellen. gramnegative anaerobier sind meist resistent. die cephalosporine der gruppe 2 sind weitgehend betalaktamasestabil. sie wirken wesentlich stärker gegen gramnegative stäbchen als die cephalosporine der gruppe 1. die breitspektrum-cephalosporine der gruppe 3 und 4 haben im vergleich zu den cephalosporinen der gruppe 1 und 2 ein breiteres spektrum, eine stärkere antibakterielle aktivität gegenüber gramnegativen stäbchen und eine unterschiedliche wirksamkeit gegen p. aeruginosa, enterobacter spp. und staphylokokken. bei einer penicillinallergie vom soforttyp sollten folgende cephalosporine nicht verabreicht werden: cefazolin, cephalexin, cefadroxil und cefamandol. patienten mit einer penicillin-spätreaktion können dagegen cephalosporine erhalten. die oralcephalosporine haben aufgrund ihrer guten wirksamkeit, verträglichkeit und einfachen verabreichung einen hohen stellenwert in der behandlung von infektionskrankheiten bei kindern. für viele indikationen sollten aber wegen der breite ihrer antimikrobiellen aktivität primär schmäler aktive substanzen (wie z. b. amoxicillin) verwendet werden. patienten mit einer penicillin-spätreaktion können oralcephalosporine erhalten. andere betalaktam-antibiotika die nur parenteral verfügbaren carbapeneme stellen eine weiterentwicklung der betalaktam-antibiotika mit sehr breitem antibakteriellem wirkspektrum dar. von klinischer bedeutung ist vor allem die wirkung gegenüber esbl-produzierenden bakterien. imipenem erfasst im grampositiven bereich sowohl enterokokken (ausnahme: e. faecium) und staphylokokken als auch gramnegative keime ähnlich den parenteralen cephalosporinen der gruppe 3 inkl. p. aeruginosa sowie anaerobier. imipenem wird zur antagonisierung nephrotoxischer metaboliten fest mit cilastatin kombiniert. es sollte nur zur behandlung schwerer infektionskrankheiten eingesetzt werden. überdosierungen und rasche applikation sind zu vermeiden (krampfanfälle). zur therapie der meningitis ist imipenem nicht geeignet. das spektrum von imipenem ist im vergleich zu penicillinen und cephalosporinen deutlich erweitert. meropenem besitzt ein wirkspektrum gegen grampositive erreger (ausnahme: enterococcus faecium), mit etwas geringerer aktivität gegen enterobacteriaceae und einer guten aktivität gegen p. aeruginosa. es ist für die therapie der meningitis zugelassen und gut liquorgängig. ertapenem ist für die therapie der ambulant erworbenen pneumonie sowie intraabdominaler und gynäkologischer infektionen zugelassen. es hat ein breites wirkspektrum gegen grampositive und gramnegative, aerobe und anaerobe bakterien inkl. s. aureus und bacteroides fragilis. enterokokken, p. aeruginosa und ein teil der acinetobacter-stämme sind resistent. es ist für kinder ab 3 monate zugelassen. kinder < 12 jahren benötigen zwei einzelgaben pro tag. aminoglykoside werden vor allem für die empirische kombinationstherapie schwerer infektionskrankheiten eingesetzt. bei schweren atemwegsinfektionen (z. b. bei patienten mit mukoviszidose) kann tobramycin bei pseudomonas-infektion zur inhalation verordnet werden. für die therapie von zns-infektionen sind aminoglykoside aufgrund ihrer schlechten liquorgängigkeit nicht geeignet. im sauren milieu (abszess) sind sie inaktiv. aminoglykoside können bei eingeschränkter nierenfunktion kumulieren, sodass eine dosisanpassung und die kontrolle der serumkonzentration erforderlich sind. auch bei normaler nierenfunktion ist die bestimmung des talspiegels vor der 3. gabe empfohlen. • gentamicin wird vor allem in der empirischen kombinationstherapie bei schweren infektionskrankheiten mit gramnegativen bakterien eingesetzt. in kombination mit penicillinen kann gentamicin synergistisch auf enterokokken, gbs und listerien wirken, wobei die klinische relevanz dieses effekts nicht sicher ist. die nebenwirkungen im kindesalter, insbesondere die ototoxizität und die nephrotoxizität, sind geringer als bei erwachsenen. • tobramycin entspricht weitgehend dem anwendungs-und wirkungsbereich von gentamicin und ist zusätzlich zur inhalativen pseudomonas-therapie bei mukoviszidose zugelassen. • amikacin gilt als reserve-aminoglykosid und sollte möglichst nur gegen gentamicin-resistente stämme von klebsiellen, enterobacter, serratia, pseudomonas aeruginosa etc. eingesetzt werden. tetrazykline sind wirksam gegen mykoplasmen, chlamydien, brucellen, rickettsien, campylobacter, vibrio cholerae, yersinien, borrelien (b. burgdorferi), spirochäten, leptospiren, francisella tularensis, burkholderia mallei und b. pseudomallei sowie einige anaerobier, grampositive und gramnegative erreger. derzeit sind in deutschland die meisten mrsa doxycyclin-empfindlich. tetrazykline werden vorwiegend in oraler form, aber auch parenteral verabreicht. die orale bioverfügbarkeit beträgt 90 %, wird aber, eingenommen in kombination mit milch und milchprodukten, wesentlich reduziert. tetrazykline sind gut liquorgängig und im allgemeinen gut verträglich. allergien sind selten, jedoch sollten die patienten aufgrund der phototoxizität starke sonnenexposition meiden. tetrazyklin-kalzium-komplexe können irreversibel im knochen und in den zähnen abgelagert werden, sodass eine anwendung bei kindern unter 9 jahren und schwangeren vermieden werden sollte. am häufigsten eingesetzt wird heute doxycyclin. aufgrund ihrer lipophilie werden tetrazykline in talgdrüsen akkumuliert und sind daher besonders gut für die therapie der acne papulopustulosa geeignet. makrolide sind gegenüber den wichtigsten erregern von atemwegsinfektionen inkl. mycoplasma pneumoniae, moraxella, legionella spp. und chlamydien wirksam. darüber hinaus sind sie u. a. aktiv gegen bordetellen, borrelien, helicobacter pylori, corynebacterium diphtheriae, erysipelothrix rhusiopathiae und ureaplasma urealyticum. weniger gut bis mäßig empfindlich sind staphylokokken, h. influenzae, campylobacter jejuni, treponema pallidum und rickettsien. clarithromycin wird auch zur kombinationstherapie von infektionen durch nichttuberkulöse mykobakterien (ntm) eingesetzt. die verträglichkeit der makrolide bei kindern ist grundsätzlich gut. an nebenwirkungen sind jedoch eine verlängerung des qt-intervalls und vielfältige interaktionen mit anderen pharmaka (u. a. theophyllin, carbamazepin, terfenadin, triazolam, midazolam, astemizol, ciclosporin a) zu beachten. aufgrund ihres wirkspektrums sind makrolide für die behandlung von weichteilinfektionen nicht geeignet. erythromycin stellt bei penicillinunverträglichkeit eine gute alternative dar. erythromycinestolat ist aufgrund seiner besseren bioverfügbarkeit den anderen erythromycinderivaten vorzuziehen. die intrazelluläre konzentration von erythromycin ist etwa 5-fach höher als die extrazelluläre, und die ausscheidung erfolgt überwiegend hepatobiliär. erythromycinlactobionat (erythrocin) kann auch i. v. verabreicht werden. kombinationen von erythromycin bzw. erythromycinethylsuccinat und bromhexin sind nicht zu empfehlen. roxithromycin und clarithromycin haben ein dem erythromycin sehr ähnliches antibakterielles wirkspektrum. jedoch sind verbesserte pharmakokinetische eigenschaften von vorteil, sodass sie niedriger dosiert werden können. indikationen sind infektionen durch bordetella pertussis, helicobacter pylori, borrelien, mycobacterium avium und bartonellen. die orale bioverfügbarkeit liegt bei 50-80 % und geht mit einer reduktion durch nahrungsaufnahme (roxithromycin) bzw. 55 % ohne reduktion durch nahrungsaufnahme (clarithromycin) einher. clarithromycin ist auch für die behandlung von kindern im 1. lebenshalbjahr zugelassen. zur i. v. gabe ist nur clarithromycin ab dem 13. lebensjahr zugelassen. azithromycin ist ein azalid und hat im vergleich zu erythromycin in vitro eine verbesserte wirkung gegenüber gramnegativen erregern (h. influenzae) und eine schwächere aktivität gegen grampositive bakterien. bei der legionellose ist azithromycin neben fluorchinolonen das mittel der wahl. nach abklingen der akuten symptome bei ehec-infektionen verkürzt es die phase der asymptomatischen ausscheidung. aufgrund besonderer pharmakokinetischer eigenschaften (halbwertszeit: > 40 h) ist eine kurzzeittherapie mit täglicher einmalgabe (atemwegsinfektionen) möglich. nachteilig dagegen ist, dass die sehr lang anhaltenden subinhibitorischen konzentrationen die entwicklung resistenter bakterien (z. b. pneumokokken, gas) fördern. der einsatz sollte sehr zurückhaltend und nur bei fehlenden alternativen erfolgen. die orale bioverfügbarkeit liegt bei ca. 40 %. clindamycin wirkt hauptsächlich auf grampositive bakterien (streptokokken, staphylokokken, corynebakterien), anaerobier, toxoplasmen und plasmodien. es ist gut schleimhautwirksam, sodass es zur eradikationsbehandlung bei rezidivierenden streptokokken-und staphylokokken-infektionen verwendet werden kann. die resistenzraten von streptokokken und staphylokokken steigen jedoch an. das haupteinsatzgebiet von clindamycin sind haut-und weichteilinfektionen, odontogene infektionen, knochen-und gelenkinfektionen sowie infektionskrankheiten durch anaerobe bakterien (z. b. aspirationspneumonie). es ist nicht liquorgängig. eine zunehmende bedeutung und die möglichkeit zur oralen therapie hat clindamycin bei camrsa-infektionen. die orale bioverfügbarkeit liegt bei 75-85 %, eine parenterale applikation ist ebenfalls möglich. bei schweren haut-und weichgewebsinfektionen durch staphylokokken oder gas, die toxinvermittelt sind, wird clindamycin in kombination mit betalaktamen eingesetzt, um die toxinproduktion rasch zu unterbinden. bezüglich der nebenwirkungen ist an ein erhöhtes risiko für eine clostridium-difficile-assoziierte enterokolitis zu denken. vancomycin ist ein reserveantibiotikum zur behandlung von infektionen durch methicillin-resistente staphylokokken-stämme, enterokokken inkl. e. faecium, streptokokken, pneumokokken inkl. penicillin-resistenter stämme, c. difficile, corynebakterien (auch c. jeikeium), listerien und grampositive anaerobier. die applikation erfolgt parenteral als infusion über mindestens 1 h (bei c.-difficile-assoziierter enterkolitis ausschließlich oral). ein red-man-syndrom kann bei zu schneller i. v. gabe auftreten, was fälschlicherweise oft als allergische reaktion interpretiert wird. bei kumulation ist vancomycin oto-und nephrotoxisch. infektionen durch methicillin-sensible staphylokokken sollten nicht mit vancomycin behandelt werden, da betalaktam-antibiotika besser wirksam und nebenwirkungsärmer sind. bei der behandlung von shunt infektionen durch grampositive erreger wird vancomycin eingesetzt. der talspiegel (vor der 3. gabe) korreliert mit der systemischen wirkung. bei kindern < 12 jahre sollte der talspiegel mindestens bei 5-10 mg / l, bei älteren kindern und schweren infektionen zwischen 15 und 20 mg / l liegen. talspiegelbestimmungen werden in der regel 2 × / woche sowie nach dosisänderungen durchgeführt und sind obligat. liegt die minimale hemmkonzentration (mhk) des erregers über 1 mg / l, ist die effektivität einer therapie mit vancomycin oft eingeschränkt, weil auch mit sehr hohen dosierungen keine ausreichende bakterizidie erreicht werden kann. teicoplanin hat ein identisches wirkspektrum wie vancomycin, jedoch mit längerer halbwertszeit und nahezu fehlender nephrotoxizität. einige kns sind gegen teicoplanin resistent. bei enterokokken, die eine vanb-resistenz tragen, ist teicoplanin wirksamer als vancomycin. teicoplanin kann, nach initial 2-mal täglicher gabe an tag 1, einmal täglich verabreicht werden, ebenso ist die i. m. gabe möglich. serumspiegelbestimmungen sind nur in besonderen klinischen situationen notwendig. bei shuntinfektion mit ventrikulitis kann es sinnvoll sein, den medikamentenspiegel im liquor zu bestimmen. auch eine intraventrikuläre gabe von teicoplanin oder vancomycin bei nosokomialer meningitis oder ventrikulitis ist möglich. das wirkspektrum der chinolone umfasst grampositive und gramnegative mikroorganismen inkl. pseudomonas aeruginosa. die fluorchinolone der gruppen 3 (levofloxacin) und 4 (moxifloxacin), die sog. atemwegschinolone, zeigen eine verbesserte wirksamkeit gegen pneumokokken inkl. penicillin-resistenter stämme und andere grampositive bakterien (z. b. s. aureus). moxifloxacin ist zudem gegen anaerobier wirksam (intraabdominale infektionen) und wird zur therapie bei multiresistenter tbc eingesetzt. in deutschland ist nur ciprofloxacin (gruppe 2) für kinder ab 5 jahren mit einer pseudomonas-infektion bei zystischer fibrose, bei kindern ab 1 jahr mit komplizierter hwi und pyelonephritis als zweittherapie und für alle kinder zur soforttherapie des milzbrands mit systemischer beteiligung und bei inhalation von b. anthracis zugelassen. irreversible schädigungen der gelenkknorpel wurden bei kindern (inkl. neugeborene > 1.000 g) und jugendlichen bislang nicht nachgewiesen. als nebenwirkungen sind zwar arthralgien beobachtet worden, diese waren aber fast immer nach absetzen der therapie reversibel, traten nicht häufiger auf als in der kontrollgruppe und entsprachen nicht den in den tierversuchen beschriebenen knorpelschäden. fluorchinolone können daher bei kindern und jugendlichen angewendet werden, wenn es für die indizierte therapie keine alternative gibt und die eltern ausreichend aufgeklärt wurden. zu den indikationen zählen infektionskrankheiten durch p. aeruginosa oder multiresistente gramnegative bakterien. hierzu gehören die gegen p. aeruginosa gerichtete eradikationstherapie oder die akute exazerbation bei zystischer fibrose, komplizierte hwi, osteomyelitis, chronisch-eitrige otitis media, schwere otitis externa, shuntinfektionen sowie infektionen des gastrointestinaltrakts durch shigellen, salmonellen, vibrio cholerae und c. jejuni. unter der behandlung mit fluorchinolonen können neurotoxische und psychiatrische nebenwirkungen auftreten. auch ist die vorbehandlung mit fluorchinolonen im kindes-und jugendalter einer der wichtigsten risikofaktoren für eine c.-difficile-assoziierte enterkolitis. im kindes-und jugendalter sollte ciprofloxacin bevorzugt werden, da es am besten untersucht ist und eine saftzubereitung zur verfügung steht. kinder unter 5 jahren sollten levofloxacin 2 × / tag erhalten. bei moxifloxacin ist die am besten geeignete dosis bei kindern noch nicht abschließend untersucht. ciprofloxacin kann als umgebungsprophylaxe mit einer einmaldosis bei invasiver meningokokken-infektion verabreicht werden. nitrofurantoin eignet sich zur behandlung der zystitis und zur infektionsprophylaxe von hwis. die substanz erreicht keine nennenswerten serumkonzentrationen und wird nur im harn ausgeschieden. sie sollte nicht verordnet werden bei säuglingen in den ersten 3 monaten und bei patienten mit niereninsuffizienz oder schwerer neuropathie. nitrofurantoin darf laut fachinfo nur verabreicht werden, wenn effektivere und risikoärmere antibiotika oder chemotherapeutika nicht einsetzbar sind. rifampicin ist gegen mycobacterium tuberculosis, m. leprae, staphylokokken und andere grampositive kokken, meningokokken, h. influenzae, chlamydien und legionellen wirksam. es kann zu einer schnellen resistenzentwicklung unter der therapie mit rifampicin kommen daher darf es nur in kombination mit anderen antibakteriell wirksamen medikamenten eingesetzt werden. eine ausnahme stellt die meningokokkenprophylaxe dar. zu beachten ist, dass sich tränenflüssigkeit (cave: kontaktlinsenträger), urin, sputum, schweiß und andere körperflüssigkeiten orange färben können. zahlreiche interaktionen mit anderen hepatisch metabolisierten arzneimitteln sind bekannt. sulfonamide haben ein wirkspektrum, das grampositive bakterien, shigellen, aktinomyzeten, toxoplasma gondii, pneumocystis jiroveci und plasmodien erfasst. aufgrund der nebenwirkungen wie appetitlosigkeit, brechreiz, zentralnervösen symptomen, schweren mukokutanen unverträglichkeitsreaktionen (stevens-johnson-syndrom und toxische epidermale nekrolyse) und möglicher nieren-und lebertoxizität sowie der hohen resistenzraten werden die sulfonamide kaum noch als monosubstanz angewendet. trimethoprim / tetroxoprim mit sulfonamiden. die synergistische kombination von trimethoprim oder tetroxoprim als folsäure-antagonisten mit sulfonamiden (im verhältnis 1 : 5) wirkt gegen zahlreiche pathogene erreger, insbesondere grampositive kokken, enterobacteriaceae inkl. salmonellen und shigellen, v. cholerae, h. influenzae, m. catarrhalis, b. pertussis, brucellen und nocardia spp. gegen enterokokken, mykoplasmen, chlamydien und legionellen ist die kombination unwirksam. für die behandlung von gas-infektionen ist cotrimoxazol (sulfamethoxazol + trimethoprim) ungeeignet. die kombinationspräparate eignen sich heutzutage nur noch zur infektionsbehandlung der harnwege bei nachweislich empfindlichen erregern. auch bei infektionen durch mrsa kann bei nachgewiesener empfindlichkeit die behandlung mit cotrimoxazol indiziert sein. trimethoprim kann zur therapie der zystitis und zur infektionsprophylaxe einer hwi eingesetzt werden. durch den fehlenden sulfonamid-anteil sind weniger nebenwirkungen zu beobachten. die dosierungen parenteral applizierter antiinfektiva in der neugeborenenzeit bis zum 3. lebensmonat sowie bei kindern, jugendlichen und erwachsenen sind in › tab. 10.51 bzw. › tab. 10.52 angegeben. amphotericin b hat ein breites wirkspektrum und ist eine wichtige substanz in der behandlung invasiver pilzinfektionen. es schließt die meisten hefen und fadenpilze ein. ausnahmen sind dermatophyten und einige seltene pilze wie aspergillus terreus, scedosporium prolificans, candida lusitaniae, trichosporon asahii und malassezia spp., die eine reduzierte empfindlichkeit gegenüber amphotericin b besitzen. amphotericin b wird oral verabreicht, kaum resorbiert und kann zur dekontamination des darms und zur topischen therapie von candida-infektionen der schleimhäute eingesetzt werden. nach i. v. gabe dissoziiert amphotericin b von seinem carrier desoxycholat und verteilt sich vorwiegend in leber, milz und knochenmark. die elimination aus dem körper dauert tage bis wochen und erfolgt in unveränderter form über den urin und die galle. die wesentlichen nebenwirkungen sind nephrotoxizität und infusionsassoziierte reaktionen wie fieber, schüttelfrost, myalgien und arthralgien. hypokaliämien sind nicht selten substitutionsrefraktär und können zum therapieabbruch zwingen. therapieindikation sind die candidämie neu-und frühgeborener und (in kombination mit flucytosin) die induktionstherapie der kryptokokkenmeningoenzephalitis. lamb ist weniger nephrotoxisch als ambd und mit weniger infusionsassoziierten reaktionen verbunden. es hat zugelassene erstlinienindikationen für alle altersstufen in der empirischen antimykotischen therapie bei fieber und granulozytopenie (3 mg / kg kg / tag in 1 ed) und in der therapie invasiver pilzinfektionen inkl. invasiver aspergillus-und candida-infektionen (3 bis max. 5 mg / kg kg / tag in 1 ed). die therapie sollte mit der vollen zieldosis unter klinischem monitoring begonnen werden und eine infusionsdauer von 1-2 h nicht unterschreiten. amphotericin-b-lipid-komplex (ablc) ablc hat eine im vergleich zu ambd verminderte nephrotoxizität, die häufigkeit der infusionsassoziierten reaktionen bleibt jedoch gleich. ablc ist ab dem 1. lebensmonat zur zweitlinienbehandlung invasiver pilzinfektionen zugelassen und eine option der primärtherapie extrakranialer mukormykosen. die empfohlene dosierung ist 5 mg / kg kg / tag in 1 ed, infundiert über 2 h. flucytosin (5-fluorocytosin; 5-fc) ist ein pilzspezifisches synthetisches basenanalogon, das ein rna-miscoding und eine hemmung der dna-synthese bewirkt. seine antimykotische aktivität ist im wesentlichen auf hefepilze beschränkt. 5-fc ist in deutschland als i. v. lösung verfügbar. es hat eine sehr niedrige eiweißbindung mit gleichmäßiger verteilung in alle gewebe-und körperflüssigkeiten inkl. liquor. die elimination erfolgt überwiegend (> 90 %) in unveränderter form durch glomeruläre filtration über den urin. aufgrund einer raschen resistenzausbildung in vitro wird 5-fc generell nur in kombination eingesetzt. die kombination mit amphotericin b ist eine etablierte, evidenzbasierte indikation zur induktionstherapie bei der kryptokokkenmeningitis und zur behandlung komplizierter invasiver candida-infektionen. in kombination mit fluconazol ist 5-fc eine alternative in der behandlung der kryptokokkenmeningitis, wenn eine amphotericin-b-basierte behandlung nicht eingesetzt werden kann bzw. eine orale behandlung erforderlich ist. unerwünschte arzneimittelwirkungen beinhalten gastrointestinale beschwerden und blutbildveränderungen (orale gabe). ein therapeutisches monitoring wird empfohlen, insbesondere bei patienten mit nierenfunktionsstörungen. die anfangsdosierung für erwachsene und pädiatrische patienten aller altersstufen beträgt 100 mg / kg kg / tag in 3-4 ed; das dosierungsziel sind plasmaspiegel zwischen 40 und 60 mg / l vor gabe. das wirkspektrum von fluconazol umfasst verschiedene candida-arten, cryptococcus neoformans, trichosporon asaihi und die endemischen dimorphen pilze. candida krusei ist intrinsisch resistent, und candida glabrata hat eine eingeschränkte empfindlichkeit. fluconazol ist oral und parenteral verfügbar. die orale bioverfügbarkeit liegt bei > 90 %. die eiweißbindung ist gering und die penetration in liquor und gewebe aufgrund der hohen wasserlöslichkeit tab. 10.51 dosierungen wichtiger parenteral applizierter antiinfektiva in der neugeborenenzeit bis zum 3 . lebensmonat (meningitisdosis in klammern) tagesdosis ( viren sind bei ihrer vermehrung auf den stoffwechsel der wirtszelle angewiesen. bei der anwendung antiviraler substanzen besteht daher die gefahr der toxischen schädigung der wirtszelle. idealerweise sollte ein virostatikum nur virusspezifische prozesse hemmen, ohne die wirtszellen zu schädigen. die höchste effektivität von virostatika wird erreicht, wenn die therapie binnen 24( -48) h nach auftreten von symptomen einsetzt. eine später begonnene therapie ist bei immunkompetenten kindern oft wirkungslos. bei einer länger anhaltenden virusreplikation, z. b. bei immundefizienten kindern oder bei virusbedingten komplikationen, kann eine therapie mit virostatika auch noch später als 48 h nach auftreten der symptome begonnen werden. viele virostatika wie famciclovir, valaciclovir, valganciclovir und ganciclovir sind für kinder und jugendliche nicht zugelassen. da der pädiater aber nicht auf die modernen virostatika verzichten kann, sollte vor der anwendung die risiko-nutzen-abwägung sehr sorgfältig geprüft werden. • aciclovir ist das erste hoch wirksame, selektive und gut verträgliche virostatikum zur systemischen therapie. das wirkspektrum umfasst hsv1 und hsv2 sowie vzv, dessen empfindlichkeit aber 10-fach schwächer ist als die des hsv. selten sind hsv-oder vzv-virusstämme gegen aciclovir resistent. wegen der geringen oralen bioverfügbarkeit von 20 % sollte aciclovir therapeutisch möglichst parenteral appliziert werden. ist dies nicht möglich, sollte es in einer hohen dosierung verordnet werden. die lokale anwendung ist nur bei herpeskeratitis von bedeutung. indikationen für aciclovir sind die behandlung von herpes-enzephalitis, eczema herpeticatum, herpes genitalis, varizellen und zoster bei immunsupprimierten patienten und patienten mit anderen risikofaktoren sowie die prophylaxe der hsv-und vzv-infektion bei exponierten immundefizienten patienten (onkologische krankheiten, organtransplantation). • valaciclovir (z. b. valtrex ® ) ist ein ester von aciclovir, das die darmzellen nach der resorption in aciclovir und valin spalten. die bioverfügbarkeit liegt bei 54 %. valaciclovir ist für immunkompetente jugendliche (≥ 12 jahre) zugelassen. • ganciclovir (cymeven ® ) ist gegen cmv 8-bis 20-fach stärker wirksam als aciclovir. gegen hsv und vzv ist es jedoch deutlich schwächer aktiv. die orale bioverfügbarkeit ist mit 5-9 % gering. ganciclovir ist viel weniger selektiv wirksam als aciclovir und hat daher auch nicht unbeträchtliche nebenwirkungen. dazu gehören knochenmarkdepression (neutropenie) sowie leber-und nierenfunktionsstörungen. indikationen für eine therapie mit ganciclovir sind vor allem lebens-und das augenlicht bedrohende cmv-infektionen bei immunsupprimierten patienten. ein therapieversuch ist auch bei einer symptomatischen konnatalen cmv-infektion (enzephalitis, hepatitis etc.) indiziert. neuraminidasehemmer hemmen selektiv das aktive zentrum der influenzavirus-neuraminidase. dadurch können keine neu gebildeten influenzaviren von der zelle freigesetzt werden. diese virostatika sind gegen alle bekannten viralen neuraminidase-subtypen (n = 9) inkl. des subtyps vom vogelgrippevirus wirksam. bei kindern können als unerwünschte nebenwirkungen abnorme verhaltensstörungen vorkommen. mit resistenten virusstämmen muss gerechnet werden. zanamivir (relenza ® ) wird 2-mal täglich über 5 tage inhaliert und ist zur behandlung und prophylaxe geeignet. in deutschland ist es für kinder ab 5 jahre zugelassen. oseltamivir (tamiflu ® ) hat eine orale bioverfügbarkeit von 80 %. es wird metabolisiert und ausschließlich renal ausgeschieden, sodass bei einer nierenfunktionsstörung eine dosisreduktion erforderlich ist. die halbwertszeit beträgt 6-10 h. oseltamivir ist für kinder ab 1 jahr zur therapie und prophylaxe zugelassen. angaben zur dosierung der beschriebenen substanzen sind › tab. 10.54 zu entnehmen. resistenz ist definiert als eine minimale hemmkonzentration eines antibiotikums gegen ein bakterium, die so hoch ist, dass auch bei der zugelassenen höchstdosierung kein therapeutischer erfolg erzielt wird. es lassen sich zwei verschiedene resistenzformen voneinander unterscheiden. • natürliche, primäre resistenz: hierbei handelt es sich um eine stets vorhandene, genetisch bedingte unempfindlichkeit einer bakterienart gegen ein antibiotikum. beispiele sind die unwirksamkeit von cephalosporinen gegen enterokokken oder von penicillin gegen pseudomonas aeruginosa. • erworbene, sekundäre resistenz: erworbenen resistenzentwicklungen liegen mutationen zugrunde. der eigentliche mechanismus besteht in einer selektion resistenter stämme, die in jeder bakterienpopulation in geringer zahl vorkommen. die selektion findet unter einwirkung eines antibiotikums statt, das die empfindlichen bakterien abtötet. darüber hinaus können resistente bakterien durch mutation oder übertragung von resistenzgenen unter einwirkung eines chemotherapeutikums herausselektioniert werden. ein beispiel hierfür ist die induktion von betalaktamasen unter der therapie mit betalaktam-antibiotika. drugmonitoring"), um eine effektive wirksamkeit zu erlangen. beispiele hierfür sind gentamicin-spitzenspiegel und vancomycin-talspiegel. bezüglich der toxizität sind die talspiegel bei aminoglykosiden zu nennen. bei der verordnung von antiinfektiva in der pädiatrie ist vorsicht geboten. stets ist eine dosierung nach körpergewicht (kg) oder körperoberfläche (kof) vorgesehen. hierbei können sich kalkulationsfehler ergeben. aus praktischen erfahrungen heraus hat sich gezeigt, dass bei kindern und jugendlichen > 40 kg kg die maximale erwachsenendosis gegeben werden kann. eine besonderheit in der pädiatrie ist, dass bestimmte infektionskrankheiten nur bei kindern und jugendlichen vorkommen (z. b. neugeborenensepsis), andere infektionskrankheiten (atemwegsinfektionen, meningitis, keuchhusten) sind viel häufiger. ferner werden antibiotika im off-label-bereich (fluorchinolone) eingesetzt, die in der erwachsenenmedizin eine weite verbreitung erfahren. etwa 75 % der antibiotika werden im ambulanten bereich verordnet. die abs-maßnahmen beziehen sich jedoch im wesentlichen auf den stationären bereich und hier insbesondere auf besonders kritische bereiche wie die neonatologie oder die pädiatrische hämato-onkologie. gerade weil atemwegsinfektionen in der pädiatrie besonders häufig sind und zunächst einmal ambulant behandelt werden, sind kenntnisse bezüglich der indikation und therapiedauer sowie der auswahl eines geeigneten antibiotikums (möglichst kein breitspektrum-antibiotikum) sinnvoll. abs-maßnahmen und schulungen sollten in analogie zum stationären bereich erfolgen. ii empfehlungen der ständigen impfkommission (stiko) am robert koch-institut: www .rki .de/de/content/kommissionen/stiko/empfehlungen/ impfempfehlungen_node .html . zur verfügung stehen die i. v. zu verabreichenden benzylpenicilline (penicillin g) und die oral zu verabreichenden magensäurestabilen phenoxypenicilline (penicillin v). die orale bioverfügbarkeit von penicillin v liegt bei 50 % und wird durch nahrungsaufnahme reduziert aminopenicilline und betalaktamase-inhibitoren • ampicillin besitzt ein erweitertes aktivitätsspektrum im vergleich zu penicillin g, vor allem im gramnegativen bereich. zusätzlich werden enterococcus faecalis (nicht aber e. faecium), haemophilus influenzae, listerien, e. coli (bis 50 % resistente stämme) • amoxicillin hat das wirkspektrum von ampicillin und ist dessen hydroxyderivat. die orale bioverfügbarkeit liegt bei 60-70 %. wird ampicillin oder amoxicillin bei der infektiösen mononukleose verabreicht durch die kombination der clavulansäure mit amoxicillin gelingt es, das spektrum auf keime zu erweitern, die aufgrund von betalaktamasebildung gegen aminopenicilline resistent sind (staphylokokken, bacteroides fragilis u sulbactam steht in parenteraler form in einem mischungsverhältnis von 2 : 1 zur verfügung. sulbactam ist ein inhibitor der meisten betalaktamasen und wird p. o. nicht resorbiert sultamicillin ist eine feste chemische verbindung von ampicillin mit sulbactam, die p. o. eine bioverfügbarkeit von 80-85 % hat > 40 kg, 125 mg / tag für kinder von 20-40 kg, und 62,5 mg / tag für kinder mit einem gewicht von < 20 kg vorgeschlagen therapiedauer beträgt 10-15 tage bei arthritis, 3-4 wochen bei osteomyelitis oder spondylodiszitis.prävention prophylaxemaßnahmen zur verhinderung sporadischer kingella-kingae-infektionen existieren nicht. erreger keuchhusten wird durch bordetella (b.) pertussis hervorgerufen, ein bekapseltes, aerobes, gramnegatives stäbchenbakterium, das über eine vielzahl von virulenzfaktoren wie toxine (pertussistoxin) und adhäsine (filamentöses hämagglutinin, pertactin) verfügt. keuchhustenähnliche symptome können auch durch b. parapertussis oder b. bronchiseptica sowie mycoplasma pneumoniae, chlamydia trachomatis, chlamydia pneumoniae und durch respiratorische viren (vor allem rsv) hervorgerufen werden. der mensch ist das einzige reservoir für b. pertussis, während b. parapertussis und b. bronchiseptica sowohl beim menschen als auch bei tieren detektiert werden. die inzidenz liegt mit starken schwankungen zwischen 10 und 41 je 100.000 einwohner. deutlich höher ist die inzidenz für pertussis bei säuglingen (10-95 je 100.000).übertragung und inkubationszeit die übertragung erfolgt durch tröpfcheninfektion bei engem kontakt mit infizierten, die mit beginn des stadium catarrhale kontagiös sind. die infektiosität hält (unbehandelt) ca. 3 wochen nach beginn des stadium convulsivum an. pertussis ist sehr ansteckend. der kontagionsindex bei ungeimpften kindern liegt bei 90 %, beim erwachsenen ist er niedriger. die inkubationszeit beträgt in der regel 7-10 (5-21) tage.klinische symptome klinisch tritt pertussis in einer ungeimpften population vor allem bei kleinkindern auf. in deutschland war zuletzt eine verschiebung der pertussisinzidenz in das frühe säuglingsalter (ungeimpft) und hin zu adoleszenten sowie erwachsenen zu beobachten. patienten mit pertussis weisen kein oder leichtes fieber auf. das klinische bild ist variabel. unter typischem keuchhusten versteht man drei stadien: 1. stadium catarrhale (leichte respiratorische symptome wie husten und rhinitis) über 1-2 wochen, gefolgt von dem charakteristischen 2. stadium convulsivum (4-6 wochen) mit den typischen, anfallsweise auftretenden hustenstößen (stakkatohusten), denen ein inspiratorisches ziehen ("keuchen") folgt. oftmals treten die hustenattacken nachts auf, und es kommt zum hervorwürgen von zähem schleim und anschließendem erbrechen. das 3. stadium wird als 3. stadium decrementi mit abklingendem husten bezeichnet. der typische keuchhusten dauert 6-12 wochen. komplikationen ergeben sich durch sekundärinfektionen wie pneumonie, otitis (durch pneumokokken oder nicht verkapselte haemophilusinfluenzae-bakterien).neugeborene und junge säuglinge erkranken schwer an keuchhusten. die klinische symptomatik kann ausschließlich mit periodischer atmung oder atempausen auffallen. auch unspezifischer husten ist ein symptom. weitere komplikationen sind krampfanfälle, pneumonien sowie eine enzephalopathie. in seltenen fällen kommt es bei säuglingen zu hyperleukozytose, hypoxämie und pulmonaler hypertension. hierbei ist die sterblichkeit deutlich erhöht.jugendliche und erwachsene erkranken unter dem bild "chronischer husten" und werden oftmals nicht diagnostiziert.diagnose diagnostisch kann keuchhusten schwierig einzuordnen sein, weil das klinische bild sehr variabel ist. bei jungen säuglingen, jugendlichen und erwachsenen gilt: an keuchhusten denken! unspezifisch kommen blutbildveränderungen (leukozytose mit lymphozytose) im stadium convulsivum vor, insbesondere bei ungeimpften säuglingen und kleinkinder, nicht jedoch bei jugendlichen und erwachsenen. die klinische verdachtsdiagnose "keuchhusten" kann durch erregeranzüchtung, pcr oder einen serologischen antikörpernachweis bestätigt werden. die mikrobiologische diagnostik von bordetella pertussis erfolgt durch untersuchung von mittels absaugung gewonnenem nasopharyngealsekret oder einem tiefen nasalen abstrich mit dacron-tupfern. es muss ein spezieller cefalexin-haltiger kohle-pferdeblut-agar verwendet werden. aufgrund der hohen sensitivität und der einfachen und schnellen durchführung hat sich die pcr zu einer säule in der diagnostik von keuchhusten entwickelt. spezifische antikörper gegen bordetella-pertussis-antigen im serum sind bei erstinfektion frühestens am übergang vom stadium catarrhale in das stadium convulsivum nachweisbar. aus diesem grund ist die serologie in den ersten 2-3 erkrankungswochen ungeeignet. wegen der schlechten abgrenzbarkeit bei geimpften kindern verbietet sich bei säuglingen und kleinkindern eine serologische diagnostik. die pertussis-toxin-elisa-antikörperbestimmung bei erwachsenen kann zur dia gnostik von pertussis herangezogen werden. insbesondere hohe igg-anti-pt-werte (≥ 100 iu / ml) im zeitraum von 3-4 wochen nach hustenbeginn können als hinweis für eine bordetella-pertussis-infektion gesehen werden. herpes zoster ist meist eine einseitige neuritis mit einem oder mehreren dermatomen, die bei kindern mit bis zu 75 % im thoraxbereich mit typischen, gruppiert angeordneten effloreszenzen und selten bei älteren kindern mit lokalen schmerzen anzutreffen ist. das übertreten der mittellinie (zoster duplex) ist selten. eine postzosterische neuralgie kommt bei kindern ebenfalls selten vor. hirnnerven können betroffen sein (zoster ophthalmicus, zoster oticus). eine fazialisparese kann wenige tage vor und nach beginn des zoster oticus auftreten (ramsay-hunt-syndrom). chronische formen bei abwehrgeschwächten patienten und rezidive sind auch im kindesalter zu beobachten.diagnostik zum nachweis einer akuten infektion stehen pcr, virusanzucht oder der immunfluoreszenztest mit monoklonalen antikörpern zur verfügung. die differenzierung von wild-und impfvirusstämmen erfolgt mittels pcr, restriktionsenzymanalyse und sequenzierung. der nachweis von spezifischen antikörpern (igg, iga, igm) kann eine akute infektion oder den impfstatus anzeigen. weitere diagnostische methoden sind der fluoreszenz-antikörpermemban-antigen-test (fama) sowie elisa und ifat zur prüfung der vzv-igg-avidität. beide verfahren gehören in den bereich der spezialdiagnostik. neugeborene von müttern mit varizellen während der perinatalperiode sind, sofern sie in der klinik verbleiben müssen, bis 28 tage post natum zu isolieren. eine prophylaxe mit vzv-immunglobulin ist möglich. die stiko empfiehlt den einsatz von vzv-immunglobulin als postexpositionelle prophylaxe für ungeimpfte personen mit negativer varizellenanamnese und kontakt zu risikopersonen, ungeimpfte schwangere ohne varizellenanamnese, immunkompromittierte patienten mit unsicherer oder fehlender varizellenimmunität, neugeborene, deren mütter 5 tage vor bis 2 tage nach der entbindung an varizellen erkrankt sind, frühgeborene ab der 28. ssw, deren mütter keine immunität aufweisen (nach exposition in der neonatalperiode), frühgeborene, die vor der 28. ssw geboren wurden (nach exposition der neonatalperiode), unabhängig vom immunitätsstatus der mutter. eine chemoprophylaxe mit aciclovir ist ab dem tag 7-9 nach exposition möglich (60-80 mg / kg kg / tag), p. o. über 5-7 tage. diese prophylaxe ist bisher nur bei immungesunden kindern erprobt.zur aktiven immunisierung steht eine impfung zur verfügung. auch nach impfung sind durchbruchsvarizellen möglich. die prävalenz wird nach einmaliger impfung mit 4-9 % der jährlich geimpften personen angegeben. auch nach zweimaliger impfung besteht kein 100-prozentiger schutz. die varizellenimpfung ist eine standardimpfung im stiko-impfkalender (› tab. 10.5). es besteht meldepflicht (ifsg). in den letzten jahren ist eine deutliche zunahme multiresistenter erreger (mre), im wesentlichen als folge einer ungezielten antiinfektiven therapie, zu beobachten gewesen. bei der auswahl von antiinfektiva, insbesondere antibiotika, kommt es auf die korrekte indikation, die auswahl eines am besten geeigneten antibiotikums sowie die angemessene dosierung und dauer der therapie an. wenn möglich, sollte die frühzeitige orale sequenztherapie initiiert und eine nicht indizierte behandlung selbstverständlich abgesetzt werden.abs in der pädiatrie unterscheidet sich wesentlich von abs in der erwachsenenmedizin. die deutsche gesellschaft für pädiatrische infektiologie (dgpi) hat unter berücksichtigung der besonderheiten bei neugeborenen, kindern und jugendlichen hinsichtlich der antiinfektiven therapie (pharmakokinetik, pharmakodynamik u. a.) eine awmf-leitlinie erstellt."antibiotic stewardship -konzeption und umsetzung in der stationären kinderheilkunde": awmf.org/leitlinien/detail/anmeldung/1/ll/048-015.html https://else4.de/0cydi im zentrum von abs in der pädiatrie steht das abs-team. das abs-team sollte aus dem ärztlichen leiter mit der fachgebietsbezeichnung pädiatrie und infektiologischer zusatzbezeichnung, einem apotheker mit klinisch-infektiologischer erfahrung und / oder weiterbildung sowie dem krankenhaushygieniker und dem mikrobiologen bestehen. ein zugriff auf eine edv-kompetenz ist wünschenswert. idealerweise ist das abs-team durch die klinikleitung mandatiert und kann entsprechende vorgaben für eine klinik erlassen.zu den abs-kernmaßnahmen gehört das verfassen einer restriktiven liste von standard-antiinfektiva für eine bestimmte klinik. reserveantibiotika oder antiinfektive therapien mit ungewöhnlicher indikation sollten immer nach rücksprache mit dem abs-team eingesetzt werden.der verbrauch der antiinfektiva sollte erfasst werden. diese verbrauchsanalysen sollten mindestens jährlich für die gesamte klinik sowie ggf. für risikobereiche (onkologie, intensivstationen, neonatologie u. a.) erstellt werden. aus der erwachsenenmedizin sind die "daily defined doses" (ddd) bekannt. hierbei wird der gesamtverbrauch durch die von der who festgelegte typische tagesdosis für einen erwachsenen (kg 70 kg) auf 100 oder 1.000 pflegetage bezogen. in der kinder-und jugendmedizin ist dieses verfahren wegen des gewichtsbezugs schwerlich möglich. dennoch kann der einsatz von ddds oder absoluten liefermengen je 1.000 patiententagen orientierende hinweise auf eine zu-oder abnahme des verbrauchs von einzelsubstanzen geben.eine geeignetere kenngröße für die pädiatrie sind die "therapietage" (days of therapy; dot) und die "therapiedauer" (length of therapy; lot), die den direkten vergleich ermöglichen. für prävalenzstudien sollte die anzahl von antibiotikaverordnungen und / oder die anzahl exponierter kinder erfasst werden.weiterhin bedeutsam für ein abs-konzept ist die regelmäßige erfassung von erregern invasiver infektionen sowie angaben zum resistenzprofil. hierbei ist die intensive und vertrauensvolle zusammenarbeit mit dem mikrobiologischen labor unerlässlich. es muss betont werden, dass lokale gegebenheiten und arbeitsabläufe eingang in interne leitlinien zu diagnostik und therapie wichtiger und häufiger infektionen haben sollten. eine allgemeingültige abs-leitlinie kann es nicht geben. zur überprüfung eines abs-programms gibt es qualitätsindikatoren (qi), die in strukturelle, organisatorische und personelle sowie prozedurale qi unterteilt werden. wissenschaftlich werden diese qi mit punktprävalenz-studien überprüft.der therapeutische erfolg des einsatzes von antiinfektiva hängt sehr wesentlich von der korrekten dosierung des jeweiligen medikaments ab. dafür ist nicht nur ein verständnis der pharmakokinetik und pharmakodynamik erforderlich, sondern auch kenntnisse bezüglich des wirkmechanismus eines antibiotikums oder antimykotikums auf den entsprechenden erreger sind vonnöten. einige antibiotika verfügen z. b. über einen "postantibiotischen effekt", manche erreichen ihre wirksamkeit über hohe spitzenspiegel oder talspiegel, die über der minimalen hemmkonzentration liegen. bei anderen substanzen wiederum ist es wichtig, die konzentration im serum zu definierten zeitpunkten zu messen ("therapeutisches key: cord-263276-keyu60in authors: zhou, weimin; lin, feng; teng, lingfang; li, hua; hou, jianyi; tong, rui; zheng, changhua; lou, yongliang; tan, wenjie title: prevalence of herpes and respiratory viruses in induced sputum among hospitalized children with non typical bacterial community-acquired pneumonia date: 2013-11-18 journal: plos one doi: 10.1371/journal.pone.0079477 sha: doc_id: 263276 cord_uid: keyu60in objective: few comprehensive studies have searched for viruses in infants and young children with community-acquired pneumonia (cap) in china. the aim of this study was to investigate the roles of human herpes viruses (hhvs) and other respiratory viruses in cap not caused by typical bacterial infection and to determine their prevalence and clinical significance. methods: induced sputum (is) samples were collected from 354 hospitalised patients (infants, n = 205; children, n = 149) with respiratory illness (cap or non-cap) admitted to wenling hospital of china. we tested for hhvs and respiratory viruses using pcr-based assays. the epidemiological profiles were also analysed. results: high rate of virus detection (more than 98%) and co-infection (more than 80%) were found among is samples from 354 hospitalised infants and children with respiratory illness in this study. of 273 cap samples tested, cmv (91.6%), hhv-6 (50.9%), rsv (37.4%), ebv (35.5%), hbov (28.2%), hhv-7 (18.3%) and rhinovirus (17.2%) were the most commonly detected viruses. of 81 noncap samples tested, cmv (63%), rsv (49.4%), hhv-6 (42%), ebv (24.7%), hhv-7 (13.6%) and hbov (8.6%) were the dominant viruses detected. the prevalence of several viral agents (rhinovirus, bocavirus, adenovirus and cmv) among is samples of cap were significantly higher than that of non-cap control group. we also found the prevalence of rsv coinfection with hhvs was also higher among cap group than that of non-cap control. conclusions: with sensitive molecular detection techniques and is samples, high rates of viral identification were achieved in infants and young children with respiratory illness in a rural area of china. the clinical significance of rhinovirus, bocavirus, adenovirus and hhv (especially cmv) infections should receive greater attention in future treatment and prevention studies of cap in infants and children. lower respiratory tract infections (primarily pneumonia) are the leading cause of death worldwide in infants and children [1, 2] . there are approximately 150 million cases of childhood community-acquired pneumonia (cap) each year [1, 3] . cap is a major cause of morbidity and mortality among children in developing countries, which is 10-50 times more common than in developed countries [1, 3] . bacteria as the principal cause of cap in children has been widely investigated [3] [4] [5] . in more than 50% of cases, however, there is still a considerable deficit in the aetiologic diagnosis resulting in unnecessary or inappropriate antibiotic prescription [2, 6] . it is clear that the involvement of viruses in cap have been underestimated due to a lack of understanding of the viral etiology in a clinical setting [7] [8] [9] [10] [11] [12] . in addition, the appropriate sample from infants and young children is critical for the aetiologic diagnosis of cap. lung itself is rarely sampled directly, and sputum, representing lower-airway secretions, can rarely be obtained from children [11, 14, 15] . among children, cap may be caused by a wide variety of microbes, including ''typical'' bacteria (e.g., streptococcus pneumonia) and atypical bacteria, mycobacterium tuberculosis and fungi. viral infections are also involved with 80% of episodes of cap in children under 2 years old and over 40% of older children [6] [7] [8] [9] [10] [11] . studies of cap have traditionally focused little on viral causes [2] . so for, very few studies have included an extensive and appropriate evaluation of the role of viruses in the aetiology of cap in developing countries, including china. in recent years, the introduction of better-quality diagnostic tests has markedly improved the ability to detect multiple viral pathogens [11] [12] [13] , shifting attention to the important role of viruses as a cause of cap [6] [7] [8] [9] [10] [11] . according to previous studies, up to two-thirds of childhood pneumonia cases are associated with a viral infection [3] [4] [5] [6] [7] [8] [9] [10] [11] . respiratory syncytial virus (rsv), influenza virus (ifv), rhinovirus (rv), human metapneumovirus (hmpv) and parainfluenza viruses (pivs) are the most common viruses associated with pneumonia [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . in addition, the roles of cytomegalovirus (cmv), other herpes viruses (hhvs), recently identified human coronaviruses (hcov-nl63 and -hku1) and human bocavirus (hbov) as causes of cap in infants and children remain controversial [3, [19] [20] [21] [22] [23] [24] [25] . so far, pathogenic profiles of hhv and its role in cap among infants and young children from rural areas have not been well characterized. the present study was undertaken to describe the profiles of hhvs and other respiratory viruses associated with hospital-based cap and non-cap among infants and young children in a rural area of china using comprehensive and sensitive molecular diagnostic techniques. all aspects of this study were performed in accordance with national ethics regulations and approved by the institutional review boards of the centre for disease control and prevention of china and the ethics committee of wenzhou medical college. the participants received written information regarding the purpose of the study and of their right to confidentiality. individual written informed consent was obtained from the parents or guardians of all participants. wenling is located in a rural area on the southeast coast of china with a sub tropical monsoon climate. it has a population of approximately 1,000,000. according to world health organisation clinical criteria [3, 8, 11] , cap was defined as the presence of pneumonic infiltrates (alveolar or parenchymal) on chest radiography with simultaneous signs and/or symptoms of acute infection in which the reading of x-ray films by specialist were blinded to the clinical results. all cap patients were also selected according to a set of necessary criteria based on respiratory symptoms (i.e., dyspnea or respiratory distress, cough, tachypnea) or evidence of parenchymal infiltrates on chest radiography. a total of 354 highquality induced sputum (is) samples (,25 squamous epithelial cells and .25 leukocytes per low-power field) were obtained from 948 hospitalised infants and young children with respiratory illness in wenling hospital from september of 2007 to april of 2008. two hundred and seventy-three samples were preselected from hospitalised children patients who diagnosed as non typical bacterial cap within 48 hrs of admission, while 81 samples from hospitalized children patients were set as a control group, whom were clinical diagnosis as non-cap patients based on chest x-ray and other respiratory signs (asthma, chronic bronchitis or cystic fibrosis) at admission. typical bacterial cap based on microbiologic tests, treatment algorithms and an elevated leukocyte count ($10 10 /l) were excluded. in addition, all patients were selected as immunocompetent at baseline and negative for hiv-1 and tb test. all the immunosuppressed or typical bacterial cap patients were excluded. the children with presumed nosocomical cap and lower-quality induced sputum (is) samples were also excluded. sputum production was induced by the inhalation of a 5.0% hypertonic saline solution; the sputum was sampled during the 1 st week after hospital admission by aspiration through the nostrils. our sputum collection method was described in detail elsewhere [14] [15] [16] . nucleic acid was extracted from 200 ml of the virus transport medium (vtm) using a qiaamp minelute virus spin kit (qiagen, germany) according to the manufacturer's instructions. polymerase chain reaction (pcr) or multiplex pcr was performed as described previously [26] [27] [28] [29] [30] for hhvs, including hsv-1 and -2, varicella zoster virus (vzv), cmv, epstein barr virus (ebv), hhv-6 and -7. adenoviruses (advs), ifv types a and b, piv types 1-3, rsv, picornaviruses (pic, including enteroviruses and rhinoviruses) using multiple rt-pcr assays [26, 27] (table s1 ); and for human coronavirus (hcov)-oc43, -229e, -nl63 and -hku1, human metapneumovirus (hmpv) using rt-pcr or and hbov using nested-pcr assays [27] [28] [29] (table s1). a positive (virus stock or dna) and negative control (vtm only) in each set pcr assay was included to survey the possibility of laboratory contamination. all the methods were reported previously and validation in our lab [26] [27] [28] [29] [30] . the amplicons of positive for pic were gel-purified for dna sequencing using a qiaquick gel extraction (qiagen, germany), according to the manufacturer's instructions. dna sequencing was performed with specific primers using an abi prism bigdye terminator cycle sequencing reaction kit (version 3.1) on an abi prism 3130 dna sequencer (applied biosystems, foster city, ca), following the manufacturer's instructions. enteroviruses (ev) or rhinoviruses were identified based on sequence alignment of amplicons. eligibility and classification of the clinical syndromes of pneumonia were determined from the original record of each item on the medical history and examination in the database. the frequency distribution of viral pathogens between cap and non-cap were analysed by the x 2 test and fisher exact test. all statistical analyses were performed with the statistical package for the social sciences (spss, version17, spss inc., chicago, il). statistical significance was assessed by tukey's test and p-values ,0.05 were considered to be statistically significant. all is samples were collected from hospitalised patients with severe cap (n = 273) and non-cap (n = 81) in wenling area between september 2007 and april 2008. the age and sex distributions are shown in in addition, we found that the prevalence of several viral agents (rhinovirus, hbov, adv and cmv) among is samples of cap were significantly higher than that of non-cap control group (p,0.05), while the prevalence of inf b (5,6.2%) among is samples of non-cap were significantly higher than that of cap group (p = 0.001). a total of 271 cap cases were positive for hhvs, accounting for about 99.3% of the hospitalised infants and young children with cap included in this study. 73 of 81 non-cap cases, however, were also identified as positive (90.1%) for hhvs. among other 15 respiratory viruses, rsv was the most dominant for both cap and non-cap groups, which was present significantly less frequent than hhvs. to further study the epidemiological profiles of virus infections, the distribution of viruses by age and season in this study were characterised (figure 1 and 2) . no significant difference was found for flua, piconavirus (enterovirus/rhinovirus), piv and hmpv among various age groups of cap cases ( figure 1a) . however, the infection rate of hbov and adv showed a peak among cap patients aged 6 months to 3 years (p,0.05). in contrast, rsv detection peaked in the infant group (0-12 months) of cap and decreased significantly with advancing age. in addition, no significant differences for hsv and cmv were observed among various age groups of cap cases ( figure 1b) . however, the rate of infection with ebv, hhv-6 and hhv-7 increased with age among cap cases (p,0.05). the infection rate was more than 73.7% for cmv, ebv, hhv-6 and hhv-7 among children older than 3 years. the distribution of viruses by age among the control group with non-cap was also investigated ( figure 1c and 1d) . no significant differences for rsv, ebv, hhv-6 and cmv were shown among various age groups of non-cap cases. however, the rate of infection with hhv-7 increased with age, which is similar to that of cap group. interestingly, co-infections were found in 241 (88.28%) of the cap cases and 65 (80.25%) of the non-cap cases (table 1) . single virus infection were detected in 31 is samples of cap cases (25 for cmv only, 5 for hhv-6 only and 1 for hbov only). in this study of co-infection, 77 patients were found to be infected with 2 viruses, 76 with 3 viruses, 48 with 4 viruses, 36 with 5 viruses, 3 with 6 viruses and 1 with 7 viruses. in addition, hhvs were the most detected co-infection agent with other respiratory viruses. among 102 rsv infections of cap cases, hhv (102 cases, 100%) and hbov (23 cases, 22.54%) were the most common concomitantly detected viruses, which were significantly higher than that of coinfection among non-cap group. the clinical manifestation of cap patients included cough, fever ($38uc), asthma and sputum. a few cases also showed signs of diarrhoea, rhinorrhoea, dyspnea and rale (data not shown). since the high virus detection rate (more than 98%) and coinfection rate (more than 80%) were among is samples from both cap and non-cap groups in this study (table 1) , it was difficult to associate the clinical symptoms of patients with cap with individual virus infection. however, the prevalence of several viral agents (rhinovirus, hbov, adv and cmv) among is samples of cap were drastically higher that of non-cap control group (p,0.05). in addition, the prevalence of rsv coinfection with hhvs (102/102, 100%) and hbov (23/102,22.54%) among cap group was significantly higher than that among non-cap controls (p,0.05).these data indicated that several viral agents (such as hbov and cmv) may contribute to the occurrence of cap. cap is more common and severe in the developing areas than developed areas [1] [2] [3] , and is a major cause of death among infants and children in rural areas [3] . previous investigations of paediatric cap in us and europe emphasised the importance of infections with common respiratory viruses (rsv, inf, piv and adv) [3, 4, 6] . the roles of hhvs and more recently identified viruses (hbov, hcov-nl63 and hcov-hku1) as causes of cap remain controversial [2, [5] [6] [7] 12, 14] . in this study, the viral prevalence in sputum specimens of childhood with non typical bacterial cap was investigated using sensitive molecular diagnostic methods for hhvs and 15 respiratory viruses, and viruses were detected in 99.6% of the children. this is not surprising considering that the samples included were highly selected for the discovery of viral etiology and addition hhvs detection in this study. to our knowledge, this is the first comprehensive study of the prevalence of hhvs in sputum samples among infants and young children with cap [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . a few reports have described the detection of dna from several hhvs in respiratory samples, with most of them focusing on immunosuppressed individuals or adults with cap [2, [19] [20] [21] [22] 31, 32] . in this study, we screened for dna of hhvs in is samples among infants and young children with cap using a sensitive multiple pcr assay; of the hhvs considered, only vzv was not detected. the highest positive rate was found for cmv infection (91.6%). cmv infection, which is usually congenital, showed no significant difference among various age groups in this study. these data are consistent with those of previous reports [2, 22, 31] . in addition, it was reported here that cmv and hhv-6 were the only detected viral agent among 25 cap cases and 5 cap cases, respectively. furthermore, the prevalence of cmv among is samples of cap were significantly higher than that of non-cap control group. the prevalence of rsv coinfection with hhvs among cap group was drastically higher than that among non-cap control (p,0.05). these data suggest an association between infection with hhvs (especially cmv) and cap in infants and children. few comprehensive studies have searched for viruses in is samples among infants and children with cap in rural areas [8, 10, 11] . in this study, 15 common and recently identified viruses associated with acute respiratory infection were screened using molecular methods. our results are consistent with previous studies conducted in china or other areas [8, 10, 11, 33] , which showed that the most-detected agent was rsv, followed by hbov, rv, hmpv, adv and piv3 in is samples from infants and children with cap [3, 11, [14] [15] [16] . rsv and hbov were also the dominant viruses detected in is samples from non-cap group. moreover, our data show a higher hbov detection rate (28.2%) compared with previous reports [23, 24] . the prevalence of hbov among is samples of cap were significantly higher that of non-cap control group. similar trends were also observed in the prevalence of rhinovirus and adv among is samples of cap when compared with non-cap control group. unlike previous data [34] , the prevalence of hcovs among is samples was significantly lower in present study. these differences might primarily due to the specimens [11, 15, 16] -is vs. a nasopharyngeal aspirate or nasopharyngeal wash. in the meantime, the impact of other factors, such as area and the duration of the study period on infection, could not be ruled out. one interesting finding of present study was that hhv coinfections were found in 70% of the cap cases, compared with rates between 15 and 45% in previous etiological studies of childhood cap [7, [35] [36] [37] . the clinical consequences of mixed infections have not been fully understood yet. evidence suggests that mixed viral infections can lead to more severe condition than individual viral infections [2, 7, [35] [36] [37] [38] . in this study, it was unable to determine whether the hhvs were reactivated from a latent reservoir after another respiratory virus infection, or if an immunosuppressed condition caused by hhv infection increases the potential risk of other respiratory virus infections. consequently, it is difficult to estimate the true association between the clinical manifestations and virus infection. at the same time, we understand that the detection of viruses in an is sample by pcr does not necessarily mean that they are the causative agents of the concomitant cap. the is samples included in this study may only represent part of hospitalized infants and children with cap in this hospital. to evaluate the real pathogenic role played by virus in hospitalized children with cap in this study, non-cap hospitalized children with chronic respiratory illness were set as control group. however, this study still has two major limitations. one limitation of this study is that viral detection from non-hospitalised children (due to limitation of ethics) and patients with bacterial cap, which would have provided a control group for this study, were not included. therefore, some viral agents detected in this study may represent asymptomatic persistence, prolonged shedding, or other situations. it is also not possible to evaluate the exact importance of each virus responsible for cap in most cases. nevertheless, it is believed that overall this study highlights the importance of hhvs (mainly cmv) and respiratory viruses in children with cap. another limitation of this study is that no samples were collected during summer, which could lead to the missing of some viral agents such as parainfluenza viruses and some enteroviruses. in summary, our study on the prevalence of hhvs and other respiratory viruses in infants and young children with cap identified a detectable virus in more than 99.6% of case participants, in which cmv, hhv-6, ebv, rsv and hbov were clearly predominant (.25%) and contributed significantly to the spectrum of cap in a rural area of china. although the hhvs were the most commonly identified pathogens in this study, which were not previously thought of as typical causes of cap in immunocopetent individuals [3] [4] [5] [6] [7] [8] 20, [38] [39] [40] [41] , further studies are required to determine the relationship of the presence of hhvs and severity of disease, thus the clinical significance of hhv infections or co-infections should receive greater attention in future treatment and prevention studies of cap in infants and children. who. global burden of disease in 2002: data sources, methods and results community-acquired pneumonia in children etiology of community-acquired pneumonia in 254 hospitalized children etiology of community-acquired pneumonia in hospitalized children based on who clinical guidelines access to a polymerase chain reaction 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epidemiologically associated with pneumonia requiring hospitalization in thailand update on rhinovirus and coronavirus infections rapid and simultaneous detection of 6 types of human herpes virus (herpes simplex virus, varicella-zoster virus, epstein-barr virus, cytomegalovirus, human herpes virus 6a/b, and human herpes virus 7) by multiplex pcr assay etiology and clinical characterization of respiratory virus infections in adult patients attending an emergency department in beijing molecular assays for detection of human metapneumovirus epidemiological profile and clinical associations of human bocavirus and other human parvoviruses characterization of human coronavirus etiology in chinese adults with acute upper respiratory tract infection by real-time rt-pcr assays the role of viruses in the aetiology of community-acquired pneumonia in adults lower respiratory tract viral infections in hospitalized adult patients molecular monitoring of causative viruses in child acute respiratory infection in endemoepidemic situations in shanghai effects of coronavirus infections in children mixed respiratory virus infections multipathogen infections in hospitalized children with acute respiratory infections viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection severe respiratory syncytial virus pneumonia associated with primary epstein-barr virus infection cytomegalovirus and herpes simplex virus effect on the prognosis of mechanically ventilated patients suspected to have ventilator-associated pneumonia cytomegalovirus pneumonia in immunocompetent host: case report and literature review cmv in critically ill patients: pathogen or bystander we thank the medical and technical staffs from the wenling hospital for their assistance and support. we also thank all the participants involve in this study for providing samples. key: cord-016932-bej10xbf authors: lum, lawrence g.; bollard, catherine m. title: specific adoptive t-cell therapy for viral and fungal infections date: 2018-06-19 journal: management of infections in the immunocompromised host doi: 10.1007/978-3-319-77674-3_20 sha: doc_id: 16932 cord_uid: bej10xbf despite advances in anti-infective agents, viral and fungal infections after hematopoietic stem cell transplantation (hsct) continue to cause life-threatening complications that limit the success of hsct. early adoptive t-cell immunotherapy studies showed that administration of allogeneic virus-specific cytotoxic t lymphocytes (vctl) can prevent and control viral infections and reconstitute antiviral immunity to cytomegalovirus (cmv) and epstein-barr virus (ebv). advances in immunobiology, in vitro culture technology, and current good manufacturing practice (cgmp) have provided opportunities for advancing adoptive cell therapy for viral infections: (1) t cells have been expanded targeting multiple pathogens; (2) vctl production no longer requires viral infection or viral vector transduction of antigen-presenting cells (apcs); (3) the source of lymphocytes is no longer restricted to donors who are immune to the pathogens; (4) naive t cells have been redirected with chimeric antigen receptor t cells (carts) or armed with bispecific antibody-armed t cells (bats) to mediate vctl activity; (5) these technologies could be combined to targeted multiple viral or fungal pathogens; and (6) pathogen-specific t-cell products manufactured from third parties and banked for “off-the-shelf” use post-hsct may soon become a reality. infections remain the leading cause of mortality and morbidity during the first 3 months after hematopoietic stem cell transplantation (hsct) [1] [2] [3] [4] . despite advances in prophylactic viral and fungal therapy to minimize the viral and fungal burden early after hsct, breakthrough viral and fungal infections remain life-threatening, and for some viral and fungal infections, there are no effective therapies [5] [6] [7] [8] [9] . vaccine strategies to induce immunity to cmv began in the 1970s but have been limited in their success [10] [11] [12] . the conditioning regimens for hsct that vary from non-myeloablative to myeloablative create an immunodeficiency that leaves the allogeneic hsct recipient susceptible to viral and fungal infections while immune reconstitution occurs during the first 6-9 months after hsct. immune reconstitution is further abrogated by intensive immunosuppression used to prevent and/or control gvhd. it is clearly established that the kinetics and rate of t-cell reconstitution are critical to controlling viral infections. factors that speed t-cell recovery will decrease the risk of viral infection during the first 3 months after hsct [2, 3, 13] . early studies showed that donor lymphocyte infusions (dli) given before t-cell reconstitution from the stem cell donor were effective for treating viral infections in hsct recipients but were associated with a high risk of gvhd [14] . since the early 1990s, investigators began to develop virus-specific cytotoxic t lymphocyte (vctl) for adoptive immunotherapy against specific targets early during immune reconstitution after hsct [15, 16] . advances in vctl therapy have benefited from (1) advances in understanding of immune responses to conserved t-cell epitopes for various pathogens [17] [18] [19] , (2) technological advances in ex vivo expansion of t cells and advances in the preparation of antigen-presenting cells [20] [21] [22] , and (3) assays that evaluate vctl activity and the mhc restriction of vctl [23, 24] . in this chapter, we review the following areas of how: (1) t cells have been expanded to target multiple pathogens; (2) vctl production no longer requires viral infection or viral vector transduction of antigen-presenting cells (apcs); (3) the source of lymphocytes is no longer restricted to donors who are immune to the pathogens; (4) naive t cells have been redirected with chimeric antigen receptor t cells (carts) to target pathogen-infected cells; (5) bispecific antibody (biab)-armed t cells (bats) can mediate vctl activity; and (6) pathogen-specific t-cell products can be manufactured by third parties and banked for "off-the-shelf" use post-hsct. we summarized the methodological approaches, clinical trials using vctl, promising preclinical studies, and early clinical trials of anti-pathogen ctls that have promise. these advances provide the rationale and impetus for future vctl adoptive immunotherapy. production of vctl as a guiding principle to decrease the risk of gvhd in allogeneic hsct recipients, strategies excluded alloreactive t cells by selecting virus-specific t cells. four major approaches were used: (1) stimulation with viral antigen(s) during ex vivo culture of donor t cells from peripheral blood mononuclear cells (pbmc), (2) direct selection of donor cells, (3) genetic modification of t cells to confer specific recognition of pathogen or pathogen-infected cells, or (4) arming of ex vivo expanded t cells with bispecific antibody to target the viral antigen ( fig. 20.1 ). numerous ex vivo culture approaches have been used to produce cytomegalovirus (cmv)-specific ctl or epstein-barr virus (ebv)-specific ctl [15, 16, [25] [26] [27] [28] [29] [30] . cmv viral-or peptide-specific stimulation in vitro expands single or multiple pathogenspecific vctl. the advantages of culture over cell selection are the generation and expansion of polyclonal vctl to clinically useful quantities of vctl from small amounts of blood [31] . however, the major disadvantages of this strategy is the daunting task of culturing and processing after stimulation to expand the vctl (up to more than 1 month) and the hlahistocompatibility requirement of finding a closely matched donor. during these longer-term cultures, the vctl may lose their capacity to self-renew and to persist in vivo, particularly after prolonged ex vivo culture [32] . it should be noted that clinical trials infusing ex vivo expanded vctl post-hsct showed prolonged persistence [33] and that ex vivo expansion using pathogen-specific stimuli decreased alloreactivity [19] . this may be due to selection of virus-specific clones and deselection of alloreactive clones. one study showed that residual alloreactivity seen in vctl is clinically insignificant [34] . the initial trials of vctl therapy required cmv lysates on apc, cmv-infected fibroblasts, or ebv-lymphoblastoid cells lines as a stimulant for expansion of donor-derived memory t cells [25, 27, 35] . the discovery of dominant and highly conserved antigens such as cmv-pp65 and adenovirus hexon and penton led to replacement of live viral stimulation with either 15-mer peptide pools spanning viral proteins or dna plasmidtransduced antigen-presenting cells [36, 37] . the newer approaches to rapidly expand and manipulate apcs enabled use of a less restricted population of donors and the targeting of an increased number of pathogens in a single culture [20, 38] . in a recent rapid vctl protocol, the addition of il-4 and il-7 leads to production of cd4+ t cells with a th 1 phenotype, whereas il-2 and il-15 tended to favor in vitro natural killer (nk) cell expansion [37] . the ideal population to adoptively transfer may be ex vivo expanded central memory t cells with a cd62l and cd45ra phenotype as these cells have a superior ability to persist in vivo after adoptive transfer [39, 40] . selection via cell capture sorting direct selection relies on cell sorting of immune donor pbmcs, usually after pulsing them with the antigen(s) of interest, to drive expansion of virus-specific t-cell clones [41] . this approach would not be viable for obtaining immune ctls from pathogen-naive donors. multimer selection is achieved by binding of hla-peptide complexes to t-cell receptors (tcrs) of known antigen specificity, followed by purification of bound cells, e.g., by magnetic column separation. alternatively, antiviral t cells expressing interferon-γ (ifn-γ) can be isolated using the gamma capture assay. direct selection methods have the advantage of rapid manufacturing time. unfortunately, these approaches require apheresis of donors in order to collect sufficient cells for sorting and processing for clinical appli-cations and pre-existing and detectable pathogenspecific t cells in the blood. multimer selection is major histocompatibility (mhc)-restricted and selects only cd8+ t cells of a limited specificity. this could possibly allow pathogen evasion and impair persistence of vctl in vivo [42] . earlier studies suggested that persistent binding of multimers to the tcr may impair t-cell function [43] . recent reversible streptamer technology for direct selection may overcome the problem of impaired function [44] . ifn-γ positive selection captures polyclonal antigen-specific cd4+ and cd8+ t cells and selects for a wider range of antigen-specific cells. combining direct selec-tion, culture expansion methods, and cytokine cocktails can optimize the selection of central memory t cells in vctl products and improve yields on targeted cellular phenotypes [37, 44] . tcr or car gene modifications t cells can be modified to redirect their specificity with retroviral and lentiviral vectors to introduce the transgenes for high-affinity tcrs or chimeric antigen receptors (cars) consisting of a single-chain variable fragments (scfvs). high-affinity tcr genes can be cloned and transduced into polyclonal t cells to generate a large population of (1) blood is obtained from donors (autologous, allogeneic, or umbilical cord blood) or is drawn or apheresis is performed to obtain a larger quantity of blood; (2) pbmcs are processed via: (a) cell selection panel using multimers with a pathogen-derived peptide associated with a type-i hla molecule or column selection after in vitro stimulation of t cells with antigens followed by binding of ifnɣ or cd154-expressing t cells with antibody-coated immu-nomagnetic beads; (b) cell expansion by stimulating the pbmc with apcs produced by antigenic peptide pools, viral transduction, or nucleofection; (c) genetic modification that involves the transfer of high-affinity pathogenspecific tcrs or cars to redirect the specificity of the t cells; and (d) polyclonal expansion of t cells for 8-14 days and arming with biabs directed at the pathogen of interest on one hand and the tcr on the other hand; (3) quality control and release testing; and (4) infusion into patients tcr pathogen-specific ctls [45] . a similar strategy was used to produce tumor-specific t cells after tcr gene transfer [18] . in contrast, cars have an extracellular region that consists of a scfv that binds to antigen, with an intracellular signaling complex composed of tcr zeta chain for first-generation cars, the tcr zeta chain and the cd28 for second-generation cars, and tcr zeta and cd28 or 41bb for third-generation cars [46] [47] [48] . the high-affinity tcr-transduced ctls have been used to target cmv-infected cells [49] , hpv-infected cells [50] , hepatitis b-infected cells [51] , hepatitis c-infected cells [52] , tuberculosis-infected cells [53] , sarsinfected cells [54] , chlamydia-infected cells [55] , and hiv-infected cells [56] . car t cells were used to target cd4 in hiv-infected cells [57] [58] [59] [60] and for recognition of β-glucans in fungi [61] . ex vivo ctl expansion is the most common method for producing clinical ctls for most clinical trials ( [25] . cmv has been the primary focus of the first virus targeted therapy trials and remains a primary focus in subsequent studies (table 20 .1). the first clinical report in which cd8 + cmvspecific ctls were isolated via tetramer selection [62] generated complete or partial clinical responses in nine patients, but there was limited data on long-term persistence of the infused cmv-specific ctls. ifn-γ column selection (gamma capture, miltenyi) to produce cmv-ctls was associated with partial and complete responses in 15 of 18 patients who were given one dose of cmv-ctls [63] . ifn-γ selection after stimulation with recombinant pp65 or an overlapping peptide pool of 15-mers covering the pp65 protein was used to produce cmv-ctl [64] . infusions of cmv-ctls administered prophylactically after stem cell transplantation successfully protected seven patients from the development of viral reactivation and disease. further, in vivo expansion of cmv-ctls was detected in 11 patients [64] . cmv-ctls from hsct donors using reversible streptamers with mhc-restricted pp65 peptides were used to successfully treat two patients with cmv reactivation after hct [44] . the strategy for using bispecific antibodies (biabs) to target cancer was nearly abandoned due to cytokine storm reactions. however, the last 10 years has seen a resurrection of interest particularly for targeting t cells to various cancer antigens. studies using retargeted t cells have been reported for her2 in breast and prostate cancer using anti-cd3 x anti-her2 biab atc [89, 90] ; egfr in colorectal, pancreatic, and lung cancer using anti-cd3 x anti-egfr biab atc [91] ; and cd20 in non-hodgkin's lymphoma using anti-cd3 x anti-cd20 biab atc [92] [93] [94] . since chemical or molecularly engineered constructs could be used to target the tcr on one hand and tumor-associated antigen (taa) on the other hand, we reasoned that cmv could be targeted by chemically heteroconjugating okt3 (anti-cd3, anti-tcr) with cytogam® (polyclonal donor-derived anti-cmv igg, designated cmvbi) to kill cmv-infected fibroblasts [95] . in this strategy shown in fig. 20 .1, anti-cd3 monoclonal antibody-activated t cells (atc) which expanded in low-dose il-2 were the t effector cells. atc alone do not kill cmvinfected targets. arming doses of cmvbi ranging from as low as 0.01 ng/10 6 atc to 50 ng/10 6 atc exhibited high levels of specific anti-cmv cytotoxicity in targets infected with cmv at multiplicities of cmv infection (moi) ranging from 0.01 to 1. the polyclonal nature of the cytogam may provide multiple antibody clones directed at multiple cmv epitopes on the cmv-infected targets leading to the increased potency at a low arming dose of cmvbi. cytotoxicity was evident at effector-to-target ratios (e:t) of 25:1, 13:1, 6:1, and 3:1 compared to unarmed atc alone. at an moi of 1.0, the mean % specific anti-cmv-specific cytotoxicities at e:t of 3, 6, and 13 were 79%, 81%, and 82%, respectively, whereas unarmed atc at the same e:ts killed <20%. unarmed atc, cytogam®, or cmvbi alone did not exhibit significant killing of uninfected or cmv-infected fibroblasts. furthermore, cultures of cmvbiarmed atc with cmv-infected targets induced cytokine and chemokine release from cmvbiarmed atc. this simple targeting strategy bypasses mhc-restricted cytotoxicity for treating viral disease in organ transplant and hsct recipients. it was shown that cmvbi atc do not react to alloantigens in vitro in a mixed lymphocyte culture, and they can be frozen and reinfused at different time points as an "off-the-shelf" drug. although promising, it is not clear from these data whether targeting cmv or other disease agents using this approach will be clinically effective. ebv-ctls have been used for prevention and treatment of post-hsct lymphoproliferative disease (ptld) as well as ebv + lymphoma. irradiated ebv-lymphoblastoid cells (ebv-lcl) were used to generate ebv-specific ctls in vitro for prophylaxis or treatment for ebv-ptld in 114 patients [27, 33] . remarkably, the first 26 patients received gene-marked ctls, and followup studies showed the gene-marked cells persisted up to 105 months after hsct (table 20 .1). hla-a2-specific pentamers and ifn-γ selection procedures were used to produce ebv-ctls. hla-a2 specific pentamers were used to produce ebv-ctls from the haploidentical mother of a patient with ebv-ptld who had received a cord blood transplantation [69] . a complete clinical response was obtained following two infusions of ebv-ctls. three of six patients with early ebv-induced ptld treated with ebv-ctls produced by ifn-γ selection achieved complete responses whereas three patients with advanced, multiorgan disease did not respond [70] . the latest strategy is to target ebv with multiviral ctl products (below) or third-party-derived ebv-ctls. most studies targeting adenovirus (adv) use multiviral ctls [21, 81, 83, 84] . a few exclusively target adv by selection technology. adv-ctls produced by ifn-γ selection was used for treatment of nine patients with drug-refractory adv infections [74] . there was in vivo ctl expansion in five of six patients and four patients cleared their disease. in all studies using cell selection, clinical benefit was observed in spite of very low doses of vctls infused (<5 × 10 4 cells/kg in most studies) [73, 74] . recent antiviral ctl therapy trials target multiple viruses (cmv, ebv, and adv as primary targets). cmv, ebv, and adv are the three leading causes of viral-associated mortality after allogeneic hsct. clinical-grade adv vector ad5f35pp65 contains the immunodominant cmv antigen pp65, providing a unique opportunity to transduce donor-derived dendritic cells or ebv-lcl to serve as apcs for the ctl cultures. triviral (cmv, ebv, and adv-specific) ctls were tested in a dose-escalation trial involving 26 patients [83] . there were no adverse effects at doses ranging from 5 × 10 6 to 1 × 10 8 cells/m 2 , and all patients were effectively protected against cmv, ebv, and adv. interestingly, although ebv-and cmv-specific ctls were detected by ifn-γ elispots, adv-ctls were not detectable except during infection. in a follow-up trial using ad5f35-transduced ebv-lcl to produce ebvand adv-ctls, 13 patients received prophylaxis or treatment for ebv and adv infections after hsct [21] . although the ctls provided protection in vivo, the adv-ctls could not be detected except in the setting of adv infection; these data suggest that levels of specific vctls below the limits of detection by ifn-γ elispots provide protection and infection induces clonal expansion. similarly, ad5f35pp65 transduced dendritic cells (dc) used to produce cmv-and adv-ctls were clinically effective in 12 patients after allogeneic hsct [84] . there were a few cases of cmv reactivation in the setting of lowdose prednisone. this approach was applied to 50 patients after allogeneic hsct with triviral (cmv, ebv, adv-specific) ctls using two methods: 10 were produced by pulsing donor dcs with the hla-a2 restricted cmv peptide nlvpmvatv and 40 were produced using ad5f35pp65-transduced donor dcs [87] . only 5 of 50 patients had cmv reactivation after ctl infusions and only 1 of 5 patients required antiviral drug therapy after steroid treatment for acute gvhd. advances in processing protocols have validated 5-mer peptide pools that include immunodominant viral antigens that replace viral transduction of apc thereby removing safety and regulatory barriers associated use of viral vectors [36] . the use of gas-permeable rapid-expansion (g-rex) bioreactors has simplified ctl culture [96] . these advances in technology led to the development of a rapid manufacturing protocol for expanding virus-specific t-cell products (vsts) that yield clinically relevant numbers of vsts in 10-12 days. further, vst products targeting multiple viral antigens have been shown to provide effective antiviral protection (against cnv, ebv and ad) in ten patients after hsct [37] . this rapid manufacturing protocol was subsequently adapted to produce five virus-specific ctls targeting ebv, cmv, adv, hhv6, and bk virus infections in a single t-cell product for patients following allogeneic hsct [88] . fourteen of 48 vst products manufactured from hsct donors recognized all 5 viral components while 35 (73%) recognized 3 or more by ifn-γ elispots. unexpectedly 22 of the donors were cmv seronegative and vsts produced predictably lacked cmv specificity. these vsts were used to treat 11 patients after hsct. the 3 patients treated prophylactically remained free of viral infections and 8 patients with 18 viral reactivations received vsts, with all experiencing partial or complete responses in their cmv, ebv, adv, or hhv6 infections. although there was intense interest in the use of ctl therapy for hiv, there was only limited success to date [97] . attempts to expand and reinfuse autologous hiv-specific ctls resulted in only transient improvements in viral load [75] . a larger number of clinical trials focused on genetically modified ctl to target hiv using transduction of a modified tcr or cars. these trials established safety, but exhibited limited antiviral efficacy [76, 77] . a major challenge for this approach is the outgrowth of escape mutants expressing alterations of the target epitope so the infected cell can no longer be targeted by the effector cells. a more successful approach has been inserting genes that would provide hiv resistance. this approach was clinically tested when antisense gene complementary to hiv env was transduced into t cells from 17 patients using lentiviral vectors [78] . the ctls persisted for 5 weeks, homed to gut-associated lymphoid tissue, and were well-tolerated with clinical toxicities. infusions of ctls in two of eight patients who underwent antiviral treatment interruption keep the viral load u ndetectable for 4 and 14 weeks. when ccr5-delta32 mutations were introduced to cd4enriched t cells through the use of a zinc-finger nuclease [79] , the ccr5-edited t cells were subsequently infused in 12 patients, and engineered t cells were detectable in the peripheral blood for up to 42 months post infusion. in six patients who underwent antiviral treatment interruption, the absolute number of gene modified cd4+ t cells decreased at a lower rate than non-modified t cells. recent studies showed that dual gene editing of cxcr4 and ccr5 via zinc-finger nucleases was successful in a t-cell line, and preclinical studies show that the t cells were highly resistant to hiv infection [98] . it is not clear whether this approach could prevent primary infection or have a clinical impact as an hiv cure strategy. there are a few studies that target other viruses with adoptive immunotherapy. the john cunningham virus (jcv) is a ubiquitous polyoma virus which can cause progressive multifocal leukoencephalopathy (pml), which occurs in immunocompromised individuals such as acquired immunodeficiency syndrome (aids), recipients of hsct or solid organ transplants, or primary immunodeficiency disorders. donorderived jcv-specific ctls were used in a 14-year-old patient with pml after prolonged steroid treatment for gvhd following hsct. cells were manufactured using 15-mer peptide pools that included jc antigens vp1 and lt and infused twice leading to clearance of jv-dna from the cerebrospinal fluid with improvements in neurologic status [80] . human papillomavirus (hpv) disease can be a late complication of hsct. peptide pools spanning the hpv e6 and e7 proteins were used to generate hpv-specific ctls from patients with oropharyngeal or cervical cancer that arise after hpv16 infection [99] . the ctls exhibited specific activity directed at hpv e6 and e7 and antitumor activity against the hpv16 cervical cancer cell line caski. adverse events after 381 infusions for 180 patients on 18 protocols by the groups at baylor college of medicine were reported [100] . side effects were limited to 24 mild adverse events observed within 6 h of infusion; nausea and vomiting were most common with 22 nonserious adverse events (fever, chills, nausea) that occurred within 24 h. no significant gvhd was attributed to ctl infusions. the only significant complications were rare reports of systemic inflammatory responses in patients with bulky ebv+ lymphomas following ebv-ctl therapy. seven cases of acute gvhd occurred in patients who had a greater degree of hla mismatch than controls after infusions of ebv-ctl. some of the cases of gvhd were attributable to reducing the corticosteroid dose prior to the cmv-ctl infusions [87] . for years, the selection or culture of anti-pathogen ctls was dependent on the presence of pathogen-specific memory t cells in the blood of donors, and, therefore, the approach could not help allograft recipients of pathogen-naive hematopoietic cell products after hsct. one strategy to address this problem is to provide "off-theshelf" pathogen-specific ctls derived from third-party donors. this strategy was first validated in a phase i trial involving 8 patients who received partially matched ebv-ctls for ptld that developed after solid organ transplantation [66, 67] and confirmed in a cohort of 33 patients in a phase ii trial [68] . the latter trial showed a response rate of 64% at 5 weeks and 52% at 6 months; the outcomes correlated with the degree of hla matching between the ctl donor and recipient. in the hsct patients, two patients with refractory ebv-ptld after cord blood transplantation (cbt) with third-party ebv-specific ctls [71] . a bank of 32 ctl lines with characterized activity against ebv, cmv, and adv were used to match for 50 patients with refractory viral infections. this strategy resulted in partial or complete antiviral responses in 74%, 78%, and 67% of those with cmv, adv, and ebv, respectively [85] . this is a marked improvement from standard therapy response rate of 13% in eight patients for whom a matched line could not be identified. despite partial hla matching at one to four loci, there were only two patients who developed grade i gvhd. clones that are responsible for gvhd have been selected against in the expansion culture and may exist at such low precursor frequencies after culture that they do not expand enough to cause clinically significant gvhd. the lower rate of response against ebv relative to cmv and adv may reflect selective expansion of t cells against immunodominant epitopes of the latter two viruses, thereby complicating the selection of an ideal third-party pathogen-specific line that fulfills the requirements of antiviral activity and mhc-restriction against multiple pathogens. the methods for producing third-party-virus-specific ctl include pentamer selection for ad, cmv, ebv [81] , and ifn-γ selection for adv-ctl [73] . a few studies reported transducing ctls with a virus-specific tcr [49, 101, 102] . a trial of transgenic ctls using a retroviral vector that expresses a cmv-specific tcr is ongoing in the united kingdom (morris e. et al. mrc# g0701703). alternatively, kumaresan et al. transduced t cells with the β-glucan receptor dectin [61] . since the carbohydrate β-glucan is found in the cell wall of most fungi [103] , investigators used its natural receptor, dectin-1, as a recognition receptor coupled to a cd28 (a key co-stimulatory molecule) and cd3-zeta transgene to initiate signaling and killing in t cells. the same group showed that the antifungal carts could mediate damage to hyphae in vitro and in vivo [61] . these novel approaches would allow creation of specific ctls from pathogennaive donors; however, they are subject to the regulatory challenges in gene transfer technology. furthermore, use of a single antifungal tcr allows for antigenic escape. a major advance in adoptive viral ctl therapy was development of virus-specific ctls from virus-naive donors. ctl could be produced from a 20% fraction from cord blood using donorderived dcs and ebv-lymphoblastoid cell line (lcl) as apc and ad5f35pp65 transduction as a source of cmv and adv antigens [20] . the resulting viral ctls exhibited specific anti-cmv, ebv, and adv ifn-γ elispots responses as well as specific 51 cr cytotoxicity with no alloreactivity. epitope mapping showed that the immunodominant epitopes recognized by cord blood-derived ctls were different from the immunodominant epitopes recognized by the cmv and ebv seropositive adult donors. the hla-a2-restricted epitope nlvpmvatv was notably absent in the cord blood-derived lines. ctls derived from cord blood were successfully infused in 12 cbt recipients in the ongoing act-cat trial (safety, toxicity and mtd of one intravenous iv injection of donor ctls specific for cmv and adenovirus, # nct00880789). recently, multiviral ctls were produced from cmv-naive adult donors using columnselected cd45ra+ naive t cells stimulated by donor dcs pulsed with cmv 15-mer peptide pools [38] . preclinical studies suggest that multiviral ctls will exhibit similar anti-cmv activity to dcs pulsed with cmv 15-mer peptide pools. ebv-ptld is a significant long-term risk in solid organ transplant recipients. rituximab can be effective, but treatment often requires reduction of immunosuppression which can lead to graft rejection. autologous ebv-ctls have been used in this setting [72] . several prophylactic infusions of autologous ebv-ctls reduced the ebv viral load without adverse reactions despite ongoing treatment with calcineurin inhibitors [65] . a heart transplant recipient who developed hodgkin's lymphoma-type ptld 8 years after transplant had remission after being treated with autologous ebv-ctls in combination with chemotherapy without alterations in his immunosuppression [72] . this observation supports the prior observations that calcineurin inhibitors block proliferation, but do not impair ctl activity. fungal infections are a major cause of morbidity and mortality in allogeneic hsct recipients, with gvhd being the major risk factor. candidal infections can range from mucocutaneous colonization of the skin and mouth to life-threatening systemic infections. aspergillus species are ubiquitous molds that cause invasive pulmonary infections as well as widespread infection including central nervous system dissemination in highly immunocompromised patients [104] . patients with inherited immunodeficiencies (e.g., chronic granulomatous disease), patients with prolonged neutropenia after repeated rounds of chemotherapy (e.g., for acute leukemia), and those receiving immunosuppression after lung transplant or allogeneic hsct are at the highest risk for mycoses [105] . the importance of t-cell immunity in defense against invasive aspergillosis and other filamentous fungi is not clear, since patients with these invasive fungal diseases usually have severe deficiencies in multiple components of the immune system. in patients with advanced aids, invasive aspergillosis is an uncommon complication and generally occurs when other forms of immune impairment (e.g., neutropenia and use of corticosteroids) are present. despite these unknowns, it may be clinically useful to target fungal infections with fungusspecific t cells after hsct. the adaptive immune response against invasive aspergillosis is believed to be orchestrated by cd4+ t cells. table 20 .2 summarizes preclinical studies that developed fungal-specific ctls against candida, aspergillus, and rhizopus (a member of the mucorales group) species. aspergillus-specific ctls were produced by stimulation of pbmc with antigens from aspergillus extracts, selection with ifn-γ secretion, and culture [106] . the ctls were predominantly cd4+, cd45r0+ memory cells that secrete ifn-ɣ in response to aspergillus and penicillium. the fungal-specific ctl enhanced hyphal damage by neutrophils and apcs. ifn-ɣ selection and stimulation with candida albicans, aspergillus fumigatus, and rhizopus oryzae extracts were used to produce multifungalspecific ctl lines, which were also nearly all cd4+ cd45ro+ hla-dr+ that exhibited activation markers of ifn-ɣ, cd154, and tnfα and enhanced oxidative activity of neutrophils when co-incubated with antigen and apcs [108] . several studies target the candida mp65 and aspergillus crf1 antigens. to produce multipathogen-specific t cells that secrete ifn-ɣ, proliferate, and kill cmv, ebv, adv, candida, and aspergillus, donor pbmcs were incubated with peptide libraries from cmv-pp65, ebv-lmp2, adv-hexon, candida mp65, and a 15-mer peptide from aspergillus crf1 [107] . however, it remains unclear what the significance of mp65 and crf1 is in antifungal immunity [117] [113] . expanded memory/effector th1 cells following stimulation with rhizopus extracts were used to generate memory/effector th1 cells for mucormycosis, and the product exhibited specificity to the original rhizopus oryzae extract as well as other mucorales species [118] . candida-specific t cells generated with cellular extracts of candida albicans released cytokines that caused hyphal damage and increased neutrophil activity against hyphae [111] . ctls produced by stimulation with inactivated conidia (spores) from aspergillus fumigatus resulted in clonal cd4+ ctls with anti-aspergillus activity by ifn-ɣ elispots [82] . these donor t-cell clones specific for aspergillus antigens were then infused in patients following haploidentical hsct. of 23 patients who developed invasive aspergillosis, 10 patients received anti-aspergillus ctls, while 13 patients did not. nine of 10 treated patients cleared their infections whereas only 7 of 13 untreated patients cleared their infections. aspergillus-specific ctls were detected in high frequencies in patients who received immunotherapy while they were barely detectable in untreated patients [82] . despite notable advances in antifungal ctls, a better understanding of the immunodominant t-cell targets that should be selected for various fungal species is needed, and standardized clinical-grade cgmp fungal antigen sources are needed to provide consistency between trials. although there have been major advances in producing pathogen-specific ctls, important questions remain regarding methods that affect potency and efficacy of the t-cell products. it is unclear whether manufacturing ctls to include more pathogens in a single culture will affect potency and specificity in the ctl cultures. although the proportions of virus-specific ctls for each virus decrease as the number of antigens increases, these effects have not seemed to impact clinical trials. ctls specific for 7 viruses (cmv, ebv, adv, bk, hhv6, rsv, and influenza) pro-duced using peptide pools for 15 antigens exhibited specific activity against all targeted viruses [37] . the question remains as to whether adding additional viral targets will skew specific cytotoxicity, alter potency for each target, induce alloreactive t cells, or compromise in vivo responses. a major challenge is achieving consistent and optimal culture conditions for generating the most effective ctl product. although multiple rounds of stimulation with antigen select and expand the specific antiviral clones, prolonged culture may lead to t-cell exhaustion. some groups have decreased production time using newer bioreactors [96] . identification of the "correct" subset of t cells for clinical use (however selected) will require well-designed randomized phase ii trials using a specific ctl product made by the same group or a common standard operating procedure (sop) in a homogeneous group of hsct patients. assays for measuring ifn-ɣ elispots and cytotoxicity need to be standardized and the timing of the studies needs to be the same. recently, a new population of healthy donors jolink [113] transgenic tcr-transduced cells tuberculosis murine cells feng [114] mhc-streptamer-enriched antigen-specific t cells listeria murine cells stemberger [115] (proof of principle) -targeting of ova-expressing parasites murine cells polley [116] transgenic trc-transduced cells chlamydia murine cells roan [55] "stem cell memory t cells" has been putatively identified -which possess characteristics ideal for use in adoptive immunotherapy. unfortunately, there are no randomized phase ii trials to date to support continued development and commercialization of clinically effective ctls. the presence of immunosuppression remains a barrier for optimal immunotherapy after allogeneic hsct and solid organ transplantation since most agents also suppress ctl functions. nearly all protocols require recipients to be receiving less than 0.5 mg/kg/day of prednisone and wait at least 30 days after anti-t-cell serotherapy to be eligible to receive ctl therapy. virtually all of the calcineurin inhibitors (cyclosporin a, tacrolimus, or sirolimus) at therapeutic doses impair ctl activity. ebv-specific ctls can be made resistant to tacrolimus by knockdown of fkbp12 via a retrovirally transduced specific sirna and exhibit anti-ebv lymphoma activity in the presence of tacrolimus [119] . similarly, ebv-specific ctls can be made resistant to both cyclosporine a and tacrolimus by mutating calcineurin [120] . the mutation does not alter the phenotype or antiviral activity of the ctls and mutated cells have a growth advantage in calcineurin inhibitors. although they have not been applied clinically, they have great potential for treating hsct and solid organ transplant recipients. there is one preclinical report of t cells used to target bacterial and parasitic infections [116] , but there are no clinical trials evaluating t-cell immunotherapy for bacterial and parasitic infections. despite numerous studies evaluating in vitro t-cell responses, there is no consensus on the role of t cells in defense against aspergillosis. infusions of anti-pathogen ctls in several hundred patients over the past several decades have been established as a safe and highly effective therapy following allogeneic hct. identifying preserved viral t-cell epitopes, probing the antigen limits in ctl monoculture, testing the clinical efficacy of immunosuppressive-resistant ctls, and improving conditions for rapid and specific expansion will further broaden the usefulness of this treatment strategy. as advances in protocols and methods for manufacture achieve acceptable clinical standards that can be supported commercially, ctl therapy may become an integral component of care offered to allogeneic hsct or immunodeficiency patients. late cytomegalovirus disease and mortality in recipients of allogeneic hematopoietic stem cell transplants: importance of viral load and t-cell immunity marked increased risk of epstein-barr virus-related complications with the addition of antithymocyte globulin to a nonmyeloablative conditioning prior to unrelated umbilical cord blood transplantation adenovirus infection rates in pediatric recipients of alternate donor allogeneic bone marrow transplants receiving either antithymocyte globulin (atg) or alemtuzumab (campath) epidemiology and outcome of invasive fungal infection in adult hematopoietic stem cell transplant recipients: analysis of multicenter prospective antifungal therapy (path) alliance registry update in management of ganciclovirresistant cytomegalovirus infection antiviral drugs for cytomegalovirus diseases rising pp65 antigenemia during preemptive anticytomegalovirus therapy after allogeneic hematopoietic stem cell transplantation: risk factors, correlation with dna load, and outcomes cidofovir for cytomegalovirus infection and disease in allogeneic stem cell transplant recipients. the infectious diseases working party of the european group for blood and marrow transplantation cd20 monoclonal antibody (rituximab) for therapy of epstein-barr virus lymphoma after hemopoietic stem-cell transplantation the history of vaccination against cytomegalovirus infusion of cytomegalovirus (cmv)-specific t cells for the treatment of cmv infection not responding to antiviral chemotherapy clinical aspects of cmv infection after stem cell transplantation cytomegalovirus and varicellazoster virus vaccines in hematopoietic stem cell transplantation infusions of donor leukocytes to treat epstein-barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation the use of anti-cd3 and anti-cd28 monoclonal antibodies to clone and expand human antigen-specific t cells restoration of viral immunity in immunodeficient humans by the adoptive transfer of t cell clones cmv pp65 and ie-1 t cell epitopes recognized by healthy subjects identification of hexon-specific cd4 and cd8 t-cell epitopes for vaccine and immunotherapy t-cell therapy in the treatment of post-transplant lymphoproliferative disease functionally active virus-specific t cells that target cmv, adenovirus, and ebv can be expanded from naive t-cell populations in cord blood and will target a range of viral epitopes cytotoxic t lymphocyte therapy with donor t cells prevents and treats adenovirus and epstein-barr virus infections after haploidentical and matched unrelated stem cell transplantation large-scale expansion of dendritic cell-primed polyclonal human cytotoxic t-lymphocyte lines using lymphoblastoid cell lines for adoptive immunotherapy analysis of cd8 t cell reactivity to cytomegalovirus using protein-spanning pools of overlapping pentadecapeptides expansion of t cells targeting multiple antigens of cytomegalovirus, epstein-barr virus and adenovirus to provide broad antiviral specificity after stem cell transplantation reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of t-cell clones from the donor a phase i-ii trial to examine the toxicity of cmv-and ebv-specific cytotoxic t lymphocytes when used for prophylaxis against ebv and cmv disease in recipients of cd34-selected/t cell-depleted stem cell transplants infusion of cytotoxic t cells for the prevention and treatment of epstein-barr virus-induced lymphoma in allogeneic transplant recipients administration of neomycin-resistance-genemarked ebv-specific cytotoxic t lymphocytes to recipients of mismatched-related or phenotypically similar unrelated donor marrow grafts immunotherapy targeting ebv-expressing lymphoproliferative diseases autologous epstein-barr virus (ebv)-specific cytotoxic t cells for the treatment of persistent active ebv infection adoptive immunotherapy for posttransplantation viral infections acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred cd8+ t cells long-term outcome of ebv-specific t-cell infusions to prevent or treat ebv-related lymphoproliferative disease in transplant recipients allogeneic virus-specific t cells with hla alloreactivity do not produce gvhd in human subjects adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virusspecific t-cell lines generation of cmv-specific t lymphocytes using protein-spanning pools of pp65-derived overlapping pentadecapeptides for adoptive immunotherapy rapidly generated multivirus-specific cytotoxic t lymphocytes for the prophylaxis and treatment of viral infections naïve t-cell-derived ctl recognize atypical epitopes of cmvpp65 with higher avidity than cmvseropositive donor-derived ctl -a basis for treatment of post-transplant viral infection by adoptive transfer of t-cells from virus-naïve donors adoptive transfer of effector cd8+ t cells derived from central memory cells establishes persistent t cell memory in primates molecular signatures distinguish human central memory from effector memory cd8 t cell subsets the role of virus-specific adoptive t-cell therapy in hematopoietic transplantation evasion of cd8+ t cells is critical for superinfection by cytomegalovirus reversible hla multimers (streptamers) for the isolation of human cytotoxic t lymphocytes functionally active against tumor-and virus-derived antigens adoptive transfer and selective reconstitution of streptamerselected cytomegalovirus-specific cd8+ t cells leads to virus clearance in patients after allogeneic peripheral blood stem cell transplantation immunobiology : the immune system in health and disease adoptive cellular therapy: a race to the finish line the promise and potential pitfalls of chimeric antigen receptors tumor-specific t-bodies: towards clinical application cmv-specific tcr-transgenic t cells for immunotherapy generating hpv specific t helper cells for the treatment of hpv induced malignancies using tcr gene transfer engineering virus-specific t cells that target hbv infected hepatocytes and hepatocellular carcinoma cell lines transduction of human t cells with a novel t-cell receptor confers anti-hcv reactivity development of genetically engineered cd4+ and cd8+ t cells expressing tcrs specific for a m. tuberculosis 38-kda antigen engineering t cells specific for a dominant severe acute respiratory syndrome coronavirus cd8 t cell epitope antigen-specific cd8+ t cells respond to chlamydia trachomatis in the genital mucosa reconstitution of anti-hiv effector functions of primary human cd8 t lymphocytes by transfer of hiv-specific alphabeta tcr genes t-cell engineering by a chimeric t-cell receptor with antibody-type specificity for the hiv-1 gp120 anti-hiv designer t cells progressively eradicate a latently infected cell line by sequentially inducing hiv reactivation then killing the newly gp120-positive cells characterization of t cell-expressed chimeric receptors with antibodytype specificity for the cd4 binding site of hiv-1 gp120 lentiviral vectors encoding human immunodeficiency virus type 1 (hiv-1)-specific t-cell receptor genes efficiently convert peripheral blood cd8 t lymphocytes into cytotoxic t lymphocytes with potent in vitro and in vivo hiv-1-specific inhibitory activity bioengineering t cells to target carbohydrate to treat opportunistic fungal infection adoptive transfer of cytomegalovirus-specific ctl to stem cell transplant patients after selection by hlapeptide tetramers adoptive transfer of pp65-specific t cells for the treatment of chemorefractory cytomegalovirus disease or reactivation after haploidentical and matched unrelated stem cell transplantation directly selected cytomegalovirus-reactive donor t cells confer rapid and safe systemic reconstitution of virus-specific immunity following stem cell transplantation reconstitution of ebv-specific t cell immunity in solid organ transplant recipients complete regression of posttransplant lymphoproliferative disease using partially hla-matched epstein barr virus-specific cytotoxic t cells treatment of epstein-barr-virus-positive post-transplantation lymphoproliferative disease with partly hlamatched allogeneic cytotoxic t cells allogeneic cytotoxic t-cell therapy for ebv-positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial a novel haploidentical adoptive ctl therapy as a treatment for ebv-associated lymphoma after stem cell transplantation effective and long-term control of ebv ptld after transfer of peptide-selected t cells successful treatment of ebv-associated posttransplantation lymphoma after cord blood transplantation using third-party ebv-specific cytotoxic t lymphocytes successful treatment of a classic hodgkin lymphoma-type posttransplant lymphoproliferative disorder with tailored chemotherapy and epstein-barr virus-specific cytotoxic t lymphocytes in a pediatric heart transplant recipient thirdparty virus-specific t cells eradicate adenoviraemia but trigger bystander graft-versus-host disease safe adoptive transfer of virus-specific t-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation safety of autologous, ex vivo-expanded human immunodeficiency virus (hiv)-specific cytotoxic t-lymphocyte infusion in hiv-infected patients prolonged survival and tissue trafficking following adoptive transfer of cd4zeta gene-modified autologous cd4(+) and cd8(+) t cells in human immunodeficiency virus-infected subjects a phase ii randomized study of hiv-specific t-cell gene therapy in subjects with undetectable plasma viremia on combination antiretroviral therapy antiviral effects of autologous cd4 t cells genetically modified with a conditionally replicating lentiviral vector expressing long antisense to hiv gene editing of ccr5 in autologous cd4 t cells of persons infected with hiv polyomavirus jc-targeted t-cell therapy for progressive multiple leukoencephalopathy in a hematopoietic cell transplantation recipient rapid salvage treatment with virus-specific t cells for therapyresistant disease transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation monoculturederived t lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals prophylactic infusion of cytomegalovirusspecific cytotoxic t lymphocytes stimulated with ad5f35pp65 gene-modified dendritic cells after allogeneic hemopoietic stem cell transplantation multicenter study of banked third-party virusspecific t cells to treat severe viral infections after hematopoietic stem cell transplantation safety and clinical efficacy of rapidly-generated trivirus-directed t cells as treatment for adenovirus, ebv, and cmv infections after allogeneic hematopoietic stem cell transplant donor-derived cmv-specific t cells reduce the requirement for cmv-directed pharmacotherapy after allogeneic stem cell transplantation activity of broad-spectrum t cells as treatment for adv, ebv, cmv, bkv, and hhv6 infections after hsct targeted t-cell therapy in stage iv breast cancer: a phase i clinical trial phase i study of anti-cd3 x anti-her2 bispecific antibody in metastatic castrate resistance prostate cancer patients anti-cd3 x anti-egfr bispecific antibody redirects t cell cytolytic activity to egfr-positive cancers in vitro and in an animal model t cells armed with anti-cd3 x anti-cd20 bispecific antibody enhance killing of cd20+ malignant b-cells and bypass complement-mediated rituximab-resistance in vitro cd20-targeted t cells after stem cell transplantation for high risk and refractory non-hodgkin's lymphoma multiple infusions of cd20-targeted t cells and low-dose il-2 after sct for high-risk non-hodgkin's lymphoma: a pilot study targeting cytomegalovirus-infected cells using t cells armed with anti-cd3× anti-cmv bispecific antibody accelerated production of antigen-specific t cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (g-rex) t-cell therapies for hiv simultaneous zinc-finger nuclease editing of the hiv coreceptors ccr5 and cxcr4 protects cd4+ t cells from hiv-1 infection human papillomavirus type 16 e6/e7-specific cytotoxic t lymphocytes for adoptive immunotherapy of hpv-associated malignancies adverse events following infusion of t cells for adoptive immunotherapy: a 10-year experience human mhc class i-restricted high avidity cd4 t cells generated by co-transfer of tcr and cd8 mediate efficient tumor rejection in vivo cord blood t cells retain early differentiation phenotype suitable for immunotherapy after tcr gene transfer to confer ebv specificity beta-glucan recognition by the innate immune system immunity to fungal infections current challenges in the diagnosis and management of invasive fungal infections: report from the 15th international symposium on infections in the immunocompromised host generation of highly purified and functionally active human th1 cells against aspergillus fumigatus generation of a multipathogen-specific t-cell product for adoptive immunotherapy based on activation-dependent expression of cd154 clinicalscale generation of multi-specific anti-fungal t cells targeting candida, aspergillus and mucormycetes clinical-scale generation of human anti-aspergillus t cells for adoptive immunotherapy robust polyfunctional t-helper 1 responses to multiple fungal antigens from a cell population generated using an environmental strain of aspergillus fumigatus generation and characterization of anti-candida t cells as potential immunotherapy in patients with candida infection after allogeneic hematopoietic stem-cell transplant cross-protective th1 immunity against aspergillus fumigatus and candida albicans characterization of the t-cell-mediated immune response against the aspergillus fumigatus proteins crf1 and catalase 1 in healthy individuals cd4+ and cd8+ t cells mediate adoptive immunity to aerosol infection of mycobacterium bovis bacillus calmette-guerin lowest numbers of primary cd8(+) t cells can reconstitute protective immunity upon adoptive immunotherapy adoptive immunotherapy against experimental visceral leishmaniasis with cd8+ t cells requires the presence of cognate antigen biochemical and immunological characterization of mp65, a major mannoprotein antigen of the opportunistic human pathogen candida albicans characterization of the cellular immune responses to rhizopus oryzae with potential impact on immunotherapeutic strategies in hematopoietic stem cell transplantation generation of epstein-barr virus-specific cytotoxic t lymphocytes resistant to the immunosuppressive drug tacrolimus (fk506) generation of ebv-specific cytotoxic t cells that are resistant to calcineurin inhibitors for the treatment of posttransplantation lymphoproliferative disease acknowledgments special thanks to the clinical coordinators for dedicating their efforts to serve the immunotherapy patients. we thank manley huang for his thoughtful reading of the chapter. the studies were supported in part by r01 ca140314 (lgl) and r01 ca182526 (lgl), translational grants #6092-09 (lgl) and #6066-06 (lgl) from the leukemia & lymphoma society, and uva cancer center support grant nci 5p30ca044579-24.lgl is a founder of transtarget, inc. cmb is supported in part by the nichd k12-hd-001399 award to mdk and cprit r01 rp100469 and nci p01 ca148600-02 awards to cmb. key: cord-340489-yo3cp5vs authors: nan title: kapitel 13 infektionskrankheiten date: 2008-12-31 journal: innere medizin doi: 10.1016/b978-3-437-42831-9.10013-0 sha: doc_id: 340489 cord_uid: yo3cp5vs zur orientierung infektionskrankheiten werden durch pathogene verursacht, die sich im wirt vermehren: ektoparasiten, helminthen, protozoen, pilze, bakterien, viren, prionen. infektionskrankheiten können alle organe bzw. organsysteme befallen. entstehung und verlauf werden durch faktoren beeinflusst, die sich grob einteilen lassen in erregerund wirtsfaktoren. die kenntnis und richtige einschätzung dieser faktoren sind entscheidend für diagnostik und therapie dieser erkrankungen. oft ist die unterscheidung zwischen infektiösen und nichtinfektiösen erkrankungen mit einer entzündlichen komponente schwer. andererseits können z. b. bei patienten mit immundefekten symptome oder zeichen trotz bestehender infektion fehlen. hautveränderungen als symptom viele infektionskrankheiten zeigen eine mitbeteiligung der haut, mit fokalen läsionen bei bakterieller endokarditis oder als exanthem bzw. enanthem bei viruserkrankungen. hautveränderungen können pathognomonisch sein, so bei meningokokkensepsis und den damit verbundenen petechialen blutungen und später großflächigen ekchymosen. hautveränderungen, auch im zeitlichen verlauf, können wichtige differentialdiagnostische hinweise liefern. der erste schritt ist die beurteilung des klinischen zustandes (› abb. 13.1). bei kritisch kranken patienten muss zunächst parallel zu supportiven maßnahmen eine rasche empirische antiinfektive therapie erwogen und ggf. begonnen werden. die diagnostik kann aber meist ohne zeitverlust in den ablauf integriert werden. infektionskrankheiten, die einen kritischen zustand eines nicht immundefizienten patienten verursachen, sind vor allem bakterielle sepsis, meningitis und bakterielle pneumonie, aber auch malaria tropica. anamnese hier sind mögliche erregerexpositionen, der zeitliche ablauf der erkrankung und die prädisposition des wirts zu beachten. • nicht für alle infektionen ist eine besondere exposition eruierbar (› tab. 13 .2). kontakte zu anderen erkrankten, reiseanamnese, nahrungsaufnahme, berufsanamnese, freizeitbeschäftigungen, tierkontakte inklusive insektenstiche, vorherige erkrankungen und deren therapie, medikamenten-, drogen-sowie sexualanamnese müssen berücksichtigt werden. • der zeitliche ablauf der krankheitsentwicklung muss geklärt werden. nahezu alle infektionserreger haben charakteristische zeitintervalle zwischen exposition und erkrankung (inkubationszeiten). so sind bakterielle erreger durch inkubationszeiten von einigen tagen gekennzeichnet, während viruserkrankungen meist inkubationszeiten von einigen wochen haben (viele ausnahmen!). zusätzlich treten bei vielen erregern saisonale häufungen auf. • die prädisposition des wirts als dritte komponente umfasst die frühere anamnese (speziell infektionen, vorgenommene impfungen), andere erkrankungen oder organschädigungen und die beurteilung des klinischen status, z. b. die integrität von haut und schleimhäuten. spezifische untersuchungen blutkulturen müssen bei allen kritisch kranken, bei allen systemisch kranken und/oder fiebernden patienten entnommen werden. hierbei sind eine rasche und frühe entnahme (vor antibiotikagabe), ausreichende blutmenge pro flasche (mind. 5-8 ml je nach system), eine ausreichende zahl von blutkulturen (mindestens zwei, je als aerob-anaerobes paar von blutkulturen) und sterile abnahme wichtig. die weitere diagnostik richtet sich nach den symptomen oder befunden, z. b. sputum-, urin-, abstrichuntersuchungen untersuchungen von organpunktaten. bei allen untersuchungen ist auf entnahmetechnik, aufbewahrung und richtigen transport zu achten. sputum z. b. muss vor der ersten gabe von antibiotika entnommen und innerhalb weniger stunden aufgearbeitet werden, ansonsten sind pathogene nicht mehr nachweisbar. der rationale einsatz von serologischen untersuchungen wie auch die aufbewahrung von serumproben zur späteren untersuchung von initial-und rekonvaleszentenserum kann zur diagnostik sinnvoll sein wie auch spezielle verfahren z. b. polymerasekettenreaktion-untersuchung einer biopsie. wirts-und pathogenitätsfaktoren beeinflussen entstehung und ablauf von infektionskrankheiten. wirtsfaktoren lassen sich einteilen in unspezifische (angeborene) und spezifische (erworbene, › tab. 13.3). zu den unspezifischen faktoren gehören barrieremechanismen von haut und schleimhäuten, aber auch mechanismen, mit denen erreger aktiv bekämpft werden können. spezifische funktionen des immunsystems sind gegen einzelne erreger gerichtet. beide systeme weisen eine vielzahl von interaktionen auf. mit pathogenität wird die eigenschaft eines mikroorganismus bezeichnet, eine erkrankung auslösen zu können. virulenz ist der grad der pathogenität innerhalb einer spezies. notwendige bedingungen für die pathogenität eines erregers sind: 1. in gewebe oder zellen anhaften oder eindringen zu können, 2. im körper zur replikation fähig zu sein. epithelbarrieren und -läsionen haut und schleimhaut haben mehrere barrieren, um ein eindringen von pathogenen zu erschweren oder vermeiden. dazu gehören neben den anatomischen auch chemische barrieren, z. b. die fettschicht der epidermis, aber auch antibakterielle substanzen, wie das in mukosalen sekreten vorhandene lysozym. monozyten und makrophagen, granulozyten und natural-killer(nk)-zellen sind die wichtigsten zellpopulationen dieses systems. die zellen des monozyten-makrophagen-systems und granulozyten weisen moleküle auf, mit denen erregerspezifische strukturen (z. b. lipopolysaccharide, bestimmte dna-sequenzen oder doppelsträngige rna) erkannt werden können. diese strukturen werden als pathogen-assoziierte molekulare pattern (pamp) bezeichnet. ein beispiel für solche rezeptoren sind die nach einer homologie mit einem rezeptor der drosophila-fliege bezeichneten tlr (toll-like-rezeptoren). die bindung von erregerspezifischen strukturen an diese moleküle führt zur raschen aktivierung einer entzündungskaskade. natural-killer-zellen sind lymphozyten, die durch antigen-antikörper-komplexe oder durch zellen, die keine mhc-(major-histocompatibility-complex)-moleküle auf der oberfläche exprimieren, aktiviert werden können. die proteine des komplementsystems können nach aktivierung im blut zirkulierende erreger lysieren. störungen des komplementsystems führen zu infektionen mit polysaccharidbekapselten erregern, z. b. pneumokokken und meningokokken. als akute-phase-reaktion wird die produktion von proteinen und peptiden bezeichnet, die nach einer infektion oder entzündungsreaktion abläuft. involviert sind makrophagen, die vor allem interleukin (il-1), tnf-α, interferon-α, il-6 und eine reihe von prostaglandinen und arachidonsäuremetaboliten produzieren. eine kooperation dieser beiden systeme findet bei jeder lokalen entzündungsreaktion statt. aktivierte granulozyten und makrophagen produzieren chemotaktische substanzen, aktivieren adhäsionsmoleküle in den lokalen endothelien und sorgen so für eine verstärke einwanderung von entzündungszellen (› abb. 13 defekte des unspezifischen immunsystems • verletzungen von epithelien begünstigen die invasion von erregern, z. b. nach zytostatischer therapie mit abschilferung der intestinalen schleimhaut. der aktive partikeltransport des flimmerepithels des respirationstraktes ist bei rauchern und bei patienten mit zystischer fibrose gestört. in beiden fällen ist die rate von infektionen des respirationstraktes deutlich erhöht. • neutropenie oder funktionsstörungen der granulozyten führen zu häufigen bakteriellen erkrankungen und invasiven mykosen. • das fehlen von natural-killer-zellen führt u. a. zu schweren infektionen mit herpesviren. die wesentlichen mechanismen sind die bildung von antikörpern durch b-zellen sowie spezifischer t-effektorzellen. beide zellsysteme haben eine hohe genetische plastizität und können eine hohe zahl spezifischer antigene erkennen. t-lymphozyten repräsentieren die spezifische zelluläre immunität. die wichtigsten funktionellen gruppen sind t-helferzellen und zytotoxische t-zellen. damit antigene durch die effektorzellen erkannt werden können, sind eine prozessierung und präsentierung durch zelluläre mechanismen notwendig. präsentation von antigen bakterielle oder virale proteine werden intrazellulär in oligopeptide zerlegt, und über mhc-(major-histocompatibility-complex)-moleküle auf der zelloberfläche präsentiert. "professionelle" antigenpräsentierende zellen nutzen mhc-klasse-ii-, alle anderen zellen mhc-klasse-i-moleküle. t-lymphozyten der kontakt von "naiven" t-helferzellen mit präsentiertem antigen führt zur aktivierung, reifung und klonalen expansion der betreffenden t-helferzellen. diese stimulieren die bildung von spezifischen zytotoxischen t-zellen und immunglobulinproduzierenden b-zellen. die zentrale rolle in der stimulierung der effektorzellen der spezifischen abwehr hat dieser zellgruppe ihren namen gegeben. die eigentlichen effektorzellen sind die zytotoxischen t-zellen. sie erkennen infizierte zellen durch die kombination der mhc-klasse-i-moleküle mit den erregerpeptiden und töten sie ab. die initale phagozytose von erregern und nachfolgende antigenpräsentation durch makrophagen mit der ausbildung einer spezifischen zellulären immunität durch t-lymphozyten ist ein beispiel für das zusammenwirken unspezifischer und spezifischer immunmechanismen. der kontakt des passenden antigens mit dem noch unreifen oberflächenimmunglobulin auf b-lymphoyzten führt zur ausbildung und selektion von plasmazellen, die entweder iga, ige oder igg mit höherer spezifität und avidität produzieren. immunglobuline können erreger (z. b. viren) und toxine neutralisieren und die phagozytose und lyse von erregern erleichtern. das spezifische immunsystem bildet außerdem ein gedächtnis für vorangegangene infektionen und reagiert mit einer besseren abwehr oder sogar immunität bei reexposition. beim erstkontakt mit dem erreger kommt es nach rascher aktivierung, reifung und expansion von t-und b-lymphozyten auch zur bildung von gedächtniszellen beider gruppen. defekte des spezifischen immunsystems können angeboren (agammaglobulinämie, kombiniertes immundefektsyndrom) und erworben (aids) sein. bei immunglobulinmangel kommt es vor allem zu infektionen mit polysaccharidbekapselten erregern, bei störungen des t-zellulären systems zu schweren infektionen z. b. mit intrazellulären erregern (z. b. toxoplasma gondii und herpesviren). viren sind obligat intrazelluläre erreger, die keinen eigenen stoffwechsel besitzen. zur replikation sind sie auf zelleigene enzyme angewiesen. zwei bedingungen müssen für virale pathogenität erfüllt sein: 1. das virus muss eine oberflächenstruktur besitzen, mit der es an eine zielzelle binden und dann eindringen kann. 2. durch die replikation in der zelle muss entweder eine störung der zellfunktion oder eine immunreaktion auf die infektion erfolgen. ein pathogenitätsmechanismus ist die möglichkeit, eine latente infektion zu erzeugen. so besitzen z. b. humane herpesviren die fähigkeit, das genom in einer inaktiven, aber reaktivierbaren form in zellen einzubauen. die mechanismen, die das gleichgewicht zwischen latenz und produktiver infektion steuern, sind nur unvollständig bekannt. die einfache kultivierung klonaler populationen und die manipulation von umgebungsbedingungen ermöglichen die untersuchung bakterieller pathogenitätsfaktoren. zusätzlich haben sequenzierung von bakteriellen genen und deren gezielte manipulation das wissen über pathogenitätsmechanismen entscheidend vermehrt. genetische regulation von pathogenitätsfaktoren neben der chromosomalen form kann dna als plasmid oder phage vorliegen. diese "mobilen" genetischen elemente erlauben eine genetische diversifizierung. chromosomale gene und externe gene können für pathogenitätsfaktoren kodieren, deren expression einer komplizierten regelung unterliegen kann. adhäsion, toxinbildung und immunevasion von bakterien wichtige pathogene mechanismen von bakterien sind adhäsion an epithelien oder die bildung von toxinen und immunevasion: • pili oder fimbrien bei escherichia coli werden nach dem identifizierten gen-p(ap)-pili genannt. die expression dieser pili wird durch umgebungsbedingungen (ph-wert und temperatur) so moduliert, dass sie vor allem in den ableitenden harnwegen produziert werden, wo sie zur besseren adhäsion führen. • häufig ist die produktion von toxinen -ebenso wie die von adhäsionsmechanismen -an plasmide oder phagen gebunden. zur produktion des diphtherietoxins muss der betreffende stamm mit einem phagen infiziert sein. andere pathogene toxine sind exotoxine von staphylococcus aureus und der gruppe-a-streptokokken, die z. b. für die toxic-shock-syndrome verantwortlich sind. • immunevasionsstrategien richten sich gegen abwehrmechanismen. beispiele: neisseria gonorrhoeae und haemophilus influenzae entziehen sich durch iga-spezifische proteasen der vernichtung auf der schleimhaut. legionella pneumophila kann nach beladung durch komplementproteine leichter in zellen eindringen. vor allem keime mit intrazellulärem vermehrungszyklus haben oft die fähigkeit, der phagozytose durch makrophagen zu entgehen. • das patientengut ändert sich mit einem ständig steigenden anteil an intensivpflegepatienten und abwehrgeschwächten patienten. • das repertoire an zur verfügung stehenden chemotherapeutika wird immer größer. • die resistenz von bakterien gegen antibakterielle chemotherapeutika nimmt sowohl quantitativ als auch qualitativ zu. jeder arzt, der eine antibakterielle chemotherapie durchführen will, muss wichtige grundprinzipien beherrschen wie auch in wesentlichen zügen das spektrum der antibakteriellen substanzen kennen. indikationsstellung antibakterielle chemotherapeutika sind ursächlich wirksame medikamente und nicht primär gegen symptome, wie z. b. fieber, gerichtet. die gabe solcher substanzen setzt also eine exakte indikationsstellung voraus, es muss mit sehr hoher wahr-scheinlichkeit eine durch bakterien verursachte infektionskrankheit vorliegen. die indikation wird naturgemäß zunächst klinisch gestellt. hierfür genügen in aller regel anamnese, befunde der klinischen untersuchung sowie einige klinisch-chemische und radiologische zusatzbefunde. gleichzeitig erfolgt die materialentnahme zur mikrobiologischen erregerdiagnose, um dadurch die indikation abzusichern. die therapie wird meist vor erhalt der endgültigen erregerdiagnose und des antibiogramms begonnen. man spricht dann von einer kalkulierten chemotherapie, d.h., es wird eine empirische therapie nur auf basis der klinischen befunde eingeleitet. hieraus sollte es in vielen fällen schon möglich sein, die zu erwartenden erreger einzugrenzen, aber auch die zu erwartende resistenzsituation sowohl generell als auch lokal zu kalkulieren. wenn dann mikrobiologische befunde -erregerdiagnose und antibiogramm -vorliegen, die mit der klinik korrelierbar sind, kann eine gezielte chemotherapie durchgeführt werden. das heißt, die kalkulierte chemotherapie muss überprüft und evtl. geändert werden. für die auswahl der chemotherapeutika müssen klinische, mikrobiologische und pharmakokinetische kriterien herangezogen werden. von seiten der klinik sind eventuelle grundkrankheiten zu berücksichtigen, ferner die infektionslokalisation und die tatsache, ob es sich um eine außerhalb (ambulant) oder innerhalb des krankenhauses (nosokomial) erworbene infektionskrankheit handelt. die zu beachtenden bakteriologischen kriterien betreffen das wirkspektrum der jeweiligen antibiotika und deren aktivität innerhalb dieses spektrums. entscheidend ist auch, ob der wirkeffekt bakterizid (keimabtötend) oder nur bakteriostatisch (proliferationshemmend) ist. eine ganze reihe von pharmakokinetischen eigenschaften der jeweiligen substanzen wie säurestabilität, enterale resorption, art der metabolisierung bzw. elimination, penetration in körperkompartimente und -gewebe beeinflusst ebenfalls in der individuellen klinischen situation die festlegung des chemotherapeutikaregimes. nicht zuletzt spielen toxikologische gesichtspunkte (s. u.) eine wichtige rolle. die durchführung der chemotherapie folgt im prinzip den allgemeinen grundsätzen der internistischen pharmakotherapie (› kap. 3), hier nur die zusätzlich besonderen aspekte der antibiotikatherapie. dosierung die dosis des chemotherapeutikums muss ausreichend hoch sein, um den gewünschten wirkeffekt sicher zu erreichen. dauer es lassen sich keine allgemein gültigen regeln aufstellen. so können unkomplizierte harnwegsinfektionen mit einer einmalgabe eines potenten antibiotikums behandelt werden, während für die therapie einer osteomyelitis eine mehrmonatige therapiedauer erforderlich sein kann. applikationsart die parenterale applikation stellt grundsätzlich den sichersten applikationsweg dar. bei schweren und schwersten infektionsverläufen ist daher dieser weg zumindest bei beginn der therapie immer zu wählen. bei einer umstellung von einer parenteralen auf eine orale therapie (= sequentialtherapie) ist darauf zu achten, ob dies mit der parenteral begonnenen substanz überhaupt möglich ist, d.h., ob sie enteral resorbierbar ist. eine orale folgetherapie mit einem anderen antibiotikum kann nur dann erfolgen, wenn es das gleiche spektrum wie das zuvor verwandte parenterale antibiotikum hat. für die orale chemotherapie ist die compliance des patienten entscheidend. applikationsintervall die pharmakodynamischen eigenschaften (= beziehung von serumspiegel, halbwertszeit und wirkaktivität gemäß minimaler hemmkonzentration) eines antibiotikums entscheiden über einmalgabe, mehrfachgabe oder dauergabe. die kombinationstherapie mit zwei oder mehreren substanzen hat in der kalkulierten chemotherapie zum ziel, ein breiteres spektrum möglicher erreger abzudecken. in der gezielten chemotherapie kann eine synergistische wirkung angestrebt werden, erwiesenermaßen sinnvoll nur für die gabe von β-lactam-antibiotika bzw. glykopeptidantibiotika mit aminoglykosiden bei grampositiven kokken als häufigste angewandte kombination. weitere gründe für die gabe einer kombination liegen vor, wenn die dosiserhöhung einer substanz aus toxikologischen gründen nicht mehr möglich ist oder bei einer mischinfektion mehrere erreger therapiert werden müssen. für die kombinierbarkeit verschiedener antibiotika gibt es keine verbindlichen regeln. "drug-monitoring" antibiotika, die bei nierenfunktionsstörungen schnell kumulieren können, wie aminoglykoside und vancomycin, müssen dann gemäß serumspiegelkontrolle dosiert werden. bezüglich allergischer und toxischer nebenwirkungen unterscheiden sich antibiotika nicht von anderen substanzen. je nach spektrum der potenziellen nebenwirkungen, das für die einzelnen chemotherapeutika sehr unterschiedlich sein kann, müssen entsprechende klinische bzw. laborchemische oder auch funktionelle kontrollen erfolgen. die besonderheiten einer antibakteriellen chemotherapie liegen darin, dass sog. biologische nebenwirkungen (folge der hauptwirkung) auftreten können: • selektion resistenter bakterien. das versagen einer antibakteriellen chemotherapie kann mehrere gründe haben. die häufigste ursache ist die primäre oder sekundäre resistenz der verursachenden bakterien. primäre resistenz bedeutet, dass alle bakterien z. b. einer spezies oder gattung gegenüber einem bestimmten antibiotikum von natur aus resistent sind. sekundäre resistenz beinhaltet, dass ein klon einer primär empfindlichen spezies durch mutation oder akquirierung eines resistenzgens (z. b. auf einem plasmid) resistent wird. unter der persistenz eines erregers versteht man das überleben des erregers am infektionsort während einer antibiotikatherapie. hierzu kommt es, wenn der erreger vorübergehend von der wachstumsphase in eine ruhephase übertritt, z. b. bedingt durch verschiedene physikalisch-chemische ursachen am infektionsort. da die meisten gebräuchlichen chemothera-peutika nur auf proliferierende keime wirken, werden sie nicht eliminiert und können daher nach absetzen der antibiotikatherapie zum rezidiv führen. die ineffektivität einer antibiotikatherapie kann natürlich auch durch einen wechsel des ätiologisch bedeutsamen erregers während der therapie bedingt sein, aber ebenso durch fehler in der durchführung der chemotherapie (s. o.). die prophylaktische gabe von antibakteriellen chemotherapeutika hat nur wenige, eingeschränkte indikationsgebiete! hierzu gehört z. b. die perioperative antibiotikagabe zur verhinderung von postoperativen wundinfektionen und septikämien, deren sinn bei bestimmten operativen eingriffen erwiesen ist. in seltenen fällen kann eine expositionsprophylaxe mit antibiotika durchgeführt werden, akzeptiert sind hier die pertussis-und die meningokokkenmeningitis-prophylaxe bei besonders gefährdeten personen, wenn in deren umgebung ein erkrankungsfall aufgetreten ist. heute steht eine große anzahl von antibakteriellen chemotherapeutika aus verschiedensten substanzgruppen zur klinischpraktischen anwendung zur verfügung. › tabelle 13.4 gibt einen orientierenden überblick über die unterschiedlichen substanzgruppen mit beispielen von einzelsubstanzen. hieraus lassen sich in geraffter form das antibakterielle wirkspektrum, wichtige pharmakologische eigenschaften und bedeutende potenzielle nebenwirkungen ablesen. diese klassifizierung folgt klinischen anwendungsgesichtspunkten und nur z. t. der exakten chemischen einteilung. wegen der großen anzahl der zur verfügung stehenden chemotherapeutika musste dabei eine auswahl erfolgen, die sich an deren praktischer bedeutung orientiert. der infektiologisch nicht spezialisierte arzt sollte sich auf ein standardrepertoire von wenigen substanzen beschränken, bei deren therapeutischem einsatz er dann eigene erfahrungen gewinnt. z u s a m m e n f a s s u n g • es steht außer zweifel, dass eine antivirale chemotherapie möglich und die gabe mancher substanzen in bestimmten klinischen situationen bereits absolut indiziert ist. • eine vielzahl von substanzen wird schon seit jahrzehnten regelmäßig in vielen laboratorien auf antivirale wirkung geprüft. aids hat zur verstärkten suche und für erheblich mehr publizität gesorgt. die wirkprinzipien werden im labor und z. t. bereits in klinischen studien anhand vieler substanzen unterschiedlicher chemischer natur untersucht. in der tat stößt z. b. die planung ausreichend kontrollierter studien zur antiviralen therapie bei aids selbst in den usa bereits auf das problem des mangels an studienfähigen patienten. th. mertens die künftigen entwicklungen in der antiviralen chemotherapie betreffen sowohl die charakterisierung neuer ziele (engl.: targets) für antivirale interventionen und die entwicklung antiviral wirksamer substanzen als auch die durch studien begründbaren empfehlungen für die anwendung vorhandener therapeutika. viren unterscheiden sich hinsichtlich struktur und vermehrungsweise grundsätzlich von allen anderen infektionserregern. sie sind für ihre vermehrung vollständig auf die energiegewinnung und syntheseleistung ihrer wirtszelle angewiesen. voraussetzung für eine antivirale chemotherapie ist somit, dass moleküle und biochemische prozesse identifiziert werden, die nur in virusinfizierten zellen vorkommen. der begriff virusselektivität beschreibt die fähigkeit einer substanz, die virusvermehrung zu hemmen, ohne die wirtszelle zu schädigen. viele viren haben strategien entwickelt, um im einmal infizierten organismus zu persistieren. in diesen fällen kann es zu infektionszuständen ohne virusvermehrung kommen (z. b. latenz der herpesviren). da alle bislang verfügbaren antiviralen substanzen im vermehrungszyklus angreifen, entziehen sich persistierende infektionen ohne virusvermehrung derzeit einer antiviralen therapie. die verfügbaren therapeutika haben ein begrenztes wirkspektrum, und es gibt bislang keine "breitbandmedikamente". antivirale therapie setzt somit eine virustypdiagnose voraus. bei akuten viruserkrankungen entscheidet darüber hinaus ein frühzeitiger therapiebeginn über den erfolg, was eine rasche diagnosestellung notwendig macht. bei patienten mit schwerster angeborener (scid), erworbener (aids) oder iatrogener (transplantation) immundefizienz ist es häufig trotz adäquater antiviraler therapie nicht möglich, die virusvermehrung zu beenden, solange es nicht zu einer verbesserung der immunsituation kommt. therapieindikation, -beginn und -dauer allgemeine regeln zur therapie sollten nur auf der grundlage klinischer studien festlegt werden (evidence-based). wesentliche allgemeine kriterien, die berücksichtigt werden müssen, sind die immunsituation des patienten, das risiko schwerer erkrankung, folgeerkrankung oder chronifizierung, die vermeidung unerwünschter arzneimittelwirkungen (uaw) und die vermeidung von resistenzentwicklung. die probleme mögen folgende fragen verdeutlichen, deren antworten z. t. in letzter zeit gegeben wurden (s. u.): muss eine akute hepatitis b oder c therapiert werden? welches ist der optimale zeitpunkt zum beginn einer antiretroviralen therapie bei hiv-infektion? wie soll man hiv-infizierte schwangere behandeln? müssen windpockenerkrankungen oder eine gingivostomatitis herpetica behandelt werden? bei schwerst immunsupprimierten wird zur verhinderung lebensbedrohlicher virusinfektionen vielfach eine antivirale prophylaxe durchgeführt. diese medikamentengabe vor beginn einer aktiven infektion führt natürlich bei etlichen patienten zur unnötigen gabe teils toxischer substanzen. der begriff der präemptiven therapie bezeichnet eine antivirale behandlung nach virologischer diagnose einer aktiven infektion ohne vorliegen von symptomen und ist abzugrenzen von der therapie einer infektionskrankheit. die therapieentscheidung erfordert den nachweis des vorteils einer vorgehensweise für die jeweilige patientengruppe. die optimale dauer der therapie ist in vielen fällen noch nicht durch studien bestimmt worden. resistenzvermittelnde mutationen treten bei jeder virusvermehrung spontan auf. bei längerfristiger anwendung antiviraler chemotherapeutika und damit vor allem bei den erheblich immunsupprimierten patienten mit langdauernder massiver virusvermehrung kann es dann relativ rasch zur selektion resistenter viruspopulationen kommen. diese virusvarianten besitzen mutationen in der viralen polymerase (rt bei hiv), der viralen kinase (herpesviren) oder anderen viralen genen, welche für zielstrukturen der antiviralen substanzen kodieren. voraussetzung für die selektion der spontan auftretenden resistenten virusvarianten ist somit virusvermehrung unter dem selektionsdruck einer antiviralen substanz. klinisch relevante virusresistenzen gegen nukleosidanaloga treten nach bisherigen erfahrungen bei kurzzeitiger therapie bzw. therapie immungesunder patienten nicht auf. vielmehr ist resistenz meist ein problem der langzeittherapie (> 1-2 monate), wenn es nicht gelingt, die produktive virusinfektion durch die therapie zu stoppen (hsv, cmv, hiv). resistenz-vermittelnde mutationen bedingen manchmal einen vermehrungsnachteil für das mutierte virus gegenüber dem wildtyp, wenn der selektionsdruck entfällt. in einigen fällen sind resistente virusvarianten (herpesviren, hiv) weniger pathogen als die wildviren. dies ist aber leider keinesfalls die regel. aufgabe der kliniker ist es, therapieversagen anhand klarer kriterien zu definieren, und den virologen obliegt es, standardisierte tests zur raschen phänotypischen oder genotypischen resistenztestung von viren bereitzustellen. manche zunächst wenig gravierende oder asymptomatische virusinfektionen können folgeerkrankungen auslösen (z. b. immunpathogenese), bei denen die viren dann keine entscheidende rolle mehr spielen und somit eine antivirale therapie zu spät kommt. ansatzpunkte für antivirale substanzen › tabelle 13.5 zeigt, dass die hemmung der virusvermehrung doch an vielen stellen möglich ist. diese frühesten vorgänge bei jeder virusinfektion, die bindung der viren an ihre wirtszelle und bei umhüllten viren die fusion mit der äußeren wirtszellmembran, lassen sich experimentell durch blockade der verantwortlichen rezeptorstrukturen auf seiten der viren oder der zellen hemmen. auch spezifische antikörper wirken auf dieser stufe der infektion. die molekularen vorgänge bei diesen frühen prozessen der infektion sind äußerst komplex und erfordern z. b. bei hiv etliche regulierte strukturelle veränderungen des viralen rezeptors. die hemmung der fusion ist bei hiv durch peptidische fusionsinhibitoren (t20), die an rezeptorstrukturen des virus binden, bereits möglich. auch die hemmung der korezeptorbindung ist denkbar. grund eines durch die strukturproteine gebildeten tiefen oberflächeneinschnittes (canyon) einlagern. dadurch wird die bindung an den zellrezeptor behindert und die zur freigabe der nukleinsäure notwendige, bei einigen virustypen ph-abhängige endosomale, intrazelluläre desintegration der viralen proteinhülle verhindert. bei einigen picornaviren wird die rezeptorbindung wenig behindert, aber das viruskapsid doch so stabilisiert, dass kein uncoating stattfinden kann. es gibt resistenz gegen diese substanzen, aber erstaunlicherweise sogar virusmutanten, die nur noch in anwesenheit dieser substanzen vermehrungsfähig sind. die faszination dieser entdeckung bestand auch darin, dass es plötzlich möglich wurde, aufgrund der kenntnis der molekularen struktur-wirkungs-beziehung antiviral wirksame moleküle sozusagen am reißbrett zu entwerfen (› tab. 13.5) . therapeutisch einsetzbare substanzen erstes und bislang einziges für die systemische therapie verfügbares medikament, das nach diesem mechanismus die picornavirus-replikation hemmt, ist pleconaril. einer der beiden wirkmechanismen von adamantanderivaten gegen influenza-a-viren beruht ebenfalls auf einer hemmung der freisetzung des ribonukleoproteins aus intrazytoplasmatischen vesikeln und des uncoatings durch blockade eines m2-virusprotein-abhängigen ionenkanals. therapeutisch einsetzbare substanzen amantadin und rimantadin stehen seit vielen jahren für die systemische therapie von influenza-a-virus-infektionen zur verfügung. die biologische besonderheit der retroviren besteht darin, dass, beginnend mit dem eindringen des viruscores (nukleokapsid) in die zelle und weiter nach der freisetzung des diploiden einzelsträngigen viralen rna-genoms, zuerst eine doppelsträngige dna hergestellt werden muss. dies geschieht in einem komplexen syntheseprozess über den zwischenzustand eines rna-dna-hybridmoleküls. zwei moleküle der hierfür notwendigen reversen transkriptase (rt) werden bei hiv im viruspartikel mitgebracht. neben der polymerasefunktion besitzt die rt in einer zweiten domäne noch eine enzymatische rnase-h-aktivität, die für die entfernung des rna-stranges vom rna-dna-hybridmolekül erforderlich ist. diese doppelsträngige dna-kopie der viralen rna wird danach als sog. provirales genom kovalent in das wirtszellgenom integriert. hierfür ist ebenfalls ein viruspartikelassoziiertes enzym, die integrase, erforderlich. substanzen zur hemmung der integration der proviralen dna eines retrovirus in das wirtszellgenom (hiv) befinden sich in der klinischen prüfung (integrasehemmer) und werden künftig möglicherweise ein weiteres standbein der antiretroviralen therapie bilden. therapeutisch einsetzbare substanzen die reverse transkriptase (rt) von hiv ist das zielmolekül für die meisten antiretroviralen medikamente. diese werden nach ihrer chemischen struktur unterteilt in nukleosidanaloge rt-hemmer und nicht nukleosidanaloge rt-hemmer (nnrti). eingriffsmöglichkeiten in virusspezifische funktionen ergeben sich während der transkription der viralen genetischen information. vorwiegend typ-i-interferone (ifn) hemmen die replikation verschiedener viren in unterschiedlichem ausmaß, wobei einer der vielfältigen mechanismen (s. u.) in der degradation viraler mrna besteht. einer der antiviralen wirkmechanismen von ribavirin beruht auf der hemmung der mrna einiger viren. therapeutisch einsetzbare substanzen verschiedene humane αund β-interferone stehen für die systemische therapie (chronische hepatitis-b-virus-und hepatitis-c-virus-infektion) und auch topische therapie (papillomaviren) zur verfügung. das relativ breit wirksame nukleosidanalogon ribavirin wird als kombinationstherapeutikum bei chronischer hcv-infektion und zur monotherapie bei rsv-und parainfluenzavirusinfektionen schwer kranker kinder, aber auch bei lassavirusinfektionen eingesetzt. abhängig von der art des vom virus in die wirtszelle eingeschleusten genoms (einzelstrang-rna/dna, doppelstrang-rna/dna) und des zur virusvermehrung erforderlichen genetischen informationsflusses bedarf es besonderer enzyme, die entweder vom virus -im partikel verpackt -mitgebracht (viruspartikelassoziiert, s. o. bei hiv) oder in der infizierten zelle synthetisiert werden (viruskodiert). beispiel für ein viruskodiertes enzym, welches in uninfizierten zellen nicht vorkommt, ist die rna-abhängige rna-polymerase der picornaviren, deren hemmung die wirkung mancher substanzen erklärt (2-[α-hydroxybenzyl]-benzimidazol, enviroxime). viele andere viruskodierte polymerasen von dna-viren unterscheiden sich von zellulären isoenzymen hinsichtlich der akzeptanz und bindung von nukleosidanaloga so weit, dass virusselektive nukleosidanaloga möglich sind. therapeutisch einsetzbare substanzen die vier derzeit gegen herpesviren einsetzbaren nukleosidanaloga, das na-phosphonat cidofovir und auch das pyrophosphatanalogon foscarnet hemmen letztlich alle die viruskodierten polymerasen. die blockierung viraler mrna durch kurze synthetische "antisense"-oligonukleotide ist eine vom konzept her sehr elegante und naturgemäß spezifische möglichkeit der hemmung der viruskodierten proteinsynthese. probleme bereiten die auswahl der geeigneten sequenzen und die chemische modifikation der oligonukleotide, die die aufnahme in die zelle ermöglichen müssen und die stabilisierung in der zelle bei erhaltener wirksamkeit sicherstellen müssen. interferone können die translationsinitiation hemmen. therapeutisch einsetzbare substanzen eingang in die therapie der zytomegalievirusretinitis hat ein intraokulär zu applizierendes antisense-phosphothioat gefunden. zur topischen therapie von herpes-simplex-virus-infektionen stehen ältere nukleosidanaloga zur verfügung mit geringerer virusselektivität. bekanntestes beispiel sind die proteasehemmer zur kombinationstherapie der hiv-infektion. der wirkmechanismus dieser substanzen beruht auf der hemmung der posttranslationalen spaltung der retroviralen gag-(gruppenspezifisches antigen) und gag-pro-pol-polyproteine durch hemmung der homodimeren "aspartatprotease" des virus. es werden unreife, nichtinfektiöse viruspartikel gebildet. auch andere posttranslational nötige modifikationen viraler proteine, z. b. glykosylierung, könnten ein ziel antiviraler substanzen sein. an dieser stelle im replikationszyklus greifen die neuen neuraminidasehemmer ein, die in der lage sind, die korrekte ausschleusung von influenzaviren zu inhibieren. während ihrer untersuchungen zur bereits bekannten interferenz von virusinfektionen entdeckten alick issacs und jean lindenmann 1957 einen übertragbaren virushemmenden faktor, den sie interferon nannten. in einem schlüsselexperiment stellten sie fest, dass zellen, die mit uv-inaktivierten influenzaviren behandelt worden waren, etwas in das gewebekulturmedium abgaben, das die infektion weiterer zellen verhinder-te. in den folgenden 50 jahren der erforschung dieses phänomens wurde ein gewaltiges netzwerk von interaktoren und interaktionen aufgedeckt. trotz vieler erkenntnisse besteht auch heute noch längst kein vollständiges bild aller zusammenhänge und wirkungen. die antivirale wirkung ist dabei nur eine von vielen, und sie ist eine folge regulatorischer funktionen der interferone in der zelle. als folge des schrittweisen erkenntniszuwachses ist auch die nomenklatur schwierig und teilweise redundant. ifn gehören nach heutiger nomenklatur zu den zytokinen. zur unterscheidung der interferone gibt › tabelle 13.6 eine übersicht. alle ifn sind relativ kleine moleküle, die in ihrer reifen form aus 165-172 aminosäuren bestehen. für die antivirale therapie spielen derzeit nur die typ-i-ifn, und hier die α-ifn, eine wesentliche rolle. die induktion kann auf verschiedenen wegen erfolgen. virusinfektionen, vor allem durch rna-viren, führen zur raschen induktion der ifn, die nach wenigen stunden wieder beendet wird. die induktion erfolgt über mehrere positiv regulatorische domänen, aber auch durch negative regulation, die zur verminderung der repressorproteine und einer gesteigerten ifn-gen-expression führt. die ifn werden von den zellen, in denen sie gebildet wurden, freigesetzt. natürliche α-ifn werden von lymphozyten, monozyten, makrophagen und einigen zelllinien gebildet. quelle für β-ifn sind fibroblasten und einige epitheliale zellen. mittlerweile werden die zur antiviralen therapie eingesetzten ifn meist als rekombinante ifn gentechnisch hergestellt. die ifn binden über spezifische zellrezeptoren an die zellen, in denen sie ihre antivirale und andere wirkungen entfalten. diese ifn-rezeptoren sind bekannt, und ihre expression unterliegt wiederum regulatorischen prozessen. viele (> 100) proteine werden durch ifn in den zellen reguliert, von denen etliche auch in die antivirale wirkung eingebunden sind. die stimulation der no-synthetase führt zur no-bildung und damit zu einer eher unspezifischen antiviralen wirkung hemmung der virusreifung ifn aktivieren eine gykosyltransferase, wodurch einerseits die notwendige posttranslationale modifikation mancher viraler proteine gehemmt und andererseits der normale ausschleusungsprozess (budding) behindert wird. bei systemischer anwendung gibt es z. t. erhebliche nebenwirkungen: neben den erwähnten zielen antiviraler therapeutika sind viele weitere denkbar. hinzuweisen ist in diesem zusammenhang auf die möglichkeit der hemmung regulatorischer proteine (z. b. tat oder rev bei hiv) oder auf die hemmung der genomreifung bei herpesviren und die genomverpackung bei im kern replizierenden umhüllten viren. die substanz wird parenteral und oral eingesetzt, auch prophylaktisch bei immunsuppression. die orale bioverfügbarkeit von acv ist allerdings nicht gut und unsicher, so dass bei schweren erkrankungen immer eine i.v. therapie angezeigt ist. die therapeutisch wirksame dosierung liegt bei dem wesentlich langsamer replizierenden vzv deutlich höher als bei hsv, was möglicherweise an der kurzen intrazellulären halbwertszeit von acv-triphosphat liegt. valaciclovir (valacv) ist ein valinester des acv. die substanz wird oral wesentlich besser resorbiert, so dass eine orale bioverfügbarkeit von ca. 60% erreicht wird. valacv wird bei der resorption und bei der ersten leberpassage praktisch vollständig in die wirksame substanz acv umgewandelt. mit einführung des valacv ist es möglich geworden, acv-serumkonzentrationen durch orale valacv-gabe zu erhalten, die ansonsten nur durch i.v. applikation von acv erreicht werden können. resistenzentwicklung acv-resistente hsv-mutanten mit mutationen/deletionen im thymidinkinase(tk)-gen und/ oder polymerase-gen können isoliert werden und sind bei immunsupprimierten u.u. klinisch relevant. tk-minus-mutanten sind nicht neuropathogen, allerdings sind resistente hsv durchaus nicht immer tk-minus-mutanten. in der regel führen tk-mutationen zu kreuzresistenz gegenüber anderen nukleosidanaloga, die durch die tk aktiviert werden müssen (s. u. und ganciclovir). resistenz aufgrund von polymerasemutationen ist seltener, führt aber meist zu breiter kreuzresistenz gegenüber nukleosidanaloga (s. u.). bvdu wird ebenfalls selektiv durch die viralen thymidinkinasen von hsv-1 und vzv zu monophosphat und diphosphat phosphoryliert. die phosphorylierung zu diphosphat kann von der hsv-2-tk nicht geleistet werden, da deren thymidylatkinase-aktivität wesentlich geringer ist, was die geringe wirksamkeit von bvdu gegenüber hsv-2 erklärt. die wirksamkeit von bvdu bei vzv-infektionen (varizellen und zoster) immunkompromittierter patienten ist durchaus sehr gut und vergleichbar der von i.v. verabreichtem aciclovir, jedoch fällt die nutzen-risiko-betrachtung insgesamt auch bei vzv-therapie zu gunsten von aciclovir aus, da bvdu eher mutagen zu sein scheint und nicht zusammen mit 5-fluorouracil (zytostatikum) gegeben werden darf. penciclovir (pcv) pcv ist strukturell dem ganciclovir sehr ähnlich und ebenfalls oral sehr schlecht resorbierbar im gegensatz zu famciclovir (fcv), einer oral sehr gut resorbierbaren substanz (bioverfügbarkeit bis > 70%). nach resorption wird fcv rasch und vollständig in pcv, das wirksame pro-drug, umgewandelt. pcv wird ähnlich wie acv durch die thymidinkinasen von hsv und vzv phosphoryliert. wesentliche indikationen genitale hsv-primärinfektionen sollten möglichst frühzeitig systemisch behandelt werden, auch mit dem ziel, möglicherweise die spätere rezidivhäufigkeit zu verringern. ähnliches gilt auch für die primäre gingivostomatitis herpetica. rekurrierende mukokutane hsv-infektionen können je nach beschwerden und beeinträchtigung, vor allem bei genitalen manifestationen (u.u. patientengesteuert), systemisch behandelt werden. in besonderen fällen mit häufigen genitalen rekurrenzen (6-10 pro jahr) oder mit schwerer psychischer beeinträchtigung kann eine suppressionsbehandlung durchgeführt werden. nach absetzen der therapie treten erneut rekurrenzen auf. topisch kann mit verschiedenen nukleosidanaloga (s. o.) behandelt werden. bei hautmanifestationen ist zwischenzeitliches betupfen der läsionen mit äther oder alkohol sinnvoll. bei schweren systemischen infektionen (enzephalitis, neugeborenensepsis) mit hsv oder vzv oder bei infektionen erheblich immunsupprimierter mit diesen viren muss mit einer sofortigen i.v. acv-therapie begonnen werden. das behandlungsergebnis hängt entscheidend von einem frühzeitigen behandlungsbeginn ab. liegt zum zeitpunkt der geburt eine hsv-infektion im geburtskanal der mutter vor, so besteht die gefahr einer konnatalen infektion des neugeborenen mit der folge einer unbehandelt oft tödlich verlaufenden hsv-sepsis (› kap. 13.5.1). in diesen fällen kann nach heutiger kenntnis eine therapie der schwangeren zur vermeidung einer schnittentbindung durchaus erwogen werden. ebenso muss die möglichkeit der antiviralen therapie in das optimale management der perinatalen vzv-infektion einbezogen werden. bei vzv-exposition eines seronegativen immunsupprimierten patienten (meist kinder) ist die sofortige gabe eines varizellen-hyperimmunglobulins (0,2 ml/kg körpergewicht) indiziert, ggf. in kombination mit antiviraler therapie. bei vzv-manifestationen bei immunsupprimierten patienten (auch zoster) ist eine antivirale therapie indiziert. es gibt durchaus gute argumente für eine generelle antivirale therapie bei varizellen, jedoch wird sie in der praxis kaum durchgeführt. in den letzten jahren hat sich gezeigt, dass acv bei cmv-erkrankungen therapeutisch nicht einsetzbar ist, dass jedoch bei prophylaktischer gabe an erheblich immunsupprimierte transplantatempfänger auch eine gewisse prophylaktische wirkung gegen cmv-erkrankungen nach endogener reaktivierung vorhanden ist. die substanz kann nur i.v. und topisch angewendet werden. pfa wird systemisch bei cmv-erkrankungen immunsupprimierter eingesetzt, wenn eine gcv-resistenz vorliegt, und stellt dann eine alternative zu cidofovir dar. pfa kann topisch erfolgreich bei herpes labialis und genitalis eingesetzt werden. bei den anderen humanen herpesviren sind z. t. gute effekte der oben genannten substanzen in vitro beschrieben worden. in klinischen studien konnte durch anwendung von acv bei ebv-infektionen auch die virusausscheidung deutlich vermindert werden, ein wesentlicher einfluss auf den krankheitsverlauf ließ sich nicht erreichen. dies gilt ebenfalls für die übrigen humanen herpesviren und hängt wohl auch mit der pathogenese der jeweiligen erkrankungen zusammen. das humane hbv ist ein sehr kleines dna-virus mit einer teilweise doppelsträngigen zirkulären dna von ca. 3200 bp. im partikel ist eine polymerase verpackt, die den während der replikation notwendigen, für ein dna-virus sehr ungewöhnlichen schritt einer reversen transkription ermöglicht. dies geschieht an einer prägenomischen rna. angesichts dieser biologischen ähnlichkeiten mit einem retrovirus und auch der homologien der hbv-polymerase mit der reversen transkriptase von retroviren ist es nicht erstaunlich, dass gegen retroviren wirksame substanzen in vitro und in vivo auch gegen hbv wirksam sind. hauptindikation für die antivirale therapie ist heute die chronische hepatitis b wegen der spätkomplikationen: leberzirrhose und hepatozelluläres karzinom. interferone natürliche und auch rekombinant hergestellte humane α-interferone inhibieren die hbv-replikation sehr effektiv und waren für etliche jahre einzige standardtherapeutika bei der behandlung der chronischen hepatitis b. dies war auch die erste indikation, bei der interferone in großem umfang zur antiviralen therapie eingesetzt wurden. virologisch und histopathologisch findet sich bei etwa einem drittel der patienten, die 6 monate lang dreimal wöchentlich 3 mio. ie α-interferone erhielten, eine deutliche besserung der chronischen hbv-infektion. die hbv-dna-konzentrationen im blut nehmen signifikant ab oder fallen -auch in abhängigkeit von der empfindlichkeit der eingesetzten nachweismethodikunter die nachweisgrenze ab. bei etwa 40% der patienten kam es zur normalisierung der transaminasen und zum therapiebedingten verlust von hbeag. leider persistiert dieser positive effekt nach beendigung der interferontherapie nur bei wenigen der ursprünglich erfolgreich behandelten patienten. andererseits machen die bekannten nebeneffekte der therapie (s. o.) eine wirkliche dauertherapie unmöglich. lamivudin (3tc) das nukleosidanalogon 3tc, welches zunächst in der hiv-therapie eingesetzt wurde, hat sich auch bei der hbv-therapie bewährt. nach einjähriger therapie mit täglich 100 mg zeigten in einer studie 60% der patienten eine histologisch nachweisbare verbesserung der hepatitis und sogar 70% eine anhaltende normalisierung der transaminasen (alt). nach fortgesetzter 2-jähriger therapie fanden sich bei einem drittel der behandelten antikörper gegen hbeag. 3tc führt nach lebertransplantation aufgrund einer hbv-induzierten zirrhose zu einer reduktion der neuinfektionen des transplantates, wobei die standardprophylaxe die fortgesetzte gabe von anti-hbs-haltigen hyperimmunglobulinpräparaten ist. verschiedene resistenzvermittelnde mutationen treten in der hbv-polymerase in sehr ähnlichen, konservierten bereichen wie bei hiv in abhängigkeit von der therapiedauer auf. die auswirkungen auf den grad der resistenz einerseits und die verbleibende vermehrungsfähigkeit der mutanten andererseits sind wie bei hiv unterschiedlich. neue nukleosidanaloga (entecavir) finden derzeit mit erfolg eingang in die therapie der chronischen hbv-infektion. langzeittherapie scheint die erfolge zu verbessern, solange keine resistenzen auftreten. es liegt nahe, dass alle in der erprobung befindlichen antiretroviralen nukleosidanaloga auch hinsichtlich ihrer hbv-wirksamkeit und kreuzresistenzen geprüft werden. hcv-infektionen stellen eine besondere therapeutische herausforderung dar, weil ein sehr hoher anteil (75-85%) der infizierten eine chronische infektion entwickelt, die dann nach jahren eines relativ symptomarmen verlaufs plötzlich in eine leberzirrhose und auch ein karzinom übergehen kann. der zunächst klinisch eher milde verlauf führt dazu, dass die therapiebereitschaft der patienten in den asymptomatischen phasen häufig nicht allzu groß ist. bei hcv handelt es sich um ein völlig anderes virus als bei hbv, um ein einzelsträngiges rna-virus mit plusstrang-polarität. das genom dieses flavivirus umfasst ca. 9000 nukleotide. ähnlich wie bei picornaviren ist das primäre translationsprodukt ein polyprotein, das bei hcv posttranslational in zehn struktur-und nicht-struktur-proteine gespalten wird. eines der nicht-struktur-proteine, das ns5a, ist offenbar für die interferonempfindlichkeit des virus verantwortlich. bei hcv unterscheidet man 6 genotypen mit verschiedenen subtypen. diese genotypen α sind geographisch unterschiedlich verteilt. die erfolgsrate einer α-interferon-therapie ist vom genotyp abhängig. in deutschland ist mit ca. 60% der genotyp 1 (subtypen a und b) am häufigsten vertreten, welcher leider die geringste ansprechrate bei α-interferon-therapie aufweist. ähnlich wie bei hiv ist die viruspopulation in einem patienten genetisch nicht einheitlich, sondern stellt ebenfalls eine quasi-spezies dar. das ausmaß der heterogenität der viruspopulation vor therapie und die veränderung unter therapie scheinen prognostische bedeutung zu besitzen. α-interferone die zunächst angewendete 6-monatige monotherapie mit dreimal 3 mio. ie α-interferon pro woche erbrachte je nach studie und bei einschluss aller genotypen nur bei etwas über 10% der patienten einen anhaltenden erfolg. dieser war definiert als mindestens 6 monate nach therapieende anhaltende normalisierung der transaminasen und verschwinden der hcv-rna aus dem blut. in den folgenden jahren konnte durch steigerung der α-interferon-dosis, durch tägliche gaben und verdoppelung der therapiedauer der erfolg verbessert werden. peg-gekoppelte interferone und kombinationstherapie mit ribavirin zwei weitere therapeutische neuerungen, einerseits die anwendung von polyethylengekoppeltem α-interferon 2a (sog. pegyliertes interferon) und andererseits die kombinationstherapie mit dem nukleosidanalogon ribavirin, konnten die ergebnisse unabhängig voneinander nochmals deutlich verbessern. durch anwendung von peg-α-ifn 2a wird bei nur einmal wöchentlicher gabe (180 mg) ein wesentlich gleichmäßigerer ifn-spiegel erreicht. der therapieerfolg liegt bei anwendung von peg-α-ifn 2a bei etwa 30% (genotyp 1) und 40% (alle genotypen) mit vergleichbaren nebenwirkungen wie nach α-ifn-therapie. die ergebnisse mit der kombinationstherapie α-ifn + ribavirin liegen in der gleichen größenordnung. auch hier lassen sich durch dosissteigerungen und verlängerte therapie verbesserungen erreichen. interessant ist die tatsache, dass frühere studien mit einer ribavirin-monotherapie zwar eine verbesserung der leberhistologie und der transaminasen gezeigt haben, aber keine verminderung der viruslast im blut. die naheliegende kombination von peg-ifn und ribavirin hat die ergebnisse weiter verbessert. eine reihe weiterer kombinationen befindet sich in der erprobung. die langzeitprognose der erfolgreich behandelten patienten ist gut und mittlerweile scheint sogar klar zu sein, dass auch chronisch hcv-infizierte patienten mit leberzirrhose, abhängig vom hcv-genotyp, gemessen an virologischen, klinischen und auch histologischen parametern, von der antiviralen therapie profitieren. therapiebestimmende faktoren derzeit sind 3 retroviren bekannt, die für die pathogenese von erkrankungen des menschen bedeutsam sind: • beide erreger von aids, hiv-1 und hiv-2 • htlv-1 als erreger der adulten t-zell-leukämie und der tropischen spastischen paraparese. zielmoleküle der meisten verfügbaren antiretroviralen therapeutika sind 2 partikelgebundene viruskodierte enzyme, die reverse transkriptase (rt) und die protease. die substanzen lassen sich einteilen in nukleosidanaloge reverse-transkriptase-hemmer, nicht nukleosidanaloge reverse-transkriptase-hemmer (nnrti) und proteasehemmer. seit der erstzulassung des ersten rt-hemmers azt in den usa im jahr 1987 sind mehr als 20 medikamente in deutschland zugelassen worden. zanamivir muss i.v. oder als aerosol verabreicht werden, wohingegen das analoge "pro-drug" oseltamivir oral verabreicht werden kann. da die beiden substanzen an unterschiedlichen stellen der funktionellen domäne der neuraminidase binden, besteht keine absolute kreuzresistenz zwischen beiden pharmaka. h5n1-infizierte patienten sind teilweise mit erfolg behandelt worden. neben den etablierten antiviralen therapeutika und therapieindikationen gibt es andere viren, bei denen die therapie am menschen erprobt wurde, aber noch eher experimentellen charakter hat. die therapie von enterovirus-und rhinovirusinfektionen ist seit mehreren jahrzehnten gegenstand wissenschaftlicher forschung und auch von therapieversuchen mit chemischen substanzen, antikörpern und interferonen. eine gruppe von sog. win-substanzen hemmt durch spezifische bindung an das viruskapsid die adsorption des virus an die zielzelle und das intrazelluläre uncoating. in den usa ist ein vertreter dieser substanzen, das pleconaril, klinisch erprobt worden. es ist gegen viele picornaviren (95%) wirksam und hat sich bei der behandlung der aseptischen meningitis und respiratorischer infektionen in kontrollierten studien bewährt. ob pleconaril bei der enterovirusmyokarditis eingesetzt werden kann, ist noch nicht klar. derzeit laufen bei dieser indikation versuche mit interferontherapie. adenoviren sind weltweit verbreitet und besitzen ein erhebliches pathogenes potenzial. insbesondere bei systemischen infektionen immunsupprimierter stellen sie ein schwieriges the-rapeutisches problem dar. zwei in vitro gegen verschiedene adenovirustypen wirksame substanzen, die für andere indikationen zugelassen sind, werden zur experimentellen therapie eingesetzt: cidofovir und ribavirin. für beide substanzen gibt es positive kasuistische mitteilungen, aber der therapeutische wert beider substanzen ist derzeit noch nicht klar. wie bereits erwähnt, zeichnet sich das nukleosidanalogon ribavirin durch ein relativ breites wirkungsspektrum aus. bereits vor knapp 20 jahren wurde es zur behandlung von rsv-infektionen der lunge bei schwer kranken intensivpflichtigen kindern zugelassen. in studien konnte bei aerosolanwendung eine senkung der letalität nachgewiesen werden. auch die intravenöse anwendung ist mit erfolg möglich, und derzeit wird die kombination von ribavirin mit einer etwa gleich wirksamen antikörpergabe evaluiert. auch beim masernvirus, einem weiteren paramyxovirus, ist über einzelne erfolge bei der behandlung von pneumonien immunsupprimierter mit ribavirin berichtet worden. interessanterweise ist ribavirin auch mit gutem erfolg bei schweren hämorrhagischen lassavirusinfektionen eingesetzt worden und gilt als therapie der wahl. auch bei dem verwandten, in südamerika vorkommenden junin-virus konnte die letalität von ca. 20 auf 2% gesenkt werden. leider ist die substanz nicht ausreichend liquorgängig, um einen therapeutischen effekt bei zns-manifestationen zu erreichen. bei der durch jc-virus hervorgerufenen progressiven multifokalen leukoenzephalopathie (pml) wurden bei gabe von cidofovir, aber auch α-interferon klinische besserungen beobachtet. eine reihe mehr oder weniger spezifisch wirksamer substanzen (auch interferone) ist für die topische therapie von papillomen erprobt worden. in jüngster zeit konnte in einer ersten studie gezeigt werden, dass auch hier eine topische applikation von cidofovir wirksam ist. die zunächst mit großen erwartungen durchgeführten studien zur behandlung der juvenilen larynxpapillomatose mit interferonen haben nicht zufrieden gestellt. diagnostik die diagnose einer katheterassoziierten infektion ist häufig schwierig. zur definitiven diagnose einer katheterassoziierten infektion ist der nachweis desselben erregers von der katheterspitze und aus der blutkultur notwendig. bei liegendem katheter kann die diagnose mit der sogenannten "differential time to positivity" (dttp) gestellt werden: im falle einer katheterassoziierten infektion wird eine aus dem katheter entnommene blutkultur mindestens 2 h früher positiv als eine gleichzeitig aus einer peripheren vene entnommene probe. als klinische hinweise können eine entzündung an der einstichstelle, fehlende andere ausgangsherde für eine bakteriämie sowie der nachweis typischer erreger, z. b. staphylokokken, angesehen werden. therapie es werden antibiotika verabreicht und der katheter entfernt. bei implantierten verweilkathetern ist vor der entfernung je nach vorliegendem pathogen ein antibiotischer behandlungsversuch gerechtfertigt. die wichtigste allgemeine maßnahme zur verhinderung nosokomialer infektionen ist das händewaschen. besonders in bereichen mit erhöhtem risiko, wie z. b. in infektions-, intensivund hämatologisch-onkologischen stationen, müssen vor und nach jedem patientenkontakt die hände gewaschen werden, 13 .3 syndrome und spezifische probleme 13 um eine nosokomiale ausbreitung von erregern zu vermeiden. daneben sind bei einigen erkrankungen spezielle maßnahmen erforderlich (s. o.). desinfektionen von medizinischen geräten sind heute durch gesetzliche bestimmungen und hygienevorschriften so geregelt, dass bei richtiger anwendung hierdurch keine erreger übertragen werden können. die liegedauer von intravasalen oder urinkathetern sollte möglichst kurz sein, d.h., jeder katheter, der nicht unbedingt benötigt wird, sollte entfernt werden. wo immer möglich, sollten therapieregime bevorzugt werden, bei denen medikamente oral statt intravenös gegeben werden. infektionen bei immunsupprimierten patienten sind häufig, verlaufen atypisch und können zu schweren komplikationen führen. art der immunsuppression und ausprägung und qualität der immunitätsfaktoren bestimmen spektrum und verlauf von infektionen wesentlich und sind für diagnostisches und therapeutisches vorgehen entscheidend. praxisfall ii eine 41-jährige frau wird wegen einer akuten myeloischen leukämie mit einer hochdosischemotherapie behandelt. am tag 10 nach beginn der chemotherapie sind die leukozyten auf < 500/μl gefallen, sie klagt über kopfschmerzen und wirkt apathisch. nach einer halben stunde steigt die temperatur auf 39,7 °c an, blutkulturen werden abgenommen und eine empirische antibiotikatherapie begonnen. der klinische zustand bessert sich rasch, die patientin ist 24 h nach deren beginn fieberfrei. zu diesem zeitpunkt ist e. coli als erreger in der blutkultur isoliert und identifziert worden. die empirische therapie ist adäquat und wird für weitere 8 tage • prognostisch ungünstigste kategorie: patienten mit lungeninfiltraten. nur bei 20-30% aller patienten finden sich positive blutkulturen, die eine gezielte antibiotische therapie ermöglichen. pilzinfektionen sind oft schwer zu diagnostizieren, da kulturelle verfahren wenig sensitiv sind. als nachweismethoden dienen heute in erster linie blutkulturen für candida-und die hochauflösende computertomografie des thorax sowie der galactomannan-test im serum für aspergillus-infektionen. therapie die behandlung eines neutropenischen patienten mit fieber muss unmittelbar nach auftreten der klinischen symptome, vor dem eintreffen mikrobiologischer befunde erfolgen. sie ist deshalb empirisch und kann später, bei erfolgter erregeridentifizierung, modifiziert werden. • initialtherapie und frühe eskalation: als primäre therapie kommen eine kombination aus einem breitspektrum-(ureido-)penicillin mit einem β-lactamase-hemmer, ein pseudomonas-wirksames cephalosporin oder ein carbapenem in frage. tritt nach 3-4 tagen keine entfieberung auf, sollte eine erneute diagnostik insbesondere mit hinblick auf pilzinfektionen erfolgen. • antimykotische therapie: wegen der gefahr von invasiven pilzinfektionen wird bei neutropenie (insbesondere bei patienten mit pulmonalen infiltraten) frühzeitig, spätestens jedoch nach 6-7 fiebertagen eine antimykotische therapie eingesetzt. zur empirischen therapie eignen sich caspofungin oder liposomales amphotericin b, beim nachweis von lungeninfiltraten ist voriconazol mittel der wahl. • fokussanierung: sie ist in der regel nur bei lokalisierten hautinfektionen möglich oder auch sinnvoll. die chirurgische therapie einer neutropenischen kolitis ist nur bei bildung von intraabdominellen abszessen oder einer offenen perforation sinnvoll, ansonsten erfolgt die therapie konservativ. prophylaxe wegen der hohen inzidenz von infektionen bei neutropenischen patienten werden verschiedene maßnahmen zur infektionsprophylaxe angewandt. diese maßnahmen zielen auf eine verminderung der endogenen keimflora und vermeidung der exposition gegenüber pathogenen organismen. • innerhalb einiger wochen erfolgt offenbar durch die immunantwort eine partielle kontrolle der hiv-replikation. die virämie nimmt ab und stellt sich auf ein individuell unterschiedlich hohes niveau ein. die höhe der virämie in dieser phase (messbar durch die quantitative bestimmung der hiv-rna) ist maßgeblicher parameter der weiteren prognose. patienten mit niedriger hiv-rna (< 10 000 genomkopien/ml blut), haben eine deutlich längere überlebenszeit als solche mit höheren werten. obwohl in dieser phase (klinische latenzphase) meist keine beschwerden vorhanden sind, besteht eine ungeheure dynamik der hiv-replikation, die schließlich zur erschöpfung des immunsystems führt. pro tag werden ca. 10 10 virionen produziert. über 99% werden dabei in den cd4 + -lymphozyten gebildet, die infolge der infektion zerstört werden. es kommt so zum stetigen abfall der t-helferzell-zahl im blut, der sich meist über viele jahre hinzieht. das verhältnis von cd4 + -zellen zu cd8 + -zellen (suppressor-und zytotoxische t-zellen) kehrt sich um (normalerweise cd4 + : cd8 + > 1). wenn die zahl der cd4 + -zellen unter eine kritische schwelle von 200/μl blut sinkt, kommt es zum auftreten von aids-typischen opportunistischen infektionen. bereits vorher können die patienten symptome aufweisen (z. b. oraler soor), die auf einen nahen zusammenbruch des immunsystems hindeuten. die hiv-infektion führt zur unspezifischen stimulation des humoralen immunsystems. dies äußert sich in einer vermehrten bildung von immunglobulinen. neben der infektion lymphatischer zellen werden auch frühzeitig langlebige makrophagen und gliazellen des zns befallen. diese zellen spielen für die dynamik der hiv-infektion keine so große rolle wie die cd4 + -lymphozyten, sind aber therapeutisch schwerer erreichbar und bereiten daher probleme. symptome je nach stadium der erkrankung treten unterschiedliche symptome auf. • akutes retrovirales syndrom: bei bis zu 50% der infizierten kommt es wenige wochen nach der ansteckung zum akuten krankheitsbild, dem sog. akuten retroviralen syndrom. wegen der klinischen ähnlichkeit mit der mononukleose wird das krankheitsbild auch als "mononukleoseähnliches syndrom" bezeichnet. typische symptome sind fieber, nachtschweiß, allgemeines krankheitsgefühl, lymphknotenschwellungen, pharyngitis und exantheme. vereinzelt treten auch schwere neurologische erkrankungen auf (z. b. guillain-barré-syndrom). während dieses akuten stadiums kommt es zum deutlichen abfall der t-helferzell-zahl, die hiv-rna im blut und damit auch die infektiosität ist sehr hoch. nach einigen tagen bis wochen bilden sich diese klinischen veränderungen wieder zurück. • asymptomatisches stadium (klinische latenz): die meisten hiv-infizierten haben über mehrere jahre keine beschwerden, die hiv-infektion wird in diesem stadium oft nur durch zufall entdeckt. als klinisches symptom können generalisierte lymphknotenschwellungen vorhanden sein (daher die frühere bezeichnung "lymphadenopathiesyndrom"). bei relativ konstantem wert der hiv-rna im plasma findet sich ein unterschiedlich rascher abfall der cd4 + -zeilen. • symptomatisches stadium: kennzeichnend ist die zunehmende immunschwäche, die sich im auftreten von opportunistischen infektionen äußert (cdc-kategorien b und c). die zahl der cd4 + -zellen ist stark abgefallen, es findet sich meist ein hoher wert der hiv-rna im plasma. die schweren aids-definierenden erkrankungen treten meist dann auf, wenn die cd4 + -zell-zahlen < 200/μl gesunken sind (› kap. 13.4.2). abb. 13.7 hiv im elektronenmikroskopischen bild. gut erkennbar ist die virushülle (pfeil) mit den knopfartig erscheinenden oberflächenantigenen, die an den cd4-rezeptor anbinden können, ferner das zylinderförmige viruskapsid (doppelpfeil), das aus dem hauptcoreprotein p24 aufgebaut ist (aufnahme: h. gelderblom, berlin). klassifikation die derzeitig gültige klassifikation kommt von den amerikanischen centers for disease control (cdc-klassifikation) und wurde zuletzt 1993 revidiert (› tab. 13.10). sie führt die aids-definierenden erkrankungen auf. ferner gibt sie eine stadieneinteilung der hiv-infektion an, die sich an klinischen und immunologischen parametern orientiert. alle patienten, die eine klinische aids-definition erfüllen, werden in die kategorie c eingestuft (› tab. 13.11). virologische diagnostik die diagnostik der hiv-infektion erfolgt meist durch nachweis virusspezifischer antikörper. als screening-test bei verdacht auf hiv-infektion dient ein eli-sa mit sehr hoher sensitivität und spezifität je > 99%. bei positivem elisa muss ein bestätigungstest erfolgen. meist ist dies ein westernblot, ggf. auch ein immunfluoreszenztest. jeder hiv-test muss nach aufklärung und mit dem einverständnis des patienten erfolgen und bei positivem ausfall durch eine 2. blutentnahme und erneute untersuchung bestätigt werden. zwischen infektion und bildung messbarer antikörper vergehen einige wochen (diagnostisches fenster). 3-6 monate nach infektion weisen fast alle infizierten antikörper auf. in der frühen phase vor einsetzen der antikörperbildung ist also bei negativem hiv-antikörpertest eine übertragung der infektion möglich. in besonderen fällen, wenn eine sichere frühzeitige entdeckung der infektion notwendig ist, kann ein direkter nachweis der viralen rna oder proviralen dna durch pcr erfolgen. diese wird z. b. bei kindern hiv-infizierter mütter eingesetzt. mit dieser oder anderen diagnostischen verfahren (nasba = "nucleic acid squence-based amplification"; bdns = "branch-chain dns) kann ferner ein quantitativer nachweis der hiv-rna (viruslast) im blut erfolgen. dieser test spielt heute eine sehr große rolle für die beurteilung der prognose und als kontrolle der antiretroviralen therapie. immunologische diagnostik die zahl der cd4-lymphozyten im blut stellt den entscheidenden immunologischen parameter für die verlaufsbeurteilung der hiv-infektion dar. die bestimmung erfolgt mittels der facs-methode. begleitende diagnostik durch die hiv-infektion können einige sekundäre laborveränderungen hervorgerufen werden. häufig finden sich anämie, leukozytopenie, thrombozytopenie und erhöhte immunglobuline. viele infizierte haben zusätzliche infektionen. besonders nach hepatitis b und c, lues, toxoplasmose und zytomegalievirus-infektion muss gesucht werden. differenzierungsmaßnahme akute hiv-infektion: klinische kategorien ( • hemmung der protease • hemmung der integration. mittlerweile stehen mehr als 20 substanzen aus verschiedenen klassen für die therapie zur verfügung (› tab. 13.12), medikamente mit weiteren neuen therapieprinzipien (ccr5-antagonisten, integrasehemmer, maturationshemmer) werden derzeit klinisch erforscht. die therapie der hiv-infektion erfolgt heute immer als kombinationstherapie. diese wird auch als "hochaktive antiretrovirale therapie" (haart) bezeichnet. typischerweise besteht sie aus der kombination von 2 nrti mit einem proteasehemmer (pi) oder einem nnrti. durch keine bisher bekannte therapie wird die vollständige elimination von hiv erreicht. daher muss eine einmal begonnene und effektive antiretrovirale therapie unbegrenzt fortgeführt werden, da es sonst erneut zur ungehemmten virusvermehrung kommt. aus unterschiedlicher motivation ist in den letzten jahren die auswirkung von therapiepausen untersucht worden. die erhofften positiven effekte (verbesserung der immunantwort gegen hiv) traten hierunter nicht ein, dagegen konnte ein vermehrtes risiko für schwerwiegende komplikationen gezeigt werden. therapiepausen sind daher nicht zu empfehlen. wiegen. bei einer cd4 + -zahl von > 500/μl wird deshalb meist zunächst abgewartet. eine gesicherte behandlungsindikation besteht für alle patienten mit einer symptomatischen hiv-infektion unabhängig von der zahl der cd4 + -zellen und der hiv-rna. eine besondere herausforderung ist die behandlung hiv-infizierter schwangerer frauen. im ersten trimenon sollte wegen des möglichen teratogenen potenzials keine antiretrovirale therapie erfolgen bzw. eine begonnene therapie ausgesetzt werden. für eine antiretrovirale therapie mit zidovudin ab der 14. schwangerschaftswoche bis zur entbindung mit anschließender 6-wöchiger nachbehandlung des kindes ist eine verringerung der vertikalen hiv-übertragung nachgewiesen. viele frauen werden heute auch in der schwangerschaft mit einer kombinationstherapie behandelt, wobei bisher kaum daten zur fruchtschädigung durch einzelne medikamente vorliegen. wegen seiner teratogenität ist efavirenz auf jeden fall kontraindiziert. therapieziele ziele einer antiretroviralen therapie sind die lebensverlängerung und eine besserung vorhandener symptome und der lebensqualität. bei asymptomatischen patienten können nur die viruslast und der immunstatus als parameter für die wirksamkeit einer therapie herangezogen werden. es sollte heute eine absenkung der hiv-rna unter die nachweisgrenze ultrasensitiver tests (< 50 kopien/ml) angestrebt werden. je nach höhe der ausgangsviruslast wird dieses ziel meist nach 3-6 monaten erreicht. durch eine effektive antiretrovirale therapie steigt die zahl der cd4 + -zellen an. normalwerte werden jedoch meist nur von patienten mit relativ guten ausgangswerten erreicht. probleme der antiretroviralen therapie diese therapie war zunächst mit erheblichen einschränkungen und belastungen behaftet ( von der akuten hiv-infektion bis zum auftreten von aids vergehen bei unbehandelten im durchschnitt ca. 10 jahre. die mittlere zeitspanne von der diagnose aids bis zum tod beträgt dann knapp 2 jahre. ein geringer anteil aller hiv-infizierten (< 5%) zeigt auch nach mehr als 10-jähriger dauer der infektion keine anzeichen eines immundefektes (long-term non-progressor). es ist bisher nicht bekannt, ob diese personen jemals an aids erkranken werden. bei allen anderen führt die hiv-infektion unbehandelt unweigerlich zur ausbildung von aids und zum tod. durch die antiretrovirale kombinationstherapie hat sich die prognose der hiv-infektion dramatisch verbessert. die meisten können damit ein normales leben führen, und die prognose ist günstig. die mittlere lebenserwartung eines patienten unter antiretroviraler therpaie lässt sich heute noch nicht sicher angeben, dürfte sich aber der normalen lebenserwartung annähern. da die hiv-infektion zur unheilbaren, tödlichen erkrankung führt und eine schutzimpfung nicht zur verfügung steht, kommt der prävention eine zentrale rolle zu. das risiko einer sexuellen übertragung der hiv-infektion kann durch vermeidung riskanter sexualpraktiken vermindert werden. die konsequente benutzung von kondomen ist eine entscheidende maßnahme zur verminderung des übertragungsrisikos. bei drogenabhängigen konzentrieren sich präventive strategien auf die suchttherapie sowie auf die kontrollierte verabreichung von ersatzdrogen ("methadon-programm"). eine weitere präventive maßnahme ist die ausgabe steriler spritzen an drogenabhängige. allgemeine vorsichtsmaßnahmen angehörige medizinischer berufe sind beim umgang mit hiv-patienten einer infektionsgefahr ausgesetzt. ein relevantes infektionsrisiko existiert allerdings nur beim kontakt mit infiziertem blut. die höchste gefahr besteht bei stichverletzungen mit hohlnadeln, die blut enthalten: in 0,2-0,5% kommt es zur übertragung von hiv. daher müssen unbedingt vorsichtsmaßnahmen eingehalten werden. am wichtigsten ist die vermeidung von prozeduren, die ein hohes verletzungsrisiko beinhalten ("re-capping" von kanülen!). scharfe gegenstände müssen immer aus dem unmittelbaren arbeitsbereich entfernt und sofort in spezielle container entsorgt werden. da eine infektion prinzipiell auch über schleimhäute und verletzte haut erfolgen kann, müssen in allen situationen, in denen blutkontakt möglich ist, schutzhandschuhe getragen werden. vorgehen bei nadelstichverletzung ist es trotz vorsichtsmaßnahmen zur nadelstichverletzung gekommen, müssen sofort folgende maßnahmen ergriffen werden: blutung anregen und die möglichst gespreizte wunde mit einem desinfektionsmittel auf alkoholbasis gründlich desinfizieren (ca. 4 min). bei hautkontakt ebenfalls desinfizieren oder gründlich mit seife und viel wasser waschen. handelt es sich um eine verletzung mit hohem risiko (injektion von größeren mengen blut, intramuskuläre verletzung mit großlumiger nadel), sollte eine antiretrovirale kombinationstherapie rasch begonnen (optimal innerhalb von 1-2 h nach verletzung) und für 28 tage durchgeführt werden. bei verletzungen mit geringerem risiko (z. b. subkutane verletzung) sollte eine individuelle beurteilung und beratung durch jemanden mit spezieller erfahrung in diesem bereich erfolgen. wichtig sind in allen situationen, in denen ein infektionsrisiko aufgetreten ist, eine psychologische betreuung der betroffenen personen und die exakte dokumentation des unfallhergangs einschließlich der hiv-tests (zeitpunkte 0, 6 wochen, 3 und 6 monate), damit ansprüche des betroffenen im falle einer infektion gewahrt bleiben. als folge der durch hiv ausgelösten immunschwäche kommt es zum auftreten so genannter opportunistischer infektionen. typisch für viele opportunistische erreger ist, dass sie weit verbreitet sind und nach einer primärinfektion, die bereits vor der hiv-infektion stattfindet, zu latenten infektionen führen. diese erreger werden erst durch die immunschwäche zu pathogenen (daher die bezeichnung opportunistisch). alle organe können von diesen infektionen betroffen werden. am häufigsten treten erkrankungen der haut, der mundhöhle, des gastrointestinaltraktes, der lunge, des auges und des nervensystems auf. einige der opportunistischen infektionen kommen bereits vor, wenn die cd4 + -zellen noch nicht maximal erniedrigt sind (zwischen 100 und 200/μl). beispiele hierfür sind die candida-ösophagitis und die pneumocystis-carinii-pneumonie. andere erkrankungen sind charakteristisch für das endstadium der immundefizienz (cd4 + -zelien < 50/μl). neben infektionen treten auch verschiedene tumoren gehäuft auf (kaposi-sarkom, non-hodgkin-lymphome). ii ein 30-jähriger, der bis dahin nie krank war, stellt sich in der notaufnahme vor. seit 3 wochen bemerkt er fieber, das in den letzten wochen täglich 39 °c überschreitet. er klagt über quälenden reizhusten mit wenig auswurf, der auch nachts sehr heftig sei. in den letzten wochen habe er 7 kg gewicht abgenommen (von 60 auf 53 kg). bis zum vortag habe er gearbeitet, was ihm in den letzten tagen sehr schwer gefallen sei. bei nachfrage gibt er an, homosexuelle kontakte zu haben, ein hiv-test sei nie durchgeführt worden. bei der untersuchung fallen eine tachypnoe (30/min) und eine ausgeprägte periphere zyanose auf. auskultation und perkussion sind unauffällig. im mund besteht ein soor. zervikal, inguinal und axillär lassen sich vergrößerte lymphknoten (bis 1,5 cm) tasten. ansonsten keine auffälligkeiten. labor: hb 10,5 g/dl; leukozyten 2800/μl; ldh 680 u/l; in der arteriellen blutgasanalyse po 2 52 mmhg, pco 2 20 mmhg, ph 7,41; sonst keine weiteren auffälligkeiten. röntgen-thorax: ausgeprägte interstitielle infiltrationen in beiden lungenflügeln, z. t. auch alveoläre verschattungen. klinische diagnose: interstitielle pneumonie, dringender verdacht auf pneumocystis-carinii-pneumonie. therapie: sofortige behandlung mit hochdosiertem cotrimoxazol und mit ceftriaxon; ferner gabe von prednison. symptomatisch verabreichung von o 2 per nasensonde, behandlung des soors mit amphotericin-b-suspension. weiterführende diagnostik: hiv-serologie im elisa und westernblot positiv; hiv-rna im plasma 820 000 copies/ml; cd4 + -lymphozyten 10/μl, cd8 + -lymphozyten 400/μl; in der bronchoalveolären lavage nachweis von pneumocystis carinii. verlauf: allmählicher rückgang der klinischen symptomatik; nach diagnosesicherung der pcp absetzen von ceftriaxon und behandlung mit co-trimoxazol über 3 wochen. danach einleitung einer antiretroviralen therapie, fortführung der co-trimoxazol-gabe in prophylaktischer dosierung und entlassung in deutlich gebessertem zustand. ii das spektrum der hautveränderungen im rahmen der hiv-infektion umfasst: • infektiöse veränderungen • allergische reaktionen • sog. idiopathische hauterkrankungen. häufig sind herpes-simplex-virus-infektionen, die als harmlose infektionen, aber auch als schwere, chronische ulzerationen imponieren können. varicella-zoster-virus-reaktivierungen (gürtelrose) treten typischerweise schon in frühen stadien der hiv-infektion auf und erstrecken sich häufig über mehrere dermatome. die behandlung der herpesvirusinfektionen kann mit aciclovir, valaciclovir, famciclovir oder brivudin erfolgen. eine andere häufige virusinfektion sind die dellwarzen (mollusca contagiosa), die sehr charakteristisch sind und oft ausgedehnte körperareale befallen (› kap. 13.5). im analbereich kommen gehäuft feigwarzen (condylomata acuminata) vor. die infektionen mit herpes-simplex-und varicella-zoster-virus werden mit aciclovir behandelt. bei dellwarzen und feigwarzen müssen kürettagen angewendet werden. bakterielle infektionskrankheiten, die bei hiv-infizierten vermehrt diagnostiziert werden, umfassen pyodermien, lues und bazilläre angiomatose. letztere wird durch die erst kürzlich entdeckten erreger bartonella henselae und quintana verursacht und äußert sich in form rötlicher, papulöser hautveränderungen. es kann ferner ein disseminierter organbefall vorkommen. die therapie erfolgt mit penicillin (lues), mit staphylokokken-wirksamen antibiotika (pyodermie) und mit makroliden (bazilläre angiomatose). allergische reaktionen und andere hauterkrankungen neben allergischen reaktionen treten gehäuft psoriasis vulgaris, seborrhoisches ekzem, xerodermie und papulöse dermatitiden auf. außer durch dermatologische lokalbehandlung bessern sich diese erkrankungen oft durch verbesserung der immunsituation im rahmen einer erfolgreichen antiretroviralen therapie. die symptomatik reicht von asymptomatischen veränderungen bis zu schmerzhaften läsionen, die eine nahrungsaufnahme fast unmöglich machen (therapie siehe oben). erkrankungen des gastrointestinaltraktes sind häufig bei fortgeschrittener hiv-infektion. im folgenden sind die häufigsten opportunistischen erkrankungen aufgeführt. hinzu kommen einige seltene infektionen und andere erkrankungen, die nicht spezifisch für die hiv-infektion sind. häufigste aids-definierende opportunistische infektion des gastrointestinaltraktes. symptome dysphagie und retrosternale schmerzen, meist auch candida-beläge in der mundhöhle. diagnostik ösophagoskopie mit dem makroskopischen bild weißer schleimhautbeläge und mikrobiologischer oder histologischer nachweis von candida. therapie fluconazol. nach erfolgreicher behandlung treten häufig rezidive auf. dann ist eine prophylaktische behandlung mit fluconazol indiziert. bei den seltenen fällen einer fluconazol-resistenz wird voriconazol eingesetzt. diese infektionen treten meist bei sehr weit fortgeschrittenem immundefekt (cd4 + -zellen < 50/μl) auf und betreffen v. a. gastrointestinaltrakt und auge. erkrankungen des gastrointestinaltraktes durch cmv können in allen abschnitten vorkommen: als ösophagitis, gastritis, enteritis, kolitis und als proktitis. symptome die ösophagitis verursacht eine dysphagie. bei der cmv-gastritis stehen oberbauchschmerzen im vordergrund. die cmv-enterokolitis manifestiert sich mit durchfällen und bauchschmerzen. bei der cmv-proktitis sind defäkationsschmerzen und blutbeimengungen im stuhl die führenden symptome. diagnostik endoskopische untersuchung mit biopsie. makroskopisch sieht man eine entzündung der schleimhaut und ulzerationen, die wie ausgestanzt wirken. histologisch ist der nachweis von intranukleären einschlusskörperchen in vergrößerten zellen (eulenaugen-zellen) typisch. die abgrenzung von anderen viralen infektionen ist durch die immunhistologie (nachweis viraler antigene) möglich. der nachweis des cmv-pp65-antigens im blut und die cmv-pcr aus dem blut geben hinweise auf die diagnose. therapie zurzeit sind 3 substanzen verfügbar: ganciclovir, cidofovir und foscarnet. sie müssen intravenös verabreicht werden und sind in ihrer wirksamkeit vergleichbar. unterschiede bestehen in den nebenwirkungen: ganciclovir ist hämatotoxisch (leukopenie, anämie), während bei foscarnet und cidofovir die nephrotoxizität im vordergrund steht. in schweren fällen können ganciclovir und foscarnet kombiniert gegeben werden kryptosporidien sind protozoen, die bei tieren und bei menschen durchfallerkrankungen auslösen können. sie werden vor allem durch kontaminiertes wasser übertragen. bei immunkompetenten kommt es zu einer milden, selbstlimitierten diarrhö. symptome hiv-patienten mit ausgeprägter immunschwäche erkranken an schwersten wässrigen durchfällen, die den patienten häufig tag und nacht quälen. infolgedessen kommt es zur auszehrung ("wasting syndrom"). diagnostik die kryptosporidien finden sich an der oberfläche des darmepithels und können dort histologisch nachgewiesen werden. nachweis von oozysten im stuhl, darstellbar mit spezialfärbungen. therapie nicht bekannt. besserung erfolgt meist mit der einleitung einer antiretroviralen therapie. symptomatische maßnahmen (loperamid, opiumtinktur) müssen häufig angewandt werden. mikrosporidien sind obligat intrazelluläre, sporenbildende protozoen. von den mehr als 1000 spezies sind bisher 5 als menschenpathogen bekannt. 2 spezies stehen bei hiv-infektion im vordergrund: enterocytozoon bieneusi infiziert darm und gallengänge. die symptome sind von denen bei der kryptosporidien-infektion nicht unterscheidbar. encephalitozoon verursacht eine allgemeininfektion. die diagnose einer mikrosporidiose erfordert spezielle techniken (fluoreszenztest) und ist aus stuhl oder abstrichen und sekreten möglich. die speziesdifferenzierung ist durch elektronenmikroskopie oder pcr möglich und von therapeuti-scher bedeutung: während encephalitozoon auf eine therapie mit albendazol anspricht, ist für enterocytozoon bieneusi keine wirksame therapie bekannt. das wasting-syndrom ist definiert durch eine gewichtsabnahme von mehr als 10% des ausgangsgewichtes, verbunden mit anhaltendem fieber oder chronischer diarrhö ohne erregernachweis. diagnostik behandelbare opportunistische infektionen müssen ausgeschlossen werden. therapie die antiretrovirale therapie führt meist zur besserung. wie alle opportunistischen infektionen sind lungenmanifestationen in den letzten jahren seltener geworden. nach wie vor stellt die pneumocystis-pneumonie als akut lebensbedrohliche infektion häufig die erstmanifestation von aids dar. die tuberkulose ist weltweit die häufigste todesursache hiv-infizierter menschen. epidemiologie weltweit ist die tuberkulose mit abstand die häufigste opportunistische infektion im rahmen der hiv-infektion. ihre größte verbreitung hat sie in den unterentwickelten ländern. aber auch in südeuropa kommt sie sehr häufig vor. an tuberkulose ist besonders zu denken bei drogenabhängigen und bei patienten aus ländern der dritten welt. die lunge ist meist betroffen, doch handelt es sich bei der tuberkulose hiv-infizierter oft um ein disseminiertes krankheitsbild mit befall unterschiedlichster organe. symptome unspezifisch mit fieber, nachtschweiß, gewichtsabnahme und husten. röntgenologisch finden sich an der lunge typische verläufe im sinne einer reaktivierung mit kavernösen veränderungen und atypische verläufe mit flächenhaften infiltraten und mediastinalen lymphknotenschwellungen. diagnostik eine tuberkulose muss bei allen pulmonalen infiltraten bei hiv-infizierten patienten ausgeschlossen werden. die definitive diagnose wird mit der sputumuntersuchung durch den nachweis säurefester stäbchen gestellt, evtl. ferner untersuchung des magensekrets oder bronchoskopie. zur unterscheidung von ubiquitären mykobakteriosen (mycobacterium-avium-komplex) ist eine mikrobiologische differenzierung nötig. therapie dieselben substanzen wie bei nicht-hiv-infizierten patienten (› kap. 10.5) meist über insgesamt 6 monate, bei komplizierten fällen auch länger. das ansprechen auf die therapie ist allgemein gut. multiresistente tuberkuloseerreger kamen in einigen amerikanischen großstädten bei hiv-infizierten gehäuft und mit sehr hoher letalität vor. in deutschland sind solche infektionen bisher nicht aufgetreten. wichtigste maßnahme zur verhütung von resistenzbildungen ist eine konsequente und effektive therapie. hervorgerufen durch den ubiquitär vorkommenden pilz (früher als protozoon klassifiziert) pneumocystis jiroveci (früher pneumocystis carinii, daher die abkürzung pcp). bereits im kindesalter besteht fast hundertprozentige durchseuchung, so dass das risiko einer erkrankung nur vom ausmaß des immundefektes abhängt. ohne prophylaktische maßnahmen beträgt die inzidenz für patienten mit cd4 + -zellen < 100/μl ca. diagnostik ein klinischer verdacht auf pcp muss sofort weiter abgeklärt werden. bei der körperlichen untersuchung finden sich häufig zyanose und tachypnoe. der auskultationsbefund ist aber typischerweise normal. im röntgenbild des thorax sieht man eine interstitielle zeichnungsvermehrung, in schweren fällen können auch alveoläre verschattungen auftreten (› abb. 13.10). obligate blutuntersuchungen sind die bestimmung der ldh (erhöht) und eine arterielle blutgasanalyse (erniedrigung des po 2 ), da beide parameter prognostische aussagekraft haben. die leukozyten sind meist nicht vermehrt. bei patienten mit den aufgeführten diagnostischen kriterien kann die klinische diagnose einer pcp gestellt werden. die definitive diagnose wird durch den erregernachweis (immunfluoreszenz oder andere färbetechniken) aus der bronchoalveolären lavage (bal) gestellt. therapie bei verdacht auf pcp muss sofort eine therapie eingeleitet werden. dies steht einer späteren diagnosesicherung nach wenigen tagen nicht im wege. mittel der wahl ist co-trimoxazol in hoher dosierung (20 mg trimethoprim, 100 mg sulfamethoxazol/kg kg und pro tag). die therapiedauer beträgt normalerweise 3 wochen. bei schweren unverträglichkeitserscheinungen (allergie) muss auf intravenös verabreichtes pentamidin ausgewichen werden. eine pcp mit arteriellem ausgangs-po 2 von 70 mmhg oder weniger muss zusätzlich mit prednison (2-mal 50 mg/d) behandelt werden, da so die letalität vermindert wird. unbehandelt ist eine pcp immer letal. auch bei optimaler behandlung handelt es sich um eine ernste erkrankung. ungünstige prognostische parameter sind eine ausgeprägte erniedrigung des po 2 und eine starke erhöhung der ldh. patienten mit beatmungspflichtiger respiratorischer insuffizienz haben eine schlechte prognose. bakterielle pneumonien treten schon bei einer helferzellzahl über 200/μl auf, werden aber mit zunehmendem immundefekt häufiger. drogenabhängige sind öfter betroffen als homosexuelle patienten. häufigste erreger sind pneumokokken, haemophilus influenzae, staphylokokken und gramnegative erreger. symptome wie bei pneumonien bei immunkompetenten personen (› kap. 10.4.1). diagnostik von der pcp ist die bakterielle pneumonie bei typischem verlauf durch raschen beginn, purulentes sputum, positiven auskultationsbefund und durch die röntgenuntersuchung abzugrenzen. es gibt aber häufig atypische verläufe, die eine klinische unterscheidung unmöglich machen. therapie bei leichtem verlauf mit cephalosporin der 2. generation bzw. ampicillin plus lactamasehemmer, bei schwerem verlauf kombinationstherapie mit cephalosporinen der 3. generation und einem makrolid oder mit einem chinolon mit pneumokokken-wirksamkeit (moxifloxacin, levofloxacin). bis zum ausschluss einer pcp sollte bei schweren pneumonien auch co-trimoxazol gegeben werden. pneumonien durch pilze kommen insgesamt selten bei hiv-infizierten vor. aspergillus-pneumonien treten im endstadium des immundefektes auf und haben eine schlechte prognose. infektionen mit kryptokokken manifestieren sich gelegentlich an der lunge, meist jedoch erst bei weiterer disseminierung als fungämie oder meningitis. andere pulmonale pilzinfektionen sind in europa sehr selten. candida-pneumonien werden bei hiv-infizierten nicht beobachtet. virale pneumonien sind ebenso sehr selten. zytomegalievirus wird zwar häufig in der lunge nachgewiesen, es handelt sich aber fast immer um eine infektion ohne krankheitswert. symptome des zentralen und des peripheren nervensystems treten im rahmen der hiv-infektion sehr häufig mit sehr vielgestaltigen ursachen auf. neurologische symptome können durch hiv selbst, durch opportunistische erkrankungen oder als unerwünschte wirkungen therapeutischer maßnahmen auftreten. die differentialdiagnose ist daher sehr schwierig. ca. 50% der bevölkerung sind mit dem protozoon toxoplasma gondii infiziert. als opportunistische infektion im rahmen der hiv-infektion tritt die toxoxplasmose aber nur bei schwer eingeschränktem immunstatus auf (cd4 + -zellen < 100/μl). als zeichen der früher erfolgten infektion finden sich antikörper im serum. bei fehlenden igg-antikörpern ist eine toxoplasmose sehr unwahrscheinlich. symptome die zerebrale toxoplasmose äußert sich in fieber, kopfschmerzen, neurologischen ausfällen und epileptischen anfällen. diagnostik nachweis von typischen veränderungen in der kernspintomografie oder computertomografie des schädels möglich (› abb. 13.11). therapie pyrimethamin und sulfadiazin. bilden sich die veränderungen hierunter zurück, ist die diagnose bestätigt. eine prophylaxe mit verminderter dosis ist wegen hoher rezidivgefahr anzuschließen. wenn sich die erkrankung unter der therapie nicht bessert, sollte eine stereotaktisch gewonnene biopsie erfolgen zum ausschluss anderer erkrankungen (z. b. lymphom). durch den hefepilz cryptococcus neoformans kann eine meningitis ausgelöst werden. diese infektion kommt v. a. in afrika und den usa häufig vor, bei uns ist sie seltener. symptome kopfschmerzen und fieber, oft über wochen progredient. diagnostik bestimmung des kryptokokken-antigens im serum mit sehr hoher sensitivität. zum ausschluss einer anderen meningitisform muss eine punktion des liquor cerebrospinalis durchgeführt werden, in dem sich die kryptokokken durch ein tuschepräparat nachweisen und kulturell anzüchten lassen. therapie amphotericin b und flucytosin und zusätzlich evtl. fluconazol. ca. 1/3 der erwachsenen patienten und 2/3 der kinder weisen symptome einer hiv-enzephalopathie auf. sie wird vermutlich durch direkte infektion des zns mit hiv verursacht und führt zu psychomotorischen störungen unterschiedlichen schweregrades. symptome das klinische bild ist sehr variabel. meist stehen konzentrations-und gedächtnisstörungen bis hin zur ausgeprägten demenz im vordergrund, aber auch epileptische anfälle und wesensveränderungen kommen vor. diagnostik nur durch den ausschluss anderer zns-manifestationen möglich. im liquor finden sich nur unspezifische veränderungen, in der kernspintomografie kann eine hirnatrophie erkennbar sein. therapie antiretrovirale behandlung. dabei gibt es verschiedenartige verläufe von der vollständigen rückbildung bis hin zur weiteren progredienz der veränderungen. die progressive multifokale leukenzephalopathie (pml) ist eine meist innerhalb von wochen bis monaten zum tode führende erkrankung des zns, die durch polyoma-viren (jc-virus) ausgelöst wird. charakteristisch sind zunehmende neurologische störungen bei meist erhaltenem bewusstsein und ausgedehnte läsionen im marklager, die sich am besten in der kernspintomographie darstellen. die diagnose kann durch pcr aus dem liquor oder durch hirnbiopsie gestellt werden. unter hochaktiver antiretroviraler therapie kommt es bei einem teil der patienten zur besserung. die cmv-enzephalitis ist eine meist rasch progredient verlaufende zerebrale infektion, die klinisch nicht von anderen enzephalitiden zu unterscheiden ist. die diagnose kann durch eine pcr aus dem liquor untermauert werden, die therapie erfolgt mit foscarnet oder ganciclovir. das zerebrale lymphom ist eine opportunistische erkrankung im endstadium der hiv-infektion. die diagnose kann heute mit hoher wahrscheinlichkeit durch den nachweis von ebv-dna im liquor cerebrospinalis mittels pcr erfolgen. eine definitive sicherung ist nur durch hirnbiopsie möglich. kurzfristige therapieerfolge können mit dexamethason erzielt werden. eine systemische chemotherapie ist nicht wirksam, und auch eine bestrahlung führt meist nur zur sehr kurzfristigen remission. bei der polyneuropathie handelt es sich um eine erkrankung, die sehr häufig in späten stadien der hiv-infektion auftritt. neben einer direkten neuropathischen wirkung des hiv kommen toxische wirkungen von medikamenten ursächlich in frage. die diagnose wird meist klinisch gestellt, elektroneurografische untersuchungen können ggf. zusätzlich erfolgen, ist die polyneuropathie medikamentös ausgelöst, kommt es nach absetzen zur besserung. andernfalls bestehen die beschwerden meist fort. die therapie ist symptomatisch (gabapentin, amitriptylin, carbamazepin). neurotoxische substanzen (d4t, ddi) müssen abgesetzt werden. die zytomegalievirus(cmv)-retinitis tritt bei patienten mit sehr schwerem immundefekt auf (cd4 + -zell-zahlen unter 50/μl). symptome verschwommenes sehen und sehminderung. unbehandelt führt die erkrankung zur erblindung. diagnostik spiegelung des augenhintergrundes mit charakteristischem befund. therapie intravenöse infusionen von ganciclovir, foscarnet oder cidofovir werden eingesetzt. viele der infektionen, die bei aids-patienten auftreten, verlaufen als disseminierte infektionen. dies gilt auch für einen teil der oben beschriebenen erkrankungen (z. b. cmv-infektion, tuberkulose). im folgenden werden diejenigen infektionen vorgestellt, die primär als generalisierte erkrankung durch den erregernachweis im blut diagnostiziert werden. in der regel manifestieren sich diese infektionen als sepsis, d.h mit klinischen symptomen (fieber, tachykardie, tachypnoe) und nachweis von bakterien im blut (bakteriämie). oftmals treten bakteriämien im zusammenhang mit intravenös platzierten kathetern auf. hier spielen vor allem staphylokokken, aber auch gramnegative keime wie pseudomonas aeruginosa eine rolle. pneumokokken-bakteriämien kommen im zusammenhang mit pneumonien vor. eine aids-definierende, selten auftretende komplikation ist die rezidivierende salmonellen-sepsis. diagnostik die blutkultur ist entscheidende maßnahme. therapie die behandlung erfolgt jeweils mit wirksamen antibiotika (antibiogramm). prognose neben der rechtzeitigen diagnose und der antibiotika-sensitivität des erregers ist der allgemeinzustand des patienten ausschlaggebend. mycobacterium avium und intracellulare bilden den mycobacterium-avium-komplex und kommen ubiquitär in der umwelt vor. bei immunkompetenten verursachen sie nur ganz selten infektionen. dagegen ist die disseminierte mac-infektion eine der schwersten infektionen bei hiv-patienten mit hochgradigem immundefekt (cd4 + -zellen < 50/μl). die aufnahme des erregers erfolgt entweder über den magen-darm-trakt oder über die lunge. hier kommt es zunächst zur kolonisierung und im weiteren verlauf zur disseminierung. der nachweis des erregers aus sputum oder stuhl beweist noch keine disseminierte infektion. symptome die disseminierte infektion äußert sich durch fieber, nachtschweiß, gewichtsabnahme, durchfälle, lymphknotenschwellungen, bauchschmerzen und eine hepatosplenomegalie. diagnostik die laborwerte zeigen meist eine anämie und eine erhöhung der alkalischen phosphatase. die diagnose lässt sich durch die anzüchtung des erregers aus der blutkultur oder anderen sterilen materialien (knochenmark, lymphknoten, leber) sichern. im gegensatz zur tuberkulose sind lungeninfiltrate durch mac selten. therapie auf die herkömmlichen antituberkulotika kann man nicht zurückgreifen, da der erreger gegen die meisten dieser mittel primär resistent ist. am wirksamsten ist eine kombination aus clarithromycin und ethambutol (eventuell zusätzlich mit rifabutin). als hiv-assoziierte tumoren (kategorie cdc c) werden das kaposi-sarkom (s. o.), das non-hodgkin-lymphom und das invasive zervixkarzinom gezählt. non-hodgkin-lymphome (nhl) treten bei ca. 5-10% aller aids-patienten auf. histologisch handelt es sich meist um hochmaligne b-zell-lymphome. ein disseminierter und extranodaler befall liegt häufig vor. symptome entsprechend dem befallsmuster: lymphknotenschwellungen und allgemeinbeschwerden (fieber, nachtschweiß) sind häufig; bei knochenmarkbefall kommt es zur panzytopenie, bei befall des magen-darm-traktes zu bauchschmerzen und gewichtsabnahme. im labor findet sich oft eine erhöhung der ldh. bei zns-befall kommt es zum auftreten neurologischer herdsymptome (anfälle, lähmungen). eine besonderheit ist das auftreten primärer zerebraler lymphome, die immer durch ebstein-barr-virus (ebv) ausgelöst sind (diagnostik durch nachweis von ebv-dna mittels pcr im liquor). therapie durchführung einer standard-chemotherapie (chop-schema: cyclophosphamid, adriamycin, vincristin, prednison). ziel ist heute die komplette remission und heilung auch in fortgeschrittenen stadien und bei fortgeschrittenem immundefekt. hierzu werden zunehmend auch aggressive therapieschemata eingesetzt. prognose nicht gut (heilung in < 50%). maligne tumoren, die durch humane papillomaviren (hpv) induziert werden, wurden bei hiv-patienten gehäuft beobachtet. hierzu zählen das zervixkarzinom der frau und plattenepithelkarzinome der analregion. außerdem wurde über vermehrtes auftreten von hodgkin-lymphomen berichtet. die prophylaxe von infektionen bereits vor deren erstem auftreten (primärprophylaxe) oder nach der ersten episode (sekundärprophylaxe) ist weiterhin eine wichtige aufgabe bei der betreuung hiv-positiver patienten, auch wenn opportunistische infektionen durch die antiretrovirale therapie insgesamt seltener geworden sind. klinisch von bedeutung ist die prophylaxe der pcp bei einer cd4 + -zell-zahl von < 200/μl mit cotrimoxazol. die beste vorbeugung aller opportunistischen infektionen ist eine wirksame antiretrovirale kombinationstherapie (haart). durch die verbesserte funktion des immunsystems ist ein schutz gegen opportunistische infektionen vorhanden. in vielen studien wurde nachgewiesen, dass patienten mit supprimierter viruslast ein sehr niedriges risiko für opportunistische infektionen aufweisen. steigen die cd4-zellen dauerhaft auf > 200/μl an, können meist primär-und sekundärprophylaxen abgesetzt werden. patienten, die trotz antiretroviraler therapie deutlich unter 200 helferzellen/μl bleiben, müssen dagegen weiter prophylaktisch behandelt werden. viren erreichen den zustand der persistierenden infektion durch unterschiedliche strategien. beim herpes-simplex-virus (humanes α-herpesvirus) kommt es z. b. nach primärinfektion an haut oder schleimhäuten mit lokaler virusvermehrung (produktive infektion) zur zunächst ebenso produktiven infektion zugehöriger sensibler ganglienzellen. im ganglion geschieht dann die "umschaltung" in eine latente infektion, die durch fehlende virusproduktion und jegliches fehlen von viruspartikeln gekennzeichnet ist. das virale dna-genom bleibt extrachromosomal bei minimaler expression einzelner gene in den ganglien. durch bestimmte triggermechanismen (sonbei zytozidaler virusinfektion kommt es am ende des virusvermehrungszyklus zum tod der wirtszelle. es gibt persistierende und nicht persistierende viren, bei denen aus der weiterhin vitalen wirtszelle nachkommen-viruspartikel ausgeschleust werden. während die folgen einer zytozidalen infektion für den organismus entsprechend dem "alles-odernichts-prinzip" wesentlich von art und anzahl der direkt zerstörten zellen abhängen, kommt es bei nichtzytozidalen virusinfektionen eher zu störungen der wirtszellregulation (z. b. embryopathie oder onkogenese) oder auch sekundär zur immunpathogenese. viele viren sind in der lage, durch gezielte modulation der wirtszell-genexpression abwehrmechanismen der zelle und des organismus (immunsystem, apoptose) zu unterlaufen (sog. immunevasion). 13 .14 und › abb. 13.13). die größe der einzelnen gruppen ist je nach erreger, prophylaktischen maßnahmen (z. b. impfungen) und therapiemöglichkeiten sehr verschieden. vereinzelt können persistierend mit hepatitis-b-virus infizierte patienten noch nach jahren die infektion beenden (serokonvertieren) und so aus der gruppe der persistierend infizierten in die gruppe der immunen wechseln. aus der gesamtpopulation (gelb) treten individuen nach infektion in die gruppe der akut infizierten (rosa). der weitere verlauf hängt davon ab, ob viren persistenzmechanismen entwickelt haben, und von der fähigkeit des wirts, eine protektive immunantwort zu bilden. die manifestationsrate bestimmt den anteil der erkrankten und die letalität den der verstorbenen. bei verlust der spezifischen immunität aufgrund einer immunsuppression können zuvor immune in die erneut infizierbare gesamtpopulation oder selten sogar bei einigen viren (z. b. hepatitis-b-virus) in die gruppe der persistent infizierten zurückkehren. viele infektionen verlaufen beim immungesunden subklinisch. primärinfektionen und rekrudeszenzen können aber auch vielfältige erkrankungen hervorrufen. patienten mit immundefekten sind durch diese viren besonders bedroht. erstaunlicherweise sind die krankheitsbilder bei primärinfektion und reaktivierung nicht nur abhängig vom ausmaß, sondern auch von der art der immunsuppression. als beispiel sei die cmv-infektion genannt, die beim immunkompetenten meist asymptomatisch verläuft. bei hiv-infizierten patienten tritt cmv dagegen in erster linie als retinitis und gastroenteritis, beim knochenmarktransplantierten patienten als interstitielle pneumonie und beim nierentransplantierten als nephritis mit gefahr der abstoßung auf. herpesvirusinfektionen sind mit ausnahme von epstein-barr-virus (ebv) und hhv-6 bis hhv-8 der therapie durch verschiedene verfügbare antivirale substanzen gut zugänglich. das herpes-b-virus des rhesusaffen ist auch hochpathogen für den menschen (enzephalitis). herpes-simplex-virus typ 1 und 2 (hsv-1, hsv-2) beschreibung und einteilung es handelt sich eigentlich eher um varianten eines serotyps, da serologisch erhebliche kreuzreaktionen bestehen. virusisolate sind relativ leicht typisierbar und inzwischen kann man auch zwischen hsv-1-und hsv-2-antikörpern unterscheiden. epidemiologie die primärinfektion mit hsv-1 findet mit 2 gipfeln in der frühen kindheit und im jungen erwachsenenalter meist oral statt. die durchseuchung erwachsener mit hsv liegt weltweit je nach sozioökonomischer situation bei 40-95%. die primärinfektion (erster kontakt eines organismus mit hsv) kann auch durch sexualkontakt, dann meist mit hsv-2, erfolgen. bei bestehender oraler hsv-1-infektion mit latenz in kranialen ganglien ist die erste infektion im genitale mit hsv-2 keine primärinfektion, sondern eine klinisch milder verlaufende exogene zweitinfektion (initiale infektion). die prävalenz von hsv-2-antikörpern ist bei erwachsenen in verschiedenen kollektiven sehr unterschiedlich, aber stets geringer als bei hsv-1. die virusvermehrung auf der schleimhaut (infektiosität) beginnt vor dem auftreten der symptome, und die virusausscheidung erfolgt durchschnittlich 7-10 tage lang (max. bis 23 tage). im gegensatz zur hsv-1-primärinfektion kommt es bei hsv-2 offenbar auch zur virämie. ätiologie und pathogenese während der virusvermehrung auf der schleimhaut werden bereits frühzeitig auch nervenendigungen infiziert. über axonalen transport ( der chronische, lokal destruierende mukokutane herpes simplex ist eine aids-manifestation; es entstehen z. b. größere perianale ulzera, die in der differentialdiagnose der klassischen venerischen infektionen durch ihre schmerzhaftigkeit von der lues abzugrenzen sind und einen belegten, weichen ulkusgrund aufweisen. die weitaus häufigste hsv-erkrankung ist der rekurrierende orale oder genitale herpes. beide haben beim immungesunden eine gute prognose, können aber zu erheblichen psychischen belastungen, bis hin zum suizid, führen. orale und genitale primärinfektionen können zu schweren erkrankungen führen, die frühzeitig und intensiv systemisch behandelt werden sollten. die rekurrierende ulzerative herpeskeratitis führt häufig zum visusverlust -ggf. mit der notwendigkeit einer hornhauttransplantation. enzephalitis, konnatale infektion und infektionen immunsupprimierter können sehr rasch zu lebensbedrohlichen erkrankungen führen und müssen daher schnellstmöglich intensiv behandelt werden. die virologische diagnose kann sehr schnell gestellt werden, wenn das geeignete material mit geeigneten methoden untersucht wird. in der akuten phase der mononukleose können 5-20% der zirkulierenden b-zellen ebv-infiziert sein (polyklonale transformation). es treten teils heterophile autoantikörper auf, was diagnostisch genutzt wird (paul-bunnell-test). im regelfall werden die ebv-infizierten b-zellen durch das intakte immunsystem (t-zellen) eliminiert, dies gelingt aber nicht vollständig, sondern es verbleiben einige latent infizierte b-zellen mit der möglichkeit der reaktivierung im späteren leben (s. u.). eine assoziation des burkitt-lymphoms mit ebv ist aufgrund molekularbiologischer und seroepidemiologischer daten gesichert. ebenso eindeutig ist der zusammenhang zwischen ebv und dem nasopharynxkarzinom (npc), das endemisch in einigen gegenden afrikas und v. a. in südchina vorkommt. mit zunehmendem alter wird das bild der infektiösen mononukleose (im) häufiger beobachtet: sie geht einher mit fieber, pharyngitis und tonsillitis mit gräulichen belägen, generalisierter oder zervikookzipital betonter lymphadenopathie, exanthem (selten enanthem), hepatitis und splenomegalie. das fieber dauert ca. 7-10 tage an und fällt wieder ab. es besteht eine kutane anergie wie beim morbus boeck, bei fortgeschrittener hiv-infektion und anderen schweren krankheitsbildern (disseminierte tuberkulose). eine restsymptomatik (subfebrile temperaturen, müdigkeit) kann monatelang anhalten. eine produktive ebv-infektion ist häufig als orale haarleukoplakie am seitlichen zungenrand bei aids und anderen schweren immundefekten nachweisbar. chronisch aktive ebv-infektionen mit lang anhaltenden, rezidivierenden organsymptomen wurden mit familiärer häufung beschrieben. es ist bislang unklar, ob in diesen fällen ein genetischer defekt oder eine besondere virusvariante verantwortlich ist. erkrankungen der blutzellen und immunorgane eine massive b-zell-proliferation mit nachfolgender kontrolle durch induzierte spezifische t-zellen gehört zum krankheitsbild der im. beim "duncan-syndrom" sind die patienten aufgrund eines genetischen defektes nicht in der lage, diese proliferation zu kontrollieren. die latente ebv-infektion wird durch nicht produktiv infizierte b-lymphozyten aufrechterhalten, die durch den nachweis von ebna-2 (ebv-nukleäres antigen) charakterisiert sind. proteine, die von latent infizierten zellen gebildet werden können, sind für die rolle des ebv in der entstehung von tumoren verantwortlich. sehr gefürchtet ist das lymphozyten-proliferationssyndrom bei immunsupprimierten nach transplantation. ebv ist in b-zell-lymphomen bei hiv-infektion, nach organtransplantation und beim morbus hodgkin nachzuweisen. vor allem in asien kommt es gelegentlich zu einer ebv-induzierten überschießenden t-zell-aktivierung, die letztlich zum hämophagozytotischen syndrom führt. weitere organbeteiligungen eine beteiligung von ebv an erkrankungen, die von infektionen des lungenepithels im rahmen einer chronisch aktiven ebv-infektion ausgehen, bis hin zur beteiligung an der idiopathischen lungenfibrose ist vielfach diskutiert worden. myokarditiden können bei im auftreten und die bestimmenden beschwerden während der rekonvaleszenz sein. die pro gnose ist insgesamt gut. die benigne hepatitis mit mäßig erhöhten transaminasen ist typisch bei der primären ebv-infektion. vielfach werden chronische ebv-reaktivierungen als ursache von anhaltenden gastrointestinalen beschwerden und gelegentlich auch hepatopathien angenommen. inwieweit ein kausaler zusammenhang besteht, ist aber meist unklar und auch schwer zu klären, da ebv-reaktivierungen auch bei anderen grunderkrankungen vorkommen. besondere klinische bedeutung hat cmv für alle immuninkompetenten (untergewichtige frühgeborene, transplantatempfänger, tumorpatienten, aids-patienten). es existieren befunde, wonach cmv evtl. auch bei immungesunden in zellen der gefäßwände durch modulation der zellulären genexpression veränderungen hervorruft, die zur entstehung der atherosklerose und zur entwicklung der restenose beitragen. bei immundefizienten und immunsupprimierten hängt die schwere der erkrankung vom ausmaß der beeinträchtigung des immunsystems ab. mit zunehmender dysfunktion der t-zellen nehmen cmv-reaktivierungen und persistierende aktive cmv-infektionen zu. diese kündigen sich noch vor der klinischen manifestation einer cmv-erkrankung durch lang anhaltende intermittierende oder kontinuierliche cmv-ausscheidung meist im urin an. asymptomatische infektionen die primärinfektion verläuft bei immungesunden älteren kindern und erwachsenen in ca. 90% asymptomatisch. symptomatische infektionen sind klinisch von einer infektiösen mononukleose nicht zu unterscheiden. endogene reaktivierungen mit virusausscheidung, die von zeit zu zeit in abhängigkeit von der aktuellen immunkontrolle der infektion durch den organismus ablaufen, werden im allgemeinen nicht bemerkt. die infektion verläuft bei reifen neugeborenen auch asymptomatisch, ist aber von einer u.u. langen cmv-ausscheidung begleitet. hno-erkrankungen ca. 8% aller klinisch diagnostizierten mononukleosen sind cmv-bedingt. die klinischen zeichen treten nach einer inkubationszeit von 20-60 tagen auf. der verlauf ist gutartig; neben fieber, lymphadenopathie, pharyngitis bzw. tonsillitis, hepatitis, splenomegalie und exanthem treten selten blutbildveränderungen (leukopenie, relative lymphozytose mit lymphomonozytären reizformen, thrombopenie) und gelegentlich eine parotitis (dd mumps) auf. cmv bei immunsuppression und aids bei aids-patienten war die infektion vor der intensiven antiretroviralen therapie (haart) am häufigsten mit einer cmv-retinitis (› abb. 13.21) assoziiert, gefolgt von gastrointestinalen erkrankungen und enzephalitis. die aktiven cmv-infektionen bei 25-90% der aids-patienten sind meist folge einer cmv-reaktivierung. die in anderen klinischen situationen bedeutsame diagnostische unterscheidung zwischen primärinfektion und reaktivierung spielt daher bei aids-patienten keine rolle. cmv-enzephalitiden wurden v. a. bei aids-patienten vor einführung von haart häufiger beobachtet. auch die interstitielle pneumonie ist eine der typischen cmv-erkrankungen, die meist bei erheblich immunsupprimierten transplantatempfängern auftritt. cmv-bedingte hepatitis ist häufig bei konnataler infektion, aber auch möglich bei virusreaktivierungen bei immunsupprimierten. gastrointestinale infektionen mit typischen, teils blutenden schleimhautulzera waren vor einführung der intensiven antiretroviralen therapie eine häufige erkrankung bei aids-patienten und werden gelegentlich bei anderen risikopatienten gefunden. ulzerationen können in allen abschnitten des gastrointestinaltraktes auftreten, vom ösophagus bis zum enddarm (› abb. 13.22). cmv spielt eine wesentliche rolle bei nierentransplantierten. neben lang anhaltenden asymptomatischen virusausscheidungen mit dem urin kommt es zu nephritiden und transplantatabstoßungen. eine primäre und sekundäre thrombozytopenie, aber auch trizytopenie ist typisches symptom bei konnataler infektion und häufig erstes symptom einer cmv-reaktivierung bei knochenmarktransplantierten patienten. intrauterine und perinatale infektionen das krankheitsbild der konnatalen zytomegalie umfasst die in › tabelle 13.25 angegebenen symptome. hinzu kommen können weitere symptome, so auch zahnbildungsschäden. cmv ist heute hauptursache einer intrauterinen infektion des fetus (0,2-2,2%). etwa 5% der intrauterin infizierten kinder zeigen das typische bild einer konnatalen zytomegalie (cid mit einschluss des zns: letalität bis 20% und häufig bleibende schäden). die prognose dieser kinder ist schlecht (gesamtletalität 11%). spätschäden (neurologische defizite, hörverlust) sind zu erwarten. weitere 5% der intrauterin infizierten haben geringfügige symptome bei der geburt, die prognose ist sehr viel besser, in 10% ist auch hier mit spätschäden zu rechnen (› tab. 13.25). fetale infektionen nach reaktivierter infektion bei der mutter führen sehr selten zu klinischen manifestationen, und wenn, dann mit deutlich schwächer ausgeprägter symptomatik als bei primärinfektionen und ohne spätfolgen. bei untergewichtigen frühgeborenen besteht auch nach postnataler infektion (z. b. durch muttermilch) ein hohes risiko, an einer schweren systemischen cmv-infektion zu erkranken. diagnostik bei verdacht auf eine cmv-pneumonie ist die röntgenologische untersuchung wichtig. für die diagnose einer aktiven cmv-infektion, aber auch für die bestimmung der prognose und für therapieindikation und -kontrolle stehen heute quantitative nachweismethoden für virale antigene und dna zur verfügung. • virusnachweis: die cmv-isolierung aus urin, bronchiallavage, speichel u.ä. ist in humanen fibroblasten möglich, sie braucht im gegensatz zu hsv aber viel länger. hier kann der nachweis von early-virus-antigen -bereits vor dem erscheinen des zytopathischen effekts in der infizierten zellkultur -die diagnostik beschleunigen (shell vial culture). eine typische histopathologische veränderung sind die eulenaugenzellen, deren nachweis zwar sehr spezifisch, aber wenig sensitiv ist (› abb. 13.23). • nachweis viraler antigene: der quantitative nachweis von cmv-pp65-antigen in polymorphkernigen leukozyten eignet sich zur früherkennung einer systemischen cmv-reaktivierung. das pp65-antigen (s. o.) ist v. a. bei systemischen infektionen immunsupprimierter während der phase der antigenämie überwiegend in polymorphkernigen leukozyten (pmnl) und zirkulierenden endothelzellen zu finden und damit von großer diagnostischer bedeutung. • das verfahren zur isolierung von hhv-6 entspricht dem vorgehen beim versuch der retrovirusisolierung. hhv-7 wurde erstmals 1990 beim gesunden isoliert. mittlerweile ist gesichert, dass in allen untersuchten populationen erwachsene zu 40-100% mit hhv-6 und hhv-7 infiziert sind, dass beide viren auch aus gesunden isoliert werden können und die durchseuchung in früher kindheit beginnt. offenbar erfolgt die hhv-7-serokonversion später. die übertragung geschieht sehr effektiv über den speichel: 50-100% aller hhv-6-infizierten scheiden hierüber das virus aus. im übrigen dienen auch hier die lymphozyten als virusreservoir. die geschichte von hhv-6 zeigt einmal mehr, wie vorsichtig man bei der ätiologischen verknüpfung des nachweises eines ubiquitären virus mit spezifischen symptomen oder syndromen sein muss. ätiologie und pathogenese ätiologisch sind hhv-6b und hhv-7 bei kindern verantwortlich für das exanthema subitum (dreitagefieber). ferner gibt es beschreibungen von schwereren krankheitsfällen. denkbar ist, dass, wie bei den anderen herpesviren, bestimmte immunologische voraussetzungen zu besonderer pathogenität führen. die klinische bedeutung von hhv-6a ist noch unklar. bei immunkompetenten kindern ist das dreitagefieber oder exanthema subitum (es) eine der klassischen "kinderkrankheiten": nach dreitägiger fieberphase kommt es gleichzeitig mit der entfieberung zum stammbetonten kleinfleckigen exanthem. das es ist häufiger begleitet von übelkeit, erbrechen und auch durchfall. bei erwachsenen kann es zum mononukleoseähnlichen krankheitsbild mit langer rekonvaleszenz kommen. bei immunsupprimierten kommen neurologische, pulmonale und hämatologische komplikationen vor. diagnostik im labor ist eine leukopenie mit relativer lymphozytose zu erkennen, eine thrombopenie kann ebenfalls vorliegen. • nachweis viraler genome: hhv-6-dna kann während der akuten infektion durch pcr leicht aus lymphozyten und speichel nachgewiesen werden: nach überstehen der primärinfektion geht die zahl der latent infizierten lymphozyten erheblich zurück, so dass die pcr im peripheren blut nur noch in 10% aller fälle ein positives ergebnis zeigt. durch quantitative pcr lässt sich ein reaktivierungsereignis diagnostizieren. • antikörpernachweis: die serodiagnostik ist in ihrer aussage durch die hohe durchseuchung von ca. 80% im 2. lebensjahr eingeschränkt. für die frische infektion kommen daher die igg-serokonversion und der igm-nachweis in frage -jedoch sind viele igm-nachweisverfahren qualitativ nicht zufrieden stellend. • virusisolierung: aus speichel und lymphozyten kann hhv-6 durch kokultivierung mit stimulierten nabelschnurlymphozyten isoliert werden: die anzucht ist auf wändig, gelingt aber auch bei gesunden virusträgern -und hier eher aus speichel als aus peripherem blut. therapie obwohl die wirksamkeit verschiedener nukleosidanaloga in vitro gezeigt werden konnte, gibt es keine guten daten zur klinischen wirksamkeit. symptomatische therapie. therapieversuche mit foscarnet oder nukleosidanaloga sind bei schweren erkrankungen immunsupprimierter patienten u.u. angezeigt. durch die entdeckung von hhv-6 und hhv-7 wurde endlich das alte rätsel des dreitagefiebers gelöst, von dem man schon seit langem annahm, dass es sich um eine infektionserkrankung handeln könnte. beim immungesunden erwachsenen kommt es gelegentlich zu schwereren, lang dauernden, mononukleoseähnlichen erkrankungen. einzelne fulminante hepatitiden wurden beobachtet. hhv-6 kann zu verschiedenen komplikationen bei immunsupprimierten patienten (pneumonie, enzephalitis) führen. beschreibung und einteilung humanes herpesvirus typ 8 (hhv-8) wurde zunächst über pcr als herpesvirusspezifische genetische information in einem aids-assoziierten kaposi-sarkom (ks) entdeckt. es ließ sich dann als freies virus aus hhv-8-assoziierten b-zell-lymphomen isolieren und als gamma-herpesvirus charakterisieren. epidemiologie antikörper gegen hhv-8 sind im elisa bei fast allen kaposi-sarkom-trägern, bei 30% der hiv-positiven homosexuellen und zum geringen prozentsatz bei blutspendern nachweisbar. damit ist hhv-8 offensichtlich nicht so verbreitet wie andere herpesviren. die antikörperprävalenz macht es wahrscheinlich, dass hhv-8 überwiegend durch sexualkontakte übertragen wird. pathogenese gamma-herpesviren wirken potenziell transformierend. hhv-8-genom wird mit der pcr inzwischen auch in kaposi-sarkomen von therapeutisch immunsupprimierten transplantationspatienten, in spontanen kaposi-sarkomen und den relativ seltenen hhv-8-assoziierten body-cavity-lymphomen nachgewiesen. kaposi-sarkome bestehen typischerweise aus einem gemisch proliferierender spindel-und endothelzellendie zur entstehung führenden mechanismen sind noch ungeklärt. enterovirusinfektionen kommen bei uns ganz überwiegend im sommer und herbst vor ("sommergrippe"). die aus-scheidung der enteroviren beginnt 2-3 tage nach infektion. sie kann einige tage lang oral erfolgen und für mehrere wochen fäkal. bei der übertragung handelt es sich generell um eine enterale "schmutz-schmierinfektion", wobei in den ländern mit hohem hygienestandard die übertragung durch rachensekrete bedeutsamer ist. sehr selten werden bei schweren immundefekten (z. b. bei agammaglobulinämie) dauerausscheider beobachtet. die paralytische poliomyelitis konnte während der letzten 30 jahre in den westlichen industrieländern durch impfung im rahmen des who-eradikationsprogramms drastisch vermindert werden (europa 1951-1955: ca. 50 000 fälle jährlich, deutschland ist seit 1990 frei von infektionen, s. u.). die seroprävalenz der hepatitis-a-antikörper hat in deutschland seit dem zweiten weltkrieg ebenfalls stark abgenommen (kriegsgeneration bis zu 80% seropositiv, studenten heute < 5%); die bedeutung der hepatitis a als reiseerkrankung (entwicklungsländer) nimmt demzufolge zu. das hepatitis-a-virus wird im gegensatz zu anderen picornaviren, die auch oral ausgeschieden werden, nur fäkal und v. a. in der späten inkubationsphase ausgeschieden. pathogenese picornaviridae führen zu unterschiedlich stark ausgeprägter zytozidaler virusvermehrung, also in der regel zu nicht persistierenden infektionen. die partikelproduktion erfolgt zunächst in epithelzellen des nasen-rachen-raums bzw. magen-darm-trakts und in den regionalen lymphknoten und findet erst danach bei einigen picornaviren in typi poliovirusinfektionen (je nach endemischer oder epidemischer situation 1-5%) unterscheidet man 3 verlaufsformen: • bei der abortiven poliomyelitis kommt es nach der inkubationszeit nur zu einer 2-5 tage anhaltenden "grippesymptomatik" (minor illness), wie sie viele enterovirustypen hervorrufen können. nach einer 2-bis 3-tägigen besserung kann es dann zu plötzlicher verschlechterung kommen (hauptkrankheit). • die meningitische poliomyelitis verläuft unter dem bild der prognostisch günstigen aseptischen meningitis, die ebenso durch viele andere enteroviren verursacht werden kann (sehr selten ist die perakute, letal verlaufende enzephalitis). zweiterkrankungen durch rhinoviren sind möglich, verlaufen aber milder. obwohl rhinovirusinfektionen bekanntlich gutartig verlaufen, besitzen sie angesichts der erkrankungshäufigkeit erhebliche ökonomische bedeutung (113 bekannte serotypen und möglichkeit der reinfektion!). jeder mensch macht viele picornavirusinfektionen durch, meist subklinisch oder als milde erkrankung. schwere krankheitsbilder kommen -auch altersabhängig -vor. picornaviren verursachen einige charakteristische erkrankungen und viele uncharakteristische symptome und syndrome. enteroviren und hepatitis-a-virus hinterlassen eine belastbare typenspezifische immunität. bei rhinoviren sind symptomatische reinfektionen bekannt. picornaviren können bei kardiomyopathien und dem juvenilen diabetes mellitus ätiologisch beteiligt sein. viele picornaviren sind leicht isolierbar. die serologie ist wenig aussagekräftig. einige der vielen tierpathogenen picornaviren können den menschen infizieren. beschreibung und einteilung adenoviren sind nackte und sehr umweltresistente ikosaedrische partikel von 70-100 nm durchmesser (› abb. 13.25). sie enthalten doppelsträngige lineare dna. im genus der mastadenoviren gibt es 6 subgenera a-f mit den zunächst serologisch definierten humanpathogenen virustypen 1-51 (hadv 1, … 51). später wurde auch eine genotypische abgrenzung nach homologie der nukleotidsequenz festgelegt. diagnostik je nach manifestation (auge, gastrointestinaltrakt) müssen andere mikrobiologische erreger abgegrenzt werden. die diagnose stützt sich auf virusisolierung und kaum auf die serologie. • virusnachweis: die meisten adenoviren sind aus rachenspülwasser, augenabstrich, stuhl, urin, liquor und anderen proben leicht in zellkulturen zu isolieren. die schwer anzüchtbaren hadv 40 und 41 werden elektronenmikroskopisch oder im antigen-elisa nachgewiesen, der auch schon eine subgenusdiagnose ermöglicht. • nachweis viraler genome: die pcr ermöglicht den nachweis der virus-dna direkt aus klinischen materialien und sogar eine genotypspezifische diagnose. • antikörpernachweis: die serologie (komplementbindungsreaktion, kbr) gestattet die diagnose einer frischen infektion bei nachweis eines antikörperanstiegs, bei gastrointestinalen infektionen kommt es aber nicht immer zu diesem antikörperanstieg. differentialdiagnose picornaviren, aber auch andere respiratorische und gastrointestinale viren. wichtig ist die frühzeitige abgrenzung zur streptokokkentonsillitis. bei schweren adenoviruserkrankungen, v. a. auch bei immunsupprimierten, ist ein therapieversuch mit cidofovir oder ribavirin möglich und indiziert. ribavirin scheint vorwiegend wirksam gegen viren des subgenus c. komplikationen bei angeborenen oder erworbenen immundefekten können adenoviren auch sehr schwere disseminierte infektionen induzieren, die lunge, gastrointestinaltrakt, leber und zns betreffen und fatal verlaufen. adeno-und rotaviren können vereinzelt auch nach ende einer akuten infektion ausgeschieden und bei gesunden nachgewiesen werden, teils gemeinsam mit enteroviren. adenovirusausbrüche auf neugeborenenstationen können sehr schwer, mit hoher letalität verlaufen. • röteln • echo-virus-9-infektion therapie und prophylaxe die therapie mit ribavirin wurde vereinzelt beschrieben und kann bei immundefekten sinnvoll sein. impfung die masern-lebendimpfung, gemäß impfkalender als mumps-masern-röteln-tripelvakzine (mmr) im 12. bis 15. lebensmonat und mit wiederholung im 6. lebensjahr, hat die zahl der masernfälle in deutschland im jahr 1996 auf 520 zurückgehen lassen. der grad der durchimpfung reicht mit 60% aber nicht aus, um die mensch-zu-mensch-übertragung ganz erlöschen zu lassen. das ziel der who, in europa bis zum jahr 2000 die masern auf weniger als 1 erkrankung/100 000 einwohner zu senken und den tod an masern auszurotten, erfordert immunitätsraten von > 95%, die mit einer einmaligen mmr-impfung nicht erreicht werden können. kürzlich ist es auch in deutschland wieder zu größeren masernausbrüchen gekommen. bei masernexposition ungeschützter personen ist ferner die passive immunisierung mit standard-serum-immunglobulin hilfreich (› kap. 13.10). angesichts der pathogenese ist es verständlich, dass die moderne masern-lebendimpfung auch vor der sspe schützt. häufigkeit masernpneumonie (als direkte folge der maserninfektion oder als folge einer bakteriellen superinfektion des geschädigten flimmerepithels) die disseminierung während der inkubation führt bei 50% zur klinisch und labormäßig fassbaren, aber prognostisch günstigen aseptischen meningitis, selten auch zur zerebellaren ataxie. eine enzephalitis ist selten und geht mit psychiatrischen und neurologischen spätschäden einher (verhaltensstörungen, krampfleiden, taubheit, retrobulbärneuritis, hydrozephalus). diagnostik der typische verlauf erleichtert die diagnose. • antikörpernachweis: serologisch lässt sich der antikörperanstieg mit hilfe der kbr nachweisen. die "kbr-antikörper" fallen allerdings 6-12 monate nach erkrankung unter die nachweisgrenze und sind daher zur immunitätsbestimmung ungeeignet. die frage der immunität wird durch nachweis virushüllenspezifischer antikörper im mumps-igg-elisa beantwortet. bei diagnose einer frischen infektion ist die untersuchung auf mumps-igm-antikörper im elisa die methode der wahl. • nachweis viraler genome: rt-pcr weist quantitativ hcv-rna nach und damit die aktive infektion. hcv-spezifische antikörper beweisen eine akute oder chronische, evtl. auch ausgeheilte infektion. der nachweis von hcv-genomen zeigt eine frische infektion, aber auch chronische carrier-zustände mit virusreplikation an; bei ausgeheilten hcv-infektionen wird die pcr negativ. die akute hcv-erkrankung ist meldepflichtig. therapie und prophylaxe therapie der chronischen hcv-infektion durch kombination von pegylierten alpha-interferonen mit nukleosidanaloga (› kap. 15.7.4) . durch untersuchung von blut-und organspendern, ggf. auch von angehörigen von hochrisikogruppen auf hcv-antikörper kann die verbreitung des virus eingeschränkt werden. die symptomatische therapie (analgetika bei arthralgien) ist möglich. impfungen sind nur gegen gelbfieber, fsme und die japanische enzephalitis verfügbar. ein impfstoff gegen dengue-virus müsste alle 4 serotypen erfassen, da teilimmunität gegen nur 1 typ negative auswirkungen (immunenhancement) bei wildvirusinfektion mit einem weiteren serotyp haben kann. komplikationen bei dengue-fieber kommt es v. a. bei sequentieller infektion durch verschiedene serotypen zu schweren krankheitsverläufen. die affenpocken verlaufen beim menschen ähnlich, meist mit viel ausgeprägterer lymphadenopathie. beim ausbruch 1996/1997 in kongo/zaire waren von 511 erkrankungen ca. 80% durch sekundäre mensch-zu-mensch-infektionen verursacht. das virus kann sich offenbar für begrenzte zeit in der fremden spezies mensch ausbreiten. andererseits war die rate an todesfällen unter den infizierten mit 1,5% viel niedriger als die noch in den 80er jahren beobachtete rate von 10%, so dass die who zurzeit eine wiederaufnahme der auch vor affenpocken schützenden vakzinierung ablehnt. haut-und schleimhauterkrankungen das molluscum contagiosum (dellwarze) ist eine harmlose, auf den menschen beschränkte infektion der epidermis, die höchstens kosmetisch bedeutsam ist. nach einer inkubationszeit (1-30 wochen) wachsen meist multiple, wachsfarbene papeln von 3-8 mm durchmesser heran, die bindegewebig gut abgegrenzt sind und nach 2-12 monaten spontan zurückgehen. die voll ausgebildeten knötchen haben zentral eine pore, aus der sämiges, weißliches material ausgepresst werden kann. dieses enthält die elektronenoptisch nachweisbaren viren. sehr häufig erkranken kinder und immunsupprimierte (aids). die übertragung, auch autoinokulation, erfolgt durch direkten kontakt oder durch gemeinsame handtuchnutzung. bei kindern findet man die veränderungen meist im gesicht und an den extremitäten, bei erwachsenen angesichts der sexuellen übertragung am genitale und dessen umgebung. dellwarzen mit längerer persistenz werden mittlerweile häufig bei aids-patienten beobachtet. melkerknotenvirus (kuh) und orf-virus (schaf) sind primär tierische poxviren, mit denen sich andere tierspezies undmeist bei beruflicher exposition -auch menschen infizieren können. kuhpocken-und melkerknotenvirus (beide sind nicht antigenverwandt) werden von tieren durch direkten kontakt auf den menschen übertragen. betroffen sind meist die hände, wobei das kuhpockenvirus vesikuläre veränderungen, das melkerknotenvirus derbe, oft geschwürig zerfallende knoten verursacht. allgemeinsymptome und lymphangitis sind bei den kuhpocken häufiger. diagnostik bei klinischem verdacht kann der erreger leicht elektronenmikroskopisch als quadervirus aus der vesikelflüssigkeit dargestellt werden. vaccinia-, affen-und kuhpockenvirus lassen sich gut auf der chorioallantoismembran anzüchten und differenzieren. der nachweis von dellwarzen bei erwachsenen ist ungewöhnlich und weist auf eine störung der immunabwehr hin; ggf. sollte eine hiv-infektion ausgeschlossen werden. therapie und prophylaxe keine spezifische antivirale therapie bekannt. die pockenimpfung gegen variola major ist nach ausrottung der humanen pocken weltweit ausgesetzt worden. die pocken waren eine der großen menschheitsseuchen und stellen die 1. infektionskrankheit dar, die durch den menschen weltweit ausgerottet wurde. beschreibung und einteilung die ehemalige familie der papovaviridae (› abb. 13.39) wurde in 2 selbstständige virusfamilien aufgeteilt, die papillomaviridae (durchmesser 55 nm, genom 8 kb) und polyomaviridae (durchmesser 45 nm, genom 5 kb). es handelt sich bei beiden um nackte, ikosaedrische partikel mit doppelsträngiger zirkulärer, superhelikaler dna. einige tierische papillomaviren induzieren tumoren, v. a. wenn sie in spezies inokuliert werden, die nicht natürliche wirte sind. epidemiologie die vermehrung der papillomaviren in konventionellen zellkulturen ist nicht möglich und eine typenspezifische serologie war nicht möglich. lange war dagegen bekannt, dass sie übertragbare warzen des menschen verursachen (› abb. 13.40). erst die molekulare genetik ermöglicht pathogenetische untersuchungen und molekulare epidemiologie. die papillomavirustypen sind so als genotypen definiert (< 50% sequenzhomologie = neuer typ). bisher unterscheidet man > 100 hpv-genotypen, die vielfach den krankheitsbildern zugeordnet werden können. polyomaviren sind in form einer latenten infektion bei den meisten menschen vorhanden. pathogenese humane papillomaviren (hpv) verursachen persistierende infektionen. die ätiologische beteiligung bestimmter hpv-typen an der entstehung anogenitaler malignome ist gesichert. die primäre infektion mit den polyomaviren bkv und jcv bleibt meist unerkannt. sie verläuft häufig als milder respiratorischer infekt und führt bei bkv zur latenz in der niere, während das eher neurotrope jcv im zns -weniger ausgeprägt auch in der niere -latent wird. symptome, verlauf und prognose asymptomatische primärinfektionen mit polyomaviren sind die regel und mit papillomaviren sehr häufig. haut-und schleimhauterkrankungen warzen entstehen nach relativ langer inkubationszeit durch produktive virusinfektion mit hpv in den epithelzellen, wobei die virusvermehrung an differenzierung und keratinisierung der zellen gebunden ist. die normalen hautwarzen sind eine selbstlimitierende erkrankung. die seltene, familiär gehäuft auftretende epidermodysplasia verruciformis, assoziiert mit hpv 20 und 36, zeigt beetartig verschiedene warzenformen, die in 30-60% in ein plattenepithelkarzinom übergehen. hno-erkrankungen die juvenile larynxpapillomatose (hpv 6, 11) ist eine hartnäckige und gefürchtete erkrankung, die möglicherweise auf einer infektion im infizierten geburtskanal der mutter beruht. die durch jcv bedingte progressive multifokale leukoenzephalopathie (pml) tritt bei schwer immunsupprimierten (maligne lymphome, v. a. morbus hodgkin, aids, transplantationspatienten) auf und spielt eine rolle in der differentialdiagnose der zerebralen non-hodgkin-lymphome und anderer demyelinisierender erkrankungen (multiple sklerose, lupus erythematodes mit zns-befall). es kommt an mehreren orten zu herden, die meist keine verdrängungserscheinungen verursachen, aber zu großen entmarkungsherden zusammenfließen können. die patienten zeigen zunehmende wesensveränderungen und kognitive störungen, die erkrankung führt 6 monate nach den ersten neurologischen ausfällen zum tode. weitere erkrankungen schwere immundefekte können zur virurie und zystitis durch bkv führen. papillomavirusinfektionen führen zu spitzen kondylomen (hpv 6, 11, 42 u. a.) und intraepithelialen dysplasien der cervix uteri und der vagina (hpv 6, 11, 16). vergleichbare dysplasien sind auch am penis möglich. diagnostik warzen und kondylome werden klinisch leicht erkannt. anders hpv-assoziierte präkanzerosen, die als epitheldysplasien charakteristische zytologische veränderungen im abstrichpräparat ergeben (› abb. 13.41). hier können dna-und rna-hybridisierung hinweisend auf latente oder aktive infektion durch bestimmte hpv-typen sein. eine pml wird zunächst nach kernspintomographie vermutet und virologisch durch jcv-pcr im liquor oder sicherer im biopsat durch pcr oder elektronenmikroskopie diagnostiziert. bkv-infektionen sind häufig mit nierenerkrankungen assoziiert und werden durch pcr leicht und spezifisch im urin nachgewiesen, so dass eine partikelisolierung entbehrlich ist. bei pml kann die niedrig dosierte chemotherapie mit cytosinarabinosid zum rückgang der symptome führen, aber nur bei relativ intakter zellvermittelter immunität. bei transplantierten mit pml ist daher die therapeutische immunsuppression zurückzunehmen -die prognose der pml bleibt insgesamt schlecht. komplikationen die kausale assoziation bestimmter hpv-genotypen (z. b. hpv 16, 18, insgesamt 40 hpv-genotypen von der cervix nachgewiesen) mit weiblichen genitalkarzinomen hat dazu geführt, dass die hpv-diagnostik mehr eingang in die vorsorgeuntersuchung bei der frau gefunden hat. der nachweis von hpv-genotypen der hochrisikogruppe führt zumindest zur engmaschigen kontrolle oder zum aktiven vorgehen bei gleichzeitigen zytologischen veränderungen. die in der westlichen hemisphäre bei pferd, rind und schwein vorkommenden vesiculoviren können als zoonose beim menschen zu grippeähnlichen infekten, myalgien und auf schleimhäuten zu herpetiformen bläschen mit hoher partikelzahl führen. pathogenese das tollwutvirus bleibt nach infektion für stunden bis wochen im bereich der eintrittspforte in der peripherie; es vermehrt sich wahrscheinlich auch in den zellen der quergestreiften muskulatur oder persistiert in makrophagen. dabei kommt es nicht zur nennenswerten protektiven immunantwort. nach eindringen in die peripheren nervenendigungen gelangt es mit dem axoplasmastrom (ca. 3 mm/h) ins zns. nach erreichen des gehirns verursacht es eine enzephalitis, die histologisch (negri-körperchen) nicht sehr ausgeprägt sein muss, und kehrt dann in verschiedene organe in der peripherie "zurück" (z. b. speicheldrüsen) und auch in verschiedene periphere nervenzellen. durch die intrazelluläre entwicklung innerhalb des nervensystems kommt es erst sehr spät zum effektiven kontakt mit dem immunsystem, so dass neutralisierende und diagnostisch verwertbare antikörper in serum und liquor anfangs fehlen können. die inkubationszeit ist umso kürzer (spanne zwischen 7 tagen und mehreren jahren; durchschnitt: 1-2 monate) und die erkrankungswahrscheinlichkeit umso höher, je näher die verletzung am zns liegt (bein: 10%, gesicht: 80%) und je schwerer sie ist. • 2. phase: neurologisch-psychiatrische symptome (verstärkte speichelsekretion, reizbarkeit). "stille wut" mit aufsteigender paralyse, "wilde wut" mit starker unruhe und charakteristischer hydrophobie in 17-80% (muskelspasmen im mund-, rachen-und larynxbereich), anfangs beim versuch zu trinken, später schon bei der visuellen wahrnehmung von wasser oder anderen akustischen und taktilen reizen. die wilde wut verläuft rascher progredient (2-7 tage) als die stille (bis 30 tage). • 3. phase: präfinales koma (3-7 tage). bei intensivmedizinischer versorgung mit beatmung kann der verlauf viel länger sein. inwieweit unterschiedliche virusstämme für verschiedene verläufe verantwortlich sind, ist noch unklar. es gibt 4 berichte über überlebte erkrankungen, wobei alle patienten vorgeimpft waren, so dass es sich eher um impfversagen handelte. die rate tatsächlich erfolgter, aber asymptomatischer infektionen ist nicht bekannt. • virusnachweis: immunfluoreszenznachweis des virusantigens im abdruckpräparat der kornea. postmortale diagnose durch genomnachweis mittels rt-pcr und histopathologisch am gehirn (negri-körperchen) oder durch immunhistologie. die virusisolierung in mäusen und neuroblastomzelllinien aus speichel ist möglich. • nachweis des viralen genoms: über rt-pcr als standardmethode • antikörpernachweis: die serologische diagnose der tollwut (ift, elisa) ist unzuverlässig. therapie und prophylaxe jede tollwutexposition bedeutet lebensgefahr und erfordert beim ungeimpften eine sofortige postexpositionelle, kombinierte aktive und passive immunisierung. nach ausbruch der erkrankung gibt es keine spezifische therapie -die rabies des menschen verläuft tödlich. virostatika zeigten keinen einfluss, doch sind zytokine wie il-12 evtl. interessant. intensivmedizinische maßnahmen wegen hypoxischem zns-ödem und gestörter thermoregulation. bei beruflicher gefährdung (u. a. tierärzte, förster) ist die aktive schutzimpfung indiziert. biss-und kratzwunden mit evtl. tollwutexposition müssen chirurgisch gereinigt, gründlich desinfiziert und mit rabies-immunglobulin umspritzt werden. präventiv lebenswichtig ist die schnelle postexpositionsimpfung (› kap. 13.10) mit inaktivierten vakzinen und bald evtl. gentechnologisch erzeugten impfstoffen. das risiko bei htlv-1-infektion, einen tumor zu entwickeln, liegt bei ca. 1% (5-10% bekommen insgesamt symptome der infektion). die bedeutung von htlv-2 für erkrankungen des menschen ist unklar, obwohl einiges für eine beteiligung bei leukämien spricht. hautmanifestationen im sinne eines kutanen lymphoms sind häufig im rahmen einer adulten t-zell-leukose (atl), an deren entstehung htlv-1 oft beteiligt ist. jedoch ist die inkubationszeit der atl mit 20-30 jahren lang. atl geht einher mit opportunistischen infektionen durch immunsuppression, lymphadenopathie, hepatosplenomegalie, lungeninfiltraten und osteolysen. das zellbild im peripheren blut kann sehr unterschiedlich sein. bei einigen verläuft die erkrankung eher unter dem bild eines lymphoms. htlv-1 ist selten ursache der tropischen spastischen paraparese (langsam fortschreitende myelopathie mit pyramidenbahnzeichen). diagnostik analog zu hiv durch antikörpernachweis und nachweis viraler rna. antikörper treten evtl. erst spät nach infektion auf. die differenzierung zwischen htlv-1 und htlv-2 bedarf manchmal zusätzlicher tests. inwieweit und in welchen ländern blutspender generell auf htlv-1 getestet werden sollten, muss immer wieder aufgrund der epidemiologischen situation geprüft werden. in › abbildung 13.12 (s. 619) ist der verlauf einer infektionskrankheit schematisch auf einer zeitskala durch die begriffe infektion und beginn der erkrankung veranschaulicht. die zeitliche differenz ist die inkubationszeit, die bei vielen infektionskrankheiten ein charakteristisches merkmal darstellt. es wurden frühzeitig erkrankungen des zentralnervensystems beschrieben, bei denen es nicht gelang, ein viruspartikel oder endogene virale nukleotidsequenzen zu identifizieren. heute gilt als sicher: prionen sind erreger von übertragbaren, chronischen, degenerativen, stets letalen erkrankungen des zentralen nervensystems. sie kommen mit ähnlichen erscheinungsformen als subakute enzephalopathien bei menschen und anderen wirbeltieren (rind, schaf, ziege, katze, hirsch, nerz u. a.) vor. beim menschen unterscheidet man folgende krankheitsbilder: • creutzfeldt-jakob-disease (cjd) • neue variante creutzfeldt-jakob-disease (vcjd) • gerstmann-sträussler-scheinker-syndrom (gss) • fatale familiäre insomnie (ffi) • kuru. bei tieren sind hier insbesondere scrapie beim schaf und die bovine spongiforme enzephalopathie (bse) beim rind aufzuführen. m e r k e allen krankheiten ist gemeinsam: • es werden keine entzündlichen prozesse, kein fieber und keine immunantwort beobachtet. • es gibt ein breites spektrum von symptomen, das für das jeweilige krankheitsbild einen charakteristischen schwerpunkt hat. • eine therapie ist gegenwärtig nicht verfügbar, alle erkrankungen führen zum tod. prionen sind nach ansicht der meisten forscher nukleinsäurefreie proteine. der name prion wurde 1982 von stanley prusiner aus der bezeichnung "proteinaceous infectious particles" abgeleitet. die assoziation von prionprotein als wesentlichem bestandteil des infektiösen agens ist zweifelsfrei bewiesen. eine alternative vorstellung geht von einer konzeptionell noch unklaren beteiligung von nukleinsäuren aus, um die existenz von varianten sowie hereditäre aspekte analog zur genetisch determinierten situation etwa in viralen systemen zu erklären. allen bisher bekannten prionen ist gemeinsam, dass es sich um glykosylierte proteine mit ca. 250 aminosäuren, entsprechend molekülmassen von 33-35 kda handelt, die von zellulären genen kodiert werden. transkription und translation sind im gesunden wie im krankhaften zustand unverändert. soweit sequenzdaten vorliegen, handelt es sich um ein evolutionär insgesamt hoch konserviertes molekül insbesondere im bereich der aminosäurepositionen 124-226. die tatsächlich vorhandenen abweichungen in der aminosäuresequenz von prionen verschiedener spezies definieren zusammen mit anderen faktoren (s. u.) die sog. speziesbarriere für eine heterologe infektion. die höhe der übertragungsbarriere ist für sequenzierte prionen im vergleich zueinander zumindest abschätzbar (unterschiede ausgedrückt als zahl der voneinander abweichenden aminosäuren: schaf -rind 7, rind -mensch > 30, maus -mensch 28). das gen für das menschliche prion (prnp) befindet sich auf dem kurzen arm von chromosom 20 und kodiert für ein primäres genprodukt prp c mit 253 aminosäuren. der index c steht für "cellular". das protein trägt am n-und am c-terminus signalsequenzen (22 bzw. 23 aminosäuren), die posttranslational durch zelluläre peptidasen entfernt werden. an das cterminale ende wird anschließend ein gpi-anker (glykosylphosphatidyl-inositol) für die befestigung in der zellmembran angehängt. diese form des prionproteins ist durch zelluläre proteasen leicht abbaubar. im gegensatz dazu lassen sich aus gehirnen von an übertragbarer spongiformer enzephalopathie (tse) erkrankten menschen und tieren isoformen des prionproteins isolieren, die trotz ihrer mit prp c identischen aminosäuresequenz wegen der spezifischen faltung unlöslich und in vitro nur bis auf den c-terminalen rest von 142 aminosäuren (positionen 90 bis 231) abbaubar sind. dieses restmolekül wird auch als prp27-30 bezeichnet und stellt den proteaseresistenten, aber immer noch infektiösen anteil von prp tse dar. die räumliche struktur von prp c enthält nach modellrechnungen drei α-helices und nur geringe β-faltblatt-bereiche, während der nicht spaltbare prp sc -anteil bis zu 30% β-faltblätter und nur einen geringen gehalt an α-helices aufweist. eine klassifizierung der prionen analog oder ähnlich derjenigen der viren gibt es gegenwärtig nicht. sinnvoll ist zurzeit lediglich die unterscheidung aufgrund der betroffenen wirte unter beachtung der tatsache, dass in tiermodellen mehr als 20 verschiedene stämme von prp sc identifizierbar sind, die sich durch die inkubationszeit, den von der krankheit betroffenen bereich der gehirne und das spektrum der klinischen symptome unterscheiden. interessant ist der befund, dass sich verschiedene klinisch definierte phänotypen von cjd verschiedenen fragmentierungsmustern nach unvollständiger proteinase-k-spaltung zuordnen ließen. fragment-und glykosylierungsmuster von cjd und bse lassen nach experimentellen übertragungen auf transgene mäuse eine definition von prionenstämmen zu. insbesondere ergaben sich nach inokulation von wildtypmäusen mit vcjd bzw. bse identische glykosylierungsmuster, d.h., die beiden krankheiten wurden mit hoher wahrscheinlichkeit durch den gleichen prionenstamm hervorgerufen. aggregatbildung und ablagerung der proteaseresistenten prp sc -moleküle werden als pathogenes prinzip angesehen, das mit dem krankheitsbild der spongiformen enzephalopathie assoziiert ist. als mechanismus der aggregation wird spontane autokatalytische bzw. durch prp sc vermittelte umfaltung zellulärer "gesunder" prp c -moleküle in die schwer abbaubaren, aggregierenden tse-prionen angenommen. im gegensatz zu viruserkrankungen kommt es nicht zum einbringen, exprimieren und vervielfältigen eines genetischen programms, sondern zur kumulativen ausbreitung einer strukturform innerhalb einer population bereits bestehender moleküle. die prionenstruktur macht krank! dies ist ein grundsätzlich neues pathogenes prinzip. creutzfeldt-jakob-disease (cjd) cjd ist die am besten bekannte tse-erkrankung, die 1920 von hans g. creutzfeldt bzw. 1921 von alfons jakob beschrieben wurde. gegenwärtig wird sie unter 4 aspekten der entstehung diskutiert als • sporadisch auftretend (spcjd) • genetisch beeinflusst (gcjd) • iatrogen hervorgerufen (icjd) • und neuerdings als variante form (nvcjd), durch aufnahme boviner prionen erzeugt. sporadisch kommt cjd weltweit mit einer inzidenz von etwa 1 fall pro 1 mio. einwohner pro jahr vor. abweichungen resultieren vornehmlich aus der nichtvergleichbarkeit der erhebungsmethoden in den einzelnen ländern. die altersgruppe der 70-bis 80-jährigen ist am häufigsten betroffen. der bisher jüngste patient in deutschland war 23 jahre alt, der älteste 88 jahre, niemals jedoch war ein kind erkrankt. beide geschlechter scheinen gleichermaßen betroffen zu sein. nach dem auftreten erster symptome (kopfschmerz, müdigkeit, schlaf-und appetitlosigkeit, depression) folgt das bild einer rasch voranschreitenden generellen enzephalopathie mit verlust der bewegungskoordination sowie mit demenz. die krankheitsdauer beträgt in etwa 65% der fälle < 6 monate. eine sichere diagnose kann bislang letztlich nur durch neuropathologische untersuchungen gestellt werden. genetisch bedingte creutzfeldt-jakob-krankheit (gcjd) familiäre häufungen von cjd sind bereits in den 30er-jahren des vorigen jahrhunderts beobachtet worden. von zentraler bedeutung scheint der polymorphismus 129 zu sein, der durch das vorkommen der aminosäuren methionin (m) oder valin (v) an der aminosäureposition 129 im prionprotein charakterisiert ist. in england liegt bei 80% der spcjd-fälle homozygotie 129mm vor, im gegensatz zu 40% in der normalbevölkerung. dagegen sind nur 10% der erkrankten heterozygot mv bei einem 50%igen anteil in der normalbevölkerung. alle bekannten nvcjd-fälle sind 129mm-homozygot (s. u.)! die aminosäureposition 129 befindet sich innerhalb des prionmoleküls an einer übergangsstelle zwischen der zweiten α-helix und dem β-faltblatt und könnte daher von wesentlichem einfluss auf die faltung des moleküls sein. das klinische bild wird bezüglich krankheitsbeginn und -dauer stark von der genetischen disposition in bezug auf die codons 129, 178 und 200 geprägt. weitere punktmutationen und insertionen sind ebenfalls von bedeutung. in den familiären fällen ist die inzidenz der erkrankung stark erhöht und geographisch auf bestimmte regionen begrenzt. so findet sich eine jüdische, aus libyen stammende population in israel mit 50-fach häufigerem auftreten von cjd. charakteristisch ist hier der aminosäureaustausch glu200lys. neben der histopathologischen abklärung ist die sequenzierung des prnp-gens zur sicherung der diagnose gcjd erforderlich. iatrogene übertragung erfolgte nach neurochirurgischen eingriffen, durch verwendung unvollständig sterilisierter neurochirurgischer geräte und elektroden, nach transplantationen von kornea und dura mater von verstorbenen sowie nach der verwendung von aus leichen gewonnenem humanem wachstumshormon (hgh) bzw. hypophysen-gonadotropin. das klinische bild entspricht demjenigen von spcjd, in die diagnose ist die krankengeschichte einzubeziehen. im jahr 1995 trat der erste todesfall auf, der einer neuen variante der cjd zuzuordnen ist. bezüglich des krankheitsbildes liegen ähnliche symptome wie bei den anderen formen vor, jedoch sind das niedrige patientenalter (28 als medianes alter für den krankheitsbeginn, gesamtintervall 14-53 jahre) sowie die epidemiologisch wichtige erkenntnis der fast ausschließlichen geographischen beschränkung auf großbritannien hervorzuheben. im mai 2002 waren weltweit 111 fälle bekannt, davon 107 in großbritannien, 3 in frankreich und 1 in irland. klinisch stehen bei krankheitsbeginn hier eher psychiatrische als neurologische symptome im vordergrund, wie depression, angst, erregung, halluzinationen und schmerz, aber auch neuropsychologische auffälligkeiten wie aphasie oder alexie. später kommen die üblichen sensorischen symptome wie ataxie, parese und demenz hinzu. im gegensatz zu spcjd finden sich neuropathologische besonderheiten. es liegen keinerlei hinweise auf familiäre häufungen vor. die übertragung erfolgt mit großer wahrscheinlichkeit durch den genuss von "infektiösen" nahrungsmitteln. durch die normale zubereitung von speisen werden prionen vermutlich nur unvollständig inaktiviert. das auftreten von nvcjd-prionen im gehirn und in den tonsillen ist ein sicheres diagnostisches merkmal. biologische typisierungen von prionen in versuchstieren sind zeitaufwändig und teuer und nicht für diagnostische zwecke geeignet. epidemiologische untersuchungen zeigten allerdings, dass bislang keine risikofaktoren wie berufszugehörigkeit (landwirte, veterinäre, schlachter, abdecker etc), essgewohnheiten oder geographische nähe zu bse-belasteten landwirtschaftlichen betrieben erkennbar sind. diese tse-erkrankung ist mit der inzidenz von einem fall unter 10 mio. einwohnern pro jahr äußerst selten und mit wenigen sporadischen ausnahmen wohl ausschließlich genetisch determiniert. der erbgang ist autosomal-dominant. im vordergrund steht eine punktmutation mit der konsequenz des aminosäureaustausches von prolin durch leucin (pro102leu). hinzu kommen weitere punktmutationen und ein spektrum von oktapeptid-insertionen, die in zusammenwirken mit dem polymorphismus an der position 129 einfluss auf die klinischpathologischen aspekte der amyloidbildung im gehirn haben. erste symptome von gss sind uncharakteristische beschwerden, wie schlafstörungen, psychische veränderungen, gedächtnisverlust, aphasie und alexie, gefolgt von dem spektrum der anderen tse-symptome, die nach völliger dezerebration einige monate bis 2 jahre nach auftreten der ersten symptome zum tode führen. das erkrankungsalter liegt zwischen 30 und 50 jahren. die diagnose erfolgt anhand der neuropathologischen befunde und ggf. durch sequenzanalysen des prnp-gens. gss ist ausschließlich als hereditär anzusehen, die vertikale weitergabe des gss-spezifischen prnp-gens sollte nicht als übertragung eines krankheitserregers bezeichnet werden. es handelt sich um eine äußerst seltene genetisch bedingte erkrankung, die 1986 zuerst bei 5 mitgliedern einer italienischen familie entdeckt wurde. der erbgang ist autosomal-dominant, scheint jedoch nur eingeschränkt penetrant zu sein, da mehrere familienmitglieder die entscheidende prnp-mutation mit der folge des aminosäureaustausches asp178asn aufwiesen, jedoch symptomlos blieben. die gleiche mutation ist auch bei der familiären form der creutzfeldt-jakob-krankheit (gcjd) von zentraler bedeutung, was zu intensiver diskussion der beiden klinisch-pathologisch sehr unterschiedlichen situationen geführt hat. auch hier ist das codon 129 von bedeutung. das zentrale klinische bild der ffi ist geprägt durch einen stark gestörten schlafrhythmus und entsprechende veränderungen in eeg-schlafmustern und endokrinen zirkadianen stoffwechselleistungen. die erkrankung tritt zwischen dem 40. und 60. lebensjahr auf und führt nach 7-18 monaten zum tode. nach zunächst uncharakteristischen stadien liefert die neuropathologische untersuchung astrogliose, vakuolenbildung und amyloidablagerungen. kuru ist der klassische fall einer horizontal übertragenen spongiformen enzephalopathie. sie wurde zuerst 1957 von gajdusek und zigas beschrieben als eine degenerative krankheit des zentralnervensystems in isolierten populationen in neuguinea. seit dem verbot des dort praktizierten rituellen kannibalismus ende der 50er jahre ist die erkrankung im verschwinden begriffen und heute praktisch ausgelöscht. homozygotie für methionin an der codonposition 129 des prnp-gens ist charakteristisch für die erkrankung, die mit hoher wahrscheinlichkeit durch die horizontale übertragung von infektiösem material eines an spontaner creutzfeldt-jakob-krankheit verstorbenen entstanden und durch die kannibalistischen beerdigungsriten epidemisch verbreitet wurde. die infektion ist vermutlich über den intestinaltrakt verlaufen. eindringen der kuru-prionen durch verletzungen während des eröffnens des leichnams und damit verbundene hautkontaminationen sowie konjunktivale und nasale schmierinfektionen sind als übertragungswege ebenso denkbar. die krankheit beginnt mit unspezifischen beschwerden und führt nach neurologischen ausfällen mit ataxie, schweren lähmungen, damit verbundener unterernährung und letztlich völliger motorischer unfähigkeit zum tode. im fall von iatrogener cjd, kuru und nvcjd ist die übertragbarkeit von infektiösen prionen sehr wahrscheinlich bzw. nachgewiesen. seit jahrzehnten wird tiermehl weltweit als zuschlagstoff in der tierfütterung eingesetzt. in großbritannien wurden ende der 70er-bis anfang der 80er jahre in verschiedenen produktionsanlagen unterschiedliche änderungen des herstellungsprozesses vorgenommen, die offensichtlich eine minderung der inaktivierungseffizienz zur folge hatten. heute wird unter dem eindruck der bse-epidemie eine 20-minütige erhitzung auf 133 °c bei 3 bar überdruck als norm gefordert. zur inaktivierung von prionen an chirurgischen instrumenten, die nicht autoklavierbar sind, wird eine einstündige behandlung mit natronlauge oder natriumhypochlorid empfohlen. um risiken inadäquater dekontaminierung zu vermeiden, wird die benutzung von lediglich einmal zu verwendendem material empfohlen. mit zunehmendem verständnis der pathogenitätsmechanismen ergeben sich hinweise auf mögliche therapiestrategien. so ist es denkbar, in den umwandlungsprozess der prp c -konformation direkt einzugreifen. behinderung von eintritt in den wirtsorganismus und transport von prp sc in das zns ist eine weitere möglichkeit. im fall tierischer erkrankungen wären genetische und züchterische maßnahmen denkbar, etwa die aufzucht von tieren, die von individuen abstammen, die künstlich negativ homozygot für das priongen (prp -/-) gemacht wurden. diese tiere sind nicht infizierbar, da sie selbst keine zellulären prionen synthetisieren können, die dann nach dem eindringen von prp sc in die pathogene konformation umgefaltet werden könnten. da die natürliche funktion des genproduktes des zellulären prp-gens und damit die folgen seines verlustes jedoch nicht bekannt sind, ist dieser weg risikoreich und vorerst nicht gangbar; beim menschen ist er sowieso ausgeschlossen. die konventionelle züchtung nicht erkrankender schafe ist gelungen und hat wohl dazu geführt, dass mittlerweile england, neuseeland und australien scrapie-frei sind. von der invasiven mykose ist die systemische mykose abzugrenzen, die nur infolge hämatogener streuung auftritt. invasive mykosen werden durch etwa 100 verschiedene pilzarten hervorgerufen. sie betreffen nahezu ausschließlich abwehrgeschwächte patienten. prädisponierende faktoren sind abdominalchirurgie, zentrale venenkatheter, störung der normalen flora durch breitspektrum-antibiotika, herabsetzung der abwehr durch kortikosteroide, immunsuppressiva und zytostatika, hiv-infektion, diabetes mellitus, transplantation solider organe oder allogener stammzellen. epidemiologie die meisten pilze leben saprophytär. einige können aber in geringer zahl auf haut und schleimhäuten sowie im darmtrakt vorhanden sein, ohne krankheitserscheinungen hervorzurufen. bei einem pilznachweis in entsprechenden materialien stellt sich daher oft die frage nach ihrer relevanz als erreger. liegen keine der oben genannten risikofaktoren vor, so ist der nachweis von hefepilzen im stuhl klinisch nicht relevant. ätiologie und pathogenese die adhärenz der pilze an die wirtszellen ist notwendige bedingung für eine infektion. sie wird durch wechselwirkungen zwischen kohlenhydrat-und proteinstrukturen der pilzzellwand und der wirtszelle verursacht. es können zell-und gewebsschädigende sekretorische proteasen und phospholipasen gebildet werden. außerdem spielen spezielle morphologische eigenschaften der pilze eine rolle, wie z. b. das "switching", der übergang von der sprosspilzform in die hyphenform bei dimorphen pilzen. für die wirtsabwehr gegen die meisten opportunistischen pilze -insbesondere bei den am häufigsten vorkommenden gattungen candida und aspergillus -sind zahl und funktion der neutrophilen entscheidend. makrophagen und monozyten wird zunehmend bedeutung beigemessen, während die t-zell-vermittelte immunität hauptsächlich für die abwehr der obligat pathogenen pilze und von cryptococcus neoformans relevant ist. diagnostik das klinische bild der meisten pilzinfektionen ist uncharakteristisch, damit ist der erregernachweis besonders bedeutend. beweisend ist die kultur aus physiologisch sterilem material (blut, liquor, biospie) oder die histologie an paraffinschnitten. in allen materialien lassen sich pilze mit optischen aufhellern (blankophoren), giemsa-färbung oder gram-färbung (grampositiv) nachweisen. für die genaue identifizierung der pilze ist die kulturelle anzüchtung erforderlich. hierfür werden als selektive medien z. b. sabouraud-agar und chrom-agar verwendet. als weitere diagnostische möglichkeiten gibt es für einige pilze antigennachweise, z. b. galactomannan für aspergillus und β-1,3-d-glucan für candida, aspergillus und andere. serologische untersuchungsverfahren weisen spezifische antikörper nach und sind generell weniger verlässlich. molekularbiologische nukleinsäurenachweise sind nicht ausreichend standardisiert und der kultur in der identifizierung des erregers unterlegen. therapie zur therapie invasiver pilzinfektionen stehen 6 substanzklassen zur verfügung, deren charakteristika in › tabelle 13.34 wiedergegeben sind. aus der gruppe der sprosspilze kommen krankheitserreger vor allem in der gattung candida vor. trichosporon und blastoschizomyces sind sehr viel seltener. candida verursacht bei schleimhautbefall weißliche beläge, den soor (engl.: thrush). dieser kann bei vorliegen von risikofaktoren zu invasiven infektionen (organbefall, fungämie) führen. eine weitere sprosspilzart, cryptococcus neoformans, verursacht nach einem flüchtigen lungeninfiltrat eine meningoenzephalitis bei abwehrgeschwächten, z. b. hiv-infizierten patienten. praxisfall ii ein 76-jähriger befindet sich zur diabeteseinstellung eher zufällig im krankenhaus, klagt über plötzlich einsetzende bauchschmerzen und wird bewusstlos. es liegt ein rupturiertes bauchaortenaneurysma vor, das notfallmäßig operiert wird. seit 7 tagen liegt er beatmet auf der intensivstation, ein perioperativ diagnostiziertes akutes nierenversagen erfordert die dialyse über einen shaldon-katheter. er fiebert auf, blutkulturen werden abgenommen, eine breitspektrumantibiotika-therapie beginnt. drei tage diagnostik im ct der lunge und des kopfes werden uncharakteristische raumforderungen gesehen, die in verbindung mit der grunderkrankung an diese differentialdiagnose denken lassen sollten. eine histologische sicherung ist zwingend erforderlich. therapie neben der radikalen und ggf. wiederholten chirurgischen sanierung besteht die medikamentöse therapie der wahl in liposomalem amphotericin b. die empfehlungen zur dosierung gehen auseinander: 3-10 mg/kg. ist der patient diabetiker, dann wird sich die nierenfunktion unter dieser therapie sehr bald verschlechtern. einzige alternative ist posaconazol 4 × 200 mg. die therapiedauer kann nicht genau abgegrenzt werden, beträgt aber zumindest 1-2 jahre. nach ende der therapie ist eine regelmäßige nachsorge nötig. verlauf und prognose der verlauf hängt von der erfolgreichen behandlung der grundkrankheit und damit im falle eines diabetes von der adhärenz des patienten ab. wird ein diabetes optimal therapiert, kann die infektion überlebt werden. die gesamtsterblichkeit beträgt 30-70%. nach 1-2 jahren kann unter engmaschiger kontrolle eine therapiepause versucht werden. • malaria tertiana und quartana: bei p. vivax-, p. ovale-und p. malariae-infektionen kommt es ungefähr 1 woche nach krankheitsausbruch zur synchronisation der parasitenvermehrung im blut, d.h., die parasiten wachsen synchron heran und zerstören gleichzeitig ihre wirtserythrozyten (p. vivax und p. ovale an jedem 2., p. malariae an jedem 3. tag). diese synchronisation bedingt die regelmäßigen und charakteristischen fieberanfälle. p. vivax und p. ovale hinterlassen sog. hypnozoiten in der leber, die monate und jahre später zu rezidiven führen können. • malaria tropica: p. falciparum neigt nicht zur synchronisierten vermehrung. eine weitere wichtige besonderheit der malaria tropica ist die veränderung der erythrozytenoberfläche durch die heranwachsenden formen von p. falciparum. die befallenen roten blutkörperchen gewinnen dadurch eine besondere affinität zum gefäßendothel. vor allem in den kapillaren bleiben sie am endothel "kleben" (sequestration) und verstopfen sie. die folge sind hypoxie und metabolitenstau im abhängigen gewebebezirk (› abb. 13.56). diese einzigartige eigenschaft der tropica-parasiten bedingt die gefährlichkeit der malaria tropica, die infolge der zunehmenden ischämie in wichtigen organen (gehirn, lunge, niere, herz) innerhalb weniger tage zum tod führen kann. • erysipel: pathognomonisches a-streptokokken-krankheitsbild, das auch tiefere hautschichten betrifft. beginnt mit lokalisiertem erythem mit schwellung, das sich rasch ausbreitet und klar vom normalen umgebenden gewebe abgrenzbar ist begleitet von hohem fieber, schüttelfrost und allgemeinem toxischem krankheitsgefühl. im gesichtsbereich ist es meist selbstlimitierend, bei anderer lokalisation kann es nur durch gezielte therapie geheilt werden. • akutes rheumatisches fieber: nach durchgemachter streptokokkenpharyngitis mit einer latenzzeit von ca. 18-20 tagen. es kommt zu fieber, schmerzhaften schwellungen der großen und mittleren gelenke und zur pankarditis (v. a. als endokarditis: endocarditis verrucosa). nach längerer latenzzeit evtl. syndrom im zns-bereich (chorea minor). andere spätfolgen sind: erythema nodosum und erythema anulare rheumaticum. man führt den gesamtkomplex des rheumatischen fiebers auf eine immunpathogenese zurück. nur bestimmte m-typen von a-streptokokken verursachen diese folgeerkrankung, solche stämme kommen zurzeit bei uns nicht autochthon vor. häufiger betroffen sind (bei uns meist türkische) patienten aus mediterranem gebiet. • akute glomerulonephritis: tritt auch nach hautinfektionen auf. gute prognose v. a. bei kindern. diagnostik einige der erkrankungen (z. b. erysipel und scharlach) sind schon klinisch pathognomonisch. wichtigste mikrobiologisch-diagnostische maßnahme ist der erregernachweis aus blutkultur und abstrich-und punktionsmaterialien. im verlauf stellt man meist serologisch eine titerbewegung der antikörper gegen streptolysin o (aso-, asl-titer) und/oder bei antikörpern gegen dnase b (adb-, streptodornase-titer) fest. faustregel: der asl-titer steigt v. a. bei infektionen im respirationstrakt, der adb-titer meist bei hauterkrankungen an. asl spielt in der diagnostik des rheumatischen fiebers nur eine geringe rolle, adb hat in der diagnostik der akuten glomerulonephritis größere bedeutung. die adb-werte können hier extrem hoch ansteigen. differentialdiagnose extrem hohe asl-bzw. adb-werte treten auch bei plasmozytomen mit entsprechender antikörperspezifität auf. differentialdiagnostisch kommen bei den pyogenen a-streptokokken-infektionen v. a. infektionen durch s. aureus in frage. dies gilt auch für das toxic shock syndrome. beim erysipel muss, v. a. bei immunsupprimierten, eine aeromonasspp.-ätiologie ausgeschlossen werden (cave: andere antibiotikatherapie!). bei streptokokken-folgeerkrankungen müssen auch autoimmunerkrankungen in die differentialdiagnose einbezogen werden. therapie penicillin g als mittel der wahl. dosierung und dauer hängen von manifestation und klinischer fulminanz ab. bei der streptokokkenpharyngitis reicht eine orale therapie über 10 tage (tagesdosis bei 6-12 mega), bei fulminanter sepsis sind tagesdosen bis zu 40 mega nötig, alternativ evtl. cephalosporine. bei penicillinallergie sind makrolidantibiotika bzw. vancomycin alternativen. bei einigen krankheitsbildern (z. b. phlegmone, fasciitis necroticans) sind ferner schnelle und offensive chirurgische maßnahmen nötig. bei streptokokken-folgeerkrankungen spielt auch die antiphlogistische behandlung eine wichtige rolle und evtl. kortikosteroidgabe. ferner ist eine rezidivprophylaxe mit penicillin oder erythromycin für mind. 1-2 jahre indiziert. bei den übrigen a-streptokokken-erkrankungen keine spezifischen prophylaktischen maßnahmen, was auch für die expositionsprophylaxe mit antibiotika bei scharlachausbrüchen gilt. hämolysierende streptokokken der serologischen gruppe b verursachen v. a. peripartale infektionen bei neugeborenen, bis 48 h post partum eine sepsis und 8-14 tage post partum meningitis. zunehmend findet man sie auch bei pyogenen infektionen geriatrischer patienten. hämolysierende streptokokken der gruppen c und g verursachen auch pharyngitis oder wundinfektion. systemisch-septische infektionen fast ausschließlich bei abwehrgeschwächten patienten. vergrünende bzw. nicht hämolysierende streptokokken haben ihren natürlichen standort im oropharynx des menschen, s. bovis im darm von mensch und tier. sie werden als orale streptokokken bezeichnet. sie verursachen: • karies und parodontitis • nativklappen-("endocarditis lenta") und spät-prothesenendokarditis (› kap. 7.9.1). bei nachweis von s. bovis in der blutkultur muss eine kolonerkrankung (karzinom, divertikulitis) ausgeschlossen werden! diagnostik erregernachweis in entsprechenden materialien. pneumokokken sterben wegen ihres starken autolysesystems auf dem transport rasch ab. wichtig sind -vor allem bei pneumonie -blutkulturen. in der meningitisdiagnostik spielen der mikroskopische erreger-und der spezifische antigennachweis (kapselpolysaccharid) eine zunehmende rolle in der spezifischen schnelldiagnostik. der antigennachweis aus sputum oder trachealsekret ist oft unspezifisch. therapie penicillin g. andernorts schon sehr häufig (spanien!) isolierte penicillin-g-resistente pneumokokken wurden bei uns bisher nur selten gefunden. stämme mit nur mäßiger empfindlichkeit gegen penicilline sind auch bei uns häufiger. eine resistenztestung ist daher durchzuführen. alternative substanzen: cephalosporine der 3. generation (z. b. cefotaxim) oder carbapeneme. es besteht die möglichkeit zur aktiven immunisierung. die vakzine beinhaltet die wichtigsten, vor allem bei septischen verlaufsformen vorkommenden kapseltypen. die impfung ist regelimpfung nach stiko für kinder und ältere menschen. epidemiologie natürlicher standort ist der darm von mensch und tier. der infektionsweg ist endogen-hämatogen nach translokation aus dem darm, auch exogene schmutz-schmierinfektionen sind möglich. klinische bilder e. faecalis ist erreger akuter harnwegsinfektionen und adnexitiden (innerhalb einer mischinfektion) der frau. von größerer bedeutung ist die enterokokkenendokarditis (ca. 10%). ferner spielen enterokokken eine rolle als wundinfektionserreger. diagnostik erregernachweis in entsprechenden materialien, v. a. in der blutkultur (endokarditis). • enterokokken-harnwegsinfektionen: aminopenicilline • enterokokkenendokarditis: auch alternativ mezlocillin. grundsätzlich immer kombinierte behandlung mit gentamicin in den ersten 2 wochen. die therapiedauer liegt bei 4-6 wochen. bei penicillinallergie oder bei stämmen mit "high-level"-resistenz (> 2000 mg/l mhk) gegen gentamicin gabe von glykopeptiden (v. a. teicoplanin). gegen glykopeptidresistente enterokokken wirken linezolid und daptomycin. die wichtigsten sind meningokokken und gonokokken der gattung neisseria. branhamella catarrhalis (früher neisseria catarrhalis, kokkoide stäbchen) gehört zur physiologischen rachenflora, kann aber infektionen des oberen und unteren respirationstrakts verursachen. moraxella-und kingella-arten (kokkoide stäb-chen) werden mitunter als opportunistische infektionserreger isoliert. selten werden veillonellen (anaerob) aus pyogenen mischinfektionen isoliert. man unterscheidet die zyklischen allgemeininfektionen typhus und paratyphus von den mit übelkeit, erbrechen und durchfällen einhergehenden enteritissalmonellosen. die übertragung erfolgt überwiegend auf oralem infektionsweg. definition die gattung salmonella hat einen sehr großen arten-bzw. serovarreichtum. es handelt sich um gramnegative stäbchen aus der familie der enterobakterien. nach heute gültiger, aber umstrittener exakter taxonomie ist der einzige humanmedizinisch bedeutende vertreter die spezies salmonella enterica bzw. die subspezies salmonella enterica subspecies enterica mit einer großen vielfalt an serovaren (zur abgrenzung von speziesnamen werden die serovare mit großbuchstaben gekennzeichnet). unter klinischen gesichtspunkten hat sich folgende einteilung bewährt: s. typhi (die abgekürzte form von s. enterica subsp. enterica serovar typhi) ist klassischer erreger des typhus, s. paratyphi (s. enterica subsp. enterica serovar paratyphi) der erreger des paratyphus, unterteilt in die gruppen a, b und c. wichtigste erreger von enteritissalmonellosen sind s. typhimurium (s. enterica subsp. enterica serovar typhimurium) und s. enteritidis (s. enterica subsp. enterica serovar enteritidis). die übrigen enteritissalmonellen werden aufgrund ihrer oberflächenantigene (o-gruppen) und geißelantigene (h-gruppen) typisiert. sie werden aufgrund der antigenformel benannt oder mit einem speziellen namen, der meist nach dem ersten nachweisort erfolgt (z. b. s. coeln). diese repräsentieren dann serovare und keine (!) spezies oder subspezies. diagnostik bei entsprechender reiseanamnese und typischem fieberverlauf lässt sich häufig klinisch die verdachtsdiagnose stellen. richtungweisend sind das auftreten von roseolen, eine leukopenie, das fehlen eosinophiler leukozyten im differentialblutbild mehrerer blutausstriche und eine relative bradykardie. die kulturelle erregerdiagnose gelingt im sehr frühen krankheitsstadium evtl. noch im stuhl, meist aber in der 1. und 2. krankheitswoche nur in der blutkultur, zur erhöhung der sensitivität sollten multiple blutkulturen abgenommen werden. die kulturelle untersuchung von knochenmark ist besonders sensitiv. am ende der 2. krankheitswoche lässt sich der erreger meist wieder aus dem stuhl isolieren. der antikörpernachweis ist für die akutdiagnostik wenig hilfreich. wegen der schwierigen isolierungsbedingungen (selektivnährböden nötig!) kann dies mehrere tage erfordern. ab ende der 1. bzw. beginn der 2. krankheitswoche kommt es zur messbaren antikörperbildung, hohe titer werden ab der 3. woche erreicht. dann kann die diagnose serologisch (widal-reaktion) bestätigt werden. bei sehr früh begonnener antibiotischer therapie kann der antikörpernachweis negativ bleiben. zur differentialdiagnose siehe auch › tabelle diagnostik erregernachweis aus stuhl, operationsmaterial und evtl. aus der blutkultur. antikörpernachweisverfahren (mikroagglutination, elisa, westernblot) spielen in der diagnostik der akuten enterokolitiden keine wesentliche rolle. bei subakut oder chronisch verlaufenden pseudoappendizitisformen und postinfektionssyndromen sind sie dagegen ausschlaggebend. therapie meist selbstlimitierende erkrankung, keine antibakterielle chemotherapie nötig. bei schwerem oder septischem verlauf behandlung mit tetrazyklinen, chinolonen, co-trimoxazol oder aminoglykosiden. definition erkrankungen durch y. pseudotuberculosis sind zooanthroponosen. ein fäkal-oraler infektionsweg wird angenommen, infektionsquelle sind infizierte tiere oder kontaminierte tierische nahrungsmittel. eine besonderheit scheint die affinität von y. pseudotuberculosis zum lymphatischen gewebe im abdominellen bereich zu sein. wichtigste klinische manifestation ist die pseudoappendizitis (mesenteriale lymphadenitis, akute terminale ileitis), die ihren häufigkeitsgipfel in der altersgruppe von 6-14 jahren hat. symptome fieber und schmerzen im rechten unterbauch. extrem selten septischer verlauf, dann v. a. bei stark abwehrgeschwächten patienten. begleitende krankheitserscheinungen sind reaktive arthritis und erythema nodosum. diagnostik erregernachweis aus schleimhautbiopsien, operationsmaterial, seltener aus stuhl. die serologische diagnostik (titerverlauf im mikroagglutinationstest) spielt jedoch eine wichtige rolle. therapie meist keine antibakterielle chemotherapie erforderlich. nur bei schwerem septischem verlauf antibiotikatherapie mit ampicillin, tetrazyklinen oder aminoglykosiden. ätiologie eine der ältesten und gefährlichsten zooanthroponosen, hervorgerufen durch y. pestis. nagetiere, v. a. ratten, sind wichtigstes erregerreservoir, flöhe die wichtigsten vektoren. epidemiologie kommt in europa nicht mehr vor. sporadische fälle werden aus dem nördlichen und südlichen afrika, aus den usa, südamerika und weiten teilen asiens berichtet. jährlich werden weltweit mehrere tausend krankheitsfälle gemeldet. wegen der relativ kurzen inkubationszeit sind auch touristisch eingeschleppte fälle extrem selten (zur meldepflicht s. ifsg nach § 6 abs. 1 nr. 1). • beulenpest: befall der regionalen lymphknoten im abflussbereich der bissstelle des infizierten flohs mit schwerer allgemeinsymptomatik • lungenpest: aerogene übertragung von mensch zu mensch mit hoher letalität. therapie gut behandelbar mit antibiotika (gentamicin oder doxycyclin). die pest verursachte in den letzten jahren nur selten größere epidemien und hat viel von ihrem früheren schrecken verloren. definition die familie der enterobacteriaceae zeichnet sich durch großen artenreichtum aus. viele gattungen besitzen auch humanmedizinische bedeutung. während den salmonellen, shigellen und yersinien überwiegend spezielle krankheitsentitäten zugeordnet werden können, ist dies für die meisten anderen gattungen nicht möglich. daher werden diese enterobacteriaceae-arten als fakultativ pathogene erreger gesondert betrachtet (› tab. 13 in speziellen fällen (z. b. zur sicheren diagnose der lues connata) ist der nachweis spezifischer igm-antikörper in der indirekten immunfluoreszenz erforderlich. bei neurosyphilis wird eine autochthone intrathekale anti-treponemen-antikörperproduktion nachgewiesen. therapie bedeutsam sind frühzeitige diagnose und therapiebeginn im 1., spätestens im 2. stadium. therapie der wahl ist penicillin g oder ceftriaxon, bei penicillinallergie doxycyclin oder azithromycin. zur vermeidung der frühen neurosyphilis muss die therapie hoch dosiert (procain-penicillin g, 2,4 mio. e i.m.) für 14 tage durchgeführt werden, um ausreichend hohe liquorspiegel zu erreichen. bei patienten mit gleichzeitiger hiv-infektion, bei tertiärer syphilis und bei neurosyphilis wird 2 × 10 mio. e penicillin i.v., alternativ 1 × 2 g ceftriaxon für 14 tage verabreicht. der verlauf der erkrankung und vor allem der effekt der therapie müssen durch regelmäßige serologische aktivitätskontrollen (vdrl-test, cardiolipin-kbr) überprüft werden. prophylaxe eine impfung existiert nicht. eindämmung der übertragungswege durch möglichst lückenlose aufklärung und überwachung der risikogruppen. abb. 13.74 syphilis. a) primäraffekt an der glans penis. b) luesexanthem im stadium ii an den fußsohlen. c) alopecia areata, ebenfalls stadium ii. infektionsschutzgesetz: kommentar und vorschriftensammlung . kohlhammer dolin: principles and practice of infectious diseases, 5 th edn ., 2 vols klinische infektiologie . urban & fischer just-nübling: antibiotikatherapie, 11 anthrax antibiotic associated colitis bacillary dysentery bartonellenerkrankungen botulism botulismus brucellosis chlamydial diseases cholera coagulase-negative staphylococci diphtherie enteric e . coli infections enteric fever enteritissalmonellose enterococci epidemic typhus erysipeloid fakultativ pathogene enterobacteracea gas gangrene/edema gonorrhoe gonorrhoea group a streptococci haem . influenzaerkrankungen hwi infektionen durch staphylokokken infektionen durch streptokokken legionärskrankheit legionellosis legionnaires' disease leprosy leptospirose leptospirosis listeriose-pneumokokken listeriosis lyme-borreliose-stadien lyme-borreliosis lyme disease meningococcal meningitis/sepsis mykoplasmenübertragung nocardiosis oculo-genital infections ornithose ornithosis pertussis pint plague pontiac fever pneumococci pseudomonas psittacosis q-fieber relapsing fever rickettsien rickettsial diseases salmonella foodborne disease salmonellose shigellose staphylococcal diseases streptococcal/enterococcal diseases swine erysipelas verfügbare zubereitungsformen aktivimpfstoffe enthalten impfantigene zur aktivierung des immunsystems mit erregerspezifischer bei einzelnen impfungen sind antikörpergrenzwerte für den optimalen impfschutz bekannt. jedoch kann nicht immer von antikörperkonzentration auf effektiven, sicheren schutz vor infektion geschlossen werden • zellvermittelte immunantwort: routinemäßig schwer zu testen. t-lymphozyten haben für die induktion humoraler antikörperantworten (z. b. gegen proteinantigene) große bedeutung und tragen zur bildung des immunologischen gedächtnisses (memory-zellen) bei. der effekt t-zell-abhängiger impfungen lässt sich z. b. durch bestimmung der masern-, mumps-und röteln-antikörper nach mmr-lebendimpfung abschätzen bei der aktiven immunisierung zum aufbau eines dauerhaften impfschutzes unterscheidet man: • lebendimpfstoffe: vermehrungsfähige, attenuierte (abgeschwächte vollkeim-impfstoff) oder in mehr oder minder reiner form immunologisch relevante antigene des erregers (spalt-impfstoff, extrakt-impfstoff, toxoid-impfstoff, subunitvakzine) durch geeignete spenderkontrolle, herstellungsverfahren von infektionskrankheiten hergestellt. beispiel: humanisierter antikörper palivizumab, zum schutz von frühgeborenen unter bestimmten indikationen (z. b. chronische lungenkrankheit) vor der infektion mit dem respiratory syncytial virus (rsv) stuhl kontaminierte) wunden, verletzungen mit gewebszertrümmerung und reduzierter sauerstoffversorgung oder eindringen von fremdkörpern jede auffrischimpfung mit td sollte anlass sein, eine mögliche indikation einer pertussis-impfung zu überprüfen und ggf. einen kombinationsimpfstoff (tdap) einzusetzen im allgemeinen werden 250 ie verabreicht, die dosis kann auf 500 ie erhöht werden; tig wird simultan mit td/t-impfstoff angewendet dosis), wenn seit der letzten impfung mehr als 10 jahre vergangen sind wenn seit der letzten impfung mehr als 5 jahre vergangen sind quelle: rki: empfehlungen der ständigen impfkommission (stiko) am robert-koch-institut/stand: juli ein patient stellt sich 8 tage nach durchführung des 4. zyklus einer ambulanten chemotherapie nach dem chop-r-schema im 14-tägigen abstand mit fieber in der notaufnahme vor. bei der untersuchung des blutbildes zeigt sich eine leukozytenzahl von 600/μl. a. welche untersuchungen werden neben einer ausführlichen körperlichen untersuchung veranlasst? wie wird der patient initial behandelt? seine körperliche leistungsfähigkeit habe abgenommen, vor allem beim treppensteigen komme er leicht "aus der puste". er wisse seit 1,5 jahren, dass er hiv-positiv sei, habe sich aber bislang nicht weiter untersuchen lassen heute habe er deswegen kaum noch etwas zu sich nehmen können. bei der körperlichen untersuchung fällt ihnen auf, dass der rachen des patienten weiße beläge aufweist, die teilweise abstreifbar sind. am hals finden sich beidseits mehrere, bis zu 2 cm im durchmesser große indolente lymphknoten, von denen der patient angibt, dass diese schon seit 1 jahr unverändert bestünden ihr verdacht bestätigt sich. was empfehlen sie dem patienten? welche sind die 3 wichtigsten antimykotika-substanzklassen? welche candida-spezies sind immer oder häufig gegen fluconazol resistent? werden sprosspilzinfektionen endogen oder exogen erworben? was ist die pathogenetische bedeutung der besiedlung der unteren atemwege mit candida species? ist die candidapneumonie eine häufige erkrankung? wie lange muss die therapie einer candidämie durchgeführt werden? 14. werden fadenpilzinfektionen endogen oder exogen erworben? welche 3 erkrankungen der lunge durch aspergillus species werden unterschieden? ein junger mann berichtet, dass er seit 4 wochen unter leibschmerzen und durchfällen, gelegentlich auch unter obstipation leide. die beschwerden seien das erste mal aufgetreten, als er noch in brasilien war, wo er für seine ethnologische doktorarbeit bei urwaldindianern material gesammelt habe. auf befragen gibt er an welche frage ist zunächst zu stellen? b. welche untersuchungen werden durchgeführt? c. wie wird der patient behandelt? d. was wird dem patienten gesagt? er gibt an, er habe seit 4 tagen fieber bis 38,9 °c, außerdem kopfschmerzen, inappetenz, abdominelle schmerzen und übelkeit. er habe keine malariaprophylaxe eingenommen, da man ihm gesagt habe, dass im januar wegen der tiefen nachttemperaturen in nordindien kein malariarisiko bestehe sie gibt an, sich seit gestern stark unwohl gefühlt zu haben, wie in letzter zeit schon öfter während ihrer regelblutung. im büro heute morgen habe sie dann begonnen stark zu schwitzen, verbunden mit hitze-kälte-wallungen, schließlich sei ihr "schwarz" vor augen geworden. befunde der orientierenden untersuchung: rr 80/60 liegend, puls 112, temperatur 39,8 °c rektal. a. wie lautet ihre verdachtsdiagnose? b. welche untersuchungen veranlassen sie zunächst? c. welche therapeutischen maßnahmen sind angezeigt? d die e.-granulosus-larve bildet in der regel eine mit flüssigkeit gefüllte zyste (› abb. 13 .62), die langsam verdrängend wächst und einen durchmesser von 20 cm und mehr erreichen kann: zystische echinokokkose.im gegensatz dazu bildet die e.-multilocularis-larve komplizierte, schwammartige, gallertig gefüllte schläuche und hohlräume von wenigen millimetern durchmesser: alveoläre echinokokkose. der parasit wächst infiltrativ destruktiv, ähnlich wie ein bösartiger tumor; metastasierungen in alle organe können vorkommen.symptome für beide echinokokkosen werden inkubationszeiten von mehreren jahren, bei der alveolären echinokokkose sogar von mehr als 10 jahren angenommen. die symptome sind von der ausdehnung des organbefalls und von der wachstumsgeschwindigkeit des parasiten abhängig. häufig sind oberbauchschmerzen, tastbarer tumor im bereich der leber mit verdrängungsgefühl oder schmerzen, seltener gallenstau und aszites. oft werden die zysten auch nur zufällig entdeckt. bei lungenbefall kommt es zu hämoptyse, atelektasen und bronchiektasen. nicht selten sind allergische reaktionen an haut und schleimhäuten, gelegentlich asthma bronchiale.diagnostik für die diagnostik der beiden echinokokkoseformen sind vor allem sonographie und computertomographie von großer bedeutung. werden zystische veränderungen in der leber oder lunge festgestellt, muss durch anwendung serologischer verfahren versucht werden, eine diagnose zu stellen. werden zwei antikörperbestimmungen unterschiedlichen aufbaus nebeneinander verwandt, so lässt sich in den meisten fällen eine klärung erreichen. die alveoläre echinokokkose lässt bei den verschiedenen bildgebenden verfahren in der regel keine zystische struktur erkennen, zentrale nekrosen im parasitengewebe können diese allerdings vortäuschen. verkalkungen sind häufig. und eiweißerhöhung; sepsis: leukozytose mit linksverschiebung, crp-, procalcitoninerhöhung) wider. therapie entscheidend ist die frühzeitige penicillin-g-therapie in hoher dosierung (20-40 mega/tag). alternativen (vor allem bei penicillinallergie) sind carbapeneme oder cephalosporine der 3. generation. die therapie dauert mind. [10] [11] [12] [13] [14] tage; von anfang an durchgeführte liquorkontrollen müssen keimfrei sein. prophylaxe eine präventive schutzimpfung für die serotypen a und c zur eindämmung von großepidemien ist möglich (ausnahme: säuglinge). gegen neisseria meningitidis typ b gibt es bisher keine impfung. expositionsprophylaxe mit rifampicin wird personen empfohlen, die intensiven kontakt zu einem erkrankten hatten (familie, kindertagesstätten). die erkrankung ist meldepflichtig (s. ifsg nach § 6 abs. 1 nr. 1). verursacht durch neisseria gonorrhoeae (gonokokken), ist sie die häufigste bakterielle geschlechtskrankheit. außerhalb der genitalschleimhaut sterben die bakterien relativ schnell ab. therapie bei erwachsenen gabe von penicillin oder chinolonen (evtl. nur eine dosis!), bei kindern cephalosporine der 3. generation. diphtherie definition erreger ist corynebacterium diphtheriae, und zwar nur diphtherietoxin-bildende stämme. die übertragung erfolgt durch tröpfcheninfektion oder direkten kontakt mit erkrankten oder gesunden keimträgern. epidemiologie 1975 gab es in deutschland zum bisher letzten mal gruppenerkrankungen bzw. kleinepidemien. einzelfälle (meist eingeschleppt aus epidemiegebieten, zurzeit russland, ukraine) kommen immer wieder vor. da bei uns der impfschutz bei älteren jugendlichen und erwachsenen (nicht durchgeführte wiederimpfungen) durch die aktive impfung stark abnimmt, ist ein epidemieartiges auftreten der diphtherie jederzeit möglich.manifest erkranken ca. 20% der infizierten. pathogenese pathogenetisch entscheidend ist das diphtherietoxin, bestehend aus den untereinheiten a (letal zytotoxisch) und b (vermittelt zelleinschleusung) und kodiert von einem prophagen.das toxin wird am ort der infektion produziert und gelangt per continuitatem oder hämatogen zu anderen gewebsbereichen bzw. organen. rachendiphtherie hauptsächliche manifestation mit rötung und schwellung von rachenschleimhaut bzw. tonsillen und schwerem allgemeinem krankheitsgefühl nach bis zu 5 tagen inkubationszeit. hohes fieber ist selten, dann oft zeichen einer primär-toxischen diphtherie. im weiteren verlauf bilden sich nach wenigen stunden weiße beläge auf der schleimhaut, aus denen die bräunlichen pseudomembranen aus fibrin, entzündungszellen und nekrotischen epithelzellen gebildet werden (› abb. 13 .70), die fest auf den wundflächen haften. eine instrumentelle ablösung führt daher zu blutungen (wichtiges diagnostisches kriterium!). von diesen pseudomembranen kann ein sehr charakteristischer fötid-süßlicher geruch ausgehen. die zugehörigen regionären lymphknoten sind deutlich geschwollen. klinischer höhepunkt nach 4-5 tagen. dieser kann im sekundär-toxischen verlauf mit spätkomplikationen, aber auch unter entfieberung in die hei lungsphase übergehen. selten hämatogene metastasierung der erreger.abb. 13 .69 meningokokken (pfeile) im liquor, umgeben von granulozyten. (aus: thomas, 1986 ). • nasendiphtherie (mit eitrig-blutiger sekretion), augendiphtherie (konjunktivitis) und nabeldiphtherie: vorrangig bei säuglingen definition grampositive fadenbakterien, die in verzweigten geflechten wachsen (strahlenpilze). man unterscheidet anaerobe (gattung actinomyces und arachnia) von aerob wachsenden aktinomyzeten (gattung nocardia). nokardien leben im erdboden, die anaeroben aktinomyzeten auf der menschlichen oropharyngealschleimhaut. therapie nach entnahme von material zur mikrobiologischen diagnostik kalkulierte therapie mit antibiotika mit wirksamkeit gegenüber enterobakterien. häufig wird hierzu ein breitspektrum-β-lactam (z. b. piperacillin oder drittgenerations-cephalosporin), ein fluorchinolon oder ein carbapenem eingesetzt (› kap. 13.2.1). grundlage für eine gezielte chemotherapie ist das ergebnis der antibiotikaresistenzprüfung. weltweit wird bei enterobakterien eine zunehmende resistenzentwicklung beobachtet, von der neben älteren substanzen (ampicillin, co-trimoxazol) zunehmend auch fluorchinolone (v. a. bei e. coli) betroffen sind. hinzu kommt das zunehmende auftreten von stämmen, die "extended spectrum"-β-lactamasen (esbls) bilden, die zur resistenz gegenüber allen β-lactam-antibiotika mit ausnahme der carbapeneme führen. besondere therapiemaßnahmen bei septischem krankheitsverlauf (z. b. gabe von antikörpern, kortikosteroiden oder aktiviertem protein c) und supportive therapiemaßnahmen werden an anderer stelle beschrieben. prävention bisher keine spezifischen maßnahmen. die verhinderung nosokomialer infektionen durch fakultativ pathogene enterobacteriaceae beruht v. a. auf präventiven hospitalhygienischen maßnahmen und dem rationalen umgang mit antibiotika. bestimmte darmpathogene e.-coli-stämme können enteritiden und kolitiden verursachen (› tab. 13 symptome 2-phasiger verlauf nach 1-bis 2-wöchiger inkubationszeit mit charakteristischen, wochenlang anhaltenden hustenanfällen im 2. stadium.diagnostik pcr-basierte nachweisverfahren zum nachweis des pathogenetisch bedeutsamen, von b. pertussis produzierten pertussis-toxins stehen im vordergrund. andere verfahren zum nachweis von b. pertussis (direkte immunfluoreszenz, kultureller erregernachweis, serologischer antikörpernachweis) sind heute von geringerer bedeutung.therapie chemotherapie mit erythromycin: geringer einfluss auf den krankheitsverlauf, verkürzt aber die erregerausscheidungsdauer. nach durchgemachter erkrankung besteht eine lang dauernde, aber nicht unbedingt lebenslange immunität.die aktive impfung erfolgt nicht mehr mit dem klassischen "ganzzell"-impfstoff, sondern mit einer "azellulären" pertussis-vakzine mit hoher effektivität und geringen nebenwirkungen (› kap. 13.10). therapie wie bei ornithose. die mögliche ätiopathogenetische bedeutung von c. pneumoniae bei der entstehung arteriosklerotischer gefäßerkrankungen und folgekrankheiten wie z. b. khk und herzinfarkt wird kontrovers diskutiert. eine rolle in der kopathogenese (entzündung!) erscheint möglich, eine monospezifische bedeutung sehr unwahrscheinlich. auf jeden fall rechtfertigt die bisher vorliegende studienevidenz keine spezifischen therapeutischen konsequenzen (antibiotikatherapie). die serovare l1-l3 von c. trachomatis sind für die klassische geschlechtskrankheit lymphogranuloma venereum verantwortlich mit manifestation im genitalbereich und den benachbarten lymphknoten.die serovare a-c von c. trachomatis, die weltweit aber vorrangig in warmen gebieten vorkommen, verursachen die in stadien fortschreitende keratokonjunktivitis, das trachom, die häufigste einzelursache für blindheit weltweit.natürliche habitate der serovare d-k von c. trachomatis sind die zervix die frau und die urethra des mannes. infektionen erfolgen daher stets von dort aus, perinatal oder durch geschlechtsverkehr.klinische bilder krankheitsbilder bei infektionen mit den serovaren d-k sind v. a. infektionen des genitaltrakts: beim mann nichtgonorrhoische und postgonorrhoische urethritis. symptome sind dysurie, urethralschmerzen und -ausfluss. komplizierend kann eine prostatitis bzw. epididymitis hinzukommen.bei der frau verlaufen infektionen oft symptomlos bzw. als urethral-oder dysuriesyndrom, mehr durch unpässlichkeit denn als richtige krankheit gekennzeichnet. daraus kann eine adnexitis bis zum tuboovarialabszess entstehen. ausgehend von ersterer kann sich eine perihepatitis (fitz-hugh-curtis-syndrom) entwickeln mit entsprechender oberbauchsymptomatik. als folge der adnexitis kann es durch verwachsungen zum tubenverschluss mit sterilität bzw. zur extrauteringravidität kommen. nach perinataler infektion kann es zur typischen chlamydienerkrankung des neugeborenen kommen, der sog. einschluss(körperchen)konjunktivitis. die entsprechende erkrankung des erwachsenen (schwimmbadkonjunktivitis) ist heute viel seltener (chlordesinfektion der schwimmbäder).bei schwer immunsupprimierten ist eine c.-trachomatis-pneumonie möglich.als komplikation nach einer c.-trachomatis-infektion gilt eine reaktive arthritis bei bevorzugter betroffenheit von hla-b27-trägern. therapie chemotherapie bei erwachsenen mit tetrazyklinen oder chinolonen (evtl. partnerbehandlung!), bei kindern mit makroliden. synonym: rickettsiosen definition rickettsia species sind kokkoide gramnegative stäbchenbakterien. die bis vor kurzem auch hierzu gerechneten coxiella species werden aufgrund neuer molekulargenetischer untersuchungen heute mit den legionellen in einer eigenen ordnung geführt. dies gilt auch für die ehrlichia species, die mit 2 anderen gattungen die familie anaplasmatacea bilden, und deren zellwand kein lipopolysaccharid und peptitglykan, sondern cholesterin enthält. alle haben einen obligat intrazellulären lebenszyklus und werden mit ausnahme von coxiella bumetii durch arthropoden übertragen. diagnostik pcr-verfahren und serologische tests. an mögliche doppelinfektionen denken (borreliose, fsme; zecken!). therapie tetrazykline, vor allem doxycyclin, und rifampicin. bartonellen sind gramnegative pleomorphe stäbchenbakterien, die früher z. t. als gattung rochalimaea in der familie rickettsiaceae geführt wurden. sie werden heute in einer eigenständigen familie (bartonellaceae) als gattung bartonella eingeordnet. im gegensatz zu den klassischen rickettsien (s. o.) können sie auf spezialmedien in etwa 1 woche kulturell angezüchtet werden. die pathogenese von bartonella-infektionen ist noch weitgehend unbekannt. eine seltene, aber gefährliche manifestation ist die neuroretinitis mit akutem mono-oder bilateralem visusverlust mit papillitis, retinaler vaskulitis und/oder makulaödem. eine weitere wichtige manifestation ist die bazilläre angiomatose mit sepsis, die nur bei immundefekten (hiv!) auftritt. charakteristisch sind generalisierte gefäßproliferationen an haut und schleimhäuten, kombiniert mit septischen zuständen.eine seltene manifestation ist die bazilläre peliosis, gekennzeichnet durch zystische, mit blut gefüllte läsionen in leber und milz.diagnostik mikrobiologisch durch mikroskopischen und kulturellen erregernachweis im spezialverfahren. wichtiger sind immunfluoreszenz-und elisa-verfahren und auch die pcr.therapie tetrazykline und makrolide. impfindikationen und -empfehlungen hängen von folgenden zielen ab:• ausrottung eines erregers (z. b. pocken, aktuell: poliomyelitis (who))• herdenimmunität: manche erreger können in einer bevölkerung nicht mehr epidemisch auftreten, wenn ein bestimmter mindestanteil der bevölkerung ausreichend immun ist.• individualschutz. die impfpolitik eines landes hängt von den epidemiologischen verhältnissen, der verfügbarkeit von impfstoffen und der impfstrategie ab. in deutschland gibt es von der ständigen impfkommission (stiko) öffentliche empfehlungen, d.h. definitionen von regel-oder standardimpfungen. eine gesetzliche impfpflicht besteht hier nicht. allgemein empfohlene standardimpfungen sollen nach einem von der stiko jährlich aktualisierten impfplan bereits im frühen säuglingsalter (ab dem vollendeten 2. lebensmonat; › tab. 13 für aktualisierungen siehe auch www.rki.de * abstände zwischen den impfungen mindestens 4 wochen; abstand zwischen vorletzter und letzter impfung mindestens 6 monate ** generelle impfung gegen pneumokokken für säuglinge und kleinkinder bis zum vollendeten 2. lebensjahr mit einem pneumokokken-konjugatimpfstoff; standardimpfung für personen ≥ 60 mit polysaccharid-impfstoff und wiederimpfung im abstand von 6 jahren *** mindestabstand zwischen den impfungen 4 wochen **** jährlich mit dem von der who empfohlenen aktuellen impfstoff ***** jeweils 10 jahre nach der letzten vorangegangenen dosis meningokokken (serogruppe c). bei erwachsenen sind auffrischimpfungen gegen tetanus und diphtherie in abständen von 10 jahren vorgesehen. für alle erwachsenen nach vollendetem 60. lebensjahr wird eine standardimpfung gegen influenza und pneumokokken empfohlen, aber selten umgesetzt. für personen ohne individuelles risiko, wird in deutschland z. b. bei reisen in länder mit endemischem auftreten der poliomyelitis eine routinemäßige auffrischung der poliomyelitisimpfung nach dem 18. lebensjahr nicht mehr empfohlen.bei den nach § 20 abs. 3 des infektionsschutzgesetzes (ifsg) öffentlich empfohlenen impfungen ist eine kostenübernahme durch die krankenkassen nicht automatisch gegeben, jedoch werden die kosten für diese schutzimpfungen in der regel nach verhandlungen über die umsetzung der stiko-empfehlungen von den verschiedenen kostenträgern übernommen (kassenleistungen nach § 23 abs. 9 sgb v). bei anerkanntem impfschaden nach einer "öffentlich empfohlenen" impfung werden die kosten zur entschädigung und versorgung durch die bundesländer übernommen.neben den standardimpfungen (regelimpfungen) und den zugehörigen auffrischimpfungen gibt es indikationsimpfungen für risikogruppen (z. b. bei bestimmten grunderkrankungen, individuell erhöhtem expositions-bzw. beruflich erhöhtem risiko). wichtig ist die kenntnis der indikationen für reiseimpfungen (z. b. cholera, fsme, gelbfieber, hepatitiden a und b, influenza, meningokokken, tollwut, typhus). die in den stiko-empfehlungen mit r gekennzeichneten reiseimpfungen werden nicht von den krankenkassen übernommen. indikationen bei den regelimpfungen ist die indikation generell gestellt. indikationsimpfungen erfolgen zum individualschutz prä-oder postexpositionell. als domäne der postexpositionellen impfung wird üblicherweise die passive immunisierung empfänglicher (nicht immuner) angesehen, z. b. standardimmunglobulingabe nach masernexposition bei schwangeren, oder hyperimmunglobulingabe nach varizellen-exposition bei immunsupprimierten oder schwangeren. postexpositionelle aktive impfungen (inkubationsimpfungen) bei immungesunden sind möglich und werden allein (z. b. masern, hepatitis a) oder als kombinierte aktiv-/passivimmunisierungen praktiziert (z. b. tollwut, hepatitis b, tetanus, ggf. hepatitis a). die verbreitete furcht vor inkubationsimpfungen hat ihre wurzeln weniger in der immunologie als in der sorge um schadenersatzansprüche bei trotz impfung schwer verlaufender bzw. nicht vermeidbarer erkrankung. impfabstände zeitliche abstände zwischen impfungen mit totimpfstoffen sind nicht erforderlich. lebendimpfungen müssen simultan oder mit 4-wöchigem abstand verabreicht werden. die empfehlungen zu zeitlichen abständen zwischen auffrischimpfungen sind sinnvolle richtschnur für individuelle impfentscheidungen. eine begonnene, aber nicht vollständig durchgeführte grundimmunisierung kann jederzeit fortgeführt und muss nicht von neuem begonnen werden ("jede impfung zählt."). zur konkreten planung und verschreibung von impfungen siehe produktinformationen der hersteller und gültige rote liste (› tab. 13 impfstoffe sinnvoll und anzustreben sind polyvalente extrakt-impfstoffe, die gereinigte kapselpolysaccharide von möglichst vielen infektionsrelevanten kapseltypen enthalten (› tab. 13.46). so gibt es eine polysaccharidvakzine aus 23 verschiedenen polysaccharidantigenen der ca. 90 bekannten pneumokokkentypen. polysaccharid-impfstoffe führen jedoch bei kindern < 2 jahren zu keiner ausreichenden immunantwort. zur bekämpfung schwerer systemischer pneumokokkeninfektionen war daher die entwicklung von pneumokokken-protein-konjugat-vakzinen ein großer fortschritt, da mit einer bei diesen konjugaten gegebenen t-zell-abhängigen immunisierung auch säuglinge effektiv gegen pneumokokken geschützt werden können. indikationen nach eindeutigen erfolgen in den usa wurde ein 7-valenter pneumokokken-konjugat-impfstoff in deutschland zunächst für die höchstgefährdeten frühgeborenen zugelassen. im juli 2006 wurde dieser erstmals als standard für alle kinder < 2 jahren empfohlen (› tab. 13.44). die verabrei-chung des pneumokokken-polysaccharid-impfstoffes (immunantwort t-zell-unabhängig) an personen > 60 jahre ist seit jahren standard. im sinne einer indikationsimpfung sollte bei kindern und erwachsenen mit erhöhter morbidität und mortalität durch pneumokokken eine immunisierung durchgeführt werden. besonders gefährdet sind patienten mit folgenden grunderkrankungen: 1. angeborene oder erworbene immundefekte mit t-und/ oder b-zellulärer restfunktion: -hypogammaglobulinämie, komplementdefekte -asplenie oder nach splenektomie -krankheiten der blutbildenden organe -zustand nach organtransplantation -sichelzellenanämie -hiv-infektion -neoplastische erkrankungen. 2. chronische krankheiten:-herz-kreislauf-krankheiten -krankheiten der atmungsorgane -diabetes mellitus und andere stoffwechselkrankheiten -chronische nierenkrankheiten -neurologische krankheiten -liquorfistel. durchführen der impfung säuglinge sollen die pneumokokken-konjugat-impfung parallel zu den anderen standardimpfungen nach vollendetem 2., 3. und 4. lebensmonat ebenso wie die 4. impfung ab vollendetem 11. lebensmonat erhalten. bei ca. 90% der vollständig immunisierten kinder in den usa lag eine schützende immunantwort gegen alle 7 verabreichten serotypen vor. auch in europa ist trotz der epidemiologischen verbreitung unterschiedlicher serotypen ein erheblicher rückgang von invasiven pneumokokken-infektionen durch die generelle einführung der konjugatvakzine zu erwarten. nebenwirkungen insgesamt gute verträglichkeit der konjugat-impfung, bisher keine berichteten bleibenden schäden. vereinzelt wurden fieberkrämpfe infolge eines raschen temperaturanstieges beobachtet. lokale schmerzen an der injektionsstelle wurden von ca. 20% der geimpften beklagt. der polysaccharidimpfstoff gegen pneumokokken ist wegen seiner zum teil erheblichen lokalen nebenwirkungen (schmerzen, schwellung) vor allem bei auffrischimpfungen in zu kurzen intervallen bekannt, gilt aber als effektiv und sicher. impfstoff von den in deutschland relevanten serogruppen a, b und c können nur a und c durch einen impfstoff erfasst werden. eine impfstoffentwicklung gegen die häufigste serogruppe b war wegen einer strukturähnlichkeit des kapselpolysaccharids mit der n-acetyl-neuraminsäure in gehirnzellen und dadurch bedingter immuntoleranz bisher nicht erfolgreich (› kap. 13.9.3). jedoch wurde in den letzten jahren u. a. in großbritannien ein meningokokken-konjugat-impfstoff gegen die serogruppe c erprobt und so eine deutliche reduktion schwerer meningokokken-typ-c-infektionen erzielt. indikationen die stiko hat im juli 2006 erstmals die impfung aller kinder zu beginn des 2. lebensjahres mit einer einmaligen meningokokken-typ-c-konjugat-gabe empfohlen. die umsetzung in der pädiatrischen praxis und der erhoffte epidemiologische erfolg mit verminderung der lebensbedrohlichen meningokokken-infektionen sind kritisch zu verfolgen. indikation zur meningokokkenimpfung gegen die serogruppen a, c, w135 und y mit einem quadrivalenten polysaccharidimpfstoff besteht bei:• besonderer gesundheitlicher gefährdung (angeborene oder erworbene immundefekte mit t-und/oder b-zellulärer restfunktion, z. b. komplementdefekte, hypogammaglobulinämie, asplenie)• gefährdetem laborpersonal • reisen in endemiegebiete (entwicklungshelfer, medizinisches personal, pilger). nebenwirkungen insgesamt gute verträglichkeit, gelegentlich lokalreaktionen, selten fieber. impfstoff der impfstoff (kühlkettenversand!) wird in embryonierten hühnereiern hergestellt und enthält daher hühnereiweiß (cave: hühnereiweißallergie!). indikationen die indikationsimpfung ist von einigen afrikanischen ländern für reisen in endemiegebiete vorgeschrieben. für reisende nach asien, die aus endemiegebieten einreisen wollen, besteht ebenfalls impf-oder quarantänezwang. die hinweise der who zu gelbfieber-infektionsgebieten sind zu beachten.die gelbfieberimpfung darf nur in von den gesundheitsbehörden zugelassenen gelbfieber-impfstellen durchgeführt werden. die impfung von kindern < 6 monaten gilt als kontraindiziert. schwangere dürfen, besonders im 1. trimenon nur bei strenger indikationsstellung geimpft werden. eine allergie gegen hühnereiweiß stellt eine kontraindikation dar, evtl. kann bei verdacht darauf durch die intrakutane gabe von 0,1 ml des lebendimpfstoffes vorgetestet werden. der impfschutz ist hervorragend (100%) und hält wahrscheinlich lebenslang an. das internationale impfzertifikat ist jedoch nur 10 jahre gültig, d.h., bei reisen in entsprechende länder ist eine wiederimpfung nach 10 jahren nötig, um den rechtlichen vorschriften zu genügen.neben der bekämpfung der vektoren (insekten) ist die impfung der einzige schutz vor gelbfieberepidemien und urbanem gelbfieber. nebenwirkungen sehr gute verträglichkeit, gelegentlich lokale rötungen, vereinzelt kurzfristige grippeähnliche symptome am 4.-6. tag nach der impfung. impfstoff die derzeit in deutschland zugelassenen impfstoffe zur oralen oder parenteralen applikation vermitteln eine schutzrate von 60-90% (kein schutz gegen paratyphusinfektionen) für 1-3 jahre. indikationen• reisen in endemiegebiete • beruflicher umgang mit infizierten oder dem erreger.kontraindikationen diese sind für den oralen lebendimpfstoff gegeben bei:• darminfektionen zum zeitpunkt der impfung • antibiotikaeinnahmen vor dem 3. tag nach beendigung der impfung • kindern im 1. lebenshalbjahr (kapsel).geimpfte scheiden für einige tage den impfstamm aus. die gleichzeitige einnahme von malariaprophylaxe, antibiotika oder laxanzien kann den impfschutz beeinträchtigen. nebenwirkungen ausgezeichnete verträglichkeit, gelegentlich leichte gastrointestinale beschwerden oder kopf-und gliederschmerzen nach den einnahmen. • reisen (entwicklungshelfer) in endemiegebiete, die indikation sollte von fall zu fall, je nach art der reise, gestellt werden.• bei choleraepidemien.durchführen der impfung die derzeit in deutschland zugelassene impfung erfolgt subkutan mit altersabhängiger dosis in form von 2 impfungen im abstand von 1-2 wochen. bei fortbestehender exposition erfolgen auffrischimpfungen im abstand von 3-6 monaten. dauer und schutzwirkung der zugelassenen choleraimpfung sind begrenzt: der schutz beträgt 40-80% für nur ca. 3 monate. daher wurde sie von der who aus den internationalen gesundheitsvorschriften im reiseverkehr herausgenommen.nebenwirkungen heftige lokale beschwerden (rötung, schwellung, schmerzhaftigkeit) sind häufig, systemische reaktionen mit fieber, kopfschmerzen, gastrointestinalen beschwerden selten. die indikationen ergeben sich aus reiseziel, aktuellen epidemiologischen bedingungen und hygienischen verhältnissen. für die zeitplanung ist entscheidend, ob lebendimpfungen und evtl. nachzuholende grundimmunisierungen oder auffrischimpfungen nötig sind (› tab. 13.49).man kann und soll bei vielen grundimmunisierungen und genug zeit bei der reiseplanung die injektionstermine der totimpfstoffe entflechten. die 3. injektion der grundimmunisierung z. b. gegen hepatitis b kann nach der rückkehr oder im reiseland erfolgen. key: cord-011030-o4jn5883 authors: hakki, morgan title: moving past ganciclovir and foscarnet: advances in cmv therapy date: 2020-01-24 journal: curr hematol malig rep doi: 10.1007/s11899-020-00557-6 sha: doc_id: 11030 cord_uid: o4jn5883 purpose of review: cmv dna polymerase inhibitors such as ganciclovir and foscarnet have dramatically reduced the burden of cmv infection in the hct recipient. however, their use is often limited by toxicities and resistance. agents with novel mechanisms and favorable toxicity profiles are critically needed. we review recent developments in cmv antivirals and immune-based approaches to mitigating cmv infection. recent findings: letermovir, an inhibitor of the cmv terminase complex, was approved in 2017 for primary cmv prophylaxis in adult seropositive allogeneic hct recipients. maribavir, an inhibitor of the cmv ul97 kinase, is currently in two phase 3 treatment studies. adoptive immunotherapy using third-party t cells has proven safe and effective in preliminary studies. vaccine development continues, with several promising candidates currently under study. summary: no longer limited to dna polymerase inhibitors, the prevention and treatment of cmv infections in the hct recipient is a rapidly evolving field which should translate into improvements in cmv-related outcomes. introduction i n 1 9 8 9 , g a n c i c l o v i r ( g c v ) b e c a m e t h e f i r s t anticytomegalovirus (cmv) agent approved by the us food and drug administration (fda) for the treatment and prevention of cmv infection and disease, followed by foscarnet (fos), cidofovir (cdv), and valganciclovir ( table 1 ). all of these agents target the cmv dna polymerase encoded by the ul54 gene (pul54) to ultimately inhibit viral dna synthesis. while these agents have dramatically reduced the burden of cmv infection in the hematopoietic cell transplant (hct) recipient [1] , their use is often limited by toxicities such as myelosuppression and renal injury, and the development of resistance [2] . therefore, agents with novel mechanisms of action and improved toxicity profiles are clearly needed. in 2017, letermovir became the first anticmv agent with a mechanism of action other than inhibition of dna polymerase activity to be approved by the fda. this review will discuss recent developments in cmv antiviral agents and non-pharmacological interventions that may augment the ability to prevent and treat cmv infections in hct recipients. cmv genomic replication involves a rolling-circle mechanism that produces multiple genomic units linked in a headto-tail manner (concatamers) [3] . the viral terminase complex cleaves concatameric viral dna into full-length genomes and then packages a single genome into the viral nucleocapsid as part of new virion formation [3] . the core terminase complex is comprised of the proteins pul51, pul56, and pul89; all three proteins are necessary for terminase function [4, 5] . targeting the terminase complex represents an attractive therapeutic option since host cellular dna replication does not require terminase functions and all three terminase proteins are individually essential for viral replication [6] . the first terminase inhibitors were the benzimidazole dribonucleosides such as bdcrb and tcrb [3] . clinical development was halted after preclinical studies demonstrated this article is part of the topical collection on stem cell transplantation unfavorable in vivo metabolism [7] . other terminase inhibitors such as gw275175x [7] and tomeglovir (bay 38-4766) [8] were not brought to clinical trials. letermovir is a 3,4-dihydroquinazoline derivative discovered to have activity against cmv by high-throughput screening of a compound library [9] . the 50% effective concentration (ec 50 ) is in the 0.004-μm range, with a selectivity index > 15,000 [9] . the identification of letermovir resistance mutations l241p and r369s in ul56 along with the finding that letermovir impaired the formation of proper unit-length viral dna genomes indicated that letermovir's mechanism of action involved targeting the terminase complex [10] . due to its mechanism of action, letermovir retains activity against cmv strains resistant to dna polymerase inhibitors but, unlike dna polymerase inhibitors, letermovir does not exhibit significant activity against hhv-6, hsv, or vzv [9, 11] . letermovir can be administered intravenously or orally, is highly (~99%) protein bound, and is eliminated via biliary excretion [12] . letermovir exerts mild-to-moderate inhibitory effects on cytochrome p450 (cyp) 3a and increases exposure to tacrolimus, sirolimus, and cyclosporine [13, 14] ; these require monitoring and dose adjustment as needed when coadministered with letermovir. the dose of letermovir should be reduced by 50% (from 480 to 240 mg/day) when coadministered with cyclosporine [13] . letermovir reduces voriconazole exposure but does not appear to affect posaconazole [15, 16] . letermovir is contraindicated in persons receiving ergot alkaloids and in persons receiving certain statins along with cyclosporine [17] . although letermovir is well tolerated in the setting of mild-to-moderate hepatic and renal impairment, it should be used with caution in severe hepatic impairment (childpugh class c) and insufficient data exist to guide dose adjustments if the creatinine clearance is < 10 ml/min [18, 19] . letermovir was safe and well tolerated in a phase 1 clinical trial [20] . a phase 2 study compared letermovir at doses of 60, 120, and 240 mg daily to placebo for the prevention of cmv reactivation in seropositive allogeneic hct recipients [21] . the incidence of virologic failure, defined as either detectable cmv infection leading to discontinuation of the study drug and administration of preemptive therapy or the development of cmv end-organ disease, was lower in the 240-mg group (6%) than in the 120-mg group (19%), the 60-mg group (21%), and the placebo group (36%). no safety concerns were identified. letermovir prophylaxis was then evaluated in a phase 3, placebo-controlled study in cmv seropositive allogeneic hct recipients [22••] . a subset of patients were categorized as cmv high-risk, including hla-a, b, or dr mismatch related donor, hla-a, b, c, and drb1 mismatch unrelated excludes overlapping toxicities with agents commonly used after hct 3 approved by the us fda (year of approval) for prevention and/or treatment 4 nd, not determined donor, haploidentical donor, cord blood transplant, ex vivo t cell-depleted graft, or graft-versus-host disease (gvhd) of grade 2 or greater requiring ≥ 1 mg/kg/day prednisone (or equivalent). letermovir prophylaxis at 480 mg/day (240 mg/ day if co-administered with cyclosporine) was begun at a median of 9 days after hct and continued through week 14 post-hct, and during this time, weekly cmv pcr monitoring was performed. preemptive therapy was initiated upon detection of viremia according to local practice, with protocol-suggested viral load thresholds of 150 copies/ml in high-risk patients and 300 copies/ml in non-high-risk patients. patients with detectable viremia prior to randomization were excluded from the primary efficacy analysis. letermovir prophylaxis met the primary endpoint of reduction in clinically significant cmv infection (requiring initiation of preemptive therapy or cmv disease) compared with placebo at 24 weeks (17.5% vs 41.8%). since cmv disease was uncommon in both groups, this endpoint was largely defined by reduction in the need for initiation of preemptive therapy; as such, the benefit of letermovir prophylaxis will depend on the viral load threshold for initiating preemptive therapy. importantly, letermovir prophylaxis was associated with a statistically significant reduction in all-cause mortality at 24 weeks, with this benefit being predominantly among high-risk patients. as cmv disease was rare and no single predominant cause of death was identified [23•] , the reason(s) for the mortality benefit at week 24 remain unclear. the reduction in mortality appeared to correlate with the prevention of cmv viremia, raising the hypothesis that the beneficial impact of letermovir prophylaxis may be related to preventing indirect negative effects of cmv infection [24] [25] [26] . adverse events including gastrointestinal effects (nausea, diarrhea), myelotoxicity, and nephrotoxicity were similar in the letermovir and placebo groups. based on these results, letermovir was approved by the fda for primary cmv prophylaxis in adult cmv seropositive allogeneic hct recipients [17] . the finding of letermovir resistance mutations in ul56 was important in elucidating its mechanism of action [10] . subsequent in vitro studies identified multiple additional ul56 resistance mutations, typically located between codons 231 to 369 [27, 28] . mutations in ul89 and ul51 conferring reduced susceptibility to letermovir have also been observed in vitro [29, 30] . letermovir-resistant mutants do not display a significant growth defect compared to wild-type cmv, even with mutations which confer complete (> 3000-fold) resistance, such as at codon c325 of ul56 [27] . resistance appears to evolve more rapidly in vitro compared foscarnet, indicative of a relatively low barrier to resistance [27] . a single case of breakthrough infection with a letermovirresistant strain containing the ul56 v236m mutation during low-dose (60 mg daily) letermovir occurred in the phase 2 prophylaxis study [31] . an analysis of resistance during the phase 3 prophylaxis study was performed, focusing on identifying resistance-associated mutations in ul56 primarily and ul89 secondarily [32•]. ul56 genotyping was successful in 50 out of 79 patients (63%) who received letermovir prophylaxis and experienced cmv infection through week 24. four ul56 resistance mutations were identified in 3 patients (6% of 50 patients analyzed). these 3 patients represented 16.7% of patients (n = 18) who experienced cmv infection while receiving letermovir prophylaxis and for whom ul56 genotyping was successfully performed. one of the 3 patients was viremic at the time letermovir prophylaxis was initiated (viral load < 151 copies/ml) and another patient missed 5 doses of letermovir. of the four ul56 resistance mutations identified, two were previously documented resistance mutations (v236m and c235w), and two were novel mutations (e237g and r369t) at positions previously demonstrated to confer resistance in vitro [27, 28] . there were no ul89 substitutions documented that had previously had been identified as conferring resistance in vitro [30]; ul51 was not analyzed. outside of these studies, cases of breakthrough infection and disease with letermovir-resistant virus have been reported in adult and pediatric hct recipients receiving letermovir primary or secondary prophylaxis [33-35]. with a novel mechanism of action and proven ability to safely and effectively prevent cmv infection after hct, letermovir represents a substantial addition to the cmv antiviral armamentarium that should demonstrably improve cmv-related outcomes in hct recipients. however, important questions remain that will require additional study, including: 1. determining the optimal duration of letermovir prophylaxis. in the phase 3 prophylaxis study, clinically significant cmv infection developed in~10% of patients (2 0% in those at high risk of cmv) between week 14, when letermovir was discontinued, and week 24 [22••] . this raises the question as to whether a longer duration of prophylaxis may be of benefit, as was found for valganciclovir after high-risk (cmv d+/r-) solid organ transplant (sot) [36] . a phase 3 clinical trial will compare 100 vs 200 days of letermovir prophylaxis in cmv seropositive allogeneic hct recipients, with the primary outcome measure being clinically significant cmv infection through week 28 post-hct (nct03930615). high-risk hct populations. high-risk patients comprised 31% of the total study population in the phase 3 prophylaxis study, with haploidentical transplant recipients comprising 14.3%, cord blood recipients 4%, and ex vivo t cell-depleted recipients 2.5% [22••] . additional study is needed, with some data already emerging [37, 38] , to define the relative benefit of letermovir prophylaxis in specific high-risk hct recipients who were relatively underrepresented in the study but for whom the benefit of letermovir prophylaxis appeared greatest. 3. determining whether there is a role for letermovir in preemptive therapy or treatment of cmv disease. the use of letermovir monotherapy for these indications is not currently recommended due to the lack of supporting data. notably, in the phase 3 prophylaxis study, 48 given their distinct mechanisms of action, the combination of letermovir with dna polymerase inhibitors represents an attractive possibility for the treatment of cmv due to the potential for additive or even synergistic antiviral activity. in one study, the combination of letermovir with the dna polymerase inhibitors ganciclovir, foscarnet, and cidofovir demonstrated only an additive, not synergistic, effect in vitro [44] . however, another study found a small degree of synergy between letermovir and brincidofovir, an oral prodrug of cidofovir [45] . more work, both clinical and in vitro, is required to address the potential utility of letermovir-based combination therapy. 4. determining the safety and efficacy of letermovir in pediatric hct recipients. studies of letermovir to date have been limited to adult patients. a phase 2b study of letermovir in pediatric hct recipients is underway (nct03940586) in order to provide much needed information pertaining to optimal dosing, safety, and efficacy in this population. maribavir is an orally available, benzimidazole l-riboside atp competitive inhibitor of the cmv ul97 kinase (pul97) [46] . this mechanism of action was revealed through selection of a resistant virus containing a mutation in ul97 [46] . pul97 is a broadly acting kinase that phosphorylates viral and host cellular proteins [47] . unlike the cmv dna polymerase pul54 or components of the terminase complex, pul97 is not absolutely essential for replication in tissue culture [6] . instead, mutant viruses deleted of the entire ul97 gene, or in which pul97 kinase activity has been abrogated, are viable but display severe growth defects [6, [48] [49] [50] [51] . the critical function(s) of pul97 that contributes to efficient cmv replication and is affected by maribavir to result in inhibition of viral replication remains poorly defined. maribavir inhibits viral egress from the nucleus to the cytoplasm through inhibition of pul97-dependent phosphorylation of the nuclear lamina component lamin a/c [52] , although the relative contribution of this to maribavir's overall antiviral activity remains to be determined. the antiviral activity of maribavir is greatly affected by cell culture conditions, with an ec 50 in human embryonic lung (hel) fibroblast cells of~0.14 μm compared with~13 μm in human foreskin fibroblast (hff) cells [53] . the reason for this difference is not entirely clear, but one possibility is that cell conditions modulate the activity of cellular kinases which can compensate for loss of pul97 activity in the presence of maribavir [53] . indeed, the addition of cellular kinase inhibitors reduces the maribavir ec 50 in hffs to values comparable to hels [53] . since maribavir inhibits pul97 activity and ganciclovir depends on pul97-mediated phosphorylation for its activity, maribavir and ganciclovir are antagonistic [53] . maribavir retains activity against most cmv strains resistant to dna polymerase inhibitors [46, 54, 55] but, similar to letermovir, is not active against other herpesviruses [55] . maribavir is available only as an oral preparation and is3 0-40% absorbed after oral administration [56] . based on studies in animals, maribavir is eliminated mainly by biliary excretion [56, 57] . maribavir clearance is not affected by renal impairment [58] . maribavir is not a significant inhibitor of major cyp enzymes and does not affect voriconazole exposure [59] . however, maribavir increases tacrolimus exposure by~50% [60] , and therefore monitoring of tacrolimus and sirolimus when co-administered with maribavir is recommended [59, 60, 61•]. phase 1 clinical trials of maribavir evaluating doses up to 1200 mg twice daily showed maribavir to be safe and well tolerated, with the most common side effects being taste disturbance and headache [56, 62] . maribavir was then evaluated in a multicenter, randomized, double-blind, placebo-controlled, dose-ranging phase 2 prophylaxis study in adult cmv seropositive allogeneic hct recipients [63] . the doses of maribavir evaluated were 100 mg twice daily, 400 mg once daily, and 400 mg twice daily to start at engraftment (between 14 and 30 days after hct) and continue for a maximum of 12 weeks after hct. the primary endpoint was the incidence and time to onset of cmv infection or disease. all doses of maribavir demonstrated reduction in cmv infection as determined by detection of pp65 antigenemia or dna pcr. cmv disease occurred in only 3 participants, all of whom were randomized to receive placebo. adverse events were more common in the group receiving 400 mg twice daily compared with lower doses of maribavir and placebo; these consisted primarily of taste disturbance (31%) and gastrointestinal disturbances (28%) (nausea, vomiting, abdominal pain, dysphagia). all doses yielded similar trough concentrations but peak plasma concentrations were~2.5× higher following 400 mg doses compared with 100 mg doses, and the 24-h drug exposure was greatest in those taking 400 mg twice daily. a subsequent phase 3 study evaluated maribavir at 100 mg twice daily compared with placebo for the prevention of cmv infection and disease in allogeneic hct recipients [64] . the dose chosen was based on the lack of a dose-dependent effect on the incidence of cmv infection and an increase in adverse effects noted at the highest dose of 400 mg twice daily in the phase 2 prophylaxis study [63] . maribavir was begun following engraftment (median of 24 days post-hct) and administered for 12 weeks after hct. disappointingly, maribavir prophylaxis failed to show a reduction in cmv infection or disease compared with placebo at either day 100 or 6 months after hct. similarly, maribavir prophylaxis at 100 mg orally twice daily did not meet noninferiority compared with oral ganciclovir for the endpoints of prevention of cmv infection or disease in high-risk (cmv d+/r−) liver transplant recipients [65] . the reason(s) for the failure of maribavir in both studies are unclear [66, 67] . with the negative results of maribavir prophylaxis in both hct and sot recipients, maribavir was not further pursued for cmv prophylaxis. contemporaneous with prophylaxis studies, maribavir was being used under individual emergency investigational new drug applications as salvage therapy in situations of resistant or refractory infection. an initial experience described 6 patients (5 sot recipients, 1 hct recipient) treated with salvage maribavir at a starting dose of 400 mg twice daily [68] . five patients had proven cmv end-organ disease and 4 patients had ganciclovir-resistant strains. viral loads at initiation of maribavir ranged from 7200 to 1,811,171 copies/ml. four patients cleared viremia after 6-41 days of maribavir; one of these patients was receiving fos concomitant with maribavir. a randomized, double-blind phase 2 study in hct and sot recipients with resistant or refractory cmv infections was subsequently performed [61•] . patients were randomized to receive 400, 800, or 1200 mg maribavir twice daily for up to 24 weeks. forty patients were randomized to each treatment dose arm; of these approximately 40% were hct recipients. 64.2% had asymptomatic infection, and end-organ disease was present in 13.3%. the median viral load at baseline was 3.7 log 10 copies/ml. eighty-six patients (72%) achieved an undetectable viral load. cmv-associated clinical manifestations improved in 24/34 (71%) patients and resolved in 6/34 (18%) by week 6 of treatment. twenty-five patients (29%) who cleared viremia subsequently experienced cmv recurrence while receiving maribavir; 13 (52%) of these were associated with the emergence of maribavir-resistant virus (discussed below). the recurrence rate was lower among hct recipients (26%) than among sot recipients (40%) despite similar rates of virologic clearance (70% vs 64%, respectively). all efficacy endpoints were similar across all doses. altered taste was the most common adverse effect (65%) but this resulted in treatment discontinuation in only one patient. overall, this study demonstrated that maribavir may be a valid option in the setting of resistant or refractory infection, but the high rate of recurrence while on therapy and the associated emergence of maribavir resistance represent cause for concern. a phase 3 study of maribavir in hct and sot recipients with resistant or refractory cmv infections is underway (nct02931539). in parallel, a phase 2, open label study comparing maribavir to valganciclovir as preemptive therapy (absence of symptomatic infection or end-organ disease) following hct or sot was performed [69•] . patients were eligible if they had a cmv dna viral load of 1000 to 100,000 copies/ml in blood or plasma. patients were assigned to receive oral maribavir 400 mg, 800 mg, or 1200 mg twice daily or valganciclovir at a 900 mg twice daily for weeks 1 through 3 followed by 900 mg once daily for up to 12 weeks. the primary efficacy endpoint was the response to treatment, defined as undetectable cmv dna in plasma within 3 weeks or 6 weeks after the start of treatment. overall, 62% and 79% of patients had an undetectable viral load within 3 weeks and 6 weeks of maribavir treatment, respectively, compared with 56% and 67% for valganciclovir. no dose-depended effect of maribavir on clearance of viremia was observed. the percentage of patients with recurrence of cmv infection at any time during the trial period was similar between maribavir and valganciclovir (22% vs 18%). similar to other studies, altered taste was the most common adverse effect of maribavir (4 0%), followed by other gastrointestinal adverse effects (nausea, vomiting, diarrhea). myelosuppression was more common in those receiving valganciclovir. a phase 3 trial of maribavir 400 mg twice daily versus valganciclovir for the treatment of first episodes of asymptomatic cmv infection in hct recipients with a plasma viral load of ≥ 1365 international units (iu)/ml and ≤ 91,000 iu/ml is now underway, with the primary outcome measure being clearance of viremia by 8 weeks of treatment (nct02927067). the ul97 mutations v353a, l397r, t409m, and h411l/n/ y emerge in vitro during maribavir selection and confer moderate-to-high level (9-fold to > 200-fold) resistance [46, 70, 71] . the first report of resistance during clinical use developed in a patient receiving maribavir as salvage therapy for cmv infection and was associated with ul97 t409m and h411y mutations [72] . resistance was not documented in either the phase 2 or 3 prophylaxis studies in hct recipients [63, 64] . genotypic analysis of maribavir-breakthrough infections from the phase 2 salvage study revealed de novo resistance mutations in 13 of 25 (52%) (t409m in 10, h411y in 3) patients; development of resistance was equal across all maribavir doses [61•] . in general, resistance mutations in ul97 that arise during ganciclovir or maribavir selective pressure do not confer cross-resistance to the other agent [54, 73] . surprisingly, however, one patient in the phase 2 salvage therapy study was retrospectively found to have a novel ul97 mutation f342y after prolonged ganciclovir exposure but prior to beginning maribavir [74] . this mutation was found to confer gcv resistance and, unique to ul97 mutations selected during gcv exposure, cross-resistance to maribavir (4.5-fold). the patient went on to develop a ul97 h411y mutation and eventually failed maribavir therapy. mutations in another cmv gene, ul27, arise under maribavir selection in vitro and also during propagation of ul97-defective strains [75] [76] [77] , suggesting that mutations in this gene represent a fundamental mechanism of compensating for lack of pul97 kinase activity. however, mutations in ul27 confer low-grade (~2-3-fold) resistance to maribavir [75] [76] [77] and have not been identified during clinical use. filociclovir (formerly "cyclopropavir") is a second-generation methylenecyclopropane nucleoside analog of 2′deoxyguanosine [78] . filociclovir, similar to gcv, is a nonobligate chain terminator of dna synthesis that requires initial phosphorylation by pul97, followed by additional phosphorylation steps performed by cellular kinases to its active triphosphate form [79] [80] [81] [82] . filociclovir ec 50 values for cmv strains are approximately 0.2-0.3 μm, which are~5fold less than for gcv [78, [83] [84] [85] . the increased potency of filociclovir compared with gcv in vitro may reflect the findings that filociclovir is a better substrate for pul97 than gcv [80, 86] and the cmv dna polymerase incorporates filociclovir-triphosphate into dna more efficiently than gcv-triphosphate [82] . filociclovir displays little cytotoxicity at concentrations required to inhibit cmv replication in a variety of cell types [81] and demonstrated efficacy in a mouse model of cmv infection [87] . in addition to cmv, filociclovir is active against hhv-6 but not hsv1, hsv2, or vzv [81] . in single-dose studies, oral bioavailability in rats and dogs ranged from 22 to 46% and 70 to 91%, respectively [88] . in single-dose rat toxicology studies, filociclovir was well tolerated up to 300 mg/kg [88] . preclinical studies and data from a single-dose (range 35-1350 mg) human study suggest that filociclovir is primarily eliminated via renal excretion [89] . an l-valine ester prodrug, valcyclopropavir, with 95% bioavailability in mice was synthesized [90] but has not been further developed for clinical use at this time. a phase 1b ascending dose (100 mg, 350 mg, or 750 mg once daily for 7 days) trial was conducted in normal, healthy volunteers [89] . no serious adverse events were reported. drug exposure plateaued around the 350 mg dose. the mean plasma concentrations exceeded the cmv in vitro 90% inhibitory concentration (ic 90 ) for doses ≥ 100 mg per day. filociclovir selection in vitro generates resistance mutations at canonical ul97 gcv resistance sites m460, h520, and c603 [2, 83, 85] . filociclovir also selects for novel ul97 mutations at positions f342 and v356, both of which individually confer cross-resistance to gcv and maribavir [51, 91] . resistance mutations in the ul54 dna polymerase also emerge under filociclovir selective pressure in vitro, some of which result in cross-resistance to gcv and/or fos [85] . conversely, filociclovir has been assayed against a variety of genotypically defined resistant cmv strains. mutation at ul97 codon l595, one of the residues commonly involved in gcv resistance [2] , confers no filociclovir resistance [83, 91] . however, mutations at the other canonical gcv resistance sites including m460, h520, c592, a594, and c603 [2] result in 3-20-fold increases in filociclovir ec 50 values [83, 91] . thus, cross-resistance between filociclovir and gcv and/or fos may occur depending on the site of mutation. brincidofovir (cmx001) is an oral lipid conjugate formulation of cidofovir with potent cmv activity [92] . in a phase 3 study in cmv seropositive allogeneic hct recipients, brincidofovir prophylaxis for 14 weeks post-hct did not meet the primary endpoint of prevention of cmv infection at week 24 compared with placebo [93•] . brincidofovir was associated with significant gastrointestinal toxicity including acute gvhd and diarrhea [93, 94] . as such, oral brincidofovir is not being further developed as an anticmv agent. in 1999, the institute of medicine, now the national academy of medicine, designated cmvas a highest priority for vaccine development [95] . this has proven a challenge, and there are no vaccines currently available for use. asp0113 was a dna vaccine encoding glycoprotein b (gb), which is capable of eliciting neutralizing antibodies, and the tegument protein pp65, which is a primary target of t cell responses [96, 97] . unfortunately, asp0113 failed to meet primary (overall mortality, cmv disease) or secondary (time to viremia and use of preemptive therapy) endpoints in a placebo-controlled, phase 3 study in hct recipients [96] . asp0113 also failed to meet the primary endpoint of reducing the risk of viremia through 1 year after transplant compared with placebo in a phase 2 study in cmv d+/r-renal transplant [98] . more recently, vaccine development has focused on incorporating the pentameric complex [96, 99] . the pentameric complex consists of gh/gl/pul128/pul130/pul131, is required for cmv entry into several clinically relevant cell types, and elicits potent neutralizing antibody responses that block entry into those cells [96, 99] . a cmv vaccine candidate (v160) incorporating the pentameric complex was constructed from the live attenuated cmv ad169 strain that was further engineered to be replication-defective in the absence of a synthetic compound called shield-1 [100] . recently, this vaccine was found to be safe and elicited robust levels of neutralizing antibodies and t cell responses when administered to cmv-seronegative subjects in a phase 1 study [101, 102] . several other candidate vaccines are currently being evaluated in phase 1 and 2 trials in adult and pediatric hct recipients [96] . adoptive immunotherapy denotes the reconstitution of cmvspecific t cell responses via the isolation, in vitro propagation, and transfusion of donor t cells to the recipient [103] [104] [105] [106] . adoptive immunotherapy has been safely used in hct recipients as an adjunct to antiviral therapy for preemptive therapy and for the treatment of refractory cmv infection, and prophylactically after hct, all in relatively small series [105, [107] [108] [109] [110] [111] [112] [113] [114] . however, the need to generate specific t cell lines for each individual patient imposes logistical limitations for broad or immediate, time-sensitive use [115] . using partially hlamatched, banked third-party cells addresses these limitations [115] . the safety and tolerability of this approach in the management of refractory cmv infection or disease has been demonstrated in several nonrandomized studies [115, 116•, 117•] . the majority of patients in these studies exhibited clinical and/or virologic responses following t cell infusion along with continued antiviral therapy. thus, the incremental benefit of the transfused t cells is unclear. randomized studies are now needed to definitively assess the benefit and safety of adoptive immunotherapy for the prevention or treatment of cmv infection in the hct recipient [118] . the utility of intravenous immune globulin (ivig) or cmv-enriched igg in the management of cmv disease is unclear due to the lack of prospective, randomized trials evaluating the benefit of adjunctive ivig compared with antiviral therapy alone. while not useful in the setting of gastrointestinal disease [119] , the addition of ivig to antiviral therapy in the management of pneumonia resulted in improved survival rates compared with historical controls in small studies [120] [121] [122] . however, a more recent, large retrospective analysis failed to demonstrate such a benefit [123] and therefore, the role of ivig in the management of cmv pneumonia remains poorly defined. ivig is not effective as prophylaxis in seronegative or seropositive hct recipients [124] [125] [126] [127] [128] [129] [130] [131] [132] . a monoclonal antibody preparation that targets both the cmv glycoprotein b (gb) and the pentameric complex is in development [133] . a previous cmv monoclonal antibody that targeted the cmv gh protein (msl-109) failed to demonstrate benefit when used as prophylaxis in hct recipients [134] . the cmv dna polymerase inhibitors gcv, fos, and cdv, while critical developments in reducing the morbidity and mortality associated with cmv infection in hct recipients, are marked by issues of toxicity and resistance that often limit their use. the approval of letermovir-a nontoxic, orally available agent with a mechanism of action distinct from dna polymerase inhibition-represents an important step in expanding the options for cmv prevention and towards the greater goal of improving outcomes after hct. additionally, the success of letermovir validates terminase inhibitors as a clinically relevant class of antiviral agents and may open the door to the development of other terminase inhibitors [135] . as agents with novel mechanisms of action such as letermovir and possibly maribavir are brought to clinical use, combination therapy for the treatment of cmv infection and disease becomes, for the first time, a possibility. in vitro studies generally support at least an additive effect, if not a synergistic one, of combining letermovir with dna polymerase inhibitors or maribavir. clinical studies are now needed to determine whether combination therapy for cmv is superior to monotherapy, as is true for the treatment of viral infections such as human immunodeficiency virus and hepatitis c virus [136, 137] . with no other agents besides maribavir and filociclovir currently in human studies, combination therapy with existing agents and perhaps with indirectly acting anticmvagents approved for other indications [138] [139] [140] [141] [142] [143] [144] [145] [146] [147] [148] [149] [150] [151] [152] [153] that are unsuitable for use as monotherapy should be considered. advances in non-pharmacologic interventions will also be important in mitigating the impact of cmv 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anticytomegaloviral kinase inhibitors protein kinase inhibitors of the quinazoline class exert anticytomegaloviral activity in vitro and in vivo utility of leflunomide in the treatment of complex cytomegalovirus syndromes publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments the author wishes to thank dr. sunwen chou and dr. lynne strasfeld for their critical review of the manuscript. conflict of interest the author declares no conflict of interest. this article does not directly involve studies with human participants or animals performed by the author. key: cord-006586-49btg9w7 authors: golfieri, r.; giampalma, e.; morselli labate, a. m.; d'arienzo, p.; jovine, e.; grazi, g. l.; mazziotti, a.; maffei, m.; muzzi, c.; tancioni, s.; sama, c.; cavallari, a.; gavelli, g. title: pulmonary complications of liver transplantation: radiological appearance and statistical evaluation of risk factors in 300 cases date: 2000 journal: eur radiol doi: 10.1007/s003309900268 sha: doc_id: 6586 cord_uid: 49btg9w7 the aim of this study was to evaluate the incidence, radiographic appearance, time of onset, outcome and risk factors of non-infectious and infectious pulmonary complications following liver transplantation. chest x-ray features of 300 consecutive patients who had undergone 333 liver transplants over an 11-year period were analysed: the type of pulmonary complication, the infecting pathogens and the mean time of their occurrence are described. the main risk factors for lung infections were quantified through univariate and multivariate statistical analysis. non-infectious pulmonary abnormalities (atelectasis and/or pleural effusion: 86.7 %) and pulmonary oedema (44.7 %) appeared during the first postoperative week. infectious pneumonia was observed in 13.7 %, with a mortality of 36.6 %. bacterial and viral pneumonia made up the bulk of infections (63.4 and 29.3 %, respectively) followed by fungal infiltrates (24.4 %). a fairly good correlation between radiological chest x-ray pattern, time of onset and the cultured microorganisms has been observed in all cases. in multivariate analysis, persistent non-infectious abnormalities and pulmonary oedema were identified as the major independent predictors of posttransplant pneumonia, followed by prolonged assisted mechanical ventilation and traditional caval anastomosis. a “pneumonia-risk score” was calculated: low-risk score ( < 2.25) predicts 2.7 % of probability of the onset of infections compared with 28.7 % of high-risk ( > 3.30) population. the “pneumonia-risk score” identifies a specific group of patients in whom closer radiographic monitoring is recommended. in addition, a highly significant correlation (p < 0.001) was observed between pneumonia-risk score and the expected survival, thus confirming pulmonary infections as a major cause of death in olt recipients. abstract. the aim of this study was to evaluate the incidence, radiographic appearance, time of onset, outcome and risk factors of non-infectious and infectious pulmonary complications following liver transplantation. chest x-ray features of 300 consecutive patients who had undergone 333 liver transplants over an 11year period were analysed: the type of pulmonary complication, the infecting pathogens and the mean time of their occurrence are described. the main risk factors for lung infections were quantified through univariate and multivariate statistical analysis. non-infectious pulmonary abnormalities (atelectasis and/or pleural effusion: 86.7 %) and pulmonary oedema (44.7 %) appeared during the first postoperative week. infectious pneumonia was observed in 13.7 %, with a mortality of 36.6 %. bacterial and viral pneumonia made up the bulk of infections (63.4 and 29.3 %, respectively) followed by fungal infiltrates (24.4 %) . a fairly good correlation between radiological chest x-ray pattern, time of onset and the cultured microorganisms has been observed in all cases. in multivariate analysis, persistent non-infectious abnormalities and pulmonary oedema were identified as the major independent predictors of posttransplant pneumonia, followed by prolonged assisted mechanical ventilation and traditional caval anastomosis. a ªpneumonia-risk scoreº was calculated: low-risk score ( < 2.25) predicts 2.7 % of probability of the onset of infections compared with 28.7 % of high-risk ( > 3.30) population. the ªpneumonia-risk scoreº identifies a specific group of patients in whom closer radiographic monitoring is recommended. in addition, a highly significant correlation (p < 0.001) was observed between pneumonia-risk score and the score), the technical quality of the transplant surgery, the type, intensity and duration of the immunosuppressive therapy and epidemiological exposure to environmental agents. because one or more of these factors are important at different points in the posttransplant course, it should be possible to predict which complications are most likely to occur at different moments [16] : an accurate chest x-ray follow-up could therefore lead us to the type of pulmonary abnormality (infectious or non-infectious) and to the possible infecting agent, thus improving the final patient outcome. the aim of the present study was to: 1. describe the radiological pattern on chest x-ray film (crx) and prevalence of non-infectious and infectious pulmonary complications during the follow-up of olt 2. verify the type of infecting pathogens and the mean time of their occurrence 3. identify the main risk factors for lung infections, and their quantification through univariate and multivariate statistical analysis, in order to evaluate the likelihood of developing pneumonia. particular emphasis was placed on the predisposing factors for pneumonia occurring during the first posttransplant month, because this is considered to be the most critical period, when technical and clinical problems are associated with fatal infections [12, 16] . many papers have been published previously on this topic [2, 3, 4, 5, 6, 7, 8, 9, 10, 11] , but, to our knowledge, the present study represents the largest series to date of radiologically demonstrated pulmonary complications following liver transplantation involving a follow-up of over 11 years. the first 300 patients who underwent olt at the surgical department and transplant centre of the university of bologna, between september 1986 and september 1997, were included in this retrospective study. among these, 269 patients received only a single graft, 29 cases were retransplanted and 2 cases received three grafts for a total number of 33 retransplantations (14 due to vascular ischaemic events, 10 for ªprimary non-functionº and 9 for acute rejection). of the 31 patients who needed retransplantation, only 12 survived (38.7 %). the medical records of all patients were reviewed from the time immediately preceding olt until death or throughout the complete follow-up (maximum 11.8 years). the following clinical data were recorded and correlated to the onset of pulmonary infections and to the outcome: 1. the mean age of the present patient population was 45.3 years (sd 12.3 years), with 105 females and 195 males. mean follow-up after the transplant was 3.4 years (median 2.7 years; interquartile range 1.2±4.8 years). two hundred fifteen olt recipients are still living (71.7 %). in addition to the six major indications listed in table 1 , the ªotherº group included the following 28 cases: 4 wilson's disease; 2 diffuse angiomatosis; 3 giant hemangiomas; 1 hemangio-endothelioma; 1 hepatoblastoma; 1 macronodular hyperplasia due to venocclusive disease; 2 crigler-najjar disease; 2 polycystic disease; 5 amyloidosis; and 7 different metabolic liver diseases. the patients' pre-operative clinical conditions, presented in table 1 , were assessed according to the united network of organ sharing (unos) classification [21] . orthotopic liver transplantation (olt) was carried out with two standard surgical techniques: the traditional technique, as first described by starlz [22] in 189 patients (63.0 %), and the ªpiggy-backº caval anastomosis technique [23] in 111 patients (37.0 %). in this population, piggy-back caval anastomosis was performed only once in each patient. selective bowel decontamination with colomycin, nystatine or gentamicin sulphate was routinely performed in the immediate pre-transplant period and continued for the 3 weeks following olt. a standard triple immunosuppressive therapy [cyclosporine a (cya)-azatioprine-steroids] was routinely used. in 34 non-responsive cases (11.3 %), monoclonal anti-t cells antibodies okt3 was administered, combined with steroids, antihistamines and lasix iv on the first day and immunoglobulins. in 29 patients (9.7 %) as an alternative to okt3 (in steroid-refractory rejection episodes or in cases of neuro-/nephrotoxicity secondary to cya) fk 506 (tacrolimus fuji, sawa, japan) alone, together with steroids and/or azatioprine was used. in 4 cases fk 506 was associated with okt3. intensive care unit (icu) risk was considered to be duration of stay in icu and days of assisted mechanical ventilation (amv). data distribution is reported in table 1 . rejection was histologically diagnosed after liver biopsy and its severity was graded from i to iv according to the current classification system (table 1 ) [24] : it was observed in 124 patients (41.3 %) and needed retransplantation in 9 cases (7.3 %). in all patients chest roentgenograms (cxr) were performed daily, as a bedside semi-erect film, in the immediate posttransplantation period and throughout the entire hospitalisation as an erect film; in the follow up, cxr was performed according to the study protocol: an erect cxr every 2 months during the first 6 months and every 6 months during the following 2 years. chest x-rays were independently reviewed by two radiologists (r. g. and e. g.) who were unaware of the clinical pa-rameters at that time: in every cxr the following parameters were evaluated and described according to the commonly used terminology, fleishner society nomenclature [25] , and quantified as described below. atelectasis was classified as slight (involvement of less than one subsegment or discoid atelectasis), moderate (involving one or more segments or lobar hypoventilation) or severe (atelectasis of one or more lobes). pleural effusion was scored as slight (in the case of loss of the sharpness of costo-phrenic sulci and diaphragmatic profiles or subpulmonary effusion), moderate (effusion involving less than 25 % of a hemithorax) or severe (involving more than 25 %, including a massive effusion with mediastinal shift). for statistical analysis, normal pulmonary patterns were considered as score 0; slight and moderate atelectasis and/or slight and moderate grades of pleural effusion were considered as score 1; and severe atelectasis and/or severe effusion and/or when lasting > 1 week were included in score 2. pulmonary oedema was classified as score 1 (interstitial: localised, basal and/or diffuse) and score 2 (alveolar: localised or diffuse). vascular pedicle width (vpw) was classified as normal when it was approximately 6 cm. hydrostatic oedema, due to hyperhydration, was considered in all cases where a non-cardiogenic oedema with homogeneous pattern was associated with an increase in vpw diameters. adult respiratory distress syndrome (ards) was defined as the acute onset of a diffuse ªpatchyº alveolar oedema documented at cxr, with no enlargement of the vascular pedicle (no clinical evidence of cardiogenic pulmonary oedema), with reduced pulmonary compliance, needing assisted ventilation at fi o 2 0.50 and, when measurable, pulmonary artery occlusion pressure of less than 18 mmhg [5, 12, 14] . infectious pneumonia was classified according to three main radiological patterns: (a) focal pulmonary consolidation; (b) nodules or rapidly growing masses (with or without central cavitation); and (c) diffuse pulmonary infiltrates (interstitial or alveolar pattern) [13, 19] . in order to identify early lung infections, control cultures from biological fluid specimens were obtained (bacteriology of sputum, bronchoscopy aspirates, pleural fluid, bile, blood) and serological samples were controlled daily. a pathogen or a potential pathogen obtained from a normally sterile site (sputum, specimen obtained through a bronchoscopy or pleural fluid) was considered responsible for pulmonary infection. the combination of a chest x-ray positive for a new or increasing infiltrate, clinical symptoms such as fever or dyspnoea, and positive cultures were considered to be pulmonary infection [5, 12] . in order to limit our observations to ªmajorº infections only, febrile episodes with no positive cultures or isolated cultural positivities without clinical±radiological positivities were considered as ªminorº infections or ªcontaminationsº and were excluded [7, 12] . when superimposed on ards, pneumonia could not be identified by cxr: the diagnosis could only be obtained by positive cultures from biological fluids. for the evaluation of the time of occurrence, in polymicrobial pneumonia, the most aggressive pathogen of the association was considered prevalent (e. g. bacterial vs viral and fungal over bacterial). the yates corrected chi-square was applied to compare proportions between different groups of patients. the putative risk factors for development of pneumonia after olt were tested by means of univariate and stepwise multivariate survival analysis based on the cox proportional hazard regression model [26] . the exponentials of the coefficients calculated by these analyses (or) were reported to quantify the effect of each putative risk factor on the hazard function. these ors estimate the fractional increase of the risk of developing pneumonia either determined by the increase of one unit in the score of the putative risk factors or determined by the presence of dichotomous risk factors. the 95 % confidence intervals (ci) of the ors were also evaluated. the same univariate and multivariate procedures were applied to analyse the mortality rate after olt. the product-limit estimate [27] was used to plot both the time course of the appearance of pneumonia and the survival after olt. statistical analyses were performed by means of the bmdp statistical software [28] running on a personal computer. a two-tailed p-value of less than 0.05 was used to define statistical significance. a summary of non-infectious pulmonary abnormalities is presented in table 2 . in the early postoperative period, pulmonary non-inflammatory changes involved 260 patients (86.7 %). atelectasis of different degrees was a common finding (73.7 %): it was lamellar or subsegmental and it accompanied pleural effusion in most cases (128 of 221, 57.9 %), whereas only in 29 cases did it prevail against effusion, appearing as lobar or multilobar atelectasis. in the majority of patients it resolved itself spontaneously within 15 days on average: in 6 of the 20 cases of severe and prolonged right lobar atelectasis, a bacterial superinfection appeared (table 2) . pleural effusion was very frequent (68.6 %) in the first week: in the majority of cases (185 cases: 89.8 %) it was slight or moderate, situated mainly or exclusively at the base of the right lung, the site of surgical manipulation, with complete resolution within 5 days on average (range 2±7 days). only in 59 cases did it last more than 7 days and in 37 of them (62.7 %) it was treated with thoracentesis. pulmonary oedema was very common in the early postoperative period. the interstitial involvement was prevalent (72.4 %). the cause was overhydration (hydrostatic oedema) in 67 cases (69.1 %) and it regressed after 3±4 days of diuretics and fluid restriction. a cardiomegaly was always absent. the radiographic pattern of interstitial pulmonary oedema was characterised by bilateral, diffuse or central ªground glassº opacities accompanied by kerley b lines. the vpw was always increased in hydrostatic oedema. in alveolar oedema a diffuse or basilar air-space consolidation was present, differing from the patchy focal opacities of interstitial pneumonia. in 12 patients (4.0 %) a disseminate pattern of ards was observed in the final phase and al-ways superimposed on sepsis complicating a pulmonary infection: the ards mortality was 100 %. the appearance of ards was characterised by an extensive patchy diffusion of air-space opacities (more extensively than the previous pneumonia infiltrates), with air bronchogram and without an increase in vpw diameters. the frequency of ards was significantly higher (p = 0.029) in retransplanted patients (4 of 31, 12.9 %) than in single graft patients (8 of 269, 3.0 %). of 34 patients who received immunosuppressive therapy with okt3 (24 due to hyperacute rejection and 10 due to the onset of cya toxicity), 21 developed acute pulmonary hydrostatic oedema with diffuse interstitial involvement. no okt3-related alveolar oedema was observed. infectious pneumonia was observed in 41 patients (13.7 %), and was fatal in 15 (36.6 %). in 10 patients a polymicrobial infection was present and in 29 a single agent was isolated (a total of 49 pathogens were isolated on biological fluids or confirmed by serological tests). in addition, in two bacterial abscesses the specific agent could not be identified. bacterial infections were the most common (26 of 41, 63.4 %) followed by viral (12 of 41, 29.3 %) and fungal (10 of 41, 24.4 %) pneumonia: the most frequent agents were gram-negative (mainly represented by pseudomonas aeruginosa as a single or coinfecting agent) isolated in 41.5 % (17 of 41), cytomegalovirus (cmv) isolated in 26.8 % (11 of 41) and candida albicans observed in 22.0 % (9 of 41) of cases, respectively. in 36.6 % of cases, pulmonary infection was the cause of death (12 with final ards). the majority of infections (38 of 41 cases, 92.7 %) had their onset within the first 2 months (ªearly infectionsº). in 32 cases (78.0 %) the pneumonia occurred during the first month, in 6 cases (14.6 %) within the second month, and in only 3 cases did the infection have later onset. all the lethal infections developed within the first 2 months after olt, 10 of which developed during the first month. twenty-seven of 38 ªearly infectionsº had onset during the icu stay (65.9 % of the whole infections); among these, 20 patients were still subjected to amv at the time of the onset of pulmonary infections and the causal agents were: pseudomonas in 11 cases; staphylococcus aureus in 5 cases; and coinfections of candida with pseudomonas and cmv in 2 cases each. in all 7 cases of infection solely by cmv, pre-olt serology was positive (high igg rate anti-cmv): 5 of these had complete recovery. two of the 3 cases of cmv pneumonia superinfected by candida were seronegative in pre-transplant screening, and both had lethal pneumonia: this confirms a more severe outcome of seronegative pre-olt patients. there was a general correlation between the type of radiographic pattern and the microorganism producing the pneumonia, since the radiological patterns observed in the 41 lung infections were: . 4) ; in 2 cases of isolated cmv and 3 cases of fungal superinfections the radiological findings were small multiple alveolar sub-segmental consolidations, associated with little pleural effusion, resembling those of bacterial pneumonia ( fig. 4 b,c) . in all cmv infections, either isolated or coinfecting agent, a previous non-infectious pulmonary abnormality was present. the only case of herpes virus pneumonia showed a diffuse symmetrical reticular interstitial thickening, without pleural effusion at cxr (fig. 5 ). fungal infections (candida, aspergillus) had late onset ± median day 18 (range 5±98 days). the radiological pattern in 4 cases was as a single pulmonary infiltrate, and the six remnants showed multiple focal rapidly growing lesions, always associated with pleural effusion. in 2 cases a central cavitation occurred, quickly evolving towards an ards syndrome with fatal outcome (figs. 3, 4 a). in all fungal infections a previous non-infectious pulmonary abnormality had been present: 7 of slightmoderate and 3 of severe entity. mortality related to pneumocystis carinii pneumonia had onset on the twenty-eighth postoperative day: cxr showed an interstitial ªground glassº pattern, mainly in the perihilar or basal lung regions (fig. 6) , which resolved after therapy in 2 weeks. univariate analysis for infections demonstrated six risk factors associated with pneumonia, as reported in ta non-infectious pulmonary abnormalities are strong risk factors for pneumonia: the increase of one unit in the score scales of both atelectasis/effusion and pulmonary oedema elevates the pneumonia-risk by three times. atelectasis and pleural effusion were observed in 97.6 % of patients who developed pneumonia. pulmonary oedema preceded pneumonia in 68.3 % of cases. among the patients who developed infections, pulmonary oedema, especially with alveolar pattern, was observed significantly (p = 0.002) more frequently (68.3 %) when compared with uninfected cases (40.9 %). these findings indicate the pre-existing pulmonary abnormalities as a local risk factor predisposing to infection. the variables which entered the stepwise procedure are shown in table 3 ; four independent risk factors for pneumonia were identified: pulmonary non-inflammatory abnormalities and oedema, both doubling the risk of pulmonary infections, and prolonged amv; on the contrary, piggy-back anastomosis reduces the risk of pneumonia as compared with traditional caval anastomosis. on the basis of the coefficients computed by the stepwise multivariate survival analysis and the pattern of the variables that entered into the procedure, a score for the risk of developing pulmonary infections was calculated for each patient: pneumonia-risk score = 0.8049 (if no piggyback) + 0.4143´amv + 1.0735´pni + 1.0244´ede, where amv, pni and ede represent the score values of mechanically assisted ventilation, pulmonary non-infectious abnormalities and oedema, respectively. patients with different risks of developing pulmonary infections were identified according to the tertile values of the pneumonia-risk score (low risk: score less than 2.25; medium risk: score from 2.25 to 3.30; high risk: score greater than 3.30). the time-course of the appearance of pulmonary infections (fig. 8) shows that the cumulative 1-year incidence of pneumonia in high-risk patients was 28.7 %, 10.3 % in medium-risk patients, and 2.7 % in low-risk patients. in univariate analysis (table 1) , a statistically significant higher risk was demonstrated in cases of ahf patients, retransplantation, immunosuppression with okt3, prolonged stay in icu and amv and in protracted pulmonary oedema; instead, surgical piggyback caval anastomosis is a factor reducing hazard. multivariate analysis taking into account all the risk factors expressed in table 1 , also including pneumonia and age, was performed. two of the four independent risk factors are represented by prolonged amv, which triples the risk, and pulmonary oedema, which doubles the risk (table 3) , whereas the piggy-back technique and a prolonged icu stay both seem to constitute a relative ªprotectionº against fatal complications, since their or were significantly less than one (0.45 and 0.43, respectively). a highly significant (p < 0.001; or = 1.66, ci = 1.23±2.23) correlation was also observed between the survival rate and the pneumonia-risk score in the same groups of patients: kaplan-meier curves depicting survival rates in pneumonia-risk scores groups are shown in fig. 9 . the expected 5-year survival rate in low-, medium-and high-risk patients is 84.1, 74.3 and 60.3 %, respectively. unlike the experience with respiratory disorders occurring after transplantation of organs such as the kidney, bone marrow, lung and heart [2, 13, 14, 15, 16, 17, 18, 19, 20] , the majority of the pulmonary complications we identified following olt were non-infectious in origin. non-infectious pulmonary complications were re[11] , respectively, in 77 % in a preliminary report of our institution [9] and in 86.7 % of the present series. these complications are directly related to surgical manipulations for diaphragmatic dissection and to the type of technique and the surgical time spent in performing caval anastomosis: the right phrenic nerve is often injured during surgery. therefore, atelectasis due to diaphragmatic hypomobility in the early postoperative period is a common finding, involving one or more lung segments, mainly on the right side [15] . reduced compliance of the pulmonary basis secondary to the increase of intravascular volume, retained secretions or compression from perioperative pleural effusion can also be responsible for postoperative atelectasis [14] . usually the recovery is complete after intense respiratory therapy, with no need for bronchial aspiration. afessa et al. [12] reported atelectasis in 74 % of patients after olt (unilateral right in 31 % and bilateral in 44 % of cases) with spontaneous resolution in 95 % of the cases after 6 weeks. similarly, in our experience, atelectasis was observed in 73.6 %, bilateral (34.6 %) or right-sided (32.0 %), and it resolved itself spontaneously within 15 days on average. a severe and prolonged right lobar atelectasis is rare [14, 15] and a superimposed bacterial pneumonia should be suspected in these cases, as it was observed in 6 of 20 patients (30 %) of the present series. pleural effusion is an expected consequence of olt, observed in 54±100 % of patients in the first postoperative week [11, 12, 14, 15] : mainly on the right side or bilateral, it is a transudate (as demonstrated by the performed thoracentesis) also named ªhepatic hydrothoraxº [15] due to residual ascites or to surgical trauma. usually less than 20 % of the effusions need thoracentesis [9, 11, 14, 15] . in duran et al.'s series, pleural effusion was noted in 61.9 % of patients, 31.4 % of them requiring thoracic tube drainage [11] . in the present series it was observed in 68.6 % of the cases, with spontaneous resolution within a week in the majority of cases and thoracentesis was required in only 18 % of them. pulmonary oedema with hemodynamic or hydrostatic origin is a common non-infectious complication of cardiac, renal, bone marrow and liver transplant. in liver transplants pulmonary oedema is due mainly to overhydration from fluid infusion, excess or massive blood transfusion during surgery, to fluid retention related to preoperative renal dysfunction or to renal failure due to cya nephrotoxicity [12, 14] . in the majority of cases, it regresses after diuretics. in our series pulmonary oedema was observed in 44.7 %, with interstitial involvement in the majority of them: the main cause was overhydration (hydrostatic oedema) and it regressed after 3±4 days of diuretic therapy. ards has been reported in the literature in from 4.5 to 17.5 % of cases following olt [3, 5, 14] . the rate of mortality approaches 80 % and the onset is generally within the first postoperative week [15] . multiple etiological factors have been described (peri-surgical events such as infraoperative hypotension, prolonged surgical time, haemorrhage and blood transfusions). despite the complexity and dura-tion of the olt surgical procedure, which often involves extensive blood transfusions, ards has rarely been described in the early postolt period. sepsis appears to be, in our and in other authors' experiences [3] , the most common cause of ards in olt patients. the incidence of ards in our experience was 4.0 %. it was always associated with sepsis from pulmonary infections and had a fatal outcome in all cases due to toxic shock syndrome. similarly, in a recent report [14] , ards was observed in 3.5 % of cases during the postolt course, sepsis was always the causal factor and, as in non-transplant patients, there was a very high mortality rate [3, 5] . retransplantation is a significant risk factor for ards, as seen in a recent series in which ards occurred in 21 % of the patients receiving multiple grafts as compared with 2.7 % of patients receiving one graft [14] . accordingly, in our series, ards was observed in 12.9 % of second graft patients and in 3.0 % of the single graft cases (p = 0.029). pulmonary infections after olt have been observed in a range from 15 to 52 % [2, 3, 4, 5, 6, 7, 8, 11] , with mortality around 40 %. these prevalences are much higher than those observed after routine hepatic surgery, as shown in approximately 25 % of cases [29] . the 13.7 % prevalence of pneumonia (lethal in 36.6 %) in the olt population of this series was similar to some previous reports [4, 7, 10] and lower in comparison with previously reported prevalences of 43.3 % of infectious events (fatal in 28 %) [11] . a wide variability among the reported experiences about rate of infections, prevalence and type of infecting agents in postolt follow-up is observed in table 4 , depending mainly on bowel decontamination, patient selection criteria for olt, immunosuppressive therapy, percentage and type of environmental agents. during the first month after olt, the majority of bacterial pneumonia is reported by almost all series [7, 8, 12] . the most frequent pathogens in our series were gram bacteria and viruses (cmv) followed by candida in 9 cases. after olt, the majority of lung infections have onset within the first 6 months of the posttransplant course. after the first month graft recipients have passed the major global risk of lethal infections (figs. 7, 8) . in our experience, in the first month 78.0 % of the infections appeared. almost all the infections (92.7 %) and all the fatal infections appeared by the end the second month (fig. 9) . the different opportunistic pathogens tend to appear at predictable times during the posttransplant course, following the timetables suggested by rubin [16] after liver transplant and recently by leung [20] after bone marrow transplant. the knowledge of the time lines of the different pathogens is helpful in the differential diagnosis. accordingly, in our series, during the first 2 weeks, bacteria and fungi were the only agents observed; subsequently (days 15±60), cmv and fungi were the most important pathogens, followed by p. carinii. the only difference in our data is the pcp onset, which is reported to occur, in the majority of cases, 3±5 months after olt and which in the sole case of our series had onset approximately the twenty-eighth day. after the second month, there is no predominant patho-gen: bacterial pneumonia from the environment, viral infections from cmv and herpes simplex and p. carinii pneumonia are common [30] . bacterial pneumonia is the most common pulmonary infection after liver, lung and cardiac transplants [7, 14, 18] . in olt, bacterial pneumonia constituted up to 50 % of pulmonary infections and arises during assisted ventilation: pre-transplant prolonged intubation, aspiration pneumonia or postoperative atelectasis and lengthy surgical procedure seems to represent promoting factors [4, 5, 6, 7, 10, 12] . in our series, bacterial infections constituted 63.4 % of all pulmonary infections. the bacteria most frequently described are enterogenic gram-and particularly pseudomonas aeruginosa, as observed in 41.5 % of our series, as single or co-infecting microorganism. all but three bacterial infections had an onset within the first month, during assisted mechanical ventilation in 52.3 %. the radiographic findings of bacterial pneumonia are believed to be identical in immunocompetent and immunocompromised patients: in the present series they appeared as dense air-space consolidations with lobar, segmental, localised or patchy distribution, usually associated with a small amount of pleural effusion. the same characteristics were also observed in two cases of candida superinfection. in patients with sepsis and bacteraemia, fulminant disease with rapid progression to ards may occur, with a pattern changing into diffuse, irregular (patchy) air-space consolidation. cytomegalovirus pneumonia is reported to be the most frequent pulmonary infection after bone marrow transplantation [16] and it has been described in up to 35 % cardiac and kidney transplants and in more than 50 % lung and lung-heart transplant recipients. in olt recipients the major cmv-infected organs are the liver, intestine and lung. a high prevalence of cmv pneumonia is, however, reported [4, 5, 7, 17] and it is confirmed in our series, where it was second in frequency, with 26.8 % overall incidence. due to frequent superinfections (three candida superimpositions in our series), the prognosis is poor. coinfection with other opportunistic pathogens has been previously described [3] , particularly with pneumocystis carinii, as observed in one case of the present series. strong antirejection therapy, such as antilymphocytic antibodies, such as okt3, has been reported to be the highest risk factor of serious cmv infections [4, 15, 31] . our experience excludes a specific cmv-promoting effect of okt3. as predictors of cmv pneumonia have also been identified: the donor seropositivity and recipient seronegativity, advanced unos status, invasive fungal diseases, abdominal reinterventions and bacteraemia, all events reducing immunological defences [30] . in our series, reactivation or reinfection mechanisms were present in the majority of seropositive recipients: preoperatively cmv-seropositive recipients have a higher percentage of reactivation infections, yet rarely develop severe infections, whereas cmv seronegative recipients have a higher risk of developing severe infections [15, 30, 32] . furthermore, as the most significant effect of primary cmv infection, a broad-based depressant effect on the host defences is described, responsible for the onset of a great number of opportunistic infections, elevating the mortality rate [15, 16] . moreover, in the present series, the immunodepressant effect of primary cmv infection and the frequent fungal superinfection was confirmed, as 3 cases of primary cmv pneumonia were superinfected by candida and led to a fatal outcome. the chest radiographic findings of cmv have been variably described as consisting of a diffuse fine reticular or a haziness pattern of interstitial pneumonia, diffuse micronodular patterns, focal air-space consolidation resembling that of bacterial pneumonia [33] or, eventually, with normal findings [20] . the majority of our cases demonstrate diffuse parenchymal haziness and in 2 cases we found a pattern of parenchymal consolidation distributed in the middle and lower lung zones, similarly to what has been observed in previous studies performed on bone marrow transplant recipients [20] : in these cases the basilar predominance has been reported as related to hematogeneous cmvdissemination, with preferential distribution to the lower lung zones, due to their greater blood flow perfusion. candida superinfections appeared as nodular or focal airspace consolidations. herpes simplex pneumonia is rarely described in liver transplant recipients. it usually has a late onset, after the first month, and is due, like cmv, to the reactivation of a latent virus, as a result of immunosuppressive therapy. the only case of herpes pneumonia observed in our series showed the same radiological pattern of uncomplicated viral pneumonia, which consisted in all cases of diffuse interstitial infiltrates, having a grossly reticular pattern. pleural effusion was absent. fungal pneumonia in solid organ transplantations is less frequent than bacterial and viral (cmv) infections, but it is by far the most severe and it has the highest mortality rate. the two main agents are usually represented by candida or, more rarely, aspergillus. these infections are frequently observed within the first 2 months [14] . the starting point of candida infection is the gastroenteric tract, colonised by this fungal agent in 30±60 % of the normal subjects, and the manipulations at the time of the transplant can cause the diffusion of the pathogens. surgical instrumentation and central catheters are the major source of aspergillus, criptococcus and mucor, which are non-endogenous agents, but are always acquired from the environment. as significant risks of postolt fungal infections, a surgical time > 12 h, intratransplant transfusional requirements, bacterial infections within the first 2 months after olt together with prolonged systemic antibiotic therapy, reinterventions and high dose immunosuppression have been cited [4, 12, 34] . accordingly, a significant correlation has been observed in our population between okt3 immunosuppression and fungal pneumonia. the incidence of candida infections in the different olt series varies from 11 to 42 % according to the type of bowel decontamination and antifungal prophylaxis performed. the usual site of infection is abdominal. the mortality rate from fungal infection after olt ranges from 70 to 90 % and is higher in fungal superinfections of pre-existing infiltrates. accordingly, in the present series, the majority of candida pneumonia superinfected bacterial or viral infections and had a fatal outcome in 70 % of the cases [12, 34] . aspergillus infection most commonly appears as pneumonia or disseminated infection with mortality rates that approach 100 %. the one observed case of aspergillosis in our series was present as an isolated pathogen with lethal pneumonia. the chest radiographic features of fungal infections consisted of multiple nodular opacities with intense alveolar infiltrates, with tendency to central cavitation and air bronchograms or nodules with hazy margins or clusters of fluffy nodules. the nodules of fungal pneumonia tended to be multiple rather than solitary, and a prevalent involvement of the upper lobes is frequently observed. pleural effusion and adenopathy may be seen [20] . in the present study, the radiological pattern of candida infections or superinfections showed multifocal areas of air space consolidations, usually not associated with pleural effusion. in those cases which do not respond to therapy, the nodules may coalesce to larger regions of consolidation and, eventually, to the typical appearance of ards. in aspergillosis the nodules were rapid growing and showed a crescent-shaped area of hyperlucency (ªair crescent signº) representing cavitation around the pre-existing dense central nodule. pneumocystis carinii pneumonia (pcp) in organ transplants has a reported incidence of 0±14 %, depending on the specific prophylaxis adopted [2, 4, 5, 6, 7, 10, 12, 14] . usually pneumocystis pneumonia has a later onset as compared with the other agents, appearing between the third and sixth month: the mortality rate is between 25 and 50 % [4] . it has rarely been observed in olt recipients (5 % of cases) and more often is a coinfection with cmv infiltrates. in this case it is associated with a higher mortality rate [4] . the radiological pattern is similar to viral pneumonia, with smooth interstitial reticular involvement or typically with central and bilateral perihilar linear processes, which progress to a homogeneous diffuse alveolar consolidation. pneumocystis carinii pneumonia sometimes appears with atypical infiltrates (focal and non-diffuse pattern) [4, 13] . the sole cases observed in our series appeared late in the postolt course (twenty-eighth day) and showed a radiological pattern of diffuse interstitial impairment, ªground glassº appearance and mild symptoms (fever). there was a complete and rapid recovery after therapy. previously reported risk factors of pulmonary infections following olt [6, 8, 15, 16, 30] included: poor preoperative unos status; technical quality and duration of the transplant surgery; surgical complications needing major surgery (retransplantation); the type, intensity and duration of the immunosuppressive therapy; infections due to immune-modulating viruses; metabolic disturbances and epidemiological exposure to environmental agents. in a previously published study on the first 100 cases of olt [9] , prolonged icu and amv, advanced unos status, okt3 immunosuppression and pulmonary oedema were identified as highly predictive risk factors for the onset of pulmonary infections. the present study, performed on a wider patient population, demonstrated, in univariate analysis, a significantly increased association of pulmonary infection with the following five risk factors: caval traditional anastomosis, retransplantation, okt3 immunosuppression, icu stay and amv duration, pulmonary non-inflammatory abnormalities, such as effusion and atelectasis, and oedema. in our multivariate analysis, the strongest independent risk factors for pneumonia were non-infectious abnormalities (atelectasis and pleural effusion) and pulmonary oedema, followed by prolonged amv, whereas piggy-back caval anastomosis is shown to be significant as a protection factor, preventing the onset of pneumonia, as compared with traditional caval anastomosis. in our experience, which differs from previous studies [6, 8, 9, 30] , basal liver disease, unos score and graft rejection do not represent statistically significant risk factors for pneumonia. about surgically related risks, traditional caval anastomosis is demonstrated in our series to be more risky than the piggy-back technique. liver transplantation with preservation of the recipient vena cava (the piggyback technique), which avoids retrocaval dissection [23] , reduces overall time of surgery, need of blood transfusions and postoperative renal failure, with earlier extubation, shorter icu and total hospital stay. due to these advantages, the follow-up of olts performed with the piggy-back technique showed, in the present study, non-infectious pulmonary abnormalities of a less severe degree, thus resulting in a significantly lower rate of pneumonia as compared with traditional caval anastomosis and a significantly reduced overall mortality rate. in multivariate analysis the piggy-back technique represents a significant factor preventing postoperative pneumonia and mortality. in accordance with the studies of the mayo clinic and other authors [6, 7] , our results confirm a significantly higher percentage of major infections after retransplantation than in a single graft. retransplant also represents a significant risk of mortality. anti-t-cell antibody (okt3) immunosuppressive therapy has previously been described as a strong risk factor for serious infection in the weeks following treatment [31, 35] . a significantly higher incidence of severe pneumonia with fatal outcome in the majority of cases has been also confirmed in our series. a further serious complication of okt3 is the onset of acute pulmonary oedema during the first 2 days of treatment, which could predispose to superinfections. this usually appears in patients already overloaded with fluid prior to treatment. in 21 of 30 cases in our series, diffuse interstitial oedema was evident, with a slight increase in vpw: clear alveolar oedema was never observed due to diuretics routinely administered in the days before the injection of okt3. a prolonged stay in icu has been reported as the only significant risk factor for the onset of infections in liver graft recipients [6] . our statistical univariate analysis confirms this as one of the major risk factors for infection: 27 of 41 infections in our series (65.9 %) developed in icu, and in 20 cases during amv (48.8 %). multivariate analysis, instead, demonstrates that the icu stay risk in the global population is paradoxically ªa protectionº against infection and mortality, and that it is only indirectly significant because it is related to the real ªmajorº risk for pneumonia and related mortality, represented by prolonged intubation and amv. protracted amv has been previously identified as a risk factor for developing nosocomial infection [15] . the high percentage of pseudomonas pneumonia observed in the present series (41.5 %) was directly related to a longer duration of amv during the icu stay. prolonged intubation and amv were shown to be, in multivariate analysis, the greatest independent risk factors for mortality. among the variables studied, the pre-existing pulmonary abnormalities ± pleural effusion, atelectasis and pulmonary oedema ± were shown to be the greatest independent predictors of pneumonia in our olt population. persistent effusion, atelectasis and pulmonary oedema triple the risk of developing infectious complications. the vast majority of infections (37 of 41) were ipsilateral and superimposed on these previously non-infectious lesions, which constitute ªlocus minoris resistentiaeº especially in nosocomial bacterial infections (pseudomonas, staphylococcus). pulmonary oedema was demonstrated, in multivariate analysis, to be the second most important predictor for mortality. by associating the selected major risk factors for pneumonia and mortality, piggy-back anastomosis, prolonged amv and icu stay, pulmonary non-inflammatory abnormalities and pulmonary oedema, we defined a pneumonia-risk score in order to identify which patients needed closer cxr and clinical screening for pneumonia. the cumulative 1-year incidence of pneumonia was 28.7 % in the high-risk score, 10.3 % in the medium-risk score, and 2.7 % in the low-risk score. the time course of the appearance of pneumonia confirms that the majority of infections had their onset within the second month, but the time of onset is more protracted as the risk score increases. in low-risk patients, all pneumonia appeared by the twentieth day after olt, whereas the pneumonia risk period is prolonged in the high-risk group, in which a higher percentage of pneumonia can appear after the first month. clinical and radiological follow-up and intensified preventive measures should therefore be prolonged according to the risk score, in order to reduce morbidity and, consequently, mortality. previously published data [7, 12] demonstrated that infections are the leading causes of death after olt. our study confirms that in all fatal infections the lung was the primary site of infection, as shown by the close correlation between pneumoniarisk score and survival: the expected 5-year survival rate differs significantly in low, medium and high pneumonia-risk populations, being 84.1, 74.3 and 60.3 %, respectively. as previously reported [20] , fungi constituted the pathogenic group most highly associated with mortality within 30 days of diagnosis (mortality 70 %) followed by bacteria (27 %) and cmv (25 %). in conclusion, in olt recipients, as in all immunocompromised patients, prophylaxis, when possible, per-sistent infection surveillance and an aggressive diagnostic and therapeutic approach help to reduce the potentially fatal impact of pulmonary abnormalities. bacteria were the most frequent (63.4 %) pathogenic group in our series, rarely fatal, followed by cmv (29.3 %) and fungi (24.4 %), the latter being associated with higher mortality rate. during the posttransplant course these agents show a predictable time of onset ± bacterial infections prevailing during the first 2 weeks, fungal agents later (median 18 days) and viral (mainly cmv) and p. carinii pneumonia appearing last. this knowledge could be an important aid to the radiological diagnosis in indicating the nature of the infiltrate and, therefore, in predisposing therapy, thus influencing the final clinical outcome. upon radiographic cxr features of the different agents, a fairly good correlation was found between the radiological cxr pattern and the microorganism, with an air-space consolidation pattern in bacterial, a nodular or focal consolidation pattern in fungal and a reticular interstitial pattern prevalent in viral and pcp pneumonia, with the only exception being cmv which could appear as focal alveolar consolidation in a minority. our study demonstrated three main independent risk factors for developing posttransplant pneumonia: prolonged amv/icu stay, pulmonary oedema and non-infectious abnormalities such as atelectasis and pleural effusion. the knowledge of the probability of the onset of infections based on the calculated pneumonia-risk score identifies the high-risk patient population, in whom closer, even daily radiographic monitoring is justified, in the early posttransplantation period, in order to control the pneumonia-risk factors. persistent or severe non-infectious pulmonary abnormalities, such as atelectasis and pleural effusions, triple the risk of pneumonia onset and therefore daily cxr monitoring is mandatory. pulmonary oedema, more frequently due to overhydration, should be surveyed and quantified by close cxr and clinically prevented, reducing postoperative water overload. furthermore, olt recipients having a high pneumonia-risk score are particularly at risk, since our data also confirms that pulmonary infections remain the leading cause of death after liver transplantation, as demonstrated by the close correlation between the pneumonia-risk score and mortality in the present series. very few studies have been done on the extensive employment of ct in early posttransplant follow-up. a recent report on ct studies of pulmonary complications after lung transplant [17] , in which a comparison with histopathological studies has been carried out, demonstrated that ct findings were not helpful in differentiating between the different parenchymal complications. when matched with proper clinical data, close cxr monitoring could be of value in orienting the diagnosis of the different pulmonary abnormalities which complicate the postoperative course after olt. a precise knowledge of the probability of the onset of the different opportunistic agents 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undergone an orthotopic liver transplant key: cord-269194-b1wlr3t7 authors: engstrom-melnyk, julia; rodriguez, pedro l.; peraud, olivier; hein, raymond c. title: chapter 5 clinical applications of quantitative real-time pcr in virology date: 2015-12-31 journal: methods in microbiology doi: 10.1016/bs.mim.2015.04.005 sha: doc_id: 269194 cord_uid: b1wlr3t7 abstract since the invention of the polymerase chain reaction (pcr) and discovery of taq polymerase, pcr has become a staple in both research and clinical molecular laboratories. as clinical and diagnostic needs have evolved over the last few decades, demanding greater levels of sensitivity and accuracy, so too has pcr performance. through optimisation, the present-day uses of real-time pcr and quantitative real-time pcr are enumerable. the technique, combined with adoption of automated processes and reduced sample volume requirements, makes it an ideal method in a broad range of clinical applications, especially in virology. complementing serologic testing by detecting infections within the pre-seroconversion window period and infections with immunovariant viruses, real-time pcr provides a highly valuable tool for screening, diagnosing, or monitoring diseases, as well as evaluating medical and therapeutic decision points that allows for more timely predictions of therapeutic failures than traditional methods and, lastly, assessing cure rates following targeted therapies. all of these serve vital roles in the continuum of care to enhance patient management. beyond this, quantitative real-time pcr facilitates advancements in the quality of diagnostics by driving consensus management guidelines following standardisation to improve patient outcomes, pushing for disease eradication with assays offering progressively lower limits of detection, and rapidly meeting medical needs in cases of emerging epidemic crises involving new pathogens that may result in significant health threats. processes, small sample volumes and can be utilised in a wide variety of applications, making it the method of choice in today's molecular laboratories. through the aid of fluorescent signalling probes to measure amplification of dna at each pcr cycle, at the point of exponential dna accumulation, real-time pcr is able to provide broader linear dynamic ranges and increased assay performance as determined by sensitivity, specificity, precision, and reproducibility. due to the consistency in signal intensity changes during the exponential growth phase of pcr, it is also easily adaptable for quantitative reporting. however, there are three properties that are uniquely associated with quantitative real-time pcr: quantification, standardisation, and lower limit resulting. the accumulation of fluorescence signal is measured at each pcr cycle of the reaction and the cycle at which this signal exceeds a predetermined background fluorescence threshold during the logarithmic phase of amplification is referred to as the cycle threshold (c t ). the c t value is inversely proportional to the viral copy number in the specimen, and through comparisons of this value to an external calibration curve or an internal quantitation standard, the initial nucleic acid target concentration can be calculated (heid, stevens, livak, & williams, 1996; livak & schmittgen, 2001) . however, accurate quantitation within each sample is hindered when relying solely on an external standard as amplification efficiencies for each individual sample may be variable and inconsistent. by utilising a standard internal reference template, with the rationale that any variable influencing amplification efficiency should example of amplification and detection of target nucleic acid by real-time pcr. affect both template and target similarly, inhibition and amplification effects are compensated for which allows for more accurate quantitation ( figure 2 ). this control can be further enhanced when incorporating an internal reference that utilises the same primer sequence as the target since any potential additional effects on pcr efficiency for each of the two targets is eliminated. thus, the competitive real-time pcr strategy is the most reliable approach for nucleic acid quantitation (diviacco et al., 1992; gilliland, perrin, & bunn, 1990; stieger, demolliere, ahlborn-laake, & mous, 1991; wang, doyle, & mark, 1989; zentilin & giacca, 2007) and is the basis for the majority of present-day virology assays. it is equally important to utilise appropriate quantitation standards, when available, to ensure accurate quantitative results, inter-laboratory correlation, and overall standardisation. standardisation of reported viral loads ensures not only interlaboratory consistency but also high clinical utility of viral load monitoring, sets the foundation for establishing clinical correlations and critical thresholds leading to better management of infections and treatments, and are critical for the development of clinical guidelines (miller et al., 2011) . with the wide availability of assay methods, viral targets, specimen type, and lack of standard reference material (hayden et al., 2012) , viral load variability across laboratories can range significantly, as high as 4.3 log copies/ml . specifically, results from proficiency testing/external quality assessment programmes as well as interlaboratory specimen exchange studies have demonstrated that there is significant variability in quantitative results for assays that lack appropriate standards quantitation of viral target using competitive quantitation standard (qs). the qs compensates for effects of inhibition and controls the preparation and amplification processes, allowing a more accurate quantitation viral target in each specimen. the competitive qs contains sequences with identical primer binding sites as the viral target to ensure equivalent amplification efficiency and a unique probe binding region that distinguishes the two amplicons. the competitive qs is added to each specimen at a known copy number and is carried through the subsequent steps of specimen preparation, reverse transcription (when applicable), simultaneous pcr amplification, and detection. viral target concentration in the test specimens is calculated by comparing the viral target signal (solid line) to the qs signal (dashed line) for each specimen and control (a, b) . in the presence of inhibitors, both qs and viral target are equally suppressed and yield accurate viral load calculations (c). 1 introduction (hayden et al., 2008; pang et al., 2009; preiksaitis et al., 2009; wolff, heaney, neuwald, stelrecht, & press, 2009 ). findings such as these reinforce the fact that with this high degree of variability and discrepancy, clinicians are unable to compare test results between two different laboratories and, further, clinically relevant cut-offs set by one test would not apply to results of another . without standardisation, the quality of patient care is dramatically impacted, preventing meaningful inter-laboratory comparison of patient results and influencing disease prevention and management programmes (kraft, armstrong, & caliendo, 2012) . this is especially critical for transplant patients, who may be initially monitored at one institution and then transferred to another for longer-term follow-up receiving results that no longer correlate. therefore, whenever possible, viral load monitoring tests must report results in iu/ml and be fully traceable to the higherorder first who international standard. they must generate highly accurate and reliable results based on a robust calibration methodology and have excellent reproducibility across the dynamic range of the test with demonstrated co-linearity to the who standard. lastly, there exist two distinct end-points with quantitative real-time pcr, which should be of consideration for result interpretation and reporting: the lower limit of detection (llod/lod) and the lower limit of quantitation (lloq/loq). these two limits are assessed differently and are not equivalent in either definition or, in some cases, their assigned values. the lod (also referred to as analytical sensitivity) represents the lowest viral load level at which !95% of tested samples are detected (clsi ep17-a, 2004); theoretically, viral levels at or below the lod are not detected !5% of the time. it differentiates between 'detectable' and 'undetectable' results. the lloq, on the other hand, is the lowest viral level that is within the linear and analytically acceptable range of the assay (clsi ep17-a, 2004) . in other words, the lloq is the lowest point at which an accurate viral load can be assigned and determines which 'detectable' sample will have a reported viral load. a common misconception is that the lod of the assay is the minimum viral level for a 'detected' result but 'undetectable' and 'detectable' viral levels are never differentiated by a single theoretical viral threshold as viral levels less than the lod may still have a high probability of being detected. this probability spans a broad range in which the lower the viral titre, the more likely the 'undetectable' result. ultimately, the statistical probability will favour the 'undetectable' result ( figure 3 ). and because the lloq can be equal or greater than the lod on some viral load assays, it is not unusual for 'detectable but below the loq' (detectable/bloq) result reporting (cobb et al., 2011) . further, the 'detectable/bloq' results should not be inferred that the actual viral concentration of the sample is between the lod and loq. the clinical demand has driven and shaped the evolution of pcr and continues to do so as we gain a greater understanding of the infections we monitor and treat. through the study of the natural history and disease progression attributed to specific viral infections, the need for sensitive, accurate, precise, reproducible, and reliable quantitative measurements of viral levels has become a necessity. with the deeper understanding of the natural history of human immunodeficiency virus (hiv) infections, it is now well understood that progressive immunosuppression and the onset and development of clinical disease are strictly associated with increasing viral burden (furtado, kingsley, & wolinsky, 1995; ho, moudgil, & alam, 1989; mathez et al., 1990; nicholson et al., 1989; schnittman et al., 1990) . thus, quantitative real-time pcr is critical for monitoring patients infected with hiv (hufert et al., 1991; mellors et al., 1995) and those undergoing antiretroviral therapy (art) to ensure viral replication is sufficiently and effectively suppressed and to monitor potential for viral resistance to the medication (dhhs hiv, 2014) . this monitoring and maintained viral suppression is absolutely necessary not only to maintain progression-free survival of hiv-infected patients but also to reduce subsequent hiv transmission (cohen et al., 2011; diffenbach, 2012) . due to the significance of viral load monitoring and maintaining viral suppression, the demand for likelihoods of different test results given different viral concentration. when the viral concentration tends to 0, the proportion of 'target not detected' increases to 1 (dotted line), increasing the likelihood of 'not detected' results. as the concentration tends to lloq (dashed line), the likelihood of 'detected but <lloq' results peaks. when the concentration tends to infinity, the proportion of quantitative results tends to 1 (solid line), resulting in a continual increase in the likelihood of 'detected: quantitative' results. at any concentration, the sum of the three types of reported results is always 100% and throughout the concentration continuum, variations in result reporting exist. as the concentration of viral levels approaches the lloq, near equal likelihood of 'detected: quantitative' and 'detected but <lloq' results are possible, as are 'not detected' results. decreasing concentrations will further shift the likelihoods and increase the chance of 'detected but <lloq' or 'not detected' results. diagram assumes lloq ¼ llod. increasingly more sensitive assays for hiv has driven the innovation of diagnostic tests and continues to push the limits of the lod and loq ever lower (glaubitz et al., 2011; sizmann et al., 2010) . additionally, the amount of viral dna in samples from either hepatitis b virus (hbv) or cytomegalovirus (cmv) chronic carriers is indicative of active viral replication in liver cells and is correlated with liver disease progression (chen, lin, et al., 2006; chen, yang, et al., 2006; emery et al., 2000; humar et al., 1999; humar, kumar, boivin, & caliendo, 2002; iloeje et al., 2006; wursthorn, manns, & wedemeyer, 2008) , highlighting the need for quantitative viral levels in chronic disease monitoring. similarly, hepatitis c virus (hcv) levels have been linked to prognosis and treatment outcomes (trepo, 2000) , treatment response (pearlman & ehleben, 2014; zeuzem et al., 2009) , as well as assessing sustained virologic response (svr), or 'cure', following treatment (pearlman & traub, 2011) . more recently, as new antiviral therapies for hcv treatment rely on targeting specific biological steps of the viral replication cycle, reliable quantitative monitoring of the viral burden at specific time-points to ensure treatment compliance and resistance emergence during treatment is recommended (au & pockros, 2014 ; aasld/idsa/ias-usa, 2014). alongside the changing clinical needs, several instrument and manufacturing innovations have been introduced to meet these newer requirements. the first pcrbased diagnostic test and the first automated system were both introduced in the early 1990s, allowing for improved standardised technique, increased efficiency, and reduction in error and contamination. in 1996, to meet the clinical needs of monitoring patients infected with hiv, the first 'personalised healthcare' diagnostic test was fda approved, that monitored whether art was working and whether a patient was on optimal treatment. entire systems were introduced in the early 2000s that automated the up-front sample preparation step, leading to further reduction of hands-on time, increased reliability and reproducibility. with the introduction of real-time pcr techniques to further enhance assay performance, the first pcr tests that allowed for simultaneous amplification and detection were introduced in 2003. throughout the decade, improvements in assays and instrumentation began pushing the boundary for hiv-1 detection, achieving greater and greater sensitivity, a feat that has proven to be incredibly relevant in understanding risks of viral load rebound and virologic failure (doyle et al., 2012; estevez et al., 2013; pascual-pareja et al., 2010) . more recently, shortly after the release of two new higher-order standards for cmv (first who international standard and nist cmv standard), fully automated real-time quantitative pcr assays were fda approved to meet laboratory and clinical demands for standardisation. not only does the instrumentation reduce assay design variability and inconsistency across laboratories, but also the assays are standardised to the who and report in international units, providing accuracy across the entire dynamic range that is only achieved by calibration and traceability to these higher-order standards. but it is not until these fda-approved tests gain widespread use will inter-laboratory agreement for cmv viral load results truly improve (kraft et al., 2012) . the objective of test innovation is to evolve and adapt to clinical and laboratory needs. as patient outcome gaps are identified, and as we gain greater understanding and insight into the clinical progression of disease and disease management, technological achievements facilitate advancements in quality of diagnostics and patient outcomes. life-threatening illnesses convert to chronic/manageable disease with the aid of viral load monitoring. and pressure exists to develop more precise, accurate, sensitive assays, which, in turn, drives the development of more efficacious drugs. this synergy demonstrates the value of diagnostics: it is an integral part of the patient care continuum. applications of quantitative real-time pcr for virology are extensive. it is especially necessary when antibody seroconversion is delayed after an acute infection and early diagnosis are essential (dibiasi & tyler, 2004; thomson et al., 2009) , in immunocompromised patients that may not have an optimal antibody response (kadmon et al., 2013) , and for the diagnosis of congenital or perinatally acquired viral infections (park, streicher, & rothberg, 1987; young, nelson, & good, 1990) . additionally, it is critical in maintaining a high level of patient care at each stage of the disease and infection (figure 4 ). screening tests are designed with one key parameter in mind: exceptional sensitivity (herman, gill, eng, & fajardo, 2002) . they must ensure that disease is not missed in a population that is generally free from risk to allow for appropriate early intervention, thereby effectively reducing mortality and morbidity. although traditional applications of real-time pcr in the continuum of care and patient management. population-based screening programmes and recommendations do not often utilise nucleic acid testing (nat) for virology targets, relying more on immunoassays and antigen testing, nats and real-time pcr assays are still integral components. donor-eligibility determination ensures that a donor is eligible to donate cells or tissues to be used as human cells, tissues, and cellular and tissue-based products (hct/ps), which can include haematopoietic stem/progenitor cell, organ, semen, and other types of donations. in part, living and cadaveric hct/p donor eligibility is granted if screening shows that the donor is free from risk factors for, and clinical evidence of, infection due to relevant communicable disease agents and diseases such as hiv type 1 and 2, hbv, hcv, and west nile virus (wnv) (fda testing, 2014) . the fda testing recommendations stipulate that hct/ps tests for hiv and hcv may include fda-licensed nat blood donor screening and that, specifically, an fda-licensed nat should be used to assess infection with wnv. despite the fact that these nat tests provide qualitative as opposed to quantitative results, the molecular technology utilised is often real-time pcr due to its enhanced accurate and reliable resulting (fda assays, 2014) . additionally, pre-emptive virology screening post-transplant may reduce subsequent complications and provide a more cost-effective management strategy (evers, 2013) . pre-emptive therapy utilises routine viral screening to initiate therapy at the first indications of viraemia, prior to clinical manifestation of disease. this strategy reduces the overall morbidity and mortality post-transplant compared to strategies in which treatment is initiated at the onset of clinical disease (sch€ onberger et al., 2010). the pre-emptive treatment model utilising routine screening by quantitative realtime pcr of asymptomatic post-transplant patients has been shown to be especially effective for paediatric patients undergoing haematopoietic stem cell transplant, who are uniquely at a high risk of cmv and epstein-barr virus (ebv) infections (evers, 2013) . this strategy and prospective screening utilising a quantitative real-time pcr assay for bk polyoma virus (bkv) is recommended as part of routine posttransplant follow-up of all kidney transplants since early identification and management of bkv infection may prevent future incidence of polyoma virus-associated nephropathy (hirsch et al., 2005; kdigo, 2009) . nats and real-time pcr are also utilised as vital parts of certain screening algorithms. the center for disease control (cdc) currently recommends that hcv rna testing be utilised for anyone who may have been exposed to hcv within the preceding 6 months. in addition, nats would identify active hcv infection among persons who have tested anti-hcv positive or those with an indeterminate antibody test indicating need for referral for further medical evaluation and care (cdc hcv, 2013). viral diagnostic tests are used to determine presence or absence of current or previous infection. because many viral infections present with similar symptoms, accurate diagnosis is critical as each requires unique and vastly different interventions and/or management strategies. historically, diagnosis of viral infections has relied on viral growth in cell culture, immunoassays, antigen assays (elisa), haemagglutination testing, and electron microscopy (krishna & cunnion, 2012) . the introduction of molecular methods including nats and real-time pcr assays has vastly improved viral diagnosis with their superior sensitivity, specificity, and rapid result reporting (emmadi et al., 2011) . nat-based infectious disease testing, providing rapid results, aids in outbreak detection (ebola), genotype identification (hcv), and identification of possible drug resistance (hiv-1), which can lead to rapid clinical therapeutic decisions and early infection control to prevent spread of disease (espy et al., 2006) . viral culture was traditionally considered to be the 'gold standard' for viral diagnosis because of increased sensitivity compared to rapid antigen-testing methods. however, a significant limitation of viral culture was a long time to result, reaching 14 days for cmv (gleaves, smith, shuster, & pearson, 1985) . improvements in culture included the introduction of shell viral culture, which reduced the result turnaround-time (tat) for cmv to 24 h. although considered to be a vast improvement, certain viruses require immediate treatment intervention to prevent life-threatening infection and therefore, a 24-h tat is simply much too long. further, reliance on viral culture for diagnostics testing introduces other pronounced drawbacks, the most noteworthy being that not all routine viruses grow in culture and that virus viability, and thus its culture ability, could be impacted by sample collection, transport conditions, or prior patient treatment. with these limitations in mind, nat testing has tremendous advantages and has replaced culture for most viruses due to its greatly reduced tat (typically less than 8 h) while still retaining high sensitivity. the applications of nat testing, and more specifically, real-time quantitative pcr technology, in viral diagnostics and confirmatory testing continue to expand. the cdc-updated recommendations for hiv testing state that specimens that are reactive on the initial antigen/antibody combination immunoassay and non-reactive or indeterminate on the hiv-1/hiv-2 antibody differentiation immunoassay should be tested with an fda-approved hiv-1 nat test (branson, 2010; branson et al., 2014) . additionally, nats have demonstrated utility in high-risk populations, in which antibody testing alone might miss a considerable percentage of hiv infections that are otherwise detectable by nat virologic tests (priddy et al. 2007; stekler et al., 2009) . particularly, immunoassays for hiv diagnosis are limited by the marked delay between infection and seroconversion, a time when hiv viral levels are at their peak ( figure 5 ). for this reason, nats are the recommended method for diagnosis of hiv during the acute phase of infection (10-50 days post-infection). hiv viral rna is the first marker to manifest itself approximately 10 days post-infection at initiation of the acute phase of infection (lindback et al., 2000) . not until 4-10 days after initial detection of hiv rna do the hivp24 antigen levels rise to detectable levels using fourth-generation immunoassays. this marked delay and the need for rapid diagnosis by the identification of hiv rna is especially critical when an early diagnosis is medically warranted. during the acute phase of the hiv infection, patients typically present with flu-like symptoms, which can often lead to a misdiagnosis. misdiagnosing those at the highest risk of infection such as prison inmates, iv drug users, and men who have sex with men (msm), also increases the risk of further disease spreading (chu & selwyn, 2010) . therefore, the use of a real-time pcr testing method that is able to identify the presence of the hiv virus at this initial stage is critical. additionally, nats are critical for early diagnosis of congenital or perinatally acquired viral infections, especially infants born to hiv-infected mothers (tang & ou, 2012) . the maternal antibodies against hiv can persist in exposed infants for up to 18 months of age, which, in turn, prevents the use of antibody-based assays for early diagnosis of infection. there is a high level of morbidity and mortality during the first 2 years of life for infected infants; therefore, it is of high importance to determine infection status quickly in the exposed infant to implement the appropriate art therapy early (van rossum, fraaij, & de groot, 2002) . in addition to hiv, additional viruses have been demonstrated to exhibit perinatal transmission including respiratory virus, cmv, herpes simplex virus (hsv), varicella zoster virus (vzv), hbv, enterovirus, rotavirus, and human papilloma virus (prober et al., 1987) . for all these, nat testing may serve as a valuable tool for early intervention, especially in this critical neonate population that lacks fully developed immune systems. in addition to their applications for hiv diagnosis, nat testing is also utilised as a diagnostic for hcv. although diagnostic hcv rna real-time pcr tests are predominately used as confirmation of a positive hcv antibody result (cdc hcv, 2012), nat testing is recommended for the confirmation of a non-reactive hcv antibody test for patients suspected of having recent hcv exposure. hcv rna can be detected as early as 2 weeks post-exposure, whereas hcv antibodies are not detectable until 8-12 weeks post-infection (ghany, strader, thomas, & seeff, 2009 ). therefore, similar to strategies outlined for hiv, nat testing is the most suitable for detection and diagnosis during the acute phase of hcv infection. further, immunocompromised patients, those with weakened immune systems can include those that are hiv positive, on immunosuppressive drugs, or undergoing chemotherapy, may lack the ability to generate an appropriate immune response required for an anti-hcv test. in these special circumstances, the cdc recommends considering hcv rna testing for diagnosing viral infections. rapid diagnostic testing is also very important for viruses with high potential to create an epidemic or outbreak. throughout history, influenza has caused many such outbreaks, the most notable being the 1918 spanish flu thought to be responsible for an estimated 50 million deaths (taubenberger & morens, 2006) . in response to the possibility of future outbreaks, the world health organization (who) has established that pcr-based influenza testing is now the first-choice diagnostic test for both humans and animals (who influenza, 2011) . the conversion to molecular tests was based on the fact that these assays are more sensitive and specific for detecting influenza viruses compared to other non-molecular methods. nat methods also have a lower likelihood of false positive or false negative results, and therefore, result interpretation is less impacted by community-based influenza prevalence (cdc influenza, 2014) . the introduction of rapid molecular testing, which can provide results in as little as 15 min, is extremely beneficial especially in hospitals, nursing homes, and chronic care facilities where early influenza identification can prevent outbreaks. the number of molecular-based diagnostic tests has expanded even further with the introduction of tests for wnv, respiratory syncytial virus, hsv, rotavirus, and even more recently, ebola. as development and improvements continue to be made to real-time pcr technology that will reduce both cost and time to result even further, the number of nat diagnostic tests will continue to increase and the applications for real-time pcr in diagnostics will also expand. once a patient is appropriately diagnosed and linked to care, partly through the aid of quantitative real-time pcr, clinicians will often consider several baseline factors, which collectively help to guide therapeutic decision. these decisions are based on a series of questions including: are treatment options available? what treatment regimen should be prescribed? for how long will the patient need to be on therapy? factors influencing these therapeutic decisions include disease complications, patient predisposition, prior treatment experience, viral genotype, viral resistance profile, and host genetic profile, among others. and depending on the viral infection, a quantitative baseline viral load may also serve an important role in helping to guide treatment decision (table 1) . over the past 25 years, pharmaceutical development and clinical trials investigating cutting-edge antiviral treatments have relied heavily on the data generated from pcr-based-and eventually quantitative real-time pcr-based-technology to determine safe and effective drug use (cobb et al., 2011; mackay, arden, & nitsche, 2002) . practice guidelines continue to reference registrational and nonregistrational studies utilising quantitative real-time pcr to guide both clinicians and laboratories in the proper implementation of treatment and testing in order to deliver the most effective personalised care to patients (aasld/idsa/ias-usa, kotton et al., 2013) . because of this extensive co-utilisation of real-time pcr, it is widely accepted as the gold-standard technology for measuring a patient's quantitative viral load before, during, and after the course of treatment. once the decision to initiate treatment for chronic hcv infection is made, several baseline factors are routinely considered (aasld/idsa/ias-usa, 2014). among these are complications like liver fibrosis stage, co-infection with hiv, hepatocellular carcinoma, end-stage liver disease, genetic variations like hcv genotype, subtype and resistance markers, and prior treatment experience and outcome. the association of baseline viral load, measured by quantitative real-time pcr, to chronic hcv treatment outcome has been well documented with earlier therapies (pegylated-interferon plus ribavirin) but, given the lack of alternative therapeutic options, has not been recommended as a therapeutic decision factor (jensen et al., 2006; pawlotsky, 2012) . historically, practice guidelines have recommended the measurement of baseline viral load to serve only as an initial time-point required for effective monitoring of treatment without any prognostic indication (yee, currie, darling, & wright, 2006) . currently, tremendous advancements in the treatment of chronic hcv infection that employ direct-acting antivirals (daas) reported cure rates (as determined by svr) of >90% for even the once most difficult to treat hcv genotype-1 patients, the most predominant in the united states. because of this high potency of these drugs across patient populations and the greater importance of numerous other factors, including hcv genotype and prior treatment experience, in determining the appropriate course of treatment, the most recent aasld/idsa practice guidelines still do not recommend a baseline quantitative viral load as a therapeutic decision factor. however, in the rapidly evolving field of hcv treatment, the recent fda approval of a fixed-dose combination drug consisting of two daas (sofosbuvir and ledipasvir) for the treatment of hcv genotype-1, the manufacturer's drug label now includes a new indication for quantitative real-time pcr. it is indicated that treatment naïve and non-cirrhotic patients with a specific baseline viral load are eligible for shortened therapy, an indication with tremendous implications. according to the prescribing information, patients with a baseline viral load below 6 million iu/ml are eligible to have shorter therapy duration of 8 weeks, much shorter than the 12-or 24-week duration for other patient populations (harvoni, 2014) . this therapeutic decision practice is the first of its kind in treatment of chronic hcv infection and is likely to be a recurring theme as daa manufacturers strive to develop high efficacy regimens requiring shorter treatment durations. additionally, shorter treatment durations are more favourable to patients and payers when considering the cost of achieving svr with daas and may improve patient drug adherence and completion of therapy (hep c online, 2014) . as much as quantitative real-time pcr helped to develop this claim for this particular regimen, this technology will also be employed by numerous laboratories to aid in this part of therapeutic decision. in contrast to chronic infection, treatment of patients presenting in the acute phase of hcv infection, within the first 6 months after exposure, is not recommended by aasld/idsa for patients in whom hcv infection spontaneously clears (aasld/ idsa/ias-usa, 2014). therefore, careful monitoring of hcv rna by a sensitive nucleic acid test is required in order to confirm spontaneous clearance, defined as hcv rna negative at two specific measurements. quantitative and qualitative real-time pcr assays are both widely used for this purpose, given their comparable sensitivity. factors influencing art decision for hiv-infected patients include determination of pregnancy, aids-defining conditions, acute opportunistic infections, low cd4 counts, hiv-associated nephropathy, potential drug interactions, co-infection with hcv or hbv, hiv resistance testing, and prior treatment experience (dhhs hiv, 2014). plasma hiv rna viral load, performed widely by quantitative real-time pcr, is also recommended as a pre-art decision factor specifically for treatment naïve patients. the department of health and human services (dhhs hiv) recommends that only art-naïve patients with a plasma hiv viral load below 100,000 cp/ml can be prescribed various regimen options, which they otherwise should be restricted from taking with higher viral load. this is primarily due to inferior virologic responses in patients with higher viral loads observed in clinical studies (sax et al., 2009) . these clinical trial studies employed quantitative real-time pcr in order to help determine this cut-off and many labs have utilised the same technology to help guide hiv-treating clinicians in this decision. in the case of chronic hbv infection, several studies have shown that hepatitis b 'e' antigen (hbeag) and high levels of hbv dna are independent risk factors for the subsequent development of cirrhosis and hepatocellular carcinoma (chen, lin, et al., 2006; chen, yang, et al., 2006; iloeje et al., 2006) . however, due to the fluctuating nature of chronic hbv infection, the prognostic utility of one high hbv dna level at a single time-point is limited. thus, hbv baseline dna viral load, along with hbeag, alanine aminotransferase (alt) levels, and fibrosis, collectively aids in the decision to treat with antiviral agents as well as which hbv antiviral regimen to choose and duration of treatment (lok & mcmahon, 2009 ). typically, patients with an hbv dna viral load >20,000 iu/ml, signs of liver disease (i.e. high alt levels and/or significant fibrosis), and loss of hbeag are considered for immediate treatment with antivirals, whereas patients <2000 iu/ml are closely monitored for viral load changes prior to treatment. patients who fall in between this range are monitored for persistent viraemia and signs of liver disease before deciding to treat. quantitative real-time pcr, therefore, plays a crucial role in the care of chronic hbv patients who, if not treated at the appropriate time with the appropriate regimen and duration, are at greater risk of liver complications. unlike treatment guidelines for hcv, hiv, and hbv, management of cmv after solid organ transplant is not associated with specific quantitative cmv viral load cutoffs in order to make therapeutic decisions (kotton et al., 2013) . this is partly due to the historical lack of an international standard and varying assay designs, which has led to poor inter-institutional correlation of quantitative nats. in addition, the widespread practice of universal prophylaxis, where cmv antiviral medication is administered to patients early in the post-transplant period and continued for a finite period of time, has diminished the clinical utility of baseline viral loads for making therapeutic decisions. however, with the recent availability of the who cmv international reference standard, the establishment of viral load cut-offs that can be applied to pre-emptive monitoring of patients prior to treatment initiation may soon become more widely accepted . until then, institutions are required to determine their own test performance characteristics and clinical cut-offs. several studies have shown that a low cmv virologic threshold (e.g. detectable viraemia) using quantitative real-time pcr should be used for starting pre-emptive therapy especially in high-risk cases where the organ donor screens positive and the receptor screens negative for cmv serology (atabani et al., 2012; couzi et al., 2012; sun, cacciarelli, wagener, & singh, 2010) . among a variety of baseline risk factors that may indicate longer cmv treatment duration, significant predictive value has been demonstrated with higher baseline viral loads where longer treatment duration may prevent cmv disease relapse (kotton et al., 2013; sia et al., 2000) . clinical trial studies supporting the recent fda approval of a quantitative real-time pcr cmv test calibrated to the who international standard also demonstrated clinical value for baseline testing of patients with cmv disease who are undergoing treatment with the anti-cmv drugs ganciclovir or valganciclovir (razonable et al., 2013) . data from this study suggested that patients with a baseline cmv viral load <18,200 iu/ml are likely to resolve cmv disease more rapidly than those who have a higher baseline viral load. further studies are needed to determine universal thresholds for pre-emptive therapy initiation and predictive value for cmv baseline viral load in defining optimal treatment duration. there exists a clear application for quantitative real-time pcr technology in baseline determination of patients with significant viral infections, and in fact, quantitative viral load determination plays a critical role in therapeutic decision for many other viral infections. high baseline viral load has been shown to correlate with advanced disease during infection with numerous viruses such as bkv, hsv-1, ebv, and adenovirus and may potentiate the need for longer duration therapies in certain scenarios (cincinnati children's hospital medical center, 2012; domingues, lakeman, mayo, & whitley, 1998; gustafson et al., 2008; randhawa et al., 2004) . after the patient's baseline assessment or pre-emptive monitoring suggests if treatment is available, which treatment regimen to choose and perhaps the duration of therapy, the patient can move on to therapeutic administration. quantitative realtime pcr has helped and continues to set the stage for decisions that potentially saves lives, reduces complications, decreases morbidity, and lessens the economic burden to both the patient and the healthcare system. serial measures of viral load serve as an individualised map of a viral infection through the estimation of the amount of virus found within an infected person. tracking viral load in the continuum of care is a vital tool used predominantly to monitor treatment response and its effectiveness, early signs of resistance emergence during therapy of chronic viral infections, and viral activation or reactivation in immunocompromised patients following bone marrow or solid organ transplantation. while the goal of treatment for chronic hcv infection is svr, patients may fail therapy due to non-response, on-treatment breakthrough, or post-treatment relapse ( figure 6 ). the early change in quantitative viral load over time may be predictive of treatment efficacy and a shorten therapy for patients who respond rapidly to treatment (yee et al., 2006) . this 'response-guided therapy' (rgt) is best exemplified during treatment of chronic hcv patients. specifically, the sooner a patient becomes hcv rna undetectable during treatment, the lower the relapse rate when treatment is shortened. conversely, the longer it takes for a patient to become hcv rna undetectable, the longer they need to remain on treatment to limit relapse. however, given the poorer efficacy of earlier regimens, not all patients who received therapy achieved svr. for this reason, 'futility rules' or 'stopping rules' were also developed, which required that failure of a patient to respond (target not detected or viral load cutoff ) by a given time-point indicated the need to immediately discontinue therapy. monitoring hcv viral loads during treatment. despite advances in treatment for hcv patients, failure to achieve svr is still a reality. patients who do not achieve svr fall into four categories: (1) null responders (black line) achieve less than 2-log decrease in hepatitis c viral load upon treatment; (2) partial responders (red line; light grey in the print version) experiences at least a 2-log decrease in hepatitis c viral load during hcv treatment but fail to proceed to an undetectable viral load level; (3) breakthrough patients (orange line; light grey in the print version) have an undetectable hcv viral load, but the virus rebounded during treatment; (4) relapsers (blue line; dark grey in the print version) have had an undetectable hcv viral load, but the virus rebounded after they completed hcv treatment. although these rgt notions were originally developed from observations made during treatment with the older therapies, peg-ifn and ribavirin, rgt was also required during treatment with the much more potent first-generation daas, telaprevir and boceprevir, and stopping rules were put in place during treatment with the second-generation daa, simeprevir (aasld/idsa/ias-usa, 2014; ghany et al., 2009; yee et al., 2006) . newer ifn-free daa regimens targeting hcv, which are better tolerated by patients and by virtue of the targets they inhibit, have a higher barrier to resistance, yield more rapidly declining viral kinetics, and, thus, do not contain treatment indications for rgt in their prescribing information (harvoni, 2014; olysio, 2014; sovaldi, 2013; viekira, 2014) . while rgt was a major driver for regular viral load monitoring during antiviral therapy, it is not the only reason to monitor hcv viral load. in the interval between baseline measurement and assessment of svr, the 2014 aasld/idsa guidelines also include recommendations for monitoring initial response (week 4 on treatment with a repeat at week 6 if detectable) and end of treatment in order to provide an assessment of drug compliance/early efficacy and predict treatment outcomes, respectively (aasld/idsa/ias-usa, 2014). in the most recent revision to these web-based guidelines, it is recommended that an hcv viral load increase of greater than 10-fold on repeat testing at week 6 (or thereafter) should prompt a discontinuation of hcv treatment. many clinicians also closely monitor and report the declining viral loads to their patients in order to demonstrate treatment efficacy, motivating patients to continue treatment and remain adherent to the drug regimen until the next follow-up appointment (fusfeld et al., 2013) . regardless of monitoring during hcv treatment for rgt, adherence/compliance, patient motivation, early treatment efficacy, etc., quantitative real-time pcr is widely used by laboratories due to its sensitivity, accuracy, and reproducibility of each consecutive viral load test. for patients infected with chronic viral infections, such as hiv, the lifelong regimen of highly active art aims to suppress hiv viral levels to near undetectable levels, ensuring progression-free survival (delay or all together prevention of the progression to aids) and reducing potential transmission. alongside monitoring immune function and immunologic efficacy through cd4 t-cell count, hiv viral levels are critical in the clinical evaluation and assessment of hiv-infected patients undergoing art. determining a patient's hiv viral load is indicated prior to entry into care, at the initiation of art, at 2-8 weeks after art initiation, and then typically every 3-4 months while on treatment: (1) to establish a baseline level of hiv viral load; (2) to establish viral response to the therapy to assess the virologic efficacy of art; and (3) to monitor for abnormalities that may be associated with antiretroviral drugs (dhhs hiv, 2014) . the baseline hiv viral load is not only linked to treatment options (sax et al., 2009) but also helps to establish the magnitude of viral load decline after initiation of art and provides prognostic information about the probability of progression to aids or death (marschner et al., 1998; murray, elashoff, iacono-connors, cvetkovich, & struble, 1999; thiebaut et al., 2000) . once treatment is initiated, the goal is to reach and maintain suppressed hiv replication as determined by undetected viral levels utilising highly sensitive nat tests, which is generally achieved within 8-24 weeks after art initiation. the need for sensitive assays to effectively assess viral suppression hinges on the need to suppress hiv replication to the extent that viral evolution and drug resistance mutations do not emerge, which typically do not occur in patients whose hiv rna levels are maintained below the llod of current real-time quantitative pcr assays (kieffer et al., 2004) . due to the introduction of more sensitive real-time pcr assays, which can detect as few as 20 viral copies/ml, natural variability in hiv viral levels over time, even in patients with effective suppression, is much more evident (lima, harrigan, & montaner, 2009; gatanaga et al., 2009; willig et al., 2010) . although controversy exists between the clinical significance of viral loads between llod and <200 copies/ ml, there are reports suggesting that this low-level viraemia is predictive of virologic rebound (doyle et al., 2012; eron et al., 2013; laprise, de pokomandy, baril, dufresne, & trottier, 2013) , virologic failure (estevez et al., 2013) , and indication of drug resistance (taiwo et al., 2010) , signifying the need for highly sensitive assays. viraemic blips, a single detectable hiv viral load (<500 copies/ml) in an otherwise seemingly suppressed patient (figure 7) , however, do not indicate subsequent virologic failure or development of resistance mutations (castro et al., 2013; lee, kieffer, siliciano, & nettles, 2006; nettles et al., 2005) . blips are not unusual (havlir et al., 2001) and appear to be more common in winter, suggesting that host-related and seasonal factors are associated with the occurrence of viraemia (van sighem et al., 2008) . on the other hand, persistent hiv rna levels !200 copies/ml are often evidence of viral evolution and accumulation of drug resistance on-treatment hiv patient monitoring. (a) hiv viral loads will fluctuate as patients are on treatment, and, in most instances, will remain 'undetectable' (at or below dotted line); viral 'blips' are not uncommon and will result in transient 'detectable' and even quantifiable results (above the dashed line). (b) virologic failure will lead to a sustained high-level viral titre that, without intervention, will increase with time. mutations (aleman, soderbarg, visco-comandini, sitbon, & sonnerborg, 2002; karlsson et al., 2004) . once treatment failure is confirmed, immediate intervention is recommended to avoid progressive accumulation of resistance mutations and effective response of new regimen (dhhs hiv, 2014), which is benefited by low hiv rna levels and/or higher cd4 cell counts (eron et al., 2013) , and even a brief interruption in therapy may lead to a rapid increase in hiv rna and a decrease in cd4 cell count and increases the risk of clinical progression (deeks et al., 2001; lawrence et al., 2003) . with the development and administration of newer drugs that target specific biological processes of hiv, routine and clinical monitoring of viral loads using a real-time quantitative pcr assay continues to be critical to predict treatment failure and early emergence of drug resistance mutations, within a timeframe that would increase subsequent treatment success. viral load monitoring is also essential when the recipient of a solid organ transplant is cmv seropositive and the decision is made to initiate treatment only once the cmv levels predictive of disease are reached. this strategy, known as pre-emptive therapy, utilises intensive monitoring for cmv activity by sensitive real-time quantitative pcr methods and short-term antiviral treatment is given only to those with significant viral counts before symptoms occur. cmv is one of the most common opportunistic pathogens that infect solid organ transplant recipients (fishman, 2007) and is associated with increased morbidity and mortality (sagedal et al., 2004; schnitzler et al., 2003) . following primary infection, the virus establishes a lifelong latent infection in several sites of the body and may reactivate in the presence of immunosuppression, such as in transplant recipients. once reactivated, cmv is able to modulate the immune system and is known to be a potent upregulator of alloantigens (razonable, 2008) , increasing the risk of chronic allograft dysfunction (reischig, 2010; sagedal et al., 2002; smith et al., 2010) and acute rejection (sagedal et al., 2004) . pre-emptive therapy reduces the incidence of cmv disease (khoury et al., 2006; reischig et al., 2008) , which has been documented as a serious problem in randomised trials upon completion of universal antiviral prophylaxis therapy (kalil, levitsky, lyden, stoner, & freifeld, 2005; lowance et al., 1999; paya et al., 2004) . long-term studies have demonstrated that patients receiving preemptive therapy, when compared to prophylaxis therapy, were less likely to develop moderate-to-severe kidney scaring and atrophy and significantly better survival of the transplanted organ (reischig et al., 2012) . however, challenges still exist around defining appropriate thresholds to initiate pre-emptive therapy (humar & snydman, 2009) . but with new standardised real-time pcr assays, widespread adoption, and utilisation of these tests, pre-emptive therapy relying in intensive viral load monitoring may become the standard for certain at-risk patients. test of cure, or end of treatment response, is assessed following a given therapeutic regimen for signs of treatment efficacy. in few cases, a quantitative viral load measurement serves as a way to establish a cure rate, but, in others, may only be used as a confirmation of virologic suppression as clinical cure may not yet be possible with current therapies or technical limitations by real-time pcr that limits the overall sensitivity of viral detection. regardless of the clinical utility for measuring a virologic suppression, quantitative real-time pcrs with their current limits of detection and limits of quantitation are valuable tools in measuring low-level viraemia and establishing undetectable viral loads. utilisation of quantitative real-time pcr to assess virologic cure is perhaps best exemplified by treatment of patients with chronic hcv. according to the aasld/ idsa guidelines, patients who have 'undetectable' hcv rna in the serum, when assessed by a sensitive pcr assay, 12 or more weeks after completing treatment, are deemed to have achieved a sustained virologic response . achieving an svr is considered a virologic cure of hcv infection since, in these patients, hepatitis c-related liver injury stops and recurrence of infection is marginal, detected in <1% of patients after 5 years post-treatment (aasld/idsa/ias-usa, 2014; manns et al., 2013) . in agreement with these guidelines, the fda recommendation to pharmaceutical daa manufacturers also stipulates that viral rna clearance at svr-12 be measured in clinical trials using an fda-approved sensitive and specific quantitative hcv rna assay (fda hcv, 2013) . according to prescribing information accompanying the current daas, the threshold of svr-12 is defined as a quantitative threshold of hcv rna <25 iu/ml at 12 weeks after the end of treatment (feld et al., 2014; kowdley et al., 2014; lawitz et al., 2013) . this is somewhat dissimilar to the aasld/idsa guidelines as 'undetected' viral levels are not equivalent to 'detected but below the limit of quantitation' (figure 3 ). but, with the benefit of high sensitivity and reproducibility, quantitative real-time pcr has a clear established role in assessment of hcv virologic cure in both clinical trials and clinical practice and is able to meet the needs for assessing svr. quantitative real-time pcr may also play a critical role in the assessment of cmv disease resolution. the consensus guidelines recommend that two consecutive negative samples be obtained with a minimum treatment course of 2 weeks before treatment is discontinued, which is thought to minimise the risk for development of resistance and disease recurrence (asberg et al., 2009; chou, 2001; sia et al., 2000) . still, some transplant centres may extend treatment (secondary prophylaxis) in patients with compartmentalised disease for as long as necessary to reduce the likelihood of recurrent cmv infection (kotton et al., 2013) . resolving cmv disease has the long-term benefits of reducing mortality, potential allograft rejection, and the risk of bacterial, fungal, or viral opportunistic infections, among many other transplantand non-transplant-specific effects (arthurs et al., 2008; fishman, 2007) . although there is currently no cure for hiv infection, highly sensitive quantitative and qualitative real-time pcr tests targeting total hiv dna and rna have been used in clinical studies for both sterilisation (elimination of hiv-infected cells) and functional (controlled hiv in the absence of art) cures (kibirige, 2013; lewin & rouzioux, 2011) . improvements in real-time pcr technology may lead to profound increases in assay sensitivity and the ability to achieve single-copy detection (1 cp/ml) may lead us to a better understanding of hiv virology and what may be needed therapeutically to achieve a cure (alidjinou, bocket, & hober, 2014) . if therapeutic strategies are one day able to achieve an hiv cure, these highly sensitive tests will no doubt play a key role in the continuum of care for patients and, most importantly, in the confirmation of cure. clinical laboratories have undergone changes to become more efficient and flexible while delivering the same high-quality results. when choosing to implement new testing, even beyond viral targets, laboratories have to consider first and foremost the performance and medical value of the test and then factors such as tat, ease of use, and cost. real-time pcr with its wide dynamic range, high specificity, and high sensitivity is considered the gold standard for the quantification and identification of a variety of targets including bacteria, fungi, viruses, or oncological mutations (klein, 2002) . furthermore, the multiplexing capability of real-time pcr increases the number of targets and information gathered from the same test, further improving laboratory workflow, tat, and costs (deshpande & white, 2012) . while novel technologies have entered clinical laboratories including mass spectrometry and next-generation sequencing, real-time pcr remains a staple and an attractive option for clinical laboratories aiming to create molecular laboratory-developed tests (ldts). in addition, pcr can quickly be adapted to provide a robust test for the identification of emerging disease and molecular testing is now able to reach beyond the clinical laboratory and further enhance healthcare (farrar & wittwer, 2015; foudeh, didar, veres, & tabrizian, 2012) . most molecular tests used in clinical laboratories are fda-approved and commercially available. there are instances, however, when a test may not be available for a specific virus or the sample type and/or clinical indication used by the laboratory differs from those of the fda-approved assay, typically leading a laboratory to design its own pcr-based test or modify existing assays. fda defines an ldt as 'a type of in vitro diagnostic test that is designed, manufactured, and used within a single laboratory' and recognises that 'ldts are important to the continued development of personalised medicine' (fda ldt, 2014) . laboratory developed tests can be grouped into three categories, fda-cleared or approved test that have been modified, tests that are not subject to fda clearance or approval, and tests for which no performance specifications have been provided by the manufacturer (e.g. analytespecific reagents or asrs) (burd, 2010; code of federal regulations, 2009 ). with alternative sample types or applications, fda-approved tests are often modified to fit the testing needs of laboratories, including alternative collection media and sample types or expanded clinical applications. as an example, a recent gap was created in the hcv-screening algorithm for the confirmation of a positive enzyme immunoassay result following the discontinuation of the only fdaapproved confirmatory test (alter, kuhnert, & finelli, 2003) . in response, the cdc published recommendation for the use of fda-approved tests detecting hcv viraemia (cdc hcv, 2013), despite the fact that most of these assays did not have specific claims for confirmatory testing; as a result, several laboratories chose to validate these assays as ldts to meet the screening needs for hcv. additionally, ldts are the only option for the identification of the aetiologic agents of viral infections that can occur in transplant patients, such as ebv, adenovirus, vzv, and bk virus, that often present with non-specific clinical manifestations (razonable, 2011) and for which fda-approved assay options are lacking. ldts are an integral part of molecular laboratory testing. whether created from the ground-up or modified from fda-approved assays, ldts are answering the clinicians' needs for information as an aid for diagnosis or treatment of patients. as with any clinical tests, ldts have to meet the minimum standards set forth by clia prior to report patient results (code of federal regulations, 2009). in july 2014, fda informed congress of the agency's ldt regulatory oversight framework (fda ldt, 2014) . fda aims to address concerns over high-risk ldts with inadequately supported claims, lack of appropriate controls, and falsification of data that may lead to inadequate treatment, possible harm to patients, and unnecessary healthcare cost. presently, there is still a high degree of uncertainty as to what the final regulation scope will be and the possible impact on molecular laboratories will have to be seen. palaeopathology confirmed the truism that humanity, since its inception, has been exposed to genetic and infectious diseases with early documentation of trachoma (8000 b.c.e.), tuberculosis (7000 b.c.e.), and pneumonia (ca. 1150 b.c.e.) (aufderheide & rodreguez-martin, 1998; hershkovitz et al., 2008; roberts & manchester, 1995; webb, 1990) . even today, emerging infectious diseases (eids) continue to appear unpredictably driven by changes in human demographic, land use, and population behaviour (lederberg, hamburg, & smolinski, 2003; sehgal, 2010; taylor, latham, & woolhouse, 2001) . these infections can be classified as either newly emerging/a previously unknown disease or re-emerging infectious diseases/a previously known disease, that reappears after a significant reduction in incidence or elimination (morens & fauci, 2013) . eids are a threat not only to human health but also to global stability and economy. efforts to monitor these eids are in place both at the global level spearheaded by the who and at the national level. in the united states, governmental agencies (department of health and human services, united states agency for international development, department of defense) are supporting activities to detect, assess, and respond to potential outbreaks. specifically, pcr and real-time pcr are easily adaptable to detect nucleic acid targets that are unique to each given pathogen, and as such, they play essential roles in the identification and detection of infectious pathogens and have been routinely used by health organisation agencies during epidemic outbreaks such as severe acute respiratory syndrome (sars), h5n1, h1n1, and ebola (shuaib et al., 2014; who influenza, 2011) . the sars epidemic appeared in november 2002, in the chinese province of guangdong before reaching the adjacent hong kong in 2003 (who sars, 2003 . this sars eventually spread to 26 countries and resulted in more than 8000 cases. in response, the cdc triggered its emergency operations centre and issued a draft genome in april 2003, 33 days after the initial who global alert (cdc sars, 2013) . soon after, real-time quantitative pcr assays were described and put in use for the diagnosis of sars (drosten et al., 2003; peiris et al., 2003) . a host of measures were taken in order to contain this epidemic, and the molecular identification and diagnosis of the infectious agent by pcr played a key role in providing critical information to address the situation and contributed to the care of the patients infected. additionally, the re-emerging 2014 ebola epidemic (cdc ebola, 2014; who ebola, 2014) started in guinea in march of 2014 before spreading to nearby west african countries and eventually reaching the united states and europe (who ebola, 2014) . at the height of the epidemic, fda issued an emergency use authorisation (eua) for the use of the first real-time rt-pcr assay (fda eua, 2014) and less than 4 months later, five additional realtime pcr tests were authorised under an eua (fda eua, 2014) to provide an early diagnosis of the ebola viral disease (cdc ebola, 2014). eids remain a constant and unpredictable threat to human health. the flexibility of real-time pcr technology continues to show how promptly it can be used for the detection of infectious agents. by providing a rapid diagnostic, real-time pcr can help in starting the appropriate treatment right away and maximise the chances of a positive outcome. the goal of point-of-care testing (poct) is to quickly obtain a test result that will be used to implement the appropriate treatment for an improved clinical outcome. by definition, poct is laboratory testing that takes place at or near the site of patients (cap poc, 2013) . the advantages of poct are an improved tat and result availability regardless of normal core laboratory hours, access to care in remote areas, and greater patient involvement. the fight against aids largely contributed to the development of poct devices with viral load capabilities (hong, studer, hang, anderson, & quake, 2004; lee et al., 2010; marcus, anderson, & quake, 2006; tanriverdi, chen, & chen, 2010; unitaid, 2014; vulto et al., 2009) . originally developed to meet the difficult conditions associated with remote places, far from any core laboratory facility often found in the developing world, the design and convenience of a portable poct device with fast turnaround and accurate results extends the reach of healthcare. with this in mind, these poct systems could easily be used in developed nations at hospitals, within clinics a physicians' offices, pharmacies, correction facilities, or mobile health units, to target pathogens that benefit from immediate actionable results, for which not only accurate but also quick results are critical (kiechle & holland, 2009 ). ultimately, test menu available on these platforms will drive its implementation as a complement for the clinical laboratory core testing. the ideal molecular poct system that includes medical value, simplicity, fast tat, and ruggedness remains an ongoing engineering challenge. however, the latest advances in microfluidics are a great example of the potential of these devices and brings the real-time pcr lab-on-a-chip closer to mainstream diagnostic use. this is an exciting time for molecular poct and the upcoming years should bring new systems and perhaps a paradigm change in the world of healthcare. as the needs of the clinicians, laboratory, and patients continue to evolve, so do the applications of molecular diagnostics and pcr. over the past decade, quantitative real-time pcr technology has been increasingly phased into clinical practice and all of the potential present-day applications of real-time pcr-based methods are enumerable. they serve to advance experimental approaches within biological fields, pushing the boundaries of what we know and what we can learn, as well as to diminish empiric medical identification and management of viral diseases. the high sensitivity of the technology has reduced risks of the most commonly transmitted transfusion illnesses and has become an integral part of managing a variety of viral infections by providing pretreatment prognostic information, therapeutic effectiveness through monitoring, and end of treatment response assessment. quantitative real-time pcr complements serologic testing by detecting infections within the pre-seroconversion window period and infections with immunovariant viruses and are able to predict therapeutic failures sooner than traditional methods, allowing for a more timely management response. real-time pcr assays can be rapidly developed in cases of emerging epidemic crises involving new pathogens that may result in significant health threats. the next few years are likely to see an even further increase in the expansion of the clinical applications of nucleic acid quantification, particularly following bone marrow and solid organ transplantation for which the newest standardised assays may provide an avenue for the development of consensus management guidelines for initiating pre-emptive anti-cmv treatment. further, with the drive towards hiv eradication and complete elimination of the 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reaction prehistoric eye disease (trachoma?) in australian aborigines world health organization. global alert and response: ebola outbreak world health organization. the use of pcr in the surveillance and diagnosis of influenza world health organization. severe acute respiratory syndrome cost ramifications of increased reporting of detectable plasma hiv-1 rna levels by the roche cobas ampliprep/cobas taqman hiv-1 version 1.0 viral load test multi-site pcr-based cmv viral load assessment-assays demonstrate linearity and precision, but lack numeric standardization: a report of the association for molecular pathology natural history: the importance of viral load, liver damage and hcc management and treatment of hepatitis c viral infection: recommendations from the department of veterans affairs hepatitis c resource center program and the national hepatitis c program office discordant human immunodeficiency virus infection in dizygotic twins detected by polymerase chain reaction. the pediatric infectious competitive pcr for precise nucleic acid quantification expert opinion on the treatment of patients with chronic hepatitis c key: cord-019009-3ngfv96u authors: gea-banacloche, juan title: risks and epidemiology of infections after hematopoietic stem cell transplantation date: 2016-02-15 journal: transplant infections doi: 10.1007/978-3-319-28797-3_6 sha: doc_id: 19009 cord_uid: 3ngfv96u infections following hct are frequently related to risk factors caused by the procedure itself. neutropenia and mucositis predispose to bacterial infections. prolonged neutropenia increases the likelihood of invasive fungal infection. gvhd and its treatment create the most important easily identifiable risk period for a variety of infectious complications, particularly mold infections. profound, prolonged t cell immunodeficiency, present after t cell-depleted or cord blood transplants, is the main risk factor for viral problems like disseminated adenovirus disease or ebv-related posttransplant lymphoproliferative disorder. understanding the epidemiology of infections after allogeneic hematopoietic stem cell transplantation (hct) is important to implement appropriate preventive strategies as well as to effectively diagnose and treat individual patients. several groups of experts and professional organizations publish guidelines that provide specifi c recommendations for prophylaxis and management of infections after hct [ 1 -8 ] , including vaccinations [ 1 , 9 , 10 ] . many of these recommendations are necessarily based on low-quality evidence and rely heavily on expert opinion. guidelines should not be followed blindly, but understood as tools that may help to provide the best possible care. risk factors for infection include individual characteristics (e.g., indication for hct, prior infections, cmv serostatus, particular genetic traits) and type of transplant (based on conditioning regimen, stem cell source, degree of hla homology, and immunosuppression). the development of graft-versus-host disease (gvhd) is frequently the decisive contributor to infectious morbidity and mortality. different indications for hct are associated with their own infectious risks. primary immunodefi ciencies (pid), hemoglobinopathies, and hematologic malignancies present different challenges. even in hematologic malignancies, the risk may vary depending on the specifi c condition: patients with chronic myelogenous leukemia (cml), acute myeloid leukemia (aml), and chronic lymphocytic leukemia (cll) present different risks based on both the biology of the disease and prior treatment. these factors should be considered when assessing individual patients. prior infections must be considered. a history of infection or colonization with a multidrug-resistant organism (mdro) like carbapenem-resistant enterobacteria (cre), extended-spectrum beta-lactamase (esbl)-producing gram-negative bacteria, vancomycin-resistant enterococcus (vre), or methicillin-resistant staphylococcus aureus (mrsa) has implications regarding optimal management of fever during neutropenia [ 6 , 11 , 12 ] , which is a common complication of hct. transplant candidates are routinely screened for serologic evidence of latent infections that may reactivate (hsv, vzv, cmv, ebv, hepatitis b and c, toxoplasmosis); some of these will be discussed later in this chapter. some transplant centers will perform screening for tuberculosis with tuberculin skin test (tst) or interferon-gamma release assay (igra), at least for patients who are considered at signifi cant risk for the disease. prior invasive fungal infections may reactivate following transplant, and secondary prophylaxis is required [ 13 -15 ] . even active fungal infection has been reported to be controllable. there are, however, cases of progression of prior aspergillosis after transplant; myeloablative conditioning, prolonged neutropenia, cytomegalovirus (cmv) disease, and graft-versus-host disease (gvhd) are risk factors [ 15 , 16 ] . as the correlates of native and adaptive immunity are better understood, genetic associations are coming to light. there is evidence that some donor haplotypes of tlr4 , the gene that encodes the toll-like receptor protein 4 (tlr4) are associated with increased risk of invasive aspergillosis after hct [ 17 ] . recipient's mutations in mbl2 , the gene that encodes mannose-binding lectin (mbl), have been associated with increased risk of infection after neutrophil recovery following myeloablative transplant [ 18 ] . other polymorphisms of mbl2 may be important for infection through a direct infl uence on the risk of developing gvhd [ 19 , 20 ] . different genotypes of activated killer immunoglobulin-like receptors (akir) in the donor have been found to protect from cmv reactivation [ 21 ] . many of these associations are preliminary and require more data to be confi rmed, but they hold the promise of a more individualized approach to infectious prophylaxis. from a practical standpoint, it is helpful to consider three distinct periods during transplant: pre-engraftment (until neutrophil recovery), early post-engraftment (from engraftment until day 100) , and late post-engraftment (after day 100) . this framework originated with myeloablative transplants, and is eminently pragmatic. the pre-engraftment phase may be accompanied by profound neutropenia and signifi cant mucositis, which results in increased risk of bacterial infections from the resident gastrointestinal fl ora, candidiasis, aspergillosis (in cases of prolonged neutropenia) and herpes simplex virus reactivation. after engraftment, with neutropenia no longer being a factor, many infections are related to the profound defect in cellular immunity caused by the conditioning regimen and the immunosuppression administered to prevent gvhd. cmv reactivation and the development of acute gvhd and its treatment play a central role during this time. the day 100 landmark derives from the standard time at which immunosuppression (e.g., cyclosporine a or tacrolimus) is frequently tapered. infections after this point would be primarily related to lack of immune reconstitution and, in the absence of gvhd, become progressively less common. not all allogeneic stem cell transplantations are the same. several characteristics of the transplant infl uence the risk of infection: the conditioning preparative regimen, the source of stem cells, the degree of hla identity between donor and recipient, and the prophylactic strategy adopted to prevent gvhd (use of t cell depletion or immunosuppressive medications). table 6 -1 summarizes the impact of these factors on infections. matching for ucb transplants focuses on three loci (hla-a, hla-b, and hla-drb1). the majority of ucb transplants are mismatched by at least one locus (often two). among transplants mismatched at two loci, mismatching at hla-c and hla-drb1 was associated with the highest risk of mortality [ 24 ] . the degree of mismatch between the donor and the recipient affects the infectious risk mainly through the likelihood of gvhd. more gvhd usually results in more infections. to prevent gvhd in a mismatched transplant, more potent immunosuppression may be required, increasing the risk of infection. it is also possible that immune reconstitution proceeds more slowly (even with the same immunosuppressive regimen) after a urd hct. these factors may result in increased risk of infections associated with t cell immunodefi ciency, like cmv, pneumocystis jirovecii pneumonia (pcp), and epstein-barr virus (ebv)-related posttransplant lymphoproliferative disorder (ptld). however, provided the number of stem cells administered is the usual (>3 × 10 6 kg −1 ), neutrophil recovery proceeds at the standard pace and there is no increased risk of neutropeniarelated infections. the problems with ucb transplants include a markedly decreased stem cell dose (often <1 × 10 5 kg −1 ) which results in prolonged neutropenia (up to 6 weeks), with the attendant risk of bacterial and fungal infections [ 27 ] . in addition, the cord blood does not have antigen-specifi c memory t cells that can expand in a thymus-independent fashion to provide protection against viruses and opportunistic pathogens. this results in high frequency of late severe infections following cord transplantation, even when the neutropenic period is shortened by coadministration of stem cells from a thirdparty donor [ 28 ]. stem cells may be given using the bone marrow, g-csfmobilized peripheral blood stem cells (pbscs), or ucb. frequently bone marrow will result in more prolonged neutropenia compared with pbsc, and increased infections during neutropenia should be expected. however, a multicenter randomized trial comparing peripheral blood stem cells with the bone marrow from unrelated donors showed no difference in the relapse or infectious mortality between both groups, but confi rmed that chronic gvhd is more common with mobilized pbsc [ 29 ] . the particular features of ucd transplants were discussed on the preceding paragraph. manipulation of the stem cells, immunosuppressive drugs, or a combination gvhd may be prevented by decreasing the amount donor t cells or by limiting t cell function with immunosuppressive agents. the stem cells, whether from the bone marrow or the periphery, may be administered unmanipulated (sometimes called "t cell replete") or enriched by cd34 selection (also called "t cell depleted"). if unmanipulated bone marrow or pbscs are used, the dose of cd3+ t cells administered with the graft varies between 24 × 10 6 kg −1 when bone marrow is used and 300 × 10 6 kg −1 when pbscs are used [ 30 ] . reductions in the amount of t cells of 2-3 log 10 are possible, and in some haploidentical transplant regimens, as few as 12.5 × 10 3 cd3+ cells are given, which still results in detectable immune reconstitution starting 2-3 months after transplant [ 31 ] . t cell depletion may minimize or altogether prevent gvhd but may result in prolonged immunodeficiency, depending on the degree of depletion. if an unmanipulated product is used, t cell depletion may be attained in vivo by using alemtuzumab or atg. these agents produce a profound depletion of t cells in vivo, and their long halflife makes them still be present and active in the recipient when the stem cell product is administered. if no in vitro or in vivo t cell depletion is used, one of a variety of immunosuppressive regimens will be given to prevent gvhd (e.g., tacrolimus + methotrexate, tacrolimus plus mycophenolate mofetil, cyclosporine a, sirolimus, posttransplant cyclophosphamide). a randomized controlled trial documented more infections in patients randomized to (moderate) t cell depletion than in the group who received pharmacologic immunosuppression [ 32 ] . t cell depletion in vivo with alemtuzumab has been associated with increased risk of infection [ 33 ] . it is possible that different pharmacological regimens may result in different infectious risks, but this has not been adequately studied. preliminary evidence suggests that a sirolimus-based regimen may result in less cmv reactivation [ 34 ] and that posttransplant cyclophosphamide result in relatively decreased risk of ptld [ 35 ]. the above categories may combine in several ways, compounding the risk of infection. these variations should be considered both when designing a regimen of anti-infective prophylaxis and when considering an individual patient who may have an infection. gvhd is the most important cause of non-relapse mortality following hct, and it is frequently complicated by infection. gvhd is categorized as acute or chronic based on its time of onset. acute gvhd develops before day 100 and is characterized by gastrointestinal disease (secretory diarrhea, nausea, vomiting), liver dysfunction, and skin rash. stages of gvhd in the skin, gut, and liver combine to give a grade (i-iv) of the severity of the disease. acute gvhd grades iii-iv is associated with signifi cant mortality. the treatment of choice is high-dose systemic corticosteroids. gvhd is associated with signifi cant immune dysregulation [ 36 , 37 ] and is frequently accompanied by cmv reactivation [ 38 ] . the combination of disruption of the gi mucosa (and sometimes skin) and high-dose corticosteroids (in addition to the immunosuppressive agents concurrently given, like tacrolimus and mmf) constitute a high-risk setting for infection. bacterial, fungal, and viral infections are common under these circumstances. chronic graft-versus-host disease (cgvhd) has been traditionally defi ned chronologically: gvhd starting after day 100. it has been classifi ed based on its relation to prior gvhd (progressive when acute gvhd continues after day 100, quiescent when there is a period of time during which the patient is free of gvhd, or de novo when chronic gvhd is the fi rst manifestation of gvhd) and its extension (limited or extensive, reformulated as clinical limited, or clinical extensive). the clinical syndrome of typical chronic gvhd is quite distinct from the acute form, and a new classifi cation focusing on the clinical characteristics of the disease as well as on the timing is being increasingly used [ 39 ] . from the standpoint of infectious diseases, the important consideration is that the presence of chronic gvhd is associated with high risk of infection [ 40 , 41 ] . multiple immune defects have been described during chronic gvhd, involving humoral and cellular immunity [ 42 , 43 ] as well as functional hyposplenism [ 44 , 45 ] . besides these abnormalities, that result in delayed immune reconstitution and poor response to immunizations, the risk is of infection is increased by the treatment of extensive cgvhd [ 41 ], which typically includes systemic corticosteroids and a variety of steroid-sparing agents. notably, cgvhd is a well-documented risk for pneumococcal infections [ 45 , 46 ] , fungal infections, and late cmv disease. however, all types of infections are more common during cgvhd, particularly during the fi rst few months [ 47 ] . when gvhd is not controlled by corticosteroids, it is called " steroid refractory ," and there is currently no universally accepted standard treatment. this situation is important from the infectious disease standpoint because patients are usually treated with a variety of highly immunosuppressive regimens (e.g., atg, cyclophosphamide, mmf, infl iximab, daclizumab, alefacept, alemtuzumab, sirolimus, visilizumab, denileukin diftitox, and others) [ 48 ] that result in a wide array of infectious complications. reactivation of cmv is very common, as are fungal infections [ 49 , 50 ] , epstein-barr virus-related ptld [ 51 ] , as well as human herpesvirus 6 (hhv-6) [ 52 ] and adenovirus [ 53 ] . there are no controlled studies to support any particular infection prevention strategy during this period of increased immunosuppression, but some authors have emphasized that early use of prophylactic antibiotics and antifungals is an essential part of a successful approach to this problem [ 54 ] . unfortunately, this is a condition for which controlled trials are unlikely to be performed, and different centers will have to decide on a particular approach of close monitoring versus prophylaxis based on local experience and published case series. in the following sections, the epidemiology of bacterial, fungal, viral, and parasitic diseases will be discussed. the implications for prophylaxis and management will be mentioned. immunizations for transplant recipients, (as well as their caregivers and immediate contacts) are discussed in chap. 48 6.6 risks and epidemiology of bacterial infections after allogeneic hct 6.6.1 early bacterial infections: pre-engraftment approximately 20% of hct recipients will experience at least one episode of bacteremia during the fi rst few weeks, and a similar proportion after engraftment [ 55 ] . these infections are usually related to either neutropenia with subsequent bacterial translocation through the gi mucosa (mucosal barrier injury laboratory-confi rmed bloodstream infection or mbi-lcbi) or the intravascular catheter (central lineassociated bloodstream infections or clabsis) [ 56 ] . the relative frequency of gram-positive and gramnegative infections during neutropenia varies in different series and with the use of prophylactic antibiotics. in some centers, the most frequent gram-positive isolates are viridans group streptococcus [ 55 ] ; this may be a function of the conditioning regimen or the patient population. enterococcus faecium , frequently vre, is another gram-positive organism that tends to cause bloodstream infection relatively early, although this seems to be rather institution dependent [ 57 ] . the gram-negative bacteria are commonly enterobacteriaceae . these infections are generally related to the disruption of the gi mucosa due to the preparative regimen. the role of reduced diversity of the microbiota with subsequent bacterial domination and ultimately bacteremia is an area of intense study [ 58 ] . the risk of bacteremia during neutropenia may be decreased by the use of prophylactic antibiotics [ 59 , 60 ] . this had been shown in multiple studies over the years, but the recommendation of using antibiotics did not become part of practice guidelines until recently. it is not clear whether this recommendation will continue amidst the increasing concern over the role of antibiotic-induced decreased microbiome diversity on the outcome of hct [ 61 ] . in this regard it is of interest that fl uoroquinolones seem to have less detrimental effects on biodiversity of the fecal fl ora than beta-lactams. levofl oxacin at a dose of 500 mg/d for patients who are going to be profoundly neutropenic for longer than 1 week is the current recommendation of the idsa [ 11 ]. following engraftment in a large study from the sloan kettering cancer center, the risk factors for post-engraftment bacteremia included acute gvhd, renal dysfunction, hepatic dysfunction, and neutropenia [ 55 ] . enterococcus (vre) and coagulase-negative staphylococcus were the most common gram-positive isolates. enterobacteriaceae and non-fermentative gram-negative bacteria (including pseudomonas , stenotrophomonas , and acinetobacter , possibly related to the indwelling catheter) were the most common gramnegative isolates. bacteremia following engraftment often happens in the setting of patients with a complicated clinical course, acute gvhd, and multiple medical problems or else is catheter related. daily bathing with chlorhexidine-impregnated washcloths decreased the risk of acquisition of mdros and development of hospital-acquired bloodstream infections in transplant recipients in a randomized trial [ 62 ] , and this practice should be considered by every transplant program. the advantages and disadvantages of active screening for colonization by resistant pathogens have not been adequately studied in hct recipients. it is likely that local epidemiology determines whether screening is an effi cacious and costeffective approach to either prevent infection or improve outcomes. a retrospective study on vre bacteremia from the sloan kettering cancer center showed that vre carriage was predictive of subsequent vre bacteremia, but failed to detect the pathogen in many patients [ 63 ] . performing surveillance cultures for resistant organisms in vulnerable patient populations is part of the cdc recommendations "management of multidrug-resistant organisms in healthcare settings, 2006" [ 64 ] , and has been vigorously advocated by some experts [ [ 66 , 67 ] . both early and late (beyond day 100) pneumococcal disease has been reported, with late infections strongly associated with active cgvhd [ 46 ] . these have been attributed to inadequate antibody production and functional hyposplenism [ 44 , 67 ] . vaccination against s. pneumoniae should be given to all hct recipients, starting 3-6 months after transplant and using the 13-valent conjugate vaccine [ 9 ] (see chap. 48 for details). four doses of the vaccine result in enhanced antibody response and tolerable side effects [ 68 ] . antibiotic prophylaxis against s. pneumoniae prophylaxis for adults with active cgvhd has been recommended [ 69 ] , although there is only weak evidence supporting its effi cacy. penicillin v-k is safe and well tolerated, but the local patterns of penicillin resistance may make other antibiotics (e.g., trimethoprim, sulfamethoxazole, azithromycin, or levofl oxacin) preferable, although their long-term safety is not well established. late bacterial infections often involve the respiratory tract. pneumonia is the most common cause of fatal late infection [ 40 , 70 ] . chronic gvhd is the risk factor most commonly identifi ed. besides s. pneumoniae , multiple other pathogens have been reported. nocardia also tends to occur late and in patients with cgvhd [ 71 , 72 ] . mycobacterial infections are uncommon and diffi cult to diagnose [ 73 ] . risk factors for the development of active tb include gvhd, corticosteroid treatment, and total body irradiation (tbi) [ 74 ] . the need for universal testing for tuberculosis is controversial, given the unknown sensitivity and specifi city of the tests in this population and the fact that tuberculosis is a relatively uncommon complication after hct (albeit still approximately three times higher than in the general population) [ invasive candidiasis follows prior colonization and favorable conditions for the yeast: disruption of the gi mucosa during chemotherapy or acute gvhd, overgrowth in the presence of broad-spectrum antibiotics, and/or presence of indwelling catheters (the catheter seems to be the main risk factor in the case of c. parapsilosis ). early studies showed that fl uconazole during the pre-engraftment period could decrease the incidence of invasive candidiasis [ 76 , 77 ] . accordingly, fl uconazole is recommended as part of the standard prophylactic regimen during the pre-engraftment period. the prevalent use of fl uconazole has resulted in substantial decrease in the incidence of infections caused by c. albicans with relative increases in the incidence of other species of candida with decreased susceptibility to this agent (e.g., c. glabrata , c. krusei ) [ 78 ] . invasive aspergillosis occurs during specifi c "at risk" periods following hct, with a fi rst peak around the time of neutropenia pre-engraftment, a second peak between days 40 and 70 (the time of acute gvhd and its treatment), and a third peak late after transplant, usually in the midst of actively treated cgvhd [ 79 ] (figure 6-1 ) . a variety of risk factors for invasive aspergillosis have been identifi ed over the years, but the most consistently found to be signifi cant in multivariate analyses are acute gvhd, chronic extensive gvhd, and cmv disease [ 80 -82 ] . systemic corticosteroids are almost always present as part of the treatment of acute and chronic gvhd. non-aspergillus mold infections (e.g., fusariosis, mucormycosis, scedosporiosis), sometimes referred to as emerging mold infections, have been reported with increasing frequency [ 83 ] . the increased use of prophylaxis with activity against aspergillus would be expected to result in a relative increase of other opportunistic mycoses like mucormycosis [ 84 ] . considering the diversity of fungal infections after transplant and the current antifungal armamentarium, it is controversial which antifungal prophylaxis is appropriate at what point during transplant. for instance, although fl uconazole is a safe and well-established intervention during the preengraftment period of myeloablative transplants [ 76 , 77 ] , it is reasonable to question how necessary it is in transplants with conditioning regimens that result in shorter neutropenia. micafungin showed to be equivalent to fl uconazole in a randomized controlled trial [ 85 ] , and the same question (what kind of transplant patient would benefi t most) applies. regarding the duration of antifungal prophylaxis, fl uconazole up to day 75 posttransplant was associated with improved survival mainly due to decreased incidence of systemic candidiasis [ 86 ] , but it is uncertain whether this strategy should be used for all patients or should be received for some selected subgroups considered at higher risk. similarly, it is reasonable to question the indication for fl uconazole during periods when the main fungal infection is aspergillosis. several randomized controlled trials have compared fl uconazole with another azole with activity against molds (itraconazole [ 87 , 88 ] , voriconazole [ 89 ] , or posaconazole [ 90 ] ) either as standard posttransplant prophylaxis or during periods of increased risk. the general conclusion of these trials is that the aspergillus-active drugs are, indeed, more effective than fl uconazole in preventing ia, but the benefi t in survival in the context of a clinical trial with careful monitoring of galactomannan antigen is hard to demonstrate [ 91 ] . the 2009 asbmt/ebmt guidelines recommend posaconazole or voriconazole as antifungal prophylaxis in the setting of gvhd and micafungin in the setting of prolonged neutropenia [ 1 ] . of note, posaconazole prophylaxis was superior to fl uconazole or itraconazole and improved survival in prolonged neutropenia in non-transplant patients [ 92 ] . now, there are even more options of mold-active prophylaxis with posaconazole delayed-release tablets, intravenous posaconazole, and the new agent isavuconazole. infections after allogeneic hct viral infections remain a challenge because newer transplant modalities result in severe prolonged t cell immunodeficiency and because the current antiviral armamentarium is very limited. multiple latent viruses may reactivate following hct [ 93 ] . the role of monitoring by pcr is well defi ned mainly for cmv. latent viral reactivation is of particular concern in recipients of cord [ 94 ] or t cell-depleted transplants. table 6 -3 presents a summary of this section. members of the herpesvirus family that have caused significant disease after transplant include hsv-1, hsv-2, vzv, ebv, cmv, and hhv-6. posttransplant complications of hhv-7 are not well defi ned, although multiple associations have been described. hhv-8 infection and disease (primary effusion lymphoma and kaposi's sarcoma) occur only infrequently after hct. hsv-1 and hsv-2 may reactivate following the preparative regimen and complicate chemotherapy-induced mucositis, so it is customary to administer prophylaxis with acyclovir or valacyclovir at least until engraftment. in patients with common recurrences, long-term suppression may be appropriate. vzv predictably reactivates following transplant (approximately 25% in the fi rst year), either as shingles, multidermatomal, disseminated, or even without a rash ("zoster sine herpete"). in patients who are at risk for vzv reactivation, the use of long-term acyclovir safely prevents the occurrence of vzv disease [ 95 , 96 ] , and currently it is recommended for at least 1 year following hct. cmv remains latent in a variety of human cells. cmvseropositive hct recipients are at risk for cmv reactivation and disease after transplant. the term "cmv infection" is used to denote the presence of cmv in the blood detected by pcr or pp65 antigenemia [ 97 ] . following reactivation, cmv may cause disease typically in the form of pneumonia and/or gastrointestinal disease (most commonly colitis). other cmv diseases like retinitis or cns involvement are rare after hct but have been described: retinitis has been associated with high cmv viral load [ 98 ] sometimes in the context of chronic gvhd and cns disease (encephalitis and ventriculitis), sometimes with resistant virus in the cns [ 99 , 100 ] . the risk for reactivation may be related to the presence of cmv-specifi c immunity in the donor. the rate of cmv infection in the donor-recipient (d/r) pairs often follows the progression d r d r d r d r -+ + + + , suggesting that cmv-specifi c memory t cells administered with the stem cells may play a role in preventing reactivation and disease. cmv infection or disease in cmv-seronegative recipients of seronegative donors (r−/d−) is rare when leucodepleted or cmv-negative blood products are used [ 101 ] . every transplant program must decide on a strategy to monitor cmv and prevent disease. depending on a variety of factors, either universal prophylaxis with ganciclovir up to day 100 or a preemptive strategy of weekly monitoring and early therapy may be used. both approaches resulted in similar overall mortality when compared in a randomized controlled trial, but universal prophylaxis was followed by more cases of late cmv disease [ 97 , 102 ] . late cmv disease has emerged as a signifi cant problem, as it occurs when patients are not being under close monitoring by the transplant center. risk factors include lymphopenia and chronic gvhd [ 103 ] . preventing late cmv disease may be accomplished by either prophylaxis with valganciclovir or the preemptive approach with weekly cmv pcr monitoring [ 104 ] . the effect of cmv serostatus of donor and recipient on overall survival is complex (for a review, see [ 105 ] and chap. 24 ). ptld is a spectrum of lymphoid proliferations that may happen after solid organ or allogeneic stem cell transplantation, usually (but not always) driven by ebv [ 106 ] . pathologically the spectrum goes from polymorphic, polyclonal tissue infi ltration of lymphocytes to monomorphic involvement with high-grade b cell lymphoma. after allogeneic hct, the proliferating cells may be from donor (most commonly) or recipient origin. this disorder is typically related to insuffi cient or abnormal t cell responses against ebv [ 107 ] , and accordingly it is more common in the setting of hla-mismatched transplants, t cell depletion, or intense immunosuppression for the treatment of gvhd [ 108 -110 ] . some cases have followed the use of alemtuzumab for in vivo t cell depletion or gvhd prophylaxis [ 110 ] , despite the fact that anti-cd52 also results in depletion of b cells and earlier had been reported to be associated with relatively less risk. interestingly, the use of posttransplant cyclophosphamide to prevent gvhd seems to be associated with lower risk of ptld [ 35 ] . monitoring of ebv viral load by quantitative pcr is now recommended in those transplants considered at high risk. preemptive management of increasing ebv viral load in patients at risk has been associated with good outcomes [ 111 ] , although it is not clear when exactly this treatment should be given. a ct/pet may be useful to localize areas amenable to biopsy (figure 6 -2 ). hhv-6 is acquired early in life, when it may cause roseola infantum and nonspecifi c febrile illnesses. it frequently reactivates following hct. using quantitative pcr, hhv-6 can often be detected in peripheral blood 2-5 weeks after transplant. most of the time the reactivation seems to be asymptomatic [ 112 ] , but a number of associations (rash, delayed engraftment, gvhd, thrombocytopenia, increased overall mortality) as well as actual clinicopathological entities (hepatitis, pneumonitis, encephalitis) have been described [ 113 -115 ] . hhv-6 is possibly the most common cause of infectious encephalitis after hct [ 116 ] . it seems to be particularly frequent after cord blood transplant. cases of encephalitis tend to be accompanied by higher viral loads of hhv-6 in plasma [ 117 ] , but the role of systematic monitoring of hhv-6 in plasma is unknown at this time, as reactivation seems much more common than disease [ 118 ] and attempts to use a preemptive strategy using foscarnet have not been successful [ 119 ] . the european conference on infections in leukemia has proposed evidence-based guidelines to address the diagnostic and therapeutic uncertainties related to this infection [ 120 ] . respiratory viruses , a heterogeneous group of virus that is responsible for most upper acute respiratory infections in normal hosts, result in signifi cant morbidity and mortality after hct, particularly during the fi rst 3 months following transplant [ 121 ] . even asymptomatic carriage of respiratory viruses at the time of transplant has been reported to result in increased risk of unfavorable outcomes [ 122 ] . besides respiratory syncytial virus (rsv) [ 123 ] , infl uenza, parainfl uenza virus (piv) [ 124 ] , rhinovirus [ 125 ] , and adenovirus, newly identifi ed viruses including metapneumovirus [ 126 ] , coronavirus [ 127 ] , and bocavirus [ 128 ] have emerged as signifi cant pathogens. these infections present signifi cant risks both acutely and in the long term. during the acute infection, hct recipients are at risk of developing viral pneumonia that sometimes progresses to respiratory insuffi ciency, mechanical ventilation and death, and also at risk of concomitant or secondary bacterial or fungal infections that are associated with increased mortality [ 124 , 129 , 130 ] . longterm, there seems to be an association between early infection (pre-day 100) with some of these viruses (most notably piv and rsv) and later development of chronic airfl ow obstruction [ 131 ] . the most signifi cant risk factor overall for progression of these infections from the upper respiratory tract to the lungs seems to be lymphopenia [ 132 ] . corticosteroid use seems to contribute to progression to pneumonia in rsv and parainfl uenza infections but not so in infl uenza [ 129 , 130 ] (see table 6 -3 ). besides its role among the community-acquired respiratory virus, adenovirus may cause disease in transplant recipients following reactivation in the gastrointestinal tract followed by dissemination and end-organ damage [ 133 ] . de novo acquisition of adenovirus may also result in disseminated disease. there are more than 60 types of human adenovirus, with dif-ferent tropisms and possibly varying susceptibilities to antiviral agents. they can cause a variety of diseases, including upper and lower respiratory tract infection, colitis, hemorrhagic cystitis (hc), nephropathy, and cns disease. systemic adenovirus disease seems to be more common in children, particularly in recipients of cord blood or t cell-depleted transplants [ 134 -136 ] . patients with gvhd on treatment with high-dose corticosteroids are also at risk (figure 6-3 ) . some studies have documented that sustained high levels of adenoviremia are associated with disease [ 137 ] . it is not known yet whether a preemptive approach with cidofovir can successfully prevent disseminated disease and death [ 133 , 138 ] . bk virus infects 90% of humans by age 12. it predictably reactivates in most patients following hct and causes hemorrhagic cystitis (hc) in a minority of them [ 139 ] . detection of high levels of bk in the peripheral blood seems to correlate with the presence of bk-induced hc [ 140 , 141 ] . in a large study from the fred hutchinson cancer research center (fhcrc), no association was found between bk virus-associated hc and lymphopenia, corticosteroid use, and gvhd-the typical risk factors for viral infections after hct [ 140 ] . in contrast, other smaller studies have found an association with gvhd. the pathogenesis of this disease remains unexplained. bk-induced nephropathy, a common problem after kidney transplant, remains infrequent after hct and does seem to be related to profound immunosuppression [ 142 ] . bk pneumonitis has also been described, but it is distinctly rare [ 143 ] . jc virus is also acquired by most people during childhood. in immunocompromised hosts, it may cause encephalitis (jc encephalitis, previously called progressive multifocal leukoencephalopathy (pml)) with multiple areas of demyelin-ation without edema detectable by mri. some studies have suggested that detectable viral load after hct may be more common than currently thought [ 144 ] . ascertaining risk factors for this disease is diffi cult because some transplant recipients may have conditions known to be associated with it and also received medications like mmf, rituximab, or brentuximab, which have been associated with pml even in the absence of allo-hct. pcp is an opportunistic infection of patients with profound cellular immunodefi ciency, and prophylaxis is recommended after hct. it is now relatively uncommon: 1.3-2.4% of patients transplanted from several series [ 145 , 146 ] most cases seem to occur relatively late, after discontinuing prophylaxis or during periods of intensive immunosuppression for the treatment of gvhd [ 147 ] . hypoxemia is characteristic at presentation. atypical radiological manifestations, including nodular infi ltrates and pleural effusions (in contrast to typical interstitial pneumonitis), are described frequently, as is the presence of co-pathogens [ 148 ] . the preferred prophylaxis is trimethoprim/sulfamethoxazole (tmp/smx) , and several dosing regimens are effective (one single-strength tablet daily, one double-strength tablet daily, or one double-strength tablet three times/week) [ 149 ] . tmp/ smx may be poorly tolerated because of hematologic toxicity, skin rash and/or gastrointestinal toxicity [ 150 ] . it is unclear which is the prophylaxis of choice if tmp/ smx cannot be used. aerosolized pentamidine is convenient, obviates the problem of compliance, and is less toxic than dapsone and better tolerated than atovaquone. however, it has been reportedly associated with more failures than dapsone [ 150 ] . dapsone seemed to be effective and well tolerated in one study [ 151 ] but not in another when it was given only three times per week [ 152 ] . dapsone should not be given to patients with g6pd defi ciency. methemoglobinemia is a well-known complication of dapsone [ 153 ] that should be considered in the presence of unexplained shortness of breath. atovaquone suspension 1500 mg/d may be used, but published experience in hsct recipients is limited [ 154 , 155 ] . atovaquone is expensive and poor tolerance has made compliance for some patients diffi cult. absorption is better in the presence of signifi cant amount of fat, and breakthroughs are well documented ( figure 6-4 ) . pcp prophylaxis is recommended at least until all immunosuppression has been stopped but it is unclear how much longer to continue it [ 156 ] . most cases of toxoplasmosis after hct represent reactivation, although rare cases of transmission with bone marrow transplant have been suspected [ 157 ] . recipients should be tested for anti-toxoplasma igg antibody, and if they are found to be positive, prophylaxis is recommended. rare cases of toxoplasmosis after hct have occurred in seronegative recipients [ 158 , 159 ] . the disease tends to occur within the fi rst 6 months after transplant, but it can happen later in the presence of persistent immunosuppression [ 160 -162 ] . the risk of toxoplasmosis varies with the type of transplant and the immunosuppression: cord blood and use of atg were found to be risk factors for disease in a prospective study [ 162 ] ; most cases in another series occurred in urd or mismatched transplants [ 107 ] . tmp/smx as given for pcp prophylaxis is considered adequate to prevent toxoplasmosis, although there have been cases on hct recipients who were receiving it [ 162 ] . the best alternative for patients who are intolerant to tmp/smx is unknown. dapsone and atovaquone showed some effi cacy in hiv-infected patients and there is increasing experience after hct [ 163 ] , although failures have been reported. other regimens include clindamycin with pyrimethamine and leucovorin, pyrimethamine with sulfadiazine, or pyrimethamine and sulfadoxine and leucovorin [ 107 ] . if a reliable quantitative pcr assay is available, frequent monitoring and preemptive treatment may be appropriate, since pcrdetected reactivation seems to precede symptoms by 4-16 days [ 162 ] . retrospective data suggest this strategy may result in improved outcome [ 164 ] . in summary, infections following hct are frequently related to risk factors caused by the procedure itself. neutropenia and mucositis predispose to bacterial infections. prolonged neutropenia increases the likelihood of invasive fungal infection. gvhd and its treatment create the most important easily identifi able risk period for a variety of infectious complications, particularly mold infections. profound, prolonged t cell immunodefi ciency, present after t cell-depleted or cord blood transplants, is the main risk factor for viral problems like disseminated adenovirus disease or ebvrelated ptld. besides all these "procedure-related" risk factors, there are individual characteristics that only now are starting to be investigated and understood. future epidemiological and basic studies will likely result in truly personalized 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transplantation early detection of toxoplasma infection by molecular monitoring of toxoplasma gondii in peripheral blood samples after allogeneic stem cell transplantation atovaquone for prophylaxis of toxoplasmosis after allogeneic hematopoietic stem cell transplantation molecular diagnosis of toxoplasmosis in immunocompromised patients: a three-year multicenter retrospective study risks and epidemiology of infections after hematopoietic stem cell transplantation key: cord-023854-w8kx5n8k authors: schuster, v.; kreth, h. w. title: virusinfektionen date: 2019 journal: p&#x000e4;diatrie doi: 10.1007/978-3-662-57295-5_14 sha: doc_id: 23854 cord_uid: w8kx5n8k dieses kapitel behandelt klinik, komplikationen, diagnostik, prophylaxe und therapie der wesentlichen virusinfektionen im kindesund jugendlichenalter, u. a. virusinfektionen der herpes-gruppe (hsv1 und 2 [u. a. schwere neonatale infektionen, herpesenzephalitis]), vzv [u. a. neonatale vzv-infektionen, herpes-zoster], ebv, cmv, hhv6–7 [dreitagefieber], hhv8 [kaposi-sarkom]. unter den virushepatititiden spielen bei kindern die hepatitis a, b (chronische hepatitis b) sowie c (chronische hepatitis b) die größte rolle. weitere bedeutsame viren sind parvovirus-b19 (ursache der ringelröteln, einer aplastischen krise oder eines hydrops fetalis), humane papillomaviren (bedeutsam v. a. genitalwarzen, larynxpapillome und karzinome), rotaviren (bedeutsame gastroenteritiserreger), fsme, hiv sowie die „typischen kinderkrankheiten“ masern, mumps und röteln. häufige erreger des oberen und unteren respirationstrakts sind rhinoviren, das rs-virus, das humane metapneumovirus (hmpv), das humane bocavirus (hbov), das humane coronavirus (hcov) sowie influenzaund parainfluenzaviren. herpes-simplex-virus-infektionen j epidemiologie infektionen mit dem herpes-simplex-virus (hsv) treten ubiquitär auf. die ansteckung erfolgt bei kindern überwiegend durch virushaltige körperflüssigkeiten (speichel) und engen körperkontakt, seltener auch durch organtransplantation. die durchseuchung von hsv-1 schwankt zwischen 30% (länder mit höherem lebensstandard) und 90% (ärmere länder). die häufigkeit von hsv-2-infektionen korreliert mit der sexuellen aktivität der jeweils untersuchten bevölkerungsgruppe. die inkubationszeit beträgt 2-12 tage. j ätiopathogenese es existieren 2 herpes-simplex-viren, typ 1 (hsv-1) und typ 2 (hsv-2): 4 infektionen mit hsv-2 sind häufig mit erkrankungen im genitalbereich assoziiert. insbesondere sind infektionen des feten oder neugeborenen meist durch hsv-2 verursacht. 4 hsv-1-infektionen sind überwiegend im gesichtsbereich lokalisiert. hsv repliziert sich in mukosazellen (v. a. rachenraum, genitalschleimhaut) . anschließend dringt das virus in die nervenendigungen von peripheren sensorischen nerven ein und wandert in ihnen retrograd bis zu den spinalen hinterstrangganglien (bei hsv-1 meist ganglion des n. trigeminus, bei hsv-2 häufig sakralganglien; . abb. 14.1). an diesem ort liegt hsv in latenter form vor (keine produktion von infektiösem virus) und persistiert lebenslang im wirt. durch verschiedene faktoren (z. b. immunsuppression, stress) kann das virus jederzeit reaktiviert werden. nach einer solchen reaktivierung "wandert" hsv anterograd über die peripheren sensorischen nerven zur mukosaoberfläche des entsprechenden dermatoms und führt dort zur bläschenbildung mit aktiver virusreplikation (herpes labialis, rekurrierender herpes genitalis). entscheidend für die immunologische bewältigung einer hsv-infektion ist die zelluläre immunität. diaplazentar übertragene hsv-neutralisierende antikörper können bei exponierten neugeborenen eine hsv-infektion u. u. verhindern oder zumindest die schwere der erkrankung abmildern. dagegen können hsv-spezifische antikörper weder rekurrierende hsv-erkrankungen noch exogene hsv-infektionen verhindern. intrauterine herpes-simplex-virusinfektion in seltenen fällen kann es zu einer diaplazentaren, hämatogenen hsv-infektion des feten (überwiegend hsv-2) kommen. betroffene kinder sind meist hypotroph (85%), fast alle zeigen kurz nach geburt ein bläschenför-. abb. 14.1 pathogenese des herpes zoster und des rekurrierenden herpes simplex. oben: nach abklingen der primärinfektion mit vzv (varizellen) oder hsv (gingivostomatitis oder primärer herpes genitalis) wandern die viren retrograd entlang der sensorischen nerven zu den spinalganglien des rückenmarks, wo die viren lebenslang persistieren. unten: durch verschiedene faktoren (z. b. immunsuppression) kann vzv und hsv reaktiviert werden. beide viren wandern anschließend entlang der sensorischen nerven zu haut-bzw. schleimhaut (dermatom), wo es zur lokalen virusvermehrung und ausbildung von bläschen kommt: herpes zoster bei vzv, herpes labialis oder genitalis bei hsv die infektion beginnt mit unspezifischen symptomen (fieber, kopfschmerzen, krankheitsgefühl) . nach 1-7 tagen kommt es zu einer progressiven neurologischen symptomatik (fokale oder generalisierte krampfanfälle, verhaltensauffälligkeiten, vigilanzstörungen) bis hin zum koma. unbehandelt versterben 70% der patienten. verschiedene genetische faktoren prädisponieren für eine herpesenzephalitis (u. a. mutationen in den genen stat1, nemo, tlr3, traf3, . bildgebende verfahren (kraniales ct oder mr) und eeg zeigen im "typischen" fall fokale veränderungen uni-oder bilateral v. a. im bereich der temporallappen. im liquor findet sich meist eine pleozytose (überwiegend lymphozyten) und eine starke ei weißerhöhung. in bis zu 85% ist der liquor als folge der ausge dehnten nekrosen im zns hämorrhagisch. im frühstadium einer hsv-enzephalitis kann der liquor noch vollkommen unauffällig sein. die aciclovirtherapie hat die letalität auf ca. 29% gesenkt. eine vollständige ausheilung ohne residualfolgen findet sich in 38% der mit aciclovir behandelten enzephalitispatienten, bei kindern liegt der prozentsatz höher. bei unbehandelten patienten mit einer herpesenzephalitis dagegen kommt es nur in 2,5% der fälle zu einer restitutio ad integrum. chronisch rezidivierende verläufe, auch bei mit aciclovir behandelten patienten, kommen gelegentlich vor. ösophagus, der gastrointestinaltrakt, der respirationstrakt (pneumonie), das zns (enzephalitis) und andere organe (leber, nieren, milz, nebennieren) betroffen. j diagnose häufig kann die diagnose einer herpesinfektion der haut oder der schleimhäute aufgrund der typischen herpeseffloreszenzen klinisch gestellt werden. in zweifelsfällen wird hsv leicht aus bläscheninhalt, schleimhautabstrichen und bioptischem material isoliert. methode der wahl für die diagnose einer hsv-enzephalitis ist der hsv-genomnachweis im liquor mittels polymerasekettenreaktion. der serologische nachweis von spezifischen hsv-antikörpern im serum oder liquor spielt in der frühdiagnostik von hsv-infektionen nur eine sekundäre rolle. bei einer unklaren enzephalitis kann der nachweis von intrathekal produzierten hsv-antikörpern am tag 7-10 nach auftreten der symptome die ursache der erkrankung nachträglich beweisen. j therapie mittel der wahl bei hsv-infektionen im kindesalter ist das nukleosidanalogon aciclovir. zur behandlung von neonatalen hsv-infektionen sowie der herpesenzephalitis wird aciclovir in einer dosierung von 3-mal 15(-20) mg/kgkg/tag i.v. (frühgeborene nur 2-mal 10 mg/kgkg/ tag i.v.) für mindestens 14, besser 21 tage eingesetzt. bei zu kurzer aciclovirtherapie einer hsv-enzephalitis (<14 tage) kann es zu rezidiven kommen. neugeborene mit hsv-infektion und neurologischer beteiligung sollten im anschluss an die i.v.-aciclovirtherapie eine orale suppressionstherapie mit 3×300 mg/m 2 kö aciclovir über 6 monate erhalten. hierdurch wird das outcome hinsichtlich der neurologischen entwicklung verbessert. > bei klinischem verdacht auf herpesenzephalitis beginne man sofort mit einer ausreichend hoch dosierten intravenösen aciclovirtherapie, ohne die endgültige labordiagnostik abzuwarten. eine stomatitis aphthosa oder ein herpes labialis beim immunkompetenten kind wird im normalfall nur symptomatisch (z. b. mit bepanthenlösung oder -salbe) behandelt. bei allen komplizierten hsv-infektionen einschließlich dem herpes genitalis ist aciclovir derzeit das mittel der wahl (i.v., oral, topisch) . bei aciclovirresistenten hsv-stämmen (immunsupprimierte patienten) kann ein therapieversuch mit foscarnet unternommen werden. für die topische behandung einer hsv-keratokonjunktivitis stehen verschiedene wirksame medikamente zur verfügung wie acicloviraugensalbe und trifluridinaugentropfen. die therapie muss immer in enger zusammenarbeit mit einem diesbezüglich erfahrenen augenarzt erfolgen. bei patienten >18 jahren kann ein genitaler herpes (primärinfektion, rezidive) auch mit famciclovir oder valaciclovir behandelt werden. j prophylaxe bei schwangeren mit aktiver genitaler herpesinfektion (sowohl hsv-erstinfektion als auch -rezidiv) am geburtstermin sollte die geburt durch kaiserschnitt erfolgen, sofern der blasensprung nicht länger als 4-6 h zurückliegt. bei frauen mit rezidivierendem herpes genitalis in der spätschwangerschaft senkt eine orale aciclovir-oder valganciclovirtherapie die häufigkeit von hsv-2-rezidiven zum zeitpunkt der geburt. mütter mit florider hsv-1-infektion dürfen nur dann stillen, wenn die brust frei von frischen hsv-effloreszenzen ist und andere aktive läsionen abgedeckt sind. familienangehörige mit floridem herpes labialis müssen beim besuch eines neugeborenen immer einen mundschutz tragen und dürfen das kind nicht küssen. die labialen herpesläsionen müssen außerdem vorher mit aciclovirsalbe abgedeckt werden. eine langzeitchemoprophylaxe mit aciclovir kann bei immunsupprimierten und transplantierten patienten die häufigkeit (und schwere) von hsv-infektionen und -reaktivierungen signifikant senken. j epidemiologie varizella-zoster-virus (vzv) kommt ubiquitär vor und ist hochkontagiös. eine krankheitshäufung findet sich in den späten wintermonaten und im frühjahr. varizellen treten vorwiegend im kindesalter auf; bis zum 16. lebensjahr sind über 90% aller kinder infiziert. die ansteckung mit vzv erfolgt meist durch direkten kontakt von mensch zu mensch, seltener aerogen. die infektiosität bei varizellen beginnt 1-2 tage vor auftreten des exanthems und endet ca. 5 tage nach exanthemausbruch (immunkompetente kinder). der herpes zoster ist weniger kontagiös als varizellen. der kontakt mit einem zosterpatienten führt bei einer seronegativen person zu windpocken. die inkubationszeit bei varizellen beträgt meist 2 wochen (10-28 tage). j ätiopathogenese vzv gehört zur gruppe der herpesviren. eintrittspforte für vzv sind die schleimhäute der oberen atemwege. nach initialer virusvermehrung tritt nach 3-4 tagen eine erste virämie auf. hierbei wird vzv in t-zellen über den blutstrom im ganzen körper verteilt. in leber und milz findet anschließend eine massive virusvermehrung statt. am tag 6-7 post infectionem (p. i.) kommt es zur 2. virämie: hierbei wird vzv auch in die peripherie zur haut und zu den schleimhäuten transportiert. infizierte haut-und schleimhautzellen gehen bei der infektion zugrunde, es bilden sich die typischen bläschen mit virushaltigem inhalt. nach abklingen der varizellen wandert vzv retrograd entlang der peripheren sensorischen nerven zu den spinalganglien des rückenmarks (n. trigeminus, thorakale ganglien u. a.), wo das virus lebenslang persistiert (. abb. 14.1). in diesen spinalganglien liegt eine latente vzv-infektion vor, d. h. es wird kein komplettes virus produziert. vzv kann bei nachlassender zellulärer immunität sowie durch noch unbekannte mechanismen jederzeit reaktiviert werden: vzv wandert nun entlang der sensorischen peripheren nerven anterograd an die hautoberfläche, wo es im bereich der betroffenen dermatome zur virusvermehrung mit bläschenbildung (herpes zoster) kommt. . abb. 14.4 herpes labialis bei einem 8 jahre altem mädchen im gegensatz zu varizellen, bei denen es im rahmen der 2. virämie zu einem schubweisen auftreten von bläschen kommt (sternenhimmelbild mit verschiedenen stadien von effloreszenzen), befinden sich die bläschen beim herpes zoster im gleichen entwicklungsstadium: es liegt ein uniformes exanthem vor. für die immunologische kontrolle einer vzv-infektion ist das zelluläre immunsystem entscheidend. vzv-neutralisierende antikörper können die schwere des verlaufs von varizellen abmildern und u. u. auch eine vzv-infektion verhindern, insbesondere dann, wenn sie vor eintritt der primären virämie verabreicht werden. > windpocken treten nur einmal im leben auf. zweiterkrankungen sind sehr selten (ca. 1-2%). meist manifestieren sich windpocken als typisches bläschenförmiges exanthem (. abb. 14.5) mit nur leichtem fieber in den ersten 2-3 krankheitstagen. die effloreszenzen treten zunächst v. a. im gesicht, am behaarten kopf, und am stamm auf, weniger häufig kommt es zu einer zentrifugalen ausbreitung auf die extremitäten. die handinnenflächen sind meist ausgespart. frisch aufgetretene bläschen, die klare virushaltige flüssigkeit enthalten, trocken rasch ein und bilden häufig krusten. daneben treten immer wieder neue bläschen auf. diese hautveränderungen entwickeln sich schubweise mit einer dauer von bis zu 8 tagen und sind oft von einem starken juckreiz begleitet. durch kratzen kann es in betroffenen hautregionen zu exkoriationen und späterer narbenbildung kommen. bei der enzephalitis (häufigkeit ca. 1:10.000), die bereits früher im verlauf der varizellen auftritt und die im allgemeinen eine schlechtere prognose hat, kommt es zu schweren krampfanfällen und bewusstlosigkeit mit exitus letalis oder ausgeprägten defektheilungen. zerebrale insulte in form von akut auftretenden hemiplegien können erst nach monatelanger latenz nach einer vzv-infektion auftreten. andere seltene komplikationen sind peri-/parainfektiöse thrombozytopenische purpura (itp), purpura fulminans, myokarditis, arthritis, nephritis und reye-syndrom. bei zellulären immundefekten und immunsuppression (organtransplantation, hiv-infektion, immunsuppressive therapie, maligne grunderkrankungen) kommt es bei kindern häufig zu schweren progressiven varizellen mit viszeraler beteiligung wie pneumonie, meningoenzephalitis, hepatitis und pankreatitis. die letalität beträgt bis zu 20%. vari zellen im 1. und 2. schwangerschaftstrimenon (v. a. in der 13.-20. schwangerschaftswoche) führen in bis zu 2% zu einem konnatalen varizellensyndrom (cvs) mit hautnarben, gliedmaßenhypoplasien, dystrophie, katarakt sowie zns-schädigungen (. abb. 14.6). varizellen während der schwangerschaft (v. a. in der 16.-33. schwangerschaftswoche) können außerdem (in über 1%) zum auftreten eines herpes zoster im ersten lebensjahr führen. ein herpes zoster bei einer immunkompetenten schwangeren dagegen führt nur extrem selten zu einer konnatalen oder neonatalen vzv-infektion. j ätiopathogenese ebv gehört zur gruppe der herpesviren. eintrittspforte für ebv ist der rachenraum (waldeyer-rachenring, tonsillen), wo das virus zu einer sog. lytischen infektion des lymphoepithelialen gewebes (b-zellen, epithelzellen) mit anschließender produktion von infektiösem ebv führt (invasive phase). im weiteren verlauf (nach ca. 2 wochen) kommt es zur virämie oder mehreren virämischen phasen. hierbei werden ebv-infizierte b-zellen über den blutstrom in andere organe (leber, milz, knochenmark, lymphknoten, evtl. zns) transportiert, erst nach einer inkubationszeit von 10-50 tagen treten die klinische symptome auf. in den im blut zirkulierenden b-zellen kommt es zunächst noch zu einer lytischen ebv-infektion mit produktion von infektiösem virus, später liegt nur noch eine sog. latente infektion vor, d. h. es werden nur noch wenige virusantigene (kernantigene ebna1-6 und membranantigene lmp1 und 2) exprimiert. diese b-zellen werden hierdurch zu lymphoblastoiden zellen "transformiert" und erwerben die fähigkeit zur unbegrenzten teilung und vermehrung (immortalisation). beim immunkompetenten menschen werden nach einer ebv-infektion rasch aktivierte zytotoxische t-zellen vom cd8+-typ gebildet, die selektiv nur die ebv-infizierten b-zellen weitestgehendeliminieren. diese aktivierten t-zellen bilden einen großen anteil der typischen "pfeiffer-zellen" (syn. lymphatische reizformen, virozyten) und der teilweise extremen lymphozytose im blutbild von patienten mit akuter infektiöser mononukleose (. abb. 14.8). nach durchgemachter ebv-infektion persistiert ebv lebenslang in ruhenden b-zellen im knochenmark. ebv kann in diesen zellen jederzeit reaktiviert werden. bei eingeschränkter zellulärer immunität (z. b. nach medikamentöser immunsuppression, aids) können sich diese b-zellen -abhängig vom ausmaß der immunsuppression -expandieren und so zu schweren lymphoproliferativen krankheitsbildern und b-zelllymphomen führen. in 70-90% tritt initial eine ausgeprägte tonsillopharyngitis mit fibrinbelägen (. abb. 14.10) auf, die in der 2. krankheitswoche meist rasch abheilt. eine splenomegalie findet sich bei 50-60% der patienten in der 2. und 3. krankheitswoche. seltener (15-25%) ist eine hepatitis mit und ohne ikterus. in 5-10% treten meist flüchtige morbilliforme exantheme auf. lymphoproliferative krankheitsbilder kinder und jugendliche mit angeborenen zellulären immundefekten (x-chromosomale lymphoproliferative erkrankung, xlp u. a.), aber auch mit erwor bener immundefizienz (organtransplantation, immunsuppressiver therapie, hiv-infektion) zeigen eine eingeschränkte immunkompetenz gegenüber ebv. hierdurch kommt es zu einer verschiebung des virus-wirt-gleichgewichts zugunsten des virus. ebv-infizierte b-zellen können daher unkontrolliert auswachsen und zu polyoligoklonalen b-zell-lymphoproliferationen bis hin zu monoklonalen malignen lymphomen führen. die häufigkeit dieser komplikationen ist direkt abhängig von der schwere der immunsuppression. ebv-assoziierte maligne erkrankungen ebv findet sich zu 100% in tumorzellen des endemischen burkitt-lymphoms und des nasopharynxkarzinoms. darüber hinaus lässt sich das virus in geringerer häufigkeit auch in anderen malignomen (m. hodgkin, b-und t-zell-lymphome) nachweisen. die rolle von ebv in der tumor entstehung und/oder -progression ist noch weitgehend unbekannt. j diagnose die infektiöse mononukleose kann meist klinisch diagnostiziert werden. labor im blutausstrich lassen sich typischerweise zahlreiche aktivierte t-lymphozyten (pfeiffer-zellen) nachweisen (. abb. 14.8). in zweifelsfällen wird bei immunkompetenten patienten die diagnose serologisch gesichert (. abb. 14.11). der mononukleoseschnelltest zum nachweis von heterophilen antikörper ist im kindesalter sehr wenig sensitiv und spielt in der pädiatrie keine rolle. die bestimmung der viruslast im blut mittels polymerasekettenreaktion ist bei immunsupprimierten patienten mit ebv-assoziierten lymphoproliferativen krankheitsbildern sinnvoll. immunsupprimierte patienten bei angeborenen immundefekten (xlp etc.) kann eine frühzeitige stammzelltransplantation (nabelschnurblut, knochenmark) zu einer immunrekonstitution führen und so spätere komplikationen durch ebv verhindern. bei auftreten von ebv-lymphomen im rahmen einer immunsuppressiven therapie führt die rechtzeitige reduktion der medikamentendosis häufig noch zu einer rückbildung der tumoren. der einsatz von monoklonalen anti-b-zellantikörpern (anti-cd20-rituximab) führt bei ebv-assoziierten lymphoproliferativen krankheitsbildern in bis zu 60% zu einer kompletten remission. die präsymptomatische therapie mit ganciclovir oder valganciclovir kann bei kindern nach organtransplantationen (v. a. lebertransplantation) wahrscheinlich die häufigkeit von ebvassoziierten lymphoproliferativen komplikationen reduzieren. bei organtransplantierten patienten kann die infusion von ebv-spezifischen zytotoxischen t-zellen des organspenders ebv-positive lymphome zur rückbildung bringen oder die neuentstehung von lymphomen verhindern (sog. "adoptiver immuntransfer"). zytomegalievirusinfektionen j epidemiologie die durchseuchungsrate mit dem zytomegalievirus (cmv) in der bevölkerung ist v. a. abhängig vom alter und lebensstandard. in deutschland sind ungefähr 50% der erwachsenen gesamtbevölkerung seropositiv für cmv. cmv-infektionen als folge von organtransplantationen treten meist nach 4 wochen bis 4 monaten auf, als folge einer bluttransfusion bereits nach 3-12 wochen. cmv wird horizontal über infektiöse körperflüssigkeiten (speichel, urin, muttermilch), blut, blutprodukte oder transplantierte organe, sowie vertikal (konnatale infektion) übertragen. cmv-positive säuglinge und kleinkinder können über wochen infektiöses cmv ausscheiden. j ätiopathogenese cmv gehört zur gruppe der herpesviren. cmv repliziert sich in epithelialen zellen der speicheldrüsen und der nieren, bei schweren generalisierten infektionen auch in leber, genitaltrakt, lungen und anderen organen. die produktive cmv-infektion führt in diesen zellen zu typischen intranukleären einschlüssen ("eulenaugenzellen"; . abb. 14.12) und zu massiver vergrößerung der infizierten zellen ("zytomegalie"). während der virämischen phase(n) findet sich cmv überwiegend zellassoziiert in der fraktion der polymorphkernigen granulozyten. nach einer primärinfektion persistiert cmv lebenslang im blut in monozyten/makrophagen sowie in anderen infizierten organen (speicheldrüsen, nieren). das virus kann bei immunsuppression jederzeit reaktiviert werden. bei der immunologischen bewältigung einer cmv-infektion spielt die zelluläre immunität (u. a. cmv-spezifische cd8+-t-zellen) eine entscheidende rolle. neutralisierende cmv-spezifische antikörper können bei einer cmv-infek tion die schwere einer cmv-erkrankung abmildern. personen die meisten cmv-infektionen verlaufen bei immunkompetenten personen asymptomatisch bzw. subklinisch. in seltenen fällen (1:1.000) manifestiert sich eine cmv-infektion als mononukleoseähnliches krankheitsbild mit ähnlichen klinischen symptomen und blutbildveränderungen (lymphozytose, atypische lymphozyten). das risiko und die schwere einer cmv-erkrankung korreliert mit dem ausmaß der immunsuppression, der virusmenge im blut und anderen faktoren, die eine reaktivierung von cmv begünstigen (akute graft-versus-host-erkrankung, infektion mit anderen herpesviren, cmv-seronegativer transplantatempfänger eines cmvpositiven organs). . abb. 14.12 eulenaugenzellen in der niere eines verstorbenen kindes mit konnataler zytomegalie . abb. 14.13 cmv-chorioretinitis bei einem 14 jahre alten patienen unter immunsuppressiver therapie (mit freundl. genehmigung von prof. dr. handrick, frankfurt/oder) konnatale cmv-infektionen die zytomegalie ist die häufigste konnatale infektion (ca. 0,2-0,4% aller neugeborenen). eine cmv-primärinfektion in der schwangerschaft führt bei ca. 7-10% der infizierten kinder zu einer schweren generalisierten cmv-erkrankung. eine cmv-reaktivierung oder -zweitinfektion in der schwangerschaft führt dagegen nur sehr selten zu einer symptomatischen zytomegalie beim kind. das risiko für eine symptomatische konnatale zytomegalie scheint direkt mit der höhe von maternalen neutralisierenden cmv-spezifischen antikörpern sowie der cmv-virusmenge im blut zu korrelieren. etwa 90% aller neugeborenen mit konnataler cmv-infektion sind bei geburt klinisch symptomfrei. ein teil dieser kinder (7-15%) kann aber später eine bleibende hörstörung entwickeln. aus diesem grund sollten bei allen kindern mit konnataler cmv-infektion wiederholt hör-und sehprüfungen veranlasst werden. eine symptomatische konnatale zytomegalie (nach cmv-erstinfektion in der schwangerschaft) ist eine multisystemerkrankung mit hoher morbidität und letalität. erkrankte kinder zeigen eine ausgeprägte intrauterine wachstumsretardierung, ikterus, hepatosplenomegalie, thrombozytopenie mit petechien (77%), pneumonie sowie schwerste zns-schädigungen (bis zu 70%) mit mikrozephalus (53%), intrazerebralen verkalkungen, chorioretinitis, späterer taub-und blindheit und geistiger behinderung. die letalität liegt bei bis zu 30%. für die einschätzung von späteren neurologischen defiziten scheint ein schädel-ct oder mrt die derzeit sensitivste methode zu sein. der nachweis von cmv-dna sowie eine eiweißerhöhung im liquor sind wahrscheinlich mit einer schlechteren prognose hinsichtlich der neurologischen entwicklung assoziiert. die schlechteste prognose haben konnatale cmv-infektionen häufig dann, wenn sie im 1. trimenon auftreten. peri-und postnatale cmv-infektionen infektionen, die durch cmv im zervikal-und vaginalsekret bzw. durch infektiöse muttermilch übertragen werden, verlaufen bei reifgeborenen, immunkompetenten kindern meist asymptomatisch oder mild. bei sehr kleinen frühgeborenen kann eine perinatale cmv-infektion (v. a. via cmv-positive muttermilch) eine schwere interstitielle pneumonie, hepatosplenomegalie und ein sepsisähnliches krankheitsbild verursachen. die letalität liegt bei 24%. j diagnose sensitive und spezifische parameter für eine floride cmv-infektion sind der quantitative nachweis des cmv-antigens pp65 oder des cmv-genoms (pcr) im blut (oder anderen körperflüssigkeiten). beide methoden erlauben eine bestimmung der viruslast und somit auch das monitoring einer virostatischen therapie mit ganciclovir bzw. valganciclovir. der "klassische nachweis" von cmv besteht in der isolierung aus verschiedenen körperflüssigkeiten (urin, speichel etc.). herpesvirus-typ-6-infektionen zu den begleitsymptomen und komplikationen, die meist schon im frühstadium (tag 1-4) auftreten, gehören gastroenteritis (55-70%), lidödeme (bis 30%), nagayama-flecken (papeln auf dem weichen gaumen und der uvula 65%), husten (50%), zervikale lymphadenopathie (30-35%), vorgewölbte fontanelle (26-30%) sowie fieberkrämpfe (5-35%). die angaben über die häufigkeit eines exanthema subitum nach einer primärinfektion mit hhv-6 schwanken stark. primärinfektionen mit hhv-6 stellen insgesamt eine häufige ursache von hochfieberhaften infekten (mit und ohne exanthem) bei kleinkindern dar. fieberkrämpfe und andere klinische manifestationen während einer hhv-6-primärinfektion kommt es nicht selten zu einer invasion des virus in das zns. bei bis zu 40% von kindern mit florider hhv-6-infektion kann das virus im liquor nachgewiesen werden, wobei entzündliche liquorveränderungen (pleozytose, eiweißvermehrung) fehlen. fieberkrämpfe treten in bis zu 35% auf. zu den seltenen neurologischen komplikationen gehören die meningoenzephalitis und das guillain-barré-syndrom. in wenigen fällen kann eine hhv-6-infektion, v. a. bei älteren kindern, auch mit einer mononukleoseähnlichen symptomatik, einer fulminanten hepatitis, einem schwer verlaufenden hämophagozytosesyndrom sowie mit der verschlimmerung einer idiopathischen thrombozytopenie (itp) assoziiert sein. organtransplantation kommt es häufig (in bis zu 80%) zu einer reaktivierung von hhv-6, die möglicherweise zu einer vermehrten transplantatabstoßung führt. nach hhv-6-infektion bzw. -reaktivierung können folgende klinische komplikationen auftreten: interstitielle pneumonie, graft-versus-host-erkrankung (gvhd) mit und ohne exanthem, enzephalopathie und knochenmarksuppression (nach knochenmarktransplantation). inwieweit diese komplikationen tatsächlich ursächlich nur durch hhv-6, oder möglicherweise erst in verbindung mit zusätzlichen infektionen (hiv, cmv und andere herpesviren) hervorgerufen werden, ist derzeit nicht bekannt. j diagnose die diagnose eines exanthema subitum kann bei typischer ausprägung klinisch gestellt werden. labor am 3. und 4. fiebertag fällt im blutbild häufig eine leukopenie mit relativer lymphozytose (bis zu 90%) auf. eine vermutete hhv-6-primärinfektion wird durch den serologischen nachweis von hhv-6-spezifischen antikörpern (anti-hhv-6-igm und/oder anstieg von anti-hhv-6-igg) bestätigt. eine hhv-6-reaktivierung bei immunsupprimierten kindern kann bei akut ansteigenden anti-hhv-6-antikörpertitern (bei bekannten ausgangstitern) vermutet werden. hhv-6-dna kann mittels pcr nachgewiesen werden (speichel, blut, plasma, liquor, urin). j therapie, prophylaxe eine spezifische therapie existiert nicht. bei hohem fieber erfolgt eine adäquate symptomatische fiebersenkung. bei (immunsupprimierten) patienten mit schweren hhv-6-assoziierten komplikationen (pneumonie, enzephalitis) ist ein therapieversuch mit foscarnet und/oder ganciclovir, zu erwägen. herpesvirus-typ-7-infektionen j epidemiologie auch herpesvirus typ 7 (hhv-7) kommt ubiquitär vor. die durchseuchung in der bevölkerung liegt z. t. bei über 90%. in den ersten 6 lebensmonaten ist eine hhv-7-infektion sehr selten, am ende des 1. lebensjahr sind bis zu 30%, am ende des 6. lebensjahrs bis zu 86% der kinder seropositiv. die primärinfektion mit hhv-7 erfolgt i. a. deutlich später als die mit hhv-6. die übertragung erfolgt über infektiösen speichel, u. a. innerhalb der familie, und möglicherweise auch über infizierte muttermilch. j ätiopathogenese hhv-7 gehört zur gruppe der herpesviren. die pathogenese ist ähnlich wie bei der hhv-6-infektion. j klinik hhv-7 ist (neben hhv-6) der erreger des exanthema subitum (dreitagefieber, roseola infantum). im vergleich zu hhv-6 scheint hhv-7 insgesamt in einer höheren frequenz zu fieberkrämpfen zu führen. das mittlere alter bei symptomatischen hhv-7-infektionen liegt bei 26 monaten, bei hhv-6-infektionen bei ca. 9 monaten. gelegentlich führt eine hhv-7-infektion bei älteren kindern auch zu einem mononukleoseähnlichen krankheitsbild. in den meisten fällen verlaufen hhv-7-infektionen allerdings subklinisch oder gehen mit einem unspezifischen fieberhaften infekt einher. j diagnose, therapie die diagnose eines exanthema subitum erfolgt bei typischer symptomatik klinisch. nur in ausnahmefällen scheint eine weitere virologische abklärung gerechtfertigt. hhv-7 kann mittels polymerasekettenreaktion im speichel, im peripheren blut, in lymphatischem gewebe und teilweise auch in der muttermilch nachgewiesen werden. der nachweis von hhv-7-spezifischen serumantikörpern erfolgt mittels indirekter immunfluoreszenz oder elisa. zu berücksichtigen ist hierbei, dass antikörper gegen hhv-7 teilweise auch mit hhv-6 kreuzreagieren können. eine spezifische therapie oder eine impfung gegen hhv-7 gibt es nicht. bei schwerem klinischem verlauf erfolgt eine entsprechende symptomatische therapie. herpesvirus-typ-8-infektionen j epidemiologie das herpesvirus typ 8 (hhv-8) kommt ebenfalls ubiquitär vor. die seroprävalenz in der bevölkerung ist in afrikanischen ländern und in japan (bis zu 100%) deutlich höher als in europa und in den usa (20-30%). hhv-8 wird überwiegend (homo)sexuell übertragen. die ansteckung von kindern und jugendlichen erfolgt wahrscheinlich über infektiösen speichel. bei nierentransplantationen kann eine transmission von hhv-8 in bis zu 10% erfolgen. j ätiologie hhv-8 gehört zur gruppe der herpesviren. hhv-8 zeigt in vitro und teilweise auch in vivo einen tropismus zu cd19+-b-zellen, zu endothelialen zellen und ganglienzellen. gefährdet sind u. a. patienten mit gleichzeitig bestehender chronischer hepatitis c. das krankheitsbild ist gekennzeichnet durch ein schnell zunehmendes leberversagen. die letalität ist hoch. j diagnose die diagnose einer hav-infektion sollte immer erwogen werden, wenn bei mehreren familienangehörigen oder anderen kontaktpersonen ein ikterus und gastrointestinale begleitsymptome auftreten. labor die diagnose eine hav-infektion wird durch den nachweis von virusspezifischen antikörpern im serum (anti-hav-igm positiv und/oder anstieg von anti-hav-igg) gesichert (. abb. 14.14). mit ausbruch der erkrankung werden anti-hav-igm-antikörper gebildet, die für 3-12 monate im blut nachweisbar sein können. die anti-hav-igg-antikörper persistieren (wahrscheinlich) lebenslang und sind ausdruck von immunität. j therapie eine spezifische therapie gibt es nicht. bettruhe und spezielle diäten haben keinen einfluss auf den krankheitsverlauf und sind nicht indiziert. symptomatische maßnahmen richten sich nach den jeweiligen beschwerden. im seltenen fall einer fulminanten hepatitis ist eine lebertransplantation zu erwägen. j prophylaxe die hepatitis-a-impfung (2 injektionen im abstand von 6 monaten; für kinder zugelassen ab dem 2. lebensjahr) kann bei rechtzeitiger gabe eine symptomatische wildvirusinfektion wirksam verhindern. sie wird allen potenziell gefährdeten immungesunden personen (u. a. seronegatives personal in medizinischen einrichtungen und kindertagestätten, reisende in regionen mit hoher hav-prävalenz, patienten mit chronischer hepatitis c) empfohlen. bereits 2 wochen nach der 1. impfung sind über 95% der geimpften geschützt. diese impfung schützt z. t. auch dann noch, wenn sie in der frühen inkubationszeit gegeben wird (sog. inkubations-oder "riegelungsimpfung"). nach einer vorausgegangenen wildvirusexposition kann durch frühzeitige einmalige gabe von immunglobulinen der ausbruch einer hepatitis a verhindert oder der verlauf abgemildert werden (postexpositionsprophylaxe). werden die immunglobuline erst nach einem intervall von 10(-14) tagen gegeben, ist keine wirkung mehr zu erwarten. im durchschnitt gelten patienten in einem zeitraum von 2 wochen vor bis 2 wochen nach ausbruch der erkrankung als ansteckend. hospitalisierte kinder mit einer hepatitis a sollten, sofern dies aus medizinischer sicht vertretbar ist, nach diagnosestellung schnellstmöglich nach hause entlassen werden. ansonsten erfolgt eine isolierung (einzelzimmer, kohortierung) für max. 2 wochen. neugeborene hbsag-positiver mütter erhalten am besten noch im kreißsaal, ansonsten innerhalb der ersten 12 h post partum simultan 0,5 ml hepatitis-b-impfstoff i.m. sowie auf der kontralateralen seite 0,5 ml/kgkg hepatitis-b-immunglobulin i.m. nach 4 wochen und 6 monaten erfolgt eine weitere impfung. geimpfte neugeborene dürfen gestillt werden. mutter und kind müssen nach geburt nicht isoliert oder voneinander getrennt werden. bei nichtimmunen personen mit fehlenden oder erniedrigten anti-hbs-antikörpern wird nach kontakt mit virushaltigem blut oder sekret schnellstmöglich (spätestens innerhalb von 12 h) hepatitis-b-immunglobulin i.m. verabreicht (postexpositionsprophylaxe). gleichzeitig sollte möglichst auch aktiv geimpft werden. in den ersten 1-3 monaten einer akuten hepatitis c kann der antikörpernachweis noch negativ verlaufen. die hcv-serologie ist daher v. a. für die diagnose von bereits einige zeit zurückliegenden oder chronischen hcv-infektionen geeignet. das hcv-genom kann mittels pcr im blut oder in der leberbiopsie nachgewiesen werden. die bestimmung der viruslast im blut (und ggf. auch im leberbioptat) sowie eine genotypisierung der vorliegenden hcv-variante sind hinsichtlich der indikation für eine interferon-α-therapie sowie für das anschließende monitoring wichtig. j therapie für eine antivirale therapie der akuten hepatitis c liegen für kinder und jugendliche bisher keine daten vor. da von einer hohen chronifizierungsrate ausgegangen werden muss, sollte wie bisher bei erwachsenen vorgegangen werden. die kombinationstherapie mit normalem oder pegyliertem (= retardiertem) interferon-α (zugelassen bei kindern ab 3 jahren) plus ribavirin ist derzeit bei kindern noch die standardtherapie der chronischen hepatitis c. prädiktoren für einen therapieerfolg sind der hcv-genotyp (v. a. typ 2 und 3), die dauer der krankheit und möglicherweise die hcv-konzentration im serum. bei erwachsenen mit chronischer hepatitis c führen neue virustatika (z. b. epclusa in kombination mit sofosbuvir und velpatasvir) zu einer ausheilung von meist über 90%. es ist zu hoffen, dass diese therapieoptionenn in einigen jahren auch bei kindern zugelassen sind. diese gehen dabei zugrunde, wobei die erythropoese kurzzeitig unterbrochen wird. eintrittspforte für parvovirus b19 ist der obere respirationstrakt. nach 4-5 tagen kommt es zu einer virämie, begleitet von einer retikulozytopenie. etwa 1 woche nach der virämie tritt meist das typische exanthem auf, möglicherweise hervorgerufen durch antigen-antikörper-komplexe. die entstehung eines hydrops fetalis nach intrauteriner parvovirus-b19-infektion wird v. a. mit der infektion von fetalen erythroblasten und der daraus resultierenden anämie erklärt. in bestimmten fällen kann möglicherweise auch die infektion des fetalen myokards mit parvovirus b19 zu einer eingeschränkten herzfunktion mit der folge eines hydrops fetalis führen. j klinik ringelröteln diese typische "kinderkrankheit" (syn: erythema infectiosum, 5. krankheit, epidemisches megalerythem, großfleckfieber, kinderrotlauf) wird in 15-20% aller frisch infizierten personen beobachtet. nach einem 2-3 tage andauernden prodromalstadium mit unspezifischen symptomen wie fieber, kopfschmerzen und schüttelfrost (zeitgleich mit der virämie) und einem anschließenden beschwerdefreien intervall von ca. 1 woche tritt im bereich der wangen ein hochrotes, leicht erhabenes konfluierendes exanthem auf (. abb. 14.19a). gleichzeitig kann eine periorale blässe wie beim scharlach bestehen. an den folgenden tagen treten . abb. 14.19 ringelröteln. a typisches konfluierendes exanthem im bereich beider wangen ("slapped cheek") mit perioraler blässe, b girlandenförmige exantheme a b an den extremitäten und am rumpf makulopapulöse effloreszenzen auf, die konfluieren. durch zentrales abblassen entstehen die typischen girlanden-netzförmigen muster (. abb. 14.19b) . in den folgenden tagen und wochen können immer wieder neue pleomorphe exantheme auftreten, teilweise provoziert durch sonnenlicht oder hohe temperatur. das allgemeinbefinden der p atienten ist meist nur wenig beeinträchtigt. andere begleitsymptome (juckreiz, kopfschmerzen, fieber, gelenkbeschwerden, bauchschmerzen) sind bei kindern eher selten. bei adoleszenten und jungen erwachsenen kommen auch vaskulitische exantheme vor mit strenger begrenzung auf die hände und füße ("handschuh-socken-syndrom"). die parvovirus-b19-assoziierte polyarthritis, die bevorzugt knie-, fuß-und die proximalen interphalangealgelenke befällt und häufiger bei mädchen und jungen frauen auftritt, ist praktisch immer selbstlimitierend. hydrops fetalis eine parvovirus-b19-primärinfektion in der schwangerschaft führt in bis zu 35% auch zu einer infektion des feten. in den allermeisten fällen ist diese fetale infektion klinisch stumm, in bis zu 5% der fälle kommt es zum abort (meist innerhalb von 3-6 wochen nach der mütterlichen infektion). die entwicklung eines hydrops fetalis nach einer mütterlichen (und fetalen) parvovirus-b19-infektion ist insgesamt selten, sie liegt bei ca. 4%. trotzdem ist die parvovirus-b19-infektion wahrscheinlich die häufigste ursache des nicht immunologisch bedingten hydrops fetalis. die fetalen komplikationen sind am höchsten bei einer parvovirus-b19-infektion zwischen der 13. und 20. gestationswoche. j klinik eine rotavirusinfektion führt bei kleinen kindern praktisch immer zu fieber, erbrechen sowie enteritis mit häufigen wässrigen, nicht blutigen stühlen. die durchfälle können bis zu 5-7 tage andauern. in der mehrzahl der fälle kommt es hierbei zu einer unterschiedlich ausgeprägten, meist isotonen, selten einer hypertonen dehydratation. ein teil der kinder zeigt zusätzliche klinische symptome wie leichte vergrößerung der zervikalen lymphknoten, otitis und rhinitis. bei immunsupprimierten kindern kann eine rotavirusinfektion chronisch und teilweise sehr schwer verlaufen. in sehr seltenen fällen können rotavirusinfektionen auch zu einer enzephalitis und zu krampfanfällen mit und ohne fieber führen. j diagnose die praktikabelste und schnellste methode zum nachweis einer rotavirusinfektion ist der rotavirusantigennachweis im stuhl (eia). follikuläre konjunktivitis häufig, meist unilateral 1-7, 9-11, 15-17, 19, 20, 22, 37 haut exantheme (morbilli-, rubelli-und roseolaform) j ätiopathogenese rhinoviren sind rna-viren aus der familie der picornaviren (pico, klein). rhinoviren infizieren v. a. die schleimhäute der nase und des oberen respirationstrakts, seltener auch die unteren atemwege. hier vermehrt sich das virus im zytoplasma. anschließend werden die neu gebildeten rhinoviren in großer menge (bis zu 106 viruspartikel pro ml nasensekret), v. a. in den ersten 3 tagen der erkältung, sowie meist noch 2-3 wochen danach ausgeschieden. während einer rhinovirusinfektion kommt es meist nur zu minimalen epithelsschäden ohne zilienverlust sowie zu einer lokalen infiltration mit granulozyten und monozyten. j klinik in den meisten fällen führt eine rhinovirusinfektion zu einer "erkältung" der oberen luftwege ("common cold") mit einer nur geringen allgemeinen klinischen beeinträchtigung. hauptsymptome sind schnupfen, husten und halsschmerzen. die regionalen zervikalen lymphknoten sind in ca. 50% vergrößert. eine meist leichte otitis media tritt in ca. 20% der fälle auf. die klinischen beschwerden sind in den ersten 3 tagen am ausgeprägtesten, in den meisten fällen bilden sie sich innerhalb einer woche, seltener nach 2 wochen zurück. vor allem bei jüngeren kindern können rhinovirusinfektionen auch zu schweren verläufen mit bronchiolitis, bronchopneumonie und obstruktiver bronchitis führen. todesfälle sind beschrieben worden. in den meisten fällen entwickeln die betroffenen kinder einen zunehmenden keuchenden husten und dyspnoe bei häufig nur leicht erhöhten temperaturen. in schweren fällen finden sich eine tachypnoe mit nasenflügelatmen und thorakalen einziehungen, eine zyanose sowie eine tachykardie. das atemgeräusch kann (als indirektes zeichen der überblähung bei atemwegsobstruktion) abgeschwächt sein. die röntgenaufnahme der lunge zeigt als typischen befund bei einer bronchiolitis eine überblähung der lungen und ggf. einige infiltrate. die blutgasanalyse ergibt häufig eine leichte bis mäßige hypoxie, eine hyperkapnie ist ausdruck der zunehmenden ateminsuffizienz, die einer dringenden intensivmedizinischen betreuung bedarf. bei früh-und neugeborenen kann eine rsv-infektion mit plötzlich auftretenden teils massiven apnoen, irritabilität, trinkverweigerung und lethargie -ohne äußere zeichen eines atemwegsinfekts -einhergehen. rsv-reinfektionen verlaufen i. a. milder und manifestieren sich häufig als oberer atemwegsinfekt oder tracheobronchitis. bei kindern mit schweren grunderkrankungen (bronchopulmonale dysplasie, herzfehler, immundefizienz) kann jede rsv-infektion zu einem lebensbedrohlichen krankheitsbild führen. j diagnose die diagnose einer rsv-infektion basiert auf der klinischen symptomatik, auf dem zeitlichen hintergrund (winterhalbjahr? bekannte rsv-epidemie?), auf der altersverteilung (kind <2 jahre) und der positiven virusdiagnostik. für den klinischen alltag sind rsv-antigen-tests zum virusnachweis in nasopharyngealsekret von großer praktischer bedeutung. daneben ist der nachweis von rsv durch anzucht in der zellkultur oder mittels pcr möglich. die serologische diagnostik spielt in der praxis keine rolle. j therapie im vordergrund steht die intensivmedizinische betreuung (u. u. kontrollierte beatmung) der meist schwer kranken kinder. symptomatisch können β 2 -sympathomimetika, racemisches epinephrin oder adrenalin verabreicht werden. kortikosteroide, theophyllin und mukolytika sind unwirksam. ribavirintherapie als virostatikum steht ribavirin zur verfügung. aufgrund unterschiedlicher, z. t. enttäuschender studienergebnisse hinsichtlich der wirksamkeit, der umständlichen inhalativen applikation sowie potenzieller nebenwirkungen (teratogenität) ist die indikation für ribavirin seit 1996 stark eingeschränkt worden. in über 90% lässt sich in tumorgewebe hpv nachweisen. zu den kanzerogenen typen gehören v. a. hpv-16 und hpv-18. j diagnose die diagnose erfolgt in den allermeisten fällen klinisch anhand der typischen manifestation (warzen, papillome). in unklaren fällen sollte immer eine histologische abklärung erfolgen. der nachweis und die typisierung von papillomaviren im biopsat erfolgt -falls überhaupt erforderlich -mit molekularbiologischen techniken. bei genitaler krankheitslokalisation sind immer auch andere geschlechtskrankheiten auszuschließen. bei kleinen kindern sollte auch an einen sexuellen missbrauch gedacht werden. larynxpapillome verursachen bei kindern eine mehr oder weniger ausgeprägte dyspnoe, die diagnose erfolgt mittels laryngo-oder bronchoskopie. j therapie vor jeder therapieentscheidung ist die hohe spontanremissionrate von warzen im kindesalter zu bedenken! hautwarzen werden durch kontaktvereisung mit flüssigstickstoff oder durch eine keratolytische und antiproliferative lokalbehandlung mit salicylsäureund 5-fluorouracil-haltigen präparaten (z. b. verrumal) behandelt. die früher durchgeführte warzenentfernung mittels elektrokauter ist wegen der schmerzhaftigkeit und der möglichen narbenbildung obsolet. filiforme warzen können chirurgisch im hautniveau exzidiert werden. larynxpapillome werden, v. a. bei entsprechenden atembeschwerden, mittels laser-oder kryotherapie behandelt. unterstützend kann zur proliferationshemmung lokal oder systemisch interferon α appliziert werden. die behandlung von genitalen warzen kann sehr schwierig sein und sollte immer von einem erfahrenen gynäkologen durchgeführt werden. die lokalbehandlung erfolgt u. a. mit podophyllinlösung (5-25%), kryotherapie sowie mittels co 2 -laser. die prodromi mit fieber und katarrhalischen symptomen (8-12 tage p. i.) signalisieren den beginn der immunologischen abwehrreaktion. so ist auch das exanthem folge der explosiven auseinandersetzung zwischen virusspezifischen t-zellen und virusinfizierten epithel-und endothelzellen. spezifische antikörper scheinen bei der überwindung der akuten phase keine rolle zu spielen. > masern gehen immer mit einer immunschwäche einher, die wochen bis monate anhalten kann. hauttests vom verzögerten typ (tuberkulintest!) werden vorübergehend negativ. außerdem kommt es durch die immunschwäche zu bakteriellen zweiterkrankungen oder zur aktivierung chronischer krankheitsprozesse. die pathogenese der para-(post-)infektiösen masernenzephalitis ist bisher nicht geklärt. histopathologisch finden sich im gehirn perivaskuläre demyelinisierungen und perivaskuläre lymphozyteninfiltrate. virale antigene oder rna-sequenzen lassen sich aber nicht nachweisen. j klinik die erkrankung beginnt mit hohem fieber und uncharakteristischen katarrhalischen symptomen, wie schnupfen, halsschmerzen, heiserkeit und bellendem husten (prodromalstadium). die patienten sind aufgrund einer konjunktivitis und einer (milden) keratitis ausgesprochen lichtscheu. gleichzeitig oder 1-2 tage später treten feine, kalkspritzerartige stippchen, bevorzugt an der wangenschleimhaut gegenüber den molaren, auf (koplik-flecken; . abb. 14.25). außerdem entwickelt sich ein fleckiges, dunkelrotes enanthem am weichen gaumen. nach leichtem fieberabfall geht das prodromalstadium 3-4 tage später unter erneutem hohem fieberanstieg in das exanthemstadium über. das makulopapulöse exanthem beginnt hinter den ohren (. abb. 14.27) und im gesicht und breitet sich weiter zentrifugal über den ganzen körper bis zu den füßen aus (. abb. 14.28). nach dem 3. exanthemtag folgt bei unkomplizierten verläufen rasche entfieberung und abblassen des exanthems. meist besteht eine generalisierte lymphadenopathie, wobei auch die hilären, paratrachealen und mesenterialen lymphknoten betroffen sind. bei ca. 50% der infizierten treten pathologische eeg-veränderungen auf, die sich später in den allermeisten fällen zurückbilden. 4 mitigierte masern treten bei jungen säuglingen auf, die noch maternelle antikörper besitzen und auch bei kindern nach gabe von immunglobulinen. 4 bei patienten mit schweren t-zelldefekten kann das exanthem völlig fehlen ("weiße masern"). es entwickelt sich eine riesenzellpneumonie, die fast immer zum tode führt. j komplikationen am häufigsten sind bakterielle sekundärinfektionen; und zwar bronchopneumonien, otitis media und diarrhö. weitere komplikationen betreffen das zentrale nervensystem. im vordergrund steht die akute masernenzephalitis mit einer häufigkeit von 1:500-1:2000. die masernenzephalitis tritt bevorzugt am 3.-9. tag nach exanthembeginn auf. typisch sind bewusstseinsstörungen (somnolenz, koma), zerebrale krampfanfälle, neurologische herdsymptome (hemiplegien, hirnnervenparesen) und gelegentlich auch myelitische symptome. die masernenzephalitis hat auch heute noch eine letalität von 30% und eine defektheilungsrate von ca. 20%. eine weitere seltenere komplikation ist die subakute sklerosierende panenzephalitis (7 abschn. 14.23.1). masern sind immer eine ernste und gefährliche krankheit. todesfälle kommen besonders im säuglingsalter, bei älteren probanden und besonders bei immundefizienten patienten vor. die krankheit verläuft besonders schwer in entwicklungsländern bei unterernährten kindern. . abb. 14.25 koplik-flecken . abb. 14.26 masernexanthem bei einem 8-jährigen jungen (hinter den ohren beginnend) . abb. 14.27 generalisiertes masernexanthem bei einem älteren säugling j diagnose im rahmen einer epidemie wird die diagnose meistens klinisch gestellt. labor ein typischer laborbefund ist die leukopenie mit erniedrigung sowohl der granulozyten als auch der lymphozyten. bei einzelerkrankungen sollte die diagnose serologisch bestätigt werden. das masernspezifische igm ist meist nach den ersten 3 exanthemtagen mittels enzymimmunoassay (elisa) nachweisbar. bei trotz impfung an masern erkrankten ist nur ein 4-facher titeranstieg im igg-elisa oder im hämagglutinationshemmtest (hht) diagnosesichernd (bei geimpften findet sich oft keine igm-antwort). in fraglichen fällen, z. b. bei immunsupprimierten patienten oder bei verdacht auf riesenzellpneumonie ist zur diagnosestellung der virusdirektnachweis (pcr oder virusisolierung) erforderlich. die diagnose einer masernenzephalitis beruht allein auf dem zeitlichen zusammenhang der enzephalitis mit einer akuten maserninfektion (igm-nachweis!), da sich im liquor in der regel weder das virus noch eine intrathekale antikörpersynthese nachweisen lassen. j diagnose wegen der ähnlichkeit mit anderen viralen und nichtviralen exanthemen ist die klinische diagnose oft schwierig. charakteristische blutbildveränderungen (leukopenie mit relativer lymphozytose und auftreten von plasmazellen) können von diagnostischer bedeutung sein. ansonsten muss die infektion serologisch bestätigt werden. beweisend sind ein 4-facher titeranstieg im hämagglutinationshemmtest (aus 2 serumproben) oder der nachweis von rötelnspezifischem igm mittels enzymimmunassay (elisa). je nach empfindlichkeit der testmethode sind spezifische igm-antikörper mitunter lange (bis zu einem jahr) im serum nachweisbar. um z wischen einer primären infektion und der (seltenen) reinfektion bei schwangeren zu unterscheiden, stehen spezielle tests zur ver fügung. bei der akuten rötelnenzephalitis findet man im liquor eine leichte lymphozytäre pleozytose. das liquoreiweiß ist normal. virale rna und oligoklonale banden lassen sich in der regel nicht nachweisen. bei mumpsmeningitis zeigt der liquor eine mäßige lymphozytäre pleozytose (10-2.000 zellen/µl) bei normalem bis leicht erhöhtem eiweiß und normalem bis leicht erniedrigtem liquorzucker. im liquor treten 2-3 wochen später virusspezifische oligoklonale mumpsantikörper auf als ausdruck einer intrathekalen immunreaktion. j therapie, prophylaxe eine spezifische therapie existiert nicht. auch eine symptomatische behandlung ist selten erforderlich. bei schweren verläufen (mumpsenzephalitis, orchitis) sind u. u. kortikosteroide indiziert. alle kinder (und noch seronegative adoleszenten und erwachsene) sollten 2-mal gegen mumps geimpft werden (7 kap. 17). spezielle immunglobuline zur passiven immunisierung stehen nicht zur verfügung. gemeinschaftseinrichtungen dürfen 9 tage nach beginn der parotitis wieder besucht werden. seit 2012 ist mumps meldepflichtig. j diagnose die diagnose wird in der regel nach klinischen symptomen und dem eeg-befund gestellt. gestützt wird die diagnose durch den nachweis von neuronalen destruktions-und glialen aktivierungsmarkern im liquor (neuronenspezifische enolase, proteine 14-3-3, s100-β-protein). die genannten marker sind allerdings nicht spezifisch für cjk. ansonsten ist der liquor unauffällig. eine definitive diagnose kann nur durch die untersuchung von hirngewebe gestellt werden. j therapie und prophylaxe es gibt bisher keine wirksame therapie. iatrogene übertragungen durch chirurgische instrumente können durch adäquate dekontaminationsmaßnahmen vermieden werden (z. b. dampfautoklavieren bei 134°c für 1 h, behandlung mit 2,5-5%iger natriumhypochloritlösung oder 1-2 n natronlauge für 1 h. prionenverseuchte nahrungsmittel dürfen auf keinen fall in den verkehr gebracht werden. humane spongiforme enzephalopathien -außer familiärhereditären formen -sind meldepflichtig. j epidemiologie tollwut ist eine weltweit verbreitete zoonose. das tollwutvirus wird durch infektiösen speichel bei kratz-und bisswunden von infizierten tieren (füchse, hunde, fledermäuse, katzen u. a.) übertragen. die jährliche inzidenz von tollwut beim menschen wird weltweit auf 40.000-100.000 fälle geschätzt. deutschland gilt derzeit als "tollwutfrei". die inkubationszeit beträgt 5 tage bis mehrere jahre. sie ist abhängig von der lokalisation der bissstelle (cave: gesicht, augenregion!) und der inokulierten virusmenge. bei kindern ist die inkubationszeit kürzer. 14.29 frühsommermeningoenzephalitis j epidemiologie die frühsommermeningoenzephalitis (fsme, zeckenenzephalitis, zentraleuropäische enzephalitis) kommt europaweit, v. a. in russland, im balkan sowie in zentral-und nordeuropa vor. die endemiegebiete in deutschland liegen hauptsächlich in bayern, baden-württemberg und im saarland. fsme-erkrankungen treten v. a. in den monaten april bis november auf. das fsme-virus wird überwiegend durch zeckenstiche übertragen. wichtigster überträger ist ixodes ricinus, der "gemeine holzbock". > in endemiegebieten sind ca. 0,1-1% der zecken durchseucht. etwa 12-15% aller fsme-erkrankungen betreffen das kindesalter. die inkubationszeit beträgt ca. 10 tage (3-28 tage). nach einer fsme-infektion besteht lebenslange immunität. j ätiopathogenese das fsme-virus gehört zur familie der flaviviren. über die pathogenese ist relativ wenig bekannt. nach der infektion kommt es zu einer starken virusvermehrung im retikuloendothelialen system und in gefäßendothelien. während der virämie (3-14 tage nach zeckenstich) können bei dem patienten grippeähnliche symptome auftreten. nach zns-invasion des fsme-virus und anschließender lokaler virusreplikation kann es in einer 2. krankheitsphase zum auftreten neurologischer symptome kommen. j klinik in 70-90% verläuft eine fsme-infektion asymptomatisch. bei 10-30% der infizierten kommt es nach meist ca. 10 tagen zu einem grippeähnlichem krankheitsbild für ca. 3-7 tage ("sommergrippe"). bei ca. 10% dieser personen tritt nach einem kurzen beschwerdefreien intervall eine 2. krankheitsphase mit fieberanstieg und folgender neurologischer symptomatik auf: 4 meningitis (60%), 4 meningoenzephalitis (30%), 4 meningoenzephalomyelitis (10%). die restitutionsphase beginnt nach etwa 3 tagen mit kontinuierlicher klinischer besserung im verlauf von 1-3 wochen. eine defektheilung (kopfschmerzen, konzentrationsstörungen, motorische lähmungen, muskelschwäche) tritt v. a. bei älteren menschen in ca. 10% auf. die letalität beträgt ca. 1% (beim östlichen fsme-virussubtyp ca. 20%). kinder und jugendliche haben in der regel leichtere (meningitische) krankheitsverläufe. j diagnose bereits anhand der anamnese (zeckenstich, endemiegebiet, jahreszeit) und der klinischen symptomatik (biphasischer krankheitsverlauf) ergibt sich der verdacht auf eine fsme-erkrankung. die diagnose wird durch den serologischen nachweis von fsme-spezifischen serumantikörpern gesichert. die liquoruntersuchung in der 2. krankheitsphase ergibt typischerweise eine lymphozytäre pleozytose (<100-5.000 zellen/mm 3 ) sowie eine liquoreiweißer höhung. j therapie, prophylaxe eine antivirale therapie existiert nicht. die behandlung ist rein symptomatisch. zur aktiven immunisierung steht ein impfstoff auch für kinder zur verfügung. > zielgruppe für eine fsme-impfung sind alle personen, die sich in endemiegebieten vermehrt im freien aufhalten. meldepflichtig sind erkrankung und tod nach einer fsme-infektion. j ätiopathogenese das "human immunodeficiency virus" (hiv) gehört zur gruppe der retroviren. es existieren 2 haupttypen (hiv-1 und hiv-2). in europa und nordamerika kommen praktisch nur hiv-1-varianten vor, in teilen afrikas findet sich auch hiv-2 bei einem großen teil der bevölkerung. hiv benutzt zur infektion einer zelle das hüllprotein gp120, mit dem es sich an den cd4-rezeptor und korezeptor (ccr5 oder cxcr4) der zielzelle (t-helferzellen, makrophagen, monozyten, gliazellen u. a.) anheftet. anschließend fusioniert die hiv-hülle mit der zellmembran, 2 rna-stränge und mehrere virusenzyme werden in das zytoplasma freigesetzt. mittels des viralen enzyms reverse transkriptase (rt) wird die rna in doppelsträngige dna "übersetzt" (provirus). diese dna wird anschließend durch das viral kodierte enzym integrase in das wirtsgenom im zellkern eingebaut (viruslatenz). durch verschiedene aktivierende faktoren kommt es zu einer ausgeprägten virusreplikation mit freisetzung von neuen hiv-partikeln in die blutbahn. der verlust der t-zellfunktion führt zu einem zunehmenden zellulären und humoralen immundefekt mit der folge von schweren opportunistischen infektionen und dem auftreten von lymphomen. j klinik die klinische symptomatik bei horizontal hiv-infizierten jugendlichen (infektion durch geschlechtsverkehr oder durch kontaminierte blutprodukte) entspricht im wesentlichen dem bild bei erwachsenen. der natürliche verlauf bei vertikal hiv-infizierten kindern variiert sehr stark. ohne therapie erkranken bereits 25-30% dieser kinder sehr früh, meist schon innerhalb des j diagnostik virologische diagnostik als sicherer nachweis einer infektion gelten wiederholt positive hiv-kulturen, wiederholte nachweise des p24-antigens oder der hiv-rna (rt-pcr). die letztere me thode erlaubt die bestimmung der viruslast und hilft mit bei der indikationsstellung für eine antiretrovirale therapie und beim therapiemonitoring. erst der wiederholte nachweis von hiv-antikörpern (elisa, immunoblot) nach dem 18. lebensmonat beweist eine hiv-infektion. bereits vorher nachgewiesene hiv-antikörper können von der mutter diaplazentar übertragen worden sein. immunologische diagnostik im rahmen einer hiv-infektion kommt es im verlauf zu einem zunehmend schweren kombinierten (zellulären und humoralen) immundefekt. im rahmen der initialen statusdiagnostik und späterer verlaufskontrollen sollten bei allen hiv-infizierten oder -exponierten kindern u. a. folgende parameter im blut bestimmt werden: 4 quantitative bestimmung der cd4 + -zellen, 4 serumimmunglobuline (igg, iga und igm), 4 impfantikörper nach erfolgter grundimmunisierung (dtp, hib, hbv, polio-salk), 4 antikörper gegen verschiedene herpesviren (nach stattgehabter infektion)! diese untersuchungen sollten möglichst immer nur am selben immunologisch ausgewiesenen speziallabor durchgeführt werden. die viruslast eines patienten muss immer mit derselben bestimmungsmethode quantifiziert werden! aids-definierenden krankheiten (kategorie c) dagegen tritt bei einem großen teil der kinder die klinische verschlechterung erst im grundschuloder schulalter auf. prädiktive parameter für eine schlechte prognose sind u. a. eine hohe viruslast im blut und ein schneller bei fortschreitender infektion und störung im immunsystem treten klinische zeichen hinzu, die schon eher an einen immundefekt denken lassen (kategorie b) weitere atemwegsassoziierte viren das humane metapneumovirus (hmpv), das erst 2001 entdeckt wurde, ist bei kindern der zweithäufigste erreger der bronchiolitis. darüber hinaus verursacht hmpv auch bronchitiden und pneumonien. die hospitalisierungsrate ist wahrscheinlich ähnlich hoch wie bei rsv-und influenza-infektionen. das humane bocavirus (hbov) wurde im sommer infektionen mit humanen coronaviren (hcov) können v. a. bei kleinkindern zu erkrankungen der unteren und oberen luftwege führen. nicht selten finden sich koinfektionen mit anderen respirationstraktviren der nachweis aller drei erreger in infektiösen körperflüssigkeiten gelingt meist mittels polymerasekettenreaktion. die therapie ist in allen fällen symptomatisch helfen meist auch bei der hiv-assoziierten autoimmunthrombozytopenie.chemoprophylaxe die früher häufigste opportunistische infektion, die pneumocystis-jirovecii-pneumonie, lässt sich durch orale gabe von cotrimoxazol (trimethoprim 150 mg/m2 kof an 3 aneinander folgenden tagen pro woche) zu fast 100% vermeiden.allgemeine schutzimpfungen nur bei einer noch asymptomatischen hiv-infektion wird eine masern-oder mmr-impfung empfohlen. auch gegen varizellen kann geimpft werden, sofern die relative cd4-zellzahl ≥25% liegt. ansonsten sollten keine lebendimpfungen erfolgen. im übrigen sollten hiv-infizierte kinder entsprechend dem impfkalender mit inaktivierten impfstoffen/ toxoiden (polio-salk-impfstoff, hepatitis b, hib, dpt) und zusätzlich gegen pneumokokken, meningokokken und influenza geimpft werden. bei kindern, die regelmäßig mit immunglobulinen behandelt werden, sind aktivimpfungen dagegen nicht mehr sinnvoll. eine hiv-infektion ist namentlich nicht meldepflichtig. es besteht nur eine anonyme labormeldepflicht. j epidemiologie influenzainfektionen sind ubiquitär. alle 3-5 jahre treten influenzaepidemien durch antigendrift der zirkulierenden influenzasubtypen bei nachlassender immunität der exponierten bevölkerung auf. in größeren zeitabständen (10-20 jahre) treten durch rekombination zwischen humanen und animalen influenza-a-virusstämmen neue subtypen auf (antigenshift), die zu schweren pandemien führen können. in unseren breiten tritt die influenzagrippe üblicherweise in den monaten dezember bis april auf. die übertragung erfolgt überwiegend durch tröpfcheninfektion (niesen, husten), seltener durch kontaktinfektion. die kontagiosität ist kurz vor ausbruch der klinischen symptome am höchsten, sie dauert bis zu 1 woche an. die inkubationszeit beträgt in der regel 2-3 tage (1) (2) (3) (4) (5) (6) (7) .j ätiopathogenese influenzaviren gehören zur familie der orthomyxoviren. es existieren 3 typen (a, b, c). influenzaviren enthalten 8 rna-segmente, die von einer hülle von strukturproteinen umgeben sind. diese enthält spikes aus hämagglutinin (h) und neuraminidase (n). gegenwärtig zirkulieren die influenza-a-subtypen h3n2 und h1n1 sowie zwei verschiedene influenza-b-virusstämme. influenzaviren führen zu einer lytischen infektion des respiratorischen epithels. hierdurch kommt es zu einem verlust der zilienfunktion, zu einer verminderten schleimproduktion sowie zur nekrose der epithelschicht. es folgt eine entzündungsreaktion mit infiltration von lymphozyten, histiozyten und granulozyten. in den folgenden 2 wochen kommt es wieder zur regeneration und erholung der epithelzellschicht. die klinische symptomatik wie fieber, abgeschlagenheit, kopf-und gliederschmerzen ist immunologisch bedingt (u. a. durch freisetzung von zytokinen).j klinik bei älteren kindern und erwachsenen führt eine influenzainfektion typischerweise zur klassischen virusgrippe mit hohem fieber, kopf-und gliederschmerzen, abgeschlagenheit, trockenem husten, pharyngitis und konjunktivitis. nach einigen tagen tritt meist entfieberung ein, gefolgt von einer bis zu wochenlangen rekonvaleszenzphase. bei 10% der patienten finden sich klinische und radiologische zeichen einer pulmonalen beteiligung.bei kleinen kindern manifestiert sich eine influenzainfektion klinisch meist nicht unterscheidbar von infektionen durch andere respirationstraktviren (rsv, adenoviren, parainfluenzaviren u. a.), unter dem bild einer bronchiolitis, einer obstruktiven bronchitis, einer pneumonie, einer akuten subglottischen laryngotracheobronchitis (infektkrupp) oder einer unspezifischen atemweginfektion. darüber hinaus können bei kleinen kindern gastrointestinale symptome (durchfall, erbrechen) auftreten. häufig kommt es auch zu fieberkrämpfen. bei kindern mit chronischen grunderkrankungen (z. b. zystische fibrose, immunsuppression) können influenzainfektionen sehr schwer verlaufen. die meisten erkrankungen verlaufen insgesamt gutartig. bei immunsupprimierten kindern können erkrankungen mit parainfluenzaviren äußerst schwer und u. u. letal verlaufen (riesenzellpneumonie).j diagnose eine spezifische diagnostik ist meist nur bei schweren krankheitsverläufen erforderlich. parainfluenzaviren können im nasopharynxsekret nachgewiesen werden (immunfluoreszenztest, elisa, rt-pcr). der serologische nachweis von parainfluenzavirusspezifischen antikörpern spielt im klinischen alltag keine rolle.j therapie, prophylaxe eine spezifische antivirale therapie oder eine impfung existieren nicht. die behandlung der klinischen beschwerden ist symptomatisch. bei immunsupprimierten kindern mit schwerer parainfluenzavirusinfektion (pneumonie) kann ein therapieversuch mit ribavirin unternommen werden. bezüglich hygienemaßnahmen gelten die gleichen empfehlungen wie für rsv-infektionen. key: cord-285433-ehnu83qe authors: sun, hongliu; qi, cai; niu, yu; kang, tengfei; wei, yongxin; jin, gang; dong, xianzhi; wang, chunhua; zhu, wei title: detection of cytomegalovirus antibodies using a biosensor based on imaging ellipsometry date: 2015-08-21 journal: plos one doi: 10.1371/journal.pone.0136253 sha: doc_id: 285433 cord_uid: ehnu83qe background: cytomegalovirus (cmv) is the most common infectious cause of mental disability in newborns in developed countries. there is an urgent need to establish an early detection and high-throughput screening method for cmv infection using portable detection devices. methods: an antibody analysis method is reported for the detection and identification of cmv antibodies in serum using a biosensor based on high spatial resolution imaging ellipsometry (bie). cmv antigen (cmv-3a) was immobilized on silicon wafers and used to capture cmv antibodies in serum. an antibody against human immunoglobulin g (anti-igg) was used to confirm the igg antibody against cmv captured by the cmv-3a. results: our results show that this assay is rapid and specific for the identification of igg antibody against cmv. further, patient serum was quantitatively assessed using the standard curve method, and the quantitative results were in agreement with the enzyme-linked immunosorbent assay. the cmv antibody detection sensitivity of bie reached 0.01 iu/ml. conclusions: this novel biosensor may be a valuable diagnostic tool for analysis of igg antibody against cmv during cmv infection screening. cmv is the most common infectious cause of mental disability in newborns in developed countries [1] . detection of cmv antibodies is effective for systematic screening for cmv infection [2] . for instance, the cmv immunoglobulin g (igg) avidity assay can help to distinguish primary from non-primary human cmv infections [3] [4] [5] . the key immune methods used for cmv antibody detection are: enzyme-linked immunosorbent assay (elisa) [6] , elecsys [7] , electrochemiluminescence immunoassay (eclia) [8] , immunofluorescence assay (ifa) [9] , flow cytometry (fcm) [10] and immunoblots [11] . additionally, new immune methods for cmv antibody detection have been developed, such as the chemiluminesent microparticle immunoassay (cmia) [12] and protein microarrays [13, 14] . however, these methods suffer from inherent limitations, such as length of testing time, the needs for expensive equipment, specialist skills, low sensitivity, and complicated sample preparation processes. for example, conventional elisa is still the main diagnostic test for cmv, and commercial cmv elisa kits are available. although elisa is often used as a comparison method for cmv antibody detection [13] , obvious shortcomings include the need of tracer label, plate washing, the indirect format of detection, and the length of time necessary for testing. thus, a rapid, simple, direct, and high-throughput method for cmv antibody detection is urgently needed. the first biosensor based on imaging ellipsometry (bie) was developed in 1995 [15, 16] . compared to the methods above, the advantages of bie are evident, e.g., high-throughput multiplexed analysis and quantitative, label-free rapid testing. previous applications of bie mainly focus on the biomedical fields [17, 18] , such as high-throughput disease diagnosis of hepatitis b virus (hbv) marker [19, 20] , the detection of avian influenza virus (aiv) [21] and antibodies against severe acute respiratory syndrome (sars) detection [22] . thus, the biosensor technology offers important tools for disease diagnosis. as such, new applications have recently been developed, including those for the analysis of the interaction between tropomyosin allergens and antibodies [23] , and the interaction between soluble n-ethylmaleimide-sensitive factor attachment receptor (snare) proteins [24] . to date, however, bie has not been applied for the detection of cmv antibody, particularly for the identification of the cmv igg and igm antibodies. the purpose of this study was to detect antibodies against cmv-3a in patient serum using a bie microarray with cmv-3a, as well as to specifically identify captured igg antibody against cmv. antibodies against cmv qualitatively were detected using bie, and then, goat antibody against human igg (anti-igg) was added to the area with the captured cmv antibody to confirm igg antibody against cmv. a standard curve representing different concentration gradients was also established for the quantitative detection of cmv antibodies. as such, the concentration of cmv antibody in serum was quantitatively detected by bie and then compared using elisas. silicon wafers were purchased from the general research institute for nonferrous metals (china). n-hy-droxysuccinimide (nhs) and 1-(3-dimethyla-minopropyl)-3-ethylcarbodiimide hydrochloride (edc), tween-20, bovine serum albumin (bsa), igg, and blocking buffer (10×, b6429) were purchased by sigma. anti-igg and a goat antibody against human igm (anti-igm) were purchased from beijing bo sheng bio-technology co. ltd. cmv-3a was purchased from galaxybio. the cmv-3a was a fusion of three segments of pp150, gp52 and pp65, which have strong antigenicity epitopes. fusion of the multi-epitope both enhances the sensitivity/specificity and reduces false negatives. thus, cmv-3a was used as the ligand in the bie assay. purified cmv antibody (pp65, c-term, rabbit, 1 mg/ml) was purchased from antibodies-online.com. patient serum samples were purchased from qilu hospital of shandong university and clinical information is listed in table a in s2 file. torch elisa kits used to analyze serum samples were purchased from medson inc. the detection process and analysis were executed according to the manufacturer's instructions. microplates were coated with native cmv antigens, highly purified by sucrose gradient centrifugation and inactivated. the solid phase was first treated with the diluted sample, and igg molecules to cmv were then captured, if present, by the antigens. after washing out all of the other sample components, bound anti-cmv igg molecules were detected by the addition of specific polyclonal anti-h-igg antibodies labelled with peroxidase (hrp) in the second incubation. the enzyme captured on the solid phase, acting on the substrate/chromogen mixture, generates an optical signal that is proportional to the amount of anti-cmv igg antibodies present in the sample. a calibration curve, calibrated against the first w.h.o international standard, makes possible a quantitative determination of the igg antibody in the patient. ne solution was prepared with nhs (0.05 m) and edc (0.2 m) in deionized water (18.3 mocm) from a milli-q plus system (millipore, bedford, ma). phosphate-buffered saline (pbs) was prepared with 140 mm nacl, 2.7 mm kcl, 10 mm na 2 hpo 4 , and 1.8 mm kh 2 po 4 (ph 7.3) in deionized water. pbst buffer was prepared with 1% tween-20 in pbs. cmv-3a (0.1 mg/ml) was prepared with pbst. bsa (10 mg/ml) was prepared with pbst. the blocking reagent was a 1:1 (vol:vol) mixture of 10 mg/ml bsa and 10× blocking buffer. purified cmv antibody, igg, anti-igg, and anti-igm were diluted to a concentration of 0.1 mg/ml with pbst. serum samples were diluted as indicated with pbst for qualitative or quantitative detection. the bie combines high spatial resolution imaging ellipsometry (figa in s1 file) with a microfluidic system (figb in s1 file) to analyze biomolecular interaction [25] . the microfluidic system is used for surface patterning and array production, as well as for solution delivery, ligand immobilization and target capture [26] . the microfluidic system has four main parts: a sample plate, multi-cell array, micro-channels and pumps. a multi-cell array was formed when a polydimethyl-siloxane (pdms) pattern mold contacted the surface of a silicon substrate, each cell having an inlet and an outlet for solution passage. the physical size of each cell was~1.5×1×0.5 mm 3 . the inlet micro-channels were placed into sample plate, and the outlet micro-channels were connected with pumps (ism939, ismatec, switzerland. www.ismatec.com) offering negative pressure. the simple channel junctions can be used in serial or parallel formats to simultaneously analyze single or multiple samples. imaging ellipsometry is a display technique for ultrathin film and surface characterization [27] , which is used to read and analyze the protein arrays made by microfluidic systems. the incident wave of polarized light irradiates the sample as a probe beam and is thereby modified resulting in a reflective or transmission beam having the ability to carry sample information, such as protein layer thickness. when imaging ellipsometry is used to detect layer thickness, the reflection intensity is represented in grayscale, and the variation in layer thickness leads to changes in the grayscale value. if the refractive index is invariant, the grayscale value is directly proportional to the thickness of the protein layer within the range of 0~30 nm layer thickness, i.e., i = kd, where i is the light intensity and d is the layer thickness [28] . under these conditions k is a constant and can be determined from a protein layer with known grayscale values and known thickness [29] . there is also a relationship between surface concentration and film thickness: surface concentration (μg/cm 2 )k×d, where k = 0.12 [27] . thus, the grayscale value directly reflects layer thickness and surface concentration. the higher the grayscale value, the thicker the layer and the higher the surface concentration. silicon wafers were cut into 20×10 mm 2 rectangles and rinsed with deionized water. after soaking in piranha solution (30% h 2 o 2 :98% h 2 so 4 = 1:3, vol/vol) for 30 min to increase the number of silanol groups on the wafer surface, and rinsing with deionized water and ethanol, the wafers were soaked in a mixture of 3-aminopropyltriethoxy-silane (aptes) and absolute ethanol (aptes:absolute ethanol = 1:10, vol/vol) and incubated for 2 h with gentle agitation. the reaction of aptes with the surface silanol groups resulted in covalent immobilization of-o-si(oh) 2 -(ch 2 ) 3 -nh 2 , forming a layer of densely packed amino groups on the surface. after rinsing three times with absolute ethanol, the wafers were incubated in a saturated solution of succinic anhydride in ethanol for 3 h with gentle agitation. the ch 2 ch 2 cooco-group of succinic anhydride reacted with the -nh 2 of -o-si(oh) 2 -(ch 2 ) 3 -nh 2 group immobilized on the surface, generating carboxyl groups (-(ch 2 ) 3 nh-co-(ch 2 ) 2 -cooh). once prepared, the wafers were stored in ethanol. the above-modified silicon wafers were used as the substrate. a piece of silicon wafer was placed on the microfluidic mold of the microfluidic system so that surfaces of the wafers were patterned to form small, regular cells in an array format. then, the carboxyl groups were activated by pumping 10 μl of ne into each cell at a flow rate of 5 μl/min and passed through the surface of the wafer. in the presence of nhs, edc transfers carboxyl groups to the sulfo-nhs ester, which reacts with the amino groups of proteins to immobilize the proteins on surface. next, the ligand was immobilized: cmv-3a as ligand or probe was pumped into each cell (10 μl per cell at 1 μl/min). third, blocking occurred by pumping the blocking reagent into each cell (50 μl per cell at 1 μl/min) and passing it through each ligand area. thus, a sensing array surface with cmv-3a was formed to catch cmv antibodies. fourth, targets were detected. pbst was used as blank control (50 μl per cell at 1 μl/min) and purified cmv antibody as a positive control (50 μl per cell at 1 μl/min) by pumping them into several of the individual cells. simultaneously, patient serum samples were also pumped into remaining cells (50 μl per cell at 1 μl/min). the cmv antibodies in the serum were captured when they interacted with the cmv-3a on the sensing surface. all cells were rinsed with pbst (20 μl per cell at 20 μl/min) between every two consecutive operation steps. finally, the microarray wafers were removed from the microfluidic system. after being rinsed with deionized water and dried with nitrogen, the wafers were analyzed using imaging ellipsometry. if the cmv antibodies in the solution or serum interacted with the cmv-3a on the surface and formed a complex, the layer in that area became thicker. the experimental results were recorded as images in grayscale, and binding of cmv antibodies resulted in a significant increase in grayscale value. to detect the specificity of antibodies against cmv, cmv-3a as ligand was immobilized on two columns (fig 1) . pbst buffer was added as blank control to two areas on the first row. simultaneously, purified cmv antibody (0.1 mg/ml) was added as positive control to two areas on the second row. normal serum without cmv antibodies was added as negative control to two areas on the third row. three patient serum samples (no. 956, 933, and 978; see table a in s2 file for sample information) were analyzed on the following rows, respectively. the increase in light reflection density at each area revealed that cmv antibodies in the samples interacted with the cmv-3a immobilized on the chip. we further analyzed the types of cmv antibodies captured by the cmv-3a (fig 2) . in the first step, igg was immobilized as ligand on the first and second columns. simultaneously, cmv-3a was immobilized on the third, fourth, fifth, and sixth columns. in the second step, sample no. 948 was added to the third and fourth columns, and sample no. 940 was added to the fifth and sixth columns. pbst buffer was added as blank control to remaining areas. in the third step, anti-igg and anti-igm were added to third and fourth rows to identify and confirm in the first step, igg was immobilized as ligand on the first and second columns. cmv-3a was immobilized on the third, fourth, fifth, and sixth columns. in the second step, pbst buffer was added as blank control to the corresponding areas in the image. sample no. 948 was added to the third and fourth columns, and sample no. 940 was added to the fifth and sixth columns. in the third step, pbst buffer was added as blank control to the first areas in every column. anti-igg and anti-igm were added to the third and fourth rows, respectively. if the antibody captured by the ligand was igg or igm. pbst buffer was added as blank control to remaining areas. to establish a calibration curve, a serum sample (no. 942, 21.8 iu/ml, see table a in s2 file) was used, and five levels of serial dilution containing 0.011, 0.043, 0.170, 0.681, and 2.725 iu/ ml of cmv antibody were prepared in pbst. cmv-3a was immobilized as ligand on two columns (fig 3) . after blocking, pbst buffer, cmv antibody and normal serum without cmv antibody were added as blank, positive, and negative controls (respectively) to the first, second, and third rows, respectively. the 0.011-, 0.043-, 0.170-, 0.681-and 2.725-iu/ml samples were added from the fourth to the eighth rows, respectively. concentration plotted on the x-axis and the variation of the grayscale value compared to blank control was plotted on the y-axis to generate the calibration curve (fig 3) . after acquiring the calibration curve, the concentrations of antibodies in samples of unknown concentration could be determined on the curve according to their grayscale values. measurements of every sample were repeated in two areas on one chip (fig 4) . commercial elisa cmv antibody kits were used as controls according to the manufacturer's instructions. for statistical analyses, the corresponding p-values were calculated with single factor analysis of variance in microsoft office excel according to the quantity of areas in images and patients' serum samples in table a in s2 file [30] . in qualitative and quantitative detection experiments, significant changes in grayscale value detection areas compared to the blank control areas and the detection areas were deemed positive signals if p-value was < 0.05. if the p was 0.05, the detection areas were deemed negative signals. in comparison of the elisa and bie data, the results of the two methods were deemed in agreement if p was 0.05. if p was < 0.05, the two methods were deemed in disagreement. the correlation coefficient (r-value) of bie and elisa was calculated with analysis of correlation coefficient in microsoft office excel (table b in s2 file). compared to blank controls areas, the purified cmv antibody and patient serum sample detection areas had markedly thicker films, with the average grayscale value displaying significant increases, while negative control areas did not (fig 1) . the mean grayscale value of blank controls was measured at 117.45 ± 0.92, and the value of the purified cmv antibody was measured at 176.8 ± 3.39. thus, the mean grayscale value of purified cmv antibody minus the mean grayscale value of blank control was 59.4 (p = 0.002). the value of the negative control was 125.9 ± 3.18. the mean grayscale value of the negative control minus the mean grayscale value of the blank control was 8.4 (p = 0.07). this indicated that there were specific interactions between the purified cmv antibody and the cmv-3a. therefore, cmv antibodies in patient samples could be captured by the cmv-3a immobilized on the substrate. indeed, the average grayscale value of the serum samples analyzed significantly increased relative to the controls (fig 1) . the mean serum value was 253.58 ± 0.49, and the mean grayscale value of serum minus the mean grayscale value of the blank control was 136.1 (p = 2.8×10 −5 ), indicating that cmv antibodies were abundant in the serum samples. this was consistent with the elisa results (table a in s2 file). when purified human igg was used as the ligand, the average grayscale value of the anti-igg detection areas significantly increased, while the anti-igm detection areas did not (left two columns in fig 2) . the mean grayscale value of the anti-igg minus the mean grayscale value of the blank control was 96.2 (p = 0.003). the mean value of the anti-igm minus the mean grayscale value of the blank control was 8.15 (p = 0.29). these data are indicative of the specific interactions between the anti-igg and the human igg immobilized on the chip, which could be used as references to determine whether the antibodies in samples are igg. compared to the cmv antibody areas, the anti-igg detection areas displayed significant increases, while the anti-igm detection areas did not (fig 2) . for example, the value of sample 948 was 156.2 ± 2.6, while the value of the anti-igg was 189.6 ± 4.4, for a difference of 33.4 (p = 0.02). however, the value of the anti-igm was 154.25 ± 0.25, and the difference was not obvious (p = 0.53). this indicated that igg cmv antibodies were captured by the chip. for sample no. 940 detection, the value of the anti-igg was 237.7 ± 11, and the increase was 77.3 (p = 0.01). the result also indicated that the content of igg in samples no. 948 and 940 was different. five concentration gradients of a serum sample (no. 942, 21.8 iu/ml, see table a in s2 file) measured in serially diluted samples were used to determine the sensitivity of the bie assay (fig 3) . we found that the change in signal intensity was consistent with the increase in cmv antibody concentration. the cmv antibody detection sensitivity levels reached 0.01 iu/ml. the grayscale values of the blank control were 124.7 ± 0.5, and the grayscale values of the dilutions as low as 0.01 iu/ml were 132.3 ± 0.1, i.e.,~6.1% greater than that of the controls (p = 0.03). repeating these tests > 10 times, our results demonstrated that the sensitivity of the assay reached 0.01 iu/ml or less. each variation in grayscale value was linked to a corresponding concentration of the cmv antibody over the range of 0.011-2.725 iu/ml, and the in the first step, cmv-3a was immobilized as the ligand on two columns. in the second step, pbst buffer was added as a blank control to two areas on the first row. simultaneously, purified cmv antibody was added as a positive control to two areas on the second row. negative serum was added as a negative control to two areas on the third row. the serial dilutions of cmv antibodies were added as analytical samples on the following rows. the same concentration was measured in two duplicate areas. quantitative detection of cmv antibodies in clinical serum 41 cmv patients (table a in s2 file) with quantitative results by elisa were tested with bie (fig 4) . for different serum samples, the changes in bie signal intensity were different. the comparison of results between the two methods is shown in fig 5. single factor analysis and correlation coefficient analysis revealed that the results were in agreement between elisa and bie (f = 1.38<f 0.05 = 3.96; p = 0.24>0.05, r = 0.7) ( table b in s2 file). high-throughput detection is suitable for mass cmv screening in women of childbearing age. when coupled with microfluidic technologies, bie can greatly improve the throughput for cmv detection on one chip. the microarrays developed here have multiple cells immobilized with different ligands for different cmv antibody subtypes. imaging ellipsometry is also suitable to high-throughput analysis. it can be used to visualize the variation in signal from all units of the microarray with high spatial resolution. patient sample analysis results from one microarray can simultaneously be identified, and the data can be obtained in several seconds using an imaging ellipsometer. presently, our method can provide simultaneous 48 reaction areas. with its enhanced throughput and lower cost, bie may be used in clinical mass cmv screenings for healthy births in the future. antibody detection is widely available for the clinical diagnosis of cmv infection. however, the identification of antibody types may help to determine the course of disease, and bie may be used as a clinical primary screening tool for cmv patients. igm is produced in large amounts early in infection (reaching a peak in the first month), and is followed by igg production [7, 31] . levels of igm decline in the months following the onset of infection, whereas igg levels persist for the rest of the patient's life [32, 33] . determining igg avidity can provide additional guidance on infection status, and low avidity igg is initially present but increases over time [34] . a positive result for igm combined with low-to-moderate igg suggests avidity a primary cmv infection within the past 3 to 4 months [35] . the visualized bie image (fig 2) could reflect types via difference in brightness, so this technology can achieve rapid screening. to date, our work is simply a demonstration for igg type antibody detection with bie. igm and other antibodies needed to screen for specific antigen can be incorporated in the future for further practical applications. presently, most methods mention the relative sensitivity (%) of detection for cmv antibodies [8, 36] , but little in provided about absolute sensitivity. compared other methods, the absolute sensitivity of bie can reach 0.01 iu/ml, and it has good resolution in the range of 0.1-1.0 iu/ml on the calibration curve for quantitative detection. the reference range for detection of cmv antibodies has previously been published. for example, elecsys (roche diagnostics) immunoassays have an equivocal range (0.5-1.0 iu/ml) of cmv igg [12] . in our experiment, elisa as a control method had a reference range of 0.4-0.6 iu/ml. therefore, the sensitivity of our bie biosensor is below the reference range and has already reached clinical standards. however, there is a substantial discordance between the elisa and the bie in some patients (i.e., 940, 956, 964, 984, and 980). samples from these patients displayed stronger signal using elisa than bie. in elisas, anti-cmv igg molecules are detected by the addition of polyclonal specific anti-h-igg antibodies labelled with horseradish peroxidase (hrp). in contrast, the bie biosensor directly identified the variation of the surface anti-cmv igg concentration after capturing target without a secondary label. thus, this label-free method may avoid some detection of cmv antibodies in patient serum using bie. in the first step, cmv-3a was immobilized as the ligand on six columns. in the second step, pbst buffer was added as a blank control to six areas on the first row. simultaneously, purified cmv antibody was added as a positive control to the last two areas on the second row. patient serum samples were added as analytical samples on the following areas, respectively. the same serum sample was measured in two duplicate areas (no.940, 959,938 no sample, p15-9, and pbst control are underlined). interference factor. in addition, the grayscale images offered uniform areas, helping to avoid false positive signals. when the correlation coefficient (r-value) and p-value were calculated after excluding the number of severe mutations (940, 956, 964, and 984), r-value = 0.93 and p = 0.8 the statistical results showed more agreement between elisa and bie. in conclusion, we have developed a label-free and multiplex screening cmv igg biosensor method. the high-throughput detection is suitable for mass cmv screening of women of childbearing age. further, our results demonstrate that the sensitivity of bie has already reached clinical diagnose standards for anti-cmv igg. thus, on the basis of anti-cmv igg detection, bie can be used for qualitative and quantitative detection of more types of cmv antibodies using a simple and fast procedure. supporting information s1 file. contains fig a. imaging ellipsometry. (a) imaging principle [23] ; and (b) laboratory prototype. fig b. prevention of maternal-fetal transmission of cytomegalovirus maternal igg avidity, igm and ultrasound abnormalities: combined method to detect congenital cytomegalovirus infection with sequelae diagnosis and management of human cytomegalovirus infection in the mother, fetus, and newborn infant new advances in the diagnosis of congenital cytomegalovirus infection maternal igg avidity, igm and ultrasound abnormalities: combined method to detect congenital cytomegalovirus infection with sequelae a simple method to quantitate ip-10 in dried blood and plasma spots clinical evaluation of new automated cytomegalovirus igm and igg assays for the elecsys analyser platform value of cmv-igm detection by electrochemiluminescence immunoassay in diag-nosis of cmv infection in pregnant women. maternal and child health care of china microtubule network facilitates nuclear targeting of human cytomegalovirus capsid the use of flow cytometry for the detection of cmv-specific antigen (pp65) in leukocytes of kidney recipients improving diagnosis of primary cytomegalovirus infection in pregnant women using immunoblots detection of human cytomegalovirus igg antibody using automated immunoassay with recombinant antigens gives uniform results to established assays using whole virus antigens multiplexed infectious protein microarray immunoassay suitable for the study of the specificity of monoclonal immunoglobulins development of recombinant antigen array for simultaneous detection of viral antibodies a biosensor concept based on imaging ellipsometry for visualization of biomolecular interactions imaging ellipsometry revisited: developments for visualization of thin transparent layers on silicon substrates development of biosensor based on imaging ellipsometry and biomedical applications biosensors for health detection of hepatitis b markers using biosensor based on imaging ellipsometry evaluation of a new ca15-3 protein assay method: optical protein-chip system for clinical application detection of avian influenza virus subtype h5 using a biosensor based on imaging ellipsometry investigation of interaction between two neutralizing monoclonal antibodies and sars virus using biosensor based on imaging ellipsometry analysis of interaction between tropomyosin allergen and antibody using a biosensor based on imaging ellipsometry analysis of interactions between snare proteins using imaging ellipsometer coupled with microfluidic array feasibility of protein a for the oriented immobilization of immunoglobulin on silicon surface for a biosensor with imaging ellipsometry a label-free protein microfluidic array for parallel immunoassays the use of the isoscope ellipsometer in the study of adsorbed proteins and biospecific binding reactions optimization of off-null ellipsometry for air/solid interfaces off-null ellipsometry revisited: basic considerations for measuring surface concentrations at solid/liquid interfaces maternal, fetal and neonatal diagnosis of congenital human cytomegalovirus infection cytomegalovirus strain diversity in seropositive women cytomegalovirus reinfections in healthy seroimmune women avidity of immunoglobulin g directed against human cytomegalovirus during primary and secondary infections in immunocompetent and immunocompromised subjects value of cytomegalovirus (cmv) igg avidity index for the diagnosis of primary cmv infection in pregnant women human cytomegalovirus detection by real-time pcr and pp65-antigen test in hematopoietic stem cell transplant recipients: a challenge in low and middle-income countries we thank qilu hospital of shandong university for clinical cmv patient samples. key: cord-014462-11ggaqf1 authors: nan title: abstracts of the papers presented in the xix national conference of indian virological society, “recent trends in viral disease problems and management”, on 18–20 march, 2010, at s.v. university, tirupati, andhra pradesh date: 2011-04-21 journal: indian j virol doi: 10.1007/s13337-011-0027-2 sha: doc_id: 14462 cord_uid: 11ggaqf1 nan patients showed rashes on face, hand and foot. ev detection carried out in vesicular fluid, stool, serum and throat swab specimens by rt-pcr of 5 0 ncr gene. serotyping was carried out by using rt-pcr of viral protein of vp1/2a junction region followed by sequencing and phylogenetic analysis using neighbor-joining-algorithm and kimura-2 parameter model of mega-4 software. overall ev positivity detected in hfmd patients from kerala, tamil nadu, west bengal and orissa states was found to be 51.6%, 66.6%, 62.5% and 71.4% respectively. typing of vp1 gene sequences indicated presence of ca-6, ev-71, echo-9 strains in kerala and ca-16 in west bengal, orissa and tamil nadu. phylogenetic analysis indicated ca-6, ev-71, echo-9 strains showed 94.8-95.7% and 95-94.4% homology with japanese, australian and french strains. however, ca-16 strains were closer to malaysian strains with 91.2-95.6% nucleotide homology. the present study documents the association of multiple types of ev's i.e., ca-6, ev-71, echo-9 and ca-16 strains contributing as prime viral pathogens in hfmd epidemics in the reported regions with new emergence of ca-6 circulating strain in kerala, india. tasgaon september 2010. sera were collected from 162 suspected hepatitis cases and there contacts and tested for anti hev igm/igg antibodies (elisa) and liver enzymes like alanine aminotransferase (alt). anti hev igm antibodies were detected in 45.7% (74/162) of the suspected cases. the overall attack rate was 0.7%. male to female ratio was 2:1. majority (60.4%) of the cases were in the age group 20-40 years and recovered without any clinical complications. weekly distribution of cases showed that the majority (79.4%, 116/146) cases occurred between 2nd and 3rd week of june. dark urine (97.5%), jaundice (93.5%), fatigue (35.9%), abdominal pain (32.6%), anorexia (29.4%), vomiting (26.5%), fever (22.8%), giddiness (14.3%), diarrhoea (12.6%) and arthalgia (3.7%) were the prominent symptoms. sera collected from 73 antenatal cases (ancs) showed anti hev igm antibody in 3. affected pregnant women had a normal outcome. a death of 32 year, male hepatitis e case was reported during the outbreak period that had cirrhosis of liver with oesophageal varices. sanitary survey revealed that water pipelines were laid down in close proximity of sewerage system, and water posts were without tap. these are the likely sources of faecal contamination of water supplies. among 17 water samples collected from various places, 5 were found to be unfit for drinking based on the routine bacteriological tests conducted at state public health laboratory, pune. no case occurred after the pipelines were repaired. this typical outbreak of hepatitis e re-emphasizes need for proper water supply/sewage disposal pipelines and adequate maintenance measures. jayanthi shastri, nilima vaidya, sandhya sawant, umesh aigal department of molecular biology, kasturba hospital for infectious diseases, mumbai, india dengue and dengue haemorrhagic fever are amongst the most important challenges in tropical diseases due to their expanding geographic distribution, increasing outbreak frequency, hyperendemicity and evolution of virulence. the gobal prevalence of dengue has grown dramatically in recent decades. who estimates 50-100 million cases of dengue virus infections worldwide every year resulting in 250,000 to 500,000 cases of dhf and 24,000 deaths each year. public health laboratories require rapid diagnosis of dengue outbreaks for application of measures such as vector control. laboratory diagnosis of dengue virus infection can be made by the detection of specific virus, viral antigen, genomic sequence and/or antibodies. currently 3 basic methods used by laboratories for diagnosis of dengue virus infection are virus isolation and characterisation, detection of genomic sequence by nucleic acid amplification technology assay and detection of dengue virus specific antibodies/antigen. molecular diagnosis based on reverse transcription (rt)-pcr s.a. one step or nested pcr, nucleic acid sequence based amplification (nasba), or real time rt-pcr, has gradually replaced the virus isolation method as the new standard for the detection of dengue virus in acute phase serum samples. several pcr protocols for detection have been described that vary in the extraction method, genomic location of primers, specificity, sensitivity and the methods to determine the products and the serotype. pcr-based dengue tests, due to the specificity of amplification, enable a definitive diagnosis and serotyping of the virus. in addition dna sequencing of the amplification product enables the virus to be genotyped, providing important information on the sources of infection. more recently tests have incorporated flurogenic probe, so called taq man technology for the specific real time detection of dengue 1-4 amplicons. product is detected by a specific oligodeoxy nucleotide probe that is labelled with 6 carboxy-fluorescein (fam). this technology offers the advantage of being both rapid and potentially quantitative. second, the detection of product by hybridisation of flurochrome labelled probes increases specificity. third, as the product is detected without the need to open the reaction tube, the risk of contamination by product carry over is minimised. the advantages of speed, contamination minimisation and reduced turn around time justify application of this assay over the currently used nested pcr assay. during the period january 2007 to october 2009, molecular laboratory received 900 samples from patients presenting with acute onset fever for dengue .6%) samples were tested positive by this method. the disease peaks in the monsoon season with a percentage of 17.5%. rapid tests, igm and igg capture elisa are popularly used tests for diagnosis of dengue infection. its utility is limited for diagnosing dengue in convalescecce (8-14 days) . specificity is also compromised due to infections with flaviviruses: japanese encephalitis and chikungunya. dengue ns1 ag elisa with its cost effectiveness, specificity and sensitivity should be considered as the test of choice for diagnosing dengue in the acute phase of illness in the developing countries. molecular diagnosis enables confirmatory diagnosis of dengue in the acute phase of the illness and is suitable for further typing methods. assistant general manager and r&d coordinator, division of quality control and r&d, bharat immunologicals and biologicals corporation ltd., village chola, bulandshahr, up vaccine development in india, though slow to start, has progressed by leaps and bounds in the past 60 years. it was dependent on imported vaccines but now it is not only self-sufficient in the production of vaccines conforming to international standards with major supplier of the same to unicef. the role of drug authorities is to enhance the public health by assuring the availability of safe and effective a2 indian j. virol. (september 2010) 21(suppl. 1):a1-a58 vaccines, allergenic extracts, and other related products. vaccine development is tightly regulated by a hierarchy of regulatory bodies. guidelines provided by the indian council of medical research (icmr) set the rules of conduct for clinical trials from phase i to iv studies as well as studies on combination vaccines. these guidelines address ethical issues that arise during a vaccine study. a network of adverse drug reaction (adr) monitoring centers along with the adverse events following immunization (aefi) monitoring program provide the machinery for vaccine pharmacovigilance. genetic modifications have been developed to develop effective and cheaper vaccines by the use of recombinant technology. to ensure safety of consumers, producers, experimental animals and environment, governments all over the world are following regulatory mechanisms and guidelines for genetically modified products. as with other industrializing countries undergoing rapid shifts, india clearly recognizes the need to restructure its regulatory system so that its biopharmaceutical industry can compete in international markets. genetic engineering approval council (geac), recombinant dna advisory committee (rdac), review committee on genetic manipulation (rcgm), institutional biosafety committees (ibsc) are responsible for development, commitment for parameters and commercialization of recombinant vaccines. to centralize and coordinate the whole system, government has taken to form two agencies to regulate the regulation laws to develop recombinant pharmaceuticals products including vaccines. the first is the creation of the national biotechnology regulatory authority (nbra), under the department of biotechnology (dbt), as part of india's long-term biotech sector development strategy. the second major initiative will affect the entire indian pharmaceutical industry. this is the replacement of most state, district, and central drug regulatory agencies with a single, central, fda-style agency, the central drug authority (cda). the cda is expected to have separate, semi-autonomous departments for regulation, enforcement, legal, and consumer affairs; biotechnology products; pharmacovigilance and drugs safety; medical devices and diagnostics; imports; quality control; and traditional indian medicines. it will set up offices throughout india and will be paid for inspection, registration, and license fees. its enforcement powers will be strengthened by a new law increasing the criminal penalties for illegal clinical trials. in the manufacturing area, though, the country has been tightening the rules and enforcement. an amendment to the regulations, ''schedule m'' of the drug and cosmetics act, now specifies the good manufacturing practice (gmp) requirements for factory premises and materials. these requirements were modeled after us fda regulations, to improve regulatory coordination between indian and us regulators. india has realized the importance of regulations in pharmaceutical specially in vaccine field but it will take several years to implementation of these. india has coordinated some of its regulatory functions with western organizations. the us pharmacopoeia established an office in hyderabad in 2007. a representative of the indian pharmaceutical lobby also recently has expressed openness to an expansion of the fda's oversight of indian manufacturing. as india expands its global drug and biologicals production, us and europe, as the world's largest drug importers, will likely expand their regulatory support in the development of the country's regulatory systems. rapid diagnosis of japanese encephalitis virus (jev) infections is important for timely clinical management and epidemiological control in areas where multiple flaviviruses are endemic. however, the speed and accuracy of diagnosis must be balanced against test cost and availability, especially in developing countries. an antigen capture enzyme-linked immunosorbent assay (elisa) for detection of circulating jev specific nonstructural protein 1 (ns1) was developed by using monoclonal antibodies (mabs) specific to recombinant (ns1). the applicability of this jev ns1 antigen capture elisa for early clinical diagnosis was evaluated with 200 acute phase serum/ cerebrospinal fluid (csf) specimens collected from different epidemics during [2007] [2008] [2009] . jev ns1 antigen was detected in circulation from day 1 to 18. the sensitivity and specificity of jev ns1 detection in serum/csf specimens with reference to reverse transcriptase pcr was 82%, and 98.9% respectively. no crossreactions with any of the other closely related members of the genus flaviviruses (dengue, westnile, yellow fever and saint louis encephalitis (sle) viruses) were observed when tested with either clinical specimens or virus cultures. these findings suggested that the reported jev specific mab-based ns1 antigen capture elisa will be a rapid and reliable tool for early confirmatory diagnosis as well as surveillance of je infections in developing countries. manmohan parida the recent emergence of a novel human influenza a virus (h1n1) poses a serious global health threat. the h1n1 virus has caused a considerable number of deaths within a short duration since its emergence. a two-step single tube accelerated rapid real-time and quantitative swine flu virus specific h1 rtlamp assay is reported by targeting the h1 gene of the novel h1n1 hybrid virus. the feasibility of swine flu h1 rtlamp for clinical diagnosis was validated with a panel of 239 suspected throat wash samples comprising 116 confirmed positive and 123 confirmed negative cases of ongoing epidemic. the comparative evaluation of h1 specific rtlamp assay with real-time rt-pcr demonstrated exceptionally higher sensitivity by picking up all the 116 h1n1 positive and 36 additional positive cases amongst the negatives that were sequence confirmed as h1n1. none of the real-time rtpcr positive samples were missed by rtlamp system. the comparative study revealed that rtlamp was 100-fold more sensitive than rtpcr with a detection limit of 1 copy number. these findings suggested that rtlamp assay is a valuable tool for rapid, real-time detection as well as quantification of h1n1 virus in acute phase throat swab samples without requiring any sophisticated equipments. because of its recurrent nature. despite considerable progress in understanding of the virus at cellular and molecular levels, the proper management of the disease in its different stages is still a dilemma particularly whether to use antiviral or steroids or both. the risk of using steroids with its attendant complications has to be weighed against the risk of progression of the disease if avoiding the use of steroids. this dilemma can be reduced to a considerable extent if basic principles of virology and pathogenesis are kept in mind. this article reviews current concepts of virological and clinical aspects of hsv keratitis to enable a broad understanding of the disease process. it is recognized several influential host factors including the fact that hsk is more common in men than women. it is observed that the ability of hsv to establish latent infection in sensory neurons and possibly cornea, but have as yet been unable to use this knowledge to prevent the disease limitations. acknowledging limitations may further stimulate application of laboratory knowledge in coping with hsk which constitutes to present major challenge in terms of management. mvo-10 study on effect of human bhsp90 in immunity of hcv core protein and hbv hbsag there are more than 500 million individuals with hepatitis b and c in the world. in spite of vaccination in the different areas there are several reports about patients who got vaccine before. also there is not efficient vaccine against of hepatitis c and one of the important problems in vaccine project is development of effective and suitable adjuvant in human vaccines. at present research we applied human bhsp90 protein as adjuvant and chaperon. this protein injected to balbc mice as adjuvant together with recombinant proteins of hcv core and hbv hbsag. then humoral and cellular immune systems of the mice were studied. core and hbsag genes were cloned into petduet-1 vector and thermal vector of pgp1-2 was used for human heat shock protein 90 expressions. the different combination of these three proteins was injected to mice and we evaluated the total igg and igg2a of mice serums after a week. two weeks after booster injection, we studied the proliferation and cytokine secretion of spleen, inguinal and popliteal lymph nodes lymphocytes in vitro and ex vivo conditions. so the core/hbsag + hsp and core + hbsag + hsp complexes induced total igg and igg2a secretion. the spleen lymphocytes proliferation were increased equal to serum igg2a level that was constant in second time bleeding with significant different to complexes with freund's adjuvant. at first il-4 and il-5 cytokines were increased and then decrease of il-4 meaned no hypersensitivity. the chaperon effect of hsp90 on structure of core and hbsag proteins was studied by cd and flourometer. it could fold the proteins after heating and unfolding. hepatitis b virus (hbv) infection is vaccine preventable global public health problem. all commercially available vaccines contain one or more of the recombinant hepatitis b envelope protein or surface antigen (hbsag). measurement of antigen responsible for immunogenicity of vaccine is central to quality assessment. the problems associated with the use of a polyclonal antibody in an assay with regard to its poorly defined nature and batch-to-batch variation has been mitigated by the use of mabs as described in this paper. the initial capture of hbsag by the mab could orientate it such that the same antibody could bind to it as a detection antibody after labeling with out steric hindrance. the development of an immuno-capture elisa (ic-elisa) to measure the hbsag content using a monoclonal antibody (mab) specific to determinant ''a'' of hbsag in the experimental vaccine formulations is being discussed. murine mabs developed against hbsag, subtype adw2 were found to cross-react with the other subtypes viz. ad and ay too. the mabs have been characterized following which, one mab hbs06 was chosen for developing ic-elisa format for the quantification of the hbsag in the final algel adsorbed vaccines. the unadsorbed hbsag was used to establish the standard curve of hbsag/a. the elisa had a sensitivity of 10 ng/ml of hbsag. the recovery rate of hbsag/a was found to be around 70% in the vaccines treated to desorb the antigen from algel. twenty seven experimental batches of monovalent hepatitis b vaccines were analyzed for the hbsag content, both by ic-elisa and a commercial kit (axsym kit, abbott laboratories, usa). the statistical analysis of ic-elisa results indicated that an experimental equation f(x) = 0.0062(x) + 0.184, could precisely estimate the amount of hbsag in the adsorbed vaccines. the amounts of hbsag recovered from the adsorbed vaccines as estimated by the ic-elisa format had a good correlation with the estimates derived from a commercial kit, which is being used by several vaccine manufacturers in india for the quality control of vaccine antigen. the varying amounts of vaccine antigens that could be recovered seemed to depend on the quality of the hbsag and the methods of hbsag adsorption to the alum gel during vaccine manufacture. epidemiology of the spread of h1n1 virus. children of school going age have become victim of this deadly virus as evident from the reporting data generated in the past few weeks. the mortality rate has also been slightly increased. the disease spread in wave pattern and presently the world is passing through the second wave of pandemic with more severity in young and otherwise health people with a predilection for lungs leading to viral pneumonia and respiratory failure. now the pandemic gained hold in the developing world affecting more severely as millions of people live under deprived conditions having multiple health problems, with little access to basic health care. current data about the pandemic from developed counties need to be very closely watched in relation to shift in virus sub type, shift of the highest death rate to younger populations, successive pandemic waves, higher transmissibility than seasonal influenza, and demographic differences etc. presently the world appears to be better prepared. vaccine is available in market in many countries. even vaccine trials are actively going on in indian population. effective antivirals are available. although till now h1n1 diagnostic centers worked with cdc/who recommended h1n1 specific primer, probes with taqman chemistry by real time pcr, efforts on the development of indigenous diagnostics, vaccines and chemoprophylaxis is going on to have a better combat against this highly infectious virus. were positive for rotavirus infection by either page or elisa methods. the available data highlights the importance of rotavirus as a cause of diarrhea in children, which is severe enough to deserve specialized care. the observed proportion of 25.5% of all diarrhea cases being associated with rotavirus falls within the range of values reported by other workers. the reported positivity varies from 10.5 to 70.7%. in our study a complete concordance of elisa and page results were observed in 194 (97%) of the 200 tested specimens. this finding closely correlates with the findings of other authors who found a 96.7-97.14% concordance results between elisa and page methods. some authors found rna-page method that is as sensitive and rapid as elisa for detecting rotavirus in stool samples of cases of diarrhea and some others proposed elisa is more sensitive than page method fond to be 100% specific. the remaining 6 (3%) samples showed conflicting results. in a lone sample in which the od value of elisa test was 0.195, this value was almost at the cutoff level, the possibility of this sample being positive by elisa test is doubtful. negative result of the same sample in page method is difficult to explain, the possibility of presence of lot of empty virus particles or due to low concentration of viral rna in the fecal specimen and insufficient extraction of viral rna could be possible. on the other hand, 5 of the samples which gave positive results by page method were negative by elisa test. these 5 samples had a typical 4-2-3-2 rna pattern. the reason for their being elisa negative thus remains unexplained, however blocking factors or the presence of inhibitory substance in stools might have been responsible. the samples containing predominantly complete particles can also give false negative results. since, the group antigen is not exposed. earlier studies have also reported page to be the most sensitive technique although some are of view that it is laborious procedure. how ever, the page system used in this study was very simple to perform and the results were available on the same day. the main requirement was of trained personnel and proper standardization of the technique. most reports states that the greatest advantage of page and silver stain method are its lack of ambiguity and the fact that it provides information about viral electropherotypes. the modified page system was thus found to be reliable, simple and rapid, no expensive reagents were required. locally available reagents from hi media were used. the cost of the chemical for page per specimen was rs. 24 approximately as compared to rs. 110 per test by confirmatory elisa. a locally produced slab gel electrophoresis system with power pack was the only equipment required. this method could be used for the routine diagnosis of rotavirus infection in the laboratory. vaccine, rapid diagnosis plays an important role in early management of patients. in this study a qc-rt-pcr assay was developed to quantify chikungunya virus rna by targeting the conserved region of e1 gene. a competitor molecule containing an internal insertion was generated, that provided a stringent control of the quantification process. the introduction of 10-fold serially diluted competitor in each reaction was further used to determine sensitivity. the applicability of this assay for quantification of chikungunya virus rna was evaluated with human clinical samples and the results were compared with real-time quantitative rt-pcr. the sensitivity of this assay was estimated to be 100 rna copies per reaction with a dynamic detection range of 10 2 to 10 10 copies. specificity was confirmed using closely related alpha and flaviviruses. the comparison of qc-rt-pcr result with real-time rt-pcr revealed 100% concordance. these findings demonstrated that the reported assay is convenient, sensitive and accurate method and has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of chikungunya virus in acute-phase serum samples. in india, measles vaccine was introduced as part of expanded programme of immunization in 1985. measles, mumps and rubella (mmr) vaccine is still not part of the national immunization schedule of india. the indian association of paediatrics (iap) recommends measles vaccine at 9 months of age and mmr vaccine at 15-18 months. however, in a recent policy update, iap committee on immunisation opined that there is a need for a second dose of mmr vaccine for providing adequate immunity against mmr. the aim of the present study was to assess the extent of sero-protection against mmr at 4-6 years of age in children who have received one dose of mmr between 12 and 24 months of age. an attempt has also been made to assess the sero-response to the second dose of mmr vaccine in 4-6 years old children. a total of 106 consecutive children between the ages of 4-6 years who had received mmr vaccine between 12 and 24 months of age and attending the immunization clinic of gtb hospital, delhi were enrolled. the vaccination status, anthropometry and physical examination findings were recorded. three ml of venous sample was again withdrawn for estimation of post vaccination antibody titre. it was observed that 20.39%, 87.38% and 75.73% children were seroprotected for mmr respectively after 2.5-4.5 year of receiving first dose of mmr vaccine. seroprotection rose to 72.62%, 100% and 100% for mmr respectively after 4-6 weeks of receiving second dose of mmr vaccine. geometric mean concentration of antibody also rose significantly in all three diseases. in view of low seroprevalence of mmr and hence high susceptibility to infection at 4-6 years of age, who have already received mmr vaccine, there is need to boost the immune responses against these three diseases by giving a second dose of mmr vaccine. baseline information on the epidemiology of viral agents causing stis and types of risk behaviour of affected persons are essential for any meaningful targeted intervention. the present study documents the pattern of viral stis in patients attending a tertiary care hospital, correlating the syndromic approach and the laboratory investigations to determine the aetiology. three hundred consecutive patients attending the sti clinic were diagnosed and categorized according to the syndromic approach of the who along with detailed history and demographic data. majority of the patients were men (53.12%) with a mean age of 24 years. men received education up to middle school. half of the female subjects were illiterate. sixty percent of the patients were married and among these, 19% were regular condom users. first sexual contact at or before 18 years of age was more in men (31% vs. 22 .22% in women). promiscuity was more among male patients who had contact with csw. genital herpes was the commonest viral sti (86/300) followed by genital wart (60/300). concomitant infection with more than one virus was seen in 35% of patients. hiv was prevalent in 10.3% of sti patients. hepatitis b, hepatitis c, herpes simplex type 1 and molluscum contagiosum were the other viral agents seen in sti clinic attendees at our centre. this disease currently prevalent in more than 100 countries world wide and annually 50-100 million people are infected with dengue virus among which 2.5-5 lakhs cases were dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) which are serious forms of dengue virus infection and due to this condition 25,000 deaths might occur annually world wide and approximately 3 million children were hospitalized for the fast 3 decades. this disease is characterized by sudden onset of high fever with sever headache, pain in the back and limbs, lymphadenopathy macuolo-papulur rash over the skin and retro-bulbar pain. early diagnosis can be established with simple and rapid lgg/1gm antibodies detection in the blood samples of the patients based on the bi-directional immunoassay system for its management and control to reduce morbidity and mortality. details will be presented. myocarditis and dilated cardiomyopathy (dcm) are common causes of morbidity and mortality both in children and adults. the most common viruses involved in myocarditis are coxsackievirus b or adenovirus. recently, the coxsackievirus and adenovirus receptor (car), a common receptor for coxsackieviruses b3, b4 and adenoviruses 2, 5 has been identified. increased expression of car has been reported in patients with dcm suggesting utilization of car by these viruses for cell entry. the present study was designed to study the expression of car in myocardial tissue of patients with dcm. formalin fixed myocardial tissues were obtained from autopsy cases. a total of 26 cases of dcm and 20 cases of controls which included non-cardiac (group-a) and cardiac disease other than dcm (group-b) were included in the study. expression of car was studied by immunohistochemical staining of myocardial tissue using car specific rabbit polyclonal antibody and biotin conjugated secondary antibody. the tissue sections were considered positive when[25% of the cell showed brown color staining by immunohistochemistry (ihc). the car positivity in dcm cases was found to be 96% (25/ 26) as compared to 30% in control group a and 40% in control group b respectively. the car positivity was significantly higher in the test group as compared to both the control groups. further car positivity in all the cellular types (myocytes, endothelial cells and interstitial cells) was found significantly higher in test group as compared to both the control groups. the expression of car was significantly higher in myocytes as compared to both endothelial and interstitial cells in all the groups. however, no significant difference was observed in car positivity between endothelial and interstitial cells. the present study highlights the increased expression of car in dcm cases with further significance of car expression in myocytes and endothelial cells. this may help further in understanding the tropism of viruses or cellular susceptibility, which in turn will help in appropriate diagnostic and therapeutic approach in management of viral myocarditis and dcm cases. food security and safety vary widely around the world, and reaching these goals is one of the major challenges, raising public concern for the wellbeing of mankind, in particular. industrialized production and processing as well as improper environmental protection have clearly shown severe limitations such as worldwide contamination of the food chain and water. contaminated water and food during the processes of production, processing and handling are essentially responsible for food and water borne viral infections/diseases. the cases of viral food borne outbreaks are on the rise, creating a threat to human health. recent researches indicate that epidemiological studies are meager to focus the frequently contaminated foods and food borne viral diseases. current paper projects the etiology of select food borne viral diseases, probable reasons for non availability of appropriate methods to detect the viruses responsible for the diseases, routes of water and food borne transmission of enteric viral infections, currently available methods of detection of select viruses and bio safety measures to prevent food borne viral infections. dietary/nutritional management in food borne viral diseases is crucial to control weakness and gastro enteric intolerance due to disease condition and antibiotic therapy. it will principally improve food intake, resulting in better nutritional status leading to optimum immune response. food borne viruses are mainly belong to rotaviruses, enteropathogenic viruses, astroviruses, adenoviruses and caliciviruses, causes acute gastroenteritis (ag) which is an important health problem. the frequency of rotavirus as a cause of sporadic cases of ag ranges between 17.3% and 37.4%. astroviruses cause ag, with a frequency ranging between 2 and 26%: outbreaks have been described in schools and kindergartens, but also in adults and the elderly. the frequency of identification of adenoviruses 40 and 41 as causes of sporadic ag in non-immuno suppressed children ranges between 0.7% and 31.5%, although there is probably underreporting because the sensitivity of conventional techniques is low. caliciviruses are separated phylogenetically into two genera: norovirus and sapovirus. norovirus is frequently associated with food-and water-borne outbreaks of ag. it is estimated that 40% of cases of ag due to norovirus are food borne. in sweden and some regions of the united states, norovirus is the first cause of outbreaks of food borne diseases. sapovirus outbreaks due to person-to-person and food borne transmission affecting both children and adults have recently been reported in countries such as canada and japan. it has been predicted that the importance of diarrhoeal disease, mainly due to contaminated food and water, as a cause of death will decline worldwide. evidence for such a downward trend is limited. this prediction presumes that improvements in the production and retail of microbiologically safe food will be sustained in the developed world and, moreover, will be rolled out to those countries of the developing world increasingly producing food for a global market. sustaining food safety standards will depend on constant vigilance maintained by monitoring and surveillance but, with the rising importance of other food-related issues, such as food security, obesity and climate change, competition for resources in the future to enable this may be fierce. in addition the pathogen populations relevant to food safety are not static. food is an excellent vehicle by which many pathogens (bacteria, viruses/prions and parasites) can reach an appropriate colonization site in a new host. although food production practices change, the well-recognized food-borne pathogens, such as salmonella spp. and escherichia coli, seem able to evolve to exploit novel opportunities, for example fresh produce and even generate new public health challenges, for example antimicrobial resistance. in addition, previously unknown food-borne pathogens, many of which are zoonotic, are constantly emerging. awareness and surveillance of viral food-borne pathogens is generally poor but emphasis is placed on norovirus, hepatitis a, rotaviruses and newly emerging viruses such as sars. it is clear that one overall challenge is the generation and maintenance of constructive dialogue and collaboration between public health, veterinary and food safety experts, bringing together multidisciplinary skills and multi-pathogen expertise. such collaboration is essential to monitor changing trends in the well-recognized diseases and detect emerging pathogens. it is also necessary to understand the multiple interactions between these pathogens and their environments during transmission along the food chain in order to develop effective prevention and control strategies. to analyse the effectiveness of these sirnas targeting rabies virus l gene, the bhk-21 cells expressing sirnas in shrna form were produced by transduction of cells with radv-l. the transduced bhk-21 cells expressing sirna were infected with rabies virus pv-11 strain. there was reduction in rabies virus multiplication as analysed by reduction in fluorescent foci forming unit (ffu) count by 51.85% (70 ffu in bhk-21 cells expressing sirna-l compared to 135 ffu in bhk-21 cells expressing negative sirna). the expression of l gene mrna was reduced by 16.11fold in rabies virus infected radv-l transduced cells compared to radv-neg transduced cells (negative control) as detected using real-time pcr. after analyzing the effectiveness of radv-l in vitro, its effectiveness was also evaluated in vivo in mice after virulent rabies challenge. the mice were inoculated with 10 7 plaque forming units (pfu) of radv-l in masseter muscle (i/m route) and challenged with 15 ld 50 rabies virus challenge virus standard (cvs) strain. the results indicated 50% protection with improved median survival from 7 to 11 days compared with group of mice treated with radv-neg. the results of this study indicated that sirnas targeting rabies virus polymerase (l) gene delivered through adenoviral vector inhibited rabies virus multiplication in vitro and in vivo. and 4 were successfully produced and purified from the infected spodoptera frugiperda (sf-9) cells using these recombinant baculovirus. the morphology of the vlps was validated by electron microscopy in comparison to the authentic bt virions. the vlps produced here were stable and were highly immunogenic with intact outer layer which is rapidly lost during normal infection of btv. these btv-vlps elicited long lasting protective immunity in vaccinated sheep against virulent virus challenge. with the use of btv-vlps it was also possible to differentiate the infected and vaccinated animals (diva). vlp-based btv vaccine has potential advantages with regard to controlling the spread of btv with multiple serotypes. it is possible to produce milligram quantities of correctly folded and processed protein complexes using this baculovirus expression system and hence it is a more promising system for producing new generation vaccines like vlp subunit vaccine against any viral diseases in large scale. peste des petits ruminants (ppr), goatpox and orf are oie notifiable diseases of small ruminants especially goat and sheep. these diseases are economically important, in enzootic countries like india and cause significant loss and are major constraints in the productivity. considering the geographical distribution of ppr, goat pox and orf infections and prevalence of mixed infection, in the present study, safety and potency of the experimental triple vaccine comprising attenuated strains of thermostable-ppr virus (pprv jhansi, p-50) grown at 40°c, high passaged goat poxvirus (gtpv uttarkashi, p100) and attenuated orf virus (orfv mukteswar, p51) was evaluated in sub-himalayan local hill goats. goats simultaneously immunized with 1 ml of vaccine consisting of either 10 3 tcid 50 or 10 5 tcid 50 of each of pprv, gtpv and orfv were monitored for clinical and serological responses for a period of 3-4 weeks post-immunization (pi) and post challenge (pc). specific immune responses i.e., antibodies directed to pprv, gtpv and orfv could be demonstrated by ppr competitive elisa kit and capripox indirect elisa, snt, respectively following immunization. all the immunized animals resisted infections when challenged with virulent strains of either gtpv or pprv or orfv on day 28 dpi, while in contact control animals developed characteristic signs of respective disease. further, ppr viral antigen could be detected by using ppr sandwich elisa kit in the excretions (nasal, ocular and oral swab materials) of unvaccinated control animals after challenge but not from any of the immunized goats. triple vaccine was found safe at dose as higher as 10 5 tcid 50 and induced protective immune response even at lower dose (10 2 tcid 50 ) in goats, which was evident from sero-conversion as well as challenge studies. the study indicated that these viruses are compatible and did not interfere with each other in eliciting immune response, paving the feasibility of use of this triple vaccine in combating these infections simultaneously. toll like receptors (tlrs), primary sensors of microbial origin, plays a crucial role in the innate immunity. till now 13 mammalian tlrs have been identified, while there is no information available on tlrs of yak. this study is part of world bank funded-icar project. yak, named bos grunniens for its distinctive vocalization and relationship with cattle, is natural habitant of extremely cold environment. when these animals comes to a lower altitude grazing land, adjacent to villages, become susceptible to the diseases of cattle, buffalo etc. thus, present study was undertaken to with genetic characterization and evolutionary lineage analysis of yak tlrs. we worked on tlr7 gene, which plays an important role in recognition of ssrna viruses. total rna was extracted from mitogen stimulated pbmcs of yak. the rt-pcr conditions were standardized for full length amplification of tlr gene 7 using specific self designed primers. the expected amplicon of 3559bps was obtained. it was cloned in pgemt-easy vector followed by transformation in e. coli top10 strain. the recombinant clones were screened, picked up for plasmid isolation and release of tlr7 was confirmed by restriction digestion. the cloned tlr7 product was sequenced and analyzed for the nucleotide and deduced amino acid sequences, and 3d structure analysis. the results revealed that yak shows more than 98% sequence homology with other bos indicus breeds and bos taurus breeds. however, identity was less than 88% with other animal species (equine, murine, feline, canine etc.). the evolutionary lineage findings cluster yak more closely with bovine species. point mutations revealed changes at 25 nucleotide positions with corresponding amino acid change at 15 positions. smart analysis of yak protein domain architecture revealed toll-interleukin i receptor (tir), leucine rich repeats (lrr) and signal peptide region. the variations in yak mainly lie in the lrr region. homology modeling revealed horse shoe shaped structure with 5 alpha helix. the additional alpha helix present in bos indicus was not detected in yak. the present study shows existence of genetic variability in tlr7 gene of yak, in particular the lrr region, which plays an important role in the pathogen recognition and the evolutionary lineage analyses shows its closeness with other bovine species. a.p. aquaculture and fisheries, tirupati in this new millennium, aquatic animal health management strategies in asia expanded and adjusted to the current disease problems faced by the aquaculture sector. this presentation will briefly discuss some of the most serious trans-boundary pathogens affecting asian aquaculture including a newly emerging disease and highlight recent regional and national efforts on responsible health management for mitigating the risks associated with aquatic animal movement. a regional approach is fundamental since many countries share common social, economic, industrial, environmental, biological and geographical characteristics. capacity and awareness building on aquatic animal epidemiology, science-based risk analysis for aquatic animal transfers, surveillance and disease reporting, disease zoning and establishment of aquatic animal health information systems to support development of national disease control programs and emergency response to disease outbreaks are needed. molecular diagnostics with emphasis towards standardization and harmonization, inter-calibration exercises and quality assurance in laboratories, accreditation program and utilization of regional resource centres on aquatic animal health will also be needed. whilst most of these strategies are directed in support of government policies, implementation will require pro-active involvement, effective cooperation and strategic networking between governments, farmers, researchers, scientists, development and aid agencies, and relevant private sector stakeholders at all levels. their contributions are essential to the health management process. generally, aquaculture plays an important role in economy as harvests from natural waters have declined or, at best, remained static in most countries. fish and shrimp, the main aquaculture product sources, have gained the most attention. many factors can cause losses in yields of fish products and infectious disease in fish and shrimp is the biggest threat to the fishery industry. shrimp and fish aquaculture has grown rapidly over several decades to become a major global industry that serves the increasing consumer demand for seafood and has contributed significantly to socio-economic development in many poor coastal communities. however, the ecological disturbances and changes in patterns of trade associated with the development of shrimp and fish farming have presented many of the pre-conditions for the emergence and spread of disease. shrimp and fish are displaced from their natural environments, provided artificial or alternative feeds, stocked in high density, exposed to stress through changes in water quality and are transported nationally and internationally, either live or as frozen product. these practices have provided opportunities for increased pathogenicity of existing infections, exposure to new pathogens, and the rapid transmission and trans boundary spread of disease. not surprisingly, a succession of new viral diseases has devastated the production and livelihoods of farmers and their sustaining communities. this review examines the major viral pathogens of farmed shrimp and fish, the likely reasons for their emergence and spread, and the consequences for the structure and operation of the shrimp farming industry. in addition, this review discusses the health management strategies that have been introduced to combat the major pathogens and the reasons that disease continues to have an impact, particularly on poor, smallholder farmers in asia. btv isolates from the same geographic region have been termed as 'topotypes' and initial observation on segment 3 nucleotide sequences identified a correlation between topotypes and genetic information. later topotyping was proposed based on segment 10, on the premise that the encoding protein ns3, which is involved in virus egress from insect cells, would lead to evolutionary fitness in parallel with the geographic distribution of the different culicoides species. further studies attempted to extend this to nucleotide sequence homology in segments 7 and 10, but failed to identify clear cut correlations or any evidence for positive selection. for example, south african isolates were found not to cluster into separate african lineage. in this study, we carried out a more extensive analysis of segment 10 sequences. our analysis showed no segregation of isolates into topographically distinct groups. instead we observed topological clustering of the clades, and we attribute this to genetic bottleneck resulting in genetic drift and founder effect leading to homogenous gene pool in a geographical area. we hypothesize that when a new virus enters a geographical area where local btv strains are already circulating, the new genes/segments would enter into a bigger gene pool. consequently, the newer incursions into a heavily endemic area tend to get diluted and disappear from the population because the rate of drift is inversely proportional to the population size, unless they are positively selected. use of live attenuated vaccine in israel, europe, south africa and usa also led to more homogenous population similar to the vaccine strains due to continuous infusion of the vaccine type genes into the gene pool. we conclude that restriction of specific strains to certain geographical areas could generate uniquely imprinted genotypes which would not only indicate origin but also predict movement of viral strains to new areas. vvo-10 viral diseases of zoonotic importance: indian context k. prabhudas pd-admas, ivri, campus, hebbal, bangalore 24 zoonoses are generally defined as animal diseases that are transmissible to humans. they continue to represent an important health hazard in most parts of the world, where they cause considerable expenditure and losses for the health and agricultural sectors. the emergence of these zoonotic diseases are very distinct, hence their prevention and control will require unique strategies, apart from traditional approaches. such strategies require rebuilding a cadre of trained professionals of several medical and biologic sciences. the article discusses virus infections that have significant zoonotic implications for india. buffalopox is a contagious viral disease affecting milch buffaloes and rarely, cows, with a morbidity rate up to 80% in the affected herd. although the disease is not responsible for high mortality, it adversely affects the productivity of the animals, resulting in large economic losses. furthermore, the disease has zoonotic implications, as outbreaks are frequently associated with human infections, particularly in the milkers. the causative agent, buffalopox virus (bpxv), is closely related to vaccinia virus. the outbreaks of febrile rash illness among humans and buffaloes were investigated in the villages of districts solapur and kolhapur of western maharashtra. clinico-epidemiological investigations of humans and buffaloes were carried out and representative clinical samples were collected respectively. the samples include vesicular fluid, scab, and blood. laboratory investigations for buffalo-pox virus (bpxv) was done by pcr on blood samples, scabs and vesicular fluid. in vitro virus isolation attempts were carried out by using vero e-6 cells. negative staining electron microscopy was also employed for detection of virus particles. a total of 166 human cases with pox lesions on hand and other body parts from village kasegaon, district-solapur and 185 cases from 20 different villages of kolhapur district were reported. besides pox lesions patients were having fever, malaise, pain at site of lesion and axillary and inguinal lymphadenopathy. in kasegaon village, attack rate in human cases was 6.6% and in buffaloes 41.9% (231/551). whereas in kolhapur area attack rate in buffaloes was 11.75% (2633/22398). bpxv was confirmed in blood, vesicular fluid and scab specimens from human cases and scab specimen from buffalo by polymerase chain reaction (pcr) method. the bpxv was also isolated from 3 different clinical specimens and further identified by pcr and electron microscopy. clinical manifestation of the disease in buffaloes from solapur district was as reported earlier like common pox lesions on teats and udders whereas the buffaloes from kolhapur district had lesions on hairless parts of ears and on the eyelids with purulent discharge. bpxv from human and buffalo cases showed similarity. vaccines have been made against several diseases and used for controlling the afflictions. however a few of them were not effective for successfully controlling the disease. the reasons for the failure are many, the major being, either the pathogen is not completely cleared from the vaccinated animal or it reemerges after changing its antigenic structure, thus making the vaccination programme less effective. in addition to this, emergences of newer diseases such as hiv the development of suitable vaccines have become a challenging task. this is especially true in the case of viral diseases. these challenges have warned the researchers ''that protection by vaccination is not that simple and strait forward approach'', and lot need to be understood in terms of host virus interaction and role of environment in perpetuating the disease. so the immediate step that was considered was the environmental safety by way using non infectious materials as vaccines. with the understanding that has been developed in molecular immunology and molecular biology and with the availability of molecular tools that have been developed through recombinant dna technology the field of vaccinology has changed dramatically to emerge as modern vaccinology. this presentation deals with the modern approaches that are being used to produce effective vaccines in the case of foot and mouth disease of cloven footed animals. the similar approach may be worked out for other viral diseases also. despite the availability of an inactivated vaccine that is noted to provide solid immunity against the disease over a short period of time, the search for an ideal vaccine, the criteria for which are; safety of the vaccine for environment, easy in its preparation, does not require a cold chain for its storage, provides longer lasting immunity, economically viable and may be able to clear the virus in case of persistent infection is on. the advent of recombinant dna technology together with the information available on the molecular biology of viruses has enabled to design the development of newer vaccines that can induce strong cellular and humoral responses. the underlying principal in the present vaccine development strategy world over is the virus antigen gene has to be expressed in the tissue and the vaccine backbone has to trigger the immune system for eliciting desired immune response. bangalore campus of ivri has been vigorously pursuing research to develop ideal vaccines for foot and mouth disease keeping above principal in mind to achieve the previously mentioned criteria. the approaches selected are to see that the virus antigen/s replicate transiently in the host. the self replicating vaccines that have been developed are pox virus vectored vaccines, alpha virus replicase based vaccines and fmdv vectored vaccines. the approach and the result obtained so far will be discussed. silkworm, bombyx mori is affected with various diseases caused by viruses viz., nuclearpolyhedrosis (bmnpv), densosnucleosis (bmdnv) and infectious flacherie (bmifv). silkworm viral diseases form major constraints for the silk cocoon production in all the sericultural countries. the losses due to silkworm diseases is estimated about 20-40% and among them viral diseases are most common. in sericulture, prophylactic measures play a vital role in the management of silkworm diseases. these include disinfection of silkworm rearing house and appliances, rearing area, rearing surroundings, silkworm egg and body, and rearing bed disinfection associated with maintenance of general hygiene and personnel hygiene. all these activities are generally carried out as rituals by using general disinfectants often with partial success. recent trends in complete management of silkworm diseases include development of silkworm hybrids evolved from disease resistant/tolerant breeds, effective eco-and user-friendly disinfectants, anti-microbial feed-supplements and use of transgenic silkworms. biotechnological breakthrough in this regard is through rna interference (rnai) approach involving dsrna mediated nuclear polyhedrosis management and this is presently pursued by apssrdi, hindupur in collaboration with centre for dna fingerprinting and diagnostics (cdfd), hyderabad. nadu and karnataka. the disease appears to be more severe in rural flocks than organized farms. our investigations revealed the morbidity, mortality and case fatality rates among rural and organised farms as 9.34%, 2.69%, 28.84% and 6.22%, 0.47%, 7.63% respectively. higher morbidity and mortality in rural areas may be due to stress factors like poor nutrition, parasitic burden, fatigue due to long walks and non availability of veterinary aid. kulkarni et al. 1992 also reported the severe bt outbreaks in rural areas of maharashtra with overall morbidity, mortality and case fatality of 32%, 8% and 25% respectively. all the south indian sheep breeds were found to be susceptible and clinical farm of the disease is evident in all of them though saravanabava (1992) reported variations in susceptibility among the indigenous sheep. trichy black and ramnad white sheep were found to be more susceptible than the vambur and mecheri sheep of tamil nadu. prevalence of bluetongue in sheep, goat and cattle appears to be high in the region. serological surveys conducted in andhra pradesh during 1991 revealed the prevalence of btv antibodies in sheep (47.5%) goats (43.56%) cattle (33%) and buffaloe (20%). similar high prevalence of btv antibodies in sheep and goats were also reported from the other states in the region. clinical disease has not been recorded in kerala though btv antibodies were recorded in sheep (13.76%) and goats (7.10%) (ravi sankar 2003) . culicoides are the known biological vectors of btv. all the culicoides species are not capable of transmitting the btv. the occurrence of the disease is related to the presence of the competent vectors in the area. jain et al. (1988) established the involvement of the culicoides in transmitting the btv by isolating the virus from culicoides at haryana, the north indian state. c. imicola and c. oxystoma were found to be prevalent in andhra pradesh and tamil nadu. narladakar et al.(1993) reported the presence of c. schultzei, c. perigrinus and c. octoni in marathwada region of maharastra. culicoid vectors are significantly affected by the climate and annual variations in the climate reflects the outcome of the disease. the monsoon season (june to dec) with the temperature ranging from 21.2 to 35.6°c appears to be favourable period for the multiplication of culicoides. the maximum no of outbreaks were recorded during the north east monsoon period (oct-dec) followed by south west monsoon period (june to sep) in the region. however, details on the distribution of the competent vectors, feeding habits and their dynamics in the region is lacking multiple btv serotypes were found to be circulating in the region. (kulkarni and kulkarni 1984; janakiraman etal. 1991; mehrotra et al. 1996) a total of 10 serotypes viz. 1-4, 8, 9, 15, 16, 18 and 23 were identified based on the virus isolations. sreenivasulu et al. 1999 isolated btv serotype 2 from an outbreak of bt in native sheep of andhra pradesh. btv serotype 9, 15 and 21 were also isolated from the outbreaks occurred in andhra pradesh. some of the isolates need to be serotyped. deshmukh and gujar (1999) isolated btv type 1 from maharashtra. following is the summary of the distribution of btv serotypes in this region. clinical picture of bt in native sheep appears to be slightly different, the major difference being that swelling of lips and face was less conspicuous. mucocutaneous borders appeared to be very sensitive to touch and bleed easily upon handling. the classical signs of cyanosis of tongue and reddening of coronary band are not the common features of the disease in native sheep. the disease was also confirmed by the virus isolation and identification. clinical disease has not been reported in cattle, buffaloes and goats in spite of high seroprevalence. in conclusion bt is established in native sheep and causes severe economic losses to the farmers. the disease is concentrated in the southern peninsula of the country. the disease is seasonal and is associated with the rain fall. multiple serotypes appear to be circulating in this region. the btv serotypes were of virulent in nature as evident by severe outbreaks. s. janardana reddy*, d. c. reddy department of fishery science and aquaculture, sri venkateswara university, tirupati 517 502 in less than three decades, the penaeid shrimp culture industries of the world developed from their experimental beginnings into major industries providing hundreds of thousands of jobs, billions of u.s. dollars in revenue, and augmentation of the world's food supply with a high value crop. concomitant with the growth of the shrimp culture industry has been the recognition of the ever increasing importance of disease, especially those caused by infectious agents. in india viral diseases have become an important limiting factor for growth of shrimp aquaculture industry. although more than 30 different viral pathogens have been identified in different species of shrimp world wide, only a few viruses have identified which are causing disease problems in cultured tiger shrimps in india, east coast of andhra pradesh, in particular. diagnostic methods for these pathogens include the traditional methods of morphological pathology (direct light microscopy, histopathology, and transmission electron microscopy), enhancement and bioassay methods, traditional microbiology, and the application of serological methods. while tissue culture is considered to be a standard tool in medical and veterinary diagnostic labs, it has never been developed as a useable, routine diagnostic tool for shrimp pathogens. the need for rapid, sensitive diagnostic methods led to the application of modern biotechnology to penaeid shrimp disease. the industry now has modern diagnostic genomic probes with nonradioactive labels for viral pathogens like infectious hypodermal and hematopoietic necrosis (ihhnv), hepatopancreatic virus (hpv), taura syndrome virus (tsv), white spot syndrome virus (wssv), monodon baculo virus (mbv), and bp. highly sensitive detection methods for some pathogens that employ dna amplification methods based on the polymerase chain reaction (pcr) now exist, and more pcr methods are being developed for additional agents. these advanced molecular methods promise to provide badly needed diagnostic and research tools to an industry reeling from catastrophic epizootics and which must become poised to go on with the next phase of its development as an industry that must be better able to understand and manage disease. within this field, shrimp immunology is a key element in establishing strategies for the control of diseases in shrimp aquaculture. research needs to be directed towards the development of assays to evaluate and monitor the immune state of shrimp. the establishment of regular immune checkups will permit the detection of shrimp immunodeficiencies but also to help monitor and improve environment quality. for this, immune effectors must be first identified and characterised. in the end, however, the assumption may be made that the sustainability of aquaculture will depend on the selection of disease-resistant shrimp, i.e. to develop research in immunology and genetics at the same time. the development of strategies for prophylaxis and control of shrimp diseases could be aided by the establishment of a collaborative network to contribute to progress in basic knowledge of penaeid immunity. however, to improve efficiency, it appears essential also to open this network to complementary research areas related to shrimp pathology, physiology, genetics and environment. bluetongue is an important viral disease of sheep causing severe economic losses to the farmers. lack of effective vaccine is the major impediments in controlling the disease. multiple serotypes were found to be circulating in the state. attempts are being made to develop the vaccine employing the available serotypes to control the disease. hence, it is essential to identify the antigenic relationship among the serotypes to identify the candidate vaccine strains to be incorporated in the preparation of vaccine. reciprocal cross neutralization test was employed to find out the r% values between btv-2, -9 and -15 which indicated the extent of antigenic relationship between the serotypes. r% value between btv-2 and btv-9 was recorded as 2.8 r% value of 3.53 and 2.8 were observed between btv-2 and -15 and btv-9 and -15 respectively. the r% values recorded in the present study revealed a weak antigenic relationship between the btv serotypes. the extent of antigenic relationship between the btv serotypes was also determined by multiple sequence alignment of the nucleotide and amino acid sequences of the reference btv serotypes 2, 9 and 15. the sequence analysis of the vp2 gene revealed a homology of 47-53% and 29-41% at the nucleotide and amino acid levels respectively. r% values obtained using reciprocal cross neutralization test with the btv-2, 9 and 15 serotypes isolated in native sheep of andhra pradesh and the genomic analysis of the reference serotypes of btv-2, 9 and 15 revealed very weak antigenic relationship and were highly divergent. diseases especially those by viral pathogens cause greater economic losses in most horticultural crop species throughout the world as compared to agricultural crops. non-genetic methods of management of these diseases include quarantine measures, eradication of infected plants and weed hosts, crop rotation, use of certified virus-free seed or planting stock and use of pesticides to control insect vector populations implicated in transmission of viruses. however, none of these measures is likely to provide an enduring solution against these diseases especially those caused by viruses due sometimes to the huge expenditure involved, but mostly to the questionable effectiveness and reliability of those methods. as key control pesticides are getting increasingly abandoned, development of alternative methods to control diseases has been a felt-need in the recent past. though breeding for disease resistance generally provides a reliable security in a long run, introgression of host plant resistance did not materialise in most important crops. non-availability of an appropriate source of resistance in inter-fertile relatives, linkage to undesirable traits, or often times polygenic nature of such sources of resistance are the stumbling blocks in breeding programs. the limitations of conventional breeding and routine cultural practices prompted the need for the development of other approaches of virus control that could be fully incorporated into traditional methods. in this perspective, the concept of pathogen-derived resistance offers an attractive strategy to evolve newer methods of virus management, by transforming crop plants with nucleotide sequences derived from the pathogen's genome. an increasing number of molecular characterisation of plant virus genomes and the stable transformation of a number of horticultural crop species have in fact opened an avenue for molecular breeding against virus pathogens. successful field-testing of genetically modified crop cultivars renders proof of their supremacy over existing cultivars. it also contributes to demonstrate their capability with regard to environmental safety with a view to winning over public concern and scepticism. in general, the eventual commercialisation transgenic lines expressing virus resistance will rely upon a host of factors including their field performance, genetic stability, public acceptance and the resolution of environmental concerns and patent related issues. as such, elaborate field trials and allied studies are now required to adapt genetically engineered horticultural crops expressing virus resistance for their implementation into practical agriculture. a few examples from current research at tnau, in india or elsewhere will be discussed in this presentation. virology unit, division of plant pathology, iari, new delhi 12 in recent times there has been greater emphasis on vegetatively propagated crops in india to help diversify the indian agriculture. fruit, flower, spice and plantation crops are important vegetatively propagated horticultural crops, which have become a driving force for economic development in several parts of india. however, most of the vegetatively propagated crops are threatened by biotic stress caused by plant pathogens in general and plant viruses in particular. plant viruses produce specific and non specific symptoms and in some cases no symptoms are produced. correct identification and diagnosis of viral diseases is first step in the management of any disease including viral diseases. there have been two major breakthroughs in virus diagnostics during last four decades. the first one was serological assay using monoclonal or polyclonal antibodies in enzyme linked immunosorbent assay (elisa) and the other one was the use of in vitro amplification of dna in polymerase chain reaction (pcr). a significant development in serological assays has been its simplification in form of user's friendly quick strip/dip stick method. the one-step lateral-flow (lf) tests have been developed for the on-site detection and identification of several plant viruses. rapid advancement in virus genome characterization has led to the development of novel approaches of nucleic acid based diagnostics which include conventional pcr, real time pcr, multiplex pcr, micro/macro arrays and biochips. pcr protocols already exist for many plant viruses of citrus, banana, apple, papaya, vegetables, ornamental and spice crops. a further advancement has led to development of realtime pcr assay which is relatively easy but requires training for diagnosticians. in real-time pcr assays, results can be available within 20 min. the nucleic acid template preparation in pcr has been simplified. membrane based dna template protocol and co-isolation of nucleic acid template preparation are novel approaches in pcr detection of virus and virus like pathogens. since many of the horticultural crops are often infected by more than one virus, their individual detection by pcr is not only expensive but also time consuming. therefore, multiplex pcr has been developed where in genome of more than one virus could be amplified and detected in the same reaction mixture. development of nucleic acid based chip is now one of the fastest and recent growing areas in the field of pathogen detection. these nucleic acid based chips have been named as dna/rna chips, biochips, genechips, biosensors or dna arrays. when it comes to applications of microarray technology for plant viruses, it is not too difficult to see the value of a method that could potentially detect a whole range of viruses using a single test. however, microarrays are unlikely to become the only method in use in a diagnostic laboratory. processing of germplasm including transgenic planting material imported for research purposes into the country. during the last two decades, a total of 49,923 samples of wheat including transgenics were imported from cimmyt (mexico), icarda (syria) and many other countries. these were grown in post-entry quarantine nursery each year at nbpgr, new delhi and the transgenic samples were grown in national containment facility of level-4 (cl-4) since its inception to ensure that no viable biological material/pollen/pathogen enters or leaves the facility during quarantine processing of transgenics. in addition, post-entry quarantine inspections of the transgenic wheat grown by indenters are also undertaken by nbpgr quarantine scientists. virus-induced gene silencing (vigs) is a technique in which viral genomes are used, usually after appropriate modifications, for transient gene silencing in plants. the mechanism behind vigs is the phenomenon called rna-interference (rnai), which is widespread in many organisms and is believed to be form of inherent defence system against intracellular pathogens, such as viruses and transposons. double-stranded rna or rna containing strong secondary structures, commonly produced during viral infections, are believed to cause triggering of rnai, which employs a battery of proteins and nucleoprotein complexes to identify and degrade specific viral transcripts. in vigs, viral genomes not causing severe symptoms, but which can accumulate and spread efficiently in the host plant are used as vectors in which a host gene is cloned and introduced into the plant. upon replication, the viral vector triggers rnai response in the host plant, which also targets the host gene, leading to its silencing and subsequently, the silenced phenotype revealing gene function in vivo. vigs has been used extensively to study gene functions in dicot plants, such as tobacco, tomato, pea, soybean, etc., using vectors derived from reference genes are commonly used as an/the endogenous normalisation measure for the relative quantification of target genes. the expression (characteristics) of seven potential reference genes was evaluated in tissues of 180 healthy, physiologically stressed and barley yellow dwarf virus (bydv) infected cereal plants. these genes were tested by rt-qpcr and ranked according to the stability of their expression (characteristics) using three different methods (two-way anova, genorm and normfinder tools). in most cases, the expression (characteristics) of all genes did not depend on the abiotic stress conditions or on the virus infections. all the genes showed significant differences in expression (characteristics) among plant species. glyceraldehyde-3-phosphate dehydrogenase (gapdh), beta-tubulin (tubb) and 18s ribosomal rna (18s rrna) always ranked as the three most stable genes. on the other hand, elongation factor-1 alpha (ef1a), eukaryotic initiation factor 4a (eif4a), and 28s ribosomal rna (28s rrna) for barley and oat samples; and beta-tubulin (tubb) for wheat samples were consistently ranked as the less reliable controls. the bydv titre was determined in two oat varieties by rt-qpcr by three different quantification approaches. statistically, there were no significant differences between the absolute and the relative quantification, or between quantification using gapdh + tubb + tuba +18s rrna and ef1a + eif4a + 28s rrna. the geometric average of gapdh, 18s rrna, tuba and tubb is suitable for normalisation of bydv quantification in barley and oat tissues. for wheat samples, a combination of gapdh, 18s rrna, tubb, eif4a and e1fa is recommended. department of microbiology, yogi vemana university, vemanapuram, kadapa 516 003 large scale production and import of propagative material poses potential risk of introducing several destructive pathogens particularly viruses and mycoplasma like organisms in our country. this demands adequate quarantine safe guards such as growing them under approved post entry quarantine facility for specific period so as to facilitate virus detection, thereby curtailing risk. when such facilities are coupled with propagation by tissue culture will ensure virus free propagative plant material. the requirement of nationwide network of post entry quarantine facility working in close collaboration with crop institutions are very much emphasized for considering import of high risk plant genera for agriculture development. present paper discusses about virus disease of quarantine importance affecting ornamental and fruit plants such as chrysanthimum, dahlia, dianthus, rosabengalensis, cattleya, cymbidium, dendrobium, lilium, citrus, vitis etc. the paper also discusses on immunodiagnostic methods of detection and methods of obtaining virus free propagative material. rice tungro occurs as epidemics in regular cycles and has been reported in the last 50 years from all the major rice growing regions of india, especially prevalent in the southern and eastern states. development of the durable resistant varieties to tungro is crucial for the management of the disease. molecular breeding, involving the use of dna markers linked to the resistant gene(s) for selection, can overcome the difficulties encountered in conventional resistant breeding programs. for successful marker-assisted selection (mas), the identification of closely linked markers through the process of gene tagging and mapping is a prerequisite. attempts have been initiated for identification of tungro resistance genes through molecular mapping and their introgression into the target varieties using marker-assisted selection at drr, hyderabad. the inheritance of resistance to rice tungro virus disease was studied in seven resistant rice cultivars with field evaluation at hot spot locations. the microsatellite markers linked to rice tungro resistance in utri merah was studied and found that resistance genes were linked to rm 336 on chromosome 7. through molecular mapping two qtl were identified controlling rtv resistance on chromosomes 7 and 2 in 'utri rajapan' explaining 40.8% and 21.6% of the phenotypic variance. in variety 'vikramarya', another two qtl for rtv resistance were detected on chromosomes 7 and 1 explaining 18.7% and 16.4% of the phenotypic variance. the closely linked markers identified in this study flanking the gene of interest through mapping will improve the efficiency and precision of introgression programs in marker assisted breeding for rtv resistance. functional characterization of these qtl for rtv resistance is under progress. there is only a limited pool of natural virus resistance in cassava against cassava mosaic geminiviruses and cassava brown streak ipomovirus hence the development of transgenic resistance in this significant crop might present an option. rna mediated resistance through the expression of inverted-repeat dsrna sequences derived from the virus genome and the modification of plant microrna to produce antiviral artificial microrna are strategies that have recently been proven very effective for induction of virus resistance (immunity) against a number of rna viruses. results from rna interference strategies against geminiviruses never resulted in immunity of transgenes. however, it suggest that viral mrna are targets of rna silencing and that the success of the strategy depends on the relevance of the target gene in the systemic spread of the virus. we have generated a number of rna silencing constructs to induce resistance against cbsv and the indian cassava mosaic viruses icmv and slcmv. due to the serious problems inherent with transformation of cassava and subsequent resistance screening, these constructs were tested for efficiency either by transient-or by transgenic expression in n. benthamiana. complete immunity was reached in transgenic n. benthamiana against cbsv using inverted repeat or amirna constructs. using different species of cbsv for resistance screening, immunity was broken, to show the minimum context for broad spectrum resistance. similarly, highly specific resistance was reached in expression of amirna. in contrast, virus resistance against icmv/ slcmv using single amirna constructs was not successful. results from the experiments to generate virus resistance against cbsv and icmv/slcmv will be shown; methods to evaluate efficiency of rnai gene constructs by transient gene expression in n. benthamiana and strategies to develop efficient resistance against rna and dna viruses in cassava will be discussed. bitter gourd (momordica charantia l.) which is also called bitter melon, balsam apple and balsam pear belongs to family cucurbitaceae. it is an important traditional vegetable of nutritive and medicinal value that is cultivated in tropical and sub-tropical asia, but is considered as a weed host reservoir for viruses in jamaica. viral disease-like symptoms were observed occurring naturally on the crops of bitter gourd grown in the fields of northern india during 2007-2009. an incidence of 78.5% of diseased plants was recorded which showed chlorotic spots and mosaic ranging from mild mottling to green blisters along with leaf smalling, leaf and fruit deformations, bud necrosis and stunted growth whereas 20.2% plants exhibited leaf curling alone or in combination with mosaic-type disease. a reduction of 34.5% in fruit yield was recorded in mosaic-like disease which could be attributed to lesser fruit setting due to bud necrosis, smaller fruit size and stunted plant growth. such plants produced deformed, notched, irregularly shaped fruits wherein pre-mature yellowing and necrosis on the anterior and posteriors ends made 22.4% fruits unfit for marketability. the dwindling yield and production of unmarketable fruits posed a major constraint for profitable cultivation of this economically important crop, thus warranting for studies on etiology and management of these diseases. the mosaic-like disease was transmitted to healthy seedlings of bitter gourd at 2-leaves stage by sap inoculation as well as by aphid viz., myzus persicae sulz. and aphis gossypii glov. initially studies were carried out to optimize protocols for efficient plant regeneration and agrobacterium-mediated transformation for nagpur sweet orange, which is a popular and elite citrus cultivar in india. organogenesis was induced in etiolated epicotyl explants of one-month-old axenically raised polyembryonic seedlings by culturing them in mt medium supplemented with 30 g/l sucrose with varying concentrations of plant hormones. it was found that bap at 1 mg/l without auxin was best for efficient shoot regeneration in citrus using epicotyl explants. a 100% regeneration frequency was obtained and multiple shoot formation was obtained from both the cut ends of all the explants. an average of 8.24 well-differentiated shoots per explant were obtained, all of which rooted normally under the influence of 1 mg/l iba. this improved regeneration protocol was utilized in standardizing agrobacterium-mediated transformation of citrus using a. tumefaciens strain eha 105, containing binary plasmid pcambia 2301 that harbors gus reporter gene and npt-ii plant selection marker gene. one-month-old epicotyl explants infected with over-night grown agrobacterium (a 600 0.6-0.8) for 15 min and co-cultured for 3 days were found to be optimum for transformation as assessed on the basis of pcr analysis and gus activity displayed by the stem and leaf sections of putative transgenics. overall transformation frequency ranged from 38 to 48%. current study focuses on the generation of citrus transgenics for ctv resistance using a. tumefaciens strain eha 105 containing binary plasmid pbinar harboring portion of coat protein gene of ctv and npt-ii gene employing the standardized protocols. several putative transgenic shoots were recovered on selection medium and they are being utilized for molecular analyses and resistance against ctv. work is also in progress on the generation of citrus transformants using rnai construct harboring ctv cp and p23 genes, singly and in conjunction. our lab was also involved in developing rice transgenics for resistance against rice tungro disease, which is one of the most important and widespread virus diseases of rice in south and southeast asia, causing an annual estimated loss in crop yield of economic losses worth millions of rupees are caused due to these diseases annually. virus diseases are frequently less conspicuous than those caused by other plant pathogens and last for much longer. this is especially true for perennial crops and those that are vegetatively propagated. one further problem with attending to assess losses due to various diseases on a global basis is that what most of the data are from small comparative trials rather than wide scale comprehensive surveys, even the small trials do not necessarily give data that can be used for more global estimates of losses. this is for several reasons, including: (1) variation in losses by a particular crop from year to year; (2) variation from region to region and climatic zone to climatic zone: (3) differences in loss assessment methodologies; (4) identification of the viral etiology of the disease; 5 variation in the definition of the term 'losses' and (6) chilli is the major vegetable and spice crop grown in thar desert areas of rajasthan. leaf curl disease (chlcd) is one of the major constrains in chilli cultivation faced by farmers and cause yield loss up to 100%. a survey was conducted in major chilli growing areas of thar desert; bikaner, nagur, jodhpur and jalore districts of rajasthan during november, 2009 to understand the present status of leaf curl disease in chilli. among the four district surveyed for chlcd, the disease incidence was recorded maximum (up to 98%) in jodhpur district followed by jolore district (up to 88%). no relation was found between the disease incidence and varieties. the major varieties grown in these area are; mehsana, rch (mandoria), haripur raipur, mathania and local cultivars. the number of whitefly was also counted in top, middle and bottom leaf of chilli grown in these areas. the average number of whitefly per plant ranged from 0.0 to 4.0. more number of whitefly (4.0) was recorded in jodhpur district and lowest (1.8) in jalore district. total dna was extracted from three leaf curl infected samples from each district and tested for the presence of begomovirus using coat protein (cp) and dna-b specific primers. all the samples were positive for cp and dna-b amplifications by pcr. the cloning and sequencing of selected cp gene and dna-b fragments are in progress. the preliminary investigations shows that the leaf curl disease of chilli is widespread in the arid region of rajasthan and may be caused by begomovirus associated with satellite dna-b. bittergourd (momordica charantia) is an important vegetable crop of kerala. the crop is affected by several diseases of which mosaic is a prominent one. a field experiment was conducted to evaluate the efficacy of potentised resistance inducing substances (ris) viz., mosaic affected bittergourd plant tissue, ash of mosaic affected bittergourd plant tissue, plumbago and salicylic acid for control of bittergourd mosaic in march 2008. ris were applied as drench and foliar spray at three potency levels twice, before flowering of the crop. the experimental crop was grown as per the package of practice recommendations in split plot design with five replications per treatment. the disease incidence, disease severity and yield of the crop were recorded. the result of the experiment shows that spraying was more effective than drenching of treatments for reducing mosaic incidence and severity. among treatments, infected plant extract at 19 potency was the most effective one for reducing mosaic incidence and it showed the maximum incubation period and minimum disease severity. the spray application of treatments produced significantly higher yield than drenching. among the treatments, ash of infected plant at 19 and 309 potency and infected plant extract at 69 potency were on par and produced comparatively higher yield. elephant foot yam (amoprhophallus paeoniifolius), colocasia (colocasia esculenta) and tannia (xanthosoma sagittifolium) are the major edible aroids cultivated in india. the elephant foot yam cultivation is gaining importance due to its high production potential, nutritional and medicinal values and good economic returns. all these aroids are vegetatively propagated and viral diseases are spreading through planting materials. ctcri has the mandate of producing healthy planting materials of these edible aroids. accurate diagnosis and identification of the virus is essential for production of healthy planting material and effective management of the disease. though occurrences of viral diseases on edible aroids in india were known in 1960s, not much attention was given for detection and identification of the virus involved. in case of elephant foot yam 5-30% mosaic incidence was observed with varying symptoms of mosaic, puckering, filiformy etc. in colocasia and tannia, 5-10% incidence was noticed. rt-pcr amplification with potyvirus group specific primers and subsequent cloning and sequencing of the amplified product has confirmed the association of dasheen mosaic virus (dsmv) with all the three edible aroids cultivated in india. the complete full length coat protein gene of dsmv infecting elephant foot yam was cloned in pgem-t vector and sequenced. further sequence analysis revealed that the cp of dsmv consisted of 942 nucleotides and the 3 0 utr comprised of 260 nucleotides. blast and phylogenetic analysis showed highest similarity of 89% with that of dsmv isolate af048981, reported from usa. the deduced amino acid sequence of cp had 92.0-98.0% identity with other dsmv isolates. blast analysis of the partial cp gene sequences of colocasia and tannia also confirmed that the virus involved is dsmv. rt-pcr analysis of large number of samples from all the three crops confirmed that the potyvirus group specific primers (mj1 and mj2) are good for rapid detection of dsmv in these crops. dsmv specific biotinylated cdna and digoxigenin labelled crna probes were also prepared and dsmv in elephant foot yam was detected through nucleic acid spot hybridization. yellow leaf disease (yld) caused by sugarcane yellow leaf virus (scylv) is a recently recorded disease in india and is found wide spread throughout country. in popular varieties, the disease incidence varied from 0 to 75.0% and attained epidemic levels under field conditions. detailed studies on the impact of yld on sugarcane revealed that the virus infection significantly reduces various cane growth parameters, cane yield and juice quality. sequence comparisons of the coat protein (cp) and movement protein (mp) of 22 scylv isolates from india and database sequences showed a significant variation between indian isolates and the database sequences both at nt and aa level in the cp/mp coding regions. the significant variation in our isolates with the database isolates, even in the least variable region of the scylv genome showed that the population existing in india is different from rest of the world. further, comparison of partial sequences encoding for orf 1 and 2 revealed that yld in sugarcane in india is caused at least by three genotypes viz., cub, ind and bra-per, of which a majority of the samples were found infected with cuban genotype (cub). the genotype ind was identified as a new genotype and this was found to have significant variation with the reported genotypes. we have identified specific primers from cp region of the virus and optimized rt-pcr conditions to diagnose the virus. this assay has been found efficient in detecting the virus in asymptomatic plants and tissue culture derived seedlings. elimination of the virus through meristem culture has been demonstrated to purify the virus from the infected planting materials and this technique needs to be adopted to supply disease-free planting materials for effective management of the disease. studies are also in progress to identify the yld-resistant sources in sugarcane germplasm to initiate breeding for yld-resistance in sugarcane. mycoviruses are viruses that infect fungi. they have been identified in all major fungal families. in the present scenario, mycoviruses are the important means of biocontrol of plant fungal pathogens. most identified fungal viruses have double stranded rna genomes, often with more than one dsrna present per virus particle, and have been spherical in shape. these viruses are mostly vesicle bound, as other viruses have protein coatings. to be a true mycovirus, they must demonstrate an ability to be transmitted-in other words be able to infect other healthy fungi through anastomosis and spores. mycoviruses lead 'secret lives', reduce the ability of their fungal hosts to cause disease in plants. this property, known as hypovirulence (hypovirulence is a term used to describe reduced virulence found in strains of pathogens), this phenomenon was first observed in cryphonectria (endothia) parasitica (chestnut blight fungus) on european castanea sativa in italy, where naturally occuring hypovirulent strains were able to reduce the effect of virulent ones. these slower growing hypovirulent strains of c. parasitica contain a single cytoplasmic element of double-stranded rna (ds rna) similar to that found in mycoviruses that was transmitted by anastomosis in compatible strains through natural virulent populations of c. parasitica. hypovirulence has also been reported in many other fungal plant pathogens, including rhizoctonia solani, gaeumannomyces gramini var. tritici, ophiostoma ulmi, sclerotinia homoeocarpa, diaporthe ambigua alternaria alternata, and fusarium sp. etc. hypovirulence has attracted attention owing to the importance of fungal diseases in agriculture and the limited strategies that are available for the control of these diseases. it reduces the use of toxic fungicides which also affect the plant growth. the symptoms resulted by the mycoviruses are reduction in growth, reduction in pigmentation and sporulation, excessive sectoring and aerial mycelial collapse. these are the consequences of alteration in complex physiological and biochemical processes involving interaction between host and virus. cassava (manihot esculenta crantz.) is the major tuber crop in peninsular india, it is grown in an area of 2.4 lakh hectares with the annual production of 6.7 million tonnes both for direct consumption and the starch grain (sago) producing industries, mainly in the southern states of tamil nadu, kerala and andhra pradesh (fao 2005) . in tamil nadu, cassava primarily produced for sago producing industries where it is considered as an industrial crop rather than food crop, so the resource rich farmers are cultivating the cassava as irrigated crop in their fertile land and the poor farmers are raising the crop under rainfed conditions. in south india in addition to cassava there is a practice of intercropping important vegetable crops like, tomato, brinjal, legumes and gourds in cassava fields since all the above mentioned crops are short duration and are money spinners for the farmers. unfortunately, the major production constraint in these vegetable crops including cassava is the geminiviruses belonging to the family of in recent years there has been growing concern regarding the standard of scientific researches in india. the strengths, weaknesses, opportunities and threats (swot) analysis on indian scientific research reviewed the progress of science during the last six decades. although the 'strengths' were highlighted in good measure, it was the list of 'weaknesses' that called for attention to upgrade the standard of research and 'opportunities' that provide scope for overall scientific growth. a comparison between india and other countries in terms of research papers published revealed that india's contribution to science has come down enormously. what ails indian science? should we compare the growth of indian science with other developed countries? what criteria should be adopted to judge the quality and standard of scientific research? how to motivate the scientists to improve their scientific output? how do motivate the scientists to improve their scientific output? how do indian journals perform in maintaining quality? this paper analyses critically the scientific journals around the world, based on the scores allotted by the national academy of agriculture sciences (naas) in 2003 and 2007 for 1460 and 1608 journals respectively. in general, the indian journals performed poorly irrespective of the disciplines with only 25-30% in the high standard. the paper dealt with the reasons for low impact factor, the anomalies in the allotment of scores to wide spectrum of the journals and the disadvantages the scientists face with the scoring system. a case study was presented of an institute with over 50 scientists whose publications were analyzed to discuss the merits and demerits of the system. the performance of the journals published by prestigious academics, societies and councils was also projected. the paper concluded with the need for enhancing the image of the country through research publications in high standard journals and the role of various scientific bodies with shore and long term measures. poster session herpes simplex virus (hsv) keratitis is a leading cause of corneal blindness throughout the world. the infection can be diagnosed by clinical manifestations but in case of atypical ocular cases, laboratory diagnosis is more helpful in timely management of disease. collection of corneal scrapings in all cases of stromal and epithelial keratitis may not be possible, but collecting tear fluid is a convenient procedure causing less discomfort to the patients. therefore, the present study was intended to evaluate the suitability of tear specimens for detecting hsv by polymerase chain reaction (pcr) and immunofluorescence (ifa). tear fluid and corneal scrapings were collected from 134 patients of suspected herpetic keratitis. hsv-1 antigen was detected by ifa using rabbit anti-hsv antibodies. pcr was performed to amplify 111 bp region of thymidine kinase (tk) coding gene and 144 bp region from dna polymerase coding gene of hsv. out of 134 patients hsv antigen was detected in 25 (18.65%) of corneal scrapings and 15 (11.19%) of tear specimens and in 12 (8.95%) patients from both the specimens. hsv gene could be amplified in 44 (32.83%) of corneal scrapings and 16 (11.94%) of tear fluids and in 13 (9.71%) patients from both the specimens. although, corneal scraping seemed to be marginally superior material for detection of hsv, tear fluid may also serve as an appropriate alternative clinical specimen, due to ease of collection and least discomfort to the patients. in either cases pcr detected higher number of hsv cases than ifa. therefore if and when feasible, both ifa and pcr should be used simultaneously on each specimen to obtain best results. cytokines play a key role in the regulation of immune responses. in hepatitis c virus infection (hcv), the production of inappropriate cytokine levels appears to contribute to viral persistence and to affect response to therapy. il-6 is produced by a variety of cells including t cells, phagocytes and fibroblast. cytokine genes are polymorphic at specific sites, and certain mutations located within coding/regulatory regions have been shown to affect the overall expression and secretion of cytokines in patients with hcv infection. to correlate the serum levels and polymorphism of il-6 gene in chronic hepatitis c patients and healthy controls. forty patients positive for hcv rna attending the medicine out patient department and wards of lok nayak hospital, new delhi as well as forty healthy controls were enrolled for the study. the serum level of il-6 was detected by using elisa. genomic dna was extracted from whole blood of hcv infected patients and healthy controls by using accuprep genomic dna extraction kit according to manufacture's instruction. the genotyping of il-6 promoter (-174 variant) was carried out by pcr and direct sequencing using the method of patricia woo et al. 1998. the serum level of il-6 was significantly down regulated in hcv infected chronic patients as compared to the healthy controls. genotyping of -174 promoter variant of il-6 was performed by pcr and direct sequencing. il-6 polymorphism in the g/g, g/c and c/c allele was non significant when compared to hcv patients and healthy controls. the il-6 serum levels were significant among hcv infected patients when compared to healthy controls. the polymorphism in the promoter region of il-6 (-174) was found nonsignificantly associated in hcv patients compared to healthy controls. in conclusion, the present study suggests that the host il-6 polymorphism alone may not play a significant role in the outcome of hcv infection. acute gastroenteritis (age) is a global health problem and has been associated with multiple etiological agents, which include bacteria, protozoa and viruses. viral gastroenteritis is considered as the second most common illness in children after upper respiratory tract infection. among enteric viruses, rota, noro, enteric adeno, astro and enterovirus are found to be associated with gastroenteritis. although, association of enteric viruses has been established in children hospitalized for age no such data is available from hospitalized children other than enteric infections. to determine the prevalence of enteric viruses circulating in hospitalized children. fecal samples, n = 292 (177 symptomatic and 115 asymptomatic for age) were collected from children \5 year of age from three different hospitals across the city of pune from june 2008 to feb. 2009. detection of group a rotavirus was carried out by using antigen captured elisa. rt-pcr and pcr was carried out for the detection of norovirus, enterovirus, astrovirus and enteric adenovirus detection by using primers targeted to rdrp gene, 5 0 ncr gene and consevered gene for serine protease and hexon gene respectively. out of 177 fecal samples tested for enteric viruses in age cases, the prevalence of rota, entero, noro, enteric adeno and astrovirus were 33.3% (59), 14.7% (26), 6.2% (11), 2.8% (5) and 1.1% (2) respectively. however, the presence of these viruses in the asymptomatic cases (n = 115) was detected at 7.8% (9), 5.2% (6), 7.8% (9), 0.86% (1) and 1.7% (2) levels respectively. mixed infections of enterovirus and rotavirus were found in both symptomatic 1.6% (3) and asymptomatic cases 0.8% (1). however, mixed infection of enterovirus with adenovirus were found only in asymptomatic cases 0.8% (1). no marked difference was observed in the seasonal pattern of all viruses in the patients with or without gastroenteritis. the findings of this study document highest circulation of rotaviruses in patients symptomatic and asymptomatic for age. the entero and noroviruses remain second most important enteric viruses in these patients. influenza in humans is a major public health concern and the understanding of its evolution in the light of its ''antigenic drift'' helps prediction of epidemics and update of yearly influenza vaccine. to antigenically characterize influenza a (h3n2) isolates and study antigenic drift during 1990 to 2009 in pune city. patients with influenza like illness were identified using a strict case definition from dispensaries located in different areas in pune and clinical samples (ns/ts) were collected after obtaining informed consent. these clinical samples were processed in vivo (in fertile eggs) and in vitro ( overall, an additional 35 (39.7%) positive cases of dengue could be detected when ns1 antigen assay was also used in the study. highest ns1 antigen positivity was encountered among the samples collected on the 3rd day of fever whereas mac elisa for anti igm antibody was positive after 4th day and gradually there was an increase in the positivity towards the convalescent phase of the disease. the results of this study indicate that ns1 antigen based elisa test can be an useful tool to detect the dengue virus infection in patients during the early acute phase of disease since appearance of igm antibodies usually occur after fifth day of the infection. concurrent use of both diagnostic assays namely ns1 antigen as well as mac elisa will improve the overall detection of dengue infection. early detection of acute dengue virus infection is crucial to provide timely information for the management of patients. human parvovirus b19, a member of the parvoviridae family, is a pathogen associated with a wide variety of diseases. most commonly, it causes childhood rash erythema infectiosum, but in some cases more serious symptoms such as persistent arthropathy, critical failures of red cell production causing transient aplastic crisis, this infection in pregnancy causes hydrops fetalis and myocarditis. traditional immunosuppressive therapy being unsuccessful, anti-viral therapy might be worthy of consideration. functional annotation would provide role of viral proteome in its survival and pathogenic mechanisms. svmprot functional family annotations of vp2 protein had deciphered its zincbinding, coat protein, outer membrane, chlorophyll biosynthesis, dna repair and calcium-binding nature. vp2 protein is having a key role in viral assembly of b19 virus and being non-homologous to human proteome, it was identified as an attractive molecular target for structure based drug discovery. the vp2 protein crystal structure was energy minimized using charmm. a structure based virtual screening method was applied using ligandfit to identify potential inhibitors of vp2 protein from chembank database and ten potential human parvovirus b19 vp2 inhibitors were proposed. prism 310 genetic analyzer. the drafting of the sequences was performed using bioedit software and were submitted in genbank. for phylogenetic interpretation denv representing the full extent of genetic diversity in denv-1, denv-2 and denv-3 were collected from genbank. neighbor joining algorithm was implemented with bootstrap value of 10,000 replicates for phylogenetic inference using mega 4.0.2. the genomic region 134 to 644 (c-prm gene junction) of denv were amplified directly from patient serum. twelve of 72 samples were positive for dengue viral rna. of these 4 were dengue type 1, 1 was dengue type 2 and 7 were dengue type 3. for molecular epidemiological survey and genotyping of the sequences more than 100 sequences from different geographical areas including sequences form previously reported north indian isolates were compared with our present data set. the critical analysis of the sequences revealed: 4 dengue type 1 sequences were clustered within sub-type 2 of genotype iii and all the 7 sequences of den-3 clustered along with genotype iii. thus, among the dengue types 1, 2 and 3 currently circulating in north india, dengue type 3, genotype iii, being the predominant one followed by, genotype iii of dengue type 1. although there is no specific treatment or vaccine available currently, the confirmative rapid diagnosis based on detection of viral nucleic acid or igm antibodies in serum, an indication of recent infection, helps in epidemiological monitoring, symptomatic treatment of patients and determining prognosis. serological detection of anti-cgv igm antibodies was performed using rapid immuno-chromatographic assay (rica) and igm-antibody capture enzyme linked immunosorbant assay (mac-elisa). eighty convalescent sera were tested by rica and 60 of them were found positive for anti-cgv igm antibodies. twenty-five anti-cgv igm antibody rica positive sera were further assayed using mac-elisa. more sera from the patients are currently being tested to compare the sensitivity of these two serological assays in anti-cgv igm antibody based early serological diagnosis of cgv infection and the findings will be presented. thus the present study was designed to evaluate the utility of multiplex pcr (mpcr) for simultaneous and rapid detection of dengue and chikungunya viral infections. seventy-two acute phase blood samples from clinically suspected dengue cases were subjected for dengue and chikungunya uniplex pcr using dengue genus specific primers and e gene specific primers for chikungunya virus as well as multiplex pcr was developed for simultaneous detection of dengue and chikungunya infection. standard strains of dengue and chikungunya virus were used as controls. 13 of the 72 clinically suspected dengue samples were found to be positive for dengue viral rna by dengue uniplex pcr as well as dengue chikungunya mpcr whereas none of the samples were positive for chikungunya virus infection by both uniplex chikungunya pcr and dengue chikungunya mpcr. the result of dengue and chikungunya uniplex pcr was found to be 100% concordant with dengue chikungunya multiplex pcr. dengue chikungunya multiplex pcr was found to be a potential rapid test to detect dengue and chikungunya viral infections simultaneously in clinical samples. sheetal malhotra, neelam marwaha, karan saluja, ratti ram sharma department of transfusion medicine, pgimer, chandigarh 160012 transmission through blood and blood products can be reduced to a great extent by efficient and reliable testing of the blood. the newer fourth generation elisa assays simultaneously detect antibodies against hiv-1 and 2 and the presence of p24 antigen and thus shorten the window period to about 14 days, as compared to 22 days with third generation elisa. to compare the hiv seroprevalance among blood donors using fourth generation elisa (antigen-antibody) versus third generation elisa (antibody) assay. this was a prospective study involving 5100 blood donors of which 3400 were voluntary donors (1700 being students and 1700 being non students) and 1700 were replacement donors. sex workers are one of the core group for transmission of sti/hiv and as a ''bridge group'' to the general population. accordingly, highest priority is given to this group in targeted intervention for prevention of hiv/aids. here we are describing one such female sex worker who was harbouring 5 concomitant sti including 4 viral sti. a 25 year old female sex worker was brought to the sti clinic of a tertiary care hospital by ngo with complaint of genital discharge for 3 days. on per speculum examination, cervix was slightly erythematous, tender with mucopurulent discharge. there was no vaginal discharge or ulcer in anogenital area. however, there was a wart at lateral wall of vagina. as per naco syndromic management guideline, treatment was given for n. gonorrhoeae, c. trachomatis and hpv. cervical swab was taken and subjected to various microbiological investigation for the detection of sti viz n. gonorrhoeae, c. trachomatis, t. pallidum, candida spp., t.vaginalis, hsv-1, hsv-2, hiv, hbv, hcv, hpv and m. contagiosum. saline wet mount showed pus cells, but no yeast cells or trophozoite of trichomonas vaginalis. gram stained smear showed more than four polymorphonuclear leucocytes in the absence of gramnegative intracellular diplococci and a presumptive diagnosis of non gonococcal urethritis was made. no organism was isolated on any culture media after appropriate incubation. cervical swab was negative for antigen of c. trachomatis. serum was tested positive for hbv, hcv, hsv-2 and t. pallidum though it was seronegative for hiv. in the present case, the female sex worker was harbouring four viral sti viz hsv-2, hbv, hcv and hpv alongwith t. pallidum. however clinically she was diagnosed and treated accurately only for genital wart while cervical discharge due to hsv-2 was misdiagnosed. it is necessary to try to test alternative approaches such as periodic presumptive therapy of viral sti, because this will not only boost up the efforts of sti control in the target group but also help in hiv control. alternatively, regular clinical and laboratory screening for viral sti may be tried. densonucleosis viruses (dnv) belong to parvoviridae family. they are the etiological agents of insect's disease known as densonucleosis, which leads to death or loss of vital functions of the infected insect. densonucleosis virus of mosquitoes has generated lot of scientific interests because of its tremendous potential in biological control and its application as a transducing vector. earlier, we have reported the isolation and characterization of a dnv from aedes aegypti mosquitoes and its prevalence among different ae. aegypti populations from india. there are reports suggesting that when aedes albopictus mosquitoes co-infected with dengue-2 and dnv, the multiplication of den-2 is suppressed. the present study focus on the effect of coinfection of ae. aegypti mosquitoes with dnv and chikungunya virus (chik). the first instar mosquito larvae were infected with dnv and the emerging dnv infected females were then infected with chikv by oral feeding. thus obtained chik infected female mosquitoes were analyzed by real time pcr for both dnv and chikv on alternate days post-infection, up to the 14th day. the data showed no significant difference in the multiplication of either of the viruses after co-infection. results suggest that chikv neither stimulates the replication of dnv nor is its own replication suppressed due to co-infection. this study forms an initial step in understanding the role played by such endogenous viruses on the vector dynamics. chandipura virus pathogenesis is manifested as encephalitis in young children with a very high mortality rate. this damage could be due to direct replication of the virus in brain parenchymal tissue or immune system mediated. this study aims at elucidating the role of brain infiltrating lymphocytes in pathogenesis using mice as the model system. mice were inoculated intracerebrally with the virus and the perfused brain tissue was used to isolate the lymphocytes. control mice were inoculated with an equal amount of media. in order to standardize the procedure for isolation of lymphocytes from brain tissue, splenocytes were processed to isolate the lymphocytes using histopaque density gradient method. methods to isolate lymphocytes from brain tissue as described by earlier workers were tested for the ease and efficiency of procedure using known suspension of lymphocytes from spleen. percoll density gradient method provided optimum yield of lymphocytes with an ease of handling. in this, brain cell suspension used to prepare 30% percoll is layered over 70% percoll prepared using media in 1:2 ratio. density gradient centrifugation is carried out at 9009g for 20 min at 15°c to obtain lymphocyte layer at the interface. leishman staining was performed to analyze the morphological characteristics of isolated lymphocytes. normal lymphocytes showed dark blue stained nucleus. some bigger sized cells with diffused nucleus characteristic of atypical lymphocytes were observed and some of the cells were surrounded by hair like structures. phenotypic characterization was carried out using flow cytometry. the presence of cd4 + , cd8 + and cd19 + cells was observed. the percentages of cd8 + , cd4 + and cd19 + cells was found to be 7.60%, 35.14% and 34.32% respectively in the lymphocytes isolated from infected animal and 5.65%, 30.27% and 3.13% respectively from control animal. hence, cd19 + cells showed maximum infiltration after infection. (santosh et al. 2008; pradeep et al. 2008 ). in the present study chikv suspected blood samples were collected and the acute phase samples were subjected to rt-pcr for the presence of virus specific rna by using the primer pair dvrchk-f/dvrchk-r as described by us earlier (naresh kumar et al. 2007 ). the convalescent phase samples were screened for chikv specific antibodies by using sd bioline chikungunya igm rapid test. six sets of primers were designed to amplify the complete nsp4 and complete structural genes of chikungunya virus. the products were further gel purified, cloned in ptz57r/t vector and the recombinant clones were sequenced and submitted to the genbank. the complete ns4gene and structural genes were compared with other available sequences in the genbank. sequence analysis results will be presented. the present study discusses these aspects in detail. . some of these phages (viz. v953, v954) showed plaques at 42°c but not at 37°c. thus they seem to be lysogenic. for propagating and increasing the titre of all the above isolates, various previously described methods were attempted, but none of these methods were satisfactory. but when siliconized glassware and plastic-ware were used, propagation was successful. we showed that siliconization of glassware and plastic-ware was essential for the propagation of our mycobacteriophage isolates v951, v952, v953, v954 and v955. also, phage dilution medium (pdm) as described by chaterjee et al. (2000) was found to be effective for picking out of the plaques made by the phages. in this way, the phage isolates were propagated up to p 3 . the various passages of the phage isolates v951, v952, v953, v954 and v955 (i.e. original, p 1 , p 2 and p 3 ) were stored at -80°c. the four major routes of transmission are unsafe sex, contaminated needles, transmission from an infected mother to her baby at birth (vertical transmission) and breast milk. screening of blood products for hiv has largely eliminated transmission through blood transfusions or infected blood products in the developed world. in 2008, globally, about 2 million people died of aids, 33.4 million were living with hiv and 2.7 million people were newly infected with the virus. hiv infections and aids deaths are unevenly distributed geographically and the nature of the epidemics vary by region. more than 90% of people with hiv are living in the developing world. there is growing recognition that the virus does not discriminate by age, race, gender, ethnicity, socioeconomic status-everyone is susceptible. however, certain groups are at particular risk of hiv, including men who have sex with men (msm), injecting drug users (idus), and commercial sex workers (csws). the present study indicates the prevalence of hiv infection among the people residing in the northern region of india predominantly among the foothills of the himalayas. the study was carried out on the patients visiting herbertpur christian hospital (a unit of emmanuel hospital association) under the integrated counselling and testing centre scheme at the respective hospital during the 2009-2010. the study indicates the screening of people groups residing in the respective area through community health schemes. the diagnosis of the hiv infection is done by three types of assays namely. the tridot method which is the rapid method of diagnosis followed by the. hiv coombs test which involves the dot immunoassay principle. the third assay is the enzyme linked immunosorbent assay (elisa). the number of patients screened during the period of september 2009 to march 2010 is 635 which include patients coming from four different states namely haryana uttarakhand uttarpradesh and himachal pradesh. the number of people who were tested positive are 8 and the number of people who were tested negative are 627. the people tested positive are sent to the higher centre for other confirmatory tests such as pcr and western blot analysis. these patients are sent for treatment and prophylaxis at a respective recognised centre in dehradun. the present study determines a consistent community hiv screening and treatment approach through diagnostics counselling and awareness programmes. classical swine fever (csf) also known as hog cholera is a highly contagious and fatal disease of swine. csf became rapidly a major issue of pig industries. it still causes important economical losses worldwide. it is considered as a major health problem of swines in india. during the month of august to october 2009 there was an outbreak of classical swine fever in bihar. from three districts darbhanga, patna and supol, total 36 numbers of different infected tissue samples like kidney, spleen and lymphnode were collected from the dead morbid/pigs. total rna was isolated from 20% homogenate of infected tissues in sterile pbs by tri-reagent (sigma, usa) according to the manufacturer's instructions and cdna was prepared by using commercial available kit. the cdna was stored frozen at -20°c until used. for the molecular detection of classical swine fever virus specific nested pcr amplification of e2 and 5 0 ntr was done along with ns5b and e rns amplification. primarily these samples were found positive with these primers. further confirmation by sequencing was done by cloning of these pcr products in pgem-t easy vector. e2 and 5 0 ntr sequences were considered for phylogentic analysis along with 20 complete available sequences of csfv. nucleotide sequence alignments were carried out using the clustalw program (dnastar) and phylogenetic tree analysis (dnastar) showed that 5 0 ntr have close proximity with taiwan strain (accession no. ay568569) and e2 shows close proximity with chinese isolate csfv-39 (accession no. af407339). peste des petits ruminants (ppr) and sheeppox are oie notifiable diseases of small ruminants especially sheep and goat. both the diseases are economically important, in enzootic countries like india and are major constraints in the productivity of animals. considering the geographical distribution of both ppr and sheep pox infections and prevalence of mixed infection, in the present study, safety and potency of the experimental duel vaccine comprising attenuated strains of thermostable-ppr virus (pprv-revati, p-50) grown at 40°c and attenuated sheep poxvirus (sppv-srinagar, p40) was evaluated in local non-descript sheep. experimental animals were grouped into four groups and each group was comprising six animals, received 100 doses (10 5 tcid 50 ), 1 dose (10 3 tcid 50 ) and 1/10th dose of vaccines and normal saline as control in 1 ml volume subcutaneously, respectively. serum samples were collected on 0, 7, 14, 21 and 28th day post vaccination. sheep simultaneously immunized with 1 ml of vaccine consisting of either 100 or 1 doses of each of pprv and sppv were monitored for clinical and serological responses for a period of 3-4 weeks post-immunization (pi) and post challenge (pc). specific immune responses i.e., antibodies directed to both pprv and sppv could be demonstrated by ppr competitive elisa kit and capripox indirect elisa, respectively following immunization. all the immunized animals' resisted infection when challenged with virulent strain of sppv (srinagar isolate at p-6) on day 28 dpi, while in contact control animals developed characteristic signs of sheeppox. the challenge of the sheep against ppr was not carried out, however, the antibody titre after immunization determined by snt and elisa, indicated that protective titre, as per earlier report on the goats. dual vaccine was found safe at higher dose and induced protective immune response even at lower dose (10 2 tcid 50 ) in sheep, which was evident from sero-conversion as well as challenge study with sppv. the study indicated that both the viruses are compatible and did not interfere with each other in eliciting immune response, paving the feasibility of use of this dual vaccine in combating both infections simultaneously. goatpox is one of the highly contagious, oie notifiable and economically important viral diseases of goats. the disease is caused by goatpox virus (gtpv) is classified of the genus capripoxvirus in the family poxviridae. the disease incurs severe economic losses in terms of high morbidity in adults and heavy mortality in young kids and is a major constraint in goat farming in india. considering the enzotic nature and economic impact of the disease, it is all important to control the infection by developing an effective vaccine. recently, vero cell based a live attenuated goat pox vaccine; using gtpv uttarkashi isolate (p60) has been developed in authors' laboratory and evaluated in goats. the vaccine was found safe, potent and immunogenic experimentally and even at field trials. the vaccine has been evaluated at large-scale at different regions of the country and found suitable for mass vaccination. however, the longevity of potency was not evaluated. therefore, a long term potency trials were studied for a period of 4 years with annual challenge by using virulent goatpox virus and sero-monitoring. a sufficient number of hill goats has been vaccinated with 1 dose of vaccine (10 2.5 tcid 50 /ml) and monitored for clinical and serological response. every year, significant number of vaccinated (n = 5) and control animals (n = 2) were used for challenge with virulent strain (2 9 10 7.0 srd 50 /ml, gtpv mukteswar). sera of pre-and post-challenged (14 dpc) animals including controls have been collected and monitored for serological response in the form of specific antibody production by snt and indirect elisa. all the vaccinated animals were protected on challenge, whereas, all unvaccinated controls developed infections. the same has been reflected in sero monitoring of collected sera. so the developed live attenuated goat pox vaccine was found safe, immunogenic and potent for a period of 4 years of immunization and suitable for mass scale vaccination in control and eradication of goat pox along with a/are suitable diagnostic tool/s in goatpox enzootic country like india. rotavirus infection in avian species varies from subclinical infections to outbreaks of diarrhea. the economic significance of rotaviral enteritis to the poultry industry has not yet been defined, but by analogy to the situation in mammals, it is likely to be significant. unlike the extensive studies performed on rotavirus infection in humans and animals, limited studies have been carried out to determine the extent of exposure of poultry birds to rotaviruses. to determine the prevalence of avian rotavirus antibodies in commercial broiler chickens. a total of 120 chicken serum samples were collected from the lairage of a poultry slaughter house where birds from four different broiler farms in and around pune city were supplied to. the serum samples were tested by an igg antibody capture elisa wherein purified chicken rotavirus ch2 was used as coating antigen. sera from specific pathogen free (spf) chick (n = 20) served as negative control in the test. cut off was calculated as mean negative control ? 3sd (standard deviation). s/co (mean sample od 450 /cut off) values above 1 (1.113-4.445) in 60% (72/120) serum samples were indicating positivity to rotavirus antibodies. the result of the study indicates exposure of the birds to avian rotavirus or similar agent that is circulating in pune city. bluetongue has become established in south india causing regular outbreaks in sheep. btv serotypes 2, 9, 15 and 21 were isolated from native sheep of andhra pradesh. the other serotypes circulating in the state need to be identified. however the major constraint is the serotype identification. to overcome the difficulties of traditional serotyping methods (neutralization tests), nucleic acid based tests are being tried. rt-pcr for serotyping was standardized using primers specific to vp2 gene of btv-2, 9 and 15 serotypes. rt-pcr resulted in 653 bp product of btv-2, 1241 bp product of btv-9 which was defined by specific primers. however non specific amplification at two different sites i.e. 700 bp and 1500 bp was noticed for btv-15. specificity of rt-pcr was evaluated. btv-2 and btv-9 specific primers could amplify only btv-2 and btv-9 respectively where as btv-15 type specific primers amplified not only btv-15 but also btv-2 and btv-9. nucleic acid sequence data obtained from btv-2 pcr product and btv-9 cloned products were specific to vp2 gene of btv-2 and btv-9 respectively. however, 700 and 1500 bp products of btv-15 were identical to vp 4 gene of btv-2, 8, 10, 11, 13 and 18 and vp 1 gene of btv-2, 8 and 10 respectively, indicating the non specific amplification of btv-15. foot and mouth disease is the most contagions and highly economically impotent disease of cloven footed animals. the disease is controlled by regular vaccination using the vaccine produced from the virus grown in the cell culture. the vaccine strain used for vaccine production is selected from the field isolates based on the adaptability and growth kinetics in bhk21 cells and antigen coverage. however the field viruses need to be passaged several times to adapt in tissue culture. passage of field viruses in tissue culture may results in development of mutants whose genetic makeup may differ from the field samples also some of the field strains may fail to adapt or may grow poorly in the tissue culture, thus the efficiency of the vaccine gets affected. structural proteins of fmdv carry the sequences which determine the serotype specificity and immunogenicity. thus one may replace the gene coding for structural proteins from the full length cdna copy of the vaccine strain that has been adapted to the tissue culture with the poly-structural protein gene (pi) so that the chimeric virus gets the serotype specificity of the field strain besides retaining the other characteristics that are needed for a vaccine virus. we have made replication competent fmdv asia i full length genome and cloned under t7 and cmv promoter separately in plasmid vectors. bam h1 sites were created for inserting pi-2a gene of other field strains. the p1-2a of type 'o' vaccine strain was amplified directly from the cattle tongue material, cloned in plasmid vector and studied the specificity by sequence analysis and gene expression. we have introduced 'o' p1-2a gene into the full length construct devoid of asia 1 structural protein gene, p1-2a. the in vitro transcribed rna in case of t7 promotered construct and plasmid dna in case of cmv promotered construct were transfected into the bhk21 cells. after the passaging the virus obtained was studied for the speciality. this approach may be used not only for rapid selection of vaccine strain and also as a repository of the cdna copy of the virus. the p1 is composed of 1a, 1b, 1c and 1d (vp4, vp2, vp3, and vp1) respectively of which the vp1 is the most immunogenic and subunit vaccine produced with vp1 alone was able to induce high level of neutralising antibodies. thus to control the disease in india polyvalent vaccine consisting of the inactivated virus of all the three serotypes are in use. however the conventional vaccines have several drawbacks which include safety and temperature sensitivity. hence alternatively sub-unit vaccines consisting of vp1 protein has been tried. however this showed limited success due to the antigenic variations occurring in the field viruses thus escaping the neutralization from the antibodies generated from single cloned protein. hence the present study was undertaken with an objective to include all the neutralizing epitopes present in the three serotypes by linking vp1 (1d) genes and produce a poly valent protein for using as poly subunit vaccine. in this study we have constructed a cassette by linking the genes of three serotypes 'o' (622 bp), 'a' (640 bp) and 'asia 1' (622 bp). these genes were cloned individually in commercially pbsk vector and confirmed by sequence analysis before linking in pc dna vector. the linked gene construct was sub-cloned in pet32 expression vector. the expression of the protein gene from the pet vector was induced with iptg and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page). a fusion protein of size 72 kda was observed in page gels. since the protein contains 6 his residues from the vector at the n-terminal end, affinity purification was carried out using nickel nitrilo-tri-acetic-acid (ni-nta) agarose matrix. the immunoreactivity of the purified protein was assayed by western blot with the anti fmdv type 'o' and 'asia 1' specific sera. the may be used as a subunit vaccine. silkworm diseases caused by viruses, bacteria, fungi and protozoans form major constraints for the silk cocoon production in all the sericultural countries and among these silkworm viral diseases viz., nuclear polyhedrosis and infectious flacherie caused by bmnpv and bmifv cause severe crop loss. the traditional disease management strategies include prophylactic measures and use of disease free silkworm eggs. the prophylactic measures such as disinfection of silkworm rearing house and appliances, egg surface, silkworm bed disinfection and rearing surroundings. the disinfectants used presently in sericulture are either formaldehyde or chlorine based products, but these chemicals are neither eco-nor user-friendly. the awareness about health hazards caused by formaldehyde and environmental pollution caused by cl 2 necessitated the development of eco-and user-friendly disinfectant products for use in sericulture. these include alternative disinfectant products developed using biodegradable chemicals and plant based ingredients by apssrdi, hindupur and central silk board for the management of silkworm diseases in india. the ideal disinfectant for sericulture would be the one which can inactivate silkworm pathogens of diverse origin and economical for sericulture. the paper discusses on the disadvantages of hcho and cl 2 based disinfectants and advantages of eco-and user-friendly disinfectant for the management of silkworm diseases especially the ones caused by viruses. the baculovirus expression vector system (bevs) is widely used for the production of high levels of properly post-translationally modified, biologically active and functional recombinant proteins and has facilitated basic biomedical research on protein structure, function, drug discovery and the roles of various proteins in disease. bevs is based on the introduction of a foreign gene into nonessential for viral replication genome region via of homologous recombination with a transfer vector containing target gene. the resulting recombinant baculovirus lacks one of nonessential gene (polh, v-cath, chia etc.) replaced with foreign gene encoding heterologous protein which can be expressed in cultured insect cells and insect larvae. insect cell-bev system is widely used to produce recombinant proteins. bevs also eliminates concerns regarding pathogens that could potentially be transmitted to humans as it is non-infectious to vertebral animals. these features make silkworm system an ideal expression and delivery package for producing proteins of medicinal importance. the efficiency, low cost and large-scale production of proteins using bevs represents breakthrough technology that is facilitating highthroughput proteomic studies. the bevs has become a core technology for cloning and expression of genes for study of protein structure, processing and function; production of biochemical reagents; study of regulation of gene expression; commercial exploration, development and production of vaccines, therapeutics and diagnostics; drug discovery research; exploration and development of safer, more selective and environmentally compatible biopesticides. utilization of silkworm larvae and pupae as bioreactor with recombinant bmnpv producing foreign proteins extends the usages of silkworms. due to its large-size and high protein synthesis ability as well as the expediency in mass culture, silkworm is considered as good candidate for producing recombinant proteins. wssbv is the causative agent of a disease, which has recently caused high shrimp mortalities and severe damage to shrimp culture. wssbv has been found across different penaeid shrimp species. in order to develop a effective diagnostic tool, a wssbv genomic library was constructed by cloning wssbv genomic dna extracted from purified virions. in the present study wssv disease free (confirmed by pcr analysis) were collected from hatcheries from different areas of guntur and prakasam districts and analysed to study the effect of various physical parameters like temperature, p h , salinity and turbidity on the prevalence of above disease. the studies on the surface water temperature revealed fluctuations in the ponds ranging between 19 to 30.2°c in diseased ponds and 25.2 to 34.5°c in healthy ponds. these results show definite influence of temperature on the prevalence of wssv. present day strategy in vaccine development is to include marker facility that helps in distinguishing antibody response due to vaccination vis-à-vis infection in vaccinated animals. such information becomes relevant for effective disease control programmes especially when using inactivated virus vaccines like foot and mouth disease (fmd). the antibodies generated in the animals, only through vaccination, is the measure of vaccine efficacy and safety. presently inactivated fmd virus (fmdv) vaccines are used to control the disease in the endemic countries like india. the quality assurance of the vaccine depends on the efficacy of the vaccine in generating protective antibody without causing subclinical disease due to improper inactivation. since protective antibody response in vaccinated animals can not be distinguished from that of infected animals one needs to assay the antibody response against non structural proteins (nsps) and the vaccine must be free of contaminated nsps. production of vaccine free of nsps requires the cumbersome method of virus purification which adds to the cost of the vaccine. alternatively one may develop a positive marker vaccine by including a foreign protein or epitope which is not expected to be present in the vaccine and the antibodies generated against which helps in detecting the vaccine related response. here we report a molecular approach by which we introduced a immuno-dominant epitope of green fluorescent protein (gfp) into the structural protein gene of foot and mouth disease virus vaccine strain asia 1 (63/72). our laboratory has produced a mini-genome of fmdv asia 1 that lacks structural protein gene (p1-2a) coding for all the structural proteins (vp1-4) of fmdv asia 1 as a vector (pcfl dasia 1). the p1-2a of the asia 1 vaccine strain was cloned separately into a plasmid vector and by successive pcr mutagenesis and cloning we have introduced nucleotide sequence corresponding to 9 amino acid epitope of gfp into p1-2a gene. gfp epitope was inserted by replacement at n-terminal region of vp-2 which is not immunogenic. the modified p1-2a was expressed in e. coli and studied. the modified p1-2a gene with gfp epitope was inserted into the pcfl dasia 1 to get full length replication competent cdna cloned under cmv promoter in pcdna (pcflasiagfp). this can be used to produce synthetic virus with gfp epitope that can generate antibodies not only against neutralizing epitopes but also against gfp epitope. presence of antibody against gfp epitope in the vaccinated animal will reveal vaccine efficacy. elisa against gfp can be used as a companion test not only for safety evaluation but also for quick evaluation of efficacy. further absence of nsp antibodies in the serum may reveal the quality of the vaccine in respect of safety. self replicating dna vaccines are developed to achieve robust immune response through enhanced antigen production and gamma interferon expression in vaccinated animals. since self replicating dna vaccines induce gamma interferon expression which helps in viral clearance such vaccines are expected to be useful to cure even the carrier and persistently infected animals. understanding the events that help in the elicitation of both the arms of immune response in vaccinated animals is necessary to understand the effectiveness of the vaccine. the work presented here deals with the immunological evaluation of a sindbis virus replicase based dna vaccine carrying linked fmdv vp1 genes in vaccinated guinea pigs. we have constructed self replicating dna vaccine vector and to the down stream of a sub genomic promoter we have inserted secretory signal followed by linked-vp1 genes of 3fmdv serotypes (o-a-asia 1) with glycine and proline bridge in between. guinea pigs were vaccinated with the construct and the sera at 28 days post vaccination were evaluated both for cellular response by studying the cd8 levels and by mtt and cytokine profiles by real time assays. the humoral response was evaluated by studying cd4 levels in the whole blood by facs analysis and serum antibody levels by snt and elisa. the animals were challenged with 100 gp infective dose of fmdv type 'o' virus lesions were scored. further the replicative efficiency of the challenge virus was studied by 3ab elisa. the results showed that all the assays except antibodies against 3ab protein have positive correlation with the protection. as expected the titre of the antibodies against 3ab protein was lower indicating that the challenge virus replication was inhibited in the vaccinated animals. the limited studies conducted by us showed that self replicating vaccine has a potentiality to emerge as potent vaccine for fmd. ganjam virus (ganjv) belongs to the genus nairoviruses (family bunyaviridae). these viruses cause diseases in livestock. it has been isolated from different animal hosts and tick vectors from india. genus nairoviruses includes a total of 34 tick-borne viruses, classified into 7 serogroups. the important serogroups are crimean congo hemorrhagic fever (cchf) and the nairobi sheep disease (nsd). the main members of the nsd group are nsd and dubge viruses. their genome consists of three segments of single stranded rna, viz. s, m and l that encodes viral nucleocapsid protein, viral glycoprotein g1 and g2 and the viral polymerase respectively. ganjv is very closely related to (nsdv). nsdv is found in east and central africa, causes very high morbidity and mortality in livestock. the present study involves phylogenetic comparison of ganjv isolates from india with other nairoviruses based on complete n gene. it will help to understand the kind of nucleotide (nt) and amino acid (aa) changes that have occurred in ganjv strains from different geographical areas. eight strains of ganjv isolated at niv during 1954-2002 from different parts of india were used in this study. virus stocks were prepared in vero e6 cell line these were used as the source of viral rna. the n gene was amplified either as a complete gene in one reaction or in fragments whenever necessary. thus obtained sequences were analyzed; annotated to get a consensus sequence, aligned against the sequence of prototype strain of ganjv and other representative nairoviruses. the nt sequences were converted to aa sequences and analysis was done at both nucleotide and amino acid levels. based on what nt or aa phylogenetic tree was constructed and compared with other nairoviruses (cchf, dugv, hazv, kupv and nsdv) where complete s segment sequences were available gen-bank database (ncbi). the phylogenetic data at both the nt and aa levels showed that all the strains of ganjv form monophyletic lineage with the nsdv. cchfv and hazv together formed another clade, whereas dugv and kupv made a separate branch in the tree. the different ganjv strains showed 9-10% difference with nsdv at the nucleotide level and 3-4% difference at the amino acid level. hazv showed 37-38% difference at the nt level and 37% difference at the aa level with ganjv as well as nsdv. the present data obtained suggests that ganjv and nsdv are minor variants of the same virus. diarrhoeal syndrome is one of the major concerns of the livestock industry. most of the diarrheic cases in animals go unnoticed and limited attention is paid on viral etiology. presence of large amount of fecal matter in animal shed acts as a source of infection for calves via drinking water, feed, or contaminated soil. keeping this in view, investigation was planned to detect the association of rotaviruses with diarrhea in dairy calves and to observe the genomic diversity among the circulating viruses in tarai area of uttarakhand. a total of 63 diarrheic fecal samples collected from instructional dairy farm, nagla, pantnagar, uttarakhand were screened during the study. samples were collected from both cow calves and buffalo calves in 0-3 months of age. for the diagnosis of rotavirus, all the fecal samples were subjected to rna-electrophoresis after nucleic acid extraction. viral genome segments were visualized by silver staining. out of the total 63 samples tested, seven were found positive in rna-page showing typical 11 genome segments migration pattern of bovine rotavirus. in the given samples prevalence of bovine rotavirus was 11.32% and 10% in cow and buffalo calves, respectively. on the basis of migration patterns of rotavirus in rna-page, group a were identified with typical 4:2:3:2 pattern. variation within movement of various genome segments among isolates of bovine rotaviruses was observed during the study that may be indicative of emergence of mutants in the circulating isolates. the vp6 gene based group a specific rt-pcr was standardized and all the isolates in this area were confirmed to be of group a type. work is in progress to genotype the bovine rotaviruses of this region based on vp7 and vp4 genes. this study emphasizes the need to explore the prevalence of bovine group a rotaviruses in different places of uttarakhand and their genetic characterization which could help in selection of control strategies for rotavirus infections. foot-and-mouth disease (fmd) is endemic in india causing enormous economic loss to the animal keepers and trade embargo with fmd free countries in livestock and animal products. rapid diagnosis of fmd is of immense importance in prevention and control of the disease. fmd is initially diagnosed clinically and confirmed by laboratory tests. virus isolation in cell culture and sandwich elisa for antigen detection are commonly practiced in laboratories. the virus isolation though is very sensitive but it can be slow and analytical sensitivity of the elisa is lower and can not be used with certain sample types. the use of molecular techniques in the diagnostic laboratory has greatly increased the speed, specificity and sensitivity of fmd diagnostic tests. molecular techniques like rt-pcr, pcr-elsa and dot hybridization can be used with more success for detecting carrier animals and animals harboring sub-clinical infection and can be applied in a wide range of clinical sample types. these techniques can be used as genus and serotype specific test including detection of particular lineage/genotypes with in the serotype. multiplex pcr has been used to differentiate serotypes of fmdv and the technique is sensitive, experimentally simpler, cost effective and less time consuming. the assay can be used for serotyping on elisa negative samples. the molecular techniques not only help in diagnosis but also useful for epidemiological studies. lineage differentiating rt-pcr has been useful in identifying different lineages of serotype asia 1 (lineage b, c and d) before proceeding with sequencing of 1d region. similarly genotype differentiating rt-pcr has been developed and used in differentiating two different genotypes of serotype a (genotype vi and vii). these assays have the potential to be applied on clinical samples directly, thereby saving much time needed for sample processing and nucleotide sequencing. recent development of real time rt-pcr methodology has allowed the diagnostic potential of molecular assays to be realised. advancement in real time pcr technology made it possible to combine several assays within a single tube which is in the progress in our laboratory. integration of these assays onto automated high throughput platforms provides diagnostic laboratories with the capability to test large numbers of samples. microarray technology was provided greater screening capabilities for pathogen detection. the microarray allows the addition of large number of oligonucleotide probes for identification of mutant pathogen and also for subtype determination. the combined properties of high sensitivity and specificity, low contamination risk, and speed has made realtime pcr and microarray technology a highly attractive alternative to conventional methods in increasing percentage of outbreaks confirmed and analyzed and for tracing the origin of fmd virus responsible for outbreaks. dna vaccines are expected to elicit both humoral and cellular responses, cellular response being long lasting. however the approach has several limitations like poor stability of dna, poor expression and risk of integration. poor expression becomes the major limitation in the case of fmd as fmdv proteins are poor immunogens. also dna vaccine vectors carrying only eukaryotic promoters elicit strong cmi response and weak humoral response. the methodology to achieve humoral response involves the expression and secretion of the expressed protein so that the antigen presenting cells will be able to process the antigen and produce humoral response. in case of fmd humoral response is as important as cellular response. the present project aims at addressing these issues; achieving higher expression and getting the protein secreted out by constructing self replicating gene vaccines for fmd and studying their efficacy. the vector for humoral immune response contains eef1 promoter, sindbis virus polymerase gene and secretory and anchoring signals. the integrity of the vectors was confirmed by sequence analysis. the linked polyvalent protein genes of fmdv serotype a, o and asia 1 were cloned into the vectors and the presence of the insert was confirmed by restriction enzyme digestion. the functionality of the constructed dna vaccine vector (pvac self rep 990) was assayed by transfecting the dna into bhk 21 cell monolayer and studying the 35 s labeled proteins in immuno-precipitation assays. the studies showed high level of expression in case of constructed vector as compared to infected virus for the specific protein. the secretion of the expressed protein was assayed by immuno-fluorescence assay and found to be positive. encouraged with these studies the preliminary studies were conducted on vaccine efficacy studies in guinea pig model. the immunized guinea pigs showed high antibody titres by snt and elisa, as compared to conventional dna vaccines (pup3cd) even at 1/10th of the dose. this approach of constructing self replicating dna vaccine for humoral response is the first report. genetically engineered microorganisms are important sources of industrial and medicinal proteins. over the past decade, plant host system has been investigated as potential host system for expressing proteins of therapeutic and diagnostic use. however concerns regarding the stability and environmental safety need to be addressed. chloroplast engineering is expected to resolve some of these issues since, plastids/chloroplasts are inherited maternally and are not disseminated through pollen. this makes plastid transformation a valuable tool for transgenic creation besides offering biological containment. since foot and mouth disease (fmd) of cloven footed animals is a major concern in the world over. foot and mouth disease (fmd) is the most feared, viral disease of the cloven footed animals causing heavy losses to the live stock industry. the disease is enzootic in many parts of the world including asia. the conventional vaccines for fmd have several limitations which include safety, temperature sensitivity and duration of immunity. attempts have been made to overcome these limitations using recombinant dna technology. amongst the newer vaccines, edible vaccines are cost effective and easy to administer. since the stability of the gene of interest is the major concern in the case of plant transgenics, marker genes are used for regular selection. the detection methods based on the available marker proteins like b glucoronidase (gus) protein/antibiotic selection are cumbersome and cost intensive. however selection based on herbicide resistance is much simpler and easy. hence in the present study, the 5-enolpyruvylshikimate-3-phosphate synthase (epsp) gene was used as a marker along with the immunogen gene of fmdv. epsp is the key enzyme in the shikimate biosynthesis pathway necessary for the aromatic amino acids production. in order to investigate the mechanism of long term immunity and the effect of protective immunity induced by cationic plg micro particle coated dna vaccination. we constructed the expression plasmid containing a foot-and-mouth disease virus (fmdv) id gene sero type a. intramuscular vaccination of guinea pigs with the micro particles coated plasmid dna induced a strong antibody response and neutralization antibodies, cellular mediated immune response which lasted 1 year. we further analyzed the persistence and expression of id gene by polymerase chain reaction and reverse transcriptase polymerase chain reaction and quantitative pcr. the results showed that id gene was present and expressed in the muscle cells up to 1 year after days post vaccination. furthermore, guinea pigs vaccinated with micro particles coated plasmid dna were protected against a challenge with fmdv virus. therefore the micro particles coated plasmid dna vaccination dose induce a protective immunity and long term humoral, cellular immuno responses against fmdv, which could be maintained by persistent expression of id gene in muscle cells. foot and mouth disease virus (fmdv) causes a highly contagious viral disease of cloven hoofed animals, which has a considerable socioeconomic impact on the countries affected. interleukin-18 (il-18) enhances the il-12 driven th1 immune response that is important in immunity against intracellular pathogens. the multiple roles of il-18 in many physiological and pathological processes have generated a great deal of interest in recent years. antiviral effects of il-18 have been reported. we evaluated the effects interleukin-18 (il-18) on the replication of fmdv in vitro in bhk-21 cells. bovine il-18 mature protein coding sequence was amplified from the bovine pbmc cells and cloned into prokaryotic expression vector pet32a. protein expressed was purified and specificity was confirmed by immunoblotting. bhk-21 cells were treated with purified expressed il-18 protein with (2 lg/ml) 4 h prior to fmd infection. cell culture supernatants were collected at 24 h post infection were subjected for elisa and virus titration assay. rna extracted from the cells was subjected to real time pcr for viral rna quantification. 2 log titer reduction was observed in the fmd virus titer in il-18 treated cells compared to the untreated cells where as virus antigen quantified by elisa has shown a reduction of 60-folds. 69-fold reduction in the fmd viral rna copy number was observed in the il-18 treated cell compared to the untreated measured by qpcr. current study demonstrated the potent anti viral activity of il-18 on fmdv by inhibiting the viral replication. these results further suggests that il-18 has the potential role of il-18 as molecular adjuvant in fmd vaccine development and development of therapeutic for fmd. foot and mouth disease is the most contagious viral disease of farm animals. control of the disease in animals is by vaccination and slaughtering of infected animals. conventional oil adjuvant vaccine has its own limitation. alternate to this genetic vaccines where the dna encoding viral antigen may be a promising approach. naked dna vaccine has limitations like poor uptake of dna by cells and more importantly by nucleus. as a result delivery of naked dna through calcium phosphate nanoparticle was attempted. calcium phosphate nanoparticle is a potential delivery agent which proved to enhance the immune response. fmdv p1-3cd ''o'' vaccine gene constructs in pcdna3.1? entrapped by the nanoparticles was prepared by using different molarity of calcium chloride and disodium hydrogen orthophosphate. the nanoparticles entrapping fmdv p1-3cd ''o'' and naked dna were presented to the guinea pigs through intramuscular injection to study the mrna expression of antigen by rt-pcr. animals were sacrificed at defined time to collect different organs and total rna was extracted. each time blood was collected to analyse the fmdv specific serum antibodies. dna vaccines presented through calcium phosphate produced transcripts in the injected muscle up to 240 days whereas naked dna up to 120 days. serum antibody levels of naked dna vaccine showed antibody titre till 60 days. whereas nanoparticle injected animals showed serum antibody till 120 days. serum neutralization titres of 1.5 were observed in calcium phosphate dna vaccines at about 28-150 days, where as naked dna sn titers were observed for short period of 30-90 days. the study clearly showed calcium phosphate nanoparticle entrapping fmdv vaccine dna may be a better delivery system for dna vaccines as it confirms availability of the antigen and persistence of antibody for longer duration than naked dna. capripox is highly infectious, contagious, and oie notifiable disease of small ruminants, caused by sheeppox and goatpox viruses which are members of capripoxvirus genus of the family poxviridae. in the present study, we analyzed the partial gene sequences of p32 protein, an immunogenic envelope protein of capripox viruses (capv) to assess the genetic relationship among different sheep pox and goat pox virus isolates from different geographical areas of the country. product of this gene has been shown to be important in attachment of capv to host cell surface receptors during viral entry and host immune response. the following virus isolates have been used in the analysis: gtpv-uttarkashi, p60, vaccine virus; gtpv mukteswar, p10, challenge virus; gtpv (akola), gtpv bareilly/00, gtpv ladakh/01 and gtpv sambalpur/82, field isolates and sppv srinagar, p40; sppv ranipet, p50; sppv-rf, p50, vaccine viruses and sppv makdhoom/07, sppv cirg/08, sppv pune/08, sppv bareilly, sppv 183/03 and sppv 125/02, field isolates. in this study, all virus isolates were confirmed by pcr amplification and analysed in pcr-restriction fragment length polymorphism (pcr-rflp) using ecori enzyme to confirm their specificity. further, the amplicons were cloned and sequenced commercially. nucleotide and the deduced amino acid (aa) sequences were compared with published sequences of the members of the genus capripox virus. sequence analysis of partial 172 bp sequence has shown high sequence identity among all indian sppv and gtpv isolates at both nt and aa levels. it revealed a 99.4-100% and 98.2 for gtpv field isolates where as, 100% for sppv field isolates at both the nt and aa levels. in general, capv isolates in this study shown 98.3-98.8 and 96.5% homology between gtpv and sppv at nt and aa levels as reported earlier. further, it revealed a unique change of g120a in all gtpv isolates resulting in formation of drai site in place of ecori and possible development of restriction enzyme specific pcr-rflp for differentiation of sppv and gtpv from field isolates. orf or contagious ecthyma is considered as non-contagious, proliferative disease and is caused by orf virus of the genus parapox virus of the family poxviridae. it is reported most commonly in sheep and goats and also a zoonotic agent. camels are also infected by orf virus and reported in camel rearing countries as a mixed infection with camel pox, the later is caused by an orthopox virus. in india, there are few reports of the orf virus infection in camels and identified by clinical signs and pcr. in this study, we identified the presence of orf virus from clinical samples of suspected case of sporadic infection in camels by serological and molecular techniques. viral dna isolated from processed scabs used initially in nested polymerase chain reaction as diagnostic pcr which successfully amplified 235 bp fragments and also sequenced to check the fidelity of the product. after confirming the infection by pcr, some of the structural and non-structural genes were amplified for sequence analysis. out of the five genes characterized, the major important one selected for sequence and phylogenetic analysis is b2l gene which is homologous to a major envelope protein p37k of vaccinia virus. full open reading frame of 1137 bp from orf b2l was amplified by pcr, cloned and sequenced commercially. nucleotide and deduced amino acid sequences of b2l were compared with other published sequences of the members of the genus papapox virus. sequence analysis shows a maximum percent identity of 94.8 and 95 (indian orf virus isolates); 94.7 and 94.5 (other orf isolates); 98.8 and 98.7 (orf-camel/jodhpur/08); 85 and 82.8 (bovine popular stomatitis virus) and finally 97.4 and 97.6 (pseudo cowpox virus) respectively at nt and aa levels. phylogenetic analysis of the isolate was also performed using the neighbour joining method in mega 4 program to know the phylogeny relatedness of the virus, which revealed that the isolate is well grouped with the jodhpur isolate and closely related to pseudo cowpox virus. it warrants further analysis of other potential genes to confirm the causative agent of the contagious ecthyma in camels as pseudo cowpox virus. chikungunya an arboviral disease is transmitted through the bite of an infected aedes mosquito. it causes a self limited febrile illness along with arthralgia and myalgia. in some cases neurological and severe hemorrhagic manifestations has been observed. chikv epidemic has been reported in africa, india, south east asian countries and during the current out break imported cases of chikv has been encountered in most of the european countries. the causative agent belongs to the genus alphavirus family togaviridae. human beings serve as the chikungunya virus reservoir host during epidemic periods. outside these periods the main reservoirs are monkeys, rodents, birds, and other unidentified vertebrates. antibodies to chikv have been detected in domestic animals. in the present study we surveyed madanapalli, palamaner, b. kotta kota and tirupati and collected a total of 67 rodent samples, 75 bovine samples; 20 sheep samples and 15 canine samples. total rna was isolated from all these samples and subjected to rt-pcr using a primer pair dvrchk-f/dvrchk-r which could amplify a 330 bp e1 gene product specific to chikungunya virus (naresh kumar et al. 2007 ). all the serum samples were further screened for chikv specific igm antibodies using commercially available ctk biotech strips. none of the samples were found positive either for chikv specific rna or chikv specific igm antibodies. more number of samples from domestic animals as well as rodents are being screened to study their possible role if any in the maintenance of chikv in nature and during the inter epidemic periods. the present study discusses these aspects in detail. petunia hybrida is widely used as experimental host plant for begomovirus identification and its characterization. hitherto, natural infection of begomovirus on petunia has not been reported in india. recently, petunia hybrida grown in and around ludhiana were found to be depicting typical symptoms caused by begomovirus. the symptoms include severe reduction in leaf size, downward curling and distorted leaves. severely infected plant became bushy, stunted and produces no flower. total genomic dna was extracted from the plants showing symptoms of begomovirus, by ctab method. the presence of virus was confirmed by using degenerated primers, designed to identify all the begomovirus prevailing in the world. to identify the strain associated with the disease, the positive samples along with healthy control were tested against different strain specific primers of tomato leaf curl virus, so far reported in india i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus. among these, only tomato leaf curl new delhi virus specific primer was able to give the desired amplicon of *1180 bp. hence, it is confirmed that the leaf curl disease of petunia hybrida is associated with tomato leaf curl new delhi virus. this disease of petunia can become a sever production constraint in coming years. from last 2 years (2008 and 2009) it was observed that some varieties of brinjal grown in rainy season, showed typical leaf curl type of symptoms. the symptoms include upward curling of the leaves, cupping, vein thickening, reduction in leaf size and distortion of leaves. the severely infected plant remains stunted and bushy, became unproductive or produces only few fruits. the disease was experimentally transmitted from naturally infected brinjal to healthy seedlings by whiteflies (bemisia tabaci) and grafting, but not by mechanical or aphid transmission. to detect the begomovirus associated, total genomic dna was extracted from the plants showing disease symptoms. the presence of virus was confirmed by using pcr based begomovirus geneus-specific primers designed by deng et al., wyatt and brown and rojas et al. these degenerated primers give the expected product size of *530, *575 and *1280 bp, respectively. core coat protein (cp) gene and dna-b was also amplified in the samples using specific primers. to identify the strain associated with leaf curl virus, dna was subjected against primers of different indian tomato leaf curl virus strain i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus, using pcr. among these, only tomato leaf curl new delhi virus primer was able to show the desired product size of *1180 bp. therefore, it was confirmed that leaf curl disease of brinjal is caused by tomato leaf curl new delhi virus in association with satellite b-dna. to identify the strain associated with the disease, all samples were further subjected to the specific primers, designed to amplify all the tomato leaf curl virus strains, so far reported from india i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus, using pcr. among these, only tomato leaf curl palampur virus specific primer was able to give the expected product size of *900 bp. this shows the association of tomato leaf curl palampur virus with leaf curl disease of calendula and marigold. thus, calendula and marigold can act as a reservoir for the tomato leaf curl palampur virus and may cause severe constrain in the production of these important ornamental plants. groundnut bud necrosis virus (gbnv) belongs to serogroup iv of the genus tospovirus in bynayaviridae family and infects several economically important crops all over india. the nucleocapsid protein (np) encoded by the small rna of gbnv encapsidates the viral rnas. apart from this structural role, the np has also been implicated in the replication, transcription, maturation and cell to cell movement. with a view to study the structure and function, the np of gbnvtomato isolate from karnataka was over expressed in e. coli and purified by ni-nta chromatography. the purified np was present as ribonucleoprotein complex and as heterogeneous mixture containing monomers, tetramers and higher order multimers. in order to determine the regions involved in oligomerization and nucleic acid binding, mutational approach was taken. n-and c-terminal deletion clones were generated (n20np, n40np, c15np and c37np), over expressed in e. coli, and were purified by a procedure identical to that used for the wild type protein. initial studies on oligomeric status suggested that in addition to n-and c-terminal regions there may be additional regions or residues which contribute to multimerization of np. the amount of rna bound to the truncated proteins was reduced in case of n20np, n40np and c15np. interestingly removal of 37 amino acid residues (natively unfolded region) from the c terminus resulted in complete loss of nucleic acid binding suggesting that the rna binding domain was located in c-terminal region of np. further np was observed to get phosphorylated in in vitro kinase assays by a kinase present in the soluble fraction of tobacco plant sap. both atp and gtp were utilized as phosporyl donors and mn 2? was the preferred metal ion which suggests that np might be phosphorylated by a ck2-like protein kinase. phosphorylation studies with n-and c-terminal truncated proteins revealed that the site of phosphorylation lies within the amino acid residues 40-239. by mass spectrometric analysis of the protein threonine-84 and serine-202 were identified as possible phosphorylation sites. a naturally occurring isolate of virus infecting gherkin (cucumis anguira l.) showing mosaic symptoms of mosaic, leaf distortion and dark green islands in the lamina was identified in the export cultivars of gherkin grown in commercial fields of kuppam rural, chittoor district, andhra pradesh. the virus infection was deadly prevalent among the field that caused a lot of economic damage to the crop that resulted in yield losses and reduced quality of fruits meant for export. symptoms of the infected fruit included blistering and malformation of the fruit. the virus infected leaf samples were collected and initial host range tests were conducted with different cucurbit species showed that the host range include propagation hosts like cucumis anguira (gherkin), cucumis sativus, cucurbita pepo, cucumis melo, langeneria vulgaris, momordica charantia and local assay host like chenopodium amaranticolor. the virus host range was only restricted to cucurbit species and chenopodium. the virus was maintained for further studies on cucurbita pepo by sap or mechanical inoculation. the virus induced mosaic, vein clearing symptoms on pumpkin. electron microscopy of the leaf dip preparations stained with 2% uranyl acetate from the pumpkin leaves showing symptoms revealed the presence of a long flexuous filamentous particle measuring 750 9 12 nm. the virus positively reacted to the polyclonal antisera of papaya ringspot virus-w, potato virus y, tobacco etch virus and also strongly reacted with the polyclonal antiserum of zucchini yellow mosaic virus in direct antigen coated-enzyme linked immunosorbent assay (dac-elisa). because of very strong reaction to polyclonal antisera of zucchini yellow mosaic virus, we tried to amplify the partial nib and cp genes of the virus along with the 3 0 utr by using two primers zy2 5 0 gctccatacatagctgag acagc3 0 and zy3 5 0 taggctttttgcaaacggagtcta at c3 0 . total rna from gherkin infected leaves was isolated using trizol ls reagent (sigma). rt-pcr was performed to obtain an amplicon of *1.2kbp, cloned into fermentas ptz57r/t vector and sequenced at mwg biotech, bangalore. sequence analysis revealed that the virus was isolate of zucchini yellow mosaic virus and was showing 98% of homology to that of the zucchini yellow mosaic virus strain b genome ay188994 and zucchini yellow mosaic virus nat genome ef062582 which were strains reported from israel. the sequence of the present study was submitted to the genbank gq482976. the results state a suspicion that the virus could have been mobilized by some infected source brought by the commercial israeli based companies into india due to poor quarantine regulations as the gherkin cultivation in these regions is chiefly supported, purchased, exported and marketed by these private companies that are based from israel. this is the first report on molecular characterisation of zucchini yellow mosaic virus infecting cucumis anguira (gherkin) from india. they also exhibited synergism with other virus which was region specific. fifty percent of the total symptomatic plant population was found be positive only for carla while remaining showed mixed infection of carla with tospo in some regions while in others carla virus was found to be associated with cmv. presence of only carlavirus was up to 10-20% incidence, without association of tospo, cmv, poty or tobamo viruses was also observed in some fields. avijit tarafdar, raju ghosh, k. k. biswas plant virology unit, division of plant pathology, indian agricultural research institute, new delhi 110012 citrus tristeza virus (ctv), a brown citrus aphid (toxoptera citricidus) transmitted closterovirus under family closteroviridae, is one of the major limiting factors in cultivation of citrus worldwide. ctv is a longest known plant virus having flexuous particle of 2000 9 11 nm in size. ctv genome is a positive sense ssrna of about 20 kb nucleotide containing 13 open reading frames (orfs) encoding 17 proteins. several biological as well as genetic variants of ctv are reported in all the citrus growing countries in the world. ctv causes decline and death of millions of citrus trees in the world. in india, ctv is a century old problem, and has killed an estimated one million citrus trees till today. in molecular and genetic level, ctv isolates from india were not fully characterized. genetic diversity and sequence divergence in ctv isolates of india are not fully established. further, evidence of recombination and causes of evolution of ctv variants in india have not been studied till date. therefore, in the present study, effort has been made to characterize several indian ctv isolates in genetic level, examine their genetic diversity, identify recombination events and analyze evolution of divergent ctv. a total number of 73 ctv isolates from different regions of india (35 from darjeeling hills, five from bangalore, 15 from delhi and 18 from vidarbha) were under taken for genetic study. two genomic regions of ctv, i.e., entire cp gene (cpg) (672 nt) and a gene fragment of 5 0 orf1a (orf1a) (404 nt) were amplified, cloned, sequenced and nucleotides were analyzed. based on cpg, indian isolates shared 88-99% nucleotide identity, and based on orf1a they shared 82-99% identity, among them. incongruence of phylogenetic relationship was observed as on sequence analysis five phylogenetic clades based on cpg, and eight clades based on orf1a, were generated suggesting the recombination events have been occurred between the sequences of indian ctv isolates. thus, to identify the potential recombination events, and determine the parental sequences in ctv isolates, six recombination detecting algorithms, namely, rdp, genconv, bootscan, maxchi, chimera and siscan were used. out of 73 indian ctv, cpg of 18 and orf1a of 47 isolates of ctv showed recombination events suggesting orf1a was more prone and fragile to rna recombination as compared to cpg. this findings indicated that high degrees of genetic diversity and incongruent relationships of indian ctv isolates are due to genetic recombination occurred, which may be the important factors in driving evolution ctv variants in india, that was also supported by a splittree decomposition analysis. b. v. bhaskara reddy, y. sivaprasad, k. rekha rani, k. raja reddy department of plant pathology, regional agricultural research station, acharya n.g. ranga agricultural university, tirupati, andhra pradesh sunflower (helianthus annus l.) is one of the most important oil seed crops in the world which ranks third in area after soyabean and groundnut. the sunflower necrosis disease (snd) is characterized by necrosis of leaves, necrosis streaks on petioles, stem, floral parts and stunted growth. the causal agent of the disease has been identified as tobacco streak virus (tsv) which belongs to genus ilarvirus of the family bromoviridae. the suspected tsv infected sunflower samples collected from chittoor district in andhra pradesh were found positive for tsv-dac elisa. total rna was extracted from sunflower using rnaeasy isolation kit (qiagen). the tsv coat protein (cp) gene, movement protein (mp) gene and replicase (rep) gene were amplified by rt-pcr with specific primers, cloned in ptz57r/t vector, sequenced and deposited in genbank (gu355899, gu355900 and gu371445). the size of cloned cp gene was 717 bp and codes for 239 amino acids. the cp gene sequence analysis revealed that the tsv-tpt infecting sunflower has 98-100% homology at nucleotide level with soybean, tagietus-tpt and okra-tn isolates and 93-99% homology at amino acid level. the movement protein gene was 615 bp and codes for 205 amino acids. the mp gene sequence analysis showed that it has 94-97% homology at nucleotide level and 92-95% at aminoacid level. chilli (capsicum annuum), the important commercial vegetable/spice of himachal pradesh, is affected by several viral diseases; of them cucumo, tospo, poty and gemini viruses are the most common genera. however, these viruses are not identified clearly and characterized fully, which are foremost needed to formulate the management strategy. therefore, in the present study, effort has been made to identify and characterize the important viruses causing diseases in chilli. in this study, several farms in major chilli growing areas of bilaspur and kangra districts in himachal pradesh were surveyed and infected plant samples were collected randomly. virus infection in these samples were detected by das-elisa using antisera to cucumber mosaic virus (cmv) and potyvirus (group specific) and through slot-blot hybridization (sbh) using cmv, iris severe mosaic poty virus (ismv), tomato spotted wilt tospo virus (tswv) and chilli leaf curl gemini virus (clcuv). based on das-elisa and sbh, the incidence of disease was estimated and ranged from 18.2 to 21.8% by cmv and 3.5 to 5.4% by potyvirus. to detect tospo and geminivirus in the infected chilli, sbh test was carried out. infected samples showed maximum virus titer in both das-elisa and sbh test were further confirmed by pcr using specific primers. desired sizes of amplicons; *540 bp, *800 bp, *570 bp and *460 bp of cmv, poty, gemini and tospo viruses, respectively, were obtained. as the present study clearly indicated that cmv appeared as a major one among the viruses infecting chilli in the hilly region of himachal pradesh, two isolates of cmv were characterized in genetic level. thus the amplified products (*540 bp) of cmv, palampur 1 and palampur 2 were cloned in pgemt cloning vector, sequenced and the sequences were submitted to ncbi database (palampur 1: acc-fm209497 and palampur 2: acc-fm209498). the sequences were then analyzed and compared with other sequences available in the data base. based on sequence analysis, it was found that present cmv isolates shared 99% nucleotide identity between them, are closely related with australian cmv isolate cmv-ly (acc-af198103) by 98% nucleotide identity. in phylogenetic tree analysis, it was observed that indian cmv isolates formed same cluster along with cmv-ly. as it is known that cmv subgroup ii comprises cmv-ly, it is concluded that the cmvs of this hilly region of himachal pradesh belong to subgroup ii. chilli is essentially a crop of the tropics and grows better in hotter regions. chlii (capsicum annuum), a member of family solanaceae is an important vegetable and spice crop of immense commercial importance. the pungency in pepper is due to an alkaloid known as capsaicine and peppers are characterized as sweet, hot or mild depending on capsaicine content. the present investigation were conducted to find out the highly resistant cultivars of capsicum annuum against cmv and tylcv among ten cultivars of chilli in agroclimatic condition of aligarh. the highest (70 and 80) percentage of infection was observed in hc-201 and kalyanpur type-1 by showing the positive reaction to cmv by elisa test. no symptoms was recorded in case of bc-16, lca-235 and jca-154 and showed negative reaction to cmv by elisa. bc-16 and lca-235 also showed negative reaction to tylcv by elisa and these were symptomless. maximum infection (70 and 80) was registered in hc-201 and c 8 , cultivar. so, the bc-16, lca-235 and jca-154 has proved highly resistant varieties against cmv and tylcv and these may be used in breeding programmes against viruses. cotton leaf curl virus belongs to the family geminiviridae, genus begomovirus. the members of this family contain circular single stranded dna molecules as their genomes. there are two kinds of begomoviruses-bipartite viruses with genomes consisting of two dna molecules designated dna-a and dna-b and the monopartite viruses which contain only dna-a but not dna-b. in monopartite viruses, the dna-a is accompanied by a small circular dna molecule called dna-b which is essential for the development of typical disease symptoms. cotton leaf curl virus is a monopartite virus and causes the cotton leaf curl disease which has emerged as a major disease of cotton in the indian subcontinent. the non-structural protein ac4 of cotton leaf curl kokhran virus-dabawali isolate (clcukv-dab) was cloned into pgex5x2 vector and overexpressed in bl21(de3)plyss e. coli cells. the overexpressed gst-ac4 protein was purified by glutathione sepharose chromatography. the purified gst-ac4 protein was found to possess atpase activity. the optimum temperature and ph for the activity were 37°c and 7.4 respectively. the atpase activity was inhibited in presence of edta, showing that it is dependent on divalent metal ions. the activity was supported by magnesium, manganese and zinc ions but inhibited in presence of calcium ions. it was also inhibited by the non-hydrolyzable atp analogue adenosine-b, c-imido triphosphate and in the presence of other nucleotides like ctp and gtp. the k m and the v max of the reaction for atp as the substrate are 1.54 mm and 95.2 nmol/min/ mg of the protein respectively. the enzyme could also utilize gtp as the substrate. the fact that ac4 is specifically an ntpase and not a general phosphatase is revealed by the finding that it does not hydrolyze p-nitrophenyl phosphate to yield yellow colour while a similar reaction carried out in parallel with alkaline phosphatase readily yields the colour. it has been suggested earlier that ac4 may be involved in cell to cell movement of the virus (rojas et al. 2001) . it is possible that by its ability to hydrolyze atp, ac4 serves to power viral movement in the plant. thirteen sugarcane yellow leaf virus isolates causing yellow midrib and irregular yellow spot pattern from six states of india were characterized by rt-pcr assays. scylv-615f and scylv-615r primers were used as forward and reverse primer pairs and the amplified products were cloned and sequenced. comparative coat protein sequence analysis confirmed that all the scylv-indian isolates were clustered into two major groups confirming the existence of two strains of scylv affecting sugarcane crops of india. in a separate experiment, the member of both of the phylogenetic groups were found to be transmitted by the sugarcane aphid, melanaphis sacchari from infected to healthy sugarcane suggesting its secondary spread in nature. the symptoms produced by the virus causing cotton mosaic disease were little bit different in both sap inoculation and under natural field condition. in natural field condition it has shown clear chlorosis type of symptoms on major leaves of plants but in sap inoculated plants veinal chlorosis and mosaic type of symptoms are found to be common. in field conditions infected plants grows erect and have less boll formation. there is no effect found on seed shape or seed size. the initial symptoms produced on cotton leaves after inoculation were wonderful. local lesions observed in second week from inoculation and then they changes to chlorotic type of symptoms and some are necrotic symptoms also. the plants at early stage are found to be affected, has less lateral branch development and hence reduction in yield production. the naturally field infected plants showing good symptoms are also difficult to identify in lateral stage of plant. because they disappear with time. the virus is very easily sap transmissible. the virus is found to be transmitted by thrips palmi and thrips tobacci in persistent manner. no seed transmission is observed. virus showed same physical properties as it shows in stem necrosis of peanut or sunflower necrosis disease. the physical properties are found to be thermal inactivation point (tip) 55-60°c, dilution end point (dep) 10 -2 to 10 -3 and longevity in vitro (liv) 5 h, virus infecting nineteen different host plants are identified belonging to five different types of families viz. malvaceae, chenopodiaceae, compositae, leguminaceae and solanaceae. however they found to produce same types of symptoms as in most of the host that have been tested before. in elisa test report it is found that the virus showing positive test only with anti serum of tsv of a cowpea and cotton but negative reaction with pbnv of cowpea and cotton which clearly denied possibility of presence of pbnv in cotton producing these kinds of symptoms elisa report clearly shows that tsv antiserum of cowpea is showing positive results with clear chlorotic types of symptoms. a powerful approach to functional genomics, and an alternative to the massive generation of transgenic plants, is the use of the recently described virus induced gene silencing (vigs) process, which allows viral vectors to knock out the function of a gene-of-interest. vigs is based on a silencing mechanism that regulates gene expression by the specific degradation of rna. as a tool for reverse genetics, vigs has many advantages over other common ways to study gene function because of the ability of viruses to replicate and move systemically within a plant. vigs can generate a phenocopy of a mutant without all the troubles of traditional methods of mutagenesis. geminiviruses with their small dna genomes and ease of inoculation through agrobacterium, are excellent candidates for vigs vector development. as a first step, the geminivirus bhendi yellow vein mosaic virus, characterized in our lab (jose and usha, virology 305: [310] [311] [312] [313] [314] [315] [316] [317] 2003) has been chosen. the satellite b dna associated with this virus has a single open reading frame (bc1). bc1 is essential for symptom development but not for replication. therefore, bc1 has been replaced by a multiple cloning site harbouring sali, xbai, bamhi, bsrgi and xhoi, initially in a cloning vector and then in the binary vector containing the partial tandem repeat of the b dna. in the place of the bc1 orf, the plant phytoene desaturase gene has been cloned and the resulting construct was used for agroinfiltration along with the partial tandem repeat clone of the begomovirus (dna a component chilli (capsicum annuum l.) plants exhibiting prominent symptoms of begomovirus like: leaf curl, vein swelling, shortening of petioles, crowding of leaves and stunting of plants were collected from rorkee, uttarakhand and dhaulpur, rajasthan, india. total genomic dna was isolated from naturally infected chilli samples and pcr was carried out with coat protein (located in dna-a) gene specific primers. as expected to the primers, *800 bp dna fragments were amplified from the infected chilli samples. to know the bipartite nature of the virus isolates, nuclear shuttle protein (located in dna-b) gene specific primers were employed which also resulted in positive amplification of *850 bp dna bands with all the coat protein tested positive samples. to ascertain the association of dnab component with the virus isolates, a set of dna-b specific primers were used which resulted in positive amplification of full length (*1.3 kb) dna bands in the chilli samples collected from rorkee, uttarakhand, however, multiple sizes bands were resulted with the samples collected from dhaulpur, rajasthan. these findings confirmed that both the virus isolates under study are bipartite begomovirus associated with dna-b satellite. the sequencing of the pcr products is under progress which analysis will be discussed. groundnut bud necrosis virus (gbnv) belonging to the genus tospovirus, which is a unique member of the family bunyaviridae, infects several economically important crops. the virus has three genomic ssrna segments namely s (ambisense), m (ambisense) and l (negative sense). the s rna codes for nucleoprotein (np) and non-structural protein (nss) from viral complimentary and viral strands respectively. many viral nonstructural proteins such as ns3 of hepatitis c virus, yellow fever virus, dengue virus, sv40 large t antigen and cytoplasmic inclusion protein of tamarillo mosaic potyvirus are known to exhibit rna/dna stimulated ntpase, dntpase and helicase activity. nss of gbnv does not have any sequence similarity with any of the above mentioned viral rna/dna helicases but has a ntp binding domain. however, it has been implicated as suppressor of gene silencing in vivo. with a view to elucidate the mechanism by which nss could act as a suppressor of gene silencing and examine the other potential roles of nss in the life cycle of the virus, the gbnv (to) nss was over-expressed in e. coli and purified by ni-nta chromatography. in vitro studies with the purified rnss suggest that it exhibits an rna stimulated ntpase activity. many of the proteins that possess the rna/ dna stimulated ntpase and datpase activity, are also shown to have atp dependent nucleic acid unwinding activity. it was therefore of interest to examine whether nss has the nucleic acid unwinding activity. the helicase assays revealed that nss has dna/rna helicase activity. helicase activity of nss was absolutely dependent on atp and mg 2? ion. nss could unwind dsdna substrate with 5 0 overhang, or 3 0 overhang. mutation of the crucial lysine in walker motif a (k189) severely affected the unwinding activity where as mutation of aspartate residue in walker motif b (d159) resulted in only 20% loss of activity. in this regard, rnss is a unique enzyme which does not have the canonical helicase motifs but can catalyze dsdna/dsrna unwinding in an atp and mg 2? dependent manner. the rnss might act as a suppressor of by unwinding the dsrna, the substrate for dicer. in addition to being a suppressor of ptgs, nss may also regulate the viral replication and transcription by modulating the secondary structure of the viral genome. this new research finding on nss might pave way for further studies on its role in viral replication and transcription. yellow vein mosaic disease of pumpkin (cucurbita moschata) poses a serious threat to the cultivation of this crop in india. the disease was found to be associated with whitefly-transmitted bipartite begomoviruses were detected in varanasi field using polymerase chain reaction (pcr) with primer design through coat protein conserved region of begomoviruses from ncbi database. all plant samples showing symptoms were infected with begomovirus. the virus species were provisionally identified by sequencing *750 bp of the viral coat protein gene (av1 ageratum conyzoides is commonly known as billygoat-weed, chick weed, goatweed and whiteweed. in india it is popularly known as bill goat weed. it is an annual herbaceous plant with a long history of traditional medicinal uses in several countries of the world and also reputed to possess varied medicinal properties including the treatment of wounds and burns. in cameroon and congo, it is used traditionally to treat fever, rheumatism, headache, and colic. during survey in and around gorakhpur in 2009, ageratum plants were found affected with the symptoms of leaf curling, mosaic mottling and leaf yellows. the infected leaf samples were processed for virus identification and association with pcr assays. total dna was extracted and pcr were performed with begomovirus specific primers (tlcv-cp). a *800 bp band was consistently amplified on 1% agarose. the pcr products were directly sequenced and sequence was submitted in genbank with the accession no. gq412352. the blast search analysis showed highest similarity of 98% with the ageratum enation virus. vernonia cinerea leaves with yellow vein symptoms were collected around crop fields in madurai. a 550 bp product amplified from total dna extracted from symptomatic leaves with degenerate primers designed to amplify a part of the av1 gene from begomoviral dna a component was cloned and sequenced. based on the above sequences, specific primers were designed and the full length dna a of 2745 nucleotides with typical genome organization of begomoviral dna a was obtained and was submitted to embl data base (acc no: am182232). the sequence comparison with other begomoviruses revealed the closest identity (83%) with emilia yellow vein virus from china and less than 80% with all known begomoviruses. the international committee on taxonomy of viruses (ictv) has therefore recognized vernonia yellow vein virus (vyvv) as a distinct begomovirus species. conventional pcr could not amplify the dna b or dna b from the infected tissue. however, the b dna (1364 bp) associated with the disease was obtained (acc no: fn435836) by the rolling circle amplification-restriction fragment length polymorphism method (rca-rflp) using phi29 dna polymerase. sequence analysis shows that dna b of vyvv has the highest identity (81%) with dna b of ageratum leaf curl disease and 58-77% with the b dna associated with other begomoviruses. infectious clones of vyvv dna a and dna b as dimers were made using the products of rca-rflp. these infectious clones will be used for agroinfection of vernonia and the results will be discussed. this is the first report of the molecular characterization of vernonia yellow vein virus (vyvv) from vernonia cinerea in india. production of bulb and seed crop of onion (allium cepa l.) is hampered by onion yellow dwarf virus (oydv) and iris yellow spot virus (iysv) with an incidence of 83.22% and 89.97% in bulb crop and 90.65% and 89.58% in seed crop, respectively in the popularly grown cv. hisar-2. four symptom-based variants of oydv designated as grade a, b, c and d produced varied types of symptoms in onion crop incurring heavy losses in bulb and seed production. iysv caused tiny hay coloured spots of different shapes and sizes on leaves and scapes which later coalesced and led to drying and lodging of scapes. the plant height, bulb weight and bulb size were 37.7 cm, 75.5 g and 24.2 cm 2 in plants infected with oydv, 39.6 cm, 79.7 g and 25.5 cm 2 in iysv infection, 35.1 cm, 68.4 g and 22.1 cm 2 due to their combined infection, as compared to 40.6 cm, 88.4 g and 27.6 cm 2 respectively, in healthy plants of bulb crop. in plants infected with oydv grade a the plant height was minimum (90.33 cm) whereas the number of umbels was maximum (9.20 umbels/pl.) but other yield parameters viz., weight/umbel (2.32 g), number of seeds/umbel (209), seed weight/umbel (0.64 g) and seed yield/plant (5.88 g) were recorded to be the lowest. the minimum reduction in plant height (100.26 cm), weight/umbel (6.72 g), number of seeds/umbel (633), seed weight/umbel (2.36 g) and seed yield/plant (11.90 g) were recorded in oydv grade d. the plant height was 98.84 cm with 5.10 umbels per plant, 4.24 g weight/umbel, 428 seeds/umbel, 1.25 g seed weight/umbel and 6.37 g seed yield/plant in iysv infected plants. the plant height (96.26 cm), umbels/plant (5.97), weight/umbel (4.60 g), number of seeds/umbel (432), seed weight/umbel (1.42 g) and seed yield/plant (7.82 g) were found to be the lowest in combined infection of oydv and iysv diseases in comparison to higher values in healthy controls (104.50 cm, 4.90, 7.84 g, 677, 2.60 g, 12.74 g, respectively). a minimum reduction in the test weight, germination and seed vigour index were found (3.06 g, 75.68% and 926) due to oydv grade a infection, whereas these were 2.92 g, 70.42% and 788 in iysv disease infected plants and 2.62 g, 70.4% and 776 in combined infection of oydv and iysv diseases in comparison to 3.84 g, 88.67% and 1276 in healthy plants. the maximum hampering of seed vigour parameters was recorded due to iysv infection. lodging of scapes caused by this disease was responsible for heavy losses in seed production and seed quality. cotton leaf curl disease is one of the major threats to cotton cultivation from northern india. survey conducted during 2009, observed the disease incidence ranged from 70 to 90% from bhatinda, abohar, fazilka, sri ganganagar, hanumanghar. in order to study genetic variability in the virus, twelve clcuv isolates were partially characterized (700 bp common region, full length av2 gene and partial sequences of ac1 and av1 gene). full length characterization of representative isolates from bhatinda, abohar, fazilka, sri ganganagar, hanumanghar is under progress. partial sequence analysis of clcuv isolates revealed that, the virus isolates collected during 2009 cropping season are closely related to cotton leaf curl burewala virus from pakistan and results were discussed. pratibha singh, h. s. savithri department of biochemistry, indian institute of science, bangalore tospoviruses, belonging to the family bunyavirideae, infect economically important plants such as groundnut, tomato, watermelon etc. they have a tripartite genome, with l, m and s segments of rna, in pseudo circular (panhandle) form. the viral genomes encode four structural proteins (l, n, g1 and g2) in the antisense orientation, and two non structural proteins nss and nsm in the sense orientation. the nsm is the only protein unique to tospoviruseses that infect plants in the bunyaviridae family and hence is proposed to be important for cell to cell movement. ground nut bud necrosis virus (gbnv), a member of the tospovirus genus, is the most prevalent virus infecting several species of leguminosae and solanaceae plants in india. total rna was isolated from gbnv infected tomato leaves and rt-pcr was performed using appropriate primers to amplify the nsm gene. the pcr product was cloned in pgex5x2 vector. the recombinant nsm clone was transformed into bl21 (de3) e. coli cells and over-expressed by induction with 0.3 mm iptg. sds-page analysis of induced and uninduced fraction revealed the presence of overexpressed protein of expected size. the soluble gst-nsm was purified by gsh sepharose affinity chromatography. purified gst-nsm was shown to interact with in vitro transcribed rna transcript by electrophoretic mobility shift assay. further nsm was shown to interact with viral encoded proteins np and nss using elisa and yeast two hybrid system. nsm was also shown to be phosphorylated in vitro by pellet fraction of plant sap. thus the recombinant gbnv nsm possesses the characteristic features of a movement protein such as nucleic acid binding, interaction with nucleocapsid protein, and ability to undergo posttranslational modification. solanum melongena, commonly called as egg plant is one of the most important vegetable crop in the world. it is cultivated widely in the tropical and sub tropical regions. several viruses such as cucumber mosaic cucumo virus (cmv), potato virus-y (pvy), potato virus-x (pvx) and tobacco ring spot virus (trsv) infect egg plant under natural conditions. in india major crop losses due to cmv infection in brinjal is 57% (fao stat-2008) . in the present study the infected leaf samples were collected from local fields of ramapuram, chandamama palli, chandragiri, madanapalli, yadhamari, durgasamudram villages in and around tirupati, were tested for cmv infection by dac-elisa with cmv antisera. the resulting positive samples were further inoculated to the raised brinjal seedlings of selected varieties through mechanical sap inoculation. different varieties of brinjal like mullabadhine, ankhur, ravya, mattigulla, casper and easter egg were used for monitoring the susceptibility to cmv infection. the mosaic symptoms were observed after 2 weeks of inoculation in all varities of brinjal except mullabadhina. among all these susceptible varities ankhur variety is selected to study induced biochemical changes such as chlorophylls, carbohydrates, proteins, nucleic acids and polyphenol oxidases in cmv infected brinjal leaves. in the infected leaves considerable reduction in chlorophyll and starch and increase in total proteins, sugars, rna and polyphenol oxidases was observed when compared to healthy leaves. the amount of total starch, protein and dna decreased to about 25, 136 and 645 lg/g respectively in infected leaves, where as sugars (75 lg/g), rna content (754 lg/g) and polyphenol oxidase activity was increased as compared to healthy leaves. the above results suggests that there is an altered concentrations of chlorophyll, proteins, nucleic acids, carbohydrates and polyphenol oxidase activity in the brinjal leaves due to the effect of cucumber mosaic cucumo virus infection. leaf analysis was found to be used as widely accepted diagnostic tool to assess the nutritional status of the vegetables. the present study deals with these aspects in detail. the total rna and dna was isolated from infected leaf samples. rt-pcr assays were performed using sugarcane yellow leaf virus (scylv) specific primers (scylv-615f and scylv-615r). the infection of scylv was detected in all the collected samples, which showed the expected size (*610 bp) amplicon during rt-pcr. in another experiment with nested pcr analysis, a phytoplasma characteristic 1.2 kb rdna pcr product were amplified from dnas of all infected samples but not in healthy sugarcane plants tested using phytoplasma universal primer pairs p1/p7 and fu3/ru5. dna extracts from plants with yellow mid rib and leaf yellows produced products of 1250 bp, which gave typical phytoplasma profiles when digested with hae iii and hha i. no pcr amplifications were produced using dna from symptomless plants. our results suggest that the yellow mid rib and leaf yellows symptoms on sugarcane varieties in uttar pradesh and uttarakhand states of india exhibiting midrib yellowing and leaf yellows symptoms is mainly caused by mixed infection of scylv and scylp. the affected clumps showed reduction in stalk height as compared to healthy fields. thirty-one sugarcane mosaic isolates belonged to sugarcane mosaic virus (scmv) and sugarcane streak mosaic virus (scsmv were collected from china and india), confirmed in indirect elisa and rt-pcr amplification with scmv and scsmv-specific primers. the amplicons (0.8 kb) from the coding region of coat protein (cp) were cloned, sequenced and compared to each other as well as to the sequences of 15 scmv isolates from sugarcane (australia, usa, china, brazil, mexico and south africa), maize (australia, china, iranian) and one scsmv isolate from sugarcane (india) in genbank. maximum likelihood and maximum parsimony analyses robustly supported two major monophyletic groups that were correlated with the host of origin: the scmv subgroup that included 18 isolates from china and only 13 isolates from india, and the scsmv subgroup that contained all isolates from india. maize dwarf mosaic virus (mdmv) and johnsongrass mosaic virus (jgmv) were not detected in any of the samples tested. a strong correlation was observed between the sugarcane groups and the geographical origin of the scmv isolates. the 11 millable sugarcane samples from china contained a virus tentatively described as sorghum mosaic virus (srmv). three isolates from nine chewing canes in fujian, yunnan and guizhou provinces of china also contained srmv, and the other 12 samples including five isolates from india was found infected with scmv. no srmv infection has been detected in sugarcane mosaic samples from india. sequence comparisons and phylogenetic analysis indicated that srmv can be considered as the most common and prevalent potyvirus infecting sugarcane in china, however in india sugarcane streak mosaic virus is dominant in causing mosaic symptoms on sugarcane. dig-labeled dna probe complementary to coat protein (cp) region of tobacco streak virus (tsv) sunflower isolate was designed for the sensitive and broad-spectrum detection of tsv isolates, the most devastating virus in india. dot-blot and tissue print hybridizations with the digoxigenin labeled probe were performed for the tsv detection at field levels. here, dot-blot hybridization was used to check a wide number of tsv isolates with a single probe and sensitivity with different sample extraction methods. the probe with cp conserved region prepared from sunflower pcr amplicon was hybridized with the tsv field isolates of gherkin, pumpkin, sunflower, marigold and globe amaranth samples because of highly conserved with little variability in cp region. the sensitivity limits were decreased from total nucleic acid to partially purified and crude extract preparations. in particular, tissue blot hybridization offers a simple, reliable procedure as dot-blot, but requires no sample processing. because there is minimal sample preparation, tissue-print hybridization could be an important component of tsv management programs. thus, the above non-radioactive labeled probe techniques can facilitate in screening the samples during tsv outbreaks and in quarantine services. savita patil, rupali sawant*, k. banerjee virology group, agharkar research institute, macs, g.g. agarkar road, pune 411 004 two mycobacterium smegmatis strains (ari lab nos. v842 and v946) were employed for the isolation of mycobacteriophages from soil and sewage samples. mycobacteriophages were isolated from soil samples collected from an area surrounding the tuberculosis (tb) ward, naidu hospital, pune, against m. smegmatis strain v842. these were numbered as v942, v943 and v944 and were isolated by using washed-cell preparation method. the bacteriophages against the other m. smegmatis strain, i.e. v946, were isolated from soil samples (collected from around tb ward, sassoon hospital, pune). some of these phages (viz.v953, v954) showed plaques at 42°c but not at 37°c. thus they seem to be lysogenic. for propagating and increasing the titre of all the above isolates, various previously described methods were attempted, but none of these methods were satisfactory. but when siliconized glassware and plastic-ware were used, propagation was successful. we showed that siliconization of glassware and plastic-ware was essential for the propagation of our mycobacteriophage isolates v951, v952, v953, v954 and v955. also, phage dilution medium (pdm) as described by chaterjee et al. (2000) was found to be effective for picking out of the plaques made by the phages. in this way, the phage isolates were propagated up to p 3 . the various passages of the phage isolates v951, v952, v953, v954 and v955 (i.e. original, p 1 , p 2 and p 3 ) were stored at -80°c. pvp-29 effect on pigments due to geminivirus infection on cowpea (vigna unguiculata) shail pande*, naveen pandey, k. shukla mahatma gandhi p. g. college gorakhpur, d.d.u. gorakhpur university, gorakhpur geminiviruses are one of the most important group of viruses causing economic losses in tropics. the symptom produced are yellowing of leaves which directly affect the pigments of diseased plants it in turn affects productivity and yield of diseased plant. cowpea vigna unguiculata is one of the important crop cultivated throughout india for its green pods which are used as vegetables and seeds are used as pulse. cowpea is affected by many viruses amongst them geminiviruses are one of the important virus on the cowpea plant. in the present study total chlorophyll content was studied in leaf of cowpea of diseased and healthy plants using arnon's method. carotenoids were also studied using ikan's method. it was found that chlorophyll content in diseased plants were lower compared to healthy plant similar results were found with carotenoids so the geminivirruses infection lowers the chlorophyll and carotenoid content in diseased plants which reduces yield of diseased cowpea plant. shweta sharma 1 , amrita banerjee 2 , j. tarafdar 2 , r. rabindran 3 , indranil dasgupta 1 * 1 department of plant molecular biology, university of delhi, south campus, new delhi; 2 bidhan chandra krishi vishwavidayalaya, kalyani, nadia, west bengal 741235; 3 tamil nadu agricultural university, coimbatore, tamil nadu 641003 rice tungro disease is an important disease of rice, caused by a joint infection by two viruses: rice tungro spherical virus (rtsv) and rice tungro bacilliform virus (rtbv) in south and southeast asia. the complex of rtbv and rtsv is transmitted by an insect vector green leaf hopper (glh). previously we reported complete genomic sequences of two geographically distinct isolates of rtbv; rtbv-wb (west bengal) and rtbv-ap (andhra pradesh) collected from the field in mid-1990s. both the sequences showed high homology all along the genome but showed divergence from previously reported southeast asian isolate i.e. rtbv-phil (philippines). to check whether a time period of a decade has resulted into variability in the genomic sequence of different isolates of rtbv in india, we cloned and sequenced the complete genome of rtbv from two geographically distinct regions of india i.e. west bengal and kanyakumari collected from the field in 2008. the complete nucleotide sequence of the dna fragments covering the whole genome of rtbv was determined using universal primers m13f and m13r and by primer walking, without any ambiguities remaining. the nucleotide sequences of overlapping clones were assembled and analyzed using the dna analysis software generunner and blastn program of ncbi. homology search at the nucleotide and amino acid level were performed using the blastn and blastp (respectively) programs of ncbi. multiple sequence alignments were performed using clus-tal-w software. sequence analysis results thus obtained showed that both the recently obtained complete genomic sequences of rtbv from two geographically distinct regions of india i.e. west bengal and kanyakumari showed very high homology (both at the nucleotide and amino acid levels) with the two previously reported rtbv isolates from india i.e. rtbv-wb (west bengal) and rtbv-ap (andhra pradesh) all along the genome. as observed earlier both the sequences diverged significantly from the southeast asian isolates. this suggests that even after the spatial and temporal difference (a time gap of approx 10 years) between the two previously reported rtbv isolates and the recently reported one, there is very little sequence variability between them. this further strengthens the earlier reports that the rtbv genomes in india are highly conserved. homology search at the nucleotide level using blastn program with the previously existing rtbv isolates revealed a very high percentage identity of 99% with the rtbv west bengal isolate and 95% with the rtbv andhra pradesh isolate. this further strengthens the earlier reports that there is not much genetic variability in the rtbv genomes in indian subcontinent. complete genomic rna sequences of two geographically distinct isolates of rice tungro spherical virus (rtsv), a member of the genus waikavirus, family sequiviridae, were determined from india. out of the two previously reported sequences, the indian isolates were closer to the resistance breaking strain rtsv-[vt6] than rtsv[phila] . between them, the indian sequences showed nucleotide as well as amino acid identities of 96%. a moderate homology was observed between the leader peptide and a putative helper component protein involved in insect transmission of the maize chlorotic dwarf virus, a closely related waikavirus, indicating its possible transmission-related function. unlike rice tungro bacilliform virus, which causes rice tungro disease jointly with rtsv, and is significantly different between isolates from india and philippines, rtsv genomes were observed to be much more conserved between isolates from the two countries. rice tungro bacilliform virus (rtbv) are believed to be the joint causative agents for the devastating tungro disease of rice prevalent in south and southeast asia [11] . rice tungro disease has become the major cause of production losses in rice during last three decades in several rice growing states of india. here, we report, for the first time the complete sequence analysis of two geographically distinct indian isolates of rtsv. we analyze the deduced protein sequences and their phylogenetic relationship with the two complete rtsv sequences from philippines as well as with other members of sequiviridae family. we provide molecular evidence that the indian isolates of rtsv are closely related to those from the philippines. we had earlier reported that rtbv isolates between india and philippines differ significantly from each other [18] . this study was undertaken in order to see whether rtsv isolates from india also show similar difference from those reported from the philippines. frequent outbreaks of tungro were reported near kanyakumari in the last 2-3 years. the present work was undertaken to clone and sequence the full-length rtbv and rtsv genomes from the infected rice plants collected from above region and to analyze the similarity of its genetic material with the existing indian isolates of rtbv and rtsv. a 1.1 kb dna fragment encoding the reverse transcriptase gene of rtbv genome was amplified and cloned in t/a vector and was sequenced commercially. homology search at the nucleotide level using blastn program with the previously existing rtbv isolates revealed a very high percentage identity of 99% with the rtbv west bengal isolate and 95% with the rtbv andhra pradesh isolate. this further strengthens the earlier reports that there is not much genetic variability in the rtbv genomes in indian subcontinent. similarly, the cp3 region of rtsv was amplified by rt-pcr and was cloned in t/a vector. recently, rice tungro disease has been reported from kanyakumari district of tamil nadu. it is important to determine the genetic nature of this isolate in order to develop resistance strategies. it is thus necessary to clone and characterize the viruses from kanyakumari and to determine the mechanism of virus resistance in transgenic lines. rice tungro disease is an important viral disease of rice. rice tungro is caused by infection by two viruses: rice tungro bacilliform virus (rtbv) and rice tungro spherical virus (rtsv). rtsv is a plant picornavirus with a 12 kb single stranded rna genome. it belongs to genus waikavirus in the family sequiviridae and is necessary for transmission of the two viruses by the leafhopper vector nephotellix virescens. rtsv rna is translated to form a large polyprotein, which is then self cleaved to form the viral proteins, including the three coat proteins, replicase, protease. studies have been conducted on rtsv from philippines. correct information of sequence variability of viral isolates to check whether different geographical conditions like those present in india select for genotypically variable strain and to design for transgenic resistance strategy, information on rtsv from india is absolutely essential. the objective of this study was to clone rtsv isolates from india and compare the genetic diversity of indian isolates from other southeast asian isolates and amongst each other. also develop strategy to impair the attack of virus-complex on rice. the achieve this, complete genomes of two isolates from india were cloned by amplifying different genes by rt-pcr and subsequently cloned in ta vectors, followed by sequencing. subsequently constructs containing cp1-3, antisense replicase, sense replicase and double stranded replicase were cloned in plant transformation vector. these constructs were used to transform aromatic rice variety from indian-pusa basmati (pb1). pcr analysis of the above plants was done to check the stable insertion of insert in the transgenics. jatropha (jatropha curcas) of the family euphorbiaceae is being grown in india as a major commercial fuel (bio-diesel) crop. jatropha is cultivated in 200 districts of 19 potential states of india. unfortunately, the cultivation of jatropha is limited by the severe mosaic disease. recently, a severe mosaic disease with significant disease incidence was observed in 2006-2009 on j. curcas grown in experimental plots of nbri and j. gossypifolia, a weed growing road side around lucknow and kathaupahadi, madhya pradesh. the disease consisted of the symptoms of severe mosaic, blistering, leaf distortion and stunting of whole plant and no fruit/seed production in severely affected plants. symptomatology and whitefly population observed on them suggested the occurrence of begomovirus infection. to detect the begomovirus infection, the total dna from leaf samples of infected jatropha plants was extracted and polymerase chain reaction (pcr) were performed using three sets of begomovirus genus specific (cpit-i/cpit-t, paliv 1978/paric 496 and paliv 722/palic 1960) primers and the expected size *800 bp, 1.2 kb and 1.2 kb amplicons were obtained which confirmed the begomovirus infection. further to identify the begomovirus/es and investigate the genetic diversity among them exists if any, the *1.2 kb amplicons were cloned and sequenced. the sequence data were deposited in the genbank database under accession nos.: gq847545 and fj346232 (from j. curcas) and eu727086 and fj177030 (from j. gossypifolia). during blast analysis gq847545 and fj346232 shared highest 95% sequence identity with each other and 84-88%% with sri lankan cassava mosaic virus (aj579307, aj607394, aj890225, aj89 0229 and aj890224) and indian cassava mosaic virus from india (ay738105) therefore, designated as two strains of jatropha mosaic india virus-lucknow. blast analysis of eu727086 showed maximum 93% similarities with croton yellow vein mosaic virus (aj507777), 82% with tomato leaf curl new delhi virus (dq629102) and 80-79% with papaya leaf curl virus (aj436992 and y15934), therefore, identified as strain of croton yellow vein mosaic virus. blast analysis of the virus isolate (fj177030) showed highest 83% identities with tomato leaf curl virus-bangalore ii (tolcv-b ii-u38239) and 82-81% with tomato leaf curl karnataka virus (tol-ckv, ay754812, fj514798), therefore, considered as new begomovirus species ''jatropha yellow mosaic india virus''. the phylogenetic analysis of gq847545 and fj346232 (from j. curcas) and eu727086 and fj177030 (from j. gossypifolia) was performed along with some selected isolates of begomovirus which showed [90% sequence identities during blast analysis. the isolate eu727086 showed closest relationship with croton yellow vein mosaic virus while fj177030 showed separate clustering of all the four begomovirus from jatropha species. during phylogenetic analysis these isolates formed three separate clusters, therefore, they were considered as three distinct begomoviruses. the above data clearly show that some genetic diversity exists among the begomoviruses infecting jatropha species in india. bitter gourd (momordica charantia l.) of the family cucurbitaceae, also known as bitter melon is extensively cultivated in north eastern region of uttar pradesh, india. it is regarded as one of the world's major vegetable crops and has great economic importance. a severe yellow mosaic disease on bitter gourd (momordica charantia) with a significant disease incidence was observed during the survey of different locations of eastern up, india in the year 2007. the whitefly (bemisia tabaci) population was also observed in the vicinity. the characteristic disease symptoms and whitefly population indicated the possibility of begomovirus infection. total dna were isolated from infected as well as healthy leaf samples. two primer pair (tlcv-cp and roja's primer) were used to study, which resulted *800 bp with tlcv-cp in 3/3 samples and *1.3 kb amplicons with roja's primer in 3/4 samples. for further identification of the begomovirus, the pcr amplicons were cloned and sequenced (genbank accession no. eu439260 and eu888908, respectively). the blastn search analysis of eu439260 indicated 99-95% identity with several isolates of tomato leaf curl new delhi virus (tolcndv). the phylogenetic analysis also showed closest relationships of the isolate (eu439260) with tolcndv isolates. based on highest sequence identity and closed relationships with tolcndv the virus isolated from bitter gourd was considered as an isolate of tomato leaf curl new delhi virus. while, blastn search analysis of eu888908 isolate, shared highest 99-97% identites with pepper leaf curl bangladesh virus (peplcbv) isolates. the phylogenetic analysis of the virus isolate with selected begomovirus isolates revealed a closest relationship with peplcbv. these results confirmed the association of peplcbv on bitter gourd. study revealed the variability of viruses on bitter gourd in eastern up, india. tobacco streak virus groundnut isolate was characterized biologically by taking six cultivars (jl24, tmv2, k6, k7, k9) and one pre-release culture (k1271) using seedlings of 7-84 days old under glasshouse conditions. there were clear differences were observed among cultivars tested regarding incubation period, percent seedling wilt and time taken to death of seedlings. k-7 was least susceptible among all the cultivars tested and it supported least virus titer (a 405 nm: 0.11-1.23). both localized (necrotic lesions on leaf, veinal necrosis, leaf yellowing, wilting) and systemic (petiole necrosis, necrotic lesions on young leaves, death of top growing buds not only on main stem but also on all primaries (side shoots), followed by stem necrosis, stunted growth, axillary shoot proliferation with small leaves having general chlorosis, peg necrosis, pod necrosis, pod size reduction, wilt of plants) symptom were observed in all cultivars tested. biological differentiation of tsv and gbnv was made by sap inoculation of both viruses separately using susceptible groundnut cultivar jl24 under glasshouse conditions. there were certain similarities and differences were observed between these viruses infecting groundnut. seed infection of tsv ranged from 18.9 to 28.9% in seeds collected from naturally infected and sap inoculated groundnut cultivars/pre-releases (jl24, tmv2, k-6, k-7, k-9 and k-1271) belonging to spanish and virginia types. tsv was detected both in pod shell and seed testa from pod samples produced by sap inoculation under glasshouse conditions. however, seed transmission of tsv was not observed in groundnut. coat protein (cp) gene of three groundnut tsv isolates (gn-ap-1-00; gn-ap2-04; gn-ap3-07) were sequenced and all the three isolates contained a single open reading frame (orf) of 717 bp nucleotide and could potentially code for 238 amino acids (aa). cp gene of tsv isolates originating from different hosts shared high degree of sequence identity both at nucleotide (97.6-100%) and amino acid (95.7-100%) levels respectively. tones grown in an area of 3.83.430 ha (fao stat2007). in india papaya is grown in nearly 80,000 ha with an annual production of 7,00,000 tones (fao stat 2007) and occupies fourth place in the world. the crop is severely affected by a number of viruses. papaya ring spot virus (prsv-p) is the most important virus. the detection of virus infection in plants has traditionally involved either bioassay on indexing plants and or immunological methods (hill 1981, torrence and jones 1981) . use of nucleic acid probes has improved the detection and sensitivity of viruses. the most common non-radioactive probes are biotynilated probes, which are very specific and sensitive. papaya ring spot virus (prsv-p) is a positive sense ssrna virus belonging to the genus potyvirus family potyviridae and transmitted by aphids. prsv-p coat protein gene region was used as template cdna for probe preparation. dot-blot hybridization with the biotin labeled probe were performed for prsv-p detection. the clarified sap of healthy and infected plants were serially diluted and spotted onto the nitrocellulose membrane, hybridized to biotin labeled probe. biotin labeled rna's are employed as probes, with a subsequent detection based on streptavidin-alkaline phosphatase conjugates. the sensitivity for viral detection of the biotin labeled probe was found to be sensitive than enzyme linked immunosorbent assay (elisa). in recent years tospovirus is causing devastating damage to the yield of vegetables in india. it infects economically important crops viz., tomato, chilli, peppers, groundnut, watermelon and various legumes. now it is emerging as severe disease in brinjal also. in order to monitor the natural occurrence and distribution of tospovirus in vegetable, surveys were conducted in the predominant brinjal growing areas of gujarat, karnataka, maharashtra and andhra pradesh during 2008-2010 incidence ranging from 5 to 10%, 0 to 80%, 1 to 40%, and 0 to 55.78% respectively. samples collected from different places of india were found positive to pbnv in direct antigen coating-enzyme linked immunosorbent assay (dac-elisa). pbnv infected brinjal plants showed mosaic mottling of leaves with leaf distortion, longitudinal streaks on the stem and necrotic rings on leaves and fruits. early infection led to severe stunting and abnormal fruiting. biological and molecular characterization of pbnv-brinjal isolates were compared with other isolates and results are discussed. for identification of virus causing mosaic symptoms on soybean various host plants were tested. plants species belonging to the different families viz. caricaceae, graminae, leguminosae, malvaceae and solanaceae were tested. the virus produced symptoms on diagnostic plant species like chenopodium album, c. quinoa, helianthus anus, phaseolus vulgaris and vigna ungiculata. among tested families the leguminosae that were the host of virus included arachis hypogea, the virus causing mosaic symptoms in soybean is inactivated between 50 and 55°c and between dilution of 10 -4 to 10 -5 . all the inoculated plants of assay host showed the symptoms at 50°c but not at 55°c. similarly local lesions produced at 10 -4 but not at 10 -5 . the virus in crude sap was infectious up to 72 h but not at 96 h at room temperature. however, the percentage infectivity decreased progressively as the aging of the sap was increased at room temperature. on the basis of reactions on diagnostic hosts pvp-38 identification and characterization of potyvirus infected chilli (capsicul annum l the virus under study caused mild mosaic and severe mottling symptom in leaves of infected plants. the dilution end point (dep) of the virus was found to be 10 -3 to 10 -4 , longevity in vitro (liv) 1-3 days at room temperature (25°c), thermal inactivation point (tip) 50-55°c. electron microscopy of purified virus preparation revealed the presence of flexuous particle of size 780 nm long and 14 nm in width with characteristic cytoplasmic inclusions: pinwheels and scrolls. the virus was transmitted by sap and by aphid myzus persicae. the host range study revealed that the host species were restricted to family chenopodiaceae and solanaceae. on the basis of above characteristic, the virus under study was identified as potyvirus associated with mild mosaic and severe mottling symptom in capsicum. phytoplasma causing grassy shoot disease and sugarcane yellow leaf viruses are important pathogens of sugarcane. these pathogens are causing severe losses in sugarcane productivity. with a view to producing virus and phytoplasma free planting material of sugarcane, experiments were undertaken using infected varieties of sugarcane growing at the farms of sugarcane research institute. apical meristems measuring about 2 mm in length, were dissected out, surface sterilized and cultured on agar gelled murashige and skoog's (ms) medium containing growth regulators for shoot induction. the established shoot cultures were multiplied through repeated subcultures on fresh media at 10-12 days interval. elimination of gsd and scylv was confirmed through molecular analysis of regenerated plants using specific primers of scylv and gsd. results revealed that apical meristem culture technique is effective in eliminating the pathogens like scylv and phytoplasma (gsd) from the infected clones. this is probably the first report on elimination of grassy shoot disease in sugarcane through meristem culture. papaya ringspot virus (prsv), which causes the most widespread and devastating disease in papaya, isolates originating from different geographical regions in south india were collected and maintained on natural host papaya. the entire coat protein (cp) gene of papaya ringspot virus-p biotype (prsv-p) was amplified by reverse transcription-polymerase chain reaction (rt-pcr). the amplicon was inserted into pgem-t vector by t-a cloning method, sequenced and sub cloned into a bacterial expression vector prset-a using directional cloning strategy. the prsv coat protein was over expressed as fusion protein in e. coli. sds-page gel revealed that cp expressed as a *40 kda protein. the recombinant coat protein (rcp) fused with 69 his-tag was purified from e. coli using ni-nta resin. the antigenicity of the fusion protein was determined by western blot analysis using antibodies raised against purified prsv. the purified rcp was used as an antigen to produce high titer prsv specific polyclonal antiserum. the resulting antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (ic-rt-pcr) assay and compared its sensitivity levels with elisa based assays for detection of prsv isolates. ic-rt-pcr was shown to be the most sensitive test followed by dot-blot immunobinding assay (dbia) and plate trapped elisa. key: cord-001938-n2d5fw2f authors: ong, david s. y.; spitoni, cristian; klein klouwenberg, peter m. c.; verduyn lunel, frans m.; frencken, jos f.; schultz, marcus j.; van der poll, tom; kesecioglu, jozef; bonten, marc j. m.; cremer, olaf l. title: cytomegalovirus reactivation and mortality in patients with acute respiratory distress syndrome date: 2016-03-01 journal: intensive care med doi: 10.1007/s00134-015-4071-z sha: doc_id: 1938 cord_uid: n2d5fw2f purpose: cytomegalovirus (cmv) reactivation occurs frequently in patients with the acute respiratory distress syndrome (ards) and has been associated with increased mortality. however, it remains unknown whether this association represents an independent risk for poor outcome. we aimed to estimate the attributable effect of cmv reactivation on mortality in immunocompetent ards patients. methods: we prospectively studied immunocompetent ards patients who tested seropositive for cmv and remained mechanically ventilated beyond day 4 in two tertiary intensive care units in the netherlands from 2011 to 2013. cmv loads were determined in plasma weekly. competing risks cox regression was used with cmv reactivation status as a time-dependent exposure variable. subsequently, in sensitivity analyses we adjusted for the evolution of disease severity until onset of reactivation using marginal structural modeling. results: of 399 ards patients, 271 (68 %) were cmv seropositive and reactivation occurred in 74 (27 %) of them. after adjustment for confounding and competing risks, cmv reactivation was associated with overall increased icu mortality (adjusted subdistribution hazard ratio (shr) 2.74, 95 % ci 1.51–4.97), which resulted from the joint action of trends toward an increased mortality rate (direct effect; cause specific hazard ratio (hr) 1.58, 95 % ci 0.86–2.90) and a reduced successful weaning rate (indirect effect; cause specific hr 0.83, 95 % ci 0.58–1.18). these associations remained in sensitivity analyses. the population-attributable fraction of icu mortality was 23 % (95 % ci 6–41) by day 30 (risk difference 4.4, 95 % ci 1.1–7.9). conclusion: cmv reactivation is independently associated with increased case fatality in immunocompetent ards patients who are cmv seropositive. electronic supplementary material: the online version of this article (doi:10.1007/s00134-015-4071-z) contains supplementary material, which is available to authorized users. abstract purpose: cytomegalovirus (cmv) reactivation occurs frequently in patients with the acute respiratory distress syndrome (ards) and has been associated with increased mortality. however, it remains unknown whether this association represents an independent risk for poor outcome. we aimed to estimate the attributable effect of cmv reactivation on mortality in immunocompetent ards patients. methods: we prospectively studied immunocompetent ards patients who tested seropositive for cmv and remained mechanically ventilated beyond day 4 in two tertiary intensive care units in the netherlands from 2011 to 2013. cmv loads were determined in plasma weekly. competing risks cox regression was used with cmv reactivation status as a time-dependent exposure variable. subsequently, in sensitivity analyses we adjusted for the evolution of disease severity until onset of reactivation using marginal structural modeling. results: of 399 ards patients, 271 (68 %) were cmv seropositive and reactivation occurred in 74 (27 %) of them. after adjustment for confounding and competing risks, cmv reactivation was associated with overall increased icu mortality (adjusted subdistribution hazard ratio (shr) 2.74, 95 % ci 1.51-4.97), which resulted from the joint action of trends toward an increased mortality rate (direct effect; cause specific hazard ratio (hr) 1.58, 95 % ci 0.86-2.90) and a reduced successful weaning rate (indirect effect; cause specific hr 0.83, 95 % ci 0.58-1. 18 ). these associations remained in sensitivity analyses. the although the burden of cytomegalovirus (cmv) disease has been well established in immunocompromised patients [1] , cmv viremia has also been described in intensive care unit (icu) patients without known prior immune deficiency. this almost exclusively results from systemic viral reactivation, and incidence rates of up to 40 % have been reported in critically ill cmv seropositive subjects [2] [3] [4] [5] [6] [7] [8] [9] . furthermore, cmv reactivation in critically ill patients has been associated with a prolonged duration of mechanical ventilation [2, 4, [9] [10] [11] [12] [13] , an increased length of stay in the icu [3, 5, 9, 10, 13] , and excess mortality [2, 4, [7] [8] [9] . nevertheless, it remains uncertain whether these findings imply that cmv reactivation is a truly independent risk factor with respect to these observed poor clinical outcomes because most studies that have assessed these associations did not adequately account for all possible sources of bias. as a consequence, cmv viremia might merely be a marker of illness severity, contributing only little to the overall burden of disease. to achieve an accurate estimation of the true effect of cmv reactivation on clinical outcome, it is crucial in observational studies to adjust for the time-dependent occurrence of cmv reactivation and the evolution of disease severity prior to its onset. moreover, the presence of competing events should be taken into account when follow-up time is censored [14] . for instance, when icu mortality is the outcome, then icu discharge is a competing risk that prohibits the event of interest from occurring first. patients with acute respiratory distress syndrome (ards) often have a long and complicated disease course in the icu, which portends a particular risk for viral reactivations [15, 16] . despite the uncertainties regarding the clinical relevance of cmv disease in immunocompetent critically ill patients, it is etiologically plausible that virus reactivation adds to the pulmonary pathology in patients with ards. in experimental murine studies, cmv reactivation caused exacerbated and prolonged cytokine and chemokine expression in lung tissues, which eventually led to increased pulmonary fibrosis compared to controls [17] . in a clinical study of open lung biopsies in ards patients with prolonged respiratory failure or in whom microbiological cultures remained negative, cmv pneumonia was found in 30 % of cases [18] . both findings suggest that cmv-related pulmonary pathology may be causally linked to the clinical disease course following ards onset, especially in the most severely ill patients who require prolonged mechanical ventilation. if cmv reactivation does contribute to poor clinical outcome in these patients, either prophylaxis or pre-emptive therapy with (val)ganciclovir may be considered. the aim of this study was to estimate the proportion of deaths that can be attributed to systemic reactivation of cmv in ards patients who are latent carriers of the virus. some results of this study have been previously reported in the form of an abstract [19] . the present study was conducted within the framework of the molecular diagnosis and risk stratification of sepsis (mars) cohort (clinicaltrials.gov identifier: nct01905033) for which the institutional review board approved an opt-out method of informed consent (protocol number 10-056c) [20] . we prospectively included consecutive adults who presented with ards to the mixed icus of two tertiary care hospitals in the netherlands between january 2011 and december 2013 and required mechanical ventilation beyond day 4 of icu admission. since data collection for our study started before publication of the berlin definition in 2012, ards was defined according to the american-european consensus conference criteria [21] : that is, the diagnosis required an acute onset of symptoms, the presence of bilateral infiltrates on chest radiography, a pulmonary artery occlusion pressure less than 18 mmhg and/or the absence of left ventricular dysfunction, and pao 2 /fio 2 ratio (p/f) less than 300. we excluded patients who had received (val)ganciclovir, (val)acyclovir, cidofovir, or foscarnet in the week before icu admission and those with known immunodeficiency [16] . immunodeficiency was defined as a history of solid organ or stem cell transplantation, infection with the human immunodeficiency virus, hematological malignancy, use of immunosuppressive medication (more than 0.1 mg prednisone per kilo for more than 3 months, more than 75 mg prednisone per day for more than 1 week, or equivalent), chemotherapy/radiotherapy in the year before icu admission, and any known humoral or cellular immune deficiency. leftover plasma, which was harvested from blood samples obtained daily as part of routine patient care, was stored at -80°c within 4 h after blood draw. cmv serostatus was determined by an enzyme immunoassay (enzygnost cmv/igg, siemens healthcare diagnostic products, marburg, germany). subsequently, in seropositive patients only, viral loads in plasma were determined by real-time taqman cmv-dna polymerase chain reaction [22] . cmv loads were determined on a weekly basis for a maximum of 30 days following study inclusion (i.e., day 5 of icu admission). for intermediary days, on which quantitative pcr was not performed, we estimated viral loads by log-linear imputation. cmv reactivation was defined as a viral load of at least 100 international units per milliliter (iu/ml), as calibrated according to the cmv world health organization (who) standard. screening for cmv was not part of routine clinical practice in either participating hospital. neither serology results nor viral loads measured as part of our study were made available to the treating physicians, and none of the included patients therefore received antiviral treatment directed against cmv. mortality was the outcome of primary interest in this study and was defined as death on mechanical ventilation before day 35 (i.e., day 30 following study inclusion). successful weaning, which is a competing event of the primary outcome, was defined as complete liberation from mechanical ventilatory support on two or more consecutive days before day 35. we considered distal end points more likely to be amenable by pre-existing comorbidities, as well as specific end-of-life practices, bed availability, and other local factors. nonetheless, in a subsequent sensitivity analysis, we used discharge and death in icu as alternative end points. for our primary analyses we used cox proportional hazards modeling, in which mortality and successful weaning were considered as competing events and cmv reactivation status was fitted as a time-dependent variable. possible confounders that were screened included all patient characteristics and therapeutic interventions listed in table 1 , and some markers of disease severity: acute physiology and chronic health evaluation apache acute physiology and chronic health evaluation, ards acute respiratory distress syndrome, copd chronic obstructive pulmonary disease, icu intensive care unit, peep positive end expiratory pressure, p/f partial pressure of oxygen in arterial blood to fraction of inspired oxygen ratio (apache) iv score, presence of septic shock, partial pressure of oxygen in arterial blood to fraction of inspired oxygen ratio, and positive end expiratory pressure (peep) setting. to account for possible confounding, we included baseline covariables that showed differences between the reactivated and non-reactivated groups at a p value of less than 0.30, and changed the crude effect estimates for either mortality or weaning by more than 10 %. we included only the strongest (possible) confounders by using these two criteria combined in order to avoid statistical overfitting (i.e., incorporating too many variables given the limited number of events). the two possible outcomes are interrelated as increased mortality may negatively impact the duration of mechanical ventilation. a competing risks analysis accommodates for this by providing two measures of association. first, the cause-specific hazard ratio (cshr) estimates the direct effects of cmv reactivation on each outcome of interest (i.e., mortality on the ventilator and successful weaning). second, the subdistribution hazard ratio (shr) estimates the risk of dying from reactivation at a given time-point, while accounting for the competing risk of successful weaning. to obtain direct estimates of cumulative risks in terms of the shr we used the fine and gray model [23] . finally, to estimate the populationattributable fraction of mortality due to cmv reactivation, we used a multi-state model (fig. s1 ), which accounts for the time of reactivation [24] . confidence intervals were calculated by bootstrap resampling [25, 26] . despite these efforts to accurately assess the effect of cmv reactivation on clinical outcomes, residual confounding may still remain, because markers of illness at baseline (which we included in all multivariable analyses) may no longer be representative of the disease state at the time of reactivation onset. thus, we performed a sensitivity analysis using marginal structural modeling to adjust for the evolution of disease severity prior to the onset of cmv reactivation (see also supplementary material) [27, 28] . such analysis first involves estimation of the daily probabilities of cmv reactivation using a multivariable logistic regression model that includes markers of disease severity on a daily basis. these probabilities are used to calculate an inversed probability weight that is then included as a summary measure of all relevant covariables in the final cox regression model. however, because marginal structural modeling requires many assumptions that are difficult to be checked, we considered this a sensitivity analysis only. data were analyzed with sas 9.2 (cary, nc, usa) and r 2.15.1 software (r foundation for statistical computing, vienna, austria; packages ''etm'', ''mstate'', ''ipw''). we enrolled 544 patients with ards who required mechanical ventilation for more than 4 days (fig. s2 ). of these 143 were excluded because of known prior immunocompromise or antiviral treatment and two were excluded because of missing samples. subsequently, 271 (68 %) patients tested seropositive for cmv and were thus included in the study. ards was of primary pulmonary origin in 158 (58 %) of these cases, whereas the remainder was of secondary etiology (e.g., associated with non-pulmonary sepsis, major surgery, or blood transfusion). cmv reactivation cmv reactivation occurred in 74 (27 %) of the included patients (table 1 ). these patients more frequently hadat the time of icu admission-concurrent septic shock, higher apache iv scores, and renal insufficiency compared to patients who never had cmv reactivation. in addition, a larger proportion of these patients were receiving high dose corticosteroid therapy during the first days in icu. the median time from icu admission to onset of reactivation was 8.5 days (interquartile range (iqr) 4-11). within the subgroup of patients acquiring cmv reactivation the proportion of individuals having relatively high viral loads (at least 1000 iu/ml) increased over time (fig. 1) . in a patient population that is selected by an icu stay of at least 5 weeks, the proportion with cmv viremia is as high as 14 of 23 patients (61 %). on day 30 after study inclusion (this was 35 days following icu admission) 52 (19 %) patients had died, 209 the quantitative pcr results were calibrated according to the cmv who standard; viral loads greater than or equal to 1000 iu/ml were denoted 'high reactivation'. viral loads of 100-999 iu/ml were denoted 'low reactivation', and undetectable loads or viral loads below 100 iu/ml were denoted 'no reactivation' (77 %) were successfully weaned, and 10 (4 %) remained still on mechanical ventilation (table 2 ). in crude analyses, patients with cmv reactivation had both a longer duration of mechanical ventilation (15 (iqr 10-26) vs. 8 (iqr 6-12) days; p \ 0.01) and higher mortality (23 of 74 (31 %) vs. 29 of 197 (15 %) patients; p \ 0.01) compared to subjects without reactivation. table 3 shows the results of the various cox survival regression analyses. baseline variables associated with reactivation status (at p \ 0.30) which changed the crude effect estimate by more than 10 % included the apa-che iv score, use of high dose corticosteroid therapy, and peep setting. in the primary multivariable adjusted analysis, cmv reactivation was no longer statistically associated with either increased mortality or a reduced rate of successful weaning. however, simultaneous effects on both the daily rates of death and weaning did reveal a significant association with overall mortality when competing risks were accounted for (shr 2.74, 95 % ci 1.51-4.79). as a post hoc sensitivity analysis, we then used marginal structural modeling to assess potential residual confounding by differences in the evolution of disease severity prior to cmv reactivation between both groups, but found very similar results (table 3) . changing the definitions of our primary end points to include all deaths in the icu (irrespective of mechanical ventilation status) and discharge (rather than successful weaning) also did not change these findings (table s1 ). furthermore, the independent association with mortality remained among subgroups of patients receiving and not receiving high dose corticosteroid therapy; shr 2.60 (95 % ci 1.29-5.25) and 3.61 (95 % ci 1.24-10.48), respectively (table s2 ). corticosteroids were mostly used for the treatment of concurrent septic shock (121 of 149 cases). figure 2 shows the predicted mortality in a hypothetical population of ards patients in which all cmv reactivation is prevented, compared to true (observed) mortality in the study population. the population-attributable fraction of icu mortality due to cmv reactivation was estimated at 23 % (95 % ci 6-41 %) by day 30, which translates into an absolute mortality difference of 4.4 % (95 % ci 1.1-7.9). data are presented as hazard ratios with 95 % ci. the causespecific hazard ratio (cshr) estimates the direct effect of cmv reactivation on clinical outcome (i.e., successful weaning or death on mechanical ventilation). the subdistribution hazard ratio (shr) is a summary measure of both separate cause-specific hazards and estimates the overall risk of dying from cmv reactivation while taking into account the competing event of successful weaning a apache iv score, use of high dose corticosteroid therapy, and peep setting b time-dependent covariables included the risk, injury, failure, loss and end-stage kidney disease (rifle) score, sequential organ failure assessment (sofa) score, presence of septic shock, and use of high dose corticosteroid therapy, which were all measured on a daily basis until 24 h prior to reactivation onset in order to explore possible causal pathways for the observed association between cmv reactivation and death, we performed a post hoc descriptive analysis of the trajectories of organ dysfunction, pulmonary and inflammatory markers over time following reactivation. in short, we compared the 74 patients having cmv reactivation with 74 non-exposed patients who were matched on baseline characteristics and their length of stay in icu at the onset of reactivation (table s3 ). in summary, the total burden of organ dysfunction was slightly higher in patients at the start of cmv reactivation compared to matched non-exposed control subjects, although individual markers of pulmonary dysfunction and inflammation were similar. more importantly, there was a clear trend towards resolution of organ dysfunction over time in nonexposed subjects that was less pronounced in patients having cmv reactivation. however, it should be emphasized that these findings should be interpreted very carefully because of the presence of informative censoring (i.e., patients who die or get discharged do not further contribute to average scores on the group level). cmv reactivation in ards patients increased the overall risk of death on the ventilator through the combined effect of subtle alterations in both the daily rates of death and successful weaning. after accounting for multiple sources of confounding, the absolute mortality that can be attributed to cmv reactivation was estimated to be 4.4 % by day 30 following study inclusion. previous findings of excess mortality have triggered debate whether antiviral prophylaxis should be used [29, 30] . however, a greater understanding of pathophysiology and clinical risk factors is necessary to select the optimal target population for such strategies. in our study, reactivation rates were 27 % in ards patients overall and 34 % among those with concurrent septic shock. the latter finding might be explained by the increased severity and duration of immune suppression that may be observed in patients with septic shock, including a pronounced depletion of t cells [31, 32] . indeed, a recent study investigating the potential use of antiviral prophylaxis based on the screening of ards patients for cmv seroprevalence found that such a strategy is unlikely to be beneficial overall, but suggested a possible benefit in a post hoc subgroup of patients with septic shock [16] . as the proportion of patients with cmv reactivation increased in time, altering the minimal length of stay in the icu as a criterion may also improve the selection of a high-risk target population. until then, a pre-emptive treatment strategy (by which patients would be screened for cmv and treated only if reactivation occurs) seems more attractive because the number of patients exposed to the toxicity of (val)ganciclovir would be reduced by 73 %. however, the effects of pre-emptive compared to prophylactic treatment on relevant patient outcomes are most likely lower, as treatment is initiated only after reactivation has already begun. intervention trials comparing prophylaxis, pre-emptive treatment, and wait-andsee strategies are necessary before any evidence-based recommendations regarding the clinical management of cmv reactivation in critically ill patients with ards can be made. our study has several strengths. first, observations were nested within a large prospective data collection initiative that included consecutive patients, thereby minimizing selection bias [20] . all ards events were diagnosed by dedicated trained observers, which minimizes information bias. moreover, we used a highly sensitive method of quantitative real-time pcr for cmv detection. most importantly, we used advanced methodologies to account for both competing risks and timedependent information in an attempt to produce unbiased estimates of the independent association between cmv reactivation and clinical outcome. this methodological approach was mainly necessary because of two reasons. first, cox regression analysis requires that censoring of survival time must be non-informative, but in our study this was clearly not the case since ards patients who are weaned and discharged from the icu alive are in a better health state than those who remain on the ventilator beyond that time point [33, 34] . furthermore, when icu mortality is the event of interest, then discharge must be regarded as a competing event as it precludes this outcome from being observed [14] . the use of the subdistribution hazard model provides a general solution to this informative censoring. second, the median time to cmv reactivation in our cohort was 8.5 (iqr 4-11) days. if ignored, such delays may cause distortion (termed immortal time bias) as nonexposed time observed before the onset of reactivation will be wrongfully attributed to the exposed time at risk, resulting in underestimation of effects associated with cmv reactivation [35, 36] . time-dependent fitting of cmv reactivation status in our regression models resolved this issue. our study also has several limitations. first, even the use of advanced methodology cannot rule out the possibility of unmeasured confounding in an observational study. therefore, it remains somewhat uncertain whether the excess mortality that we observed can be fully attributed to cmv reactivation, or whether other unknown factors-including other viral reactivations [8, 37]-may also be involved. second, the principle of multivariable analysis to adjust for confounders is to statistically 'force' exposed and non-exposed patients to be similar in all aspects of disease aside from their reactivation status. however, in a dynamic icu setting, during which critically ill patients continuously deteriorate and improve over time, it is very difficult to verify whether such adjustment was successful. we performed marginal structural modeling as a sensitivity analysis to assess the possible impact of variations in the evolution of disease severity between patients on our effect estimates, yet found very similar results as in our primary analysis. third, we measured systemic cmv reactivation in plasma but did not collect information about concurrent viral loads in the lungs. this study, therefore, provides no insight into either the prevalence or relevance of pulmonary cmv reactivations. of note, previous studies have shown that pulmonary reactivation may occur without the concurrent viremia [4, 38, 39] . furthermore, we focused exclusively on the occurrence of reactivation while patients were on mechanical ventilation (primary analysis) or in the icu (sensitivity analysis), as we considered these to be the most relevant time windows to potentially treat or prevent cmv reactivation in the icu. however, because of this deliberate focus we cannot provide information about possible episodes of reactivation that may have occurred later. likewise we did not investigate the occurrence of reactivations after day 35 in the icu. thus, this study only provides insight into the short-term effects of systemic cmv reactivation in ards patients in settings in which screening or antiviral prophylaxis is not part of routine clinical practice. in conclusion, systemic reactivation of cmv in immunocompetent ards patients is common and independently associated with death in the icu. these findings support the need for future studies to better predict cmv reactivation as well as to evaluate the efficacy of treatment strategies directed against cmv reactivation in these patients. human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments active cytomegalovirus infection is common in mechanically ventilated medical intensive care unit patients virological and immunological features of active cytomegalovirus infection in nonimmunosuppressed patients in a surgical and trauma intensive care unit looking for biological factors to predict the risk of active cytomegalovirus infection in nonimmunosuppressed critically ill patients cytomegalovirus seroprevalence as a risk factor for poor outcome in acute respiratory distress syndrome pulmonary cytomegalovirus reactivation causes pathology in immunocompetent mice a contributive result of openlung biopsy improves survival in acute respiratory distress syndrome patients cytomegalovirus reactivation in critically ill patients with acute respiratory distress syndrome interobserver agreement of centers for disease control and prevention criteria for classifying infections in critically ill patients the american-european consensus conference on ards. definitions, mechanisms, relevant outcomes, and clinical trial coordination diagnosing herpesvirus infections by real-time amplification and rapid culture a proportional hazards model for the subdistribution of a competing risk attributable mortality due to nosocomial infections. a simple and useful application of multistate models use of multistate models to assess prolongation of intensive care unit stay due to nosocomial infection the time-dependent bias and its effect on extra length of stay due to nosocomial infection marginal structural models and causal inference in epidemiology attributable mortality of ventilator-associated pneumonia: a reappraisal using causal analysis treating hsv and cmv reactivations in critically ill patients who are not immunocompromised: pro treating hsv and cmv reactivations in critically ill patients who are not immunocompromised: con sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy transient cd8-memory contraction: a potential contributor to latent cytomegalovirus reactivation adjusting for time-varying confounding in the subdistribution analysis of a competing risk modeling the effect of time-dependent exposure on intensive care unit mortality immortal time bias in critical care research: application of timevarying cox regression for observational cohort studies effectiveness of inhaled corticosteroids in chronic obstructive pulmonary disease: immortal time bias in observational studies coreactivation of human herpesvirus 6 and cytomegalovirus is associated with worse clinical outcome in critically ill adults immunological insights into the pathogenesis of active cmv infection in non-immunosuppressed critically ill patients detection of herpesvirus ebv dna in the lower respiratory tract of icu patients: a marker of infection of the lower respiratory tract? acknowledgments we thank huberta dekker (department of medical microbiology, university medical center utrecht, the netherlands) for her logistical support in this project, and the participating icus and research nurses of the two medical centers for their help in data acquisition. this work was supported by the center for translational molecular medicine (http://www.ctmm.nl), project mars (grant 04i-201). jk received a personal fee from becton-dickinson. the sponsor did not play a role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.the mars consortium also includes the following per key: cord-010130-28bt3x25 authors: crocchiolo, r.; bramanti, s.; vai, a.; sarina, b.; mineri, r.; casari, e.; tordato, f.; mauro, e.; timofeeva, i.; lugli, e.; mavilio, d.; carlo‐stella, c.; santoro, a.; castagna, l. title: infections after t‐replete haploidentical transplantation and high‐dose cyclophosphamide as graft‐versus‐host disease prophylaxis date: 2015-03-26 journal: transpl infect dis doi: 10.1111/tid.12365 sha: doc_id: 10130 cord_uid: 28bt3x25 background: recently, a platform of t‐cell replete haploidentical hematopoietic stem cell transplantation (haplo‐hsct) using post‐transplant cyclophosphamide (cy) has shown high reproducibility and acceptable safety profile. method: this prospective cohort analysis allowed us to collect data on infections among 70 consecutive recipients of haplo‐hsct affected by various hematologic malignancies. results: after a median follow‐up of 23 months, cumulative incidence of viral infections was 70% (95% confidence interval [ci] 59–81) at 100 days and 77% (95% ci 67–87) at 1 year; 35 of 65 patients at risk had cmv reactivation (54%) and the rate of polyomavirus‐virus‐associated cystitis was 19% (13/70). cumulative incidence of bacterial and fungal infections at 1 year were 63% (95% ci 51–75) and 12% (95% ci 4–19), respectively. of note, only 1 invasive fungal infection occurred beyond 1 year after transplant (day +739). conclusion: in conclusion, despite a high rate of viral infections in the early period, present data suggest a satisfactory infectious profile after t‐cell replete haplo‐hsct using post‐transplant cy. these results may help clinicians to improve both prophylactic and therapeutic antimicrobial strategies in this emerging haploidentical setting. one of the major limitations of hematopoietic stem cell transplantation from haploidentical donor (haplo-hsct) is the impaired immune reconstitution owing to extensive immunosuppression necessary to overcome human leukocyte antigen disparity. despite important advances over the last decades, infections are still mostly responsible for toxicity and non-relapse mortality among transplanted patients, owing to prolonged immunosuppression related, or not, to chronic graft-versus-host disease (gvhd) (1, 2) . a platform for t-cell replete haplo-hsct using posttransplant cyclophosphamide (cy) (3) showed a low treatment-related mortality (trm) and a high feasibility with an acceptable safety profile. this type of haplo-hsct seems to compare favorably with t-cell depleted methods, in terms of infectious complications (3, 4) . to further explore this topic, we report herein the incidence of infections in a single-center cohort of 70 consecutive patients receiving t-cell replete haplo-hsct with post-transplant cy at our center, in order to provide useful information about the post-transplant period after this type of emerging transplant platform. data on patients with hematologic malignancies who underwent haplo-hsct between april 2009 and april 2014 at humanitas cancer center (milan, italy) were prospectively collected using electronic patients' charts. conditioning regimens were myeloablative, reducedintensity, or non-myeloablative. non-myeloablative conditioning consisted of cy 14.5 mg/kg/day intravenously (i.v.) on days à6 and à5, fludarabine 30 mg/ m 2 /day i.v. on days à6 to à2, and total body irradiation (tbi) 200 cgy on day à1. two reduced-intensity regimens were administered: (i) thiotepa 10 mg/kg/ day i.v. on day à6, fludarabine 30 mg/m 2 /day i.v. on days à5 to à2, cy 30 mg/kg/day i.v. on days à5 and à4, and tbi 200 cgy on day à1; or (ii) thiotepa 5 mg/ kg/day i.v. on days à6 and à5, fludarabine 50 mg/m 2 / day i.v. on days à4 to à2, and busulfan 3.2 mg/kg/day i.v. on days à4 and à3, as modified from sanz et al. (5) . the myeloablative regimen consisted of thiotepa 5 mg/ kg/day i.v. on days à6 and à5, fludarabine 50 mg/m 2 / day i.v. on days à4 to à2, and busulfan 3.2 mg/kg/day i.v. on days à4 to à2. all patients received unmanipulated bone marrow or mobilized peripheral stem cells. gvhd prophylaxis was performed with cy 50 mg/kg/day on days +3 and +4; tacrolimus 1 mg/day i.v. from day +5 (to reach a concentration of 5-15 ng/ml), or cyclosporine 3 mg/ kg/day i.v. from day +5 (to reach a concentration of 100-200 ng/ml), both up to day +100 and then tapered up to day +180, unless gvhd occurred; and mycophenolate mofetil 15 mg/kg 39 a day orally from day +5 to +35. infections were defined according to european society for blood and marrow transplantation (available at: http://www.ebmt.org/contents/about-ebmt/who-we-are/scientificcouncil/documents/idwpdefiniti ons.pdf), including microbiologically documented viral, bacterial, or fungal infections with or without laboratory and/or radiologic features consistent with organ involvement. cytomegalovirus (cmv) reactivation (or de novo infection) and disease were diagnosed as reported elsewhere (6) , and invasive fungal infections (ifis) were classified according to the definitions of the eortc/msg consensus group (7); only proven or probable ifis were recorded. data were recorded as of june 3, 2014 for all patients. the study was approved by the local institutional review board. antimicrobial prophylaxis was started during the conditioning regimen and consisted of acyclovir 500 mg/ m² 3 times in a day; levofloxacin 500 mg/day; and cotrimoxazole 2 tablets per day until day à2, and then 1 tablet every other day was resumed after hematologic reconstitution. antifungal prophylaxis was performed with an echinocandin (either caspofungin or micafungin 50 mg/day) until day +5, when itraconazole (200 mg/day i.v.) was administered, unless contraindicated; otherwise the echinocandin was maintained. after september 2013, the echinocandin was maintained for all patients. acyclovir, levofloxacin, and antifungal prophylaxis were administered until engraftment occurred. twice weekly blood polymerase chain reaction (pcr) cmv monitoring was started at day +15 until day +100 and weekly until day +180, or when clinically indicated. weekly epstein-barr virus (ebv) monitoring by pcr was started at day +15 up to day +100, or when clinically indicated. all other tests were performed whenever indicated. piperacillin-tazobactam alone or in combination with an aminoglycoside was administered as empirical therapy for febrile neutropenia, unless previous colonization for resistant bacteria was documented; in this case, an appropriate antibacterial agent was delivered. the same was true when a suspected bacterial infection occurred, with the exception of pneumonia for which linezolid was added, in combination with the abovecited antibacterial drug(s). first-line preemptive therapy for cmv infection/reactivation was with intravenous ganciclovir, whereas foscarnet was administered if the patient was in aplasia. ebv reactivation and polyomavirus-related hemorrhagic cystitis were treated by rituximab and cidofovir, respectively. threshold of cmv viremia for the initiation of therapy was 3300 copies/ml; threshold of ebv viremia was 10,000 copies/ml. analysis of circulating lymphocytes was performed at regular intervals whenever available, at days +7, +14, +21, and +28, and every month afterward. the following transplant infectious disease 2015: 17: 242-249 monoclonal antibodies and combinations were used: cd45, cd3/cd4, cd3/cd8, cd19, cd16/cd56 (beckman coulter, fullerton, california, usa), to quantify t, b, and nk cell compartments at the different time points studied. categorical variables were expressed as absolute numbers with respective percentage and continuous variables as the median with the respective range. cumulative incidence of viral, bacterial, or fungal infections was calculated using competing risk analysis (8) starting from the day 0 to the day of the first infection; death was considered as the competing event. infection incidence was also expressed as the events/1000 patient-days (pt-days) for each time period within 1 year after transplant, with intervals defined as follows: days 0-30, 31-100, 101-180, and 181-365. owing to the paucity of late events, data beyond day +365 were collected singularly and classified according to pathogen group. neutrophil engraftment was defined as the first of 3 consecutive days with a persistent count >0.5 9 10 9 /l; platelet engraftment was defined as the first of 3 consecutive days with a persistent count >20 9 10 9 /l (https://portal. ebmt.org/sites/clint2/clint/documents/statguidelines_ oct2003.pdf). the kaplan-meier method was used to compute overall survival (9); cumulative incidence of trm and acute and chronic gvhd were calculated using competing risk analysis (8) . death because of documented infection was defined as infection-related death. logrank test was used to compare the incidence of infections with flow cytometry results. seventy consecutive adult patients undergoing haplo-hsct were identified. the main patient and transplant characteristics are shown in table 1 . fifty-five patients (79%) were affected by lymphoma, and 81% of patients underwent haplo-hsct in partial or complete remission. only 5 patient/donor pairs were cmv dà/rà. as concerns antifungal prophylaxis, 5 patients did not receive itraconazole owing to moderate increase in pre-transplant liver function tests (n = 1) or reduction in left ventricular ejection fraction (n = 4); 11 more patients did not receive itraconazole because haplo-hsct was performed after september 2013. three patients received secondary antifungal prophylaxis with voriconazole because of previous pulmonary aspergillosis. median follow-up of living patients was 23 months (range 1-60) from day of stem cell infusion. at last follow-up, a total of 224 documented infectious events occurred among 67 of 70 patients, with a median of 3 events/patient (range 1-10); 55% were of viral origin (n = 123), 40% bacterial (n = 89), 5% fungal (n = 11). cumulative incidence of first viral infection was 70% (95% confidence interval [ci] 59-81) and 77% (95% ci 67-87) at day +100 and +365, respectively; at day +365, the incidence of bacterial infections was 63% (95% ci 51-75), and that of ifis was 12% (95% ci 4-19) (fig. 1) . in 54% (35 of 65 patients at risk) at least 1 cmv reactivation developed; of these, 26 patients had 1 cmv reactivation, and 5, 2, 1, and 1 patients had a total of 2, 3, 4, and 7 cmv reactivations, respectively. two non-fatal (1 colitis, 1 pneumonia) and 1 fatal (pneumonia) cmv diseases occurred. no primary cmv infections occurred in the 5 cmv dà/rà patient/donor pairs. polyomavirus-related hemorrhagic cystitis was observed in 13 patients (19%): 10 were caused by bk virus and 3 by jc virus. importantly, no ebv-related lymphoproliferative disorders occurred so far. forty-five patients (64%) presented with at least 1 documented bacterial infection: 10 (14%), 21 (30%), and 14 (20%) patients had an infection by grampositive, gram-negative, or both types of bacteria, respectively. eleven ifis were detected in 9 patients: n = 6 probable invasive aspergillosis (pneumonia in 5 patients and sinusitis in 1), n = 5 invasive candidiasis, all by non-albicans candida (2 candidemias, 2 colitis, and 1 hepatosplenic candidiasis); median of occurrence was 62 days from haplo-hsct (range 0-739). among the 3 patients receiving secondary antifungal prophylaxis, only 1 non-albicans candida colitis was observed at day +77 in 1 patient. no ifis occurred under active gvhd. notably, only 2 ifis occurred beyond day +180: 1 pulmonary aspergillosis at day +191, and 1 candidemia at day +739, this latter in a patient who was under salvage treatment for post-transplant relapse. details of etiologies are reported in table 2 . we did not observe significant differences of infectious events according to conditioning regimen administered (data not shown). when considering the timing of all episodes, bacterial infections occurred mostly between day 0 and +30, whereas viral infections/ reactivations between days +31 and +100, with 11.08 bacterial events/1000 pt-days between day 0 and +30, and 15.15 viral events/1000 pt-days between days +31 and +100 (fig. 2) . the overall incidence of viral events between day 0 and day +180 was 8.8 events/1000 ptdays. a total of 13 bacterial and 13 viral infections were observed after 1 year from transplant. engraftment rate was 91% (64 of 70 patients), with a median of 20 days (range 14-49) and 27 days (range 16-115) for neutrophil and platelet recovery, respectively. four patients died before engraftment (on days +13, +22, +33, and +48 because of gram-negative sepsis, multiorgan failure, progressive disease, and bacterial pneumonia, respectively) and 2 presented primary antibody-linked graft failure, and are alive at last follow-up, after autologous reconstitution (days +530 and +834). cumulative incidence of acute grade 2-4 and 3-4 gvhd was 23% (95% ci 10-40) and 4% (95% ci 0-9) respectively; chronic gvhd was 8% (95% ci 1-27). two-year overall survival was 48% (95% ci 35-58) and trm was 26% (95% ci 12-38); 18 patients (26%) relapsed or progressed after haplo-hsct. infection-related deaths were 9% (6/70, occurring between days +13 and +113; bacterial pneumonia = 2, gram-negative sepsis = 1, cmv pneumonia = 1, h1n1 pneumonia = 1, and jc virus-related progressive multifocal leukoencephalopathy = 1). the other, non-infectious, causes of trm were heart failure (n = 4), secondary malignancy (n = 2 myelodysplastic syndrome, n = 1 esophageal cancer), multiorgan failure (n = 3), thrombotic microangiopathy (n = 1), and acute hepatitis (n = 1). at last follow-up, 34 patients are alive and 28 of them are in complete remission. immunophenotypic analysis reveals a progressive increase in all lymphocyte subset counts from day +7 through later post-transplantation time points. we found a trend toward less viral infection incidence among those patients who have a total lymphocyte count >2/mm 3 at day +28: hazard ratio 0.67, p = 0.10. no other associations were observed. lymphocyte subsets and number of patients analyzed are shown in figure 3 . in the present analysis, we described infectious complications after unmanipulated, t-cell replete haplo-hsct using post-transplant cy in 70 consecutive patients and found, aside from a high incidence of viral infections/reactivations, especially in the early posttransplant period, a quite low incidence of late bacterial infections, together with a very low incidence of ifis after day +180 (2 events in the overall 11 observed). present findings confirm that the infectious profile is better in t-cell replete vs. t-cell depleted haplo-transplantation (10); the lower incidence of infections observed after day +100 may reflect a partial and quite effective restoration of antimicrobial immunity during the post-transplant period in this type of haplo-hsct. importantly, the low rate of chronic gvhd seen in our cohort is likely to contribute to this phenomenon, as chronic gvhd is known to be a major risk factor of late morbidity and mortality (11) . nevertheless, we found an unexpected 26% trm incidence, higher than that originally reported with post-hsct cy (3); this may be a result of the inclusion of patients with more advanced disease in the haplo-hsct program at our center. as concerns viral infections, our results are in line with previous publications in the setting of t-cell replete haploidentical transplants. ciurea et al. (12) reported 10.8 events/1000 pt-days within the first 6 months from transplant (vs. 8.8 events/1000 pt-days between day +0 and +180 in our hands), and raiola et al. (13) found that 62% of patients presented with a viral infection in the first year. the 54% of cmv reactivations found here was comparable to the 38-50% reported in similar haploidentical settings (3, 12, 13); the slightly higher incidence that we found here may be explained by the longer follow-up in our series. the polyomavirus-associated cystitis rate of 19%, which is lower than that reported in the myeloablative setting (14) , is likely because of the different conditioning regimens in our cohort, although a role played by the different gvhd prophylaxis cannot be excluded; indeed, bk virus nephropathy was found to be more frequently associated with tacrolimus than with cyclosporine in recipients of kidney allografts (15) . here, a quarter of the patients (18/70) received cyclosporine as gvhd prophylaxis. interestingly, we confirm the lack of ebv-related lymphoproliferative disorders, as recently reported also by kanakry et al. (16) . we found a 12% incidence of fungal infections, none of them fatal; it is important to note that fatal episodes of fungal infections are among the major limiting toxicities associated with t-cell depletion in haplo-hsct (17, 18) ; we cannot exclude a role played by the use of anti-mold prophylaxis during marrow aplasia, although it is difficult to draw definitive conclusions owing to the lack of a true control arm in our study, and to the low number of ifis. of note, we observed only 2 ifis 6 months after transplant. concerning bacterial infections, we may explain the low prevalence of late bacterial infections (i.e., beyond 1 year) by the surprising 8% incidence of chronic gvhd; in fact, the risk of bacterial events remained low in the absence of late immunosuppressive therapy (11) . with a median follow-up of 23 months, we observed 13 late bacterial events in a total of 31 patients having at least 1 year of observation (last observation day is 60 months after haplo-hsct). all this information argues in favor of the fact that giving a t-cell replete graft without deep in vivo t-depletion (i.e., with anti-thymocyte globulin or alemtuzumab) and with post-transplant cy allows a satisfactory infectious profile after transplant. the posttransplant high-dose cy permits naive and non-activated memory cells to reconstitute the immune system later on (19, 20) , enabling patients to be protected from late infectious events. the same mechanism probably also explains the high viral reactivation incidence found in the first 6 months, owing to the low number of adoptively transferred memory t cells in the early phase after transplantation (lugli e. et al., unpublished data). we acknowledge that potential selection bias may be present in the study, as we cannot exclude the possibility that some non-severe or very late infections were not captured because of incomplete reporting. however, all patients were followed at the same institution; therefore, it is unlike that clinically relevant infectious complications were missed; moreover, diagnostic procedures and prophylactic measures were similar for all patients, thus contributing to the accuracy of diagnosis of the infectious events. in conclusion, the present single-center data on 70 consecutive patients receiving t-cell replete haplo-hsct with post-transplant cy confirm a high rate of viral infections before day +100 and a lower incidence of infections afterward, suggesting a satisfactory although non-optimal immune reconstitution after this type of transplantation. future comparisons with other haploidentical platforms and/or other alternative stem cell sources (i.e., cord blood), as well as investigations of novel strategies of transfer of immunity are warranted. furthermore, the present data may provide useful information in an attempt to improve control of infections by adequate prophylaxis and/or antimicrobial therapy in the early post-transplant period, after use of the emerging transplant platform of haplo-hsct. reduced mortality after allogeneic hematopoietic cell transplantation long-term survival and late deaths after allogeneic hematopoietic cell transplantation hla-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide outcomes of related donor hla-identical or hla-haploidentical allogeneic blood or marrow transplantation for peripheral t cell lymphoma cord blood transplantation from unrelated donors in adult with high-risk acute myeloid leukemia definitions of cytomegalovirus infection and disease in transplant recipients revised definitions of invasive fungal disease from the european organization for research and treatment of cancer/invasive fungal infections cooperative group and the national institute of allergy and infectious diseases mycoses study group (eortc/msg) consensus group estimation of failure probabilities in the presence of competing risks: new representations of old estimators nonparametric estimation from incomplete observations infectious complications in cord blood and t-cell depleted haploidentical stem cell transplantation risk factors for late infections after allogeneic hematopoietic stem cell transplantation from a matched related donor improved early outcomes using a t cell replete graft compared with t cell depleted haploidentical hematopoietic stem cell transplantation unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning haploidentical transplantation using t cell replete peripheral blood stem cells and myeloablative conditioning in patients with high-risk hematologic malignancies who lack conventional donors is well tolerated and produces excellent relapse-free survival: results of a prospective phase ii trial ast infectious diseases community of practice. bk polyomavirus in solid organ transplantation absence of post-transplantation lymphoproliferative disorder after allogeneic blood or marrow transplantation using posttransplantation cyclophosphamide as graft-versus-host disease prophylaxis full haplotype-mismatched hematopoietic stem-cell transplantation: a phase ii study in patients with acute leukemia at high risk of relapse infusion of suicidegene-engineered donor lymphocytes after family haploidentical haemopoietic stem-cell transplantation for leukaemia (the tk007 trial): a non-randomised phase i-ii study high-dose cyclophosphamide for graft-versus-host disease prevention thymic t-cell development in allogeneic stem cell transplantation thanks: we thank all personnel working in the hematology and transplantation unit at humanitas cancer center for their remarkable contribution in patients' care and assistance to their families.author contributions: r.c. designed the study, performed data analysis, and wrote the manuscript; s.b., b.s., f.t., and e.m. provided clinical care; a.v. collected data and performed statistical analysis; r.m., e.c., and i.t. provided laboratory and microbiological data; e.l. and d.m. critically revised the manuscript; c.c-s., a.s., and l.c. provided clinical care and critically revised the manuscript.conflict of interest: all authors declare no financial conflict of interest. key: cord-023669-3ataw6gy authors: masur, henry title: critically ill immunosuppressed host date: 2009-05-15 journal: critical care medicine doi: 10.1016/b978-032304841-5.50056-x sha: doc_id: 23669 cord_uid: 3ataw6gy nan as the population of patients with cancer, organ transplants, vasculitides, and human immunodefi ciency virus (hiv) infection has grown, intensivists are seeing more and more patients with altered immunity. these patients may come to the intensive care unit (icu) because of life-threatening opportunistic infections, or they may develop life-threatening infection while in the icu for an unrelated problem. intensivists must recognize how these patients differ from immunologically normal patients in terms of clinical presentation and management of these infections. this chapter emphasizes the important ways in which immunosuppressed patients differ from immunologically normal individuals in terms of infectious complications. clearly, however, immunosuppressed patients also develop complications from their underlying diseases and the drugs used to treat these underlying processes. these noninfectious complications are not the focus of this chapter but are reviewed in chapter 81. patients who are at increased risk for infectious complications because of a defi ciency in any of their host defense mechanisms are referred to as compromised hosts. patients in icus are almost universally compromised either by virtue of their underlying disease or by virtue of the invasive devices utilized to support and monitor them. patients are termed immunocompromised or immunosuppressed if their defect specifi cally involves immune response. often, patients who have defi cient infl ammatory response (e.g., neutropenia) are grouped into the category of immunocompromised or immunosuppressed, although technically they have a different category of defi cient host response. patients in icus are often immunosuppressed as a result of their underlying disease, therapy, or nutri-tional status. this chapter focuses specifi cally on patients who are immunocompromised or immunosuppressed. the microbial complications that any patient develops are determined by general, nonspecifi c barriers; innate immunity; acquired specifi c immunity; and environmental exposures. nonspecifi c barriers include anatomic barriers such as intact skin and mucous membranes; chemical barriers such as gastric acidity or urine ph; and fl ushing mechanisms such as urinary fl ow or mucociliary transport. organisms that breach these barriers encounter nonspecifi c and innate host factors termed the acute phase response. acute phase responses include trigger molecules and effector molecules. organisms also encounter acquired specifi c immune response systems including mononuclear phagocytes and antibodies. 1 infections that occur may result from normal fl ora that colonize mucosal or cutaneous surfaces. infections may result from abnormal fl ora that have invaded or replaced normal fl ora because of environmental exposures, disrupted barriers, or selective pressure of antimicrobial agents. table 54-1 lists organisms that cause disease when specifi c anatomic defenses are disrupted in individuals with normal microbial fl ora. infections may also result from common defects in the infl ammatory or immunologic systems; examples are detailed in table 54 -2. [1] [2] [3] [4] [5] [6] [7] [8] [9] infl ammatory and immunologic barriers can be disrupted by the primary disease process (e.g., tumor can invade the bone marrow, immunologic abnormalities associated with aplastic anemia or collagen vascular disease can destroy cells either in the bone marrow or the periphery). infl ammatory and immunologic mechanisms can also be disrupted by drugs. cytotoxic drugs, for instance, can reduce neutrophil number and function. certain monoclonal antibodies can destroy lymphocyte populations or interfere with cytokine attachment to receptor sites. some agents such as corticosteroids have multiple effects on neutrophils, lymphocytes, and soluble factors. infections may result from organisms that are usually not pathogenic, but become opportunistic because of poor host defense mechanisms. opportunistic infections are defi ned as those that occur with enhanced frequency or severity in a specifi c patient population compared with a normal patient population. pneumocystis jiroveci, for example, never causes disease in immunologically normal individuals but can cause frequent episodes of pneumonia in certain immunosuppressed patients. candida can cause mild mucosal disease in normal patients receiving antibacterial drugs but causes more frequent and more severe mucositis when patients have impaired cell-mediated immunity. recognition of which host defense mechanisms are disrupted enables the clinician to focus diagnostic, therapeutic, and prophylactic management and optimize patient outcome. for instance, if a patient presents with severe hypoxemia and diffuse pulmonary infi ltrates, a health care provider who recognizes a prior splenectomy as the major predisposition to infection would focus the diagnostic evaluation and the empiric therapy on streptococcus pneumoniae and haemophilus infl uenzae. by contrast, if the patient's major predisposition to infection were hiv infection with a cd4+ t lymphocyte count below 50 cells/µl, the health care provider would focus on pneumocystis jiroveci and s. pneumoniae; if a cytomegalovirus (cmv)-negative patient's major predisposition were a recent allogeneic stem cell transplant from a cmv-positive donor, then cmv would be a prime consideration. [2] [3] [4] [5] [6] [7] [8] [9] immune competence should ideally be measurable by objective laboratory parameters. in fact, the risk for opportunistic infection in patients with hiv infection can be assessed by clinical laboratories with a high degree of accuracy by measuring the number of circulating cd4+ t lymphocytes. 5 the susceptibility of cancer patients to opportunistic bacterial and candida infections can be assessed by measuring the number of circulating neutrophils. 7, 10, 11 the predisposition of patients with certain congenital immunodefi ciencies can be assessed by measuring serum immunoglobulin levels. 12 unfortunately, however, for a large number of immunodefi ciencies, no objective laboratory measures have been validated as predicting the risk of infection. moreover, laboratory measures must be interpreted in context. cd4+ t lymphocyte counts have great prognostic value in patients with hiv infection but not in most other patient populations; neutrophil counts are relevant in all patient populations, but low counts are associated with disrupted mucosal surfaces compared with those with intact mucosa. thus laboratory parameters must be interpreted in the context of the patient's underlying disease-risk is not always easily manageable by measuring one laboratory parameter. most importantly, most patients have multiple overlapping predispositions to infection. knowledge of the infectious complications associated with specifi c diseases, specifi c immune defects, and specifi c laboratory abnormalities is helpful for predicting and managing infectious complications. however, a specifi c diagnosis should be established in each patient: knowledge of the immune defect helps guide empiric therapy or helps determine therapy if a diagnostic procedure is not safe to perform. immunocompromised patients, by defi nition, are susceptible to a broader array of pathogens than immunocompetent patients. understanding the specifi c immune defect can be enormously helpful in understanding the likely location and source of infection. however, the immune defect must be assessed in the context of the specifi c disease: the clinical manifestations of hiv infection, for instance, are quite different from the clinical manifestations of patients with other diseases that alter cellmediated immunity such as lymphoma. the immune defect must also be interpreted with the understanding that predisposition to infection is usually multifactorial: in addition to neutropenia or lymphocyte depletion, patients often have impaired mucosal barriers, poor ciliary function, or breaches in their skin (i.e., from catheters) that can increase their risk of infection. effective management of opportunistic infections requires understanding of several basic tenets of care. 1. diseases may present with subtle symptoms and signs, and patients are predisposed to deteriorate precipitously. because immunocompromised patients may lack infl ammatory and/or immunologic mediators, the clinical manifestations of infections are often less prominent and less impressive than immunocompetent patients with similar complications. thus clinicians must recognize that even subtle changes in skin color, catheter site appearance, chest radiograph, or abdominal examination may warrant an aggressive diagnostic evaluation and early institution of broad-spectrum empiric therapy. although all icu patients demand prompt attention and vigorous diagnostic and therapeutic management, many types of immunosuppres-sion can be associated with especially precipitous clinical deterioration despite their innocuous presentation. 2. fever is not invariably present when patients are infected. although fever is not invariably present in any patient population with infection, immunosuppressed patients are notorious for developing infection in the absence of fever. thus infection must be considered as part of the differential diagnosis among patients with afebrile syndromes that might not appear to be infectious. conversely, patients with fever may not have infection: fever may be a manifestation of the underlying disease, an allergic response to a drug, or an underlying neoplastic or collagen vascular disease. 3. diagnostic evaluation needs to be prompt and defi nitive. as indicated earlier, patients with life-threatening infection may present with subtle symptoms and signs that progress rapidly: these early manifestations merit aggressive attempts to defi ne the anatomy of the lesion and the causative microbial pathogen. because the spectrum of potential pathogens includes a wide array of microorganisms (e.g., viruses, fungi, protozoa, or bacteria), clinicians must be certain that appropriate specimens are obtained and the appropriate microbiologic and histologic tests are ordered to identify common, as well as uncommon or unusual, pathogens. invasive diagnostic techniques such as bronchoalveolar lavage or tissue biopsies should be performed with less hesitancy than in immunologically normal patients. patients often have enhanced risk factors for invasive procedures, such as thrombocytopenia, coagulation factor defi ciencies, or compromised organ function. however, the benefi t of defi nitive diagnosis often outweighs these risks when the procedures are performed by experienced operators. 4. the threshold for initiating broad-spectrum empiric therapy should be low. because patients can deteriorate rapidly and because they are susceptible to such a wide array of microbial pathogens, clinicians should have little hesitation in instituting empiric antimicrobial therapy. this therapy must be directed at the full range of bacterial, fungal, viral, protozoal, and helminthic infections to which patients are predisposed. this therapy should be administered promptly, preferably within an hour of suspecting an infectious process. clinicians should initiate comprehensive regimens: antimicrobial agents can be discontinued or reduced when culture results and clinical events clarify the scenario. 5. foreign bodies and infectious foci should be addressed. patients may need careful imaging to be certain that they do not have an obstructed viscus or localized collection that should be drained. such imaging is appropriate even when signs or symptoms are unimpressive. similarly, patients often have multiple intravascular catheters that may need to be removed, as discussed in chapter 51. 6. consideration should be given to augmenting the immune or infl ammatory response. there may be opportunities to augment immunologic or infl ammatory responses by administering pharmacologic or biologic agents such as granulocyte colony-stimulating factor (g-csf) or intravenous immunoglobulin. [12] [13] [14] [15] eliminating immunosuppressive drugs or reducing the dose can also improve the patient's prognosis. 7. effi cacy and toxicity of therapy should be assessed serially. icu patients characteristically require attentive monitoring to assure the adequacy and safety of therapy. immunocompromised patients often have multiple prior and concurrent insults to their renal and hepatic function, and they often receive multiple drugs that can produce drug-drug interactions. thus monitoring the pharmacokinetics and assessing potential toxicities are especially important in these patient populations. moreover, because response to therapy may be less robust than in immunocompetent patients, antigen titers or pcr titers, as well as serial imaging studies, can be important to assure the adequacy of the management plan. therapy must often be continued longer than in immunologically normal patients. cytotoxic therapy-induced neutropenia is a major predisposition to infection. 7, 11 counts below 500 cells/mm 3 (the total of polymorphonuclear neutrophils and bands) increase susceptibility to infection in a linear fashion (i.e., the lower the neutrophil count, the greater the degree of susceptibility). the absolute neutrophil count is not the only factor that determines susceptibility, however, because some patients with cyclic neutropenias, druginduced neutropenias, or hiv-induced neutropenias, for example, are not nearly as susceptible to infection as are cancer patients receiving cytotoxic therapy. other important contributors to susceptibility, in addition to the absolute neutrophil count, are the duration of neutropenia, the functional capability of neutrophils, the integrity of physical barriers such as the skin and gastrointestinal mucosa, the patient's microbiologic environment (endogenous and exogenous fl ora), and the status of other immune mechanisms. for example, a patient with vancomycin-induced neutropenia during therapy for a staphylococcal infection may not develop any complications if the neutropenia is brief and defense mechanisms are otherwise intact. a patient with hiv-induced neutropenia may have prolonged or even lifelong neutrophil counts below 500/µl yet suffer few serious bacterial complications. 14 the presence of intact physical defense barriers is a major difference compared with cancer patients, whose skin and mucous membranes are disrupted by cytotoxic therapy in which the skin and gastrointestinal tracts are portals of entry for infections that are not controlled by diminished host immunologic or infl ammatory defenses. thus the patient with hiv infection is usually at a much lower risk for a bacterial infection than is a cancer patient, despite a comparable neutrophil count. in the 1960s and 1970s, aerobic gram-negative bacilli such as escherichia coli, klebsiella pneumoniae, and pseudomonas aeruginosa predominated as pathogens in neutropenic patients. anaerobic bacteria and aerobic gram-positive cocci were recognized less commonly. aerobic gram-negative bacillus infections were also associated with a poorer outcome than infections from gram-positive cocci. given the spectrum of pathogenic organisms that were seen in that era, combination therapy was usually advocated. 11,16-24 a number of reasons were proposed to justify combination therapy: (1) broad coverage of potential pathogens; (2) prevention of emergence of resistance; and (3) synergy. in general, these principles are reasonable concepts on which to base a preference for using combination therapeutic regimens. however, no study unequivocally demonstrated that combination therapy provided better outcomes than did monotherapy, assuming that both study arms contained drugs that had activity against the causative organism. in addition, predicting synergy proved diffi cult. 25 in the 1990s the spectrum of causative pathogens in neutropenic patients shifted from a predominance of gram-negative bacilli to a majority of gram-positive cocci including streptococci, staphylococci (including oxacillin-resistant staphylococcus aureus), and enterococci (including vancomycin-resistant enterocci). 20, 24, 26 the development of potent broad-spectrum β-lactam and quinolone drugs in the 1980s and 1990s has provided single agents that can probably provide comparable outcomes to combination therapy when used empirically or specifi cally. in the current era the choice of single or combination regimens is based predominantly on the spectrum of organisms that needs to be covered rather than attempting a strategy of trying to obtain more potency through additive or synergistic combinations. 10, 27 promptly initiating broad-spectrum antibacterial therapy for all cancer patients who are febrile and who are neutropenic (neutrophil count <500/mm 3 ) as a result of cytotoxic chemotherapy is standard practice. 7,10,27 for febrile neutropenic patients who have no apparent source of infection, there is no evidence that the initial antibacterial regimen is any more effective if a broad-spectrum antibacterial regimen consisting of two or more drugs is used instead of a single broad-spectrum antibacterial drug. for stable "low-risk" patients outside the icu, an oral regimen is now considered a reasonable approach. 7, 10, 17 such oral regimens would not be used for inpatients in most circumstances and would not be appropriate for high-risk or unstable patients. 7, 10 antifungal and antiviral drugs are generally not used empirically when neutropenic patients are initially treated unless there is a specifi c reason to have a high suspicion for a fungal or viral process. historically, an infectious cause of fever has been found in about two thirds of febrile, neutropenic cancer patients. when a specifi c causative organism is identifi ed, antimicrobial therapy is modifi ed to include an agent or agents determined to be active by in vitro susceptibility tests and that penetrate to the site of the infection. 10 combination therapy is advocated by some authorities for the specifi c (compared with empiric) therapy of either gram-positive or gram-negative bacteria, although, as noted earlier, there are little data for most pathogens that indicate that a combination regimen produces a better outcome than an appropriate single agent. therapy is generally not narrowed in terms of spectrum, however, because alteration of broad-spectrum coverage to focused therapy has been associated with more complications (e.g., "breakthrough bacteremias") unless the neutropenia resolves. whenever fever persists, therapy has generally been continued during the entire course of neutropenia because cessation of antimicrobial therapy has been associated with recurrent bacteremia resulting from the initial causative organism or a newly identifi ed pathogen. a 10-to 14-day course of antibacterial therapy is usually the minimum recommended if a causative infection is identifi ed. therapy is usually stopped promptly when the neutrophil count exceeds 1000 cells/µml if fever resolves and no source was ever identifi ed. empiric antibacterial therapy has been a successful strategy for reducing morbidity resulting from bacterial processes but has been associated with the emergence of fungal infections, as well as resistant bacterial pathogens. candida and aspergillus organisms, in particular, have become major causes of morbidity and mortality over the past 2 decades. these fungal processes can be diffi cult to diagnose because they are not always associated with detectable fungemia. the emergence of fungi as important pathogens, especially in patients with prolonged neutropenia, has led to the recommendation that empiric antifungal therapy be added to neutropenic patients who do not have an identifi ed bacterial process and who do not defervesce within 4 to 7 days of empiric antibacterial therapy. 7,10 fluconazole or an amphotericin b compound (e.g., liposomal amphotericin b) are often used, although echinocandins or certain other azoles such as voriconazole are being used by some investigators and clinicians. [28] [29] [30] [31] as patients receive chemoprophylaxis with quinolones and/or azoles during periods of intense neutropenia or immunosuppression, breakthrough pathogens are more and more likely to be resistant to the prophylactic agents. 32, 33 thus empiric regimens must be chosen with keen attention to the drugs that patients have received in the recent past, as well as pathogens they have previously been colonized or infected with. 34 patients with fever and neutropenia require aggressive diagnostic efforts to identify the cause of fever so that the appropriate antimicrobial agent is used and appropriate procedures (e.g., surgical drainage, removal of foreign body such as a catheter) can be performed. regular physi-cal examination is necessary to identify sites that merit more focused investigation: with impaired infl ammatory response, fi ndings on examination may be subtle. knowledge of the specifi c immunologic defect is important so that when cultures of blood, sputum, urine, or other appropriate body fl uids or body sites are performed, special microbiologic approaches can be used to detect viruses, fungi, helminths, protozoa, and bacteria. imaging studies are also important because intra-abdominal, intrathoracic, intracerebral, or musculoskeletal processes can be clinically subtle and may not be associated with identifi able organisms in the bloodstream. a growing array of antigen, nucleic acid, and gene detection systems including polymerase chain reaction and microarray gene assays are being investigated to facilitate diagnosis. some antigen or nucleic acid detection systems for blood or other body fl uids can be useful for detecting cryptococcus, histoplasma, hepatitis b and c, hiv, mycobacteria, pneumococci, and legionella. some of these approaches, despite their promising initial reports, are not yet clinically practical because of their level of sensitivity, specifi city, or the cost or expertise required to perform them adequately. careful attention to antimicrobial susceptibility patterns is also important. patients are exposed to repeated courses of antimicrobial agents. patients come into contact with contaminated environments in a variety of health care settings. resistance is no longer an issue exclusively for aerobic gram-negative organisms but is a concern for anaerobes, gram-positive cocci, viruses, fungi, and protozoa. clinicians must recognize that pathogens may be resistant when they are acquired by the patient, or they may become resistant during therapy if there is an inducible resistance mechanism or drug concentrations are not adequate to inhibit or kill the organism. a broad-spectrum agent used as monotherapy for febrile, neutropenic patients should have activity against aerobic gram-positive cocci and aerobic gram-negative bacilli including p. aeruginosa. 7, 10, 19, 35 potential drugs for this indication include certain cephalosporins (e.g., cefepime), carbapenems (e.g., imipenem or meropenem), and βlactam/β-lactamase combination agents (e.g., piperacillintazobactam). ceftazidime is an option chosen by some, but its poor activity against gram-positive cocci has caused some clinicians to use other agents. 18 intensivists must recognize, however, that these monotherapy regimens may not be appropriate in an icu. patients in icus, by defi nition, are either unstable hemodynamically or have a potentially life-threatening process such as diffuse pneumonia or are "fragile" because of concurrent processes. thus combination regimens are preferred by many authorities in icu settings, even though no study clearly documents superior outcomes from such combination regimens. the decade that started in 2000 is an era when microbial resistance is becoming an increasingly important problem for many types of bacteria including aerobic grampositive cocci and anaerobes, as well as aerobic gramnegative bacilli. multiple drug empiric regimens are more likely than monotherapy regimens to include an agent with activity against the offending pathogen(s). thus in a situation in an icu when failure to use an active drug is more likely to be lethal than in other settings, and when enhanced potency is a logical goal, combination therapy is prudent as an initial management strategy. thus adding vancomycin or linezolid or daptomycin for better grampositive coverage, adding a quinolone for better gramnegative bacillus coverage, and adding metronidazole to cefepime would be prudent in this patient population pending results of initial diagnostic studies. of note, however, is that although this strategy is logical, no study has shown convincingly that such an approach improves outcome. 22 a substantial number of febrile, neutropenic patients fail to improve in terms of fever or other manifestations. failure to improve may result from poor immune response, a need for drainage or necessity to remove foreign bodies, the use of drugs without activity against the causative organism, or a noninfectious process including drug allergy (i.e., fever resulting from a drug including an antimicrobial agent). the potential causative processes need to be aggressively reassessed on a regular basis by physical examination, history, cultures, and imaging techniques. most centers add antifungal therapy empirically at day 4 or day 7 of therapy if patients remain febrile. 10, 27, 29, 36, 37 fluconazole, liposomal amphotericin b, caspofungin, or voriconazole may be used: in some situations fl uconazole would be less attractive either because the patient has received fl uconazole prophylaxis or because molds are suspected. 28, 38, 39 the toxicity profi le of amphotericin b, even in its liposomal form, has led many clinicians to prefer voriconazole or one of the echinocandins (i.e., caspofungin, micafungin, or anidulafungin). 30, 40, 41 after empiric antimicrobial therapy is initiated, the optimal duration of therapy is a complex issue that depends on the type and severity of the infectious process and the duration and severity of immunosuppression, especially the neutropenia. if a causative bacterium is identifi ed, a minimum of 7 to 10 days of therapy is generally advocated, with at least 3 to 4 days being administered after neutropenia has resolved. longer courses may be required in certain settings. the duration of antifungal therapy is a complex issue and depends on the specifi c mycosis, the location and extent of disease, and the patient's immune status. 15 this is discussed in chapter 53. the use of combination therapy for fungal diseases remains controversial. 42, 43 a common problem in febrile, neutropenic patients is managing indwelling intravascular lines. [44] [45] [46] in general, these lines can be left in place initially if examination of the site reveals no indication of infection. blood cultures should be drawn through the catheter. although some experts advocate drawing a culture through each port of each catheter, obtaining this many blood cultures is often not feasible. if a patient is hemodynamically unstable and fails to respond promptly to fl uid administration, it is prudent to remove the line in case an infected catheter is the source of the sepsis. failure to remove the foreign body in this situation probably increases the likelihood of an unfavorable outcome. should blood cultures become positive and should the suspicion be high that the catheter is the source, antibacterial therapy may be successful in some settings (e.g., if the pathogen is a bacteria that is relatively sensitive to antibacterial therapy), thus avoiding the need to remove the catheter. situations suggesting that catheter removal is necessary include hemodynamic instability despite aggressive fl uid resuscitation, tunnel infection, or infections resulting from fungi or relatively antibiotic-resistant bacteria such as p. aeruginosa. a major determinant of prognosis is the immunologic status of the patient. prompt return of neutrophil number to normal improves the outcome. the use of g-csf or granulocyte-monocyte colony-stimulating factor (gm-csf), if not contraindicated by the underlying disease, can improve clinical status by hastening the return of neutrophil numbers and function. [12] [13] [14] [15] 47 granulocyte transfusions have not been proved useful in most clinical settings because of the inability to administer a large number of cells with adequate frequency. 48 the manipulation of immune response with cytokines, cytokine inhibitors, or immunoglobulins is the subject of considerable investigation: such interventions may reduce the duration of fever or the incidence of infections when used empirically, but in no setting have they been clearly shown to improve survival when administered after an infection has been documented. an algorithm for managing fever in neutropenic patients is provided in figure 54 -1. table 54 -3 suggests modifi cations of standard empiric regimens in certain common clinical scenarios. given the experience with frequent and severe infectious complications in cancer patients with neutropenia, it has been logical to attempt to prevent infection. 33 most microorganisms causing disease in this patient population arise from endogenous gastrointestinal, cutaneous, or respiratory fl ora. total protected environments probably reduce frequency of infection, but this approach is expensive and inconvenient. trying to prove a consistent benefi cial impact on survival has been diffi cult, and thus such isolation is rarely used anymore. some experts are enthusiastic about placing patients in positive pressure rooms so that pathogens do not enter via particles and droplets from outside the room. this type of isolation has not clearly improved outcome, however, and is not a standard of care. prophylactic bacterial therapy has also been controversial. 32 systemic antibacterial prophylaxis and systemic antifungal prophylaxis have been shown in some studies to reduce the number of infections, but their lack of effect on patient survival, their cost, and their impact on the emergence of resistance have made many clinicians reluctant to use them. selective gastrointestinal decontamination has not consistently improved survival and thus is not recommended by most authorities in the united states. antipneumocystis prophylaxis is, in contrast, highly effective in susceptible populations. prophylaxis for cmv is highly effective in well-defi ned, high-risk patients (e.g., some recipients of organ transplants who are either sero-positive for cmv or who are seronegative but received a graft from a seropositive donor). 2, 4, 49 strategies that reduce the period of immunologic susceptibility (e.g., reduce the duration of neutropenia), such as adding g-csf to a regimen or reducing the intensity of chemotherapeutic regimens, are promising. because so many patients are receiving highly active antiretroviral therapy (haart), opportunistic infections are not complicating the course of hiv infection to the same degree that they did in the 1980s and early 1990s. [50] [51] [52] [53] opportunistic infections continue to occur, however, in three groups of hiv-infected patients: (1) those who are unaware of their hiv status until they develop a clinical syndrome; (2) those who are unable or unwilling to receive appropriate therapy; and (3) those who fail haart and opportunistic infection prophylaxis. although haart has dramatically reduced the incidence of opportunistic infections, a surprisingly large fraction of patients either never respond virologically and immunologically or lose their response within the fi rst 12 to 24 months of therapy. these patients, most of whom have dominant viral quasispecies that are highly resistant to currently licensed antiretroviral drugs, will likely experience immunologic decline over the next few years and will again become more susceptible to opportunistic infections. severe necrotizing mucositis or gingivitis add specifi c antianaerobic agent (e.g., metronidazole, meropenem, imipenem, or piperacillin-tazobactam) plus agent with activity against streptococci; consider acyclovir. ulcerative mucositis or gingivitis add acyclovir and anaerobic coverage. add fl uconazole or caspofungin; consider adding acyclovir. pneumonitis, diffuse or interstitial add trimethoprim-sulfamethoxazole and azithromycin or levofl oxacin or moxifl oxacin (plus broad-spectrum antibiotics if the patient is granulocytopenic). perianal tenderness include anaerobic agents such as metronidazole, imipenem, meropenem, or piperacillin-tazobactam. abdominal involvement add antianaerobic agent (e.g., metronidazole, meropenem, imipenem, or piperacillin-tazobactam). patients with hiv infection develop clinical disease as a result of three basic processes: the direct effect of hiv on specifi c organs (e.g., cardiomyopathy, enteropathy, dementia); immunologically mediated processes (e.g., glomerulonephritis, thrombocytopenia); or opportunistic infections and tumors that are enabled by hiv-induced immunosuppression. hiv appears to cause direct organ damage. 50,53-57 this damage may be mediated by cytokines, lymphocytes, monocytes, or infl ammatory cells. cardiomyopathy, for example, can be a profound and lethal process that can lead to icu admission or complicate other processes. 55 when patients present with or develop pulmonary manifestations such as shortness of breath or diffuse bilateral infi ltrates on chest radiograph, cardiogenic causes must be considered. hiv also causes a diffuse pneumonitis, 56 profound encephalopathy, 54 and a diffuse enteropathy. 57 patients with compatible syndromes need a comprehensive evaluation to look for other specifi c opportunistic infections or tumors, especially those that can be specifi cally treated. in all of the hiv-caused syndromes, hiv as the etiology remains a diagnosis of exclusion. the institution of antiretroviral therapy appears to be benefi cial for patients with susceptible isolates, although data regarding such effects for these hiv-related entities are largely anecdotal. hiv-related thrombocytopenia and anemia appear to be immunologically mediated. 58, 59 both can be severe: platelet counts below 10,000/mm 3 and hemoglobins below 10 g/dl can be seen with the expected complications. these disorders are related to the development of antigenantibody complexes and may improve dramatically with the institution of antiretroviral therapy and a decline in viral load. for thrombocytopenia, intravenous immunoglobulin (or anti rhd antibody), corticosteroids, or splenectomy may also be useful. hemolytic anemia can also be severe: hemoglobin levels below 5 g/dl can be seen. the most prominent manifestations of hiv continue to be the opportunistic infections and tumors that occur as a consequence of hiv-induced immunosuppression. the cd4+ t lymphocyte cell number is a useful marker for predicting the occurrence of opportunistic infections in patients with hiv infection. 5, 9 this relationship of cd4+ t lymphocyte count to the occurrence of opportunistic infection continues to be as valid in the era of haart as it was before the licensing of the fi rst antiretroviral agent, zidovudine, in 1987. [60] [61] [62] figure 54 -2 demonstrates the typical relationship of cd4+ t lymphocyte counts to the occurrence of opportunistic infections. knowledge of this relationship permits the focusing of diagnostic, therapeutic, and prophylactic management. for instance, if a patient with hiv infection and a cd4+ t lymphocyte count of 700 cells/µl presents with diffuse pulmonary infi ltrates, the diagnostic evaluation and empiric antimicrobial regimen should focus on s. pneumoniae; h. infl uenzae; mycoplasma, legionella, and chlamydia organisms, as well as common community-acquired viruses. in contrast, if the same patient had a cd4+ t lymphocyte count of 50 cells/µl, the evaluation and empiric regimen would focus on pneumocystosis and cmv, although the previously mentioned processes that occur at high cd4+ t lymphocyte counts can also occur at lower cd4+ t lymphocyte counts. keeping in mind that cd4+ t lymphocyte counts are useful predictors of susceptibility to infection is important, but they are not perfect. occasionally, patients will develop opportunistic infections at "uncharacteristically" high cd4+ t lymphocyte counts. for instance, 5% to 10% of cases of pneumocystosis occur at cd4+ t lymphocyte counts greater than 200 cells/µl. 61 clinical parameters can provide additional clues; for example, oral candidiasis, a previous opportunistic infection, a prior episode of pneumonia, or high viral load are independent risk factors for the occurrence of pneumocystis jiroveci carinii pneumonia (pcp), and logically for other infections as well. 9 a frequent question is whether an hiv-infected patient's prior cd4+ t lymphocyte count nadir affects the likelihood of an opportunistic infection occurring if haart has stimulated a cd4+ t lymphocyte count rise. specifically, if a patient has a cd4+ t lymphocyte count of 400 cells/µl while receiving haart and that patient's cd4+ t lymphocyte count was 50 cells/µl before haart, is that patient at greater risk for developing an opportunistic infection than another patient whose current cd4+ t lymphocyte count is 400 cells/µl but whose nadir before haart was 250 cells/µl? the data suggest that these two patients have comparable risk (i.e., the current cd4+ t lymphocyte count is the most important predictor of risk and the earlier nadir has only minor infl uence on opportunistic infection susceptibility). 62 in evaluating the differential diagnosis of infectious syndromes in patients with hiv (and in every other patient population as well), geography is an important part of the history. tuberculosis is always a concern because of the extraordinary susceptibility of hiv-infected patients for developing active disease. 63 in many urban settings in the united states, each pulmonary evaluation should include smears and cultures for m. tuberculosis, both to diagnose the appropriate cause of the pulmonary dysfunction and to assist in determining what respiratory precautions are appropriate. in some areas of the country, such as the ohio river valley and indianapolis, histoplasmosis is as common as pneumocystosis in causing diffuse pulmonary infi ltrates. 64 in the southwestern united states, coccidioidomycosis must be recognized as a cause of pulmonary infi ltrates. the clinical presentations of tuberculosis, histoplasmosis, coccidioidomycosis, and other processes such as cmv can be clinically indistinguishable from pcp. thus for patients with pulmonary infi ltrates in an icu, prolonged empiric therapy is discouraged in favor of vigorous efforts to establish a specifi c diagnosis. hiv-infected patients are admitted to icus for several major syndromes: respiratory insuffi ciency, cerebral dysfunction, septic shock, hepatic or renal failure, and drug toxicities. 50 however, patients with hiv infection also come to icus for routine procedures and routine postoperative care. in those situations their management ordinarily requires no extraordinary measures, with two exceptions. first, the staff must be fully aware of how hiv is transmitted, the danger of injuries resulting from sharp objects, and the procedure for managing injuries involving sharp objects contaminated with blood or other biologic fl uids from infected or potentially infected patients. 65 second, drug interactions involving drugs used during procedures and certain antiretroviral drugs can have important clinical consequences. 66, 67 many of the protease inhibitors and the non-nucleoside reverse transcriptase inhibitors that are now the backbone of antiretroviral therapy can inhibit or enhance the metabolism of drugs that depend on the cytochrome p450 system. thus the half-lives of certain analgesics, sedatives, and hypnotics can be prolonged in hiv-infected patients who are taking ritonavir, for example. this pharmacokinetic effect is also relevant for a host of other therapeutic agents used in the icu and may affect their effi cacy or safety. clinicians need to be familiar with these interactions when selecting new therapies for procedures or for clinical entities. patients with hiv infection can develop severe pulmonary dysfunction because of common community-acquired pathogens such as s. pneumonia, legionella, mycoplasma, and chlamydia; adenovirus; infl uenza; or respiratory syncytium virus, as well as other opportunistic viruses and fungi. thus the diagnostic evaluation needs to be comprehensive, emphasizing direct smears of sputum or bronchoalveolar lavage. it is important to recognize that the clinical presentations produced by many causative agents can be similar. for instance, histoplasmosis, tuberculosis, and nonspecifi c interstitial pneumonitis can present identically to pcp. 50, 61, 63, 64, 68 thus although empiric diagnosis and empiric therapy may be reasonable as initial approaches to some patients with hiv infection and mild pneumonitis, such an approach is usually not appropriate for patients in an icu. evaluation of induced sputum is the fi rst step in the diagnostic approach to pcp. sensitivity can be 80% to 95% at many hospitals (at some institutions the yield is considerably lower). 69 specifi city should be 100% in an experienced laboratory. other pathogens, including mycobacteria, fungi, and routine bacteria, can be identifi ed in sputum as well. for intubated patients, respiratory secretions obtained by deep intratracheal suctioning are also likely to be useful, although they have not been as carefully studied as induced sputum. should the diagnosis not be established by evaluation of sputum or intratracheal secretions, bronchoscopy should be performed. bronchoalveolar lavage should diagnose almost 100% of cases of pcp, even if patients have received 7 to 10 days of empiric therapy. 70 a diagnosis of pcp is established by visualizing one or more clusters of organisms. diagnostic criteria for other opportunistic infections are reviewed in chapters 12 and 43. in patients with hiv, cmv merits special mention. culture of sputum or bronchoalveolar lavage does not provide useful information because patients with cd4+ t lymphocyte counts below 100 cells/µl will predictably have cmv present in their secretion independent of whether or not pulmonary disease is present. 71 a diagnosis of cmv pneumonia in this patient population is suggested by cytology and confi rmed by the presence of multiple inclusion bodies in lung tissue obtained by transbronchial or open lung biopsy. similarly, mycobacterium avium complex (mac) and hsv can often be found in respiratory secretions, but these organisms almost never cause pneumonia in patients with hiv infection. in other patient populations they can clearly cause pneumonia, but the dearth of cmv, mac, and hsv pneumonia in this patient population emphasizes the point that it is important to know from published literature what the clinical likelihood is for different microbial processes. fungal pneumonias other than pcp are generally diagnosed by direct microscopy or culture of respiratory secretions (sputum or lavage). candida organisms almost never cause pneumonia in patients with hiv infection. the frequency of cryptococcus, histoplasma, blastomyces, and coccidioides organisms as causes of pneumonia depends on the geographic exposure of the patient. among these mycoses, antigen detection techniques can be useful for fi nding cryptococcus and histoplasma organisms. mycobacteria frequently infect the respiratory tract of patients with hiv infection. as noted earlier, m. avium complex almost never causes pulmonary dysfunction in this patient population. when acid-fast bacilli are seen (as opposed to cultured) in respiratory secretions or tissue, m. tuberculosis is almost always the pathogen; m. kansasii and other mycobacteria less commonly cause disease. screening all patients with acid-fast bacillus smears is important for preventing transmission of tuberculosis and text continued on p. 1126 should be considered as part of a routine respiratory evaluation for patients with radiographic infi ltrates in most areas of the united states. therapy of opportunistic infections is summarized in table 54 -5. 72 while awaiting a specifi c diagnosis, it is reasonable to initiate empiric therapy in patients ill enough to merit admission to an icu. for patients with a cd4+ t lymphocyte count greater than 250 to 300 cells/µl, azithromycin and ceftriaxone or azithromycin and ampicillin-sulbactam would be reasonable choices. for patients with cd4+ t lymphocyte counts below 200 to 250 cells/ µl, levofl oxacin or moxifl oxacin plus trimethoprimsulfamethoxazole or pentamidine plus levofl oxacin or moxifl oxacin would be potential regimens. if pcp is documented, trimethoprim-sulfamethoxazole is always the drug of choice in patients who can tolerate it. table 54-5 lists alternatives for sulfa-intolerant individuals. regardless of which specifi c antipneumocystis regimen is used, corticosteroid therapy is indicated for any patient who presents with an oxygen pressure (po 2 ) below 70 mm hg or an alveolar-arterial gradient higher than 30 mm hg. [73] [74] [75] [76] patients with an initial po 2 lower than 70 mm hg are the subgroup with substantial mortality for whom corticosteroids have been shown to provide a survival benefi t. corticosteroids may provide more rapid and perhaps more complete resolution of pulmonary manifestations in patients who present with better pulmonary function, but survival in this population is so high that clinical trials have not been able to show survival benefi t. some experts are concerned that corticosteroid use will be associated with reactivation of latent infections such as cmv or tuberculosis. however, reactivation of life-threatening infections has not been associated with this corticosteroid regimen. how should a patient with aids-associated pcp be managed if there is no improvement, or if there is deterioration, after 5 to 10 days of therapy? the median time to improvement in clinical variables is 4 to 8 days; therefore, changes in therapy are probably not warranted before 5 to 10 days. at that point the accuracy of the diagnosis should be reassessed: consideration should be given to repeat bronchoscopy with transbronchial biopsy to determine if cmv, fungi, mycobacteria, or a nosocomial bacterial process is present. noninfectious processes such as congestive heart failure or tumor (e.g., kaposi's sarcoma) must also be considered. if pneumocystosis is the only causative process that can be identifi ed, corticosteroids should be added to the regimen if they have not been already. whether switching from one antipneumocystis agent to another or whether adding a second agent is helpful has not been determined by clinical trials. some human pneumocystosis isolates are resistant to sulfonamides, but such testing is available only in a few research centers. most clinicians add parenteral pentamidine to trimethoprim-sulfamethoxazole. parenteral trimetrexate or clindamycin-primaquine could be used as salvage regimens as well. patients who have not improved after 14 to 21 days of therapy with specifi c chemotherapy plus corticosteroids have an exceedingly poor prognosis. should patients with aids-related pcp be intubated and provided with mechanical ventilation? mortality for such patient populations was 70% to 80% in several series in the early 1980s. [77] [78] [79] [80] since that era, supportive care has improved, and treatment modalities for concurrent infectious and noninfectious processes have become more effective. patient selection for ventilatory support is probably also improving. patients who have multiple active opportunistic infections, substantial weight loss, and no response to 14 days of therapy have a worse prognosis than ambulating patients who develop respiratory failure the third day of therapy. thus decisions about icu support for patients with hiv infection and respiratory failure need to be individualized on the basis of a realistic assessment of prognosis, the availability of resources, and the preference of the individual patient. a frequent question for any hiv-infected patient in the icu is whether antiretroviral drugs should be continued or initiated during the critical or life-threatening illness. although there is no specifi c study of various strategies, most authorities discourage the use of antiretroviral drugs in the icu because of drug interactions and drug toxicities. in addition, the initiating haart can be associated with dramatic "immune reconstitution" syndromes that can complicate the process that brought the patient to the icu. [81] [82] [83] finally, almost all antiretroviral drugs that are commercially available are oral: in most situations it is better to discontinue all antiretroviral drugs for a few days or weeks or months rather than risk poor absorption and suboptimal serum levels. the latter would enhance the emergence of drug-resistant hiv. an important cause of admitting hiv-infected patients into the icu is either seizures or altered mental status. either can result from infectious or neoplastic processes caused by meningeal disease or parenchymal involvement. the differential diagnosis of meningeal disease includes pneumococcal and staphylococcal meningitis, cryptococcal meningitis, tuberculous meningitis, and lymphomatous meningitis, as well as involvement from other endemic mycoses and common community-acquired viral and bacterial processes. 24,84 diffuse central nervous system parenchymal disease can be caused by hiv itself, by progressive multifocal leukoencephalopathy, and occasionally by herpes viruses such as cmv or herpes simplex virus. focal mass lesions may be caused by toxoplasmosis or lymphoma. less often, tuberculosis, fungi, conventional bacterial abscesses, nocardia, and other tumors are the cause of focal lesions. these lesions can be diffi cult to distinguish clinically and radiologically. the cd4+ t lymphocyte count can help narrow the differential diagnosis, but csf or brain tissue is usually necessary for defi nitive diagnosis. the routine therapies for many of these processes are outlined in table 54 -5. toxoplasmosis deserves particular mention because of its frequency. [85] [86] [87] toxoplasmosis occurs mainly in patients with hiv infection who have cd4+ t lymphocyte counts below 100 cells/µl, have a positive igg antibody titer against toxoplasma, and who have not been receiving trimethoprim-sulfamethoxazole or dapsone prophylaxis. patients present with altered cognition, focal motor or sensory defi cits, or seizures. lesions may be unifocal or multifocal. they usually enhance with contrast, but this is not invariably true. for patients who fi t the profi le for high risk of toxoplasmosis, and with a compatible presentation, it is reasonable to establish an empiric diagnosis and institute specifi c therapy with sulfadiazine plus pyrimethamine or, for patients unable to tolerate sulfa, clindamycin plus pyrimethamine. corticosteroids may be needed for patients with considerable intracerebral edema or elevated intracranial pressure. antiseizure medication is usually instituted only after a seizure has occurred rather than prophylactically. most patients improve clinically and radiologically within 7 to 10 days. if patients fail to improve, a stereotactic needle biopsy is appropriate, especially because the prevalence of lymphoma is increasing. organisms can be diffi cult to see in brain specimens obtained by this technique. patients with hiv infection develop hypotension resulting from the same types of disorders as with non-hiv infected individuals-sepsis from a primary infection or a wound or device (especially an intravascular access device), fl uid depletion from vomiting or diarrhea, and hemorrhage from a gastrointestinal lesion are examples of common causes. the evaluation of hypotension in a patient with hiv infection must take into account factors particular to this patient population: it is susceptible to opportunistic infections; it undergoes many procedures that can be associated with infectious complications; and it receives an array of drugs, some of which have cardiovascular effects. thus evaluating hypotension in this patient population requires a comprehensive and thorough approach. a differential diagnosis of the major causes is shown in table 54 -6. adrenal function always deserves special attention because several viral processes, fungal and mycobacterial diseases, hiv, and drugs can suppress the adrenal axis and either cause hypotension or exacerbate it. patients with hiv infection typically receive several antimicrobial agents to reduce the likelihood they will acquire opportunistic infections. 9 primary prophylaxis is the term used to indicate strategies that reduce the likelihood of an initial episode of a disease process. secondary prophylaxis is the term used to indicate strategies that prevent recurrences or relapses. chronic suppressive therapy is identical to secondary prophylaxis: this refers to regimens that are continued after the initial therapeutic course to prevent relapses. all patients with hiv infection and cd4+ t lymphocyte counts below 200 cells/µl typically receive antipneumocystis prophylaxis. trimethoprim-sulfamethoxazole is the regimen of choice. patients who actually take this drug have very few breakthroughs of pcp and receive considerable protection against toxoplasmosis and certain routine bacterial infections. alternative regimens include monthly dapsone, weekly dapsone-pyrimethamine, or daily aerosol pentamidine. prophylaxis against m. avium complex is recommended for patients with cd4+ t lymphocyte counts under 100 cells/µl; clarithromycin and azithromycin are currently the drugs of choice. 9 many clinicians also use fl uconazole or acyclovir prophylaxis to reduce the frequency of fungal and viral processes, respectively, although this is not recommended because of issues of cost, pill burden, and the emergence of resistant pathogens. isoniazid prophylaxis is important for any patient with a tuberculin skin test that shows more than 5 mm of induration or a history of substantial recent exposure. 9 transmission of tuberculosis from patients to other patients, from patients to staff, or from staff to patients is an urgent concern in icus. patients with hiv infection are extraordinarily susceptible to tuberculosis. thus an infected patient poses a substantial risk, especially when hospitalized for pneumonia or when undergoing procedures at high risk for producing aerosols such as intubation, bronchoscopy, sputum induction, or aerosol pentamidine treatment. identifying potentially infected patients early and placing them in appropriate isolation until their tuberculosis status is fully examined is important. in many centers, patients with syndromes compatible with pulmonary or upper airway tuberculosis are maintained in isolation at least until three specimens of respiratory secretions have been examined for tuberculosis. hiv-infected health care practitioners need to carefully assess their risk of acquiring tuberculosis by their exposure in the icu. transmission of hiv is an issue that requires attention in the icu. 65 no evidence exists that hiv-infected health care professionals can infect patients, regardless of what procedure they perform, outside of two unusual events. hiv patients pose a risk to health care professionals, however. this risk can be substantially reduced by education, by strict monitoring for compliance with universal precautions, and by having proper equipment. almost all hiv transmission in an occupational setting occurs as a result of injuries involving sharp instruments (e.g., needles, scalpels). the risk of such injuries is about one case of hiv transmission per 250 injuries, but the likelihood of transmission in an individual accident depends on the amount of viremia at the time of the accident (late-stage patients generally have more circulating virus than do early-stage patients) and the nature of the accident. most authorities recommend immediate prophylaxis if a signifi cant injury occurs involving an hiv-infected patient. considerable debate exists over the optimal choice of drugs and the optimal duration of therapy, but it is clear that initiating therapy within a period of hours rather than days is best. many authorities now advocate a haart regimen for any situation when the patient and health care provider determine that therapy is appropriate, and continue that for 4 to 6 weeks. increasingly, icus are caring for organ transplant recipients, either in the period immediately after the procedure or during a crisis that occurs days, weeks, months, or years after engraftment. managing each type of organ transplant recipient has unique features depending on whether bone marrow, kidney, heart, lungs, liver, or other organs are transplanted. 2, 6 laboratory monitoring provides useful predictive information about the status of cellular immunity, humoral immunity, and neutrophil number and function. ultimately, however, clinical experience is necessary with each type of organ transplant and each immunosuppressive regimen to predict the most likely pathogens, when they most characteristically occur in relation to the transplant procedure, and what infl uence each immunosuppressive therapy has. an example of the temporal pattern of infectious complications after bone marrow transplantation is shown in figure 54 -3. 9 although such fi gures are useful conceptually, however, the immunosuppressive regimens are changing rapidly, and such fi gures may be misleading when applied to current transplantation protocols. organ transplant recipients share a complex interaction between immunosuppression and infection. immunosuppression is usually necessary in allogeneic transplantation to permit graft survival. the more potent the immunosuppression, the more likely infection is to occur. strategies that use antimicrobial agents (drugs, vaccines, and other biologic products) aggressively may reduce the risk of and damage from infection in a manner that allows more potent immunosuppression and better graft survival. such approaches may include prophylactic antibacterial and antiretroviral treatment, as well as prompt empiric therapy for emerging febrile episodes. patients receiving hematopoietic stem cell transplantation (hsct) or solid organ transplants are often receiving antimicrobial prophylaxis. acyclovir for hsv, valacyclovir for cmv, fl uconazole for yeast, voriconazole for yeast and molds, trimethoprim-sulfamethoxazole for pcp, and quinolones for bacteria are used in various combinations at different transplant programs. these agents dictate which organisms will break through to cause disease, and what their antibiotic susceptibility patterns will be. several pathogens deserve special mention. cmv is one of the most prominent pathogens for solid organ and bone marrow transplant recipients. [88] [89] [90] most disease is secondary (i.e., disease results from reactivation of a previously acquired, latent infection) in a seropositive organ recipient. in urban areas of the united states, 60% to 70% of the population is seropositive for cmv, and thus 60% to 70% of the transplant recipients will have latent infection that could potentially be reactivated. some cmv seronegative patients acquire primary infections from a cmvinfected organ or from cmv-infected blood or blood products. a few cmv seropositive individuals develop superimposed cmv disease from cmv acquired through a seropositive donor. laboratory monitoring of patients for evidence of cmv disease by using a dna amplifi cation assay, or surveillance of cmv antigen in buffy coat smears, is an important feature in efforts to reduce morbidity and mortality resulting from cmv. 89, [91] [92] [93] [94] [95] intensivists need to understand how to interpret these assays in terms of starting empiric, pre-emptive, or defi nitive therapy. strategies to reduce the frequency of cmv disease with acyclovir, intravenous or oral ganciclovir (or oral valganciclovir), the investigational agent proganciclovir, or immune globulin are used by many programs. cmv disease can cause substantial morbidity and mortality including fever, hypotension, pneumonitis, hepatitis, glomerulitis, enteritis, and allograft injury. the availability of ganciclovir, foscarnet, and cidofovir has enabled these conditions to be treated successfully in many instances, although all three of these drugs are associated with substantial toxicity. whether immune globulin (either immune globulin or specifi c hyperimmune globulin) adds anything to the potency of therapeutic regimens is not clear, although these products are usually administered when they are available. pcp has been reported in recipients of most types of organ transplants. most organ transplant programs use pcp prophylaxis. 6, 33, 96 trimethoprim-sulfamethoxazole is usually the prophylactic agent of choice because it is more effective than other agents, is well tolerated, and reduces the frequency of urinary tract infections and other potential complications (e.g., disease resulting from nocardia, s. pneumoniae, and haemophilus organisms). fungal infections have been common, but the causative pathogens are changing because of changes in prophylactic regimens. with the use of fl uconazole, candida albicans infections became less common. molds, especially aspergillus, became more important pathogens, as did fl uconazole-resistant candida. some programs are now using voriconazole prophylaxis. for such patients, mucormycosis and non-albicans candida are becoming more prominent causes of morbidity. thus clinicians must know what antifungal prophylaxis has been used in order to anticipate which complications will occur. mold infections can be diffi cult to diagnose: serum galactomannan assays can yield specifi c information, but the test has low sensitivity. mold infections almost never cause fungemia. thus diagnosis depends on cultures, which can be highly suggestive if obtained from sources such as bronchoalveolar lavage or biopsy. viral respiratory infections require particular mention because some are treatable and most are transmissible. community-acquired respiratory viruses such as adenoviruses, coronaviruses, or infl uenza can occur in immunocompetent or immunosuppressed patients. when respiratory infections occur in immunocompromised patients, health care professionals need to be certain that a transmissible virus is not the cause because of the potential to infect other patients, families, or hospital staff. of the respiratory infections, rsv deserves special attention in hsct patients. although rsv can, like other community-acquired viruses, cause disease in any patient population, it is especially lethal in solid organ, bone marrow, and stem cell transplants. thus rsv must be specifi cally sought in this patient population, as well as their visitors and health care providers, so that it does not spread to highly susceptible patients. similarly, when caring for immunosuppressed patients, attention to mycobacterium tuberculosis is important because this pathogen can also spread to other patients, families, and hospital staff. with more immigrants in the united states and more patients having travel exposure, m. tuberculosis needs to be considered in the differential diagnosis and specifi cally sought by gene probe, smear, or culture where appropriate. diagnosis and therapy of opportunistic infections and nosocomial infections should follow the guidelines given in chapters 43, 51, and 54. in choosing therapies, attention must be focused on the toxicities of antimicrobial agents and how they infl uence the outcome of the transplanted organ. in addition, drug interactions are important, especially with cyclosporine. drugs that alter hepatic metabolism, such as rifampin, rifabutin, and fl uconazole, can have substantial infl uence on cyclosporine levels and thus need to be used with careful pharmacologic attention. finally, clinicians must recognize that new immunosuppressive regimens and changing prophylactic regimens are changing the spectrum of infectious complications. as mentioned earlier, fungal infections are increasingly likely to be caused by species other than c. albicans: non-albicans candida, fusarium, and rhizopus are recognized with increasing frequency. similarly, prophylaxis with valganciclovir is reducing cmv disease and pushing disease that does occur later and later in relation to the transplant procedure. viruses such as hhv-6 and bk virus are causing disease. thus clinicians need to look for changing spectrum of pathogens, as well as changing manifestations if the morbidity and mortality caused by infection is to be managed optimally. ■ knowledge of a patient's specifi c defects in immunologic and infl ammatory response helps predict which opportunistic pathogens are most likely to occur. ■ icus are increasingly successful in enabling immunosuppressed patients to survive acute crises, especially if the defect in immunologic or infl ammatory function is reversible over time or by replacement therapy. ■ for neutropenic patients, gram-positive cocci are becoming more frequent than gram-negative bacilli as causes of life-threatening illness. ■ resistance to antimicrobial agents is becoming a major problem including bacteria (e.g., vancomycin-resistant enterococci and penicillin-resistant pneumococci), fungi (e.g., fl uconazole-resistant candida organisms), as well as pcp, and viruses (e.g., acyclovir-resistant herpes simplex and ganciclovir-resistant cmv). ■ in neutropenic patients, combination therapy should be considered when treating any life-threatening bacterial process. ■ a substantial fraction of hiv-infected patients with pcprelated respiratory failure can survive mechanical support and be discharged from the hospital. ■ adjunctive corticosteroid therapy is indicated for respiratory failure related to pcp. ■ tuberculosis is a concern in any immunologically abnormal individual with pulmonary disease but is a special concern in hiv-infected patients. tuberculosis in these cases often warrants respiratory isolation until appropriate specimens are evaluated for mycobacteria. ■ organ transplant recipients develop opportunistic infections at relatively predictable points depending on the type of transplantation and the specifi c immunosuppressive regimen used. innate (general or nonspecifi c) host defense mechanisms infections in solid organ transplant recipients infectious diseases associated with complement defi ciencies infection in organ-transplant recipients cd4 counts as predictors of opportunistic pneumonias in human immunodefi ciency virus (hiv) infection recent advances in the diagnosis and management of infection in the organ transplant recipient infections in recipients of hematopoietic stem cell transplantation bacteremia and fungemia in patients with neoplastic disease guidelines for the preventing opportunistic infections among hiv-infected persons-2002 recommendations of the u.s. public health service and the infectious diseases society of america guidelines for the use of antimicrobial agents in neutropenic patients with cancer fever in immunocompromised patients use of intravenous immunoglobulin in human disease: a review of evidence by members of the primary immunodefi ciency committee of the american academy of allergy, asthma and immunology colony-stimulating factors for chemotherapy-induced febrile neutropenia: a meta-analysis of randomized controlled trials neutropenia, neutrophil dysfunction, and bacterial infection in patients with human immunodefi ciency virus disease: the role of granulocyte colony-stimulating factor update of recommendations for the use of white blood cell growth factors: an evidence-based clinical practice guideline monotherapy with meropenem versus combination therapy with ceftazidime plus amikacin as empiric therapy for fever in granulocytopenic patients with cancer. the international antimicrobial therapy cooperative group of the european organization for research and treatment of cancer and the gruppo italiano malattie ematologiche maligne dell'adulto infection program a double-blind comparison of empirical oral and intravenous antibiotic therapy for low-risk febrile patients with neutropenia during cancer chemotherapy monotherapy for fever and neutropenia in cancer patients: a randomized comparison of ceftazidime versus imipenem treatment of febrile neutropenia with cefepime monotherapy additional anti-gram-positive antibiotic treatment for febrile neutropenic cancer patients fever in the pediatric and young adult patient with cancer. a prospective study of 1001 episodes gram-positive infections and the use of vancomycin in 550 episodes of fever and neutropenia ceftazidime monotherapy for empiric treatment of febrile neutropenic patients: a meta-analysis staphylococcus epidermidis: an increasing cause of infection in patients with granulocytopenia antibiotic synergism and response in gram-negative bacteremia in granulocytopenic cancer patients vancomycin-resistant enterococcal infections an evidence-based evaluation of important aspects of empirical antibiotic therapy in febrile neutropenic patients effi cacy of caspofungin against invasive candida or invasive aspergillus infections in neutropenic patients antifungal therapy in patients with fever and neutropeniamore rational and less empirical? caspofungin versus liposomal amphotericin b for empirical antifungal therapy in patients with persistent fever and neutropenia a randomized, double-blind comparative trial evaluating the safety of liposomal amphotericin b versus amphotericin b lipid complex in the empirical treatment of febrile neutropenia. l amph/ablc collaborative study group levofl oxacin to prevent bacterial infection in patients with cancer and neutropenia guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients. recommendations of cdc, the infectious disease society of america, and the american society of blood and marrow transplantation breakthrough zygomycosis after voriconazole treatment in recipients of hematopoietic stem-cell transplants beta-lactam antibiotic therapy in febrile granulocytopenic patients. a randomized trial comparing cefoperazone plus piperacillin, ceftazidime plus piperacillin, and imipenem alone risk assessment and riskbased therapeutic strategies in febrile neutropenia randomized, double-blind clinical trial of amphotericin b colloidal dispersion vs. amphotericin b in the empirical treatment of fever and neutropenia voriconazole versus amphotericin b for primary therapy of invasive aspergillosis review of treatment of zygomycosis with posaconazole in a patient with acute myeloid leukemia intravenous and oral itraconazole versus intravenous amphotericin b deoxycholate as empirical antifungal therapy for persistent fever in neutropenic patients with cancer who are receiving broad-spectrum antibacterial therapy. a randomized, controlled trial anidulofungin: a new echinocandin with a novel profi le combination antifungal therapy for invasive aspergillosis treatment of solid organ transplant patients with invasive fungal infections: should a combination of antifungal drugs be used? prevention of intravascular catheter-related infections guidelines for the management of intravascular catheter-related infections guidelines for the prevention of intravascular catheter-related infections eortc guidelines for the use of granulocyte-colony stimulating factor to reduce the incidence of chemotherapyinduced febrile neutropenia in adult patients with lymphomas and solid tumours return of granulocyte transfusions metaanalysis: the effi cacy of strategies to prevent organ disease by cytomegalovirus in solid organ transplant recipients 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with the acquired immunodefi ciency syndrome. members of the actg 077p/anrs 009 study team toxoplasmic encephalitis in aids preemptive ganciclovir therapy to prevent cytomegalovirus disease in cytomegalovirus antibody-positive renal transplant recipients. a randomized controlled trial prevention of fungal and hepatitis virus infections in liver transplantation antiviral drugs for cytomegalovirus in transplant recipients: advantages of preemptive therapy randomised trial of effi cacy and safety of oral ganciclovir in the prevention of cytomegalovirus disease in livertransplant recipients. the oral ganciclovir international transplantation study group whole blood real-time quantitative pcr for cytomegalovirus infection follow-up in transplant recipients prophylactic versus preemptive oral valganciclovir for the management of cytomegalovirus infection in adult renal transplant recipients benefi ts of cytomegalovirus prophylaxis in solid organ transplantation posttransplant microbiological surveillance should prophylaxis for pneumocystis carinii pneumonia in solid organ transplant recipients ever be discontinued? key: cord-003376-2qi4aibx authors: van de groep, kirsten; nierkens, stefan; cremer, olaf l.; peelen, linda m.; klein klouwenberg, peter m. c.; schultz, marcus j.; hack, c. erik; van der poll, tom; bonten, marc j. m.; ong, david s. y. title: effect of cytomegalovirus reactivation on the time course of systemic host response biomarkers in previously immunocompetent critically ill patients with sepsis: a matched cohort study date: 2018-12-18 journal: crit care doi: 10.1186/s13054-018-2261-0 sha: doc_id: 3376 cord_uid: 2qi4aibx background: cytomegalovirus (cmv) reactivation in previously immunocompetent critically ill patients is associated with increased mortality, which has been hypothesized to result from virus-induced immunomodulation. therefore, we studied the effects of cmv reactivation on the temporal course of host response biomarkers in patients with sepsis. methods: in this matched cohort study, each sepsis patient developing cmv reactivation between day 3 and 17 (cmv+) was compared with one cmv seropositive patient without reactivation (cmvs+) and one cmv seronegative patient (cmvs−). cmv serostatus and plasma loads were determined by enzyme-linked immunoassays and real-time polymerase chain reaction, respectively. systemic interleukin-6 (il-6), il-8, il-18, interferon-gamma–induced protein-10 (ip-10), neutrophilic elastase, il-1 receptor antagonist (ra), and il-10 were measured at five time points by multiplex immunoassay. the effects of cmv reactivation on sequential concentrations of these biomarkers were assessed in multivariable mixed models. results: among 64 cmv+ patients, 45 could be matched to cmvs+ or cmvs− controls or both. the two baseline characteristics and host response biomarker levels at viremia onset were similar between groups. cmv+ patients had increased ip-10 on day 7 after viremia onset (symmetric percentage difference +44% versus −15% when compared with cmvs+ and +37% versus +4% when compared with cmvs−) and decreased il-1ra (−41% versus 0% and −49% versus +10%, respectively). however, multivariable analyses did not show an independent association between cmv reactivation and time trends of il-6, ip-10, il-10, or il-1ra. conclusion: cmv reactivation was not independently associated with changes in the temporal trends of host response biomarkers in comparison with non-reactivating patients. therefore, these markers should not be used as surrogate clinical endpoints for interventional studies evaluating anti-cmv therapy. electronic supplementary material: the online version of this article (10.1186/s13054-018-2261-0) contains supplementary material, which is available to authorized users. cytomegalovirus (cmv) reactivation is observed in 14-41% of intensive care unit (icu) patients without known prior immune deficiency [1] [2] [3] and is associated with increased morbidity and mortality [4] [5] [6] . in a previous study, we estimated that the population-attributable fraction of icu mortality due to cmv reactivation was 23% in patients with acute respiratory distress syndrome (ards) [7] . in a subsequent study among patients with septic shock, we found an effect of cmv reactivation on icu mortality only in patients with concurrent epstein-barr virus reactivation [8] . although multiple studies point toward a causal relationship, definitive proof that cmv reactivation worsens clinical outcome is lacking, as most data are also compatible with a scenario in which cmv reactivation is merely a marker of immune suppression in this patient group. based on previous studies in icu patients, there is a clear pathophysiological link between inflammation and immune suppression on the one hand and the subsequent risk of cmv reactivation on the other [9] [10] [11] [12] [13] . markers reflecting impaired functioning of natural killer cells and cytotoxic t cells were predictive of cmv reactivation [10, 11] . furthermore, bacterial sepsis and corticosteroids have been identified as clinical risk factors for cmv reactivation [9, 12, 13] . however, less is known about the reverse association and thus the effects of cmv reactivation on the immune system. direct cytotoxic effects of cmv on organs have been observed primarily in immunocompromised hosts [14] but also in previously immunocompetent patients in the icu [15] . moreover, indirect immune-modulating effects are assumed to play a role in the pathogenicity of cmv [13, [16] [17] [18] . in vitro analysis revealed multiple mechanisms encoded within the genome of cmv that may contribute to a non-specific inhibition of both cellular and humoral immunity [19] . observational clinical studies yielded conflicting results comparing levels of multiple inflammatory markers in patients with and without cmv reactivation [1, 11, 20] . however, these studies analyzed biomarker responses only immediately upon icu admission and thus could not assess potential immunological effects due to the onset of cmv reactivation. nevertheless, cytokine levels were used as a primary (surrogate) endpoint in a recent placebo-controlled randomized control trial in which prophylactic antiviral treatment with ganciclovir failed to reduce interleukin-6 (il-6) levels [21] . hence, definite proof of immune-modulating effects induced by cmv remains to be demonstrated. naturally, such an effect can be demonstrated only after onset of cmv reactivation. therefore, this longitudinal study aimed to investigate whether the temporal course of seven host response biomarkers, including both pro-and anti-inflammatory cytokines, in previously immunocompetent icu patients with sepsis differs between patients with and without cmv reactivation. this matched cohort study was performed among patients who had been included in two previous studies conducted within the molecular diagnosis and risk stratification of sepsis (mars) cohort [7, 8] . for this study, we included sepsis patients who presented with either concomitant ards or septic shock to the mixed icus of two university medical centers in the netherlands between january 2011 and june 2014 and had remained in the icu beyond day 4. exclusion criteria were cmv seronegative patients with cmv viremia (thus a primary infection) during their icu stay and known immunodeficiency or anti-viral treatment in the week before icu admission. the institutional review boards of both study centers approved an opt-out method of informed consent (protocol number 10-056c). from this parent cohort, we selected patients with an onset of cmv reactivation between day 3 and 17 in the icu. these patients with viremia were matched to two control groups consisting of patients without viremia on any day of icu admission. first, we matched patients with reactivation in a 1:1 ratio to cmv seropositive patients without reactivation (further referred to as "primary comparison"). second, we matched patients with reactivation in a 1:1 ratio to cmv seronegative patients without cmv viremia (further referred to as "secondary comparison"). this secondary comparison was intended mainly to confirm results of the primary comparison; the rationale was that any finding suggestive for an effect of cmv reactivation should also become apparent when compared with seronegative patients who are not at risk for cmv reactivation. matching criteria to reduce confounding were length of stay until reactivation (determines t = 0), sequential organ failure assessment (sofa) score at t = 0 (± 2 points), age (± 10 years), sex, and high-dose corticosteroid use during 4 days prior to t = 0 (that is, more than 250 mg hydrocortisone or equivalent). patients were also matched on hospital and calendar day of icu admission (± 365 days) in order to reduce possible influences of variation in sample workup and biobank storage duration [22] . the optimal matching result was retrieved by selecting the largest sample size after 1000 random iterations of the matching procedure. leftover plasma, obtained daily as part of routine patient care, was stored at −80°c and used to determine cmv serostatus at icu admission. subsequently, cmv load in blood was measured weekly, and for intermediary days, on which quantitative polymerase chain reaction was not performed, we estimated viral loads by log-linear imputation (see electronic supplementary materials of [7] ). cmv viremia was defined as at least 100 international units (iu) per milliliter. this cutoff value was similar to the ones used in previous studies [7, 8] . results of cmv viral load measurements in plasma performed for this study were not made available to the treating physicians, and none of the included patients received anti-cmv treatment. to map the immune response, we measured a panel of host response biomarkers in samples derived from five time points: day of viremia onset (t = 0), 2 days prior (t = −2), and after viremia onset at day 3, 7, and 10 (sample availability depended on length of stay in the icu). a multiplex luminex immunoassay was performed by using edta plasma and included the following proteins: il-6, il-8, il-18, tumor necrosis factor-alpha (tnf-α), tnf-related apoptosis-inducing ligand (trail), interferon-gamma (ifn-γ), ifn-γ-induced protein-10 (ip-10), neutrophilic elastase, granzyme-b, il-1 receptor antagonist (ra), and il-10. based on the results of a pilot run using 82 samples obtained from 15 ards patients without sepsis at icu admission (whom were not included in this study), we excluded ifn-γ, tnf-α, trail, and granzyme-b from the final panel because the levels of these biomarkers were below the lower limit of detection in more than 70% of the samples. of note, in this pilot run, cmv reactivation was not associated with detectability of the four excluded biomarkers. measurements of biomarkers were performed by using an in-house developed and validated multiplex immunoassay (iso9001 certified) based on luminex technology (xmap, luminex, austin, tx, usa). the assay was performed as described previously [23] . in short, thawed edta plasma samples (60 μl) were diluted 1:1 in high performance elisa (hpe) buffer (sanquin, the netherlands) and centrifuged through filtration columns to remove debris. then non-specific heterophilic immunoglobulins were pre-absorbed from all samples with heteroblock (omega biologicals, bozeman, mt, usa). next, samples were incubated with antibody-conjugated magplex microspheres for 1-h at room temperature with continuous shaking and this was followed by 1-h incubation with biotinylated antibodies and 10-min incubation with phycoerythrin-conjugated streptavidin diluted in hpe buffer. acquisition was performed with the flexmap 3d system (bio-rad laboratories, hercules, ca, usa) in combination with xponent software version 4.2 (luminex). data were analyzed by 5-parametric curve fitting using bio-plex manager software, version 6.1.1 (bio-rad laboratories). univariable analyses were performed to compare patients and disease characteristics for matched groups with and without cmv reactivation using chi-squared, wilcoxon rank sum, or fischer exact tests as appropriate. measured host response markers were natural log-transformed concentrations in picograms per milliliter for all analyses. symmetric percentage differences were calculated for each patient at the different time points. this delta percentage reflects the relative change from the measurement 2 days prior to cmv reactivation until the follow-up measurement [24] . we performed additional multivariable analyses by using generalized linear mixed models to assess the effect of cmv reactivation on the time course of each individual biomarker. in the mixed model analyses, we assessed whether baseline biomarker levels were comparable between matched groups (that is, coefficient for cmv reactivation) as well as the effect of cmv on the course of the biomarker levels over time (that is, coefficient for interaction term between time and cmv reactivation). a priori we chose to model the established immune markers il-6 and il-10. based on the observed divergence in the symmetric percentage differences over time between groups, we conducted the multivariable analyses also for the pro-inflammatory chemokine ip-10 and the anti-inflammatory cytokine il-1ra. since not all cmv reactivation patients were included in both comparisons, we performed separate mixed model analyses for the primary and secondary comparisons. thus, in total, eight models were built (for each of the four biomarkers in each of the two comparisons). for each model, sofa score at t = 0 (that is, the day of reactivation) and age were included as confounders since we used a range (instead of an exact value) as matching criteria for these co-variables. for the fixed part of th emodels a polynomial term for time was evaluted (that is, quadratic time effect). furthermore, a random intercept and a rondom slowe were evaluted for each model. restricted maximum likelihood estimation (reml) was used to generate unbiased variance estimates for the final models [25] . different ways to model the time course for each host response marker were compared by using the likelihood ratio test and akaike's information criterion. to take multiple testing into account and reduce the risk of spurious findings, we performed all statistical testing against a p value of 0.01 and used a confidence interval of 99%. bonferroni adjustment was deemed inappropriate and too conservative as the different measurements performed over time within a single patient and hence the tests were highly correlated with each other. analyses were performed by using either sas enterprise guide 7.1 (sas institute, cary, nc, usa) or r version 3.3.2 (r foundation for statistical computing, 2015; used packages "lme4", "lmetest"). forty-five (70%) of 63 eligible patients with cmv reactivation during icu day 3-17 could be included after matching (fig. 1) . twenty-eight patients were matched to a seropositive patient as well as a seronegative, nine to only seropositive, and eight to only seronegative, respectively. this resulted in a study population of 118 unique patients, divided into a primary comparison (that is, 37 with cmv reactivation matched to 37 cmv seropositive without reactivation) and a secondary comparison (that is, 36 with cmv reactivation matched to 36 cmv seronegative). patient and disease characteristics at icu admission were comparable between matched groups and are presented in table 1 . in the patients with cmv reactivation, median peak level of cmv dna load was 404 iu/ml (interquartile range (iqr) 214-1370). median length of stay in the icu until reactivation was 9 days (iqr 6-11), which was influenced by the used inclusion criterion (that is, viremia onset between day 3 and 17 in the icu). of the 118 unique sepsis patients included, 81 (69%) presented to the icu with septic shock and 80 (68%) patients had ards during the first week of icu admission. in the primary comparison, the median icu length of stay was 16 days (iqr 10-21) for patients with cmv reactivation versus 14 days (iqr [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] for subjects without reactivation (p = 0.90). this was 16 days (iqr 11-21) versus 19 days (iqr [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] in the secondary comparison (p = 0.21), respectively. hospital mortality was 57% for patients with cmv reactivation and 46% for the matched patients without reactivation in the primary comparison (p = 0.35). in the secondary comparison, this was 58% versus 47% (p = 0.35), respectively. baseline levels of measured host response markers were comparable between patients with and without reactivation, both at t = −2 (that is, 2 days prior to viremia onset) and at t = 0 (that is, day of reactivation onset) ( table 2 ). in general, this remained the case for each marker up to 10 days after cmv reactivation; the exceptions were median il-10 levels (which were significantly higher on day 10) and median il-6 levels (which were significantly lower on day 7) in patients with cmv reactivation compared with controls (additional file 1: table s1 ). however, these differences were not consistent across both primary and secondary comparison. time trends of various markers within patients were described by symmetric percentage differences relative to their levels 2 days prior to cmv viremia onset (fig. 2 for primary comparison, additional file 1: figure s1 for secondary comparison). for ip-10 and il-1ra, differences in time trends were observed between patients with and without reactivation in both comparisons. patients with cmv reactivation had a more pronounced increase of ip-10 (median percentage difference of 44% versus −15%) and decrease of il-1ra (median percentage difference of −41% versus 0%) on day 7 after viremia onset compared with cmv seropositive patients without reactivation. for the secondary comparison, with cmv seronegative patients, similar differences in trends were observed for ip-10 (+37% versus +4%) and il-1ra (−49% versus +10%), respectively. of importance, sample size decreased over time because of death or icu discharge with a minimum of 11 per patient group after 10 days (additional file 1 table s1 ). in the multivariable mixed model analyses, cmv reactivation did not significantly affect the baseline levels of il-6, ip-10, il-10, and il-1ra (table 3) . a significant decrease over time was observed in all patients for il-6 in both the primary and secondary comparison and for il-10 in the primary comparison only, respectively. however, cmv reactivation did not significantly affect the time trend of any of the four analyzed biomarkers. we performed an explorative study to compare time trends of host response biomarkers in patients with reactivation that were matched to non-reactivating control patients who were either seropositive or seronegative for cmv. although we initially observed differential trends of il-1ra and ip-10 in the crude analysis, these differences did not remain in the linear mixed model analysis the hypothesis of an immune-modulating effect of cmv is based on the observation of increased mortality and morbidity in patients with viremia without organ manifestation of cmv disease [13, 19] . proposed mechanisms of such indirect pathogenicity are autoantibody production, enhanced inflammation, vascular damage, and cmv-induced immunosuppression [17] . based on this hypothesis and an observed association between plasma markers and mortality in patients with ards [26] , il-6 was used as a surrogate endpoint in a recent randomized controlled trial that evaluated the safety of preventive antiviral treatment in icu patients [16] . our finding that cmv reactivation is not associated with modified il-6 dynamics questions the suitability of il-6 as an endpoint in clinical trials evaluating preventive therapy for cmv reactivation in icu patients. furthermore, time trends of other immunological biomarkers were not robustly affected by cmv reactivation. our study has several strengths. first, to our knowledge, this is the first study with serial measurements of the immune response following (instead of prior to) cmv reactivation. second, our study design included two matched control groups. because of the used matched cohort design, we could include only 45 out of 63 patients with cmv reactivation but this loss was compensated by the ability to include controls that were more comparable to those patients. sepsis patients in the icu are known to be very heterogeneous [27, 28] ; thus, the matching reduced in theory both confounding and unwanted variation by extraneous factors. third, by using mixed model analyses, we accounted for correlation of measurements performed within one patient by the use of random effects, which increased the statistical power to identify differences between patient groups. moreover, this type of analysis takes into account the considerable loss to follow-up of patients and allowed us to estimate an average trend over time based on available data. our study also has some limitations. first, this was an explorative study evaluating multiple host response biomarkers. we chose a lower p value threshold of significance in order to decrease the risk of spurious findings due to multiple testing, but false-negative findings remain an accessory risk to keep in mind also when considering our study sample size. unfortunately, a formal sample size calculation for this kind of statistical analysis was not possible. nevertheless, we postulate that possible immunomodulating effects of cmv reactivation il-18 6.5 (5.9-6.9) 6.4 (5.9-6.6) 0.46 6.3 (5.9-6.7) 6.4 (5.9-6.7) 0.77 ip-10 6.5 (6.0-7.0) 6.3 (5.9-6.8) 0. seem at most to be rather limited in these patients because no large differences in biomarker levels between matched groups were observed. second, we analyzed host response biomarkers as standalone markers, which is probably a simplification of the complex immune response. however, large sample sizes are required to perform more advanced network analyses, and the integration of time series in such analyses, to our knowledge, has not been conducted before. we also measured only the plasma concentrations. since cmv pneumonitis could be an important mediator of the pathological effect of cmv reactivation in critically ill patients, bronchoalveolar lavage samples may be additionally informative but were not available [16, 29] . finally, we did not evaluate all potentially relevant biomarkers for cmv reactivation; thus, future studies are needed before an immunomodulating effect of cmv can be ruled out with certainty as an important pathological mechanism in previously immunocompetent icu patients. this study could not demonstrate an independent immunomodulating effect of cmv reactivation in patients with sepsis. this finding does not lend support for the use of immunological markers as surrogate endpoints for clinical outcome in interventional studies of prophylactic or pre-emptive cmv therapy in icu patients. additional file 1: table s1 . absolute levels of host response markers during follow-up by cytomegalovirus (cmv) reactivation status. figure s1 . ethics approval and consent to participate all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional or national research committee (or both) and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. for this study, an opt-out informed consent method was approved. not applicable. cytomegalovirus reactivation in a general, nonimmunosuppressed intensive care unit population: incidence, risk factors, associations with organ dysfunction, and inflammatory biomarkers cytomegalovirus reactivation and associated outcome of critically ill patients with severe sepsis cytomegalovirus reactivation in critically ill immunocompetent patients prevalence and mortality associated with cytomegalovirus infection in nonimmunosuppressed patients in the intensive care unit association between cytomegalovirus reactivation and clinical outcomes in immunocompetent critically ill patients: a systematic review and meta-analysis cytomegalovirus infection in critically ill patients: a systematic review cytomegalovirus reactivation and mortality in patients with acute respiratory distress syndrome epidemiology of multiple herpes viremia in previously immunocompetent patients with septic shock active cytomegalovirus infection is common in mechanically ventilated medical intensive care unit patients interferon-gamma production by natural killer cells and cytomegalovirus in critically ill patients evaluation of cytomegalovirus (cmv)-specific t-cell immunity for the assessment of the risk of active cmv infection in non-immunosuppressed surgical and trauma intensive care unit patients human cytomegalovirus infections in nonimmunosuppressed critically ill patients cytomegalovirus reactivation in icu patients human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments a contributive result of open-lung biopsy improves survival in acute respiratory distress syndrome patients cmv in critically ill patients: pathogen or bystander cytomegalovirus-induced immunopathology and its clinical consequences reactivation of multiple viruses in patients with sepsis the 'indirect' effects of cytomegalovirus infection virological and immunological features of active cytomegalovirus infection in nonimmunosuppressed patients in a surgical and trauma intensive care unit effect of ganciclovir on il-6 levels among cytomegalovirus-seropositive adults with critical illness: a randomized clinical trial cytokine assays: an assessment of the preparation and treatment of blood and tissue samples effect of anticoagulants on 162 circulating immune related proteins in healthy subjects statistics notes: percentage differences, symmetry, and natural logarithms using the general linear mixed model to analyse unbalanced repeated measures and longitudinal data lower tidal volume ventilation and plasma cytokine markers of inflammation in patients with acute lung injury why have clinical trials in sepsis failed? unexplained mortality differences between septic shock trials: a systematic analysis of population characteristics and control-group mortality rates immunological insights into the pathogenesis of active cmv infection in non-immunosuppressed critically ill patients we thank the department of medical microbiology and the multiplex core facility of the laboratory for translational immunology for their logistical support and performance of measurements, the participating icus and research nurses of the two medical centers for their help in data acquisition, and all members of the molecular diagnosis and risk stratification of sepsis all authors declare that they have no conflicts of interest related to the subject matter. key: cord-288721-3bv3aak6 authors: schneider, annika; kurz, sandra; manske, katrin; janas, marianne; heikenwälder, mathias; misgeld, thomas; aichler, michaela; weissmann, sebastian felix; zischka, hans; knolle, percy; wohlleber, dirk title: single organelle analysis to characterize mitochondrial function and crosstalk during viral infection date: 2019-06-11 journal: sci rep doi: 10.1038/s41598-019-44922-9 sha: doc_id: 288721 cord_uid: 3bv3aak6 mitochondria are key for cellular metabolism and signalling processes during viral infection. we report a methodology to analyse mitochondrial properties at the single-organelle level during viral infection using a recombinant adenovirus coding for a mitochondrial tracer protein for tagging and detection by multispectral flow cytometry. resolution at the level of tagged individual mitochondria revealed changes in mitochondrial size, membrane potential and displayed a fragile phenotype during viral infection of cells. thus, single-organelle and multi-parameter resolution allows to explore altered energy metabolism and antiviral defence by tagged mitochondria selectively in virus-infected cells and will be instrumental to identify viral immune escape and to develop and monitor novel mitochondrial-targeted therapies. mitochondria are crucial for cellular energy metabolism, critically involved in the coordination of signalling processes within cells and orchestrate induction of apoptotic cell death 1, 2 . besides this, cell-autonomous defence mechanisms during viral infection link innate immune sensing of infection and inflammation at the level of mitochondria 3, 4 . the research in the recent years has expanded our knowledge about the different roles of mitochondria. for the different functions mitochondrial shape and motility, but also size, are important and are highly dynamic processes 5 . mitochondrial shape and size are continuously changed during the dynamics of mitochondrial fusion and fission and mitochondrial turnover is controlled by mitophagy 5 . viruses modify the host cell to create an ideal ambience, which includes metabolic support for viral gene expression and replication. such modifications of cellular metabolism and structure of viruses can also affect mitochondria. there are more and more reports about viruses known to influence mitochondrial dynamics. viruses known to enhance mitochondrial fission are hepatitis b virus (hbv), hepatitis c virus (hcv) and epstein-barr virus [6] [7] [8] [9] . viruses, which interfere with or enhance mitophagy are hbv, hcv and measles virus [6] [7] [8] 10 . sars coronavirus is reported to enhance the fusion of mitochondria 11 . but the influence of viral infection on mitochondrial membrane potential and stress response has not been addressed in detail because of methodological constraints. so far, analysis of mitochondria and their functions relied mostly on bulk analysis of mitochondrial populations analysed ex vivo. in infected tissues where both, infected and non-infected cells are simultaneously present, it is very difficult to discriminate between mitochondria from infected versus healthy non-infected cells. this may be achieved by serial tissue sections analysed by electron microscopy, where viral particles could be visualized. however, this is a very time demanding process yielding results with little statistical power. we therefore aimed to develop a technology, where high numbers of single mitochondria and their function can be analysed in the context of viral infection in order to characterize changes induced by viral infection. we chose the liver, and more specifically hepatocytes, as viral infection increases size and mitochondrial fragility of liver mitochondria. we first aimed to determine the influence of viral infection of the liver on the size of liver mitochondria by flow cytometry. to that end, we established a reference curve using polystyrene microparticles with defined sizes (0.88 µm, 1,34 µm and 3 µm). forward scatter analysis of these polystyrene microparticles revealed clear demarcation of the differently sized microparticles and a direct linear correlation of forward scatter results with microparticle size (r 2 = 0.99) ( fig. 2a) , consistent with earlier reports that forward scatter measurements directly correlate with microparticle size down to 0.5 µm 20, 21 . the flow cytometric analysis revealed that mitochondria isolated from healthy non-infected liver ranged in size from 0.8 µm up to 1.4 µm (fig. 2b ) assuming that isolated mitochondria are spherical in morphology, which is indicated by electron microscopy (see fig. 1b ). since mitochondria from hepatocytes are much larger than those from non-parenchymal liver cells or immune cells, we assume that mitochondria ≥0.8 µm in size are derived from hepatocytes. mitochondria purified from virus-infected livers had a slightly higher mean size compared to healthy liver (1.04 ± 0.06 µm compared to 0.97 ± 0.04 µm, respectively) and ranged in size from 0.8 µm up to 3 µm (fig. 2b) . infection with recombinant replication-deficient adenoviruses is a well-established preclinical model system to study hepatotropic infections [22] [23] [24] . however, to confirm the results we repeated the experiments by infection with wildtype replication-competent lymphocytic choriomeningitis virus (lcmv). also after lcmv-infection, we detected an increase of mitochondrial size confirming the results obtained after adenoviral infection (supp. fig. 1a ). in order to investigate whether innate immunity generated during viral infection, was responsible for this increase in mitochondrial size, we induced a type i interferon response by application of poly i:c 25 . flow cytometric analysis of mitochondria isolated after poly i:c application did not reveal any differences in their size compared to the control groups suggesting other mechanisms (supp. fig. 1b) . the exact determination of the size of single mitochondria now opened the possibility to use this information for further analysis. next, we evaluated mitochondrial functionality by determining the mitochondrial membrane potential using the potentiometric dilc 1 (5) fluorescence dye. dose titration experiments of the dilc 1 (5) dye demonstrated a dose-dependent increase in fluorescence intensity in purified mitochondria (supp. fig. 1c ). upon addition of the electron chain uncoupling agent cccp, we found a profound reduction in dilc 1 (5) fluorescence (fig. 2c ) demonstrating that flow cytometric determination of changes in dilc 1 (5) fluorescence reflected mitochondrial membrane potential. by flow cytometric analysis we observed a significant decrease in the mitochondrial membrane potential of mitochondria isolated from virus-infected vs. healthy livers after either adenoviral or lcmv infection compared to healthy controls ( fig. 2d and supp. fig. 1d ). in contrast, we did not detect changes in the membrane potential after innate immune stimulation by poly i:c (supp. fig. 1e ). yet, the size of mitochondria may influence dilc 1 (5) signal intensity. indeed, we found a direct correlation between mitochondrial size and dilc 1 (5) staining ( fig. 2e and supp. fig. 1f ) suggesting that larger mitochondria purified from virus-infected livers should show higher dilc 1 (5) fluorescence intensity. we therefore compared mitochondria with the same size isolated from healthy or virus-infected livers. such direct comparison demonstrated that mitochondria of the same size from healthy vs. virus-infected livers showed a remarkable decrease in the membrane potential of mitochondria from infected livers (fig. 2f ) and suggested that viral infection caused changes in mitochondrial functionality. mitochondria also function to take up calcium from the cytosol and thereby coordinate cellular function 26 , which can also serve as a stress test. when challenged with high concentrations of calcium (100 µm), mitochondria isolated from virus-infected livers are much more fragile shown by time-dependent loss of membrane potential and change of their morphology indicated by decrease in side-scatter (fig. 2f ). this accurately detects mitochondrial swelling after loss of membrane potential following ca 2+ challenge which is also detected by bulk analysis with a classical stress test by adding ca 2+ and detection of loss of membrane potential by rh123-fluorescence and swelling by measuring optical density at 540 nm (supp. fig. 1g ) 18 . consistent with the loss of membrane potential and changes in side-scatter signals, we detected loss of mitochondrial integrity after www.nature.com/scientificreports www.nature.com/scientificreports/ calcium challenge. number of viable mitochondria detected per second by flow-cytometry declined after calcium challenge, consistent with loss of mitochondrial integrity, and did so much faster in samples from virus-infected livers (fig. 2f ). comparing mitochondria with different sizes, it became evident that larger mitochondria are more fragile and disappeared more rapidly after ca 2+ -challenge (fig. 2f ). taken together, here we detected an increase in size and a decrease in membrane potential as well as mitochondrial fragility of liver mitochondria after viral infection. however, since both, non-infected as well as infected hepatocytes are present in livers after adenoviral infection (see fig. 1b ), current protocols for isolation and analysis yield a mixture of mitochondria derived from healthy as well as infected hepatocytes. this makes it necessary to develop a methodology, by which mitochondria from healthy and virus-infected hepatocytes can be separated in order to characterize changes in mitochondrial function specifically in virus-infected cells. we generated a recombinant adenovirus expressing the fluorescent protein dsred fused to a mitochondrial localization sequence (ad-cmv-mitorl) that accumulates and selectively labels mitochondria within infected cells (supp. fig. 2 ). we combined this mitochondrial labelling in infected cells with a multispectral flow cytometric single organelle measurement of isolated mitochondria. upon infection of hepatocytes with ad-cmv-mitorl in vitro we detected mito-dsred-fluorescence in mitochondria using confocal microscopy (fig. 3a) . since ad-cmv-mitorl also codes for luciferase, we detected in vivo bioluminescence of the liver after infection, thus www.nature.com/scientificreports www.nature.com/scientificreports/ confirming successful infection of hepatocytes in vivo (fig. 3b ). this allowed us to test whether mitochondria from ad-cmv-mitorl-infected hepatocytes (mito-dsred + mitochondria) could be distinguished from those of non-infected hepatocytes (mito-dsred − mitochondria) within the same liver. after density-gradient purification, mitochondria isolated from virus-infected livers were counterstained with mitotracker green and analysed by flow cytometry allowing discrimination of mito-dsred + mitochondria from mito-dsred − mitochondria from the same liver (fig. 3c ). mito-dsred + mitochondria from virus-infected hepatocytes had a mean size of 1.19 ± 0.06 µm as compared to mito-dsred − mitochondria from healthy hepatocytes with a mean size of 0.96 ± 0.01 µm (fig. 3d ). this confirmed the results obtained from mitochondria isolated from non-infected livers and further demonstrated a more pronounced size difference when mitochondria from virus-infected could be distinguished at the single organelle level from those of healthy hepatocytes. this was most likely related to a relative underestimation of size for mitochondria from virus-infected livers due to contamination with mitochondria from non-infected cells that are smaller than hepatocyte mitochondria. yet, we cannot formally exclude that mito-dsred localizing to mitochondria after infection with ad-cmv-mitorl may have contributed to the size difference. the almost identical forward scatter results and size of mito-dsred − mitochondria compared to mitochondria isolated from non-infected livers (fig. 3d) indicated that there was no influence of viral infection in neighbouring hepatocytes on mitochondrial size after isolation. differences in size of mitochondria may have also an influence on other parameters detected by flow cytometry and we therefore systematically measured mitochondrial autofluorescence from 450 to 800 nm using a spectral flow cytometer. as expected, we detected increased fluorescence at 590 nm in mito-dsred + mitochondria, where the maximum of dsred fluorescence emission (590-650 nm) is expected 27 . interestingly, we detected higher autofluorescence signals between 500 and 550 nm as well as above 650 nm in mito-dsred + mitochondria (fig. 3e) . as the strength of autofluorescence may be influenced by the size of mitochondria, we analysed autofluorescence signals against the size of isolated mitochondria (fig. 3f ). we found that autofluorescence intensity between 430 and 550 nm directly correlated with mitochondrial size, which may explain the higher autofluorescence observed in larger mito-dsred + mitochondria. together, these data demonstrate the usefulness of single-organelle analysis by flow cytometry in combination with in vivo mitochondrial labelling in virus-infected hepatocytes to exactly determine physical parameters such as size or autofluorescence. mitochondrial crosstalk enables changes in membrane potential. since discrimination of mitochondria isolated from virus-infected compared to non-infected hepatocytes was reliably achieved using flow cytometry, we proceeded to test for changes in mitochondrial functionality upon infection. we assumed that the difference in membrane potential detected between mitochondria isolated from virus-infected livers compared to non-infected livers (see fig. 2 ) has previously been underestimated, and that our method would allow to more specifically discriminate mitochondria from virus-infected hepatocytes compared to non-infected hepatocytes. we determined whether mito-dsred + differed from mito-dsred − mitochondria with respect to dilc 1 (5) fluorescence intensity. to our surprise, we found that the dilc 1 (5) signal was similar for all sizes of mito-dsred + compared to mito-dsred − mitochondria (fig. 4a ). since dilc 1 (5) fluorescence was homogenous in all mitochondria isolated from ad-cmv-gol infected livers (see fig. 1 ), although they consisted of a mixture of mitochondria from infected and non-infected hepatocytes, we wondered whether there was an exchange of molecules between mitochondria. therefore, we mixed dilc 1 (5)-labelled mitochondria with non-labelled mitochondria and by time-dependent flow cytometric analysis found that dilc 1 (5) fluorescence decreased in pre-labelled and increased in un-labelled mitochondria reaching an equilibrium of intermediate fluorescence intensity within 30 seconds (fig. 4b) . however, mito-dsred was not exchanged between mitochondria, because we found clearly distinct dsred staining of mitochondria isolated from ad-cmv-mitorl-infected livers, and mito-dsred − mitochondria showed the same absent dsred fluorescence intensity as mitochondria isolated from non-infected livers. in order to further evaluate mitochondrial functionality, we challenged mitochondria with ca 2+ as stress test and performed time kinetic measurements of dilc 1 (5) fluorescence and side-scatter of mito-dsred + and mito-dsred − mitochondria isolated from ad-cmv-mitorl infected livers. remarkably, the differences in mitochondrial characteristics observed when comparing mitochondria isolated from infected livers to mitochondria from non-infected livers (see fig. 2f ) where not present any more when comparing mito-dsred + to mito-dsred − mitochondria originating from the same liver. in fact, loss of dilc 1 (5) fluorescence, decrease in side scatter and mitochondrial events were the same for mito-dsred + mitochondria as compared to mito-dsred − mitochondria (fig. 4c) . when in direct physical contact with mito-dsred + mitochondria, also mito-dsred − mitochondria showed the same fragility as mitochondria from virus-infected hepatocytes. there, was still a small difference in the large mitochondrial group after calcium stimulation and flow cytometric analysis of the ssc and dilc 1 (5) which could be explained by the fact that 5 to 10 minutes after calcium stimulation the number of events was drastically reduced. only approximately 10% from the initial number of events are still detectable (shown by number of events/s). because of the statistical variation the conclusions at later time points has to be taken with caution. interestingly, also mixing of dilc 1 (5) labelled mitochondria isolated from either ad-cmv-golor lcmv-infected with those from healthy uninfected livers yielded in rapid loss of mitochondrial membrane potential to that measured in mitochondria from infected livers (fig. 4d and supp. fig. 2 ) taken together these data demonstrate that mitochondria which are in close physical proximity exchange information leading to changes in mitochondrial membrane potential but not in mitochondrial size. here, we describe the influence of viral infection on the phenotype and function of mitochondria employing a new methodology combining spectral flow cytometry with virus-encoded markers to simultaneously evaluate multiple mitochondrial parameters at the level of single organelles. most studies involve confocal microscopy to detect mitochondria, which is also available in an automated manner to quantify large datasets of mitochondria 28 . www.nature.com/scientificreports www.nature.com/scientificreports/ while most of these microscopic studies are performed in cell cultures to explore mitochondrial dynamics at the level of single cells, there are only few reports specifically detecting tagged mitochondria in tissues for ex vivo or in vivo analysis 29, 30 . since in vivo microscopic analysis of mitochondria requires a complex experimental setup, is rather time consuming and does not allow for analysis of large numbers of mitochondria, we aimed to establish a methodology to evaluate mitochondria directly ex vivo following isolation from virus-infected tissue. so far, most of available methods analyse properties of mitochondria ex vivo at the level of mitochondrial populations, www.nature.com/scientificreports www.nature.com/scientificreports/ such as extracellular flux analysis, western blot analysis, calcium uptake or swelling assays. beyond visualization by microscopy, flow cytometry has emerged as technology to characterize mitochondria 18, 31, 32 . however, mitochondria isolated from virus-infected tissues can be derived from both, virus-infected cells as well as healthy cells, which may skew the experimental results. we therefore generated recombinant adenoviruses containing a mito-dsred expression cassette to selectively label mitochondria of infected cells. fusion of a fluorescent marker to mitochondrial target sequences has previously been reported to reliably and specifically label mitochondria as shown by confocal microscopy 33, 34 . we combined virus-encoded mito-dsred labelling of mitochondria to separate mitochondria of virus-infected cells from those originating from healthy cells, with the power of multi-parameter analysis by spectral flow cytometry. using this methodology, we provide evidence that mitochondria can be reliably separated from virus-infected cells and that viral infection led to an increase in size as well as a decrease of mitochondrial membrane potential. such changes in biophysical and functional properties of mitochondria were not triggered by innate immunity following recognition of infection through microbe-associated pattern recognition receptors indicating other reasons for these changes, which still have to be defined. time kinetic measurements of single mitochondria by flow cytometry further allowed us to detect a previously unknown mitochondrial cross-talk that involves rapid exchange of small molecules like the potentiometric dye dilc 1 (5) . such exchange of molecules among mitochondria required physical contact, occurred within seconds and did not include mitochondrial matrix-embedded proteins. this indicates a dynamic regulation of mitochondrial properties by cell autonomous mechanisms that require further investigation. taken together, the combination of mitochondrial labelling through mito-dsred together with single organelle analysis using spectral flow cytometry is ideally suited to further unravel biophysical and functional properties of mitochondria as well as mechanisms and consequences of mitochondrial interconnectivity in virus-infected cells. given the important role of mitochondria in cellular metabolism, anti-viral defence, cell signalling and cell death, the multiparametric analysis of single mitochondria opens new avenues to explore these complex mitochondrial functions in more detail in virus-infected cells. mice. c57bl/6 j mice were purchased from charles river (sulzfeld, germany). mice were maintained under specific pathogen-free (spf) conditions in the central animal facility of the klinikum rechts der isar, in accordance with the guidelines of the federation of laboratory animal science association. animal experiments were approved by the animal care commission of bavaria. male mice between the ages of 6-10 weeks were used. the expression cassette for cloning into recombinant adenovirus consists of the genes for the fluorescent protein dsred linked to a mitochondrial targeting sequence and cbg99-luciferase separated by p2a linker sites from the porcine teschovirus 1 followed by a bgh poly(a) signal. gene expression was driven by the ubiquitous minimal cmv-promoter (ad-cmv-mitorl). ad-cmv-gol generation has been reported before 23 . recombinant second generation serotype 5 adenoviruses were generated using the gateway ® technology from thermofisher as described before 23 . briefly, expression cassettes with cmv promotor, dsred linked to the mitochondrial targeting site, cbg99-luciferase and the bgh poly(a) signal were synthesized (eurofins genomics, germany) and cloned into gateway ® pentr ™ 11 dual selection vector (thermofisher scientific, germany). recombination of pentr ™ with expression cassette into pad/pl-dest ™ gateway ® vector (thermofisher scientific, germany) was performed in vitro via the lr clonase ® enzyme mix (thermofisher scientific, germany). the obtained pad/pl-dest ™ with expression cassette was linearized using the paci restriction enzyme and the resulting adenoviral dna was transfected with lipofectamine 2000 (thermofisher scientific, germany) into hek293 cells (crl-1573 ™ ; atcc, usa). cell debris and supernatant were harvested when complete detachment of the cells occurred. this suspension was freeze/thawed, centrifuged and used for further infection of hek293 cells. cells from several cell culture dishes were harvested and resuspended in tris-buffer and freeze/thawed three times. cell debris was removed by centrifugation and supernatant purified by a two-step cscl gradient ultracentrifugation. the band containing adenovirus was harvested and dialyzed. virus titer was determined via adenovirus hexon titration. hek293 cells were infected with serial dilutions of purified adenovirus. after 35 to 40 hours, cells were fixed with methanol, and virus infected cells were stained with anti-hexon antibody (anti-hexon 2297hrp, acris, germany) and detected via dab (dako, usa). the infected cells were counted and the titer was calculated. bioluminescence imaging. imaging of luciferase expression in infected mice was monitored by ivis lumina lt-series iii instrument (perkinelmer las, germany). five minutes before measurement mice have been anesthetized with 2.5% isofluran and treated intraperitoneally with 100 mg/kg bodyweight d-luciferin-k-salt (pjk gmbh, germany). isolation of mitochondria from murine liver tissue. heparin/nacl (300 u/150 µl) was injected i.p. into the mouse 5 minutes prior to preparation. mice were sacrificed and livers were perfused via portal vein for 1 minute with pbs to remove blood. liver was removed and weighed, and the liver was rinsed with isolation buffer www.nature.com/scientificreports www.nature.com/scientificreports/ (220 mm mannitol, 80 mm sucrose, 10 mm hepes, 1 mm edta, ph 7.4). the whole isolation procedure was performed on ice and in ice-cold isolation buffer. the tissue was rinsed with 1 ml isolation buffer and cut with a blunt end scissor into small pieces. the liver fragments were resuspended in 1 ml isolation buffer supplemented with 0.5% bsa and protease inhibitor (protease inhibitor cocktail, edta-free, roche, switzerland) per 0.1 gram of weighted organ and homogenized in a potter-elvehjem with 3 strokes at 800 rpm. the homogenate was transferred to cooled 50 ml falcon and centrifuged at 600 x g for 10 minutes to remove nuclei, intact cells and cellular debris. the supernatant was transferred to a glass tube and centrifuged at 4000 x g for 10 minutes to sediment mitochondria. the received crude pellet was gently dislodged with a glass pestle from the side of the glass tube. mitochondrial purification by density gradient centrifugation. mitochondria were purified as previously described 35, 36 . in brief, a discontinuous percoll density gradient was used for mitochondrial purification. crude mitochondria were resuspended in ip-buffer (300 mm sucrose, 5 mm tes, 0.2 mm egta, ph 6.9), loaded on a percoll density gradient (60%, 30% and 18% diluted in ipp buffer: 300 mm sucrose, 10 mm tes, 0.2 mm egta, 0.1% w/v bsa, ph 7.2) and separated at 9000 × g for 10 minutes. the phase containing mitochondria between 60% and 30% percoll-layer was recovered with a glass pipette and transferred to a 30 ml glass tube, resuspended in 15 ml ip-buffer and centrifuged for further 10 minutes at 9000 × g. the pellet was washed again in 10 ml ip-buffer and centrifuged at 9000 × g for 10 minutes to get rid of remaining percoll. the supernatant was removed and mitochondrial pellet was dislodged from the side of the glass tube. the received mitochondria were resuspended in 100 µl ip-buffer and kept on ice. determination of protein concentration. the protein content in the mitochondrial preparations was determined using the dc tm protein assay kit (bio rad laboratories, germany). the assay was performed according to the manufacturer´s protocol. four different bsa-dilutions reaching from 0.25 mg/ml to 1.5 mg/ml in ip-buffer were used as standards. the optical density was measured at 750 nm with a multiplate reader (infinitem100 pro, tecan, germany). determining mitochondria by flow cytometry. mitochondria were diluted to 10 µg protein per µl in ice-cold mitochondrial staining buffer msb (0.2 m saccharose, 10 mm mops-tris, 5 mm succinate, 1 mm phosphoric acid, 10 µm egta). the different mitochondrial probes were diluted in msb, mixed with the mitochondrial dilution in a 1:1 ratio and incubated at room temperature for 20 minutes. the cell permeable carbocyanine-based mitotracker green probe (mtg, 200 nm), which contains a mildly thiol-reactive chloromethyl moiety, was used to selectively stain all undamaged mitochondria regardless of the membrane potential. dilc1(5) (100 nm), a cationic carbocyanine dye, was used to measure the membrane potential of isolated mitochondria. mitochondria were pelleted at 9000 x g for 2 minutes and washed once in ice cold pbs. mitochondrial pellet was resuspended in msb to a final concentration of 10 µg/µl and stored on ice for analysis. immediately before analysis, samples were diluted in ice-cold and filtered pbs to the final analysis concentration of 0.05 µg/µl. samples were analysed using the spectral cell analyzer sp6800 (sony biotechnology inc, japan). the sample flow rate was set to record about 1500 events per second. as mitochondrial uncoupling by the protonophore cccp is well known to dissipate mitochondrial membrane potential (mmp), 5 µm cccp (sigma-aldrich, st. louis, missouri, usa) was used as a positive control for membrane potential dependence of diic 1 (5) (biotium, hayward, usa). the mitochondrial permeability transition (mpt), a process characterized by a large increase of permeability of the inner mitochondrial membrane (imm), leading to an influx of solutes with a molecular weight less than 1.5 kda and water into the mitochondrion, is a ca 2+ -induced process. the influx of solutes and water leads to swelling of mitochondria. in mpt-measurements 100 µm ca 2+ in msb was added to induce swelling and samples were analyzed immediately after administration and every following 5 minutes for 45 minutes in total. cyclosporina (sigma-aldrich, st. louis, missouri, usa) inhibiting mpt and thereby reversing the effect of ca 2+ , was added at a concentration of 5 µm. mitochondrial size was determined using polystyrene particle size standard beads (flow cytometry grade, spherotech) in three sizes: 0.88 μm, 1.34 μm and 3 μm. beads of each size were separated via ultrasound, vortexed and 20000 beads/size were added per ml filtered pbs. immediately before analysis, mitochondria were diluted in bead mixture to the final analysis concentration of 0,05 µg/ml. data were analysed using flowjo software (version 10, flowjo, oregon, usa). western-blot. 30 µg of protein per sample was loaded onto 4-20% mini-protean ® tgx stain-free ™ precast gels (bio rad laboratories, münchen) and separation was performed within a gel chamber filled with 1x sds electrophoresis buffer at 100 v for 1 to 2 hours. after separation, proteins were blotted using the trans-blot ® turbo ™ mini pvdf transfer packs (bio rad laboratories, germany). proteins were transferred onto membranes at 2.5 a for 30 minutes using the trans-blot turbo ™ (bio rad laboratories, germany). membranes were blocked with 10% milk in tbs-t (tbs + 0.1% tween-20) for 1 hour at room temperature, washed three times with tbs-t and incubated with primary antibodies in 5% bsa in tbs-t overnight at 4 °c. the membranes were washed three times with tbs-t and incubated for 4 hours at room temperature with hrp-coupled secondary antibodies in 10% milk powder in tbs-t. blots were washed three times and developed using cheluminate-hrp picodetect (applichem gmbh, germany), which was evenly distributed on the membrane. the luminescence was detected for up to 20 minutes using the imaging-system chemidoc tm xrs (bio rad laboratories, germany). to visualize www.nature.com/scientificreports www.nature.com/scientificreports/ several proteins on the same blot, primary and secondary antibodies were removed by incubating membranes for 45 minutes at 50 °c in stripping buffer containing ß-mercaptoethanol. subsequently membranes were washed three times with tbs-t and incubated as previously described with primary and secondary antibodies. following primary antibodies were used: adenine nucleotide translocator (ant) (santa cruz biotechnology usa), cytochrome-c-oxidase (cox iv), cytochrome-c (cyt-c), glyceraldehyde 3-phosphate dehydrogenase (gapdh), glucose-regulated-protein 78 (grp78), histon 2b (h2b), voltage dependent anion channel (vdac) (all cell signaling technology, usa), lysosome-associated membrane protein 2 (lamp2) (thermo fisher scientific, usa), peroxisomal membrane protein 70 (pmp70) (origene technologies, usa), following secondary antibodies were used: rabbit anti-goat hrp (santa cruz biotechnology, usa), mouse anti-rabbit hrp, goat anti-mouse hrp (jackson immunoresearch, uk). histology. mouse livers were fixed for 48 hours in 4% paraformaldehyde. dehydrated livers (leica asp300s, germany) were embedded in paraffin. serial 2 µm-thin sections were prepared with a rotary microtome (hm355s, thermofisher scientific, usa) and subjected to histological and immune-histochemical analysis. hematoxylin-eosin (he) staining was performed on deparaffinized sections with eosin and mayer's haemalaun according to standard protocol. immunohistochemistry was performed using a bondmax rxm system (leica, wetzlar, germany, all reagents from leica) with primary antibody against egfp (a-11122, diluted 1:500 in antibody diluent, invitrogen, thermofisher scientific, usa). slides were deparaffinized, pre-treated with epitope retrieval solution 1 for 30 minutes. bound antibody was detected with a polymer refine detection kit without post primary reagent and visualized with dab as a dark brown precipitate. counterstaining was done with hematoxyline. electron microscopy. tissues were fixed in 2.5% electron microscopy grade glutaraldehyde in 0.1 m sodium cacodylate buffer ph 7.4 (science services, munich, germany), postfixed in 2% aqueous osmium tetraoxide 37 , dehydrated in gradual ethanol (30-100%) and propylene oxide, embedded in epon (merck, darmstadt, germany) and cured for 48 hours at 60 °c. semithin sections were cut and stained with toluidine blue. ultrathin sections of 50 nm were collected onto 200 mesh copper grids, stained with uranyl acetate and lead citrate before examination by transmission electron microscopy (zeiss libra 120 plus, carl zeiss nts gmbh, oberkochen, germany). pictures were acquired using a slow scan ccd-camera and item software (olympus soft imaging solutions, münster, germany). statistics. student's t tests were calculated using graphpad prism software. significance was set at p < 0.05 and denoted as *p < 0.05, **p < 0.01, ***p < 0.001 and ***p < 0.0001. all results are expressed as the mean ± standard deviation (sd). the data within this manuscript are available from the corresponding author upon reasonable request. mitochondrial control of cellular life, stress, and death mitochondrial signaling pathways: a receiver/integrator organelle mechanisms of mavs regulation at the mitochondrial membrane identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 mitochondria: dynamic organelles in disease, aging, and development hepatitis b virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis hepatitis c virus triggers mitochondrial fission and attenuates apoptosis to promote viral persistence hepatitis c virus induces the mitochondrial translocation of parkin and subsequent mitophagy epstein-barr virus latent membrane protein-2a alters mitochondrial dynamics promoting cellular migration mediated by notch signaling pathway mitophagy enhances oncolytic measles virus replication by mitigating ddx58/rig-i-like receptor signaling sars-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/ traf3/traf6 signalosome living in the liver: hepatic infections correlated morphometric and biochemical studies on the liver cell. i. morphometric model, stereologic methods, and normal morphometric data for rat liver bioluminescence imaging allows measuring cd8 t cell function in the liver isolation of mitochondria from cultured cells and liver tissue biopsies for molecular and biochemical analyses analysis of mitochondria by flow cytometry real-time flow cytometry analysis of permeability transition in isolated mitochondria flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death measurement of mitochondrial mass by flow cytometry during oxidative cell sizing: a light scattering photometer for rapid volume determination overcoming limitations of microparticle measurement by flow cytometry tnf-induced target cell killing by ctl activated through cross-presentation outcome of anti-viral immunity in the liver is shaped by the level of antigen expressed in infected hepatocytes perforin inhibition protects from lethal endothelial damage during fulminant viral hepatitis reduced type i interferon production by dendritic cells and weakened antiviral immunity in patients with wiskott-aldrich syndrome protein deficiency calcium uptake mechanisms of mitochondria biochemistry, mutagenesis, and oligomerization of dsred, a red fluorescent protein from coral deep analysis of mitochondria and cell health using machine learning multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo in vivo imaging of disease-related mitochondrial dynamics in a vertebrate model system flow cytometry of isolated mitochondria during development and under some pathological conditions why to compare absolute numbers of mitochondria analysis of mitochondrial dynamics and functions using imaging approaches strategies for imaging mitophagy in high-resolution and high-throughput a semi-automated method for isolating functionally intact mitochondria from cultured cells and tissue biopsies progressive stages of mitochondrial destruction caused by cell toxic bile salts a chrome-osmium fixative for electron microscopy this work was funded by the deutsche forschungsgemeinschaft (dfg, german research foundation) -projektnummer 272983813 -trr 179 to d.w and p.k. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-44922-9.competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-004591-2hchnlwb authors: wicker, s.; friedrichs, i.; rabenau, h.f. title: seroprävalenz von antikörpern gegen schwangerschaftsrelevante virale infektionserreger bei mitarbeiterinnen im gesundheitswesen date: 2012-07-25 journal: bundesgesundheitsblatt gesundheitsforschung gesundheitsschutz doi: 10.1007/s00103-012-1509-0 sha: doc_id: 4591 cord_uid: 2hchnlwb healthcare workers (hcws) are exposed to infectious diseases throughout the course of their work. the concerns of pregnant hcws are considerable because certain otherwise mild infections may affect fetal development. we studied 424 pregnant hcws at the university hospital frankfurt between march 2007 and july 2011. serological tests were carried out for varicella zoster virus (vzv), measles, mumps, rubella (mmr), cytomegalovirus (cmv) and parvovirus b19. our overall seroprevalence data with regard to vzv, mmr, cmv and parvovirus b 19 corresponded to the general population. however, physicians demonstrated lower seroprevalence towards the two non-vaccine-preventable diseases (cmv: 37.5% [ki 27.4–48.5]; parvovirus b19: 69.3% [ki 58.6–78.7]) compared with nurses (cmv: 53.4% [ki 46.1–60.6], parvovirus b19: 75.1% [68.4–81.1]). it was striking that, only one in five of the study population showed igg antibodies against all of the six pregnant-relevant viral diseases tested, of the physicians as few as one in six. a routine exclusion from the workplace due to non-immunity would mean that it would not be possible to employ the majority of pregnant staff in healthcare and childcare. originalien und übersichten in den vergangenen jahren sind in deutschland wiederholt behördliche be schäftigungsverbote für schwangere mit arbeiterinnen ausgesprochen worden, so fern diese in der kinderbetreuung oder im gesundheitswesen tätig und gegenüber schwangerschaftsrelevanten infektionen (z. b. mumps oder parvovirus) nicht im mun waren [1, 2, 3, 4, 5] . die diesbezügli chen regelungen in den einzelnen bun desländern sind uneinheitlich und führ ten in den vergangenen jahren zu kontro versen diskussionen und rechtsstreitig keiten [2] . ziel der vorliegenden arbeit war es, die seroprävalenz von antikörpern gegen schwangerschaftsrelevante virale infek tionserreger bei schwangeren medizi nischen beschäftigten zu erfassen und vor dem hintergrund der gesetzlichen grundlagen und der infektiologischen relevanz aus arbeitsmedizinischer sicht zu bewerten. es sollte geklärt werden, ob sich die seroprävalenzdaten beim medi zinischen personal von denen in der all gemeinbevölkerung unterscheiden und ob unterschiedliche berufsgruppen unter umständen erhöhte seroprävalenzen auf weisen. das gesetz zum schutz der erwerbs tätigen mutter -mutterschutzgesetz (muschg) -hat unter anderem das ziel, die werdende mutter und ihr kind vor gesundheitlichen gefahren am arbeits platz zu schützen. die verordnung zum schutze der mütter am arbeitsplatz (muscharbv) konkretisiert die maß nahmen zur verbesserung der sicherheit und des gesundheitsschutzes schwange rer arbeitnehmerinnen, wöchnerinnen und stillender arbeitnehmerinnen. be merkenswerterweise gelten sowohl das muschg als auch die muscharbv nur für frauen, die in einem arbeitsverhält nis stehen, nicht jedoch beispielswei se für selbstständige oder für studen tinnen. im hinblick auf eine potenzielle infek tionsgefährdung dürfen nach § 4 abs. 2 nr. 6 des muschg werdende mütter nicht "mit arbeiten, bei denen sie infol ge ihrer schwangerschaft in besonderem maße der gefahr, an einer berufskrank heit zu erkranken, ausgesetzt sind oder bei denen durch das risiko der entste hung einer berufskrankheit eine erhöh te gefährdung für die werdende mutter oder eine gefahr für die leibesfrucht be steht" beschäftigt werden. maserninfizierte schwangere haben im vergleich zu nichtschwangeren unter umständen ein erhöhtes pneumonie risiko und können vermehrt stationäre aufenthalte aufweisen [6, 7] . akute ma serninfektionen um den geburtstermin können schwere neonatale infektionen bewirken. in deutschland besitzen wahr scheinlich mehr als 80-90% der frauen im gebärfähigen alter eine ausreichende masernimmunität [6, 8] . mumpsinfektionen verlaufen bei schwan geren nicht schwerer als bei nichtschwan geren, es finden sich keine hinweise auf eine erhöhte kongenitale defektrate bzw. frühgeburtrate [1, 6] . daten aus den al ten bundesländern belegen eine seroprä valenz von mumpsantikörpern von 77% bei frauen in medizinischen und erziehe rischen berufen [6] . daten aus den neu en bundesländern zeigen hingegen eine seroprävalenz von 96% bei schwangeren frauen [8] . das rötelnvirus kann während der ge samten schwangerschaft vertikal auf die leibesfrucht übertragen werden. häu figkeit und schwere der kindlichen fehl bildungen hängen vor allem vom zeit punkt der mütterlichen infektion ab. das hauptrisiko für das vollbild der röteln embryopathie (herzfehlbildung, kata rakt, innenohrschwerhörigkeit, zns schädigungen) ist primär auf die ersten 11 schwangerschaftswochen (ssw) be schränkt [1, 6, 9] . die rötelnembryopa thie ist nach vorgaben des infektions schutzgesetzes (ifsg) meldepflichtig. seit 2001 wurden in deutschland insgesamt 10 kongenitale infektionen gemeldet [10] . allerdings muss von einer untererfas sung der rötelnembryopathiefälle aus gegangen werden, da einerseits schwan gerschaftsabbrüche aufgrund von röteln infektionen in deutschland nicht erfasst werden und andererseits rötelnbedingte hörstörungen oftmals erst im kleinkin desalter klinisch manifest und zu diesem zeitpunkt nicht mehr als rötelnbedingt erkannt werden. nach einführung der rötelnimpfung sank der anteil der frau en im gebärfähigen alter ohne röteln antikörper sukzessive ab und liegt zurzeit bei unter 3% [6] . das varizellazostervirus (vzv) kann während der gesamten schwangerschaft von einer infizierten schwangeren auf das ungeborene übertragen werden. als folge einer vzvinfektion kann es bis zur 21. schwangerschaftswoche (ssw) zum kongenitalen varizellensyndrom kom men [11] . erkrankt die schwangere 5 ta ge vor bis 2 tage nach der geburt an vzv kann dies zu lebensbedrohlichen dissemi nierten neonatalen varizellen führen. die wichtigste mütterliche komplikation ist die vzvpneumonie, deren letalität oh ne kausale therapie bei bis zu 45% liegt. die seroprävalenz von vzvigg bei frau en im gebärfähigen alter bewegt sich in deutschland bei etwa 96-97% [8, 12] . zytomegalievirus (cmv)infektionen sind weltweit die häufigste ursache für fe tale virusinfektionen und können zu an geborenen defekten des zns mit hör und sehstörungen sowie zu mentalen re tardierungen führen [13, 14, 15, 16, 17] . bei einer primärinfektion der mutter liegt die rate für fetale infektionen bei ca. 40-50%, bei bereits vor konzeption cmvpositi ven schwangeren bei 0,5-2%, nur weni ge ihrer neugeborenen sind jedoch sym ptomatisch infiziert [1, 18] . die seroprä valenz von cmvigg ist länder sowie al tersabhängig und steht im zusammen hang mit dem sozioökonomischen sta tus. sie liegt bei frauen im gebärfähigen alter in deutschland bei ca. 45% [15, 19] . die serokonversionsrate bei schwangeren liegt weltweit bei ungefähr 2% [20] . werden schwangere vor der 20. ssw mit parvovirus b19 infiziert, kann es bei sero negativen schwangeren zu spontanabor ten, totgeburten und beim feten unter anderem zum hydrops kommen [21, 22] . hinweise auf fetale fehlbildungen als fol ge der parvovirusb19infektion finden sich nicht [5] . die parvovirusb19sero prävalenz liegt bei frauen im gebärfähi gen alter in deutschland bei ca. 70% [4, 23, 24] . . it was striking that, only one in five of the study population showed igg antibodies against all of the six pregnant-relevant viral diseases tested, of the physicians as few as one in six. a routine exclusion from the workplace due to non-immunity would mean that it would not be possible to employ the majority of pregnant staff in healthcare and childcare. healthcare workers · maternity protection law · pregnancy · occupationally acquired infections gezeigt werden, dass patienten und medi zinisches personal sowohl auslöser von infektionsketten sein als auch an solchen infektionen erkranken und versterben können [25] . so in deutschland sind jedoch in den vergangenen jahren wiederholt beschäf tigungsverbote für schwangere, die gegen schwangerschaftsrelevante erreger nicht immun waren, ausgesprochen worden [2] . in unserem kollektiv war nur bei je der fünften schwangeren von einer im munität gegenüber den 6 untersuchten schwangerschaftsrelevanten infektions erregern auszugehen -bei den ärztinnen war es sogar nur bei ungefähr jeder sechs ten der fall. damit erhält die frage einer ggf. "überprotektiven" einschränkung durch das muschg und die muscharbv neben der individuellen relevanz auch eine volkswirtschaftliche. anders als in deutschland sehen die amerikanischen gesundheitsbehörden und die anderer staaten keine notwen digkeit, bei parvovirusb19 oder cmv negativen schwangeren ein beschäfti gungsverbot auszusprechen [43, 44] . das berufliche bedingte infektionsrisiko wird in diesen fällen nicht höher eingestuft als das infektionsrisiko der allgemeinbevöl kerung. epidemiologische daten zu nosoko mialen infektionsübertragungen zwi schen dem medizinischen personal, vom personal zum patienten und von patient zu patient werden in deutschland nur unvollständig erhoben. diese daten sind jedoch wichtig, um letztendlich aussa gen über die infektionsgefährdung am arbeitsplatz treffen und in der folge an gemessene schutzmaßnahmen (beispiels weise für schwangere beschäftigte oder im rahmen pandemischer geschehen) im plementieren zu können [45] . es konnte gezeigt werden, dass so wohl krankenhaushygienische maßnah men (z. b. häufiges händewaschen bzw. händedesinfektion) als auch die auf klärung über infektionswege zu niedri geren cmvserokonversionsraten füh ren [46, 47, 48] deutsche vereinigung zur bekämpfung von viruskrankheiten (dvv) (2006) virale infektionen und infektionsschutz in der schwangerschaft rechtliche rahmenbedingungen für den schutz von schwangeren und von nasciturus vor infektionsgefahren im arbeitsleben dargestellt anhand des parvovirus b19 (ringelröteln) urteil bverwg 5 c 11 current epidemiological aspects of human parvovirus b19 infection during pregnancy and childhood in the western part of germany parvovirus-b-19-infektion in der schwangerschaft masern, mumps und röteln in der schwangerschaft measles in pregnancy: maternal morbidity and perinatal outcome antikörper gegen impfpräventabler erkrankungen bei schwangeren und deren neugeborenen world health organization (who) (2011) rubella vaccines: who position paper rki) (2010) infektiologisches jahrbuch meldepflichtiger krankheiten für 2009. röteln, konnatale röteln preventing congenital varicella syndrome with immunization cytomegalovirus infections: occupational risk for health professionals molecular epidemiology of primary human cytomegalovirus infection in pregnant women and their families epidemiology of human cytomegalovirus (hcmv) in an urban region of germany: what has changed? problems and challenges in the diagnosis of vertical infection with human cytomegalovirus (cmv): lessons from two accidental cases diagnosis of and screening for cytomegalovirus infection in pregnant women passive immunization during pregnancy for congenital cytomegalovirus infection zytomegalievirus(cmv) durchseuchung und häufigkeit von cmv-primärinfektionen bei schwangeren frauen in deutschland cytomegalovirus seroconversion rates and risk factors: implications for congenital cmv risk of fetal hydrops and non-hydropic late intrauterine fetal death after gestational 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based on seroprevalence occupational risk for primary cytomegalovirus infection among pediatric health-care workers is primary cytomegalovirus infection an occupational hazard for obstetric nurses? a serological study human parvovirus b19 infection in healthcare workers parvovirus b10 outbreak in a children's ward prevalence of cytomegalovirus infection among health care workers in pediatric and immunocompressed adult units cytomegalovirus is not an occupational risk for nurses in renal transplant and neonatal units. results of a prospective surveillance study cytomegalovirus infection among employees of a children's hospital. no evidence for increased risk associated with patient care nosocomial cytomegalovirus infections within two hospitals caring for infants and children molecular epidemiology of cytomegalovirus in a nursery: lack of evidence for nosocomial transmission centers for disease control (cdc) (2010) cmv: people who care for infants and children monitoring major illness in health care workers and hospital staff washing our hands of the congenital cytomegalovirus disease epidemic prevention of child-to-mother transmission of cytomegalovirus among pregnant women does hygiene counselling have an impact on the rate of cmv primary infection during pregnancy? results of a 3-year prospective study in a french hospital mit eindringlichen bildern und texten verdeutlicht "hausparty", welche folgen zu hoher alkoholkonsum haben kann. der spot "fischen" ist teil der bekannten aidspräventionskampagne "gib aids keine chance". im mittelpunkt steht die botschaft: kondome schützen. auf humorvolle art und weise erzählt der spot eine geschichte, bei der neben einem verliebten jungen paar auch ein fisch eine tragende rolle spielt. beide spots werden im kino und im fernsehen eingesetzt. die bzga-kampagnen zur alkoholund aidsprävention werden seit mehreren jahren vom verband der privaten krankenversicherung e key: cord-018785-tcr5xlf8 authors: nambiar, puja; silibovsky, randi; belden, katherine a. title: infection in kidney transplantation date: 2018-06-27 journal: contemporary kidney transplantation doi: 10.1007/978-3-319-19617-6_22 sha: doc_id: 18785 cord_uid: tcr5xlf8 infection is an important cause of morbidity and mortality after kidney transplantation. it has been estimated that 70% of kidney transplant recipients will experience an infection episode within the first 3 years after transplantation (dharnidharka et al. 2007). after cardiovascular disease, infection is the second leading cause of death in recipients with allograft function (snyder et al. 2009). the immunosuppressive therapy required to prevent organ rejection places the kidney transplant recipient at increased risk for donor-derived, nosocomial, and community-acquired infections as well as reactivation of latent pathogens. pretransplant screening, immunizations, and optimal antibacterial and antiviral prophylaxis can help to reduce the impact of infection. awareness of the approach to infection in the transplant recipient including diagnostic and management strategies is essential to optimizing outcomes. a total of 17,600 kidney transplants were performed in the united states in 2013. as the incidence of acute rejection has declined, the probability of graft and patient survival continues to improve (usrds 2015) . infection, however, remains an important cause of morbidity and mortality after kidney transplantation. it has been estimated that 70% of kidney transplant recipients will experience an infection episode within the first 3 years after transplantation (dharnidharka et al. 2007 ). after cardiovascular disease, infection is the second leading cause of death in recipients with allograft function (snyder et al. 2009 ). the immunosuppressive therapy required to prevent organ rejection places the kidney transplant recipient at increased risk for donor-derived, nosocomial, and community-acquired infections as well as reactivation of latent pathogens. the kidney transplant recipient's net state of immune suppression and epidemiologic exposures determine the risk for infection at a given time. a traditional timeline has been used to predict patterns of infection after organ transplantation. this timeline has been altered in recent years with changes in immunosuppressive therapy and the routine use of antibacterial and antiviral prophylaxis. treatment for acute rejection and coinfection with viruses such as cytomegalovirus (cmv) and epstein-barr virus (ebv) may also alter predictable patterns of infection (fishman 2007) . the basic concepts of the traditional timeline, however, are still used to establish a differential diagnosis for infection at varied intervals posttransplantation (fig. 1) . within the first month, infections are noted to include those related to surgical complications, nosocomial exposures, and donor-derived pathogens. multidrug-resistant organisms including methicillin-resistant staphylococcus aureus (mrsa), vancomycin-resistant enterococcus (vre), and carbapenem-resistant enterobacteriaceae (cre) are important considerations, as is clostridium difficile. urinary tract infections are common within the first 6 months. opportunistic infections are more likely to occur 1-6 months after transplantation, reflecting the greater impact of immune suppression during this time. reactivation of latent pathogens such as polyoma virus bk, hepatitis c virus (hcv), and mycobacterium tuberculosis may also occur. prophylaxis for pneumocystis jiroveci, herpes viruses including cmv, and hepatitis b virus (hbv) makes these infections less common during this time period. beyond 6 months, the degree of immune suppression for most patients decreases. risk remains, however, for community-acquired infection, environmental exposures, recurrent infection, and the late presentation of viral infection, in particular cmv, once prophylaxis has been discontinued (fishman 2007; karuthu and blumberg 2012) . interventions can be undertaken to reduce the impact of infection after kidney transplantation. pretransplant screening of donors and recipients for infection that can be transmitted with organ donation or reactivated in an immune suppressed recipient is essential for optimizing transplant outcomes. guidelines for pretransplant screening are available from the american society for transplantation (fischer et al. 2013) , kidney disease: improving global outcomes (kdigo 2009) and the us public health service (seem et al. 2013) . recommended screening tests for donors and recipients are listed in table 1 . screening of living donors is performed prior to transplantation with varied timing. if there is a hiv(þ) consider if hiv is well controlled hcv: anti-hcv and hcv nat hcv(þ) hcv(à) reject, may be a consideration in the future hcv(à) hcv(þ) consider, hcv(þ) candidates should have a liver biopsy, improved outcomes if hcv is treated pretransplant hcv(þ) hcv(þ) consider (as for dà/rþ) hbv: hbsag, hbsab and hbcab (igm/igg); hbv nat (center dependent) sag(à), cab(à) sag(à), cab(þ), sab(þ/à) accept, vaccinate sab(à) candidates consider, with prophylaxis posttransplant sag(à), cab(þ) sag(à), cab (þ/à), sab (þ/à) accept if donor is cigm(à) and vaccinate sab(à) candidates, offer prophylaxis posttransplant if sab(à) or lost; reject if donor is cigm(þ) sag(þ), cab(þ) sag(à), cab (þ/à), sab (þ/à) reject cmv igg cmv(þ) or (à) cmv(þ) accept; will need posttransplant prophylaxis or preemptive therapy cmv(þ) cmv(à) accept; high risk for cmv infection, will need posttransplant prophylaxis ebv igg ebv(þ) or (à) ebv(þ) accept ebv(þ) ebv(à) accept; at risk for primary ebv and ptld, monitor posttransplant hsv 1/2 igg hsv(þ) hsv(þ) or (à) (fischer et al. 2013) . deceased donor screening, in contrast, is under time constraints and is usually performed within hours of transplantation in coordination with organ procurement organizations. infection with hiv, hbv, and hcv may not be detected in the early stages of infection. many transplant centers now perform more sensitive rapid molecular testing on potential organ donors including nucleic acid amplification (nat) testing for hiv, hbv, and hcv. a comprehensive medical and social history on potential organ donors is required in order to identify risk factors for blood borne pathogens. in efforts to expand the pool of available organs, recipients may consent to receipt of a kidney from a nat negative donor who is deemed "high risk" for blood borne infection based on identified risk factors. recipients of such organs are monitored posttransplantation with testing for hiv, hbv, and hcv between 1 and 3 months and for hbvagain at 12 months (fischer et al. 2013; seem et al. 2013; kovacs et al. 2014; len et al. 2014 ). use of hcv-and hbv-positive organs can be considered in respective positive recipients. furthermore, in 2013 the hiv organ policy equity act lifted a long-standing ban on allowing hivpositive organs to be donated to hiv-positive recipients (mgbako et al. 2013; muller et al. 2015) . donors who have active bacterial infection at the time of kidney procurement may transmit infection to the recipient. screening for bacterial infection in kidney donors includes assessing for urinary tract infection and bacteremia. urine and blood culture data are reviewed. if a kidney donor is known to have a urinary tract or systemic infection with a virulent organism such as staphylococcus aureus, pseudomonas aeruginosa, or candida species, the organ recipient is usually treated with a 10-14 day course of targeted antimicrobial therapy since these bacteria can compromise vascular and urinary anastomoses leading to mycotic aneurysms, anastomotic, and organ failure (fischer et al. 2013 ). allograft contamination can occur during organ procurement or processing. interpretation of organ preservation fluid cultures is challenging. the risk of transmission of infection to the organ recipient from contaminated preservation fluid, however, is low (fischer et al. 2013; len et al. 2014 ). candidates for kidney transplantation should have their vaccine status reviewed and updated in accordance with recommendations issued by the advisory committee on immunization practices with the centers for disease control and prevention (cdc 2012). while vaccinations in end stage renal disease patients may be less effective and durable than in healthy patients, a better response can be anticipated prior to transplantation than after (janus et al. 2008; kausz and pahari 2004) . special consideration should be given to vaccination for pneumococcus, influenza, and hbv. two pneumococcal vaccines are currently licensed for use in the united states: the 13-valent pneumococcal conjugate vaccine (pcv 13, prevnar 13) and the 23-valent-pneumococcal-polysaccharide vaccine (ppsv 23, pneumovax 23). current guidelines recommend that unvaccinated patients with chronic renal failure receive pcv 13 followed at least 8 weeks later by ppsv 23 (kobayashi et al. 2015) . a second dose of ppsv 23 is recommended 5 years after the first dose (cdc 2012). influenza vaccination should be administered annually. there are a number of influenza vaccine formulations available. live attenuated influenza vaccination (flumist) is not recommended in chronic kidney disease patients. an inactivated vaccine option should be used (cdc 2012) . a high dose inactivated influenza vaccine is now available and was shown to induce a higher antibody response than traditional vaccines in adults over the age of 65 (diaz-granados et al. 2014) . the use of this vaccine in transplant candidates and recipients is currently under investigation. transplant candidates not immune to hbv should receive high dose hbv vaccination (40 micrograms antigen per dose) due to decreased response rates with standard dosing (huprikar et al. 2015) . human cytomegalovirus-human herpes 5 (cmv), a member of the family herpesviridae, is an opportunistic pathogen occurring in 20-60% of solid organ transplant recipients (brennan 2001) . cmv is a cause of significant morbidity and mortality in this population (mwintshi and brennan 2007) . the incidence of cmv in the renal transplant population is estimated to be between 8% and 32% (patel and paya 1997) . renal transplant patients are at lower risk for primary cmv compared with other organ transplant recipients owing to a lower burden of latent virus in renal allograft tissue. the risk factors for development of cmv disease include donor seropositivity/recipient seronegativity(dþ/rà), use of induction immunosuppression (antilymphocyte antibodies), donor age >60 years, simultaneous kidney-pancreas transplantation, treatment for acute rejection, impaired transplant function, and concurrent infection from other viruses (like ebv and hhv-6 and 7) (de keyzer et al. 2011) . cmv-seronegative recipients of cmv-seropositive donors (d + /r à ) are at the highest risk, whereas d + /r + or d à /r + transplantations are considered to be moderate risk with d à /r à being lowest risk, with an incidence of cmv disease <5% (de keyzer et al. 2011) . immunosuppressive drugs also influence the incidence and severity of cmv disease. for instance, cyclosporine increases the risk of cmv disease, whereas use of sirolimus seems to have a protective effect (san juan et al. 2008) the use of antilymphocyte antibody (antithymocyte globulin or muromonab-cd3) is associated with a two to fivefold increase in the rate of cmv, but basiliximab and daclizumab do not seem to increase its incidence (de keyzer et al. 2011) . cmv infection may occur in solid organ transplantation recipients as primary infection when a cmv seronegative individual receives cells latently infected with cmv from a seropositive donor, donor-derived reinfection, or reactivation of latent recipient infection (patel and paya 1997) . the following definitions are commonly used in the transplant literature to differentiate cmv infection from cmv disease. cmv infection is evidence of cmv replication regardless of symptoms, and cmv disease is evidence of cmv infection with symptoms, such as viral syndrome, leukopenia, thrombocytopenia, or invasive tissue disease (e.g., pneumonitis, hepatitis, retinitis, gastrointestinal disease) (humar and snydman 2009) . cmv disease and even asymptomatic cmv infection have been shown to be independent risk factors for reduced graft survival and overall mortality beyond 100 days posttransplantation . infection with cmv has been implicated in acute allograft dysfunction and chronic allograft nephropathy (mclaughlin et al. 2002; tong et al. 2002) . cmv disease is also associated with posttransplant lymphoproliferative disorder (ptld), posttransplant diabetes mellitus, and transplant artery stenosis (pouria et al. 1998; hjelmesaeth et al. 2004; manez et al. 1997) . cmv infection can occur as acute infection between the first and 6 months following transplant, when immunosuppression is at its maximum or as delayed infection from reactivation of latent virus after antiviral prophylaxis has completed, later in the first year. given the significant effect of cmv on patient outcomes, prevention plays an important role. serologic screening for cmv should be performed on both donor and recipient prior to transplant to categorize high risk patients. several cmv vaccine candidates are under investigation although none are currently available. universal prophylaxis involves giving antivirals to those recipients at risk posttransplant before the onset of infection, whereas in preemptive therapy patients are monitored at regular intervals and started on antivirals when there is early evidence of replication prior to onset of clinical disease. chemoprophylaxis in high risk patients (dþ/rà) has shown to reduce the incidence of cmv disease by 60% and has decreased cmv associated mortality and opportunistic infection (hodson et al. 2005) . preemptive therapy in high risk patients based on cmv viral load monitoring has not shown reduction in acute rejection or all-cause mortality (strippoli et al. 2006) . a randomized controlled trial by kliem et al. in 2008 comparing oral ganciclovir chemoprophylaxis with viral load monitoring revealed improved graft survival in those who received ganciclovir chemoprophylaxis (kliem et al. 2008) . a recent cochrane review from 2013 concluded that the efficacy of preemptive therapy compared with prophylaxis to prevent cmv disease remains unclear due to significant heterogeneity between studies and that additional head-tohead studies are required to determine the relative benefits and harms of preemptive therapy and prophylaxis to prevent cmv disease in solid organ transplant recipients (owers et al. 2013) . standard prophylactic guidelines recommend therapy in dþ/rà, dþ/rþ, and dà/rþ using oral ganciclovir or valganciclovir for a minimum of 3 months posttransplant and 1-3 months after treatment of rejection with antilymphocyte therapy (humar and snydman 2009; kotton et al. 2013 ). valganciclovir has replaced ganciclovir because of better bioavailability, lower pill burden, and reduced availability of oral ganciclovir (paya et al. 2004 ). the optimal length of prophylaxis is unknown, but recent trials have shown that 6 months of prophylaxis is more effective in decreasing the incidence of cmv disease in dþ/ rà kidney transplant recipients (humar et al. 2010; doyle et al. 2006) . current guidelines recommend dosing valganciclovir at 900 mg daily (adjusted for renal dysfunction) if tolerated in dþ/ rà recipients. some centers have successfully treated patients with half of this dose (450 mg daily) with less drug toxicity. however, dþ/rà recipients may be at higher risk of breakthrough infection and the development of resistance with this lower dosing strategy (kotton et al. 2013) . the diagnosis of cmv disease can be made by several techniques including cmv antigenemia assay, nucleic acid testing (nat), serology, antibody testing, viral culture, and histopathology. nat is generally more sensitive than antibody testing or culture. higher values by nat are suggestive of cmv disease and weekly viremia testing can be used to monitor response to therapy. the interlaboratory variability of nat is expected to be reduced with the recent establishment of international standards, intended to be used in the standardization of nucleic acid amplification technique (nat)-based assays for hcmv (karuthu and blumberg 2012) . patients with gastrointestinal and neurologic cmv disease often fail to exhibit cmv viremia and histopathology is necessary to establish diagnosis in these instances. treatment of active cmv disease requires a combination of immunomodulation, antiviral therapy with or without adjuvant therapy and if possible, reduction of immunosuppression (kotton et al. 2013; green et al. 2004 ). the mainstay of therapy is intravenous ganciclovir. the victor trial (valcyte in cmv disease treatment of solid organ recipients) demonstrated oral valganciclovir was not inferior to intravenous ganciclovir in mild to moderate cmv disease in solid organ transplant recipients (asberg et al. 2009 ). the current guidelines recommend renally adjusted intravenous ganciclovir 5 mg/kg twice daily or oral valganciclovir, 900 mg twice daily for mild cmv disease (kotton et al. 2013) . in severe cmv disease, intravenous ganciclovir is preferred with reduction of immunosupression despite the increased risk of rejection (de keyzer et al. 2011) . the use of adjuvant therapy with cmv-specific hyperimmune globulin or standard intravenous immunoglobulin may be considered in individuals with hypogammaglobulinemia, severe systemic infection, or in failure to respond to standard therapy (humar et al. 2010) . cmv resistance to ganciclovir has been noted in renal transplant recipients due to mutations in ul 97, the gene responsible for the first phosphorylation step in ganciclovir activation and ul 54, the gene responsible for dna polymerase (limaye et al. 2000) . cmv resistance should be considered when patients have worsening disease or persistent, unchanged viremia at 2 weeks of therapy and in such cases, genotype testing for mutations of the genes encoding ul 97 and ul 54 should be performed (weikert and blumberg 2008). treatment options for drug resistant cmv include the use of high dose ganciclovir, foscarnet, and cidofovir; however, no clinical trial data are available regarding optimal therapy options for resistant cmv. the use of novel agents including leflunomide and artesunate has been attempted as salvage therapy with varying success. several new antiviral treatment options are currently under investigation including maribavir and brincidofovir (an oral prodrug of cidofovir with less nephrotoxicity) for use in the treatment of drug resistant cmv (limaye et al. 2000) . epstein barr virus -human herpesvirus 4 (ebv) is a ubiquitous gamma herpes virus that remains latent in lymphocytes following primary infection. it is responsible for posttransplant lymphoproliferative disorder (ptld) which increases morbidity and mortality in the transplant population. approximately 62-79% of ptld cases have been associated with ebv (karuthu and blumberg 2012) . ptld most commonly occurs in the first year posttransplant (cockfield et al. 1993) . the risk factors for ptld include ebv naïve recipients who receive ebv seropositive organs, active primary ebv infection, younger recipient, coinfection by cmv and other viruses, prior splenectomy, second transplant, acute or chronic graft versus host disease, immunosuppressive drug regimen (okt3 or polyclonal antilymphocyte antibody), and the type of organ transplanted. kidney transplant recipients are at lower risk compared with other types of transplants and have an incidence of approximately 1-3% (gulley and tang 2010; allen et al. 2009; taylor et al. 2005) . the majority of symptomatic ebv infections in renal transplant recipients are primary infection likely related to transmission of donor virus. ebv disease can be asymptomatic or presents with a nonspecific febrile syndrome, lymphadenopathy, hepatosplenomegaly, atypicalþ lymphocytosis, hematologic disorders including anemia, leukopenia, thrombocytopenia, and organ-specific diseases like gastroenteritis, hepatitis, or pneumonitis (allen et al. 2009 ). ptld typically follows primary infection and frequently presents as a rapidly enlarging mass in the grafted organ, lymph nodes, bone marrow, or extranodal sites (manez et al. 1997) . ptld is divided into four major histopathologic subtypes as per the world health organization (who): early lesions, polymorphic ptld, monomorphic ptld, and classical hodgkin lymphoma type ptld. definitive diagnosis of ptld requires histopathologic confirmation by tissue excision biopsy with immunologic cell typing, cytogenetics, immunoglobulin gene rearrangements, and ebvspecific staining (allen et al. 2009 ). staging is performed by histologic types (monoclonal versus polyclonal, t cell versus b cell) and location (allograft, other organs, metastasis) (weikert and blumberg 2008) . clinical management of ptld typically involves reduction of immunosuppression which can lead to remission in 23-86% of the patients (weikert and blumberg 2008). antiviral therapy with acyclovir or ganciclovir is controversial and no evidence supports its efficacy (taylor et al. 2005) . rituximab (monoclonal antibody to cd20) is commonly used for treatment of ptld in recipients who failed reduction of immunosuppression alone (allen et al. 2009 ). in isolated graft ptld, surgical resection is an option (weikert and blumberg 2008) . in patients that fail the above strategies, ifn and ivig have been used with varying success and cytotoxic chemotherapy with radiation remains salvage therapy (green et al. 2004 ). there is no standardized therapy to prevent ptld. kdigo guidelines recommend monitoring ebv viral load in high risk renal transplant patients within the first week after transplant, then at least monthly for 3-6 months and then every 3 months for the rest of the first posttransplant year. additional viral load monitoring is recommended after treatment for acute rejection in high risk groups (children, ebv dþ/rà). outcomes with ptld in renal transplant patients vary according to the site involved. patients with isolated graft involvement have a 5-year survival of 68% compared with those patients with ptld extending beyond the allograft whose survival varied between 36% and 38% (weikert and blumberg 2008). human herpesvirus 1herpes simplex virus types 1 and 2 (hsv)and human herpesvirus 3varicella zoster virus (vzv)are alpha herpes viruses. hsv 1 has a seroprevalence of 60% in the adult population, while hsv 2 has a seroprevalence of 15% and vzv rates can be as high as 90% (green et al. 2004 ). the incidence of hsv disease in renal transplant recipients is approximately 53% and vzv 4-12% (patel and paya 1997) . hsv may cause primary infection following which the virus remains latent in the sensory nerve ganglia or more commonly causes reactivation infection. hsv may be seen as early as in the first posttransplant month in the absence of prophylaxis. hsv infection usually presents with oral or genital mucocutaneous lesions, occasionally pneumonitis, tracheobronchitis, esophagitis, hepatitis, encephalitis, or disseminated infection (green et al. 2004) . vzv causes localized dermatomal or multidermatomal or disseminated zoster with or without visceral involvement (pneumonitis, hepatitis, pancreatitis, encephalitis). pretransplant screening for prior vzv infection should be performed, and naïve patients should be vaccinated with live attenuated varicella vaccine before transplant whenever possible in order to avoid primary vzv infection posttransplantation (fehr et al. 2002) . since vzv is a live vaccine, it should not be given if transplant is expected within 4-6 weeks in order to avoid active shedding of virus at the time of transplant. posttransplant prophylaxis is recommended with acyclovir, valacyclovir, or ganciclovir (in those who need cmv prophylaxis) for approximately 1-3 months posttransplant in order to avoid hsv and vzv reactivation (green et al. 2004) . diagnosis of hsv and vzv infection can be made with pcr or direct fluorescence antibody for hsv from vesicular lesions, csf, or visceral tissue samples. serologies are rarely helpful in active infection owing to high seroprevalence. kdigo guidelines recommend that renal transplant recipients who develop less severe hsv or vzv infections can be treated with an appropriate oral antiviral agent (e.g., acyclovir, valacyclovir, or famciclovir), and those with systemic infection should be treated with intravenous acyclovir and a reduction in immunosuppressive medication and subsequently switched to an appropriate oral antiviral agent (green et al. 2004 ). the use of foscarnet, cidofovir, or topical trifluridine may be considered in patients with acyclovir resistant virus with careful monitoring of renal functions (kotton and fishman 2005; tan and goh 2006) . human herpesvirus 6 and human herpesvirus 7 (hhv 6 and hhv 7) are ubiquitous with high seroprevalence in adults. these viruses are common causes of fever in children and remain latent in lymphocytes following primary infection. hhv 6 uses the cd46 molecule as its receptor but may also infect other cell types, such as monocytes, and epithelial and endothelial cells. hhv 7 uses the cd4 molecule as its receptor and is more strictly lymphotropic. infection occurs as a result of reactivation in the first 4 weeks following transplant often in recipients not on cmv prophylaxis (singh and carrigan 1996) . clinical manifestations include fever, rash, hepatitis, interstitial pneumonitis, encephalitis, leukopenia, and myelosuppression. owing to its immunomodulatory effects, it is hypothesized that hhv 7 may act as a cofactor for hhv 6 and cmv reactivation, while both hhv 6 and hhv 7 may act as cofactors in the pathogenesis of cmv disease and acute rejection (kidd et al. 2000; chapenko et al. 2000; dockell and paya 2001) . the diagnosis of hhv 6 and hhv 7 is made by tissue immunohistochemistry or nat testing of peripheral blood lymphocytes. treatment includes reduction in immunosuppression and ganciclovir, but cidofovir and foscarnet have also been utilized (green et al. 2004; kotton and fishman 2005; dockell and paya 2001) . hhv 8 is associated with primary effusion lymphoma, kaposi's sarcoma, and multicentric castleman's disease. infection can be acquired as primary through the allograft or through reactivation of latent virus (diociaiuti et al. 2000; regamy et al. 1998 ). hhv 8 causes kaposi's sarcoma, the most common presentation in renal transplant recipients, through upregulation of vascular endothelial growth factor (vegf) receptor f1 k1/kdr in endothelial cells (stallone et al. 2005) . treatment includes reduction in immunosuppression and cytotoxic chemotherapy. sirolimus, an immunosuppressive drug used in renal transplant patients is thought to inhibit not only the production of vegf but also dampens its effect on endothelial cells (stallone et al. 2005 ). bk polyomavirus (bkv) and jc polyomavirus (jcv) belong to the family polyomaviridae. bkv is responsible for causing polyomavirus associated nephropathy (pvan) in 95% of cases and jcv in less than 5% of the cases. pvan occurs in 1-10% of patients with renal transplantation and causes renal allograft loss in 10-80% of cases dadhania et al. 2008) . the risk factors for bkv associated pvan include the use of potent immunosuppressive regimens, caucasian race, older age, diabetes mellitus, cadaveric renal transplant, and combined kidney and pancreas transplant trofe et al. 2003) . bkv is known to cause interstitial nephritis, ureteral stenosis, and ureteral stricture of the allograft kidney most commonly occurring within the first 3-4 months after renal transplant patients when immunosuppression is at its highest (randhawa and brennan 2006) . jcv less commonly causes pvan and is more frequently associated with progressive multifocal leukoencephalopathy (pml), a demyelinating disorder of the white matter presenting as neurologic impairment and dementia (phillips et al. 2004) . diagnosis of bkv includes the use of viral load assays (blood, urine), detection of viral cytopathic effect (decoy cells), nat, bkv-specific antibody, or histopathology (hariharan 2006) . kdigo guidelines recommend screening all renal transplant recipients for bkv with quantitative plasma nat at least monthly for the first 3-6 months after transplantation, then every 3 months until the end of the first posttransplant year, whenever there is an unexplained rise in serum creatinine, and after treatment for acute rejection. the guidelines suggest reducing immunosuppressive medications when bkv plasma nat is persistently greater than 10,000 copies/ ml (107 copies/l) (kdigo 2009). sustained high bk viremia in spite of reduction in immunosuppression may need additional antiviral therapy, although data regarding optimal treatment options are unknown. there are limited data regarding the effectiveness of leflunomide and/or cidofovir or the use of fluoroquinolones or ivig for treatment of bkv infection (randhawa and brennan 2006; josephson et al. 2006) . to date there is no effective treatment for pml. patients with allograft loss due to pvan have undergone successful retransplantation (hariharan 2006) . patients with chronic renal failure, in particular those receiving hemodialysis, are at increased risk for contracting hepatitis b virus (hbv). the prevalence of hepatitis b surface antigen (hbsag)positive patients has declined because of hbv vaccination, strict segregation of hbsag-positive patients in dialysis units, improved screening of blood products, and the use of erythropoiesis stimulating agents (karuthu and blumberg 2012) . approximately 2-10% of patients with a history of hbv prior to transplant will reactivate posttransplant (weikert and blumberg 2008) . serial monitoring of hbv dna every 3-6 months is required after transplantation as liver enzyme levels do not reflect infection status and elevated viral loads suggest resistance to therapy (levitsky et al. 2013) . in a meta-analysis conducted by fabrizi and his colleagues, hbsag seropositivity was an independent risk factor for allograft loss and posttransplant death (fabrizi et al. 2005) . the treatment options currently approved for chronic hbv include: ifn alpha, pegylated ifn, lamivudine, entecavir, telbivudine, tenofovir, and adefovir (fabrizi et al. 2005; chan et al. 2002; chang et al. 2010) . kdigo recommends that interferon treatment generally be avoided because of the high associated incidence of rejection. tenofovir or entecavir are preferable to lamivudine, to minimize the development of drug resistance, unless medication cost requires that lamivudine be used. during therapy with antivirals, hbv dna and alt levels should be measured every 3 months to monitor efficacy and to detect drug resistance. all hbsagpositive renal transplant recipients should receive prophylaxis with tenofovir, entecavir, or lamivudine. hbsag-positive patients with cirrhosis should be screened for hepatocellular carcinoma every 12 months with liver ultrasound and alpha feto-protein. patients who are negative for hbsag and have hbsab titer <10 miu/ml should receive booster vaccination to raise the titer to >100 miu/ ml (kdigo 2009). hepatitis c virus (hcv) infection has been increasingly recognized in end stage renal disease patients (esrd). donor-derived hcv may uncommonly occur after transplantation. screening of patients with esrd and testing renal transplant patients for newly acquired hcv should include nat (levitsky et al. 2013) . hcv-positive donors can be considered for hcv-positive recipients and possibly will be considered for hcv-negative recipients in the future given improved treatment options for cure of hcv that could be administered post transplant. hcv-infected renal transplant recipients have decreased survival and increased complication rates. posttransplant complications include glomerulonephritis (gn), posttransplant diabetes mellitus, and accelerated progression to cirrhosis with fibrosing cholestatic hepatitis (morales et al. 2010) . liver biopsy within 6-12 months of transplantation and subsequent biopsies are required for evaluation of liver disease posttransplant as 20-51% of patients may have normal liver enzyme levels with abnormal histologic features (ashry ahmed gheith 2011). hcv-infected recipients should be tested for proteinuria every 3-6 months, and patients with new onset proteinuria should undergo allograft biopsy (kdigo 2009) . the effect of immunosuppression on the progression of hcv-related liver injury and the management of immunosuppression in the hcvinfected renal transplant recipient remain uncertain. thus, it is preferable to treat hcv in transplant candidates prior to transplantation given the potential for improved outcomes with successful hcv treatment and the complications associated with treatment posttransplant. patients with a sustained virologic response to pretransplant treatment have a reduced risk for hcv recurrence and decreased posttransplant gn (domınguez-gil and morales 2009). options for treatment include interferon/ peginterferon alone or in combination with ribavirin. the risk of toxicity with the addition of ribavirin has limited the use of combination therapy in chronic kidney disease (ckd) patients. the availability of direct acting hcv protease and polymerase inhibitors has sparked new enthusiasm for treating hcv-infected ckd patients and studies are ongoing evaluating the use of these agents in ckd. if treatment cannot be given prior to transplant, kdigo recommends monotherapy with standard interferon for hcv-infected renal transplant recipients in whom the benefits of antiviral treatment clearly outweigh the risks (kdigo 2009). the use of direct acting hcv antivirals posttransplantation can also be considered and will likely be preferred in the future given improved tolerance and efficacy with these agents with an understanding that drug interactions with calcineurin inhibitors may occur.a study looking at 20 hcv-positive kidney transplant recipients (60% treated pre-transplant with interferon unsuccessfully) treated with direct acting antivirals posttransplant found that 100% cleared the virus and had a sustained virologic response at 12 weeks. the most common agents used were sofosbuvir and simeprevir (sawinski et al. 2016 ). human immunodeficiency virus (hiv) belongs to the family of retroviridae. with the introduction of antiretroviral therapy (art) in the mid-1990s, the incidence of hiv related deaths has been reduced. renal diseases related to hiv infection include hiv associated nephropathy (hivan), immune complex diseases, and thrombotic microangiopathy (frassetto et al. 2009 ). a total of 10% of patients with hiv develop hivan and it remains an important complication of hiv infection, progressing rapidly to end stage renal disease (esrd) (shahinian et al. 2000) . a large prospective clinical trial examining outcomes among 150 hiv + kidney transplant recipients reported 3-year patient and graft survival rates of 88.2% and 73.7%, respectively, which were similar to survival rates among a cohort of unmatched elderly (>65 years) hivnegative (hiv à ) kidney recipients (stock et al. 2010 ). the candidates for transplantation include those with well-controlled hiv infection with undetectable viral loads, cd4 >200 cells per microliter, and absence of untreatable infections or malignancies (blumberg et al. 2009 ). the most significant complications in this patient population posttransplant include increased rejection rates (up to 25%), managing drug interactions between art and immunosuppressive therapy and complications related to cardiovascular risk factors and hepatitis coinfection (blumberg et al. 2009 ). the choice of art should be based on susceptibility results and if possible, the use of protease inhibitors should be avoided owing to significant drug interactions with this class of art. with regards to immunosuppressive therapy, the use of thymoglobulin may result in prolonged depression of cd4 counts, whereas monocloncal anti-il2 receptor antibodies, such as basiliximab/daclizumab, have been shown to increase cd4 cell counts (ciuffreda et al. 2007; carter et al. 2006) . the risks of antilymphocyte therapy should be balanced with the risks of rejection in hiv-infected recipients. of note, hiv-positive donors can now be considered in hivpositive recipients. the various respiratory viruses that cause infection affecting the renal transplant patient population include adenovirus, respiratory syncytial virus (rsv), influenza, parainfluenza, human metapneumovirus, rhinovirus, and coronavirus (green et al. 2004) . clinical manifestations include upper respiratory tract infection, bronchitis, and pneumonia. in addition to respiratory illness, adenovirus is known to cause gastroenteritis, hemorrhagic cystitis, pancreatitis, meningoencephalitis, necrotizing hepatitis, and nephritis/renal dysfunction in renal transplant recipients (pham et al. 2003; alsaad et al. 2007) . infection with these viruses may also be associated with rejection (weikert and blumberg 2008) . prevention involves hand hygiene and the use of droplet precautions for those suspected of having infection. influenza vaccination is recommended prior to transplant and yearly following transplant. treatment of respiratory viral infection involves supportive care and antiviral medications. influenza can be treated with oseltamivir or zanamavir. ribavirin is approved for the treatment of rsv. adenovirus infection is treated with reduction of immunosuppression with consideration of cidofovir (ison 2006) . emerging viral pathogens include newly recognized viruses or previously known viruses that are either increasing or threatening to increase in incidence. some of the emerging viruses causing infections in renal transplant population include human t-cell leukemia virus type 1 (htlv-1), hepatitis e virus (hev), measles virus, rabies virus, lymphocytic choriomeningitis virus (lcmv), dengue virus (denv), west nile virus, and zika virus. case reports of adult t-cell leukemia (atl) following renal transplantation in htlv-1-positive patients have been documented, though in a case series of renal transplant recipients with long-term follow-up, no cases of atl or htlv-1-associated myelopathy (ham) developed (nakamura et al. 2005; tanabe et al. 1998) . hev may induce kidney injury with significant reduction in glomerular filtration rate. glomerular injuries such as membranoproliferative glomerulonephritis have been described in kidney transplant patients with acute and chronic hev infections (kamar et al. 2012) . the incidence of measles in transplant recipients is unclear. cases of subacute measles encephalitis (sme) have developed in renal transplant recipients. the clinical course is one of deteriorating mental status and treatment refractory seizures (waggoner and deresinski 2013) . worldwide, vector-borne viral disease is increasing in incidence and can be transmitted with blood products and organ transplantation. fatal cases of dengue have been reported within the first month following renal transplant (waggoner and deresinski 2013) . west nile virus has also been reported in transplant recipients with a high incidence of neuroinvasive disease and poor outcomes. zika virus is also now a concern. cases of donor-derived rabies in the sot population have been reported. patients typically developed encephalitis between 1 and 2 months posttransplant, and all symptomatic reported patients died (srinivasan et al. 2005) . cases of lcmv causing severe disease in organ transplant patients have been documented. the 4 clusters of lcmv infection occurred in the united states and involved kidney, liver, and lung transplants; symptoms included fever, abdominal pain, nausea, diarrhea, and altered mental status (srinivasan et al. 2005; barry et al. 2008; fischer et al. 2006) . two renal transplant recipients survived lcmv infection. ribavirin has been employed in some cases, though the benefits remain unclear (waggoner and deresinski 2013) . data regarding the incidence, screening and treatment options of the above-mentioned emerging viruses are limited. given the risk of donor-derived viral transmission, organs should not be accepted from donors with unexplained febrile or neurologic illness. in unclear cases, the risk of donor-derived infection should be balanced with the benefit of the transplant. bacterial infections after renal transplantation can be due to surgical complications at the time of transplantation, nosocomial infection, immunosuppression, or community-acquired infection. donorderived bacterial infections from the transplanted kidney or blood stream can occur as well. about 47% of kidney transplant recipients develop bacterial infections (patel and paya 1997) . occurring any time posttransplantation, urinary tract infections account for the overwhelming majority of these infections and are the most common bacterial infections prolonging or leading to re-hospitalization (wyner 1994) . enterococci, staphylococci, enteric gram-negative organisms, and p. aeruginosa are the most common bacteria isolated (wyner 1994) . bacterial pneumonia, postoperative wound infections, and bacteremia or sepsis, although less common, also prolong or lead to rehospitalizations after transplantation (karuthu and blumberg 2012) . common bacterial pathogens for these infections are gram-negative organisms, including multidrug resistant bacteria; gram positive organisms, including methicillin-resistant staphylococcus aureus (mrsa), and vancomycin-resistant entercococci (vre), as well as organisms more typically seen in immunocompromised patients such as listeria. months after the operation, bacterial pathogens include streptococcus species, mycoplasma, legionella, listeria, salmonella, and nocardia. trimethoprim-sulfamethoxazole (tmp-smx) prophylaxis has been shown to reduce the incidence of some of these infections. increased antimicrobial resistance, urgency of treatment, drug interactions, and toxicities, as well as the risk for clostridium difficile colitis all contribute to the complex decision making required for antimicrobial management. risk factors for urinary tract infection after transplantation are a prolonged period of hemodialysis before transplant, prolonged bladder catheterization, female sex, deceased donor transplant, kidney-pancreas transplant with bladder drainage, uretero-vesical stents, and an increased immunosuppressed state (karuthu and blumberg 2012; lapchik et al. 1992) . prophylaxis to lower the risk of infection after transplant with trimethoprim-sulfamethoxazole is routine (karuthu and blumberg 2012) . controversy regarding the exact dosing and duration of prophylaxis exists. typically it is given at a dose of 160 mg trimethoprim and 800 mg of sulfamethoxazole daily for 6-12 months (kdigo 2009 ). trimethoprim-sulfamethoxazole reduces the risk of uti and bacteremia (karuthu and blumberg 2012; patel and paya 1997) . symptoms of uti include frequency, urgency, and dysuria as well as nausea and vague abdominal complaints. some patients are asymptomatic. escherichia coli is the most common pathogen and an increasing number of pathogens are multidrug resistant. sensitivity testing is required. treatment of asymptomatic bacteriuria in the renal transplant recipient is controversial and is not routinely recommended (coussement and abramowicz 2013) . although not well studied, since utis in renal transplant patients are complicated, 7-14 days of antibiotics is a typical duration. removal of stents and catheters as well as drainage of abscesses are frequently required to prevent relapse and for cure. surgical wound infections, occurring at a rate of 3-4%, usually present within the first 4 weeks after transplant (ramos et al. 2008) . obesity, urine leaks, re-operation through the original incision, diabetes, high creatinine levels in plasma, and prolonged bladder catheterization are risk factors for wound infections (humar et al. 2001; khoury and brennan 2005) . improved organ procurement, preservation, and surgical techniques along with preoperative antibiotics all reduce the risk of subsequent postoperative wound infection. bacterial organisms causing these types of infections may be nosocomial and multidrug-resistant making antibiotic treatment difficult due to limited options or toxicities. source control with good wound care is critical in the management of these types of infections. although pneumonia is the most common bacterial infection in all solid organ transplant recipients, its incidence is lowest in those who have received a kidney (khoury and brennan 2005) . occurring early in the posttransplant period, cmv infection and rejection treatment with antilymphocyte preparations increase the pneumonia risk. hospital-acquired pneumonia due to resistant pathogens, such as mrsa, and extended spectrum beta lactamase (esbl) or carbapenemresistant (cre) gram-negative organisms are increasing in incidence and sometimes require nephrotoxic agents for treatment. communityacquired pneumonia can occur any time after transplantation and the incidence of communityacquired pneumonia specifically due to streptococcus pneumoniae can be lowered with vaccination. bacteremia and sepsis are most commonly due to a urinary source, followed by lung, wound, and abdomen (khoury and brennan 2005) . intravenous catheters also play a role. diabetes mellitus and posttransplant dialysis increase the incidence of sepsis which decreases the survival rate in these patients (abbott et al. 2001) . prompt treatment with broad spectrum antibiotics followed by rapid de-escalation to pathogen-specific therapy based on sensitivities is required. removal of foreign bodies such as intravenous catheters and stents is also necessary for cure. nocardia is a rare infection seen in the renal transplant recipient occurring in less than 4% of patients (wilson et al. 1989 ). trimethoprim-sulfamethoxazole prophylaxis used after transplant to prevent pneumocystis jiroveci pneumonia (pcp) likely prevents nocardia infection as well. nocardia asteroides is the most common species and causes pulmonary infections, including cavitary lesions and pleural effusions (patel and paya 1997) . other common sites of infection, due to dissemination, are central nervous system (cns) and cutaneous. all patients with nocardia should be evaluated for cns disease. allograft rejection, high-dose prednisone, azathioprine, instead of cyclosporine based immunosuppression, and neutropenia are risk factors for this infection (patel and paya 1997) . diagnosis is made by the identification of branching and beading rods on gram and modified acid fast staining and cultures of infected sites. antimicrobial susceptibility testing should be performed on all isolates. high dose trimethoprim-sulfamethoxazole sometimes in combination with amikacin is the treatment of choice, but allergic reactions and other side effects sometimes limit their use. alternatives include imipenem, minocycline, and ceftriaxone, but choices should be based on susceptibilities and site of infection (spelman 2016) . nocardia infections can relapse and prolonged therapy up to a year is recommended followed by chronic suppressive therapy (spelman 2016; arduino et al. 1993 ). listeria monocytogenes is a bacterial organism that is transmitted most commonly during summer and early fall to humans via the gastrointestinal tract from contaminated dairy products, raw vegetables, and meat. although more common during the first 2 months after transplantation, infection may occur at any point, and risk is increased with rejection therapy (patel and paya 1997) . infections involving the central nervous system, such as meningitis and meningoencephalitis, are most common and present with headaches, fever, meningismus, altered mental status, and possibly focal neurologic deficits including cranial nerve palsies and seizures (patel and paya 1997) . cerebrospinal fluid examination typically reveals a pleocytosis, mostly polymorphonuclear leukocytes, decreased glucose, and elevated protein, but as the name implies, a mononuclear predominance may occur instead. gram staining has a low sensitivity and may be negative or reveal gram positive bacilli which may be confused with diphtheroids. other sites of infection include bacteremia, pneumonia, endophthalmitis, and septic arthritis. while trimethoprim-sulfamethoxazole, used for p. carinii prophylaxis, may also prevent infection with listeria, the treatment of choice is intravenous ampicillin and gentamicin for up to 8 weeks in those with cns infections to prevent relapses. gentamicin is usually continued for a shorter duration, about 2 weeks if kidney function is stable. (gelfand 2016) . trimethoprim-sulfamethoxazole is an alternative treatment for those who are allergic to penicillin. decreasing immunosuppressive agents is sometimes, but not always necessary. legionella infections in renal transplant recipients most commonly occur early in the posttransplantation period, but can be seen any time, especially during episodes of rejection. legionella pneumophila is the most common species to infect humans, and although more commonly community-acquired, nosocomial transmission occurs (patel and paya 1997) . most infections are pulmonary including pneumonia, and abscess with cavitation. symptoms are typical of lung infections but also may include headache and diarrhea. a legionella urinary antigen test and culture of lower respiratory secretions on selective media are used for diagnosis. empiric treatment for legionella is appropriate while waiting for results. quinolone antibiotics, such as levofloxacin, are preferred over macrolides in renal transplant patients because of drug interactions between macrolides and immunosuppressive medications. initially given intravenously, quinolone antibiotics can be quickly deescalated to oral treatment when the patient has defervesced. renal transplant patients, especially those who are severely ill at presentation, should receive 21 days of treatment (yu 2016) . along with pcp and listeria, as noted above, prophylaxis with trimethoprim-sulfamethoxazole may also prevent legionella infection. immunosuppression increases the risk of developing mycobacterium tuberculosis (tb) disease. although the majority of tuberculosis infections in renal transplant recipients occur in the first 18 months, tb can occur any time after transplantation (khoury and brennan 2005) . its overall incidence is lower in the united states when compared to the rest of the world, and foreign-born recipients are at greatest risk. having a high index of suspicion is important in renal transplant patients because presentation can be atypical and pretransplant screening with tuberculin skin tests or ifn-gamma release assays are unreliable in chronic kidney disease patients due to anergy. extra-pulmonary sites of infection and disseminated disease occur in about a third of cases (karuthu and blumberg 2012). laryngeal, meningeal, skeletal, cutaneous, intestinal, and renal infections are examples of extra-pulmonary disease. fevers are common, but sweats and weight loss may be absent (patel and paya 1997) . screening prior to transplant should include a history regarding prior exposures, and treatment for tb, as well as a chest x-ray and urine afb culture. prophylaxis with isoniazid or rifampin should be offered to patients prior to transplantation with a history of inadequately treated tb, an abnormal chest x-ray suggestive of prior tb exposure, a positive ppd or ifn gamma assay, contact with someone with active tb, or a kidney from a ppd-positive donor in order to minimize reactivation disease after transplantation (khoury and brennan 2005) . patients receiving treatment for latent tb may undergo renal transplantation and complete their defined course afterwards with special attention to potential drug interactions and toxicities (karuthu and blumberg 2012) . diagnosis of tb after renal transplantation often requires a biopsy of the infected site with stains for acid fast bacilli and cultures for sensitivity testing. treatment of active disease after transplantation requires multiple drugs and should follow the american thoracic society, center for disease control, and infectious disease society of america guidelines (mmwr 2003) . special attention to drug toxicities and interactions with immunosuppressive agents is required. rifampin, in particular, decreases cyclosporine levels and increases the risk for rejection. fungal infections in kidney transplant recipients occur less frequently than in other solid organ transplant recipients. most present within the first 6 months after transplantation (hagerty et al. 2003 ) and can represent primary, reactivated, or donor-derived infection. those associated with geographic and environmental exposures include histoplasmosis, coccidioidomycosis, blastomycosis, and paracoccidioidomycosis. others are considered opportunistic and include infections such as candida, aspergillus, and cryptococcus (karuthu and blumberg 2012) . broad spectrum antibiotics, corticosteroids, diabetes mellitus, rejection therapy, cmv infection, and duration of pretransplant dialysis are risk factors (khoury and brennan 2005) . esophageal candidiasis, urogenital candidiasis, and pneumonia are the three most common sites of fungal infections in these patients (abbott et al. 2001) . clinical presentation may be nonspecific and diagnosis difficult due to testing limitations. positive cultures may represent colonization rather than infection with pathogens such as candida and aspergillus. cultures, antigen assays, serum galactomannan assays, and radiography may be helpful, but are not always diagnostic. subsequently, biopsy with pathology and cultures is considered the gold standard for diagnosing fungal infections (karuthu and blumberg 2012) . drug interactions and toxicities as well as immune reconstitution, due to lowering of immunosuppressive medications, further complicate the management of fungal disease in these patients and require expert advice (karuthu and blumberg 2012) . pneumocystis jiroveci (formerly pneumocystis carinii (pcp), protozoa) is a pathogen currently considered a fungus based on nucleic acid and biochemical analysis. presenting as pneumonia with interstitial infiltrates on chest x-ray within the first year after transplantation in those not receiving prophylaxis, mortality may be high. nonproductive cough and shortness of breath with rapid progression to hypoxia is a classic presentation. diagnosis is based on silver staining of deep respiratory specimens from induced sputum, bronchoalveolar lavage, or transbronchial biopsy (martin and fishman 2013) . the treatment of choice is high dose trimethoprim-sulfamethoxazole for 21 days with corticosteroids in hypoxic patients (partial pressure of oxygen of <70 mmhg on room air) tapered over 14 days. atovaquone or clindamycin plus pyrimethamine are alternative agents (martin and fishman 2013) . trimethoprim-sulfamethoxazole prophylaxis for 6-12 months after transplantation is highly effective in preventing this infection and should be administered to all renal transplant patients if tolerated. frequently used alternatives for prophylaxis in allergic patients include dapsone (if glucose-6 phosphate dehydrogenase levels are normal) and atovaquone. infection remains an important concern in patients undergoing kidney transplantation. attention to pretransplant screening of the potential organ donor and recipient is essential to optimizing transplant outcomes. advances in the management of transplant-related infections include the increasing use of rapid molecular diagnostic testing as well as improvements in the approach to prophylaxis and treatment. ongoing challenges include the need for prolonged immunosuppression to prevent organ rejection, drug-drug interactions, and the management of resistant and emerging pathogens. continued awareness of the risks, timing, and presentation of infection posttransplant and strategies to reduce its impact will contribute further to progress in the field of kidney transplantation. hospitalizations for bacterial septicemia after renal transplantation in the united states epstein-barr virus and posttransplant lymphoproliferative disorder in solid organ transplant recipients late-onset acute haemorrhagic necrotizing granulomatous adenovirus tubulointerstitial nephritis in a renal allograft nocardiosis in renal transplant recipients undergoing immunosuppression with cyclosporine long-term outcomes of cmv disease treatment with valganciclovir versus iv ganciclovir in solid organ transplant 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experience and risk factor analysis in a single center should we treat asymptomatic bacteriuria after renal transplantation? epidemiology of bk virus in renal allograft recipients: independent risk factors for bk virus replication human cytomegalovirus and kidney transplantation: a clinician's update risk factors for hospitalization for bacterial or viral infection in renal transplant recipients-an analysis of usrds data efficacy of high-dose versus standard-dose influenza vaccine in older adults hhv 8 in renal transplant recipients human herpesvirus-6 and-7 in transplantation transplantation in the patient with hepatitis c 24-week oral ganciclovir prophylaxis in kidney recipients is associated with reduced symptomatic cytomegalo-virus disease compared to a 12-week course polyomavirus disease in renal transplantation: review of pathological findings and diagnostic methods hbsag seropositive status and survival after renal transplantation: meta-analysis of observational studies disseminated varicella infection in adult renal allograft recipients: four cases and a review of the literature transmission of lymphocytic choriomeningitis virus by organ transplantation ast infectious diseases community of practice (2013) screening of donor and recipient in solid organ transplantation infection in solid organ transplant recipients renal transplantation in patients with hiv clinical manifestations and diagnosis of listeria monocytogenes infection guidelines for the prevention and management of infectious complications of solid organ transplantation using epstein-barr viral load assays to diagnose, monitor, and prevent posttransplant lymphoproliferative disorder fungal infections in solid organ transplant patients bk virus nephritis after renal transplantation polyoma-virus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations asymptomatic cytomegalovirus infection is associated with increased risk of new-onset diabetes mellitus and impaired insulin release after renal transplantation antiviral medications for preventing cytomegalovirus disease in solid organ transplant recipients ast infectious diseases community of practice: cytomegalovirus in solid organ transplant recipients are wound complications after a kidney transplant more common with modern immunosuppression the efficacy and safety of 200 days valganciclovir cytomegalovirus prophylaxis in high-risk kidney transplant recipients solid organ transplantation from hepatitis b virus-positive donors: consensus guidelines for recipient management adenovirus infections in transplant recipients vaccination and chronic kidney disease polyomavirus-associated nephropathy: update on antiviral strategies hepatitis e virus and the kidney in solid-organ-transplant patients common infections in kidney transplant recipients the value of vaccination in chronic kidney disease kdigo clinical practice guideline for the care of kidney transplant recipients infectious complications in kidney transplant recipients: review of the literature prospective study of human betaherpesviruses after renal transplantation. association of human herpesvirus 7 and cytomegalovirus co-infection with cytomegalovirus disease and increased rejection improvement in long-term renal graft survival due to cmv prophylaxis with oral ganciclovir: results of a randomized clinical trial intervals between pcv13 and ppsv23 vaccines: recommendations of the advisory committee on immunization practices (acip) viral infection in the renal transplant recipient transplantation society international cmv consensus group: international consensus guidelines on the management of cytomegalovirus in solid organ transplantation selecting suitable solid organ transplant donors: reducing the risk of donor-transmitted infections risk factors for nosocomial urinary tract and postoperative wound infections in renal transplant patients: a matched-pair case-control study recommendations for screening of donor and recipient prior to solid organ transplantation and to minimize transmission of donor-derived infections infectious diseases community of practice (2013) viral hepatitis in solid organ transplant recipients emergence of ganciclovir-resistant cytomegalovirus disease among recipients of solid-organ transplants posttransplant lymphoproliferative disease in primary epstein-barr virus infection after liver transplantation: the role of cytomegalovirus disease american society of transplantation and american society of transplant surgeons, pneumocystis pneumonia in solid organ transplantation cytomegalovirus seromismatching increases the risk of acute renal allograft rejection allowing hiv-positive organ donation: ethical, legal and operational considerations renal transplantation in patients with hepatitis c virus antibody. a long national experience hiv-positive-to-hiv-positive kidney transplantation-results at 3-5 years prevention and management of cytomegalovirus infection in solid-organ transplantation prognosis of htlv-i-positive renal transplant recipients pre-emptive treatment for cytomegalovirus viraemia to prevent cytomegalovirus disease in solid organ transplant recipients infections in solid-organ transplant patients valganciclovir solid organ transplant study group: efficacy and safety of valganciclovir vs. oral ganciclovir for prevention of cyto-megalovirus disease in solid organ transplant recipients fatal disseminated adenovirus infections in immunocompromised patients polyoma nephropathy and progressive multifocal leukoencephalopathy in a renal transplant recipient cmv infection is associated with transplant renal artery stenosis incisional surgical site infection in kidney transplantation bk virus in transplant recipients: an overview and update transmission of human herpesvirus 8 infection from renal-transplant donors to recipients impact of early cytomegalovirus infection and disease on longterm recipient and kidney graft survival resitra network of the spanish study group of infection in transplantation (2008) impact of current transplantation management on the development of cytomegalovirus disease after renal transplantation successful treatment of hepatitis c in renal transplant recipients with direct-acting antiviral agents phs guideline for reducing human immunodeficiency virus, hepatitis b virus, and hepatitis c virus transmission through organ transplantation prevalence of hiv-associated nephropathy in autopsies of hiv-infected patients human herpesvirus-6 in transplantation: an emerging pathogen rates of first infection following kidney transplant in the united states up to date transmission of rabies virus from an organ donor to four transplant recipients sirolimus for kaposi's sarcoma in renal transplant recipients outcomes of kidney transplantation in hiv-infected recipients preemptive treatment for cytomegalovirus viremia to prevent cytomegalovirus disease in solid organ transplant recipients viral infections affecting the skin in organ transplant recipients: epidemiology and current management strategies long-term results in human t-cell leukemia virus type 1-positive renal transplant recipients post-transplant lymphoproliferative disorders (ptld) after solid organ transplantation the association of viral infection and chronic allograft nephropathy with graft dysfunction after renal transplantation polyomavirus in kidney and kidney-pancreas transplant recipients epidemiology of kidney disease in the unites states. national institutes of health, national institute of diabetes and digestive and kidney diseases in: safdar a (ed) principles and practice of transplant infectious diseases nocardial infections in renal transplant recipients the evaluation and management of urinary tract infections in recipients of solid organ transplants treatment and prevention of legionella infection key: cord-017012-yl0vanuh authors: herberg, jethro; pahari, amitava; walters, sam; levin, michael title: infectious diseases and the kidney date: 2009 journal: pediatric nephrology doi: 10.1007/978-3-540-76341-3_52 sha: doc_id: 17012 cord_uid: yl0vanuh the kidney is involved in a wide range of bacterial, viral, fungal, and parasitic diseases. in most systemic infections, renal involvement is a minor component of the illness, but in some, renal failure may be the presenting feature and the major problem in management. although individual infectious processes may have a predilection to involve the renal vasculature, glomeruli, interstitium, or collecting systems, a purely anatomic approach to the classification of infectious diseases affecting the kidney is rarely helpful because most infections may involve several different aspects of renal function. the kidney is involved in a wide range of bacterial, viral, fungal, and parasitic diseases. in most systemic infections, renal involvement is a minor component of the illness, but in some, renal failure may be the presenting feature and the major problem in management. although individual infectious processes may have a predilection to involve the renal vasculature, glomeruli, interstitium, or collecting systems, a purely anatomic approach to the classification of infectious diseases affecting the kidney is rarely helpful because most infections may involve several different aspects of renal function. in this chapter, a microbiologic classification of the organisms affecting the kidney is adopted. although they are important causes of renal dysfunction in infectious diseases, urinary tract infections and hemolytic uremic syndrome (hus) are not discussed in detail because they are considered separately in chapters 54 and 48 respectively. elucidation of the cause of renal involvement in a child with evidence of infection must be based on a careful consideration of the geographic distribution of infectious diseases in different countries. a history of foreign travel; exposure to animals, insects, or unusual foods or drinks; outdoor activities such as swimming or hiking; and contact with infectious diseases must be sought in every case. the clinical examination should include a careful assessment of skin and mucous membranes and a search for insect bites, lymphadenopathy, and involvement of other organs. a close collaboration with a pediatric infectious disease specialist and hospital microbiologist will aid the diagnosis and management of the underlying infection. a tantalizing clue to the pathogenesis of glomerular disease is the marked difference in the incidence of nephrosis and nephritis in developed and underdeveloped areas of the world. in several tropical countries, glomerulonephritis (gn) accounts for up to 4% of pediatric hospital admissions; the incidence in temperate climates is 10-to 100-fold less. this difference might be explained by a complex interaction of several different factors, including nutrition, racial and genetically determined differences in immune responses, and exposure to infectious diseases. a growing body of evidence, however, suggests that longterm exposure to infectious agents is a major factor in the increased prevalence of glomerular diseases in developing countries. renal involvement in infectious diseases may occur by a variety of mechanisms: direct microbial invasion of the renal tissues or collecting system may take place in conditions such as staphylococcal abscess of the kidney as a result of septicemic spread of the organism or as a consequence of ascending infection; damage to the kidney may be caused by the systemic release of endotoxin or other toxins and activation of the inflammatory cascade during septicemia or by a focus of infection distant from the kidney; ischemic damage may result from inadequate perfusion induced by septic shock; the kidney may be damaged by activation of the immunologic pathways or by immune complexes resulting from the infectious process. in many conditions, a combination of these mechanisms may be operative. in the assessment of renal complications occurring in infectious diseases, the possibility of druginduced nephrotoxicity caused by antimicrobial therapy should always be considered. the nephrotoxic effects of antibiotics and other antimicrobial agents are not addressed in this chapter but are covered in chapter 53. bacterial infections associated with renal disease and the likely mechanisms causing renal dysfunction are shown in > impaired renal function is a common occurrence in systemic sepsis (1) . depending on the severity of the infection and the organism responsible, the renal involvement may vary from insignificant proteinuria to acute renal failure requiring dialysis. the organisms causing acute renal failure as part of systemic sepsis vary with age and geographic location and also differ in normal and immunocompromised children. in the neonatal period, group b streptococci, coliforms, staphylococcus aureus, and listeria monocytogenes are the organisms usually . responsible. in older children, neisseria meningitidis, streptococcus pneumoniae, and s. aureus account for most of the infections. in people who are immunocompromised, a wide range of bacteria are seen, and, similarly, in tropical countries other pathogens, including haemophilus influenzae, salmonella species, and pseudomonas pseudomallei, must be considered. where h. influenzae type b vaccine has been introduced, however, the incidence of severe systemic infections due to this organism has shown a sharp fall. systemic sepsis usually presents with nonspecific features: fever, tachypnea, tachycardia, and evidence of skin and organ underperfusion. the pathophysiology of renal involvement in systemic sepsis is multifactorial (1, 2) . hypovolemia with diminished renal perfusion is the earliest event and is a consequence of the increased vascular permeability and loss of plasma from the intravascular space. hypovolemia commonly coexists with depressed myocardial function because of the myocardial depressant effects of endotoxin or other toxins. the renal vasoconstrictor response to diminished circulating volume and reduced cardiac output further reduces glomerular filtration, and oliguria is thus a consistent and early event in severe sepsis (1, 3) . a number of vasodilator pathways are activated in sepsis, including nitric oxide and the kinin pathways. this may lead to inappropriate dilatation of vascular beds. vasodilation of capillary beds leading to warm shock is common in adults with sepsis due to gram-negative organisms but is less commonly seen in children, in whom intense vasoconstriction is the usual response to sepsis. if renal underperfusion and vasoconstriction are persistent and severe, the reversible prerenal failure is followed by established renal failure with the characteristic features of vasomotor nephropathy or acute tubular necrosis. other mechanisms of renal damage in systemic sepsis include direct effects of endotoxin and other toxins on the kidney, and release of inflammatory mediators such as tumor necrosis factor (tnf) and other cytokines, arachidonic acid metabolites, and proteolytic enzymes (3) . nitric oxide (no) is postulated to play a key role in the pathophysiology of renal failure in sepsis. whether the renal effects of increased no are beneficial or harmful remains unclear. trials of selective no synthetase inhibition did not offer any advantages over saline resuscitation (4) . no in endotoxemia is possibly beneficial because it maintains renal blood flow and glomerular filtration. activation of coagulation is an important component of the pathophysiology of septic shock and may contribute to renal impairment. activation of multiple prothrombotic and antifibrinolytic pathways occurs, together with downregulation of antithrombotic mechanisms such as the protein c pathway. treatment with activated protein c has been shown to improve outcome of adult septic shock, but has not been confirmed to have benefit in pediatric sepsis, and may carry a risk of bleeding particularly in infants (5) . the renal findings early in septic shock are oliguria, with high urine/plasma urea and creatinine ratios, low urine sodium concentration, and a high urine/plasma osmolarity ratio. once established, renal failure supervenes, and the urine is of poor quality with low urine/ plasma urea and creatinine ratios, elevated urine sodium concentration, and low urine osmolarity. proteinuria is usually present, and the urine sediment may contain red cells and small numbers of white cells (1) . the management of acute renal failure in systemic sepsis depends on early diagnosis and administration of appropriate antibiotics to cover the expected pathogens. . in addition, management is directed at improving renal perfusion and oxygenation. volume replacement with crystalloid or colloid should be undertaken to optimize preload. central venous pressure or pulmonary wedge pressure monitoring is essential to guide volume replacement in children in severe shock (1, 2) . the use of low-dose (2-5 pg/kg/min) dopamine to reduce renal vasoconstriction together with administration of inotropic agents such as dobutamine or epinephrine to improve cardiac output may reverse prerenal failure. early elective ventilation should be undertaken in patients with severe shock. if oliguria persists despite volume replacement and inotropic therapy, dialysis should be instituted early, because septic and catabolic patients may rapidly develop hyperkalemia and severe electrolyte imbalance. in most children who develop acute renal failure as part of systemic sepsis or septic shock, the renal failure is of short duration, and recovery can be expected within a few days of achieving cardiovascular stability and eradication of the underlying infection. occasionally, renal cortical necrosis or infarction of the kidney may result in prolonged or permanent loss of renal function. meningococcus n. meningitidis continues to be a major cause of systemic sepsis and meningitis in both developed and underdeveloped parts of the world (6) . in developed countries, most cases are caused by group b and y strains, particularly after introduction of meningococcal c vaccination, whereas epidemics of meningococcus groups a, c and w135 continue to occur in many underdeveloped regions of the world (6, 7) . infants and young adults are most commonly affected, but cases in adolescents and young children are also common. there are two major presentations of meningococcal disease (6) : meningococcal meningitis presents with features indistinguishable from those of other forms of meningitis, including headache, stiff neck, and photophobia. lumbar puncture is required to identify the causative agent and distinguish this from other forms of meningitis. despite the acute nature of the illness, the prognosis is good, and most patients with the purely meningitic form of the illness recover without sequelae. meningococcemia with purpuric rash and shock is the second and more devastating form of the illness. affected patients present with nonspecific symptoms of fever, vomiting, abdominal pain, and muscle ache. the diagnosis is only obvious once the characteristic petechial or purpuric rash appears. patients with a rapidly progressive purpuric rash, hypotension, and evidence of skin and organ underperfusion have a poor prognosis, with a mortality of 10-30%. adverse prognostic features include hypotension, a low white cell count, absence of meningeal inflammation, thrombocytopenia, and disturbed coagulation indices (8) . renal failure was seldom reported in early series of patients with meningococcemia, perhaps because most patients died rapidly of uncontrolled septic shock. with advances in intensive care, however, more children are surviving the initial period of profound hemodynamic derangement, and renal failure is more often seen as a major management problem. approximately 10% of children with fulminant meningococcemia develop renal failure, which usually occurs 24-48 h after the onset of illness (9) . the pathophysiology of meningococcal septicemia involves the activation of cytokines and inflammatory cells by endotoxin (6, 7) . mortality is directly related to both the plasma endotoxin concentration and the intensity of the inflammatory response, as indicated by levels of tnf and other inflammatory markers (10) . patients with meningococcemia have a profound capillary leak leading to severe hypovolemia. loss of plasma proteins from the intravascular space is probably the major cause of shock (11) , but myocardial suppression secondary to il-6 production is also important (12) . intense vasoconstriction further impairs tissue and organ perfusion, and vasculitis with intravascular thrombosis and consumption of platelets and coagulation factors is also present (6) . oliguria is invariably present in children with meningococcemia during the initial phase of the disorder. this is prerenal in origin and may respond to volume replacement and inotropic support. if cardiac output cannot be improved and renal underperfusion persists, established renal failure supervenes. occasionally, cortical necrosis or infarction of the kidneys occurs. children with meningococcemia should be aggressively managed in a pediatric intensive care unit, with early administration of antibiotics (penicillin or a third-generation cephalosporin), volume replacement, hemodynamic monitoring, and the use of inotropic agents and vasodilators. if oliguria persists despite measures to improve cardiac output, elective ventilation and dialysis should be instituted early (6, 7) . because activation of coagulation pathways occurs, severe acquired protein-c deficiency may result and is usually associated with substantial mortality (13) . protein c is a natural anticoagulant which also has important antiinflammatory activity. despite evidence for impaired function of the activated protein c pathway in meningococcal diseases (14) , and adult trials suggesting benefit of activated protein c administration in septic shock (prowess trial) (15) , pediatric trials of activated protein c showed no clear benefit, and were associated with increased risk of intracranial bleeding in very young infants (5) . the role of apc therapy in pediatric sepsis remains unclear. most patients who survive the initial 24-48 h of the illness and regain hemodynamic stability will ultimately recover renal function even if dialysis is required for several weeks. the least common presentation of meningococcal sepsis is chronic meningococcemia. patients with this form of the illness present insidiously with a vasculitic rash, arthritis, and evidence of multiorgan involvement. the features may overlap those of henoch-schonlein purpura or subacute bacterial endocarditis (sbe), and the diagnosis must be considered in patients presenting with fever, arthritis, and vasculitic rash, often accompanied by proteinuria or hematuria. response to antibiotic treatment is good, but some patients may have persistent symptoms for many days resulting from an immunecomplex vasculitis. staphylococcal infections may affect the kidneys by direct focal invasion during staphylococcal septicemia, forming a renal abscess; by causing staphylococcal bacteremia; or by toxin-mediated mechanisms, as in the staphylococcal toxic shock syndrome. staphylococcal abscess. staphylococcal renal abscess presents with fever, loin pain and tenderness, and abnormal urine sediment, as do abscesses caused by other organisms (16) . the illness often follows either septicemia or pyelonephritis. the diagnosis is usually considered only when a patient with clinical pyelonephritis shows an inadequate response to antibiotic treatment. the diagnosis is confirmed by ultrasonography or computed tomographic scan, which shows swelling of the kidney and intrarenal collections of fluid. antibiotic therapy alone may result in cure, but if the patient remains unwell with evidence of persistent inflammation despite use of appropriate antibiotics, surgical intervention may be required. percutaneous drainage under ultrasonographic or computed tomographic scan guidance is often effective and may avoid the need for a more direct surgical approach (16, 17) . staphylococcal toxic shock syndrome. the staphylococcal toxic shock syndrome is a systemic illness characterized by fever, shock, erythematous rash, diarrhea, confusion, and renal failure. the disorder was first described by todd et al. in 1978 in a series of seven children (18) . during the 1980s, thousands of cases were reported in the united states. most cases were in menstruating women, in associated with tampon use. although most cases worldwide are seen in women and are associated with menstruation, children of both sexes and of all ages are affected (19) . the illness usually begins suddenly with high fever, diarrhea, and hypotension, together with a diffuse erythroderma (20) . mucous membrane involvement with hyperemia and ulceration of the lips and oral mucosa or vaginal mucosa, strawberry tongue, and conjunctival injection are usually seen. desquamation of the rash occurs in the convalescent phase of the illness. confusion is often present in the early stages of the illness and may progress to coma in severe cases. multiple organ failure with evidence of impaired renal function, elevated levels of hepatic transaminases, thrombocytopenia, elevated cpk and disseminated intravascular coagulation (dic) is often seen. according to cdc criteria, the diagnosis is made on the basis of the clinical features of fever, rash, hypotension, and subsequent desquamation along with deranged function of three or more of the following organ systems: gastrointestinal (gi), mucous membranes, renal, hepatic, hematologic, central nervous system, and muscle. other disorders causing a similar picture, such as rocky mountain spotted fever, leptospirosis, measles, and streptococcal infection, must be excluded. the staphylococcal toxic shock syndrome is now known to be due to infection or colonization with strains of s. aureus that produce one or more protein exotoxins (21) . most cases in adults are associated with toxic shock toxin i; in children, many of the isolates associated with the syndrome produce other enterotoxins (a to f). the staphylococcal enterotoxins appear to induce disease by acting as superantigens (22) , which activate t cells bearing specific v beta regions of the t-cell receptor; this causes proliferation and cytokine release (23) . the systemic illness and toxicity are believed to result largely from an intense inflammatory response induced by the toxin. the site of toxin production is often a trivial focus of infection or simple colonization, and bacteremia is rarely observed. renal failure in toxic shock syndrome is usually caused by shock and renal hypoperfusion. in the early stages of the illness, oliguria and renal impairment are usually prerenal and respond to treatment of shock and measures to improve perfusion. in severe cases and in patients in whom treatment is delayed, acute renal failure develops as a consequence of prolonged renal underperfusion, and dialysis may be required. in addition to underperfusion, direct effects of the toxin or inflammatory infectious diseases and the kidney mediators may also contribute to the renal damage. recovery of renal function usually occurs, but in severe cases with cortical necrosis or intense renal vasculitis, prolonged dialysis may be required. the management of staphylococcal toxic shock syndrome depends on early diagnosis and aggressive cardiovascular support with volume replacement, inotropic support, and, in severe cases, elective ventilation. if oliguria persists despite optimization of intravascular volume and administration of inotropic agents, dialysis should be commenced early (20) . anti-staphylococcal antibiotics should be started as soon as the diagnosis is suspected and the site of infection identified. initial empiric antimicrobial therapy should include an anti-staphylococcal antibiotic effective against betalactamase-resistant organisms and a protein synthesisinhibiting antibiotic such as clindamycin to stop further toxin production (24) . if there is a focus of infection such as a vaginal tampon, surgical wound, or infected sinuses, the site should be drained early to prevent continued toxin release into the circulation. the intravenous administration of immune globulins may be considered when infection is refractory to several hours of aggressive therapy, an undrainable focus is present, or persistent oliguria with pulmonary edema occurs (24) . with aggressive intensive care, most affected patients survive, and renal recovery is usual, even in patients who have had severe shock and multiorgan failure. relapses and recurrences of staphylococcal toxic shock syndrome occur in a proportion of affected patients because immune responses to the toxin are ineffective in some individuals. panton valentine leucocidin (pvl) producing staphylococcal infection: in recent years there have been increasing reports of severe staphylococcal disease, associated with shock and multiorgan failure, caused by strains of staphylococci producing the pvl toxin. panton-valentine leukocidin (pvl) is a phage-encoded toxin, which profoundly impairs the host response due to its toxic effect on leucocytes (see review (25) ). pvl producing strains are associated with tissue necrosis and increased propensity to cause abscesses in lung, bone, joint, and soft tissue infections. perinephric abscesses have been reported (26) . there are increasing numbers of children and adults admitted with fulminant sepsis, and shock due to pvl producing strains, and renal failure is a significant component of the multiorgan failure. in addition to intensive care support, antibiotic treatment of pvl strains should include antibiotics which reduce toxin production, such as clindamycin, linezolid or rifampicin, as well as vancomycin if the strain is resistant to methicillin. beta-lactam antibiotics should be avoided, as there is some data to suggest that pvl toxin production can increased by these antibiotics under some conditions (27) . immunoglobulin infusion may also be of benefit. aggressive surgical drainage of all collections requires close consultation with orthopedic and surgical teams. the group a streptococci (gas) are a major worldwide cause of renal disease, usually as poststreptococcal nephritis. however, in addition to this post-infection immunologically mediated disorder, in recent years there have been increasing reports of gas causing acute renal failure as part of an invasive infection with many features of the staphylococcal toxic shock syndrome (28) . acute poststreptococcal glomerulonephritis. acute poststreptococcal gn (apsgn) is a delayed complication of pharyngeal infection or impetigo with certain nephritogenic strains of gas. different strains can be serotyped according to the antigenic properties of the m protein found in the outer portion of the bacterial wall. apsgn after pharyngeal infection is most commonly associated with serotype m12. in contrast, in apsgn after impetigo, serotype m49 is most commonly identified (29) . on occasions, other serotypes and non-typeable strains have been described as causing gn. the pathology and pathogenesis of the disorder is discussed in detail in chapter 30. apsgn has a worldwide distribution. epidemiologic differences are observed between pharyngitis-associated and impetigo-associated streptococcal infections. pharyngitis-associated apsgn is most common during school age and has an unexplained male/female ratio of 2:1. it occurs more often in the cooler months, and familial occurrences are commonly described. the latent period is 1-2 weeks, in notable contrast to impetigo-associated cases, which have a latent period of 2-6 weeks. in many developing countries, children have chronic skin infections, and it may be difficult to establish the latent period with accuracy. impetigoassociated cases are more common in the warmer months, sex distribution is equal, and children tend to be younger. introduction of a nephritogenic strain into a family often results in the occurrence of several cases within that family, and in some cases, attack rates of up to 20% have been described (30) . the incidence is linked to poor socioeconomic conditions. renal involvement in apsgn can be mild, and in many patients, the disease may not be manifested clinically. studies of epidemics with nephritogenic strains of streptococci have shown that up to 50% of those infected had subclinical evidence of renal disease (30, 31) . in a typical case a sudden onset of facial or generalized edema occurs. hypertension is usually modest but is severe in 5% of cases, and occasionally may lead to encephalopathy or left ventricular failure. the urine is smoky or tea colored in 30-50% of cases. pallor, headache, backache, lethargy, malaise, anorexia, and weakness are all common nonspecific features. the urine volume is decreased. proteinuria is present (up to 100 mg/dl), and microscopy shows white cells, red cells, and granular and hyaline casts. urea, electrolyte, and creatinine levels are normal in subclinical cases but show features of acute renal failure in severe cases. it may be possible to culture gas from the skin or the throat in some patients. other evidence of infection with a gas can be obtained through the antistreptolysin-o titer (asot), which is increased in 60-80% of cases. early antibiotic treatment can reduce the proportion of cases with elevated asot to 30%. anti-deoxyribonuclease b and anti-hyaluronidase testing has been shown to be of more value than asot in confirming group a streptococcal infection in impetigo-associated cases. measurement of anti-m protein antibodies is of more value for epidemiologic purposes than for the diagnosis of individual cases (31) . decreased c3 and total hemolytic complement levels are found in 90% of cases during the first 2 weeks of illness and return to normal after 4-6 weeks. penicillin should be given to eradicate the gas organisms. erythromycin, clindamycin, or a first-generation cephalosporin can be given to patients allergic to penicillin. antibiotic treatment probably has no influence on the course of renal disease but will prevent the spread of a nephritogenic strain (32) . close contacts and family members who are culture-positive for gas should also be given penicillin, although antibiotic treatment is not always effective in eliminating secondary cases. recurrent episodes are rare, and immunity to the particular nephritogenic strain that caused the disease is probably lifelong. antibiotic prophylaxis is therefore unnecessary. most studies suggest that the prognosis for children with apsgn is good, with more than 90% making a complete recovery. however, 10% of cases may have a prolonged and more serious course with long-term chronic renal failure (33) . other streptococci. apsgn has also been described after outbreaks of group c streptococcus infection (34) . this has occurred after consumption of unpasteurized milk from cattle with mastitis. patients developed pharyngitis followed by apsgn. endostreptosin was found in the cytoplasm of these group c strains, and during the course of the illness, patients developed anti-endostreptosin antibodies. this antigen has been postulated to be the nephritogenic component of gas. in addition, strains of group g streptococci have been implicated in occasional cases of apsgn (35) . isolates possessed the type m12 protein antigen identical to the nephritogenic type m12 antigen of some group a streptococcal strains. streptococcal toxic shock syndrome and invasive group a streptococcal infection. since 1988, there have been several reports of an illness with many similarities to the staphylococcal toxic shock syndrome, occurring in both children (36) and adults, associated with invasive group a streptococcal disease (32, 37, 38) . patients with this syndrome present acutely with high fever, erythematous rash, mucous membrane involvement, hypotension, and multiorgan failure. unlike staphylococcal toxic shock syndrome, in which the focus of infection is usually trivial and bacteremia is seldom seen, the streptococcal toxic shock syndrome is usually associated with bacteremia or a serious focus of infection such as septic arthritis, myositis, or osteomyelitis (36, 38) . laboratory findings of anemia, neutrophil leukocytosis, thrombocytopenia, and dic are often present, together with impaired renal function, hepatic derangement, and acidosis. acute renal failure requiring dialysis occurs in a significant proportion of cases. it is not clear why there are increasing numbers of cases with invasive disease caused by gas, nor why there has been an emergence of streptococcal toxic shock syndrome, and indeed a similar syndrome caused by some pseudomonas and klebsiella strains. the most common antecedent of invasive gas disease is varicella infection, with the streptococcal infection developing after the initial vesicular phase of the disease is subsiding. strains causing toxic shock syndrome and invasive disease appear to differ from common isolates of gas in producing large amounts of pyrogenic toxins that may have superantigenlike activity. another important mechanism is the production by invasive gas of an il8 protease. il8 serves as a molecular bridge between receptors on neutrophils and the vascular endothelium. cleavage of this protein prevents neutrophil attachment to the endothelium, and results in uncontrolled spread of the bacteria through the tissues (39) . in severe cases necrotizing fasciitis occurs with extensive destruction of the subcutaneous tissues, and is often associated with multiorgan failure. the pathophysiology of streptococcal toxic shock syndrome and that of invasive disease is similar in that superantigen toxins that induce release of cytokines and other inflammatory mediators play a role in both conditions. however gas toxic shock is usually more severe, carries a higher mortality, and is more often associated with focal collections or necrotizing fasciitis. treatment of streptococcal toxic shock syndrome depends on the administration of appropriate antibiotics, aggressive circulatory support, and treatment of any multiorgan failure. surgical intervention to drain the infective focus in muscle, bone, joint, or body cavities is often required. antibiotic therapy with beta-lactams should be supplemented by treatment with a protein synthesisinhibiting antibiotic, such as clindamycin, and it is suggested that this limits new toxin production (40, 41) . a number of new therapies are in development. firstly, pooled intravenous immunoglobulins are now in widespread use in the treatment of toxic shock, particularly when caused by streptococcus (42, 43) . the role of steroids remains unclear, with their hemodynamic benefit set against the detrimental effects of hyperglycemia secondary to gluconeogenesis. (44) . the benefit of insulin therapy to control hyperglycemia is unclear. a recent study in adults found that intensive insulin therapy increased the risk of serious adverse events (45) . in contrast to adult patients, in children with severe sepsis, the use of activated protein c (drotrecogin) cannot be recommended, as in a multicenter trial, fatality was increased in the treatment group (5) . recovery of renal function occurs in patients who respond to treatment of shock and the eradication of the infection. leptospirosis is an acute generalized infectious disease caused by spirochetes of the genus leptospira (46) . it is primarily a disease of wild and domestic animals, and humans are infected only occasionally through contact with animals. most human cases occur in summer or autumn and are associated with exposure to leptospiracontaminated water or soil during recreational activities such as swimming or camping. in adolescents and adults, occupational exposure through farming or other contact with animals is the route of infection. the spirochete penetrates intact mucous membranes or abraded skin and disseminates to all parts of the body, including the cerebrospinal fluid (csf). although leptospires do not contain classic endotoxins, the pathophysiology of the disorder has many similarities to that of endotoxemia. in severe cases, jaundice occurs because of hepatocellular dysfunction and cholestasis. renal functional abnormalities may be profound and out of proportion to the histologic changes in the kidney (47) . renal involvement is predominantly a result of tubular damage, and spirochetes are commonly seen in the tubular lesions. the inflammatory changes in the kidney may result from either a direct toxic effect of the organism or immunecomplex nephritis. however, hypovolemia, hypotension, and reduced cardiac output caused by myocarditis may contribute to the development of renal failure. in severe cases, a hemorrhagic disorder caused by widespread vasculitis and capillary injury also occurs (47, 48) . the clinical manifestations of leptospirosis are variable. of affected patients, 90% have the milder anicteric form of the disorder, and only 5-10% have severe leptospirosis with jaundice. the illness may follow a biphasic course. after an incubation period of 7-12 days, a nonspecific flu-like illness lasting 4-7 days occurs, associated with septicemic spread of the spirochete. the fever then subsides, only to recur for the second, ''immune,'' phase of the illness. during this phase, the fever is low grade and there may be headache and delirium caused by meningeal involvement, as well as intense muscular aching. nausea and vomiting are common. examination usually reveals conjunctival suffusion, erythematous rash, lymphadenopathy, and meningism. the severe form of the disease (weil's disease) presents with fever, impaired renal and hepatic function, hemorrhage, vascular collapse, and altered consciousness. in one series the most common organs involved were the liver (71%) and kidney (63%). cardiovascular (31%), pulmonary (26%), neurologic (5%), and hematologic (21%) involvements were less common (49) . vasculitis, thrombocytopenia, and uremia are considered important factors in the pathogenesis of hemorrhagic disturbances and the main cause of death in severe leptospirosis (50) . urinalysis results are abnormal during the leptospiremic phase with proteinuria, hematuria, and casts. uremia usually appears in the second week, and acute renal failure may develop once cardiovascular collapse and dic are present (48) . the clinical features of leptospirosis overlap with those of several other acute infectious diseases, including rocky mountain spotted fever, toxic shock syndrome, and streptococcal sepsis. the diagnosis of leptospirosis should be considered in febrile patients with evidence of renal, hepatic, and mucous membrane changes and rash, particularly if a history of exposure to fresh water is found. diagnosis can be confirmed by isolation of the spirochetes from blood or csf in the first 10 days of the illness or from urine in the second week (48) . the organism may be seen in biopsy specimens of the kidney or skin or in the csf by dark-field microscopy or silver staining. serologic tests to detect leptospirosis are now sensitive and considerably aid the diagnosis. immunoglobulin m (igm) antibody may be detected as early as 6-10 days into the illness, and antibody titers rise progressively over the next 2-4 weeks. some patients remain seronegative, and negative serologic test results do not completely exclude the diagnosis. in one series levels of igm and igg anticardiolipin concentrations were significantly increased in leptospirosis patients with acute renal failure (50) . leptospirosis is treated with intravenous penicillin or other beta-lactam antibiotics. the severity of leptospirosis is reduced by antibiotic treatment, even if started late in the course of the illness (51) . supportive treatment with volume replacement to correct hypovolemia, administration of inotropes, and correction of coagulopathy is essential in severe cases. dialysis may be required in severe cases and may be needed for prolonged periods until recovery occurs. infection with s. pneumoniae is one of the most common infections in humans and causes a wide spectrum of disease, including pneumonia, otitis media, sinusitis, septicemia, and meningitis. despite the prevalence of the organism, significant renal involvement is relatively rare but is seen in two situations: pneumococcal septicemia in asplenic individuals or in those with other immune deficiencies presents with fulminant septic shock in which renal failure may occur as part of a multisystem derangement. the mortality from pneumococcal sepsis in asplenic patients is high, even with early antibiotic treatment and intensive support. the second nephrologi syndrome associated with s. pneumoniae is a rare form of hus. in 1955, gasser and colleagues described hus as a clinical entity in children, and they included two infants with pneumonia among the five patients they described (52) . hus associated with pneumococcal infection is induced by the enzyme neuraminidase released from s. pneumoniae (53, 54) . thomsen-friedenrich antigen (t antigen) is present on the surface of red blood cells, platelets, and glomerular capillary endothelia against which antibodies are present in normal serum. neuraminidase causes desialation of red blood cells, and possibly other blood cells and endothelium, by the removal of terminal neuraminic acid, which leads to unmasking of the t antigen. the resultant widespread agglutination of blood cells causes intravascular obstruction, hemolysis, thrombocytopenia, and renal failure. results of the direct coombs test are frequently positive, either from bound anti-t igm or from anti-t antibodies. the diagnosis of thomsen-friedenrich antibody-induced hus should be suspected in patients with acute renal failure, thrombocytopenia and hemolysis after an episode of pneumonia or bacteremia caused by s. pneumoniae. fragmented red blood cells will usually be present on blood film. association with s. pneumoniae is defined by culture of pneumococci from a normally sterile site within a week before or after onset of signs of hus. clues to a pneumococcal cause, in addition to culture results, include severe clinical disease, especially pneumonia, empyema, pleural effusion, or meningitis; hemolytic anemia without a reticulocyte response; positive results on a direct coombs test; and difficulties in abo crossmatching or a positive minor crossmatch incompatibility (55) . however, when renal disease is seen in the context of severe pneumococcal infection, it is important to maintain a broad diagnostic perspective, because the occurrence of acute tubular necrosis due to septic shock and dic is well described (56, 57) . therapy for this syndrome should be with supportive treatment and antibiotics (usually a third generation cephalosporin); dialysis may be required if renal failure occurs. because normal serum contains antibodies against the thomsen-friedenrich antigen, blood transfusion should be undertaken with washed red blood cells resuspended in albumin rather than plasma (53, 54) . exchange transfusion and plasmapheresis have been used in some patients, with the rationale that these procedures may improve outcome by eliminating circulating neuraminidase (53, 57, 58) . intravenous igg has been used in a patient and was shown to neutralize neuraminidase present in the patient's serum (59) . in comparison to patients with the more common diarrhea-associated hus, s. pneumoniae-induced hus patients have a more severe renal disease. they are more likely to require dialysis. their long-term outcome maybe affected by the severity of the invasive streptococcal disease itself, and a significant proportion of surviving patients (30-70%) develop end-stage renal failure (60, 61) . a recent review of uk cases found an eightfold increase in early mortality as compared to diarrhoea-induced hus (62) . gastrointestinal infections (escherichia coli, salmonella, campylobacter, yersinia, shigella, vibrio cholerae) the diarrheal diseases caused by escherichia coli, salmonella, shigella, campylobacter, vibrios, and yersinia remain important and common bacterial infections of humans. although improvements in hygiene and living conditions have reduced the incidence of bacterial gastroenteritis in developed countries, these infections remain common in underdeveloped areas of the world, and outbreaks and epidemics continue to occur in both developed and underdeveloped countries. renal involvement in the enteric infections may result from any of four possible mechanisms. regardless of the causative organism, diarrhea results in hypovolemia, abnormalities of plasma electrolyte composition, and renal underperfusion. if severe dehydration occurs and is persistent, oliguria from prerenal failure is followed by vasomotor nephropathy and established renal failure. e. coli, shigella, and salmonella (particularly salmonella typhi) may invade the bloodstream and induce septicemia or septic shock. acute renal failure is commonly seen in infants with e. coli sepsis but is also reported with klebsiella, salmonella, and shigella infections. its pathophysiology and treatment were discussed previously. enteric infections with e. coli, yersinia, campylobacter, and salmonella have been associated with several different forms of gn, including membranoproliferative gn (mpgn), interstitial nephritis, diffuse proliferative gn, and iga nephropathy (63) (64) (65) . in typhoid fever, gn ranging from mild asymptomatic proteinuria and hematuria to acute renal failure may occur (64, (66) (67) (68) . renal biopsy findings show focal proliferation of mesangial cells, hypertrophy of endothelial cells, and congested capillary lumina. immunofluorescent studies show igm, igg, and c3 deposition in the glomeruli, with salmonella antigens detected within the granular deposits in the mesangial areas. in the iga nephropathy after typhoid fever, salmonella vi antigens have been demonstrated within the glomeruli. yersinia infection has been reported as a precipitant of gn in several studies (65, 69) . transient proteinuria and hematuria are found in 24% of patients with acute yersinia infection, and elevated creatinine levels in 10%. renal biopsy reveals mild mesangial gn or iga nephropathy. yersinia antigens, immunoglobulin, and complement have been detected in the glomeruli. yersinia pseudotuberculosis is well recognized as one of the causes of acute tubulointerstitial nephritis causing acute renal failure, especially in children; patients have histories of drinking untreated water in endemic areas (70) (71) (72) . the illness begins with the sudden onset of high fever, skin rash, and gi symptoms. later in the course, periungual desquamation develops, mimicking kawasaki disease. elevated erythrocyte sedimentation rate, c-reactive protein level, and thrombocytosis are noticeable, and mild degrees of proteinuria, glycosuria, and sterile pyuria are common. acute renal failure, which typically develops 1-3 weeks after the onset of fever, follows a benign course with complete recovery. renal biopsy mainly reveals findings of acute tubulointerstitial nephritis. antibiotic therapy, although recommended, does not alter the clinical course, but reduces the fecal excretion of the organism (73, 74) . hus is characterized by three distinct clinical signs: acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia. it was first described in 1955 and was associated with infection by shiga toxin-producing shigella dysenteriae. a major breakthrough in the search for the cause of hus occurred in the 1980s when karmali et al. reported that 11 of 15 children with diarrheaassociated hus had evidence of infection with a strain of e. coli that produced a toxin active on vero cells (75) . in diarrhea-associated hus in the united states and most of europe, e. coli 0157:h7 is the most important of these strains. e. coli 0157:h7 occurs naturally in the gi tract of cattle and other animals, and humans become infected through contaminated food products. most outbreaks have been associated with consumption of undercooked meat, but unpasteurized milk and cider, drinking water, and poorly chlorinated water for recreational use have also been implicated as vehicles for bacterial spread. hus is discussed in detail in chapter 67. the global epidemic of mycobacterium tuberculosis is growing. several factors have contributed to this increase, including the emergence of the human immunodeficiency virus (hiv) infection epidemic, large influxes of immigrants from countries in which tuberculosis (tb) is common, the emergence of multiple-drug-resistant m. tuberculosis, and breakdown of the health services for effective control of tb in various countries. it is generally estimated that, overall, one-third of the world's population is currently infected with the tb bacillus. there are more than 8 million cases of tb, which result in the death of approximately 2 million people each year. furthermore, 5-10% of people who are infected with the tb bacillus develop tb disease or become infectious at some time during their lives (76, 77) . after respiratory illness in children, mycobacteria are widely distributed to many organs of the body during the lymphohematogenous phase of childhood tb (78) . tubercle bacilli can be recovered from the urine in many cases of miliary tb. hematogenously-spread tuberculomata develop in the glomeruli, which results in caseating, sloughing lesions that discharge bacilli into the tubules. in most cases, the renal lesions are asymptomatic and manifest as mycobacteria in the urine or as sterile pyuria. tuberculomata in the cortex may calcify and cavitate or may rupture into the pelvis, discharging infective organisms into the tubules, urethra, and bladder. dysuria, loin pain, hematuria, and pyuria are the presenting features of this complication, but in many cases, the renal involvement is asymptomatic, even when radiologic and pathologic abnormalities are very extensive. continuing tuberculous bacilluria may cause cystitis with urinary frequency and, in late cases, a contracted bladder (79) . the intravenous urogram is abnormal in most cases. early findings are pyelonephritis with calyceal blunting and calyceal-interstitial reflux. later, papillary cavities may be seen, indicating papillary necrosis. ureteric strictures, focal calcification, hydronephrosis, and cavitation may also be seen. renal function is usually well preserved, and hypertension is uncommon. in some cases, either the infection itself or reactions to the chemotherapeutic agents may result in renal failure with evidence of an interstitial nephritis (79) (80) (81) . classic symptomatic renal tb is a late and uncommon complication in children, rarely occurring less than 4 or 5 years after the primary infection, and therefore is most commonly diagnosed after adolescence (78, 80) . adult studies have shown that 26-75% of renal tb coexists with active pulmonary tb and 6-10% of screened sputumpositive pulmonary tb patients have renal involvement. the diagnosis is established by isolation of mycobacteria from the urine or by the presence of the characteristic clinical and radiographic features in a child with current or previous tb. renal tb is treated with drug regimens similar to those used for other forms of tb, with isoniazid, rifampicin, pyrazinamide and ethambutol administered initially for 2 months, and isoniazid and rifampicin then continued for a further 7-10 months. late scarring and urinary obstruction may occur in cases with extensive renal involvement, and such patients should be followed by ultrasonography or intravenous urogram. mycobacteria, both m. tuberculosis and atypical mycobacteria, have also emerged as important causes of opportunistic infection in immunocompromised patients undergoing dialysis and in patients undergoing renal transplantation. the possibility of mycobacterial disease must be considered in patients with fever of unknown origin or unexplained disease in the lungs or other organs. results of the mantoux test are often negative, and diagnosis depends on maintaining a high index of suspicion and isolating the organism from the infected site. renal involvement has been well documented in both congenital and acquired syphilis, with an estimated occurrence of 0.3% in patients with secondary syphilis and up to 5% in those with congenital syphilis (82, 83) . the most common manifestation of renal disease in congenital syphilis is the nephrotic syndrome, with proteinuria, hypoalbuminemia, and edema. in some patients, hematuria, uremia, and hypertension may be seen. the renal disease is usually associated with other manifestations of congenital syphilis, including hepatosplenomegaly, rash, and mucous membrane findings. nephritis in congenital syphilis is usually associated with evidence of complement activation, with depressed levels of clq, c4, c3, and c5. histologic findings are a diffuse proliferative gn or a membranous nephropathy. the interstitium shows a cellular infiltrate of polymorphonuclear and mononuclear cells (84) . immunofluorescent microscopy reveals diffuse granular deposits of igg and c3 along the glomerular basement membrane (gbm). mesangial deposits may also contain igm. on electron microscopy, scattered subepithelial electron-dense deposits are seen, with fusion of epithelial cell foot processes (84) . good evidence exists that renal disease is due to an immunologically mediated reaction to treponemal antigens. antibodies reactive against treponemal antigens can be eluted from the glomerular deposits, and treponemal antigens are present in the immune deposits. treatment of both congenital and acquired syphilis with antibiotics results in rapid improvement in the renal manifestations (82, 84) . renal involvement is surprisingly rare in mycoplasma pneumoniae infection considering the prevalence of this organism and its propensity to trigger immunologically mediated diseases such as erythema multiforme, arthritis, and hemolysis. acute nephritis associated with mycoplasma infection may occur 10-40 days after the respiratory tract infection (85, 86) . renal histopathologic findings include type 1 mpgn, proliferative endocapillary gn, and minimal change disease (87) . antibiotic treatment of the infection does not appear to affect the renal disease, which is self-limited in most cases (85, 86) . since its recognition in 1976, legionnaires' disease, caused by legionella pneumophila, has emerged as an important infectious diseases and the kidney cause of pneumonia. the disease most commonly affects the elderly but has been reported in both normal and immunocompromised children (97, 98) . renal dysfunction occurs in a minority of patients (98) . patients who develop renal impairment present with oliguria and rising urea and creatinine levels. they are usually severely ill, with bilateral pulmonary infiltrates, fever, and leukocytosis. shock may be present, and the renal impairment has been associated with acute rhabdomyolysis with high levels of creatine phosphokinase and myoglobinuria. renal histologic examination usually shows a tubulointerstitial nephritis or acute tubular necrosis (98, 99) . the pathogenesis of the renal impairment is uncertain, but the organism has been detected within the kidney on electron microscopy and immunofluorescent studies, which suggests a direct toxic effect. myoglobinuria and decreased perfusion may also be contributing factors, however. mortality has been high in reported cases of legionnaires' disease complicated by renal failure. treatment is based on dialysis, intensive care, and antimicrobial therapy with erythromycin (98) . steroid therapy may be effective for tubulointerstitial nephritis (99) . the rickettsial diseases are caused by a family of microorganisms that have characteristics common to both bacteria and viruses and that cause acute febrile illnesses associated with widespread vasculitis. with the exception of q fever, all are associated with erythematous rashes. there are four groups of rickettsial diseases: 1. the typhus group includes louse-borne and murine typhus, spread by lice and fleas, respectively. 2. the spotted fever group includes rocky mountain spotted fever, tick typhus and related mediterranean spotted fever and rickettsial pox, which are spread by ticks and mites, with rodents as the natural reservoir. 3. scrub typhus, which is spread by mites. 4. q fever, which is spread by inhalation of infected particles from infected animals. rickettsial diseases have a worldwide distribution and vary widely in severity, from self-limited infections to fulminant and often fatal illnesses (88) . in view of the widespread vasculitis associated with these infections, subclinical renal involvement probably occurs in many of the rickettsial diseases. however, in rocky mountain spotted fever, tick typhus, and q fever, the renal involvement may be an important component of the illness. rocky mountain spotted fever is the most severe of the rickettsial diseases (89, 90) . the onset occurs 2-8 days after the bite of an infected tick. high fever develops initially, followed by the pathognomonic rash, which occurs between the second and sixth days of the illness. the rash initially consists of small erythematous macules, but later these become maculopapular and petechial, and in untreated patients, confluent hemorrhagic areas may be seen. the rash first appears at the periphery and spreads up the trunk. involvement of the palms and soles is a characteristic feature (88) . headache, restlessness, meningism, and confusion may occur together with other neurologic signs. cardiac involvement with congestive heart failure and arrhythmia are common. pulmonary involvement occurs in 10-40% of cases. infection is associated with an initial leucopenia, followed by neutrophil leukocytosis. thrombocytopenia occurs in most cases. histopathologically, the predominant lesions are in the vascular system (91) . rickettsiae multiply in the endothelial cells, which results in focal areas of endothelial cell proliferation, perivascular mononuclear cell infiltration, thrombosis, and leakage of red cells into the tissues. the renal lesions involve both blood vessels and interstitium, and acute tubular necrosis may occur. acute gn with immune-complex deposition has been reported (92) , but in most cases the pathology appears to be a direct consequence of the invading organism on the renal vasculature (90, 93) . renal dysfunction is an important complication of rocky mountain spotted fever. elevation of urea and creatinine levels occurs in a significant proportion of cases, and acidosis is common. prerenal renal failure caused by hypovolemia and impaired cardiac function may respond to volume replacement and inotropic support, but acute renal failure may subsequently occur, necessitating dialysis. rocky mountain spotted fever is diagnosed by the characteristic clinical picture, the exclusion of disorders with similar manifestations (e.g., measles, meningococcal disease, and leptospirosis), and detection of specific antibodies in convalescence. culture of rickettsia rickettsii, immunofluorescent staining, and polymerase chain reaction (pcr) testing of blood and skin biopsy specimens are available only in reference laboratories. antibiotics should be administered in suspected cases without awaiting confirmation of the diagnosis (93) . doxycycline is the drug of choice for children of any age. chloramphenicol is also effective (94) . intensive support of shock and multiorgan failure may be required in severe cases, and peritoneal dialysis or hemodialysis may be required until renal function returns. before the advent of specific therapy, mortality was 25%. today the overall mortality in the united states is still 5-7%. death predominantly occurs in cases in which the diagnosis is delayed. q fever is caused by coxiella burnetii and has a worldwide distribution, with the animal reservoir being cattle, sheep, and goats. human infection follows inhalation of infected particles from the environment. the clinical manifestations range from an acute self-limited febrile illness with atypical pneumonia to involvement of specific organs that causes endocarditis, hepatitis, osteomyelitis, and central nervous system disease (95) . proliferative gn may be associated with either q fever endocarditis, rhabdomyolysis or a chronic infection elsewhere in the body (96) . renal manifestations range from asymptomatic proteinuria and hematuria to acute renal failure, hypertension, and nephrotic syndrome. renal histologic findings are those of a diffuse proliferative gn, focal segmental gn, or mesangial gn. immunofluorescent studies reveal diffuse glomerular deposits of igm in the mesangium, together with c3 and fibrin. c. burnetii antigen has not been identified within the renal lesions. treatment of the underlying infection may result in remission of the renal disease, but prolonged treatment may be required for endocarditis. tetracycline has been used in conjunction with rifampicin, co-trimoxazole, or a fluoroquinolone. nephritis has been reported in association with the presence of a wide range of microorganisms that cause chronic or persistent infection (> table 52 -2) (63, 100). it is likely that any infectious agent that releases foreign antigens into the circulation, including those of very low virulence, can cause renal injury either by deposition of foreign antigens in the kidney or by the formation of immune complexes in the circulation, which are then deposited within the kidney. nephritis is most commonly seen in association with intravascular infections such as sbe or infected ventriculoatrial shunts, but it is also seen after focal extravascular infections; ear, nose, and throat infections; and abscesses. renal involvement is one of the diagnostic features of bacterial endocarditis. virtually all organisms that cause endocarditis also produce renal involvement ( > table 52 -2). although endocarditis caused by bacteria is the most common and is readily diagnosed by blood culture (100), unusual but important causes of culture-negative endocarditis include q fever (101) and legionella infection (102) . in the immunocompromised individual, opportunistic pathogens such as fungi and mycobacteria are important causes. the usual renal manifestations of sbe are asymptomatic proteinuria, hematuria, and pyuria. loin pain, hypertension, nephrotic syndrome, and renal failure may occur in more severe cases. the renal lesions occurring in endocarditis are variable, and focal embolic and immune-complex-mediated features may coexist (100, 103, 104) . embolic foci may be evident as areas of infarction, intracapillary thrombosis, or hemorrhage. more commonly, there is a focal necrotizing or diffuse proliferative gn. immunofluorescent studies show glomerular deposits of igg, igm, iga, and c3 along the gbm and within the mesangium. electron microscopy reveals typical electron-dense deposits along the gbm and within the mesangium (100, 103, 104) . early reports suggested that the renal lesions were caused by microemboli from infected vegetations depositing in the kidney, a hypothesis supported by the occasional presence of bacteria within the renal lesions. most subsequent evidence, however, indicates that immunologic mechanisms rather than emboli are involved in the pathogenesis in most cases: bacteria are rarely found within the kidney, and renal involvement occurs with lesions of the right side of the heart, which would not be likely to embolize to the kidney. immune complexes containing bacterial antigens are present in the circulation, and both bacterial antigens and bacteria-specific antibodies can be demonstrated within the immune deposits in the kidney. serum c3 level is usually low, and complement can be found within both the circulating and the deposited immune complexes. these features all support an immune-complex-mediated pathogenesis of the renal injury (100, 103, 104) . treatment of the endocarditis with antibiotics usually results in resolution of the gn and is associated with the disappearance of immune complexes from the circulation and return of c3 levels to normal. the prognosis of the renal lesions in sbe generally depends on the response of the underlying endocarditis to antibiotics or, in cases of antibiotic failure, to surgical removal of the infective vegetations (105) . in patients previously treated by shunting for hydrocephalus, there is a well-documented association of gn with infected ventriculoatrial shunts. this condition is another example of an immune-complex nephritis similar to that seen in endocarditis (106) . coagulase-negative staphylococci are the causative organisms in 75% of cases. the clinical and pathologic findings are similar to those in sbe. presenting features are proteinuria, hematuria, and pyuria, and they may progress to renal failure. immune complexes containing the bacterial antigens and complement are present in the serum, and c3 is depressed. histologic findings are those of a diffuse mesangiocapillary gn. immunofluorescent microscopy demonstrates deposits of immunoglobulin and c3 along the gbm, and bacterial antigen can be demonstrated in the renal lesions (107) . the prognosis for the renal lesion is good if the infection is treated early. this usually involves removal of the infected shunt and administration of appropriate antibiotics (106, 108) . the possible progression to end-stage renal disease requires frequent nephrologic monitoring of patients with ventriculoatrial shunts (106) . there are a few reports in the literature of a similar renal complication occurring in chronic infection of ventriculoperitoneal shunts. gn has been reported after chronic abscesses (63), osteomyelitis, otitis media, pneumonia, and other focal infections ( > table 52 -2). acute renal failure has been the presenting feature of focal infections in various sites, including the lung, pleura, abdominal cavity, sinuses, and pelvis. many different organisms have been responsible, including s. aureus, pseudomonas, e. coli, and proteus species. this is probably another example of immunecomplex gn. c3 level is decreased in approximately onethird of reported cases, and immunofluorescent studies reveal diffuse granular deposits of c3 in the glomeruli of all reported instances, with a variable presence of immunoglobulin. the renal lesion is that of mpgn and crescentic nephritis. the renal outcome is reported to be good with successful early treatment of the underlying infection. the role of viral infections in the causation of renal disease has been less well defined than that of bacterial infections. clearly defined associations of renal disease have been made with hepatitis b virus (hbv), hepatitis c virus (hcv), hiv, and hantaviruses, but the role of most other viruses in the pathogenesis of renal disease is not clearly defined. most viruses causing systemic infection may trigger immunologically mediated renal injury. with increasing application of molecular techniques, it may be that a significant proportion of gns currently considered to be idiopathic will ultimately be shown to be virus induced. in children with immunodeficiency states and those undergoing renal transplantation, viruses such as cytomegalovirus (cmv) and polyoma virus have been recognized to be associated with nephropathy. since the discovery of hepatitis b surface antigen (hbsag) in 1964, hepatitis virus has been shown to infect more than 5% of the world's population and is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma worldwide. some 350 million people have hbsag in the circulation (who figures). the infection is most common in africa and the orient, where it is acquired in childhood by vertical transmission from infected mothers or by horizontal transmission from other children or adults. in developed countries, transmission in adults occurs more often by blood product exposure, sexual contact, or intravenous drug use. the epidemiology of hbv infection in children is changing following the widespread use of effective vaccination at birth, in both developed and developing countries. hbv is a complex dna virus with an outer surface envelope (hbsag) and an inner nucleocapsid core containing the hepatitis b core antigen (hbcag), dna polymerase, protein kinase activity, and viral dna. incomplete spherical and filamentous viral particles consisting solely of hbsag are the major viral products in the circulation and may be present in concentrations of up to 1014 particles per ml of serum. hepatitis b e antigen (hbeag) can be released from hbcag by proteolytic treatment and may be found in the circulation either free or complexed to albumin or igg antibodies. the presence of hbeag correlates with the presence of complete viral particles and the infectivity of the individual (109) . infection with hbv may result in either a self-limited infectious hepatitis followed by clearance of the virus and complete recovery, or a chronic or persistent infection in which the immune response is ineffective in eliminating the virus. chronic hbv infection with continued presence of viral antigens in the circulation caused by an ineffective host immune response provides the best-documented example of immunologically mediated renal injury caused by persistent infection (110) . development of chronic hbv infection is positively associated with infection at a younger age, particularly in infancy, where the rate of chronic infection is up to 90%. in contrast, the likelihood of an acute, symptomatic illness increases with age, to a level where approximately 40% of infections are symptomatic in children above 15 years. in the early prodromal phase of hbv hepatitis, before the onset of jaundice, some patients develop fever, maculopapular or urticarial rash, and transient arthralgias or arthritis. occasionally, proteinuria, hematuria, or sterile pyuria are observed. the syndrome usually lasts 3-10 days and often resolves before the onset of jaundice (110, 111) . there have been no histologic studies of the renal changes during this prodromal period. since 1970, numerous reports have linked hbv infection with polyarteritis nodosa (pan). most of these cases have been in adults, but the disorder has also been reported in children (112, 113) , where it is estimated that approximately one third of pan cases are caused by hbv (114) . hbv pan appears to be uncommon in africa and the orient, where infection is usually acquired in childhood, and has declined in incidence following the introduction of hbv vaccination (115) . hbv pan presents weeks to months after a clinically mild hepatitis but may occasionally predate the hepatitis. after a prodromal illness, frank vasculitis affecting virtually any organ appears. abdominal pain, fever, mononeuritis multiplex, and pulmonary and renal involvement may occur. the renal involvement may appear as hypertension, hematuria, proteinuria, or renal failure (see chapter 61) . laboratory investigations reveal a florid acute-phase response, leukocytosis, and anemia. transaminase levels are usually elevated, and hbsag is present in the circulation. the pathology consists of focal inflammation of small and medium-sized arteries, with fibrinoid necrosis, leukocyte infiltration, and fibrin deposition. renal pathology may be limited to the medium-sized arteries or may coexist with gn (110, 116) . circulating immune complexes containing hbsag and anti-hbs antibodies are usually present in the circulation (110, 116) . c3, c4, and total hemolytic complement levels are depressed. hbsag, igg, and igm antibodies to hbv and c3 have been identified by immunofluorescence in the blood vessels (116) . a positive anca excludes hbv-pan (115) . although most evidence suggests that the pathogenesis involves an immunecomplex-mediated vasculitis, autoantibody or cell-mediated vascular injury may coexist. if the condition is untreated, the mortality is high (112) . most studies suggest that steroids or immunosuppressants help to suppress the vasculitis but potentially predispose to chronic infection . (110, 112) . successful treatment of hepatitis b-associated pan with nucleoside analogues such as lamivudine or newer anti-viral drugs, either alone or in combination with interferon-alpha and conventional immunosuppressive therapy, has been reported (117) (118) (119) . hbv is now the major cause of membranous gn (mgn) in children worldwide. the proportion of patients with mgn caused by hbv is directly related to the incidence of hbsag in the population, with 80-100% of all cases of mgn in some african and oriental countries being associated with hbv (110, 120) (see chapter 26) . hbv mgn usually presents in children aged 2-12. there is a striking male predominance; in the united states, 80% of patients are males (121) . the virus is usually acquired by vertical transmission from infected mothers or horizontally from infected family members. unlike adults with hbv mgn, children do not usually have a history of hepatitis or of active liver disease, but liver function test results are generally mildly abnormal. liver biopsy specimens may show minimal abnormalities, chronic persistent hepatitis, or (occasionally) more severe changes (121) . the renal manifestations are usually of proteinuria, nephrotic syndrome, microscopic hematuria, or (rarely) macroscopic hematuria. hypertension occurs in less than 25% of cases, and renal insufficiency is rare. hbsag and hbcag are usually present in the circulation, and hbe antigenemia is seen in a high proportion of cases. occasionally, hbsag may be found in the glomeruli but is absent from the circulation. c3 and c4 levels are often low, and circulating immune complexes are found in most cases. immunohistologic study reveals deposits of igg and c3 and (less commonly) igm and iga in subepithelial, subendothelial, or mesangial tissue. hbv particles may be seen on electron microscopy, and all the major hepatitis b antigens, including hbsag, hbcag, and hbeag, have been localized in the glomerular capillary wall on immunofluorescence. immunologic deposition of hbv and antibody in the glomerular capillary wall is clearly involved in the glomerular injury, but the underlying immunologic events are incompletely understood (110, 122) . passive trapping of circulating immune complexes may be involved, but the circulating immune complexes containing hbsag are usually larger than would be expected to penetrate the basement membrane. hbsag and hbcag are anionic and are therefore unlikely to penetrate the glomerular capillary wall. in contrast, hbeag forms smaller complexes with anti-hbe antibodies and may readily penetrate the gbm. this may explain the observation that hbeag in the circulation frequently correlates with the severity of the disease (110) . an alternative mechanism for immunemediated glomerular injury is the trapping of hbv antigens by antibody previously deposited in the kidney. anti-hbe antibodies are cationic and may readily localize in the glomerulus and subsequently bind circulating antigen and complement. the third possibility is that the depositions of hbv and antibodies are consequences of glomerular injury by cellular mechanisms or autoantibodies. little evidence supports this view at present (110) . a transgenic mouse model of hbv-associated nephropathy has been developed, in which hbsag and hbcag is expressed in liver and kidney, particularly tubular epithelial cells, without viral replication. in these mice, gene expression analysis revealed upregulation of acute-phase proteins, particularly c3, although measurable serum c3 levels were reduced. this supports the notion that local persistent expression of hbv viral proteins contributes to hbv-associated nephropathy (123) . hbv infection has been associated with a variety of other forms of gn in both adults and children. in one small series in children, mpgn was found to be equal in incidence to mgn in the spectrum of hbv-associated gns (124) . both mpgn and mesangial proliferative gn may be triggered by hbv. in several countries where hbv is common, the proportion of patients with these forms of nephritides who test positive for hbv greatly exceeds the incidence of positivity in the general population (122) . as with mgn and hbv-associated pan, circulating immune complexes and localization of hbv antigens in the glomeruli have been reported in both mpgn and mesangial proliferative gn, and it is likely that similar mechanisms are occurring (110, 125) . several other forms of gn have been associated with hbv, including iga nephropathy, focal glomerulosclerosis, crescentic nephritis, and systemic lupus erythematosus, but the evidence for these associations is less consistent than for the entities discussed earlier (125) . hbv is normally cleared as a result of cell-mediated responses in which cytotoxic t cells and natural killer cells eliminate infected hepatocytes. it is not surprising, therefore, that the administration of steroids and immunosuppressive agents either may have no effect on hbv disease or may increase the risk of progressive disease (126) . children with hbv mgn have a good prognosis, and two-thirds undergo spontaneous remission within 3 years of diagnosis. steroid therapy does not appear to provide any additional benefit (110, 120, 110) . antiviral therapy with pegylated interferon-alpha and lamivudine shows promise in facilitating clearance of hbv, and in some cases, elimination of the infection with antiviral therapy in both children and adults is associated with improvement or resolution of the coexisting renal disease. there is considerable effort being put into the development of newer anti-viral agents which avoid the common problems of resistance associated with lamivudine (127-129). hcv is an enveloped, single-stranded rna virus of approximately 9.4 kb in the flaviviridae family. there are six major hcv genotypes. hepatitis c is a common disease affecting approximately 400 million people worldwide. in the united states, 4.1 million persons are estimated to be anti-hcv positive, and 3.2 million may be chronically infected (130) . an estimated 240,000 children in the united states have antibody to hcv and 68,000-100,000 are chronically infected (131) . children become infected through receipt of contaminated blood products or through vertical transmission. the risk of vertical transmission increases with higher maternal viremia and maternal co-infection with hiv. acute hcv infection is rarely recognized in children outside of special circumstances such as a known exposure from an hcv-infected mother or after blood transfusion. most chronically infected children are asymptomatic and have normal or only mildly abnormal alanine aminotransferase levels. although the natural history of hcv infection during childhood seems benign in the majority of instances, the infection can take an aggressive course in a proportion of children, leading to cirrhosis and end-stage liver disease during childhood. the factors responsible for this more aggressive course are unidentified (131) . even in adults, the natural history of hcv infection has a variable course, but a significant proportion of patients will develop some degree of liver dysfunction, and 20-30% will eventually have end-stage liver disease as a result of cirrhosis. the risk of hepatocellular carcinoma is significant for those who have established cirrhosis. hepatitis c is currently the most common condition leading to liver transplantation in adults in the ''western world.'' gn has been described as an important complication of chronic infection with hcv in adults. the clinical presentation is usually of nephrotic syndrome or proteinuria, hypertension, or hematuria, with or without azotemia (132) . mpgn, with or without cryoglobulinemia, and mgn are most commonly described. isolated case reports of other, more unusual patterns of glomerular injury, including iga nephropathy, focal segmental glomerulosclerosis, crescentic gn, fibrillary gn, and thrombotic microangiopathy, have also been associated with hcv infection (132, 133) . glomerular deposition of hepatitis antigens and antibodies has been described and is believed to play a role in pathogenesis. cryoglobulinemia is a common accompaniment of gn that is associated with the depression of serum complement levels (132) . renal failure may develop in 40-100% of patients who have mpgn (132, 134) . the presence of virus-like particles as well as viral rna within the kidney sections of patients with hcv-associated glomerulopathies has been reported (135) . the diagnosis should be suspected if glomerular disease is associated with chronic hepatitis, particularly with the presence of cryoglobulins, but renal biopsy is necessary to establish a definitive diagnosis. hcv infection is relatively common in children with end-stage renal disease and is an important cause of liver disease in this population. acquisition of hcv infection continues to occur in dialysis patients because of nosocomial spread (136) . elevation of transaminase level is not a sensitive marker of infection in children and hcv enzymelinked immunosorbent assay or pcr testing should be used to increase sensitivity (137) . hcv-infected renal transplant recipients had higher mortality and hospitalization rates than other transplant recipients (138) , and hcv infection has been reported to be associated with de novo immune-mediated gn, especially type 1 mpgn, in renal allografts, resulting in accelerated loss of graft function (139, 140) . no large randomized, controlled trials of treatment of children with chronic hepatitis c have been performed, although one study (peds-c) is currently recruiting patients into a trial of pegylated interferon +/ã� ribavirin (141) . small heterogeneous studies of interferon monotherapy have reported sustained virologic response rates of 35-40% (131) . in adults, improvement of proteinuria and renal function often follows interferon-alpha treatment (132, 134) , but relapses are common after cessation of treatment. combination of interferon with ribavirin in infectious diseases and the kidney patients with chronic liver disease has been shown to increase the rate of sustained response in these patients (142) . as yet, however, there are few data regarding the use of combination therapy with interferon and ribavirin in children. moreover, interferon-alpha therapy is associated with acute or subacute renal failure in more than one-third of the patients with renal transplants (143) . hepatitis c may be complicated by systemic mixed cryoglobulinemic (mc) vasculitis, and in some cases by a polyarteritis nodosa (pan)-type non-cryoglobulinemic vasculitis (144) . treatment with interferon-a (ifn-a) and ribavirin mostly is associated with an improvement of vasculitic symptoms. in some cases, exacerbation and rarely new onset of vasculitis of the peripheral nervous system have been described after this treatment. in fulminant cases immunosuppressive therapy with steroids, and cyclophosphamide, or rituximab may be needed to control life threatening vasculitis prior to antiviral treatment (144) . cytomegalovirus cmv is one of the eight human herpes viruses. transmission of the virus requires exposure to infected body fluids such as breast milk, saliva, urine, or blood. individuals initially infected with cmv may be asymptomatic or display nonspecific flu-like symptoms. after the initial infection cmv, like all herpes viruses, establishes latency for life but will be periodically excreted by an asymptomatic host. cmv replicates within renal cells, and on biopsy samples from immunocompromised hosts, viral inclusions can be visualized by light microscopy in cells of the convoluted tubules and collecting ducts (145) . glomerular cells and shed renal tubular cells may have characteristic inclusions, but clinically evident renal disease is rare and is seen virtually only in immunocompromised or congenitally infected children (145, 146) . the clinical manifestations of cmv-induced renal disease in congenitally infected infants are variable and range from asymptomatic proteinuria to nephrotic syndrome and renal impairment. in congenital cmv infection, histologic changes of viral inclusions commonly occur in the tubules. in addition, proliferative gn has been reported, with evidence on electron microscopy of viral immune deposits in glomerular cells (146, 147) . in cmv-infected immunocompromised patients, immunecomplex gn has been documented with mesangial deposits of igg, iga, c3, and cmv antigens within glomeruli. eluted glomerular immunoglobulins have been shown to contain cmv antigens (148) . cmv is the most common viral infection after kidney transplantation. experience with pediatric kidney transplant recipients suggests a 67% incidence of cmv infection (149) . the direct and indirect effects of cmv infection result in significant morbidity and mortality among kidney transplant recipients. cmv-negative patients who receive a cmv-positive allograft are at risk for primary infection and graft dysfunction. patients who are cmv seropositive at the time of transplantation are also at risk of reactivation and superinfection. tubulointerstitial nephritis is a well-characterized pathologic feature of renal allograft cmv disease, which can be difficult to distinguish from injury caused by rejection. histologic evidence of endothelial cell injury and mononuclear cell infiltration in the glomeruli has been reported (148) . cmv glomerular vasculopathy in the absence of tubulointerstitial disease, causing renal allograft dysfunction, has also been reported (150) . beyond the acute allograft nephropathy associated with cmv viremia, cmv is known to cause chronic vascular injury. this may adversely affect the long-term outcome of the allograft and may be the explanation for the observed association with chronic allograft nephropathy (151) . newer techniques for rapidly diagnosing cmv infection are becoming widely available and include shell vial culture, pp65 antigenemia assay, pcr, and the hybridcapture rna-dna hybridization assay for qualitative detection of cmv pcr. quantitative plasma pcr testing (pcr viral load) is increasingly used for diagnosis and monitoring of cmv viremia in renal transplant recipients. antiviral agents that have been shown to be effective against cmv include ganciclovir, valganciclovir, foscarnet, and cidofovir. ganciclovir remains the drug of choice for treating established disease. intravenous ganciclovir therapy is preferred in children because of the erratic absorption of oral ganciclovir. major limitations of ganciclovir therapy are the induction of renal tubular dysfunction and bone marrow toxicity, principally neutropenia and thrombocytopenia. dosage adjustments are necessary for recipients with renal dysfunction. oral valganciclovir is now used for cmv prophylaxis post-transplant (152) . use of other antiviral agents such as foscarnet and cidofovir is limited because of nephrotoxicity and difficulty of administration. a number of reports have demonstrated the effectiveness of high-titer cmv immune globulin therapy in reducing severe cmv-associated disease when used in combination with ganciclovir (149, 153) . the association of varicella with nephritis has been known for more than 100 years since henoch reported on four children with nephritis that occurred after the appearance of varicella vesicles. varicella, however, is rarely associated with renal complications (154) . in fatal cases with disseminated varicella and in the immunocompromised individual, renal involvement is more common. cases in which varicella infection caused gn in renal transplant recipients have been reported (155) . histologic findings in fatal cases include congested hemorrhagic glomeruli, endothelial cell hyperplasia, and tubular necrosis. in mild and nonfatal cases and in non-immunocompromised individuals, varicella is occasionally associated with a variety of renal manifestations, ranging from mild nephritis to nephrotic syndrome and acute renal failure (156) . histologic findings include endocapillary cell proliferation, epithelial and endothelial cell hyperplasia, and inflammatory cell infiltration (154) . rapidly progressive nephritis has also been reported. immunohistochemical studies reveal glomerular deposition of igg, igm, iga, and c3. on electron microscopy, granular electron-dense deposits have been found in the paramesangial region, and varicella antigens may be deposited in the glomeruli. the features suggest an immune-complex nephritis. elevated circulating levels of igg and iga immune complexes and depressed c3 and c4 levels support this possibility (154) . fulminant disseminated varicella and varicella in immunocompromised patients should be treated with intravenous acyclovir. renal involvement is common during acute infectious mononucleosis, usually manifesting as an abnormal urine sediment, with hematuria in up to 60% of cases. hematuria, either microscopic or macroscopic, usually appears within the first week of the illness and lasts for a few weeks to a few months. proteinuria is usually absent or low grade. more severe renal involvement with proteinuria, nephrotic syndrome, or acute nephritis with renal failure is much less common. acute renal failure may be seen during the course of fulminant infectious mononucleosis with associated hepatic failure, thrombocytopenia, and encephalitis. it is usually caused by interstitial nephritis that is likely the result of immunopathologic injury precipitated by epstein-barr virus (ebv) infection. however, the identification of ebv dna in the kidney raises the possibility that direct infection might play a role (157) . the renal involvement must be distinguished from myoglobinuria caused by rhabdomyolysis, which may occur in infectious mononucleosis, and from bleeding into the renal tract as a result of thrombocytopenia. renal histologic findings in ebv nephritis are an interstitial nephritis with mononuclear cell infiltration and foci of tubular necrosis. glomeruli may show varying degrees of mesangial proliferation. on immunohistochemical study, ebv antigens are seen in glomerular and tubular deposits. the prognosis for complete recovery of renal function is good. treatment with corticosteroids may have a role in the management of ebv-induced acute renal failure and may shorten the duration of renal failure (158) . ebv-associated post-transplantation lymphoproliferative disease is a recognized complication in renal transplant recipients. latent infection of ebv in renal proximal tubular epithelial cells has recently been described as causing idiopathic chronic tubulointerstitial nephritis (159) . the herpes simplex virus (hsv) causes persistent infection characterized by asymptomatic latent periods interspersed with acute relapses. as with other chronic and persistent infections, immunologically mediated disorders triggered by hsv are well recognized, and it is perhaps surprising that hsv has rarely been linked to nephritis. acute nephritis and nephrotic syndrome have been associated with herpes simplex encephalitis. renal histology shows focal segmental gn with mesangial and segmental deposits of igm, c3, and hsv antigens. as with other herpes viruses, hsv has been suggested as a trigger for iga nephritis, mpgn, and membranous nephropathy. elevated levels of hsv antibodies have been reported in patients with a variety of forms of gn, but no conclusive evidence exists of an etiologic role for hsv (160) . adenovirus and enterovirus, are unrelated ubiquitous pathogens that infect large proportions of the population annually and yet are rarely associated with renal disease. the literature contains scattered reports of acute nephritis after infection with each of these viruses. adenovirus is a major cause of hemorrhagic cystitis and was implicated as the cause of hemorrhagic cystitis in 23-51% of children with this disorder (161) . boys are affected more often than girls, and hematuria persists for 3-5 days. microscopic hematuria, dysuria, and frequency may occur for longer periods. adenovirus types 11 and 21 are the usual strains isolated. picornaviruses, including enteroviruses, echovirus and coxsackieviruses, have been linked with acute nephritis and acute renal failure associated with rhabdomyolysis. coxsackie b virus can be isolated in urine. direct infection of kidney cells is supported by in vitro work demonstrating lytic infection of human podocyte and proximal tubular epithelial cell cultures, although different strains exhibit variable degrees of nephrotropism. renal damage in vivo may have both a direct lytic mechanism and an immune-complex basis (162) . in the newborn, enteroviruses cause fulminant disease with dic, shock, and liver failure, and acute renal failure may occur. renal involvement from measles virus is uncommon, although measles virus can be cultured from the kidney in fatal cases. an acute gn has been reported to follow measles with evidence of immune deposits containing measles virus antigen within the glomeruli. the nephritis is generally self-limiting (163) . mild renal involvement is common during the acute phase of mumps infection. one-third of children with mumps have abnormal urinalysis results, with microscopic hematuria or proteinuria. mumps virus may be isolated from urine during the first 5 days of the illness, at a time when urinalysis findings are abnormal. plasma creatinine concentrations usually remain normal, despite the abnormal urine sediment, but more severe cases in adults have been associated with evidence of acute nephritis with impaired renal function. renal biopsy specimens demonstrate an mpgn with deposition of iga, igm, c3, and mumps virus antigen in the glomeruli, which suggests an immune-complex-mediated process (164) . despite the increasing availability of interventions to limit vertical hiv transmission, an estimated 1,500 children renal involvement in hiv infection was first described in 1984 in adults (165) (166) (167) and in children (168) , and renal involvement occurs in 2-15% of hiv-infected children in the united states (169) (170) (171) . since the development of highly active antiretroviral therapy (haart), however, the incidence of end-stage renal disease in hiv infection in both adults and children in industrialized countries has declined, but it is predicted that the dramatic decline in aids-related deaths will lead to an ageing population of hiv-infected individuals who will be at risk of non-hiv related renal problems, such that the numbers of hiv-positive esrd patients will increase in the united states (172) . hiv infection is associated with a number of renal pathologies. hiv-associated nephropathy (hivan) is a syndrome of glomerular and tubular dysfunction, which can progress to end-stage renal failure. it is discussed more fully below. glomerular syndromes other than hivan include mgn that resembles lupus nephritis and immune-complex gn, with iga nephropathy and hcv-associated mpgn being the most common forms. there have also been several case reports of amyloid kidney (171, 173, 174) . the kidneys may be affected by various other mechanisms. opportunistic infections with organisms such as bk virus (bkv) that give rise to nephropathy and hemorrhagic cystitis have been reported in association with hiv infection (175) . systemic infections accompanied by hypotension can cause prerenal failure leading to acute tubular necrosis. acute tubular necrosis has also been reported in hiv patients after the use of nephrotoxic drugs such as pentamidine, foscarnet, cidofovir, amphotericin b, and aminoglycosides. intratubular obstruction with crystal precipitation can occur with the use of sulfonamides and intravenous acyclovir. indinavir is well recognized to cause nephropathy and renal calculi (176) . mpgn associated with mixed cryoglobulinemia and thrombotic microangiopathy/atypical-cal hus in association with hiv infections have been reported (177, 178) . hivan is characterized by both glomerular and tubular dysfunction, the pathogenesis of which is not entirely known. hivan is a clinico-pathologic entity that includes proteinuria, azotemia, focal segmental glomerulosclerosis or mesangial hyperplasia, and tubulointerstitial disease (171) . in adults in the united states, there is a markedly increased risk of nephropathy among african american persons with hiv infection. this appears to be true in children as well, but the data are sparse. the spectrum of hivan seems to be coincident with the degree of aids symptomatology. it is thought that hivan can present at any point in hiv infection, but most patients with hivan have cd4 counts of less than 200 ã� 10 6 /200 cells/ml, which suggests that it may be primarily a manifestation of late-stage disease (179) . although a spectrum of clinicopathologic entities including mesangial hyperplasia, focal segmental glomerulosclerosis, minimal change disease, and systemic lupus erythematosus nephritis has been described, the classic pathologic feature of hivan is the collapsing form of focal and segmental glomerulosclerosis (180) . in the affected glomeruli, visceral epithelial cells are hypertrophied and hyperplastic, and contain large cytoplasmic vacuoles and numerous protein resorption droplets. there is microcystic distortion of tubule segments, which contributes to increasing kidney size. podocyte hyperplasia can become so marked that it causes obliteration of much of the urinary space, forming ''pseudocrescents'' (173) . capillary walls are wrinkled and collapsed with obliteration of the capillary lumina. the interstitium is edematous with a variable degree of t-cell infiltration (181) . the bowman capsule can also be dilated and filled with a precipitate of plasma protein that represents the glomerular ultrafiltrate. one of the most distinctive features of hivan, however, is the presence of numerous tubuloreticular inclusions within the cytoplasm of glomerular and peritubular capillary endothelial cells (173) . immunofluorescence testing is positive for igm and c3 in capillary walls in a coarsely granular to amorphous pattern in a segmental distribution (180, 181) . the presence of the hiv genome in glomerular and tubular epithelium has been demonstrated using complementary dna probes and in situ hybridization. proviral dna has been detected by pcr in the glomeruli, tubules, and interstitium of micro dissected kidneys from patients who had pathologic evidence of hivan, but it has also been detected in the kidneys of hiv-positive patients with other glomerulopathies (182) . a combination of both proliferation and apoptosis of renal cells may cause the loss of nephron architecture. apoptosis has been demonstrated in cells in the glomerulus, tubules, and interstitium of biopsy specimens from hiv-positive patients with focal segmental glomerulosclerosis. in addition, the role of various cytokines and growth factors, specifically transforming growth factor beta (tgf-beta), in the development of sclerosis has been studied (183, 184) . transgenic murine models provide some of the strongest evidence for a direct role of hiv-1 in the induction of hivan. these mice do not produce infectious virus but express the hiv envelope and regulatory genes at levels sufficient to re-create the hivan that is seen in humans (183) . serial deletion experiments have concluded that the nef and vpr genes are necessary though not sufficient for hivan pathogenesis. additional factors such as genetic predisposition are thought to explain the fact that african americans have a far greater likelihood of developing hivan than other racial groups, and that hivan is more likely in patients with a family history of esrd. hivan can manifest as mild proteinuria, nephrotic syndrome, renal tubular acidosis, hematuria, and/or acute renal failure (168) (169) (170) (171) . nephrotic syndrome and chronic renal insufficiency are late manifestations of hivan. children with hivan are likely to develop transient electrolytic disorders, heavy proteinuria, and acute renal failure due to systemic infectious episodes or nephrotoxic drugs. early stages of hivan can be identified by the presence of proteinuria and ''urine microcysts'' along with renal sonograms showing enlarged echogenic kidneys. urinary renal tubular epithelial cells are frequently grouped together to form these microcysts, which were found in the urine of children with hivan who had renal tubular injury (171) . advanced stages of hivan typically present with nephrotic syndrome with edema, heavy proteinuria, hypoalbuminemia, and few red or white blood cells in urinary sediments. hypertension may be present, but usually blood pressure is within or below the normal range. hivan in adults follows a rapidly progressive course, with end-stage renal disease developing within 1-4 months, but in children this rapid progression does not necessarily occur. definitive diagnosis of hivan should be based on biopsy results, and biopsy should be performed if significant proteinuria is present, because in approximately 50% of hiv-infected patients with azotemia and/or proteinuria (>1 g/24 h) who undergo renal biopsy, the specimen will have histologic features consistent with other renal diseases (179) . when available, haart should be given to children with symptomatic hiv disease. specific treatment of hivan remains controversial. several studies have looked at the role of haart, angiotensin i-converting enzyme (ace) inhibitors, steroids, and even cyclosporin with somewhat encouraging results. however, as yet no randomized case-controlled trials have been undertaken. most of the studies have been small and retrospective, and many have included patients both with and without renal biopsy-proven hivan. cyclosporin has been used to treat hivan in children with remission of nephrotic infectious diseases and the kidney syndrome (169) . similar responses have been reported to treatment with corticosteroids in various studies (185) (186) (187) (188) . ace inhibitors have been used with encouraging results (189) . the general regimen used to treat patients with hiv, including haart, should be applied to children with hivan. the dosages of some medications must be adjusted to the patients glomerular filtration. there are reports of spontaneous regression of hivan with supportive management and treatment with haart, particularly with regimes containing protease inhibitors (190) (191) (192) (193) . it should be emphasized that the improvement reported with other modalities of treatment such as corticosteroids, cyclosporin, and ace inhibitors always occurs when these agents are given in conjunction with antiretroviral therapy. the kidneys of transgenic mice have been found to have elevated levels of tgf-beta messenger rna and protein (184) . furthermore, gene expression analysis on tubular epithelial cells from a patient with hivan found upregulation of several inflammatory mediator genes downstream of interleukin 6 and of the transcription factor nfkb (194) . several other therapeutic options have been suggested, aimed specifically at the presumed role of tgfbeta in the pathogenesis of hivan. treatment directed at its synthesis using gene therapy to block tgf-beta gene expression is being explored. therapy directed at decreasing the activity of tgf-beta using anti-tgf-beta antibodies or other inhibitory substances is also an area of investigation. in addition, blocking renal receptors for chemokines such as rantes (regulated upon derivation, normal t cell expressed and secreted), interleukin-8, and monocyte-chemoattractant protein-1 has been proposed as another possible treatment alternative (195) . in the haart era, the outlook for hiv patients with esrd has improved, but these patients fare worse than esrd patients without hiv (196) . most reports of hivinfected patients on hemodialysis have shown poor prognosis, with mean patient survival times ranging from 14-47 months. mortality is therefore still close to 50% within the first year of dialysis. in general, improved survival is associated with younger age at initiation of hemodialysis and with higher cd4 counts. access complications such as infection and thrombosis tend to occur at a higher rate in hiv-infected hemodialysis patients. cross infection with hiv in dialysis patients is very rare. no patient-topatient hiv transmission has yet been reported in a hemodialysis unit in the united states, although several such cases have occurred in south america (195, 197) . peritoneal dialysis is an alternative for hiv-infected patients. the incidence of peritonitis varies across studies, but some studies did report a higher incidence of pseudomonas and fungal peritonitis in the hiv-positive population (195) . infections with unusual organisms such as pasteurella multocida, trichosporon beigelii, and mycobacterium avium intracellulare complex have also been reported. several studies, however, have suggested that there is no significant difference between the hiv-infected and non-hiv-infected populations. of note is that virus capable of replication in vitro has been recovered from the peritoneal dialysis effluent, and it can be recoverable for up to 7 days in dialysis bags at room temperature and for up to 48 h in dry exchange tubing (195) . previously, long-term dialysis had been thought to be preferable to renal transplantation, primarily because of the concern that the immunosuppressive therapy required after transplantation could promote progression of hiv/ aids. a multicenter prospective study has been addressing these questions (198) . data so far indicate that the outcome for liver and kidney transplantation is not considerably different from patients without hiv, with good graft persistence, and a low rate of development of opportunistic infections in those with well-controlled hiv and relatively high cd4 counts (199) . the human polyoma viruses are members of the papovavirus family and have received increasing attention as pathogens in immunocompromised patients. they are nonenveloped viruses ranging in size from 45-55 nm, with a circular, double-stranded dna genome that replicates in the host nucleus. the best-known species in this genus are the bkv, the jc virus (jcv), and the simian virus sv40. bkv was first isolated from the urine of a 39-year-old man who developed ureteral stenosis 4 months after renal transplantation (200) . the name of the virus refers to the first patients initials, which is also true of jcv. bkv establishes infection in the kidney and the urinary tract, and its activation causes a number of disorders, including nephropathy and hemorrhagic cystitis. bkv-associated nephropathy has become an increasingly recognized cause of renal dysfunction in renal transplantation patients (201) (202) (203) (204) (205) . jcv establishes latency mainly in the kidney, and its reactivation can result in the development of progressive multifocal leukoencephalopathy. there are a few reports of nephropathy in association with jcv infection (see references in (206) ), but bkv poses a much bigger problem in this regard. recent studies have reported sv40 in the allografts of children who received renal transplants and in the urine, blood, and kidneys of adults with focal segmental glomerulosclerosis, which is a cause of end-stage renal disease and an indication for kidney transplantation (207) . seroprevalence rates as high as 60-80% have been reported among adults in the united states and europe. the peak incidence of primary infection (as measured by acquisition of antibody) occurs in children 2-5 years of age. bkv antibody may be detected in as many as 50% of children by 3 years of age, and in 60-100% of children by 9 or 10 years of age; antibodies wane thereafter. bkv infection may be particularly important in the pediatric transplantation population, in whom primary infection has a high probability of occurring while the children are immunosuppressed (208) . primary infection with bkv in healthy children is rarely associated with clinical manifestations. mild pyrexia, malaise, vomiting, respiratory illness, pericarditis, and transient hepatic dysfunction have been reported with primary infection. investigators hypothesize that after an initial round of viral replication at the site of entry, viremia follows with dissemination of the virus to distant sites at which latent infection is established. the most frequently recognized secondary sites of latent infection are renal and uroepithelial cells. secondary infection has been reported to cause tubulointerstitial nephritis and ureteral stenosis in renal transplantation patients. it may be that renal impairment in immunocompromised patients and in non renal solid organ transplant recipients is found to be frequently associated with bkv infection. the reported prevalence of bkv nephropathy in renal allografts is between 1 and 8% (201, 202, 205, 209, 210) . asymptomatic infection is characterized by viral shedding without any apparent clinical features. viruria, resulting from either primary or secondary infection, can persist from several weeks to years. tubulointerstitial nephritis associated with bkv in renal transplant recipients is accompanied by histopathologic changes, with or without functional impairment. ''infection'' and ''disease'' must be differentiated carefully. bkv infection (either primary or reactivated) can progress to bkv disease, but will not always do so (208) . furthermore, not all cases of bkv disease lead to renal impairment. however, infection can progress to transplant dysfunction and graft loss, although the diagnosis may be complicated by the coexistence of active allograft rejection. bkv nephritis is reported to have a bimodal distribution, with 50% of bkv-related interstitial nephritis cases occurring 4-8 weeks after transplantation and the remainder of patients developing disease months to years after transplantation (211) . allograft failure is due mainly to extensive viral replication in tubular epithelial cells leading to frank tubular necrosis (203) . although damage is potentially fully reversible early in the disease, persisting viral damage leads to irreversible interstitial fibrosis. tubular atrophy and allograft loss has been observed in 45% of affected patients (203, 212) . in most cases, bkv nephropathy in adult renal transplant recipients represents a secondary infection associated with rejection and its treatment. in children, however, primary bkv infection giving rise to allograft dysfunction may occur (208) . the definitive diagnosis of bkv nephropathy requires renal biopsy. histopathologic features include severe tubular injury with cellular enlargement, marked nuclear atypia, epithelial necrosis, denudation of tubular basement membranes, focal intratubular neutrophilic infiltration, and mononuclear interstitial infiltration, with or without concurrent tubulins. this constellation of histologic features, particularly severe tubulitis, is often misinterpreted as rejection, even by the experienced pathologist. the presence of well-demarcated basophilic or amphophilic intranuclear viral inclusions, primarily within the tubular and parietal epithelium of the bowman capsule, can help distinguish bkv disease from rejection (202, 203, 205) . additional tests such as immunohistochemistry, pcr analysis, or electron microscopy of biopsied tissue aimed at the identification of bkv may be required. a practical diagnostic approach for identifying bkv in renal transplant patients is summarized in > table 52-3. bkv infection may cause ureteral obstruction due to ureteral ulceration and stenosis at the ureteric anastomosis. bkv-associated ureteral stenosis has been reported in 3% of renal transplant patients and usually occurs between 50 and 300 days after transplantation. ulceration due to inflammation, proliferation of the transitional epithelial cells, and smooth muscle proliferation may lead to partial or total obstruction. high-level bkv replication is implicated in acute, late-onset, long-duration hemorrhagic cystitis after bone marrow transplantation (213) . there are two case reports in children of renal carcinomas arising in the transplanted kidney in association with bk virus nephropathy. it remains unclear whether infectious diseases and the kidney bk virus itself has oncogenic potential in the transplant setting, but this is possible given that the big t antigen (t-ag) expressed by polyomavirus family viruses has been shown to have the ability to disrupt chromosomal integrity (214, 215) . whether patients with asymptomatic viremia or viruria need specific therapeutic intervention is not certain. review of the literature suggests that careful reduction of immune suppression, combined with active surveillance for rejection, will result in clinical improvement. reduction in immunosuppression may precipitate episodes of acute cellular rejection, which need to be judiciously treated with corticosteroids. the outcome of bkv nephropathy is unpredictable, and stabilization of renal function may occur regardless of whether maintenance immunotherapy is altered or not (216) . some reports favor the use of cidofovir. cidofovir has important nephrotoxic side effects in the usual therapeutic dosage recommended for the treatment of cmv infection, and for bkv nephropathy a reduced dosage regime is generally used. the efficacy of cidofovir in reducing viremia has been demonstrated (see review in (210)). however, spontaneous clearance of viral infection after reduction of immunosuppression (without cidofovir) has also been reported. there are also case studies of the use of leflunamide. presence of bkv by pcr or decoy cells in urine signifies bkv replication. decoy cells are caused by infection of the urinary epithelial cells with human polyoma viruses. the nuclei are enlarged and nuclear chromatin is completely homogenized by viral cytopathic effect. positive pcr results for bkv viruria and presence of decoy cells have poor predictive value. specificity is increased if >10 cells/ cytospin along with presence of inflammatory cells. presence of antibody is usually indicative of previous infection; however, positive results for bkv dna pcr on serum signifies bk viremia. bkv pcr testing of plasma has proven to be a sensitive (100%) and specific (88%) means to identify bkv-associated nephropathy in adults. viral load has also been used to monitor infection and clearance. however, because primary infection occurs in childhood, it might not be applicable to the pediatric population. the definitive diagnosis of bkv nephropathy requires renal biopsy. histopathology might mimic rejection or drug toxicity. however, characteristic findings have been described. electron microscopy and immune staining are helpful in confirming the diagnosis. pcr assays of viral load in tubular cells have been reported to be a sensitive marker for diagnosis and monitoring. viral hemorrhagic fever involves at least 12 distinct rna viruses that share the propensity to cause severe disease with prominent hemorrhagic manifestations ( > table 52 -4) . the viral hemorrhagic fevers, widely distributed throughout both temperate and tropical regions of the world, are important causes of mortality and morbidity in many countries. most viral hemorrhagic fevers are zoonoses (with the possible exception of dengue virus), in which the virus is endemic in animals and human infection is acquired through the bite of an insect vector. aerosol and nosocomial transmissions from infected patients are important for lassa, junin, machupo, and congo-crimean hemorrhagic fevers, and marburg and ebola viruses (217) . viral hemorrhagic fevers have many clinical similarities but also important differences in their severity, major organs affected, prognosis, and response to treatment. in all viral hemorrhagic fevers, severe cases occur in only a minority of those affected; subclinical infection or nonspecific febrile illness occurs in the majority. fever, myalgia, headache, conjunctival suffusion, and erythematous rash occur in all the viral hemorrhagic fevers (218) . hemorrhagic manifestations range from petechiae and bleeding from venepuncture sites to severe hemorrhage into the gi tract, kidney, and other organs. a capillary leak syndrome, with evidence of hemoconcentration, pulmonary edema, oliguria, and ultimately shock, occurs in the most severely affected patients (218) . renal involvement occurs in all the viral hemorrhagic fevers, proteinuria is common, and prerenal failure is seen in all severe cases complicated by shock. however, in congo-crimean hemorrhagic fever and hemorrhagic fever with renal syndrome (hfrs), an interstitial nephritis, which may be hemorrhagic, is characteristic, and renal impairment is a major component of the illness. dengue is caused by a flavivirus that is endemic and epidemic in tropical america, africa, and asia, where the mosquito vector aedes aegypti is present (219). classic dengue is a self-limited nonfatal disease; dengue hemorrhagic fever and dengue shock syndrome, which occur in a minority of patients, have a high mortality if not aggressively treated with fluids. after an incubation period of 5-8 days, the illness begins with fever, headache, arthralgia, weakness, vomiting, and hyperesthesia. in uncomplicated dengue the fever usually lasts 5-7 days. shortly after onset a maculopapular rash appears, sparing the palms and the soles, and is occasionally followed by desquamation. fever may reappear at the onset of the rash. in dengue hemorrhagic fever and dengue shock syndrome, the typical febrile illness is complicated by hemorrhagic manifestations, ranging from a positive tourniquet test result or petechiae to purpura, epistaxis, and gi bleeding with thrombocytopenia and evidence of a consumptive coagulopathy. increased capillary permeability is suggested by hemoconcentration, edema, and pleural effusions (219) . in severe cases, hypotension and shock supervene, largely as a result of hypovolemia. renal manifestations include oliguria, proteinuria, hematuria, and rising urea and creatinine. acute renal failure occurs in patients with severe shock, primarily as a result of renal underperfusion. however, glomerular inflammatory changes may also occur. children with dengue hemorrhagic fever show hypertrophy of endothelial and mesangial cells, mononuclear cell infiltrate, thinning of basement membranes, and deposition of igg, igm, and c3. electron microscopy shows viral particles within glomerular mononuclear cells (220) . the diagnosis of dengue is made by isolation of the virus from blood or by serologic testing. there is no specific antiviral treatment, and management of patients with dengue shock syndrome or dengue hemorrhagic fever depends on aggressive circulatory support and volume replacement with colloid and crystalloid (221, 222) . with correction of hypovolemia, renal impairment is usually reversible, but dialysis may be required in patients with established acute renal failure. yellow fever is caused by a flavivirus, and is transmitted by mosquito bites, typically aedes species. it remains an important public health problem in africa and south america. renal manifestations are common and include albuminuria and oliguria. over the next few days after first manifestation of infection, shock, delirium, coma, and renal failure develop, and death occurs 7-10 days after onset of symptoms. laboratory findings include thrombocytopenia and evidence of hemoconcentration, rising urea and creatinine levels, hyponatremia, and deranged liver function test results. pathologic findings include necrosis of liver lobules, cloudy swelling and fatty degeneration of the proximal renal tubules, and, often, petechiae in other organs. the oliguria appears to be prerenal and is due to hypovolemia; later, acute tubular necrosis supervenes. at present, there is no effective antiviral agent for yellow fever. . congo-crimean hemorrhagic fever, first recognized in the soviet union, is now an important human disease in eastern europe, asia, and africa (223) . severely affected patients become stuporous or comatose 5-7 days into the illness, with evidence of hepatic and renal failure and shock. proteinuria and hematuria are often present. the disease is fatal in 15-50% of cases. the virus is sensitive to ribavirin, but in one small trial of i.v. ribavirin versus supportive treatment only, there was no significant improvement in outcome in the treatment group (224) . rift valley fever is found in many areas of sub-saharan africa. in humans, most infections follow mosquito bites or animal exposure. the infection may present as an uncomplicated febrile illness, with muscle aches and . (226) . clinical entities include korean hemorrhagic fever, nephropathia epidemica in scandinavia, and epidemic hemorrhagic fever in japan and china. in general, hfrs due to hantaan, porogia, and belgrade viruses is more severe and has higher mortality than that due to puumala virus (nephropathia epidemica) or seoul virus. hantaan is predominant in the far east, porogia and belgrade in the balkans, and puumala in western europe; seoul has a worldwide distribution (225) . the clinical features of the disease vary. the incubation period is 4-42 days. although hfrs occurs with the same clinical picture in children as in adults, both incidence rates and antibody prevalence rates are very low in children under 10 years of age. men of working age make up the bulk of clinical cases (226) . mild cases are indistinguishable from other febrile illnesses. in more severe cases, fever, headache, myalgia, abdominal pain, and dizziness are associated with the development of periorbital edema, proteinuria, and hematuria. there is often conjunctival injection, pharyngeal injection, petechiae, and epistaxis or gi bleeding. the most severely affected patients develop shock and renal failure. the disease usually passes through five phases: febrile, hypotensive, oliguric, diuretic, and convalescent. laboratory findings include anemia, lymphocytosis, thrombocytopenia, prolonged prothrombin and bleeding times, and elevated levels of fibrin degradation products. liver enzyme levels are elevated, and urea and creatinine levels are elevated during the oliguric phase. proteinuria and hematuria are consistent findings. the renal histopathologic findings are those of an interstitial nephritis with prominent hemorrhages in the renal medullary interstitium and renal cortex. acute tubular necrosis may also be seen. immunohistochemical analysis reveals deposition of igg and c3, and the gbm, mesangial, and subendothelial deposits may be seen on electron microscopy (227) . recovery from hantavirus-associated disease is generally complete, although chronic renal insufficiency is a rare sequela of hfrs. in mildly affected patients, the disease is self-limiting and spontaneous recovery occurs. however, in severe cases, with shock, bleeding, and renal failure, dialysis and intensive circulatory support may be required (228) . mortality rates vary depending on the strain of virus; rates are 5-15% for hemorrhagic fever and renal syndrome in china and significantly lower for the milder finnish form associated with the puumala virus strain. ribavirin is active against hantaan viruses in vitro, and clinical trials indicate that both mortality and morbidity can be reduced by treatment with this antiviral agent if it is administered early in the course of illness. dosages of 33 mg/kg followed by 16 mg/kg every 6 h for 4 days and then 8 mg/kg every 8 h for 3 days have been used (229) . lassa fever is a common infection in west africa, caused by an arenavirus, and usually manifests as a nonspecific febrile illness. in 10% of cases, a fulminant hemorrhagic disease occurs. in severe cases, proteinuria and hematuria are usually present, and renal failure may occur. ribavirin is effective in decreasing mortality. as in other hemorrhagic fevers, intensive hemodynamic support and correction of the hemostatic derangements are important components of therapy (230) . junin and machupo viruses, the agents of argentine and bolivian hemorrhagic fever, respectively, cause hemorrhagic fevers with prominent neurologic features and systemic and hemorrhagic features similar to those of lassa fever. oliguria, shock, and renal failure occur in the most severe cases. marburg and ebola viruses have been associated with outbreaks of nosocomially transmitted hemorrhagic fever. both viruses cause fulminant hemorrhagic fever. onset is with high fever, headache, sore throat, myalgia, and profound prostration. an erythematous rash on the trunk is followed by hemorrhagic conjunctivitis, bleeding, impaired renal function, shock, and respiratory failure. the mortality rate is high. renal histopathologic findings in fatal cases are of tubular necrosis, with fibrin deposition in the glomeruli. there is no specific treatment for these disorders. the important role played by a number of other recently characterized viruses is only now being recognized, as improved molecular diagnostic techniques allow identification of hitherto unrecognized viruses. two examples of recently described viruses are metapneumovirus (237) and bocavirus (238) . while both have significant prevalence, and may make an important contribution to the burden of childhood viral infection, as yet there are no reports indicative of significant renal pathology in association with these infections. influenza virus has been linked with nephritis and acute renal failure. an emerging infectious disease is avian flu, caused by highly pathogenic h5n1 strains which have hitherto been confined to an avian reservoir, and there have been several outbreaks of infection in humans, particularly in the first part of this decade. commonly, these patients develop a flu-like illness with prominent respiratory and gastrointestinal symptoms. renal failure may develop alongside multi-organ failure in the context of acute respiratory distress syndrome (231) . as yet, there is no clear correlation of degree of initial renal insufficiency, and outcome (232) . there is little data available on treatment, but based on the known resistance patterns of h5n1 strains, oseltamivir and zanamivir are the preferred agents to be used for treatment of infection with h5n1. severe acute respiratory syndrome (sars) is a newlyemerged infectious disease which was first seen in south china in 2002. it is caused by a sars coronavirus (sars cov). predominantly, it causes a viral pneumonia, with diffuse alveolar damage; it has considerable mortality (233) . renal effects are not generally significant in the pathophysiology of sars. however, sars cov has been found in kidney tissue at post-mortem (234) (235) . sars cov enters cells via angiotensin converting enzyme 2 (ace2) (236) , and it is thought that the invasion of kidney tissue reflects the virus' tropism for ace2, which is expressed on kidney cells. chronic exposure to infectious agents is a major factor in the increased prevalence of glomerular diseases in developing countries. malaria is the best-documented parasitic infection associated with glomerular disease, but other parasitic infections including schistosomiasis, filariasis, leishmaniasis, and possibly helminth infections may also induce nephritis or nephrosis. malaria is estimated to cause up to 500 million clinical cases of illness and more than 1 million deaths each year (239) . the association of quartan malaria and nephritis has been well known in both temperate and tropical zones since the end of the nineteenth century. epidemiologic studies provide the most conclusive evidence for a role of plasmodium malariae in glomerular disease (240, 241) . chronic renal disease was a major cause of morbidity and mortality in british guiana in the 1920s. the frequent occurrence of p. malariae in the blood of these patients led to detailed epidemiologic studies that implicated malaria as a cause of the nephrosis. after the eradication of malaria from british guiana, chronic renal disease ceased to be a major cause of death in that country (240) . the link between malaria and nephrotic syndrome was strengthened by studies in west africa in the 1950s and 1960s that demonstrated a high prevalence of nephrotic syndrome in the nigerian population (242) . the pattern of nephrotic syndrome differed from that in temperate climates, with an older peak age, extremely poor prognosis, and unusual histologic features. the incidence of p. malariae parasitemia in patients with the nephrotic syndrome in nigeria was vastly in excess of that occurring in the general population, whereas the incidence of plasmodium falciparum parasitemia was similar to that in the general population. the age distribution of nephrotic syndrome also closely paralleled that of p. malariae infection (242) . in some affected patients, circulating immune complexes and immunoglobulin, complement, and antigens were present in the glomeruli that were recognized by p. malariae-species antisera. there is now a view that the patterns of childhood renal disease described in the last century may no longer be representative of the current situation. the variable patterns of renal disease throughout africa may no longer reflect a dominant role for ''malarial glomerulopathy,'' and the relative causative role of tropical infections in nephropathy remains an unanswered question (243) . most patients have poorly selective proteinuria and are unresponsive to treatment with steroids or immunosuppressive agents. the characteristic lesions of p. malariae nephropathy are capillary wall thickening and segmental glomerular sclerosis, which lead to progressive glomerular changes and secondary tubular atrophy (242) . cellular proliferation is conspicuously absent. electron microscopy shows foot-process fusion, thickening of the basement membrane, and increase in subendothelial basement membrane-like material. immunofluorescent studies show granular deposits of immunoglobulin, complement, and p. malariae antigen in approximately one-third of patients. in addition to the histologic pattern, termed quartan malaria nephropathy, p. malariae infection is associated with a variety of other forms of histologic appearance, including proliferative gn and mgn (244) . although quartan malaria nephropathy has been clearly linked to p. malariae infection in nigeria, a number of studies from other regions in africa have not revealed the typical histopathologic findings described in the nigerian studies (245) . furthermore, quartan malaria nephropathy may be seen in children with no evidence of p. malariae infection or deposition of malaria antigens in the kidney. this, together with the fact that antimalarial treatment does not affect the progression of the disorder, raises the possibility that factors other than malaria might be involved in the initiation and perpetuation of the disorder. although there is undoubtedly a strong association between p. malariae infection and nephrotic syndrome on epidemiologic grounds, the direct causal link is not proven. most likely, a number of different infectious processes, including malaria, hepatitis b, schistosomiasis, and perhaps other parasitic infections that cause chronic or persistent infections and often occur concurrently in malaria areas, may all result in glomerular injury and a range of overlapping histopathologic features. the prognosis for the nephrotic syndrome in most african studies has been poor, regardless of whether the histologic findings were typical of quartan malaria nephropathy or whether p. malariae parasitemia was implicated. treatment with steroids and azathioprine is generally ineffective, and a significant proportion of patients progress to renal failure. p. falciparum appears to be much less likely to cause significant glomerular pathology. epidemiologic studies have failed to show a clear association between p. falciparum parasitemia and the nephrotic syndrome. whereas renal failure appears to be a common complication of severe malaria in adults, it seldom occurs in children. renal biopsy specimens from adult patients with acute p. falciparum infections who have proteinuria or hematuria show evidence of glomerular changes, including hypercellularity, thickening of basement membranes, and hyperplasia and hypertrophy of endothelial cells (246) . electron microscopy reveals electron-dense deposits in the subendothelial and paramesangial areas. deposits of igm, with or without igg, are localized mainly in the mesangial areas. p. falciparum antigens can be demonstrated in the mesangial areas and along the capillary wall, which suggests an immune-complex gn. the changes, generally mild and transient, are probably unrelated to the acute renal failure that may complicate severe p. falciparum infection (246) . heavily parasitized erythrocytes play a central role in the various pathologic factors (247) . renal failure occurring in severe p. falciparum malaria is usually associated with acidosis, volume depletion, acute intravascular hemolysis or heavy parasitic infection that leads to acute tubular necrosis. recent studies have confirmed an important role for volume depletion in children with severe falciparum malaria, who characteristically have evidence of tachycardia, tachypnoea, poor perfusion and in severe cases hypotension (248) . volume expansion with either colloid or crystalloid results in improvement in hemodynamic indices and reduction in acidosis (249) . there is growing evidence that volume expansion with albumin is associated with a better outcome than saline or synthetic colloids (250, 251) . treatment with antimalarials, correction of hypoglycemia and infectious diseases and the kidney electrolyte imbalance, and volume expansion reduces mortality to less than 5%. although renal failure is usually associated with infection by p. falciparum, acute renal failure has been described with plasmodium vivax infection and mixed infections (252) . the term blackwater fever refers to the combination of severe hemolysis, hemoglobinuria, and renal failure. it was more common at the start of the twentieth century in nonimmune individuals receiving intermittent quinine therapy for p. falciparum malaria. blackwater fever has become rare since 1950, when quinine was replaced by chloroquine. however, the disease reappeared in the 1990s, after the reuse of quinine because of the development of chloroquine-resistant organisms. since then, several cases have been described after therapy with halofantrine and mefloquine, two new molecules similar to quinine (amino-alcohol family) (253) . renal failure generally occurred in the context of severe hemolytic anemia, hemoglobinuria, and jaundice. the pathophysiology of the disorder is unclear; however, it appears that a double sensitization of the red blood cells to the p. falciparum and to the amino-alcohols is necessary to provoke the hemolysis. histopathologic findings include swelling and vacuolization of proximal tubules, necrosis and degeneration of more distal tubules, and hemoglobin deposition in the renal tubules. recent studies indicates a better outcome with earlier initiation of intensive care and dialysis combined with necessary changes in antimalarial medications. schistosomiasis affects 200 million people living in endemic areas of asia, africa, and south america (254) . the infection is usually acquired in childhood, but repeated infections occur throughout life. schistosoma japonicum is found only in the orient, whereas schistosoma haematobium occurs throughout africa, the middle east, and areas of southwest asia. schistosoma mansoni is widespread in africa, south america, and southwest asia. human infection begins when the cercarial forms invade through the skin, develop into schistosomula, and move to the lungs via the lymphatics or blood. they then migrate to the liver and mature in the intrahepatic portal venules, where male:female pairing takes place. the adult worm pairs then migrate to their final resting site -the venules of the mesenteric venous system of the large intestine (s. mansoni) or in the venules of the urinary tract (s. haematobium). the females release large numbers of eggs, which may remain embedded in the tissues, embolize to the liver or lungs, or pass into the feces or urine. clinical manifestations may occur at any stage of the infection. cercarial invasion may cause an intense itchy papular rash. katayama fever is an acute serum sicknesslike illness that occurs several weeks after infection, as eggs are being deposited in the tissues. deposition of the eggs in tissues results in inflammation of the intestines, fibrosis of the liver, and portal hypertension. with s. haematobium, chronic inflammation and fibrosis of the ureters and bladder may lead to obstructive uropathy (255) . renal manifestations of schistosomiasis occur most commonly in s. mansoni infection. schistosomal nephropathy usually presents with symptoms including granulomatous inflammation in the ureters and bladder, but glomerular disease (probably on an immune-complex basis) may also occur. renal disease usually occurs in older children or young adults with long-term infection, but serious disease may also occur in young children (255) . the early renal tract manifestations of schistosomiasis are suprapubic discomfort, frequency, dysuria, and terminal hematuria. in more severe cases, evidence of urinary obstruction appears. poor urinary stream, straining on micturition, a feeling of incomplete bladder emptying, and a constant urge to urinate may be severely disabling symptoms. the fibrosis and inflammation of ureters, urethra, and bladder may be followed by calcification and may result in hydroureter, hydronephrosis, and bladder neck obstruction. renal failure may ultimately develop, and there is a suspicion that squamous cell carcinoma of the bladder may be linked to the chronic infective and inflammatory process. secondary bacterial infection is common within the obstructed and inflamed urinary tract (254) . the hepatosplenic form of s. mansoni infection may be accompanied by a glomerulopathy in 12-15% of cases, manifested in the majority as nephrotic syndrome (256) . histopathologic findings include mesangioproliferative gn, focal segmental glomerulosclerosis, mesangiocapillary gn, mgn, and focal segmental hyalinosis (257) . immune complexes may be detected in the circulation of these patients, and glomerular granular deposition of igm, c3, and schistosomal antigens are seen on immunofluorescence. usually schistosoma-specific nephropathy is a progressive disease and is not influenced by antiparasitic or immunosuppressive therapy (258) , but isolated case reports of remission after treatment with praziquantel have been reported (259) . the diagnosis is confirmed by the detection of schistosoma eggs in feces, urine, or biopsy specimens. eggs are shed into the urine with a diurnal rhythm, and urine collected between 11 am and 1 pm is the most useful. urinary sediment obtained by centrifugation or filtration through a nuclepore membrane should be examined. in cases in which studies of urine and feces yield negative results in patients in whom the diagnosis is suspected, rectal biopsy specimens taken approximately 9 cm from the anus have a high diagnostic yield for both s. mansoni and s. haematobium infection. biopsy of liver or bladder may be required to establish the diagnosis. antibodies indicating previous infection can be detected using enzyme-linked immunosorbent assay or radioimmunoassay. the tests are sensitive but lack specificity and may not differentiate between past exposure and current infection. praziquantel is the drug of choice for treatment of schistosomiasis. a single oral dose of 40 mg/kg is effective in s.haematobium and s. mansoni infection and is usually well tolerated. the alternative drug for s. mansoni infection is oxamniquine. complete remission of urinary symptoms may occur in renal disease of short duration, but in late disease with extensive fibrosis, scarring, and calcification, obstructive uropathy and renal failure may persist after the infection has been eradicated. there are reports of a drastic decrease in the number of severe hepatosplenic forms of s. mansoni infection after mass treatment of the population in endemic areas with oxamniquine. this also reduced schistosomal nephropathy (256) . visceral leishmaniasis is a chronic protozoon infection characterized by fever, hepatosplenomegaly, anemia, leukopenia, and hyperglobulinemia. proteinuria and/or microscopic hematuria or pyuria have been reported in 50% of patients with visceral leishmaniasis (260) . acute renal failure in association with interstitial nephritis has also been reported (261) . renal histologic analysis in patients with visceral leishmaniasis reveals glomerular changes, with features of a mesangial proliferative gn or a focal proliferative gn, or a generalized interstitial nephritis with interstitial edema, mononuclear cell infiltration, and focal tubular degeneration. immunofluorescence reveals deposition of igg, igm, and c3 within the glomeruli, as well as electron-dense deposits in the basement membrane and mesangium on electron microscopy (260) . circulating immune complexes together with immunoglobulin and complement deposition in the glomeruli suggests an immune-complex cause. renal disease in leishmaniasis is usually mild and may resolve after treatment of the infection. renal dysfunction may be associated with treatment for visceral leishmaniasis with antimony compounds. proteinuria is more common in filarial hyperendemic regions of west africa than in nonfilarial areas. renal histologic analysis has shown a variety of different histopathologic appearances; the most common is diffuse mesangial proliferative gn with c3 deposition in the glomeruli (262) . renal biopsy specimens also demonstrate large numbers of eosinophils in the glomeruli, and microfilariae may be seen in the lumen of glomerular capillaries. filarial antigens have been detected within immune deposits within the glomeruli. echinococcus granulosus causes chronic cysts within a variety of organs. in addition, nephrotic syndrome in association with hydatid disease has been reported. membranous nephropathy, minimal change lesions, and mesangiocapillary gn have been described in association with hydatid disease (263, 264) . immunofluorescence reveals deposits of immunoglobulin, complement, and hydatid antigens within the glomeruli. remission of nephrotic syndrome has been reported with treatment by antiparasitic agents such as albendazole (263, 264) . few reports have been published of renal disease occurring in patients with trypanosomiasis. the trypanosomal antigens can induce gn in a variety of experimental animals (265) . nephrotic syndrome has occasionally been reported as a manifestation of congenital toxoplasmosis. dissemination of previously latent toxoplasma infection in patients undergoing treatment with immunosuppressive drugs has been increasingly recognized in recent years. reactivation of toxoplasmosis or progression of recently acquired primary infection should be considered in patients undergoing renal transplantation or immunotherapy for renal disease who develop unexplained inflammation of any organ. fungal infections of the kidneys and urinary tract occur most commonly as part of systemic fungal infections in patients with underlying immunodeficiency, as focal urinary tract infections in patients with obstructive lesions, or as a result of indwelling catheters. although candida infection is the most common fungal infection in both immunocompromised and non immunocompromised hosts, virtually all other fungal pathogens may invade the renal tract during severe immunocompromise. urinary infection with candida albicans is most commonly a component of systemic candidiasis in patients who are severely immunocompromised. systemic candidiasis is also seen in premature and term infants with perinatally acquired invasive candidiasis. presentation is usually with systemic sepsis, fever or hypothermia, hepatosplenomegaly, erythematous rash, and thrombocytopenia. systemic candidiasis may be seen on ophthalmologic investigation as microemboli in the retina. the first clue to the underlying diagnosis may be the presence of yeasts in the urine (266) . candida involvement of the urinary tract may affect all structures including the glomeruli, tubules, collecting system, ureters, and bladder. microabscesses may form within the renal parenchyma, and large balls of fungi may completely obstruct the urinary tract at any level. acute renal failure caused by systemic candidiasis or obstruction of the renal tracts with fungal hyphae is a wellrecognized complication of systemic candidal infection (266, 267) . indwelling catheters (which form a nidus for persistent infection) should be removed. successful treatment of non-obstructing bilateral renal fungal balls by fluconazole either alone or in combination with liposomal amphotericin b has been reported (268, 269) . in the presence of obstruction, however, percutaneous nephrostomy to relieve the obstruction with antegrade amphotericin b irrigation, coupled with systemic antifungal therapy, is the mainstay of treatment (267) . amphotericin b is the most effective antifungal agent, but it is not excreted in the urine. local irrigation via nephrostomy provides good results, however. for treatment of urinary tract candidiasis, it is usually combined with fluconazole or 5-flucytosine, both of which are excreted in high concentrations in the urine. treatment is required for weeks to months to ensure complete elimination of the fungus, and the ultimate outcome is largely dependent on whether there is a permanent defect in immunity. in 1983, levin et al. first described hemorrhagic shock and encephalopathy, which appeared to be distinct from previously recognized pediatric disorders (270) . other cases have subsequently been reported from several centers in the united kingdom, europe, israel, the united states, and australia, and the syndrome is now recognized as a new and relatively common severe childhood disorder (271) . hemorrhagic shock and encephalopathy usually affects infants in the first year of life, with a peak onset at 3-4 months of age. a prodromal illness with fever, irritability, diarrhea, or upper respiratory infection occurs 2-5 days before the onset in two-thirds of cases. affected infants develop profound shock, coma, convulsions, bleeding and evidence of dic, diarrhea, and oliguria. laboratory findings include acidosis, falling hemoglobin and platelet levels, elevated urea and creatinine levels, and elevated levels of hepatic transaminases. despite vigorous intensive care, the prognosis is poor, and most affected infants die or are left severely neurologically damaged (271, 272) . a small number of patients have been reported to survive without residual sequelae. the renal impairment appears to be largely prerenal in origin, and when aggressive volume replacement and treatment of the shock results in improved renal perfusion, rapid improvement in renal function is usually observed. in patients with profound shock unresponsive to initial resuscitation, vasomotor nephropathy supervenes and dialysis may be required. myoglobinuria in association with hemorrhagic shock and encephalopathy has been reported. following the description of the mucocutaneous lymph node 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shock and encephalopathy: reflections about a new devastating disorder that affects normal children haemorrhagic shock and encephalopathy: clinical, pathologic, and biochemical fea-mks use of protein-c concentrare, heparin, and haemodiafiltration in meningococcus-induced purpura fulminans quantitative viral load monitoring and cidofovir therapy for the management of bk virus associated nephropathy in children and adults antiviral therapy and prophylaxis for influenza in children infectious diseases and the kidney key: cord-016478-gpl0zbvd authors: barry, maura; chandra, sunandana; hymes, kenneth b. title: cytopenias in transplant patients date: 2018-12-08 journal: principles and practice of transplant infectious diseases doi: 10.1007/978-1-4939-9034-4_10 sha: doc_id: 16478 cord_uid: gpl0zbvd anemia, leukopenia, thrombocytopenia, as well as pancytopenias can be seen following solid organ transplant. varying patterns of cytopenia can be seen based on the drugs used in the posttransplant period, infections encountered by the individual, as well as the individual’s immune response and bone marrow function. the chapter discusses the main causes of anemia, leukopenia/neutropenia, and thrombocytopenia. the differential diagnosis for anemia after solid organ transplant includes hemolysis, drug toxicities, iron deficiency, infection, posttransplant lymphoproliferative disorder, graft-vs.-host disease, and hemophagocytic syndrome. etiologies for leukopenia and neutropenia include drug toxicities and infection, and etiologies for thrombocytopenia include drug toxicities, infections, autoimmune events such as immune thrombocytopenic purpura, and underlying causes such as persistent portal hypertension and splenomegaly. mismatched abo solid organ transplantation is often employed due to the shortage of transplantable organs. three different groups of abo incompatibility can be found in transplantation: minor, major, and bidirectional. complications arising from minor abo-mismatched solid organ transplants include the passenger lymphocyte syndrome (pls) [6] . recipients of minor abo-incompatible transplantation express abo antigens that are not expressed in the donor and may result in a graft-versus-host (gvh) reaction, including delayed hemolysis of recipient red blood cells [7] . passenger lymphocyte syndrome occurs when antibodies that are produced by the donor b-lymphocytes result in a primary or secondary immune response against the recipient's abo and rh antigens. the severity of hemolysis depends on the level of red cell isoagglutinins in the donor tissue that are passively transferred with the organ and the subsequent rise in antibodies in the transplant recipient that occurs 1-3 weeks posttransplant and usually resolves within 3 months posttransplant [7] . in rare instances, pls can occur due to non-abo/rh antibodies if the organ had been previously sensitized to other red cell antigens in the setting of pregnancy or transfusion [8] [9] [10] [11] . pls occurs more frequently in the heart and lung transplants and less frequently in liver and kidney transplants [7] . there are numerous drugs that are often used in the solid organ transplant setting that can cause myelosuppression, including anemia, through a variety of pathophysiologic mechanisms. a number of immunosuppressants with various pharmacologic mechanisms of action are used to prolong graft and recipient survival. the immunosuppressants mycophenolate mofetil and tacrolimus have been shown to cause anemia in renal transplant recipients [12] . one-year posttransplant, renal transplant patients with anemia who are on mycophenolate mofetil have a lower rates of survival and higher rates of cardiovascular death [13] . sirolimus, another immunosuppressant, may result in greater myelosuppression compared to mycophenolate mofetil [14] . sirolimus and calcineurin inhibitors such as tacrolimus and cyclosporine have been shown in renal and lung transplant recipients to cause hemolytic anemia, thrombotic thrombocytopenic purpura, and atypical hemolytic uremic syndrome [15] [16] [17] . the calcineurin inhibitors have been shown to cause anemia ranging from 1% to 5% in european trials to 38-47% in us trial [18] . the antimetabolite azathioprine, a purine-analog drug, can also cause cytopenias. mycophenolate mofetil, tacrolimus, azathioprine, and anti-thymocyte globulin have all shown to cause pure red cell aplasia [19, 20] . primaquine and dapsone are used for pcp treatment, and both can result in hemolysis in glucose-6-phosphatedehydrogenase-deficient patients, which is not restricted to solid organ transplant settings. in patients with low body weight or renal failure, dapsone may induce a hemolytic anemia and produce methemoglobinemia even if the g6pd levels are normal [21] [22] [23] . ribavirin and interferon can cause bone marrow suppression in liver transplant recipients who are being treated with recurrent hepatitis c virus [24] . ribavirin is used in treating respiratory syncytial virus after transplant in both the oral and inhaled formulations, both of which can cause bone marrow suppression [25] . in patients who are co-infected with human deficiency virus (hiv) and hepatitis c virus, myelosuppression can be seen with the anti-retroviral medication, azt, and the anemia can be exacerbated with the coadministration of ribavirin. the antibiotic trimethoprimsulfamethoxazole can also cause myelosuppression including anemia. valganciclovir has been reported to cause bone marrow failure in renal transplant patients who received this antiviral as prophylaxis [26] . newer immunosuppressants have been developed which allow for the sparing of steroids and calcineurin inhibitors, the latter of which can cause chronic nephropathy. these newer agents include alemtuzumab, a human anti-cd52 antibody that depletes t-and b-cells, daclizumab, a human anti-cd25 antibody that targets the il-2 alpha subunit, and anti-thymocyte globulin (atg). alemtuzumab has been reported to be associated with red cell aplasia, autoimmune hemolytic anemia, and idiopathic thrombocytopenia purpura in pancreas transplant patients [27] . iron deficiency is often overlooked in transplant patients. in renal transplant patients, those with a hematocrit of less than 30% have iron studies checked only 40% of the time [28] . perioperative bleeding and frequent phlebotomies for labo-ratory studies can contribute to iron deficiency anemia. anemia of chronic disease is also frequently seen in the transplant population due to chronic inflammation, abnormal erythropoietin production due to allograft nephropathy after renal transplant. drugs such as ace inhibitors that are often used in chronic kidney disease are also associated postkidney transplant anemia [1] . numerous infectious etiologies that can occur during the posttransplant immunosuppressed period have been shown to cause myelosuppression including anemia. parvovirus b19, a single-stranded dna virus, has been known to cause red cell aplasia with anemia, reticulocytopenia, and erythroid maturation arrest [29] . elevated parvoviral b19 titers have been found by pcr in lung transplant recipients who had anemia with other etiologies that were ruled out [30] [31] [32] . cytomegalovirus (cmv) infection as well as its first-line therapies, ganciclovir and valganciclovir, can be associated with bone marrow suppression. also tuberculosis, histoplasmosis, epstein-barr virus (ebv), human herpes virus-6, and human herpes virus-8 infections can be associated with bone marrow suppression and pancytopenia [33] . posttransplant lymphoproliferative disorder that can be seen with immunosuppressive therapy can also be associated with pancytopenia. posttransplant lymphoproliferative disorder (ptld) which includes the spectrum of infectious mononucleosis, ebvdriven polyclonal lymphocyte proliferation, and non-hodgkin's lymphoma can be seen with solid organ transplantation [29] . ptld is due to the impaired ebvspecific cytotoxic t-cell activity that allows for recipient b cells that have latent ebv infection to expand. ptld can result in bone marrow infiltration and pancytopenia, as well as cause autoimmune hemolytic anemia. the severity of ptld depends on the level of immunosuppression and usually occurs within the 1st year after transplant. graft-versus-host disease (gvhd) is rarely seen after sot and is due to the engraftment and proliferation of allograftassociated lymphocytes in the immunosuppressed transplant recipient causing an immune-mediated response toward hla-unmatched host tissue. risk factors for the development of gvhd includes the volume of lymphoid tissue that is transplanted and therefore is seen more with small bowel and liver transplants, in those over 65 years of age and with hla mismatch between donor and recipient [34, 35] . gvhd, in contrast to the development of ptld, occurs early after sot, on the order of weeks to months depending on the type of solid organ transplant. the clinical presentation usually includes fever, rash, diarrhea, and cytopenias, and diagnosis is made by histologic confirmation of affected tissue. hemophagocytic syndrome is a systemic inflammatory disease that can include the symptoms of fever, hepatosplenomegaly, lymphadenopathy, pancytopenia, rash, jaundice, cough, dyspnea, cachexia, and neurologic dysfunction and can often occur in response to a precipitant, such as infection [29] . this syndrome is a result of aberrant immune response of abnormal t-cell activation leading to hemophagocytosis by activated, nonmalignant macrophages that secrete numerous cytokines including interleukin (il)-1, il-6, il-12, and tumor necrosis factor-alpha in the bone marrow, liver, lymph nodes, and spleen, resulting in a "cytokine storm" [29] . acquired hemophagocytic syndrome has been documented in the renal, liver, heart, and pancreaskidney solid organ transplants. there have been cases of hemophagocytic syndrome due to disseminated histoplasmosis in renal transplant recipients which were diagnosed by bone marrow biopsy [36] . leukopenia can be defined as having a white blood cell (wbc) count of less than 3000-4000 cells/μl, with neutropenia defined as an absolute neutrophil count (anc) <500/ mm 3 by the infectious diseases society of america [37] . leukopenia is commonly seen after solid organ transplantation and can be caused by noninfectious and infectious etiologies. it can signal an underlying infection or disease process, such as posttransplant lymphoproliferative disorder (ptld). it also increases the risk of developing further complications such as opportunistic infection and can require reduction of immunosuppression, increasing the risk of graft rejection. while there is no data to suggest a clear independent relationship between leukopenia and graft rejection, the complications of leukopenia mentioned above provide ample reason to investigate the etiology of the decreased white cell count. solid organ transplant recipients are at risk for developing infections due to their medically induced immunodeficiency following transplant, required to prevent rejection of the transplanted organ. noninfectious causes of leukopenia include drugs that are often used in transplant settings. numerous immunosuppressants can cause leukopenia, but given their use in combination, it is difficult to elucidate each agent's individual role in incidence and management. in one retrospective study of adult kidney and pancreas transplantations, the incidence of either leukopenia or neutropenia was 58%, with the first episode occurring at a mean of 91 days posttransplant [38] . one of the most common immunosuppressants, azathioprine, is a purine analog that causes an antimetabolite effect. azathioprine may result in leukopenia in a dose-dependent manner, as well as based on the duration of treatment. the leukopenia that results from azathioprine is usually reversible upon dose-reduction or drug discontinuation. the leukopenia, often occurring late in the course of therapy, can be related to low or absent levels of s-methyl-transferase (tpmt) activity, which metabolizes 6-mercaptopurine, and can result in increased myelotoxicity [39] . drugs that result in the depletion of t cells, such as thymoglobulin and alemtuzumab, can also lead to leukopenia in 10-14% of patients [40] . the immunosuppressant, mycophenolate mofetil (mmf), reversibly and noncompetitively inhibits the enzyme, inosine monophosphate dehydrogenase, the rate-limiting enzyme for de novo purine synthesis during lymphocyte proliferation [29] . mmf can result in leukopenia in 13-35% of patients. the myelosuppression of mmf is dose-dependent and is related to the trough level of the active metabolite, mycophenolic acid; however, brief discontinuations of the drug can lead to organ rejection, especially in the era of steroid-sparing regimens [41, 42] . the calcineurin inhibitors such as cyclosporine, tacrolimus, and sirolimus can also lead to cytopenias, including leukopenia. some of these agents can cause leukopenia as one of many symptoms of infection. for example, leukopenia (and often thrombocytopenia as well) have been observed as a sign/ symptoms of infection with pathogens such as adenovirus, coronavirus, lymphocytic choriomeningitis virus (lcmv), parainfluenza, ehrlichiosis, and measles [43] [44] [45] . in areas endemic for the disease, dengue infection also causes both leukopenia and thrombocytopenia in patients after solid organ transplant [46] . fungal infections such as histoplasmosis can cause disseminated organ infiltration, with the bone marrow being a common area of involvement, resulting in decreased hematopoiesis and cytopenias [33] . parvovirus b19, much better known for its role in causing both acute and chronic anemia in solid organ transplants, is also reported to cause acute and chronic leukopenia in approximately 37.5% of solid organ as well as hematopoietic stem cell transplant recipients who develop the infection [32, 47] . an acute infection with hhv-8 can present with fever, splenomegaly, and leukopenia (as part of a pancytopenia), with bone marrow biopsy revealing hypocellularity, plasma cell infiltration, and evidence of viral infection by immunohistochemical staining [33] . a retrospective analysis of liver and kidney transplant recipients was performed to assess the relationship between leukopenia and positive hepatitis b and c serologies. the investigators found that there was no significant correlation between leukopenia and hepatitis c infection, but that the incidence of leukopenia in those with active hepatitis b infection was 7.4%. they posited that, similar to other viruses, infection with hepatitis b virus could lead to "decreased or ineffective leukocyte production in the bone marrow…shifts of cells from the circulation to the marginal blood pools… [and] also produce peripheral destruction of white blood cells due to immune and nonimmune processes" [48] . cytomegalovirus infection is the most well-known transplant-related infection to cause cytopenias, with leukopenia found in approximately 20% of infected transplant recipients and with most of the data and research conducted in kidney transplant populations [49] . infection with cmv has direct effects on the bone marrow, inhibiting hematopoiesis by affecting both the bone marrow stroma and the stem cells and hematopoietic precursors [33, 50] . cmv disease (acute symptomatic infection) is most often seen in the first 6 months, particularly during the first 3 months posttransplant, and presents with constitutional complaints such as fever, abdominal pain, diarrhea, and respiratory symptoms along with cytopenias [33, 49, 51] . however, in heart transplant patients, a subclinical infection during the 1st year where infected individuals are asymptomatic has also been associated with leukopenia, with the most significant reductions occurring in the neutrophil and monocyte populations and preservation of the lymphocyte counts [52] . an added challenge when addressing cmv infection and leukopenia results from the frequent finding that the treatments for the disease can result in further leukopenia (discussed in "noninfectious etiologies of leukopenia" section). additional diagnoses to consider when assessing the etiologies of leukopenia, as well as pancytopenia, with regard to infection are hemophagocytic syndrome (hps) which is associated with cmv, ebv, hhv-6, hhv-8, and histoplasmosis, as well as ebv-associated ptld [29, 33] . as in all cases of thrombocytopenia, when evaluating a finding of low platelets in a patient after sot, it must be determined whether the primary problem is one of impaired production in the bone marrow or if it is a matter of consumption or sequestration outside the marrow. infections and drugs are known to suppress megakaryocyte production in the marrow, such as cytomegalovirus and trimethoprimsulfamethoxazole (tmp-smx). additionally, both infections and medications as well as auto-and alloimmune processes can lead to destruction of platelets despite adequate production of megakaryocytes in the bone marrow. solid organ transplant recipients are at risk for developing infections due to their immunosuppression, and viral infections in particular are a potential contributor to the development of thrombocytopenia following solid organ transplant. detailed discussions of these infections are found in other chapters of this book, but their involvement in thrombocytopenia is discussed below. in addition to the viral infections that contribute to thrombocytopenia, it is important to remember that thrombocytopenia can be a sign of bacterial infection and sepsis most often in the context of disseminated intravascular coagulation (dic). appropriate workup to rule out infection is among the first steps in examining thrombocytopenia in a solid organ recipient. the virus of particular concern in regard to platelet count in transplant patients specifically is cytomegalovirus, though thrombocytopenia due to other viruses has also been described, often among a constellation of systemic symptoms. cytomegalovirus can cause thrombocytopenia both by decreasing production of and through destruction of platelets. studies have shown that cmv can impair megakaryocyte production in its early stages by infection of stromal cells, which interferes with growth factor production, as well as by directly infecting myeloid cells [50] , similar to cmvrelated leukopenia. the other reported etiology of thrombocytopenia from cmv is due to intravascular destruction of platelets by cmv-associated thrombotic microangiopathy (tma), with a clinical picture resembling that of thrombotic thrombocytopenic purpura (ttp)/atypical hemolytic uremic syndrome (ahus), consisting of varying degrees of coombs-negative hemolytic anemia, thrombocytopenia, acute kidney injury, fever, and neurological findings. while this etiology is more often identified as a drug-related phenomenon, particularly due to the immunosuppressants required to prevent organ rejection (see next section), there have been multiple case reports associating cmv infection as a trigger of tma in the posttransplant setting [53, 54] . this has been noted with particular frequency in the renal transplant literature, where both de novo and recurrent forms of ahus were associated with cmv infection in renal transplant recipients. however, particularly in the patients with "de novo" disease, it is possible that cmv may be directly driving the thrombotic microangiopathy, rather than solely by the complementmediated events of ahus. mechanisms thought to be underlying cmv's endothelial effects include activation of cmv-specific cytotoxic immune responses and induction of primitive endothelial dysfunction as well as direct infection of endothelial cells by cmv [55, 56] . however, some investigators question how significant a contributor cmv actually is to thrombotic microangiopathy in transplant patients. in a review of tma among lung transplant recipients by hachem and colleagues, an analysis of 24 patients who were diagnosed with tma following lung transplantation revealed that only 4 patients had evidence of cmv infection, and additionally that there were 229 incidences of cmv viremia among the 237 lung transplant patients who did not develop tma. additionally, in their univariate and multivariate analyses, neither cmv viremia nor serologic status was identified as a risk factor for tma in the study population [57] . infection with epstein-barr virus often results in conditions associated with pancytopenia, such as ptld and hemophagocytic syndrome, both of which are described in greater detail in previous sections of this chapter (see "leukopenia and anemia" sections). ebv should be considered as a possible infectious etiology during the workup of thrombocytopenia, particularly if other systemic signs or symptoms are present. other infectious etiologies that have thrombocytopenia among the constellation of presenting symptoms that have been described in organ transplant recipients include coronavirus, particularly sars, lymphocytic choriomeningitis virus (lcmv), and hhv-6, though the thrombocytopenia is unlikely to be the primary issue at presentation [58] . parvovirus b-19 and polyoma bk virus infection have also been associated with development of ahus [55, 59] . it is also important to note that chronic infection with hepatitis c can be an etiology for thrombocytopenia both in and outside the context of solid organ transplantation. the etiology of thrombocytopenia in the setting of hepatitis c infection can be due to hepatocellular damage including fibrosis and/or cirrhosis affecting thrombopoietin (tpo) production, hypersplenism due to portal hypertension, bone marrow suppression, immune dysfunction, and development of platelet autoantibodies [60] . additionally, treatment for hepatitis c with interferon is known to cause thrombocytopenia. there are numerous noninfectious etiologies of thrombocytopenia that have been identified in sot patients. many pharmacologic agents have been implicated in the development of thrombocytopenia following sot through varying mechanisms, such as tma, decreased megakaryocyte production, and auto-and allo-immune mechanisms of platelet destruction. the drugs most strongly associated with decreased platelet counts due to thrombotic microangiopathy are the calcineurin inhibitors (ci) cyclosporine and tacrolimus. calcineurin inhibitor induced tma often occurs within weeks following sot, and the cis are thought to cause direct endothelial injury and platelet aggregation, although the specific mechanism has not been identified. when this is identified, numerous case studies in multiple different organ systems (lung, liver, kidney solid organ transplant) have reported that changing from one ci to another (tacrolimus to cyclosporine or vice versa) or to another class of medication such as sirolimus or mycophenolate mofetil can prevent further episodes of tma from occurring [61] [62] [63] [64] . however, the addition of the mtor inhibitor sirolimus to a calcineurin inhibitor also increases the chance of developing tma [57, 65] . ganciclovir and valganciclovir are used in prophylaxis and treatment of cmv. both are known to have myelosuppressive effects, particularly on granulocytes and platelets, but generally there is rapid recovery of counts following withdrawal of the medication. one of linezolid's most well-known adverse effects is thrombocytopenia, with the package insert reporting a rate of 3% in adults. other studies have reported rates of grades iii-iv thrombocytopenia of approximately 5.2% [66] . no mechanism has been identified for linezolid-related thrombocytopenia, though some evidence suggests that it is an immune-mediated phenomenon [67] . the medication is frequently used in the treatment of vancomycin-resistant enterococcus (vre), which has been an infection seen in transplant patients, as well as non-transplant patients, with increasing frequency. a multicenter compassionate use trial published in 2003 showed that it was an effective drug in treating vre, which was identified as having a mortality rate of up to 83%, with the authors reporting a 62% survival rate after treatment with linezolid. thrombocytopenia was the main adverse effect of treatment, seen in 4.7%, but did not necessitate the cessation of therapy [68] . a second study in liver transplant patients treated with linezolid for vre infec-tion showed a similar treatment efficacy and again reported no cases (0/46 patients) requiring cessation of therapy due to severe thrombocytopenia, and furthermore found no correlation between treatment duration and platelet counts [69] though other articles advise caution when using linezolid for extended time periods [70] . thus, while it may or may not require any intervention or change in treatment plan, it should be considered as part of the differential diagnosis when assessing thrombocytopenia. heparin-induced thrombocytopenia is an additional drugrelated event that can occur in the setting of solid organ transplant. assessment of this as a possible etiology for thrombocytopenia follows the same algorithm as it would for any patient receiving heparin. the probability of the thrombocytopenia being related to heparin use would be based on the 4ts whether the timing (>10 days following start of heparin use or sooner if heparin was used previously), degree of fall (>50% decrease from baseline), presence of thrombosis, and lack of alternate explanations for the thrombocytopenia suggest that heparin could be the causative agent [71] . studies reveal that hit is an uncommon occurrence in liver transplant recipients, and that thrombotic events and hit antibody positivity were not well correlated [72, 73] . case studies in renal transplant patients have reported some incidences of hit posttransplant and graft-failure related to hit, in part related to previous exposure to heparin in hemodialysis [74, 75] . hit antibody immunoassays are often sent if a patient develops thrombocytopenia and has received heparin at any time during the hospitalization. however, the high sensitivity but low specificity of the test results in overdiagnosis of heparin-induced thrombocytopenia exposes patients to unnecessary risks associated with therapeutic anticoagulation. chaturvedi and colleagues examined this phenomenon at cleveland clinic and found that utilizing the 4ts algorithm to first rule out patients at low risk for hit was a safe, reliable, and cost-effective [76] . therefore, we recommend that immunoassays for hit antibodies be utilized only in those patients whose 4t scores suggest intermediate or high probability of heparin-induced thrombocytopenia. if a hit antibody immunoassay is sent once a patient is determined to be of intermediate/high risk for hit, it is important to understand how this test is interpreted. the immunoassay detects the presence of antiplatelet factor 4 (pf4) antibodies in patient serum and is interpreted by optical density (od). a higher reported od correlates to a higher titer of the antibody and is more strongly suggestive of a diagnosis of hit. as mentioned previously, elisas for hit have a high sensitivity (meaning a negative test can rule out the diagnosis) but a low specificity, underscoring the need to first confirm a high pretest probability. immune thrombocytopenic purpura (itp) is characterized by very low platelet counts, petechiae and bruising, as well as mucosal bleeding, due to opsonization of platelets in the circulation. occurrence of itp following solid organ transplant has been documented particularly in the liver transplant literature, with the cases attributed to either autoimmune itp, at times precipitated by an identified infectious etiology such as tuberculosis [77] or alloimmune etiologies. the literature reports that chronic renal disease and renal transplant in patients with itp are noted to be very rare [78] , and thus most of our knowledge of itp as an etiology following transplant is from the liver transplant literature. one study reported a case series of eight patients who developed itp following orthotopic liver transplantation (olt), with a mean time of presentation of itp since olt of 5.4 years [79] . these cases were all felt to be autoimmune cases, as there was no history of itp in the donors. this case series also presented a review of the previous literature on itp after olt, and they noted two distinct time patterns of itp presentation, early (within 3 months) or late (>3 months). the authors note that it has been proposed that the earlyonset presentation may be due to passive transfer of antibodies from the donor to the recipient. those that developed late-onset itp were felt to have developed the antibodies independently of their donors [79] . additionally, studies have reported on development of alloimmune thrombocytopenic purpura, with antibodies introduced from donors with a history of itp [79, 80] . one case study described a case where a donor liver was obtained from a donor who had died after a cerebral hemorrhage secondary to itp. the recipient developed itp within 3 days of transplant and subsequently expired after developing portal vein thrombosis. the authors attributed the death to itp in that they were unable to anticoagulate but were providing blood products that may have resulted in increased likelihood of thrombosis. it is also possible, however, that the donor was producing procoagulant antibodies, as approximately 20-25% of patients with itp also have antiphospholipid antibodies [81, 82] . based on this event, the authors recommended excluding cadaveric transplants from donors whose death is attributed to itp [80] . particularly in liver transplant patients, thrombocytopenia is often seen prior to transplant, and generally approximately 50% of transplant recipients develop worsening thrombocytopenia within 2 weeks following transplant. this acute decrease generally resolves within the 1st month after trans-plant, and if thrombocytopenia persists, another etiology should be sought [79, 83, 84] . thrombocytopenia following liver transplant can also be attributed to residual portal hypertension or hypersplenism, if either of these conditions persist following transplant. however, it is important to remain vigilant to other causes particularly drug-related and infectious etiologies that could cause a drop in the platelet count. additionally, while case reports exist of tma with low adamts13 levels attributed to inhibitors present in the blood [85] , this is not a common phenomenon, and etiologies of tma mentioned previously (infectious and drug-related) would be much more likely. in most cases, treatment of the underlying etiology of the thrombocytopenia will result in improvement 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counts in cytomegalovirus infected heart transplant patients: impact of acute disease versus subclinical infection spectrum of cytomegalovirus-induced renal pathology in renal allograft recipients de novo thrombotic microangiopathy induced by cytomegalovirus infection leading to renal allograft loss new insights into postrenal transplant hemolytic uremic syndrome thrombotic microangiopathy and cytomegalovirus in liver transplant recipients: a case-based review thrombotic microangiopathy after lung transplantation emerging viruses in transplantation: there is more to infection after transplant than cmv and ebv de novo thrombotic microangiopathy after kidney transplantation: clinical features, treatment, and long-term patient and graft survival chronic hepatitis c-associated thrombocytopenia: aetiology and management hematologic disorders after solid organ transplantation. ash educ program book tacrolimus associated localized thrombotic microangiopathy developing in early stage after renal transplantation tacrolimus-associated thrombotic microangiopathy in a lung transplant recipient tacrolimus-induced thrombotic microangiopathy in orthotopic liver transplant patients: case series of four patients increased risk of thrombotic microangiopathy in patients receiving a cyclosporin-sirolimus combination analysis of linezolid-associated hematologic toxicities in a large veterans affairs medical center mechanisms for linezolid-induced anemia and thrombocytopenia linezolid in the treatment of vancomycinresistant enterococcus faecium in solid organ transplant recipients: report of a multicenter compassionate-use trial efficacy and safety of linezolid in liver transplant patients thrombocytopenia associated with linezolid therapy evaluation of pretest clinical score (4 t's) for the diagnosis of heparin-induced thrombocytopenia in two clinical settings incidence of heparin-induced thrombocytopenia type ii and postoperative recovery of platelet count in liver graft recipients: a retrospective cohort analysis heparin-induced thrombocytopenia after liver transplantation heparin-induced thrombocytopenia type ii: a serious hazard in preemptive renal transplantation: a case report successful preemptive renal retransplantation in a patient with previous acute graft loss secondary to hit type ii: a case report and review of literature over-testing for heparin induced thrombocytopenia in hospitalized patients immune thrombocytopenic purpura induced by intestinal tuberculosis in a liver transplant recipient renal transplantation and idiopathic thrombocytopenic purpura: two case reports immune thrombocytopenic purpura following liver transplantation: a case series and review of the literature transmission of idiopathic thrombocytopenic purpura during orthotopic liver transplantation thrombotic risk in patients with immune thrombocytopenia and its association with antiphospholipid antibodies clinical implications of elevated antiphospholipid antibodies in adult patients with primary immune thrombocytopenia thrombocytopenia after liver transplantation thrombocytopenia after liver transplantation inhibitors of adamts13: a potential factor in the cause of thrombotic microangiopathy in a renal allograft recipient a phase 1 trial of eltrombopag in patients undergoing stem cell transplantation after total body irradiation romiplostim treatment allows for platelet transfusion-free liver transplantation in pediatric thrombocytopenic patient with primary sclerosing cholangitis key: cord-006713-io9yp1y2 authors: wrede, c. e.; holler, e. title: intensivmedizinische betreuung von patienten nach stammzelltransplantation date: 2007 journal: intensivmed notfallmed doi: 10.1007/s00390-007-0774-x sha: doc_id: 6713 cord_uid: io9yp1y2 within the hematologic therapy procedures, stem cell transplantation (sct) represents the most extensive and invasive intervention. those patients have certain risks for several bacterial, viral, as well as fungal infections during the different stages of transplantation. especially in allogenic transplantation, discrimination of non-infectious, mostly immunologic complications like graft-versus-host reactions or vod (veno-occlusive disease) is crucial, and often represents a therapeutic challenge. an adequate intensive care therapy of these patients can only be achieved with the knowledge of the specific complications of sct. this review starts with an overview of the sct stages with their corresponding infectious and noninfectious complications, followed by the discussion of organ specific pulmonary, renal, cardiac, gastrointestinal, hepatic and neurological complications of stem cell transplantation. " abstract within the hematologic therapy procedures, stem cell transplantation (sct) represents the most extensive and invasive intervention. those patients have certain risks for several bacterial, viral, as well as fungal infections during the different stages of transplantation. especially in allogenic transplantation, discrimination of non-infectious, mostly immunologic complications like graft-versus-host reactions or vod (veno-occlusive disease) is crucial, and often represents a therapeutic challenge. an adequate intensive care therapy of these patients can only be achieved with the knowledge of the specific complications of sct. this review starts with an overview of the sct stages with their corresponding infectious and noninfectious complications, followed by the discussion of organ specific pulmonary, renal, cardiac, gastrointestinal, hepatic and neurological complications of stem cell transplantation. " key words intensive carestem cell transplantationallogen -autologgraft versus host " zusammenfassung innerhalb der hämatologischen therapieverfahren stellen stammzelltransplantationen (szt) den umfassendsten eingriff dar. in verschiedenen phasen der transplantation haben diese patienten erhöhte risiken für unterschiedliche bakterielle und virale infektionen sowie pilzinfektionen. insbesondere bei allogen transplantierten patienten ist die abgrenzung nichtinfektiöser, überwiegend immunologischer komplikationen, z. b. graft-versus-host-reaktionen oder eine vod (veno-occlusive disease) notwendig, was oft eine diagnostische herausforderung darstellt. eine adäquate intensivmedizinische therapie solcher patienten kann nur unter kenntnis der spezifischen komplikationen nach szt erfolgen. diese zusammenstellung gibt zunächst einen überblick über die verschiedenen phasen der szt mit den entsprechenden infektiösen und nichtinfektiösen komplikationen, und geht anschließend einzeln auf die organspezifischen pulmonalen, renalen, kardialen, gastrointestinalen, hepatischen und neurologischen komplikationen nach szt ein. die stammzelltransplantation stellt innerhalb der hämatologisch/onkologischen therapieverfahren den umfassendsten und belastendesten eingriff dar: die zur ausschaltung der grunderkrankung und der hämatopoiese eingesetzte hochdosischemotherapie, ggf. unter einbeziehung der ganzkörperbestrahlung, beruht auf dem prinzip der dosissteigerung und kann neben einer regelhaften schleimhauttoxizität an den epithelialen barrieren zu zusätzlichen spezifischen organtoxizitäten vor allem an lunge, leber und niere führen. diese toxizitäten werden durch die im anschluss an die hochdosistherapie einsetzende bis zu vierzehntägige knochenmarksaplasie verstärkt. durch das fehlen von neutrophilen in dieser phase werden infektiöse, durch das fehlen von thrombozyten blutungs-komplikationen begünstigt, die sich jeweils mit den schleimhautschäden potenzieren können. während bei der transplantation patienteneigener, kryokonservierter und nach auftauen reinfundierter stammzellen (sogenannte autologe stammzelltransplantation) mit dem anwachsen der stammzellen das komplikationsrisiko deutlich abnimmt, kommt bei der transplantation fremder, sog. allogener stammzellen von hla-identischen geschwistern und hla-gematchten fremdspendern als wichtiger risikofaktor für komplikationen eine durch immunologische reaktionen bedingte und mindestens über 3-4 monate anhaltende immundefizienz dazu. diese immundefizienz erklärt sich einerseits durch den neuaufbau der spezifischen immunabwehr durch t-und b-zellen, die wesentlich länger als die erholung der neutrophilen benötigt. zusätzlich müssen bei der allogenen transplantation zur prophylaxe der immunologischen hauptkomplikation, der akuten graft-versus-host reaktion, immunsuppressiva eingesetzt werden, die den immundefekt verstärken. je nach hla-kompatibilität kommt es bei allogener szt trotz dieser prophylaxe dennoch nach dem anwachsen der spenderzellen bei 20-40% der patienten zur klinischen graft-versus-host erkrankung (gvhd) [14] , die zu schweren entzündungen an haut, darm und leber führen kann. bei schwerer ausprägung kann die gvhd selbst zu lebensbedrohlichen komplikationen führen (wie z. b. darmblutungen oder leberversagen). zusätzlich müssen aber zur behandlung weitere immunsuppressiva (corticosteroide, t-zell-antikörper) hochdosiert eingesetzt werden, die den immundefekt drastisch verschlechtern und das auftreten opportunistischer infektionen mit viren und pilzen vor allem an der lunge begünstigen. auch noch jahre nach allogener szt stellen immundefekte und organbeteiligungen im rahmen der chronischen gvhd einen risikofaktor für lebensbedrohliche infektionen dar. während früher als stammzellquelle überwiegend direkt aus den beckenkämmen gewonnenes knochenmark transplantiert wurde, werden heute bei der autologen szt in nahezu 100%, bei der allogenen transplantation in über 70% mit hilfe von g-csf in das periphere blut mobilisierte blutstammzellen eingesetzt [16] . eine weitere, heute vor allem bei kindern zum einsatz kommende stammzellquelle stellt nabelschnurblut dar. auf grund der besonderen pathophysiologie der komplikationen nach szt führten diese schon immer häufiger als andere therapieverfahren zu organdysfunktionen, die eine intensivmedizinische intervention nötig machten. je nach zentrumsspezifischer indikation zur verlegung von der transplantationsstation auf die intensivstation schwanken die angaben der patienten, die nach szt irgendwann im verlauf eine intensivmedizinische betreuung benötigen, zwischen 20 und 40% aller transplantierten patienten. nach großen übersichten steht dabei bei der mehrzahl der patienten die respiratorische insuffizienz mit der indikation zur invasiven diagnostik sowie zur nicht-invasiven und invasiven beatmung im vordergrund: in der literatur wird die respiratorische insuffizienz als aufnahmegrund bei 60-70% aller szt patienten angegeben [2, 46] , in einer eigenen auswertung liegt der anteil von patienten, die nach allogener szt wegen respiratorischer insuffizienz auf die intensivstation verlegt werden, bei 116/132 (87%) (auswertung aus dem klinikum großhadern, lmu münchen, zeitraum 1990-1996 und dem klinikum der universität regensburg, zeitraum 1998-2003) der verlegten patienten. gerade patienten mit respiratorischer insuffizenz nach szt stellen aber auf grund der komplexen pathophysiologie und der daraus resultierenden schwere der pulmonalen schädigung eine extreme belastung und herausforderung dar: ihre prognose ist gegenüber patienten, die aus anderen gründen verlegt wurden, deutlich schlechter, die therapie auf grund der notwendigkeit einer breiten antiinfektiösen, immunsuppressiven und supportiven therapie extrem komplex und aufwändig. in den ersten systematischen übersichten zur intensivtherapie bei szt-patienten wurde die prognose mechanisch beatmeter patienten nach allogener szt mit < 2% langzeitüberleben angegeben, so dass immer wieder die berechtigte frage nach dem nutzen intensivmedizinischer maßnahmen bei szt patienten gestellt wurde. vor diesem hintergrund erscheint eine kritische wertung des aktuellen stands der intensivmedizinischen behandlung stammzelltransplantierten patienten von besonderer bedeutung: grundsätzlich ist hervorzuheben, dass autolog transplantierte patienten auf grund des wegfalls der immunologischen komponente seltener intensivmedizinisch behandelt werden müssen und in der regel wegen der aussicht auf stabilisierung nach erholung der blutwerte eine bessere prognose haben [3] . aber auch für allogen transplantierte patienten zeigt sich im verlauf der letzten jahre eine eindeutige verbesserung der prognose nach intensivmedizinischer behandlung inkl. mechanischer beatmung, die mehrzahl der aktuellen arbeiten gibt ein 6 monats-oder längeres überleben für diese patientengruppe an, welches jetzt bei 15-20% liegt [3, 23, 37, 47] . dies konnte auch anhand der daten des klinikums der universität regensburg bestätigt werden (s. abb. 1). für diese entwicklung können mehrere ursachen verantwortlich gemacht werden: 1) durch den zunehmenden einsatz von blutstammzellen konnte die phase der schweren neutropenie bei der mehrzahl der patienten um einige tage gegenüber der transplantation von stammzellen aus dem knochenmark verkürzt werden [44] . 2) heute wird bei risikopatienten in zunehmendem maße eine so genannte dosis-redu-zierte hochdosischemotherapie bzw. konditionierung eingesetzt, so dass die komponente der direkten organtoxizität reduziert werden konnte [31] : so konnte in einigen arbeiten gezeigt werden, dass sich insgesamt die inzidenz pulmonaler komplikationen durch die neuen konditionierungsverfahren senken lässt. 3) die infektionsdiagnostik, -prophylaxe und -therapie konnte erheblich verbessert werden, insbesondere durch die verfügbarkeit weniger toxischer, aber aspergillus-wirksamer antimykotika und die breite einführung der cmv-surveillance, die eine präemptive therapie mit virustatika erlaubt und damit das fortschreiten zur cmv-pneumonie verhindert. auf der anderen seite haben diese entwicklungen aber auch dazu geführt, dass sich das spektrum der zur intensivmedizinischen behandlung führenden komplikationen verändert hat: sie treten heute weniger in der akutphase im ersten monat nach transplantation, sondern häufiger im weiteren verlauf monate und jahre nach szt bei auftreten der gvhd oder nach erneuter adoptiver zelltherapie mit retransplantation auf. dies bedeutet aber auch, dass der patient zu diesem zeitpunkt häufiger nicht mehr im behandelten zentrum, sondern auch in einer nicht hämatologischen einrichtung primär versorgt werden muss. gleichzeitig haben die o. g. veränderungen in den transplantationsverfahren dazu geführt, dass heute auch ältere patienten bis zu ei in der pre-engraftment-phase sind bakterielle infektionen häufig, wobei gram-negative erreger wie klebsiella, escherischia coli und pseudomonas aufgrund der kürzeren neutropenie-zeit und prophylaktischen maßnahmen wie die darmdekontamination und antibiotikaprophylaxe seltener geworden sind. daher nimmt der anteil grampositiver erreger, z. b. mrsa, streptococcus viridans und enterokokken zu [6] . generell ist bei dem auftreten von fieber nach entsprechender diagnostik eine initialtherapie mit cephalosporinen der 3. generation oder carbapenemen sinnvoll, bei instabilen patienten ist die hinzunahme von aminoglykosiden (und ggf. antifungal wirkenden substanzen) zu empfehlen [33] , diese therapie sollte nach erhalt eines antibiogramms angepasst werden. aufgrund der reduzierten immunabwehr von patienten nach stammzelltransplantation sind opportunistische infektionen häufig. beteiligte virale infektionserreger sind cytomegalie-viren (cmv), herpessimplex-viren (hsv), varizella-zoster-viren (vzv), respiratory-syncytial-viren (rsv), das humane herpes simplex virus 6 (hhv-6) und adenoviren. während die hsv-infektionen eher früh in der preengraftment-phase auftreten, finden sich cmv-infektionen eher in der postengraftment-phase und in der späten phase, insbesondere bei vorliegen einer gvhd. in dieser phase treten auch vermehrt vzv-infektionen auf. in der behandlung von virusinfektionen muss zwischen einer sogenannten preemptiven therapie, die aufgrund eines virusnachweises bei asymptomatischen patienten begonnen wird, und der therapie einer virusbedingten erkrankung unterschieden werden. die cmv-pneumonitis mit hoher mortalität war vor der einführung der preemptiven therapie (s. u.) die häufigste virale infektion in den ersten 100 tagen nach allogener transplantation, während mittlerweile die häufigkeit der cmv-pneumonie eher in den späteren phasen nach dem 100. tag ansteigt. risikofaktoren sind die gabe cmv-positiver blutprodukte bei cmv-negativem empfänger, neutropenie, gvhd, bestrahlung, und die gabe von anti-thymozytenglobulin. die diagnose einer cmv-pneumonie gründet sich auf die klinik mit dyspnoe, fieber und respiratorischer insuffizienz. im konventionellen röntgen-thorax finden sich diffuse interstitielle veränderungen (s. abb. 2), höhere diagnostische aussagekraft hat ein high-resolution ct mit nachweis von interstitiellen nodulären veränderungen. zur sicherung der diagnose sollte eine bronchoalveoläre lavage (bal) mit direkter immunfluoreszenz oder cmv-pcr durchgeführt werden. die therapie besteht in der frühzeitigen gabe von ganciclovir in kombination mit immunglobulinen, alternativ kann auch foscarnet eingesetzt werden [47] . wesentliche nebenwirkungen von ganciclovir sind eine neutropenie, cerebrale krämpfe und nephrotoxizität. zu den viren des respirationstraktes, die schwere pulmonale infektionen bei stammzelltransplantierten patienten hervorrufen können, zählen rsv, adenoviren, influenza-und parainfluenza-virus. zu den allgemeinen maßnahmen der prävention für diese patienten gehört die vermeidung von kontakten zu personen, die unter infekten der atemwege leiden, und die influenza-impfung von angehörigen, medizinischem personal sowie dem patienten selbst vor sowie 6 monate nach der transplantation [1] . respiratorische symptome bis hin zur pneumonie können durch rsv verursacht werden, wobei die infektion typischerweise innerhalb der ersten 4 wochen nach transplantation auftritt. die mortalität bei pneumonie ist hoch, weswegen eine frühzeitige diagnostik mittels nasenflüssigkeit oder bronchoalveolärer lavage angestrebt werden sollte. obwohl keine kontrollierten randomisierten studien zur therapie existieren, bestehen die empfehlungen in einer frühzeitigen gabe von ribavirin (in aerosol-form oder intravenös) und die gabe hochtitriger immunglobuline [1] . weitere respiratorische viren wie influenza und parainfluenza können ebenfalls schwere pneumonien verursachen, sind jedoch seltener als die rsv-infektionen. die therapie einer influenza besteht in der frühzeitigen gabe von neuraminidase-hemmern, z. b. oseltamivir, bei auftreten von symptomen des oberen respirationstrakts, um das auftreten von pneumonien zu vermeiden. eine spezifische therapie für parainfluenza besteht nicht. adenoviren können respiratorische infektionen einschließlich pneumonien sowie eine gastroenteritis, meningoencephalitis, hepatitis und hämorrhagische zystitis verursachen, risiken sind die allogene transplantation, das vorliegen einer gvhd und der adenovirus-status des spenders [41] . die datenlage erlaubt derzeit keine sichere beurteilung des spontanverlaufs der erkrankung, da die publizierten mortalitätsraten zwischen 8 und 71% schwanken, wobei ein pulmonaler befall mit einer schlechteren prognose einhergeht [28] . zur medikamentösen behandlung von adenovirus-infektionen existieren bislang nur publikationen von kleinen serien. sowohl ribavirin als auch cidofovir wurden therapeutisch und zur preemptiven therapie bei adenovirus-nachweis eingesetzt, eine generelle therapieempfehlung kann aufgrund der publizierten daten nicht gegeben werden. latente humane herpesvirus 6 (hhv-6)-infektionen finden sich bei nahezu allen stammzelltransplantierten patienten. eine reaktivierung findet häufig 30n pilzinfektionen candida-spezies besiedeln normalerweise den verdauungstrakt, und können bei reduzierter immunabwehr, insbesondere bei gvhd, neutropenie, steroidtherapie, unter breitspektrumantibiotika und bei kritisch kranken patienten zu einer candidämie und sepsis führen. primäre pulmonale infektionen sind selten, häufiger kommt es zu einer hepatischen oder splenischen infektion. die häufigkeit einer systemischen candida-infektion ist durch die prophylaxe mit fluconazol (s. u.) reduziert worden [29] . während candida albicans normalerweise sensibel für fluconazol ist, können resistente stämme wie candida glabrata und candida krusei selektiert werden, die weitere therapie muss dann mittels caspofungin oder liposomalem amphotericin b (ambisome) erfolgen. die invasive aspergillose ist die häufigste pilzinfektion und tritt sowohl während der neutropenischen phase als auch bei allogen transplantierten patienten in der späten phase unter immunsuppressiver therapie der gvhd auf. primärer ort der infektion ist meist die lunge, wobei die bildgebung unspezifisch ist und mit diffusen pulmonalen infiltraten als auch mit lokalisierten infiltraten einhergehen kann, die einschmelzen können (s. abb. 3). weitere befallene organe können das gehirn, leber, niere, darm und haut sein. sputumproben und bal mit aspergillus-nachweis haben eine hohe spezifität, aber nur eine geringe sensitivität, während der nachweis von aspergillus-antigen im serum vermutlich eine höhere sensitivität besitzt. die therapie der wahl besteht in der gabe von voriconazol, bei nichtansprechen kann caspofungin oder liposomales amphotericin b gegeben werden. hierunter werden ansprechraten von etwa 50% erreicht [20] angesichts des zunehmenden langzeiteinsatzes aspergilluswirksamer azole muss heute auch mit einer zunahme anderer mykosen (z. b. infektionen mit fusarien und mukor) gerechnet werden, so dass die spezifische erregerdiagnose auch bei typischer bildgebung von großer bedeutung ist. die pneumocystis jirovecii pneumonie (pcp) ist seit einführung der pcp-prophylaxe selten geworden. radiologisch finden sich bihiläre diffuse infiltrationen, die diagnostische sensitivität mittels bal ist hoch. die therapie besteht in der hochdosierten gabe von trimethoprim-sulfmethoxazol. n antiinfektiöse prophylaxe aufgrund der häufigkeit und schwere von infektionen in der neutropenischen phase nach stammzelltransplantation werden eine routinemäßige pcp-prophylaxe mit trimethoprim-sulfmethoxazol und eine antifungale prophylaxe mit fluconazol durchgeführt, die zu einer reduktion der mortalität führen [1] . vor allem bei der allogenen transplantation wird in der phase der neutropenie, aber auch bei schwerer darm-gvhd eine darmdekontamination mit lokal oder systemisch wirksamen antibiotika (z. b. mit chinolonen) durchgeführt, um einerseits die gefahr gramnegativer infektionen zu reduzieren, andererseits die experimentell gesicherte interaktion von darmflora und gvhd zu modulieren. bei hsv-positiven empfängern wird zudem aciclovir gegeben. die inzidenz von klinisch relevanten cmv-infektionen konnte durch die gabe von gancyclovir [15] oder valgancyclovir [8] in cmv-positiven patienten reduziert werden. eine preemptive therapie wird hierbei begonnen, wenn aufgrund des monitorings von pp65-antigen oder cmv-pcr aufgrund eines titer-oder kopienanstieges eine infektion wahrscheinlich ist [9] . pulmonale komplikationen nach szt lassen sich einerseits nach ihrer genese (nicht-infektiös vs. infektiös), anderseits nach dem zeitraum nach szt, in dem sie auftreten, klassifizieren (tab. 2). innerhalb der infektiösen komplikationen bieten allerdings nur die bakteriellen infektionen eine klar abgrenzbare entität: interstitielle pneumonien durch viren wie cmv, hhv6 oder respiratorische viren sind häufig schwierig von immunologischen und toxischen komplikationen zu unterscheiden, zumal diese viren immunologische reaktionen wie die gvhd triggern können und umgekehrt die immunsuppression, aber auch bestimmte proinflammatorische mediatoren die virusreaktivierung fördern [21] . die bei patienten mit fortgeschrittener gvhd und nach längerem intensivaufenthalt besonders häufigen pilzinfektionen propfen sich umgekehrt häufig auf primär nicht infektiöse pulmonale komplikationen auf: vor diesem hintergrund ist eine rasche und möglichst standardisierte diagnostik bei jedem patienten mit respiratorischer insuffizienz nach szt anzustreben, die einerseits ein thorax-ct mit high resolution modus, andererseits eine bal mit einer erregerdiagnostik auch auf atypische erreger, insbesondere auch viren (cmv, hhv6, adenoviren, entsprechend der jahres-zeit auch rsv, influenza und parainfluenza) beinhalten sollte. in den ersten 30 tagen nach transplantation stehen zunächst auf grund der neutropenie pulmonale komplikationen im rahmen bakterieller infektionen, insbesondere sepsis-assoziiertes ards, toxische lungenkomplikationen durch die chemotherapie und perakute immunreaktionen wie das capillary leakage syndrom im vordergrund. kommt es zwischen dem 10. und 20. tag nach szt zum engraftment der neutrophilen und damit zum "anschoppen" von entzündungszellen in der lunge, stellen das peri-engraftment respiratory distress syndrom (perds) und die diffuse alveoläre hämorrhagie (dah) als endotheliale manifestation spezifische komplikationen dar: während die alveoläre hämorrhagie häufig bronchoskopisch diagnostiziert wird, ist für das perds die enge zeitliche zuordnung zum raschen leukozytenanstieg entscheidend. die prognose des perds ist günstiger, da sie bei einem teil der patienten rasch auf eine zusätzliche hochdosierte korticosteroidtherapie (2-10 mg/kg prednisolon-äquivalent) anspricht, auch bei der dah stellen hochdosierte korticosteroide neben der spezifischen therapie das mittel der wahl dar [4] . nach dem engraftment zwischen tag 30 und 180 stellen virale interstitielle pneumonien und das idiopathische pneumoniesyndrom typische komplikationen dar, wobei es mit zunehmend genauerer pcrbasierter diagnostik von viren häufig schwierig ist, diagnostisch beide krankheitsbilder klar zu trennen. eine weitere differenzierung könnte hier nur durch die transbronchiale oder besser offene lungenbiopsie erreicht werden, die sich in dieser phase nach szt aber häufig auf grund noch erniedrigter thrombozytenwerte und einer generell erhöhten blutungsneigung verbietet. werden viren wie cmv oder influenza gefunden, so steht zunächst die spezifische antivirale therapie im vordergrund. in der pathogenese des idiopathischen pneumonie-syndroms zeigt sich zumindest im tiermodell eindeutig eine beteiligung der gvhd, die zytokinblockade kann hier experimentell und in ersten klinischen pilotstudien die prognose verbessern [11] . insgesamt sind aber die kriterien für den additiven beginn einer hochdosierten immunsuppressiven therapie extrem unscharf definiert, so dass allenfalls ein kurzfristiger versuch mit hochdosierten korticosteroiden auch differentialdiagnostisch eingesetzt werden kann. gerade bei intensiv immunsupprimierten patienten mit schwerer gvhd ist zu bedenken, dass die ursache der respiratorischen insuffizienz im verlauf des intensivaufenthalts wechseln kann: insbesondere sekundäre pulmonale pilzinfektionen komplizieren häufig sowohl bei viraler pneumonie als auch bei ips den weiteren verlauf. bei langzeitpatienten nach allogener szt (> tag 180) ist die lunge direktes zielorgan immunologischer reaktionen: obstruktive veränderungen im sinne eines bronchiolitis obliterans syndroms [10, 43] treten hier als manifestation der chronischen gvhd auf. kommt es zur respiratorischen globalinsuffizienz mit häufig extremer hyperkapnie, so ist die beatmung und vor allem das weaning dieser patienten extrem schwierig. die mehr als restriktive infiltration auftretende bronchiolitis obliterans organzing pneumonia (boop) ist vom bos durch typische ct-veränderungen abgrenzbar und spricht im gegensatz zum schweren bos besser auf eine intermittierend hoch-dosierte (1-2 mg/kg mp-äquivalent) steroidtherapie an. entsprechend der zugrundeliegenden pathophysiologie stehen nach autologer szt vor allem die in den ersten 30 tagen auf dem boden neutropenischer infektionen und toxischer schädigung entstehenden lungenkomplikationen im vordergrund; vor allem nach konditionierung mit ganzkörperbestrahlung und bei intensiv vorbehandelten patienten kann aber auch nach autologer szt ein über 3-6 monate anhaltender t-zell-defekt auftreten, der dann auch virale pneumonien mit grundsätzlich identischem erregerspektrum wie nach allogener szt begünstigt. [24] . eine ausreichende wässerung und harnalkalisierung verringert das risiko. eine hämorrhagische zystitis kann ebenfalls über eine obstruktion der ableitenden harnwege zu einem akuten nierenversagen führen. die hämorrhagische zystitis kann entweder früh in der pre-engraftment-periode durch cyclophosphamid oder bestrahlung induziert, oder in der post-engraftment-phase durch virusinfektionen wie cmv, bk-virus oder adenovirus vermittelt sein, wobei die allogen transplantierten patienten ein höheres risiko aufweisen [18] . die prophylaxe besteht in einer ausreichenden wässerung, und der verwendung von mesna bei cyclophosphamid-gabe. bei auftreten einer stärkeren hämorrhagischen cystitis stehen therapeutisch die verbesserung der gerinnung, z. b. durch thrombozytengabe, die verhinderung der blasentamponade und ureterverlegung durch wässerung und anlage eines blasenspülkatheters im vordergrund, ggfs. ergänzt durch eine spezifische antivirale therapie. bei schwerer blutung werden interventionelle verfahren eingesetzt, unterstützend hat sich eine östrogentherapie bewährt [19] . die häufigste ursache des akuten nierenversagens in der pre-engraftment-phase ist die veno-occlusive disease (vod) der leber [48] . diese erkrankung ist durch eine sinusoidale dysfunktion mit konsekutivem verschluss der kleinen lebervenolen mit leber-venenstauung und leberfunktionseinschränkung gekennzeichnet [25] . die von der leber freigesetzten mediatoren wie prostaglandine und leukotriene führen, analog zu einem hepatorenalen syndrom, zu einer renalen vasokonstriktion. zur therapie der vod wurde r-tpa und defibrotide (prociclide®) eingesetzt [25] , eine spezifische therapie des nierenversagens bei vod ist nicht beschrieben [35] . kardiale komplikationen sind mit 19% aller intensiv-verlegungen stammzelltransplantierter patienten häufig [47] , meist führt eine dekompensierte herzinsuffizienz zur intensiv-aufnahme. die ursache hierfür ist oft die intensive wässerung in der pre-engraftment-phase, verbunden mit einer häufig auftretenden nierenfunktionseinschränkung (s. o.). aufgrund des zunehmenden alters der transplantierten patienten ist auch eine vorbestehende herzinsuffizienz häufiger, was das risiko für eine dekompensation erhöht. eine kardiotoxizität von chemotherapeutika, z. b. von anthracyclinen, cyclophosphamid, paclitaxel, etoposid und cisplatin, könnte ebenfalls zu einer herzinsuffizienz beitragen. dass die transplantation zu einer klinisch signifikanten schädigung des herzens führt, ist jedoch eher unwahrscheinlich, da in einer studie mit 96 allogen und 52 autolog stammzelltransplantierten patienten kein unterschied in der linksventrikulären ejektionsfraktion vor und durchschnittlich 60 tage nach transplantation gefunden wurde [27] . kardiale arrhythmien treten im rahmen der komplikationen wie elektrolytentgleisungen oder sepsis auf, die häufigste rhythmusstörung ist die absolute arrhythmie bei vorhofflimmern. jedoch sind auch fatale bradyarrhythmien durch das stammzell-kryokonservierungsmittel dmso beschrieben worden [5] . die therapie der arrhythmien entspricht dem medizinischen standard. endokarditiden sind eher selten und wurden in 1,3% der stammzelltransplantierten patienten festgestellt, wobei 75% erst post mortem diagnostiziert wurden. bemerkenswert ist die hohe rate an pilzbedingten endokarditiden mit candida und aspergillus in dieser studie [26] . gerade auf grund der höheren altersgrenze für die stammzelltransplantation muss heute bei akuten ereignissen die kardiale ischämie in die differentialdiagnose mit einbezogen werden, wobei die therapeutischen möglichkeiten bei gleichzeitiger thrombopenie meist eingeschränkt sind. während in der neutropenischen pre-engraftment phase die neutropenische enterocolitis (siehe kapitel infektionen nach stammzelltransplantation) im vordergund steht, stellen vor allem bei allogener transplantation nach dem engraftment enteritiden eine häufige komplikation dar: hier ist die exakte erregerdiagnose wichtig, neben clostridium difficile können eine vielzahl viraler erreger (cmv, hhv6, vzv, adenovirus, aber auch typische enteroviren wie rotaviren und noroviren) zu schweren und lang anhaltenden enteritiden führen. die wichtigste differentialdiagnose der infektiösen gastroenteritis stellt die intestinale gvhd dar [19, 45] : wegen der gegensätzlichen therapeutischen konsequenzen bei infektiöser und gvhd-bedingter enteritis ist, wenn immer möglich, eine histologische diagnosesicherung mit erregerisolierung aus der biopsie anzustreben. die schwersten folgekomplikationen der intestinalen gvhd stellen die intestinale pseudoobstruktion und schwere gastrointestinale blutungen dar. da die entzündung bei gvhd in der regel langstreckig und diffus ist, stehen zunächst systemische maßnahmen wie supportive therapie, die adäquate thrombozytensubstitution und die spezifische therapie der gvhd im vordergrund. endoskopische blutstillungsverfahren sind meist nur bei lokalisierten prozessen erfolgreich, im einzelfall kann hier auch eine chirurgische intervention nach sicherung der lokalisierten blutungsquelle indiziert sein. da die intestinale gvhd häufig mit einem schweren faktor xiii mangel einhergeht, sind erfolgreiche behandlungen durch faktor xiii substitution beschrieben, ebenso wie in einzelfällen der einsatz von aktiviertem faktor vii [17, 38] . andererseits ist gerade die fortgeschrittene intestinale gvhd als prognostisch extrem ungünstig zu werten, so dass der einsatz entsprechender maßnahmen vor allem unter berücksichtigung der bereits erfolgten immunsuppressiven vortherapien und nach gemeinsamer abwägung der gesamtprognose erfolgen sollte. in der preengraftment-phase treten hepatische komplikationen am häufigsten im rahmen der sepsis und des assoziierten organversagens auf, leichte formen dieser septischen hepatopathie werden im nadir der leukozyten bei entsprechender entzündungskonstellation häufig beobachtet [30] . viele der prophylaktisch eingesetzten medikamente bei stammzelltrans-plantation sind lebertoxisch, dies gilt insbesondere für die calcineurininhibitoren ciclosporin und tacrolimus sowie nahezu alle antimykotika. die wichtigste spezifische leberkomplikation ist in den ersten 30 tagen nach szt die veno-occlusive disease (vod), die heute auch als sinusoidales obstruktions-syndrom (sos) bezeichnet wird [13] . durch eine kombination aus konditionierungsbedingter (z. b. busulfan-gabe) und inflammatorischer endothelschädigung kommt es zur gerinnungsaktivierung in den lebersinusoiden bis hin zum kompletten verschluss mit konsekutiven leberzellnekrosen. diuretika-refraktäre gewichtszunahme, schmerzhafte hepatomegalie und neu aufgetretener aszites mit im verlauf zunehmendem bilirubinanstieg sind die wegweisenden klinischen zeichen [22] , die duplexsonographie ergibt in der frühphase häufig keine spezifischen befunde. diagnostisch beweisend sind letztlich nur ein deutlich erhöhter lebervenenverschlussdruck sowie die leberbiopsie. bei auftreten in der thrombopenischen phase sowie typischerweise bei vod auftretendem sekundären thrombozytenverbrauch ist die biopsiegewinnung risikoreich und sollte wenn möglich transjugulär erfolgen. als laborparameter kann hier die bestimmung des pai-1 hilfreich sein, da die vod immer mit einer mehr als fünffachen erhöhung dieses parameters einhergeht [39] . die schwere vod hat mit einer hohen wahrscheinlichkeit eines leber-und konsekutiven multiorganversagens eine extrem schlechte prognose. neben der supportiven therapie des leberversagens (inkl. nierenersatzverfahren) wurde früher die thrombolyse mit hohem blutungsrisiko eingesetzt, heute ist, wie bereits im kapitel renale komplikationen beschrieben, die konsequente applikation des antikoagulatorischen und als pai-1-antagonist wirkenden defibrotide (prociclide®) die aussichtsreichste therapie [40] , die mit geringeren blutungskomplikationen einhergeht. neben einer späten vod treten nach dem engraftment als spezifische leberkomplikationen die akute und chronische gvhd der leber, die allerdings selten das isoliert führende gvhd-symptom ist, sowie hepatitiden vor allem durch virale erreger wie cmv, hhv-6, adenoviren und andere viren auf. auch hier ist bei massiv erhöhten leberwerten häufig nur die leberbiopsie wegweisend und voraussetzung für eine adäquate therapie. da szt-patienten selbst bei weitgehend normalisiertem blutbild ein deutlich erhöhtes risiko punktionsbedingter blutungen haben, ist der transvenöse zugang für die biopsie zu empfehlen. neurologische komplikationen treten nach szt in 20-30% der patienten auf und sind bei etwa 6% aller verlegungen von szt-patienten das führende symptom [42] : toxische enzephalopathien werden insbesondere unter calcineurininhibitoren wie ciclosporin und tacrolimus beobachtet, sie können sich akut als schweres klinisches bild mit generalisierten krampfanfällen, aber auch schweren wesens-und vigilanzveränderungen und paresen manifestieren. die kernspintomographie kann hier durch den nachweis spezifischer marklagerveränderungen neben dem nachweis hoher oder hochnormaler serumspiegel wegweisend sein, die symptome sind nach reduzierung der dosis oder umstellung der immunsuppression meist reversibel. als neue medikamentös induzierte encephalopathie sind neurologische nebenwirkungen des azols voriconazol anzuführen, das auf grund seiner hohen zns-gängigkeit häufig zu visuellen halluzinationen, gelegentlich aber auch zu schweren wesensveränderungen führen kann. cerebrovaskuläre komplikationen treten bei szt-patienten auf grund einer erhöhten thromboseneigung häufiger als in der normalbevölkerung auf. als eine wichtige differentialdiagnose muss bei schwer immunsupprimierten patienten und/oder multiplen vaskulären herden auch an eine manifestation einer angioinvasiven aspergillose gedacht werden. intracerebrale blutungen sind insbesondere bei refraktärer thrombopenie möglich, bei patienten mit einer anamnese gehäufter liquorpunktionen treten öfter chronische subduralhämatome auf, die sich dann unter thrombopenie sekundär verschlechtern. die cerebrale beteiligung bei der transplantations-assoziierten mikroangiopathie ist eher selten, da diese meist bereits früher auf grund systemischer symptome oder befunde diagnostiziert wird. mit wesens-und vigilanzveränderungen gehen die vor allem nach allogener transplantation auf grund des protrahierten immundefekts beobachteten encephalitiden einher: hier ist die rasche radiologische diagnostik inklusive der kernspintomographie entscheidend. temporallappenveränderungen treten vor allem im rahmen von herpesvirusinfektionen (hsv, hhv6) auf, auch die cerebrale toxoplasmose zeigt häufig typische veränderungen. neben cmv, vzv können auch adenoviren zur encephalitis führen, die spezifische liquordiagnostik ist hier wegweisend. eine schwerwiegende, da häufig irreversible komplikation stellt die mulitfokale leukoenzephalopathie dar. zeigen sich im liquor hinweise auf eine beteiligung von jc oder bk viren, so kann im einzelfall eine therapie mit cidofovir versucht werden. während erreger wie nocardien oder listerien in der regel gut behandelt werden können, stellt die cerebrale beteiligung bei pilzinfektionen wie aspergillus oder mucor in der regel eine schwerwiegende komplikation dar, die nur bei patienten mit gleichzeitiger stabilisierung der gvhd und möglichkeit der weitgehenden reduktion der immunsuppression erfolgreich behandelt werden kann. eine adäquate diagnostik und therapie von komplikationen nach szt ist für die intensivmedizinische behandlung dieser patienten essentiell, und kann zu einer verbesserung der prognose führen. hierbei ist die kontinuierliche bettseitige interdisziplinäre zusammenarbeit von hämatologen und intensivmedizinern von vorteil. guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients major complications following hematopoietic stem cell transplantation intensive care unit support and acute physiology and chronic health evaluation iii performance in hematopoietic stem cell transplant recipients outcome of diffuse alveolar hemorrhage in hematopoietic stem cell transplant recipients adverse events occurring during bone marrow or peripheral blood progenitor cell infusion: analysis of 126 cases pre-and post-engraftment bloodstream infection rates and associated mortality in allogeneic hematopoietic stem cell transplant recipients pre-and post-engraftment bloodstream infection rates and associated mortality in allogeneic hematopoietic stem cell transplant recipients valganciclovir is safe and effective as pre-emptive therapy for cmv infection in allogeneic hematopoietic stem cell transplantation surveillance of cytomegalovirus infection in haematopoietic stem cell transplantation patients airflow obstruction after myeloablative allogeneic hematopoietic stem cell transplantation tumor necrosis factor-alpha neutralization reduces lung injury after experimental allogeneic bone marrow transplantation neutropenic enterocolitis toxic injury to hepatic sinusoids: sinusoidal obstruction syndrome (veno-occlusive disease) acute graft-vs-host disease: pathobiology and management early treatment with ganciclovir to prevent cytomegalovirus disease after allogeneic bone marrow transplantation ebmt activity survey 2004 and changes in disease indication over the past 15 years factor xiii replacement in stem cell transplant (sct) recipients with severe graft-versus-host disease of the bowel: report of an initial experience hemorrhagic cystitis after allogeneic bone marrow transplantation in children: clinical characteristics and outcome estrogen as treatment of hemorrhagic cystitis in children and adolescents undergoing bone marrow transplantation voriconazole versus amphotericin b for primary therapy of invasive aspergillosis cytokines, viruses, and graft-versus-host disease venoocclusive disease of the liver following bone marrow transplantation changing outcomes for children requiring intensive care following hematopoietic stem cell transplantation cryopreservation and infusion of autologous peripheral blood stem cells hepatic veno-occlusive disease (sinusoidal obstruction syndrome) after hematopoietic stem cell transplantation characteristics and outcome of patients developing endocarditis following hematopoietic stem cell transplantation cardiac systolic function before and after hematopoietic stem cell transplantation treatment of adenovirus infections in the immunocompromised host prolonged fluconazole prophylaxis is associated with persistent protection against candidiasis-related death in allogeneic marrow transplant recipients: long-term follow-up of a randomized, placebo-controlled trial advances in prevention and treatment of hepatic disorders following hematopoietic cell transplantation hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graft-versus-tumor effects management of infectious complications in the hematopoietic stem cell transplant recipient management of infectious complications in the hematopoietic stem cell transplant recipient acute renal failure in hematopoietic cell transplantation acute renal failure in hematopoietic cell transplantation acute renal failure independently predicts mortality after myeloablative allogeneic hematopoietic cell transplant outcome of critically ill allogeneic hematopoietic stem-cell transplantation recipients: a reappraisal of indications for organ failure supports recombinant activated factor vii in treatment of bleeding complications following hematopoietic stem cell transplantation diagnosis of hepatic venoocclusive disease by plasminogen activator inhibitor-1 plasma antigen levels: a prospective analysis in 350 allogeneic hematopoietic stem cell recipients multi-institutional use of defibrotide in 88 patients after stem cell transplantation with severe veno-occlusive disease and multisystem organ failure: response without significant toxicity in a high-risk population and factors predictive of outcome adenoviral infection after allogeneic stem cell transplantation (sct): report on 130 patients from a single sct unit involved in a prospective multi center surveillance study neurological complications of hematopoietic cell transplantation risk factors for bronchiolitis obliterans in allogeneic hematopoietic stem-cell transplantation for leukemia allogeneic bone marrow transplantation vs filgrastim-mobilised peripheral blood progenitor cell transplantation in patients with early leukaemia: first results of a randomised multicentre trial of the european group for blood and marrow transplantation early and late gastrointestinal complications after myeloablative and nonmyeloablative allogeneic stem cell transplantation critical care considerations of hematopoietic stem cell transplantation outcome and prognostic factors of hematopoietic stem cell transplantation recipients admitted to a medical icu acute renal failure in the setting of bone marrow transplantation key: cord-004059-furt6xcn authors: hraiech, sami; bonnardel, eline; guervilly, christophe; fabre, cyprien; loundou, anderson; forel, jean-marie; adda, mélanie; parzy, gabriel; cavaille, guilhem; coiffard, benjamin; roch, antoine; papazian, laurent title: herpes simplex virus and cytomegalovirus reactivation among severe ards patients under veno-venous ecmo date: 2019-12-23 journal: ann intensive care doi: 10.1186/s13613-019-0616-6 sha: doc_id: 4059 cord_uid: furt6xcn background: herpesviridae reactivation among non-immunocompromised critically ill patients is associated with impaired prognosis, especially during acute respiratory distress syndrome (ards). however, little is known about herpes simplex virus (hsv) and cytomegalovirus (cmv) reactivation occurring in patients with severe ards under veno-venous extracorporeal membrane oxygenation (ecmo). we tried to determine the frequency of herpesviridae reactivation and its impact on patients’ prognosis during ecmo for severe ards. results: during a 5-year period, 123 non-immunocompromised patients with a severe ards requiring a veno-venous ecmo were included. sixty-seven patients (54%) experienced hsv and/or cmv reactivation during ecmo course (20 viral co-infection, 40 hsv alone, and 7 cmv alone). hsv reactivation occurred earlier than cmv after the beginning of mv [(6–15) vs. 19 (13–29) days; p < 0.01] and after ecmo implementation [(2–8) vs. 14 (10–20) days; p < 0.01]. in univariate analysis, hsv/cmv reactivation was associated with a longer duration of mechanical ventilation [(22–52.5) vs. 17.5 (9–28) days; p < 0.01], a longer duration of ecmo [15 (10–22.5) vs. 9 (5–14) days; p < 0.01], and a prolonged icu [29 (19.5–47.5) vs. 16 (9–30) days; p < 0.01] and hospital stay [44 (29–63.5) vs. 24 (11–43) days; p < 0.01] as compared to non-reactivated patients. however, in multivariate analysis, viral reactivation remained associated with prolonged mv only. when considered separately, both hsv and cmv reactivation were associated with a longer duration of mv as compared to non-reactivation patients [29 (19.5–41) and 28 (20.5–37), respectively, vs. 17.5 (9–28) days; p < 0.05]. co-reactivation patients had a longer duration of mv [58.5 (38–72.3); p < 0.05] and icu stay [51.5 (32.5–69) vs. 27.5 (17.75–35.5) and 29 (20–30.5), respectively] as compared to patients with hsv or cmv reactivation alone. in multivariate analysis, hsv reactivation remained independently associated with a longer duration of mv and hospital length of stay. conclusions: herpesviridae reactivation is frequent among patients with severe ards under veno-venous ecmo and is associated with a longer duration of mechanical ventilation. the direct causative link between hsv and cmv reactivation and respiratory function worsening under ecmo remains to be confirmed. herpes simplex virus (hsv) and cytomegalovirus (cmv) belong to the herpesviridae family and are characterized by an often asymptomatic primo-infection generally during childhood followed by a latency phase. in immunocompromised subjects, herpesviridae are common viral causes of opportunistic infections. but hsv and cmv reactivations are also frequently reported in intensive care unit (icu) non-immunocompromised patients [1, 2] . reactivation ranges from 13 to 64% and 15 to 45% for hsv and cmv, respectively [3, 4] . herpesviridae reactivation in immunocompetent icu patients is associated with poorer outcome [5] . hsv pulmonary reactivation has been described to be associated with a longer mechanical ventilation (mv) duration, icu stay and mortality [2, 6, 7] . cmv reactivation is also associated with a higher mortality, mv duration and icu length of stay [8] . in particular, cmv has been identified as a cause of persistent acute respiratory distress syndrome (ards) [9] and has also been shown to increase the mortality in ards patients [10] . however, despite these associations, the debate on the proper pathogen role of herpesviridae rather than being a witness of patients' severity is still ongoing. studies failed to demonstrate that cmv prophylaxis was able to decrease il-6 plasma levels in cmv seropositive critically ill patients [11] or to decrease mortality [12] . the role of herpesviridae pre-emptive treatment among icu patients has been recently evaluated in a randomized controlled trial (rct) (nct 02152358). the data concerning hsv showed that preemptive acyclovir did not decrease the duration of mv although a trend towards lower mortality was found in treated patients [13] . the most frequent risk factors for cmv and hsv reactivation in the icu are patients severity, sepsis, prolonged mv [14] , high-dose corticosteroid therapy, acute renal failure or massive transfusion [15] , with a strong association for mv and sepsis [16] . patients under veno-venous extracorporeal membrane oxygenation (vv ecmo) for severe ards [17] often combine several or all of these risk factors [18] . despite the uncertainties regarding the exact role of herpesviridae reactivation in immunocompetent critically ill patients, it might add to the pulmonary pathology in patients with ards. in experimental studies, cmv reactivation led to increased pulmonary fibrosis [19] and accessing bacterial pneumonia [20] . these findings suggest that herpesviridae-related pulmonary pathology may be causally linked to the clinical disease course following ards onset, especially in the most severely ill patients who require prolonged mechanical ventilation, and might particularly concern patients under ecmo. however, despite the tight link that seems to exist between herpesviridae, mechanical ventilation and ards, no study has investigated the occurrence of hsv and/or cmv reactivation in patients under vv ecmo. in this study, we aimed to assess the frequency of herpesviruses reactivation during ecmo course and to determine its impact on patients' prognosis. we conducted an observational, retrospective study in a medical icu (ards and ecmo referee center) at the marseille university hospital between december 2011 and april 2017. patients aged 18 or more, hospitalized in the icu for severe ards requiring a vv ecmo for 2 days or more were included. hsv and/or cmv reactivation (see definition below) occurring after ecmo insertion was screened for these patients. patients with immunosuppression (immunosuppressive treatments including corticosteroids > 0.5 mg/kg/day prednisoneequivalent within 30 days prior to inclusion, severe neutropenia < 0.5 g/l of neutrophils, hiv seropositivity, bone marrow or solid organ transplantation), antiviral therapy against hsv and/or cmv prior to inclusion, or hsv/cmv reactivation known at the time of ecmo insertion were excluded. at the time of the study, hsv and cmv screening were routinely performed twice weekly in all patients under mv. hsv reactivation was defined by a positive qualitative throat sample (virocult ® ) pcr. cmv reactivation was defined by a positive quantitative blood pcr with a copy number > 500/ml. when a broncho-alveolar lavage (bal) was performed for suspicion of ventilator-associated pneumonia, hsv and cmv pcr were systematically realized in bal and blood. cmv viral loads were converted in iu/ml and qualified as "high reactivation" for viral loads greater than or equal to 1000 iu/ml or "low reactivation" for viral loads of 100-999 iu/ml [10] . cmv antigenemia was also researched in case of reactivation suspicion. the following data were retrospectively recorded from the patients' medical file: age, sex, simplified acute physiologic score ii (saps ii) [21] , sequential organ failure assessment (sofa) score [22] , presence of co-morbidities, presence of previous immunosuppression, cause of ards, date of mv initiation, date of ecmo implementation, other organ failure associated with ards during icu stay (in particular need for catecholamines or renal replacement therapy), blood transfusion, post-aggressive pulmonary fibrosis (defined by an alveolar procollagen iii higher than 9 µg/l) [23] , time of hsv/cmv reactivation, delay between mv and hsv/cmv reactivation, delay between ecmo and hsv/cmv reactivation, duration of mv (from the day of intubation to the day of mv weaning), ecmo duration (from the day of ecmo implementation to its removal or death), ecmo-free days at day 28, ventilator-free days (vfd) at day 28, icu length of stay [from the day of icu admission (in the first icu if the patient was referred from another hospital) to discharge], hospital length of stay [from the admission to hospital (in the original hospital if the patient was referred from another hospital) to discharge to home or to rehabilitation ward], icu and hospital mortality, acyclovir or ganciclovir treatment after reactivation under ecmo. statistical analysis was performed using ibm spss statistics version 20.0 (ibm spss inc., chicago, il, usa). first, a univariate analysis was performed. data were expressed as mean ± the standard deviation or median with interquartile range for the quantitative variables, and as numbers and percentages for the categorical variables. patient characteristics and clinical outcomes were compared to the viral reactivation status of the patients or antiviral treatment. groups were compared using the chi-square or fisher's exact test for categorical characteristics, and using the student's t test or mann-whitney u test for continuous ones, as appropriate. then a multivariate analysis was performed to assess the independent effect of viral reactivation on different outcomes. multiple linear regression was used to construct models. variables that were marginally significant (p < 0.10) in the univariate analysis, and that had clinical relevance were included in the regression models. beta coefficients and their p values were presented. a two-sided p value less than 0.05 was considered statistically significant. during the study period, 181 patients were admitted to our icu for severe ards requiring a vv ecmo for 2 days or more (see flowchart, fig. 1 ). of these, 58 patients were excluded because of immunosuppression (44 patients), hsv/cmv reactivation at the time of ecmo implementation (10 patients) or acyclovir/ganciclovir treatment before ecmo (4 patients). among the 123 patients included, 67 patients (54%) experienced hsv and/or cmv reactivation during the icu stay and 56 (46%) were free from hsv/cmv reactivation at the time of icu discharge or death. population's characteristics are presented in table 1 . patients with hsv/cmv reactivation had a longer mv before ecmo than non-reactivated patients (p < 0.01). mean cmv viral loads (in blood or bal) were 6916 ± 8934 iu/ml with a high reactivation for 25 (93%) patients. clinical outcomes are presented in table 2 . patients exhibiting hsv/cmv reactivation received more transfusion [11 (7-21.5) vs. 10 (6-15) red cells pellets; p = 0.05]. pulmonary fibrosis, diagnosed by an alveolar procollagen iii > 9 µg/l, was not different between both groups. in univariate analysis, hsv/cmv reactivation was associated with a longer duration of mechanical ventilation [34 (22 when separating patients according to hsv, cmv, and co-reactivation (hsv and cmv), we found that hsv reactivation was associated with a longer duration of in multivariate analysis (table 5) , only hsv reactivation remained independently associated with a longer duration of mv and hospital length of stay but a shorter icu stay. thirty-four patients (51%) received an antiviral treatment (acyclovir or ganciclovir) during ecmo course. no difference in clinical outcomes was found between treated and untreated patients except a trend towards longer duration of mv for treated patients (additional file 1: table s1 ). multivariate analysis evaluating, after adjustment on patients' severity and length of mv and ecmo duration before reactivation, the clinical impact of hsv/ cmv reactivation. the coefficient designates the number of days by which the different endpoints are affected data are presented as median and interquartile range or absolute value and percentage a p < 0.05 compared with non-reactivation group until today, no data have been published concerning herpesviridae reactivation in icu patients under vv ecmo for severe ards. in this retrospective study covering a 5-year period, we found that hsv/ cmv reactivation was frequent and concerned more than half non-immunocompromised patients, which is higher than that described in previous studies including all icu patients [4, 8, 14] . this might be explained by several reasons: the use of pcr to diagnose reactivation with a higher sensitivity than older technics, the age of our cohort of patients (with a high probability of seropositivity for hsv and cmv at icu admission) and the frequency of sepsis with a probable induced "immunoparalysis" [24] . in our cohort, hsv reactivation occurred earlier than cmv reactivation and the median time of reactivation for both viruses was comparable to what is described in "non-ecmo" patients [25] . cmv viral loads in blood and bal were high in almost all patients. elevated cmv viremia is associated with a higher risk of death or prolonged hospitalization [4] . patients included were comparable except for the duration of mv before ecmo that was longer in the reactivation group. it is well known that mv is a risk factor for herpesviridae reactivation with a strong association for cmv [16] . we found that herpesviridae reactivation was associated with a prolonged mv, this association persisting in multi-variate analysis. we also found in these patients a prolonged ecmo duration, icu, and hospital stay, although not confirmed in multivariate analysis. in a recently published meta-analysis, li et al. [8] showed that cmv reactivation was associated with an increase of 9 days in mv and a 12 days increase in icu stay. these results confirmed those published by limaye et al. [4] , which showed that cmv viremia among icu patients was associated with a higher risk of death or prolonged icu stay > 30 days. similarly, in a case-control study [15] , cmv reactivation was associated with a prolonged duration of mv and icu stay. in a specific population of ards patients, ong et al. [10] demonstrated that patients with cmv reactivation had a 15 (10-26) days median duration of mv as compared to 8 (6-12) days for non-reactivated patients. icu length of stay was also longer [16 (11-28) vs. 9 (7) (8) (9) (10) (11) (12) (13) (14) days] for reactivated patients. same results have been published concerning hsv reactivation [2, 6] , especially during ards. our findings suggest that herpesviridae reactivation is associated with worse outcomes for ards patients including when they are under ecmo. when examining the impact of each virus separately, we found that both hsv and cmv were associated with a prolonged mv, and also ecmo duration for hsv. coreactivation had a negative effect not only on mv and ecmo duration but also on icu and hospital stay, as compared to patients free from reactivation or with only one virus. hsv was independently associated with a longer duration of mv and hospital length of stay but, surprisingly, a shorter icu stay. these results highlight the potential negative role of hsv in ards patients under ecmo. very recently, luyt et al. [13] showed that preemptive treatment with acyclovir, compared to placebo, for mechanically ventilated patients with oropharyngeal hsv reactivation, was not associated with shorter mv duration. however, a trend towards lower day-60 mortality was observed in the acyclovir group. in our cohort, more than half of the patients were treated after the diagnosis of viral reactivation. treatment with acyclovir or ganciclovir did not improve the outcomes, with a trend for longer duration of mv in the sub-group of treated patients. these results might be explained by the fact that anti-viral treatment was decided by clinicians more frequently in case of worsening respiratory status, persisting fever or end-organ hsv/cmv disease, and so reserved for the most severe patients. we did not find any increase in renal failure in patients receiving antiviral drugs, which was also noticed in luyt et al. 's study [13] . however, we cannot exclude any other side effects. our study has some limitations. first, the retrospective design of our cohort, counterbalanced by the important number of patients included during this 5-year period. second, the applicability of our results to the general population of patients under ecmo must be considered cautiously considering the high rate of patients treated with antiviral drugs after reactivation. however, in non-emco patients, routine screening of herpesviridae has been reported as well as the use of antiviral treatment despite the lack of recommendation [3, 15, 16] . third, few patients developed an isolated cmv reactivation. this precludes to conclude clearly on the specific impact of cmv in our cohort of patients. fourth, our methods do not prevent competing risks. in particular, the difference in mv duration between reactivated and non-reactivated patients might have been influenced by the high mortality reported. however, this mortality was similar in both groups and the difference of mv duration persisted when considering only the patients discharged alive from the icu. finally, despite the statistical association, it is not possible to conclude whether herpesviridae reactivation is directly responsible for worse clinical outcomes or if it is a consequence and a witness of the severity of the disease, as in non-ecmo populations [3] . herpesviridae reactivation is frequent among patients with severe ards under veno-venous ecmo and is associated with a prolonged mechanical ventilation. this association is present for hsv as well as cmv and also for co-reactivation. the direct causative link between hsv and cmv reactivation and respiratory function worsening under ecmo remains to be confirmed. supplementary information accompanies this paper at https ://doi. org/10.1186/s1361 3-019-0616-6. additional file 1: table s1 . clinical outcomes according to anti-viral treatment. abbreviations ards: acute respiratory distress syndrome; bal: broncho-alveolar lavage; cmv: cytomegalovirus; ecmo: extracorporeal membrane oxygenation; hiv: human immunodeficiency virus; hsv: herpes simplex virus; icu: intensive care unit; il-6: interleukine 6; iu/ml: international units/milliliter; mv: mechanical ventilation; pcr: polymerase chain reaction; saps ii: simplified acute physiologic score ii; sofa: sequential organ failure assessment; vfd: ventilator free days; vs.: versus; vv: veno-venous. active cytomegalovirus infection is common in mechanically ventilated medical intensive care unit patients herpes simplex virus lung infection in patients undergoing prolonged mechanical ventilation cytomegalovirus reactivation in icu patients cytomegalovirus reactivation in critically-ill immunocompetent patients cytomegalovirus and herpes simplex virus effect on the prognosis of mechanically ventilated patients suspected to have ventilator-associated pneumonia herpes simplex virus load in bronchoalveolar lavage fluid is related to poor outcome in critically ill patients is acyclovir effective among critically ill patients with herpes simplex in the respiratory tract? cytomegalovirus infection and outcome in immunocompetent patients in the intensive care unit: a systematic review and meta-analysis a contributive result of open-lung biopsy improves survival in acute respiratory distress syndrome patients cytomegalovirus reactivation and mortality in patients with acute respiratory distress syndrome effect of ganciclovir on il-6 levels among cytomegalovirus-seropositive adults with critical illness: a randomized clinical trial safety and efficacy of antiviral therapy for prevention of cytomegalovirus reactivation in immunocompetent critically ill patients: a randomized clinical trial acyclovir for mechanically ventilated patients with herpes simplex virus oropharyngeal reactivation: a randomized clinical trial prevalence and mortality associated with cytomegalovirus infection in nonimmunosuppressed patients in the intensive care unit cytomegalovirus infection in critically ill patients: associated factors and consequences cytomegalovirus infection in immunocompetent critically ill adults: literature review formal guidelines: management of acute respiratory distress syndrome long-term neurocognitive outcome is not worsened by of the use of venovenous ecmo in severe ards patients pulmonary cytomegalovirus reactivation causes pathology in immunocompetent mice cytomegalovirus reactivation enhances the virulence of staphylococcus aureus pneumonia in a mouse model a new simplified acute physiology score (saps ii) based on a european/north american multicenter study the sofa (sepsis-related organ failure assessment) score to describe organ dysfunction/failure. on behalf of the working group on sepsis-related problems of the european society of intensive care medicine type iii procollagen is a reliable marker of ards-associated lung fibroproliferation early herpes and ttv dnaemia in septic shock patients: a pilot study cytomegalovirus reactivation and associated outcome of critically ill patients with severe sepsis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we acknowledge mrs claudine marion and sabine depetris for their help.authors' contributions sh, eb, cg, ma, gp, gc and bc collected and analyzed the data. sh, eb, cg, ar and lp analyzed and interpreted more precisely the data. cf, al, sh and cg performed the statistical analysis. sh, eb and lp wrote the manuscript. all authors read and approved the final manuscript. the authors received no funding for this work. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. patients and their relatives were informed of the possibility of the use of medical data for retrospective studies and did not manifest opposition. the study was approved by the "portail d'accès aux données de santé de l' assistance publique des hopitaux de marseille" ("règlement général pour la protection des données" registration number 2019-132). not applicable. the authors declare that they have no competing interests. key: cord-001690-cn21fgug authors: franceschi, valentina; parker, scott; jacca, sarah; crump, ryan w.; doronin, konstantin; hembrador, edguardo; pompilio, daniela; tebaldi, giulia; estep, ryan d.; wong, scott w.; buller, mark r.; donofrio, gaetano title: bohv-4-based vector single heterologous antigen delivery protects stat1((-/-)) mice from monkeypoxvirus lethal challenge date: 2015-06-18 journal: plos negl trop dis doi: 10.1371/journal.pntd.0003850 sha: doc_id: 1690 cord_uid: cn21fgug monkeypox virus (mpxv) is the etiological agent of human (mpx). it is an emerging orthopoxvirus zoonosis in the tropical rain forest of africa and is endemic in the congo-basin and sporadic in west africa; it remains a tropical neglected disease of persons in impoverished rural areas. interaction of the human population with wildlife increases human infection with mpx virus (mpxv), and infection from human to human is possible. smallpox vaccination provides good cross-protection against mpx; however, the vaccination campaign ended in africa in 1980, meaning that a large proportion of the population is currently unprotected against mpxv infection. disease control hinges on deterring zoonotic exposure to the virus and, barring that, interrupting person-to-person spread. however, there are no fda-approved therapies against mpx, and current vaccines are limited due to safety concerns. for this reason, new studies on pathogenesis, prophylaxis and therapeutics are still of great interest, not only for the scientific community but also for the governments concerned that mpxv could be used as a bioterror agent. in the present study, a new vaccination strategy approach based on three recombinant bovine herpesvirus 4 (bohv-4) vectors, each expressing different mpxv glycoproteins, a29l, m1r and b6r were investigated in terms of protection from a lethal mpxv challenge in stat1 knockout mice. bohv-4-a-cmv-a29lgd(106)δtk, bohv-4-a-ef1α-m1rgd(106)δtk and bohv-4-a-ef1α-b6rgd(106)δtk were successfully constructed by recombineering, and their capacity to express their transgene was demonstrated. a small challenge study was performed, and all three recombinant bohv-4 appeared safe (no weight-loss or obvious adverse events) following intraperitoneal administration. further, bohv-4-a-ef1α-m1rgd(106)δtk alone or in combination with bohv-4-a-cmv-a29lgd(106)δtk and bohv-4-a-ef1α-b6rgd(106)δtk, was shown to be able to protect, 100% alone and 80% in combination, stat1((-/-)) mice against mortality and morbidity. this work demonstrated the efficacy of bohv-4 based vectors and the use of bohv-4 as a vaccine-vector platform. monkeypox virus (mpxv) is an orthopoxvirus with a broad-host range capable of infecting many animal species [1] . in humans, mpxv causes a disease very similar to the closely related variola virus, the etiological agent of smallpox: a rash (a 2-4 week period where macules develop and form papules, vesicles and pustules) which is preceded by a 10-14 day incubation, followed by a 1-3 day interval characterized by a prodromal fever, malaise and severe lymphadenopathy of the neck, inguinal and axillary regions [2] [3] [4] . the more virulent strains of mpxv (from the congo basin region) can induce mortality rates of~10% [5] [6] [7] [8] . the main clinical difference between human mpx and smallpox is the presentation of lymphadenopathy in the former [9, 10] . mpxv is endemic to central and west africa with increasing numbers of human infections being reported. in 2003, the transmissibility of mpxv became acutely obvious when an outbreak occurred in the united states and mpxv was transmitted to humans from prairie dogs [11, 12] . the global vaccination campaign that eradicated smallpox utilized vaccines that are also efficacious against mpxv and was successful because there is no animal reservoir for variola [13] . indeed, prior to the eradication of smallpox, the existence of mpxv was unknown and it is likely that many cases of human mpx were reported as smallpox. cases of human mpx are increasing in africa, this may be due to several factors: 1) increasing interactions between humans and infected animals through environmental degradation; 2) the cessation of routine vaccination against smallpox; 3) an increase in human susceptibility to the virus; 4) an increase in animal-human and human-human transmissibility of the virus; and 5) adaption of the virus to new host species that co-exist in, or close to, human geographical regions [2, [14] [15] [16] [17] [18] . the high mortality rate and morbidity rate of mpxv in humans makes it an important emerging disease for study. to date, no antiviral has been fda-approved for the treatment of human mpx. vaccines against smallpox have demonstrated good protection against mpxv in animal models and anecdotally in humans [1] ; however, most first-and second-generation smallpox vaccines are associated with at least some level of morbidity and a large proportion of the population are contraindicated to vaccination [19] . third-generation vaccines, such as mva, have been shown to be safer in non-human primates but require 2 the a29l, m1r and b6r orfs, were amplified from monkeypox dna (strain v79-i-005; accession: hq857562.1) using primer pairs listed in table 1 . the sense primer contains at its 5 0 -end the nhei restriction site along with the kozak's sequence preceding the atg, for a better translation initiation, whereas the antisense primer contains at its 5 0 -end a sali restriction site for the in-frame cloning of the orf with a gd106 tag peptide present in pcmv-igk-vp2-gd 106 plasmid vector. pcmv-igk-vp2-gd 106 has the human cytomegalovirus immediate early gene (cmv) promoter, the immunoglobulin kappa light chain leader sequence (igk), the bluetongue virus vp2 orf, the gd 106 epitope of bovine herpesvirus 1 glycoprotein d, successfully used as a tag during the cloning, and the bovine growth hormone polyadenylation site [37] . the 3 orfs were cut with nhei/sali and cloned into pcmv-igk-vp2-gd 106 cut with the same enzymes. this strategy allowed the substitution of the igk-vp2 sequence with those of the 3 orfs to generate pcmv-a29lgd 106 , pcmv-m1rgd 106 and pcmv-b6rgd 106 . to generate ptk-cmv-a29lgd 106 -tk, pcmv-a29lgd 106 was cut with bamhi, treated with t4 dna polymerase for blunt ending and cut with nhei; a29lgd 106 (442 bp) was excised from the pcmv-a29lgd 106 and inserted into the shuttle vector pint2egfp [25] cut with nhei/smai to replace gfp orf with the chimeric protein. pint2egfp contains two bohv-4 thymidine kinase gene homology regions flanking the green fluorescent protein (gfp) expression cassette driven by the cmv promoter, cutting with nhei/smai the gfp orf was replaced with that of a29lgd 106 . to generate pef1α-m1rgd 106 and pef1α-b6rgd 106 , the cmv promoter of pcmv-m1rgd 106 and pcmv-b6rgd 106 was substituted with that of the human elongation factor 1 alpha (ef1α). ef1α was amplified by pcr from pwpi plasmid vector (addgene plasmid #12254). pwpi was first linearized with pmei, then the pcr reaction was carried out with 0.25 μm of a couple of primers (ef1α-ndei-sense and ef1α-nhei-antisense; table 1 ) in a final volume of 50 μl containing 10 mm tris-hydrochloride ph 8.3, 0.2 mm deoxynucleotide triphosphates, 3 mm mgcl2, 50 mm kcl and 5% dmso. each cycle of 35, consisted of denaturation at 94°c for 1 min, primer annealing at 50°c for 1 min and elongation for 90 sec with 1u of pfu dna polymerase at 72°c. the 1237 bp ef1α amplification product was checked in 1% agarose gel and visualized after ethidium bromide staining in 1× tae buffer (40 mm tris-acetate, 1 mm edta). ptk-ef1α-m1rgd 106 -tk and ptk-ef1α-b6rgd 106 -tk constructs were obtained by subcloning ef1α-m1rgd 106 and ef1α-b6rgd 106 expression cassettes, cut with ndei/mlui and blunted-end with t4 dna polymerase, from pef1α/m1rgd 106 and ef1α/b6rgd 106 respectively in smai cut pint2 [38] . all enzymes were purchased from thermo scientific. confluent hek293t cells were seeded into 6 well plates (3x10 5 cells/well) and incubated at 37°c with 5% co 2 ; when the cells were sub-confluent the culture medium was removed and the cells were transfected with ptk-cmv-a29lgd 106 -tk, ptk-ef1α-m1rgd 106 -tk or ptk-ef1α-b6rgd 106 -tk using polyethylenimine (pei) transfection reagent (polysciences, inc.). briefly, 3 μg of dna were mixed with 7.5 μg pei (1mg/ml) (ratio 1:2.5 dna-pei) in 200 μl of dulbecco's modified essential medium (dmem) at high glucose percentage (euroclone) without serum. after 15 min at rt, 800 μl of medium without serum was added and the transfection solution was transferred to the well and left on the cells for 6 h at 37°c with 5% co 2 in air, in a humidified incubator. the transfection mixture was then replaced with fresh medium (emem, with 10% fbs, 50 iu/ml of penicillin, 50 μg/ml of streptomycin and 2.5 μg/ml of amphotericin b) and incubated for 24 h at 37°c with 5% co 2 . bohv-4-a, bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk were propagated by infecting confluent monolayers of bek and vero cells at a multiplicity of infection (moi) of 0.5 50% tissue culture infectious doses (tcid 50 ) per cell and maintained in medium with only 2% fbs for 2 h. the medium was then removed and replaced with fresh emem containing 10% fbs. when the cytopathic effect (cpe) occurred in the majority of the cell monolayer (*72 h post infection), the virus was prepared by freezing and thawing cells three times and pelleting the virions through a 30% sucrose cushion, as described previously [39] . virus pellets were then resuspended in cold emem without fbs. tcid 50 were determined with bek cells by limiting dilution. a plaque-purified isolate of the mpxv strain zai-79 [40] was purified through a sucrose cushion [41] and propagated in bs-c-1 cells [42] . virus infectivity was estimated as described previously [43] . briefly, virus suspensions were serially diluted in pbs +1% fcs (fetal clone ii, hyclone), absorbed to monolayers for 1 hour at 37°c, and overlaid with a suspension of 1% carboxyl methyl cellulose in dmem +5% fcs. after 2 days at 37°c, virus plaques were visualized and virus inactivated by the addition to each well of a 0.3% crystal violet/10% formalin solution. protein cell extracts were obtained from a 6-well confluent plate of hek293t transfected with pint2/cmva29lgd 106 , pint2/ef1αm1rgd 106 or pint2/ef1αb6rgd 106 and from 25-cm 2 confluent flasks of bek cells or vero cells infected with bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk by adding 100 μl of cell extraction buffer (50 mm tris-hcl, 150 mm nacl, and 1% np-40; ph 8). a 10% sds-page gel electrophoresis was used to analyze cell extracts containing 50 μg of total protein, after protein transfer in nylon membranes by electroblotting, the membranes were incubated with bovine anti gd 106 monoclonal antibody (clone 1b8-f11; vrmd, inc., pullman, wa) antibody, probed with horseradish peroxidase-labelled anti-mouse immunoglobulin antibody (sigma) and visualized by enhanced chemiluminescence (ecl kit; pierce). recombineering was performed as previously described [44] with some modifications. five hundred microliters of a 32°c overnight culture of sw102 containing bac-bohv-4-a-kanagalkδtk, were diluted in 25 ml luria-bertani (lb) medium with or without chloramphenicol (sigma) selection (12.5 mg/ml) in a 50 ml baffled conical flask and grown at 32°c in a shaking water bath to an od 600 of 0.6. then, 10 ml were transferred to another baffled 50 ml conical flask and heat-shocked at 42°c for exactly 15 min in a shaking water bath. the remaining culture was left at 32°c as the uninduced control. after 15 min the two samples, induced and uninduced, were briefly cooled in ice/water bath slurry and then transferred to two 15ml falcon tubes and pelleted using 5000 r.p.m. (eppendorf centrifuge) at 0°c for 5 min. the supernatant was poured off and the pellet was resuspended in 1ml ice-cold ddh 2 o by gently swirling the tubes in ice/water bath slurry. subsequently, 9 ml ice-cold ddh 2 o were added and the samples pelleted again. this step was repeated once more, the supernatant was removed and the pellet (50 μl each) was kept on ice until electroporated with gel-purified fragments (*3.3, *4.4 and *4.6 kb respectively for tk-cmv-a29lgd 106 -tk, tk-ef1α-m1rgd 106 -tk and tk-ef1α-b6rgd 106 -tk) obtained by cutting ptk-cmv-a29lgd 106 -tk, ptk-ef1α-m1rgd 106 -tk and ptk-ef1α-b6rgd 106 -tk with clai/pvuii (thermo scientific). an aliquot of 25 μl (~200 ng) was used for each electroporation in a 0.1 cm cuvette at 25 μf, 2.5 kv and 201o. after electroporation, for the counter selection step, the bacteria were recovered in 10 ml lb in a 50 ml baffled conical flask and incubated for 4.5h in a 32°c shaking water bath. bacteria serial dilutions were plated on m63 minimal medium plates containing 15 g/l agar, 0.2% glycerol, 1mg/l d-biotin, 45 mg/l l-leucine, 0.2% 2-deoxy-galactose and 12.5 mg/ml chloramphenicol. all the complements for m63 medium were purchased from sigma. plates were incubated 3-5 days at 32°c; then several selected colonies were picked up, streaked on mcconkey agar indicator plates (difco, bd biosciences) containing 12.5 g/ml of chloramphenicol and incubated at 32°c for 3 days until white colonies appeared. white colonies were grown in duplicate for 5-8h in 1ml of lb containing 50 mg/ml of kanamycin (sigma) or lb containing 12.5 mg/ml of chloramphenicol. only those colonies that were kanamycin negative and chloramphenicol positive were kept and grown overnight in 5ml of lb containing 12.5 mg/ml of chloramphenicol. bac dna was purified and analyzed through hindiii restriction enzyme digestion for tk-cmv-a29lgd 106 -tk, tk-ef1α-m1rgd 106 -tk and tk-ef1α-b6rgd 106 -tk fragment targeted integration, was separated by electrophoresis overnight in a 1% agarose gel, stained with ethidium bromide, capillary transferred to a positively charged nylon membrane (roche), and cross-linked by uv irradiation by standard procedures [28] . hybridization with digoxigenin-labeled probes confirmed the identity of specific restriction fragments. the 353, 573, 977 bp amplicons for a29l, m1r and b6r probes were generated by pcr with the primers: a29l sense/antisense, m1r sense/antisense, and b6r sense/antisense listed in table 1 , as previously described [29] . original detailed protocols for recombineering can also be found at the recombineering website (http://recombineering.ncifcrf.gov). bek or bekcre cells were maintained as a monolayer with complete dmem growth medium with 10% fbs, 2 mm l-glutamine, 100 iu/ml penicillin and 10 mg/ml streptomycin. when cells were sub-confluent (70-90%) they were split to a fresh culture vessel (i.e., every 3-5 days) and were incubated at 37°c in a humidified atmosphere of 95% air-5% co2. bac dna (5 μg) was electroporated in 600 μl dmem without serum (equibio apparatus, 270 v, 960 mf, 4-mm gap cuvettes) into bek and bekcre cells from a confluent 25-cm 2 flask. electroporated cells were returned to the flask, after 24h the medium was replaced with fresh medium, and cells were split 1:2 when they reached confluence at 2 days post-electroporation. cells were left to grow until the appearance of cpe. bek cells were infected with bohv-4-a, bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk at a m.o.i. of 0.1 tcid50/cell and incubated at 37°c for 4 h. infected cells were washed with serum-free emem and then overlaid with emem containing 10% fbs, 2 mm lglutamine, 100 iu/ml penicillin, 100 mg/ ml streptomycin and 2.5-mg/ml amphotericin b. the supernatants of infected cultures were harvested after 24, 48, 72 and 96 h, and the amount of infectious virus was determined by limiting dilution on bek cells by the tcid50 method. eight-week old female 129 stat1 -/mice were bred in-house and housed in filter-top microisolator cages and fed commercial mouse chow and water ad libitum. the randomized mice were housed in an animal biosafety level 3 containment area, with 5 mice/group. animal husbandry and experimental procedures were approved by the institutional animal care and use committee. mice were monitored every day until the termination of the experiment. bohv-4s vectors were injected intraperitoneally (ip) in a total volume of 300 μl with dmem used as a vehicle. for vaccinations with one vector, injections were comprised of 100 #x03bc;l of vector + 200 μl of vehicle. for the combination injections, 100 #x03bc;l of each vector was included for a total of 300 #x03bc;l injections were given as a primary vaccination at t = 0 days and as a booster vaccination at t = 23 days (see table 2 ). each vector was injected at a modified vaccinia ankara (mva) (a gift from the niaid-nih, bethesda, md) was provided at a dose of 2x10 8 plaques forming units (pfu)/ml and was injected in 0.1 ml between the skin and underlying layers of tissue in the scapular region on the backs of mice. mice were anesthetized with 0.1 ml/10 g body weight of ketamine hcl (6 mg/ml) and xylazine (0.5 mg/ml) by intraperitoneal injections. anesthetized mice were laid on their dorsal side with their bodies angled so that the anterior end was raised 45°from the surface; a plastic mouse holder was used to ensure conformity [45] . mpxv was diluted in pbs without ca 2+ and mg 2+ to the required concentration and slowly loaded into each nare (5 #x03bc;l/nare). mice were subsequently left in situ for 2-3 minutes before being returned to their cages. paired t-tests (tailed) were used to compare means between groups of mice. mortality rates were compared using the fisher's exact test. blinded lesion pictures were measured qualitatively using a scoring system ranging 0-4 in severity. p values <0.05 were considered statistically significant. among the approximately 200 genes that comprise the monkeypox virus genome, only few genes encoding antigenic proteins-that are known-are able to elicit a neutralizing antibody response in vaccinated animals. among these antigens, a29l, m1r and b6r orthologs were selected as candidate antigens to be delivered by bohv-4 based-vector. a29l, m1r and b6r orfs were amplified by pcr from a cosmid library and sub-cloned in frame with a tag peptide, gd106 [46] (fig 1a, 1e and 1h) , which was contained in a mammalian expression vector plasmid construct. m1r and b6rgd 106 tagged orfs were placed under the transcriptional control of the ef1α promoter (fig 1f and 1i ), whereas a29lgd 106 tagged orf under the transcriptional control of the cmv promoter ( fig 1b) . so generated expression cassettes (cmv-a29lgd 106 , ef1α-m1rgd 106 and ef1α-b6rgd 106 ) were validated, in terms of protein expression, by transient transfection in 293t cells and western-immunoblotting with a monoclonal antibody directed against the gd 106 tag peptide. a29l, m1r and b6rgd 106 tagged antigens were all well expressed in the whole cell extracts of the transiently transfected cells (fig 1c, 1g and 1j), further a29lgd 106 was also secreted (fig 1d) in the supernatant of the transiently transfected cells. construction of bohv-4s-based vector expressing a29l, m1r and b6rgd106 tagged antigens an apathogenic strain of bohv-4 cloned as a bac was used to create a bohv-4-a-based vector [28] . the tk bohv-4-a genome locus was utilized as the integration site for the cmv-a29lgd 106 , ef1α-m1rgd 106 and ef1α-b6rgd 106 expression cassettes. the bohv-4 tk genomic region is strongly conserved among bohv-4 isolates [47] , ensuring the stability of the genomic locus from potential recombination when foreign dna sequences are inserted in. indeed, the bohv-4 tk genomic locus has been interrupted by the insertion of foreign dna sequences of different size, without interfering with viral replication in vitro and loss of heterologous protein expression [25,27-29,31,32,34]. cmv-a29lgd 106 , ef1α-m1rgd 106 and ef1α-b6rgd 106 expression cassettes were first sub-cloned into pint2, a shuttle vector plasmid containing 2 bohv-4 tk sequences [25] , to generate ptk-cmv-a29lgd 106 -tk, ptk-ef1α-m1rgd 106 -tk and ptk-ef1α-b6rgd 106 -tk targeting vectors. restriction enzyme linearized targeting vectors were used for heat-inducible homologous recombination sw102 e. coli containing pbac-bohv-4-a-kanagalkδtk [28, 39, 48] (fig 2a) to generate pbac-bohv-4-a-cmv-a29lgd 106 δtk, pbac-bohv-4-a-ef1α-m1rgd 106 δtk and pbac-bohv-4-a-ef1α-b6rgd 106 δtk. selected clones were first analyzed by hindiii restriction enzyme digestion and then by southern blotting (fig 2b) . because heat-inducible recombination in sw102 e. coli and repeated passages could establish altered bacterial phenotypes due to an aberrant recombenases transcription, sw102 e. coli carrying pbac-bohv-4-a-cmv-a29lgd 106 δtk, pbac-bohv-4-a-ef1α-m1rgd 106 δtk and pbac-bohv-4-a-ef1α-b6rgd 106 δtk were serially cultured over for 20 passages and checked by hindiii restriction enzyme digestion. no differences among restriction patterns at various passages were detected (s1 fig), thus ensuring the stability of the clones. infectious viable bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk were obtained by electroporating pbac-bohv-4-a-cmv-a29lgd 106 δtk, pbac-bohv-4-a-ef1α-m1rgd 106 δtk and pbac-bohv-4-a-ef1α-b6rgd 106 δtk dna into bek and bekcre cells. the only difference was that the recombinant viruses reconstituted from electroporated bekcre resulted in depletion of the bac plasmid backbone containing the gfp expression cassette, as shown by the loss of gfp expression (fig 3a, 3e and 3h ). because the time necessary to reconstitute the viable recombinant bohv-4s was different among them, it was of interest to know if the foreign antigens, encoded by the expression cassette integrated into the viral genome, could have a detrimental effect on the viral replication. therefore, the growth characteristics of bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk were compared with that of the parental virus, bohv-4-a. although bohv-4-a-cmv-a29lgd 106 δtk and bohv-4-a-ef1α-m1rgd 106 δtk demonstrated a slower replication kinetics respect to bohv-4-a, they reached the same viral titer at the end-point (~10 6 ) (fig 3b and 3f) . whereas bohv-4-a-ef1α-b6rgd 106 δtk replication was drastically impaired, a 2 log reduction of the viral titer end-point (~10 4 ) respect to bohv-4-a was observed ( fig 3i) ; however, transgene expression was well detected in the whole cell extract of bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk infected cells (fig 3c, 3g and 3j). further, a29lgd 106 glycoprotein was found to be expressed as a secreted protein in the supernatant of bohv-4-a-cmv-a29lgd 106 δtk infected cells (fig 3d) . in vivo efficacy testing of bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk to test the efficacy of the vectors in vivo, we sought to determine if they could protect mice against a lethal challenge with mpxv. several murine strains have been developed as models of mpxv, in this study we utilized the 129 stat1 -/strain. thirteen cages were prepared with 5 mice/cage ( table 2) . cages 1 and 2 were un-vaccinated. cage 3 was vaccinated with vehicle (dmem without fbs). cages 4 and 5 were vaccinated with mva where cage 4 received a primary injection of vaccine at t = 0 days and a vehicle booster at t = 23 days, and cage 5 cages 12 and 13 were also vaccinated following the above regimen; however, these mice received a combination (combo) of the 3 bohv-4 vectors. bohv-4 vectors were injected ip in a total volume of 0.3 ml. there was no apparent morbidity-as measured visually-or weightloss recorded in any of the mice (s2 fig). at t = 50 days, mice in cages 2-13 were challenged with 2x10 5 pfu/mouse of mpxv. mortality rates are shown in fig 4. as expected, the mva vaccinated mice in cages 4 (mva/ veh) and 5 (mva/mva) were 100% protected against challenge. mice in cage 9 (m1r/m1r) were also 100% protected (p = 0.004); and although mice in cage 13 (combo/combo) experienced 1 death, they were still 80% protected against the mpxv challenge (p = 0.02). when comparing weight-change (fig 5) , we found that mice in cage 5 (mva/mva) did not lose weight compared to the pbs control (cage 1) and that mice in cage 4 (mva/veh) only lost weight (5%) on days 6, 7 and 8 post challenge. we also found that mice in cages 10 (b6r/ veh), 11 (b6r/b6r), 12 (combo/veh), and 13 (combo/combo) had significantly reduced weight-loss from day 8 post challenge (15%, 15%, 11%, and 15% on day 8, respectively), even though mice in cage 13 were 80% protected against the challenge. some protection from weight-loss was also afforded to mice in cages 8 (m1r/veh) and 9 (m1r/m1r) on days 13, 14 and 15 post challenge (15%). these data indicate that the presence of m1r is required to protect the mice against challenge, and that a booster vaccination is required. it also indicates that a combination of the 3 vectors improves protection against weight-loss. although m1r could provide protection, it was inferior to that provided by vaccination with mva. the data also indicate that although b6r does not provide protection against mortality, it does provide protection against weightloss in surviving mice (see above). the aim of the current study was to ascertain the potential utility of bovine herpesvirus 4 (bohv-4)-based vectors as safe, potent, large-capacity vaccine vectors for category a agents-mpxv for this specific case. this study demonstrated protection in the stat1 -/model and consideration should be given to also evaluating the vectors in cast/eij mice which have also been established as a murine model of monkeypox [49, 50] . ultimately, this study could pave the way for further studies in other animal models such as prairie dogs (cynomys ludovicianus) [51] [52] [53] , the 13-lined ground squirrel (spermophilus tridecemlineatus) [54, 55] and non-human primates (reviewed in [1] )-all of which have been extensively used as models of human monkeypox-and ultimately in human protection studies. in fact, since mpxv also infects humans and causes clinical-signs very similar to smallpox, it is classified as a category a select agent [56] . primarily for ethical but also for cost reasons, c57bl/6 stat1 knockout mice (stat1 (-/-) ) were chosen as an in vivo model before progressing to gene delivery and immunogenicity studies in non-human primates. stat1 (-/-) miceare highly sensitive to mpxv and the disease course in mpxv infected stat1 (-/-) mice, characterized by weight loss and death by day 10 post infection-is similar to that observed in wild-type mice infected with mousepox/ectromelia virus (ectv), the etiology agent of mousepox-and probably the best small animal model of smallpox [35, 57] . moreover it was revealed that antiviral therapy could protect mice to a degree similar to that of vaccination with dryvax or mva, supporting the use of stat1 (-/-) mice as a reliable model to evaluate orthopoxvirus prophylactics and therapeutics [35] . stat1 (-/-) mice were found to be sensitive to a wide number of pathogens due to the loss of stat1, a key factor responsible for type 1 and 2 interferon (ifn) signaling [58] [59] [60] [61] [62] [63] . bohv-4-based vectors in wild type and immunocompromised mice behave as replicating incompetent viral vectors, showing absence of pathogenicity [31,32, [64] [65] [66] and bohv-4 replication is strongly impaired in vitro after the treatment of bohv-4 infected cells with ifnγ [67] . considering that ifn-γ is an activator of stat1, the potential pathogenicity of bohv-4-based vectors in stat1 (-/-) mice was a concern. however, intra peritoneal bohv-4 inoculated stat1 (-/-) mice did not show any overt clinical sign, detrimental effect or pathology correlated to challenge. a29l, m1r, and b6r mpxv antigens were selected, as candidate antigens to be delivered by bohv-4-based vectors, as they are orthologous to a27l, l1r and b5r vaccinia virus (vv) antigens respectively. a27l is a 14 kda protein thought to be involved in vv entry events [68] . l1r is a 23-29 kda myristoylated surface protein involved in a yet-to-be-identified post viral attachment and pre-fusion events [69] . whereas b5r is a 42 kda glycoprotein found on the surface of the virus [70, 71] and is involved in cell surface glycosaminoglycan-mediated disruption of the viral outer membrane [72] . further, they were shown to be able to elicit a protective immune response in mice and non-human primates when formulated in combination as a subunit vaccine consisted of purified proteins, plasmid dna vaccines, recombinant adenovirus and alphavirus replicons [73] [74] [75] [76] [77] [78] [79] . since the purpose of this study was to determine the capability of bohv-4-based viral vectors to protect stat1 (-/-) mice against a lethal mpxv infection, the first concern was the generation of optimized expression cassettes to be integrated into the bac-bohv-4-a genome that were able to efficiently express a29l, m1r and b6r antigens. because no antibodies are available for a29l, m1r and b6r proteins, a short in-frame sequence coding for a tag peptide (gd 106 ) was provided at the 3 0 end of their orfs and this allowed their expression to be monitored by western immunoblotting. initially a29l, m1r and b6r tagged orfs were customized under the transcriptional control of the cmv promoter; however, the only orf to be efficiently expressed was a29l. for this reason, the cmv promoter of the m1r and b6r expression cassette was substituted with the human ef1α promoter which induced expression. the reason why the cmv promoter did not work with the orfs of m1r and b6r has not been determined. another interesting observation was the presence of a29l protein into the supernatant of the transfected cells despite the absence of a canonical signal peptide within the primary sequence of the protein as deduced by different signal peptide prediction software (http://www.csbio.sjtu.edu.cn/bioinf/signal-3l/; http:// www.cbs.dtu.dk/services/signalp/; http://phobius.sbc.su.se/) [80, 81] . in fact, the a29l protein analysis by secretomep (http://www.cbs.dtu.dk/services/secretomep), a sequence based method for the prediction of mammalian secretory proteins targeted to the non-classical secretory pathway, included a29l within the group of non-classical secreted proteins like fibroblast growth factors, some interleukins and galectins. bohv-4 is considered a virus without a clear disease association, the existence of a bohv-4 potentially pathogenic biotype cannot be absolutely excluded when a virus is going to be exploited as a gene delivery vector. bovine herpesvirus 4 (bohv-4) has been most consistently associated with uterine disease in postpartum cattle and bohv-4 infection is often identified concurrently with bacteria that cause uterine diseases [82, 83] . the association between bohv-4 infection and uterine disease has been difficult to establish. it was suggested that there may be an association with bacterial endometritis which leads to secretion of prostaglandin e2 (pge2) and then stimulation of viral replication by pge2, tnf-α and lipopolysaccharide (lps)-which causes further endometrial tissue damage and inflammation [84] [85] [86] . for this reason, a putative non-pathogenic strain of bohv-4 (bohv-4-a) isolated from the cell milk fraction of a healthy cow whose genome was cloned as a bacterial artificial chromosome (pbac-bohv-4-a) [28] was used. cmv-a29lgd 106 , ef1α-m1rgd 106 and ef1α-b6rgd 106 were integrated into the tk locus of pbac-bohv-4-a, and proved to be stable through passages in e. coli sw102 and recombinant viable bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk, bohv-4-a-ef1α-b6rgd 106 δtk were successfully reconstituted in bekcre cells. when bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk were characterized in terms of replication kinetics, a reduction of replication was observed for bohv-4-a-ef1α-b6rgd 106 δtk and this was attributed to a partial toxic effect induced by the abundant expression of b6rgd 106 -which was also observed in transfections of cells with ef1α-b6rgd 106 expression cassette. this latter observation excluded a potential detrimental effect induced by the topological location of the foreign dna in the bohv-4 genome. despite their replication characteristics, all three recombinant bohv-4s abundantly expressed their transgene in infected cells. in vivo protection studies determined that m1r protected against a lethal mpxv challenge. one hundred % protection was achieved when the vectors were administered twice (prime followed by booster), although the m1r expression vector was not superior to vaccination with mva as measured by weight-loss. protection was also afforded to mice when the vectors were administered in combination (combo) as a prime and booster. these mice experienced less weight-loss than mice vaccinated with m1r alone. this finding is surprising as the other vectors included in the combo were not protective when administered individually, although we did find protection against weight-loss when mice were treated with b6r alone. nevertheless, our studies reveal that protection can be afforded even when a small number of mice are used. further studies should be considered that increase the dose of vector administered to the mice. also, since the combination of all 3 vectors gave 80% protection against mortality and morbidity, various vector permutations should be considered to elucidate the most efficacious combination and ratio of vectors. no overt clinical-signs were observed following vaccination with the prime or booster injection, suggesting low immuno-reactivity and therefore possibly low-levels of adverse events in nhps and humans. although second generation live vaccines, such as acam2000, provide the most robust immune response, they are quite reactogenic and induce some level of morbidity in all vaccines. furthermore, a significant portion of the human population are contraindicated to vaccination with first-and second-generation vaccines [19] . mva is a non-replicating vaccine that has demonstrated efficacy in many animal trials. the main draw-back to mva is its relatively low immunogenicity, meaning that booster administrations are usually required for 100% protection against morbidity and mortality [87, 88] . future studies could reveal that vectors' studied here could be used as alternatives to mva. in summary, our findings have demonstrated that bohv-4 based vectors can be used as vaccines to protect against a lethal mpxv challenge in mice. our studies utilized stat1 (-/-) mice; however, other strains have demonstrated sensitivity to mpxv, namely the caste/eij strain [49, 50, 89] . future studies should consider evaluating the protection of these vectors in this strain also. this work provides a "proof-of-concept" for the bohv-4-based vector as a potential vaccine for category a agents. future and ongoing studies are focused on the design of bohv-4-based vectors expressing antigens from other category a pathogens, as well as an assessment of protection in non-human primate. a review of experimental and natural infections of animals with monkeypox virus between 1958 and 2012 human monkeypox: an emerging zoonotic disease diagnosis and management of smallpox status of human monkeypox: clinical disease, epidemiology and research reemergence of monkeypox: prevalence, diagnostics, and countermeasures outbreaks of disease suspected of being 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challenge safety, immunogenicity and efficacy of modified vaccinia ankara (mva) against dryvax challenge in vaccinia-naive and vacciniaimmune individuals susceptibility of the wild-derived inbred cast/ei mouse to infection by orthopoxviruses analyzed by live bioluminescence imaging we would like to thank alfonso rosamilia for technical assistance. key: cord-006841-3u56erru authors: einsele, hermann; bertz, hartmut; beyer, jörg; kiehl, michael g.; runde, volker; kolb, hans-jochen; holler, ernst; beck, robert; schwerdfeger, rainer; schumacher, ulrike; hebart, holger; martin, hans; kienast, joachim; ullmann, andrew j.; maschmeyer, georg; krüger, william; niederwieser, dietger; link, hartmut; schmidt, christian a.; oettle, helmut; klingebiel, thomas title: infectious complications after allogeneic stem cell transplantation: epidemiology and interventional therapy strategies: guidelines of the infectious diseases working party (agiho) of the german society of hematology and oncology (dgho) date: 2003-09-10 journal: ann hematol doi: 10.1007/s00277-003-0772-4 sha: doc_id: 6841 cord_uid: 3u56erru the risk of infection after allogeneic stem cell transplantation is determined by the underlying disease, the intensity of previous treatments and complications that may have occurred during that time, but above all, the risk of infection is determined by the selected transplantation modality (e.g. hla-match between the stem cell donor and recipient, t cell depletion of the graft, and others). in comparison with patients treated with high-dose chemotherapy and autologous stem cell transplantation, patients undergoing allogeneic stem cell transplantation are at a much higher risk of infection even after hematopoietic reconstitution, due to the delayed recovery of t and b cell functions. the rate at which immune function recovers after hematopoietic reconstitution greatly influences the incidence and type of post-transplant infectious complications. infection-associated mortality, for example, is significantly higher following engraftment than during the short neutropenic period that immediately follows transplantation. early post-transplantation period (pre-engraftment) febrile episodes in the early phase after allogeneic stem cell transplantation are in the vast majority of cases caused by infections [21] . after full conditioning regimens, almost all patients develop severe neutropenia and almost all patients develop neutropenic fever as an early clinical sign of infection. however, other symptoms of infection may masked by severe neutropenia. therefore, the source of infection can only rarely be identified on clinical grounds or with the help of imaging techniques. the few, non-infectious causes of neutropenic fever during the early post-transplantation period include transfusion of blood products, administration of immunoglobulins, drug-induced fever (e.g. cytosinarabinoside, amphotericin b, bleomycin, g-csf), allergies, and acute gvhd reaction, which can cause fever within days after transplantation. the risk of developing an infection in the early posttransplantation period is mainly determined by the duration and the severity of neutropenia. other risk factors for infectious complications are extensive mucosal damage as a result of the conditioning treatment, bacterial colonization, local fungal and viral infections, reactivation of infections that have been acquired during previous neutropenic periods, and finally the use of central venous catheters. the number of stem cells in the graft and the type of gvhd prophylaxis are factors which determine the rate of hematopoeitic reconstitution and may therefore also influence incidence and severity of infections during the early post-transplantation period. bacterial and fungal infections after allogeneic transplantation in neutropenia often take a life-threatening course. bacterial pathogens account for about 90% of infections during this phase. bacteremias are documented in 16-31% of patients after allogeneic transplantation, the majority (65-75%) being caused by gram-positive pathogens. infections due to gram-negative bacteria are less frequent. gram-negative infections, however, are generally associated with a significantly higher morbidity and mortality. the most frequent gram-positive pathogens are coagulase-negative staphylococci, corynebacteria, and alphahemolytic streptococci. gram-positive infections are mainly associated with central venous catheters and most frequent in patients with severe mucositis. particularly bacteremias with viridans streptococci such as streptococcus mitis are associated with a toxic shock syndrome or ards in 10% of affected patients and result in high mortality. in contrast, gram-negative pathogens are believed to enter the bloodstream via damaged mucosa of the gastrointestinal tract in patients with severe gastrointestinal mucosal damage. viral infections frequently occur in the early period after transplantation. in seropositive patients without adequate antiviral prophylaxis, hsv infections can be documented in more than 70% of patients. however, since in most centers aciclovir prophylaxis is routinely given to all patients after allogeneic stem cell transplantation, disseminated hsv infections rarely occur. in recent years, an increase in infections with respiratory viruses such as respiratory syncytial virus (rsv), parainfluenza virus, influenza virus, adenovirus, and rhinovirus have been reported [22] . after an initial infection of the upper respiratory tract, these viruses can subsequently lead to interstitial pneumonia causing substantial mortality. diagnostic procedures in patients with neutropenic fever initial diagnostic procedures follow the guidelines that have been described in the manuscript "antimicrobial therapy of unexplained fever in neutropenic patients" [10] . s176 microbiological diagnostic procedures as indicated by the symptoms of infection: if evidence of microorganisms has been documented in blood, urine, or csf culture, a surveillance culture should be obtained after treatment to document the efficacy of microbiological eradication. since conventional chest x-ray is insensitive, and has only a low negative predictive value for detecting pulmonary infiltrates in neutropenic patients, spiral or high-resolution computed tomography of the lungs should be obtained early to establish the cause of fever in neutropenic patients and particularly in those not responding to the initial therapy [8] . antimicrobial therapy in patients with neutropenic fever after allogeneic stem cell transplantation when an empiric antibacterial therapy is selected in patients with neutropenic fever, the local hospital resistance of pathogens must be considered. fever of more than 38.3c or fever of 38.0c lasting for an hour, or that reoccurs within 24 hours, should result in immediate broad-spectrum antibacterial treatment. microbiologic identification of an underlying pathogen is only possibly in about one third of all patients. therefore, it has become accepted practice to initiate broad-spectrum antimicrobial treatment on the basis of clinical or radiological signs or symptoms. in order to avoid ineffective empirical regimens, only combination treatments with documented broad-spectrum activity against enterobacteriaceae, pseudomonas aeruginosa, staphylococci, and streptococci should be used. clinical trials that investigated single agent regimens in patients with neutropenic fever included only few patients after allogeneic stem cell transplantation. therefore, the efficiency of single agent treatment e.g. with cefepim, ceftazidime, or a carbapenem, has not been sufficiently validated to date in the allogeneic setting and can not be recommended. for example, patients with severe mucositis should not receive single agent ceftazidime, because of the risk of a bacteremia with viridans streptococci. in these latter patients, the initial empiric treatment regimen should contain an antibiotic proven to be effective against streptococci and staphylococci, such as a broad-spectrum penicillin in combination with a b-lactamase inhibitor or a glycopeptide [15] . in the case of skin infections or venous catheter infections, addition of vancomycin or teicoplanin to the initial empiric regimen should be considered. however, administration of glycopeptide antibiotics should be discontinued early after a few days, if no multi-resistant gram-positive bacteria have been identified. if aminoglycosides are administered as part of the initial empiric regimen, regular surveillance of serum drug levels is required. to avoid excessive nephrotoxicity from additional nephrotoxic medication (e.g. ciclosporin, aciclovir etc.), daily surveillance of serum creatinine levels is mandatory, if aminoglycosides are used. modification of empiric antimicrobial regimens in patients with neutropenic fever after allogeneic stem cell transplantation when the causative agent of an infection has been identified, antibacterial therapy should be adapted according to the resistance pattern of the pathogen. however, narrowing the spectrum of the initial empiric treatment should be avoided, as identification of a pathogen does not exclude the presence of a polymicrobial infection. in the absence of a clinical response within 72-96 hours, the initial empiric regimen also requires modification. if steroids have been used over prolonged periods, or if steroids are given at a dose of >2 mg/kg/day, systemic antimycotic treatment should be administered as part of the second-line treatment. antimycotic treatment is also recommended as part of the first or second-line treatment as soon as pulmonary infiltrates occur. antibiotic treatment may be discontinued if all of the following conditions are met: 1. afebrile for at least 48 hours 2. negative cultures 3. imaging techniques without evidence of an infection 4. no clinical evidence of an infection 5. neutrophil count above 1000/ml it is mandatory to perform further microbiologic screening if the patient continues to be febrile. if infections have been microbiologically proven, it is advisable to repeat the initial diagnostic procedures, in s177 order to document the microbiological response (e.g. blood cultures, csf cultures, urine cultures, stool cultures, bronchial secretions in case of ventilated patients, smears). reduction of the antimicrobial spectrum (e.g. by discontinuation of aminoglycosides) can be acceptable in individual patients, depending on clinical response and the occurrence of drug toxicity. patients after allogeneic stem cell transplantation are at the highest risk of developing localized as well as systemic fungal infections. in this patient population, the incidence of life-threatening systemic mycoses can be as high as 15%, or more. some of the risk factors that contribute to this high incidence are listed in table 1 [19] . depending on the local epidemiological environment, candida and aspergillus species are the most frequent pathogens of systemic fungal infections in patients after allogeneic stem cell transplantation. fever that is unresponsive to broad-spectrum antibiotic treatment is frequently the first, and only, symptom of a systemic fungal infection. in the case of pulmonary aspergillus infections, pleuritic chest pain, cough, or hemoptysis may also occur. blood cultures may sometimes be positive for candida species, but rarely for aspergillus species. aspergillus spp. that is found in clinical specimens from neutropenic patients may indicate a systemic infection with this pathogen [24] . however, the sensitivity of screening for systemic aspergillus infections by culturing techniques is low. the significance of bronchoalveolar lavage in the diagnosis of pulmonary fungal infections is therefore still disputed. when fungi are found in bal specimens, it may be difficult to distinguish between contamination with fungi from the oropharynx and true invasive pulmonary infection. even in invasive pulmonary aspergillosis, cultures from bal are often negative. unfortunately, all serological procedures that have been established for detection of systemic fungal infections so far, have a low sensitivity and often also a low specificity for the detection of systemic mycoses. new serologic techniques may improve this situation. a new elisa assay for the detection of galactomannan in serum as well as the polymerase chain reaction (pcr) for the identification of fungispecific dna are currently being evaluated [4, 5, 12, 18] . in recent years, imaging techniques have also been increasingly used for the diagnosis of systemic fungal infections. if a systemic fungal infection is clinically suspected, imaging techniques should be used early. especially with the help of computed tomography, characteristic findings of invasive fungal infection may be present often before such alterations are seen with conventional radiological examinations [8] . in the rare case of hepatosplenic candidiasis, characteristic changes may also be identified by ultrasound sonography of the liver and spleen. because of its broad anti-fungal activity, intravenous amphotericin b deoxycholate is still the current standard in the treatment of patients with suspected or documented fungal infections after allogeneic stem cell transplantation. empiric treatment should be initiated as soon as the presence of a systemic fungal infection is suspected. the recommended dose of amphotericin b for empirical therapy is 0.5-0.7 mg/kg/day. this dosage is also used as a therapeutic dose in documented invasive candida infection. a higher dose of 1-1.5 mg/kg/day should be given, in case of a pulmonary infiltrate or if an invasive pulmonary aspergillosis is suspected. antifungal treatment has to be continued until neutrophil recovery and disappearance of all signs of an acute infection are achieved. recently, new lipid formulations of amphotericin b have been approved for the treatment of systemic fungal infections. in several clinical trials, the new amphotericin b formulations proved to be equipotent to conventional amphotericin b deoxycholate and could be administered at higher dosages of 3-4 mg/kg/day. the advantage of these liposomal formulations compared to conventional amphotericin b desoxycholate, is the much lower rate of acute side effects, especially of nephrotoxicity [20] . however, the excessive costs of these preparations limit their clinical usefulness. it is therefore recommended to change to a lipid formulation of amphotericin b, in case of severe clinical nephrotoxicity (e.g. creatinine >2.5 mg/ dl), intolerance, or inefficacy of amphotericin b desoxycholate. new azoles with broad antimycotic activity or new generations of drugs such as the echinocandines or pneumocandins, are currently being investigated in clinical trials [9] and might become alternatives to amphotericin b for the treatment of systemic fungal infections in recipients of an allogeneic stem cell graft in the future. intermediate post-transplantation period (from hematopoietic reconstitution to day + 100 after transplantation) after engraftment, a severe combined quantitative and functional deficiency in the t and b lymphocyte compartment persists despite full hematopoietic reconstitution. if t cell depletion has been used, or if incompatibility in the major histocompatibility antigens between the recipient and the donor had to be accepted, these immunodeficiencies are prominent for prolonged periods after transplantation. these deficiencies manifest as disorders in t helper cell function, immunoglobulin synthesis, but also in an impaired cytotoxic t cell response. in spite of normalization of cell counts, disturbances of granulocyte functions also persist, e.g. impairment of chemotaxis and phagocytosis. in 74% of all allogeneic stem cell transplantation patients, infections develop after day +50. in the majority of patients, these infections are triggered by viruses such as cmv or other viral infections such as hhv6, rsv, adenovirus, vzv, and ebv. in 14% of patients, bacteremias can be documented after hematopoietic engraftment. the mortality rate as a result of bacteremias in allogeneic stem cell recipients is comparable in the periods before and after engraftment. the spectrum of pathogens in patients with documented bacteremias shows that gram-positive pathogens (47% staphylococci) are responsible for about 75% of all infections. in contrast to the microbiologically documented infections in patients before hematopoietic engraftment of only 5-10%, the focus of bacterial infection can be identified in more than 50% of patients after hematopoietic engraftment [21, 23] . catheter infections are responsible in more than 30% of bacteremias during the postengraftment period. chills occurring within the first hour after intravenous drug administration may be the first sign of a catheter infection. other frequent sources of infection during the intermediate post transplant period, are pneumonias, especially caused by streptococcus pneumoniae, klebsiella species, and pseudomonas aeruginosa. cases of pyogenic arthritides with salmonella eneritidis and staphylococcus aureus have also been reported. late infections after allogeneic stem cell transplantation (after day + 100 following transplantation) in the late post transplant period, immune reconstitution is usually advanced, particularly in patients who have received a transplant from an hla-identical family donor. these patients often show full hematopoietic reconstitution and early immune reconstitution. if no gvhd occurs, immunosuppressive treatment is usually discontinued. in these patients who do not demonstrate a graft-versus-host reaction, who require no immunosuppressive therapy, who usually have a cd4-count of >200 per ml blood, and whose serum immunoglobulins levels are in the normal range, infectious complications rarely occur. these patients can be considered as immunocompetent and they are no longer at an increased risk of opportunistic pathogens, so that no intensive antimicrobial therapy is required. however, depending on a number of clinical risk factors, chronic gvhd may occur in 30% of patients, or more, which is characterized by a severe combined cellular and humoral immunodeficiency. due to mucosal damage, functional deficiencies of granulocytes (especially impaired chemotaxis), functional asplenia and qualitative as well as quantitative t and b cell deficiencies, a significantly increased susceptibility to infections must be assumed in patients with chronic gvhd. in these patients, bacterial infections of the upper and lower respiratory tract constitute a main cause of death. life-threatening infections are typically caused by encapsulated bacteria such as streptococcus pneumoniae or haemophilus influenzae. sinusitis, otitis media, and pharyngitis are common manifestations of such infections in this late post-transplantation period. patients who present at least one of the above mentioned risk factors, should receive immediate antibacterial treatment at the earliest signs of infection. if several of the above mentioned risk factors are present, antibiotic prophylaxis with an oral penicillin or a macrolide antibiotic is recommended. if fever occurs in a patient later than 100 days after allogeneic stem cell transplantation, the upper and lower respiratory tract (bronchitis, pneumonia, sinusitis), and bacteremias have to be considered as specific foci of infections. particularly pneumonias and bacteremias constitute the majority of life-threatening infections, which occur in 20% of all patients after day + 50 after allogeneic stem cell transplantation [14] . in a recent analysis, pulmonary infections were documented after day + 100 in approximately 50% of patients with cgvhd, in 21% of patients without cgvhd, and in only 2% of patients after autologous stem cell transplantation [9] . the incidence of infections is particularly high in patients who received a graft from an unrelated donor. frequently, herpes zoster or less often visceral manifestations of a vzv infection are identified (see chapter on varicella infection). an important pathogen of interstitial pneumonias in the late phase after allogeneic stem cell transplantation is pneumocystis carinii. without specific prophylaxis, about 30% of patients with chronic gvhd develop pneumocystis carinii pneumonia (pcp). late infections after stem cell transplantations constitute an important factor for morbidity and can take a fatal course in 4-15% of patients. a. viral infections the evident increase of infections caused by respiratory viruses can be explained by more intensive screening, improved culturing methods and transplantation procedures with prolonged and intensified immunosuppression. respiratory viruses may be acquired prior to sct, so that clinical manifestations can already develop within the first weeks after transplantation. often, infections result from direct contact with infected family members, doctors, or nurses. in this clinical setting, rsv is most often identified, followed by rhinoviruses, parainfluenza virus type 1 and 3, and influenza virus type a [22] (for diagnostic procedures see table 2 .) adenovirus infections can either be caused by primary infections, reinfections, or by virus reactivation. excretion of adenoviruses in urine and throat washings has been documented in 3.8-20.9% of patients after allogeneic stem cell transplantation. adenovirus diseases, however, occur in only 0.95-6.5% of patients [6] . the intensification of immunosuppression or t cell depletion after stem cell transplantation has also led to an increase of adenovirus infections. adenoviruses can first be detected by culture techniques around day 44 (day 13-199) after transplantation, demonstrating a predominance of serotypes 11, 34, and 35. clinical manifestations of adenovirus infections in patients after allogeneic stem cell transplantation that have been reported so far include pneumonia, hepatitis, cystitis, diarrhea, and also disseminated disease (for diagnostic procedures see table 3 ). the mortality rate of these infections is about 60%. there have been reports about successful treatment of adenovirus infections with cidofovir or ribavirin. successful treatment of rsv infection has been reported with either inhalative or intravenous ribavirin and/or monoclonal antibodies. herpes simplex virus infection after allogeneic stem cell transplantation is associated with a high morbidity and leads to a substantial degree of oral mucositis, especially during the early post transplant period. before the introduction of aciclovir prophylaxis, 80% of the seropositive patients excreted hsv within the first 50 days (median second and third week) after transplantation, mainly due to reactivation of persisting virus. aciclovir prophylaxis from day 0 to day 30 reduced the hsv reactivation rate by 75% within the first 100 days after transplantation. clinical manifestations of the hsv infection after stem cell transplantation include skin manifestations, urogenital infections, esophagitis, keratitis, and infrequently, also pneumonias, hepatitides, or encephalitides (see also table 4 ). diagnostic measures should be initiated depending on the manifestation and may either consist of virus culture from throat washings, urine, skin lesions, or mucosal swabs, or antigen detection, or viral dna pcr (especially in csf specimens) ( table 4) . treatment of hsv infections with aciclovir (see also table 4 ) is very effective. aciclovir-resistant herpes simplex virus isolates have been identified in only about 6% of patients treated with aciclovir, in about 2% during primary and in about 9% during secondary prophylaxis. it is important to note that the documentation of persisting excretion of hsv during treatment with aciclovir treatment does not necessarily imply resistance to this drug. therefore, if possible, a sensitivity test should be performed. infections with aciclovir-resistant hsv can be accompanied by local or disseminated manifestations [11] . the treatment of choice in acyclovir-resistant hsv infections is foscarnet. prior to the introduction of aciclovir prophylaxis, vzv reactivations developed in 30-50% of adults and in 25% of children within the first 6 months (median 5 months) after allogeneic stem cell transplantation. acute and chronic gvhd were identified as the major risk factors for reactivation. eighty-four percent of vzv infections in adult patients manifest as localized herpes-zoster. disseminated or visceral vzv infections occur in only 13-25% of patients. if dissemination or visceral involvement occurs, the mortality of such vzv is high. during aciclovir prophylaxis until at least day +30, vzv infections can only be documented in approximately 16% of allogeneic stem cell transplant recipients. visceral manifestations may even precede cutaneous manifestations. in recent years, antiviral treatment has significantly improved the prognosis of vzv infections. intravenous administration of aciclovir (310 mg/kg) within the first 24-48 hours after clinical manifestation shortens the duration of skin manifestations and often also prevents dissemination as well as the incidence of post-herpetic neuralgia [13] . the mortality rate associated with vzv pneumonia, however, is still high. if a vzv infection occurs within the first 9-12 months after stem cell transplantation, treatment is indicated. this also applies for a longer period in patients who receive immunosuppression for acute or chronic gvhd. infections with cytomegalovirus (cmv) are one of the main causes of infection-associated mortality after allogeneic stem cell transplantation. if no antiviral prophylaxis is administered, cmv infection occurs between 30 and 100 days after stem cell transplantation (for diagnostic procedures see table 5 ). cmv infection occurs in approximately 60-70% of cmv-seropositive patients or seronegative patients who receive transplants from a seropositive donor. manifestations of cmv disease are pneumonia, gastroenteritis, hepatitis or retinitis. the mortality of cmv disease has not decreased, in spite of a combination treatment with ganciclovir and cmv hyperimmunoglobulin. despite the fact that an initial response can be documented in about 60-80% of patients, only 31% of patients survive a cmv-induced interstitial pneumonia for more than 3 months. in recent years, antiviral prophylaxis and preemptive therapy based on highly sensitive screening tools have succeeded in substantially reducing the incidence of cmv disease in the early phase after stem cell transplantation (to 2-4%). however, both prophylaxis and early intervention have led to a late increase in cmv infections after day +100 post transplantation. especially in patients with chronic gvhd, the incidence of late cmv disease, which occurs a median 156 days after transplantation, has increased. due to the high mortality of cmv disease, intensive efforts are being made to prevent its occurrence. in cmvseronegative patients who receive a transplant from a seronegative stem cell donor, this can be achieved by transfusion of cmv-seronegative and leukocyte-depleted blood products. the probability of developing a cmv infection can thus be reduced to 3-6%, and the occurrence of cmv disease can be reduced to 0.5-1.5%. in seropositive patients, several different strategies exist. first cmv reactivation and the development of a cmv disease may be prevented by prophylactic administration of antiviral agents. since there is a higher incidence of secondary bacterial and mainly fungal infections due to secondary neutropenia and possible ganciclovir-associated immunosuppression, ganciclovir prophylaxis does not show any advantage in respect to survival despite the marked reduction in the rate of cmv infection and disease. an alternative strategy is early intervention, which means that ganciclovir treatment is initiated when cmv is first identified in clinical specimens (bronchoalveolar lavage, throat washings, blood, or urine). a significant reduction in the incidence of cmv disease as well as in cmv-associated mortality was achieved with this strategy. however, in approximately 30-35% of patients, ganciclovir-associated secondary neutropenia developed. since culture methods demonstrate only a low sensitivity, cmv diseases may occur in 12-13% of patients even before the virus is detected by conventional culture assays. today, due to the widespread application of more sensitive screening tools (pcr, antigenemia), virus infection can be detected and treated earlier. this reduces the incidence of cmv disease to 3-6% of patients [3] . the treatment strategies of cmv disease are presented in tables 6 and 7 . after allogeneic stem cell transplantation, the incidence of ebv-associated lymphoproliferative disorders in hlaidentical family donors is only around 0.45%, but may increase to about 1.4% in family donors who are not fully hla-matched [25] . when t cell depletion methods are performed, the incidence of ebv-associated lymphoproliferative disorders increases substantially. with t cell depletion in combination with stem cell grafts from unrelated donors, the incidence of ebv-associated lymphoproliferative disorder increases even further, to an incidence as high as 29% [25] . ebv-associated lymphoproliferative syndrome is associated with very high amounts of ebv-dna in the peripheral blood. ebv reactivation in the absence of lymphoproliferative disease is observed more frequently. however the association of ebv reactivation with symptomatic disease has not been systematically evaluated. donor lymphocyte infusions have been the only treatment of ebv-associated lymphoproliferative syndrome after allogeneic stem cell transplantation so far. more recently, rituximab, an anti-cd20 antibody, has been successfully administered in a small number of patients. since ebvassociated lymphoproliferative disorders have a high proliferation rate and require immediate treatment, donor lymphocyte infusions have to be initiated promptly in order to be successful. thus, monitoring ebv-dna load in the peripheral blood of patients who received t-cell depleted grafts, grafts from unrelated and/or mismatched donors, or who have other risk factors is essential. if the viral load increases significantly, it is advisable to initiate treatment without delay (for diagnostic and therapeutic approach see table 8 ). donor lymphocyte infusion ebv-specific t cell lines/clones anti-cd20 antibodies (e.g. rituximab) before the introduction of pcp prophylaxis, the incidence of pneumocystis carinii pneumonia in allogeneic stem cell transplant recipients amounted to approximately 6.8% [17] . seventy-five percent of patients with pcp develop dyspnea, cough, and fever as clinical signs of the infection. in 58% of patients, conventional chest radiography shows bilateral infiltrations, typically with sparing of the periphery of the lungs. in 15% of the patients, however, conventional chest x-rays may be normal or may show only minimal abnormalities. in a retrospective analysis, the immunofluorescence staining of bronchoalveolar lavage specimens was identified as an accurate method of diagnosis of pneumocystis carinii pneumonia in the majority of patients. pcr is increasingly used as diagnostic procedure for pcp, but this has not yet become a standard procedure. pneumocystis carinii pneumonia is treated with high dosages of trimethoprim-sulfamethoxazole (tmp/smx) (20/100 mg/kg/ p.o. or i.v. in 3 or 4 divided doses per day) for 2-3 weeks. alternatively, it may be possible to administer trimethoprim 20 mg/kg/day in four doses combined with dapson 1100 mg p.o. for 3 weeks or pentamidin (pentacarinate) 3-4 mg/kg daily by the intravenous route. another alternative is atovaquone (meprone) 2750 mg p.o. for 3 weeks. despite high-dose tmp/smx treatment or intravenous administration of pentamidin, the mortality rate for pneumocystis carinii pneumonia is 89% within the first 6 months in patients after allogeneic stem cell transplantation, and still as high as 40%, if pcp occurs more than 6 months after stem cell transplantation. c. toxoplasmosis toxoplasmosis can occur as early as day +30 after allogeneic stem cell transplantation. it presents as pneumonia, perimyocarditis, encephalitis with focal-neurological signs or convulsions as well as chorioretinitis. the incidence in a large retrospective analysis at the fhcrc in seattle amounted to 0.3% in 4312 examined patients, with a local seroprevalence of 17% [16] . in areas with a higher seroprevalence for toxoplasma gondii (e.g. in france with a 70% seroprevalence), the incidence of toxoplasmosis after allogeneic stem cell transplantation is as high as 2-3% [2] . risk factors for the toxoplasma infections are the serostatus of the patient and the extent of immunosup-pression. particularly in areas with a high seroprevalence, determination of toxoplasma serostatus prior to allogeneic stem cell transplantation should be mandatory. if a toxoplasma infection is clinically suspected, serum, liquor, and bronchoalveolar lavage specimens should be screened, to see if toxoplasma can be detected. however, sensitivity of morphological methods is limited. if the clinical suspicion is high, computed tomography of the chest as well as a ct/mri scan of the cns should be performed. in some centers, qualitative and quantitative pcr techniques are being evaluated in respect of their usefulness in screening for clinical infections via the demonstration of toxoplasma dna. however, despite first encouraging results in high risk patients, this technique is not yet part of routine diagnostic standards at present. the treatment of choice in toxoplasma infections after allogeneic stem cell transplantation consists of sulfadiazin (and pyrimethamin) for 3-6 weeks. dosage: -sulfadiazin: 4-8 g/day in 4 separate doses p.o. (~100-150 mg/kg kg), -pyrimethamin: 2100 mg p.o. as loading dose on day 1, then 50-75 (100) mg/day (~1 mg/kg) -folinic acid: 10-15 mg/day p.o. as a supplement to reduce hematological toxicity. in case of chorioretinitis or increased intracranial pressure, additional corticosteroid therapy is recommended. alternatives for sulfadiazin if sulfonamides are not tolerated: -clindamycin: 4600 mg/day p.o. or i.v. in combination with pyrimethamin (see above) in case of cerebral and ocular toxoplasmosis successful treatment has been reported with: successful modification of cytomegalovirus disease in allogeneic marrow transplant recipients toxoplasmosis in bone marrowtransplant recipients: report of seven cases and review pcrmonitoring after bmt to reduce the incidence of cmv disease and the duration and side effects of antiviral therapy detection and identification of fungal pathogens in blood by using molecular probes prediction of invasive pulmonary aspergillosis from colonisation of lower respiratory tract before rnarrow transplantation increasing incidence of adenovirus disease in bone marrow transplant recipients risk factors for developing ebvrelated b cell-lymphoproliferative disorders (blpd) after non-hla-identical bmt in children pneumonia in febrile neutropenic patients and in bone marrow and blood stem-cell transplant recipients: use of high-resolution computed tomography antifungal agents in the 1990s. current status and future developments antimicrobial therapy of unexplained fever in neutropenic patients aciclovir-resistant herpes simplex causing pneumonia after marrow transplantation comparison of an enzyme immunoassay and a latex agglutination system for the diagnosis of invasive aspergillosis in bone marrow transplant recipients a prognostic score for postherpetic neuralgia in ambulatory patients late infections after allogeneic bone marrow transplantations: comparison of incidence in related and unrelated donor transplant recipients fever in immunocompromised patients toxoplasma gondii infection in marrow transplant recipients: a 20-year experience pneumocystis carinii pneumonitis following bone marrow transplantation detection of antigen in sera of patients with invasive aspergillosis: intra-and interlaboratory reproducibility. the dutch interuniversity working party for invasive epidemiology of aspergillus infections in a large cohort of patients undergoing bone marrow transplantation liposomal amphotericin b for empirical therapy in patients with persistent fever and neutropenia. national institute of allergy and infectious diseases mycoses study group infection in the bone marrow transplant recipient community respiratory virus infections among hospitalized adult bone marrow transplant recipients unique risk factors for bacteraemia in allogeneic bone marrow transplant recipients before and after engraftment significance of isolation of aspergillus from the respiratory tract in diagnosis of invasive pulmonary aspergillosis. results from a three-year prospective study epstein-barr virus lymphoproliferation after bone marrow transplantation key: cord-016990-ot1wi3xi authors: zaki, sherif r.; paddock, christopher d. title: viral infections of the lung date: 2008 journal: dail and hammar&#x02019;s pulmonary pathology doi: 10.1007/978-0-387-68792-6_11 sha: doc_id: 16990 cord_uid: ot1wi3xi the lungs are among the most vulnerable to microbial assault of all organs in the body. from a contemporary vantage, lower respiratory tract infections are the greatest cause of infection-related mortality in the united states, and rank seventh among all causes of deaths in the united states.2,3 from a global and historic perspective, the scope and scale of lower respiratory tract infection is greater than any other infectious syndrome, and viral pneumonias have proven to be some of the most lethal and dramatic of human diseases. the 1918–1919 influenza pandemic, perhaps the most devastating infectious disease pandemic in recorded history, resulted in an estimated 40 million deaths worldwide, including 700,000 deaths in the u.s.4 the global outbreak of severe acute respiratory syndrome (sars) during 2003, although considerably smaller in scale, resulted in 8098 cases and 774 deaths5 and is a dramatic contemporary example of the ability of viral pneumonias to rapidly disseminate and cause severe disease in human populations. the lungs are among the most vulnerable to microbial assault of all organs in the body. from a contemporary vantage, lower respiratory tract infections are the greatest cause of infection-related mortality in the united states, and rank seventh among all causes of deaths in the united states. 2 ,3 from a global and historic perspective, the scope and scale of lower respiratory tract infection is greater than any other infectious syndrome, and viral pneumonias have proven to be some of the most lethal and dramatic of human diseases. the 1918-1919 influenza pandemic, perhaps the most devastating infectious disease pandemic in recorded history, resulted in an estimated 40 million deaths worldwide, including 700,000 deaths in the us. 4 the global outbreak of severe acute respiratory syndrome (sars) during 2003, although considerably smaller in scale, resulted in 8098 cases and 774 deaths s and is a dramatic contemporary example of the ability of viral pneumonias to rapidly disseminate and cause severe disease in human populations. although viruses are commonly identified causes of pneumonia of infants and young children, they are relatively infrequently recognized as agents of communityacquired pneumonia in adults. 6 in several large series that investigated a microbiologic cause of community-acquired pneumonia, viral etiologies were identified in only 9% to 18% of cases (table 11 .1).7-12 however, it is likely that viral pneumonias are underrecognized and underdiagnosed for various reasons. although some agents may cause distinct cytopathology or inclusions (e.g., adenoviruses, herpesviruses, and paramyxoviruses), many important pathogens (e.g., influenza viruses) do not, and none of these agents are resolved specifically in tissue by routine histologic stains. viruses require live cells for cultivation, and are generally more difficult than bacteria to isolate from clinical samples. for some viral pneumonias, 426 the pathogen appears to initiate a cascade of destructive host responses that continue or progress even in the absence of the specific infectious agent, and in these patients the etiologic agent may be absent from host tissues at the time of autopsy.13 thirty to sixty percent of community-acquired pneumonias are etiologically undetermined, 14 and it is entirely possible that viruses directly cause more episodes of pneumonia than currently appreciated. because viral infections of the lower respiratory tract often precede bacterial pneumonias, viruses may indirectly exert considerable influence on the cumulative morbidity and mortality of infectious pneumonias. 15 , 16 the mechanisms by which viruses may facilitate bacterial invasion of the respiratory tract are complex and varied. certain viruses cause the death of ciliated respiratory epithelium and thereby disrupt normal ciliary activity. viruses may also inhibit the phagocytic or bactericidal activities of neutrophils, t lymphocytes, and alveolar macrophages, and predispose the host to secondary infections. certain gram-positive and gram-negative bacteria adhere to and colonize virus-infected epithelium more readily than to noninfected cells by various hypothetical mechanisms, including alteration and induction of receptors at the host-cell surface and changes in the extracellular environmenty-19 finally, viral infections of the lung may exacerbate noninfectious pulmonary conditions (e.g., asthma and chronic obstructive pulmonary disease) and indirectly contribute to aggregate morbidity and mortality associated with these conditions. 20 although influenza viruses remain the most frequently identified cause of viral pneumonia in adults (table 11 .1), the diversity of agents identified as causes of viral pneumonias has expanded considerably. several newly recognized viral pneumonias have been identified since 1992 that are among the most feared and lethal of all emerging infections, including those caused by hantaviruses, nipah virus, and sars corona virus (co v). !3,21-23 certain causes "rubella (2), rhinovirus (1) , mixed viral infections (3) . source: adapted from greenberg. 6 of viral pneumonia, particularly those that occur in vulnerable patient cohorts, have diminished during this same interval. by example, the u.s. incidence of varicella pneumonia has dropped by two thirds since universal childhood vaccination for varicella was implemented in 1995, 24, 25 and advances in the clinical management of transplant recipients have reduced the incidence of cytomegalovirus (cmv) pneumonia?6 also occurring during the last decade has been the development and use of powerful molecular techniques that have unveiled the identity of historic pathogens (e.g., the hini "spanish" influenza a virus),27.28 and have facilitated the rapid characterization of emerging agents (e.g., sars-co v). 13 it should be noted that the disease manifestations of several of these agents (e.g., human parainfluenza virus [hpiv] , respiratory syncytial virus [rsv] , and influenza) are often confined to the upper airway and are not invariaorb rsv hpiv adeno cmv varicella other' 10 0 3 0 0 3 27 1 4 3 0 0 0 7 2 0 0 1 0 19 5 5 0 0 1 7 8 13 15 0 0 0 37 5 11 5 0 0 3 21 (11) 32 (16) 30 (15) 1 (0.5) 1 (0.5) 6 (3) ably associated with pneumonia. with some of these pathogens (e.g., influenza viruses, rsv, hantaviruses, and hpiv viruses) respiratory disease is the primary manifestation. for other agents, such as measles, nipah virus, and herpesviruses, typically the lungs are involved as part of a multisystem syndrome. the diagnosis of viral pneumonia, suspected by patient history and clinical manifestations, also can be supported histopathologically, and the general pattern of histopathologic lesions may suggest a specific diagnosis. many viruses can be identified in lung by examining the tissue response and cytopathic changes. some of these viruses cause recognizable tissue reaction patterns including necrotizing tracheobronchitis, bronchiolitis, and interstitial pneumonia. a summary of the key diagnostic histopathologic and ultrastructural features for the most common viral pathogens that cause a majority of pulmonary infections is provided in tables 11.2 and 11.3. yes (cytoplasmic; ill-defined) yes (nuclear and cytoplasmic) pulmonary tissue reaction necrotizing bronchiolitis; smudge cells; dad severe edema, early dad interstitial pneumonia; dad; occasional multinucleation interstitial pneumonia; dad; cytomegaly dad; necrosis and rare multinucleation dad; necrosis and rare multinucleation dad; necrotizing bronchiolitis interstitial pneumonia with multi nucleation dad dad; interstitial pneumonia with occasional multinucleation necrotizing bronchiolitis, interstitial pneumonia with occasional multinucleation recently recognized; human pathology not well described interstitial pneumonia with multinucleation; dad only certain viruses can cause cytopathic changes that are sufficiently distinct to enable the pathologist to recognize a specific diagnosis on routine histologic examination of lung specimens. with the availability of special figure 11 .1. negative stain electron microscopic images of different viruses that can cause pulmonary infections. a. adenovirus. adenoviruses are protein-shelled icosahedral-shaped nonenveloped viruses that measure approximately 70 to 90nm in diameter. two of the viruses are stain penetrated revealing the dna-containing nucleoprotein. b. sin nombre virus. sin nombre virus, the causative agent of hantavirus pulmonary syndrome, belongs to the genus hantavirus in the family bunyaviridae. the envelopes of hantaviruses are checkerboard in appearance, and particles measure 90 to 150nm in diameter. c. herpes simplex virus. the stain has penetrated the envelope of several of these herpesvirus particles, delineating the icosahedral-shaped nucleocapsids, which measure 90 to 100nm in diameter. d. cytomegalovirus. another herpesvirus; one virus particle (left) is intact while two nearby particles are stainpenetrated and show the viral nucleocapsids. the nucleocapsid of the upper right particle shows a central core that harbors the dna of the virus. e. influenza virus. influenzaviruses belong to the family orthomyxoviridae; viral particles are pleomorphic s.r. zaki and cd. paddock diagnostic techniques, such as immunohistochemistry (ihc) and in-situ hybridization (ish), many viruses can be detected in formalin-fixed, paraffin-embedded tissue samples even if specific viral inclusions cannot be found in histologic examination of tissue sections. among the techniques, ihc utilizing specific antibodies can be routinely performed on formalin-fixed tissue and can enhance the pathologist's accuracy in identifying organisms in tissue specimens. in addition to histologic pattern recognition, ihc, and ish in tissue, several other diagnostic tests are available to aid the pathologist. cell culture techniques, serology, polymerase chain reaction (pcr), and electron microscopy (em) all play vital roles in the diagnosis of these infections. while histologic techniques can be an excellent means of demonstrating organisms, tissue culture isolation remains essential for definitive identification of the virus. when a viral pneumonia is suspected, samples of lung tissues should be evaluated by cell culture, which has the advantage of being a nonbiased method for screening purposes that does not rely on the availability of specific antibodies or probes. electron microscopy offers the same utility as a broad scope diagnostic tool and has been especially critical in outbreaks of unknown etiology. it played a critical role during the hendra and nipah virus outbreaks in 1994 and 1999, respectively, and more recently, in the early recognition of a novel coronavirus associated with sars in 2003 and in the diagnosis of emerging transplant-associated infections. 13.22.29-33 the advantage of this approach is that viral particles may be demonstrated by negative stain or thin section em, either directly in clinical material or after amplification in cell culture. like culture, em is not limited by narrow specificity of reagents or by prior clinical bias ( fig. 11 .1). and can be filamentous or spherical in shape. the evenly spaced spikes cover the entire virus surface and contain both the hemagglutinin and neuraminidase surface glycoproteins. f. human metapneumovirus. these paramyxoviruses are heterogeneous in size and shape, and range in size from 150nm to 1 j.lm in diameter. g. parainfluenza virus. another paramyxovirus, the viral nucleocapsid, with its typical herringbone appearance, can be seen both within the stain-penetrated particle as well as partially extruded from the virion. an important feature that can help distinguish between paramyxovirinae (parainfluenza and measles viruses) and pneumovirinae (human metapneumovirus and respiratory syncytial virus) is the diameter of the nucleocapsids, which measure 18nm and 14nm, respectively. h. severe acute respiratory syndrome (sars) coronavirus. these 80-to 100-nm particles are named for the characteristic crown-like fringe on the surface. scale bars, 100nm. (a,b,d,f,h: courtesy of c. humphrey; c,g: courtesy of e. palmer; e: courtesy of ea. murphy, all at centers for disease control and prevention, atlanta, ga.) adenoviruses were first cultured and identified during the early 1950s by investigators searching for etiologic agents of acute respiratory infections. the initial adenovirus isolate was made serendipitously from adenoid tissues obtained from children during efforts to establish a primary human adenoid cell line. 34 a related virus was identified the following year by investigators studying respiratory disease in military recruits?5 these agents were subsequently named adenoviruses after the original source of tissue from which the prototype strain was identified. 36 adenoviruses are non enveloped viruses with a single, linear, double-stranded dna genome that is contained within an icosahedral capsid that measures 70 to 90nm in diameter (fig. ilia) . the capsid is comprised of seven known polypeptides, including the hexon capsomere, which contains group-specific antigenic determinants. 37 adenoviruses are a ubiquitous and diverse group of viruses found naturally in the upper respiratory tracts and gastrointestinal systems of humans, other mammals, and birds. most adenoviruses infect mucosal epithelium, although some pathogens of animals are trophic for endothelial cells, and endothelial infection has been identified in some immunocompromised humans. 38 adenoviruses are represented by at least 51 serotypes on the basis of resistance to neutralization by antisera to other known adenovirus serotypes, and comprise six subgroups or subgenera (a through f) that are distinguished by differential hemagglutination with erythrocytes from various animal species. 37 ,39ao more than 50% of the known adenovirus serotypes are associated with human diseases of the upper and lower respiratory tract, conjunctiva, urinary tract, intestine, and occasionally heart, liver, and central nervous system. the others are rarely encountered and mayor may not act as pathogens in recognizable disease. 37 it is estimated that approximately 5% to 10% of all pneumonias in infants and young children are caused by adenoviruses. 41 ,42 most pediatric cases of adenovirus pneumonia occur between 6 months and 5 years of age, and serotypes 3, 7, and 21 (all members of the b subgenus), are the most common causes of pneumonia in this patient cohort. 43 -45 serotypes 3 and 7 are particularly pathogenic adenoviruses that can cause disseminated and often fatal disease in previously healthy children. 46 in adults, pneumonia is generally associated with serotypes 3,4, and 7.47 periodic epidemics of adenovirus pneumonia in young adults have been identified, particularly among military recruits. 48 ,49 in a manner similar to other pathogens, adenoviruses take advantage of impaired or destroyed immune systems to establish persistent and disseminated infections in immunocompromised hosts. in this patient cohort, the s.r. zaki and cd. paddock case fatality rate of adenoviral pneumonia approaches 60%, compared with an approximately 15% mortality in immunocompetent patients. 37 immunocompromised patients are also susceptible to a broader range of different adenovirus serotypes. by example, the commonly recognized serotypes in normal children account for only about 50% of the adenovirus serotypes reported for children with congenital immune deficiencies. 37 .4 6 because some adenoviruses establish latency in lymphoid tissues and the kidneys of their host, it is believed that many, possibly most, cases of clinical disease caused by adenoviruses in immunocompromised patients are reactivated infections. 37 the lungs of patients with adenovirus pneumonia are typically heavy and edematous, and the bronchi are generally filled with mucoid, fibrinous, or purulent exudates. the histopathologic findings ( fig. 11 .2) include necrotizing bronchitis and bronchiolitis with extensive denudation ofthe surface epithelium,particularly in medium-sized (1 to 2mm in diameter) intrapulmonary bronchi ( fig. 11 .2a). affected airways may be occluded by homogeneous eosinophilic material, mixed inflammatory cells, detached epithelium, and cellular debris. the lamina propria of bronchi and bronchioles is typically congested and infiltrated by predominantly mononuclear inflammatory cell infiltrates. bronchial serous and mucous glands are also often involved and show necrosis and mixed infiltrates. 5o as the infection progresses, there is involvement of the pulmonary parenchyma, forming bronchocentric necrosis with hemorrhage, neutrophilic and mononuclear cell infiltrates, and karyorrhexis. these findings generally occur against a background of exudative diffuse alveolar damage, with filling of the air space by macrophages, fibrin, and detached pneumocytes, and hyaline membrane formation. 51 patients with fatal pneumonia may develop disseminated intravascular coagulopathy and demonstrate fibrin thrombi in vessels of the lungs, kidney, heart, adrenals, and central nervous system (see fig. 4 .20 in chapter 4). 48 adenoviruses form intranuclear inclusions in respiratory epithelial cells of the trachea, bronchi, and bronchioles, in the acinar cells of bronchial glands, and in alveolar pneumocytes, and are generally most abundant at the viable edges of necrotic foci. by using the hematoxylin and eosin (h&e) stain, early inclusions appear as small, dense, amphophilic structures surrounded by a cleared zone and peripherally marginated chromatin, similar to herpetic inclusions. as the cellular infection progresses, the inclusion becomes larger (as large as 14/-lm in some cells) and more basophilic, and the margins of the nuclear membrane become blurred to form the characteristic "smudge cell" (fig. 11 .2b,c).5o,51 tracheal aspirates of patients with adenovirus pneumonia may show distinctive features on cytologic preparations that include cells with fine strands of chromatin that radiate from a central inclusion to the marginated chromatin at the nuclear membrane ("rosette cells"), and cells with foamy, "honeycomb" nuclei, as well as typical smudge cells (see fig. 7 .45 in chapter 7). 52 various methods can be used to diagnose adenovirus infections that include antigen detection, cell culture, electron microscopy, molecular assays, and serology. direct detection techniques that identify the common group-reactive hexon antigen in tissues or body fluids include fluorescence antibody assays and enzyme immunoassays.53 immunohistochemistry staining methods have been used successfully to detect adenovirus-infected cells in formalin-fixed, paraffinembedded tissues using various commercially available, adenovirus group-specific antibodies ( fig.11 .2e,f).38,51 electron microscopy of adenovirus-infected tissues reveals a paracrystalline array of virions ( fig. 11 .2d ). 54, 55 most adenoviruses can be isolated in cell culture from bronchial washings, tracheal aspirates, or lung biopsy specimens during the early stage of the illness and grow well in various cell lines, including human embryonic kidney, hela, and hep-2 cells. 47 cell cultures infected with adenoviruses exhibit a relatively characteristic cytopathologic effect, described as a "cluster of grapes," within 3 to 5 days after inoculation. serotyping of the isolate is accomplished by using hemagglutination inhibition and neutralization tests with hyperimmune type-specific animal antisera. 56 molecular assays, particularly gene amplification using pcr, and ish methods, have been developed to detect adenovirus nucleic acid in respiratory secretions and in formalin-fixed, paraffin-embedded tissues. 51 ,57,58 broad-range, sensitive assays that can detect any adenovirus amplify common genomic sequences (e.g., the hexon gene region). other more specific assays detect specific adenovirus types with unique genomic sequences. 59 serologic assays include tests for groupspecific antibodies (e,g., complement fixation and enzyme immunoassays), or type-specific antibodies (e.g., neutralization and hemagglutination-inhibition assays). pitfalls associated with serologic testing for adenoviruses include occasional rises in heterotypic antibodies when typespecific assays are used, and relatively low sensitivity demonstrated by complement fixation assays. 59 human hantaviral diseases are caused by a group of closely related, trisegmented, negative-sense rna viruses of the genus hantavirus, of the family bunyaviridae. 60 -62 members of the genus hantavirus have similar morphologic features. 63 ,64 virus particles are 70 to 130nm in diameter and generally appear spherical to ovoid, although pleomorphic forms may be seen. a lipid envelope containing glycoprotein spikes surrounds a core consisting of the genome and its associated proteins (nucleocapsids) s.r. zaki and cd. paddock arranged in delicate tangles of filaments showing occasional granulation. the presence of characteristic inclusion bodies in thin section electron microscopy and a unique grid-like pattern on negative-stain electron microscopy differentiate these viruses from other members of the family bunyaviridae ( fig. 11.1b) . 65, 66 the severity and disease type largely depends on the viral serotype. two categories of hantavirus-associated illnesses are described: hemorrhagic fever with renal syndrome (hfrs) for disease in which the kidneys are primarily involved, and hantavirus pulmonary syndrome (hps) for disease in which the lungs are primarily involved. 67 -69 the isolation of the first recognized hantavirus (hantaan virus, named for the river in south korea), and its subsequent identification as the causative agent of hfrs was reported in 1978. 70 in 1993, the deaths of several previously healthy individuals due to a rapidly progressive respiratory disease in the southwestern united states were etiologically linked to a previously unrecognized hantavirus. clinically, the disease differs from hfrs in its pronounced pulmonary involvement and higher mortality rates and is known as hps. 23.69,71.72 hantavirus-associated diseases primarily affect blood vessels and result in different degrees of generalized capillary dilatation and edema. 73 in contrast to severe hfrs where abundant protein-rich, gelatinous retroperitoneal edema fluid is found, all hps patients have large bilateral pleural effusions and heavy edematous lungs. 7 in fatal far eastern hfrs, a distinctive triad of hemorrhagic necrosis can be seen in the renal medullary junctional zone, cardiac right atrium, and anterior pituitary. 75, 78 however, in patients with hps, hemorrhages are rare, and ischemic necrotic lesions, except those attributed to shock, are not seen. 72, 74 histologically, morphologic changes of the endothelium are uncommon but, when seen, consist of prominent and swollen endothelial cells. vascular thrombi and endothelial cell necrosis are rare. in hfrs, the most severe and characteristic microscopic lesions involve the kidney; however, an interstitial pneumonitis can also be seen in some fatal cases. in contrast, the microscopic changes of north and south american hps are principally seen in the lung and spleen. 72 ,74 the lungs show a mild to moderate interstitial pneumonitis characterized by variable degrees of edema and an interstitial mononuclear cell infiltrate comprised of a mixture of small and enlarged mononuclear cells with the appearance of immunoblasts ( fig.11 .3a). focal hyaline membranes composed of condensed proteinaceous intraalveolar edema fluid, fibrin, and variable numbers of inflammatory cells are observed ( fig. 11 .3b). typically, neutrophils are scanty and the alveolar pneumocytes are intact with no evidence of cellular debris, nuclear fragmentation, or hyperplasia. in fatal cases, with a prolonged survival interval, tissues show features more characteristic of the exudative and proliferative stages of diffuse alveolar damage. lung biopsies taken from patients who survive their illness appear similar with proliferated reparative type ii pneumocytes, severe edematous and fibroblastic thickening of the alveolar septa, and severe air-space disorganization with distorted lung architecture (see chapter 4) . other characteristic microscopic findings in hps cases include variable numbers of immunoblasts within the splenic red pulp and periarteriolar white pulp, lymph nodal paracortical zones, hepatic portal triads, and peripheral blood ( fig. 11.3d,e) . similarly, in severe hfrs cases, large mononuclear cells can be present in the spleen, lymph nodes, blood, and hepatic portal triads. 75 . 76 electron microscopic studies of hps lung tissue demonstrate infection of endothelial cells and macrophages. 69 . 72 the virus or virus-like particles observed are infrequent and extremely difficult to identify in autopsy tissues because of the considerable degree of viral pleomorphism and the postmortem deterioration of tissues. however, typical hantaviral inclusions are seen more frequently and their identity can be confirmed by immunolabeling ( fig. 11 .3f,g). similar inclusions are observed in epithelial cells in hfrs and are considered to be ultrastructural markers of hantavirus-infected cells. 63 .79.8o using immunohistochemistry, viral antigens are found primarily within capillary endothelium throughout various tissues in both hps and hfrs. in hps, marked accumulations of hantaviral antigens are in the pulmonary microvasculature and in splenic and lymph nodal follicular dendritic cells (fig. 11 .3c).72 despite the extensive endothelial cell accumulations of hantaviral antigens, there is little ultrastructural evidence of cytopathic effect. hantavirus pulmonary syndrome should be suspected in cases of adult respiratory distress syndrome (ards) without a known precipitating cause among previously healthy individuals. the level of suspicion should be particularly high when patients have a known exposure to rodents in areas where peromyscus maniculatus or other reservoirs of hantavirus are found. physicians need to differentiate hps from other common acute respiratory diseases, such as pneumococcal pneumonia, influenza virus, and unexplained ards. the diagnosis of hps, suspected by patient history and clinical manifestations, can also be supported histopathologically. although there is no single pathognomonic lesion that would permit certain histopathologic diagnosis of hps, the overall constellation of histopathologic hematologic findings suggests the diagnosis.72.7 4 diseases that need to be distinguished pathologically from hps include a relatively large number of different viral, rickettsial, and bacterial infections, as well as various noninfectious disease processes. virus-specific diagnosis and confirmation can be achieved through serology, pcr for hantavirus rna, or ihc for hantaviral antigens. 2 1,72 serologic testing can detect hantavirus-specific immunoglobulin m or rising s.r. zaki and cd. paddock titers of immunoglobulin g in patient sera and is considered the method of choice for laboratory confirmation of hps. immunofluorescent assays and enzyme-linked immunosorbent assays (elisas), which demonstrate the presence of specific antihantaviral antibodies, are currently used as rapid diagnostic tests and provide results within a few hours. recently, synthetic hantaviral nucleocapsid proteins have been used to improve the sensitivity and specificity of serologic assays. these proteins are more available than inactivated hantaviral antigens. 81 . 82 polymerase chain reaction detects viral rna in blood and tissues and is extremely useful for diagnostic and epidemiologic purposes. hantaviral rna can also be detected in formalin-fixed, paraffin-embedded archival tissue by reverse-transcriptase (rt)-pcr.83 immunohistochemistry testing of formalin-fixed tissues can be used to detect hantavirus antigens, and is a sensitive method to confirm hantaviral infections. 72 it has a unique role in the diagnosis of fatal hps cases when serum samples and frozen tissues are unavailable but formalinfixed autopsy tissues are obtainable. 84 • 85 severe acute respiratory syndrome the causative agent of sars is an enveloped, positivestranded rna virus that is a member of the genus coronavirus, of the family coronaviridae. corona viruses have the largest genomes of all rna viruses and replicate by a unique mechanism that results in a high frequency of recombination. maturation of sars coronavirus (sars-co v) is similar to features previously described for other coronaviruses. 8 6-9 0virions form by alignment of the helical nucleocapsids along the membranes of the endoplasmic reticulum or golgi complex and acquire an envelope by budding into the cisternae. the cellular vesicles become filled with virions and progress to the cell surface for release of the virus particles; large numbers of particles remain adherent to the plasma membrane at the cell surface. severe acute respiratory syndrome was recognized during a global outbreak of severe pneumonia that began in late 2002 in guangdong province, china, and gained prominence in early 2003 as cases were identified in more than two dozen countries in asia, europe, north america, and south america. the disease causes an influenza-like illness with fever, cough, dyspnea, and headache, and in severe cases it can cause death in humans. person-toperson transmission, combined with international travel of infected persons, accelerated the worldwide spread of the illness. 5 • 13 . 91 several reports have described diffuse alveolar damage with various levels of progression and severity as the main histopathologic findings in sars patients ( fig. 11 .4a,b)y·92-98 lungs typically show changes described for the proliferative phase of diffuse alveolar damage, with hyaline-membrane formation, desquamation of epithelial cells, fibrin deposit in the alveolar space, and hyperplasia of type 2 pneumocytes. increased mononuclear infiltrate in the interstitium can be seen in some cases. other findings identified in some patients included focal intra alveolar hemorrhage, necrotic inflammatory debris in small airways, and organizing pneumonia. in addition, multinucleated syncytial cells may be seen in the intraalveolar spaces of some patients who died 14 days or more after onset of illness ( fig. 1l .4c). infection with some coronaviruses, including sars-co v, is known to induce cell fusion in culture producing syncytial cells similar to those sometimes observed in lungs of patients who die from sars. these cells contain abundant vacuolated cytoplasm with cleaved and convoluted nuclei, but without obvious intranuclear or intracytoplasmic viral inclusions. the ish and ihc studies of tissues from sars patients have identified corona virus infection of upper airway bronchiolar epithelium ( fig. 11 .4d_f).92,99-101 infected ciliated columnar epithelial cells can be seen focally in lining epithelium of trachea and larger bronchi ( fig. 11 .4e). many of these infected cells slough from the epithelium and can be observed by using ish within the bronchial lumen. abundant viral antigens can also be found distributed focally in parenchyma of lungs of some patients and are seen predominantly in the cytoplasm of pneumocytes, in occasional macrophages, and in association with intra alveolar necrotic debris and fibrin (fig. ll.4d). double-stain studies indicate that most sars-co v-infected cells are type 2 pneumocytes. double-stain studies also detected viral nucleic acids with a distribution similar to that seen in ihc studies, mainly in pneumocytes and in some macrophages.looelectron microscopic examination of lung tissues selected from areas with abundant ihc staining shows numerous coronavirus particles and nucleocapsid inclusions. virions are seen in cytoplasmic vesicles and along the cell membranes of pneumocytes, in phagosomes of macrophages, and associated with fibrin in alveolar spaces (fig.ll.4g-i). because corona virus particles may be confused morphologically with other non viral cellular components, definitive ultrastructural identification can be achieved by using immunogold labeling electron microscopy. the primary histopathologic lesions seen in the lungs of patients who die from sars are somewhat nonspecific and can also be seen in acute lung injury cases caused by infectious agents, trauma, drugs, or toxic chemicals. 102 multinucleated syncytial cells similar to those seen in some sars patients can also be found in a number of virus infections, including measles, parainfluenza viruses, rsv, and nipah virus infections. 102 -104 in an early study of four human sars patients,13 viral antigens were not detected in the lung by ihe. the most likely explanation is that all patients in the study had a clinical course aver-s.r. zaki and cd. paddock aging more than 2 weeks. for many virus infections, viral antigens and nucleic acids are cleared within 2 weeks of disease onset by the host immune response. it is also possible that the pulmonary damage associated with sars is not caused directly by the virus, but represents a secondary effect of cytokines or other factors induced by the virus infection. similarly, in influenza virus infections, viral antigens are seen predominantly in respiratory epithelial cells of large airways and are only rarely identified in pulmonary parenchyma despite concomitant and occasionally severe interstitial pneumonitis. !os in recent reports by shieh et al. 100 and chong et al.,92 the temporal relationship between the duration of illness and clearance of sars-co v in human lung tissue was examined. viral antigens and nucleic acids were detected only in pulmonary tissues of patients who died early in the disease. the development of specific ihc, ish, and immunoelectron microscopy (iem) assays to identify sars-co v in formalin-fixed, paraffin-embedded samples has facilitated the assessment of the cellular tropism of sars-co v infection in human lung tissues. localization of sars-co v in the lung occurs mainly in the cytoplasm of pneumocytes, primarily type 2, and occasionally in alveolar macrophages ( fig. ll.4f ). type 2 pneumocytes are known to secrete pulmonary surfactant, resulting in reduced surface tension and preservation of the integrity of the alveolar space. these cells also play an important role in tissue restitution following lung damage. moreover, there is mounting evidence to support their contribution to the development of acute inflammatory lung injury following exposure to biological or chemical agents. additional studies are needed to further define the role of type 2 pneumocytes and alveolar macrophages in sars-co v infection. cynomolgus macaques inoculated with sars-cov develop pathologic findings of pneumonia and have been proposed as an animal model. 106 haagmans et a1. 107 showed extensive sars-co v antigen expression in experimentally infected macaques 4 days after infection. the antigens were mainly in alveolar lining epithelial cells with morphologic characteristics of type 1 pneumocytes, indicating type 1 pneumocytes are the primary target for sars-co v infection early in the disease. type 1 pneumocytes normally represent 90% of the alveolar epithelial cell volume and are easily damaged during pulmonary infections or other types of injury. in a more recent study on nonhuman primates,108 evidence of infection of type 1 pneumocytes in addition to some type 2 pneumocytes and macrophages was found. small animal models, such as rodents, would be very useful for evaluating vaccines, immunotherapies, and antiviral drugs, and we have recently identified the mouse as an animal model for this purpose. 10 9 in those studies, microscopic examination of trachea, bronchus, lung, thymus, and heart on day 2 after infection revealed mild and focal peri bronchiolar mononuclear inflammatory infiltrates with no significant histopathologic change in other organs. viral antigens and nucleic acids were focally distributed in bronchiolar epithelial cells, and virions were found in these same areas by ultrastructural analysis. data suggest that sars-co v replicates in mice to a titer sufficient to evaluate vaccines and antiviral agents. the mouse and other small animal models l1o might also be used to test the ability of the virus to replicate and cause disease and facilitate identification of host-immune mechanisms that contribute to the resolution of sars-co v infection. cytomegaloviruses (cmv) comprise a distinct and ancient group of herpesviruses that are widely distributed in nature, share similar growth characteristics in cell culture, and cause cellular enlargement and form distinctive inclusions in infected cells. these cytopathic changes, identified by early pathologists in the salivary glands of children dying from various unrelated diseases,111-113 led to the early designation of cytomegalic inclusion disease many years before the causative agent was isolated in the mid-1950s. the name cytomegalovirus was proposed in 1960 to reflect the cytopathic changes caused by these viruses. 114 cytomegaloviruses are highly host-specific, and various mammalian hosts, including nonhuman primates, rodents, and domesticated animals, are infected with their own distinct cmy. in this context, human cmv is stringently species-specific and, with rare exception, only infects cells of human origin. ll5 several cell types are permissive for cmv replication, including alveolar pneumocytes, vascular endothelium, fibroblasts, monocytes, dendritic cells, and exocrine and endocrine glandular epithelial cells.ll6 cytomegalovirus is a ~-herpesvirus with the largest genome (230 kilobase pair [kbp]) of all the herpesviruses known to infect humans. the double-stranded linear dna genome is contained within a 90 to 100nm icosahedral capsid and is surrounded by an amorphous material known as the tegument. these components are enclosed in a lipid bilayer envelope that is derived from the host cell nuclear or golgi membranes and contains several vir ally encoded glycoproteins necessary for infection of other cells. mature enveloped virions range from 150 to 200nm, making cmv one of the largest viruses that infect humans (fig. i1.1d ).ll7 the structure of cmv is typical of other human herpesviruses, but demonstrates some subtle ultrastructural differences from other viruses in this group including greater pleomorphism of the lipid envelope and dense body inclusions in the cytoplasm of infected cells.ll8 437 cytomegalovirus is a ubiquitous human pathogen, and in north america infects approximately 50% to 90% of the population.ll7 most of these infections are inapparent, although some cases of primary infection in otherwise healthy individuals result in a self-limited mononucleosis syndrome similar to that caused by epstein-barr virus; it is estimated that 20% to 50% of cases of heterophile-negative mononucleosis, and 8% of all cases of mononucleosis, are caused by cmy' 119 pulmonary involvement in cmv mononucleosis is infrequent and occurs in approximately 6% of these casesyo congenitally acquired cmv infection has various deleterious effects on the fetus, including mental retardation, neurologic abnormalities, sensorineural hearing loss, and retinitis, and in one series pulmonary involvement occurred in 64 % of symptomatic infants. 121 like all herpesviruses, cmv remains with its host for life after primary infection and establishes latency in various cell types, including vascular endothelial cells, monocytes and macrophages, neutrophils, and renal and pulmonary epithelial cells. 122 activation of viral replication occurs in persons with severely compromised immunity. patients with advanced hiv disease and recipients of hematopoietic stem cell or lung transplants are particularly at risk of developing cmv pneumonia. before the use of cmv screening and effective antiviral prophylaxis regimens, 10% to 30% of all patients undergoing allogeneic bone marrow transplantation for leukemia, and 15% to 55% of solid organ transplants, developed cmv pneumonia with case fatality rates of greater than 80% in some series. 26 ,123,124 the relatively high frequency of cmv pneumonia in lung transplant recipients may be a correlate of animal model data that indicate the lungs are a major site of latent cmv infection. 125 before the use of ganciclovir as therapy for cmv disease in aids patients, the case fatality rate for cmv pneumonia in this patient cohort was 75% when cmv was the only pathogen identified. mixed infections with cmv and another pathogen had an even worse prognosis, and the case fatality rate for patients with pulmonary disease caused by cmv and pneumocystis jiroveci (formerly carinii) was 92%. 126 cytomegalovirus pneumonia can show various histopathologic patterns (fig. 11 .5). extensive intra alveolar hemorrhage with scattered cytomegalic cells and relatively few inflammatory cell infiltrates may occur ( fig. 11 .5a).127 in a similar manner, extensive involvement of the alveolar epithelium with minimal inflammation or overt evidence of parenchymal injury has also been described. 128 other patterns include multifocal lesions with mixed inflammatory cell infiltrates, hemorrhage, necrosis, and cytomegalic cells, or a diffuse, predominantly mononuclear cell infiltrate, interstitial pneumonitis with intraalveolar edema and fibrin deposition, and diffusely distributed cytomegalic cells (fig. 11 .5e).129-131 the cytomegalic changes of cmv-infected cells are evident by standard h&e staining and are virtually pathognomonic of active cmv infection. the cells are enlarged (25 to 40!j,m) and contain amphophilic to deeply basophilic intranuclear and intracytoplasmic inclusions ( fig. 11 .5b,e). the single intranuclear inclusion is composed of viral nucleoprotein and assembled caps ids, and is a large (up to 20 !j,m), round to ovoid body with a smoothly contoured border that is generally surrounded by a clear halo that gives the inclusion a distinctive owl'seye appearance. the host cell nucleolus is often retained in the inclusion. 131 cytoplasmic inclusions are small (1 to 3!j,m), granular bodies that appear after the intranuclear inclusion is well developed and are not uniformly present in all cmv-infected cells (fig. 11 .5b). these inclusions represent a mixture of virions and various cellular organelles, and increase in size and number as the infection progresses. 132 unlike the intranuclear inclusion, the cytoplasmic inclusions stain with periodic acid-schiff stain and are deeply argyrophilic with gomori's methenamine silver stain.!33 cytomegalovirus pneumonia is defined by the presence of signs or symptoms of pulmonary disease combined with the detection of cmv in bronchoalveolar lavage (bal) fluid or lung tissue samples. in this context, detection methods that support this definition include virus isolation, histopathologic observation of cytomegalic cells, ish, or ihc stains (fig. 11 .5f). detection by pcr alone is considered too sensitive for the diagnosis of cmv pneumonia and is insufficient for this purpose. 134 cytomegalovirus is most often cultured in human diploid fibroblasts such as human embryonic lung and human foreskin fibroblasts. it grows slowly in conventional cell culture, and the cytopathic effect is generally not detected until the second week or longer after inoculation. for this reason, a shell vial method using centrifugation to enhance infectivity has become the standard isolation technique and can usually yield diagnostic results within 48 hours. 135 for centuries, the term herpes, derived from the greek erpein (to crawl), was used in medicine to describe any spreading cutaneous lesion. by the end of the 19th century, investigators surmised that the herpetic lesions of the lips and genitalia were manifestations of a single infectious agent, and recognized that it was a disease distinct from herpes zoster. 136.137 as with all human herpesviruses, hsv is a large, enveloped, double-stranded dna-containing virion with an icoshedral nucleocapsid approximately 100 to 1l0nm in diameter, composed of 162 cap somers. the nucleocapsid is surrounded by an amorphous, sometimes asymmetric material (the tegument) that is surrounded by a thin, trilaminar envelope that contains numerous glycoprotein spikes (fig. 11.1c ). the assembly of hsv begins in the nucleus of its host cell and the virus acquires its envelope as the capsid buds through the inner lamella of the nuclear membrane.120.l38 two serologic types are recognized and each is most frequently associated with particular disease syndromes; however, either serotype may cause any of the aggregate clinical syndromes. herpes simplex virus-1 causes gingivostomatitis, pharyngitis, esophagitis, keratoconjunctivitis, and encephalitis, and is the serotype most commonly associated with adult hsv pneumonia. herpes simplex virus-2 typically infects genital sites such as the penis, urethra, vulva, vagina, and cervix, and is the serotype associated with approximately 80% of disseminated disease and pulmonary infections in newborn infants. 136 all herpesviruses have the ability to persist in an inactive state for varying periods of time and then recur spontaneously following undefined stimuli associated with physical or emotional stress, trauma to nerve roots or ganglia, fever, immunosuppression, or exposure to ultraviolet radiation. 138 during the primary infection, hsv replicates at the portal of entry (typically oral or genital mucosae), and infects sensory nerve endings. the virus is transported centripetally along peripheral sensory nerves to central axons and finally to nerve cell bodies in the trigeminal, sacral, and vagal ganglia, where it replicates briefly before becoming latent.139-141 antiviral drugs have no effect on latent infection with hsv. following cues that initiate viral reactivation, hsv replicates in sensory ganglia and is transported centrifugally along sensory nerves to epithelial cells on mucosal surfaces. 138 reactivation of hsv from the trigeminal ganglion is associated with asymptomatic excretion of virus in saliva, and with the development of herpetic ulcers on the vermillion border of the lip, oral mucosa, or external facial skin. 142 newborn infants, severely immunosuppressed or burned patients, and patients with severe trauma are at greatest risk of developing hsv pneumonia.143-146 lower respiratory tract disease in neonates is most commonly associated with disseminated herpetic infections. disseminated hsv infection in the newborn was first described in 1935 as "hepatoadrenal necrosis"147 because of the prominent and frequent necroses that occur in the livers and adrenal glands of affected neonates. 148 most cases of neonatal disease represent primary hsv infections and are acquired during parturition from hsvinfected mothers. the incidence of neonatal hsv infection is approximately 1 in 3200 deliveries, and disseminated disease develops in approximately 25% of infected neonates. 149 in disseminated infections, signs and symptoms appear a mean of 5 days after birth (range, 0 to 12 days), and approximately 40% to 50% of these patients develop pneumonia. in the pre antiviral era, 85 % of neonates with disseminated disease died from the infection. with early diagnosis and high-dose acyclovir therapy, mortality has been reduced to approximately 30%?6,138,149 the disease can be exceedingly difficult to diagnose in a timely manner as only 10% of mothers of affected infants have clinically apparent hsv infection at the time of delivery.15o neonates that survive severe disseminated disease may develop hepatic and adrenal calcifications evident on abdominal radiographs. 151 in adults, infection of the respiratory tract with hsv may be associated with disseminated herpetic infection, but is more commonly identified as an isolated disease manifestation resulting from reactivation of latent herpetic infections in the oropharynx. herpetic tracheobronchitis is an ulcerative process characterized by large areas of denuded mucosal epithelium and fibrinopurulent exudate containing necrotic cells with densely eosinophilic cytoplasm. despite extensive tissue damage, cells with intranuclear inclusions may be sparse, and, when identified, are found most often at the margins of the ulcerated epithelium or occasionally in the mucous glands subjacent to ulcerated surfaces. ls2 aspiration of viruscontaining secretions into the lower respiratory tract is believed to be the most frequent cause of pulmonary infection with hsv; however, oral lesions may be absent in patients with herpetic laryngotracheobronchitis and bronchopneumonia. 153 disease can be also associated with airway trauma caused by tracheal intubation or from hematogenous dissemination of hsv. 144 ,1s2,1s4 chest radiographs of hsv pneumonia generally show ill-defined nodular or reticular densities of various sizes scattered in both lung fields. during the early stages of disease, these nodules measure 2 to 5 mm and are best seen in the periphery of the lungs. as the disease progresses, these lesions coalesce and enlarge to form more extensive infiltrates. 137 herpetic infections of the airways and lung are characteristically difficult to diagnose clinically and hsv pneumonia was not described as a distinct clinical entity until 1949.155 several studies attest to the relative infrequency with which this diagnosis is considered in patients with respiratory disease. for example, none of the 15 cases of hsv disease of the middle and lower respiratory tract identified in a review of autopsies at brooke army medical center during 1965 to 1968 were suspected prior to autopsy. 143 in a 1982 review of 20 culture-confirmed cases of hsv pneumonia, none of the patients had been diagnosed prior to death and all 16 had oral herpetic lesions at the time of death.144 recent investigations also suggest that lower respiratory tract disease caused by hsv may be more common than currently appreciated. in a study from sweden, hsv was cultured from proa diagnosis of tracheobronchitis and pneumonia is best established histologically (fig. 11.6 ). because lower respiratory tract hsv infections are often focused in the tracheobronchial tree, open lung biopsy may be less sensitive than bronchoscopy.154 herpetic lesions show extensive necrosis and karyorrhectic debris and are associated with hemorrhage and a sparse-to-moderate neutrophilic infiltrate (fig. 11.6a,b) . intranuclear inclusions are best appreciated in cells at the leading edge of necrotic foci ( fig. 11 .6b,c). inclusions appear either as homogeneous, amphophilic, and glassy (cowdry type b inclusions), or as eosinophilic with a halo separating the inclusion from the nuclear membrane (i.e., cow dry type a inclusions). 158 cowdry type b inclusions contain actively replicating virus. type a inclusions, considered noninfectious and devoid of viral nucleic acid or protein, represent the nuclear "scar" of hsv infection. 136 ,141 other changes associated with hsv, including multi nucleation and nuclear molding, and ballooning degeneration of the cytoplasm, are more frequently associated with squamous epithelium and are seldom encountered in the lung. because of the high frequency of hepatic and adrenal involvement with disseminated hsv infection in young children ( fig. 11 .6f,g), liver biopsy has been suggested as a diagnostic technique in this patient cohort. 148 commercially available antibodies exist for ihc detection of hsv in tissues (fig. 11.6d ).ls9 virus isolation remains an important diagnostic method; however, because hsv can be isolated from oropharyngeal secretions and occasionally from the lower respiratory tract of patients who lack overt pulmonary disease, virologic cultures must be interpreted in the context of complementary clinical, radiographic, and histopathologic findings as much as possible. cell culture systems susceptible to hsv include vero cells and foreskin fibroblasts. cytopathic effects generally develop within 24 to 48 hours after cultures are inoculated with infectious specimens. suitable specimens include scrapings made from mucocutaneous lesions, tracheobronchial aspirates, or bal specimens. in infants with evidence of hepatitis, it may also be useful to obtain duodenal aspirates for hsv isolation. 138 polymerase chain reaction methods that amplify hsv dna from clinical specimens, including tissue and blood, can be particularly useful by specifically distinguishing between hsv-l or hsv-2 infections.138,149 varicella-zoster virus (vzv), also known as human herpesvirus 3 (hhv-3) , is a human a-herpesvirus most closely related to hsv. it has a linear, double-stranded dna genome with approximately 125 kbp that encodes more than 70 proteins. the icosahedral nucleocapsid is indistinguishable in appearance from other herpes viruses. the nucleocapsid and the tegument are surrounded by a lipoprotein envelope derived from the host cytoplasmic membranes. the enveloped viral particle is pleomorphic to spherical in shape and 180 to 200nm in diameter. 16o the primary infection is initiated by inoculation of respiratory mucosa by the virus through infectious aerosols or by direct contact of skin lesions of patients with varicella or herpes zoster. after a primary viremia in the reticuloendothelial system, and secondary viremia in circulating mononuclear cells, the virus is disseminated to the skin, where it initiates a vesicular rash, and back to mucosal sites in the lungs. the release of infectious virus into respiratory droplets is a pathogenic characteristic that distinguishes vzv from other human herpesviruses. the attack rate for previously uninfected household contacts exposed to varicella is approximately 90%. for less sustained exposure, it is estimated to be approximately 10% to 30%. 160 during primary infection with vzv, viral replication in keratinocytes (fig. 11.7 a) and vascular and lymphatic endothelial cells of the superficial dermis produces the generalized, pruritic, vesicular rash of varicella commonly known as chickenpox. varicella-zoster virus also establishes latent infection within satellite cells and neurons of the trigeminal and dorsal root ganglia and can reactivate under various conditions to cause herpes zoster, a painful vesicular rash commonly referred to as shingles. 161 the origin of the term chickenpox is speculative, but believed to derive from gican, an old english term for itching. the term shingles originates from the medieval latin cinguls, or girdle, and alludes to the partial encircling of the trunk by the rash of herpes zoster. 137 ,160 varicella-zoster virus is ubiquitous in human populations around the world, and humans are the only known host. during the prevaccine era in the u.s., approximately 4 million cases, 4000 to 9000 hospitalizations, and 50 to 140 deaths were reported annually.25.162 the risk of severe illness during primary or recurrent vzv infection appears to depend more on host factors rather than a particular viral strain. chickenpox is considered a relatively benign infection in children, but adult patients are approximately 25 times more likely than children to develop pneumonia. the greatest risk of severe disease and pneumonia occurs in those patients with chronic lung disease, immunesuppressing conditions, neonates, and pregnant women. 163 varicella-zoster virus-related deaths have declined sharply in the u.s. since universal childhood vaccination was implemented in 1995. 24 ,25 s.r. zaki and cd. paddock varicella pneumonia was first described in the medical literature in 1942 164 and is the most frequently reported complication of chickenpox in adult patients. 24 ,165 pneumonia occurs in approximately 10% to 15% of adults infected with vzv. 166 ,167 the occurrence of pneumonia during herpes zoster is rare, and limited primarily to profoundly immunosuppressed patients, particularly bone marrow transplant recipients. m varicella-zoster virus pneumonia generally develops within 2 to 7 days following the onset of rash and may be characterized by fever, cough, tachypnea, chest pain, and hemoptysis. 26 .168 hypoxemia is common and may be severe. radiographically, the lungs show multiple, scattered, defined, nodular densities. untreated adult varicella pneumonia is fatal in approximately 10% of cases, but mortality is as high as 25% to 40% in certain high-risk cohorts, including pregnant women, transplant recipients, and neonates. 137 ,lfj9-171 massive pulmonary hemorrhage is a frequent terminal event. gross examination reveals lungs that are generally two to three times heavier than normal, firm, and plumcolored. there are often multiple necrotic and hemorrhagic lesions on the visceral pleura that resemble the pox lesions of skin. 169 ,172,173 pox may also be seen on the parietal pleura, although pleural effusions are uncommon and rarely prominent. 163 ,173 the trachea and bronchi are generally edematous and erythematous with occasional vesicles on the mucosal surfaces, and there may be lobular consolidation of the lungs. microscopically, pulmonary involvement consists primarily of interstitial pneumonitis and diffuse miliary foci of necrosis and hemorrhage in the pulmonary parenchyma that involve alveolar walls, blood vessels, and bronchioles ( fig. 11.7b,d) . 172 other findings may include intraalveolar collections of edema, fibrin, or hemorrhage, diffuse alveolar damage, and septal edema. 1m ,168.173 virally infected cells with intranuclear inclusions may be identified in respiratory epithelial cells, pneumocytes, interstitial fibroblasts, or capillary endothelium ( fig. 11 .7e,f).167 eosinophilic intranuclear inclusions and multinucleated syncytial cells may be difficult to locate but are best identified at the edges of necrotic foci (fig. 11.7c ). in cases of disseminated disease, similar necrotizing hemorrhagic lesions and occasional viral cytopathic changes in epithelial cells or fibroblasts may be observed in other tissues and organs, including esophagus, pancreas, liver, renal pelves, ureters, urinary bladder, spleen, bone marrow, thymus, lymph nodes, adrenal glands, and brain. 172,174 in those patients who recover from severe vzv pneumonia, some necrotic parenchymal foci may mineralize, and can be identified by chest radiograph years later as miliary, 1 to 5 mm, nodular opacities. microscopically, the lesions are characterized as discrete collections of dense fibrous connective tissue that surround multiple, small, calcified bodies. the periphery may include a cellular zone of fibroblasts and occasional giant cells.175 a radiographic survey of 16,894 persons identified pulmonary calcifications in eight (1.7%) of 463 patients who had chickenpox during adulthood compared with only eight (0.05%) of the remaining 16,431 who did not report having varicella as an adult. 176 a positive history of varicella predicts immunity in >95% of persons. 160 because pulmonary symptoms most often occur several days following the onset of the characteristic rash of varicella, a pathologic diagnosis is seldom required for a real-time diagnosis of vzv pneumonia. however, hematopoietic cell transplant recipients may present with signs of visceral dissemination and pneumonia 1 to 4 days before the localized cutaneous eruption of herpes zoster appears, and lower respiratory tract disease has been described in the absence of skin lesions, particularly in neonates and bone marrow transplant recipients. 26 ,137,177 commercially available antigen detection kits can be used for rapid diagnosis of cutaneous vzv infection. epithelial cells are scraped from the base of a newly formed vesicle, applied to a slide, and stained by using fluorescein-conjugated vzv monoclonal antibodies to detect specific viral proteins in the specimen. in a similar manner, the tzanck test uses wright-giemsa stain to demonstrate multinucleated giant cells in these specimens; however, this test does not differentiate between hsv and vzv, and false-negative results are common. commercially available antibodies are also available for ihc detection of vzv in tissue specimens ( fig. 11 .7e,f); however, relatively few laboratories are able to provide well-validated assays. some commercial laboratories offer pcr amplification to detect viral nucleic acid in clinical specimens. isolation of the virus in cell culture remains the reference standard for the diagnosis of vzv. in human melanoma cells, an excellent substrate for vzv isolation, the average time for visible cytopathic effect from the virus is 3 to 5 days.178 infectious vzv is usually recoverable from the clear fluid of cutaneous vesicles of varicella for approximately 3 days after the appearance of these lesions and for approximately 1 week from herpes zoster lesions. the lungs are the most common organ from which vzv is isolated at autopsy, but isolates have also been obtained from heart, liver, pancreas, gastrointestinal tract, brain, and eyes. 160 influenza is derived from the term influentia, meaning epidemic in the italian form of latin, originally used because epidemics were thought to result from astrologic or other occult influences. influenza is a highly contagious, acute respiratory illness with a spectrum of clinical illness ranging from asymptomatic or mild disease with rhinitis or pharyngitis to primary viral pneumonia with s.r. zaki and cd. paddock fatal outcome. influenza may also be associated with a broad range of other disorders affecting the heart, brain, kidneys, and muscle. influenzaviruses belong to the orthomyxoviridae family, which consists of four genera that include the two important influenza viruses types a and b associated with significant human disease. 179 ,180 influenza a viruses are further classified into subtypes based on the antigenicity of their hemagglutinin (ha) and neuraminidase (na) surface glycoproteins. only one type of ha and one type of na are recognized for influenza b. influenza a occurs in both pandemic and interpandemic forms. fortunately, pandemics, defined as worldwide outbreaks of severe disease, occur infrequently. interpandemic influenza, although less extensive in its impact, occurs virtually every year. the epidemiologic pattern of influenza in humans is related to two types of antigenic variation of its envelope glycoproteins, namely antigenic drift and antigenic shift. during antigenic drift, new strains related to those circulating in previous epidemics evolve by accumulation of point mutations in the surface glycoproteins. this enables the virus to evade the immune system leading to repeated outbreaks during interpandemic years. antigenic shift occurs with the emergence of a "new" potentially pandemic, influenza a virus that possesses a novel ha alone or in combination with a novel na. there are 16 recognized ha subtypes and nine na subtypes of influenza a virus. viruses from all ha and na subtypes have been recovered from aquatic birds, but only three ha subtypes (hi, h2, and h3) and two na subtypes (nl and n2) have established stable lineages in the human population since 1918. since 1997, widespread avian infection with influenza a (h5n1) and associated clusters of human disease have aroused concern about the threat of a pandemic, and attention has been appropriately focused on control measures to deal with such an event. all influenzaviruses have a segmented, negative-sense rna core surrounded by a lipid envelope. influenzavirus particles are pleomorphic. among isolates that have undergone a limited number of passages in cell culture or eggs, more filamentous than spherical particles are seen. spherical morphology becomes dominant when the virus is extensively passaged in the laboratory. a 10-to 12-nm layer of ha (rod-shaped) and na (mushroomshaped) spikes project radially on the surface of the influenza a and b viruses. hemagglutinin facilitates entry of virus into host cells through its attachment to sialic-acid receptors. because neutralizing antibodies are directed against this antigen, it is a critical component of current influenza vaccines. neuraminidase, the second major antigenic determinant, catalyzes the cleavage of glycosidic linkages to sialic acid and the release of progeny virions from infected cells. accordingly, it has become an important target for drug inhibitors such as oseltamivir and zanamivir. the m2 surface component and channel of influenza a (not present in influenza b virus) regulates the internal ph of the virus and is blocked by the antiviral drug amantadine. influenzaviruses are spread person-to-person primarily through the coughing and sneezing of infected persons. the typical incubation period for influenza is 1 to 4 days, with an average of 2 days. adults can be infectious from the day before symptoms begin through approximately 5 days after illness onset. children can be infectious for ~1o days, and young children can shed virus for several days before their illness onset. severely immunocompromised persons can shed virus for weeks or months. uncomplicated influenza illness is characterized by the abrupt onset of constitutional and respiratory signs and symptoms (e.g., fever, myalgia, headache, malaise, nonproductive cough, sore throat, and rhinitis). among children, otitis media,nausea,and vomiting are also commonly reported with influenza illness. respiratory illness caused by influenza is difficult to distinguish from illnesses caused by other respiratory pathogens on the basis of symptoms alone. influenza typically resolves after 3 to 7 days in most patients, although cough and malaise can persist for >2 weeks. among certain persons, influenza can exacerbate underlying medical conditions (e.g., pulmonary or cardiac disease), lead to secondary bacterial pneumonia or primary influenza viral pneumonia, or occur as part of a co-infection with other viral or bacterial pathogens. young children with influenza infection can have initial symptoms that mimic bacterial sepsis. more than 20% of children hospitalized with influenza can have febrile seizures. influenza has also been associated with encephalopathy, transverse myelitis, reye syndrome, myositis, myocarditis, and pericarditis. the risks for complications, hospitalizations, and deaths from influenza are higher among persons aged ~65 years, young children, and persons of any age with certain underlying health conditions than among healthy older children and younger adults. influenza-related deaths can result from pneumonia or from exacerbations of cardiopulmonary conditions and other chronic diseases. the histopathologic features of nonfatal and fatal influenza have been well described and include necrotizing bronchitis, thrombosis, interstitial inflammation, hemorrhage, hyaline membrane formation, and intra alveolar edema (figs. 11.8a,b, 11 .9a,c, and 11.10a). 105, [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] [191] the pathology is more prominent in larger bronchi, and inflammation may vary in intensity in individual patients, viral inclusions cannot be identified by light microscopy (fig, 11 .8d), secondary bacterial infections with organisms such as streptococcus pneumoniae (group a streptococcus [gas]), staphylococcus aureus, and haemophilus influenzae may occur as a complication in about 50% to 75% of fatal cases and make it difficult to recognize the pathologic changes associated with the primary viral infec-445 tion ,190,192,193 the histopathologic features in other organs may include myocarditis, cerebral edema, rhabdomyolysis, and hemophagocytosis (figs, 11.8h and 11.9e,f), immunohistochemistry and ish assays demonstrate that viral antigens and nucleic acids are usually sparse and are primarily seen in the bronchioepithelial cells of larger bronchioles (figs. 11.8c,e,f and 11.9d) . 105.189,190 antigens are more readily identified in patients who die within 3 to 4 days of onset of illness. recent studies suggest that unlike human influenza viruses, avian virus h5n1 preferentially infects cells in the lower respiratory tract of humans, resulting in extensive damage of the lungs with minimal pathology in the upper respiratory tract (fig. 11.10a,c) . this may help explain why the h5n1 avian influenza virus is so lethal to humans but so difficult to spread from person to person. these studies show that the avian virus preferentially binds to the a-2,3-galactose receptors, which are found only in and around the alveoli. this is in contrast to the human influenzaviruses that preferentially bind to the a-2,6-receptors, which are found throughout the respiratory tract from the nose to the lungs. 194.195 in birds and other animals, viral antigens can be detected in the lung as well as a variety of extrapulmonary tissues (fig. 11.10b) . the diagnosis of influenza, suspected by history and clinical manifestations, can also be supported histopathologically. however, because of the absence of any characteristic viral inclusions and because the overall pathologic features of influenza may resemble other viral, rickettsial, and certain bacterial infections, an unequivocal diagnosis can be made only by laboratory tests such as viral culture, direct fluorescent antibody and rapid antigen assays, serology, and ihc. 190 ,196,197 measles measles (rubeola) is an infectious, acute febrile viral illness characterized by upper respiratory tract symptoms, fever, and a maculopapular rash. the causative agent, a member of the genus morbillivirus, of the family paramyxoviridae,198,199 is an enveloped virus that contains a negative sense, single-stranded rna genome of 16,000 nucleotides. other human pathogens in this family include parainfluenza, mumps, and respiratory syncytial viruses. measles virions are pleomorphic, generally spherical, enveloped particles from 120 to 250 nm in diameter. the virus is morphologically indistinguishable from other members of the paramyxoviridae family when viewed by negative contrast electron microscopy. a lipid envelope surrounds a helical nucleocapsid composed of rna and protein. two transmembrane glycoproteins, hemagglutinin (h) and fusion (f), are present in the envelope and appear as surface projections. these proteins mediate viral attachment and figure 11.8. influenza a. a. alveolar damage in a patient with fatal influenza a showing prominent congestion with intraalveolar macrophages and fibrin deposits. b. extensive ulceration of respiratory epithelium in a large airway of a patient with fatal influenza a. the lamina propria shows florid vascular congestion and focal hemorrhage, and predominantly mononuclear inflammatory cell infiltrates. c-e. immunohistochemical staining (c,e) of influenza virus a hemagglutinin antigens in the cytoplasm of residual respiratory epithelial cells of a large airway. this same focus of extensively infected respiratory epithelium (d) demonstrates that, unlike many other viral respiratory pathogens, influenza viruses do not elicit specific cytopathic effects in infected cells. f. in-situ hybridization assay demon-• fusion with respiratory epithelium. they are also believed to playa role in virus maturation through their interaction with the matrix (m) protein, which, in turn, is thought to interact with the nucleocapsid structure. 2oo measles is a highly communicable disease of worldwide distribution. before the introduction of measles vaccines, epidemics occurred about every 2 to 5 years when the percentage of nonimmune members of a population reached critical levels. recently, epidemics have occurred in cycles of about 10 years. in small and isolated communities, measles circulation may cease altogether unless it is reintroduced. if introduced in non immune populations, the disease tends to be more severe and may involve more than 90% of the population because of the highly infectious nature of the virus. although still a significant problem in underdeveloped countries, measles infection became uncommon in the us. after the development and widespread use of an effective measles vaccine. however, a recrudescence of measles infection occurred in several large us. urban centers in recent years, associated with reduced use of the vaccine among children and young adults. during the peak of this activity (i.e., between 1989 and 1991), greater than 50,000 measles cases and approximately 150 measlesassociated deaths were reported. 20l measles virus is highly contagious, spread by aerosols and droplets from respiratory secretions of acute cases.202-204 less frequently, contaminated fomites are involved in transmission. a person with acute measles is infective from just before onset of symptoms to defervescence of fever. in developed countries, likely settings for exposure to measles virus are infectious disease clinics, pediatric emergency rooms, and physicians' offices. 20s children are usually infected by 6 years of age, resulting in lifelong immunity, and almost all adults are immune. clinical infection in children younger than 9 months of age is generally uncommon because of passive protection afforded the infant by the transfer of maternal antibodies. however, with the resurgence of measles in the us. came the realization that most women of childbearing age 447 strating active replication of influenza a in respiratory epithelium in a large airway. g. electron micrograph of influenza a particles (arrows) attached to the cilia of a rodent tracheal epithelial cell. viral ribonucleoproteins are evident in the central aspect of the particles. the hemagglutinin and neuraminidase surface glycoproteins make up the peripheral spike layer. (courtesy of f.a. murphy.) h. rhabdomyolysis in a patient with fatal influenza a. bar, 100nm. a,b,d,h, h&e; c,e, immunoalkaline phosphatase stain, naphthol fast-red, and hematoxylin counterstain; f, digoxigenin-iabeled probe followed by immunoalkaline phosphatase staining, naphthol fast-red. and hematoxylin counterstain; g, uranyl acetate, lead citrate stain. acquired immunity to measles through vaccination and not through natural infection. lower levels of maternal antibodies were found to be transferred from an immunized mother to her infant; thus, a substantial number of measles cases occurred in children younger than 1 year of age in the us. in recent years. after an incubation period of about 1 to 2 weeks, the prodromal phase of measles begins with fever, rhinorrhea, cough, and conjunctivitis. koplik's spots, which are small, irregular red spots with a bluish-white speck in the center, appear on the buccal mucosa in 50% to 90% of cases shortly before rash onset. an erythematous maculopapular rash begins on the face 3 to 4 days after prodromal symptoms and usually spreads to the trunk and extremities. the symptoms gradually resolve, with the rash lasting for approximately 6 days, fading in the same order as it appeared. although recovery is rapid and complete in most cases, complications can arise as a result of continued and progressive virus replication, bacterial or viral superinfections, or abnormal host immune response. 204.206.207 the most common complications are secondary bacterial pneumonia and otitis media. 206 other complications include febrile convulsions, encephalitis, liver function abnormalities, chronic diarrhea, and sinusitis. several pulmonary and central nervous system (cns) syndromes that are often fatal have been described. death occurs in about 1 of every 1000 measles cases; however, the risk of death and other complications is substantially increased in infants, adults, malnourished and immunocompromised individuals, persons with underlying illnesses,and nonimmunized populations in underdeveloped countries.208-212 the first step in measles infection is attachment of the virus to cd46 cell surface receptors on the respiratory epithelium.213 adhesion and fusion of the virus to the respiratory epithelium is mediated by both the hand f viral glycoproteins. 214 this stage is followed by local replication in respiratory mucosa and draining lymph nodes. a primary viremia follows, with dissemination two types of multinucleated giant cells have been described in patient tissues during measles infection. 215 ,216 the reticuloendothelial giant cell (warthin-finkeldey) appears first during the incubation period and is seen in 449 the nucleus of a pneumocyte in the lung of a patient with fatal avian influenza. unlike other influenza viruses that cause disease in humans, h5n1 preferentially infects alveolar epithelial cells, and causes relatively minimal pathology in the upper airway. a, h&e; b,c, immunoalkaline phosphatase stain, naphthol fast-red, and hematoxylin counterstain. different lymphoid tissues throughout the body. the second type is the epithelial giant cell, which has been observed in the epithelium of essentially every major organ. the onset of the rash temporally coincides with the appearance of detectable serum antibody to measles virus. interestingly, virus replication and giant cell formation cease with the appearance of rash. t-cell immunity is essential in the process of viral clearance from lymphoid tissue and respiratory tract. while children with congenital agammaglobulinemia respond normally to measles virus infection, patients with cell-mediated immunodeficiency develop severe disease that presents as giant-cell pneumonia or encephalopathy in the absence of an exanthem. [208] [209] [210] [211] 217, 218 immunity to the f surface glycoprotein is necessary to prevent the spread of measles infection. 219 an atypical measles syndrome characterized by pulmonary consolidation with pleural effusions and hilar adenopathy has been reported in children exposed to wild-type measles virus who had previously received the killed-measles virus vaccine.220-222 recipients of the formalin-inactivated vaccine have a good antibody response to the h protein, but antibodies to the functional region of the f protein and to the nucleoprotein are weak or absent. it has been suggested that the lack of a functional f antibody response may playa role in virus spread. 223 the pathologic features of measles have been well described and several references containing detailed morphologic descriptions are recommended. 13 1,209.224-230 the typical morbilliform skin lesions, koplik's spots, and measles lymphadenitis are seldom seen by the surgical pathologist since the clinical diagnosis is usually apparent. histopathologic changes in the skin include mild congestion, edema, and a predominantly mononuclear infiltrate surrounding small vessels of the dermis, as well as other nonspecific features. occasional diagnostic multinucleated epithelial giant cells with eosinophilic cytoplasmic and nuclear inclusions are observed. 228 .231 pathognomonic reticuloendothelial multinucleated giant cells can be observed in appendix specimens from patients mistakenly operated on for acute appendicitis before the emergence of diagnostic koplik's spots and rash. these cells, which have been reported in various lymphoreticular tissues throughout the body, are typically large and contain from a few to occasionally up to 100 nuclei. these cells do not usually contain viral inclusions. the lymphoid tissues are typically hyperplastic, and the architecture is partially or totally obliterated by diffuse proliferation of immunoblasts. [232] [233] [234] a focal or generalized interstitial pneumonitis, similar to that seen in many other viral infections, is seen in the lungs of measles patients. histopathologic features seen include various degrees of peribronchial and interstitial mononuclear cell infiltrates, squamous metaplasia of bronchial endothelium, proliferation of type ii pneumocyte alveolar lining cells, and intraalveolar edema with or without mononuclear cell exudates and hyaline membranes. secondary changes, such as bacterial or viral superinfection, or organizational changes may alter the original pathology. the hallmark of the disease is the formation of multinucleated epithelial giant cells (fig. ll.lla,b) . these cells, which are often numerous, are formed by fusion of bronchiolar or alveolar lining epithelial cells (fig.ll.l1a) . in contrast to the reticuloendothelial giant cells, these cells generally contain characteristic nuclear and cytoplasmic inclusions. the intranuclear inclusions are homogeneous, eosinophilic, and surrounded by a slight indistinct halo (fig. 11.11c,d) . the cytoplasmic inclusions are deeply eosinophilic, vary in size, and some form large masses with a "melted tallow" appearance ( fig. 11.11d ). these giant cells may undergo degenerative changes with progressive loss of cytoplasm, increasing basophilia, and shrinkage of nuclei. the presence of measles virus in these giant cells may be demonstrated by immunofluorescent,235.236 ihc,21o.237 and ish techniques (fig.ll.lle,f) . these giant cells can also be seen in extra pulmonary tissues (fig. 11.11 g,h) . the diagnosis of typical cases of measles can usually be made on the basis of clinical signs and symptoms. other causes of a similar rash, but without other features of measles, include rubella, dengue virus, enteroviruses, and drug reactions, especially to ampicillin. the typical case of measles giant-cell pneumonia generally presents little diagnostic difficulty for the surgical pathologist. the presence of giant cells with both intranuclear and intracytoplasmic inclusions in a setting of interstitial pneumonitis is highly specific for measles infection. however, multinucleated giant cells are not seen in all cases of measles pneumonia and their absence should not exclude the j diagnosis. other viral and rickettsial agents may also cause a similar interstitial pneumonitis, but without the typical giant cells, and should be differentiated. 229 as previously noted, the histopathologic features in measles pneumonia can be somewhat variable,226 and secondary bacterial and viral infections may modify the histology, further complicating the pathologic diagnosis ( fig. 11.5d ).207.229 other viral pathogens, such as respiratory syncytial virus,238 parainfluenza,239.240 vzv,241 and a recently discovered hendra virus,242 as well as granulomatous diseases of the lung, may give rise to pneumonia with giant cells and should also be considered in the differential diagnosis. however, these clinical entities can be distinguished by history, histopathologic features, and laboratory tests. immunohistochemistry237.243,244 or ish 243 ,244 tests demonstrate viral antigens or nucleic acids in the majority of cases. laboratory confirmation is useful to avoid possible confusion with other rash-causing illnesses. diagnostic laboratory procedures consist of direct detection of either the virus or the viral antigens, usually by indirect immunofluorescence or by serologic methods using hemagglutination inhibition, neutralization, or enzyme immunoassay. specimens for serologic testing consist of acute-and convalescent-phase serum pairs. antibody appears within 1 to 2 days after onset of rash, and titers peak approximately 2 weeks later. alternatively, the presence of specific immunoglobulin m (igm) antibody can be used to diagnose recent infection. 245 human parainfluenza viruses (hpivs) are second only to rsv as a cause of lower respiratory tract disease in young children. human parainfluenza viruses are negative-sense, nonsegmented, single-stranded, enveloped rna viruses that possess fusion and hemagglutinin-neuraminidase glycoprotein "spikes" on their surface (fig. 11 .ig). the four serotypes of hpiv belong in the family paramyxoviridae, subfamily paramyxovirinae, and genera respirovirus (hpiv-l and -3) and rubulavirus (hpiv-2 and -4). the virions are variable in shape and size, ranging from 150 to 300nm. 246 human parainfluenza viruses are spread from respiratory secretions through close contact with infected persons or contact with contaminated surfaces or objects. infection can occur when infectious material contacts mucous membranes of the eyes, mouth, or nose, and possibly through the inhalation of droplets generated by a sneeze or cough. human parainfluenza viruses are unstable in the environment (surviving a few hours on environmental surfaces), and are readily inactivated with soap and water. they are ubiquitous, and infect most s,r. zaki and cd. paddock people during childhood. the highest rates of serious hpiv illnesses occur among young children. serologic surveys have shown that 90% to 100% of children aged 5 years and older have antibodies to hpiv-3, and about 75% have antibodies to hpiv-l and -2. the different hpiv serotypes differ in their seasonality, with hpiv-l causing biennial outbreaks of croup in the fall and hpiv-2 causing annual or biennial fall outbreaks. human parainfluenza virus-3 peak activity occurs during the spring and early summer months each year, but the virus can be isolated throughout the year. similar to rsv, hpivs can cause repeated infections throughout life, usually manifested by an upper respiratory tract illness (e.g., cold and sore throat). human parainfluenza viruses can also cause serious lower respiratory tract disease with repeat infection (e.g., pneumonia, bronchitis, and bronchiolitis), especially among the elderly, and among patients with compromised immune systems. each of the four hpivs has different clinical and epidemiologic features. the most distinctive clinical feature of hpiv-l and hpiv-2 is croup (i.e., laryngotracheobronchit is ); hpiv-l is the leading cause of croup in children, whereas hpiv-2 is less frequently detected. both hpiv-l and -2 can cause other upper and lower respiratory tract illnesses. human parainfluenza virus-3 is more often associated with bronchiolitis and pneumonia. human parainfluenza virus-4 is infrequently detected, possibly because it is less likely to cause severe disease. the incubation period for hpivs is generally from 1 to 7 days. 247 most hpiv infections cause a mild, self-limited illness; however, hpiv-3 infections are an important cause of bronchiolitis, croup, and pneumonia that may be lifethreatening in infants and newborns. 247 human parainfluenza virus infections are also increasingly being recognized as an important cause of severe morbidity and mortality in immunocompromised patients. 20 ,248--252 the mortality of bone marrow transplant patients with hpiv-3 infection has been reported to be as high as 60%. 249, 253, 254 in patients with severe hpiv infection, multinucleated giant cells derived from the respiratory epithelium may be seen in association with an interstitial pneumonitis and organizing changes (fig. 11.12a,b ) . 239,240.249,255-260 these giant cells, which may contain intracytoplasmic eosinophilic inclusions, (fig. 11.12c) , have also been reported in extrapulmonary tissues such as kidney, bladder, and pancreas. 258 other viral causes of giant cell pneumonia, including measks, rsv, vzv, and hsv, should be considered in the histopathologic differential and laboratory testing, including ihe, can be useful in making this differentiation possible ( fig. 11.12d) . diagnosis of infection with hpiv scan also be made by virus isolation, direct detection of viral antigens py enzyme-linked immunoassay (eia) or immunofluorescent assay (ifa) in clinical specimens, detection of viral rna by rt-pcr, demonstration of a rise in respiratory syncytial virus (rsv) is the most common cause of bronchiolitis and pneumonia among infants and children under 1 year of age. the causative agent is a negative-sense, nonsegmented, single-stranded, enveloped rna virus. the virion is variable in shape and size and ranges from 120 to 300 nm. respiratory syncytial virus is a member of the family paramyxoviridae, subfamily pneumovirinae, in the genus pneumovirus, and can be 453 ing poorly defined cytoplasmic eosinophilic inclusions. d. parainfluenza virus antigens in giant cells localized by using immunohistochemistry. a-c, h&e; d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. further distinguished genetically and antigenically into two subgroups, a and b. the subgroup a strains are usually associated with more severe infections. two surface glycoproteins, g and f, are present in the envelope and mediate attachment and fusion with respiratory epithelium. the f protein also mediates coalescence of neighboring cells to form the characteristic multinucleated syncytial giant cells for which the virus name is derived. 265 respiratory syncytial virus is spread from respiratory secretions through close contact with infected persons or contact with contaminated surfaces or objects?66 infection can occur when infectious material contacts mucous membranes of the eyes, mouth, or nose, and possibly through the inhalation of droplets generated by a sneeze or cough. respiratory syncytial virus is unstable in the environment, surviving a few hours on environmental surfaces, and is readily inactivated with soap and water. in temperate climates, rsv infections usually occur during annual community outbreaks, often lasting several months, during the late fall, winter, or early spring months. the timing and severity of outbreaks in a community vary from year to year. respiratory syncytial virus spreads efficiently among children during the annual outbreaks, and most children will have serologic evidence of rsv infection by 2 years of age. illness begins most frequently with fever, runny nose, cough, and sometimes wheezing. during their first rsv infection, between 25% and 40% of infants and young children have signs or symptoms of bronchiolitis or pneumonia, and 0.5% to 2% require hospitalization. most children recover from illness in 8 to 15 days. the majority of children hospitalized for rsv infection are under 6 months of age. respiratory syncytial virus also causes repeated infections throughout life, usually associated with moderate-to-severe cold-like symptoms; however, severe lower respiratory tract disease may occur at any age, especially among the elderly or among those with compromised cardiac, pulmonary, or immune systems. the major histopathologic changes described in fatal rsv infections include necrotizing bronchiolitis and interstitial pneumonia (fig. 11.13a,b) .238,267-272 bronchiallumina and airways are usually filled with necrotic debris and inflammatory cells. these findings may be accompanied by various degrees of diffuse alveolar damage (fig. 11.13e ), organizational changes, and secondary bacterial superinfection. giant cell pneumonia is a feature seen in some cases ( fig. 11.13c,d) . the multinucleated giant cells contain irregular, intracytoplasmic, eosinophilic inclusions surrounded by a clear halo. these inclusions are extremely difficult to identify with any degree of certainty and are only seen in about half the cases (fig. 11.13d) . other viral causes of giant cell pneumonia should be considered in the histopathologic differential and laboratory testing, including ihc,271,273,274 can be useful in making this differentiation possible ( fig. 11.13b,f) . diagnosis of rsv infection can also be made by virus isolation, direct detection of viral antigens in clinical specimens by eia or ifa, detection of viral rna by rt-pcr, demonstration of a rise in rsvspecific serum antibodies, or a combination of these approaches (see also fig. 7.43, chapter 7) .264,275-281 in 2001, van den hoogen et a1. 282 described the identification of this new viral agent from clinical specimens obtained from patients with respiratory illness, which 455 they designated human metapneumovirus (hmpv). it is a negative-sense, nonsegmented, single-stranded, enveloped rna virus. the virion is variable in shape and size, ranging from 150 to 300nm (fig. 11.1f ). it has been categorized in the family paramyxoviridae, subfamily pneumovirinae, genus metapneumovirus, based on genomic sequence and gene constellation. human metapneumovirus can be further distinguished genetically and antigenically into two subgroups, a and b. similar to rsv, hmpv infection is ubiquitous and occurs during infancy and early childhood, with annual epidemic peaks occurring in the winter and spring months in temperate regions. seroprevalence studies reveal that 25% of all children aged 6 to 12 months have antibodies to hmpv; by age 5 years, 100% of patients have evidence of past infection. like rsv, hmpv has been associated with a wide spectrum of respiratory illnesses.283-287 the patient may be asymptomatic, or symptoms may range from mild upper respiratory tract illness to severe bronchiolitis and pneumonia. although rsv, hpiv-l, and hpiv-3 have been definitively linked to cases of lower respiratory tract disease in infants and young children, the relative contribution of hmpv remains undetermined. like rsv and the hpivs, studies suggest that hmpv may also contribute to respiratory disease in elderly adults and the immunocompromised. 288-29o histopathologic descriptions of features of hmpv infections are few and have not been well described. [291] [292] [293] [294] this is partly related to interpreting the clinical significance of virus detection in context with the ubiquitous nature of the virus. virus detection in such cases is usually made by culture isolation of the virus from upper airways or by pcr assays performed on nasopharyngeal aspirates or bal washings. in nonhuman primates viral antigens are observed in ciliated epithelial cells, type 1 pneumocytes, and alveolar macrophages. this distribution is associated with mild, multifocal, erosive, and inflammatory changes in airways, and an increased number of foamy macrophages in alveoli. 291 the bal specimens collected from patients within a few days of a positive hmpv assay show degenerative changes and cytoplasmic inclusions within epithelial cells, multinucleated giant cells, and histiocytes. the intracytoplasmic inclusions are ill-defined, eosinophilic structures that measure 3 to 4 ~m. 293 lung biopsy or autopsy tissue obtained and examined later in the disease show chronic airway inflammation, intraalveolar foamy and hemosiderin-laden macrophages, and acute and organizing lung injury including areas of diffuse alveolar damage with hyaline membrane formation and foci of a bronchiolitis obliterans/organizing pneumonia like reaction ( fig. 11 .14). in such cases, typical multinucleated giant cells or viral inclusion cannot be identified. 292 -294 in-situ hybridization studies on limited number of human cases human metapneumovirus is difficult to identify with commonly used viral diagnostic procedures. the virus replicates slowly in primary and tertiary monkey kidney cell lines, and the cytopathic effect can be difficult to discern. commercial monoclonal antibody reagents to hmpv are not widely available. most hmpv studies have been conducted using rt-pcr assays or by demonstration of a rise in hmpv-specific serum antibodies. two novel paramyxoviruses, hendra and nipah, have been recently identified in australia and malaysia; these viruses have been associated with acute febrile encephalitis and respiratory tract disease. both infections are zoonotic. hendra was first identified in 1994 when patients who came in close contact with sick horses developed an influenza-like illness. two patients died with pneumonitis and multiorgan failure. 30 the closely related nipah virus was identified during an outbreak in malaysia and singapore during 1998-1999 that included more than 250 patients. patients presented with a severe acute encephalitic syndrome, but some also had significant pulmonary manifestations. 22 ,295-302 most of the patients had a history of contact with pigs, most of them being pig farmers. in bangladesh in 2001 and 2003, outbreaks of nipah encephalitis occurred. 303 ,304 similar to the malaysian outbreak, the most prominent symptoms were fever, headache, vomiting, and an altered level of consciousness. respiratory illness was much more common in the bangladesh cases, however, with 64 % having cough and dyspnea. the reason for increased involvement of the respiratory tract in this outbreak is not known. epidemiologic and laboratory investigations identified fruit bats of the pteropus genus as asymptomatic carriers of hendra and nipah viruses and possible animal reservoirs. [305] [306] [307] [308] [309] hendra and nipah viruses belong to the recently designated genus henipavirus within the family paramyxoviridae, subfamily paramyxovirinae. both viruses are nonsegmented, negative-stranded rna viruses composed of helical nucleocapsids enclosed within an envelope to form roughly spherical, pleomorphic virus particles.3io-312 the structure of their genome is consistent with the other members of the subfamily. 30 histopathologic findings in fatal cases of hendra and nipah infections are similar with varying degrees of cns and respiratory tract involvement. 104 ,313-315 findings include a systemic vasculitis with extensive thrombosis, endothelial cell damage, necrosis, and syncytial giant cell formation in affected vessels ( fig. 11.15a ,b,f). plaques with various degrees of necrosis, in association with inclusion-bearing neurons, can be found in both the gray and white matter of the cns (fig. 11.15e ). multinucleated giant cells with intranuclear inclusions can occasionally be seen in lung, spleen, lymph nodes, and kidneys ( fig. 11 .15c,g). in the lung, vasculitis and fibrinoid necrosis can be seen in majority of cases. fibrinoid necrosis often involves several adjacent alveoli and is frequently associated with small vessel vasculitis. the multinucleated giant cells with intranuclear inclusions are usually noted in alveolar spaces adjacent to necrotic areas. histopathologic changes of bronchiolar epithelium are uncommon; rarely, the large bronchi may show transmural inflammation and ulceration. widespread presence of nipah virus antigens can be seen by ihc in endothelial and smooth muscle cells of blood vessels as well as in various parenchymal cells (fig. 11.15d ). the diagnosis of nipah virus infection, suspected by patient history and clinical manifestations, can be supported by characteristic histopathologic findings. from a diagnostic standpoint, perhaps the most unique histopathologic finding is the presence of syncytial and parenchymal multinucleated endothelial cells. this feature 457 occurs in only approximately one fourth of the cases and cannot be used as a sensitive criterion for the diagnosis of henipah virus infections; furthermore, these cells can also be seen in measles virus, rsv, hpiv, herpesviruses, and other infections. unequivocal diagnosis can be made only by laboratory tests such as ihc, cell culture isolation, pcr, or serology.l04,304,316-319 the parvoviruses are small (18 to 26nm) naked viruses that possess a single-stranded dna genome and require actively dividing cells to complete the viral replication cycle. in 2005, allander et a1. 320 identified a new parvovirus (genus bocavirus) associated with lower respiratory tract infections in children; however, there is no information as yet that describes specific pulmonary pathology attributed to this newly identified agent. on the other hand, human parvovirus b19, a member of the genus erythrovirus, has long been known to cause human disease and has been well studied. 321 ,322 the most commonly recognized manifestation of b19 infections is erythema infectiosum, and approximately one third of the cases of maternal parvovirus infections result in intrauterine parvovirus b19 infections. this places the fetus at increased risk for severe anemia, hydrops, and death. hydrops fetalis is the most commonly recognized complication of intrauterine parvovirus infection, accounting for 4% to 18% of all cases of nonimmune hydrops. cases tend to be clustered during community outbreaks of erythema infectiosum. 321 . 3 22 pathophysiologic effects and histopathologic findings are a result of the tropism of b19 parvovirus for erythroid precursor cells. villi from placentas from patients with bl9-associated nonimmune hydrops are edematous, and fetal capillaries show numerous nucleated erythroid precursors, some containing parvovirus inclusions. the infected cells with eosinophilic "ground glass" intranuclear inclusions and ring-like margination of nuclear chromatin are easily recognized and in the context of a hydropic fetus are pathognomonic of b19 infection. the liver is the major site of blood production in the fetus and the principal organ affected by intrauterine b19 infection. inclusion-bearing nucleated erythrocytes can also be frequently identified in the lung, making histopathologic examination of this organ a worthwhile endeavor for confirming the diagnosis of intrauterine bi9 infection ( fig. il.i6a,b) .323-332 however, these cells may be infrequent and irregularly distributed, requiring examination of multiple sections and use of ihc to confirm the diagnosis (fig.l1.16c,d) . the combination of fever and hemorrhage can be caused by different viruses, rickettsiae, bacteria, protozoa, and fungi. however, the term viral hemorrhagic fever (vhf) is usually reserved for systemic infections characterized by fever and hemorrhage caused by a special group of viruses transmitted to humans by arthropods and rodents. the vhfs are febrile illnesses characterized by abnormal vascular regulation and vascular damage and are caused by small, lipid-enveloped rna viruses. this syndrome can be caused by viruses belonging to four different families that differ in their genomic structure, replication strategy, and morphologic features (table 11 .4). arenaviruses, bunyaviruses, and filoviruses are negative-stranded, 459 in the nucleus and cytoplasm of erythroid precursors by using immunohistochemistry (ihe) (same patient as in a,b). a,b, h&e; c,d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. whereas fiaviviruses are positive-stranded rna viruses. hemorrhagic fever viruses are distributed worldwide, and the diseases they cause are traditionally named according to the location where they were first described. the oldest and best known is yellow fever virus; others include lassa fever, lymphocytic choriomeningitis, ebola, and dengue viruses. viral hemorrhagic fevers share many common pathologic features, although the overall changes vary among the different diseases. the similar pathologic and immunopathologic findings in cases of vhf suggest that microvascular involvement and instability is an important common pathogenic pathway leading to shock and bleeding in many instances. infection of the mononuclear phagocytic system and endothelium are thought to play c s.r. zaki and cd. paddock 11 .17. ebola virus hemorrhagic fever. a. pulmonary congestion and lack of inflammation. b. numerous filamentous ebola virus inclusions are seen within hepatocytes in association with hepatocellular necrosis. c. ebola virus-infected intra alveolar macrophages as seen by colorimetric in-situ hybridization using digoxigenin-labeled probes. d. viral anti-gens are seen in endothelial cells and other interstitial cells in this lung section. a,b, h&e; c, digoxigenin-labeled probes followed by immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain; d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. figure 11.18 . yellow fever. a. pulmonary congestion in fatal case of yellow fever associated with vaccination. b. yellow fever antigens in the pulmonary interstitium of the same patient. in natural infections, yellow fever antigens are usually not seen outside the liver in fatal cases. in contrast, in vaccine-associated cases viral antigens can be found in a variety of extrapulmonary a critical role in the pathogenesis of vhfs through the secretion of physiologically active substances, including cytokines and other inflammatory mediators (figs. 11.17c,d, 11.18b, 11.19b,c, and 11.20b). at autopsy common findings include widespread petechial hemorrhages and ecchymoses involving skin, mucous membranes, and internal organs. however, in many hf patients manifestations of bleeding may be minimal or absent. effusions, occasionally hemorrhagic, are also frequently seen. widespread, focal, and sometimes massive necrosis can be commonly observed in all organ systems and is often ischemic in nature. necrosis is usually most prominent in the liver and lymphoid tissues. the most con-461 organs, including heart, lung, and spleen. c. extensive midzonal hepatic necrosis characteristic of yellow fever. d. abundant antigens of yellow fever virus are observed in midzonal area of hepatic lobule in this immunohistochemical preparation. a,c, h&e; b,d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. sistent microscopic feature is found in the liver and consists of multifocal hepatocellular necrosis with cytoplasmic eosinophilia, councilman bodies, nuclear pyknosis, and cytolysis (figs. 11.17b and 11.18c). inflammatory cell infiltrates and necrotic areas are usually mild and, when present, consist of neutrophils and mononuclear cells. commonly observed histopathologic changes in the lung include various degrees of hemorrhage, intra alveolar edema, interstitial pneumonitis, and diffuse alveolar damage (figs. 11.17 a, 11.18a, 11.19a, 11.20a, and 11.21a). several references containing detailed pathologic descriptions in human cases are recommended. 29.72.333-35o .' a. pulmonary hemorrhage and diffuse alveolar damage in a patient who was infected through organ transplantation. note that, unlike this case, which occurred due to immunosuppression, lcmv infections are rarely fatal and usually resolve with no specific treatment. b. abundant lcmv antigens in areas of the diagnosis of vhf should be suspected in patients with appropriate clinical manifestations returning from an endemic area, particularly if there is travel to rural areas during seasonal or epidemic disease activity. the diagnosis suspected by history and clinical manifestations can also be supported histopathologically, and the overall pattern of histopathologic lesions may suggest a specific diagnosis. however, because of similar pathologic features seen in vhf and a 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west nile virus in the united states acknowledgments. the authors gratefully acknowledge the invaluable assistance of gillian genrich, of the cdc, for her comments and help with organization of the references, cynthia goldsmith for her comments, and mitesh patel, of the cdc, for his help with preparation and assembly of the images. key: cord-015139-s7ox0h4f authors: stockschläder, m.; montemurro, m.; kiefer, t.; dölken, g. title: atemwegsinfektionen bei immunsupprimierten personen date: 2003 journal: bundesgesundheitsblatt gesundheitsforschung gesundheitsschutz doi: 10.1007/s00103-002-0536-7 sha: doc_id: 15139 cord_uid: s7ox0h4f pulmonary infections in immunocompromised patients remain a diagnostic and therapeutic challenge.new methods for the early detection of fungal and viral diseases might allow an immediate and specific therapy, thereby lowering the high mortality associated with such infections.the development of new drugs will hopefully lead to more effective treatment while decreasing the incidence of adverse effects. interdisciplinary team work between internal medicine, radiology, surgery, microbiology, and virology is essential for the successful management of these patients. pul monale veränderungen finden sich häufig bei immunsupprimierten patienten und können durch die grunderkrankung, deren behandlung, infektionen, tumoren oder kombinationen dieser ursachen bedingt sein. infektiöse lungenveränderungen müssen abgegrenzt werden von nichtinfektiösen ursachen wie lungenödem, diffuser alveolärer hämorrhagie, fibrosierung, hypersensitivitätsreaktion, alveolarproteinose, bronchiolitis obliterans und ards ("adult respiratory distress syndrome"). die dauer, ursache und intensität der immunsuppression bestimmen das muster der infektionen. defekte hautund schleimhautbarrieren des patienten durch intensive chemotherapie oder bestrahlung prädisponieren zum eindringen von pathogenen keimen. die neutropenie stellt einen gesicherten risikofaktor für bakterielle und bei längerer dauer opportunistische infektionen dar. störungen der humoralen immunität prädisponieren zu infektionen mit streptokokken und bekapselten bakterien (haemophilus influenzae, neisseria meningitidis, pseudomonas aeruginosa), störungen der zellulären immunität zu infektionen mit viren, protozoen wie toxoplasma gondii, pilzen, bakterien oder helminthen. eine spender-gegen-wirt-reaktion ("graft versus host disease"=gvhd) oder immunsuppressive therapie erhöhen ebenfalls das risiko für pulmonale infektionen [1] . diese arbeit stellt eine übersicht zu einem thema dar, das wegen der hete-rogenität der formen der immunsuppression an dieser stelle nicht in seiner ganzen breite behandelt werden kann und somit eine fokussierende beschränkung erfordert. der inhaltliche schwerpunkt liegt deshalb auf transplantierten patienten, jedoch werden auch wichtige aspekte bei hiv-infizierten patienten dargestellt. für die behandlung einiger infektionen liegen nicht für jedes patientenkollektiv studien vor. eine übertragung der ergebnisse zwischen verschiedenen patientenkollektiven ist jedoch wegen der unterschiedlichen form und dauer der immunsuppression nicht unproblematisch. im folgenden werden prinzipielle aspekte der diagnostik und therapie infektiöser pulmonaler erkrankungen beschrieben. auf spezifische diagnostische und therapeutische modalitäten wird in den abschnitten zu bakteriellen, viralen und pilzinfektionen gesondert eingegangen. leitsymptome pulmonaler infektionen sind fieber, husten und dyspnoe, die jedoch auch bei ausgeprägten infektiösen lungenveränderungen fehlen können. fieber bei immunsupprimierten patienten bedarf einer sofortigen die diagnostik pulmonaler infektionen stellt eine ganz besondere herausforderung dar. nicht selten sind therapeutische maßnahmen erforderlich, bevor die diagnostik abgeschlossen ist bzw. deren ergebnisse vorliegen. serologische untersuchungen sind nur in einzelfällen hilfreich. blutkulturen sollten immer angefertigt werden. hochrisikopatienten erfordern eine regelmäßige überwachung mittels polymerasekettenreaktion (pcr) auf zytomegalievirus (cmv). auch bei der toxoplasmose kann eine pcr-analyse des blutes oder des bronchiallavageats diagnostische bedeutung erlangen [4] . die sputumdiagnostik ist bei nachweis von pneumocystis carinii und mycobacterium tuberculosis, bei allogen transplantierten patienten auch von aspergillus wegweisend. beim nachweis säurefester stäbchen sollte über kultur und dna-analyse eine exakte differenzierung der mykobakterien erfolgen. die röntgenuntersuchung des thorax kann wertvolle hinweise geben, stellt jedoch nicht jede infektion in der frühphase oder der neutropenie dar. auch bei fehlenden radiologischen hinweisen einer pulmonalen infektion sollten routinemäßig weiterführende untersuchungen angeschlossen werden. rundherde in der lungenperipherie finden sich häufig bei pilzinfektionen. eine gleichförmige, bilaterale, perihilär betonte und interstitielle zeichnungsvermehrung, die später mit kleinfleckigen verschattungen einhergeht, kann typisch für die pneumocystis-carinii-pneumonie (pcp) sein. bei cmv-infektionen findet man häufig eine kombinationen von milchglasartiger, interstitieller verschattung mit später disseminierten fleckförmigen knötchen, wobei die unter-und mittelfelder betont betroffen sind [5] . die computertomographie des thorax ist sehr aussagekräftig und sollte so früh wie möglich erfolgen. zu bevorzugen ist die hochauflösende technik ("high resolution-ct"=hrct). bei normalen hrct ist eine manifeste pulmonale infektion unwahrscheinlich [ nach allogener bszt werden das infektionsrisiko und der schweregrad der infektion mit den möglichen komplikationen ganz wesentlich mitbestimmt durch das stadium der grunderkrankung, die vorbehandlung, die transplantationsmodalität (z. b. grad der hla-identität zwischen spender und empfänger, t-zell-depletion), die rekonstitution der hämatopoese und das auftreten einer gvhd. die zeit nach der allogenen bszt wird in 3 phasen mit z. t. typischen infektionen eingeteilt. aufgrund der hochdosierten chemo-/ strahlentherapie kommt es in den ersten 30 tagen nach der transplantation zu einer ablation der hämatopoese mit schwerer peripherer zytopenie. der immunologische defekt ist vorwiegend in der neutropenie begründet. die generalisierte mukositis bedeutet multiple eintrittspforten für erreger. in dieser phase werden häufig diffuse beidseitige lungeninfiltrate aufgrund einer toxischen lungenschädigung durch chemotherapie und strahlentherapie in zeitlicher assoziation mit fieber beobachtet. differenzialdiagnostisch sind pulmonale blutungen bei erniedrigten thrombozy-tenzahlen, pulmonale stauung infolge kardiotoxischer chemotherapie, hepatische vod ("veno-occlusive disease"), "capillary leak syndrome" und ards zu erwägen (übersicht 1). häufigste infektiöse erreger von pneumonien in dieser phase sind bakterien (90%) und pilze. von tag 30 bis tag 100 steigt die rate der infektiösen komplikationen aufgrund der abgeschwächten zellulären immunität, die b-und t-zellen betrifft, stark an. die akute gvhd verstärkt und verlängert die bereits bestehende immunsuppression. diese phase (>100 tage) ist geprägt von einer kombinierten störung der zellulären und humoralen immunität, der beeinträchtigung des retikuloendothelialen systems und dem auftreten der so genannten chronischen gvhd. • interstitielle pneumonie (radiologisch) • nachweis von cmv in der bal mit hilfe von zellkulturverfahren zierte, risikoadaptierte antivirale prophylaxe" mit ganciclovir empfohlen [52] . die therapiestrategien "universelle prophylaxe" und "preemptive treatment" der cmv-erkrankung gelten auch für patienten mit iatrogener immunsuppression nach organtransplantation. die dauer der frühinterventionstherapie sollte bei patienten nach transplantation von soliden organen oder allogenen blutstammzellen von der dauer und art der immunsuppression und den ergebnissen der virologischen überwachungsuntersuchungen abhängen. die späte cmv-erkrankung nach blutstammzelltransplantation, insbesondere nach dosisreduzierter konditionierung und bei verstärkter immunsuppression wegen drohender abstoßung eines transplantierten soliden organs, wird zunehmend häufig beobachtet. sie erfordern eine prophylaxe mit intravenösem oder oralem ganciclovir oder valganciclovir. nach längerer ganciclovirexposition muss mit ganciclovir-resistenten viren gerechnet werden [53] . die therapie der hsv-pneumonie besteht in der gabe von aciclovir 3-mal 5-10 mg/kg/tag über 14-21 tage. in 2-10% der fälle können aciclovir-resistente viren isoliert werden [54] . therapie der wahl ist in diesen fällen foscarnet (3mal 60 mg/kg/tag) [55] . seltene schwere viszerale komplikationen der vzv-infektion werden mit aciclovir 10-15 mg/ kg/tag für etwa 14 tage behandelt. die therapie der rsv-pneumonie (mortalität 60-80%) besteht in der gabe von ribavirin in aerosolform (20 mg/ml für 18 stunden/tag) oder ribavirin i.v. 25 mg/kg/tag als initialdosis, dann 3-mal 5 mg/kg/tag in kombination mit immunglobulinen i.v.0,2-0,4 g/kg/ tag therapeutisch wird bei der cmv-pneumonie ganciclovir (2-mal 5 mg/kg/ tag) meist in kombination mit hohen dosen intravenöser immunglobuline eingesetzt [56] . grundsätzlich verlangt die therapie der cmv-infektion/erkrankung mit ganciclovir, foscarnet oder cidofovir eine so genannte "induktionsphase", in der hohe dosen des virostatikums die virusreplikation rasch vermindern,und eine erhaltungstherapie,in der niedrigere dosen die cmv-reaktivierung und das fortschreiten der cmv-erkrankung verhindern,z.b.initial ganciclovir 5 mg/kg alle 12 stunden für 14 tage und anschließend 5 mg/kg/ tag. vor der proteaseinhibitor-basierten "highly active antiretroviral therapy" (haart) konnten cmv-erkrankungen, z. b. die cmv-retinitis, bei 85% der aids-oder hiv-infizierten patienten mit geringer cd4-zahl beobachtet werden [57] . unter haart kann die erhaltungstherapie nach etwa 6 monaten bei anstieg der cd4 + -t-zellen abgebrochen werden.aufgrund der niedrigen bioverfügbarkeit bei oraler gabe muss ganciclovir bei cmv-retinitis in einer dosierung von 1,5 g 3-mal/tag gegeben werden [58] . eine alternative ist das seit kurzer zeit zur verfügung stehende, oral zu verabreichende, gut resorbierbare valganciclovir. die antivirale resistenz ist vor allem ein problem der langzeitbehandlung. toxoplasma gondii ist ein häufiger erreger opportunistischer infektionen. immunsupprimierte patienten sollten eine exposition vermeiden. prophylaktisch wirksam sind trimethoprim-sulfamethoxazol (tmp/smz) oder eine kombinationen aus pyrimethamin/leucovorin mit sulfadiazin oder dapson. beide regime wirken auch gegen pneumocystis-carinii-infektionen [59, 60] . pneumocystis carinii ist ein extrazellulärer, im alveolarraum persistierender, einzelliger pilz, der bei immunsupprimierten patienten schwere pneumonien verursachen kann. patientengruppen mit ausgeprägter immunsuppression sollten eine prophylaxe erhalten. als standardprophylaxe gilt tmp/smz in der bevorzugten dosierung von 960 mg/ tag. alternativ kann tmp/smz in einer dosierung von 480 mg/tag oder 960 mg 3-mal/woche verabreicht werden. weitere möglichkeiten bestehen in der prophylaktischen regelmäßigen pentami-din-inhalation oder oralen applikation von dapson. häufig muss die tmp/ smz-prophylaxe aufgrund von nebenwirkungen beendet werden [60, 61] . pulmonale symptome bei normalem röntgenbild lassen an eine pneumocystiscarinii-pneumonie (pcp) denken. in diesem fall sind die untersuchung des induzierten sputums, evtl. eine bal und möglicherweise eine tbb angezeigt. die pneumocystis-carinii-pneumonie wird mit hoch dosiertem intravenösem tmp/ smz, intravenösem pentamidin oder mit clindamycin/primaquin behandelt. eine kürzlich veröffentlichte metaanalyse legt für die "salvage"-therapie eine behandlung mit clindamycin/primaquin nahe [62] . die inzidenz von pilzerkrankungen bei immunsupprimierten patienten hat in den letzten jahren zugenommen [63, 64] . in einer retrospektiven analyse wurden bei ca. 20-50% der verstorbenen patienten mit hämatologischen neoplasien und bei 19% der patienten mit aids autoptisch hinweise für eine pilzinfektion gefunden [65] .nach knochenmark-/ blutstammzelltransplantation kommt es bei ca. 11-18% der patienten zu einer candidainfektion [66] und bei bis zu 11% der patienten zu einer invasiven aspergillusinfektion [67] . bei neutropenischen patienten hängt das risiko, eine pilzinfektion zu entwickeln, entscheidend von der schwere und dauer der neutropenie ab [68] . candida albicans und aspergillus fumigatus sind für ca. 70-80% der systemischen pilzinfektionen bei onkologischen patienten verantwortlich, während mucor-und cryptococcusinfektionen bei diesem patientenkollektiv nur selten nachgewiesen werden. pilzinfektionen nach organtransplantationen treten mit einer inzidenz von 5% (nierentransplantation), 25% (lungentransplantationen) und 40% (lebertransplantation) auf, wobei 80% durch candida-und aspergillusspezies hervorgerufen und in den ersten 2 monaten nach transplantation manifest werden [69] [72] . candidainfektionen (soor und soorösophagitis) sind die häufigsten pilzinfektionen bei hiv-infizierten patienten. candidapneumonien sind eine rarität.vor dem hintergrund einer wirksamen und konsequenten antiretroviralen therapie werden deutlich weniger pilzinfektionen beobachtet. die klinische symptomatik der invasiven aspergillusinfektion der lunge ist in der frühphase häufig uncharakteristisch (husten 92%, brustschmerzen 76%, hämoptysen 54%). der direkte nachweis von aspergillus in blutkulturen gelingt nur selten, ein histologischer nachweis durch biopsien kann wegen der thrombozytopenie nur selten erbracht werden. häufig finden sich bei der invasiven aspergillusinfektion der lunge röntgenologisch rundlich-fleck-förmige bronchopneumonische infiltrate, die multifokal besonders in der peripherie lokalisiert sind. aber auch infarktpneumonische bilder bis hin zu einem unauffälligen röntgenbefund werden beobachtet. frühzeitig sollte bei patienten mit verdacht auf eine ipa ein hrct des thorax (übersicht 4) veranlasst werden [73] . invasive aspergillusinfektionen manifestieren sich häufig als unscharfe verschattungen ("milchglasartig"), nekrotisierende bronchopneumonie mit sekundärer ausbildung von lungeninfarkten, abszessen und höhlenbildungen. pilzsequestrationen, einschmelzungen und das so genannte "halo"-zeichen werden als lisa ("lesions with imaging suggestive of aspergillosis") bezeichnet und haben einen 82-90% positiv-prädiktiven wert für eine invasive aspergillusinfektion der lunge [74] . invasive methoden, vor allem die fob mit bal, haben ebenfalls einen festen stellwert. die sensitivität der bal liegt bei 14-60% [74] . bei blutstammzelltransplantierten patienten ist jedoch die signifikante komplikationsrate der fob (0-40%), bei z. t. nur geringer diagnostischer aussagekraft (31-89%), zu berücksichtigen [75] . sensitive nachweismethoden auf der basis aspergillus-und candida-spezifischer polymerasekettenreaktion (pcr) werden derzeit entwickelt und hoffentlich zu einer sensitiven und spezifischen frühdiagnostik invasiver mykosen führen [78, 79] . eine pilzprophylaxe scheint sinnvoll für patienten, deren erwartete neutropeniedauer (0,1-0,5×10 9 /l) mehr als zwei wochen beträgt oder die eine schwere neutropenie (<0,1×10 9 /l) von mehr als einer woche entwickeln. patienten, die allogen blutstammzelltransplantiert werden, sollten als hochrisikopatienten ebenfalls eine pilzprophylaxe erhalten. optimale hygiene inklusive händewaschen sind von entscheidender bedeutung [63] . fluconazol ist erfolgreich zur prophylaxe von pilzinfektionen während der behandlung von akuten leukämien und im rahmen der knochenmarktransplantation eingesetzt worden [66] . die zahl disseminierter candida-albicansund -tropicalis-infektionen nimmt signifikant ab, jedoch haben patienten unter fluconazolprophylaxe ein 7-fach erhöhtes risiko, eine infektion mit fluconazolresistenten candida-krusei-hefen zu entwickeln. zusammenfassend kann festge-stellt werden,dass für allogen stammzelltransplantierte patienten und hochrisikopatienten, die sich einer autologen bszt unterziehen, eine prophylaxe mit fluconazol (400 mg/tag) empfohlen wird [63] .aspergillusinfektionen werden hierdurch allerdings nicht mit erfasst. da die meisten infektionen durch inhalation von schimmelpilzkonidien entstehen, ist die unterbringung von hochrisikopatienten in "laminar air flow"-(laf-)räumen mit einer "high-efficiency particulate air"-(hepa-)filtration sinnvoll und effektiv [80] . niedrig dosiertes i.v.-amphotericin b (0,1-0,25 mg/ kg kg/tag) [81] und inhalatives amphotericin b können als prophylaxe nicht empfohlen werden [82] . prophylaktisch eingesetztes liposomal verkapseltes amphotericin b (1 mg/ kg kg/tag) führte bei transplantationspatienten zu einer reduktion der fungalen kolonisation und einer nicht signifikanten reduktion bewiesener pilzinfektionen, aber nicht zu einem überlebensvorteil [83] . intravenöses niedrig dosiertes liposomales amphotericin b (2 mg/ kg kg/tag, 3-mal/woche) reduzierte weder die zahl der pilzinfektionen noch die mortalität. itraconazol als orale lösung hat sich als effektiv in der pilzprophylaxe bei neutropenischen patienten in 3 studien bewährt [84, 85] . wichtig scheinen adäquate itraconazolspiegel zu sein, die mindestens (250-) 500 ng/ml betragen sollten. in der prophylaxe können diese spiegel nach applikation der oralen lösung bei 86-100% der patienten erst nach ca. 12 tagen erreicht werden [84] . nach intravenöser gabe werden effektive spiegel in der mehrzahl der fälle (97%) bereits nach 2 tagen erreicht [86] . bezüglich der dauer der prophylaxe sollte bei folgenden patientengruppen eine langfristige prophylaxe erwogen werden: patienten unter langfristiger hoch dosierter steroidtherapie, patienten nach "hla-mismatch"-allogener bszt, patienten mit einer gvhd, patienten, die eine hochpotente kombinationschemotherapie erhalten haben (insbesondere fludarabin-haltige kombinationstherapien), patienten, die eine langfristige chemotherapie erhalten, und patienten, die eine myeloablative chemotherapie nach vorausgegangener invasiver pilzinfektion erhalten sollen. da bei onkologischen patienten pilze autoptisch häufig nachweisbar waren und systemische pilzinfektionen häufig als unmittelbare todesursache identifiziert werden konnten, wird seit vielen jahren das konzept einer empirischen antimykotischen therapie bei klinisch vermuteten systemischen candida-oder aspergillusinfektionen verfolgt [87] . dies gilt insbesondere für hochrisikopatienten, die während der granulozytopeniephasen fieber unbekannter ursache entwickeln und nicht auf eine mehrtägige (72-96 stunden) breitspektrumantibiotikatherapie ansprechen [87] .studien belegen,dass die empirische gabe von amphotericin b (0,6-1,0mg/kg kg/tag) zu einer erhöhten überlebenswahrscheinlichkeit führt. liposomales amphotericin b (1-3 mg/kg kg/tag) ist möglicherweise eine bessere alternative,da es im vergleich zu amphotericin b zu signifikant weniger bewiesenen refraktären ("breakthrough") pilzinfektionen kommt und bei allogen transplantierten patienten deutlich weniger neue ("emergent") pilzinfektionen auftreten [88, 89] . außerdem ist liposomales amphotericin deutlich besser verträglich, jedoch um ein vielfaches teurer. für granulozytopenische patienten mit persistierendem fieber unklarer ursache,normalem röntgenthoraxbild,lee-reraspergillusanamnese,negativenüberwachungskulturen für aspergillus und geringem aspergillusinfektionsrisiko konnten studien eine gleiche effektivität von intravenösem amphotericin b (0,5-0,8 mg/kg kg/tag) und intravenösem fluconazol (400 mg/tag) belegen [90] . fluconazol führte zu deutlich weniger nebenwirkungen. bei patienten mit lungeninfiltraten in der febrilen neutropenie ist jedoch wegen fehlender aspergilluswirksamkeit von fluconazol bereits initial konventionelles oder liposomales amphotericin b zu empfehlen. in der empirischen antifungalen therapie bei neutropenischen patienten scheint itraconazol mindestens so effektiv wie amphotericin, führt jedoch zu deutlich weniger nebenwirkungen [86] . zur behandlung der candidapneumonie wird amphotericin b (>0,7 mg/kg kg/tag) allein oder in kombination mit 5-flucytosin [100(-150) mg/kg kg/tag] [91] empfohlen. eine effektive alternative ist bei ausgeschlossener resistenz gegenüber fluconazol (cave: c. krusei und c. glabrata) und zuvor nicht stattgehabter fluconazolprophylaxe die behandlung mit dem triazol fluconazol [92] . amphotericin (<1,5 mg/kg kg/tag) sollte jedoch das mittel der ersten wahl bei klinisch instabilen patienten und patienten mit einer organmykose bleiben [64] . bei der seltenen c.-lusitaniae-infektion muss mit einer geringeren empfindlichkeit, evtl. resistenz gegenüber amphotericin b, gerechnet werden. für die ösophageale candidiasis bei hiv-positiven patienten belegen studienergebnisse die effektivität von itraconazol, das ebenso erfolgreich eingesetzt wurde wie fluconazol [93] . die behandlung der invasiven aspergillose bei immunsupprimierten patienten stellt weiterhin eine herausforderung dar. für die invasive aspergillusinfektion der lunge wird amphotericin b weiterhin in der behandlung als "goldstandard" angesehen, ist jedoch zum teil mit erheblicher toxizität verbunden.amphotericin b wird in dosen von 1 (-1,5) mg/kg kg/tag für die erstbehandlung empfohlen [22] . es kann mit einem ansprechen von ca. 30-50% gerechnet werden [94] . für patienten, die bereits nephrotoxische medikamente wie cyclosporin a oder aminoglykosidantibiotika erhalten, stellt das liposomale amphotericin b (ambisome 1-5 mg/kg kg/tag) eine sinnvolle alternative für die erstbehandlung dar [88] , wobei es in einer dosierung von 1 mg/kg kg/tag bis 4 mg/kg kg/tag wohl gleich wirksam ist. patienten, die unter behandlung mit amphotericin b eine niereninsuffizienz entwickeln, können mit liposomalem amphotericin b erfolgreich weiterbehandelt werden [95] . patienten mit invasiver aspergillose können möglicherweise neben amphotericin b bei gleicher effektivität alternativ mit dem aspergillus-wirksamen triazol itraconazol behandelt werden [96, 97, 98] . eventuell ist die kombination von amphotericin b mit itraconazol bei offensichtlich fehlendem antagonismus wirksamer. itraconazol bietet sich auch für die sekundärprophylaxe nach erfolgreicher amphotericin-b-behandlung an [96] . itraconazol steht als orale (kapsel und saft) und intravenöse darreichungs-form zur verfügung.bei der ipa kann mit einem ansprechen von ca. 32-60% der patienten gerechnet werden, wobei patienten nach allogener transplantation generell ein schlechteres ansprechen zeigen als neutropenische patienten, patienten nach organtransplantationen oder aids-patienten [96, 99] . patienten mit lisa (fokale läsionen, s. oben) sollten zunächst medikamentös behandelt werden. zeigt sich bei wöchentlich durchgeführten ct-kontrollen eine besserung, so sollte die therapie weiter konservativ erfolgen. eine vorübergehende verschlechterung ist trotz effektivität der therapie möglich. sollte diese jedoch fortschreiten, insbesondere wenn die läsion die integrität der pulmonalarterien zu gefährden droht, sollte eine operation -sofern vertretbar -durchgeführt werden. über eine chirurgische entfernung sollte auch vor durchführung einer allogenen transplantation nachgedacht werden [100] .bei hämoptysen ist je nach schweregrad die unmittelbare operation indiziert [74] .aspergillome in präexistenten und nichtmykotischen höhlen müssen jedoch von aspergillomen unterschieden werden, die durch eine invasive lungenaspergillose nach einschmelzung und demarkation entstehen. die behandlung der cryptococcusinfektion besteht aus einer systemischen gabe von amphotericin b in kombination mit flucytosin (100-150 mg/kg kg/tag). die kombinationstherapie hat sich der alleinigen gabe von amphotericin b als überlegen erwiesen. caspofungin gehört zu 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amphotericin b for treatment of invasive aspergillosis niaid mycoses study group multicenter trial of oral itraconazole therapy for invasive aspergillosis an eortc multicentre prospective survey of invasive aspergillosis in haematological patients: diagnosis and therapeutic outcome. eortc invasive fungal infections cooperative group analysis of compassionate use itraconazole therapy for invasive aspergillosis by the niaid mycoses study group criteria intravenous itraconazole followed by oral itraconazole in the treatment of invasive pulmonary aspergillosis in patients with hematologic malignancies, chronic granulomatous disease, or aids lung resection for invasive pulmonary aspergillosis in neutropenic patients with hematologic diseases a randomized double-blind study of caspofungin versus amphotericin for the treatment of candidal esophagitis randomized, double-blind, multicenter study of caspofungin versus amphotericin b for treatment of oropharyngeal and esophageal candidiases in vitro antifungal activities of voriconazole and reference agents as determined by nccls methods: review of the literature treatment of fluconazole-resistant candidiasis with voriconazole in patients with a randomized, double-blind, double-dummy, multicenter trial of voriconazole and fluconazole in the treatment of esophageal candidiasis in immunocompromised patients voriconazole versus amphotericin b for primary therapy of invasive aspergillosis voriconazole compared with liposomal amphotericin b for empirical antifungal therapy in patients with neutropenia and persistent fever key: cord-018331-ovmtz4sb authors: dancygier, henryk title: viral infections by nonhepatotropic viruses date: 2010 journal: clinical hepatology doi: 10.1007/978-3-642-04519-6_10 sha: doc_id: 18331 cord_uid: ovmtz4sb nan containing intranuclear viral inclusions (cowdry a: inclusion surrounded by a clear halo; cowdry b: homogeneous, ground glass like inclusion) are seen [12] . typically there is only a minimal infl ammatory response. in neonatal hsv hepatitis multinucleated giant cells (giant cell hepatitis) occur. the viruses may be demonstrated by electron microscopy, immunocytochemistry, and by dna-in situ hybridisation. however, the light microscopic appearance is so characteristic that these sophisticated techniques are not required to diagnose hsv hepatitis. hepatitis is a rare complication of hsv infection, but when it occurs it usually presents as a fulminant disease with a high mortality rate of up to approximately 80% [15, 21] . hsv hepatitis in pregnant women (usually caused by hsv type 2) occurs in the late second and in the third trimester. the disease is heralded by nonspecifi c infl uenza-like symptoms, right upper quadrant pain, and eventually signs of hepatic encephalopathy. indeed, the fi rst case of hsv infection associated fulminant liver necrosis in adults was described in a pregnant woman. extended parenchymal necrosis in hsv hepatitis leads to marked elevation of aminotransferase levels up to several thousand u/l (ast > alt) and to a coagulopathy demonstrated by a prolongation of prothrombin time. in contrast to aminotransferases, serum bilirubin concentration usually is only slightly (£ 5 mg/dl) elevated. pregnancy specifi c liver diseases, such as acute fatty liver, hellp-syndrome and cholestasis of pregnancy should be considered in the differential diagnosis (see section xxi). the fatality rate of hsv hepatitis in pregnancy is high, approximately 40-50% for mother and child. therefore, in pregnant women, hsv infection must be excluded in every case of acute hepatitis. liver biopsy is the defi nitive diagnostic test (often a transvenous approach is necessary). additionally, vaginal, cervical and pharyngeal smears should be obtained [18] . opportunistic hsv hepatitis in patients after solid organ transplantation is not as frequent as cmv and ebv infection, but usually occurs earlier after the transplant than cmv and ebv hepatitis [21] . hsv infection in these patients is mostly due to reactivation of latent virus rather than to a de novo infection. hsv hepatitis requires immediate treatment. acyclovir (30 mg/kg body weight i.v. daily) is life saving in many patients. infections with the human herpesviruses 6 and 7 are ubiquitous in childhood. rarely, especially in children, both viruses may cause a fulminant hepatitis during a primary infection. viral reactivation in immunosuppressed patients after organ transplantation may also be responsible for hepatitis [4] . the laboratory fi ndings are nonspecifi c, and are characterized by elevation of aminotransferases, cholestatic enzymes, leuko-and thrombocytpenia. the viruses may be isolated from peripheral blood lymphocytes, and may be identifi ed by negative contrast and thin-section electron microscopy, dna-hybridization, and immunofl uorescence [38] . human herpesvirus 8 causes kaposis's sarcoma (ks), and is linked with two other neoplasms, a b cell non-hodgkin's lymphoma (body cavity based lymphoma) and multicentric castleman's disease (mcd). the liver is frequently involved in visceral ks, predominantly in hiv infected persons with advanced immunodeficiency, and more rarely after organ transplantation. peliosis hepatis, perisinusoidal fi brosis and nodular regenerative hyperplasia have been described in few cases of mcd. a disseminated varicella-zoster virus (vzv) infection is rare. it may occur in children within the context of chickenpox, while in adults immunosuppression with reactivation of vzv is the usual cause. hepatic involvement with varicella (varicella hepatitis) is uncommon and predominantly affects immunosuppressed hosts, such as transplant recipients, cancer and aids patients, but also normal hosts. histologically, focal liver cell necrosis as well as massive widespread hepatic necrosis with intranuclear hepatocellular inclusions and multinucleated giant cells at the periphery of necrotic parenchyma is seen. the lesions resemble those of hsv hepatitis. clinically varicella hepatitis may manifest as a symptomatic or subclinical aminotransferase elevation coincident with the onset of varicella, or, especially in the immunocompromised host, as fulminant hepatic failure leading to death [20, 31] . the varicella skin rash may precede, appear coincident with, or follow the onset of hepatitis, but varicella hepatitis with fulminant failure with widespread visceral dissemination in the absence of a rash has also been documented in bone marrow transplant recipients [32] . in fulminant hepatic failure aminotransferase levels reach several thousand u/l, with levels of ast being generally higher than alt. reye's syndrome has been reported to be preceded by a vzv infection in approximately 10% of patients. a diffuse microvesicular steatosis, vomiting and signs of a hepatic encephalopathy are characteristic of reye's syndrome [23] . therapy of varicella hepatitis is early high dose acyclovir (30 mg/kg body weight i.v. daily) or liver transplantation in fulminant cases with organ failure [27, 37] . congenital cytomegalovirus (cmv) infection may be due to intrauterine or peripartal contagion. histologically steatosis, focal liver cell necrosis, mononuclear infl ammatory infi ltrates, and occasionally multinucleated giant cells (neonatal hepatitis) are seen. the typical intranuclear inclusions surrounded by a clear halo impart the cells an "owl's eye" appearance. they are found in hepatocytes, bile duct epithelia and in endothelial cells. the affected cells are enlarged. intrauterine cmv infection may result in biliary atresia. cmv hepatitis in the immunocompetent host clinically resembles hepatitis of infectious mononucleosis [14] . the liver and spleen are enlarged and the clinical manifestations are mild, with mild increases of aminotransferase and bilirubin levels. often hepatitis is anicteric. occasionally alkaline phosphatase and ggt may be markedly elevated (up to >1,000 u/l), which, however, does not portend a serious prognosis. the course of cmv hepatitis is self-limited, and chronic hepatitis does not ensue. isolated cases of budd-chiari syndrome and portal vein thrombosis associated with cmv hepatitis have been reported [34, 35] . compared to viral hepatitis a, b and c, cmv hepatitis is characterized by prolonged fever, splenomegaly, atypical lymphocytosis, milder elevations of aminotransferases and milder histopathological alterations. histologically, focal liver and bile duct injury, lymphocytic sinusoidal infi ltrates, and occasionally noncaseating histiocytic granulomas are seen (fig. 64.1) . thus, cmv hepatitis should be included in the differential diagnosis of granulomatous hepatitis [3] . viral inclusions or immunocytochemically detectable viral antigens usually cannot be demonstrated. in cmv hepatitis in the immunocompromised host viral inclusions may be found in the absence of an infl ammatory reaction. if such inclusions are accompanied by hepatocellular injury and by lymphocytic infi ltration, hepatitis may be attributed to cmv infection. cmv hepatitis after liver transplantation usually manifests 1-4 months after the operation, either as a de novo infection of the donor liver through blood transfusion or as reactivation of a latent cmv infection in the recipient due to postoperative immunosuppressive therapy. clinically the disease may be asymptomatic or resemble infectious mononucleosis, with mild elevation of aminotransferases, leuko-and thrombocytopenia. it must be differentiated from a rejection reaction. the histological appearance of cmv hepatitis in a transplanted liver is characteristic. focal accumulations of neutrophils form so-called microabscesses or a necrotic hepatocyte is surrounded by a mixed infl ammatory cell infi ltrate ("microgranuloma"). furthermore, the nuclear inclusions described above are present. viral antigens may be demonstrated by immunocytochemistry. the portal infl ammatory infi ltrate varies in density. in contrast to cellular rejection, in cmv hepatitis neither an endothelitis nor a cholangtis are seen. patients with cmv hepatitis after liver transplantation have an increased risk of developing a vanishing bile duct syndrome. therapy of cmv hepatitis with ganciclovir (5 mg/ kg i.v. bid) is usually successful. epstein-barr virus (ebv) causes infectious mononucleosis. it infects and transforms b lymphocytes, and is associated with the development of hairy leukoplakia, certain lymphomas and nasopharyngeal carcinoma. approximately 5% of patients with infectious mononucleosis develop jaundice and 15% have elevated serum aminotransferases. ebv hepatitis generally is a mild hepatitis accompanying a generalized ebv infection [16] . its clinical manifestations are overshadowed by systemic signs and symptoms of infectious mononucleosis. jaundice in a patient with ebv infection mostly is due to autoimmune hemolytic anemia and not to hepatitis. in the vast majority of cases ebv hepatitis is self-limited. fulminant courses with liver failure are extremely rare [17, 33] . they occur predominantly in x chromosomal inherited lymphoproliferative syndrome (duncan's syndrome) and in lymphoproliferative diseases after organ transplantation. on histologic examination the portal tracts are heavily infi ltrated by atypical lymphocytes and plasma cells. the infl ammatory infi ltrate spills over through the limiting plate to the lobular parenchyma. ebv antigens may be demonstrated in lymphocytes by immunocytochemistry and by in situ hybridization. the sinusoids are infi ltrated either diffusely or in the form of small aggregates by mononuclear cells. intrasinusoidal lymphocytosis often has a characteristic "indian-fi le" appearance ( fig. 64.2) . liver cells are usually only mildly affected with scattered apoptotic bodies or foci of parenchymal necrosis fi lled with lymphocytes. hepatocellular injury is clearly less pronounced than in acute viral hepatitis a or b. regenerative changes and mitoses may be prominent. a steatosis or non-caseating, fi brin-ring granulomas rarely occur. cholestasis is not part of the typical microscopical picture of ebv hepatitis, and if present should prompt one to search for granulomas [6] . the main histological differential diagnosis of ebv hepatitis is from leukemia or lymphoma. adenovirus infection may cause severe hepatitis with liver failure in children and in immunosuppressed adults [1, 19] . pathology and clinical manifestations resemble that of hsv hepatitis. coxsackie b and echoviruses [36] . coxsackie virus, and more rarely echoviruses may cause a severe, hemorrhagic-necrotizing hepatitis in newborns. the clinical manifestations in adults are milder, generally refl ecting an acute cholestatic hepatitis. histologically centrilobular cholestasis and ballooned hepatocytes are seen. the portal and sinusoidal infl ammatory infi ltrates are composed of mononuclear cells and neutrophil leukocytes. liver involvement in measles virus infection is mild and self-limited [25] . the histological alterations are nonspecifi c, showing "hepatocellular unrest," nuclear vacuolization and a mild intrasinusoidal lymphocytosis. isolated cases with clinically severe hepatitis and multinucleated giant cells have been documented, however, their paramyxovirus etiology has not been proven unequivocally [29] . rubella virus may cause hepatitis in the newborn within the context of the congenital rubella syndrome. focal hepatocellular necrosis, signs of cholestasis and a mild chronic infl ammatory portal infi ltrate are seen. in isolated cases extensive parenchymal necrosis has been described. intrauterine infection with the rubella virus may cause biliary atresia. rubella infection in adults may be accompanied by a mild anicteric, sublinical or asymptomatic hepatitis with slightly elevated aminotransferases [28] . arthropode transmitted flavi-and bunyaviruses cause diseases that are characterized by disseminated intravascular coagulation with extended hemorrhages, and are therefore denominated hemorrhagic fevers. liver injury in all arbovirus infections shows common features and is characterized by variably large areas of parenchymal necrosis and microvesicular steatosis, with a relatively mild infl ammatory reaction. if the patient survives, scavenger and regenerative processes dominate the histological picture. the yellow fever virus belongs to the genus of fl aviviruses. the disease is endemic in africa and in south america. it manifests acutely with fever, myalgias and headaches that are followed by jaundice after a few days. death is due to liver and renal failure. the histopathological appearance depends on the stage of the disease. confl uent, centrilobular parenchymal necrosis, scattered apoptotic bodies (classic councilman bodies), and eosinophilic intranuclear inclusions that are arranged concentrically around the nucleolus (torres bodies) characterize the acute stage. in contrast to the marked parenchymal injury the infl ammatory response is scant. surviving hepatocytes show microvesicular steatosis and ballooning. regeneration is evidenced by hepatocellular hyperplasia and multinucleated hepatocytes [10] . international travel to endemic areas is a major risk factor for both primary and secondary dengue infection. the primary infection manifests as an exanthematic, infl uenza-like illness. hemorrhagic fever is caused by reinfection with different serotypes of dengue virus (den 1-4) . dengue remains a diagnostic challenge, given its protean nature, ranging from a mild febrile illness to profound shock. dengue shock syndrome has an estimated mortality rate close to 50%. liver involvement appears to occur more frequently when infections involve den-3 and den-4 serotypes. the liver is interspersed with extended, partly confl uent areas of hemorrhagic parenchymal necrosis. the infl ammatory reaction is mild. the surviving hepatocytes show a microvesicular steatosis. fulminant liver failure is extremely rare in adults, but has been reported in single cases [11] . if the patient survives, diffuse parenchymal calcifi cations may be the only sign of previous liver involvement [7] . certain types of hantavirus cause hemorrhagic fever with a renal syndrome (hfrs), while others are responsible for the hantavirus pulmonary syndrome (hps). primary involvement of the liver does not occur in either syndrome. in chinese patients with acute hepatitis of unknown etiology hantavirus infection has been discussed as a possible cause [26] . arenaviruses cause lassa fever and hemorrhagic fevers in argentina and bolivia. lassavirus infection is endemic in central and west africa. fever, pharyngitis, diarrhea and a hemorrhagic diathesis characterize the clinical picture. pain in the right upper quadrant may supervene. there is a marked rise of serum aminotransferases, while jaundice is rare. the mortality rate is approximately 30%. the liver has a mottled appearance caused by apoptotic hepatocytes (councilman-like bodies) and hemorrhagic necrosis of groups of liver cells which may coalesce forming bridging necrosis. liver injury is accompanied by a marked hyperplasia of kupffer cells and by lipofuscin deposits in hepatocytes. cholestasis and steatosis are lacking. the viral particles can easily be demonstrated by electron microscopy [5] . these viruses cause hemorrhagic fevers in south america. the clinical picture and pathological liver fi ndings correspond to those of lassa fever. marburg virus disease is a highly contagious, febrile infection. it is associated with disseminated intravascular coagulation, hemorrhages, and shock. the mortality rate is 20-25%. the pathology of the liver corresponds to that of lassa fever. ebola fever resembles clinically and pathologically marburg virus infection. mortality rate is close to 50%. parvovirus b19 is the only human pathogenic parvovirus, causing erythema infectiosum (fi fth disease) in children. epidemiologic data suggest that parvovirus b 19 may also cause an acute hepatitis in children [39] . adults with parvovirus b 19 infection, especially patients with underlying hemoglobinopathies (e.g. sickle cell disease) or other erythrocytic disorders (e.g. hereditary spherocytosis) may develop transient aplastic crisis. combined with aplastic anemia massive hepatic necrosis and acute liver failure may occur [22] . parvovirus b 19 may be demonstrated by pcr in liver tissue. human coronaviruses have long been known to cause the common cold. in 2002 the severe acute respiratory syndrome (sars) was described for the fi rst time. it is caused by sars coronavirus (scov) that is genetically dissimilar from known human or animal coronaviruses. the disease presents with fever, infl uenza-like symptoms, dry cough, atypical pneumonia, and diarrhea. the liver is involved in up to 60% of cases and infection of the liver by scov was verifi ed for the fi rst time in 2004 by demonstrating scov-rna in liver tissue. in approximately 25% of patients aminotransferases are elevated (alt: 200-900 iu/l). histological examination shows signs of hepatocyte injury, such as ballooning and apoptosis accompanied by mild to moderate lobular lymphocytic infi ltrates. numerous mitoses probably denote regenerative activity [2, 13] . adenovirus hepatitis in an immunsuppressed adult patient sars-associated viral hepatitis caused by a novel coronavirus: report of three cases cytomegalovirus granulomatous hepatitis human herpesvirus-6 and -7 in transplantation the pathology of lassa fever case report: severe cholestatic jaundice induced by epstein-barr virus infection in the elderly dengue virus induced hepatitis with chronic calcifi c changes herpesvirus infection of the liver acute hepatitis due to herpes simplex virus in an adult a clinicopathological study of human yellow fever fulminant liver failure secondary to haemorrhagic dengue in an international traveller herpes simplex hepatitis in apparently immunocompetent adults severe acute respiratory syndrome and the liver clinical and histological features of cytomegalovirus hepatitis in previously healthy adults herpes simplex virus hepatitis: case report and review structural and functional abnormalities of liver in infectious mononucleosis severe hepatitis caused by epstein-barr virus without infection of hepatocytes herpes simplex virus hepatitis in pregnancy. two patients successfully treated with acyclovir disseminated adenovirus infection with hepatic necrosis in patients with human immunodefi ciency virus infection and other immunodefi ciency states varicella-zoster virus hepatitis and a suggested management plan for prevention of vzv infection in adult liver transplant recipients herpes simplex virus hepatitis after solid organ transplantation in adults parvovirus b19 as a possible causative agent of fulminant liver failure and associated aplastic anemia grade i reye's syndrome. a frequent cause of vomiting and liver dysfunction after varicella and upper-respiratory-tract infection hepatitis from non-hepatotropic viruses acute hepatitis in an adult with rubeola high prevalence of hantavirus infection in a group of chinese patients with acute hepatitis of unknown aetiology successful acyclovir therapy of severe varicella hepatitis in an adult renal transplant recipient intrahepatic lymphocyte subpopulations in acute hepatitis in an adult with rubella syncytial giant-cell hepatitis. sporadic hepatitis with distinctive pathological features, a severe clinical course, and paramyxoviral features five cases of fulminant hepatitis due to herpes simplex virus in adults subclinical hepatic changes in varicella infection visceral varicella zoster infection after bone marrow transplantation without skin involvement and the use of pcr for diagnosis liver failure and epstein-barr virus infection acute partial budd-chiari syndrome and portal vein thrombosis in cytomegalovirus primary infection: a case report a case report and literature review of portal vein thrombosis associated with cytomegalovirus infection in immunocompetent patients hepatitis associated with myocarditis: unusual manifestations of infection with coxsackie group b, type 3 fulminant hepatic failure following varicella-zoster infection in a child. a case report of successful treatment with liver transplantation and perioperative acyclovir brief report: primary human herpesvirus 6 infection in a patient following liver transplantation from a seropositive donor human parvovirus b19 infection associated with acute hepatitis key: cord-003085-7krf1yxz authors: li, xi; huang, yongbo; xu, zhiheng; zhang, rong; liu, xiaoqing; li, yimin; mao, pu title: cytomegalovirus infection and outcome in immunocompetent patients in the intensive care unit: a systematic review and meta-analysis date: 2018-06-28 journal: bmc infect dis doi: 10.1186/s12879-018-3195-5 sha: doc_id: 3085 cord_uid: 7krf1yxz background: cytomegalovirus (cmv) infection is common in immunocompetent patients in intensive care units (icus). however, whether cmv infection or cmv reactivation contributes to mortality of immunocompetent patients remains unclear. methods: a literature search was conducted for relevant studies published before may 30, 2016. studies reporting on cmv infection in immunocompetent patients in icus and containing 2 × 2 tables on cmv results and all-cause mortality were included. results: eighteen studies involving 2398 immunocompetent patients admitted to icus were included in the meta-analysis. the overall rate of cmv infection was 27% (95%ci 22–34%, i(2) = 89%, n = 2398) and the cmv reactivation was 31% (95%ci 24–39%, i(2) = 74%, n = 666). the odds ratio (or) for all-cause mortality among patients with cmv infection, compared with those without infection, was 2.16 (95%ci 1.70–2.74, i(2) = 10%, n = 2239). moreover, upon exclusion of studies in which antiviral treatment was possibly or definitely provided to some patients, the association of mortality rate with cmv infection was also statistically significant (or: 1.69, 95%ci 1.01–2.83, i(2) = 37%, n = 912,). for cmv seropositive patients, the or for mortality in patients with cmv reactivation as compared with patients without cmv reactivation was 1.72 (95%ci 1.04–2.85, i(2) = 29%, n = 664). patients with cmv infection required significantly longer mechanical ventilation (mean difference (md): 9 days (95% ci 5–14, i(2) = 81%, n = 875)) and longer duration of icu stay (md: 12 days (95% ci 7–17, i(2) = 70%, n = 949)) than patients without cmv infection. when analysis was limited to detection in blood, cmv infection without antiviral drug treatment or reactivation was not significantly associated with higher mortality (or: 1.69, 95%ci 0.81–3.54, i(2) = 52%, n = 722; or: 1.49, i(2) = 63%, n = 469). conclusion: critically ill patients without immunosuppression admitted to icus show a high rate of cmv infection. cmv infection during the natural unaltered course or reactivation in critically ill patients is associated with increased mortality, but have no effect on mortality when cmv in blood. more studies are needed to clarify the impact of cmv infection on clinical outcomes in those patients. electronic supplementary material: the online version of this article (10.1186/s12879-018-3195-5) contains supplementary material, which is available to authorized users. human cytomegalovirus (cmv) is a prototypic member of the β herpes virus subfamily [1] . the prevalence of cmv seropositivity in human populations is roughly 50-95% [2] [3] [4] and highest amongst older people [5] . cytomegalovirus infection induces innate immune responses (eg. natural killer cells) and adaptive immunity (eg. cd4+/cd8+ t cells). however, the virus can evade host detection by expressing genes that interfere with both the innate and adaptive immune systems. eventually, cmv is able to establish latency in which either the host fails to eliminate the virus or the virus cannot replicate. however, cmv can become reactivated during periods of host immune suppression [6] . it is well known that cmv infection is common in canonical immunodeficiency patients, such as those with human immunodeficiency virus infection, solid organ or stem cell transplantation and patients undergoing chemoor radiotherapy [7] [8] [9] . with the development of more sensitive detection method, the rate of cmv detection is high in intensive care units (icus) [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . however, so far, there is no convincing research to support the use of antiviral treatment when critically ill but immunocompetent patients present with cmv infection. furthermore, whether cmv is a contributor or simply a bystander to the severity of illness remains under debate [26] [27] [28] . whether cmv infection is associated with increased mortality in immunocompetent icu patients remains controversial [13] [14] [15] [16] . a previous meta-analysis published in 2009 demonstrated that cmv infection was associated with a higher mortality rate, nearly twice that observed in patients without cmv infection [29] . however, this study did not consider the influence of antiviral drugs on clinical outcomes. moreover, many clinical studies about cmv have been reported in recent years. thus, to acquire a better understanding of the potential role of cmv infection in contributing to mortality in critically ill patients, especially those not receiving antiviral agents and cmv detected in blood, we performed a meta-analysis of data available in the literature, focusing on the outcome in immunocompetent icu patients with cmv infection. a literature search for relevant publications included within the electronic databases pubmed, embase and the cochrane library was performed using combinations of the keywords "cytomegaloviruses", "salivary gland viruses", "herpes virus", "cytomegaloviral infection", "hhv5", "intensive care", "critical care", "critical illness", "mechanical ventilation", and "pulmonary ventilator". all searches were updated on may 30, 2016 . no language restriction was enforced. we also consulted relevant reference articles and searched using google scholar. two researchers (lx and hyb) performed data extraction independently, and any discrepancies were addressed by discussion and reevaluation until consensus was achieved. observational studies were eligible if they reported on cmv infection in immunocompetent patients in the icu, and if a 2 × 2 table could be constructed based on cmv results and all-cause mortality. all patients were over 18 years of age. the systematic review included only studies in which all patients were tested for cmv. an episode of cmv infection was defined by one of the examination cmv viral culture, polymerase chain reaction (pcr), cmv antigen (pp65) in blood, tracheal aspirates, urine, or a combination of these. a case was defined by the presence of reactivation, where the patient had cmv infection and was seropositive. immunocompetent patients were defined as those patients who did not receive a solid organ or hematopoietic stem cell transplant, did not receive immunosuppressive treatment, did not have human immunodeficiency virus infection, did not have primary immunodeficiency, and did not receive chemotherapy or radiotherapy before icu admission. we obtained information on basic study characteristics (author, year of publication, country of origin, study period, setting, and study design), characteristic population, the site and detection method of sample, cmv seropositivity, cmv infection incidence, all-cause mortality, length of icu/hospital stay, length of mechanical ventilation, and administration of antiviral drugs. the newcastle-ottawa scale, developed for evaluating the quality of observational studies (additional file 1: table s1 ) [30] , was used to assess the validity of included studies. continuous variables are reported as mean or median values and categorical variables are reported as n (%). meta-analytic pooling was performed for outcome variables with a logit transformation approach, reporting results as summary point estimates (95% confidence interval, ci). we used the mantel-haenszel method to obtain odds ratios (ors) and 95% ci. when only the median, range, or interquartile range of length of mechanical ventilation and the length of icu stay were reported, we used simple formulas to estimate the mean and standard deviation [31] . between-study heterogeneity was examined using the i 2 measure of inconsistency and the chi-square test of heterogeneity. to evaluate publication bias, we constructed a funnel plot and used the egger test. sensitivity analyses of the begg's test were additionally conducted to ascertain the robustness of our findings. all meta-analyses were performed with r software (version 3.3.3 for windows) and spss 18 (ibm, armonk, ny, usa). the initial database search identified 1846 potentially relevant studies. following this, assessment of the full text yielded 17 studies suitable for analysis. another publication was incorporated after examining references from the extracted articles [23] . consequently, our meta-analysis consisted of 18 articles (fig. 1) , including one case-control [20] and 17 cohort studies [10-19, 21-25, 32] . most studies were conducted in the united states and europe, except one cohort study in egypt [12] , and were published between 1990 and 2016 (table 1) . overall, the studies were well done, with a median score of 7 (range 6-8) on the newcastle-ottawa scale for appraising the quality of observational studies. a total of 2398 patients were included, having been admitted to the icu for a variety of reasons, with a median age of 59 years. the median period of prospective studies was 24 months, ranging broadly from 3 to 78 months. all studies used cmv blood assays, and 6 studies also assayed sputum samples. most studies indicated that the frequency of sample collection was once a week. in our analysis, the methods used to assess cmv infection were virus culture, pp65 antigen detection and pcr detection of cmv dna in ten, three and two studies, respectively, and combinations of two diagnostic methods in the remaining three studies. as shown in fig. 2 , the overall detection rate of cmv was 27% (95% ci 22-34%, i 2 = 89%, n = 2398). as compared with patients without cmv infection, the all-cause mortality of patients with cmv infection was significantly higher (or: 2.16; 95% ci 1.70-2.74, i 2 = 10%, n = 2239) (fig. 3a) . when analysis was limited to cmv detection in blood, there was still statistical significance in mortality rate between patients with cmv infection (or: 2.15, 95% ci 1.48-3.15, i 2 = 34%, n = 1441) compared with patients without infection (additional file 2: figure s1 ). to rule out the impact of antiviral drugs on patients with cmv infection, four studies in which patients received antiviral drugs during their icu stay and eight studies that did not specify the use of antiviral drugs were excluded. the remaining six studies of patients without antiviral treatment during the course of icu stay were analyzed [10, 13, 18, 19, 21, 33] . the difference in mortality rates between patients with cmv infection remained significant (or: 1.69, 95% ci 1.01-2.83, i 2 = 37%, n = 912) compared with patients without infection (fig. 3b) . when analysis was limited to cmv detection in blood, there was no statistical significance in mortality rate between patients with cmv infection (or: 1.69, 95% ci 0.81-3.54, i 2 = 52%, n = 722) as compared with patients without infection (additional file 3: figure s2 ). the mean difference in mechanical ventilation days and duration of icu stay was an increase of 9 days (95% ci 5-14, i 2 = 81%, n = 875) and 12 days (95% ci 7-17, i 2 = 70%, n = 949), respectively, between patients with and without cmv infection ( fig. 4a and b) . when analysis was limited to cmv detection in blood, there was still a statistically significant difference in length of mechanical ventilation and icu stay between patients with cmv infection as compared with patients without infection (md: 7 days (95% ci 3-11, i 2 = 77%, n = 547) and md: 9 days (95% ci 4-13, i 2 = 66%, n = 547)), respectively (additional file 4: figure s3 and additional file 5: figure s4 ). the cmv seropositivity rate, which represents previous infection, was 71% (95% ci 68-75%, i 2 = 35%, n = 1242) in immunocompetent icu patients (fig. 5a) . patients with cmv reactivation, which represents cmv detected among seropositive patients, was 31% (95% ci 24-39%, i 2 = 72%, n = 666) (fig. 5b) . the or for mortality in patients with cmv reactivation as compared with patients without cmv reactivation was 1.72 (95% ci 1.04-2.85, i 2 = 29%, n = 664) (fig. 5c) . but for patients of cmv infection in blood, the reactivation was not associated with higher mortality (or: 1.49, 95% ci 0.46-4.28, i 2 = 63%, n = 469) (additional file 6: figure s5 ). we also analyzed the rate of cmv and mortality thought categorized by the detection methods (additional file 7: figure s6 , additional file 8: figure s7 : additional file 9: figure s8 and additional file 10: figure s9 ). we used the egger test to detect publication bias. there was no publication bias either in the overall cmv prevalence analysis (t = 1.1264, p = 0.2766) or in the all-cause cmv mortality analysis (t = − 1.3418, p = 0.1984). we also used begg's test to detect sensitivity analysis, and the results showed that the analyses were robust. in this meta-analysis, we have demonstrated that cmv infection frequently present in critically ill immunocompetent patients at icu admission. the overall rate of cmv infection was 27%, which was higher than the 17% presented in a previous meta-analysis [29] , because eight recent studies detecting cmv infection by pcr assay were included in our meta-analysis [10-15, 32, 33] . polymerase chain reaction has been demonstrated to be the most sensitive method of cmv detection [34] , but even so, the cmv infection rate may still be underestimated because we chose only the studies containing 2 × 2 tables on cmv results and all-cause mortality. we excluded studies where either the rate of cmv infection or mortality was zero and we also excluded some studies with a 0% infection rate that used early monitoring of cmv, often fewer than 7 days after admission to the icu [26, [35] [36] [37] . we believe this could have led to underestimation of the cmv infection rate because the transition to cmv infection requires time for the complete lytic virus cycle to develop from the latent phase [38] . we found that the detection rate of cmv by culture, pp65 and pcr was 13, 22 and 34%, respectively. desachy for cmv infection were obtained in a median of 4 days by pcr compared with 11 days by pp65 antigen detection after onset of sepsis [36] . therefore, pcr facilitates earlier diagnosis of an episode of cmv infection than any other method. we then analyzed the association between cmv positivity and mortality, stratified by detection method. we also found that patients with cmv infection detected by pcr had higher mortality than patients without cmv infection (or: 2.07, 95% ci 1.59-2.70, i 2 = 40%, n = 1441). however, when compared with other methods, the association with mortality was marginally less strong using pcr. we may think that viral burden of cmv is determinant of pathogenesis, and higher cmv loads is correlated with progression of some cmv infection disease [39, 40] . the presence of cmv seropositivity, representing previous infection, was found in 71% of immunocompetent icu patients and the incidence of cmv reactivation was high, observed in 31% of seropositive patients in our meta-analysis. there are several factors that might explain the high prevalence. first of all, the rate of cmv seropositivity increases with advancing age [5] and in our analysis, the median age was 59 years. second, to inhibit the reactivation of cmv, as many as 10% of all peripheral cd4+ and cd8+ t cells are constantly required for immune surveillance to maintain functional latency [41] . sepsis is associated with immunoparalysis, as apoptosis of cd4+ and cd8+ t cells is increased [42, 43] . furthermore, some patients in the icu may be immunosuppressed after trauma and major surgery [44] . in addition, treatments commonly received in the icu, such as massive transfusion, corticosteroids, or catecholamines may transiently compromise host immunity [45] . it has also been reported that the use of heart-lung machines can lead to temporary systemic immunosuppression [46] . therefore, patients in the icu may show transient immunoparalysis [47] , potentially resulting in the observed cmv reactivation. third, some inflammatory cytokines including tumor necrosis factor alpha and interleukin-1β, can stimulate reactivation of latent cmv [48] . thus, significant numbers of immunocompetent patients harboring latent virus are susceptible to cmv reactivation during critical illness. when the mortality analysis was limited to cmv detection in blood, cmv infection without antiviral drug treatment or reactivation was not significantly associated with higher mortality. this maybe explained that the presence of high peripheral levels of functional cmv-specific cd4+ and cd8+ t cells in immunocompetent patients, which can suppress cmv during episodes of reactivation [26] . it was observed that cmv infection was not associated with mortality in cmv colitis. in steroid-refractory patients with ulcerative colitis, cmv was found in the colon by histopathology, which is also not associated with adverse clinical outcomes [49] . indeed, there has been no research to demonstrate that immunocompetent critically patients with cmv infection could benefit from antivirus therapy. and there are a number of side effects of antiviral drugs, such as hematologic complications (neutropenia, anemia and thrombocytopenia), renal dysfunction, mental disorders [50] . therefore, giving antiviral drugs to critically ill patients should be considered cautiously in terms of advantage-disadvantage ratio. to address this issue, there are two ongoing, blinded, randomized placebo-controlled clinical trials of an antiviral drug with activity against cmv in critically ill patients in the icu (nct 01335932, nct 02152358). patients with sepsis have the highest incidence of cmv infection [22] . early in 1990's, bacterial sepsis was considered to trigger cmv reactivation [26] . the reactivation associated with sepsis was consequence of inflammatory stimulation, transient immune compromise, and maybe involving some component of epigenetic regulation of viral dna [26] . there are five limitations in this study. first, we observed large heterogeneity in many of our analyses. however, little or no heterogeneity was observed in the meta-analysis of mortality outcome. second, most studies were not blind, thus reducing the reliability of the results. third, lack of sufficient data on clinical parameters (eg: severity of illness, cause of icu admission, comorbidity) meant that stratified analyses based on such clinical characteristics were not possible. fourth, the definition of the state of cmv infection was inconsistent and maybe restrictive to capture the dynamics of cmv infection. as such, we could not conduct meta-analysis with outcome data and this is a major limitation of our meta-analysis. finally, one [10] cannot discount the effect of unmeasured confounders given the observational nature of the body of evidence comprising this meta-analysis. cytomegalovirus: pathogen, paradigm, and puzzle seroprevalence of cytomegalovirus (cmv) and risk factors for infection in adolescent males cytomegalovirus seroconversion rates and risk factors: implications for congenital cmv cytomegalovirus seroprevalence in the united states: the national health and nutrition examination surveys 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herpesvirus 6 variant a occurs in critically ill immunocompetent hosts reactivation of human herpesvirus type 6 in multiple organ failure syndrome evaluation by polymerase chain reaction of cytomegalovirus reactivation in intensive care patients under mechanical ventilation cytomegalovirus infection in patients with bacterial sepsis diagnostic approaches to cytomegalovirus infection in bone marrow and organ transplantation a real-time taqman pcr for routine quantitation of cytomegalovirus dna in crude leukocyte lysates from stem cell transplant patients broadly targeted human cytomegalovirus-specific cd4+ and cd8+ t cells dominate the memory compartments of exposed subjects sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy sepsis: a roadmap for future research acquired immunologic deficiencies after trauma and surgical procedures immunologic abnormalities in patients receiving multiple blood transfusions pediatric cardiac surgery with cardiopulmonary bypass: pathways contributing to transient systemic immune suppression sir isaac newton, sepsis, sirs, and cars lipopolysaccharide, tumor necrosis factor alpha, or interleukin-1beta triggers reactivation of latent cytomegalovirus in immunocompetent mice cytomegalovirus in inflammatory bowel disease: pathogen or innocent bystander? treating hsv and cmv reactivations in critically ill patients who are not immunocompromised: con our findings suggests that there is a high incidence of cmv seropositivity and cmv infection in critically ill patients without immunosuppression. this study suggest that cmv infection without antiviral drug treatment or reactivation in critically ill patients is associated with increased mortality, and is not associated with mortality when cmv infection is detected in blood. further research is necessary to determine the full role of cmv in this vulnerable patient demographic. additional file 1: table s1 . the newcastle-ottawa scale (pdf 17 kb) additional file 2: figure s1 availability of data and materials all data generated or analyzed during this study are included in this published article.authors' contributions lx conducted the literature search, extracted data, performed statistical analysis, and drafted the manuscript. hyb conducted the search, extracted the data, and revised the manuscript. xzh performed the statistical analysis and edited the manuscript. zr conducted the literature search and extracted data. lxq interpreted the data. mp designed the study, interpreted data, and revised the manuscript. lym conceived and designed the study and revised the manuscript. all authors have read and approved the final manuscript. key: cord-016267-idrc1sdh authors: ljungman, per title: viral infections in hematopoietic stem cell transplant recipients date: 2009-11-27 journal: allogeneic stem cell transplantation doi: 10.1007/978-1-59745-478-0_29 sha: doc_id: 16267 cord_uid: idrc1sdh viral infections are important as causes of morbidity and mortality after allogeneic stem cell transplantation (sct). severe viral infections are more common after unrelated and mismatched donor sct and in particular after haploidentical sct. b-cell function and specific antibodies are the main defense mechanisms against infection with exogenous viruses, thus reducing the risk for reinfection in already seropositive individuals. on the other hand, t-cell function in particular cytotoxic t-cell function is the main mechanism for preventing severe viral disease and also for the control of viruses such as herpesviruses that can cause latency and thus reactivate in an immunocompromised individual. the immune defects in sct-patients are frequently complex with defects in cytotoxic t-lymphocyte, helper t-lymphocyte, nk-cell, and b-lymphocyte functions. t-cell dysfunction is usually most important early after sct while deficient b-cell reconstitution can remain for many years after sct. furthermore, since loss of specific antibodies occurs frequently over time after allogeneic sct, this will also increase the risk for reinfections with previously encountered viruses such as measles or varicella-zoster virus (vzv) and allow reactivation of viruses controlled by antibodies such as hepatitis b virus (hbv) [1, 2]. viral infections are important as causes of morbidity and mortality after allogeneic stem cell transplantation (sct). severe viral infections are more common after unrelated and mismatched donor sct and in particular after haploidentical sct. b-cell function and specific antibodies are the main defense mechanisms against infection with exogenous viruses, thus reducing the risk for reinfection in already seropositive individuals. on the other hand, t-cell function in particular cytotoxic t-cell function is the main mechanism for preventing severe viral disease and also for the control of viruses such as herpesviruses that can cause latency and thus reactivate in an immunocompromised individual. the immune defects in sct-patients are frequently complex with defects in cytotoxic t-lymphocyte, helper t-lymphocyte, nk-cell, and b-lymphocyte functions. t-cell dysfunction is usually most important early after sct while deficient b-cell reconstitution can remain for many years after sct. furthermore, since loss of specific antibodies occurs frequently over time after allogeneic sct, this will also increase the risk for reinfections with previously encountered viruses such as measles or varicella-zoster virus (vzv) and allow reactivation of viruses controlled by antibodies such as hepatitis b virus (hbv) [1, 2]. many different techniques have been developed for diagnosis of viral infections. a summary is shown in table 29 -1. during recent years, important advances have been made through the use of rapid nucleic acid testing improving sensitivity and thereby making specific diagnosis and monitoring of viral infections feasible. the most commonly used technique is polymerase chain reaction (pcr) especially when used for determining viral load. other techniques such as the hybrid capture assay, nasba or branched-dna have been applied and have shown good sensitivity and high specificity. the source of the specimen, the timing of collection in relation to onset of symptoms, the rapidity and method of delivery to the laboratory, and the clinical and epidemiological data provided to the laboratory are important factors that directly affect the likelihood of successful isolation and/or identification of a viral pathogen. cytomegalovirus (cmv) remains one of the most important complications to allogeneic bone marrow and stem cell transplantation. cmv can cause multi-organ disease after sct including pneumonia, hepatitis, gastroenteritis, retinitis, and encephalitis. cmv disease can occur both early and late after transplantation [3] [4] [5] [6] . seropositivity of the patients remains a risk factor for transplant related mortality in unrelated transplant patients despite major advances in early diagnosis and management [7-9] seronegative patients with seropositive stem cell donors develop primary cmv infection in about 30% and have an increased mortality in bacterial and fungal infections [10] . in a study using the ebmt registry database, cmv seropositive patients receiving seropositive unrelated donor grafts had improved survival and reduced trm compared to those receiving seronegative grafts and a similar result was found in a single center study [11, 12] . the mechanism for this positive effect was hypothesized to be the transfer of cmv-specific donor cells with the grafts. other studies have, however, failed to find this correlation and therefore, it remains controversial [13] . other identified risk factors include acute and chronic gvhd and the use of mismatched or unrelated donors. sirolimus as prophylaxis against acute gvhd has been reported to result in a lower risk for cmv reactivation [14] . the mechanism behind this reduced risk is unknown. cmv might also be one factor in the pathogenesis of chronic gvhd [15, 16]. since the prognosis of established cmv disease is still poor, preventive measures are very important. the available strategies can be divided into prevention of a primary infection, recurrence of cmv (prophylaxis), and prevention of development of disease when a reactivation has occurred (preemptive therapy). serology should be performed before sct in both patients and donors. patients who are cmv seronegative before sct should if possible be transplanted from a cmv seronegative donor [17] . to reduce the risk of cmv transmission from blood products, blood products from cmv seronegative donors or leukocyte depleted blood products should be used, as cmv is mainly harbored in the leukocyte fraction [18] [19] [20] . which strategy is preferable is still not definitively settled [21, 22] . in many centers, and even in entire countries, leukocyte filtration is obligatory for all blood products and no study has in a controlled fashion compared the benefit of use of seronegative blood to that of already filtered blood products. iv immune globulin has at best a minor effect and has therefore been replaced by other more effective strategies. high doses of acyclovir and valacyclovir can reduce the risk for cmv [27, 28] . however, these studies were performed before the widespread use of growth factors such as g-csf and ganciclovir induced neutropenia was a problem in both studies. valganciclovir is the product of ganciclovir giving similar blood levels as i.v ganciclovir but no study has evaluated its efficacy as a prophylactic agent. preemptive therapy based on early detection of cmv has become the most commonly used strategy for prevention of cmv disease after allogeneic sct [29, 30]. as early identification of patients at risk for developing viral disease reduces virus-related morbidity and mortality, monitoring with sensitive techniques such as antigenemia or quantitative pcr is indicated in all allogeneic sct patients. viral load monitoring seems to be of importance for assessing the risk for cmv disease or the efficacy of antiviral therapy [31] [32] [33] [34] [35] . ganciclovir is the most used drug for preemptive therapy. valganciclovir has been studied in several uncontrolled studies and in a small randomized pharmacokinetic study and gives higher drug exposure compared to i.v. ganciclovir and similar efficacy [36] . both drugs are associated with bone marrow suppression and renal toxicity. antiviral resistance can develop on the basis of mutations in the cmv genes, which the drugs inhibit. however, virus mediated antiviral resistance is quite rare in sct patients while "clinical" resistance based on rapid replicating virus in the severely immunocompromised patients is quite common especially early after initiation of antiviral therapy. increasing antigenemia or cmv dna is therefore commonly not a sign of antiviral resistance and does not necessitate change of therapy [32, 37] . although foscarnet is as effective as ganciclovir [38] , it is more commonly used today as a second line drug. foscarnet is associated with renal toxicity and electrolyte disturbances. the duration of preemptive therapy has varied in the published studies. one strategy is to continue therapy until day 100 after sct [39] while the other possibility is to treat until the indicator test becomes negative, usually resulting in a shorter duration of therapy [38, 40] . also the combination of ganciclovir and foscarnet has been used [41, 42]. cidofovir (3-5 mg/kg per week) has also been used as a second-line agent but is associated with renal toxicity [43] [44] [45] . case reports have been published of treatment with leflunomide or artesunate in patients failing other antiviral therapies [46-48]. appropriate diagnostic procedures should be undertaken in patients suspected to have cmv end-organ disease [49] . the prognosis in patients with established cmv disease is still poor [3, 50] . standard therapy of cmv pneumonia has been intravenous ganciclovir combined with high dose immune globulin but this standard was questioned by the results of an uncontrolled study suggesting that the advantage of adding immune globulin is limited with no improvement in survival over ganciclovir therapy given alone [50] . for patients with cmv disease other than pneumonia, the addition of immune globulin does not seem to be beneficial [51] . a retrospective survey reported that cidofovir could salvage nine of 16 patients with cmv pneumonia failing therapy with ganciclovir, foscarnet, or the combination [43]. the lack of specific immunity to cmv, both regarding cytotoxic t-cell (ctl) response and helper t-cell response to cmv, has been associated with a high risk for cmv disease [3, 5, 52, 53]. monitoring of cd8 and/or cd4 cmv specific t-cells has therefore been studied and different techniques can be applied including detection by tetramers containing immunodominant peptides from cmv or measurement of peptide-specific intracellular cytokine staining herpes simplex virus (hsv) can cause local and rarely disseminated infections after sct. serology is useful for determining the risk for reactivated hsv infection and should be performed before transplantation. the manifestations in transplant patients can be atypical causing generalized inflammation and pain from the mucous membranes without classical vesicular or ulcerative lesions. generalized and invasive disease can occur but encephalitis is not more frequent in immunocompromised compared to immune competent patients. acyclovir prophylaxis is indicated in all hsv seropositive sct recipients [67] . the duration of antiviral prophylaxis should be at least during the aplastic phase but a longer duration should be considered in patients with gvhd or a history of frequent reactivations before the transplantation [67]. a recent study has shown a 2-year probability for hsv disease of 32% when acyclovir was given for 30 days compared to 0% if prolonged prophylaxis was given [68] . acyclovir resistant virus strains are still quite rare but seem to be more common in high risk patients such as unrelated donor transplants or patients with severe gvhd [69-71]. however, the risk was reported to be very low in patients receiving prolonged prophylaxis [68] . the recommended drug for acyclovir-resistant hsv is foscarnet [72-74]. two reports have described mutants resistant to both acyclovir and foscarnet [69, 70] . currently, the only available antiviral drug available for treatment of double resistant hsv is cidofovir. a primary vzv infection (varicella) is an uncommon but severe complication in sct patients [75] . seronegative patients are at risk for developing varicella and preventive measures are therefore indicated. the risk is highest in children but cases of varicella-like disease in seropositive adults becoming seronegative after sct have been described. serology is therefore important to identify patients at risk for varicella and should be performed in all patients before and at regular intervals after sct. varicella-zoster immune globulin is the recommended prophylactic measure in seronegative patients if it can be given within 4 days after a household or other type of close exposure [67] . another option is prophylaxis with acyclovir or valacyclovir but there are no published data regarding effectiveness. reactivated vzv infection -herpes zoster -develops in approximately one third of the sct recipients in the absence of prophylaxis [78] [79] . severe and fatal cases have also been reported after allogeneic sct with reduced conditioning. herpes zoster is usually dermatomal but disseminated and potentially fatal infections with visceral involvement can occur [75] . the clinical picture might be atypical with gastro-intestinal, liver, or cns disease occurring in the absence of skin lesions. the risk of herpes zoster is highest between 3 and 6 months after transplantation and decreases thereafter steadily over the first 2 years after sct [80] . therefore, the duration of antiviral prophylaxis must be long to be effective. a rebound phenomenon occurs when prophylaxis is given for 6 months [81, 82] but does not exist if prophylaxis is given for 12 months [83] . longer prophylaxis reduces the rates even further especially in patients with chronic gvhd [80]. the recommended therapy for primary varicella, disseminated herpes zoster, or localized zoster developing early after sct or in patient on treatment for gvhd is intravenous acyclovir 10 mg/kg (or 500 mg/m 2 ) three times daily. for localized dermatomal herpes zoster occurring late after sct especially on patients of immunosuppression, clinical experience suggests that oral therapy with acyclovir, famciclovir, or valaciclovir is effective in the majority of patients [84, 85]. epstein-barr virus (ebv) is frequently detected after allogeneic sct [86] [87] [88] [89] [90] . however, only a few case reports have suggested that it directly causes significant disease such as meningo-encephalitis [91] . ebv induced post-transplant lymphoproliferative disease (ptld) is a serious complication to allogeneic sct. although the incidence of ebv-ptld is generally lower than 2% following allogeneic sct, it may increase up to 20% in patients with risk factors such as mismatched donor sct, the use of an ebv positive donor to an ebv negative recipient, t-cell depletion, atg therapy, and other forms of intensified immunosuppression for prevention and treatment of gvhd [92, 93] . cord-blood sct recipients receiving reduced intensity conditioning including atg were reported to have a high risk for ebv associated complications [94] . ebv-ptld usually occurs during the first 3 months after sct although it can present later. ptld usually presents during the first months after sct as a polymorphic polyclonal lymphoproliferation that may result in monoclonal malignant lymphoma if left untreated. ebv dna load monitoring in peripheral blood has been studied as a predictor for ebv-ptld but the variations in the "in house" developed assays and the use of different sample types such as whole blood, serum/plasma, or pbl make it difficult to draw firm conclusions [88, [95] [96] [97] . the usefulness of viral load monitoring depends on the likelihood for a patient developing ptld. the positive predictive values vary greatly between different studies [98] with the highest for patients having risk factors for ebv-ptld [95, 96, 98] . despite these uncertainties, monitoring of viral load seems to be a valuable tool especially in high risk patients. as many different techniques using different materials and primers exist, no cut-off viral load for initiating therapy can be recommended. however, rapid increase in viral load has been suggested to be associated with a high risk for ebv-ptld. the first management option in a patient at high risk for ptld is, if possible, to reduce the immunosuppression. antiviral therapy might lower the ebv viral load but whether this influences the risk for ptld is doubtful. rituximab has been used as "preemptive therapy" in several patient series with good results but no controlled data exist [88, 94, 98, 99] . another prophylactic option is to give infusions with ebv specific ctl [100] [101] [102] . there is no established therapy for treatment of ptld. rituximab has been used after both solid organ and sct [90, [103] [104] [105] [106] . cloned ebv specific donor t-cells [100, 102] , partially hla-matched allogeneic donor ctl [107] , and unspecific donor lymphocyte infusions have also been used as treatment of ptld [108] . human herpes virus type 6 (hhv-6) exists in two subtypes (a and b) that differ from each other in 4-8% of the dna. subtype b is the cause of exanthema subitum in childhood. hhv-6 infection is very common early in life; hence the rate of seropositivity in older children and adults is more than 95%. serology is therefore not helpful in patient management. there is no "gold standard" diagnostic test for hhv-6 infection but quantitative pcr has been used to better define of the contribution of hhv-6 to post transplant complications [109] [110] [111] . a possible confounding factor is that the hhv-6 genome can be integrated into cellular dna resulting in high levels of hhv-6 dna in blood samples including pbl [112, 113] . the best documented clinical manifestations of hhv-6 are encephalitis and bone marrow suppression. hhv-6 has a propensity for the cns and although hhv-6 dna can occasionally be detected in the csf of asymptomatic sct recipients [114, 115] , the combination of symptoms of encephalitis with detection of hhv-6 dna is suggestive of hhv-6 disease of the cns. approximately 35 case reports have been published [110, [114] [115] [116] [117] [118] [119] [120] [121] [122] [123] [124] [125] [126] [127] [128] . a summary of published information around these cases regarding patient characteristics, diagnostic findings, and outcome of hhv-6 cns disease in sct patients is shown in table 29 -2. lethargy, confusion, convulsions, and decreased consciousness are the predominant clinical manifestations of hhv-6 encephalitis. focal neurological findings have been reported but are less common. magnetic resonance imaging can show abnormalities but it can also be normal. these changes included multiple, non-enhancing, low attenuation lesions in the gray matter. eeg usually shows diffuse changes. the prognosis is poor unless the encephalitis is treated with antiviral drugs. both ganciclovir and foscarnet have been reported being effective against hhv-6 meningo-encephalitis after sct (table 29. 2) [114, 129] . another possible manifestation of hhv-6 is bone marrow suppression or delayed engraftment as hhv-6 can infect hematological progenitor cells and reduce colony formation [87, 110, 130-132]. respiratory viruses including respiratory syncytial virus (rsv), parainfluenza viruses, coronaviruses, rhinoviruses, and influenza a and b are widespread in the community with major seasonal variations. recently several new viruses have been discovered including bocavirus and two papovaviruses that can cause respiratory disease. the epidemiological situation in the local community has been shown to influence the risk for infection in the sct patients. this at least partly explains the variation in frequencies of diagnosed infections between different studies [133] [134] [135] [136] . respiratory viruses can be spread nosocomially through immune competent staff and patient relatives and outbreaks of both rsv and parainfluenza infections have been documented in transplant units [137] [138] [139] [140] [141] . thus, infection control measures including isolation of symptomatic patients, use of sensitive diagnostic procedures, and as far as possible avoidance of exposure to infected persons including family and staff are important in the management of respiratory infections. the influence of respiratory virus infections on transplant related mortality has been estimated by a study rsv has been the cause of outbreaks in sct patients forcing closure of transplant units [136, [144] [145] [146] . in a prospective survey, the overall mortality in patients with a rsv lower respiratory tract infection was 30% and the rsv associated mortality 17% [133] . more recently, the impact of rsv seems to have been reduced [134, 147] there is no established therapy for rsv. in a small randomized trial there was no difference between patients receiving ribavirin or no therapy regarding the risk for progression to pneumonia, but there was a tendency for a greater viral load decrease in ribavirin treated patients (p = 0.07) [151] . in an uncontrolled study, 4 of 14 patients treated with the combination of ribavirin and high dose iv immune globulin developed pneumonia [152] . in the prospective ebmt survey, no regimen was superior to any other [133] . in a small phase i study of the rsv monoclonal antibody palivizumab, three patients were treated for an upper respiratory tract infection and none developed lower respiratory tract disease [153] . only uncontrolled phase ii treatment studies of rsv pneumonia have been reported. there are no proven benefits with any drug or combination, but patients treated when ventilator dependent usually have dismal outcome [136] . ljungman et al reported similar outcomes with ribavirin given intravenously and as aerosol [133] . influenza is an important problem to consider in sct recipients. the mortality has been reported to be around 15% in untreated patients [133, 160] . the mortality is highest in patients developing pneumonia [161] . fatal influenza infections can occur several years after an allogeneic sct in particular in patients with chronic gvhd [133] . the primary mode for prevention of influenza is vaccination and should be given to all transplant patients from 4 months after transplantation and yearly while the patients are immunosuppressed [67, 162] . the antibody responses have been poor when vaccinations are performed early after sct [163, 164] but clinical protection might still be achieved [165] . vaccination of family members and hospital staff to reduce the risk for transmission of influenza is recommended [133] . the possibilities for prevention with antiviral agents include today mainly the neuramidase inhibitors (zanamivir and oseltamivir). no controlled study has been performed in sct patients, but in an uncontrolled study oseltamivir was given to 41 patients with influenza of whom two developed pneumonia and none died [166] ). in another series 6 of 34 untreated patients, one of eight treated with rimantadine, and none of nine patients treated with oseltamivir developed pneumonia [161] . one concern is the reported rapid development of oseltamivir resistant influenza viruses. metapneumovirus is a paramyxovirus causing upper and lower respiratory tract infections in children. martino et al found in a prospective study an incidence of 5% in allogeneic and 3% in autologous sct recipients [135] . forty-four percent of the patients with metapneumovirus infections in allogeneic sct recipients developed pneumonia. fatal infections have been reported [167] . the impact of other respiratory viruses including rhinoviruses, coronaviruses, and the recently discovered boca-and respiratory papovaviruses needs further study. no therapy exists for any of these recently discovered respiratory viruses. adenoviruses cause a number of clinical syndromes in immune competent individuals that are usually mild and self-limiting, but more severe manifestations have also been reported. although 51 distinct adenovirus serotypes have been identified, most human diseases are associated with only one-third of these types. adenovirus infections can result in morbidity and mortality after allogeneic sct. the frequencies of adenovirus infections vary between studies. overall, there is a higher frequency in children. flomenberg et al. reported a frequency of 31% in children compared to 14% in adults [168] . in a study using pcr monitoring of pediatric sct recipients, lion et al. reported that 27% of the patients had adenovirus dna detected [169] while hoffman et al. in a study of pediatric sct recipients detected adenovirus in 47% of the patients [170] . other reports give frequencies of 3-29% [171] [172] [173] [174] [175] [176] . the factors influencing the detection frequency seem to be the age of the population studied, whether the study was prospective or retrospective, and the diagnostic technique used, but there also seems to be a center effect with some centers experiencing a major adenovirus problem while it is rather rare in other centers. the most serious disease manifestations are pneumonia, encephalitis, and fulminant hepatitis. however, hemorrhagic cystitis and gastroenteritis seem to be more common. the most commonly recognized risk factors for adenovirus disease in allogeneic sct recipients are younger age, t-cell depletion, gvhd, the use of mismatched and unrelated donors, the use of unrelated cord blood grafts, and adenovirus detected from more than one site [168, 170-174, 177, 178] . identification of adenovirus in peripheral blood has also been associated with an increased risk for adenovirus disease [169] . there is no established either prophylaxis or therapy for adenovirus infections in sct recipients. ribavirin has been used in case reports with varying outcome [173, [179] [180] [181] [182] [183] [184] [185] . morfin et al. reported that the in vitro efficacy varied among different subgenera of adenovirus with group c isolates being more sensitive in vitro to ribavirin compared to other subgenera [186] . this might explain some of the inconsistencies in the treatment results with ribavirin. cidofovir might have effect against adenovirus infections but no controlled studies have been performed in sct recipients. reported results have been varying but it seems probable that cidofovir has an anti-adenoviral effect in many patients but it alone cannot give long-term control as development of a specific t-cell response is necessary [187] [188] [189] [190] [191] [192] [193] . similar to cmv and ebv, ctl based immunotherapy is under development for adenovirus [194, 195] . in patients who are hbsag positive before transplantation there does not seem to be an obvious increased risk for severe liver complications after transplantation [196, 197] and long-term survival is similar in hbv-positive and negative patients [198] . patients who are anti-hbs positive at the time of transplant can during long-term follow-up become hbsag and hbv-dna positive and also develop a flare of acute hepatitis because of loss of specific antibodies to hbv [2, [199] [200] [201] [202] . in a seronegative recipient, the use of an hbv antigen positive marrow donor should if possible be avoided as the risk for transfer of hbv is high and hepatitis is likely to develop [203] . if a seropositive donor must be used, vaccination of the patient before transplant would be logical as patients who are antibody positive to hbv before transplant are less likely to develop severe liver complications [196, 204] . hbv specific immune globulin can be given to the patient before transplantation [196] . lamivudine has been used in sct patients to prevent reactivation [205] [206] [207] [208] [209] [210] [211] . patients with hepatitis c virus (hcv) and abnormal liver function tests were reported having an increased risk for hepatic vod [212, 213] . if the stem cell donor is hcv rna positive the risk for transmission to the patient is very high [214] . therefore, the use of an hcv positive donor should be avoided if alternatives exist. hcv-infected long-term survivors of allogeneic sct have a high risk for development of liver cirrhosis [215, 216] . therapy with interferon together with ribavirin using similar dose and duration as in non-transplant individuals seems to be safe and effective although no controlled study exists [217] [218] [219] . papovaviruses are a group of dna-viruses with two members -jc-virus and bk-virus -that can be pathogenic in sct patients. jc-virus is the agent causing progressive multifocal leukoencephalopathy (pml) and bk-virus has been implicated in hemorrhagic cystitis and nephropathy in transplant recipients. both bk-and jc-viruses are excreted in the urine of many patients after transplant. papovaviruria has been associated with hemorrhagic cystitis although there is no absolute correlation. higher viral loads in urine, mutations in a viral gene, and bk-viremia have been correlated to hemorrhagic cystitis [220] [221] [222] [223] [224] [225] [226] . however, also transplant factors such as allogeneic rather than autologous sct, myeloablative conditioning, and the use of mismatched or unrelated donors have also been shown to correlate to hemorrhagic cystitis [223, 224, 227, 228] . thus, the pathogenesis of hemorrhagic cystitis seems to be multifactorial [229] . cidofovir has in small uncontrolled studies been reported to be effective against polyoma virus-associated hc [230, 231] . there is no established therapy for pml although cidofovir and ara-c have been given with varying results. measles can be fatal in immunocompromised hosts [232, 233] and severe cases have been reported in sct recipients [234, 235] . most patients will lose immunity during extended follow up and are therefore vulnerable to infection [236] . vaccination against measles has been shown to be safe in patients without gvhd or ongoing immunosuppression. the seroconversion rates varied between 54 and 100% [1, 237, 238] . parvovirus b19 exhibits a marked tropism for human bone marrow and replicates only in erythroid cells. occasional case reports of protracted parvovirus infections have been published after stem cell transplantation [239, 240] . rotavirus infections mostly affect otherwise normal children below 3 years of age. reinfection in adults can occur. the symptoms are usually diarrhea and vomiting. in bmt recipients, gastroenteritis caused by rotavirus has been described [241] . electron microscopy and elisa can confirm the diagnosis. there is no proven effective treatment, although two cases described by kanfer et al. [242] appeared to respond to oral immunoglobulin (6 g daily for 5 days). coxsackie a1 virus infection with diarrhea and a significant mortality has been reported in bmt patients [241] . the diagnosis can be obtained with virus isolation from stool, cerebrospinal fluid, secretions from nose and pharynx, tears, and urine and by serology. prolonged enteroviral infection has been described in a bmt recipient who developed pericarditis and heart failure posttransplant [243] . although no formal study has been performed, it seems likely that these outbreaks are associated with the epidemiological situation in the community and awareness of the local situation can be of value. west nile virus can be transmitted by blood products or from the stem cell donor and has been associated with severe diseases including fatal outcome after sct [244] [245] [246] [247] [248] . viral infections remain important challenges for the physician taking care of sct patients. this includes "old" pathogens that 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a retrospective study by the italian group for blood and marrow transplantation progressive disappearance of anti-hepatitis b surface antigen antibody and reverse seroconversion after allogeneic hematopoietic stem cell transplantation in patients with previous hepatitis b virus infection a case-controlled study on the use of hbsag-positive donors for allogeneic hematopoietic cell transplantation a prophylactic approach for bone marrow transplantation from a hepatitis b surface antigen-positive donor lamivudine treatment for chronic replicative hepatitis b virus infection after allogeneic bone marrow transplantation lamivudine therapy for a hepatitis b surface antigen (hbsag)-positive leukemia patient receiving myeloablative chemotherapy and autologous stem cell transplantation successful long-term control with lamivudine against reactivated hepatitis b infection following intensive chemotherapy and autologous peripheral blood stem cell transplantation in non-hodgkin's lymphoma: experience of 2 cases limited efficacy of lamivudine against hepatitis b virus infection in allogeneic hematopoietic stem cell transplant recipients extended lamivudine therapy against hepatitis b virus infection in hematopoietic stem cell transplant recipients fatal fulminant hepatitis b after withdrawal of prophylactic lamivudine in hematopoietic stem cell transplantation patients lamivudine prophylaxis and treatment of hepatitis b virus-exposed recipients receiving reduced intensity conditioning hematopoietic stem cell transplants with alemtuzumab the role of hepatitis c and b virus infections as risk factors for severe liver complications following allogeneic bmt: a prospective study by the infectious disease working party of the european blood and marrow transplantation group hepatitis c virus infection and bone marrow transplantation: a cohort study with 10-year follow-up marrow transplantation from hepatitis c virus seropositive donors: transmission rate and clinical course cirrhosis of the liver in long-term marrow transplant survivors long-term outcome of hepatitis c infection after bone marrow transplantation long-term effects of hepatitis c virus infection in allogeneic bone marrow transplant recipients alpha-interferon treatment of chronic hepatitis c after bone marrow transplantation for homozygous beta-thalassemia treatment of chronic hepatitis c virus in allogeneic bone marrow transplant recipients rapid quantification and differentiation of human polyomavirus dna in undiluted urine from patients after bone marrow transplantation quantification of polyoma bk viruria in hemorrhagic cystitis complicating bone marrow transplantation overrepresentation of point mutations in the sp1 site of the non-coding control region of bk virus in bone marrow transplanted patients with haemorrhagic cystitis the incidence of hemorrhagic cystitis and bk-viruria in allogeneic hematopoietic stem cell recipients according to intensity of the conditioning regimen association between a high bk virus load in urine samples of patients with graft-versus-host disease and development of hemorrhagic cystitis after hematopoietic stem cell transplantation bk dna viral load in plasma: evidence for an association with hemorrhagic cystitis in allogeneic hematopoietic cell transplant recipients bk virus infection in hematopoietic stem cell transplant recipients: frequency, risk factors, and association with postengraftment hemorrhagic cystitis incidence, clinical outcome, and management of virus-induced hemorrhagic cystitis in children and adolescents after allogeneic hematopoietic cell transplantation clinical course and treatment of haemorrhagic cystitis associated with bk type of human polyomavirus in nine paediatric recipients of allogeneic bone marrow transplants polyomavirus bk infection in blood and marrow transplant recipients treatment of bk virus-associated hemorrhagic cystitis and simultaneous cmv reactivation with cidofovir lowdose cidofovir treatment of bk virus-associated hemorrhagic cystitis in recipients of hematopoietic stem cell transplant fatal measles infection in children with leukemia severe measles in immunocompromised patients clinical features of measles in immunocompromised children measles in bone marrow transplant recipients during an outbreak in sao paulo long-term immunity to measles, mumps, and rubella after allogeneic bone marrow transplantation early measles vaccination in bone marrow transplant recipients response to measles, mumps and rubella vaccine in paediatric bone marrow transplant recipients successful treatment of severe aplastic anemia associated with human parvovirus b19 and epstein-barr virus in a healthy subject with allo-bmt parvovirus b19 transmitted by bone marrow infectious gastroenteritis in bone-marrow-transplant recipients severe rotavirus-associated diarrhoea following bone marrow transplantation: treatment with oral immunoglobulin prolonged enteroviral infection in a patient who developed pericarditis and heart failure after bone marrow transplantation west nile virus encephalopathy in an allogeneic stem cell transplant recipient: use of quantitative pcr for diagnosis and assessment of viral clearance west nile encephalitis in 2 hematopoietic stem cell transplant recipients: case series and literature review fatal west nile virus encephalitis following autologous peripheral blood stem cell transplantation west nile virus encephalitis causing fatal cns toxicity after hematopoietic stem cell transplantation key: cord-305085-bv7udg9k authors: lawrence, robert m. title: chapter 13 transmission of infectious diseases through breast milk and breastfeeding date: 2011-12-31 journal: breastfeeding doi: 10.1016/b978-1-4377-0788-5.10013-6 sha: doc_id: 305085 cord_uid: bv7udg9k nan a large body of evidence clearly demonstrates the protective effects of breastfeeding and documents the transmission of specific infections to infants through breast milk. the fear and anxiety that arise with the occurrence of any infectious disease are even greater in the situation of the breastfeeding mother-infant dyad. uncertainty and lack of knowledge often lead to proscribing against breastfeeding out of fear, which then deprives the infant of the potential protective, nutritional, and emotional benefits of breastfeeding exactly at the time when they are most needed (see the discussion of immunologic benefits of human milk in chapter 5). decisions concerning breastfeeding in a mother with an infectious illness should balance the potential benefits of breastfeeding versus the known or estimated risk for the infant acquiring a clinically significant infection via breastfeeding and the potential severity of the infection. documenting transmission of infection from mother to infant by breastfeeding requires not only the exclusion of other possible mechanisms of transmission but also the demonstration of the infectious agent in the breast milk and a subsequent clinically significant infection in an infant that was caused by a plausible infectious process. the first step is to establish the occurrence of a specific infection (clinically or immunologically evident) in a mother and demonstrate the persistence of the infectious agent such that it could be transmitted to the infant. isolation or identification of the infectious agent from the colostrum, breast milk, or an infectious lesion of the breast is important but not necessarily proof of transmission to an infant. epidemiologic evidence of transmission must be considered, including identifying characteristics of the organism that relate an isolate from an infant to the maternal isolate. infectious organisms can reach the breast milk either by secretion in the fluid or cellular components of breast milk or by contamination of the milk at the time of or after expression. a reasonable mechanism of infection via breast milk should be evident and proved through either animal or human studies. demonstration of a subclinical or clinically evident infection in an infant should follow these outlined steps. exclusion of other possible mechanisms of transmission (exposure to mother or other persons/ animals via airborne, droplet, arthropod, or vector modes of transmission or through direct contact with other infectious fluids) would complete the confirmation of transmission of infection via breastfeeding. it is essential to exclude prenatal or perinatal transmission of infection to a fetus/infant, but doing this can often be difficult. clinical case reports or studies confirming the isolation of an infectious agent from the milk are important. to determine a reasonable estimate of the risk for infection via breast milk, larger epidemiologic studies are needed that compare infection rates in breastfed infants versus formula-fed infants, robert m. lawrence addressing the issues just identified. timing of breastfeeding is important relative to the timing of maternal infection and to the presence of a pathogen in colostrum or breast milk. the duration of breastfeeding is another important variable to consider in the estimate of risk because shedding of a pathogen in breast milk may be intermittent. these considerations are only some of the variables to be taken into account, in general, to assess the risk for transmission of an infectious agent from mother to infant via breast milk or breastfeeding. efforts to prove transmission of infection in a particular maternal-infant dyad can be just as difficult and must consider many of the same factors. this chapter focuses on a discussion of specific, clinically relevant, infectious agents and diseases, with reasonable estimates of the risk for infection to infants from breastfeeding. the basic tenet concerning breastfeeding and infection is that breastfeeding is rarely contraindicated in maternal infection. 243 the few exceptions relate to specific infectious agents with strong evidence of transmission and to the association of an infant' s illness with significant morbidity and mortality. the risk or benefit of breastfeeding relative to immunization of a mother or infant is discussed for certain microorganisms. appendix d addresses drugs in breast milk and includes table d-1, on antiinfective agents, and chapter 5 reviews how breastfeeding may protect against infection. chapter 21 addresses specific concerns relating to banked breast milk and includes standards developed by the human milk banking association of north america to guide the appropriate handling of banked human milk relative to possible infectious agents. isolation precautions have undergone some revisions in terminology and conceptualization. 143 understanding that the transmission of microorganisms can occur with a known infection and with unrecognized sources of infection, recommendations have been made for standard precautions to be applied to all patients to protect health care workers from potentially infectious body fluids. additionally, precautions based on the predominant modes of transmission have been recommended to protect against infection through the airborne route, direct contact, or contact with droplets. although these precautions are intended to be used in clinical situations to protect health care workers, they may be applied in certain situations to the mother-infant dyad to prevent transmission of infectious agents from one to the other or to other hospitalized mothers and infants. these precautions are useful most often when a mother and infant are still hospitalized. the use of such precautions within the home is not meant to limit breastfeeding. they are intended to allow breastfeeding in the majority of cases and to facilitate the continuation of breastfeeding with some additional safeguards in certain situations, after short temporary periods of stopping breastfeeding, and when to safely use expressed breast milk (see appendix f). standard precautions include preventing contact with blood, all body fluids, secretions and excretions, nonintact skin, and mucous membranes by (1) careful handwashing before and after every patient contact; (2) use of gloves when touching body fluids, nonintact skin, or mucous membranes or any items contaminated with body fluids (linens, equipment, devices, etc.); (3) use of nonsterile gowns to prevent contact of clothing with body fluids; (4) use of masks, eye protection, or face shields when splashing with body fluids is possible; and (5) appropriate disposal of these materials. standard precautions should be applied to all patients regardless of actual or perceived risks. the centers for disease control and prevention (cdc) does not consider breast milk a body fluid with infectious risks and thus these policies do not apply to breast milk. (see section on misadministration of breast milk later in this chapter as a possible exception to this concept.) in considering breastfeeding infant-mother dyads and standard precautions, body fluids other than breast milk should be avoided, and only in specified situations should breast milk also be avoided. in general, clothing or a gown for the mother and bandages, if necessary, should prevent direct contact with nonintact skin or secretions. avoiding infant contact with maternal mucous membranes requires mothers to be aware of and understand the risks and to make a conscious effort to avoid this type of contact. the use of gloves, gowns, and masks on infants for protection is neither practical nor appropriate. the recommendations concerning the appropriateness of breastfeeding and breast milk are addressed for specific infectious agents throughout this chapter. human immunodeficiency virus (hiv) infection is an example of one infection that can be prevented by the use of standard precautions, including avoiding breast milk and breastfeeding. the recommendations concerning breastfeeding and hiv and the various variables and considerations involved are discussed later. airborne precautions are intended to prevent transmission via droplet nuclei (dried respiratory particles smaller than 5 mcm that contain microorganisms and can remain suspended in the air for long periods) or dust particles containing microorganisms. airborne precautions include the use of a private room with negative-air-pressure ventilation and masks at all times. in the case of pulmonary tuberculosis (tb), respiratory protective devices (requiring personal fitting and seal testing before use) should be worn. airborne precautions are recommended with measles, varicella or disseminated zoster, and tb. breastfeeding in the presence of these maternal infections is prohibited for the infectious period. this is to protect against airborne transmission of the infection from the mother and to allow the infant to be fed the mother' s expressed breast milk by another individual. the exception to allowing breast milk would be local involvement of the breast by varicella-zoster lesions or mycobacterium tuberculosis, such that the milk becomes contaminated by the infectious agent. transmission via droplets occurs when an individual produces droplets that travel only a short distance in the air and then contact a new host's eyes, nose, mouth, or skin. the common mechanisms for producing droplets include coughing, sneezing, talking (singing or yelling), suctioning, intubation, nasogastric tube placement, and bronchoscopy. in addition to standard precautions applied to all patients, droplet precautions include the use of a private room (preferred) and a mask if within 3 feet (0.9 m) of the patient. droplet precautions are recommended for adenovirus, diphtheria, respiratory infections, haemophilus influenzae, neisseria meningitidis or invasive infection, influenza, mumps, mycoplasma, parvovirus, pertussis, plague (pneumonic), rubella, and streptococcal pharyngitis, pneumonia, or scarlet fever. the institution of droplet precautions with a breastfeeding mother who has these infections should be specified for each particular infection. this may require some period of separation for the infant and mother (for duration of the illness, for short-term or complete treatment of the mother, for the infectious period) with use of expressed breast milk for nutrition in the interim. prophylactic treatment of the infant, maternal use of a mask during breastfeeding or close contact combined with meticulous handwashing, and the mother's avoidance of touching her mucous membranes may be adequate and reasonable for certain infections. contact precautions are meant to prevent transmission of infection via direct contact (contact between the body surfaces of one individual with another) and indirect contact (contact of a susceptible host with an object contaminated with microorganisms from another individual). contact precautions include cohorting or a private room, gloves and gowns at all times, and handwashing after removal of gown and gloves. contact precautions are recommended for a long list of infections, such as diarrhea in diapered or incontinent patients with clostridium difficile infection, escherichia coli o157:h7, shigella, rotavirus, hepatitis a, respiratory illness with parainfluenza virus or respiratory syncytial virus (rsv), multidrug-resistant (mdr) bacteria (e.g., enterococci, staphylococci, gramnegative organisms), enteroviral infections, cutaneous diphtheria, impetigo, herpes simplex virus (hsv) infection, herpes zoster (disseminated or in immunocompromised individuals), pediculosis, scabies, staphylococcus aureus skin infection, viral hemorrhagic fevers (e.g., ebola, lassa), conjunctivitis and abscesses, cellulitis, or decubitus that cannot be contained by dressings. 94 for a breastfeeding infant-mother dyad, implementation of precautions for each of these infections in a mother requires meticulous attention to gowning and handwashing by the mother and a specialized plan for each situation. each of these transmission-based precautions can be used together for organisms or illnesses that can be transmitted by more than one route. they should always be used in conjunction with standard precautions, which are recommended for all patients. the red book: report of the committee on infectious diseases by the american academy of pediatrics (aap) 96 remains an excellent resource for infection control guidelines and recommendations to prevent transmission in specific situations and infections. routine culturing of breast milk or culturing breast milk to screen for infectious agents is not recommended except when the milk is intended as donor milk to another mother' s child directly or through human milk banks. see chapter 21 for specific bacterial count standards for raw donor milk and for pasteurization of donor milk. breastfeeding and the expression of or pumping of breast milk (referred to as expressed breast milk) for later use are not sterile activities. in general expressed breast milk should not contain large numbers of microorganisms (less than 10 4 for raw milk and less than 10 6 for milk to be pasteurized), nor should it contain potential pathogens such as s. aureus, β-hemolytic streptococci, pseudomonas species, proteus species, or streptococcus faecalis or faecium. few studies have examined "routine" culturing of milk and the significance of specific bacterial colony counts relative to illness in infants. the studies have been primarily concerned with premature or low-birth-weight (lbw) infants who remain hospitalized and are commonly fed via enteral tubes. a study from canada tested 7610 samples of milk for use in 98 preterm infants. 242 the study did not identify any adverse events in the infants attributed to organisms growing in the milk samples, and routine bacteriological testing of expressed breast milk was not recommended. a study from chicago examined gram-negative bacilli in the milk used in premature infants. 48 samples were tested before feeding and from the nasogastric tubes during feeding. milk samples from before feeding were less likely to contain gram-negative bacilli (36%) than milk samples from the nasogastric tubing (60%). feeding intolerance was observed when there were more than 10 3 colony-forming units per milliliter (cfu/ml), and episodes of sepsis were identified when the bacterial counts in the milk were greater than or equal to 10 6 cfu/ml. this study recommended the routine bacteriologic testing of expressed breast milk. another study from arkansas focused on contamination of feeding tubes during administration of expressed breast milk or formula. 277 ten infants in the neonatal intensive care unit (nicu) were exposed to greater than 10 5 gram-negative bacteria in their feeding tubes. the three infants who were fed expressed breast milk with contamination at greater than 10 5 organisms remained well, but the seven formula-fed infants with high levels of bacterial contamination in the feeding tubes developed necrotizing enterocolitis. the gram-negative bacteria with high level contamination in the feeding tubes were either enterobacter or klebsiella in all cases. many nicus consider 10 5 to 10 6 cfu/ml as the significant bacterial count for gram-negative bacilli in breast milk that places premature and lbw infants at greater risk for infection. even less data are available concerning specific bacterial colony counts for gram-positive organisms and the risk to the infant. generally less than 10 3 gram-positive organisms per ml of milk is considered acceptable, with only case reports and no controlled trials to support this cutoff. when the presence of an infectious illness in an infant and/or the breastfeeding mother' s breast when breast milk is seriously considered as a possible mechanism of transmission to the infant, culturing breast milk to identify the organism may be warranted and useful. more important than hurrying to culture breast milk is the careful instruction of mothers on the proper technique for collecting expressed breast milk, storing it, and cleaning the collection unit. the reinforcement of proper technique from time to time, especially when a question of contamination arises, is equally important. many small reports comment on the contamination of breast milk with different collection methods. relative comparisons suggest decreasing contamination of expressed breast milk when collected by the following methods; drip milk, hand pumped milk, manual expression, modern electric pumped milk. one group from malaysia published results showing no difference in contamination between milk collected by electric pump versus manual expression when collected in the hospital. expressed breast milk collected at home by breast pump had higher rates of contamination with staphylococci and gram-negative bacteria. 46 discussion continues about the need to discard the first few milliliters of milk to lower bacteria numbers in expressed breast milk without any evidence to suggest if this is truly necessary. 62, 337 no evidence shows that cleansing the breast with anything other than tap water decreases the bacterial counts in cultured expressed breast milk. 414 if an infant is directly breastfeeding, collecting milk for culture by manual expression and trying to obtain a "midstream" sample (as is done with "midstream" urine collection for culture) is appropriate. if an infant is being fed expressed breast milk, collecting and culturing the milk at different points during collection (utilizing the same technique the mother uses [manual expression, hand pump, or electric pump]) and administration is appropriate. this might include a sample from immediately after collection, another of stored expressed breast milk, and a sample of milk from the most recent infant feeding at the time the decision to culture is made. please see box 13-1 for the basic steps in culturing expressed breast milk. interpretation of such culture results can be difficult and should involve a pediatric infectious disease expert, a microbiologist, and hospital epidemiologist. additional organism identification is often required, utilizing antibiogram patterns or molecular fingerprinting by various techniques to correlate a bacterial isolate from breast milk with an isolate causing disease in infant or mother. misadministration of breast milk, also known as misappropriation, breast milk exposure, and accidental ingestion of breast milk, and other terms, is a medical-legal issue when it occurs in a hospital. this scenario occurs when one infant receives breast milk from another mother by mistake. this occurrence can be very distressing to the families (recipient patient, recipient parent, and donor mother) and medical staff involved. the actual risk for transmission of an infectious agent to an infant via a single ingestion of expressed breast milk (the most common occurrence) from another mother is exceedingly low. in this scenario, the cdc recommends treating this as an accidental exposure to a body fluid, which could be infectious. 84 bacterial, fungal, or parasitic infection from the one exposure is highly unlikely. the concern is about viral pathogens, known to be blood-borne pathogens, which have been identified in breast milk and include but are not limited to hepatitis b virus (hbv), hepatitis c virus (hcv), cytomegalovirus (cmv), west nile virus, human t-cell lymphotropic virus (htlv), and hiv. most hospitals have protocols for managing the situation from both the infection control/prevention and the medical-legal perspectives. these protocols advise informing both families about what occurred, discussing the theoretical risks of harm from the exposure, and reviewing test results and/ or recommending testing to determine the infectious status of each mother relative to the above mentioned viruses. hcv is not a contraindication to breastfeeding and west nile virus infection in lactating women is rare. 74, 177 neither infection has a documented effective form of prevention or acute treatment. testing either mother (donor or of recipient infant) for these agents is not warranted. prenatal testing for hiv is more commonplace throughout the world. the incidence of hiv among women of childbearing age is low, although it varies significantly by geographic location, and the hospital or locale-specific incidence would be important to know to estimate risk. most women and medical staff are aware that hiv can be transmitted by breastfeeding; therefore breast milk from hiv-positive women is rarely if ever stored in hospitals. the risk for transmission of hiv via breastfeeding is due to the volume of feedings over months (estimated at 400 to 500 feedings in the first 2 months of life) compared with the small "dose of exposure" from one or two "accidental feedings." transmission of hiv from a single breast milk exposure has never been documented. immunologic components in breast milk, along with time and cold of storage, inactivate the hiv in expressed breast milk. for these reasons, the risk for transmission of hiv via expressed breast milk consumed by another child is thought to be extremely low. htlv-i/ii infection in childbearing women is uncommon except in certain geographic regions (japan, africa, the caribbean, and south america). transmission of htlv via breast milk does occur and, like hiv, appears to be related to the volume and duration of breastfeeding. limiting the duration of breastfeeding is effective in decreasing transmission. 407, 409, 446 freezing and thawing expressed breast milk decreases the infectivity of htlv-i. 11 in areas of low prevalence, a positive test in a mother should be suspected to be a false positive test, and retesting with both antibody and polymerase chain reaction (pcr) testing should be performed. for these reasons the transmission of htlv-i/ii via accidental expressed breast milk exposure is thought to be extremely low. although the majority of women are cmv positive by childbearing age and cmv transmission occurs via breastfeeding, the risk for cmv in a full-term infant is low. premature or lbw infants are at greater risk for developing disease with cmv infection. freezing expressed breast milk (at −20° c) for 3 to 5 days significantly decreases the infectivity of cmv. here again the risk for cmv transmission from a single accidental exposure to cmv-positive expressed breast milk is extremely low. with a discussion of theoretical risk should be a discussion of possible preventive interventions, such as vaccination or antimicrobial postexposure prophylaxis. if donor mothers are positive for hbv, it is appropriate to give recipient infants hepatitis b virus immunoglobulin (hbig) and hbv vaccines if they have not already received them. if a box 13-1. culturing breast milk 1. wash hands as per routine. 2. wash breast with warm tap water and a clean washcloth. 3. manually express breast milk ("midstream" collection is not required) or attach breast pump flange (previously cleaned as per routine) for collection and collect milk. 4. place a 3 to 5 ml sample of expressed breast milk in a sterile container with a nonleakable top. 5. deliver to the labatory in less than 1 hour or refrigerate at 4° c until delivery. before sending samples to the viral lab or for nucleic acid/ po lymerase chain reaction (pcr) testing, confirm that the laboratory will accept and process the sample as requested and that the appropriate collection container and prelaboratory management of the specimen are utilized. 6 324 it may also be appropriate to consult a pediatric infectious disease specialist. additional important components of the hospital-based protocols for managing accidental expressed breast milk exposure include ongoing psychosocial support for the families and staff, documentation of medical discussions with the families, investigative steps, consents and interventions, and the demonstration of ongoing infection control efforts to prevent additional events of misadministration of breast milk. microorganisms produce a whole spectrum of clinical illnesses affecting mothers and infants. many situations carry the risk for transmission of the involved organism from a mother to the infant, or vice versa; in general, however, infants are at greater risk because of such factors as inoculum size and immature immune response. as always, an infection must be accurately diagnosed in a timely manner. empiric therapy and initial infection control precautions should begin promptly based on the clinical symptoms and the most likely etiologic agents. when dealing with a maternal infection, clarifying the possible modes of transmission and estimating the relative risk for transmission to the infant are essential first steps to decision-making about isolating a mother from her infant and the appropriateness of continuing breastfeeding or providing expressed breast milk. breastfeeding infrequently is contraindicated in specific maternal infections. 243 often the question of isolation and interruption of breastfeeding arises when symptoms of fever, pain, inflammation, or other manifestations of illness first develop in a mother and the diagnosis is still in doubt. a clinical judgment must be made based on the site of infection, probable organisms involved, possible or actual mechanisms of transmission of these organisms to the infant, estimated virulence of the organism, and likely susceptibility of the infant. additionally, by the time the illness is clearly recognized or diagnosed in a mother, the infant has already been exposed. given the dynamic nature of the immunologic benefits of breast milk, continuation of breastfeeding at the time of diagnosis or illness in a mother can provide the infant protection rather than continued exposure in most illnesses. stopping breastfeeding is rarely necessary. many situations associated with maternal fever do not require separation of mother and infant, such as engorgement of the breasts, atelectasis, localized nonsuppurative phlebitis, or urinary tract infections. appendix f lists a number of clinical syndromes, conditions, and organisms that require infection control precautions in hospitals. this appendix also includes short lists of possible etiologic agents for these conditions and appropriate precautions and recommendations concerning breastfeeding for different scenarios or organisms. this chapter considers specific infectious agents that are common, clinically significant, or of particular interest. bacillus anthracis, a gram-positive, spore-forming rod, causes zoonotic disease worldwide. human infection typically occurs due to contact with animals or their products. three forms of human disease occur: cutaneous anthrax (the most common), inhalation anthrax, and gastrointestinal (gi) disease (rare). person-to-person transmission can occur as a result of discharge from cutaneous lesions, but no evidence of human-to-human transmission of inhalational anthrax is available. no evidence of transmission of anthrax via breast milk exists. standard contact isolation is appropriate for hospitalized patients or patients with draining skin lesions. the issue of anthrax as a biologic weapon has exaggerated its importance as a cause of human disease. the primary concerns regarding anthrax and breastfeeding are antimicrobial therapy or prophylaxis in breastfeeding mothers and the possibility that infant and mother were exposed by intentional aerosolization of anthrax spores. the cdc published recommendations for treatment and prophylaxis in infants, children, and breastfeeding mothers. 72 the recommendations include the use of ciprofloxacin, doxycycline, amoxicillin, and several other agents without discontinuing breastfeeding. little available is information on ciprofloxacin and doxycycline in breast milk for prolonged periods of therapy or prophylaxis (60 days) and possible effects on infants' teeth and bone/cartilage growth during that time period. depending on the clinical situation and sensitivity testing of the identified anthrax strain, other agents can be substituted to complete the 60-day course. the cdc has approved the use of ciprofloxacin and doxycycline for breastfeeding women for short courses of therapy (less than several weeks). simultaneous exposure of infant and mother could occur from primary aerosolization or from spores "contaminating" the local environment. in either case decontamination of the mother-infant dyad' s environment should be considered. breastfeeding can continue during a mother' s therapy for anthrax as long as she is physically well. open cutaneous lesions should be carefully covered and, depending on the situation, simultaneous prophylaxis for the infant may be appropriate. considerable justifiable concern has been expressed because of the reports of sudden infant death from botulism. infant botulism is distinguished from food-borne botulism from improperly preserved food containing the toxin and from wound botulism from spores entering the wound. infant botulism occurs when the spores of clostridium botulinum germinate and multiply in the gut and produce the botulinal toxin in the gi tract. 17 the toxin binds presynaptically at the neuromuscular junction, preventing acetylcholine release. the clinical picture is a descending, symmetric flaccid paralysis. not every individual who has c. botulinum identified in the stool experiences a clinical illness. the age of infants seems to relate to their susceptibility to illness. the illness is mainly in children younger than 12 months of age; the youngest patient described in the literature was 6 days old. 17 most children become ill between 6 weeks and 6 months of age. the onset of illness seems to occur earlier in formula-fed infants compared with breastfed infants. when a previously healthy infant younger than 6 months of age develops constipation, then weakness and difficulty sucking, swallowing, crying, or breathing, botulism is a likely diagnosis. the organisms should be looked for in the stools, and electromyography may or may not be helpful. in a group reviewed by arnon et al, 19 33 of 50 patients hospitalized in california were still being nursed at onset of the illness. a beneficial effect of human milk was observed in the difference in the mean age at onset, with breastfed infants being twice as old as formula-fed infants with the disease. the breastfed infants' symptoms were milder. breastfed infants receiving iron supplements developed the disease earlier than those who were breastfed but unsupplemented. of the cases of sudden infant death from botulism, no infants were breastfed within 10 weeks of death. all were receiving iron-fortified formulas. in most cases, no specific food source of c. botulinum can be identified, but honey is the food most often implicated, and corn syrup has been implicated in infants older than 2 months of age. honey may contain botulism spores, which can germinate in the infant gut. however, botulin toxin has not been identified in honey. it has been recommended that honey not be given to infants younger than 12 months of age. this includes putting honey on a mother' s nipples to initiate an infant' s interest in suckling. arnon 18 reviewed the first 10 years of infant botulism monitoring worldwide. the disease has been reported from 41 of the 50 states in the united states and from eight countries on four continents. the relationship to breastfeeding and human milk is unclear. in general the acid stools (ph 5.1 to 5.4) of human milk fed infants encourage bifidobacterium species. few facultative anaerobic bacteria, or clostridia, existing as spores, are present in breastfed infants. in contrast, formula-fed infants have stool phs ranging from 5.9 to 8.0, with few bifidobacteria, primarily gram-negative bacteria, especially coliforms and bacteroides species. c. botulinum growth and toxin production decrease with declining ph and usually stops below ph 4.6. breast milk also contains additional protective immunologic components, which purportedly have activity against botulinum toxin. 269 the relationship between the introduction of solid foods or weaning in both formula-fed and breastfed infants and the onset of botulism remains unclear. for a breastfed infant, the introduction of solid food may cause a major change in the gut with a rapid rise in the growth of enterobacteria and enterococci followed by progressive colonization by bacteroides species, clostridia, and anaerobic streptococci. feeding solids to formula-fed infants minimally changes the gut flora as these organisms already predominate. although more hospitalized infants have been breastfed, sudden-death victims are younger and have been formula fed, which supports the concept of immunologic protection in the gut of a breastfed infant. much work remains to understand this disease. clinically, constipation, weakness, and hypotonicity in a previously healthy child constitute botulism until ruled out, especially with recent dietary changes. at this time, no reason exists to suspect breastfeeding as a risk for infant botulism, and some evidence suggests a possible protective effect from breastfeeding. breastfeeding should continue if botulism is suspected in mother or infant. brucella melitensis has been isolated in the milk of animals. foods and animals represent the primary sources of infection in humans. brucellosis demonstrates a broad spectrum of illness in humans, from subclinical to subacute to chronic illness with nonspecific signs of weakness, fever, malaise, body aches, fatigue, sweats, arthralgia, and lymphadenitis. in areas where the disease is enzootic, childhood illness has been described more frequently. the clinical manifestations in children are similar to those in adults. 259 infection can occur during pregnancy, leading to abortion (infrequently), and can produce transplacental spread, causing neonatal infection (rarely). the transmission of b. melitensis through breast milk has been implicated in neonatal infection. 259, 260 there have been eight cases of brucellosis in infants that were possibly associated with breastfeeding, but brucella was not isolated from the breast milk in any of those cases.* one case of brucellosis in an infant caused by breast milk transmission, with b. melitensis isolated from the breast milk, before antibiotic treatment was given to the mother has been documented. 415 additionally, brucella melitensis has been cultured from women with breast lumps and abscesses. 295 only one of six women described in this report was lactating at the time of diagnosis, and no information about the infant was given. brucellosis mastitis or abscess should be considered in women presenting with appropriate symptoms and occupational exposure to animals, contact with domestic animals in their environment, or exposure to animal milk or milk products (especially unpasteurized products). the breast inflammation tends to be granulomatous in nature (without caseation) and is often associated with axillary adenopathy; occasionally systemic illness in the woman is evident. treatment of brucellosis mastitis or abscess should be treated with surgery or fine needle aspiration as indicated and 4 to 6 weeks of combination antibiotic therapy with two or three medications. temporary interruption of breastfeeding with breast pumping and discarding the milk to continue stimulation of milk production is appropriate. breastfeeding should then continue after an initial period of 48 to 96 hours of therapy in the mother. acceptable medications for treating the mother while continuing breastfeeding include gentamicin, streptomycin, tetracycline, doxycycline, trimethoprim-sulfamethoxazole, and rifampin (see appendix d). chlamydial infection is the most frequent sexually transmitted disease (std) in the united states and is a frequent cause of conjunctivitis and pneumonitis in an infant from perinatal infection. the major determinant of whether chlamydial infection occurs in a newborn is the prevalence rate of chlamydial infection of the cervix. 364 chlamydial immunoglobulin a (iga) has been found in colostrum and breast milk in a small number of postpartum women who were seropositive for chlamydia. no information is available on the role of milk antibodies in protection against infection in infants. 389 it is not believed that chlamydia is transmitted via breast milk. use of erythromycin or tetracycline to treat mothers and oral erythromycin and ophthalmic preparations of tetracyclines, erythromycin, or sulfonamides to treat suspected infection in infants are appropriate during continued breastfeeding. separating infants from mothers with chlamydial infections or stopping breastfeeding is not indicated. simultaneous treatment of mothers and infants may be appropriate in some situations. corynebacterium diphtheriae causes several forms of clinical disease, including membranous nasopharyngitis, obstructive laryngotracheitis, and cutaneous infection. complications can include airway obstruction from membrane formation and toxinmediated central nervous system (cns) disease or myocarditis. the overall incidence of diphtheria has declined even though immunization does not prevent infection but does prevent severe disease from toxin production. fewer than five cases are reported annually in the united states. transmission occurs via droplets or direct contact with contaminated secretions from the nose, throat, eye, or skin. infection occurs in individuals whether they have been immunized or not, but infection in those not immunized is more severe and prolonged. as long as the skin of the breast is not involved, no risk for transmission exists via breast milk. no toxin-mediated disease from toxin transmitted through breast milk has been reported in an infant. breastfeeding, along with chemoprophylaxis and immunization of affected infants, is appropriate in the absence of cutaneous breast involvement (see appendix f). maternal infection with neisseria gonorrhoeae can produce a large spectrum of illness ranging from uncomplicated vulvovaginitis, proctitis, pharyngitis, conjunctivitis, or more severe and invasive disease, including pelvic inflammatory disease, meningitis, endocarditis, or disseminated gonococcal infection. the risk for transmission from mother to infant occurs mainly during delivery in the passage through the infected birth canal and occasionally from postpartum contact with the mother (or her partner). risk for transmission from breast milk is negligible, and n. gonorrhoeae does not seem to cause local infection of the breasts. infection in neonates is most often ophthalmia neonatorum and less often a scalp abscess or disseminated infection. mothers with presumed or documented gonorrhea should be reevaluated for other stds, especially chlamydia trachomatis and syphilis, because some therapies for gonorrhea are not adequate for either of these infections. with the definitive identification of gonorrhea in a mother, empiric therapy should begin immediately, and the mother should be separated from the infant until completion of 24 hours of adequate therapy. treatment of the mother with ceftriaxone, cefixime, penicillin, or erythromycin is without significant risk to the infant. single-dose treatment with spectinomycin, ciprofloxacin, ofloxacin, or azithromycin has not been adequately studied but presumably would be safe for the infant given the 24-hour separation and a delay in breastfeeding without giving the infant the expressed breast milk (pump and discard). doxycycline use in a nursing mother is not routinely recommended. careful preventive therapy for ophthalmia neonatorum should be provided, and close observation of the infant should continue for 2 to 7 days, the usual incubation period. empiric or definitive therapy against n. gonorrhoeae may be necessary depending on an infant' s clinical status and should be chosen on the basis of the maternal isolate' s sensitivity pattern. the mother should not handle other infants until after 24 hours of adequate therapy, and the infant should be separated from the rest of the nursery population, with or without breastfeeding. haemophilus influenzae type b can cause severe invasive disease such as meningitis, sinusitis, pneumonia, epiglottitis, septic arthritis, pericarditis, and bacteremia. shock can also occur. because the increased utilization of the h. influenzae type b conjugate vaccines, invasive disease caused by haemophilus has decreased dramatically, more than 95%, in the united states. most invasive disease occurs in children 3 months to 3 years of age. older children and adults rarely experience severe disease but do serve as sources of infection for young children. children younger than 3 months of age seem to be protected because of passively acquired antibodies from the mothers, and some additional benefits may be received from breast milk. transmission occurs through contact with respiratory secretions, and droplet precautions are protective. no evidence suggests transmission through breast milk or breastfeeding. evidence supports that breast milk limits the colonization of h. influenzae in the throat. 185 in the rare case of maternal infection, an inadequately immunized infant in a household is an indication to provide rifampin prophylaxis and close observation for all household contacts, including the breastfeeding infant. expressed breast milk can be given to an infant during the 24-hour separation after the mother' s initiation of antimicrobial therapy, or if the mother' s illness prevents breastfeeding, it can be reinitiated when the mother is able (see appendix f). although uncommon in the united states, leprosy occurs throughout the world. this chronic disease presents with a spectrum of symptoms depending on the tissues involved (typically the skin, peripheral nerves, and mucous membranes of the upper respiratory tract) and the cellular immune response to the causative organism, mycobacterium leprae. transmission occurs through long-term contact with individuals with untreated or multibacillary (large numbers of organisms in the tissues) disease. leprosy is not a contraindication to breastfeeding, according to jeliffe and jeliffe. 202 the importance of breastfeeding and urgency of treatment are recognized by experts who treat infants and mothers early and simultaneously. no mother-infant contact is permitted except to breastfeed. dapsone, rifampin, and clofazimine are typically and safely used for infant and mother regardless of the method of feeding (see appendix d). listeriosis is a relatively uncommon infection that can have a broad range of manifestations. in immunocompetent individuals, including pregnant women, the infection can vary from being asymptomatic to presenting as an influenza-like illness, occasionally with gi symptoms or back pain. severe disease occurs more frequently in immunodeficient individuals or infants infected in the perinatal period (pneumonia, sepsis, meningitis, granulomatosis infantisepticum). although listeriosis during pregnancy may manifest as mild disease in a mother and is often difficult to recognize and diagnose, it is typically associated with stillbirth, abortion, and premature delivery. it is thought that transmission occurs through the transplacental hematogenous route, infecting the amniotic fluid, although ascending infection from the genital tract may occur. 122 early and effective treatment of a woman can prevent fetal infection and sequelae. 206, 257 neonatal infection occurs as either early-or late-onset infection from transplacental spread late in pregnancy, ascending infection during labor and delivery, infection during passage through the birth canal, or, rarely, during postnatal exposure. no evidence in the literature suggests that listeria is transmitted through breast milk. treatment of the mother with ampicillin, penicillin, or trimethoprim-sulfamethoxazole is not a contraindication to breastfeeding as long as the mother is well enough. expressed colostrum or breast milk also can be given if the infant is able to feed orally. the management of lactation and feeding in neonatal listeriosis is conducted supportively, as it is in any situation in which an infant is extremely ill, beginning feeding with expressed breast milk or directly breastfeeding as soon as reasonable. n. meningitidis most often causes severe invasive infections, including meningococcemia or meningitis often associated with fever and a rash and progressing to purpura, disseminated intravascular coagulation, shock, coma, and death. transmission occurs via respiratory droplets. spread can occur from an infected, ill individual or from an asymptomatic carrier. droplet precautions are recommended until 24 hours after initiation of effective therapy. despite the frequent occurrence of bacteremia, no evidence indicates breast involvement or transmission through breast milk. the risk for maternal infection to an infant after birth is from droplet exposure and exists whether the infant is breastfeeding or bottle feeding. in either case the exposed infant should receive chemoprophylaxis with rifampin, 10 mg/kg/dose every 12 hours for 2 days (5 mg/kg/dose for infants younger than 1 month of age), or ceftriaxone, 125 mg intramuscularly (im) once, for children younger than 15 years of age. close observation of the infant should continue for 7 days, and breastfeeding during and after prophylaxis is appropriate. the severity of maternal illness may prevent breastfeeding, but it can continue if the mother is able, after the mother and infant have been receiving antibiotics for 24 hours. a period of separation from the index case for the first 24 hours of effective therapy is recommended; expressed breast milk can be given during this period. respiratory illness caused by bordetella pertussis evolves in three stages: catarrhal (nasal discharge, congestion, increasing cough), paroxysmal (severe paroxysms of cough sometimes ending in an inspiratory whoop, i.e., whooping cough), and convalescent (gradual improvement in symptoms). transmission is via respiratory droplets. the greatest risk for transmission occurs in the catarrhal phase, often before the diagnosis of pertussis. the nasopharyngeal culture usually becomes negative after 5 days of antibiotic therapy. chemoprophylaxis for all household contacts is routinely recommended. no evidence indicates transmission through breast milk, with similar risk to breastfed and bottle-fed infants. in the case of maternal infection with pertussis, chemoprophylaxis for all household contacts, regardless of age or immunization status, is indicated. in addition to chemoprophylaxis of the infant, close observation and subsequent immunization (in infants older than 6 weeks of age) are appropriate. despite chemoprophylaxis, droplet precautions and separation of mother and infant during the first 5 days of effective maternal antibiotic therapy are recommended. expressed breast milk can be provided to the infant during this period. staphylococcal infection in neonates can be caused by either s. aureus or coagulase-negative staphylococci (most often s. epidermidis) and can manifest in a wide range of illnesses. localized infection can be impetigo, pustulosis in neonates, cellulitis, or wound infection, and invasive or suppurative disease includes sepsis, pneumonia, osteomyelitis, arthritis, and endocarditis. s. aureus requires only a small inoculum (10 to 250 organisms) to produce colonization in newborns, most often of the nasal mucosa and umbilicus. 193 by the fifth day of life, 40% to 90% of the infants in the nursery will be colonized with s. aureus. 126 the organism is easily transmitted to others from mother, infant, family, or health care personnel through direct contact. outbreaks in nurseries were common in the past. mothers, infants, health care workers, and even contaminated, unpasteurized, banked breast milk were sources of infection. 298, 326 careful use of antibiotics, changes in nursery layout and procedures, standard precautions, and cohorting as needed decreased the spread of s. aureus in nurseries. now the occurrence of methicillin-resistant s. aureus (mrsa) is again a common problem, requiring cohorting, occasionally epidemiologic investigation, and careful infection control intervention. there are numerous reports of mrsa outbreaks in nicus.* the significance of colonization with staphylococcus and the factors leading to development of disease in individual patients are not clear. the morbidity and mortality related to s. aureus infection in neonates is well described. 192, 195, 219 management of such outbreaks has been reviewed. 147, 250 little has been written about the role of breastfeeding in colonization with s. aureus in nicus, wellbaby nurseries, or at home. mrsa is an important pathogen worldwide. community-acquired mrsa is different from hospital-acquired mrsa. community-acquired mrsa is usually defined as occurring in an individual without the common predisposing variables associated with hospital-acquired mrsa, lacking a mdr phenotype (common with hospital-acquired mrsa), frequently carrying multiple exotoxin virulence factors (such as panton-valentine leukocidin toxin), as well as carrying the smaller type iv staphylococcal cassette cartridge for the meca gene on a chromosome (hospital-acquired mrsa carries types i-iii staphylococcal cassette cartridge) and as being molecularly distinct from the common nosocomial strains of hospital-acquired mrsa. community-acquired mrsa is most commonly associated with skin and soft tissue infections and necrotizing pneumonia and less frequently associated with endocarditis, bacteremia, necrotizing fasciitis, myositis, osteomyelitis, or parapneumonic effusions. community-acquired mrsa is so common, it is now being observed in hospital outbreaks. 24, 144, 164, 358 community-acquired mrsa transmission to infants via breast milk has been reported. 34, 144, 210, 253, 286 premature or small-forgestational-age infants are more susceptible to and at increased risk for significant morbidity and mortality due to mrsa due in part to prolonged hospitalization, multiple courses of antibiotics, invasive procedures, and intravenous (iv) lines, their relative immune deficiency due to prematurity and illness, and altered gi tract due to different flora and decreased gastric acidity. therefore colonization with mrsa may pose a greater risk to infants in nicus in the long run. full-term infants develop pustulosis, cellulitis, and soft tissue infections, but rarely has invasive disease been reported. 82, 132, 298 fortunov et al 132 from texas reported 126 infections in term or late-preterm previously well infants including 43 with pustulosis, 68 with celluliltis or abscesses, and 15 invasive infections. family history of soft tissue skin infections and male sex were the only variables associated with risk for infection; cesarean delivery, breastfeeding, and circumcision were not. 132 nguyen et al 298 reported mrsa infections in a well-infant nursery from california. the eleven cases were all in full-term boys with pustularvesicular lesions in the groin. the infections were associated with longer length of stay, lidocaine injection use in infants, maternal age older than 30 years, and circumcision. breastfeeding was not an associated risk factor for mrsa infection. 298 the question of the role of circumcision in mrsa outbreaks was addressed by van howe and robson. 426 they reported that circumcised boys are at greater risk for staphylococcal colonization and infection. 426 others report that s. aureus carriage in infants (and subsequent infection) is most likely affected by multiple variables including infant factors (antibiotics, surgical procedures [circumcision being the most common], duration of hospital stay as a newborn), maternal factors (previous colonization, previous antibiotic usage, mode of delivery, length of stay), and environmental factors (mrsa in the family or hospital, nursery stay versus rooming-in, hand hygiene).* gerber et al 147 from the chicago area published a consensus statement for the management of mrsa outbreaks in the nicu. the recommendations, which were strongly supported by experimental, clinical, and epidemiologic data, included using a waterless, alcohol-based hand hygiene product, monitoring and enforcing hand hygiene, placing mrsa-positive infants in contact precautions with cohorting if possible, using gloves and gowns for direct contact and masks for aerosolgenerating procedures, cohorting nurses for care of mrsa-positive infants when possible, periodic screening of infants for mrsa using nares or nasopharyngeal cultures, clarifying the mrsa status of infants being transferred into the nicu, limiting overcrowding, and maintaining ongoing instruction and monitoring of health care workers in their compliance with infection control and hand hygiene procedures. evaluation of the outbreak could include screening of health care workers and environmental surfaces to corroborate epidemiologic data and laboratory molecular analysis of the mrsa strains if indicated epidemiologically. the use of mupirocin or other decolonizing procedures should be determined on an individual basis for each nicu. s. aureus is the most common cause of mastitis in lactating women. 317, 394, 395, 436 recurrence or persistence of symptoms of mastitis is a well described occurrence and an important issue in the management of mastitis. communityacquired mrsa has been associated with mastitis as well. 342 pasteurization, s. aureus was not detected in any of the 6820 samples of expressed breast milk. colonization of one infant with mrsa was identified, but no mrsa infections were identified in any of the hospitalized infants in the nicu during the 18 months of the study. 26 novak et al 300 identified mrsa in 57 of 500 samples (11%) of expressed fresh-frozen milk from 500 different donors from five brazilian milk banks. only 3 of the 57 samples were positive with high-level bacterial counts of mrsa: greater than 10,000 cfu/ml. these were the only samples that would not have been acceptable by bacteriological criteria according to brazilian or american criteria for raw milk use. they did not investigate other epidemiologic data to identify possible variables associated with low or high level contamination of expressed breast milk with mrsa. 300 management of an infant and/or mother with mrsa infection relative to breastfeeding or use of breast milk should be based on the severity of disease and whether the infant is premature, lbw, very-lowbirth-weight (vlbw), previously ill, or full term. full-term infants who themselves or their mothers develop mild to moderate infections (impetigo, pustulosis, cellulitis/abscess, mastitis/breast abscess, or soft tissue infection) can continue breast feeding after a short period of interruption (24 to 48 hours). during this time, pumping to maintain the milk supply should be supported, an initial evaluation for other evidence of infection should be done in the maternalinfant dyad, the infected child and/or mother should be placed on "commonly" effective therapy for the mrsa infection, and ongoing observation for clinical disease should continue. the mother and infant can "room-in" together in the hospital, if necessary, with standard and contact precautions. culturing the breast milk is not necessary. empiric therapy for the infant may be chosen based on medical concerns for the infant and the known sensitivity testing of the mrsa isolate. appropriate antibiotic choices include short-term use of azithromycin (erythromycin use during infancy [less than 6 weeks of age], or breastfeeding associated with an increased risk for hypertrophic pyloric stenosis), sulfamethoxazoletrimethoprim (in the absence of g6pd deficiency and older than 30 days of age), clindamycin, and perhaps linezolid for mild to moderate infections. infants in nicus (premature, lbw, vlbw, and/ or previously ill), who themselves or their mothers have a mrsa infection, should have the breast milk cultured and suspend breastfeeding or receiving breast milk from their mother until the breast milk is shown to be culture negative for mrsa. the infant should be treated as indicated for their infection or empirically treated if symptomatic (with pending culture results) and closely observed for development of new signs or symptoms of infection. pumping to maintain the milk supply and the use of banked breast milk are appropriate. the infant should be placed on contact precautions, in addition to the routine standard precautions. the infant can be cohorted with other mrsa-positive infants with nursing care cohorted as well. for the mother with mrsa infection, she should be instructed concerning hand hygiene, the careful collection, handling, and storage of breast milk, contact precautions to be used with her infant, and the avoidance of contact with any other infants. the mother can receive several possible antibiotics for mrsa that are compatible with breastfeeding when used for a short period. if the mother remains clinically well, including without evidence of mastitis, but her breast milk is positive for mrsa greater than 10 4 cfu/ml, empiric therapy to diminish or eradicate colonization would be appropriate. various regimens have been proposed to "eradicate" mrsa colonization, but none have been proven to be highly efficacious. these regimens usually include systemic antibiotics with one or two medications (rifampin added as the second medication), nasal mupirocin to the nares twice daily for 1 to 2 weeks with routine hygiene, with or without the usage of hexachlorophene (or similar topical agent or cleanser) for bathing during the 1 to 2 week treatment period. there is no clear information concerning the efficacy of using similar colonization eradication regimens for other household members or pets in preventing recolonization of the mother or infant. before reintroducing the use of the mother' s breast milk to the infant at least two to three negative breast milk cultures should be obtained after completion of therapy. routine screening of breast milk provided by mothers for their infants in nicus for the presence of mrsa is not indicated in the absence of mrsa illness in the maternal-infant dyad, an mrsa outbreak in nicus, or a high frequency of mrsa infection in a specific nicu. one case of staphylococcal scalded skin syndrome was reported by katzman and wald 208 in an infant breastfed by a mother with a lesion on her areola that did not respond to ampicillin therapy for 14 days. subsequently the infant developed conjunctivitis with s. aureus, which produced an exfoliative toxin, and a confluent erythematous rash without mucous membrane involvement or nikolsky sign. no attempt to identify the exfoliative toxin in the breast milk was made, and the breast milk was not cultured for s. aureus. the child responded to iv therapy with nafcillin. this emphasizes the importance of evaluating mother and infant at the time of a suspected infection and the need for continued observation of the infant for evidence of a pyogenic infection or toxin-mediated disease, especially with maternal mastitis or breast lesions. this case also raises the issue of when and how infants and their mothers become colonized with s. aureus and what factors lead to infection and illness in each. the concern is that staphylococcus can be easily transmitted through skin to skin contact, colonization readily occurs, and potentially serious illness can occur later, long after colonization. in the case of staphylococcal scalded skin syndrome or toxic shock syndrome (tss), the primary site of infection can be insignificant (e.g., conjunctivitis, infection of a circumcision, or simple pustulosis), but a clinically significant amount of toxin can be produced and lead to serious disease. toxic shock syndrome can result from s. aureus or streptococcus pyogenes infection and probably from a variety of antigens produced by other organisms. tss-1 has been identified as a "superantigen" that affects the t lymphocytes and other components of the immune response, producing an unregulated and excessive immune response and resulting in an overwhelming systemic clinical response. tss has been reported in association with vaginal delivery, cesarean delivery, mastitis, and other local infections in mothers. mortality rate in the mother may be as high as 5%. the case definition of staphylococcal tss includes meeting all four major criteria: fever greater than 38.9° c, rash (diffuse macular erythroderma), hypotension, and desquamation (associated with subepidermal separation seen on skin biopsy). the definition also includes involvement of three or more organ systems (gi, muscular, mucous membrane, renal, hepatic, hematologic, or central nervous system); negative titers for rocky mountain spotted fever, leptospirosis, and rubeola; and lack of isolation of s. pyogenes from any source or s. aureus from the cerebrospinal fluid (csf). 368 a similar case definition has been proposed for streptococcal tss. 451 aggressive empiric antibiotic therapy against staphylococci and streptococci and careful supportive therapy are essential to decreasing illness and death. oxacillin, nafcillin, first-generation cephalosporins, clindamycin, erythromycin, and vancomycin are acceptable antibiotics, even for a breastfeeding mother. the severity of illness in the mother may preclude breastfeeding, but it can be reinitiated when the mother is improving and wants to restart. standard precautions, but allowing breastfeeding, are recommended. staphylococcal enterotoxin f has been identified in breast milk specimens collected on days 5, 8, and 11 from a mother who developed tss at 22 hours postpartum. 428 s. aureus that produced staphylococcal enterotoxin f was isolated from the mother' s vagina but not from breast milk. infant and mother lacked significant antibody against staphylococcal enterotoxin f in their sera. the infant remained healthy after 60 days of follow-up. staphylococcal enterotoxin f is pepsin inactivated at ph 4.5 and therefore is probably destroyed in the stomach environment, presenting little or no risk to the breastfeeding infant. 35 breastfeeding can continue if the mother is able. coagulase-negative staphylococcal infection (the predominant isolate is staphylococcus epidermidis) produces minimal disease in healthy, full-term infants but is a significant problem in hospitalized or premature infants. factors associated with increased risk for this infection include prematurity, high colonization rates in specific nurseries, invasive therapies (e.g., iv lines, chest tubes, intubation), and antibiotic use. illness produced by coagulasenegative staphylococci can be invasive and severe in high-risk neonates, but rarely in mothers. there are reports of necrotizing enterocolitis associated with coagulase-negative staphylococcus. at 2 weeks of age, for infants still in the nursery, s. epidermidis is a frequent colonizing organism at multiple sites, with colonization rates as high as 75% to 100%. serious infections with coagulase-negative staphylococci (e.g., abscesses, iv line infection, bacteremia/sepsis, endocarditis, osteomyelitis) require effective iv therapy. many strains are resistant to penicillin and the semisynthetic penicillins, so sensitivity testing is essential. empiric or definitive therapy may require treatment with vancomycin, gentamicin, rifampin, teicoplanin, linezolid, or combinations of these for synergistic activity. transmission of infection in association with breastfeeding appears to be no more common than with bottle feeding. as with s. aureus infection control includes contact and standard precautions. occasionally, during presumed outbreaks, careful epidemiologic surveillance may be required, including cohorting, limiting overcrowding and understaffing, surveillance cultures of infants and nursery personnel, reemphasis of meticulous infection control techniques for all individuals entering the nursery, and, rarely, removal of colonized personnel from direct infant contact. s. epidermidis has been identified as part of fecal microbiota of breastfed infants. 203 s. epidermidis has also been identified in the breast milk of women with clinical evidence of mastitis. 107 nevertheless, s. epidermidis is rarely associated with infection in full-term infants. conceivably breast milk for premature infants could be a source of s. epidermidis colonization in the nicus. the other factors associated with hospitalization in a nicu noted previously presumably play a significant role in both colonization and infection in premature infants. the benefits of early full human milk feeding potentially outweigh the risk for colonization with s. epidermidis via breast milk. 348 ongoing education and assistance should be provided to mothers about the careful collection, storage, and delivery of human breast milk for their premature infants. 353 s. pyogenes (β-hemolytic group a streptococcus [gas]) is a common cause of skin and throat infections in children, producing pharyngitis, cellulitis, and impetigo. illnesses produced by gas can be classified in three categories: (1) impetigo, cellulitis, or pharyngitis without invasion or complication; (2) severe invasive infection with bacteremia, necrotizing fasciitis, myositis, or systemic illness (e.g., streptococcal tss); and (3) autoimmune-mediated phenomena, including acute rheumatic fever and acute glomerulonephritis. gas can also cause puerperal sepsis, endometritis, and neonatal omphalitis. significant morbidity and mortality rates are associated with invasive gas infection; mortality rate is 20% to 50%, with almost half the survivors requiring extensive tissue débridement or amputation. 347 infants are not at risk for the autoimmune sequelae of gas (rheumatic fever or poststreptococcal glomerulonephritis). transmission is through direct contact (rarely indirect contact) and droplet spread. outbreaks of gas in the nursery are rare, unlike with staphylococcal infections. either mother or infant can be initially colonized with gas and transmit it to the other. in the situation of maternal illness (extensive cellulitis, necrotizing fasciitis, myositis, pneumonia, tss, mastitis), it is appropriate to separate mother and infant until effective therapy (penicillin, ampicillin, cephalosporins, erythromycin) has been given for at least 24 hours. breastfeeding should also be suspended and may resume after 24 hours of therapy for the mother. group b streptococcus (gbs, streptococcus agalactiae) is a significant cause of perinatal bacterial infection. in parturient women, infection can lead to asymptomatic bacteriuria, urinary tract infection (often associated with premature birth), endometritis, or amnionitis. in infants, infection usually occurs between birth and 3 months of age (1 to 4 cases per 1000 live births). it is routinely classified by the time of onset of illness in the infant: early onset (0 to 7 days, majority less than 24 hours) and late onset (7 to 90 days, generally less than 4 weeks). infants may develop sepsis, pneumonia, meningitis, osteomyelitis, arthritis, or cellulitis. early-onset gbs disease is often fulminant, presenting as sepsis or pneumonia with respiratory failure; three quarters of neonatal disease is early onset. type iii is the most common serotype causing disease. transmission is believed to occur in utero and during delivery. colonization rates of mothers and infants vary between 5% and 35%. postpartum transmission is thought to be uncommon, although it has been documented. risk factors for early-onset gbs disease include delivery before 37 weeks' gestation, rupture of membranes for longer than 18 hours before delivery, intrapartum fever, heavy maternal colonization with gbs, or low concentrations of anti-gbs capsular antibody in maternal sera. 95 the common occurrence of severe gbs disease before 24 hours of age in neonates has lead to prevention strategies. revised guidelines developed by the aap committees on infectious diseases and on the fetus and newborn 95 have tried to combine various variables for increased risk for gbs infection (prenatal colonization with gbs, obstetric and neonatal risk factors for early-onset disease) and provide intrapartum prophylaxis to those at high risk ( figure 13 -1) the utilization of these guidelines and intrapartum prophylaxis across the united states has decreased the incidence of early-onset disease by approximately 80%. in 2005, the incidence of early-onset disease was 0.35 cases per 1000 live births. 95 late-onset gbs disease is thought to be the result of transmission during delivery or in the postnatal period from maternal, hospital, or community sources. dillon et al 112 demonstrated that 10 of 21 infants with late-onset disease were colonized at birth, but the source of colonization was unidentified in the others. gardner et al 141 showed that only 4.3% of 46 children who were culture negative for gbs at discharge from the hospital had acquired gbs by 2 months of age. anthony et al 15 noted that many infants are colonized with gbs, but the actual attack rate for gbs disease is low and difficult to predict. acquisition of gbs through breast milk or breastfeeding is uncommon. cases of late-onset gbs disease associated with gbs in the maternal milk have been reported. 58, 214, 313, 366, 438 some of the mothers had bilateral mastitis, at least one had delayed evidence of unilateral mastitis, and the others were asymptomatic. it was not clear when colonization of the infants occurred or when infection or disease began in the infants. the authors discussed the possibility that the infants were originally colonized during delivery, subsequently colonized the mothers' breasts during breastfeeding, and then became reinfected at a later time. butter and demoor 56 showed that infants initially colonized on their heads at birth had gbs cultured from their throat, nose, or umbilicus 8 days later. whenever they cultured gbs from the nipples of mothers, the authors also found it in the nose or throat of the infants. byrne et al 58 presented a review of gbs disease associated with breastfeeding and made recommendations to decrease the risk for transmission of gbs to infants via breastfeeding or breast milk. some of their recommendations included confirming appropriate collection and processing procedures for gbs cultures 370 in medical facilities to decrease false-negative cultures, reviewing proper hygiene for pumping, collection, and storage of expressed breast milk with mothers, reviewing the signs and symptoms of mastitis with mothers, and utilizing banked human milk as needed instead of mother' s milk. when a breastfed infant develops late-onset gbs disease, it is appropriate to culture the milk. (see discussion of culturing breast milk earlier in this chapter.) consider treatment of the mother to prevent reinfection if the milk is culture positive for gbs (greater than 10 4 cfu/ml), with or without clinical evidence of mastitis in the mother. withholding the mother' s milk until it is confirmed to be culture negative for a pathogen is appropriate and should be accompanied by providing ongoing support and instruction to the mother concerning pumping and maintaining her milk supply. serial culturing of expressed breast milk after treatment of the mother for gbs disease or colonization would be appropriate to insure the ongoing absence of a pathogen in the expressed breast milk. there are reports of reinfection of the infant from breast milk. 23, 225 eradication of gbs mucosal colonization in the infant or the mother may be difficult. some authors have recommended using rifampin prophylactically in both the mother and infant at the end of treatment to eradicate mucosal colonization. 23 (see chapter 16 for management of mastitis in the mother.) a mother or infant colonized or infected with gbs should be managed with standard precautions 94 while in the hospital. ongoing close evaluation of the infant for infection or illness and empiric therapy for gbs in the infant are appropriate until the child has remained well and cultures are subsequently negative at 72 hours. occasionally, epidemiologic investigation in the hospital will utilize culturing medical staff and family members to detect a source of late-onset gbs disease in the nursery. this can be useful when more than one case of late-onset disease is detected with the same serotype. cohorting in such a situation may be appropriate. selective prophylactic therapy for colonized infants to eradicate colonization may be considered, but unlike gas or staphylococcus infection, gbs infection in nurseries has not been reported to cause outbreaks. no data support screening all breastfeeding mothers and their expressed breast 4 cbc including wbc count with differential and blood culture. 5 applies only to penicillin, ampicillin, or cefazolin and assumes recommended dosing regimens. 6 a healthy-appearing infant who was ≥ 38 weeks' gestation at delivery and whose mother received ≥ 4 hours of iap before delivery may be discharged home after 24 hours if other discharge criteria have been met and a person able to comply fully with instructions for home observation will be present. if any one of these conditions is not met, the infant should be observed in the hospital for at least 48 hours and until criteria for discharge are achieved. milk for gbs as a reasonable method for protecting against spread of gbs infection via expressed breast milk. selective culturing of expressed breast milk may be appropriate in certain situations. the face of tuberculosis (tb) is changing throughout the world. in the united states the incidence of tb rose during 1986 through 1993 and has been declining since then. 60 increased rates of tb were noted in adults between 25 and 45 years of age, and because these are the primary childbearing years, the risk for transmission to children increased. tb during pregnancy has always been a significant concern for patients and physicians alike. 340 it is now clear that the course and prognosis of tb in pregnancy are less affected by the pregnancy and more determined by the location and extent of disease, as defined primarily by chest radiograph, and by the susceptibility of the individual patient. untreated tb in pregnancy is associated with maternal and infant mortality rates of 30% to 40%. 365 effective therapy is crucial to the clinical outcome in both pregnant and nonpregnant women. tb during pregnancy rarely results in congenital tb. any individual in a high-risk group for tb should be screened with a tuberculin skin test (tst). no contraindication or altered responsiveness to the tst exists during pregnancy or breastfeeding. interpretation of the tst should follow the most recent guidelines, using different sizes of induration in different-risk populations as cutoffs for a positive test, as proposed by the cdc. 68 figure 13 -2 outlines the evaluation and treatment of a pregnant woman with a positive tst. 398 treatment of active tb should begin as soon as the diagnosis is made, regardless of the fetus' gestational age, because the risk for disease to mother and fetus clearly outweighs the risks of treatment. isoniazid, rifampin, and ethambutol have been used safely in all three trimesters. isoniazid and pyridoxine therapy during breastfeeding is safe, although the risk for hepatotoxicity in the mother may be a concern during the first 2 months postpartum. 391 congenital tb is extremely rare if one considers that 7 to 8 million cases of tb occur each year worldwide and that less than 300 cases of congenital tb have been reported in the literature. as with other infectious diseases presenting in the perinatal period, distinguishing congenital infection from perinatal or postnatal tb in infants can be difficult. postnatal tb infection in infancy typically presents with severe disease and extrapulmonary extension (meningitis, lymphadenopathy, and bone, liver, spleen involvement). airborne transmission of tb to infants is the major mode of postnatal infection because of close and prolonged exposure in enclosed spaces, especially in their own household, to any adult with infectious pulmonary tb. potential infectious sources could be the mother or any adult caregiver, such as babysitters, day care workers, relatives, friends, neighbors, and even health care workers. the suspicion of tb infection or disease in a household with possible exposure of an infant is a highly anxiety-provoking situation ( figure 13 -3). although protection of an infant from infection is foremost in everyone' s mind, separation of the infant from the mother should be avoided when reasonable. every situation is unique, and the best approach will vary according to the specifics of the case and accepted principles of tb management. the first step in caring for the potentially exposed infant is to determine accurately the true tb status of the suspected case (mother or household contact). this prompt evaluation should include a complete history (previous tb infection or disease, previous or ongoing tb treatment, tst status, symptoms suggestive of active tb, results of most recent chest radiograph, sputum smears, or cultures), physical examination, a tst if indicated, a new chest radiograph, and mycobacterial cultures and smears of any suspected sites of infection. all household contacts should be evaluated promptly, including history and tst with further evaluation as indicated. 68 continued risk to the infant can occur from infectious household contacts who have not been effectively evaluated and treated. an infant should be separated temporarily from the suspected source if symptoms suggest active disease or a recent tst documents conversion, and separation should continue until the results of the chest radiograph are seen. because of considerable variability in the course of illness and the concomitant infectious period, debate continues without adequate data about the appropriate period of separation. 278 this should be individualized given the specific situation. hiv testing and assessment of the risk for mdr tb should be done in every case of active tb. sensitivity testing should be done on every mycobacterium tuberculosis isolate. table 13 -1 summarizes the management of the newborn infant whose mother (or other household contact) has tb. initiation of prophylactic isoniazid therapy in the infant has been demonstrated to be effective in preventing tb infection and disease in the infant. therefore continued separation of infant and mother is unnecessary after therapy in both mother and child has begun. 114 the real risk to an infant requiring separation is from airborne transmission. separation of the infant from a mother with active pulmonary tb is appropriate, regardless of the method of feeding. however, in many parts of the world, after therapy in the mother and prophylaxis with isoniazid in the infant has begun, the infant and mother are not separated. with or without separation, the mother and infant should continue to be closely observed throughout the course of maternal therapy to ensure good compliance with medication by both mother and infant and to identify, early on, any symptoms in the infant suggestive of tb. tuberculous mastitis occurs rarely in the united states but does occur in other parts of the world* and can lead to infection in infants, frequently involving the tonsils. a mother usually has a single breast mass and associated axillary lymph node swelling and infrequently develops a draining sinus. tb of the breast can also present as a painless mass or edema. involvement of the breast can occur with or without evidence of disease at other sites. evaluation of extent of disease is appropriate, including lesion cultures by needle aspiration, biopsy, or wedge resection and milk cultures. therapy should be with multiple anti-tb medications, but surgery should supplement this, as needed, to remove extensive necrotic tissue or a persistently draining sinus. 16 neither breastfeeding nor breast milk feeding should be done until the lesion is healed, usually 2 weeks or more. continued anti-tb therapy for 6 months in the mother and isoniazid for the infant for 3 to 6 months is indicated. in the absence of tuberculous breast infection in the mother, transmission of tb through breast milk has not been documented. thus even though temporary separation of infant and mother may occur pending complete evaluation and initiation of adequate therapy in the mother and prophylactic isoniazid therapy (10 mg/kg/day as a single daily dose) in the infant, breast milk can be expressed and given to the infant during the short separation. breastfeeding can safely continue whether the mother, infant, or both are receiving anti-tb therapy. anti-tb medications (isoniazid, rifampin, pyrazinamide, aminoglycosides, ethambutol, ethionamide, p-aminosalicylic acid) have been safely used in infancy, and therefore the presence of these medications in smaller amounts in breast milk is not a contraindication to breastfeeding. although conflicting, reports indicate that breastfeeding by tst-positive mothers does influence infants' responses to bacille calmette-guérin notes: 1 further workup should always include evaluation of tb status of all other household (or close) contacts by tuberculin skin testing (tst), review of symptoms, physical examination, and chest x-ray (cxr). sputum smears and cultures should be done as indicated. 2 separation should occur until interpretation of cxr film confirms absence of active disease, or, with active disease, separation should continue until individual is no longer considered infectious: three negative consecutive sputum smears, adequate ongoing empiric therapy, and decreased fever, cough, and sputum production. separation means in a different house or location, not simply separate rooms in a household. duration of separation should be individualized for each case in consultation with tb specialist. 3 this assumes no evidence of breast involvement, suspected tb mastitis, or lesion (except in status 5, when breast involvement is considered). risk to infant is via aerosolized bacteria in sputum from the lung. expressed breast milk can be given even if separation of mother and infant is advised. 4 tst positive, no symptoms or physical findings suggestive of tb, negative cxr film. 5 prophylactic therapy: isoniazid 10 mg/kg/day, maximum 300 mg for 6 months; pyridoxine 25 to 50 mg/day for 6 months. empiric therapy: standard three-or four-drug regimens for 2 months, and treatment should continue for total of 6 months with isoniazid and rifampin when organism is shown to be sensitive. suspected multidrug-resistant (mdr) tb requires consultation with tb specialist to select optimum empiric regimen and for ongoing monitoring of therapy and clinical response. vaccine, the tst, and perhaps the m. tuberculosis bacillus. despite efforts to identify either a soluble substance or specific cell fractions (gamma/delta t cells) in colostrum and breast milk that affect infants' immune responsiveness, no unified theory explains the various reported changes and no evidence has identified a consistent, clinically significant effect. 39, 213, 319, 367 viral infections arboviruses arboviruses were originally a large collection of viruses grouped together because of the common mode of transmission through arthropods. they have now been reclassified into several different families: bunyaviridae, togaviridae, flaviviridae, reoviridae, and others. they include more than 30 human pathogens. these organisms primarily produce either cns infections (encephalitis, meningoencephalitis) or undifferentiated illnesses associated with fever and rash, severe hemorrhagic manifestations, and involvement of other organs (hepatitis, myalgia, polyarthritis). infection with this array of viruses may also be asymptomatic and subclinical, although how often this occurs is uncertain. some of the notable human pathogens include bunyaviridae (california serogroup viruses), hantavirus, hantaan virus, phlebovirus (rift valley fever), nairovirus (crimean-congo hemorrhagic fever), alphavirus (western, eastern, and venezuelan equine encephalomyelitis viruses, chikungunya virus), flavivirus (st. louis encephalitis virus, japanese encephalitis virus, dengue viruses, yellow fever virus, tick-borne encephalitis viruses), and orbivirus (colorado tick fever). other than for crimean-congo hemorrhagic fever and for reported cases of colorado tick fever associated with transfusion, direct person-to-person spread has rarely been described. recent outbreaks of chikungunya virus infection in reunion island and in india described infection in young infants probably secondary to vertical spread from mother to infant transplacentally. 146, 339, 422 a few cases of early fetal deaths were associated with infection in pregnant women. the cases of vertical transmission occurred with near-term infection in the mothers, and the infants developed illness within 3 to 7 days of delivery. 146, 339 no evidence for transmission via breast milk or breastfeeding is available. little evidence indicates that these organisms can be transmitted through breast milk. the exceptions to this include evidence of transmission of two flaviviruses via breast milk, west nile virus, and yellow fever vaccine virus. standard precautions are generally sufficient. with any of these infections in a breastfeeding mother, the severity of the illness may determine the mother' s ability to continue breastfeeding. providing the infant with expressed breast milk is acceptable. (see the discussion of west nile virus and yellow fever vaccine virus later in this chapter.) in general, treatment for these illnesses is supportive. however, ribavirin appears to decrease the severity of and mortality from hantavirus pulmonary syndrome, hemorrhagic fever with renal failure, and crimean-congo hemorrhagic fever. ribavirin has been described as teratogenic in various animal species and is contraindicated in pregnant women. no information is available concerning ribavirin in breast milk, with little information available on the use of iv or oral ribavirin in infants. arenaviruses are single-stranded ribonucleic acid (rna) viruses that infect rodents and are acquired by humans through the rodents. the six major human pathogens in this group are (1) lymphocytic 6 sensitivity testing should be done on any positive culture. 7 isoniazid 10 mg/kg/day for 3 to 9 months depending on mother's or contact's status; repeat tst at 3 months and obtain normal cxr in infant before stopping isoniazid. before beginning therapy, workup of infant for congenital or active tb may be appropriate. this workup should be determined by clinical status of infant and suspected potential risk, and may include tst after 4 weeks of age, with cxr, complete blood count, and erythrocyte sedimentation rate, liver function tests, cerebrospinal fluid analysis, gastric aspirates, sonography/computed tomography of liver/spleen, and chest if congenital tb is suspected. 8 breastfeeding is proscribed when separation of mother and infant is indicated because of risk for aerosolized transmission of bacteria. expressed breast milk given to infant via bottle is acceptable in absence of mastitis or breast lesions. 9 consult with tb specialist about mdr tb. empiric therapy will be chosen based on the most recent culture sensitivities of index patient or perhaps suspected source case, if known, as well as medication toxicities and other factors. 10 tb mastitis usually involves a single breast with associated axillary lymph node swelling and, infrequently, a draining sinus tract. it can also present as a painless mass or edema of breast. 11 with suspected mastitis or breast lesion caused by tb, even breast milk is contraindicated until lesion or mastitis heals, usually 2 weeks or more. 12 patient has a documented, recent tst conversion but has not been completely evaluated. evaluation should begin and cxr done and evaluated in less than 24 hours to minimize separation of this person from infant. further workup should proceed as indicated by symptoms, physical findings, and cxr results. choriomeningitis virus, (2) lassa fever virus, (3) junin virus (argentine hemorrhagic fever), (4) machupo virus (bolivian hemorrhagic fever), (5) guanarito virus (venezuelan hemorrhagic fever), and (6) sabia virus. the geographic distribution of these viruses and the illness they cause are determined by the living range of the host rodent (reservoir). the exact mechanism of transmission to humans is unknown and hotly debated. 25, 69, 131 direct contact and aerosolization of rodent excretions and secretions are probable mechanisms. lymphocytic choriomeningitis virus is well recognized in europe, the americas, and other areas. perinatal maternal infection can lead to severe disease in the newborn, but no evidence suggests transmission through breast milk. 28, 224 standard precautions with breastfeeding are appropriate. lassa fever (west africa) and argentine hemorrhagic fever (argentine pampas) are usually more severe illnesses with dramatic bleeding and involvement of other organs, including the brain. these fevers more frequently lead to shock and death than do the forms of hemorrhagic fever caused by the other viruses in this group. person-to-person spread of lassa fever is believed to be common, and transmission within households does occur. 212 this may relate to prolonged viremia and excretion of the virus in the urine of humans for up to 30 days. 330 the possibility of persistent virus in human urine, semen, and blood after infection exists for each of the arenaviruses. the possibility of airborne transmission is undecided. current recommendations by the cdc 69 are to use contact precautions for the duration of the illness in situations of suspected viral hemorrhagic fever. no substantial information describes the infectivity of various body fluids, including breast milk, for these different viral hemorrhagic fevers. considering the severity of the illness in mothers and the risk to the infants, it is reasonable to avoid breastfeeding in these situations if alternative forms of infant nutrition can be provided. as more information becomes available, reassessment of these recommendations is advisable. a vaccine is in clinical trials in endemic areas for junin virus and argentine hemorrhagic fever. preliminary studies suggest it is effective, but data are still being accumulated concerning the vaccine' s use in children and pregnant or breastfeeding women. cytomegalovirus (cmv) is one of the human herpesviruses. congenital infection of infants, postnatal infection of premature infants, and infection of immunodeficient individuals represent the most serious forms of this infection in children. the time at which the virus infects the fetus or infant and the presence or absence of antibodies against cmv from the mother are important determinants of the severity of infection and the likelihood of significant sequelae (congenital infection syndrome, deafness, chorioretinitis, abnormal neurodevelopment, learning disabilities). 234 about 1% of all infants are born excreting cmv at birth, and approximately 5% of these congenitally infected infants will demonstrate evidence of infection at birth (approximately five symptomatic cases per 10,000 live births). approximately 15% of infants born after primary infection in a pregnant woman will manifest at least one sequela of prenatal infection. 96 various studies have detected that 3% to 28% of pregnant women have cmv in cervical cultures and that 4% to 5% of pregnant women have cmv in their urine. 120, 172 perinatal infection certainly occurs through contact with virus in these fluids but usually is not associated with clinical illness in fullterm infants. the lack of illness is thought to result from transplacental passive transfer of protective antibodies from the mother. postnatal infection later in infancy occurs via breastfeeding or contact with infected fluids (e.g., saliva, urine) but, again, rarely causes clinical illness in full-term infants. seroepidemiologic studies have documented transmission of infection in infancy, with higher rates of transmission occurring in daycare centers, especially when the prevalence of cmv in the urine and saliva is high. cmv has been identified in the milk of cmv-seropositive women at varying rates (10% to 85%) using viral cultures or cmv deoxyribonucleic acid (dna) pcr. 172, 301, 397, 430 cmv is more often identified in the breast milk of seropositive mothers than in vaginal fluids, urine, and saliva. the cmv isolation rate from colostrum is lower than that from mature milk. 172, 396 the reason for the large degree of variability in identification of cmv in breast milk in these studies probably relates to the intermittent nature of reactivation and excretion of the virus in addition to the variability, frequency, and duration of sampling of breast milk in the different studies. some authors have hypothesized that the difference in isolation rates between breast milk and other fluids is caused by viral reactivation in cells (leukocytes or monocytes) in the breast leading to "selective" excretion in breast milk. 301 vochem et al 430 reported that the rate of virolactia was greatest at 3 to 4 weeks postpartum, and yeager et al 455 reported significant virolactia between 2 and 12 weeks postpartum. antibodies (e.g., secretory iga) to cmv are present in breast milk, along with various cytokines and other proteins (e.g., lactoferrin). these may influence virus binding to cells, but they do not prevent transmission of infection.* several studies have documented increased rates of postnatal cmv infection in breastfed infants (50% to 69%) compared with bottle-fed infants (12% to 27%) observed through the first year of life 120, 281, 397, 430 in these same studies, full-term infants who acquired cmv infection postnatally were only rarely mildly symptomatic at the time of seroconversion or documented viral excretion. also, no evidence of late sequelae from cmv was found in these infants. postnatal exposure of susceptible infants to cmv, including premature infants without passively acquired maternal antibodies against cmv, infants born to cmv-seronegative mothers, and immunodeficient infants, can cause significant clinical illness (pneumonitis, hepatitis, thrombocytopenia).* in one study of premature infants followed up to 12 months, vochem et al 430 found cmv transmission in 17 of 29 infants (59%) exposed to cmv virolactia and breastfed compared with no infants infected of 27 exposed to breast milk without cmv. no infant was given cmv-seropositive donor milk or blood. five of the 12 infants who developed cmv infection after 2 months of age had mild signs of illness, including transient neutropenia, and only one infant had a short increase in episodes of apnea and a period of thrombocytopenia. five other premature infants with cmv infection before 2 months of age had acute illness, including sepsis-like symptoms, apnea with bradycardia, hepatitis, leukopenia, and prolonged thrombocytopenia. 430 vollmer et al 431 followed premature infants with early postnatal cmv infection acquired through breast milk for 2 to 4.5 years to assess neurodevelopment and hearing function. none of the children had sensorineural hearing loss. there was no difference between the 22 cmv-infected children and 22 matched premature control cmv-negative infants in terms of neurologic, speech and language, or motor development. 431 neuberger et al 296 examined the symptoms and neonatal outcome of cmv infection transmitted via human milk in premature infants in a case-control fashion; 40 cmv-infected premature infants were compared with 40 cmv-negative matched premature infants. neutropenia, thrombocytopenia, and cholestasis were associated with cmv infection in these infants. no other serious effects or illnesses were found directly associated with the infection including intraventricular hemorrhage, periventricular leukomalacia, retinopathy of prematurity, necrotizing enterocolitis, bronchopulmonary dysplasia, duration of mechanical ventilation or oxygen therapy, duration of hospital stay or weight, gestational age, or head circumference at the time of discharge. exposure of cmv-seronegative or premature infants to cmv-positive milk (donor or natural mother' s) should be avoided. 379 various methods of inactivating cmv in breast milk have been reported, including holder pasteurization, freezing (−20° c for 3 days), and brief high temperature (72° c for 10 seconds). 120, 135, 155, 393, 455 one small, prospective study suggests that freezing breast milk at −20°c for 72 hours protects premature infants from cmv infection via breast milk. sharland et al 379 reported on 18 premature infants (less than 32 weeks) who were uninfected at birth and exposed to breast milk from their cmv seropositive mothers. only one of 18 (5%) infants became positive for cmv at 62 days of life, and this infant was clinically asymptomatic. this transmission rate is considerably lower than others reported in the literature. cmvseronegative and leukocyte-depleted blood products were used routinely. banked breast milk was pasteurized and stored at −20° c for various time periods and maternal expressed breast milk was frozen at −20° c before use whenever possible. the infants received breast milk for a median of 34 days (range 11 to 74 days) and they were observed for a median of 67 days (range 30 to 192 days). breast milk samples pre-or postfreezing were not analyzed by pcr or culture for the presence of cytomegalovirus. 379 buxmann et al 57 demonstrated no transmission of cmv in 23 premature infants receiving thawed frozen breast milk until 33 weeks (gestational age + postnatal age) (less than or equal to 31 weeks gestational age) born to 19 mothers who were cmv-igg negative. cmv infection was found in five premature infants of 35 infants born to 29 mothers who were cmv-igg positive and who provided breast milk for their infants. three of the five children remained asymptomatic. one child development a respirator-dependent pneumonia and the second developed an upper respiratory tract infection and thrombocytopenia in association with their cmv infections. 57 yasuda et al 454 reported on 43 preterm infants (median gestational age 31 weeks) demonstrating a peak in cmv dna copies, detected by a real-time pcr assay, in breast milk at 4 to 6 weeks postpartum. thirty of the 43 infants received cmv dna-positive breast milk. three of the 30 had cmv dna detected in their sera, but none of the three had symptoms suggestive of cmv infection. much of the breast milk had been stored at −20° c before feeding, which the authors propose is the probable reason for less transmission in this cohort. 454 lee et al 248 reported on the use of maternal milk frozen at −20°c for a minimum of 24 hours before feeding to premature infants in a nicu; 23 infants had cmv-seropositive mothers and 39 infants had cmv-seronegative mothers. two infants developed cmv infection, which was symptomatic. they were both fed frozen thawed milk from cmv-seropositive mothers. 248 others have reported individual cases of cmv infection in premature infants despite freezing and thawing breast milk. 268, 314 simple freezing and thawing of breast milk does not completely prevent transmission of cmv to premature infants. the efficacy of freezing and thawing breast milk for varying lengths of time to prevent cmv infection in premature infants has not been studied prospectively in a randomized controlled trial. eleven of 36 neonatal units in sweden (27 of which have their own milk banks) freeze maternal milk to reduce the risk for cmv transmission to premature infants. 314 a prominent group of neonatologists and pediatric infectious disease experts in california who recognize the significant benefits of providing human milk to premature and lbw infants recommend screening mothers of premature infants for cmv igg at delivery and, when an infant' s mother is cmv igg positive at delivery, using either pasteurized banked human milk or frozen then thawed maternal breast milk for premature infants until they reach the age of 32 weeks. 445 in consideration of the low rates of cmv virolactia in colostrum 169, 397 and the predominant occurrence of virolactia between 2 and 12 weeks (peak at 3 to 4 weeks) postpartum, 430,455 they reasonably propose beginning colostrum and breast milk feedings for all infants until the maternal cmv serologic screening is complete. they appropriately recommend close observation and follow-up of premature infants older than 3 weeks of age for signs, symptoms, and laboratory changes of cmv infection until discharge from the hospital. 445 cmv-seropositive mothers can safely breastfeed their full-term infants because, despite a higher rate of cmv infection than in formula-fed infants observed through the first year of life, infection in this situation is not associated with significant clinical illness or sequelae. dengue viruses (serotypes dengue 1 to 4) are flaviviruses associated primarily with febrile illnesses and rash; dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. the mosquito aedes aegypti is the main vector of transmission of dengue virus in countries lying between latitudes 35 degrees north and 35 degrees south. more than 2.5 billion people live in areas where transmission occurs; dengue virus infects over 100 million individuals a year and casuses approximately 24,000 deaths a year. 159, 163 although dengue hemorrhagic fever and dengue shock syndrome occur frequently in children younger than 1 year of age, they are infrequently described in infants younger than 3 months of age. 167 there are also differences in the clinical and laboratory findings of dengue virus infection in children compared with adults. 222 boussemart et al 49 reported on two cases of perinatal/prenatal transmission of dengue and discussed eight additional cases in neonates from the literature. prenatal or intrapartum transmission of the same type of dengue as the mother was confirmed by serology, culture, or pcr. phongsamart et al 333 described three additional cases of dengue virus infection late in pregnancy and apparent transmission to two of the three infants with passive acquisition of antibody in the third infant. sirinavin et al 386 reported on 17 cases in the literature of vertical dengue infection, all presenting at less than 2 weeks of age, but no observations or discussion of breast milk or breastfeeding as a potential source of infection were published. watanaveeradej et al 439 presented an additional three cases of dengue infection in infants documenting normal growth and development at follow-up at 12 months of age. it has been postulated that more severe disease associated with dengue disease occurs when an individual has specific igg against the same serotype as the infecting strain in a set concentration, leading to antibody-dependent enhancement of infection. the presence of preexisting dengue serotype specific igg in an infant implies either previous primary infection with the same serotype, passive acquisition of igg from the mother (who had a previous primary infection with the same serotype), or perhaps acquisition of specific igg from breast milk. watanaveeradej et al 439 documented transplacentally transferred antibodies against all four serotypes of dengue virus in 97% of 2000 cord sera at delivery. follow-up of 100 infants documented the loss of antibodies to dengue virus over time with losses of 3%, 19%, 72%, 99%, and 100% at 2, 4, 6, 9, and 12 months of age, respectively. no evidence is available in the literature about more severe disease in breastfed infants compared with formula-fed infants. no interhuman transmission of dengue virus in the absence of the mosquito vector and no evidence of transmission via breast milk are known. only one report of a factor in the lipid portion of breast milk, which inhibits the dengue virus, is available, and no evidence for antibody activity against dengue virus in human breast milk is known. 127 breastfeeding during maternal or infant dengue disease should continue as determined by the mother' s or infant' s severity of illness. epstein-barr virus (ebv) is a common infection in children, adolescents, and young adults. it is usually asymptomatic but most notably causes infectious mononucleosis and has been associated with chronic fatigue syndrome, burkitt lymphoma, and nasopharyngeal carcinoma. because ebv is one of the human herpesviruses, concern has been raised about lifelong latent infection and the potential risk for infection to a fetus and neonate from the mother. primary ebv infection during pregnancy is unusual because few pregnant women are susceptible. 149, 189 although abortion, premature birth, and congenital infection from ebv are suspected, no distinct group of anomalies is linked to ebv infection in fetus or neonate. also, no virologic evidence of ebv as the cause of abnormalities was found in association with suspected ebv infection. culturing of ebv from various fluids or sites is difficult. the virus is detected by its capacity to transform b lymphocytes into persistent lymphoblastoid cell lines. pcr and dna hybridization studies have detected ebv in the cervix and in breast milk. one study, which identified ebv dna in breast milk cells in more than 40% of women donating milk to a breast milk bank, demonstrated that only 17% had antibody to ebv (only igg, no igm). 204 another study examining serologic specimens from breastfed and bottle-fed infants showed similar seroprevalence of ebv at 12 to 23 months of age (36/66 [54.5%] and 24/43 [55.8%]) in the breastfed and bottle-fed children, respectively. 236 the question of the timing of ebv infection and the subsequent immune response and clinical disease produced requires continued study. differences exist among the clinical syndromes that manifest at different ages. infants and young children are asymptomatic, have illness not recognized as related to ebv, or have mild episodes of illness, including fever, lymphadenopathy, rhinitis and cough, hepatosplenomegaly, or rash. adolescents or young adults who experience primary ebv infection more often demonstrate infectious mononucleosis syndrome or are asymptomatic. chronic fatigue syndrome is more common in adolescents and young adults. burkitt lymphoma, observed primarily in africa, and nasopharyngeal carcinoma, seen in southeast asia, where primary ebv infection usually occurs in young children, are tumors associated with early ebv infection. 246 these tumors are related to "chronic" ebv infection and tend to occur in individuals with persistently high antibody titers to ebv viral capsid antigen and early antigen. the questions of why these tumors occur with much greater frequency in these geographic areas and what cofactors (including altered immune response to infection associated with coinfections, immune escape by ebv leading to malignancy, or increased resistance to apoptosis secondary to ebv gene mutations) may contribute to their development remain unanswered. 21, 289 it also remains unknown to what degree breast milk could be a source of early ebv infection compared with other sources of ebv infection in an infant' s environment. similar to the situation of postnatal transmission of cmv in immunocompetent infants, clinically significant illness rarely is associated with primary ebv infection in infants. more data concerning the pathogenesis of ebv-associated tumors should be obtained before proscribing against breastfeeding is warranted, especially in areas where these tumors are common but the protective benefits of breastfeeding are high. in areas where burkitt lymphoma and nasopharyngeal carcinoma are uncommon, ebv infection in mother or infant is certainly not a contraindication to breastfeeding. marburg and ebola viruses cause severe and highly fatal hemorrhagic fevers. the illness often presents with nonspecific symptoms (conjunctivitis, frontal headache, malaise, myalgia, bradycardia) and progresses with worsening hemorrhage to shock and subsequent death in 50% to 90% of patients. personto-person transmission through direct contact, droplet spread, or airborne spread is the common mode of transmission. however, the animal reservoir or source of these viruses in nature for human infection has not been identified. attack rates in families are 5% to 16%. 330 no postexposure interventions have proved useful in preventing spread, and no treatment other than supportive is currently available. a recent report documented the presence of ebola virus in numerous body fluids including in breast milk. one acute breast milk sample on day 7 after the onset of illness and a "convalescent" breast milk sample on day 15 from the same woman were positive for ebola virus by both culture and pcr testing. 30 in the same study, saliva remained virus positive for a mean of 16 days after disease onset, urine was positive for a mean of 28 days, and semen for a mean of 43 days after the onset of disease. no information is available concerning the risk for transmission of these viruses in breast milk or additional risks or benefits from breastfeeding. contact precautions are recommended for marburg virus infections and contact and airborne precautions for ebola virus infection. given the high attack and mortality rates, these precautions should be carefully instituted and breastfeeding not allowed. if any other suitable source of nutrition can be found for an infant, expressed breast milk should also be proscribed for the infant of a mother with either of these infections for at least 3 weeks postrecovery. the diagnosis of hepatitis in a pregnant woman or nursing mother causes significant anxiety. the first issue is determining the etiology of the hepatitis, which then allows for an informed discussion of risk to the fetus/infant. the differential diagnosis of acute hepatitis includes (1) common causes of hepatitis, such as hepatitis a, b, c, and d; (2) , igm anti-hbcag, anti-hcv) as the initial diagnostic tests. simultaneous consideration of other etiologies of acute liver dysfunction is appropriate depending on a patient' s history. if the initial diagnostic tests are all negative, subsequent additional testing for anti-hepatitis d virus (hdv), hcv rna, hepatis g virus (hgv) rna, anti-hepatis e virus (hev), or hev rna may be necessary. if initial testing reveals positive hbsag, testing for anti-hdv, hbeag, and hbv dna is appropriate. these additional tests are useful in defining the prognosis for a mother and the risk for infection to an infant. during the diagnostic evaluation, it is appropriate to discuss with the mother or parents the theoretic risk for transmitting infectious agents that cause hepatitis via breastfeeding. the discussion should include an evaluation of the positive and negative effects of suspending or continuing breastfeeding until the exact etiologic diagnosis is determined. the relative risk for transmission of infection to an infant can be estimated and specific preventive measures provided for the infant (table 13-2) . hepatitis a virus (hav) is usually an acute selflimited infection. the illness is typically mild, and generally subclinical in infants. occasionally, hav infection is prolonged or relapsing, extending 3 to 6 months, and rarely it is fulminant, but hav infection does not lead to chronic infection. the incidence of prematurity after maternal hav infection is increased, but no evidence to date indicates obvious birth defects or a congenital syndrome. 372, 464 hav infection in premature infants may lead to prolonged viral shedding. 349 transmission is most often person to person (fecal-oral), and transmission in food-borne or water-borne epidemics has been described. transmission via blood products and vertical transmission (mother to infant) are rare. 440 transmission in daycare settings has been clearly described. infection with hav in newborns is uncommon and does not seem to be a significant problem. the usual period of viral shedding and presumed contagiousness lasts 1 to 3 weeks. acute maternal hav infection in the last trimester or in the postpartum period could lead to infection in an infant. symptomatic infection can be prevented by immunoglobulin (ig) administration, and 80% to 90% of disease can be prevented by ig administration immune serum globulin within 2 weeks of exposure. hav vaccine can be administered simultaneously with ig without affecting the seroconversion rate to produce rapid and prolonged hav serum antibody levels. transmission of hav via breast milk has been implicated in one case report, but no data exist on the frequency of isolating hav from breast milk. 440 because hav infection in infancy is rare and usually subclinical without chronic disease and because exposure has already occurred by the time the etiologic diagnosis of hepatitis in a mother is made, no reason exists to interrupt breastfeeding with maternal hav infection. the infant should receive ig and hav vaccine, administered simultaneously. hepatitis b virus (hbv) infection leads to a broad spectrum of illness, including asymptomatic seroconversion, nonspecific symptoms (fever, malaise, fatigue), clinical hepatitis with or without jaundice, extrahepatic manifestations (arthritis, rash, renal involvement), fulminant hepatitis, and chronic hbv infection. chronic hbv infection occurs in up to 90% of infants infected via perinatal and vertical transmission and in 30% of children infected between 1 to 5 years of age. given the increased risk for significant sequelae from chronic infection (chronic active hepatitis, chronic persistent hepatitis, cirrhosis, primary hepatocellular carcinoma), prevention of hbv infection in infancy is crucial. transmission of hbv is usually through blood or body fluids (stool, semen, saliva, urine, cervical secretions). 94 vertical transmission either transplacentally or perinatally during delivery has been well described throughout the world. vertical transmission rates in areas where hbv is endemic (taiwan and japan) are high, whereas transmission to infants from hbv carrier mothers in other areas where hbv carrier rates are low is uncommon. 399 transmission of hbv to infants occurs in up to 50% of infants when the mothers are acutely infected immediately before, during, or soon after pregnancy. 462 hbsag is found in breast milk, but transmission by this route is not well documented. beasley 31 and beasley et al 32 demonstrated that although breast milk transmission is possible, seroconversion rates are no different between breastfed and nonbreastfed infants in a long-term follow-up study of 147 hbsag-positive mothers. hill et al 176 followed 101 breastfed infants and 268 formula-fed infants born to women who were chronically hbsag positive. all infants received hepatitis b immunoglobulin at birth and a full series of hepatitis b vaccine. none of the breastfed infants and nine of the formulafed infants were positive for hbsag after completion of the hbv vaccine series. breastfeeding had occurred for a mean of 4.9 months (range 2 weeks to 1 year). transmission, when it does happen, probably occurs during labor and delivery. another report from china followed 230 infants born to hbsag-positive women. the infants received appropriate dosing and timing of hbig and hbv vaccine. at 1 year of age, anti-hbs antibody was present in 90.9% of the breastfed infants and 90.3% of the bottle-fed infants. 437 risk factors associated with immunoprophylaxis failure against vertical transmission of hbv include hbeag-seropositive mothers and elevated hbv dna "viral loads" in the mothers. 392 in 2009 the aap committee on infectious diseases stated that "that breastfeeding of the infant by a hbsag-positive mother poses no additional risk for acquisition of hbv infection by the infant with appropriate administration of hepatitis b vaccine and hbig." 96 screening of all pregnant women for hbv infection is an essential first step to preventing vertical transmission. universal hbv vaccination at birth and during infancy, with administration of hepatitis b immunoglobulin (hbig) immediately after birth to infants of hbsag-positive mothers, prevents hbv transmission in more than 95% of cases. breastfeeding by hbsag-positive women is not contraindicated, but immediate administration of hbig and hbv vaccine should occur. two subsequent doses of vaccine should be given at appropriate intervals and dosages for the specific hbv vaccine product. this decreases the small theoretic risk for hbv transmission from breastfeeding to almost zero. when acute peripartum or postpartum hepatitis occurs in a mother and hbv infection is a possibility, with its associated increased risk for transmission to the infant, a discussion with the mother or parents should identify the potential risks and benefits of continuing breastfeeding until the etiology of the hepatitis can be determined. if an appropriate alternative source of nutrition is available for the infant, breast milk should be withheld until the etiology of the hepatitis is identified. hbig and hbv vaccine can be administered to the infant who has not already been immunized or has no documented immunity against hbv. 400 if acute hbv infection is documented in a mother, breastfeeding can continue after immunization has begun. acute infection with hcv can be indistinguishable from hepatitis a or b infection; however, it is typically asymptomatic or mild. hcv infection is the major cause of blood-borne non-a, non-b hepatitis (nanbh). chronic hcv infection is reported to occur 70% to 85% of the time regardless of age at time of infection. sequelae of chronic hcv infection are similar to those associated with chronic hbv infection. bortolotti et al 47 the two commonly identified mechanisms of transmission of hcv are transfusions of blood or blood products and iv drug use. however, other routes of transmission exist because hcv infection occurs even in the absence of obvious direct contact with significant amounts of blood. other body fluids contaminated with blood probably serve as sources of infection. transmission through sexual contact occurs infrequently and probably requires additional contributing factors, such as coinfection with other sexually transmitted agents or high viral loads in serum and other body fluids. studies of transmission in households without other risk factors have demonstrated either low rates of transmission or no transmission. the reported rates of vertical transmission vary widely. in mothers with unknown hiv status or known hiv infection, the rates of vertical transmission were 4% to 100%, whereas the rates varied between 0% and 42% in known hiv-negative mothers. 113 these same studies suggest that maternal coinfection with hiv, hcv genotype, active maternal liver disease, and the serum titer of maternal hcv rna may be associated with increased rates of vertical transmission. 263, 307, 461 the correlation between hcv viremia, the hcv viral load in a mother, and vertical transmission of hcv is well documented. 288, 355, 406, 456 the clinical significance and risk for liver disease after vertical transmission of hcv are still unknown. the timing of hcv infection in vertical transmission is also unknown. in utero transmission has been suggested by some studies, 125 whereas intrapartum or postpartum transmission was proposed by ohto et al 308 when they documented the absence of hcv rna in the cord blood of neonates who later became hcv rna positive at 1 to 2 months of age. more recently, gibb et al 150 reported two pieces of data supporting the likelihood of intrapartum transmission as the predominant time of vertical transmission: (a) low sensitivity of pcr for hcv rna testing in the first month of life with a marked increase in sensitivity after that for diagnosing hcv infection in infants and (b) a lower transmission risk for elective cesarean delivery (without prolonged rupture of membranes) compared with vaginal or emergency cesarean delivery. 150 another group, mcmenamin et al, 275 analyzed vertical transmission in 559 mother-infant pairs. the overall vertical transmission rate was 4.1% (18/441), with another 118 infants not tested or lost to follow-up. comparison of the vertical transmission rate was no different for vaginal delivery or emergency cesarean in labor versus planned cesarean (4.2% vs. 3.0%). this held true even when mothers had hepatitis c rna detected antenatally (7.2% vs. 5.3%). the authors did not support planned cesarean delivery to decrease vertical transmission of hepatitis c infection. no prospective, controlled trials of cesarean versus vaginal delivery and the occurrence of vertical hepatitis c transmission are available. the risk for hcv transmission via breast milk is uncertain. anti-hcv antibody and hcv rna has been demonstrated in colostrum and breast milk, although the levels of hcv rna in milk did not correlate with the titers of hcv rna in serum. 36, 162, 256, 355 nevertheless, transmission of hcv via breastfeeding (and not in utero, intrapartum, or from other postpartum sources) has not been proven in the small number infants studied. transmission rates in breastfed and nonbreastfed infants appear to be similar, but various important factors have not been controlled, such as hcv rna titers in mothers, examination of the milk for hcv rna, exclusive breastfeeding versus exclusive formula feeding versus partial breastfeeding, and duration of breastfeeding.* zanetti et al 461 including 23 infants whose mothers were seropositive for hcv rna. eight infants in that study were infected with hcv, their mothers had both hiv and hcv, and three of these eight infants were infected with both hiv and hcv. the hcv rna levels were significantly higher in the mothers coinfected with hiv compared with those mothers with hcv alone. overall, the risk for hcv infection via breastfeeding is low, the risk for hcv infection appears to be more frequent in association with hiv infection and higher levels of hcv rna in maternal serum, no effective preventive therapies (ig or vaccine) exist, and the risk for chronic hcv infection and subsequent sequelae with any infection is high. it is therefore appropriate to discuss the theoretic risk for breastfeeding in hcv-positive mothers with the mother or parents and to consider proscribing breast milk when appropriate alternative sources of nutrition are available for the infants. hiv infection is a separate contraindication to breastfeeding. additional study is necessary to determine the exact role of breastfeeding in the transmission of hcv, including the quantitative measurement of hcv rna in colostrum and breast milk, the relative risk for hcv transmission in exclusively or partially breastfed infants versus the risk in formulafed infants, and the effect of duration of breastfeeding on transmission. the current position of the cdc is that no data indicate that hcv virus is transmitted through breast milk. 83 therefore breastfeeding by a hcvpositive, hiv-negative mother is not contraindicated. infants born to hcv rna-positive mothers require follow-up through 18 to 24 months of age to determine infants' hcv status, regardless of the mode of infant feeding. infants should be tested for alanine aminotransferase and hcv rna at 3 months and 12 to 15 months of age. alanine aminotransferase and anti-hcv antibody should be tested at 18 to 24 months of age to confirm an infant' s status: uninfected, ongoing hepatitis c infection, or past hcv infection. hepatitis d virus (hdv) is a defective rna virus that causes hepatitis only in persons also infected with hbv. the infection occurs as either an acute coinfection of hbv and hdv or a superinfection of hbv carriers. this "double" infection results in more frequent fulminant hepatitis and chronic hepatitis, which can progress to cirrhosis. the virus uses its own hbv rna (circular, negative-strand rna) with an antigen, hdag, surrounded by the surface antigen of hbv, hbsag. hdv is transmitted in the same way as hbv, especially through the exchange of blood and body fluids. hdv infection is uncommon where the prevalence of hbv is low. in areas where hbv is endemic, the prevalence of hdv is highly variable. hdv is common in tropical africa and south america as well as in greece and italy but is uncommon in the far east and in alaskan inuit despite the endemic occurrence of hbv in these areas. 390 transmission of hdv has been reported to occur from household contacts and, rarely, through vertical transmission. no data are available on transmission of hdv by breastfeeding. hdv infection can be prevented by blocking infection with hbv; therefore hbig and hbv vaccine are the best protection. in addition to hbig and hbv vaccine administration to the infant of a mother infected with both hbv and hdv, discussion with the mother or parents should include the theoretic risk for hbv and hdv transmission through breastfeeding. as with hbv, once hbig and hbv vaccine have been given to the infant, the risk for hbv or hdv infection from breastfeeding is negligible. therefore breastfeeding after an informed discussion with the parents is acceptable. hepatitis e virus (hev) is a cause of sporadic and epidemic, enterically transmitted nanbh, which is typically self-limited and without chronic sequelae. hev is notable for causing high mortality rate in pregnant women. transmission is primarily via the fecal-oral route, commonly via contaminated water or food. high infection rates have been reported in adolescents and young adults (ages 15 to 40 years). tomar 416 reported that 70% of cases of hev infections in the pediatric population in india manifest as acute hepatitis. maternal-neonatal transmission was documented when the mother developed hepatitis e infection in the third trimester. although hev was demonstrated in breast milk, no transmission via breast milk was confirmed in the report. five cases of transfusion-associated hepatitis e were reported. 416 epidemics are usually related to contamination of water. person-to-person spread is minimal, even in households and day care settings. although ig may be protective, no controlled trials have been done. animal studies suggest that a recombinant subunit vaccine may be feasible. 344 hev infection in infancy is rare, and no data exist on transmission of hev by breastfeeding. no evidence of clinically significant postnatal hev infection in infants or of chronic sequelae in association with hev infection and no documented hev transmission through breast milk is available. currently no contraindication exists to breastfeeding with maternal hev infection. ig has not been shown to be effective in preventing infection, and no vaccine is available for hev. hepatitis g virus (hgv) has recently been confirmed as a cause of nanbh distinct from hepatitis viruses a through e. several closely related genomes of hgv, currently named gbv-a, -b, and -c, appear to be related to hcv, the pestiviruses, and the flaviviruses. epidemiologically, hgv is most often associated with transfusion of blood, although studies have identified nontransfusion-related cases. hgv genomic rna has been detected in some patients with acute and chronic hepatitis and a small number of patients with fulminant hepatitis. gbv-c/hgv has also been found in some patients with inflammatory bile duct lesions, but the pathogenicity of this virus is unconfirmed. hgv rna has been detected in 1% to 3% of healthy blood donors in the united states. 8 feucht et al 128 described maternal-to-infant transmission of hgv in three of nine children. two of the three mothers were coinfected with hiv and the third with hcv. none of these infants developed signs of liver disease. neither the timing nor the mode of transmission was clarified. lin et al 255 reported no hgv transmission in three mother-infant pairs after cesarean delivery and discussed transplacental spread via blood as the most likely mode of hgv infection in vertical transmission. wejstal et al 442 reported on perinatal transmission of hgv to 12 of 16 infants born to hgv viremic mothers, identified by pcr. hgv did not appear to cause hepatitis in the children. 442 fischler et al 130 followed eight children born to hgv-positive mothers and found only one to be infected with hgv. that child remained clinically well, while his twin, also born by cesarean delivery and breastfed, remained hgv negative for 3 years of observation. five of the other six children were breastfed for variable periods without evidence of hgv infection. ohto et al 309 examined hgv mother-to-infant transmission. of 2979 pregnant japanese women who were screened, 32 were identified as positive for gbv-c/hgv rna by pcr; 26 of 34 infants born to the 32 hgv positive women were shown to be hgv rna positive. reportedly, none of the infants demonstrated a clinical picture of hepatitis, although two infants had persistent mild elevations (less than two times normal) of alanine aminotransferase. the viral load in mothers, who transmitted hgv to their infants, was significantly higher than in nontransmitting mothers. infants born by elective cesarean delivery had a lower rate of infection (3 in 7) compared with infants born by emergency cesarean delivery (2 of 2) or born vaginally (21 of 25) . in this study, hgv infection in breastfed infants was four times more common than in formula-fed infants, but this difference was not statistically significant because only four infants were formula fed. the authors report no correlation between infection rate and duration of breastfeeding was seen. testing of the infants was not done frequently and early enough routinely through the first year of life to determine the timing of infection in these infants. 309 schröter et al 371 reported transmission of hgv to 3 of 15 infants born to hgv rna positive mothers at 1 week of age. none of 15 breast milk samples were positive for gbv-c/hgv rna, and all of the children who were initially negative for hgv rna in serum remained negative at follow-up between 1 to 28 months of age. 371 the foregoing data suggest that transmission is more likely to be vertical, before, or at delivery rather than via breastfeeding. the pathogenicity and the possibility of chronic disease due to hgv infection remain uncertain at this time. insufficient data are available to make a recommendation concerning breastfeeding by hgv-infected mothers. herpes simplex virus types 1 and 2 (hsv-1, hsv-2) can cause prenatal, perinatal, and postnatal infections in fetuses and infants. prenatal infection can lead to abortion, prematurity, or a recognized congenital syndrome. perinatal infection is the most common form of infection (1 in 2000 to 5000 live births, 700 to 1500 cases per year in the united states) and is often fatal or severely debilitating. the factors that facilitate intrapartum infection and predict the severity of disease have been extensively investigated. postnatal infection is uncommon but can occur from a variety of sources, including oral or genital lesions and secretions in mothers or fathers, hospital workers and home caregivers, and breast lesions in breastfeeding mothers. a number of case reports have documented severe hsv-1 or hsv-2 infections in infants associated with hsvpositive breast lesions in the mothers. 116, 161, 338, 403 cases of infants with hsv gingivostomatitis inoculating the mothers' breasts have also been reported. in the absence of breast lesions breastfeeding in hsv-seropositive or culture-positive women is reasonable when accompanied by careful handwashing, covering the lesions, and avoiding fondling or kissing with oral lesions until all lesions are crusted. breastfeeding during maternal therapy with oral or iv acyclovir can continue safely as well. inadequate information exists concerning valacyclovir, famciclovir, ganciclovir, and foscarnet in breast milk to make a recommendation at this time. breastfeeding by women with active herpetic lesions on their breasts should be proscribed until the lesions are dried. treatment of the mothers' breast lesions with topical, oral, and/or iv antiviral preparations may hasten recovery and decrease the length of viral shedding. human herpesvirus 6 (hhv-6) is a cause of exanthema subitum (roseola, roseola infantum) and is associated with febrile seizures. hhv-6 appears to be most similar to cmv based on genetic analysis. no obvious congenital syndrome of hhv-6 infection has been identified, although prenatal infection has been reported. 118 seroepidemiologic studies show that most adults have already been infected by hhv-6. therefore primary infection during pregnancy is unlikely, but reactivation of latent hhv-6 infection may be more common. no case of symptomatic hhv-6 prenatal infection has been reported. the significance of reactivation of hhv-6 in a pregnant woman and the production of infection and disease in the fetus and infant remains to be determined. primary infection in children occurs most often between 6 and 12 months of age, when maternally acquired passive antibodies against hhv-6 are waning. febrile illnesses in infants younger than 3 months of age have been described with hhv-6 infection, but infection before 3 months or after 3 years is uncommon. various studies involving serology and restriction enzyme analysis of hhv-6 isolates from mother/infant pairs support the idea that postnatal transmission and perhaps perinatal transmission from the mothers are common sources of infection. one study was unable to detect hhv-6 in breast milk by pcr analysis in 120 samples, although positive control samples seeded with hhv-6-infected cells did test positive. 119 given the limited occurrence of clinically significant disease and the absence of sequelae of hhv-6 infection in infants and children, the almost universal acquisition of infection in early childhood (with or without breastfeeding) and the absence of evidence that breast milk is a source of hhv-6 infection, breastfeeding can continue in women known to be seropositive for hhv-6. human herpesvirus 7 (hhv-7) is closely related to hhv-6 biologically. primary infection with hhv-7 occurs primarily in childhood, usually later in life than hhv-6 infection. the median age of infection is 26 months, with 75% of children becoming hhv-7 positive by 5 years of age. 63 seroprevalence of hhv-7 antibody has been reported to be 80% to 98% in adults, and passive antibody is present in almost all newborns. 306, 408 like hhv-6, hhv-7 infection can be associated with acute febrile illness, febrile seizures, and irritability, but in general it is a milder illness than with hhv-6 with fewer hospitalizations. virus excretion of hhv-7 occurs in saliva, and pcr testing of blood cells and saliva are frequently positive in individuals with past infection. 463 congenital infection of hhv-6 was detected via dna pcr testing in 57 of 5638 of cord blood samples (1%), but hhv-7 was not detected in any of 2129 cord blood specimens. 165 hhv-7 dna was detected by pcr in 3 of 29 breast milk mononuclear cell samples from 24 women who were serum positive for hhv-7 antibody. 137 in the same study, small differences were seen in the hhv-7 seropositive rates between breastfed infants and bottle-fed infants at 12 months of age (21.7% versus 20%), at 18 months of age (60% versus 48.1%), and at 24 months of age (77.3% versus 58.3%, respectively,). none of these differences were statistically significant. given that, in general, hhv-6 infection occurs earlier than hhv-7 infection in most infants and that hhv-6 is rarely found in breast milk, it seems unlikely that hhv-7 in breast milk is a common source of infection in infants and children. the infrequent occurrence of significant illness with hhv-7 infection, with the absence of sequelae except in patients who had transplantation surgery at older ages and the common occurrence of infection in childhood argue, that no reason to proscribe against breastfeeding for hhv-7 positive women exists. human papillomavirus (hpv) is a dna virus with at least 100 different types. these viruses cause warts, genital dysplasia, cervical carcinoma (types 6 and 11), and laryngeal papillomatosis. transmission occurs through direct contact and sexual contact. laryngeal papillomas are thought to result from acquiring the virus in passage through the birth canal. infection in pregnant women or during pregnancy does not lead to an increase in abortions or the risk for prematurity, and no evidence indicates intrauterine infection. hpv is one of the most common viruses in adults and one of the most commonly sexually transmitted infections. diagnosis is usually by histologic examination or dna detection. spontaneous resolution does occur, but therapy for persistent lesions or growths in anatomically problematic locations is appropriate. therapy can be with podophyllum preparations, trichloroacetic acid, cryotherapy, electrocautery, and laser surgery. interferon is being tested in the treatment of laryngeal papillomas, with mixed results. 109 prevention against transmission means limiting direct or sexual contact, but this may not be sufficient because lesions may not be evident and transmission may still occur. rintala et al 346 examined the occurrence of hpv dna in the oral and genital mucosa of infants during the first 3 years of life. hpv dna was identified in 12% to 21% of the oral scrape samples and in 4% to 15% of the genital scrape samples by pcr. oral hpv infection was acquired by 42% of children, cleared by 11%, and persisted in 10% of children; 37% of the children were never infected. they did not report on breast milk or breastfeeding in that study. the question of the source of the infection remains undetermined. the breast is a rare site of involvement. 110 hpv types 16 and 18 can immortalize normal breast epithelium in vitro. 441 hpv dna has been detected in breast milk in 10 of 223 (4.5%) of milk samples from 223 mothers, collected 3 days postpartum. 361 no attempt was made to correlate the presence of hpv dna in breast milk with the hpv status of an infant or to assess the "viral load" of hpv in breast milk or its presence over the course of lactation. a second study found dna of cutaneous and mucosal hpv types in 2 of 25 human milk samples and 1 of 10 colostrum samples. 64 no reports of hpv lesions of the breast or nipple and documented transmission to an infant secondary to breastfeeding are available. no increased risk for acquiring hpv from breast milk is apparent, and breastfeeding is acceptable. even in the rare occurrence of an hpv lesion of the nipple or breast, no data suggest that breastfeeding or the use of expressed breast milk is contraindicated. measles is another highly communicable childhood illness that can be more severe in neonates and adults. measles is an exanthematous febrile illness following a prodrome of malaise, coryza, conjunctivitis, cough, and often koplik spots in the mouth. the rash usually appears 10 to 14 days after exposure. complications can include pneumonitis, encephalitis, and bacterial superinfection. with the availability of vaccination, measles in pregnancy is rare (0.4 in 10,000 pregnancies), 148 although respiratory complications (primary viral pneumonitis, secondary bacterial pneumonia), hepatitis, or other secondary bacterial infections often lead to more severe disease in these situations. prenatal infection with measles may cause premature delivery without disrupting normal uterine development. no specific group of congenital malformations have been described in association with in utero measles infection, although teratogenic effects of measles infection in pregnant women may rarely manifest in the infants. perinatal measles includes transplacental infection when measles occurs in an infant in the first 10 days of life. infection from extrauterine exposure usually develops after 14 days of life. the severity of illness after suspected transplacental spread of virus to an infant varies from mild to severe and does not seem to vary with the antepartum or postpartum onset of rash in the mother. it is uncertain what role maternal antibodies play in the severity of an infant's disease. more severe disease seems to be associated with severe respiratory illness and bacterial infection. postnatal exposure leading to measles after 14 days of life is generally mild, probably because of passively acquired antibodies from the mother. severe measles in children younger than 1 year of age may occur because of declining passively acquired antibodies and complications of respiratory illness and rare cases of encephalitis. measles virus has not been identified in breast milk, whereas measles-specific antibodies have been documented. 1 infants exposed to mothers with documented measles while breastfeeding should be given immunoglobulin (ig) and isolated from the mother until 72 hours after the onset of rash, which is often only a short period after diagnosis of measles in the mother. the breast milk can be pumped and given to the infant because secretory iga begins to be secreted in breast milk within 48 hours of onset of the exanthem in the mother. table 13 -3 summarizes management of the hospitalized mother and infant with measles exposure or infection. 148 mumps is an acute transient benign illness with inflammation of the parotid gland and other salivary glands and often involves the pancreas, testicles, and meninges. mumps occurs infrequently in pregnant women (1 to 10 cases in 10,000 pregnancies) and is generally benign. mumps virus has been isolated from saliva, respiratory secretions, blood, testicular tissue, urine, csf in cases of meningeal involvement, and breast milk. the period of infectivity is believed to be between 7 days before and 9 days after the onset of parotitis, with the usual incubation period being 14 to 18 days. prenatal infection with the mumps virus causes an increase in the number of abortions when infection occurs in the first trimester. a small increase in the number of premature births was noted in one prospective study of maternal mumps infection. 383 no conclusive evidence suggests congenital malformations are associated with prenatal infection, not even with endocardial fibroelastosis, as originally reported in the 1960s. perinatal mumps (transplacentally or postnatally acquired) has rarely if ever been documented. natural mumps virus has been demonstrated to infect the placenta and infect the fetus, and live attenuated vaccine virus has been isolated from the placenta but not from fetal tissue in women vaccinated 10 days before induced abortion. antibodies to mumps do cross the placenta. postnatal mumps in the first year of life is typically benign. no epidemiologic data suggest that mumps infection is more or less common or severe in breastfed infants compared with formula-fed infants. although mumps virus has been identified in breast milk and mastitis is a rare complication of mumps in mature women, no evidence indicates that breast involvement occurs more frequently in lactating women. if mumps occurs in the mother breastfeeding can continue because exposure has already occurred throughout the 7 days before the development of symptoms in the mother and secretory iga in the milk may help to mitigate the symptoms in the infant. human parvovirus b19 causes a broad range of clinical manifestations, including asymptomatic infection (most frequent manifestation in all ages), erythema infectiosum (fifth disease), arthralgia and arthritis, red blood cell (rbc) aplasia (less often decreased white blood cells or platelets), chronic infection in immunodeficient individuals, and rarely myocarditis, vasculitis, or hemophagocytic syndrome. vertical transmission can lead to severe anemia and immune-mediated hydrops fetalis, which can be treated, if accurately diagnosed, by intrauterine transfusion. inflammation of the liver or cns can be seen in the infant, along with vasculitis. if the child is clinically well at birth, hidden or persistent abnormalities are rarely identified. no evidence indicates that parvovirus b19 causes an identified pattern of birth defects. postnatal transmission usually occurs person to person via contact with respiratory secretions, saliva, and rarely blood or urine. seroprevalence in children at 5 years of age is less than 5%, with the peak age of infection occurring during the schoolage years (5% to 40% of children infected). the majority of infections are asymptomatic or undiagnosed seroconversions. 417 severe disease, such as prolonged aplastic anemia, occurs in individuals with hemoglobinopathies or abnormal rbc maturation. attack rates have been estimated to be 17% to 30% in casual contacts but up to 50% among household contacts. in one study of 235 susceptible pregnant women, the annual seroconversion rate was 1.4%. 223 no reports of transmission to an infant through breastfeeding are available. excretion in breast milk has not been studied because of limitations in culturing techniques. rat parvovirus has been demonstrated in rat milk. the very low seroconversion rate in young children and the absence of chronic or frequent severe disease suggest that the risk for parvovirus infection via breast milk is not significant. the possibility of antibodies against parvovirus or other protective constituents in breast milk has not been studied. breastfeeding by a mother with parvovirus infection is acceptable. poliovirus infections (types 1, 2, and 3) cause a range of illness, with 90% to 95% subclinical, 4% to 8% abortive, and 1% to 2% manifest as paralytic poliomyelitis. a review by bates 29 from 1955 of 58 cases of poliomyelitis in infants younger than 1 month of age demonstrated paralysis or death in more than 70% and only one child without evidence of even transient paralysis. more than half the cases were ascribed to transmission from the mothers, although no mention was made of breastfeeding. breastfeeding rates at the time were approximately 25%. prenatal infection with polioviruses does cause an increased incidence of abortion. prematurity and stillbirth apparently occur more frequently in mothers who developed paralytic disease versus inapparent infection. 188 although individual reports of congenital malformations in association with maternal poliomyelitis exist, no epidemiologic data suggest that polioviruses are teratogenic. also, no evidence indicates that live attenuated vaccine poliovirus given during pregnancy is associated with congenital malformations. 89, 170 perinatal infection has been noted in several case reports of infants infected in utero several days before birth who had severe disease manifesting with neurologic manifestations (paralysis) but without fever, irritability, or vomiting. additional case reports of infection acquired postnatally demonstrate illness more consistent with poliomyelitis of childhood. these cases were more severe and involved paralysis, which may represent reporting bias. 89 no data are available concerning the presence of poliovirus in breast milk, although antibodies to poliovirus types 1, 2, and 3 have been documented. 270 in this era of increasing worldwide poliovirus vaccination, the likelihood of prenatal or perinatal poliovirus infection is decreasing. maternal susceptibility to poliovirus should be determined before conception and poliovirus vaccine offered to susceptible women. an analysis of the last great epidemic in italy in 1958 was done using a population-based case-control study. 336 in 114,000 births, 942 infants were reported with paralytic poliomyelitis. a group of matched control subjects was selected from infants admitted to the hospital at the same time. using the dichotomous variable of never breastfed and partially breastfed, 75 never-breastfed infants were among the cases and 88 among the control group. the authors determined an odds ratio of 4.2, with 95% confidence interval of 1.4 to 14, demonstrating that the risk for paralytic poliomyelitis was higher in infants never breastfed and lowest among those exclusively breastfed. because by the time the diagnosis of poliomyelitis is made in a breastfeeding mother, the exposure of the infant to poliovirus from maternal secretions has already occurred, and because the breast milk already contains antibodies that may be protective, no reason exists to interrupt breastfeeding. breastfeeding also does not interfere with successful immunization against poliomyelitis with oral or inactivated poliovirus vaccine. 71 the occurrence of human t-cell leukemia virus type i (htlv-i) is endemic in parts of southwestern japan, 66, 105, 207, 450 the caribbean, south america, 156 and sub-saharan africa. htlv-i is associated with adult t-cell leukemia/lymphoma and a chronic condition with progressive neuropathy. the progressive neuropathy is called htlv-i associated myelopathy or tropical spastic paraparesis. 136 other illnesses have been reported in association with htlv-i infection including dermatitis, uveitis, arthritis, sjögren syndrome in adults, and infective dermatitis and persistent lymphadenitis in children. transmission of htlv-i occurs most often through sexual contact, via blood or blood products, and via breast milk. infrequent transmission does occur in utero or at delivery and with casual or household contact. 291 seroprevalence generally increases with age and varies widely in different regions and in populations of different backgrounds. in some areas of japan, seropositivity can be as high as 12% to 16%, but in south america, africa, and some caribbean countries the rates are 2% to 6%. in latin america seropositive rates can be as high as 10% to 25% among female sex workers or attendees to std clinics. 156 in blood donors in europe, the seroprevalence of htlv-i has been reported at 0.001% to 0.03%. the seroprevalence in pregnant women in endemic areas of japan is as high as 4% to 5% and in nonendemic areas as low as 0.1% to 1.0%. htlv-1 is not a major disease in the united states. in studies from europe the seroprevalence in pregnant women has been noted to be up to 0.6%. these pregnant women were primarily of african or caribbean descent. 138 htlv-i antigen has been identified in breast milk of htlv-i positive mothers. 220 another report shows that basal mammary epithelial cells can be infected with htlv-i and can transfer infection to peripheral blood monocytes. 254 human milk from htlv-i positive mothers caused infection in marmosets. 221, 453 htlv-i infection clearly occurs via breastfeeding and a number of reports document an increased rate of transmission of htlv-i to breastfed infants compared with formula-fed infants.* ando et al 12, 13 in two separate reports demonstrated a parallel decline in antibodies against htlv-i in both formula-fed and breastfed infants to a nadir at approximately 1 year of age and a subsequent increase in antibodies from 1 to 2 years of age. the percentage of children seropositive at 1 year of age in the breastfed and formula-fed groups, respectively, was 3.0% and 0.6%, at 1.5 years of age it was 15.2% and 3.9%, and at 2 years of age it was 41.9% and 4.6%. a smaller group of children followed through 11 to 12 years of age demonstrated no newly infected children after 2 years of age and *references 9, 10, 12, 13, 178-180, 407. no loss of antibody in any child who was seropositive at 2 years of age. 12, 13 transmission of htlv-i infection via breastfeeding is also clearly associated with the duration of breastfeeding. 407, 409, 446, 447 it has been postulated that the persistence of passively acquired antibodies against htlv-i offers some protection through 6 months of life (table 13-4) . other factors relating to htlv-i transmission via breast milk have been proposed. yoshinaga et al 460 presented data on the htlv-i antigen producing capacity of peripheral blood and breast milk cells and showed an increased mother-to-child transmission rate when the mother' s blood and breast milk produced large numbers of antigen-producing cells in culture. 460 hisada et al 183 reported on 150 mothers and infants in jamaica, demonstrating that a higher maternal provirus level and a higher htlv-i antibody titer were independently associated with htlv-i transmission to the infant. ureta-vidal et al 421 reported an increased seropositivity rate in children of mothers with a high proviral load and elevated maternal htlv-i antibody titers. various interventions have been proposed to decrease htlv-i transmission via breastfeeding. complete avoidance of breastfeeding was shown to be an effective intervention by hino et al 180, 181 in large population of japanese in nagasaki. avoiding breastfeeding led to an 80% decrease in transmission. breastfeeding for a shorter duration is another effective alternative. ando et al 11 showed that freezing and thawing breast milk decreased the infectivity of htlv-i. sawada et al 363 demonstrated in a rabbit model that htlv-i immunoglobulin protected against htlv-i transmission via milk. it is reasonable to postulate that any measure that would decrease the maternal provirus load or increase the anti-htlv-i antibodies available to infants might decrease the risk for transmission. the overall prevalence of htlv-i infection during childhood is unknown because the majority of individuals do not manifest illness until much later in life. the timing of htlv-i infection in a breastfeeding population has been difficult to assess because of passively acquired antibodies from the mother and issues related to testing. furnia et al 139 in areas where the prevalence of htlv-i infection (in the united states, canada, or europe) is rare, the likelihood that a single test for antibody against htlv-i would be a false positive test is high compared with the number of true positive tests. repeat testing is warranted in many situations. 66 quantification of the antibody titer and the proviral load is appropriate in a situation when mother-to-child transmission is a concern. a greater risk for progression to disease in later life has not been shown for htlv-i infection through breast milk, but early-life infections are associated with the greatest risk for adult t-cell leukemia. 402 the mother and family should be informed about all these issues. if the risk for lack of breast milk is not too great and formula is readily available and culturally acceptable, then the proscription of breastfeeding, or at least a recommendation to limit the duration of breastfeeding to 6 months or less, is appropriate to limit the risk for htlv-i transmission to the infant. freezing and thawing breast milk before giving it to an infant might be another reasonable intervention to decrease the risk for transmission, although no controlled trials document the efficacy of such an intervention. neither ig nor antiviral agents against htlv-i are available at this time. human t-cell leukemia virus type ii (htlv-ii) is endemic in specific geographic locations, including africa, the americas, the caribbean, and japan. transmission is primarily through intravenous drug use, contaminated blood products, and breastfeeding. sexual transmission occurs but its overall contribution to the prevalence of htlv-ii in different populations remains uncertain. many studies have examined the presence of htlv-i and ii in blood products. pcr testing and selective antibody tests suggest that about half of the htlv seropositivity in blood donors is caused by htlv-ii. htlv-ii has been associated with two chronic neurologic disorders similar to those caused by htlv-i, tropical or spastic ataxia. 258 a connection between htlv-ii and glomerulonephritis, myelopathy, arthritis, t-hairy cell leukemia, and large granulocytic leukemia has been reported. mother-to-child transmission has been demonstrated in both breastfed and formula-fed infants. it appears that the rate of transmission is greater in breastfed infants.* htlv-ii has been detected in breast milk. 174 nyambi et al 304 reported that htlv-ii transmission did correlate with the duration of breastfeeding. the estimated rate of transmission was 20%. the time to seroconversion (after the initial loss of passively acquired maternal antibodies) for infected infants seemed to range between 1 and 3 years of age. 304 at this time avoidance of breastfeeding and limiting the duration of breastfeeding are the only two possible interventions with evidence of effectiveness for preventing htlv-ii mother-to-child transmission. 207 with the current understanding of retroviruses, it is appropriate in cases of documented htlv-ii maternal infection to recommend avoiding or limiting the duration of breastfeeding and provide alternative nutrition when financially practical and culturally acceptable. mothers should have confirmatory testing for htlv-ii and measurement of the proviral load. infants should be serially tested for antibodies to htlv-ii and have confirmatory testing if seropositive after 12 to 18 months of age. further investigation into the mechanisms of transmission via breast milk and possible interventions to prevent transmission should occur as they have for hiv-1 and htlv-i. human immunodeficiency virus type 1 (hiv-1) is transmitted through human milk. refraining from breastfeeding is a crucial aspect of preventing perinatal hiv infection in the united states and many other countries. the dilemma is the use of replacement feeding versus breastfeeding in countries where breastfeeding provides infants with significant protection from illness and death due to malnutrition or other infections. the question of the contribution of breastfeeding in mother-to-child hiv-1 transmission is not a trivial one when one considers the following: 3. the who estimates that 2.7 million people were newly infected with hiv-1 in 2007, with children younger than 14 years old making up 370,000 of that 2.7 million. (this number has declined due to increasing access to interventions to prevent mother-to-infant transmission. availability of antiretroviral therapy for prevention of mother-to-child hiv transmission in developing countries in 2007 was estimated to reach 33% of the mothers who needed it.) 419 4. breastfeeding contributes an estimated 10% to 20% increase in the overall mother-to-child transmission rates, over and above intrauterine and intrapartum transmission, when no specific interventions to prevent transmission via breastfeeding are utilized. 5. despite a dramatic increase in the number of people receiving antiretroviral therapy in developing countries (3 million), this represented only 31% of the individuals who needed treatment. 419 the evidence of hiv transmission via breastfeeding is irrefutable. multiple publications summarize the current evidence for hiv transmission via breastfeeding in the literature. 232, 341, 420 since 1985, case reports have documented hiv transmission via breast milk to children around the world. 182, 198, 249, 465 primary hiv infection in breastfeeding mothers, with the concomitant high viral load, is associated with a particularly high rate of hiv transmission via breast milk. palasanthiran et al 322 estimated that risk at 27%.large observational studies have demonstrated higher rates of hiv transmission in breastfed infants of mothers with chronic hiv infection compared with formula-fed infants. 43, 108, 124 a systematic analysis of published reports estimated the additional risk for perinatal hiv transmission due to breastfeeding to be 14% (95% confidence interval 7% to 22%). 117 more recently published cohort studies similarly attributed additional risk for hiv transmission due to breastfeeding at 4% to 22% over and above the risk from prenatal and intrapartum transmission. 38, 104, 121 laboratory reports demonstrate the presence of cell-free virus and cell-associated virus in breast milk as well as various immunologic factors that could block or limit infection.* a dose-response relationship has been observed, correlating the hiv viral load in human milk as well as a mother' s plasma viral load with an increased transmission risk for the breastfed infant. 335, 345, 351, 373 many of the potential risk factors associated with human milk transmission of hiv is higher the longer the duration of breastfeeding. 108, 251, 282, 290, 424 maternal characteristics related to transmission of hiv via human milk include younger maternal age, higher parity, lower cd4+ counts, higher plasma viral loads, and breast abnormalities (mastitis, abscess, or nipple lesions). characteristics of human milk that relate to a higher risk for transmission include higher viral load in the milk, lower concentrations of antiviral substances (lactoferrin, lysozyme), and lower concentrations of virus-specific cytotoxic t-lymphocytes, levels of various interleukins (il-7, il-15), 434, 435 secretory iga, and igm. mixed breastfeeding is also associated with a higher risk for hiv transmission compared with exclusive breastfeeding. 99, 100, 410 the measurable benefits of breast milk versus the relative risk for hiv transmission to the infant due to exclusive breastfeeding (with optimization of other factors to decrease hiv transmission) have been reported in a couple of studies. 97, 229 the measurable benefits of receiving breast milk versus the relative increased risk for hiv transmission will need to be determined in a prospective fashion in different locales. 247 a number of potential interventions to prevent breastfeeding transmission of hiv-1 can be utilized (box 13-3) . the simplest and most effective is the compete avoidance of human milk. this is a practical solution in places like the united states and other countries where replacement feeding as well as other strictly medical interventions are feasible and reasonable, and the risk for not providing breast milk to the infant is negligible. in resourcepoor situations, where the risk for other infections is high without the benefits of breast milk, exclusive breastfeeding is appropriate, with any other reasonable and culturally acceptable interventions to decrease hiv transmission via breast milk. potentially effective interventions include exclusive breastfeeding, early weaning versus breastfeeding for longer durations, education, and support to decrease the likelihood of mastitis or nipple lesions. 191 other possible interventions include treating a mother with antiretroviral therapy for her own health (cd4 counts less than 350) or prophylactically to decrease the human milk viral load, treating an infant prophylactically for a prolonged period of time (6 weeks to 6 months) to protect against transmission via breastfeeding, treating the milk itself to decrease the viral load (by pasteurization or other methods), 316, 318 treating acute conditions in mothers and infants (e.g., mastitis, breast lesions, infant candidiasis), and enhancing an infant' s own defenses via vitamins, immunization, or antiretroviral therapy. some of these may not be feasible in certain settings such as pasteurization or maternal antiretroviral therapy. others may not be culturally acceptable, such as treating expressed breast milk before giving it to an infant or even exclusive breastfeeding. significant data demonstrate the advantage of breastfeeding, even for hiv-infected or hivexposed infants. the complete avoidance of breastfeeding in certain situations may lead to increased risk for illness and death due to other reasons besides hiv transmission. 106 a study from kenya showed improved hiv-1-free survival rates in a formula-fed group of children born to hiv-positive mothers, but the breastfed and formula groups had similar mortality rates (24.4% versus 20.0%, respectively) and similar incidences of diarrhea and pneumonia in the first 2 years of life. 272 no difference in the two groups was seen in the prevalence of malnutrition, but the breastfed infants had better nutritional status in the first 6 months of life. arpadi et al 20 recommend additional nutritional interventions to complement breastfeeding in this population after 6 months of age. two reports from zambia document the benefit of exclusive breastfeeding for decreasing late hiv transmission and the lower mortality at 12 months in infants who had continued breastfeeding rather than had discontinued breastfeeding at 4 months of age. 229, 385 in malawi, hiv-infected and hiv-exposed infants who were breastfed (exclusive breastfeeding for 2 months and mixed feeding after that) had lower mortality at 24 months than those who were not breastfed. 405 a report from botswana examined breastfeeding plus infant zidovudine prophylaxis for 6 months versus formula feeding plus infant zidovudine for 1 month; this study showed a decreased risk for vertical transmission with formula feeding, but also increased cumulative mortality for the hiv-infected infants at 7 months of age who were in the formula-fed group. 411 a study from south africa examining the use of vitamin a also demonstrated less morbidity in hiv-infected children who were breastfed than not breastfed. 102 other abstract reports have shown increased morbidity in hiv-infected children due to diarrhea, gastroenteritis, and hospitalization after weaning from breastfeeding. 205, 226, 315, 413 exclusive breastfeeding in most areas of the world is essential to infant health and survival, even in the situation of maternal hiv infection. 97, 99, 100, 229 the duration of exclusive breastfeeding is crucial to decreasing the risk for hiv infection in infants versus the risk for malnutrition and other infections with early weaning. in the mashi study in botswana, thior et al 411 evaluated infants randomized to breastfeeding plus infant zidovudine for 6 months or formula feeding plus 1 month of infant zidovudine. the cumulative infant mortality was significantly higher at 7 months for the formula-fed group but at 18 months it was similar between the two groups. the breastfed infants were more likely to become hiv infected despite the 6 months of zidovudine prophylaxis. 411 becquet et al 33 analyzed data from cote d'ivoire for 2001 to 2005; 47% of the hivexposed infants were breastfed for a median of 4 months, and 53% were formula fed and observed for 2 years. no significant difference in the rate of hiv infection was seen in the two groups, and no significant difference between the two groups was seen for morbid events (diarrhea, acute respiratory infections or malnutrition) or hospitalization or death. the authors attributed these good outcomes to effective nutritional counseling and care, access to clean water, and the provision of a safe and continuous supply of breast milk substitute. 33 coovadia et al 97 studied exclusive breastfeeding in the first 6 months of life as an intervention in south africa. of the exclusively breastfed infants, 14.1% at 6 weeks of age and 19.5% at 6 months of age were hiv infected. breastfed infants who also were fed solids or formula milk were more likely to acquire infection than exclusively breastfed infants. the cumulative mortality at 3 months of age was markedly lower for exclusively breastfed infants (6.1%) versus 15.1% in the infants receiving mixed feedings. kuhn et al 230 examined the effects of early, abrupt weaning on hiv-free survival of 958 children in zambia. infants were randomly assigned to two different counseling programs that advised either abrupt weaning at 4 months or prolonged breastfeeding. in the weaning intervention group, 69% of mothers stopped breastfeeding by 5 months compared with a median duration of breastfeeding of 16 months in the control group. the study found no significant difference in hiv-free survival at • women and their health care providers need to be aware of the potential risk for transmission of hiv infection to infants during pregnancy and in the peripartum period and through breast milk. • documented, routine hiv education and routine testing with the consent of women seeking prenatal care are strongly recommended so that each woman knows her hiv status and the methods available both to prevent the acquisition and transmission of hiv and to determine whether breastfeeding is appropriate. • at delivery, education about hiv and testing with the consent of women whose hiv status during pregnancy is unknown are strongly recommended. knowledge of a woman' s hiv status assists in counseling on breastfeeding and helps each woman understand the benefits to herself and her infant of knowing her serostatus and the behaviors that would decrease the likelihood of acquisition and transmission of hiv. • women who are known to have hiv infections must be counseled not to breastfeed or provide their milk for the nutrition of their own or other' s infants. • in general, women who are known to be hiv seronegative should be encouraged to breastfeed. however, women who are hiv seronegative but at particularly high risk for seroconversion (e.g., injection drug users and sexual partners of known hiv-positive persons or active drug users) should be educated about hiv with an individualized recommendation concerning the appropriateness of breastfeeding. in addition, during the perinatal period, information should be provided on the potential risk for transmitting hiv through breast milk and about methods to reduce the risk for acquiring hiv infection. • each woman whose hiv status is unknown should be informed of the potential for hiv-infected women to transmit hiv during the peripartum period and through breast milk and the potential benefits to her and her infant of knowing her hiv status and how hiv is acquired and transmitted. the health care provider needs to make an individualized recommendation to assist the woman in deciding whether to breastfeed. 24 months in the two groups (83.9% versus 80.7%). children already infected by 4 months of age had a higher mortality if they were assigned to the early weaning group (73.6% versus 54.8%). additional analysis showed that in mothers with less severe hiv disease early weaning was clearly harmful to the infant. 231 arpadi et al 20 studied the growth of hiv-exposed, uninfected children who were exclusively breastfed for 4 months with rapid weaning to replacement foods or exclusively breastfed until 6 months and then continued breastfeeding with complementary foods. weight-for-age z scores dropped markedly in both groups from 4 to 15 months of age but less so in the continued breastfeeding group. length-for-age z score also dropped dramatically, but was not influenced by continued breastfeeding. even in this hiv-exposed, uninfected group of children, additional nutritional interventions are essential to complement breastfeeding beyond 6 months of age. 20 in recent years the discussion around preventing hiv transmission via breastfeeding and in the number of studies examining the important issues have increased. 98, 233, 283 the fact that intrapartum and perinatal transmission of hiv from mothers to infants has decreased markedly due to the increased utilization of antiretroviral therapy during pregnancy, delivery, and postnatally for prevention emphasizes the importance of now working harder to decrease breast milk transmission of hiv. in considering different possible interventions to decrease mother-infant hiv transmission, it is crucial to reemphasize the goals of optimizing maternal health and survival and optimizing infant health and survival at the same time. a laboratory report shows that mothers receiving highly active antiretroviral therapy (haart) while breastfeeding do have decreased whole breast milk hiv-1 viral loads (23/26 mothers had less than 50 copies/ml) compared with mothers who did not receive haart (9/25 with less than 50 copies/ml). however, the whole milk hiv-1 dna load (measured as "undetectable" at less than 10 copies/10 6 cells) was not significantly different in the haart (13 of 26 mothers)] and non-haart (15 of 23) groups. 378 hiv-1 dna is incorporated into cells found in breast milk. another group showed significantly lower hiv rna levels in the breast milk of women treated with nevirapine, zidovudine, and lamivudine compared with women not receiving antiretroviral therapy. 152 the use of maternal haart seems to decrease hiv-1 transmission via breastfeeding. one group working in mozambique, malawi, and tanzania working with mother-infants pairs receiving haart as prevention during pregnancy compared one cohort (809 mother-infant pairs) who received supplementary formula and water filters for the first 6 months of life with a second cohort (251 motherinfant pairs) breastfeeding exclusively and the mothers receiving haart for the first 6 months. the cumulative incidence rate of hiv infection at 6 months of age was 2.7% for the formula-fed infants and 2.2% for breastfed infants. through 6 months of age no apparent additional risk for late postnatal transmission of hiv was observed. 323 the petra study team working in tanzania, south africa, and uganda examined the efficacy of three shortcourse regimens of zidovudine and lamivudine in preventing early and late hiv transmission in this predominantly breastfeeding population. 332 there were four regimens: a, zidovudine and lamivudine starting at 36 weeks' gestation plus intrapartum medication and 7-days' postpartum treatment; b, same as a without the prepartum component; c, intrapartum zidovudine and lamivudine only; d, placebo. at week 6 the hiv transmission rates were 5.7% in group a, 8.9% in group b, 14.2% in group c, and 15.3% in group d. at 18 months the hiv infection rates were 15% in group a, 18% in group b, 20% in group c, and 22% in group d. although a measurable decrease in transmission at 6 weeks of age was observed, limited protection was seen at 18 months of age. an observational study from tanzania compared maternal haart for 6 months with exclusive breastfeeding and abrupt weaning at 5 to 6 months of age with a historical control of the same feeding schedule without the postnatal maternal haart. in the treatment group the cumulative hiv transmission was 4.1% at 6 weeks, 5.0% at 6 months, and 6.0% at 18 months of age. the cumulative hiv infection or death rate was 8.6% at 6 months and 13.6% at 18 months of age. the cumulative risk for hiv transmission was 1.1% between 6 and 18 months. the hiv transmission in this treatment group was half the transmission rate in the historical control group. 218 another study in sub-saharan africa with 6 months of maternal haart and exclusive breastfeeding for 6 months demonstrated 94% hiv-free survival at 12 months of age; the maternal and infant mortality rates for the treated mother-infant pairs were significantly lower than the country' s maternal and infant mortality rates. 264 antiretroviral therapy prophylaxis for infants is another investigated intervention to decrease hiv transmission via breastfeeding. in a study from cote d'ivoire comparing different groups over time, infants received zidovudine (zdv) alone as zdv prophylaxis, a single dose of nevirapine (nvp), and 7 days of zidovudine (zdv) as nvp/ zdv prophylaxis, or a single dose of nevirapine plus zidovudine and lamivudine (3tc) for 7 days as nvp/zdv/3tc prophylaxis. formula feeding (ff) was compared with exclusive shortened breastfeeding (esb) upto 4 months of age and prolonged breastfeeding (pb). the cumulative transmission rates at 18 months were 22.3% in 238 infants in the zdv + pb group, 15.9% in 169 infants in the nvp/zdv + esb group, 9.4%, in the 195 infants in the nvp/zdv +ff group, 6.8% in the 198 infants in the nvp/zdv/3tc + esb group, and 5.6% in the 126 infants in the nvp/zdv/3tc + ff group. 252 kumwenda et al 235 working in malawi demonstrated decreased hiv transmission with breastfeeding and two different infant prophylaxis regimens. at 9 months of age, they observed a 10.6% occurrence of hiv transmission for infants receiving a single dose of nevirapine plus 1 week of zidovudine compared with 5.2% in the group receiving a single dose of nevirapine plus 1 week of zidovudine plus 14 weeks of daily nevirapine, and 6.4% in the group receiving a single dose of nevirapine plus 1 week of zidovudine plus 14 weeks of nevirapine and zidovudine. 235 in the mitra study in tanzania in which the median time of breastfeeding was 18 weeks, the hiv transmission rate at 6 months in the infants who received zidovudine plus 3tc for 1 week plus 3tc alone for breastfeeding through 6 months of age was less than 50% of the transmission rate for those infants receiving only 1 week of zidovudine plus 3tc. 217 a summary of three trials in ethiopa, india, and uganda compared a single dose of nevirapine at birth for infants with 6 weeks of daily nevirapine in predominantly breastfed infants whose mothers were counselled regarding feeding per the who/ unicef guidelines. at 6 months 87 of 986 infants in the single-dose group and 62 of 901 in the extended-dose group were hiv infected, which was not statistically significant. the authors suggested that a longer course of infant antiretroviral prophylaxis might be more effective. 388 the potential effect of breastfeeding on the hivpositive mother needs to be adequately assessed in relation to the mother's health status. from uganda and zimbabwe mbizvo et al 271 reported no difference in the number of hospital admissions or mortality between hiv-positive and hiv-negative women during pregnancy. in the 2 years after delivery the hiv-positive women had higher hospital admission (approximately two times increased risk) and death rates (relative risk greater than 10) than hiv-negative women. 271 chilongozi et al 90 reported on 2292 hiv-positive mothers from four sub-saharan sites followed for 112 months. serious adverse events occurred in 166 women (7.2%); 42 deaths occurred in the hiv-positive women, and no deaths occurred in 331 hiv-negative women. 90 several studies have examined breastfeeding relative to mothers' health and reported conflicting results. the first study from kenya demonstrated a significantly higher mortality rate in breastfeeding mothers compared with a formula-feeding group in the 2 years after delivery. the hypothesized explanation offered by the authors for this difference was increased metabolic demands, greater weight loss, and nutritional depletion. 294 a second study from south africa showed an overall lower mortality rate in the two groups with no significant difference in mortality rate in the 10 months of observation. 101 kuhn et al 227 reported no difference in mortality at 12 months after delivery between 653 women randomly assigned to a short breastfeeding group (326 women, median breastfeeding duration 4 months, 21% still breastfeeding at 12 months) and a long breastfeeding group (327 women, 90% breastfeeding at 5 months, 72% breastfeeding at 12 months, median 15 months). the hiv-related mortality rates were high (4.9%), but not associated with prolonged lactation. 227 walson et al 433 followed 535 hiv-positive women for 1 to 2 years in kenya. the mortality risk was 1.9% at 1 year and 4.8% at 2 years of follow-up. although less than 10% of women reported a hospitalization during the 2 years, they experienced various common infections (pneumonia, diarrhea, tb, malaria, stds, urinary tract infections, mastitis). breastfeeding was a significant cofactor for diarrhea and mastitis but not for pneumonia, tb, or hospitalization. 433 in summary, breastfeeding of infants by hiv-positive mothers does lead to an increased risk for hiv infection in the infants. much remains to be understood about the mechanisms of hiv transmission via breast milk and the action and efficacy of different interventions to prevent such transmission. the complete avoidance of breastfeeding is a crucial component for the prevention of perinatal hiv infection in the united states and many other countries. in resource-poor settings, where breastfeeding is the norm and where it provides vital nutritional and infection protective benefits, the who, unicef, and the joint united nations programme on hiv/ aids (unaids) recommend education, counseling, and support for hiv-infected mothers so they can make an informed choice concerning infant feeding. mothers choosing to breastfeed should receive additional education, support, and medical care to minimize the risk for hiv transmission and to optimize their own health status during and after breastfeeding. mothers choosing to use replacement feedings should receive parallel education, support, and medical care for themselves and their infants to minimize the effect of the lack of breastfeeding. good evidence now shows that antiretroviral prophylactic regimens for mothers or infants while continuing breastfeeding does decrease postnatal hiv transmission. early weaning is associated with increased morbidity and mortality. further carefully controlled research is indicated to adequately assess the risks and benefits to infants and mothers of prolonged breastfeeding with antiretroviral prophylaxis for either or both mothers and infants. along with this, hiv testing rates must be improved at the same time as increased availability and access to antenatal care, hiv prevention services, and hiv medical care for everyone must be increased. the availability and free access to antiretroviral medications must also improve. the decision about infant feeding for hivpositive mothers remains a difficult one, but this is slowly changing with increasing options. the goals remain 100% hiv transmission prevention, optimal maternal health and survival, and long-term infant health and survival. human immunodeficiency virus type 2 (hiv-2) is an rna virus in the nononcogenic, cytopathic lentivirus genus of retroviruses. it is genetically closer to simian immunodeficiency virus than to hiv-1. the clinical disease associated with hiv-2 has similar symptoms to hiv-1 infection but progresses at a slower rate to severe immunosuppression. hiv-2 is endemic in western africa and parts of the caribbean and found infrequently in europe and north and south america. 190, 305 it is transmitted via sexual contact, blood, or blood products and from mother to child. routine testing for hiv-2 is recommended in blood banks. antibody tests used for hiv-1 are only 50% to 90% sensitive for detecting hiv-2. 65 specific testing for hiv-2 is appropriate whenever clinically or epidemiologically indicated. vertical transmission occurs infrequently. ekpini et al 121 followed a large cohort of west african mothers and infants: 138 hiv-1 positive women, 132 hiv-2 positive women, 69 women seropositive for both hiv-1 and 2, and 274 hiv seronegative women. a few cases of perinatal hiv-2 transmission occurred, but no case of late postnatal transmission was observed. 121 it is probable that hiv-2 transmission via breast milk is less common than with hiv-1, but insufficient data support that the risk for transmission is zero. mothers who test positive for hiv-2 should be tested for hiv-1, and guidelines for breastfeeding should follow those for hiv-1 until additional information is available. rabies virus produces a severe infection with progressive cns symptoms (anxiety, seizures, altered mental status) that ultimately proceeds to death; few reports of survival exist. rabies occurs worldwide except in australia, antarctica, and several island groups. in 1992 more than 36,000 cases of rabies were reported to the who, a number that is probably a marked underestimate of the actual cases. 67 between 1990 and 2003, 37 cases of human rabies were reported in the united states. 70, 78 postexposure prophylaxis is given to thousands of patients each year. rabies virus is endemic in various animal populations, including raccoons, skunks, foxes, and bats. because of aggressive immunization programs, rabies in domesticated dogs and cats in the united states is uncommon. the virus is found in the saliva and tears and nervous tissue of infected animals. transmission occurs by bites, licking, or simply contact of oral secretions with mucous membranes or nonintact skin. many cases of rabies in humans now lack a history of some obvious contact with a rabid animal. this may be a result of the long incubation period (generally 4 to 6 weeks, but can be up to 1 year, with reports of incubation periods of several years), a lack of symptoms early in an infectious animal, or airborne transmission from bats in enclosed environments (caves, laboratories, houses). person-to-person transmission via bites has not been documented, although it has occurred in corneal transplants. 44 rabies viremia has not been observed in the spread of the virus. no evidence exists indicating transmission through breast milk. in the case of maternal infection with rabies, many scenarios can occur before the onset of progressive, severe cns symptoms. the progression and severity of maternal illness can preclude breastfeeding, but separation of an infant from the mother is appropriate regardless of the mother' s status and method of infant feeding (especially to avoid contact with saliva and tears). breastfeeding should not continue when the mother has symptoms of rabies, and the infant should receive postexposure immunization and close observation. an infant may received expressed breast milk, but the expression must occur without possible contamination with saliva or tears from the mother. depending on the scenario, the nature of a mother' s illness, the possible exposure of an infant to the same source as the mother, and the exposure of a child to the mother, postexposure immunization of an infant may be appropriate. a more common scenario is a mother' s apparent exposure to rabies (without exposure for the infant), necessitating postexposure immunization of the mother with rabies vaccine. in the majority of cases, in the absence of maternal illness, breastfeeding can reasonably continue during the mother' s five-dose immunization series in 28 days. in a rare situation in which apparent exposure of mother and infant to rabies occurs together, postexposure treatment of both mother and infant should be instituted, and breastfeeding can continue. respiratory syncytial virus (rsv) is a common cause of respiratory illness in children and is relatively common in adults, usually producing milder upper respiratory tract infection in adults. no evidence indicates that rsv causes intrauterine infection, adversely affects the fetus, or causes abortion or prematurity. rsv does produce infection in neonates, causing asymptomatic infection, afebrile upper respiratory tract infection, bronchiolitis, pneumonia, and apnea. mortality rate can be high in neonates, especially in premature infants and ill full-term infants, particularly those with preexisting respiratory disease (hyaline membrane disease, bronchopulmonary dysplasia) or cardiac disease associated with pulmonary hypertension. rsv is believed to be transmitted via droplets or direct contact of the conjunctiva, nasal mucosa, or oropharynx with infected respiratory secretions. documentation of rsv infection is rarely made in adults, and spread from a mother or other household contacts probably occurs before a diagnosis can be made. therefore risk for rsv transmission from breast milk is probably insignificant compared with transmission via direct or droplet contact in families. in nurseries, however, it is appropriate to make a timely diagnosis of rsv infection in neonates to isolate infants from the others and prevent spread in the nursery. ribavirin is not recommended for routine use. it is infrequently used in patients with potentially life-threatening rsv infection. rsv infection should be suspected in any infant with rhinorrhea, nasal congestion, or unexplained apnea, especially in october through march in temperate climates. prophylaxis against rsv with rsv-specific immunoglobulin iv (rsv-igiv) during this season for infants at highest risk for severe disease is appropriate. debate surrounds the topic of the effect of passively acquired antibodies (in infants from mothers before birth) against rsv on the occurrence and severity of illness in neonates and infants. it appears that a higher level of neutralizing antibody against rsv in neonates decreases the risk for severe rsv disease. 153, 239 some controversy remains concerning the measurable benefit of breastfeeding for preventing serious rsv disease. 3, 54, 115 some studies have shown benefit and others no effect. controlling for possible confounding factors (e.g., smoking, crowded living conditions) in these studies has been difficult. at this point, no reason exists to stop breastfeeding with maternal rsv infection; a potential exists for benefit from nonspecific factors in breast milk against the rsv. infants with rsv infection should breastfeed unless their respiratory status precludes it. rotavirus infections usually result in diarrhea, accompanied by emesis and low-grade fever. in severe infections the clinical course can include dehydration, electrolyte abnormalities, and acidosis and can contribute to malnutrition in developing countries. generally, every child will have at least one episode of rotavirus infection by 5 years of age. 157 in developed countries, rotavirus is often associated with diarrhea requiring hospitalization in children younger than 2 years of age, but rarely associated with death. worldwide rotavirus is the leading cause of diarrhea-related deaths in children younger than 5 years old. estimates suggest that in children younger than 5 years old rotavirus infection leads to more than 100 million occurrences of diarrhea, 2 million hospital admissions, and 500,000 deaths each year. 157 fecal-oral transmission is the most common route, but fomites and respiratory spread may also occur. spread of infection occurs most often in homes with young children or in daycare centers and institutions. in hospitalized infants or mothers with rotavirus infection, contact precautions are indicated for the duration of the illness. no evidence indicates prenatal infection from rotavirus, but perinatal or postnatal infection from contact with the mother or others can occur. no case of transmission of rotavirus via breast milk has been documented. breast milk does contain antibodies to rotavirus for up to 2 years. human milk mucin has been demonstrated to inhibit rotavirus replication and prevent experimental gastroenteritis. 457 the mechanisms of rotavirus immunity are not well understood. they are thought to be multifactorial with cell-mediated immunity limiting severity and the course of infection, while humoral immunity protects against subsequent infections. innate and adaptive responses at the level of the mucosa are probably the most important. 134 exclusive breastfeeding may decrease the likelihood of severe rotavirus-related diarrhea by as much as 90%. 93, 377 although breastfeeding does not prevent infection with rotavirus, it seems to decrease the severity of rotavirus-induced illness in children younger than 2 years old. 93, 123, 184 at least one study suggested that this may represent simply the postponement of severe rotavirus infection until an older age. 93 one study suggested that protection against rotavirus rapidly declines upon discontinuation of breastfeeding. 356 this delay in rotavirus infection until the child is older may be beneficial in that the older child may be able to tolerate the infection or illness with a lower likelihood of becoming dehydrated or malnourished. continuing breastfeeding during an episode of rotavirus illness with or without vomiting is appropriate and often helpful to the infant. no reason to suspend breastfeeding by a mother infected with rotavirus is apparent. two rotavirus vaccines (rotateq and rotarix) have been licensed for use in more than 90 countries, but less than 20 countries have routine immunization programs. additional types of rotavirus vaccines are undergoing study in various countries, specifically examining the efficacy of the vaccines in low and medium income countries. 444 some of the explanations for the slow implementation of an effective vaccine globally include differences in protection with specific vaccines in high income countries compared with low or medium income countries, the unfortunate association with intussusception in the united states, the delayed recognition of the significant rotavirus-related morbidity and mortality, and the cost of the new vaccines. the question of variable efficacy of the specific rotavirus vaccines in developed and developing countries remains an important one. several trials are examining this issue and attempting to address factors such as maternal transplacentally transferred antibodies, breastfeeding practices (especially immediately before immunization with a live oral rotavirus vaccine), stomach acid, micronutrient malnutrition, interfering gut flora, and differences in the epidemiology of rotavirus in different locations. 327 evidence indicates that maternal immunization with rotavirus vaccine can increase both transplacental acquisition of antibodies and secretory iga in breast milk. 334 additionally, oral rotavirus vaccines have been able to stimulate a good serologic response in both formula-fed and breastfed infants, although the antigen titers may need to be modified to create an optimal response in all infants. 86 the actual protective effect of these vaccines in different situations and strategies will require measurement in ongoing prospective studies. congenital rubella infection has been well described, and the contributing variables to infection and severe disease have been elucidated. the primary intervention to prevent congenital rubella has been to establish the existence of maternal immunity to rubella before conception, including immunization with rubella vaccine and reimmunization if indicated. perinatal infection is not clinically significant. postnatal infection occurs infrequently in children younger than 1 year of age because of passively acquired maternal antibodies. the predominant age of infection is 5 to 14 years old, and more than half of those with infections are asymptomatic. postnatal rubella is a self-limited, mild viral infection associated with an evanescent rash, shotty adenopathy, and low-grade transient fever. it most often occurs in the late winter and spring. infants with congenital infection shed the virus for prolonged periods from various sites and may serve as a source of infection throughout the year. contact isolation is appropriate for suspected and proved congenital infection for at least 1 year, including exclusion from day care and avoidance of pregnant women, whereas postnatal rubella infection requires droplet precautions for 7 days after the onset of rash. rubella virus has been isolated from breast milk after natural infection (congenital or postnatal) and after immunization with live attenuated vaccine virus. both iga antibodies and immunoreactive cells against rubella have been identified in breast milk. breastfed infants can acquire vaccine virus infection via milk but are asymptomatic. because postpartum infection with this virus (natural or vaccine) is not associated with clinically significant illness, no reason exists to prevent breastfeeding after congenital infection, postpartum infection with this virus, or maternal immunization with rubella vaccine. severe acute respiratory syndrome (sars) is a term that could be applied to any acute serious respiratory illness caused by or associated with a variety of infections agents; since 2003, however, it has been linked with sars-associated coronavirus (sars-cov). in the global outbreak of 2002 to 2003, more than 8400 probable cases of sars and more than 800 deaths occurred. more than the actual number of affected individuals or its associated mortality rate (approximately 10% overall, and closer to 50% in persons older than 65 years of age), it was what we did not know about this new unusual illness, and the tremendous publicity surrounding it, that made sars such a sensation. we now know the cause of this illness, known as the sars-cov. sars-cov was shown not to be closely related to the previously characterized coronavirus groups. 265, 350 despite intense international collaboration to study the illness and the virus, many things are not known, such as the degree of infectiousness, the actual period of transmissibility, all the modes of transmission, how many people have an asymptomatic infection compared with those with symptoms or severe illnesses, how to make a rapid diagnosis of confirmed cases, and where it originated. at least 21 cases of probable sars in children have been described in the literature. 42, 187, 380, 387 in general, the illness in children is a mild, nonspecific respiratory illness, but in adolescents and adults it is more likely to progress to severe respiratory distress. it has been reported that children are less likely to transmit sars than adults. 187 the overall clinical course, the radiologic evolution, and the histologic findings of these illness are consistent with the host' s immune response playing a significant role in disease production. five infants were born to mothers with confirmed sars. the infants were born prematurely (26 to 37 weeks), presumably due to maternal illness. although two of the five infants had serious abdominal illnesses (other coronaviruses have been associated with reported outbreaks of necrotizing enterocolitis), the presence of sars-cov could not be demonstrated in any of these infants. 380 no evidence of vertical transmission of sars is available. the mode of feeding for any of the reported cases of young children with sars or the infants born to mothers with sars was not mentioned. as with other respiratory viruses predominantly transmitted by droplets, transmission via breast milk is an insignificant mode of transmission, if it occurs at all. the benefits of breastfeeding being what they are, mothers with sars should continue breastfeeding if they are able, or expressed breast milk can be given to an infant until the mother is able to breastfeed. in this era of worry about biologic terrorism, smallpox is an important concern. the concern for infants (breastfed or formula-fed) is direct contact with mothers or household members with smallpox. smallpox is highly contagious in the household setting due to person-to-person spread via droplet nuclei or aerosolization from the oropharynx and direct contact with the rash. additional potential exposures for infants include the release of a smallpox aerosol into the environment by terrorists, contact with a smallpox-contaminated space or the clothes of household members exposed to an aerosol, and infection via contact with a mother' s or a household member' s smallpox vaccination site. these risks are the same for breastfed and formulafed infants. no evidence for transmission of the smallpox virus via breast milk exists. a contact is defined as a person who has been in the same household or had face-to-face contact with a patient with smallpox after the onset of fever. patients do not transmit infection until after progression from the fever stage to the development of the rash. an exposed contact does not need to be isolated from others during the postcontact observation period (usually 17 days) until the person develops fever. temperature should be monitored daily in the exposed contact. personal contact and breastfeeding between mother and infant can continue until the onset of fever, when immediate isolation (at home) should begin. providing expressed breast milk for the infant of a mother with smallpox should be avoided because of the extensive nature of the smallpox rash and the possibility of contamination (from the rash) of the milk during the expression process. no literature documents transmission of the smallpox virus via expressed breast milk. the other issue for breastfeeding infants is the question of maternal vaccination with smallpox in a preexposure event vaccination program. children older than 1 year of age can be safely and reasonably vaccinated with smallpox in the face of a probable smallpox exposure. smallpox vaccination of infants younger than 1 year of age is contraindicated. breastfeeding is listed as a contraindication to vaccination in the preevent vaccination program. it is unknown whether vaccine virus or antibodies are present in breast milk. the risk for infection due to contact or aerosolization of virus from a mother' s smallpox vaccination site is the same for breastfed and formula-fed infants. the advisory committee on immunization practices also does not recommend preevent smallpox vaccination of children younger than 18 years old. 443 a report documents tertiary contact vaccinia in a breastfeeding infant. 140 a united states military person received a primary smallpox vaccination and developed a local reaction at the inoculation site. despite reportedly observing appropriate precautions, the individual' s wife developed vesicles on both areolae (secondary contact vaccinia). subsequently, the breastfeeding infant developed lesions on her philtrum, cheek, and tongue. both the mother and infant remained well and the infections resolved without therapy. culture and pcr testing confirmed vaccinia in both the mother' s and the infant' s lesions. the breast milk was not tested. 140 in a review from 1931 to 1981, sepkowitz 375 reported on 27 cases of secondary vaccinia in households. the cdc reported 30 suspected cases of secondary/tertiary vaccinia with 18 of those cases confirmed by culture or pcr. the 30 cases were related to 578,286 vaccinated military personnel. this is an incidence of 5.2 cases per 100,000 vac cinees and 7.4 cases per 100,000 primary vaccinees. 79 in a separate report on the civilian preevent smallpox vaccination program, 37,802 individuals were vaccinated between january and june 2003, and no cases of contact vaccinia were reported. 77 the risk for contact vaccinia is low. the risk is from close or intimate contact. in the above-mentioned case, the risk for the infant was contact with the mother' s breasts, the inadvertent site of her contact vaccinia. breastfed and formulafed infants are equally at risk from close contact in the household of a smallpox vaccinee or a case of secondary vaccinia, and separation from the individual is appropriate in both situations. if the breast of the nursing mother is not involved, expressed breast milk can be given to the infant. tt virus (ttv) is a recently identified virus found in a patient (tt) with posttransfusion hepatitis not associated with the other hepatitis-related viruses a through g. ttv has been described as an unenveloped, circular, single-stranded dna virus. 311 this virus is prevalent in healthy individuals, including healthy blood donors, and has been identified in patients with hepatitis. ttv dna has been detected in infants of ttv-positive and ttvnegative mothers. ohto et al 310 reported no ttv dna was detected in cord blood from 38 infants, and it was detected in only 1 of 14 samples taken at 1 month of age. they noted an increasing prevalence from 6 months (22%) to 2 years (33%), which they ascribed to acquisition via nonparenteral routes. in comparisons of the ttv dna in ttvpositive mothers and their ttv-positive infants, 6 of 13 showed high level nucleotide sequence similarity, and 7 of 13 differed by greater than 10%. 310 schröter et al 371 reported on ttv dna in breast milk examined retrospectively. notably, ttv dna was detected in 22 of 23 serum samples of infants at 1 week of age, who were born to 22 women viremic for ttv dna. twenty-four women who were negative for ttv dna gave birth to 24 children who were initially negative for ttv dna and remained negative throughout the observation period (mean 7.5 months, range 1 to 28 months). ttv dna was detected in 77% of breast milk samples from ttv viremic women and in none of the breast milk samples from ttv-negative women. no clinical or laboratory evidence of hepatitis was found in the 22 children who were observed to be ttv dna positive during the period of the study. 371 other authors have reported ttv in breast milk detected by pcr. they describe the absence of ttv dna in infants at 5 days and 3 months of age, and 4 of 10 infants were positive for ttv dna at 6 months of age, suggesting the late acquisition of infection via breastfeeding. 197 tt virus is transmitted in utero and is found in breast milk. no evidence of clinical hepatitis in infants related to ttv infection and no evidence for a late chronic hepatitis exist. given the current available information, no reason to proscribe breastfeeding by ttv-positive mothers is compelling. certainly more needs to be understood concerning the chronic nature of this infection and the possible pathogenesis of liver disease. no documented evidence indicates that women with breast cancer have rna of tumor virus in their milk. no correlation between rna-directed dna polymerase activity has been found in women with a family history of breast cancer. rna-directed dna polymerase activity, a reserve transcriptase, is a normal feature of the lactating breast. 91, 129, 352 epidemiologic data conflict with the suggestion that the tumor agent is transmitted through the breast milk. the incidence of breast cancer is low among groups who had nursed their infants, including lower economic groups, foreign-born groups, and those in sparsely populated areas. 262 the frequency of breast cancer in mothers and sisters of a woman with breast cancer is two to three times that expected by chance. this could be genetic or environmental. cancer actually is equally common on both sides of the family of an affected woman. if breast milk were the cause, it should be transmitted from mother to daughter. when mother-daughter incidence of cancer was studied, no relationship was found to breastfeeding. sarkar et al 360 reported that human milk, when incubated with mouse mammary tumor virus, caused degradation of the particular morphology and decreased infectivity and reverse transcriptase activity of the virions. they suggest that the significance of this destructive effect of human milk on mouse mammary tumor virus may account for the difficulty in isolating the putative human mammary tumor agent. sanner 359 showed that the inhibitory enzymes in milk can be removed by special sedimentation technique. he ascribes the discrepancies in isolating virus particles in human milk to these factors, which inhibit rna-directed dna po lymerase. the fear of cancer in breastfed female offspring of a woman with breast cancer does not justify avoiding breastfeeding. breastfed women have the same breast cancer experience as nonbreastfed women, and no increase is seen in benign tumors. daughters of breast cancer patients have an increased risk for developing benign and malignant tumors because of their heredity, not because of their breastfeeding history. 280, 287 unilateral breastfeeding (limited to the right breast) is a custom of tanka women of the fishing villages of hong kong. ing et al 194 investigated the question, "does the unsuckled breast have an altered risk for cancer?" they studied breast cancer data from 1958 to 1975. breast cancer occurred equally in the left and the right breasts. comparison of patients who had nursed unilaterally with nulliparous patients and with patients who had borne children but not breastfed indicated a highly significantly increased risk for cancer in the unsuckled breast. the authors conclude that in postmenopausal women who have breastfed unilaterally, the risk for cancer is significantly higher in the unsuckled breast. they thought that breastfeeding may help protect the suckled breast against cancer. 194 others 274 have suggested that tanka women are ethnically a separate people and that left-sided breast cancer may be related to their genetic pool and not to their breastfeeding habits. no mention has been made of other possible influences, such as the impact of their role as "fishermen" or any inherent trauma to the left breast. 274 in 1926, lane-claypon 240 stated that a breast that had never lactated was more liable to become cancerous. nulliparity and absence of breastfeeding had been considered important risk factors for breast cancer. macmahon et al 262 reported in 1970 that age at first full-term pregnancy was the compelling factor, and the younger the mother, the less the risk. in a collective review of the etiologic factors in cancer of the breast in humans, papaioannou 325 concludes, "genetic factors, viruses, hormones, psychogenic stress, diet, and other possible factors, probably in that order of importance, contribute to some extent to the development of cancer of the breast." 325 wing 449 concluded in her 1977 review on human milk and health that "in view of the complete absence of any studies showing a relationship between breastfeeding and increased risk of breast cancer, the presence of virus-like particles in breast milk should not be a contraindication to breastfeeding." henderson et al 173 gradually, studies have appeared challenging the dogma. brinton et al, 52 mctiernan and thomas, 276 and layde et al 245 showed the clearly protective effects of breastfeeding. another example is a study conducted to clarify whether lactation has a protective role against breast cancer in an asian people, regardless of confounding effects of age at first pregnancy, parity, and closely related factors. 458 in a hospital-based case-control study of 521 women without breast cancer, statistical adjustment for potential confounders and a likelihood ratio test for linear trend were done by unconditional logistic regression. total months of lactation regardless of parity was the discriminator. regardless of age of first pregnancy and parity, lactation had an independent protective effect against breast cancer in japanese women. 458 although breast cancer incidence is influenced by genetics, stress, hormones, and pregnancy, breastfeeding clearly has a protective effect. "there is a reduction in the risk of breast cancer among premenopausal women who have lactated. no reduction in the risk of breast cancer occurred among postmenopausal women with a history of lactation," according to newcombe et al, 299 reporting a multicenter study in 1993. varicella-zoster virus infection (varicella/chickenpox, zoster/shingles) is one of the most communicable diseases of humans, in a class with measles and smallpox. transmission is thought to occur via respiratory droplets and virus from vesicles. varicella in pregnancy is a rare event, although disease can be more severe with varicella pneumonia, and can be fatal. congenital varicella-zoster virus infection occurs infrequently, causing abortion, prematurity, and congenital malformations. a syndrome of malformations has been carefully described with congenital varicella-zoster virus infection, typically involving limb deformity, skin scarring, and nerve damage, including to the eye and brain. 148 perinatal infection can lead to severe infection in infants if maternal rash develops 5 days or less before delivery and within 2 days after delivery. illness in infants usually develops before 10 days of age and is believed to be more severe because of the lack of adequate transfer of antibody from the mother during this period and transplacental spread of virus to the fetus and infant during viremia in the mother. varicella in a mother occurring before 5 days before delivery allows sufficient formation and transplacental transfer of antibodies to the infant to ameliorate disease even if the infant is infected with varicella-zoster virus. mothers who develop varicella rash more than 2 days after delivery are less likely to transfer virus to the infant transplacentally; they pose a risk to the infants from postnatal exposure, which can be diminished by the administration of varicellazoster ig to the infant. postnatal transmission is believed to occur through aerosolized virus from skin lesions or the respiratory tract entering the susceptible infant's respiratory tract. airborne precautions are therefore appropriate in the hospital setting. infants infected with varicella-zoster virus in utero or in the perinatal period (younger than 1 month of age) are more likely to develop zoster (reactivation of latent varicella-zoster virus) during childhood or as young adults. postnatal varicella from nonmaternal exposure can occur but is generally mild when it develops after 3 weeks of age or when a mother has passed on antibodies against varicella-zoster virus via the placenta. severe postnatal varicella does occur in premature infants or infants of varicella-susceptible mothers. when a mother' s immune status relative to varicella-zoster virus is uncertain and measurement of antibodies to varicella-zoster virus in mother or infant cannot be performed promptly (less than 72 hours), administration of vzig 81 or ivig to the infant exposed to varicella or zoster in the postnatal period is indicated. ideally a mother' s varicella status should be known before pregnancy, when varicella virus vaccine could be given if indicated. varicella-zoster virus virus has not been cultured from milk, but varicella-zoster virus dna has been identified in breast milk. 459 antibody against varicella-zoster virus has also been found in breast milk. 270 breast milk from mothers who had received the varicella vaccine in the postpartum period was tested for varicella-zoster virus dna. varicella dna was not detected in any of the 217 breast milk samples from the 12 women, all of whom seroconverted after vaccination. 45 one case of suspected transfer of varicella-zoster virus to an infant via breastfeeding has been reported, but virus may have been transmitted by respiratory droplet or exposure to rash before the mother began antiviral therapy. 459 isolation of an infant from the mother with varicella and interruption of breastfeeding should occur only while the mother remains clinically infectious, regardless of the method of feeding. as soon as the infant has received varicella-zoster ig, expressed breast milk can be given to an infant if no skin lesions involve the breasts. persons with varicella rash are considered noninfectious when no new vesicles have appeared for 72 hours and all lesions have crusted, usually in 6 to 10 days. immunocompetent mothers who develop zoster can continue to breastfeed if the lesions do not involve the breast and can be covered because antibodies against varicella-zoster virus are provided to the infant via the placenta and breast milk and will diminish the severity of disease, even if not preventing it. conservative management in this scenario would include giving an infant varicella-zoster ig as well (see table 13 -5). 331 it is estimated that 150 to 300 asymptomatic cases of west nile infection occur for every 20 febrile illnesses and for every one case of meningoencephalitis associated with west nile virus. west nile fever is usually a mild illness of 3 to 6 days' duration. the symptoms are relatively nonspecific, including malaise, nausea, vomiting, headache, myalgia, lymphadenopathy, and rash. west nile disease is characterized by severe neurologic symptoms (e.g., meningitis, encephalitis, or acute flaccid paralysis, and occasionally optic neuritis, cranial nerve abnormalities, and seizures). children are infrequently sick with west nile virus infection and infants younger than 1 year of age have rarely been reported. 331 the case-fatality rate for 2003 in the united states was approximately 2.5%, but has been reported as high as 4% to 18% in hospitalized patients. the case-fatality rate for persons older than 70 years of age is considered to be higher, 15% to 29% among hospitalized patients in outbreaks in romania and israel. 331 the primary mechanism of transmission is via a mosquito bite. mosquitoes from the genus culex are primary vectors. the bird-mosquito-bird cycle serves to maintain and amplify the virus in the environment. humans and horses are incidental hosts. the pathogenesis of the infection is believed to occur via replication of the virus in the skin and lymph nodes, leading to a primary viremia that seeds secondary sites before a second viremia causes the infection of the cns and other affected organs. 59, 111 transmission has been reported in rare instances during pregnancy 7,73 via organ transplant 199 and percutaneously in laboratory workers. 75 a study of west nile virus infection in pregnancy documented four miscarriages, two elective abortions, and 72 live births. cord blood samples were tested in 55 infants and 54 of 55 were negative for anti-west nile virus igm. three infants had west nile virus infection, which could have been acquired congenitally. three of 7 infants who had congenital malformations might have been caused by maternal west nile virus infection based on timing in pregnancy, but no evidence of west nile virus etiology is conclusive. 312 west nile virus transmission occurs via blood and blood product transfusion, 186 and the incidence has been estimated to be as high as 21 per 10,000 donations during epidemics in specific cities. 40 no evidence of direct person-to-person transmission without the mosquito vector has been found. one case of possible west nile virus transmission via breastfeeding has been documented. 74 the mother acquired the virus via packed rbc transfusions after delivery. the second unit of blood she received was associated with other blood products from the same donation causing west nile infection in another transfusion recipient. eight days later the mother had a severe headache and was hospitalized with fever and a csf pleocytosis on day 12 after delivery. the mother' s csf was positive for west nile virus-specific igm antibody. the infant had been breastfed from birth through the second day of hospitalization of the mother. samples of breast milk were west nile virus-specific igg and igm positive on day 16 after delivery and west nile virus-specific igm positive on day 24. the same milk was west nile virus rna positive by pcr testing on day 16, but not on day 24 after delivery. the infant tested positive for west nile virus-specific igm in serum at day 25 of age, but remained well without fever. no clear-cut exposure to mosquitoes for the infant were reported. the cord blood and placenta were not available to be tested. igm antibodies can be found in low concentrations in breast milk, but this is not common or as efficient as the transfer of iga, secretory iga, or igg into breast milk. a review of west nile virus illness during the breastfeeding identified six occurrences of breastfeeding during maternal west nile virus illness. 177 five of the six infants had no illness or detectable antibodies to west nile virus in their blood. one infant developed a rash and was otherwise well after maternal west nile virus illness, but was not tested for west nile virus infection. two infants were identified who developed west nile virus illness while breastfeeding, but no preceding west nile virus infection was demonstrated in their mothers. two other breastfeeding infants developed west nile virus-specific antibodies after their mothers acquired west nile virus illness in the last week of pregnancy, but congenital infection could not be ruled out. live virus was not cultured from 45 samples of breast milk from mothers infected with west nile virus during pregnancy, but west nile virus rna was detected in two samples and 14 samples had igm antibodies to west nile virus. 177 the above data suggest that west nile virus infection through breastfeeding is rare. to date evidence of significant disease due to west nile virus infection in young breastfeeding children is lacking. at this time, no reason exists to proscribe breastfeeding in the case of maternal west nile virus infection if a mother is well enough to breastfeed. as with many other maternal viral illnesses, by the time the diagnosis is made in a mother, the infant may have already been exposed during maternal viremia and possible virolactia. the infant can and should continue to receive breast milk for the potential specific and nonspecific antiviral immunologic benefits. yellow fever virus is a flavivirus which is transmitted to humans by infected aedes and haemogogus mosquitos in tropical areas of south america and africa. large outbreaks occur when mosquitos in a populated area become infected from biting viremic humans infected with yellow fever virus. transmission from the mosquitos to other humans occurs after an incubation period in the mosquito of 8 days. direct person-to-person spread has not been reported. illness due to yellow fever virus usually begins after an incubation period of 3 to 6 days, with acute onset of headache, fever, chills, and myalgia. photophobia, back pain, anorexia, vomiting, and restlessness are other common symptoms. the individual is usually viremic for the first 4 days of illness until the fever and other symptoms diminish. liver dysfunction and even failure can develop as can myocardial dysfunction. cns infection is uncommon but symptoms can include seizures and coma. medical care should include intensive supportive care and fluid management. one case of congenital infection after immunization of a pregnant woman with the attenuated vaccine strain has been reported. one of 41 infants whose mothers had inadvertently received the yellow fever virus vaccine during pregnancy developed igm and elevated neutralizing antibodies against the yellow fever virus without any evidence of illness or abnormalities. 418 a more recent study 404 from brazil examined inadvertent yellow fever virus immunization during pregnancy during a mass vaccination campaign in 2000; 480 pregnant women received the yellow fever virus at a mean of 5.7 weeks' gestation, the majority of whom did not know their pregnancy status at the time. seroconversion occurred in 98.2% of the women after at least 6 weeks after vaccination. mild postvaccination illness (headache, fever, or myalgia) was reported by 19.6% of the 480 women. the frequency of malformations, miscarriages, stillbirths, and premature deliveries was similar to that found in the general population. at the 12-month follow-up point, 7% of the infants still demonstrated neutralizing antibodies against yellow fever virus, but after 12 months only one child was still seropositive. 404 transmission of the yellow fever vaccine virus through breastfeeding was recently reported from brazil. 85 the mother was immunized during a yellow fever epidemic in a nonendemic area in brazil; 15 days after delivering a healthy female infant (39 weeks' gestational age) the mother received the 17dd yellow fever vaccine, and 5 days later the mother reported headache, malaise, and low-grade fever that persisted for 2 days. the mother continued breastfeeding and did not seek medical care for herself. at 23 days of age the infant became irritable, developed fever, and refused to nurse. the infant developed seizures and subsequent evaluation of the infant demonstrated an abnormal csf and ct of the brain showed bilateral areas of diffuse low density suggestive of inflammation and consistent with encephalitis. yellow fever-specific igm antibodies were identified in the infant' s serum and csf. reverse-transcriptase polymerase chain reaction (rt-pcr) testing of the csf also demonstrated yellow fever virus rna identical to the 17dd yellow fever vaccine virus. breast milk and maternal serum were not tested for yellow fever virus. 85 yellow fever virus, wild or vaccine type, has not been identified in human breast milk, although another flavivirus, west nile virus, has been detected in milk from a few lactating women with west nile virus infection. 177 (see the section on west nile virus.) yellow fever vaccine-associated neurologic disease occurs at different rates in different age-groups, including 0.5 to 4.0 cases per 1000 infants younger than 6 months of age. 285 the 17d-derived yellow fever vaccines are contraindicated in infants younger than 6 months of age. since 2002, the advisory committee on immunization practices has recommended, based on theoretical risk, that yellow fever vaccine be avoided in nursing mothers, except when exposure in highrisk yellow fever endemic areas is likely to occur. 76 no case of transmission of yellow fever virus from an infected mother to her infant via breastfeeding or breast milk has been reported. published information on the severity of yellow fever virus infection in infants younger than 1 year of age, potential protection from passively acquired antibodies, or protection from breast milk is limited. no information on a differential risk in breastfed versus formula-fed infants is available. given the well documented method of transmission of yellow fever virus via mosquitos, and the lack of evidence of transmission via breast milk, it makes more sense to protect all infants against mosquito bites than to proscribe breastfeeding, even in the mother infected with yellow fever virus. continued breastfeeding or use of expressed breast milk will depend on a mother's health status and ability to maintain the milk supply while acutely ill. if another source of feeding is readily available then temporarily discarding expressed breast milk for at least 4 days of acute illness in the mother is a reasonable precaution. lyme disease, as with other human illnesses caused by spirochetes, especially syphilis, is characterized by a protean course and distinct phases (stages) of disease. lyme borreliosis was described in europe in the early twentieth century. since the 1970s, tremendous recognition, description, and investigation of lyme disease have occurred in the united states and europe. public concern surrounding this illness is dramatic. lyme disease is a multisystem disease characterized by involvement of the skin, heart, joints, and nervous system (peripheral and central). stages of disease are identified as early localized (erythema migrans, often accompanied by arthralgia, neck stiffness, fever, malaise, and headache), early disseminated (multiple erythema migrans lesions, cranial nerve palsies, meningitis, conjunctivitis, arthralgia, myalgia, headache, fatigue, and, rarely, myocarditis), and late disease (recurrent arthritis, encephalopathy, and neuropathy). the varied manifestations of disease may relate to the degree of spirochetemia, the extent of dissemination to specific tissues, and the host' s immunologic response. the diagnosis of lyme disease is often difficult in part because of the broad spectrum of presentations, inapparent exposure to the tick, and the lack of adequately standardized serologic tests. culture of the spirochete, borrelia burgdorferi, is not readily available. enzyme-linked immunosorbent assay (elisa), immunofluorescent assay, and immunoblot assay are the usual tests. pcr detection of spirochetal dna requires additional testing in clinical situations to clarify and standardize its utility. gardner 142 reviewed infection during pregnancy, summarizing a total of 46 adverse outcomes from 161 cases reported in the literature. the adverse outcomes included miscarriage and stillbirth (11% of cases), perinatal death (3%), congenital anomalies (15%), and both early-and late-onset progressive infection in the infants. silver 384 reviewed 11 published reports and concluded that lyme disease during pregnancy is uncommon, even in endemic areas. although the spirochete can be transmitted transplacentally, a significant immune response in the fetus is often lacking, and the association of lyme infection with congenital abnormalities is weak. 401, 448 little published information exists on whether b. burgdorferi can be transmitted via breast milk. one report showed the detection of b. burgdorferi dna by pcr in the breast milk of two lactating women with untreated erythema migrans, but no evidence of lyme disease or transmission of the spirochete in the one infant followed for 1 year. 369 no attempt to culture the spirochete was made, so it is not possible to determine if the detectable dna was from viable spirochetes or noninfectious fragments. in that same study of 56 women with untreated erythema migrans who had detectable b. burgdorferi dna in the urine, 32 still had detectable dna in the urine 15 to 30 days after starting treatment, but none had it 6 months after initiating therapy. ziska et al 466 reported on the management of nine cases of lyme disease in women associated with pregnancy; seven of the nine women were symptomatic at conception and six received antibiotics throughout pregnancy. follow-up of the infants showed no transmission of lyme disease, even in the seven infants who had been breastfed. 466 the lack of adequate information on transmission of b. burgdorferi via breast milk cannot be taken as proof that it is not occurring. if one extrapolates from data on syphilis and the treponema pallidum spirochete, it would be prudent to discuss the lack of information on the transmission of b. burgdorferi via breast milk with the mother or parents and to consider withholding breast milk at least until therapy for lyme disease has begun or been completed. if the infection occurred during pregnancy and treatment has already been completed, an infant can breastfeed. if infection occurs postpartum or the diagnosis is made postpartum, infant exposure may have already occurred. again, discussion with the mother or parents about withholding versus continuing breastfeeding is appropriate. after prenatal or postnatal exposure, an infant should be closely observed and empiric therapy considered if the infant develops a rash or symptoms suggestive of lyme borreliosis. treatment of mother and infant with ceftriaxone, penicillin, or amoxicillin is acceptable during breastfeeding relative to the infant' s exposure to these medications. doxycycline should not be administered for more than 14 days while continuing breastfeeding because of possible dental staining in the neonate. continued surveillance for viable organisms in breast milk and evidence of transmission through breastfeeding is recommended. a large body of information is available on various "lyme vaccines" used in dogs, but these vaccines are only partially protective and must be repeated yearly. preliminary information suggests that a vaccine for use in humans safely produces good serologic responses, but protective efficacy has not been demonstrated, and no information exists on its use during pregnancy or breastfeeding. syphilis is the classic example of a spirochetal infection that causes multisystem disease in various stages. both acquired syphilis and congenital syphilis are well-described entities. acquired syphilis is almost always transmitted through direct sexual contact with open lesions of the skin or mucous membranes of individuals infected with the spirochete, treponema pallidum. congenital syphilis occurs by infection across the placenta (placentitis) at any time during the pregnancy or by contact with the spirochete during passage through the birth canal. any stage of disease (primary, secondary, tertiary) in a mother can lead to infection of the fetus, but transmission in association with secondary syphilis approaches 100%. infection with primary syphilis during pregnancy, without treatment, leads to spontaneous abortion, stillbirth, or perinatal death in 40% of cases. similar to acquired syphilis, congenital syphilis manifests with moist lesions or secretions from rhinitis (snuffles), condylomata lata, or bullous lesions. these lesions and secretions contain numerous spirochetes and are therefore highly infectious. postnatal infection of an infant can occur through contact with open, moist lesions of the skin or mucous membranes of the mother or other infected individuals. if the mother or infant has potentially infectious lesions, isolation from each other and from other infants and mothers is recommended. if lesions are on the breasts or nipples, breastfeeding or using expressed milk is contraindicated until treatment is complete and the lesions have cleared. spirochetes are rarely identified in open lesions after more than 24 hours of appropriate treatment. penicillin remains the best therapy. evaluation of an infant with suspected syphilis should be based on the mother' s clinical and serologic status, history of adequate therapy in the mother, and the infant' s clinical status. histologic examination of the placenta and umbilical cord, serologic testing of the infant' s blood and csf, complete analysis of the csf, long bone and chest radiographs, liver function tests, and a complete blood cell count are all appropriate given the specific clinical situation. treatment of the infant should follow recommended protocols for suspected, probable, or proven syphilitic infection. 96 no evidence indicates transmission of syphilis via breast milk in the absence of a breast or nipple lesion. when a mother has no suspicious breast lesions, breastfeeding is acceptable as long as appropriate therapy for suspected or proven syphilis is begun in the mother and infant. giardiasis is a localized infection limited to the intestinal tract, causing diarrhea and malabsorption. immunocompetent individuals show no evidence of invasive infection, and no evidence exists that documents fetal infection from maternal infection during pregnancy. giardiasis is rare in children younger than 6 months of age, although neonatal infection from fecal contamination at birth has been described. 22 human milk has an in vivo protective effect against giardia lamblia infection, as documented by work from central africa, where the end of breastfeeding heralds the onset of giardia infection. 145 this has been reaffirmed in undeveloped countries around the world. the protective effect of breast milk has been identified in the milk of noninfected donors. 151 the antiparasitic effect does not result from specific antibodies but rather from lipase enzymatic activity. the lipase acts in the presence of bile salts to destroy the trophozoites as they emerge from their cysts in the gi tract. hernell et al 175 demonstrated that free fatty acids have a marked giardiacidal effect, which supports the conclusion that lipase activity releasing fatty acids is responsible for killing g. lamblia. g. lamblia have also been reported to appear in the mother' s milk, and the parasite has been transmitted to newborns via that route. the exact relationship of breastfeeding to transmission of g. lamblia and the effect on infants continue to be studied, even though symptomatic infection in breastfed infants is rare. 151 one report from the middle east suggests that even partial breastfeeding is protective against infection with intestinal parasites, including cryptosporidium and giardia lamblia. 41 breastfeeding by mothers with giardiasis is problematic mainly because of the medications used for therapy. metronidazole' s safety in infants has not been established, and little information is available on quinacrine hydrochloride and furazolidone in breast milk. paromomycin, a nonabsorbable aminoglycoside, is a reasonable alternative recommended for treatment of pregnant women. breastfeeding by a mother with symptomatic giardiasis is acceptable when consideration is given to the presence of the therapeutic agents in the breast milk. hookworm infection, most often caused by ancylostoma duodenale and necator americanus, is common in children younger than the age of 4 years, and there is at least one report on infantile hookworm disease from china. 374 this publication from the chinese literature reports hundreds of cases of infantile hookworm disease that include the common symptoms of bloody stools, melena, anorexia, listlessness, and edema. anemia, eosinophilia and even leukemoid reactions occur as part of the clinical pictures in young children. they also note at least 20 cases of hookworm diseases in newborn infants younger than 1 month of age. in the discussion of infantile hookworm infection, they note four routes of infection: direct contact with contaminated soil, "sand-stuffed" diapers, contaminated "washed/wet" diapers, and vertical equal to transmammary transmission or transplacental transmission. they postulated that infection of infants before 40 to 50 days of age would most likely be due to transplacental transmission and infection before environmental contact would most likely be due to transmammary transmission. ample evidence is available in veterinary medicine of transmammary spread of helminths. 302, 382 at least two reports suggest the possibility of transmammary transmission of hookworms in humans. setasuban et al 376 described the prevalence of necator americanus in 128 nursing mothers as 61% and identified n. americanus in breast milk in one case. nwosu 303 documented positive stool samples for hookworms in 33 of 316 neonates (10%) at 4 to 5 weeks of age in southern nigeria. the majority of neonatal infections were due to ancylostoma duodenale although necator americanus is more prevalent in that area of nigeria. examination of colostral milk did not demonstrate any hookworm larvae. 303 additional epidemiologic work is necessary to determine the potential significance of transmammary spread of helminths in humans, and more careful examination of breast milk as a source of hookworm infection is required before reasonable recommendation are possible. malaria is recognized as a major health problem in many countries. the effect of malaria infection on pregnant and lactating women and thus on the developing fetus, neonate, and growing infant can be significant. the four species of malaria, plasmodium vivax, p. ovale, p. malariae, and p. falciparum, vary in the specific aspects of the disease they produce. p. vivax exists throughout the world, but p. falciparum predominates in the tropics and is most problematic in its chloroquine-resistant form. malaria in the united states is most often seen in individuals traveling from areas where malaria is endemic. the parasite can exist in the blood for weeks, and infection with p. vivax and p. malariae can lead to relapses years later. transmission occurs through the bite of the anopheline mosquito and can occur via transfusion of blood products and transplacentally. congenital malaria is rare but seems to occur more often with p. vivax and p. falciparum. it usually presents in the first 7 days of life (range 1 day to 2 months). it may resemble neonatal sepsis, with fever, anemia, and splenomegaly occurring in the most neonates and hyperbilirubinemia and hepatomegaly in less than half. malaria in infants younger than 3 months of age generally manifests with less severe disease and death than in older children. possible explanations include the effect of less exposure to mosquitoes, passive antibody acquired from the mother, and the high level of fetal hemoglobin in infants at this age. 22 the variations in the infection rates in children younger 3 months of age during the wet and dry seasons support the idea that postnatal infection is more common than congenital infection. no evidence indicates that malaria is transmitted through breast milk. the greatest risk to infants is exposure to the anopheline mosquito infected with malaria. the main issues relative to malaria and breastfeeding are how to protect both mothers and infants effectively from mosquitoes and what drugs for treating malaria in mothers are appropriate during lactation. protection from mosquito bites includes screened-in living areas, mosquito nets while sleeping, protective clothing with or without repellents on the clothes, and community efforts to eradicate the mosquitoes. chloroquine, quinine, and tetracycline are acceptable during breastfeeding. sulfonamides should be avoided in the first month of an infant' s life, but pyrimethamine-sulfadoxine (fansidar) can be used later. mefloquine is not approved for infants or pregnant women. however, the milk/plasma ratio for mefloquine is less than 0.25, there is a large volume of distribution of the drug, high protein binding of the drug limits its presence in breast milk, and the relative importance of breastfeeding in areas where malaria is prevalent shifts the risk/benefit ratio in favor of treatment with mefloquine. the single dose recommended for treatment or the onceweekly dose for prevention allows for continued breastfeeding with discarding of the milk for short periods after a dose (1 to 6 hours). maternal plasma levels of primaquine range from 53 to 107 ng/ml, but no information is available on levels in human milk. primaquine is used in children, and once daily dosing in the mother would allow discarding milk with peak levels of drug. therefore breastfeeding during maternal malaria even with treatment is appropriate with specific medications. strongyloides stercoralis is a nematode (roundworm). most infections are asymptomatic, but clinically significant infection in humans can include larval skin invasion, tissue migration, intestinal invasion with abdominal pain and gi symptoms, and a loeffler-like syndrome due to migration to the lungs. immune-compromised individuals can develop dissemination of larvae systemically, causing various clinical symptoms. humans are the principal hosts, but other mammals can serve as reservoirs. infection via the skin by filariform larvae is the most common form of transmission; ingestion is an uncommon occurrence. transmammary transmission of strongyloides species has been described in dogs, ewes, and rats. 211, 302, 382 only one report of transmammary passage of strongyloides larvae in humans is available. in 76 infants younger than 200 days of age, 34% demonstrated the presence of strongyloides fuelleborni on stool examination. the clinical significance of this was not elucidated. strongyloides larvae was identified in only one sample of milk from 25 nursing mothers. 53 in the absence of an understanding of the clinical significance of strongyloides in the stools of young infants, given the lack of exclusion of the most common mechanism of transmission (through the skin) in the single report and the apparent infrequent evidence of these larvae in human milk, it is difficult to make any recommendations concerning breastfeeding and strongyloides. toxoplasmosis is one of the most common infections of humans throughout the world. the infective organism, toxoplasma gondii, is ubiquitous in nature. the prevalence of positive serologic test titers increases with age, indicating past exposure and infection. the cat is the definitive host, although infection occurs in most species of warmblooded animals. postnatal infection with toxoplasmosis is usually asymptomatic. symptomatic infection typically manifests with nonspecific symptoms, including fever, malaise, myalgia, sore throat, lymphadenopathy, rash, hepatosplenomegaly, and occasionally a mononucleosis-like illness. the illness usually resolves without treatment or significant complications. congenital infection or infection in an immunodeficient individual can be persistent and severe, causing significant morbidity and even death. although most infants with congenital infection are asymptomatic at birth, visual abnormalities, learning disabilities, and mental retardation can occur months or years later. the syndrome of congenital toxoplasmosis is clearly defined, with the most severe manifestations involving the cns, including hydrocephalus, cerebral calcifications, microcephaly, chorioretinitis, seizures, or simply isolated ocular involvement. the risk for fetal infection is related to the timing of primary maternal infection, although transmission can occur with preexisting maternal toxoplasmosis. 241 in the last months of pregnancy the protozoan is more readily transmitted to the fetus, but the infection is more likely to be subclinical. early in pregnancy the transmission to a fetus occurs less frequently but does result in severe disease. treatment of documented congenital infection is currently recommended, although duration and optimal regimen have not been determined, and reversal of preexisting sequelae generally does not occur. 343 prevention of infection in susceptible pregnant women is possible by avoiding exposure to cat feces or the organism in the soil. pregnant or lactating women should not change cat litter boxes, but if they must, it should be done daily and while wearing gloves. the oocyst is not infective for the first 24 to 48 hours after passage. mothers can avoid ingestion of the organism by fully cooking meats and carefully washing fruits, vegetables, and food preparation surfaces. 94 in various animal models, t. gondii has been transmitted through the milk to the suckling young. the organism has been isolated from colostrum as well. the newborn animals became asymptomatically infected when nursed by an infected mother whose colostrum contained t. gondii. only one report has identified t. gondii in human milk, and some question surrounds the reliability of that report. 241 transmission during breastfeeding in humans has not been demonstrated. breast milk may contain appropriate antibodies against t. gondii. given the benign nature of postnatal infection, the absence of documented transmission in human breast milk, and the potential antibodies in breast milk, no reason exists to proscribe breastfeeding by a mother known to be infected with toxoplasmosis. trichomonas vaginalis is a flagellated protozoan that can produce vaginitis (see chapter 16 for a discussion of vaginitis) but frequently causes asymptomatic infection in both men and women. the parasite is found in 10% to 25% of women in the childbearing years. it is transmitted predominantly by sexual intercourse, but it can be transmitted to the neonate by passage through the birth canal. this parasite often coexists with other stds, especially gonorrhea. infection during pregnancy or while taking oral contraceptives is more difficult to treat. some evidence suggests that infection with and growth of the parasite are enhanced by estrogens or their effect on the vaginal epithelium. no evidence indicates adverse effects on the fetus in association with maternal infection during pregnancy. occasionally female newborns have vaginal discharge during the first weeks of life caused by t. vaginalis. this is thought to be influenced by the effect of maternal estrogen on the infant' s vaginal epithelium and acquisition of the organism during passage through the birth canal. the organism does not seem to cause significant disease in a healthy infant. no documentation exists on transmission of t. vaginalis via breast milk. the difficulty encountered with maternal infection during lactation stems from metronidazole (flagyl), the drug of choice, being contraindicated for infants. case reports describe treatment of neonates with metronidazole without adverse effect. although topical agents containing povidone-iodine (betadine) or sodium lauryl sulfate (trichotine) can be effective when given as douches, creams, or suppositories, metronidazole remains the treatment of choice. the aap advises using metronidazole only with a physician' s discretion and considers its effect on a nursing infant unknown but possibly a concern. the potential concerns are metronidazole' s disulfiram-like effect in association with alcohol, tumorigenicity in animal studies, and leukopenia and neurologic side effects described in adults. on the other hand, metronidazole is given to children beyond the neonatal period to treat serious infections with various other parasites, such as entamoeba histolytica. the current recommendation for lactating women is to try local treatment first, and if these fail, then to try metronidazole. a 2-g single-dose treatment produces peak levels after 1 hour, and discarding expressed breast milk for the next 12 to 24 hours is recommended. if this treatment also fails, a 1-g twice-daily regimen for 7 days or a 2-g single daily dose for 3 to 5 days is recommended, with discarding of breast milk close to the dose and timing of feedings distant from the dose. infants who exclusively breastfeed are presumed at greater risk from exposure to metronidazole than those who are only partially breastfed. candida consists of multiple species. the most common species affecting humans include c. albicans as the dominant agent and c. tropicalis, c. krusei, and c. parapsilosis, as well as many other uncommon species. in general, candida exists as a commensal organism colonizing the oropharynx, gi tract, vagina, and skin without causing disease until some change disrupts the balance between the organism and the host. mild mucocutaneous infection is the most common illness, which can lead to vulvovaginitis, mastitis, or, uncommonly, oral mucositis in a mother, and thrush (oral candidiasis) and candidal diaper rash in an infant. invasive candidal infection occurs infrequently, usually when a person has other illness, impaired resistance to infection (hiv, diabetes mellitus, neutropenia; decreased cell-mediated immunity in premature infants or lbw or vlbw infants), or disrupted normal mucosal and skin barriers and has received antibiotics or corticosteroids. invasive disease can occur through local spread, and may occur more often in the genitourinary tract (urethra, bladder, ureters, kidneys), but usually develops in association with candidemia. the bladder and kidney are more frequently involved, but when dissemination occurs via candidemia, a careful search for other sites of infection should be made (e.g., retina, liver, spleen, lung, meninges). 279 transmission usually occurs from healthy individuals colonized with candida through direct contact with them or through contact with their oral or vaginal secretions. intrauterine infection can occur through ascending infection through the birth canal but is rare. no distinct syndrome of congenital candidal infection exists. most often an infant is infected in passing through the birth canal and remains colonized. postnatal transmission can occur through direct contact with caregivers. the mother and infant serve as an immediate source of recolonization for each other, especially during the direct contact of breastfeeding. for this reason, an infant and breastfeeding mother should be treated simultaneously when treating thrush, vulvovaginitis, diaper candidiasis, or mastitis. colonization with this organism usually occurs in the absence of any clinical evidence of infection. simultaneous treatment should occur even in the absence of any clinical evidence of candida infection or colonization in the apparently uninvolved individual of the breastfeeding dyad. no well-controlled clinical trials define the most appropriate or most effective method(s) of treatment for candidal infection in breastfeeding mother-infant pairs. the list of possible treatment products is extensive and includes many anecdotal and empirical regimens. in the face of this absence of data, brent 51 conducted a survey of members of the academy of breastfeeding medicine concerning the respondents' approach to diagnosis and treatment of thrush in the breastfeeding dyad. most of the respondents relied on the history and physical examination of the infant, but only a third rated the examination of the mother as very important in making a diagnosis. only 7% reported using laboratory testing to make the diagnosis. twentyone percent of the respondents reported using only oral nystatin for the infant when the mother was asymptomatic. almost half treated the infant and the mother with topical nystatin, and 13% used oral nystatin for the infant and oral fluconazole for the mother when the mother had breast pain. less than 5% used oral fluconazole for both infant and mother, and other therapies were used by about 15% of the respondents. for recurrence of persistence of the thrush, more respondents reported treating the mother or both the infant and mother with fluconazole, and almost a quarter reported using other therapies. considerable discussion of mammary candidosis/candidiasis, the clinical diagnosis of candidal involvement of the breast, the significance of pain with breastfeeding, and the presence or absence of candida albicans in milk samples is ongoing. 14, 133, 166 this topic will continue to be debated because additional prospective studies are necessary to clarify specific issues. data are inadequate to make specific recommendations about various clinical situations regarding candida and breastfeeding. clinical practice will vary with experience, especially for the more problematic clinical situations. some general guidelines follow. (see chapter 16 for a discussion of mastitis.) the treatment of mucocutaneous candidiasis should probably begin with a topical agent, such as nystatin, clotrimazole, miconazole, econazole, butaconazole, terconazole, or ciclopirox. treatment should continue for at least 2 weeks, even with obvious improvement in 1 or 2 days. failures most often result from inadequate therapy involving the frequency of application, careful washing and drying before application, or, in the case of diaper candidiasis, decreasing the contact of the skin with moisture. nystatin oral suspension is less effective for the treatment of oral candidiasis in infants, now compared with the past, supposedly due to increasing resistance. 154 gentian violet (diluted to 0.25% to 1.0%) applied to the breast or painted onto an infant' s mouth is being recommended more frequently. other topical preparations have been recommended for the mother' s breast including mupirocin, grapefruit seed extract, or mixtures of mupirocin, betamethasone ointments, and miconazole powder. controlled clinical trials for efficacy and toxicity are not available. when good adherence to the proposed regimen with topical agents fails, or when infant or mother are severely affected by pain and decreased breastfeeding, systemic therapy is appropriate. fluconazole and ketoconazole are the most commonly used systemic agents for oral or diaper candidiasis and vulvovaginitis or mastitis. fluconazole has a better side effect profile than ketoconazole, and more data are available concerning its safe use in children younger than 6 months of age and even neonates and premature infants. 87, 154, 209 fluconazole is not currently approved for use in infants younger than 6 months of age. for severe invasive infections in infants, amphotericin b with or without oral flucytosine, iv fluconazole, voriconazole or caspofungin are reasonable choices in different situations. use of itraconazole in infants has not been adequately studied to date. maternal use of fluconazole during breastfeeding is not contraindicated because only a small amount of medicine compared with the usual infant dose reaches the infant through breast milk. amphotericin or caspofungin therapy in mothers is also not contraindicated because these are both poorly absorbed from the gi tract. whenever a mother is treated for candidal mastitis or vulvovaginitis, the infant should be treated simultaneously, at least with nystatin oral suspension as the first choice of medication. any predisposing risk factors for candidal infection in mothers and infants should be reduced or eliminated to improve the chance of rapid, successful treatment and to decrease the likelihood of chronic or recurrent disease. for mothers, such interventions might include decreasing sugar consumption, stopping antibiotic use as soon as possible, and consuming some form of probiotic bacteria, such as acidophilus (in yogurt, milk, or pill form), to reestablish a normal colonizing bacterial flora. for infants, breastfeeding can enhance the growth of specific colonizing bacterial flora such as lactobacillus, which can successfully limit fungal growth. breastfeeding should continue with appropriate support and problem-solving with a professional who is knowledgeable about breastfeeding. hiv-1, hiv-2, htlv-i, and htlv-ii are the only infectious diseases that are considered absolute contraindications to breastfeeding in developed countries. when the primary route of transmission is via direct contact or respiratory droplets/particles, temporary separation of mother and infant may be appropriate (whether the infant is breastfed or formula fed), but expressed breast milk should be given to the infant for the organism-specific immunologic benefits in the mother' s milk. in most instances, by the time a specific diagnosis of infection is made for a mother, the infant has already been exposed to the organism and providing expressed breast milk to the infant should continue. (refer to appendix f for specific exceptions, such as lassa fever.) regarding antimicrobial therapy for mothers and continued breastfeeding, the majority of the medications commonly used in adults can be used to treat the same infection in infants. the additional amount of medication received by infants via breast milk is usually insignificant. in almost all instances, an appropriate antimicrobial agent for treating mothers that is also compatible with breastfeeding can be chosen. unless the risk to infants for transmission of an infectious agent via breast milk that leads to a clinically significant illness in the infants is documented, breastfeeding should continue. measles antibodies in the breast milk of nursing mothers spectrum of breast tuberculosis respiratory syncytial virus infection among young children with acute respiratory tract infection in iraq probable breast milk borne brucellosis in a young infant breast milk transmission of cytomegalovirus (cmv) infection congenital and perinatal cytomegalovirus infections intrauterine west nile virus: ocular and systemic findings the cloning and clinical implications of hgv and hgbv-c bottle feeding can prevent transmission of htlv-i from mothers to their babies transmission of adult t-cell leukemia retrovirus (htlv-i) from mother to child: comparison of bottle-with breastfed babies effect of freezethawing breast milk on vertical htlv-i transmission from seropositive mothers to children long-term follow up study of vertical htlv-i infection in children breastfed by seropositive mothers long-term followup study of htlv-i infection in bottle-fed children born to seropositive mothers the yeast connection: is candida linked to breastfeeding associated pain? epidemiology of group b streptococcus: maternal and nosocomial sources for acquisition tuberculosis and pregnancy and tuberculous mastitis infant botulism infant botulism: anticipating the second decade protective role of human milk against sudden death from infant botulism growth faltering due to breastfeeding cessation in uninfected children born to hiv-infected mothers in zambia other viral infections of the fetus and newborn protozoan and helminth infections (including pneumocystis carinii) recurrent group b streptococcal disease in infants: who should receive rifampin? methicillin-resistant staphylococcus aureus sccmec type iv: nosocomial transmission and colonisation of healthcare workers in a neonatal intensive care unit stringent precautions are advisable when caring for patients with viral hemorrhagic fevers prevalence of methicillin-resistant staphylococcus aureus in expressed breast milk in a neonatal intensive care unit transmision de brucelosis por lactancia materna: presentacion de dos casos congenital lymphocytic choriomeningitis virus infection in twins poliomyelitis in pregnancy, fetus and newborn assessment of the risk of ebola virus transmission from bodily fluids and fomites transmission of hepatitis by breastfeeding 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has a history of chickenpox, she may return home. without a history, she should be tested for varicellazoster virus antibody titer.* if test is positive, she may return home. if test is positive, she may return home. if test is negative, varicella-zoster ig † is administered and she is discharged home. 2. neonate: may be discharged home with mother if mother has history of varicella or is varicella-zoster virus-antibody positive. if mother is susceptible, administer varicella-zoster ig to infant and discharge home or place in protective isolation. b. mother has no history of chickenpox; exposed during period 6-20 days antepartum. ‡ ‡if exposure occurred less than 6 days antepartum, mother would not be potentially infectious until at least 72 hours postpartum. §considered noninfectious when no new vesicles have appeared for 72 hours and all lesions have crusted. ¶dosage of acyclovir for pregnant woman is 30 mg/kg/day; for seriously ill infant with varicella, 750 to 1500 mg/m 2 /day. elisa, enzyme-linked immunosorbent assay; fama, fluorescent antibody to membrane antigen; la, latex agglutination. key: cord-016903-z2vqfq98 authors: herndler-brandstetter, dietmar; grubeck-loebenstein, beatrix title: the efficacy of vaccines to prevent infectious diseases in the elderly date: 2007 journal: immunosenescence doi: 10.1007/978-0-387-76842-7_10 sha: doc_id: 16903 cord_uid: z2vqfq98 infectious diseases still represent a major challenge to human progress and survival. especially elderly persons are more frequently and severely affected by infectious diseases and they display distinct features with respect to clinical presentation and treatment. although vaccinations are considered a vital medical procedure for preventing morbidity and mortality caused by infectious diseases, the protective effect of vaccinations is abrogated in elderly persons. this is due to a decline in the functions of the immune system referred to as immunosenescence. the first part of this chapter will therefore summarize the status quo of the efficacy of vaccines in preventing morbidity and mortality caused by typical infectious diseases in the elderly, such as influenza, pneumonia and tuberculosis. the second part will then elucidate the underlying age-related mechanisms which may contribute to the decreased efficacy of vaccines. based on the complex mechanisms involved in immunosenescence, strategies will be outlined which may be succesfful in enhancing protective immune responses following vaccination in elderly persons. with respect to the current demographic development in many countries, including the european union and the united states of america, infectious diseases in geriatric patients are becoming an increasingly important issue. infections in elderly persons are not only more frequent and more severe, but they also have distinct features regarding clinical presentation, microbial epidemiology and treatment. urinary tract infections, lower respiratory tract infections, skin and soft tissue infections, infective endocarditis, bacterial meningitis, tuberculosis and herpes roster appear to have a higher prevalence in elderly persons. in developed countries like the united states, pneumonia, influenza and septicemia are ranked among the ten major causes ofdeaths in people aged 6s years and older. 1 the reasons for the increased susceptibility to infectious diseases include epidemiological elements, imrnunosenescence, malnutrition and age-dependent anatomical alterations. infectious diseases still represent a major challenge to human progress and survival as they are responsible for about 20% ofall deaths in the world. this is not only related to microbial and viral factors but also to social and environmental determinants, such as social upheaval, urbanization, air travel, natural disasters and climate change.' newly emerging infectious diseases include acquired immune deficiency syndrome (aids), hepatitis c, several hemorrhagic fevers, severe acute respiratory syndrome (sars) and avian influenza. the resurgence ofseveral other infectious diseases is supported by the increased occurrence ofmultiple drug-resistant microorganisms such as staphylococcus aureus, mycobacterium tuberculosis, escherichia coli and streptococcuspneumoniae. *corresponding author: beatrix grubeck-loebenstein-institute for biomedical aging research, austrian academy of sciences, rennweg 10, 6020 innsbruck, austria. email: beatrix.grubeck-loebenstein@oeaw.ac.at immunosenescence, edited by graham pawelec. ©2007 landes bioscience and springer science+business media. altogether, this represents an enormous economic burden on health care systems all over the world. for instance, the annual costs ofmedical care for treating infectious diseases in the united states alone is about $120 billion and for treating antimicrobial-resistant infections it may be as high as $5 billion. ' a great success story was the implementation oflarge-scale vaccination strategies that led to the eradication of smallpox in 1980 4 and to a drastic reduction ofpoliomyelitis, tetanus, diphtheria, measles, pertussis and meningitis. presently, vaccinations are still considered the most cost-effective medical procedure for preventingmorbidity and mortality caused by infectious diseases. 26 different infectious diseases can be prevented by vaccinations and 61 vaccines are being developed according to a 2004 survey by the pharmaceutical research and manufacturers ofamerica," the new candidate vaccines are intended to provide protection against diseases caused by rotavirus, herpes zoster and papilloma virus and will be available from 2007 onwards ( table 1) . but also improved vaccines against influenza, pneumonia and tuberculosis are currently being tested in clinical trials ( table 1 ). this chapter now outlines the relevance ofvaccines to fight infectious diseases in old age and how age-related changes within the immune system contribute to the decreased efficacy of vaccines. it also discusses the progress made in the development of vaccines with improved immunogenicity in elderly persons. outbreaks of deadly infectious diseases such as ebola, marburg, sars or the h5nl avian influenza regularly alert the world, whereas there is not much public attention paid to infectious diseases that cause substantial morbidity and mortality among the elderly population. for instance, influenza, invasive streptococcus pneumoniae infection, urinary tract and skin infections have a higher prevalence in elderly persons," old individuals may also fail to respond sufficiently to therapy and frequently suffer from opportunistic infections, recurrent infections with the same pathogen or reactivation oflatent diseases, such as those caused by mycobacterium tuberculosis or the varicella zoster virus. there are no vaccines available for many infectious pathogens that are frequent in elderly subjects and existing vaccines are underused and often do not assure such an effective protection as in young subjects. the following paragraphs will highlight the most important infectious diseases which threaten the elderly population and will provide information on epidemiology, vaccine availability and efficacy,vaccination coverage and general health authority recommendations. influenza is a highly contagious, acute viral respiratory disease that causes significant morbidity and mortality. the annual outbreaks affect approximately 5-20% ofthe population worldwide with 3-5 million cases ofsevere illness and up to one million deaths each year. especially elderly people and persons that are chronically ill or otherwise immunocompromised are at enhanced risk. for example, during influenza epidemics, barker and mullooly reported two deaths per 100,000 healthy people below 65 years ofage compared with 797 per 100,000 in those over 65 with two or more high-risk conditions? in contrast to measles, smallpox and poliomyelitis, influenza is caused by viruses that undergo continuous antigenic variation and possess an animal reservoir. therefore, we are recognizingannual epidemics that have been interruptedby three pandemics (spanish influenza, hinl, 1918-1919; asian influenza, h2n2, 1957-1958 andhongkonginfluenza,h3n2, 1968), caused by new influenza virus strains with increased virulence. influenza viruses are enveloped viruses containing eight single-stranded rna segments which encode for viral proteins, such as hemagglutinin (ha), neuraminidase (na), matrix protein (ml) and nucleoprotein (np) (fig. 1) . influenza viruses belong to the family orthomyxoviridae and are divided into three genera, influenza virus a, b and c, based on antigenic differences in two oftheir structural proteins, m and np. disease symptoms caused by influenza c are rare whereas influenza b often causes sporadic outbreaks, especially in residential communities like nursing homes. influenza a viruses are further divided into subtypes according to the antigenicity oftheir major envelope glycoproteins, ha and na. with at least is different hemagglutinin and 9 different neuraminidase subtypes, there is con siderable antigenic variation among influenza viruses. the human influenza viruses are currently limited to three hemagglutinin (h i, h2 and h3) and two neuraminidase subtypes (n 1 and n2), whereas birds are the predominant hosts for the other subtype strains. ha initiates viral infection by binding to sialic acid residues on the carbohydrates ofglycoproteins present on epithelial cells ofthe respiratory trace. therefore, high-affinity iga and igg antibodies against ha may preven t infection from influenza virus. in contrast, na cleavesthe sialic acid from viral and cellular proteins to promote the release of newly synthesized influenza viruses from the infected host's plasma membrane. although antiviral drugs with moderate efficacy are available, active immunization represents the most vital element in th e prophylaxis ofinfluenza disease. however, the frequently occurring antigenic driftrequires an annual modification ofthe vaccinecomponents according to the recommendations ofthe who. therefore, vaccination has to be repeated annually to ensure protection against the circulating influenza strains. but vaccination coveragediffers largelywithin european countries. in 2002, the rate ofvaccinedistribution washighest in spain, belgium,the netherlands, united kingdom and germany (between 18.1 and 20.3%) and lowest in poland, czech republic, lithuania and latvia (between 1.9 and 7.1%). canada and the united states had the highest rate ofvaccine distribution, being 32.8 and 28.9%, respectively. remarkably,70% ofus citizens aged 65 and abovehave been vaccinated against influenza."although there are severalvaccinesavailable, the efficacyofmanyvaccinesin preventing influenza diseasein elderlypersons isonly around 56%.9 especiallyveryold and frail persons show a decreased response to influenza vaccines.p'ihe reduced vaccine efficacyis due to low levelsofiga and iggantibodies, delayed peak antibody titers and shortened maintenance of titers after vaccination. nevertheless, immunization in elderly people has been shown to be safe,cost-effective and associated with reduced rates ofhospitalization and influenza-relateddeaths.i1,12in particular, the efficacyofinfluenzavaccination to reduce mortality in elderlypeople is greater after repeated annual vaccination than after first administration. 13presently, influenza vaccines can be classifiedin split-virus, subunit, virosomal and live-attenuated vaccines ( table 2 ). split-virus vaccines are used since the 1980s, are cheap and offer a good protection for children above 6 months of age and adults. recently, subunit vaccines with new adjuvants have been developed (fluado, addigrip') that show an increased immunogcnicity, a favorable safety profile and may be more suitable for the vaccination of elderly persons.'! additionally, invariant antigens, such as m 1 and np, may also play an important role in protection and could be used in vaccines to induce long-lasting immunity to a variety of different influenza strains. another strategy to enhance immunogenicity may the use ofvirosomes that are nontoxic , biodegradable lipid-basedantigen-presentation systems." virosomal influenzavaccines, such asinflexalv', infiuvac plus'and invivac'have been on the market in several european countries for a number of years. they display a high immunogenicity and a similar safety profile in elderly persons compared with inactivated influenza vaccines.f" furthermore, new live-attenuated and subunit influenza vaccines ate currently in clinical trials that promise to have an increased efficacy ofprotection (table 1) . especially live-attenuated influenza vaccines ate believed to elicit strong tcel! responses and should be able to enhance antibody levels after vaccination. importantly, before administration of live-attenuated vaccines to elderly or immunocompromised persons, an acceptable safety profile has to be demonstrated. irrespective ofthe improvement ofinfluenza vaccines for elderly persons, vaccination of children is clinically effective'" and high vaccination coverage among pupils has demonstrated to induce herd immunity and to reduce mortality in older adults. streptococcus pneumoniae is an important cause of invasive clinical manifestations such as bacterial pneumonia, meningitis and septicemia, particularly in young children and the elderly. 80 to 90% ofdeaths associated with streptococcus pneumoniaeinfection occur in people aged 60 years and above. several further groups at higher risk ofinvasive pneumococcal disease have been defined, including individuals with splenic dysfunction, immunosuppression, chronic pulmonary or cardiac disease, diabetes mellitus and chronic liver disease. generally, antibiotic therapy has to be initiated as soon as possible to reduce the risk ofcomplications due to pneumonia, meningitis or sepsis. nonetheless, 50% ofall deaths occur within the first 48 hours despite adequate antibiotic therapy. this may be due to the increased occurrence of multiple drug-resistant pneumococcal strains. in 2002, the proportion ofpenicillin-resistant streptococcus pneumoniaewas reported to be 53% in france and more than 25% in israel, poland, romania and spain." after recovering from pneumococcal infection, people are not necessarily immune, because there are about 90 different serotypes and immunity will be guaranteed only to the strain that has caused the infection. currently, pneumococcal vaccines are available that include up to 23 strains which are responsible to cause disease in almost 90% ofall cases ( table 2 ). these vaccines offer protection against invasive pneumococcal disease in 65% of the general elderly population 22.23whereas in elderly persons with high risk factors, the protective effect of vaccination seems to be only moderate. although many european countries recommend the administration of pneumococcal vaccines to all those >65 years ofage, vaccination coverage among the elderly population is very low. this may be due to the high costs ofthe vaccine and its unsatisfying efficacy in elderly people. but more immunogenic vaccines are currently in different phases ofclinical trials (table 1 ) and promise to be more efficient in old age. additionally, implementing pneumococcal vaccination for children may decrease the incidence ofpneumococcal disease in the elderly by reducing transmission and possibly accomplishing herd immunity.-" each year, about 8 million people are infected worldwide with the tubercle bacillus mycobacterium tuberculosis and 1.6 million ofthem die. the eu25 has a tuberculosis (tb) burden ofmore than50,000 new casesper year,with the highest incidences in latvia, lithuania and estonia (50-100 cases/ 100,000). the risk ofdeveloping a disease following tb infection is about 5-10% during lifetime and individuals above 65 years ofage have a four-fold increased risk ofdeveloping tb than the average population," tb is also frequently diagnosed with delay due to an atypical manifestation in old age. this may lead to an increased morbidity and mortality and to a spreading ofthe disease, in particular within institutionalized elderly persons.p further difficulties include the increased emergence of new, multiple drug-resistant strains with higher rransmissibiliry," the poor efficacy of the current bacille calmette guerin (bcg) vaccine in protecting adults and elderly people from pulmonary infection" and the increased risk oftb co-infection in hiv positive patients." however, in the past few years, several tb vaccine candidates have entered phase i clinical trials, including adjuvanted subunit vaccines as well as improved live recombinant strains of the current bcg vaccine (table 1 ). all these vaccine candidates are supposed to induce an effective and sustainable cellular immune response which is thought to be crucial to protect the host from an intracellular pathogen such as mycobacterium tuberculosis. 30 primary infection with the varicella zoster virus (vzv) causes chickenpox which is usually a mild disease in childhood. the virus then persists in a latent form in sensory ganglia until its reactivation which results in the clinical manifestation of herpes zoster (shingles). between 13 and 26% ofpersons with herpes zoster develop complications, such as postherpetic neuralgia." postherpetic neuralgia also increases with age with a prevalence of 50% in people aged 70 years and above. the incidence and severity ofherpes zoster increase with age, because vzv reactivation is associated with a progressive decline in cell-mediated immunity to vzvy.·33 routine vaccination ofchildren using a tetravalent vaccine that protects against measles, mumps, rubella and varicella will soon be available (table 1 ) and may reduce the incidence of chickenpox as well as the reactivation ofvzv in later life. since 1995, a live-attenuated oka strain vzv vaccine is on the market that has shown clinical efficacy in preventing children from chickenpox." however, the currently available vzv vaccines have not been proven to adequately boost t-cell responses in older adults and to prevent reactivation ofherpes zoster. recently, a vaccine that may prevent herpes zoster virus reactivation has been submitted for registration. this live-attenuated vzv vaccine has been developed to prevent reactivation of herpes zoster in the elderly.3s.36this is of particular importance, because the elderly population has not been vaccinated against but may have been frequently infected by vzv. for instance, more than 90% ofadults in the united states have had chickenpox. as a consequence, it is estimated that up to 800,000 people in the united states suffer from shingles each year and the incidence is expected to increase as the population ages. thus, reactivation ofherpes zoster and its clinical manifestations represents a serious health burden to the growing elderly population and could be counteracted by potent vaccines. the cytomegalovirus (cmv), a b-herpesvirus, has also been shown to persist throughout life until its reactivation as a result ofimmune suppression or deficiency. cmv infection is quite common and affects 60-100% ofthe adult population, dependingon the area. the cmvis transmitted via person-to-person contacts but immunocompetent subjects mostly do not recognize infection as it causes no or few unspecific symptoms. however, a cmv infection represents a severe health problem in immunocompromised persons (e.g.,due to immunosuppressive disease, chemotherapy or transplantation) or in a fetus as a result of congenital infection. research results over the past decade suggest that cmv favors an accelerated aging of the immune system as cmv infection is chronic and the organism is forced to continuously prevent virus reactivation.f'" despite the high frequency of cmv-specific cd8+ tscells, the virus usually can not be eliminated by the immune system. this is because the virus has evolved several mechanisms to escape the host's immune defense." for instance, cmv encodes for a type ofproteins called immunoevasins that modulate the presentation ofviral peptides or directly suppress cellular immune responses. hence, the accumulation of cmv-specific t-cells substantially constricts the diversity of the t-cell repertoire" and leads to the production ofproinflammatory cytokines, such as gamma interferon and tumor necrosis factor alpha.'? this imbalance in the cytokine production profile may not only promote the pathogenesis of age-related diseasesf but leads to a decreased production of antibodies following influenza vaccination in elderly persons.fa few antiviral substances including ganciclovir, valganciclovir, foscarnet and cidofovir are available to prevent cmv infection in immunocompromised patients. but antiviral therapy is limited by its severe adverse reactions, such as neutropenia, nephrotoxicity, hypocalcemia and seizures. another strategy is the adoptive transfer ofdonor-derived cmv-specific cd4+ and cd8+ t-cells that may restore the host's immunity against cmv. 44 despite the need ofa safe and potent vaccine that prevents cmv disease, no vaccine candidate has yet entered the market. a few vaccines against cmv are currently in phase iiii clinical trials (table 1) . active immunization against cmv could reduce the incidence ofneonatal infections as well as complications in immunocompromised persons and may prevent cmv-associated premature aging ofthe immune system when applied early in life. pertussis (whooping cough) is a highly contagious respiratory system infection caused by the bacterium bordetella pertussis and rarely by b.parapertussis, b. bronchiseptica or other pathogens. each year, more than 20 million cases ofpertussis are reported worldwide, 90% ofwhich occur in developing countries, with an estimated 200,000 to 300,000 fatalities. the implementation of routine childhood vaccination against pertussis has reduced the high mortality rate among children. although most infants are being immunized against pertussis in industrialized countries, immunity usually fades during adolescence. consequently, a significant rise in pertussis incidence has been noticed in adolescents and adults." however, the reported pertussis cases in adults and elderly people are likely to be underestimated because symptoms ofdisease may be characterless and make clinical diagnosis difficult. among nonvaccinated elderly people the attack rate ofpertussis is high (53%) and up to 10% ofelderly persons may die from intracranial bleeding while they are symptomatic for pertussis." regular booster immunizations should thus be considered for adults and elderly persons, which is indispensable to remain protected from disease. tetanus is acquired via environmental exposure to the spores of clostridium tetani,which are present in soil worldwide. the disease is caused by a potent neurotoxin produced by the bacterium in dead tissue, e.g.,dirty wounds. diphtheria is a bacterial disease caused by corynebacterium diphtheriac and is transmitted from person to person through close physical contact. the public health burden ofboth diseaseshas been low in developed countries due to routine immunization. however, outbreaks ofdiphtheria have been reported in the independent states ofthe former soviet union, algeria, china, iraq, sudan, thailand and other countries. thus, maintaining high vaccination coverage is important to prevent the outbreak of new diphtheria epidemics. although vaccines that prevent from tetanus and diphtheria have been used for routine immunization for a long time all over the world, few studies exist that document their efficacyin elderly people. the vaccination coverage among elderly subjects is decreasing in several european countries and up to 40% of appropriately vaccinated elderly persons do not have protective tetanus-specific antibody concentrations. 47-49therefore, public health authorities ofsome european countries have recommended five instead often year booster vaccination intervals for people over 60 years ofage. additionally, strategies should be developed to draw public attention to the problem ofimmunizations in the elderly, to inform general practitioners and to increase vaccination acceptance. the increasing mobility ofelderly persons recognized worldwide is accompanied by an enhanced risk to encounter new antigens. this may be ofconcern because elderly persons possess a limited t-cell repertoire that may not guarantee full responsiveness to a wide variety ofnew antigens (see below for details). nevertheless, in vitro experiments have demonstrated that naive t-cells from elderly persons can still be stimulated by neoantigens, at least to the recombinant etr protein of tbe virus and rabies virus." based on an assessment of the risks for travel-related diseases, including the destination, the type of journey and the duration, vaccination is recommended to protect from typhoid and yellow fever, hepatitis a and b, japanese encephalitis, tick-borne encephalitis (tbe) or rabies. but elderly persons should also check whether they have followed the recommended booster intervals of routine immunizations, e.g., against tetanus, diphtheria, poliomyelitis, measles or influenza. tbe is caused by a virus that is primarily transmitted to humans by infected ticks. there are three genetically closely related subtypes of the tbe virus known (european, siberian and far eastern subtype). tbe is among the most dangerous neuro-infectious diseases in europe and asia and is responsible for up to 12,000 casesoftbe annually, most ofthem occurring in russia, czech republic and the baltic states." up to 30% ofadults with clinically confirmed tbe infection develop meningitis or meningoencephalitis and the lethality oftbe in europe is up to 1%.yet,there is no specific therapy available and, therefore, active immunization with inactivated whole virus provides the only efficient protection from tbe disease ( table 2 ).52 importantly, more and more tbe cases are reported in people over 50 years ofage and vaccination coverage in this population is lower than average. therefore, future strategies should increase the vaccination coverage among elderly persons and assure that they stick to regular booster intervals. anyhow, regular boosters should be given throughout life as this may favor the maintenance oflong-lastinghumoral immunity against tbe53and may decrease the risk ofimmunization failures in the elderly. hepatitis a is an acute disease ofthe liver caused by the hepatitis a virus (hay), a nonenveloped virus belonging to the picornaviridae family. each year, an estimated 1.5 million cases ofhepatitis a occur worldwide. hay infection induces life-long immunity and is usually asymptomatic in young children, whereas adults frequently experience symptomatic disease. hay is acquired directly from infected persons by close contact or by the consumption ofcontaminated drinking water, vegetables, fruits or shells. hay vaccination is recommended when traveling to tropic and subtropic countries that have an increased risk of infection. for instance, the risk ofhepatitis a infection ofpersons traveling to developing countries was estimated to be 3 to 20 cases per 1000 persons per month ofstay,varying with destination, living conditions and age." improved sanitary standards in developed countries have reduced the opportunity for environmental exposure to hay and have lowered the overall incidence ofinfection. paradoxically, susceptibility to the virus increased because of the decrease in natural immunity. consequently, less than 20% ofpersons born after 1945 have a natural immunity against hay'54 in contrast to hepatitis b and c that may lead to the manifestation of a chronic infection, clinical illness after hepatitis a infection is usually mild in young individuals. but increasing age represents an enhanced risk ofsevere infection and mortality rates are about 2% for persons over 40 and 4% for those over 60 years ofage." several vaccines against hepatitis a are available (table 2 ) and a study of773 adults showed that immunogenicity and safety profiles between 'iwinrix' and havrix' are comparable.56 but there is some evidence oflower antibody titers with advanced age. for instance, the seroconversion rates 8 months after two doses ofhavrix' were found to be 85% and 60% for adults < 35 years and >35 years, respectively.57after the recommended immunization schedule with twinrix'.seroprotection was 92% and 63% for adults <40 years and >60 years, respectively.58 therefore, it may be useful to measure hay antibodies in elderly persons, as in the case ofvaccination failure, boosters have shown to be effective." it is further recommended that the vaccine is given at least 3 to 4 weeks before travel due to a slower onset ofthe antibody response in elderly individuals. 59 another travel vaccine is directed against yellow fever (yf), which is endemic in tropic regions ofafrica and south america. yf is transmitted by the bite ofinfective aedes aegypti and other mosquitoes that bite during daylight hours in regions below 2500 meters ofaltitude. most infections lead to an acute illness characterized by fever, muscular pain, headache, anorexia, nausea and/or vomiting, often with bradycardia. after a few days, about 15% ofpatients progress to a second phase, with resurgence offever,development ofjaundice, abdominal pain and haemorrhagic manifestations. halfofthese persons die 10-14days after the onset ofillness. the who estimates that a total of200,000 cases ofyf occur each year, with about 30,000 deaths. 6oyf also represents a significant risk to more than 3 million travelers that visit yf-endemic areas each year. neonates and elderly individuals demonstrate the highest mortality when infected by the yf virus. as there is no specific antiviral treatment against yf available yet, vaccination is the only way to protect persons from yf disease. the currently available vaccine contains a live-attenuated 17d strain virus ( table 2 ) and has been shown to be safe and highly potent." however, due to the increased use in international travelers, it has become evident that advanced age might be a risk factor for serious adverse effects and even deam. 62 compared with persons aged 25-44 years, individuals aged <75 had an 18-fold greater risk to experience serious adverse events after vaccination. the rate for systemic illness requiring hospitalization or leading to death after yf vaccination was reported to be 3.5 per 100,000 among people 65 to 75 years of age and 9.1 per 100,000 for people more than 75 years. furthermore, there are no studies available that demonstrated the efficacy of the yf vaccine in elderly persons. accordingly, recommendations and manufacturing standards have been modified to increase vaccine safety in elderly persons. although the benefit-risk ratio still favors the vaccination ofpeople at high risk for infection and outlines the vaccine's fundamental role in disease prevention and control, efforts to improve safety and to ensure vaccine efficacy in elderly persons are ofurgent need. the term immunosenescence refers to a complex remodeling ofthe immune system in old age and may contribute significantly to morbidity and mortality in the elderly. thymic involution, telomere shortening, t-cell signal transduction changes, alterations in the interaction of the innate and adaptive immune response, impaired dna repair and antioxidant mechanisms as well as persistent antigenic stress may all be factors contributing to immunosenescence. although perturbations ofinnate immune system components have been described, much ofthe decrease in immunoresponsiveness seen in elderly people is associated with changes in t-cell responses. this is due to the continuous loss offunctional thymic tissue with increasing age. 63.64the thymus, the central lymphoid organ, is responsible for the maturation and selection ofso-called naive t-cells that regenerate the peripheral tcell pool and retain the capability ofthe immune system to respond to a variety ofdifferent pathogens. in old age, the number ofnaive t-celis decreases while the number ofantigen-experienced 'tcells increases. 65 .66 these antigen-experienced t-celis include a substantial proportion of senescent memory-effector t-cells that accumulate in elderly persons. senescent memory-effector tcelis display phenotypic (loss ofcostimulatory molecules such as cd28 and cd40l) as well as functional changes (altered cytokine production profile, decreased proliferative response, shortened telomeres, increased resistance to programmed cell-death and restricted t-cell diversity).67.68ofparticular importance, the senescent cd8+cd28-memory-effector t-cell population predominantly produces the pro-inflammatory cytokine gamma interferon (ifny), but does not produce interleukin 2 (il-2) and the anti-inflammatory, b-cell stimulating cytokine il-4. 43 recent data also support the hypothesis that chronic infection with the cytomegalovirus, ã -herpesvirus, may lead to a decrease in the size ofthe naive and early memory cd8+ t-cell pool, but to an increase in the number of dysfunctional, ifny-producing cd8+cd28-memory-effector tvcells (fig. 2) , 37 one clinical consequence of the accumulation of cd8+cd28-t-cells is an impaired generation of protective antibody levels after vaccination. 43.69 furthermore, the age-dependent increase in the level ofpro-inflammatory cyrokines may lead to ubiquitous chronic inflammatory responses in old age 42 and may therefore support the development of age-related chronic diseases, such as atherosclerosis," rheumatoid arthritis" and alzheimer's disease. 72, 73 although individuals maintain a relatively constant total number ofperipheral b-celis during aging, each b-cell subset comprises severeperturbations in size,dynamics and repertoire. the alterations affecting the bvcell subsets are due to a decreased generation ofb-cell precursors, such asearly lymphoid precursors and pro-bvcells, cell-intrinsic as well as micro-environmental disturbances are both likely to contribute to the decreased output ofpro-bscells. furthermore, alterations in environmental factors also impair overall v(d)j recombinase activity among pro-bvcellst'whlch accounts for the limited b-cell repertoire frequendy detected in elderly persons." although no decrease in overall serum immunoglobulin levels have been observed during aging, the antibodies generated in old age are oflower affinity due to a shift in antibody isotypes from igg to igm,76 of particular importance, b-celis from elderly individuals are stimulated 70% less efficiendy by follicular dendritic cells than bvcells from young subjects," suggesting loss ofb-cell function, in part due to the decreased expression ofcostimulatory molecules, such as cd40 or cd27,78 impaired t-cell-mediated immunity as well as defects in antigen presentation by antigen presenting cells there is a tremendous need to increasethe protective effect ofvaccines in the elderly. research of the last decade has provided new insights into the molecular mechanisms ofthe immune response in old age, which can now be used for th e development of potent vaccines. in the past, vaccines were primarily designed to elicit a strong humoral immune response . however, vaccines in elderly persons may be more effective ifthe stimulate innate immune components and the generation of long-lived memory t-cells. currently,severalstrategies are being pursued to increase immunogeniclry, to minimize adverse side effects and to increase vaccine acceptance by introducing needle-free injection devices. proven and promisingvaccine technologies are used to design conjugate, subunit, live vector, dna and live-attenuated vaccines (table 3) .80 while live-attenuated vaccines (e.g.• against varicella, measles or yellow fever) stimulate numerous immune components and display enhanced immunogenicity, conjugate and subunit vaccines (e.g., against influenza) are often supplemented with adjuvants" to ensure their protective effect. generally, adjuvants can be divided into antigen delivery systems (cationic microparticles, proteasomes and virus-like particles) and immune potentiators (e.g.• cytokines). these adjuvants may overcome the proposed age-related functional decline ofinnate immune responses by targeting pattern-recognition receptors, such as the recently identified toll-like receptors or nucleotide-bindingoligomerization domain proteins." the enhanced activation ofthe innate immune system may also improve antigen processing and presentation leading to more potent t and b-cell responses and to sustained immunological memory. vaccines supplemented with the dna of cytokines (e.g., il-2 , il-? il-i2, il-is or il-21 ), chemokines or costirnulatory molecules may magnify immune responses by generating more and long-lived memory t_ceiis83-85and may overcome immunodominance.'" in addition to improve vaccine efficacy, a modification of vaccination strategies for elderly persons has been supported by the results of several vaccination trials. for instance, a decreased response and a shortened duration ofprotective immunity following booster immunization is a characteristic feature of old age. 87thus , several european health authorities have recommended five-year vaccination intervals for tetanus, diphtheria, pertussis and pneumonia. increased public awareness of regular booster vaccinations in adults should be enforced, as these immunization regimes may be essential to maintain the ability to respond to recall antigens in old age. recent result s also indicate that long-lasting protection but also a good booster effect can be expected even a long time after the last vaccination, when a live-attenuated vaccine (e.g., polio vaccine) is used for primary immunization in early life. new delivery systems that make use oftiny micro-needles or non-injectable application devices (nasal , oral, transcutaneous) may further increase vaccination acceptance, especially in the case of influenza as this vaccination has to be repeated annually. infectious diseases in elderly persons are becoming an increasingly importan t issue. an utmost need represents the development ofmore immunogenic vaccines for the elderly. the improvement of specific vaccine types regarding immunogenicity and tolerability, th e addition of adjuvants. the design ofnew delivery systems as well as specific immunization regimes should all contribute to an enhanced efficacy ofvaccines in elderl y persons. further improvements may comprise the adjustment ofvaccination intervals in old age, the increase in vaccine acceptance and vaccination coverage as well as raising people's awareness to stick 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adjuvants targeting the innate immune response with improved vaccine adjuvants augmentation and suppression of immune responses to an hiv-l dna vaccine by plasmid cytokine/ig administration coimmunization with an optimized il-15 plasmid results in enhanced function and longevity of cd8 t-cells that are partially independent of cd4 t-cell help il-21 influences the frequency, phenotype and affinity of the antigen-specific cd8 t-cell response adjuvant il7 or il-15 overcomes immunodominance and improves survival of the cd8+ memory cell pool insufficient protection for healthy elderly adults by tetanus and tbe vaccines immunizations in the elderly: do they live up to their promise the authors wish to acknowledge the support ofthe austrian science fund and the austrian green cross society for preventive medicine. key: cord-018659-rxzy6k3b authors: danziger-isakov, lara; munoz, flor m.; estabrook, michele title: posttransplant complications and comorbidities date: 2018-01-08 journal: solid organ transplantation in infants and children doi: 10.1007/978-3-319-07284-5_71 sha: doc_id: 18659 cord_uid: rxzy6k3b infectious complications cause significant acute morbidity and mortality after pediatric lung transplantation. with the lung graft in direct communication with the environment, it is susceptible to a variety of bacterial, fungal, and viral pathogens. appreciation for pretransplant risk factors in addition to perioperative and posttransplant exposures is necessary to anticipate, diagnose, and treat infections in this population. further, epidemiologic associations between infection and chronic allograft dysfunction have been reported and suggest consequences of infectious events may have substantial impact. infectious complications cause significant acute morbidity and mortality after pediatric lung transplantation. with the lung graft in direct communication with the environment, it is susceptible to a variety of bacterial, fungal, and viral pathogens. appreciation for pretransplant risk factors in addition to perioperative and posttransplant exposures is necessary to anticipate, diagnose, and treat infections in this population. further, epidemiologic associations between infection and chronic allograft dysfunction have been reported and suggest consequences of infectious events may have substantial impact. bacteria · cytomegalovirus · infectious complication · nontubercular mycobacteria · pediatric lung transplantation · respiratory virus bacteria account for about 50% of infections post lung transplant with pneumonia being most frequent. other sites include nosocomial central lineassociated bacteremia, urinary tract, and surgical site infections. the greatest risk is within the first year after transplantation, particularly in the first 3-6 months. donor infection and recipient airway colonization are also risk factors (speich and van der bij 2001; aguilar-guisado et al. 2007; parada et al. 2010; burguete et al. 2013; yun et al. 2015) . one of the largest studies of primarily adults found that 75% of infections occurred within the first year posttransplant and 42% occurred within the first 3 months. the majority, 48%, was bacterial (parada et al. 2010; burguete et al. 2013) . another study showed similar results but found that bacteremia, both primary and catheter associated, was the most common infection in the first month after transplant with pneumonia becoming most frequent after 2 months. multidrug-resistant bacteria including methicillin-resistant staphylococci, vancomycin-resistant enterococci, and carbapenem-resistant or extended-spectrum betalactamase producing gram-negative bacilli were involved in 66% of infections. bacterial infections were significantly more common in those colonized with multidrug-resistant gram-negative bacilli than those who were not (yun et al. 2015) . a pediatric study of 42 children and 49 lung transplants found that half of the infections were bacterial with 42% occurring within 3 months after transplant and 80% in the first year. the lung was the most common site (72%) and pseudomonas aeruginosa was the most common organism. bacterial infections were felt to contribute to pulmonary dysfunction (bronchiolitis obliterans) but were not the primary cause of death (metras et al. 1999) . recent data from the registry of the international society for heart and lung transplantation reported that non-cytomegalovirus infection was the cause of death in 24% of lung transplant recipients in the first year after transplant (benden et al. 2013) . one of the largest studies of pneumonia in 236 lung transplant recipients (aguilar-guisado et al. 2007) found that the most common etiology was bacterial in 83%. gram-negative bacilli accounted for 60% with pseudomonas aeruginosa being the most frequent isolate in 25%, followed by acinetobacter baumannii in 14%. staphylococcus aureus was the etiology in 14%. the probability of 1-year survival was significantly higher in those recipients who did not have an episode of pneumonia (aguilar-guisado et al. 2007 ). late-onset communityacquired pneumonia with streptococcus pneumoniae also occurs (de bruyn et al. 2004 ). survival after lung transplantation is limited by the high incidence of chronic lung allograft dysfunction (clad) that has two forms: bronchiolitis obliterans syndrome (bos) and restrictive allograft syndrome (ras). the role of infection in the development of clad has recently been reviewed (martin-gandul et al. 2015; gregson 2016) . while acute infection with community-acquired respiratory viruses is recognized as a risk, pseudomonas aeruginosa and staphylococcus aureus are increasingly recognized as well. one study of lung transplant recipients with cystic fibrosis found that loss of colonization with pseudomonas was protective against the development of bos (gottlieb et al. 2009 ). two further studies found that infections due to gram-positive bacteria, primarily staphylococcus aureus, increased the hazard risk for bos (gupta et al. 2009; valentine et al. 2009 ). the underlying allograft inflammatory state in the setting of infection also appears to be important in determining the development of clad (gregson 2016). cystic fibrosis (cf) is a common, underlying diagnosis in children who undergo lung transplantation. the registry of the international society for heart and lung transplantation reported that half of children <10 years of age and 70% of children aged 11 through 17 years had cf (benden et al. 2013) . cf-specific bacterial pathogens including multidrug-resistant (mdr) or pan-resistant bacteria persist in the paranasal sinuses and upper airways and can be a cause of posttransplant pneumonia. pseudomonas aeruginosa is most common, but other organisms include stenotrophomonas maltophilia, achromobacter xylosoxidans, and burkholderia cepacia complex (shoham and shah 2013) . in lung transplant recipients, there is increasing resistance in gram-negative bacilli including extended-spectrum beta-lactamase, ampc betalactamase, and carbapenemase (shoham and shah 2013; van duin and van delden 2013) . pseudomonas aeruginosa infection occurs in up to 80% of patients with cf and bronchiectatic lung disease, and pretransplant colonization is a significant risk factor for infection after transplant. mdr p. aeruginosa has a prevalence rate from 10% to 45% in patients with cf (shoham and shah 2013) . survival posttransplant in patients colonized with pan-resistant p. aeruginosa before transplant was similar to those with sensitive organisms at 1 year (88% vs. 96%) but lower at 3 years (63 vs. 91%) (hadjiliadis et al. 2007; shoham and shah 2013) . however, the 2006 update of the international guidelines for the selection of lung transplant candidates stated that colonization with multidrug or pan-resistant p. aeruginosa was not a contraindication because it has not been shown to significantly affect shortterm survival (orens et al. 2006) . a recent study in lung transplant recipients with cf reported that infection with pan-resistant achromobacter xylosoxidans and stenothrophomonas maltophila also did not reduce survival after lung transplantation (lobo et al. 2015) . burkholderia cepacia complex (bcc) is comprised of several different species that colonize the respiratory tract in 15-22% of patients with cf. most infections are caused by b. cenocepacia (genomovar iii) and b. multivorans (genomovar ii). pretransplant colonization with b. cenocepacia has been associated with increased posttransplant mortality (relative risk 8.4) with one study reporting 1-year survival of 29% compared to 92% in those uninfected and is considered by many centers as a contraindication to transplant (shoham and shah 2013) . recipients colonized with b. multivorans did not have decreased survival while b. gladioli had an increased mortality risk but not as high as b. cenocepacia. bcc has an 80% prevalence of multidrug resistance (shoham and shah 2013) . methicillin-resistant staphylococcus aureus (mrsa) has been increasingly recognized as a significant bacterial pathogen post lung transplantation. a study of lung transplant recipients found that 18% had s. aureus infection in the first 90 days with 62% being methicillin sensitive (mssa) and 35% being mrsa. the site of infection was pneumonia 48%, tracheobronchitis 26%, and bacteremia 12%. colonization before transplant with mrsa was a risk factor for mrsa infection posttransplant but was not associated with increased mortality at 30 and 90 days post onset of infection (shields et al. 2012) . a second study had a calculated incidence rate of mrsa of 76 cases per 1000 transplant years with the median onset of 3 months posttransplant. the most common site was the lower respiratory tract and 31% of mrsa infections were associated with bacteremia. the direct mortality after mrsa infection was 17.6% (manuel et al. 2009 ). nontuberculous mycobacteria (ntm) are ubiquitous bacteria found in environmental sources including water, soil, plants, and animals. exposure is felt to be from the environment but more recently patient-to-patient transmission has been proposed for m. abscessus complex (bryant et al. 2013) . pretransplant infection is confined primarily to the lungs, with abnormal parenchyma such as cystic fibrosis or bronchiectasis being a risk factor. posttransplant infection can involve asymptomatic colonization, invasive lung disease, skin and soft tissue infection, and central lineassociated bacteremia (griffith et al. 2007; keating and daly 2013; smibert et al. 2016) . ntm isolation from respiratory cultures in lung transplant candidates is common particularly in those with cystic fibrosis. one study (chalermskulrat et al. 2006 ) reported a 20% colonization rate with 45% of isolates being m. avium complex (mac) and 41% m. abscessus. isolation after transplant is also common from 13 to 22% with mac accounting for about 70%. invasive disease after transplant is much less common, however, occurring in fewer than 5% of lung transplant recipients (chalermskulrat et al. 2006; chernenko et al. 2006; huang et al. 2011; knoll et al. 2012) . pretransplant colonization has been associated with an increased risk of posttransplant ntm infection as well as invasive disease but only for m. abscessus (chalermskulrat et al. 2006) . while ntm isolation and disease particularly with m. abscessus is associated with increased complications post lung transplant, it has not been associated with increased mortality and is not considered an absolute contraindication to transplantation (chalermskulrat et al. 2006; knoll et al. 2012; qvist et al. 2013) . case reports of two adolescents with cystic fibrosis and pretransplant m. abscessus infection showed that when antibiotic therapy led to afb stain negativity at the time of transplant, the outcome was favorable even in the face of positive cultures (zaidi et al. 2009 ). diagnosis of ntm disease is based on criteria published by the american thoracic society/ infectious diseases society of america that include clinical and microbiological criteria (griffith et al. 2007) . compatible symptoms and radiological changes consistent with ntm infection with other etiologies excluded must be accompanied by: positive culture from at least two sputum samples, positive culture from one bronchial lavage or wash, or lung biopsy with consistent pathology and positive culture. treatment depends on accurate identification and susceptibility testing of the organism. guidelines are available and consultation with an infectious disease expert is recommended (griffith et al. 2007; keating and daly 2013) . obtaining cultures of respiratory, blood, urine, and wound samples with accurate identification and determination of drug sensitivity is critical in the treatment of bacterial infection post lung transplant. consultation with a transplant infectious diseases physician and pharmacist is recommended when designing antibiotic therapy for multi-and pan drug-resistant organisms to maximize effectiveness and minimize toxicity. removal of sources of infection such as central venous lines and drainage of focal fluid collections should be undertaken when feasible. there are no well-conducted studies that have addressed the optimal choice of agent, duration, and efficacy of antibiotic prophylaxis for lung transplantation. in the absence of positive cultures from the donor or recipient, prophylactic regimens of 48-72 h and no longer than 7 days are recommended (bratzler et al. 2013 ). in recipients with cf, broad-spectrum antibiotics are administered at the time of transplant and are selected to cover the pretransplant bacterial pathogens and associated resistance patterns (hirche et al. 2014) . most centers treat recipients with a history of p. aeruginosa infection with two-drug antipseudomonal therapy for 2-3 weeks postoperatively to reduce the risk of pneumonia and colonization of the allograft (shoham and shah 2013). large multicenter prospective studies of adult sot recipients reported that the most common fungal organisms in lung recipients were aspergillus (63%), candida (23%), and other molds (10%) while zygomycosis, cryptococcosis, and endemic fungi were uncommon (neofytos et al. 2010 ). more recent data suggest an emergence of non-aspergillus mold infections (neofytos et al. 2013; chong et al. 2015) . pediatric studies have reported an incidence of pulmonary fungal infection from 10.5 to 20%, with aspergillus being the most common organism in two studies (danziger-isakov et al. 2008; liu et al. 2009b) . a single center study of 55 lung transplant recipients (liu et al. 2009b) found a higher incidence of posttransplant fungal colonization (60%) compared to adult patients (30-40%). however, posttransplant colonization was not associated with invasive pulmonary disease, and pulmonary fungal infections were not associated with chronic allograft rejection or death (liu et al. 2009b) . a larger retrospective multicenter study with patients transplanted in 1988-2006 found tacrolimusbased immunosuppression, cytomegalovirus sero-mismatch, age over 15 years, and prior episode of rejection greater than a2 were risks for pulmonary fungal infection, but the study did not investigate colonization as a risk factor (danziger-isakov et al. 2008) . additionally, pulmonary fungal infection was independently associated with decreased 12-month survival. mortality for proven and probable infection was 38 and 11%, respectively, similar to what has been reported for adults (danziger-isakov et al. 2008) . bronchial airway anastomotic complications occurred in 14% of 214 pediatric lung transplant recipients in a single center cohort, and this complication was associated with prior episodes of posttransplant fungal infection (choong et al. 2006) . these studies indicate that fungal infection in pediatrics significantly impact posttransplant morbidity and potentially mortality. while candida species isolated from respiratory secretions may represent normal commensal flora, invasive infections due to candida species have been reported in pediatric lung transplant recipients. in addition to bronchial anastomotic infection, pleural infection, pulmonary fungal infections, and bloodstream infections appear in the pediatric literature (danziger-isakov et al. 2005; danziger-isakov et al. 2008; liu et al. 2009b ). non-albicans species including c. krusei, c. glabrata, c. parapsilosis, and c. dubliniensis can all cause disease but may have differing antibiotic susceptibilities. identification to the species level is important to facilitate optimum treatment especially as non-albicans species have been associated with increased mortality (andes et al. 2016). aspergillus species cause posttransplant infections including tracheobronchitis with anastomosis infection, invasive pulmonary disease, and disseminated disease (hosseini-moghaddam and husain 2010). risk factors for invasive disease include ischemia at the anastomosis site, single lung transplant, hypogammaglobulinemia, placement of bronchial stent, cmv infection, and colonization (robertson et al. 2009; hosseini-moghaddam and husain 2010; chong et al. 2015) . as with candida species, speciation of aspergillus is important. while a. fumigatus causes the majority of disease, other species including a. niger, a. terreus, a. flavus, and a. ustus appear to be increasing in prevalence especially with the use of inhaled amphotericin as prophylaxis (hosseini-moghaddam and husain 2010; peghin et al. 2016) . prompt diagnosis of invasive aspergillus infection is imperative to improve outcome; however, newer diagnostics have not been specifically evaluated in pediatric lung transplantation. in pediatric cancer patients, the sensitivity and specificity of galactomannan (gm), beta-dglucan, and pcr-based assays were highly variable (lehrnbecher et al. 2016) . in adult lung transplant recipients, the serum gm assay has excellent specificity but poor sensitivity while bronchoalveolar lavage gm appears more promising for diagnosis with a sensitivity of 88-100% and specificity of 89-90% depending on the cutoff used for diagnosing invasive pulmonary aspergillosis (husain et al. 2004; pasqualotto et al. 2010; luong et al. 2011 ). further, a pan-aspergillus real-time pcr assay also performed well with a sensitivity and specificity of 100% and 93%, respectively (luong et al. 2011). as newer diagnostics emerge, their utility in pediatric lung transplantation will need assessment. histoplasmosis, blastomycosis, and coccidioidomycosis are endemic fungi with restricted geographical distribution. they are found in the environment as molds and the route of infection is inhalation of spores. posttransplant disease with these organisms is rare in adults and has not been reported in the pediatric lung transplant literature to date (neofytos et al. 2010; assi et al. 2013 ). treatment of invasive fungal infection in pediatric lung transplant recipients should include input from an infectious diseases specialist particularly regarding drug choice and dosage. several national and international guidelines present treatment recommendations for invasive fungal infections (pappas et al. 2016; patterson et al. 2016) . new antifungal agents have emerged in the past decade including second-generation azole medications and echinocandins (lewis 2011). while these agents are improving outcomes related to fungal infections, clinicians must pay careful attention to therapeutic drug monitoring, interactions with immunosuppressive agents (both calcineurin inhibitors and mtors), and medication side effects to reduce potential complications. despite the significant morbidity and mortality associated with fungal infections following lung transplantation, there are not established guidelines for prophylaxis. in pediatrics, the impact of prophylaxis to prevent colonization and progression to infection is uncertain. several small, nonrandomized clinical trials in adult recipients have demonstrated efficacy ranging from 80 to 100% (hosseini-moghaddam and husain 2010; brizendine et al. 2011). three main approaches have been used in lung transplant recipients: universal prophylaxis, targeted prophylaxis, and pre-emptive therapy. universal prophylaxis is given to all recipients immediately post transplantation while targeted prophylaxis is given to patients with known risk factors (neoh et al. 2011) . further, response to positive cultures on routine posttransplant bronchoscopy may prompt initiation of pre-emptive therapy, but the optimal response to positive bal cultures is unclear (avery 2011). while inhaled amphotericin has recently been linked to a decrease in posttransplant aspergillus, amphotericin-resistant strains have emerged indicating that intervention is not benign (peghin et al. 2016) . a recent world-wide survey of antifungal prophylaxis (neoh et al. 2011 ) showed a highly variable approach with the majority (58%) using universal prophylaxis that primarily focused on preventing aspergillus infections. a survey of centers performing pediatric lung transplantation (50% exclusively pediatric) revealed an equally variable approach. universal prophylaxis is provided in 28% of centers, while 48% use targeted prophylaxis primarily to patients with cystic fibrosis or pretransplant fungal colonization (mead et al. 2014) . the focus of prophylaxis includes both aspergillus and candida species with most centers reporting the use of either voriconazole or inhaled amphotericin. additionally, the duration of prophylaxis is widely distributed from 3 to 6 months to more than 12 months. the optimal approach for fungal prophylaxis in pediatric lung transplant recipients is undefined and there are sparse data for this population to guide recommendations. the introduction of preventative antiviral regimens has improved the natural history of cytomegalovirus (cmv) after adult lung transplantation (patel and paya 1997; zamora et al. 2004; chmiel et al. 2008) ; however, cmv remains associated with increased morbidity and mortality after transplantation (husni et al. 1998; monforte et al. 2001; ruttmann et al. 2006; chmiel et al. 2008) . to improve the clarity of cmv reporting in the literature, specific definitions have been suggested and updated with diagnostic evolution (humar and michaels 2006; husain et al. 2011; ljungman et al. 2017) . cmv infection refers to the presence of active replicating virus by any method without associated symptoms. cmv syndrome includes the presence of virus plus one or more associated symptoms including fever, fatigue/malaise, leukopenia, atypical lymphocytes, thrombocytopenia, or transaminitis. those with evidence of tissue invasion are defined as end-organ cmv disease. newer definitions take into account the availability of quantitative cmv pcr testing, but a specific viral load in bal to determine cmv pneumonitis has not yet been established (ljungman et al. 2017 ). cytomegalovirus (cmv) occurs in approximately 30% of pediatric lung transplant recipients (danziger-isakov et al. 2003b; danziger-isakov et al. 2009 ) and is associated with decreased survival in this population (metras et al. 1999; danziger-isakov et al. 2003b; danziger-isakov et al. 2009 ). the largest multicenter study from the international pediatric lung transplant collaborative identified cmv donor seropositivity, a2 rejection, and transplant in the earliest era of transplantation (1985) (1986) (1987) (1988) (1989) (1990) (1991) (1992) (1993) as increased risks for cmv. progression from cmv infection to disease occurred in 22% (danziger-isakov et al. 2009 ). interestingly, cmv developed in 7% of cmv dà/rà recipients and induction therapy increased the risk for cmv in this group (danziger-isakov et al. 2009 ). the optimal preventative regimen against cmv remains uncertain in pediatric lung transplant recipients. controversies include choices around the use of universal prophylaxis, risk-based prophylaxis, or pre-emptive therapy and duration of prevention strategy (danziger-isakov et al. 2003a) . as the merits and potential disadvantages in the limited population of pediatric transplant recipients are difficult to discern, extrapolation from the adult lung transplant population directs current practice (kotton et al. 2013) . data from adult lung transplantation indicates that prolonged prophylaxis (9-12 months) with valganciclovir has both short-and long-term benefits preventing cmv events and decreasing risk for bronchiolitis obliterans syndrome (finlen et al. 2011; mitsani et al. 2010; palmer et al. 2010 ). pre-emptive therapy is not currently recommended for high-risk cmv d+/rà lung transplant recipients (kotton et al. 2013; . antiviral complications including nephrotoxicity, bone marrow suppression, gastrointestinal manifestations, and the development of viral resistance mutations must be considered when developing prevention strategies (mitsani et al. 2010; danziger-isakov and mark baillie 2009) . a study in pediatric transplantation showed safety and efficacy of an oral valganciclovir dosing algorithm, but no pediatric lung transplant recipients were enrolled (vaudry et al. 2009 ). pediatric studies have assessed long-term intravenous ganciclovir and the adjunctive use of cmv hyperimmune globulin (cmvig) (spivey et al. 2007; ranganathan et al. 2009 ). in a study of nine pediatric lung transplant recipients, 12 weeks of intravenous ganciclovir was feasible, safe, and effective prevention, although cases of catheter-related bloodstream infections did occur when the catheters remained in place beyond the 12-week ganciclovir administration period (spivey et al. 2007 ). cmvig administration as part of a prevention regimen was associated with a threefold decrease in cmv infection but did not impact the incidence of cmv disease or other posttransplant morbidities and mortality in a multinational retrospective study (ranganathan et al. 2009 ). each institution should assure that a consistent prevention strategy and adequate monitoring are in place (kotton et al. 2013 ). cmv monitoring is an integral part of any prevention strategy and potentially allows identification of cmv infection prior to the development of cmv symptoms or end-organ disease. viral culture or a pp65 antigenemia assay has been replaced by more sensitive polymerase chain reaction (pcr) (weinberg et al. 2000) . as interassay and intercenter variability has been reported for pcr testing even in controlled research settings; utilization of a consistent assay is crucial so that results can be compared for an individual subject over time (pang et al. 2009; rychert et al. 2014 ). based on multicenter retrospective evaluation of pediatric lung transplant recipients (danziger-isakov et al. 2009 ), the highest risk period for cmv infection occurs during the first 6 weeks after discontinuation of prophylaxis; thus, appropriate monitoring should occur during this high-risk period. additionally, evaluation for cmv should occur with new onset symptoms suspicious for cmv infection including fever, fatigue, and lymphadenopathy even in cmv dà/rà patients. increased frequency of cmv surveillance is suggested during periods of increased immunosuppression, but not limited to cytolytic therapy for refractory rejection, plasmapheresis, or prolonged lymphopenia (kotton et al. 2013) . additional monitoring for cmv-specific immunity continues to develop (westall et al. 2008; kumar et al. 2009; snyder et al. 2011; manuel et al. 2013b ) and may be employed in the future to personalize cmv prevention strategies. treatment of cmv infection and disease relies primarily on antiviral therapy and, if possible, decreasing immunosuppression. a multicenter randomized clinical trial of predominantly adult kidney transplant recipients reported noninferiority of oral valganciclovir compared to intravenous ganciclovir for nonlife-threatening cmv disease; however, no pediatric patients were enrolled (asberg et al. 2007 ). current recommendations from the transplant society consensus statement include the use of intravenous ganciclovir for pediatric-aged patients as first-line therapy with acknowledgement that some experts would use oral valganciclovir for cmv infection (kotton et al. 2013) . oral ganciclovir, acyclovir, famciclovir, or valacyclovir should not be used to treat cmv. adjunctive therapy with immunoglobulins for severe pneumonitis (either intravenous immunoglobulin or cmvig) can be considered (kotton et al. 2013) . resistant cmv is rarely reported in pediatric transplant recipients (martin et al. 2010; kim et al. 2012 ), but concern may prompt consideration for alternative antiviral therapy including high-dose ganciclovir, foscarnet, and cidofovir (kotton et al. 2013) . newer antiviral agents are under investigation as options for either prevention or treatment including brincidofovir, letermovir, and maribavir. emerging data on the adoptive transfer of cmv-specific t-cells and the use of small-molecule drugs such as sirolimus, leflunomide, and artesunate may alter the future of treatment, but currently no data related to these interventions exist for pediatric lung transplant recipients. human herpes virus 6 and 7 epidemiology and risk human herpes virus (hhv) 6 and 7 are ubiquitous, common viruses that cause mild infections in young children so frequently that by 5 years of age, practically all children have been infected. there are two types of hhv-6, and although the epidemiology of hhv-6a is not clearly defined, hhv-6b is the most common cause of pediatric infections. young infants, especially those under 2 years of age, are at risk for community-acquired or nosocomial hhv-6 infection after solid organ transplantation, while infection may also be acquired through the allograft or as a reactivation of a prior infection in older children. overall, symptomatic or significant infection with hhv-6 after lung transplantation is uncommon, and the overall incidence in solid organ transplant recipients has been reported to be less than 1% . hhv-7 infection seems to be common, but its clinical manifestations are less well characterized. the most typical disease manifestation of hhv-6 infection is roseola infantum (also known as exanthem subitum or sixth disease), a classic febrile illness in young children where the resolution of a high fever of short (3-5 days) duration is followed by the appearance of a characteristic erythematous rash. while young children may present with roseola after lung transplantation, the most likely clinical manifestation in these patients may be a nonspecific febrile illness that may or may not be associated with an erythematous diffuse rash, hepatitis, pneumonia, encephalitis, and leukopenia. hhv-7 coinfection with hhv-6 is reported frequently, and hhv-7 infection alone appears to be asymptomatic or associated with milder clinical manifestations. diagnosis hhv-6/7 infection is confirmed by detection of the virus in otherwise sterile samples (blood, csf, tissue) by nucleic acid identification (pcr) or consistent histopathologic changes. quantification of viral load might be helpful to assess the progression of viremia. however, there is no known clinically relevant viral load threshold to predict progression or severity of disease. immunohistochemical staining is available in some laboratories and might be helpful to determine the presence of infection in specific organs. however, hhv-6/7 latency in human cells may result in the identification of these viruses in samples without correlation with infection. the first step in the treatment of suspected or confirmed hhv-6/7 infection in immunocompromised solid organ transplant patients is decreasing immunosuppression to allow the host's immune system to control the virus. there are no specific antivirals recommended for treatment of hhv-6/7. however, antiviral activity has been described with ganciclovir and its oral form valganciclovir, foscarnet, and cidofovir. consultation with an infectious diseases expert for the antiviral management of these infections is recommended. there are no vaccines available for the prevention of hhv-6/7 infections. suppression may be observed with antivirals used after transplantation for cmv, such as ganciclovir and valganciclovir; however, specific antiviral prophylaxis for hhv-6/7 is not recommended. hand hygiene is the most effective method to prevent transmission. hhv-8, known as the cause of kaposi's sarcoma, is an oncogenic virus associated with a variety of malignancies (primary effusion lymphoma and castleman disease) and other disease syndromes such as febrile illness, bone marrow suppression, hemophagocytosis, and multiorgan failure in highly immunocompromised patients, including transplant recipients . however, the incidence of hhv-8 infection and disease in children is very rare in the united states. residence in hhv-8 endemic areas is a risk factor, as is receipt of an organ from a donor coming from an endemic area. hhv-8 serology is not routinely obtained in solid organ recipients or donors. as with other human herpesviruses, latency can be established. decreasing immunosuppression is recommended, while treatment of associated malignant disease may also include surgical debulking, cytotoxic chemotherapies, and antivirals (for which the efficacy has not been established). adenoviruses commonly cause self-limited respiratory and gastrointestinal infections in immunocompetent children, but infections can be severe in lung and other solid organ transplant recipients. adenovirus infections are more common in pediatric than in adult transplant recipients. primary adenovirus infections may be acquired after transplantation in young children, while reactivation of latent infection or infection from the transplanted organ may occur in older children and adolescents (florescu et al. 2013) . lung transplant patients are at particularly high risk for complicated respiratory tract infection though inhalation of infected aerosol particles or direct contact transmission from infected individuals. gastrointestinal infection may occur via fecal-oral transmission. most infections occur in the first few months after transplantation, or during periods of enhanced immunosuppression. nosocomial and community exposures may be the source of infection. clinical manifestations of adenovirus depend on the organ affected. adenovirus infection can result in severe respiratory disease in lung transplant recipients, including rapidly progressive, necrotizing and potentially fatal pneumonia, as well as development of chronic lung disease with fibrosis and bronchiectasis (liu et al. 2010) . adenovirus may also cause gastroenteritis, hepatitis, meningoencephalitis, and disseminated disease with multiorgan involvement (florescu et al. 2013) . asymptomatic infection, defined as the identification of adenovirus in clinical specimens by nucleic acid detection (pcr) or culture, has been reported more commonly in adults. in children, persistent and rising viremia should be considered a concerning sign of end organ infection and risk for disseminated disease. graft failure may result from acute adenovirus infection after lung transplantation (doan et al. 2007 ). the diagnosis of adenovirus infection requires the presence of consistent clinical symptoms and the identification of adenovirus by viral culture, molecular methods, direct antigen detection, or characteristic histopathology. most adenovirus serotypes (with the exception of adenovirus 40 and 41 which cause gastroenteritis) can be isolated in cell culture; however, diagnosis by pcr is more commonly used and available. the sensitivity of rapid antigen detection tests is variable and not reliable in immunocompromised hosts. while adenovirus can be identified in respiratory secretions, stool, and urine, these are places where prolonged shedding after infection may occur. therefore, the diagnosis of acute infection is more reliable when viral identification is associated with consistent clinical symptoms, or when adenovirus is found in otherwise sterile specimens such as blood and cerebrospinal fluid or in tissues. rising viremia and detection of virus in two or more sites is considered consistent with invasive adenovirus disease. a viral load cutoff or threshold does not exist to predict the progression of disease or its outcome. however, higher and/or persistent viral loads are concerning for progressive or disseminated disease and typically indicate the need to intervene. decreasing the level of immunosuppression to allow for the host's immune response to handle the virus is the most important treatment strategy to manage adenovirus infections in young solid organ transplant patients. in certain cases, antiviral treatment may be useful, if instituted with the guidance and follow-up of a pediatric infectious diseases specialist. while there are no approved adenovirus-specific antivirals, some agents such as cidofovir have activity against adenovirus and have been used empirically for treatment. however, use of this agent is limited by its propensity to cause nephrotoxicity and bone marrow suppression. close follow up and monitoring for these side effects is recommended. the standard dose of cidofovir in children is 5 mg/kg once weekly. however, more frequent, lower dosage (1 mg/kg three times per week) and pre-and post-dose hydration have been used in an attempt to reduce the risk of renal toxicity (doan et al. 2007) . treatment is usually continued until resolution of viremia and/or symptoms, with close monitoring for side effects. other antivirals have been evaluated for treatment of adenovirus, including a lipid conjugate of cidofovir (cmx001, chimerix inc.), which is administered orally and has a lower risk for nephrotoxicity; however, its use remains experimental at this time. lung transplant patients with severe infection may have hypogammaglobulinemia, and in these cases, administration of intravenous immunoglobulin (ivig) for replacement has been used, although its benefit has not been proven (mawhorter and yamani 2008 ). an effective novel treatment strategy has been developed with the use of antigen-specific cytotoxic t lymphocytes (ctl) directed against adenovirus in hematopoietic stem cell transplant recipients; ctls have not been evaluated in lung or other pediatric solid organ transplant recipients (leen et al. 2009 ). prolonged shedding after resolution of the acute infection may occur; therefore, strict hand hygiene and disease prevention strategies need to be implemented. there are no licensed vaccines for the prevention of adenoviruses. epidemiology and risk pediatric solid organ transplant recipients and particularly lung transplant recipients are at increased risk of medical complications and mortality when acquiring common respiratory viral infections (manuel et al. 2013a) . common respiratory viruses that circulate with well-described seasonality in the united states include influenza virus, respiratory syncytial virus (rsv), human metapneumovirus, human rhinovirus, parainfluenza viruses, coronaviruses, and other respiratory viruses that are being more frequently described, such as bocaviruses. lung transplant recipients are at risk for community and nosocomial exposures during the typical time of circulation of these viruses. infection with respiratory viruses may also increase the risk for secondary bacterial pneumonia and other bacterial or fungal infections, particularly in the first few months after transplant (liu et al. 2009a) . after an acute lower respiratory virus infection, the risk for graft rejection or chronic allograft dysfunction may increase as shown in adult lung transplant recipients; however, this is controversial and has not been shown in pediatric lung transplant recipients to date (liu et al. 2010; liu et al. 2009a; vu et al. 2011) . although upper respiratory infections may present similarly in solid organ transplant recipients as in immunocompetent hosts, progression to lower respiratory tract disease manifestations with tachypnea, cough, abnormal breath sounds, hypoxemia, and respiratory failure is a concern in lung transplant recipients. clinical deterioration due to respiratory viruses is more frequently reported in the period of highest immune suppression shortly after transplant. prompt diagnosis with viral detection using nucleic acid amplification methods (pcr) is recommended in immunocompromised hosts. viral cultures could be obtained but are not as useful given that results are delayed in comparison with pcr. pcr platforms that test for multiple viruses at the same time are most helpful in lung transplant recipients. rapid antigen detection tests are no longer recommended for influenza due to their variable sensitivity; but they could still be useful for the diagnosis of rsv. respiratory secretions including nasal wash or swabs, naropharyngeal aspirates, and tracheal or broncheoalveolar lavage can be used. these viruses do not tend to be associated with viremia. supportive measures must be instituted promptly in lung transplant recipients with progression to lower respiratory tract disease. the need for invasive mechanical ventilation or other higher level of supporting measures such as extracorporeal membrane oxygenation (ecmo) is not uncommon in patients with severe disease. specific antiviral treatment is available for influenza a and b infection. immediate initiation of neuraminidase inhibitor (oseltamivir and zanamivir) therapy in lung transplant patients with fever and/or other respiratory symptoms during the period of influenza circulation may decrease the risk of complications and death associated with influenza. although influenza antivirals are usually preferred within 48 h of the onset of clinical symptoms, lung and other solid organ transplant patients have improved outcomes even with later treatment initiation (kumar et al. 2010 ). in some cases, a more prolonged duration of antiviral therapy has been used given these patient's immune-suppressed status and prolonged viral shedding. intravenous peramivir (also a neuraminidase inhibitor) is now licensed for adults, with clinical studies underway in children and adolescents. intravenous administration might be preferred in patients who have inadequate enteral absorption and who are severely ill with influenza. ribavirin, an aerosolized antiviral with in vitro activity against rsv, parainfluenza, human metapneumovirus, and other viruses, is fda approved but not routinely recommended for treatment of these infections due to lack of definitive efficacy. however, ribavirin has been used early in the course of rsv and other respiratory virus infections, as well as in more severe cases of respiratory disease, in lung transplant patients due to its antiviral effects. no randomized controlled trials have been performed although data from adult lung transplantation has indicated a potential response to aerosolized, intravenous, and oral ribavirin (glanville et al. 2005; pelaez et al. 2009; li et al. 2012 ). an inhaled small-interfering rna that targets rna (aln-rsv-001) has also been investigated as a therapy for rsv in adult lung transplantation showing potential reduction in bronchiolitis obliterans syndrome after rsv infection (zamora et al. 2011; gottlieb et al. 2016) . utilization of an rsv antibody preparation (monoclonal antibody) along with antiviral treatment in severe cases has been reported to reduce rsv-associated mortality in some cases (chavez-bueno et al. 2007 ). similar to the management of other viral infections, decreasing immune suppression is advisable when respiratory viral infections are identified. influenza immunization prior to and/or after transplantation for the recipient and all close contacts and family members is recommended to prevent infection and severe disease. inactivated influenza vaccine should be administered ideally prior to the start of the season, to ensure optimal protection. however, after transplant, and in some patients prior to transplant depending on their underlying diagnosis or need for chronic steroid or other medication use, the immune responses to vaccination might be suboptimal in lung and other solid organ transplant recipients. therefore, vaccination of close contacts and avoidance of contact with sick individuals become important measures for prevention of infection (avery et al. 2013) . prophylactic antivirals may also help decrease the risk of infection and complications in exposed unvaccinated or unprotected transplant recipients. there are no other vaccines available for the prevention of respiratory infection in most pediatric lung transplant recipients. however, palivizumab, a monoclonal antibody against rsv, can be used during the rsv season in young children less than 2 years of age who are lung transplant recipients, immunosuppressed, or who have underlying chronic lung or hemodynamically unstable heart disease (american academy of pediatrics committee on infectious diseases and american academy of pediatrics bronchiolitis guidelines committee 2014). all lung and solid organ transplant patients with suspected or known respiratory viral infections need to be isolated from other patients using standard contact and droplet precautions. posttransplant, infections remain a significant factor causing both morbidity and mortality in pediatric lung transplant recipients. pathogens are diverse including bacteria, fungi, and viruses with timing of events dependent on time from transplant. all events can have both immediate and long-term consequences in this at-risk population. prevention, identification, and early intervention for infectious events can improve outcomes after pediatric lung transplantation. lung infections in pediatric lung transplantation: experience in 49 cases cytomegalovirus disease among donor-positive/recipient-negative lung transplant recipients in the era of valganciclovir prophylaxis nebulized amphotericin b prophylaxis for aspergillus infection in lung transplantation: study of risk factors epidemiology and outcome of invasive fungal infections in solid organ transplant recipients epidemiology, risk factors, and outcomes of clostridium difficile infection in kidney transplant recipients antifungal prophylaxis in lung transplantation-a world-wide survey international guidelines for the selection of lung transplant candidates: 2006 update -a consensus report from the pulmonary scientific council of the international society for heart and lung lransplantation extended valganciclovir prophylaxis to prevent cytomegalovirus after lung transplantation: a randomized, controlled trial interlaboratory comparison of cytomegalovirus viral load assays clinical practice guideline for the management of candidiasis: 2016 update by the infectious diseases society of america early and late infections in lung transplantation patients diagnosis of invasive aspergillosis in lung transplant recipients by detection of galactomannan in the bronchoalveolar lavage fluid infections in solid-organ transplant recipients practice guidelines for the diagnosis and management of aspergillosis: 2016 update by the infectious diseases society of america 10 years of prophylaxis with nebulized liposomal amphotericin b and the changing epidemiology of aspergillus spp. infection in lung transplantation efficacy of oral ribavirin in lung transplant patients with respiratory syncytial virus lower respiratory tract infection nontuberculous mycobacterial disease is not a contraindication to lung transplantation in patients with cystic fibrosis: a retrospective analysis in a danish patient population cytomegalovirus immunoglobulin decreases the risk of cytomegalovirus infection but not disease after pediatric lung transplantation human herpesviruses 6, 7 and 8 in solid organ transplant recipients cytomegalovirus in solid organ transplantation hypogammaglobulinemia: incidence, risk factors, and outcomes following pediatric lung transplantation combined cmv prophylaxis improves outcome and reduces the risk for bronchiolitis obliterans syndrome (bos) after lung transplantation multicenter comparison of laboratory performance in cytomegalovirus and epstein-barr virus viral load testing using international standards staphylococcus aureus infections in the early period after lung transplantation: epidemiology, risk factors, and outcomes impact of multidrug-resistant organisms on patients considered for lung transplantation mycobacterium abscessus complexa particular challenge in the setting of lung transplantation polyfunctional cytomegalovirusspecific immunity in lung transplant recipients receiving valganciclovir prophylaxis epidemiology and management of infections after lung transplantation safety and efficacy of prolonged cytomegalovirus prophylaxis with intravenous ganciclovir in pediatric and young adult lung transplant recipients effect of etiology and timing of respiratory tract infections on development of bronchiolitis obliterans syndrome multidrug-resistant gram-negative bacteria infections in solid organ transplantation valganciclovir dosing according to body surface area and renal function in pediatric solid organ transplant recipients (2013) (6) key: cord-007575-5ekgabx5 authors: luby, james p. title: southwestern internal medicine conference: pneumonias in adults due to mycoplasma, chlamydiae, and viruses date: 2016-01-14 journal: am j med sci doi: 10.1097/00000441-198707000-00007 sha: doc_id: 7575 cord_uid: 5ekgabx5 pneumonias in adults due to mycoplasma, chlamydiae, and viruses are a common clinical problem. these microorganisms contribute to the etiologies in 6–35% of all cases of pneumonia and are the sole pathogens in 1–17% of hospitalized cases. important trends and developments in the field include (1) the emergence of a chlamydia psittaci strain (twar) that is passaged from human to human, causes a mycoplasma-like illness, and that is relatively resistant to erythromycin, (2) the recognition of respiratory syncytial virus as a pathogen in nursing home outbreaks and in immunosuppressed adults, the continuing high lethality of fully developed influenza pneumonia, (4) the efficacy of acyclovir and adenine arabinoside in limiting the complications of varicella-zoster virus infections, and (5) the increasing frequency of pneumonia caused by cytomegalovirus and the severity of this disorder in highly immunosuppressed patients. developments in the rapid diagnosis and therapy of respiratory syncytial virus infections with an aerosolized antiviral drug in children may pave the way for comparable advances in difficult pneumonias in adult patients. southwestern internal medicine conference: pneumonias in adults due to mycoplasma, chlamydiae, and viruses by james p. luby, md abstract: pneumonias in adults due to mycoplasma, chlamydiae, and viruses are a common clinical problem. these microorganisms contribute to the etiologies in 6-35% of all cases of pneumonia and are the sole pathogens in 1-17% of hospitalized cases. important trends and developments in the field include (1) the emergence of a chlamydia psittaci strain (twar) that is passaged from human to human, causes a mycoplasma-like illness, and that is relatively resistant to erythromycin, (2) the recognition of respiratory syncytial virus as a pathogen in nursing home outbreaks and in immunosuppressed adults, (3) the continuing high lethality of fully developed influenza pneumonia, (4) the efficacy of acyclovir and adenine arabinoside in limiting the complications of varicella-zoster virus infections, and (5) the increasing frequency of pneumonia caused by cytomegalovirus and the severity of this disorder in highly immunosuppressed patients. developments in the rapid diagnosis and therapy of respiratory syncytial virus infections with an aerosolized antiviral drug in children may pave the way for comparable advances in difficult pneumonias in adult patients. key population-based studies on the incidence ofpneumonia have been performed infrequently. in one study, during an 8-year interval from december 1, 1963 , through november 30, 1971 , foy and her colleagues determined the incidence of pneumonia in a prepaid medical health insurance plan comprising more than 100,000 members in seattle, washington. 1 they found that total pneumonia rates varied yearly and ranged between 7.2 and 16.8 cases/1,000 population/year. only 15% of the cases seen by the physicians caring for this group were hospitalized; 85% of the total number of cases were managed as outpatients. in adults, 35% of all cases of pneumonia were associated with cultural and/or serologic evidence of mycoplasma and/or viral infection. total rates for all cases of pneumonia increased during influenza a2 epidemic years. the highest rates were generally found in the winter quarter, followed by rates occurring during the spring quarter ( figure 1 ). the major viral and mycoplasma agents contributing to the etiology of pneumonia in this study were influenza a virus, and mycoplasma pneumoniae, followed by a smaller number of cases due to adenoviruses, influenza b virus, respiratory syncytial virus (rsv), and parainfluenza viruses. most of the parainfluenza virus infections were caused by types 2 and 3, but no attempt was made to ascertain the exact contribution of specific agents involved because of antigenic overlap in the complement fixation test. it was recognized that the majority of pneumonias associated with influenza a were related to bacterial suprainfection. rates for all pneumonia were highest in young children, followed by a peak in pneumonia due to m. pneumoniae in the 30-40-year age group. pneumonia due to influenza a virus increased in incidence above the age of 60 years. of interest is their finding that sometimes pneumonia was associated with laboratory evidence of infection with more than one respiratory nonbacterial agent. the severity of disease did not appear different between patients with a single infection and those with multiple infections, as measured by duration of illness and hospitalization rates. in individual reports, however, it has been suggested that in certain individuals, multiple infections can sometimes lead to a more severe course than would have been predicted by infection with a single agent. 2 overall rates for pneumonia as determined in this study were similar to those observed in the national health survey. in houston, texas, during the years 1975-1978, adult hospitalizations for pneumonia increased sharply during influenza a epidemics but did not change much during influenza b epidemics, a finding that was also seen in the seattle study (figure 2) . 3 although hospitalizations for pneumonia did not increase in houston during the 1976-1977 influenza b epidemic, an increased number of patients hospitalized with complications due to influenza b virus infection was seen in dallas, texas, at this time. 4 the etiology of community-acquired pneumonia in 54 adult outpatients in sweden has been determined. 5 using rises in antibody titer between acute and convalescent sera to determine etiology, these investigators found evidence of mycoplasma pneumoniae infection in 37%; streptococcus pneumoniae in 9%; hemophilus in{luenzae in 12% (6% type band 6% nontypeable); influenza a virus in 6%; chlamydia psittaci in 4%; and influenza b virus, parainfluenza 3 virus, respiratory syncytial virus, and adenovirus in 2% each. multiple infections occurred in several patients and there was no serologic evidence of infection with a particular microorganism in 41 %. the etiologic agents of community-acquired pneumonia in adult patients hospitalized for their disease can be examined (table 1) . six recent studies were selected for analysis because they had a worldwide authorship and because an attempt had been made to estimate the contribution made by both bacterial and nonbacterial agents.6-11 bacterial 46 etiologies contributed most significantly to the problem of community-acquired pneumonia in adult hospitalized patients. the most frequent microorganisms were streptococcus pneumoniae followed by staphylococcus aureus, haemophilus in{luenzae, legionella pneumophila, and other gram-negative bacteria. the role of anaerobic bacteria in the etiology of pneumonia was not studied systematically in five of the series. a nottingham, england, study accentuated the role of l. pneumophila in the etiology of pneumonia and showed the propensity of this microorganism to be associated with specific geographic sites. the major nonbacterial agents implicated in these studies included m. pneumoniae, influenza a and b viruses, adenoviruses, rsv, parainfluenza viruses, varicella, and c. psittaci. all of these latter agents could be seen as single pathogens but influenza a and b viruses, adenoviruses, rsv, parainfluenza viruses, and varicella virus also were associated with bacterial pneumonia. nonbacterial agents contributed from 6 to 35% to the etiologies of all cases. an indication of the approximate incidence of nonbacterial pneumonia without bacterial suprainfection can be ascertained in five of the series and ranged from 1 to 17% of the cases. the association of influenza a and b viruses, rubeola virus, and varicella virus with bacterial suprainfection has been well established. recently, bacterial suprainfection has been shown to occur in adults who have evidence of infection with adenoviruses and rsv. the frequency of bacterial suprainfection in association with m. pneumoniae infections is difficult to ascertain. it has been considered the concept of primary atypical pneumonia was set forth in an article by hobart a. reimann in 1938. 12 the major bacteria-causing pneumonia were known at that time with the exception of l. pneumophila. the clinical entity of psittacosis had been elucidated. influenza a virus had been grown in ferrets by laidlaw, andrews, and smith. reimann described eight cases of what he called atypical pneumonia, which he thought was due to a filterable virus. the description of cases allows a view of untreated primary atypical pneumonia. it now seems probable that m. pneumoniae was the etiologic agent in most of his cases. the illness often began in-the american journal of the medical sciences sidiously with fever, headache, and pharyngitis. with descent of the disease into the respiratory tract, the larynx became involved and hoarseness was present. finally, laryngotracheobronchitis and pneumonia occurred. a troublesome cough developed in the patient that could not be alleviated and was only slightly productive. in some patients, a pulse-temperature dissociation occurred. during the course of the disease, which often lasted several weeks, patients became dyspneic and cyanotic. two of the patients became delirious and had central nervous system dysfunction during the course of the infection. on physical examination, the patient was flushed and had evidence of pharyngitis. the physical examination of the chest usually revealed scattered rales without striking evidence of consolidation; in one patient a large pleural effusion was present. the white blood count was only modestly elevated. chest radiographs revealed mottled or diffuse areas of infiltration. attempts to isolate pathogenic bacteria and influenza a virus were unsuccessful in establishing an etiology for this syndrome. reimann considered diagnoses such as typhoid, psittacosis, and epidemic influenza, but the history in none of these cases was consistent and influenza virus could not be recovered. to summarize reimann's words, "the infection occurred in adults and began as a mild infection of the respiratory tract; this was followed by severe diffuse atypical pneumonia and in two cases by the symptoms of encephalitis. dyspnea, cyanosis, hoarseness, cough without sputum, drowsiness, and profuse sweating were the chief characteristics. the disease lasted several weeks." in 1943, finland found elevated cold agglutinin titers in cases of atypical pneumonia. eaton later isolated the agent in embryonated eggs, and chanock and colleagues were able to grow it on defined media and demonstrate it to be a mycoplasma. the entity of primary atypical pneumonia became well known and later was defined as pneumonia that did not clear with penicillin or sulfonamides, or nonbacterial pneumonia, or pneumonia with no sputum or a mucoid sputum without a predominant organism on gram's stain. we recognize today that the clinical entity of primary atypical pneumonia has multiple etiologies, particularly m. pneumoniae, but also c. psittaci, the twar strain ofc.psittaci, chlamydia trachomatis, q-fever, and viruses such as adenoviruses, rsv, influenza viruses, and para-48 influenza viruses. l. pneumophila infections often are considered in the differential diagnosis. ls early bacterial pneumonia also should be considered when the patient is first seen. in retrospect, persons with this diagnosis are often excluded from series of cases to focus specifically on the nonbacterial nature of the problem. in pertinent geographic areas, acute histoplasmosis and coccidioidomycosis may present like primary atypical pneumonia. major attempts to identify the etiologic agent on clinical grounds have been made, but the exact diagnosis usually depends on laboratory determination of the offending agent. in one study of 150 patients of all ages, 50 with viral pneumonia, 50 with mycoplasma pneumonia, and 50 with bacteremic i:meumococcal pneumonia were compared. 14 the best discriminating variables were the c-reactive protein determination, the presence or absence of predisposing disease or previous antibiotic treatment, the erythrocyte sedimentation rate, the presence of lymphocytosis, and the band neutrophile count. signs of an upper respiratory tract infection and the presence or absence of auscultatory abnormalities also aided significantly in the discrimination. determinations favoring bacteremic pneumococcal pneumonia included predisposing disease, a short duration of illness before hospitalization, alcoholism, the absence of signs of an upper respiratory tract infection, high c-reactive protein determinations and erythrocyte sedimentation rates, no prior antibiotic treatment, total leukocyte counts exceeding 15,000, relative lymphocyte counts less than 35%, relative band neutrophile counts greater than 20%, abnormal auscultatory findings, and the presence of lobar consolidation on chest radiograph. differentiation between viral and mycoplasma pneumonia could not be made easily. however, symptoms of mycoplasma pneumonia before hospitalization lasted a longer time and these patients were more likely to have received antibiotic treatment in the interval before hospitalization. patients with mycoplasma pneumonia were more likely to have lobar consolidation on chest radiograph than those with viral pneumonia, but in this study no distinction could be made between mycoplasma pneumonia and bacteremic pneumococcal pneumonia on the basis of roentgenographic findings alone. in another study comparing community-acquired pneumonias, mycoplasma pneumonia tended to occur at an earlier age than legionnaire's disease, pneumococcal pneumonia, or psittacosis.l 5 homogeneous shadowing on chest radiograph was more common in legionnaire's disease and pneumococcal pneumonia than mycoplasma pneumonia. pleural effusions were uncommon in all groups but occurred most commonly in bacteremic pneumococcal pneumonia as did multilobe disease on presentation. hilar lymphadenopathy occurred only in mycoplasma pneumonia. roentgenographic resolution was fastest in mycoplasma pneumonia, intermediate in psittacosis and nonbacteremic pneumococcal pneumonia, and slowest in legionnaire's disease and bacteremic pneumococcal pneumonia. deterioration on chest radiograph after hospital admission characterized legionnaire's disease and bacteremic pneumococcal pneumonia. because the differential diagnosis of primary atypical pneumonia at a clinical level includes pneumonia due to m. pneumoniae, chlamydial species, q fever, adenoviruses, rsv, influenza viruses, parainfluenza viruses, as well as l. pneumophila infections and early bacterial pneumonia, therapy should include an antibiotic to which the majority of these the american journal of the medical sciences luby microorganisms are susceptible. chlamydial species are more susceptible to tetracycline than erythromycin. tetracycline is effective against rickettsiae but not for l. pneumophila infections. up to 4% of pneumococcal isolates are resistant to tetracycline. a reasonable antibiotic choice is erythromycin at an equivalent dose of 30 mglkg of erythromycin base per day for 10-14 days. if legionnaire's disease is diagnosed, a higher dose of erythromycin may be necessary. if a chlamydial or rickettsial etiology is recognized, tetracycline at a dose of 2 gmiday should be given. occasionally patients with proven m. pneumoniae have been treated with erythromycin, failed to respond to therapy, but subsequently responded to a course of tetracycline therapy.ls conversely, some patients with m. pneumoniae infections have responded to erythromycin after a suboptimal response to tetracycline therapy. viruses may cause primary atypical pneumonia; however, antibiotic treatment in these instances is useless, does not prevent suprainfection, and may actually change the nature of the bacterial species suprainfecting the patient. antibiotic therapy seems reasonable in this syndrome, however, because it is usually impossible to differentiate clinically between mycoplasma pneumonia and an entity such as adenovirus pneumonia in the adult. advances in rapid laboratory diagnosis may be able in the future to influence treatment options but these techniques are still under development, are expensive, and are not widely available. mycoplasmas, the smallest free-living microorganisms, are cell-wall deficient, but have no relationship to cell-wall deficient bacteria with which they were once confused. m. pneumoniae attaches to the mucosal epithelium of the respiratory tract through a specific protein that enables the microorganism to adhere to neuraminic acid residues on respiratory epithelial cells. if mycoplasmas cannot attach, there is no damage to the host. upon adherence, mycoplasmas are able to generate hydrogen peroxide and superoxide anion, resulting in injury to epithelial cells. because infection occurs commonly in children younger than 5 years of age, although disease is rare at this time of life, mycoplasmas may induce disease primarily by immunopathologic mechanisms. l7 . lb in experimental animals not primed by prior mycoplasma exposure, inflammatory changes occur only after a long interval. with reinfection, inflammatory changes occur more briskly. the extrapulmonic manifestations of my coplasma infection have never been explained completely, but there are reports demonstrating m. pneumoniae in sites such as cerebrospinal fluid and blood. l9 alternatively, immunopathologic reactions may be the primary mechanism involved. path-ologically, the disease in man is characterized by tracheobronchial, bronchiolar and septal lymphoplasmocytic infiltrates, luminal exudates rich in polymorphonuclear leukocytes, bronchiolar and alveolar cell metaplasia, and occasionally diffuse alveolar injury.2o the bronchiole appears to be the major site of attack. the microorganisms colonize the nasopharynx and transmission of infection occurs only by close contact. especially conducive to the transmission of m. pneumoniae are situations in which persons are housed in closed quarters, such as military platoon barracks or family unit dwellings. in families, there is a high attack rate and cases continue to occur over a 3-4-month interva1. 21 . 22 the cumulative attack rate of mycoplasma infections in families may approach 90% (figure 3 ).21 mycoplasma carriage is not affected by antibiotic therapy, thereby allowing the family epidemic to continue. mycoplasma disease occurs throughout the year but is particularly frequent during fall and winter. increased numbers of cases occur with a 3-5-year periodicity. although pneumonia may occur soon after infection, the disease is usually manifested as an upper respiratory tract infection progressively descending into the lung. pharyngitis progresses into laryngitis followed by tracheobronchitis, and, finally, pneumonia. hoarseness and dysphonia may be present. middle ear involvement may occur with bullous myringitis, which usually heals without scarring. occasionally, otitis may lead to tympanic membrane perforation. sinus involvement is frequent but usually asymptomatic. the cough is often intractable and usually only slightly productive of a mucoid sputum that contains mainly polymorphonuclear leukocytes but no predominant bacterial microorganism on gram's stain. when pneumonia develops, the patient has an elevated temperature and, occasionally, a 'temperature-pulse dissociation. headache, irritation, a flushed facies, myalgias, and arthralgias are common. 2 3-26 on physical examination, the patient is febrile, appears flushed, and usually has physical evidence of pharyngitis. hemorrhagic bullous myringitis may be present in up to 5% of cases. physical findings on chest examination usually are limited to scattered rales, wheezes, and rhonchi and are often localized to the lung bases. evidence of consolidation is not striking, although m. pneumoniae infections can cause lobar pneumonia. 26 the white count is usually elevated with a shift to the left, but rarely exceeds 15,000 white blood cells/mm 3 and the neutrophile band count is usually less than 10%.14 chest radiograph reveals peribronchial infiltrates with accentuation of interstitial markings in adjacent lung segments, patchy alveolar infiltrates usually localized to the lower lobes, especially on the left, and occasionally hilar lymphadenopathy. 27 more than one lobe may be involved and a confluent lobar infiltrate may be present in some patients ( figure 4 ). less commonly, there is a diffuse interstitial infiltrate and rarely an x-ray picture indistinguishable from the adult respiratory distress syndrome. 28 . 29 without therapy, the disease course usually lasts approximately 3 weeks, but may extend up to 7 weeks. 12 extrapulmonic manifestations of mycoplasma infection often are a clue to the diagnosis and include bullous myringitis; neurologic disturbances suggesting encephalitis or aseptic meningitis and, rarely, transverse myelitis; arthritis; myopericarditis; hepatic dysfunction; splenomegaly; and skin eruptions.~u a stevens-johnson syndrome may occur. japanese workers have described typical cases of pityriasis rosea that followed mycoplasma infection. 32 the cerebrospinal fluid (csf) may be abnormal with an increased number of cells and an elevated protein concentration. hemolytic anemia may be present resulting from antibody directed against the i antigen on the red-cell membrane. 31 almost all patients recover completely after mycoplasma infection but cigarette smokers may have prolonged abnormalities in diffusion capacity.33 individual case reports have described pulmonary fibrosis, bronchiolitis obliterans, and bronchiectasis following m. pneumonia. 3~6 glomerulonephritis with continuing renal dysfunction also has been reported. 37 • 38 the diagnosis is established by culture of the microorganism or the demonstration of a fourfold rise in antibody by complement fixation or other serologic test. a single high complement fixation test antibody titer (~1: 128) may be used as presumptive evidence of infection. cold agglutinin antibody titers at low level are norispecific but very high values (~1 : 128) also can be used to support the diagnosis. treatment consists of the administration of either erythromycin or tetracycline as outlined in the therapy of primary atypical pneumonia. the patient usually responds, but it should be remembered that there are reports of inadequate resolution of the disease and the necessity to switch to the alternate drug to achieve more rapid clinical improvement. antibiotic therapy does not eliminate the carrier state. immunity is relatively short-lasting and documented episodes of repeated mycoplasma infection have been reported. a vaccine against m. pneumoniae, given present priorities, appears only a hopeful future development. mycoplasma pneumonia. an 18-year-old man was well until 8 days before admission into the hospital, at which time he developed fever, a sore throat, and a nonproductive cough. his oral temperature reached 40° c. a "pounding" headache developed. the cough persisted and became productive of a mucoid sputum. oral penicillin was prescribed but did not alleviate his symptoms. physical examination on admission into the hospital revealed a young man who was confused about time and uncertain about recent events. the oral temperature was 38.8° c and the pulse rate was 100. the pharynx was described as normal. chest examination revealed harsh breath sounds with bilateral inspiratory rales, especially on the right, anteriorly and inferiorly. there was no egophony or decreased fremitus. rhonchi were present more on the right than left. hepatosplenomegaly was present. laboratory examination revealed 12,200 white blood cells with 70% polymorphonuclear cells, 28% lymphocytes, one monocyte, and one eosinophile. the serum aspartate aminotransferase was 140 (normal <40). arterial blood gases on room air showed a ph of 7.55, pc02 of29, and p02 of 45. a lumbar puncture was performed that showed 31 white blood cells, 95% of which were mononuclear cells. the csf glucose was 69 mg/dl and the simultaneous plasma glucose 155 mg/dl. the patient was treated initially with intravenous penicillin for presumed pneumococcal pneumonia and partially treated bacterial meningitis. his condition deteriorated but finally he was placed on erythromycin therapy at the advice of a consultant. mycoplasma complement fixation test titers rose from less than 1: 8 to 1: 64. comment. encephalitis, hepatosplenomegaly, and mild hepatic dysfunction were the extrapulmonic manifestations of mycoplasma disease. typical of mycoplasma pneumonia were the long duration of illness before admission into the hospital, prior antibiotic administration, presence of a sore throat, physical examination of the chest, and characteristics of the sputum. psittacosis was first described by ritter in switzerland in 1879 as a disease of the lungs in patients in contact with sick psittacine birds. later, in 1929-1930, a pandemic of psittacosis occurred involving psittacine birds exported from south america. the clinical manifestations were described fully and the epidemiology was established, leading to control measures that have kept psittacosis or the better, more inclusive term, ornithosis, at a low level of occurrence. occasionally migrant birds can carry c. psittaci, and persons dealing with them may develop ornithosis. more importantly now, ornithosis is an occupational hazard to the farmer who manages poultry such as ducks and turkeys.17,lb,40,41 clinically, patients with ornithosis have headache, fever, pulse-temperature dissociation, pneumonia, hepatic function abnormalities, and hepatosplenomegaly. intra-alveolar inflammatory changes predominate in ornithosis with interstitial changes being secondary and less prominent. the chest radiograph reflects this and lobar consolidation may be seen. when lung involvement is minor, the disease can be diagnostically confusing, and present as a fever of undetermined origin. granuloma formation can be found in both the liver and the bone marrow and may be a diagnostic clue. ornithosis can be diagnosed by serologic tests with a chlamydial common group antigen by either complement fixation or the enzyme-linked immunoabsorbent assay (eia). treatment is with tetracycline for 10-14 days. chlamydia trachomatis can cause an afebrile pneumonia-like syndrome in young infants beginning at the age of 1-3 months, and is characterized by an afebrile state, failure to gain weight, and a staccato-like cough. on examination, there are rales, expiratory wheezing, and evidence of hyperaeration of the lungs. chest radiograph usually reveals diffuse interstitial pneumonia and hyperaerated lung fields. laboratory determinations show a modest eosinophilia and hyperglobulinemia. upon this identification, the infants can be treated with oral erythromycin syrup for 3 weeks with benefit. recently, c. trachomatis has been isolated from the lower respiratory tract of immunosuppressed patients with pneumonia, although four of the six patients reported and the only ones tested did not show a serologic response to that microorganism. 42 cases of community-acquired pneumonia in normal adults also have been reported with serologic evidence of infection with c. trachomatis. 43 fifty-two patients were studied and seven were found to have definite or suggestive serologic evidence of infection. 52 the seven ranged in age from 22 to 77. the chest radiographs of these patients have been analyzed and the infiltrates were found to be patchy and characteristically streaky with areas of plate atelectasis. there was no particular localization to a single lobe and three patients had radiographic evidence ofmultilobar involvement. 44 further studies need to be done to corroborate these reports and determine the frequency with which lung involvement occurs. in finland, an epidemic of mild pneumonia has been related to a newly described strain of c. psittaci, capable of being passaged from human to human. this epidemic occurred in adolescents and young adults and had a point prevalence of pneumonia of 15-19 cases/1,000 students at the time of x-ray survey. 45 the contribution of this particular strain of c. psittaci, designated the twar strain from tw-183 and ar-39, the first two isolates, has been examined best during a 2.5-year study at the university of washington. infected students usually presented with a mild pneumonia that simulated mycoplasma infection and was often associated with pharyngitis and laryngitis. 46 in this study, the twar strain of c. psittaci caused 12% of the pneumonias in the student population. the twar strain of c. psittaci was isolated from the students, and serial sera showed conversion to the common chlamydial group antigen by complement fixation tests. microimmunofluorescence tests revealed specific reactions to the twar strain of c. psittaci. the microorganisms isolated from the students formed typical inclusion bodies in tissue culture, were not stained by iodine, and were considered typical of c. psittaci. the clustering of cases had an epidemiology that suggested human-to-human transmission. bird-to-human transmission could not be demonstrated in any of the cases. treatment with tetracycline shortened the course, but, occasionally, patients did not respond to 1 gm of erythromycin given for 5-10 days. this new strain of c. psittaci was isolated from the patients and serologic reactions to specific antigens were demonstrated. the evidence linking c. trachomatis to lung disease has either been by isolation alone or just by serologic testing. further studies similar to the one in seattle need to be performed to link c. trachomatis to lung disease. it is clear, however, that a new strain of c. psittaci exists and can cause disease commonly. the disease due to this microorganism can be diagnosed by complement fixation or eia tests using chlamydial group antigen. specialized laboratories can isolate the organism and also perform microimmunofluorescence tests. a major new development in the evaluation of patients with primary typical pneumonia is the emergence of this c. psittaci strain that is capable of being passaged from human to human, and that may not have the desired response to erythromycin treatment. adenoviruses are ubiquitous nonenveloped dna viruses that colonize the human nasopharynx and are transmitted to other persons by close contact. types 4 and 7 are recognized for their capacity to produce epidemics in military recruit populations. because the transmission of this group of viruses is dependent upon close human contact, disease is often produced in the home or the military recruit barracks. pathogenetically, lung infection usually follows pharyngitis and a movement of the disease process down the respiratory tract. although most cases of pneumonia are not severe, cases coming to autopsy show that the tracheobronchial mucosa is denuded of the normal epithelial structures down to the basal layer. squamous metaplasia occurs along with interstitial space thickening due to the presence of chronic inflammatory cells. alveolar edema and mononuclear cell infiltrates are present. as with m. pneumoniae infections, infiltrates are often peribronchial or peribronchiolar in distribution. nuclear inclusion bodies or nuclei with a smudged appearance may be found in epithelial cells. clinically, the disease often begins with pharyngitis associated with fever and anterior cervical lymphadenopathy with or without conjunctivitis, then involves the tracheobronchial tree, and, finally, the parenchyma of the lung. pneumonia is most common in infants, young children, and military recruits. in military recruit populations, mycoplasma and adenovirus pneumonia have been found to be indistinguishable clinically except for an increased luby frequency of exudative pharyngitis with adenovirus infection. physical examination reveals pharyngitis and rhinitis and scattered rales and rhonchi. 47 evidence of consolidation is infrequent but occasionally lobar consolidation can occur, as can a pleural effusion 48 ( figure 5 ). virus rarely has been isolated from pleural fluid. 2 fatal cases of adenovirus pneumonia can occur in infants, immunosuppressed patients, and rarely in normal persons. 4 9-53 in these cases, the pneumonia is progressive with the development of diffuse bilateral alveolar infiltrates and hypoxemia requiring ventilator assistance for its correction. as the infiltrates progress, leukopenia ensues with marked lymphocytopenia. rhabdomyolysis occurs along with evidence of disseminated intravascular coagulation and renal failure. terminally, the patient becomes obtunded. bacterial suprainfection can be associated with adenovirus pneumonia. suprainfecting species of. bacteria include s. pneumoniae, group a strepto-· cocci, h. influenzae, s. aureus, and group y neisseria meningitidis. 54 in military recruits, an increased frequency of group y meningococcal suprainfection has been observed because these microorganisms commonly colonize the nasopharynx in this population. administration of antibiotics during the course of the adenovirus pneumonia does not prevent bacterial suprainfection. although most persons recover uneventfully from adenovirus pneumonia, occasional patients have residual abnormalities such as restrictive lung disease, bronchiectasis, or bronchiolitis obliterans. 55 ,56 extrapulmonic manifestations of adenovirus infection include pharyngitis, conjunctivitis, pericarditis, arthritis, skin rashes, and hepatic dysfunction. reye's syndrome has been described during the course of adenovirus pneumonia. 57 the occurrence of one or more of these manifestations during the course of pneumonia can lead the physician to order appropriate diagnostic tests to make a specific etiologic diagnosis. virus can be isolated from the nasopharynx, sputum, or endotracheal secretions. antigen can be detected by immunofluorescence tests, eia, or dna hybridization within epithelial cells derived from the respiratory tract. 58 these latter tests are specific but at the present time less sensitive than viral culture. there is no specific therapy for the infection. oral live attenuated vaccines are available against types 4 and 7 adenoviruses, and these are used now in the military to prevent epidemic disease. fatal adenovirus pneumonia. a 32-year-old man with an unremarkable past history except for hypertension was controlled on medication. two weeks before admission he developed a nonproductive, hacking cough and began to have dyspnea, which increased to the time of admission. on physical examination he appeared in moderate respiratory distress. oral temperature was 38.1° c, pulse rate was 100/min, blood pressure was 1701106, and respiratory rate was 30/min. the oropharynx was described as normal. scattered rhonchi and rales were heard diffusely through the lungs. a summation gallop was heard at the cardiac apex. laboratory examination revealed a white blood count of 5,100 with 82% polymorphonuclear cells, 11% band forms, 3% lymphocytes, and 4% monocytes. arterial blood gases on room air showed a ph of 7.44, pco. of 31, and po. of 56. ekg showed left ventricular hypertrophy. chest radiograph revealed an enlarged cardiac silhouette with patchy alveolar infiltrates ofthe entire right lung and left lower lobe. the patient was started on erythromycin 500 mg every 6 hours intravenously. he continued to spike temperatures to 40· c. cefamandole and tobramycin were added to his antibiotic therapy. two days after admission, the creatine phosphokinase value rose to 16,420 and the next day was 37,576. his creatinine rose to 4.8 mg/dl. his heart was enlarged on radiograph and the pulmonary infiltrates continued to increase. his mental status gradually deteriorated and he was transferred to the intensive care unit. the white blood count was 3,700 with 12% lymphocytes. he developed evidence of disseminated intravascular coagulation and died on the eighth hospital day of respiratory insufficiency. postmortem examination revealed changes of viral pneumonia, with some epithelial cells showing intranuclear inclusions and the appearance of "smudged" nuclei, an enlarged heart due to idiopathic myocardial disease, only minimal pathologic evidence of hypertension, and findings of disseminated intravascular coagulation. electron microscopy oflung sections revealed adenovirus. the adenovirus complement fixation test titer rose from less than 1:8 to 1:64. comment. the patient had a 2-week febrile period before admission, and pulmonary infiltrates progressed on antibiotic therapy. he developed leukopenia, lymphocytopenia, rhabdomyolysis, disseminated intravascular coagulation, and acute renal failure. his illness occurred in the setting of idiopathic myocardial disease, and it is possible that mild, chronic, left ventricular failure might have predisposed him to severe adenoviral pneumonia similar to the manner in which cardiac failure augments influenza pneumonia. pneumonia due to respiratory syncytial virus. respiratory syncytial virus is the predominant respiratory tract viral pathogen of infancy and young childhood. infection in adults usually results in no symptoms or a mild upper respiratory tract illness such as the common cold. it is now recognized that immunosuppressed patients and elderly persons can develop pneumonia because of rsv and that it can be severe and complicated by bacterial suprainfection. 5 9-61 furthermore, since immunosuppressed and elderly persons may aggregate in hospitals and nursing homes, these institutions are often sites of acquisition of infection. an epidemic of pneumonia and febrile respiratory illness took place in los angeles in february-march, 1979. 62 forty of 101 residents were affected, 22 having pneumonia. eight persons died for a case-fatality rate of 20%. other such outbreaks have been recorded. nosocomial acquisition ofrsv is very difficult to prevent. hospital personnel become colonized and may have no or mild respiratory tract symptoms. transfer of virus can occur by patient to personnel to patient transmission or directly from the personnel themselves. hands and fomites become contaminated by respiratory secretions and virus is spread to patients by direct contact with these sources. the pathology of pneumonia due to rsv is similar to that of other viral pneumonias; however, epithelial cells with intracytoplasmic inclusion bodies can be seen. the x-ray appearance of the pneumonia can be that of a diffuse interstitial process or have interstitial and patchy alveolar infiltrates in the lower lobes, or have an appearance indistinguishable from the adult respiratory distress syndrome. pneumonia due to rsv in immunosuppressed and elderly persons is a newly described phenomenon, but one that may be of increasing importance. it is also important because rsv infections can be diagnosed early by antigen detection techniques (immunofluorescence or eia) and because effective therapy has been developed recently. antigen detection tests for rsv now equal or exceed the efficacy of viral cultures for diagnosis of infection. respiratory syncytial virus infections in infancy now have been treated successfully with aerosolized ribavirin. 63 • 64 this therapy is indicated for infants and children with lower respiratory tract involvement with rsv who are exceptionally ill or who may have congenital heart disease or bronchopulmonary dysplasia. with aerosol delivery by oxygen tent, hood, or mask, concentrations of ribavirin are quite high in the upper and lower respiratory tract and exceed the minimum inhibitory concentration necessary to inhibit the growth of the virus in tissue culture. to achieve this concentration by oral administration of the drug, unacceptable toxicity would be en-countered. this toxicity would include bone marrow depression and particularly anemia related both to maturation arrest and, to a lesser extent, hemolysis. this latter event occurs because ribavirin triphosphate can accumulate in erythrocytes, having a halflife greater than 40 days, and interferes with the formation of guanosine triphosphate. aerosolized ribavirin therapy is expensive, but is presently approved by the food and drug administration for the therapy of complicated rsv infections in infants and young children. it represents the first example of an effective drug for treating a significant lower respiratory tract viral infection. it is conceivable that this technology could be applied to influenza infections. influenza and rubeola will be covered in detail but it is now recognized that other rna viruses can cause lower respiratory tract involvement in adults. these viruses include the parainfluenza viruses, respiratory enteroviruses such as coxsackie b viruses and coxsackie virus a21, rhinoviruses, and coronaviruses. 65 the magnitude of the problem, however, appears to be limited. documented instances of severe lower respiratory tract infection due to parainfluenza ii and iii viruses have occurred, however. there is presently no accepted therapy for these latter infections, although aerosolized ribavirin has been used successfully to control persisting parainfluenza virus infections of the lower respiratory tract in immunodeficient children. influenza a virus is the cause of pandemics and epidemics that occur every or every other year. all influenza a viruses possess a common group complement fixation test antigen, the nucleoprotein antigen. influenza a viruses differ in the antigenic character of the hemagglutinin and neuraminidase. the h1n1 strain of influenza a circulated in the world from 1918-1919 through 1957, when asian influenza strains (h2n2) became predominant. these strains circulated until 1968-1969, when hong kong influenza (h3n2) appeared; strains of this virus continue to be transmitted. h1n1 strains again began to circulate during 1976 and they continue to do so. influenza b strains have the same common complement fixation test antigen, and this differs from that of influenza a virus. the hemagglutinin and neuraminidase of influenza b virus are less prone to change; pandemic disease due to this virus does not occur and the interepidemic interval is longer than that of influenza a, namely, every 3-4 years. serious morbidity due to influenza a virus occurs because of host factors such as age, underlying disease, and immunosuppression; because immunity wanes with time; and because influenza a viruses are constantly changing their antigenic character. in pandemic years, when both the hemagglutinin and neuraminidase change concomitantly, the american journal of the medical sciences luby there is a tendency for more serious disease to occur than if just one of the surface proteins changes. this is well illustrated by the 1918-1919 and 1957 pandemics. influenza a viruses may, on certain occasions, be more virulent. in the 1918-1919 epidemic the pneumonia rate in persons from the ages of 25 to 40 years was approximately 10% of those who had influenza. 66 this and other facts have been cited to indicate the virulence and striking pneumotropism of the virus that led to 20 million deaths occurring throughout the world during the pandemic. influenza b viruses are more likely to cause disease in younger persons and only occasionally do epidemics occur in which there is excess mortality. pathogenetically, influenza virus attaches to cells of the respiratory epithelium and enters by a process termed "receptor-mediated endocytosis." the virion is uncoated in the endosome by fusion with the membrane of this structure, a process requiring an acidic ph. the particle then undergoes a cytoplasmic and a nuclear stage of replication. virion rna is capped and polymethylated in the nucleus so that the rna message now can be recognized by the cell and translated at the ribosome. 67 in the process of replication, the virus rapidly destroys respiratory tract epithelial structures in order to compromise natural defense mechanisms of the lung, such as mucous production and ciliary activity. in cases of severe pneumonia the epithelium of the trachea and bronchi are destroyed down to the basal layer and then metaplasia occurs, leaving the respiratory tract coated with a layer of squamous cells. there is involvement of bronchiolar structures and an intense peribronchiolar inflammatory process. in uncomplicated influenza, small airways are commonly affected, producing diffuse dysfunction in these structures, mild hypoxemia, and a compensated respiratory alkalosis. 68 . 69 in severe influenza pneumonia, there is alveolar cell destruction and disruption of the alveolar-capillary membrane resulting in hemorrhage into the alveoli along with edema, a mononuclear cell infiltrate, and the presence ofhyaline membranes. thickening of the interstitium occurs with a chronic inflammatory cell infiltrate. the process can be fulminant, occurring coincident with the onset of illness, or it can be more protracted, leading to the occurrence of progressive infiltrates over 5-7 days. when an adult respiratory distress syndrome-like picture is produced, influenza pneumonia has a high case-fatality rate, which may approximate 75%.70.71 not all influenza a pneumonia is this severe, however, and there are cases in which only an interstitial or bronchopneumonic process is apparent and the disease simulates m. pneumonia, except that in influenza the leukocyte count tends to be normal or decreased. 72.73 influenza pneumonia can coexist with bacterial suprainfection or bacterial suprainfection can occur alone. the offending bacterial pathogens may vary between pandemics; in 1889-1890, h. in{luenzae evidently was a major pathogen. in the 1918-1919 pandemic, the group a streptococcus was considered a major pathogen; more recently, s. pneumoniae has been the most common offending agent followed by s. aureus andh. in{luenzae. occasionally, other gram-negative bacteria may be involved. influenza b virus can cause a similar spectrum of pulmonary disease, but the number of patients involved is fewer. the hospitalization rate for lower respiratory tract disease nearly always increases during influenza a epidemics. this rate tends not to increase during influenza b epidemics, although total hospitalizations may be increased during this period; like influenza a virus, influenza b virus can cause a variety of disease processes outside the lung. these include myopericarditis, rhabdomyolysis, disseminated intravascular coagulation, nervous system disturbances such as encephalitis, reye's syndrome, the landry-guillan-barre : syndrome, the stevens-johnson syndrome, and others. 4 • 74 . clinically, the patient with influenza virus pneumonia has the sudden onset off ever, prostration, and myalgias followed shortly by dyspnea. blood-tinged sputum may be produced. the dyspnea progresses until hospitalization and ventilatory support are required. the illness can also assume a more protracted course leading to progressive interstitial and alveolar infiltrates over a week ( figure 6 ). some pa-tients simply have viral pneumonia with pulmonary dysfunction but do not need ventilator assistance. complicating bacterial suprainfection may coexist with viral pneumonia or more commonly presents after an afebrile interval, during which the patient appears to be recovering from the primary infection. morbidity and mortality are greatest in elderly persons, in those with chronic disease states such as chronic obstructive pulmonary disease, chronic congestive heart failure or diabetes mellitus, and in immunosuppressed patients. morbidity due to influenza a and b viruses is not limited to these groups, however. women in the third trimester of pregnancy also may have an increased rate of developing influenza pneumonia and death due to this disease process. 75 in renal transplant recipients who contract influenza a, illnesses are often prolonged, with the development of viral pneumonia, bacterial suprainfection, and myopericarditis. there may be loss of the renal allograft due · to the combination of these disease processes. 76 influenza a and b virus infections are diagnosed by serial titer rises in a suitable serologic test such as the complement fixation or the hemagglutination inhibition test. a single complement fixation test titer :::1: 128 has been shown to correlate highly with recent influenza b infections. 4 virus can be grown from the nasopharynx or endotracheal secretions by inoculation of the specimen into rhesus monkey kidney or madin-darby canine kidney tissue culture. embryonated eggs sometimes need to be used for figure 6 . influenza pneumonia. the radiograph on the lett shows combined influenza pneumonia with lett lower lobe consolidation indicating bacterial supra infection. the radiograph on the right shows diffuse alveolar infiltrates in the course of serologically documented influenza a infection. bacterial cultures from endotracheal secretions consistently showed no growth. optimal recovery of virus. virus may be able to be identified within 72 hours by using immunofluorescence. direct detection of antigen by immunofluorescence or eia can be applied to appropriate secretions, but these tests are not yet as sensitive as viral culture. 77 amantadine and rimantadine are two compounds that have both prophylactic and therapeutic efficacy against influenza a but not influenza b virus. they act by preventing uncoating of influenza a virus, perhaps by preventing the development of an acidic ph so that the envelope of the virion cannot fuse with the endosomal membrane. at the dosage given, 100 mg twice a day, amantadine has more central nervous system side effects and the dose has to be adjusted with renal failure. 78 the dose of rimantadine does not have to be adjusted with renal dysfunction because the compound is metabolized in the body. a study sponsored by the national institutes of health is underway evaluating whether rimantadine can be used effectively in the therapy of hospitalized patients with influenza a, and would include patients with influenza a virus pneumonia. ribavirin has in vitro efficacy against both influenza a and b viruses. it has multiple sites of action including interference with the formation of guanosine triphosphate and deoxyguanosine triphosphate and prevents placement of the polymethylated cap structure on the influenza a viral rna message. with the aerosolization of ribavirin, high concentrations of the drug can be produced within the respiratory tract but serum levels are low. 79 ,8o attempts most likely will be made in the future to treat influenza a virus pneumonia with aerosolized ribavirin or a combination ofribavirin and rimantadine. influenza b virus pneumonia may be able to be treated with aerosolized ribavirin. vaccines exist for both influenza a and b viruses, and standard medical care necessitates yearly immunization of elderly patients or those with underlying medical conditions. a recent emphasis of the public health service is to have medical personnel also immunized yearly, since they are. exposed to persons with influenza, may develop that illness themselves, and may then transmit the infection to sick patients within the hospital. nosocomial influenza pneumonia. a 55-year-old alcoholic man was admitted to the hospital on january 30 with alcoholic liver disease, macrocytic anemia, and symptoms of bladder neck obstruction. he was a heavy smoker and had evidence of chronic obstructive pulmonary disease. 'lwelve days after admission into the hospital, he developed fever to 39.2° c while awaiting a urologic procedure. he "felt terrible" with myalgias and developed a cough, mild diarrhea, and dyspnea. chest examination revealed diffuse rales and rhonchi. chest radiograph showed new interstitial infiltrates, more prominent on the right. the sputum was mucoid. the diagnosis of pneumonia was made and the diag-the american journal of the medical sciences luby nosis of congestive heart failure with pulmonary edema considered. however, his heart examination revealed no gallop sounds and his neck veins were not distended. he was placed on ampicillin, became afebrile after 5 days, and his dyspnea improved with low flow oxygen by face mask. influenza a complement fixation test titer on a single convalescent serum specimen was 2::1: 256. comment. influenza virus pneumonia occurred during hospitalization. the pneumonia cleared with symptomatic therapy. the public health service now recommends widespread immunization of medical personnel in an attempt to prevent nosocomial acquisition of influenza. although predictions were made that rubeola would be eradicated in this country during the early 1980s, this has not been achieved. in dallas, texas, during 1986 more than 150 cases of rubeola occurred. this marked a resurgence of cases after a relatively disease-free interval after 1971, when a large epidemic occurred in dallas, causing more than 1,000 cases, including three deaths. as a consequence ofthis and other epidemics, in 1971 texas adopted a law requiring the compulsory immunization of children against measles, mumps, rubella, poliomyelitis, diphtheria, and tetanus. present rubeola vaccines are at least 95% effective, but universal immunization of the preschool child is not practiced, particularly in lower socioeconomic class population groups. furthermore, rubeola virus has been found to violate the concept of herd immunity, a major principle on which eradication was based, because outbreaks occur in high schools and colleges in which a large percentage of the population has been immunized. an inactivated vaccine was available from 1962 through 1965. children who received the inactivated vaccine may have developed atypical measles on exposure to rubeola virus. following this, an attenuated, live strain of measles virus was used as vaccine but the high sidereaction frequency necessitated the concomitant administration of 'v-globulin. it is now recognized that the concurrent use of 'v-globulin sometimes rendered immunization ineffective. many children were immunized before the age of 12 months; for effective immunization to occur, vaccine must be given after the age of15 months. as a consequence of the lack of universal preschool immunization and difficulties related to the vaccine, there now exist two population groups who may be nonimmune with respect to rubeola, ie, preschool children, and adolescents and young adults. a few years ago rubeola epidemics were common in military recruits. following the occurrence of these outbreaks, recruits now routinely undergo serologic testing, and if antibody to rubeola virus is not detected by either hemagglutination inhibition or indirect immunofluorescence tests, live attenuated vaccine is given. this practice essentially has stopped the occurrence of these outbreaks in the military. persons who received the inactivated vaccine can develop atypical measles. the first cases of this new syndrome were misdiagnosed as rocky mountain spotted fever. they were confused with this disease because the rash began on the extremities and spread inward to involve the trunk. the rash could be maculopapular, vesicular, or petechial. in atypical measles, pulmonary involvement consists of nodular infiltrates, lobar consolidation, and the occurrence of pleural effusions. hilar lymphadenopathy may also present in these patients. s1 • s2 they have an anamnestic response in antibody production with the infection. mild eosinophilia may also be present and the virus cannot be recovered from the nasopharynx. in young adults with typical measles, approximately 5% develop clinical evidence of pneumonia. radiographic evidence of pneumonia, however, may be seen in up to 50% of the patients. the pneumonia is usually characterized by diffuse bilateral interstitial or fine reticulonodular infiltrates, particularly affecting the lower lobes. bacterial suprainfection occurs in as many as 30% of cases of recognized viral pneumonia. the types of bacteria causing infection may be determined by the circumstances in which disease occurs, such as military recruit populations. recognized pathogens include h. influenzae, s. pneumoniae, group a streptococci, group y meningococci, and s. aureus. bacterial suprainfection generally occurs between the fifth and tenth days after the rash and is heralded by clinical worsening, new or different lung infiltrates, or changes in sputum characteristics or the white blood count. antibiotic treatment of viral pneumonia does not prevent bacterial suprainfection. in immunodeficient children, measles pneumonia occurred without a rash and pathologically was called giant-cell pneumonia. it is now recognized that these children lacked cell-mediated immunity and had depressed and delayed antibody production. s3 intact cell-mediated immunity is essential for rash production. in fatal rubeola pneumonia, the entire tracheobronchial tree may be denuded of cells down to the basal layer; squamous metaplasia of the cells occurs; there is widening of the interstitial space with edema and inflammatory cells; and alveoli are filled with edema, hyaline membranes, and mononuclear cells. in addition, giant cells containing multiple nuclei are found within the tracheobronchial epithelium. extrapulmonic manifestations of measles occur and include otitis media, sinusitis, encephalitis, and the common presence of hepatic dysfunction in young adults, mostly consisting of mild elevations of the serum aspartate aminotransferase and lactic dehydrogenase. there presently is no specific therapy. some public health authorities now think that reimmunization with measles, mumps, and rubella vaccines should be 58 given before or when the child enters high school. medical personnel not sure of their rubeola immunity should have that status assessed by determination of specific antibody. varicella-zoster virus can produce pneumonia during the course of varicella or disseminated herpes zoster and can be a severe disease that can lead to mortality. varicella in childhood is not usually associated with viral pneumonia, but bacterial suprainfection can occur, necessitating appropriate antibiotic treatment. in immunosuppressed children, however, pure viral pneumonia can occur in association with varicella. in adults, there is a tendency for the virus to affect the lung relatively commonly during varicella. fifteen to twenty percent of all adults with varicella may have x-ray evidence of pneumonia but only about 5% require hospitalization. most frequently in adults, varicella pneumonia is not complicated by bacterial suprainfection; however, this can occur, particularly when patients require intubation. the virus reaches the lung both by passage down the respiratory tract and by hematogenous seeding because the rash is occurring at the same time as the pneumonia. initially, the pneumonic process appears as nodular infiltrates, 1-4 mm in diameter associated with an interstitial inflammatory infiltrate. a4 the lesions are more dense toward the hilum and are the counterpart in the lung of the pox occurring on the skin (figure 7) . peribronchial inflammatory infiltrates, hilar adenopathy, and pleural effusions may occur. the reticulonodular interstitial infiltrate can progress to widespread alveolar damage and diffuse pulmonary parenchymal infiltrates. pathologically, the pneumonia resembles influenza pneumonia except that areas of coagulative necrosis can be seen. although these necrotic areas generally clear during the course of clinical disease, they can become calcified, and the radiograph shows a picture of miliary calcifications. it has been shown that this area of coagulative necrosis can become surrounded by an inflammatory infiltrate and resemble a granuloma. a5 fibrous tissue envelopes the necrotic granulomatous process and the lesion eventually calcifies. another process occurring in varicella pneumonia is destruction of the epithelium of the trachea and bronchi. in cases in which the illness is protracted, the development of a thick, fibrinopurulent crust may occur over the lower pharynx, larynx, and upper trachea. this thick crust can cause respiratory embarrassment and can pose a problem for intubation. disseminated herpes zoster can cause the same processes in the lung. most normal adults recover from varicella pneumonia without difficulty, but there can be substantial respiratory morbidity and morfigure 7 . varicella pneumonia. the chest radiograph on the lett is from the case report described in the protocol. the close-up film on the right in another pallent shows peribronchial infiltrates and multiple 1-4-mm rounded opacities in the right lung field. tality in immunosuppressed patients or in women during the third trimester ofpregnancy. 86 clinically, the patient with varicella-zoster virus pneumonia presents with a rash followed by cough and dyspnea. the sputum is initially white and modest in amount, but can become hemorrhagic. the process can be complicated by the development of chest pain and pleural effusions, which are often blood tinged and related to the presence of pox on the pleural surface. extrapulmonic manifestations of varicella occur and consist of the characteristic skin rash, otitis media with bacterial suprainfection, myopericarditis, hepatic dysfunction, and encephalitis. reye's syndrome can complicate the course ofvaricella. there can be an associated glomerulonephritis and varicella virus can occasionally induce frank arthritis. in caring for patients with varicella-zoster virus pneumonia, it is important to realize that the external appearance of the patient or his apparent well-being may disguise underlying hypoxemia. if efforts are not made to diagnose and correct the hypoxemia, the patient may become confused, perform inappropriate activity, and become more hypoxemic. ventilator support may become necessary. deaths in varicella pneumonia occur because of respiratory insufficiency, the development of tension pneumothoraces, bacterial suprainfection, or progressive pulmonary fibrosis. two antiviral compounds, adenine arabinoside (ara-a) and acyclovir (acv), have been proven to be efficient in the treatment of significant, complicated varicella-zoster virus infections. adenine arabinoside inhibits viral dna polymerase and is given at a dosage of 10 mglkg over a 12-hour period for at least 5 days. the dosage could be increased to 15 mglkg the american journal of the medical sciences but the majority of experience with varicella-zoster virus infections is with 10 mglkg/day. the drug is sparingly soluble so that 2 ml of vehicle are required for each milligram of drug administered to the patient. at a dose of 10 mglkg, bone marrow suppression does not usually occur. if the dose is not decreased in the setting of hepatic and renal dysfunction, central nervous system disturbances, which consist of insomnia, hallucinations and tremulousness, may occur. these central nervous system manifestations usually fade with stopping the drug and rarely lead to death, but can persist for a protracted period after the drug has been discontinued. acyclovir also has been used to treat varicella-zoster virus infections. it inhibits viral dna polymerase and also acts as a chain terminator. its dosage is 500 mg/m 2 every 8 hours for at least 7 days. the only significant problem with the administration with acyclovir in this setting is the production of an obstructive nephropathy, due to salting out of the drug in the collecting tubules ofthe kidney. this is usually easily managed by administration of a fluid bolus, a diuretic, or mannitol. a comparison of the two drugs in complicated varicella-zoster virus infection has been made. 87 . 88 one group found that acyclovir was more efficacious; the other study determined that ara-a was equally as effective. because the administration of ara-a requires an increased volume of fluid and can be associated with central nervous system side effects, some authorities now consider acyclovir the drug of choice in the treatment of complicated infections due to varicella-zoster virus. oral acyclovir is absorbed poorly by the gastrointestinal tract and its efficacy in uncomplicated herpes zoster is apparent only when 800 mg are given 5 times a day for at least a 5-day period. there has been no experience with oral acv in treating varicella pneumonia. future modes of therapy may include combining ara-a with acv or administering one or other of the drugs with an interferon preparation. alpha-interferon also has been shown to be effective as therapy in complicated varicella-zoster virus infections but its use has been superceded by acv and ara-a. case report varicella pneumonia. a 39-year-old man was exposed to his two children with chicken pox. two days before admission he developed a rash, then dyspnea. on physical examination, a typical varicella rash was present. he was in severe respiratory distress. rales were present diffusely over both lung fields. the chest radiograph revealed bilateral extensive alveolar infiltrates. arterial blood gases showed a ph of 7.42, pc02 of 32, and a p02 of 24. he was intubated and begun on positive end-expiratory pressure (peep) with a fi0 2 of 70%. he was started on intravenous acyclovir 500 mg/m2 every 8 hours. he improved and was able to be extubated after 5 days. comment. severe varicella pneumonia responded to intravenous acyclovir while his oxygenation was maintained on peep with a high fi02. herpes simplex virus can cause a necrotizing bronchopneumonia in neonatal infections and can also cause pneumonia in severely immunosuppressed adult patients. the largest series of patients with herpes simplex virus pneumonia was reported from seattle in bone-marrow transplant recipients 'and consisted of 20 patients with either a focal pneumonia (12 patients) or a diffuse interstitial pneumonia (eight patients). 89 the focal pneumonia was found associated with herpetic esophagitis and tracheitis and probably resulted from contiguous spread of herpes virus to the lung parenchyma. diffuse interstitial pneumonia most probably resulted from hematogenous dissemination of virus to lung. pathologically, the process can be one of a necrotizing bronchopneumonia or of a widening of the interstitial space in the lung associated with diffuse alveolar injury. in these highly immunosuppressed patients, both bacterial and fungal suprainfection occurred and it was difficult to sort out which process was responsible for what proportion of lung damage. acyclovir has been shown to be an effective treatment of complicated herpes simplex virus infections in immunosuppressed patients. its dose in the usual patient is 250 mg/m 2 every 8 hours for at least 7 days, but if the process has been ascertained to be a rapidly progressive herpetic pneumonia, the dose could be increased to 10 mglkg every 8 hours until a therapeutic response had been obtained. with the development of potent antiviral chemotherapy, there is a need to consider and diagnose herpetic pneumonia. specific diagnosis can only be accomplished readily by lung biopsy. 60 although involvement of the lung in infectious mononucleosis due to epstein-barr virus must be considered a rare occurrence, recent studies and case reports demonstrate that it probably can happen. 90 . 91 careful attention should be given to possible coexisting mycoplasma and other viral infections, particularly since the former can be treated. radiographic abnormalities may consist of hilar adenopathy, strand-like parenchymal infiltrates, diffuse bilateral pneumonia, and a picture consistent with primary atypical pneumonia. although epstein-barr virus is susceptible to acyclovir, there are no reports treating lung involvement with this drug. cytomegalovirus (cmv) rarely causes pneumonia in normal adults as part of the cmv mononucleosis syndrome. 92 however, it is more common for cmv to induce pneumonia in normal hosts than epstein-barr virus. of 443 patients with communityacquired pneumonia, 18 had virologic,2 pathologic,2 or serologic 14 evidence of cmv infection. 93 ten of these 18 patients were not immunosuppressed. in five of the ten, cmv was the only pathogen. the remaining five patients had one or more coexisting infections; c. trachomatis in two, m. pneumoniae in one, epstein-barr virus in one, and bacteria in three, both aerobic and anaerobic. cytomegalovirus more commonly causes pneumonia in immunosuppressed patients. it occurs particularly in renal, heart, liver, and bone-marrow transplant recipients. it is now becoming an increasing problem in patients with aids. in the transplant recipient experiencing a primary cmv infection, the virus most probably reaches the lung parenchyma through the hematogenous route, and the first finding is that of a reticulonodular infiltrate and the presence of 1-4 mm opacities ( figure 8 ). pathologically, these focal areas usually consist of necrotic tissue, hemorrhage and alveolar damage with edema, a mononuclear infiltrate, and typical cytomegalic cells. the process can extend leading to diffuse interstitial and alveolar infiltrates. an attempt has been made to separate the foregoing process from that of an insidiously developing interstitial pneumonia that occurs more commonly in reactivated infections and has a better prognosis. 94 in bone marrow transplant recipients, diffuse interstitial pneumonitis due to cmv is much more common in patients receiving allogeneic transplants, and the case-fatality rate approximates 90%. 95 in some renal transplant recipients, the pneumonic process can be focal and does not have to be exceptionally severe. 96 small pleural effusions can occur occasionally. in other renal transplant recipients, however, cmv pneumonia can progress rapidly and lead to death as part of a widely disseminated infectious process. 97 in cardiac transplant recipients a variety of pulmonary opportunistic suprainfections has been well documented to occur in the course of cmv pneumonia. 98 typical microorganisms causing suprainfections include p. carinii and nocardia species. in patients with aids, there may be co-existing infection with pneumocystis. cure of the pneumocystis can be effected by drugs, leaving cmv as the major pulmonary pathogen. in patients with aids, rapid development of cmv pneumonia can occur and lead to the death of the patient. extrapulmonic manifestations of cmv in the renal transplant recipient include fever, malaise, hepatic dysfunction, splenomegaly, leukopenia, and an increase in serum creatinine. 96 with extensive cmv dissemination in heavily immunosuppressed patients, including those with aids, extrapulmonic manifestations include gastrointestinal ulceration with bleeding and perforation, hepatic dysfunction, adrenal cortical involvement, and central nervous sytem dysfunction. cytomegalovirus can be thought of as an immunosuppressive viral agent, and infection with this microorganism may lead to further immunosuppression with consequent bacterial, fungal, and parasitic suprainfection. clinically, patients with cmv pneumonia complain of dyspnea with a nonproductive cough. there can be an associated pleurisy. the process can be transient or can extend to respiratory insufficiency necessitating ventilatory support. therapy of fully developed cmv pneumonia has been shown not to be effective and this includes the use of the adenine arabinoside, acyclovir, ganciclovir (dhpg), and combinations of interferon with all the above. ganciclovir has been successful in achieving an antiviral effect in the lung, yet has not improved outcome in the american journal of the medical sciences bone marrow transplant recipients with cmv pneumonia. 99 in an occasional renal transplant recipient who has the potential of a good immune response to the virus, the cmv illness that can include localized pneumonia might be benefited by the judicious use of ganciclovir. attempts at preventing cmv pneumonia have included donor selection, avoidance of white blood cell transfusions, and prophylactic administration of alpha-interferon or 'y-globulin preparations given before and through the first 60 days after transplantation. alpha-interferon does prevent cmv viremia in the renal transplant recipient; 'y-globulin protects partially against cmv pneumonia if white blood cell transfusions have not been given. studies are in progress trying to make this latter effect more consistent, and consist of determining whether total antibody content is the necessary component or whether the effect necessitates the presence oflarge quantities of neutralizing antibody. on a priority basis, live, attenuated cmv vaccine development has been curtailed for the immediate future. a 40-year-old homosexual man presented to the hospital with fever, cough, and an erythematous rash. he had been followed in clinic with aids-related complex with lymphadenopathy, thrush, lymphopenia, anergy, diarrhea, and a positive antibody test to human immunodeficiency virus. at the time of his acute terminal illness he had a temperature of 38.5° c and had a diffuse, erythematous pruritic rash over the trunk and upper legs. the admission chest radiograph was interpreted as normal. on the second hospital day, the patient became delirious, had a worsening cough, and developed severe dyspnea. arterial blood gases on room air revealed a ph of 7.50, pco. of 30, and a po. of 44. chest radiograph now revealed bilateral diffuse reticulonodular infiltrates. he was started on sulfatrimethoprim but had a respiratory arrest and died. postmortem examination revealed cmv pneumonia without evidence ofpneumocystis. lung viral cultures rapidly grew cmv, with the cytopathic effect being present the second day. comment. explosive illness in a patient with arc revealed only cmv at autopsy and on viral culture of the lung. viral and mycoplasmal pneumonia in a prepaid medical care group during an eight-year period primary atypical pneumonia in a family due to concomitant mycoplasma pneumoniae and adenovirus type 7 infection viral pneumonia as a cause and result of hospitalization severe illness with influenza b strannegard 0, trollfors b: etiology of community-acquired pneumonia in out-patients etiologies and characteristic features of pneumonias in a municipal hospital etiology of community-acquired pneumonia in patients requiring hospitalization adult community-acquired pneumonia in central london virological investigations in adults with acute pneumonia hospital study of adult community-acquired pneumonia acute community-acquired pneumonias an acute infection of the respiratory tract with atypical pneumonia. a disease entity probably caused by a filtrable virus causes of atypical pneumonia: results of a 1-year prospective study differential diagnosis of viral, mycoplasmal and bacteraemic pneumococcal pneumonias on admission to hospital comparative radiographic features of community acquired legionnaires' disease, pneumococcal pneumonia, mycoplasma pneumonia, and psittacosis mycoplasma pneumonia: failure of erythromycin therapy lung infections caused by viruses, mycoplasma pneumoniae, and rickettsiae pneumonias due to rickettsiae, chlamydiae, viruses and mycoplasma neurologic disease associated with mycoplasma pneumoniae pneumonitis. demonstration of viable mycoplasma pneumoniae in cerebrospinal fluid and blood by radioisotopic and immunofluorescent tissue culture techniques open lung biopsy in myco-62 plasma pneumoniae pneumonia mycoplasma pneumoniae infection in families pulmonary involvement in mycoplasma pneumoniae infection in families the clinical spectrum and diagnosis of mycoplasma pneumoniae infection clinical features of mycoplasmal pneumonia in adults mycoplasmal pneumonias in the community hospital. the "unusual" manifestations become common lobar pneumonia caused by mycoplasma pneumoniae radiographic appearances of mycoplasma pneumonia mycoplasmal pneumonia and adult respiratory distress syndrome: a complication to be recognized acute respiratory failure due to atypical pneumonia the protean manifestations of mycoplasma pneumoniae infection in adults weekly clinicopathological exercises: case 39-1983 pityriasis rosea gibert and mycoplasma pneumoniae infection abnormalities in lung function following clinical recovery from mycoplasma pneumoniae pneumonia van der straeten m: mycoplasma pneumonia with fulminant evolution into diffuse interstitial fibrosis bronchiolitis obliterans due to mycoplasma pneumoniae mycoplasma pneumonia complicated by bronchiectasis mycoplasmal pneumonia associated with mesangiocapillary glomerulonephritis type ii (dense deposit disease) mycoplasma pneumoniae pneumonia associated with iga nephropathy new york city medium for enhanced recovery of mycoplasma pneumoniae from clinical specimens principles and practice of infectious diseases psittacosis. a diagnostic challenge isolation of chlamydia trachomatis from the lower respiratory tract of adults chlamydia trachomatis infection in adults with community-acquired pneumonia chlamydia trachomatis pneumonia in adults: radiographic appearance an epidemic of mild pneumonia due to an unusual strain of chlamydia psittaci a new chlamydia psittaci strain, twar, isolated in acute respiratory tract infections clinical features of adenoviral pneumonia in air force recruits lobar pneumonia associated with adenovirus type 7 fatal pneumonia associated with adenovirus type 7 in three military trainess fatal adenovirus pneumonia in a young adult associated with adv-7 vaccine administered 15 days earlier fatal disseminated adenovirus infection in a renal transplant recipient adenovirus infections in patients undergoing bone-marrow transplantation fatal disseminated adenovirus 11 pneumonia in an agammaglobulinemic patient bacterial pneumonia complicating adenoviral pneumonia bronchopneumonia with serious sequelae in children with evidence of adenovirus type 21 infection diffuse pneumoniti~ due to adenovirus type 21 in a civilian association of adenovirus type 16 with reye's-syndrome-like illness and pneumonia rapid diagnosis of respiratory adeno-vi~us infections in young adult men fatal haemorrhagic pneumonia in an adult due to respiratory syncytial virus and staphylococcus aureus respiratory syncytial virus pneumonitis in adults case report: presumed respiratory syncytial virus pneumonia in three immunocompromised adults an outbreak of respiratory syncytial virus pneumonia in a nursing home for the elderly ribavirin treatment of respiratory syncytial viral infection in infants with underlying cardiopulmonary disease ribavirin aerosol treatment of bronchiolitis due to respiratory syncytial virus infection in infants coronavirus infections of man associated with diseases other than the common cold influenza: the newe acquayantance orthormyxo-and paramyxoviruses and their replication, in fields bn sanfordjp: pulmonary function in uncomplicated influenza pulmonary mechanics after uncomplicated influenza a infection studies on influenza in the pandemic of 1957-1958. ii. pulmonary complications of influenza severe influenza virus pneumonia in the pandemic of 1968-1969 mycoplasma and influenza pneumonia in a series of 214 adults the leukocyte response during viral respiratory illness in man acute myocarditis in influenza a infections. two cases of non-bacterial myocarditis, with isolation of virus from the lungs fatal influenza a pneumonia in pregnancy epidemic renal transplant rejection associated with influenza a victoria rapid diagnosis of primary influenza pneumonia pharmacokinetics of amantadine hydrochloride in subjects with normal and impaired renal function ribavirin small-particle aerosol treatment of influenza ribavirin small-particle aerosol treatment of infections caused by influenza virus trains anictoriaj7/83 (h1n1) and bltexas/1/84 measles pneumonia. bacterial suprainfection as a complicating factor measles pneumonia in young adults. an analysis of 106 cases isolation of measles virus at autopsy in cases of giant-cell pneumonia without rash report of seven cases and a review of literature persistent pulmonary granulomas after recovery from varicella pneumonia varicella pneumonia complicating pregnancy treatment of varicellazoster virus infection in severely immunocompromised patients comparative trial of acyclovir and vidarabine in disseminated varicella-zoster infections in immunocompromised patients coreyl: herpes simplex virus pneumonia. clinical, virologic, and pathologic features in 20 patients diffuse pneumonia and acute respiratory failure due to infectious mononucleosis clinical, virologic, and serologic evidence of epstein-barr virus infection in association with childhood pneumonia pneumonia associated with rising cytomegalovirus antibody titres in a healthy adult does cytomegalovirus playa role in communityacquired pneumonia? cytomegalovirus pneumonia in bone marrow transplant recipients: miliary and diffuse patterns nonbacterial nonfungal pneumonia following marrow transplantation in 100 identical twins disease due to cytomegalovirus and its long-term consequences in renal transplant recipients. correlation of allograft survival with disease due to cytomegalovirus and rubella antibody level clinical characteristics of the lethal cytomegalovirus infection following renal transplantation diagnosis of cytomegalovirus pneumonia in compromised hosts activity of 9-[2-hydroxy-1-(hydroxymethyl)-ethoxymethyl]guanine in the treatment of cytomegalovirus pneumonia key: cord-015922-5wwy0m2k authors: marty, francisco m.; baden, lindsey r. title: infection in the hematopoietic stem cell transplant recipient date: 2008 journal: hematopoietic stem cell transplantation doi: 10.1007/978-1-59745-438-4_19 sha: doc_id: 15922 cord_uid: 5wwy0m2k nan there are three key elements of the hsct procedure that determine the type and timing of the infectious risk profile after transplantation [1, 7] : 1, 7 a. the duration of neutropenia and mucosal injury which is a function of the conditioning regimen selected (myeloablative or not) and the stem cells' procurement (cord or adult; peripheral or bone marrow acquisition among adult donors). b. the strategy chosen to prevent gvhd among allogeneic recipients. t cell depletion and other t cell manipulation procedures lead to delayed recovery of lymphocyte function and provide a specific immune deficiency profile. c. the occurrence and severity of acute and chronic gvhd and its treatment [1, 7] . the temporal course of infection following hsct can be divided into three time periods (fig. 19-1 ) [1, 3, 4, 7] : 1. conditioning to engraftment the duration of this period has become dynamic and depends on the conditioning regimen itself, the source and dose of stem cells infused and whether growth factors are used. it usually ranges from five (with nonmyeloablative transplants) to 30 days (with bone marrow or umbilical cord blood transplants). the combination of profound granulocytopenia and mucositis with myeloablative conditioning makes the patient particularly vulnerable to bacterial and candidal infections. in addition, infection present in the transplant recipient pre-transplant may be amplified by the granulocytopenic state and deficiencies of t and b-cell numbers and function. thus, control of pre-transplant infection is needed before initiating the conditioning regimen. prior to engraftment (both with autologous and allogeneic transplants), approximately 50 percent of patients will have fever of unknown origin, with bloodstream infection in ~12.5 percent and pneumonia in ∼10 percent. the risk of an invasive mold infection is related to the duration of neutropenia and the environmental strategy used in a transplant center. 2. engraftment to post-transplant day 100 during this time period viral infections, particularly cytomegalovirus (cmv) and the other herpes group viruses, are the major concerns. the occurrence, severity and treatment modalities selected for acute gvhd further modulates and increases the risk of herpesvirus infections, especially cmv and epstein-barr virus (ebv), and invasive mold infections [8] [9] [10] [11] . 3. more than 100 days post-transplant in the absence of gvhd, the incidence of infection decreases significantly, with varicella zoster virus (vzv), pneumocystis jiroveci (formerly carinii) pneumonia (pcp) and pneumococcal infection being the primary problems of this time period. routine use of prophylaxis, such as with trimethoprim/sulfamethoxazole and acyclovir, significantly decreases the occurrence of pcp and herpesvirus infections, respectively. in addition, late or relapsing cmv infection may manifest during this time. if gvhd is present, it is typically treated with significant augmentation of immunosuppressive therapy such as with high-dose corticosteroids and monoclonal antibodies. patients in this last category (gvhd under treatment) are at particular risk for invasive mold infection, cmv reactivation, pcp and other common and opportunistic pathogens. there are four modes in which antimicrobial therapy can be administered to the hsct patient [4] : 1. a therapeutic mode, in which antimicrobial therapy is prescribed for the treatment and eradication of identified microbes causing clinical illness. 2. a prophylactic mode, in which antimicrobial therapy is prescribed to an entire population before an event to prevent clinically important infection. for such a strategy to be successful, the infection(s) being targeted must be important enough to justify the intervention, and the antimicrobial therapy prescribed must be nontoxic and inexpensive enough to justify the intervention. by far the most effective antimicrobial prophylactic strategy is low-dose trimethoprim-sulfamethoxazole, which has virtually eliminated the occurrence of pneumocystis jiroveci, listeria monocytogenes, nocardia sp, and toxoplasma gondii in patients who adhere to the regimen. other prophylactic strategies commonly utilized in hsct patients include acyclovir to prevent herpes simplex virus (hsv) and vzv reactivation, fluoroquinolones [5] to prevent gram-negative sepsis and fluconazole to prevent yeast infection. 3. an empiric mode, in which antimicrobial therapy is administered in response to a symptom complex. in this context, empiric antimicrobial therapy is initiated during the period of profound granulocytopenia in response to fever +/− rigors or subtle signs of sepsis (unexplained hypotension, tachypnea, an ongoing volume requirement, or acidosis). in the patient deemed not to be a therapeutic emergency, initial therapy is usually aimed at aerobic gramnegative bacilli (e.g., the enterobacteriaceae and pseudomonas aeruginosa). a variety of drugs have been utilized for this purpose, depending in part on the nature of particular problem organisms found at a given medical center. advanced spectrum beta-lactams (e.g., ceftazidime, piperacillin or imipenem), either alone or together with an aminoglycoside or a fluoroquinolone, are the mainstays of this approach. thus, empiric therapy is based on an algorithm rather than on microbiologic or other studies. 4. a preemptive mode, in which antimicrobial therapy is prescribed to a proportion of patients deemed to be at particularly high risk because of clinical/epidemiologic information or the isolation of microbial pathogens. examples of preemptive therapy in hsct are the molecular surveillance of cmv linked to deployment of ganciclovir or, more recently, the use of galactomannan monitoring for initiation of anti-aspergillus antifungal treatment [12] . given the nature, duration and severity of host defense defects present in hsct patients, it is not surprising that bacterial infection is a regular feature of the post-transplant course. the most common involved sites include blood stream (often catheter-related), lung, gastrointestinal tract and skin/soft tissue. the greatest rate of bacterial infections occur during the period prior to engraftment; this rate is a product of granulocytopenia, mucositis that permits the translocation of bacteria and yeast from the oral cavity and gut into circulation, and the presence of vascular access devices that traverse the skin and serve as direct conduits into the systemic circulation. thus, the primary mucocutaneous barriers to infection are compromised, and the absence of granulocytes only amplifies the susceptibility of the patient [1, 4, 7] . in an attempt to decrease bacterial infections during the neutropenic period, especially those due to gram-negative bacilli, strategies of prophylactic antimicrobial use have been studied, including the use of trimethoprimsulfamethoxazole and fluoroquinolones. some studies, most recently with levofloxacin, have demonstrated benefit in decreasing the occurrence of fever and microbiologically-confirmed bacterial infections [13] [14] [15] . however, significant concerns regarding this approach have been raised given that no mortality benefit has been demonstrated, the emergence of resistant organisms, and the impairment this widespread antimicrobial approach has on the use of quinolones in future oral outpatient management. thus, in many transplant centers, an empiric antibacterial regimen targeting pseudomonas and other enterobacteriaceae in response to fever or other infectious syndromes remains a preferred approach. whereas gram-negative bacteremia was the major cause of blood stream infection 15 to 30 years ago, today gram-positive organisms are the most frequent cause of positive blood cultures. the possible reasons for this shift are many: the widespread use of fluoroquinolones, with their potent activity against gram-negative bacteria, as prophylaxis during this period; the presence of indwelling central venous catheters for prolonged periods; and the widespread use of systemic anti-gram-negative therapy all contribute to the gram-positive predominance. the bacteria isolated during the preengraftment period, then, include staphylococci (especially coagulase-negative staphylococcus), viridans streptococci, enterococci and corynebacteria, with fewer isolates of enterobacteriaceae or pseudomonas aeruginosa being identified. an increasing problem in the hsct population is antibiotic resistant organisms, particularly vancomycin-resistant enterococci, methicillin-resistant staphylococcus aureus, and resistant gram-negative bacilli (such as extended spectrum β-lactamase producing klebsiella and chromosomal inducible βlactamase producing enterobacter species) [1, 4, 6, 7, [16] [17] [18] [19] [20] . the typical approach for the severely granulocytopenic patient at present is the initiation of empiric antibacterial therapy in response to an unexplained fever or other signs of sepsis. what remains controversial is what the regimen should be. since clinical deterioration can occur rapidly with untreated gram-negative sepsis in the granulocytopenic patient, anti-gramnegative therapy is always employed. the traditional approach of a β-lactam (e.g., piperacillin) plus an aminoglycoside is still favored by some experts, although nephrotoxicity from the aminoglycoside has led to the trial of other approaches, including the substitution of a fluoroquinolone for the aminoglycoside, or the prescription of a single advanced spectrum drug such as ceftazidime, cefepime, imipenem or meropenem. if fluoroquinolone prophylaxis has been utilized, then its use as a therapeutic agent may be diminished. empiric fluoroquinolone monotherapy is inferior to other regimens, and if pure aerobic gram-negative agents are utilized, (e.g., aztreonam, aminoglycosides) due to confirmed severe beta-lactam hypersensitivity, then the addition of empiric gram-positive coverage that targets aerobic and anaerobic streptococci of the gastrointestinal tract should be considered. use of extended interval (once-daily dosing) aminoglycoside administration may be safer and as effective. the second area of controversy is whether empiric gram-positive treatment should be initiated at the same time, given the preponderance of grampositive infection. as there is typically time to evaluate culture data and deploy targeted gram-positive antimicrobial therapy rather than empiricism, vancomycin should rarely be required empirically. furthermore, empiric gram-positive coverage is not associated with better outcomes [21] . indications for the immediate initiation of vancomycin as part of the empiric therapy regimen include the following [6, 16, [18] [19] [20] [21] : catheter-related sepsis is likely because of evidence of infection at the insertion site (or within the tunnel), severe illness such as shock and/or respiratory distress are present, the patient is at particular risk for seeding of a prosthetic device (e.g., a prosthetic valve, a hip prosthesis, etc.), or the empiric gram-negative coverage exclusively covers aerobic gram-negative rods -such as the combination of aztreonam and gentamicin. vancomycin or other anti-staphylococcal agents should be started if cultures become positive for gram-positive cocci. in our experience, vancomycin can be discontinued safely in patients in whom vancomycin was started empirically, but in whom blood cultures remain negative after 48 to 72 hours and there is no specific syndrome, such as cellulitis, that requires treatment with vancomycin. on the other hand, empirical treatment against gram-negative organisms should be continued until resolution of neutropenia, whether fevers resolve or not [22] . the emergence and persistence of multidrug-resistant organisms should guide local practice in a dynamic fashion. indwelling long-term catheters remain a feature of the early post-transplant period to provide chemotherapy, nutritional and blood product support until stable engraftment. routine anti-gram-positive antimicrobial therapy is not required just because a central catheter is in place for the prevention and management of catheter-related infections [21, 23] . the use of antimicrobial-coated catheters should be studied in this population, especially when non-tunneled catheters need to be used. nonantimicrobial-based strategies to prevent bacterial infections during the neutropenic period include the systematic use of hand hygiene and the use of mask and gloves by health care personnel and family members. other nonantimicrobial strategies which may be beneficial in preventing infections, but have not been tested in hsct, include the use of palifermin to prevent mucositis [24] in patients undergoing myeloablative conditioning. after engraftment, the risk of bacterial infections depend on the community exposures to common and opportunistic bacteria (e.g., nocardia, rhodococcus, listeria), the presence of acute and chronic gvhd, the degree of b-cell reconstitution and the use of trimethoprim-sulfamethoxazole prophylaxis. patients with chronic gvhd are at risk for invasive infection from encapsulated organisms, particularly streptococcus pneumoniae, haemophilus influenzae and neisseria meningitidis. it is postulated that the combination of b lymphocyte dysfunction secondary to the conditioning regimen and the effects of gvhd and its treatment have resulted in the loss and failure to develop an opsonizing antibody to these organisms, particularly streptococcus pneumoniae. in addition, for at least one to two years post-transplant, hsct patients have an inadequate response to pneumococcal vaccine. as igg levels are often low for some time after hsct, they should be routinely monitored, with replacement being considered when the igg level falls below 500 mg/ml [25, 26] . in addition, antimicrobial prophylaxis, such as with low-dose trimethoprimsulfamethoxazole (one single strength tablet daily for pcp prophylaxis), may afford further protection against this problem [1, 3] . there are several classes of viral infection of particular importance in the hsct recipient: those due to herpesviruses (cmv, ebv, hsv, vzv and human herpesvirus-6 [hhv-6]); those due to hepatitis viruses (e.g., hepatitis b [hbv]); those due to respiratory viruses (e.g., influenza, rsv, parainfluenza, adenoviruses, and others), and those due to polyoma viruses. the human herpesviruses share a number of characteristics that make them particularly successful pathogens in hsct recipients [1, 4, 27] : 1. latency once infected with a herpesvirus, one is infected for life, with a circulating antibody (seropositivity) in the absence of active viral replication being the classic marker for latent infection. reactivation from latency may be triggered by tumor necrosis factor (tnf), with the catecholamines epinephrine and norepinephrine and proinflammatory prostaglandins also playing a role. thus, the virus may be reactivated by such processes as sepsis, gvhd, allogeneic reactions, okt3 and antilymphocyte globulin. once a replicating virus is present, medications such as cyclosporine, tacrolimus and prednisone may significantly amplify the viral replication. 2. cell association these viruses are highly cell-associated, meaning that transmission occurs through intimate person-to-person contact, or transfusion or transplantation of latently or actively replicating cells from a seropositive donor. humoral immunity is, hence, less important than cell-mediated immunity. indeed, the key host defense is accomplished by major histocompatibility complex (mhc)-restricted, virus-specific, cytotoxic t cells, just that component of host defense most affected by gvhd and its treatment. 3. oncogenesis herpesviruses, such as ebv and hhv-8, play a direct role in oncogenesis-causing post-transplant lymphoproliferative disease (ptld) and kaposi's sarcoma, respectively. herpesviruses may also play an indirect role in oncogenesis with symptomatic cmv disease, increasing the incidence of ebv-associated ptld severalfold. 4. indirect effects in addition to the direct causation of infectious disease syndromes, human herpesviruses, particularly cmv, have indirect effects that are clinically important. it is believed that cytokines, chemokines and growth factors produced in response to viral replication may be responsible for these effects. they include, in addition to the modulation of oncogenesis, increasing the net state of immunosuppression so that the risk of opportunistic infection is increased. this last point is particularly important, as a variety of experiments have shown that gvhd and infection are closely linked by the production of these mediators. that is, there is a bidirectional trafficking of mediators between these two processes. the clinically most important direct effects of cmv in the hsct recipient are pneumonia and gastrointestinal disease. before effective antiviral treatment became available, cmv pneumonia occurred in 20 to 30 percent of seropositive recipients and had an associated mortality around 80 percent [28] . cmv commonly causes fever in the absence of preemptive treatment, and end-organ disease (hepatitis, bone marrow dysfunction, retinitis, and encephalitis) may occur. among allogeneic hsct recipients, the risk of cmv reactivation (60-80%) and end-organ disease is greatest in the seropositive recipient who receives a graft from a cmv seronegative donor (cmv d−/r+), likely due to the loss of native immunity during the transplant process and immune reconstitution with a cmv naïve allograft [29, 30] . patients who are cmv d+/r+ have cmv reactivation (50-60%) and disease risk that is similar to or slightly lower than that of the cmv d−/r+ patient. patients who are cmv d+/ r− have a lower risk of cmv infection (10-30%) and disease, but higher than cmv d−/r− patients (<5%). the risk of cmv infection in this latter group has been greatly decreased by use of leukoreduced blood products or by exclusive use of cmv negative products when available [31] . the risks of cmv reactivation and disease among autologous hsct recipients is minimal (<1%) [1, 4] . another major risk factor for the development of cmv reactivation and disease is the occurrence, severity and treatment of acute gvhd [29, 30] . other potential factors associated with an increased risk of cmv reactivation and disease are reception of t cell-depleted or cord blood allograft, whether the donor is unrelated or mismatched, or donated bone marrow (instead of peripheral stem cells), and whether the conditioning regimen was myeloablative [29, 32] . the most widely used therapy for clinical cmv disease is ganciclovir, which can be administered either intravenously or orally in the form of a prodrug, valganciclovir, with an acceptable bioavailability profile (~50-60%). typically, the parenteral form is administered until the patient is able to tolerate oral therapy. gastrointestinal absorption of valganciclovir, even in the setting of mild to moderate gi gvhd, has been demonstrated to be adequate [33, 34] . duration of treatment depends on the clinical response and the nature of the recovery of native immune function. in the case of serious illness, particularly pneumonia, anti-cmv hyperimmune globulin can be considered as adjunctive therapy. despite these efforts, the mortality from cmv pneumonia remains high. the major toxicity of ganciclovir is myelosuppression, so that great effort is placed in monitoring these patients closely and adjusting doses appropriately [1, 4] . occasionally, g-csf support may be required to preserve an acceptable neutrophil count and to allow adequate therapy of a serious cmv infection. while certain medications, such as atg and okt3, are likely to induce cmv reactivation, others like sirolimus may inhibit this [29] . current strategies are based on preventing cmv disease through prophylaxis or preemption. prophylaxis with ganciclovir from the time of engraftment until at least day 100 post-transplant has been studied in randomized trials [35, 36] . although cmv viremia and disease were prevented, there was no overall benefit of this strategy due to secondary bacterial and fungal infections related to ganciclovir-induced neutropenia. alternatively, a preemptive strategy is employed in which patients are monitored weekly for viremia through either a pcr assay for cmv dna or an antigenemia assay. positive results are linked to initiating ganciclovir or other antiviral drugs active against cmv. typically, these assays turn positive several days to weeks prior to the onset of clinical disease, permitting the use of effective preemptive therapy [1, 4, 29, 33, [37] [38] [39] [40] [41] . a preemptive approach significantly decreases the amount of prophylactic medication used, thus minimizing medication-associated toxicity. in the pre-ganciclovir era, cmv disease typically occurred during the first three months post-transplant. increasingly, with the widespread use of a prophylactic or preemptive antiviral strategy, breakthrough occurs much later, typically one to three months after the cessation of the antiviral therapy. risk factors for late cmv disease include chronic gvhd, low cd4-t cell counts, and cmv infection before day 100. relapse or the emergence of ganciclovirresistant virus also can occur, particularly in the face of high viral loads and inadequate courses or dosing of ganciclovir. foscarnet is the preferred drug in this setting or when further potential myelosuppression with ganciclovir is not advisable. the experience with cidofovir use in the hsct population is limited. both foscarnet and cidofovir are potentially nephrotoxic and should be administered with caution [1, 4] . studies are examining the emerging strategies for the management of cmv infection and the use of cmv vaccines in donors and recipients, adoptive immunotherapy for patients with refractory or relapsing cmv infection and the use of maribavir for prophylaxis. the major recognizable clinical effect of ebv in the hsct patient is in the pathogenesis of ptld. following the recovery from primary ebv infection (>95% of the adult population), ongoing lytic infection of b-cells occurs in the oropharynx, with latent infection of b-cells in the peripheral blood and lymphoid tissues. these latently infected cells can be transformed and immortalized, resulting in polyclonal proliferation. in the normal seropositive individual, these cells are kept in check by a specific cytotoxic t cell response. in the presence of immunosuppressive therapy, this surveillance system is inhibited in a dose-related fashion, thus permitting continued b-cell proliferation. such ongoing proliferation results in particular clones being favored and the potential for developing cytogenetic abnormalities, which leads to the development of a truly malignant process-ptld [1, 4, 27, 42] . the spectrum of clinical disease seen with ptld is quite broad, ranging from a mononucleosis-like process or a polyclonal proliferation of lymphocytes that usually responds to decreasing immunosuppressive therapy, to a monoclonal, highly malignant b-cell lymphoma. the mononucleosis-like process is seen particularly in children with primary post-transplant ebv infection. the clinical presentation is one of fever, sore throat, cervical adenopathy and tonsillar hypertrophy and inflammation. unlike b-cell lymphoma in the normal host, in the transplant patient, particularly the adult, the process can be extranodal. thus, presentations may include central nervous system (cns) invasion (from involvement of the meninges to focal cerebral lesions), liver, lung and bone marrow diseases. not uncommonly, involvement of the gut (particularly the small bowel) may lead to recognition of the ptld, with a clinical presentation of small bowel obstruction, perforation, or occult gastrointestinal bleeding. disseminated, multi-organ disease is quite common in the hsct patient [1, 4, 41, 42] . risk factors for developing ptld include: primary ebv infection in association with high-dose immunosuppression; interventions such as t cell depletion, umbilical cord blood transplant and the systemic administration of anti-thymocyte globulin increase the risk significantly; and intensive immunosuppression that results in suppression of the key host defense against ebv-transformed cells (mhc-restricted, ebv-specific, cytotoxic t cells) significantly increases the risk of ptld. in addition to the host characteristics mentioned, high ebv viral loads correlate with an increased risk of ptld. it has been suggested that ebv viral load surveillance in peripheral blood be carried out in high risk patients (those with primary ebv infection, anti-t cell antibody therapy for gvhd, hla-mismatched or t cell-depleted hsct recipients), with decreased immunosuppression +/− antiviral therapy (acyclovir or ganciclovir) carried out in the setting of high viral loads [1, 4, 41, 42] . treatment of ptld remains controversial. all patients with diagnosed ptld should have a significant decrease in immunosuppressive medications. many centers also prescribe antiviral therapy. patients not responding to these measures are usually treated with an anti-b-cell monoclonal antibody (rituximab, an anti-cd20 monoclonal antibody) [43, 44] . after that, therapies have ranged from anti-lymphoma chemotherapy to alpha-interferon and intravenous gamma globulin. hsv infection prior to the introduction of acyclovir was a major problem in the hsct recipient. occurring in the preengraftment period, hsv infection greatly exacerbated the severity of mucositis. not only were ulcers observed in the oral cavity and anogenital areas, ulcerations of the esophagus, stomach and intestine were also observed. hsv pneumonia was also noted, with rare cases of cutaneous dissemination and encephalitis. the current standard of care is to test all candidates for hsct for an antibody to hsv, with seropositive individuals then placed on antiviral prophylaxis, beginning prior to hsct. effective agents for hsv prophylaxis include acyclovir (intravenous or oral), valacyclovir or famciclovir. recurrence of hsv may occur later in the course, and should again be treated with an acyclovir regimen, with repeated episodes justifying long-term prophylaxis. acyclovir resistance is uncommon in this situation, but can occur, and requires treatment with foscarnet [1, 4] . all patients and donors should have serologic testing for vzv prior to transplant. seronegative individuals post-transplant should avoid exposures to vzv, but if such an exposure occurs, valacyclovir or varicella hyperimmune globulin should be promptly initiated. before universal prophylaxis with acyclovir became standard, an estimated 40 percent of hsct patients developed active vzv, with a median time of onset being five months post-transplant. the great majority of these patients had zoster, but approximately 20 percent had a more generalized process resembling primary varicella. a significant concern was visceral involvement in the setting of disseminated disease as well as neurologic complications such as myelitis or encephalitis [1, [45] [46] [47] [48] . prophylaxis with acyclovir in the early period post-transplantation substantially decreases the occurrence of herpesvirus infections, including vzv, and is rarely, if ever, seen during acyclovir prophylaxis. prophylaxis is typically given for the first year post-allogeniec transplantation. vzv reactivation is often seen three months post-discontinuation of prophylaxis. as the vzv vaccine is a live attenuated viral vaccine, its use is contraindicated for at least two years posttransplantation, and unless a research study or close follow-up is involved, should be omitted. hhv-6 is a β-herpesvirus (as is cmv) whose role in post-transplant complications is being defined. in the great majority of instances, hhv-6 primary infection occurs by the third year of life, with a seroprevalence rate of 90 percent at one year, and close to 100 percent at three years [49, 50] . the clinical effects associated with primary hhv-6 infection include exanthem subitum (roseola), and a form of encephalitis. in hsct patients, bone marrow suppression, especially delayed platelet engraftment, and encephalitis have been associated with hhv-6 type b. the encephalitis typically occurs one to two months after transplantation and is associated with profound memory loss, especially short-term memory, and mri changes in the mesial temporal lobes (limbic encephalitis) [51, 52] . the highest risk patients for this complication are male, umbilical cord blood recipients for whom the attack rate may be as high as 10 to 20 percent. detecting hhv-6 dna in the blood of allogeneic hsct recipients is a common phenomenon occurring transiently in 40 to 60 percent of patients, yet encephalitis is a rather infrequent occurrence (1-2%). as obtaining brain biopsies is not usually feasible early after transplantation, the diagnosis of hhv-6 encephalitis is currently achieved by developing an acute limbic encephalitis syndrome, confirmed with mri imaging of the brain and by the detection of hhv-6 in the csf [52] . it remains unclear what the treatment of choice for this virus is. one approach that we currently favor is to use foscarnet. it is possible that anti-cmv preventative strategies with ganciclovir may have a beneficial effect on this virus as well [1, 53] . hsct recipients are at significant risk for infection with respiratory viruses circulating in the community. these infections can occur at any time in the post-transplant course, and can be acquired in the community or during hospitalization from infected staff, family and friends. overall, an estimated 10 to 20 percent of hsct patients will become infected in the first year posttransplant, with the potential for this figure to rise significantly in the setting of a community-wide outbreak [54] . the dilemma for the clinician is how to prevent these infections, as there is a far higher rate of progression to pneumonia (viral and/or bacterial or fungal superinfection), which carries a far higher morbidity and mortality than what is observed in the general population. in addition, antiviral therapy for these agents is in its infancy. it is important to attempt to make an etiologic diagnosis. avoiding exposure to infected individuals by systematic infection control measures in both family members and friends, but most importantly in health care workers, is the best preventative strategy available [55] [56] [57] [58] . although rsv can be acquired by inhaling an aerosol, direct contact with infected secretions is the usual mode of spread between individuals. in the hsct patient, both adult and pediatric, rsv is a cause of significant morbidity and mortality. the illness begins with the signs and symptoms of a viral upper respiratory tract infection (rhinorrhea, sinus congestion, sore throat and/or otitis media), that may progress to pneumonia, especially if the virus is acquired in the preengraftment phase. as with influenza, pneumonic syndromes can be due to rsv itself, but in our experience it is more frequently due to secondary bacterial and fungal infections. the advent of rapid rsv diagnosis by antigen detection in nasopharyngeal swabs has resulted in the recognition that rsv is a significant pathogen for both adults and children, particularly in immunosuppressed patients. optimal antiviral management, however, remains unclear. there are reports that aerosolized ribavirin +/− anti-rsv polyclonal or monoclonal antibody may have therapeutic benefit, but this remains unproven. there is also interest in prophylaxis with an anti-rsv antibody, although there have been no trials in hsct patients [55] [56] [57] [58] [59] . as with rsv, the incidence of influenza infection in hsct patients reflects the level of influenza activity in the community. the impact of this virus on infected hsct recipients is demonstrated by the following statistics: ~60 percent of the patients with influenza develop pneumonia and ~25 percent of patients with influenza pneumonia die of progressive respiratory failure. when influenza is identified as a pathogen, use of a neuraminidase inhibitor (oseltamvir or zanamavir) or an amantadate (amantadine or rimanatine) should be considered. the neuraminidase inhibitors are attractive in this setting as they are effective against both influenza a and b and antiviral resistance occurs more slowly compared with amantadine use. annual influenza vaccination should be considered, but its benefit is attenuated; indeed, it is probably fair to say that maximal benefit from vaccination occurs when the vaccine is administered to health care workers, family, friends and other contacts of the patient. when an infection is diagnosed, early treatment should be considered [58, 60] . there are more than 50 serotypes of adenovirus and nearly all have been described to cause human disease. adenovirus disease post-transplantation is likely due to both a newly acquired virus and viral reactivation. the most common adenovirus-associated illness post-transplantation is hemorrhagic cystitis which has been described in a recent report to occur in up to 42 percent of patients in the first year post-transplantation [61] . the overwhelming majority of cases are asymptomatic and require no intervention [62] . occasionally the severity of hemorrhage or bladder-associated pain is so great that intervention is required. other important adenovirus-associated syndromes include hepatitis and pneumonitis which may be fatal in the early post-transplant period. in the late post-transplant period adenovirus gastroenteritis may occur which is often a self-limited illness; however, severe disease has been described especially in patients requiring significant levels of immunosuppression for gvhd. therapeutic options for adenovirus are limited. the role of the antiviral cidofovir is controversial with mixed results having been reported [63] . decreasing immunusuppression and attempting reconstitution of the native host immune response is critical. the role for other adjunctive therapies, such as ivig, is unproven, but can be considered in severe cases. avoiding exposure to new infection, as with all community-acquired pathogens, is central to optimal care. parainfluenza, rhinoviruses, metapneumovirus and coronaviruses are all capable of causing lower respiratory tract infection in hsct recipients. of these many viruses, parainfluenza virus type iii is especially associated with a high mortality [64, 65] . again, specific therapy is not available, emphasizing infection control strategies in the hospital setting and avoiding individuals with respiratory tract complaints at home. when upper respiratory tract complaints occur in hsct patients, a diagnosis should be made, utilizing rapid diagnostic techniques (e.g., antigen detection assays or nucleic acid testing). preemptive therapy, when available, should be initiated, while immunosuppressive therapy diminished and isolation from other hsct patients should be accomplished. bk and jc viruses are the two important species in this family of viruses with a genitourinary and cns predilection, respectively. approximately 60 to 80 percent of adults have been infected with one or both of these viruses, typically in childhood. with immunosuppression, reactivation occurs which may lead to disease. bk virus is associated with hemorrhagic cystitis in the early post-transplant period. this virus is commonly found in the urine and rarely requires any therapeutic intervention. jc virus is the etiologic agent of progressive multifocal leukoencephalopathy (pml) which is a rare, but severe post-transplant complication. pml involves the white matter and presents with focal neurologic symptoms associated with the specific area of the cns where the lesion(s) occur. diagnosis requires correlating the clinical presentation, radiographic findings (typically by contrast-enhanced mri imaging) and csf pcr results for jc virus. control of jc virus is associated with an intact cell-mediated immune response. therapy for polyomavirus infection is quite limited with minimizing immunsuppression, when possible, being critical. the role of cidofovir is controversial with mixed results being reported. the use of quinolones for bk viruria is controversial at best and we do not recommend this practice [66] . although the use of gatifloxacin was advocated by some authors, this drug is no longer available. leflunomide administration has been used by some for treatment of bk in renal transplant patients, but no randomized trial data exists to support or recommend its use in either kidney or hsct recipients at this time. hepatitis b and c viruses may cause chronic infection which often leads to eventual significant liver dysfunction. given the high global prevalence of these viruses, it is prudent to screen for past or current infection prior to transplantation. when ongoing infection is found, careful assessment of liver function and a pre-transplant liver biopsy should be considered to assess for occult cirrhosis, as this may influence peri-transplant management [67] . hbv infects approximately 350 million people worldwide chronically, and substantially more have had prior resolved infection. the use of the hbv vaccine as a routine childhood immunization will likely decrease the number of chronically infected individuals over the next several decades. the advent of nucleic acid detection technology has allowed a more precise mechanism to detect active hbv replication compared with antigen-(for surface and e) only methods. for patients with evidence of prior hbv exposure (hbv core antibody positive), it is important to consider hbv reactivation in the setting of post-transplant liver dysfunction and to differentiate this from other causes such as hepatic gvhd or medication toxicity, although reactivation initially occurs in the setting of normal liver tests. the best strategy for surveillance post-transplantation remains to be defined. some recommend routine surveillance for hbv reactivation post-transplantation, whereas others would suggest antiviral prophylaxis. it is important to be aware that old resolved infections, including those with hepatitis core and surface antibody, but without antigen or hbv dna detected, are at risk for reactivation (seroreversion) post-transplantation, especially in the setting of high levels of immunosuppression [68, 69] . several therapeutic options have become available over the last several years and include lamivudine [70] [71] [72] , adefovir [73] , entecavir [71, 72] and telbivudine. other agents such as tenofovir and emtricitabine also have excellent anti-hbv activity. use of these agents requires careful consideration to minimize the risk for the emergence of resistant virus, which may be as high as 10 percent per year for lamivudine, but is less than 1 percent for adefovir and entecavir. epidemiologic studies suggest that more than 170 million people worldwide have been infected with hepatitis c virus (hcv) and the majority (approximately 85%) are chronically infected [67] . over several decades, chronic hcv infection is associated with progressive hepatic fibrosis, liver failure, and hepatoma. this process is accelerated in certain immunocompromised patients including hsct recipients [74, 75] . it is important to assess patients for seropositivity to hcv prior to transplantation and in those who are found seropositive, to assess the hcv viral load, genotype and liver pathology. the presence of elevated liver enzymes in the setting of hcv before allogeneic hsct has been associated with an increased incidence of vod [76] . a more precise profiling of the hcv-infected patient, including liver biopsy, should be considered to better define the extent of the hcv-induced liver disease, and to optimize the conditioning regimen and frequency of surveillance posttransplantation. treatment of hcv is limited and typically requires use of an interferon and ribavirin which are likely to be poorly tolerated in the early post-transplant setting. in patients who are infected, it is prudent to counsel them to avoid hepatotoxins, receive the hepatitis a and b vaccines, and minimize the risk of transmission to close contacts. there are three categories of fungal pathogens that can infect the hsct patient: a) the classic opportunistic fungi, which cause >90 percent of the invasive fungal infections that occur in the hsct patient -candida, aspergillus and cryptococcus being the most important of these infections; b) the geographically restricted systemic mycoses caused by blastomyces dermatitidis, coccidioides immitis and histoplasma capsulatum; and c) invasive infection due to the so-called "newly emerging fungi" -fusarium, the zygomycetes and such dematiaceous fungi as scedosporium, scopulariopsis and dactylaria [4] . candida is a major cause of fungal bloodstream infection during the preengraftment phase of hsct. although there is the possibility that the portal of entry can be vascular access catheters, it is believed that translocation of candida species across gut mucosa damaged by the pre-transplant conditioning regimen is the major route of access to the bloodstream in the granulocytopenic patient [77, 78] . in the past, c. albicans and c. tropicalis accounted for virtually all of the candida bloodstream infections. the incidence of candidemia was ∼11 to 16 percent (with a median time to onset of two weeks post-transplant), resulting in a high rate of tissue invasion and an attributable mortality of nearly 40 percent [79, 80] . with the introduction of empirical antifungal therapy or fluconazole prophylaxis (400 mg/day) during the preengraftment period, the incidence of candidemia has been significantly decreased, hepatosplenic candidiasis has become quite rare, and the attributable mortality has been significantly decreased. fluconazole-resistant candida sp, c. krusei and c. glabrata, have emerged as not uncommon causes of candidemia in hsct patients, as have the other non-albicans candida species [1, 4, [81] [82] [83] [84] [85] [86] [87] . it is also important to recognize that other species of yeast (e.g., trichosporon sp, blastoschyzomyces capitatus, saccharomyces cereviseae and rhodotorula sp.) can cause clinical syndromes identical to those observed with invasive candidiasis (bloodstream infection, infection metastatic to the skin and subcutaneous tissues, as well as other sites, including hepatosplenic disease identical to that caused by candida species) [88] . in an era of increased use of echinocandins for prophylaxis [89] and empirical antifungal treatment [90] , these organisms [88, 91] and echinocandin-resistant candida sp, especially c. parapsilopsis, have become emerging causes of fungemia in the hsct units [1, 4, 82] . invasive fungal disease has been most commonly caused by aspergillus sp, with a. fumigatus, a. flavus, a. terreus, a. niger and a. nidulans being the most common causes of invasive aspergillosis. the portal of entry for 90 percent of cases of invasive aspergillosis is the lungs, with the nasal sinuses and the skin accounting for virtually all of the remaining cases. there are two major host defenses that are mobilized in response to inhalation of the aspergillus spores -granulocytes and cell-mediated immunity, specifically cytotoxic t cells. the importance of these mechanisms is demonstrated by the clustering of cases of invasive aspergillosis at two timepoints in the posttransplant course: preengraftment when profound granulocytopenia is present, with the incidence of invasive aspergillosis increasing steadily as the period of granulocytopenia is extended, and after the diagnosis of gvhd and the treatment of this adverse event. indeed, these late cases of invasive aspergillosis have become more common than the preengraftment cases. mortality rates have traditionally been high in patients who developed invasive aspergillosis in either time period [1, 4, 92, 93] . the clinical syndromes caused by aspergillus invasion reflect the pathologic consequences of the vasculotropic nature of this mold. the three major consequences of the vascular invasion that characterizes aspergillus invasion include hemorrhage, infarction and metastatic disease. initial clinical complaints include persistent fever, chest pain, tachypnea, hypoxemia and hemoptysis, as well as symptoms related to metastases. before the availability of noninvasive fungal markers (galactomannan and β-glucan) and aggressive imaging with spiral chest computerized tomographic (ct) scanning, 50 percent or more of patients experience disseminated infection at the time of first diagnosis, accounting for the high mortality observed in allogeneic hsct recipients. a particular problem is infection in the cns, where mortality historically has approached 100 percent. metastases can present any site, but particularly important is the skin, as innocent appearing skin lesions can lead to early recognition of the disease, and should be aggressively biopsied [4] . definitive diagnosis of invasive mold infections, including invasive aspergillosis, is usually accomplished by biopsying the site of abnormality. early diagnosis is the key to effective therapy [94] . sputum or bronchoscopic samples rarely yield mold on culture. in recent years, considerable effort has been made to find other technology that will lead to an earlier and timely diagnosis. the ones that have been incorporated into practice are the systematic measurement of aspergillus antigens and serial chest ct imaging. monitoring the serum of hsct patients for galactomannan or β-glucan is now commercially available and has been incorporated into the current diagnosis guidelines [95, 96] . the detection of circulating fungal dna in the blood by pcr [97] remains experimental. findings on chest ct, in particular the halo sign ( fig. 19-2) , are associated in the neutropenic patient with invasive aspergillosis (although other pathogens can cause the same radiologic finding: fusarium and other vasculotrophic molds and nocardia asteroides being examples of this). european groups have been advocating protocol serial chest ct scans to find such pathology as a guide to early diagnosis [98] . if prevention fails, then early diagnosis is the key to the patient's survival [4, 92, 93] . given the limitations of current diagnostic techniques and the significant morbidity associated with invasive fungal infection, two strategies of antimicrobial use are commonly deployed in the hsct patient. the first is prophylactic fluconazole use during the initial transplant period, which has been shown to decrease fungal infections [80] in one study, and overall mortality fig. 19-2 . computerized tomographic scan of the chest in a patient with a "halo sign" due to invasive aspergillosis. note that halo signs most commonly occur in granulocytopenic hsct recipients with invasive aspergillosis. however, it must be emphasized that a halo sign is occasionally seen in patients with nocardia, scedosporium, fusarium and other forms of pneumonia. the patient was treated with voriconazole monotherapy during neutropenia during consolidation chemotherapy for aml and his treatment was continued through allogeneic transplantation. [79] in another, when started on day 0 until engraftment [80] or day +75 [79] . it is important to note that a high background rate of candida infections was noted in both of these reports and may not represent the experience of other transplant centers. echinocandins may be an alternative to fluconazole prophylaxis during this risk period [89] . the second common strategy is empiric antifungal therapy in neutropenic patients with persistent fever without a source, despite broad-spectrum antimicrobial therapy for >96 hours [99, 100] . in this setting the primary concern is both candida and invasive mold infection, especially aspergillus [2] . the traditional antifungal therapy utilized as empiric therapy is an amphotericin product [101, 102] . caspofungin use in this setting has become common because of the favorable side effect profile of this class of agents, but at the expense of a more limited fungal spectrum [90] . other echinocandins (micafungin, anidulafungin) are likely to be similarly effective, but no randomized comparisons with these latest drugs have been performed. the role of voriconazole in this setting is controversial [102] . when treating invasive aspergillosis several approaches should be considered simultaneously: 1) antifungal therapy, 2) reverse or minimize the host immune defects (decrease corticosteroids, increase neutrophils), 3) control permissive viral infections (e.g., cmv) and 4) consider surgical excision, if possible. voriconazole has become a cornerstone of therapy for invasive aspergillus infections, though the management of potential side effects is substantial [103] [104] [105] . whether the combination of therapeutic agents (polyenes, azoles and echinocandins) increases the therapeutic benefit has yet to be determined [106] . increasing experience suggests that voriconazole alone is sufficient in most cases for a successful outcome in invasive aspergillosis and has decreased the morbidity and mortality of this infection [11, 106] . another significant risk period for invasive fungal infections (ifi) is in the setting of significant gvhd, such as grade iii or iv, and its therapy [9] . in this setting, posaconazole (versus fluconazole) prophylaxis has recently demonstrated some benefit in preventing ifi compared to fluconazole (5.3% versus 9.0%, p = 0.07) and in preventing probable or proven invasive aspergillosis (2.3% versus 7.0%, p = 0.006 -interestingly, these results were largely driven by results from galactomannan assay testing) [107] [108] [109] . posaconazole has activity against the zygomycetes as well as aspergillus sp [110] [111] [112] . when an azole is used in this patient population, careful assessment of drug interactions, both with the initiation and cessation of therapy, is critical. therapy for the emerging fungi fusarium and scedosporium should be guided by in vitro sensitivity testing done locally or at regional reference laboratories, but voriconazole use should be considered. when therapy for the endemic mycoses is indicated, initial treatment (induction therapy) with an amphotericin preparation should be considered, followed by a prolonged course of consolidative therapy with an oral azole. cryptococcal disease should be treated initially with an amphotericin preparation, cns involvement should be excluded by cerebrospinal fluid sampling, and the use of flucytosine should be considered if present. pneumocystis jiroveci, formerly carinii, is a ubiquitous environmental organism which is an important cause of pneumonia in patients who are immunosuppressed, such as those who have undergone an hsct, on chronic prednisone (typically >20mg per day) or with advanced hiv infection. pcp infection typically presents as an interstitial pneumonitis with marked hypoxemia. severe infection can be life threatening. fortunately, universal prophylaxis of high risk patients during the high risk periods with trimethoprim-sulfamethoxazole has markedly decreased this complication. however, intolerance to prophylaxis, use of second line prophylaxis agents (e.g., dapsone, pentamidine, or atovaquone), poor medication compliance or failure to re-institute prophylaxis in the setting of augmented immunosuppression (e.g., treatment of gvhd) are common reasons why cases still occur. several important issues must be addressed after successful hsct to minimize infectious complications. first, it is important to avoid exposure to pathogens, especially when the immunosuppressive therapy to prevent gvhd is the highest. this includes avoiding gardening and soil exposures, mold exposures such as cleaning out damp basements or smoking marijuana, individuals with active respiratory infections, especially children, and avoiding enteric pathogens. second, optimal treatment or monitoring for latent infections such as herpesviruses, hepatitis viruses and prior granulomatous diseases (e.g., mycobacterium tuberculosis). those patients with a positive test for latent tuberculosis should receive secondary prophylaxis, which typically is begun within one month post-transplantation, after the acute regimen toxicities associated with transplantation have subsided, when screening and preventive treatment have not occurred previous to hsct. the first line therapy for secondary prophylaxis is isoniazid for nine months. however, in patients with significant hepatic dysfunction or peripheral neuropathy alternative regimens need to be considered. rifamycin-based regimens are difficult given the potential hepatotoxicity, as well as the significant drug interactions, especially with concomitant use of a calcineurin or an azole. a quinolone, such as levofloxacin, with ethambutol may be considered. when a mycobateriologically static regimen is chosen, the duration of therapy often must be extended with some using this combination for 18 months as secondary prophylaxis (table 19-2) . third, optimizing vaccinations for routine pathogens such as diphtheria, tetanus, pertussis, influenza and pneumococcus (table 19-3) . this optimal timing of re-vaccination depends on the nature of the transplant, with earlier re-vaccination schedules being considered in the nonmyeloablative setting. fourth, prophylaxis for pcp, which is typically continued for approximately one year or until the immunosuppressive medications are tapered off. the optimal medication to use for pcp prophylaxis is trimethoprimsulfamethoxazole which offers some protection for a variety of other important pathogens including pneumococcus, hemophillus influenza, nocardia sp., toxoplasma, listeria, salmonella sp., and other enteric bacterial pathogens. if trimethoprim-sulfamethoxazole is not tolerated due to significant renal dysfunction or bone marrow suppression, then alternative agents for pcp prophylaxis include dapsone, atovaquone or aerosolized pentamidine; however, none of these second line agents afford the broad microbial protection which trimethoprim-sulfamethoxazole affords. and lastly, herpes group viral prevention which should include acyclovir to prevent hsv and vzv and systematic monitoring for cmv, in the allogeneic setting, with early use of a cmv active antiviral if evidence for cmv activation or disease is observed. prophylaxis must be re-assessed in the setting of persistent or augmented immunosuppression, such as in the setting of clinically significant gvhd, regardless of time since hsct. medication doses may need to be adjusted for renal dysfunction. * trimethoprim-sulfamethoxazole affords modest protection for a broad array of potential environmental and community pathogens including: nocardia sp, toxoplasmosis, pneumococci, h influenza, listeria, shigella, and slamonella sp. ** alternatively preemptive monitoring with serial viral antigen or viral load assays can be considered. *** for those with evidence of prior hbv infection (e.g., hepatitis b core antibody positive), consider monitoring hbv viral load for evidence of reactivation periodically. if reactivation is detected then consider treatment if persistent hbv viremia detected. specific hbv antiviral therapy is discussed in the text. **** pre-transplant secondary prophylaxis for mtb is preferred. ***** decision for systemic azole prophylaxis should be based on local epidemiology of invasive fungal infections. consider posaconazole prophylaxis in the setting of significant gvhd (e.g., grade 3 or 4) and its therapy. drug interactions must be carefully managed both with initiation and cessation of azole therapy. an important aspect of antimicrobial therapy in the hsct patient is the management of drug interactions, especially between antimicrobial agents (e.g., azoles, macrolides) and the immunosuppressive medications (e.g., calcineurin inhibitors, sirolimus) used to prevent and treat gvhd. there are three important categories of interaction to pay particular attention to, two of which are related to the major route of drug metabolism for the calcineurin inhibitors, hepatic cytochrome p450 enzymatic metabolism. these interactions are as follows: 1) certain antimicrobial agents (most notably the macrolides [eryth romycin>clarithromycin>azithromycin] and the azoles [ketoconazole>itraco nazole>voriconazole>fluconazole]) will downregulate the metabolism of the calcineurin inhibitors, resulting in elevated blood levels of active drug, and an increased risk of nephrotoxicity, as well as over-immunosuppression and an increased incidence of opportunistic infection; 2) certain antimicrobial agents (such as rifampin and rifabutin) upregulate metabolism of the calcineurin inhibitors, leading to a fall in blood levels and an increased risk of gvhd, and 3) therapeutic blood levels of the calcineurin inhibitors, when combined with such drugs as amphotericin b, aminoglycosides and vancomycin, can cause significant renal toxicity. hsct has become one of the great success stories of modern medicine. it is the therapy of choice for an increasing number of conditions, including a variety of cancers, bone marrow failure states, congenital immunodeficiencies, metabolic disorders and even as a means for introducing new genes. the major hurdle in most of these attempts, however, remains infection. bacterial and fungal sepsis, as well as herpes group viral infection and community-acquired respiratory virus infection threaten the well-being of these patients. there are two phases of the post-transplant course when the patient is at particular risk: preengraftment with profound granulocytopenia and mucositis, and post-engraftment when gvhd and its therapy render the patient vulnerable to both fungal and viral infection. new preventative strategies are being formulated involving both prophylaxis and preemptive therapy. similarly, new non-culture diagnostic approaches that rely on antigen detection or pcr detection of microbial dna are being developed. new therapies, both antiviral and antifungal, have emerged. these should prompt much more effective prevention and therapeutic strategies. infection in hematopoietic stem cell tansplantation quantitative relationships between circulating leukocytes and infection in patients with acute leukemia guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients preventing opportunistic infections after hematopoietic stem cell transplantation: the centers for disease control and prevention, infectious diseases society of america, and american society for blood and marrow transplantation practice guidelines and beyond. hematology / the education program of the invasive aspergillus infection: possible nonward common source within the hospital environment septicemia and shock syndrome due to viridans streptococci: a case-control study of predisposing factors introduction to marrow and blood stem cell transplantation infectious complications associated with alemtuzumab use for lymphoproliferative disorders infliximab use in patients with severe graftversus-host disease and other 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intravenous ganciclovir in allogeneic stem cell transplant patients with graft-versus-host disease of the gastrointestinal tract oral valganciclovir leads to higher exposure to ganciclovir than intravenous ganciclovir in patients following allogeneic stem cell transplantation early treatment with ganciclovir to prevent cytomegalovirus disease after allogeneic bone marrow transplantation ganciclovir prophylaxis to prevent cytomegalovirus disease after allogeneic marrow transplant cytomegalovirus pp65 antigenemia-guided early treatment with ganciclovir versus ganciclovir at engraftment after allogeneic marrow transplantation: a randomized double-blind study polymerase chain reaction monitoring reduces the incidence of cytomegalovirus disease and the duration and side effects of antiviral therapy after bone marrow transplantation successful modification of a pp65 antigenemia-based early treatment strategy for prevention of cytomegalovirus disease in allogeneic marrow transplant recipients 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of primary human herpesvirus 6 infection mr imaging of human herpesvirus-6-associated encephalitis in 4 patients with anterograde amnesia after allogeneic hematopoietic stem-cell transplantation post-transplant acute limbic encephalitis: clinical features and relationship to hhv6 human herpesvirus 6 variant b infection in adult patients after unrelated cord blood transplantation community respiratory virus infections among hospitalized adult bone marrow transplant recipients phase 1 evaluation of the respiratory syncytial virus-specific monoclonal antibody palivizumab in recipients of hematopoietic stem cell transplants detection of rhinovirus rna in bronchoalveolar lavage in hematopoietic stem cell transplant recipients with pneumonia rhinovirus infections in myelosuppressed adult blood and marrow transplant recipients community respiratory virus infections in transplant recipients palivizumab prophylaxis to prevent respiratory syncytial virus mortality after pediatric bone marrow transplantation: a decision analysis model influenza infections after hematopoietic stem cell transplantation: risk factors, mortality, and the effect of antiviral therapy hemorrhagic cystitis in adults after unrelated cord blood transplantation: a single-institution experience in japan assessment of disseminated adenovirus infections using quantitative plasma pcr in adult allogeneic stem cell transplant recipients receiving reduced intensity or myeloablative conditioning cidofovir for adenovirus infections after allogeneic hematopoietic stem cell transplantation: a survey by the infectious diseases working party of the european group for blood and marrow transplantation parainfluenza virus infections after hematopoietic stem cell transplantation: risk factors, response to antiviral therapy, and effect on transplant outcome respiratory disease due to parainfluenza virus in adult bone marrow transplant recipients ciprofloxacin decreased polyoma bk virus load in patients who underwent allogeneic hematopoietic stem cell transplantation influence of human immunodeficiency virus infection on the course of hepatitis c virus infection: a meta-analysis a long-term follow-up study on hepatitis b surface antigen-positive patients undergoing allogeneic hematopoietic stem cell transplantation effectiveness of prophylactic anti-hbv therapy in allogeneic hematopoietic stem cell transplantation with hbsag positive donors peginterferon alfa-2a, lamivudine, and the combination for hbeag-positive chronic hepatitis b entecavir versus lamivudine for patients with hbeag-negative chronic hepatitis b a comparison of entecavir and lamivudine for hbeag-positive chronic hepatitis b long-term therapy with adefovir dipivoxil for hbeag-negative chronic hepatitis b long-term outcome of hepatitis c infection after bone marrow transplantation hepatitis reactivation and liver failure in haemopoietic stem cell transplants for hepatitis b virus (hbv)/hepatitis c virus (hcv) positive recipients: a retrospective study by the italian group for blood and marrow transplantation hepatitis c virus infection is a risk factor for liver failure from veno-occlusive disease after bone marrow transplantation guidelines for treatment of candidiasis should vascular catheters be removed from all patients with candidemia? an evidence-based review efficacy and safety of fluconazole prophylaxis for fungal infections after marrow transplantation-a prospective, randomized, double-blind study a controlled trial of fluconazole to prevent fungal infections in patients undergoing bone marrow transplantation infections in the neutropenic patient-new views of an old problem. hematology / the education program of the fungal infections in granulocytopenic patients: current approaches to classifications, diagnosis candidemia in immunocompromised patients infections due to resistant candida species in patients with cancer who are receiving chemotherapy prospective study of candida endophthalmitis in 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patients who are refractory to or intolerant of conventional therapy: an externally controlled trial we would like to thank robert h rubin, m.d. for the many years of advice and teaching, and for critically reviewing this work. key: cord-001079-v01vwu00 authors: thoden, j.; potthoff, a.; bogner, j. r.; brockmeyer, n. h.; esser, s.; grabmeier-pfistershammer, k.; haas, b.; hahn, k.; härter, g.; hartmann, m.; herzmann, c.; hutterer, j.; jordan, a. r.; lange, c.; mauss, s.; meyer-olson, d.; mosthaf, f.; oette, m.; reuter, s.; rieger, a.; rosenkranz, t.; ruhnke, m.; schaaf, b.; schwarze, s.; stellbrink, h. j.; stocker, h.; stoehr, a.; stoll, m.; träder, c.; vogel, m.; wagner, d.; wyen, c.; hoffmann, c. title: therapy and prophylaxis of opportunistic infections in hiv-infected patients: a guideline by the german and austrian aids societies (daig/öag) (awmf 055/066) date: 2013-09-14 journal: infection doi: 10.1007/s15010-013-0504-1 sha: doc_id: 1079 cord_uid: v01vwu00 introduction: there was a growing need for practical guidelines for the most common ois in germany and austria under consideration of the local epidemiological conditions. materials and methods: the german and austrian aids societies developed these guidelines between march 2010 and november 2011. a structured medline research was performed for 12 diseases, namely immune reconstitution inflammatory syndrome, pneumocystis jiroveci pneumonia, cerebral toxoplasmosis, cytomegalovirus manifestations, candidiasis, herpes simplex virus infections, varizella zoster virus infections, progressive multifocal leucencephalopathy, cryptosporidiosis, cryptococcosis, nontuberculosis mycobacteria infections and tuberculosis. due to the lack of evidence by randomized controlled trials, part of the guidelines reflects expert opinions. the german version was accepted by the german and austrian aids societies and was previously published by the arbeitsgemeinschaft der wissenschaftlichen medizinischen fachgesellschaften (awmf; german association of the scientific medical societies). conclusion: the review presented here is a translation of a short version of the german–austrian guidelines of opportunistic infections in hiv patients. these guidelines are well-accepted in a clinical setting in both germany and austria. they lead to a similar treatment of a heterogeneous group of patients in these countries. although opportunistic infections (ois) in human immunodeficiency virus (hiv)-infected patients have become rare in industrialized countries [1] , patients continue to present with advanced hiv disease and hiv-related ois. patients (so-called ''late presenters'') are often unaware of their hiv infection or have not received antiretroviral treatment. they present at a late stage and when their overall health status is already poor [2] . diagnosis and therapy of these ois remain a challenge. the aim of the recommendations presented here is to develop general and practical guidelines for the treatment and prophylaxis of the most common ois in germany within the framework of local epidemiological conditions. the tables in the different sections of the guidelines represent a summary of the therapeutic guidelines. with regard to diagnosis, the authors refer to the appropriate literature. at the time the guidelines were approved some articles were only available as congress abstracts; if these were published as peer-reviewed article at a later date, the published articles were cited. the kaad (clinical aids working group germany) guidelines conform to the international guidelines of the u.s. centers for disease control and prevention (cdc) (http:// www.cdc.gov/mmwr) [3] and guidelines formulated by the awmf (association of the scientific medical societies in germany) in the overlapping fields dermatology and neurology (http://www.uni-duesseldorf.de/awmf/ll/). members of other medical societies and the austrian aids society have also participated and have been consulted (see appendix). some of the following recommendations go beyond the approved use of drugs. in many cases, data from randomized controlled trials (rcts) are missing, and evidence is based on practical and clinical experiences not presented in published studies (expert opinion). in addition, we advise always checking interactions and toxicities of the applied drugs as these factors cannot be described in detail within the scope of this guideline. for the treatment of bacterial pneumonia, which is similar in hiv-positive and hiv-negative patients, the appropriate guidelines should be referred to. the indication for antiretroviral therapy (art) in germany is based on the guidelines by the german and austrian aids societies (daig and ö ag, respectively). however, general recommendations regarding when to start art with mostly art-naïve patients in the setting of an (acute) oi cannot be given. in the case of candidiasis, herpes virus infections or, for example, cryptosporidiosis, the immediate start of art is uncomplicated; in the case of progressive multifocal leukoencephalopathy (pml) it is even necessary and recommended. the situation is more difficult in cases of pneumocystis jiroveci pneumonia (pcp), cerebral toxoplasmosis, cytomegalovirus (cmv)-retinitis, tuberculosis (tb), atypical mycobacteriosis, and cryptococcosis. we refer to the corresponding sections of these guidelines. the recommendations given here represent the consensus of the guideline consensus group. the recommendations referring to medical therapies might involve off-label therapies that have not been officially approved. this is due to the lack of data from rcts on hiv-infected patients with oi. in such cases, the recommendation often refers to data on hiv-negative persons or personal experience (expert opinion). it should also be noted that drug-drug interactions or toxicities need to be excluded in each single case. the kaad was given the task to develop guidelines for the treatment and prophylaxis of oi by the daig in march 2010. the members of the daig, ö ag, and other german medical societies (in total 24 societies represented; see appendix) were asked to participate in the consensus process. the members formed small interest groups (n = 3-10 members) covering the different chapters of these guidelines. a first version was sent out in march 2010 based on the corresponding chapters of the digital version (http://www.hivbook.com). the different groups were free to base their chapters on this proposal after review of the relevant literature or to create new chapters. via an email system these new chapters were put together until the groups reached a consensus on a final draft. four weeks before a consensus conference in cologne on 25 june 2010, these drafts for all 12 chapters were sent out to all members of all groups and to all daig members with the request for suggestions for changes. the submitted suggestions for changes which were received were then sent out to the members prior to the meeting. during the consensus conference all suggestions were discussed and voted on separately. finally, each single chapter and the whole guideline proposal were voted on separately. there was an agreement of 100 % on the whole proposal between all members of the guideline group. in a third step the cologne proposal was sent out via email to all members of the daig four weeks prior to a daig member assembly in munich (17 march 2011) for comment. only minor revisions were asked for. the guidelines were again put to vote during the meeting. during the final vote the guidelines received 36 positive unanimous votes and were agreed on in the current version as the daig/kaad oi guidelines. the german version (long version) of these guidelines was submitted to the awmf on 30 august 2011 and was published online on 8 november 2011 (http://www.awmf. org/leitlinien/detail/ll/055-006.html). ö ag approved these guidelines on 9 november 2011. immune reconstitution inflammatory syndrome the immune system is expected to recover following initiation of art. some patients, however, show a paradoxical reaction. with widely varying symptoms, this pattern of disease is defined as immune reconstitution disease, immune reconstitution syndrome, or immune reconstitution inflammatory syndrome (iris) [4] [5] [6] [7] . different clinical case definitions exist [8, 9] manifestations of iris are diverse and range from unspecific symptoms, ois to autoimmune diseases, and malignomas [10] . regarding ois, the physician must differentiate between symptomatic relapse of a prior infection (paradoxical iris) and infections first appearing on art (unmasking iris). data on the incidence of iris vary widely, ranging between 10 and 23 % of all patients at initiation of an art [10] [11] [12] [13] . a prospective study showed an incidence rate in germany of 24.8 % [14] . an international meta-analysis showed a total incident rate of 16.1 % for iris, with the highest rates for iris uveitis, followed by tb, cryptococcal meningitis, pml, and rarer cases of kaposi's sarcoma or varizella zoster virus (vzv) infections [13] . the greatest risk factor would appear to be a low cd4 t-cell count of \50 cells/ll [12, 15] . patients starting an art with a cd4 t-cell count of \200 cells/ll and especially those who have a high viral load require close monitoring. patients with\50 cd4 t-cells/ll should also be tested for a latent mycobacterial infection (by culture). a large prospective trial [16] showed no difference for the development of an iris when art was initiated immediately after patients had started an oi therapy (patients with tb were excluded from the trial). in this study, corticosteroids were often given on initiation of art in a high number of pcp cases, which possibly suppressed some iris cases. for tb and cryptococcosis, however, several studies showed an higher incidence of an iris when art was initiated early [17] [18] [19] . corticosteroids are useful in cases of tb-iris [20] . steroid therapy for 2-6 weeks is recommended for cryptococcal-iris (increase of intracerebral pressure). the use of non-steroidal anti-inflammatory drugs (nsaids) and thalidomide was recommended in some studies, but a general recommendation can not be given for these agents [21] . art should only be interrupted in very severe cases. results of the swiss hiv cohort study prove that consequent isoniazid (inh)-prophylaxis in hiv patients with latent tb significantly reduces the risk of a relapse [22] . in general, prognosis for an iris is good and the mortality rate is not higher than that for patients without an iris [23] . pneumocystis jiroveci pneumonia is the most frequent oi in germany and appears predominantly in hiv-infected patients with advanced immunodeficiency (cd4 t cells \200/ll). if there clinical-radiological findings suggest pcp, therapy should be initiated immediately without awaiting results of a bronchoalveolar lavage. a mild pcp [bga: partial pressure of oxgen (po 2 ) [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] can be treated in outpatient medical care. if ventilation becomes necessary, non-invasive methods (continuous positive airway pressure) are beneficial if applied at an early stage [24] . with respect to the treatment of artnaïve patients, several experts believe that the initiation of art can be delayed until acute treatment is completed. however, one rct has shown advantages of an early start [16] . acute therapy should be given at least for 21 days, if necessary longer. the treatment of choice is a combination of trimethoprim and sulfamethoxazole (tmp/smx, cotrimoxazole). oral application of tmp/smx is only recommended in mild cases, but this therapy can be also considered after initial improvement during intravenous therapy. positive effects with lower doses of tmp/smx have been observed in some case reports, but data from controlled trials are missing [25] . all severe cases should be treated intravenously in hospital. in cases of respiratory insufficiency [po 2 \ 70 mmhg or alveolar-arterial oxygen tension difference (aado 2 ) c 35 mmhg on room air], most experts recommend (5)-10 days of adjuvant administration of prednisolone [approx.1 mg/kg body weight as a single dose or split dose twice daily (bid)]. with prednisolone, mortality risk of severe pcp can be reduced by half and significantly fewer patients require mechanical ventilation [26] . compared to tmp/smx, all alternative therapies are less effective. in the event of intolerance or sulfonamide allergy, intravenous pentamidine (4 mg/kg once daily (qd) for 14-21 days is recommended as a second choice; this agent is however more toxic and the dose may therefore have to be reduced after 5 days (2 mg/kg). treatment with inhaled pentamidine can be attempted in mild cases of pcp [27, 28] ; however, reports on experience with this approach are conflicting [29] [30] [31] . instead of pentamidine, the administration of atovaquone suspension or a combination of trimethoprime and dapsone or clindamycin and primaquine is possible [test for glucose-6-phosphate dehydrogenase (g6pd) deficiency!]. data are only available for mild to moderate pcp [32] [33] [34] . primaquine is no longer approved for use in germany, but it is available through international pharmacies. it can only be applied if there are no other alternatives and requires increased efforts in educating patients. according to a meta-analysis, the combination of clindamycin plus primaquine is the most successful therapy if cotrimoxazole therapy fails [35] ; this combination appears to be more effective than pentamidine alone [36] . patients with \200 cd4 t-cells/ll (or \14 % of total lymphocytic count) or a previous pcp require prophylaxis. the therapy of choice is tmp/smx, which also has a protective effect against bacterial infections and cerebral toxoplasmosis [37, 38] . daily administration is possibly more effective than three doses a week [39] . in cases of moderate cutaneous allergic reactions, desensitization is possible [40] . monthly pentamidine inhalations are a welltolerated alternative [41, 42] . a suitable inhalation system should be chosen and an inhalative ß-sympathomimetic should be administered beforehand. other options are dapsone [41, 42] and atovaquone, both of which have proved to be similarly effective as tmp/smx, dapsone, and pentamidine in two multi-center trials [43] [44] [45] . atovaquone, however, proved inferior to tmp/smx in another study [32] . pcp prophylaxis can be discontinued after successful immune reconstitution on art to c200 cd4 t-cells/ll for at least 3 months [46] [47] [48] [49] . only a few cases of reoccurring pcp have been reported for discontinuation at [200 cd4 t-cells/ll [50, 51] . if the hiv rna is well suppressed, [100 cd4 t-cells/ll may be sufficient to discontinue prophylaxis [52] . however, larger trials would be needed to submit a general recommendation regarding discontinuation for these patients. the recommendations concerning therapy and prophylaxis of pcp are summarized in table 1 . the incidence of cerebral toxoplasmosis has decreased to less than a quarter of that during the earlier years of the hiv epidemic, [53] . nevertheless, it remains the most important neurological oi in hiv-infected patients in europe [54] . cerebral toxoplasmosis almost always results from a reactivation of a latent infection with toxoplasma gondii. extracerebral manifestations are rare. standard therapy is a combination of pyrimethamine and sulfadiazine, which is effective in 75-89 % of cases [55, 56] . an equivalent alternative is pyrimethamine and clindamycin [55, 57] . tmp/smx is also possible, with the same doses as used in pcp [58, 59] . tmp/smx proved to be as effective as sulfadiazine/pyrimethamine in two rcts on ocular and cerebral toxoplasmosis [60, 61] . a cochrane review showed no superiority of any one specific regimen [62] . for pyrimethamine, a ''loading dose'' within the first few days has been used since the first studies [56] . however, the efficacy of this approach has not been proven. due to the myelotoxicity of pyrimethamine, it is important to add folinic acid (not folic acid) from the start [63] . other alternatives are atovaquone/pyrimethamine [64] or azithromycin/pyrimethamine [65] ; however, data are limited. acute therapy lasts for a period of at least 4 (to 6) weeks-longer for alternative therapies. in most cases, empiric treatment of toxoplasmosis is initiated upon identification by radiographic testing. any improvement or clinical deterioration should be evaluated clinically and by magnetic resonance imaging (mri) scanning during therapy (after 14 days). in the case of progression, an alternative diagnosis (i.e., cerebral lymphoma, tuberculoma) and a brain biopsy should be considered. maintenance therapy with a reduced dosage should be initiated when lesions have resolved at least by 50 %, the clinical course has improved, and contrast enhancement has been reduced or eliminated. art should be initiated as soon as possible. in cases of increased intracranial pressure or extensive edema, steroids can be given (dexamethasone, 3-4 9 4-8 mg/day). the choice for steroid therapy must be considered carefully as steroids distort possible differential diagnoses. for example, primary cerebral lymphomas also respond to steroids, and in the case of therapeutic failure, the validity of a potential biopsy can be reduced with steroids. antiepileptic therapy is indicated if epileptic attacks occur. due to rare interactions with art, gabapentin, pregabalin, and levetiracetam are applied. levetiracetam is also available as infusion. a distinction must be made between exposure prophylaxis, primary prophylaxis, and secondary prophylaxis after cerebral toxoplasmosis. • exposure prophylaxis: immunoglobulin g (igg)-negative patients should avoid eating raw or undercooked meat. an increased risk due to proximity to cats has not been proven [66] . stricter measures of hygiene should be followed. however, the importance of this recommendation under effective art is questionable. • primary prophylaxis: igg-positive patients with \100 cd4 t-cells/ll require primary prophylaxis. the drug regimen of choice is tmp/smx. in cases of allergy, desensitization may be considered [40] . see above for alternatives. primary prophylaxis can be discontinued if cd4 t-cell count is[200 cells/ll for at least 3 months. • secondary prophylaxis: in the absence of immune reconstitution, patients require lifelong secondary prophylaxis, usually consisting of half the dose needed for acute therapy [67] . clindamycin is presumably less suitable as secondary prophylaxis as it cannot cross the intact blood-brain barrier [63] . tmp/smx also seems less effective for secondary prophylaxis. however, it may be considered because it is simple. a higher dose than that for pcp is definitely required [68, 69] . prophylaxis may be discontinued safely if initial therapy has led to radiological resolution and if there is an immune reconstitution of [200 cd4 t-cells/ll for at least 3-6 months [31, [70] [71] [72] . the recommendations on therapy and prophylaxis of cerebral toxoplasmosis are summarized in table 2 . in germany, seroprevalence of cmv infection in the adult population is 50-70 %. the risk of a reactivation of cmv infection increases when the cd4 t-cell count is \100 cells/ll. in addition to cmv retinitis, impairment of other end-organs may occur. due to the limited data on cmv manifestations, the same systemic therapy is recommended in these latter cases as for cmv retinitis [73] . international guidelines are also available for this approach [3] . all patients with manifest cmv infection should start art immediately. the cmv-specific immune response is restored [74] , leading to a reduction of cmv viremia [75] and delaying progression of an existing cmv retinitis or its recurrence [76, 77] . in addition to art, a cmv-specific therapy should be initiated at the time of diagnosis. therapy of cmv retinitis can be performed locally or systemically. a local therapy alone does not provide protection against dissemination of infection in the contralateral eye or other organs, but it can be considered if systemic drug toxicity is high. for systemic therapy, four substances are available: gancyclovir, foscarnet, cidofovir, and valgancyclovir (valgcv). the reader is referred to the product information on these substances for the respective side effects. valgancyclovir is the only drug that can be administered orally. it is almost completely hydrolyzed to gancyclovir after resorption in the gastrointestinal tract [78, 79] . gancyclovir and foscarnet are both recommended as first choices for treating cmv retinitis even though foscarnet proved to be superior in pre-art times [80, 81] . the side effects of both drugs differ, but the response rates to therapy are similar with both substances [81] [82] [83] . as foscarnet must be administered via a central catheter, the administration of gancyclovir is easier and often preferred. valgancyclovir has proven to be effective in a comparative study and has the advantage of being less complicated to administer than intravenous infusion. intravenous treatment, however, may be necessary if foveal infections occur with acute risk of impairing visual acuity. in these cases, gancyclovir and foscarnet are equally recommended for first-line therapy. treatment with both agents consists of an induction therapy followed by life-long maintenance therapy. induction therapy usually lasts for at least 2-3 weeks until lesions resolve. without sufficient art, selection of resistant cmv mutations is frequent and accumulates as the infection progresses [84, 85] . several authors recommend valgcv for first-line therapy based on the results of a prospective randomized trial with valgcv and parenteral gancyclovir [86] and on those of studies on the pharmacokinetics of ganciclovir, with both showing similar results after the administration of valgcv [78, 79, 86] . other studies on the pharmacokinetics of gancyclovir following the administration of valgancyclovir either lack a comparison with parenteral gancyclovir [78] , or the administered doses were too low to show bioequivalence of valgcv and gancyclovir [79] . in summary, a clear recommendation in favor of valgcv cannot be given at the present time. in the presence of sight-threatening lesions, the panel strongly recommends against treatment with valgancyclovir due to the lack of clear evidence. some experts recommend a combination therapy of gancyclovir and foscarnet in full doses for acute sightthreatening lesions. maintenance therapy with valgcv can be initiated after lesions have completely resolved [87] ; however, this recommendation also lacks data. without sufficient art, a relapse is likely to occur, even under maintenance therapy with valgancyclovir. if lesions (zone ii and iii) are more anterior, therapy with valgcv may be attempted with weekly monitoring of the fundus. cidofovir has not been tested in controlled trials against gancyclovir or foscarnet. compared to a delayed therapy, cidofovir significantly slows down the progression of the infection [88] ; however, cidofovir is not recommended as first-line therapy due to its side effects. it does remain an important agent in the treatment of progredient cmv retinitis under gancyclovir or foscarnet therapy. sufficient art is crucial for a successful therapy of cmv retinitis. patients with progredient cmv retinitis on a gancyclovir regimen can be treated successfully with foscarnet or a combination of foscarnet and gancyclovir [89] . a good response is obtained in many cases with treatment with cidofovir, and this drug can therefore be an alternative. if foscarnet should fail, gancyclovir or a combination of gancyclovir and foscarnet can be effective. here too, therapy with cidofovir can prevent further progression. gancyclovir implants can still be effective after therapy failure under systemic gancyclovir or foscarnet due to the significantly higher intraocular gancyclovir concentration produced by the implants [90] . however, there is no protection against further spread of the infection to other organs or to the contralateral eye [91] [92] [93] . extraocular manifestations are always treated in the same way as a cmv retinitis, although only a few studies support this recommendation. in the presence of a cmv encephalitis or ventriculitis, clinical experience and smaller case studies indicate that a combination therapy with gancyclovir and foscarnet is superior to monotherapy [94] [95] [96] [97] [98] [99] . due to the toxicity of this therapy, the diagnosis should be confirmed. • primary prophylaxis: gancyclovir prophylaxis for cmv retinitis with a cd4 t-cell count of \50 cells/ll is effective, but this is usually too toxic. fundoscopy every 3 months is recommended but not necessary in the opinion of most experts (especially at a cd4 t-cell count of [100 cells/ll). • a dose-reduced secondary prophylaxis should be initiated, preferably with oral valgcv after about 3 weeks of acute therapy and after lesions have formed scars [87] . discontinuation of secondary prophylaxis to avoid side effects as soon as possible is recommended and feasible [77, 100, 101]-however, not before at least 6 months of maintenance therapy and immune reconstitution at a cd4 t-cell count of[100-150 cells/ ll. a small study showed that discontinuation after 18 months of art/maintenance therapy is already safe at a cd4 t-cell count of [75/ll [101] . in the first stage after discontinuation, patients undergo an ophthalmology control at least once a month. the required duration of a recurrence prophylaxis is not clear, nor is it as yet known for how long recurrences with other organ manifestations should be monitored. duration should therefore be handled as for cmv retinitis. the recommendations on therapy and prophylaxis of cmv manifestations are summarized in table 3 . from the roughly 200 candida species only about 15 different species are encountered in clinical daily practice. the most frequent species by far is c. albicans. clinical response to fluconazole of infections caused by c. albicans and candida parapsilosis is mostly good, whereas that to infections caused by c. glabrata or c. krusei is poor or totally missing. primary in vitro resistance of c. albicans to azoles is rare [102] . secondary resistance development under long-term azole therapy (fluconazole) was frequently observed in the pre-highly active art (haart) era. for the treatment of oral and vulvovaginal candidiasis, the reader is referred to the respective awmf guidelines [103, 104] . esophageal candidiasis (thrush) does not require an endoscopy to confirm the diagnosis in the presence of a typical clinical course and a mouth sore. the imidazole antimycotics, such as clotrimazole and the hydroxypyridone ciclopirox olamine, are suitable for local therapy of cutaneous candidiasis. if the immune status of the patient is good and/or in the case of a first episode of an oral candidiasis (oc), topical antimycotics, such as suspensions or pastilles (nystatin, amphotericin b, miconazole), are more inexpensive therapy options, although inferior to a therapy with fluconazole [105] [106] [107] . however, adherence is restricted with topically effective suspensions/pastilles. alternatives are mucoadhesive applications, although these are clearly more expensive. oral therapy with systemical azole derivatives (fluconazole, itraconazole, posaconazole, voriconazole) show a more rapid response, provide longer protection against recurrences, and are tolerated better by patients [108] [109] [110] [111] . fluconazole can be considered the drug of choice for oc and esophageal candidiasis. a once-daily oral therapy (100 mg for 5-14 days) has been established as the standard for oc [112] . single doses of up to 750 mg fluconazole have been tested in a small patient group (mostly without art) and was considered to be equivalent to a 14-day therapy. this therapy, however, should be confined to patients with compliance problems, as data on late relapses are limited [113, 114] . esophageal candidiasis is usually treated for 10-14 days with doses of 200-400 mg fluconazole qd. patients presenting with severe dysphagia can initially be treated intravenously and switched to oral application as symptoms improve. if fluconazole resistance has been detected, treatment with other azole derivatives is usually still effective and should be attempted before parenteral therapy is initiated (e.g., with echinocandin). traconazole, voriconazole, and posaconazole have demonstrated clinical efficacy for cases of fluconazole refractory oropharyngeal and esophageal candidiasis [115] [116] [117] [118] . all azole derivatives require a double dose on the first day of the regimen (loading dose). therapy with a higher dose of fluconazole (b800 mg/day & 12 mg/kg/day) or an antimycotic combination therapy [119] can be considered, but data are insufficient. therapy failure and/or fast relapses occur most frequently in patients with poor immune status (\100 cd4 t-cells/ll). data from randomized studies have shown that echinocandins (caspofungin, micafungin or anidulafungin) are as effective and well tolerated as fluconazole for the treatment of candida esophagitis [120] [121] [122] . however, application should be restricted to azole refractory infections with clear fluconazole resistance [120, 123, 124] . art should be initiated immediately if chronic recurring oropharyngeal/esophageal candidiasis is present and at the latest if resistance problems occur. azole refractory candidiasis as well as azole-resistant strains can disappear with sufficient immune reconstitution as a consequence of art [125, 126] . regular change of toothbrush and thorough cleaning of dentures are a basic recurrence prophylaxis for oc. oc in hiv-infected children and adults can be treated and relapses prevented by applying disinfecting mouth rinses containing chlorhexidine 0.12 % 1-29 daily for a 90-day period [127, 128] . in the pre-haart era, secondary prophylaxis or life-long therapy with fluconazole led to significant reductions of chronic recurring oropharyngeal candidiasis-but it has also led to the development of secondary resistance [129, 130] . in a randomized study comparing secondary prophylaxis after oc with intermittent therapy on oc recurrence, relapses and infections of systemic candidiasis were reduced by the long-term prophylaxis. however, no survival benefit has been demonstrated for any candidiasis prophylaxis [131] . primary prophylaxis is not recommended, and indications for secondary prophylaxis should be restricted to individual case. the recommendations on therapy and prophylaxis of candidiasis are summarized in table 4 . herpes simplex virus (hsv) infections are frequent in hiv-infected patients. chronic and atypical courses are possible especially in the setting of severe immune deficiency (\100 cd4 t-cells/ll). organs such as the esophagus, central nervous system (cns), eyes, and respiratory tract may also be affected. in these cases and with persistence of lesions for a period of[4 weeks, hsv infection is an aids-defining illness. topical treatment with acyclovir is adequate for patients with a good immune status and only discrete oral lesions. pencyclovir cream is probably as effective [132] . genital herpes lesions do not respond as well to topical treatment. for systemic treatment against hsv-1 and hsv-2, the drug of choice is still acyclovir. resistance is rare [133] , and healing of lesions can be accelerated by the therapy [134] . severe cases with organ involvement should be treated intravenously. as hsv levels are lower in the cns than in plasma, the dose to treat encephalitis should be increased. valacyclovir (valacv) and famcyclovir are equally effective alternatives to acyclovir [135, 136] . however, they are not approved for patients with immune deficiency and should only be applied if response to acyclovir fails [137] . for uncomplicated genital herpes lesions, shorter regimens of 2 days of 500 mg famcyclovir may be as effective, provided there is no immune deficiency [138] . according to the opinion of some experts, brivudine is an alternative for hsv-1 and vzv, although contraindicated for immunosuppressed patients and only approved for the treatment of vzv. however, results from controlled studies with hiv-infected patients are not available. in cases of painful mucocutaneous lesions, a local anesthetic can also be applied. treatment with foscarnet for several weeks may be helpful in exceptional cases, especially if lesions remain refractory to standard treatment [139] . primary prophylaxis is not recommended. an earlier meta-analysis in which acyclovir was found to reduce the risk of both hsv and vzv disease by more than 70 % [140] must be viewed in the context of art today. nevertheless, long-term treatment with low-dose acyclovir or valacv can still be effective treatments for recurrent hsv [141, 142] . the risk of hiv transmission, which is increased threefold by genital hsv-infection [143] , is not reduced by treatment with acyclovir [144] [145] [146] . between 70 and 90 % of patients with symptomatic hsv-2 infection and at least 20-50 % of patients with symptomatic hsv-1 infection experience recurring episodes within the first year. possible causes are local trauma, uv exposure, fever, and immune suppression. a long-term prophylaxis for at least 6 months is recommended for frequent recurrences. this prophylaxis can prevent further episodes in 70-80 % of cases. the recommendations on therapy and prophylaxis of genital hsv infections are summarized in table 5 . patients infected with hiv are at increased risk for vzv infection. multisegmental zoster or zoster generalisatus are often observed with low cd4 t-cell counts. chronic courses with ulcerating forms and involvement of other organs are rare. pneumonia or cns involvement should be considered. a monosegmental zoster can be treated with oral acyclovir. famcyclovir and valacv are alternatives. each complicated, multisegmental or facial zoster should be treated intravenously for 10-14 days. after clinical improvement is evident, a switch to oral therapy is possible. zoster neuralgia occurs less frequently in hiv-negative patients treated with the alternative drugs valacv, famcyclovir, and brivudine than when treated with acyclovir table 5 therapy and prophylaxis of genital herpes simplex virus infections a therapy/prophylaxis drug therapeutic regimen acute therapy (duration: 7-10 days), daily doses first choice acyclovir (3-) 5 9 400 mg p.o. severe cases 3 9 5-10 mg/kg i.v. 5 9 800 mg p.o. alternatives valacyclovir 2-3 9 1,000 mg or 3 9 500-1,000 mg (expert opinion) therapy for recurrent herpes simplex infection virus episodes acyclovir 3 9 400 mg p.o. for 5-10 days valacyclovir 2 9 1,000 mg for 5-10 days famciclovir 2 9 500 mg for 5-10 days long-term prophylaxis (duration: at least 90 days) acyclovir 2-3 9 400-800 mg valacyclovir 2 9 500 mg famciclovir 2 9 500 mg a daily doses [147] . however, according to a cochrane analysis, the results of this study are not clear [148] . brivudine is not licensed for the treatment of immunocompromised patients. acyclovir resistance is rare and most frequently observed under long-term therapy [149, 150] ; in these cases, foscarnet (3 9 40 mg/kg) or cidofovir (5 mg/kg, maximum 375 mg 19/week) can be given. early concomitant and monitored pain management with nsaids and/or other opiates in combination with amitriptyline and/or pregabalin is important. for further information on zoster pain, the reader is referred to the awmf guidelines. varicella vaccination seems to be fairly safe and effective for patients with a cd4 t-cell count of [400/ll [149] . vaccination should be considered if vzv serology is negative. in individuals with negative serology and exposure to vzv, administration of hyperimmunoglobulin may be attempted. long-term primary prophylaxis is usually not effective; however, a long-term low-dose therapy can be considered in the presence of persistent recurring episodes. the recommendations on therapy and prophylaxis of vzv-infections are summarized in table 6 . progressive multifocal leukoencephalopathy (pml) is a severe demyelinating disease of the cns caused by the john cunningham virus (jcv). prognosis for pml was poor in the pre-haart era, with the median interval between the onset of the first symptoms and death being 3-6 months. with effective art, there are significantly fewer cases of disease progression, and even complete remission seems possible [151] . nevertheless, mortality of patients with pml remains high at 50 %, albeit art [152] . there is no specific pml treatment with proven efficacy; consequently, the mainstay of therapy is immune reconstitution. as such, priority remains on the initiation and optimization of an art. treating physicians are recommended to apply intracerebral penetrating agents. a successful immune reconstitution accounts for a significant reduction in mortality [151] [152] [153] [154] [155] [156] . after initiation of art, a paradoxical worsening of clinical symptoms in terms of an immune reconstitution inflammatory syndrome (iris) has been observed in approximately 16-23 % of pml cases. administration of corticosteroids for pml-iris has only been described in case studies [157] , and evidence of a benefit was not provided. given the slight difference in the 1-year survival rate for pml patients and pml-iris patients [158] , the use of corticosteroids or a temporary discontinuation of art must be weighed up against the risks of a possible decline of the jcv-specific immune response. several supportive immunomodulatory approaches have been tested, but to date there is no convincing evidence for the efficacy of treatments with immunoglobulin, interleukin-2 (il-2), or il-a [159] . therapeutic regimens aimed at inhibiting jcv replication have also been attempted, but as yet relevant evidence supporting the clinical use of drugs such as cytosine arabinoside is not available [153, 160] . antiviral treatment with acyclovir, cidofovir, ganciclovir, brivudin, ribavirin, foscarnet and the combination therapy foscarnet and zidovudine have also proven to be ineffective [161] . recently, 5-ht2a inhibitors and/or serotonin receptor antagonists have been discussed for pml treatment. in vitro data for the suppression of jcv replicates via 5ht(2a)r inhibitors are contradictory [162] [163] [164] [165] [166] [167] . results from controlled clinical studies are missing. based on promising in vitro data [168] a phase i/ii study of mefloquine was initiated-only be stopped due to a lack of efficacy. in summary, specific treatments for pml cannot be recommended outside clinical trials. there is no prophylaxis. exposure prophylaxis is also not possible. the recommendations for therapy and prophylaxis of pml are summarized in table 7 . cryptosporidiosis is a parasitic intestinal disease with fecal-oral transmission, mainly caused cryptosporidium parvum (two other frequent types: c. hominis and c. [169] . infection of the biliary tract leading to sclerosing cholangitis is frequent, particularly among patients with severe immunodeficiency, but may be reversible with immune reconstitution [170] [171] [172] (level of evidence c). other rare manifestations are infections of the pancreatic duct and pulmonary infections [173, 174] . successful immune reconstitution under art can lead to complete resolution of clinical crytosporidiosis [175, 176] . symptomatic treatment with loperamide and/or tincture of opium should be given. octreotide (off label) can also be applied. sufficient hydration is important and infusions may even be required. no specific treatment has been validated [177] . rifaximin is promising, as first studies with aids patients show [178] ; however, results from randomized studies are still missing. nitazoxanide was found to be effective in a small randomized study in immunocompetent patients [179] . however, this drug is not approved for aids patients and showed no effects in a double-blind randomized study in hiv-infected children with cryptosporidiosis [180] . paromomycin has been found to have favorable effects on diarrhea [181] . however, a double-blind randomized study showed no benefit compared to placebo [182] . in a cochrane review for the prevention and treatment of cryptosporidiosis, paromomycin did not reduce diarrheal frequency permanently [177] . there is no generally accepted prophylaxis, although a protective effect of rifabutin and clarithromycin has been reported from retrospective studies. azithromycin showed no effect [183] . the usual hygienic measures (gloves) are usually adequate. patients do not need to be isolated. however, accommodation with other immunosuppressed patients should be avoided. the recommendations on therapy and prophylaxis of cryptosporidiosis are summarized in table 8 . for further information, refer to guidelines by the cdc for cryptosporidiosis in hiv-infected patients (cdc 2009; http://www.cdc.gov/mmwr) [3] . cryptococcosis occurs much more frequently in africa, the usa, and southeast asia than in europe. bird droppings (especially of pigeons) are presumably a key reservoir, but a direct transmission between humans has not been observed. although transmission occurs via inhalation, pulmonary symptoms or lung infiltration are only seen in 30-40 % of cases of hiv-infected patients. cryptococcosis infection is often followed by disseminated disease in hiv patients with severe immunodeficiency (\100 cd4 t-cells/ ll) and often involves the cns ([75 %, meningitis) [21] . recommended treatment for a cryptococcal meningitis is the combination regimen of amphotericin b deoxycholate (amb-d; 0.7-1.0 mg/kg/day i.v.) and flucytosine (100 mg/kg/day i.v. or p.o. if available), divided into four doses a day. acute therapy should be given for at least 14 days. if clinical response is good, a switch to monotherapy with fluconazole (400 mg/day) for another 8 weeks is possible [21] . liposomal amphotericin is slightly more effective than conventional amb-d and provides an alternative, if amb-d is not well tolerated [184] . monotherapy with fluconazole as initial treatment in hiv-infected patients is not sufficient, even with higher daily doses of 800-2,000 mg. thus, it is only considered as an option in countries with limited resources [21, 185] . in the pre-haart era, a triple combination therapy with amb-d, flucytosine, and fluconazole was favored for the treatment of cryptococcal meningitis in germany [186] . however, in one randomized study, the triple combination was not more effective than a combination with amb-d and flucytosine or amb-d and fluconazole or a monotherapy with amb-d [187] . the combination with amb-d and fluconazole is an alternative in regions with limited resources where flucytosine is not available. in a small study in thailand, a higher dose of fluconazole (800 mg/day) combined with amb-d (0.7 mg/kg/day) was more effective than monotherapy with amb-d alone or a regimen of amb-d ? fluconazole (400 mg/day). other combination therapies (e.g. fluconazole ? flucytosine) are possible alternatives, but lack sufficient data [188] . itraconazole plays no role in primary therapy and is less effective than fluconazole in maintenance therapy [189] . monotherapy with posaconazole showed a response rate of up to 50 % in a small case study on refractory diseases and therefore provides an alternative for this indication [190] . efficacy of voriconazole in salvage therapy is still not clear [191] . echinocandines show no in vitro effect against c. neoformans. in the case of an iris when art is initiated during antimycotic treatment, additional treatment with corticosteroids (0.5-1.0 mg/kg/day prednisolone equivalent) is required [21] . in refractory treatment situations, additional administration of c-interferon might be useful in individual cases [192] . treatment success is monitored based on the clinical course and repeated lumbal punctures. patients should have their intracranial pressure measured at time of diagnosis. if the intracranial pressure is very high, several punctures should be made in the first week until it is reduced to b20 cm. in individual cases, cerebrospinal fluid (csf) drainage can be considered to reduce the intracranial pressure if there are no contraindications [21] . for mild, isolated cryptococcal pneumonia (negative csf diagnosis), monotherapy with fluconazole (400 mg/day) is possible. treatment should continue for 6-12 months. severe cases of pneumonia with or without acute respiratory distress syndrome (ards) should be treated the same way as meningitis (see above). art-naïve patients at the time of diagnosis should start an art after a 2wo-week induction therapy with antimycotics. however, an optimal time for initiation of art is not yet clearly defined. primary prophylaxis can not be recommended to hivinfected patients in germany due to lack of a clear survival benefit [193] . after acute therapy of cryptococcal meningitis, secondary prophylaxis should be introduced. fluconazole (200 mg/day) is the regimen of choice and is also more effective than itraconazole [21] . secondary prophylaxis can be discontinued after at least 6 months maintenance therapy with sufficient immune reconstitution ([100 cd4 t-cells/ll and hiv-rna below detection limit for over 6 months). the risk of a relapse is high if maintenance therapy is discontinued too early [194] . the recommendations on therapy and prophylaxis of cryptococcosis are summarized in table 9 . human immunodeficiency virus-associated infections of nontuberculous mycobacteria (ntm) have declined in countries where art is available [195] [196] [197] . in addition to disseminated ntm diseases, which develop almost exclusively in the setting of severe cd4 t-cell depletion (\50 cd4 t-cells/ll) and which are mainly ([90 %) caused by the mycobacterium avium complex or m. intracellulare (mycobacterium avium intracellure complex, mai), incidences of ntm-iris as well as pulmonary ntm diseases are also observed. pulmonary ntm is frequently caused by other species, such as m. kansasii, m. xenopi, m. malmoense, and m. abscessus. for further information on diagnosis, the reader is referred to the american thoracic society criteria [198] . due to the ubiquitous occurrence of ntm, pre-exposure prophylaxis is not possible and an isolation of infected patients is not necessary. some specialists, however, recommend a screening of generalized mai infections in patients with cd4 t-cell counts of\50/ll prior to initiation of an art. given here are only recommendations for the mai therapy. with respect to ntm species other than mai, the reader is referred to the appropriate literature [198] or advised to consult experts (ntm-net). a combination treatment of macrolide (clarithromycin or azithromycin) and ethambutol plus/minus rifabutin is recommended [198] . rifabutin is preferred to rifampicin due to its in vitro efficacy against mai and its lower interaction potential. following the publication of data showing that rifabutin could be omitted from the treatment regimen [199] , another randomized study demonstrated a survival benefit with the triple combination clarithromycin, rifabutin, and ethambutol compared to clarithromycin with either ethambutol or rifabutin-the mortality rate was halved in the treatment arm receiving this triple (clarithromycin-containing) combination [200] . the doses for rifabutin must occasionally be adjusted to the art regimen [201] . clarithromycin increases the rifabutin serum level, while rifabutin decreases the clarithromycin level. treatment duration with rifabutin has not yet been determined in studies; however, experts recommend discontinuing rifabutin after a few weeks and with clinical improvement. the daily doses for clarithromycin should not exceed 2 9 500 mg, as a higher mortality risk has been described for patients receiving higher dosages [202, 203] . azithromycin can be administered instead of clarithromycin, as these two drug are comparably effective in combination with ethambutol, with slightly more rapid sterilization of blood cultures with clarithromycin [196, 199, 204] . as macrolides are the cornerstone of therapy, the development of resitance to macrolides must be avoided, and monotherapy with macrolides should not be administered. in the case of intolerance, alternative substances, such as fluoroquinolone, amikacin, cycloserine, dapsone, linezolid, or mefloquine, are available. however, clinical evidence for the treatment of mai infections with these alternative substances is still insufficient. in the case of ntm-iris, the extent and duration of an antimycobacterial therapy are not clear. it is possible that partial virus suppression is enough for a ntm-specific immune reconstitution [205] . it is easier to evaluate the clinical response to localized ntm infections. in cases of localized lymphadenitis and skin manifestations, therapy duration of 6 months is recommended after patients are culture-negative. if the clinical response is good and cd4 t-cells continue to increase under a still effective art, the regimen can be reduced after 3 months to a recurrence prophylaxis with a macrolide for a further 3 months. patients with abdominal localization have a poorer response and require a more aggressive and longer therapy [206, 207] . additive corticoid therapy has symptomatic indications. the treatment of patients with pulmonal ntm diseases not deriving from an iris are based on the guidelines for non-hiv-infected patients [198] . in the usa, placebo controlled trials for clarithromycin, azithromycin and rifabutin showed that primary prophylaxis significantly reduced mai-morbidity and -mortality in severely immunocompromised patients [208] [209] [210] [211] . all these studies, however, were undertaken in the pre-ha-art era. in addition, mai-infections are less frequent in europe, so that only a few patients receive primary prophylaxis [212] . due to the declining incidences since the introduction of art, primary prophylaxis can only avoid a small number of diseases [197] . ntm-associated iris can also not be prevented by prophylactic drugs [206] . therefore primary prophylaxis is not recommended in germany. after treatment of a disseminated mai-infection, patients lacking other art options, should receive secondary prophylaxis with a macrolide, provided cd4 t-cell count is under 50 cells/ll. weekly doses of azithromycin are convenient and efficacy is comparable to daily rifabutin [208] . secondary prophylaxis or maintenance therapy can be discontinued under an art and if patients are without symptoms and cd4 t-cell count is [100/ll for 6 months. the recommendations concerning therapy and prophylaxis of disseminated mai-diseases are summarized in table 10 . globally, tb is the most prevalent hiv-associated opportunistic infection. in germany, tb is rare. hiv-infected patients are affected by tb independent of their cd4 t-cell count [213] , although incidences increase with advanced immunodeficiency [214] . uncomplicated cases of tb can successfully be treated with a standard therapy regimen over a period of 6 months, regardless of hiv status. first-line drugs are rifampicin, inh, ethambutol, pyrazinamide, and streptomycin, with inh and rifampicin being the most effective. tb should always be treated with a combination of four drugs in the initial phase to prevent drug resistance. standard initial phase therapy is a 2-month course of rifampicin, inh, ethambutol, and pyrazinamide, followed by a continuation phase therapy of 4 months rifampicin and inh. in individual cases, such as incompliance, it may be necessary to extend the standard treatment duration to c9 months, especially if sputum cultures are still positive after 2 months. recurrences after successful therapy appear more frequently in hiv-infected patients [215] . if standard therapy has not been initially applied, treatment should always last for at least 9 months. alternatively, ethambutol, streptomycin, and reserve drugs such as ofloxacin or moxifloxazin, cycloserine, and linezolid may be administered. since this treatment is no different from that for multiresistant tb, these patients should be treated in specialized centers. adverse effects occur frequently with anti-tb therapy (refer to individual drug information for side effects, necessary testing, and drug interactions). severe side effects are observed more often in hiv-infected patients than in hiv-negative patients [216] . antiretroviral therapy significantly reduces the morbidity and mortality rate in hiv-infected patients [217] . a 6-month tb standard therapy achieves similar success in both hiv-infected and hiv-negative patients [218] . although a large retrospective and a large open-label, randomized trial showed a survival benefit with simultaneous art and anti-tb treatment, this approach proves to be difficult in practice due to overlapping drug interactions and side effects [219] . for tb meningitis, side effects are more frequent during the first 2 months of therapy if art and anti-tb therapy are initiated simultaneously. in this case, a delay of art by 2 months is possible without risking a higher mortality [220] . with regard to other forms of tb, 25-60 % of patients develop an iris in the first 3 months of art treatment [221] . a consensus on a uniform case definition of tb-iris was reached in 2008 [9] , which we refer to in the chapter on iris of this guideline. adherence to simultaneous hiv and mycobacterium tb treatment is difficult to achieve due to the high pill burden and overlapping toxicities. both rifampicin and protease inhibitors (pis) are metabolized by cytochrome p450 3a. concomitant therapy is therefore not recommended [222, 223] (table 11 ). rifabutin can be combined with boosted unboosted protease inhibitors are no longer recommended due to insufficient plasma levels. consider tdm pis, however the dose must be adjusted (table 12 ). it may be useful to determine serum levels [201] ; however, this approach has not been tested in clinical research with clear endpoints. recommendations can be given for first-line art therapy with tenofovir (tdf), tdf ? emtricitabine (ftc), and ftc plus efavirenz in combination with rifampicinbased tb therapy. alternatives are other efavirenz-based regimens (without adjustment of dose) with rifabutin [222] . to date, clinical data on combinations of rifamycin with new drugs, such as darunavir, raltegravir, or maraviroc, are limited. due to the strong inducing potential of cytochrome p450 3a, pis should be avoided and maraviroc should only be given under close observation. rifampicin also induces the enzyme ugt1a1, leading to increased glucoronidation and reduced plasma levels of raltegravir [224] . no interactions have been reported with tenofovir and t-20 [225] . the recommendations on the adjustment for combination of art/rifampicin in tb therapy are summarized in table 12 . treatment of active tb has clinical priority over art. in patients with \100 cd4 t-cells/ll, simultaneous treatment of both infections is indicated [222, 226] . however, even in this situation it is recommended to start tb therapy first for 2 weeks before initiating art to prevent possible side effects. for patients with 100-350 cd4 t-cells/ll, art can be delayed for 2 months until the anti-tb drugs can be reduced for the continuation phase. there is no evidence for an optimal timing of art when the cd4 t-cell count is [350 cells/ll [222] . the results of a large randomized trial indicate that mortality rate in patients with 200-500 cd4 t-cells/ll is reduced when art is initiated during tb therapy [219] . for hiv patients with\50 cd4 t-cells/ll, the results of a recent study show a benefit of a delayed art. the decision should be made carefully under consideration of the situation of each single patient [17, 227] . hiv-infected patients already on a successful art should remain on art, although the regimen may need to be modified [226] . the recommendations for co-administering art with rifabutin are summarized in the statement that adherence is the most important factor for therapeutic success and to avoid resistant tb strains. the world health organization (who) recommends a directly observed therapy for these patients. latent tuberculosis infection (ltbi) is defined by a positive mycobacterium tb-specific immune response in the tuberculin skin test (tst) or an interferon gamma release assay (igra) in the absence of active tb. clear values for a positive mycobacterium tb-specific immune response in hiv-infected patients do not exist. patients are not infectious as the tb is not active. however, hiv-infected patients with ltbi carry a higher risk of developing active tb. according to guidelines for the treatment of ltbi by the cdc [228] , hivinfected patients with a tst of [5 mm should be given treatment with inh for 9 months. this probably also applies to patients with positive igra test results, but convincing data are still missing [229] . alternatively, a 4-month course of rifampicin can be given. a 2-month course of rifampicin and pyrazinamide is no longer recommended, as it has been associated with significantly higher toxicities in hiv-negative patients [230, 231] . in 2006, 2.2 % of all tb patients showed multidrug resistance (at least resistance against inh and rifampicin). among these, 5 % were hiv-infected [232] . in addition to incidences of multidrug resistance (mdr), incidences of extensive drug resistance (xdr) were reported in at least 58 countries in 2010 [233] . xdr tb is defined by the who as tb which is additionally resistant to fluoroquinolones and at least one of the injectable drugs amikacin, capreomycin, or kanamycin. due to the complex therapy and an overall poor prognosis, patients with mdr/xdrtb should be treated in specialized centers. 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2015-03-20 journal: bone marrow transplant doi: 10.1038/bmt.2015.27 sha: doc_id: 313474 cord_uid: 1gux1gsi nan introduction: the incidence of cgvhd after allogeneic sct is higher when peripheral blood stem cells are used as stem cell source. there is a strong need for preventing cgvhd after asct without increasing the risk of relapse. materials (or patients) and methods: we performed a multicenter, multinational, open-label, randomized study comparing anti-lymphocyte globulin (atg-fresenius s ) 10 mg/kg on day -3, -2 and -1 with no atg in patients with aml (n ¼ 110) or all (n ¼ 45) in 1 st complete remission (cr; n ¼ 139) or 2 nd cr (n ¼ 16) who received peripheral blood stem cells from their hla-identical sibling after standard tbi (12 gy)/ccclophosphamide (120 mg/kg) or busulfan (16 mg/ kg)/cy (120 mg/kg) based myeloablative conditioning regimen. standard gvhd prophylaxis consisted of cyclosporine a and a short course of mtx (10 mg/m 2 on day þ 1, þ 3, þ 6 and þ 11). the primary study aim was to compare the cumulative incidence of cgvhd at 2 years after asct. results: out of 161 randomized patients from 27 centers and 4 nations 6 were withdrawn before conditioning and asct due to leukemia progression, or cancellation of the donor. 155 patients were analyzed for safety and efficacy; 83 were randomized to atg and 72 to non-atg. the treatment groups were comparable regarding recipient and donor age and sex, cmv serostatus, disease (aml vs all), 1 st or 2 nd cr. the median time to leukocyte (41.0x10e9/l) and platelet (4 20x 10e9/l) engraftment was significantly delayed in the atg group (18 vs. 15 days, po 0.001 and 20 vs 13 days, po0.001). the incidence of acute gvhd grade i-iv was 25% for the atg arm and 35% for the non-atg arm (p ¼ 0.32 ) and for severe grade iii/iv acute gvhd 2% and 8%, respectively (p ¼ 0.2). the cumulative incidence of cgvhd at 2 years was 32% (95% ci 22-47%) in the atg and 69% (95% ci 51-74%) in the non-atg arm (po0.0001). regarding limited and extensive cgvhd the ci at 2 years was 26% vs 53% (p ¼ 0.002) and 19% vs 53% (po0.001), respectively. there was no difference in infectious complications, cmv and ebv reactivation between both arms. the cumulative incidence of therapy related mortality at 2 years was 14% (95% ci 8-24%) for the atg arm and 12% (95% ci 6-22%) for the non-atg arm (p ¼ 0.60), resulting in 2 year relapse-free and overall survival of 59% (95%ci 48-69%) and 74% (95% ci 63-82%) for the atg group and of 65% (95% ci 51-75%) and 78% (95% ci 66-86%) for the non-atg group (p ¼ 0.46 and p ¼ 0.21, respectively). chronic gvhd free survival at 2 years was 50% for the atg and 23% for the non atg arm (po 0.001). a composite endpoint cgvhd and relapse-free survival at 2 years was in favor for the atg treated patients (30% vs. 17%, p ¼ 0.005). conclusion: this is the first randomized cgvhd prevention study providing evidence that atg-fresenius s (3x 10 mg/kg) is highly effective in preventing limited and extensive cgvhd in hla-identical sibling pbsc transplantation when used within a myeloablative preparative conditioning regimen. the use of atg-fresenius did not result in an obvious increase of infectious complications and relapse, resulting in similar overall survival rates, but improved cgvhd/relapse free survival. introduction: the establishment of large transplant registries and introduction of novel statistical techniques have paved the way for large scale data analysis. nevertheless, contemporary tools for risk prediction of transplant related mortality (trm) following allogeneic (allo) hematopoietic stem cell transplantation (hct) are of limited clinical use, owing to a sub-optimal predictive accuracy. apart from inherent procedural uncertainty, methodological factors impeding prediction might be attributed to the statistical methodology used, number and quality of features collected, or simply the population size. using an in-silico approach (i.e. iterative computerized simulations), based on machine learning (ml) algorithms, we aimed to define prediction limiting factors of day 100 trm and rank variable contribution. ml is a field of artificial intelligence dealing with the construction and study of systems that can learn from data, rather than follow explicitly programmed instructions. commonly applied in complex data scenarios, such as financial settings, it may be suitable for outcome prediction of hct. materials (or patients) and methods: study population was a cohort of 28,236 adult acute leukemia allo-hct recipients from the ebmt-alwp. twenty four variables were analyzed, including recipient, donor and transplant characteristics. study design involved two phases. the first, focused on development and comparison of several ml based prediction models of day 100 trm. in the second phase, a repetitive computerized simulation was applied. factors necessary for optimal prediction were explored: algorithm type, size of data set, number of included variables, and performance in specific subpopulations. models were assessed and compared on the basis of the area under the receiver operating characteristic curve (auc). results: six ml based prediction models for day 100 trm were developed on the entire dataset. optimal aucs ranged from 0.65-0.68 . depending on the algorithm used for prediction model development, the in-silico experimental system yielded the following results: predictive performance plateaued on a population size ranging from n ¼ 5647-8471; a range of 6-12 ranked variables, selected by a separate feature selection algorithm, were necessary for optimal prediction; disease status and donor type were consistently top ranking variables. predictive performance of models developed for specific subpopulations, ranged from 0.59 to 0.67 for patients in second complete remission and patients receiving reduced intensity conditioning respectively. conclusion: we present a novel experimental system for assessment of prediction boundaries in hct. the present approach has clinical implications. we show that predictive performance of day 100 trm is unlikely to improve with the data routinely gathered on hct recipients, as different algorithms reach approximately the same performance. in addition, an exhaustive search for variable importance, reveal that few variables "carry the weight" with regard to predictive influence. predictive performance converged when sampling more than 5000 patients, reflecting the importance of large registry studies. overall, it seems we have reached a point of predictive saturation. improving predictive performance will likely require additional types of input like genetic, biologic and procedural factors. disclosure of interest: none declared. introduction: allogeneic stem cell transplantation (sct) can provide long-term disease control in selected patients with relapsed refractory nhl. restricted availability of a matched sibling donor limits its use especially for patients with rapidly progressing disease in whom unrelated donor search cannot be awaited. because data of alternative donor transplants in nhl is sparse, we aimed to compare outcome of haploidentical and cord blood transplants with conventional relatedand matched unrelated donor transplats for nhl. materials (or patients) and methods: information of patients with mantle cell lymphoma (mcl), dlbcl, t-cell lymphoma (tcl) and follicular lymphoma (fl) who received an sct from a sibling donor (sib), 10/10 matched unrelated donor (mud), haplo-identical donor (haplo) or cord blood (cord) between 2007 and 2012 was downloaded from the ebmt database. results: 2798 patients with nhl were identified in the ebmt database meeting the inclusion criteria. 2065 received a transplant from a sib, 447 from a mud, 167 from cord (18 mcl, 36 dlbcl, 43 fl, 70 tcl) and 119 from a haplo donor (16 mcl, 30 dlbcl, 22 fl, 51 tcl) . active disease (po0.01) and karnofsky index (ki) below 80% was also more common in the haplo group (p ¼ 0.02). other variables were balanced. median follow-up after sct for all patients was 27 months (ci 25 to 29). relapse incidence after conventional transplants (sib, mud) and alternative donor transplants (haplo, cord) was not significantly different within the whole group (haplo: hr 1.2 95% ci 0.9-1.8 p ¼ 0.23; cord: hr 1.1 95% ci 0.7-1.4, p ¼ 0.74 ) and across all studied disease entities. in contrast, nrm incidence was not significantly different between sib and mud, but significantly higher with alternative donor transplants (haplo: hr 1.8 95% ci po0.001 ; cord: hr 1.9 95% ci po0.001) . with the exception of fl where mud in addition to haplo and cord transplants had a significantly higher nrm incidence than sib transplants. because patients who received a haplo transplant had more commonly active disease at transplant and worse ki, we performed multivariate modeling of relapse-and nrm incidences adjusting for the above mentioned covariates. no different relapse incidences between donor groups was observed. nrm incidence in contrast, was significantly higher in mud (reference sib, hr , p ¼ 0.033) and cord (reference sib, hr , p ¼ 0.001) but not in haplo transplants. most interestingly, acute gvhd incidence was significantly increased in mud compared to sib (p ¼ 0.003) transplants but not in haplo (p ¼ 0.08) or cord (p ¼ 0.34) transplants. multivariate adjustment for diagnosis (mcl, dlbcl, fl, tcl), remission prior to sct, ki (kio90% vs. 490%) and conditioning intensity (ric vs. mac) did not reveal worse os for haplo but a worse os for cord (hr po0.0003 introduction: retrospective studies in mds/saml suggest that reducing the intensity of the conditioning regimen prior to allogeneic stem cell transplantation reduces the risk of nonrelapse mortality but is associated with a higher risk of relapse, but prospective randomized studies for mds are lacking so far. materials (or patients) and methods: within the chronic malignancies working party (cmwp) of ebmt, we performed a prospective randomized trial comparing a busulfan based (busulfan 8 mg/kg orally or equivalent dosis intravenously (iv) plus fludarabin 180 mg/m 2 ) reduced intensity conditioning regimen (ric) and a standard myeloablative busulfan (busulfan 16 mg/kg orally or equivalent dosis iv plus cyclophosphamide 120 mg/kg) based regimen (mac) in patients with mds or saml (o20% blasts). between may 2004 and december 2012, 129 patients were included from 18 centers and 7 nations and 127 could be analysed. major inclusion criteria were: mds (according to fab: ra, rars, raeb, raeb-t), cmml, and saml, blasts less than 20%, matched related or unrelated donor (hla 8/8, 1 mismatch was allowed), age 18 -60 years (for unrelated) and 18 -65 years (for hla-identical sibling). included patients were stratified according related vs unrelated donor and blast countoor4than 5%. the primary endpoint of the study was 1 year non-relapse mortality.the median age of the patients was 51.4 years (r.19-64y) . donors were hla-identical sibling (n ¼ 33), matched unrelated (n ¼ 74) and mismatched related or unrelated (n ¼ 20). the patients were well distributed in both arms regarding age, gender, ipss risk profile, number of blasts at transplantation, donor source and mismatch donor. results: graft failure occurred in 1 after mac and 2 after ric. median time to leukocyte (4 1.0 x 10e9/l) occurred after 14days after mac and 15days after ric and and platelet (420x10e9/l) engraftment occurred after 14 days after mac and 15 days after ric, respectively. acute gvhd ii-iv was noted in 28% after ric and 31% after mac. chronic gvhd was seen in 64% after ric and 63% after mac. the cumulative incidence (ci) of non-relapse mortality (nrm) after 1 year was 17% (95% including 12 pediatric/adolescent cases o18 years), 74 with myeloablative conditioning (67%; 34 tbi and 40 non-tbibased), 64 from hla-identical siblings (58%), and 9 using ex vivo t-cell depletion. in addition to the above matching criteria, cases and controls were also balanced for other factors such as donor gender and gender mismatch, cmv serostatus, in vivo t-cell depletion and karnofsky's performance status. compared to hiv-neg, hiv-pts had lower rates of neutrophil engraftment (92.6% vs 97.5%, p ¼ 0.03), higher incidence of grade iii-iv acute gvhd (21% vs 13%, p ¼ 0.05), higher nrm at day 100 (17% vs 11%, p ¼ 0.04) and 2 years (33% vs 23%, p ¼ 0.04), and similar incidence of relapse (29% vs 25%, p ¼ 0.96). overall, hiv-pts had poorer pfs (39% vs 52%; p ¼ 0.03; hr 1.42 [1.04-1.94] ) and os (47% vs 59%; p ¼ 0.004; hr 1.60 [1.16-2.22 ]) at 2 years than hiv-neg cases.outcomes within hiv-pts were comparable for myeloablative vs reduced intensity conditioning and among peripheral blood, bone marrow and cord blood stem cell sources (data not shown). finally, while hiv-pts' outcomes were comparable for allo-hct from hla-identical siblings and alternative donors (os: 48% vs 40%; p ¼ 0.38) , the use of such alternative donors in hiv-pts was less common than in hiv-neg hct recipients (42% vs 50%, p ¼ 0.02). conclusion: this study showed that the outcome of allo-hct is poorer in hiv-pts than in the general population, primarily driven by higher nrm, and in keeping with their inferior overall life expectancy despite haart. even so, allo-hct is feasible in hiv-pts with hematologic indications, with a 47% os at 2 years. in view of the current reduced use of alternative donors despite comparable results to matched sibling donors, hiv-pts requiring an allo-hct should be granted access to donor search and consideration for transplantation at the same level as hiv-neg counterparts. these data are key to inform allo-hct strategies in hiv-pts, including its investigational use to eradicate hiv infection. disclosure of interest: none declared. introduction: haplo-hsct is a therapeutic option for patients with high risk hematologic neoplasms with the advantages of quick availability, easy programation and logistics, and a committed donor. it has shown promissing results in patients diagnosed with relapsed or refractory hodgkin lymphoma (hl) at least comparable to allogeneic transplant from siblings or unrelated donors (burroughs lm et al. biol blood marrow transplant 2008; 14:1279 -1287 . materials (or patients) and methods: we retrospectively evaluate the results of haplo-hsct with iv busulfan (bux) based ric regimens (fludarabine 30 mg/m2 x5 days (-6 to -2), cyclophosphamide14,5 mg/kg x2 days (-6 to -5) , bux 3,2 mg/ kg x 1 (bux1)or 2 days (bux2) on days -3 to -2) and gvhd prophylaxis based on ptcy (50 mg/kg on days þ 3 and þ 4) and a calcineurin inhibitor plus mycophenolate from day þ 5 performed in geth centers to patients diagnosed with relapsed or refractory hl. results: from march-2009, 43 haplo-hsct have been performed in patients diagnosed with relapsed or refractory hl in 11 geth centers. median age was 31 years (17-53), 67% were males and all were in advanced phases of their disease, after a median of 4 prior treatment lines (2) (3) (4) (5) (6) (7) (8) . autologous hsct was previously employed in 79%, and allogeneic hsct in 7%. five patients (11.5%) have received more than 2 prior transplants. disease status at haplo-hsct evaluated by pet was complete remission in 14 (32%) and persistent disease in 29 (68%). bone marrow was employed in 11 (26%) and peripheral blood in 32 (74%), without t-cell depletion in all cases. the haploidentical donor was patients mother (20), father (3), siblings (19) or daughter (1) . the ric regimens employed were bux1 in 14 (32.5%) and bux2 in 29 patients (67.5%). median neutrophils engraftment was day þ 18 (13-44) and platelets 420 k was day þ 26 (13-150). graft failure with autologous reconstitution happened only in 1 patient (2.5%). the day þ 100 cumulative incidence (ci) of non-relapse mortality (nrm) was 7% (3/43) and 16% (7/43) at 1 year posttransplant. the day þ 100 ci of grade ii-iv acute gvhd was 43%, and grade iii-iv was 14.5%. chronic gvhd ci was 26.5% at 1 year, being extensive in 6%. after a median follow-up for survivors of 13 months , the event-free survival (efs) was 59.5% and overall survival (os) was 84%. the 1-year ci of relapse or progression was 25%. factors related with better 1year efs were cr prior to haplo-hsct (93% vs 45%; p ¼ 0.017) and receiving less than 4 treatment lines prior to haplo-hsct (100% vs 51.5%; p ¼ 0.018). no significant differences were seen when comparing bux1 against bux2 in terms of nrm, efs or os. conclusion: haplo-hsct with ptcy and bux based ric conditioning in relapsed or refractory hl patients, renders long-lasting remissions with acceptable toxicity and gvhd, obtaining better results in those transplanted in cr and with less than 4 treatment lines prior to haplo-hsct. disclosure of interest: none declared. introduction: b-lineage acute lymphoblastic leukemia (all) is the most common childhood cancer. although this disease can be successfully treated in 80% of patients, prognosis for primary refractory or relapsed disease is very poor. even after allogeneic stem cell transplantation (sct), relapse rates are considerable and correlate significantly with persistent minimal residual disease (mrd) prior to and after sct. mrd constellations represent favorable effector-target ratios and thus might be optimally suited for immunotherapeutic intervention with therapeutic antibodies. materials (or patients) and methods: we developed an fcoptimized cd19 antibody (4g7sdie) and produced it in pharmaceutical quality. 4g7sdie mediates markedly enhanced antibody-dependent cellular cytotoxicity (adcc) through its improved capability to recruit fcgriiia bearing effector cells including nk cells and gd t cells. 4g7sdie was applied on compassionate use to mrd-positive pediatric patients with relapsed or refractory all (cr1 n ¼ 3, zcr2, n ¼ 11). results: side effects such as headache and fever were negligible. in all patients complete cd20 þ b-cell depletion was observed during therapy. after discontinuation of 4g7sdie therapy b cell counts recovered rapidly to normal levels. in 9/14 patients mrd was reduced byz1 log or fell below mrd-detection threshold of 10 -4 over the course of treatment. 2/9 responders were receiving additional treatment. 6 patients relapsed, 1 patient died of cns chemotherapy associated toxicity and 1 patient died of late posttransplant sepsis. 6 patients have been in sustained remission for 264-1115 days (median follow-up 720 days). in initial cytotoxicity screenings, performed in an allogeneic setting, significantly increased lysis of all blasts by pbmc after adding 4g7sdie was observed. nk cells and gd t cells were identified as main effector cell populations. cd19 expression on patient blasts was confirmed by quantitative flow cytometry (mean 1.71x10 4 molecules/ cell, ± 0.54x10 4 ). cytotoxicity assays using patient pbmc on autologous blasts confirmed sustained functionality of patient effector cells over the course of 4g7sdie treatment. cytotoxicity assays were performed using pbmc from transplanted patients obtained at different time points of 4g7sdie treatment. lysis of autologous all blasts was increased when 4g7sdie or autologous patient serum taken after antibody infusion was added. after infusion of 20 mg/m 2 -40 mg/m 2 4g7sdie serum half-life was 20 h -43 h. serum levels of 4g7sdie remained above saturating concentrations ofz700 ng/ml (ec 50 ¼ 65 ng/ml) until the following application in the bi-weekly treatment cycle. notably, in 2/2 analyzed patients under 4g7sdie therapy, a down-modulation of cd19 surface expression on the leukemic blasts was observed. in vitro antigenic shift assays on patient blasts showed considerable but very heterogeneous shift of cd19 surface expression. furthermore, a positive correlation between cd19 surface expression levels and 4g7sdie mediated lysis was observed. these observations hint at in vivo tumor escape mechanisms and moreover indicate selective pressure exerted by immunotherapy with 4g7sdie, underlining its therapeutic potential, but also delineating possible limitations. conclusion: promising anti-leukemic effects of the 4g7sdie antibody have been observed in vitro and in vivo. we are currently preparing a clinical phase i/ii trial. disclosure of interest: none declared. introduction: allogeneic hct with myeloablative conditioning is considered a standard of care for adults with high risk acute lymphoblastic leukemia (all). however, with improving results of conventional-dose chemotherapy and the introduction of novel agents on one hand, and the improvement in transplantation techniques on the other, the indications for allohct require re-evaluation, taking into account patient-and procedure-related factors associated with the risk of nonrelapse mortality (nrm). the aim of this study was to analyze the results of myeloablative allohct treatment for patients with all according to recipient age and donor type. materials (or patients) and methods: 4859 patients treated with allohct in first complete remission during the period 1993-2012 were included. the outcomes were analyzed for the periods 1993-2002, 2003-2007 and 2008-2012 , in various age groups, separately for hla matched sibling (msd, n ¼ 2681) and unrelated donors (urd, n ¼ 2178). results: for msd-allohct recipients treated during the period 2008-2012, the following two-year probabilities of os were obtained: 76% for the 18-25 years old (y.o.) group, 69% for both the 26-35 y.o. and 36-45 y.o. groups and 60% for the 46-55 y.o. group. the incidence rates of nrm were 12%, 11%, 15% and 24%, respectively for those same age groups. in comparison with the 1993-2007 period, significant improvements were observed for all age groups except for the 26-35 y.o. patients. the improved survival rates were a consequence of reduced nrm and a tendency towards a reduced risk of relapse. among urd-allohct recipients, the os was 66% (18-25 y.o.) , 70% (26-35 y.o.) , 61% (36-45 y.o.) , and 62% (46-55 y.o.) , while the respective incidence rates of nrm were 21%, 20%, 21% and 19%, the improvement of os over time was documented for 36-45 y.o. (p ¼ 0.005) and 46-55 y.o. (0.0007 ) patients due to the reduced incidence of nrm with no significant effect on relapse. in a multivariate analysis adjusted for disease-, patient-, donorand procedure-related factors, transplantations performed for the period 2008-2012, when compared to 1993-2007 , were associated with significantly reduced risks of the overall mortality (hr ¼ 0.72, p ¼ 0.0003), treatment failure (either relapse or nrm, hr ¼ 0.77, p ¼ 0.002), and nrm (hr ¼ 0.73, p ¼ 0.01) and showed a trend towards reduced risk of relapse (hr ¼ 0.81, p ¼ 0.06). the overall mortality was reduced for transplants with tbi-based compared to chemotherapy-based conditioning (hr ¼ 0.71, p ¼ 0.005) as a result of reduced risk of relapse (hr ¼ 0.55, p ¼ 0.00004). type of donor (msd vs. mud) had no significant effect on survival (hr ¼ 0.9, p ¼ 0.24). conclusion: results of allohct for adults with all improved significantly over time in all age groups, mainly due to the reduction of nrm. importantly, results obtained with matched unrelated transplants were comparable to sibling transplants. total body irradiation should still be considered as the preferable type of myeloablative conditioning for all. disclosure of interest: none declared. cmv seronegativity is associated with a 20% decrease in 1-year survival in patients undergoing reduced intensity sibling-donor transplantation for treatment of myeloid malignancy d. j. lewis 1,* , c. holmes 1 , k. peggs 2 , a. peniket 3 , m. nikolousis 4 , s. nagra 5 , g. pratt 1,4 , c. craddock 1,5 , r. malladi 1,5 , p. moss 1, 5 1 school of cancer sciences, university of birmingham, birmingham, 2 university college hospital, london, 3 oxford university hospital, oxford, 4 birmingham heartlands hospital, 5 centre for clinical haematology, university hospital birmingham, birmingham, united kingdom introduction: reduced intensity (ric) allogeneic stem cell transplantation is a highly effective treatment for acute myeloid leukaemia and the immunological 'graft versus leukaemia' effect is believed to be a major mechanism of disease control. cytomegalovirus reactivation has been suggested to reduce the rate of disease relapse in acute myeloid leukaemia (aml). we investigated the influence of cmv serostatus on the clinical outcome of patients who underwent t cell depleted ric allografts for myeloid malignancy, and went on to examine reconstitution of lymphoid subsets. materials (or patients) and methods: we studied patients who underwent ric allografts for aml (n ¼ 272) and mds (n ¼ 82) from four uk centres, with fludarabine and melphalan conditioning þ /-alemtuzumab. overall survival was calculated. relapse rate and non-relapse mortality were calculated with competing risk analyses. results: the median overall survival was 2.17 years. the relapse rate of the entire cohort was 23% at 1 year with a nonrelapse mortality of 22%. the overall survival for the 'cmv at risk' group was 64.7% at 1 year compared to 57.9% for cmv (-/-). this difference was most marked in patients transplanted from sibling donors (n ¼ 140); the overall survival at 1 year was 79% in cmv-at-risk patients (n ¼ 99) compared to only 59% in the cmv seronegative group (n ¼ 41) (p ¼ 0.027). this 20% increment in survival was due to a 37% reduction in the rate of disease relapse in patients that were cmv-at-risk (1 year relapse rate of 21% versus 33%). there was a non-significant trend towards improved overall survival in those that experienced cmv reactivation amongst the cmv-seropositive patients (1 year os 83% versus 61%, p ¼ 0.19) , mainly due to a reduction in the rate of relapse (1 year relapse rate 17% versus 28%). because of this large difference in relapse risk, we went on to examine the effects of cmv serostatus and alemtuzumab use on reconstitution of lymphocyte subsets at 3 months post transplant. alemtuzumab led to 5-fold decrease in the t cell count at 3 months compared to transplants in which t cell depletion was not used. however, within this alemtuzumab group, positive cmv serostatus in the donor or recipient was associated with a relative 7 fold and 35 fold increase in the cd3 þ and cd8 þ t cell count compared to cmv seronegative pairs. indeed, cd8 þ t cells were virtually undetectable at this time point in cmv seronegative transplant/donor pairs. conclusion: cmv seropositivity is markedly beneficial for patients who undergo a sibling donor reduced intensity allograft for myeloid malignancy and in whom alemtuzumab is used for conditioning. this effect is most likely to be due to the profound influence of chronic viral replication on boosting t cell immune reconstitution in the early post transplant period. cmv seronegative patients with aml should be considered at risk of impaired survival for certain subgroups of stem cell transplant. disclosure of interest: none declared. introduction: allogeneic stem cell transplantation is the only curative option for patients with high risk acute myeloid leukemia (aml) and for those experiencing relapse. either matched sibling donor (msd) or unrelated donor (ud) is indicated. materials (or patients) and methods: with the aim to compare the outcomes of both strategies we have retrospectively analysed 1554 adults with aml receiving either msd (n ¼ 961) or ud (n ¼ 593) in ebmt centers from 2000-2012. for ud, 481 were 10/10 hla matched and 112 were 9/10. median follow up was 28 (range 3-157) months. there were statistical differences between the 2 groups. compared to msd recipients, ud transplants were older (49 vs 52 years, p ¼ 0.001), were performed more recently (2009 vs 2006, p ¼ 0.001) , had longer interval between diagnosis to transplant (10 vs 9 months, p ¼ 0.001), had more often secondary aml (13% vs 19%, p ¼ 0.002) and were transplanted with higher proportion of cmv negative donors (38% vs 37%, p ¼ 0.001). peripheral blood stem cells (pbsc) was used as graft source in 90% of patients in both groups, p ¼ 0.14. conditioning regimen was more frequently myeloablative for patients transplanted with a msd (61% vs 46%, p ¼ 0.001). msd received more often busulfan and cyclophosphamide as mac (16%) or a tbi based regimen (18%). for ud, bu-fludarabine was the most frequent conditioning used (19%). results: cumulative incidence (ci) of neutrophil engraftment was similar (93% vs 92% for msd vs ud, respectively, p ¼ 0.07). grade ii-iv acute gvhd was 26% vs 30% (p ¼ 0.11 ) and ci of chronic gvhd was 25% vs 24% (p ¼ 0.9) for msd and ud, respectively. for msd and ud respectively, ci of relapse (ri) was 57% vs 49%, p ¼ 0.001; ci of non-relapse mortality (nrm) was 23% vs 24%, p ¼ 0.24. probability of leukemia-free survival (lfs) at 2 years was 21% vs 26%, p ¼ 0.001, and overall survival (os) was 26% vs 33% p ¼ 0.004, respectively. chronic gvhd as time-dependent variable was associated with lower ri (hr 0.78 , p ¼ 0.05), higher nrm (hr 1.71 , p ¼ 0.001), and higher os (hr 0.69, p ¼ 0.001). according to hla-match for msd, 10/10 ud and 9/10 ud, ci of relapse (ri) was 57% vs 50% vs 45%%, p ¼ 0.001; ci nrm was 23% vs 23% vs 29%%, p ¼ 0.26 and probability of lfs at 2 years was 21% vs 27% vs 25%, p ¼ 0.003, respectively. in a multivariate analysis adjusted for differences between the 2 groups, ud was associated with lower ri (hr 0.76, p ¼ 0.001) and higher lfs (hr 0.83, p ¼ 0.001) compared to msd. when analyzing according to hla-match, there was no differences for patients transplanted with an ud 9/10 or a 10/10 for ri (hr 0.77, p ¼ 0.10) , and lfs (hr 0.92, p ¼ 0.53) . the other factors independently associated with better outcomes were the interval between diagnosis and transplant (ri hr 0.62, po0.001) , and lfs (hr 0.67, po0.001) . conclusion: unrelated donor transplant was associated with better lfs due to lower ri compared to msd for high-risk patients with aml transplanted in first relapse. there were not differences for the ud 9/10 match probably due the graftversus-leukemia effect in the setting of patients transplanted with active disease. disclosure of interest: none declared. introduction: allogeneic reduced intensity transplantations (rict) in elderly patients (pts) with aml in cr1 has become a commonly used treatment modality. however, no prospective studies to date have been reported to support use of this strategy. the aim of this prospective, multicenter study is to compare outcomes of patients receiving rict with patients being treated with conventional chemotherapy. in 2012 an amendment allowed also the use of matched unrelated donors. the study is currently ongoing in 8 countries with a total of 250 pts included. only pts included as per the original protocol are accounted for in this interim analysis which focuses on safety. materials (or patients) and methods: the study was designed by the transatlantic leukemia group (tralg) consisting of centers associated with the swedish and canadian bmt groups, and centers from germany, norway, finland and new zealand. patients should have intermediate or high risk aml in cr1 and not being eligible for a myeloablative transplant due to age or comorbidities, and have at least one potential sibling donor. date of inclusion was defined as the date of hla-typing of the first sibling, and pts were allocated to the donor (d) or no-donor group (nod) based on hla match. overall survival at 3 yrs is the primary endpoint; secondary endpoints include rfs, gvhd, non-relapse (nrm) and transplant related (trm) mortality. conditioning consisted in the vast majority of patients of fludarabine 150 mg/m 2 iv and busulphane 8 mg/ kg po or 6.4 mg/kg iv. gvhd prophylaxis was cya/methotrexate or cya/mmf. the study started to accrue patients in 2004. results were analyzed on an intent to treat basis. results: from 2004 pts were included in sweden (n ¼ 62), canada (n ¼ 56), germany (5) , norway (13), new zealand (5) and finland (5) . 12 patients were excluded from analysis; 3 favorable risk, 9 protocol violations and 134 pts; 60 females, 74 males, median age 62 (51-74) yrs, 21 saml, were allocated to the d group (n ¼ 75), or the nod group (n ¼ 59). in the d group, 16 pts (21%) did not reach transplant, 15 of these died (12 after relapse, 3 other causes). cytogenetic risk groups were categorized as per eln criteria. the distribution of total disease risk categories was 25% adverse, 66% intermediate, 9% unknown. median follow-up of surviving pts at dec 6 th 2014 was 4.6 (2.6 -10.1 ) yrs after inclusion. ninety-one pts relapsed (75%). kaplan-meier estimates of os and rfs in the whole group at 3 yrs were 45% and 37% respectively. for adverse and intermediate risk pts os and rfs at 3 yrs were 36% and 21%, and 51% and 44%, respectively. in the transplanted pts acute gvhd grade 0-1 occurred in 73% of pts, grade 2-4 in 20%. extensive gvhd occurred in 42%, limited in 15%. 89 pts (66%) have died, 17 without and 72 after aml relapse. in the nod group 5 (8%) pts died from non-relapse causes. in the d group, 3 pts died before transplant, while the trm was 9/59 (15%) with causes of death: 6 gvhd, 3 other. conclusion: selected patients with aml in cr1 tolerate rict or standard management with low mortality. disease control remains a major issue. this multicenter prospective protocol will continue to accrue patients until relevant conclusions can be drawn comparing a rict to standard treatment. the current rapid inclusion of pts and participation of new sites in australia, greece and estonia will help to complete the study. disclosure of interest: none declared. introduction: the wilms' tumor gene 1 (wt1) is overexpressed in 480% of acute myeloid leukemias (aml) and myelodysplastic syndromes (mds) and proved to be a good marker for minimal residual disease (mrd) monitoring. although allogeneic haematopoietic stem cell transplantation (allo-hsct) is the most effective treatment for aml/mds, disease recurrence remains a major problem. after allo-hsct, quantitative wt1 monitoring can represent a useful tool to detect mrd and tailor immunotherapeutic strategies. materials (or patients) and methods: we included in this retrospective analysis 111 pts with aml/mds (99 and 12 respectively) overexpressing wt1 and allotransplanted in our institution from 12/2007 to 01/2014. twenty two pts were transplanted from a matched related donor, 26 from a matched unrelated donor, 57 from a mismatched related donor and 6 from cord blood units. 101/111 pts received a reduced toxicity conditioning treosulfan-based. at the time of transplant, 73/111 pts (66%) were in cr, while 38/111 (34%) had active disease. median follow up (fu) of our cohort was s10 599 days (range, 55-2538 days). wt1 mrd monitoring was performed by rq-pcr and considered positive for values4250 copy numbers of wt1 per 10e4 abl [1] . after allo-hsct, detection of positive wt1 was followed by immunomodulatory therapeutic interventions according to the time from transplant, the presence of active graft-versus-host disease (gvhd) and the general clinical conditions: tapering and/or discontinuation of immunosuppressive drugs (is), donor lymphocytes infusions (dli), administration of hypomethylating agents. median time to disease relapse was calculated from the time of detection of wt1 value above the threshold. results: at day 30 post allo-hsct, 109 out of 111 pts (98%) were in cr. forty-five out of 109 (41%) cr pts had wt1 levels persistently negative during follow up (fu) and 23/45 remained in cr at the last fu. sixty-four out of 109 pts (59%) had at least one increase of bm wt1 levels above the threshold during observation and were evaluated for preemptive treatment. in 45/64 (70%) is was tapered until suspension and/or dli þ /-azacytidine was administered. median time from first wt1 positive value to treatment was 30 days (range, 15-45 days). in 16 out of these 45 pts (35%) wt1 level normalized, whereas 29 pts (65%) progressed to overt disease. all the 16 that normalized wt1 remained in cr. no grade iii or iv acute gvhd was observed, while severe chronic gvhd occurred in 2/45 pts (4%). median time to disease relapse for the 45 treated pts was 116 days (range, 20-951 days). in 11/64 pts (17%) with a post allo-hsct wt1 positivity, the presence of an active form of acute or chronic gvhd prevented from applying further immunotherapy strategies. among these pts, 5 remained in cr, 6 relapsed. median time to disease recurrence for these 11 pts with gvhd was 156 days (range, 32-204 days). finally in 8/64 pts (13%) concurrent clinical issues (e.g. active infection) did not allow any attempt to prevent relapse and 4/8 relapsed. median time to disease relapse for untreated pts was 35 days (range, 15-82 days). conclusion: in our series, pre-emptive immune-modulatory maneuvers targeting wt1 levels proved to be feasible and safe and of potential clinical benefit to postpone overt relapse in high risk aml/mds pts post allo-hsct. introduction: allogeneic stem cell transplantation (sct) with both myeloablative (mac) and reduced intensity conditioning (ric) is effective therapy in aml. several studies have shown that leukemia-free survival (lfs) is similar after sct with mac and ric. however, there is paucity of data on the long term outcome (beyond 10 years) following ric due to the relative recent introduction of this approach. the alwp of ebmt published in leukemia 2005 the largest study until that time, comparing the outcome of aml patients (pts) given ric (n ¼ 315) and mac (n ¼ 407). the median follow-up was 13 months. in multivariate analysis, non-relapse mortality (nrm) was significantly lower and relapse rate was significantly higher after ric resulting in similar lfs. materials (or patients) and methods: in order to better predict long-term outcome and late events we have now updated sct outcomes in a larger cohort of pts, age z50 years (n ¼ 1423) after sct from matched sibling donors, in the years 1997-2005 with a median follow up of 8.3 years (0.1-17) . results: 722 pts were given ric and 701 mac regimens. the median age at sct was 57 (50-75) and 54 (50-72) years, respectively (po0.001). 21% of ric recipients had advanced disease at sct compared to 25% of mac recipients. the percentage of pts in cr1 and cr2/ later cr was 62% and 17% compared to 63% and 12%, respectively (p ¼ 0.02). ric recipients were more likely to receive pbsc (92% vs 73%, po0.001 ) and in vivo t-cell depletion (33% vs 12%, po0.001). 16% and 20% had poor-risk cytogenetics, respectively (p ¼ 0.19 1.4, po0.001 ) and poor cytogenetics (hr 1.7, po0.005) . the conditioning regimen did not predict 10-year lfs. nrm rates were higher after mac than ric throughout the late post sct course, while relapse rates were only mildly decreased at the late phases. in pts surviving leukemia-free 2 years after sct the subsequent nrm was 15% and 9%, respectively (p ¼ 0.03). subsequent relapse rates were 14% and 9% (p ¼ 0.12) and lfs was 71% and 73%, respectively (p ¼ 0.76). in pts surviving leukemia-free 5 years after sct, subsequent nrm was 9% and 4%, respectively (p ¼ 0.06). subsequent relapse rates were 5% and 6% (p ¼ 0.53), and lfs was 86% and 90%, respectively (p ¼ 0.27). conclusion: lfs remains similar after ric and mac even 10 years after sct. the trend for excess nrm with mac remains throughout the late course, however excess relapse after ric is more obvious in the early phases. pts remaining leukemia-free 5 years after sct can expect excellent subsequent outcome with both regimens. long-term follow-up studies (beyond 10 years) are of significant importance when assessing sct outcomes. disclosure of interest: none declared. myeloablative busulfan based strategies in transplantation particularly of children with non-malignant diseases. a reduction in short term mucosal and hepatic (sos) toxicity and the absence of long term pulmonary toxicity have been demonstrated. it is unclear, however, whether this reduction of toxicity is accompanied by an equal or inferior myeloablative capacity compared to busulfan based myeloablative regimens. materials (or patients) and methods: we performed a retrospective analysis of consecutive patients with nonmalignant diseases transplanted in 4 german pediatric transplant centers (hamburg, hannover, munich and ulm) with a treo based regimen (treo and fludarabin (flu) ± thiotepa (tt)±serotherapy) in the period between january 1st 2000 and june 30th 2013. results: we identified 153 patients with inborn errors including primary immunodeficiencies, hemoglobinopathies, hemophagocytic lymphohistiocytosis, mucopolysaccharidosis and osteopetrosis. the median age at transplantation was 4.8 years (0.1-22) . all pts received treo/flu. tt was added in 102 of these pts, serotherapy in 139 pts. the os after 2 years was 89%, the efs 81%. the incidence of agvhd 1ii-1iv was 19% and 5.4% for cgvhd 11-13. primary engraftment of donor cells was present in 97% of patients. however, mixed chimerism at any time point was found in 59% and disease recurrence in 11%. an additional cellular therapy (act: stem cell boost, dli, or 2nd transplant) was applied in 28% of patients. act was more often needed after transplantations from mmfds and muds than from msd/mfds (p ¼ 0.08). there was a significant difference in patients who received alemtuzumab as serotherapy early (d-13 to d-4) versus late (d-3 or later) during conditioning, with 32% act in the early and 52% act in the latter group (p ¼ 0.02). cell source (bm or pbsc) and addition of tt did not affect the cumulative incidence (ci) of act. conclusion: there is an excellent overall survival and efs with treo based conditioning in the transplantation of patients with non-malignant diseases. despite a good primary engraftment, a high rate of mixed chimerism, disease recurrence and need for additional cellular therapy was observed. interestingly, early administration of serotherapy was correlated with a higher probability of stable donor engraftment and has to be considered as a relevant factor for transplant outcome. a randomized controlled prospective study comparing conditioning regimens with treo against busulfan in nonmalignant diseases is needed. disclosure of interest: none declared. long-term outcomes of reduced-intensity conditioning allogeneic stem cell transplantation for adult high-risk acute lymphoblastic leukemia in first complete remission s. lee 1,* , k. introduction: reduced-intensity conditioning (ric) allogeneic stem cell transplantation (sct) has emerged as an option designed to lower nonrelapse mortality (nrm) for older patients and those with comorbid conditions. however, the role of ric-sct in adult acute lymphoblastic leukemia (all) remains unclear because the interpretation of transplantation outcome is mainly limited by the small sample size, short follow-up duration, various regimens for conditioning and graft-versus-host disease (gvhd) prophylaxis, and the heterogeneity of the criteria used to select patients for ric-sct. previously, we conducted a phase 2 trial of ric-sct in adults with high-risk all and showed the potential role of this strategy, especially in patients in first complete remission (cr1). here, we report on the updated results of ric-sct by analyzing 93 consecutive adult high-risk all transplants in cr1. materials (or patients) and methods: during the period between 2000 and 2012, 93 consecutive patients in cr1 (median age, 51 years [range, 15-65 years]) were given an identical ric regimen consisting of fludarabine (150 mg/m 2 in total) and melphalan (140 mg/m 2 in total). the indications for ric-sct were advanced age (z50 years; n ¼ 53; 57.0%) and comorbid conditions (n ¼ 40; 43.0%). graft sources were peripheral blood stem cells (n ¼ 91; 53 8/8-matched sibling donor, 15 8/8-matched unrelated donor, 23 7/8-matched unrelated donor) and bone marrow (n ¼ 2; 1 8/8-matched unrelated donor, 1 7/8-matched unrelated donor). the median time-to-transplantation was 161 days (range, 106-291 days). gvhd prophylaxis was attempted by administering calcineurin inhibitors (cyclosporine for sibling donor transplants, tacrolimus for unrelated donor transplants) plus methotrexate. antithymocyte globulin (2.5 mg/kg in total) was administered to the patients who received allele-mismatched unrelated donor grafts. if residual leukemia was detected in the absence of gvhd at 3 months after transplantation, calcineurin inhibitors were rapidly discontinued. results: fifty-two patients developed grade ii to iv acute gvhd (42 grade ii, 6 grade iii, 4 grade iv). the cumulative incidence of acute gvhd at 100 days was 55.9%. of the 87 patients who survived for at least 100 days with sustained engraftment after transplantation, 61 developed chronic gvhd (30 limited, 31 extensive), resulting in a 5-year cumulative incidence of 65. 6% . after a median follow-up of 60 months (range, 24-174 months), the cumulative incidence of relapse (cir) and nrm at 5 years were 28.1% and 22.9%, respectively, and the 5-year disease-free survival (dfs) and overall survival (os) rates were 54.4% and 60.4%, respectively. within the cohort of ph-negative all transplants (n ¼ 43), the 5-year cir, nrm, dfs, and os rates were 26.4%, 15.5%, 61.7%, and 67.4%, respectively. in a subgroup of ph-positive all transplants (n ¼ 50), the 5-year cir, nrm, dfs, and os rates were 29.7%, 28.9%, 48.6%, and 55.0% respectively, and for these patients, minimal residual disease kinetics (early-stable molecular response vs late molecular response vs poor molecular response) and the presence of chronic gvhd were closely related to cir and dfs. conclusion: our data suggest that ric can be considered as a reasonable choice for providing a long-term disease control for adult high-risk all patients in cr1. disclosure of interest: none declared. substitution of tbi by intravenous busulfan for elderly aml/mds patients within the flamsa-ric protocol is feasible and yields comparable results m. schleuning 1,* , d. judith 1 , i. burlakova 1 , h. baurmann 1 , r. schwerdtfeger 1 , g. stuhler 1 1 centre for hematopoietic cell transplantation, dkd helios klinik, wiesbaden, germany introduction: the flamsa-ric protocol is a highly effective conditioning protocol for high risk myeloid leukaemia patients (pts). however, many of these pts are beyond the age of 60. in this population the use of total body irradiation (tbi) might be toxic, even with the reduced dose of 400 cgy, as described in the original flamsa-ric protocol. in an attempt to further reduce toxicity for elderly (460y) or comorbid pts we substituted tbi by intravenous busulfan (ivbu; 8 x 0.8 mg/kg bw) within the flamsa-ric protocol. materials (or patients) and methods: retrospective study to analyze the results of ivbu in comparison to those achieved in pts receiving the classical flamsa-ric protocol with tbi during the same time period. results: from november 2006 to october 2012 173 pts with high-risk aml or mds received an allogeneic stem cell transplant after flamsa-ric conditioning. eighty-three pts (median age 47y) received tbi and ninety pts (median age s12 64y) received ivbu. in the tbi group 76 pts suffered from aml and 7 pts from mds and in the ivbu group diagnoses were aml in 74 and mds in 16 pts. unfavourable cytogenetics or molecular genetics were found in 64% of tbi and 50% of ivbu pts, respectively. in the tbi group 43% were transplanted with active disease as compared to 71% in the ivbu group. in both groups stem cell grafts from unrelated donors were used in approximately 75% of pts. all pts engrafted. after a median follow-up of 5.5 y for surviving pts the probability of leukaemia-free survival at 5 y after transplant is 40% in the tbi group and 35% in the ivbu group (p ¼ 0.133). twentyseven relapses occurred in the tbi and 21 in the ivbu group. non-relapse mortality (nrm) for the tbi cohort was 10% and for the ivbu group 24% at day þ 100 (p ¼ 0.052) and 33% and 41% during the entire observation period (p ¼ 0.004). the difference in nrm is probably owing to the older age of the pts in the ivbu group and was due to more infectious complications. no significant difference in survival was observed in mud or sibling transplants in either group. conclusion: in conclusion, substitution of tbi by ivbu is feasible with no enhanced relapse rates observed and should be further evaluated in prospective clinical trials also for younger pts. introduction: to ascertain the therapeutic potential of non-tbi-based conditioning for cd34 þ hpc-selected, t celldepleted allografts, we conducted a trial comparing our standard regimen, arm (a) 1375 cgy hftbi þ thiotepa,5 mg/ kg/day x 2 days þ cyclophosphamide 60 mg/kg/day x 2 days vs. arm (b) busulfex 0.8 mg/kg/6 h x12 (dose adjusted) þ melphalan 70 mg/kg/day x 2 þ fludarabine 25 mg/m 2 /day x5 and arm (c) clofarabine 20 mg/m 2 /day x 5 þ melphalan 70 mg/m 2 /day x 2 þ thiotepa 5 mg/kg/day x2, as preparation for t-cell depleted cd34 þ pbsc transplants from gcsfmobilized leukocytes fractionated with the clinimacs cd34 þ reagent system. materials (or patients) and methods: primary endpoints were engraftment, gvhd, transplant-related mortality (trm) and 2 yr os and dfs (confer table) . stratification of pts to arms a (standard), b or c was based on the patient's disease, disease stage and clinical factors such as age, prior therapy or comorbidities enhancing risks of tbi. arm b was the non-tbi arm predominantly used for myeloid and arm c for lymphoid malignancies. prior to transplant, recipients of hla-matched or non-identical transplants received rabbit thymoglobulin at 2.5 mg/kg/day x2 or 3 days respectively, to prevent graft failure. no gvhd drug prophylaxis was given post transplant. results: a total of 215 consecutive patients, accrued between 5/13/2010 and 11/20/2014, were analyzed (84 in arm a, 103 in arm b, 28 in arm c). these pts have been followed for a median of 19.7 months. donors were related or unrelated and hla-matched for 73% of the patients and 1-2 hla alleles disparate for 27%. median age for the entire group was 47.8 years, with older pts predominating in the non-tbi groups (medians arm a, 31.2 yrs; arm b, 58.8 yrs; arm c, 24 yrs). the cd34 þ pbsc transplant provided a mean dose of 9.7 x 10 6 cd34 þ progenitors/kg (range 1.4 -89.7 ) and 4.5x10 3 cd3 þ t-cells/kg (range 0.6-25.3 ). all pts engrafted; but 2 pts (1.9%) in arm b experienced late graft failure, one of whom was reconstituted after a secondary graft. overall the incidence of grade ii-iv acute gvhd was 6%, 6% and 7% for arm a, b and c respectively. trm at 1 year was 9% in arm a, and 15% in arm b and 16% in arm c. two year os and dfs for each arm are: arm a -62.2% and 56%; arm b -68% and 59% and 48% and 48% for arm c. for the 115 pts who received standard risk transplants (i.e., pts with high risk forms of aml, all or nhl in 1 o cr, aml in 2 o cr, mds ra/rcmd, cml in 1 o cp or mm in cr1), 2 year os and dfs are: arm a -60% and 59%; arm b -74% and 65%; arm c -50% and 50%, with relapse rates at 2 yrs of arm a -23%, arm b -10.4%, and arm c -11.5%. cumulative incidence of relapse (cir) stratified by risk group reveals that the probability for relapse is significantly higher for the pts with high risk disease (p o0.0005), whereas the cumulative incidence of non-relapse mortality (nrm) is comparable (p ¼ 0.36)( figure 1 ). the median time to relapse has not been reached in either group. the estimates of relapse at 1 year were 23.2% (95% ci, 16.5-30.0% ) for high risk pts and 11.6% (95% ci, 6.7 -16.6%) for the other group. the estimated 2-year cir was 31.4% (95% ci, 23.5-39.4%) for the group at high risk and 14.6% (95% ci, as measured from allosct prior to dli bm progression and focal progression patterns were highly similar with cumulative incidences of bm progression of 23%±13% and 23%±13%, and of focal progression of 19% ± 12% and 21% ± 12%, after 12 and 24 months post-transplant, respectively. in contrast, as measured from dli bm progression and focal progression patterns showed strong dissimilarity: at 12 and 24 months after dli cumulative incidences of bm progression were 14%±13% and 17%±14% respectively, whereas focal progression was 48% ± 19% and 62% ± 18%, respectively, illustrating a potent immunological response in bm with only limited effect of dli on focal lesions. conclusion: disease progression patterns of multiple myeloma after tcd-ric allosct diverged from initially similar bm and focal progression patterns in the absence of alloimmune responses towards disease control in bm with focal progression after dli. this finding illustrates failure of donor lymphocytes to target extra-medullary/focal disease in multiple myeloma. disclosure of interest: none declared. engineered t cells modified to express a cs-1-specific chimeric antigen receptor (car) confer anti-myeloma activity in vitro and in pre-clinical in vivo models introduction: adoptive immunotherapy with t cells that were modified by gene-transfer to express tumor-targeting chimeric antigen receptors (cars) has therapeutic potential in advanced b-cell malignancies. we are pursuing the glycoprotein cs1 (slamf7/cd319) as candidate target for car t cells in multiple myeloma (mm), due to its restricted high level expression on malignant plasma cells in a significant proportion of mm patients. here, we evaluated the anti-mm function of t cells that we modified with cs1-specific cars in vitro and pre-clinical in vivo models. materials (or patients) and methods: we constructed two cs1-specific cars with antigen-binding domains derived from the huluc63 and luc90 mabs that target distinct cs1 epitopes, each comprising a signaling module of cd3zeta and a cd28 co-stimulatory domain, and encoded both constructs in lentiviral vectors for gene-transfer. results: cd8 þ and cd4 þ t-cell lines expressing the huluc63 and luc90 cs1-cars could be readily generated from healthy donors and mm patients (n ¼ 5/3), and propagated and expanded in vitro with similar kinetics as t cells expressing a cd19-specific car that we included as a reference. in functional experiments, cd8 þ t cells expressing either of the cs1-cars conferred specific high level lysis of mm lines (mm1.s, nci-h929 and opm-2), primary mm, k562 that had been stably transfected with cs1, but not native cs1-negative k562. we also detected high level production of ifng and il-2 (cd44cd8), and productive proliferation after stimulation with cs1 þ target cells, with significantly superior anti-mm reactivity mediated by the huluc63 compared to the luc90 cs1-car construct. we confirmed the anti-mm efficacy of cs1-car modified t cells using a xenograft model in immunodeficient mice (nsg/mm1.s). mice were inoculated with fireflyluciferase labeled mm1.s myeloma by tail vein injection and 14 days later, when mice presented with disseminated disease, administered a single dose (i.v.) of a cell product consisting of equal proportions of cd8 þ and cd4 þ t cells modified with either the optimal cs1-car, the cd19-specific car or mock transduced. we observed rapid and durable complete rejection of established mm from bone marrow and resolution of extramedullar mm manifestations in all of the mice treated with cs1-car t cells (n ¼ 4), whereas mice treated with cd19-car t cells, or control t cells had to sacrificed due to progressive disease (n ¼ 4/4). of interest, in this in vivo model, we observed similarly effective anti-mm responses mediated by cs1-car t cells that had been derived from healthy donors and mm patients. conclusion: our data suggest the potential of t cells expressing cs1-specific cars to confer anti-mm activity in clinical settings. the experience with anti-cs1 mab huluc63, that as single agent has only minute anti-mm activity, indicates targeting this molecule will be safe and not be associated with toxicity to normal tissues. we observed stronger anti-mm reactivity with cs1-cars targeting a proximal (huluc63) rather than a distal (luc90) epitope on cs1 protein, in line with our previous observation that the targeted epitope on a given antigen affects tumor recognition of car t cells. experiments to analyze the function of our cs1-cars against panels of primary mm in our nsg model are ongoing to inform our efforts of clinically translating car t-cell therapy in this entity. 4;14) , t(14;16), del17p by fish and/or del13q by karyotyping]. all pts had to achieve at least a partial response from preceding salvage chemotherapy (n ¼ 32) or second salvage auto hsct (n ¼ 12). pts underwent allo tcd hsct with busulfan (0.8 mg/kg x 10 doses), melphalan (70 mg/m 2 x 2 days), fludarabine (25 mg/m 2 x 5 days) and rabbit atg (2.5 mg/ kg x 2 days). tcd was performed by positive cd34 selection (isolex) followed by rosetting with sheep erythrocytes for the initial 13pts (2008-09) and by cd34 þ enrichment by the miltenyi device in 31pts thereafter, achievingo10 4 cd3 þ /kg for all grafts. none of these pts received immuno suppressive therapy post tcd hsct. pts with 10/10 hla matched donors were also eligible to receive low doses of dli (5x10e 5 -1x10e 6 cd3 þ /kg) no earlier than 5mos post allo hsct. results: 44 pts with a median follow up of 24.8 mos (range: 11.1-81.2 mos) of survivors are reported, median age 56 years (range 32-69). all pts engrafted promptly (median d þ 10, range þ 9 -þ 12).trm (grade ii-iv) at 12mos is 18% (95% ci: 8% -31%). acute gvhd was 2% (95% ci: 0% -11%) and chronic gvhd was not observed in any pt. the overall survival (os) and progression-free survival (pfs) with their 95% confidence intervals (ci) are shown in table 1 . factors associated with worse outcome were disease status and number of previous treatments prior to tcd hsct. (1) . in this study, we have analyzed the molecular consequences of del(8)(p21), an abnormality we and others have previously shown to have an adverse impact on survival of mm patients (2) (3) (4) . materials (or patients) and methods: in a cohort of 140 patients that were diagnosed with mm between 2001 and 2012, we have investigated the clinical impact of del(8)(p21) on time to progression (ttp) and overall survival (os). moreover, response rate of 84 patients to 1 st line bortezomib treatment was investigated. we have also analyzed the expression profiles of genes located near the 8p21 region in patients with and without del(8)(p21). additionally, we have analyzed the in vitro response of primary mm cells with and without the deletion to bortezomib-mediated killing and sensitization to trail/apo2l-triggered apoptosis in an attempt to understand why mm patients carrying 8p21 deletion respond poorly to bortezomib treatment. results: we found that mm patients carrying del(8)(p21) deletion had significantly shorter ttp compared to patients without the deletion (p ¼ 0.011) and most importantly these patients had significantly shorter os compared to patients without the deletion (p ¼ 0.001). in a cohort of 84 patients, we observed that patients with del(8)(p21) (n ¼ 24) responded poorly to bortezomib, 50% showing no response while 90% of patients without the deletion (n ¼ 60) responded to bortezomib treatment. in vitro analysis revealed that mm cells from patients with del(8)(p21) show higher resistance to bortezomib treatment possibly due to upregulated expression of genes such as ptk2b, ccdc25, rhobtb2, nfkb, myc and bcl2 while showing downregulated levels of tp53 and scara3 when compared to mm cells without the deletion. furthermore, we have observed that mm cells with del(8)(p21) express higher levels of the decoy death receptor, trail-r4 and fail to upregulate the pro-apoptotic death receptors trail-r1 and trail-r2 that are located in the 8p21 region. as a result, mm cells with del(8)(p21) were largely resistant to bortezomib and trail/apo2l-mediated apoptosis. conclusion: substantiating the clinical outcome of the patients, our data provides a potential explanation regarding the poor response of mm patients with del(8)(p21) to bortezomib treatment. furthermore, our clinical evaluation suggests that including immunomodulatory agents such as lenalidomide in the treatment regimen may help to overcome this negative effect, providing an alternative thought in planning treatments of patients with del(8)(p21). introduction: autologous stem cell transplantation is the standard treatment in patients with multiple myeloma (mm). however, there is discrepancy over the optimal mobilization regimen. therefore a randomized study was conducted to compare cellular composition of the collected grafts as well as early hematopoietic and immune recovery in mm patients receiving g-csf with or without low-dose cyclophosphamide for mobilization of blood grafts after induction with lenalidomide, bortezomib and dexamethasone. materials (or patients) and methods: thirty patients with mm were included into this prospective multicenter study. there were 16 males and 14 females with a median age of 62 years (range 43-70). fourteen patients were mobilized with cyclophosphamide plus g-csf (arm a) whereas sixteen patients were mobilized with g-csf alone (arm b). melphalan 200 mg/m 2 was used as high-dose therapy and patients having graft cd34 þ cell countso3 x 10 6 /kg (measured before freezing) were scheduled to receive g-csf after the graft infusion. cryopreserved graft samples were analyzed with a flow cytometry for t and b cells (cd3/cd8/cd45/cd19) as well as for nk cells (cd3/cd16 þ cd56/cd45). also cd34 þ cell subclasses were analyzed (cd34/cd38/cd133/cd45). complete blood counts were evaluated on day þ 15 and one month post-transplant and a flow cytometry for blood lymphocyte subsets (t, b, nk) was performed one month after the graft infusion. results: the blood grafts in arm a contained significantly higher amounts of cd34 þ cells and the grafts of the arm b contained significantly higher proportion of primitive cd34 þ cd133 þ cd38cells and t, nk and b lymphocytes ( table 1) . the median amount of infused cd34 þ cells was comparable between the arms (3.9 â 10 6 /kg in group a vs. 3.1 â 10 6 /kg in group b, p ¼ 0.056). the number of platelets was slightly lower in the group b at d þ 15 (p ¼ 0.094) otherwise the course of early hematological and immune recovery was comparable between the groups. the use of g-csf alone instead of a combination with cyclophosphamide seems to enrich the blood grafts with significantly higher number of t and b lymphocytes and a higher proportion of more primitive stem cells. the hematological and immune recovery was comparable between the arms. the possible effects of graft composition in long-term patient outcomes will be further evaluated in the ongoing goa study (graft and outcome in autologous stem cell transplantation). disclosure of interest: none declared. introduction: pet is a useful tool that allows deeper assessment of response beyond that measured by m protein levels. it has been reported to predict outcome following both asct. to be able to integrate pet-ct negativity to internationally accepted response criteria the cut-off level needs to be validated by independent investigators. this prospective study was initiated to elucidate the prognostic role of pet-ct in the asct setting utilizing the cut-off found in our patients in ankara university (3.35 ) comparing with those initially reported by barlogie et al (3.6 ) and zamagni et al (4.2) materials (or patients) and methods: 85 consecutive patients diagnosed and transplanted in ankara university with pre-and post-asct fdg-pet-ct imaging were included. patients were: median age 56.6 þ /-8.8 (m/f: 45/40), iss i/ii/iii: 37/33/15, renal impairment (8,2%), bone involvement (94,1%), del13q (40.9%), t (4;14) and/or p53(26.5%), ldh high(10,5%), induction with bortezomib (72,9%). pre-asct clinical response 3 vgpr: 52.9%, post-asct clinical response 3 vgpr:77.5%. overall survival (os): median: 33 months (4.2-141 months). pasw statistics for windows program was used for statistical analysis. results: as reported previously roc analysis revealed 3.35 as a significant cut-off level (p ¼ 0.005; os). pet-cr was defined fdg uptake less than 4.2 or 3.35 depending on the analysis. post-asct pet (44.2) was predictive for pfs (p ¼ 0.05) but not os (p ¼ 0.096) . however pet (43.35 ) was predictive for os (p ¼ 0.037) but not pfs. depending on the cut-off more (suv 3 4.2: 43/64) or less (suv 3 3.35 : 29/64) patients met the criteria for pet-negativity (or remission) following asct. expert pet assessment resulted with pet-cr 39/85 similar to the suv 3.35 frequency. as shown in figure patients to converted to cr after asct (positive/negative group) displayed a better pfs than those who had reached cr prior to asct. this analysis was significant if cut-off was 3.35 but not 4.2. expert assessment was also able to differentiate patients with better prognostic features. 0.0-0.49 ). 23 leukaphereses were analized for mrd: the median plasma cells value was 0,03% (0,00-0,7%); in 6 pts pcs wereo0,01% (cut off for mrd negativity). conclusion: mobilization with cy-bor-dx þ g-csf and borbased asct is safe and effective in elderly mm patients. this schedule allows the collection of an adequate dose of cd34 þ cells, with a very low rate of mobilization failure (2%), also in elderly mm pts. a low rate of clonal pcs contamination in the harvest was also observed. this approach allows to perform asct in most elderly pts, achieving high response rate and promising outcome with a short term treatment:20 weeks compared to the non-asct programs (54 weeks in the vmp program). disclosure of interest: none declared. mica expression levels were investigated in n ¼ 180 gut biopsies with sybr green s qrt-pcr. histological grades of the gastrointestinal gvhd (gi gvhd) were determined by the pathology department at the university clinic, regensburg and severity was grouped by assigning an apoptotic score (0 ¼ absence of apoptosis, 3 ¼ maximum apoptosis). a protein biochip array (evidence investigator s , randox) was utilised for measuring mica serum levels and evaluated in n ¼ 129 samples from allo-hsct patients collected at pretransplantation, day-7, day þ 14, day þ 28 and 3 months post transplantation. results: our analysis showed that the methionine allele in rs1051792 was associated with an increased risk of relapse (p ¼ 0.029). the same allele was also found to be associated with a reduced overall survival (p ¼ 0.041) which was more severe for non-t cell-depleted allo-hsct (p ¼ 0.001). vice, versa, the presence of the valine allele was associated with the development of agvhd (p ¼ 0.044). in the gut, mica expression was investigated in patients treated with low doses of steroids (r 20 mg/kg), as high dose steroid treatment strongly suppressed mica expression. higher levels of mica were associated with an apoptotic score ¼ 0 (no apoptosis) (p ¼ 0.044) and the absence of active gi gvhd (p ¼ 0.046). increased soluble mica levels at 3 months post-transplantation were significantly associated with agvhd (p ¼ 0.0123). conclusion: mica molecules have been shown to play prominent roles in immune processes and therefore are also potential agvhd biomarkers. in this study, we showed that the methionine (mica-129met) allele was associated with the incidence of relapse while the valine (mica-129val) allele was associated with an increased risk agvhd. a low overall survival for patients who did not have had the t cell depletion treatment was also associated with the presence of the methionine (mica-129met) allele. in the gut of patients treated with low doses of steroid, mica gene expression levels were higher with the absence of gvhd. this may indicate that the isoforms are able to meditate nk-cell and t cell inactivation, and down-regulate nkg2d with high levels of soluble mica contributing to the development of agvhd. eleven patients needed gvhd treatment: 4 pts received ganciclovir iv (gcv 5 mg/kg/12 h/14 days), 7 pts valganciclovir per os (vgcv 900 mg/12 h/14 days). both gcv and vgcv were effective in control clinical manifestations of gvhd in a median of 13 days (range 7-52) and resulted in a significant reduction in numbers of circulating tk-cells, without reduction of cd3 þ tk-negative lymphocytes resulting in no effect on long-term immune reconstitution. in five patients additional concomitant treatment with low-dose steroid (prednisone o0.5 mg/kg per day for a median of 2 weeks) was given. a pt who presented severe gut and liver gvhd and one pt who received at transplantation an high dose of unmanipulated lymphocytes (5.4x10 5 /kg) -were succesfully treated with a combined therapy of prednisone and cyclosporine or rapamicine in association with gcv. one patient developed a severe classic de novo c-gvhd, with sclerodermatous lichenoid skin and mouth features plus moderate dry-eye symptoms that was successfully treated with vgcv and a transient course of mycophenolate mofetil (2 g per day) over a 2 months period. no cases of quiescent or progressive c-gvhd was observed after a median follow-up of 679 days (range 139/4035). conclusion: in our long-lasting clinical application of haploidentical tk-cells, an effective induction of immune reconstitution and a complete control of gvhd, provided a long-term immunosoppressive therapy free survival in absence of gvhd related deaths or longterm complications. introduction: despite major improvements in allogeneic hematopoietic cell transplantation (allo-hct) over the last decades, severe corticosteroid-refractory acute and chronic graft-versus-host-disease (gvhd) still remains a life-threatening complication characterized by high mortality rates (40-70%). since preclinical and early clinical evidence indicated anti-inflammatory effects of ruxolitinib, we collected the outcome data from multiple stem cell transplant centers using the jak1/2 inhibitor ruxolitinib as salvage treatment in patients suffering from corticosteroid-refractory gvhd. materials (or patients) and methods: a total of 13 stem cell transplant centers in germany, france, switzerland and united states reported outcome data from 52 patients who received ruxolitinib for corticosteroid-refractory gvhd (skin, mucosa, intestine, liver, lung, musculoskeletal) between 01/ 2012 and 12/2014. patients were classified as having acute (n ¼ 32) or chronic (n ¼ 20) gvhd. the median number of previous gvhd-therapies was 4 for acute gvhd (range: 1-7) and 3 for chronic gvhd (range: 2-10). results: the overall response rate was 84.3% (27/32) in acute gvhd comprising 10 crs (31.2%) and 17 prs (53.1%). in chronic gvhd the overall response rate was 80% (16/20). clinical improvement was rapid with a median time to response of 1.5 (1-4) weeks and 1 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) week after initiation of ruxolitinib treatment in acute and chronic gvhd, respectively. all responders were in persistent remission at last follow-up. the median follow-up was 18 (2-58) and 13 (2-70.5) weeks for acute and chronic gvhd patients, respectively. non-responders (acute gvhd: 5/32, chronic gvhd: 4/20) received other salvage therapies. cytopenias (anemia, leukopenia or thrombocytopenia) and cmv reactivation were observed during the time of ruxolitinib treatment in both acute (18/32, 56.2% and respectively 11/32, 34.3% ) and chronic (4/20, 20% and respectively 3/20, 15%) gvhd patients, sometimes however cytopenias already preceded ruxolitinib treatment. ruxolitinib treatment was stopped or reduced in 2 patients because of cytopenia. cmv was controlled by antiviral therapy in all patients. 3 ruxolitinib responders died, one because of leukemia relapse 4 weeks after ruxolitinib was stopped, and two from gvhd progression. in one of the patients who died, ruxolitinib was stopped due to impossibility of oral drug application. conclusion: overall, these data collected in multiple centers using different strategies for gvhd prophylaxis and treatment suggest that ruxolitinib is a very promising agent in the treatment of corticosteroid-refractory acute or chronic gvhd and may be successfully used to treat a major subset of patients beyond 2nd line of gvhd treatment. a prospective randomized multicentre clinical trial testing therapeutic jak1/ 2 inhibition as salvage treatment in gvhd is planned to verify the efficacy of ruxolitinib and to identify potential biomarkers that may be predictive for response. disclosure of interest: none declared. transplant-associated renal microangiopathy is associated with a high risk of refractory acute gvhd and characterized by a specific biomarker signature introduction: there is increasing evidence that endothelial damage is involved in the pathogenesis of steroid-refractory graft-versus-host disease (refgvhd). recently, serum soluble st2 (suppressor of tumorigenicity, il-33 receptor), an independent risk factor of cardiovascular death, has been identified as a risk factor of refractory gvhd. however, the pathomechanism of endothelial cell dysfunction which is associated with mortality from gvhd is yet poorly characterized. renal transplant-associated microangiopathy (tma) is another endothelial complication of allosct, and its association with severe gvhd and with biomarkers of endothelial damage (st2, scd141 (soluble thrombomodulin)), and endothelial function (vegf) is investigated in this study. materials (or patients) and methods: evidence for renal tma was studied in a cohort of 508 patients who underwent allosct between 2002 and 2013 at our institution and who have provided informed consent for this observational study. criteria to diagnose renal tma included an otherwise unexplained 50% rise in creatinine and lactate dehydrogenase (ldh) levels (or a pre-existing ldh above 400 u/l), a 50% drop in platelet counts (or a pre-existing platelet count below 50/nl) and at least 4% schistocytes. cytokines were measured in sera taken prior to allosct and on the indicated days thereafter and stored at minus 80c. statistical analyses were performed using spss19 and included a cumulative incidence analysis of causespecific hazards and the non-parametrical median test of independent probes. results: both renal tma and refgvhd were rare complications after allosct but were significantly associated with each other (tma only 18/508 (3.5%), refgvhd only 26/508 (5.1%), both 20/508 (3.9%), chi 2 0.000). median time intervals from allosct to renal tma and refgvhd were 1.93 (0.23-60.16 ) months and 1.23 (0.16-12.4 ) months respectively. in the overlap group, gvhd onset usually occurred before renal tma (0.69 months, -55.4 to 11.2) nrm rates were significantly increased in all three cohorts but approached 100% in patients with both complications. serum stm levels as well as soluble st2 levels increased between transplantation and day 100/day 200 after allosct in all cohorts, refgvhd, tma and both. in contrast, vegf levels (day 100) were significantly lower specifically in patients with tma with or without refgvhd, but not in patients with refgvhd without tma. conclusion: this study identifies renal tma as an endothelial cell dysfunction associated with extremely high mortality rates in the context of gvhd. the absence or presence of renal tma defines two separate subsets of refgvhd with a different prognosis. biomarkers of endothelial damage or vulnerability, such as stm, st2, and vegf can help to dissect tma and refgvhd, and might be useful to identify and guide management of patients with endothelial dysfunction who are at high risk of fatal complications after allosct. disclosure of interest: none declared. introduction: snps of the key cytokines and chemokines involved in the pathogenesis of agvhd have become an object of major interest recently. here we present snps rs3774937 cc/ct/tt and rs3774959 aa/ag/gg of the nf-kb1 gene in association with agvhd. materials (or patients) and methods: in our single-center study we analyzed 70 patients allografted for the following hematological malignancies: aml (37%), all (14%), cll (13%), mds (13%), cml (6%), nhl (6%), imf (3%), cmml (4%), and other hematological disorders (4%) between 2009-2014. the median age of the study group was 51 (21-62) years. patients were allografted after myeloablative (11%), non-myeloablative (14%) and reduced (intensity/toxicity) conditionings (74%). gvhd prophylaxis was done by solo cyclosporine-a (80%), cyclosporine-a with mycophenolate mofetil (19%) and cyclosporine-a with short-methotrexate (1%). ''in vivo'' t-depletion with thymoglobuline was used in 64% of recipients. patients were allografted from hla identical donors (related 46%) with median age 37 (18-63) years. the female donor/male recipient combination represented 14% of all pairs. the grafts contained median 4.6 (2.8-8.0 ) x106 cd34 þ cells/kg and median 6.8 (1.9-27.1) x108 mnc /kg. snps analysis was done from genomic dna isolated from edta-treated peripheral blood. genotyping was performed with sequenom massarray platform using allele-specific maldi-tof mass spectrometry assay (sequenom, san diego, ca, usa). primers were designed using the sequenom snp assay design software version 3.0 for iplex reactions. univariate analysis was performed to find significant difference in agvhd among the patient groups with different nf-kb1 profiles. the asymptotic pearson's chi-square test was used in cross tabulation with significance level set to 0.05. results : out of 70 patients 46 patients (66%) are still alive, 24 patients (34%) died (12 died of transplant-related complications). the median post-transplant follow-up was 1.6 (0.1-4.5) years. agvhd developed in 21 patients (30%), grade iii-iv in 7 of them (10%). both nfkb1 snps were completely correlated in the sense that knowing the genotype of one fully determined the genotype of the second one (5 patients did not follow this correlation and were excluded from the analysis). consequently, we defined a common predictor nf-kb1 which codes the information carried by both nf-kb1 snps in the following way: all patients carrying the rs3774937 cc genotype were also positive for rs3774959 aa allele and were marked as nf-kb1 ¼ i, all patients carrying the rs3774937 ct genotype were also positive for rs3774959 ag allele and were marked as nf-kb1 ¼ ii and finally all patients carrying the rs3774937 tt genotype were also positive for rs3774959 gg allele and were marked as nf-kb1 ¼ iii. the nf-kb1 profile was found to be significantly associated (p ¼ 0.002) with agvhd in the following way: patients in the nf-kb1 ¼ i group are more probable to suffer from agvhd than in the nf-kb1 ¼ iii group. conclusion: this is the first report showing the association of nf-kb1 gene snps with agvhd. the transcription factor nf-kb has been implicated in the regulation of cellular stress and inflammatory signals. according to our pilot data patients with inherited genetic abnormalities of the nf-kb1 gene may be prone to agvhd. patients.the two groups -control/treatment -were well balanced in terms of diagnosis (p ¼ 0.9), and disease phase (p ¼ 0.4) : the most frequent diagnosis was aml (n ¼ 75), followed by all (n ¼ 38) and mds (n ¼ 10). the median age for control/treatment was 46 years (1-69) vs 38 yers (0, , (p ¼ 0.06); the proportion of patients over 50 years was 51% in the control and 49% in treatment group (p ¼ 0.8). the donor type in the control/treatment arms was as follows: hla identical siblings n ¼ 36/n ¼ 34, unrelated cord blood (cb) n ¼ 6/n ¼ 2, unrelated donor (ud) n ¼ 36/n ¼ 42, and haploidentical family donors (haplo) 6/7 (p ¼ 0.4). skin biopsies.a skin biopsy before randomization, was not mandatory: it was performed in 38 patients. gvhd was diagnosed as proven, probable and possible respectively in s23 21%, 32% and 29%of the patients.these different reports were equally distributed in treatment and controls (p ¼ 0.7). results: the cumulative incidence of acute gvhd grade ii (primary end point), was 50% in controls and 35% in treatment patients (p ¼ 0.02). this difference was maintained in different subgroups. the ci of transplant related mortality (trm) was 18% (control) vs 27% (treatment) (p ¼ 0.1), despite a non significant younger median age in treatment patients. excess mortality in the treatment arm was due to an excess incidence of infections. actuarial 1 year survival was 80% (control) vs 78% (treatment) (p ¼ 0.1). cases of death in the control/treatment groups were as follows: gvhd 10% -13%; infection, 4%4 8%; interstitial pneumonia, 2% -0%; toxicity, 1% -2%; leukemia relapse 19% -18% (p ¼ 0.3). there was no significant difference in trm among different centers (p ¼ 0.5) progression of gvhd and skin biopsies.the proportion of patients progressing to gvhd grade ii þ was simlar in proven, probable, possible gvhd (62%, 50%, 45% ) (p ¼ 0.7). however the proportion of gvhd related deaths was 37% for proven gvhd, and 8% for probable/possible gvhd. introduction: graft versus host disease (gvhd) is a common and severe complication after allogeneic stem cell transplantation. during pregnancy, the placenta and the fetal membranes function as an immunological barrier, protecting the fetus from the mother's immune system. we have isolated stromal cells from the decidual layer of term placentas. decidual stromal cells (dscs) are of maternal origin and strongly inhibit the alloreactivity of t-cells in vitro. the effect is mainly contact dependent, and decreases the production of several key cytokines involved in the cytokine storm promoting the continuation of gvhd. materials (or patients) and methods: to investigate the effect of dscs on acute gvhd we enrolled 34 patients diagnosed with acute gvhd and clinically non-responsive to standard therapy. the protocol was modified after 17 patients, the dscs were then thawed and infused in infusion solution with albumin instead of ab-plasma and given repeatedly and earlier upon diagnosis. this led to the formation of two treatment groups (group 1 n ¼ 17 and group 2 n ¼ 17), which were compared to matched historical controls (n ¼ 54). we also performed a retrospectively corrected analysis of steroid refractivity. results: group 1 received a median of 1 infusion on day 11 after standard treatment compared to group 2, who received a median of 2 infusions (p ¼ o0,05) on day 7 (ns). no adverse events related to the treatment were observed. at 4 weeks after treatment, 60% of the patients in group 1 hade responded to the treatment. in contrast, all patients in group 2 responded (p ¼ o0,05). all patients in the treatment groups received fungal prophylaxis. the overall cumulative survival (os) at one year was 28% for the controls as compared to 47% for group 1 and 80% for group 2 (p ¼ o0.001). when the groups were corrected for steroid-refractivity, the os was 3% for the controls, 38% for group 1 and 70% for group 2(p ¼ o0.001). in the steroid-refractory control group (n ¼ 32), the risk of dying from gvhd at one year was 81%, whereas no patients in the steroid-refractory group 2 died from gvhd(n ¼ 13, p ¼ o0.001). in the last 19 months, no patients have died from acute gvhd at our center. in conclusion, dscs might be an effective treatment of acute gvhd. the infusion should be prepared in albumin, given as early as possible and in repeated doses. to confirm these striking findings, we will extend the followup time and enroll more patients in our study. disclosure of interest: none declared. oral session: stem cell source and donor type o041 unmanipulated haploidentical stem cell transplantation after reduced intensity or ablative conditioning regimen for the treatment of acute leukemia-a report from the acute leukemia working party of the ebmt m. introduction: haploidentical hematopoietic stem cell transplantation(haplo-hsct)is feasible option for patients with acute leukemia(al)at high risk of relapse who do not have hla-matched related or unrelated donors. haplo-hsct was associated with severe acute graft-versus-host disease (agvhd) in unmanipulated transplants and a high incidence of graft rejection in t-cell depleted transplants because of the high frequency of t cells that recognized major class i or ii hla disparities between donor and recipient. two approaches were developed to overcome these problems:megadose of t-cell depleted hematopoietic progenitor cells without any posttransplant immunosuppression and t-cell replete grafts with innovative pharmacological prophylaxis of agvhd.posttransplant cyclophosphamide(ptcy)is regarded as a gvhdspecific immunosuppressant in adults but its feasibility is unknown in children. purpose: to evaluate the feasibility and outcome of haplo-hsct in children and adolescents with acute leukemia in active disease depending on conditioning regimens and methods of harvesting haplo-graft. primary end points: overall survival (os), transplant-related mortality (trm). secondary end points: engraftment rate, agvhd, cgvhd, relapse rate. materials (or patients) and methods: 56 patients(range from 1-21y.o. median 9 y.o.)with al(all-32pts, aml-24pts)in progressive disease (pd)(cytoreduction chemotherapy (ctx) prior conditioning regimen-15pts, without-41pts) were analysed.mac þ atg regimen based on giac protocol received 20pts, mac þ ptcy 50 mg/kg on d þ 3, þ 4-5pts, ric þ atg regimen based on flu-18pts, ric þ ptcy 50 mg/kg on d þ 3, þ 4-13pts. all pts received prophylaxis of agvhd based on csa-30pts, tac-8pts, tac þ sir-18pts. g-csf-primed t-cell replete bm was used as a graft source in 33pts (median cd34 þ cells 4,7x10 6 /kg), g-csf mobilized peripheral blood (cd34 þ selected by clinimacs, miltenyi biotec) and g-csfprimed bm-23pts (median cd34 þ cells 11,3x10 6 /kg). [o042] results: 3-year os was 33,3%. ptso9y.o. had significantly higher os vs pts49y.o. 46,7% and 18,5% respectively (p ¼ 0,01). 3-year os in pts receiving ctx prior conditioning regimen was 50% vs 26,8% in pts with leukemic burden(p ¼ 0,02). significantly difference were observed in 3year os in pts transplanted g-csf-primed t-cell replete bm 45,5% vs 13% in pts after g-csf mobilized peripheral blood and g-csf-primed bm (p ¼ 0,03). pts receiving ric þ ptcy had 3-year os 61,5%, ric-11,1%, mac-35%, mac þ ptcy-20% (p ¼ 0,06). trm after haplo-hsct in pts with al in pd was 38%. engraftment was sustained in 82,1% pts. full donor chimerism was achieved in 73,2% pts on d þ 30. median anc engraftment (40,5x10 9 /l) d þ 19,plt recovery(420x10 9 /l) d þ 17. cumulative incidence of grade 2-4 agvhd was 27,3%,cgvhd-23,9%. cumulative incidence of relapse was 39,3%. conclusion: haplo-hsct g-csf-primed unmanipulated bm is an effective method of achieving remission with good sustained engraftment rate in children and adolescents with resistant disease. ric regimen followed by t-cell replete haplo-hsct with ptcy on d þ 3, þ 4 was associated with good os, low incidences of gvhd and trm. before any definitive conclusions can drawn, a randomized study is required. disclosure of interest: none declared. the detection of donor specific anti-hla antibodies in recipients of unmanipulated haploidentical blood and marrow transplantation is predictive of poor graft function introduction: our previous study suggest that choosing young, male, non-inherited maternal antigen-mismatched donors is reasonable following unmanipulated haploidentical blood and marrow transplantation (hbmt). recently, a correlation between the presence of donor-specific anti-hla antibodies (dsa) and graft failure has been demonstrated in haploidentical transplant settings. in our protocol, approximately 99% patients can achieve sustained, full donor chimerism. however, poor graft function (pgf) remains one of complications after unmanipulated hbmt. therefore, we determined the effect of dsa on primary pgf in order to provide further evidence for donor selection. materials (or patients) and methods: three hundreds and fourty-five patients with hematological diseases receiving hbmt were enrolled in this prospective study. the median age of the patients was 26 years (range, 2-58 years). these patients were randomly selected as training group (n ¼ 173) and validation group (n ¼ 172). peripheral blood serum were collected pre-conditioning regimens. dsa were determined using the luminex-based assay. results: in all 345 patients, the percentages of dsa positive cases were 11.3% (39/345). the incidence of dsa in female patients was higher than that of male cases (16% vs. 8%, p ¼ 0.020). in the training set, a cutoff value of dsa (mfi ¼ 2000) were developed. multivariate analysis showed that the presence of dsa (patients with mfiz2000 vs. cases with mfiz2000) was associated with primary pgf. in the validation set, the association of dsa with primary pgf following transplantation was also confirmed. the association of pgf with inferior overall survival (os) was demonstrated both in the training group and in the validation group. in all 345 patients, the median time to platelet recovery in dsa positive (mfiz2000) patients was slower than that of dsa negative ones (25 days vs. 18 days, p ¼ 0.004). the incidence of primary pgf and primary graft failure was 5.5% and 0.9%, respectively. dsa positive patients experenced higher incidence of primary pgf (31% vs. 3 introduction: transplacental trafficking of maternal and fetal cells during pregnancy establishes long-term, reciprocal microchimerism in both mother and child because of exposure of the two immune systems to the non-self alloantigens (maloney et al., j clin invest. 1999 ). studies show the immune system in the mother is capable of being sensitized by paternal histocompatibility antigens. for example, antibodies directed against paternal hla-antigens (van rood et al., nature. 1958 ) and t lymphocytes directed against paternal major (van kampen et al., hum immunol. 2001 ) and minor histocompatibility antigens (verdijk et al., blood. 2004) are frequently detected in multiparous women. we previously demonstrated mother/child immune interactions positively influenced the outcome of mother to child hla haploidentical t cell-depleted hematopoietic transplantation. in a series of adult and pediatric patients we demonstrated mother donors conferred protection against leukemia relapse and improved transplant related mortality (trm), which was largely due to infection, and improved survival (stern et al., blood 2008). materials (or patients) and methods: the kaplan-meier method evaluated leukemia-free survival. cumulative incidence estimates were used for relapse and trm, as they are competing risks. multivariate analysis assessed the impact of diverse variables on transplantation outcomes. results: we analyzed the outcomes of 238 adult acute leukemia patients after t cell-depleted haploidentical transplantation. when compared with transplantation from all other family members, transplantation from mother donors was associated with significantly lower trm (largely infectious) (27% vs 50% from all other donors, p ¼ 0.01). multivariate analyses demonstrated transplantation from mother donors was an independent factor predicting improved survival (hazard ratio 0.41, 95% confidence interval 0.12 to 0.95, p ¼ 0.03). in an attempt to elucidate the mechanism, we analyzed donor t cell repertoires that were specific for cmv antigens presented by recipient antigen-presenting cells (by elispot and by limiting dilution cloning). unlike all other donor/recipient pairs, mothers possessed cmv-specific cd8 cell clones that killed child's and father's cmv-pulsed dendritic cells (dcs). such clones were nonalloreactive as they did not kill the child's or father's non-cmv-s26 pulsed dcs. mothers also possessed cd4 t cell clones that produced ifn-gamma in response to child's and father's cmvloaded dcs. such clones were non-alloreactive as they did not respond to child's or father's non-cmv-pulsed apcs. thus, mothers possessed a t cell repertoire that recognized cmv antigens also when presented by the unshared, father's, hla haplotype. in fact, they showed twice as many t cells that recognized cmv antigens presented by the child's apcs than all other donor/recipient pairs (po0.05). conclusion: therefore, pregnancy resulted in the generation of an additional t cell repertoire that specifically recognized pathogen antigens presented by the unshared paternal hla haplotype antigens on the child's apcs. apparently, upon mother to child t cell-depleted hematopoietic transplantation, such repertoire expands over time and helps reduce infectious mortality. further studies are needed to elucidate the mechanisms underlying mother t cell selection/education by paternal hla haplotype antigens on the child's apcs. disclosure of interest: none declared. uni-directional and bi-directional non-permissive hla-dpb1 t cell epitope group mismatches have similar risk associations in 10/10 matched unrelated donor hct , was analyzed after separating uni-directional from bi-directional non-permissive mismatches. non-permissive mismatches were defined as unidirectional hvg when the donor but not the patient carried an hla-dpb1 allele from a tce group not present in the patient, and vice versa as uni-directional gvh. bi-directional nonpermissive mismatches were present when none of the hla-dpb1 alleles in patient and donor were from the same tce group. the associations with clinical endpoints of overall survival (os), transplant related mortality (trm), relapse, acute gvhd (agvhd) and chronic gvhd (cgvhd) were studied using multivariate proportional hazards methods. results: the number of transplants with permissive or nonpermissive uni-directional hvg, uni-directional gvh and bidirectional mismatches was 1537, 527, 529 and 143, respectively. in the trm analysis, non-permissive uni-directional hvg (hr 1.32, p ¼ 0.001) and gvh mismatches (hr 1.28, p ¼ 0.005) had significantly higher relative risks (rr) of trm compared to the permissive group. the bi-directional group had similar rr (hr 1.35 , p ¼ 0.05). in pairwise comparisons, there were no statistical differences between the uni-and the bi-directional non-permissive groups for any of the outcomes tested. introduction: there are several alternative sources of donor stem cells available for patients (pts) who need an allo-sct and especially for those who lack a hla-matched donor. outcomes of mismatched-unrelated-donor (mm-urd) transplant have recently improved, and a comparison between matched and mm-urd sources in a uniform cohort of pts has not been performed after ric regimens. materials (or patients) and methods: pts, aged z50 year, who underwent fully matched or mm-urd ric pbsct or bmt from 2000-2012 were included in the study. all donors were hla-matched (10/10) or mismatched at one or two-loci (9/10 or 8/10). the kaplan-meier-estimator, the cumulative incidence function and cox proportional hazards regression models were used where appropriate. results: in total 3197 pts receiving matched or mismatched ric-urd allo-sct were included in the study (aml 2947, all 250). 2370 10/10 hla-matched pts were compared with recipients receiving 9/10 (n ¼ 712) or 8/10 (n ¼ 115) after ric mm-urd allo-sct. median age of 10/10, 9/10 and 8/10 hlamatched recipients were 60 years. higher female donor to male recipients were in 8/10 cohorts (20%) compared to 10/10 (12%) and 9/10 (14%) group (p ¼ 0.02). more pts with cr2 and advanced-disease were among 9/10 and 8/10 cohort compared to 10/10 matched donor recipients (cr2 24, 24 and 20%; advanced disease 26, 25, 24% respectively; p ¼ 0.04). also higher percentage of pts with secondary leukemia were in 8/10 cohorts compared 10/10 and 9/10 matched donor (35, 27, 25%; p ¼ 0.07). percentages of engraftment (97%, 95%, 97%, p ¼ 0.16) were no different between the 3 groups. acute gvhd grade ii-iv was 27%, 33%, 33%, and grade iii-iv 10, 13, and 10%, respectively for 10/10, 9/10 and 8/10 matched donors, respectively (p ¼ 0.003 and 0.03). in univariate analysis, 2-year survival rate was significantly higher for pts receiving 10/10 donor ric-urd allo-sct in cr1 compared to 9/10 or 8/10 mm-urd (os: 55%, 46%, 46%, p ¼ 0.01; lfs: 51%, 43%, 45%, p ¼ 0.03, respectively). however, among the cr2 and advanced disease groups there were no differences in outcome between fully matched or mm-urd (9/ 10 or 8/10) donor (cr2 þ : os 49%, 43%, 48%, p ¼ 0.30; lfs 42%, 37%, 45%, p ¼ 0.36; advanced-disease: os 38%, 31%, 31%, p ¼ 0.39; lfs 32%, 26%, 28%, p ¼ 0.50, respectively). there was no difference in ri between the 3 groups and nrm was higher after fully matched donor compared to mm-urd (9/10 or 8/10) only in pts with cr1 diseases (p ¼ 0.02). multivariate analysis showed higher nrm after 9/10 (hr 1.33, p ¼ 0.001) compared to fully matched donor and no difference in nrm between 9/10 vs. 8/10 mm-urd. there was no difference in ri between 10/10 vs. 9/10 or 9/10 vs. 8/10 mm-urd donor. os and lfs were superior after fully matched donor vs. 9/10 mm-urd (os: hr 1.24, p ¼ 0.001; lfs: hr 1.19 , p ¼ 0.002). however, there was no difference in adjusted os and lfs between 9/10 vs. 8/10 mm-urd ric allo-sct. chronic gvhd rate was not different between matched or mm-urd allo-sct groups. conclusion: despite the limitations of a retrospective registrybased study, our analysis shows no significant outcome difference between 9/10 and 8/10 mm-urd allo-sct after ric regimen in patient aged z50 year. in the absence of prospective data, we conclude that mm-urd ric allo-sct is a therapeutic option for acute leukemia pts not having fully matched donor. disclosure of interest: none declared. introduction: the use of minors as hsc donors is medically and legally accepted and is increasing. however, there is a lack of understanding of the physical and psychosocial effects of pediatric hsc donation. the goal of this investigation was to longitudinally investigate hrqol in this group. materials (or patients) and methods: participants were related pediatric donors (n ¼ 105) who donated at domestic u.s. centers between 4/10 and 5/13. data were collected from donors and their parents via structured telephone interviews at pre-donation, and 4 weeks and 1 year post-donation. a healthy age/gender matched pediatric sample was generated from existing data for normative comparisons. interviews gathered socio-demographics, psychosocial characteristics, and multidimensional hrqol using the well-validated pediatric quality of life inventory (pedsql) which produces a total score and physical, emotional, social, school and psychosocial subscores. t-tests were used to compare hrqol from donor self-report, parental proxy-report, and the normative sample across the three assessment points. mixed logistic models were used to examine the effects of pre-donation variables on post-donation hrqol. results: donors were 5-17 yrs (median ¼ 11 yrs) and all but one were sibling donors. most parental respondents were mothers (74%; median age 40 yrs), 87% were married, and 36% had at least a bachelor's degree. donor vs proxy. across all hrqol domains except emotional functioning and at all three assessment time points, donor self-reported hrqol was significantly lower than that reported by parental proxies. donor vs norm. at pre-donation, as compared to the normative sample, donors reported significant hrqol deficits across multiple subdomains and in total hrqol (t ¼ -2.76,po.01). at 4 weeks post-donation, donors reported deficits in physical functioning (t ¼ 5.99, po.001 ) and total hrqol (t ¼ -2.82,po.01). at 1 year post-donation, donors reported deficits in physical (t ¼ -2.51,po.05) and school functioning (t ¼ -2.12,po.05). donors at hrqol risk. across the three assessment time points, 21%, 19%, and 17% of donors respectively had self-reported pedsql total scores below the standard cutoff indicating significant clinical risk of poor hrqol -scores below the cutoff are similar to those of chronically ill children. sixteen percent, 13%, and 5% were below the cutoff at only one, two, or all three assessments respectively. the youngest donors (5-7 yrs) were at significantly greater risk of being below the cutoff than were their older counterparts with 37%, 39% and 82% of this group below cutoff at each of the three assessments respectively. in multivariable analyses, the pre-donation factor most strongly and consistently associated with below cutoff pedsql scores at 4 weeks and 1 year post-donation was pre-donation donor self-reported pedsql score (likelihood ratio: 9.17,po.01; 15.54,po.001). conclusion: these findings suggest that there may be significant hrqol deficits among pediatric hsc donors and that in this particular context, parents are not able to accurately report those deficits. these findings also indicate that research to identify predictors of poor hrqol and the development of interventions to screen and address hrqol deficits are urgently needed. disclosure of interest: none declared. introduction: in allo hct patients (pts) with disseminated adv disease, mortality is reported to be up to 80%. antiviral treatment (tx) usually consists of iv cidofovir (cdv), which has a significant risk of nephrotoxicity. bcv is an orally-available, lipid-conjugate of cdv with no evidence of nephrotoxicity in clinical trials. the pilot portion of the phase 3 advise (cmx001-304) study was initiated in march 2014 to enroll b100 allo hct and other immunocompromised adv pts with, or at risk of progression to, disseminated adv disease, to guide the final study design. as of 10nov2014, 73 subjects have been enrolled and entered into the database, including 60 allo hct pts (48 with disseminated adv disease), 7 solid organ transplant pts and 6 ''other'' pts. preliminary safety and virologic results for the 48 allo hct pts with disseminated disease are described. materials (or patients) and methods: all subjects receive open-label bcv 100 mg (z50 kg) or 2 mg/kg (o50 kg) twiceweekly for 12 wks, extendable up to 24 wks for pts at high-risk of relapse, and are followed for 24 wks post-tx. adv dna viral load (vl) in plasma is measured using a quantitative pcr test (limit of detection [lod] 2 log 10 c/ml). results: baseline (bl) characteristics for the 48 subjects are: median (range) age 12 (0.7, 69) y, 65% o18 y; 69% male; median (range) plasma adv vl 4.6 (o lod to 7.6 ) log 10 c/ml (n ¼ 45); 42% adv positive by qualitative pcr in respiratory secretions, 60% in urine, 65% in stool; 29% with cmv in plasma, 6% ebv in plasma and 42% bkv in urine; 42% received prior iv cdv. as of 25nov2014, 5 subjects had completed tx and 21 had discontinued tx prematurely. the most common reasons for tx discontinuation were death (n ¼ 11) and adverse event ([ae] n ¼ 4). median (range) tx duration was 38 (1, 141) days (n ¼ 45). virologic response in cdv-naïve and exposed subjects with detectable plasma adv vl at bl are summarized in the table. in subjects with positive adv pcr at bl, 65% (13/ 20) cleared adv in respiratory secretions, 55% (16/29) in urine and 48% (15/31) in stool. through 08dec2014, 40% (19/48) of allo hct subjects with disseminated adv disease had died, with a median 71-day observation period for living subjects. no death was attributed to bcv. aes leading to permanent tx discontinuation attributed to bcv were vomiting and abdominal pain in 1 subject, and acute gvhd in 1 subject. median (range) change in adv vl from bl (log 10 c/ml) median (range) time to minimum on-tx (days) proportion ?3 log 10 reduction in adv vl or to undetectable at nadir minimum on-tx last on-tx cdv-naive (n=23) -2.0 (-5.1, 0.5) 1.8 (-5.1 , +2.1) 15 (3, 106) 57% (13/23) cdv-exposed (n=18) 1.5 (-5.4 , +0.6) 1.2 (-5.4 , +0.6) 15 (4, 77) 72% (13/18) s29 conclusion: the observed mortality rate was 40% for allo hct pts with disseminated adv disease in advise, which is lower than literature rates reported for this pt population (50-80%; ison 2006, sandkovsky 2014). bcv showed potent virologic activity in cdv-naïve and exposed pts with no new safety concerns. these preliminary data support expansion of the pilot portion to a definitive phase 3 study. introduction: the guidelines for immunization of hematopoietic stem cell transplant (hsct) recipients recommend 3 doses of anti-pneumococcal conjugate vaccine (pcv) from 3-6 months after transplant, followed by a dose of polysaccharide 23-valent (ppv23) vaccine at 12 months in case of no chronic graft-versus-host disease (gvhd), or an additional pcv dose in case of gvhd. however, due to lack of long-term data, there is no recommendation for boosts after 12 months. our goal was to assess the retainment of anti-pneumococcal antibodies in allogeneic hsct recipients vaccinated 10 years ago. materials (or patients) and methods: in 2009, the idwp published the results of the idwp01 trial that compared the immune response assessed one month after 3 doses of pcv7, started either at 3, or at 9 months after myeloablative hsct 1 . additionally, all patients received 1 dose of ppv23 at 12 or 18 months after transplant. all surviving patients had been assessed for anti-pneumococcal antibodies against the vaccine-serotypes 24 months after hsct. this study was the basis of the current guidelines for anti-pneumococcal immunization after allogeneic hsct. the present study included 30 surviving patients from the idpw01, who were assessed for antibody levels against the 7 pcv7-antigens and against 2 of the ppv23antigens (pn1 and pn5), between 8.3 and 11 years after transplant, i.e. 6 to 9 years after the last assessment in the initial study. the mean age was 39 y (18-55), and 18/30 had acute leukemia. only 7 had chronic gvhd (limited: 6, extensive: 1) and 2 had suffered a leukemia relapse. eleven (37%) had received an additional dose of ppv23 at a mean time of 6.5 years (2-11 years) after transplant, according to local procedure. the rates of persistent responses to all 7 antigens of pcv7 were 65.5% for an ab cut-off of 0.15 mg/ml, and 40% for a cut-off of 0.50 mg/ml. compared to the response rate at 24 months after transplant, these rates were not significantly decreased but showed important serotype-specific variability. similar findings were observed for pn1 and pn5 antibody levels. neither the recipient or donor age, donor type, source of stem cells, gvhd, nor the administration of an additional dose of ppv23 (given to 11/30 patients) influenced the maintenance of the response. the timing of the initial vaccination was the only parameter influencing the long-term response; patients who were vaccinated lately after hsct (from 9 months) had a significantly better maintenance of the response than patients vaccinated early (from 3 months) after transplant. the 3 patients who were not responders at 24 months and who received an additional dose of ppv23 at 39, 40 and 72 months after transplant, respectively, did not respond. conclusion: in long-term hsct survivors without severe chronic gvhd vaccinated against s pneumoniae according to the current guidelines, the specific immunity is not fully maintained a decade later. patients, who received an additional ppv23 dose after 24 months post-transplant, do not seem to benefit from this boost. boosts with pcv should be explored. so far, the optimal schedule of anti-pneumococcal vaccination in hsct recipients after 12 months remains to be established. references : early cytomegalovirus reactivation -a potential factor for early robust t cell reconstitution and possibly a prognostic factor for agvhd after hsct p. r many previous studies have shown that agvhd puts patients at risk of cmv reactivation, most likely due to more intensive immunosuppression. however, recent studies and case reports also show that that cmv-r could be a risk factor for agvhd. here, we studied the effect of cmv-r on t cell reconstitution in patients with and without agvhd. materials (or patients) and methods: 106 cmv r þ /d þ patients transplanted 2005-2013 in our institution were included in this study. all the patient samples were monitored for the cmv viral load (cmvpp65 expressing cells/400,000 leukocytes) and t cell reconstitution (cd3, cd4, cd8 and all available hla specific cmv tetramers for each patient) within the first 100 days after hsct. patients were subdivided into five groups: group 1: no agvhd but cmv-r (no-agvhd-cmv-r), group 2: agvhd after cmv-r (agvhd-after-cmv-r), group 3: agvhd before cmv-r (agvhd-before-cmv-r), group 4: agvhd but no cmv-r (agvhd-no-cmv-r) and group 5: no agvhd-and no cmv-r (no-agvhd-no-cmv-r). results: the characteristics for onset of cmv reactivation and agvhd in the different subgroups are provided in table 1 . cd3, cd4, cd8 and cmv specific t cells were analyzed on day 50±10days after hsct. in order to investigate the potential influence of cmv-r in the absence of agvhd on t cell reconstitution, we compared the t cell numbers in the groups cmv-r þ /-subsequent agvhd (i.e. group 1 þ 2) with group 5 (no-agvhd-no-cmv-r). we found significantly more cd3 (p ¼ 0.021) and cd8 t cells (p ¼ 0.0057) in groups 1 þ 2 compared to group 5. there were no differences in t cells between the groups with cmv-r þ /subsequent agvhd (i.e. groups 1 þ 2) compared to the groups with agvhd þ /-subsequent cmv-r (i.e. groups 3 þ 4). moreover, there were no differences in t cell numbers between the groups with agvhd þ /-subsequent cmv-r (i.e. groups 3 þ 4) compared to group 5. to study the impact of cmv-r on t cell reconstitution in patients with subsequent agvhd we compared the t cell numbers in group 2 (agvhd-after-cmv-r) with group 1 (no-agvhd-after-cmv-r). there were significantly more cd4 t cells (p ¼ 0.0041) and a trend for more cd3, cd8 and cmv-ctls in group 2 (agvhd-after-cmv-r) compared to group 1 (no-agvhd-after-cmv-r). subsequently, we compared the potential influences of cmv-r and of agvhd on t cell reconstitution. we found significantly more cd3 (p ¼ 0.0138) and cd8 t cells (p ¼ 0.0125) in group 2 (agvhd-after-cmv-r) compared to group 4 (agvhd-no-cmv-r). moreover, we studied the overall potential influence of cmv-r in the presence of agvhd on t cell reconstitution. we found a trend for more t cells (cd3, cd4, cd8 and cmv-ctls) in group 2 (agvhd-after-cmv-r) compared to group 3 (agvhdbefore-cmv-r). in conclusion, patients with agvhd after cmv-r had considerably more cd4 t-cells on day 50 compared to patients with cmv-r but no agvhd and significantly more cd3 and cd8 t cells compared to patients with agvhd but no-cmv-r. these results suggest that early cmv-r enhances overall t cell reconstitution which could be a potential risk factor for developing agvhd after hsct. single and double cbt were used in 289 and 159 cases, respectively. tbi was part of the conditioning regimen in 263 cases (59%). in vivo t-cell depletion by atg was used in 61% of patients. at least one pcr with a viral load 43 log/ml of blood was sufficient to define hhv-6 reactivation after the graft. the impact of hhv-6 reactivation on cbt outcomes has been studied as a time-dependent variable. in multivariate analysis, hhv-6 was independently associated with graft failure (hr: 1.44 conclusion: our study confirms that hhv-6 reactivation is a risk factor for graft failure in cbt recipients after myeloablative conditioning regimen. this result has to be confirmed prospectively and in the setting of reduced-intensity conditioning cbt. this paves the way also to test prospectively the indication of ganciclovir or foscarnet use as anti-hhv-6 prophylaxis in cbt recipients. disclosure of interest: none declared. introduction: candida is the second more frequent cause of invasive fungal infection in haematological immunocompromised hosts, especially in the patients who undergo an haematopoietic stem cell transplantation (hsct). the aim of this study was to analyse retrospectively the outcome of patients with candida infections acquired in the first 100 days after allogeneic hsct. materials (or patients) materials (or patients) and methods: in prospective study 173 allohsct recipients were included from dec 2012 to jul 2013. the median age was 34 y.o., males -54%. most of pts had high-risk acute leukemia (70%). allohsct from mud were performed in 57%, mrd -24%, haplo -11%, mmud -8%, predominantly with ric (80%). eortc/msg 2008 criteria for diagnosis and response to therapy were used. since 2011 active diagnostic strategy, including bronchoscopy with bal, in pts with ct-scan lung lesions before allohsct has been introduced to the routine practice. ''active ia'' is the ia diagnosed just before hsct. results: incidence of ia before allohsct was 22,5% (n ¼ 39/ 173). according to eortc/msg 2008 criteria 92% of pts had probable ia and 8% proven ia. the main sites of infection were lungs -95%, central nervous system -3%, and colon -3%, other localizations were observed mostly in a combination with lung involvement: sinuses -5%, spleen -3%, and liver -3%. antifungal therapy before allohsct was administrated in 69% pts (voriconazole -95%, other -5%) with the median duration of therapy -2 months. complete response to antifungal therapy was registered in 10 (26%) pts, partial response or stabilization in 17 (43%), and ''active ia'' in 12 (31%) pts. after allohsct all pts received antifungal therapy with voriconazole (first line -31%, continuation of treatment -43%, and secondary prophylaxis -26%). median length of treatment was 166 days (37-394). cumulative incidence of relapse or progression of ia after allohsct was 12,4% (n ¼ 6). relapse of underlying disease was the main risk factor for the relapse or progression of ia after allohsct (6% vs 33%, p ¼ 0,007). progression of ia after allohsct was treated with voriconazole 600 mg per day (n ¼ 1) and combination vori þ caspo (n ¼ 3). relapse of the ia after allohsct was treated with voriconazole 400 mg per day (n ¼ 2). no toxicity of the antifungal treatment was registered. complete response was achieved in 4 pts, and stabilization -2. 12-weeks overall survival (os) after the start of antifungal therapy was 67%. two pts died with the progression of the underlying disease. 100days os after allohsct was 70%, 1-year os after allohsct was 57%. there was no significant difference in os in pts with or without ia before allohsct. conclusion: incidence of the ia before allohsct was 22,5%. cumulative incidence of relapse or progression of ia after allohsct in pts with proven and probable ia before allohsct was 12,4%. relapse of the underlying disease was the main risk factor for relapse or progression of ia after allohsct. secondary prophylaxis with voriconazole should be used in pts with ia before allohsct. relapse or progression of ia after allohsct didn't impair os. ia is not a contraindication for allohsct. disclosure of interest: none declared. pt. 12 patients had acute kidney injury which was managed conservatively without the need for renal replacement therapy. 3/6 survivors have normal lft's, 2 patients have a residual mild increase in transaminases due to cgvhd, whereas 1 patient has a moderate increase in lft's due to cgvhd. conclusion: busulphan based conditioning were the most important risk factor for vod. myelofibrosis had a strong trend towards causing vod (p-0.06). early intervention with defibrotide along with supportive management was able to completely resolve vod in most of the cases and the 100 day mortality was only 3/13 (23%). only 1 death was directly attributed to vod and 1 death each due to sepsis and biopsy proven drug induced liver failure. disclosure of interest: none declared. validation of two new prognostic scores to predict nonrelapse mortality in patients undergoing reduced-intensity conditioning allogeneic hematopoietic cell transplantation p. barba introduction: in 2014, 2 new pretransplant predictive models of non-relapse mortality (nrm) for patients undergoing allogeneic hematopoietic cell transplantation (all-hct) have been created based on modifications of the hct comorbidity index (hct-ci) and the ebmt score. the first model (hct-ci/ age) consisted of the addition of an extrapoint for patients440 years to the hct-ci (sorror et al. jco.2014 ). the other model developed by the alwp of the ebmt combined 16 categories of the hct-ci and the ebmt score into an integrated score (versluis et al. leukemia. 2014 ). none of these models have been validated in independent cohorts. materials (or patients) and methods: we analyzed the predictive capacity of these new models and compared it with the hct-ci and the ebmt score in a population of reducedintensity conditioning allo-hct (allo-ric) consecutively transplanted patients in 2 spanish centers during an 11 year period (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) . the scores of all models were calculated by a single investigator as originally defined. risk-groups stratification was also performed as originally defined, except for the ebmt score in which patients were divided into low-(score 0-3), intermediate-(score 4-5) and high-risk (scores 6) according to percentiles 33, 66 and 100. for the hct-ci/age patients scoring 0 points (n ¼ 3) were grouped with those scoring 1-2 points. the predictive capacity of all models was calculated by means of the harrell's c-statistic and were compared by calculating a z-score and p values from the estimated standard error. results: a total of 232 patients were included. median age at hct was 55 years (range 18-71). most patients received allo-hct from hla identical sibling donors (n ¼ 170, 73%) mainly for acute myeloid leukemia and myelodisplastic syndromes (n ¼ 73, 32%). median follow-up for survivors was 5.5 years (range 0.3-11) . the median hct-ci/age and the alwp model scores were 4 (range 0-14) and 5 (range 1-15), respectively. the median hct-ci, ebmt scores were 3 (range 0-13) and 5 (range 1-7), respectively. risk group distribution of patients according to each model is summarized in introduction: systemic inflammatory response syndrome (sirs) is defined as an inflammatory state induced by infections or toxic damages. sirs is diagnosed when two or more of the following criteria are met: body temperatureo36 1 c or438 1c, heart rate490 beats/minute, tachypnea420 breaths/minute or paco2o32 mmhg, leukocyteso4000 cells/ mm 3 or412000 cells/mm 3 or presence of410% immature neutrophils. the goal of this study was to assess the incidence of sirs early after an allogeneic stem cell transplantation (allosct) (from day 0 to hematopoietic recovery) and evaluate whether sirs may influence the occurrence of acute s36 graft-versus-host disease (agvhd) and non-relapse mortality (nrm introduction: pure red cell aplasia (prca) after allogeneic hematopoietic stem cell transplantation (hsct) is a relatively rare complication after major abo incompatible allogeneic transplant. although reduced intensity transplant was a potential risk factor for prca, the impact of stem cell source has not been fully evaluated. we conducted a retrospective risk factor analysis for developing prca in 163 major abo incompatible transplant including 106 cord blood transplantation (cbt) and 74 reduced-intensity conditioning. materials (or patients) and methods: we reviewed the medical records of 668 adult patients who underwent allogeneic hsct for the first time at the toranomon hospital from 2006 to 2013. prca after hsct was defined as anemia with low reticulocyte counts (o1%) in peripheral blood for more than 60 days after transplantation in association with neutrophil engraftment and a lack of erythroid precursors in bone marrow. results: one-hundred and sixty-three patients with major or bi-directional abo incompatibility who achieved neutrophil engraftment and survived more than 60 days after hsct were included in this study. seventy four patients received reducedintensity conditioning, 106 patients underwent cbt, 39 did bone marrow transplantation (bmt) and 18 did peripheral blood stem cell transplantation (pbsct). reticulocyte engrafted (reticulocyte 4 ¼ 1%) in 160 patients with a median time of 29 days after hsct during this study, which was significantly longer after cbt compared to bmt/pbsct (31 days vs. 26 days, p ¼ 0.008). in 9 patients, reticulocyte count remained o1% beyond 60 days post-transplant, 5 of whom were diagnosed as prca with a cumulative incidence of 3.1%. prca was not observed in cbt patients, and the cumulative incidence of prca was significantly lower after cbt compared to bmt/pbsct (0% vs. 8 is the only curative treatment in fa. immune reconstitution after hsct is increasingly recognized as a critical determinant of morbidity and mortality in hsct. the aim of the study was to better understand the kinetics of immune reconstitution in children with fa who underwent allogeneic hsct after a fludarabine based reduced intensity conditioning regimen. materials (or patients) and methods: in this study, lymphocyte subgroups of children who underwent hsct were evaluated before hsct and 1, 3, 6, 12, and 24 months after hsct. children with fa (n:21) comprised the study group and children with non-malignant diseases (n:36) comprised the control group. in addition to classical lymphocyte subgroups; activated t lymphocyte subgroups including cd8/57( þ ), cd8/56( þ ), cd3/hla-dr( þ ), cd4/25( þ ), cd4/ 28( þ ) t lymphocytes were evaluated in study and control groups. results: when absolute levels of lymphocyte subgroups were evaluated in children with fa, cd3( þ ) lymphocyte count returned to pre-hsct levels at 12 months. cd4( þ ) t lymphocyte count reached to pre-hsct levels at 24 months. cd8( þ ) t lymphocyte count returned to pre-hsct levels at 3 months. cd19( þ ) b lymphocyte count turned to pre-hsct levels at 3 months. cd4/8 ratio returned to pre-hsct levels at 12 months. cd16/56( þ )cd3( þ ) nk-t and cd16/56( þ )cd3(-) nk lymphocytes returned to pre-hsct levels within 1 month after hsct. among hsct related complications; acute gvhd developed in 2/21 (9.5%) children in study group and in 10/36 (27.7%) children in control group. on the other hand, chronic gvhd developed in 1/21 (4.7%) children in study group and in 5/36 (13.8%) children in control group. when specific subgroups reflecting lymphocyte activation were evaluated in study and control groups; activated cd8/ 56( þ ) nk lymphocyte and cd8/57( þ ) t lymphocyte count returned to pre-hsct levels at 3 months in both groups. while activated cd3/hla-dr( þ ) t lymphocyte count returned to pre-hsct levels at 1 months in both groups; activated cd3/ hla-dr( þ ) t lymphocyte count was higher at 1, 6, and 12 months in control group. activated cd4/25( þ ) t lymphocytes returned to pre-hsct levels at 12 months in study group and those returned to pre-hsct levels after 24 months in control groups. cd4/25( þ ) activated t lymphocyte count was higher at 24 months in study group (pr0.05). cd4/28( þ ) activated t lymphocytes reached pre-hsct levels at 12 months in control group and at 24 months in study group. besides, cd4/28( þ ) activated t lymphocyte count was higher at 6 and 12 months in control group (pr0.05). in this study, we show that the kinetics of recovery of the lymphocytes subgroups in children with fa after hsct follows those patterns also described for children with other diseases: early recovery of nk cells (1 month), followed by effector cytotoxic t cells (3 months) and b cells (3 months) , and finally, cd4( þ ) t-helper cells (24 months). high levels of cd3/dr( þ ) activated t lymphocyte count at 1, 6, and 12 months and high levels of cd4/28( þ ) t lymphocyte count at 6 and 12 months in control group are attributable to the high frequencies of acute and chronic gvhd in control group than those of study group. disclosure of interest: none declared. introduction: although hla haploidentical hsct has been largely employed in children with life-threatening nonmalignant disorders, the survival of patients given this type of allograft has been reported to be inferior to that of patients transplanted from a compatible unrelated volunteer (uv). we implemented a novel method of ex vivo t-and b-cell depletion based on the selective elimination of ab þ t cells and b cells. we herein report an update of 31 children with non-malignant disorders who were given this type of allograft. materials (or patients) and methods: twenty-two patients were males and 9 females, median age at hsct being 3.5 years (range 0.3-13.2) . nine patients had severe combined immunedeficiency (scid), 8 fanconi anemia (fa), 4 severe aplastic anemia (saa), 2 thalassemia major, 2 hemophagocytic lymphohistiocytosis (hlh) and 1 each immunedeficiency with polyendocrinopathy enteropaty x-linked (ipex), kostmann syndrome, hyper ige syndrome, osteopetrosis, swachmann-diamond syndrome and congenital amegakaryocytic thrombocytopenia (camt). all patients were transplanted from 1 of the 2 parents (21 from the mother and 10 from the father), the median number of cd34 þ and ab þ t cells infused being 22.57 x 10 6 /kg and 4 x 10 4 /kg. the original conditioning regimen consisted of treosulphan and fludarabine (flu) þ thiotepa in 13 (9 scid, 1 ipex, 1 camt, 1 kostmann syndrome and 1 swachmann-diamond syndrome), flu and cyclophosphamide þ single dose tbi in 12 (8 fa and 4 saa) and busulphan, flu and thiotepa in 6 (2 thalassemia, 2 hlh, 1 osteopetrosis and 1 hyper ige syndrome). no patient received immunosuppression after hsct. all patients received fresenius rabbit atg (4 mg/kg/day) on days -5 through -3 before allografting and rituximab (200 mg/m 2 ) to prevent ebv-related ptld on day -1. results: all patients but 6 engrafted, the median time to reach neutrophil and platelet recovery being 13 days (range 9-23) and 9 days (range 7-40), respectively. the 6 patients (2 with saa and 1 each with thalassemia, fa, hlh and osteopetrosis) who had primary graft failure were successfully re-transplanted (2 from the same parent, 3 from the other relative and 1 from an 1-hla locus disparate uv figure 1a ). in the latter group, agvhd started median 21 days after hsct and was zgrade iii and steroid refractory in 3/6 cases. a higher agvhd rate was also observed in male patients who received a graft from a female donor (f-4m mismatch) compared to all other sex-matches (5/9 vs. 2/16, p ¼ 0.02). in multivariate cox regression analysis, both omission of mtx and f-4m mismatch were associated with agvhd occurrence. in line with this, the protective effect of mtx was most pronounced in the subcohort of patients with a f-4m mismatch: grade ii-iv agvhd occurred in 5/5 f-4m mismatched non-mtx recipients whereas 0/4 f-4m mismatched mtx recipients developed agvhd (p ¼ 0.005). all relapse occurred in 9/25 patients (36%). we did not observe differences in relapse rate between non-mtx recipients and mtx recipients (4/11 vs. 5/14, p ¼ 0.97, figure 1b) . although non-mtx recipients were more often transplanted for all in 2 nd remission, pre-transplantation minimal residual disease (mrd) status did not differ between the groups. in line with earlier reports, mrd positivity was the most important risk factor for all recurrence. in mrd positive patients, the omission of mtx and agvhd occurrence did not prevent relapse after hsct. conclusion: mtx prophylaxis reduced the occurrence of severe agvhd, without compromising relapse free survival in pediatric all patients after t cell replete hla-identical bone marrow transplantation. prevention of agvhd reduces morbidity and the need for high-dose immunosuppressive agents, allowing for alternative immunotherapy-based therapeutic interventions in individuals at high risk for disease recurrence. introduction: hematopoietic stem cell transplantation (hsct) has contributed to improved outcome in childhood acute leukemia (al). however, post-hsct relapse is associated with a dismal prognosis and its optimal treatment remains unclear. we aimed to compare patients' related factors and treatment strategy, in case of relapse or progression post-allogeneic hsct in children with al in a recent ten-year period. materials (or patients) and methods: a total of 334 children who received a first allogeneic hsct for all or aml from january 2000 to december 2009 experienced a relapse or progression thereafter. they were treated in the 33 centers of the sfgm-tc, among them 279 cases were analysable. primary endpoint was overall survival (os) after diagnosis of relapse or progression post first hsct whatever the treatment post relapse was. . lymphocyte (sub)populations were analysed frequently post transplantation by flow cytometry. cd3 þ t-cell recovery was defined as appearance ofz100 cells/ml, b-cell and nk cells recovery asz50 cells/ml. the median active atg serum concentration at time of t-cell or b-cell reappearance was 0.03 au/ml, the maximum level was 1 au/ml. in 25% of the patients, nk cells re-appeared when the median active atg level was higher (0.16 au/ml) up to a maximum level of 7 au/ml (see figure) . for alemtuzumab the median concentration at t-cell recovery was 0.008 mg/ml, max. 0.2 mg/ml, but in 80% of the patients nk cells reappeared at a higher concentration up to 2.2 mg/ml. for both drugs, t-cell recovery was significantly correlated with the serum level of the drug (po0.001 for atg, and po0.01 for c1h), whereas a significant correlation was absent for nk cells. conclusion: atg and alemtuzumab are both able to deplete t-, b-, and nk cells. for both drugs, the exposure is highly variable even in patients with an equal weight receiving the same dose. here, we report that t-cell recovery is closely related with serotherapy exposure. nk-cells are the first cells that re-appear post hsct . all of the patients received the same ric regimen based on the use of fludarabine in combination with melphalan and antithymocyte globulin (atg). prophylaxis against gvhd was achieved via cyclosporine and methylprednisolone. results: all patients were engrafted. the median times to neutrophil and platelet engraftments were 11 days (range: 8-33), and 22 days (range: 10-67), respectively. patients underwent hsct from hla matched sibling donors (n ¼ 9), full matched other related donors (n ¼ 6), unrelated matched donor (n ¼ 1) and unrelated mismatched donor (n ¼ 3). the source of graft were peripheral blood (n ¼ 11), bone marrow (n ¼ 6) and cord blood (n ¼ 2 materials (or patients) and methods: twenty-nine patients with miop were treated by hsct at the university childreń s hospitals in paris (n ¼ 9, since 2008) and ulm (n ¼ 20, since 1998). miop was caused by mutations in tcirg1 (n ¼ 20), clcn7 (n ¼ 5), snx10 (n ¼ 1), rank (n ¼ 2), and fermt3 (n ¼ 1); age at transplant was between 2 and 72 months (median 6 months); donors were haploidentical family donors (n ¼ 16), mud (n ¼ 7), phenoidentical relatives (n ¼ 4), and msd (n ¼ 2). thiotepa and serotherapy was added to the busulfan and fludarabine based regimen in all patients with donors other than msd. results: all but 6 patients showed a primary and sustained engraftment; 4 of 6 patient, who rejected their haploidentical graft, could be rescued by a second graft from the second parent. severe vod, which had to be treated by ascites puncture, was seen in 2 patients only. only one case of gvhd4 12 and no case of chronic gvhd was observed. cause of death in 4 patients were liver toxicity in conjunction with cmv and fungal infection after prolonged aplasia (mud transplant at age of 1 month) and complications in conjunction with engraftment failure ( introduction: bmt is the only proven curative treatment available for haemoglobinopathies. however, the number of patients who can benefit is seriously restricted by the lack of hla-matched related donors not suffering from the condition and the limited number of unrelated donors available for the ethnic groups in which these conditions are prevalent. in order to expand the donor pool, haploidentical transplantation with a post-infusion of stem cells cyclophosphamide approach has been developed for young adults, but whilst well tolerated it has resulted in relatively high rates of rejection and the need for a prolonged period of immunosuppression 1 . materials (or patients) and methods: 12 consecutive parental haploidentical transplants (11 for sickle cell disease and one for b halassaemia major) were performed at st. mary's hospital, london, from june 2013 to november 2014. the median age was 9.5 years of age (range 3 to 14). all patients lacked a suitable hla-matched related donor and an unrelated search had not identified a 10/10 or 9/10 donor. endogenous haemopoieis was suppressed with hypertransfusions, hydroxycarbamide 30 mg/kg and azathioprine 3 mg/kg for at least two months pre-transplantation. the conditioning included fludarabine 150 mg/m 2 , thiotepa 10 mg/kg was added, cyclophosphamide 29 mg/kg, tbi 2 gy and atg (thymoglobulin) 4.5 mg/kg. gvhd prophylaxis was provided with cyclophosphamide 50 mg/kg on days þ 3 and þ 4, mmf and sirolimus. the minimum follow-up was 40 days post-transplantation and half of the patients are 4150 days post-transplantation and have completed all treatment. the source of stem cells was g-csf primed bone marrow in all cases, aiming 48 x 10 8 tnc/kg. results: all patients engrafted, though one patient subsequently suffered secondary graft failure following macrophage activation syndrome and died. the median neutrophil engraftment was 17 days (range 16 to 19). all 11 surviving patients are cured from the manifestations of the original disease. none of the patients suffered vod, though infectious complications occurred at a higher rate than seen for related transplants for the same conditions at our institution. five patients had no evidence of acute or chronic gvhd. five patients developed stage 1 acute gvhd (median presentation day þ 36, range 21 to 66) responding to topical steroids; one patient suffered skin gvhd stage 3 on day þ 35 and one patient gut gvhd stage 4 on day þ 24, both treated with msc. all patients responded to first line treatment with no recurrence of disease. all patients but one achieved 490% donor fraction both in whole blood and t cells. one patient requires immunosuppression beyond day þ 180 with stable 59% donor fraction in whole blood and 10% in t cells. introduction: bone marrow transplantation (bmt) offers a definitive cure for thalassemia in over 90% of low-risk children with a matched related donor. many centers currently incorporate thiotepa in busulfan-or treosulfan-based bmt regimens for thalassemia. this combination, however, may permanently impair fertilty in most patients. in the era of increasingly effective supportive care in which many thalassemia patients may have children, this is concerning. very longterm follow up studies have shown how the standard bu-cy regimen may be associated with birth rates comparable to the control population (la nasa et al. blood 2013). this study retrospectively compares bmt outcomes in two groups of low risk patients (defined as livero2cm and ageo12y) with severe thalassemia (st) (defined as a thalassemia syndrome with spontaneous hemoglobino7 g/dl), receiving oral busulfan (14 mg/kg), cyclophosphamide (200 mg/kg) and either thiotepa (10 mg/kg) (tt-bu-cy) or rabbit atg (fresenius 16 mg/kg or thymoglobulin 4 mg/kg total doses from day -12 to -10) (atg-bu-cy) as preparative regimen. standard cyclosporine and short-term methotrexate plus low dose methylprednisolone were used for gvhd prophylaxis. materials (or patients) and methods: this is a retrospective multicentre comparative study of the safety and efficacy of substituting tt with atg in low-risk st patients undergoing matched-related bmt. between january 2009 and july 2013, a group of 35 patients were transplanted after conditioning with tt-bu-cy, while between august 2013 and july 2014, 30 patients were conditioned with atg-bu-cy. results: the actuarial overall survival in the tt-bu-cy and atg-bu-cy groups is 91.3% and 93.4%; thalassemia-free survival is 88.4% and 93.4% at a median follow up of 23.5 and 3.1 months respectively, with no statistically significant difference by logrank test. conclusion: substituting thiotepa with atg in the standard bu-cy context seems safe and effective. higher fertility rates are expected for patients on the atg-bu-cy regime. disclosure of interest: none declared. materials (or patients) and methods: data has been collected retrospectively from 234 infants with scid who received transplants at 8 different centers over a 20-year period . the differences between groups were compared by using chi-square or fisher's exact test, where appropriate. a p value of less than 0.05 was considered statistically significant. results: 145 boys (62%) and 89 (%38) girls with scid whose ages ranged between 0.25-176 months (median 5 months) at the time of diagnosis were transplanted. parental consanguinity was identified in 171 (76%) of 224 infants. 72% of the patients had received bcg vaccination before diagnosis. b þ and b-phenotypes were detected in 41.8% (n ¼ 97) and 51.3% (n ¼ 119) respectively, while ada deficiency was recognized in 4.7%, rd (reticular dysgenesis) in 2.2% of the cases. rag1, jak3, rag2 and artemis defects were the leading genotypes among the patients with molecular diagnosis (42.3%; n ¼ 101). out of 234, 114 patients (48.7%) had either a matched sibling or a family donor, while 81 (34.6%) and 39 (16.7%) children received haploidentical (mmfd) and mud transplants respectively. the hsct source was bone marrow in 113 (48.3%), peripheral blood in 89 (38%) and cord blood in 32 (13.7%) of the patients. among a total of 24 (10.25%) retransplants, 18 received a second transplant while 6 children received a boost only. 153 children survived, 80 died and 1 was lost to followup. the overall survival rate was 65.7% over a 20 years period. it increased from 54% (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) to 69% (p ¼ 0.052) during the latter 10 years (2005-2014) and even to 72,9% during the last 4 years (2010-2014). the survival rates with relation to donor types were as follows: msd ¼ 85.7% (n ¼ 77), mrd ¼ 70.3% (n ¼ 37), haploidentical 47.5% (n ¼ 80) and mud 59% (n ¼ 39). age at diagnosis significantly (r5 months or 45months) influenced the survival rate of the patients (p ¼ 0.002). immunophenotype did not seem to have an effect on survival rate and immunoglobulin (ig) requirement following hsct did not differ between b þ vs b-phenotypes (p4 0.05). conclusion: this is the first multicenter study with the largest data obtained from scid patients transplanted in turkey. the median age at diagnosis was 5 months, b-phenotype and rag were the most common among other defects. age at diagnosis (45 months), and donor type (haploidentical) (po0.01) were two major factors significantly related to poor outcome. expanded donor availability, advances in intensive care facilities, diversity of transplantation centers and specialized teams are among major factors contributing to the longterm outcomes of hsct. however, newborn screening is of paramount importance in ensuring early diagnosis and timely transplantation thus improving the survival of scid patients in turkey. disclosure of interest: none declared. oral session: stem cell mobilization & regenerative medicine haematology, bmt unit, hospital clínico universitario virgen de la arrixaca, imib, university of murcia, 2 surgery, hospital clínico universitario virgen de la arrixaca, 3 haematology, bmt unit, hospital clínico universitario virgen de la arrixaca, imib, university of murcia, murcia, spain introduction: amniotic membrane (am) is a non-tumorigenic tissue attributed with various biological properties (low immunogenicity, anti-inflammatory, anti-fibrotic and antimicrobial effects) related to its ability to synthesize and release cytokines and growth factors. am, that is usually discarded after birth, is in our experience an easily obtained tissue which processing, storage and management can be included in the daily routine of the cryobiology laboratory. ma can be used as a ''biologic bandage'' for healing management of chronic wounds in diabetic and non-diabetic patients. in our hospital there is an ongoing clinical trial to study the use of am to improve epithelialization (nct01824381). here, we describe the results obtained prospectively after the compassionate use of cryopreserved human amniotic membrane allografts in 6 patients with chronic diabetic foot ulcers. materials (or patients) and methods: am was obtained from healthy mothers who had programmed an elective caesarean operation for obstetric reasons after they signed the informed consent. donors were screened by reviewing their medical records and by performing laboratory test to discard transmissible disease agents. am processing was done under sterile conditions in the gmp facility; the process involves: washing the am to eliminate blood traces, cut am into several fragments, sew each fragment on an impregnated dressing sheet and introduce them on cryopreservation bags adding the cryoprotectant solution based on human albumin, tc-199 medium and 9% dmso. the am fragments were storaged at -1961c and delivered once we the negative viral serology of the donor was confirmed 3 months later. cryopreserved am was applied to six consecutive patients with diabetic foot ulcers under a compassionate use program of the diabetic foot unit from may to november 2014. wound size reduction and rates of complete healing were evaluated. results: patients were aged between 45 and 65 years. they were affected by grade ii diabetic foot ulcers in the wagner ulcer classification scale that had failed previous treatments for periods between 2 months and 4 years. am was applied weekly or every ten days until complete healing or partial reepithelialization of the ulcers. a median of 5 (3-8) cryopreserved am fragments were applied for an average treatment period of 45 days. the mean size of the ulcer was reduced by 76%.l wreduced in size by 76%. at last follow-up, 4 out 6 patients have total epithelialization of the ulcer. no adverse events related to its application were observed. conclusion: our results show that the application of cryopreserved amniotic membrane is a feasible and safe treatment in complex diabetic foot ulcers. more rapid healing may decrease clinical operational costs and prevent long-term medical complications. furthermore, the treatment achieves re-epithelialization of long evolution wounds that were not reached with conventional therapies. disclosure of interest: none declared. bone as a regulator of human hematopoietic stem cell (hsc) trafficking: study of biochemical markers of bone remodeling and angiogenic cytokines during hsc mobilization, in patients with lymphoma and myeloma p. tsirkinidis 1,* , e. terpos 2 , g. boutsikas 1 , a. papatheo-dorou 3 introduction: bone is not considered just a structural, supportive tissue of bone marrow, but an hsc-niche regulator. data regarding the role of bone turnover in hsc mobilization in humans are scarce. the aim of the present study was to study bone remodeling and vessel equilibrium during hsc mobilization in lymphoma and multiple myeloma patients. materials (or patients) and methods: forty-six patients (32 lymphoma and 14 multiple myeloma) were studied. serum samples were collected at two time points: before mobilization (pre-mobilization sample) and on the day of hsc collection, which coincided with the peak circulating cd34 counts (collection sample). in 19/46 patients, 3 additional serum samples were collected, between mobilization and collection. the following molecules were measured by elisa in patients' sera: 1) bone resorption markers: carboxyterminal telopeptide of collagen type 1 (ctx), aminoterminal telopeptide of collagen type 1 (ntx), tartrate resistant acid phosphatase isoform 5b (tracp-5b), 2) bone formation markers: bone alkaline phosphatase (balp), osteocalcin (osc), osteopontin (opn), 3) the osteoblastogenesis inhibitor dickkopf-1 (dkk-1) 4) the osteoclastic regulators: receptor activator nf-kb ligand (rankl), osteoprotegerin (opg), 5) angiogenic cytokines: angiopoietin-1 (angp1), angiopoietin-2 (angp2), angiogenin (ang). values were compared with non-parametric methods. patients who had either a collection of cd34 þ cells o2.0x10 6 /kg, or a circulating cd34 count peak o20/ml were considered poor mobilizers. results: the comparison of the aforementioned molecules between the pre-mobilization and collection samples revealed: balp (p ¼ 0.000) and opn (p ¼ 0.049) increased significantly, while osc, a marker of bone turnover, and dkk-1 decreased significantly (p ¼ 0.000 and p ¼ 0.041, respectively). these findings reveal a significant increase of bone formation during mobilization. rankl (p ¼ 0.000) and opg (p ¼ 0.000) increased significantly, leading to an increase of rankl/opg ratio (p ¼ 0.000), consistent with osteoclastic activation. however, there was no evidence of increased osteoclastic activity, as ctx decreased significantly (p ¼ 0.026), while both ntx and tracp-5b did not change. angp-1 showed a dramatic reduction (p ¼ 0.000), while angp-2 increased (p ¼ 0.000), resulting in a significant decrease of the angp-1/angp-2 ratio, a finding indicating vessel destabilization during mobilization. these results were further supported by the intermediate measurements, which showed a straightforward alteration of bone metabolism early in hsc mobilization. poor mobilizers had significantly higher ctx levels both at premobilization (p ¼ 0.004) and collection samples (p ¼ 0.001), higher ntx levels at collection (p ¼ 0.02), lower angp-1 premobilization (p ¼ 0.004) and higher osc at collection (p ¼ 0.000) compared to good mobilizers. thus, ctx, ntx s48 and angp-1 pre-mobilization levels may serve as reliable predictors of poor mobilization. conclusion: our study showed for the first time in humans, that bone plays a dynamic role during hsc mobilization: bone formation and vessel destabilization are the two major events and osteoblasts seem to be the orchestrating cells during this process. osteoclasts are stimulated, but not fully active. moreover, some of these markers may identify poor mobilizers. disclosure of interest: none declared. introduction: allogeneic bone marrow transplantation is a curative treatment for leukemia and genetic disorders. although some patients are cured of their underlying illness, they are at risk of developing potentially fatal graft-versus-host disease (gvhd). a recent phase 3 randomized trial conducted by the canadian bmt group (n ¼ 230) comparing the impact of g-csf mobilized peripheral blood (pb) to bone marrow (bm). a representative group of 85 donor samples were evaluated and identified that high concentrations of cd56 bright nk cells in the donor product is strongly associated with a lack of development of both acute and chronic gvhd (odds ratio 0.11 ; p ¼ 0.0002) in g-csf stimulated bm. we found that the cd56 bright nk population was cd335 (nkp46) positive with comparable expression of cd337 in both sub-populations consistent with regulatory nk cells (nk reg ). we hypothesized that alternate strategies utilizing the mobilizing agent, plerixafor, may enrich for cd56 bright nkp46 cells in pb thus reducing the risk of gvhd and circumventing the need of harvesting donor cells from the bm. materials (or patients) and methods: we performed a pilot study to examine the impact of plerixafor, that blocks the sdf-1 cxcr4 interaction, in mobilizing cd56 bright nk reg cells in the pb and bm, to determine the whether we can optimize a donor source with the highest potential for post transplant tolerance, resulting in a lack of both agvhd and cgvhd. we recruited 9 healthy adult human volunteers. five subjects received one dose of plerixafor at 240 micrograms/kg/day and pb and bm samples were harvested prior to, at 4, and 24 hours after plerixafor administration. the subsequent 4 participants each received 4 daily doses of granulocyte colony stimulating factor (g-csf) at 5 micrograms/kg/day for 4 days, followed by a dose of plerixafor on day 5. pb and bm samples were collected prior to g-csf, after g-csf, and at 4 and 24 hours after plerixafor administration. we used multi-parametric flow cytometry to identify cd56 bright nk cells. the phenotype of cd56 bright nk cells was cd56 bright cd335 þ (nk46 þ ) cd3 -cd16perforingranzyme b -. results: we observed a significant rise in pb nucleated cells at both 4 and 24 hours after plerixafor administration with a peak increase at 4 hours. plerixafor alone induced a significantly higher proportion of cd56 bright nk reg cells in pb when compared to g-csf for 4 days (2.5 vs. 0.94 fold, p ¼ 0.041) and g-csf for 4 days followed by plerixafor treatment (2.5 vs. 0.43 fold, p ¼ 0.011). moreover, cells collected 4 hours after plerixafor alone had a significant increase in the proportion of cd56 bright nk reg cells (2.5 vs. 0.89 fold, p ¼ 0.031) in pb compared to bm. this study suggests that plerixafor alone is able to mobilize a high number of cd56 bright nk reg cells in pb harvested after 4 hours. we were not able to increase that population in a similar manner by using g-csf alone or g-csf plus plerixafor. these findings suggest that allogeneic donor mobilization of peripheral blood may give a product with a low rate of gvhd potentially superior to g-csf stimulated marrow and warrants further testing in a randomized clinical trial. disclosure of interest: none declared. introduction: only small studies have compared performance of the novel spectra optia apheresis system (terumo bct) with the widely used com.tec (fresenius healthcare) and the late cobe spectra (terumo bct) device in allogeneic stem cell collections. our collaborative working group compared performance data of all 3 devices from two collection centers to analyze device-as well as center-specific performance parameters. materials (or patients) and methods: we analyzed 5288 firstday apheresis collections in g-csf-stimulated healthy donors that were performed in cologne (cgn) using spectra optia mnc (n ¼ 1818), cobe spectra mnc (n ¼ 877), or com.tec (n ¼ 1657) and in dü sseldorf (dus) using spectra optia mnc (n ¼ 63) or cobe spectra mnc (873). peripheral blood and product samples were analyzed in center-specific laboratories. results: in both centers, irrespective of the apheresis device, similar yields of cd34 þ cells per kg donor bodyweight were reached. in cgn, collection rates (collected cd34 þ cells per kg donor bodyweight per cd34 þ cell count in peripheral blood before apheresis) were similar between the three apheresis systems. the continuously collecting cobe spectra yielded more cd34 þ cells over time (cr per h: 0,034 ± 0,009) than the discontinuously collecting apheresis systems 030±0, 009, po0, 001) and spectra optia mnc (0,025 ± 0,008, po0,001). the com.tec collected cd34 þ cells with highest efficiency (ce2: 63 ± 15% vs 61 ± 16%, po0,001 and 55 ± 15%, po0,001 for spectra optia mnc and cobe spectra, respectively). in comparison to cobe spectra (178±49 min), procedure times were longer when using com.tec (199±50 min, po0, 001) or spectra optia mnc (237±58 min, po0,001) systems. the product purity, measured as percentage of mnc was highest in products collected with the spectra optia mnc (79 ± 15% vs 76 ± 10%, po0,001 vs 75 ± 14%, po0,001 for cobe spectra and com.tec, respectively). the relative differences between cobe spectra and spectra optia collection performance parameters were similar in the dus apheresis center: shorter procedures with higher cr per h for cobe spectra, higher mnc purity in spectra optia mnc products. absolute results differed between the two centers. conclusion: the highest collection efficiency (ce2) was seen when the com.tec device was used. however, this was accompanied by low product purity. compared to any other apheresis device that has been analyzed in this study the novel spectra optia allows collection of higher mnc purity apheresis products from allogeneic donors. however, this was associated with a significant prolongation of procedure timethe major disadvantage of this device. the cobe spectra collects more cells over time by allowing higher mean inlet flow rates despite inferior collection efficiency. therefore, a continuous collection procedure on the spectra optia device that allows high inlet flow rates would be an ideal collection setting in terms of collection efficiency and collection rate per unit of time. disclosure of interest: none declared. introduction: our group has previously established the safety and effectivity in terms of cd34 þ cell recovery and viability of the automated washing of cryopreserved hematopoietic progenitors (hp) with the sepax â device (biosafe) using normal saline supplemented with 2.5% albumin (nsa), as well as the absence of infusion-related events and of a negative impact on engraftment dynamics (sánchez-salinas et al. 2012 ). in our present study we compare this solution with a ready-to-use free of human derived components solution: 6% hydroxyethyl starch 130/0.4 in 0.9% sodium chloride injection (voluven â , fresenius kabi). materials (or patients) and methods:: 411 peripheral blood hp units apheresis cryopreserved using autologous plasma plus 9% dmso corresponding to 158 autologous peripheral blood hp transplants were studied. after rapid thawing in a water bath at 371c, an automatic wash with the sepax (2 washes cycle) was performed using either nsa (229 units) or voluven (182 units). nucleated cell levels determined by an hematology analyzer, flow cytometry cd34 þ cell counts, trypan blue cell viability and granulocyte macrophage (gm-cfu) and erythroid (e-bfu) colony forming cell cultures were performed on aliquots collected prior to and after the washing technique. statistical analysis was performed using descriptive statistics and a simple-measures anova. results: the mean total nucleated cell (tnc) and cd34 þ cell recovery was 75,12% ±14,66, and 113,18% ±57,76 respectively for nsa and 79,08% ± 14,75 and 110,02% ± 44,02 for voluven. the mean gm-cfu and e-bfu cell recovery was 163,80% ± 152,64 and 144,62% ± 176,61 respectively for nsa and 187,59% ± 232,48 and 141,82% ± 148,75 for voluven. the mean viability recovery was 102.02 ±17,79 for nsa and 101,59 ±15,04 for voluven without differences between both solutions (p 0,795). there were no significant differences between both solutions in none of this parameters in spite of the tnc significant loss (p o0,001), there were no significant differences between the pre and post-washing cd34 þ cell numbers (p ¼ 0,146), gm-cfu (p 0,051), e-bfu (p 0,952) or viability (p 0,537). in contrast with the 40% of untoward reactions recorded in our historical data of 226 dmso containing cell infusions, we observed just three adverse effect with the washed cells with voluven (1,6%) and none with nsa. one patient experienced a epileptic fit related to the infusion speed, another two suffered grade 1 nausea and transient hipotension respectively. median time to neutrophil engraftment (4500 cells/ml) and platelet engraftment (420.000 cells/ml) for nsa were 11,29 ± 3,32 and 13,19 ± 6,8 days respectively and 12,02 ± 3,01 and 14,77 ± 14,87 for voluven. when comparing both solutions, there were no significant differences in neutrophil engraftment (p 0,169) or in platelet engraftment (p 0,376). conclusion: both nsa and voluven are equally effective for washing cryopreserved hp, ensuring a good cd34 þ cell recovery and preserving their viability and engraftment potential. both solutions avoid the dmso infusion related adverse events. as so, voluven constitutes an excellent alternative free of human-derived products and ready-to-use solution to our previous nsa standard washing solution. references: sánchez-salinas a. et al. transfusion. 2012 nov; 52(11) :2382-2386 disclosure of interest: none declared. introduction: ruxolitinib is a jak inhibitor that was recently approved for treatment of primary and secondary mf and shows impressive symptom control by suppression of inflammation. ruxolitinib is also a promising drug for treatment of acute and chronic gvhd. the immune-modulatory effects of ruxolitinib are at least in part due to an inhibitory effect on dendritic cell biology (heine et al., blood 2014) . dendritic cells (dcs) are important antigen-presenting cells. upon antigen contact they migrate into the draining lymph nodes to prime t cells. the aim of this study was to define the impact of the jak inhibitor ruxolitinib on dc migration. materials (or patients) and methods: cd14 þ cells isolated from human buffy coats were differentiated for seven days in the presence of gm-csf and il-4 to modcs and finally matured with lps. murine bone marrow-derived dcs (bmdcs) were generated by flushing bone marrow from femur and tibia of mice, plating the cell in medium containing gm-csf and maturing the cells with lps. migration of dcs was analyzed in transwell assays or dynamically by time-lapse microscopy within three dimensional collagen gels towards ccl-19 gradients. signaling events were analyzed by western blot to evaluate changes in phosphorylation levels. results: 2d migration of ruxolitinib-exposed dc is dosedependently reduced in vitro. by analyzing the migratory phenotype of human modcs within three dimensional collagen gels, ruxolitinib-exposed dcs are still able to sense chemokine gradients and form lamellipodia at the leading edge of the cell, whereas the retraction of the uropod is inhibited. additional in vivo studies could show that the jak inhibitor potently reduces homing of bmdcs into draining lymph nodes in mice. sirna knockdown experiments revealed that this inhibitory effect is jak1-and jak2-independent. on a molecular level we could show a reduced phosphorylation of rho-associated protein kinase (rock) in ruxolitinib-treated modc upon ccl-19 stimulation. finally, the migration phenotype of modc exposed to ruxolitinib could be mimicked by the rock inhibitor y-27632. conclusion: rhoa family members are key proteins controlling important steps of cell migration, such as protrusion formation at the front of the cell and consequently cell polarization. rock is a downstream effector of rhoa and leads to stabilization of the actin cytoskeleton via cofilin and actomyosin ii contraction by the myosin light chain. the observed reduction of rock phosphorylation may reveal an important mechanism of ruxolitinib-induced inhibition of dc migration. our current efforts focus on the validation of rock as offtarget jak1/2-independent target kinase of ruxolitinib as potential mediator of the observed effects, which may at least in part also explain the potential therapeutic impact of ruxolitinib for therapy of gvhd. introduction: alemtuzumab, a monoclonal cd52-antibody, is used for t-cell depletion (tcd) in the context of allogeneic hematopoietic stem cell transplantation (hsct) to prevent graft versus host disease (gvhd). when we applied this approach in a clinical trial, nearly half of our patients developed acute gvhd (agvhd) overall grade i-ii1. since regulatory t cells (treg) play a major role in controlling gvhd, we hypothesized that they might be functionally impaired in our patients after alemtuzumab based treatment and further investigated on these cells. materials (or patients) and methods: we analyzed peripheral blood samples of 20 patients with agvhd, 10 patients with chronic gvhd, and 12 patients who never developed gvhd after alemtuzumab-mediated tcd. treg were identified as cd3 þ cd4 þ cd25 þ cd127-or cd3 þ cd4 þ cd25 þ foxp3 þ and subsets described by expression of cd52. since cd52 is linked to the membrane by a glycosylphosphatidylinositol (gpi)-anchor, we used flaer to stain for gpi-anchors themselves. to further investigate treg activation, we stained additional markers: cd39, cd44, gitr, cxcr3, ccr5, ctla-4, garp and granzyme. to observe how treg reconstitute after hsct, we analysed samples from patients with agvhd at different time points after transplantation and correlated our findings with the clinical course of gvhd. treg function was evaluated in cfse-suppression-assays: patient derived ex vivo treg were facs-sorted according to their cd52 expression and incubated with proliferating cfsestained cd4-effector t cells from healthy donors. reduced cd4-proliferation was used as indirect marker for the suppressive function of the cd52 positive and negative treg. results: patients with agvhd showed significantly elevated percentages of cd52 negative treg: mean 55.3% (range 34.4-79.7% ) in comparison to only 10.1% (range 1-21.3%) in patients with chronic our without gvhd. by flaer-staining we confirmed that loss of cd52 correlates with the absence of gpi-anchors on the cell surface, these cells do also lack other gpi-anchored proteins (e.g. cd56, cd59). patients who overcame agvhd over time reconstituted gpianchor positive treg, whereas gpi-anchor negative treg remained the dominant population in patients with ongoing agvhd. the fraction of activated garp positive treg was mainly detected among the gpi-anchor positive treg population. all other markers showed a heterogeneous expression profile with a tendency towards lower expression on gpi-anchor negative treg. suppression-assays showed a higher suppressive capacity of gpi-anchor positive ex vivo treg in contrast to gpi-anchor negative treg of the same patient. conclusion: cd52 negative / gpi-anchor negative treg reconstituted in patients after alemtuzumab mediated tcd and mainly persisted in patients with acute gvhd. these treg were less likely to express the activation marker garp. functional assays demonstrated that gpi-negative treg were functionally impaired -in patients developing acute gvhd, these impaired treg represented the major treg-population. our preliminary data suggest that gpi-anchor negative treg, like other gpi-anchor negative t-cell subpopulations (previously shown), are functionally altered. we hypothesize that this might promote acute gvhd. these gpi-anchor negative treg could be useful to diagnose and guide immunosuppressive therapy in patients with acute gvhd. disclosure of interest: none declared. introduction: we previously showed that enhanced human invariant nkt (hinkt) lymphocytes recovery after allogeneic stem cell transplantation was associated with a reduced risk of acute graft versus host disease (agvhd). using a humanized mouse model of gvhd, we aimed to determine whether hinkt cell subsets could be involved in the regulation of allogeneic immune response and to elucidate their mechanisms of action. materials (or patients) and methods: human inkt were obtained by in vitro expansion of total peripheral blood mononuclear cells (pbmc) from healthy donors followed by electronic sorting of cd4 þ and cd4 -inkt subsets among the cd3 þ cd1d-tetramer positive total inkt population. xenogeneic gvhd was assessed in 2 gy irradiated nod/scid/ gamma-c -/-(nsg) mice previously injected with hpbmc alone or hpbmc enriched with cd4 -(pbmc þ inkt4 -) or cd4 þ (pbmc þ inkt4 þ ) hinkt cells. the effects of hinkt lymphocytes on the survival and phenotype of human monocyte derived dendritic cells (hmo-dc) were assessed by flow cytometry (on the expression of annexine v, propidium iodide, and that of cd86, cd80, cd40, respectively). the effects of cd4or cd4 þ hinkt cell subsets on the expression of the t cell activation marker cd25 and on the cytokine expression profile of cd4 þ t lymphocytes was assessed in vivo in the nsg model and in vitro, during the allogeneic mixed lymphocyte reaction (mlr). results: in vivo, nsg mice transplanted with pbmc þ inkt4cells showed a significantly prolonged overall survival in comparison to nsg mice transplanted with pbmc alone (p ¼ 0.001) or with pbmc þ inkt4 þ inkt cells (p ¼ 0.01). improved survival observed with hpbmc þ inkt4cells was associated with lower clinical and histological gvhd scores. in vitro, at 2 inkt/ 1 dc (2 n) ratio, hcd4 -inkt cells significantly increased the apoptosis of mature hmo-dc compared to the cd4 þ inkt subset (p ¼ 0.001). hmo-dc apoptosis in the presence of cd4 -inkt cells was contact dependent (21% in contact vs. 3% in transwell, po0.0001), occurred as early as 4 hours after cell-contact and was associated to the degranulation of cd4 -inkt cells as shown by cd107 expression. while h inkt cd4lymphocytes could inhibit the maturation of hmo-dc in vitro, their cd4 þ counterpart strongly induced high levels of expression of cd80, cd86 and cd40 on mo-dc in a contact dependent way. when allogeneic mlr was performed, in the presence of cd4 -inkt lymphocytes, proliferating alloreactive t cd4 þ lymphocytes showed a lower median fluorescence intensity of cd25 (p ¼ 0.02), and intracellular inf-g (p ¼ 0.005) il-17 (p ¼ 0.002) and il-21 (p ¼ 0.021) in comparison to mlr without addition of inkt cells. compared to nsg mice injected with pbmc alone, mice injected with pbmc þ inkt4cells showed lower cd25 expression on circulating t cd8 lymphocytes (p ¼ 0.001), and reduced intracellular expression of il-17 on circulating t cd4 lymphocytes (p ¼ 0.01). conclusion: these results are in line with our clinical observations and suggest that h cd4 -inkt cell subset could directly modulate the allogeneic immune response by downregulating antigenic presentation and subsequently t cell activation and th1/th17 cytokine production. the nsg preclinical mouse model suggest that developing a cellular therapy based on inkt cd4lymphocytes might be interesting for the prevention of human agvhd. disclosure of interest: none declared. in vivo expansion of host type regulatory t cells via a selective tnfr2 agonist protects from acute gvhd medicine ii, wuerzburg university hospital, 2 pathology, wuerzburg university, wuerzburg, germany introduction: cd4 þ foxp3 þ regulatory t cells (tregs) suppress graft-versus-host disease (gvhd) after allogeneic hematopoietic stem cell transplantation (allo-hct). by controlling the magnitude of the alloresponse tregs still allow for efficient anti-leukemia (gvl) effects of transplanted conventional t cells in preclinical mouse models. current clinical study protocols for donor tregs in the treatment or prophylaxis of gvhd rely on their ex vivo expansion and infusion in high numbers. here we present a fundamentally novel strategy for inhibiting gvhd based on the in vivo expansion of recipient tregs prior to allo-hct, exploiting the crucial role of tumor necrosis factor receptor 2 (tnfr2) in treg biology. materials (or patients) and methods: to expand tregs in vivo we developed a highly selective tnfr2 agonist (that would not bind to tnfr1) and treated allo-hct recipients two weeks before allo-hct. expansion of radioresistant host type tregs, alloresponses of conventional t cells, and tumor progression were monitored with bioluminescence imaging, fluorescence microscopy, and flow cytometry in different mhc major mismatch mouse models (c57bl/6, h-2 b 4balb/c, h-2 d and fvb/n, h-2 q 4c57bl/6, h-2 b ) of gvhd and gvl. results: in vitro, this new tnfr2-agonist expanded natural tregs from wild type but not from tnfr2-deficient mice. accordingly, a human variant of this tnfr2-specific agonist expanded human tregs in vitro. in vivo treatment of healthy mice with the murine tnfr2-agonist significantly increased treg numbers in secondary lymphoid organs and peripheral tissues, particularly in the gastrointestinal tract, a prime target of acute gvhd. consequently, pre-treatment of recipient mice with this novel tnfr2-agonist expanded host-type radiation resistant tregs prior to allo-hct in two models across mhc barriers. tnfr2agonist pre-treatment resulted in significantly prolonged survival and reduced gvhd severity when compared to tnfr2-deficient recipients or untreated allo-hct recipients. this was accompanied by reduced donor t cell proliferation and infiltration into gvhd target organs. in vivo depletion of host derived tregs completely abrogated the protective effect of tnfr2-agonist pre-treatment. while in vivo tnfr2-agonist pre-treatment protected allo-hct recipients from gvhd, antitumor effects of transplanted t cells remained unaffected in two different murine b cell lymphoma models. conclusion: our novel strategy demonstrates that the expansion of host tregs by selective in vivo tnfr2-activation significantly improves the outcome after allo-hct and results in prolonged tumor-free survival. disclosure of interest: none declared. introduction: we reported that mir-155 expression is upregulated in donor t cells during agvhd and mice receiving mir-155 knock-out (ko) donor splenocytes do not exhibit lethal gvhd and have improved survival as compared to mice receiving wild type (wt) splenocytes. while we showed that mir-155 does not affect the alloreactive or homeostatic proliferative potential of t cells, a significant decrease in the expression of the homing receptors ccr5, cxcr4, and s1p1 were found on mir-155-ko t cells, suggesting that the loss of mir-155 could impair the migration of these cells to the peripheral target organs resulting in less agvhd. here, we further investigate the impact of mir-155 expression in t cell migration and elucidate the t cell population responsible for agvhd modulation. materials (or patients) and methods: lethally irradiated balb/c or b6d2f1 recipients were infused with t cell depleted bone marrow (bm) cells (5x10 6 ) and gfp expressing mir-155 ko or gfp-b6 wt t cells (1x10 6 ). transplants with cd4 þ (2x10 6 ) only t cell subsets were performed to identify the lymphocyte population that contributes to the mir-155 mediated effects on agvhd. results: on days 7, 14 and 21 post transplant, recipient mice were sacrificed, and tissues harvested in order to study the kinetics of mir-155 ko t cell migration following allogeneic hsct. there was a dramatic decrease in t cell infiltration of peripheral organs (peyer's patches, liver, lung and skin) in recipients of mir-155-ko t cells as compared to wt t cells as evidenced by confocal microscopy of gfp labeled donor cells. we reasoned that these effects could be due to the modulation of ccr5 and other chemokine receptors by mir-155. we found that a recently described long noncoding rna (lincr-ccr2-5 0 as) located in the vicinity of several chemokine receptor encoding genes including ccr1, ccr2 and ccr5 and that is involved in chemokine regulation and t cell migration has 3 conserved potential mir-155 binding sites. we then set out to determine if mir-155 regulates the expression of this lncrna in relationship with ccr5 expression. there was a significant decrease in ccr5 mrna expression in mir-155-ko versus wt donor t cells obtained from recipient mice at the time of agvhd, but no significant difference in the levels of lincr-ccr2-5 0 as. a luciferase reporter assay, however indicate that there was an interaction between mir-155 and lincr-ccr2-5 0 as. our result does not exclude the possibility that mir-155 might influence the activity rather than lincr-ccr2-5 0 as levels, a hypothesis that is currently being tested. to identify the lymphocyte population involved in mir-155 mediated modulation of agvhd we performed a b6 into f1 transplant using cd4 þ t cells alone as the source of donor t cells. median survival of recipients of t cell depleted bm þ wt cd4 þ t cells (n ¼ 12) was 48 days as compared to 100% survival of all recipient mice of mir-155 ko cd4 þ t cells (n ¼ 12) on day 100, (p ¼ 0.02). recipients of mir-155 ko cd4 þ t cells exhibited also significant lower agvhd clinical (po0.01) and pathological scores (po0.01) than wt recipients. conclusion: our data suggest that mir-155 may exert its modulating effects in agvhd by affecting t cell migration. our results also point to the cd4 þ t cell subset seems to play an important role in the mir-155 regulation of agvhd. experiments are currently underway to determine the role of mir-155 in regulatory t cells and cd8 þ t cell subsets in the modulation of agvhd. disclosure of interest: none declared. oral session: solid tumors introduction: hdct has a recognized indication in the salvage setting of advanced gct and is steadily utilized worldwide. while the prognostic impact of response to prior lines of ct (i.e. definition of chemoresistance) is ascertained, that of response to induction/mobilization ct preceding single or multiple hdct cycles is unknown. we present the results of a retrospective study of the ebmtstwp. materials (or patients) in the multivariable model for pfs, tumor primary (overall p value ¼ 0.039), igcccg category (overall p value ¼ 0.033), and progression to induction (hr: 2.02, 95%ci: 1.31-3.12 , p ¼ 0.001) were significantly prognostic. the latter was also significantly prognostic for os (hr: 2.36, 95%ci, po0.001) together with the transplant setting (overall p value ¼ 0.014). results for response to induction ct were confirmed in the models that included missing data. the c-index of the model was 0.62 for pfs and 0.63 for os. conclusion: progression to induction ct prior to hdct was independently and significantly associated with shorter pfs and os, while response or progression to prior ct lines was not. this information could have important implications to refine patient eligibility to transplantation and enhance the prognostic risk grouping. these data need external validation. disclosure of interest: none declared. kir-ligand incompatibility in the graft-versus-host direction improves progression-free survival in patients with primary high risk neuroblastoma after umbilical cord blood transplantation with nonmyeloablative conditioning y. takahashi introduction: donor nk cells expressing inhibitory killer cell immunoglobulin-like receptors (kirs), which do not recognize their cognate ligands ('kir ligands') on recipient targets, are free from hla inhibition, resulting in a decreased incidence of relapse and improved the outcome after hla haploidentical stem cell transplantation (hsct) (ruggeri, blood 2007) or umbilical cord blood transplantation (ucbt) (willemze, leukemia 2009) in leukemia patients. venstrom reported that significant association between kir/hla genotypes predictive of missing kir ligands have a better outcome without allogeneic hsct following anti gd2 monoclonal antibody therapy in patients with high risk neuroblastoma (venstrom, clin cancer res 2009). these observations led us to start the clinical trial of allogeneic ucbt from kir ligand incompatible donor for patients with high risk neuroblastoma in 2008. materials (or patients) and methods: eligibility criteria of this study were newly diagnosed stage iv neuroblastoma patients with one of the following 1) chemo-resistant disease defined as mibg positive score after 4 courses of induction chemotherapy, 2) older age defined as 10 years old and older at diagnosis or 3) mycn amplification defined as greater than 10 copies per tumor cell. kir ligand mismatched ucbt donor was prospectively selected from the japan cord blood bank network according to the genotyping of hla-c or b as previously reported (willemze, r. leukemia 2009). we scheduled ucbt with reduced intensity conditioning regimen about three months after conventional high dose chemotherapy followed by autologous peripheral blood stem cell transplantation. reduced intensity conditioning regimen for cbt was flu, l-pam 140 mg/m2 and tbi 2 gy. tacrolimus and methotrexate were used for gvhd prophylaxis. single inhibitory kir expressed nk cells with no corresponding recipient's hla were monitored by flowcytometry before and after ucbt to access the expansion of alloreactive nk cells in vivo. we retrospectively analyzed the outcome of 82 patients with high risk neuroblastoma treated in nagoya university hospital between april 1982 and june 2014. results: fifty four patients were treated before dec. 2007 . after this study started in jan. 2008 , 15 patients underwent kir ligand incompatible cbt who match eligibility criteria (7 chemo-resistant, 6 mycn amplification and 2 older age) and 13 patients received standard treatment. all patients achieved engraftment in ucbt group and no patients developed grade iii or more acute gvhd and chronic gvhd. only two patients died in this group because of bu related lung toxicity. surprisingly, no patient in this group relapsed with the median follow-up period of 51 (6-71) months. on the other hand, cumulative incidence of relapse in others was 51.0%. 3year progression free survival in cbt group was significantly better than in others (83.6% vs 40.7%, p ¼ 0.048). single inhibitory kir expressed nk cells significantly expanded after cbt (p ¼ 0.0009). finally, multivariate analysis revealed only kir ligand incompatible cbt and stage iii were significant better covariate factors for relapse. conclusion: this is the first report that kir ligand incompatible allogeneic ucbt significantly reduced the incidence of relapse in high risk neuroblastoma. disclosure of interest: none declared. better prognosis for brca-mutated breast cancers treated with high-dose chemotherapy and autologous hematopoietic progenitor cell transplantation. a singleinstitution retrospective study l. boudin 1, 2, 3, * , a. goncalves 1, 2, 4 , j. m. extra 1, 2 , r. sabatier 1, 2 , h. sobol 2, 5 , f. eisinger 2, 5 , j. moretta 2, 5 , c. tarpin 1, 2 , j. camerlo 1, 2 , b. calmels 2, 6 , c. lemarie 2, 6 , a. granata 2, 6 , 4, 7 , f. bertucci 1, 2, 4 , a. madroszyk 1, 2 , p. viens 1, 2, 4 , c. chabannon 2, 4, introduction: hereditary brca genetic mutation is responsible for approximately 2-3% of breast cancers (bc).the objective of this study was to compare the outcome of brca-mutated (brca mut ) versus brca wild type or unknown (brca wt/uk ) bc following treatment with high dose chemotherapy (hdc) and autologous hematopoietic progenitor cell transplantation (act). materials (or patients) and methods: all female patients treated for breast cancer (bc) with hdc and act at institut paoli-calmettes between 2003 and 2012 were included. patients were divided into 2 groups depending on the indication of hdc with act: metastatic breast cancer (mbc) or high risk breast cancer (hrbc) including inflammatory breast cancer (ibc) and/or massive lymph node involvement (lni). all patients were examined for the presence of known brca 1 or 2 mutations. information regarding patient, tumor characteristics and treatment was also collected. the main objectives were the analysis of overall survival (os) and progression-free survival (pfs) according to the brca mutation status in the two groups. survival curves were generated using kaplan-meier method and compared using log-rank test. results: a total of 377 patients were included, 235 mbc and 142 hrbc (73 lni and 69 ibc). among mbc and hrbc, 10 (6 brca1, 4 brca2) and 5 (3 brca1, 2 brca2) patients were brca mut , respectively. in mbc, median age was respectively 36 (range 29-53) and 42 years (range 24-61) for brca mut and brca wt/uk ; 70% of brca mut patients had triple negative (tn) bc subtype (her2-negative and hormonal receptor-negative) versus only 21% in brca wt/uk individuals. in brca mut and brca wt/uk median number of metastatic sites was 2 and 1, with 90% and 69% visceral metastases, respectively. in hrbc, median age for brca mut and brca wt/uk was 36 (range 28-58) and 49 (range 20-62), respectively; 60% of brca mut had tn bc subtypes versus 24,6% of brca wt/uk . ibc represented 49% of hrbc in brca wt/uk and 40% in brca mut . in mbc, 90% (all except one) of brca mut remained alive at 5 years; the median overall survival (os) was not reached, compared with a median os of 3.62 years for brca wt (ci95% ¼ 3.07-4.68), (p ¼ 0.048 log-rang test); median progression free survivals (pfs) were 26,4 months (cl95% ¼ in brca mut patients versus 15,6 months (cl 95% ¼ 11. 64-18.48) , and in brca wt/uk patients (p ¼ 0.073 log-rank test). in hrbc, 8 years probabilities of os were respectively 1 (all patients alive) vs for brca mut and brca wt/uk individuals, while 8 years probabilities of disease free survival (dfs) were ) and , respectively (p ¼ 0.374 log-rank test). conclusion: the prognostic impact of brca mutation in bc remains controversial. in this series of mbc and hrbc, we have reported an outstanding survival outcome in a small subset of patients with documented brca mutation. in spite of a higher proportion of tn and more aggressive features, brca mut bc had a better outcome than their brca wt/uk counterpart, the difference reaching statistical significance in mbc population. hypersensitivity to dna-damaging agent, possibly due to defect in homologous recombination associated with brca mutation, could explain these results. despite the low numbers of cases in our series, hdc with act, may be considered as an option for brca mut , women with advanced bc. disclosure of interest: none declared. introduction: paclitaxel-based regimens are now commonly employed for second or third-line salvage therapy for gct. this might have an impact on the results with subsequent salvage hdct in these patients (pts). the ebmt-stwp is sponsoring a retrospective study on the outcomes of hdct administered in the last 10 years. hence, we aimed to study outcomes with hdct after relapse to paclitaxel-ct to identify the level of chemoresistance in these pts. materials (or patients) and methods: data have been collected from ebmt registry, including 24 european centers. eligibility included adult male patients (pts) with gct, and treatment with second or further-line hdct between the years 2002 and 2012. both paclitaxel used in prior ct lines of therapy and in induction-mobilization regimens pre-hdct were considered. multivariable cox regression analyses (mva) were undertaken to evaluate the association of prespecified factors (site of tumor primary, igcccg category, response to induction ct, response to prior lines [chemosensitive vs chemorefractory], line of hdct, year of hdct) including prior-paclitaxel therapy with progression-free (pfs) and overall survival (os). results: since 10/2013, 442 pts have been registered, 324 of them were suitable for present analysis. 165 pts (51%) received hdct in second-line, 102 (31%) in third and 57 (18%) beyond the third-line. hdct regimens were as follows: 177 (55%) hd-cbdca-vp16 (ce), 41 (12%) adding ifosfamide (cei), 106 (33%) other mixed regimens. 76 (23%) were taxane-containing. 120 pts received a single hdct course, 99 pts double and 104 multiple courses (1 missing). median follow up was 36 months (iqr: 19-70). prior paclitaxel was significantly associated with shorter os in the univariable model (p ¼ 0.032). however, on mva prior paclitaxel-therapy was not significantly prognostic for both pfs and os, as shown in the table. a separate model evaluated the interaction between prior paclitaxel-therapy and taxane-containing hdct: no significant interaction was found (p ¼ 0.221 1.18-5.76 .017 conclusion: the administration of paclitaxel-based regimens before hdct did not affect pfs/os. results were confirmed when excluding pts who were administered taxane-containing hdct. line of hdct was not significantly prognostic too. there is no evidence to disallow patients who have been treated with taxanes in second or third line to receive hdct as futher salvage therapy. these data need external validation. disclosure of interest: none declared. favorable outcome of a cohort of metastatic breast cancer patients treated with high dose chemotherapy and autologous transplantation at a single institution does not change the prognostic significance of histopathological subtypes l. boudin introduction: studies of high dose chemotherapy (hdc) in breast cancer (bc) often lack of biomarker information, notably the human epidermal growth factor receptor 2 (her2) status. the objective of this study was to evaluate the outcome of patients affected with different subtypes of bc following treatment with hdc and autologous hematopoietic progenitor cell transplantation (act). materials (or patients) and methods: all female patients treated for metastatic breast cancer (mbc) with hdc and act at institut paoli-calmettes between 2003 and 2012 were included. patient, tumor and treatment characteristics were collected. patients were categorized in three subtypes based on hormonal receptor (hr) and her2 status of the primary tumor: luminal (l), (hr þ /her2-), her2 (her2 þ , any hr), and triple negative (tn) (her2-and hr-). main study endpoints were overall survival (os) and progression-free survival (pfs) categorized by bc subtypes. treatment related mortality (trm) was also evaluated. survival curves were generated using kaplan-meier method and compared using log-rank test. results: a total of 231 mbc patients are included; median age was 47 (range 24-61); metachronous and synchronous mbc were 64% and 36%, respectively. 96% patients received hdc with act as part of their first-line treatment following diagnosis of metastases. median number of metastatic sites was 1 (range 1-6), including 69% of visceral metastases. l, her2 and tn subtypes were 61, 18 and 21% of patients, respectively. all but one her 2 patients (98%) received trastuzumab during the metastatic phase of the disease before (88%) and/or after (91%) oral session: acute leukemia 2 introduction: the outcome of patients 455 years old with all is poor with no clear recommendations regarding the indications for hct. these patients are usually considered ineligible for myeloablative allohct and offered transplantation with reduced intensity conditioning (ric). autologous hct is an alternative. however, the role of both treatment options has not been established so far. the aim of this study was to retrospectively compare results of ric-allohct and autohct in all 455 years old and to identify factors affecting outcome. materials (or patients) and methods: 267 patients treated with ric-allohct from either hla-identical sibling (n ¼ 154) or matched unrelated donor (n ¼ 113) and 179 treated with autohct in first complete remission between 2000 and 2011 have been included in this analysis. median age in both groups was 60 (55-74)y and 60 (55-76)y, while median interval from diagnosis to hct was 5.9 months and 6.6 months, respectively. the proportion of ph( þ ) all among those with reported cytogenetics was 71% and 66%, respectively. results: with a median follow-up of 33 months, the probability of os at two years was 44% for ric-allohct and 57% for autohct (p ¼ 0.02), while lfs rates were 34% and 41%, respectively (p ¼ 0.06). relapse incidence at two years was comparable for ric-allohct and autohct (42% vs. 48%, p ¼ 0.39) while non-relapse mortality was significantly reduced for autohct (23% vs. 11%, respectively, p ¼ 0.002). the advantage in favor of autohct was significant for ph(-) all (os: 61% vs. 38%, p ¼ 0.02; lfs: 54% vs. 21%, p ¼ 0.005) while no significant differences could be observed for ph( þ ) all (os: 55% vs. 47%, p ¼ 0.6; lfs: 42% vs. 35%, p ¼ 0.4). in a multivariate analysis adjusted for recipient age and gender as well as interval from diagnosis to transplantation the use of autohsct was independently associated with reduced risk of mortality (hr ¼ 0.69, p ¼ 0.01), treatment failure (hr ¼ 0.76, p ¼ 0.03) and non-relapse mortality (hr ¼ 0.39; p ¼ 0.0004) with no effect on relapse incidence (hr ¼ 0.98, p ¼ 0.88). in the ric-allohsct subgroup lfs was negatively affected by female donor/male recipient combination (hr ¼ 1.64, p ¼ 0.01). lfs rates for both sibling and mud transplants were comparable (32% vs. 35%, p ¼ 0.18). the use of peripheral blood cells compared to bone marrow was associated with reduced risk of relapse (hr ¼ 0.5, p ¼ 0.03). in the autohsct setting there was a tendency to higher risk of treatment failure with increasing recipient age (hr ¼ 1.05, p ¼ 0.06). other variables including type of conditioning (tbi-based vs. chemotherapy-based) did not affect survival in any of the study cohorts. conclusion: considering poor overall prognosis of all patients 455 years old, results of both ric-allohct and autohct appear enhancing and both types of transplantation may be considered valuable treatment options. potential advantage of autohct as suggested by results of our analysis should be further explored including data on disease-related prognostic factors and the status of minimal residual disease. disclosure of interest: none declared. introduction: the karyotype of the leukemic cells at diagnosis is one of the strongest prognostic factors in acute myeloid leukemia (aml). but the major cytogenetic risk categorizations were based on the large clinical studies of conventional chemotherapy for aml. in this retrospective study, we analyzed the influence of the cytogenetics of aml at diagnosis on the outcomes of allogeneic hematopoietic cell transplantation (hct). materials (or patients) and methods: from the database of jshct, we extracted the data of adult patients with aml who receive first hct between 2006 and 2010. a total of 4,241 recipients were included. additional survey for the recipients who had been reported to have cytogenetic abnormalities at diagnosis (n ¼ 2,384) confirmed the data of karyotype of 1,360 cases. results: cytogenetics at diagnosis were categorized into (a) normal karyotype (41.5%), (b) inv(16) or t(16;16) (2.9%), (c) t(8;21) (9.1%), (d) 11q23 abnormality (4.9%), (e) complex karyotype (4 ¼ 3 abnormalities) (12.3%), (f) monosomal karyotype (mk) (6.3%), and others. overall survival (os) and cumulative incidence of relapse at 2 years of all cases were 48.2% and 37.5%, respectively. these recipients were classified into 4 groups: favorable (fav) included (b) (n ¼ 121); intermediate (int), (a), (c), t(9;11) and others (n ¼ 3,169); unfavorable-1 (unf-1), (d) except t(9;11), and (e) except (f) (n ¼ 404); unfavorable-2 (unf-2), (f) (n ¼ 265). adjusted os at 2 years were 65.2% in fav, 53.2% in int, 37.8% in unf-1 and 24.2% in unf-2 ( figure) . risk of overall mortality (rom) and risk of relapse (rr) were compared among these four groups, adjusted by age, sex, disease status at transplantation and stem cell source. in fav group, rom (hr 0.71, , p ¼ 0.0038) and rr (0.44, 0.27-0.71 , p ¼ 0.0007) were significantly lower than in int group. on the other hand, compared with int group, recipients in unf-1 and unf-2 group had significantly (po0.001) higher rom (unf-1 1.48, 1.29-1.70 ; unf-2 2.22 1.93-2.57 ) and rr (unf-1 1.65, 1.41-1.92 ; unf-2 2.35, 1.94-2.73 ). these results showed that the karyotype of leukemic cells at diagnosis was also one of the powerful prognostic factors in hct for aml. but the impact of each cytogenetic presentation on the outcomes of transplantation may have some differences from that of chemotherapy. to improve this risk stratification system, molecular abnormalities of aml at diagnosis are also considered for the influence on outcomes of hct. disclosure of interest: none declared. late intensification with autologous hsct after nonmyeloablative beam conditioning has shown to be effective approach in adults with t-cell acute lymphoblastic leukemia treated by non-intensive protocol: results of the rall study group e. parovichnikova 1,* , l. group is conducting the all-2009 trial where the main principle is non-intensive but non-interruption treatment and autologous hsct after beam followed by maintenance in t-cell all pts without hla-identical donors (ebmt 2014, op-009). the autologous hsct was planned for all patients as late high dose consolidation (on the 20-22 weeks of the protocol). in patients with early and late t-cell immunophenotype having hla-identical siblings allogeneic bmt was an option. from nov, 2008 , till nov, 2014 , 30 centers enrolled 264 all patients. in 6phenotype was unknown (2,2%). b-cell precursor phenotype was diagnosed in 62,6% (n ¼ 166), t-cell precursor phenotypein 34,8% (n ¼ 91); biphenotypic al -in 0,4% (n ¼ 1). t-cell all patients were young -28 y (16-56); male gender prevailed -33 f/58 m. t-all subtypes distribution in our study was as follows: 44 (47,8%) patients had early t-all (t-i/ii), 36 (40,1%)thymic (t-iii), 11 (11,1%) -mature (t-iv). for the whole group medians were: hb-112 g/l (42-180), l -22,3*10/ 9 l (0,5-313), plt -90*10/ 9 (5-943), b/m blasts -74,9% (0-99), ldh -995 iu (131-12 000). no born marrow involvement was detected in 4 pts, b/m blasts 5-25% -in 10 (so t-lbl -14,5%). cytogenetics was available in 63,2% of pts (n ¼ 55/87), 45,5% of them (n ¼ 25/55) had normal (nk), 25 pts -abnormal karyotype, 5 pts (9%) had no mitosis. mediastinum involvement was registered in 48/87 (54,5%) and cns disease -in 11/87 (12,5%). results: induction and follow-up data were available in 87 pts. cr was achieved in 78/87 (89,8%): after prephase ¼ 12, after 1 st ind.phase ¼ 45, after 2 nd ind.phase ¼ 21. induction death occurred in n ¼ 5 (5,7%). primary resistance and even progression during induction was registered in n ¼ 4 (4,5%). 28 pts proceeded to autologous hsct, almost all (n ¼ 26) were successfully harvested at a median time of 20 weeks. 2 pts were poor mobilizers and bone marrow exfusion was carried out. no data on mrd level at time of harvesting is available so far. auto-hsct was applied at a median 6 months from cr. allogeneic hsct from sibling donors was performed in 6 cr pts (4 with ti/ii and 2 with t-iv). no deaths in allo-hsct group have occurred. the land mark analysis at 6 months for chemotherapy group (n ¼ 27) and at day of hsct for transplanted groups was done. disease free survival constituted for chemotherapy -55% at 5 years, and 100% -in both transplanted groups. none of the transplanted patients relapsed so far. the overall and diseasefree survival in the whole cohort of t-cell all patients was 58% and 68%. in a multivariate analysis only autologous hsct influenced dfs. conclusion: our study demonstrates that t-all may be treated by non-intensive, but non-interruption approach. even without high dose consolidation 5-years os and dfs (land mark analysis) constituted 55%. autologous hsct with beam conditioning after mild induction/consolidation followed by prolonged maintenance seems to add benefit to the overall optimistic results decreasing the relapse rate from 34% to 0% within 5 years. disclosure of interest: none declared. with a median follow up from time of hla typing of 14 months (range, 0.3-97) for all patients and 30 months (range, 3-97) for survivors, the 1-and 8-year probability of survival for all 225 eligible aml patients was 68±3% and 40±4%, respectively. at the same time points, for the 172 transplanted patients the probability of survival from time of graft was 60 ± 4% and 43 ± 4%, respectively. the probability of survival was particularly dismal for the 37 patients transplanted in advanced disease phase (4±4% at 4 yrs), whereas for the 135 patients transplanted in early (cr1 þ cr2) phase the 8-yrs probability of survival was 54±5% and, by excluding the low number of cb recipients, it was 55 ± 7% for 56 hla id sib, 58 ± 8% for 40 hrd and 68 ± 9% for 28 mud recipients (p ¼ ns). conclusion: rtn policy allows a donor identification in 94% of all evaluable aml patients and provides an allogeneic transplant to 86% of them with no substantial differences in s58 terms of long-term survival between initially eligible and definitively transplanted patients or by comparing the different donor stem cell sources. transplant results should be given following information on the specific transplant policy and only the itt analysis allows to know the real impact of each transplant program. disclosure of interest: none declared. materials (or patients) and methods: oral pan was administered in two sequential schedules, either thrice weekly (tiw) every week (a) or every other week (b). arm a, in which pan was started at a dose of 10 mg tiw and escalated to 30 mg tiw using a 3 þ 3 design was followed by arm b, in which pan doses increasing from 20 mg tiw to 40 mg tiw were investigated. pan was initiated between day þ 60 and þ 150 after hsct and given for up to 1 year. eligibility criteria included: ancz1,000/ml, plateletsz75,000/ml, adequate organ function and no severe gvhd. dlt was defined as prolonged g4 hematologic toxicity or any non-hematologic toxicityzg3 unrelated to disease progression within 28 days of the first pan dose. results: 24 pts (21 aml, 3 mds), median age 52 yrs (range, 21-71), are evaluable for mtd. cytogenetics were classified as low (n ¼ 2), intermediate-1/2 (n ¼ 11) or adverse risk (n ¼ 11) according to eln criteria. pan was started a median of 98 days (range, 60-147) after hsct from a mrd (n ¼ 8), mud (n ¼ 10) or mismatched donor (n ¼ 6) which was performed in active disease (n ¼ 17, median bone marrow blasts 23%, range, 8-77), cr1 (n ¼ 5) or cr2 (n ¼ 2). the rp2d was 20 mg tiw in arm a and 30 mg tiw every other week in arm b based on 5 dlts: fatigue g3 at 20 mg, colitis and nausea/emesis g3 each at 30 mg (arm a), diarrhea and headache g3 at 40 mg each (arm b). pan-related or unrelated g3/4 aes were reported in 20 of 24 pts (83%) and included hematologic toxicity (50%), laboratory alterations (42%), infections (29%), constitutional (29%) and gastrointestinal symptoms (25%) and neurologic, pulmonary, pancreatic/hepatobiliary or vasculary toxicity (4-8% each). aes were rapidly reversible after interruption and/or dose reduction (n ¼ 6) and no study-related deaths occurred. fifteen pts (63%) have completed one year of pan, 9 pts discontinued treatment prematurely after 7-217 days due to aes (n ¼ 8) or relapse (n ¼ 1). prophylactic or preemptive dlis (1-6) were administered to 15 pts (63%). eleven pts (45%) developed mild (n ¼ 6), moderate (n ¼ 3) or severe (n ¼ 2) chronic gvhd. patients died of aml (n ¼ 3) or severe chronic gvhd (n ¼ 1 introduction: our previous research has provided the evidence that an autoreactive immune system can be ?reset? into a healthy, tolerant state by immunoablative treatment to eradicate pathogenic effector cells, followed by transplantation of hematopoietic progenitor cells (hsct). here, we present the clinical and serologic responses and long-term immune reconstitution in 20 patients with severe ads for up to 15 years after receiving immunoablation and asct. materials (or patients) and methods: since 1998, 20 patients with refractory autoimmune diseases (sle, n=10; ssc, n=4, ms, n=2; polychondritis, n=1; panniculitis, n=1, granulomatosis with polyangiitis, n=1, and chronic inflammatory demyelinating polyneuropathy, n=1) received a cd34 + -selected autologous stem cell transplantation after immunoablation with atg (neovii, formerly fresenius) and cyclophosphamide as part of a prospective monocentric phase i/ii clinical trial. autoantibody titers were evaluated with elisa, peripheral tand b lymphocyte subsets immunophenotyped using multicolor flow cytometry. results: with a median follow-up of 12 years after immune reset (range, 24 months to 16 years), 15 of 20 patients (75%) achieved a progression-free survival (pfs), defined as survival without major organ failure. 50% of these patients showed durable clinical remissions for up to 15 years despite discontinuation of immunosuppressive treatment, while 25% of patients had a stabilization of their underlying disease under maintenance therapy. disease relapse occurred in three sle patients (at 18, 36, and 80 months, respectively), two of whom died later from uncontrolled disease and infection, respectively. 3 of 20 patients died from infection (n=2) or cardiac failure (n=1). anti-dsdna antibodies completely normalized in all sle patients and ana significantly declined from a median titer of 1:5120 at baseline to 1:160 six months after transplantation in patients with connective tissue diseases. recovery of recent thymic emigrants (rtes) was completed between 12 and 24 months after immune reset, reaching on average 3.6 to 5.1 times the baseline levels. recurring foxp3 + tregs had significantly higher expression levels for cd31 and cd45ra compared to age-matched healthy controls, indicating their recent thymic origin. numeric recovery of the naÿve b cell compartment was completed within 1 year after immune reset in responding patients. conclusion: these data confirm our assumption that the reprogramming of an autoreactive immune system into a juvenile and self-tolerant immune system is feasible and associated with long-term remissions. our findings propose that chronic autoimmunity is not an end point depending on continuous treatment with specific anti-inflammatory agents, but may be cured by combining specific targeting of autoreactive memory and effector cells with a reactivation of thymic activity. a future challenge is to make this therapeutic approach attractive for a larger number of patients by reducing the rate of severe infections. this may be achieved by either using a more selective graft purging, e.g., depletion of t cell receptor alpha/beta and cd19 + cells from apheresis products with the clinimacs device, or an adoptive transfer of microbe-or virus-specific memory t and/or b cells. disclosure of interest: none declared. in the present report we summarize the long term effects of transplantation. materials (or patients) and methods: the mobilization protocol included cyclophosphamide and g-csf. the conditioning consisted of cyclophosphamide (50 mg/kg/day on days -5,-4,-3, -2 prior to transplantation) and antithymocyte globulin (thymoglobulin -of 0.5 mg/kg/day given on day -5, and 1.0 mg/kg/day given on days -4,-3, -2 and -1) except for three patients, who received atg fresenius at adjusted doses. results: one of the transplanted patients died during neutropenia due to the sepsis and its complications. the mean time of observation of remaining 23 patients as of december 2014 was 57 months (range 33 -80 months). the independence of exogenous insulin after the transplantation was achieved in 20 of them. three patients were lost to follow up. the median time without exogenous insulin for 20 patients in follow up was 30 months (range 0-80 months). four out of 20 (20%) remain in remission of diabetes (exogenous insulin free) with median follow up of 54 months (range 34-80 months). notably, three patients in the series were treated with atg fresenius (due to transient problems with thymoglobulin availability) -the median follow-up of these patients is 65 months with 58 months of remission of diabetes. the treatment led to significant reduction of hba1c and good glycemic control in patients. no attempt was made to repeat procedure in relapsing patients. the hsct leads to remission of new onset t1dm in majority of patients but the response in most of them is limited to average of 30 months. patients receiving atg fresenius tended to have longer remission but small number prevented drawing conclusions. there is a need to develop procedure to either prolong response or to effectively treat the relapse of diabetes. disclosure of interest: none declared. hematopoietic stem cell transplantation for refractory crohn's disease: feasibility and toxicity j. introduction: autologous hematopoietic stem cell transplantation (hsct) is a salvage treatment for severe refractory crohń s disease (cd) patients. we evaluated feasibility and toxicity of autologous hsct for refractory crohn's disease (cd). materials (or patients) and methods: in this prospective study, refractory cd patients with an aggressive course despite medical treatment, impaired quality of life and no surgical options were included. hematopoietic stem cells were mobilized with cyclophosphamide and g-csf and collected by leukapheresis from pheripheral blood. in a second step, conditioning regimen with cyclophosphamide and rabbit antithymocite globulin (ratg) was used and previously collected stem cells were infused. toxicity and complications during the procedure and within first-year after transplantation were evaluated, along with the impact on safety after the introduction of supportive measures over the study. results: twenty-six patients were enrolled. during mobilization, 16 patients presented febrile neutropenia, including 1 bacteremia and 2 septic shocks. neutropenia median time after mobilization was 5 days. five patients withdrew from the study after mobilization and 21 patients entered into the conditioning phase. hematopoietic recovery median time for neutrophils (40.5x10 9 /l) was 11 days and for platelets (420x10 9 /l) was 4 days. ninety-five percent of patients suffered febrile neutropenia and 3 patients presented worsening of perianal cd activity during conditioning. among non-infectious complications, 6 patients presented antithymocyte globulin reaction, 12 patients developed mucositis and 2 patients had hemorrhagic complications. changes in supportive measures over the study, particularly antibiotic prophylaxis regimes during mobilization and conditioning, markedly diminished the incidence of severe complications. during the first 12-month follow-up, viral infections were the most common observed complications, and one patient died due to systemic cytomegalovirus infection. conclusion: autologous hsct for refractory cd patients is feasible but extraordinary supportive measures need to be implemented. we consider that this procedure should only be performed in highly-experienced centers. disclosure of interest: none declared. autologous haematopoietic stem cell transplantation in aggressive multiple sclerosis: a uk cohort from two centres j. clay 1,* , p. 0.5 (range 0.5-4.5) , whilst the remaining 4 had a deterioration of 0.5-1.0 materials (or patients) and methods: 25 cb units (cbu) were frozen and thawed across the participating banks, according to local sops; pre-freezing and post-thaw samples were assessed for clonogenic potential (cfu) and cd34 þ recovery and viability through standard ishage protocol (si) (brocklebank am 2001). a modified ishage gating strategy (mi) was developed by reduction of the debris gate and by extension of lympho-monocytes gate in order to detect cd34 þ events with abnormal physical parameters (cd34 þ gate). facs sorting was performed to collect cd45 þ /cd34 þ events included inside (p8) and outside(p9) the si cd34 gate. sorted cells were then characterized by fc, confocal microscopy analysis and cfu assay in order to confirm that cd34 þ cells detected out of the si cd34 gate are not an artifact. confocal microscopy analysis was performed by labeling with a cd34 ab recognizing a different epitope and marked with a nuclear counterstain. results: si and mi strategies showed no significant discrepancies when determining cd34 þ absolute number and viability in pre-freezing cbus. the recovery of viable cd34 þ cells assessed by si or mi in the thawed products did not show any differences statistically significant, whilst the recovery of total cd34 þ cells was significantly lower in si (58,3 ± 18%) than in mi (80,1±19.7%)-analyzed samples (mean±sd, po0,05 with pair t test). this difference resulted in a lower cd34 þ viability with mi than si (79,5% ± 15.9 vs 96,7% ± 3%, respectively, po0.02). fc post-sorting analysis showed that the sorted p8 (si cd34 gate) population was cd45 dim cd34 þ cd133 þ cd5 -7-aad-, whereas p9 (mi extended cd34 gate) showed the same markers expression but a 7-aad þ staining, therefore determining such events as cd34 þ dead cells. this data was confirmed by confocal microscopy analysis. finally, p8 events showed a 67% clonogenic efficiency, whereas p9 events were not able to generate any colony. conclusion: a cd34 þ count after cbu thawing lower than the fresh sample is often reported, usually with a high rate of viability. we showed that the standard methodology for cd34 þ counting leads to the exclusion of a number of dead cd34 þ cells, thus resulting in a viability overestimation. we propose here a modification of the ishage counting standard for thawed samples, which provides a more reliable assessment of cd34 þ viability. the content of pdc in bm grafts had a significant beneficial effect on post-transplant survival, an association not seen in gpb grafts 1 . the 20% improved survival seen in recipients of more donor bm pdc was due to decreased deaths from graft rejection and acute and chronic gvhd. donor bm pdcs in murine bmt favorably modulated t-cell activation, decreasing gvhd through gamma-interferon-dependent upregulation of ido, while preserving gvl effects 2 . the present study tested whether the biological activity of pdc from different graft sources are explained by the differential expression of homing receptor molecules or immuneregulatory pathway genes. materials (or patients) and methods: facs isolated pdc from allogeneic stem cell grafts obtained from 10 bm, 8 gpb, 4 umbilical cord blood (ucb) and 2 following mobilization with plerixafor (plxpb) samples. expression of chemokine receptor, integrin and selectin were measured by flow cytometry. gene expression on flow cytometry-sorted pdc was analyzed with illumina chips. differentially expressed genes using a 2-sided t-test comparing groups of replicate samples with po0.05 were selected for exploratory analyses of regulatory pathways. mhc mismatched mouse h2b-h2k transplants compared outcomes with pdc from ccr7 ko vs wt donor. indirect presentation of allogeneic peptides by pdc and classical dc was studied using h2b-h2 b/d transplants with h2kd peptide recognition by tea transgenic donor t-cells. results: pdc from bm and plxpb grafts had higher cd62l expression but lower ccr7 and cxcr3 expression than pdc from gpb and ucb grafts. high cxcr4 and low ccr9 expression were seen in pdc from all graft sources. while differences in ccr7 expression between pdc from bm vs gpb grafts suggested homing differences might explain observed differences in trm associated with the pdc content of the allograft, donor pdc from ccr7 ko mice had equal ability to modulate gvhd as pdc from wt mice. pdc in grafts from both human and mice were phenotypically immature and were relatively ineffective in activating t-cells through indirect presentation of alloantigen peptide, indicating that immune regulatory effects might be more important than antigen presentation. supporting this hypothesis, gene expression patterns of human pdc from bm showed lower expression levels of genes related to adaptive immunity, and higher [o108] expression of genes associated with innate immunity, than pdc from gpb, ucb, or plxpb grafts. conclusion: functional differences in immuneregulatory genes explain some of the observed differences in transplant outcome when stratifying recipients based upon between the content and source of donor pdc. bm pdc appear to be polarized towards activation of innate immunity while pdc from other graft sources are more polarized towards antigen presentation. novel approaches of stem cell mobilization, such as plerixafor, may increase the content of immature pdc enriched for expression of that might be effective in recapitulating the beneficial effects of bm pdc with respect to preventing early posttransplant transplant related mortality. references : results: with a median follow-up of 43, 43 and 49 months in the 10/10 urd, 9/10 urd and ucb, hematopoietic recovery was slower in ucb compared to urd recipient: the cumulative incidence of neutrophils recovery at day 28 and platelet recovery 450 â 10 9 /l at day 180 were respectively 73% and 56% in ucb against 96% and 85% in 10/10 urd and 95% and 75% in 9/10 urd (po0.0001). while there was no significant difference in the day 100 cumulative incidence of grade 2-4 agvhd: 31% in 10/10 urd, 39% in 9/10 urd and 32% in ucb, the 2 years cumulative incidence of cgvhd was significantly lower in ucb recipients: 16% against 37% in 10/10 urd (po0.0001) and 29% in 9/10 urd (p ¼ 0.004). in multivariate analysis, there was no statistically significant difference in nrm between ucb recipients and 9/10 recipients (hr 1.58, p ¼ 0.13) or 10/10 recipients (hr 1.35, p ¼ 0.25) . in multivariate analysis, the cumulative incidence of relapse/progression was significantly lower in 10/10 urd recipients compared to ucb recipients (hr 0.60, p ¼ 0.02) and there was also a trend towards a decreased incidence of relapse in the 9/10 urd recipients (hr 0.62, p ¼ 0.07). compared to ucb transplants there was no significant difference in pfs after 9/10 urd (hr 1.17, p ¼ 0.29 ) and 10/10 urd (hr 1.10, p ¼ 0.49 (table 1) . results: there were no significant differences in pre-transplant characteristics between both groups ( table 1) . the 30day cumulative incidence of neutrophil engraftment was similar in both groups (93% vs 100%), with a median time of 16 and 17 days, respectively (p ¼ 0.28). similarly, the 60-day cumulative incidence of platelet engraftment was 70% vs 76%, in a median time of 39 and 35 days, respectively (p ¼ 0.11). four cases among the haplo-cord group showed primary cb graft failure (3 of them showing third party donor cells engraftment) who were rescued by a second cb transplant in two cases and haplo-sct in two cases. there were no graft failures in the haplo-sct group. grade ii-iv acute gvhd was more frequent in the haplo-sct group (20% vs 41%, p ¼ 0.06) and chronic gvhd showed a higher tendency in the haplo group (18% vs 35%, p ¼ 0.24). with a median follow-up of 51 months (10-122) in the haplo-cord group and 19 months in the haplo-sct group, os was 52% and 78% (p ¼ 0.15), respectively. efs was 41% vs 72% (p ¼ 0.06), relapse rate was 22% vs 20% (p ¼ 0.71) , and trm was 22% vs 8% (p ¼ 0.16), respectively. conclusion: in our experience, albeit differences in follow-up and limited number of patients, myeloablative single cord supported by hla-mismatched cells and haploidentical transplantation with post-transplant cyclophosphamide both offer valid alternatives for patients with acute leukemia. engraftment rates are similar in both groups as well as relapse rate. early toxic mortality is higher in the haplo-cord group while gvhd rates seem to be higher in the haplo-sct patients. further analysis including larger series and longer follow-up are needed to confirm these observations. disclosure of interest: none declared. introduction: studies on conditioning regimens prior to autologous stem cell transplant (asct) in lymphomas generally present a major pitfall, the miscellaneous mix of histologies composing the series, resulting in a reduced statistical power when focusing on a specific subset. we previously demonstrated (visani et al, blood 2011) the safety of a new conditioning regimen with bendamustine, etoposide, cytarabine, and melphalan (beeam) prior to asct in resistant/relapsed lymphoma patients (eudractnumber2008-002736-15). the regimen showed long-lasting significant antilymphoma activity, with a 3-year pfs of 75%. materials (or patients) and methods: we thus designed a single histology, phase ii study to evaluate the efficacy of the beeam conditioning in resistant/relapsed diffuse large b-cell non-hodgkin lymphoma (dlbcl) patients. the study was registered at at european union drug regulating authorities clinical trials (eudract) n. 2011-001246-14. the primary endpoint of the study is to evaluate the 1-year complete remission rate. fixing the lowest acceptable rate as 55% and the successful rate as 70%, with a significance level a ¼ 0.05 and a power 1-b ¼ 0.90, the sample size was estimated in 88 patients. results: until now, 53 patients (median age 54 years, range 19-69) were enrolled. at the time of writing, we have data available for 44 patients with resistant/relapsed diffuse large b cell non-hodgkin lymphoma were consecutively enrolled in the study. briefly, 33/44 patients had advanced stage disease (iii-iv), 14 were primary refractory and 30 had relapsed after a median number of 2 lines of therapy (range: 2-3). eleven patients had 1 or more relevant comorbidities (range: [1] [2] [3] [4] [5] . 22 patients were in ii or subsequent cr after salvage therapy, whereas 19 were in pr and 3 had progressive disease. a median number of 5.90x10 6 cd34 þ /kg cells (range 2,8-9,42) collected from peripheral blood was reinfused to patients. all patients engrafted, with a median time to anc40.5x10 9 /l of 10 days. median times to achieve a platelet count 420x10 9 /l and 450x10 9 /l were 12 and 16 days respectively. ten out of 44 patients presented a fever of unknown origin (23%), whereas 21 patients (47%) presented a clinically documented infection. all patients received g-csf after transplant for a median time of 8 days (range: 8-13). one patient died due to an incomplete hematological recovery after transplant, producing an overall transplant related mortality of 2.7%. thirtyeight out of 44 patients are evaluable up to now for response to treatment. 31/38 (81.5%) obtained a cr, 3/38 a pr, whereas 4/38 did not respond to therapy. after a median follow-up of 12 months after transplant (range 2-30), 4/38 patients were refractory, 7/38 relapsed, and 27/38 (71%) are still alive, in continuous cr. conclusion: in conclusion, this data provide evidence that the beeam regimen is safe and has promising efficacy in resistantrelapsed aggressive diffuse large b cell lymphoma. pts with hl at risk of relapse post-asct (clinicaltrials. gov #nct01100502). the primary results showed that bv significantly improved progression-free survival (pfs) per independent review vs. placebo. efficacy analyses by investigator review were performed in subgroups as these data may provide useful information for making treatment decisions. materials (or patients) and methods: the aethera trial is a phase 3, randomized, double-blind, placebo-controlled study. after asct, pts received bv 1.8 mg/kg q3wk or placebo for up to 16 cycles. pts were enrolled in 1 of 3 high-risk categories: refractory to frontline (fl) therapy, relapse o12 mos after fl therapy, and relapse z12 mos after fl therapy with extranodal disease. the primary endpoint was pfs per independent review. pfs by investigator was also evaluated. results: 329 pts were randomized at 78 sites in the us and europe. median age was 32 yrs (range, 18-76) and 53% were male. asct conditioning regimens were beam: 61%, cbv: 11%, or other regimens: 28%. 6% of pts received radiation as part of their transplant regimen. all pts had completed the treatment phase as of aug 2013. subgroup analyses by demographics, stratification factors, pt characteristics, and number of risk factors showed that pfs by investigator was improved across all groups; the hazard ratio comparing bv to placebo was o1 for all analyses (table 1) . in an ad hoc analysis, pts with increasing numbers of risk factors for progression had progressively greater pfs benefit with bv vs. placebo. the most common adverse events (aes) in the bv arm (vs. placebo) were peripheral sensory neuropathy (56% vs. 16%) and neutropenia (35% vs. 12%). a higher incidence of herpes simplex and zoster infections were observed with bv (11%) vs. placebo (3%); otherwise, opportunistic infections were rare and balanced between the arms. the incidence of pulmonary toxicity (by meddra smq) was low, but slightly higher with bv vs. placebo (5% vs. 3%). two pts died r40 days of last dose of bv; cause of death was acute respiratory distress syndrome. response to salvage therapy pre-asct cr 123 0.78 (0.42, 1.46 ) pr 113 0.44 (0.26, 0.76 ) sd 93 0.40 (0.23, 0.71) hl status after frontline therapy refractory 196 0.55 (0.37, 0.83 ) relapse o12 months 107 0.45 (0.27, 0.93 ) relapsez12 months with extranodal disease 26 0.30 (0.08, 1.16) 42 treatments pre-asct 149 0.32 (0.19, 0.54 ) b symptoms after failure of frontline therapy 87 0.29 (0.15, 0.55) extranodal disease at time of pre-asct relapse 107 0.37 (0.20, 0.68) risk factors a z1 329 0.50 (0.36, 0.70 ) z2 280 0.40 (0.28, 0.57 introduction: although high-dose chemotherapy (hdc) is the gold standard for the treatment of many relapsed or refractory lymphomas, the outcome is still unsatisfactory in some subsets of patients with adverse prognostic features. we treated 111 high-risk patients with a tandem strategy associating debulking with hdc followed by autologous stem cell transplantation (asct) and subsequent allogeneic sct (tandem auto-allo) materials (or patients) and methods: adult patients consecutively treated at two centers were included. criteria for receiving tandem auto-allo were: hl and nhl refractory to first-line therapy, less than cr after first salvage treatment, relapse after prior asct, multiple relapses, histology of transformed follicular, mantle-, t-and nk-cell lymphoma and documented infections during hospital stay in 30 (27%), without significant differences between beam and hd-mel groups. among the 65 patients with active disease before asct, the overall response rate was 86% (n ¼ 56 responders) and those in cr were 34 (52%). no difference was observed in terms of response in the beam and hd-mel groups (p ¼ 0.28). allogeneic sct donor was either hla-identical (n ¼ 86), mud 9/10 (n ¼ 2), haploidentical (n ¼ 20) or cord blood (n ¼ 3). 3-y os of entire cohort was 68% (95% ci: 59-77), 3-y pfs was 61% (52-70), rates of acute gvhd grade 2-4 and chronic gvhd were 28% and 38% respectively. trm rate was 18% (n ¼ 20). no difference between beam and hd-mel group was observed for os (73% and 64% respectively, p ¼ 0.40) or trm (19% and 13%, p ¼ 0.44). of note, os of patients in cr before and after asct and os of those obtaining cr after asct was superimposable ( figure 1) figure) . a multivariate analysis revealed that late allo-hsct was a unfavorable prognostic factor for os with a statistical significance (hazard ratio, 1.46 ; 95% ci, 1.01-2.11; p ¼ 0.04 (3) or complete response after autosct after a third line of chemotherapy (9 pts). donors of allosct were hla identical siblings in 38% of patients, matched unrelated in 35% and haploidentical donors in 27% of patients. median age at transplant was 33 (range, 17-60). at allosct, 43% of patients were in cr, 30% were in pr, 27% in sd or pd. 83% patients performed allosct having relapsed o12 months from autosct or with primary refractory disease, 17% having relapsed 412 months after autosct. results: median follow-up of surviving patients was 4.8 years (range, 0.5-10.5) . overall survival (os) was 76% at 1 year, 59% at 3 years and 55% at 5 years of follow-up. in multivariate analysis, only disease status before allosct significantly impacted the os (p ¼ 0.003, hr 1.6, ci95% 1.2-2.2) , whereas donor and timing or relapse/refractoriness did not change os (p ¼ 0.149, hr ¼ 1.3, and p ¼ 0.501, hr ¼ 1.3, respectively better control of the disease: this has occurred together with changes in donor type (from unrelated to family haploidentical), in gvhd prophylaxis (from mtx cya atg to post transplant cyclophosphamide), and in the conditioning regimen, combining thiotepa and intravenous busulfan; in addition dipss was somewhat lower in more recent patients, and also spleens were smaller. we believe these data suggest that alternative donor grafts are currently a therapeutic option for patients with mf. disclosure of interest: none declared. long follow-up data show that survival rate after allogeneic stem cell transplantation in patients with cll is higher for younger patients because of significantly lower nrm during the whole follow-up period: a retrospective study of the cmwp m. van gelder 1,* on behalf of cmwp . among pediatric pts, 28% were aged 0-23 months, 52% were aged 2-11y, and 20% were aged 12-16y. day þ 100 survival data by age group are available for 526 pts post-hsct and 62 pts post-ct (table) , with rates of svod post hsct of 55% in pediatric pts and 50% in adults. hpse has been shown to be involved in inflammation and may therefore play an important role in the pathogenesis of vod of the liver. the purpose of this study was to identify an association between hpse gene single nucleotide polymorphisms (snps) and vod of the liver after allogeneic hsct in childhood. materials (or patients) and methods: 160 children (median age, 14 years) who underwent allogeneic bone marrow (n ¼ 91) or peripheral blood stem cell transplantation (n ¼ 69) in a single center and their respective donors were genotyped of hpse for rs4693608 and rs4364254 using taqman real-time polymerase chain reaction. the donor was hla-matched unrelated in 63% of transplants and hlaidentical related in 25% of transplants. conditioning regimen was myeloablative in all cases and based on busulfan in 46% of transplants or total body irradiation in 33% of transplants. two forms of post-transplant immunosuppression predominated, cyclosporine a and methotrexate in 50% of transplants and cyclosporine a alone in 30% of transplants. results: 160 donor/patient pairs were analyzed. cell samples from the patient were available in 155 cases and from the donor in 153 cases. genotype ag of hpse rs4693608 snp was found in 82 patients (53%), aa in 49 patients (32%), and 24 patients were homozygous for gg (15%). analysis of hpse rs4364254 snp revealed a similar distribution for tc (n ¼ 69, 44%) and tt (n ¼ 68, 44%) and a frequency of 18 patients (12%) for cc. vod of the liver was observed in 12/160 patients (8%). if vod of the liver was diagnosed all of our patients were treated with defibrotide as early as possible. altogether, 4/12 patients with vod of the liver (33%) died of vod, whereas 8/ 12 patients with vod of the liver (67%) survived vod. early medical intervention is most probably the reason for this high cure rate. patients with hpse genotypes gg or ag of rs4693608 (g4a) had a significantly reduced incidence of vod of the liver on day 100 after hsct compared to patients with genotype aa (5% vs. 14%, p ¼ 0.038). in addition, incidence of vod in patients with genotype cc or ct of rs4364254 (c4t) was significantly decreased in comparison to patients with genotype tt (2% vs. 15%, p ¼ 0.004). interestingly, no patient with genotype cc developed vod. because both snps co-occur in vivo, we generated subsets: aa-tt, gg-cc and a group with remaining snp combinations. we found significant differences between all three patient groups (p ¼ 0.035). patients with aa-tt showed the highest incidence of vod of the liver (17%), while vod was not observed in patients with gg-cc (0%) and residual combinations were numerically in-between (5% allogeneic, n ¼ 42) were considered at risk of hbv reactivation for the following criteria of positivity: 1) donor and/or recipient anti-hbc and/or 2) anti-hbe and/or 3) anti hbs þ /-. lamivudine prophylaxis was given to 51 out of 58 patients (88%). overall, 4 patients, 1 autologous and 3 allogeneic recipients, developed hbv reactivation at a median time of 40 months (range, 28-53) after hsct. of these 4 patients, 3 were at risk of reactivation and 2 of them were not receiving prophylaxis. one was hbv-negative at the time of pretransplant screening. the cumulative incidence of hbv reactivation was 4,9% for patients at risk and 1%.for the entire hsct population studied. one patient reactivated hbv infection during lamivudine prophylaxis. hbv isolate genotype was studied in all reactivated patients. two isolates showed hbsag escape mutations (123 n, 124y, 126i, 145 k, 145 r, 144e, r122k, t140s) and in 1 lamivudine drug resistance mutations (181 s and l801l), 3 of them resulted hbv genotype d and one resulted genotype f. conclusion: the low rate of reactivation in our cohort is probably due to the accuracy of pre-transplant donor/ recipients screening and to the extensive prophylaxis program. however, a case of hbv reactivation occurred in a patient negative at serological screening (sero-negative hbv occult infection). the detection of immune-escape hbsag mutations, associated with lack of recognition by neutralizing immune activity and by diagnostic assay in most hbvreactivated patients, supports the role of these mutations in the process of immune-suppression driven hbv reactivation. disclosure of interest: none declared. introduction: polyomavirus (pv) hemorrhagic cystitis (hc) is a severe complication after haploidentical hematopoietic stem cell transplantation (haplo hsct). in the setting of solid organ transplantation, the use of tacrolimus (fk) has been associated with higher risk of pv-hc compared with cyclosporine a (csa). the aim of our study was to investigate risk factors of pv-hc in haplo-hsct recipients, with particular focus on immunosuppressive agent used as graft-versus-host disease (gvhd) prophylaxis. materials (or patients) and methods: we retrospectively analyzed the incidence, risk factors and outcome of pv-hc in 149 consecutive adult patients (pts) undergoing haplo hsct due to hematological malignancies between 2009 and 2014 at our two institutions. all hscts were t-cell replete and included post-transplant high-dose cyclophosphamide as part of gvhd prophylaxis. pv-hc was defined as pv detection in urine by pcr testing in association with clinical symptoms of hc without other concurrent genitourinary conditions. variables tested as potential risk factors to develop a pv-hc were: conditioning regimen, age, diagnosis, stem cell source, levofloxacin prophylaxis, acute gvhd, interval between diagnosis and hsct, pre-hsct therapy lines, previous subdiaphragmatic radiotherapy, n. of cd34 þ cells infused, neutrophil and platelet engraftment, fk vs csa-based immunosuppressive regimen. results: main pts' characteristics are shown in table 1 . after a median follow-up time of 13 months from transplantation, ten (7%) pts developed a pv-hc. pv-hc occurred in 6/52 (12%) pts receiving fk and 4/97 (4%) pts receiving csa (p ¼ 0.10). etiology was bk virus in 9 (90%) cases and jc virus in one (10%) case. median time of pv-hc diagnosis was 30 (7-68) days post-hsct. in the multivariate analysis, myeloablative (mya) or reduced-intensity conditioning (ric) regimens (hr ¼ 4.25, 95% ci: 1.18-15.33 ; p ¼ 0.03) and fk-based immunosuppression (hr ¼ 3.78, 95% ci: 1.05-13.64 ; p ¼ 0.04) were the only two independent factors associated with pv-hc. regarding treatment, two pts received cidofovir therapy, one pt benefited from immunosuppression tapering, whereas only supportive therapy was required for the remaining 7 pts. notably, all pts achieved complete remission of symptoms. 3.9 (0.8-14.0) relapse who would benefit from further treatment intensification early after transplant. introduction: the prognosis for patients with hematologic malignancies (hm) who relapse after allohct is dismal, and immune checkpoint modulation with ctla4 blockade is a novel strategy to enhance the graft versus tumor (gvt) effect. materials (or patients) and methods: this is a phase i/ib study of the ctla4 blocking antibody ipilimumab to treat patients with any hm histology who relapse after allohct. the primary objectives are to determine the mtd and evaluate safety. secondary objectives include a preliminary evaluation of efficacy and changes in immune cell phenotype. ipilimumab was given at 3 mg/kg or 10 mg/kg iv q3 weeks for 4 cycles of induction, followed by maintenance dosing q12 weeks up to 1 year. disease-specific response criteria were assessed at the mid-point (7 weeks), end of induction (13 weeks), and throughout maintenance. immunophenotyping was performed by 8-color flow cytometry and analyzed by facsdiva. results: twenty-three patients are enrolled to date. in the phase i portion, 6 patients were treated at 3 mg/kg and 7 patients were treated at 10 mg/kg. an mtd was not reached, and 10 patients subsequently enrolled in the phase ib expansion cohort at 10 mg/kg. the median number of prior therapies excluding transplant was 3 (range 2-11), and 14 patients had received prior therapy for their post transplant relapse. histologies included chl (n ¼ 7), aml (n ¼ 7), nhl (n ¼ 4), and mds (n ¼ 2), and 1 patient each had mm, mpn, and all. the median age at enrollment was 55 (range 22-75). immune-related adverse events (iraes) were observed in 4 patients, and included itp (n ¼ 1), diarrhea (n ¼ 2), pneumonitis (n ¼ 3), and colitis (n ¼ 1), all of which were generally reversible with steroids with most patients resuming ipilimumab dosing. three dlts have been observed, including 2 cases of chronic gvhd (both liver, mild) and one patient death due to presumed sepsis in the context of severe iraes, including grade 3 colitis and grade 4 pneumonitis. nine patients remain on treatment, and 11 patients discontinued due to progressive disease, 2 patients due to cgvhd, and 1 patient with trm. in an interim efficacy analysis, 7/19 (36.8%) patients evaluable for response had anti-tumor activity with clinical benefit. four patients achieved formal responses by disease-specific criteria, including a chl patient with pr with dramatic reduction in nodal and extranodal disease with a complete marrow response at 7 weeks (baseline 90% involvement), a mm patient with a pr with near resolution of a lung plasmacytoma, and two patients with aml who achieved cr by 7 weeks. the median follow-up time among survivors is 5.4 months, and 6 month overall survival is currently 57%. immunophenotyping studies revealed that the ratio of regulatory t cells to conventional t cells decreased between 24% and 41% after ipilimumab treatment. conclusion: multiple doses of ipilimumab given to patients with relapsed hm after allohct were generally well-tolerated, with cgvhd and iraes observed in a small number of patients. anti-tumor activity was observed, both in lymphoid malignanices and for the first time in myeloid malignancies, including 2 patients with aml who achieved cr. the ratio of regulatory to conventional t cells decreased with ctla4 blockade. the phase ib expansion cohort at 10 mg/kg continues to accrue and will provide additional efficacy, safety, and correlative data. disclosure of interest: none declared. adverse risk cytogenetic (p ¼ 0.04). in patients who relapsed between 6 and 12 months post-transplant more than one course of chemotherapy to achieve cr1 (p ¼ 0.02), adverse risk cytogenetics (p ¼ 0.05) and flt3 itd positivity (p ¼ 0.00002) all predicted for relapse. finally only cmv positivity predicted for relapse risk for relapse more than 12 months post-transplant (p ¼ 0.05). conclusion: this study demonstrates for the first time that a complex interaction of disease specific and transplant factors determine the kinetics of relapse post-transplant. in addition to identifying that heterogenous factors related to transplant characteristics and disease biology determine the timing of disease relapse these data confirm the importance of early intervention post-transplant in patients allografted for aml and and will assist in the design of novel therapeutic strategies. disclosure of interest: none declared. introduction: bone marrow (bm) is the recommended stem cell source in hematopoietic stem cell transplantation (hsct) for bone marrow failure (bmf). recent large studies in aplastic anemia showed that the use of peripheral blood stem cells (pbsc) increased the risk for chronic graft-versus-host disease without reducing graft failures and lead to an inferior survival after hsct. however, a substantial proportion of hsct is still performed with pbsc. ease of collection and a more rapid engraftment might favor pbsc in the short term and more background information to clarify the situation is warranted. materials (or patients) and methods: the current global practice of hsct for bmf was studied for potential macroeconomic factors associated with the selection of stem cell source. introduction: the outcome of alternative donor hematopoietic stem cell transplantation (hsct) for the patients with severe aplastic anemia (saa) who lack an hla-matched familial donor has improved recently. this study was aimed to compare the treatment outcome of immunosuppressive therapy (ist) with alternative donor hsct in children with saa. materials (or patients) and methods: medical records of saa patients who received ist and/or alternative donor hsct from umbilical cord blood (ucb), haploidentical peripheral blood stem cells (pbsc), unrelated bone marrow (ubm), or unrelated pbsc (upbsc) between june 1996 and december 2012 were reviewed, and data were analyzed retrospectively. the kaplan-meier method was used to calculate the estimated survival rates which were compared with log-rank test. results: of 59 patients with saa, 42 patients received ist and/ or alternative donor hsct. nineteen patients received ist as an initial treatment modality, of whom 13 patients failed to respond at 6 months from the initiation of treatment. thirtyfour patients received alternative donor hsct either following ist (n ¼ 11) or as frontline therapy (n ¼ 23; 11 ubmt, 11 upbsct, 8 ucbt, 4 haploidentical pbsct). the failure-free survival rate (ffs) of ist group was 31.6%, while that of frontline hsct group was 91.3% (po0.001). patients who received hsct following ist showed an inferior event-free survival rate as compared with those who received frontline hsct (50.9% vs 91.3%; p ¼ 0.015). conclusion: the outcome of alternative donor hsct in our cohort was higher than usually expected, especially in those who received hsct without prior ist. these results suggest that frontline alternative donor sct might be a better treatment option than ist for children with saa who lack an hla-matched familial donor. disclosure of interest: none declared. increased risk of development of malignancies in adult patients treated with antithymocyte globulin as first line treatment for aplastic anaemia during a follow-up period of thirty years. j. v. d introduction: adult aplastic anemia (aa) is considered to be an immune-mediated disease with bone marrow aplasia and pancytopenia. patients can be treated with either hematopoietic stem cell transplantation (hsct) or immunosuppressive therapy (ist) with antithymocyte globulin (atg) and achieve long-term survival. treatment related long term toxicity and outcomes should guide the decisions about first line therapy. a long-term toxicity of both treatments is the development of malignancies. follow-up studies until 15 years after ist in aa patients showed cancer development in up to 25% of patients. analysis of cohorts with a longer duration of follow up are lacking and the incidence of malignancies in aa patients was never compared with a control population and the contribution of second line hsct to this risk is not clear. we assessed the long-term cumulative incidence of malignancies in a cohort of adult patients with aa who were treated with atg as first line treatment at leiden university hospital between 1980 and 2008 with hsct and death as competing risks and compared this incidence to cancer development in the general dutch population. materials (or patients) and methods: 93 patients treated with first line atg between 1980 and 2008 were entered in this single-centre retrospective cohort study. primary endpoint was the cumulative incidence of malignancies with death and second line treatment with hsct as competing risks. secondly, the cumulative incidence in our cohort was compared to that of the sex-and age matched general dutch population derived from the dutch cancer registration, using standardized incidence ratio (sir). furthermore, several patient-dependent (age), disease-dependent (severity of disease) and treatmentdependent (addition of ciclosporin to atg) variables were assessed as possible prognostic factors for cancer development, using cumulative incidence curves, gray's test and the cox proportional hazard model for the cause-specific hazard. spss statistics 20.0 and r were used for all data analyses. ethical approval for data collection and data analysis was obtained. results: median length of follow-up of surviving patients without hsct was 17.4 years (range 0.2-32.2 years) , with a cumulative malignancy rate of 29% after 30 years. the incidence of malignancies was increased compared with the general dutch population (sir: 3.35 (95% ci: 1.98-5.08 )). the cumulative incidence of developing mds/aml was 6% after 30 years (occurring at 5-17 years after start of treatment). in the multivariate analysis, age at time of ist was significantly associated with a higher malignancy rate (hr: 1.52 per 10 years; p ¼ 0.01). hr for addition of ciclosporin was1.87 (p ¼ 0.25) and hr for nsaa vs (v)saa hr was 1.65 (p ¼ 0.32)). the risk of the development of malignancies in aa patients treated with atg is increased compared to the risk in the age and sex matched dutch population for both solid and hematologic malignancies. we are not able to discern whether the increased risk is due to the treatment with ist or due to the biology of the disease. long follow up studies in age matched aa patients receiving hsct are needed to compare this long term risk between both treatments. these results underscore the recommendation in international guidelines to follow all patients with aa after initial ist lifelong. disclosure of interest: none declared. 1 hematopoietic stem cell transplantation, 2 molecular biology, dmitriy rogachev center for pediatric hematology, oncology and immunology, moscow, russian federation introduction: results of matched unrelated and, to a lesser extent, haploidentical transplantation in severe aplastic anemia have improved over the last decade. the major factors behind this improvement are high-resolution hla-typing, intensified immune suppression with fludarabine and alemtuzumab and modern supportive care. we implemented a new graft manipulation method, tcr-alpha/beta depletion, to further improve gvhd control and overall results of transplantation in a group of saa patients, refractory to two courses of atg/csa immune suppression. materials (or patients) and methods: twenty six patients with saa, median age 11,4 (2,5 -22) years, 16 male/ 10 female, received mud (20) or haploidentical (6) transplantation with tcr alpha/beta and cd19 depletion between 01. 09.2012 and 01.09.2014 . patients received a median of 2 atg/csa courses. core preparative regimen included cyclophosphamide 25 mg/ kg x4, fludarabine 30 mg/kg x 5, atg (horse) 25 mg/kg x 4 and total lymphoid irradiation 6 gy total dose. one patient received alemtuzumab instead of atg due to anaphylaxis. in all cases g-csf-mobilized pbsc were used as graft source. tcr-alpha/beta and cd19 depletion were performed with clinimacs plus according to manufacturer's recommendations. final graft contained 10x106 of cd34 þ /kg and 17x103 of tcra/b þ /kg. post-transplant immune suppression included short methotrexate and tacrolimus till day þ 45-60. results: primary engraftment was registered in all patients, median 15 days for neutrophils and 14 for platelets. secondary graft failure/rejection developed in 2 patients, both transplanted from mud. both patients were salvaged with a second mud transplantation. acute gvhd was registered in 4 patients, grade 2 skin in 2 and grade 3 skin þ gut in 2 patients. cumulative incidence of agvhd grade 2-3 was 15%(95%ci:6-38). chronic gvhd was registered in 1 patient, cumulative incidence 4%(95% ci:0,7-30). two patients (1 from each cohort) died of cmv pneumonia at 4 and 8 months respectively. in both cases cmv sero status was d-/r þ . at 2year follow-up cumulative incidence or trm is 12%(95%ci:0,3-46), km estimte of overall survival is 88%(95%ci:72-100), 92%(95%ci:76-100) in the mud group, 67%(95%ci:13-100) in the haplo group. conclusion: tcr alpha/beta depletion is a highly effective method of gvhd prophylaxis in saa patients. use of this platform makes alternative donor transplantation in saa a safe therapeutic option. further improvement will depend on new strategies to control viral infections. disclosure of interest: none declared. tp:129 (34) vs 173 (36) cm/s, respectively; (po0.001), and were decreased more significantly following hsct than on tp: mean(sd) d: -42 (31) vs þ 6 (34), respectively. the percentage of patients with normal velocities was significantly higher post-hsct (29/32) than in the tp arm (16/32) (p ¼ 0.001) conclusion: this prospective national trial comparing tp vs. hsct in sca-patients with a history of abnormal velocities shows for the first time that hsct significantly results in a greater decrease in velocities than tp, and has very little toxicity. these results suggest hsct is the treatment of choice for sca-children with a history of abnormal-tcd and genoidentical donor. disclosure of interest: none declared. introduction: busulfan (bu) is the backbone of the conditioning regimen for patients with sickle cell anemia (sca) undergoing bmt. patients with sca might predispose to transplant-related neurological and pulmonary toxicities due to pre-existing disease-related cerebrovascular and lung injury. bu therapy appears to be an important contributing factor in this context. to date, no studies have evaluated the pharmacokinetic (pk) parameters of intravenous bu (ivbu) for subsequent doses in patients with sca. materials (or patients) and methods: we studied ivbu pk parameters and clinical outcomes of 36 children with sca undergoing bmt from hla matched siblings. the median age of patients was 10.4 (range, 1.7-17.1 ) years. conditioning regimen consisted of bu/cy200/atg (n ¼ 12) or flu/bu/cy200 (n ¼ 24). six patients (17%) had stroke, 11 (31%) exhibited gliosis due to previous brain injury, 13 (36%) had repeated acute chest syndrome, and 8 (22%) patients were on chronic transfusions. ivbu was administered every 6 h for 4 days with pk-guided dose adjustment to target a conservative area under the concentration versus time curve (auc) range of 900-1350 mmol*min. the role of glutathione s-transferase (gst) polymorphisms has also been investigated. results: all patients had sustained engraftment. a repeated measures anova showed that the first-dose bu clearance (4.24 ml/min/kg) was significantly higher than after dose 5 (3.70 ml/min/kg; po0.0005), dose 9 (3.58 ml/min/kg; po0.0005), and dose 13 (3.42 ml/min/kg; po0.0005). such differences in clearance have never been described in patients with sca. after the first-dose, 69% of patients achieved the target range.none of the patients developed hepatic vod. no patient developed grade z3 toxicity. there was no association between gst polymorphism and the pk of bu. we adapted a new dose-adjustment strategy targeting exposures to the lower end (900 mmol*min) of the auc range after the first dose of bu to avoid unnecessary dose increases on subsequent days due to differences in clearance. this strategy enabled most patients (90%) to maintain the auc within therapeutic range following dose adjustments. a 3-year probability of os and sickle-cell free survival were 91% (95% ci, 75-97%). there was no correlation between any bu pk parameters and survival, toxicity, acute gvhd, chronic gvhd, and transplant-related mortality. conclusion: this study found that the pk behavior of ivbu in children with sca is characterized by significantly higher bu clearance after the first-dose compared with subsequent daily doses. this finding allowed us to adapt a new dose adjustment strategy after the first dose of bu, which effectively prevented subsequent dose readjustments. conservative auc range and targeting exposures to the lower end of the range after the first dose was associated with negligible toxicity, and high engraftment and sickle cell-free survival rates. disclosure of interest: none declared. however, the probability to find a suitable door is only 25-50%. since most of these patients (pts) could not find the potential donor, we would like to investigate the haploidentical donor hsct (hapo-sct) in thalassemia. materials (or patients) and methods: between jan 2013 and december 2014, a total of 116 severe thalassemia patients (pts) underwent hsct in our center. sixty five pts underwent mrd-hsct, 33 pts underwent mud-hsct and 18 pts underwent haplo-hsct. for haplo-hsct, 10 subjects were male and 8 were female. the median age was 16 yrs (range; 2-22). thirteen of 18 received stem cells from mother and 5 from father. ten of 18 were high risk class 3. these high risk class 3 pts received hydroxyurea 20 mg/kg/d for at least 3 months prior to hsct. all pts received 2 courses of pre-transplant immunosuppression (ptis) consisting of fludarabine (flu) 40 mg/m 2 /d together with dexamethasone (dex) 25 mg/m 2 /d for 5 days. after 2 courses of ptis, all pts received a reducedtoxicity conditioning (rtc) regimen consisting of thymoglobulin 1.5 mg/kg/d (d-11 to d-9) , flu 35 mg/m 2 /d i.v. (-7 to -2) each dose immediately followed by busulfan (bu) 130 mg/ m 2 once daily i.v. (d-7 to d -4) gvhd prophylaxis consisted of cyclophosphamide (cy) 50 mg/kg/d (d þ 3 to d þ 4). tacrolimus or sirolimus was given for 6 months to 1 yr started together with mycophenolate mofetil on d þ 5, the latter was quickly tapered after 2 months. t-cell repleted peripheral blood stem cells (pbsc) were given to all pts, targeting a cd34 þ dose of 7-16 x 10 6 cells/kg. results: sixteen of 18 were engrafted with full donor chimerism (100%) while 2 pts suffered graft failure. however, these 2 pts received second transplant on day þ 30 with minimal conditioning regimen and additional pbsc after which they achieved full donor chimerism. the median time to neutrophil engraftment was 18 days (range; 14 -22) . six pts had acute gvhd gr i, 3 grade ii and 1 gr iv. only one had extensive chronic gvhd. at this time, 17 of 18 pts survive thalassemia-free and have sustained full donor chimerism (100%). event free survival (efs) and overall survival (os) rates are 95%. the median follow up time is 12 months (range 4-20). the efs rates among mrd, mud and haplo-hsct in our center are 88%, 82% and 95% respectively (p ¼ 0.46). conclusion: haploidentical hsct for high risk thalassemia pts with our novel approach is safe and should be considered as modality to secure thalassemia-free survival with a low risk for graft rejection and treatment-related mortality. in view of our results, we suggest that all thalassemia pts even those with high risk class 3 features should be offered the chance for cure with hsct. disclosure of interest: none declared. introduction: allogeneic hematopoietic stem cell transplantation (hsct) is a curative option for many patients with hematological malignancies. graft versus host disease (gvhd) mediated by donor alloreactive t cells remains a major obstacle and limits its wider application. it is now well established that t regulatory cells (tregs) are critical for the maintenance of self-tolerance, and a deficiency or dysfunction in these cells is thought to be a key factor in the development and progression of gvhd. numerous murine studies have shown the benefit of adoptive transfer of tregs in the setting of hsct. notably, the success of this approach is improved if the transferred tregs are specific for antigens expressed by the host. a robust method to generate homogeneous populations of alloantigen specific tregs in humans would be a major step towards realizing the goal of using these cells to suppress gvhd. traditional approaches to generate antigen specific t cells include repetitive cycles of re-stimulation with antigen in vitro, sorting of tetramer positive cells, or over-expression of a t cell receptor for a specific antigen. these methods produce limited cell numbers and require modification for each individual patient depending on their own tissue type and that of the transplanted patient. we hypothesized that a more efficient approach to generating antigen specific tregs would be to genetically engineer them to express alloantigen-specific chimeric antigen receptors (cars). materials (or patients) and methods: we generated an hla-a2 specific car by cloning the immunoglobulin heavy and light chain variable regions from an anti-hla-a2 antibodysecreting hybridoma. these regions were fused into a single chain antibody, which was then linked to intracellular signalling domains from human cd28 and cd3zeta. surface expression and antigen-specificity of the a2-car was confirmed by flow cytometry to detect expression of a myc epitope tag on the extracellular portion of the car and binding to an hla-a2 tetramer, respectively. cd4 þ cd25 hi cd45ra þ naïve tregs (ntregs) and cd4 þ cd25 lo cd45ra þ naïve tconv (ntconv) cells were sorted from the blood of hla-a2donors, stimulated with artificia apcs and transduced with lentivirus encoding the a2-car or a control car specific for her2. results: transduced tregs maintained their expected phenotype, including high levels of foxp3, ctla-4, cd25 and helios, and low levels of cd127 and il-2 compared to tconv cells. to test antigen specificity, the transduced t cell lines were stimulated with k562 cells expressing hla-a2 or her2: only k562 cells expressing hla-a2 stimulated proliferation of a2-car expressing tregs and tconv cells. car-stimulated tregs also suppressed the in vitro proliferation of car-stimulated tconv cells. to demonstrate the in-vivo suppressive capacity of a2-car tregs, nsg mice were reconstituted with hla-a2 þ pbmcs in the absence or presence of different ratios of control-transduced or a2-car transduced tregs. preliminary results suggest that a2-car tregs have a superior ability to delay the onset of gvhd compared to polyclonal tregs. conclusion: human tregs can be efficiently transduced to express functional, alloantigen-specific cars that confer antigen-specific specific suppression of effector t cells both in-vitro and in-vivo. these data supports the feasibility of this strategy to re-direct tregs for transplant-related therapies. disclosure of interest: none declared. car spacers including ngfr domains allow efficient t-cell tracking and mediate superior antitumor effects m. casucci 1,* , l. falcone 1 , b. camisa 2 , f. ciceri 3 , c. bonini 2 , a. bondanza 1 1 innovative immunotherapies unit, 2 experimental hematology unit, 3 clinical hematology and bone marrow transplantation unit, san raffaele hospital scientific institute, milano, italy introduction: chimeric antigen receptors (cars) frequently include an igg1-ch2ch3 spacer conferring optimal flexibility for antigen engagement and allowing the selection and tracking of car-expressing t cells. a serious drawback of ch2ch3-spaced cars is however their interaction with fcg receptors (fcgrs). indeed, this antigen-independent binding may lead to the unintended elimination of cells expressing these receptors (mainly phagocytes), foster the development of non-specific immune reactions and drastically decrease the efficacy of the strategy due to the premature clearance of transduced t cells in vivo. materials (or patients) and methods: we designed and constructed novel car backbones by substituting the igg1-ch2ch3 spacer with regions from the extracellular portion of the low-affinity nerve-growth-factor receptor (lngfr), differing for the length and potential binding to ngf. in particular, we used our recently developed cd44v6-specific car as a model for comparing the antitumor activity of the different lngfr-based designs both in vitro and in vivo. results: after transduction, all constructs could be identified on the t-cell surface using anti-lngfr antibodies, indicating that they were correctly processed, mounted on the cell membrane and still recognized by anti-ngfr antibodies. as a consequence, all the lngfr-based spacers allowed selecting car-t cells with immune-magnetic beads coupled to anti-ngfr antibodies, without interfering with their expansion and functional differentiation after activation with cd3/cd28 beads plus il-7 and il-15. most importantly, lngfr-spaced car-t cells maintained potent cytotoxic, proliferative and cytokine-release activity in response to cd44v6-expressing leukemia and myeloma cells, while lacking antigen-independent recognition through the fcgrs. noticeably, even at supraphysiological ngf concentrations, the lngfr-spaced cd44v6-car.28z car t cells were not induced to proliferate, indicating the absence of signaling via soluble ngf. strikingly, lngfrspaced car-t cells better expanded and persisted in vivo compared to ch2ch3-spaced car-t cells and mediated superior antitumor effects in a well-established tumor disease model. interestingly, we demonstrated that the premature disappearance of ch2ch3-spaced car-t cells was due to engulfment by mouse phagocytes, a phenomenon not occurring with lngfr-spaced t cells. in conclusion, we have demonstrated that the incorporation of the lngfr marker gene directly in the car sequence allows for a single molecule to work as a therapeutic and as a selection/tracking gene and shows an increased efficacy/safety profile compared to the igg1-ch2ch3 spacer. disclosure of interest: none declared. university children's hospital, würzburg, 10 clinic of pediatric oncology, heinrich-heine-university düsseldorf, 11 university children's hospital, essen, 12 university children's hospital, münster, 13 department of pediatrics, jena university hospital, germany introduction: viral infections represent an important cause of morbidity and mortality in immunosuppressed patients post hematopoietic stem cell transplantation (hsct). as viral infections often remain refractory to pharmacologic treatment, alternative treatment strategies such as immunotherapy are required. adenovirus (adv) is the predominant diseasecausing pathogen in pediatric hsct. materials (or patients) and methods: in a clinical trial we analyzed safety and efficacy of ex vivo adoptive t-cell transfer (act) with hexon-specific t cells, predominantly of t helper -1 phenotype. thirty patients suffering from chemo-refractory adv disease or viremia after hsct were treated with advspecific t cells generated by ifn-g capture technique. results: adv-specific t cells were successfully isolated in 100% of cases using the adenoviral hexon antigen and were directly infused into the patients without further ex vivo expansion steps. adv-specific t-cell grafts were composed of a mixture of naïve, central memory, effector memory and effector t-cell populations with a predominance of late effector stages, indicating proliferation and effector potential of the transferred t cells. in all thirty patients, act was feasible without acute toxicities or significant onset of graft-versus-host disease. act led to antiviral immunity in vivo up to 6 months with viral control, resulting in complete clearance of viremia in 86% of patients with antigen-specific t-cell responses. efficacy of adoptive t-cell transfer was independent of the initial t-cell dose. after a follow up of 6 months post act, overall survival was markedly increased in responders (mean: 122 days, 15 survivors) as compared to non-responders who all died shortly after act (mean: 24 days, no survivors). adv-related mortality was 100% in non-responders compared to 9.5% in responders (z1 log reduction of dna copies/ml post act), indicating a strong correlation between virus-specific immunity and virus control. conclusion: in conclusion, ex vivo act of adv-specific t helper -1 cells was well tolerated and led to successful and sustained restoration of t-cell immunity, correlated with virological response and protection from virus-related mortality. this cellular immunotherapy is a short-term available and broadly applicable treatment. disclosure of interest: none declared. [o164] putkonen 6 , t. kuittinen 1 , j. pelkonen 2,7 , p. mäntymaa 7 , k. remes 6 , v. varmavuo 1 , e. jantunen 1 o094 o096 comparison of ric-allohct and autohct for 455 years old patients with acute lymphoblastic leukemia: an analysis from acute leukemia working party of the ebmt s united kingdom, 7 erasmus mc-daniel den hoed cancer centre double-blind, placebo-controlled phase 3 study of brentuximab vedotin in patients at risk of progression following autologous stem cell transplant for united states, 4 szent istvan & szent laszlo corporate hospital hematology & stem cell dept united states, 6 department of bone marrow transplantation & oncohematology, maria sklodowska-curie institute of oncology united states, 10 istituto nazionale dei tumori, milano, 11 irccs azienda ospedaliera universitaria united states introduction: the aethera trial was initiated to evaluate whether brentuximab vedotin (bv) can prevent progression in conclusion: bv improved post-asct pfs across all subgroups of pts. pts with more risk factors had the most benefit from bv. aes were consistent with the known safety profile of bv references: 1. lion t. adenovirus infections in immunocompetent and immunocompromised patients european guidelines for diagnosis and treatment of adenovirus infection in leukemia and stem cell transplantation: summary of ecil-4 adoptive transfer and selective reconstitution of streptamer-selected cytomegalovirus-specific cd8 þ t cells leads to virus clearance in patients after allogeneic peripheral blood stem cell transplantation lowest numbers of primary cd8 þ t cells can reconstitute protective immunity upon adoptive immunotherapy donor ebv status has introduction: epstein-barr virus (ebv) has been a major cause of post-transplant lymphoproliferative disorder after allogeneic stem cell transplantation (hsct). the impact of the donor (d) and recipient (r) serologic status on survival, relapse-free survival, relapse incidence, non-relapse mortality and incidence of graft-versus-host disease was unknown so far. objective: we analyzed the influence of the donor's and recipient's ebv status on allo-hsct transplant outcomes. materials (or patients) and methods: 11,364 allo-hscts performed due to acute leukemia ebv-seropositive donors also had no influence on relapse-free survival 57), and non-relapse mortality hsct recipients receiving grafts from ebv-seropositive donors had higher risk of acute gvhd after adjusting for confounders (donor type, conditioning, stem cell cource, patient age, gender match, t-cell depletion, year of transplant), d þ serostatus had an impact on development of acute gvhd p ¼ 0.09, and for cgvhd all patients engrafted, with a median time to neutrophil and platelet recovery of 18 days (13-45) and 16 days (9-100), respectively. post-hsct recovery of lymphocyte subsets was broad and fast: median time to cd34100/ml was 28 days, to cd44200/ml 41 days and to cd1940/ml 41 days. circulating t cells comprised naïve and memory subsets, with a recovery of cd31 recent thymic emigrants (rtes) from day 30. all patients had a significantly higher proportion of rtes at day 30 and 180 compared to their pre-hsct levels, suggesting an improvement in their thymic function after hsct. with a median follow-up for living patients of 15 months (5-24), the 1-year cumulative ci of nrm and relapse were 17% and 35%. three of the 11 acute leukemia relapses were hla-loss variants. notably, one was observed for the first time in all. ci of agvhd grade ii-iv and iii-iv at 6 months were 17% and 7%, while 1-year ci of cgvhd was 20% conclusion: myeloablative haplohsct with pbsc, pt-cy and sirolimus is a valid option for patients with aggressive/ advanced disease. the acceptable rates of gvhd and nrm as well as the favorable immune reconstitution profile open the way for combining it with novel immunomodulatory/ cellular therapies to improve dfs in patients at high risk for o142 factors determining the kinetics of disease relapse after allogeneic stem cell transplantation (allo-sct) for acute myeloid leukaemia (aml): a survey from the acute leukaemia working party of the ebmt c o143 a novel quantitative pcr approach targeting insertion/ deletion polymorphisms (indel-pcr) for chimerism quantification: finally high sensitivity and quantification capacity together a post-hematopoietic stem cell transplantation (sct) chimerism monitoring is important to assess engraftment, anticipate relapse and provide information on the development of graft versus host disease, facilitating therapeutic intervention. the aim of this study was to test the technical efficacy and clinical utility of a novel quantitative pcr approach targeting insertion/deletion polymorphisms of note, analysis of artificial mixtures provides evidence of significantly (z2 logs) higher sensitivity by indel-pcr (0.01%) than by str-pcr (1%, table 1). moreover, indel-pcr shows unprecedented quantification capacity (table 1). out of the 113 samples analyzed, 29 were positive and 6 negative by both methods, while 78 were positive only by indel-pcr (95% with o1% recipient). hematological relapse occurred in 5 patients, molecular relapse/persistence in 2 patients. all of them presented a positive indel-pcr (with increasing %r in 4/ 5) and a negative str-pcr result in the sample before relapse (table 2). the 12 patients in complete remission, although presented positive indel-pcr, showed stable or decreasing %r chimerism dynamics in 10/12 (data not shown). conclusion: this novel indel-pcr is a simple and accurate technique that, in comparison with the current gold standard str-pcr, shows very good concordance and provides higher rates of informative loci per patient, as well as unprecedented combined sensitivity and quantification capacity introduction: the immune recovery after cd34 þ cell selection is slow and patients tend to remain susceptible to opportunistic infections for several months after hsct. to hasten and improve post-transplant immune reconstitution broad repertoire various strategies under this approach, a rapid immunological reconstitution and very promising outcome have been reported in pediatric patients. with the aims of confirming these results even in adults, we have recently launched this programme and here we report our preliminar clinical data in 22 leukemia patients we have so far treated over the past 26 months. materials (or patients) and methods: twenty-two patients, median age 44 years (range 19-67), with aml (n ¼ 16), all (n ¼ 5), mds (n ¼ 1) entered the study. eight patients were in cr1, 3 in cr2, and 11 in advanced-stage disease at transplant. conditioning consisted of atg 1,5 mg/kg from day -13 to day -10, treosulfan 12gr/sqm from -9 to -7, fludarabine 30 mg/sqm from -6 to -2 and thiotepa 5 mg/kg on days -5 and -4. no patient received any post transplantation pharmacologic prophylaxis for gvhd. ten mg/kg g-csf was used to mobilize pbpcs from one-haplotype mismatched donors (3 mothers median cd4 þ cell/ml counts at 30, 60 and 90 days since the transplant were 36, 80 and 110, respectively. cmv antigenemia reactivation occurred in 6 cases (in 2, cmv serology was unfavourable). no patients has so far developed cmv disease. invasive fungal disease was prevented in all cases using l-amb-based prophylaxis over the neutropenic phase. overall,10 patients have so far died (7 relapse,3 non-hematologic causes). 12 survive (10 diseasefree,2 in early relapse) at a median follow-up of 13 months (range 2-24) (fig.1). conclusion: the infusion of ab/cd19-depleted grafts was safe and effective also in adult setting, resulting into rapid engraftment and fast immunological reconstitution haploidentical stem cell transplantation in very poor risk cytogenetics acute myeloid leukemia: results in 40 consecutive patients cytogenetic prognostic risk was defined according to the revised medical research counsil (mrc) classification, from diagnostic bone marrow samples with standard methods and in accordance with international system of human cytogenetics guidelines. os and disease free survival (dfs) were calculated using the kaplan-meier methods. results: median age of the patients at time of transplant was 50 years (range 28 to 67).cytogenetics:chromosome 7 abnormalities 16 pts, monosomal karyotype 3 pts and complex karyotyping 21 pts non relapse mortality (nrm) was 17% at 1 year after transplant.estimated lfs from day þ 30 after transplant was 34.5% and 28% at 3 years. conclusion: haploidentical stem cell transplant (haplo-sct) is a valid treatment option for the patients with very poor risk aml. references: 1 haploidentical, unmaipulated, primed bone marrow for pts with high risk hematologic malignancies haploidentical transplantation using t cell replete pbsc and myeloablative conditioning in pts with high risk hematologic malignancies who lack conventional donors kodera 7 on behalf of the worldwide network of blood and marrow transplantation o149 graft depletion of tcralpha/beta and cd19 in matched unrelated and haploidentical transplantation for severe aplastic anemia: high survival with low incidence of graftversus-host disease and transplant-related mortality m at enrollment all patients were on tp and paired analysis showed that mean (sd) maximum velocities had significantly decreased (po0.001) under tp:169 (46) cm/s vs 219 (26) cm/s at tp initiation. following hla-typing, 35 without genoidentical donor were included in the transfusion arm and 32 with genoidentical donor were transplanted in 6 hsct-centers. during the 12 months follow-up, no stroke was observed but one patient in the tp arm experienced a hyperammonemic reversible coma, without mri/mra alteration. in the hsct arm, all patients successfully engrafted, one grade ii and two grade iii acute gvhd, and no chronic gvhd were observed. complications were seizures (n ¼ 2), cmv (n ¼ 9) or ebv replications (n ¼ 5), hemorrhagic cystitis (n ¼ 4), aspergillosis (n ¼ 1), prolonged but reversible thrombopenia (n ¼ 2), transitory hemolytic anemia (n ¼ 1) granda ospedale maggiore policlinico, 5 hematology and bone marrow transplantation unit metabolic syndrome (ms) is defined as a clustering of five factors including (1) hyperglycaemia (2) hypertriglyceridaemia; (3) low hdl cholesterol; (4) hypertension; (5) obesity (high waist circumference or body mass index (bmi)). it is associated with raised risk of cardiovascular disease (cvd) and is increasingly recognised in patients after hct and revised guidelines for long-term hct survivors recommend screening for ms. previous studies have been small and the definition of ms variable, although harmonised criteria are now agreed materials (or patients) and methods: this was an ebmt approved cross-sectional, non-interventional study of consecutive hct patients aged 18 þ years and a minimum of 2 year post-transplant attending routine follow-up hct and/or late effects clinics in 9 centres. centres completed med c forms incorporating routine recording of the ms parameters (given above) as well as performance status (ecog); evidence of cardiovascular events using the harmonised definition of ms (at least 3/5 factors), the prevalence of ms was 47.2%. there was no difference in time since hct but there was a significant difference in prevalence by age at diagnosis, hct, follow-up (all po0.001 with increasing age). as expected, statistical differences (po0.001) between patients with and without ms were observed for bmi routine screening and early intervention may reduce the risk of cardiovascular events in hct survivors, and should ideally be tested in a randomised controlled trial setting. meanwhile, screening and management of reversible features of the metabolic syndrome should be robustly integrated within routine hct long-term follow-up care. references: disclosure of interest: none declared. introduction: the introduction of less toxic conditioning regimens for hematopoietic cell transplantation (hct) has led to an increase in eligible patients, although their benefit on patient's perceived wellbeing remains unclear. we aimed to prospectively study patients' quality of life (qol) and emotional wellbeing (ew) in consecutive hct recipients depression and anxiety (hads), and sleep quality (psqi) at pre-hct, at hospital discharge (hd) and at 3 months post-hct. results: out of 223 transplanted patients, 191 (86%) consented to participate. those who refused (n ¼ 32, 14%) more frequently had active disease at hct (31% vs. 21% for pr/cr, p ¼ 0.032) and/or had a prior hct among included patients (57% men; median age 53 83 received an auto-hct (n ¼ 79 at hd; n ¼ 52 at þ 3 m) 55 received an allo ric-hct (n ¼ 41 at hd; n ¼ 33 at þ 3 m) at baseline, clinically significant depressive symptoms were reported by 5% of the patients, with a slight increase (7%) at hd and at þ 3 m (p ¼ 0.058). again, there was a strong interaction between depressive symptoms and hct groups in the early post-hct phase (p ¼ 0.002); depression decreased in auto-hct after hd and, on the contrary, it increased in the same time-point in mac-hct (p ¼ 0.041). borderline differences were seen between auto-and ric-hct (p ¼ 0.062) but not between ric-and mac-hct (p ¼ 0.827). clinically significant anxiety was observed at baseline in 14% of the patients and significantly decreased at the time of hd (6%) and afterwards (5%) conclusion: mac-hct recipients reported the greatest impairment in the parameters studied; other variables such as gender, age and baseline ew/qol should be considered for specific psychological/clinical follow-up and eventual intervention unit of molecular and functional immunogenetics, 4 unit of hematology and bone marrow transplantation conflict with: scientific consultant of molmed s.p.a. o161 erbb2-chimeric antigen receptor (car) modified cytokineinduced killer (cik) cell intervention for refractory solid tumors after allogeneic stem cell transplantation m already at day 10 of culture, up to 90% of transduced cells showed surface expression of the erbb2-car (n ¼ 4). there were no significant phenotypic differences (cd3 þ /cd56 þ /cd4 þ /cd8 þ /tcr-a/b and tcr-g/d) between unmodified, empty control vector and erbb2-car transduced cik cells. erbb2-car cik cells efficiently lysed erbb2-overexpressing breast carcinoma cells (mda-mb 453) in a 3 hour short-term cytotoxicity (europium release) assay in vitro. compared with unmodified and empty-vector transduced cik cells e:t; 46.0% vs. 18.1% specific lysis, n ¼ 4; po0.019). long-term cytotoxicity analysis (16 h, brightfield imaging cytometry) demonstrated comparable results even at low effector to target ratios of 1:1 (36.3% vs. 11.8% specific lysis, n ¼ 3). comparable results for shortand long time cytotoxicity could be obtained for all other tested rhabdomyosaroma cell lines in vitro. conclusion: erbb2-car engineered cik cells are highly specific and efficient against erbb2-antigen expressing tumor cell lines in vitro. our experiments may help to develop an approach for improved treatment of patients with high-risk adoptive t-cell immunotherapy with hexon-specific thelper-1 cells as a treatment for refractory adenovirus infection after allo-sct -safety and efficacy results from a anna children's hospital university children's hospital, frankfurt, 5 dr. von haunersches kinderspital introduction: allogeneic hematopoietic cell transplantation (hct) offers the chance of cure for patients with nontransformed follicular lymphoma (fl) but is associated with the risk of non-relapse mortality (nrm). the aim of this study was to identify subgroups of fl patients who benefit from hct. materials (or patients) and methods: the minimum essential a data of 146 consecutive patients who received hct for fl between 1998-2008 were extracted from the database of the german registry ''drst''. diagnosis of fl was verified by contact with reference pathologists. results: the median patient age at time of transplantation was 48 years (range 29-71). prior to allogeneic hct 90/146 patients (62%) had undergone autologous hct. at time of hct, 110 patients (77%) had sensitive disease while 33 patients (23%) had chemorefractory disease (rd). engraftment was achieved in 99% of evaluable patients. day 100 nrm was 16%. the median follow-up of surviving patients was 9.1 years (range 3.6-15.7) . estimated 1, 2, 5, 10-year overall survival (os) was 67%, 60%,53%, and 48%, respectively. the corresponding estimates for efs were 63%, 53%, 47%, and 40%, respectively. 40% of the 33 patients with rd at time of transplantation survived long-term. of the n ¼ 116 patients with documented cr after hct only n ¼ 17 (15%) relapsed. only two late relapses (beyond year 3) were diagnosed among the 77 patients with a follow-up45 years. patients with chronic gvhd (irrespective of stage) had a lower risk of relapse, if transplanted in cr, and a higher chance to achieve cr, if transplanted in pr or with pd (no chronic gvhd: 19/48, chronic gvhd: 11/75, p ¼ 0.0019). therefore a reduced intensity conditioning approach might be considered in future prospective trials. hsct is most successful prior to leukemic transformation. given implications for treatment decisions and donor selection gata2 mutation screening should be performed on all patients with molecularly undefined mds and bmf disorders and potential related donors. disclosure of interest: none declared. pre-transplant weight loss predicts non-relapse mortality and relapse rates in patients with myelodysplastic syndrome after allogeneic stem cell transplantation a. radujkovic 1, * introduction: we have recently provided evidence that weight loss and minor metabolic changes prior to allosct were able to predict relapse and death of acute myeloid leukemia patients using data from two independent patient cohorts. this retrospective study investigated the influence of pre-transplant weight loss on the outcome of mds patients after allosct in three independent cohorts. materials (or patients) and methods: a total of 111 patients (59% male) with a median age of 52 years were included into the analysis. patients have been diagnosed with mds according to who criteria and received an allosct between 2000 and 2012 in three different german referral centers (heidelberg, dresden and berlin). weight data were raised from medical records by three independent researchers in three independent institutions. weight loss (expressed in percent) was calculated on the basis of recorded weight data at the time of allosct and the maximum weight in the time period of 3-6 months prior to allosct. the mds who subtype was ra(rs)/rcmd in 31 patients (28%), raeb1 in 30 patients (27%) and raeb2 in 49 patients (45%). according to ipss 34%, 45% and 21% of the patients were in the risk groups intermediate-1, intermediate-2 and high, respectively. the majority of the patients (n ¼ 72, 65%) was previously untreated. nineteen patients (17%) and 14 patients (13%) received hypomethylating agents and induction type chemotherapy prior to allosct, respectively. thirty-one patients (28%) received transplants from related donors, 59 patients (53%) from matched unrelated donors and 21 (19%) from mismatched unrelated donors. ninety-three patients (84%) received reduced intensity conditioning and 18 patients (16%) received standard myeloablative conditioning. survival times were measured from date of allosct. overall survival (os), relapse-free survival (rfs), relapse incidence and non-relapse mortality (nrm) were calculated from date of allosct to the appropriate endpoint. cox regression analysis was applied for os, rfs, relapse and nrm. relapse and nrm were considered as competing risks. results: estimated median follow-up at the time of analysis of surviving patients was 36 months. a total of 34 (31%) patients experienced weight loss 42% with 17 (15%) patients losing more than 5% weight in the period of 3-6 months prior to allosct. patient, disease and transplant characteristics did not differ between patients with weight loss (42%, n ¼ 34) and those without (n ¼ 77). in multivariate analysis, weight loss and donor type were independently associated with shorter os and rfs (po0.001 and po0.05, respectively). nrm was predicted by donor type (p ¼ 0.006), ipss (p ¼ 0.015) and pre-transplant weight loss (p ¼ 0.014) in multivariate analysis. furthermore, weight loss was also an independent predictor of relapse (cause-specific hr 11.52 95%ci po0.001) . in a mixed effect model with weight loss as outcome only ipss prior to allosct had significant impact on weight loss (p ¼ 0.046 introduction: hla-c-encoded kir ligands (c1/c2) have been identified as important factors for the outcome of unrelated allogeneic hsct: in a previous retrospective study cml recipients bearing at least one c2 ligand showed worse survival when compared to c1c1 recipients (hr 5.9 , po0.01), especially when peripheral blood progenitor cells (pbpc) were used or in advanced disease stages. these initial findings were confirmed in a second cohort in advanced aml/cml, (but not in mds or all/nhl) receiving pbpc. notably, hla-c allele matching contributed differentially to the transplantation outcome: it was beneficial in c1 patients, but was detrimental in c2 recipients (increased trm, hr 3.5, po0.012 ; increased relapse, hr 2.7, p ¼ 0.06). we hypothesized that c1 patients have a high frequency of immuno-competent nk cells (icnk) enabling eradication of residual disease due to the genetically hard-wired sequential acquisition of kir receptors during early reconstitution phase post hsct, with c1-specific nk cells emerging first. alloreactive t cells -resulting from hla-c mismatch-might thus not have an additional beneficial effect in c1 patients and might even be detrimental (e.g. increased gvhd), but may serve an important function in relapse control in c2 patients. the lack of disease control in hla-c-matched c2 patients would thus be explained by a combination of insufficient numbers of icnk cells in the early phase and the lack of alloreactive t cells later post hsct. consequently, this group exhibited poorest clinical outcome of all four groups defined by recipients kir ligands and hla-c allele matching in the investigated cohorts. materials (or patients) and methods: the aim of the present retrospective study was to determine the influence of hla-c allele matching on the background of hla-c encoded kir ligand status in a large patient cohort (n ¼ 7327, provided by cibmtr). statistical analysis was performed by cibmtr. patients received unrelated allografts between 1988 and 2009 , with the majority of patients after 2000 (70%). 30% of the recipients were younger than 30y, 67% younger than 40y. 70% of patients had early, 5% intermediate, and 25% advanced disease (aml 40%, all 21%, cml 22%, mds 17%). 56% received bone marrow, 44% pbpc. 80% received a myeloablative conditioning. endpoints were os, agvhd ii-iv and iii-iv, extensive chronic gvhd, relapse, dfs, and nrm. due to multiple comparisons and multiple endpoints, po0.01 was considered as significant. all models were adjusted for significant clinical covariates. stratification was used in cases of non-proportional hazards. patient-donor pairs were classified according the recipient hla-c kir ligand expression (c1c1, or c2 ( ¼ c1c2 or c2c2)) and the degree of the hla-c allele match: results: introduction: hepatic veno-occlusive disease (vod), also called sinusoidal obstruction syndrome, is a potentially fatal complication of hematopoietic stem cell transplantation (hsct). severe vod (svod), clinically characterized by multiorgan failure (mof), has been associated with a 480% mortality rate; it may develop in a substantial number of highrisk patients (pts). defibrotide (df), a sodium salt of complex single-stranded oligodeoxyribonucleotides, is thought to protect injured endothelium and to restore thrombo-fibrinolytic balance. in a phase 3 trial in svod, df improved complete response rate and survival at day þ 100 post hsct vs historical controls, with a favorable safety profile. in the european union, df is approved for treatment of severe hepatic vod in hsct therapy in adults and children. in the us, df is available through an expanded access, protocol-directed treatment ind (t-ind) collecting data on safety/efficacy in children and adults with svod and non-severe vod post hsct or post chemotherapy (ct). the original t-ind protocol required vod diagnosed by baltimore criteria (total bilirubin z2.0 mg/dl with z2 of hepatomegaly, ascites, or 5% weight gain) plus mof (renal and/or pulmonary) following hsct; the study was amended to include non-severe vod (ie, materials (or patients) and methods: in a single center retrospective study 382 patients who underwent allogeneic hematopoietic stem cell transplantation (hsct) for various diseases (51% acute leukemia) were genotyped for cyp 1b1 (c432g) expression and their influence on outcome was analyzed. genotyping of cyp 1b1 (c432g) was performed by real-time pcr.results: 169 patients (44%) were genotyped as homozygous wild-type (wt) gene c/c, 157 (41%) as heterozygous genotype c/g and 56 (15%) as homozygous gene mutated g/g. calculated genotype frequencies did not differ from that reported earlier by other studies for caucasians. patients' demographic and treatment characteristics showed no difference between the three groups except that cyp 1b1 cc was more common in females 52% than in males 38% (po0.02).five-year estimate for overall survival (os) was 58 þ 4% for the cc group and 48 þ 3% for the c/g-and g/g groups (po0.036). surprisingly, this difference was primarily evident in males (po0.009), where the group with cyp gene mutations did significantly worse (3-year estimate for os: 65 þ 5% vs. 47 þ 4%), whereas it was virtually absent in females (p ¼ 0.99). trm and rr were higher for the group with mutated genes in regard to the group with wt gene (although not significant). 2006 . in phylogenetic analysis of the e3 gene, a two-step pcr amplification of almost the entire adenovirus e3 gene was performed using primers designed from the known sequence of the hadv a31 reference strain (am749299.1). results: all 9 patients had been admitted to the ward, but the two last patients (patient 8 and 9) had no timely connection to other known hadv a31 cases ( figure) . in addition, four of the patients (1, 5, 6 and 7) made visits to the out-patient clinic on the same day as one or several other hadv positive patients.sequencing the hexon gene resulted in 100% homology between the patient samples but also to the reference strains of hadv a31 (accession number ab330112. 1 (cmv) is an important cause of morbidity after allogeneic hematopoietic stem cell transplantation (hsct). the latent virus reactivates in immune-compromised patients, due to both post-hsct immunosuppressive therapy and impaired t-cell reconstitution/function. here we report the impact of mismatches in hla-molecules between donor and recipient on cmv-reactivation and cmv-specific immune reconstitution. materials (or patients) and methods: this retrospective study included 752 patients, who received a transplant from a matched related donor (mrd n ¼ 234), matched unrelated donor (mud n ¼ 384), mismatched unrelated donor (mmud n ¼ 115) or mismatched related donor (mmrd n ¼ 19). hlatyping (10/10) of patients and donors was conducted via highresolution multiplexed pcr. blood samples were routinely monitored for cmv pp65 antigen expressing cells per 400,000 leukocytes. cmv-cytotoxic lymphocytes (cmv-ctl) reconstitution was analyzed in the blood from 246 patients at days þ 50, þ 100, þ 200, þ 300 after hsct, using 6 hla-cmv tetramers. results: the fisher's exact test was used to analyze the data. the outcome was that hla mismatch (class i or ii) showed significant influence on recurrent (multiple) cmv reactivations (mcmv-r) (po0.001). we analyzed the relative risk (rr) in the subgroups with different levels of hla-matching for 1cmv-r and mcmv-r. the group transplanted from mrd served as reference (ref. ) . shortly, we found significantly higher risk for mcmv-r in the mmud group (rr 2.6, 95% ci 1.63-4.15 , p ¼ 0.0001). in the mrd 24 (10%) patients had mcmv-r, while in the mud 59 (15%), in the mmrd 3 (16%) and in the mmud group 31 (27%) had mcmv-r. furthermore, we investigated the mean numbers of cmv-ctl/ ml of blood in the groups with different levels of hla-match. we divided cmv-ctl levels into 3 ranges: o1, 1-10 and 410 cmv-ctl/ml. in the mmud group we observed a trend for an increased risk for the lack of cmv-ctl (25 (48%) patients; rr 1.5 , 95% ci 0.96 to 2.38, p ¼ 0.07) compared to 21 (32%) patients from the mrd group. significantly less patients (17; 33%) had more than 10 cmv-ctl from the mmud group (rr 0.6, 95% ci 0.39 to 0.95 thus the focus of the present study was the selection of hadvstreptamer þ t-cells and ebv-streptamer þ t-cells.materials (or patients) and methods: cells from leukapheresis healthy donors were prepared in large (1 à 6 â 10 9 ) and small (25 x 106) cell batches. whereas the larger batch was directly labelled with streptamers to select hadv and/or ebvspecific t-cells (large-scale), the smaller batch was used to generate in vitro virus-specific t-cell lines before streptamerlabelling for streptamer selection (small-scale). isolation of hadv-and/or ebv-specific t-cells was performed using the clinimacs device.results: the purity of hadv-streptamer þ t-cells among cd3 þ cells, obtained from large-scale selection was only 7.6%, but reached up to 56% when hadv-and ebvstreptamers were applied simultaneously. a further increase in purity of hadv-specific t-cells reaching up to 98% was achieved by small-scale selection. all final products fulfilled the microbiological and chemical release criteria. ifn-g-response indicating functional activity was seen in 6/9 hadv and 2/3 ebv large-scale selections and in 2/3 hadv small-scale selections.conclusion: the use of hadv-streptamers for clinical applications is feasible particularly when combined with other streptamers or when performed after a previous in vitro expansion period. in this cohort of 149 t-cell replete haplo hscts using post-transplant high-dose cyclophosphamide, we found that a higher intensity of conditioning (mya or ric vs non-mya) as well as the use of more immunosuppressive calcineurin inhibitor (fk vs csa) were both significantly associated with a higher incidence of pv-hc. results: in cd34 þ lin -cd10cells, 1609 probes were deregulated between patients without agvhd and patients with agvhd (1560 of this probe were up-regulated and 49 were down-regulated, po0.05, fold change41.5) . in cd34 þ lin -cd10 þ cd24progenitors, 987 probes were deregulated between patients without agvhd and patients with agvhd (941 of this probe were up-regulated and 46 were downregulated, po0.05, fold change41.5). 273 probes were deregulated in both cd34 þ lin -cd10 þ cd24and cd34 þ lin -cd10populations. genes from ribosome protein biogenesis, translation machinery (eef1d, eef1g, eif3k) and cell cycle (ccnd1, cdk6) were over-expressed in cd34 þ lin -cd10 þ cd24and in cd34 þ lin -cd10populations from patients without agvhd compared with those from patients affected by agvhd and from healthy donors. expressions of genes from the oxidative phosphorylation metabolic pathway (ndufs2, sdha, atp5a1) and genes involved in stress resistance (btg2, mgst3, hpx) were specifically increased in cd34 þ lin -cd10 þ cd24lymphoid progenitors and not in cd34 þ lin -cd10non-lymphoid progenitors from patients without agvhd compared with patients suffering from agvhd and from healthy donors. we show for the first time that circulating lymphoid t-cell progenitors undergo profound changes in metabolism favoring energy production and response to stress after allo-hsct in humans. these mechanisms are abolished in case of agvhd, indicating a persistent cell-intrinsic defect in addition to the impact of agvhd on the bone marrow environment. disclosure of interest: none declared. introduction: post transplant interventions such as donor lymphocyte infusion (dli) or administration of pharmacological agents, represent important novel strategies with the potential to reduce the risk of disease relapse after allo-sct in acute myeloid leukaemia (aml). such approaches are critically dependent on timely intervention post-transplant but despite this the factors determining the kinetics of disease relapse in patients allografted for aml have not been defined. materials (or patients) and methods: 1052 adults who received an allo-sct for aml in first complete remission (cr1) between 2000 and 2012 were studied. 544 patients were transplanted using a sibling donor and 508 from an adult matched-unrelated donor. 538 patients received a myeloablative conditioning (mac) regimen and 514 a reduced intensity (ric) regimen. a series of landmark analyses were performed at 3, 6 and 12 months in order to identify prognostic factors of relapse for patients alive and well at the beginning of each time interval. the probabilities of relapse were calculated by using the cumulative incidence estimator to accommodate for death as a competing risk. factors predicting relapse were studied using cox regression model including time dependent variables. a backward stepwise procedure was used for variable selection. results: with a median follow-up of 26 months, 244 patients relapsed. the 3 year cumulative incidence of relapse was 26% [95%ci: 23-28]. overall 84% of patients destined to relapse did so within the first year post-transplant. the overall factors predicting disease relapse for the whole population were more than one course of chemotherapy to achieve cr1, flt3 itd positivity, adverse risk cytogenetics, shorter interval from cr1 to transplant. the occurrence of acute gvhd grade ii or greater (p ¼ 0.05) and chronic gvhd (p ¼ 0.03) were both associated with a lower risk of relapse. using landmark analyses the factors determining relapse at different stages post transplant were observed to differ. in the first 3 months post-transplant the significant factors determining relapse risk were: patient age (p ¼ 0.03), prolonged interval from diagnosis to cr1 (p ¼ 0.05), flt3 itd positivity (p ¼ 0.002), adverse risk cytogenetics (p ¼ 0.02) and use of in vivo t cell depletion (p ¼ 0.003). the only factors observed to determine relapse risk between 3 and 6 months post-transplant were introduction: the number of haematopoietic stem cell transplants being performed worldwide is increasing as is interest in side effects. despite the increase in published data on late effects in the last decade, data on very long term survivors (425 years) is lacking. in this study we describe the outcome of all the patients transplanted at our centre more than 25 years ago. materials (or patients) and methods: between june 1979-january 1990, 216 patients had received allogeneic sct for haematological malignancies at the hammersmith hospital. most patients (180/216) were transplanted for cml with cy/ tbi conditioning and the majority were in chronic phase at sct (n ¼ 140). at the time of analysis in december 2014, 151/216 (70%) patients had died. of 65 presumed survivors, 14 patients had moved abroad and an additional 13 patients were considered lost to follow up as they had had no contact with our centre within 5 years of the study. of the remaining 41 patients, detailed follow up information was available for 34. results: the majority of deaths (94/151) occurred within 2 years of sct. a further 27 patients died between 2-10 years after sct the most frequent cause of death being relapse (17/ 27) and infection (5/27). between 10-20 years after sct there were 18 deaths; the most frequent causes were relapse (n ¼ 4), second malignancy (n ¼ 4) and gvhd (n ¼ 4). between 20-35 years there have been 9 deaths and the most frequent causes were second malignancy (n ¼ 3) and respiratory (n ¼ 2). the latest recorded relapse was at 14.8 years. for 34 survivors for whom we had detailed follow up information the median follow up time was 29y 10 months (range 25y 8 months -35 years 7 months). the median age at follow up was 61y (range 45-80). 11/34 patients had had a diagnosis of cancer at the following sites: skin (bcc or melanoma) n ¼ 5, oral or tongue n ¼ 3, oesophagus n ¼ 1, breast n ¼ 1 and a further patient had had testicular and bladder cancer. 16/34 patients had dyslipidaemia and 14/34 were being treated for hypertension. 5/34 were diabetic and 12/34 were hypothyroid. of 15 male patients, 3 had low testosterone levels requiring treatment. 8/34 had vascular complications including three with ischaemic heart disease one of whom also had pvd, two with venous thrombosis, one with a tia and two patients with renovascular disease. dxa data was available for 25/34 of these patients and bmd was recorded as low (osteopenia or osteoporosis) in 12/25. conclusion: we conclude that late deaths more than 20 y after sct are more likely to be due to second malignancy than relapse. appropriate screening identifies a large number of abnormalities in the surviving patients most of which are amenable to treatment. disclosure of interest: none declared. the aim was to evaluate the survival and late toxicities, defined as any disease condition other than lymphoma occurring after at least 6 months after asct. the median patient age at asct was 52 years (range, 20-70). all patients relapsed after at least one chemotherapy line (previous treatments range 1-8), 26% of them received radiotherapy. at asct, 76% of patients were in cr, 15% in pr, 1% stable, 8% in progression. full dose beam was given to 87% of patients while 13% received dose reductions for comorbidities. results: the median follow-up was 5.4 years (range 0.5-12.2) . the 5-year os and pfs were 81 and 69% (median not reached for both). the non-lymphoma-associated mortality was 5%, 8% and 9% at 3, 5 and 7 years of follow-up. the os was impacted in multivariate analysis by disease status before asct (p ¼ 0.001, hr 2.2, ci95% 1.4-3.6 ), and radiotherapy (p ¼ 0.017, hr 6.5, ci95% 1.4-30.7) . pfs was impacted by female gender (p ¼ 0.029, hr 0.4, ), pre-transplant disease status (p ¼ 0.001, hr 1.9, ), and radiotherapy (trend, p ¼ 0.064, hr 2.7, . none of the factors analyzed impacted late non-lymphoma-associated mortality, except for a trend given by age (p ¼ 0.075). late toxicities after beam asct occurred in 61% of patients, and included infection (32% -most frequently pulmonary), hypogammaglobulinemia (30%), pulmonary complications (21% -mostly reduction of pulmonary function tests scores), metabolic syndrome (17%), cardiovascular complications (12%), second tumors (10%), hypothyroidism (8%), diabetes (5%), chronic kidney failure (4%), hepatitis b reverse seroconversion (2%) and ocular complications (1%). the cumulative incidence of second tumors was 1, 6, and 10% at 3, 5, and 7 years of follow-up, and reached a plateau of 16% at 10 years of follow-up. 17 patients had a second cancer, of whom 12 had a solid tumor (skin [4] , colorectal, prostate, lung [2 each], breast and oropharingeal [1 each]), 5 a hematologic tumor (secondary mds or aml [4] or nhl [1] ). age (p ¼ 0.013, hr 1.5 per year, ci95% 1.0-2.1) , and male gender (p ¼ 0.043, hr 0.05 favorable for female sex, ci95% 0.0-0.9) , increased risk of a second tumor. of 13 patients who died without lymphoma, 6 died of second tumors, 2 died of cardiovascular complications, 2 of late infections, and 3 for other causes. in multivariate logistic regression, the incidence of second tumors was associated with age (p ¼ 0.04), and there was a trend for patients receiving radiotherapy for late cardiovascular complications (p ¼ 0.07). conclusion: beam conditioning is associated with a 61% crude incidence of late effects, mostly infections, hypogammaglobulinemia, and pulmonary complications. the most important preventive measures for late mortality could be the screening for cancer, especially for older patients, screening for heart disease particularly for patients receiving radiotherapy, and prompt and aggressive treatment of late infections. disclosure of interest: none declared. introduction: the advent of highly active antiretroviral therapy (haart) in 1996 had led to a suppression of hiv viral load, to an improved immune function resulting in a significant reduction of opportunistic infections and hiv related morbidity and mortality. consequently, more intensive treatments, including autologous stem cell transplantation (asct), have been extended also to the hiv-positive population. however, in the literature data are scarce concerning the long-term events (incidence of lymphoma relapse, of second cancers and aidsdefining conditions) in hiv-positive patients (pts) affected by relapsed lymphoma who underwent asct. materials (or patients) and methods: we treated 36 hivpositive pts affected by relapsed/refractory lymphomas with asct consecutively in our cancer center. ten pts died during or early after asct due to progressive disease (4 pts), chemotherapy toxicity (1 pt) and infection (5 pts). we analyzed the post-transplantation long-term data of 26 hiv-positive lymphoma pts, reaching a complete response after asct. eighteen pts were male (69%) and 8 pts were female. our cohort of pts included 17 non-hodgkin's lymphomas (nhl) and 9 hodgkin's lymphomas (hl), respectively. twenty-two pts (85%) received one, and 4 pts received two second-line chemotherapy regimen before asct, respectively. the majority of the pts were submitted to a single (beam conditioning regimen) and only two pts to a tandem asct procedure (high dose melphalan followed by beam conditioning regimen). all pts received haart concomitantly to cancer treatment. results: two pts experienced a lymphoma relapse, after 4.27 and 3.08 years from asct, respectively. three pts presented with a secondary malignancy (1 pt an anal squamous cell cancer, 1 pt a squamous cell carcinoma of the larynx and 1 pt a cin2, respectively), with a median time of 3.01 years from asct. eight pts had opportunistic infections (oi): 2 pts developed a pneumocystis carinii pneumonia, 1 pt a cytomegalovirus pneumonia, 1 pt a mycobacterium avium complex pneumonia, 1 pt a herpes simplex chronic ulcer, 3 pts cutaneous relapsing herpes zoster, respectively. the median time of oi appearance was 0.25 years (iqr: 0.11-2.33 ). two pts died: one of lymphoma relapse, the other of car accident. with a median of 6-years follow up (iqr: 4.55-9.87 ) the os and pfs of the entire sample of pts were 91% and 36% at 10 years, respectively. our results may be summarized as follows: 1) 24 out of 26 pts are still alive and in long-term complete remission after asct. these data confirm the long-term efficacy of asct in hiv-positive pts affected by relapse/ refractory lymphoma. 2) the appearance of oi is earlier than that of second malignancies after asct. 3) the secondary malignancies developed by our pts are non-aids-defining cancers, in agreement with the increased incidence reported by the literature in the haart-era and at least two cases are linked to a viral pathogenesis (hpv for both anal cancer and cervical cancer precursor lesion). 4) both oi and second malignancies in our pts series were successfully managed and cured and the only long-term death occurred due to lymphoma relapse. disclosure of interest: none declared. introduction: allogeneic hematopoietic cell transplantation (hct) is an effective therapeutic option for high risk hematological malignancies; 80% of those who survive the first 2 years are expected to become long-term survivors. the prevalence of chronic health conditions approaches 75% among hct survivors and that for severe or life-threatening conditions exceeds 20%. 1 materials (or patients) and methods: a standardized followup of hct survivors is applied at our center, according to jacie standards. here we report the analysis of data collected between nov 2013 and nov 2014 in 249 adult patients (ptsmedian age at follow-up 54y -r19-81) who underwent an hct between 1992 and 2013 at our institution. data on 7 items were prospectively collected in an institutional database. a written consent was given by pts allowing the use of medical records for research in accordance with the declaration of helsinki. results: overall 40% of pts received an haplo, 30% a mud and 30% a match related hct; 13 pts deceased in the last year (7 because of disease relapse, 4 of late major infectious complication, 2 of second cancer). at a median follow-up of 4y (r1-22; cumulative follow-up 1277y) we observed: -chronic graft-versus-host-disease (c-gvhd): at a median follow-up of 3y (r1-21) 61 (25%) pts are presenting c-gvhd features. according to nih 2004 consensus criteria 15 cases were classified as mild, 25 moderate, 21 severe. median number of involved organs 3 (r1-4), 32 pts were experiencing skin lesions, 38 eyes impairment, 23 mouth alterations.-late infectious manifestation: 54 (22%) pts present late infections, 4 pts deceased. pneumonia was reported in 22 pts, varicella zoster virus reactivation in 12, encephalitis in 5 (3 virus related, 2 toxoplasma related), hepatitis in 4, ebv reactivation in 2.-second cancers: second malignancies were diagnosed in 32 (13%) pts, 5 pts are actually under work-up for diagnosis. nonmelanoma skin cancer was the most frequent diagnosis (17 cases); 3 pts were diagnosed with cervix cancer, 2 with prostate cancer, 2 with lung cancer and 2 with bladder cancer. single cases of thyroid, parathyroid, colon, gastric, kidney, larynx, endometrial and breast malignancies were also reported. all pts were treated according to standard policy for general population, 30/32 are alive.-thyroid dysfunction: 38 (15%) pts presented overt hypothyroidism.-cardiovascular diseases: arterial diseases were reported in 17 pts, atrial fibrillation in 5 and cardiomyopathy in 2 pts -overall 10%.-metabolic syndrome (ms): 84 (34%) pts were presenting features of ms (3/5 features among hypertension, dyslipidemia -raised triglycerides and lowered high-density lipoprotein cholesterol-, raised fasting glucose and central obesity). -secondary hemosiderosis: iron overload was documented (with mri and blood parameters) and treated in 38 pts (15%). according to donor source no difference were observed (chisquare test -p ns) except for higher incidence of moderate/ severe gvhd incidence in match related hct (p 0.0097) as compared to alternative hct. conclusion: hct survivors are at a defined relevant risk of developing long-term complications that have a direct impact on quality of life, morbidity and mortality. 1 introduction: long-term survivors of allogeneic hsct now form an expanding and unique patient population with often complex physical and psychological late effects (le) and associated unmet needs. despite international guidelines 1 , optimal delivery models of le services are unclear from clinical, organisational and economic viewpoints. materials (or patients) and methods: in order to scope current models of care for le service delivery within the uk, we undertook a survey of the 27 nhs adult allogeneic hsct centres during 2014. centres were invited to participate in an online survey composed of 30 questions examining service organisation, multi-disciplinary team (mdt) provision, access to other specialist services and patient engagement. results: a 100% response rate was achieved from programme directors or delegated specialist staff. around half of centres also treated patients r18 years and all centres had achieved or were working towards jacie accreditation. in 480% of centres, the le service was led, coordinated and delivered by consultant medical staff, with the remainder being nurse-led. most centres (490%) provided follow-up in a dedicated allograft or le clinic for the first year, but thereafter attrition resulted in b50% of patients being followed after 5 years, and b30% after 10 years. most centres had easy access to medical specialities necessary for le management, but specialist interest in long-term hsct complications was uncommon. only 18% of centres had access to a le mdt, often limited to patients o25 years. despite specific jacie competency s91 standards, a third of centres had held no le educational event in the previous three years. most centres (70%) had an sop for long-term monitoring and le management, with the focus predominantly on physical le with only 28% including a formal psychological screening assessment. only 39% of centres had audited the performance of the sop. screening for endocrinopathies, iron overload and cardiovascular complications was near universal, but access to mammography and cervical smear testing was more limited. revaccination rates were high, but only 23% of centres routinely tested antibody responses. despite recommendations, most (59%) centres never used standard templates 2 to communicate le risk to gps or referring consultants. only 41% of centres had a patient support group accessible to hsct survivors with equivalent numbers having undertaken patient satisfaction surveys related to le service provision. the most commonly perceived barriers to implementation of le services were funding of psychological and other clinical staff and extra investigation costs.conclusion: this survey has demonstrated variation and limitations in the provision of long-term follow up of allogeneic hsct survivors within the uk nhs. although patients are seen in specialist clinics and have access to other specialities, there are limitations in sustaining long-term screening, mdt working, education, audit and patient engagement, as well as perceived barriers to resourcing staff and investigations. further work is warranted to optimise effective, sustainable and affordable models of care for delivery of le services in this expanding specialised patient population. references: 1. majhail et al. biol 2 hematology department, hospital sant pau, 3 hematology department, hospital vall d'hebrón, 4 epidemiology department, hospital de sant pau, 5 hematology department, hospital del mar, barcelona, spain in vivo and to unravel the requirements for their long-term persistence directly in humans. materials (or patients) and methods: we studied the immune system of 10 patients who underwent haploidentical hsct and infusion of donor lymphocytes transduced to express tk suicide gene (median dose: 1.9x10 7 cells/kg) for high-risk hematologic malignancies. in case gvhd, proliferating tk-cells were promptly eliminated upon ganciclor (gcv) administration with complete resolution of the adverse reaction without immunosuppressive treatments. results: at a median follow-up of 7 years after hsct (range 2-12.3), a complete recovery of nk cells, b lymphocytes and ab or gd t cells was observed. the cd8 þ and cd4 þ t cell compartment of tk patients were characterized by level of naïve and memory cell comparable to age and sex matched healthy controls. tk-cells were detected in all patients, at low levels (median ¼ 4 cells/ul), even in patients treated with gcv. ex vivo selection of pure tk-cells confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory t cells to persist for years. importantly, gcv sensitivity was preserved in long-term persisting tk-cells, independently from their differentiation phenotype. longitudinal follow up revealed that tk-cells circulated in patients at stable levels and displayed a conserved phenotype comprising effector memory (t em ), central memory (t cm ) and stem memory (t scm ) t cells. the low level of ki-67 positivity suggested the maintenance of a pool of gene-modified memory cells through homeostatic proliferation. polyclonality was demonstrated by sequencing among tk-cells of thousands of diverse tcrs with a broad usage of v and j alpha and beta genes. the number of tk-cells persisting at the longest follow-up did not correlate with the amount of infused cells, but instead with the peak of tk-cells measured within the first months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory t cells. accordingly, we documented the persistence of cmv and fluspecific tk-cells only after post-transplant cmv reactivation or after flu infection. we observed that the number of infused t scm cells positively correlated with early tk-cell expansion and with their long-term persistence, suggesting that t scm might play a privileged role in the generation of a long-lasting immunological memory. conclusion: after infusion, gene-modified memory t cells persist for up to 12 years within a physiological immune system. antigen exposure and a t scm phenotype were associated with long-term persistence of infused tk-cells. further studies on tk-cell tcr repertoire and vector integrations are currently being performed to elucidate the in vivo dynamics of infused memory t cells. use of zoledronic acid after tcrab/cd19-depleted haploidentical transplantation to enhance gd t cells anti-leukemia effect p. merli introduction: hsct is a potentially curative option for a number of malignant disorders; however, up to 30% of patients lack a suitable hla-matched either related or unrelated donor. in order to optimize haploidentical transplantation, we recently developed a new method of graft manipulation (i.e. tcrab/cd19 negative selection), which retains in the final product large numbers of effector cells, namely nk and tcrgd lymphocytes. relapse remains the main cause of treatment failure. based on preclinical data showing bisphosphonates-mediated improvement of tcrgd cells-blast killing through accumulation of phosphoantigens, we started a prospective trial based on post-transplant infusion of zoledronic acid, with the aim of enhancing tcrgd cells anti-tumor effect. materials (or patients) and methods: enrolled in the study were 35 pediatric patients (median age at transplantation 10.3 years, range 1-18) affected by either all and aml (26 and 9 patients, respectively) at very-high risk for relapse/trm due to disease status (cytogenetic/molecular characteristics, lack of remission or previously failed hsct). all of them underwent a tcrab/cd19-depleted hsct from an hla-haploidentical donor (one of the two parents). according to the model of kir/kir ligand mismatch, 13 patients were transplanted from an nk-alloreactive donor. the median number of infused gd þ t cells was 7.9 x 10 6 /kg (range 0.9-42.7) . zoledronic acid was administered monthly at the dose of 0.05 mg/kg per dose (maximum dose 4 mg), starting from day þ 30 after transplantation. patients underwent zoledronic acid infusions, together with oral calcitriol and calcium supplementation, in the outpatient unit. results: a total of 102 infusions were administered with a mean of 2.9 infusions per patient (range [1] [2] [3] [4] [5] ; only one episode of symptomatic hypocalcemia (at first administration) occurred and was rapidly corrected with parenteral calcium supplementation. none of the patients experienced de novo onset or worsening of previously developed acute gvhd, this finding supporting the observation that gd t-lymphocytes do not cause gvhd. in the study period, six patients relapsed and 2 died due to infectious complications. with a median follow up of 9 months (range 4-22) the 2-year kaplan-meyer estimate of os and lfs were 88.1% (se 6.6) and 62.2 (se 10.1), respectively ( figure 1a) . the cumulative incidence of relapse and trm were 31.3% and 6.6%, respectively (figure 1b) . repeated infusions of zoledronic acid (i.e. more than 3) seem to offer an advantage in terms of dfs (87.5% vs 48.6%, p ¼ 0.13), although the difference was not statistically significant (figure 1c and d) .conclusion: these data suggest that the infusion of zoledronic acid after tcrab/cd19-depleted haplo hsct is safe. repeated infusions of zoledronic acid after haploidentical hsct seems to be more effective in preventing leukemia recurrence. more patients and a longer follow-up are needed to establish the efficacy of this approach. disclosure of interest: none declared. inducible t-cell receptor expression in precursor t-cells for leukemia control introduction: the co-transplantation of hematopoietic stem cells (hs) with those that have been engineered to express tumor-reactive t cell receptors (tcrs) and differentiated into precursor t cells (prets) may optimize tumor reduction. since expression of potentially self-(tumor-) reactive tcrs will lead to negative selection upon thymic maturation, we investigated whether prets forced to express a leukemia-reactive tcr under the control of a tetracycline-inducible promoter would allow timely controlled tcr expression thereby avoiding thymic negative selection. materials (or patients) and methods: using lentiviral vectors, murine lsk cells were engineered to express a tetracyclineinducible tcr directed against a surrogate leukemia antigen. tcr-transduced lsk cells were co-cultured on t cell development-supporting op9-dl1 cells to produce prets. lethallyirradiated b6/ncrl recipients received syngeneic t celldepleted bone marrow and 8 â 10 6 syngeneic or allogeneic (b10.a) tcr-engineered prets. an otherwise lethal leukemia cell (c1498) challenge was given 28 days later. results: after in vivo maturation and gene induction up to 70% leukemia free survival was achieved in recipients of syngeneic tcr-transduced prets (po0.001) as shown in figure 1 a. importantly, transfer of allogeneic gene-manipulated prets increased the survival of recipients (po0.05) without inducing graft versus host disease (gvhd). nontransduced prets provided significantly lower leukemia protection being not significantly superior to the pbs controls. the progenies of engineered prets gave rise to effector and central memory cells providing protection even after repeated leukemia challenge. in vitro transduction and consecutive expansion of mature t cells required at least 40 â 10 6 cells/ recipient to mediate similar anti-leukemia efficacy, risking the development of severe gvhd if of mismatched origin, and providing no long-term protection. importantly, while transgene induction starting immediately after transplant forced cd8 þ t cell development and was required to obtain a mature t cell subset of targeted specificity, late induction favored cd4 differentiation and failed to produce a leukemiareactive population due to missing thymic positive selection. conclusion: co-transplanting tcr gene-engineered prets is of high clinical relevance since small numbers of even mismatched hs can be transduced at a reasonable cost, expanded in vitro, stored if needed, and provide potent and long lasting leukemia protection. disclosure of interest: none declared. key: cord-015389-vwgai4k9 authors: nan title: publication only date: 2009-03-25 journal: bone marrow transplant doi: 10.1038/bmt.2009.50 sha: doc_id: 15389 cord_uid: vwgai4k9 nan introduction & objectives: literature states that human postnatal dental pulp stem cells (hdpscs) have the ability to differentiate to osteoblastic cells. the purpose of this paper is to present the results obtained in the differentiation of hdpscs with three different media and to compare their osteogenic ability. materials & methods: human dental pulp was extracted from teeth of healthy adult subjects aged 21 to 45 years. the pulp was gently removed and immersed in a digestive solution for 1 h at 37cº. after digestion, cells were cultured and adherent cells were isolated. after the second pass the cells were placed in three different 75 fl asks with three classes of differentiation media. medium 1: osteodiff (miltenyi®); medium 2: alpha-mem supplemented with 15% fetal bovine serum (fbs), 100 u/ml penicillin, 0.1 mg/ml streptomycn, and 0.25 mg/ml amphotericin b; medium 3: alpha-mem medium, supplemented with 20% fbs, 100 mm 2 p-ascorbic acid, 2 mm l-glutamine, 100 u/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 mg/ml amphotericin b. flasks were incubated at 37ºc in a 5% co2 and the medium changed twice a week for 35 days. to quantify the different amount of mineralized nodules the absorbance rate was used. results & discussion: hdpscs were obtained at a good rate and differentiated with any of the three media into osteoblastic cells that developed mineralization nodules (clusters), as revealed by alizarin red staining. this staining was signifi cantly more intense with medium 1 than medium 2 and medium 3 (absorbance values 1.107, 0.576 and 0.325 respectively). conclusions: this study demonstrates the ability of hdpscs to differentiate into osteoblasts. the medium 1 (osteodiff medium, miltenyi®) , was the best to differentiate these cells to the osteogenic lineage. long-term haematopoietic reconstitution and clinical evaluation of autologous peripheral blood stem cell transplantation after cryopreservation of cells at -80°c in a mechanical freezer for longer than 6 months l. calvet, a. cabrespine-faugeras, n. boiret-dupre , e. merlin, c. paillard, m. berger, j.-o. bay, o. tournilhac, p. halle chu (clermont-ferrand, fr) controlled-rate freezing in 5 or 10% of dmso and storage in the nitrogen is the standard technique for cryopreservation of hematopoietic progenitor cells (phs). the main inconveniences are its high cost and dmso toxicity. many teams try to reduce dmso infused by phs concentration before cryopreservation or wash before infusion. however, labor intensive increases the cost and not free of cell loss. we developed an easier and cheaper technique, the cryopreservation of the phs at -80°c, an uncontrolled rate freezing with 2.5% hes, 1% albumin and only 3.5% of dmso allowing infusion without wash. this technique preserves the functional capacities of phs, can produce successful engraftment and reduces toxicity during infusion. does the cryopreservation of the phs at -80 °c allow a long-term hematopoietic reconstitution and clinical course even if storage is greater than 6 months? 239 patients who had undergone 325 autografts (204 adults, 121 children) were studied. the median storage time of the 445 phs cryopreserved was 1.7 months [0. with 9.7% (43/402) preserved more than 6 months (median 13,7 [6-136] ). the median recovery of nucleated cells and cd34+ cells were similar, for the preserved phs <or > 6 months (71% versus 70% , p=0.44) and (104% versus 91% , p=0.11), respectively. only mild infusion-related toxicity was observed in 29.8% (nauseas/vomiting 8.6%, shivers 4.7%). median time to reach 0.5x109/l granulocytes (pn), 20 and 50x109/l platelets (pl) were 13 , 12 and 15 days respectively. delay to reach hematopoietic reconstitution was similar between phs preserved < or > 6 months except for pl > 20x 109/l. this delay was signifi cantly longer for phs kept > 6 months 12 versus 14 [6-46] (n=0.015) with a correlation between cd34+ cells dose and the number of days need to reach 20x109/l pl. in order to assess long term hematopoietic reconstitution, only patients without other treatment (n=128) were studied at 3, 6 and 12 months. median values were 150, 168 and 185x109/l for the platelets and 2,37, 2,43 and 2,8 x109/l for the pn at 3, 6 and 12 months respectively. mortality at 100 post-autograft days was of 5.5%. median overall survival was 54 months and 3 years survival rate was of 55%. the long term hematopoietic reconstitution was satisfactory. this easier and cheaper cryopreservation method leads to successful engraftment even if phs had been cryopreserved more than 6 months. improve mobilization in these patients have been described. another exciting option for these patients is the new cytokine, amd3100. this agent is an inhibitor of sdf1 binding to cxcr4 and appears to promote mobilization of cd34+ cells into the circulation. the use of this amd3100 in combination with g-csf in patients unable to collect adequate cd34+ cells with g-csf alone was recently reported in 280 patients with lymphoma and multiple myeloma (mm) . in this study g-csf was given at a dose of 10 mcg/kg per day and amd3100 was started at 240 mcg/kg on day 4 of mobilization. in contrast, clinical studies showed that aml, cll and pcl cells may also be mobilized by amd3100 via cxcr4 inhibition. due to these concerns, aml, cll and pcl patients are excluded from amd3100 trials. we here report 8 patients (3 female/5 male) with non hodgkins lymphoma (n=4), mm (n=3) and germ cell cancer (n=1) who failed stem cell mobilization after chemotherapy and g-csf administration (patient characteristics table 1 ). patients received 2 x 5 µg/ kg daily of g-csf for 4 days followed by 240 µg/kg of amd3100 given subcutaneously 10-11 hrs before collection on day 5. our aim was to assess the effect of amd3100 on the mobilization of cd34+ cells. administration of g-csf and amd3100 were continued daily until end of collection cycle. adequate collection of cd34+ cells (2.6 and 5.54 x 106 cd34+ cells/kg) were achieved in 5 patients. in 2 patients additional bone marrow collection were performed, 1 patient failed mobilization with amd3100. until now 4 patients underwent autologous transplantation with 1.48, 2.6, 3.35 and 3.58 x 106 cd34+ cells/kg respectively and achieved sustained leukocyte and platelet engraftment. in conclusion, amd3100 in combination with g-csf was generally safe and offers a new treatment to collect cd34+ cells for autologous transplant from poor mobilizers. due to the reported mobilization of leukemic cells, amd3100 should be restricted to patients with lymphomas, mm and solid tumors. evaluating the effect of substance p on expansion of human umbilical cord blood cd34+ haematopoietic stem cells in a serum-free media s. shahrokhi (1) , m. ebtekar (1) , k. alimoghaddam (2) , m. kheirandish (3) , a. pourfathollah (1) , a.r. ardjmand (4), a. ghavamzadeh (2) (1)tarbiat modares university (tehran, ir); (2) hematology, oncology and bone marrow transplantation research center (tehran, ir) ex vivo expansion of cord blood hematopoietic stem cells has been progressively interested as alternative sources for stem cell transplantation. using different combination of growth factors especially cytokines has been investigated in most reports, but there are little evidence about regulatory roles of other factors including neuropeptides in this way, then we choose substance p (sp) to evaluate its effect on expansion. material and methods: cd34+ purifi ed from umbilical cord blood by macs, were cultured in a serum-free liquid culture system. different concentration of sp used in combination with cytokine cocktail of scf, fl, tpo, il3 and il6. phenotypic and functional analysis of the cells produced in culture, was performed by fl owcytometry. count and percentage of cd34+ cells were compared in different groups of treated cells. results: ex vivo expansion cultures of cd34+ cells of ucb were signifi cantly increased, in cells cultivated in "sp + cytokine cocktails" group compared cytokine groups alone. conclusion: consideration of the role of other growth factor such as sp along with cytokines, may enable us to overcome the diffi culties before us in ex vivo expansion of cord blood cells. our studies indicate that sp could act as a superior supplement for expansion of ucb-hsc cytokine cocktails. additional studies are needed to establish the functional activity of expanded ucb-hsc as well as the effects of substance p. standard protocols for cryopreservation of peripheral blood progenitor cells (pbpc) use rate-controlled freezing and storage in liquid nitrogen, which are both time-consuming and expensive. in the last 11 years we used a simplifi ed method (galmes et al 1995) consisting of storage in a mechanical freezer at -80ºc, with dmso as the sole cryoprotectant. this study evaluates the safety of this approach, in terms of infusion-related toxicity and hematopoietic reconstitution, in 385 consecutive autologous transplantations performed from 4/97 to 9/08 in 348 patients (median age 46; underlying disease: lymphoma in 178, myeloma in 131, acute leukaemia in 17, breast cancer in 22). after mobilization with g-csf ± chemotherapy (usually cyclophosphamide 1.5 g/m²) pbpc were collected in a cs3000+ separator (fenwall), mixed in autologous plasma and dmso (to a fi nal concentration of 10%) and frozen in plastic bags (cryocyte, fenwall) at -80ºc. median cd34+ count was 3.6x106/kg and median storage duration was 32 days (6-564). infusion-related toxicity was frequent (25%) and generally mild (transient hypoxemia, broncospasm, hypertension or arrhythmia, and abdominal pain, nausea or diarrhea) but there were 2 cases of acute congestive heart failure and 1 anaphylactic shock (probably related to dmso). engraftment to 500 neutrophils and 20,0 platelets/ul occurred on days +11 and +14 (median). bacteremia occured in 25% transplantations, and grade 3 or 4 toxicity in 20%. median hospitalisation duration was 19 days. mortality at day +30 and +100 was 0.5 and 2.8% respectively. an engraftment delay beyond d+60 was seen in 2 cases. there were no secondary graft failures. with a median follow up of 37 months, 66% patients are alive. these results confi rm the feasibility and safety of this simpler and cheaper cryopreservation methodology. belarus y. isaikina, n. minakovskaya, o. aleinikova belarusian center for ped oncohematology (minsk, by) introduction: recent studies suggest that cotransplantation of mesenchymal stem cells (mscs) can improve the engraftment of allogeneic hematopoietic stem cells and prevent graft-versus-host disease (gvhd) due to their immunomodulatory properties. we analyzed the clinical effect of msc infusion on day +30 after hsct for prophylaxis of gvhd and applying of mscs for treatment of severe steroid-resistant gvhd. patients and methods: eight pts after allogeneic hematopoetic stem cell transplantation (hsct) underwent mscs infusions (median age of pts was 11 years, male/female: 6/2) between 2006 and 2009. diagnoses included:all-4, aml-1, aa-2, mds-1.gvhd prophylaxis for pts with all, mds consist of csa and mtx 10 mg/m² (n=3); for pts with aa -csa+mmf; for pts with aml -csa and mtx 10 mg/m² (n=4). for the treatment of gvhd all pts received metylprednisolon 1-2 mg/kg. mscs were prepared applying technique of expansion in vitro from bone marrow of hla-identical siblings, haplo-identical and haplo-nonidentical family donors and unrelated donors. four pts received mscs once and four -twice. for three pts mscs was used for prophylaxis of gvhd on day +30 after hsct and the median dose was 1,0(0,7-1,5)x106/kg and fi ve pts received mscs for treatment of steroid-resistant gvhd with medium time of mscs infusion after hsct 126(110-151) days and the dose was 2,2(1,3-3,7)x10 6 /kg. results: there was no evidence of early and late side effect of msc infusion. one patient died from pulmonary gvhd 1 month after cotransplantation mscs and seven pts-alive. all pts (n=3), who received mscs on day +30 for prophylaxis gvhd developed grades ii-iv gvhd and needed the secondary mscs infusion and the median time between mscs infusions were 120(90-150) days. four pts out of fi ve with steroid-resistant gvhd showed signifi cant improvement of clinical sign of gvhd that allowed reducing immunosuppressive therapy and stopping the steroids. conclusion: our experience demonstrates the absence of positive gvhd prophylactic effi cacy when infusion of mscs was done on day +30. however, we observed decreasing of gvhd grades from iii-iv to 0-ii, when mscs were used as treatment of steroid-resistant gvhd. clinical characteristics of early-onset acute graft-versushost disease after allogeneic haematopoietic stem cell transplantation t. yamashita, y. najima, t. kikuchi, h. muto, c. sakurai, w. munakata, m. yamamoto, k. ohashi, h. sakamaki, h. akiyama tokyo metropolitan komagome hospital (tokyo, jp) acute graft-versus-host disease (gvhd) is one of the major factors that have infl uence on the outcomes of allogeneic hematopoietic stem cell transplantation (hsct). traditionally, acute gvhd has been defi ned as a syndrome after neutrophil engraftment within the fi rst 100 days following hsct. but in our practice, we sometimes encounter acute gvhd that may occur s367 both early, even before engraftment, and late, beyond day 100. the latter has been defi ned as "late-onset acute gvhd", but the former may not be clearly identifi ed yet. in this retrospective study, we evaluated the incidence, clinical manifestations and outcomes of "early-onset acute gvhd", defi ned as that occurring before engraftment after transplantation, among 117 consecutive myeloablative allogeneic hscts at our hospital. of 117 patients, the median age was 40 years. ninety-three percent of patients received allogeneic hsct for hematologic malignancies. thirty-eight percent of patients received an hlamatched related donor transplant, 40% received hla-matched unrelated donor grafts and 19% received hla-mismatched unrelated donor grafts. the stem cell source was bone marrow in 82% of patients and peripheral blood in 18%. the conditioning regimen was tbi-based for 34% of patients and 60% received busulfan-based conditioning. forty-three percent (n=50) of the 117 cases developed grade ii-iv acute gvhd. of these, 30 (60%) cases were described as early-onset acute gvhd (group e). other 20 cases of acute gvhd occurred after engraftment (group c). the median onset date of acute gvhd is day 10 in group e and day 28 in group c. grade iii-iv acute gvhd was seen in 27% of group e and in 35% of group c (p=0.34). the frequency and severity of each involvement site were comparable in both groups. major primary therapy for acute gvhd was mpsl 2-2.5mg/kg/day, but 41% cases in group e were refractory for this primary therapy and 18% in group c (p=0.05). three-years overall survival (oas) was 58% in group e and 49% in group c (p=0.83). in group c, oas of 19 cases without gi symptoms was 71%, whereas oas of 11 cases with gi involvement was 36% (p=0.02). in group c, oas was not affected by with or without gi-gvhd (p=0.89). in conclusion, early-onset acute gvhd accounts for a substantial proportion of acute gvhd after allogeneic hsct. patients with early-onset acute gvhd tend to be refractory to steroid therapy and will have poor prognosis if gi involvement exists. contrast enhanced ultrasound sonography in intestinal acute graft-versus-host disease e. benedetti (1) a 20 year old female with high risk acute b cell leukemia received a fully ablative peripheral blood stem cell transplant from a 1 allele (at the b locus) mismatched unrelated donor. conditioning consisted of cy/tbi and gvhd prophylaxis of cyclosporine (csa) and short course mtx. on day +19 she developed steroid refractory (biopsy proven) acute skin gvhd. photopheresis was started with major skin improvement. on day +102 she developed nausea, vomiting and profuse diarrhea. standard endoscopy with gastric biopsies showed gvhd. infections were ruled out. a trans-abdominal sonography (ta-us) revealed mucosal oedema and thickening of the terminal ileum (5.1 mm) and the ascending colon. moreover, pillcam capsule endoscopy showed mucosal oedema, erosions and lymphagectasies. infl iximab at 10mg/kg was added and, after 2 doses, despite a major clinical improvement, her terminal ileum was still thickened. to investigate if this thickening was associated with residual active gvhd she underwent a contrast enhanced ultrasound sonography (ceus) using a linear phased-array 7.5-mhz transducer. a sulphur hexafl uoridebased with a phospholipid shell microbubble contrast agent (sonovue®, bracco) was injected i.v. as a bolus (2.4 ml) followed by 5 ml saline fl ush. sonovue® is a blood pool second generation contrast agent. ceus showed an intense and sustained enhancement in the arterial phase involving the whole ileum wall with a late phase wash out. such enhancement pattern has been previously described in active crohn disease. given the clinical improvement, infl iximab was discontinued to reduce the risk of infections. however, as ceus revealed active gvhd she continued on budesonide, beclometasone, csa and prednisone. forty days later her abdominal symptoms had completely resolved and a ta-us showed a normal terminal ileum. four months later her intestinal gvhd (confi rmed by colon biopsies) fl ared. ceus was performed on descending colon (most involved intestinal tract by standard ultrasonography) and showed intense arterial phase enhancement with late phase wash out. rituxan and mmf were added with slow resolution of symptoms and normalisation of us features. in conclusion ceus showed residual gvhd activity despite the improved clinical symptoms. moreover, good concordance with clinical symptoms and standard colonoscopy when gvhd fl ared was also shown. further prospective studies are needed to evaluate its usefulness in monitoring intestinal gvhd. extensive chronic graft-versus-host disease is a frequent complication after peripheral blood stem cell transplantation -results of long-term follow-up d. stamatovic, l. tukic, b. balint, o. tarabar, m. elez, g. ostojic, b. todoric zivanovic, z. tatomirovic, o. tasic, b. cikota, m. malesevic, s. marjanovic military medical academy (belgrade, rs) introduction: many studies have compared effi cacy of allogeneic stem cell transplantation (sct) from peripheral blood (pb) with bone marrow (bm), but fi nal conclusion concerning this treatment modality is still not well defi ned. aim: to compare effi cacy of pbsct with bmt in the treatment of hematological malignancies with respect to engraftment, transfusion need, frequency and severity of acute and late complications and overall survival (os). methods: we have analyzed 132 patients (pts), median age 27 years (9-52), m/f 84/48, with various hematological diseases (saa-18, cml-31, aml-29, all-38, mds-8, mm-2, mh-2, granulocytic sarcoma-2) in whom we perfomed allogeneic sct from 1989 till 2008. in 15 pts we perfomed secondary allogeneic sct in due to graft rejection (2) or relapses (13 pts). pts were divided into two groups concerning sc origin-69 pts in bm group and 63 pts in pb group. all pts had hla-dr sibling transplant (5 singeneic, 121 fully matched, 4 mismatched and 2 haploidentical). sc were collected from bm up to standard method and from pb with one apheresis after fi ve days aplication of granulocytic growth factor. all pts have received unmanipulated suspension of sc. conditioning were adjusted to primary diseases and gvhd prophylaxis was mostly combination of cyclospirine a and metothrexate. prevention of infections were standard. results: pts with sc originate from pb have received signifi cantly more mononuclear cells (10,07±7,31 vs 2,33± 0,79, p<0,001) in comparisson with bm. engraftment was more rapid (p<0,001) in the pb group approximately for 6 days. transfusion requirements were much higher in bm group (p<0,01). those pts had more frequent oropharingeal mukositis grade 3-4 (33,33% vs 9,5%, p<0,05). there were no difference in the incidence of acute (44,4% vs 49,2%, ns) or chronic gvhd (38,6% vs 54,5%, ns). pts with pbsct had signifi cantly more frequent extensive cgvhd (29,5% vs 12,4%, p<0,05). there were no difference considering trm (10,1% vs 15,1%, ns) or relapses (21,7% vs 22,2%, ns). pts with bmt had better overall survival but with no statistical signifi cancy. conclusion: results of this analysis mostly corresponds with other studies showing that pbsct have rapid engraftment and less acute complications. pbsct is connected with more frequent extensive chronic gvhd that is potentialy fatal, making results of this particular treatment option less better. future will bring defi nite estimation of pbsct effi cacy. a preliminary study of human natural killer t-cell recovery post allogeneic stem cell transplantation b. rees (1) , r. morse (1) , s. robinson (2) , j. hows (1) , c. donaldson (1) (1)centre for research in biomedicine, university of the west of england (bristol, uk); (2)university hospitals bristol nhs foundation trust (bristol, uk) natural killer t cells (nkt), defi ned by their cell surface immunophenotype cd3+, v alpha 24+, v beta 11+ and their specifi c activation pathway by the glycolipid alpha-galactosyl ceramide are a unique and small (0.01-0.1%) subset of lymphocytes. these cells may play a key role in the cure of leukaemia after stem cell transplantation (sct) through activation of the graft versus leukaemia (gvl) effect. they have the ability to stimulate both innate and adaptive immune responses through cytokine production and the activation of 'classical' t, b and natural killer (nk) cells. campath, a complement fi xing monoclonal antibody targets the cd52 antigen expressed by t, b and nk cells and may be used in vitro and/or in vivo for donor lymphocyte depletion during stem cell transplantation. our previous work has shown that cd3+, v alpha 24+, v beta 11+ nkt cells also express the cd52 antigen and so are also susceptible to damage by campath. twelve patients (median age 45.5 years, range 21 -57) on the bmt unit, university hospitals bristol were recruited. diagnoses were aml (3), all (1), anll (1), cml (2), mds (2), nhl (2) , and hd (1) . seven received reduced intensity conditioning, 4 tbi and 10/12 received campath. all patients received adult stem cells, 4 from matched siblings, 8 from unrelated donors. nine survived more than 1 year, including the patient with hd who relapsed 6 months post autologous sct and is alive 20 months post matched unrelated sct. the normal range for nkt cell numbers in adult blood was established, mean 0.71 x 106/l (sd 0.92) (n=18). cells stained with cd3-pecy5, v alpha 24-fitc and v beta 11-pe were analysed using the becton dickinson facs vantage se cell sorter with cell quest software. recovery of nkt cells was studied up to 18-24 months post transplant, with mean levels of 0.10 ± 0.04 x 106/l. all individual values were below those in the normal adult population. recovery of other lymphocyte subsets was comparable with those reported in previous studies. nk cells recovered to within their normal range 3 to 6 months post sct, cd8 t cells numbers were within the normal range by approximately 6 months and cd4 t cells only attained values in their normal reference range by 18 months. the slow recovery of nkt-cells has not been previously reported and this may contribute to a reduced gvl effect. n. nakano, a. kubota, m. tokunaga, y. takatsuka, s. takeuchi, t. itoyama, a. utsunomiya imamura bun-in hospital (kagoshima, jp) background: adult t-cell leukemia/lymphoma (atll) has a poor prognosis because of its chemo-resistance. many chemotherapeutic regimens have been created but none of them have shown suffi cient results. we proposed allogeneic stem cell transplantation (allo-sct) for atll patients and showed an improved survival rate. however, relapse or progression of atll is one of the major limiting factors of survival in post sct patients. objectives: in order to establish a better treatment strategy for poor responders after sct for atll, we analyzed the outcome of relapse or progression cases after allo-sct. we paid special attention to the graft versus atll (gvatll) effect. methods: there were 33 atll patients in which allo-sct was performed in imamura bun-in hospital (ibh) from june 1998 to november 2007. twenty seven cases survived over 90 days after sct. sixteen of the 27 patients relapsed. using data in medical records of ibh, we analyzed transplant characteristics and the outcome of these 16 patients retrospectively. results: disease status at sct was cr in 2 pts, 2 pr, 5 sd, and 7 pd. eight patients received conventional stem cell transplantation (cst) and the other eight patients received reducedintensity stem cell transplantation (rist). fourteen patients in 16 obtained remission (9 cr and 5 pr), but the remaining 2 did not (1 sd and 1 pd) after sct. the sites of relapse or progression in 16 were skin in 10 patients, 6 lymph node, 7 peripheral blood, 3 central nervous system, and 1 bone. all patients discontinued immunosuppressants after relapse or progression. eleven patients obtained remission. especially, in 6 out of 11 patients, remission was obtained only by discontinuation of immunosuppressants, and the time to remission after discontinuation of immunosuppressants was between 1 to 14 days. twelve patients were complicated with acute gvhd (grade i-iv). twelve patients died after sct. the causes of death were disease progression of atll in 5 patients, 3 acute gvhd, 3 infectious complications, and 1 interstitial pneumonia. four patients who were complicated with acute gvhd survived over 24 months. conclusions: a certain number of patients obtained remission only by the discontinuation of immunosuppressants. four patients survived more than 2 years with their complication of acute gvhd. these results suggest that the gvatll effect after sct exists and plays an important role in longer survival for poor responders of post allo-sct in atll patients. adoptive immune transfer in paediatric and young adult patients with refractory malignancies p. sovinz, w. schwinger, h. lackner, m. benesch, a. moser, c. urban medical university graz (graz, at) background: patients with metastatic malignancies refractory to or relapsing after conventional ± high-dose chemotherapy have a poor prognosis. graft-versus-tumor (gvt) effects have been reported in small numbers of patients for various solid tumors. patients and methods: eight pediatric and young adult patients (male: female = 3:5; age 1.9 to 22 years) underwent 9 allogeneic hematopoietic stem cell transplantations (allohsct). diagnoses were relapsed/ refractory neuroblastoma (n=3), second relapse of hodgkin's disease, refractory mediastinal large-b-cell-lymphoma, metastatic ewing sarcoma/ osteosarcoma /wilms tumor, respectively. five patients had received high-dose chemotherapy with autologous stem cell rescue. conditioning regimens consisted of fl udarabine (n=8) combined with melphalan ±atg (n=2) or melphalan/thiotepa/okt3 (n=5) or treosulfan/thiotepa/okt3 (n=1); and treosulfan/melphalan (n=1). haploidentical donors (parents, n=6) underwent 2 aphereses: one product was cd3/19 depleted, the other cd34selected; grafts from matched donors (siblings:n=2, unrelated: n=1) were not manipulated. median cd34-number was 12.8 x 106/kg; median cd3-number in haploidentical grafts was 6.35x 104/kg. in the absence of graft-versus-host disease (gvh) immunosuppression was stopped median on day +37. to date, a median of 7 donor lymphocyte infusions (dli; 1-66; dose range:2.5x104 to 3x106) were given to 7/8 patients, starting on median day + 50. results: neutrophil engraftment (>1.0x 109/l) was achieved median on day +9. acute gvh of the skin (i-ii) developed in 3 patients, of skin+liver (iii) in one; chronic gvh occurred in 3 patients (skin:n=3, gut:n=1) there was no transplant-related mortality; 6/8 patients survive for a median of 310 days (range: 64-777) in complete (cr; n=2) or partial remission (pr; n=3) with ongoing regression (disease status not yet evaluated: n=1). two patients who were transplanted in disease progression showed partial response after allohsct but eventually died of progressive disease on day +84 (mediastinal large-b-cell-lymphoma) and +126 (neuroblastoma, after the second allohsct). conclusions: eight heavily pretreated pediatric and young adult patients with poor-prognosis metastatic malignancies tolerated the conditioning regimens well. all patients showed at least transient partial response to allohsct ±dli; six patients in partial remission or better before allohsct survive in cr or pr with evidence of further tumor regression. cmv infection in seropositive patients with haematologic malignancies after allogeneic peripheral blood stem cell transplantation t.-d. tan koo foundation sun yat-sen cancer center (taipei, tw) objective: to investigate the incidence and outcomes of cmv infection in our seropositive population patients after allotransplant as compared with other western patients. we also investigate the impact of post-transplant occurrence of acute graft-vs-host disease and the use of anti-thymocyte globulin upon the outcome of our patients. methods: 68 cmv seropositive patients of various hematologic malignancies underwent allogeneic peripheral blood stem cell transplantation at our institute between march 2001 and november 2008. we used weekly cmv pcr to monitor cmv infection following neutrophil engraftment until day +90 or when any infectious complication occurred. when two consecutive pcrs were positive with >1000 copies present or cmv was found histopathologically, we treated patients with intravenous ganciclovir 5mg/kg q12h for 14 to 21 days. results: 68 patients (median age 38.5, 19~59) of various hematologic malignancies including aml (n=28), cml (n=10), all (n=9), nhl (n=14), hl (n=4), myeloma (n=2), myelodysplastic syndrome (n=1), underwent myeloablative or non-myeloablative allotransplant (51 vs 17). the source of stem cells includes related (48 patients), unrelated (16 patients), and umbilical cord blood stem cell (4 patients). cmv infection or reactivation rate was 21.3% (13 in 61) with median date of occurrence ranges +15 to +267 days with the median of +45 days and the immediate cmv-related mortality rate was 23.1% (4 in 13). the incidence of cmv infection in patients with grade 0~i vs ii~iv acute gvhd are 6.25% vs 42.31%, respectively, with risk ratio 11 (p=0.0039). the occurrence of cmv infection in patients with or without the use of anti-thymocyte globulin use was 26.67% vs 20.0%, respectively, with risk ratio 1.46 (p=0.59). the 5-year event-free survival and overall survival of our patients with or without cmv infection are 38.5% vs 72.2%(p=0.015), and 38.5% vs 73.9%(p=0.004), respectively. conclusions: our cmv seropositive patients do not have higher incidence of cmv infection or reactivation than other lower seropositive patients reported in the western world. there is an increased incidence of cmv infection in the patients who suffer from grade ii~iv acute gvhd, and there are signifi cant differences in efs and os between patients with or without cmv infection. on the contrary, the impact of atg use in our patients is not clear. objectives: patients after hematopoietic stem cells transplantation (sct) have markedly increased susceptibility to moulds infections. according to recent data, the moulds of fusarium spp are emerging as human pathogens associated with significant morbidity and mortality in immunocompromised patients. in current report we are describing disseminated invasive fungal infections caused by fusarium incarnatum in three recipients of allogeneic hematopoietic stem cells, a pathogen not earlier reported for such patients. methods: blood samples were analyzed using automatic bact/ alert system. the culture and identifi cation were performed according to conventional microbiological procedures. the sabouraud agar was used for strain's isolation and the samples were incubated in 30°c for 10 days. the cream to nut-brown mould's colonies were suggestive for fusarium incarnatum. also the microscopic analysis of direct samples revealed microand macroconidias typical for fusarium genus. results: the 46-years-old male and a 28-years-old female patients, with relapsed and refractory acute myelogenous leukemia (aml) have been treated by allogeneic sct from matched unrelated donors after myeloablative conditioning. the third patient, a 51-years-old woman with hodgkin's lymphoma relapsed after autologous sct was transplanted from hla-matched sibling donor after reduced intensity conditioning. all patients suffered from neutropenic fever which did not respond to broad-spectrum antibiotics and fl uconasole. the appearance of nodular, painful skin lesions with characteristic dark red colour and central necrotic area in later stadium suggested skin microembolism caused by infectious microorganism. the mycological analysis confi rmed fusarium incarnatum as a pathogen. i.v. voriconazole in standard doses was started as soon as invasive fungal infection was suspected. the two female patients responded well to voriconazole with gradual resolution of fever and skin lesions. this corresponded with neutrophil engraftment. the male patient with aml died of disseminated fusariosis (autopsy confi rmed) before achieving engraftment. conclusions: we identifi ed fusarium incarnatum as a new mould pathogen which can cause disseminated fatal infections in immunocompromised patients and sct recipients. although the voriconazole was proven to be an effective agent to treat these patients, the hematological recovery seems to be a prerequisite factor needed to survive the disseminated fusariosis. background: infections are the most common complications of stem cells transplantation and chemotherapy induced neutropenia. bacterial infections predominate during the early stage after transplantation. during this phase deep neutropenia and central venous catheter are the most important risk factors. because of high rate of mortality due to gram-negative bacteria, prophylaxis against this microorganisms is mandatory, but this strategy offer gram-positive predomination in all sites of isolation. despite low rate of mortality due to gram-positive bacteria, infections caused by streptococcus today became a real problem. material and methods: during a 8 years period we have performed 144 stem cells transplantation in 134 patients with different hematological malignancies(aml: 74; all: 6; cml: 7; cll: 1, nhl: 13; hodgkin diseases: 16; multiple myelomas: 24; aplastic anaemia: 1;myelofi brosis:1 ewing sarcoma: 1; male:78 female 66. median age: 34 years (12-63). in order to monitoring local micro-fl ora we perform in all patient two times a week: blood-culture, sputum, urine-culture, and simples from central venous catheters. cultures were performed using standard microbiological tools. patients were treated in sterile room conditioned with hepa fi lters, gram-negative prophylaxis with ciprofl oxacine 1,0gr. per day, low bacterial diet. results: gram-positive cocci were predominantly isolated microorganisms (70%), then gram-negative bacteria (20%) and fungi (10%). the most frequent isolated bacteria was staphylococcus coagulaza negative, from central venous catheter, while streptococcus pneumonia was the most common bacteria isolated after day +12, predominantly from sputum. meticillin resistant staphylococcus aureus (mrsa) was isolated in 10% from all gram positive bacteria. we have no vancomicyn-resistant enterococcus isolation. conclusion: the epidemiological pattern of bacterial infection continues to evolve globally and locally at the institutional level, as do patterns of susceptibility and resistance. these trends are often associated with local treatment practices and have a signifi cant effect on the nature of empirical antibiotic prophylaxis and therapy. in our center gram positive bacteria were isolated predominantly. gram-positive prophylaxis is doctrinary used in some centers, but there is a problem with gram-positive resistance. heptavalent pneumococcal vaccination may be reasonable choice. background: invasive fungal infections (ifi) are an important life-threatening complication after allogeneic hematopoietic stem-cell transplant (ahsct). risk factors that further increase the risk of ifi in these patients include prolonged neutropenia, graft failure, immunosupression and graft-versus-host-disease (gvhd). aim: to evaluate the effi cacy and safety profi le of posaconazole as prophylaxis of invasive fungal infection after ahsct. material and methods: in patients at high risk who received posaconazole for prophylaxis we analyzed the incidence of ifi during the treatment period. demographic, clinical, laboratorial and radiologic variables of all patients were studied including age, gender, underlying disease and it´s status at allogeneic transplantation, presence of gvhd, treatment with steroids, adverse events, galactomannan antigen in plasma and high resolution computed tomography (ct-scan). adverse events were also analyzed. results: from a total of 44 patients received posaconazol 37 patients were included in the study, among them 34 received ahsct. during the treatment period there were no proven ifi reported. probable ifi were reported in 1 patient. no serious adverse events related to treatment were reported. during the observational period the overall mortality was 21% (8 patients) and none of them died due to ifi. 19 patients (51,4%) were receiving steroids during the treatment period and none of them developed ifi. the incidence of global gvhd was 65%. acute gvhd incidence was 46%. 3 patients had galactomannan positive and ct-scan were performed in all of them without found ifi in any case. conclusions: posaconazole prophylaxis is a useful and safe approach in order to prevent ifi avoiding systemic antifungal treatment in patients who had undergone ahsct. mucormycosis are an emerging form of invasive fungal infections (ifi) with high mortality rate (60%). early treatment contributes to improve prognosis. posaconazole is a broad spectrum azole that prevents ifi in patients with aml and in patients receiving an immunosuppressive treatment for gvhd. we describe two cases of mucormycosis (cunninghamella bertholletiae) in patients receiving posaconazole prophylaxis. the fi rst received allogeneic haematopoietic stem cell transplantation with reduced-intensity conditioning for myeloma in relapse. because of grade ii cutaneous gvhd, corticosteroids s371 were added to ciclosporine 2 months later associated with posaconazole prophylaxis. however, the patient developed a digestive gvhd. at this date, cunninghammella bertholletiae was found in bronchioalveolar lavage cultures. amphotericin b was added. the patient died with disseminated infection. autopsy confi rmed multiple pulmonary lesions of mucormycosis. the second patient was hospitalised with aml for induction therapy. posaconazole was introduced on the fi rst day. ten days after, a febrile episode occurred without documentation. liposomial amphotericin b was substituted. five days later, mucormycosis was identifi ed in skin biopsy. despite anti-fungal treatment associating amphotericin b and posaconazole, he died 2 months later with disseminated infection. residual concentrations of posaconazole were assessed retrospectively by hplc, using sera conserved at a temperature of 4°c (therapeutic residual plasma concentration: 0.5 and 1 mg/l). for the fi rst patient, the serum concentration was below detection threshold (<0.1mg/l). for the second patient, two sera were collected at prophylaxis and curative treatments (0.5 and 0.6 mg/l, respectively). in both cases, the pathogens were susceptible to posaconazole (in vitro minimal inhibitory concentrations values). our second patient had probably been imunocompromised for several months (long-lasting neutropenia preceding the onset of aml, and history of diabetes). our fi rst patient had an intestinal gvhd with major diarrhoea, which was likely responsible for the very low (undetectable) levels measured when mucormycosis was diagnosed. in conclusion, our report stresses out the necessity to closely evaluate the use of broad spectrum prophylactic antifungal therapy. the prophylaxis in patients with gvhd and/or diarrhea must be used with caution. we recommend to systematically monitor posaconazole levels at least in these cases. inhalation of mold spores can lead in immunocompromised patients to an invasive disease and pneumonia. invasive fungal infection (ifi) has still a high mortality rate. mold-dna can be detected by a polymerase chain reaction (pcr) based method. using it for the bronchio-alveolar lavage (bal) can help to detect an ifi in an early stage. the pcr can discriminate between different mold species and directs the treatment. in our study on 23 patients, a mold pcr from bal was conducted in addition to routine diagnostics. the pcr with primers specifi c for mitochondrial aspergillus-dna and ribosomal 18s dna for zygomycetes. our results show that mold pcr is more sensitive than standard fungal diagnostics. based on these pcr results, an intensifi ed therapy was undertaken successfully. hence, mold pcr from bal is a useful additon of the microbiological investigations. the mould pcr allows the proof of a zygomycosis at an early stage and thereby ensures successful treatment. further investigations are to show if computer-tomography of the lung combined with mold pcr are suffi cient to diagnose for sure a pulmonal mold infection. introduction: cartilage hair hypoplasia (chh)is a rare autosomal recessive disorder caused by mutations in the ribonuclease rna-processing rmrp complex. hsct has resulted in immune restoration, yet fails to correct the chondrodysplasia. we describe a patient with chh and combined immune deficiency who developed granulomatous infl ammation. treatment with anti-tnf-alpha monoclonal antibodies (moab) caused reactivation of jc virus with ensuing progressive multifocal leukoencephalopathy (pml). case report: at age 4y a female chh patient (63c>t and 70 a-g mutation in rmrp) with combined immune defi ciency developed painful non-caseating granulomas. no infectious agent was identifi ed and antibiotic therapies failed. finally at age 17y anti-tnf-á moab(infl iximab) was started with partial response. after the 3rd administration she developed a debilitating intentional tremor of the right hand. mri t2 and flair showed demyelination in the right cerebellum. jc virus pcr was (+)in blood and in cerebrospinal fl uid (csf) and (pml) was diagnosed. 4 weekly administrations of cidofovir, followed by two-weekly administrations for 1 month resulted in a partial response. cidofovir was continued two-weekly. 7 months after diagnosis of pml, hsct with a 9/10 unrelated donor was performed with reduced intensity conditioning according to ebmt-esid guidelines. there was neutrophil engraftment at d+10 and stable donor chimerism of >95% at d+30. at d+60, the patient complained of dizziness, with evidence of a cerebellar syndrome. mri and csf polyoma virus copies were stable. at d+87, she presented with hypertensive encephalopathy including convulsions reminiscent of posterior reversible encephalopathy. discontinuation of ciclosporine led to resolution of the encephalopathy. however, pml progressed despite restoration of t cell function, with increasing cerebellar and brain stem symptoms including ataxia, dysarthria, aphasia, n. facialis and n. glossopharyngeus paralysis with corresponding mri imaging and increase in jc virus pcr copies in the csf. despite intensifi cation of cidofovir treatment, trials of steroids, fl uoroquinolones, mirtazapine, lefl unomide as well as high dose ivig and cytarabine iv, the neurodegeneration was progressive and the patient died of respiratory failure at d+205. conclusion: we describe the fatal course of pml due to jc virus reactivation in a patient with chh, despite successful hsct in terms of myeloid engraftment and restoration of t cell function. a. tomaszewska (1), b. nasilowska-adamska (1), t. dzieciatkowski (2), b. marianska (1) (1 introduction: viral infections still are a serious diagnostic and therapeutic problem in patients undergoing alternative donor transplants. betaherpesviruses (hhv5, hhv6, hhv7) are recognized pathogens in this group of patients. we report a case of hhv6 encephalitis complicated by guillain-barré syndrome (gbs) in a hematopoietic stem cell transplant (hsct) recipient with preceding reactivation of cmv infection. methods: a 43 year-old-man with a history of chronic myeloid leukemia underwent hsct from a matched unrelated female donor in october 2006. sero-status for cmv was igg positive in the recipient and igg negative in his donor. on the day +70 patient developed acute graft-versus-host disease successfully treated with iv methylprednisolone. in march 2007 he was admitted to our unit due to cmv infection reactivation. he started pre-emptive therapy with iv gancyclovir. after 2 weeks of treatment he revealed high fever, uroschesis, paraparesis, impaired consciousness and generalized epileptic seizure. computed tomography of his brain was normal. a lumbar puncture revealed pleocytosis (24/µl) and elevated level of protein (213.2 mg/dl). investigation of cerebrospinal fl uid (csf) by pcr for infective causes of patient's neurological decline including hsv t.1/2, vzv, adenovirus, cmv and dna candida and aspergillus were negative as well as csf culture, real-time pcr revealed in his csf presence of hhv6 dna. according to these fi ndings and neurological status of our patient we made a diagnosis of an hhv6 encephalitis complicated by gbs. the therapy with foscarnet (all symptoms revealed during pre-emptive therapy with gancyclovir) and ivig was started. due to gbs diagnosis we performed 5 procedures of plasmapheresis. we observed gradual improvement in neurological status. after discharging home the therapy was continued with cidofovir given once a week during four weeks. at present, 1.5 year after this episode, the patient remains in a good condition without cmv and hhv6 reactivation, with slight neurological defi ciency. conclusions: betaherpesviruses are emerging pathogens in the hsct setting and may cause central nervous system disease. gbs is a very rare complication among stem cell transplant recipients and usually has been attributed to infection. our successfully diagnosed and treated case of hhv6 neuroinfection complicated by gbs suggests that hsct recipients with cns signs and symptoms should have their csf investigated for hhv6 as well as other pathogens. zygomycosis is a rapidly growing systemic fungal infection, commonly fatal, despite intensive antifungal treatment. it almost always occurs among patients with an immunosuppressive background, diabetes mellitus, prolonged neutropenia, recent chemotherapy and an excessive iron overload. iron is essential for the growth, development and virulence of many fungi, and particularly of the zygomycetes, which are incapable to grow under iron-deprived conditions. we report on a 38-year old male patient, who at the age of 33 was diagnosed with cd10+ b-cell acute lymphoblastic leukemia and achieved a cr following chemotherapy of hyper-cvad type. the patient remained relapse-free for almost 3 years, but when he relapsed, he was treated with the g-mall protocol and a second cr was obtained after 2 cycles of treatment. at that point a fully matched related pbsc allograft, obtained from his 34-year old sister was offered. he engrafted on day+15, and the post-transplant period was complicated by cmv reactivation and mild chronic gvhd. the patient relapsed on day+367 and he was treated with high dose cytosine arabinoside days 1-4 and 24-h infusional mitoxandrone on days +5 and +6. during the aplastic phase he was complicated by histologically proven, extensive left rhinocerebral and pulmonary zygomycosis, with left facial nerve paresis. at that time point he had a transferring saturation of 95% and ferritin 10583 ng/ml. the patient was refractory to initial treatment was surgical debridement and a combination of liposomal amphotericin-b and posaconazole. since no signifi cant improvement was obtained despite a second surgical intervention, deferasirox 30 mg/kg of body weight was added to his antifungal regimen. following 10 weeks of treatment with the triple combination fever was rapidly subsided, as did both, nasal and facial symptoms and lesions. the pulmonary lesions were clearly improved. transferrin saturation decreased to 32% and ferritin to 572 ng/ml. unfortunately, chemotherapy produced a minor response and 2 months later leukemia reappeared. the patient fi nally succumbed from pulmonary hemorrhage, following salvage treatment with clofarabine and cyclophosphamide, without any sign or symptom of recurrence of his previous zygomycosis. introduction: despite the relatively high transplant-related mortality (trm), the management of the end-life care is poorly understood issue and the problems of providing palliative care to patients submitted to stem cell transplantation (sct) may be underestimated. in this regard, the use of palliative sedation therapy (pst) in the sct setting remains a major concern. patients (pts) and methods: in order to address this issue, a retrospective study on the use of pst in our tertiary sct unit was performed. search criteria were: death and previous sct. data regarding symptoms, symptoms control and use of pst were collected. we identifi ed 18 dead pts. last line of therapy before death was sct and a salvage treatment given for a post sct relapse in 11 and 7 patients respectively. near the death, 12/18 patients experienced a total of 18 refractory symptoms and in 6 cases more than one of them was present. intractable symptoms were: excruciating dyspnoea in 8 (67%), agitated delirium in 6 (50%), severe pain in 2 (17%) and massive bleeding in 2 (17%). results: pst was started in all 12 patients, at a median of 2 (1 -4) days before death. the most used sedative drug was midazolam, that was administered to 9/12 pts as single agent and in 2 cases in association with promazine; 1 pt received the latter agent alone. at the start of pst, 8 pts with pain were receiving parenteral morphine. symptoms control was adequate in 12 cases (complete and partial symptoms control in 9 and 2 respectively) and not adequate in 1. conclusion: pst is a controversial issue in palliative medicine, although it has been clearly claimed that when it has the intent to provide symptom relief, pst should be considered a proportionate intervention. sct failure represents a so strongly discouraging event to determine diffi culties to recognize end life status. as a consequence, the risk of an inadequate symptoms assessment and of an inappropriate palliation should be considered. in our experience, in a patient closed to the death, when other treatments failed to relieve the intolerable suffering from refractory and otherwise intractable symptoms, pst represented a valid palliative care option by a reduction in patient consciousness, using appropriate drugs carefully titrated to the patient's comfort. adequate symtom control was obtained in more than 80% (11/12) of pts. an internal operative protocol is under construction to improve those results. donor lymphocyte infusion as therapy for persistent pure red cell aplasia following major abo-incompatible stem cell transplantation a. lübking, i. winqvist, s. lenhoff lund university (lund, se) pure red cell aplasia (prca) after abo-mismatched allogeneic stem cell transplantation (sct) is not uncommon. however, spontaneous remissions within 6 months are frequent. we here report a case of long-lasting prca refractory to multiple therapies that eventually responded to donor lymphocyte infusion (dli). a 36 year old woman received peripheral blood cells from an unrelated hla-identical donor following myeloablative conditioning six months after diagnosis of aml. there was a major abo incompatibility between recipient (0+) and donor (b+). engraftment of granulocytes (>0,5x109/l) and platelets (>20x109/l) was noted on day 25 and 30 respectively. due to the absence of reticulocytes, bone marrow analysis was performed on day 50 showing the total absence of erythroid precursors. initial treatment with steroids, erythropoetin and withdrawal of immunosuppressive therapy was not successful. four doses of rituximab were given from day 265 without any effect. starting on day 545 immunoabsorbtion on three consecutive days was performed followed by methylprednisolone, cyclophosphamide and immunoglobulin infusions. although the igg and igm antidonor isoagglutinins were reduced from 1:128 to1:4 and 1:1 respectively, the prca persisted. from day 638 she received 4 doses of dli within 6 months in escalating doses (1, 5, 15 and 24 million cd3/kg). three months after last dli she developed signs of a mucosal gvhd accompanied by moderate eosinophilia. concomitantly, stable reticulocytosis occurred from day 924 and she became transfusion independent. since residual recipient b-and plasma cells are presumed to be responsible for production of anti-donor-isoagglutinins causing prca, inducing gvhd by withdrawal of immunosuppressive therapy or dli might be a reasonable option. there are previously published cases of successful dli treatment for prca, but in many cases dli was given relatively shortly after transplantation, i.e. when spontaneous remission still was possible and the time between dli and reappearance of reticulocytes varied. in our case stable reappearance of reticulocytes occurred concomitant with signs of gvhd. we therefore fi nd our case highly suggestive of that inducing gvhd with dli can overcome post-sct prca refractory to almost all other therapy options. cystatin c level as a marker of renal function in haematopoietic stem cell transplantation h. muto, k. ohashi, m. ando, r. hanajiri, t. kikuchi, w. munakata, c. sakurai, m. yamamoto, t. kobayashi, t. yamashita, h. akiyama, h. sakamaki tokyo metropolitan komagome hospital (tokyo, jp) hematopoietic stem cell transplantation (hsct) recipients have an increased risk of acute kidney injury (aki) or chronic kidney disease (ckd). however, serum creatinine level may underestimate the prevalence of these renal complications because of decreased lean body mass or concurrent liver disease, which was frequently observed in a hsct setting. cystatin c measurement may be more sensitive for detecting impaired kidney function. we retrospectively reviewed the medical records of 95 hsct (75 allogeneic and 20 autologous) recipients who had at least one chance to monitor serum cystatin c level during last 2 years in our institution, and evaluated cystatin c as a possible new marker which can predict subsequent renal dysfunction. the occurrence of aki was defi ned by the rifle classifi cation and ckd staging was based on kdoqi criteria. of 95 transplant recipients, 35 patients developed aki after median 48 days (range 0-664 days) after hsct, while worsening ckd stage was observed in 24 patients during observational periods. cystatin c level was not infl uenced by autologous transplant (p=0.311), but signifi cantly elevated after allogeneic transplantation (p<0.001). pretransplant advanced disease status also had an infl uence on cystatin c level before transplantation (p=0.004) multivariate analysis disclosed that the use of calcineurin inhibitor was a major cause of cystatin c elevation (odds ratio 7.09, p=0.017). there was also a strong inverse correlation between cystatin c and estimated gfr (r=-0.749, p<0.001). proportional hazard modeling analysis revealed that the episode of aki after transplantation were a great risk for substantially worsening ckd stage (hazard ratio 19.5, p<0.001). cystatin c measurement could be a useful clinical tool to identify hsct recipient at increased risk for ckd. control of severe bleeding from acute gvhd by treatment with tranexamic acid j. hasenkamp (1) acute graft-versus-host disease (agvhd) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. 50% of the cases with intestinal agvhd are refractory to standard treatment regimen. these patients suffer frequently from severe agvhd grades 3 to 4 including massive gastro-intestinal bleedings. we report from clinical courses of two cases treated with tranexamic acid for diffuse, life-threatening gastro-intestinal bleedings caused by steroid-refractory agvhd. the agvhd was confi rmed by biopsy and histopathology. immunosuppression consisted of tacrolimus, mycophenolate mofetil, prednisolone and second line treatment with alemtuzumab. one patient received additionally extra-corporal photopheresis and mesenchymal stem cells. global coagulation and factor xiii plasma levels were kept in normal ranges by substitution. thrombocytopenias were compensated by adequate transfusion of cell separated thrombocytes concentrates. bloody stool volumes of 3 and 5 kg in 24h lead to dropped hemoglobin levels despite massive transfusion of erythrocyte concentrates. because of this persistent, diffuse gastro-intestinal bleedings, both patients were treated additionally with 500 mg tranexamic acid i.v. every 8h. after three infusions of tranexamic acid the bleedings in both patients stopped. treatment with tranexamic acid was discontinued without reoccurrence of the bleedings. there were no adverse events of tranexamic acid observed. local hyperfi brinolysis in the gastro-intestinum may contribute to bleedings from tissue damage caused by agvhd. tranexamic acid is indicated for prophylaxis and treatment of bleedings by systemic and local hyperfi brinolysis after e.g. surgery or plasminogen activator treatment. abortion of hyperfi brinolysis can contribute to stabilization of coagulation. prophylaxis or control of severe agvhd is preferred for prevention of hemorrhage. however, tranexamic acid is a treatment option in otherwise unmanageable gastro-intestinal bleeding caused by agvhd. further studies are desired to charge the signifi cance of tranexamic acid in this indication. a low or high body mass index is not predictive for outcome following allogeneic haematopoietic stem cell transplantation j. auberger, j. clausen, b. kircher, g. gastl, d. nachbaur innsbruck medical university (innsbruck, at) objectives: recently it was hypothesized that a low (<20) bodymass index (bmi) is signifi cantly correlated with an increased transplant-related mortality, decreased survival and relapsefree survival after allogeneic sct (k le blanc, haematologica 2003;88:1044). patients: 208 patients receiving a fi rst allogeneic transplant were studied. underlying diagnoses were acute myeloid leukemia (aml) (n=71), acute lymphoblastic leukemia (all) (n=41), lymphoma (n=11), and other diseases (n= 75). median patient age at time of transplant was 45 (range, 18-76) years. 108 patients were grafted from an hla-identical sibling donor and 110 patients received grafts from volunteer unrelated donors. conditioning was myeloablative in 60 and of reduced intensity in the remaining 148 patients. results: overall survival for the entire cohort was 34% (24%-45%,95% confi dence interval, ci). there was a trend for a poorer outcome in patients with <25% and >75% percentile bmi (i.e. bmi ≤ 21 and ≥ 27) (os 48% vs 34%, p=0.1 log rank test) due to a higher non-relapse mortality in this patient cohort (37% vs. 30%). these differences were observed in both, the myeloablative as well as reduced intensity transplant cohorts. the bmi had no infl uence on relapse incidence in either patient cohort. conclusion: by deviding patients into percentiles bmi had no signifi cant impact on outcome and non relapse mortality neither following myeloablative nor following reduced intensity allogeneic stem cell transplantation. autoimmune thyroiditis after haplo-identical stem cell transplantation for severe combined immunodefi ciency f. dogu (1) introduction: thyroid dysfunction is a well known complication in survivors of hematopoietic stem cell transplantation, and is reported after tbi as well as radiation-free conditioning. the most common disorders after radiation free conditioning are euthyroid sick syndrome(ets) and compansated hypothyroidism. autoimmune thyroiditis is rarely reported after hsct in children and it has never been described after hsct for scid. here we report an autoimmune thyroiditis developed 9 months after the third haploidentical stem cell transplantation for scid. case: a 5-months-old girl was referred to clinic with the diagnosis of t-b-nk+ scid. as she didn't have a fully matched sibling donor and her clinical condition was unstable she received peripheral blood stem cell transplantation(pbsct) from his haploidentical father after cd34+ cell selection without conditioning. engraftment wasn't achieved on day +28 and she received second haploidentical cd34+ selected pbsct from her mother. third transplantation was performed 2 months after the second one, due to graft failure and this time she received bu/cyclo for conditioning and csa for gvhd prophylaxis. myeloid and platelet engraftments were achieved on day+14 and +18 respectively. grade i acute gvhd developed on +26 and treated with corticosteroid for ten days. she was discharged on day+55 with full donor chimerism. thyroid hormone levels which were normal before hsct revealed compansated hypothyroidism at posttransplant 9 months in a routine follow-up visit. elevated antithyroid proxidase (53 iu/ml) and anti-thyroglobulin (478 iu/ml) titers were all consistent with the diagnosis of autoimmune thyroiditis(hashimoto). levothyroxin treatment was started. since the thyroid hormone levels were normal and antithyroid antibodies were negative in her mother, the transfer of autoimmune disorder was excluded. conclusion: regular screening of thyroid functions is important and necessary to detect and treat thyroid illness, especially in young children following hsct. once-daily intravenous busulfan as myeloablative reduced-toxicity conditioning regimen in haematopoietic stem cell transplantation s. santarone, e. di bartolomeo, p. bavaro, p. di carlo, p. olioso, g. papalinetti, p. di bartolomeo bmt center (pescara, it) postulating favorable antileukemic effect with reduced toxicity and improved safety, we used i.v. busulfan (bu) associated with either cyclophosphamide (cy) or fl udarabine (flu) as conditioning therapy for hematopoietic stem cell transplantation (hsct) in 14 patients affected by aml (n=8), mds (n=4), all (n=1) and thalassemia major (n=1) between may 2006 and june 2008. patient age was 1-61 (median 28) years. five of them were older than 50 years. nine patients received flu at a dose of 30 mg/m2/day for 4 days (from -5 to -2) immediately followed by bu given in single i.v. administration over 3 hours at a dose of 3,2 mg/kg day for 4 days (total dose 12,8 mg/kg). five patients received the same dosage of bu from day -7 to day -4 followed by cy 60 mg/kg/day from -3 to -2. donors were hlaidentical (n=6) or 1 antigen mismatched siblings (n=2) and 6 were matched unrelated (mud). the graft-versus-host disease (gvhd) prophylaxis included cyclosporine and short course methotrexate for all patients with the addition of antithymocyte globulin for the mud transplants. eleven patients received bone marrow cells (median dose of nucleated cells 3,76 x108/ kg, range 3,1-10,9) and 3 were given peripheral blood stem cells (median dose 7,4 x106/kg cd34+ cells, range 5,7 -7,7). all patients achieved primary engraftment. the median time to 0.5 x109/l neutrophils and 25 x109/l platelets was 18 (range, 15-32) and 15 days (range, 12-25) respectively. chimerism studies revealed that 13 of 14 were complete chimeras (100% donor) at 1 year post-hsct. acute gvhd was observed in 5 patients (grade i in 2, grade ii in 2, grade iii in 1). two patients had mild to moderate chronic gvhd. there was no death due to the transplant procedure. the transplant-related complications were limited. grade iii who hepatic toxicity occurred in 3 patients, hemorrhagic cystitis in 2, moderate oral mucositis in 1 and a single episode of seizures in 1. six patients developed cmv reactivation between day 6 and 47 post-transplant (median, day 32). two patients relapsed and died. as of december 15 2008, 12 patients (86%) are alive and disease-free after a median follow-up of 250 days (range, 130-641) . although the small number of patients does not permit any fi nal conclusion, our hsct protocol treatment confi rms that i.v. bu, associated either with flu or cy, is a well tolerated reduced-toxicity myeloablative conditioning regimen and deserves further study with more patients and longer follow-up. background: down's syndrome (ds) is associated with higher incidence of both haematological and non haematological neoplastic diseases, if compared with general population. reduced susceptibility to chemo-and radiotherapy and the frequent comorbidities limit the use of high dose treatments, especially required in adult patients. case report: a 19 year old male with ds developed an acute myelogenous leukaemia, fab m2, aml1/eto rearranged, in september 2003. he received standard induction treatment obtaining complete remission (cr), consolidation therapy and, in february 2004, autologuos transplantation with bu-mel conditioning regimen using mobilized peripheral blood stem cells. patient relapsed in february 2007, at the age of 23, was treated with mec schedule, obtaining a second cr. hla typing showed the presence of an identical sibling. a full clinical evaluation revealed mild reduction of the ejection fraction due to corrected congenital fallot tetralogy (ef=55%) and a pulmonary hypertension. a reduced intensity conditioning regimen was proposed, consisting of thiotepa (5 mg/kg iv /d x 2 dd) and fludarabine (25 mg/kg iv /d x 5 dd) followed by allogeneic stem cell reinfusion in june 2007. standard gvdh prophylaxis was given; engraftment was achieved at day +16 for anc >0,5 x 109 /l and +13 for plt >50 x 109/l; grade 4 who was recorded for liver toxicity and grade 1 for mucosal toxicity. full donor chimerism was documented at day +30. the patient developed stage 1 agvhd; however, 4 months after transplantation relapse was diagnosed with immunological features of all. immunosoppression was suspended, although blast percentage increased rapidly to 100% and a salvage therapy with all active drugs was started. discussion and conclusion: few data are reported on allogeneic stem cell transplantation in adult patients with ds. this is the fi rst case of ric allosct in a ds adult. those sporadic data do not allow conclusions about outcome on ds adult. a retrospective analysis on large database and a prospective study would be useful to address this issue, helping physicians on treating adult ds pts, when immunogenic effect of allosct play a crucial role to prevent relapse. severe immune hemolysis and pure red cell aplasia after haploidentical non-myeloablative allogeneic stem cell transplantation g. nair, a. mischo, g. stüssi, u. schanz university hospital (zurich, ch) haploidentical stem cell transplantation (sct) offers potential cure to patients without hla-identical donor. recently nonmyeloablative conditioning regimens with in-vivo t-cell depletion have been introduced. severe immune hemolysis rarely occurs after hla-identical sct, but little is known about the occurrence after haploidentical sct. here, we describe 5 patients receiving haploidentical sct for high-risk or relapsed aml (3), cml after 2 blast crises (1), and as a rescue therapy in a patient with all after primary graft failure following hla-identical mud sct. all patients were in morphological remission at the time of sct. conditioning regimen included fl udarabine (30mg/qm x 4 days), cyclophosphamide (500mg/qm x 4 days), and alemtuzumab (20mg x 5 days) for in-vivo t-cell depletion. gvhd prophylaxis comprised mycophenolate mofetil (day 1-28) and cyclosporine (day -1-60) and all patients received prophylactic antibiotic treatment. g-csf was administered until hematologic recovery. peripheral blood sct was performed over 1-3 days with a median number of 8.12 x 106 (7.97-9.82) cd34 positive cells. the early posttransplant course was uneventful, the median time of aplasia was 3 (0-6) days. acute gvhd occurred in 4/5 patients (i: 2; ii:1; iv:1). four patients experienced 9 posttransplant infectious complications (3 cmv, 3 bk, 2 fungal infections, one pulmonary infection). two patients experienced severe immune hemolytic anemia and concomitant pure red cell aplasia in the bone marrow 4 and 10 months after sct. in both patients relapse was diagnosed shortly before or after the onset of hemolysis. the direct antiglobulin test was positive for igg and c3d. the serum of both patients reacted with all cells in a 11 cell antibody search panel without evidence for cold-reacting antibodies and no antibody specifi city could be evaluated. one patient was treated with steroids, ivig, rituximab, and high-dose cyclophosphamide, but eventually died due to fatal hemolysis. the second patient is currently being treated with steroids, ivig and high dose cyclophospamide with a marked reduction of hemolytic activity. the remaining three patients are currently in complete remission without evidence of hemolysis. in conclusion, nonmyeloablative conditioning regimens in haploidentical sct offer new possibilities for patients without a hla-identical donor. however, physicians should be aware of the potentially fatal complication of severe immune hemolysis. one of the major side effects poorly tolerated, especially in children, is represented by emesis post-chemotherapy. the use of antiemetic during chemotherapy (three to four doses for day) is necessary to reduce this complication. in this work was evaluated using a single dose of palonosetron intravenous for the prevention of nausea and vomiting secondary to chemotherapies. methods: since 2006 we have used the palonosetron in 28 pediatric patients of which 19 males and 9 females, undergoing bone marrow transplantation, 15 allogeneic (both sibling that mud) and 13 autologous. the median age is 10 years (range 1-18) and the median weight is 42 kg (range 8-79 kg). the diseases in young patients are reported in table 1 , the conditioning transplantation are listed in table 2 . the dosage used, including scientifi c literature data, was 5 mcg /kg body weight. the palonosetron was considered effective when the emesis was not more than 2 episodes in 24 hours and nausea no more than 2nd grade. results: it was encouraging, having achieved a good control of nausea and/or vomiting induced by chemotherapy, in fact, only seven patients (25%) was necessary to resort to a second dose of antiemetic, in four of seven (14% of total) was repeated the success with palonosetron a distance of four days after the fi rst dose using the same dosage. in 15 patients (53%) has not been no emetic episode while in the remaining group (22%) episodes were occasional and not have needed any treatment. in all patients was not noted any adverse event or side effect. conclusions: our experience, although on a small sample, it suggests that palonosetron can be considered an effective drug in preventing the nausea induced by chemotherapy, is also a drug that not have adverse events, so well-tolerated and easily manageable, it is necessary a single dose within 24 hours before the start of chemotherapy, not least the assessment of the reduction in costs compared to conventional antiemetic. allogeneic or autologous haematopoietic stem cell transplantation (hsct) is an established mode of treatment of different diseases. loss of protective immunity to pathogens has been consistently demonstrated in patients referred to hsct. impairment of humoral and cell-mediated immunity is commonly seen after transplantation. the degree of immunodefi ciency is determined by many factors, particularly by the type of disease and transplant, the presence of graft-versus-host disease (gvhd) or ongoing immunosuppressive treatment. the aim of the study was to evaluate 1) immunogenicity of a revaccination schedule in pediatric hsct recipients 2) quality of recipient immune reconstitution and protection against ordinary pathogens. patients and methods: twenty one patients (pts) 1.4-22 (average 7.8) years old, 13 boys and 8 girls after autologous (11, 52%) and allogeneic (10, 48%) hsct were included in revaccination program. indications to hsct were: solid tumors -11, hematological malignancies -5, immunodefi cency states -3 and aplastic anemia 2 pts. time interval between hsct and begining of vaccination protocol was 0.8-4 (av. 1.5) years. vaccines used in protocol were as follows: diphtheria and tetanus toxoids, pertussis (for patients <7 years old), hbv, vzv, haemophilus infl uenzae type b conjugate, 23-valent pneumococcal polysaccharide, inactivated infl uenza, inactivated polio and attenuated measles-mumps-rubella vaccines. plasma samples to determine specifi c antibodies by elisa tests were collected before and after vaccinations. results: with the exception of one patients presented with repeated fevers, lymph nodes enlargement, muscles and joints pain, no important side effects of vaccinations were observed. a meningococcial meningitis developed in one patient who refused vaccinations. plasma antibody concentrations before and after vaccinations were as follows: antidiphteria (0-300, mean 62.5; 100-5800, mean 1838), antitetanous (0-500, mean 133; 826-5500, mean 3483) and antihbv (0-135, mean 33; 317-1000, mean 532) iu/ml. conclusions: 1) systemic immunization is necessary at appropriate time intervals following transplantation to re-establish immunity. 2) a signifi cant increase of antibodies titer after hbv, diphtheria and tetanus toxoids was detected. 3) vaccinations in patients after hsct are effi cient and well tolerated. 4) a delay in begining of vaccination can result in life threatening complications. ministry of science rp, grant number 501/g/640. according to the world bank data, released in the 2008 report, romania has an upper-middle-income economy. the hematopoietic stem cell transplantation (hsct) program started in romania in 2001 and more than 200 transplants (auto and allo) were performed. we analyzed the outcome for 26 patients who underwent an allogeneic hematopoietic stem cell transplantation from matched related donor for acute leukemia (24 patients) and aplastic anemia (2 patients). for 20 of the patients the procedure was performed in romania and for 6 patients abroad. for both categories the follow-up after transplant was done in hematology units in romania. the overall survival was 14.69 months, with the longest survival of 60 months and respectively shortest outcome for less than one month. on the 1st november 2008, there were 10 patients alive, between 1 and 60 months from the procedure, with a median survival of 27 months. sixteen patients died, the median survival being 6 months after transplant. four out of 16 patients died during the fi rst month after transplant, and a total of 9 patients died during the fi rst 6 months after transplant. the transplant related mortality was 53.84%, 38.46% died due to relapsed disease and 15.38% died of graft failure. for these results, there could be incriminated the irregular and inadequate drugs and reagents supplies in the romanian health system, an ineffi cient follow-up system and registry and home-care facilities defi ciencies in romania. in conclusion, the gross national income (gni) per capita and the human development index (hdi) are very important factors for the outcome of recipients of hematopoietic stem cell. background: umbilical cord blood stem cell transplantation has many advantages over bone marrow transplantation or peripheral blood stem cell transplantation. but, there are some problems to be solved in order to be applied to adults. the main problem is limitation of volume, which can be collected from one placenta was only between 80ml and 120ml. to overcome this problem, the ex vivo expansion of cryopreserved umbilical cord blood stem cells is needed. the object of this study was to evaluate the effect of cryopreservation on ex vivo expansion potential and viability of umbilical cord blood stem cells. methods: after normal delivery, cord blood was drawn from umbilical cord vein and was used to evaluate the mononuclear cell count, the cell viability and clonogenic capacity of cord blood stem cells before and after cryopreservation. results: before cryopreservation, the mononuclear cell count of umbilical cord blood was 2.92 ± 1.08 x 106/ml, cell viability was 92 ± 2.88%, total colony count was 101.5 ± 23.74 and percentages of cfu-gm, cfu-gemm, bfu-e were 29.5 ± 5.80%, 21.0 ± 1.45% 24.8 ± 5.0%, respectively. the mononuclear cell count of umbilical cord blood cryopreserved for 28 days was 1.42 ± 0.42 x 106/ml and cell viability was 66 ± 3.87%. total colony count of umbilical cord blood cryopreserved for 28 days was 52.5 ± 12.13 and percentages of cfu-gm, cfu-gemm, bfu-e were 28.0 ± 3.45%, 27.2 ± 6.52%, 45.3 ± 4.99%. but, there were few colony count which could be observed after cryopreserving for 7 days. conclusion: there was no difference of clonogenic capacity of umbilical cord blood stem cells before and after cryopreservation. the cell viability of umbilical cord blood stem cells was decreased after cryopreservation but there was no difference between umbilical cord blood cryopreserved for 7 days and 28 days. therefore, it is possible that suffi cient umbilical cord blood stem cells could be obtained by ex vivo expansion of cryopreserved umbilical cord blood in order to be used for adult patient. objective and methods: combined hematopoietic stem cell transplants (hsct) plus solid organ transplants (sot) have been rarely reported. the majority of patients with a previous history of liver transplants were children that underwent hsct for aplastic anemia after viral hepatitis. here we report an adult patient who received a cord blood hsct after a preceding liver transplantation. results: in 1993 a 42 year old man required orthotopic liver transplantation for cirrhosis after b viral hepatitis. in april 2006 acute myeloid leukaemia m1 citotype , normal karyotype, flt-itd positive was diagnosed and a fi rst complete remission was reached after 2 induction and consolidation cycles. at that time the patient was not considered eligible for a transplant program due the previous history of sot. in february 2008 the patient relapsed and came to our centre: he was treated with high-dose cytosine-arabinoside chemotherapy, that was complicated by a pulmonary aspergillosis , but reached a second complete remission. we decided to start a cord blood donor search, since siblings were not available and he could not wait for an unrelated donor search. a cord blood with hla locus a allelic mismatch and locus c antigenic mismatch was identifi ed. patient's comorbidity index according sorror at transplant was 5. in may 2008 a preparative regimen containing treosulfan, fludarabine and atg fresenius was administered and 1,2 x 105/kg cd 34+ cells were reinfused. grade i mucositis and grade ii hepatoxicity were observed. a bacterial pneumonia and cmv reactivation occurred at day 6 and at day 34 respectively and both rapidly resolved. a neutrophil count > 1 x 109/l was reached at day 19 and platelet counts > 20 and > 50 x 109/l platelet count were reached at day 36 and day 43 respectively. no acute and chronic gvhd were observed. a 100% donor chimerism has been reached in whole peripheral blood and in cd3+ cells since 28 days onwards. no minimal residual disease has been detected by marrow immunophenotyping and by wt-1 gene expression until last follow-up, at day 171. conclusion: to our knowledge this is the fi rst report of a successful cord blood allogeneic hsct in an adult patient with a history of liver transplantation. this case might encourage physicians to propose allogeneic hsct by any stem cell source to patients with high-risk haematological diseases, who had previous liver or other sot's. double unit cord blood transplantation(cbt) has been established as an alternative source of donor cells for allogeneic haematopoietic stem cell transplantation (hsct). we reported here an interesting case of long-term mixed full donor chimerism during the regular follow-ups of one year after hsct. a 20year-old woman with acute lymphoblastic leukaemia in second complete remission received two units of cord blood after a myelo-ablative conditioning regimen. the cord blood units were hla 4/6 identical with the recipient (2b mismatches and 1a+1b mismatches). total nuclear cell doses infused were respectively 1.8x107 and 1x107, whereas the cd34+cells number was identical in the 2 cbus and the cd3+cells number was higher (x2) in the fi rst one (table1). neutrophil recovery was observed at day 34 and platelets engraftment at day 55 after cbt. only one event of acute graft versus host disease (gvhd)grade i was reported at day 49. currently the patient does not have chronic gvhd and is disease free. analysis of chimerism was performed by str-pcr or rq-pcr on whole blood and specifi c lineage cells (cd3+, cd15+ and cd19+). follow-up was done at 3, 6, 9 and 12 months post transplant. full donor chimerism (fd) was achieved on day 60. each of the two units contributed at different levels to the donor chimerism in specifi c lineage cells: whole blood and cd15 were about 50% cbu1/cbu2, cd19 cells were preferentially from cbu2 origin (65%), and cd3 cells were preferentially from cbu1 origin (75%). this mixed origin of donor cells was detected early and was constant during regular follow-ups. usually, recipients of double unit cbt were engrafted predominantly with one of the 2 units after 4 months. the mechanism of a long-term mixed full donor chimerism is still unknown for our patient. kir ligand analysis showed an absence of mismatch in gvh direction between recipient (c1-c2) and each cb unit (c1-c1 and c2-c2) while there was a mismatch between the 2 units. in this case, a state of full tolerance settled down between the various lineages, either immune mediated interaction between host/graft or between graft/graft could explain s378 this chimerism pattern, but it will have to be clarifi ed: a specifi c study of treg cells is in progress. optimising cd34 yields in pbsch: a comparative analysis of 4 mobilisation regimens c. black, t. elston, m. streetly, m. kazmi guy's hospital (london, uk) cyclo/g-csf (cyclophosphamide/ granulocyte-colony stimulating factor) has been the mobilisation regime of choice when collecting peripheral blood stem cells (pbscs) for transplantation yet pbsc harvests post chemotherapy produce effi cacious yields. this data seeks to compare and inform current mobilisation strategies in this centre. dhap (p=0.018, and ara-c (p=0.001, t-test) therapy yielded signifi cantly better cd34 results compared to cyclophosphamide. mm patients mean cd34 for cyclo mobilisation (n=11) were 2.18 x 10 6 /kg (range: 0.38-5.42), g-csf only (n=3) 2.62 x 10 6 /kg (range: 1.21-4.95), and ara-c ( n=31) 14.15 x 10 6 /kg (0.86-74.62). myeloma patients post ara-c yielded signifi cantly more cd34+ cells (p=0.001) compared to cyclo than those mobilised with g-csf only. nhl patients mean cd34 harvest results for cyclo mobilisation (n=6) were 2.94 x 10 6 /kg (range: 0.16-7.86), g-csf only (n=7) 1.62 x 10 6 /kg (range: 0.71-3.09), and dhap (n=17) 16.08 x 10 6 /kg (range: 0.55-64.31). cd34 yield of nhl patients mobilised with cyclo compared with those harvested post dhap, a signifi cantly higher harvest result was noted (p= 0.020) than those mobilised with g-csf only (p=0.328). paired mm data (n=6) compared patients fi rst mobilised with cyclo-gsf and post ara-c, (p=0.002, paired t-test). this study suggests that harvesting of patients post ara-c, or post dhap is valuable, giving greater cd34 yields than traditional agents and should be considered. paired data also indicates that ara-c could be used for effective second mobilisation. can type of delivery infl uence cord blood units' quality? g. pucci, a. pontari , d. marcuccio, i. bova, r. monteleone, d. princi, g. gallo, a. dattola, e. spiniello, c. garreffa, t. moscato, p. iacopino ao bianchi melacrino morelli (reggio calabria, it) the cord blood banks use the total nucleated cell (tnc) number as principle to proceed or not to cryopreservation of the cord blood (cb) units. we know that tnc and cd34-positive cells infused on unrelated umbilical cord blood transplantation in haematological disease are fundamental for the engraftment of haematopoietic stem cells (hsc) background: immunomagnetic cd34+ selection is a procedure used both for autologous grafts to perform cellular purging and for allogenic transplant. in aploidentic transplant the purpose of cd34+ is to reduce the quantity of cd3+ and cd19+ cells so as to reduce the incidence rate of the graft versus the host disease (gvhd). aims: in this study we have valued the purity and the cellular recovery after immunomagnetic selection performed with clini-macs automatic system (miltenyi biotec, germany); a group of concentrates has been selected after incubation manually performed, while another group has been submitted to incubation and to the subsequent washings using an automated system (cytomate (baxter oncology, chicago il). methods: in our study we subjected 63 peripheral blood stem cells (pbsc) concentrates taken from 16 donors with microcythemia to immunomagnetic cd34+ selection, in order to perform aploidentic grafts on children affected by beta-thalassemia major. 46 concentrates have been submitted to washings pre and postincubation and to incubation using the automatic system cytomate, while 17 concentrates have manually been worked. cell count of nucleated cells (nc) was performed using an electronic cell counter while cd34+, cd3+, and cd19+ were quantifi ed using fl ow cytometry. results: the following table shows the results. conclusion: immunomagnetic selection in microcythemic donors determines according to our experience, a less recovery in comparison to the data reported in literature, nevertheless in our study results evident, even though casuistry is not very ample, that the use of an automatic system for the washing and the incubation of the cellular concentrates has determined a greater recovery and a greater purity in comparison to the procedures manually performed. allogeneic transplantation from hla identical family donor is a common therapeutic approach in patients with intermediate risk aml in fi rst cr. we present an unusual onset of acute leukemia in a female patient that was a healthy donor of pbsc (previously mobilized with g-csf 10mcg/kg) for her hla identical sister with diagnosis aml (fab-m4). the transplantation was preformed in january 2004 with myeloablative bu-cy conditioning and conventional cy+mtx gvhd prophylaxis. at day +35 acute gvhd gr i/ii was observed and resolved with addition of median dose of corticosteroids. in a period of 4 years after this occasion, acute leukemia (aml-m2 no cytogenetic abnormalities) was diagnosed in the donor and treatment with chemotherapy was started. the induction chemotherapy was provided with dae regimen. with 2 consecutive cycles cr was achieved. the patient followed consolidation treatment with hd-arac and anthracikline. further treatment with allogeneic transplantation was on schedule and the source of stem cells would be taken under consideration. can a person with aml and 4 years surviving in a complete remission become a donor of its own donor (hla dna identical) with the same diagnosis is a question that has to be resolved in a higher number of patients. clinical outcome and characteristics of donor graft failure in 25 patients with haematopoietic disease given donor cell boost, second allogeneic transplantation or no treatment r. ahmed nacer, m. benakli, f. mehdid, r. belhadj, n. rahmoune, m. baazizi, a. talbi, f. kaci, r.m. hamladji pierre and marie curie center (algiers, dz) introduction: donor graft failure (gf) is a life-threatening complication of allogeneic hematopoietc stem cell transplantation(hsct), determined when anc had not reached 0,5 109/l by day 21 (primary gf) or when anc decreased irreversibly after engrafment (secondary gf). frequency of gf is variable in function to hematopoietic disease. material and methods: from may 1998 to december 2007, 811 patients (pts) underwent allogeneic hsct from hla-identical sibling donor. 758 pts are appraisable for this study and gf was diagnosed in 25 pts (3,29%) : primary gf: 18 pts, secondary gf: 7 pts with median time engrafment to ; median age at transplant 18 years (5-49); sex ratio (m/f) 1,5; hematologic non malignant disease (hnmd; n: 17): aplastic anemia: 11/198 (5,6%), major athalassemia: 6/15(40%) and hematologic malignant disease (hmd): 8/492 (1,6%); 15 pts had received more than 20 transfusions before allograft; abo incompatibility between donor/recipient was seen in 11 d/r pair; median interval from diagnosis to transplant 39 months ; 2 pts was multiparous; 24 pts received myeloablative conditioning regimen (mcr ) and one pt reduced intensity conditioning (ric); 18 pts received peripheral blood stem cell (pbsc) and 7 pts bone marrow transplant (bmt). the chimerism testing was performed in 8 cases: predominant host population (host population: 90-100%) in 6 pts and mixed (host population < 85%) in 2 pts. 14 pts were given donor cell boost with no additional conditioning with median time gf to treatment 108 days (30-559) and fi ve pts a second hsct within one a third hsct (mcr:5; ric:1) with median time gf to treatment 296 days (69-683). 6 pts had not been treated. at november 2008 maximal follow up is 127 months and minimal 11 months. results: 10 pts are alive (40%) within 6 pts (donor cell boost:5 pts; second hsct:1 pt) with success engraftment(donor population:100%) after median follow up 101 months (70-124) and 4 pts (second hsct:1 pt; no treatment:3 pts) with autologous reconstitution (donor population:0%). 15 pts died (60%) within 9 pts after given donor cell boost, 3 pts after second hsct and 3 pts before given donor cell boost. os at 9 years is 45%. conclusion: gf is rare but serious and concern hnmd more than hmd. better outcome can be obtained after chimerism testing study to choice treatment : predominant donor population will have donor cell boost and predominant host population second hsct with another donor. mixed haematopoietic chimerism: how the initial dynamics of mixed chimerism correlate with later chimerism status k assing (1), c. heilmann (2) (1)herlev university hospital (herlev, dk); (2)university hospital, rigshospitalet (copenhagen, dk) background: the natural history of mixed (hematopoietic) chimerism (mhc) has been extensively studied in hematopoietic stem cell recipients, in order to fi nd determinants for relapse or graft rejection. methods enabling quantitative prediction of later mhc status have not been devised. methods: 21 recipients, receiving hematopoietic stem cells due to non-clonal disorders and displaying at least 5% donor chimerism at minimum one time point, were serially tested for whole blood chimerism over a median period of 2.6 years (range: 0.7-5.6 years). relative changes in the host fraction (termed alfa4) between the median time points: 3.3 and 17.1 weeks post-transplantation were correlated with later mhc status. the predictivity of alfa4 values for later mhc outcome was assessed in a linear regression model. findings: all recipients engrafted. subsequently, 66.6% became mixed chimera and 28.6% achieved complete donor chimerism. weekly chimerism fl uctuations prior to six months posttransplantation (12.0% points; 0.0-192.3% points) exceeded those after six months time (0.9% points; 0.0-192.3% points, p<0.001). at seventeen weeks, alfa4 values correlated with endpoint mhc levels at 2.6 years (r = 0.87, p<0.001). negative alfa4 values predicted (95% confi dence intervals) the presence of less than 30% host cells, while alfa4 values between: 0.0-107.7, were predictive of mhc with ≤ 70% host cells. the only recipient experiencing rejection (4.8%) displayed the largest alfa4 value and had a predicted mhc outcome of 99.8% host cells (95% ci: 79.3-120.2%). interpretation: we have devised a simple mathematical method enabling us, early post-transplantation, to predict later mhc status and thus determine at an early time point, where intervention is needed in order to prevent rejection or poor graft function. feasibility of out-patient autologous stem cell transplantation for malignant haematologic disorders a. ghavamzadeh, a. allahyari, k. alimoghaddam, a. karimi, r. aboulhasani, a. manookian, m. asadi, a.r. shamshiri hematology-oncology and sct research center (tehran, ir) introduction: high-dose chemotherapy with autologous stem cell support is utilized for the treatment of a variety of malignancies including non-hodgkin's lymphoma, hodgkin's lymphoma, and acute leukemias. the aim of this study was to explore the feasibility and safety of performing autologous stem cell transplantation (asct) on an out-patient basis. material and methods: total of 8 patients affected by malignant hematologic disorders(4 cases of hl, 2 cases of nhl, 2 cases of aml) with median age of 25 y (range :16-41 y) and in complete remission and without medical problem were selected. they received conditioning regimen (ceam for nhl and hl, busulfan and etoposide for aml) and stem cell infusion in hospital. the day after sct, patients were discharged and followed by outpatient sct team; include a general physician, staff nurse and care giver during their neutropenic period, and to be rehospitalized in the case of febrile neutropenia, after sepsis workup and performing chest x-ray, they were received the fi rst dose of antibiotic in hospital and treatment continued in their home. results: median time for wbc recovery was11 days (range: 8-13 days), median time for plt recovery was 15 days (range: 11-66 days), median number of transfused single donor plt was 2.5 units (range: 1-9 unit). mucusitis grade 3 was seen in 2 patients, median duration of neutropenic fever was 6 days (range: 0-10 days), 3 patients was rehospitalized because of the neutropenic fever, median duration of rehospitalization in these patients was 5 days, median follow up of patients was 130 days (range: 20-200 days), all patients were alive and in complete remission. conclusion: results show that out-patient autolgous sct in malignant hematologic disorders (hl, nhl, and aml) is feasible and its complication is manageable. haplo-identical sct as a salvage therapy in haematological malignancies: a single-centre experience o. paina, y. stankevich, i. kazantsev, n. stancheva, a. golovacheva, e. babenko, a. alyanskiy, n. ivanova, e. semenova, p. krugliakov, d. polyntzev, l. zoubarovskaya, b. afanasyev spb state i. pavlov medical university (st. petersburg, ru) background: allogeneic hematopoietic stem cell transplantation (allo-hsct) is the one of curative option for patients (pts) with acute leukemias, though its usage is often limited by lack of matched related donor or the time required for search of unrelated one. usage of haploidentical donors allows to avoid these problems and to perform allo-hsct in time. patients and methods: 24 very high risk pts underwent haploidentical sct: all -10 (42%) pts, aml 11 (44%) pts, jmml -1pt, cml-1 pt, and resistant neuroblastoma-1 pt. the total number of resistant/in progression pts was 16 (66%), 8 (33%) pts were in remission. 19 (79%) pts were children (age 1-18), 5 (21%) were adults (age 21-47). in all cases reduced intensity conditioning regi-mens (ric) were used: fl udarabine and atg with addition of different alkylating agents (busulphan, melphalan or thiotepa). sources of hsc -peripheral blood stem cells (pbsc) and bone marrow. for pbsc cd34+ positive selection clinimacs was used. the mean cd34+ count was 12,8x106 /kg (1, 7) . in 20 pts agvhd prophylaxis consisted of csa and short course of mtx with or without mmf. in 4 pts tacrolimus and mmf were used. in 2 pts at d-1 used mesenchymal stem cells (msc) from third -party donors were used prevention of agvhd, in 3 pts, msc were used for treatment of acute gvhd. results: the incidence and severity of agvhd weren't higher, than in other types of allohsct: 6 (25%) pts had grade iii-iv agvhd with skin and gut involvement, one pt died. when mcs using in conditioning regimen agvhd, i stage was observed. treatment of agvhd with msc was successful: in 3 pts in 2 cr. the toxicity of the conditioning regimen was acceptable, 6 (25%) developed grade ii-iii organ toxicity. 5 (21%) pts had invasive aspergillosis and 8 (33%) pts of them had cmv reactivation. the 1-year os is 62%, with mediam observation terms of 4,6 months (1 to 12 months). 5 pts died in relapse and 3 in cr (infection -1pt, another failed to engraft and acute gvhd of the gut). conclusions: haploidentical hsct with ric is characterized by acceptable toxicity and agvhd control, stable engraftment. it proved to be a good option for the group of pts with poor prognosis. randomized clinical trials are necessary for estimation of therapeutic effect of mscs in haploidentical hsct pts. results: there were 38 donation requests involving 37 donors (18 females,19 males). one donor was contacted but declined to donate dli. one donor donated twice for the same recipient. for 1/37 donors no details of donation are available. the median age at time of donation was 43.8 years (range 12-68 years). there were no failed collection procedures. 6/37 donors experienced mild citrate toxicity. 2/37 donors had a vasovagal episode, but both recovered rapidly and collection was able to be completed.1 donor required central access for dl collection: she had also previously required central access for pbsc donation. a median 1.88 donor blood volumes was processed (range 1.30-2.34). no late donor complications were reported. in total, 36 donors had dl collected. among the 36 prospective recipients (15 female; 21 male), indications for dl were: mixed chimerism(n=17); residual disease(n=3); molecular relapse(n=10); clinical relapse(n=2); ebv reactivation(n=1); pancytopenia of uncertain cause(n=1); no data(n=2). 29 of 34 patients for whom data were available (85%) actually received dl infusions. for the remaining 5, reasons for not proceeding were: spontaneous improvement in blood counts; death from ebv; death from relapsed disease; development of gvhd prior to dli; and spontaneous resolution of mixed chimerism. an escalating-dose regimen was used at 3-monthly intervals depending on response: the median number of doses reinfused was 2 (range 1-5). 9 patients (31%) developed gvhd s381 following dli. the dli was successful in treating the stated indication in 18 patients (46%). there were 10 recipient deaths: relapsed disease(n=4), infection(n=3), gvhd(n=2) and progressive ebv(n=1). only the two gvhd deaths were considered dl-related. conclusions: our single-centre experience confi rms that dl are frequently an effective treatment for mixed chimerism or early relapse post-hsc transplant, and that donor experiences are generally good. although requirement for dl is itself an adverse prognostic factor following hsc transplant, 46% of recipients had a successful outcome. nowadays, haematopoietic stem cell transplantation (hsct) remains the single curative approach to the treatment patients (pts) with the resistant primary and secondary aml. these pts have extremely poor prognosis with the level of relapse at least 40% and the risk of trm 45-60%. as known, high level of blasts to the moment of transplantation infl uences on dfs and os. patients and methods: at the russian children research hospital between october 2005 and june 2008 32 hsct were made in 27 refractory aml pts (20m/7f). the median age was 11 (1-18) years. fab-type: m0 -2 pts, m2 -4 pts, m3 -1 pt, m5 -5 pts, m6 -4 pts, m7 -7 pts, mx -4 pts. primary refractory aml was diagnosed in 8 pts and secondary refractory -in 19 pts. 15 kids were transplanted from msd, 8 pts -from mud (2 mmud) and 9 pts -from mmfd (8 haplo-pbsc) with the usage of cd3/cd19-depletion (7 pts) or cd34-selection (1 pt) of the graft. the median level of blasts in bone marrow prior to hsct was 18% (6% -98%). the myeloablative conditioning regimens were used in 29 hsct and non-myeloablative regimen -in 3 second hsct. 11 pts received double-phase conditioning regimens. a median dose of cd34±cells was 8 (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) x 106/kg. 14 pts received dli on median day 63 (8 -119); 4 pts received prophylactic dlis and 10 pts received treated dlis due to increasing of mrd level or the mixed chimerism. results: the engraftment level was 89% with a median time to neutrophil recovery 15 days (8-33) and to platelets recovery -18 days (11-132). 84% pts achieved cr to day 30, 3 pts had the progression of disease, one pt died before engraftment, and the rejection was documented in 1 pt. acute gvhd developed in 17 pts (55%), chronic gvhd -in 8 (44%) from 18 pts who were alive to day 100 after hsct. 14 pts had the liver toxicity grade 2-3 and two pts had the pulmonary toxicity grade 3-4 (who-classifi cation). trm level was 22%. relapse was diagnosed in 10 pts (dfs 48%). at this time 12 pts are alive in cr, 16 pts died (6 -from relapse, 9 -from gvhd and sepsis and one pt -from dag). os for all pts was 17% with a median time 8 months (2 -37) . but after double-phase conditioning regimen os and dfs were 41% both and trm was 0%. overall rfs depended on the presence of gvhd, the type of donor, the using of dli. conclusion: our results show that even for very high risk aml pts hsct may be performed successfully without the significant increasing of trm particularly with prophylactic dlis. a.a. hamidieh, a. ghavamzadeh, m. jahani, k. alimoghaddam, a. mosavi, m. iravani, b. bahar, a. khodabandeh, m. jalili hematology-oncology and sct research center (tehran, ir) objective: hematopoietic stem cell transplantation (hsct) has been extensively used in the treatment of pediatric leukemia. this is follow-up report of pediatric patients (<15 years) with acute myeloid leukemia (aml) and acute lymphoblastic leukemia (all) whom transplanted in hematology-oncology and stem cell transplantation research center, tehran, iran. methods: 142 pediatric patients 85 boys and 57 girls (median age=11 years) with acute leukemia (96 patients with aml and 46 patients with all) received hsct between 1991 and 2008. the most common conditioning regimen was cyclophosphamide + busulfan. they have received allogeneic (51 aml/40 all) or autologous (45 aml/6 all) hsct from bone marrow (26 aml/9 all), peripheral blood (68 aml/37 all) or cord blood (2 aml). donor type for allogeneic transplantation in 84 patients(47 aml/37 all) were stem cell from hla matched siblings, 5 patients (3 aml/2 all) received from other related (hla-matched confi rms with high resolution method) and 2 patients(aml) from other related with one or more than one antigen mismatch. prophylaxis regimen for graft-versushost disease (gvhd) was cyclosporine a and methotrexate or cyclosporine a alone. results: 104(73.2%) patients are alive, 38(26.8%) patients died (27 aml/11 all). the most common cause of death was relapse of disease in 81.6%. among patients who received allogeneic transplantation acute gvhd occurred in 65% and chronic gvhd in 15.5%. two years overall survival and disease free survival of aml patients were 70% and 67% respectively. two years overall survival and disease free survival of all patients were 74% and 60% respectively. no statistical difference between aml group and all. conclusions: our results of overall survival and disease free survival are compatible with literatures. autologous haematopoietic stem cell transplantation with busulfan and etoposide as conditioning regimen for acute myelogenous leukaemia patients s. mousavi, k. alimoghaddam, f. khatami, m. jahani, m. iravani, b. bahar, a. khodabandeh, a. jalali, a. ghavamzadeh hematology-oncology and stem cell transplantation research center (tehran, ir) introduction: acute myelogenous leukemia(aml) is a potentially lethal disease. hematopoietic stem cell transplantation(hsct) has increased disease free survival (dfs) and overall survival (os) of patients more than conventional treatment. the result of autologous hsct in patients without suitable donor is near to allogeneic transplantation. we performed autologous transplantation with busulfan and etoposide as conditioning regimen for patients who didn't have suitable donor. methods: since january 2003 until oct 2008, 108 patients received autologous transplantation. we included all children and adult with aml in fi rst or second complete remission without suitable donor and end organ failure who can tolerate high dose chemotherapy. mobilization regimen was cyclophosphamide 2g/m² for one day and g-csf 10µg/kg for 7 days. we have done stem cell harvesting when patient's white blood cell (wbc) count raised to 1000/µl then the patients received oral busulfan 4mg/kg(from -4 to -1) and etoposide 15mg/kgiv (from -4 to -3) as conditioning regimen. after that patients have transplanted with their peripheral blood stem cells. results: median age at time of transplantation was 25.5 years (age range:4-68), male/female:57/51. 94 patients were in fi rst complete remission and 14 patients were in second complete remission. median infused mononuclear cells was 5.11 * 10 8 /kg despite of the progress in the treatment of acute myeloid leukemia allogeneic hematopoietic stem cell transplantation (hsct) remains the single curative approach to the treatment of resistant aml. these patients (pts) have extremely poor prognosis with the level of relapse 70-80% and the risk of trm 45-70%. high level blasts to the moment of transplantation infl uences on dfs and os. the usage double-phase conditioning regimens with the aim to reduce the level of blasts maximally to the transplantation date can improve dfs and os without the elevation of trm. patients and methods: we reviewed the records of 12 refractory aml pts (8 m/4 f) who underwent hsct at the russian children research hospital between october 2005 and june 2008. the median age was 5 (1-18) years. fab-type: m0 -2 pts, m5 -3 pts, m6 -2 pts, m7 -3 pts, mx -1 pt and secondary aml -1 pt. primary refractory aml was diagnosed in 7 pts and secondary refractory -in 5 pts. 4 kids were transplanted from msd, 2 pts -from mud, 1 pt -from mmud and 5 pts -from mmfd with cd3/cd19-depletion of graft. the median level of blasts in bone marrow prior to hsct was 58% (6-98). first phase included flam (3 pts), flag/go (2 pts), ham (3 pts), flae (1 pt), go (1 pt) or dacogen (1 pt). mylotarg doses were 3-5 mg/m². the interval between 1-st and 2-nd phase of conditioning protracted 1-12 days. second phase of conditioning was treosulfan-(9 pts) or bu-based (3 pts) . a median dose of cd34±cells was 8,5x10 6 /kg. seven patients received dli on median day 63 (44 -119). results: engraftment was documented in all patients with a median time to neutrophil recovery 17 days (8-33) and to platelets recovery -18 days . 11 patients achieved cr to day 30 and one patients had pr. acute gvhd developed in 6 patients, chronic gvhd -in 3 from 9 patients who were alive to day 100. 5 patients had the liver toxicity grade 2-4, and one patient had the pulmonary toxicity grade 3-4. trm level was 8,3%. relapses were diagnosed in 6 patients (dfs 33%). nowadays 5 pts are alive in cr, 1 pt has bm-relapse, 6 pts died (4 -of the relapse, 1 pt -of viral infection, 1 pt -of vod). os amounted 47% with a median time 6 months. rfs depended on the presence of gvhd and type of donor. conclusion: our results show that the usage of double-phase conditioning regimen generally doesn't increase the level of toxicity and trm and allows to achieve the long-term survival in pts with very high risk aml. allogeneic haematopoietic stem cell transplantation for acute myelogenous leukaemia in other than fi rst complete remission status k. alimoghaddam, f. khatami, a. jalali, m. jahani, s. mousavi, m. iravani, a. khodabandeh, b. bahar, a. alimohammadi, n. bahar, a. ghavamzadeh hematology-oncology and stem cell transplantation research center (tehran, ir) introduction: for patients with aml in whom an initial remission cannot be achieved or for those who have a relapse after chemotherapy, stem cell transplantation from an hla-identical sibling offers the best chance for cure. this study reports the result of 18 years allogeneic hsct for aml in other than fi rst complete remission status. patients and methods: from march 1991 until november 2008, 108 aml patients, 55 male and 53 female with median age of 27 years old (range: 3-56 yrs) received allogeneic hsct. the status of them before transplantation was other than cr1 (including second cr, third or more cr, relapse and primary induction failure). source of hematopoietic stem cells was 97 peripheral blood, 10 bone marrow, and 1 cord blood. result: median time to absolute neutrophil count ≥0.5 * 10 9 l was 12 days post hsct and median time to platelet count ≥20 * 10 9 l was 17 days post hsct. forty-two recipients developed acute graft versus host disease (gvhd) and twenty-four developed chronic gvhd. at present 73 (68%) patients are alive. the most common cause of death was relapse. median follow up period was 10 month (range: 1-99 month). six month disease free survival (dfs) and overall survival (os) were 64% (se=5%) and 75% (se=4%), respectively. 2 year dfs and os were 50% & 64% (se=5%). conclusion: allogeneic hsct for aml in other than fi rst complete remission could be advice and can improve the result of treatment in these high-risk patients. outcome of haematopoietic stem cell transplantation for patients with acquired aplastic anaemia at a cancer center, amman, jordan: experience of a young hsct program in a developing country f. abdel-rahman, i. al-sadi, a. badeeb, h. el taani, a. ahmed, r. rihani, a. al zaben, m. sarhan king hussein cancer center (amman, jo) purpose: to evaluate the outcome of hsct in patients with acquired aplastic anemia at khcc. patients and methods: between (3/2003-10/2008) ,15 patients had allogenic hsct for aplastic anemia. there were 9 adults (60%), and 6 children (40%), with a median age of 20 years (range:5-52 years). there were 10 patients (67%) with severe aplastic anemia and 5 patients (33%) with very severe disease. the source of stem cells was bone marrow in 13 patients (87%), and peripheral blood in 2 patients (13%). the median time from diagnosis to transplantation was 86 days. among the group, 12 patients had a full hla matched-related donor, one had 5/6 matched related donor and 2 had 3/6 donor. the conditioning regimens were cyclophosphamide +antithymocyte globulin (atg) in 10 patients, and different conditioning in the other 5 patients. results: the main end points of the study are overall survival for the whole group, and overall survival according to the age, severity of the disease, occurrence of graft versus host disease, and degree of hla match. the median duration of follow up was 5.5 months (1.1-47.2 months) . the median time for the wbc engraftment was engrafted the wbc or the platelets, and 2 patients never engrafted the platelets. the median survival for the whole group was 10.6 months. from the 15 patient, 8 patients are still alive. from the 7 deaths, 6 patients died from sepsis and one from massive gi-bleeding secondary to gut gvhd. from the 9 adult patients 6 are alive (57%), while from the 6 pediatric patients 2 are alive (33%) from the 10 patients with severe aplastic anemia 5 are alive while from the 5 patients with very severe disease 3 are alive. from the 12 patients who had transplant from 6/6 hla matched related donor, 8 are alive (67%). the three other patients who received mismatched graft died. acute gvhd was associated with increased mortality. six of nine patients who develop gvhd died while only 1 out of 6 patients who did not develop gvhd died. four patients had second transplant, two of them are still alive. conclusion: the important predictors of the outcome are: 1-degree of hla match: survival 67% in 6/6 hla match, versus 0% for the mismatch transplant. 2-occurrence of gvhd: survival is 83% in patients without gvhd, and 33% in patients with gvhd. therefore, the most important factor for predicting survival is the degree of hla match. our plan is not to transplant aa from mismatched donors except according to an international study protocol. (1) diabetes type 1 is caused by immune destruction of insulinproducing b cells of pancreas. it has recently been shown that immunoablation combined with transplantation of autologous hematopoietic stem cells may alter the course of the disease and alleviate exogenous insulin requirement [voltarelli et al. jama, 2007; 297:1568 -1576 . we report a 28 year old patient with an early diabetes type 1 (typical clinical course, presence anti-gad antibodies, diagnosis 6 weeks prior to study inclusion) with sustained presence of c-peptide in the blood, in good clinical condition without other serious comorbidities who has been chosen for treatment after signing informed consent for study protocol earlier accepted by local bioethics committee. treatment consisted of plasmapheresis followed by mobilization with cyclophospamide (2 g/m²) and granulocyte colony stimulating factor (g-csf) analogues at 10 µg/kg from day +1. three x 106 cd34+ cells/ kg were obtained by leukapheresis and were later used for transplantation without further selection. conditioning regiment consisted of cyclophosphamide (50 mg/kg for days -5 to -2 each) with atg (thymoglobuline 4,5 g /kg over days -5 to -1). and was followed by transfusion of collected peripheral blood cells on day 0. results: the transplantation was performed on the 1.05.2008. patient engrafted on day +13. during the cytopenic period no major complications were observed. the patient insulin requirement was: 0,47 iu/kg -before moblization, 0,36 iu/kg on the transplantation day, 0,17 iu/kg on engraftment. insulin was discontinued shortly after the regeneration (+24). glucose monitoring showed normal glucose levels without the need for insulin injections from that day on. hba1c levels at diagnosis were 13,8%, 5,2% after 3 months from transplantation and 5,7% 6 months after the transplantation. continuous glucose monitoring was performed around 5 months after the transplant and showed normal values (glucose 75 -135 mg/dl) -fi gure 1. intravenous glucose tolerance test showed normal values of glucose levels after 120 minutes, however the 1st phase of insulin secretion was not present . conclusion: this case support the notion that immunoablation followed by autologous stem cell transplantation in patients with early diabetes type 1 may at least temporary alleviate insulin requirement with excellent control of glycemia. introduction: the allogeneic stem cell transplantation (hsct) represent an effective curative treatment in cml but treatmentrelated morbidity and mortality can be substantial. with the era of bcr-abl kinase inhibitor the place of sct is in discuss for children. we report the results of myeloablative allogeneic hsct underwent in 25 patients (pts) under 18 years of age. material and methods: from december 1998 to april 2007 (101 months period) 25 pts under 18 years of age with cml (chronic phase: 21, accelerated phase: 4) underwent myeloablative allogeneic hsct from hla-identical sibling donors; median age at transplant 13 years (4-17); sex ratio m/f 0,66; median interval from diagnosis to transplant 13 months (4 to 39). all patients received chemotherapy based conditioning regimen: tutshka (n:19), tutshka with additional vp16 (n : 3) and santos (n: 3). 21 pts received peripheral blood stem cell with median cd34+ cell 7,77 10 6 /kg body weight (bw), 3 pts bone marrow transplant with median nuclear cell ( nc ) 3,97 10 8 /kg bw, one pt blood cord transplant with 3,5 10 7 nc/ kg bw. graft-versus-host disease (gvhd) prophylaxis consisted of association ciclosporin and methotrexate. the molecular bcr-abl transcripts diagnosis concerned 14 pts (m(b2a2): 8, m(b3a2): 5 and double transcripts m (b2a2; b3a2):1). molecular monitoring of disease using real-time quantitative polymerase chain reaction (rq-pcr) concerned 11 pts. at 31 july 2008 maximal follow-up is 128 months and minimal 16 months. results: the median time of aplasia was 14 days ( 6-35). eighteen pts (72%) are alive in complete hematological remission (complete molecular remission: 8; major molecular remission: 3; no evaluated: 7) after median follow-up time 61 months . acute gvhd occurred in 7 pts (28%) with 6 grade ii-iv, and chronic gvhd in 10 pts (43,5%) with 5 extensive. disease relapse occurred in 7 pts (28%) within 2 are in complete remission with imatinib. seven pts died (32%): acute gvhd grade iv (n : 2, trm: 8%) ; relapse (n: 5; 24%). the os and event free survival (efs) at 9 years are respectively 66% and 60%. conclusion: our results confi rm that trm is low in young pts and the mayor problem is still relapse disease. the relapse after graft can be treated with bcr-abl kinase inhibitor (2 pts in our study). the question about using allogeneic hsct or bcr-abl kinase inhibitor in children with cml is still open. reduced-intensity conditioning allogeneic stem cell transplantation in advanced chronic lymphocytic leukaemia. the impact of conditioning regimen on the non-relapse mortality. a single-centre experience j. el-cheikh, c. oudin, l. wang, c. faucher, s. furst, d. blaise institut paoli calmettes (marseille, fr) purpose: a unicentric retrospective study to determine the transplant related toxicity in patients with advanced chronic lymphocytic leukemia (cll) after reduced intensity conditioning hematopoietic stem-cell transplantation (hsct) including or not antithymoglobuline (atg). patients and methods: we studied 19 patients with progressive or relapsing chronic lymphocytic leukemia (cll) treated with hematopoietic stem cell transplantation (hsct) in our cancer centre of marseille. 13 males and 6 females, (median age: 60 years). all patients received a reduced intensity conditioning regimen. we compared 11 patients (58%) receiving a non myeloabaltive conditioning including atg with fl udarabine, busulfan (atg group) to 8 patients (42%) receiving fl udarabine, total body irradiation (tbi 2gy) and anti cd20 without atg (non atg group). 14 patients (74%) had a matched related and 5 patients (26%) a matched unrelated donors. graft-versus-host disease (gvhd) prophylaxis consisted of cyclosporine alone in the atg group or a combination with mycophenolate mofetil (mmf) in the non atg group. results: after a median follow-up of 29 months, 13 patients (68%) still alive and in complete remission (to date). mrd was monitored in those patients with cr; all patients achieved a molecular cr. 13 patients had acute and/or chronic gvhd, (70% in atg group vs 30% in non-atg group). at the last follow up 6 patients died (32%), and the cause of death in all of them was the treatment related complications (infections and/ or gvhd); the trm at 100 days was 0%; 26% at one year and 32% at three years of transplantation. (21% in atg group vs 11% in the non atg group); overall survival (os) at three years was 52% in the atg group vs 66% in the non atg group. the os at one and three years was 69% and 59% respectively. fig1. in conclusion: despite the small effective, we can conclude that hsct after reduced conditioning is effective and has the capacity to induce a long term complete remissions, the real impact of atg should be revaluated on further large multicentric studies. dasatinib: optimal bridge to stem cell transplant in chronic myeloid leukaemia blast crisis a. gozzini, s. guidi, c. nozzoli, b. bartolozzi, r. saccardi, b. scappini, a. bosi bmt unit (florence, it) pts presenting cml-bc have a survival of 3-6 months and scarce response to imatinib. dasatinib (bms-354825) is an oral, multi-targeted kinase inhibitor, currently being used in pts with imatinib-resistant advanced cml or relapsed/refractory ph+ all. most of these pts will be evaluated for sct, even though for them this curative therapy showed higher incidence of gvhd, vod and trm. we report here fi ve pts affected from cml-lb who received dasatinib prior to allosct. donors were matched siblings (2), matched unrelated (2) or blood cord unit (2) . 4 were male and 2 female with a median age of 36,4 (18-55) years. first line therapies included chemotherapy (vcr) plus high dose imatinib. all pts after 2-5 months from diagnosis received dasatinib 70mg bid. t315i mutation occurred in 3 patients, y253 and e255k in 2 patients, and a non codifi ed mutation in 1 patient. dasatinib induced complete hematological response (chr) in 4 pts, and complete (n=2) and partial cytogenetic response (pcyr) (n=1) prior to sct. 3 patients did not achieved a complete haematological response presenting 25% marrow blasts and 65% respectively prior to sct. all pts were conditioned with myeloablative protocol. gvhd prophylaxis consisted of csa and mtx (n=4) or micofenolate association until +30 (n=2). pts received a mobilized peripheral blood stem cell graft with 3,52-11,04x 10 6 cd34+ cells/kg (n=4) and cord blood unit with 0,1x10 6 cd34+ cells/kg (n=2). dasatinib was stopped 6 days before transplant procedure. 6/6 pts successfully engrafted reaching anc >0.5x10 9 /l on day +19 (11-37) and plt >20x10 9 /l on day +21 (11-50). dasatinib was introduced again in 2 patients 30 days after sct. one of them stopped therapy because of haematological toxicity after 2 weeks. 5/6 patients presented chimerism was 97-100%. transplant related toxicities were grade i/ii. no pts developed hyperbilirubinemia or vod. hyperacute extensive gvhd (gr iii) was observed in only 1 pts at +9. five patients are alive, all of them in complete molecular response with a median follow-up of 9.3 (4-19) months, 1 died of agvhd. we may conclude that in pts undergoing sct following dasatinib there is no evidence of adverse effect on sct outcome, organ toxicities. larger studies and longer follow-up are obviously indicated to confi rm our preliminary results. both t315i positive patients are alive in chr. dasatinib represents an effi cient bridge to transplant to improve the outcome of this subset of patients. inmatinib combined myeloablative allogenetic haematopoietic stem cell transplantation for advanced chronic myeloid leukaemia y. luo, y. tan, j. shi, x. han, g. zheng, x. zhu, x. lai, h. huang zhejiang uninversity school of medicine (hangzhou, cn) improved strategies are needed to treat patients with advanced chronic myeloid leukemia (cml) in order to reduce the need for lifelong therapy. we treated 14 patients with advanced cml (5 in ap, 9 in bc) with myeloablative allogeneic stem cell transplantation (allo-sct) combined with pre-transplantation imatinib. the donors included hla-matched and 1-locus mismatched unrelated volunteers (n=7), and hla-matched siblings (n=7). graft-versus-host disease (gvhd) prophylaxis consisted of cyclosporine, mycophenolate mofetil and short-term methotrexate. 6 out of 14 (42.9%) evaluable patientsdeveloped ii-iv agvhd, 3(21.4%) patients suffered from agvhd grade iii-iv. two patients suffered from intensive chronic gvhd. after a median follow-up of 19 months (range 3¨c47 months), the overall survival was (71.4%) 10/14. the ten patients were all in molecular remission. imatinib combined with allo sct could provide a safe, well-tolerated therapeutic option for patients with advanced cml. this conclusion needs to be tested in prospective randomized clinical trials. p-beam group. both groups did not differ in terms of time of hospital stay, days of iv antibiotics, mucositis and infections. with the median follow-up of 6 (5-10) years, the probability of overall survival at 6 years equaled 83% for p-beam and 63% for chopp-cbv group (p= 0.2). the probability of progressionfree survival was 65% and 50%, respectively (p=0.2). conclusions: p-beam and chopp-cbv protocols followed by autopbsct are effective and well-tolerated salvage therapies for patients with advanced hl. prolonged administration of the therapy seems to be appropriate for this group of patients. towards safer autotransplants in patients with non-hodgkin's lymphoma: cardiac pre-evaluation, angiotensin-converting enzyme inhibition in patients with decreased left ventricular function, antimicrobial prophylaxis and vigilant supportive care e. jantunen, s. hämäläinen, t. kuittinen, k. penttilä, m. pyörälä, a. juutilainen, i. koivula, t. nousiainen kuopio university hospital (kuopio, fi) autologous stem cell transplantation (asct) for nhl is associated with an early non-relapse mortality rate of 3-5% most commonly due to sepsis. during 1996-2006 160 nhl patients received asct at our department. seventeen patients (9%) experienced severe sepsis and nine (4.5%) died due to septic shock. severe sepsis was caused by gram-negative bacteria including pseudomonas in a signifi cant proportion of the patients (hämäläinen et al. scand j infect dis 2008). subclinical anthracycline cardiomyopathy may be important in regard to the development of severe sepsis in some nhl patients. since january 2008 we have applied prospectively cardiac pre-evaluation (radiocardiography), angiotensin converting enzyme inhibition in patients with decreased left ventricular ejection fraction (lvef) (< 50%), ciprofl oxacin prophylaxis and start with ceftazidime plus tobramycin in patients with neutropenic fever in nhl patients undergoing asct. febrile patients are observed closely with measurements of pro-brain type natriuretic peptide (bnp) and c-reactive protein (crp) for three days. also blood pressure, blood oxygen saturation, hydration and diuresis are monitored. until nov 2008, altogether 14 patients with nhl (10 m, 4 f) with a median age of 55 years (range 28-65) have received beam followed by pb infusion according to this protocol. lvef was < 50% in six patients (43%) pre-transplant and they received enalapril during the peritransplant period. neutropenic fever was observed in 12 patients (86%). no cases with gram-negative bactereamia or severe sepsis have been observed. the median peak crp value was 139 mg/l (23-333) and was reached in a median of two days after rise of fever. serum bnp values were above normal limit in 3/12 patients with fever on day 0. elevated bnp values were observed in 4/10 patients on day 1, in 9/12 patients on day 2, and in 7/9 patients on day 3, respectively. whether severe sepsis or early deaths could be prevented with this approach remains to be seen in upcoming years with larger number of patients. outcome of refractory/relapsed patients affected by hodgkin's lymphoma treated with or without peripheral blood stem cells autografting: a single-centre experience f. angrilli, s. falorio, f. fioritoni, s. santarone civic hospital (pescara, it) introduction: despite a high curability rate, 10 to 40% of patients (pts) affected by hodgkin lymphoma (hl) fail to respond or relapse after front-line treatment with polychemotherapy alone or combined with radiotherapy. the treatment of choice for refractory or early relapsed pts is high-dose chemotherapy (hdc) followed by peripheral blood stem cells autografting (pbsca), while late relapsed pts may be treated with either conventional therapy or hdc plus pbsca. methods: from 1999 to december 2007, 179 untreated pts with hl have been admitted in our institution. after front-line therapy, 168 (93%) pts obtained a complete remission (cr) and 11 pts (7%) were refractory to standard treatment. overall, 11 pts relapsed within 12 months after diagnosis of hl, while 6 pts experienced late relapse. the aim of this retrospective study is to evaluate the outcome of the our 28 refractory/relapsed pts according to the type of salvage treatment. twenty-six pts received as salvage treatment 3-6 courses of igev (iphosphamide, gemcytabine, vinorelbine), 1 patient 6 courses of coppebvcad (cyclophosphamide, carmustine, melphalan, epirubicin, vinvristine, vinblastine, prednisone) and 1 patient 6 courses of abvd (doxorubicin, bleomycin, vincristine, dacarbazine). today, 27 pts completed salvage chemotherapy. of them, 19 (8 with refractory hl and 11 with relapsed disease) have been submitted to pbsca. conditioning regimen consisted of beam in all cases. results: 15 pts were male and 13 female (m/f ratio 1,15). median age was 34 years (range 16-59). overall, 17 pts obtained a cr (63%) and 10 pts had progressive disease (37%). in particular the cr were 14 (73%) in the group of the pts receiving pbsca and 3 (33%) in the other pts (p <0.05). one patient died in cr of beam toxicity prior pbsca and 10 pts died of progressive hl. after a medium follow-up of 24 months, overall survival was 63% for the pts who received pbsca and 38% for those who received conventional treatment (p=0.05). conclusions: our data confi rm the benefi t of hdc plus pbsca both in relapsed and in refractory patients with hl. nevertheless, a portion of refractory or early relapsed pts fail to respond to pbsca and died of hl. for these pts tandem pbsca or allogeneic stem cell transplantation should be proposed, especially if they are not in cr prior to pbsca. t cell lymphoma is a heterogeneous group of aggressive lymphomas associated with poor prognosis with standard chemotherapy and autologous hematopoietic progenitor cells transplantation (hpct) is offered as consolidation in fi rst remission or at relapse. in this study we conducted a retrospective analisis of 16 patients underwent hpct from december 1993 to august 2008. seven patients had diagnosis of peripheral t-cell lymphoma, four patients of systemic anaplastic large cell, and fi ve patients of linfoblastic lymphoma. five patients were transplanted in fi rst complete or partial response, ten patients in second or beyond complete or partial response and one patients in second refractory disease. median age was 36,5 years; seventy-fi ve percent preesented advanced (iii-iv) ann arbor stage, 50% had b symptoms, 50% had high lactate dehydrogenase. with a median follow-up of 73 months from diagnosis and 31,5 months from transplantation, the 5-year progression-free survival (pfs) and overall survival (os) were 37,5% and 31,2% respectively. based on these preliminary results the hpct as consolidation therapy may offer a durable survival benefi t. the chimeric anti-cd20 monoclonal antibody rituximab offers new therapeutic options in the treatment of b-cell nhl (non-hodgkin's lymphoma). the addition of rituximab to chop (cyclophosphamide, doxorubicin, vincristine, and prednisolone) or cvp (cyclophosphamide, vincristine, and prednisolone) regimen was found to signifi cantly improve the response rates and survival in patients with untreated diffuse large b-cell lymphoma (dlbcl) and is now considered as the standard therapy option. rituximab also has been shown to improve response rates when combined with salvage chemotherapy. there are few studies regarding the effects of rituximab on mobilization. we compared the effi cacy of rituximab plus eshap (etoposide, metil prednisolone, cytosine arabinoside, cisplatin) with eshap alone as mobilization regimen in 34 (40%) hodgkin's and 50 (60%) non-hodgkin's lymphoma patients. 26 (30%) patients were dlbcl. 43 (51%) relapsed and 21 (25%) refractory patients were involved. 19 (23%) patients were treated with r-eshap and 65 (77%) patients with eshap regimen. 228 aphaeresis were evaluated. median number of aphaeresis was 2.64 days for r-eshap patients and 2.71 days for eshap patients. median number of mononuclear cell aphaeresis was 4.75*10 8 per kg (kilogram) and 6.83*10 8 per kg respectively. total number of cd34+ cells was 15.58*10 6 per kg in the r-eshap group and 17.75*10 6 per kg in the eshap group. toxicities were similar in both groups. there were no engraftment delays in the r-eshap group. so we conclude that r-eshap is effective and feasible as eshap regimen for mobilization. total number of cd34+ cell aphaeresis was slightly lower in the r-eshap group but did not have an effect on engraftment. prospective randomized studies are needed to evaluate whether rituximab really decreases mobilization adequacy or not. no benefi t of autologous stem cell transplantation as consolidation for high and high-intermediate risk diffuse large b-cell lymphoma in 1.cr after r-chop therapy -a single-centre experience m. karas, k. steinerová, p. jindra, d. lysák, s. vokurka, v. vozobulová, m. schützová, l. mohammadová, v. koza charles university hospital pilsen (pilsen, cz) objectives: the role of high-dose therapy (hdt) and autologous stem cell transplantation (asct) for patients (pts) with high and high-intermediate (h/hi) risk diffuse large b-cell lymphoma (dlbcl) in 1.cr was not clearly defi ned especially after addition of rituximab (r) to fi rst line chemotherapy (cht) and the use of rituximab also as maintenance therapy. therefore, we retrospectively analysed outcome of pts treated in our transplant centre with hdt and asct for h/hi risk dlbcl in 1.cr after 6-8 cycles of r-chop-21 chemotherapy and we compared their outcome with a control group of pts with h/hi risk dlbcl in 1.cr treated only with chemoimmunotherapy. patients and methods: between 2003 and 2008 (median follow-up 38 months, range 13-64 months) 17 consecutive pts with median of age 48 years (range 24-63 years) with h/hi risk dlbcl in 1.cr after 6-8 cycles of r-chop-21 underwent hdt (beam) and asct. the median of time from diagnosis to asct was 8 months (range 5-13 months). source of stem cells was peripheral blood and median of infused cd34+ cells was 4,36x10 6 /kg (range 3,28-9,94x10 6 /kg). the control group consisted of 11 consecutive pts with h/hi risk dlbcl in 1.cr treated only with chemoimmunotherapy (6-8 cycles of r-chop-21, 45% maintenance therapy with rituximab). the control group except for the older age did not differ in any prognostic parameters. results: in the transplanted group 15 pts (88%) are alive in cr. 2 pts (12%) relapsed and died. no patient died due to transplant-related mortality (trm). the estimated probabilities of 4-years disease-free survival (dfs) and overall survival (os) were 87% and 86%. in the chemoimmunotherapy treated group 10 pts (91%) are alive in cr. 1 patient (9%) relapsed and died. the estimated probabilies of 4-years dfs and os were 75% and 67%. we did not observe between both groups any significant difference in cumulative relapse incidence (p=1,00), dfs (p=0,91) and os (p=0,89). conclusion: our data suggest that hdt with asct in pts with h/hi risk dlbcl in 1.cr after r-chop chemotherapy was well-tolerated with no trm death but in comparison with pts treated only with chemoimmunotherapy we did not observe any improvement of outcome among transplanted pts. of course relatively lower number of evaluated pts and retrospective type of analysis could infl uence our results and only prospective randomized studies can fi nally defi ne the role of frontline hdt with asct for h/hi risk dlbcl in 1.cr after chemoimmunotherapy. kyrcz-krzemien medical university of silesia (katowice, pl) background and aims: autologous peripheral blood stem cell transplantation (autopbsct) is widely used for the treatment of poor-risk patients with hodgkin's lymphoma (hl), however, the optimal preparative regimen has not been established. we assumed that patients with advanced hl may benefi t from receiving intensive pre-transplant therapy with prolonged administration of cytostatics and the addition of oral drugs, such as procarbazine or chlorambucil. therefore, we modifi ed the commonly used beam and cbv protocols by incorporating oral agents and prolonging the distribution of the total doses to 7 and 9 days, respectively. the goal of this pilot study was to evaluate safety and effi cacy of those regimens. patients and methods: 33 patients (20 males and 13 females, median age 27 years, range 17-63) with relapsed hl were included in this study. previous therapy consisted of median 3 (2-5) lines of treatment and 11 (3-32) chemotherapy cycles results: 1/19 patients died due to septic complications in chopp-cbv group, whereas no procedure related mortality was observed among patients treated with p-beam. all remaining patients engrafted. time to achievement anc >0.5 g/l was signifi cantly shorter in p-beam vs. chopp-cbv group: 14 (10-34) days vs zevalin-beam conditioning in transformed follicular lymphoma; acceptable toxicity and possible therapeutic benefi t a hdt-sct) using beam conditioning has become standard therapy for relapsed fl, however recurrent disease especially in transformed follicular lymphoma (t-fl) remains the commonest cause of death. the addition of zevalin (ibritumomab tiuxetan), a cd20 targeted radiolabelled antibody to beam is safe and may improve the effi cacy of hdt we analysed 5 patients aged 42 to 58 with advanced stage t-fl who had received a median of 4 (range 3-4) lines of therapy prior to zevalin-beam sct. the median time from diagnosis to hdt-sct was 43 months (range 17-55.7) and all patients had chemosensitive disease in partial remission bcnu 300mg/m 2 , etoposide 200mg/m 2 , cytarabine 200mg/m 2 , melphalan 140mg/m 2 ) from day -7 to -1. the median stem cell dose was 2 the 3 remaining patients remain stable at a median of 16 months (range 4 -23) post-sct conclusion: the zevalin-beam protocol is as well tolerated as standard beam conditioning. the disease free survival in this small cohort of high risk patients with t-fl is encouraging but needs longer follow-up rituximab or not? a historical comparison of eshap and r-eshap as mobilisation regimen in 84 patients non-myeloablative allogeneic stem cell transplantation in patients with high-risk lymphoma: a multicentre experience g. console (1), g. irrera (1), m. martino (1) a. meliadò (1), c. rigolino (1), t. del vecchio (1), o. iacopino (1), m.c. cannatà (1), p. scaramozzino (1), i. bova (1), d. marcuccio (1), c. stelitano (2), s. molica (3), r. cantaffa (3), l. nocilli (4), a. mele (5) pugliese-ciaccio" (catanzaro, it); (4)osp 44 patients (pts) (23 females and 21 males), median age 43,1 years (range 18-67) underwent nst for high risk hodgkin disease (hd, 14 cases) and non hodgkin lymphoma (nhl, 30 cases) conditioning regimens consisted of fludarabine, thiotepa and cyclophosfamide in 30 cases, tli and atg in 5 cases, fludarabine and cyclophosfamide in 4 cases, fludarabine and thiotepa in 1 case, fludarabine, melphalan, thiotepa and atg in 1 case, campath-1, fludarabine, melphalan and tbi were employed in 2 cases. in 1 case tbi and fludarabine. cyclosporine-a (cya) and methotrexate (mtx) were used as gvhd prophilaxys in 35 cases, in 2 campath-1 and moftil micofenolate (mmf) were combined and in 7 cases cya and mmf were used. a mean number of 5.41x10 6 /kg cd34+ cells (range 2,1-8,7) were infused. pts received a mean of 3.04 (range 0-22) packed red blood cells after a median follow up of 33,2 months, (range 2-84), 31 pts are still alive (22 in cr,4 pr,5 in relapse), experienced cgvhd (4 cutaneos w.h.o. grade1-2,1 pneumonial w.h.o. grade 1,1 liver w.h.o 4). 3 pts died for liver agvhd,1 patient died for cerebral vasculitis at 18 months to the transplant,1 patient died for ards at 2 months from transplant. 1 pts for acute bacterial pneumonia. 2 pts for mof respectively at 1 and 2 months from transplant, 1 for aptt, at 18 months from transplant. 1 patient for interstitial pnemoniae at 20 months to the transplant, 3 pts died for disease recurrence at 10, 17and 36 months post-transplant respectively here we report on a single centre, retrospective analysis evaluating the outcome of patients (pts.) with dlcl treated with high-dose chemotherapy and autologous (auto) or autologous-allogeneic (auto-allo) hsct. patients and methods: in total, 22 (66,7%) male pts. and 11 (33,3%) female pts. with dlcl underwent auto (26 pts.) or autoallo hsct (7 pts) between 01.01.1994 and 30.06.2007. 19 pts. received auto hsct as part of fi rst-line therapy (group 1). in 14 pts auto (7pts, group 2) or auto-allo (7pts., group 3) hsct was performed as second-line therapy. the median patient age was 49 years 1: 3/6/10, group 2: 0/6/1, group 3: 0/2/5. patients who received auto hsct as fi rst-line therapy (group 1) tended to have a better median os (1374 vs. 272d, p=0.232), rfs (1245 vs. 95 d, p=0.025) and 5-ysr-os (65% vs. 45%, p=0.382) compared to pts of group 2. furthermore patients of group 1 had a signifi cant better os and 5-yrs-os in the auto-allo group 6 of 7 pts died (5 pts died from severe infection with multiorgan failure and 1 patient from relapse of disease). in contrast, none patient died from trm after second auto hsct, but 2 died from progressive disease and 1 pt from relapse. conclusion: the survival of patients with relapse of dlcl could not be improved by using the therapeutic approach of auto-allo hsct compared to an auto hsct based regime, due to the high trm in the auto-allo group. however, for interpretation of these results some facts have to taken into account since year 2000, 10 patients with primary myelofi brosis (8 females and 2 males age 29-55y median 46.5) received allo hsct (7 sib and 3 unrelated donors matched at allele level). according to the dupriez prognostic system: 2,6,2 patients were in high, intermediate an low risk of the disease. the diagnosis was proved by trephine biopsy, which revealed that all patients were at advanced stage of fi brosis, all patients had splenomegaly and abnormal blood smear with the presence of erythroblasts. the length of the disease duration was from 7 to 36 months (median 19). six patients were transfusion dependent because of anaemia and thrombocytopenia, three patients were on steroids and six on hydroxycarbamide. splenectomy prior to transplantation was performed in two patients. two patients received myeloablative conditioning (busulfan 16mg/ kg cyclophosphamide 120 mg/kg) and eight reduced intensity conditioning (busulfan 8 mg/kg, fludarabine 120 -150 mg/m² or melphalan 120-140 mg/m² and low dose atg). all patients were transplanted with pbpc with cd34 dose from 1.8 to 11.7 x 10 6 /kg (median 6.2 x 10 6 /kg). two patients died due to transplant toxicity (one with additional ebv reactivation and sepsis and one with vod symptomatology). in other patients toxicity was mild and there was no agvh exceeding grade i. two patients transplanted with major blood group incompatibility developed prca. plasmapheresis and erythropetin were successfully employed in those patients. finally all surviving patients reconstituted haematologically. a trephine biopsy performed 1 months post transplant documented the process of bone marrow remodeling with a normal picture six months post transplant. all patients except one had full chimerism. eight out of ten patients are alive and with normal hematopoesis during observation period from 1 to 104 months (median 24). the post transplantation course was similar in patients having and lacking jak 2 mutation. in conclusion haematopoetic stem cell transplantation in primary osteomyelofi brosis is associated with rather low risk and results in sustained haematological recovery. nutritional assessment of children undergoing haematopoietic stem cell transplantation for primary immunodefi ciency or severe autoimmune disease m. slatter, c. ferguson, e. rogerson, a. yurasova, p. askew, t. flood, m. abinun, a. cant, s. bunn, j a major challenge post hsct is adequate nutrition, as poor nutritional status adversely affects outcome. patients undergoing hsct for pid often fail to thrive pre-hsct due to underlying disease. we aimed to document nutritional intake of pid children undergoing hsct at our centre. 15 children who underwent hsct for pid or severe autoimmune (ai) disease from april 2007 -january 2008 were evaluated. the following prospective data was collected: diagnosis, age, donor, conditioning, presence of infection and growth. nutritional intake, biochemical indices, use of antiemetics and complications were documented on admission, after 2 weeks, then monthly until day 0, +7, +14, +21, +28, +42 then monthly until discharge home. patient characteristics: 8 patients had scid, 5 had other pid. 2 had severe ai disease. age at transplant ranged from 2 months to 16.6 years (median 8 months). 9 were ≤ 1yr. 8 had unrelated (2 cords), 5 matched family, 1 matched sibling and 1 haploidentical donor. all had chemotherapy conditioning -4 bu/cy, 3 flu/melph, 7 treo/flu, 1 treo/cy. results: all received supplementary feeding via nasogastric (14) or percutaneous jejunal tube (1) . only 2 required total parenteral nutrition, 1 with severe ai disease and 1 with persistent norovirus enteritis. all received at least 1 anti-emetic. 4 had viral enteritis -2 norovirus, 2 adenovirus. in 14 patients for whom adequate data was available, all had a reduction in calorie and protein intake in the 2-3 weeks following hsct, because of fl uid restriction. 2 had grade ii skin gvhd, none developed gut gvhd. 7 had mucositis requiring morphine. only 1 patient lost weight overall from time of admission to discharge, one had static weight, but 13 gained weight, by time of discharge. further evaluation of nutritional indices is required. the time around hsct is the most challenging to support adequate nutrition. careful nutritional assessment of patients undergoing hsct is critical and should direct nutritional support. patients should be optimised nutritionally prior to hsct, as the high metabolic demands around the time of hsct are unlikely to be met over the immediate transplant period. thalidomide+dexamethasone and partly b, b+dexamethasone, b+adriamycin+ dexamethasone (pad). the b group had a post-tx maintenance therapy with b 4 weeks: 1,3 mg 2 iv doses weekly + dexamethasone 20 mg 4 days. results: length of survival times (os) without and with b were signifi cantly different. further analysis of the curves in complete remission indicated 100% survival probability and 90% disease free survival (dfs) in 19 patients in a 50-month period. in the very good partial remission (vgpr) group (12 pts) the os was 100%, however, the dfs was only 60%. the survival curves were signifi cantly worse when tx was made in partial remission (os: 55%, dfs: 50% by 23 pts). conclusions: 1. the author›s data support the fi nding that lasting survival can be expected when tx is performed in cr or vgpr. 2. in the interest of this, in cases of a more aggressive disease, the fi rst line pad protocol before tx is the best therapy. after the tx a consolidation therapy with b+dexamethasone is very useful. 3. in a slightly less aggressive disease or with accompanying diseases a thalidomide+dexamethasone fi rst therapy may also be possible. 4. tx performed in partial remission maybe dangerous. at this time needed put in "the therapy arsenal". acute renal failure in myeloma patients during mobilisation procedures for autologous transplantation a. pivkova (1) during the last 30 years blood cell separation, generally referred to aphaeresis, has established a central role in both blood donor programmes and therapeutics. the technological advances in aphaeresis equipment have made procedures safer, faster and more effective. we present 3 cases (2 males and 1 female) with multiple myeloma treated at our department during 2007 until 2008. initial chemotherapy treatment was provided with thalidomide based regimens (c-thal dex 4 cycles or thaldex in 4 cycles) or 4 cycles of vad in one patient. all 3 patients before diagnosis and during initial treatment had normal and stable renal function. after completing remission in all, mobilisation of pbsc was preformed with g-csf 10mcg/kg in duration of 5 days. the number of wbc count prior collection was median 42 x10 9 /l (30-51) with median lymphomonocyte percent 13, 43 (4-22). aphaeresis was preformed at day 5 with cobe spectra cell separator and large volume aphaeresis. in all 3 patients after fi nishing the fi rst procedure we registered increase of renal degradation products in the serum during the fi rst 6 hours post aphaeresis and complete anuria which revealed in acute renal failure (renal type) treated with haemodialysis in several consecutive occasions. one month after resolving the renal impairment the patients continued with second mobilisation procedure with the same regimen and obtained a minimal mnc count of 2,0x10 8 /kg. autologous transplantation followed by melphalan reduced dose conditioning 100mg/m². engraftment was registered for ne>0,5x10 9 /l and plt >20x10 9 /l on median day + 10 (8 to 12). the patients had no need for blood transfusions. all 3 are in cr med 7 mths (3) (4) (5) (6) (7) (8) (9) (10) (11) after transplant. in one patient 4 months after, a double transplant was preformed. concerning the small group of patients, we can evaluate the possible impact of large volume aphaeresis in the renal impairment in these patients or the infl uence of cytokine mobilised cells on renal tubules. key: cord-015365-iqdi99pd authors: nan title: 25th annual meeting of the austrian society of transplantation, transfusion and genetics graz, october 19–21, 2011 date: 2011 journal: eur surg doi: 10.1007/s10353-011-0041-z sha: doc_id: 15365 cord_uid: iqdi99pd nan grundlagen. parvovirus b19 (pvb19) als ursache einer schweren aplastischen anämie (saa) oder eines myelodysplastischen syndroms (mds) ist bisher erst selten beschrieben. es existieren auch kaum berichte über stammzell-transplantationen (sct) bei diesen krankheitsbildern. methodik. bei 6 patienten mit saa (n ¼ 4) oder mds (n ¼ 2) wurde bei diagnosestellung eine frische infektion mit pvb19 mittels positivem nachweis von igm, igg und pcr nachgewiesen. alle patienten erhielten eine sct unter dem schutz repetitiver intravenöser immunglobulin (ivig)-gaben. das virologische monitoring mittels pcr erfolgte in 5 von 6 fällen in der akutphase wöchentlich, danach monatlich bis zur hämatologischen rekonstituierung. ergebnisse. die 4 patienten mit saa erhielten knochenmark eines hla-identen geschwister-spenders (n ¼ 3) oder hla-idente hochgereinigte, periphere stammzellen eines unverwandten spenders (n ¼ 1). ein patient entwickelte nach sct eine chronische, hypoplastische anämie, die bis zur vollständigen blutbild-regeneration am tag 186 andauerte. der posttransplantations-verlauf der ü brigen 3 patienten war komplikationslos mit einem leukozyten-engraftment nach 8-31 tagen und einer hämatologischen rekonstituierung nach 36-112 tagen. die 2 patienten mit mds wurden nach konditionierung mit thiotepa, fludarabin und atg einer sct mit unverwandten spendern (1x knochenmark, 1x periphere hochgereinigte stammzellen) unterzogen. nach zögerlichem leukozyten-engraftment dauerte es 1260 bzw. 119 tage bis zur blutbild-normalisierung, pvb19 wurde in der pcr bis zum tag 686 bzw. 139 nachgewiesen. schlussfolgerungen. eine erfolgreiche sct bei diesen patienten ist möglich, sofern eine engmaschige monitorisierung der pvb19-pcr und eine konsequente repetitive ivig-therapie erfolgen. der transplantationsverlauf kann durch ein zögerliches engraftment und eine prolongierte "poor graft function" kompliziert werden. retrospective study to test ferritin serum levels as biomarker for graft-versus-host disease-associated non-infectious inflammatory reaction in 117 children after hematopoietic stem cell transplantation background. myelodysplastic syndromes (mds) are a heterogeneous group of stem cell disorders characterized by bone marrow dysplasia, peripheral cytopenia, and an enhanced risk to transform to acute myeloid leukemia (aml). in most patients, treatment options are limited to supportive care and palliative cytoreduction. however, in a group of patients, intensive therapy can be offered. the only established curative treatment approach for these patients is haematopoietic stem cell transplantation (sct) . methods. in the present study, we retrospectively analyzed a cohort of 60 adult patients (33 males, 27 females) with mds (n ¼ 28) or mds transforming into secondary aml (n ¼ 33), who underwent sct at our institution between 1988 and 2010. fifty-one patients had an hla-identical related transplant donor, and 9 had an hla-matched unrelated donor. the median age at time of sct was 44 years (range: 18 to 68 years). according to the who classification, 4 patients had ra, 1 rars, 3 rcmd, 1 rcmd-rs, 6 raeb-1, 12 raeb-2, 1 cmml, and 32 had aml following raeb at sct. conditioning consisted of chemotherapy plus total body irradiation (55/60 patients) or chemotherapy alone (5/60 patients). graft versus host disease (gvhd) prophylaxis consisted of a combination of low-dose methotrexate and cyclosporine a (37/60 patients) or cyclosporine a plus mycophenolat mofetil (23/60 patients). results. patients were followed up with a median observation time of 16 months (range: 1-218). currently, 34 patients (57%) are alive. of the 26 patients who died, post-transplantation relapse occurred in 12 patients, and 14 patients died of treatment-related causes (multi-organ failure, n ¼ 7; sepsis, n ¼ 4; haemorrhage, n ¼ 3). in multivariate analysis we identified pre-transplantation ferritin as a significant adverse prognostic variable for survival in our mds patients. in contrast, the overall outcome after sct was independent of ipss risk categories or the who classification. conclusions. based on these data and similar published data we recommend to select patients with mds or saml for sct who are considered candidates for sct according to the presence of pre-transplantation iron-overload. significantly worse survival of patients with chronic graft-versus-host disease (cgvhd) defined according to the national institutes of health (nih) consensus criteria and thrombocytopenia data and expert opinion. for both acute as well as cgvhd new definitions were proposed. we performed a prospective study on all consecutive patients undergoing allogeneic hematopoietic stem cell transplantation (hct) since 2005 to assess the prognostic impact of the new cgvhd staging criteria. methods. one hundred seventy-eight patients (85 males, 93 females) with a median age of 40 years alive on day þ 100 after hct with myeloablative (n ¼ 110) or reduced-intensity (n ¼ 68) conditioning and a related (n ¼ 37) or unrelated (n ¼ 141) stem cell donor were enrolled into the study. starting on day þ 100 after hct all patients were assessed clinically every 3 months in the outpatient clinic for cgvhd activity according to the nih consensus criteria (filipovich ah et al, bbmt 2005; 11:945-956) . results. one hundred twenty-six (71%) patients experienced acute gvhd grades i to iv including 11 with recurrent, 11 with persistent, and 10 with late-onset acute gvhd after a median of 18 (range, 9-120) days after hct. one hundred-fifteen patients (65%) experienced cgvhd after a median of 151 (range, 82-510) days after hct. eighty-nine patients (77%) had classic cgvhd and 26 (23%) overlap syndrome. probability of overall survival (os) at 3 years for late-acute gvhd, chronic classic and overlap cgvhd were 69%, 83%, and 73%. three-year os for mild, moderate and severe cgvhd at onset were 93%, 79%, and 62.5% and significantly different between mild and severe (p ¼ 0.007). patients with progressive onset type of cgvhd had significantly worse three-year survival compared to de novo and quiescent (54.5% vs. 89.5%, 84%, p < 0.01). three-year os was also significantly worse in patients with platelet counts below 100 g/l at onset of cgvhd (35% vs. 86%, p < 0.0001). conclusions. this prospective analysis supports the importance of distinguishing late-acute gvhd from cgvhd and indicates a prognostic value of thrombocytopenia and progressive onset type of cgvhd for hct outcomes. current concepts suggest a major role for innate immunity in the initiation of acute graft-vs.-host-disease (agvhd) as well as in crohn's disease. identical single nucleotide polymorphisms (snp) have been described as risk factors for both diseases. recently, the noncoding c/t polymorphism rs2274910 in intron 3 of the intelectin 1 (itln1) gene (human lactoferrin receptor) has been associated with crohn's disease. we retrospectively typed this polymorphism in aml patients and their donors. a pilot study of 19 patients in cr1 transplanted from hla identical sibling donors after myeloablative conditioning was confirmed in a second cohort including 40 other aml patients. a total of 59 consecutive patients (median age 43 yrs (18-63); 25/34 cr1/advanced disease; 40 myeloablative and 19 reduced intensity conditioning; 54 peripheral stem cells) were tested. t-alleles were found at a frequency of 28.8% in recipients and 29.7% in donors. 50.8% of patients had a cc genotype. in the pilot study, 2 out of 12 patients with a cc genotype versus 5 out of 7 patients with a t-allele had acute intestinal agvhd (p ¼ 0.045). these results were confirmed in a second cohort. acute intestinal gvhd was found in 3/18 patients (16.7%) with a cc genotype and 12/ 22 patients (54.5%) with a t-allele (p ¼ 0.014). in a combined analysis (n ¼ 59) we found intestinal agvhd in 58.6% of patients with a t-allele versus 16.7% of patients with a cc genotype (p < 0.001). the lower incidence of agvhd grades ii-iv in patients with a cc genotype (p ¼ 0.019) was only due to less intestinal agvhd while no difference was found with regard to skin or liver agvhd. we did not see any association between donor genotypes and agvhd. the strong association (rr 3.52; 95% ci 1.5-8.2) between this snp and the incidence of intestinal agvhd is in accordance with reports about snps in other genes associated with both agvhd and crohn's disease. our results further support the concept of the importance of the gut associated innate immune system in the initiation of agvhd. grundlagen. klinische studien zeigen, dass sich minorhistokompatibilitätsantigen (mhag)-spezifische spender t-zellen positiv in der therapie eines leukämischen relapses nach hlaidenter hämatopoetischer stammzelltransplantation (szt) auswirken. der prädiktive wert der mhag-typisierung für das auftreten eines graft-versus-leukämie effektes oder von komplikationen nach szt ist jedoch nach wie vor umstritten. auch langzeitstudien über den einfluss von mhag-mismatches auf das ü berleben der patienten fehlen. deshalb haben wir in der vorliegenden studie unterschiede in zehn verschiedenen mhag in insgesamt 217 patienten und ihren hla-identen familienoder fremdspendern untersucht und sie mit ü berleben und komplikationen nach allogener szt korreliert. methodik. die bestimmung der mhag wurde mittels allelspezifischer polymerase-kettenreaktion mit hilfe eines kommerziell erhältlichen mhag-typisierungskits durchgeführt. die wahrscheinlichkeit des ü berlebens (bis zu 22 jahre nach szt) wurde mittels kaplan-meier ü berlebensanalyse, die komplikationsraten mittels cumulativer inzidenz berechnet. ergebnisse. von den 217 patienten/spenderkombinationen wiesen 119 keinen mhag-mismatch, 67 einen mhag-mismatch und 31 zwei oder mehrere mhag-mismatches auf. sämtliche patienten mit einem ha-1-, ha-2-, ha-8-(hla-a2-restringiert) oder ha-3-(hla-a1-restringiert) mismatch zeigten eine geringere rezidivrate als patienten ohne den entsprechenden mhag-mismatch. ein ha-2-und ein ha-3-mismatch waren jedoch mit einer erhöhten inzidenz an akuter gvhd und daher mit einer geringeren ü berlebensrate verbunden. patienten/spenderkombinationen mit einem hy-mismatch profitierten mit einem leichten ü berlebensvorteil (48 % vs. 40 %). interessanterweise wirkten sich mehrere mhag-mismatches positiv auf das langzeitüberleben aus. vier jahre nach szt betrug das gesamtüberleben von patienten ohne mismatch 44 %, von patienten mit einem mismatch 43 %, von patienten mit 2 mismatches 55 % und von patienten mit 3 mismatches 75 % (allerdings nur 8 patienten). in ü bereinstimmung mit diesem ergebnis betrug die inzidenz der nonrelapse-mortalität von patienten ohne mismatch 50 %, von patienten mit einem mismatch 54 % und von patienten mit zwei bzw. drei mismatches 36 % bzw. 14 %. eine erklärung für dieses ergebnis könnte der graft-versus-leukämie effekt sein, der durch die erhöhte anzahl an spender t-zellen induziert wird, die verschiedene hämatopoetisch-restringierte mhag auf den residuellen leukämiezellen des patienten erkennen und diese zerstören. schlussfolgerungen. die bestimmung von mhag in patient und spender vor der szt wäre eine sinnvolle maßnahme zur zusätzlichen risikoabschätzung von komplikationen, vor allem aber ü berleben nach szt. "stuhltransplantation" als erfolgreiche therapie bei schwerer idiopathischer enterocolitis grundlagen. die normale humane darmflora umfasst etwa 100 billionen bakterien, von denen der großteil mit konventionellen methoden nicht kultivierbar ist. störungen in der zusammensetzung der darmflora scheinen bei der entstehung von darmentzündungen und infektionen eine wichtige rolle zu spielen. die fäkale bakterientherapie (stuhltransplantation) zielt auf die wiederherstellung einer gestörten darmflora durch ü bertragung von stuhl eines gesunden in den darm eines erkrankten. diese therapie wird erfolgreich bei therapierefraktären clostridium difficile infektionen eingesetzt. fallbericht und methodik. eine 16-jährige patientin wurde aufgrund eines schädelhirntraumas intensivmedizinisch behandelt. unter einer mehrfachen, antibiotischen therapie in kombination mit steroiden entwickelte die patientin eine schwere enterocolitis mit ausgeprägter diarrhoe (bis zu 7 litern stuhl pro tag). die histologischen und endoskopischen befunde der darmentzündung zeigte das bild einer graft versus host disease (gvhd) artigen entzündung. eine abklärung auf mögliche ursachen insbesondere auf infektiöse erreger blieb negativ. im verlauf nach 8 wochen kam es zu einer spontanen besserung der entzündung im dünndarm, im dickdarm persistierte jedoch die schwere colitis. in den histologischen proben aus dem colon zeigte sich ein fast kompletter verlust des epithels. als ursache der colitis wurde eine störung der darmflora durch die antibiotische therapie angenommen und deshalb bei der patientin eine stuhltransplantation durchgeführt. als stuhlspender wurde die gesunde mutter der patientin herangezogen, diese wurde zuvor auf übertragbare infektionskrankheiten getestet. der stuhl wurde mit kochsalzlösung verflüssigt und filtriert. im anschluss wurde bei der patientin eine coloskopie durchgeführt und insgesamt 400 ml des flüssigen spenderstuhls in das ileum und colon appliziert. ergebnisse. die stuhltransplantation führte bei der patientin in der endoskopischen kontrolle nach 1 woche zu einer rapiden besserung vor allem des histologischen befundes. ebenso kam es zu einem schrittweisen rückgang des stuhlvolumens mit normalisierung der stuhlfrequenz nach 6 wochen. in der kontrolle nach 2 monaten war der endoskopische und histologische befund im colon normal. im weiteren beobachtungszeitraum nach 5 monaten ist es zu keiner neuerlichen reaktivierung der idiopathischen enterocolitis gekommen. schlussfolgerungen. die gvhd-artige idiopathische enterocolitis nach antibiotikatherapie scheint durch eine persistierende störung der darmflora mitbedingt zu sein. die stuhltransplantation war bei der beschriebenen patientin mit diesem schweren krankheitsbild eine sehr effektive therapiemaßnahme. tissue specific epigenetic memory: differentiation capacity of mesenchymal stem cell is restricted to their tissue of origin human mesenchymal stem and progenitor cells (mspcs) are currently evaluated in clinical trials for bone and marrow regeneration and their immune modulation potential. mspcs from virtually all tissues appear indistinguishable regarding immunephenotype and multipotent differentiation capacity in vitro. improvements of limited clinical efficiency are hampered by a lack of understanding mspc functionality in vivo. here we demonstrate that the capacity of in vivo endochondral bone formation followed by infiltration of hematopoietic components can be used as a surrogate to determine in vivo mspc-multipotentiality. mspcs from bone marrow (bm), adipose tissue (at) and umbilical cord (uc) have been isolated. comparative analysis of immune-phenotype, adipo-, chondro-and osteogenic differentiation potential in vitro as well as expression analysis of key mesenchymal lineage genes were performed. epigenetic profiling of mspcs was done using a methylation array. in vivo differentiation capacity was tested by transplanting mspcs subcutaneously into immune-compromised mice. the developmental sequence of chondrogenic and osteogenic as compared to perivascular mesenchymal tissue formation was analyzed. formation of a marrow niche with establishment of the complete host hematopoiesis was studied. secondary transplants of mspcs were performed and analyzed equally. mspcs from all tissues analyzed show an almost identical immune-phenotype using a mspc marker profile. osteo-and adipogenic differentiation potential in vitro as well as gene expression can not distinguish tissuespecific mspcs. mspcs from all tissues except bm lack in vitro chondrogenic differentiation potential using stringent 3d-chon-drogenesis assays followed by appropriate histological staining. in vivo studies could strengthen these findings, because only bmderived mspcs were able to generate bone through an endochondral ossification process with subsequent invasion of mouse marrow. these results correlate with the epigenetic status of the cells since bm-derived mspcs cluster separately in principalcomponent-analysis (pca), whereas mspcs from all other tissues cluster together. bm was the only tissue containing mspcs with multipotent differentiation capacity. this is reflected by a bmspecific epigenetic profile that differs from all other tissues analyzed. since cartilage formation is an essential developmental process important for bone generation and hematopoiesis attraction, an epigenetic predisposition of bm-mspcs to undergo endochondral ossification makes these cells unique for bone and marrow regeneration purposes. grundlagen. die hemmung des renin-angiotensin-systems (ras) entweder mit ace-hemmern oder mit angiotensin-rezeptor-blockern (arb) verlangsamt die progression unterschiedlicher chronischer nierenerkrankungen und der chronischen transplantatdysfunktion. ras-blockierung kann auch durch eine direkte renin-inhibierung erreicht werden, die aber infolge einer verminderten produktion der protektiven angiotensin ii-spaltprodukte wie z. b. angiotensin (ang)(1-7) zusätzliche effekte haben kann. methodik. im fischer-lewis nierentransplantationsmodell der ratte wurde der effekt von aliskiren (10 mg/kg/tag) auf die entstehung der chronischen transplantatdysfunktion im vergleich zu einer behandlung mit vehikel oder mit dem arb candesartan als bekannter hemmer der chronischen transplantatdysfunktion untersucht. analysiert wurden die histologie der niere und der verlauf der proteinurie, der serum-spiegel von ang (1-7) und der spiegel von angiotensinogen im harn als indikator für die intrarenale ras-aktivität. ergebnisse. im gegensatz zu candesartan verminderte aliskiren weder die klinischen (proteinurie, kreatinin-clearence) noch die histologischen zeichen (glomerulosclerose, interstitielle fibrose, makrophagen-infiltration) der chronischen transplantatdysfunktion. candesartan verbesserte im vergleich zu vehikel-und aliskiren-behandelten gruppen sowohl die proteinurie als auch den histologieschaden. die mit aliskiren behandelten ratten zeigten einen verminderten serum-spiegel des protektiven ang(1-7) und einen höheren harn-angiotensinogen-spiegel im vergleich zu den mit candesartan behandelten tieren. schlussfolgerungen. der renin-blocker aliskiren verminderte das fortschreiten der chronischen transplantatdysfunktion nicht. der fehlende schutzeffekt hängt wahrscheinlich entweder mit der verminderten produktion des protektiven ang(1-7) oder mit einer ineffektiven intrarenalen ras-blockierung zusammen. kein unterschied im gehalt an energiereichen phosphaten in organen von lebendspendern, hirntoten spendern und "non-heartbeating" spendern im tiermodell schlussfolgerungen. zusammenfassend sind unterschiedliche mirna-signaturen mit zellulärer oder humoraler abstoßung sowie dgf assoziiert. die identifizierten mirnas und target-gene werden neue sichtweisen auf die molekulare regulation von transplantatabstoßung und transplantatversagen erlauben und so den weg für neue therapieansätze ebnen. darüber hinaus könnten die identifizierten mirnas und target-proteine in zukunft als neue diagnostische marker eingesetzt werden. correlation of recipient factors with the course of lymphocytes after alemtuzumab induction in renal transplantation background. alemtuzumab, an anti-cd52 t-cell and b-cell depleting monoclonal antibody, used as induction therapy in renal transplantation (kts). the recovery of lymphocytes after alemtuzumab induction has been investigated in a number of trials, however, the clinical course after ktx has not been correlated with lymphocyte recovery. herein, we correlate the outcome as well as recipient factors with lymphocyte recovery after induction with alemtuzumab. methods. single center retrospective analysis of 225 patients/consecutive kidney transplantations between 01/ 2004 and 12/2010 which received alemtuzumab as induction agent (one dose of 30mg). patients were devided into 4 groups according to lymphocyte recovery at 4 points of time (pre-tx, 1-3 weeks post-tx, 3 weeks-3 months post-tx and 3-6 months after ktx). the relevance of recipient-characteristics was analyzed. delayed kidney graft function (dgf) was defined as requirement for more than one dialysis within the first week after ktx. statistical analysis was performed with spss 17.0 software (spss inc., chicago, il, usa). analysis of variance for repeated measurements with measurement time as withinsubject factor and with age, cmv status, dgf status as between subject factors were performed. results. median age of all recipients was 49.63 years, 65.33% were male. mean lymphocyte counts were 22.8 ae 9.41% pre-tx, 2.61 ae 3.11% between week 1 and 3, 6.98 ae 6.7% between 3 weeks and 3 months after tx and 18.20 ae 11.48% at later time points. among all factors analyzed, dgf, cmv status and age showed a significant correlation with lymphocyte counts. dgf occurred in 27.56% of the recipients. the lymphocyte-counts in the dgfgroup were significantly higher, 10.7% vs. 13.13% (p ¼ 0.036) post-tx. cmv-status of the recipient influences the quantity of lymphocytes pre-tx significantly (p ¼ 0.009). age showed a significantly influence on the lymphocyte count 3 months post-tx (p ¼ 0.032). conclusions. cmv-status and age have a significant impact on lymphocyte recovery after alemtuzumab induction therapy. lymphocyte counts early after transplantation represent a prognostic factor for kidney function early after transplantation. a detailed analysis of phenotype and function of lymphocytes after alemtuzumab induction together with a correlation with the clinical course is warranted. belatacept-based immunosuppression in de novo liver transplant recipients: 1-year experience from a phase ii study background. calcineurin inhibitors contribute to substantial toxicities in liver transplant (lt) patients that can lead to renal dysfunction/failure and cardiovascular (cv) disease. this phase ii study evaluated belatacept versus tacrolimus (tac) in de novo lt recipients. methods. 250 lt recipients were randomized to belataceptbased (high dose [hd] or low dose [ld]) or tac-based regimens. all patients received steroids for the first 3 months. results. demographic characteristics were similar among groups; 46% of patients were hcv positive. the primary endpoint (composite of acute rejection [ar], graft loss [gl] , and death by month 6) occurred more frequently in belatacept groups. by year 1, more deaths and gl occurred with belatacept ld vs. other groups. causes of early (by 6 weeks) death and gl were mostly due to postoperative complications; thereafter causes included sepsis, ptld, and multi-organ failure. ar was more common with belatacept-based regimens compared to tac þ mmf. by 1 year, mean cgfr was 15-34 ml/min higher in belatacept vs. tac groups. lower blood pressure and less neurotoxicity were observed with belatacept treatment. 2 ptld cases and 1 pml case (hd group) occurred in the belatacept groups. conclusions. belatacept hd group had comparable efficacy relative to tac alone but was less effective compared to tac þ mmf. belatacept ld group was less effective with more deaths and gl observed compared to both tac-based groups. belatacept provided improved renal function with less cv/metabolic and neurotoxicities vs. tac-based regimens. ptld and pml were observed with belatacept. the optimal dose/regimen of belatacept in lt is yet to be determined. cmv late phase-induced mtor activation is essential for efficient virus replication in human m2-polarized macrophages human cytomegalovirus (cmv) remains one of the most important pathogens following solid-organ transplantation, potentially leading to cmv disease, allograft dysfunction, acute and chronic rejection and opportunistic infections. mounting evidence indicates that mammalian target of rapamycin (mtor) inhibitors may decrease the incidence of cmv infection in renal transplant recipients. here we aimed to elucidate the molecular mechanisms of this effect by employing a human cmv (hcmv) infection model in human macrophages, since myeloid cells are the principal in vivo targets of hcmv and the major viral source during early cmv disease. here we demonstrate that polarization of macrophages into m1 and m2 macrophages resulted in highly divergent host cell permissiveness for hcmv with optimal infection susceptibility in m2 versus m1 macrophages. employing an ultra-high purified hcmv stock (tb-40/e) selective rapamycinindependent induction of ifn-transcripts, but no proinflammatory cytokines or early signalling events including mapk and mtor signalling could be detected. assessment of viral gene expression and mtorc1 activity during the course of macrophage infection revealed that rapamycin significantly suppressed cmv replication 3 and 5 days post infection, while cmv proliferation in fibroblasts was largely unaffected by mtor-blockade. analysis of mtorc1 activation and late phase viral proteins including pul-44 and pp65 signified an exquisite mtorc1 dependency of protein synthesis during the late phases of viral replication. collectively, these data indicate that mtorc1 is essential for virus replication during late phases of the viral cycle in myeloid cells which might explain the potent anti-cmv effects of mtor inhibitors after organ transplantation. background. brain death (bd) triggers inflammatory signals and graft injury. clinically, reduced organ quality as a result of brain death contributes to inferior graft and patient survival. regulatory t-cells are primarily known for their control of the immune response. we analyzed cd25þ/foxp3þ t-cell subpopulations in recipients of either brain dead and native donor hearts. to dissect the role of donor immune competent cells in communicating inflammatory signals following bd we performed syngeneic cardiac transplants from wildtype and immune deficient donors. methods. hearts from brain dead wild type (bd/wt) c57bl6 mice and rag2/double knockout (bd/ko) mice were procured 3 hrs following brain death induction and transplanted into wt recipients. hearts originating from native c57bl6 mice served as controls. by 3 days, t-lymphocyte subpopulations (cd4, cd8, cd4/cd25/foxp3) were assessed by flow cytometry and graft-specific changes were assessed by ihc and rt-pcr. results. blood pressure remained stable following bd induction and intragraft cell infiltration was comparable in all groups (p ¼ n.s.). cardiac isografts from bd/wt donors demonstrated pronounced cellular infiltrates; only few cells infiltrated hearts from living wt donors (p ¼ 0.025). of note, cd4/cd25/foxp3 regulatory t-cells counts were significantly elevated in recipients of bd/wt grafts (p ¼ 0.04). recipients of bd/ko hearts featured reduced cd4 þ t-cell infiltrates, but comparable foxp3 levels (p ¼ n.s.). in contrast, bd/ko heart recipients showed significantly lower il-6, ifn-, and tnf--levels (p ¼ 0.03, 0.005, 0.01). conclusions. inflammatory events after brain death are counterbalanced by significantly elevated rates of cd4/cd25/ foxp3 reg. t-cells. less pronounced consequences of brain death in hearts originating from immune deficient donors suggest a critical role of immune competent cells in communicating inflammatory signals. lipocalin-2 as a therapeutic agent in chemotaxis during ischemia and reperfusion injury following solid organ transplantation background. neutrophil gelatinase-associated lipocalin (ngal/lcn-2) expression is associated with ischemia/reperfusion injury (iri) following transplantation and correlates with polymorphonuclear cell infiltration. to get insight into the regulatory role of lcn-2 during iri the expression of different chemokines and adhesion molecules were analyzed in a murine heart transplantation model. methods. the murine heterotopic heart transplant model also implying exogenous i.p. application of lcn-2 was used for in vivo experiments. the mrna expression of the chemokines mip-2, lix, kc, mcp-1, il-6 and ccl-6 and their receptors cxcr2 and ccr2 as well as icam-1 was analyzed by qpcr. immunohistochemistry was performed in heart sections and correlated with neutrophil infiltration at various time points (2, 12, 24 and 48 h). results. significant lower granulocyte infiltration and serum creatinine kinase levels during iri were observed in the lcn-2-/transplants correlating with a stable icam-1 expression compared to the lcn-2 wt setting (>5fold expression at 2 h of reperfusion). in the early phase of reperfusion (2 h) mcp-1, kc, lix and mip-2 showed a lower expression pattern in the lcn-2-/transplants with delayed upregulation at 12 h (lix, mip-2). after i.p. lcn-2 application no significant difference in apoptosis was observed. the number of infiltrating granulocytes was reduced after application of the iron-loaded lcn-2 compared to iron-free lcn-2. conclusions. our data point to a possible chemotactic role of lcn-2 which may also affect the expression of particular chemokines in the early phase of iri. the role of the iron binding capacity of lcn-2 in chemotaxis during iri is still unknown. understanding these regulatory mechanisms will be crucial to establish treatment strategies for iri during solid organ transplantation. glomeruläre effekte des mtor-inhibitors rapamycin in der nephrotoxischen serumnephritis grundlagen. der mtor inihibitor rapamycin ist ein immunosuppressivum, welches klinische anwendung zur prävention von abstossungsreaktionen bei nierentransplantierten patienten gefunden hat. einer der limitierenden faktoren bei der klinischen anwendung ist das de novo auftreten von proteinurie. ziel der arbeit war es, die glomerulären veränderungen der rapamycininduzierten proteinurie in einem experimentellen modell der nephrotoxischen serumnephritis (nts) näher zu untersuchen. methodik. nts wurde in c57bl/6 mäusen induziert und die gabe von rapamycin in einer dosis von 0,5 mg/kg 14 tage nach induktion begonnen. die mäuse wurden am tag 35 sakrifiziert und glomeruli zur genexpressionsanalyse isoliert. außerdem wurde mittels immunhistochemie die glomeruläre expression von t-zell-(cd4), makrophagen-(f4/80, cd68), podocyten-(nephrin), sowie endothelzellmarkern (cd31) untersucht. ergebnisse. mäuse, die mit rapamycin ab tag 14 behandelt wurden, entwickelten eine signifikant erhöhte proteinurie im vergleich zu den kontrolltieren. dies war mit einer verstärkten glomerulären infiltration von cd4-positiven (mittlere intraglomeruläre positive zellen/50 glomeruli 4,43 ae 0,68 vs 2,23 ae 0,57; p < 0,05), f4/80-positiven (8,2 ae 1,58 vs. 3,64 ae 0,74; p < 0,05) und cd68-positiven zellen (18,80 ae 3,05 vs. 9,15ae 1,61; p < 0,05; n ¼ 13 rapamycin, n ¼ 12 vehikel) assoziiert. keine unterschiede fanden sich in der glomerulären färbung von nephrin und cd31. real-time pcr der aus glomeruli gewonnenen rna zeigte, dass die rapamycin-behandlung zu einer verstärkten expression der proinflammatorischen zytokine il-6 (12,0 ae 5,2 vs. 1,0 ae 0,1; p < 0,05) und tnf-(3,4 ae 1,0 vs. 1,0ae 0,1; p < 0,05) führte, während sich hierbei keine hinweise auf eine schädigung der endothelzellen ergab. außerdem fand sich eine gesteigerte glomeruläre expression des transkriptionsfaktors foxp3. bei tx-patienten/innen findet man eine bis zu 200-fache erhöhung der sog. ,non-melanoma skin cancer'-gruppe und eine 5-fache steigerung der melanom-inzidenz im vergleich zur normalbevölkerung. das klinische bild dieser neoplasien ist nicht selten atypisch, das verhalten aggressiv und die prognose ungünstig. die intensität bzw. dauer der immunsuppression und die wahl der immunsuppressiven medikamente in verbindung mit der aktuellen und zurückliegenden uv-exposition korreliert dabei mit der tumorentstehung. besonders wichtig sind regelmäßige nachsorge-untersuchungen post transplantation, welche in speziellen dermatologischen ambulanzen durchgeführt werden sollten. hierbei stehen heute moderne hauttumor-therapieformen (,targeted' therapy und immunmodulatoren) zur verfügung. background. we compared steady state pharmacokinetics of mycophenolate mofetil -myfenax + (teva) and cellcept + (roche) -in stable kidney transplant recipients (ktr). methods. this was an international, multi-centre, randomized, open-label, two-treatment, two-sequence crossover study with a 3-months follow-up. we included ktrs at least 12 months post-transplantation with stable renal graft function for at least 3 months. the maintenance treatment consisted of mmf in combination with tacrolimus with or without steroids. at the end of the two treatment periods 6-hour or 12-hour pharmacokinetic studies of mycophenolic acid (mpa) were performed. results (0-6h) , and cmin of mpa were within the bioequivalence margins and cmax was marginally outside of these margins in this set of patients. the numbers and types of adverse events were not different between the two treatments. conclusions. the steady state pharmacokinetics of mpa as well as adverse events are comparable for myfenax and cellcept in tacrolimus treated stable kidney transplant recipients. belatacept in de novo kidney transplant recipients -10-year experience in a single center background. belatacept is a co-stimulation blocker which has recently been approved as immunosuppressive therapy in renal transplant recipients. here we assess the outcome of patients that have been treated with belatacept for 10 years after kidney transplantation. methods. in our center, 22 patients were enrolled in the phase ii multi-center belatacept trial that started in 2001. patients were randomly assigned to belatacept-(n ¼ 14) or cya-(n ¼ 8) based immunosuppression (all patients received basiliximab, mmf and steroids). in this retrospective analysis we report the outcome of the belatacept group as of june 2011. patient and graft survival, the incidence of acute rejection, kidney function (calculated gfr; mdrd [ml/min/1.73 m 2 ]) and cardiovascular risk profiles (triglycerides and cholesterol) are presented. results. at an average of 9.2 (8.5-10.1) years after kidney transplantation 9 out of 14 belatacept (64%) patients are still on therapy. five patients discontinued the study: 1 due to lack of efficacy (atg-resistant rejection), 1 due to ptld, 1 due to pneumocystis carinii pneumonia and 2 patients withdrew consent (1 and 3.5 years after transplantation with a functioning graft; gfr: 65.4 and 46.05 ml/min/1.73 2 ). three patients died: the patient with ptld and the patient with pneumocystis carinii pneumonia shortly after discontinuation and the patient with lack of efficacy 6 years later due to cardiac arrest. two patients developed biopsy-proven acute rejection (2/14; 14.3%) (banff iia, banff iib). in 8 out of 9 patients kidney function remained stable over time: mean gfr 12 months after kidney transplantation 59.64 (sd 10.6), mean egfr at the time of the last visit: 59.32 (sd 11.2). one patient had an impaired kidney function with a gfr of 45.03 at the last visit (gfr at month 12: 68). mean serum triglycerides and cholesterol at 12 months after transplantation were 167 (sd 70) and 207 (sd 34) respectively, and changed little over time (mean triglycerides: 159 (sd 44) and cholesterol: 201 (sd 44) at the last visit). conclusions. in this selected group of patients enrolled in a phase ii trial, continuous use of belatacept therapy for 10 years is associated with stable long-term graft function in a high percentage of patients. the innsbruck hand transplant program: update at 11 years after the first transplant background. we describe here the outcome after two bilateral hand, one bilateral forearm and one unilateral hand transplantation at 11/8/5 and 2 years after transplantation. methods. four patients received a bilateral hand (n ¼ 2), a bilateral forearm (n ¼ 1) or a unilateral hand transplant between march 2000 and july 2009. induction therapy with atg (n ¼ 2) or alemtuzumab (n ¼ 2) was followed by tacrolimus, prednisolone, mmf (n ¼ 3) or tacrolimus and mmf (n ¼ 1) maintenance is. later, sirolimus/everolimus was added under simultaneous withdrawal (n ¼ 2) or dose reduction (n ¼ 1) of tacrolimus (n ¼ 1) or mmf (n ¼ 1). steroids were avoided in one and withdrawn in two patients. results. range of motion reached up to 70% of normal with a grip strength of 2-10 kg. hand function correlated well with time after transplant and amputation level. intrinsic hand muscle function recovery and discriminative sensation were observed in all patients. complications included cmv infection, fungal infection, hypertension, hyperglycemia, transient creatinine increase and headache. three, six, four, and one rejection episode were successfully treated with steroids, anti-cd25, anti-cd52 antibodies and/or intensified maintenance is. skin histology at current shows no or mild perivascular lymphocytic infiltrates without signs of progression. vessels are patent without signs for luminal narrowing or intimal proliferation. conclusions. the overall functional outcome and patient satisfaction after bilateral hand, bilateral forearm and unilateral hand transplantation are highly encouraging. all patients are now free of rejection with moderate levels of is. renal function efficacy measures following conversion from calcineurin inhibitors to sirolimus after cardiac transplantation in patients with renal insufficiency methods. this was a randomized (1:1), comparative, multinational, phase 4 study of patients 1-8 years posttransplant. patients on cni therapy with gfr 40-90 ml/min/1.73 m 2 were eligible for inclusion. exclusion criteria included acute rejection (ar) within 3 mos or proteinuria >500 mg/d. primary end point was 1-yr change from baseline in creatinine clearance (clcr). results. a total of 114 patients were randomized and treated (srl 57; cni 57); mean baseline gfr was $57 ml/ min/1.73 m 2 . in the itt locf analysis, 1-yr adjusted mean change from baseline in clcr was þ 4.0 and à1.2 ml/min/ 1.73 m 2 for srl vs. cni, respectively (p < 0.001); for the ot analysis, values were þ4.7 and à2.1, respectively (p < 0.001). ar rates were numerically higher in the srl group; there was 1 ar with hemodynamic compromise in each group. a significantly higher (33.3% vs. 0%; p < 0.001) treatment discontinuation rate due to aes was seen in the srl group. most common treatment-emergent aes that were significantly higher in the srl group were diarrhea (28.1%), rash (28.1%) and infections (47.4%). conclusions. conversion to srl from cni therapy resulted in improved renal function in cardiac transplant recipients with renal insufficiency, but was associated with an attendant ar risk and higher discontinuation rate due to aes. background. atg-induction therapy after heart transplantation is still controversial and used only by less than 50% of centers. moreover, there exist no data about the optimal dosage of atg-induction. the aim of this study is to compare different doses of atg-induction. methods. between 1996 and 2009 586 cardiac transplants were performed in our center. 523 (89%) patients with full data sets were included in the analysis. the median age was 56 years, 21% (n ¼ 112) were female. the median follow-up-time was 98 months. patients were divided into 3 different groups according to total atg dose: group a: ¼ 4.5 mg/kg vs. group b: 4.5-7.5 mg/ kg vs. group c: >7.5 mg/kg. survival, incidence of rejection, infection, graft vasculopathy and cancer were compared by kaplan-meier-analyses (log rank test). results. there was better early (12 m) and late (150 m) survival in group b (a: 80%, 43%; b: 90%, 65%; c: 88%, 58%; p ¼ n.s.), however, the difference was not significant. freedom from treated acute rejection was better in group b (88%) compared to a and c (79%, 80%, p ¼ 0.08). signs of histological rejection were significantly different between the groups (a: 25%, b: 18%, c: 33%; p ¼ 0.03). group b had the lowest incidence of severe infection (a: 37%, b: 21%, c: 51%; p < 0.01). cmv infection incidence was higher in group c (35%) compared to groups a, b (20%, 23%; p < 0.01). there was no significant difference in freedom from graft vasculopathy between the groups (a: 91%, b: 85%, c: 79%; p ¼ n.s.). the incidence of cancer was similar in all atg groups (a: 3%, b: 7%, c: 11%; p ¼ n.s.). conclusions. different doses of atg-induction seem to have a significant impact on the outcome of heart transplantation. there is a strong need for more studies on optimization of atg therapy. nighttime kidney transplantationa risk or a need? background. kidney transplantation is performed as emergency surgery also as a nighttime procedure to reduce cold ischemia time und therefore improve outcomes after kidney transplantation. however, surgical procedures performed during nighttime in the context of 24 hour shifts and sleep deprivation have been associated with a higher rate of complications than elective surgery. the aim of this retrospective analysis is to determine the impact of nighttime surgery on the outcome after kidney transplantation. methods. all kidney transplants from cadaveric donors performed at our center from january 2000 through january 2010 (n ¼ 873) were included in this retrospective study and grouped according to the time of surgery: daytime (from 8 a.m. to 8 p.m., n ¼ 608) versus nighttime (from 8 p.m. to 8 a.m., n ¼ 265). statistical analysis compared patient and graft survival in the two groups, rate of delayed graft function and acute rejection as well as surgical complications. results. 5-year patient survival rates of 87% and 85% and 10-year patient survival rates of 73% and 69% in daytime and nighttime kidney transplants did not show any significant difference. also, graft survival at 5 and 10 years did not differ significantly (85% versus 85% and 69% versus 60%) in the two groups. delayed graft function occurred in 31% of daytime transplants compared to 37% of nighttime procedures (p ¼ 0.06). acute allograft rejection was observed in 23% of daytime graft recipients compared to 18% of nighttime graft recipients (p ¼ 0.13). furthermore, nighttime procedures were associated with a 23% risk of surgical complications which was not significantly different from daytime surgery (22%, p ¼ 0.7). conclusions. nighttime kidney transplants are not associated with a higher surgical complication rate than daytime procedures. nighttime kidney transplant procedures have the same outcome as daytime transplants and are necessary to keep cold ischemia time as short as possible. ursolic acid a constituent of dwarf elder (sambucus ebulus) inhibits surface expression of endothelial adhesion molecules and prevents intimal hyperplasia in an in vivo model for bypass surgery background. in a recent study for the identification of compounds capable of inhibiting vcam-1 expression we isolated ursolic acid (ua) from extracts of dwarf elder. herein, we analyse the mechanism of action and the in vivo applicability of the compound in a setting for venous bypass graft intimal hyperplasia. methods and results. our analyses indicate that ua does not interfere with the adhesion molecule expression pathway at or upstream of nfk-b, as nfk-b translocation and nfk-b mediated transcription were not affected by ua. however, ua inhibited adhesion molecule protein synthesis suggesting an inference with the translation of adhesion molecules. in an in-vivo rat model for vein graft disease, ua-containing sambucus extracts inhibited endothelial vcam-1 expression, induced cell death in smooth muscle cells, and inhibited intimal hyperplasia in a rat model for venous bypass graft disease. conclusions. our results suggest that the inhibition of vcam-1 expression constitutes a valuable target for drug discovery in the field of cardiovascular research. ua may be an interesting agent in the prevention of vein graft disease and graft failure. the killer-cell immunoglobulin-like receptor (kir) genotype correlates with acute kidney failure in the early post-liver transplantation period acute renal failure (arf) is a major complication following liver transplantation (lt) leading up to chronic end-stage renal disease. the etiology of post-lt impairment is multifactorial, but it is suggested that e.g. during ischemia initial insults provoke morphological and functional changes within the vascular and tubular epithelium. because it has been demonstrated that ischemic arf can occur in the absence of classical t cell function and that natural killer (nk) cells can kill syngeneic tubular epithelial cells in vitro, we aimed to elucidate the role of nk cells and their receptors in the context of early post-liver transplant ari and arf more precisely. patients with impaired kidney function (serum creatinine >1.2 mg/dl, n ¼ 13) illustrated heightened peripheral nk cell frequencies prior to lt compared with patients showing stable renal function (n ¼ 9) (17.22% ae 10.56 versus 12.98% ae 9.09). we further tested retrospectively 89 liver transplant recipients for their killer-cell immunoglobulin-like receptor (kir) genotype and the risk of ari/arf. during the first week post-lt, ari occurred in 12% and arf in 22% of the patients. ari was a significant risk factor for acute rejection (p ¼ 0.0009) and arf led to elevated serum creatinine levels (>1.2 mg/dl) at the time of hospital discharge (p ¼ 0.008). interestingly, significantly less patients having a homozygous kir haplotype a/a (char-acterized by the presence of only one activating kir gene) displayed a stable early postoperative kidney function, compared to patients with a kir haplotype b/x (more than one activating receptor) (p ¼ 0.025, odds ratio 2.3, ci ¼ 1.3-3.9). moreover, the absence of kir2dl2/ds2 genes significantly influenced the risk of arf (p ¼ 0.05). a multivariate regression model of clinical and genomic risk factors for ari/arf confirmed a link between the kir haplotype a/a and post-lt acute renal failure (p ¼ 0.04). in summary, we observed a higher percentage of nk cells prior to lt in patients with impaired renal function and identified the kir haplotype a/ a as an independent genetic risk factor for arf within the first postoperative week. our data provide new aspects of an innate immune response within the setting of post-lt kidney injury and failure. background. new-onset diabetes after transplantation (nodat) is a serious complication after kidney transplantation affecting graft and patient survival. currently, no guidelines exist about the ideal management of renal transplant patients with impaired glucose tolerance (igt, blood glucose >140 and < 200 mg/dl) who are at highest risk for developing manifest diabetes. here, we studied the efficacy and safety of vildagliptin and pioglitazone in patients with igt after renal transplantation. methods. after routine oral glucose tolerance testing (ogtt) in our center, 48 stable renal transplant patients with igt were recruited in this double blind, prospective study and randomized 1 : 1 : 1 to receive either 50 mg vildagliptin, 30 mg pioglitazone or placebo once daily for 3 months. after treatment ogtts were performed and hba1c levels along with body mass index (bmi), metabolic and safety parameters were evaluated. results. so far 45 patients have finished the study. 2 hour glucose values improved in all three groups with the greatest mean improvement in the pioglitazone group. fasting glucose values also showed the strongest improvement in the pioglitazone group as well as hba1c (vildagliptin: 0.079% ae 0.249%, pioglitazone: à0.153% ae 0.318%, placebo: þ0.056% ae 0.294%). bmi increased in the pioglitazone group and decreased in the vildagliptin and placebo group. the difference in hba1c between pioglitazone and placebo reached marginal statistical significance at p ¼ 0.05. safety analysis showed no significant influences on glomerular filtration rate (gfr). pioglitazone led to significant reductions in gpt and gamma-gt as well as increases in hdl. adverse events occurred at similar rate in all three groups and were generally mild and reversible. conclusions. vildagliptin and pioglitazone are both safe and effective in patients after renal transplantation. however, only pioglitazone led to significant improvements in glycemic control. furthermore, pioglitazone led to improvements in gfr and lipid metabolism besides causing the largest weight gain. while further studies with longer observation periods are urgently required, pioglitazone seems to possess efficient beta-cell protective potency. clinical relevance of preformed complement-and non-complement-fixing hla alloreactivity in cardiac transplantation s. mahr 1 , m. wahrmann 2 , a. zuckermann 1 , g. böhmig 2 background. there is increasing evidence for a role of alloantibody-mediated rejection in organ transplantation. solid phase hla antibody detection using bead array technology may help identify patients at risk of rejection and graft loss. methods. in this retrospective monocentric cohort study we evaluated 229 consecutive heart transplant recipients (transplantation between 2000 and 2006; immunosuppression: atg induction and calcineurin inhibitor-based maintenance therapy) for the presence of preformed (complement-and non-complement-fixing) hla alloantibodies. sera obtained immediately before transplantation were screened by flowpra, and test-positive sera were subjected to luminex-based single antigen testing including a test modification for detection of in vitro c4d deposition. results. seventeen recipients (7.3%) were found to have preformed igg type donor-specific alloantibodies (dsa), five of them with c4d-fixing capability. the presence of dsa was related to retransplantation and previous pregnancies, but not associated with prior implantation of a ventricular assist device. evaluating clinical endpoints, we found an association between dsa and acute rejection (>grade 1a according to the ishlt 1990 grading system; no dsa: 17%; [igg]dsa: 33%; [igg/c4d] dsa: 60%). however, in our study cohort, sensitization had no effect on long-term survival rates (5-year transplant survival: 72% vs. 92% vs. 80%) or rates of chronic transplant vasculopathy (20% vs. 18% vs. 20%). moreover, none of the dsa-positive patients was in need of extracorporeal membrane oxygenation, and the duration of post-transplant intensive care did not differ between groups. conclusions. in conclusion, our data point to a relationship between preformed donor-specific alloreactivity and acute rejection. however, possibly as a result of our local immunosuppressive regimen, which also includes induction therapy with a depleting anti-lymphocyte antibody, such reactivity did not influence long-term allograft outcomes. background. so far, no data is available on the use of everolimus in pediatric cardiac transplantation. we present the first results of everolimus in pediatric heart transplantation. methods. from 2000 to 2008, htx was performed in 86 children aged below 17 years. 52 of these children received continuously either mycophenolat mofetil (mmf) (group a, n ¼ 28) with standard cyclosporine a (csa) exposure or were randomized to receive everolimus (eve) (group b, n ¼ 24) with reduced csa exposure in combination with steroids. during a follow-up period of 24 months, efficacy with regard to acute rejection, allograft vasculopathy and survival as well as safety with regard to myelosuppression, infection, tumors, lipids and body growth was studied. results. mean post htx csa exposure was significantly lower in eve treated children compared to mmf at month 6 (167 ng/ml versus 284 ng/ml), month 12 (mean 134 ng/ml versus 235 ng/ml) and month 24 (79 ng/ml versus 76 ng/ml). mean daily dose of eve was 1.16 ae 0.56 mg/m 2 body surface area resulting in trough levels of 4.8 ae 1.3 ng/ml by 24 months of follow-up. two cases in each group encountered biopsyproven acute rejection >grade 2r (7% versus 8%; p ¼ 0.043). cardiac allograft vasculopathy at month 12 was detected in 4 children (16%) with eve compared to 18% in mmf. kidney function deteriorated in both groups during the first month, recovering after 24 months to a glomerular filtration rate of 95.1 ae 0.3 ml/min/sqm for eve versus 88.9 ae 5.2 ml/min/sqm for mmf. rates of myelosuppressive disorders (anaemia, leukopenia, thrombocytopenia), infections and hyperlipidaemia was low and comparable in both groups. children with eve did not encounter inferiority in body growth. 1 child with eve died following intractable ventricular fibrillation during myocardial biopsy compared to 5 children with mmf (2 deaths due to allograft rejection). conclusions. everolimus with reduced csa combined with steroids in paedriatic cardiac transplant recipients enables high efficacy and is comparatively safe to immunosuppression with mmf and standard exposure of csa. transplantation of a minor-mismatched skin graft elicits a rapid humoral response including the induction of antigen-specific ige background. sensitization to major and minor antigens is a critical problem in transplantation medicine with antibodies of igm, iga and igg isotypes have been demonstrated towards major (i.e. mhc) and minor (i.e. non-mhc) antigens. in a new transgenic mouse model we investigated humoral antigenspecific responses towards a highly immunogenic non-mhc antigen. a transgenic mouse expressing the well-characterized major grass pollen allergen phl p 5 ubiquitously on the cell surface was generated. methods. a phl p 5-transgenic balb/c mouse was generated by pronuclear injection integrating phl p 5 (and gfp) fused to a leader peptide and a transmembrane domain. splenocytes (2.5â106 per mouse), cell extracts containing mainly membrane proteins and recombinant (r) phl p 5 [5 mg ae al(oh)3 per mouse] were injected subcutaneously into naïve balb/c mice (n ¼ 8 per group). furthermore tail skin of phl p 5-transgenic mice was grafted onto naïve balb/c (n ¼ 13 in two independent experiments). the phl p 5-specific humoral response was determined in sera by elisas and t-cell proliferation assays were assessed. results. surprisingly, a prompt rejection of phl p 5-transgenic skin was elicited (within 8-10 days), accompanied by a strong phl p 5 specific antibody response including phl p 5-specific ige. additionally to the skin grafted group, mice receiving splenocytes or rphl p 5 plus adjuvant showed a comparable response in terms of phl p 5-specific ige throughout the whole follow-up (week 1, 2, 3, 5, 7, 9, 11) suggesting an unusually strong immune response to cell or tissue-bound phl p 5. furthermore phl p 5-specific igg isotypes, iga and igm were induced. besides splenocyte-proliferation assays showed phl p 5 specific t-cell responses in all groups of mice that showed strong humoral responses. conclusions. the high immunogenicity of tissue-bound phl p 5 may represent a new stringent model for studying humoral responses towards non-mhc antigens. notably, the immune reaction included a rapid ige response in this model. the influence of recipient age on chimerism-based tolerance induction k. hock 1,2 , r. oberhuber 2,3 , y.-l. lee 2 , t. wekerle 1 , s. g. tullius 2 background. immune senescence substantially alters alloreactivity. higher frequencies of memory t cells (tmem) found in older recipients are considered a substantial barrier to tolerance induction. tolerance induction through mixed chimerism holds promise for clinical translation but has only been investigated in young recipients so far. as the average recipient age has increased substantially in clinical organ transplantation, we investigated the consequences of recipient age on the outcome of costimulation blockade based allogeneic bone marrow transplantation (bmt) for the purpose of mixed chimerism and tolerance induction. methods. young (2 months; weighing approx. 20 grams) and old (12 months; 25 g) recipients (c57bl/6) were treated with 3 or 1 gy total body irradiation (tbi, d-1) and received adjusted to the body weight 20â10 6 and 25â10 6 un-separated balb/c bm cells (d0) and co-stimulatory blockade with anti-cd154 mab (d0) and ctla4ig (d þ 2) was administered. lymphocyte subsets and cytokine production were compared between young and old naïve mice and multilineage chimerism was followed by flow cytometry. results. old mice contain significantly more cd4 (p < 0.05) and cd8 (p < 0.001) memory t cells (cd44highcd62llow), early activated cd4 t cells (cd4 þ cd69 þ ; p < 0.01), less cd4 and cd8 t cells and comparable amounts of regulatory t cells (tregs; cd4 þ cd25 þ foxp3þ; p ¼ n.s. vs. young animals). moreover, older cd4 and cd8 t cells release more ifn-(cd4: p < 0.05), il-2 (not cd8 t cells), il-6 (cd4: p < 0.05), il-10 and tnf-. chimerism developed earlier in old recipients: within one week most older recipients became chimeric following an irradiation with 3 gy tbi and co-stimulatory blockade (17/18 vs. 9/18 chimeras, d þ 7 and 16/17 vs. 12/17, d þ 14). old recipients became even chimeric with a reduction of the total body irradiation to 1 gy. in sharp contrast, none of the young recipients became chimeric under those conditions (4/8 vs. 0/8 chimeras in young recipient, p < 0.05, d þ 30). conclusions. recipient age is linked to a faster donor bm engraftment and chimerism. moreover, chimerism is attainable with a lower dose of irradiation. those results support the clinical relevance of the chimerism strategy for tolerance induction. background. in allogeneic bone marrow transplantation (bmt) donor t cells have pleiotropic effects. surprisingly, cotransplanting high doses of donor t cells with donor bm causes rejection of donor bm despite costimulation blockade. therefore, in the present study we investigate the molecular mechanisms responsible for this seemingly paradoxical phenomenon. methods. recipients (c57bl/6) were treated with 3 gy tbi (d-1) and received approximately 20â106 unseparated balb/c bm cells (d0) and costimulation blockers anti-cd154 mab (d0) and ctla4ig (d þ 2). 30â10 6 balb/c, cb6f1 (balb/câb6), irradiated balb/c or c3h cd4 t cells (isolated by macs) were co-transplanted in addition. groups either received anti-il-6, anti-ifn-, anti-lfa1 mab or rapamycin. multilineage chimerism was followed by flow cytometry and cytokine release was analyzed. results. co-transplantation of 30â10 6 cd4 t cells but not cd8 t cells triggered rapid bm rejection of donor bm under costimulation blockade within one week in an otherwise successful protocol (0/13 vs. 17/20 chimeras, p < 0.001). the levels of il-6, ifn-, il-17a (p < 0.05) and tgf-were found to be higher in mice treated with additional donor t cells. the neutralization of il-6, but not of ifn-resulted in a significantly higher rate of chimerism induction compared to controls (5/7 vs. 0/5 chimeras; p < 0.05). the injection of cb6f1 or irradiated balb/c cd4 t cells did not abrogate chimerism (5/6 and 4/5 vs. 0/4 chimeras with balb/c t cells; p < 0.05), whereas c3h cd4 t cells induced bm rejection (0/5 vs. 9/9 chimeras bmt, p < 0.001). the additional treatment with rapamycin or anti-lfa1 overcame the negative effect of donor t cell injection (5/5 and 6/6 vs. 0/4 chimeras; p < 0.01). conclusions. the abrogation of bm engraftment through cotransplantation of donor cd4 t cells involves il-6, requires proliferative capacity of the co-transplanted t cells and needs to recognize the recipient as allogeneic. neutralisation of il-6, rapamycin and anti-lfa1 overcome the effect of co-transplanted donor cd4 t cells and offer potential targets for therapeutic intervention in costimulation blockade-resistant rejection. background. mixed chimerism is an effective strategy for the induction of transplantation tolerance, however, translation from murine models to the clinical setting is challenging. one hurdle is the high frequency of alloreactive memory t-cells (tmem) found in the (pre)clinical setting, which is not present to a similar degree in mouse models. to better model this clinical reality, we have developed a murine model in which the transfer of 3â10 7 t-cells from sensitized mice inhibits the induction of chimerism and tolerance in a well-characterized costimulation blockade-based bone marrow transplantation (bmt) model. here, we evaluated whether treatment with clinically approved drugs (rapamycin, anti-lfa-1mab) can overcome costimulationresistant rejection triggered by memory cells and tried to gain insight into the underlying mechanisms. methods. t-cells sensitized towards donor (3â10 7 tmemenriched t-cells) were adoptively transferred into groups of naïve b6 mice prior to bmt (d-7). bmt recipients received 1gy total body irradiation (d-1), 20â10 6 fully mismatched balb/c bone marrow cells (d0) and costimulation-blockade consisting of anti-cd154mab (1 mg, d0) and ctla4ig (0.5 mg, d þ 2). groups additionally received rapamycin (0.1 mg/d, d-1/0/2) or anti-lfa-1mab (0.1 mg/d, d-1/2). multilineage chimerism and deletion of donor-reactive t-cells were followed by flow-cytometry and in vitro tolerance was assessed by mlr. transferred t-cells were followed in vivo using cd45.1/2 alleles as congeneic markers in peripheral blood (d-2/12) and lymphoid organs (d7). ifngamma-production upon stimulation with donor antigen was measured by elispot and intracellular flow-cytometry. results. rapamycin and anti-lfa-1 were able to overcome costimulation-blockade resistant rejection and induced stable multi-lineage chimerism in pre-sensitized recipients (0/7 control vs. 7/7 rapa, p < 0.001; 3/7 anti-lfa-1, p ¼ 0.2; representative for multiple experiments). chimeras showed central and peripheral deletion of donor-reactive t-cells and donor-specific hyporesponsiveness in vitro. the frequency of transferred t-cells decreased over time, but less so in the rapamycin-treated group. ifngamma-production was reduced by rapa or anti-lfa-1 treatment. conclusions. rapamycin and anti-lfa-1 overcome the additional t-memory-cell barrier for tolerance induction, although rapamycin seems to be more potent in tmem tolerization in this model. the trend that elimination of tmem is lower in the rapamycin-treated group and central deletion is more pronounced early after bmt suggests different mechanisms for tmem tolerization. lunchsymposium virusinfektionen in der organ-und stammzelltransplantation 035 diagnostik von virusinfektionen in transplantationspatienten patienten nach organ-oder knochenmarkstransplantationen werden immunsuppressiv behandelt und weisen daher ein anderes spektrum von potentiell gefährlichen virusinfektionen auf als immunkompetente personen. vor allem viren, die im normalen wirt nur leichte oder asymptomatische infektionen verursachen und latent im organismus verbleiben, wie herpesviren (zytomegalievirus, epstein-barr virus etc.) oder auch polyomaviren (bk-, jc-virus), können unter immunsuppression hoch replizieren und zu schweren und potentiell tödlichen infektionen führen. um schwere infektionen oder reaktivierungen durch verschiedene viren zu verhindern, wird heute routinemäßig in regelmäßigen abständen im verlauf nach transplantation der direkte und quantitative nachweis verschiedener viren (vor allem von zytomegalievirus) mittels pcr im blut, aber auch in verschiedenen anderen patientenmaterialien durchgeführt. ziel der virusdiagnostik ist es hier nicht so sehr eine klinisch relevante virusinfektion nachzuweisen, sondern vielmehr das vorhanden-sein einer signifikanten virusreplikation frühzeitig, noch vor krankheitsbeginn zu erkennen. das ist dann auch die basis für die "präemptive" antivirale therapie, die gegeben wird, wenn die viruslast eine bestimmte höhe überschreitet, aber noch bevor klinische symptome auftreten. die festsetzung der virologischen grenzwerte fü r den einsatz der präemptive therapie ist aber eine große herausforderung, da vor allem herpesviren wie zytomegalievirus (cmv) oder epstein-barr virus (ebv), aber auch polyomaviren auch ohne jede krankheitsrelevanz im organismus nachgewiesen werden können. neben den quantitativen pcr ergebnissen hängt der einsatz einer präemptiven therapie auch von bestimmten weiteren aspekten ab, wie vom donor/recipient virus-serostatus, dem transplantierten organ oder vom material in dem man die virusnukleinsäure nachweist. daher ist ein enges zusammenspiel von virologischer befunderstellung, interpretation und der kenntnis der klinischen aspekte notwendig, um eine optimale virologische kontrolle der einzelnen patienten zu erreichen. antivirale therapie bei patienten nach organ-oder hämatopoetischer stammzelltransplantation klinische abteilung für pädiatrische hämato-/onkologie, universitätsklinik für kinder-und jugendheilkunde, medizinische universität graz, graz, österreich zahlreiche humanpathogene viren persistieren nach primärinfektion im körper und können unter geschwächter abwehrlage reaktiviert werden. zu diesen zählen die herpesviren herpes simples 1 und 2 (hsv 1, 2), cytomegalievirus (cmv), epstein-barr-virus (ebv), varicella zoster virus (vzv) und hhv-6 sowie das parvovirus b 19 (pvb19), das adenovirus (adv), die hepatitisviren b (hbv) und c (hcv) und die polyomaviren bk-virus (bkv) und jc-virus (jcv). diese und andere viren (z. b. enteroviren) können bei empfängern von organoder stammzelltransplantationen durch primärinfektion oder reaktivierung zu schweren krankheitsverläufen ("virus-sepsis") und immunologischen prozessen wie transplantatabstoßung oder graft-versus-host disease führen. neben unspezifischen maßnahmen wie reduktion der immunsuppression, gabe von immunglobulinen und immunmodulation mittels interferon, wurde in den letzten jahren eine zunehmende zahl antiviraler substanzen verfügbar. deren antiviraler effekt beruht auf unterschiedlichen mechanismen: hemmung der aufnahme der viren in die wirtszelle (amantadin, pleconaril), inhibierung der replikation des viralen genoms (aciclovir, ganciclovir, cidofovir, foscarnet, ribavirin), unterdrückung der freisetzung von viruspartikeln aus der wirtszelle (oseltamivir, zanamivir). all diese substanzen wirken virustatisch und nicht viruzid, können also die virusvermehrung hemmen, nicht jedoch die viren gänzlich eliminieren. während einige substanzen relativ spezifisch in die replikationsmechanismen einzelner viren eingreifen (aciclovir, ribavirin, oseltamivir, zanamivir), zeigen andere substanzenmeist jene mit breiterem wirkspektrum (cidofovir, foscarnet) -auch eine vermehrte aktivität innerhalb humaner, molekularer mechanismen, was zu einer beträchtlichen zahl unerwü nschter arzneimittelwirkungen fü hrt (v. a. nephro-, myelo-, neurotoxizität). handelsname dna-viren rna-viren ciprofloxacin** ciproxin þ (in vitro) wirkspektrum ausgewählter virustatika (ohne anti-retrovirale substanzen) wirksamkeit entspricht nicht den zugelassenen indikationen * experimentell, nicht verfügbar ** experimentell einige substanzen haben im tierversuch ein carcinogenes (ganciclovir, cidofovir) und/oder teratogenes (ganciclovir, cidofovir, ribavirin) potenzial gezeigt. unter zu geringer dosierung und/oder mangelhafter medikamenteneinnahme kann es zu viralen mutationen und -daraus resultierend -zur resistenzentwicklung kommen. tabelle 1 gibt einen ü berblick über die wichtigsten virustatika und deren wirkspektrum. viele dieser substanzen sind jedoch für die anwendung im "transplantations-setting" nicht offiziell zugelassen. in zukunft sind weitere, teilweise dzt. noch experimentell eingesetzte therapieoptionen zu erwarten: so scheint ciprofloxacin als gyrasehemmer auch gegen polyomaviren wirksam zu sein, mit cmx001 wird eine wirksamere und nebenwirkungsärmere weiterentwicklung von cidofovir derzeit klinischen tests unterzogen. reaktivierungen von persistierenden virusinfektionen sind häufige komplikationen nach transplantation. je nach risikokonstellation reicht das klinische spektrum von einem harmlosen infekt bis zu einem lebensbedrohlichen infektionsverlauf. die häufigsten pathogene sind cytomegalievirus (cmv), epstein-barr virus (ebv) und adenoviren (adv). die möglichkeiten der virostatischen pharmakotherapie sind meist limitiert. alle drei virusinfektionen haben gemeinsam, dass fü r eine elimination eine suffiziente t-zellimmunität notwendig ist. darauf baut die rationale eines adoptiven immuntransfers auf, bei dem ag-spezifische t-zellen von einem gesunden spender in einen erkrankten empfänger infundiert werden. hierbei ist das therapieziel eine adaptive immunität im empfänger zu induzieren. im rahmen einer risikoadaptierten therapie wurde bei refraktären virusreaktivierungen nach transplantation dieses therapieverfahren angewendet. eine ag-spezifische t-zellantwort konnte im empfänger gegen immundominante epitope (pp65 fü r cmv, hexonprotein fü r adv und ebna1 fü r ebv) induziert werden um den empfänger vor virus-assoziierten komplikationen zu schü tzen. background. we investigated the safety and efficacy of t and b cell depleted peripheral stem cells from full haplotype mismatched parental donors in pediatric patients. methods. use of the clinimacs system and cd3/cd19 coated magnetic microbeads resulted in a 4 log depletion of t cells and allowed to cotransfuse high numbers of donor nk cells (median: 107â10 6 /kg). tbi or busulfan based myeloablative regimens or a melphalan based intensity reduced regimen were used. all patients underwent intensive pretreatment according to current study protocols; 41/106 already received previous allogeneic transplantations. the diagnoses were: acute leukemias and mds (n ¼ 60), solid tumors (n ¼ 32) and nonmalignant diseases (n ¼ 14). results. primary engraftment occurred in 89% of patients. after tli based reconditioning and second haploidentical stem cell donation, final engraftment was achieved in 100%. median time to reach >500 neutrophiles/ml and independence from platelet substitution was 10 (8-15) and 9 (5-59) days respectively. 35% of patients had no gvhd, 36% had grade i, 23% had grade ii and 4% had grade iii. chronic limited and extensive gvhd was observed in 8 and 11%. transplant related mortality was 0% at day 100 and 8% at one year. event free survival at 3 years was 66% for patients with leukemias in any cr and 80% for patients with nonmalignant diseases. over all survival at 2 years was 20% for patients with solid tumors. relapse or progression were the major causes of death. thus, pilot studies with il-15 stimulated grafts and posttransplant donor-nk cell infusions were initiated and are currently ongoing. conclusions. transplantation of cd3/cd19 depleted haploidentical stem cells resulted in a fast recovery of neutrophiles and platelets. engraftment rates similar to that of patients with myeloablative standard conditioning and positive selected stem cells could be achieved, possibly due to a graft facilitating effect of cotransfused nk cells. the regimen helped to minimize trm, despite intensive pretreatment (including previous transplantation). however, relapse remains a major problem and further immunotherapeutic elements have to be evaluated. antibody based immunotherapy combined with haploidentical stem cell transplantation for high risk neuroblastoma background. pediatric patients with relapsed metastatic neuroblastomas have a poor prognosis and additional therapeutic strategies are warranted. we present preliminary results with haploidentical stem cell transplantation and posttransplant immunotherapy with an anti-gd2 monoclonal antibody (ch14.18 cho). methods. t and b cell depleted haploidentical stem cells in combination with a toxicity reduced conditioning regime (melphalan, thiotepa, fludarabine and okt3, now substituted by low dose atg + fresenius) were used for transplantation. in a subsequent clinical trial, we evaluate the feasibility and safety of antibody infusions against the neuroblastoma antigen gd2: 6 cycles of mab ch14.18/cho (20 mg/m 2 infusion for 5 days; in cycles 4-6, 1â10 6 units/m 2 interleukin 2 (il-2) is given additionally on days 6, 8 and 10). results. haplotransplantation without subsequent antibody infusions resulted in a 2-year over all survival of 28%. trm at day 100 was 0%, single cause of death was relapse/ progression. thus, transplantation itself seems to be not sufficient for most patients but may be a platform for further immunotherapies on the basis of antibody dependent nk cell mediated cytotoxicity and complement lysis. 12 patients have been enrolled in our ch14.18 study and received a total of 42 antibody cycles. side effects were: inflammation symptoms (pain 42/42, fever 36/42 and crp elevation 35/42 cycles), which decreased or disappeared by increasing cycle numbers; accommodation disturbance (n ¼ 3), seizures (n ¼ 2), loss of weight (n ¼ 2). during antibody infusions, endogenous secretion of il-2 in the patients was significantly increased (880 u/ ml prior vs. 1580 u/ml post), which resulted in an also significantly increased stimulation of natural killer (nk) cells in peripheral blood (measured by cd69 expression). predominantly adcc exerting nk cells (with expression of the fc-gamma receptor cd16) were stimulated. in 3 out of 5 investigated patients, nk cell and complement mediated anti-tumor effects against neuroblastoma cells were detectable. 5 patients finished the protocol so far and were evaluable after 6 cycles. 3/5 patients responded and reached a complete remission or improved their partial remission, 2 patients progressed. conclusions. preliminary results of our ongoing study suggest an anti-tumor effect of the donor derived immune system in vitro and in vivo. sct from a matched sibling donor (msd) is the treatment of choice for children with saa, however limited by donor availability. immunosuppression (is) as the other option has the disadvantage of high treatment failures. 7 patients with saa without a msd and refractory to previous is, median age 11 years (5-16), median interval from diagnosis to sct 12 months (3-155) and median number of transfusions before sct 55 (18-116) underwent alternative donor sct. donors were matched unrelated (mud) (n ¼ 4), mismatched unrelated (n ¼ 2) and haploidentical (n ¼ 1). conditioning regimens contained cyclophosphamide and either muromonab-cd3 or anti-thymocyte globulin (atg) in 7/7, thiotepa (tht) in 6/7, fludarabine (flu) in 5/7 and total lymphoid irradiation in 2/7 patients. grafts were either cd34þ selected and/or cd3/19 depleted using the miltenyi clinimacs device. median yield of purified cd34þ cells was 10.17â10 6 /kg (7.85-24.3) and median cd3þ number was 5.5â10 4 /kg (0.84-10). all patients had wbc-engraftment on median day 10 (8-12). there was no gvhd prophylaxis in 2 patients and either cyclosporine-a or mycophenolate mofetil up to day þ 60 in 5 patients. 1 mud-hsct recipient who had 6 is courses and the longest interval to hsct (13 yrs) and who received the highest cd 3þ dose (10â10 4 /kg) developed gvhd (grade ii) on day þ 125 with progressive kidney failure due to microangiopathic hemolytic anemia. all children are alive with a median follow up of 20 months (8-149) with stable complete engraftment and stable chimerism of median 98.5% (90.65-100). conclusions. long interval to transplant, multiple transfusions and long term immunosuppression before sct are associated with poor alternative donor sct-outcomes in saa. cyclophosphamide conditioning including flu, tht and atg but without tli, high doses of purified cd34þ cells and/or cd3/19 depletion may prevent graft rejection and gvhd and hasten engraftment. this may facilitate the decision for the earlier use of unrelated stem cells preventing complications from prolonged and multiple immunosuppressive therapies and presumably increasing the survival of patients with saa without a matched sibling donor. extracorporeal photopheresis in patients with high bleeding risks background. for anticoagulation in extracorporeal photopheresis (ecp) to prevent clotting in the extracorporeal circuit, the manufacturer's recommendation is the use of heparin (normally 10,000 units). however, patients with acute graft-versushost disease (agvhd) after allogeneic hematopoietic stem cell transplantation are at high risk for bleeding complications due to low platelet counts, intestinal lesions from agvhd or impaired hepatic function. for these patients, alternative anticoagulation using acid citrate dextrose (acd-a) has been infrequently used in small patient cohorts. methods. we investigated the safety and efficacy of this approach in 94 consecutive patients (43 male, 51 female) with agvhd (45% with gut gvhd) undergoing ecp with acd-a anticoagulation at a single institution 2 to 3 times per week on a weekly basis until complete resolution of agvhd. a total of 1242 ecp procedures were analyzed with respect to side effects and changes in haemoglobin and platelet levels. moreover, in a proportion of ecp treatments activated partial thromboplastin time (aptt) was monitored. priming of the separator was performed with 1,000 units of heparin followed by anticoagulation with acd-a at a ratio of 1:10 during the procedure. results. ecp was tolerated well by all patients. in only 0.2% of procedures mild citrate reactions seen as transient paresthesias were observed but resolved without the need for calcium substitution. in no case bleeding complications were noted during or after citrate anticoagulation. during ecp, aptt levels increased marginally from a median of 32.1 sec. (range, 25.3 to 44.5 sec) before to a medium of 35.4 sec. (range, 25.4 to 54.7 sec.) after ecp, respectively. at start of ecp 51% of patients had platelet counts <100 g/l and 26% <50 g/l, respectively. haemoglobin and platelet counts decreased by 11% and 14%, respectively. conclusions. in conclusion, citrate anticoagulation during ecp is a feasible and safe alternative for patients with high risk for bleeding complications, especially for those after allogeneic hematopoietic stem cell transplantation. allogeneic stem cell transplantation with reduced intensity conditioning in patients with therapy-related myeloid neoplasms background. therapy-related myeloid neoplasms (t-mns) occur as late complication after cytotoxic therapy for malignant and non-malignant disorders. in most patients with t-mns, allogeneic haematopoietic stem cell transplantation (hsct) is the only potentially curative approach. performed with myeloablative conditioning this option is associated with a high transplantrelated mortality (trm). here we report our results in using reduced intensity conditioning (ric) in these patients. methods. between july 2000 and february 2011, 18 patients (male: 5; female: 13; median age: 48.5 years; range: 28-66) with t-mns underwent ric hsct either from a matched sibling (n ¼ 8) or a matched unrelated (n ¼ 7) or one antigen mismatched unrelated donor (n ¼ 3). primary disorders were solid tumors and haematologic malignancies in 9 patients each. patients presented with t-mds (n ¼ 4), t-aml (n ¼ 13) and t-mds/mpn (n ¼ 1). cytogenetic analysis revealed clonal aberrations in 15/18 patients. nine patients were transplanted in 1st cr, one in 2nd cr, five in pr and three in relapsed/refractory disease. furthermore, five patients showed persistant primary disease. conditioning regimen consisted of fludarabine/melphalan (n ¼ 15) or fludarabine/ low-dose tbi (n ¼ 3). gvhd prophylaxis consisted of cyclosporine and mycophenolate mofetil and additionally anti-thymocyte globulin in unrelated transplantations. results. after a median follow-up of 31 months (range: 3-97) 9 patients are alive and in cr. causes of death were: relapse (n ¼ 4), sepsis/multiorgan failure (n ¼ 4) and gvhd (n ¼ 1). acute and chronic gvhd was observed in 6 and 5 patients, respectively. the estimated os is 53.8% at one and 47.9% at three years; the dfs is 48.1% at one and three years, respectively. of 5 patients transplanted with active primary neoplasms, 2 are alive at 1 year showing cr of both primary and secondary neoplasms which was associated with the development of chronic gvhd. conclusions. the results of ric hsct in these patients with poor risk disease are encouraging with respect to trm, relapse rate and overall survival. our data show a more favourable outcome compared with previous reports using a myeloablative conditioning. furthermore, allogeneic hsct should be considered as a potentially curative strategy for patients with t-mns and persistant primary malignancy. the bone component of cta gives rise to donor hscs which migrate to recipient thymus and differentiate to mature t cells background. composite tissue allotransplantation (cta) is immunologically unique in that it represents the only type of graft to include a vascularized functional bone marrow component. here we studied if the bone component of a composite tissue graft represents the source of hscs that differentiate in the thymus and thereby reconstitute a functional immune system (cd3þ t cells in peripheral blood/lymphoid organs) in immuno-deficient b6/scid recipients rather than originate from donor mature passenger t cells that expand in the host. methods. b6 (wt/nude) murine composite tissue grafts (osteomyocutaneous or myocutaneous) were transplanted heterotopically to b6 (wt/scid) recipients using a non-suture cuff technique for revascularization. flow cytometry of peripheral blood (cd3þ, cd19þ) was performed at pod 7, 14, 21, 28, 56. in addition, histopathology (h&e) and immunohistochemistry of tissues was performed at indicated time points. to assess immunocompetence, allogeneic skin grafts (balb/c) were transplanted to either naïve b6/nude, naïve b6/scid or b6/scid mice that prior received a b6/nude cta. results. the surgical success rate was 85% in all groups. as expected no cd3þ cells and no rejection of skin allografts were detected in b6/nude and b6/scid controls. b6/scid mice that received b6/nude osteomyocutaneous flaps demonstrated b and t cell immunity from pod 7 and 21 respectively. the percentage of cd3þ and cd19þ cells within peripheral blood mononuclear cells steadily increased to 57.7% and 17.1% respectively at pod 56. allogeneic skin allografts were rejected 2 weeks after transplantation. however, no b and t cell reconstitution was observed in b6/scid mice receiving b6/nude myocutaneous flaps (without bone component). conclusions. the vascularized bone marrow component of cta provides an effective source of hscs to restore immunocompetence in t-and b-cell deficient mice. this might also contribute to chimerism induction and maintenance after cta and facilitate the clinically observed immunoprivilege of ctas. early in vivo signaling events involved in neo-vasculogenesis via stem cell transplantation using proteomic profiling background. the precise mechanisms regulating human neo-vasculogenesis and organ regeneration are still unclear. it has been shown that neo-vasculogenesis can be induced in immunocompromised nod/scid/il-2-receptor gamma-knockout (nsg) mice in vivo by transplantation of human bone marrowderived mesenchymal stem and progenitor cells (hmspcs) and umbilical cord-derived endothelial colony-forming progenitor cells (hecfcs), whereas transplantation of pure hmspc or hecfc cells lack the capacity to form stable functional vessels (reinisch et al., blood, 2009) . understanding the activity of the mediators of neo-vasculogenesis would provide us with tools to develop strategies for therapeutic intervention as well as angiogenesis and regenerative applications. methods. autologous pairs of mspcs and ecfcs were transplanted subcutaneously in matrigel plugs at a ratio of 20:80 into nsg mice and implants were harvested 1 day post transplantation to investigate proteomic profiling using kam 1.3 antibody microarray (www.kinexus.ca), testing over 800 signaling and phospho-proteins. the state of vessel formation and stability of the transplants were verified by immunohistochemistry of the explants 2 and 8 weeks post transplantation. results. protein microarray data analysis revealed significant alteration in the expression and activity of components 1) regulating apoptosis, mitotic check point control, and centrosome structure; 2) modulating glycolysis and the coordinated expression of cyto-protective genes; and 3) mediating cell adhesion, migration and tissue invasion. selected targets are currently being validated by western blotting to allow for the development of novel therapeutic intervention strategies. a detailed expression and interactive network analysis of targets will be presented and discussed. conclusions. our data confirm that more than one purified cell type is needed for tissue engineering in vivo and suggest that composite cellular transplantation may be useful for future therapeutic strategies. proteomic profiling unraveled at least three distinct but partially overlapping signaling networks involved in the complex process of vascular regeneration. understanding the origin and activity of the mediators of vessel formation and repair would provide us with tools to develop and further optimize novel cell transplantation strategies. oxygen sensing of mesenchymal stem and progenitor cells promotes neo-vasculogenesis in vivo background. vascular regeneration requires a stringent interaction of somatic endothelial colony-forming progenitor cells (ecfcs) with mesenchymal stem and progenitor cells (mspcs). hypoxia in ischemic tissue is a key factor driving the revascularization machinery. because ecfcs, despite hypoxic stimulation, only form patent vessels in vivo in the presence of mspcs, we hypothesized that mspcs play a decisive role in oxygen sensing during vasculogenesis. methods. adult human ecfcs were isolated directly from whole venous blood and mspcs from human bone marrow aspirates. pooled human platelet lysate entirely replaced fetal bovine serum during cell culture. progenitor cell phenotype, long-term proliferation, molecular cellular response, wound repair as well as migratory and vasculogenic functions were monitored under reduced oxygen levels (5% o 2 ), severe hypoxia (1% o 2 ) and standard culture conditions (20% o 2 ). ecfc and mspc interaction in vivo and the influence of protein synthesis were studied in immune-deficient nsg mice after subcutaneous injection with matrigel. immune histochemistry and tunel assays were performed on explants in the time course after transplantation. results. in vitro ecfc and mspc proliferation was reduced with declining oxygen levels, while the absolute colony number remained unchanged. ecfc vascular wound repair and vascular-like network formation in vitro improved with escalating oxygen supply. ecfcs stabilize hypoxia-inducing factor-1 (hif-1) only under 1% o 2 , while mspcs stabilize hif-1 under 1% o 2 as well as 5% o 2 conditions. in a mouse model, injected ecfcs underwent apoptosis after 1 day and attracted mouse leucocytes. in vivo co-cultured ecfcs and mspcs formed perfused human vessels 7 days after transplantation. perivascular cells, but not ecfcs, in vivo were positive for hif-1. inhibition of mspc, but not ecfc, protein synthesis and hif-1 stabilization prior to co-implantation blocked vessel formation. conclusions. these data indicate that hypoxic ecfcs alone are not able to function in vitro and form patent vessels in vivo. mspcs react to the low oxygen environment and promote ecfcs to form vessels at least in part by rescuing ecfcs from hypoxia-induced apoptosis. this suggests that oxygen sensing mspcs are a key factor in stem cell transplantation and regenerative medicine. background. low sensitivity and specificity rates for the detection of invasive pulmonary mycosis result in a major cause of mortality among immunosuppressed children. we sought to determine the accuracy of percutaneous computed tomographyguided biopsy in children with cancer and hematopoetic stem cell transplantion. methods. we retrospectively reviewed 17 imaging-guided percutaneous biopsies of 17 children for suspicious lesions detected by computed tomography. thirteen were being treated for hematologic malignancies, three for solid tumors, one for immunodeficiency; 47% had received allogeneic bone marrow or peripheral stem cell transplants. the accuracy of the percutaneous lung biopsy was determined by subsequent surgical resection, autopsy, or clinical course. results. histopathological studies showed 11 biopsy specimens with septate hyphae, indicating a mold, including 6 with aspergillus, 4 with mycoraceae, 1 with aspergillus and mycoraceae colonies in culture; 2 specimens revealed candida, 1 saccharomyces. the remaining 3 biopsies revealed bronchiolits obliterans pneumoniae. invasive pulmonary mycosis was detected by percutaneous biopsy with 100% sensitivity and 100% (14/14) specificity. percutaneous biopsy results influenced the surgical decision in 21% (3 of 14) and changed the treatment option in 78% of the cases. pneumothorax complicated the biopsy in one patient. conclusions. percutaneous computed tomography-guided biopsy is an accurate technique for the diagnosis of invasive pulmonary mycosis in children. it reveals the local epidemiology, correctly determines the therapeutic anti-mycotic agent and influences the choice of prophylaxis for invasive mycosis. methods. we now report for the first time a modified implantation technique by cannulating the right atrium in order to reach better hemodynamics for right side support. to place the heartware system in the area of the right atrium we have created a cavity (8â8 cm) in the right-sided pericardium using a ptfe membrane. results. the design of the heartware device enables a quick and less invasive implantation. the small size of the pump allows for intrapericardial placement even in biventricular support. to prevent compromise of the right atrium it is necessary to build a cavity in the pericardium for pump placement. conclusions. adequate pump flow was observed during total support time. the presented implantation technique allows a safe and elegant bridging-to-transplant in htx candidates representing biventricular failure. use of cardiopulmonary bypass for lung transplantation: institutional experience background. the use of cardiopulmonary bypass (cpb) for lung transplantation is still judged controversial. however, in 30% of lung transplantations cpb support during the surgical procedure is required for hemodynamic and/or respiratory instability or for repair of intracardiac pathologies. this study aimed to determine if the use of cpb has an effect on survival. methods. a retrospective review of 190 lung transplants in 180 patients (mean age: 59 years ) with different lung pathologies who underwent bilateral or single lung transplantation between november 1993 and june 2011 was performed. 75 patients (39.4%) in whom elective as well as emergent cpb was necessary were compared with 115 cases (60.6%) without cpb. results. the indications for lung transplantation were chronic obstructive pulmonary disease (copd, n ¼ 111), fibrosis (n ¼ 32), alpha-1 antitrypsin deficiency emphysema (n ¼ 13) and others (n ¼ 34). the follow up period ranged from 2 days to 14 years. total mean survival was 980 days (range 0 to 5276 days). there was no significant difference in mortality between the cpb and the control group (p ¼ 0.723). the comparison between copd patients and other indications for lung transplantation also revealed no significant difference in survival (p ¼ 0.11). there was also no difference between copd patients in whom surgery was performed with cpb or without (p ¼ 0.676). survival rates in patients over 60 years were similar to patients under 60 independent from the use of cpb (p ¼ 0.676). there was also no significant difference in the usage of cpb between single versus bilateral lung transplantation (p ¼ 0.44). conclusions. this study revealed no increase in mortality after lung transplantation when cardiopulmonary bypass was required. when cpb appears necessary it should be employed right away to avoid hemodynamic compromise or severe reperfusion edema due to unacceptable pulmonary arterial pressures. erfahrungsbericht aus dem deutschen herzzentrum berlin successful bridging to heart transplantation using the levitronix centrimag system and duraheart lvad in a patient with resuscitation related liver injury n. reiss 1 , l. kizner 2 , u. schulz 2 1 department of cardiac surgery heidelberg, universitätsklinikum heidelberg, germany; 2 heart center north rhine-westphalia, ruhr-university of bochum, bad oeynhausen, germany background. mortality rates from cardiogenic shock after acute myocardial infarction remain extremely high. efforts have been made to develop ventricular assist devices capable of providing complete hemodynamic support in this situation. mechanical circulatory assistance represents an evident problem when bleeding complications occur. we report a very rare case of successful bridging to heart transplantation despite severe resuscitation related liver injury. methods. a 54-year-old male patient underwent failed percutaneous coronary intervention of lad with consecutive prolonged resuscitation. a levitronix centrimag system was implanted via femoral vessels for rapid hemodynamic stabilization. an acute laparotomy was necessary because of severe injury of the left lobe of the liver. during laparotomy the abdominal cave was tamponated by multiple compresses. three times relaparotomy was necessary to achieve final hemostasis. after four weeks of levitronix centrimag support the system was switched to duraheart lvad for long-term assistance as a bridge-to-transplant. results. successful heart transplantation was performed after complete recovery and mobilization of the patient 320 days after the disastrous and hopeless initial situation. conclusions. the present case demonstrates that successful bridging to heart transplantation is possible even in cases of severe bleeding complications. special attention is given to the thin line between bleeding complication and necessary anticoagulation because of mechanical circulatory assist. background. advagraf (tacrolimus extended-release capsules) has been tested in de-novo liver and renal transplant recipients. in cardiac transplantation there exists only data on conversion of tacrolimus to advagraf. the aim of this analysis was to evaluate efficacy and safety of de novo use of advagraf in cardiac transplant patients. methods. 10 patients received advagraf after atg induction therapy, 8.1 ae 3.3 days after cardiac transplantation. mean patient age was 40.1 ae 16.4 years. all patients received also mycophenolate and steroids. follow-up was 4.4 ae 1.7 months. dosages, tacrolimus drug levels (tac) and creatinine (crea) levels were recorded 1, 2 weeks and 1 to six months post transplant. clinical events were defined as acute rejection, infection type, new onset diabetes mellitus (nodm) and renal dysfunction. results. six-month survival was 100%. two patients were converted to cyclosporine due to nodm 1 and 3 months post transplant. no rejection episodes were recorded during follow-up. two infections were documented 6 months after transplant (bacterial pneumonia and cmv infection). advagraf starting dose was 6.2 ae 1.9 mg/d. crea before start was 1.14 ae 0.34 mg/dl. first measured tac levels at steady state (5 days post drug start) were 7.8 ae 3.7 ng/ml. until the end of the first month advagraf was slowly increased to 10.1 ae 1.9 mg/d with tac levels of 10.7 ae 2.8 ng/ml and crea of 1.22 ae 0.31 mg/dl. three and six month drug doses were 9.3 ae 2.11 and 8.2 ae 0.8 mg with corresponding tac levels of 11.2 ae 2.9 and 11.2 ae 1.3 ng/ml. crea was 1.47 ae 0.22 and 1.49 ae 0.32 mg/dl. there were no events of renal failure. conclusions. advagraf de-novo therapy shows acceptable efficacy and safety early after cardiac transplantation. atginduction therapy might be responsible for a low risk of acute rejection despite lower early tac levels. usage of if-inhibitors in regulation of heart rate after heart transplantation background. due to denervation of the heart from n. vagus, patients physiologically have a heart rate of 90-100 beats per minute (bpm) after heart transplantation. inhibiting the funnychannels (if) of the pacing-cells of the sinus node reduces ionic-influx and spontaneous depolarization and hereby decreases the heart rate. to improve myocardial oxygenation by extending diastolic time, reducing the heart rate is an important goal for these patients. objective of the current study is to show the efficacy of the if-inhibitor ivabradine (procoralan + ) on the transplanted heart. methods. this is a single-center retrospective study including 143 patients, who underwent heart transplantation from 1985 to 2011 at our center in vienna and who were in need of a heart rate regulating therapy after transplantation. only patients in stable conditions and with sinus rhythm were treated with ivabradine. an average dose of 10 mg per day was used to control the heart rate. analyzed data were collected during routine checkups in our long-term outpatient-clinic and during ergometry examination by periodical measurements of heart-rate, bloodpressure and ecg. results. mean age at the time of transplantation was 51.3 ae 11.9 years, 117 patients (81.8%) were male (age 16-71 years) and 26 patients (18.2%) were female (age 14-68 years). before establishing the procoralan + therapy, patients had an average heart rate of 96.2 ae 11.9 bpm. with the procoralan + therapy, patients had an average heart rate of 83.1 ae 9.5 bpm. after an average time of 1.8 ae 1.4 years, usage of ivabradine has reduced the heart rate by 14.9 ae 9.4%. in 7 cases (4.8%) we had to terminate therapy because of gastro-intestinal side effects and in 4 cases (2.7%) even under lowest ivabradine dosage patient developed bradycardia. conclusions. using ivabradine after heart transplantation is a safe way to lower heart rate and could become a new indication for if-inhibitors. the individual dosage for each patient has to be found in augmenting the initial daily dosage of 5 mg slowly to the optimum dosage of 10 mg per day. comparison between referral and explant diagnoses in lung transplant recipients: discrepancies and additional findings background. lung transplantation is a widely accepted therapeutic option for a range of pulmonary conditions in which the diagnosis is often based on clinical data or on limited biopsy material. posttransplantation complications and recurrence of underlying disease may be related to the primary disease, and an accurate diagnosis is therefore essential. methods. a pathologic review was performed on 1056 primary lung transplantations over a period of 22 years (1998 to 2010). diagnoses of native lungs were compared with referral diagnoses to assess the presence of discrepancies or expanded results like malignancies or infections. results. 73 (7%) cases presented a different or expanded diagnosis. discrepancies between referral diagnosis and histopathology were found in 34 of 1054 cases (3%). the highest percentage of discordance was depicted in chronic obstructive lung diseases (12 of 344), with the final diagnosis of uip (n ¼ 4), chronic interstitial fibrosis (n ¼ 4), silicosis (n ¼ 2), lam (n ¼ 1) and sarcoidosis (n ¼ 1). 16 patients who were referred with the diagnosis of an interstitial lung disease had predominantly emphysema (n ¼ 12), bronchiectasis (n ¼ 2) and histiocytosis x (n ¼ 2). expanded results included aspergillus (n ¼ 11) and mycobacterial (n ¼ 16) infections, carcinomas (n ¼ 10), cystic adenomatoid dysplasia (n ¼ 1) and carcinoid (n ¼ 1). however, short-and long-term survival was not different in patients with different diagnoses, malignancies or implanted infections. interestingly all mycobacterial infections and all malignancies occurred in patients with copd. conclusions. on account of this high rate of discrepancies and its possible influence on survival, frequently repeated clinicopathologic investigations should be performed during the waiting list period. background. induction therapy with alemtuzumab, followed by lower than conventional intensity post-transplant immunosuppression, has been associated with reduced morbidity and mortality in abdominal and heart transplantation. we performed a prospective randomized trial in lung recipients receiving alemtuzumab in conjunction with reduced immunosuppression compared with patients receiving thymoglobulin in association with routinely dosed immunosuppression. methods. 60 lung tx recipients were prospectively randomized in two groups: group a received alemtuzumab in conjunction with early lower-dose tacrolimus, lower-dose steroids, and half-dose mycophenolate mofetil, compared with group b receiving thymoglobulin in association with routinely dosed immunosuppression. survival analyses examined patient and graft survival, freedom from acute cellular rejection (acr), lymphocytic bronchiolitis, bronchiolitis obliterans syndrome (bos), kidney function, infectious complications and post-transplant lymphoproliferative disorder (ptld). results. there were no significant differences in 6-and 12month survival (alemtuzumab 96% vs. atg 93% and 93% vs. 96%, respectively, p ¼ ns). acute cellular rejection episodes ¼ a2 within the first tx year were significant lower in group a (alemtuzumab 0 vs. atg 0,33; p ¼ 0.019), lymphocytic bronchiolitis was not different between the groups (cumulative b scores group a 2.9 ae 2.7 vs. group b 3.2 ae 2.3 per patient, p ¼ 0.74). there were no significant differences in bacterial (group a 2. conclusions. alemtuzumab induction in conjunction with reduced immunosuppression significantly reduces higher grade rejection rates with comparable survival results and infectious complications to high-dose standard immunosuppression. the incidence of early bos was higher after alemtuzumab-induction. background. cardiac allograft vasculopathy (cav) is a major cause of allograft failure in long-term heart transplant patients. even without typical angina symptoms and diffuse lesions, cav resembles atherosclerosis, but shows more concentric rather than eccentric intimal proliferation. both, proximal and distal portions of the coronary tree are involved. cardiac angiographies are periodically performed in the routine long-term follow up after heart transplantation. methods. cardiac angiographies and interventions have been performed on 19 patients (17 male and 2 female) without any complications and substantial advantage in this cohort of patients. 6 patients already had previous interventions. mean time after transplantation was 11.4 ae 5.8 years. results. follow-up angiography showed no in-stent restenosis in the patients with previous interventions after a mean time of 36 ae 18 months. 3 patients were in need of interventions, 2 of them in ladp3 with endeavour stents and 1 patient in rca1 with cypher stent. no complications have been observed. conclusions. radial angiography is a safer method for routine follow-up for patients after heart transplantation, especially in long-term follow-up, as femoral approach often leads to complications. tation and repeated antigen immunoadsorption (glycosor-babo) until the isoagglutinine titre was less than 1:8. in addition intravenous immunoglobulin (intratect, biotest pharma) was administrated at a dose of 1.0 g/kg body weight after each immunoadsorption. a standard immunosuppressive regimen (tacrolimus with a target level of 10 to 12 ng/ml, mycophenolate mofetil at a dose of 1 g twice a day and prednisolone) with il-2 antibody induction was started approximately two weeks prior to kidney transplantation. results. a total of seven abo incompatible kidney transplantations were performed. patients' characteristics are summarised in table 1 . all patients showed an immediate graft function and their serum creatinine decreased to a median level of 1.69 mg/dl (range: 1.09-3.39 mg/dl) on day 7. in one patient there was an early loss of graft function due to thrombotic microangiopathy despite rescue treatment with bortezomib and eculizumab leading to graft loss on day 21. after a median follow up of 3.7 months, serum creatinine averaged 1.7 mg/dl (range: 1.25-2.27 mg/dl). in the performed renal biopsies there was no evidence of relevant acute or chronic graft injury, all allografts showed c4d positive immunostaining. conclusions. in conclusion, our small case series demonstrated that perioperative isoagglutinine removal in combination with rituximab is an effective and safe protocol for abo incompatible transplantation. therefore we offer abo incompatible kidney transplantation to patients as alternative to cadaveric kidney transplantation to shorten the waiting time on dialysis. background. amyloidosis is a protein misfolding disorder where conformationally changed proteins are pathologically deposited as abnormal insoluble fibrils in distinct tissues potentially leading to morphological and functional disintegration. heredi-tary amyloidosis is a rare autosomal-dominant disorder arising from mutations in several genes like in the fibrinogen a-chain that affect various organs. mutations in the fibrinogen a-chain have been discussed to lead to a predominant renal deposition of amyloid generally leading to esrd. methods. here, we describe a large spanish family with 101 members affected by the rare fibrinogen r554l mutation and isolated renal amyloidosis. results. most patients presented with late-onset gross proteinuria and a variable course of decline of renal function. in contrast to other reported fibrinogen a mutations, extrarenal involvement or accelerated arteriosclerosis employing cardiac magnetic resonance imaging, carotid ultrasound and tissue biopsy was not observed indicating a highly selective process of renal amyloid deposition. histological analysis revealed almost complete obliteration of the glomerular and specifically the mesangial architecture despite moderately reduced kidney function. treatment with angiotensin-converting enzyme and subsequently angiotensin-ii receptor inhibitors did not significantly affect proteinuria. two affected members underwent renal transplantation with the longest surviving graft exhibiting 13 years of survival and graft failure due to surgical and infectious problems, but recurrence of amyloidosis. conclusions. collectively, these results introduce a new perspective on this atypical fibrinogen mutation with significant pathophysiological and clinical impact also with regard to novel therapeutical approaches. methods. 217 enteric drained whole pancreas transplants (ptx) in 208 patients performed at our center during a seven year period were retrospectively analysed. prophylactic immunosuptype ii dm was defined as requirement of insulin or oral antidiabetics in the presence of c-peptide production to maintain blood glucose levels <200 mg/dl. results. actuarial patient, pancreas and kidney graft survival at one year were 96.4%, 88.5% and 94.8%. at last follow up, two patients were lost, 30 patients died, 26 pancreatic grafts and 16 renal allografts were lost. a total of 17 patients were c-peptide positive at the time of ptx; three patients were excluded from analysis due to early graft loss (n ¼ 2) and dm type ii due to partial insufficiency of a previous pancreatic allograft (n ¼ 1). of the 14 study patients (13 men/1 woman with a median age of 56 [range 41-62] years, two died [invasive fungal infection 1, intracerebral bleeding 1]), another two lost their pancreatic graft [hyperacute rejection 1, chronic rejection 1]) but none their renal graft. rejection rate was 28.6%, infection rate 64% and surgical complication rate 50%. median serum creatinine at 5 years follow up was 1.3 (range 0.9-3.6) mg/dl and hba1c was median 3.2 (range 0.9-6.1)%. conclusions. pancreas transplantation seems to be a good therapeutic option of selected patients with type ii dm. due to the fact that these patients are older than their counterparts with type i dm and the fact that we found these patients to have a high infection rate, less intensive immunosuppressive regimens are recommended. ergebnisse. von allen 1064 nierentransplantierten waren 132 patienten (12 %) typ-1-oder typ-2-diabetiker und hatten 113 patienten (11 %) bereits ptdm. aus n ¼ 307 ogtts ergab sich ptdm bei 29 patienten (9 %) und igt bei patienten 62 (20 %). signifikante prädiktoren für ptdm und/oder igt beinhalteten höheres lebensalter, immunsuppression mit tacrolimus, höheres c-reaktives protein und niedrigeres serum-albumin. ogtts mit bestimmung von insulin und c-peptid (n ¼ 105) zeigten im vergleich mit 1357 ogtts nicht-transplantierter patienten (datensatz g. pacini) eine erhöhte insulinsensitivität (ogis: 414ae 67 vs. 316ae 53 mlâmin -1 âm -2 ; isicomp: 6,6 ae 4,5 vs. 3,9ae 2,4, beide p < 0,001). diese ergebnisse waren in sensitivitäts-analysen gematchter ogtts (nach alter, bmi, geschlecht, 2h-blutzucker im ogtt) konsistent, des weiteren hatten nierentransplantierte mit normoglykämie (2h-blutzucker im ogtt < 140 mg/dl) ebenso wie mit hyperglykämie (2h-blutzucker im ogtt 140-199 mg/dl bzw. >200 mg/ dl) signifikant bessere insulinsensitivität als die normalbevölkerung ohne oder mit igt bzw. typ-2-diabetes. die regressions-kurven der insulin-ausschüttung und des insulinogenen index, aufgetragen gegen den 2h-blutzucker im ogtt, wiesen zwischen transplantierten und nicht-transplantierten eindeutige unterschiede auf, mit besonders starkem abfall der insulinsekretion bei nierentransplantierten mit 2h-blutzucker-werten >200 mg/dl (insulin-area under the curve (auc) 3,4 ae 1,9 vs. 6,1 ae 3,4 mu/ml 2h, p ¼ 0,002). eine validierung aller verwendeter ogtt-indizes erfolgte anhand 23 euglykämisch-hyperinsulinämischer clamps. schlussfolgerungen. insulin-sekretionsverlust könnte das dominante problem bei der entwicklung eines ptdm nach nierentransplantation sein und verlangt nach therapeutischen strategien zum schutz der betazellen. hämoglobinvariabilität ist assoziiert mit mortalität nach nierentransplantation ergebnisse. die hazard ratio (hr) fü r mortalität und transplantatversagen stieg signifikant mit der hämoglobinvariabilität an. im linearen modell betrug der anstieg fü r die mortalität 2,35 (95 % konfidenzintervall 1,75-3,17; p < 0,001), fü r das funktionelle transplantatversagen 2,45 (1,76-3,40; p < 0,001). in einem klinischen expertenmodell, das fü r die verwendung von esa, hämoglobin, alter bei der transplantation, diabetes, tage an der dialyse, glomeruläre filtrationsrate, abstoßung und jahr der transplantation adjustiert wurde, war die hämoglobinvariabilität mit der mortalität assoziiert (hr: 2,11; 1,51-2,94; p < 0,001), jedoch nicht fü r das funktionelle transplantatversagen (hr: 1,34; 0,93-1,93; p ¼ 0,121). ein ähnliches ergebnis erhielten wir beim purposeful selection modell: die hämoglobinvariabilität war signifikant assoziiert mit mortalität (hr: 2,63; 1,70-4,08; p < 0,001) und nicht signifikant mit dem funktionellen transplantatversagen (hr: 1,27; 0,63-2,54; p ¼ 0,509). schlussfolgerungen. hämoglobinvariabilität unter esa therapie nach nierentransplantation ist mit erhöhter mortalität assoziiert. influence of recipient cyp3a5 genotypes on the pharmacokinetics of tacrolimus in liver transplant recipients background. tacrolimus, which is widely used in liver transplant recipients, shows high interindividual variability in pharmacokinetics. it is primarily metabolized by hepatic cytochrome p450 3a4 and 3a5 (cyp). cyp3a5 is also expressed in the kidney and in the intestine. in this study, we evaluated the influence of recipient cyp3a5 genotypes on the pharmacokinetics of tacrolimus in patients after liver transplantation. methods. patients after liver transplantation with tacrolimus maintenance therapy were included into the study. cyp3a5 genotypes of the recipients were established. clinical and laboratory data at various time points (1 month, 3 months, 1 year and 3 years after start of medication with tacrolimus) were evaluated retrospectively. doses and trough levels of tacrolimus were noted and a screening of all concomitant medications with possible cyp3a5 interaction was performed. spss for windows (release 14.0; spss, inc) was used for statistical analyses. continuous variables were analyzed by anova-test and presented as means ae standard deviation, variables deviating from normal distribution were analyzed by rank sum test and presented as median and range. categorical variables are presented as percentages and were compared by chi-square test. the criterion for statistical significance was p < 0.05. results. ninety-two liver transplant recipients participated in the study. cyp3a5 genotypes were successfully determined in all subjects and did not deviate from the hardy-weinberg equilibirum. 86% of the patients carried the cyp3a5 *3/*3 genotype and were thus classified as cyp3a5 non-expressors. 14% carried the cyp3a5 *1/*3 genotype and were classified as heterozygous expressors. no homozygous cyp3a5 expressor (*1/*1) was found. neither tacrolimus dose nor levels were significantly different between cyp3a5 expressors and non-expressors at any point of time conclusions. the pharmacokinetics of tacrolimus in patients after liver transplantation is not influenced by the recipient cyp3a5 genotype. it is mostly the hepatic metabolism that contributes to the excretion of tacrolimus. the donor cyp3a5 genotype might be useful to predict the tacrolimus pharmacokinetics. background. due to predisposing factors like massive ascites or kachexia incisional hernias occur in about 30% of patients after ltx. following ltx the immunosuppressive regimen has been associated with the development of incisional hernias. appropriate closure techniques are still controversially discussed. the usage of mesh grafts is feasible in those patients as direct closure often results in hernia reccurrence. methods and results. we would like to present a patient who suffered from an incisional hernia 6 months following ltx. the hernia was repaired using a mesh graft (permacol + ) which was placed in a sublay technique. the patient primarily recovered and was dismissed on day 7 following hernial repair. after 14 days he presented with fever and general illness. an abdominal ct scan revealed a fluid formation above the mesh graft. open drainage had to be performed. the healing of the wound was impaired with the permacol + graft completely visible in the wound. vac was applied directly on the permacol + patch. the wound closed completely without any sings of reinfection or peritonitis until now. an abdominal ct scan 4 months showed no pathologies. conclusions. the usage of a permacol + patch seems likely to repair incisional hernias after ltx whereas severe wound infections might occur after the placement of biological grafts due to immunosuppression. in our patient a vac treatment was applied and managed the developed infection well even if it had to be placed directly on the patch. grundlagen. empfänger-desensibilisierung mittels blutgruppen-spezifischer immunadsorption (abo-ia) ist derzeit das mittel der wahl, um eine erfolgreiche transplantation über die abo-blutgruppen-barriere zu ermöglichen. zu diesem zweck wäre der einsatz von regenerierbaren, nicht-antigen-spezifischen (semiselektiven) adsorbern ebenfalls denkbar, aber die effizienz dieser immunadsorptionsmaterialien wurde in diesem zusammenhang noch nicht getestet. methodik. acht mit abo-ia behandelte transplantationskandidaten und 39 patienten, die mit verschiedenen indikationen außerhalb der abo-inkompatiblen transplantation mit semiselektiver immunadsorption behandelt wurden, wurden in die studie inkludiert. die semiselektive behandlung basierte auf ia mit protein a, peptid-ligand und anti-human immunglobulin antikörpern. antikörper-muster (igg, igg1-4 subklassen, igm und komplement-fixierende reaktivitäten) wurden mit durchflusszytometrie und konventionellen agglutinationstests analysiert. ergebnisse. die sensitive durchflusszytometrie-analyse der mit abo-ia desensibilisierten transplantationskandidaten ergab eine profunde, aber auch oft unvollständige reduktion der abo-blutgruppen-spezifischen antikörper (anti-a/ b). persistierende komplement-oder auch nicht-komplement-fixierende reaktivitäten konnten jedoch nicht mit abstoßung oder mit c4d-ablagerung in kapillaren von protokoll-biopsien assoziiert werden. beim vergleich zwischen semiselektiver und selektiver ia im rahmen einer einmaligen behandlungssitzung stellten sich die semiselektiven adsorber als effizienter in der reduktion von anti-a/b igg heraus (reduktion der mediane auf 28 % der ausgangswerte versus 59 % bei abo-ia, p < 0,001). im gegensatz dazu wurden blutgruppen-spezifisches igm (74 % versus 30 %, p < 0,001) und igg3 (72 % versus 42 %, p < 0,05) weniger gut dezimiert, ohne erkennbare unterschiede zwischen den getesteten semiselektiven adsorber-typen. eine analyse nach vier konsekutiven ia-sitzungen zeigte, dass die unterlegene effizienz nicht durch wiederholte behandlungen wettgemacht werden konnte. schlussfolgerungen. unsere beobachtung einer limitierten adsorptionskapazität bezü glich bestimmter blutgruppenspezifischer immunglobulin-(sub-)klassen lässt zu erhöhter vorsicht bei der verwendung von semiselektiven immunadsorptionstechniken bei abo-inkompatibler nierentransplantation raten. new immunosuppressive strategies for preserving renal function and improving outcome j. m. g. boira the prevention of acute rejection (ar) has been the main goal of new immunosuppressants (is) introduced in renal transplantation in the last decades. nowadays -with the use of calcineurin inhibitors as mainstay is -the rejection rates are low. however the nephrotoxicity of these agents contribute to the chronic allograft injury which may result in graft loss in the long term. new is agents should effectively prevent ar and preserve renal function. new biologic and small molecule agents are devoid of nephrotoxicity and interfere with crucial mechanism of allo-immune response. during the presentation recent data on new agents and regimes under development in the prevention of ar will be discussed. das ziel ist es daher, zum einen die empfänger t-lymphozyten zu eliminieren oder zu tolerisieren und zum anderen die spender t-lymphozyten zu tolerisieren. eine methode, die sich zur eliminierung und tolerisierung der empfänger t-lymphozyten in der knochenmarktransplantation bewährt hat, ist, neben der medikamentösen therapie, die total nodale lymphknotenbestrahlung. damit können abstoßungsreaktionen selbst bei hla-nichtidenten knochenmarktransplantationen weitgehend verhindert werden. zur behandlung der gvhd hat sich in der allogenen knochenmarktransplantation die extracorporale photopherese (ecp) bewährt, die durch im einzelnen nicht vollkommen aufgeklärte mechanismen eine toleranz induziert und somit in der lage ist, die gvhd ohne intensive immunsuppression zu mildern oder auch ganz zu eliminieren. anhand von fallbeispielen wird diskutiert werden, dass die total nodale lymphknotenbestrahlung und ecp auch in der organtransplantation eine rolle spielen könnten, da die mechanismen der toleranz oder abstoßung sich prinzipiell nicht von einer knochenmarktransplantation unterscheiden. plenarsitzung iv: immun-und organtoleranz/toleranzinduktion ii 067 approaches to tolerance: mouse to man harvard medical school, division of transplantation, massachusetts general hospital, boston, ma, usa over the past two decades, non-specific immunosuppression (i.s.) incorporating polyclonal and monoclonal antibodies, calcineurin inhibitors (cni), mtor inhibitors, anti-metabolites and steroids have greatly improved early allograft survival rates. however, the rate of chronic allograft loss has decreased only modestly over that observed earlier. following kidney transplantation, for example, the rate of chronic attrition is discouragingly similar to that observed two decades ago, generally resulting from "chronic rejection" or, perhaps more accurately, "chronic allograft nephropathy". the relentless progression of similar histopathologic processes is manifested as vasculopathy in the coronary arteries of heart allografts (cav) and in lung transplants as obliterative bronchiolitis (ob) despite the long-term administration of currently available i.s. thus, the ultimate goal of i.s. for transplant recipients is the development of long-term donor-specific nonresponsiveness which leaves responses to other foreign stimuli intact. successful induction of "transplant tolerance" would avoid the complications of chronically administered i.s. and reduce the risks of chronic rejection. numerous approaches employing both peripheral and central tolerance mechanistic strategies have been successfully developed in rodent models. currently, several of these approaches appear to be clinically applicable, having now proved to be effective in large animal models and preliminary clinical trials. the most successful to date has been induction of hematopoietic mixed chimerism. the mixed chimerism approach, an initially presumed central tolerance strategy, has been extensively studied in our laboratories, first in rodent, non-human primate, and porcine models, and now in human recipients of renal allografts. using a non-myeloablative conditioning regimen followed by donor bone marrow (dbm) transplantation, we observed successful production of mixed chimerism in non-human primate renal allograft recipients, followed by normal kidney function with no evidence of chronic rejection for periods as long as thirteen years after discontinuing all i.s. in vitro studies of these recipients as well as of humans suggest that both central (clonal deletion) and peripheral (regulatory cells) mechanisms are involved. encouraged by the pre-clinical studies, we have to date extended this approach to 20 human renal allograft recipients. ten individuals with esrd secondary to multiple myeloma received a nonmyeloablative preparative regimen consisting of perioperative cyclophosphamide, equine atg, csa, and thymic irradiation. the recipients underwent hla-matched kidney transplantation followed by iv infusion of dbm and subsequent donor lymphocyte infusions (dli) to enhance the anti-myeloma effect. transient lymphohematopoietic chimerism was achieved in all patients and only two reversible rejection episodes have occurred with a follow-up of 1 to 13 years after discontinuing i.s. longterm control of the underlying malignancy was better than with any other currently available treatment, but 4 of these individuals required additional therapy for recurrent myeloma. ten patients with esrd and no myeloma received hla-mismatched kidneys after a similar conditioning regimen except that medi-507, an anti-cd-2 mab, was used in place of atg and dli was not used. transient mixed chimerism and apparent allograft tolerance has been achieved in 7 of these recipients. these 7 patients have received no i.s. for periods of 2-9 years. two allografts were lost: acute humoral rejection (1); thrombotic micro angiopathy (1) and the last recipient has been returned to chronic i.s. after developing acute rejection when i.s. was withdrawn. these observations plus similarly encouraging observations in several clinical trials elsewhere suggest that tolerance induction may become a more widely applicable clinical reality. experimental rationale for using hematopoietic stem cell transplantation for tolerance induction it was recognized a long time ago that an individual that shares hematopoietic cells with another, becomes tolerant to this individual. thus, a recipient of a successful donor bone marrow (bm) transplant (bmt) becomes tolerant toward the donor. triggered by this observation experimental studies have been systematically addressing why and how donor bm transplantation achieves tolerance. the concept of mixed chimerism -in which a substantial donor hematopoietic cell proportion co-exists with the recipient hematopoietic repertoire following donor bmt -has emerged as a promising bmt-based tolerance strategy. mixed chimerism leads to a particularly robust and durable type of tolerance. central clonal deletion of immature donorreactive t cells was identified as a key mechanism of tolerance in mixed chimeras and seems to be in large part responsible for the robust nature of tolerance achieved. peripheral clonal deletion of mature donor-reactive t cells and non-deletional (i.e. regulatory) mechanisms also contribute to tolerance induction in some models. overall, donor bmt emulates many of the physiologic mechanisms controlling self-tolerance, extending them to donor antigens. while clinical translation of the mixed chimerism approach has been achieved in pilot trials, large-scale application in routine clinical practice awaits minimally toxic bmt regimens. myelosuppressive and cytotoxic pre-treatments commonly employed in 'conventional' bmt recipients would be unacceptable in organ transplant recipients. therefore, efforts to develop advanced, non-toxic murine bmt protocols are ongoing. the use of costimulation blockers and, more recently, adjunctive t regulatory cell therapy have yielded dramatically less toxic regimens which hold promise for clinical translation. aspergillus und seltene schimmelpilzinfektionen. die inzidenz der aspergillose bei der allogenen stammzelltransplantation beträgt zwischen 8-25 %. aspergillussporen kommen ubiquitär in der umwelt vor; ungefilterte luft aus belü ftungssystemen und staub bilden die hauptinfektionsquellen. aspergillussporen werden exogen durch inhalation erworben. die reduktion der sporenlast in der umgebung von patienten "at risk" ist die wichtigste prophylaktische maßnahme. der klinisch wichtigste vertreter ist aspergillus fumigatus. die letalität invasiver aspergillosen liegt bei 70-90 %. zu den anderen schimmelpilzinfektionen zählen die zygomykosen (absidia-, mucor-und rhizopus-spezies), die insbesondere bei patienten mit diabetes mellitus oder zunehmend bei immunsupprimierten patienten unter voriconazol therapie auftreten. die letalität beim immungeschwächten ist annähernd 100 %. bei empfängern solider organtransplantate konnte durch neuerungen in der chirurgie und intensivierung der immunsuppression das transplantatü berleben verbessert werden. mit bis zu 15 % ist die inzidenz bei herzund lungentransplantation am höchsten. nach lungentransplantation treten neben der typischen pulmonalen aspergillose gehäuft ulzerierende tracheobronchitis und aspergillose der bronchusanastomose auf. das geringste risiko einer invasiven aspergillose besteht bei nierentransplantierten mit 1 % inzidenz, die mortalität ist generell hoch. institut für hygiene, mikrobiologie und umweltmedizin, medizinische universität graz, graz, österreich pilzinfektionen spielen bei immunsupprimierten, vor allem bei patientinnen und patienten nach organ-und stammzelltransplantationen, eine steigende rolle. dabei reicht das spektrum der verursachenden pilze von hefen der gattung candida zu schimmelpilzen der gattung aspergillus, wobei auch eine zunahme von infektionen mit anderen schimmelpilzen wie den zygomyzeten, fusarien, scedosporium/pseudallescheria und anderen zu beobachten ist. um eine zielgerichtete therapie einleiten zu können, ist eine schnelle und genaue diagnostik unumgänglich, da die einzelnen pilzarten teils unterschiedliche antimykotika-empfindlichkeitsspektren aufweisen. neben den klassischen kulturmethoden, die trotz ihrer relativen "langsamkeit" nach wie vor unverzichtbar sind, wurden in den letzten jahren techniken entwickelt, die teilweise innerhalb weniger stunden ergebnisse bringen können -vorausgesetzt, sie werden richtig angewandt und interpretiert. dazu zählen insbesondere bildgebende verfahren, molekularbiologische methoden und antigen-nachweise. idealerweise beruht die diagnose einer pilzinfektion auf der kombination mehrerer techniken, man spricht auch von einer puzzle-diagnostik. die einzelnen techniken zur diagnose von pilzinfektionen in der organ-und stammzelltransplantation sollen vorgestellt und deren vor-und nachteile diskutiert werden. therapie von pilzinfektionen in der organ-und stammzelltransplantation universitätsklinik für innere medizin, medizinische universität graz, graz, österreich pilze verursachen bei immunsupprimierten infektiöse komplikationen, die von oberflächlichen infektionen der haut/ schleimhäute bis zu systemischen, lebensbedrohlichen organinfektionen oder fungämien reichen. auslöser dieser infektionen sind meist candida-arten, aspergillus-arten, andere schimmelpilze wie zygomyceten, und seltene andere pilze. zur therapie der systemischen infektionen stehen polyene (z. b. amphotericin b, lipid-assoziierte amphotericin b präparate), azole (z. b. fuconazol, itraconazol, voriconazol, posaconazol) und echinocandine (z. b. caspofungin, anidulafungin, micafungin) zur verfügung. fluconazol ist ausschließlich gegen candida arten wirksam. die anderen oben genannnten antimykotika sind in vitro sowohl gegen candida-als auch aspergillus-arten wirksam. für die therapie von candida-infektionen eignen sich abhängig von der lokalen epidemiologie fluconazol, echinocandine oder amphotericin b-präparate. für die therapie von aspergillus-infektionen eignen sich voriconazol, posaconazol, amphotericin b-präparate oder echinocandine. für zygomkosen stehen amphotericin b-präparate und posaconazol zu verfügung. für seltene pilzinfektionen sind adjuvante therapeutika (wie z. b. flucytosin) vorhanden. für den behandlungserfolg der schweren lebensbedrohlichen infektionen ist der zeitpunkt des therapiebeginns und das spektrum der antifungalen therapie relevant. es werden empirische, kalkuliert-präemptive und zielgerichtete therapien unterschieden. bei machne pilzinfektionen sind chirurgische sanierungen zusätzlich zur antimykotischen therapie notwendig. die einzelnen therapioptionen zur behandlung von pilzinfektionen in der organ-und stammzelltransplantation sollen vorgestellt und deren vor-und nachteile diskutiert werden. erlebenüberlebenund dann? lebensqualität nach herztransplantation klinische abteilung für herzchirurgie, universitätsklinik für chirurgie, medizinische universität wien, wien, österreich aus wissenschaftlicher perspektive ist der begriff "lebensqualität" ein nicht direkt beobachtbares konstrukt, das die bewertung des physischen, psychischen und sozialen zustandes einer person in seiner zusammenschau bezeichnet. eine gute annäherung kann auch durch den begriff "lebenszufriedenheit" erfolgen. im vortrag soll vor allem auf langzeit-lebensqualitätsstudien und die psychische verarbeitung der herztransplantation bezug genommen werden: wie ändert sich das leben der patientinnen nach einer oft schicksalhaften schweren erkrankung und lebenserhaltenden transplantation? in einer prospektiven studie unserer abteilung über lebensqualität ein, fünf und 10 jahre nach herztransplantation zeigte sich z. b. eine schere zwischen konstant durchaus guter lebensqualität in physischer und zunehmend abnehmender lebensqualität in physischer hinsicht (v. a. depression, labilität und dysphorie). es werden gründe für diese differenz diskutiert, lösungsmöglichkeiten besprochen. ferner ist die frage interessant, ob persönlichkeitsfaktoren einfluß haben auf langzeitüberleben und lebensqualität bzw. -zufriedenheit: wer lebt länger, wer lebt besser? es werden klinisch-psychologische studien vorgestellt, die belegen, dass ein bestimmtes persönlichkeitsmuster (typ d, denollet 1996) für die drei am häufigsten durchgeführten pädiatrischen organtransplantationen (niere, leber, herz) wird der jeweils aktuelle forschungsstand referiert. darüber hinaus werden zwei spezifische problembereiche in der pädiatrischen organtransplantation dargestellt, das problem der compliance/adherence und der transition von der kinder-und jugendmedizin in die erwachsenenmedizin. die studien unterscheiden sich hinsichtlich der operationalisierung der lq, des studiendesigns, der dauer des follow-ups und des altersbereichs der kinder. prospektive längsschnittstudien mit größeren stichproben in multizentrischer zusammenarbeit unter einbeziehung möglichst spezifischer erhebungsmethoden sind erforderlich, um die postoperative entwicklung der kinder besser beurteilen zu können. vor dem hintergrund einer neuen ä ra der immunsuppressiven therapie (steroidminimierung, individualisierte therapie) kann hier zukünftig eine weitere steigerung der lq erwartet werden. background. autologous stem cell transplantation (asct) is a curative procedure for a variety of haemato-oncological malignancies and depends on the transplantation of sufficient cd34þ cells (threshold of >2â10 6 /kg) to ensure sustained engraftment. in the last decade, peripheral blood stem cells (pbsc) have completely replaced bone marrow (bm) as preferred stem cell graft in asct. however, a number of patients fail to mobilize the threshold of >2â10 6 /kg cd34þ cells. in these poor mobilizers, bm harvest was the only possibility to collect sufficient numbers of stem cells so far. recently, the chemokine-receptor antagonist plerixafor has shown to enhance stem cell mobilization in patients who demonstrated with previous mobilization failure with an overall success rate of about 70%. we here report 4 patients with mobilization failure in whom g-csf primed bm as well as plerixafor primed pbsc were collected. methods. four heavily pretreated patients (median of 10 chemotherapy cycles, range 8-17) with non-hodgkin lymphoma (nhl, n ¼ 3.) and germ cell cancer (n ¼ 1) presented with mobilization failure after 1-7 mobilization attempts. in the 3 nhl patients bm was harvested after unsuccessful pbsc collection with plerixafor. in the patient with germ cell tumor before plerixafor primed pbsc collection was done after insufficient bm harvest. results. in the 3 nhl patients plerixafor enhanced pbsc revealed an insufficient number of cd34þ cells (i.e. 0.64-1.15â10 6 /kg cd34þ cells in 2-3 leukaphereses procedures). therefore bm was harvested after g-csf stimulation. however, again we failed to obtain enough cd34þ cells in all of them (0.16 to 0.62â10 6 /kg cd34þ cells). in the patient with germ cell cancer a collection of cd34þ cells from the bm was carried out as first salvage procedure after the failure of conventional mobilization (0.9â10 6 /kg cd34þ cells). thus, in a next attempt pbscs were mobilized with g-csf and plerixafor resulting in a successful harvest (3.36â10 6 /kg cd34þ cells in one leukapheresis). conclusions. reviewing our cases one might speculate that a bm harvest after failure of stem cell mobilization with g-csf and plerixafor does not yield in sufficient cd34þ cell numbers. transient elastography (fibroscan) for the prediction of liver toxicity following autologous or allogeneic hsct j. auberger 1,2 , i. graziadei 3 , w. vogel 3 , j. clausen 1,2 , d. grundlagen. lebertoxizität oder das sinusoidale obstruktions-syndrom (sos) gehören zu den häufigsten organtoxizitäten nach zytoreduktiver hochdosis-chemo-/radiotherapie bei autologer oder allogener hsct. die transiente elastographie (fibroscan, fs) ist eine neue, schnell zugängliche und nicht-invasive methode eine leberfibrose ü ber die lebersteifigkeit (ls) zu bestimmen und wird bereits erfolgreich bei patienten mit hepatitis c und alkoholischer lebererkrankung eingesetzt. methodik. zwischen april 2009 und oktober 2010 wurden 67 patienten fs-messungen während der routine-abdomensonographie im rahmen der transplantvoruntersuchungen vor autologer (n ¼ 37) oder allogener (n ¼ 30) transplantation zugeführt. die konditionierung bestand aus hd mel (140-200 mg/m 2 ), beam oder anderen krankheitsspezifischen hochdosisschemata im rahmen autologer transplantationen. allogene transplantationen wurden nach myeloablativer konditionierung (mac, n ¼ 18) oder in reduzierter intensität, hauptsächlich buflu basiert (ric, n ¼ 12) durchgeführt. die lebertoxizität wurde anhand des maximalen serumbilirubins, der anzahl der tage mit einem bilirubin >2 mg/dl und der prozentualen, maximalen gewichtszunahme bis zum tag þ20 definiert. ergebnisse. das maximale serum bilirubin vor tag þ20 betrug 1,18 (range, 0,44-11,72) mg/dl, der mediane zeitpunkt mit einem serumbilirubin !2 mg/dl war 10,5 (range, 1-20) tage und die mediane prozentualen gewichtszunahme war 5,2 % (range, 0,1-17,2). lebertoxizität wurde häufiger nach mac vs. ric konditionierung beobachtet, wenngleich nicht statistisch signifikant (50 % vs. 33 %, p ¼ 0,25). patienten, die eine immunsuppression (is) mit csa/mtx erhielten, zeigten häufiger anzeichen einer lebertoxizität verglichen mit pat., die csa/mmf oder keine is erhielten (83 % vs. 18 %). die mediane ls vor transplantation war 5,4 kp (range, 2,3-62,7). die ls korrelierte signifikant mit der got und gpt vor transplantation in der gesamten kohorte und mit gpt, ggt und ap bei pat. mit mehr als 10d bilirubin >2 mg/dl bis zum tag þ 20. zwischen der ls vor hsct und zeichen der lebertoxizität (definiert als max. serumbilirubin, anzahl der tage mit bilirubin >2 mg/dl und prozentualer gewichtszunahme) nach hsct konnte keine signifikante korrelation hergestellt werden. schlussfolgerungen. die nicht-invasive transiente elastographie (fibroscan) ist nicht wegweisend, um patienten zu identifizieren, die ein hohes risiko für die entwicklung einer lebertoxizität nach autologer oder allogener transplantation aufweisen. norovirus associated hemophagocytic lymphohistiocytosis (hlh) after unrelated bone marrow transplantation (bmt) in a boy with refractory aml g. kropshofer, c. salvador, r. crazzolara, b. meister background. hlh is a potentially life threatening macrophage activating syndrome which leads to massive hyperinflammation through an exaggerated but ineffective immune response. diagnosis after bmt is complicated by other inflammatory syndroms like engraftment syndrome, capillary leak syndrome, venoocclusive disease and systemic infections. methods and results. we report on a meanwhile 24month-old boy with aml (m4) relapse. already in phase of maintenance chemotherapy he acquired a gastrointestinal infection with norovirus. reinduction chemotherapy was performed with flag/daunoxome and flamsa -he never reached a complete remission. so we decided to perform an allogeneic bmt (mud) in aplasia. conditioning regimen consisted of iv busulfan, cyclophosphamide and melphalan. there were no early complications despite his ongoing norovirus infection. leukocyte engraftment was obtained on day þ20. bone marrow at day þ28 showed a nearly normocellular bone marrow and full donor chimerism. at day þ40 the patient impressed with persistent fever despite antimicrobial therapy, his general status deteriorated. he developed cns symptoms with vertical nystagmus, increasing hepatosplenomegaly and respiratory insufficiency. laboratory findings showed hyperferritinemia (45000 mg/l), a subsequent pancytopenia, hypofibrinogenemia, elevated d-dimer (34000 mg/l) and hypertriglyceridemia (723 mg/dl). intensive care was necessary including high doses of catecholamines. subsequent bone marrow aspiration revealed an aplastic bone marrow and hemophagocytosis. the patient underwent ventilation support for 17 days. treatment included dxm 10 mg/m 2 /d for 14 days, then dxm was tapered to 5 mg/kg/d. etoposide (150 mg/kg) was given three times until now. considering the long aplastic phase and the ongoing hemophagocytosis we decided to give him stem cell support (remaining frozen bone marrow from the same donor) without reconditioning. four days later we went on with a monoclonal anti-cd25 antibody (simulect + ). the boy improved but therapy is still ongoing. the outcome at the moment is unclear. conclusions. hlh still is a rarely seen complication after bmt, therefore early diagnosis is necessary to start therapy promptly. the role of monoclonal anti-cd25 antibody and anti tnf-antibody as part of therapy is unclear yet. up to now there is no report of norovirus triggering hlh. therapeutic effect of allogeneic bone marrow mesenchymal stem cell transplantation on liver chronic damage in rats methods. 75 male rats divided in 3 groups in which chronic hepatitis was modeled by means of ccl 4 . 1st (n ¼ 25) received single transplantation of 2.5â10 6 msc on 3rd day after modelling. in 2nd (n ¼ 25) msc were injected twice in a dose of 2.5â10 6 cells on days 3 and 10 after modelling. 3rd group was control group (cg, n ¼ 25). biochemical indices: alt, ast, alp; cytokines (ifn-, tnf-, il-4, il-10) were studied during 5 weeks. liver histology were studied after 7, 14, 21, 28, 60, 90 days. immunohistochemical methods: -sma, caspase-3, afp and pcna were used. results. at the 1-3 weeks the ast, alt and alp were increased in all groups, but in the cg level of indices above was higher. analysis of morphologic changes in liver resulted in following findings: on 7th and 14th day liver fatty degeneration was in all groups, but its appearance was stronger in the 1st and especially in cg. in 60 and 90 days the fibrosis became more severe in cg, but after msc application a resorption of fibrosis occurred (especially in 2nd). it is important to note that connective tissue forming in liver after msc application had two-phase dynamics: at first an area of connective tissue sharply increased and then gradually decreased. it was found out that the -sma and caspase 3 expression after msc application had also two-phase dynamics: at 1-2 weeks these indices were increased and then (3-4 weeks) began to decrease, becoming lower than in cg. at first weeks cytokines disbalance was identified in all groups, but in msc groups cytokines disbalance was less expressed on 5th week. afp and pcna expression rates were higher in msc groups, than in cg up to end of examination. conclusions. mscs transplantation for therapy of liver damage can reduce liver fibrosis, but this process has two-phase dynamics (firstly increasing and then decreasing of fibrosis) which can explain the contradictory information on the cell therapy of chronic hepatitis. background. treatment of chronic liver failure in pretransplant period is an actual problem of medicine. this investigation was undertaken for working out of biounit containing viable liver cell (lc) and multipotent mesenchymal stromal cells (mmsc), which could support the functioning and recovered regeneration of damaged liver for a long time. methods. chronic liver damage was modeled on wistar rats by means of 0.3 ml ccl 4 on 100 g rat weights within 6 weeks. adult wistar rats were also used as donor cells. isolated cells were cultivated in 10 days: at first mmsc during 7 days then the same mmsc together with lc in 3 days on cytodex. suspension of lc 2.5-4.0â10 6 cells/cm 3 and mmsc 0.5-0.8â10 6 cells/ cm 3 was applied on biodegradable matrixes "sphero + gel". formed biounits were transplantated into rat liver. survival of transplanted cells and liver morphology were investigated in 365 days after transplantation by using hematoxylin and eosin staining, masson's trichrome staining. cell viability and cellular phenotype were investigated by trypan blue staining and immunohistochemically (cytokeratine 18 and mitochondrial antigen). results. before biounit transplantation liver damage was characterized by: fatty, lymphoid-cellular infiltration and proliferation of histioblasts and macrophages; porto-portal sclerosis; hydropic dystrophia and focal necrosis of hepatocytes. in 365 days after biounit transplantation it was detected: viable hepatocytes, neogenic plethoric vessels, neogenic plethoric bile duct in biounit. liver damage (dystrophia, safe structure of a liver, beam structure, fatty vacuoles inside hepatocytes and other indices) were significant less after biounit transplantation than in control group without biounit transplantation. restoration of hepatic lobe structure was better in studied group. conclusions. our preliminary studies demonstrate that in biounit after transplantation into damage liver there is formation of morphologic structures of liver (viable cell, vessels, bile ducts); other words the used method allows to carry out organotypic liver remodeling into biounit and to support the function of the damaged liver. thereby the suggested method is a perspective one and can be used as technology of building intracorporeal biounit for the long-term auxiliary supporting of the damaged organs. liposomales amphotericin b als prophylaxe invasiver pilzinfektionen nach hämatopoietischer stammzelltransplantation bei pädiatrischen und adoleszenten patienten photericin b (lamb) sind ein breites antifungales spektrum, eine lange eliminations-halbwertszeit, und im vergleich zu den neuen azolen weniger medikamenten-interaktionen. methodik. im zeitraum 2009-2010 wurden bei 20 konsekutiven patienten (medianes alter: 11,4 jahre; m:w ¼ 8:12) insgesamt 23 hszt durchgeführt: 12 autologe (davon 3 tandem-transplantationen) und 11 allogene szt, wegen soliden tumoren (n ¼ 7), rezidivierter schwerer aplastischer anämie (n ¼ 5), malignen hämatologischen erkrankungen (n ¼ 5), mb. hurler (n ¼ 2) bzw. mb crohn (n ¼ 1). autolog transplantierte patienten erhielten im median 7,46â10 6 /kg cd34þ zellen/kg und erreichten am median tag þ 9,5 das leukozytäre engraftment. allogene stammzellquellen waren periphere stammzellen (cd34þ selektioniert und/oder cd3/19 depletiert) in 9 und knochenmark in 2 fällen; spender waren hla-idente fremd-(n ¼ 9) bzw. geschwisterspender (n ¼ 1) und ein haploidenter elternteil. die mediane stammzellzahl lag bei 15,37â10 6 cd34þzellen/kg; das leukozytäre engraftment erfolgte im median am tag þ 11. bei 5 patienten trat eine graft-versus-host-erkrankung (gvh) auf, davon einmal eine gvh iv. die lamb-prophylaxe wurde in einer dosis von 5 mg/kg in 3-tägigen (n ¼ 15) oder 2-tägigen (n ¼ 8) intervallen gestartet, bei 2 patienten mit refraktärer aml am tag à46 bzw. à42, bei allen anderen patienten im median am tag à3. die patienten erhielten im median 11, prophylaktische dosen bis zu median tag þ20,5 (þ11 bis þ43). ergebnisse. bei 8 patienten wurden wegen fieber oder crp-anstieg die infusionsintervalle verkürzt. eine intermittierende candida-kolonisation wurde durch wöchentliche ü berwachungskulturen bei 9 patienten detektiert. wöchentliche galaktomannan-test blieben bei 17 patienten durchgehend negativ, 2 patienten hatten je ein einzelnes positives testergebnis; keiner dieser 19 patienten entwickelte während einer medianen nachbeobachtungszeit von 10 monaten eine invasive pilzinfektion. eine patientin mit gvh iv unter multimodaler immunsuppression und wiederholt positiven galaktomannan-tests entwickelte trotz kontinuierlicher präemptiver therapie mit lamb alternierend mit caspofungin präfinal eine aspergillus-pneumonie und verstarb am tag þ140. bei keinem patienten mußte lamb wegen infusionsassoziierter nebenwirkungen abgesetzt werden; die hauptnebenwirkung war eine hypokaliämie, die in 20/23 anwendungen substitutionsbedürftig war. schlussfolgerungen. die intermittierende antifungale prophylaxe mit lamb nach autologer bzw. allogener szt war bei 19/20 patienten erfolgreich. die nebenwirkungen beschränkten sich auf eine substitutionsbedürftige hypokaliämie. background. late complications including diabetic vasculopathy, retinopathy and neuropathy are still serious problems in the treatment of diabetes mellitus. whole pancreas transplantation or human islet cell transplantation are alternatives but limited due to the organ shortage and lifelong immunosuppression. the use of immune isolated porcine islet cells (pic) could offer a feasible alternative. in this study, the safety and applicability of three possible implantation sites for sodium cellulose sulfate (scs) microencapsulated pic were evaluated in five healthy beagle dogs. methods. five healthy, male castrated beagle dogs were used for this study. scs microencapsulated pic were implanted in the subcutaneous tissue, the omentum and the gastric submucosa. surgery time and intraoperative complications were monitored. implantation in the omentum and gastric submucosa was performed laparoscopic assisted. all animals were continuously monitored including clinical examinations and complete blood counts for possible occurring side effects. on day à3, 30 and 90 a glucagon stimulation test was performed to assess -cell function and glucose metabolism. on day à3, 1, 3, 12, 30, 60 and 90 fasting glucose and insulin were measured. blood samples were taken to check a possible porcine endogenous retrovirus (perv) transmission. biopsies of all implantation sites were obtained for histopathology on day 90. results. no technical complications occurred at the subcutis and omentum site. at the gastric submucosa only a limited amount of cells could be implanted. delayed wound healing at the subcutaneous implantation site was observed in three out of five dogs. no further side effects occured during the study period. transmission of perv was not detected. glucagon stimulation tests showed a trend towards a faster response to a glucagon stimulus and an increased insulin peak. histopathology results have not been completely evaluated yet. conclusions. first results show that scs microencapsulated pic do not cause any harmful side effects to the recipients concerning application. no transmission of perv was detected. the outcome of histopathology in combination with the clinical results will display the efficacy of this treatment and the expected differences in transplantation sites. background. heart failure following acute myocardial infarction (ami) is a major cause of morbidity and mortality. our previous observation that injection of apoptotic peripheral blood mononuclear cells (pbmc) was able to restore cardiac function in a rat acute ischaemia model prompted us to study the effect of soluble factors derived from apoptotic pbmc on ventricular remodelling after ami. methods. cell culture supernatants derived from irradiated apoptotic peripheral blood mononuclear cells (aposec) were collected and injected as a single dose intravenously after myocardial infarction in an experimental ami rat model and in a porcine reperfused ami model. mri and echocardiography were used to quantitate cardiac function. immunohistology and flowcytometry were used to analyse the cellular components. analysis of soluble factors present in aposec was performed with proteome membrane arrays and activation of signalling cascades in human cardiomyocytes by aposec in vitro was studied by immunoblot-analysis. results. intravenous administration of aposec resulted in a reduction of scar extension in both models. hearts explanted from animals infused with aposec evidenced less myocardial necrosis after 24 hrs. troponin-i release was less than in animals treated with medium as control. in the porcine ami model aposec led to an improvement of ejection fraction (57.0% vs. 40.5%, p < 0.01), cardiac output (4.0 vs. 2.4 l/min., p < 0.001) and a reduced infarction size (12.6% vs. 6.9%, p < 0.02) as determined by mri. administration of aposec in the rat ami model caused increased presence of cd68 þ macrophages and c-kitþ endothelial progenitor cells (epc) in the infarcted myocardium within 72 hrs. exposure of human cardiomyocytes with aposec in vitro triggered activation of pro-survival signalling-cascades (akt, p38 mapk, erk1/2, creb, c-jun) and anti-apoptotic gene products (bcl-2, bag1). conclusions. intravenous infusion of aposec attenuated myocardial remodelling in both models of experimental ami. this effect seems to be due to the activation of pro-survival signalling cascades in the affected cardiomyocytes and to a higher presence of regenerative cells (epcs, macrophages) within the ischaemic tissue. aposec represents a "biological" which prevents myocardial infarction by causing peri-infarct conditioning and stimulation of regenerative effects in the hypoxic myocardium. anti-thymocyte globulin (atg) reduces damage caused by ischaemia and preserves cardiac function after experimental myocardial infarction background. myocardial infarction (mi) is one of the leading causes of death in the western world. consequent inflammatory reactions initiate and sustain remodeling of the damaged myocardium, which can worsen the outcome additionally. our previous findings suggest that apoptotic peripheral blood mononuclear cells (pbmcs), injected intravenously after mi in rats, can attenuate inflammation and subsequently improve cardiac function after mi. in this study we sought to investigate, if anti-thymocyte globulin (atg) as a therapeutic agent inducing apoptosis in white blood cells, evidences similar effects after experimental mi in rats. methods. to study effects of atg in vitro, we incubated whole blood and pbmc cell cultures with this polyclonal antibody for 24 hours. elisa was utilized to assess changes in the secretion profile of various cytokines. furthermore, we conducted an in vivo study in an experimental rat model of myocardial infarction. rodents were injected with atg (10 mg/rat) after ligation of the left anterior descending (lad) artery. untreated and sham operated animals served as controls. short term effects were evaluated by immunohistology after three days. echocardiography and assessment of infarction size by planimetry depicted the outcome after six weeks. results. cytokines and chemokines held responsible for cardioprotective and neo-angiogenic effects (e.g. il-1ra, il-8, mcp-1) were significantly elevated in atg groups over controls in vitro. histology of in vivo experiments confirmed these data, as atg treated animals had a reduced area of necrosis three days after ami (10.7% vs. 20.6%), smaller infarct scars after six weeks and an increased infiltration of macrophages. echocardiography revealed a treatment advantage of atg, as rodents enrolled in this study group evidenced an ejection fraction of 52.35% ae 1.96% compared to 42.91% ae 2.22% in controls (n ¼ 13, p < 0.001). conclusions. via the mechanism of apoptosis, atg induces a plethora of pro-angiogenic and immune-modulatory factors. secretion of this ensemble of cytokines and chemokines attracts macrophages/monocytes into the infarcted area, and attenuates inflammation after myocardial infarction. in conclusion, interaction of these factors can significantly reduce infarction size and improve left ventricular function after experimental myocardial infarction in rats. secretion of cytokines and chemokines by peripheral blood mononuclear cells is triggered by coagulation products these mediators systemic in immune activation/sepsis, wound healing, autoimmune-diseases, artherosclerosis and myocardial infarction. however, cytokines and chemokines are usually not considered to be very stable after blood collection, which might therefore alter test. results. thus, the aim of the pilot study was to obtain better knowledge about stability of these mediators in blood samples for interpretation of test results. methods. venous blood was taken from healthy probands (n ¼ 7) using different blood tubes (serum, heparin plasma and edta plasma). blood tubes were either centrifuged initially within 20 minutes after venipuncture and kept frozen at à80 until further testing or were stored at 4 c, at room temperature (rt) or at 37 for up to 24 hours. samples were evaluated for il-1ß, il-6, tnf-and for selected chemokines such as interleukin-8, epithelial neutrophil-activating protein 78 (ena-78) and granulocyte chemotactic peptide-2 (gcp-2) using commercially available enzyme-linked immunosorbent assay (elisa) kits. results. interestingly all examined mediators rise when samples were stored above room temperature for more than 4 hours in serum tubes. the rise of serum chemokine and cytokine levels culminated in a 79-fold increase for il-6 (p < 0.0081), a 22-fold increase for ena-78 (p < 0.0006) and a 17-fold increase for gcp-2 (p < 0.0026) compared to basic values. serum levels of il-1 and tnf-where not detectable at basic samples but rise up to 1157 pg/ml (mean il-1, p < 0.03) and 488 pg/ml (mean tnf-, p < 0.03). conclusions. these data indicate that the most chemokine and cytokine levels remain stable when analysed within a short interval after venipuncture. when tubes were exposed to temperatures higher than 24 (rt), levels of measured chemokines increased dramatically. background. rabbit antithymocyte globulin (ratg) is widely used as induction agent in solid organ transplantation. beside depletion of circulating lymphocytes there is a growing body of evidence suggesting that ratg may also play a pivotal role in modulating the immune system. as blood circulating endothelial cells (cecs) and circulating hematopoietic progenitor cells (cpcs) represent two minute fractions (cecs: 0.1% to 6.0% and cpcs: 0.01-0.20%) of blood mononuclear cells that are thought to play important roles in tissue vascularisation, the study of both cell types is currently suggested as surrogate markers for numerous pathologies. especially the noninvasive endothelial evaluation as an early index of vascular injury following kidney transplantation has been already demonstrated. methods. we used four surface markers to identify viable cecs as cd31bright, cd34dim, cd45-, cd133-and viable cpcs as cd34bright, cd133þ, cd45dim, cd31 þ cells in the peripheral blood of liver transplanted recipients (n ¼ 28) until day 20 post transplantation via facs-analysis. results. an induction of cecs was exclusively observed for ratg-treated patients (n ¼ 17) increasing from 0.56% ae 0.98% pre transplantation to 1.83% ae 1.85% at day 1-2 post transplantation compared with control patients receiving standard immunosuppression (n ¼ 11) (p < 0.04). in addition, the induction of cpcs was even more pronounced illustrating an increase in ratg treated patients from 0.20% ae 0.26% pre transplantation to 1.55% ae 1.75% at day 1-2 post transplantation (p < 0.001). a significant elevation of blood cpcs is still detectable at day 5 (p ¼ 0.0379 compared with controls) and starts to decline at day 10 post transplantation. conclusions. in summary we illustrated that both cecs and cpcs were detectable in numbers that allows kinetic monitoring of these cell types post transplantation and that ratg treatment results in a transient induction. as clinical correlations between the concentration of these two populations and the effect of immunosuppressive regimens has been already proven, validation of these cell populations as biomarkers in the setting of solid organ transplantation remains to be determined. background. lipocalin-2 (lcn-2) has been described as a marker and potential positive modulator of inflammation during ischemia/reperfusion injury (iri) following solid organ transplantation. data on lcn-2 expression during allograft rejection have been missing so far. methods. sera of 68 patients undergoing orthotopic liver transplantation were collected preoperatively and postoperatively from day 1 to 15. lcn-2 was analyzed by elisa and expression levels were correlated with parameters of allograft rejection. lcn-2 expression was further correlated with preexistent malignancy, postoperative renal failure and various immunosuppressive regimens. results. lcn-2 serum levels were elevated 3 to 7 fold immediately after liver transplantation due to iri and also increased prior to clinically diagnosed acute rejection, however not statistically significant (p > 0.05) but closely related to an elevation of routinely used laboratory parameters. conclusions. our data suggest lcn-2 to be a chemoattractant stimulus for infiltrating immune competent cells into the allograft following solid organ transplantation. it is an inflammatory marker which is upregulated during acute graft rejection and its elevation prior to routinely used parameters of acute rejection might be an important tool for therapeutic intervention. effect of oxidative stress and endotoxin on albumin in brain death background. albumin binds and detoxifies endotoxin in healthy people. oxidative stress leads to protein oxidation and thus to impaired binding properties of albumin. this, in combination with increased gut permeability leads to appearance of endotoxin in the systemic circulation and further to impaired organ function. we hypothesise that these processes occur in serum of brain-dead organ donors. methods. endotoxin was determined with an adapted limulus amoebocyte lysate assay. albumin fractions and binding capacity were determined by hplc. flowcytomixtm was used for determination of cytokine levels. results. eighty-four brain-dead organ donors were enrolled and categorized by the length of intensive care unit (icu) stay. albumin binding capacity for dansylsarcosine was reduced in brain-dead patients compared to controls. endotoxin positivity in 16.7% of donors was associated with decreased binding capacity in donors and worse survival of recipients. lengths of icu stay increased albumin oxidation. in addition, il-6, il-8, il-10 and il-1 levels were elevated in patients, whereas ifn-levels were within the normal range. conclusions. we conclude that oxidative stress and systemic endotoxemia is present in brain-dead organ donors what might affect recipient survival. high endotoxin levels might be due to increased gut permeability and decreased binding capacity of albumin influenced not only by higher albumin oxidation. evidence of lymphoid neogenesis in skin biopsies of human hand allografts during rejection background. excessive production of reactive oxygen species (ros) is a major contributor to the development of ischemia-reperfusion injury (iri) in the course of solid organ transplantation. in particular mitochondria-derived ros are critical for the initiation and progression of iri, which restricts the pool of donor organs and results in elaborate follow up treatments. in various in vivo (ir) and in vitro (hypoxia/reoxygenation, hr) models we observed a consistent pattern in the activation of key intracellular signaling pathways. most strikingly the use of p38 specific inhibitors prevented mitochondrial ros production and cell death. here we further dissected the contribution of p38 to ir-and hr-induced damage and provide first evidence for a therapeutic benefit of p38 inhibition by birb-796 in vivo. methods. kidney transplantation and kidney clamping in the rat were used for the induction of iri. h/r was analyzed in hl-1 cardiomyocytes and primary mefs. intracellular signaling was monitored by using phosphorylation specific antibodies. mitochondrial ros levels were determined by imaging of mitotracker red cm-h2xros. ros/nos-induced tissue damage was visualized by 3-nitrotyrosine specific antibodies. to assess acute kidney injury (aki) hsp70 expression was monitored by immunoblotting, serum creatinine and urea were determined, and serum cystatin c and ngal concentrations were measured by elisa. results. the expression patterns for all p38 isoforms were established in hl-1 cells and sirna-mediated knockdown of the predominant isoform (p38alpha) reduced ros production, confirming the critical role of p38. preliminary data suggested the requirement of mapkap kinase 2 (mk2) rather than the transcription factor atf-2 downstream of p38. as observed in other settings reperfusion following kidney clamping or transplantation was marked by a profound increase in the activity of p38, its upstream kinases mkk3/6 and the putative effector mk2. application of birb-796 prevented deterioration of kidney function following ir based on reduced serum creatinine, urea, cystatin c and ngal levels in animals treated with the inhibitor. p38 inhibition also protected from oxidative damage. thus the inhibition of p38 prevents key processes, which are essential for the development of iri. conclusions. inhibiting p38 signaling during ir and hr may provide a potent strategy for limiting iri. the role of constitutively expressed nitric oxide synthases in ischemiareperfusion-injury background. single shot donor therapy with the essential nitric oxide synthase cofactor (nos) tetrahydrobiopterin (h4b) was shown to attenuate ischemia-reperfusion-injury-related pancreatitis in a murine pancreas transplantation (ptx) model. since underlying mechanisms of tetrahydrobiopterin-mediated protection are still controversially discussed, we aimed to investigate, whether the two constitutively expressed nos-isoforms represent its major targets using endothelial (enos-/-) and neuronal nos (nnos-/-) knockout mice. methods. in a heterotopic ptx-model syngeneic c57bl6 mice (wild-type, enos-/-and nnos-/-) were used as donorrecipient pairs. non-transplanted animals served as controls. following a reperfusion time of 4 h, graft microcirculation was analyzed by intravital fluorescence microscopy. parenchymal damage as well as peroxynitrite-formation were assessed by h&e-staining and immunohistochemistry. h4b levels were determined by high-performance liquid chromatography (hplc). finally, all groups where tested for recipient survival. results. compared to non-transplanted controls, prolonged cit significantly worsened microcirculation in untreated wildtype and enos-/-grafts (p < 0.05), whereas no deterioration was measured in untreated nnos-/-. while h4b-pre-treatment significantly restored capillary blood flow in wild-types and enos-/-(p < 0.01), no further beneficial effect was observed in nnos-/-(p > 0.05). in contrast to untreated wild-type and enos-/-graft, nnos-/-grafts developed minor parenchymal damage following prolonged cit, which could be slightly ameliorated by tetrahydrobiopterin pre-treatment. there weren't any significant differences between the analyzed groups regarding intragraft nitrotyrosine formation. while h4b pre-treatment extended survival of all recipients, significantly prolonged recipient survival was also achieved if donors were untreated nnos-/-(p < 0.01). conclusions. these observations in pancreatic grafts lacking nnos suggest, that instead of the endothelial isoform, it is the neuronal isoform to be crucially involved in the promotion of ischemia-reperfusion-injury in this model, representing therefore the major target of tetrahydrobiopterin mediated protection. nutrazeutika optimieren die organfunktion in einem rattennieren-ischämie-reperfusionsmode p. gehwolf 1 , f. m. struller 1 , a. kostron 1 , m. wolzt 2 , f. bach 3 , l. otterbein 3 , b. wegiel 3 , j. pratschke 1 , r. öllinger 1 tion, and angiogenic sprouting in vitro, and angiogenesis in a chicken embryochorioallantoic membrane assay. 5 0 -methoxy leoligin was consequently analyzed in an in vivo rat mi model. the novel compound potently stimulated angiogenesis in the peri-infarction zone and led to a significant increase in the cardiac ejection fraction (plus 20% 28 days after mi) in animals treated with 5 0 -methoxy leoligin. based on microarray analyses followed by knockdown and over-expression experiments in vitro cyp26b1 was identified as the central players in 5 0 -methoxy leoligin-induced angiogenesis induction. conclusion. the data presented herein indicate that 5 0methoxy leoligin induces angiogenesis in the peri-infraction area after mi via upregulation of cyp26b1. 5 0 -methoxy leoligininduced capillarisation is capable of partially restoring cardiac function after mi. therefore this novel compound could be able to positively influence post mi myocardial remodeling and even induce myocardial recovery. this or other pharmacological strategies to induce neoangiogenesis could preserve organ function and avoid several patients ending on the waiting list for cardiac transplantation. background. a major reason of vein graft failure after coronary artery bypass grafting is neointimal hyperplasia. several clinical studies revealed that the patency rates of arterial conduits are better than those of venous conduits. in the current study, we aimed to improve the patency rate of experimental vein grafts by induction of graft arterialisation by transplantation of homologous skeletal myoblasts. methods. for the experiments we isolated skeletal myoblasts from c57/bl6 mice. syngenic c57/bl6 mice underwent interposition of the vena cava inferior from donor mice into the common carotid artery of recipient mice. we used 2 experimental groups: one control group, where we applied only the control medium without any cells on the vein grafts and a treatment group where we applied skeletal myoblast periadventially on the grafts. after 4 weeks of follow up veins were harvested, embedded in paraffin and underwent histological evaluation. results. 4 weeks after surgery the neointimal thickness in the control group was 56 mm (15-60), whereas in the treatment group we found neointimal thickness levels of 183 mm (152-204) (p < 0.001). apart from that we could show that cells remain on the outside of the vein wall even after 4 weeks. conclusions. application of skeletal myoblasts does not induce vascular regeneration after vein graft failure and does not reduce neointimal hyperplasia. moreover application of these cells leads to a severe induction of neointimal hyperplasia in a murine vein graft model. this might be due to secretion of growth factors by the cells. background. based on international recommendations combined immunosuppression is used after kidney transplantation in children. a common regimen is based on steroids, mycophenolat-mofetil and cyclosporin a with il-2 antagonist induction. one third of the patients had to be switched from cyclosporin a to tacrolimus, due to dermatologic cosmetic side effects. thus, tabic was established as a novel immunosuppressive regimen. aim of the present registry is to perform quality assurance and to evaluate a risk/benefit analysis of the tabic-regimen compared to previous immunosuppressive regimens in children undergoing kidney transplantation at our center. methods. in this observational single-center study, 32 consecutive patients undergoing kidney transplantation between december 2005 and october 2011 at our center, were enrolled. patients who were not treated according to the tabic-scheme served as a historic control. the end-point was a combination of death and graft loss. results. 5 patients (16%) had to be switched from tacrolimus to rapamycin due to side effects. furthermore, 5 patients (16%) suffered from acute rejection, but no graft loss was observed. within follow-up, no patient reached the combined end-point (100% kidney transplant as well as 100% patients survival). a kaplan-meier-analysis revealed a significantly better outcome for patients treated with the tabic-scheme compared to controls (5 years event free survival, tabic vs. non-tabic: 100% vs. 79%, p ¼ 0.048). conclusions. our data show that the tabic-regimen is efficient and safe compared to cyclosporin a based immunosuppression. tacrolimus is associated with a significantly better graft and patient survival. therefore, it should be considered as standard therapy after kidney transplantation in children. falsely elevated tacrolimus concentrations in two kidney allograft recipients using the affinity column-mediated immunoassay (acmia) method: identification of igg isotype rheumatoid factor as causative endogenous antibodies background. therapeutic monitoring of tacrolimus is currently performed employing acmia in several transplant centers. here we report on two renal transplant patients displaying excessive tacrolimus levels either not corresponding to oral drug dosage or without taking the drug at all. various antibodies, such as anti ds-dna antibodies or heterophilic antibodies, have been identified in previous cases of falsely elevated tacrolimus measurements and have been suggested as a possible cause for interaction with the tacrolimus assay. methods and results. in our observations, both patients were on a stable, long-term tacrolimus-based immunosuppressive regimen. in the first patient the elevated tacrolimus trough levels were only observed after loss of graft function (6 years posttransplant) while the patient was not longer on tacrolimus, as the patient was on hemodialysis again. the second patient currently still has a functioning graft, although he is taking minute amounts of tacrolimus. all rheumatoid and immunologic tests (including anti ds-dna antibodies) were negative, except igg isotype rheumatoid factors, which proved to be highly positive in both patients. conclusions. our results show that certain patients with positive igg isotype rheumatoid factor can exhibit false-positive tacrolimus trough levels when using the acmia assay. it can therefore be concluded that abnormally high drug levels should be verified by mass spectrometry methods. further studies are underway to determine whether non-transplanted patients with positive rheumatoid factors also display falsely elevated tacrolimus values and also if this can be suppressed after preincubation with immunoglobulin blocking reagents specific for the igg isotype rheumatoid factor. background. the impact of vesicoureteral reflux after renal transplantation has been a matter of debate in the past with small studies reporting controversial results. the aim of the present study was to evaluate the effect of early post-transplantation vur on long-term renal allograft performance, incidences of urinary tract infections (uti), rejection rates, graft and patient survival in a large cohort series. methods. retrospective study of 646 consecutive renal transplant patients with routine post-transplantation vesicocystourethero-graphy (vcug). uretero-neocystostomy was performed in an antirefluxive fashion using extravesical submucosal tunneling. vcug was performed prior to discharge according to the international grading system and reviewed by an independent radiologist and urologist. results. overall, 263 of the 646 (40.7%) kidney transplant recipients were diagnosed with vur by vcug at discharge. patients had vur grade i, ii, iii and iv in 7.9%, 19.8%, 10.2%, and 2.8%, respectively. no grade v reflux was seen. vur showed no significant impact on death-censored graft survival, patient survival, proteinuria or uti. patients with vur had a significantly lower gfr at one year after transplantation than patients without vur (60 vs. 52 ml/min/1,73 m 2 , p ¼ 0.021), this difference was no longer seen at 3 and 5 years. conclusions. although vur is a common finding in patients after renal transplantation, it has no impact on death-censored graft survival, patient survival, proteinuria or uti and only a short term effect on renal graft function that was no longer seen in long term follow up. grundlagen. der einfluss von primär unentdeckten hepatozelluären karzinomen (hcc) auf das ü berleben nach lebertransplantation wird in der literatur kontroversiell diskutiert. ziel dieser studie war es, die rate von okkulten karzinomen in unserem kollektiv an äthyltoxischer zirrhose erkrankter und für diese indikation transplantierter patienten der letzten 10 jahre zu erheben und den einfluss auf das rezidivfreie uund gesamtüberleben nach lebertransplantation zu untersuchen. methodik. retropektive analyse von 285 wegen äthyltoxischer zirrhose gelisteter und transplantierter patienten im zeitraum von 1998 bis 2008. neben den baseline parametern wurden alle explanthistologien ausgewertet. ergebnisse. dreizehn patienten (4,5 %) zeigten ein hcc in der explanthistologie. drei patienten waren außerhalb der mailand kriterien und 19 von 27 herden waren größer als 1 cm. die wartezeit der patienten betrug im median 2,7 monate. nach 2004 gab es keine okkulten hccs in unserem kollektiv. ein ü berlebensnachteil fü r patienten mit okkultem hcc gegenü ber der normalgruppe konnte in diesem kollektiv nicht gezeigt werden. schlussfolgerungen. die kurze wartezeit der patienten mit okkultem hcc sowie die tumorgröße legt nahe, dass bei diesen patienten bereits vor der listung das hcc unentdeckt blieb, während es später mit verbesserung der radiologischen technik sowie längerer wartezeit im rahmen des screenings entdeckt wurde. rein deskriptiv zeigte sich in unserem kollektiv kein ü berlebensnachteil für patienten mit okkultem hcc. ist die ungeplante reoperation nach nierentransplantation ein indikator für organverlust? k. huber 1 , a. krause 1 , o. gangl 1 , w. enkner 2 , r. oberbauer 2 , r. függer 1 ö sophagusvarizen (grad ii; >5 mm unter insufflation) während der olt aufgetreten sind und mittels ballontamponade behandelt wurden. vergleicht man die beiden patientenkollektive (mit und ohne krampfadern), so ist das ergebnis statistisch nicht signifikant: chi-quadrat-test, p ¼ 0,61. alle patienten erhielten im median 2 einheiten erythrozytenkonzentrate (bereich 0 bis 70 einheiten), 7 einheiten fresh frozen plasma (bereich von 0 bis 60 einheiten) und 0 thrombozytenkonzentrate (bereich 0 bis 4 einheiten). obwohl patienten mit krampfadern eine signifikant längere prothrombinzeit und eine geringere thrombozytenzahl aufwiesen, bestand gegenü ber patienten ohne varizen kein unterschied in der verwendung von blutprodukten. schlussfolgerungen. die tee scheint bei olt patienten mit dokumentierten ö sophagusvarizen eine allgemein sichere methode zur kontrolle der herzfunktion zu sein. entwicklung des anteils an leberspendern mit erweiterten spenderkriterien innerhalb der letzten dekade an der universitätsklinik für transplantationschirurgie wien grundlagen. die anzahl der jährlich durchgeführten lebertransplantationen ist von der anzahl der gemeldeten organspender abhängig. allerdings eignet sich nicht jeder organspender als leberspender. ziel dieser studie ist es, die evolution der leberspender innerhalb der letzten 10 jahre zu verfolgen. methodik methodik. im zeitraum von jänner 1994 bis juli 2011 wurden an der medizinischen universität wien, abteilung für transplantation 22 patienten, davon 18 männlich und 4 weiblich, mit einem medianen durchschnittsalter von 51 jahren kombiniert leber und niere transplantiert. inkludiert wurden bei dieser retrospektiven analyse nur einzeitig transplantierte patienten. die indikationen zur lebertransplantation waren wie folgt verteilt: virushepatitis (n ¼ 6), malignom (n ¼ 4), metabolische zirrhose (n ¼ 5), oxalose (n ¼ 3), sklerosierende cholangitis (n ¼ 1), hämochromatose (n ¼ 1), citrullinämie (n ¼ 1) und zystische zirrhose (n ¼ 1). die nierentransplantation erfolgte aufgrund von: chronischer niereninsuffizienz o. n. b. (n ¼ 7), glomerulonephritis (n ¼ 4), metabolischer nephropathie (n ¼ 4), oxalose (n ¼ 3), pyelonephritis (n ¼ 1), hantavirus (n ¼ 1), iga-nephropathie (n ¼ 1) und cyclosporin-induziert (n ¼ 1). 72 % (n ¼ 16) der patienten waren zum zeitpunkt der transplantation dialysepflichtig, davon 13 an der hämodialyse und 3 erhielten peritonealdialyse. ergebnisse. die patienten waren durchschnittliche 121 tage gleichzeitig für leber und niere gelistet und der mediane meld (model for end-stage liver disease) score betrug bei transplantation 22. die 1-und 5-jahresüberlebensrate betrug bei unseren patienten 81 % und 67 %. unmittelbar postoperativ verstarben 3 patienten, davon 2 an einer sepsis und einer in folge einer primären nichtfunktion der leber. schlussfolgerungen. die klnt stellt an unserem zentrum eine gute therapieoption bei terminaler leber-und nierenerkrankung dar. laut eltr beträgt die 1-und 5-jahresüberlebensrate für alleinige orthotope lebertransplantation 78 % bzw. 64 % und ist daher mit unseren resultaten vergleichbar. die hohe perioperative mortalität der klnt spricht für ein rezent diskutiertes zweizeitiges vorgehen, um einen möglichen organverlust zu vermeiden. orthotopic liver transplantation (olt) under the use of a protective filter against phototoxicity in a patient suffering from erythropoietic protoporphyria (epp) with liver cirrhosis background. erythropoietic protoporphyria (epp) is a rare condition arising from a deficiency in the enzyme ferrochelatase, leading to abnormally high levels of protoporphyrin in the tissue. apart from increased photosensitivity leading to acute and chronic skin changes, in a small number of cases protoporphyrin deposits and pigment loading of the hepatocytes cause liver cirrhosis. in these patients liver transplantation is the therapy of choice. nevertheless intestinal ulceration, bleeding and ultimately multiorgan failure can occur if no protective measures are taken against phototoxic injury during surgery. methods and results. we would like to present the case of a 36-year-old patient who was admitted to our department for liver transplantation. epp had been diagnosed 32 years ago. the patient suffered from typical skin changes and cholestatic liver cirrhosis had been histologically confirmed. olt was performed successfully using protective measures to prevent phototoxic injury to the abdominal organs. a flexible yellow filter omitting wavelengths below 470 nm was applied to operating room luminaires to avoid phototoxic injury while maintaining visual colour perception of the surgeons. the immediate postoperative period was without any complications related to the epp. seven months after transplantation the patient is in good general health and liver function tests show good results. conclusions. olt is a suitable treatment for patients suffering from epp with hepatic involvement and cholestatic cirrhosis if protective measures against phototoxic injuries are used during surgery. günstige ergebnisse nach schweren immunologischen und infektiösen komplikationen nach dünndarm-transplantation: ein fallbericht c. bösmüller 1 , a. weißenbacher 1 , m. biebl 1 , r. öllinger 1 , s. schneeberger 1 , f. aigner 1 , s. weiß 1 , r. oberhuber 1 , c. ensinger 2 , j. pratschke 1 uneventful course after transplantation with only three mild acute rejection episodes within 11 years. due to a fracture of the right allograft radius requiring surgery at 10.5 years posttransplant immunosuppression (is) was switched from sirolimusmonotherapy to prednisone. one month thereafter the patient experienced metabolic deterioration (hypothyroidism, hyperlipidemia and hyperglycemia) and developed an exanthema and pruritus on the trunk and the upper extremities, including both the recipient's own skin and the allograft skin. bp was diagnosed upon direct immunofluorescence of skin biopsies (linear c3 and igg deposits along the basement membrane zone) and detection of serum autoantibodies against the bp antigen 2 (bp180nc16a: 142 u/l). histology showed a partial separation along the dermalepidermal junction and a mild perivascular infiltration consisting of lymphocytes, eosinophils and granulocytes. cell counts were within normal range. no donor specific antibodies were detected. skin lesions disappeared under intensified therapy with prednisone. while conversion of is and metabolic deterioration might have contributed to development of bp after hand transplantation, the pathomechanism remains unclear. belatacept treatment for 2 years after liver transplantation is not associated with overt immunomodulation background. belatacept is a costimulation blocker with immunomodulatory properties in the experimental setting. moreover, the liver graft itself has been claimed to have 'tolerogenic' properties. the belatacept multi-center phase ii liver transplantation trial was terminated during the long-term extension period on the recommendation of the data monitoring committee. this situation gave us the unique opportunity to evaluate the minimum immunosuppression required in liver transplant recipients after >2 years of treatment with belatacept. methods. in our center all belatacept patients (n ¼ 4) were switched to mmf monotherapy (2â1 g/day) after discontinuation of belatacept. we prospectively assessed the occurrence of acute rejection and evaluated kidney function (calculated gfr; mdrd) over time in comparison to tacrolimus-treated patients enrolled in the control groups of the multi-center trial (n ¼ 4). results. the mean period from transplantation to withdrawal of belatacept was 30 months (range 25-35). gfr at the time of withdrawal was more than 60 ml/min/1.73 m 2 in the belatacept group (4/4; mean: 101 ml [range 89-114]), whereas 3 out of 4 patients in the control group had a gfr below 60 ml/min/ 1.73 m 2 with a mean of 57.92 ml/min/1.73 m 2 (range 36-98) (p ¼ 0.026). five months after belatacept discontinuation kidney function declined on average by 19.22 ml (range à45 to þ3,5) (follow-up ongoing). after belatacept withdrawal all 4 patients developed 3-fold elevated liver enzymes (asat, alat) within 10.3 weeks after eot (7-14) (biopsies were not performed, mostly due to lack of consent.). patients were therefore switched to triple therapy with corticosteroids, cnis and mmf. graft dysfunction resolved within one to three weeks after switch. conclusions. consistent with results from the multi-center trial, patients treated with belatacept showed better kidney function compared to those treated with cnis. mmf monotherapy following withdrawal of belatacept is associated with a high risk of rejection. thus belatacept has no obvious immunomodulatory effect in liver transplant recipients that would be sufficient to allow a high success rate of minimization strategies. ausgezeichnete blutdruckkontrolle nach nierentransplantation durch antihypertensive mehrfachtherapie -entwicklungen der letzten 20 jahre grundlagen. patienten mit terminalen lebererkrankungen entwickeln gehäuft ö sophagusvarizen auf basis einer portalen hypertension. ziel dieser studie ist es, die inzidenz von ö sophagusvarizen und deren korrelation mit dem grad der lebererkrankung bei patienten, die auf der leberwarteliste stehen, zu errechnen. methodik. hierfür wurden die daten von 512 patienten, die zwischen 2002 und 2010 an der universitätsklinik für chirurgie lebertransplantiert wurden, analysiert. patienten, die die lebertransplantation aufgrund eines akuten leberausfalls benötigten und solche, bei denen die daten unvollständig waren, wurden aus der studie ausgeschlossen. somit umfasst die studie 396 patienten (77 %). die ergebnisse werden median in der 25. und der 75. perzentile angegeben. ergebnisse. die lebertransplantierten waren im median 54 (48-60) jahre alt. im median lagen 227 tage (125-368) zwischen der letzten ö sophagogastroduodenoskopie und der lebertransplantation. 287 patienten (72,5 %) zeigten varizen: 130 (32,8 %) varizen grad i ( < 5 mm bei insufflation) und 157 (39,6 %) var-izen grad ii (>5 mm bei insufflation). red spot signs fand man bei 40 patienten (10,1 %). 82,2 % der varizen lokalisierten sich im ö sophagus, 4,2 % im magen und 13,6 % im ö sophagus und magen. patienten mit ösophagealen varizen zeigten signifikant niedrigere serum-natrium werte und thrombozytenzahlen und signifikant höhere ammoniak-spiegel, prothrombinzeiten und meld-scores als solche ohne varizen. schlussfolgerungen. ö sophagusvarizen treten gehäuft bei patienten mit terminalen lebererkrankungen auf und werden von einer verschlechterung der labor-und meld-werte begleitet. background. a project called transplant procurement management (tpm) was created in barcelona, spain. the aim of this project is to provide profound knowledge and skills for health care professionals, working in the transplantation field with the final purpose of promoting and increasing donation rates. we evaluated the effects of this program on donor outcome and the organ procurement in a single center analysis. methods. number of donor procurements over a 1-year period, from june 1st, 2010 to may 31st, 2011, evaluated at the transplant center of the medical university vienna, the sixmonth period before and after participation in the tpm-course (nov 22nd to nov 26th, 2010) . results. in the 1st evaluation period from june 1st to nov 30th, 2010 74 potential donors were reported in our center out of which 31 became actual donors. within the 43 missed donors 23 did not fulfill brain dead criteria. moreover twelve donors could not be realized because of their co-morbidities and eight due to opposition against organ donation. in the comparable 2nd period from dec 1st to may 31st, 2011 we registered an increase of 42 actual donors out of 69 potentials. thirteen donors were missed because of unaccomplished brain dead criteria. moreover eight donors were lost due to their co-morbidities and six because of opposition against organ donation. however during this evaluation period the donor rates increased for about 35 %. the procurement of transplantable kidneys increased from 60 to 66, transplantable lungs increased from 12 to 13 and in liver procurement, we could even register an increase from 13 to 22 transplantable organs. only in heart procurement we registered a decrease from 16 to 13 transplantable organs. these results show a remarkable effect of this training program as well as the fluctuation of potential donors and the diverse potential of organ recovery during this year. conclusions. after all training programs in organ donation are an interesting field and seem to increase organ donation rates. efforts have to be made to provide and spread profound knowledge and skills for health care professionals, working in the transplantation field. pneumonia and had to be admitted. the patient with bos ii died due to acute respiratory distress syndrome (ards). both patients received no vaccination. the other two ltrs, one with bos i the other with bos ii and active vaccination protection were treated with oseltamivir at the time of the first clinical assessment, without any further respiratory complications. therapy with oseltamivir was only started with clinical assessment within the first 48 hours. conclusions. our review shows a causal link between preexisting bos, vaccination state and the severeness of respiratory symptoms after infection with community acquired respiratory viruses and the clinical effectiveness of seasonal influenza vaccination especially in bos patients. a. scheed 1 , a. aliabadi 2 , p. jaksch 1 , s. taghavi 1 , w. klepetko 1 , a. zuckermann 2 bei 836 patienten mit funktionierendem transplantat ein jahr nach der transplantation, die in unterschiedlichen perioden 1990/1991 (n ¼ 129) sowie die im rahmen von patientenselbstmessungen erhobenen blutdruckwerte analysiert bei patienten, die auf der leberwarteliste stehen, korrelieren ösophagogastrale varizen mit dem grad der lebererkrankung r. schwarzer 1 , m. thum 1 , r. karatosic 2 , u. burger-klepp mark 1 of recipient-, donor-and transplantcharacteristics were analyzed using uni-and multivariate analyses kinder mit hoch ungünstigem svm hatten höhere 24h-rr werte. die rr-differenz aus klinik-rr und 24hrr war jedoch höher bei kindern mit günstigem svm. es konnte kein zusammenhang zwischen svm und klinik-rr gefunden werden. schlussfolgerungen. unsere ergebnisse zeigen, dass stressverarbeitungsmechanismen bei der blutdruck-regulation nach ntx eine rolle spielen attitude towards xenotransplantation of patients prior and after human organ transplantation v 53 i iberer, f. 30 43 ö ttl, k. 41 p pacini druck: holzhausen druck gmbh, 1140 wien ö sterreich. -verlagsort: wien. -herstellungsort: wien. printed in austria p. b. b.= =erscheinungsort: wien= =verlagspostamt 1201 wien ergebnisse. unter konsequenter systemischer und topischer therapie mit liposomalem amphotericin b gefolgt von voriconazol, und reduktion der immunosuppression (tacrolimusspiegel um 10 ng/ml, cortison 10 mg), war die pneumonie rückläufig bis zum 9. po. monat. im 12. po. monat ist die patientin in gutem allgemeinzustand mit klinisch-bioptisch bestätigter stabiler transplantatfunktion, ausreichender oraler ernährung, stabilem körpergewicht, normaler leukozytenzahl nach beherrschten infektionen (zystitis, lidabszeß, herpesstomatitis).schlussfolgerungen. unter sorgfältiger anpassung der immunosuppression und konsequenter antimikrobieller therapie konnten eine stabile transplantatfunktion und lebensqualität erreicht werden nach schwerer akuter abstoßungsreaktion, cmv-transplantatenteritis und aspergilluspneumonie. bullous pemphigoid 11 years after bilateral hand transplantation background. expression of peripheral-node-addressin (pnad) on endothelial cells indicates presence of tertiary lymphoid organs (tlo) in chronic autoimmunity and allograft rejection. we herein investigated the expression of pnad in skin biopsies of human hand allografts for evidence of tlo after composite tissue allotransplantation.methods. 167 skin biopsies of 11 hand allografts were collected over 10 years and assessed by he-histology and immunohistochemistry using antibodies for pnad, cd3, cd4, cd8, cd20, c4d, cd68, lfa-1, icam-1, e-selectin, p-selectin, ve-cadherin, hla-dr, psoriasin, ido and foxp3. levels of pnad expression was assessed semiquantitatively (% of pnadþ vessels: 0, 1, 2, 3 and pnad staining intensity: 0, 1, 2, 3) and correlated with rejection grade, characterization of the infiltrate, expression of adhesion molecules and time after transplantation.results. rejection ranged from grade 0 to iv (mean score: 0.79 ae 1.05). upon rejection, expression of pnad was increased in endothelial cells (grade 0 : 0.24 ae 0.48 vs. all grades of rejection: 0.44 ae 0.62). most often pnad expression was only found in few vessels (1-10%). pnad staining intensity was increased the higher the grade of rejection (grade 0: 0.38 ae 0.76; grade i: 0.41 ae 0.74; grade ii: 0.67 ae 0.80; grade iii: 0.73 ae 0.91; grade iv: 0.50 ae 0.58). intense pnad-staining was associated with more cd4þ and cd8 þ infiltrating t-cells, but less b-cells and macrophages, compared to mild pnad staining intensity (cd4 þ cells 49.00% ae 29.89%; cd8 þ cells 31.00% ae 22.34%; cd20 þ b-cells 0.50% ae 1.54%; cd68 þ macrophages 0.57 ae 0.60 vs. cd4 þ cells 37.35% ae 40.82%; cd8þcells 27.35% ae 35.93%; cd20 þ b-cells 0.94% ae 2.02%; cd68þmacrophages 0.67 ae 0.66). pnad expression correlated well with cd3þcells and cd20þbcells. poor correlation was found for expression of adhesion molecules, ido and foxp3, except for lfa-1þ infiltrating cells. pnad expression was observed at all time-points after transplantation; however, staining intensity was enhanced very early and late after transplantation.conclusions. pnad expression in endothelial cells is increased in skin biopsies of human hand allografts indicating presence of tlo. further investigations are needed to enlighten the role of pnad and tlos in composite tissue allotransplantation. grundlagen. die zum hämabbau notwendige induzierbare hämoxygenase-1 (ho-1) wirkt stark antiinflammatorisch und schützt vor ischämie-und reperfusionsschäden (irs). eine klinische anwendung im sinne einer ho-1-induktion ist aufgrund der hepatotoxizität der experimentell verwendeten ho-1-induktoren nicht möglich. ziel der studie war es, natürlich vorkommende nutrazeutika auf ihr potential zur ho-1 induktion zu untersuchen.methodik. verschiedene nutrazeutika wurden in hinblick auf die ho-1-induktion mittels pcr getestet. in einem etablierten ratten-nierenarterien-klemmmodell wurde ein irs gesetzt, nierenfunktionsparameter und ho-1 expression wurden zu festgelegten zeitpunkten bestimmt, histologische untersuchungen wurde durchgefü hrt. die nutrazeutika wurden 24 h vor ischämie und unmittelbar nach reperfusion oral appliziert.ergebnisse. zwei der getesteten nutrazeutika führten zu einer starken hochregulation (resveratrol: 11-fach, ginseng: 17fach) der ho-1 expression. im nierenarterien-klemmmodell kam es nach 48 stunden zu einem anstieg des serum-kreatinin von 0,38 mg/dl ae 0,07 auf 3,06 mg/dl ae 0,86 in den kontrolltieren. die applikation von beiden nutrazeutika in einer dosierung von 10 mg/kg (resveratrol, 48h-kreatinin 0,54 mg/dl ae 0,23) bzw. 30 mg/kg (ginseng, 48h-kreatinin 0,53 mg/dl ae 0,06) verhinderte dramatisch die einschränkung der nierenfunktion (p < 0,0001 für beide vs. kontrolle). die kompetitive antagonisierung durch snpp (5 mg/kg/kg) konnte diesen positiven effekt vermindern. histologische und immunhistochemische auswertungen unterstützen die ergebnisse.schlussfolgerungen. die anwendung von fü r menschen ungefährlichen nutrazeutika stellt eine ausgezeichnete möglichkeit dar, die ho-1 zu induzieren und den irs zu minimieren. the novel compound 5 0 -methoxy leoligin increases the ejection fraction of infarcted rat hearts by promoting cyp26b1dependent angiogenesis background. in times of organ shortage strategies to recovery damaged organs seem to be valuable options to face this problem. cellular based strategies to recover ischemic myocardium have shown promising experimental results but the clinical evidence is scarce. especially the lack of angiogenesis in cardiac peri-infarction and infarction areas is one of the most important problems in functional cardiac recovery after myocardial infarction (mi). while searching for possible pharmacological strategies to stimulate angiogenesis after mi we conducted a screen for plant compounds with pro-neoangiogenetic properties.methods and results. 5 0 -methoxy leoligin, a compound isolated from the roots of leontopodium alpinum (edelweiss) potently stimulated endothelial cell migration, tube forma-grundlagen. ungeplante reoperationen dienen als indikator der qualitätskontrolle in der chirurgie. daten für die nierentransplantation liegen noch nicht vor.methodik. retrospektive analyse von 320 nierentransplantationen zwischen 9/2002 und 12/2010. die anzahl der ungeplanten reoperationen und ursache, mortalität, kreatininspiegel 1 monat und 1 jahr post tx und die art der immunsuppression in bezug zum organverlust wurden als mögliche risikofaktoren evaluiert.ergebnisse. in der analyse unseres patientenkollektives (320 nierentransplantationen über 100 monate) zeigt sich eine 14 % ige reoperationsrate, dies entspricht 46 reoperationen, wobei bei 16 % der reoperierten mehr als eine reoperation notwendig war. ursächlich dafür waren vordergründig gefäßkomplikationen (30 %) gefolgt von hämatomausräumungen und wundinfektionen, welche einer operativen sanierung bedurften (28 %). seltener kam es zu reoperationen aufgrund urologischer ursachen (20 %), lymphozelen und abdomineller komplikationen (je 6,5 %). von insgesamt 40 organverlusten waren 17 bei reoperierten patienten (das entspricht 43 % der reoperierten patienten) und 23 (9 %) bei nicht reoperierten patienten zu verzeichnen (p < 0,001). in der subanalyse der komplikationen, welche zu reoperationen führten, sind gefäßkomplikationen die häufigste ursache für vorzeitigen organverlust (64 %, das entspricht 9 von 14 reoperationen vs. 33 % bei lymphozelen, 33 % bei abdominellen komplikationen, 31 % bei weichteilproblemen und 11 % bei urologischen komplikationen). in bezug zur immunsuppression konnten keine statistisch signifikanten unterschiede erhoben werden. ein erhöhtes serum-kreatinin nach 1 monat (kreatinin >2) fand sich bei 42 % der reoperierten vs. 16,5 % der nicht reoperierten patienten (p < 0,001). nach einem jahr war das kreatinin bei 37 % der reoperierten patienten >2, hingegen nur bei 16,5 % der nicht reoperierten patienten (p ¼ 0,005 ergebnisse. varizen wurden mittels ö sophago-gastroskopie in 287 (72,5 %) von 396 untersuchten patienten dokumentiert, wobei nur in einem fall schwere blutungen aus case report. 11 years after bilateral hand transplantation a patient developed a bullous pemphigoid (bp), which is an autoimmune blistering skin disease, characterized by autoantibodies targeting the type xvii collagen component of hemidesmosomes in the skin basement membrane zone. this is the first report on an autoimmune complication in a patient after vascularized composite allotransplantation (vca). the patient showed a quite background. the mounier-kuhn syndrome (mks) is a rare disease characterized by a pathological dilation of the trachea and the bronchial system. the etiology of the disorder remains elusive but genetic alterations and degradation of elastic fibers are thought to be involved in the pathogenesis. no causative treatment is available although transplantation is an option for endstage disease. here, we describe a patient suffering from mks who received a lung transplant at our department.methods. a 39-year-old male never-smoker presented at our department for evaluation for a lung transplantat. the patient suffered from mounier-kuhn syndrome (mks) with greatly dilated trachea and bronchi. since a familial clustering of mounier-kuhn syndrome is discussed in the literature, we performed a chromosomal analysis and an array-cgh to search for genetic abnormalities. at the time of transplantation we collected samples from the bronchi and performed hematoxylin and eosin (he), elastic van-gieson's (evg) and immunohistochemical stains. specimens of main bronchi from the donor lung harvested for transplant served as control.results. the chromosomal analysis and array-cgh revealed that the patient's genome was completely unremarkable, the karyogram as well as the genome hybridization showed no significant gain or loss in known encoding regions. through the histological evaluation we found considerably lower amounts of elastic and collagen fibers in the submucosal layer in the patient compared to healthy controls. furthermore inflammatory infiltrates were present in the connective tissue throughout the whole histological specimen. since chronic inflammation is known to result in tissue remodeling we performed immunohistological staining of different matrix metalloproteinases. mmp1, mmp2, mmp3 and mmp9 were detected in mks tissue, but were totally absent in control bronchi.conclusions. based on these findings we hypothesize that the pathophysiology of mks is of a chronic inflammatory type leading to tissue destruction and a loss of elastic fibers, possibly due to upregulation of mmp. the triggering mechanism(s) for this inflammatory reaction remain elusive, but since mks has been reported to be associated with autoimmune diseases, an autoimmune reaction against components of the major airways could be one possible explanation. background. lung transplant recipients (ltrs) are uniquely predisposed in developing severe complications associated with community acquired respiratory viral infections (carv). we report the outcomes of influenza infections in a cohort of 82 screened lung transplant recipients at our center.methods. data were collected from december 2010 to march 2011 on using real-time polymerase chain reaction (pcr) from nasal secretion. during this period 245 patients frequented our out patient department for thoracic surgery. all ltrs (n ¼ 82) with respiratory symptoms were screened. there were 9 (10, 9%) confirmed cases. h1n1 infection was diagnosed in 5, influenza b in 4 lung transplant recipients, median age 36 (26-65) years, with a median of 6 (1,1-12) years post lung transplantation.results. all patients with bos grade 0 (bronchiolitis obliterans syndrome, n ¼ 5) were treated symptomatically alone, with no further impact on their lung function. two patients of this group were vaccinated for seasonal influenza. among the group of patients with pre-existing bos (n ¼ 4), two lung transplant recipients, one with bos i, the other with bos ii developed grundlagen. eine vielzahl von medikamenten mit potentiell toxischen nebenwirkungen auf den respirationstrakt sind bekannt. zytotoxische substanzen wie methotrexat oder bleomycin und andere pharmaka wie amiodaron oder diverse antibiotika werden am häufigsten als ursache fü r eine medikamentös induzierte interstitielle lungenerkrankung genannt.methodik und ergebnisse. wir berichten den fall eines 64jährigen patienten, der im oktober 2010 wegen dilatativer kardiomypathie mit terminaler herzinsuffizienz eine orthotope herztransplantation erhielt. zu diesem zeitpunkt bestand radiologisch bereits der verdacht auf eine amiodaron-induzierte lungenfibrose mit funktionell reduzierten werten: vc 3,49 l (73 %), fev1 3,28 l (86 %), mef50 6,52 l (135 %) und tlc 5,88 l (84 %). der postoperative verlauf gestaltete sich von kardialer seite komplikationslos, allerdings war der patient respiratorisch stark limitiert. der weaningprozess gestaltete sich aufgrund einer lobärpneumonie protrahiert. auch auf der normalstation fiel bei geringsten belastungen die sauerstoffsättigung auf bis zu 87 %. die lungenfunktion 2 monate nach htx ergab eine restriktive ventilationsstörung mit vc 1,33 l (37,9 %), fev1 1,29 l (34,3 %), mef50 3,3 l (71,2 %), tlc 2,76 (39,4 %), pao2 52,8 % und paco2 38,7 %. zur histologiegewinnung wurde nach inkonklusiver bronchoskopie eine offene lungenbiopsie durchgeführt, welche das vorliegen einer lungenfibrose vom uip-typ ergab. die kontroll-echokardiographien zeigten durchwegs eine gute rechtsund linksventrikuläre funktion, allerdings verschlechterte sich der respiratorische zustand des patienten zunehmend, sodass er "high urgent" zur lungentransplantation gelistet werden musste. ein größen-und blutgruppenkompatibles spenderorgan wurde verfügbar und eine bilaterale lungentransplantation mit veno-arterieller ecmo unterstützung durchgeführt. die ecmo konnte am ende der operation bei hämodynamisch und respiratorisch stabilen verhältnissen wieder explantiert werden. die ischämiezeit der rechten lunge betrug 340 minuten und links 460 minuten. am 19. postoperativen tag konnte der patient auf die normalstation verlegt und nach weiteren 23 tagen aus dem krankenhaus entlassen werden.schlussfolgerungen. entgegen der allgemeinen erwartung, dass eine fibrose unter immunsuppressiver therapie ein geringes risiko fü r progression hat, zeigte sich in diesem fall eine dramatische entwicklung nach der herztransplantation. deshalb sollte eine lungenfibrose vor einer htx genau abgeklärt und engmaschige postoperative kontrollen durchgefü hrt werden. invasive mycoses after heart transplantation: outcome and long-term prognosis t. haberl 1 , d. hutschala 2 , c. pelanek 1 , a. aliabadi 1 , g. laufer 1 , a. zuckermann 1background. although orthotopic heart transplantation has become a routine procedure in treatment of end-stage cardiac failure, postoperative mycosal infections are still a serious risk for the patients. objective of the current study is to show the influence of various parameters on the severity of infection and the outcome for patients.methods. this is a single-center retrospective study including 338 patients who underwent heart transplantation from 2000-2008 at our center in vienna. the analyzed data were collected during the post transplant course in the intensive care unit (icu). mycoses were diagnosed by cultures of blood, bronchoalveolar lavages (bal) and smear tests of central arterial and venous catheters, wounds and drainages.results. mean age at time of transplantation was 49 years, 260 patients (76.9%) were male (age 5-71 years) and 78 patients (23.1%) were female (age 3 weeks-76 years). 90 patients (34%) acquired an invasive mycosal infection with candida species (94.3%), aspergillus (3.8%) and pneumocystis carinii (1.9%). diagnosis was performed in 24.1% by cultures of blood, in 23.5% by bal, 18.8% by smear tests of vena-cava catheters and in 11.1% from pulmonal artery catheters. in 77.6% treatment with a single antimycosal therapy was sufficient. 20.9% of patients were in need of a double-drug therapy and 1.5% needed a triple-drug-therapy. patients with mycosal infection had an median stay in the icu of 16 days (1st quartile: 7.25, 3rd quartile: 24.75) with a median of 4.5 (q1: 0, q3: 7) respirator-days compared with patient without infection who had median 9 (q1: 4, q3: 16) days in icu with 1 (q1: 0, q3: 2) respirator-days. 100% of the patients with mycosis compared to 49% of the patients without mycosis developed or already had bacterial co-infection (p < 0.001). the 1-year survival of patients with mycosis was 63.3% compared to 248 patients without infection where 1-year survival was 83.3% (p < 0.001). average time to first fungal infection was 7.6 ae 5.3 days.conclusions. invasive mycosal infection acquired by immune suppressed patients after heart transplantation is associated with prolonged time of stay on the icu as well as higher rates of co-infections and a significant increase of mortality post transplant.background. xenotransplantation is a potential strategy to overcome the shortage of human donor organs. since this technique has a major medical and psychological impact on patients and their family and friends, the attitude of patients currently waiting for organ transplantation is important.medhods. therefore we conducted a survey on the attitude towards xenotransplantation of patients on the waiting list and already transplanted patients. patients received detailed information before being asked to fill in the questionnaire.results. we found that 65% would accept xenotransplantation, irrespective of gender, education level or if the patients were on the waiting list or already transplanted. the most common concern was transmission of diseases or genetic material, followed by psychological concerns and ethical issues. more patients had a positive attitude towards accepting cell or tissue transplantation as compared to whole organs. pig pancreas islet cell transplantation is generally well accepted, patients with diabetes mellitus show even higher acceptance rates than patients without diabetes.conclusions. xenotransplantation seems to be well accepted in patients who are potential future candidates for organ transplantation. informing patients about the current status of research tended to decrease acceptance rates slightly. key: cord-006856-b1w25ob5 authors: nan title: 19th meeting of the austrian society of transplantation, transfusion, and genetics, october 26–28, 2005 date: 2005 journal: eur surg doi: 10.1007/s10353-005-0216-6 sha: doc_id: 6856 cord_uid: b1w25ob5 nan background. nowadays lung transplantation is an established standard procedure for the treatment of most end-stage respiratory disorders. in the last years, like for all solid-organ transplantations, the demand for donor lungs exceeded significantly the existing organ pool. thus, most specialised centers face a high mortality on the waiting list. this retrospective study compares the outcome of marginal versus ideal donor lungs. methods. we performed a retrospective analysis of 98 consecutive primary lung transplantations from 94 donors from 1/2001 to 12/2002 . recipients were divided in two groups (standard versus "extended") according to the donor lung acceptance criteria (age, >55 years; pao 2 at fio 2 /peep 5, <300 mmhg; positive tobacco anamnesis [>20 packages per year]; inhalation of noxious agents; presence of infiltration on chest x-ray or purulent secretions at bronchoscopy). results. twenty-three donors (24.5%) were extended. twenty-six recipients (26.55%) received organs from extended donors. according to our data, differences in intubation times, icu stay, and hospital stay were not statistically significant. furthermore, postoperative bleeding rates were comparable as well as bronchial anastomotic complications. we encountered no significant statistical difference in the 3-month (standard 88.89% vs. extended 92.31%) and 1-year (standard 81.94% vs. extended 84.62%) survival between the two groups. conclusions. our study suggests that the use of selected marginal donor lungs has no influence on the outcome after transplantation. background. female donor gender has been described to be an independent risk factor for primary graft failure. we performed this study to evaluate the impact of donor gender on outcome and complications after lung transplantation. methods. we retrospectively reviewed the impact of donor gender on outcome of 163 primary lung transplant recipients (93 recipients were male [57%], 70 were female [43%]) from january 2001 to december 2003. recipients were stratified whether they received a female-or male-donor organ. both groups were compared with regard to duration of intubation time, icu stay, postoperative complications and survival. both groups were comparable with regard to mean age, indications, and mean waiting list time. results. mean time until extubation was 8 days in the group receiving organs from male donors and 16 days in the group receiving organs from female donors (p = 0.041). mean icu stay of 12 days for the male-donor recipient group was significantly shorter than that for the female-donor recipient group, 20 days (p = 0.044). 3-month survival rates were comparable in both groups: 89.53% (male-donor recipi-lunge 19th meeting of the austrian society of transplantation, transfusion and genetics background. nowadays lung transplantation is an established therapeutic option for most end-stage respiratory diseases and one of the fastest-growing solid-organ transplantation procedures in the world, reflected by the enormous demand for lungs. this retrospective review of the vienna lung transplantation group demonstrates data about waiting time and mortality rate for various end-stage respiratory diseases for the years 2003 and 2004. methods. . different variables specific for pretransplant period such as probability of transplantation, death on the waiting list, and time till transplantation were analysed for most frequent end-stage respiratory diseases. results. we found no significant changes in the average mortality rate (constantly 8%) on the waiting list during the studied period. however, patients suffering from pph have the highest probability of dying while being listed for lung transplantation (2003, n = 2 [20%] ; 2004, n = 2 [14.3%]), followed by patients with idiopathic fibrosis (2003, n = 3 [15%]; 2004, n = 4 [11.4%] ). subsequently the next subgroup is represented by cystic fibrosis patients, who are characterized by a moderate mortality rate (2003, n = 1 [3.8%] ; 2004, n = 1 [5%]). in contrast, patients who were suffering from copd showed the lowest probability of dying (2003, n = 0 [0%]; 2004, n = 1 [2.9%]). the average waiting time for the observed 2 years amounts to 97.5 days (2003, 99 ± 98 [min, 0; max, 464] days; 2004, 96 ± 96 [min, 0; max, 400] days). regarding the disease-specific waiting time, pph patients had to wait a longer period (133 ± 128 days) for their transplantation than patients with other diagnosis. conclusions. waiting time and mortality on the waiting list are showing remarkable differences within the disease-specific subgroups. v05 increased recipient vegf serum level is a risk factor for severe reperfusion edema after lung transplantation k. krenn, s. taghavi, w. klepetko, s. aharinejad laboratorium für kardiovaskuläre forschung, zentrum für anatomie und zellbiologie, medizinische universität wien, wien, österreich background. primary graft dysfunction (pgd) due to ischemia-reperfusion injury is a severe complication in lung transplantation (ltx) . therapeutic strategies are limited and there exist no preoperative markers to predict the risk for reperfusion-induced edema. vascular endothelial growth factor (vegf) is a key regulator of vascular permeability. methods. preoperative vegf serum levels were measured by elisa for 76 patients undergoing ltx. underlying diseases in ltx patients were copd (n = 22), cystic fibrosis (n = 15), idiopathic pulmonary fibrosis (n = 11), emphysema (n = 6), primary pulmonary hypertension (n = 6), sarcoidosis (n = 4), and others (n = 12). the ischemia time of the grafts and the blood gas parameters in donors were comparable. reperfusion edema was diagnosed and scored by characteristic changes in chest radiographs and deteriorating blood gases according to the guidelines of the international society for heart and lung transplantation, grading pgd from 0 to 3 (0, none; 1, only radiographic evidence; 2, moderate, pao 2 /fio 2 ratio of 200-300; 3, severe, pao 2 /fio 2 ratio of <200 or ecmo support necessary). results. grade 3 pgd occurred in 15%, grade 2 in 23%, grade 1 in 43%, and grade 0 in 19% of the patients. the preoperative vegf serum levels were significantly higher in patients with pgd grade 2 and 3 versus those without clinically relevant pgd (grade 0 and 1) following ltx (p = 0.0007). preoperative recipient vegf serum levels significantly predicted pgd in receiver operating characteristic analysis (p = 0.0002, auc = 0.755, ci = 1.001-1.003). conclusions. preoperative serum vegf levels in patients awaiting ltx could identify those at risk for reperfusion-induced edema following transplantation. background. airway stenoses are well known after lung transplantation although most occur due to surgical problems of the bronchial sutures. we wanted to analyse the use of endobronchial stenting in stenoses not related to the bronchial anastomoses. methods. we performed a retrospective analysis in 12 patients after bilateral lung transplantation with consecutive stent placement aside the bronchial anastomoses. the indication for stent implantation was central bronchial stenoses due to bronchomalacia, granulation tissue with bronchial wall destruction, and endoluminal stenosis with airflow limitation or occurrence of segmental or lobar atelectasis. we used boston ultraflex (23 of 26), rüsch polyflex (2 of 26), rüsch dynamic (1 of 26) stents. results. in 12 lung allograft recipients, we implanted in total 26 stents between 2 and 72 month after bilateral lung transplantation. the predominant locations were the right (20%) and left lower lobe bronchus (25%) and the bronchus intermedius (33%). the patient had granulation tissue proliferation due to ischemia and/or chronic bacterial or fungal bronchitis. only one patient had concomitant anastomoses dehiscence. the stents remained in situ from 1 day to 850 days after placement. in 18 of 26 stent placements no severe complications occurred. stent migration was observed in 6 of 26; severe granulation tissue that revealed further interventional treatment, in 4 patients. one patient died because of necrotizing vasculitis and letal hemoptysis. all other stents are still in place (10 of 26), were in place at the time of death due to other reasons (4 of 26), or were explanted regularly (5 of 26). conclusions. endobronchial stent placement is an effective treatment for bronchial stenoses that are not related to the bronchial anastomoses. complications occurred in about 40% of all stents. v07 preoperative oral corticosteroids predict the risk of late postoperative bleeding and perioperative mortality in lung transplantation conclusions. patients listed for lung transplantation with high-dose preoperative oral cs intake have a significantly increased risk of late postoperative bleeding. the perioperative mortality and the probability of 1-year survival of recipients with late bleeding are severely affected compared with patients with no or early bleeding. background. the efficacy of induction therapy after lung transplantation remains controversial and data on its use are limited. we hypothesized that induction therapy would have an impact on incidence of early rejection after lung transplantation and may cause a higher rate of infectious complications during the first 6 months post transplantation. methods. all patients who underwent lutx between jannuary 2003 and march 2004 and received induction therapy with rabbit antithymocyte globulin (2.5 mg/kg/d for 3-6 days) were analysed retrospectively. basic immunosuppression consisted of tacrolimus, mycophenolate mofetil, and prednisone. primary end point was patient survival after 6 months. secondary end point was histologically proven grade i or higher grade of rejection within the first 6 months, incidence of infectious episodes and bronchiolitis obliterans syndrome (bos). a total of 29 adult patients who underwent single (n = 2), heart-lung (n = 2), or bilateral lung (n = 26) transplantation entered the study. female, 10 (35%); male, 19 (65%). mean age was 37 ± 12.9 years (range, 19.3-59.9 years). results. the 6-month survival using kaplan-meier analysis was 92%; 27 patients are alive, two died 151 and 160 days after transplantation. one patient was retransplanted 3 days after primary transplantation because of graft dysfunction. follow-up ranged from a minimum of 151 days to 492 days (mean, 279 ± 85 days). 14 patients had one histologically proven rejection episode. rejections were graded ai for 10 patients, grade ai-ii for 2, and grade aii for 2 patients. 7 rejection episodes were treated with iv methylprednisone, no recurrent or ongoing rejections were observed. incidence of bacterial infections requiring treatment was 21/100 patients days. 2 cmv infections, 1 non-cmv viral infection, and 4 fungal infections (2 candida, 2 aspergillus) were diagnosed and treated. no patient developed signs of bos of grade ≥1. no lymphoma occurred. conclusions. this retrospective analysis suggests that induction therapy with atg in combination with tacrolimusbased triple immunosuppressive regimen results in excellent survival rates and a low rate of acute early rejections. however, this high immunosuppression efficacy was paralleled by a considerably high rate of bacterial infections during the first 6 postoperative months. rin inhibitor based quadruple drug is. an extensive infectious monitoring was used in this cohort. results. one-year patient survival was 71.4%, perioperative rejection rate 35%. infection incidence during first hospitalization was 79.6% (1.3 episodes per transplant): pneumonia, 74%; sepsis, 13%; wound infection, 17%; hsv/vzv, 28%; uti, 2%. during follow-up, cmv-associated complications were observed in 50% of patients including cmv infection (n = 9), cmv disease (n = 16). there were nine patients with cmv syndrome, ten patients with cmv graft pneumonitis and two patients with cmv gastrointestinal disease. excluding the 3 retransplants and the 2 perioperative deaths, the incidence of aspergillosis was 27%, six patients with aspergillus tracheobronchitis and seven patients with invasive disease. a total of 33 patients died during follow-up, 28 from infectious complications. as part of our centers' microbiological monitoring, 2773 specimens (51/transplant) were collected. these specimens were taken on 1276 observation days. 1426 investigated specimens were sterile and in 993 specimens microorganisms could be isolated (354 normal flora, 639 pathogens). a total of 1155 pathogens were identified: 673 gram-positive cocci, 177 gram-negative rods, 155 pseudomonas/acinetobacter, 124 candida. >60% of staphylococcus aureus and 55% of coagulase-negative staphylococci were methicillin resistant. other multiresistant organisms were e. faecium (no vre), n = 75; corynebacterium jk, n = 2; stenotrophomonas maltophila/burkholderia cepacia, n = 31. conclusions. infection remains the most common complication and the most common cause of death following lung transplantation. further refinement of infectious prophylaxis is required to improve results. background. valgancyclovir is routinely used for prophylaxis against cytomegalovirus (cmv) reactivation in lung transplant recipients. problems may arise due to its myelotoxic properties. it is unclear whether cytotect ® , a human igg cmv antibody preparation, offers similar protection against cmv reactivation without causing neutropenia. methods. we report on a female patient (63 years) who received a double lung transplant in 2002. cmv status was d+r+. four months postoperatively, she developped cmv pneumonitis and gastroduodenitis and was treated with gancyclovir and cytotect. thereafter, valgancyclovir was instituted but had to be stopped repeatedly because of neutropenia; each withdrawal of valgancyclovir prompted further reactivation episodes as detected by pp65 monitoring. in july 2004, she was started on cytotect, 150 mg q.d. over five days, followed by weekly doses of 50 mg for twelve weeks. results. cytotect was tolerated without side effects. of note, leucocyte counts remained within normal limits. since she has been started on cytotect, she has not experienced any further episodes of cmv reactivation until to date. conclusions. the observations made in this patient suggest that long-term therapy with cytotect ® has the potential to prevent cmv reactivation in selected patients who do not tolerate valgancyclovir due to its myelotoxic side effects. v12 die kombinationsprophylaxe verbessert die cmv bedingte morbidität und mortalität und reduziert das risiko der bronchiolitis obliterans (bos) nach lungentransplantation e. ruttmann, c. geltner, b. bucher, h. ulmer, d. höfer, h. b. hangler, s. semsroth, h. bonatti, r. margreiter, g. laufer, l. müller klinische abteilung für herzchirurgie, universitätsklinik für chirurgie, medizinische universität innsbruck, innsbruck, österreich grundlagen. die opportunistische cmv-infektion stellt ein schwerwiegendes problem nach lungentransplantation dar. ziel dieser untersuchung war, den einfluss der cmv-kombinationsprophylaxe mittels ganciclovir und cmv-hyperimmunglobulinen (cmv-ig) bezüglich patientenüberleben, cmv-reaktivierung, klinischer cmv-erkrankung und entwicklung der bronchiolitis obliterans (bos) zu evaluieren. methodik. eine konsekutive serie von 68 cmv-hochrisikopatienten (d+/r-, d+/r+) mit einem minimalen followup von mindestens 1 jahr post-transplant wurden analysiert. dreißig patienten (44,1 %) erhielten eine alleinige ganciclovir-prophylaxe für 3 monate (kontrollgruppe), 38 transplantationsempfänger (55,9 %) erhielten eine zusätzliche prophylaxe mit cmv-ig (cytotect ® , biotest pharma) in 5 dosen während des 1. postoperativen monats (studiengruppe). das mediane follow-up betrug 16,5 monate in der kontrollgruppe und 23,8 monate in der studiengruppe (p = 0,54). ergebnisse. insgesamt 5 cmv-assoziierte todesfälle (16,7 %) ereigneten sich in der kontrollgruppe, jedoch keiner in der studiengruppe (p = 0,014). in der kontrollgruppe wurden 13 fälle mit klinischer cmv-erkrankung beobachtet (43,3 %), in der studiengruppe 5 patienten (13,2 %) (p = 0,007). zusätzlich zeigte sich ein signifikant verbessertes patientenüberleben in der studiengruppe (log-rank, p = 0,01). die 1-jahresfreiheit von cmv-reaktivierungen betrug 52,1 % in der kontrollgruppe und 71,5 % in der studiengruppe (logrank, p = 0,027). die 3-jahresfreiheit von bos war signifikant höher in der studiengruppe (54,3 % vs. 82 %, log-rank, p = 0,024). schlussfolgerungen. eine zusätzliche cmv-hyperimmunglobulinprophylaxe senkt die cmv-assoziierte morbidität und mortalität. weiters kann das auftreten der bos mittels augmentierter cmv-prophylaxe reduziert werden. durch die dadurch reduzierte morbidität ist die kosteneffizienz gegeben. v13 extracorporeal photoimmune therapy (ecp) with uvadex in conjunction with standard therapy compared to standard therapy alone for the prevention of rejection in lung transplantation patients p. jaksch, r. knobler, b. schlechta, s. guth, w. klepetko klinische abteilung für herz-thoraxchirurgie, universitätsklinik für chirurgie, medizinische universität wien, wien, österreich background. extracorporeal photopheresis has been shown to be beneficial in acute and chronic rejection in heart transplant patients and has also been used in lung transplant recipients with acute rejection or bronchiolitis obliterans. methods. we performed a prospective study to document the efficacy of photoimmune therapy in the prevention of acute rejections in the first 12 months after lung transplantation. 12 lung transplant recipients with copd were randomized in 2 groups. group a (6 pat) received in total 16 ecp treatments (starting 2 weeks after tx) and group b without ecp or other kind of induction therapy, both groups receiving standard triple immunosuppression with tacrolimus, mycophenolate, and steroids. surveillance bronchoscopies with biopsies were performed after 2, 4, 8, 12, 26, and 52 weeks. primary objectives were acute biopsy-proven rejections of ishlt grade >1, secondary objectives were number of infections (cmv, bacterial, fungal, viral non-cmv, tuberculosis, parasitic), patients and graft survival. results. demographics in both groups were similar (gender, age, underlying disease, cmv mismatch, and type of tx). fev 1% and mef 50% values after 1 year were equal (88.3 ± 7.9% vs. 90 ± 28. 1 and 113.5 ± 41.7% vs. 114.5 ± 42.2%, respectively) . the number of rejections in group a (with ecp) was lower than in group b (0.17 ± 0.41 vs. 0.83 ± 0.75, p = 0.094), as well as the median rejection grade (0.16 ± 0.4 for group a vs. 0.66 ± 0.51 for group b, p = 0.092). after one year post tx, all patients are alive, none developed bo(s) within the first year post tx. conclusions. our preliminary data show a clear trend towards reducing the number and severity of acute rejections in lung transplant recipients. the number of infections was similar in both groups. adding ecp to a standard triple-drug immunosuppressive regimen seems to be a safe and efficient tool in reducing rejection rates without increasing the rate of bacterial, fungal, or viral infection. grundlagen. erhöhte natriumspiegel bei organspendern können mit der bildung von reperfusionsödemen und transplantatdysfunktion assoziiert sein. unklarheit besteht allerdings über die klinischen auswirkungen von erhöhtem spender-natrium nach herztransplantation (htx). in dieser studie wurde der einfluss von hohem spender-natrium auf die frühund 1-jahresmortalität nach htx in einem großen patientenkollektiv analysiert. methodik. es wurden 3157 herztransplantationen der eurotransplant-region aus dem zeitraum jänner 1997 bis dezember 2001 analysiert. entsprechend der spender-natrium-spiegel (sns) wurde das kollektiv in drei gruppen unterteilt: gruppe a, <160 mmol na + je liter n = 2903; gruppe b, 160-170 mmol na + je liter n = 218; gruppe c, ≥170 mmol na + je liter n = 54. eine kaplan-meier-überlebensanalyse und eine multivariate analyse bezüglich des einflusses des sns wurden durchgeführt. endpunkte waren die mortalität ein monat und ein jahr nach htx. ergebnisse. der sns hatte in der univariaten analyse keinen einfluss auf die frühmortalität und einen grenzwertig signifikanten einfluss auf die 1-jahres-mortalität (p = 0,06). in der multivariaten analyse war dieser effekt nicht signifikant (p = 0,2). die kombination aus hohem spenderalter mit hohem sns hatte jedoch in der multivariaten analyse einen hochsignifikanten einfluss auf die früh-und 1-jahresmortalität (p = 0,004). schlussfolgerungen. diese daten zeigen, dass hohe sns mit einer erhöhten früh-und 1-jahresmortalität nach htx einhergehen. diese ergebnisse stehen im gegensatz zu früheren arbeiten mit geringeren patientenzahlen. vor allem die kombination aus höherem spenderalter und hohem sns zeigt einen deutlichen risikoanstieg. herzen von spendern mit einem natriumspiegel von >170 mmol/l sollten nicht elektiv transplantiert werden, bei gleichzeitig hohem spenderalter sollte das herz nicht verwendet werden. die organempfänger wurden entsprechend dem spenderalter in 2 gruppen geteilt: gruppe 1, <35 a, n = 12; gruppe 2, >35a, n = 10. die gruppen waren hinsichtlich organischämiezeit, geschlechts-und cmv-mismatch sowie lipidstatus, nierenfunktion und medikamentöse therapie (immunsuppresion, ace-hemmer, statine, ca-antagonisten) 1 jahr post-htx vergleichbar. pv1 war in gruppe 1 tendenziell niedriger als in gruppe 2 (25,2 ± 17,3 mm 3 vs. 34 ± 22 mm 3 ; p > 0,05). umgekehrt wies gruppe 1 im verlauf des 1. jahres nach htx einen zuwachs an plaquevolumen auf, während in gruppe 2 eine abnahme (∆pv, 2,6 ± 17,6 mm 3 vs. 3,0 ± 10,2 mm 3 ; p > 0,05) festgestellt wurde. von den oben angeführten risikoparametern zeigte lediglich der triglyzeridspiegel 1 jahr post htx eine signifikante korrelation mit ∆pv (r = -0,613, p = 0,002). ∆pv und spenderalter waren nicht miteinander korreliert. schlussfolgerungen ergebnisse. die mortalität beträgt 0 %. wegen gastrointestinaler beschwerden (übelkeit, erbrechen) mussten 3 patienten (12%) auf ein anderes immunsuppressives schema umgestellt werden. bei 2 patienten (everolimus-talspiegel, >8 ng/ml) zeigte sich eine schwere pneumonie (pseudomonas), welche stationär behandelt wurde. es gab keine stationäre behandlung wegen cmv-infekten. die nierenfunktion war in allen patienten stabil (mittleres crea, 1,84 ± 0,85), außer in 2 patienten, welche bereits vor der umstellung erhöhte kreatininwerte zeigten und in denen sich eine weitere erhöhung der kreatininwerte (+20,25%) feststellen ließ. eine bei den meisten patienten auftretende hyperlipidämie konnte unter erhöhung der statintherapie eingestellt werden. in den routinemäßig durchgeführten endomyocard-biopsien fanden sich einen monat nach umstellung und danach keine akuten zellulären abstoßungen mit grad von >1b nach dem ishlt-grading. schlussfolgerungen. certican erwies sich als sicher und verträglich, die umstellung auf das neue immunsuppressivum war in allen patienten komplikationslos. everolimus-talspiegel von 8 ng/ml scheinen ausreichend, höhere spiegel könnten das infektionsrisiko erhöhen. bezüglich der nierenfunktion bleibt abzuwarten, wie sich ein cyclosporin-a-talspiegel von 60-80 ng/ml auswirkt. eine aussage bezüglich der cav steht zu diesem zeitpunkt noch aus. v17 20 jahre herztransplantation in wien (eine retrospektive über 1000 transplantationen) a. zuckermann empfänger-und spenderalter sind im laufe der jahre signifikant gestiegen (recipient age: 42,5 ± 11,8 vs. 52,0 ± 13,8, p < 0,05; donor age, 28,8 ± 10,3 vs. 35,2 ± 12,9, p < 0,01) . mehr patienten wurden präoperativ stationär aufgenommen (28 % vs. 44 %), jedoch hat sich die zahl der intensivpflichtigen patienten signifikant reduziert (21 % vs. 7 %). die zahl der patienten, die zur transplantation "gebridged" werden, ist ebenfalls massiv angestiegen (28 vs. 63 %, p < 0,01). innerhalb dieser gruppe hat die gruppe der patienten mit mechanischer herzunterstützung am stärksten zugenommen (7 % vs. 19 %, p < 0,01). pharmakologisches bridging (55 % vs. 42 %) und aicd (38 % vs.40 %) blieben stabil. pharmakologisches bridging wird heutzutage vermehrt mit prostaglandinen und levusimendan als mit dopamin oder dobutamin durchgeführt. mehr patienten sind am herzen voroperiert (28 % vs. 52 %, p < 0,01), patienten warten länger auf die transplantation (75,9 ± 91,6 vs. 289,8 ± 368,8, p < 0,01) . trotzdem hat sich die mortalität auf der warteliste stark verbessert (27,6 % vs. 12,0 %, p < 0,01), was ein klares zeichen der verbesserten überbrückungsmaßnahmen ist. ischämiezeiten sind ebenso angestiegen (129,3 ± 49,8 vs. 190 ,0 ± 49,2, p < 0,01) wie die liegezeiten auf der intensivstation (4,7 ± 5,7 vs. 7,6 ± 7,7, p < 0,01). dies ist ein klares indiz dafür, dass heute ältere, kränkere und komplexere patienten transplantiert werden. diese veränderungen sind international bei allen zentren zu be-obachten. dies hat dazu geführt, dass mit der zunehmenden erfahrung und verbessertem überleben die nachbeobachtungszeit der patienten stark gestiegen ist und damit die behandlung dieser patienten kostenintensiver geworden ist, was aber mit der guten lebensqualität der herztransplantierten patienten zu rechtfertigen ist. v18 neoplastic diseases after heart transplantation: a retrospective study d. kammerstätter, a. aliabadi, j. ankersmit, d. dunkler, g. wieselthaler, e. wolner, m. grimm, a. zuckermann klinische abteilung für herz-thoraxchirurgie, universitätsklinik für chirurgie, medizinische universität wien, wien, österreich background. prolonged immunosuppression after solidorgan transplantation is associated with an increased risk for development of neoplasms. the purpose of this analysis was to investigate neoplasm incidence and outcome in patients with induction therapy. methods. 921 cardiac recipients were included in this retrospective analysis. all patients received antibody induction therapy with either polyclonal-atg or monoclonal antibodies. neoplasms were devided into 3 groups: (1) skin cancer, (2) ptld, (3) other neoplasms. overall incidence of neoplasms, patient survival after diagnosis of neoplasms were analysed by the kaplan-meier method. results. a total of 143 tumors were diagnosed at a mean follow-up of 59.6 ± 51.2 months after cardiac transplantation. freedom from neoplasms was 96.7%, 86.6%, and 71.5% after 1-, 5-, and 10-year respectively. 5-year survival after diagnosis of tumor was 50.3%. 65 patients developed skin cancers after 62 ± 40.6 months. 1-and 5-year survival after diagnosis was 96% and 71% respectively. there were 7 tumor-related deaths in this group. 19 patients developed ptld 40.7 ± 33.7 months after transplantation. 1-and 5-year survival was 63% and 40% with 12 deaths associated with the neoplasm. in the third group, a total of 59 patients were included. this group consisted of lung cancer (n = 17), abdominal cancer (n = 15), urogenital cancer (n = 7), and other tumors (n = 20). neoplasms were diagnosed at an average follow-up of 53.5 ± 42.7 months. 1-year and 5year survival was 57% and 25%. 31 deaths were associated with tumor. conclusions. although all patients received antibody induction therapy, overall incidence of neoplasms was comparable to centers using no induction therapy. especially incidence of ptld was low. as long-term survival after cardiac transplantation increases steadily and the risk of cancer increases continuously, patients in long-term follow-up should be checked for malignant diseases on a routine basis. background. while the predictive value of n-terminal pro-b-type natriuretic peptide (nt pro-bnp) in nontransplant cardiac patients is well recognized, its value in heart transplantation (htx) is incompletely understood. certain graft factors (e.g., isolated diastolic dysfunction) may affect both, nt pro-bnp levels and peak exercise tolerance. we therefore hypothesized a relationship between these variables in long-term htx recipients. methods. we measured nt pro-bnp levels of 27 htx patients before a symptom-limited upright bicycle exercise test was performed. graft function was stable in all patients and there were no signs of rejection. patients were divided according to a cut-off value of 70% exercise tolerance predicted normal into "low" and "normal" htx fitness groups. results. in 12 patients (11 m, 1 f; 57 ± 10 years; 6.5 ± 4 years posthtx; donor age, 36 ± 10 years; bmi, 28.6 ± 2.8 kg/m 2 ; creatinine clearance, 42 ± 10 ml/min) peak exercise tolerance was "low" (93 ± 25 w), while in 15 patients (11 m, 4 f; 57 ± 11 years; 7.4 ± 3.6 years posthtx; donor age, 32 ± 10 years; bmi, 26.6 ± 2.7 kg/m 2 ; creatinine clearance, 42 ± 8 ml/min) it was "normal" (136 ± 37 w). in the "low" htx fitness group, nt pro-bnp levels were 702 ± 778 pg/ml, in the "normal" htx fitness group, 324 ± 250 pg/ml (p = 0.08 between groups). regression analysis of peak exercise tolerance, achieved percentage of exercise tolerance predicted "normal", and renal function with nt pro-bnp levels failed to demonstrate a significant relationship. conclusions. the findings confirm previous studies that nt pro-bnp levels are increased in asymptomatic long-term htx recipients. a larger sample size is warranted, however, to support the hypothesis that nt pro-bnp might be a useful indicator to predict physical fitness in these patients. calcineurin inhibitor therapy is an important cause of renal dysfunction in heart transplant patients. sirolimus (srl) is a novel immunosuppressive (is) drug without nephrotoxic side effects. however, cases of proteinuria associated with srl have been reported following renal transplantation. in cardiac transplantation the potential incidence of proteinuria is not known. 29 long-term cardiac transplant patients (age, 60.9 ± 7.3 years) were switched from cyclosporine-based immunosuppression to a srl-based is 8.8 ± 4.5 years after transplantation. concomitant is consisted of mycophenolate-mofetil with or without steroids. two patients died 14 and 37 months post switch due to infection. both patients were dialysis dependent at time of death. one other patient was switched back to cni-based is due to interstitial nephritis. 24 h collections of urine were performed in all patients before switch and at several time points post switch to measure proteinuria. proteinuria increased significantly from 0.45 ± 1.0 mg/dl (median, 0.17) pre switch to 1.03 ± 2.04 mg/dl (median, 0.21) post switch (p = 0.017). proteinuria of 0.21-1.0 mg/dl was seen in 21% of patients before switch and in 28% after switch. proteinuria of >1.0 mg/dl was seen in 10% of patients before switch and in 24% after switch (n.s.). three patients developed severe proteinuria (>3.5 mg/dl) after switch. one died on dialysis, one was switched back to cni and one still remains on srl. in conclusion, proteinuria may develop in cardiac transplant patients after switch to srl-based is. srl seems to have a potential adverse renal effect in these patients. srl should be used cautiously with close monitoring for proteinuria or increased renal dysfunction. v21 early growth-response factor-1 is involved in cellular injury of transplanted hearts background. we have shown a persistent mitochondrial pathology in patients with idiopathic dilative (dcm) but not ischemic (icm) cardiomyopathy following cardiac transplantation. early growth response factor (egr)-1 that is stimulated by cytokines and hypoxia is suggested to induce inflammation and tissue injury. whether egr-1 mediates the persistent cellular pathology in hearts transplanted to dcm patients is unknown. methods. egr-1 and hypoxia-inducible factor-1 (hif-1) gene expression was examined in left ventricular biopsies of explanted failing hearts in 28 icm and 42 dcm patients, as well as in 12 donor grafts before reperfusion (control), at 10, 30, 60 minutes after reperfusion, and at 1, 2, 3, 4, 6, 12 posttransplant weeks, using real-time rt-pcr. hif-1 binding activity was examined using emsa. results. egr-1 myocardial gene expression was upregulated in dcm and icm (p < 0.01). hif-1 mrna levels were unchanged in both groups, whereas hif-1 binding activity was increased in icm only (p < 0.01) vs. controls. egr-1 and hif-1 myocardial expression increased during reperfusion in donor grafts (p < 0.01) vs. control hearts. in icm group, graft egr-1 and hif-1 expression returned to and remained at the baseline level of control hearts one week after transplantation. in contrast, in dcm group, egr-1 levels remained significantly upregulated during the follow-up period in transplanted hearts (p < 0.01), although hif-1 expression returned to the control baseline level one week after transplantation. conclusions. chronic hypoxia specifically triggers hif-1 binding activity in icm, and reperfusion upregulates egr-1 and hif-1 mrna expression in heart grafts. the persisting egr-1 overexpression in grafts transplanted to dcm patients could mediate mitochondrial impairment. targeting egr-1 overexpression that bypasses hif-1 might be beneficial to counteract acute reperfusion-induced injury, and the chronic cellular pathology in cardiac grafts transplanted to dcm patients. introduction. giant-cell myocarditits (gcm) is a rare and frequently fatal disorder of unknown origin that is defined histopathologically as diffuse myocardial necrosis with multinucleated giant cells in the absence of sarcoid-like granuloma. patients usually have ventricular arrhythmias or congestive heart failure. although a variety of systemic disorders have been seen in association with giant cell mycocarditis, most cases occur in previously healthy adults. conclusive diagnosis requires histologic analysis of myocardial tissue obtained by endomyocardial biopsy (emb). congestive heart failure (chf) is the most common cardiac presentation (75%), with sustained, refractory ventricular tachycardia. the case presented here is that of a 65-old-man suffering of gcm in whom an extracorporeal membrane oxygenation (ecmo) had to be implanted to overcome cardiogenic shock. antithymocyte gobuline (ratg, thymoglobuline, sangstat), respectively ciclosporine (inn: cyclosporine), mycophenolate and steroids were utilized to bridge the time to complete myocardial recovery. we are reporting the first successful implantation and bridging to myocardial regeneration in a patient suffering of gcm by means of ecmo and initiation of immunomodulating drugs including polyclonal ratg. clinical summary. a 65-year-old man was admitted to a public hospital because of thoracic pain, positive heart enzymes, and a highly pathologic electrocardiogramm. echocardiography demonstrated a pericardial effusion and reduced left ventricular function (lvf, ef 15%). performed angiography evidenced and a high-grade stenosis of the left anterior descending (lad) which led to stent implantation. despite stent placement, the clinical condition worsened leading to cardiogenic shock including incipient shock liver. in this clinical condition the patient was transferred to our institution. in addition, the patient developed malign ventricular tachycardia (lowen iv) and had to undergo repetitive defibrillation. in this clinical scenario it was decided to implant a femoral veno-arterial ecmo. the patient's metabolic data improved noticeably; however, due to repeated ventricular tachycardia, the patient had to be defibrillated multiple times (max. 30×/d). to define exact cardiac pathology, we performed a heart biopsy with the pathology of gcm. immunosuppressive therapy with cyclosporine (50 mg/d), mycophenolate and prednisolon (250 mg/d) was initiated. in addition, rabbit atg (ratg, thymoglobuline, sangstat) at the dosage of 75 mg/d was added to the therapy. this drug therapy was continued for 5 days. of note is the fact that with initiation of ratg, cardiac fibrillation episodes abated immediately. routinely performed echo-cardiography (tee) revealed an improvement in ventricular function, and one week after ecmo support, we were able to wean the patient from extracorporeal support. moreover, a routine biopsy after 14 days revealed complete remission of gcm in the heart tissue. an intracardial defibrillator (icd) was additionally implanted. three months after emergency admission to our department the patient was discharged. continuous medication of prednisolon 5 mg/d, mycophenolat mofetil 500 mg/d, and plavix 75 mg/d was prescribed. 12 month after initial event the patient describes nyha class i heart function and echocardiography reveals an moderate impairment of heart function (ef 55%). immunosuppressive with low-dose steroid and mycophenolate drug regimen is continued as the patient describes no side effects. discussion. this report adds to the available knowledge of giant-cell myocarditis by providing that (a) ecmo implantation is feasible if the patient is demonstrating acute haemodynamic deterioration because of biventricular dilation and medically intractable ventricular fibrillation and (b) after verified histological diagnosis of gcm immunemodulation with cyclosporine/mycophenolate with additional application of ratg is feasible with favourable outcome. in various studies, patients with gcm were treated with immunosuppression (cyclosporine, steroids, murine monoclonal antibodies [okt-3]) and even assist device as bridge to transplantation. heart transplantation has shown to be successful as method of treatment. autoimmune diseases and its mechanisms were suggested to be involved in the pathogenesis of gcm. most recently, a novel mechanism of action of immunoglobulin was proposed to be due to anti-inflammatory activities through the inhibitory fc receptos (fcrs). it has been suggested that t-cell-mediated autoimmune diseases is the result of inappropriate antigen presentation of either a self-antigen or an antigen with the capacity to mimic a self-antigen in the peripheral lymphoid tissues. relevant to this novel application of ratg, polyclonal suspensions like igm/g, ivig containing fc receptors and have been demonstrated to be beneficiary in autoimmune myocarditis. in an elegant study by shioji et al. ivig was highly effective in ameliorating experimental myocarditis. however it has to be mentioned that immunoglobulin treatment failed to ameliorate myocarditis. in respect to our patient suffering of rcm fc containing ratg provided remarkable clinical benefit. background. the criteria for liver donation have been widely extended due to the increasing waiting time and waiting list mortality. marginal donors provide an upcoming option to diminish the number of waiting patients. methods. the criteria for marginal were icu stay of >7 days, age of >65 years, bmi of >27, alcohol or oral drug abuse, infection, hypernatremia (na, >150 mg/dl), high liver enzymes (ast, alt 2 times the normal), liver parenchymal damage and metabolic diseases. from 01/00 until 06/05 our center reported 55 donors, who fulfilled at least 3 of the above criteria. results. all livers were transplanted either in standard or in piggyback technique with a cold ischemic period between 130 minutes and 12 hours. the mean recipient age was 45 (13-75) years. 6 livers were used for hu patients, 5 for a retransplantation, 2 were implanted in combination with kidneys, and 37 organs were transplanted electively. primary diseases were cryptogenic cirrhosis, hepatocellular carcinoma, post-hepatitis cirrhosis, polycystic liver disease, scleroting bile ducts, hepatic artery thrombosis, and acute liver failure. 2 month after transplantation, 54 recipients were alive, 1 died 1 month after tx not transplant related. 16 livers were implanted at our center in piggyback technique with retrograde reperfusion. 15 patients were elective patients in good or moderate condition, 1 patient was a hu patient suffering a hepatic artery thrombosis. the initial graft function was good (got, <1000 mg/dl pod 1) in 8, moderate (got, 1000-2000 mg/dl pod 1) in 5, and delayed (got, >2000 mg/dl pod 1) in 3 cases, all patients survived. conclusions. marginal livers are eligible for transplantation. delayed graft function has to be taken into account. hospital between january 1993 and december 2003. we employed the local registry of the department of transplant surgery, where variables of all patients are routinely and prospectively recorded. primary outcome was initial graft function, secondary outcome was patient survival. results. cumulative number of marginal donor criteria was significantly and linearly associated with an increased rate of primary dysfunction (p = 0.005). in patients with more than three cumulative marginal donor criteria the rate of primary dysfunction was 36 percent. patient survival was not influenced by the cumulative number of donor criteria (log-rank test, p = 0.81). independent marginal donor criteria to predict primary dysfunction were cold ischemia time of >10 hours (or, 0.56; 95% ci, 0.32 to 0.98) and donor peak serum sodiumof >155 meq/l (or, 0.44; 95% ci, 0.26 to 0.77), as assessed in a multivariate regression model. conclusions. the use of marginal liver donors with more than three marginal donor criteria shows deleterious effects on initial graft function. noteworthy, patient survival was not associated with marginal donor criteria, which may be explained by early and successful retransplantation of liver recipients with primary nonfunction. ergebnisse. alle transplantate zeigten eine gute initiale organfunktion, der transaminasenverlauf und das gesamtbilirubin, gemessen am 1. postoperativen tag, am 7. postoperativen tag und bei entlassung (mittelwerte mit standardabweichung) waren wie folgt: got 1334 u/l (±848), 43 u/l (±13), 25 u/l (±9); gpt 877 u/l (±245), 420 u/l (±649), 54 u/l (±40); ggt 118 u/l (±86), 355 u/l (±232), 232 u/l (±205); gesamtbilirubin 5,1 mg/dl (±2,2), 3,5 mg/dl (±2,5), 1,9 mg/dl (±0,9) . background. meld score is a useful tool in predicting mortality in patients awaiting liver transplantation. its capacity to predict patient survival seems to be relatively poor and is still discussed controversially. the purpose of the study was to analyse the impact of alterations of the meld score during waiting time on the posttransplant survival rate. additionally, the impact of donor quality on posttransplant survival was investigated. methods. 242 adult patients were transplanted between 1997 and 2003 because of end stage liver disease without malignancy. the meld scores at time of listing (meld on) and of transplantation (meld tx) were gathered. additionally the delta-meld was calculated from listing to transplantation. results. a high meld tx showed only a trend to poorer survival. patients who died within the 1 st year after transplantation showed a significant increase in the meld score during waiting time (p < 0.01). patients with a delta-meld higher than 4 during waiting time had a 50% 1-year mortality after transplantation, patients with a meld increase not higher than 4 had only a 23.1% 1-year mortality (p < 0.01). patients with a meld score higher than 24 who received a marginal graft showed a trend to poorer posttransplant survival. conclusions. patients with a substantial increase of the meld score during waiting time had a significantly poorer 1year posttransplant survival. in contrast, the meld score at time of listing or transplantation had no impact on the posttransplant survival rate. the use of marginal grafts in patients with a higher meld score has to be evaluated carefully. there is no significant difference of serum sodium levels in ltx candidates with or without ascites crease in serum creatinine is a late event in patients with ascites and is not directly reflected within the meld formula. for this purpose we compared patients who died on the waiting list with patients who finally were transplanted. the impact of serum sodium and ascites on death on the waiting list was evaluated. methods. from 1997 to 2005, 621 adult patients with end-stage liver disease were listed for orthotopic liver transplantation (olt). only patients without hepatoma who died on the waiting list (123 patients) or were finally transplanted (300 patients) were evaluated. in addition to the meld score, the serum sodium and the ascites were investigated at time of listing and of coming off the list (transplantation or death). results. transplanted patients had a significantly lower meld on (p < 0.01) than the patients who died while on the waiting list. patients who died while on the waiting list had a significant increase in the meld score during waiting time (p < 0.01). patients who underwent transplantation showed a stable meld score during their waiting time. refractory ascites and spontaneous bacterial infection were evaluated as independent risk factors for death on the waiting list as well as the meld on. 47.2% of the patients (58 of 123 patients) who died on the waiting list were suffering from ascites, in contrast to only 28.7% of the transplanted patients (86 of 300 patients). there was no significant difference in the mean meld on between the patients who were suffering from ascites and those who were not (p = 0.72). nor was any significant difference found in the meld off (p = 0.77). the serum sodium of patients suffering from ascites showed no significant difference to those who showed no signs of ascites. conclusions. ascites was evaluated as independent risk factor for death on the waiting list. no significant difference in the serum sodium levels were found between patients suffering from ascites or not. therefore complications of portal hypertension should be treated adequately and rigorously, especially in patients with lower meld scores. renal failure is an established risk factor for impaired patient outcome after orthotopic liver transplantation (olt). as the endothelin pathway is known to be involved in the development of acute renal failure (arf), we designed a study to clarify its role in arf following olt. 20 consecutive patients with intact kidney function scheduled for their first olt were prospectively studied. plasma big endothelin-1 (et-1) levels were measured before surgery, after graft reperfusion, and on the first and second postoperative days. according to postoperative gfr, patients were assigned to the acute renal dysfunction group (ardf) and the non-ardf group. each patient's gfr was estimated according to the four variable formula used in the modification of diet in renal disease before surgery, daily within the first postoperative week, and at 1, 3, 12 and 24 months after surgery. postoperative mean big et-1 lev-els correlated significantly with the maximum percent decrease of gfr within 3 days after olt (p < 0.01). the proportion of patients who developed ardf was significantly correlated to mean postoperative big et-1 quartiles (p < 0.01). in the ardf group, the percent decrease of gfr within 24 months was significantly higher (p < 0.05) as compared to the non-ardf group. in conclusion, patients who develop acute renal dysfunction immediately after olt do not fully recover to baseline regarding long-term kidney function. short-term gfr was significantly correlated with postoperative big et-1 plasma levels, suggesting renal dysfunction is mediated by the activated endothelin system. background. with improved survival of liver transplant recipients, chronic renal failure has become an important cause of morbidity and is associated with a high mortality. serum creatinine is widely used as marker for renal function, but it depends on various nonrenal factors and major changes will occur late in the course of progressive renal impairment. we evaluated cystatin c and urine microscopy for detection of renal dysfunction in patients after liver transplantation. methods. from november 2003, 70 liver transplant recipients at various intervals from liver transplantation were included to our follow-up. every three months we investigated serum creatinine, urea, renal creatinine clearance and cystatin c as marker for renal function. furthermore urinary sediment was examined by urinary test, automated urinary sediment analyser, and urine microscopy. in patients with reference to renal deterioration we tried to decrease immunosuppressive therapy, to optimize the blood pressure, and to discontinue nephrotoxic medication. infections were detected early by urine microscopy and treated, even when there was no clinical appearance and the urinary test was negative. results. the results of our study showed that concerning the renal function, cystatin c is more sensitive than creatinine and creatinine clearance. the microscopy of the urine sediment showed the highest sensitivity compared with the other methods. concerning damages of the kidney, urine microscopy offered the best possibility to identify the etiology. during the follow-up and after adequate and early therapy, fifteen patients (21.4%) showed an amelioration of renal function after a few months. in 3 patients (4.2%) there was a marked deterioration. two of them had to receive a higher dose of immunosuppressive therapy and one had an infection which was treated with nephrotoxic medication. conclusions. the early identification of renal failure and its etiology are necessary in patients after liver transplantation. the results of our study confirmed cystatin c as early prognostic marker for patients with renal dysfunction. in combination with urine microscopy, renal dysfunction could be detected in time and renal function could be protected with an adequate therapy. background. hcv-infected patients and their grafts have short-term survival rates similiar to other indications. recent data on long-term outcome are contradictory, showing a trend towards poorer outcome in patients transplanted for hcv cirrhosis. in this study we present a retrospective analysis of our experience with patients who underwent liver transplantation (lt) due to hcv-associated end stage liver disease. methods. patients' charts were reviewed for survival, histologically defined hcv recurrence, genotype, presence of cirrhosis, donor and recipient age as well as type of immunosuppression (cyclosporine and tacrolimus; azathioprine and mycophenolate mofetil). survival was analysed by the kaplan-meier procedure, the influence of baseline variables was analysed by binary logistic regression. results. between 1986 and october 2004, 162 patients were transplanted for hcv-related cirrhosis. ten pts. received one and 3 pts. two relts. in 3 (23%) pts. recurrent hepatits c was the cause for relt, in 10 vascular and/or biliary complications. hepatitis b coinfection was present in 6 patients. median follow-up was 44 months (range, 0.6-221). the overall, 1-, 2-, 3-, 5-, and 10-year survival rates were 86%, 81%, 78%, 71%, and 59%, respectively, which were comparable to other indications. the probability of recurrent hcv was 34%, 51%, 62%, 70%, and 83% after 1, 2, 3, 5, and 10 years, respectively, post lt. nine (6.6%) pts. developed cirrhosis after a median of 50 months (28-116). hcv recurrence did not negatively influence patient and graft survival. concerning genotype, cmv status, presence of hcc before lt, rejection episodes, immunosuppression, donor and recipient age only immunosuppression had a significant effect on survival. in cyclosporine-treated patients (lt after 1995) 1-, 2-, and 5-year survival rates were 79%, 72%, 64% compared to 93%, 91%, 77%, respectively, for tacrolimus-based regimens (p = 0.04, log rank test). conclusions. on the basis of our data, the overall survival of hcv transplanted patients were similiar to other indications. recurrent hcv infection did not influence patient and graft survival. immunosuppression may have an impact on survival in hcv-positive recipients, but optimal regimens need to be better defined by prospective studies. the advent of highly sensitive molecular techniques has revealed the possible persistence of hepatitis b virus (hbv) genomes in hbsag-negative patients with or without serologic markers of previous infection, a state called occult hbv infection. the highest prevalence of such infection has been shown in patients infected with hepatitis c virus (hcv). some studies suggested that occult hbv infection might favor or accelerate the progression of hcv infection towards cirrhosis. hcv infection recurs almost in all patients after liver transplantation (lt). about 5-10% of lt patients develop a fibrosing cholestatic hcv recurrence, which is associated with a very poor outcome. no specific risk factor for this pattern of recurrence has been described so far. the aim of this study was to determine the prevalence of occult hbv infection in patients presenting with fibrosing cholestatic hcv recurrence after lt. between 1986 and 2004, 151 hcv patients (104 m; 47 f) underwent lt at our institution. the mean follow-up was 51 months (range, 1-228 months). the diagnosis of recurrence was based on biochemical and histologic parameters. genotype 1 was predominant (75%), followed by 2 (15%), 3 (8%), and 4 (3%). eleven patients (7.3%; 10 m, 1 f) developed a fibrosing cholestatic pattern of recurrence characterized by highly elevated cholestatic parameters and typical histologic findings. also in this group, genotype 1 was predominant (n = 7), three had type 3, and one type 4. serum hbv dna was tested with the taqman test (roche, austria). five patients were hbsag negative, whereas six had serologic markers (antihbc positive). interestingly, hbv dna could not be detected in the sera of any of these patients with fibrosing cholestatic hcv recurrence. the actuarial 1-, 2-, 5-, and 10-year survival rates of all hcv patients were 83%, 79%, 68%, and 60%. hcv recurrence did not show a negative impact on patient and graft survival; however, the outcome of the patients with the fibrosing cholestatic pattern was less favorable. seven out of 11 patients died due to hcv recurrence, one patient had to be retransplanted secondary to recurrent disease. only three patients are alive with a functioning first allograft. this study indicates that occult hbv infection is not associated with fibrosing cholestatic hepatitis c recurrence after lt. background. the use of grafts from hepatitis b core antibody (anti-hbcab)-positive donors for liver transplantation (lt) is associated with the risk of de novo hepatitis b. patients who test positive for anti-hbcab pretransplant are also theoretically at risk to develop graft hepatitis b. methods. the outcome of 467 consecutive lts performed in 402 individuals between 1998 and 2001 was retrospectively analyzed with regard to the presence of anti-hbcab in donors and recipients. patients with hepatitis b and recipients of known anti-hbcab-positive grafts received hbig/lamivudine prophylaxis. results. a total of 111 recipients (28%) tested positive for anti-hbcab including 24 patients (7%) with hbv-associated cirrhosis and three patients with fulminant hepatitis b. a total of 20 allografts from anti-hbcab-positive donors were utilized, 14 of those (70%) were allocated to anti-hbcab-positive recipients. anti-hbcab-positive recipients were significantly more likely to have concomitant hcv (62% vs. 34%, p < 0.0001) and hepatocellular carcinoma (30% vs. 14%, p < 0.002). anti-hbcab-positive donors were more frequently non-caucasian (60 vs. 24%, p < 0.001) and cmv seropositive (85% vs. 65%, p < 0.035). survival of anti-hbcab-positive individuals and recipients of allografts from anti-hbcab-positive donors did not differ from their anti-hbcab-negative counterparts. there were no reported cases of recurrent hepatitis b in anti-hbcab recipients or patients receiving lt for hbv-associated liver disease. three cases of de novo acute posttransplant hepatitis b were identified, one being acquired during unprotected intercourse and two being transmitted through the graft. the two with graft-transmitted disease were anti-hbcab negative, treated initially with lamivudine and were switched to adefovir due to the emergence of ymdd mutants. conclusions. the frequency of anti-hbcab-positive recipients in our series was higher than expected. these individuals seem at minimal risk for posttransplant hepatitis b. recurrence of hbv after lt in the setting of hbig/lamivudine prophylaxis is extremely rare with 5-year median follow-up. the risk of transmission of hbv through anti-hbcab-positive livers despite prophylaxis cannot be neglected and the emergence of an ymdd mutant is of particular concern. anti-hbcab-positive grafts should be preferably given to anti-hbcab-positive recipients. v36 the response to preoperative transarterial chemoembolisation predicts outcome of patients with hepatocellular carcinoma after liver transplantation division of gastroenterology and hepatology, department of internal medicine, medical unversity of innsbruck, innsbruck, austria liver transplantation (lt) is the only curative therapy for patients with early-stage hepatocellular carcinoma (hcc). the impact of prelt chemoembolisation (ce) on patient survival and incidence of hcc recurrence has been controversially discussed and remains undetermined. the aim of this study was to evaluate the efficacy of ce prior to lt in hcc patients with regard to tumor recurrence and patient outcome. between 1984 and 2004, 167 hcc patients (142 m; 25 f) underwent lt at our institution. the underlying liver disease was viral hepatitis in 87 (hcv 69, hbv 18), alcoholic liver cirrhosis in 49, and other diseases in 18 patients. according to child-pugh classification, 75 patients presented with child a, 79 with stage b and 9 with stage c. hccs were diagnosed according to the easl guidelines. on the basis of prelt radiology, 23 patients were diagnosed with stage i, 79 stage ii, 34 stage iii, and 31 stage iv according to the modified uicc criteria. ce was performed in 120 patients prior to lt. in 47 patients no treatment was performed prior to lt. patients underwent multiple cycles of ce (mean, 1.6 ce/patient). ce response prior to ce was assessed by ct scan. on explant histology, complete response with no evidence of vital tumor was found in 38 of 120 (32%) patients, 55 (46%) patients showed a partial remission (tumor necrosis, >50%), and 27 (22%) patients showed a poor response or even progression. the intention-totreat analysis showed that patients with early-stage hcc showed an excellent survival with a 1-, 5-, and 10-year rate of 98%, 69% and 69%, respectively. ce prior to lt had no positive effect on overall patient outcome and rate of recurrence. however, patients with complete response to ce, on the basis of both pre lt and post lt histology, had a significantly bet-ter long-term survival (1-, 5-, and 10-year rates of 98%, 91%, and 91%) and rate of recurrence compared to those with partial or no response, but only in patients within milan and not san francisco criteria. the 1-, 5-, and 10-years survival rates of patients with advanced hcc were 95%, 67%, and 35%. hcc recurrence was found in 24 patients, 11 of 24 presented with advanced stage (iii and iv). only 13 had undergone ce prior to lt, and 9 of those were nonresponders to ce. this study indicates that response to prelt ce may predict the outcome of hcc patients after lt. patients with early-stage hcc, who responded to ce pre lt, showed an excellent outcome with 5-and 10-year survival rates around 90%. patients with early-stage hcc and only partial or no response to ce had a higher risk of recurrence of hcc after lt, but outcome was still favorable compared with advanced-stage tumors regardless of ce response. the roles of the regenerative factors hepatocyte growth factor (hgf), transforming growth factor a (tgf-a), and of vascular endothelial growth factor (vegf) were described in the context of hypertrophy and regeneration after liver resection but not well known in the transplantation situation. in the recipients of 63 consecutive liver transplantations with a graft survival of >2 weeks, the factors hgf, tgf-a and vegf were determined postoperatively (day 1, 3, 5, 7, 10, 14) by an el-isa in the serum and correlated to graft survival (kaplan-meier). the median concentrations of hgf were constant in total during the observation period (day 1, 2591 pg/ml; day 7, 2434 pg/ml; day 14, 2490 pg/ml). an individual increase to levels above 4000 pg/ml in the middle of the observation period correlated to a significantly decreased one-year graft survival (54% vs. 85%). similar was the course of tgfa. an increase from the median concentration of 39 pg/ml to levels above 80 pg/ml was observed in the context of a decreased primary function. regarding vegf, an almost linear increase of the concentration from 60 pg/ml via 177 pg/ml to 424 pg/ml (day 1, 7, and 14) was observed. here it became obvious, that an extensive increase of the vegf concentration correlated to a good transplant function. under the premise that the systemically determined concentrations were in relevant correlation to the secretion and thus to the local concentration in the liver, it can be concluded that vascular regeneration induced by vegf substantially contributes to graft survival, whereas a temporal increase of hgf and tgfa rather has to be interpreted as an indicator of an injured graft with a decreased functional prognosis. in total there are about 1650 patients on dialysis in bh and percentage of transplanted is 9.24. the aim of the paper is to analyse survival of grafts, patients, and grafts and patients over a 5-year period of living-related kidney transplantation history at university medical center tuzla. the results obtained were compared with the results of the centers with great number of transplants. methods. recipients and donors as family members were admitted after informative discussions and after the test results had been obtained from primary health care laboratories. the protocol was the standard one, in accordance with the recommendations of esot and eurotransplant. donors were tested first and then recipients. lumbotomy was done for nephrectomy. colins solution was used for kidney rinsing and perfusion, reconstruction of blood vessels was done as well as kidney biopsy. then, kidneys were stored at +4°c and grafts implantations were done on the contralateral iliac fossa. terminolateral anastomosis between external iliac artery and vein was done. implantation of ureters was done by modified lich-gregore technic, with or without dj stent. all patients received basic immunosuppressive protocol. the peculiarity was introduction of basiliximab (simulect) in therapy on the first and fourth postoperative day. descriptive statistics was done using spss software for windows 8.0. survival is presented by kaplan-meier curve. results are shown as means with standard deviations (sd). results. the first living-related kidney transplantation was done at university medical center tuzla on 15. 09.1999 09. . until 15.09.2004 . as many as 52 transplants have been done. donors were related to recipients as follows (in parentheses, mean age [years] with sd): mother, n = 24 (55.0 ± 11.3); father, n = 15 (61.4 ± 6.9); sister, 5 (44.5 ± 6.4); brother, n = 6 (46.0 ± 2.9); others, n = 2 (38.5 ± 5.5). table 1 shows mean dtpa clearance rate in donors. average separate dtpa clearance rates were within normal limits. as a rule, left-side donor nephrectomy was done; and in five cases, right-side nephrectomy. three donors showed borderline dtpa clearance rates of about 40 ml/min. bladder neck sclerosis was found in one patient and the air expansion of the bladder had to be done in the other one, at least to achieve its minimal capacity. two renal arteries were found in 5 patients (3 mothers, 1 father, 1 sister), two arteries were found with left kidney transplantation. termino-lateral anastomosis was done in 2 cases. the average age of the donors was 51.08 ± 6.58 years. as many as 16 donors were over 60 years old in our transplants, which reflected in our results. the average donor age of 36 males and 16 females was 32.4 ± 8.1 years. the data on hypertension before transplants were not reliable. the average hemodialysis time was 21.48 ± 10.36 months. the most common recipients' diseases were chronic glomerulonephritis, pyelonephritis, interstitial nephritis, nondefined renal disease, diabetes mellitus, systemic lupus, vesico-ureteral reflux. there were only 8 biopsies done before transplantation so that their histological background was known. the average serum creatinine after 5 years is 153.56 ± 24.65 µmol/l. cumulative graft survival after 5 years is shown by kaplan-meier curve: graft survival, 75.1%; patients survival, 83.9%; graft and patients survival, 67.1%. conclusions. the results on 5year living-related kidney transplantation at university medical center tuzla, bosnia and herzegovina, are similar or identical to the results of the developed centers in the world. the existing program has got to be improved, especially with respect to the donor selection from the standpoint of biological and chronological age. the experience gained so far is the basis for the development of cadaveric kidney transplantation in bosnia and herzegovina. background. shortage of available organs has increased the demand for living kidney donation. whereas donation for relatives is well accepted, there remains some controversy in the setting of emotionally related donation. there might be a survival benefit for grafts donated by relatives due to better hla matching. a retrospective analysis of 162 living donor kidney transplants which were performed at the university hospital of innsbruck was made. we divided the transplants in two groups according to the date of transplant: group 1 (1975-1993), 41 transplants; group 2 (1994-2005) , 89 transplants. the time period from 1994 until 2004 was analyzed in detail and data additionally compared to a cohort of 940 cadaveric renal transplants. during these eleven years, five liv-ing donations were carried out in patients who were not eligible to receive a cadaveric graft within eurotransplant. results. overall there were 128 lrt (53 siblings, 70 parents, 5 off-springs) and 34 ert (22 spouses, 12 others). mean donor age was 45 years (range, 21-70); mean recipient age was 40 (range, 2-72) years. in group 1 only 2 transplants were emotionally related (5%), whereas in group 2 the proportion increased to 25%. graft survival of living donated kidneys was better when compared with cadaveric kidneys (95% versus 92% at one and 89% and 83% at five years), but the difference did not reach statistical significance (p = 0.06). lrts and erts produced equal outcome. overall graft loss rate after a median follow-up of six (range, 0.5-11.5) years was 11% (lrts) vs. 16% (erts). the rejection rate was slightly higher in the lrt group with 31% vs. 22% (p > 0.05, n.s.). ten living donated grafts were lost and seven recipients of a living donated graft died. causes of death were cardiac ischemia (n = 1), pulmonary embolism (n = 1), fungal infections (n = 1), suicide (n = 1), and 3 causes were not specified. conclusions. the frequency of living-related and unrelated kidney donation has increased during the past two decades at our institution. equal results for cadaveric grafts were achieved when compared to lrt and erl. background. the 20s proteasome plays an important role in the nonlysosomal pathway of intracellular protein degradation and apoptosis, thus being a possible marker for ischemic and reperfusion injuries. the aim of this study was to monitor the proteasome levels in patients receiving kidney transplants to detect a relationship with the return of renal function. methods. we examined 12 patients with end stage renal disease, receiving kidney transplants: blood samples were collected intraoperatively and postoperatively on 5 consecutive days and 20s proteasome levels were measured for each sample with a sandwich elisa as described by dutaud et al. anesthesia and immunosuppressive medications were standardized, creatinine clearance and urine output were assessed daily on a routine basis. results. patients who had no adequate urine output (430 ± 300 ml) after the 4th postoperative day had significantly higher proteasome levels intraoperatively than patients with high urine output (4032 ± 1076 ml; 1.7 ± 1.5 µg/ml low vs. 0.5 ± 0.4 µg/ml high urine output, means with standard deviations, p = 0.02). this difference in proteasome levels seemed to even out during the follow-up period. conclusions. patients with impaired renal function after kidney transplant had significantly higher proteasome levels intraoperatively. a higher plasma level of proteasomes intraoperatively may therefore be a negative prognostic marker for postoperative return of renal function of the transplanted kidney. background. dendritic cells (dcs) are the most potent antigen-presenting cells and are pivotal for initiating allograft immunity. recently, however, particular dc subsets have also been implicated in allogeneic tolerance induction. campath-1h (alemtuzumab) is a novel t-cell-depleting antibody that is currently under investigation for the use in allogeneic organ transplantation and may confer tolerogenic properties. here we study the effect of alemtuzumab on peripheral dc subsets in kidney transplant patients under fk506 monotherapy in comparison to patients under conventional triple therapy. methods. patients receiving their first renal allograft were recruited within the tacam trial and randomly assigned to receive either alemtuzumab as induction agent followed by fk506 monotherapy (n = 7) or to receive conventional immunosupression consisting of fk506, mycophenolate mofetil and steroids (n = 7). absolute numbers of peripheral blood dcs and their different subpopulations were assessed by four-colour, single-platform fluorocytometry at the day before and 1, 4, 12, and 24 weeks after the transplant procedure, respectively. peripheral dcs were identified as hla-dr + and lineage-negative cell population. the respective dc subpopulations were cd11c + dcs (myeloid dcs or dc1), cd123 + dcs (plasmacytoid dcs or dc2), cd11c + and further, bdca1 + and bdca3 + dcs. results. induction with campath-1h led to a strong and sustained reduction of the total number of peripheral dcs compared to controls. while the absolute number of peripheral dcs in control patients recovered within 6 months after transplantation, campath-1h-treated patients exhibited a profound reduction of their circulating dcs. interestingly, a prominent shift of the ratio of myeloid to plasmacytoid dc subsets (dc1/dc2 ratio) was observed as early as one month after transplantation in campath-1h-treated patients. conclusions. employment of campath-1h as induction therapy in renal transplant patients is associated with a peculiar alteration of the peripheral dc repertoire. since plasmacytoid dcs have been linked to tolerance induction, the presented data suggest that the use of campath-1h in solidorgan transplantation creates an opportunity to safely introduce novel strategies to achieve alloantigen-specific hyporesponsiveness. background. detection of c4 complement split product c4d in peritubular capillaries represents a valuable diagnostic marker for antibody-mediated rejection (amr). numerous studies have demonstrated inferior allograft function and survival in c4d-positive as compared with c4d-negative kidney allograft recipients. however, anecdotal data implicate that in selected cases stable long-term function can be maintained despite detectable c4d deposits. as recently dicussed in the literature, c4d deposits could also reflect a state of graft acceptance (accommodation), rather than rejection. aim. the objective of this study was to investigate individual clinical outcomes in a large cohort of c4d-positive kidney transplant recipients. our emphasis was thereby to identify and characterize a subpopulation of c4d-positive recipients with only mild graft dysfunction and stable long-term graft function. methods. this retrospective analysis focused on 74 out of 878 adult kidney transplant recipients (transplantation between between 1999 and 2003) included on the basis of a positive c4d result early after transplantation. results. at the time of c4d-positive biopsy (median, 12 days post-tx) median serum creatinine was 6 mg/dl (range, 1.1 to 7 mg/dl) with 49% of the patients dialysis-dependent. according to our local standard, patients with severe graft dysfunction (n = 15) were subject to immunoadsorption treatment (ia). in another 20 highly sensitized recipients, c4d-positive graft dysfunction was diagnosed during or after pre-emptive peritransplant ia. furthermore, intense treatment included antilymphocyte antibody therapy (32%) and/or high-dose steroids in a high proportion of c4d-positive recipients (46%). analyzing all 74 c4d-positive recipients, 1-month post-biopsy se-rum creatinine was 2.1 mg/dl (30% of the patients dialysis-dependent). 1-year graft and patient survival was 73% (serum creatinine, 1.84 mg/dl). in a subsequent subanalysis we focused on twelve patients (two were biopsied under ia/atg induction) with only mild to moderate graft dysfunction (serum creatinine, 1.4 to 2.45 mg/dl) at the time of c4d-positive biopsy (banff borderline lesion in five, and banff i rejection in two biopsies). within this patient subgroup, we were able to identify five recipients in whom stable long-term graft function could be achieved following steroid bolus therapy only, without further therapeutic measures. in this particular subgroup excellent 1-year allograft function (serum creatinine levels between 1.0 to 1.8 mg/dl) was observed. conclusions. our results demonstrate that in the majority of patients peritubular capillary c4d deposits are associated with severe graft dysfunction necessitating aggressive treatment. nevertheless, in a small subgroup of recipients stable graft function for a long period of time can be achieved without intense therapy despite capillary c4d deposition in biopsy. background. combined kidney pancreas transplantation (ptx) evolved as excellent treatment for diabetic nephropathy with infections remaining common and serious complications. methods. 217 consecutive enteric drained ptxs performed from 1997 to 2004 were retrospectively analyzed with regard to bloodstream infection. immunosuppression consisted of antithymocyteglobuline induction, tacrolimus, mycophenolic acid, and steroids for the majority of cases. standard perioperative antimicrobial prophylaxis consisted of pipercillin/tazobactam in combination with ciprofloxacin and fluconazole. results. one-year patient, pancreas and kidney graft survival were 96.4%, 88.5%, and 94.8%, surgical complication rate was 35%, rejection rate 30%, and rate of infection 59%. in total, 46 sepsis episodes were diagnosed in 35 patients (16%) with a median onset on day 12 (range, 1-45) post transplant. sepsis source was intra-abdominal infection (iai) (n = 21), a contaminated central venous line (n = 10), wound infection (n = 5), urinary tract infection (n = 2), and graft transmitted (n = 2). nine patients (4%) experienced multiple sepsis episodes. overall, 65 pathogens (iai sepsis, 39; line sepsis, 15; others, 11) were isolated from blood. gram-positive cocci accounted for 50 isolates (77%): coagulase negative staphylococci (n = 28 [43%]) (nine multiresistant), staphylococcus aureus (n = 11 [17%]) (four multiresistant), enterococci (n = 9 [14%]) (one e. faecium). gram-negative rods were cultured in twelve cases (18%). patients with blood borne infection had a two-year pancreas graft survival of 76.5% versus 89.4% for those without sepsis (p = 0.036), patient survival was not affected. conclusions. sepsis remains a serious complication after ptx with significantly reduced graft but not patient survival. the most common source is iai. background. mobilized allogeneic peripheral blood stem cells (pbsc) are increasingly used as graft source instead of bone marrow. although the short-term safety profile of recombinant human granulocyte colony-stimulated factor (rhg-csf) seems acceptable, minimal data exist regarding long-term safety. methods. we reviewed data of 196 allogeneic pbsc donors (siblings, n = 159; unrelated donors, n = 37) with respect to side effects of rhg-csf administration, adverse events of leukapheresis and late effects. written informed consent was signed before pbsc mobilization and collection. donors (m/f, 119/77) had a median age of 40 years (range, 11-72). they received rhg-csf at a dose of 2 times 5 µg/kg of body weight a day for 4 consecutive days starting on day 1. routinely, pbsc collection was started on day 5. on condition that donors' white cell count exceeded 70.000/µl or cd34-positive cells exceeded 50/µl, pbsc harvest was started on day 4. rhg-csf was administered until end of the apheresis period unless white blood cell count did not exceed 70.000/µl. pbsc were collected with a continuous-flow blood cell separator processing 3-3.5 times total blood volume. citrate was used as anticoagulation and in the majority of donors a continuous calcium infusion (2.25 mmol [89.4 mg] of calcium per h) was given. reinfusion of autologous platelet-rich plasma from pbsc collection was performed in donors with post-donation thrombocytopenia of <100 g/liter if further collection were necessary or in donors with platelet counts of <80 g/liter, respectively. pbsc harvest procedures were repeated until the predicted cd34 + cell yield of 4 × 10 6 /kg of body weight of recipient was collected. however, not more than 3 consecutive collections were performed. flowcytometric analyses were performed using a becton-dickinson facs-scan or facs-calibur, respectively. for follow-up we assessed peripheral blood counts, electrolytes, lactic dehydrogenase (ldh), alkaline phosphatase (ap), total protein, albumin, on days 1, 7, 30, 100, 365 and then yearly for 5 years. donors received a questionnaire for evaluation of medical history and quality of life, results and evaluation are pending. results. the main adverse events related to rhg-csf administration were bone pain (63 of 196, 32%), myalgia (61 of 196, 31%), headache (44 of 196, 22%), fatigue (36 of 196, 18%), sleep disturbance (30 of 196, 15%) and were rated as moderate to severe by 35% of the donors. due to continuous calcium infusion, incidence of citrate toxcicity was low (34 of 196, 17%) and consisted only of mild paraesthesia. in 10 of 196 (5%) donors post donation platelet count decreased below threshold and required reinfusion of autologous rich plasma. eighty-eight of 196 donors (44%) were lost for follow-up. eighty-two were sibling (82 of 159, 52%) and 6 (6 of 37, 16%) were matched unrelated donors, respectively. from the remaining 108 donors, unrelated donors (31 of 37, 84%) had a median of 4 check-ups (range, 1-8) during the first 100 days (median; range, 1 day to 3 years) after donation, whereas siblings (77 of 159, 48%) had a median of 2 check-ups (range, 1-6) during the first 30 days (median; range, 1 day to 5 years), respectively. two donors (1%), both siblings, were hospitalized within 2 weeks after donation due to severe enteritis and subarachnoidal haemorrhage. the latter donor never had platelet counts below 100 g/liter after pbsc donation and recovered without neurological deficit. follow-up of peripheral blood counts showed a loss of platelets during donation and early post-donation period and a decrease of white blood cells 7 days after donation, both returning to normal within 30 days after, respectively. ldh and ap showed a significant increase during pbsc mobilization, they returned to normal within 1 week after donation, other blood parameters remained unaffected. conclusions. due to the fact that we observed hospitalization of 2 donors within 14 days after pbsc collection, a close monitoring of donors in the early post-donation period seems advisable. although reported anomalies in medical history of donors beyond 30 days after pbsc harvest could not be associated with rhg-csf administration, a regular followup for at least 5 years should be considered. with the particular focus on donor safety, a standardized approach to data collection of follow-ups to monitor short-and long-term effects in all centers should be established. background. donor-recipient sex mismatch is an established risk factor for poor outcome after allogeneic myeloablative hematopoietic stem cell transplantation (hsct). the risk of transplant-related mortality (trm) due to graft-versushost disease (gvhd) is higher in male recipients of female stem cells compared with female patients receiving a graft from a female donor. with longer follow-up, however, the graft-versus-leukemia (gvl) effect due to hy minor histocompatibility antigen mismatch may predominate. the contribution of donor-patient sex on outcome after nonmyeloablative hsct, however, has not been examined in detail yet. methods. we therefore analyzed a single-center cohort of 72 high-risk patients transplanted with a related or unrelated stem cell graft after nonmyeloablative conditioning for outcome (acute and chronic gvhd, trm, relapse, and survival). results. of the 72 patients, 19 male patients received a graft from a female donor, 21 males a graft from a male donor. sixteen female donors were transplanted with a male donor and 16 with a female donor. around 30% of the patients with a sex-mismatched donor received stem cells with an hla mismatch compared to 10% of the patients without sex-mis-stammzellen matched donor, the other clinical differences were similar between all groups. the highest cumulative incidence for acute and chronic gvhd was detected in male patients receiving a stem cell graft from a male donor (52.4% and 53.3%, respectively). the highest relapse incidence (55.6%) was detected in male patients transplanted with a female donor. this was borderline significant (p = 0.0845) to female patients receiving a female graft (20% relapse incidence) and argues against an effective anti-hy response in this patient cohort. the mean cumulative incidence for trm was 57.3%; however, female recipients receiving a graft from a female donor displayed an unexpectedly high incidence for trm (87.5%) which could not be explained by clinical characteristics. the overall survival of 41% 2.5 years after transplant in this group, however, was not different from male patients receiving a female graft (43.7% at 1.4 years after transplant). the overall survival from male patients with a male donor was slightly lower (33% at 2.5 years after transplant) compared with female patients transplanted with male stem cells (47.4% at 1.4 years after transplant). conclusions. these data, analyzed in a small cohort of patients, show that a sex mismatch between patient and donor may have a negative impact also on outcome after nonmyeloablative hsct. however, studies with larger and homogeneous cohorts have to be performed to prove these findings. between 1995 and 2004, 20 patients with chronic lymphoid leukemia (cll), binet stage b or c (n = 19) or a with risk factors (n = 1) with a median age of 53 (range, 27-62) years underwent autologous (n = 11) or allogeneic (n = 9) hematopoietic stem cell transplantation (hsct) at the medical university of vienna. the median time from diagnosis to hsct was 29 (range, 6-77) months. eleven patients underwent autologous stem cell transplantation (asct) in partial remission (n = 9) or complete remission (cr) (n = 2) and received bcell-depleted peripheral blood stem cell (pbsc) grafts. nine patients with refractory disease (n = 6) or chemosensitive relapse (n = 3) underwent allogeneic hsct with unmanipulated bone marrow (n = 3) or pbsc (n = 6) from sibling (n = 8) or unrelated (n = 1) donors. in the majority, conditioning therapy consisted of total-body irradiation (tbi) of 12-13.2 gy and cyclophosphamide. three patients underwent reduced-intensity conditioning (ric) with fludarabine and tbi of 2 gy. graft-versus-host disease (gvhd) prophylaxis consisted mainly of cyclosporine (csa) and methotrexate for myeloablative and csa and mycophenolate mofetil for ric-hsct. complete clinical remission was attained in 10 patients (91%) after asct and in 6 (67%) after allogeneic hsct. molecular remission assessed by immunoglobulin heavy chain gene (igh) rearrangement pcr was attained in 8 patients after asct and 5 after allogeneic hsct. in seven patients we noticed persistence of the igh rearrangement, six of these patients died of disease progression or relapse 1-29 months after asct (n = 2) or allogeneic hsct (n = 4). after a median follow-up of 58 (range, 21-122) months, nine autologous (82%) and four allogeneic (44%) graft recipients are alive and 10 patients (asct, n = 6; allogeneic hsct, n = 4) are in clinical and molecular remission. the median time to clinical relapse was 20 (range, 1-102) months. treatment-related death occurred only in one patient 27 months after myeloablative hsct. probability of overall survival is 82% after asct and 42% after allogeneic hsct. in summary, all cll patients with long-term cr after asct and allogeneic hsct also attained sustained molecular remission of the igh rearrangement. . the incidences of the observed genotypes in this small group of patients and donors tested were in the range as published: il10 with 54% c/c, 41% c/a, 5% a/a; nod2 with 12% recipient or donor; 5% recipient and donor, and mpo with 56% g/g, 43% g/a, 1% a/a. since the patient population was very heterogeneous concerning diagnosis (34 aml, 12 all, 9 nhl, 6 cml, 5 mds, 4 saa, 4 solid tumors, 3 mm, 2 omf, 1 cll, and 1 et), course of disease, and condition regimen, patients were divided into four groups for evaluation: 23 with aml and identical high-dose induction therapy (bu/cy), 23 with reduced condition regimen, 6 with unrelated donors, and 29 others. concerning all 81 patients, 19 of them (23%) died through trm (3 aml, 6 reduced, 10 others), but none of them was at high risk determined by nod2 polymorphism (mutated donor and recipient) and only one determined by mpo a/a genotype (aml). four patients (3 reduced, 1 others) with nod2 mutations in donor and recipient dna did not die from trm. twenty-three patients developed severe (grade 3 or 4) acute gvhd (7 aml, 5 reduced, 11 others), 10 of them had il10 c/c genotype (5 aml, 2 reduced, 3 others) and in three patients nod2 was mutated in donor and/or recipient dna (1 aml, 2 others). on the other hand, 34 patients with il10 c/c genotype and 13 patients with nod2 mutations in donor and/or recipient dna developed no or mild gvhd only. in conclusion, so far we were not able to find a correlation between gvhd/trm risks and polymorphisms of il10, nod2, and mpo. this might be due to the small number and the heterogeneity of the patients; however, a panel of additional snps may increase the accuracy of risk assessment prior to allogeneic sct. background. allogeneic stem cell transplantation is a curative therapy for patients with lymphoproliferative disorders as a result of the intensity of the conditioning regimen and the application of a graft-versus-lymphoma effect. however, conventional conditioning regimens have been associated with a 24-61% risk of transplant-related mortality (trm) in advanced hodgkin's lymphoma (hl). in an attempt to reduce the high trm reported with allografting in lymphoma, reduced-intensity regimens have been investigated. methods. four patients between the age of 34 and 44 years underwent allogeneic peripheral blood stem cell (pbsc) transplantation (sct) from hla-identical sibling or unrelated donors at our institution. age, sex, manifestation of disease, and donor were as follows: patient 1-37 years, female, pulmonary bulk, sibling; patient 2-34 years, male, pulmonary bulk, unrelated donor; patient 3-42 years, male, pulmonary bulk, sibling; patient 4-44 years, male, abdominal bulk, sibling. all patients had received multiple courses of polychemotherapy (table 1 ) and local radiotherapy prior to sct. patients 1 and 4 had undergone autologous stem cell transplantation. we administered a reduced-intensity conditioning consisting of beam (bcnu, etoposide, cytarabine and melphalan) over 6 days. to permit durable engraftment in the allogeneic setting, patients received additional pretransplant immunosuppression with an anti-cd52 antibody (campath) at a total dose of 50 mg over 5 days and graft-versus-host disease (gvhd) prophylaxis of cyclosporine a. pbsc of donors were collected after g-csf stimulation at 10 µg/kg of body weight given for 4 days. results. patient 2 rejected his unrelated-donor graft and received an autologous stem cell infusion 44 days thereafter resulting in sustained hematologic recovery (anc, >0.5 g/l on day 10). all other patients engrafted uneventfully (anc, >0.5 g/l on days 14-16; platelets, >20 g/l on days 10-13). none of the patients showed evidence of acute or chronic gvhd. two patients achieved complete donor chimerism in myeloid and lymphoid cell populations, another one had prolonged mixed chimerism and was given a donor leukocyte infusion of 1 × 10 6 cd3-positive cells per kg. one patient experienced a cmv reactivation on day 51 after sct and a sarcoidosis on day +150. three months after sct, all patients showed marked regression of disease. after a follow-up of 5 to 11 months (median, 8 months), 2 patients experienced progression undergoing salvage chemotherapy. time to progres-sion was 5 and 6 months, respectively. two patients remained progression free for 7 and 11 months, respectively. overall survival is 5 to 11 months. conclusions. our data demonstrate that this regimen was well tolerated with a low risk of gvhd and transplant-associated morbidity. an increased dosage of campath could be considered to prevent rejection in unrelated-donor transplantation. longer follow-up and larger patient numbers are warranted for assessment of the efficacy of campath-beam with regards to durable remissions. background. as long-term survivors of hematopoietic cell transplantation (hct) become more numerous, studies addressing the issue of long-term follow-up are necessary. in this study, we report on the quality of life (qol) of hct patients who were alive at least at 5 years after transplantation in comparison with an age-and sex-matched sample of healthy controls assessed in the same time period and the same geographical region. methods. the eortc-quality of life questionnaire (eortc-qlq c30) was sent by post to 39 hct survivors. thirty-four patients answered the questionnaire. patients were compared with 68 healthy controls from the same geographical region. patients and controls completed the eortc in the same time period. results. mann-whitney u tests identified significantly lower qol on the dimensions of physical and social functioning and on the financial impact symptom scale. conclusions. patients who had survived their hct for more than 5 years did generally well in terms of global qol. this is consistent with kiss et al. (2002) who found that cml patients who were alive at least 10 years after hct report lower physical functioning in comparison with healthy controls. problems in the areas of social functioning and financial difficulty can possibly be addressed by intensive rehabilitation processes integrating patients, family members, and significant others. interdisciplinary (medical, psychological, and social) treatment of patients should not come to an end after the acute phase of the illness but should continue during checkups following transplantation. background. bone marrow transplantation (bmt) together with costimulation blockade can reliably induce mixed chimerism and tolerance in certain experimental models. natural killer t cells play an important role in the induction of tolerance in several transplantation and autoimmunity models. it has been reported, for instance, that activation of nk t cells by α-galactosylceramide (α-gal) can prevent the onset and recurrence of autoimmune type i diabetes. in recent experiments we wanted to investigate the role of nk t cells in a model of tolerance induction through bmt with costimulation blockade. to delineate the role of donor and recipient nk t cells, we used nk-t-cell-deficient mice (jα281-/-c57bl/6 and jα281-/-balb/c) as recipient and/or donor strain. we also evaluated whether in vivo stimulation of nk t cells with α-gal has a beneficial effect. methods. c57bl/6 mice and c57bl/6 jα281-/-received a total-body irradiation (tbi) of 3 gy or 1 gy (day -1), approximately 20 × 10 6 fully mismatched balb/c or balb/c jα281-/-bone marrow cells (day 0) and costimulation blockade consisting of anti-cd154 mab (mr1, day 0) and ctla4ig (day +2). groups were additionally treated with α-gal or a vehicle (5 µg on day -1, +2, +7, +14, and +21). multilinage chimerism and skin graft survival were followed for more than 120 days. results. with our standard protocol, 12 of 18 mice developed long-term multilinage chimerism and permanent donor skin graft survival. when using recipients deficient in nkt cells, 5 of 5 became chimeric and showed long-term skin graft survival; using nkt knockouts as donors 6 of 6 and using nkt knockouts both as recipients and donors 5 of 6 became chimeric (p = n.s. for all comparisons). unexpectedly, stimulation of nk t cells by α-gal (using wild-type recipients and donors) prevented chimerism induction after 3 gy tbi (0 of 8 vs. 4 of 6 and 4 of 4 in the vehicle group, p < 0.01) and did not promote engraftment after 1 gy tbi (0 of 8 vs. 0 of 8). conclusions. nk t cells do not play a critical role in tolerance induction after bmt with costimulation blockade. their stimulation with α-gal even prevents induction of mixed chimerism and tolerance. background. phospholamban (plb) is a critical regulator of sarcoplasmic reticulum ca 2+ -atpase activity and myocardial contractility. in this study we investigated the role of plb phosphorylation in ischemia and reperfusion following cardiac transplantation. methods. gene expression of plb was investigated in a syngeneic heterotopic cardiac transplant model in mice. grafts underwent 10 h of cold ischemia or were tranplanted immediately after harvest. gene expression was analysed at various time points employing dna microarray and rt-pcr. for in vitro experiments, hl-1 cardiomyocytes were submitted to a protocol of global normothermic hypoxia for 6 h and reoxygenation in the absence or presence of the ca 2+ /calmodulin kinase ii inhibitor aip (1 µm) or the beta-adrenergic blocker dl-propranolol (1 µm) vs. beta-adrenergic stimulator isoproterenol (1 µm). results. at 24 h, gene expression of plb was diminished by 14.1 and 3.6-fold in groups with and without ischemia, respectively. basal phosphorylation of plb at ser16 (protein kinase a site) and at thr17 (ca 2+ /calmodulin kinase ii site) was present in cultured cardiomyocytes and heart lysates. in the mouse system, increase in plb phosphorylation is observed during early (up to 10 min) reperfusion. thereafter, plb phosphorylation drops below that of control levels. addition of aip diminishes reperfusion-induced thr17 phosphorylation; propranolol significantly decreases ischemia-induced ser16 phosphorylation. in contrast, isoproterenol enhances plb-ser16 and thr17 phosphorylation. conclusions. ischemia regulates phospholamban by two different mechanisms, decrease in expression levels and alterations in the phosphorylation of critical regulatory sites. modulation by aip and dl-propranolol will help for investigation of the role of pbl phosphorylation in ischemia and reperfusion in cardiac transplantation in the future. background. the p38 mitogen-activated protein kinase (p38-mapk) pathway plays a crucial role in pathological events like oxidative stress, inflammation, and abnormal cellular proliferation, resulting in activation of several signalling cascades, involving overexpression of tumor necrosis factor alpha (tnf-α). tnf-α is known to play an important role in chronic rejection. currently, pharmacological inhibitors of p38-mapk are being tested clinically for the treatment of experimentelle transplantation chronic inflammatory diseases such as rheumatoid arthritis, morbus crohn and psoriasis as well as inhibition of vascular smooth muscle cell (vsmc) proliferation after balloon angioplasty. to date, there are no findings that address the role of p38-mapk in the context of chronic allograft vasculopathy, which is characterized by vascular lesions in the graft that consist of concentric myointimal proliferation, resulting in deterioration of allograft function and organ loss. the purpose of this study was to understand the role of p38-mapk in abnormal vsmc proliferation associated with chronic rejection and to investigate the potential therapeutic role of a specific inhibitor of p38-mapk activation in chronic allograft vasculopathy. methods. in vivo, a mouse model of heterotopic aorta transplantation in an allogenic setting has been used. aortas were allografted into recipient mice by a carotid artery cuff technique, using c57bl/6 (h-2 b ) mice as donors and balb/c (h-2 b ) mice as recipients. four weeks after transplantation, the aortic segments were harvested and immunofluorescence was performed using anti-vsmc-α-actin and anti-phospho-p38 antibodies. tnf-α serum levels were measured by elisa. in vitro, vsmcs were isolated from c57bl/6 aortas. expression levels of total and phosphorylated forms of p38-mapk as well as key cell cycle regulators were detected by western blot. immunocytochemistry was performed with primary antibodies directed against phospho-p38-mapk. proliferation of vsmcs was measured by [ 3 h]thymidine incorporation in the presence or absence of sb 203580, a specific inhibitor of p38-mapk activation. cell cycle progression was monitored by dna content analysis; apoptosis by the annexin v binding assay. cell lysates were probed with antibodies directed against cyclindependent kinase 2 (cdk2) and yin yang 1 (yy1). results. in vivo, 4 weeks after the transplant aortic segments were significantly narrowed due to neointimal hyperplasia. the neointimal lesions mainly consisted of vsmcs and showed profound activation of p38-mapk as demonstrated by immunofluorescence. further, serum tnf-α levels were significantly increased even 4 weeks after allogeneic aortic transplantation, suggesting an important role of p38 activation in this model. in vitro, stimulation of vsmcs with 10% fcs resulted in a rapid increase of phosphorylated forms of p38-mapk (8.0 ± 0.7 fold increase) when compared with the nonstimulated quiescent state. immunocytochemistry showed higher levels of phospho-p38-mapk in the nuclei as well as in the cytosol after stimulation. sb 203580 in a dose-dependent manner significantly inhibited vsmc proliferation, which was due to inhibition of cell cycle progression at the g 0 /g 1 phase. we did not observe apoptosis in the sb 203580-treated vsmcs at 20 µm, the highest dose being tested. blockade of p38-mapk activation decreased protein levels of cdk2 and the transcription factor yy1, which plays an important role in vsmc dna replication and protein synthesis. conclusions. p38 mapk activation appears to play an important role in an in vivo allogeneic model of aorta transplantation as well as in vsmc proliferation in vitro, blocking of which prevents the cells from entering the s phase of the cell cycle, thus abrogating cell proliferation. targeting p38-mapk might become a potent strategy in the treatment of vascular proliferative diseases like chronic allograft vasculopathy. background. ctla4ig, a costimulation blocker which is currently under advanced clinical development, has been used for years as part of mixed chimerism protocols. recent data suggest that ctla4ig also functions via modulation of tryptophan metabolism by upregulating indoleamine 2,3-dioxygenase (ido) through b7 signals. we thus investigated the role of ido in our ctla4ig-based mixed chimerism protocol. methods. c57bl/6 mice received a total body irradiation (tbi, d -1) of 3 gy, approx. 20 × 10 6 fully allogeneic balb/c bone marrow cells (d 0) and costimulation blockade consisting of anti-cd154 mab (1 mg, d 0) and ctla4ig (0.5 mg, d 2). different groups of mice were additionally implanted with 1-methyltryptophan pellets on d -1 (1-mt, which is a competitive inhibitor of ido, 7-day release at 0.9 mg/h) or on d -1 and d 6 (14-day release) or placebo pellets. macrochimerism and deletion of donor-reactive cells were followed by flow cytometry. levels of tryptophan, kynurenine, and 1-mt were measured at several timepoints in serum by hplc. results. 8 of 10 mice which received bmt, tbi, mr1 plus ctla4ig but only 2 of 10 mice without ctla4ig developed lasting multilineage chimerism (p < 0.05, measured at week 32 post bmt). 8 of 10 mice with ctla4ig treatment accepted donor skin grafts for more than 150 days, whereas only 1 of 10 mice, which got no ctla4ig injection accepted donor skin long-term (p < 0.05; representative data from two separate experiments), demonstrating that ctla4ig is critical for our protocol. 3rd-party grafts were promptly rejected in all groups. long-term multilineage chimerism developed in 6 of 8 mice with 1-mt treatment, which is not significantly different from treatment with placebo pellets or standard protocol alone (7 of 11, pooled data from both groups). 2-week treatment with 1-mt also did not lead to a significant difference in the rate of multilineage chimerism (4 of 5 with 1-mt vs. 4 of 7 without 1-mt). deletion of donor-reactive t cells was neither blocked nor enhanced by treatment with 1-mt. the kynurenine-to-tryptophan ratio in serum was similar in groups with (11.8 ± 2.7) and without (12.1 ± 3.3) ctla4ig (p = n.s., measured on d 3). substantial serum levels of 1-mt were detected in mice with 1-mt treatment but not in untreated mice, indicating that ido was indeed inhibited in the 1-mt groups. conclusions. ctla4ig plays an essential role for tolerance induction in this model but its mechanisms of action does not critically depend on ido. background. induction of antigen-specific tolerance remains the ultimate goal in clinical organ and cell transplantation, as it would eliminate the need for continuous immunosuppression. one strategy leading to tolerance induction against a transplanted organ consists of infusing blood from the organ donor, an approach known as donor-specific transfusion (dst). although the mechanisms underlying tolerance induction by dst are not fully understood, clonal deletion of alloreactive t cells and generation of immunoregulatory cd25 + cd4 + t cells are important in the process. it is well established that expression of heme oxygenase-1 (ho-1) can promote the survival of transplanted organs. however, the mechanisms underlying this effect remain to be elucidated. we hypostesized that ho-1 is required for tolerance induction involving dsts and that ho-1 can magnify the tolerogenic effect of dsts. methods. allograft survival has been tested by using a well established model of costimulatory blockade (anti-cd40l-ab) plus dst (day -7) in c57bl/6 (h-2 b ) heart allografted balb/c (h-2 d ) wt and ho-1 ko mice. further, ho-1 activity has been induced (by copp) or suppressed (by znpp) in b6af1 (h-2 k/d,b ) mice receiving dba/2 (h-2 d ) allografts plus dst on day 0 or day -7. donor-specific tolerance was tested by challenging the mice with a second dba/2 or third-party fvb (h-2 q ) allograft. leukocytes (depleted or undepleted of cd25 + cd4 + t cells) from mice carrying allografts for >100 days were adoptively transferred to sublethally irradiated b6af1 mice receiving dba2 heart allografts. rna levels of foxp3, tgf-β, il-10 and ctla-4 have been assesed by using rt-pcr. cd25 + cd4 + t cells have been enumerated by flow cytomerty. results. anti-cd40l-ab plus dst treatment resulted in balb/c recipients tolerizing fully mismatched c57bl/6 allografts (n = 6). however, by using ho-1-deficient recipients, this effect was abrogated, in that c57bl/6 allografts have been rejected in a similar manner as in untreated wt recipients (mst = 17.5 d, n = 4). further, dst plus copp (in contrast to dst plus znpp) treatment resulted in donor-specific tolerance of dba/2 allografts in b6af1 recipients (n = 7). tolerant animals showed significantly increased percentage of cd25 + cd4 + t cells and increased levels of foxp3, tgf-β, il-10, and ctla-4 mrna. adoptively transferred b6af1 leukocytes retrieved from the lymph nodes and spleens transferred to sublethally irradiated b6af1 recipients of dba2 allografts led to subsequent allograft loss (mst = 16.7 d, n = 6); in contrast, transfer of leukocytes of tolerant (copp plus dst treated) mice led to indefinite allograft survival in this model. however, when those leukocytes were depleted of cd25 + cd4 + t cells, allografts were immediately rejected (mst = 17.7 d, n = 5). conclusions. ho-1 in a graft recipient can be critical for long-term graft survival and for induction of tolerance. this mainly is mediated by generation of cd25 + cd4 + t cells (t regs). modulation of ho-1 expression and activity may be used therapeutically to promote graft tolerance. background. human immunoglobuline (ivig) has been advocated in the treatment of acute rejection in allograft transplantation and treatment of sepsis. mechanisms describing the immune modulatory activity are however scarce. we sought to investigate immune suppressive properties of pooled human immuneglobuline (ig), unspecific fc and fab fragments and their respective ligands by allogeneic blastogenesis assays. methods. human mixed lymphocyte reactions (mlr) were performed. in detail peripheral blood mononuclear cells (pbmcs) were purified from 5 donors by ficoll density gradient centrifugation (amersham biosciences, buckinghamshire, england). 100,000 cells per well were stimulated with 100,000 radiated pbmc (60 gy) and incubated for 5 days at 37°c together with unspecific igg, igm (both sigma-alderich, st. louis, mo), anti-cd32 (lab vision, fremont, ca) and anti-cd64 (chemicon, temecula, ca) in a dose-dependent fashion. before harvesting cells were pulsed for 18 h with [ 3 h]thymidine (3.7 × 10 4 bq/well) and the [ 3 h]thymidine uptake was measured in a liquid scintillation counter. results. the addition of pooled human igg and igm to allogeneic stimulated cells both resulted in a significant and dose dependent decrease of proliferation, although the suppressive properties of igm was greater as compared to igg (both, p < 0.0001). to investigate whether this effect was partly related to fc receptor involvement we blocked cd32 and cd64 on antigen presenting cells (apcs), known receptors in the activation of mlr. surprisingly, this assay demonstrated that sole fc blocking (high-and low affinity fc receptors) resulted in a significant downregulation of allogeneic response in vitro (both, p < 0.001). conclusions. our data evidence that the addition of unspecific immune globulines results in a reduction of proliferation in in vitro allogeneic blastogenesis assays and is partly fc receptor dependent. these results corroborate the clinical success of ivig and pooled igm in the treatment of solid organ allograft rejection and cautions the utilization of immune globulines in immune suppressed septic patients. die interaktion professioneller antigen-präsentierender zellen mit allogenen t-zellen resultiert in der ausbildung der sog. immunologischen synapse (is), welche für die effiziente aktivierung von t-zellen und damit letztlich für die transplantatabstoßung essentiell ist. im rahmen der is werden der t-zellrezeptor/cd3-komplex, kostimulatorische wie adhäsionsmoleküle und komponenten des zytoskeletts in die kontaktzone des mhc-tcr/cd3-komplexes der is rekrutiert. der einfluss von gängigen immunsuppressiva, die zur prävention der allogenen organabstoßung klinisch verwendet werden, auf die is-evolution ist bislang unbekannt. in dieser studie zeigen wir erstmals, dass kalzineurinhemmer wie csa oder fk506, aber nicht mtor-inhibitoren, selektiv die rekrutierung des tcr/cd3-komplexes in die is blockieren. ein ähnlicher effekt wurde für kortikosteroide, aber nicht mycophenolat beobachtet, was auf eine essentielle rolle des calcineurin-nf-κb-signalweges für die tcr/cd3-regulation im rahmen der t-zellaktivierung hinweist. interessanterweise blockierte das neue immunsuppressivum fk778 nicht nur die tcr/cd3-, sondern auch die lfa-1-und f-aktin-rekrutierung in die is. die bedeutung dieser globalen interferenz mit der is-ausbildung für die spezifische immunantwort muss in weiterführenden studien geklärt werden. diese daten zeigen, dass klassiche immunsuppressiva nicht nur simple blocker der zytokinsynthese sind, sondern schon sehr früh die t-zell-apz-interaktion stören und damit einen weiteren immunologischen mechanismus besitzen, der ihre klinische potenz erklärt. background. ischemia (i) and reperfusion (r) trigger a series of events, which culminate in severe injuries to the affected organs in organ transplantation. cell death, metabolic alterations, and inflammation result in impairment of shortand long-term function. the group of mitogen-activated protein kinases (mapks) are central regulators of these events, they have been implicated through aberrant activation in many pathophysiological settings ranging from autoimmune diseases, cancer, to i/r-associated organ damage. methods. intracellular signaling pathways were analysed in vivo and in vitro employing a cardiomyocyte cell line and a murine heart transplant model. hl-1 cardiomyocytes are a cardiac muscle cell line derived from at-1 mouse atrial cardiomyocytes. syngeneic cardiac transplants were carried out us-ing male inbred balb/c mice, hearts were transplanted heterotopically into the neck of recipients. results. in summary, (i) reoxygenation was characterized by a dramatic increase in the activity of all mapks at the end of the observation period; (ii) growth factor abrogation together with hypoxia (h) and reoxygenation (r) had a substantial effect on the course of signaling events; (iii) signaling processes in response to ischemia and reperfusion in vivo are in line with observations made in cardiomyocytes. conclusions. data obtained so far in our study demonstrate the suitability of the chosen experimental approaches for investigation of i/r-associated alterations in intracellular signaling and cellular responses. preliminary data suggest that h/r treatment of hl-1 results in significant apoptotic cell death, the intracellular signaling pathways involved are therefore currently analyzed. background. ischemia and reperfusion (i/r) in cardiac transplantation results in inflammation and cell death. to gain further insight into the regulation of these processes, we investigated the role of lipocalin-2 (24p3, ngal, uterocalin), a potential regulator of the acute inflammatory response and cellular apoptosis in vitro and in vivo. methods. c57bl/6 hearts were heterotopically transplanted to syngeneic recipients immediately and after 10 hours of prolonged cold ischemia with the grafts harvested at various timepoints (2 min, 2 h, 12 h, 24 h, 48 h, 10 d) after transplantation. gene expression analysis on mrna extracts was performed employing cdna microarray and rt-pcr. protein synthesis was investigated by western blotting and apoptosis by using tunel assay. for the identification of the cellular source of lipocalin-2 and its function in i/r-associated cell death, the effect of recombinant lipocalin-2 was investigated in the hl-1 cardiomyocyte cell line. hl-1 cardiomyocytes undergoing ischemia/reoxygenation as well as purified mononuclear cells and granulocytes were analyzed for lipocalin-2 expression by rt-pcr. results. in the cardiac transplants, high levels of lipocalin-2 gene and protein expression were detected at 12 h of reperfusion, whereby transcription was higher in groups without cold ischemia (22-versus 8.8-fold). he staining demonstrated dense mononuclear infiltrates, cellular edema, and small focal necrosis in groups with and without prolonged cold ischemia. upregulation of gene transcription was confirmed by pcr. apoptotic cells were first detectable at day 2 and peaked 10 days after transplantation. expression of lipocalin-2 was also detected in hl-1 cells by rt-pcr and western blotting following i/r, demonstrating for the first time the presence of lipocalin-2 in this cell lineage. lipocalin-2-transfected hl-1 cardiomyocytes showed a higher cell viability especially under ischemic condition. megalin, known as the lipocalin-2 receptor, was detected in hl-1 cells by rt-pcr. conclusions. this study demonstrates the time-dependent expression of lipocalin-2 in a cardiomyocyte culture as well as in transplanted hearts in response to ischemia and reperfusion/reoxygenation. lipocalin-2 is suggested to target cardiomyocytes with ameliorating effects on cell viability during ischemia. obvious alterations in lipocalin-2 expression at the protein level suggest a possible involvement of lipocalin-2 during i/r-induced cell death probably as a self-limiting process for inflammation. background. protease activation as well as inflammatory responses contribute to organ damage in response to ischemia and reperfusion. in this study we investigated the role of the protease inhibitor slpi in ischemia and cardiac transplantation. methods. hearts from slpi knockout mice (slpi-/-) were heterotopically transplanted to slpi-/-recipients. grafts underwent 10 hours of cold ischemia (ci) prior to transplantation or were transplanted immediately. c57bl/6 wild-type isografts (wt) undergoing the same procedure served as controls. in selected groups, 200 µg of recombinant slpi (rslpi) were added to the preservation solution or given i.v. after reperfusion. after evaluation of graft function, hearts were removed at 15 min, 12 h, 24 h, and 10 days. morphology was investigated by histology, slpi gene expression was analysed using quantitative rt-pcr. slpi protein expression was studied by immunohistochemistry (ihc). slpi, tnf-α, tgf-β, and nf-κb, cathepsin g, and elastase activity were analysed employing elisa and western blot. results. at 15 min, recovery of graft function was normal in wt and slpi-/-mice transplanted without ci (4.0 ± 0.0). in contrast, slpi-/-hearts transplanted after 10 h of ci showed no or marginal recovery of organ function (0.6 ± 0.65). at 24 h, cardiac function in slpi-/-(2.8 ± 0.89) was less when compared with wt (3.36 ± 0.55). single administration of rslpi i.v. had no effect; however, when slpi was added to the preservation solution, organ function comparable to wt mice was observed (3.36 ± 0.55). a mild mononuclear cell infiltrate and small focal necrosis where found in all groups at 24 h. at 10 days, postischemic inflammation as well as myocyte necrosis were significantly higher in the slpi-/group (2.5 ± 0.5 vs. 1.8 ± 0.4 and 1.6 ± 0.5 vs. 0.2 ± 0.4). myocyte vacuolisation as a sign of sublethal ischemic injury was present at high level in slpi-/-mice undergoing ci only. slpi gene expression was detected in wt mice at 12 and 24 h after reperfusion. gene transcription at 12 h was significantly higher after prolonged ci (7.99 vs. 1.57 orders of magnitude) and was associated with significantly decreased nf-κb, tgfβ, and tnf-α activity. slpi protein was first observed at 24 h, high levels of slpi protein were found at 10 days. slpi-positive cells were mainly identified as macrophages (ihc). high intragraft levels of slpi activity were found early as well as 10 days after application of recombinant protein. high slpi levels correlate with decreased cathepsin g early and decreased nf-κb, tgf-β, and elastase activity late after reperfusion. conclusion. herein we demonstrate that slpi has a substantial effect in prevention of inflammation and myocyte damage in response to ischemia and reperfusion of the heart via inhibition of nf-κb, tgf-β, and elastase. in addition, slpi seems to be crucial for recovery of organ function early after heart transplantation -inhibition of protease activity seems to be the underlying mechanism. perfusion with rslpi ex vivo represents a promising therapeutic option for modulating the destructive processes of postischemic inflammation while preserving its restorative nature. methodik. die studie wurde an 5 hausschweinen durchgeführt. die tiere wurden in allgemeinnarkose versetzt und intubiert. alle hämodynamischen daten wurden invasiv gemessen. die parameter (abp, pap, zvd und lap) wurden wie in der klinischen praxis mit einem hp-patientenmonitor registriert und gespeichert. das herzzeitvolumen (co) wurde mit hilfe eines flowmeters der firma transsonic systems inc. gemessen. die hämodynamischen ausgangswerte von 4 patienten vor ecmo wurden retrospektiv mit den experimentellen daten verglichen. ergebnisse. in der frühphase des tierversuchs nach erfolgter abklemmung blieb ein eindeutiger anstieg des pap bei 4 von 5 versuchstieren aus. auch ein rascher abfall des co war nicht zu beobachten. auch der pvr zeigte keine signifikante veränderung. die pap/ap-druckratio reagierte sehr rasch und in allen fällen mit einem anstieg auf das ereignis. von 4 patienten, die mittels ecmo erfolgreich therapiert worden waren, hatten 3 eine pap/ap-ratio über 0,3, ein patient hatte eine pap/ap-ratio von 0,26. die patienten mit pap/ap über 0,3 wurden mittels va-ecmo therapiert, der patient mit pap/ap von 0,26 mittels vv-ecmo. schlussfolgerungen. isolierte veränderungen der pulmonalen hämodynamik repräsentieren offenbar nur bedingt den schweregrad der vorhandenen beeinträchtigung. als wichtiger parameter für eine rasche einschätzung des grades der kompromittierung kann die druckratio zwischen pulmonalem und systemischem kreislauf angesehen werden. als leicht zu erhebender parameter könnte die pap/ap-ratio eine entscheidungsgrundlage für die zu wählende ecmo-konfiguration darstellen. nach den bisherigen erfahrungen wäre die indikationsgrenze bei einer pap/ap-ratio von etwa 0,3 zu ziehen. background. pancreas transplantation in the mouse is an extremely demanding procedure and severe technical problems have limited its widespread use. since the mouse, however, would be a good model for the study of various transplantation-related problems, such as ischemia-reperfusion injury or graft pancreatitis, we designed a new surgical strategy for cervical heterotopic vascularized pancreas transplantation using a cuff technique. methods. male syngeneic c57bl6 (h-2 b ) mice (n = 27) 10-to 12-week-old were used as size-matched donor and recipient pairs. recipients were intraperitoneally injected with 312.5 mg of streptozotocin per kg in order to become hyperglycemic (blood glucose, >300 mg/dl) and transplantation was performed 4 days later. recipient operation: the right external jugular vein (ejv) and common carotid artery were dissected free. by using a polyethylen cuff (od, 0.63 mm) it became possible to evert the artery over the cuff body and finally fix the vessel with 8-0 silk ligatures. similarly, the ejv could be everted over a 0.94 mm cuff. donor operation: after a complete midline incision the pancreas was isolated using a no-touch technique on a segment of the aorta, including the celiac axis and the superior mesenteric artery. the venous outflow was provided by the portal vein. all grafts were flushed with 4 °c saline solution. implantation: the graft was placed in the right cervical region and vascular anastomoses completed by pulling the pv over the ejv cuff and the donor aortic segment over the carotid cuff and held in place with a 8-0 silk ligature. after releasing venous and arterial clamps, all grafts immediately returned to their normal pink color with the arterial stump pulsating. results. out of 27 recipients, surviving over 50 days, 2 animals died from haemorrhage (survival rate, 93%). donor operation lasted 40 ± 5 min and dissection of recipient vessels took 20 ± 4 min. implantation time was 4 to 6 min, resulting in a total pancreas ischemia time of 33 ± 6 min. no thromboembolic complications at the cuff side were observed. preoperative glucose levels were 518 ± 59 mg/dl and could all be normalized by po day 1 (88 ± 13 mg/dl). histopathological examination on po day 10 and 30 showed almost normal islet cell and acinar architecture of all grafts. conclusions. for the first time a method of cervical heterotopic pancreas transplantation using a non-suture cuff technique in the mouse is described. major advantages are a short ischemia time, lack of arterial thrombosis or venous stenosis, and short operation time, and thus a very high survival rate. this model is especially applicable for investigating preservation, reperfusion injury, and graft pancreatitis. background. as human islet transplantation is limited by the lack of sufficient numbers of human donor organs, xenotransplantation with the use of porcine islet cells seems to be a promising therapeutic option to cure diabetes. in order to achieve sufficient numbers of viable islet cells, better protocols for organ preservation, isolation, and purification are needed. recent studies showed that the two-layer method (tlm) of pancreas preservation prior to isolation significantly improved islet yield. the tlm oxygenates pancreata and activates metabolism to generate atp and leads to resuscitation of ischemically damaged organs. another possibility to achieve a higher partial pressure of oxygen levels in fluids is the use of hyperbaric oxygenation (hbo). the aim of this study was to assess the influence of preoxygenated preservation solutions on the porcine pancreas. methods. university of wisconsin solution (uw), celsior, perfadex, custodiol, and a preservation solution especially designed for this study on the basis of ketoglutarate are oxygenated with 100% oxygen for 50 minutes at 1.5 bar using a hyperbaric chamber. porcine pancreata are harvested at a local slaughter house and stored in preoxygenated and not oxygenated preservation solutions at 4 °c for 4 hours. tissue cuts are performed to assess the occurrence of apoptosis and to determine the oxidative stress. atp-to-adp ratio is measured and immunohistochemistry is performed. results. it is feasible to preoxygenate preservation solutions. the oxygen levels can be raised up to 100 times in the preservation solutions using hbo and can be maintained for at least 12 hours after hbo treatment. mda and carbonylated protein levels are not significantly elevated in organs stored in preoxygenated solutions. atp-to-adp ratio as a sign of viability is significantly higher in organs stored in preoxygenated solutions. conclusions. preoxygenation of preservation solutions using 100% oxygen and a hyperbaric chamber is feasible. hbo has a positive impact on porcine organ preservation. as ischemically damaged islet cells are likely to undergo cell death or lose functionality due to hypoxia, the use of preoxygenated preservation solutions is a promising method to achieve better yields after islet isolation and transplantation. alternative zur humanen pankreastransplantation und inselzelltransplantation stellt die xenotransplantation von porcinen inselzellen dar. um xenogene zellen erfolgreich transplantieren zu können, müssen sie vom immunsystem des empfängers abgeschirmt werden. die verwendung von mikrokapseln scheint eine vielversprechende methode dazu zu sein, wobei natrium zellulose sulfat (nacs) in graz verwendet wird. darüber hinaus muss eine ausreichende anzahl an vitalen zellen mit hoher reinheit aus dem porcinen pankreas isoliert werden. methodik. porcine pankreata werden von einem lokalen schlachthof erhalten. die organe werden kanüliert, und ein enzymgemisch aus neutraler protease und kollagenase nb wird infundiert. die digestion erfolgt in einer modifizierten ricordi-kammer mechanisch und enzymatisch, und die anschließende purifikation wird mit einem ficoll-dichtegradienten durchgeführt. vitalität der zellen wird mit fluorescein-diacetat/propidium jodid, mtt und der bestimmung der atp/adp-ratio überprüft. die reinheit wird mit einer dithizon-färbung festgestellt. insulinkonzentrationen werden mittels elisa detektiert, der dna-gehalt der inselzellen mit dem dneasy-kit festgestellt. inselzellen werden in kooperation mit der firma austrianova mit nacs mikroverkapselt. ergebnisse. die organe wurden durchschnittlich 35 min digestiert, es wurde eine reinheit von 96% und eine zellzahl von durchschnittlich 2 × 10 5 zellen isoliert. beste resultate wurden mit dem lymphoprep tm -dichtegradienten erzielt. isolierte porcine inselzellen überleben bei 37 °c im durchschnitt 12 tage in vivo und produzieren glucose-abhängig insulin. die funktionalität der zellen bleibt über den gesamten zeitraum erhalten. mikroverkapselung mit nacs ist machbar. schlussfolgerungen. die isolation porciner inselzellen ist eine viel versprechende methode den mangel an humanen spenderorganen in der pankreastransplantation und humanen inselzelltransplantation zu umgehen. nacs als verkapselungsmaterial ist weniger immunogen und weitaus biokompatibler als alle bisher verwendeten materialien und die mikroverkapselung xenogener inselzellen mit nacs scheint eine innovative methode zur therapie des diabetes mellitus zu sein. background. to achieve its full potential, transplantation of pancreatic islets has to overcome a number of obstacles. one of the obstacles are the still lacking read-out parameters to assess the quality of human islets after the isolation procedure and prior to transplantation. being able to predict the functional potential of the pancreatic islets after isolation or even short-term culture would greatly enhance the success of islet transplantation. therefore one of the primary challenges in islet transplantation is to identify and understand the changes taking place in islets after isolation or culture. life confocal microscopy is a powerful tool to identify such changes in living islets not achievable by use of fixed-cell techniques. methods. islets were isolated according to the method of ricordi et al., using a continuous ficoll gradient. 3 fluorescent dyes, dichlorodihydrofluorescein diacetate (dcf), tetramethylrhodamine methyl ester (tmrm), and fluorescent wheat germ agglutinin were used to assess either overall oxidative stress, time-dependent mitochondrial membrane potentials, or localisation of oligosaccharides. confocal microscopy was performed with an microlens-enhanced nipkow disk-based confocal system ultraview rs (perkin elmer, wellesey, ma, usa) mounted on an olympus ix-70 inverse microscope (olympus, nagano, japan). results. with the above described confocal system we were able to identify differences in the localisation and amount of oligosaccharides in endocrine vs. exocrine cells of freshly isolated pancreatic islets. the staining pattern changed during the course of culture, suggesting a remodeling of cell surface oligosaccharides. the study of the mitochondrial membrane potential proved to be very useful in order to early identify damaged or stressed cells and thereby gain insights into the vitality of the isolated islets. conclusions. this makes us believe that, especially in the light of the many other fluorescent dyes which can be used as subcellular markers, a combination of these with a powerful live confocal imaging system will be of great value for a better islet assessment after isolation and culture. background. therapeutic drug monitoring (tdm) of immunosuppressants is a well established concept supporting the work of clinicians from the historical onset of the use of these drugs. within the last years the combination of hplc (highperformance liquid chromatography) with tandem mass spectrometry (ms/ms) provides an alternative to antibody-based immunoassays. this new analytical method to quantify immunosuppressants affords a robust and rapid separation technique with high selectivity. up to now, the quantification of wholeblood levels of cyclosporin a, fk 506 (tacrolimus), and 42-o-(2-hydroxyethyl)-rapamycin (everolimus) at our institute have relied upon three different, indirect assays from different companies. however, these systems have drawbacks caused by cross reactions with active and inactive drug metabolites, fluctuations in assay performance, and comparatively high prices. in contrast, hplc-ms/ms platforms are now becoming frequently used to measure circulating drug levels and their metabolites with lower associated costs and higher specificity, accuracy, and precision. methods. the setup chosen at our laboratories consists of two independent mass spectrometers (one machine at the zimcl, a backup machine at biocrates) and allows a sample throughput of about 25 samples per hour. all immunosup-varia pressants currently monitored in whole blood can be quantified within one analysis from a sample volume of less than 100 µl. the sample workup (cell lysis and protein precipitation) is performed in a bar-code-supported parallel setup, minimizing the risk of sample mix-up. an online solid-phase extraction (spe) strategy has been chosen to reduce matrix interferences and ion suppression. daily calibrations and quality control samples performed with certified reference materials assure a high level of accuracy and precision. results. the hplc-ms/ms assay established for cyclosporin a, tacrolimus, everolimus, and sirolimus covers the analyte concentration range needed for tdm (e.g., up to 2000 mg/ml for cyclosporin a). intraday and interday repeats of the assay and data from quality control measurements were sufficiently accurate and precise (all cvs, <15%; bias, <15%). comparison of the quantitative results did show a linear relationship between antibody-based immunoassays and the hplc-ms/ms-derived data with bias values (ca. 10% for cyclosporin a and ca. 30% for tacrolimus on the basis of bland-altmann plots) in agreement with the literature. conclusions. the established hplc-ms/ms platform will allow replacement of the current antibody-based assays. the major advantage of this technique is the ability to simultaneously acquire absolute quantitative concentrations of each of the therapeutic drugs administered from one sample. therefore, the requirement for different sample preparation schemes or parallel measurements on different analytical instruments are no longer needed. the high sample throughput also assures timely tdm data report to the ward. a significant reduction of costs is now expected due to the lower consumable expenses in hplc-ms/ms assays. background. a clinical trial towards improved safety of the application of everolimus (certican) in heart transplantations (crad001a2403, data collection started in august 2004 and is ongoing) allowed us to compare therapeutic drug monitoring (tdm) data (e0 and e2 levels) measured on two different platforms -an lc-ms/ms method in an external laboratory and a immunochemical method recently established at our institute. methods. lc-ms/ms measurements of everolimus were performed in a reference laboratory. the immunochemical measurements were performed with a fluorescence polarization immunoassay (innofluor certican immunoassay by seradyn) measured on a tdx instrument from abbott. method comparison included analysis of the time series of the patients (n = 7, 65 observation pairs) and the quality control samples of the immunochemical assay gathered over a year as well as inter-and intra-assay data comparisons based on bland-altmann plots and regression data. results. comparison of the quantitative results obtained from the lc-ms/ms and the immunochemical assay showed good agreement between these methods. the bias between the methods (bland-altmann plot) was found to be rather small, with the immunochemical method measuring in average approximately 5% (±25%) lower values as the lc-ms/ms method, which is in good agreement with current reports. the coefficient of variation (cv) (measured over several months) of the innofluor quality control measurements was <7% for the medium (ca. 12 ng/ml) and high (ca. 25 ng/ml) quality control samples, whereas the lower quality control level (ca. 4.5 ng/ml) showed an increased cv (<13%). however, especially during the phase of method establishment, higher cv values and bias deviations have been found conclusions. the method comparison did show that the immunochemical everolimus assay provided by seradyn is a good alternative to hplc-ms/ms measurements. the assay bias was found to be rather low and the assay uncertainty was within acceptable ranges. however, a stringent quality control network must be provided to assure stable assay performance over time. grundlagen. die inzidenz der endokarditis beträgt ca. 6/100 000 einwohner pro jahr in der gesamtbevölkerung. obwohl die immunsuppression das auftreten systemischer infektionen begünstigt, sind endokarditisstudien an transplantationsempfängern nicht verfügbar. ziel dieser epidemiologischen studie war, das auftreten der endokarditis und ihrer risikofaktoren nach organtransplantation zu evaluieren. methodik. insgesamt 2556 patienten, welche sich zwischen 1989 und 2004 einer soliden organtransplantation an unserem zentrum unterzogen, wurden untersucht. der mbds unserer klinik wurde zum patientenscreening herangezogen. ergebnisse. insgesamt wurden im beobachtungszeitraum 27 endokarditisfälle beobachtet. neun endokarditisfälle (33,3 %) konnten erst post mortem mittels autopsie diagnostiziert werden, 8 patienten (29,6 %) konnten durch alleinige antibiotische therapie geheilt werden. insgesamt 10 transplantationsempfänger (37,1 %) mussten sich einem kardiochirurgischen eingriff unterziehen. die gesamtmortalität betrug 44,4 % (12 patienten). staphylococcus aureus konnte in 16 fällen (59,3 %) und pilze konnten in 4 fällen als ursächliche keime isoliert werden. die inzidenz der endokarditis in transplantationsempfängern beträgt 1 % (95 % ci, 0.67-1.49) und zeigt ein 171-fach erhöhtes risiko verglichen mit der gesamtbevölkerung. schlussfolgerungen. die endokarditis stellt ein signifikantes problem nach organtransplantation dar und ist mit einer exzessiv hohen mortalität assoziiert. eine erhöhte aufmerksamkeit ist daher indiziert, da wir durch den einsatz der transösophagealen echokardiographie vermehrt derartige fälle in der zukunft diagnostizieren werden. ein 47-jähriger patient erhält 1995 eine herztransplantation aufgrund einer dilatativen kardiomyopathie bei altersentsprechender nierenfunktion. die immunsuppression besteht aus cyclosporin a und mycophenolat mofetil. innerhalb der ersten drei monate nach der operation steigt das kreatinin auf 2,0 mg/dl an, bleibt dann aber konstant. ab 2003 nimmt die nierenfunktion weiter ab, weshalb im august 2004 wegen des verdachtes auf eine chronische calcineurininhibitor-nephropathie auf sirolimus und mmf umgestellt wird. das serum-kreatinin beträgt zu diesem zeitpunkt 3,0 mg/dl. zwei monate später ist das serum-kreatinin auf 7,0 angestiegen, der inzwischen 57-jährige patient leidet unter einer renalen anämie, hyperphosphatämie und hypokalziämie. im harn finden sich leukozyten und erythrozyten, die allerdings nicht deformiert sind (keine dysmorphen erythrozyten und akanthozyten), sowie eine geringgradige proteinurie (300 mg/24 h). in der harn-polyacrylamidgel-elektrophorese zeigt sich vor allem alpha-1-mikroglobulin als indikator einer tubulointerstitiellen schädigung. nach erneuter umstellung der immunsuppression von sirolimus/mmf auf niedrigdosis-cyclosporin/mmf kommt es zum raschen rückgang des serum-kreatinins, der harnbefund sowie die elektrolyte normalisieren sich, einzig die erythropoetin-substitution muss beibehalten werden, um den hämoglobin-zielwert von 11 g/dl zu erreichen. acht wochen nach der zweiten therapieumstellung führen wir eine nierenbiopsie durch. es findet sich eine akute interstitielle nephritis ohne glomeruläre schädigung. unseres wissens ist dies der erste dokumentierte fall einer akuten interstitiellen nephritis auf sirolimus. background. group milleri streptococci (gms) are a heterogeneous group of streptococci including the species streptococcus intermedius, s. constellatus, and s. anginosus. due to their ability of producing toxins, they tend to cause chronic intra-abdominal and intrathoracic abscesses, which are difficult to treat, as gms are able to escape conventional antibiotic therapy. aim. evaluation of epidemiology, clinical significance, and impact on the outcome in all solid-organ recipients with gsm infections during a 4-year period. patients and methods. retrospective analysis comprising 45 solid-organ recipients with gms. results. between 2001 and 2004, 45 solid organ recipients (88 isolates) including 34 liver, four kidney and pancreas, one kidney, two small bowel, three combined liver and kidney, and one combined kidney and small bowel transplant re-cipients developed infection with gms. in 42 cases, gms caused intra-abdominal infection; in two cases, pleural empyema; and in one case, soft tissue infection. in only one case, gms were cultured from blood. in 54 of the 88 specimens (61%) which grew gms, also other pathogens could be isolated. gms frequently caused recurrent cholangitis (n = 17) associated with anastomotic and anastomotic biliary strictures. these cases were managed by repeated stenting or surgical intervention and prolonged antibiotic therapy. no patient died directly related to gms infection. all responded to combined surgical and antibiotic treatment. one pancreas graft was lost due to erosion haemorrhage associated with an abscess. all isolated strains of gms were susceptible to penicillin g, carbapenems, and clindamycin, whereas cephalosporins and quinolones showed intermediate activity or resistance in some cases and gms in general were found resistant to aminoglycosides. conclusions. gms are frequent pathogens in transplant surgery and are capable of causing difficult to treat infections. their prevalence in transplant surgical site infection thus far might have been underestimated. therefore, we recommend empiric antibiotic treatment for sufficiently long time in combination with surgical intervention when necessary. background. by using intensified immunosuppressive protocols the incidence of immunological complications after solid-organ transplantation has constantly declined. however, the incidence of some infections, in particular complicated fungal infections, seems to increase. candida krusei (ck) is resistant to fluconazole and recently this pathogen has been more commonly isolated in severe infections, particularly in the immunocompromised host. methods. between 1. 1. 2004 and 30. 6. 2005 , a total of 400 solid-organ transplants were performed at the innsbruck medical university. this included 150 renal, 70 liver, 50 pancreas, 5 intestinal, 30 cardiac, 20 lung, and 10 islet transplants. prophylactic immunosuppression consisted of calcineurin inhibitor-based triple drug therapy for the majority of cases. antifungal prophylaxis was given to all pancreatic and intestinal recipients (fluconazole 400 mg per day) and to all patients considered at high risk for filamentous-fungal infections (ambisome 3 mg/kg). results. a total of five patients with ck infection were identified within this series (2%). five patients developed infection with candida tropicalis and three with candida glabrata. within this time period, another two patients with ck infection were identified, one had a pleural empyema following esophageal perforation and the other was treated with a ventricular assist device and developed pulmonary infection with ck. both patients were treated on the transplant intensive care unit. the transplant patients were three pancreas, one liver, and one lung recipient. all three pancreas recipients were diagnosed intra-abdominal infection, the liver recipient had an ischemic cholangiopathy with ck cholangitis and required retransplantation, and the lung recipient developed postoperative hemorrhage and subsequently ck pleural empyema. all patients presented also with other infectious complications. treatment of ck infection consisted in four cases of caspofungin or voriconazole or combination of the two, and in one case, ck was isolated from infected hematoma and did not require antifungal therapy after surgical removal. all infected collections were evacuated either surgically (n = 3) or through pig-tail drainage (n = 2). ck infection was successfully managed in all cases and patients are currently alive, only one pancreas graft was lost. conclusions. candida krusei infections now represent frequent severe complications in solid-organ recipients. however, rapid diagnosis and treatment with new antifungal agents such as voriconazole, caspofungin, or ambisome allow successful therapy of these infections. solid-organ recipients seem to be at increasing risk to acquire non-albicans candida infections. background. after solid-organ transplantation immunosuppression (is) and concomitant infection with cmv, ebv, hhv-6, -8, or papillomavirus put patients at risk for developing malignant diseases (14% cumulative risk at 10 years; adami et al. 2003) . posttransplant lymphoproliferative disorders (ptld) and skin cancers are known to have the highest incidence in immunocompromised patients. reports on colorectal cancer after different types of organ transplantation are rare and the incidence was 0.01% to 3.9% and did not differ from that of the normal population. we analysed our database with regard to the incidence and course of colorectal malignancies following treatment and introduction of tor inhibitor-based is. methods. medical records of solid-organ transplants performed between 1986 and 2005 at our center were analysed retrospectively. in a total of 3568 patients 247 heart, 118 lung, 2074 kidney, 757 liver, 367 pancreas, and 9 combined heart-lung transplantations were performed (27 small bowel and 4 hand transplantations not included). immunosuppressive therapy consisted of triple therapy comprising steroids, azathioprine/mmf, and cya/tac. some of them received induction therapy with antithymocyte globulin. results. a total of 206 patients (5.72%) developed malignancies. of them, 9 patients (1 female, 8 male; median age at diagnosis, 66.1 years; 2 kidney, 3 heart, 4 liver recipients) had colorectal malignancies (0.25%) during a mean follow-up period of 7.3 years. on average, diagnosis was made 5.8 years after transplantation. four carcinomas were located in the rectum or at the rectosigmoid junction and five were colon cancers (five pt3, one pt2, and three pt1 stages). r0 resection was performed in all 9 patients plus radio-and/or chemotherapy in all t3 stages. five patients (55%) died 7.2 years post transplant due to cardiovascular disease (n = 4) and recurrent tumor disease (n = 1). the 1-year survival rate was 67% for t3 and 100% for t1 rectal cancers, 50% for t3 and t1 each and 100% for the only t2 colon cancer. three anal neoplasms (one ain iii°, two anal cancers, pt1 and pt2; median age at diagnosis, 50 years) developed on average 7.2 years after transplantation (0.08% vs. 0.001% in the general population) with a 100% 1-year survival rate. all patients were switched to rapamycin or everolimus after completion of primary therapy. conclusions. the incidence of anal but not of colorectal cancers in our transplant patient population differed from that of immunocompetent patients of corresponding age (0.08% vs. 0.001% and 0.25% vs. 0.2% tyrol tumor registry and 0.3% seer). the 1-year survival rate was significantly decreased in the transplant group with t3 tumors (67% [rectum] and 50% [colon] vs. 80%). potential antineoplastic effects of rapamycin and overall less immunosuppression long-term may improve prognosis of colorectal malignancies following transplantation. background. solid-organ transplantation is the treatment of choice for terminal organ failure. due to the scarcity of organs, the mandate to utilize extended-criteria donors has dramatically increased over time and achievable results are good. this harbors the risk of transmission of infections from the donor to the recipient. aim. an overview on the magnitude of possible transmittable diseases that could accompany donor organs is given. possible preventive strategies are discussed. overview. the faith of an organism that is transmitted by a donor allograft depends on the virulence and the quantity of the transmitted pathogen and the site of infection and furthermore is impacted by prophylactic steps undertaken during donor procurement, organ perfusion, implantation, and post transplant course and lastly depends on the ability of the recipient to control the infection by the immune system. in the best case, the microorganism is cleared before it can do any harm; however, in the worst case scenario, transmitted pathogens can lead to fatality. there is a multitude of pathogens that can potentially be transmitted by an allograft including viruses, bacteria, fungi, and protozoa. there are microorganisms that can be transmitted by any type of graft and there are pathogens which due to a certain tropism can be transmitted by certain organs only. there are common pathogens such as cmv and ebv as opposed to extremely rare pathogens such as lyssa, west nile fever, or lymphochoriomenigitis virus. intracellular pathogens are preferably targeted by mhc restricted cytotoxic t-cell reaction. as in organ transplantation hla matching is in general not performed, donor-derived antigen-presenting cells cannot be recognised by recipient-specific cd8 + lymphocytes. therefore, either donor-specific cytotoxic t cells must function as counterparts or alternative tar-gets must be defined by the immune system. bacterial and/or fungal contamination must be considered in lung transplantation (bronchial stump), pancreas transplantation (duodenal segment), and small bowel allografts. cardiac, renal, and liver grafts can be considered sterile but can become contaminated during procurement. rare transmitted organisms are toxoplasma gondii, trypanosoma cruzii, and schistosoma mansoni. conclusions. when looking at allocation of latently virus-infected organs, they should be preferably given to recipients who have antibodies against the particular agent. in terms of bacterial or fungal contamination of graft and/or preservation solution, routine monitoring seems mandatory in order to assure quality. keeping blood and serum samples from donors should be carried out from an epidemiological and legal standpoint. bacterial and fungal prophylaxis should be used to prevent not only recipient-but also donor-derived pathogens. antiviral prophylaxis is standard for cmv and hbv, for all other viruses no final recommendations are available. methods. the aim of the current review was to investigate the impact of several risk factors on ptld. therefore, patients developing ptld after heart transplantation at our institution were screened. results. 8 patients have been identified with ptld, all cases occurring during the last decade. mean age at ptld onset was 46.4 ± 20.0 years and time between transplant and development of ptld was 26.4 ± 30.4 months. there were 5 ebv-associated ptlds, 2 non-ebv-associated, cd20-negative b-cell lymphomas, and 1 t-cell lymphoma. immunosuppression at ptld onset was calcineurin inhibitor based (cyclosporine a, 5 patients; tacrolimus, 3 patients). initial immunosuppression included atg induction. six patients received perioperative antiviral prophylaxis with either valgancyclovir/gancyclovir (n = 4) or acyclovir (n = 2) in combination with anti-cmv hyperimmunoglobulin (n = 1). two patients experienced a total of five episodes of acute rejection (ishlt ii°), all were treated with bolused steroids. four patients are still alive (50%), three of them in current remission of ptld, one patient is under therapy recently. median survival was 27 months in survivors and 3.4 months in nonsurvivors. conclusions. these data show that ptld is associated with a high mortality rate. the majority of ptlds are ebvassociated. therefore, screening for ebv infection and prophylactic treatment may help to prevent a potentially fatal consequence of heart transplantation. background. late acute cellular rejection is associated with a decrease of survival and the development of cav. new immunosuppressice drugs have been introduced into clinical practice. everolimus, as one of these, has shown to be safe in cardiac transplantation. we report of our experience with everolimus in heart transplant recipients who developed late cardiac rejection. methods. patients with a history of previous rejection episodes who experienced cardiac rejection after at least 2 months postoperative were switched to an everolimus, cyclosporine a, and corticosteroid-based immunosuppressive regime. all patients already received statins and antihypertensive medication before. everolimus, cyclosporine a trough levels and laboratory values were controlled monthly. drug administration was adapted to an everolimus trough level between 3 and 8 ng/ml, mean maintenance dosage was at 0.25 to 1.5 mg/day. death, safety, side effects, biopsy-proven acute rejection, laboratory values and blood levels were evaluated retrospectively. results. 4 cardiac allograft recipients (2 male, 2 female), at a median of 1473.25 days post orthotopic heart transplantation (ohtx) (65-3045), received 1 mg to 1.5 mg everolimus per day. in a follow-up period of at least 2 month (2-10) mortality was 0%. the drug was well tolerated and no acute cellular rejection greater than grade 1a (ishlt grading) was observed after two month. in one patient raised cholesterol values and in two others elevated triglyceride levels were seen but were controlled with higher statine therapy. no obvious raised creatinine values were seen with certican ® . conclusions. in conclusion, conversion to an everolimus-based immunosuppressive regimen after late cardiac rejection is safe and effective. no major side effects were observed. p02 collagen iii in transplanted heartdonor or recipient derived m. pichler, b. tessaro, d. kniepeiss, r. kleinert, g. hoefler institute of pathology, medical university of graz, graz, austria background. transplantation of donor hearts is often associated with progressive development of interstitial myocardial fibrosis and alterations in composition and organisation of the extracellular matrix. changes in cardiac interstitial collagen network are thought to contribute to abnormal stiffness and loss of function of the myocardium. fibroblasts are the main producers of type i and type iii collagens, the major in-poster terstitial collagens found in the heart. in transplanted hearts, intragraft fibroblasts may consist of two cell populations. donor-derived fibroblasts preexist in donor organs, whereas hostderived fibroblasts may progressively immigrate as mesenchymal progenitors from the circulation to the allograft. purpose of the study. to determine the contribution of these two distinct fibroblast populations to progression of myocardial fibrosis, we studied endomyocardial biopsies over a time period of some years from a male patient who had received a heart from a female donor. methods. in these sex-mismatched patients two frequent genetic polymorphisms at the collagen iii locus were determined by polymerase chain reaction-based restriction enzyme digestion, both in donor allograft and in the corresponding explanted recipient heart. on the basis of differences of the collagen genotype in donor and recipient tissue, we selected 10 endomyocardial biopsies by hematoxylin eosin staining covering an overall follow-up period of nine years since the transplantation event. immunohistochemistry, chromogene in situ hybridisation, and population-specific collagen expression using single nucleotide polymorphisms were used to determine the course of fibrosis. results. we developed an analytical system generally applicable to measure population-specific differences of collagen iii synthesis in transplanted organs. the amounts of interstitial collagen type i and type iii increased in a time-dependent manner within cardiac allograft. years after transplantation, a number of y-chromosome-positive recipient-derived cells consisted of noninflammatory spindle-shaped fibroblast-like cell types. conclusions. our data confirm the existence of a substantial number of fibrosis-mediating immigrated recipient-derived fibroblasts in cardiac allografts. furthermore, this suggests a potential, future therapeutic approach for reduction the cardiac fibrosis process. methods. between 1993 and 1998 a total of 32 lung transplants (31 patients) and six combined heart-lung transplants were performed at our center. immunosuppression consisted of atg induction, cyclosporine a, azathioprine, and steroids. all patients with rti underwent a meticulous micro-biological screening and underwent bronchoscopy with bronchiolo-alveolar lavage (bal) and transbronchial biopsies (tbb). rsv was detected from bal specimens by an immunoassay. results. a total of five lung recipients (31% of 19 survivors) and one of the two alive heart-lung recipients developed rsv infection of the lung allograft. all cases were observed during an rsv epidemic in children in the area between october 1998 and may 1999. the patients were 47-55 years old, all were females. one patient experienced two episodes. onset of rsv was median 3 (range, 4-29) months post transplant. clinical symptoms included cough (6 of 7), rhinitis (6 of 7), and fever (4 of 7). deterioration of lung function occurred in six of the seven episodes with one deteriorating to respiratory failure requiring ventilator support. two individuals developed pulmonary infiltrates. in all patients, immunosuppression was significantly tapered, 86% required hospitalization and antibiotic therapy. the patient with rsv recurrence received inhalative ribavirin therapy during the second episode. all patients recovered and survival of this cohort was 86% after 1 year and 72% at 4 years after rsv infection. none of the patients developed bronchiolitis obliterans syndrome (bos) or rejection during follow-up. conclusions. rsv is a severe but benign complication following lung transplantation with a wide range of clinical presentations. routine use of antiviral treatment is not necessary; however, reduction in the level of immunosuppression is required. no long-term effects were observed in this cohort. a. stamatelopoulos 1,2 , s. guth 2 , a. abrahim 2 , g. m. marta 2 , l. tsourelis 3 , p. jaksch 2 , c. konaris 4 , s.taghavi 2 , w. klepetko 2 due to cni nephrotoxicity in a substantial number of ntx patients. we therefore studied the effects of a switch from a cni regimen (cni + mpa + p) to a dual regimen of sirolimus (srl) plus prednisolon (p) in 26 pts with moderately impaired kidney function (s-crea, 1.5+0.4 mg%) due to either cni toxicity or clinical evidence for chronic allograft disease. 13 pts received regimen 1 consisting of srl 12 mg dosage on day 1 and from days 2 to 5 srl 4 mg plus csa at half of maintenance dosage. from day 6 on, pts received srl 8 mg and csa was withdrawn. mpa or aza dose was continued halfed for 4 to 6 weeks. p was kept constant. target level for srl was 12-20 ng/ml. another 13 pats received regimen 2 consisting of srl 12 mg and csa withdrawal on day 1 and from days 2 to 6 srl 6 mg was administered for a target level of 7 to 10 ng/ml. mpa or aza were continued halfed for 4 to 6 weeks, p was kept constant. with regimen 1 there were 6 dropouts due to adverse events, whereas with regimen 2 only 2 dropouts occurred. 45% of the patients showed a decrease of s-crea after 2-year observation period, 20% were unchanged and only 25% showed an increase. overall, there was a slight but significant increase of cholesterol and triglycerides, whereas other parameters were unchanged. conclusions. switch from cni containing immunosuppression to a dual regimen of sirolimus plus prednisolon results in an improved kidney function after 2 years in the majority of pts. a regimen of sirolimus with target levels of 12 to 20 ng/ml and an overlap of immunosuppression shows a high rate of adverse events and is associated with a 3-fold dropout rate as compared to a regimen with a target level of 7-10 ng/ml. background. certain anatomical variations mainly concerning the portal system preclude living donor liver transplantation (ldlt). to the best of our knowledge, two left lateral segments with two arteries have never been transplanted so far. case report. a 6-month-old girl was diagnosed with endstage liver cirrhosis secondary to biliary atresia and therefore scheduled for ldlt. preoperative evaluation of the donor including ct with 3-d reconstruction revealed normal vascular supply of the liver with a left and a right hepatic artery. during donor operation, two tiny arteries with a diameter of 2 mm for segments ii and iii were identified. they were found to arise from the left hepatic artery right behind the bifurcation of the proper hepatic artery, which made it impossible to preserve a common trunk. venous and portal venous reconstruction was performed in an end-to-end fashion. the left graft artery was directly anastomosed to the proper hepatic artery with 8/0 pds interrupted sutures using a microscope. the 2nd graft artery was revascularised with the help of saphenous vein from the recipient as interposition graft to the gastroduodenal artery but failed. therefore, the trunk of the inferior mesenteric vein was used for reconstruction with 8/0 pds with excellent outcome. under fk-based immunosuppression, postoperative course was uneventful with both arteries patent. conclusions. multiple arteries for the left lateral segments are not a contraindication for paediatric ldlt. the inferior mesenteric vein can be used as an interposition graft. combined renal-pancreas transplantation is an established treatment for patients with diabetic nephropathy, producing excellent results. we report on a patient who suffered from long-standing multiple sclerosis and underwent successful combined renal-pancreas transplantation. post transplant course was complicated by yeast intra-abdominal infection, which was treated with antifungal agents. using tacrolimus and sirolimus, both grafts are well functioning at two years and the patient is without any symptoms of disseminated encephalitis and does not require specific medication for multiple sclerosis. for diabetic patients suffering from multiple sclerosis, pancreas transplantation offers an excellent treatment option. whether normalisation of carbohydrate metabolism or chronic immunosuppression or both lead to complete response of multiple sclerosis is not clear. background. until recently, the peripheral blood stem cell (pbsc) donation procedure was used only infrequently among unrelated allogeneic donors. nowadays, both related and unrelated donors are expected to consider this alternative donation. to promote pbsc donation both safety and well-being of healthy unrelated volunteer donors must be protected and data are to be collected to establish the long-term safety of g-csf stimulation. methods. from 2000 to 2004, 16 pbsc aphereses on unrelated allogeneic donors have been carried out in our center. all pbsc donors were treated with 5 µg of g-csf per kg twice a day from day -4 to day -1. aphereses were performed using peripheral venous access on day 0 and -if indicatedon day 1. since 2003, all donors have annually received a questionnaire about their actual state of health and medication, as well as their physical and mental conditions. detailed questions concerned donors' anamnesis for epistaxis, bruises, thrombosis or embolia, as well as infections and fever, night sweat, and weight loss of unclear origin. results. 11 male and 2 female donors (81%) with an average age of 44.59 (29-56) years responded to the questionnaire. the observation periods were between 3 and 53 months (mean, 28.75 months) after g-csf stimulation. 5 pbsc donors (38%, all male) reported that they had been severely ill during the observation period: one donor developed an exostosis of the 5th rib, one was operated on an umbilical hernia, one suffers from recurrent articular and muscle pain accompanied by night sweat and weight loss, one has a chronic compensated renal failure, one had diarrhoe and a common cold and suffered from fatigue, nausea, sleeping problems, and circulatory disorders. one female donor recognized dizziness and an increased tendency for bruises as well as paraesthesia in both arms. the disorders these donors reported occurred between 3 and 22 months after g-scf stimulation. all the other pbsc donors (62%) have never been severely ill or under medical observation. no donor had fever of unclear origin, phlebitis, thrombosis, or embolia, no donor recognized an increased tendency for epistaxis. three donors need medication they did not have before g-csf stimulation, which are to lower blood lipids and anti-inflammatory ones. one donor estimates that he is getting ill more easily than others, all the other donors feel themselves in best physical and mental condition. background. hematopoietic stem cell transplantation (hsct) has been successfully performed in patients with otherwise incurable malignant diseases. however, relapse after hsct is one of the main reasons for treatment failure and further therapeutic strategies with acceptable toxicity are warranted. since myeloablative (ma) conditioning after prior hsct has been associated with high treatment-related mortality (trm), reduced-intensity conditioning (ric) regimens have been developed as salvage therapies for these patients. so far, encouraging results have been achieved with ric; however, a direct comparison with standard conditioning has never been performed. therefore, we retrospectively analysed these two conditioning strategies in patients experiencing relapse after prior stem cell grafting. methods. we analyzed 45 patients with relapsed disease (acute myeloid leukemia, n = 16; indolent lymphoma, n = 9; multiple myeloma, n = 7; chronic myeloid leukemia, n = 4; myelodysplastic syndrome, n = 2; chronic lymphocytic leukemia, n = 2; acute lymphocytic leukaemia, n = 2; aggressive lymphoma, n = 1; hodgkin's disease, n = 1; ovarian cancer, n = 1) after prior hsct who received either reduced-intensity or myeloablative conditioning for allogeneic hsct between 1986 and 2005. ric consisted of fludarabine 90 mg/m 2 and total-body irradiation (tbi) of 2 gy according to the seattle protocol (n = 18) or alemtuzumab in combination with the beam regimen (n = 1). myeloablative therapy consisted of cyclophosphamide (cy) and tbi of 12 to 13 gy (n = 13), cy plus busulfan (bu) (n = 4), c plus antithymocyte globulin plus bu (n = 2), or bu alone (n = 7). donors were syngeneic in 4, related in 21, and unrelated in 20 patients. stem cell source was bone marrow in 13 (29%) and peripheral blood in 32 (71%) patients. for graft-versus-host disease (gvhd) prophylaxis, 22 patients received cyclosporine a (csa) plus mycophenolate mofetil, 16 csa plus methotrexate, 2 mtx alone, and 1 csa alone. results. all patients achieved complete hematopoietic engraftment by day 28 after stem cell transplantation with complete donor chimerism. all patients conditioned with ric presented complete donor chimerism of t cells, myeloid progenitor cells, and nk cells 28 to 81 days after hsct. the in-cidence of acute gvhd was 37% and comparable in both groups consisting of grade i in 6 patients, grade ii in 3, grade iii in 6, and grade iv in 2. fourteen patients died after ma conditioning of acute gvhd (n = 2), infections (n = 5), or severe toxicity (n = 7), while only one patient died due to infection after ric. probability of transplant-related mortality (trm) at 1 year after hsct was with 54% significantly (p = 0.001) higher in patients given myeloablative conditioning compared to 5% after ric. incidence of therapy requiring chronic gvhd was with 64% versus 33% significantly (p = 0.001) higher in patients who received ric. response rates were comparable between patients who received ric or ma conditioning (90% versus 100%). relapses occurred in 37% of patients after ric and 32% after ma conditioning. after a median follow-up of 44.5 (range, 8-204) months 3 (11%) of patients of the ma group and 11 (58%) of the ric group are currently alive. probability of survival at 1 and 4 years after hsct is with 73% versus 24% and 48% versus 12% significantly (p = 0.008) higher after ric than after myeloablative conditioning. conclusions. allogeneic stem cell transplantation is a highly effective treatment option in patients relapsing after prior hsct. durable hematologic engraftment and sustained complete remissions can be achieved in patients with otherwise poor prognosis. since transplant-related mortality of dose-reduced conditioning is in comparison to myeloablative hsct considerably lower, overall survival can be significantly improved with this new treatment modality. background. patients with comorbidities such as organ dysfunctions or preexisting infections experience a high treatment-related mortality which makes them ineligible for conventional conditioning therapy. for these patients, reduced-intensity conditioning (ric) is an option which offers the benefits of an allogeneic transplant with lower extramedullary toxicity. we report on 8 pediatric and young adult patients who were considered for ric because of severe cumulative pretreatment or substantial comorbidities treated at our institution between 2001 and 2005. methods. eight patients (median age, 11 years; range, 1-28 years; m, 4; f, 4) diagnosed with aml (cr 1, n = 4; cr 2, n = 1), all (cr 3, n = 1), alps (n = 1), and relapsed ewing's sarcoma (n = 1). 7 of 8 patients were planned to receive ric, in one patient the regime was changed during conditioning due to an acute viral infection. all patients received fludarabine (n = 7, 150 mg/m 2 ; n = 1, 60 mg/m 2 ) combined with melphalan (n = 6, 100-140 mg/m 2 ), busulfan (n = 1, 13 mg/kg), or total-body irradiation (n = 1, 2 gy). 3 patients received atg (45-60 mg/kg), two patients campath (40 mg/m 2 ). post-transplant immunosuppression consisted of cyclosporine a in all patients combined with mycophenolate mofetil (n = 1) or methotrexate (n = 2). 7 of 8 patients received hla identical grafts (sibling, n = 3; hla identical mother, n = 2; mud, n = 2), one patient received a c-locus mismatched graft. stem cell sources were bone marrow in 6 patients and peripheral blood stem cells in 2 containing a median of 5.13 × 10 6 cd34 + cells per kg of body weight of the recipient. results. all patients had primary engraftment; the median time to neutrophil recovery (n > 1.0 × 10 9 /l) was 15 days. complete donor chimerism as evidenced by vntr was achieved in median on day +22 (+11 to +28). acute mild graftversus-host disease (gvhd) of the skin occurred in 5 of 8 patients and responded to prednisolone; one patient required additional immunosuppression with mmf. one patient progressed to extensive chronic gvhd; one patient developed chronic gvhd of the skin, another patient shows clinical evidence of chronic gvhd of liver and gut. the remaining 3 patients showed no gvhd at all. 5 of 8 patients were positive for one or multiple viruses on routine viral pcr monitoring, requiring virostatic treatment. no treatment-related mortality occurred. the patient with ewing's sarcoma died 4 months posttransplant from tumor progression; the patient with extensive chronic gvhd died of sepsis on day +400. all 5 patients with aml are in remission. the patient with alps is still positive for various autoantibodies. 3 patients developed a posttransplant macrophage-activation syndrome (mas). all of them required a stem cell boost because of pancytopenia. conclusions. ric followed by allogeneic hsct was successful in terms of achieving primary engraftment and complete donor chimerism as well as avoiding transplant-related mortality in all patients; in our patients with myeloid malignancies it was also successful in terms of maintaining stable remission. as for posttransplant problems, we encountered acute gvhd of the skin in 5 of 8 patients, three of whom developed chronic gvhd; and viral infections in 5 of 8 patients, three of whom developed mas and eventually required a stem cell boost. background. due to an aggressive course mantle cell lymphoma is characterized by poor prognosis with a median survival of 3 years and only 10-15% long-time survivors. recent improvements in therapy have been made by combined immunochemotherapy and intensification with high-dose chemotherapy. methods und results. ten patients (4 female, 6 male) with a median age of 56 (49-65) years were treated with rituximab plus chop for four or six cycles. furtheron, patients received 2 or 3 cycles of claeg-d (3 days cladribine 0.2 mg/kg, ara-c 1.5 g/m 2 , etoposid 60 mg/m 2 , and on day 1 daunoxome 80 mg/m 2 ) including stem cell collection. as a result, 5 out of 7 newly diagnosed patients reached cr1 and 2 pr. out of 3 patients treated after first relapse, 2 reached again cr and 1 pr. autologous transplantation was performed at a median of 9 (5-11) months after diagnosis. high-dose conditioning consisted of beam chemotherapy plus the addition of 4 doses of rituximab (days -9 and -1 of the conditioning regimen and days +48 and +55 after transplantation). four patients could not receive the rituximab therapy at days +48 and +55 due to complications. a median number of 5.71 × 10 6 cd34+ cells (1.65-21.21 ) per kg were reinfused. all patients had a short haematologic regeneration time (granulocytes, <0.5 g/l day +10 [9-11]; platelets, day +13 [10-17]) and received a median number of 3 erythrocyte (2-6) and 2 platelet (2-5) concentrates. all patients suffered from mucositis grade i-ii, 3 of 10 had emesis grade i-ii. infectious complication of short duration appeared in 7 patients (4 fuo, 2 pneumonia, and 1 sepsis) early after transplantation. one patient developed late complications (hypothyreosis on day +88 and sarcoidosis on day +105). following transplantation, all patients reached clinical and molecular cr lasting for a median time of 35 (1-53) months. two patients relapsed 17 and 26 months after transplantation. despite continuous salvage immunochemotherapy, one of these patients died 34 months after transplantation, the other one is still in pr (+48 months). median observation periode after diagnosis is 46 (11-105) months. conclusions. treatment intensification with immunochemotherapy and high-dose consolidation is accompanied by acceptable toxicity and seems to be an effective treatment procedure for mantle cell lymphoma. background. bone marrow transplantation (bmt) together with costimulation blockade can reliably induce mixed chimerism and tolerance. in recent studies, we showed that regulation by cd4 + cd25 + t cells plays an important role in this model. stimulation of cd4 + cd25 + t regs by an anti-cd3 mab can inhibit and reverse the outbreak of certain autoimmune diseases, and the maintenance and function of t regs was demonstrated to critically depend on il-2. we thus investigated if stimulation of regulatory cells by anti-cd3 or interleukin-2 facilitates bm engraftment and reduction of tbi in our model. methods. c57bl/6 mice received a total-body irradiation (day -1) of 3 gy or 1.5 gy, approximately 20 × 10 6 fully mismatched balb/c bone marrow cells (day 0) and costimulation blockade consisting of anti-cd154 mab (mr1, day 0) and ctla4ig (day +2). additional groups received further treatment with (n = 4-8 per group): (1) anti-cd3 mab (5 µg, day 0 to +4), (2) il-2 (4000 ie/day, day 0-27), (3) rapamycin (0.3 mg/kg/day, day 0-27), and (4) rapamycin and il-2 (day 0-27). multilinage chimerism and skin graft survival were followed for more than 120 days. results. with our standard protocol, 10 of 10 (3 gy) and 8 of 14 (1.5 gy) mice developed long-term multilinage chimerism and accepted donor skin grafts permanently. while the majority of mice treated with anti-cd3 and 3 gy (7 of 8), rapamycin plus 1.5 gy (4 of 6), and il-2 and rapamycin plus 1.5 gy (4 of 5) developed multilinage chimerism and longterm skin graft survival, groups treated with anti-cd3 plus 1.5 gy tbi (0 of 8, p < 0.01) and il-2 plus 1.5 gy (2 of 6, p = 0.1) showed markedly reduced rates of chimerism and tolerance. we investigated if these negative effects might be correlated with a cytokine shift caused by anti-cd3 treatment, but we did not observe such a shift towards th1 or th2 (as measured on day 13 post-bmt). conclusions. unexpectedly neither anti-cd3 nor il-2 had a positive effect in this model, in some groups anti-cd3 treatment even showed a negative effect. thus, other strategies for augmenting the effect of regulatory cells need to be developed. the role of minor antigen disparities for the induction of mixed chimerism and tolerance through bmt plus costimulation blockade s. bigenzahn 1 , i. pree 1 , p. nierlich 1 , e. selzer 2 , f. mühlbacher 1 , t. wekerle 1 long-term donor skin grafts looked macroscopically intact in the b10.d2 group but showed shrinking and scabs in the balb/c group. conclusions. minor antigen disparities pose a substantial barrier for the induction of mixed chimerism and tolerance. for increased clinical relevance, tolerance models should preferably use strain combinations including major plus minor mismatches. p20 immunohistochemical and confocal analysis of pancreatic tetranectin d. pirkebner 1 , m. hermann 1 , a. draxl 1 , w. mark 2 , r. margreiter 2 , p. hengster 2 background. islet transplantation is not yet widely used in part because of the shortage of human islet cells. gaining detailed knowledge of the physiological basis governing the processes of differentiation of pancreatic stem or progenitor cells that have the capacity to self-renewal and to generate differentiated beta cells is instrumental for the ambitious goal of engineering new pancreatic islets in order to cure type i diabetes. the aim of our study was to cultivate and characterize a pancreatic cell population expressing tetranectin (tn). the ability of tn to bind plasminogen indicates that it may have a role in regulating pericellular proteolysis and proteolytic activation of latent forms of metalloproteinases and growth factors. methods. islets were isolated according to the method of ricordi et al. (1992) using a continuous ficoll gradient. immunohistochemistry and immunofluorescence were performed with a monoclonal antibody against human tetranectin (amino acids 17-181 of human tetranectin monomer; antibodyshop, grusbakken, denmark). to determine in vitro cell proliferation, cells were labeled with brdu (roche, basel, switzerland). results. we describe the localization of tn-positive cells in the human pancreas and their growth in vitro. interestingly, individual positive cells are present within the exocrine acini. we were able to isolate human and murine islets and cultivate these tetranectin-positive cells under adherent and nonadherent conditions as shown by immunohistochemistry and confocal immunofluorescence. the possibility to culture these cells is a first step towards their better characterisation. conclusions. together with its above mentioned ability to bind plasminogen-like hepatocyte growth factor and tissuetype activator, tn may thereby play an important role in the survival of islets after islet transplantation. as we could show, tn positive cells can be isolated and maintained in culture after human islet isolation, thereby providing the possibility to further clarify their role and function in vivo as well as in the course of islet transplantation. p21 fty720 interferes with effector functions and downregulates protein expression of s1p1 and s1p4 in human dendritic cells tures with fty720/fty720-p-treated dc illustrated a cytokine production profile with a lower ifn-? (22% vs. 25% relative reduction) and a higher il-4 (64% vs. 92% relative increase), indicating a shift from th1 toward th2 differentiation as previously evinced for s1p. dc yields, phenotypic differentiation into idc and mdc (besides a minor reduction in cd18 surface expression), as well as the investigated mechanisms of idc antigen uptake (bacterial phagocytosis, mannose receptor-mediated, and fluid-phase endocytosis), were not affected at therapeutically relevant concentrations. conclusions. we conclude that treatment of human dc with fty720 and fty720-p interferes with dc effector functions that are essential for dc to serve their pivotal duty as professional antigen-presenting cells and that dc can therefore be added to the potential list of target cells of fty720. impairment of dc migration and th1-priming capacity due to downmodulation of dc-expressed s1p 1 and s1p 4 might represent a new aspect in the overall mechanism of action and hence contribute, in part, to fty720-mediated immunosuppression. the ibal-fresenius: bioartificial liver innsbruck (ibal) utilizing fresenius standalone, rotating bioreactor m. wurm, v. lubei, p. hengster in order to establish a suitable environment for cultivation of hepatocytes serving as bioartificial liver (bal), we were testing and further developing fresenius standalone, rotating bioreactor. to create optimal conditions for cultivation of hepatocytes, a special environment of nearly gravity-free, low shear force and high mass transfer is needed. furthermore, basic parameters like stability of heating, gas exchange, and sufficiency of nutrition have to be evaluated allowing utilization of various breeding chambers. in summary, we have established a standalone bioreactor capable of quick mass transfer between small and big chambers and external media supply, in nearly gravity-free environment minimizing shear force thus allowing for cultivation of various cell types. background. laparoscopic donor nephrectomy is a less invasive alternative to open nephrectomy for living kidney donation. this study presents the results of the first 58 laparoscopic donor nephrectomies in our center. methods. from june 2000 to may 2005, 58 patients underwent laparoscopic donor nephrectomy for living-related renal transplantation. patient demographic, intraoperative, and postoperative parameters and complications, as well as renal allograft outcome, were evaluated. results. 58 patients (40 female and 18 male) donated their left kidney. the mean donor age was 45 years (24-69 years). the mean surgical time was 233 ± 54 minutes. mean warm ischemia time was 179 ± 63 seconds. patients could be discharged from hospital after a mean time of 5.8 ± 1.5 days. in four patients (6.9%) conversion to open surgery had to be performed. reasons for conversions were lack of operative progression in two cases, in one case venous bleeding, and in one case lesion of the renal artery. there were no reoperations required in the donors. in the recipients, 3 (5.2%) delayed graft functions and 1 (1.7%) primary nonfunction were observed. mean serum creatinine level in the recipients was 1.3 mg/dl 3 months after transplantation. conclusions. laparoscopic live donor nephrectomy is safe for the donor and the transplant kidney. we believe that offering this technique for living renal donation can safely and effectively increase the pool of donor organs available to patients with end-stage renal disease. background. liver transplantation is the treatment of choice for end-stage acute and chronic liver failure. some liver diseases are associated with diseases of the intestinal tract such as primary sclerosing cholangitis and inflammatory bowel disease. moreover, post transplant immunosuppressive agents might cause colonic diseases and there is an abundance of opportunistic pathogens that can manifest in the large intestine. aim. we retrospectively analyzed the incidence and spectrum of colonic disorders in a cohort of 402 liver recipients and determined the impact of these complications on survival. methods. a total of 467 consecutive lts in 402 individuals were performed between 1998 and 2001 at the mayo clinic, jacksonville, florida. standard immunosuppression consisted of tacrolimus, mycophenolic acid, and rapidly tapered steroids. results. there were 29 patients transplanted for psc and 19 were also diagnosed with inflammatory bowel disease (ulcerative colitis, n = 16; crohn's disease, n = 3), with eight having undergone colonic resection prior to lt. in four patients, colitis persisted post transplant. six patients had a history of colonic resection for malignant (n = 3) or infectious diseases. five patients had pre-transplant endoscopic polypectomy. combined colon resection and transplantation were done in 2 patients; one with peritonitis and multiple colonic perforations during retransplantation and the other for ischemic colitis leading to fulminant liver failure. in another case a preexisting transverse colostomy had to be reinforced. there were 32 cases of clostridium difficile-associated enterocolitis. nine patients developed cmv gastrointestinal complications with three cases of colitis, one leading to perforation, intra-abdominal sepsis and death. two patients developed sigmoid diverticulitis and one appendicitis. colonic polyps were endoscopically removed in seven patients and three patients were diagnosed with colorectal cancer (one cecal, two rectal cancers), which all were surgically treated. chronic unexplained diarrhea was observed in fifteen patients, which led to withdrawal of mycophenolic acid. one patient developed a hemorrhage of the terminal ileum/cecal region in the course of intra-abdominal sepsis and was treated by endovascular embolization of the ileocolic artery. four patients had ongoing ulcerative colitis. one herpetic rectal ulcer and two perianal hsv-associated lesions were diagnosed. two patients developed hemorrhoids requiring surgical interventions, and two patients had perianal fistulas. conclusions. the frequency of colonic disorders in our series was higher than expected, with infections accounting for majority of cases. the high incidence of clostridial colitis warrants improvement in screening and preventive measurements. screening for polyps pre-transplant and annually post-transplant might be recommended. background. bartonella has been identified as causative agent of cat scratch disease but is also inflicted in other diseases in the immunocompromised host. case reports. we describe two cases of bartonella henselae-associated diseases in liver transplant recipients who both had contact with cats. the first recipient developed localized skin manifestation of bacillary angiomatosis in association with granulomatous hepatitis. he tested positive for igg antibodies against bartonella henselae. the second patient developed axillary lymphadenopathy, with biopsy showing necrotizing granulomatous inflammation and pcr studies were positive for bartonella henselae dna. her serology for bartonellosis showed a fourfold rise in antibody titers during her hospitalization. both patients responded to treatment with azithromycin in combination with doxycyclin. these were the only cases within a series of 467 liver transplants in 402 patients performed during a four-year period. conclusions. although bartonellosis is a rare infection in lt recipients, one should consider this disease in patients presenting with fever, cns symptoms, skin lesions, lymphadenopathy or hepatitis in particular if contact with cats is reported. background. new immunosuppressive protocols and advanced surgical technique resulted in a major improvement in the outcome of pancreatic transplantation. by reducing the incidence of immunological complications using intensified immunosuppressive protocols, the incidence of some infections, in particular complicated fungal infections, might increase. methods. 217 enteric drained whole-pancreas transplants (ptx) performed at the innsbruck university hospital between march 1997 and october 2004 were retrospectively analysed. prophylactic immunosuppression consisted of atg induction, tacrolimus, mmf, and steroids for the majority of cases. perioperative antimicrobial prophylaxis consisted of amoxicillin/clavulanic (30 ptx), pipercillin/tazobactam (157 ptx), and others (30 ptx). 168 patients additionally received fluconazole. results. actuarial patient, pancreas and kidney graft survival at one year were 96.4%, 88.5% and 94.8%, rejection rate was 30%. within this series, a total of 13 patients developed invasive fungal infections. of those, four had aspergillosis, one zygomycosis, and the remaining ten were caused by yeast. two patients had aspergillosis and later pulmonary infection with candida albicans and candida glabrata. the zygomycosis was the only fungal infection that was diagnosed post mortem and this patient had received pretreatment with caspofungin for non-albicans candida wound infection. one patient died due to aspergillosis following his second pancreas retransplant. three cases of aspergillosis were successfully treated using liposomal amphotericinb in one and a combination of caspofungin and voriconazol in two cases. this combination was also used in a patient who developed intra-abdominal infection with candida krusei. the remaining infections were due to candida albicans including six cases of intra-abdominal infection, one urinary tract infection, and one mucocutaneous candidiasis. type ii diabetics were found at increased risk for fungal infection. conclusions. fungal infections represent frequent and life-threatening complications after ptx. they are amongst the most common causes of graft loss and death. non-albicans candida strains are increasingly isolated and the incidence of filamentous fungal infections has increased during the study period parallel to a decreasing rejection rate. c57bl/6 (h-2 b ) mice received a total-body irradiation (tbi, d-1) of 3 gy and costimulation blockade consisting of anti-cd154 mab (1 mg, d0) vß11 + cd4-cells, p < 0.05). donor skin acceptance fty720 is the first agent in a new class of immunomodulators termed sphingosine-1-phosphate (s1p) human dc, which are known to express mrna for s1p 1 , s1p 2 , s1p 3 and s1p 4 , have not been described so far. methods. to elucidate for the first time the influence of s1p receptor agonists on human monocyte-derived dc (mo-dc), we used therapeutically relevant concentrations (2-200 ng/ml) of fty720 and its phosphorylated metabolite fty720-p and investigated their effects on dc surface marker expression (lineage markers, costimulatory and adhesion molecules, chemokine receptors), protein levels of s1p receptors and dc effector functions: antigen uptake ccl19/elc; or fty720-p-treated mdc (53% vs. 49%; untreated dc, 72%) 24-25 datenkonvertierung und umbruch: manz crossmedia druckerei ferdinand berger & söhne gesellschaft m. b. h., 3580 horn, österreich. -verlagsort: wien. -herstellungsort: horn. printed in austria p. b. b. / erscheinungsort: wien / verlagspostamt 1201 wien -fachkurzinformation rapamune 1mg bzw. 2mg überzogene tablette; rapamune 1 mg/ml bzw. 1mg/ 1ml bzw. 2 mg/2ml lösung zum einnehmen wirkstoff: sirolimus zusammensetzung: 1 tablette enthält 1 mg bzw. 2 mg sirolimus. 1 ml lösung enthält 1 mg sirolimus. 1beutel zu 1 ml bzw. 2 ml enthält: 1 mg bzw. 2 mg sirolimus. sonstige bestandteile: tablettenkern: laktose-monohydrat, macrogol, magnesiumstearat, talkum, tablettenüberzug: macrogol engmaschige überwachung der sirolimus-talspiegel im vollblut bei: leichter bis mittelgradiger leberfunktionsstörung; gleichzeitiger verabreichung starker cyp3a4-induktoren oder -inhibitoren sowie nach deren absetzen; bei absetzen oder deutlicher dosisreduktion von ciclosporin. begrenzte exposition gegenüber sonnen-und uv-strahlung bei patienten mit einem erhöhten risiko für hautkrebs. antimikrobielle prophylaxe gegen pneumocystis carinii pneumonie während der ersten 12 monate nach der transplantation sowie eine zytomegalievirus (cmv)-prophylaxe über 3 monate nach der transplantation ( insbesondere für patienten mit einem erhöhten risiko für eine cmv-erkrankung ) empfohlen. in kombination mit einem hmg-coa-reduktaseinhibitor oder fibrat überwachung auf entwicklung einer rhabdomyolyse und anderen nebenwirkungen dieser präparate. bei kombinierter gabe mit ciclosporin nierenfunktion überwachen, ggf. bei erhöhten serumkreatininspiegeln eine angemessene dosisanpassung erwägen. vorsicht bei gleichzeitiger anwendung von anderen substanzen, die bekanntermaßen eine schädigende wirkung auf die nierenfunktion haben erhöhte laktat-dehydrogenase (ldh), arthralgie, akne, infektion des harntraktes gelegentlich: pankreatitis, lymphom/lymphoproliferative erkrankung nach transplantation, panzytopenie. die immunsuppression erhöht die anfälligkeit, lymphome oder andere bösartige neubildungen, vor allem der haut, zu entwickeln fachkurzinformation zu inserat von umschlagseite 2 p08 liver transplantation for patients with hepatitis b-related liver disease: a single-center experience l. hinterhuber, i. w. graziadei background. liver transplantation (lt) is the only effective therapy for end-stage liver disease due to hepatitis b (hbv). before introduction of passive immunoprophylaxis with hepatitis b immunoglobulin (hbig) and new antiviral nucleoside analogues, hepatitis b recurrence was seen in the majority of the patients resulting in an inferior graft survival. in this study we analyzed the different clinical courses of hbv recurrence, the impact of hbv recurrence on patient survival, and potentially contributing factors for long-term outcome of hbv patients after lt.methods. between 1983 and 2003, 57 out of 692 patients (50 m, 7 f; mean age, 51 years) were transplanted secondary hbv cirrhosis at our center. the mean follow-up was 44 months (range, 1-246 months). immunosuppression (is) consisted of cya/fk 506, prednisolone and/or azathioprine/mmf. fourteen patients received no hbig (prior to 1993), 14 patients received hbig alone, and 29 patients hbig in combination with lamivudine (lam).results. the actuarial overall 1-, 5-, 10-year survival rates were 83%, 69%, and 65%, comparable to those of other indications. patients with combined prophylaxis showed the best survival rates (88%, 77%, 77%), compared to patients treated with hbig (82%, 51%, 51%) and patients without treatment (76%, 70%, 63%). five patients required reltx, one patient two reltx. in total, 17 patients (33.5%) developed recurrent hbv infection after lt: 50% (6 of 12) in the non-hbig group, 42% (6 of 14) in the hbig mono, and 18% (5 of 28) in the combined prophylaxis group. four of the five patients in the hbig/lam group were hbv dna positive prior to lt, two presented with lam mutants. hbv recurrence, however, did not negatively impact patient outcome. all patients with recurrent disease were treated with antivirals (famcyclovir, lam, adefovir). forty-seven percent of patients responded to the treatment and remained hbv dna negative. only one patient was retransplantated due to hbv recurrence. no possible risk factors for overall survival were found to be significant.conclusions. our study showed that patients with combined hbig/lam prophylaxis had excellent long-term survival. recurrent hbv in the allograft could be effectively treated in the majority of patients and did not influence longterm survival. background. liver failure is associated with reduced synthesis of clotting factors, consumptive coagulopathy, and platelet dysfunction. the aim of the study was to evaluate the effects of liver support using the molecular adsorbent recirculating system (mars) on the coagulation system in patients at high risk of bleeding.methods. 61 mars treatments in 33 patients with acute liver failure (n = 15), acute on chronic liver failure (n = 8), sepsis (n = 5), liver graft dysfunction (n = 3), and cholestasis (n = 2) have been studied. standard coagulation tests, standard thromboelastography (teg), heparinase-modified and abciximab-fab-modified teg were performed immediately before and 30 minutes after commencement of mars and after the end of mars treatment. to all patients, prostaglandin i2 was administered extracorporeally. 17 patients additionally received unfractioned heparin.results. three moderate bleeding complications in three patients, requiring 3-4 units of packed red blood cells, were observed. all were sufficiently managed without interrupting mars treatment. although there was a significant decrease in platelet counts (median, 9 g/l; range, -40 to 145 g/l) and fibrinogen concentration (median, 15 mg/dl; range, -119 to 185 mg/dl) with a consecutive increase in thrombin time, the platelet function, as assessed by abciximab-fab-modified teg, remained stable. mars did not enhance fibrinolysis.conclusions. mars treatment appears to be well tolerated in patients with marked coagulopathy due to liver failure. although mars leads to a further decrease in platelet count and fibrinogen concentration, platelet function, measured as contribution of the platelets to the clot firmness in teg, remains stable. according to teg-based results, mars does not enhance fibrinolysis. methodik. ein 69 jahre alter patient war vor 13 jahren aufgrund einer post-hepatitischen zirrhose (phcc) lebertransplantiert worden. nun war es im verlauf der letzten zeit zu einer deutlichen gewichtszunahme gekommen, sodass der patikey: cord-005478-5iu38pr6 authors: nan title: the 45th annual meeting of the european society for blood and marrow transplantation: physicians – oral session date: 2019-07-03 journal: bone marrow transplant doi: 10.1038/s41409-019-0562-9 sha: doc_id: 5478 cord_uid: 5iu38pr6 nan methods: study aim was to evaluate the schedule of ist given in combination with pt-cy as gvhd-prophylaxis post-haplo for acute leukemia (al) and reported to the alwp/ebmt registry. patients were divided into 3 groups: received cyclosporine a-mycofenolate-mofetil(csa-+mmf) initiated at day+1 (group-1, n=124) or csa +mmf both started at day+5 (group-2, n=170) and tacrolimus + mmf from day+5 (group-3, n=215). transplants were performed from 2006-2017 and median follow up is 21 months (range 11-36). pt-cy was given on day+3 and day+5 in group-1 and on day+3 and day+4 in group-2 and 3. results: acute myeloid leukemia (aml) was the most common indication for haplo (76%) and approximately 45% of patients were transplanted in cr1. there were some differences among groups: patients in group-1 were younger (median age 46 years, p< 0.02) were transplanted in more recent year (2015, p< 0.001), received more frequently a regimen based on tbf (thiotepa, fludarabine and busulfan) (83%, p< 0.001) and bone marrow (bm) as source of stem cells (77%, p< 0.001), with no atg (100%, p< 0.001). probability of os at 2 years was 59%, 48% and 44%, for the 3 groups, respectively, p=0.15. probability of lfs and grfs at 2 years were 52% and 46%, 43% and 36%, 39% and 33%, for the 3 groups, respectively, (lfs p=0.05, grfs p=0.01. overall the cumulative incidence (ci) of grade ii-iv acute gvhd was 18%, 39% and 25%, for the 3 groups, respectively, p< 0.001, and the ci of chronic gvhd was 23%, 21% and 25%; p=0.28. the ci of relapse at 2 years was 26%, 37% and 35% (p=0.01) and background: patients with acute myeloid leukaemia (aml) often achieve remission but subsequently die of relapse driven by chemotherapy resistant leukemic stem cells (lscs). here we hypothesized that lscs must also escape immunosurveillance to initiate and maintain cancer and investigate the interplay with nkg2d, a danger detector expressed by cytotoxic lymphocytes such as natural killer (nk) cells that recognizes stress-induced ligands (nkg2dl) of the mic and ulbp protein families on aml cells. methods: 175 de novo aml were stained with antibodies against mica, micb and ulb2/5/6 or an nkg2d-fc chimeric protein recognizing pan-nkg2dl. nkg2dl pos and nkg2dl neg aml cells sorted from the same patient were analysed in colony forming assays, leukemogenesis assays in nsg mice, by rnaseq, gene expression arrays, qrt-pcr and targeted next generation sequencing. aml cells co-cultured or not with nk cells (control or anti-nkg2d pre-treated) were co-stained for additional stem/immunological markers. parp1 expression was analysed by qrt-pcr and immunoblot, and binding to nkg2dl promoters by chromatin immunoprecipitation. parp1 inhibition (parpi) in aml cells was performed in vitro or in vivo using sirnas or inhibitors (ag-14361, veliparib) . results: heterogeneous nkg2dl expression was detected among leukemic cells of the same patient (fig. 1a) . interestingly, when compared to nkg2dl pos subpopulations, nkg2dl neg aml cells isolated from the same patient showed immature morphology, enhanced in vitro clonogenicity (39±47 colonies vs. 1±4, p< 0.001, n=32 aml patients) and selective abilities to initiate leukemia (nkg2dl neg , 64/70, 91%; nkg2dl pos , 0/78, 0%; p< 0.00001, fig. 1b , n=19 aml patients) and survive chemotherapy in nsg mice devoid of functional nk cells. in mice, nkg2dl neg aml cells generated both nkg2dl pos and nkg2dl neg progeny of which again only latter was able to induce leukemia in re-transplant assays. similar leukemia-specific mutations were detected in nkg2dl neg compared to nkg2dl pos aml cells from the same aml but published lsc (fig. 1c ), hsc and 17genes stemness score signatures were specifically enriched in nkg2dl neg subfractions. nkg2dl neg cells enriched for lscs defined by alternative markers (cd34 + , cd38 -, gpr56 + ) but could identify cells with functional and molecular lsc activity also in cd34 non-expressing aml (n=11 analyzed patients). nkg2dl expression was repressed by parp1 recruitment at nkg2dl promoters. parp1 inhibition (parpi) induced nkg2dl surface expression in lscs and co-treatment with parpi and nk cells (but not with either alone) suppressed leukemogenesis in patient derived xenograft (pdx) models (fig. 2c ) cotransplanted with nk cells. low nkg2dl surface or high parp1 mrna expression associated with poor outcome in aml patients. furthermore, nkg2dl neg and cd34 + lscs showed reduced expression of other immune stimulatory molecules (e.g. cd112, cd155, cd80, cd86) and different expression of immune or inflammatory response gene signatures (gsea). conclusions: these data indicate that lscs escape nk cell recognition by selectively suppressing the surface expression of nkg2dl and other immunostimulatory molecules. absence of nkg2dl can identify lscs across genetic aml subtypes (including cd34 negative amls). this lsc specific mechanism of immune evasion could be overcome by treatment with parp1 inhibitors, which in conjunction with functional nk cells holds promise to eradicate lscs and promote immune-mediated cure of aml. disclosure: c.l.: sanofi, novartis, otsuka (consultancy); roche (research funding) background: in contrast to imatinib, data on the use of 2 nd and 3 rd generation tyrosin kinase inhibitors (tki) in the treatment of minimal residual disease (mrd), molecular and hematological relapse (mr/hr) after allogeneic stem cell transplantation (sct) in philadelphia chromosome positive (ph+) acute lymphoblastic leukemia (all) are scarce. methods: we performed a retrospective, ebmt registry based analysis, including patients with documented use of 2 nd or 3 rd generation tki given for persisting mrd, mr or hr after allosct in 2006 -2016 choice of tki, efficacy, and toxicity of tki and patient outcome were analysed. results: 140 patients (female 58, male 82) were identified, out of which 136 had also received a tki (78% imatinib) before allosct. median age at transplant was 48 years (18-66), 81% were transplanted in first complete remission (cr1), 51% of the patients were in molecular cr. conditioning was myeloablative in 71% and reduced in 29%, donors were matched siblings (48%), unrelated (41%), haploidentical (6%) and cord blood (5%). after allosct, 111 patients developed hr, 23 mr and 6 had persisting mrd. for treatment, patients received dasatinib (n=104), nilotinib (n=18) and ponatinib (n=18). median interval between diagnosis of persisting mrd or mr/hr and first application of a tki was 10 days, median duration of tki treatment was 154 days (range 4 -2193). fifty-eight patients were treated with tki only (dasatinib, n=50, nilotinib, n=3, ponatinib, n=5) , while 82 received additional treatment such as dli, chemotherapy, or second allosct. main toxicities of dasatinib were effusion, edema, or other pulmonary complaints (10 -15% of patients) and infections (13%). no particular side effects were reported for nilotinib and ponatinib (no vascular events). dose reduction of tki was required in 32%. response rates were 71% (entire cohort) and 72% (patients receiving tki only). for the entire cohort, 2-and 5-year overall survival (os) from first application of tki was 49% and 33%. two-year os was comparable in patients treated for persisting mrd/mr and for hr (48% and 50%). among patients treated with tki only, 2/5-year os was 38%/33%. rate of cgvhd was 11% for the whole population and 14% for the tki alone cohort. conclusions: the use of 2 nd and 3 rd generation tki, given alone or in combination with other therapies for treating persisting mrd, mr or hr after allosct in ph+ all was not associated with increased toxicities. dasatinib was the most frequently used drug. outcome compared favorably with published results on relapse after allosct in ph negative all, suggesting that treatment with tki could improve survival after post-transplant relapse, even when given as single therapy. type of relapse did not influence response rates and outcome. disclosure: nothing to declare multi-state modelling of the interplay between remission-induction chemotherapy and consolidation with allosct in newly diagnosed aml patients reduced rate of relapse in the clofarabine arm, discovering a significant difference between the treatment arms in the hazard of relapse only after allosct (hr 0.65, 95% ci 0.46-0.94, p-value = 0.02), and not before allosct (hr 0.81, . in addition, we found increased nrm in the clofarabine arm before allosct (hr 1.95, 95% ci 1.15-3.31, p-value = 0.01), and not after allosct (hr 0.80, 95% ci 0.50-1.28, p-value = 0.34). these effects are statistically significantly different (interaction test hr 0.41, 95% ci 0.20-0.83, p-value = 0.01). at two years after registration, 20.4% (95% ci 16.6-24.1) of the patients in the control arm, and 19.9% (16. 1-23.6 ) in the clofarabine arm were alive relapse-free in cr without allosct, while 20.9% (16.7-25.1) and 26. 8% (22.3-31.4 ) were alive relapse-free after allosct. conclusions: presented results suggest that the rate of relapse after allosct is lower among patients whose induction therapy includes clofarabine. these results could possibly be explained by higher rate of mrd negativity achieved in the clofarabine arm before proceeding to allosct. we also observed a higher nrm rate in cr before allosct in the clofarabine arm, indicating that the favorable effect of clofarabine on relapse may be compromised by toxicity. clinical trial registry: hovon 102 study is registered at netherlands trial registry #ntr2187 disclosure: nothing to declare haploidentical transplant with post-transplant cyclophosphamide for t-cell acute lymphoblastic leukemia: outcome strongly correlates with disease status; a report from the ebmt acute leukemia working party background: partial tandem duplication of mll (mll-ptd) is an infrequent mutation in aml which produces a number gain of 5' acetyltransferase domains of kmt2a protein as a result of a repeated exon 3-9/10 gene sequence. mll-ptd leads to a disturbed histone acetylation and upregulation of determined hox genes. mll-ptd aml defines a specific aml entity, distinguishable from cases of aml with mll rearrangement, with a characteristic pattern of co-mutations, including a high association with flt3-itd (q-y sun et al., leukemia 2017) . prognosis of mll-ptd-aml is remarkably poor, with initial chemoresistance and high relapse rate; as a consequence, allogeneic hematopoietic cell transplantation (allohct) in early phase is recommended to overcome its high-risk prognostic impact (jp patel et al., nejm 2012; v grossmann et al, blood 2012) . nonetheless, studies focusing on transplant outcome have not been previously addressed. methods: for this purpose, we analysed the outcome of mll-ptd aml adult patients reported to ebmt who had received an allohct from matched related or unrelated donors in cr1 during the period 2000-2016. molecular screening of mll-ptd was performed locally, but the presence of this mutation was verified specifically by a focused questionnaire among participating centres. results: overall, we identified 58 patients fulfilling inclusion criteria (median age, 52.8 years; 29/58 female patients). most patients harboured an intermediate risk cytogenetics (50/56; 89% of available) and 14 (14/54, 26% of available) patients presented with concomitant flt3-itd. donor was a matched sibling in 27 transplants (47%) and an unrelated donor in the remaining cases. conditioning was myeloablative (mac) in 35 procedures (61%) and reduced intensity in 22 (40%). in vivo t-cell depletion was used in 36 (64%) of transplants. at two years, cumulative incidence of relapse (cir) was 33% (95% ci: 21-45), and non-relapse mortality (nrm) 16% (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) ; 100-day acute gvhd of grade iii-iv was 7% (2-16) and 2-yr chronic gvhd was 38% (extensive, 13%, 6-23). two-year overall survival (os) was 56% (45-71), with leukemia-free survival (lfs) and graft-and relapse-free survival (grfs) of 51% (40-65) and 39% (27-58) , respectively (figure) . multivariate analysis did not identify any prognostic factor for lfs; notably, presence of flt3-itd, conditioning regimen intensity or donor type did not influence outcome. conclusions: these results support the potential clinical benefit of allohct performed in cr1 in patients with mll-ptd aml, with a decreased relapse risk compared to previously reported series, suggesting the existence of a tangible graft-versus-leukemia effect (gvl) in this setting. disclosure: nothing to declare o017 safety and efficacy of reduced intensity conditioning regimen combined with anti-thymocyte globulin and post-transplantation cyclophosphamide as graft versus host disease prophylaxis for acute myeloid leukemia background: we aimed to evaluate the safety and efficacy of the use of reduced intensity conditioning regimen (ric) combined with anti-thymocyte globulin (atg) and posttransplant cyclophosphamide (ptcy) for graft versus host disease (gvhd) prophylaxis in patients diagnosed with acute myeloid leukemia (aml). methods: one hundred four adult patients were included. all patients received the same ric regimen including fludarabine (30mg/m2/day day -5 to -2), busulfan (3.2kg/ m2/day day -3 and -2), and total body irradiation (200 cgy) (day -1) combined with rabbit-atg (4.5 mg/kg: day -3 to -1), ptcy (50mg/kg/day: day +3,+4), and cyclosporine. unmanipulated peripheral blood stem cells were infused. last follow-up was november 2018. median follow-up was 19 months (range 5-35). results: findings are summarized in figure 1 . one year overall survival (os) progression-free survival (pfs) and non-relapse mortality (nrm) was 67.7% (95% ci 58.6-76.8), 60.8% (95% ci 51. 1-70.5 ) and 16.4% (95% ci 6. 9-25.8) respectively. main causes of death were relapse (18%) and infection (13%). three patients had residual disease prior-transplant and they had a significant worst os (p=0.000) and rfs (p=0.002). patients who had karnofsky performance status (kps) ≤80% had a significantly worse os (p=0.004) and pfs (p=0.014). the achievement of ≥95% chimerism of background: indications for hematopoietic stem cell transplantation (hsct) for adults with all evolve over time and vary among countries. methods: the goal of this study was to assess general trends in the number of various types of hscts performed between years 2001 and 2015 in europe. data reported to the ebmt registry were used for this analysis. in addition, we evaluated hsct rates with respect to the incidence of all in selected european countries. results: altogether, 15346 first allogeneic (n=13460) or autologous (n=1886) hscts were performed in the period 2001 -2015 . comparing years 2013 -2015 and 2001 -2003 , the number of allohscts performed in first cr increased by 136%, most prominently for transplantations from unrelated (272%) and mismatched related donors (339%). the number of hscts from matched sibling donors increased by 42%, while the number of autohscts decreased by 70%. the increase of the use of allohsct, irrespective of the disease stage, was stronger for ph-pos (166%) than ph-neg all (38%). among patients aged >55 years, the number of allohsct increased by 559% while among younger adults (18-55 years), by 59%. between 2001 and 2003 , peripheral blood was used as source of stem cells in 61% cases of allohsct, compared to 84% between 2013-2015. the use of bone marrow decreased from 38% to 16%, respectively. the proportion of allohsct with reduced-intensity conditioning (ric) increased from 6% to 27%. among myeloablative transplantations, regimens based on total body irradiation were the preferable option (app. 80% over the whole study period). in contrast, among ric regimens, the use of chemotherapy predominated (84% between 2013-2015) . in most of analyzed individual countries, the estimated rates of allohsct (no. hsct per 100 newly diagnosed all) for patients in cr1 increased over time. however, the values for a period 2013-2015 varied strongly, being highest in finland (57.9), followed by the netherlands (35.4) and sweden (35.4) while lowest in russia (2.6) . conclusions: results of our analysis indicate continued trend to increased use of allohsct for adults with all, which may be attributed to increasing availability of unrelated donors. however, it may also be speculated that the introduction of tyrosine kinase inhibitors allowed higher proportion of patients with ph-pos all proceeding to transplantation. finally, the implementation of ric regimens contributed to wider use of allohsct among older adults. limitations of the analysis include any assumptions made regarding all incidence for the specified time period and possible variation in reporting to the ebmt registry from different countries over time. year type of hsct 2001 hsct -2003 hsct 2004 hsct -2006 hsct 2007 hsct -2009 hsct 2010 hsct -2012 hsct 2014 hsct -2015 disclosure: sg has received honoraria for amgen. as and sw are employees of amgen and own shares in amgen. mm has received honoraria for speaking for amgen and pfizer. outcome of allogeneic-hsct in adult patients with phpositive-all in the era of tki: a retrospective analysis of università degli studi e ospedale maggiore policlinico di milano, milano, italy, 33 genova, italy, 34 azienda socio sanitaria territoriale papa giovanni xxiii, bergamo, italy background: we conducted a retrospective, nationwide analysis to describe the clinical outcome of adult patients with philadelphia chromosome-positive acute lymphoblastic leukemia (ph+all) undergoing allogeneic hematopoietic stem cell transplantation (hsct) after being treated with a tki based therapy. methods: a total of 441 patients were included in the study. the median age at hsct was 44 (range: 18-70). all 441 patients (100%) received tki before hsct (performed between 2005-2016) . of these patients, 404 (92%) were in cytologic complete remission (cr) while 37 (8%) had an active disease at the time of hsct. molecularly measurable residual disease (mrd) was negative in 147 patients (36%) at the time of hsct. the donor was unrelated in 46% of cases. the prevalent source of stem cells was peripheral blood (70%). the conditioning regimen was myeloablative in 82% of cases (tbi-based in 50%) and included atg in 51% of cases. results: with a median follow-up after hsct of 39.4 months (range: , the probability of overall survival (os) at 1, 2 and 5 years was 69.6%, 61.1%, and 50.3%, respectively, with a median os of 62 months. progression free survival (pfs) at 1, 2 and 5 years was 60.2%, 52.1% and 43.7%, respectively. os and pfs were significantly better in patients with cr and mrd-negativity at the time of transplant compared to patients with cr but mrd-positive (50% os not reached vs. 36 months, p=0,015; 50% pfs not reached vs 26 months, p=0.003). the cumulative incidence of relapse (cir) at 5 years was significantly lower in patients with cr and .4%, p=0.001). the non relapse mortality (nrm) after 1, 2, and 5 years was 19.1% (95%ci: 15.5-22.9), 20.7% (95%ci: 17-24.7), and 24.1% (95%ci: 20-28.5), respectively. the subgroup of patients with mrdnegative both at hsct and at 3rd month after hsct had a better outcome (5 year os 70%). conversely, the 37 patients who underwent hsct with active ph+all had a median os and pfs of 7 and 5 months, respectively. background: haploidentical (haplo) donors have expanded patient transplant access. however, outcome of patients with acute lymphoblastic leukemia (all) undergoing allogeneic stem cell transplant (asct) with haplo donors in argentina has not been reported. we aimed to analyze the outcome of asct in patients with all, particularly results with haplo donors. methods: we collected data from patients with an all diagnosis who underwent asct in first complete remission (cr1) and subsequent remissions (cr2+) in 15 centers in argentina, affiliated to gatmo, between 2008 and patients that underwent asct with matched donors (sibling and unrelated) and haplo donors (with post-transplant cyclophosphamide) were included. both donor categories were compared in terms of overall survival (os), nonrelapsed mortality (nrm) and cumulative incidence of relapse (cir). graft versus host disease (gvhd) was also evaluated. multivariate analysis was performed by cox regression for os and fine-gray for ci of competing events. a further propensity score (ps) adjustment was performed by donor group. results: in a 10-year period, 236 patients were included (mean age 31y; range 16-64; male 63.1%); 188 (80%) during last 5 years. all phenotype was b (79%) and t (21%). at diagnosis, 47/236 (20%) had cns involvement and 75/236 (32%) were philadelphia chromosome positive. asct was performed in cr1 (n=126; 53%) and in cr2+ (n=110; 47%) after a median time from all diagnosis to asct of 9 and 26 months, respectively. comorbidity index (hct-ci) was 0-1 in 199/236 (90%). donors were matched (n=175; 74%; 146 related and 29 unrelated) and haplo (n=61; 26%). conditioning regimen was myeloablative in 215/236 (91%; 170 patients with total body irradiation), and this conditioning was more frequent in matched (95%) than haplo (79%) (p=0.001) donors. two-years os was 54 % (95%ci 46-60) for the entire population; 55% (95ci 47-63) for matched donors and 49% (95% ci 34-62) for haplo donors (p=0.350). in the multivariate analysis, pretransplant status (cr1 vs cr2+; hr 2.06, p< 0.001), cns status at diagnosis (yes vs no; hr 1.70; p=0.019) and unrelated donors (yes vs no; hr 1.77; p=0.036) were independently associated with os; donor category had not impact in the os. by adjusting the ps term (roc area 0.787), no difference was found by donor category. twoyears nrm was 24% (95%ci 18-31) for matched and 22% (95%ci 12-33) for haplo (p=0.999) donors; older donors (p=0.049) and unrelated donors (p=0.001) were associated with higher nrm. two-years cir was 25% (95%ci 19-33) for matched and 38% (95%ci 24-51) for haplo (p=0.137) donors; only male donors were associated with higher cir (p=0.031). ci of grade 3-4 acute gvhd was 20% vs 17% (p=0.784) and chronic gvhd was 35% vs 27% (p=0.057) for matched and haplo donors respectively. in both groups, matched and haplo donors, the half of deaths were due to relapse. background: relapse is the most important cause for treatment failure in pediatric b-precursor acute lymphoblastic leukemia (bcp-all) occurring in 10-20% of patients. mechanisms of ineffective graft-versus-leukemia (gvl) effects or t-cell responses against all remain to be investigated. methods: we analyzed parameters of immunosurveillance in bone marrow (bm) samples of 100 pediatric patients to identify potential mechanisms of t-cell suppression. expression of co-stimulatory/ co-inhibitory molecules was analyzed to identify implications for gvl. expression was correlated with clinical outcome (8 years mean followup) . t-cell immunoglobulin and mucin-domain containing-3 (tim-3) overexpression and crispr/cas9-mediated knockout (ko) in primary t cells were performed to analyze its role for anti-leukemic t-cell functionality. to induce an interaction of t cells with leukemic blasts, anti-cd19/-cd3 bispecific t-cell engager (bite) was added and t-cell activation/ proliferation were analyzed. fold change (fc) was created by comparing levels of t-cell activation/ proliferation before vs. after co-culture. transcriptome analysis of primary bm samples identified expression levels of known tim-3 inducers. results: flow cytometric analyses of 100 bcp-all samples showed increased tim-3 expression on cd4 + bm t cells at initial diagnosis in patients with relapse in the course of disease. multivariate analysis confirmed 7-fold increased relapse risk in tim-3 high (n=37) vs. tim-3 low expressing (n=37) patients. pd-1 expression on bm t cells alone had no impact on relapse-free survival (rfs), whereas patients with high percentage of tim-3/pd-1 double positive cd4 + bm t cells showed significantly decreased rfs (87.8% vs 41.7%). co-culture experiments revealed that tim-3 is induced in primary t cells by contact with leukemic cells (mean tim-3 expression 51.1% vs. 29 .7% on t cells with vs. without addition of leukemic cells, n=3). transcriptome analysis was performed to identify expression levels of known tim-3 ligands/ inducers in bm samples with high vs. low tim-3 expression. no significant differences in expression levels of high-mobility-group-protein b1 (hmgb1), carcinoembryonic antigen-related cell adhesion molecule 1 (cea-cam1) or galectin 9 were observed. known tim-3 inducers il-12, il-15, il-27, il-7 or transforming growth factor beta 1 (tgf-β1) were not differentially expressed indicating that another mechanism must be responsible for tim-3 overexpression. tim-3 overexpression and crispr/ cas9-mediated tim-3 ko were performed to analyze functional relevance of tim-3 expression in an in vitro leukemia model. t cells of healthy donors were co-cultured with leukemic cells and anti-cd19/-cd3 bite to induce anti-leukemic t-cell response. tim-3 ko t cells showed significantly increased activation compared to wildtype t cells (fc of cd69 expression 5.0 vs. 3.2, n=3) . in contrast, proliferation of tim-3 overexpressing t cells was significantly impaired (fc 1.6 vs. 2.3, n=3) , whereas tim-3 ko t cells showed higher proliferation levels compared to controls (fc 6.5 vs. 2.4, n=3) . conclusions: tim-3 expression on cd4 + bm t cells is a strong predictor for pediatric bcp-all relapse and is induced by t-cell interaction with leukemic cells. tim-3 expression decreases anti-leukemic t-cell activation and proliferation and thus constitutes a new mechanism of immune escape and potentially insufficient gvl effects in pediatric bcp-all. targeting the tim-3 axis can be of interest to improve future immunotherapy of advanced bcp-all. disclosure: nothing to declare. abstract already published. multiparameter flow cytometric minimal residual disease before myeloablative allogeneic hematopoietic cell transplantation in acute myeloid leukemia influences patients survival in first and second complete remission background: the growing evidence from the literature strongly suggest that multiparameter flow cytometric (mfc) minimal residual disease (mrd) assessment in aml can be used to risk-stratify patients at the time of allogeneic hematopoietic stem cell transplantation (allohct). we sought to determine the significance of mfc-mrd status in patients with aml in first or second complete remission (cr) treated with myeloablative conditioning (mac) allohct at six centers of the polish adult leukemia group (palg). methods: mrd was assessed by 6-color (8-color since 2017) mfc performed on bone marrow aspirates obtained as routine assessment before allohct. all consecutive patients undergoing mac allohct were included in the analysis if they underwent pre-hct mfc-mrd analysis from may 2013 until january 2018. the abnormal population was quantified as a percentage of the total cd45 + white cell events. residual disease at a ≥ 0.1% level was considered mrd-positive (mrd+) . results: we identified 83 adult patients (median age 41 years, range 19-64) with aml undergoing allohct from either hla-identical sibling (n=30) or unrelated donor (n=53), in cr1 (n=72) or cr2 (n=11), who were conditioned with i.v. busulfan given in myeloablative dose (9,6-12,8 mg/kg) in combination with flu (n=46) or cy (n=37). gvhd prophylaxis consisted of calcineurin inhibitor combined with mtx plus atg in allohct from unrelated donors. positive mrd status before allohct was detected in 30/72 (42%) pts in cr1 and 6/11 (55%) pts in cr2. the mrd(-) and mrd(+) groups did not differ in terms of gender, age, eln cytogenetic and molecular genetic risk, first and second cr, conditioning regimen, hsc source, and type of donor. the 3-year overall survival (os) for mrd(-) and mrd(+) patients were 77% and 49% (log-rank p= 0.023). the respective 3-year leukemia-free survival (lfs) were 61% and 37% (log-rank p=0.012). in univariate and multivariate cox proportional hazard model the only significant adverse prognostic factors for lfs were mrd(+) (hr 2.45, p=0 .025) and high eln genetic risk (hr 3.70, p=0.0009) . the same factors significantly influenced os [hr 2.48, p=0.036 and hr 3.47, p=0 .004 for mrd(+) and high eln risk, respectively] . conclusions: our findings confirm that pre-transplant residual disease at a ≥ 0.1% level assessed by mfc is independent risk factor for both lfs and os in patients undergoing allohct. in addition, the results of our study show that mac allohsct outcomes in patients with aml in first and second cr are significantly influenced by both mfc-mrd status and eln cytogenetic and molecular genetic risk. [[o023 image] 1. leukemia-free survival according to pre-transplant minimal residual disease level] disclosure: nothing to declare o024 second allogeneic stem cell transplantation in acute lymphoblastic leukemia patients in second complete remission or relapse: a study on behalf of the alwp / ebmt background: second allogeneic transplantation (hsct2) is a therapeutic option for patients (pts) relapsing (rel) after first hsct (hsct1) however most of the available data is in acute myelogenous leukemia (aml) and there is very limited data on hsct2 in patients (pts) with all. therefore, the alwp of the ebmt performed a large registry analysis to study the outcome of hsct2 in pts with all. methods: we studied 245 pts receiving hsct2 as a salvage treatment between the years (y) 2000-2017 for rel following hsct1 in cr1. median follow-up of surviving pts was 58 months (iqr: 24-98). results: 142 pts (58%) were males and median age at hsct2 was 34.6 years (range: 18-74). median time from hsct1 to hsct2 was 463 (63-5482) days (d) and from rel to hsct2 it was 114 (15-348) d. at the time of hsct2 157(64 %) pts were in cr2 while 88 (36%) had advanced disease.101 (41%) pts received sibling donor (msd) and 144 (59%) unrelated donor (ud) hsct2 (10/10-63; 9/10-10, missing hla-71). in 34% of pts with available data the hsct2 was performed from the same donor. the majority of pts with available mrd data transplanted in cr2 (64/84 pts) were mrd negative pre-hsct2. karnofsky performance status was >90 in 60% of pts. 93% were transplanted with pb graft. 204 (83%) pts received chemotherapy based conditioning (reduced intensity 63%, myeloablative 37%) while it was tbi based in 41(17%) pts. 109 (52 %) pts received in vivo t-cell depleted (tcd) grafts. 94% of the pts engrafted. acute graft-versus-host disease (agvhd) ≥ ii and ≥ iii-iv occurred in 33% and 17% of the pts. incidence of 2y total and extensive chronic gvhd was 38% and 19%, respectively. main causes of death were leukemia recurrence in 54%, gvhd in 18% and infections in 19%. at 2 and 5 y, the cumulative incidence of nrm, ri, lfs, os and grfs were 24% & 26 %: 56% & 62%, 20% & 12 %, 30% & 14 % and 12% & 7%, respectively. in mva no factor predict nrm. in multivariate analysis, ri was independently associated with time from hsct1 to rel, agvhd ≥ii after hsct1 and in vivo tcd and lfs the prognostic factors were time from hsct1 to rel and kps≥90 at hsct2. factors associated with os were age (per 10 years), time from hsct1 to rel, ric at hsct1, kps>90 at hsct2 and ud vs msd. longer time internal from rel to hsct2 and in vivo tcd was associated with inferior cgvhd. lastly for grfs the prognostic factors were time from hsct1 to rel, agvhd ≥ii after hsct1, ric at hsct2 and kps≥90 at hsct2. conclusions: results of hsct2 in all pts with rel or cr2 are poor with only 14% os and 7% grfs at 5 y with very high ri of 62%.the prognostic factors are similar to those previously reported for hsct2 in aml. the future goals are to prevent and treat relapsed all by mrd driven novel monoclonal antibodies and car-t cell therapy. disclosure: nothing to declare. aplastic anaemia outcomes of allogeneic stem cell transplantation (hsct) for older patients (> 50 years) with severe aplastic anaemia using alemtuzumab-based ('fcc') regimen: king's college hospital experience background: treatment of older patients with severe aplastic anaemia (saa) is problematic with poor long-term survival after treatment with antithymocyte globulin (atg) and/or ciclosporin (csa). use of fludarabine, low dose cyclophosphamide (cy) and atg ('fcatg') conditioning suggests better outcomes among older patients transplanted from matched sibling donors compared to high dose cy/ atg conditioning, but gvhd remains a serious concern. we have transplanted saa patients aged > 50 years, predominantly from unrelated donors, using alemtuzumabbased ('fcc') regimen. methods: from our fcc saa database of 65 patients, 27 aged > 50 years were transplanted between 2007-18. median age was 61 years (range 51-71); 12 aged 50-59 and 15 aged ≥ 60 years. donor was matched sibling (msd) in 6 (22%), 10/10 matched unrelated (mud) in 18 (66%), 9/ 10 unrelated (mmud) in 3 (12%). conditioning was fludarabine 30mg/m 2 x 4, cy 300mg/m 2 x 4, alemtuzumab 0.2mg/kg daily from day -7 to -3. post graft immune suppression was csa alone. 2gy tbi was added to fcc for mmud hsct. all patients received peripheral blood (pbsc) as stem cell source. 10/27 (36%) patients were hla alloimmunised. pb telomere length (tl) by multiplex qpcr measured in 17 patients, was < 1 st centile in 3 (17%), < 10 th centile in 2 (11%) and normal in 12 (70%) patients. first line hsct was performed in 2/6 (38%) msd and 3/21 (12%) among unrelated donors. hct comorbidity index (htc-ci) score was 0-1 in 10 (37%); 2 in 7 (25%) and >2 in 10 (37%). results: three patients had invasive fungal infection at time of hct and died day +14 to 21, and one patient died at 11 months from multiorgan failure with recurrent parainfluenza virus and cmv. median cd3 chimerism was 71% (1-100), 77% (23-100) and 60% (22-98) at day +100, iyr and 3yr post hsct. one late graft failure at 6 months was associated with low csa blood levels, and was followed by successful 2 nd transplanted with no gvhd. 5-year os was 86%, compared to 96% among 27 patients aged < 50years (p=0.14). os was 89% and 82% for patients aged 50-59 and ≥ 60 year, respectively, p=0.8. os for msd, mud and 9/10 mmud was 100%, 80% and 100%, respectively. htc-ic score of >2 was associated with worse os of 72% compared to 94% with score < 2, p=0.13. outcomes were comparable irrespective of telomere length (84% vs 80% for normal vs short telomere, p=0.76). cumulative rates of acute and chronic gvhd were 5% and 12%, respectively. all cases of acute gvhd were confined to skin and grade i/ii only, and no cases of severe chronic gvhd. 17 (62%) patients needed dose reduction of csa with addition of mycophenolate due to renal dysfunction. rates of cmv and ebv reactivation were 25% and 27% respectively, with no cmv or ebv disease. conclusions: fcc conditioning regimen enabled high survival and low risk of gvhd among older patients with hct-ci score < 2 and who did not have established invasive fungal disease at time of hsct. clinical trial registry: not applicable disclosure: no conflicts of interest to declare haploidentical transplantation with post-transplant cyclophosphamide (haplo-ptcy) for 71 patients with acquired or inherited bone marrow failure syndromes (bmf): the experience from curitiba, brazil background: availability of unrelated donors as well as time to find a donor and the costs related to graft acquisition are important limitations in countries with ethnical minorities and fewer resources. methods: we describe the experience of 78 transplants in 71 patients(pts) with acquired or inherited bmf submitted to an haplo-ptcy transplantation between 04.2008 and 08.2018. the median age was 9ys, 70% were male and 94% were cmv positive. haplo-ptcy was the 1 st transplant for 62 pts, second or third for 9pts. diagnosis: fanconi anemia (fa,n=48), acquired severe aplastic anemia (asaa,n=10), telomere diseases (n=6); other inherited bmf (n=7). all pts had failed prior therapies and 96% had previous blood transfusions. the majority received a ric regimen with low dose tbi (n=69, 97%). donors were father(n=28), mother(n=31), other relatives(n=12). bone marrow was the stem cell source in 70pts. all pts received gvhd prophylaxis that included ptcy followed by cyclosporine and mycophenolate mofetil. fa pts received a modified preparatory regimen and ptcy at a total dose of 50mg/kg (n=32) or 60mg/kg (n=16) while other bmf received 100mg/kg results: fa pts: 14 pts did not receive atg in the preparatory regimen and all engrafted, despite the presence of donor specific antibodies(dsa) in 2 pts. three pts had aml and 2 are in remission 3 and 6ys after transplant. 7pts died due to gvhd (n=5); toxoplasmosis/cmv pneumonia (n=1) or relapse (n=1). 7/14 pts are alive with a median follow-up(fu) of 6.7ys. in the subgroup of fa pts receiving r-atg(n=34), 3pts presented primary or secondary graft failure(gf), none had dsa and all died despite a 2 nd haplo-ptcy with different donors. 6pts had advanced disease and 4 are in remission at the last fu. 26/34pts are alive at a median of 4 ys after transplant. eight pts died due to gvhd (n=4); rsv pneumonitis (n=1) and gf (n=3). all 10 pts transplanted with asaa are alive and fully engrafted at a median of 5.6ys after transplant and none developed gvhd, nine out of 13pts transplanted for other inherited bmf are alive at a median of 3ys after transplant. gf occurred in 4pts, all received a 2 nd haplo-ptcy from different donors and 2 are alive and engrafted. 4pts died due to gvhd (n=1), gf (n=2) and tma (n=1). altogether cmv reactivation occurred in 51pts (72%), at a median of 31 days (range:15-90) and hemorrhagic cystitis in 31pts (44%) at a median of 44 days (range:8-65). after transplant. conclusions: haplo-ptcy for pts with acquired or inherited bmf should be offered for those who need an immediate transplant but lack a matched donor. 70% of pts are alive at a median fu of 3ys but gvhd is a major complication for pts with inherited bmf, especially fa. new approaches to gvhd prophylaxis and treatment are needed in order to improve quality of survival for these pts. disclosure: nothing to declare relationship between plasma rabbit anti-thymocyte globulin level and response to immunosuppressive therapy in patients with severe aplastic anemia: results of a multicenter, prospective, randomized study background: patients with acquired aplastic anemia (aa) who do not have hla-matched donors receive immunosuppressive therapy (ist) with anti-thymocyte globulin (atg). previous studies have suggested several variables that predict response to ist. however, no studies have investigated the plasma atg level as a variable. in this study, we assessed the relationship between plasma rabbit atg (r-atg) level and response to ist in patients with severe aa. methods: patients with severe aa who required initial ist were enrolled from may 2012 to october 2017. the ist regimen included r-atg (thymoglobulin®, sanofi, cambridge, 2.5 or 3.5 mg/kg/day for 5 days) and cyclosporine a (6 mg/kg/day for minimum 6 months). plasma r-atg level was measured using a rabbit igg elisa kit on days 14 and 28. response rate was defined as complete and partial responses at 6 months. receiver operator characteristic curves were generated to discriminate between response and no response to ist. results: a total of 81 patients (aged 1.7-67.9 years) were randomized; 43 and 38 patients received 2.5 and 3.5 mg/kg of r-atg, respectively. in the 2.5 mg group, the response rate was 63%, which was comparable with that in the 3.5 mg group (58%) (p = 0.820). plasma r-atg level greatly varied in both groups. median r-atg level on days 14 and 28 after ist was 15.2 (0.0-97.7) and 1.8 (0.0-74.9 μg/ml), respectively, which was not significantly different between two dosages of atg groups (day 14, p = 0.498; day 28, p = 0.404). according to the r-atg level, response rates were significantly higher in the group with higher r-atg level than in that with lower atg level (day 14, 88% vs. 52%, respectively; p = 0.006 and day 28, 79% vs. 46%; p = 0.005) (figure) . cut-off levels at days 14 and 28 were 21.6 and 4.8 μg/ml, respectively. the vast majority (90%) of patients with levels higher than cut-off levels on day 14 responded to ist. in multivariate analysis, higher atg levels at day 28 were independent favorable predictors of response to ist at 6 months (or = 0.29; 95% ci: 0.09-0.93; p = 0.037). there were no significant differences in the kinetics of lymphocyte subsets among patients treated with different dosages of atg. however, higher atg level was associated with lower cd4+ t and regulatory t cell numbers for the entire 6-month period. conclusions: the present data indicate interindividual variability in plasma r-atg level. higher atg level resulted in improved response to ist and correlated with prolonged immune reconstitution. individualized dosing of atg via a pharmacokinetic model may improve the response rate to ist and reduce the number of patients who require allogeneic stem cell transplantation following ist. clinical background: radiation and dna alkylating agents used in hematopoietic cell transplantation (hct) can cause organ damage, malignancy and death. these risks are heightened in patients with genetic bone marrow failure (bmf) syndromes driven by defects in cellular proliferation or dna repair, including dyskeratosis congenita (dc), which arises from impaired telomere maintenance. we hypothesized that proliferative defects in hematopoietic cells of patients with bmf and very short telomeres might permit myeloid engraftment following hct without the need for radiation or dna alkylating agents. we conducted a multi-center prospective trial (nct01659606) evaluating engraftment after hct without these agents. methods: we enrolled bmf patients with genetic validation of dc or lymphocyte telomere length < 1 st percentile by flow-fish. we performed hct using bone marrow allografts from related or unrelated donors matched at 7 or 8 of 8 hla alleles after a preparative regimen consisting of only fludarabine and alemtuzumab. graft versus host disease (gvhd) prophylaxis consisted of cyclosporine a and mycophenolate mofetil. the primary endpoint of the trial was donor myeloid engraftment, defined as an absolute neutrophil count ≥500 cells/μl by day +42 and donor myeloid chimerism >50% by day +100. [[o029 image] 1. engraftment and survival after radiation-and alkylator-free hct for bmf with very short telomeres] results: twenty patients (age 1.7-31.5 years old at hct) received treatment between august 2012 -october 2018 at 7 institutions. eighteen of the 20 patients received unrelated donor grafts (15 matched, 3 single-allele mismatched). primary myeloid engraftment was achieved in 19 of 20 patients (95%) at a median 22 days post-hct (range 1-35 days). the single patient with primary graft failure had dc-related liver disease and hypersplenism; in this case, splenectomy at day +47 promptly revealed donor myeloid engraftment. of the other 19 patients, 16 had sustained myeloid engraftment, with a median post-hct follow-up of 21 months (range 1-74 months). three patients had secondary graft failure. two of these had early graft rejection and underwent successful repeat hct using higher intensity regimens. the third patient maintained high donor chimerism after primary engraftment but developed severe neutropenia in the setting of multiple viral reactivations, and died of a fungal infection 90 days post-hct. there was one other death, due to dc-related gastrointestinal complications 19 months post-hct. none of the 16 patients who engrafted durably under the protocol regimen had acute gvhd. four had chronic gvhd (3 limited, 1 extensive), treated successfully with limited courses of topical or oral steroids. conclusions: we conclude that this radiation-and alkylator-free hct conditioning regimen is an effective strategy for bmf in patients with dc or very short lymphocyte telomeres. eliminating dna damaging agents may reduce hct complications including gvhd and enable transplant in patients with high-risk comorbidities. clinical background: the standard treatment of acquired aplastic anemia (aa) is either intensive immunosuppressive therapy (ist) or allogeneic hematopoietic cell transplantation (hct). as supportive measures, red blood cells and platelet transfusions are the mainstay of therapy and patients are often multitransfused, which in turn can lead to anti-human leukocyte (hla) alloimmunization. in acquired aa the rate of hla-alloimmunization has previously shown a higher frequency in patients with aa compared to hematological malignancies. however, these results date back before the general introduction of leukoreduction of blood products and photochemical pathogen reduction of platelet components, and are based on cell-based assays. in recent years, leukoreduction and pathogen reduction of blood products became standard in switzerland and the solid-phase assay (luminex® technology) is now widely available to test for hla-antibodies, allowing a more extensive and detailed characterization of hla-antibodies. with these techniques, less is known on the exact incidence of hla-antibodies and their magnitude, associated cofactors and its impact on treatment outcomes in acquired aa. methods: we retrospectively investigated 54 aa patients treated with ist (n=44) and/or hct (n=25) at the university hospital of basel and the university children's hospital of basel (switzerland) regarding hla antibodies since the introduction of testing with the luminex® at our center in 2008. at least one hla antibody measurement before and/or after therapy was available per patient. all patients received leukoreduced blood products and as of 2011 platelets treated with intercept® (uv+ amotosalen). results: overall, hla-antibodies were detected in 40 (74%) patients with a higher rate of hla alloimmunziation by severity of aa (p< 0.01). the median number of hlaantibodies in each patient before therapy (i.e. ist or hct) was 3 (iqr 0-25). in patients undergoing hct hlaantibodies were more frequent before treatment start as compared to patients with ist treatment (median 13 (iqr 0-45) versus 2.5 (iqr 0-16.5), p< 0.05). differences between treatments remained after adjusting for all covariates (p< 0.01). there was no statistically significant difference regarding the hla antibody mean fluorescence intensity (mfi) between the two treatment forms (overall mean mfi of 1580 +/-2075). the highest mean hlaantibody mfi before therapy was 4747 (+/-7221) with a maximum of 24020. females showed a significantly higher number of hla-antibodies (p< 0.01) and also higher mean mfi (p< 0.05). furthermore, the number of pregnancies was associated with higher numbers of hla-antibodies (p< 0.01), however the number of transfusions did not have significant impact on hla-antibody number and mfi. regarding outcome, there was no significant association between the number of hla-antibodies and engraftment as well as bleeding events. conclusions: hla-alloimmunization is still frequent in patients with acquired aa but today number of pregnancies and gender seem to be more important for development of hla-alloimmunization than number of transfusions. interestingly, patients treated by hct show a higher rate of hla-alloimmunization before treatment start in comparison to ist, thereby emphazing the importance of blood management and donor selection in hct in acquired aa as hla-antibodies can cause platelet refractoriness and can represent donor-specific antibodies in the setting of mismatched hct (e.g. haploidentical). disclosure: nothing to declare. health-related quality of life in systemic sclerosis before and after autologous hematopoietic stem cell transplant -a systematic review background: autologous hematopoietic stem cell transplantation (ahsct) for severe rapidly progressive systemic sclerosis (ssc) allows significant regression in skin and lung fibrosis and improvements in overall and event free survival up to 7 years after transplant. we undertook this study to synthesize the evidence on changes in healthrelated quality of life (hrqol) associated with ahsct for ssc. methods: autologous hematopoietic stem cell transplantation (ahsct) for severe rapidly progressive systemic sclerosis (ssc) allows significant regression in skin and lung fibrosis and improvements in overall and event free survival up to 7 years after transplant. we undertook this study to synthesize the evidence on changes in healthrelated quality of life (hrqol) associated with ahsct for ssc. results: the search returned 656 articles. eight were selected: 3 uncontrolled phase i or ii trials, 2 cohort studies and 3 rct (assist, astis, scot). hrqol data from 289 ssc patients treated with ahsct and 125 with intravenous cyclophosphamide (cyc) as a comparator were extracted. hrqol was assessed using the health assessment questionnaire-disability index (haq-di) (n=275 patients), the short-form health survey (sf-36) (n=249 patients) and the euroqol 5 dimensions (eq-5d) (n=138 patients). hrqol was analyzed as a secondary outcome in all studies. quality of the data was assessed as high. haq-di improved significantly more with ahsct compared to cyc (-0.58 vs -0.19, p=0.02 at 2 years in astis; 53% vs 16% improved at 4.8 years in scot). scores also improved pre-post ahsct in the uncontrolled studies (ranging from -0.6 to -1.7 points at one year (all p< 0.05), and up to -1.5 points at 7.5 years (p< 0.001)). sf-36 physical component summary scores were significantly better in subjects treated with ahsct compared to cyc (between group differences ranging from 26 points at one year in assist and 6.1 points at 2 years in astis (p=0.01); 56% vs 15% improved at 4.8 years in scot (p=0.02)). similar differences pre-post treatment scores were also reported in an uncontrolled study (increase of 20 points, p< 0.0001). in assist, there was a trend for the sf-36 mental component summary score to improve in the ahsct arm (46 vs 58, p=0.07) and worsen in the cyc arm (56 vs 42, p=0.04) at one year. there were no significant differences between the ahsct and cyc arms in astis and scot with 2.0 and 4.8 years of follow-up, respectively. post-treatment scores improved significantly compared to pre-treatment in an uncontrolled study (from 51 to 64 points, p=0.005). astis showed a significant difference in the index-based utility score of the eq-5d (0.29, p< 0.001) and a non-significant difference in the general health visual analogue scale (6.7, p=0.36) at two years, in favour of ahsct compared to cyc. conclusions: although there is heterogeneity in the reported data, ahsct in ssc was consistently associated with marked and sustained improvement in hrqol. this analysis provides additional compelling data for the role of ahsct in ssc when assessing patient's point of view. clinical trial registry: we hypothesised that ahsct induces a rebooting of thymic function, resulting in the re-development of a functional, tolerant immune system. we aimed to examine cellsurface and dna markers of recent thymic emigrants (rte's) longitudinally in a cohort of ms patients post-ahsct in order to identify markers that correlate with a durable treatment response. methods: peripheral blood mononuclear cells (pbmncs) were collected from patients enrolled in the phase ii trial at st vincent's hospital sydney for ahsct in ms (moore et al, jnnp in press). a multicolour flow cytometry panel to optimally identify rte's was performed on 10 patient samples at 0, 6, 12, 24 and 36 month timepoints, allowing us to track changes in thymic output longitudinally following ahsct. dna markers of thymic function -sj:b trec ratio was performed in the same cohort of patients, on the same bio-banked sample to enhance the validity of observed changes. statistical analysis was performed with graphpad prism. results: a sustained, significant increase in rte's and sj:b trec was detected between pre-transplant and 24 month post-transplant specimens (p = 0.024). in patients where similar analysis was able to be performed at 36 months a trend to increased markers of thymic output was observed. a correlation between rte's and sj:b trec was observed (r=0.70, p = 0.003). contrary to other publications in the field, trec as a marker of thymic output did not appear to be lower when patients were analysed by age (< 30 yrs vs. >30 yrs). greater thymic output as determined by sj:b trec was observed at all timepoints in patients who had evidence of sustained disease remission as opposed to patients who experienced disease relapse. conclusions: we have seen evidence that sustained thymic reactivation is a component of immune reconstitution following ahsct for ms. previous studies have only demonstrated thymic activity at 12 months but this study confirms that thymic activity remains prominent at 24 and even 36 months. this thymic regeneration may contribute to a durable clinical response in patients with ms post hsct. clinical background: allogeneic hsct offers the potential replacement of an aberrant immune system. this retrospective study assessed long-term outcomes of this strategy in patients treated for severe autoimmune diseases (ads), reported to the ebmt registry. methods: among the total 126 patients who received allogeneic hsct between 1997-2014, we received detailed questionnaires on long-term outcomes from 64 patients. the diagnosis of ad was hematological (n=21) and nonhematological (n=43), among pediatric (=45) and adult (=19) populations. the median age of patients at hsct was 11.14 years (pediatric: median 8.07 years, range 1.22-17.77); adult : median 31.30 years, range 21.43-51.57). all patients were refractory to previous immunosuppressive therapies (median of 4 lines of treatments, range 1-13), and eight of them received a previous autologous transplant. the graft source was pbscs in 38, bm in 24, and cord blood in 2 patients. donors were as follows; 41% mrd, 42% mud, 9% mmrd, 5% syngeneic and 3% cord blood. conditioning was mac in 35 and ric in 29 patients. serotherapy with atg was given in 16 patients, while 30 patients received alemtuzumab. post-transplant gvhd prophylaxis was cyclosporine-based for the majority of patients (n=54). results: median follow-up was 67 months (range 7.9-189 months). toxicity profile was similar to allogeneic hsct in other contexts. the incidence of grades ii-iv acute gvhd was 16.4% (95% ci: 8.4 -26.8 ) at 100-days; severe acute gvhd was reported in 6.5% of them. cumulative incidence of chronic gvhd was 32.5% (95% ci: 20. 8 -44.8) at 5-year; extensive manifestations were reported for 58% of chronic gvhd. overall graft rejection rate was 4.9%. seven secondary ad and one case of new malignancy (lymphoma) occurred. viral reactivations were reported in a total of 33 patients, including cmv (n=14), ebv (n= 10), adenovirus (n=5), bk virus (n=3), hsv (n=2), hhv6 (n=3) and vzv (n=2). seven cases of invasive fungal infection were reported (one aspergillosis and three candidiasis). ten bacterial infections (only 4 patients developed infection from gram-negative bacteria) and four pneumonia were observed. at the last follow-up complete clinical response was obtained in 69.4% of patients, while partial remission was reported in 6.5%. relapse incidence (ri) was 21.9% (95% ci: 11. 8 -33.9 ) at the last follow-up. post-hsct autoimmune disease specific treatment was required for 12 patients. in subgroup analysis among different diseases, the os rates were similar between immune cytopenia and other ads. at 5 years, os was 76% (95% ci: 64. 8 -87.3) , nrm was 13.3% (95% ci: 6.1 -23.2) and pfs was 64.8% (95% ci: 52.1 -77.6), with no differences between immune cytopenia (73.8%) and other ads (64.3%). by multivariate analysis, only one prognostic factor remained significantly associated with long-term outcomes: a more recent year of transplant (better os, p=0.007; lower chronic gvhd, p=0.047). conclusions: this large retrospective survey of the ebmt registry confirms the potential of allogeneic hsct to produce long-term disease remission in a large proportion of refractory ads, with acceptable toxicities and nrm. results: twenty-seven patients were evaluated before and at 6 months after transplant, 22 of which were additionally evaluated at 12 months. at 6 and 12 months after ahsct, patients presented significant improvement of mrss (p< 0.01), mip (p< 0.01), mep (p< 0.01), 6mwt distance (p=0.02), and physical (p< 0.01) and mental (p=0.02) components of the sf-36, when compared to pretransplant evaluations. no changes were observed in fvc after treatment. despite a transient decline in dlco at 6 months (p< 0.01) after transplant, dlco levels at 12 months were not different from baseline (pre-transplant). significant correlations were observed between the 6mwt distance and physical component score of quality of life (r=0.62, p< 0.01). no significant correlation was observed between pulmonary function and the other evaluated variables (mrss, respiratory muscle strength, physical capacity and quality of life). conclusions: ahsct significantly improves the functional status of ssc patients in the first year of follow-up. although the pulmonary function remained stable after ahsct, there was significant increase in the physical capacity and quality of life of patients. these results can be interpreted as positive outcomes of ahsct for ssc. disclosure: nothing to declare. model of multidisciplinary and multicenter approach for hsct for children with multiple sclerosis: long background: hsct for children with multiple sclerosis (ms) proved effectiveness and safety. it is required to improve the results by analysis of long-term follow-up and late effects. several challenges identified in multidisciplinary collaboration for successful treatment as well as a problem of switching these patients to the adult healthcare. we aimed to create a model of organization of help for children with severe refractory multiple sclerosis based on multidisciplinary and multicenter approach. methods: fifteen children with ms received autologous hsct (ahsct) from january 2010 to may 2018. gender: females -11, males -4. mean length of ms prior ahsct was 22.7±8.4 months and mean age of ms debut was 12.7 ±2.1 years old. all patients had severe refractory ms treated with corticosteroids, interferons, plasmapheresis and mitoxantron with negative results. these patients presented signs of neuroinflammation. mean baseline edss before the start of mobilization was 4.8±1.4. procedures included mobilization with the help of cyclophosphamide and filgrastim and conditioning: cyclophosphamide 200 mg/kg and hatg 160 mg/kg. all patients received at least 2 x 10˄6/kg cd34 + hematopoietic stem cells (mean 4.3 x 10˄6±2.6 x 10 ˄6). we analysed the incidence and nature of late effects in patients with at least one year of follow-up (based on the standard protocol for late effects). fertility preservation proposed for patients. ahsct as well as pre-and posttransplant care was done by multidisciplinary team involved both transplant and neurological team. technology of transfer patients to adult center for post-transplant observation was identified. results: all patients survived. mean time to engraftment was 11 ±1.6 days. eleven patients experienced culture negative fever, one patient -cystitis, and one patient had cmv reactivation within 100 days of the transplant. no patient experienced an edss increase post-hsct above baseline, and all patients improved. mean improvement of edss was 2.8±1.2 during the first 60 days after ahsct (fast recovery). in-time transplanted patients improved better. improvement confirmed by immunological data (increasing of immune regulation index and t-regs in comparison with the baseline). median follow-up period was 48 months (8-93 months) . four patients (26.7%) experienced exacerbations (both neurological and mri) in median of 2 years (1-3 years) after ahsct. no onsets of secondary autoimmune disease and malignancies was seen. cardiocascular late effects were seen in 6 patients and endocrine -in 3 patients (all females). all these late effects were successfully treated. patients after the age of 18 years old were transferred to partnering adult center. this center uses the same protocol for hsct (in adults) and posttransplant observation. detailed scheme of transfer developed. conclusions: ahsct for pediatric patients with severe refractory ms appears to be safe and effective method and in-time hsct can significantly improve the outcomes. most of the patients did not experienced exacerbations. late effects found in patients were successfully treated. we provide a best care for these patients in both childhood and adulthood by transferring them to adult center. thus, a unique multicenter and multidisciplinary model of care for children with severe refractory ms was found. disclosure background: neuromyelitis optica spectrum disorder (nmosd) is an inflammatory central nervous system disorder characterized, despite immunotherapy treatments, by life-long, severe, and disabling attacks of optic neuritis and myelitis. the aim is to determine if autologous nonmyeloablative hematopoietic stem cell transplantation could be an alternative treatment option. methods: following stem cell mobilization with cyclophosphamide (2 g/m 2 ) and filgrastim, patients were treated with cyclophosphamide (200 mg/kg) divided as 50 mg/kg intravenously (iv) on day -5 to day -2, ratg (thymoglobulin) given iv at 0.5 mg/kg on day -5, 1 mg/kg on day -4, and 1.5 mg/kg on days -3, -2, and -1 (total dose 6 mg/kg), and rituximab 500 mg iv on days -6 and +1. unselected peripheral blood stem cells were infused on day 0. aqp4-igg antibody status was determined by clia validated elisa or flow cytometry assays. cell killing activity was measured using a flow cytometry based complement assay. results: twelve (eleven aqp4-igg positive) patients were treated with a median follow-up of 54 months. ten patients are more than five years post-transplant. at five years, 80% of patients were relapse-free off all immunosuppression (p< 0.001). at one and five years after hsct, edss improved from a baseline mean of 4.3 to 2.8 (p<0.001) and 3.25 (p<0.001), respectively. nrs improved after hsct from a baseline mean of 69.5 to 83.8 at one year (p<0.001) and 85.9 at five years (p<0.001). the sf-36 quality of life total score improved from mean 34.2 to 55.1 (p=0.03) and 62.1 (p=0.001). aqp4-igg serostatus converted to negative in nine patients and complement activating and cell killing ability of patient serum was switched off. two patients remained aqp4-igg seropositive (with persistent cell killing ability) and relapsed within two years of hsct (p< 0.01). conclusions: prolonged drug-free remission with aqp4-igg seroconversion to negative following nonmyeloablative autologous hsct warrants further investigation in larger randomized controlled trial. clinical trial registry: identifier: nct00787722 clinicaltrials.gov disclosure: nothing to declare o038 abstract already published. autoimmune haemolytic anaemia after haematopoietic stem cell transplantation in children: a french multicenter study background: autoimmune cytopenias (aic) are a rare but serious complication of haematopoietic stem cells transplantation (hsct). the auto immune haemolytic anaemia (aiha) is the most frequent of these complications in paediatrics and is difficult to treat. so far, there has been no nationwide post transplantation aiha study. methods: this observational, retrospective and multicentric study focused on french paediatric cases of aiha after hsct between january 2007 and january 2018. data was collected from national reference databases and direct interview of physicians. the inclusion criteria were patients between 0 to 18 years old who developed an aiha or an evans syndrome after hsct. data concerning patient, primary diagnosis, hsct procedure, pre-transplant viral status, blood group compatibility, characteristics of cytopenia, delay transplant-cytopenia, graft vs host reaction. laboratory characteristics and therapies were analyzed as well as the response to first, second or third line treatments. results: 2856 paediatric hsct were performed in 16 french paediatric centers between 2007 and 2018. among them, 36 children developed an aic: 30 aiha, 1 pancytopenia and 5 evans syndromes. the median age at hsct was 4,9 years old (0, [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] 8) . average delay between transplantation and aic was 192 days (20-600). the median follow-up after transplant was 35,8 months (5-94) . 23 patients were transplanted for non-malignant disease (12 benign hemopathies, 11 immunodeficiencies) and 13 for a malignant one. stem cell source was bone marrow for 50% of patients, peripheral blood stem cells for 29% and cord blood for 12%. the donor was unrelated for 65% of hsct, matched related for 15% and 12% of procedures were haploidentical. 76% of patients received als as part of their conditioning regimen, it was myeloablative in 65 % (tbi based for 6%) and reduced intensity in 20%. direct anti-globulin test (dat) was positive to igg +/-c3d for 52% of patients, 16 patients had a blood group incompatibility (8 minor and 8 major). 64% of patients had an acute gvh and chronic gvh on time of aiha was find in 12% of patients. for 70% of patients the first line treatment was a combination of steroids (2mg/kg/day), intravenous immunoglobulin and rituximab (375mg/m 2 per week). for 41% of patients a second or third-line treatment was required (imurel, cellcept, cyclophosphamide, sirolimus, campath, bortezomib). 8 patients died (4 relapse, 2 infections and 1 severe anaemia, 1 multiple organ failure). conclusions: this is the first study describing precisely the aiha post hsct in pediatrics in france along with the various treatments used. it's a rare but severe complication with multiple risk factors associated with a high mortality rate and no standardized therapy at this time. further studies would allow us to understand the disease better in order to prevent its occurrence and treat it more efficiently. disclosure: nothing to declare background: the prognosis of relapsed/refractory acute lymphoid leukemia (all), non hodgkin lymphoma (nhl) or chronic lymphocytic leukemia (cll) is very poor, particularly in patients relapsing after autologous (autohct) or allogeneic hematopoietic cell transplantation (allohct). in the last decade, several chimeric antigen receptor anti-cd19 (car19) constructs have been developed. two of them (tisa-cel and axi-cel) are already approved by the fda and ema for all and nhl. however, the availability and affordability of these commercial carts remains a challenge in europe. methods: the first academic pilot clinical trial (clinicaltrials.gov nct03144583) using our fully academic (a3b1:cd8:4-1bb:cd3z) car19 was approved by the spanish agency of medicines on may/2017. eligibility criteria included r/r all (adult and pediatric), nhl and cll who had failed standard available therapy. the primary objective of the study was safety, and secondary objectives were response rate and its duration (progressionfree survival). here we report an updated analysis of this trial. results: as of december/2018 we have included 35 patients in the study. of these, we performed lymphoapheresis to 34 and we processed the cells of 33, although for the moment we have only infused to 25 of them. the diagnoses of these 25 patients were all in 20 (13 adults), nhl in 4 and cll in 1. median age was 23 years and 40% were women. of the 20 patients with all, 17 had relapsed after allohct, while 3/4 patients with nhl had relapsed after autohct. after conditioning with fludarabine (90 mg/ m 2 ) and cyclophosphamide (900 mg/m 2 ), we infused 0.4-5 x10 6 ari-0001 cells /kg, first as a single infusion (first 19 patients, cohort 1), and then in 2-3 fractions (last 6 patients, cohort 2). we have observed 5 (20%) cases of severe cytokine release syndrome (crs) and the non-relapse mortality (nrm) was 12% (3/25) . these deaths were due to crs (2) and pseudomembranous colitis (1), and all happened in cohort 1. we had no cases of severe neurotoxicity, and grade ii neurotoxicity was only seen in 2 patients. of the 14 patients with active all at inclusion, 11 had enough follow-up for efficacy analysis, and all of them achieved a cr (9 of them with negative mrd). with a median follow-up of 8.7 months (range, 1.3-14.8) , relapses were observed on days +223 (cd19+), +105 and +284 (cd19-), leading to a progression-free survival of 53% at 12 months. in patients with nhl/cll we have documented cr in 2, one of them (nhl) relapsing at day +259. one nhl patient has not been evaluated yet and the other 2 patients did not respond and have died due to their lymphoma. the cll patient, refractory to 5 lines of treatment, achieved a cr, which is maintained at day +377. conclusions: treatment with ari-0001 cells is effective with a response rate of 78.5% in all (pfs of 53% at 1y) and 50% in nhl/cll. we also observed cases of severe crs in 20% and a nrm of 12% of patients, though dose fractioning seems to improve safety profile. still, longer median follow-up and further patient inclusions are needed. clinical background: bcma car-t cells have demonstrated substantial preclinical and clinical activity for relapsed/ refractory multiple myeloma (rrmm) patients. in different clinical trials, the overall response rate (orr) varied from 50% to 100%. complete remission (cr) rate varied from 7% to 60%. previous studies indicated higher car-t cell expansion in vivo achieved better remission. here we developed a bcma car-t cell product manufactured via lentiviral vector-mediated transduction of activated t cells to express a second-generation car with 4-1bb costimulatory domain. methods: our trial (chictr1800017404) was initiated to evaluate the safety and efficacy of autologous bcma car-t treatment for rrmm. the enrolled rrmm patients either had received at least 3 prior treatment regimens, including a proteasome inhibitor, an immunomodulatory agent, anti-cd38 monoclonal antibody or were double or triple-refractory, and have over 30% bcma expression. patients were subjected to a lymphodepleting regimen with flu daily for 3 days (30mg/m 2 , d4 to d2) and cy (500mg/ m 2 , d-3 to d-2) prior to the car-t cell infusion (d0) at a dose range of 1-5 x10 6 car+ cells/kg. results: as of the data cut-off date (nov 28th, 2018), 17 patients, median age 61.8 (49 to 75) years old, with a median of 4.9 (0.4 to 10.8) years since mm diagnosis, were infused with bcma car-t cells. patients had a median of 14 prior regimens (range 4 to 24) ( figure 1a ). all the 17 patients were eligible for initial evaluation of early clinical response with a median observation period of 12 (0.9 to 40) weeks. orr was 100%. all the patients achieved mrd negative in bone marrow by flow cytometry within 1-2 weeks after car-t infusion. partial response (1 pr, 5.9%), very good pr (6 vgpr, 35.3%), and complete response (10 cr, 58.9%) within 12 weeks post car-t infusion were achieved. durable responses from 4 weeks towards the data cut-off date were found in 15/17 patients (88.2%). the disease progressed in 1 patient from pr by week 5 ( figure 1b ). one patient died of severe infection by day 9. all patients had detectable car-t expansion by flow cytometry from day 3 post car-t cell infusion. expansion peaks were found between day 6 to day 17. the peak car-t cell expansion in cd3+ lymphocytes of peripheral blood (pb) varied from 35% to 95% with a median percentage of 83.5% ( figure 1c ). the peak absolute number of car-t cells in pb varied from 530 to 23000 per μl with a median number of 10300 per μl. cytokine release syndrome (crs) was reported in all the 17 patients, including 2 with grade 1, 4 with grade 2 and 11 with grade 3. car-t -related aes were 9 pancytopenia (52.9%), 14 fever (82.3%), 8 nausea (47.1%), 1 heart failure (5.8%). no patient died of crs complication. conclusions: our data showed bcma car-t treatment is safe with prominent efficacy. we also observed high expansion level and long term persistence of bcma car-t cells contribute to potent anti-myeloma activity. these initial data provide strong evidence to support the further development of this anti-myeloma cellular immunotherapy. clinical trial registry: chictr1800017404 disclosure: nothing to declare chimeric antigen receptor (car)-mediated bcl11b suppression in lymphoid progenitor cells propagates natural killer-like cell development background: the transcription factor b cell cll/lymphoma 11b (bcl11b) is indispensable for t lineage development of lymphoid progenitors. pre-fabricated t cell products, allowing for a wider choice of effectively targetable antigens, being applicable to a wider range of patients, and minimizing the risk of long-term sequalae from on target/off tumor effects would be highly desirable. here we hypothesized that antigen receptor engineering of hematopoietic stem cells would fundamentally impact lymphoid progenitor cell fate and as a consequence biological properties thereby allowing to generate an ubiquitously available lymphoid cell product for targeted immunotherapy across mhc barriers. methods: murine hematopoietic stem cells were transduced with a broad panel of 1 st and 2 nd generation murine cars containing different costimulatory domains and numbers of active immune receptor-based activation motifs (itams) against cd19 in a lentiviral backbone and consecutively differentiated into lymphoid progenitors using the op9-dl1 feeder layer system. resulting products were comparatively assessed in vitro and in vivo (facs, functional assays, microarray gene transcript analysis, western blotting, timely controlled transgene expression, leukemia challenge experiments, in vivo lymphoid depletion experiments) upon co-transplantation into a mhc-mismatched myeloablative transplantation model for relapsed leukemia. results: car expression on early ex vivo generated lymphoid progenitors suppresses bcl11b ( figure 1b ) and leads to decreased t cell-associated gene expression. concomitantly, suppressed bcl11b permits lymphoid progenitors to acquire nk cell-like properties ( figure 1a ) and upon adoptive transfer into hematopoietic stem cell transplant recipients they differentiate into carinduced killer cells (carik) that mediate potent antigendirected antileukemic activity across mhc barriers ( figure 1c ,d). a cd28, but not 4-1bb, costimulatory domain and active itams are critical for a functional carik phenotype. conclusions: these results give important insights into differentiation of lymphoid progenitors driven by synthetic car transgene-expression and inform the potential use of ex vivo generated carik as an "off-the-shelf" product for targeted immunotherapy. disclosure: m.v.d.b has ip licensing with seres therapeutics and juno therapeutics. m.v.d.b. has also received honorariums from flagship ventures, novartis, evelo, seres, jazz pharmaceuticals, therakos, amgen, merck & co, inc., acute leukemia forum (alf), and dkms medical council (board) and research support and has stock options with seres therapeutics. a.g. has received research support from aprea therapeutics and infinity therapeutics. all remaining authors have no conflict of interest. [[o043 image] 1. figure 1 : car-induced killer (carik) cells provide strong anti-leukemia effects in vivo.] (a) c57bl/6 (b6) recipients received 3 x 10 6 b6 tcd-bm only or additionally with 8 x 10 6 syngeneic im1928z1lymphoid progenitors. numbers of nk1.1 + carik cells and frequencies of cd3 + tcrβ + progeny within the tom+ gate in spleens on day 28 (im1928z1, n= 5; itom, n= 4). statistics was performed by using students t-test (twotailed). data represent means ± s.e.m. (b) western blot analysis for bcl11b in lysates from itom lymphoid progenitors, im1928z1-lymphoid progenitors or b6 wt thymocytes. (c) recipients co-transplanted with either syngeneic (syn) or mhc class i and ii mismatched (allo) im1928z1-expressing lymphoid progenitors (n= 10 mice, respectively) received 1.2 x 10 6 c1498-mcd19 leukemic cells on day 21 after transplantation and monitored for survival. (d) car-lymphoid progenitor-co-transplanted mice were treated with weekly i.p. injections of either an anti-nk1.1 antibody (clone: pk136; 200μg/dose) or with pbs for control (n= 10 per group). all mice received 1.2 x 10 6 c1498-mcd19 cells on day 21 after transplantation and were monitored for survival. survival curves were compared using mantel-cox (log-rank) test. significant differences are indicated by *p < 0.05, ***p < 0.001, ****p < 0.0001. characterization of an hla-dpb1 specific t-cell receptor for adoptive immunotherapy sebastian klobuch 1 , elisabeth neidlinger 1 , carina mirbeth 1 , wolfgang herr 1,2 , simone thomas 1, 2 background: hla-dpb1 mismatches occur in up to 80% of allogeneic hematopoietic stem cell transplantations from hla 10/10 matched donors and were shown to be associated with a decreased risk of leukemia relapse. therefore, targeting hla-dpb1 mismatched antigens by donor t cells seems to be an attractive strategy to enhance graft-versusleukemia effect. we recently established a reliable method to generate and isolate hla-dpb1 mismatch reactive t cells receptors (tcr). tcr-modified t cells showed leukemia eradication of primary human aml blasts in a xenogeneic nod/scid/il2rgc -/-(nsg) mouse model. however, human fibroblasts used as surrogate cells for graft-versus-host (gvh) reactivity were also recognized by hla-dpb1-specific t cells upon ifn-γ pretreatment, which up-regulates hla-class ii expression on these cells. in this project, we aim at the isolation of tcrs recognizing mismatched hla-dpb1 only on hematopoietic cells, which might lower their risk for gvhd. methods: naive-enriched cd4 t cells were stimulated with autologous dendritic cells expressing allo-hla-dpb1 alleles upon rna transfection. to drive the outgrowth of cd4 t-cell populations expressing 'cd4-independent' tcr which allow the redirection of both cd4 and cd8 t cells, we blocked the cd4/hla interactions by the addition of cd4 binding antibody. allo-hla-dpb1 reactive cultures were analyzed for their recognition of primary aml blasts in ifn-γ elispot as well as 51 chromium-release assays. highly reactive cd4 t cell populations were further analyzed for their ifn-γ secretion against nonhematopoietic cells. tcrs from most promising cd4 t cell clones with an hla-dpb1 specific recognition of hematopoietic but not non-hematopoietic cells were isolated and further analyzed. results: two cd4-independent t cell clones with reactivity to the hla-dpb1*03:01 mismatch allele specifically lysed hla-dpb1*03:01 + primary aml blasts. most importantly, one of these t cell clones did not show ifn-γ secretion upon co-culture with ifn-γ pretreated primary fibroblasts. therefore, we isolated this tcr and transferred it into cd4 and cd8 t cells from healthy donors. tcr dp03 re-directed cd4 and cd8 t cells specifically recognized and lysed primary aml blasts from several hla-dpb1*03:01 + patients in vitro. again, cells of non-hematopoietic origin (fibroblasts) were not recognized even after ifn-γ pretreatment and hla-dp upregulation. to optimize tcr expression and activity, we exchanged the constant domains of the tcr by their murine counterparts. this modification not only led to a higher ifn-γ production and lysis of aml blasts, but also induced recognition of ifn-γ pretreated fibroblasts. however, tumor cell lines overexpressing hla-dpb1*03:01 were also recognized by cd4 t cells engineered with the wild-type or murinized tcr dp03 , suggesting that recognition of hematopoietic and non-hematopoietic cells is rather triggered by the avidity between the t cell and the target cell than by the tcr target epitope. conclusions: in conclusion, our data suggest that allo-hla-dpb1 specific tcrs are powerful therapeutic off-theshelf reagents in allogeneic t-cell therapy of leukemia. the isolation of allo-hla-dpb1 specific tcr without crossreactivity to non-hematopoietic cells might be one strategy to avoid hla-dpb1 specific gvh reactivity upon inflammatory situations (e.g. viral infections). however, our data also indicate that finding of the most suitable tcr candidate is challenging. disclosure: nothing to declare. abstract already published. infusion of memory t cell (cd45ra-depleted) dli improves cmv-specific immune response early after abt cell-depleted hsct: first results of a prospective randomized trial background: abt cell depletion effectively prevents severe gvhd in mismatched hsct, but in a proportion of cases delayed immune recovery leads to increased infection risk and nrm. we've shown in a pilot study that infusion of low-dose memory t cells (cd45-ra depleted) is safe after engraftment among recipients of ab t cell-depleted grafts (pmid:29269793). we initiated a prospective trial to directly test the efficiency of this approach. we report here an interim result of a prospective, randomized, single-centre trial (nct02942173). methods: a total of 100 paediatric patients with malignant disorders (all, n=56; aml, n=30; nhl, n=8; acute mixed lineage leukemia, n=5 and mpd, n=1) were enrolled between october 2016 and september 2018. patients were randomly assigned to receive cd45ra-depleted dlis (experimental arm), n=54, or not (control arm), n=46. median age at hsct was 8.9 years, m:f ratio -42:58. the conditioning consisted of either treosulfan (n= 50) or tbi (n= 50) in combination with fludarabine and thiotepa. gvhd prophylaxis included tocilizumab at 8 mg/kg at day 0, abatacept at 10 mg/kg at day 0, +7, +14 and +28, and bortezomib at 1,3 mg/m 2 at days -5, -2, +2, +5. neither antithymocyte globulin nor calcineurin inhibitors were used. donors were hla-haploidentical (n=94) or matched (n=6). all donors and 76% of the recipients were cmv seropositive. pmbc grafts were split and tcrαβ/cd19 depletion and cd45ra depletion were performed with clinimacs prodigy. the median dose of cd34+ cells was 10x10 6 /kg, αβt cells -28x10 3 /kg. in the experimental arm memory dlis were infused on day 0 at 25x10 3 /kg and on days +30, +60, +90, +120 at 50x10 3 /kg. in the control arm 8 patients received dli after engraftment to prevent relapse (n=6) or treat infections (n=2). primary endpoints were the cumulative incidence (ci) of cmv viremia (>500 copies/ml) by day +100 and the ci of acute gvhd grade ≥ ii. results: median follow-up for survivors was 1 year (0,2-2). engraftment of wbc and platelets was achieved in 99 pts, one patient died at day +8. wbc and platelets engrafted at a median of 11 days and 14 days, respectively. the incidence of cmv viremia was 45% (36-56) overall, 41% (30-56) in the experimental arm vs. 50% (38-67) in the control arm, p=ns. the ci of agvhd ≥ grade ii was 10% (6-18) overall, 10% (4-23) in the experimental arm vs. 9% (4-24) in the control arm, p=ns. two patients died, one per treatment arm, resulting in 2% (0-14) ci of trm at 1 year among the whole cohort. causes of death were preengraftment bloodstream infection and disseminated adenovirus infection. patients randomized to experimental arm acquired anti-cmv reactivity significantly earlier, according to ifn-g elispot assay on day +30 after hsct (p=0,0001). conclusions: co-infusion of donor-derived memory dli with the αβ t cell-depleted graft is safe and improves recovery of virus-specific immune responses. replacement of atg with targeted blockade of cd28/cd80 costimulation and il-6 receptor does not compromise engraftment and gvhd control, and is associated with low rate of non-relapse mortality. [[o046 image] 1. disclosure: nothing to declare o047 conditioning prior to cd19-specific car (19-28z) t cells predicts response and survival in pediatric relapse/refractory (r/r) b-all background: cd19-specific car t cells have demonstrated clinical benefit in patients with r/r b-cell acute lymphoblastic leukemia (b-all). several factors have been associated with response including conditioning chemotherapy, cd4/8 ratio, and post infusion car t cell expansion. methods: we studied the feasibility of a multi-center trial of a msk-developed cd19-specific car (19-28z) for the treatment of r/r b-all, the toxicity following infusion, and performed predictor analysis for optimal response. pediatric and young adult patients with r/r b-all were eligible for infusion. patients received a cyclophosphamidebased (cy) conditioning of high dose (hd; 3g/m2) or low dose (ld; 1.5g/m2) chemotherapy. outcomes of interest were complete response (cr), overall survival (os). variables considered were conditioning regimen (hd-cy vs ld-cy), pre-treatment disease burden (mrd vs morphologic), complete remission (cr) status, absolute lymphocyte count (alc) change, and in vivo car t cell expansion. results: 25 patients were included; 17 patients received hd-cy and 8 received ld-cy prior to car t cell infusion. among evaluable patients (n=24), cr or cr with incomplete count recovery was demonstrated in 94% and 38% for hd-cy vs ld-cy cohorts respectively (p=0·01). os was superior in the hd-cy cohort as compared to the ld-cy cohort (median os = not reached; nr) vs. 4·7 months (p=0·004; figure 1 ). lymphodepletion (delta alc: prior to and following cy) was significantly higher in the hd-cy cohort as compared to the ld-cy cohort (p< 0·001; figure 2 ). the in vivo car t cell expansion (peak car t cell vector copy number/ml) in peripheral blood was higher in the hd-cy cohort as compared to the ld-cy cohort (p=0·01; figure 2 ). to less extent, disease burden prior to treatment with conditioning chemotherapy and car t cells impacted response. disease response was 93% (13/14) in low disease burden group (mrd-cohort) compared to 50% (5/10) in the high disease burden group (morphologic cohort) (p=0·05). os was also superior in the low disease burden group (median os = nr) compared to high disease burden group (median os = 4.3 months; p=0·01). combined response for hd-cy/mrd was 100% (12/12), hd-cy/morphologic 75% (3/4), ld-cy/mrd 50% (1/2), and ld-cy/morphologic 33% (2/6). grade iii/ iv toxicity occurred in 32% (8/25) of patients including severe cytokine release syndrome (scrs) in 16% of patients and severe car-associated neurotoxicity in 28% of patients. conclusions: in this preliminary analysis we demonstrate that dose intensity of conditioning chemotherapy positively correlated with response and overall survival for patients treated with car t cells and confirms, to a lesser extent, pre-treatment disease burden impacts both response and overall survival. clinical trial registry: nct01860937 disclosure: the authors acknowledge william lawrence and blanche hughes foundation provided funding for the conduct of this study with juno therapeutics providing funding for an extension cohort of patients. k.j.c. has received research support from juno therapeutics; has consulted, participated in advisory boards, or participated in educational seminars for juno therapeutics, and novartis. r. j.b. m.s. i.r. are co-founder, stock holders, and consultants for juno therapeutics. c.s.s. has received research support from juno therapeutics; has consulted or participated in advisory boards for juno therapeutics, kite and novartis. early and late hematologic toxicities in children and adults treated with cd19-car t cells elad jacoby 1,2 , shalev fried 1,2 , abraham avigdor 1,2 , bella bielorai 1,2 , amilia meir 1 , michal besser 1,2 , jacob schachter 1,2 , avichai shimoni 1,2 , arnon nagler ,1,2 , amos toren 1, 2 background: autologous t cells transduced with cd19directed chimeric antigen receptors show notable remission rate in advanced patients, leading to approval by the fda and ema for treatment of relapsed and refractory acute lymphoblastic leukemia (all) and non-hodgkin lymphoma (nhl). the most common adverse events reported are cytokine-release syndrome (crs) and neurotoxicity. here we study and profile of hematologic toxicity of patients following locally produced car t cells. methods: we studied the first 38 patients treated on a phase 1b/2 clinical trial using cd19 car-t cells for b-cell malignancies, focusing on hematologic toxicities (neutropenia, thrombocytopenia and anemia), from the initial lymphodepleting regimen till progression or an additional treatment was administered. cytokine levels were studied using milliplex map, human cytokine/chemokine panel ii. results: between july 2016 and march 2018, 38 patients were enrolled on the trial, and 35 patients who received car-t cells and survived more than 21 days were included in this analysis. neutropenia, thrombocytopenia and anemia occurred frequently (94%, 80% and 51%, respectively) after car t cell infusion, and were associated with a prolonged or biphasic nature: in 93% of patients hematologic toxicity occurred or were ongoing after 21 days from cell infusion, and in 52% (neutropenia) and 44% (thrombocytopenia), two trough levels were noted, the second trough occurring after day +21. later events of cytopenia, following more than 42 days from car infusion and in absence of further therapy, occurred in 62% (neutropenia), 44% (thrombocytopenia) and 17% (anemia requiring prbc transfusion) of patients. we identified a strong correlation between the late hematologic toxicities (thrombocytopenia and neutropenia, p=0.018, thrombocytopenia and anemia, p< 0.0001, anemia and neutropenia p=0.05). on univariate analysis, factors affecting late cytopenia were prior hsct (p=0.0015, 0.0083 and 0.02 for anemia, thrombocytopenia and neutropenia respectively) and higher crs grade p=0.003, 0.018 and 0.04 for late anemia, thrombocytopenia and anemia respectively). diagnosis (all vs nhl) or age were not correlated to the incidence of early or late post-car cytopenia. to further study potential causes of late events in patients who were in remission, and in absence of signs of hemophagocytosis, patients' serum was analyzed for chemokine panel at different time points. as expected, serum thrombopoietin levels were correlated with the platelet count. we observed that only in late events (more than 21 days from infusion) sdf-1 serum levels correlated to neutrophil count (r 2 =0.3, p=0.04). conclusions: cytopenia are common events following cd19 car t cell therapy, and may have a prolonged and even biphasic course. patients at risk include those following a recent hsct or with high grade crs. late neutropenia events which occurred later than expected recovery from conditioning chemotherapy and following resolution of crs, were correlated with serum sdf1 levels, similar to prior observations with late onset neutropenia related to rituximab (dunleavy, blood 2005 background: acute myeloblastic leukemia (aml) represents 40% of all leukemias of western countries, being the second malignant hemopathy in pediatric population. in the last decades, the survival rate has maintained around 60%, being relapse the main problem. it has been highlighted the role of immune system for the control of aml and new therapeutic strategies have been developed. in this setting, the use of alloreactive natural killer (nk) cells could play a key role not only in the context of hematopoietic stem cell transplantation (hsct) but also as adoptive immunotherapy in patients with minimal residual disease. in this project we proposed including the cellular therapy with haploidentical activated and expanded nk cells as adjuvant therapy in pediatric patients affected by aml in complete remission and without indication of hsct. methods: it is a multicentre, open, prospective and no randomized phase ii clinical trial, to evaluate the efficacy and safety of allogenic nk cells infusion from haploidentical donor after a lympho-ablative chemotherapy in pediatric patients affected by low and intermediate risk aml in first complete cytological remission. patients were treated according to spanish protocol (sehop 2002) including 2 inductions plus 2 consolidation cycles. after chemotherapy patients received the infusion of activated and expanded nk cells (nkae) after a lympho-ablative treatment based on cyclophosphamide 60mg/kg and fludarabine 125mg/kg. nk cells were obtained from 250 ml of peripheral blood from haploidentical donors selected based on alloreactivity of kir inhibitors and kir activators receptors. nk cells were activated and expanded for 3 weeks trough co-culture with the cellular line k562-mbil15-41bbl suitable for clinical use in humans (good manufacturing practices, gmp). results: at this time 14 products in 7 patients have been infused, with the following characteristics: nk cells (cd45 + cd56 + cd3 -) 86.64 ± 11.36%; t lymphocytes (cd45 + cd56 -cd3 + ) 2.94 ± 3.55%. one product was rejected for quality criteria. a mean of 51.14 ± 53.99 x 10 6 nk cells/kg and 0.69 ± 0.35 x 10 6 t cells/kg were infused. median age of the patients were 7 years (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) . treatment and infusions were well tolerated. the main adverse event was neutropenic fever in one patient. after a median follow up of 530 days (371-813) all the 7 patients are alive. only one patient showed relapse at day +307 after nk cells infusion and was submitted to an hsct from a matched unrelated donor. conclusions: the preliminary results of this trial suggest that nk cells infusion as consolidative strategy in pediatric patients with aml, is feasible and secure. the therapeutic effect should be confirmed in a larger cohort of patients. clinical trial registry: clinicaltrials.gov (nct02763475) and eudract (2015-001901-15) . disclosure: authors have no conflict of interest. the trial was funded by "fundación mutua madrileña" grant o050 tcrαβ/cd19 depleted and il-15 stimulated donor cell infusions can exert anti-tumor effects after allosct without inducing gvhd in vitro and in vivo background: natural killer (nk) cells and γδ t-cells have been shown to play a significant role in gvl effects after allogeneic stem cell transplantation (sct). both cell types are not restricted by mhc molecules which makes them unlikely to elicit graft versus host disease (gvhd) even in mismatched sct and therefore are suited also for cell-based posttransplant immunotherapy. incubation with cytokines strongly enhances their spontaneous and antibody dependent cellular cytotoxicity (adcc). methods: thus, we investigated the efficacy of a combination of il-15 stimulated nk-and γδ t-cells from mismatched donors in vitro and based on urgent medical need in ultra high risk patients. results: in vitro data: spontaneous cytotoxic activity and adcc with tcrαβ/cd19 depleted pbmcs from healthy donors against ls (neuroblastoma) and nalm-6 (leukemia) target cells after overnight stimulation with il-15 was significantly increased compared to non-stimulated cells (e: t ratio 20:1; ls cells: 43% and 61% vs 19%; nalm-6 cells: 44% and 68% vs 26%). the results for cd107a assays are in line with the cytotoxicity approaches. nk and γδ t-cells showed a significantly stronger stimulation after il-15 incubation +/-adcc, as measured by secreting cytokines (cd107a, infγ, tnfα). moreover, 7 patients at extremely high risk for relapse received nk-/ γδ t-cell infusions after previous transplantation from haploidentical (n=5) or matched donors (n= 2); diagnoses: t-all, relapse after 1 st sct n=1; b-lineage-all, cr after 2 nd sct, n=3; aml, relapse after 1 st sct, n=1; relapsed neuroblastoma, pr after 2 nd sct, n=1. for that purpose magnetic microbeads and the clin-imacs ® device were used for tcrαβ/cd19 depletion of leukapheresis products from the original donors. afterwards, remaining cells (nk, γδ t and myeloic cells) were stimulated with il-15 (10 ng/ml) overnight. efficacy of the stimulation procedure was measured in 3 leukapheresis products: after incubation with target cells (ls) both, nkcells as well as γδ t-cells showed a significant increase in cd107a secretion compared to non-stimulated effectors (n=4) from healthy donors (nk-cells: 75% vs 12%; γδ tcells: 44% vs 2%). a median number of 12.4x10 6 nk-cells and 1.66x10 6 γδ t-cells and 1.08x10 3 residual tcrαβ cells/ kg were infused. four patients had received therapeutic mabs (anti-gd2, anti-cd19 or anti-cd38) within 48 hours after cell infusion to induce adcc. side effects were treatable cytokine release syndrome in 3 patients; no gvhd occurred. infused cells could be tracked by cd69 expression for up to 26 days in peripheral blood. outcome: 2 patients with t-all and neuroblastoma responded and achieved another cr, 2 patients maintained their previous cr for up to now 55/222 days; 3 patients progressed/relapsed after 26/69/89 days. additional therapies were applied in 4/7 patients. 4 patients are alive (median follow up: 248 days after infusion), 2/4 are disease free. conclusions: infusion of a combination of nk/ γδ tcells can increase anti-tumor effects and adcc with appropriate therapeutic mabs without inducing gvhd even after mismatched sct. further investigations are needed to evaluate the role of this cellular therapy. disclosure: nothing to declare o051 abstract already published. identifying chimeric antigen receptor (car) centers and car activity in europe, a survey on behalf of the cellular therapy & immunobiology working-party (ctiwp) of ebmt background: the use of chimeric antigen receptor (car) t cells has shown outstanding efficacy in patients with relapsed/refractory b-cell lymphoid malignancies, as reported by many groups in united states (us) and china. car-t cell based activity in europe is still at early stages. few european academic and pharma-sponsored clinical trials are currently opened to inclusions, and access to the two ema-approved autologous car-t products remains limited for the majority of european centers, since they must undergo a tedious and non-harmonized qualification process imposed by the manufacturer as part of the drug label. improved awareness of car-t centers and ongoing car-t activities in europe are essential to promote european networking activities, improve competitiveness of eu medical centers, and develop clinical trials, education, accreditation, and registration/long-term follow-up, thus fulfilling the needs of stakeholders. methods: a survey questionnaire was prepared by the committee members of the ebmt cellular-therapy-&-immunobiology-working-party (ctiwp). questions were formulated to identify car-t centers and car-t based activity in europe, and to know the characteristics of centers and the organization for implementing a clinical car-t program. using monkey survey, the survey was sent to 566 ebmt-centers. results: 123 centers replied to the survey. 340 patients (all 45%, nhl 32%, mm 10%, cll 5%, aml 1.6%, solid tumors 7%) have been treated so far in 31 ebmt centers from 12 countries. most of these centers are jacie-accredited (90% for clinical activities; 93% for cell collection activities). 54 additional centers reported plans to start car-t cell administration within the following 6-months. most patients were treated in the setting of academic clinical trials (58.6%l) or pharma sponsored trials (36%l). few patients (7%) received marketed car-t cells. pcr (36%) and flow-cytometry (60%) were used to monitoring the persistence of car-t cells after infusion. cytokine levels were measured in 78% of the cases, and ngs was performed in 45% of the centers. half of the centers reported to use "point of care" manufacturing. patient management was mainly carried out by bone marrow transplant (bmt) physicians (85%). however, 77% of the centers reported to have also implemented a "car-t team", as a multidisciplinary team managed by a coordinator, with weekly clinical meetings. in more than 90% of the centers, there were specific rules for transferring patients to the intensive-care-unit. in contrast, only 40% of the centers had a nurse coordinator for management assistance of these patients. 77% of the centers plan to expand the capacity of their own department for car-t program. conclusions: this survey is the first attempt to gather information about car-t cell activity in europe. this first results confirms active and growing involvement and high quality standards of european centers having car-t cell program. the expanding use of these complex atmp approaches could be facilitated through harmonization of the clinical practice, shared analysis of good quality data, and by a centralized european clinical trial office. the cellular therapy car-t form is readily available to capture in more detail safety and efficacy of this intervention and will allow sharing in a transparent process registry data with stakeholders. methods: this is an open-label, phase 1 study (chictr1800017404). eligible patients had rrmm, confirmed as bcma-positive by flow cytometry or pathological examination. autologous t cells transduced with a bcmatargeted chimeric antigen receptor (car-bcma t cells) are used(1×10^7/kg car+t cells), following pre-infusion treatment--intravenous administration and lymphodepletion conditioning (fludarabine and cyclophosphamide). the primary outcome measure is incidence of adverse events (aes) especially clinical events, including crs, neurotoxicity and coagulopathy, et al. additional outcome measures are duration and expansion of bcma car t cells. results: 19 patients (median age of 61, range 41 to 71) with rrmm have received treatment. the median %pc in bm at enrollment was 30.23% (range 2% to 72%). 4/19 had high tumor burden, defined as ≥ 50% bone marrow plasma cells. 3/19 had prior autologous stem cell transplant. as of data cut-off (dec 1,2018), crs grade 1-2 occurred in 8 patients, grade 3-4 occurred in 11. surprisingly, not a few patients developed coagulation disfunction, manifesting elevated d-dimer, prolonged aptt, pt, tt, and decreased fibrinogen, with 0 case of organ bleeding. laboratory values correlating with crs reaching grade 3-4 (n=11) compared to those with milder crs (n=8) included peak ferritin (mean: 31095.23 vs 1665.53 ug/l, p< 0.005), peak il-6 (mean: 8001.07 vs 266.92 pg/ml, p< 0.005), peak il-10 (mean: 324.89 vs 43.81 pg/ml, p< 0.01), and peak ifn-γ(mean: 2936.46 vs 58.21 pg/ml, p< 0.05). no significant difference was seen in peak crp, il-2 and tnf-α. patients with crs 3-4 had a higher △aptt (mean: 38.86 vs 12.09 s, p< 0.01), △d-dimer (mean: 44509.18 vs 13347.75 ug/l, p< 0.05) and △fibrinogen (mean: -1.68 vs -0.34 g/l, p< 0.05) compared to those with milder crs. there was no significant difference in △pt and △tt.the changes of d-dimer and aptt were in line with the indictors of crs, such as crp, il-6 and il-10 in some individual cases. the significant correlation between aptt and crp (p< 0.05) appeared on 17 patients (89.5%), while the significant correlation between il-6 and aptt as well as between il-6 and d-dimer only appeared on 7 patients (36.84%). conclusions: early safety and efficacy results of bcma car t therapy in rrmm are encouraging. aes of coagulation were observed. changes of aptt, d-dimer and fibrinogen of patients with high crs level were more significant than those with milder crs. on the individual level, aptt correlated with crp in the course of therapy. given extensive cross-talk between inflammation and coagulation, coagulation-related indictors are more convenient for monitoring crs. also, our study suggests the importance in diagnosis and early management for coagulopathy to avoid crs-related mortality. clinical trial registry: chictr1800017404 http:// www.chictr.org.cn/abouten.aspx disclosure: nothing to declare o054 abstract already published. point-of-care production of cd19 car-t cells in an automated closed-system bioreactor: report of first clinical experience background: cd19 car-t cell products are approved as therapy for advanced b-cell malignancies. the predominant manufacturing model is a centralized industrial-type production process. an alternative approach to car-t cell production and delivery to the patient is via a point-of care manufacturing process. methods: between february 2018 to november 2018 a total of 15 pts with relapsed/refractory b-cell malignancies (4 female, 11 male[gr1], median age 12 y) were screened, 10 pts were enrolled for single center, phase i-ii trial of safety and clinical efficacy of automatically produced cell therapy product, 5 were eligible for compassionate use of therapy. seven patients had relapsed b-all after hsct, 6 pts had refractory relapse after chemotherapy, 1 pt had refractory induction failure, 1 pt had refractory primary mediastinal bcell lymphoma (pmbcl). eight patients received previous blinatumomab infusion. eight patients had high disease burden >5% blast cells in bm, median 89% (14-100), 6 pts had minimal residual disease (mrd) in bm. the clinimacs prodigy t cell transduction (tct) process was used to produce cd19 car-t cells from fresh patientderived leukapheresis product. automatic production included cd4/cd8 selection, stimulation with macs gmp t cell transact, transduction with lentiviral (second generation cd19.4-1bb zeta) vector (lentigen, miltenyi biotec company) and expansion over 10 days in the presence of texmacs gmp medium and macs gmp il-7/il-15 combination. final product was administered without cryopreservation after fludarabine/cyclophosphamide preconditioning. all patients received prophylactic tocilizumab at 8 mg/kg. results: all production cycles were successful. median transduction efficiency was 59% (32-75). median expansion of t cells was 36 fold (18-49). cd4/cd8 ratio in the final product was 0,6. the cell products were administered at 1*10 5 /kg of car-t cells in 5 pts, 5*10 5 /kg in 5 pts, 1*10 6 /kg in 5 pts. cytokine release syndrome occurred in 8 (53%) pts: grade i in 5 pts, grade ii in 2 pts, grade iv in 1 pts. car-t cell related encephalopathy occurred in 4 (26%) pts, including one fatal brain edema. grade i neurotoxicity had 2 pts, grade ii -1 pt, grade v -1 pt. four patients were admitted to the intensive care unit (icu). three patients died until day 28 (2 due to sepsis, 1 due to fatal brain edema and sepsis, on autopsy in the brain vessels of this patient were found klebsiella pneumoniae emboli) twelve patients were evaluable for response at day 28. three pts had persistent leukemia, without evidence of car-t expansion. mrd-negative responses were achieved in 8 cases among 11 evaluable cases with bone marrow involvement. patient with pmbcl had a decrease in metabolic activity on pet/ct scan. cd19(-) relapse after initial response was registered in 1 case at 109 day. conclusions: production of car-t cells with the clinimacs prodigy tct process is a robust option that provides a point-of-care manufacturing approach to enable rapid and flexible delivery of car-t cells to patients in need. robustness and consistency of this approach provides a solid basis for multi-center academic trials in the field of adoptive cell therapy. disclosure: nothing to declare background: in the last decade, several prognostic scoring models such as the international prognostic scoring system (ipss), the dynamic ipss (dipss) or dipss-plus have been developed for diagnosed primary myelofibrosis (pmf) and are currently used for decision finding regarding allogeneic stem cell transplantation. furthermore, the prognostic relevance of mutation profile resulted in a mutation-enhanced system (mipss70) in transplant-age pmf patients (70 years or younger). for secondary myelofibrosis, the mysec-pm was developed. while all scoring systems were developed in nontransplant populations and may be useful in decision finding regarding transplantation, uncertainty remains regarding usefulness of these systems to predict posttransplant outcome and thus counseling patients whether to proceed to transplant. methods: bone marrow or peripheral blood samples were obtained before transplantation and mutations were detected using next-generation sequencing. the following myelofibrosis-associated genes were sequenced: jak2, calr, mpl, asxl1, idh1/2, cbl, dnmt3a, tet2, sf3b1, srsf2, u2af1, ezh2, tp53, nras, kras, runx1, and flt3. cytogenetic analysis and reporting were performed according to the international system for human cytogenetic nomenclature criteria using standardized techniques. we examined 361 myelofibrosis patients, of whom 260 had pmf and 101 smf at time of transplantation. a training cohort of 205 patients was used to create a clinical-molecular transplant scoring system (mtss) predicting survival from cox models, internally validated by use of bootstrap and cross-validation. model discrimination was measured by the concordance index (c). the final mtss was externally validated in a cohort of 156 patients and was furthermore applied to posttransplant non-relapse mortality as a secondary objective. results: multivariable analysis on 5-year survival identified age ≥ 57 years, karnofsky performance status < 90%, platelet count < 150 x 10 9 /l and leukocyte count > 25 x 10 9 /l at time of transplantation, hla-mismatched unrelated donor, asxl1 mutated and calr-/mpl-unmutated genotype being independent prognostic factors for outcome. the uncorrected concordance index for the final survival model was 0.723 (0.713-0.733), and bias-corrected indices were similar. a weighted score of 2 was assigned to transplantation from an hla-mismatched unrelated donor and an calr-/mplunmutated genotype, whereas a score of 1 was assigned to older age, leukocytosis, thrombocytopenia, asxl1 mutation, and poor performance status. the mtss consisted of four distinct risk groups showing 5-year survival in the validation cohort of 83% (71-95%) for low (score 0-2), 64% (53-75%) for intermediate (score [3] [4] , 37% (17-57%) for high (score 5), and 22% (4-39%) for very high risk (score > 5), respectively (p < 0.001). increasing score was predictive of non-relapse mortality (p < 0.001) and remained applicable to pmf ( conclusions: we show here that this internally and externally validated mtss accurately discriminated different risk for death and may improve counseling patients with respect to transplant compared with currently existing systems, as well as facilitate design of clinical trials in the transplant setting. disclosure: nothing to declare. response to up-front azacitidine in juvenile myelomonocytic leukemia (jmml): interim analysis of the prospective european multicenter study aza-jmml-001 background: jmml is a chemotherapy-resistant neoplasia of early childhood. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative therapy, saving approximately 50% of these children. relapse is the major cause of treatment failure, with chemotherapy prior to hsct being notably unsuccessful. novel therapies controlling the disorder prior to hsct are urgently needed. methods: we conducted a phase 2, multicenter, open-label study to evaluate pharmacodynamics, safety, and antileukemic activity of azacitidine monotherapy prior to hsct in patients with newly diagnosed jmml. azacitidine was administered at 75 mg/m 2 /day intravenously on days 1-7 of a 28-day cycle for 3 to 6 cycles. the primary endpoint was the number of patients with clinical complete remission or clinical partial remission (cpr) at cycle 3 day 28 (c3d28); secondary endpoints included overall survival following hsct. results: eighteen jmml patients (13 ptpn11-, 3 nras-, 1 kras-, 1 nf1-mutated) aged 0.2-7.0 years were enrolled. median (range) wbc, platelet count and spleen size were: 19.7 (4.3-59.0) × 10 9 /l, 28 (7-85) × 10 9 /l, and 4 (2-14) cm below the costal margin, respectively. dna methylation class (lipka et al. nat comm 2017; n=17) was high, intermediate, or low in 10, 5, and 2 patients, respectively. sixteen patients completed 3 cycles of therapy and 5 of them completed 6 cycles. two patients discontinued treatment before completing 3 cycles due to disease progression. six patients (33%) experienced ≥ 1 grade 3 or 4 manageable adverse event, consistent with the known azacitidine safety profile. eleven patients (61%) achieved cpr at c3d28 and 7 had progressive disease either at c3d28 or prior. importantly, 8 of the 15 patients who needed platelet transfusions before or shortly after treatment initiation did not require transfusions at the time of hsct. seven of these 8 platelet responders had normalized their platelet count (≥ 130 × 10 9 /l). palpable spleen size decreased in 11 responders by a median of 3.5 cm after 3 cycles and ranged from 0-2 cm below the costal margin after 6 cycles. sixteen patients received allo-hsct from a family or compatible unrelated donor following a busulfan-(n=15) or treosulfan-based (n=1) preparative regimen after a median of 57 days (36-112) from last azacitidine dose. thirteen transplanted patients were leukemia-free at median follow-up of 15.7 months (0.1-31.7) after hsct. two children (both high methylation class) given hsct relapsed after the allograft. sixteen of the 18 patients were alive at a median follow-up of 19.8 months (2.6-37.3). one patient who discontinued treatment before cycle 3 died from disease progression, and 1 non-responder died from graft failure. conclusions: this study shows azacitidine monotherapy was well tolerated in children with newly diagnosed jmml. although the long-term advantage of azacitidine therapy remains to be fully assessed, both the decrease in spleen size and significant platelet responses observed demonstrate that the drug was effective in jmml and provided clinical benefit to jmml patients in this study. this clinical trial has shown that azacitidine therapy prior to hsct may be considered for patients with jmml. clinical trial registry: nct02447666 disclosure: charlotte niemeyer is a member of a board of directors or advisory committee and provides consultancy for celgene. claudia rössig is a member of a board of directors or advisory committee at amgen, eusapharm, roche, celgene, novartis, pfizer, bms; honoraria from amgen, roche, pfizer. andré baruchel provides consultancy (includes expert testimony) with celgene, novartis, servier, jazz pharma; research funding and honoraria from shire; honoraria from novartis, jazz pharma. susana rives has received honoraria (for talks in industria sponsored satellite symposia) from novartis, jazz pharma, baxalta, shire, servier; speaker's bureau at novartis, jazz pharma, baxalta, shire, servier, amgen, erytech pharma; membership on an entity´s board of directors or advisory committees at novartis; received travel and accommodation expenses for medical congresses from novartis, jazz pharma, baxalta, shire, servier, amgen, erytech pharma. marco zecca has received honoraria from chimerix and jazz pharma. marry m. van den heuvel-eibrink received honoraria from celgene (consultation fee). bouchra benettaib, noha biserna, jennifer poon, mathew simcock, meera patturajan are employees of and hold stock or other equity ownership in celgene. christian flotho, daniel lipka, jan starý, karsten nysom, gérard michel, thomas kilngebiel, franco locatelli, giuseppe basso, concetta micalizzi, irith baumann, markus schmugge liner have nothing to disclose. ruxolitinib before allogeneic hematopoietic stem cell transplantation (hsct) in patients with myelofibrosis: a preliminary descriptive report of the jak allo phase ii study marie robin 1,2 , raphael porcher 3 , corentin orvain 4 , background: allogeneic hematopoietic stem cell transplantation (hsct) is the only curative treatment for patients with myelofibrosis; os is from 30 to 60% depending on age, comorbidities, disease status and type of donors. jak1/2 inhibitors have been reported to decrease constitutional symptoms and spleen size in one half of patients with a possible advantage of os as compared to best current treatment. retrospective studies show that ruxolitinib has been used in patients before hsct with apparent good tolerance. methods: in 2012, we initiated a phase ii prospective french collaborative (filo and sfgm-tc) trial testing the role of ruxolitinib given before allogeneic hematopoietic transplantation in patients with myelofibrosis. patients could be included if they were intermediate or high risk according to lille score or ipss. patients had to start ruxolitinib after inclusion and were transplanted in case a donor has been identified within 4 months. primary aim was progression-free survival at one year after hsct. results: 76 patients could be included. 53 (70%) had a primary myelofibrosis and 23 (30%) had a myelofibrosis secondary to essential thrombocythemia (n=10) or polycythemia vera (n=13). at time of inclusion, 43 (57%) patients had constitutional symptoms, 72 (95%) had palpable splenomegaly, 48 (63%) patients had hemoglobin < 10 gr/dl, 23 (30%) patients had peripheral blast cell count > or = at 1% and 16 (21%) had thrombocytopenia < 100 g/l. median follow-up was 31 months. at 4 months, one patient has died, 11 patients had no donor, 18 had an hla matched sibling donor, 31 had an hla matched unrelated donor and 15 had a 9/10 hla mismatched unrelated donor. 59 (78%) patients with a donor at 4 months could be transplanted of whom 18 underwent a splenectomy before transplantation. conditioning regimen was fludarabine 90mg/m2 in combination with melphaln 140mg/m2. gvhd prophylaxis was cyclosporine and mycophenolate mofetil with atg in unrelated donor. partial response under ruxolitinib was 26% while the whole complete response incidence was 46% at one year. os and pfs at 2 years were 68% (95% ci: 59-80) and 53% (95% ci: 43-66). os was significantly better in patients with hla matched sibling donor as compared to patients without a donor or with an unrelated donor (p=0.008, figure 1 ). cumulative incidence of acute gvhd on day 100 was 66% of whom 44% had grade 3-4 acute gvhd. cardiogenic shock generally associated with severe sepsis or "sepsis like" syndrome occurred in 7 patients: 2 patients were not transplanted because of this complication while in the 5 other patients cs occurred within the 15 days after transplantation. three renal failures secondary to tumor lysis syndrome were declared just after conditioning regimen initiation. figure 1 . overall survival in jak allo patients. conclusions: os was very good in patients with hla matched related donor and significantly better than in patients with an unrelated donor. more analyses are currently ongoing to determinate the potential role of cytokine release in peri-transplantation time, as well as specific myelofibrosis and quality of life questionnaires assessing the impact of ruxolitinib in patients. background: while cytogenetics may influence outcome in primary myelofibrosis (pmf) from diagnosis which lead to several prognostic systems incorporating different cytogenetic risk classifications, its definitive role specific after allogeneic stem cell transplantation is still unclear. here, we aim to evaluate the role of currently existing cytogenetic risk classifications included in the dynamic international prognostic scoring system (dipss)-plus and in the mutation-enhanced system (mipss70-plus version 2.0). methods: in this multicenter retrospective study, we examined 149 pmf patients undergoing allogeneic stem cell transplantation. current cytogenetic risk stratifications used in dipss-plus and mipss70-plus version 2.0 were evaluated. according to dipss-plus, an unfavorable karyotype includes +8, -7/7q, i(17)q, -5/5q,12p-, inv (3), and 11q23 rearrangements while mipss70-plus version 2.0 consisted of very high risk (vhr: single/multiple abnormalities of -7, i(17q), inv(3)/ 3q21, 12p-/12p11.2, 11q-/11q23, +21, or other autosomal trisomy, not including +8/+9), favorable (normal karyotype or sole abnormalities of 13q-, +9, 20q-, chromosome 1 translocation/duplication or sex chromosome abnormality including -y), and unfavorable (all other abnormalities). results: the median follow-up period of all 149 pmf patients was 56 months and the median time from diagnosis to transplant was 26 months. after five years, 51 (34%) patients had died. the median age at transplant was 57 years and 63% of patients were male. most allografts were applied using peripheral blood (97%) and were received from an hla-matched unrelated donor (42%), followed by mismatched unrelated (34%), matched related (23%), and mismatched related or haploidentical (1%). reduced intensity conditioning was applied to 75% of patients. splenectomy before transplant was undergone in 15% and ruxolitinib was received by 27%. frequencies according to driver mutation genotype were: calr (19%), jak2 (59%), mpl (5%), and triple negative (17%). asxl1 mutation was present in 38% while 64% had more than two mutations overall. 94 (63%) patients had normal karyotype. most frequent abnormalities were trisomy 8 in 11 (7%), trisomy 9 in 5 (3%), deletion in 20q in 17 (11%), deletion in 13q in 11 (7%), chromosome 7 in 5 (3%), chromosome 5 in 5 (3%), and chromosome 1 translocation/duplication in 2 (1%). more than two abnormalities were present in 9 (6%) patients. high cytogenetic risk category according to dipss-plus was present in 24 (16%) while 12 (8%) had vhr and 25 (17%) had unfavorable risk according to mipss70-plus version 2.0. regarding outcome, an unfavorable karyotype according to dipss-plus showed 5year os of 62% (52-72%) vs 63% (57-70%), with causespecific hazard being 1.16 (p=0.69). 5-year relapse and nonrelapse mortality rates were not significantly different showing 9% and 33% for unfavorable karyotype vs 17% and 28% (p=0.31 and 0.52). with respect to the three-tiered classification of mipss70-plus version 2.0, 5-year os of vhr was 74% (62-86%) and unfavorable was 52% (42-62%) vs favorable risk of 64% (58-70%), with cause-specific hazard of 1.09 (p=0.74). relapse and non-relapse mortality were 9% and 17% for vhr, 4% and 48% for unfavorable, and 19% and 26% for favorable risk (p=0.19 and 0.48). conclusions: current cytogenetic risk stratifications do not predict outcome in pmf after allogeneic stem cell transplantation. [[o060 image] 1. background: the aim of this study was to assess the outcome of patients with myelofibrosis allografted before and after 2010, in two transplant centers (genova san martino and rome gemelli). methods: we have studied 147 patients, divided in two groups: 2001-2010 (n=59) and between 2011 and 2018 (n=88). the age was significantly older in the most recent group (53 vs 58 years, p< 001), and there was a greater use of alternative donors (38% vs 80%, p< 0.0001), and more patients with dipss-r high score (40% vs 47%, p=0.1). the transplant score (based on transfusions >20 and splee size >22 cm) was intermediate-high risk in 82% and 58% (p< 0.01) of patients respectively. the conditioning regimen was a combination of thiotepa, busulfan fludarabine (tbf) in 1% and 87% of patients grafted before and after 2010 (p< 0.00001). the transplant risk score (ts) was based on spleen size (>22 cm) and pre-graft transfusion (>20) as previously described (bone marrow transplant. 2010 mar; 45(3) :458-63) results: outcome. the cumulative incidence (ci) of acute grade ii-iv (25%) and of chronic gvhd (18%) was comparable in the two time periods. the 5 year ci of non relapse mortality (nrm) was reduced overall from 37% to 20% (p=0.01) and the 5 year ci of relapse from 47% to 11% (p=0.00006). as a consequence the overall 5 year actuarial survival has improved from 35% to 64% (p=0.003). predictive factors. the following factors predicted survival in multivariate cox analysis: high risk transplant score (hr 2.5, p=0.01), high risk dipss-r (hr 2.0, p=0.01), the use of tbf (hr 0,50, p=0.02), alt donor (ht 1.8m p=0.04), donor age >29 years (hr 1.8, p=0 .06) and abo match (hr 0.64, p=0.08). dipss and transplant score. when combining dipss-r (high) and transplant score (int2-high) we could identify 3 groups : score 0 (dipss not high and ts not high) (n=40) score 1 (either high dipss or high ts) (n=51), score 2 (both dipss a ts high risk) (n=55) . nrm was 9%, 27% 40% in these 3 group (p=0.0004), relapse was 17%, 29%, 25% (p=0.1) and 5 year survival was 78%, 55%, 20% (p< 0.00001). in the current transplant era (>2010) the 5 year disease free survival of these 3 groups is 78%, 70%, 35% (p< 0.00001). abo matching further increases dfs for score 0 patients. nrm mortality for these 3 groups is currently 6%, 16%, 36% (p=0.01). conclusions: the 5 year survival of allografts in patients with myelofibrosis has improved overall from 35% before 2010, to 64% beyond 2010, despite the current use of 80% alt donors. predictive factors are the transplant score (transfusions and spleen size), dipss-r and the use of 2 alkylating agents (tbf), the latter being the major change in the 2 transplant eras. patients received ruxolitinib before the transplantation, 7 were transfusion-dependent. all the patients underwent pbsc infusion, except one who received bmsc. in 7 cases the source was a sibling donor, in 9 a mud, in 2 a hla-mismatched ud (1 mismatch). conditioning regimen was mostly based on the combination of singlealkylating agent and fludarabine (12 bu/flu; 3 mel/flu/ bcnu; 1 mel/flu; 1 treo/flu) with atg infusion. six patients received a fully myeloablative schema, 12 reduced-intensity conditioning. only one patient received conditioning with tbi. results: before the transplant, a panel of molecular analysis based on next-generation-sequencing revealed, in addition to the mpl mutation, alterations in asxl1 (22%), srsf2 (11%), sf3b1 (5.5%), ezh2 (11%), idh1 (5.5%), idh2 (5.5%), tet2 (33.5%), tp53 (5.5%). after the transplant, the incidence of acute gvhd was 72%; only 3 patients (16.5%) experienced acute gvhd grade 3-4. chronic gvhd was registered in 50% of cases (9 patients: 8 mild, 1 moderate, 0 severe). relapse occurred in only 1 case. nrm incidence was 16.5% of cases, occurring in the first year after transplant. with a median follow up of 55 months, 5-year overall survival was 83.5%, and 5-years pfs reported the same value, beeing the only relapse at >10 years after transplantation. the relapse occurred in the only patient who harbored mutations in both asxl1 and ezh2 genes. conclusions: these retrospective data suggest that the particular group of mpl-mutated myelofibrosis may have a good outcome after stem cells transplantation. in particular, our data revealed very low rate of disease relapse (5.5%) in comparison with the available historical controls regarding myelofibrosis in toto. [ background: a significant proportion of cml patients undergoing allogeneic stem cell transplantation (allo-hsct) restart tki following transplant to prevent relapse. there is however no data to support if tki can be discontinued following allo-hsct and whether such patients can safely discontinue tki remains controversial. practices varies among transplant centres depending on countrywide practices, centre experience, duration of molecular remission, patients 'wish and analysis of retrospective data on the outcome of patients who discontinue tki after transplant may provide further insight to help elaborating future guidelines. the present study objective is to investigate the outcome of patients with cml who discontinue tki therapy after restarting tki following allo-hsct. this retrospective study may be helpful to support future guidelines. methods: through the ebmt database of patients who received an allo-hsct between march 2004 and september 2013, we identified 81 cml patients who restarted tki treatment post allo-hsct and stopped it after at least 3 months of tki therapy. results: out of 81 patients who discontinued tki, 21 were in first chronic phase (cp1) at the time of transplant, 31 in second or third chronic phase (cp1 and cp2) and 28 in accelerated or blastic phase (ap/bp) or primary refractory disease. one patient had missing data at the time to transplant. allo-hsct conditioning was of reduced intensity (ric) in 27 patients and myeloablative (mac) in 54 patients, including tbi in 29 patients. tki therapy was (re)started in all patients after a median time from transplant of 4.4 months (range, 0.4 to 57.6 months) for a duration of 9.7 (range, 3.1-62.3 months). after a median time from tki discontinuation of 65.8 months (range 51.8-71.1), 5-years progression free survival (pfs) and overall survival (os) were 51% (95%ci 39 to 62%) and 62% (95%ci 51 to 73%) respectively. patients in cp1 at the time of transplant had a significantly higher 5-years os compared to those in cp2/ cp3 or ap/bp, 100% vs 37% (95%ci 17 to 58%) and 59% (95%ci 39 to 80%) respectively (p< 0.001, figure 1 ). causes of death (cod) in the cp2/3 and ap/bp groups were relapse (12/31 and 5/28 respectively) and nrm (4/31 and 4/28 respectively, 1 missing cod in each group). conclusions: post-transplant tki discontinuation appears safe in patients who receive an allo-hsct while still in first chronic phase. however, such approach in patients who transform to advanced phases before allo-hsct remains a matter of concerns given the high incidence of post allo-hsct relapse. further analysis to identify reason for restating tki post-transplant and for subsequent discontinuation will be performed after further data is collected through the ongoing data quality initiative (dqi) in cml. [ background: allogeneic stem-cell transplantation (allo-sct) is a well-established treatment modality for high-risk hematopoietic malignancies. however, the optimal intensity of myeloablation with a reduced-toxicity conditioning regimen to decrease relapse rate after allo-sct without increasing trm has not been well established. thiotepa is an alkylating compound with antineoplastic activity and immunosuppressive properties, as well as the ability to penetrate the blood brain barrier. thiotepa has become an integral part of the thiotepa, busulfan iv (busilvex), and fludarabine (tbf) conditioning regimen, which is being used with increasing frequency, particularly for haploidentical and cord-blood transplants. however, few studies have focused on analyzing the effect of thiotepa dose in the tbf conditioning. methods: the aim of this study was to evaluate the optimal dose of thiotepa, as part of the tbf conditioning for allo-sct in adults with aml transplanted in complete remission (cr), by comparing the transplantation outcomes of patients who received 5 mg/kg thiotepa and 2 days of busilvex (6.4 mg/kg) (t1b2f) versus those who received 10 mg/kg thiotepa with 2 days of busilvex (t2b2f) or 3 days of busilvex (9.6 mg/kg) (t2b3f) using a large dataset from the ebmt registry. results: we identified 639 aml patients allotransplanted between january 2009 and june 2018 from matched related or unrelated donors or t replete haplo-identical donors. 127 patients (20%) received (t1b2f); 113 patients (18%) received (t2b2f); the remaining 399 patients (62%) received (t2b3f). all the patients were in cr at transplant. median follow-up was 20 months (iqr: 9-37). outcomes are summarized in the table 1. acute gvhd grade ii-iv was 15%, 17% and 19% (p=0.14) respectively. at 2 years the non-relapse mortality (nrm) was 22%, 25% and 21% respectively (p=0.62); the relapse incidence (ri) was 18%, 19% and 17% (p=0.37) respectively; the leukemia free survival (lfs) was 60% vs 56% vs 63% (p=0.21) respectively and the overall survival (os) was 67% vs 62% vs 67% (p=0.56) in the 3 groups, respectively. the 2year grfs was 50%, 43%, and 55% respectively (p=0.02). in multivariate analysis, acute gvhd was higher for patients receiving t2b2f (p=0.01; hr 2.25) or t2b3f (p=0.02; hr 2.05) as well as for patients receiving transplant from haploidentical donor or peripheral blood stem cells, whereas nrm was higher for older patients (p=0.001; hr 1.56), patients receiving t2b3f (p=0.008; hr 2.28) or haploidentical transplant (p=0.009; hr 2.2). importantly, os was lower for older patients (p=0.001; hr 1.4) or for patients receiving t2b3f (p=0.004; hr 2.09). the multivariate analysis was adjusted to all the different factors between the 3 groups. conclusions: t2b2f is associated with higher incidence of acute gvhd compared to t1b1f whereas t2b3f associated with higher nrm, a higher incidence of acute gvhd and a lower os compared to t1b1f. with the limitation of the retrospective nature of registry data, these results suggest that a lower dose-intensity of thiotepa and busilvex in the tbf regimen in general may yield better results in aml patients transplanted in complete remission. background: thethiotepa-busulfan-fludarabine (tbf) based conditioning regimen is widely used in t-cell repleted haploidentical transplantation (haplo) with post-transplant cyclophosphamide. however, the use of anti-thymocyte globulin (atg) has not been well established. it decreases the incidence of graft versus host disease however some claim that it's at the cost of increased relapse. we conducted this multi centric study to compare the outcomes of patients who underwent haplo with tbf conditioning regimen with atg to those without. methods: this is a multicentric retrospective study. data was collected from 4 centers, the american university of beirut medical center, hospital saint antoine paris, institute paoli calmette marseille, and humanitas research hospital milan. we included all consecutive adult patients who underwent haplo with tbf conditioning. the conditioning consisted of thiotepa 5 mg/kg per day infused on days -7 and/or -6, fludarabine 30 mg/m2 infused on day -5 to day -2; and busulfan 130 mg/m2 infused on day -5 to day -3. graft versus host disease (gvhd) prophylaxis consisted of post transplantation cyclophosphamide 50 mg/ kg per day on day +3 and day +5, cyclosporine on day +6 and readjusted according to level, and mycophenolate mofetil 500 mg every 6 hours beginning on days +6 to +28 or +35 depending on the center. patients who received atg received a dose of 2.5 mg/kg per day. results: we included a total of 268 patients, 69 of whom (26%) received atg (group 2) as part of the conditioning chemotherapy. patients who received atg had a younger median age compared to the second group without atg (group 1) (53 and 58 years respectively; p value 0.004). (63% vs 61%) of each group had acute leukemia, and (71% vs 70%) were in complete remission at the time of transplant, while 47 patients (24%) in the group 1 had progressive disease at transplant. 151 patients (56.5%) had an intermediate disease risk index (dri). in the atg group, 59 patients (30%) compared to 50 (73%) in the other group received 5mg/kg thiotepa, while 140 (70) and 19 (27%) received 10mg/kg respectively. peripheral blood stem cells were the most common graft source in both groups (83% and 88% respectively). at a median follow-up of 15.4 months, patients receiving atg had a statistically significant decreased risk of acute graft versus host disease (agvhd) (rr 0.47; p value 0.031), and non-relapse mortality (nrm) at 24 months (rr 0.5; p value 0.027). atg also resulted in higher progression and overall survival at 24 months, which was not statistically significant (66.2% and 59.8%; p value 0.168, with 76.6% and 67.8%; p value 0.056 respectively). conclusions: atg as part of the pre-transplantation conditioning leads to significant reduction in agvhd and nrm at 24 months without significant effects on pfs or os. disclosure background: high-risk leukemia is associated with poor prognosis and inferior outcome. in elderly or comorbid patients allogeneic sct with myeloablativ conditioning regimen as the most effective treatment option is not available. sequential regimen combining cytoreductive therapy with ric has shown high antileukemic activity for high-risk patients with acceptable toxicity profile. this study is based on the observation that antileukemic effects have been described previously for the nucleoside analogue clofarabine. methods: the trial was designed as an investigatorinitiated prospective, multicenter, open-label, two-arm, parallel-group phase ii study comparing clarac to flamsa regimen. flamsa regimen consists of fludarabine (30 mg/ m2, days -13 to -10), amsacrine (100 mg/m2, days -13 to -10) and cytarabine (2000 mg/m2, days -13 to -10). clarac regimen consists of clofarabine (30 mg/m2, days -16 to -12) and cytarabine (1000 mg/m2, days -16 to -12). both cytoreductive therapies were combined with bu/cy (busulfan, 4 x 0.8mg/kg, days -6 to -5 and cyclophosphamide 60 mg/kg, days -4 to -3). as gvhd-prophylaxis atg, csa and mmf were used. patients with high risk aml or advanced mds (ipss ≥ int2) with contraindication for conventional conditioning or refractory to induction therapy were eligible. primary objective was to demonstrate that event-free survival is improved by clarac instead of the flamsa. secondary objectives were overall and relapse-free survival, mortality rate, safety profiles and cardiac toxicity. results: between 2011 and 2017, 62 patients were recruited, 2 patients did not meet the in-/exclusion criteria. a total of 60 were randomized with 30 patients each in the clarac and flamsa group. mean time to event was 656.6 ± 84.6 days for flamsa and 565.6 ± 49.2 days for clarac, respectively (p=0.177, figure 1 ). in total 38 of the adverse events were serious with fatal outcome of 3 patients in the clarac and 4 patients in the flamsa group. cardiac toxicity was observed in 26 patients in the clarac treatment arm, whereas 27 patients were affected in the flamsa treatment arm (p=0.730). overall survival for clarac was numerically, but not statistically inferior to flamsa (p=0.134). a part of 16/30 (53.3%) patients died until the end of the study in the clarac treatment arm, whereas only 12/30 (40.0%) died in the flamsa treatment arm (p=0.134). conclusions: this study did compare two different conditioning regimens for allogeneic stem cell recipients with high risk aml/mds. 62 patients have been included and 60 were randomized. the treatment arms were wellbalanced at study baseline for relevant covariates. superiority of the clarac treatment regimen over the flamsa regimen could not be demonstrated. consistently hazard ratios for event free survival, overall survival and relapsefree survival were in favor of the control group with flamsa treatment. no differences were found regarding cardiac toxicity, rate of engraftment, or chimerism. regarding general safety parameters, no relevant differences between the two treatment strategies were found. clinical trial registry: background: currently, there is no direct evidence to recommend specific conditioning intensities in myelofibrosis undergoing allogeneic stem cell transplantation. moreover, recent risk stratifications for diagnosed myelofibrosis (mf) included specific mutation profiles as prognostic factors. using clinical-molecular data from four different centers from germany and france, we sought to determine whether molecular genetics have an impact on outcome after reduced intensity (ric) and myeloablative conditioning (mac) stem cell transplantation in mf. methods: previously published methods were used to sequence myelofibrosis-associated genes (i.a. calr, jak2, mpl, asxl1, srsf2, ezh2, idh1/2, dnmt3a, tet2, tp53). risk stratifications according to dynamic international prognostic scoring system (dipss), mutation-enhanced system (mipss70), genetic inspired prognostic system (gipss), prognostic model for secondary myelofibrosis (mysec-pm), cytogenetics as well as other clinical and transplant-specific variables were included in analyses. cox model with hazard ratios (hr) was used for survival (os) and cumulative incidence for relapse and non-relapse mortality (nrm). risk ratios (rr) were obtained for subgroup analysis. results: the study included 361 mf patients (260 primary and 101 secondary mf) of whom 230 received ric and 131 mac. the median follow-up was 61 months in ric and 73 months in mac and the median age was 57 and 54 years. patients receiving ric were at higher risk according to dipss, mipss70, and mysec-pm, whereas frequencies of driver mutation genotype (calr, jak2, mpl, triple negative) as well as asxl1 mutation were similar. hla-mismatched unrelated donors were more frequent in ric. most common conditioning regimens for ric were bu/flu (83%) and flamsa (12%), and flu/mel (37%), treo/flu and tbi/flu (21%, respectively) for mac. no significant difference was found for ric versus mac with respect to os (62% vs 57%; p=0.35), relapse (16% vs 13%; p=0.60) or nrm (28% vs 30%; p=0.68). early relapse within five months was increased after ric (7% vs 3%). including molecular variables, ric showed higher but not significantly different os rates in patients with < 3 mutations, in triple negative driver mutation genotype, asxl1 mutation ( figure 1 ). when evaluating patients with an asxl1 mutation, those 88 patients who had only one asxl1 or one additional mutation seemed to benefit from ric showing 5-year os of 65% vs 20% for mac (p=0.001), whereas no difference was identified when more than two additional mutations were present (57% vs 65%; p=0.27) . furthermore, in patients with an asxl1 mutation and one additional driver mutation (calr, mpl, jak2), 5year os was significantly different showing 63% in ric vs 18% in mac (p=0.005). regarding current risk stratifications, ric showed significantly improved os only in high risk dipss and mysec-pm, whereas no difference was found regarding the remaining systems such as dipss-plus, mipss70 and gipss ( figure 1 ). conclusions: the evaluation of different conditioning intensities in mf did not favor ric or mac regarding currently existing risk stratifications. with respect to molecular genetics, a proportion of patients specifically harboring up to two mutations including asxl1 may benefit from ric with respect to os. [ background: sequential conditioning regimens (sr) have shown substantial activity in relapsed/refractory acute myeloid leukemia (r/raml). the original prototype sr is the flamsa-cytbi regimen. various modifications of this protocol have been developed in recent years (see table 1 ). in the current study, we compared the outcomes of patients suffering from r/raml that had received their first allogeneic stem cell transplantation (allosct) after conditioning with a sr. methods: adult patients with r/raml who had received their first allosct following sr conditioning between 2000 and 2017 and were reported to the ebmt registry were analyzed. patients were grouped according to the type of sr used as shown in table1. the flamsa-cytbi group served as comparator for all others. the primary endpoint was leukemia-free survival (lfs). secondary endpoints were overall survival (os), relapse incidence (ri), nonrelapse mortality (nrm), refined graft-versus-host-diseasefree, relapse-free survival (grfs), acute (a)gvhd and chronic (c)gvhd. multivariate analysis was done using cox regression. results: patients' characteristics are detailed in table 1 . there were more patients with transformed nhl in the beam (18%vs7%). the median time to neutrophil count > 0.5 g/l and platelet counts > 50 g/l were 10 (4 -24) and 10 (1-124) days for beeam and 12 (1-34) and 12 (9-50) days for beam, respectively. twenty-nine (22%) patients in the beeam and 35 (15%) patients in the beam groups relapsed after a median time to relapse of 6 and 9 months, respectively. after a median follow-up from transplant of 33 months, 54 (15%) patients died, 24 (18%) in the beeam and 30 (13%) in the beam groups, respectively. the main causes of death was lymphoma in 31 patients, (beeam:13, beam:18), infections in 16, (beeam:7, beam:9), secondary cancers in 2 patients (beeam). there were no significant differences between beeam and beam regimens for os: hr=1.02 [(0.55-1.86 conclusions: in this matched pair analysis, beeam and beam resulted in equivalent nrm, lfs and os suggesting that both conditioning regimens may reasonably be employed in patients with dlbcl. the higher relapse rate following beeam requires further evaluation. a prospective randomized study will be required to confirm the equivalence of the two regimens. [[o071 table] 1. background: inflammatory bowel diseases (ibd) are thought to increase the risk and severity of graft-versus-host disease (gvhd) and non-relapse mortality (nrm) after allogeneic hematopoietic stem cell transplantation (allo-hsct). thus, ibd have been included in pre-transplant comorbidity risk scores, although formal analysis of allo-hsct outcomes in patients with ibd are lacking. methods: with this background, we designed an ebmt registry retrospective case-controlled analysis to assess outcomes of allo-hsct in patients with ibd. the aim was to compare the incidence of gvhd, nrm and overall survival (os) after allo-hsct in the 2 groups of patients. each patient with ibd was matched with 3 controls according to following factors: patient sex, patient age, disease, intensity of conditioning, donor type and hla disparity, cell source and year of transplant. group comparisons were done using logrank test or gray test for competing risks outcomes. results: between 2011 and 2015, 175 patients with ibd who underwent allo-hsct for a hematologic malignancy were reported to ebmt. the cohort comprised 94 males and 81 females, with a median age of 53 years (range, 18 to 69 years) at the time of transplantation. the most frequent malignancies in the ibd group were acute leukemia (106 patients; 61%) and myelodysplastic/myeloproliferative neoplasm (42 patients; 24%). the donor was an identical sibling for 66 patients (38%), and a matched unrelated donor for 56 patients (32%). 93 patients (53%) received a myeloablative conditioning regimen while 82 patients (47%) received a reduced-intensity conditioning regimen. with a median follow-up of 37 months (range, 32-45) for the patients with ibd and 36 months (range, 33-39) for controls, the cumulative incidence of grade ii-iv acute gvhd was 27% for the patients with ibd and 28% for controls (hazard ratio (hr) for ibd versus controls, 0.95; 95% ci, 0.66 to 1.36; p=0.77). the cumulative incidence of extensive chronic gvhd at 36 months was 25% in patients with ibd and 17% in controls (hr, 1.54; 95% ci, 1.03 to 2.28; p=0.03). nrm at 36 months was 27% for the patients with ibd and 25% for controls (hr, 1.07; 95% ci, 0.76-1.52; p=0.68). the relapse incidence at 36 months was 32% in patients with ibd and 33% in patients without ibd (hr, 1.19; 95% ci, 0.94-1.49; p=0.82). os at 36 months was 47% for the patients with ibd and 49% for matched controls (hr, 1.04; 95% ci, 0.81-1.33; p=0.79). finally, gvhd-free relapse-free survival (grfs) at 36 months was 29% for the patients with ibd and 34% for matched controls (hr, 1.19; 95% ci, 0.94-1.49; p=0.14). conclusions: we report the largest matched-controlled study of allo-hsct in patients with ibd conducted so far. contrary to our expectations, we found no significant differences in acute gvhd, nrm or os between the ibd group and controls. however, ibd patients had significantly more extensive chronic gvhd than the control group. our results suggest that allo-hsct should not be contraindicated if ibd alone is considered a comorbidity. however, ibd patients have a higher risk for developing severe forms of chronic gvhd, which could considerably impair their long-term quality of life. clinical background: the easix (endothelial activation and stress index) score is associated with non-relapse mortality (nrm) and overall survival (os) after reduced intensity (ric) allohct (luft et al, lancet haematol 2017). we aimed to validate the prognostic ability of easix in a cohort of both unmodified and cd34-selected allohct. methods: between april 2008 and december 2016, 509 adult patients (pts) underwent unmodified ric or non-myeloablative (nma) allohct (n=149) with uniform gvhd prophylaxis of sirolimus/tacrolimus and low/ dose mtx or myeloablative conditioning (mac) allohct using ex vivo cd34-selection (n=360) with the clinimacs cd34 reagent system (miltenyi biotech) as gvhd prophylaxis. the easix formula (ldh*creatinine/thrombocytes) was calculated at multiple timepoints (pre-allohct, day 30, day 100 and onset of acute gvhd). a log transformation using base 2 (log2) was applied to all easix variables to reduce skew. a one unit increase in log2 easix is associated with a doubling (one-fold increase) of easix on the original scale. relapse and death or relapse, were considered competing risks for nrm and acute gvhd, respectively. results: median age was 55.6 years (19.6-78.7) and most pts were male (59%). most pts had myeloid malignancies (72%) and received mac (70%). with a median follow up of 4.8 years (0.7-10) among survivors, 1 and 3-year os were 79.9% (95% ci, 76.2-83.2) and 63.2% (95% ci, 5.7-67.3), respectively. the 1 and 3-year nrm were 13.2% (95% ci, 10.4-16.3) and 22.2% (95% ci, 18.6-25.9), respectively. the 1-year cumulative incidence of grade 1-4, 2-4 and 3-4 acute gvhd was 44.4% (95% ci, 40.0-48.7), 33.0% (95% ci, 29.0-37.1) and 10.4% (95% ci, 8.0-13.3), respectively. causes of death in 211 pts at last follow up included relapse (34%), gvhd (25%) and infection (20% higher easix score at d30, d100 and at onset of acute gvhd was significantly associated with increased risk of death and nrm (table 1 and figure 1). conclusions: higher easix scores at day 30, day 100, and at onset of acute gvhd are associated with higher nrm and inferior os with a more prominent association in calcineurin inhibitor-based unmodified allohct compared to cd34-selected allohct. endothelial damage is an important contributor to poorer outcomes after allohct and easix formula provides an easy complimentary tool to predict outcomes in these patients. background: subsequent malignant neoplasms (smns) are one of the most important complications of hematopoietic stem cell transplantation (hsct) and result in considerable morbidity and mortality. the reported rate of smns after hsct in adults ranges between 1-11% at 10-years. there is limited data on smns after pediatric, adolescent and young adult (aya) hsct, where the potential years of life gained is greater than among older adults. the objective of this study was to assess the incidence and types of smns in a cohort of survivors of childhood and aya hscts that were performed for malignant indications. methods: all hsct patients (age 0-30 years at time of transplant) who survived at least 2-years after hsct in the province of ontario, canada between 1994 and 2017 were identified from 3 transplant centers. clinical data were linked to provincial administrative databases and the ontario cancer registry that identifies cancer cases based on pathology reports and electronic patient records. results: four-hundred and forty-six 2-year allogeneic hcst survivors were included in this study. of them, 36 (8%) developed 45 smns at a median follow up of 13.1 years (range: 0.25-22.8 years). the 10-year cumulative incidence of smn was 4% (2.1-6.3%) and the 20-year cumulative incidence of smn was 18% (12.4-24.6%). several patients developed more than 1 smn. the most common smns were: papillary carcinoma of the thyroid (n=11); secondary leukemia/lymphoma (n=10); squamous cell carcinoma of the skin/oral mucosa (n=9); and adenocarcinoma of colon/lung (n=5). ten other types of smns were found including sarcoma, melanoma, nerve sheath tumor and breast cancer. nine survivors died at a median of 7.7 months after smn diagnosis. the 10-year cumulative incidence of smn for 190 acute lymphoblastic leukemia survivors who received total-body irradiation was 5% (2.4%-10.6%). conclusions: our findings corroborate the observation that children and aya who undergo allogeneic hsct are at a significant risk for developing smn. careful observation in the survivorship period is required for potential early detection. clinical 1979 and 1998 and who survived at least 20 years post-hct while continuing follow-up at our centre. we documented performance status, comorbidities, number of medications and occurrence of secondary malignancies at 20 years, as well as survival following the 20-year time-point. results: the median age of the cohort at 20 years post-hct was 56 years (range 37-77), 157 (91%) of patients underwent transplant using a related donor. eighty patients (46%) underwent hct for cml, 44 (25%) for aml, 14 (8%) for all, 12 (7%) for aplastic anemia, 23 (13%) for other indications. bone marrow grafts were used in 163 (94%) patients. myeloablative conditioning was used in 163 (94%) patients. individual comorbidities were categorized into five major groups: endocrine 79 (46%), cardiac 59 (34%), secondary cancer 46 (27%), psychosocial 39 (23%), and other organ dysfunctions 94 (54%). at the 20 year mark, median karnofsky performance status was 100 (range 30-100). no comorbidities were seen in 24 (14%) patients. the most frequent individual comorbidities were dyslipidemia (n=54, 31%), hypertension (n=54, 31%), osteoporosis (n=31, 18%) and hypothyroidism (n=28, 16%). at the time of the 20-year post-hct follow-up, the median number of medications patients were taking was 2 (range 0-25). follow-up data after the 20-year mark was available for 146 (84%) patients. median follow-up of survivors after the 20-year mark was 59 months (range 4-219 months). fiveyear overall survival of the 146 patients was 92% (95%ci 85-96%) and at 10 years was 89% (95% ci 79-94%). when grouped by age at the 20-year mark, there was no significant difference in 5-year os survival between ages 35-49 (n=48, 5-year os 91%), 50-60 (n=58, 5-year os 95%) and 61-75 (n=40, 5-year os 87%) (p=0.90). when grouped by the number of concurrent comorbidities, there was a significant difference in os between the groups with 0-1 (n=54), 2-3 (n=53) and ≥4 comorbidities (n=39) (10-year os 98%, 87% and 67% respectively, p=0.0001, figure 1 ). when grouped by the number of medications patients were on at the 20-year mark, there was a borderline significant difference between the groups on 0-1 (n=61), 2-4 (n=46) and ≥5 (n=39) different medications (10-year os 93%, 93% and 76% respectively, p=0.05). conclusions: long-term allogeneic hct recipients may develop a number of long-term comorbidities that negatively influence survival even past the 20-year mark. these findings warrant the continuous long-term medical followup of allogeneic transplant patients, regardless of age or time that has lapsed post-hct. background: no standard procedure is in use to predict sinusoidal obstruction syndrome/venoocclusive disease (sos/ vod). recently the sos/vod cibmtr clinical risk score (age, karnofsky, sirolimus, hepatitis b/c, conditioning regimen, disease type) has been established using the cibmtr database (biol blood marrow transplant. 2018; 24:2072) . the endothelial activation and stress index (easix), based on the simple formula 'ldh(u/l) x creatinine(mg/dl) / thrombocytes (10 9 /l)', has been proven to predict mortality after gvhd (lancet haematol 2017; 4:e414) . the aim of the current study was to test prediction of sos/vod by easix compared with the cibmtr score in two independent european cohorts. methods: sos/vod was defined according to the 2016 ebmt criteria. the capacity of easix and of the cibmtr score for predicting sos/vod was tested retrospectively in 556 consecutive adult patients undergoing allosct at a single institution between 2001 and 2013 (training cohort). the primary endpoint was prediction of sos/vod by the cibmtr score or by easix-d0 (easix measured at the day of allosct). results were validated in an independent cohort of 446 adult allosct recipients from another single institution transplanted between 2013 and 2015. incidence of sos/vod was assessed by uni-and multivariable cox regression analyses using age, easix and the cibmtr score as confounders. results: sos/vod was diagnosed in 35 patients (6.3%, median onset day +8) in the training cohort and in 45 patients (10.1%, median onset d +9) in the validation cohort, respectively. in the training cohort, increasing easix-d0 was significantly associated with sos/vod incidence on multivariate analysis (hr per log2 increase 1.27, 95%ci 1. 03-1.57, p=0.023) . similarly, easix-d0 predicted the incidence of sos/vod in the validation cohort (hr per log2 increase 1.35, 95% ci 1.14-1.59, p< 0.001). also, the cibmtr score showed an association with sos/vod incidence which was however significant only in the training cohort (hr per log2 increase 1.55, 95% ci 1.09-1.62, p=0.016) but not in the validation cohort, (hr per log2 increase 1.43, 95% ci 0.98-2.08, p=0.060). these results are visualized by comparing easix-d0 and cibmtr-vod scores in patients grouped according to later vod development within the observation period ( figure 1 , kruskal-wallis tests; 1a-b easix-d0 and 1c-d cibmtr risk score). the association of easixd0 with vod incidence was independent from cibmtr score. conclusions: easix-d0 is very easy to test and predicted sos/vod in two separate cohorts of allosct recipients independent of the cibmtr-vod score. patients with high easix-d0 scores might be candidates for clinical evaluation of intensified strategies to prevent sos/vod after allosct. [[o076 image] 1. figure 1 ] disclosure: the authors thank jazz pharmaceuticals for providing financial support that was used for data collection. the company had no active part in this project, did not have access to data and was not involved in analyses or writing/editing of this abstract. early hyperbilirubinaemia without sos/vod -a link between endothelial dysfunction and early mortality after allogeneic transplantation thomas luft 1 , david schult 1 , joshua majer-lauterbach 1 , sihe jiang 2 , aleksandar radujkovic 1 , peter dreger 1 , olaf penack 2 1 university hospital heidelberg, heidelberg, germany, 2 charité universitätsmedizin berlin, berlin, germany background: endothelial dysfunction is a risk factor for early mortality after allogeneic stem cell transplantation (allosct) and is linked to transplant-associated thrombotic microangiopathy (tam). similar to tam, diagnosis of sinusoidal obstruction syndrome / venoocclusive disease (sos/vod) is based on consensus criteria, and several scores have been proposed that include earlier or later stages of the diseases. early hyperbilirubinaemia occurs frequently after allosct. the pathophysiology is often elusive, and only in a subset of allosct recipients with hyperbilirubinaemia sos/vod is identified as the underlying mechanism. the aim of the current study was to explore clinical impact and pathophysiological context of early hyperbilirubinaemia without sos/vod. methods: in two independent cohorts of 864 and 446 patients, serum bilirubin levels before allosct and on days 0, 3, 5, 7, 14, 21 and 28 were retrospectively retrieved. sos/vod was defined according to the 2016 ebmt criteria. patients with at least one bilirubin value of >3mg/dl between days 0-28 were grouped as "early hyperbilirubinaemia". we assessed overall survival (os), non-relapse mortality (nrm) and time to relapse (ttr) with and without early hyperbilirubinaemia depending on coincident sos/vod, tam, and refractory acute gvhd, and we investigated the impact of statin-based endothelial cell prophylaxis (pravastatin plus ursodeoxycholic acid). serum bilirubin levels were correlated with the endothelial activation and stress index (easix, 'ldh(u/l) x creatinine(mg/dl) / thrombocytes(10 9 /l)'), measured before allosct (easix-pre) or on the day of transplantation (easix-d0). similarly, bilirubin levels were correlated with the sos/vod cibmtr clinical risk score, and with serum markers of liver damage (alanine transaminase, alt, gamma-glutamyltransferase, ggt) and endothelial cell distress markers (angiopoietin-2). results: early hyperbilirubinaemia was diagnosed in 156 (18%) and 141 (32%) patients of the training and validation cohort, and vod diagnostic criteria were met by 23% and 30% of patients with early hyperbilirubinaemia, respectively. in all patients, early hyperbilirubinaemia was associated with increased nrm and os (nrm, training: hr 2.03, 95%ci 1.47-2.80, p< 0.001; nrm validation: hr 2.12, 95%ci 1.48-3.06, p< 0.001). increased nrm in recipients with hyperbilirubinaemia without sos/vod was independent from tma or refractory acute gvhd ( figure 1 ). easix-pre and easix-d0 correlated with day 0-28 bilirubin in both cohorts. easix-pre, easix-d0 and the cibmtr-vod score predicted risk of early hyperbilirubinaemia. however, only easix predicted risk of nrm in patients without sos/vod. endothelial protection with statins and ursodeoxycholic acid was associated with reduced incidence of and reduced nrm after early hyperbilirubinaemia. furthermore, pre-transplant angiopoietin-2 correlated with early hyperbilirubinaemia, whereas alt and ggt did not. conclusions: early hyperbilirubinaemia represents a novel risk factor for nrm independent of tma and refractory acute gvhd, even in patients not meeting the diagnostic criteria for vod. the endothelial relationship of this condition is underlined by the observation that angiopoietin-2, easix-pre and easix-d0 but not markers of liver damage associate with the incidence of early hyperbilirubinaemia. therapeutic strategies aiming at normalization of endothelial dysfunction after allosct are attractive. as a first example, our data demonstrate reduced incidence of early hyperbilirubinaemia and reduced nrm thereafter in allosct recipients prophylactically treated with statins and ursodeoxycholic acid in the peri-transplant period. [[o077 image] 1. figure 1 background: accumulating evidence has suggested complement activation in transplant-associated thrombotic microangiopathy (ta-tma), mainly in the pediatric setting. to further understand its pathogenesis in adults, we hypothesized that both complement and neutrophils are activated in ta-tma as previously observed in distinct thrombotic disorders. methods: we enrolled adult ta-tma (international working group/iwg criteria), acute and/or chronic graftversus-host-disease (gvhd) and control hematopoietic cell transplantation (hct) recipients in a 1:1:1 ratio (january 2015-june 2018). complement activation was detected in patient sera and plasma with the modified ham test (mham), soluble c5b-9 and activated c3 fragments; while neutrophil activation with neutrophil extracellular traps (nets) using extracellular dna and myeloperoxidase/ mpo-dna. results: we studied 10 ta-tma, 10 gvhd and 10 control patients. ta-tma patients suffered from severe acute and/or extensive chronic gvhd. ta-tma presented at median +109 (9-930) day post-transplant. full donor chimerism was evident in all patients. no significant difference in transplant characteristics was observed among groups, except for the significantly lower gvhd rate in the control group. c5b-9 and mham levels were significantly increased in ta-tma compared to gvhd (p=0.004 and p=0.006, figure a ) and control patients (p=0.001 and p=0.005, figure b ), while no significant difference was observed in activated plasma c3 levels in the plasma. in the multivariate analysis, c5b-9 levels were an independent predictor of ta-tma diagnosis (p=0.026). we next sought to determine the cutoff value of c5b-9 that distinguishes ta-tma from other hct recipients. in the receiver-operating characteristic curve, we found a significantly high area under the curve (0.823, p=003). values higher than 321.5 ng/ml conferred a specificity of 70% with a very high sensitivity of 91.7% for ta-tma diagnosis. regarding nets, both extracellular and mpo-dna were significantly increased in patients with ta-tma compared to gvhd (p< 0.001 and p=0.042, figure c ) and control patients (p< 0.001 and p=0.029, figure d) . interestingly, increased complement activation markers (c5b-9 and mham) were strongly associated with mpo-dna (r 2 =0.461, p=0.001 and r 2 =0.403, p=0.018, respectively) and extracellular dna (r 2 =0.555, p=0.001 and r 2 =0.502, p=0.002, respectively). lastly, we studied changes of complement and neutrophil activation in 3 patients that received complement inhibition by eculizumab. despite delayed initiation in the first two patients (28 and 18 days post ta-tma diagnosis respectively), we observed laboratory response including evidence of reduced hemolysis, schistocytosis and transfusion needs. both extracellular dna and c5b-9 levels were significantly reduced post 2 doses of eculizumab (p< 0.001). however, all patients succumbed to complications of endstage renal disease and infections after a median of 3 (2) (3) (4) (5) (6) (7) (8) doses of eculizumab. conclusions: our findings demonstrate for the first time a crosstalk between complement and neutrophils in adult ta-tma. in addition, we were able to set a cut-off c5b-9 value for distinguishing complement activation in unselected patients diagnosed with the iwg criteria. although complement inhibition by eculizumab seems to hinder this pathophysiological process, further studies are needed to clarify changes and identify optimal therapeutic targets in this complex setting. [[o078 image] 1. background: hepatic vod/sos with multi-organ dysfunction (mod; typically, renal or pulmonary) may be associated with >80% mortality. defibrotide is approved for treating severe hepatic vod/sos post-hematopoietic stem cell transplantation (hsct) in patients aged >1 month in the european union, and for hepatic vod/sos with renal or pulmonary dysfunction post-hsct in the united states and canada. per prescribing guidelines, defibrotide 25 mg/kg/day (4 divided doses) is recommended for ≥21 days. this pooled analysis examined time to complete response (cr) of vod/ sos and mod symptoms relative to day of defibrotide initiation in patients who received defibrotide 25 mg/kg/day. methods: time to cr and safety data were pooled from 2 studies that included patients with vod/sos and mod post-hsct who were treated with defibrotide: a phase 3 trial (n=102) and a phase 2, randomized dose-finding trial (n=74 receiving 25 mg/kg/day). duration of therapy in patients who discontinued due to cr in an expanded-access program (t-ind) in vod/sos patients with and without mod post-hsct (n=1000) was analyzed separately due to differences in the patient population and data monitoring protocol (eg, cr data by day were not collected). vod/sos diagnosis was defined by baltimore criteria/biopsy for the phase 2 and 3 studies; in the t-ind, modified seattle criteria also was permitted. minimum recommended treatment duration was ≥14 days (phase 2) or ≥21 days (phase 3, t-ind). results: the pooled phase 2 and 3 trials had 60 patients with cr (n=34 and n=26, respectively) and 116 patients without cr (n=40 and n=76, respectively). of 60 patients with cr ( figure) in the phase 2 and 3 trials, the median time to cr was 24.5 days (range: 7-123); of these, 53.3% achieved cr after 4 or more weeks of treatment. in the t-ind, 390 patients discontinued treatment due to cr (median time to discontinuation, 22 days after initiation of defibrotide; range, 2-64), with 57 patients (14.6%) discontinuing on or after week 4 of treatment. in the phase 2 and 3 studies (n=176), 58 patients (33%) had treatment-related adverse events (traes); most common were gastrointestinal (gi) hemorrhage (4.0%), epistaxis (4.5%), hypotension (5.7%), and pulmonary alveolar hemorrhage (5.7%). in the t-ind (n=1000), 210 patients (21.0%) had ≥1 trae; most common were pulmonary hemorrhage (4.6%), gi hemorrhage (3.0%), epistaxis (2.3%), and hypotension (2.0%). conclusions: among patients with cr in the phase 2 and 3 studies, a significant number of patients achieve cr (40%) after 4 weeks of persistent treatment, highlighting the importance of continued therapy as per label indication. support: jazz pharmaceuticals figure. time to cr among 60 patients achieving cr in defibrotide phase 2 and 3 studies] trial registry: clinicaltrials.gov: nct00003966, nct00358501, and nct00628498 disclosure: paul g. richardson has served on advisory committees and as a consultant, and has received research funding from jazz pharmaceuticals. angela r. smith and leslie lehmann have nothing to disclose. nancy a. kernan received grants from gentium during the conduct of the study, and her research was supported by the national cancer institute of the national institutes of health under award number p30 ca008748; the content is solely the responsibility of the author and does not necessarily represent the official views of the national institutes of health. she has a research grant from jazz pharmaceuticals. robert ryan and william tappe are employees of jazz pharmaceuticals and hold stock and/or stock options in jazz pharmaceuticals plc. stephan a. grupp has served on a steering committee and as a consultant to jazz pharmaceuticals. sperm counts and prevalence of testosterone substitution at long-term follow-up after myeloablative allogeneic hsct in childhood background: little is known about the long-term effects of pediatric hsct on the male reproductive axis. we investigated sperm counts and prevalence of testosterone substitution twenty years after pediatric hsct and aimed at identifying risk factors for azoospermia and testosterone substitution. methods: this cross-sectional follow-up study included two national cohorts of adult males (≥ 18 years) treated with myeloablative allogeneic pediatric hsct before age 17 between 1980-2010 in denmark and finland. the study included medical history; physical examination including testicular volume and screening for chronic graft-versushost-disease (cgvhd); sex hormones and a semen sample. cumulative (pre-hsct and hsct) gonadal irradiation including tbi and cumulative cyclophosphamide equivalent doses (ced) were estimated from patient files. results: 98/181 (54%) of eligible patients (age 18-47 years) participated with a median (range) follow up time of 18 years. 76% had malignant diagnoses and 74% were treated with tbi-conditioning. 91 delivered a semen sample. results of semen and sex hormone analyses are listed in table 1. 30/98 (31%) had detectable sperm counts (0.010-524.8 million), of these 15 were treated with chemotherapy only and 15 with tbi (2 with 2 gy tbi and 13 with 10-12 gy tbi including 5 with gonadal shielding). in patients with detectable sperm in the ejaculate, increase in sperm counts was associated with time from hsct (β=6.6 million per year 95%ci ((-0.18)-13.32), p=0.056, time range 9-32 years) indicating late spermatogenic recovery. testicular irradiation was a strong risk factor for azoospermia (or=6.2 95%ci (2.0-20.7), p< 0.001) and testosterone substitution (or=5.0 95%ci (1.3-28.3), p=0.012) and no patients with cumulative testicular irradiation doses >12 gy had detectable sperms, figure 1 . pre-pubertal stage at hsct was a risk factor for later testosterone substitution (or=10.4 95%ci (1.5-453), p=0.0060). risk of testosterone substitution was associated with time from transplantation (or for +1 year 1.1 95%ci (1.0-1.2), p=0.025). cumulative ced adjusted for testicular irradiation was not a risk factor for azoospermia or testosterone substitution, nor was cgvhd. [[o080 image] 1. figure 1] conclusions: late spermatogenic recovery is possible 10-30 years following myeloablative hsct but depends on cumulative testicular irradiation dose; azoospermia was present in all patients treated with >12 gy. pre-pubertal stage at hsct increases the risk for later testosterone substitution supporting the hypothesis that pre-pubertal leydig cells are more sensitive to irradiation than more mature ones. additionally, the risk of testosterone substitution increased with time from transplantation indicating a potential early androgen insufficiency in male hsct recipients. thus, close follow-up and focus on cumulative irradiation doses are needed. disclosure background: hematopoietic stem cell transplantation (hsct) has become a standard component of therapy for several malignant indications. as hsct may improve survival for some cancers, the risk for late complications is of increasing concern. the frequency of hospitalizations can serve as a proxy measure of severe morbidity. however, knowledge regarding late hospitalizations is limited. the objectives of the study were to describe health care utilization as measured by hospitalizations beyond 2 years following transplant in survivors of pediatric/adolescent and young adult (aya) hsct for a malignant indication. methods: we linked data from 3 ontario hsct centers and provincial health care utilization data housed at institute for clinical evaluative sciences (ices) to describe all hospitalizations and their indications. we also described intensive-care unit admissions. the study population included ontario residents with cancer age 0-30 years who underwent hsct between 1992 and 2015, who survived more than 2 years from transplant (index date). hospitalizations were described from the index date until dec 2017 or death. results: the cohort consisted of 446 survivors who were followed for a median of 11.4 years from index date (interquartile range (iqr) 2.6-18.5). indications for transplant included: acute lymphoblastic leukemia (n=190, 42.6%); myeloid malignancy (n=231, 51.8%); and lymphoma (n=25, 5.6%). of these, 150 (33.6%) received a related-donor bone-marrow hsct. at the time of hsct, ages were: 0-9 (n=132, 29.6%); 10-18 (n=137, 30.7%); and 19-29 (n=177, 39.7%) years. there were 269 patients (60%) with at least 1 hospitalization beyond 2 years from hsct. there were a total of 1879 hospitalizations, resulting in a hospitalization rate of 0.43 per follow-up year. average length of hospital stay was 6.1 days. a total of 97 intensivecare unit admissions were documented among 57 (12.8%) patients. the most common indications for hospitalization were: graft-versus-host disease (gvhd) (n=486, 25.9%), relapse (n=303, 16.1%), infection (n=186, 9.8%), orthopedic procedures/fractures (n=90, 5.4%), benign neoplasm (n=96, 5.3%) and subsequent malignant neoplasm (n=83, 4.7%). among those who did not relapse, 190/320 (59%) were hospitalized. at 10 years following hsct, the proportion of patients hospitalized was 10.5%. an underlying diagnosis of acute myeloid leukemia (aml) (p=0.001) and chronic gvhd (p=0.014) were associated with increased rate of hospitalization. in the follow-up period, 73 (16.4%) patients died. conclusions: we identified a high rate of late hospitalization in pediatric/aya survivors who underwent hsct for a malignant indication, even among those without relapse. a diagnosis of aml and chronic gvhd were associated with increased risk for hospitalization. careful observation in the survivorship period is required for potential prevention of hospitalization. clinical background: engraftment syndrome (es) is a clinical complication characterized by inflammatory signs and symptoms occurring during neutrophil recovery after stem cell transplantation (sct). its incidence varies depending on the clinical criteria used. the objective of this study was to analyze the incidence, clinical characteristics, risk factors and clinical outcomes of es after haploidentical-sct with post-transplant cyclophosphamide (haplosct) in a single center. methods: 105 consecutive haplo-sct performed between 2010-2016 in our center were retrospectively reviewed. gvhd prophylaxis was performed with cyclophosphamide 50mg/kg/day days +4 and +5, mmf and csa from day +5. g-csf was started in all cases from day +5 until engraftment. 11 cases were excluded from the analysis (9 due to death before engraftment and 2 due to primary graft failure). maiolino and spitzer´s diagnostic criteria were used to define es. results: characteristics of the 94 transplant included are shown in table 1 . the es incidence was 27.6%, with median time to diagnosis of 17 days (iqr, (15) (16) (17) (18) (19) . median time to neutrophil engraftment in the es cohort was 17 days (iqr, (14) (15) (16) (17) (18) (19) (20) . fever (100%) and skin rash (77%) were the most frequent clinical findings. there were other 5 cases of fever and skin rash during the peri-engraftment period with a final diagnosis of gvhd considering clinical outcome. 18 patients (69%) received high doses of corticosteroids, with favorable response in 78% of cases. of note, 28% cases also needed intensive supportive care. there were no deaths secondary to es. univariable analysis showed a higher risk of es with use of brother/sister as cell donor (61% cases of es; p=0,003). no other risk factors were identified. no association was noted with acute or chronic gvhd. there was no significant difference in nrm, overall survival and progression-free survival between es and non-es patients. conclusions: in our experience, es is a frequent complication of haplosct with post-transplant cyclophosphamide. to our knowledge, this is the largest study including only haploidentical sct. most cases of es had a self-limited course or good response to corticosteroids, and there were no associated mortality. however es can progress to multi-organ dysfunction with need of intensive supportive care. in our analysis, incidence of es was higher with the use of sibling haplo-donors, and further studies are needed to confirm these results. we have not found relationship between es and gvhd. specific biomarkers may contribute to an early identification of this entity in order to install therapeutic measures. disclosure: nothing to declare features and outcome of early cardiac toxicity associated with post-transplant cyclophosphamide in allogeneic stem cell transplantation background: data on risk factors and incidence of early cardiac events (ece) after post-transplant cyclophosphamide (pt-cy) are scarce. thus, we compared clinical outcomes between patients who received pt-cy and patients who did not in a cohort study including all consecutive patients allografted in our center. methods: we analyzed all ece occurring within 3 months after hsct in 331 patients. transthoracic echocardiography and ekg were performed before hsct, at day +90 and in case of ece. prior to transplant, 276 patients (83.4%) had at least one cardiovascular risk factor, 72 (21.8%) cardiovascular disease history and 31 (9.4%) left ventricular systolic dysfunction (lvsd) (defined by left ventricular ejection fraction < 53%). median age was 55 years (range, 15-76) and 60.4% of patients were males. patients were transplanted for aml (n=153, 46%), all (n=49, 15%), lymphoma (n=44, 13%), multiple myeloma (n=8, 2.4%), mds (n=35, 11%) and mpn (n=42, 13%). disease risk index was high or very-high in 93 patients (28%). conditioning regimen were mac (n=131, 40%), ric (n=85, 26%) or sequential (flamsa-like) (n=115, 35%). donors were matched related (n=89, 27%), unrelated (n=124, 37%) or haploidentical (n=118, 36%). stem cell source was peripheral blood (n=310, 94%) or bone marrow (n=21, 6%). gvhd prophylaxis included cyclosporine in all patients associated with mycophenolate mofetil (n=267, 81%), short courses of methotrexate (n=23, 7%) and/or antithymocyte globulin (n=312, 94%). in the pt-cy group, 136 patients received pt-cy at 50 mg/kg/day for at least 1 day and 100 patients for 2 days, including 13 patients with unrelated donor and 6 patients with matched related donor, either because of hla-mismatch or renal insufficiency, or inclusion in a clinical trial. results: in univariate analysis, cumulative incidence of ece was 21.3% in the pt-cy group and 8.2% in the no pt-cy group (p< 0.001). the main complication was lvsd (15% of patients in the pt-cy group and 3% in the no pt-cy group, p< 0.001). other ece included acute pulmonary edema (n=13, 4%), arrhythmia (n=12, 4%), pericarditis (n=7, 2%) and coronary artery syndrome (n=3, 1%) in the whole patient group. ece resolved in 38 patients (78%). cardiovascular risk factors and the cumulative doses of anthracycline were not significantly associated with the incidence of ece. in multivariate analysis, the main factors associated with ece were the use of pt-cy [hr=2.8, 95% ci 1. after a median follow-up of 36.5 months (iqr, 32-40), the 2-year cumulative incidences of nrm, relapse, os and dfs were 28% vs. 22%; 23% vs. 19%; 56% vs. 63%; 49% vs. 59% in the pt-cy and no pt-cy groups, respectively (p values non significant). at last-follow-up, 136 patients have died. the main causes of death were disease relapse (n=51), gvhd/infection (n=48) and ece (n=8). conclusions: incidence of ece is significantly higher in the pt-cy group. in elderly patients or with a history of pretransplant cardiac event, an alternative to pt-cy should be considered to prevent ece. disclosure: nothing to declare severe iron overload, measured by liver mri at the preallo-hsct, significantly impaired the long-term outcome of the procedure methods: once approved by the clinical trials and ethics committee, a liver mri was systematically offered to patients who were admitted to undergo an allo-hsct in our center. among the 131 pts consecutively transplanted between june 2015 and july 2018, 100 pts signed the informed consent and underwent a pre-hsct mri to assess the hepatic iron load. a signal intensity ratio (sir) method was employed for the measurement. results: 64 pts were male, and 36 were female. median age was 54 years (range: 12-69). the baseline diseases were: 37 aml, 24 nhl, 16 all, 8 mds, 8 cmpd, 6 mm, and 1 bmf. 67 underwent alternative donor transplants (56 unrelated, and 11 haplo-identical), and 33 hla-id family donor transplants. stem cell source was pb in 96, and bm in 4 cases. conditioning regimen was intensive in 51 pts, and ric in 49; no non-myeloablative allo-hsct were performed. based on hepatic iron overload at pre-hsct mri, the patients were classified into the following groups: 23 pts (23%) showed severe io (lic > 80 micromol/g or 4.5 mg/g), 25 pts (25%) moderate io (lic 36-80 micromol/g or 2.1-4.4 mg/g) and 52 pts (52%) no significant io (lic < 36 micromol/g or 2.1 mg/g). as shown in table 1, majority of patients with severe iron overload had been heavily transfused, and had a high pre-hcst ferritin level. surprisingly, pre-hsct chelation had been employed only in 3 pts (13%) of this group. overall mortalities at days +100, +180 and +365 in the global series, and in severe versus non-severe io group are reflexed in table 2. conclusions: 1) our data shows that pre-allo-hsct iron-overload correlates with previous prbc transfusion load; 2) it also makes evident that io is an important risk factors for post-transplant mortality. 3) our real-life study reflects that only a minority of heavily transfused pts had received chelation therapy previously to the allo-hsct. 4) considering the relevance of pre-allo-hsct iron overload, we strongly suggest to referring physicians to employ chelation therapy for patient candidates to transplant during the treatment of the underline disease. background: neurologic complications (ncs) are associated with relevant morbidity and mortality after allo-hsct. the aim of this study was to analyze the incidence, characteristics, risk factors and outcomes of patients developing ncs after allo-hsct. methods: we evaluated 971 consecutive adult patients (>17 years) who underwent allo-hsct at our center between january 2000 and december 2016. we collected data on neurological symptoms, diagnostic methods, time of onset and cause. nc was defined as any neurological event that occurred after starting the conditioning regimen and before relapse. nc due to central nervous system (cns) infections o neoplastic infiltration were excluded. we considered both cns and peripheral nervous system (pns) complications. results: the current series comprised 467 allo-hsct from matched sibling donor (msd), 381 from umbilical cord blood (ucb), 49 matched unrelated donor (mud) and 74 haploidentical donor (haplo). median age was 41 years and most patients had acute leukemia (63%). median follow-up of surviving patients was 71 months (range, 11-213). there were differences in median follow-up according to the donor source, being longer in ucb and msd, 87 and 67 months respectively, and shorter in mud and haplo, 23 and 24 months respectively (p< 0.0001). overall, ncs were documented in 149 cases, 64 after msd transplants, 74 after ucb, 9 after mud, and 5 after haplo. cns complications (68%) were more common than pns events. the most frequent nc was encephalopathy (31%) followed by myopathy (13%) 1-year and 5-year cumulative incidence of ncs was 11% and 13%, respectively. 5-year cumulative incidence was 11% after msd, 17% after ucb, 16% after mud, and 6% after haplo transplants (p=0.014). conclusions: ncs are common and diverse after allo-hsct. ncs were more frequent in recipients allografted from alternative donors, recipients older than 40 years, and in those developing gvhd. cns complications, but not pns, are associated with poor os. disclosure: nothing to declare. abstract already published. using ciclosporin's area under the curve (auc) to predict risk of acute kidney injury in non-myeloablative haematopoietic stem cell transplantation vaidie julien 1 , jean-baptiste woillard 1 , stéphane girault 1 , pierre marquet 1 , franck saint-marcoux 1 , arnaud jaccard 1 , pascal turlure 1 background: non myeloablative allogeneic stem cell transplantation (hsct) by limiting toxicity, can be proposed to elderly patients. however, acute renal injuries related to anti-calcineurin, which are frequent in this population, can negatively impact the outcome. currently, the exposure indexes to follow and the target to use are not consensually established. however, using the area under the curve (auc) for therapeutic drug monitoring (tdm) is theoretically the best method to describe the patient's exposure. the primary objective of this study was to determine an auc target for ciclosporin associated to the occurrence of acute kidney injury in hsct patients. methods: we retrospectively studied all consecutive patients who received a non-myeloablative allogeneic stem cell transplantation at limoges university hospital from june 2009 to december 2015. patients received fludarabine 30 mg/m2/day between d-6 and d-2 before allograft and busulfan 3.2 mg/kg/day at d-4 and d-3. gvh prophylaxis consisted in rabbit anti-lymphocyte serum at the dose of 2.5 mg/kg at d-2 and d-1, and ciclosporin at the beginning dose of 3mg/kg per os twice a day. mycophenolate mofetil was added for patients with hla-matched or mismatched unrelated donors. tdm of ciclosporin was done based on trough concentration (c0) twice a week with concomitant renal evaluation using creatininemia. dose adjustments were done in according to the sfgm-tc recommendations and renal tolerance. ciclosporin's auc was evaluated at day 1, day 14 and day 28 after allograft using bayesian estimation from a limited sampling strategy and a population pharmacokinetics model previously published. the association between ciclosporin auc and acute kidney injury was investigated using a joint model. a roc curve was then constructed to investigate an auc threshold associated with the best sensibility/specificity ratio for acute kidney injury (aki . interestingly, a very low correlation was observed between ciclosporin c0 and auc (r² =0.55 for the overall period). additionally, an higher intra-individual variability was observed with c0 than auc (coefficient of variation= 36% and 26% respectively). conclusions: we report in this study that a ciclosporin auc=5.15 mg*h/l could be used as a high threshold for aki. new evaluations of auc in prospective studies are needed to better define the relevance of this marker in clinical practice. disclosure background: human cord blood (cb) provides an attractive source of hematopoietic stem cells for allogeneic transplantation of patients with a variety of diseases. a sufficient hematopoietic stem cell (hsc) dose, currently measured as cd34+ cells/kg recipient, is essential for successful engraftment. nevertheless, the frequency of true hsc within the cd34+ population and the dynamics of their clonal offspring remain poorly understood and may differ between donors. methods: here, we use cellular barcoding and multiplexed high-throughput sequencing to determine the frequency of repopulating cells among cd34+ cells from 20 individual human cb donors, and to quantify their contribution to each of the blood lineages over time in murine nod/scid/il2ry -/-(nsg) xenografts. results: in total, we detected a median of 14.3 (range 5-168) clones in blood and 15.0 (range 1.7-85) clones in bone marrow, corresponding to hspc frequencies of 1:100 to 1:1000. the number of retrieved clones correlated to barcoded cd34+ cell dose (spearman r=0.85, p=0.001), yet could vary up to four-fold between mice transplanted with the same cell dose. clonal patterns in blood early after transplant differed markedly from those at later time points, and became increasingly deterministic over time. the majority of clones displayed multilineage output, yet clones with bias towards lymphoid or myeloid lineages were also present. similar to recent data on murine hsc clones, human cb clones were distributed asymmetrically across different bone marrow sites. conclusions: in conclusion, the frequency of nsgrepopulating cells among cord blood cd34 + cells is low, and highly variable between individual cb donors. heterogeneity in hsc frequency, proliferation and lineage fate decisions may contribute to (non-)engraftment upon hsct. future research will be aimed at identifying the underlying mechanisms guiding hsc behavior upon transplantation and expanding our findings to human hsct recipients. [[o089 image] 1. background: chronic granulomatous disease (cgd) is a rare genetic immune disorder that leaves patients susceptible to life-threatening infections, chronic inflammation and often long hospital stays. x-linked cgd (x-cgd) is caused by mutations in cybb encoding the gp91phox subunit of the phagocyte nadph-oxidase, which regulates cell ph and ionic content for efficient microbial killing. allogeneic hematopoietic stem cell transplant (hsct) has been a potentially curative approach for x-cgd patients, but is often complicated by lack of hla-matched donors and risks of graft versus host disease, graft rejection, and procedure-related fatality. previous attempts at autologous ex vivo gene therapy for x-cgd using gammaretroviral vectors have met with limited efficacy due to transient engraftment of gene corrected cells, gene silencing, and mutagenic activation leading to myelodysplasia. here we report on 9 patients with a history of severe x-cgd-related complications, who received autologous ex vivo gene therapy (gt) using a novel self-inactivating lentiviral vector (g1xcgd lv) designed to limit the risk of mutagenesis through preferential expression of the missing g91phox subunit in mature myeloid cells. methods: similar trials of gt with g1xcgd lv were initiated in the uk (n=3, plus 1 compassionate use patient) and in the usa (n=5, 3 sites). all patients had histories of severe, persistent infections, and inflammatory disease. g-csf and plerixafor-mobilized cd34+ selected hematopoietic stem and progenitor cells were transduced ex vivo with g1xcgd lv. subjects received near-myeloablative conditioning with single agent busulfan, targeted to net area-under-the-curve of 70,000 ng/ml*hr. freshly prepared or cryopreserved quality-tested gene-modified cells, manufactured on-site, were administered intravenously. primary endpoints were efficacy, as determined by percent of oxidase positive granulocytes by dihydrorhodamine [dhr] flow cytometry, and safety at 12 months. results: we report results for 7 patients (2-27 years) with 1-2.5 years of follow-up; 2 additional patients were treated but died within 3 months of gt from complications deemed related to pre-existing disease-related co-morbidities (severe pulmonary disease, anti-platelet antibodies). gt was welltolerated, only one serious adverse reaction (a systemic inflammatory process at engraftment of functional neutrophils) was reported as possibly related to gt. patients experienced typical conditioning-related transient neutropenia, thrombocytopenia and mucositis. there has been no molecular evidence for clonal dysregulation or gene silencing through cpg dinucleotide methylation. followup demonstrated sustained stable persistence of 12-46% oxidase (+) neutrophils for >12 months in 6/7 surviving patients (one, who remains clinically well, had a decline to < 5% after 3 months) [ figure] . these patients have maintained restoration of biochemical function and immunity (defined as ≥10% of oxidase (+) by dhr) as of december 2018. patients have been well, without new x-cgd-related infections, and 4 are successfully weaned off prophylactic antibiotics. conclusions: these results are the first demonstration of effective autologous lentiviral gt at 12 months in severely affected x-cgd patients without evidence of genotoxicity. corrected neutrophil function has been observed in 6 patients for >12 months and has been associated with significant clinical improvement, freedom from infections, and resolution of chronic inflammation. results are supportive of extended clinical trials evaluating the safety and efficacy of g1xcgd lv-based gene therapy. updated results from the ongoing northstar-2 (hgb-207) trial of lentiglobin gene therapy in patients with transfusion-dependent β-thalassemia and non-β 0 /β 0 genotypes franco locatelli 1 , alexis thompson 2,3 , janet kwiatkowski 4,5 , suradej hongeng 6 , john porter 7 , martin sauer 8 , adrian thrasher 9 , isabelle thuret 10 , heidi elliot 11 , ge tao background: allogeneic hematopoietic stem cell (hsc) transplantation is potentially curative for patients with transfusion dependent β-thalassemia (tdt); however, it is associated with risks of morbidity and mortality and is limited by donor availability. gene therapy has the potential to be an effective treatment option for patients with tdt, but without some of these limitations. lentiglobin gene therapy contains autologous cd34+ hscs transduced ex vivo with the bb305 lentiviral vector encoding β-globin with the t87q amino-acid substitution. lentiglobin is being studied in patients with tdt and non-β 0 /β 0 genotypes in the ongoing, phase 3 northstar-2 study (hgb-207; nct02906202). methods: northstar-2 is enrolling patients with tdt who had a history of ≥100 ml/kg/year of red blood cells (rbcs) or ≥8 rbc transfusions/year. to generate drug product (dp), autologous cd34+ cells were collected by apheresis after g-csf and plerixafor mobilization and transduced with the bb305 lentiviral vector. patients received myeloablative conditioning with single-agent busulfan before dp infusion. primary endpoint was the proportion of patients achieving transfusion independence (ti, weighted average hemoglobin [hb] ≥ 9 g/dl without rbc transfusions for ≥ 12 months). patients are followed for 2 years and offered participation in a long-term followup study. statistics are presented as median (min-max). results: as of 14 september 2018, 16 patients (age 19 years; 9 patients ≥ 18 years) have been treated (follow-up 9.3 [0.7-20.4] months). patients received a cell dose of 7.7x10 6 (5.0-19.4) cd34+ cells/kg with a dp vector copy number of 3.1 (2.1-5.6) vector copies/diploid genome and 82% (53-90%) of cells were transduced. baseline liver iron content (lic) was 6.4 (1.0-41.0) mg fe/g dw. outcomes by age and baseline lic will be reported. times to neutrophil and platelet engraftment were 19 (13-32) and 44.5 (20-84) days, respectively; 1 patient (1.0month follow-up) and 4 patients (1.0-2.1 months follow-up) had not achieved neutrophil and platelet engraftment, respectively, at time of analysis. of 11 patients with ≥ 3 months of follow-up, 10 (6 ≥ 18 years old) stopped chronic rbc transfusions and two have achieved ti. at last study visit, total hb in these 10 patients was 11.1-13.3 g/dl consisting of 7.7-10.6 g/dl gene therapy-derived hb, hba t87q . one treated patient had no rbc transfusions for 11 months, then re-initiated transfusions due to low hb. non-hematologic grade ≥ 3 adverse events post-infusion in ≥ 3 patients included stomatitis, febrile neutropenia, epistaxis, pyrexia, and veno-occlusive liver disease (vod). one grade 3 event of serious prolonged thrombocytopenia after platelet engraftment was considered possibly related to lentiglobin. the three grade 4 serious vod events were attributed to myeloablative conditioning ( table 1 ). the events resulted in extended hospitalization and resolved following defibrotide treatment. there were no deaths or graft failure and no evidence of vector-mediated replication of competent lentivirus or clonal dominance. conclusions: in northstar-2, 10/11 patients with tdt and non-β 0 /β 0 genotypes treated to date produced sufficient gene therapy-derived hb, hba t87q , to stop chronic transfusions following lentiglobin gene therapy. total hb in patients off rbc transfusions remains stable at > 11 g/dl. the safety profile of lentiglobin remains generally consistent with myeloablative busulfan conditioning. background: metachromatic leukodystrophy (mld) is an ultra-rare and devastating demyelinating lysosomal storage disease caused by mutations in the arylsulfatase a (arsa) gene, currently with no approved treatment. we report an interim analysis of the safety and efficacy results of 20 early-onset mld subjects treated with experimental autologous, ex-vivo, lentiviral-mediated hematopoietic stem cell gene therapy (hsc-gt) followed for up to 8 years posttreatment, as part of an ongoing, open-label, study. the study has completed the enrollment period; follow up visits are currently on-going. methods: gt consists of a formulation of autologous cd34 + cells transduced ex vivo with a self-inactivating lentiviral vector encoding for the human arsa gene and administered intravenously after busulfan conditioning. twenty early-onset mld subjects (pre-symptomatic or early symptomatic) were enrolled and treated (9 late infantile [li] and 11 early juvenile [ej]). co-primary efficacy endpoints were improvement in gross motor function measure (gmfm) score (10%) and a significant increase in arsa activity in peripheral blood mononuclear cells (pbmcs), evaluated 24 months after treatment. safety endpoints include engraftment failure and long-term safety and tolerability of lentiviral-transduction. results: 18/20 subjects are alive after a clinical follow-up of 3-8 years. two ej subjects, treated after onset of symptoms, died due to rapid disease progression 8-and 15months post-treatment. there was no treatment-related mortality, no evidence of abnormal clonal expansion, and no adverse events related to the medicinal product. durable and stable engraftment of gene-corrected cells were observed beginning 1-month post-treatment, with persistent vector copy number in cd34 + bone marrow cells and pbmcs throughout the follow-up for all 18 subjects. reconstitution of arsa activity in the hematopoietic system was observed in both populations (li and ej), stabilizing at normal to supranormal levels within three months. arsa activity in csf showed a similar pattern; normal levels were observed 9-12 months post-treatment, demonstrating effective enzymatic production in the central nervous system (cns). the majority of li and ej subjects treated before the onset of overt symptoms showed normal motor development, stabilization of motor dysfunction or a significant delay in disease progression, as measured by gmfm total score and gross motor function classification (gmfc)-mld. cognitive function (measurements included performance and verbal iq scores) was maintained within normal range for most subjects, independent of their symptomatic status at the time of treatment. improvement or stabilization of central demyelination and peripheral nervous system (pns) abnormalities were observed in most subjects treated. conclusions: this interim analysis demonstrates that hsc-gt continues to be a safe and well-tolerated treatment for all mld subjects treated with a clinical follow-up ≤8 years. all subjects achieved high levels of multi-linage engraftment, polyclonal hematological reconstitution and central and peripheral arsa activity reconstitution within or above normal levels. patients treated prior to symptom onset achieved a sustained clinical benefit in motor and cognitive function as well as on instrumental biomarkers of pns and cns demyelination, suggesting that autologous, ex-vivo hsc-gt is a highly promising therapeutic approach for li and ej mld pre-symptomatic subjects. further research is needed to support the benefit:risk profile in ej patients. clinical trial registry: nct01560182 https://www.clinicaltrials.gov/ct2/show/nct01560182? term=nct01560182&rank=1 disclosure: the san raffaele telethon institute for gene therapy (sr-tiget) is a joint venture between telethon and ospedale san raffaele (osr). ada-scid gene therapy (strimvelis) was licensed to glaxosmithkline (gsk) in 2010 and received european marketing authorization in 2016. alessandro aiuti is the pi of the ada-scid long-term follow up clinical trial sponsored by gsk. strimvelis was licensed to orchard therapeutics (otl) in april 2018. lentiglobin gene therapy in patients with sickle cell disease: updated interim results from hgb-206 background: β-globin gene transfer may reduce or eliminate complications in patients with sickle cell disease (scd). lentiglobin gene therapy (gt) comprises drug product (dp) made from autologous hematopoietic stem cells (hscs) transduced with the bb305 lentiviral vector (lvv) encoding β-globin with an anti-sickling t87q substitution (hba t87q ). the safety and efficacy of lentiglobin gt in adults with scd is being evaluated in a phase 1 study, hgb-206 (nct02140554). patients were initially treated with dp made from bone marrow harvested (bmh) hscs (group a, fully enrolled), then from dp made from bmh hscs but using a refined manufacturing process (group b, fully enrolled), and subsequently from plerixafor mobilized hscs (group c, currently enrolling). methods: adults with severe scd (history of recurrent vaso-occlusive crisis, acute chest syndrome, stroke, or tricuspid regurgitant jet velocity of > 2.5 m/s) were enrolled. autologous cd34+ cells, collected by bmh or apheresis following mobilization with 240 μg/kg plerixafor, were transduced with bb305 lvv. after myeloablative busulfan conditioning (area under the curve goal of 5000 [range 4400 -5400] μm*min daily), patients were infused with the transduced cells and monitored for safety and efficacy. summary statistics are median (min-max). results: as of may 15, 2018, 22 patients had hscs collected, 18 patients had dp manufactured and 15 patients were treated. eleven patients (9 in group a, 2 in group b) underwent bmh and 12 patients (1 in group b [who also had bmh], 11 in group c) underwent mobilization/ apheresis. median of 4.3 (0.1-10.8) x 10 6 and 10.4 (3.8 -21.6) x 10 6 cd34+ cells/kg were collected per bmh (n=26) and per mobilization cycle (n=17), respectively. eighteen grade 3 adverse events (aes) in 6 patients were attributed to bmh and 5 grade 3 aes in 3 patients to mobilization/apheresis. dp and treatment characteristics are shown in table 1 . dp characteristics were improved in group b and group c vs group a. the safety profile post-dp infusion was consistent with myeloablative conditioning and underlying scd; most common non-hematologic grade ≥ 3 aes were stomatitis, febrile neutropenia, and vasoocclusive pain. no grade ≥ 3 dp-related aes, graft failure, veno-occlusive liver disease, replication competent lentivirus detection or clonal dominance were reported. at last visit (table 1) , hba t87q levels were higher in group b (3.2-7.2 g/dl) vs group a (0.5-1.2 g/dl). in 4 group c patients at the 3-month visit, hba t87q (4.1 [3.2-6 .0] g/dl) levels were equal to or exceeded hbs levels (3.3 [2.8-3.8 ] g/dl). in 1 group c patient at the 6-month visit, hba t87q was 8.8 g/dl and total hb was 14.2 g/dl. conclusions: these data support the safety and feasibility of plerixafor-mediated hsc collection in patients with scd. hgb-206 protocol changes have improved lentiglobin dp characteristics yielding higher hba t87q levels. additional data will determine the clinical effect of increased hba t87q /hbs ratios. background: the prognosis of most patients with chemotherapy-refractory or multiply-relapsed cd30+ (a cell membrane protein) non-hodgkin's lymphoma (nhl) or hodgkin lymphoma (hl) still remain poor. targeting cd30 with monoclonal antibodies in hl and anaplastic large cell lymphoma (alcl) was shown to induce remarkable clinical activity; however, occurrence of adverse events (mainly neuropathy) may result into treatment discontinuation in many patients. immunotherapeutic approaches targeting cd30 by chimeric antigen receptor (car) has been demonstrated to be of value in two independent clinical trials (pmid: 27582488) (pmid:28805662), although clinical benefit was sub-optimal. methods: we designed two 3 rd generation, clinical-grade retroviral vectors carrying the cassette anti-cd30 singlechain variable fragment linked via cd8 hingetransmembrane domain, to the signaling domains of two costimulatory domains, namely either cd28/4-1bb or cd28/ox40 and cd3-ζ. the inducible caspase-9 (icasp9) safety switch was also included in the constructs with the goal of promptly controlling undue toxicity. as a selectable marker, we added, in frame with the car molecule, a peptide derived from cd34 antigen. the in vitro anti-tumor efficacy was evaluated by using karpas299, l428 or hdml-2, in both short-term cytotoxic assay (represented by cr 51 release assays) and long-term co-cultures (7 days). cytokine profile upon antigen stimulation was characterized, as well as tcell exhaustion and memory marker profile. to assess the expansion, persistence, and antitumor effect of car.cd30 t cells in vivo, we used a nsg mouse model engrafted i.v. with human lymphoma cell lines (karpas299 and l428) genetically modified with ff-luciferase, this allowing the monitoring of tumor growth by ivis imaging system. persistence of car.cd30 t cells was evaluated every 15 days, together with a deep characterization of memory profile and policlonality of persisting t cells. results: independently from the costimulatory domains cd28/ox40 or cd28/4-1bb, the generated retroviral vectors showed similar transduction efficiency of t cells (86.50±5.08% and 79.30±5.33%, respectively). nevertheless, car.cd30 incorporating cd28.ox40 costimulatory domains was associated with more stable expression of the car over time, during extensive in vitro culture (84.72 ±5.30% vs 63.98±11.51% car+ t cells at 30 days after transduction; p=0.002). this finding was also associated with the evidence that car.cd30-cd28.ox40 t cells showed a superior anti-lymphoma in vitro activity as compared to car.cd30-cd28.41bb t cells, when challenged at very high tumor/effector ratio (8:1). moreover, antigen-specific stimulation was associated to high levels of th1 cytokine production, with car.cd30-cd28.ox40 t cells secreting a significantly higher amount of ifnγ (8306.03±3745.85 pg/ml), il2 (13492.68±5837.77 pg/ml) and tnfα (17661.00±11113.27 pg/ml) as compared to car.cd30-cd28.41bb t cells (6617.81±3025.67 pg/ ml, p= 0.040; 7616.67±4464.06 pg/ml, p=0.008; 5824.63 ±1823.73 pg/ml, p=0.02; respectively). in nsg mouse lymphoma models, we proved that car.cd30-cd28. ox40 t cells had an extensive superior anti-tumor control than car.cd30-cd28.41bb t cells, leading to a significant reduction of bioluminescence at day 45 (3.32x10 6 vs 2.29x10 10 , p=0.04) and an increased overall survival of the treated mice (60% vs 10%, at 180 days, p=0.0014). conclusions: overall, these data indicate that, in the context of car.cd30 t cells, the costimulatory machinery of cd28.ox40 is crucial for improving both persistence and ultimately the antitumor efficacy of the approach. disclosure: nothing to declare background: acute graft-vs-host disease (agvhd) is a serious complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). less than 50% of patients (pts) achieve sustained responses with first-line corticosteroid (cs) treatment. retrospective studies demonstrated clinical benefit with the janus kinase (jak)1/jak2 inhibitor ruxolitinib (rux) in pts with steroid-refractory (sr) agvhd. here we present 6-month follow-up data from patients enrolled in reach1 (nct02953678), a phase 2 trial evaluating rux plus cs in sr agvhd. methods: reach1 was a single-arm, open-label, multicenter study. eligible pts were aged ≥12 years and developed grade ii-iv sr agvhd following allo-hsct from any donor source for hematologic malignancies. sr agvhd was defined as gvhd that progressed after 3 days or had not improved after 7 days of primary treatment with methylprednisolone ≥2 mg/kg/d (or equivalent), development of gvhd in another organ after receiving ≥1 mg/kg/d methylprednisolone for skin or skin plus upper gastrointestinal gvhd, or inability to tolerate cs taper. pts received rux 5 mg twice daily (bid), with optional increase to 10 mg bid in the absence of cytopenias. the primary endpoint was day 28 overall response rate (orr), and the key secondary endpoint was 6-month duration of response (dor). orr was defined as the proportion of patients demonstrating a complete response (cr), very good partial response, or partial response. results: the study enrolled 71 pts. median age was 58 years, and 49.3% were male. treatment was ongoing in 11 pts (15.5%) at data cutoff (2 jul 2018) . at day 28, orr was 54.9% (cr, 26.8%). responses were observed irrespective of agvhd grade and sr criteria. best orr at any time during treatment was 73.2% (cr, 56.3%). median (range) time to response was 7 (6-49) days. median dor with minimum 6 months follow-up was 345 days for both day 28 responders ( figure 1 ) and for pts who had a best overall response at any time during treatment. four pts (5.6%) had malignancy relapse. overall, nonrelapse mortality at 6 months was 44.4%; results varied by day 28 response (day 28 responders, 21.2%; other responders, 64.1%; nonresponders, 78.9%). median overall survival had not been reached for day 28 responders. the most frequently reported hematologic treatment-emergent adverse events (teaes) were anemia (64.8%), thrombocytopenia (62.0%), and neutropenia (47.9%). cytomegalovirus infection (12.7%), sepsis (12.7%), and bacteremia (9.9%) were the most frequently reported infections. fatal rux-related teaes were sepsis and pulmonary hemorrhage (1 pt each) and were attributed to both rux and cs. conclusions: in this first prospective trial of rux in sr agvhd, orr was 54.9% by day 28 and 73.2% at any time during treatment. responses were rapid and durable. the ae profile was consistent with expectations for rux and pts with sr agvhd. rux represents a promising therapeutic strategy. background: chronic graft-versus-host disease (cgvhd) remains a major complication after allogeneic hematopoietic cell transplantation (allo-hsct). over the last decade, clinical success in patients with cgvhd has been hampered by the lack of insight into the complex pathobiological mechanisms of the disease and the paucity of specific therapeutic targets. although, it is now evident that the clinical manifestations of cgvhd are the result of a highly complex immune pathology involving both donor b cells and t cells as well as other cells. current work on immune cells involved in cgvhd pathobiology is limited by the number of parameters that conventional flow cytometry (fcm) can analyze because of cell autofluorescence and fluorescent dye spectral overlap. mass cytometry time-offlight (cytof) substitutes rare earth elements for fluorophores to label antibodies, which allows simultaneous measurements of more than 40 parameters in single cells without correction for spectral overlap, and empowers us to understand cgvhd biology at the single-cell level. methods: we used mass cytometry with extensive antibody panels to perform in-depth immune profiling of peripheral blood samples from 34 patients following allo-hsct, in which 11 patients were without cgvhd, 7 patients experienced moderate cgvhd and 16 patients experienced severe cgvhd. the involved organs in patients with cgvhd are skin, liver and lung. results: we simultaneously stained cells with 42 antibody panels created for this study. the t cell panel was designed to identify different populations of naive, memory, effector, regulatory, and exhausted t cells. the panels also included markers for the identification of b cells, natural killer cells, nkt cells, dc cells, plasma cells, granulocytes, and myeloid cells. in 4 million measured cells, we identified 40 immune cell phenotypes, in which there were 22 t cell phenotypes, 6 b cell phenotypes, 6 monocyte phenotypes and 6 granulocyte phenotypes. to generate a comprehensive view of the immune ecosystem of cgvhd, we generated two-dimensional maps of the data using the dimensionality reduction algorithm t-sne. this analysis showed a strong overlap between cgvhd of moderate and severe grades, but seperation from patients without cgvhd. seven immune compositions were identified to be cgvhd-associated. five distinct immune cells were correlated with specific cgvhd-involved organs (skin or lung), thereby presenting an in-depth human atlas of the immune cells in this disease. conclusions: this study revealed potential biomarkers and targets for immunotherapy of cgvhd and validated cytof as a valuable tool that can be used for immune profiling of cgvhd. disclosure: nothing to declare o100 t cell costimulation blockade with abatacept for acute graft-versus-host disease prevention in matched and mismatched unrelated donor transplantation: results of the first phase 2 trial background: we performed a phase 2 trial in adults and children to test abatacept for agvhd prevention ('aba2'), based on our promising preclinical and pilot patient data. methods: aba2 had 2 cohorts: a) hla mismatched ('7/ 8', n= 43), a single-arm study with pre-specified cibmtr matched analysis (vs cni+mtx or cni+mtx+atg). b) hla-matched ('8/8', n= 142) , randomized double-blind, comparing cni+mtx+placebo vs cni+mtx+aba ('aba'). abatacept dosing was 10mg/kg on d -1, +5, +14, +28. aba2 was designed as a screening phase 2 trial, with relaxed type 1 error (0.2) and standard type 2 error (0.2). power analysis assumed reduction of gr 3-4 agvhd from 30%-->10% in 7/8s and 20%-->10% in 8/8s. median follow up = 708 days (7/8s) and 369 days (8/8s) . results: reduced grade 3-4 agvhd: aba was associated with decreased d180 gr 3-4 agvhd. in 7/8s, gr 3-4 agvhd =2.5% (aba) vs 31% (cni+mtx) and 22% (+atg), (1-sided p =0.001, 0.005). in 8/8s, gr 3-4 agvhd =6.85 % in aba vs 14.6% in placebo, (1-sided p =0.068). reduced grade 2-4 agvhd in 8/8s: aba was associated with decreased d180 gr 2-4 agvhd. in 7/8s, gr 2-4 agvhd =42% (aba) vs 54% (cni+mtx) and 45% (+atg, 1-sided p =0.098, 0.25). in 8/8s, gr 2-4 agvhd =44.5% in aba vs 62.3% in placebo (1-sided p =0.004). chronic gvhd: for 7/8s, 1 yr cgvhd =38.8% (aba) vs 43.5% (cni+mtx) and 35.5% (+atg, p =0.4, 0.99). in 8/8s, data collection is ongoing. safety indicators: there was no difference in neutrophil or platelet engraftment, cmv and ebv reactivation between aba and controls. cumulative incidence of relapse in 7/8s at 2 yr 9.4% (aba) vs 20.6% (cni+mtx) and 23.4% (+atg) (p=0.115, 0.085). in 8/8s, at 1 yr, it was 13.8% (aba) vs 20.5% (placebo, p =0.7). statistically significant survival advantage in 7/8s: for 7/8s, 1 yr non-relapse mortality (nrm) = 10.5% (aba) vs 32.7% (cni+mtx) and 26% (+atg, p =0.024, 0.365). for 8/8s, nrm = 7.1% vs 14.6% at 1 yr (p =0.5). severe agvhd free survival at 6 months for 7/ 8s =97% (aba) vs 55% (cni+mtx) and 59% (+atg, p =0.001, 0.006). for 8/8s = 89.0% (aba) vs 76.8% (placebo, p = 0.049). for 7/8s, relapse-free survival (rfs) = 73.7% (aba) vs 38.7% in cni+mtx and 48.7% in +atg (p =0.001, 0.027). for 8/8s, rfs = 79.1% for aba vs 64.9% (placebo, p =0.38). for 7/8s, overall survival (os) =71% (aba) vs 47.5% (cni+mtx) and 58% (+atg, p =0.01, 0.145). for 8/8s, os =83.2% (aba) vs 76.6 (placebo, p =0.32). conclusions: our results suggest that short-course aba can safely prevent agvhd without compromising relapse. despite the modestly sized study, the comparative event size for 7/8s was high enough that the protective effect of aba against gr3-4 agvhd was highly significant. for 8/ 8s, there was a statistically significant improvement for gr 2-4 gvhd and a trend toward an advantage in all parameters. for both cohorts, severe agvhd free survival was statistically-significantly improved. these results are the first to demonstrate efficacy of in vivo t cell costimulation blockade in preventing agvhd. background: in the nih cgvhd diagnostic classification, patients with gvhd after 3 months are classified as either late agvhd or cgvhd. to date, this is only a clinical classification, with no biological differences identified. recently, the pbmtc 1202/ applied biomarker in late effects of children and adolescent (able) study completed accrual of 302 pediatric allogeneic hematopoietic cell transplantation (hct) patients. we used day 100 biomarkers to identify biological differences between cgvhd and late agvhd. methods: the pbmtc1202/able study with 26 centers in canada, us, and europe prospectively collected peripheral blood samples at 3, 6, 12 month post hct and at the onset of cgvhd in 302 children. a comprehensive analysis for previously identified cgvhd immune cell markers by flow and cytokines by elisa on plasma and streck tubes shipped overnight and centrally evaluated at bc children's hospital. clinical data was collected centrally with a thorough central clinical adjudication by the pbmtc study committee. of those enrolled, 228 were evaluable at day 100 and classified as a) late agvhd (n = 58), b) cgvhd (n = 44), and c) controls that did not develop cgvhd (n = 132). univariate analysis was performed comparing late agvhd, cgvhd, and no gvhd controls. significant differences were defined as a biomarker with both a roc auc ≥0.60 and p value ≤0.05 compared to controls. results: the profile of cgvhd included a cluster of abnormalities in memory and transitional b cells, conventional naïve and follicular helper t cells, and a loss of both recent thymic emigrant regulatory t cells and cd56 bright nk regulatory cells. four inflammatory cytokines, st2, aminopeptidase n, cxcl9 and mmp3 (see table 1 ) were increased. patients clinically identified as late agvhd had a more restricted biomarker pattern of limited b cell abnormalities and st2. conclusions: late acute gvhd is limited to restricted b cell and elevation of st2. cgvhd is characterized by the identical b cell abnormalities but with the additional loss of regulatory function in cd56bright nkregs and rte treg cells. with the loss of regulatory function in cgvhd, there is an increase in cd21 lo b cells, follicular t helper cells, and additional cytokines. these prognostic markers findings may suggest therapeutic targets that differ for late agvhd compared to cgvhd. background: we are reporting the outcome of 69 patients with steroid refractory acute graft versus host disease (sr-gvhd), treated with an anti-cd26 monoclonal antibody (begelomab r ). methods: twenty-eight patients were enrolled in two pilot studies eudract no. 2007-005809-21 and no. 2012-001353-19 , whereas 41 patient were treated on a multicenter follow up compassionate use of the antibody. the median age of the patients was respectively 42 and 44 years. at the time of anti-cd26 treatment, gvhd was overall recorded as grade ii in 8 patients, grade iii in 33 and grade iv in 28 patients. in the pilot sudies patients had failed 1 line of treatment, wheas in the follow up compassionate use, patients had failed one line (n=18), two lines (n=11), three lines (n=11) or 4 lines of treatment (n=1). results: there were no adverse events attributable to the antibody. day 28 response was recorded in 75% and 63% in the pilot studies and follow up patients. response in grade ii gvhd was evaluable only in the pilot studies (57%); response in grade iii gvhd was recorded in 80% and 83% patients in the two groups; response in grade iv gvhd was recorded in 66% and 56% of patients in the two groups. overall there were 60% responses for skin and liver stage 3-4, and 70% responses for gut stage 3-4 gvhd. the cumulative incidence of non relapse mortality (nrm) at 6 months was 28% and 38%. for day 28 responders, this figure was 19% and 22%, for non responders it was 57% and 66% in the two groups. the overall survival at 1 year was 50% for the pilot studies and 33% for the follow up patients. conclusions: in conclusion, begelomab induces a high remission rate on day+28 in patients with sr-gvhd, including a significant proportion of patients wih severe gut and liver gvhd. clinical background: cgvhd is characterized by an imbalance between effector and regulatory arms of the immune system that results in overproduction of inflammatory cytokines including il-17 and il-21. moreover, a persistent reduction in the number of regulatory t (treg) cells limits the ability of the immune system to recalibrate this pro-inflammatory environment. kd025 is an orally available rho-associated coiled-coil kinase 2 (rock2) selective inhibitor. in vitro data suggest that kd025 modulates immune homeostasis by shifting the th17/treg balance towards a treg phenotype. methods: kd025-208 is an open-label phase 2a study in patients with steroid-dependent cgvhd after no more than 3 prior lines of treatment. three cohorts (c1: 200mg qd (n=17), c2: 200mg bid (n=16), and c3: 400mg qd (n=21)) were enrolled. the primary endpoint is overall response rate (orr), defined per the 2014 nih consensus criteria. results: as of 13-september-2018, the median duration of treatment was 37, 33 and 27 weeks for c1, c2 and c3, respectively. the median age was 52 years (range 20-75) and median time from cgvhd diagnosis to kd025 treatment was 19 months. 67% of patients had received ≥2 prior lines of therapy and 48% had ≥4 organs involved at baseline. 20 patients remain on treatment with kd025, with median duration of treatment of 89 weeks (n=6), 68 weeks (n=3) and 34 weeks (n=11) for each cohort, respectively. the orr was 65% in c1, 63% in c2 and 52% in c3. responses were rapid, with 75% of responders achieving a response at the first assessment (8 weeks). among responders, 82%, 50% and 36% have sustained responses for ≥20 weeks in each cohort, respectively. responses were observed across all affected organ systems, including crs in upper gi, lower gi, esophagus, mouth, skin, joints/fascia, eyes, and liver. two patients with lung cgvhd achieved pr. 69% of patients achieved reductions in corticosteroid dose and 7 patients discontinued corticosteroid treatment while receiving kd025. 72% of responders achieved a clinically meaningful improvement (≥7-point reduction) in the lee symptom scale (lss) score. kd025 has been well tolerated. commonly reported aes (≥20% patients) were urti, ast/alt elevations, fatigue, nausea and diarrhea. grade ≥3 aes occurring in >3 patients were ggt elevations (n=6) and hyperglycemia (n=4). no saes were considered related to study drug. two patients discontinued treatment due to aes considered possibly related to kd025 (headache, diarrhea). three fatal events occurred (relapse of leukemia; lung infection; cardiac arrest); none were considered related to kd025. no increase in incidence of infection was observed. consistent with the postulated kd025 mechanism of action, th17 cells decreased and treg cells increased in patients receiving kd025 background: antithymocyte globulin (atg) treatment significantly decreases later development of chronic graftversus-host disease (cgvhd). one phase 3 trial evaluating atg was the canadian bmt group (cbmtg) 0801 study that found that atg treatment resulted in significantly less cgvhd and dependence on immunosuppressive treatment at 1 year. the exact mechanism by which atg decreases cgvhd is not known. we hypothesized that using known prognostic day 100 cgvhd biomarkers in the wellcontrolled cbmtg 0801 trial represented an optimal approach to understand atg's biological impact on cgvhd in humans. methods: a separately developed cbmtg cgvhd biomarker study opened while cbmtg 0801 was ongoing and accrued 40 patients (n = 25; atg treated and n = 15 controls) of the 203 cbmtg 0801 patients. samples were collected at 3, 6, and 12 months and at the onset of cgvhd and evaluated at bc children's' hospital research institute, vancouver, bc. patients were evaluated for day 100 immune cellular markers previously associated with later cgvhd including: a) naive helper t (th) cells, b) recent thymic emigrant (rte) th cells; c) cd21 low b cells; d) cd56 bright nkreg cells; and e) treg cells. frequencies in the atg treated and control group were evaluated to detect significant difference using non-parametric t-test mann-whitney test. results: patients of this subpopulation (aged 16-70 years) were shown to be representative of the larger cbmtg 0801 study population. the atg treated group had a significant decrease in total t cells, rte th cells, and naïve th cells at day 100 compared to the control population (p < 0.0001 each population -see table 1 ). atg treatment had no impact on tregcells, cd19+ b cells, and cd21lowb cells but there was a significant increase in cd56bright nkreg cells (p < 0.0001). we evaluated the ratio of naïve th effector cells to the regulatory nkregcell population and saw a >100 fold difference the atg treated group (naïve th cell:nkregcell ratio = 0.06) compared to untreated patients (ratio of 9.2; p < 0.0001). in this small population we found that the naïve th cell:nkregcell ratio was also high prognostic for later development of cgvhd (1.37 vs. 0.13; p < 0.0001). conclusions: these results suggest that atg's major mechanism of action is related to its ability to simultaneously inhibit naïve th cells and enhance cd56 bright nkreg cells after transplantation. while these results require confirmation, they support strategies that target the ratio of nk reg cell and naive cd4+ t cells to modulate cgvhd. clinical trial registry: nct01217723 disclosure: none of the authors have any conflicts of interest to declare o105 immune reconstitution -based score at diagnosis of cgvhd predicts gvhd severity and overall-survival: a novel prognostication tool for gvhd treatment tailoring background: allogeneic stem cell transplantation (hsct) survivors are at a relevant risk of developing chronic gvhd (cgvhd), which importantly affects quality of life and increases morbidity and mortality. early identification of patients at risk of development of severe cgvhd related morbidity would be a relevant tool to tailor preventive strategies. we have previously demonstrated the role of immune reconstitution (ir) as predictive biomarker of occurrence of cgvhd. the aim of this study was to evaluate the prognostication power of ir at cgvhd onset through a new ir-based score. methods: we analyzed clinical data from 383 adult patients consecutively undergoing first allogeneic hsct transplant between january 2011 and december 2016 at our institution. a written consent was given for the use of medical records for research. patients were divided into a test cohort (307 pts) and a validation cohort (76 pts). median follow-up for surviving patients was 4 years.we built a cox multivariate models for os in patients with cgvhd of any severity. variables included in the models were: patient age (according to median value), r-dri score, type of donor (matched related donor vs matched unrelated vs haploidentical), main gvhd prophylaxis platform (atg-based vs ptcy-based vs neither of the two), ir values (cd4, cd19, nk, iga, igm according to median values) at cgvhd diagnosis, history of prior agvhd, karnofsky ps, plt < 100.000/μl, alc< 1000/μl, eos < 500/μl.once we identified the variables independently predicting os by multivariate analysis, we derived a formula for a prognostic risk index by using the β coefficients found in the model. each patient was then assigned a score and we defined three groups of os risk (low, intermediate and high) by dividing the score into three classes using the first and third quartiles. finally, to evaluate predictive performance of the ir-score we calculated the receiver operating characteristics (roc) curve via the area under the curve (auc), to summarize the ir-score ability to correctly classify events and non-events. results: 115 patients (87 test-cohort, 28 validationcohort) were evaluated for cgvhd and outcome. our multivariate model defined the variables independently predicting os at cgvhd onset: cd4+ count >233/ mcl, nk count < 115/mcl, igm < 0.45 g/l, karnofsky ps < 80%. final score was calculated as follows: 2,4 (if cd4 >233/ mcl) + 2,1 (if nk < 115/mcl) + 2,1 (if igm < 0,45) + 4,3 (if karnofsky < 80). low risk patients were defined as having a score ≤2.4, intermediate >2,4 and ≤4.5, high risk >4.5. the 3y-os for low risk patients was 96%, for intermediate 76% and for high risk 27% in the test-cohort and 100%, 66% and 35% in the validation-cohort ( figure 1a-b) . the roc curve analysis supports the validity of the ir-score in our cohort of patients -auc 85.5%, with 95% confidence intervals higher than 50%. furthermore ir-score was able to stratify across nih-severity classification (figure 1c). conclusions: immune-reconstitution score at diagnosis of cgvhd predicts gvhd severity and overall-survival. irscore could be adopted to identify patients at high risk and modulate cgvhd treatments accordingly. disclosure: chiara bonini has research contract with intellia therapeutics. the other authors declare that they have no conflicts of interest. haploidentical transplantation with sirolimus-based gvhd prophylaxis and unmanipulated pbsc graft: background: haploidentical transplantation has emerged as a viable option for patients lacking a fully matched donor. we firstly explored the association of sirolimus and atg, later followed by sirolimus with pt-cy as gvhd strategy. herein, we describe long-term outcomes of haploidentical hsct using sirolimus-based gvhd prophylaxis. methods all patients received sirolimus and mmf as gvhd backbone prophylaxis plus atg in 203 patients, and pt-cy in 151. conditioning regimen was based on treosulfanfludarabine; recipients of pt-cy transplants were more likely to receive a regimen intensified by a 2 nd alkylating agent (melphalan or thiotepa). median follow up was longer in atg group (70 vs 26 monhs, p< 0.01). there were no differences in dri. results: the majority of patients reached the neutrophil (89% in atg group vs 86% in pt-cy group) and platelet (76% vs 77%) engraftment within 30 days after hsct. immune-reconstitution was broad and fast, reaching more than 100/ml cd3+ t cells within a median of 35 vs 33 days. the two groups were similar in terms of survival and main transplant outcomes. in the atg group, the cumulative incidence of grades ii-iv and iii-iv acute gvhd at 100 days was 26% and 20%. corresponding rates after pt-cy were 35% and 20%. the cumulative incidence of overall and severe chronic gvhd was 31% and 10% at 3 years in atg group and 42% and 16% after pt-cy .the cumulative incidences of relapse and nrm in atg group were respectively 41% and 31% at 3 years. corresponding rates after pt-cy were 35% and 27%. in atg group, 3-year os was 36%, while grfs was 24%. the corresponding probabilities after pt-cy were 44% and 24%. the only difference reported was a better pfs in favour of pt-cy (38% vs 29%, p=0.04 conclusions: extended follow-up in 354 patients confirms sirolimus-based gvhd prophylaxis as feasible and safe in haploidentical hsct based on unmanipulated pbsc graft. both atg and ptcy association to sirolimus provide an effective prevention of gvhd and translate into a similar long-term overall survival. a significant advantage of sir-pt-cy on relapse rate warrants further investigation. background: steroid-refractory graft-versus-host disease (sr-gvhd) is still responsible for high mortality in patients undergoing allogeneic stem cell transplantation; a number of agents is currently available in case of steroid-refractoriness, yet there is so far no consensus about a standard second-line treatment and overall survival (os) remains poor.α1-antitripsyn (αat) is a circulating 52-kda serine protease inhibitor found to enhance the production of anti-inflammatory cytokines and to favor the expansion of regulatory t-cells; it has therefore been tested in situations of altered tolerance and disproportionate inflammation, including gvhd. two studies showed that treatment of sr acute gvhd with αat is feasible and effective. methods: we retrospectively analyzed a series of patients who received exogenous αat for sr acute gastrointestinal gvhd or overlap gvhd with acute gut features. sr-gvhd was defined and graded according to standard criteria. αat was administered intravenously at a loading dose of 90 mg/kg at day 1 followed by 30 mg/kg /day every other day for a total of 8 doses. response to treatment was defined according to published criteria; os was estimated with the kaplan-meier method.a panel of 46 cytokines and immune cell subsets were measured before treatment and once weekly during treatment by a luminex assay and by flow cytometry, respectively. results: sixteen patients were treated for gut gvhd between september 2016 and march 2018. median age was 50 years (range 18-56). αat was administered at a median time of 104 days from transplantation (range 38-215) and of 65 days from gvhd onset (range 12-198) . acute gvhd was scored as grade ii in 28% of patients, grade iii in 66%, grade iv in 6%. sixty-seven percent of patients had already received one or more lines of treatment other than steroids, including ruxolitinib, etanercept, atg; orr was 44% with a cr rate of 27%; median time to best response was 21 days (range 6-26), with a continued orr at day 56 of 39%. the overall rate of gastrointestinal responses was 61%. median follow-up of living patients was 440 days (range 84-602); median os was 138 days and 1-year os was 48% (95% ci 26% -74%).the most common infectious event was cmv reactivation (29%); 2 grade 3-4 infectious complications were recorded. there was no quantitative deficiency of blood aat levels before treatment (1.72 g/l ± 0.46); blood αat rose significantly during and after treatment. baseline αat level didn´t differ between responding and nonresponding patients. a cytokine profile was evaluable in 12 patients; no statistically significant increase or decrease in cytokine plasma concentration after αat infusion was observed. surprisingly, a decrease in circulating t regs after exposure was found (p=0.002), regardless of patients´responding status. conclusions: treatment with αat was safe and effective in a cohort of sr-agvhd high risk, pre-treated patients and should be considered as a possible alternative. changes in the cytokine milieu and t-cell subsets shown in murine models were not observed in a real-life setting. table 1 . peripheral blood was used as graft source in 87% of the patients in the atg group and in 78% in the pt-cy group. gvhd prophylaxis consisted in atg 2mg/m 2 days -4 to -2, mtx days +1, +3, +6 and +11, and csa from day -1 in the atg group. the pt-cy group received cyclophosphamide 50 mg/kg/d on days +3 and +4, followed by either csa or tacrolimus and mycophenolate mofetil (mmf) from day +5 in 30 patients (42%), cyclophosphamide on days +3 and +5 combined with csa or tacrolimus from day 0 in 26 patients (36%), or cyclophosphamide on days +3 and +5 combined with tacrolimus and sirolimus from day +5 in 16 patients (22%). cumulative incidence at 100 days of grade ii-iv (67% vs 46%, p=0.02) and iii-iv (41% vs 3%, p=0.002) acute gvhd, were significantly higher in the mtx-csa group ( figure 1a ). there were no differences in the 2-year cumulative incidence of chronic moderate to severe gvhd between the atg and the pt-cy group (30% vs 24%, p=0.6). after a median follow-up of 90 months for the atg group and 26 months for the pt-cy group, 2-year overall survival (os) was higher in the pt-cy group (45% vs 60%) although not statistically significant (p=0.09) ( figure 1b) . we found no differences between both cohorts in 2-year event-free survival (efs) (46% and 50%, p=0.55) and the composite endpoint of gvhd-free and relapse-free survival (gfrs) (38% vs 40%, p=0.253). the 2-year cumulative incidence of relapse was significantly higher in the pt-cy group (26% vs 6.6%, p=0.04) and non-relapse mortality (nrm) at 2-years was higher in the atg group (40% vs 22%) but not statistically significant (p=0.06). conclusions: in our experience, in spite of the limited number of patients, gvhd prophylaxis using pt-cy combined with additional immunosuppression after mud hsct, using mostly peripheral blood as graft source, reduced the cumulative incidence of agvhd compared to standard prophylaxis with mtx-csa. prospective studies with longer follow-up are needed to confirm these observations. disclosure: nothing to declare o109 abstract already published. methods: 44 consecutive patients who underwent allo-sct for hematological malignancies between january 2016 and august 2018 were included in this prospective singlecentre protocol. all patients had at least one baseline risk factor predicting development of severe gvhd (e.g. hla mismatch, fem-to-male sex mismatch). pt-cy in combination with a second immunosuppressive drug was used as gvhd prophylaxis. results: patients characteristics are summarized in table 1 . median age was 56 (range, 19-67) years, with 6 male patients (14%) receiving a graft from a female donor. more frequent allo-sct indications were acute leukemia and mds (54.5%) followed by nhl (18%). eleven patients (25%) were transplanted in advanced status. donor was a sibling, matched unrelated or mismatched unrelated in 34%, 25% and 41% respectively. seven patients (16%) received a myeloablative conditioning regimen including tbi (8 or 13.5 gy) while the remaining (84%) received a ric/rtc regimen based on fludarabine, busulfan or melphalan +/-thiotepa (3mg/kg). median follow-up for survivors was 365 days (range:64-959). median time to neutrophil and platelet engrafment were +23 (12-36) and +22 (10-50) days, respectively (g-csf not routinely used). early toxicity was low, without cases of thrombotic microangiopathy, only 2 cases of drug-related renal failure (4.5%) and 1 case of possible vod. before the introduction of mini-thiotepa in the ric protocols (flubu/ flumel) there was 1 case of primary graft failure (gf) and 5 cases of late graft failure (6/28; 21%); 4 cases were successfully regrafted with the mini-thiotepa ric. there have been no further cases of gf after its introduction (n=8 evaluable cases + 4 second salvage allorics). additional potential risk factors for gf were cd34+ ≤3x10e6/kg (p=0.07) and a high lymphocyte count at stem cell infusion (p=0.06). the ci of grade 2-4 acute gvhd at day + 120 was 28% (95%ci: 14-48) with only 2 cases of refractoriness to steroids. of the 37 evaluable patients, only 1 developed moderate chronic gvhd leading to a 1yr-ci of 3%. nrm was 10.5% at 1yr and the ci of relapse was 31% (95% ci: 21-40). all relapses occurred in patients with intermediate/ high rdri. 1yr-os was 76% and the estimated 1-year dfs was 63.6%. conclusions: these outcomes confirmed the feasibility of both ric and mac allo-sct using pt-cy as soc with a single immunosuppressive drug in patients at high risk of gvhd. an important observation was the high rate of gf with "classical" alloric platforms (flubu/flumel), which appears to be lower after introducing the mini-thiotepa. although these data need confirmation in larger cohorts, the current results suggests that pt-cy may pave the way to improving the quality of life of transplant survivors by markedly reducing severe gvhd. protection of the endothelium during steroid-refractory gvhd background: clinical data demonstrated that endothelium related factors predict mortality after the diagnosis of agvhd, suggesting that the endothelium may be involved in the pathobiology of steroid-refractory agvhd (sr-agvhd) (j clin oncol. 2018 mar 10;36 (8) methods: intestinal biopsies from patients after allosct. murine agvhd models balb/c→b6, b6→bdf and lp/ j→b6 with and without steroid treatment. immunostaining, electron microscopy, light sheet fluorescence microscopy (lsfm), facs. in vivo and in vitro assays for endothelial dysfunction. treatment with phosphodiesterase type 5 inhibitor (pde5) in sr-agvhd models. results: we found a significant higher percentage of apoptotic vessels in duodenal and colonic mucosa biopsies of patients with grade iii-iv agvhd compared to no gvhd ( figure 1a ). in murine experimental agvhd, we detected severe microstructural endothelial damage and reduced endothelial pericyte coverage accompanied by reduced expression of endothelial tight junction proteins leading to increased endothelial leakage in agvhd target organs. during intestinal agvhd, colonic vasculature structurally changed, reflected by increased vessel branching and vessel diameter ( figure 1b) . we analysed human biopsies and murine tissues from sr-gvhd vs. naive (untreated) agvhd and found significantly lower lymphocyte infiltration in sr-gvhd, demonstrating low inflammatory activity ( figure 1c ). our findings suggest that endothelium-related and t cell independent mechanisms play a previously unrecognized role during sr-agvhd, providing the rationale for t-cell independent treatment strategies. as a first example for such an approach, we tested the endothelium-effective pde5 inhibitor sildenafil and found reduced apoptosis as well as improved metabolic activity of endothelial cells in vitro. in accordance, sildenafil treatment resulted in improved survival and reduced target organ damage during experimental sr-agvhd ( figure 1d ). conclusions: we show profound endothelial involvement after allo-hsct and demonstrate that endothelialprotection with sildenafil ameliorates sr-agvhd, providing a novel non-immunosuppressive treatment approach. these results can serve as rationale for translational development of endothelium-based therapies for sr-agvhd. disclosure: the authors declare no confilct of interest relevant to this study o113 abstract already published. the therapeutic effect of immune-modifying microparticles in an acute graft-versus-host disease model john galvin 1 , sara beddow 2 , stephen miller 2 1 university of illinois chicago, chicago, il, united states, 2 northwestern university, chicago, il, united states background: inflammatory monocytes are recruited to target organs during acute graft versus host disease (agvhd). as seen in other autoimmune disorders, inflammatory monocytes play an important role in antigen presentation and cytokine production. these actions allow for a sustained activation and proliferation signal to t-cells. previous studies have shown that imp treatment in mouse models of colitis, encephalitis, myocardial infarction and peritonitis markedly reduced monocyte accumulation in the affected end-organs --promoting tissue repair; reducing disease symptoms and increasing survival. therefore, our objective was to test clinical outcomes after imp treatment in a mouse model of agvhd. methods: murine agvhd model: balb/c mice were given 800 cgy total body irradiation, irradiated balb/c mice were transplanted with 5×10 6 c57bl/6 bone marrow cells and 1×10 6 c57bl/6 spleen cells via tail vein.imp treatment: imps were made with plga (phosphorex inc, hopkinton ma) was administered to the recipient mice (1.4 mg/kg body weight) by iv daily starting from day 5 to day 10 after bone marrow transplantation (bmt). pbs at the same volume was used as vehicle control. in vivo bioluminescence imaging: mice were given an intraperitoneal injection of luciferin (150 mg/kg body weight) and then anesthetized and imaged using the ivis imaging system (xenogen). imaging data were analyzed and quantified with living image software (xenogen). results: imp treated mice had significantly less severe acute gvhd symptoms (average score of 2.48) than the untreated bm+sp group (average score 3.96) starting at the time of imp treatment (days 5-10) and remained with significantly reduced symptoms for the 30 day course (figure 1 ). imp treatment also rescued bm+sp mice from agvhd associated mortality with a 30-day overall survival of 62% compared to 4% in the untreated bm+sp group (figure 2 ). intestinal tissue from the imp treated mice compared to the bm+sp mice demonstrated less evidence of agvhd (an average score of 1.25 and 2.75, respectively). hepatic tissue from the imp treated mice compared to the bm+sp mice demonstrated less evidence of agvhd (an average score of 1.5 and 2.42, respectively). imp treatment also significantly reduced inf-γ levels in the intestinal tissues of treated mice compared to untreated bm +sp mice. in the mice infused with lymphoma cells (a20-luc), imp treatment reduced agvhd symptoms and death while preserving the gvl effect. conclusions: our results demonstrate that imps significantly reduce symptoms and mortality in a murine model of agvhd while preserving gvl. the reduction in inflammatory monocytes with imps leads to a reduction in inflammatory cytokines, hepatic lymphocyte infiltration and intestinal mucosal denudation. these findings highlight the potential of imp therapy as a specific and potentially safe treatment in acute gvhd. [[o114 image] 1. figure 1 background: despite significant improvements in the supportive sickle cell disease (scd) causes substantial morbidity and mortality. allogeneic hematopoietic stem cell transplantation (hsct) is currently the only curative option but is only offered if a matched sibling donor (msd) is available. with a msd availability of < 20% t cell depleted hsct from a haploidentical donor (t-haplo-hsct) is a potential alternative. methods: 29 patients (pts) with advanced stage scd (asscd) were transplanted with a cd3 + /cd19 + or αβ/cd19 + depleted t-haplo-hsct (20 pts, median age 13 years, range 3-31 years) or with bone marrow (bm) from a msd, 9 pts, median age 14, range 9-25 years). indication for hsct was asscd with multiple scd related complications. all pts underwent exchange transfusion before hsct. in all pts the conditioning regimen consisted of treosulfan, thiotepa, fludarabine and atg. immunosuppression was carried out with cyclosporine a or tacrolimus and mycophenolate mofetil. the control group received a msd bm allograft. results: in the t-haplo-sct group the pts received a cd3 + or αβ + t-and cd19 + b-cell depleted peripheral stem cell allograft with 13,1 x 10 6 cd34+ cells/kg body weight (range 8,1 to 77,9) . all pts with a median follow up of 22 months (range 4-57 months) in the msd group and 18 from 20 pts with a median follow up of 17 months (range 5-58 months) in the t-haplo-sct group are alive. engraftment was achieved in all pts with stable chimerism over 90%, except for 4 pts with a stable mc in the t-haplo sct group and 1 patient in the msd group, but complete engraftment of red cell precursor in the bm. all pts are off immunosuppression with a stable almost complete chimerism. the conditioning regimen was well tolerated with no case of high-grade transplant related morbidity. the post-transplantation complications were comparable in both groups. one patient developed after severe rotavirus gastroenteritis a severe cmv pneumonitis and succumbed to an uncontrolled cmv pneumonitis. one patient in the t-haplo sct group suffered from a late graft failure and developed a macrophage activation syndrome. he died in a septic event.none developed a glucksberg grade iii-iv agvhd and in the t-haplo sct group 4 pts (20%) and in the msd group 2 pts (22%) developed a steroid sensitive mild to moderate cgvhd with symptoms of fasciitis, oral as well as mild cutaneous gvhd. in both groups no severe or steroid refractory cgvhd was observed. conclusions: our results demonstrate increasing evidence for the safety and efficacy of cd3 + /cd19 + or αβ + /cd19 + depleted haploidentical hsct in asscd. the treosulfan based conditioning regimen was an excellent alternative to busulfan with a low incidence of transplant related morbidities and therefore most suitable for pts with scd. these results open the option of a curative therapy for almost all scd pts without a msd. disclosure: nothing to declare. abstract already published. hematopoietic stem cells o117 abstract already published. comparison of outcomes post allogeneic hematopoietic cell transplantation using fresh versus cryopreserved peripheral blood stem cell grafts background: cryopreservation is routine practice with autografts, however in the allogeneic hct setting the effects of cryopreservation have not been thoroughly investigated. we sought to compare allogeneic hct outcomes using fresh versus cryopreserved grafts in a large single centre cohort. methods: between 2003 and 2017, we retrospectively reviewed 951 consecutive adult patients who underwent allogenic peripheral blood hct at our centre. outcomes assessed included platelet (≥20x10e9/l) and neutrophil (≥0.5x10e9/l) engraftment, occurrence of acute graft-versushost disease (gvhd) in the first 100 days, overall survival (os), cumulative incidence of relapse (cir) and non-relapse mortality. results: median follow up of survivors was 47 months (range 4-177 months). fresh grafts were received by 525 patients, 426 received cryopreserved grafts, median age at hct was 53 and 54 years respectively. transplant indication was myeloid malignancy in 711 (75%), lymphoid in 229 (24%) patients. myeloablative regimens were used in 506 (53%) patients. the majority of fresh grafts were from unrelated donors (82%) while most cryopreserved grafts were from matched related donors (90%). in vivo t-cell depletion was performed in 65% of fresh and 18% of cryopreserved transplants. median time to neutrophil engraftment for fresh versus cryopreserved grafts was 15 and 15 (10-48) days respectively, while median time to platelet recovery was 16 (12-186) and 17 days, respectively. for fresh versus cryopreserved grafts, grade ii-iv acute gvhd was seen in 51% and 54%, respectively (p=0.42) while grade iii-iv acute gvhd was seen in 28% of patients in both groups (p=0.96). on univariate analysis, os for the entire cohort at 2 years was 50% (95%ci 47-54%) and at 5 years was 40% (95%ci 36-43%). two and 5 year os was 47% (95%ci 43-52%) and 39% (95%ci 34-44%) respectively for fresh grafts and 54% (95%ci 49-59%) and 41% (95%ci 36-46%) respectively for cryopreserved grafts (p=0.21). cumulative incidence of relapse (cir) of the entire cohort at 2 years was 18% (95%ci 16-21%) and at 5 years was 21% (95%ci 19-24%). two and 5 year cir was 16% (95%ci 13-20%) and 19% (95%ci 15-22%) respectively for fresh grafts and 21% (95%ci 17-25%) and 25% (95%ci 21-29%) respectively for cryopreserved grafts (p=0.02, figure 1 ). [[o118 image] 1 . figure 1] multivariable analysis for os verified no significant difference between fresh versus cryopreserved grafts (p=0.25). for cir however, cryopreservation was the only independent predictor of relapse (hr 1.43 for cryopreserved, 95%ci 1.07-1.91, p=0.02), while for nrm cryopreservation was not an independent predictor of increased risk (p=0.06). when the multivariable analysis was repeated for related donor transplants only (n=474, 385 cryopreserved grafts, 89 fresh), this confirmed the independent increased relapse risk for cryopreserved grafts (hr 2.30, p=0.01) . conclusions: we confirmed on univariate and multivariable analysis that there is no significant difference in os between allogeneic transplants performed with fresh versus cryopreserved peripheral blood stem cell grafts, however there is a significant increase in relapse risk associated with cryopreservation. disclosure: nothing to declare. background: high dose post-transplantation cyclophosphamide(ptcy) used in haploidentical transplantation (haplo-sct) has demonstrated to be highly effective in acute and chronic gvhd prophylaxis; however it is associated with high relapse rates. anti-t-lymphocyte globulin (atg-fresenius®) is also effective as gvhd prophylaxis but its benefit in overall survival (os) and relapse free survival (rfs) is unclear. the aim of this study was to compare the effectiveness of two gvhd prophylaxis regimen employed in high risk transplantation: ptcy-haplo-sct and low doses of atg-f used in peripheral blood(pb) and mismatched transplantation. the primary endpoint was to evaluate the incidence and severity of agvhd and cgvhd. as secondary endpoints we analysed the os, rfs and grfs (considering as events severe agvhd, systemic therapy-requiring cgvhd, relapse or death). we also evaluated mortality related to transplantation(trm) and post-transplant complications. methods: we retrospectively analysed 111 allo-sct performed in our institution between 2012 and 2017. we analysed two cohorts: 49 haplo-sct with ptcy (50 mg/ kg, days +3,+4) followed by tacrolimus and mycophenolate (mmf); and 62 pb and/or mismatched transplants with low dose atg-f (7 mg/kg days -3,-2,-1) associated to calcineurin inhibitors starting on day -1, with short course mtx (days +1,+3,+6) or mmf. in both cohorts, mmf was stopped on day +28 and calcineurin inhibitors were tappered on day +50. comparing both groups, we found differences in diagnosis (lymphoproliferative disorders 16 vs 4,p=0.003), high dri-score (18 vs 11, p=0.04), previous transplantation (12 vs 5,p=0.02), reduced-intensity conditioning regimen (36 vs 20,p< 0.001) and bone marrow as stem cells source (38 vs 9, p< 0.001). results: median time to neutrophil engraftment was similar in both groups: 17 vs 16 days. conversely, median time to platelet recovery was longer in ptcy cohort (33vs18 days, p=0.016). there were no differences in agvhd incidence (ptcy 30.6% vs atg 36.4%) or severe agvhd (ptcy 4.1% vs atg 9%, p= 0.852). the global cgvhd incidence was pcty 56.1% vs atg 66%. mild, moderate and severe cgvhd incidence was 31.7%, 19.5% and 4.8%, for ptcy vs 28%, 26% y 12% for atg(p=0.475).with a median follow-up of 27 months (28 months for ptcy and 22 months for atg) the os at 12 and 24 was: pcty 67.8 % and 61%, and atg 68.8% and 59.9%,p=0.971). rfs (12 and 24 months) was: 61% and 57.9% for ptcy, and,69% and 54% for atg(p=0.839). grfs at 12 and 24 months was 46.5% and 42.9%, for ptcy patients and 40.7% and 33% for atg patients (p=0.433). ptcy cohort seemed to develop more relevant non-infectious complications, but there were no differences among infectious complications. image 1. trm was similar in both cohorts: pcty 18.4% vs atg 22.5% (p=0.250). we neither found differences in early toxic mortality (< 100 days): pcty 16.3% vs atg 14.5% for atg (p=0,421). conclusions: regardless the different transplant scenarios in which they were used, ptcy and low dose atg seem to be equally effective in the prophylaxis of severe forms of acute and chronic gvhd, offering similar grfs. moreover,they show similar early toxicity and rate of infectious events. disclosure: we have nothing to disclose. conclusions: emapalumab treatment promoted disease control, blunting the exacerbated immune response typical of the disease, with a favorable safety and tolerability profile. the results of the study also suggest that emapalumab may contribute to optimize post-transplant outcome of patients given hsct. based on these data, emapalumab received marketing authorization in the us from the food and drug administration for the treatment of patients with primary hlh with refractory, recurrent or progressive disease or intolerance with conventional therapy. background: allogeneic hematopoietic stem cell transplantation (hsct) is a potentially curative treatment for some inherited disorders, including selected primary immunodeficiencies (pids). in the absence of a wellmatched donor, hsct from a haploidentical family donor (hifd) may be considered. various approaches are being developed to mitigate the risks of graft failure and graftversus-host disease (gvhd) and to speed-up immune reconstitution. among those, high-dose, post-transplant cyclophosphamide (ptcy) is increasingly used in adult recipients. however, data on ptcy in children and those with inherited disorders in particular are scarce. methods: we reviewed the outcome of 27 children transplanted with hifd and ptcy for pid (n= 22) or osteopetrosis (n= 5) in a single center. median (range) age was 1.5 years (0.2 to 17)). patients in our series had major risk factors for poor post-hsct outcome such as active viral infections at the time of transplantation (n=9), ebvrelated lymphoproliferation in partial remission (n=3), previous kidney transplant (n=1). hsct with ptcy was a primary (n=21) or a rescue procedure after graft failure (n=6). conditioning regimen was myeloablative in most primary hscts and non-myeloablative in all rescue procedures. results: after a median follow-up of 17.3 months, 24 of the 27 patients engrafted. twenty-one patients are alive and have been cured of the underlying disease. the two-year overall survival rate was 77.7%. the cumulative incidences of acute gvhd grade ≥ ii, chronic gvhd and autoimmune disease were 49%, 26.6%, and 26.5%, respectively. there were only two cases of grade iii acute gvhd, all cases of cgvhd were limited and allowed to stop systemic immune-supression, autoimmunity consisted in 2 autoimmune hemolytic anemia in remission at last follow up, 1 vitiligo and 2 thyroiditis. the cumulative incidence of blood viral replication and life-threatening viral events were 58% and 15.5%, respectively. there was evidence of early t cell immune reconstitution including early anti-viral responses. conclusions: in the absence of an hla-identical donor, hifd hsct with ptcy is a viable option for patients with life-threatening inherited disorders. disclosure: nothing to declare background: griscelli syndrome (gs) is a very rare autosomal recessive disease, characterized by skin hypopigmentation and silvery-gray hair. gs type 2 (gs2) patients suffer immunodeficiency and potentially fatal episodes of macrophage activation known as accelerated phases during early childhood. the only curative treatment modality for these patients is allogeneic hematopoietic stem cell transplantation (hsct). we report the outcome of hsct in 35 children with gs2. to date, this is the largest cohort of gs patients who have undergone transplantation at a single center. methods: we retrospectively reviewed 35 consecutive patients with gs2 who underwent hsct at our institution between january 1993-december 2017. median age at diagnosis and at transplant was 0.3 (0-12) and 0.9 (0.3-15.1) years respectively. prior to hsct, 28 (80%) and 16 (46%) patients had life-threatening accelerated phases (hlh) and cns involvement respectively. all such patients were treated with chemotherapy and achieved remission at the time of hsct. the source of grafts was matched related marrows in 19 (54.3%) pts and partially mismatched unrelated cord in 14 (40%) pts. two patients received haploidentical and matched unrelated marrows respectively. conditioning regimens were myloablative doses of busulfan/cyclophosphamide and busulfan/fludaribine in 27 (77%) and 8(23%) patients respectively. all patients received gvhd prophylaxis. growth factors were administered in 33 (94.3%) and median cd34 dose used was 5.46 (2.14-11.94) 10^6 and 5.45 (1.8-13 .5) 10^5 per kg of body weight for marrow and cord respectively. results: post infusion rate of engraftment was 88.6% (31 cases) with median time to neutrophil and platelets recovery were 15 (8-30) and 29 (14-61) days respectively. cumulative incidence of acute gvhd was 28.6% (10), with an overall grade of i, ii and iv as 20%, 60% and 20% respectively. the post-transplant course was complicated by cmv infection, ebv viremia and veno-occlussive disease in 14(40%), 7(20%) and 7(20%) patients respectively. chimerism studies at the last contact were available for 24 patients. full donor cell chimerism (100%) was seen in 16 (67%) of the transplanted patients. post-transplant two patients experienced disease reactivation at a median time 45.5 (28-63) days. with a mortality rate of 37.1% (13) and a median follow-up time of 87.7 months, five-year cumulative probability of overall survival (os) for our cohort of patients was 0.625 ± 0.083. transplant related mortality counted as death within day 100 was 28.6% (10). prominent causes of death were septic shock followed by ards. cumulative probability of five years overall survival was significantly better in those who did not have hlh prior to sct (1.0±0.0 vs. 0.529±0.096, p-value: 0.037). of the 16 patients with neurological involvement before hsct, 8 survived with residual sequelae in 3 patients. os at five years was 0.500±0.125 and 0.729±0.104(p-value: 0.328) in pts with and without cns involvement at presentation respectively conclusions: hsct in patients with gs is potentially curative with long-term, disease-free survival. early hsct before the development of the accelerated phase showed better result. [ background: primary immunodeficiencies (pids) are heterogeneous inborn disorders resulting in impaired cellular and/or humoral responses. pid present a wide range of clinical manifestations, ranging from infections to autoimmune, inflammatory and/or malignant complications. allogeneic stem cell transplantation (allosct) is curative for pediatric pid with an excellent safety. in adults on the other hand, this therapeutic approach remains controversial. methods: this is a retrospective, monocentric study of 26 consecutive adult patients with pid who underwent an allosct between 2011 and 2018. the objective was to assess the feasibility, effectiveness and safety of this procedure. results: twenty-two (85%) of 26 patients presented an inherited t or b cell deficiency and 4 (15%) a phagocyte impairment. twenty (77%) patients had a genetic diagnosis. besides infectious complications, 8 (31%) patients had an history of lymphoma and 13 (50%) an history of autoimmune/ inflammatory complication. the median age at transplant was 24 years (range 17-37). twelve (46%) patients received a myeloablative conditioning (mac), 12 (46%) a reduced intensity conditioning (ric) and 2 (8%) had no conditioning (second transplant for severe combined deficiency). mac included fludarabine (flu)/buslfan (bu, dosage: 12.8 mg/kg) based regimen (n=11) and bu (12.8 mg/kg)/cyclophosphamide (cy) based regimen (n=1). ric included based flu/bu (range dosage: 6.4-9.6 mg/kg) regimen (n=9), flu/cy based regimen (n=1), thiotepa/flu/bu regimen (n=1) and low dose tbi based regimen (n=1). among the 24 patients who received a conditioning, 4 (17%) received alemtuzumab and 11 (46%) received antithymocyte globulin as part of the conditioning. the stem cell source was bone marrow for 8 (31%) and peripheral blood stem cells for 18 (69%). the donor was matched related for 10 (38%), matched unrelated for 12 (46%), mismatch unrelated for 2 (8%), and haplo-identical for 2 (8%). all assessable patients had a successful engraftment. eight (31%) presented a grade ii-iv acute gvhd (one grade iii, 0 grade iv), and 5 (23%) a chronic gvhd (2 limited and 3 extensive). with a median follow-up of 29 months (3-90 months) post-transplant, the 3-years overall survival (os) was 80.8%. the transplant related mortality was 19.2% with 5 deaths. among them, both patients transplanted with an haplo-identical donor died. all deaths occurred in the firstyear post-transplant. thus, the use of an haploidentical donor was associated with an adverse outcome. conversely, neither the conditioning nor the stem cell source was associated with a worse outcome. except one patient with an history of aggressive b cell lymphoma who relapsed few months after allosct, no patient with an history of lymphoma relapsed. after a salvage treatment, the relapsing patient remained in complete remission 5 years later. at last follow-up, all surviving patients had a stable, mixed donorrecipient or full donor chimerism without any sign of active infection. conclusions: allosct in the pid setting is an effective therapeutic with an acceptable toxicity and should be considered in case of severe infectious, inflammatory and/or malignant complications in young adult patients with pid when an appropriate donor is available. disclosure methods: we analyzed the results of allogenic hsct with tcrαβ/cd19 graft depletion in 148 patients with various pid (excluding classic scid) who received hsct from may 2012 to september 2018 in our center. the median age at hsct was 3,5 years (range 0,43-17,63). 112 patients received hsct from matched unrelated, 31haploidentical donors, 5 -siblings. the conditioning regimens included: fludarabin (flu) 150mg/m2 with 1 alkylator (treosulfan (treo) 36-42g/m2) in 33 patients, with 2 alkylators (treo 36-42g/m2 with melphalan 140mg/m2 or thyotepa 10mg/kg) in 74. twenty-five patients received 2 alkylators with addition of g-csf 30mg/kg and plerixafor 24mg/kg. seventeen patients with nijmegen breakage syndrome (nbs) received reduced intensity conditioning with busulfan 4mg/kg or treo 30mg/m2, cyclophosphamide 40mg/kg and flu 150mg/m2. in all but 2 patients serotherapy was used: 10 patients -horse atg -90mg/kg, 133 -rabbit atg (thymoglobulin) 5mg/kg, 3 -1mg/kg anti cd52 monoclonal antibodies. in 55 patients two or more immunosuppressive drugs were used after transplantation (combination of calcineurin inhibitor (cni) with short course of methotrexate, or mycophenolate mofetil, or abatacept), 66 patients received one immunosupressive agent (cni). from november 2017 no posttransplant immunosuppression was used (27 patients). primary end points were: incidence of acute gvhd, graft failure (gf) and transplant related mortality (trm). because of the supposed influence of conditioning regimen on gf and trm incidence, to the analysis of gf and trm were included only 74 patients with similar conditioning regimen with 2 alkylators (excluding patients with nbs, with 1 alkylator and addition of g-csf and plerixafor in conditioning regimen). median follow up after hsct was 1,95 years (range 0,04-6,3 years). results: overall survival (os) in 148 patients was 0,85 (95% ci 0,79-0,91). cumulative incidence (ci) of acute gvhd in patients with two and more immunosupressants was 0,164 (95% ci 0,09 -0,3), with one -0,17 (95% ci 0,1 -0,29), with no immunosupression -0,11 (95% ci 0,04 -0,32), p=0,843. ci of graft failure was: in patients with two and more immunosuppressive agents (n=25) -0,24 (95% ci 0,12 -0,48), with one (n=32) -0,17 (95% ci 0,1 -0,29), without immunosuppression (n=17) -0,06 (95% ci 0,01 -0,44), p=0,172. ci of trm in patients with two and more immunosuppressants was 0,22 (95% ci 0,1 -0,47), with one -0,1 (95% ci 0,02 -0,28), without immunosuppression-0,26 (95% ci 0,09 -0,73), p=0,42. conclusions: we conclude that in our group of pid patients presence or absence of immunosuppression after hsct with tcrαβ/cd19 graft depletion and its extent made no significant difference on the incidence of acute gvhd, graft failure and trm. considering these results hsct with tcrαβ/cd19 graft depletion without posttransplant immunosupression could be recommended for patients with pid. disclosure background: lps-responsive beige-like anchor protein (lrba) deficiency is a severe primary immunodeficiency with a variable clinical phenotype, including features overlapping with common variable immunodeficiency, autoimmune lymphoproliferative syndrome, and immune dysregulation polyendocrinopathy enteropathy x-linked syndrome and an association with lymphoma. recent findings strongly support hsct in patients with severe presentation of lrba deficiency. however, there are no up-to-date follow-up and survival data of patients not undergoing transplantation beyond previous publications of large cohort studies. this study was conducted to increase the knowledge on transplant experience and to elucidate the clinical course of both transplanted and untransplanted patients with lrba deficiency under various targeted treatment modalities. methods: we performed an international inborn errors working party-and european society for immunodeficiencies-wide survey in 2018 to collect information about the hsct experience and the clinical course of both transplanted and untransplanted patients with lrba deficiency. we included an assessment of the disease activity and treatment responses with a specially developed immune deficiency and dysregulation activity (idda) score, which weighs the sum of organ involvement (scored by 0-4) by days of hospitalization and performance scores, taking into account the type of therapy and response per treatment phase, which we herewith introduce. data were obtained in accordance with the declaration of helsinki and an institutional review board review by means of retrospective chart review by local physicians. results: we analyzed the data of 68 lrba deficient patients from 23 centers, including 35 unpublished patients. 20 patients of our cohort underwent hsct between 2005 and 2018. overall survival of patients undergoing transplantation (median fu 30 months) was 70% (14/20 patients); all deaths were due to transplant-related mortality and occurred within 3 months of hsct. 83.3% (40/48) of untransplanted patients (2-68 years) are currently alive. out of the surviving transplanted patients (n=14) 6 are in complete remission, 4 in good partial remission (no treatment) and 4 in partial remission (receiving therapy), implying that 10 out of 14 transplanted patients (71.4%) are currently without treatment. only 5 out of 40 living untransplanted patients (12.5%) do currently not receive therapy. most commonly used drugs in lrba deficiency were steroids (27.7%), sirolimus (18.8%) and abatacept (18.8%). disease activity, measured by idda score, was significantly lower in patients treated with abatacept (p< 0.0001). no immunosuppression-associated malignancy was detected in our cohort. analyzation of the idda score of all living patients revealed significantly lower disease activity in patients transplanted and alive, than in those who did not undergo hsct (p= 0.01). conclusions: in conclusion our findings do, concordant with previous results, support hsct, especially in patients with a severe phenotype of lrba deficiency. however, the question of hsct indication and optimal time point cannot yet be generally resolved. in terms of conventional treatment, abatacept is clearly favorable in untransplanted patients. nevertheless, it is not available in many countries and the long-term dependency on this treatment with its associated potential risks remains. finally, our newly introduced idda score for assessment of disease activity may also prove useful for other syndromes with immune dysregulation. disclosure: nothing to declare background: cytomegalovirus (cmv), is a major complication of allogeneic hematopoietic stem cell transplant (hct) recipients. to overcome morbidity associated with cmv infection, antivirals are given, and while they successfully suppress viremia, they do so in the absence of immune reconstitution. a different approach is to use a vaccine to bolster immunity to cmv early post-hsct and sustain it at least until the patient is immune reconstituted. we selected a highly attenuated, non-proliferating viral vector referred to as modified vaccinia ankara (mva) to insert 3 immunodominant cmv antigens pp65, ie1-exon4, and ie2-exon5. this novel vaccine (triplex) demonstrated excellent tolerability and immunogenicity in a clinical study of 24 healthy adults (la rosa, et al. blood 2017) . these findings prompted us to conduct a test of vaccine protective efficacy in at-risk hct recipients in a multi-center, randomized and placebo-controlled phase 2 clinical trial (nct02506933). methods: eligible patients were cmv-seropositive and undergoing hct for hematologic malignancies from matched related/unrelated donors without the use of exvivo or in-vivo t cell depletion. the randomization (1:1) was stratified by donor serostatus and hct center. patients were enrolled pre-hct, and on d28 were requalified for eligibility based on lack of prior cmv reactivation, ≥grade 3 gvhd, high-dose steroids, or other significant transplantrelated morbidity. after randomization patients received triplex or placebo injections on d28 and d56 post-hct and followed for one year. the primary endpoint is defined as any cmv reactivation [≥500 cmv genome copies (gc)/ ml], low-level reactivation prompting antiviral therapy, or cmv disease prior to d100 post-hct. based on the objective of reducing reactivation from 30% to 10% or from 40% to 15% on the vaccine arm, a sample size of 102 was determined to provide at least 90% power at a one-sided 0.10 level of significance by log-rank test. results: the patient clinical/hct characteristics were balanced between the vaccine arm (n=51) and placebo arm (n=51). the vaccine was well tolerated with no significant difference in grade 3-4 aes or saes in both arms. triplex displayed potent immunogenicity, as many vaccine recipients with either cmv-positive or -negative donors showed strong reconstitution of both cd4 and cd8 cmv-specific immunity that initiated soon after the first injection and was elevated for at least 100d post-hct. most notably there was a reduction of primary endpoint-defining reactivation through d100 in the vaccine arm (5 events, 9.8%) compared to the placebo arm (10 events, 19.6%, p=0.08). greater detail regarding protocol-specified secondary endpoints including time to viremia, its duration, frequency and duration of antiviral drug treatment, late cmv viremia, time to engraftment and incidence of acute and chronic gvhd, relapse and non-relapse mortality will be presented. conclusions: in summary, triplex was well tolerated in seropositive hct recipients, and was significantly better than placebo in preventing viremia cases accompanied by improved cmv-specific t cell reconstitution. background: due to the risk of invasive pneumococcal disease (ipd) after allogeneic transplant, the ecil7 guidelines recommend 3 doses of pneumococcal conjugate vaccine (pcv) starting 3 months after transplant, followed by either one polysaccharide 23-valent vaccine (psv23) dose or a 4th pcv in case of chronic gvhd. however, no data strongly support recommendations from the 2 nd year. the aim of this study was to assess the anti-pneumococcal antibody (ab) concentrations, years after different schedules of vaccination. methods: patients who received allogeneic hct more than 18 months ago and were seen in consultation between jan-nov 2018 were screened for igg ab concentrations for the 7 pneumococcal serotypes shared by pcv7, pcv13, and psv23 (4, 6b, 9v, 14, 18c, 19f, 23f) , using modified enzyme linked immunosorbent assay (wernette et al.2003) . patients were considered protected if their ab concentrations were > 0.35μg/ml for the 7 serotypes tested. from 2001, all patients received 3 pcvs during the 1 st year of transplant, then either one dose of psv23 or (from 2009) a 4 th pcv. for patients transplanted for more than 12 months when pcv became available, we locally recommended > 2 pcv and one psv23. results: sixty-one patients were assessed at a median of 8.3 years (range: 1.7-39.1) after transplant. the mean age was 53 years (range: 21-79), m/f sex ratio 0,85. most patients had acute leukemia (32, 52.5%) or lymphoproliferative diseases (13, 21.5%). the donor was hla-identical in 42 (69%) cases. conditioning regimen was myeloablative in 37 (61%), reduced-intensity or non-ma in 12 (19.5%) each. none patient had experienced ipd since transplant nor had received immunoglobulins for at least 6 months or rituximab for at least 23 months at time of assessment. fifty-four patients (88.5%) received 3 consecutive pcv: 46 (75.5%) during the first 12 months and 8 (13 %) thereafter. five (8%) patients received 2 pcv and only one patient (1.5%) received a unique dose of pcv. the 1 st pcv was administered at a median of 3.5 (range: 3-201) months after hct. fifty-seven (93.5%) patients received one psv23 at a median of 13 months (range: 0.9-385) and 33 (54%) of them received a 2nd psv23 thereafter. finally, 39/ 61 (64%) patients received the recommended program. the overall protection rate was 29/61 (47.5%) at a median time of 8.3 years after hct. no differences were observed between patients who received the recommended program (18/39, 46%) and the others (11/22, 50%). however, this latter group was often vaccinated > 10 years after hct and therefore not protected for years, but may have developed a better response to only one or 2 pcv. in all groups, the ab concentrations were heterogeneous from one serotype to an other. recipient age, donor age, underlying disease, conditioning, donor type, previous gvhd, lymphocyte count or gammaglobulin serum levels were not associated with seroprotection. conclusions: although the early antipneumococcal vaccination program is now well established, half of the patients vaccinated according to the guidelines were not protected anymore years later. prospective studies are needed to establish an optimal long-term programme. disclosure: nothing to declare , letermovir vs. placebo) and was well tolerated overall. we evaluated risk factors associated with development of cs-cmvi to further inform the clinical use of letermovir prophylaxis and future trial designs. methods: the 495 participants without detectable cmv dna at randomization (primary efficacy population) were analyzed. cs-cmvi was defined as cmv viremia requiring antiviral preemptive therapy (pet) or cmv diseasepatients without cs-cmvi who died or withdrew from the trial on or before week 24 were censored at the time of those events for this analysis. potential risk factors for cs-cmvi by week 24 post hct were examined using univariate and multivariate cox proportional hazards models. candidate covariates were included in the multivariate cox model if they were associated with cs-cmvi in the univariate analysis or had been previously identified in the literature as significant risk factors. to avoid collinearity, the trial high-risk for reactivation of cmv stratification covariate was replaced with an updated covariate that only included patients who underwent hct with a mismatched donor, cord blood, ex-vivo t-cell depletion, or received alemtuzumab. the haploidentical hct and matched unrelated donor type were considered separately from the trial high-risk cmv categories. graft-versus-host disease (gvhd) and systemic glucocorticoid exposure were modeled as time-dependent covariates. race was dichotomized into asian and non-asian. the effect of study treatment was not included in the model due to nonproportional hazards. instead, letermovir treatment was used as a stratification variable. hazard ratios and 95% confidence intervals (ci) were calculated. results: there were 128 cs-cmvi events (25.9%) among 495 patients by week 24 post-hct for an incidence rate of 0.19/100 patient-days (95% ci, 0. 16-0.22 conclusions: donor cmv seronegativity, haploidentical hct, gvhd, glucocorticoid use, atg use, and asian race conferred significant risk of cs-cmvi through week 24 post-hct in a phase 3 study of letermovir prophylaxis. these results identify hct patient groups that would benefit the most from letermovir prophylaxis. methods: 769 adult patients (age 18-60) with de novo aml in complete remission (cr) perusing t-cell replete haplo-hsct with atg+g-csf protocol were consecutively enrolled at peking university people's hospital between 2010 and 2016. all patients were evaluated for donor-patient cmv serostatus before hsct. cmv dna emia were positive for >1000 copies/ml cmv by pcr tests on peripheral blood (pb). refractory cmv infection was defined as cmv dnaemia lasting for >2 weeks in spite of treatment. pb samples of 8 patients with refractory cmv infection (4 relapse and 4 without aml relapse) were sequencing for cmv derived small rna. cmv-mir-us4-1/ul-148d were then chosen as target mirnas and tested by stem-loop taqman qpcr in consecutive patients (n=157). cumulative incidence of relapse (cir) and treatment related mortality (trm) were calculated using competing risks, cox model was tested with patient age, sex, wbc count at diagnosis, cr status, courses to achieving cr, cytogenetic risk group, minimal residual diseases before hsct, cmv infection, cmv-mir-us4-1/ ul-148d expression. us4-1/ul-148d were further explored for the association with t cells and natural killer (nk) cells reconstitution and target mrna validation. results: in the total cohort of 769 patients, the 3-year overall survival (os) and leukemia-free survival (lfs) were 75.3 % and 78.4%, cir and trm were 15.9% and 8.8%. cmv infection occurred in 71.5% recipients at a median time of 4 weeks after haplo-hsct, and the duration was 2 weeks. 49.7% recipients experienced refractory cmv infection. cmv infection (yes vs. no, refractory vs no) did not affect cir in univariate analysis, while in multivariate analysis, cytogenetic risk group was identified as the only independent prognostic factor affecting cir (hr 1.72, 95% ci, p=0.032 ). in the cohort testing for cmv microrna, 35.0% patients were identified expression of us4-1, 40.1% patients for ul-148d, while 29.3% patients had co-expression. the relapse incidence was significantly lower in patients with vs. without co-expression of mir-us4-1/ul-148d. co-expression of mir-us4-1/ul-148d was the only independent risk factor for reducing cir (hr 0.414, 95% ci, 0.165-1.036; p=0.019). us4-1/ul-148d as also found to promote reconstitution of nkg2c+ nks. luciferase assay identify plzf as target of us4-1 while klrc1 targeted by mir-ul-148d, which suggest potential role of shifting nkg2c/nkg2a balance to nkg2c domination. conclusions: the present study is the first prospective trial to evaluate cmv infection on relapse for aml patients following t-cell replete haplo-hsct with atg+ g-csf protocol. our results suggested cmv-mir-us4-1/ul-148d co-expression rather than cmv infection reduces the relapse incidence, the stronger gvl effect might be associated with strengthening nkg2c+ nk cells reconstitution and alloreactivity via plzf/klrc1 pathway. [[o129 image] 1. mir-us4-1/ul-148d reduces relapse in aml after haplo-hsct] clinical trial registry: chictr-och-10000940 disclosure: nothing to declare o130 a randomised, placebo-controlled phase 3 study to evaluate the efficacy and safety of asp0113, a first-inclass, dna-based vaccine in cmv-seropositive allogeneic haematopoietic cell transplant recipients adjudicated cmv-specific avt or adjudicated cmv eod and mortality through 1 year post transplant. results: overall, 514 patients were randomized, of whom 501 received ≥1 dose of randomized treatment (asp0113 n=246; placebo n=255). there was no statistically significant difference in the proportion of patients who achieved the primary endpoint between the asp0113 (n=87 [35.4%]) and placebo (n=77 [30.2%]) groups, respectively (odds ratio 1.27; 95% confidence interval p=0.205 ). there were no statistically significant differences between groups for any secondary endpoints (table) . the incidence of teaes was similar between groups, except for a greater incidence of drugrelated teaes in the asp0113 group (n=194 [78.9%] ) compared with placebo (n=74 [29. 0%]) attributed to injection site-related teaes. mean t-cell response to pp65 increased over time in both groups and was significantly greater with placebo compared with asp0113 (p=0.027). there was no statistically significant difference between groups for mean gb-specific antibody response. conclusions: asp0113 did not demonstrate efficacy in the reduction of overall mortality and cmv eod through 1 year post transplant. asp0113 demonstrated a similar safety profile to placebo, with the exception of injection siterelated teaes that were more frequent in the asp0113 group. participants in this study will be followed up to 5.5 years post transplant for long-term safety assessments. disclosure: jm: personal fees and non-financial support from astellas, basilea, cidara, f2g, gilead, merck and hsct) and 893 autologous hsct (auto-hsct). during the observation period 40 probable and proven rare ifd (eortc/msg 2008 criteria) cases were diagnosed in children and adults with hematological malignances and non-malignant hematological diseases after allo-hsct (n=30), auto-hsct (n=2), and chemotherapy (n=8). the median age was 23 (2-59) y.o., males -60%(n=24). the median follow up time for rare ifd cases was 3 months; for survivors -30 months. results: incidence of rare ifd in hsct recipients was 1,5%, it was higher after allo-hsct (1,6%) than auto-hsct (0,2%) (p< 0,01). in eight patients, this complication developed after ct and four of them proceed to allo-hsct. the most frequent underlying diseases were acute lymphoblastic leukemia (33%) and acute myeloid leukemia (30%). the median time of onset of rare ifd after allo-hsct was 104 (21-1057) days, auto-hsct -138 (60-216), after start of . etiology of rare ifd was identified by culture in 65% cases: rhizopus spp. -27%, paecilomyces spp. -7%, fuzarium spp. -5%, malassezia furfur -5%, trichosporon asahii -3%, scedosporium apiosperium -2%, scopulariopsis gracilis -2%, rhizomucor pusillus -2%, lichtheimia corymbifera -2%, mix rare ifd with rhizopus spp. + paecilomyces spp. -5%, paecilomyces spp. + fuzarium spp. -5%. 35% cases (mucormycosis) were diagnosed by microscopy. in 45% cases rare ifd developed after or in combination with invasive aspergillosis, and 2 patients had both preexisting invasive aspergillosis and co-infection with mucormycosis. the main site of infection were lungs (82%), the main clinical symptom -febrile fever (95%). antifungal therapy was used in all patients: lipid amphotericin b -30%, lipid amphotericin b + caspofungin -22,5%, voriconazole -17,5%, posaconazole -12,5%, lipid amphotericin b + posaconazole -10%, and echinocandins -7,5%. surgery was used in 10% patients. overall survival at 12 weeks from the diagnosis of rare ifd was 50%. the 12-weeks overall survival was better in patients after ct and auto-hsct (80%) than allo-hsct (42%), p=0,048. conclusions: the incidence of rare ifd in hsct recipients was 1,5% and depends on type of transplantation. rare ifd is a late complication after chemotherapy and hsct and usually develops after or in combination with invasive aspergillosis. higher incidence and worst prognosis rare ifd is observed in allo-hsct recipients. disclosure: nothing to declare o133 incidence and outcome of kaposi sarcoma after hsct: a retrospective analysis on behalf of idwp background: kaposi's sarcoma (ks) is an angioprolipherative disease which occurs in immunosuppressed patients, often associated with infection by human herpes . in solid organ transplantation (sot) ks is relatively common, the risk being 60-500 folds higher than that of general population and representing around 23% of secondary cancers. also hematopoietic stem cell transplantation (hsct) is a risk factor for the development of ks but until now only few case reports were published. we assessed retrospectively the incidence, the clinical characteristics, and the outcome of ks in the ebmt database. methods: the cases of ks were identified by ebmt registry (promise) and by inviting all 569 ebmt centers to notify ks cases. the clinical features, type of therapy, survival rate and causes of death were retrieved from of promise or, if lacking, by specific case report form sent to participating centers. the center response rate was 74/ 569 (13%). results: fourteen centers reported 17 patients with ks, all ks were diagnosed from 2004 to 2017, but one case that was diagnosed in 1987. the analysis was limited to 13/17 patients because ks was diagnosed before hsct in 4 patients: they were 13 patients, 3 females and 10 males who developed ks after allo hsct (10) and auto hsct (3); moreover, ks occurred after a second hsct in 2 patients and after a third hsct in 1 patient. the search of hhv8 in tumour tissue was done in 9 cases and resulted positive in 8/ 9. the underlying disease were: 46% leukemia, 23% lymphoma, 15% myeloma, 8% myelodysplastic syndrome (mds), 8% bone marrow failure. the source of stem cells was bone marrow in 4 patients (31%) and peripheral blood in 9 patients (69%). the median age at ks diagnosis was 49.7 years (range 6. 3-61.4) . considering the number of hsct performed in the participating center from 2004 to 2017, the incidence rate was 0.17% in allogeneic transplantations (9/5345), 0.05% in autologous transplantations (3/5857) and 0.11% (12/11202) in the whole group. the interval of time between hsct and the development of ks was 7 months (range -0. 3-61.2) . the organ involvement was: 62% skin (8 patients), 31% lymph nodes (4), 8% gingival (1) . apart from withdrawal of immunosuppression, 2 patients received chemotherapy, 3 patients received radiotherapy, and 1 patient received radio and chemotherapy; moreover 5 patients received antiviral treatment with ganciclovir or foscarnet. eight patients (62%) are alive whereas 5 patients (38%) died at a median time of 8.1 months, range 0.5-12.3. the causes of death were infection in 2 cases, secondary malignancy/ptld in 2 and relapse/progression in 1 whereas no case of death directly associated to ks. conclusions: ks is a rare complication of the immunosuppressive status related to hsct that generally occurs within the first year after hsct. the low prevalence and the rarity of this complication do not justify the adoption of screening program for hhv-8. on the other hand, the role of the virus in febrile status in immunosuppressed patients and the risk factors for the development of ks are not well known. disclosure background: autologous stem cell transplantation (auto-sct) is considered the standard treatment for patients with relapsed or refractory (r/r) hodgkin lymphoma (hl). for those with high-risk disease, an alternative consolidation strategy with allogeneic sct (allo-sct) could be a potential option to improve the outcome. however, allo-sct with a reduced-intensity conditioning (ric) needs around 3 months for the graft-versus-lymphoma effect (gvl) to develop, thus in patients with an aggressive hl the disease might progress before this happens. in this setting, a tandem auto-ric-sct approach has the potential of combining cytoreduction to keep the lymphoma under control and the potential benefit of a gvl effect. to better understand the safety and efficacy of a tandem auto-ric-sct approach we conducted a retrospective analysis of patients treated with this strategy between january 2004 and december 2015 and reported to the ebmt registry. methods: patients were included if they had received an auto-sct followed by a planned ric-sct in < 6 months with no disease relapse between the procedures. the primary endpoint was progression-free survival (pfs) after the tandem procedure. secondary endpoints were overall survival (os), cumulative incidence of non-relapse mortality (nrm), incidence of relapse (ir) and graft versus host disease (gvhd). results: one-hundred and thirty patients [58% male, median age at auto-sct, 30 years (range: 18-65)] fulfilled the inclusion criteria. the median time between diagnosis and auto-sct was 16 months (range: 2-174) and the median number of lines prior to auto-sct 2 (2) (3) (4) . disease status at auto-sct was complete response in 32%, partial response in 27% and the remaining 41% were transplanted with active disease. the median time from auto to allo-sct was 3 months (1-6). forty percent underwent an identical sibling allo-sct, 39% unrelated and 21% haplo. tbi was used in 35% of the patients as a part of ric. gvhd prophylaxis was cyclosporine-methotrexate in 36% of the patients, cyclosporine-micofenolate mofetil in 16% and post-transplant cyclophosphamide in the remaining 17%. 91% of the patients engrafted after ric-sct. after a median follow-up of 44 months (6-130), 33% of the patients background: allogeneic stem cell transplantation (allo-sct) is a valid option in patients with refractory/relapsed lymphoma but gvhd remains the major cause of mortality and morbidity. calcineurin-inhibitors (cni) combined with methotrexate or atg is the conventional strategy for preventing gvhd, resulting in an incidence of cgvhd of 41-60%. post-transplant cyclophosphamide (pt-cy) reduces the risk of severe cgvhd and improves survival in acute leukemia patients receiving allo-sct from matched sibling (msd) or unrelated donors (ud). the aim of this retrospective registry-based study was to compare pt-cy-based gvhd prophylaxis to standard prophylaxis in the setting of msd or ud for patients with lymphoma. methods: three thousand eight hundred sixty-four lymphoma patients undergoing an hla -id sct(hla identical stem cell transplantation) registered in promise were included in the study (table 1) . outcomes between pt-cy vs no-pt-cy were compared a) with a multivariate cause-specific cox model adjusted on ric/mac, donor type, source of stem cell, age of the patient, donor gender, patient gender, diagnosis and, disease status at sct; and b) by matching one pt-cy patient (118) with two no-pt-cy patients (217) using the same covariates. results: in univariate analysis, comparing pt-cy and no-pt-cy, the 100-day ci of grade 2-4 agvhd was 26% and 27% (p=0.7), the 1-year ci of non-relapse mortality (nrm) was 12% and 16% (p=0.6) and the 3-year relapse incidence (ri) was 38% and 33% (p=0.2), respectively. the 1-year ci of cgvhd was 32% vs 42% (p=0.1), 3-year pfs for pt-cy and no-pt-cy was 43% and 46% (p=0.5) and 54% and 57% (p=0.9) , respectively. in multivariate analysis, prophylaxis with pt-cy was not associated with a reduced risk of agvhd, overall or extensive cgvhd, nrm, ri, nor with an improved pfs or os. likewise, in the matched-pair analysis pt-cy did not impact on any of these outcomes. conclusions: this study demonstrates that in lymphoma patients who underwent an hla-id sct, gvhd prohphylaxis strategies employing pt-cy-based achieve equivalent transplant outcomes to those seen with cni-based strategies. myeloablative conditioning may contribute to disease control after stem cell transplantation in blastic plasmacytoid dentric cell neoplasia background: blastic plasmacytoid dentric cell neoplasia (bpdcn) is a rare and clinically aggressive hematopoietic malignancy, which preferentially involves the skin, the bone marrow, and, occasionally, the lymph nodes. it mainly affects elderly patients and has a poor prognosis with conventional chemotherapy. the treatment for this condition is heterogeneous, some patients treated with lymphoma-type regimens and others receiving intensive acute leukemiatype chemotherapy. although preliminary case series suggest that hematopoietic stem cell transplantation (sct) could provide sustained disease control in patients with bpdcn, the role of sct and potential graft-versus-tumor effects (gvt) in this condition is yet undefined. methods: between 2009 and 2017 26 patients were included in an ebmt prospective non-interventional study (nis) on the value of sct in bpdcn. these were compared with 133 patients with bpdcn registered with the ebmt during the same time period outside the nis. no differences with regard to overall survival (os), line of treatment or year of transplant was observed between patients included in the nis and the remainder from the ebmt database, and they were therefore analyzed together. results: one hundred and forty-two patients were treated with an allogeneic sct (allosct) and 17 patients with autologous or syngeneic sct (autosyn). the median follow-up was 17 months with no differences between the allosct and autosyn groups. disease status at sct was complete remission (cr) in 88%, and 90% patients received the sct as part of first-line treatment. two-year os after autosyn was 70% (95% ci 46-99%), whereas it was 65% (95% ci 53-83%) following allosct. of those patients who received an allosct, 71% were transplanted from an unrelated donor (ud), and 29% from an identical sibling donor (sib). reduced intensity conditioning (ric) was used in 47% and myeloablative conditioning (mac) in 53% of the patients. in allotransplanted patients, multivariate competitive risk analysis of nrm and relapse incidence considering age, conditioning intensity, line of treatment, remission status prior to allosct and donor type revealed a statistically significant reduction of relapse incidence (p=0.02, hr 0.2 ci 0.1-0.7) without increased nrm for patients who received mac compared to ric. multivariate cox regression analysis for os considering the same co-variates confirmed that mac was associated with a significantly reduced mortality risk (p=0.05, ci 0.4, hr 0.1-0.99) along with being in cr at allosct (p< 0.001, hr 9.1, . conclusions: this study confirms on a large data set that sct is an effective and potentially curative treatment for patients with bpdcn. the superiority of mac and the efficacy of autosyn suggest that apart from gvt, highdose therapy might be an important contributor to long-term disease control in this condition. clinical background: allogeneic hematopoetic cell transplantation (allo-hct) is a curative therapy for patients with relapsed/ refractory and high-risk non-hodgkin lymphoma (nhl). however, no large studies have evaluated the trends in the utilization of allo-hct in elderly nhl patients (≥65 years). using the cibmtr registry we report here a time trends analysis of allo-hct use in elderly nhl subjects methods: we identified 727 nhl patients (≥65 years of age) undergoing a first allohct during 2000-2015 in the united states (u.s.). study cohort was divided into the following time-periods for analysis; 2000-2005 vs. 2006-2010 vs. 2011-2015 . primary outcome was overall survival (os). secondary outcomes included progression-free survival (pfs), relapse/progression (r/p) and non-relapse mortality (nrm). results: baseline patient characteristics are shown in table1. during the three study time-periods median patient age (67-68-year), use of reduced-intensity conditioning regimens and proportion of patients with chemosensitive disease remained stable. in the most recent era (2011-2015) a higher proportion of patients had t-cell nhl, history of prior autografts, good performance status (kps 90-100) and high hct-ci, while fewer subjects received a hlamatched sibling hct. the cumulative incidence of day100 grade 2-4 acute graft-versus-host disease (gvhd) for 2000-2005 vs. 2006-2010 vs. 2011-2015 cohorts was 25% vs. 35% vs. 31% respectively (p=0.47). the cumulative incidence of chronic gvhd at 1 year was 21% vs. 34% vs. 31%, in similar order (p=0.07). the 4-year probabilities of nrm and r/p of 2000-2005 vs. 2006-2010 vs. 2011-2015 time-periods were 34% vs. 29% vs. 30% (p=0.69) and 48% vs. 40% vs. 40% (p=0.39), respectively (figure) . the 4-year probabilities of pfs and os (2000 -2005 vs. 2006 -2010 vs. 2011 -2015 showed significantly improved outcomes in the most recent time-periods as following: 17% vs. 31% vs. 30% (p=0.02) and 21% vs. 42% vs. 44% (p< 0.001), respectively (figure) . on multivariate analysis, compared to the 2000-2005 cohort, the 2011-2015 cohort showed a 28% reduction in the risk of mortality (rr=0.72, 95%ci=0.52-1.008, p=0.056). the most common cause of death was relapse of primary disease in all time-periods. conclusions: utilization of allo-hct has steadily increased in elderly nhl patients in the u.s. since 2000. in the recent years despite decline in the use of hlamatched sibling donors and transplanting elderly patients with higher hct-ci and more heavily pretreated disease, survival outcomes have improved. age alone should not be a determinant for allo-hct eligibility in nhl. methods: four medical experts who had managed patients with dlbcl using different car t-cell therapy protocols and products independently reviewed the extracted adverse event data from the case report forms and re-graded crs using the more commonly used lee scale (lee, blood, 2014) and the nt grading for encephalopathy (modified car t related encephalopathy syndrome (mcres) (neelapu, nature reviews in clinical oncology, 2018) while blinded to the original trial grading and other experts' grading. re-grading assessments and disagreements concerning the assigned lee and nci ctc grades were later discussed and reconciled among reviewers during a live meeting. as per the investigational charter, in cases that could not be reconciled, the most conservative final assessment of any reviewer determined the final grading for any individual case. results: crs: 64 of 111 (58%) patients were originally recorded as crs by penn scale, and 63 were regraded as crs by lee criteria. 18 of 64 subjects received anticytokine therapy overall. with this blinded reassessment, more patients were categorized as grade 1 (lee vs penn: 26 vs 17), fewer patients as grades 2 and 3 (18 vs 23 and 10 vs 15, respectively) and the same number of patients as grade 4 (9 vs 9). nt: results were compared with data on nt determined by nci ctc criteria of tisagenlecleucel, in which nt was broadly defined as the occurrence of any nervous system or psychiatric ae (eg, anxiety, dizziness, headache, peripheral neuropathy, and sleep disorder). 68 of 111 subjects were identified as having nt by ctc criteria: 34 (30.6%) patients were identified as having grade 1/2 nt, 11 (9.9%) patients as having grade 3 nt,& 5 (4.5%) patients as having grade 4 nt. evalution by mcres grading system revealed low rates of encephalopathy/delirium: 5 (4.5%) patients had grade 1/2 nt, 6 (5.4%) patients had grade 3 nt, and 8 (7.2%) patients had grade 4 nt. no grade 5 events were seen and the presence of crs was associated with higher likelihood of concomitant nt. conclusions: these results demonstrate difficulties in identifying cross protocol toxicity assessments. harmonized grading scales being developed for future studies will facilitate comparison of the safety profiles of different car t-cell and other immune effector cell products. further analyses are ongoing of these data with the new asbmt consensus crs guidelines. recent studies show that mouse bone marrow tregs localize in the hematopoietic stem cell (hsc) niche, where they contribute to hscs maintenance and promote donor engraftment and b cell lymphopoiesis. we are investigating if human tregs promote b cell reconstitution and immunity in preclinical models and in haplo-hct patients. methods: b cell reconstitution was analysed monthly by facs in bone marrow (bm) and peripheral blood (pb) samples from 51 patients who underwent either treg/tcon haplo-hct (33 patients), or t-cell depleted haplo-hct (8 patients) or haplo-hct with high dose post-transplant cy (ptcy, 10 patients). diagnosis was acute leukemia in 39 patients, lymphoma in 9 and multiple myeloma in 3. pb total immunoglobulin (ig) and anti-cytomegalovirus (cmv) igm were also monitored together with cmv viremia. for the mouse model, donor derived human treg and cd34 + hematopoietic stem cells (hscs) were coinfused in sublethally irradiated (2 gy) immune-deficient nsg mice and donor engraftment and b cell reconstitution were analysed in mouse pb twice a month by facs. results: b cell reconstitution was faster after treg/tcon haplo-hct when compared to other haplo-hct protocols. b cell counts were higher in pb of patients that received treg/ tcon haplo-hct (p = .02) and were comparable to those of healthy subjects by 4 months after transplant (131±121 cells/ mm 3 , fig.1a ). we could detect early frequencies of cd34 + cd38 + cd10 + cd127 + common lymphoid progenitors, cd45 + cd10 + cd38 + cd19 -pre/pro-b, cd45 + cd10 + cd38 + cd19 + pre-b, and pro-b cells in the bm of these patients, that resulted in an increased production of cd38 + cd19 + cd5 -igm + immature b cells, cd38 + cd19 + cd5 + igm + transitional b cells and cd19 + cd20 + mature b cells. we used a mouse model of xenotransplantation to understand whether donor b cell reconstitution in treg/ tcon haplo-hct is boosted by a treg-mediated effect on donor human hscs. we found that infusion of human tregs facilitated donor hsc engraftment. hsc-derived mature b cells were rapidly abundant and easily detectable 60 days after hsc infusion in pb of treg-treated animals. to evaluate donor b cell function we analysed ig production in response to cmv reactivation in transplanted patients. post-transplant hypogammaglobulinemia was rapidly corrected in treg/tcon haplo-hct patients. total igm were higher compared to other haplo-hct protocols and reached normal levels by 3 months after transplant (152 ±194 mg/dl, fig.1b ). cmv reactivation rate was similar among haplo-hct protocols (~73%), but it occurred later after treg/tcon immunotherapy (45+/-17 days vs 33 +/-11 days). new production of anti-cmv specific igm was documented in 60% of cmv seropositive patients 76 +/-33 days after treg/tcon haplo-hct, while anti-cmv specific igm were undetectable after other haplo-hct protocols within the first 6 months after transplant. [ background: allogeneic cell transplantation (hsct) success prediction is partly based on minimal residual disease (mrd) and hematopoietic chimerism testing. we developed a droplet digital pcr platform (dpcr) for the simultaneous detection of mrd and hematopoietic chimerism. methods: a panel of 12 deletion/insertion polymorphic markers and frequent molecular targets used for mrd testing: npm1, runx1-runx1t1 (t 8;21), dnmt3a, mll-ptd, cbfß-myh11 (inv 16), kras, mll-af10, idh 1/2, ckit, bcr-abl (t9; 22), evi1 and wt1 expression were included in a single dpcr platform for mrd and hematopoietic chimerism analysis. a total of 225 patients were evaluated with a mean follow-up of 653 days (range: 30-5895 days). results: hematopoietic chimerism analysis revealed mixed chimerism (mc) in 93 patients (41% of all patients). mc was detected more frequently in patients with reduced intensity conditioning (68%) when compared with full conditioning (47%). the mean percentage of host derived dna in peripheral blood was 13% (range: 0.1-90%). three different patterns of mc were observed: increasing, decreasing or stable mc. in those patients with stable or decreasing mc (n=46), as well as patients with complete donor chimerism (n=132) the molecular targets used for mrd monitoring were not detectable. in 47 out of 93 patients, increasing mc was detected. this group of patient showed in addition either a positive mrd marker, increased wt1 expression or both. in 37 patients with increasing mc, a positive mrd marker and increased wt1 expression hematologic relapse of the underlying disease was observed. in patients with increasing mc and a positive mrd marker, we analysed whether mc or the molecular target for mrd was first detected. in 40% of the cases mc was detected before the molecular marker used for mrd assessment, while in 13% of the patients mrd positivity was detected before mc. in the remaining patients (47% of the patients) mc and mrd positivity were detected simultaneously. the mean time between either mc or mrd detection in peripheral blood and relapse was 116 days (range: 15-385 days). patients with increasing mc and mrd positivity, whether or not they responded to treatment, showed a similar kinetic pattern for the chimerism and mrd markers. in those patients that responded to molecular relapse treatment (n=10) the mean time to achieve complete donor chimerism or mrd pcr negativity was 305 days and 294 days respectively. conclusions: the combination of mrd and chimerism markers in a dcpr platform represents a sensitive and accurate diagnostic tool for the comprehensive assessment of the molecular remission status after hsct. in addition, by using the developed dpcr platform costs and turnaround times can be reduced. disclosure background: minimal residual disease (mrd) monitoring can help to indicate impending relapse of acute leukemia after allogeneic hematopoietic stem cell transplantation (allo-hsct). because impending relapse can be altered with early detection of low-volume disease and timely therapies, preemptive intervention is a reasonable option for patients with mrd which can spare those in remission from further therapy. chemotherapy plus donor leukocyte infusion (chemo-dli) is the most important preemptive intervention, but it may lead to several complications, such as severe graft-versus-host disease (gvhd) and pancytopenia. hypomethylating agents (hmas) represent another potential preemptive intervention, but it only delayed the time to hematologic relapse and the long-term efficacy may be unsatisfactory. thus far, few studies had identified the longterm efficacy of preemptive intervention with drugs in patients with mrd after allo-hsct.two prospective studies (nct02027064 and nct02185261) reported that preemptive interferon-α (ifn-α) treatment can help clear minimal residual disease (mrd) and prevent relapse after allogeneic hematopoietic stem cell transplantation (allo-hsct). in this extension study, we aimed to identify the long-term clinical outcomes of preemptive ifn-α treatment in acute leukemia patients who were mrd positive after allo-hsct (n=118). methods: mrd was monitored by multiparameter flow cytometry (mfc) and taqman-based reverse transcriptionreal time polymerase chain reaction (pcr). a patient was considered to be mrd-positive when a single bone marrow sample tested positive by pcr or mfc. recombinant human ifn-α-2b injections were administered subcutaneously for 6 cycles (twice or thrice weekly in every 4-week cycle). results: the 4-year cumulative incidence of total chronic graft-versus-host disease (cgvhd) and severe cgvhd after ifn-α treatment was 58.5% (95% ci, 49.5-67.5%) and 10.2% (95% ci, 4.7-15.7%), respectively. the 4-year cumulative incidence of relapse and non-relapse mortality (nrm) after ifn-α treatment was 16.1% (95% ci, 9.4-22.8%) and 5.6% (95% ci, 1.1-10.1%), respectively. the 4year probabilities of disease-free survival and overall survival (os) after ifn-α treatment were 78.3% (95% ci, 70.6-86.0%) and 84.0% (95% ci, 77.1-90.9%). in multivariate analysis, severe acute gvhd was associated with a higher risk of nrm and poorer os, and mild to moderate cgvhd was associated with a lower risk of relapse and better survival. conclusions: these data confirmed that preemptive ifnα treatment showed long-term efficacy in patients who were mrd-positive after allo-hsct. clinical trial registry: the study was registered at http://clinicaltrials.gov as #nct02185261 and #nct02027064. disclosure: the authors declare no competing financial interests. a phase ii clinical trial of leuprolide for enhancement of immune reconstitution after ex vivo cd34+ cell-selected allogeneic hct with tbi-based conditioning gnrh agonist use, which has been associated with thymic cellular degeneration in mouse (velardi, jem 2014). as direct gnrh antagonism might circumvent this effect, a follow-up phase ii trial evaluating peri-transplant degarelix for enhancement of immune reconstitution is in progress. background: in allogeneic hematopoietic stem cell transplantation (allo-hsct) recipients, cytomegalovirus (cmv) reactivation and disease are frequent causes of morbidity and mortality, that may be evaded by cmv-specific t cell reconstitution. methods: we designed a prospective, single-center observational study to assess if the kinetic and quality of cmv specific t-cell reconstitution impact the incidence and severity of cmv reactivations. we report data on the first 54 consecutive patients affected by hematological malignancies receiving allo-hsct followed by cyclophosphamide and rapamycin between december 2017 and august 2018. patients received allo-hsct from family (sib-lings=9; hla haploidentical=21), unrelated hla matched (n= 23) donors or cord blood (n=1). the cmv serostatus of host (h) and donor (d) pairs was: h + /d + (n=36, 67%), h + /d -(n=17, 31%) and h -/d + (n=1, 2%); h -/dwere excluded. cmv dnaemia was assessed weekly in whole blood (wb). absolute numbers of polyclonal and cmv-specific t cells were quantified by flow cytometry using trou-count™ tubes (bd) and dextramer® cmv-kit (immu-dex), respectively, in the graft and fresh wb at days -7, +30, +45, +60, +90, +120, +150 and +180. dextramers permit the identification of cmv-specific lymphocytes restricted for several hla class i molecules: a*01:01/ *02:01/*03:01/*24:02 and b*07:02/*08:01/*35:01. these alleles allowed the longitudinal evaluation of 48 (89%) patients. results: at a median follow-up of 150 days post-hsct, 28 (58%) patients experienced a cmv-related clinically relevant event (cre, median +50 days), including 7 patients (15%) with cmv disease. for each time-point, we compared the absolute number of cmv-specific lymphocytes in patients experiencing or not a subsequent cre. at +45 days, we observed lower cmv-specific cd8 + t cells in patients prone to reactivate cmv than in not reactivating patients (median cmv-specific cd8 + cells/ ml= 0.125 vs 1.69, p=0.026). furthermore, patients with any dextramer positivity at +45 days displayed a lower incidence of cre compared with subjects who were negative (cre probability: 0.55 vs 0.83, p=0.038). patient stratification based on different thresholds of dextramerpositive cells confirms the inverse association between cre and cmv-specific immunity (cre incidence in patients with: 0 cells/ml=0.83, < 1 cells/ml= 0.66, ≥ 1 cells/ml= 0.36; p=0.046). we observed a higher cre incidence in cmv h + /dpairs than in h + /d + (0.89 vs 0.44, p=0.03). taking advantage of the hla mismatched-hsct setting, we then dissected cmv-specific t-cell response according to hla restriction elements (h/d=shared n=40, drestricted n=12, h-restricted n=19). in h + /d + pairs, we observed a fast and similar kinetic of reconstitution of cmv-specific lymphocytes restricted by h/d and d hlas. conversely, in h + /dpairs, we detected only cmv-specific cd8 + lymphocytes restricted for h/d haplotypes. hostrestricted cells remained undetectable for the first 150 days after hsct. conclusions: when the donor is cmv seropositive, a rapid and effective reconstitution of cmv-specific d-and h/d-restricted memory t cells occurs. if the donor is cmv seronegative, only h/d-restricted lymphocytes are observed early after allo-hsct in h + /dpairs. these findings indicate that cmv reactivation can prime h/d-restricted t cells presumably educated in the donor thymus; conversely, d-and h-restricted donor-derived lymphocytes have not yet undergone neither cross-priming nor thymic education, which might be required for full protection from cmv. disclosure: c.b. received research support from molmed s.p.a. and intellia therapeutics. l.b. is employed by immudex aps. non of the other authors has any relevant conflict of interest to disclose. o148 5-azactidine is safe and effective therapy for prevention of disease relapse in high-risk patients with acute myeloid leukemia and myelodysplastic syndrome following allogeneic stem-cell transplantation ivetta danylesko 1 , noga shem-tov 1 , adriana del-giglio 1 , ronit yerushalmi 1 , arnon nagler 1 , avichai shimoni 1 background: allogeneic stem-cell transplantation (sct) is curative approach in patients with aml or mds. however, disease recurrence is the major cause of treatment failure. 5azacitidine has been used in standard treatment of mds and also in aml patients not eligible for standard chemotherapy. there is limited data on the safety and efficacy of azacitidine after sct. we explored the use of low-dose azacitidine for prevention and treatment of relapse of aml/ mds after sct. methods: patients in cr after sct who were considered to be at high-risk for relapse were given azacitidine at 32mg/m 2 for 5 days every 28 days, planned for 2 years. patients with overt relapse after sct were given 32-75 mg/ m 2 for 5 days until progression. results: ninety-four patients, median age 64 years (24-77) were given azacitidine after sct from hla-matched sibling (n=29), matched-unrelated (n=64) or haploidentical (n=1) donors. diagnosis was aml (n=66) or mds (n=28). the conditioning regimen was myeloablative (n=56) or reduced-intensity (n=38). 22 patients were given prophylactic azacitidine; 15 were in cr after sct but at high-risk for relapse due to active disease (n=7) or positive minimal-residual disease (mrd) (n=3) prior to sct, or poor-risk cytogenetics (n=5). seven patients were given azacitidine as secondary prevention after achieving cr with chemotherapy for post-transplant relapse. 19 patients were given azacitidine pre-emptively for mixed-chimerism, positive mrd after sct or early relapse (< 10% marrow blasts). patients in the combined prophylactic/ preemptive group (n=41) started azacitidine in a median of 2.4 months (1.2-14.9) after sct and received a median of 8 courses . 4 patients were also given donor-lymphocyte infusions (dli) concomitantly with azacitidine. 23 are still on therapy, 12 died or progressed, 2 stopped after long remission, 3 stopped due to patient request and only 1 discontinued due to toxicity. 17 of 22 patients given prophylactic azacitidine remained in cr. 16 of 19 patients given azacitidine preemptively achieved cr and 10 remained in cr. with median follow-up of 14 months , 30 of the 41 patients in the prophylactic/ pre-emptive group are alive and 11 died. the estimated 3-year os and pfs are 58% (95%ci, 29-86) and 50% (95%ci, 27-74), respectively. the expected pfs in this group of high-risk patients is less than 20%. 53 patients were given azacitidine for overt relapse after sct. patients were given a median of 3 courses (2-36) and 16 were also given dli. 10 patients achieved cr, 10 stable disease and 33 progressive disease. 5 patients are still on therapy, 44 died or progressed and 4 had to discontinue due to hematological toxicity. with median follow-up of 15 months , 12 are alive and 5 are progression-free. and 7% (95%ci, 0-13), respectively. conclusions: azacitidine is safe and effective therapy when used prophylactically in high-risk aml/mds patients to prevent relapse or preemptively to treat mrd or early relapse after sct. azacitidine maintenance may improve outcome in this high-risk patient group and should be further explored. results of azacitidine treatment in overt post-transplant relapse are limited. disclosure: nothing to declare novel mass cytometry analysis identifies reciprocal changes in nkreg and cd4 em as the dominant early immune reconstitution signature associated with subsequent acute gvhd after ric-ahst background: treatment failure after allogeneic haematopoietic stem-cell transplantation (ahst) using reducedintensity conditioning (ric) results from either too much alloreactivity and harmful acute graft-versus-host disease (agvhd) or not enough (reducing graft-versus-tumour effects). studies have identified individual reconstituting immune cell subsets associated with development of clinical alloreactivity but the functionally dominant parameters remain unknown. we therefore used mass cytometry (ms) technology to determine multiple parameters simultaneously to identify dominant cellular immune reconstitution signatures associated with development of clinical alloreactivity after ahst. methods: phenotypic markers identifying >30 t, b and nk cell subsets known to influence alloreactivity were combined in a single ms panel. peripheral blood from 52 patients with haematological cancer was analysed at d+30 after t-replete hla-matched ric-ahst using uniform conditioning. test samples were spiked with cd45barcoded healthy control cells. three complementary high-dimensional analytic tools were used to identify immune signatures in cd45 + lineage + cells across the whole cohort and identify differences between patients grouped by subsequent occurrence of agvhd. results: significant batch effects were effectively reduced with a novel r-based algorithm normalising data to control cells. unsupervised clustering analysis using phenograph and flowsom algorithms identified 24 and 40 phenotypically distinct clusters respectively. diversity analysis demonstrated lower cluster diversity (p=0.06) in the 20 patients who subsequently developed agvhd consistent with perturbation of phenotypic clusters in these patients. comparison of individual cluster abundance identified 2 cluster groups significantly different between patients who subsequently developed agvhd and those who did not. a cluster containing cells with a cd56 bright cd16 neg cd27 +/regulatory/tolerant nk cell (nkreg) phenotype was significantly reduced in patients who subsequently developed agvhd using both phenograph and flowsom algorithms (p< 0.001). a differentiating cell population with this phenotype was also identified in forward analysis using the citrus algorithm. notably, this reduction in nkreg was accompanied by a significant increase in abundance of a cluster of ccr5 + cd45ra -ccr7 -cd4 effector memory t-cells (tem) in patients who subsequently developed agvhd. there was a significant negative correlation (p=0.01) between nkreg and tem. in contrast there was no inverse correlation between cd4 regulatory t-cells and cd4em. these changes were independent of clinically significant cmv reactivation. finally, we determined the impact of time to agvhd on this novel immune signature. reciprocal changes in nkreg and cd4 tem abundance were more significant in patients who developed agvhd before d60, consistent with a dynamically evolving immune signature. conclusions: we show proof-of-concept that a novel pipeline can be applied to ms data to measure multiple immune reconstitution parameters after ahst. importantly, this pipeline identified concomitant loss of nkreg and increase of cd4 tem in patients who subsequently developed agvhd. this is consistent with loss of nk cell-mediated control of alloreactive cd4 tem cells as the dominant immune process preceding the development of agvhd after ric-ahst. our data provide mechanistic insight into evolution of alloreactivity and support the development of strategies to maintain or expand nkreg numbers early post-transplant to reduce harmful agvhd. [[o149 image] 1. dominant immune reconstitution signature early after ric-ahst associated with subsequent acute gvhd] multiple myeloma o150 abstract already published. impact of high-risk cytogenetics in newly diagnosed multiple myeloma undergoing upfront stem cell transplantation: a study from the ebmt chronic malignancies working party background: current consensus identifies t(4;14), t(14;16), t(14;20), gain and/or deletion in chromosome 1, and del(17/ 17p) as high-risk cytogenetics in newly diagnosed multiple myeloma (ndmm). however, evidence on outcome of specific abnormalities after transplantation as first-line treatment is limited. we analyzed high-risk ndmm patients reported to the european society for blood and marrow transplantation (ebmt) registry undergoing upfront stem cell transplantation. methods: upfront transplantation was defined as first autologous transplant within 12 months from mm diagnosis. survival and cumulative incidence were calculated from date of first transplant (95% confidence interval). end points were progression-free survival (pfs), overall survival (os), relapse and non-relapse mortality (nrm). cox model with hazard ratios (hr) was used for multivariable os analyses and cumulative incidence method for relapse incidence and nrm. results: within the ebmt registry, 623 high-risk ndmm patients according to cytogenetics underwent single autologous (n=446), tandem autologous (n=105), autologous-allogeneic (n=72) stem cell transplantation between 2000 and 2015. the median follow-up of all patients was 58 months (95% ci, 52-63 months), the median age was 59 years (range, 25-76 years) and the median time between diagnosis and transplantation was 5.6 months (range, 2.2-11.7 months). frequencies according to cytogenetic were: del(17) (n=333, 54%), t (4;14) (n=344, 55%), gain or deletion in chromosome 1 (n=85, 14%), t(14;16) (n=19, 3%). two or more cytogenetic abnormalities were documented in 143 patients (23%). 56% of patients were male and most patients had igg (53%) or iga (26%) paraproteins. a karnofsky performance status < 90% had 29% of patients while frequencies according to international staging system (iss) i/ii/iii were 21%/57%/22%. complete remission (cr) at time of transplantation was achieved by 18%. in univariable analysis, presence vs absence of del (17) conclusions: in ndmm patients with at least one highrisk cytogenetic abnormality undergoing upfront transplantation, outcome was similar between del(17) and t(4;14) while the presence of two or more high-risk cytogenetic abnormalities showed significantly worse os compared with only one high-risk abnormality. disclosure: nothing to declare. the role of renal impairment at diagnosis in multiple myeloma undergoing autologous transplantation. a retrospective analysis of the cmwp background: renal impairment (ri) is frequent in newly diagnosed myeloma patients and is considered to be a risk factor for worse overall survival. with active myeloma therapy renal function often improves or even normalises. however, it is unclear whether renal impairment at diagnosis is a persisting biological risk factor or rather a potentially reversible organ complication. methods: from the ebmt calm study database all myeloma patients having received a first autologous transplant between 2008 and 2012 with information on renal function both at diagnosis and at transplant were extracted. renal function was classified according to the calculated glomerular filtration rate (gfr) rate as normal ("normal", gfr > 50 ml/min), moderately impaired ("moderate", gfr 30-50 ml/min) or severely impaired ("severe", gfr < 30 ml/min). categorial variables were tested by chi-square test. os was determined from transplantation and calculated by kaplan meier with logrank testing. results: 1905 patients fulfilled the selection criteria and were included. at diagnosis, 1447 patients had normal, 184 moderate and 274 severe ri. median age at diagnosis was 58, 60 and 59 years in the ri subgroups. genetic information was available in only a subgroup of patients. t(4;14) was present in 14%, 22% and 13% respectively and del17 was found in 7%, 8% and 4%. bortezomib-based induction therapy was given in 53%, 58% and 64% of cases (p=0.018). os differed significantly between the ri groups with a median of 84, 72 and 62 months, respectively (p< 0.001, fig 1) . in contrast renal function at transplant had no impact on os with a median of 78 months (no ri at transplant), 79 months (moderate ri at transplant) and not reached (severe ri at transplant). most of the 274 patients with severe ri at diagnosis had improved their renal function by the time of transplantation. however, this did not positively impact on os: patients with no ri at transplant had a median os of 37 months (n=132), while it was 65 months for moderate ri (n=71) and not reached in patients transplanted with persisting severe ri (n=71,p< 0.001). conclusions: from this large analysis including almost 2000 myeloma patients renal impairment at diagnosis has been found to be a risk factor for os, while renal function at transplant did not impact on post transplant survival. these findings support the safety and efficacy of autologous transplantation in patients with severe ri at transplant. on the other hand improving renal function between diagnosis and transplant does not seem to improve prognosis.. our analysis supports the notion that ri at diagnosis appears to be a surrogate parameter for a more aggressive disease course. disclosure: nothing to declare fig.1 oerall survival according to ri at diagnosis] analysis of outcomes in patients with myeloma who had a second allohct either for disease relapse or graft failure: an ebmt cmwp study background: the options for patients with myeloma (mm) who relapse or develop graft failure after an allosct are limited. a second allohct is occasionally feasible though is high-risk. we performed a retrospective analysis to assess outcomes in this cohort. methods: data on patients with mm who underwent a second allohct at ebmt centres between 1994 and 2017 were obtained from the ebmt registry. results: a total of 273 patients (165 m,108 f) with mm (51% igg, 22% iga, 23% lc) underwent a second allosct. the median (range) age at the first allohct was 48.5 (20.1-67.9) years and 78.1% were >/=pr. when comparing the indications for the 2 nd allohct -relapsed mm (74%) or graft failure (26%) -patients with graft failure were significantly more likely to have received a mismatched (related/unrelated) or unrelated donor for the 1 st allosct (58% vs. 31%) (p=0.001), were more commonly female (57% vs. 38%) (p=0.018) and were more likely to have had a ric as opposed to a mac allosct (79% vs. 59%) (p=0.015). the median (range) interval between the 1 st and 2 nd allohcts was 3.5 (7.7-66.6) months in cases of graft failure and 40.5 (68.6-170.4) months for those who had relapsed. at a median (95% ci) follow-up of 107.2 (84.9 -141.7) months following the second allohct, overall survival (os) was 41% (35-47%) at two years and 26% (20-32%) at five years. there was no difference in os at five years based on the indication for allohct: 25% (17-32%) for relapse and 32% (19-45%) for graft failure (p=0.595) (figure 1 ). neutrophil engraftment following the 2 nd allohct was achieved by day +28 in 94% (90-98%) and 80% (69-92%) of the relapse and graft failure patients, respectively. the cumulative incidence of agvhd (ii-iv) and cgvhd (at five years) following the 2 nd allohct was 27% (21-33%) and 39% (32-47%), respectively.relapse-free survival was 12% (7-17%) at 5 years, 7% (2-13%) in those transplanted for disease relapse and 22% (10-35%) in those transplanted for graft failure (p=0.061). the cumulative incidence of relapse and nrm at five years was 64% (57-71%) and 24% (18-30%), respectively. the five-year os following the second allosct was 14% (1-28%) for those relapsing between 12 and 24 months after the first allohct and 31% (21-40%) for those relapsing later than 24 months (p=0.07).on univariate analysis, os at five years was superior in patients who had had hla identical sibling donors as opposed to other donor sources: 32% (24-40%) vs. 17% (9-25%) (p< 0.001). on multi-variate analysis, donor source (hla identical sibling vs. other) remained a predictive factor for os (p=0.014). conclusions: in this high-risk mm cohort, one quarter of patients remained alive five years after the 2 nd allohct with similar outcomes seen following disease relapse and graft failure. however, the relapse-free survival rate was low in those transplanted for relapsed mm. later relapses after the first allohct appear to fare better and the best outcomes are seen using matched sibling donors. a 2 nd allohct therefore remains an option to be considered for selected mm patients. disclosure: nothing to declare minimal residual disease (mrd) ratio before and after autologous stem cells transplantation (asct) in multiple myeloma (mm) riccardo boncompagni 1 , michela staderini 1 , chiara nozzoli 1 , elisabetta antonioli 1 , barbara accogli 1 , riccardo saccardi 1 1 careggi university hospital, florence, italy, background: in the last ten years, multiparametric flow cytometry (mfc) has been standardized and routinely applied for the detection of mrd as a prognostic factor in mm patients across different lines of therapy. we assessed the mrd carried out before and after asct in a series of consecutive mm patients in order to investigate whether the ratio of the two determinations might increase the prognostic potential. methods: we collected bone marrow samples for mrd assessment at the end of induction therapy and 3 months after asct from 61 mm patients treated between 2013 and 2017 achieving at least a very good partial remission (vgpr) with a bortezomib-based induction therapy, according to the most recent international myeloma working group (imwg) criteria (kumar s et al, lancet oncol 2016) . mfc-determined mrd was evaluated according to euroflow recommendations (kalina t et al, leukemia 2012) . all patients were examined with 18fluorodeoxyglucose positron emission tomography/computed tomography (fdg-pet/ct) scan before and after the asct. results: post-induction therapy mrd was found predictive of post-asct mrd status. indeed, patients transplanted in a mrd positive status had a significantly increased risk to maintain a mrd positivity status after transplantation (odds ratio -or -15,053, p = 0,002). detection of post-asct mrd had a negative impact on median pfs (28 months vs not reached respectively, p = 0,001). in cox-regression analysis, a complete remission status (cr) with an undetectable mrd after the asct resulted to be the major protective factor from relapse (hazard ratio -hr -0,012, p = 0,005), while patients with a detectable mrd before and after the asct had the worse pfs (22 months, hr 2,958; p = 0,029). risk analysis showed 3 different pfs risk groups: "high" for the patients with mrd detectable before and after the asct, "intermediate" for patients with mrd positivity before the asct who achieve a negativity after, and "low" in the case of mrd undetectable before and after. in our study, response evaluated by fdg-pet/ct showed no correlation with pfs. conclusions: multiparametric flow cytometry is a relatively recent method to assay mm mrd, and its role in mm therapeutic path is still under investigation. according to our data, a detectable mrd after the asct is a major relapse risk. interestingly we found that it can be early predicted by the post-induction mrd status and its negativization after asct has a modest impact on this. therefore, we support the concept of treatment escalation when a cr is not reached after the induction treatment, in order to undergo to the asct in the best possible response. however, double mrd determination before and after vtd, with ≥60% of patients achieving a best response of ≥vgpr. the orr in patients receiving 'other' induction therapies was 71% and the ≥vgpr rate was 29%. finally, following auto-sct, the orrs for patients receiving vtd induction were around 80%, 60%, and 33% in lines 2-3, 4, and 5+, respectively. conclusions: this analysis provides prospective, realworld data on therapy of patients with mm receiving auto-sct. vtd is the most widely used induction regimen prior to auto-sct. moreover, the response rates are in line with reported rates in phase iii clinical trials. while other induction regimens are being developed, vtd is likely to remain a standard of care, because access to novel agents will continue to vary greatly from country to country due to factors such as affordability, local guidelines/restrictions, and regulatory decisions. background: car-t cell therapy against the cd19 antigen is a breakthrough treatment for patients with relapsed/ refractory (r/r) b-cell non-hodgkin lymphoma (nhl). despite impressive outcomes, non-response and relapse with cd19 negative disease remain challenges. through dual b-cell antigen targeting of cd20 and cd19, with a first-in-human bispecific lentiviral car-t cell (lv20.19car), we attempt to improve response rates while limiting relapses due to cd19 antigen loss. production was optimized with point of care automated manufacturing using the clinimacs prodigy, a compact gmp compliant tabletop device in an iso7 clean room. methods: patients were treated on our phase 1 dose escalation + expansion trial (nct03019055) to demonstrate feasibility of point of care manufacturing and safety of a bispecific 41bb/cd3z lv20.19car t cell for adults with r/r b-cell nhl. safety was assessed by incidence of dose limiting toxicities (dlts) within 28 days postinfusion. dose was escalated in incremental 3+3 fashion with a starting dose of 2.5 x 10 5 cells/kg and a target cell dose of 2.5 x 10 6 cells/kg. lymphodepletion was with fludarabine 30 mg/m 2 x 3 days and cyclophosphamide 500 mg/m 2 x 1 day. patients received either fresh uncryopreserved car-t cell infusions (n= 7) or cells thawed (n= 3) after cryopreservation. results: 10 patients have completed treatment: 9 patients in dose escalation and 1 patient in dose expansion. median age was 55 years (46-67) and histology included dlbcl in 4 patients, mcl in 4 patients, and cll in 2 patients. in dose escalation, 3 patients were treated at 2.5 x 10 5 cells/kg, 3 patients at 7.5 x 10 5 cells/kg, and 3 patients at 2.5 x 10 6 cells/kg with no dlts to report. no patient experienced grade 3-4 cytokine release syndrome (crs) or grade 3-4 neurotoxicity (ntx) allowing start of a dose expansion cohort at the 2.5 x 10 6 cells/kg level. in total, 6 patients had grade 1-2 crs and 3 patients had grade 1-2 ntx. mean time to crs was day +9 post-infusion and no patient required icu level care. 4 patients required 1-2 doses of tocilizumab. the day 28 overall response rate for all patients was 80% with 5/10 achieving a complete response (cr) and 3/10 achieving a partial response (pr). all patients in cr remain in remission, the longest 15 months from treatment. car-t persistence is demonstrated in figure 1 . two patients had progressive disease (pd) at day 28 and 2 patients with pr, eventually progressed. all progressing patients underwent repeat biopsy, and all retained either cd19 or cd20 positivity. target dose lv20.19 car t cells were produced in all patients indicating 100% feasibility of our manufacturing process. conclusions: phase 1 results from the first-in-human bispecific lv20.19 car t clinical trial demonstrate that near patient manufacturing and infusion of 2.5 x 10 6 cells/ kg is safe for further investigation with no dlts among treated patients. point of care production logistics aided the administration of fresh car-t cells in the majority. with limited toxicity and 60% sustained response in this relapsed refractory population, this approach to car-t production and dual b-cell targeting merits further investigation. o159 car-t cell therapy bridging to allogeneic hematopoietic cell transplantation for patients with refractory and relapsed acute lymphoblastic leukemia jia chen 1,2 , yi fan 1,2 , yang xu 1,2 , suning chen 1,2 , huiying qiu 1,2 , xiaowen tang 1,2 , yue han 1,2 , chengcheng fu 1,2 , depei wu 1, 2 background: refractory and relapsed (r/r) acute lymphoblastic leukemia (all) always leads to a dismal outcome. allogeneic hematopoietic cell transplantation (hct) is the only potentially curative modality for r/r all, but the long-term survival post-hct remains unsatisfying. car-t cell therapy targeting to cd19 produces promising response for r/r all patients, but the recurrence is the major concern. we investigated the effectiveness of a tandem protocol using car-t cell therapy followed by allogeneic hct. methods: we conducted a prospective study to enroll the patients with r/r all. major inclusion and exclusion criteria are: 1) definitely diagnosed as all; 2) primary refractory (failed to achieve cr after induction) patients or relapsed patients with no response for salvage therapy; 3) with an available donor for allogeneic hct; 4) without severe organ dysfunction or uncontrolled infection. patients enrolled received car-t cell therapy targeting to cd19 with a total dose of 5~10×10 6 /kg of recipient weight, and the preparative regimen before car-t cell infusion consisted of fludarabin and cyclophosphamide. after the evaluation at 30 days post car-t cell infusion, patients started the allogeneic hct procedure using a myeloablative conditioning (modified bu/cy). the control group consisted of patients with r/r all and underwent allogeneic hct without car-t therapy in the same time frame. results: totally 24 patients were enrolled in this study from december, 2016 through april, 2018, including 17 primary refractory patients and 7 relapsed patients (details in table 1 ). twenty patients (83.3%) achieved cr after car-t cell therapy, and 6 patients (25%) developed grade 2 or higher crs. no irreversible toxicities emerged and all the patients moved to the hct procedure. when comparing with the control group (figure 1) , the 1-year cumulative incidence of relapse was 14.8 ± 8.0% for the trial group versus 46.4 ± 12.4% for the control group (p = 0.090), and 1-year overall survival was 71.4 ± 17.1% versus 54.0 ± 11.4% (p = 0.067). conclusions: we concluded that car-t cell therapy bridging to allogeneic hct is a promising approach for r/r all patients, which leads to both high response and low risk of relapse. besides, car-t cell therapy is a safe modality as salvage treatment for r/r all patients, which had little negative impact for following hct. methods: to test the hypothesis that haploidentical hct would be a valid option for high-risk pediatric aml patients lacking a matched donor, we designed a diseasespecific, multi-centre study. we retrospectively analyzed 179 consecutive patients under 18 years with high-risk aml underwent matched sibling donor (msd) (n=23) or haploidentical donor (hid) hct (n=156) between july, 2013 and dec, 2017. a 1:3 ratio matched pair analysis was implemented with the following matching factors: cytogenetic risk, disease status (cr1/cr2/>cr2), age and sex of patients, sex of donor, and graft type. results: all patients achieved myeloid recovery with a median time of 15d and 13d for msd cohort and hid group (p=0.002). the cumulative incidence of grade ii-iv acute graft-versus-host-disease (gvhd) in msd cohort (13%) was significantly lower than in hid group (35%, p=0.048); the incidence of chronic gvhd was comparable between the two groups. the cumulative incidence of relapse in msd cohort (39%) was significantly higher than in hid group (16%, p=0.037); the incidence of nrm was 0 and 10% (p=0.12), respectively. the 3-year overall survival (65% versus 75%, p=0.68) and leukemia free survival (61% versus73%, p=0.29) were comparable in msd-hct compared with hid-hct. in a multivariate analysis, hid-hct remained a significant factor for reduced relapse rate (hr 0.259(0.092-0.731), p= 0.011) in comparison with msd-hct. in subgroup analysis for patients with known cytogenetics and transplanted in the first complete remission (n=58), the cumulative incidence of relapse in msd cohort (50%, n=14) was significantly higher than in hid group (9%, n=44, p=0.001); and leukemia free survival (50% versus 81%, p=0.021) were significantly lower in msd-hct compared with hid-hct. in a multivariate analysis among these subgroup of patients, hid-hct remained a significant factor for increased lfs (hr 0.304(0.102-0.905), p= 0.032) in comparison with msd-hct. conclusions: in conclusion, unmanipulated haploidentical-hct achieves outcomes comparable to those of isd-hct for high-risk pediatric aml patients and even exerts greater gvl effect in some circumstances. such transplantation was proved to be a valid alternative treatment for high-risk pediatric aml patients lacking a matched donor. larger prospective studies are needed to confirm these findings. disclosure: nothing to declare. background: allogeneic hematopoietic stem cell transplantation (hsct) is the only curative option for patients with beta thalassemia major. although limited study in the literature has evaluated the impact of age on success of transplantation, more data are needed. in this study, we aimed to evaluate the effect of age of the patients on transplant outcome in cases who underwent hsct with the diagnosis of thalassemia major. methods: all cases who underwent stem cell transplantation with thalassemia major were included. all thalassemia major patients with a median age of 7 years (range 7 month,17.7 years) underwent allogeneic hsct using myeloablative conditioning regimen. cyclosporine and methotrexate were used as gvhd prophylaxis. in total, 169 patients underwent hsct at age younger than 7 and 159 patients underwent at age older than 7 years and all patients were assigned to two different groups according to transplantation age. the distribution of donor type and stem cell sources by age groups is shown in table 1 . no statistical difference was found between the two age groups in terms of donor type and stem cell source. patients in two different age groups were compared with cox regression analysis in terms of overall survival, thalassemia free survival and thalassemia-gvhd free survival. results: a total of 299 patients; 162 patients under 7 years of age, 137 patients aged 7 and over, were engrafted and remained transfusion independent with full or mixed chimerism. four patients, two from each age group, did not engrafted and had primary rejection. four patients under seven years of age developed secondary rejection, whereas in the group of patients older than 7 years of age, 17 patients experienced secondary rejection (p< 0.05) . a total of 42 patients developed acute gvhd (12.8 %)and their rates were similar in both age groups (10.6% vs 15%). chronic gvhd development rates in two group was also similar (9% vs 7%). the median follow-up time was 34 months (range 0.5-93 months). the 3 -year overall survival rates (os), thalasemia-free survival rates (tfs), thalassemia-gvhd free survival rates (dfs) were shown in the table 2. both thalassemia-free survival and thalassemia-gvhd free survival were higher in patients who underwent transplantation under seven years of age . there was no difference in overall survival. conclusions: the results of our study show that the rates of rejection are high, thalassemia free and thalassemia / gvhd free survival are low in patients who underwent stem cell transplantation over seven years of age. in the light of successful transplantation results from unrelated donors, the delay in age of transplantation in thalassemia patients should also be avoided. disclosure: nothing to declare o167 hla-haploidentical transplantation with regulatory and conventional t-cell adoptive immunotherapy in pediatric patients with high-risk acute leukemia background: post-transplant relapse is still a major cause of treatment failure in high-risk acute leukemia (al) patients. in order to separate the gvl effect from gvhd, we investigated the role of a thymic-derived cd4 + cd25 + foxp3 + regulatory t cells (tregs). the perugia center reported results from 69 adult high-risk al patients who received an hla haploidentical t-cell-depleted hematopoietic transplant and adoptive immunotherapy with donor tregs and conventional t cells (tcons) (and no posttransplant pharmacologic immunosuppressive gvhd prophylaxis) (di ianni et al., blood 2011 , martelli et al. blood 2014 . adoptive immunotherapy with tregs and tcons prevented post-transplant leukemia relapse and largely protected patients from gvhd. in this report we present a pediatric cohort of high risk leukemia patients who received a haploidentical treg/tcon-based hematopoietic transplant. methods: twelve pediatric patients, median age of nine years (range, 5-19) with high-risk acute leukemia underwent hla-haploidentical stem cell transplantation with regulatory and conventional t-cell adoptive immunotherapy between september 2016 and december 2017. eleven had all (three ph+), one secondary aml. seven patients were transplanted in cr1 (3 ph+ all, 1 all in cr after second-line induction, 1 all with extramedullary leukemia, 1 all with t(19;11), 1 secondary aml after medulloblastoma), two patients in cr2, three in cr3. median time from diagnosis to transplantation was 18.5 months (range, 5-48), median time from relapse to especially for aml pts. our analysis suggests that early αβ t cell recovery is associated with a relatively low nonrelapse mortality and relapse rate. disclosure: nothing to declare background: enhancing stem cell performance can improve the results of hematopoietic stem cell transplant (hsct) for diseases in which engraftment is unpredictable, and in patients receiving pre-hsct conditioning regimens of progressively decreasing intensity. in a series of preclinical studies, we explored the use of tat-myc, a chimeric recombinant protein, to improve the performance of hematopoietic stem cell grafts. methods: tat-myc recombinant protein encompasses 9 amino acids from the n-terminal nuclear localization domain of hiv-tat, coupled to the entire coding sequence of c-myc, with an appended histidine tag to aid in protein purification. the construct was expressed in bacteria and purified to pharmacological grade purity under glp conditions. results: brief (1 hour) culture of fibroblasts or hematopoietic cells in medium containing 10 microgram/ ml tat-myc results in rapid nuclear localization of the recombinant protein, whence it disappears within 48 hours as measured by western blot. flow cytometric assays have been validated to measure the uptake of tat-myc recombinant protein into nucleated marrow cells. marrow homing of tat-myc recombinant protein-treated murine marrow increased 5-fold as compared to that of control cells. incubation of activated murine t cells with tat-myc recombinant protein conferred resistance to granzyme b cytotoxicity, but did not protect cells from effects of cyclophosphamide. tat-myc recombinant protein-treated marrow could be serially transplanted in mice for three generations. murine bone marrow harvested from 5fluorouracil treated mice and briefly incubated with tat-myc recombinant protein outcompeted control marrow when transplanted in sub-lethally irradiated immunedeficient mice even at ratios of 1:9 treated:control cells. t-and b-cell reconstitution following transplantation in immune deficient mice was superior following tat-myc vs control incubation of murine marrow. engraftment of human umbilical cord blood (ucb) cells in sub-lethally irradiated immune deficient mice was markedly improved following tat-myc incubation as compared to that of control ucb cells. the transforming potential tat-myc protein was extensively explored. tat-myc culture hematopoietic cells did not display aneuploidy in cytogenetic or spectral karyotypic analyses. intramuscular injection of 10 micrograms of tat-myc protein in p53 +/mice for 12 consecutive weeks did not result in tumor formation. to exaggerate potentially transformative effects of tat-myc protein, murine marrow was co-incubated with both tat-myc and tat-bcl2 recombinant proteins prior to transplantation into irradiated immune deficient mice. mice were followed for 24 weeks and none developed malignancies. serially transplanted marrow incubated with both tat-myc and bcl-2 proteins did not result in malignancies in recipient mice. of >500 mice that have been exposed to tat-myc recombinant protein in our experiments, none has developed a tumor. conclusions: in preclinical studies, brief incubation with tat-myc recombinant protein enhances homing, engraftment and immune reconstitution of murine and human cells in recipient mice following transplantation. brief exposure to the recombinant protein (as opposed to transduction of the myc gene) does not cause malignant transformation of cells. we are currently developing clinical trials using tat-myc protein to enhance engraftment following hsct. disclosure: greg bird, brian turner, thomas payne, yosef refaeli are employees and or shareholders in taiga biotechnologies. jerry stein has received laboratory support from taiga biotechnologies graft γδ t-cell receptor sequencing identifies public clonotypes associated to hsct efficacy in aml patients and unravels cmv impact on repertoire distribution lucas cm arruda 1 , ahmed gaballa 1 , michael uhlin 1,2,3 relapse patient group had an increased proportion of long cdr3 sequences (54-57 nucleotides) compared to relapse patients [0.41%vs0.27% (p=0.02) and 0.11%vs0.04% (p=0.04)]. grafts from cmv-positive donors presented significantly reduced diversity (inverse simpson's di: 30.70vs81.21, p=0.02), decreased proportion of cdr3 sequences having 24, 27, 42 and 51 nucleotides [0.27%vs0.61% (p=0.01), 0.57%vs1.12% (p=0.02), 5.11%vs7.54% (p=0.007) and 0.61%vs2.1% (p=0.04)], and an increase of sequences with 30-39 nucleotides (5.32%vs4.13%, p=0.03). hyperexpanded clones took up 2.5 times more space in the cmv positive grafts (49.33% vs19.38%, p=0.007), who presented a skewed non-gaussian distribution. for all samples, the segments trgv9 and trgjp were the most frequent. nonrelapsing group received grafts with lower usage of trgv4, trgv5 and trgjp2 segments and higher usage of trgjp1 compared to relapse patients [4.13%vs7.46% (p=0.04), 1.12%vs4.77% (p=0.02), 3.02%vs5.52% (p=0.02) and 6.62%vs3.23% (p=0. 02) ]. cmv-positive donor grafts presented a lower trgv2 and trgjp expression (6.29%vs8.99%, p=0.04, and 30.99% vs60.63%, p=0.01) as well as a higher trgjp1 gene usage (11.04%vs3.90%, p=0.04). the v9-jp combination was the most frequent pairing in all samples. non-relapse patients received grafts with lower usage of the pairing v4-j2, v5-j2, v8-jp2 [3.12%vs6.57% (p=0.04), 1.06% vs4.38% (p=0.02) and 1.33%vs1.91% (p=0.03)] and higher usage of v2-jp1 pairing (1.28%vs0.36%, p=0.03) than relapse patients. the tcr usage of the sequence pairs v2-j2, v2-jp2, v4-jp2, v9-jp, and v9-jp2 was lower in cmv-positive grafts [4.62%vs6.94% (p=0.04), 0.59%vs1.33% (p=0.04), 0.31%vs0.09% (p=0.02), 30.75%vs60.30% (p=0.02) and 0.07%vs0.29% (p=0.01)]. we identified 12 public clones shared exclusively between the grafts received by non-relapsing patients in addition to four private over-represented sequences exclusively present in grafts given to nonrelapse patients, taking from 2.00% to 6.23% of the trg repertoire and longer than 45 nucleotides. we also identified five private over-represented and one public cdr3 sequence associated to cmv infection. additionally, cmv-positive grafts presented the highest percentage or repertoire taken by private over-represented clones, ranging from 13.72% to 41.61%. conclusions: our findings show that the trg composition is not associated to agvhd incidence, cmv infection reshapes the trg repertoire and several public sequences are associated to clinical remission. disclosure: the authors have nothing to disclose. background: pediatric patients with high-risk alveolar rhabdomyosarcoma (arms) above the age of 10 years cannot be cured by conventional therapies. immune cells targeting erbb2 with a chimeric antigen receptor (car) were recently considered for these patients. cytokineinduced killer (cik) cells already capable of natural killer (nk)-like anti-tumor capacity additionally redirected with an erbb2 car may provide overall disease control in these high-risk tumors. methods: erbb2-car modified cik cells were generated from conventional cik cells (wt-cik) by lentiviral gene transduction on day 4 of culture. the codon-optimized car sequence consists of an igg heavy-chain signal peptide, an erbb2-specific antibody fragment scfv (frp5) and a modified cd8α hinge region, as well as cd28 transmembrane and intracellular domains and a cd3ζ intracellular domain. 1x10 5 luciferase gene-transduced rh30 (arms) cells were engrafted in immunodeficient nod/scid/γc -(nsg) mice. mice were randomly selected into 5 different treatment groups (dbps on day +1, 2.5x10 6 wt-cik or erbb2 car-cik cells on days +1 and +36, 2.5x10 6 wt-cik or erbb2 car-cik cells on days +22 and +57). mice were monitored by bioluminescence imaging (bli) until day +100. tumor engraftment and immune cell homing at tumor sites were analyzed by facs, chimerism and immunohistochemistry analyses. results: human rms xenografts were established in all mice treated with dbps only. control-mice showed a median survival of 62 days. human rms was identified in all analyzed organs, with the highest tumor burden seen in livers of dbps-treated mice. mice injected with wt or erbb2-car cik cells on days +1 and +36 showed a significant improved (p < 0.014 and p < 0.01) disease-free survival, respectively. furthermore, no signs of tumor engraftment were shown by bli in erbb2 car-cik cell treated mice while some of the mice treated with wt-cik cells developed positive tumor signals between weeks 7 and 10. in 4 out of 6 (64%) wt-and in all (8 of 8, 100%) car-cik cells treated mice no residual tumor cells were identified by pcr-based analysis. in contrast, tumor cells were detectable in all mice with delayed anti-tumor treatment applied on day +22 and +57. however, tumor growth was lower in these groups. correspondingly, bli showed delayed tumor engraftment in mice with wt-and even more with car-cik cell treatment given on day 22. treatment on day 22 resulted in a significantly improved survival of erbb2-car cik cell treated mice (p < 0.01), while survival was not improved after wt-cik cell infusion (p > 0.07). within all treatment groups, immune cells were detected by chimerism and facs analyses. facs analyses showed a significant increase of nk-like t cells (p < 0.01 and < 0.05, wtand erbb2-car cik cells). additionally, a higher, but not significant, amount of effector memory and stem cell memory t cells were detected. conclusions: these pre-clinical in vivo results indicate that erbb2-car redirection of cik cells improves both homing and nk-like cytotoxicity of cik cells in the presence of erbb2-positive tumors, implying that this therapy may represent a step forward in the treatment of patients with resistant, relapsed and advanced rms. disclosure: michael merker, juliane wagner, vida meyer, thomas klingebiel, winfried s. wels and eva rettinger have nothing to declare. peter bader declares the following potential conflicts of interest: novartis (consultancy: included expert testimony, speaker bureau, honoraria), medac (research funding, patents and royalties), riemser (research funding), neovii (research funding), amgen (honoraria) . genesis -a phase iii randomized double-blind, placebocontrolled trial, evaluating safety and efficacy of bl-8040 and g-csf in mobilization of hcs's for autologous transplantation-multiple myeloma hemda chen 1 , zachary d. crees 2 , keith stockerl-goldstein k 2 , abi vainstein 1 , ella sorani 1 , osnat bohana-kashtan 1 , john f dipersio 2 1 biolinerx, tel aviv, israel, 2 washington university in st. louis, st. louis, wa, united states background: cxcr4 mediates retention of hematopoietic stem cells (hscs) in the bone marrow (bm) niche. bl-8040, a novel, high affinity cxcr4 antagonist is a potent mobilizer of hscs to the peripheral blood with numerous potential clinical applications, including mobilization of cd34+ cells for autologous hsc transplantation (auto-hsct) in multiple myeloma (mm). this study aims to evaluate the efficacy of single dose bl-8040 plus g-csf in mobilization of ≥6.0x10 6 cd34+ cells/kg in up to 2 apheresis sessions for auto-hsct in mm. methods: a phase iii study composed of an open-label, single-arm lead-in part1 followed by a randomized, doubleblinded, placebo-controlled part2. eligible mm patients age 18-78 will receive g-csf (10 μg/kg; sc) daily for up to 8 days and one dose of bl-8040 (1.25 mg/kg; sc) or placebo on day 4 followed by up to 2 apheresis sessions; and if needed a second dose of bl-8040 or placebo on day 6 followed by up to 2 apheresis sessions. progressive disease at time of ldc did not respond although car-t cells could be seen morphologically under the microscope. this might be explained by multidrug related phenomenon protecting refractory leukemia from car-t cell attack. conclusions: commercial available car-t cell product tisagenlecleucel (kymriah®) showed high efficacy in r/r-all patients to re-induce cr. clinical trial registry: commercial available car-t cell product tisagenlecleucel (kymriah®) showed high efficacy in r/r-all patients to re-induce cr. disclosure: pb: novartis (consultancy: included expert testimony, speaker bureau, honoraria); medac (research funding, patents and royalties); riemser (research funding); neovii (research funding); amgen (honoraria) . aj: novartis and bluebird: (consultancy) . all other author declare no coi. the main causes of death were sct-related in 53% and disease in 35%. 36-months pfs, os, ir and nrm were 53% (44-63), 72% (64-80), 34% (25-43) and 13% (8-20), respectively. cumulative incidence of grade 3-4 acute gvhd at 100 days after ric-sct was 10% (5-16) and chronic gvhd at 36 months 48% pfs and ir were influenced by patient sex (p=0.025 and 0.04) and disease status at allo-sct 039) and stem cell source (p=0.04); acute grade 3-4 gvhd by donor type (p=0.037) and chronic gvhd by allo-sct conditioning (p=0.016) and donor sex (p=0.01). conclusions: this is the largest series analysing the efficacy and safety of a tandem auto-ric-sct approach in r/r hl. the low nrm and ir with promising pfs and os suggest that this might be an effective post-transplant cyclophosphamide-based gvhd prophylaxis compared to standard prophylaxis in patients with lymphoma receiving hla identical transplantation: a retrospective study from the lwp of ebmt luca castagna 18 ebmt lymphoma worky party clinical trial registry: nct02445248 disclosure: richard t. maziarz: honoraria, membership of advisory committee and research funding employment: oregon health & science university (ohsu); the potential conflict of interest re: consultant services to and payment from novartis has been reviewed and managed by dava oncology; honoraria and research funding: genentech; membership on an entity's board of directors or advisory committees membership on an entity's board of directors or advisory committees and research funding: novartis honoraria and membership on an entity's board of directors or advisory committees: nordic nanovector. vadim v. romanoff: employment: novartis employment: novartis. james signorovitch: employment: analysis group, which received research funding from novartis. solveig g. erickson: employment: novartis. david g. maloney: research funding other: scientific advisor: kite pharma, novartis disclosure: nothing to declare o155 multiple myeloma treatment in real-world clinical practice: a focus on induction regimens prior to autologous stem cell transplantation from the prospective, multinational, non-interventional emmos study cic, ibmcc (usal-csic) 13 state budget healthcare institution of moscow. city outpatient clinic 68 of healthcare use of unmanipulated hla-haploidentical donor transplants (haplo-hsct) is constantly increasing in the last years. few cases of haplo-hsct using posttransplant-cyclophosphamide (pt-cy) for pediatric patients were reported by single center and registry studies, with an incidence of grade ii-iv agvhd ranging from 30% to 40% and cgvhd approaching up to 40%, although with low incidence of extensive disease. methods: we investigated the outcomes of children (< =18y) undergoing haplo-hsct using pt-cy as gvhd prophylaxis disease status at haplo-hsct was cr1 for 31%, cr2 43% and advanced for 26%. poor-risk cytogenetics was reported in 36% of aml, and 11% had ph+ all all patients received pt-cy, in association with tacrolimus/mmf in 33% or csa/mmf in 28% or engraftment rate was 89% with 22 patients experiencing graft failure. cumulative incidence (ci) of day-100 acute gvhd grade ii-iv and grade iii-iv were 33% and 14% respectively, and ci of 2-y chronic gvhd was 20% (9% extensive disease). 2-y ci of nrm was 19% and relapse 41%. disease recurrence and infections were the most common causes of death. 2y-os and lfs were 49% and 40%. 2y-os was 53% and 47% (p=0.91) for aml and all; it was 80%, 47% and 22% (p< 0.001) for patients transplanted in cr1, cr2 and advanced disease. for 2y-lfs, no significant difference was found according to the type of conditioning regimen (39% macchemotherapy-based, 43% mac-tbi based and 38% for ric, p=0.97). the use of pbsc was associated with higher ci of grade os: cr2 vs cr1 conclusions: pt-cy is effective in preventing severe gvhd in children with leukemia receiving an unmanipulated haploidentical-donor transplant. disease status remain the most important factor for outcomes. the use of pbsc as stem cell source increases the risk of grade ii-iv agvhd. the effect of long-term complications, and morbidity related to gvhd results: all patients achieved primary, sustained fulldonor engraftment (median neutrophils engraftment 14 days, range 9-19; median platelets 15 days, 9-19). five patients (42%) developed ≥ grade 2 agvhd (2/5 had concomitant hcv hepatitis and developed liver agvhd), none developed cgvhd. the immune recovery was good in all patients despite immune suppressive therapy in patients with agvhd causes of nrm were: 1 agvhd, 1 invasive aspergillosis, 1 thrombotic microangiopathy. nine of the 12 patients are alive at a median follow-up of 19 months (2-22 months), cgvhd/ leukemia-free survival is 75%. conclusions: these preliminary data in 12 very high risk pediatric patients showed that hla-haploidentical transplantation with regulatory and conventional t-cell adoptive immunotherapy russian federation background: relapse, gvhd and associated non-relapse mortality are the main obstacles to successful hsct in children with leukemia. αβ t cell depletion was developed to prevent gvhd and improve immune reconstitution in recipients of mismatched grafts either melphalan (n=41) or thiophosphamide (n=31) or etoposide (n=16) were added, fludarabine was used in all pts. two types of gvhd prophylaxis were used: type 1 (n=18): hatg 50 mg/kg and post-hsct tacro/mtx, type 2 (n=71): thymoglobulin(ratg) 5mg/kg the median dose of cd34+ cells was 8 x10 6 /kg, aβ t cells -16 x10 3 /kg. results: five patients (5,6 %) died before engraftment due to septic event. primary engraftment was achieved in all evaluable pts (100%) with full donor chimerism. among the whole cohort the ci of gvhd grades ii -iv and iii -iv was 26-74), p=0,002. ratg was also effective in prevention of cgvhd: ci at 2 year after hsct was 15% vs 33%, p=0,08. 3-year ptrm was in the group with available immune reconstitution data (n=68) αβ t cell recovery at day +30 was associated with a trend to decreased incidence of relapse, ci of relapse was 29% (95% ci:15 -54) in those with αβ-t cell count < median vs 15 % (95% ci: 6-38) in those with αβ-cell count >median italian bone marrow donor registry hematopoietic stem cell transplantation (hsct) from hla-a, -b, -c and -drb1-matched unrelated donors (8/8 ud) is performed across hla-dpb1 barrier in more than 85% of cases. clinically tolerable (permissive) mismatches (mm) at hla-dpb1 locus have been classified by different immunogenetic models. here we compare the prognostic value of these models ii) a similar model subdividing alleles in 4 tce groups (tce4, crocchiolo iii) differences in "delta functional distance" scores of 12 polymorphic aminoacids in hla-dpb1 peptide-binding groove (crivello while the first three models were applicable to all 382 hla-dpb1 mm patients, the latter was restricted to 229 of them. the tce4 model appeared the most restrictive one, with only 36% of mm considered to be permissive. median follow-up was 3.2 y. results: hla-dpb1 permissive (p) mm pairs defined by tce4 model (n=135) had superior 3-y overall survival (os) and gvhd-free & relapse-free survival (grfs) compared to non-permissive (np) mm (n=247) (60±8% vs 49±7% cgvhd (hr 1.6, p .03) and extensive cgvhd (hr 3.6, p< .01). the predicted hla-dpb1 mismatched allele expression in the recipient was associated with 100-d ci of grade≥2 agvhd: 32±10% in high expression (n=76) versus 16±6% in low expression (n=153), p< .01. this was confirmed in multivariate analysis for grade≥2 agvhd (hr 2.2, p< .01), however, without higher hazards for trm and overall mortality. the overlap among the four models and their adjusted hr for os is shown in figure 1. conclusions: functional hla-dpb1 matching is of prognostic value in 8/8 ud-hsct outcomes. in our cohort, tce4 appears superior to other models in predicting survival and stratifying risks of trm and cgvhd however, it remains unclear how outcomes of patients with all treated with a haploidentical donor (haplo) compare with hla matched unrelated donor (mud) transplants. methods: we, therefore retrospectively compared outcomes of 506 patients with all who underwent haplohct with ptcy, reported from the participating centers (hit-rc and ebmt) from 01/2005 to 6/2018, with a matched cohort of 1012 patients (1:2) who underwent mud-hct and were reported to ebmt. patients were matched for sex, age at transplant (≤40 or >40), disease type (b-all vs. t-all), disease stage (cr1 vs. cr2 vs. other), disease risk (high vs. others), philadelphia chromosome status (positive vs negative), and conditioning regimen (mac vs. ric/nma) in multivariable analysis, os, pfs, nrm, and relapse rate were not statistically different between patients receiving hct from haplo or mud, regardless of the intensity of the conditioning regimen; (table 1). conclusions: in conclusion, in this large retrospective analysis, outcomes of patients with all undergoing transplant from a haploidentical donor with post median follow-up was 14,6 months and was similar in both cohorts (p=0.78). 2-year overall survival (os) was 41.5% for the all patients and did not differ between transplants from a ird or a crd (36% vs 47%, p=0.33; table i). 2-year progression-free survival (pfs) and gvhd/relapse-free survival (grfs) was 39% and 37%, respectively. 1-year non-relapse mortality (nrm) was 22% and was similar between ird and crd (p=0.62, table i). 6-months cumulative incidence of grade 2-4 acute gvhd, grade 3-4 acute gvhd and 1-year moderate-severe chronic gvhd was 23%, 8% and 19%, respectively. again, there was no difference between crd and ird transplants in terms of grade 2-4 acute gvhd and moderate-severe chronic gvhd (table i) conclusions: our results confirm previous findings that a crd haploidentical transplant is a viable option for haplo-sct when a first-degree donor is available. main long-term outcome are not different in this matched pair analysis. extending our analysis to a larger cohort of patients receiving a crd is warranted to confirm our preliminary results. references: 1 disclosure: the authors have no conflict of interest to disclose stem cell mobilization, collection and engineering o173 a recombinant chimeric protein, safely enhances graft performance following hematopoietic stem cell transplantation median follow-up time for survivors was 27 months. results: overall survival (os) and relapse-free survival (rfs) rates at 2 years after transplantation were 49% 37%, respectively. cumulative incidences of non-relapse mortality (nrm) and relapse at 2 years were 21% and 43%, respectively. in multivariable analysis, performance status (ps) over 1 (ps>1 vs ps < =1, hr 2.49, p=0.005) and lymphoma progression at transplantation (cr/cr-u/pr vs others, hr 1.95, p=0.043) showed significant negative impacts on os. cbt was strongly associated with better os compared to unrelated bmt/pbsct (hr 0.34, p=0.003) and comparable to related bmt/pbsct. lymphoma control status at transplantation was significantly associated with relapse (relapse/induction failure vs cr/cr-u/pr, hr 2.61, p=0.017). poor ps over 1 at transplantation (ps>1 vs ps < =1, hr 3.35, p=0.023) and reduced-intensity conditioning (ric) regimen (ric vs myeloablative, hr 3.54, p=0.025) were associated with higher risk of nrm. conclusions: allo-hsct could improve overall survival of patient with mature t-or nk-cell lymphomas, if performed at appropriate timing with good disease control of partial remission (pr) or better. cord blood unit could be a favorable alternative donor source when related donors are not available. on the other hands, ric regimen did not decrease the risk of nrm in allo-hsct for mature t-and nk-cell lymphoma patients in our setting. out study also suggested that major problem of allo-hsct is still a high frequency of relapse after transplantation. better disease control is mandatory to improve the outcomes of allo-hsct for mature t-or nk fabio ciceri 1 , luca vago 1 , katharina fleischhauer 5 unite´de recherche mixte en sante´(umr_s) 938, inserm netherlands, 11 institute of hematology and blood transfusion ptcy is largely adopted as gvhdprophylaxis backbone in haploidentical transplantation. the encouraging results prompted investigations to assess ptcy also in unrelated donor (ud) setting. high-resolution hla-matching contributes to ud-hsct success; however, due to the selective in-vivo deletion of alloreactive t-cells, ptcy could modulate hla-matching impact on ud-hsct. methods: we compared the outcomes of acute leukemia patients receiving 10/10 (n=431) and 9/10 (n=234) hlaallele matched ud-hsct with ptcy gvhd-prophylaxis table 1 illustrates patients' characteristics. the power to detect a 2-years 10% difference grfs between the 2 groups was 83% (alpha 2-sided=5%). results: outcome endpoints at 2 years were not different between ±7%, p=0.5; lfs: 56±5% and 55±7%, p=0.7; os: 63±5% and 60±8%, p=0.9, respectively). the 100-day ci of grade≥2 and grade≥3 agvhd were comparable for 10/10 and 9/10 ud (31±5% and 28±6%, p=0.4 and 10±3% and 9 likewise, the 2-y ci of cgvhd and extensive cgvhd were similar between 10 the 2-y ci of trm was 19±4% after 10/10 and 17±5% after 9/10 ud-hsct (p=0.4). relapse incidence at 2-y was 25±5% for 10/10 and no interaction was found between donor type and additional atg use. variables associated with grfs were active disease (hr 2.1 compared to 1 st cr, p< 10 -5 ) and karnofsky ps≥90% (hr 0.6, p< 10 -5 ). conclusions: in the present series of acute leukemia patients transplanted with ptcy, we report comparable survival with 9/10 and 10/10 hla-matched ud-hsct, across all disease stages, suggesting that this platform could alleviate the detrimental effect of single hla-allele mismatching. these results warrant prospective comparative trials of ptcy versus standard use of atg-based gvhd disease status: cr1 disclosure: nothing to declare o178 myeloablative conditioning for first allogeneic hsct in pediatric all: ftbi or chemotherapy? -an update of the retrospective multicenter ebmt-pdwp study rose-marie hamladji 14 , cristina diaz de heredia 15 , elena skorobogatova 16 czech republic, 4 hôpital robert debré and paris-diderot university methods: this update was done to extend the time of follow-up (fu, date of analysis: 01/oct/2018). to compare outcomes of ftbi vs cht-conditioning, we performed a retrospective ebmt-registry study. children aged 2-18 years (y) after mac for first allo-hsct of bm/ pbsc from msd/ud in cr1/cr2 between 2000-2012 were included. propensity-score-weighting was used to control pretreatment imbalances of observed variables. this statistical method ensured that analyzed groups differed only in the parameter under investigation (here: conditioning) busulfan/cyclophosphamide/ etoposide (bu/cy/eto) was the most frequently applied cht-regimen in cr1 (66 (31%)) and bu/cy in cr2 (68 (32%)). the remaining conditionings bu/ cy (68) or other-chemo (143) with median-fu of 6.2, 5.2 and 5.8 y. in weighted univariate analysis, 5-y-os was 31.1% after other-chemo, 43.5% after bu/cy and 58.8% after ftbi. in weighted cox-model, pts having received other-chemo had a higher risk to experience an event compared to ftbi (hr=2.00, p=< .0001). 5-y-lfs was 25 coxmodel, pts having received bu/cy and other-chemo had a higher risk to experience an event compared to ftbi (hr=2.06, p=.006; hr=2.13, p< .0001). 5-y-nrm (range: 18.3% (bucy) to 21.1% (other-chemo)) did not show significant differences in weighted cox-model. conclusions: this recent study-update ensured a substantial fu. we confirmed the clear superiority of ftbiconditioning compared to cht with regard of lfs and ri for all-pts undergoing allo-hsct in cr2. for pts in cr1 we could not find significant differences between ftbi and cht-conditioning. these retrospective findings are currently re with data monitoring committee (dmc) review after each cohort. part2 will include 177 patients randomized 2:1. results: part1 enrolled 11 patients, median age following these promising results, dmc recommended early continuation to part2 of the phase iii trial. conclusions: the genesis lead-in results demonstrate bl-8040 is a potent mobilizer of hscs, with potential to improve mobilization rates while minimizing mobilizationrelated healthcare costs. clinical trial registry: nct03246529 disclosure: hemda chen -medical director, biolinerx abi vainstin -vp of medical affairs, biolinerx ella sorani-vp of r&d, biolinerx osnat bohana-kashtan-project manager markus y. mapara 1 , john f. tisdale 2 , julie kanter 3 , janet l. kwiatkowski 4,5 , lakshmanan krishnamurti 6 , manfred schmidt 7 , alexandra l. miller 8 , francis j. pierciey jr. 8 background: phlh is a rare, genetic, hyper-inflammatory syndrome driven by high production of interferon (ifn)-γ. emapalumab (ni-0501) is a monoclonal antibody that neutralizes ifn-γ and is developed as a treatment for hlh.methods: this open-label phase 2/3 study (nct01818492, data cut-off is july 20 2017) includes 34 patients ≤18 years old with phlh based on genetic confirmation, family history, or presence of ≥5/8 hlh-2004 diagnostic criteria. patients were treatment-naïve (7) or had failed previous conventional hlh therapies (27) . emapalumab (1 mg/kg intravenously every 3-4 days, increased up to 10 mg/kg based on clinical and laboratory response parameters) was administered with dexamethasone (5-10 mg/m 2 /day with tapering permitted). treatment duration was 8 weeks with possible shortening to a minimum of 4 weeks or extension up to allogeneic hematopoietic stem cell transplantation (hsct). the primary endpoint, overall response rate (orr), was objectively assessed based on normalization or ≥50% improvement in pre-defined criteria: fever, splenomegaly, cytopenias, hyperferritinemia, fibrinogen and/or d-dimer levels, central nervous system (cns) abnormalities, with no sustained worsening of scd25 levels. an exact binomial test at one-sided 0.025 significance level was used to evaluate the null hypothesis that orr be at most 40%. following completion of the study, patients entered an extension phase (nct02069899).results: patients (median age 1.0 yr, range 0.1-13 yr) entered the study with broad spectrum of phlh clinical abnormalities, >30% with cns involvement. mutations in phlh-associated genes were present in 79% of patients. orr was significantly higher than the pre-specified null hypothesis, thus meeting the primary efficacy endpoint (table) . response rates based on investigator's clinical judgement were 70.6% and 70.4% in the two groups.emapalumab was safe and well tolerated. mild to moderate infusion-related reactions occurred in 27% of patients. infection caused by pathogens potentially favored by ifn-γ neutralization occurred in 1 patient (disseminated histoplasmosis, resolved with treatment). no off-target effects were observed. most patients proceeded to hsct (50% of patients received myeloablative conditioning and 50% reduced intensity conditioning) with favorable outcome (engraftment in 86% and 89% of patients, respectively) and 90% of patients receiving hsct were alive at 1 year post transplant.background: asp0113 is a first-in-class, dna-based vaccine designed for the prevention of cytomegalovirus (cmv) infection; it contains two plasmids encoding glycoprotein b (gb) and phosphoprotein 65 (pp65). this study aimed to investigate the efficacy, safety and immunogenicity of asp0113 compared with placebo in allogeneic haematopoietic cell transplant (allo-hct) recipients.methods: this was a randomised, double-blind, placebocontrolled phase 3 study. cmv-seropositive allo-hct recipients received five intramuscular 1 ml doses of 5 mg/ ml asp0113 or placebo (phosphate buffered saline) in a 1:1 ratio on days −14 to −3, 14 to 40, 60±5, 90±10 and 180 ±10, relative to the day of transplant (day 0). plasma cmv viral load was determined through 1 year and analysed at the central laboratory. treatment-emergent adverse events (teaes) were recorded through 30 days after the last randomized treatment injection. immunogenicity was measured by t-cell responses to pp65 and gb-specific antibody levels through 1 year after the first randomized treatment injection. the primary endpoint was the proportion of patients with a composite of all-cause mortality and adjudicated cmv end-organ disease (eod) through 1 year post transplant. secondary endpoints were cmv viraemia rate, adjudicated cmv-specific antiviral therapy (avt) rate, a composite of protocol-defined cmv viraemia and adjudicated cmv-specific avt use, first occurrence of background: haploidentical stem cell transplantation (haplo-sct) with post-transplant cyclophosphamide (pt-cy) represents a potential curative strategy for patients with hodgkin lymphoma (hl) when a matched related or unrelated donor is not available 1 . while bone marrow (bm) was originally the preferred stem cell source, more recently peripheral blood stem cell (pbsc) is more often used. some retrospective studies suggest that the risk to develop acute and chronic graft-versus-host-disease (gvhd) is higher with pbsc than bm, while pbsc may reduce the risk of relapse 2 . here we analyzed the effect of stem cell source in 91 hl patients receiving haplo-sct with pt-cy, with the aim to evaluate if the final outcome is modified by the use of pbsc or bm.methods: from april 2009 to january 2017, 91 consecutive patients with poor prognosis hl received a haplo-sct with pt-cy either from a pbsc (n=38) or bm (n=53). the two cohorts were similar for most characteristics, but the pbsc group had more patients with an unfavorable hematopoietic stem cell transplant comorbidity index (hct-ci) score ≥3 (p=0.002) and had received a non myeloablative conditioning (nmac; p=0.001).results: cumulative incidence of neutrophil>500/ul at day +30 and of platelet >20000/ul at day +60 were 96% (95% ci: 89-98) and 96% (95% ci: 88-99), respectively, with no significant differences between the pbsc and bm cohorts. with a median follow-up of 22.8 months, there was no difference between pbsc and bm graft in terms of cumulative incidence of grade 2-4 acute gvhd (29% vs 21%, p=0.3), grade 3-4 acute gvhd (3% vs 4%, p=0.7) and moderate-severe chronic gvhd (9% vs 7%, p=0.7). this was also confirmed by multivariate analysis. in the whole population, the 2-year overall survival (os), 2-year progression-free survival (pfs) and 1-year gvhd/relapse free survival (grfs) rates were 67%, 58% and 58%, respectively. we observed a trend for improved os (74% vs 62%, p= 0.07) and pfs (62% vs 56%, p= 0.1) for recipients of pbsc relative to bm cells, but pre-transplant disease status was the only significant variable by univariate analysis (table i) . by multivariate analysis, pre-transplant active disease status, transplant from a bm and hct-ci ≥ 3 remained the only independent predictors of adverse outcome in terms of os; pfs and grfs (table i) . nonrelapse mortality was not affected by graft source both by univariate and multivariate analysis, while pre-transplant disease status was the only variable affecting the chance of disease relapse.conclusions: overall these data suggest that pbsc is associated with better outcome, in terms of os, pfs and grfs, relative to bm cells as graft source for patients undergoing haplo-sct with pt-cy. in addition, the risk of acute and chronic gvhd is not increased after pbsc relative to bm graft.references disclosure: the authors have no conflict of interests to disclose o139 consensus crs and neurotoxicity regrading of "juliet": phase ii prospective study of tisagenlecleucel therapy in patients with relapsed/ refractory large b cell lymphoma background: t-cell depletion using ex vivo cd34+ cell selection reduces gvhd risk after allogeneic hct, but delayed immune reconstitution, particularly t-cell reconstitution, has limited improvement in survival. sex steroids negatively impact lymphopoiesis, likely by thymic atrophy, and our preclinical models have shown that androgen blockade with the gnrh agonist leuprolide enhances thymopoiesis (goldberg, ji 2009; velardi, jem 2014) . we hypothesized that peri-hct leuprolide could improve immune reconstitution among recipients of cd34selected hct.methods: this was a phase ii clinical trial of leuprolide in cd34-selected myeloablative allogeneic pbsct for hematologic malignancies in patients aged 18-60 (nct01746849). all participants received conditioning with tbi 1375cgy, thiotepa 10mg/kg, and cyclophosphamide 120mg/kg; antirejection prophylaxis with rabbit atg 5-7.5mg/kg; and palifermin 60mg/kg/d on days -13 to -11 and 0 to +2. patients received a 3-month depot of leuprolide 11.25mg 2-6 weeks before conditioning and a second depot 3 months later. primary endpoint was an absolute cd4+ count >=200 by 6 months post-hct. patients who died, relapsed, or otherwise did not have flow data available at day +180 were excluded from primary endpoint analysis but included in outcome analyses. we excluded flow data after secondary cell infusions (dli, ctls, second hct, cd34+ cell boost) but followed recipients of these interventions for survival analysis. descriptive statistics summarized absolute levels of lymphocyte subset counts at select time intervals. a kruskal-wallis rank sum test compared counts among patients who received leuprolide/palifermin, historical controls who received palifermin alone, and historical controls who received neither. kaplan-meier functions estimated os/ rfs. cumulative incidence functions estimated gvhd/ nrm.results: thirty-two patients received at least one dose of leuprolide. median age was 39 years (range 21-57). twenty-six(82%) had acute leukemia, 3(9%) mds/mpn, and 3(9%) cml. all but one had an 8/8-matched donor. at median follow-up of 24 months among survivors (range 7-61), estimated 2y os was 74%(95%ci 60-91%) and rfs 62%(95%ci 46-83%). ci of trm at 2y was 10%(95%ci 2-24%). ci of grade iii-iv acute gvhd was 3%(95%ci 0.2-14%). of 27 patients with evaluable flow data, 30% achieved a cd4+ count >=200 by 180+/-30 days, not significantly different from historical controls. median lymphocyte counts at 180+/-30 days did not differ significantly among groups (table; figure) .conclusions: this phase ii study did not demonstrate significant quantitative improvement in immune reconstitution after leuprolide with palifermin in recipients of tbibased cd34-selected hct. tcr sequencing to identify possible improvement in t-cell diversity in this cohort is forthcoming. one potential explanation for these results lies in the initial surge in sex steroid levels immediately after background: the prospective, multinational, noninterventional emmos study aimed to document, and describe real-world treatment regimens and disease progression in patients with mm at different stages of the disease.methods: adult patients initiating any new mm therapy between 2010 and 2012 were eligible. a multi-staged patient/site recruitment model was applied to minimize selection bias, and enrolment was stratified by country, region, and practice type. patients' medical/disease features, treatment history and remission status were recorded at baseline, and prospective data on treatment, efficacy and safety were collected electronically every 3 months until 2 years after last enrolment. responses were investigatorassessed. overall findings from emmos were previously reported. here, we are presenting additional analyses focusing on the induction regimens used in the subgroup of patients who proceeded to auto-sct frontline.results: a total of 2358 patients (775 with stem-cell transplant [sct] and 1583 without) were enrolled. patient demographics/baseline characteristics were as expected. of 380 recipients of sct after enrolment, 299 (79%) underwent auto-sct frontline. 90% of the auto-sct patients were aged ≤65 years. among these 299 frontline auto-sct patients, the majority had a single transplant (87%). the most frequent induction regimen was bortezomibthalidomide-dexamethasone (vtd; n=95; 32%), bortezomib-dexamethasone (vd; n=56; 19%), bortezomib-cyclophosphamide-dexamethasone (vcd; n=49; 16%), doxorubicin-bortezomib-dexamethasone (pad; n=26; 9%), and cyclophosphamidedexamethasone-thalidomide (ctd; n=26; 9%). only 1% of patients received a bortezomib-lenalidomidedexamethasone (vrd) induction regimen, while lenalidomide was shown to be the most frequently used agent in lines 2 and 3 at time of relapse. in the vtd subgroup, most patients received 100 mg thalidomide dose during induction. the majority of administration schedule was based on 21 days cycles, while a few other schedules were seen corresponding to 35 days cycles or to delay due to adverse events or other specific reasons. the most prevalent number of vtd cycles was 3 and 4 (42% and 38%, respectively). lower or higher number of cycles was only marginal (6% and 14%, respectively). out of the 95 patients with vtd induction, 39% achieved cr as best response, 28% ncr or vgpr and 16% pr. after auto-sct, the best overall response rates (orr) at any time during frontline therapy were >85% for those patients whose treatment included 24 university medicine greifswald, greifswald, germany background: we explored the effect of dinutuximab beta (ch14.18/cho) on outcome within the hr-nbl1/siopen trial population by comparing an era prior immunotherapy availability.methods: the analysis cohort consists of the immunotherapy population (ip) (2009-2013) and a matched control population (cp) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) . all patients had rapid cojec induction, up to two tvd courses and high-dose chemotherapy (bumel or cem) followed by autologous stem cell reinfusion (hdc/ascr) within 9 months since diagnosis; local control included surgery and local radiotherapy (21gy) followed by maintenance with six cycles of isotretinoin. ip patients had additional five cycles of dinutuximab beta short-term infusions with or without subcutaneous interleukin-2. cp patients had to be part of the hdc randomization. the median time between ascr and initiation of immunotherapy was 109 days in the ip. only patients without progressive disease at this landmark time point were included in the cp. median follow-up was 5.8 years (iqr: 4.2 to 8.2 years) for 844 eligible patients.results: the 5y-efs of the ip (378 patients) was 57% ±3% compared to 42%±2% for the cp (466 patients; p< 0.001). both populations were balanced for sex, age, stage 4, mycn amplification and response prior hdc. multivariate analysis showed an independent higher risk for the cp (p=0.0002, hr 1.573), for cem (p=0.0029; hr 1.431), a response < cr prior to maintenance therapy (p=0.0043, hr 1.494) and for >1 metastatic compartment at diagnosis (p< 0.001, hr 2.665). after adjustment for risk factors, the benefit of immunotherapy was confirmed in bumel-(p=0.0066; hr 1.439) and in cem-treated patients (p=0.0107; hr 2.334).conclusions: results suggest a patient benefit from dinutuximab beta based immunotherapy with or without il2 within the hr-nbl1 trial.clinical trial registry: the trial was registered with clinicaltrials.gov (number nct01704716) and eudract (number 2006-001489-17) . https://www.siopen-r-net.org disclosure: the academic data supported apeiron to obtain the dinutuximab beta product licensure in may 2017 in the european union (ema). siopen and ccri had an agreement in place with apeiron regarding the provision of academic data. ruth ladenstein and holger lode acted as consultants for apeiron on behalf of siopen for the ch14.18/cho development.the other authors declared no conflicts of interest. 19 out of 24 (79%) received serotherapy with alemtuzumab (n=12) or atg (n=6) before viral reactivation. where possible immunosuppression was withdrawn in combination to bcv therapy. viral load was detected in blood by pcr as copies/ml. data on response to bcv was divided into complete response (cr) with undetectable virus in blood and resolution of symptoms, partial response (pr) with at least 1 log drop in viral load after bcv, no response (nr) with no change in the viral load and stationary disease and progressive disease (pd) with evidence of at least a log rise in viral load by pcr or organ disease progression.results: the median viral load at the start of bcv was 5.4 million copies/ml (range: 4142-85 million copies/ml). bcv was used as a first line treatment in 8 cases and as second line in 16 cases after failure of first line therapy (n=8), toxicity (n=5) or both(n=3).13/24 (54%) patients had evidence of viral induced organ disease at time of bcv administration; 11 adv disease (encephalitis, pneumonitis, hepatitis and colitis), 1 ganciclovir resistant cmv retinitis and 1 bk haemorrhagic cystitis. 19/24 (79%) patients achieved either a cr (n=15) or pr (n=4) and 8/13(61%) patients with organ disease achieved a cr.two patients with adv disease and pr received donor derived cytotoxic t lymphocytes to achieve cr. at a median follow-up of 20 months (range: 7-53.8), patients who were in cr or pr did not show any evidence of viral reactivation after bcv discontinuation despite no evidence of immune reconstitution (ir). four patients had evidence of disease progression with significant rise in viral load while on bcv therapy and all 4 died. the patient with ganciclovir resistant cmv attained cr of cmv retinitis.among 22 patients with adv viraemia ± disease; 18 (81%) achieved either cr (n=14) or pr (n=4). nine cases had concomitant bk± cystitis at the time of bcv therapy and all had nr. toxicity was observed in 4/24 cases; renal impairment (n=1), transaminitis (n=1) and diarrhoea (n=2). median cd3, cd4 and cd8 were persistently low both pre-bcv and at the end of treatment; 100 cells/ul vs 290 cells/ul, 40 cells/ul vs 70 cells/ul and 20 cells/ul vs 120 cells/ul; p=0.2, p=0.34, p=0 .5 respectively (figure 1 ). at last follow-up, 15/24 (63%) were alive. of these, viral infection related mortality was 4/24 (17%).conclusions: bcv is an effective and well tolerated treatment in immune compromised patients with adv infection with a response rate of 80%.[[o163 image] 1. background: although the impact of donor graft composition on clinical outcome after hematopoietic stem cell transplantation (hsct) has been studied, little is known about the role of intra-graft γδ t-cell receptor (tcr) repertoire on clinical outcome following hsct. using high-throughput sequencing we sought to analyze the tcr γ-chain (trg) repertoire of γδ t-cells within donor stem cell grafts and address its potential impact on clinical response in the corresponding patients.methods: we analyzed twenty peripheral blood stem cell grafts from matched unrelated donors and classified as cmv-positive/negative. the respective acute myeloid leukemia recipients were followed for disease relapse and acute graft-vs-host disease (agvhd) development post-hsct. γδ t-cells were isolated using magnetic beads and the gdna extracted for next-generation sequencing (immunoseq, adaptive biotechnologies). trg characteristics were assessed using vdjtools, vdjviz, tcr and immunoseq analyzer platforms.results: deep sequencing showed similar median total/ unique reads in all grafts as well as similarly low unique cdr3 trg ratios. grafts presented multiple clonal overrepresentations, with no differences on tcr richness between patient groups. grafts received by the non-background: prognosis of mature t-and nk-cell lymphomas remains poor despite development of novel therapeutic agents. accordingly, these lymphomas are still good candidates of allogenic hematopoietic stem cell transplantation (allo-hsct) to achieve long-lasting remission of the diseases. however, the analysis of transplantations for these lymphomas is scarce, mainly due to the rarity of these lymphomas. hence, we analysed the data of these transplantations operated in 15 different japanese institutions as a multi-institutional joint research and examined factors that affected the outcomes in aims of figuring out better transplant strategies against these lymphomas.methods: a total of 116 patients who received allo-hscts for mature t-and nk-cell lymphomas (ptcl-nos, n=38; nk/t cell lymphoma nasal type, n=22; aitl, n=20; alcl, n=12; eatl, n=8; other lymphomas, n=16) from 1998 to 2016 in 15 institutions were examined. median age at transplantation was 50 (range, 18-69). forty patients received transplantation from related bone marrow transplantation (bmt) / peripheral blood stem cell background: children, adolescents and young adult patients with all with second relapse, relapse after allogeneic sct or patients with primary refractory disease have a poor prognosis with conventional treatment concepts. in this patient group several studies using second generation cd19 chimeric antigen receptor t-cells (car-t cells) demonstrated high efficacy with two year survival rates of up to 65%. recently, two different car-t cell products were approved by the fda and in 2018 also by the ema in europe: tisagenlecleucel (kymriah®) for the treatment of patients with b-precursor all who are i) refractory, ii) in second relapse or iii) who relapsed after allogeneic sct (relapsed/refractory all; r/r all) as well as for diffuse large cell lymphoma (dlbcl) and axicabtagen ciloleucel (yescarta®) for the treatment of b-cell lymphoma. here we report our first results using commercially available car-t-cell product tisagenlecleucel (kymriah®) in patients with all which were treated by the university hospital for children and adolescents frankfurt am main (n=9), the department of medicine iii, university hospital lmu munich (n=1), and the von hauner kinderspital, lmu munich, germany (n=1).methods: between october and december 2018 eleven patients received apheresis for car-t cell generation. nine patients suffered from r/r c-all, and two from r/r bprecursor all. eight patients had relapsed after allogeneic hsct, one patient each suffered from first r/relapse, second r/relapse or from primary r/all. in 9/11 (82%) patients car-t cell production was successful after one and in 2 patients (18%) after a second apheresis.median patients' age was 16.7 years (1.1-25.4 ). between apheresis and start of lymphodepleting chemotherapy (ldc), 10/11 patients received low dose chemotherapy according to the frapostall protocol (willasch et al. 2016 ) and one patient was treated with inotuzumab. production slots were immediately available, resulting in turn-around-time from apheresis to product delivery of 3-4 weeks. disease status at start of ldc was cr w/o mrd (n=4), cr mrd pos. (n=2), cri (n=1), persistence of blasts (n=2), and disease progression (80-100% blasts, n=2). ldc consisting of flu-cyc was given to 9/10 patients, one patient did not receive ldc.results: car-t cells could be transfused to 10/11 patients at a median dose of 1.5 mio/kgbw (0.145 mio 8.5). in one patient, in whom a second viral transduction procedure was necessary; neither ldc nor car-t cell transfusion could be given because of diseases progression and deterioration of the patient's general condition. cytokine release syndrome (crs) grade i was observed in one patient; 8/10 patients did not develop crs. cytokine related encephalopathy syndrome (cres) grade ii was observed in 1/10 patients. at day +28 8/10 patients (80%) achieved mrd negative cr. the two patients with key: cord-005487-vac061r8 authors: nan title: physicians abstracts: ebmt 2010 date: 2010-04-07 journal: bone marrow transplant doi: 10.1038/bmt.2010.40 sha: doc_id: 5487 cord_uid: vac061r8 nan b cells have been demonstrated to present antigen to t cells in vivo. cd40-activation dramatically improves antigen presentation by normal and malignant b cells and has therefore been studied as an approach to generate autologous "non-artifi cial" antigen presenting cells for active immunotherapy. furthermore, cd40-b cells have recently been shown to expand tumorantigen and viral specifi c ctl as well as regulatory t cells and are therefore of great interest for post-transplant immunotherapy. human b cells when activated via cd40-l/il-4 can be expanded from small amounts of peripheral blood in 12-14 days. cd40-activated b cells can prime naïve cd4 + and cd8 + t cells, expand memory t cells and express important surface homing molecules. nevertheless, it remains unclear whether cd40-activated b cells migrate to secondary lymphoid organs (slo) in vivo to attract and interact with t cells. to address this question we established a methodology to generate murine cd40-activated b cells. at day 14 of culture, these cells are >95% cd19 + and cd80/86/mhci/mhciihi. murine cd40-activated b cells present a 'homing phenotype'; migrate toward slo chemokines such as ccl19, ccl21 and cxcl13; and induce t-cell chemotaxis in vitro. upon cd40l activation, b cells up-regulate ccr7 while down-regulating cxcr5 expression which suggests direction of activated b cells toward the b-zone/t-zone boundary. we compared the homing of gfp + cd40-activated b cells to resting gfp + b cells and show for the fi rst time that cd40-activated b cells home to slo significantly more effi ciently than resting b cells. furthermore, cd40activated b cells localize in the b-cell areas, and a signifi cant fraction move to the b-t boundary close to the t-cell zone over time. to dissect t-cell-apc interactions on a single cell we analyzed three-dimensional migration in collagen matrix using time-lapse videomicroscopy. interestingly, antigen-loaded cd40-activated b cells differ from immature and mature dc by displaying a rapid migratory pattern undergoing highly dynamic, short-lived (7.5 min) and sequential interactions with cognate t cells. taken together, these data revealed that cd40-activated b cells can home to secondary lymphoid organs and interact dynamically with t cells thus underlining their potential as cellular adjuvant for cancer immunotherapy. long-term follow-up of upfront tandem autologous -ric (reduced intensity conditioning) allogeneic transplantation versus autologous transplantation (nmam2000) in multiple myeloma g. gahrton, b. björkstrand, s. iacobelli, u. hegenbart, a. gruber, h. greinix, l. volin, f. narni, p. musto, m. beksac, a. bosi, g. milone, p. corradini, h. goldschmidt, t. de witte, c. objectives: treatment of multiple myeloma with allogeneic hematopoietic stem cell transplantation is controversial. the nmam2000 study compares tandem autologous (asct)/ reduced intensity conditioning (ric) allogeneic transplantation (ricallo) to only asct or asct in tandem (asct2) in a prospective study based on genetic randomization. patients and methods: out of 358 myeloma patients accrued during 2001-2005 from 26 ebmt centres 109 with an hlaidentical sibling donor were allocated to tandem asct/ricallo and 249 without to asct only. previously untreated patients with at least stable disease after vad (vincristine, doxorubicine, dexamethasone)-like induction treatment were included at the time of conditioning for asct. single (n = 145) or tandem (n = 104) asct was optional in the asct arm. conditioning for asct was melphalan 200 mg/m 2 , and for ricallo fl udarabine 30 mg/m 2 x 3 plus tbi 2 gy. the two treatment groups were well matched for standard prognostic parameters and response status at asct. results: on an intention to treat basis the cumulative 24 and 60 months non-relapse-mortality (nrm) was 12 % and 16% with asct/ricallo and 3% and 4% with asct respectively (p = 0.0001 (gray test)). the cr rate within 60 months was 52% with asct/ricallo and 41% with asct (p = 0.0009: improved in time (fine/gray model)). at 60 months after asct relapse/progression rate was 49% and 78% (p = 0.0014: reduction in time fine/gray model)), pfs 35% and 18% (p = 0.0012 reduction of risk in time (cox)) and os 65% and 58% (p = 0.0048 reduction in time (cox)) (at 84 months 60% and 22%) for asct/ricallo and asct respectively. a comparison between those patients who received a second allo (n = 91) versus a second auto (n = 104) relapse/progression rate at 60 months from second transplant was 43% and 78% and pfs 39% and 19% in asct/ricallo and auto2 respectively. information about the chromosome 13 deletion (del(13q14)) was present in 214 patients. in 92 patients with the deletion os at 60 months was 69% and 55%, and pfs 31% and 11% for asct/ricallo and asct respectively. gvhd in ricallo was as expected (agvhd grade 0-1: 80%; grade 2-4: 20%; cgvhd limited: 36%; extensive: 27%). conclusion: the risk of myeloma relapse is lower with tandem asct/ricallo as compared to asct or tandem asct, both in an intention to treat analysis and in patients that received the correct treatment. nrm is higher, but considered acceptable in view of the improved pfs and os with tandem asct/ricallo. a fi ve biomarker panel predicts acute graft-versus-host disease s. paczesny (1) , d. bickley (1) , s. choi (1) , j. crawford (1) , t. braun (1) , s. pitteri (2) , j. hogan (2) , p. reddy (1) , s. hanash (2) , j. ferrara* (1) , j. levine* (1) (1)university of michigan (ann arbor, us); (2)fred hutchinson cancer research center (seattle, us) * have contributed equally to this work current evidence suggests that a composite panel of four biomarker proteins (il2ra, tnfr1, il8, hgf) is diagnostic and prognostic for acute graft-versus-host disease (gvhd). elafi n is a biomarker that has also been found to be diagnostic for gvhd of the skin, as well as prognostic for overall survival (os). we sought to evaluate whether these fi ve biomarkers are able to predict future occurrence of gvhd, non-relapse mortality (nrm) and os. we measured by sequential elisa, levels of these fi ve proteins in plasma collected prospectively from 485 allogeneic hct patients randomly divided into training (149 gvhd-, 175 gvhd + ) and validation (74 gvhd-, 87 gvhd + ) sets. we obtained samples 3-14 days before onset of gvhd (median day 29) and at equivalent time points in patients without gvhd. there were no signifi cant differences between sets in age, conditioning intensity, donor source, hla match or gvhd grade between training and validation sets. the median day of sample acquisition was day 20 and day 21, respectively. logistic regression determined that a linear combination of the fi ve proteins levels predict the occurrence of acute gvhd in the training set. the receiver operating characteristic curves of each of the fi ve individual biomarkers are shown in figure 1 with an area under the curve (auc) for the composite panel of 0.77 (95%ci: 0.72-0.82). when this model was applied to samples of the validation set, the corresponding auc was 0.76 (95%ci: 0.69-0.84). this 5-biomarker panel therefore discriminated between patients who later developed gvhd and those who did not. proportional odds logistic regression models determined that the panel gave prognostic information regarding the eventual maximum grade of gvhd (p<0.001 in training set, p = 0.05 in validation set). given this correlation, we next divided the patients into high and low risk groups based on their predicted probability of developing gvhd (high risk ≥ 0.7 and low risk < 0.7) and analyzed these groups for differences in nrm, relapse mortality and os. the differences in 1 year nrm and os between groups were signifi cant in both the training and validation sets (table 1 ). when adjusted for other known risk factors (age, conditioning intensity, donor source, and hla match), the difference in os remains signifi cant (p = 0.02) in both sets. in conclusion, a 5-biomarker panel can predict gvhd before any clinical manifestation and provides prognostic information including long term survival. s3 their interaction with dendritic cells. recent clinical data has indicated that the content of plasmacytoid dendritic cells (pdc) in an allograft is associated with the risk of relapse following allogeneic bone marrow transplantation (bmt). we have previously shown that the addition of donor pdc to a graft comprised of purifi ed hsc and t-cells led to enhanced th1 activation of donor t-cells with improved gvl activity without increasing gvhd in multiple murine bmt model systems (li and waller ji 2009 ). here we studied the mechanism that donor pdc augment gvl without increasing gvhd, and present a novel model for regulation of post-transplant immunity. methods: donor hematopoietic stem cells (hsc), t-cells and pdc in the allograft were rigorously purifi ed using immuno-magnetic selection and high-speed fl uorescent-activated cytometry (facs) in the h2b b6->h2k b10.br transplant model. donor cell subsets purifi ed from wild type (wt) or interferon gamma knock-out(ko),(ifn-gko), interferon gamma receptor ko (ifn-grko), and indolamine 2,3 dioxygenase (ido) ko (ido-ko) were combined to determine the role of the ifn-g and ido in the separation of gvhd from gvl in murine bmt. results: in the b6->b10.br bmt model, the addition of 50,000 donor pdc to a graft comprised of 3,000 hsc and 300,000 t-cells lead to th1 immune polarization and enhanced proliferation of donor t-cells with higher levels of donor t-cell chimerism and improved gvl activity without increasing gvhd. the absence of allo-reactivity was dependent upon donor t-cell synthesis of ifn-g and the presence of ifn-gr on donor pdc. co-culture of t-cells with syngenic pdc in one-way mlr lead to up-regulation of ido in donor pdc. increased gvhd after transplanting pdc from ido ko donors in combination with wt t-cells and hsc demonstrated the central role of ido in the initiation of counter-regulatory effects that limit gvhd. wt t-cells and pdc lead to the generation of donor-derived t-reg in bmt recipients that limited gvhd. pdc from ido-ko donors had markedly reduced numbers of donor t-reg. conclusions: donor pdc regulate the initial activation and gvl activity of donor t-cells and subsequently generate t-reg that limit gvhd. the dynamic regulation of the activation and alloreactivity of donor t-cells by donor pdc suggest novel clinical approaches to enhance gvl activity without gvhd. j. peccatori (1) , d. clerici (1) , a. forcina (1) , m. bernardi (1) , r. crocchiolo (1) , c. messina (1) , m. noviello (1) , s. mastaglio (1) , f. giglio (1) , s. malato (1) , m.t. lupo stanghelllini (1), s. marktel (1) , a. assanelli (1) , m. battaglia (1) , a. ferraro (1) , s. rossini (1) , m.e. bernardo (2) , a. bondanza (1) , m. g. roncarolo (1) , c. bonini (1) , f. locatelli (1) , f. ciceri (1) ( background and aim: rapamycin, in contrast to calcineurin inhibitors, allows t regulatory cell (t-regs) proliferation while inhibits effector t cell expansion. we investigated the safety of infusion of t-cell repleted unmanipulated pbsc from haploidentical donor with a combination of rapamycin, mycophenolate and atg as gvhd prophylaxis, to preserve early t-regs function (trramm study, eudract 2007-5477-54) . patients and methods: since 2007, fi fty-nine patients (pts) underwent sct for aml (39 pts), all (9), mds (3), cml-bc (4), nhl (2) or hd (2) . median age was 50 years (range 14-69). at transplantation 7 pts were in early phase, and 52 in advanced phase. median comorbidity index (ci) score was 1 (0-5). the conditioning regimen included treosulfan (14 g/m 2 for 3 days), fludarabine (30 mg/m 2 for 5 days) and an invivo t and b-cell depletion by atg-fresenius (10 mg/kg for 3 times) and rituximab (a single 500 mg dose). all pts received allogeneic unmanipulated pbsc from an hla-haploidentical related donor. gvhd prophylaxis consisted of rapamycin (target level 8-15 ng/ml, till day + 60) and mmf (15 mg/kg tid till day + 30). results: all patients engrafted and all but eight were in disease remission at fi rst marrow evaluation on day + 30. cumulative incidence of grade 2-4 and grade 3-4 agvhd were 29 and 13% respectively. only 12 patients developed cgvhd. cumulative incidence of trm and relapse incidence were 25% and 44% respectively. after a median follow-up of 8 months, projected os at 1 year is 43%. immunoreconstitution was fast and sustained with a median 221 circulating cd3 + cells/μl (range 43-1690) from day 30. the immune-reconstitution was polarized towards central memory (cd45ra-cd62l + cells 32.7% ± 4.8), il-2 producing cells (il-2 + cells 26.2% ± 5.3). we detected high levels of cd4 + cd25 + cd127-foxp3 + t regulatory cells (up to 30% of circulating cd4 + t lymphocytes) starting from day 30. these cells were able to suppress in vitro proliferation of autologous effector cells demonstrating to be regulatory t cells. conclusions: rapamycin-mycophenolate-atg are effective as gvhd prevention in t-cell replete unmanipulated haploidentical peripheral sct and are associated with an early t-cell immunoreconstitution characterized by the in-vivo expansion of earlydifferentiated t cells and t-regs. further studies are warranted to gain insight on the role of rapamycin as platform for exploitation of t-regs in allogeneic hsct from mismatched donors. direct interaction of human nk cells with aspergillus fumigatus induces a th1 immune response and provokes signifi cant fungal killing but not via their usual cytotoxic pathways m. bouzani, m. ok, o. kurzai, h. einsele, j. loeffl er wurzburg university (wurzburg, de) objectives: invasive aspergillosis (ia), caused mainly by aspergillus fumigatus (af), is a highly devastating disease for immunosuppressed subjects. host's defence is principally confi ned to innate effector cells like alveolar macrophages, neutrophils and dendritic cells. in our study, we questioned the possible interaction of af with another potent component of the innate immunity, the natural killer (nk) cells. methods: human nk cells were isolated after magnetic depletion of the peripheral blood of volunteers and they were used after 24h priming with 500 u/ml recombinant human interleukin 2, rhil2. interferon gamma (ifn-g) and tumor necrosis factoralpha (tnf-a) regulation were assessed after nk-af coculture. fungal damage was investigated through plate killing assays. to investigate the infl uence of rhil2 on nk cells, plate killing experiments were performed with resting and primed nk cells. transwell permeable membranes, nk cell granule depletion (treatment with strontium chloride), surface expression of degranulation markers cd107a/b and neutralization of nk death ligands (tnf-related apoptosis-inducing ligand [trail] and fasl) by blocking antibodies were used to evaluate the means of interaction. results: fungal germlings induced towards nk cells a th1 immune response with upregulation of ifn-g and tnf-a (p<0.05). nk cells displayed strong fungicidal effect against germlings (p<0.05), but they were inactive against conidia. priming with rhil2 (p<0.05) and direct effector-pathogen contact (p<0.001) were required for their interaction. the cytotoxic effect was not attributed neither to the release of perforingranzyme, nor to the engagement of nk cell death ligands. conclusion: human nk cells are stimulated in vitro by af, which triggers a th1 immune response and causes important fungal killing. this interaction requires priming of nk cells with rhil2 and conditions of direct contact between nk cells and fungus. interestingly, nk cells do not mediate their cytotoxic effect via perforin -granzyme pathway, neither through the engagement of death ligands, suggesting that another pathway is involved in nk cell -af interaction. our study attributes to nk cells anti-aspergillus properties, suggesting them as a future potential immunotherapeutic tool against ia. objective: to conduct a survey on indications, effi cacy and safety of growth hormone (gh) treatment in children and ado-lescents after haematopoietic stem cell transplantation (hsct) within the ebmt. methods: in this retrospective survey using a two step approach, we asked (1st questionnaire) for information on endocrine follow-up, the use of gh, reasons for not using gh, indications for gh treatment, number of patients treated with gh and diagnostic tests to diagnose gh defi ciency (ghd); and (2nd questionnaire, to follow) data on growth, diagnosis of ghd, interval between hsct and gh treatment, dose schedules, other endocrine defi ciencies. results: 1st questionnaire: 53 centres replied until 15.11.2009: endocrine follow-up (n = 50) was performed by the hsct-clinic and an endocrinologist in 34%, the hsct-clinic in 12%, an endocrinologist in 38%, a specialist in another clinic in 10% and the hsct-and another clinic in 6%. 63% (33/52) centres treated patients after hsct with gh, whereas 37% (19/52) did not. of the centres reporting, 3/10 centres from italy used gh, whereas 7/10 did not. in contrast, 6/7 centres from france used gh whereas 1/7 did not. german centres reported the use in 3/6 centres. all centres from finland (2), great britain (2), spain (2) and switzerland (2) reported the use of gh. indications for gh treatment were growth failure in 6% (2/32), growth failure or ghd in 50% (16/32) and ghd only in 44% (14/32). reasons for not using gh were: no indication in 74% (14/19), risk of side effects in 11% (2/19) and lack of fi nancial support in 5% (1/19) . the numbers of patients treated in centres was less than 5 patients in 28% (9/32), 5 to 10 patients in 31% (10/32), 10 to 20 patients in 22% (7/32) and more than 20 patients in 19% (6/32). diagnostic tests for ghd used were the insulin-test (n = 17), arginin-test (n = 14), spontaneous gh-secretion (n = 10), clonidin-test (n = 10) and ghrh-test (n = 9). conclusions: in the majority of centres the endocrine follow-up was performed by an endocrinologist. gh treatment was regularly used for the treatment of growth failure after hsct. about 50% of the centres used gh treatment for growth failure due to ghd only. in centres not using gh the main reason was that they saw no indication for gh treatment. only a minority did not use gh due to the risk of side effects. the insulin-test, arginintest and spontaneous gh-secretion were the major tests used for the diagnosis of ghd. a. rovo, m.t. van lint, m. aljurf, n. salooja, g. sucak, a. hunter, m within nonmalignant complication after hematopoietic stem cell transplantation (hsct) gonadal dysfunction with absence of sperm production leading to defi nitive infertility is a common long-term sequela. young age at hsct and longer interval time since hsct has been associated as a favourable factor for spermatogenesis recovery. the total body irradiation (tbi) used as part of the conditioning regimen plays a central role in posttransplantation infertility. we assessed in a retrospective multicenter study on a large cohort of male survivors, the probability of sperm recovery after hsct, and evaluated associated factors for fertility recovery. male patients in which at least one seminal fl uid analysis was performed after hsct being in complete remission and in whom the results were available were the target population. ninety centers reporting to the ebmt were asked to participate, 23 accepted and so far 15 centers contributed with 217 patients. the median age at hsct and at time of 1st sperm fl uid analysis (sfa) was 23 and 29 (5-64) years respectively. the median time interval between hsct and sfa analysis was 54 months . 206 (93%) received an allogeneic hsct, 169 (73%) had bone marrow as source of stem cells, 201 (94%) received a myeloablative conditioning and 149 (67%) received tbi (with a median doses of 12 gy (7.5-14.4) as part of the conditioning. during the follow-up 130 recipients (63%) had any grade of acute gvhd, 128 (64%) any type of chronic gvhd (cgvhd); 23 (17%) from them had ongoing cgvhd at sfa time. presence of at least one spermatozoa in sfa was considered as recovery of spermatogenesis. spermatozoa were detected in the semen in 58/217 (27%) patients; 19 (9%) of them showed normozoospermia. in the univariate analysis following associated factors were signifi cant (table 1) : younger age at hsct, longer time interval between hsct and sfa, conditioning without tbi, patients without ongoing cgvhd. in the multivariate analysis, absence of tbi (p<0.00001), longer time interval (p = 0.002), absence of ongoing cgvhd at sfa (p = 0.037) were signifi cant. in conclusion, this is the largest population of male survivors evaluating spermatogenesis after hsct. in this study we confi rm the established factors which infl uence recovery of spermatogenesis such as age (univariate analysis), time interval between hsct to sfa and tbi. here for the fi rst time evidence for graft versus testis effect can be demonstrated. introduction: development of leukemia or myelodysplasia derived from donor cells termed donor cell leukemia (dcl) is a rare but severe complication following allogeneic hematopoietic transplantation. the estimated incidence is low ranging from 124 cases of dcl per 100,000 transplants (mostly myeloablative conditioning, retrospective analysis) to 5,000 per 100,000 transplants (non-myeloablative, single institution experience). although about 60 cases have been reported in the literature, very little is known about pathogenesis and clinical management of donor cell leukemia. factors proposed to be involved in malignant transformation and development of dcl include immunoregulatory dysfunction, immunosuppression, host environment triggering malignant processes, replicative stress and epigenetic reprogramming as well as antigenic or viral stimulation. due to the increasing number of case reports throughout the last few years the late effects working party decided to analyze and evaluate the experiences with donor cell leukemia within the ebmt centers. methods: a fi rst, short questionnaire will be sent to all ebmt centers asking for observed, proven or suspected cases of dcl. centers reporting one or several cases will be asked to complete a second, more detailed questionnaire. the aim of the study is to identify and analyze a suffi cient number of cases to answer the following questions: how is the incidence of dcl evolving? is development of dcl associated with viral status before and viral reactivation/infection during as well as after transplantation? is there any infl uence of graft source and donor type (bone marrow vs. peripheral blood stem cells vs. cord blood and related vs. unrelated)? is the risk of malignant transformation of grafted cells increasing with donor age? is there any association with the form and extent of pretreatment (radiotherapy and chemotherapy) implicating a role of damaged microenvironment in the development of dcl? and fi nally, is dcl also observed as a very late complication of hematopoietic stem cell transplantation? conclusion: the answers to these questions will help to further characterize donor cell derived leukemia and provide new insights into leukemogenesis in general. a. crotta, a. tichelli, a. ruggeri, i. ionescu, a.l. herr, k. boudjedir, e. gluckman, v unrelated cord blood transplant (ucbt) is associated with a reduced incidence of chronic gvhd when compared to other sources of stem cells, however risk factors analysis for incidence is scarce in the literature. we retrospectively analyzed 1257 patients (pts), 755 children (age≤18) and 502 adults, receiving fi rst single (n = 1080) or double ucbt (n = 177) in ebmt centers, between 1995 and 2009 , for malignant and non-malignant diseases, who survived at least 100 days from transplantation with neutrophils recovery and without relapse or autologous reconstitution. median age was 12 years (0. , most common disease was acute leukemia (60%). 11% of units were hla-identical (antigen level for hla-a and b, allelic for drb1), while 41% and 48% had 1 or 2-3 mismatches, respectively. median tnc infused was 3.7x10 7 /kg. conditioning regimen was myeloablative (mac) in 75% of cases (tbi>6gy in 30%), ric in 25%. busulphan-based conditioning was used in 41% and atg was added in 75% of cases. csa + steroid was the most common gvhd prophylaxis (50%); in children prednisone was used in 70% of pts, mycophenolate mofetil was used in combination in 52% of adults. median follow-up was 28 months (3-157). incidence of cgvhd at 2y was 29±1% and 45±2% in children and adults respectively (p<0.001). due to statistical difference between children and adults, risk factors analyses were performed separately. gvhd was extensive in 42% of children and in 52% of adults. out of 412 pts who developed cgvhd, 219 had previously agvhd (126 out of 212 children and 93 out of 200 adults). in univariate analysis in children, factors associated with increased incidence of gvhd were: age (>5y), advanced status of disease at transplant (>cr2) and number of hla disparities; cgvhd was 25±2% and 36±4% (p = 0.03) in 6-5/6 cord blood unit and 4-3/6 respectively. in multivariate analysis only hla disparities (hr = 1.95, p<0.001) and status of disease at transplant (hr = 1.74, p = 0.006) were associated with increased incidence of gvhd. in adults, higher incidence of cgvhd was associated with male sex, advanced disease at ucbt and use of mac regimen. in multivariate analysis only the status at transplant was independently associated with cgvhd (hr = 1.46, p = 0.03). in conclusion, incidence of cgvhd was lower in children than in adults. status of disease at transplant was associated with increased incidence of gvhd in children and adults. number of hla disparities was associated with increased cgvhd only in children. in ms, progression free survival has been reported to range from 50 to 70% at 5 years after hsct. the most frequent conditioning regimen adopted in europe is the association of beam and atg, an intermediate-intensity scheme able to completely suppress mri activity for at least 2 years in most of patients. mortality has dropped in the last years from a relevant 7,3% to 1.2%, due to a better transplant management and patients selection. in rapidly progressive ssc 5-years mortality is estimated to be 40-50% [11] . the most commonly used conditioning regimen in europe is cyclophosphamide (200 mg/kg) and atg. in these patients, progression free survival after hsct has been showed to be above 50% at 5 years. furthermore, the use of high intensity conditioning regimens does not seem to offer special advantages over lower intensity schedules for this disease. the distribution of transplants per year shows a drop of activity in sle and especially in infl ammatory arthritis, following the introduction of new biological agents after 2000. the emergence of pharmacological resistance in long-term treatments may suggest a possible renewal of the activity also in this subset of patients. in chronic infl ammatory bowel diseases (ibd) an increasing activity has also been shown in the last 6 years. hsct is able to provide a high rate of long-term, immunosuppression-free remissions in ad patients resistant to conventional therapies. appropriate selection of patients is crucial for providing the best risk-benefi t equipoise. evidence of effectiveness and careful assessment of toxicity is expected from ongoing and future prospective clinical trials. data reported to the ebmt registry at december 2009 102 european activity in multiple sclerosis r. martin (1) , g. mancardi (2) intensive immunosuppression followed by autologous transplantation of hematopoietic stem cells (ahsct) has been investigated as a possible strategy for the treatment of severe autoimmune disorders, including multiple sclerosis (ms) . it has been demonstrated that ahsct leads to the elimination of self reactive t cells and complete or almost complete renewal of the immune repertoire. immune system renewal has been demonstrated at the level of the t cell receptor repertoire in cd4 + and cd8 + t cells and in subsets of nk-and t cell population. according to the database of the european blood and transplantation group (ebmt) registry, more than 400 ms cases have been treated with ahsct. the retrospective survey, carried out in 2006, of all the patients recorded in the registry, with a median follow up of more than 3 years, showed that improvement or stabilization of neurological conditions occurred in 63% of cases. transplant related mortality was very high (7,3%) in the 1995-2000 period, but subsequently dropped to 1.3% in the 2001-2007 years. the results appear to be particularly impressive in rapidly evolving severe ms cases unresponsive to conventional therapies. in these cases, defi ned also as malignant forms of ms, numerous european groups convincingly demonstrated the capacity of ahsct to suppress the progression of the disease, with an unexpected relevant improvement of the neurological conditions lasting for a long period of time. the establishment of ahsct as a rescue therapy in highly active ms cases unresponsive to other conventional therapies represents a very important step in ms clinical management, and the above mechanistic studies strongly support the rationale of this approach. despite these advances, the acceptance of ahsct as a rational treatment escalation remains low among neurologists, and this is probably one of the main reasons why the phase 2 study (astims), comparing ahsct vs. mitoxantrone with a mri primary endpoint, had to be terminated due to recruitment diffi culties. based on this experience, a new phase iib/iii study is currently being designed. this study will compare ahsct with best available and approved therapy in the group of highly active relapsing-remitting ms patients, who failed fi rst-line therapy and one escalation step. the main goals of this trial will be to establish effi cacy in a controlled trial and to address important mechanistic questions. the status of the current discussion toward the trial design as well as strategies to recruit interested centers with ms-specialized neurologists and experienced transplant physicians at one location and potential mechanisms of funding will be presented. systemic sclerosis is a potentially life-threatening chronic autoimmune disease characterized by skin thickening, vasculopathy, and visceral involvement, mainly of lungs, gut, heart and kidneys. the clinical manifestations are diverse and refl ect a spectrum of subsets, ranging from limited to diffuse disease. diffuse cutaneous systemic sclerosis (dcssc) generally runs a more aggressive disease course, requiring intensive immunosuppressive therapy. haematopoietic stem cell transplantation (hsct) has resulted in long-term improvements of skin thickening, stabilization of organ involvement, but at the expense of mainly cardiopulmonary toxicity which has resulted in death in 5-8% of cases according to a recent analysis by the ebmt working party autoimmune diseases. to assess whether the benefi ts outweigh the risks, a prospective randomized controlled trial is nearing completion comparing hsct with iv pulse cyclophosphamide in which 156 dcssc patients have been enrolled in 27 centres. the primary endpoint is event-free survival, defi ned as survival until death or development of major organ failure during 2 years follow-up. formal analyses of safety and effi cacy will be done in 2011. interim safety analyses have lead to changes in the transplant protocol and eligibility criteria to also target patients with very early disease. the rationale of the trial is that effective intervention in very early disease is needed to induce long-term remissions. all patients are followed-up for at least 7 years. in anticipation of the results from the astis trial, a new smallscale clinical trial will be done to comprehensively investigate the mechanism of action of hsct in ssc focusing on its effects on biomarkers of fi brosis, autoimmunity, infl ammation and vasculopathy. it is hoped that the results of the astis trial and those of the north american scot trial will provide solid data to decide on the design of a subsequent randomized trial. if astis and scot indeed prove that hsct is superior over conventional chemotherapy, then one option would be to modify the trans-plant regimen to reduce the risk of cardiopulmonary toxicity and/or enhance the intensity of the control arm. crohn's disease (cd) is characterized by chronic infl ammation in segments of the digestive tract leading to tissue damages. uncontrolled infl ammation is associated with excessive immune responses towards the microbiota. progress has been recently made in the management of cd, with increased use of immunosuppression and biologic therapies. early optimized use of these treatments in patients with poor prognosis may provide a satisfactory control of the disease in the majority of patients. however, a fraction of cd patients experiences severe disease refractory to medical management, which may result in progressive tissue damages, need for surgery and chronic disability. the fi rst evidence of effectiveness of hematopoietic stem cell transplantation (hsct) was reported with the observation that patients with cd, who underwent allogenic or autologous hsct for a haematological or solid malignancy, experienced long-lasting remission of their ibd. although few cases regarding autologous hsct in concomitant cd and malignant diseases have been reported, collected data have shown similar results to allogeneic transplantation in terms of remission. beyond case reports, autologous hsct as primary treatment for cd has been investigated in two single-centre phase ii studies. despite the low number of patients and their limited follow-up, autologous hsct was shown to have the capacity to induce complete clinical and endoscopic remission, with impressive results. the astic trial, designed to assess its effi cacy and tolerability, is ongoing. included patients undergo mobilization of stem cells, and are then randomized to transplantation after one month or after one year. autologous hsct may represent a therapeutic option in cd patients with refractory disease, and could change the natural history of the disease, inducing in some cases long term remission. haematopoietic cell transplantation for autoimmune diseases: ebmt/cibmtr collaboration m. pasquini (milwaukee, us), r. saccardi (florence, it) high dose immunosuppressive therapy with autologous haematopoietic cell rescue has the potential to halt the autoimmune process and result in a medication-free period. since 1996, more than 1000 patients in europe and 400 patients in north and south america who underwent transplantation for autoimmune disease (aid) have been registered in the ebmt and cibmtr databases. despite this activity, questions related to the timing of transplantation and what aid benefi ts more from transplantation remain unanswered. the development of a collaborative strategy has the potential to answer some of these questions and assist the development of prospective studies that can advance the fi eld. following this strategy the cibmtr and ebmt autoimmune disease committees joined forces in 2007 to expand the study portfolio and establish an infrastructure to facilitate future studies. the most common aid indication for transplantation is multiple sclerosis (ms). data collection from both registries was harmonized into a single disease-specifi c form developed by a group of investigators affi liated to both committees. collaborative project to focus on long term outcomes after transplant for ms was developed, especially to determine factors associated with prolonged disease-free intervals. two in person discussions on the topic leveraged the development of an international phase iii clinical trial with the objective to compare transplant with best medical treatment in a population with rapidly progressive ms. the next collaborative initiative is to focus on the second most common indication, systemic sclerosis (ssc). we plan to start harmonizing disease-specifi c forms and a collaborative study focused in this disease. lastly, as the majority of patients present to transplantation with advanced aid, studying patients with coexistent aid who present for allogeneic transplantation, represent an opportunity to understand the graft-versus-autoimmunity effect. thus, the cibmtr is planning to identify these patients at time of registration and subsequently enrolled them on a prospect cohort study to evaluate the impact of graft-versus-host disease on aid. outcomes database on transplant for aid are currently underutilized due to the heteroneity of diseases, scarcity of comprehensive level disease-specifi c information and loose integration with non transplant specialist. bringing the ebmt and cibmtr together in the last 3 years, helped engage investigators, including neurologists and rheumatologist to develop collaborative studies. an update on autologous stem cell transplantation in severe systemic lupus erythematosus and in early diabetes type 1 d. farge, r. cervera, d. jayne, a. voskuyl, j.m. van laar, a. doria, m. mosca, d. boumpas, r. saccardi, e. snarski, j.f objectives: to present the experimental and clinical rational for the ongoing ebmt or nih trials for ahsct in severe sle and in early diabetes type i. background: since 1996, autologous hematopoietic stem cell transplantation (ahsct) has been used successfully in severe systemic lupus erythematosus (sle) and early insulin type i dependent diabetes (t1d) to induce durable remission. in sle bilag a, the nih or eurolupus cyclophosphamide (cy) protocols and mycophenolate mofetil (mmf) for induction are associated with 20% failure, 50% relapse and 10-15 % death at 10 yrs. among 85 sle out of 900 ebmt pts with fi rst ahsct in 2009, 3 years pfs was 54 % (95% ci: 42%-66%) with 87% (95% ci: 79%-95%) overall survival (farge d haemetologica 2009 ). higher remission rates and also some relapses, were shown in the north american experience (burt r, jama, 2006) with the possibility of resetting the autoimmune response and inducing tolerance in sle after hsct and then in new onset type i diabetes (voltarelli and burt et al, jama, 2007) . in susceptible strains of mice, allogeneic hsct prevents insulitis and development of type 1 dm. in 2008, follow-up of the 23 ahsct for t1d with in brasil, showed that 14/21 remained insulin free (and normal hba1c) with longest insulin free follow-up beyond 4 yrs and no correlation with pre-hsct c-peptide, but ahsct must be performed within 3 months of diagnosis. eight patients with t1d (<6 weeks from diagnosis, c-peptide positive, anti gad -antibodies positive) treated in poland using 200mg/cy conditioning and ahsct plus antithymocyte globulin (atg genzyme) had no major complications after 6 (2-15) mths median follow-up and 8/8 pts became independent from exogenous insulin within 24 ( + 6-+ 60) days after ahsct. acarbose may have positive effects on the duration of remission of t1d after ahct. planned studies: these were the basis for 1) the multicenter, ebmt approved astil phase ii study, to analyse the effi cacy of ahsct followed by mmf as maintenance for severe sle 5 yrs since diagnosis with sustained or relapsed active bilag a sle after at least 6 months of best standard local therapy among 30 patients. after cy 4 g/m 2 mobilisation, unselected ahsct will use cy (200 mg/kg body wt in 4 daily doses) plus rabbit atg and maintenance by mmf (2 g/day). the primary effi cacy endpoint will be the proportion of patients alive who achieve clinical success at 12 months and maintenance of this status until 24 months after inclusion; 2) the nih approved phase i/ii study of ahsct in 15 newly-diagnosed t1d pts within 3 months of diagnosis, and a positive antibody to an islet cell autoantigen and fasting c-peptide > 0.2 nmol/l. to compare cy 4 g/ m 2 mobilization, unselected ahsct using cy (200 mg/kg body wt in 4 daily introduction: graft versus leukemia (gvl) effect of hematopoietic stem cell transplantation (hsct) is mostly based on donor t cell-mediated alloreactivity. this is particularly relevant in the context of hsct from family haploidentical and matched unrelated donors (mud), in which mismatched hla molecules are potent targets for donor t cells. still, upon in vivo selective pressure by donor t cells, leukemia can undergo genomic rearrangements which result in loss of the patient-specifi c hla, a mechanism which our group recently demonstrated to be frequently responsible for leukemia relapse after haploidentical hsct (vago et al., nejm, 2009 ). methods: 113 consecutive patients who underwent a partially hla-matched transplantation for myeloid malignancies from 2002 to present, were included in our analysis. for 76 patients the donor was family haploidentical, and for 37 was unrelated and mismatched for a median of 2/12 hla alleles. all patients received donor t cells as part of the hsct protocol. post-transplantation follow-up comprised monthly bone marrow examination, with short tandem repeat (str) chimerism analysis and hla typing performed in parallel. in cases of relapse with suspected loss of the mismatched hla, str chimerism and hla typing were performed also on purifi ed leukemic blasts. results: disease relapse occurred in 31 and 9 patients after haploidentical and mud transplantation, respectively. after haploidentical transplantation, 11/31 relapses (35.5%) were due to mutated leukemic blasts which had lost the patientspecifi c hla haplotype. interestingly, 1 of the 9 relapses after mud transplantation occurred through the same mechanism, in a patient who had received two subsequent transplants from hla-c-mismatched donors. upon detection of the mutated leukemic blasts, 9 of these 12 patients were enrolled to receive a subsequent hsct from a different donor, mismatched for the remaining hla haplotype. conclusions: our data consolidate the clinical relevance of this escape mechanism from the gvl effect mediated by alloreactive donor t cells. screening for these mutants should be encouraged in patients who relapse after partially hla-mismatched hsct, to guide therapeutic strategies, avoid predictably ineffi cacious donor t cell add-backs, and quickly enroll the patients to a salvage transplant. to improve this approach, novel diagnostic and therapeutic tools are warranted, to provide earlier molecular detection and specifi c targeting of these mutants. impact of allogeneic stem cell transplantation on prognosis of patients with high-risk aml: results of the aml shg 295 and 01/99 trials k. wagner, g. heil, m. zucknick, d. hoelzer, o.g. ottmann, h. martin, m. lübbert, j. finke, w. heit, w. fiedler, l. kanz, g. schlimok, h. kirchner, a. raghavachar, w. brugger, a. ganser, j objective: patients with high risk acute myeloid leukemia (aml) have a dismal prognosis after chemotherapy. allogeneic stem cell transplantation (allosct) in fi rst complete remission (cr) is widely recommended for these patients but its impact on outcome is uncertain. methods: in the prospective multicenter trials aml 295 and 01/99, we treated 825 adult patients with aml (except apl) up to 60 years. all patients received intensive double induction and early consolidation chemotherapy. high risk patients were defi ned by either bad response to induction i (persistent bone marrow blasts on day 15 after start of therapy) or high risk karyotype (all karyotypes other than normal or cbf-leukemias). all high risk patients with a matched related donor (mrd) were scheduled for an allosct as late consolidation. patients without a family donor in the 295 trial should receive an autologous stem cell transplantation, whereas in the 01/99 trial an allosct from matched unrelated donors (mud) was recommended. results: median follow up of the patients is 78 months. 393 of the patients fulfi lled the high risk criteria. median overall survival (os) of these patients is 17 months and the six-year os is 30%. in multivariate analysis, a complex or monosomal karyotype (hr 1.82; 95% ci 1.36-2.43) and age above the median of 47 years (hr 1.53; 95% ci 1.18-1.98) were identifi ed as adverse prognostic factors for os. 234 of the 393 high risk patients (59.5%) achieved a cr. median relapse-free survival (rfs) is 12.8 months and six-year rfs is 33%. leukocytes above the median (hr 1.65; 95% ci 1.18-2.29) and a complex/ monosomal karyotype (hr 1.61; 95% ci 1.06-2.44) were identifi ed as independent prognostic factors for rfs. of the 234 patients who achieved cr, 97 received an allosct from mrd (n = 65) or mud (n = 32) in fi rst cr. median os of the transplanted patients is not reached and six-year os is 57%. median rfs after allosct is 100 months and six-year rfs is 53%. outcome did not differ between mrd and mud allosct. the impact of allosct on prognosis was analyzed in a multivariate cox-model with allosct included as a time-dependent covariable. in this analysis, allosct in fi rst cr signifi cantly improved os (hr 0.48; 95% ci 0.34-0.69) and rfs (hr 0.48; 95% ci 0.33-0.69). conclusion: allosct from matched related or unrelated donors in fi rst cr improves overall and relapse-free survival of patients with high risk aml. cord blood transplantation for adults with acute lymphoblastic leukaemia -a survey of outcomes conducted by eurocord and the acute leukaemia working party of the ebmt d. purtill (1) cord blood transplantation (cbt) is considered an option for adults with acute lymphoblastic leukaemia (all) for whom stem cell transplantation is indicated. however, little information is available regarding outcomes of this procedure in this group of patients. we conducted a retrospective review of the eurocord database in order to describe outcomes of adults treated with single or double cbt for all at ebmt centres from 2000 until 2008. two-hundred and thirty six patients with a median age of 30 years (18-62 years) were included. most were transplanted in fi rst complete remission (cr1) (n = 101) or cr2 (n = 75). median white cell count at diagnosis was 13.9x10 9 /l (0.6-624) and 56% (n = 56) of patients in cr1 had t(9;22) or t (4;11). double cord blood transplantation was performed for 73 patients (31%). overall median total nucleated cell dose at freezing was 3.9x10 7 /kg (1.45-8.81 ) and it was 4.5 (2.1-8.8 ) and 3.4x10 7 /kg (1.5-7 .0) respectively for double and single transplants. most patients received cord units with one (n = 65) or two (n = 133) hla disparities. overall cumulative incidence (ci) of neutrophil recovery was 74±3% and acute graft-vs-host disease was 33±3%. transplant-related mortality (trm) was 41±3% and 35±3% for patients in cr1 and cr2 respectively, and 63±3% for the 60 remaining patients with more advanced disease (cr3, relapsed or refractory disease). ci of relapse were 20±6%, 27±8% and 33±11% respectively for cr1, cr2 and advanced disease. at the median follow-up of 2 years, leukaemia-free survival (lfs) was 30±3% for the whole population and 39±9% for cr1, 38±6% for cr2 and 4±3% for advanced disease. lfs for patients in cr1 with t(9;22) or t(4;11) was 39±7% and for double cbt it was 38±7%. on multivariate analysis, being in cr1 or cr2 at transplant was the only factor associated with improved lfs (hr 0.25, 95%ci 0.08-0.42, p<0.001). when this group was analysed separately, the use of a reduced intensity conditioning (ric) regimen was associated with improved lfs (57±8% vs. 32±5%, hr = 0.53, 95%ci 0.27-0.79, p = 0.01). in particular, the 27 patients who received the combination of cyclophosphamide, fl udarabine and tbi (2-6 gy) had an lfs of 64±10%. cord blood is an alternative source of stem cells for allogeneic transplantation for adults with high risk all. results with ric and double cbt are encouraging and should be further investigated in a larger series of patients. superior long-term survival with a high rate of allogeneic stem cell transplantation in aml (non-apl) patients below 60 years of age g. juliusson (1) introdution: allogeneic stem cell transplantation (allosct) prevents relapse in aml, but the indication in intermediate risk is unclear and depends on the transplant-related mortality. the swedish acute leukemia registry (blood 2009; 113:4179) includes 3878 patients (pts) with acute leukemia diagnosed 1997-2006 covering 98% of all cases in sweden, with 9 million inhabitants. using this population-based registry, we evaluated the sct rates in aml, and the long-term outcome. allo-sct rates in different swedish regions were also evaluated. transplantation data was individually validated in 2008, and survival was updated through the national population registry. patients and results: of 797 adult aml (non-apl) pts <60 yrs (median 51 yrs), 304 (38%) had an allosct (23% with secondary aml) in cr1 (n = 206; 26%), or at later stages (n = 92; 12%), with decreasing rates by age ( figure 1 ). donors were unrelated in 52%. median follow-up was 6.2 yrs, and 170 (56%) of allografted pts were alive. treatment-related mortality (trm, i.e., death in cr), and aml deaths according to age, donor, and status at allosct is shown in figure 2 . estimated 5-yr and 10-yr os was 41% and 35%, respectively. these results could be compared to recently published large aml studies from ecog, eortc-gimema, uk mrc and gamlcg, with allosct rates of 5-15% and 5-yr os of 18-38%. the health care region se, which had the highest allosct rate (61% of aml pts <60 yrs) had a 5-yr os of 52% for the total aml population, as compared to the other swedish regions with a mean allosct rate of 35% and a 5-yr os of 40% (log rank, p = 0.04). the difference in allosct rate was mainly due to more allografts among older pts: in the age cohort 50-59 yrs, 42% of cr1 pts underwent allosct yrs in region se as compared to 15% in the rest of sweden, associated with a more pronounced survival benefi t (p = 0.007). conclusions: this is a real world population-based study on allosct rates, treatment-related mortality and long-term survival in aml pts. our results indicate that allosct is performed in sweden with relatively low total non-relapse mortality, and with a moderate relapse rate. the association found between a high allosct rate in pts 50-59 yrs and a better survival is intriguing, is suggestive of a positive impact of a more frequent transplantation practice in this age cohort, and supports the need for prospective studies. role of the graft-versus-leukaemia effect after reducedintensity conditioning allogeneic stem cell transplantation as treatment for acute myeloid leukaemia: a survey from the acute leukaemia working party of the ebmt f. baron, m. labopin, m. mohty, n. basara, d. niederwieser, n. milpied, j.j. cornelissen, c. malm, l. vindelov, d. blaise, j.j.w.m. janssen, e. petersen, g. socie, v previous studies have observed a lower risk of relapse in patients (pts) who experienced chronic gvhd after ric allo-sct versus in those who did not. the objective of the current study was to further investigate the association between chronic gvhd and relapse in a large cohort of pts given ric allo-sct as treatment for aml. data from 1188 aml pts in fi rst or second cr transplanted between 2000 and 2008 following a ric regimen at ebmt affi liated centers were analyzed. patients were given pbsc from hla-identical sibling (mrd, n = 879), or from hla-matched unrelated donors (mud, n = 309). median pt age at transplantation was 55 yrs in pts given grafts from mrd, versus 57 yrs in those given grafts from mud. the impact of chronic gvhd on relapse risk, non-relapse mortality (nrm) and leukemia-free survival (lfs) was assessed by time-dependent multivariate cox models and in a landmark analysis. three-yr incidences of relapse, nrm and lfs were 35 ± 2%, 14 ± 2%, and 50 ± 2%, respectively, while 2-yr incidence of chronic gvhd was 49 ± 2%. in a landmark analysis at 18 months after allo-sct, 5-year relapse rates were 10 ± 2% versus 19 ± 3% for patients with or without chronic gvhd (p = 0.04), respectively. in multivariate cox models, cr2 versus cr1 (p = .003), pt age >55 yrs (p = .008), alemtuzumab use in the ric (p = .048), tbi-based ric (p = .006), high-risk cytogenetics (p = .001), and absence of chronic gvhd (p = .015) were each associated with higher risk of relapse. factors associated with high nrm were mud versus mrd (p = .003), grade ii-iv acute gvhd (p<.001), and chronic gvhd (p = .002). factors associated with lower lfs were cr2 versus cr1 (p = .003), pt age >55 yrs (p = .007), alemtuzumab use in the ric (p = .012), and high-risk cytogenetics (p = .003). in conclusion, in this cohort of aml patients transplanted in remission, chronic gvhd was associated with a lower risk of relapse while profound in-vivo t cell depletion with alemtuzumab was associated with higher relapse rate suggesting that gvl effects play a role in preventing aml relapse in patients given ric allo-sct. in spite of the presence of gvl effect, chronic chvd was associated with higher nrm and therefore there was no net impact on lfs. strategies to decrease nrm related to chronic gvhd should be further investigated in order to keep the benefi ts from gvl effect and improve lfs. finally, the impact of chronic gvhd duration and severity on lfs need to be studied. allogeneic stem cell transplantation in acute myeloid leukaemia with normal karyotype: a risk factor analysis in 247 patients, based on molecular markers and stage at transplantation t. pfeiffer (1) , m. schleuning (2) cytogenetically normal acute myeloid leukaemia (cn-aml) represents a heterogenous disease, that can be subdivided by molecular analysis. only limited data is available on sct in different molecular subgroups, particularly in advanced stage. therefore, we retrospectively analyzed data on 247 pts. with cn-aml, who uniformly had received the flamsa-ric regimen for sct in 14 european centres. pts. suffered from de novo aml (76%), saml/mds (21%), and taml (4%). median age was 52y (19-71). donors were matched or mm family, and matched or mm unrelated donors in 30%, 2%, 50% and 18%, respectively. sct was performed in untreated disease (6%), primary induction failure (pif,23%), 1st complete remission (cr1,14%), and >cr1 (57%). median follow-up of survivors was 19 mo. overall survival (os) and leukaemia-free-survival (lfs) at 2y from sct was 51% and 47%. the stage at sct was the most important factor (p = .001 for os, <.001 for lfs). results were promising after sct in cr1 (2y os/lfs 76%), and pif (2y os/lfs 69%), but were inferior after sct in untreated disease (2y os/lfs 34%), or >cr1 (2y os 42%, lfs 34%). information on molecular markers was available in 183 pts. (74%). as suggested (schlenk, nejm 2008) , analysis was based on 2 subgroups: 22 pts. with isolated npm1 mutation (npm1mut), and 161 pts. with other genotypes (flt3-itd, n = 66; or wildtype flt3/wildtype nmp1 [flt3wt/npm1wt], n = 95). pts. with npm1mut had a 4y os/lfs of 75/63% with identical outcome, when transplanted in pif, cr1, or >cr1. pts. with other genotypes showed an os/lfs of 51%/48% at 2y and of 40%/39% at 4y, without differences among pts. with flt-itd and flt3wt/npm1wt. however, in this subgroup, outcome was highly dependent on the stage at sct, with excellent results after sct in cr1 (2y os/lfs: 76%) or pif (2y os/lfs: 75%/74%), but inferior outcome after sct >cr1 (2y os/lfs 38%/33%; p = .004 for os, .001 for lfs). conclusion: allosct following flamsa-ric produces excellent survival in pts. with cn-aml, particularly when performed in cr1. encouraging results in pif support an early sct, regardless of molecular subgroup, when cr is not reached after double induction. in npm1mut, sct in advanced disease achieved identical results as in early stage, supporting the delay of sct until relapse has occurred. in contrast, pts. with flt3-itd or flt3wt/npm1wt achieved signifi cantly worse results when transplanted >cr1, arguing in favour of sct in cr1 for this molecular subgroup. comparison of outcomes after allogeneic sct for adult patients with aml in remission using either i.v. busulfan plus cyclophosphamide or tbi plus cy in the myeloablative conditioning regimen: an-alwp-ebmt survey a. nagler (1) , m. labopin (2) , a. shimoni (1) tbi/cy and oral bu/cy are the most common myeloablative regimens for adults with aml with comparable survival and relapse probabilities. intravenous (i.v.) bu in contrast to oral bu has more predictable pharmacokinetics and a more favorable toxicity profi le. outcomes with i.v. bu/cy, thus, may be superior to those achieved with tbi/cy. in order to address these issues, the alwp of the ebmt performed a survey comparing i.v. bu/cy to tbi/cy as conditioning regimen for adult pts. with aml undergoing allosct. overall, 1479 allosct were analyzed: bu/cy -332, tbi/cy -1147. median age was 40 (range, 18-60) and 39 (range, 18-65) years and median transplant year was 2007 (2000 -2007) and 2004 (2000 -2007) for the bu/cy and tbi/cy groups, respectively (p<0.0001). disease status at allosct was cr1 -81% vs. 80% and cr2-19% vs. 20% for the bu/cy and tbi/cy groups, respectively (ns). wbc count at diagnosis was 15x109/l in both groups. cytogenetic: good -6% and 8%, intermediate -26% and 42% and poor risk -6% and 6%, respectively (na -44%-62% of pts). 76% and 82% of both groups underwent allosct from sibling donors, while 24% and 22% from mud, respectively. follow up was 15(0.5-96) and 41 mo. for both groups, respectively. 75% and 47% of the bu/cy and tbi/cy groups received pbsc, while 25% and 53% received bm grafts, respectively (p<0.0001). engraftment was similar in the bu/cy vs. the tbi/cy groups, 97% and 97%, respectively. cumulative incidence of nrm at 2y was 16 + 2% and 18±1%, respectively (p = 0.46). acute gvhd both >ii-iv and iii-iv were signifi cantly lower in the bu/cy vs. the tbi/ cy groups: 21% and 32%and 8.5% and 19.5%, respectively (p<0.0001). while, vod was signifi cantly higher in the bu/cy vs. the tbi/cy groups, 7.8% vs. 1.7%, respectively (p<0.001). two year relapse incidence was comparable, 25 + 3% and 21 + 1% for bu/cy vs. tbi/cy, respectively (p = 0.73). lfs was comparable, as well, 59 + 3% and 61 + 2%, respectively (p = 0.73). in multivariate analysis poor prognostic factors for lfs were: poor cytogenetics (p≤10-4), disease status (cr2 vs. cr1) (p≤10-4), age >40y (p = 0.001), mud vs. sib. donor (p = 0.037) and pb vs. bm grafts (p = 0.042). in conclusion, in this retrospective ebmt survey, outcomes of allosct including engraftment, trm, rr and lfs was comparable, while acute gvhd probability was signifi cantly lower and vod probability signifi cantly higher in adult pts. with aml in cr using myeloablative i.v bu/cy vs. tbi/cy conditioning regiments, respectively. higher incidence of relapse with higher doses of cd34 + cells from leukapheresis products infused in adults with acute myelocytic leukaemia autografted during the fi rst remission n.c. gorin, m.m. labopin, d. blaise, f. witz, t. de witte, g. meloni, m. attal, d. carreras, v. rocha on behalf of the alwp purpose: the cell source for autologous stem cell transplantation has shifted from bone marrow (bm) to peripheral blood (pb). for patients with aml in cr1, we previously showed that the risk of relapse was greater with pb than bm and a poorer outcome was associated with a shorter interval from cr1 to pb transplantation (≤80 days) (jco 2009 in press). leukemic and normal progenitors bear the cd34 + antigen and can be mobilized together; we questioned whether there was a relation linking the doses of cd34 + cells infused to the outcome. methods: out of 1262 patients autografted with pb more than 80 days post cr1 and reported to the ebmt registry using medb form, the dose of cd34 + cells infused was available in 772. results: the cd34 + cell doses were categorized by percentiles to divide the whole group into fi ve categories containing the same number of patients. we identifi ed the fi fth percentile (>7.16 × 10 6 /kg) as the cut off point for relapse incidence (ri) and leukemia-free survival (lfs). patients receiving the highest dose had a higher ri (57 ± 4%, vs. 44 ± 2%; p = 0.008) and a lower lfs (34±4% vs. 49 ± 2%; p = 0.007). in a multivariate analysis adjusted for differences, ri was higher in patients receiving the highest cd34 + cell dose ((hazard ratio [hr], 1.48; 95% ci, 1.12-1.95; p = 0.005).and the lfs was worse (hr, 0.72; 95% ci, 0.55-0.93; p = 0.01). conclusion: for patients with aml in cr1 autografted with pb, risk of relapse is greater and lfs is lower in those receiving the highest doses of cd34 + cells. oral session 2: early side effects o122 dna transfer from donor to host cells as a mechanism for epithelial chimerism and genomic alterations after allogeneic haematopoietic cell transplantation m. waterhouse (1) , m. themeli (2) , h. bertz (1) , n. zoumbos (2) , j. finke (1) , a. spyridonidis (2) (1)albert ludwigs university of freiburg (freiburg, de) ; (2)university hospital patras (patras, gr) research in the fi eld of allogeneic hematopoietic cell transplantation (allo-hct) revealed hidden consequences of the co-existence of two genetically distinct populations in the transplant recipient. first, epithelial cells with donor-derived genotype emerge and second, epithelial tissues of the host acquire genomic alterations. we asked whether horizontal transfer of donor-dna to host epithelium is operating in (and linking) both phenomena. 176 buccal samples were obtained from 71 allotransplanted patients and analyzed with microsatellite markers for the presence of donor-dna and microsatellite instability (msi). the presence of graft-derived hematopoietic cells in the samples was excluded by immunocytochemistry. the results were associated with clinical data. genomic instability induction and dna transfer in epithelial cells by allogeneic lymphocytes was assessed in vitro. we found a high contribution of donor-dna (mean 26.6%) in buccal samples in 61 out of 68 evaluable patients. in addition, 32% of the samples were positive for msi (msi + ). the extent of donor-dna was signifi cantly correlated with the occurrence of genomic alterations (p<0.05). the age of the patient and a female-to-male transplantation were signifi cantly correlated with msi. there was a trend for increasing risk of msi for patients who experienced severe graft-versushost disease. during follow up secondary malignancy was diagnosed in 5 patients (14%) who exhibited msi but only in 1 (3%) with no msi. by applying a time-dependent statistical analysis we found that the probability of secondary tumor development was signifi cant higher in the msi + as compared to the msipatients (p = 0.024, hazard ratio 6.68, 95% . in an in vitro model, we demonstrated that apoptotic lymphocytes may not only induce genomic alterations but also transfer dna through a phagocytotic process to co-cultured epithelial cells. the ingested lymphocytic dna was also incorporated into the nucleus and integrated into the genome of the recipient epithelial cells, resulting in the creation of hybrid chromosomes. our results indicate that continuous charge of the transplant recipient with apoptotic donor-dna and its illegitimate integration into host epithelium may come in light as epithelial cells with donorderived genome or genomic instability events and may provide a new model for elucidating protean clinical manifestations after allo-hct, such as secondary malignancy. the haematopoietic cell transplantation-specifi c co-morbidity index and non-relapse mortality after reduced intensity conditioning: fi nal analysis from the alwp of ebmt in aml patients in fi rst complete remission m. mohty, m. labopin, n. basara, j.j. cornelissen, r. tabrizi, c. malm, j. perez-simon, a. nagler, n. kröger, b. rio, r. martino, m. eder, k. bilger, d. bunjes, g. socié, d. blaise, e. polge, v the current study was designed to test the performance of the adapted charlson comorbidities index (ci; so-called "sorror index") and its association with outcomes among a cohort of 345 patients (i) aged >50 y.; (ii) diagnosed with a single disease entity, aml in cr1, and (iii) who underwent a ric allo-sct reported to the ebmt registry between 1999 and 2006. in this series, the median year of allo-sct was 2004, and the median age was 58 y. (range, 50-76) . 32% of patients needed more than one induction course to achieve cr1. a fl udarabinebased ric regimen was used in 64% of patients, while 31% of patients received low-dose tbi as part of their ric, and 6% received other non-specifi ed ric regimens. 76% of the patients received allo-sct from an hla-matched sibling donor. based on score calculated with hazard ratios (hr) estimated on the population studied, 161 patients (47%) had a ci score of 0, 96 patients (29%) had a score of 1, and 49 (14%) had a ci score of 2. the remaining 39 patients (11%) had ci scores of 3 or more. in this cohort, 2 years overall and leukemia-free survival rates were 64±3% and 54±3% and the 2 years relapse and non-relapse mortality (nrm) cumulative incidences were 32±2%, and 15±2% respectively. the 2 years nrm incidences according to comorbidities score 0, 1, 2 and 3 + were 9±2%, 15±4%, 18±5% and 31±7%, respectively. in multivariate models (adjusted for recipient age, donor type, use of tbi or not, and cytogenetics risk group) comorbidities such as moderate active liver disease, obesity, prior history of renal dysfunction, and prior history of severe liver disease were associated with the highest hrs for 2 years nrm (varying from 2.11 to 2.76) and 2 years cumulative incidences of nrm varying from 22% to 44%, whereas previous solid tumor, diabetes, rheumatologic abnormalities, moderate pulmonary diseases, cardiac abnormalities (other than arrhythmia and valve disease) were associated with the lowest hrs (varying from 0.2 to 1.0) with 2 years cumulative incidences of nrm varying from 5 to 17%. results from this large study performed in a single disease entity and homogeneous allo-sct setting, suggest that the hematopoietic cell transplantation-specifi c ci is a simple, informative and useful tool for capturing pre-transplant comorbidities, and for predicting nrm after ric allo-sct for aml in cr1 in patients aged >50 y. such index may be used for clinical trials and patient counselling before ric allo-sct. fibrin glue directly applied on damaged bladder mucosa during cystoscopy is highly effective to treat severe, refractory, haemorrhagic cystitis after allogeneic transplant m.c. tirindelli (1) (1) background: hemorrhagic cystitis (hc) occurring after hematopoietic stem cell transplant (hsct) signifi cantly affects quality life of patients, prolongs hospitalization and in some cases can become a life-threatening complication. its management has not been established. fibrin glue (fg) is a hemostatic agent derived from human plasma with proven effi cacy in repairing damaged tissues. study design and methods: this study included patients who met the following criteria: grade ≥3 hc not responding to hyperhydration, bladder irrigation, antiviral treatments and transfusion support. fg was obtained using vivostat system, an automatic method for processing and applying fg. during conventional cystoscopy and maintaining bladder distension by a co2 insuffl ator, fg was accurately sprayed through a specifi c applicator on bleeding mucosa. fg polymerized on contact and set over several days. the response to the treatment was defi ned complete (cr) for disappearance of hematuria, partial (pr) for at least one grade regression of hc and no response (nr). results: from jan 06 to oct 09, 626 patients undergoing an autologous (n = 428) or allogeneic (n = 198) hsct were registered at the rtn. no autologous hsct recipients developed hc of severe grade, whereas 18 of 198 patients (9%) undergoing an allogeneic hsct met the criteria to enter the study. these 18 patients (6 m, 12 f) with a median age of 32.5 years (range, 18-53) had been submitted to a hsct from hla identical sib. (n = 4), unrelated cb (n = 4), mud (n = 2) or related haploidentical donor (n = 8) for different hematological malignancies. all patients, deeply immunosuppressed with positive bkv viruria >7x10 6 copies/ml, developed a very severe hc, refractory to all current treatments including antiviral therapy. at time of fg application, hc persisted for a median of 16 days (range, 7-65) and was grade 3 and 4 in 14 and 4 patients, respectively. the number of fg applications was 1 in 15 patients, 2 in 2 and 3 in 1 patient for a median of 11 ml (range, 6.3-16.2) of glue. the treatment was successful in 16 out of 18 patients (89%). all 14 patients with grade 3 hc responded and the response was complete in 12 (86%) and partial in 2 (14%), while of the 4 patients with grade 4 hc: 1 achieved cr, 1 pr and 2 nr. no patient died of hc. conclusions: fg therapy is a feasible, safe, easy repeatable, not invasive, small time consuming, lightly expensive and highly effective procedure in treating severe, refractory posttransplant hc. glutamine-enriched intravenous total parenteral nutrition doesn't improve mucositis and clinical outcome after stem cell transplantation for childhood malignancies. a prospective double-blind controlled study on behalf of aieop group c. uderzo (1) , e. marrocco (1) , p. rebora (1) , f. cichello (1) , m. bonetti (1) , s. cesaro (2) , s. varotto (2) , p. verlato (2) introduction: high dose chemo-radiotherapy followed by stem cell transplantation (sct) constitutes the principal cause of post-transplant severe mucositis and related complications. intravenous glutamine-enriched solution (ges) has been advocated as one of the support treatment capable to improve marked body protein wasting, oxidative stress which result in a mucosal damage, often "primum movens" of infectious diseases beginning from g.i.tract damage. patients and methods: 118 patients (79 males, median age at sct 8.1 years) with haematological malignancies have been treated from june 2005 to june 2008 with high dose chemotherapy and/or tbi followed by allogeneic sct (35 match related,65 match unrelated,18 mismatch.patients were randomly and double-blinded assigned to undergo either for total parenteral nutrition (tpn) or for ges tpn at the dose of 0,4 g/kg/day from day of sct until the end of tpn. the principal endpoint of the study was the mucositis assessment according to who criteria.the evaluation of the clinical,haematological and laboratory parameters served to perform the evaluation of secondary end-points.statistical analysis was set up to demonstrate a 20% difference in terms of mucositis incidence and severity with a power of 80% (alfa = 0.05; two-sided tests). results: both study groups were comparable for age, gender, diagnosis and type of sct. the ges patients (60) were clinically similar to the controls at the entry; however, they didn't experienced a reduction of rate and grade of mucositis, with an odd ratio of 0.98(0.26-3.63) adjusted for the type of sct. neither the type of analgesic treatment nor the duration of opiod treatment, were statistically different. days of both different tpn, length of hospital stay, trm at day + 180,acute or chronic gvhd, incidence of severe infectious diseases, immunological recovery, progression of malignancies were similar in both groups. nutritional status at the beginning and at the end of both types of tpn didn't show any difference at univariate analysis. no toxicity of ges was observed. conclusions: the current study is one of the few designed to demonstrate the utility of ges on mucositis in the setting of children undergoing sct for malignancies. ges failed to infl uence incidence and severity of mucositis after sct and didn't offer any advantage on clinical outcome, as claimed by other retrospective studies often performed in adult transplanted patients. clinical and genetic risk assessment for overall survival in haematopoietic stem cell transplantation a. dickinson (1) objectives: non-hla gene polymorphisms and clinical risk factors impact on outcome after hsct. age, stage of the disease, time from diagnosis to transplant, histocompatibility and donor and patient gender combination are key transplant risk factors for hsct. we have assessed the impact of non hla gene polymorphisms on the ebmt risk score for overall survival in a eurobank cohort of 915 hla identical sibling and matched unrelated donor (mud) transplants. methods: hsct patients with acute leukaemia (aml + all (al)) (n = 463), cml (n = 187) plasma cell neoplasia (n = 120) or lymphoma (n = 145) and their donors from 8 transplant centres were genotyped for non-hla polymorphisms within the tumour necrosis factor receptor ii (tnfrsf1b), estrogen receptor (esr1), vitamin d receptor (vdr), interleukin (il) 6 (il6), il-1 receptor antagonist (ilrn), interferon gamma (ifng) and il 4 (il4) genes. genetic factors were assessed using the log rank test (p value < 0.2). candidate factors were included in addition to the ebmt risk score in a stepwise cox regression procedure to select fi nal genes. predictive value of the model for overall survival was assessed through the concordance (c) index and prediction error curves. results: in the whole cohort the presence of allele c in the donor il-6 genotype (snp il6-17) was associated with lower survival time (especially in the cml sub group p = 0.025) and improved the prediction of the ebmt risk score. in the al subgroup the absence of patient il4 (any t) and presence of il1rn (any c) (high risk group) were associated with lower survival time (compared to the remaining patients (low risk group)), and taken together also improved the predictive value of the ebmt risk score. figures 1a and b show the lower probability of survival (and increased trm) in the high risk versus low risk group. conclusions: this study confi rms the importance of non-hla genotyping for risk assessment in allogeneic hsct. improvement of fi t of the ebmt risk score presents a powerful novel tool to assess this impact of gene polymorphisms in a complex heterogeneous hsct population. in a confi rmatory study the ebmt risk score was associated with survival for an al patient group. assessment of patient il4 (any t) and il1rn (any c) in this second cohort is in progress. background: cognitive impairments have been found among patients receiving chemotherapy and hsct. however, studies with representative samples are rare, but needed to clarify the short and long term impact of different conditioning regimens and hsct. in the present multicenter trial, we assessed the prevalence and course of cognitive dysfunctions in patients with hematological diseases undergoing allogeneic hsct. moreover, the role of neurotoxic intensity of conditioning regimens was investigated. methods: 102 patients (61% male) with mixed hematological cancers (41% acute myeloid leukemia) at an average of 48 years of age were assessed before (t0), 100 days after (t1) and one year following (t2) allogeneic hsct. 36% of the participants received intense neurotoxic conditioning regimens. a battery of neuropsychological tests using computer-and paperpencil-based methods was used covering the domains of attention, memory, executive and psycho-motor function. measures assessing self-perceived cognitive impairments and psychological distress were additionally applied. results: compared to published test norms at each assessment time point, signifi cant impairments in several neuropsychological test parameters across all cognitive domains were found. at baseline, patients had impaired cognitive functions in 50% of the test parameters. no signifi cant change in the prevalence of cognitive impairments was observed over time except for a mild increase in psycho-motor dysfunction at t2. patients who were classifi ed as having intense neurotoxic conditioning were more likely to show impairments in the domain attention at t1 (p<0.05, d = 0.53) and showed a different time course of performance in attentional tasks compared to patients with mild neurotoxic conditioning (p<0.01, eta² = 0.05). conclusions: a remarkable amount of patients was classifi ed as having cognitive impairments prior to hsct. possible explanations include the impact of the hematological cancer disease, invasive treatments applied prior to conditioning and hsct, and treatment related distress. a decline in cognitive functioning was primarily limited to psycho-motor functions. however, subgroups of patients and in particular those with intense neurotoxic conditioning regimens might be at greater risk for developing short term cognitive impairments particularly in the domain attention. acknowledgement of funding: this study was supported by a research grant from the german josé carreras leukemia foundation. clinical outcomes of second allogeneic haematopoietic stem cell transplantation for acute leukaemia and myelodysplastic syndrome relapsing after fi rst allogeneic transplantation t. yamashita, t. kikuchi, i. kamiya, r. hanajiri, y. nagata, s. wakabayashi, k. taoka, t. kobayashi, k. ohashi, h. sakamaki, h. akiyama tokyo metropolitan komagome hospital (tokyo, jp) background: allogeneic hematopoietic stem cell transplantation (hct) is an effective therapy in acute leukemia and myelodysplastic syndrome (mds). but even after hct, these diseases recur in some cases and prognosis of these patients is very poor. second allogeneic hct (hct2) may be one of the most effective therapies in some patients who relapsed after fi rst allogeneic hct (hct1). because of the heterogeneity of hct2, it is often diffi cult to fi nd useful prognostic factors and appropriate strategies. in our single-center study, we retrospectively analyze the clinical data of 465 patients with acute myeloid leukemia (aml), acute lymphoblastic leukemia (all) and mds who received hct1 in our hospital to investigate the factors that affect clinical outcomes of hct2. patients and methods: we included patients with aml, all and mds who received hct1 between 1986 and 2008 in our hospital. probability of overall survival (oas) and event-free survival (efs) were calculated using the kaplan-meier method. cumulative incidence rates were calculated using standard methods for hematopoietic recovery, acute and chronic gvhd, relapse and non-relapse mortality (nrm). multivariate analysis was performed with variables that can affect the clinical outcomes using cox proportional-hazards regression models. results: a total of 465 patients were eligible for this analysis. five years oas of hct1 is 50.8%. 5 years cumulative incidence of relapse (ri) 30.5% and 138 patients relapsed after hct1. relapsed patients group was signifi cantly lower incidence of chronic gvhd (p = 0.008). in this patients group, hct2 was performed for patients with aml (n = 19), all (n = 11), mds (n = 4). myeloablative conditioning was used in 28 cases. five years oas of hct2 was 15.2%, and oas and efs of myeloabative hct2 was 24.4% and 13.1%, respectively. five years ri was 57,2% and trm was 30.7%. multivariate analysis showed that 1) relapse to hct2 over 180 days, 2) age at hct2 under 33 years, 3) cr at hct2, 4) related donors are signifi cant favorable prognostic factors for oas of hct2. conclusion: our study shows that myeloablative hct2 brought sustained remission in a proportion of patients with aml, all and mds who relapsed after hct1. age and disease status at hct2 are important factors that infl uence the clinical outcomes. this study also suggests that the duration from relapse after hct1 and hct2 have impact on regimen-related toxicity of hct2. hepatic dysfunction is frequent and diverse before and after allo-ric and has a major impact on the transplant outcomes p. barba (1) introduction: hepatic dysfunction is one of the most frequent and less studied organ impairments in the setting of allogeneic stem cell transplantation (allo). to analyze the impact of pretransplant liver function on the outcome of allo-reduced intensity conditioning (allo-ric) and to determine the incidence, characteristics and risk factors of hepatic injury after allo-ric we conducted a retrospective study in two spanish centers. patients and methods: we analyzed 281 adult patients receiving an allo-ric. the median follow-up for survivors was 5 years (rg 0.2-10). ric consisted in fl udarabine 150 mg/m 2 plus melfalan 70-140 mg/m 2 or busulfan 8-10mg/kg. pretransplant indicators of liver injury analyzed were aspartate aminotransferase (ast), alanine aminotransaminase (alt), gammaglutamyl transpeptidase (ggt), alkaline phosphatase (ap), total bilirubin (bil), prothrombin time (pt) and the category of severe hepatic disease according to hct-comorbidity index (hct-ci_hep). posttransplant severe hepatic injury (shi) was defi ned as maximal total serum bilirubin (bil) level > 4 mg/dl or encephalopathy of hepatic origin (according to hogan, blood, 2004) . results: pretransplant liver laboratory tests abnormalities were found in 51 (18%) patients. we observed high levels of ast, alt, ggt, ap, bil, pt and hct-ci_hep in 5 (2%), 16 (6%), 26 (9%), 21 (7%), 14 (5%), 2 (1%) and 27 (9%) patients, respectively. among the pretrasplant indicators of liver injury analyzed, hct_ci hep showed to be the best predictor of transplant outcomes. in multivariate analysis (mva), hct-ci_hep high risk patients showed higher 100-days and 2-years nrm (hr 3.1 [95%ci 1.5-6.2] p = 0.002 and hr 1.9 [95%ci 1-3.7] p = 0.04, respectively), lower overall survival (os) (37% vs. 53%, p = 0.04), higher grade 2-4 agvhd p<0 .001]), higher grade 3-4 agvhd and a trend to higher cgvhd (1.6 [95%ci 0.9-2.8] p = 0.06). after allo-ric, a total of 182 patients (65%) developed grade iii-iv liver toxicities. a total of 74 (26%), 111 (40%), 160 (57%) and 51 (19%) patients developed grade iii-iv levels of ast, alt, ggt, and bil at a median time of 194 (rg 0-1629), 146 (rg 0-1932), 195 (rg 0-3126) and 74 days (rg 0-783), respectively. sixty-seven patients (24%) developed shi at 5 years with a cumulative incidence of 19% (95%ci 15-25). main causes of shi were gvhd (n = 36, 54%) and pharmacological toxicity (n = 7, 10%). in mva, risk factors for shi were unrelated donors (hr 3. a hallmark of acute graft-versus-host disease (agvhd) is the selective attack of certain tissues, namely the gastro-intestinal tract, liver and skin but not others such as the pancreas and kidneys. imaging techniques such as whole body in vivo bioluminescence imaging, dynamic multiphoton-laser-scanningmicroscopy and ultramicroscopy utilized by others and our own group help to elucidate the elusive mechanisms underlying alloreactive t cell traffi cking. here we dissected the molecular mechanism that direct alloreactive t cells via vascular endothelial cells to target tissues in a murine mhc major mismatch hematopoietic cell transplantation mouse model. as a strategy we employed endothelial ligand blocking antibodies during the agvhd effector phase after alloreactive t cells leave secondary lymphoid organs. immunohistochemical analysis of target and non-target tissues revealed organ dependent upregulation of infl ammation induced endothelial ligands. the identifi ed surface molecules subsequently served as therapeutic targets. we learned that during the agvhd effector phase multiple endothelial ligands need to be blocked simultaneously to avert agvhd target manifestation. blocking of single molecules such as madcam-1, vcam-1, e-selectin, p-selectin alone could not prevent alloreactive t cell traffi cking to the intestinal tract, liver and skin. when we treated transplanted mice with the combination of two or even three antibodies we observed a reduction of alloreactive t cells. however, only when we blocked four targets simultaneously, namely madcam-1, vcam-1, e-selectin, p-selectin in combination we could prevent gvdh target manifestation in the skin, in the liver and the small intestines. from these data we conclude that alloreactive t cell homing depends on the recruitment by infl ammation induced endothelial ligands of target tissues. after clonal expansion of alloreactive t cells simultaneous blocking of several endothelial ligands was required to effi ciently abrogate agvhd in target tissues. our data indicate that t cell homing remains highly attractive for therapeutic interventions. our results also point out that several pathways should be targeted simultaneously in order to prevent agvhd. distinct temporal and spatial roles for host conventional and langerhans cells in the development of graft-versushost injury f. fallah-arani, h. goold, b.r. flutter, j. sivakumaran, c.l bennett, r. chakraverty royal free and ucms (london, uk) background: following bone marrow transplantation, donor t cell reactivity can be confi ned to the lympho-haematopoietic system or, in the presence of infl ammation, can additionally extend to peripheral tissues leading to graft-versus-host disease (gvhd). in this study, we have explored the role of individual host dendritic cell (dc) subsets in regulating these processes using models involving inducible depletion following donor t cell transfer to allogeneic chimeras. methods: balb/c thy1.1 + t cells were transferred to established allogeneic chimeras generated following reconstitution of lethally irradiated b6 mice with a mix of balb/c and b6.cd11c-dtr bone marrow. cd11c.dtr mice express a primate diptheria toxin receptor (dtr) under the control of the cd11c promoter. in the resulting chimeras, host cd11c + conventional dc (cdc) were depleted at the time of donor t cell transfer by injections of diphtheria toxin (dt). in experiments to test the role of host langerhans cells (lc), balb/c thy1.1 + splenocytes were transferred to balb/c + b6 > b6.langerin. dtr, where co-treatment with dt leads to depletion of host, epidermal lc. in each of the above settings, infl ammation was induced in some chimeras by systemic or local application of a toll-like receptor (tlr) agonist at the time of t cell transfer. results: absence of host cdc at the time of delayed t cell transfer abrogated accumulation of donor cd8 cells in recipient spleens and reduced in vivo cytotoxicity against host target b cells. this was associated with a lack of conversion to full donor chimerism, demonstrating a requirement for host cdc in priming the lympho-haematopoietic graft-versus-host response in the steady state. we then explored the role of host cdc and lc in a model of cutaneous gvhd, where local gvhd is induced following application of a tlr agonist (imiquimod). maximal imprinting of e-selectin ligand expression upon donor cd8 cells within the draining lymph node required the presence of host cdc. however, even in their absence, donor t cells were able to access the epithelium and cause injury. selective depletion of host lc had no effect on imprinting or tissue infi ltration of donor t cells. however, the capacity of donor t cells to induce gvhd was severely impaired in this case. conclusions: these data show that host cdc are required for gvh reactivity in the steady state. in contrast, in a model of skin gvhd, host cdc are dispensable whereas lc have a unique post-priming role. failure to imprint donor cd8 memory and exhaustion may explain loss of gvt responses following donor leukocyte infusions b.r. flutter (1) , f. fallah-arani (1), s. sivakumaran (1) , c.l. bennett (1) , s. ghorashian (1) , g. freeman (2) , m. sykes (2) (1)university college london (london, uk); (2)harvard medical school (boston, us) background: donor t cell alloreactivity can be co-opted to deliver graft-versus-tumour (gvt) responses following donor leukocyte infusions (dli) given after bone marrow transplantation (bmt). however, the major reason for treatment failure following dli is relapse, suggesting a long-term failure of antitumour immunity. a distinctive element to the gvt response is that the antigens (ag) to which the response is directed are cleared from the haematopoietic system but continue to be expressed by non-haematopoietic cells. in this study, we have explored the infl uence of non-haematopoietic ag upon the antihost cd8 t cell response in a model of delayed dli. methods: b6 cd45.1 t cells were given after 8 weeks to allogeneic chimeras where ag was ubiquitous (b6 + bdf1 >bdf1) or restricted to the haematopoietic compartment (b6 + bdf1 >b6). in each setting, direct priming of the initial t cell response by bdf1 ag-presenting cells resulted in the eradication of bdf1 haematopoietic cells. however, in b6 + bdf1 >bdf1 chimeras, ag was still present in peripheral tissues, whereas in b6 + bdf1 >b6 chimeras, ag was cleared completely. the donor t cell response was tracked at timed intervals following transfer in both sets of chimeras. results: the continued presence of non-haematopoietic ag led to a failure to sustain donor cd8 cytotoxic and effector cytokine responses by 60d following transfer and this was associated with a failure to establish a central memory (t-cm) population. in contrast, where ag was absent in peripheral tissues, functional t-cm populations were preserved. the failure to generate donor t-cm in chimeras with ubiquitous ag expression occurred at two distinct levels. firstly, residual dli-derived cd8 t cells demonstrated a pd-1 hi/cd127 lo/ ifn-g neg 'exhausted' signature. consistent with this, cd8 functions were partially restored by in vivo antibody blockade of the pd-1 pathway. secondly, during the initial phase (<14d) of the response, we observed a lack of memory precursor formation when ag was ubiquitous. thus, when ag was ubiquitous, the capacity of activated, post-mitotic, cd8 t cells to establish recall immunity following transfer to ag-free hosts was much reduced. conclusions: these data demonstrate that alloantigen within non-haematopoietic tissues is not ignored, but rather blocks memory imprinting and drives eventual cd8 t cell exhaustion. while these effects may lessen the risk of gvhd, they might also impair the durability of the gvt response. background: adoptive transfer (at) of tcr-gene transduced t cells has become a promising therapeutic tool, however, limited in vivo survival and the development of an unresponsive state often termed 'exhaustion' has hampered reproducible clinical success. here we demonstrate that a) upregulation of pd-1 on donor-derived tcr-engineered t cells is associated with a loss of gvl effects after late at and b) pd-l1 blockade after hematopoietic stem cell transplantation restores robust gvl-effects. methods: we chose a mhc-mismatched allogeneic bmt model (b10.a mice (h-2a) into c57bl/6 (h-2b) mice) and a retrovirally encoded tcr (ot-1 anti-ovalbumin) that was introduced into b10.a-derived donor t cells for at after mhc-mismatched hct. after hct, leukemia-bearing (c1498-ova) mice (syngeneic hct-recipients served as reference) received at and were monitored for gvhd, gvl, and in vivo t cell persistence/ functionality. for pdl-1 blocking experiments pdl-1 antibodies (clone 10f.9g2) were used. results: 1) up to 1x10 9 tcr-transduced cd8 + t cells were generated in vitro within 7 days using a retroviral vector system linking the genes for the á-and â-chain via a 2a sequence. 40% of cd8 + t cells coexpressed the respective tcr chains vá2 and vâ5. 2) the introduced tcr mediated comparable specifi c cytotoxicity against c1498-ova in vitro being functional on autologous and allogeneic t cells. 3) after early at transduced t cells rescued up to 60% of mice in a dose dependent manner. (p = 0.001-0.01 versus mock-transduced controls). 4) after late adoptive transfer (day 56) autologous t cells peaked in numbers by day 3 after at and provided strong gvl-effects. in contrast, numbers of allogeneic tcr-modifi ed t cells declined rapidly within the peripheral blood and increasingly expressed pd-1. lower frequencies of allogeneic t cells in the periphery translated into nearly abolished gvl effi cacy upon leukemia challenge 3 days after at (p = 0.004). 5) pd-l1 blockade in vivo after allogeneic hct restored gvl-effi cacy of adoptively transferred t cells raising leukemia free survival from 0% to 60% (p = 0.001 compared to isotype controls) without increasing gvhd rates. conclusion: at with tcr-engineered t cells after allogeneic hct can decrease the risk of gvhd and provide potent gvl effects. however, increased expression of pd-1 on adoptively transferred t cells is associated with decreased gvl effi cacy and can effectively be reverted by pd-l1 blockade. allogeneic hematopoietic stem cell transplantation (allo-hsct) is a curative treatment for many hematologic malignancies or hematopoietic dysfunction syndromes in adult patients, but the application is still limited due to major complications, such as severe graft versus host disease (gvhd). diagnosis of agvhd is based on clinical features and biopsies. we have shown earlier that agvhd can be diagnosed using a proteomic pattern established by screening with capillary electrophoresis (ce) and mass spectrometry (ms). the agvhd-specifi c proteomic pattern has been evaluated prospectively and blinded on more than 960 samples collected from more than 141 patients undergoing allo-hsct at hannover medical school (mhh) and 7 additional clinics. since 2007 a new diagnostic proteomic pattern for agvhd was developed, using a higher resolution ms. thus, 192 patients from mhh and 133 patients from 4 collaborating clinics were subjected to double screening for development and prospective evaluation of the ms-17-agvhd-specifi c pattern. the majority of the patients included was transplanted for hematological malignancies (n = 313), 12 for hematopoietic failure syndromes (2 pnh, 9 saa) . conditioning regimens included reduced intensity conditioning regimens (ric) for about 60% of the patients of mhh, as well as standard conditioning regimens (mainly tbi + cy or busulfan + cy). gvhd-prophylaxis was cyclosporine a (csa) and mycophenolate (mmf) or csa metothrexate (mtx), as appropriate. in addition, about 80% percent of the patients received atg (antithymocyte globulin) prior to hsct. the new agvhd-specifi c pattern, ms_17, consisting of 17 differentially excreted proteins and peptides was developed and prospectively evaluated in parallel for the last 2 years. prospective and blinded evaluation of the patients included in this analysis for early recognition of patients at risk for agvhd development revealed the correct classifi cation of patients developing agvhd about 7 days (range: 2-21 days) prior to the development of clinical symptoms for agvhd with a sensitivity 76% and specifi city of about 85%. pre-emptive therapy ad ministered upon positivity of the protemic patterns reduced the incidence and severity of agvhd (p = 0.04). thus, a multicenter study has been initiated in germany to test the effi cacy and safety of pre-emptive therapy. hhv6 and acute gvhd c. pichereau, k. desseaux, a. janin, r. peffault de latour, m. robin, p. ribaud, s. chevret, g. socié hôpital saint louis (paris, fr) background: previous studies have suggested that hhv6 infection is correlated with acute gvhd following allogeneic hsct, but whether hhv6 triggers, is associated with, or is a differential diagnosis of gvhd is unknown. methods: 414 patients received an allogeneic hsct at the saint louis hospital (paris) between january 2004 and december 2007. whenever acute gvhd was suspected, hhv6 rq pcr and organ biopsy was performed. cumulative incidence of hhv6 reactivation was estimated using competing risks approaches. predictive factors of increased risk of reactivation or acute gvhd were assessed using cause-specifi c hazard cox models. effect of reactivation on further occurrence of acute gvhd was tested by introducing a time-dependent variable. results: 376 pts were tested for hhv6: 229 (61%) were male, median aged 40 years . 300 (80%) had malignancies and 76 suffered from non malignant disorders (aplastic anemia, sickle cell disease, thalassemia). 188 pts were grafted with related donors (50%). the conditioning regimen was myeloablative in 237 cases (63%). the source of hsct was bone marrow in 161 cases, peripheral blood in 157 cases and cord blood in 58 cases. 240 pts (64%) developed acute gvhd. hhv6 was detected in 100 pts, 66 of whom developed gvhd. hhv6 reactivation was signifi cantly associated with cord blood graft (59% versus 21%, p<0.0001) and unrelated transplant (39% versus 14%, p = 0.007). gvhd was signifi cantly associated with previous hhv6 reactivation (hazard ratio, 1.63; 95% confi dence interval, 1.01-2.62; p = 0.04). tissue biopsies were available for 64 of the 100 hhv6 infected pts (32 skin and 32 gut samples). there were no signifi cant difference between pts who had a biopsy and pts who hadn't, except for the incidence of acute gvhd (75% versus 50%, p = 0.01). 24 pts who had no or poor pathological evidence of gvh (grade 0 or 1), later on developed severe clinical acute gvhd (grade iii and iv), suggesting the role of hhv6 as a trigger of severe gvhd. beside this, 10 pts who didn't have clinical and histopathological gvhd (grade 0) showed a significant lymphoid infi ltrate on their biopsy. immunohistochemical analysis are ongoing, that could provide the evidence of "pure" hhv6-related skin rash after hsct. conclusion: this study suggests the role of hhv6 as a trigger of acute gvhd, particularly in its severe manifestations. beside this, it confi rms that hhv6 infection is a differential diagnosis of acute gvhd in a signifi cant proportion of patients. prochymal® improves response rates in patients with steroid-refractory acute graft-versus-host disease involving the liver and gut: results of a randomized, placebo-controlled, multicentre phase iii trial in gvhd p.j. martin (1) , j.p. uberti (2) background and methods: steroid-refractory acute gvhd (sr-gvhd) remains a signifi cant and life-threatening complication of allogeneic hematopoietic cell transplantation (hct). prochy-mal® (mesenchymal stem cells, msc, derived from unrelated volunteer adult donors) was evaluated in addition to standard of care, including institutionally selected second line treatment, in a randomized (2:1) trial in patients with sr-gvhd (protocol 280). patients received 8 infusions of 2 x 10 6 msc/kg over 4 weeks (or volume equivalent for placebo), with an additional 4 infusions administered weekly after day 28 in patients who had a partial response, defi ned as improvement in at least one organ without progression in others. the primary endpoint was durable complete response (dcr) for ≥ 28 days. additional prospectively defi ned outcomes included responses in patients by organ involvement. patients and results: 244 patients with sr-gvhd (skin involvement n = 144, gastrointestinal involvement n = 179, liver involvement n = 61) were enrolled and treated on a 2:1 basis: prochymal (n = 163), placebo (n = 81). there were no signifi cant differences in age, pre-transplant conditioning, graft source, hla-matching or second-line therapy between treatment arms. for the prochymal and placebo arms, respectively, the grades of gvhd at entry were b (22% vs. 26%), c (51% vs. 58%), and d (27% vs. 16%). the respective dcr rates were 35% vs. 30% (p = 0.3) in the intent-to-treat population and 40% vs. 28% (p = 0.08) in the per protocol population. results for secondary endpoints are shown in table 1. patients with gvhd affecting all 3 organs had overall complete or partial responses rate of 63% vs. 0% (n = 22, p<0.05, fisher's exact test) at day 28. patients treated with prochymal had less progression of liver gvhd at weeks 2 and 4 respectively (32% vs. 59%, p = 0.05; and 37% vs. 65 %, p = 0.05). the incidence of infections was not different between arms. incidence rates were 9% vs. 8% for recurrent malignancy, 1.8% vs. 2.5% for infusional toxicity, and 0.6% vs. 4.6% for ae-related discontinuation in the prochymal and placebo arms, respectively. conclusion: gvhd with liver or gut involvement is a life-threatening complication of hct. these results suggest that the addition of prochymal produced signifi cant improvement without additive toxicity in patients with sr-gvhd involving visceral organs. s18 o137 treatment of steroid-refractory acute gvhd with mesenchymal stem cells improves outcomes in paediatric patients. results of the paediatric subset in a phase iii randomized, placebo-controlled study p. szabolcs (1) , g. visani (2) background: successful treatment of steroid-refractory acute graft-versus-host disease (sr-gvhd) following allogeneic hematopoietic cell transplantation remains a signifi cant challenge. because of their immunomodulatory properties and safety profi le, adult mesenchymal stem cells (mscs) have been proposed as a treatment for sr-gvhd. intravenous allogeneic msc therapy (prochymal) for sr-gvhd was independently evaluated in the pediatric subset of a double-blind, placebocontrolled study. methods: pediatric patients (<18 years old) with grade b-d sr-gvhd were randomized to receive either prochymal or placebo in addition to standard of care, including institutionally selected second line agent. patients received 8 infusions of 2 × 10 6 cells/kg for 4 weeks (or volume equivalent for placebo), with 4 more infusions weekly in the case of a partial response (pr). the primary endpoint was durable complete response (cr 28 days); secondary endpoints included incidence of cr, pr and progression through 100 days, survival, and safety. results: twenty-eight children were randomized to prochymal (50% male, 79% caucasian) or placebo (71% male, 71% caucasian), with a median age of 7 yrs (range 1-15) and 10 years (range 1-18), respectively. the dominant transplant graft source was cord blood (71% prochymal, 57% placebo), with mostly unrelated donors (93% vs. 79%, respectively). the median duration of agvhd prior to enrollment was 20 days for prochymal and 8 days for placebo (p<0.05). at baseline, agvhd grades b:c:d were 3:8:3 for both arms. for prochymal, organ involvement was 64% skin, 43% gi, and 36% liver. for placebo patients, organ involvement was 57% skin, 79% gi, and 29% liver. durable cr was 64% for prochymal and 43% for placebo (p = 0.5). prochymal improved rates of cr and or (table) . the median time to cr was 25 days vs. 63 days. the safety data showed no infusional toxicity and no evidence of prochymal leading to ectopic tissue. there were no aes leading to discontinuation of therapy. conclusion: in a sr-gvhd population in which 79% of patients had grade c/d disease, the addition of prochymal to standard of care resulted into a faster and better cr. in view of their increased response rates and a well-tolerated safety profi le, mscs appear to be a safe and effective therapy in the treatment of pediatric patients with sr-gvhd. aim: response rate after induction and after asct. patients and methods: td consisted of thalidomide 200 mg/d and dexamethasone 40 mg on days 1-4 and 9-12 at 4-week intervals for 6 cycles. vtd was identical to td plus velcade 1.3 mg/m² on days 1,4,8,11 of each cycle. combination chemotherapy plus velcade consisted of 4 cycles of vbmcp/vbad followed by 2 cycles of velcade. results: as of december 31, 2008, 306 patients (median age: 57 yrs, m 156, f: 150; igg 183, iga 71, light chain 43, others 9) entered the study. 55 of 253 (22%) had high-risk cytogenetics (t(4;14), t(14;16) and/or 17p deletion). all 306 patients (td: 104, vtd: 102 and vbmcp/vbad/velcade: 100) were evaluable for response and toxicity to induction therapy. the cr plus vgpr rate was signifi cantly higher with vtd than with vbmcp/vbad/velcade (59 vs. 37%, p = 0.003) and td (59 vs. 28%, p<0.001). the progressive disease (pd) rate was higher with td than with vtd (22 vs. 8%, p = 0.005). in patiens with high-risk cytogenetics vtd was associated with a higher cr rate than td and vbmcp/vbad/velcade (42 vs. 0%, p = 0.003 and 23 vs. 0%, p = 0.04, respectively) as well as with a lower pd rate (0 vs. 37%, p = 0.003 and 0 vs. 23%, p = 0.04, respectively). the incidence of > grade 2 thrombotic events was higher with td than with vtd (8% vs. 1%, p = 0.01) and grade >2 peripheral neuropathy was higher with vtd than with td or vbmcp/ vbad/velcade (14 vs. 1% vs. 0%, p<0.001). 218 patients were evaluable for response after asct. the cr rate was higher with vtd (52%) than with vbmcp/vbad/velcade (49%) and td (37%) although the differences did not reach statistical signifi cance. the estimated os at 2 yrs. was 82% with no significant differences among the 3 arms. ttp and pfs were shorter with td (p = 0.05 and p = 0.012, respectively). summary: 1) induction with vtd produced a higher cr + vgpr rate, 2) in patients with high-risk cytogenetics, td resulted in a signifi cantly lower cr and higher pd rate than bortezomibcontaining regimens, 3) the pfs was shorter with td and 4) asct increased the cr rate in 23%, 21% and 26% in the td, vtd and vbmcp/vbad/velcade arms, respectively. interim analysis of a phase iii, prospective randomized trial of melphalan stem cell source was pbsc in 93% pts. 15% pts received myeloablative allogeneic sct as part of a planned tandem strategy, whereas 85% received a non myeloablative conditioning. overall response at allo sct was 84% and included complete remission (cr 19%), very good partial remission (vgpr 9%), partial remission (pr 56%). results: all pts except 3 had sustained donor engraftment. among pts, 36%, 13% and 20% achieved cr, vgpr and pr respectively at day 100. median follow-up from allo sct was 37 months (range 1-122). event-free survival (efs) was 58% (49-66) at 3 years. cumulative incidence of transplant related mortality (trm) at 100 days was 10% and was mostly related to pneumonia. a single line of treatment before tandem auto/allo sct was associated with improved 3-year-efs (p = 0.02). 28% pts experienced grade i to ii acute graft-versus-host-disease (gvhd) and 13% grade iii to iv. 11% pts had limited and 18% extensive chronic gvhd. whereas grade i to ii acute gvhd was associated to better 3-year-efs (p<0.0001), extensive chronic gvhd had no statistically signifi cant impact on efs. moreover the absence of deletion 13 was not associated with a better efs. conclusion: our results suggest that the number of treatment lines received before tandem auto/allo sct is an important issue, with an improved efs for pts treated by a single line before tandem. whereas better control of acute gvhd might further improve survival, impact of the deletion 13 on outcome seems limited. purpose: the outcome of mm patients treated with low dose tbi and allogeneic related sct following autologous sct has been shown to be superior to that from two autologous sct (gahrton et al.; bruno et al.) . since related donors are available for approximately 30% of patients only, the outcome of allogeneic unrelated sct in patients without a related donor was compared to that of patients with a related donor in an intention to treat analysis. patients and methods: mm patients (n = 44) with a median age of 51 (range 32-65) years were treated at the university of leipzig with autologous sct followed by allogeneic sct. autologous stem cells were mobilised with cyclophosphamide and transplanted after melphalan 200 mg/m 2 on day -3. allogeneic sct was performed at a median of 5 (range 2-11) months after autologous sct using fludarabine (30 mg/m 2 /d from days -3 to -1) and tbi (2 gy on day 0) followed by cyclosporine (6.25 mg/kg twice daily from day -1) and mycophenolate mofetil (15 mg/kg daily from day 0). patients received an unrelated (n = 22) or related (n = 22) stem cell graft. results: patients with unrelated donors (n = 22) showed hematological toxicity comparable to those of related donors. all patients had stable hematopoietic engraftment with t-cell chimerism of 100% 3-6 months after sct. with a median follow up of 41 (range 2-87) months overall survival (os) at 5 years was 51±12% and 52±13% (p = 0,19) in patients with unrelated and related donors, respectively. progression free survival (pfs) was 34±12% at 5 years for patients with an unrelated compared to 14±8% for patients with a related donor. this difference was due to a decreased relapse incidence (ri) in patients with unrelated donors (54±12%) compared to related donors (84±9%). non relapse mortality was 22±9% in the whole population with no difference between the two groups. of the 22 patients having received allogeneic unrelated sct, 7 (32%) are currently in cr, 2 (10%) in vgpr, 3 (14%) in pr and 1 (5%) has stable/progressive disease, with no difference between related and unrelated sct. conclusion: our data confi rm the feasibility of autologous sct followed by low-dose tbi conditioning regimen and unrelated sct. pfs seems to be higher and ri lower in unrelated compared to related sct. these feasibility data provide the basis for a phase iii study comparing auto-allo unrelated to auto-auto sct. background: poems syndrome is a rare paraneoplastic syndrome resulting from a clonal plasma cell proliferation producing a small monoclonal protein usually lambda type restricted. this syndrome is a multisystemic disease, its major clinical feature being a progressive peripheral neuropathy. single osteosclerotic lesions should be treated with radiation therapy only. in widespread disease, high-dose therapy followed by autologous hematopoietic stem cell rescue (asct) has shown to be an effective therapeutic option in short series, but with significant morbidity. patients and methods: between december 1999 and september 2009, 19 patients with poems syndrome (12 female/7 male) were treated with melphalan-200 (16 patients) or melphalan-140 (3 patient) followed by asct at 9 spanish institutions. median age was 54 years (range: 26-67). all of them presented a m protein lambda type (iga: 16; igg 3), peripheral polyneuropathy (18), osteosclerotic lesions (12), organomegaly (16), endocrinopathy (7), skin lesions (18), extravascular volume overload (14), papilledema (6), polycythemia (2) and thrombocytosis (12). four patients had pulmonary hypertension, two portal hypertension, and three castleman disease. four patients were previously untreated. the median number of prior therapies was 2 (range, 0-4). median time from diagnosis to asct was 8 months (range, 2-95). results: no transplant-related-mortality (trm) was observed. after a median follow-up of 45 months (range, 3-89), one patient has died of progression 90 months post-asct. five patients presented an engraftment syndrome and two a primary graft failure resolved, one with a back-up infusion on day + 26 and the other presented a delayed engraftment. sixteen patients were evaluable for response, 8 achieved a complete hematologic response (cr) (if-), 7 a near-cr (negative electrophoresis but if positive) and one died of progression 90 months post-asct. clinical improvement was observed at 4-6 months post-transplantation. all patients had a signifi cant organic improvement. the four patients with severe pulmonary hypertension and the two with portal hypertension improved of both pressures. conclusions: in this series, asct proved to be a highly effective therapy for patients with widespread poems syndrome. despite no trm was observed, these patients may have a delayed hematopoietic recovery and may develop an engraftment syndrome that must be diagnosed/treated promptly. a salvage treatment containing novel agents consolidated by allogeneic stem cell transplantation with reducedintensity conditioning improves outcome of multiple myeloma patients failing autologous transplantation f. patriarca (1), h. einsele (2) objectives: allogeneic stem cell transplantation (allo-sct) employing reduced intensity conditioning (ric) is a feasible procedure in selected patients with relapsed multiple myeloma (mm), but its effi cacy is still a matter of debate. we investigated the role of ric allo-sct in mm pts who relapsed after auto-sct and were then treated with a salvage therapy based on novel agents. our study was structured similarly to an intention to treat analysis and included only those pts undergoing a hlatyping immediately after the failure of auto-sct. the cohort of pts having a donor (donor group) was compared with the one not having a suitable donor (no donor group). patients and methods: one hundred thirty-six consecutive pts were retrospectively evaluated. fifty-seven found a donor and 50 (88%) underwent an allo-sct: 16 identical sibling (32%), 34 mud (68%). conditioning regimens were fl udarabine, melphalan±thiotepa in 21 patients (42%), fl udarabine + 2 gy tbi in 15 cases (30%), fl udarabine and cyclophosphamide in 8 patients (16%) and fl udarabine and treosulfan in the remaining 6 cases (12%). seven pts having a donor did not receive allo-sct for progressive disease or severe comorbidities. median age of donor group was signifi cantly younger than no donor group (53 versus 60 years, p = 0,000045). the 2 groups were balanced with regard of time between auto-sct and relapse, treatment of fi rst relapse, duration of salvage treatment, and quality of response after salvage treatment. results: the median follow-up for all patients was 15 months (1-97) after relapse. the median time to progression-freesurvival (pfs) and overall survival (os) for all patients were 16 and 25 months, respectively. two-year pfs was 41% in the donor-group and 18% in the no-donor group (p = 0.0003). twoyear os was 57% in the donor-group and 49% in the no-donorgroup (p = 0.08). in multivariate analysis the availability of a donor was statistically signifi cant for pfs (p = 0.003); moreover a signifi cant difference in outcome was observed comparing patients who achieved cr + vgpr versus pr versus sd/pd after salvage treatment (p = 0.05, p = 0.002, p = 0.0001 for pfs, p = 0.01, p = 0.001, p = 0.0001 for os). conclusions: this study comparing salvage treatment with novel agents consolidated by ric allosct versus salvage treatment alone after a failed auto-sct provides evidence for a signifi cant pfs benefi t and a trend for a prolonged os in mm patients having a donor. grouping patients based on d and non-d kir2ds4 allelic status revealed individuals homozygous for kir2sd4(d) had significantly shorter duration of fever, compared to those heterozygous or homozygous for kir2ds4 (non-d), (p = 0.003). this held true on multivariate analysis. no signifi cant association was seen between kir2ds4 genotype and neutrophil engraftment time or microbial isolates. of note, there was a signifi cant association between kir2dl3 positivity and gram-ve bacteremia (p = 0.017). these data support a role for kir genotype and infectious complications post-asct. kir genotyping may aid risk assessment of infectious complications post-asct and optimization of anti-infectious prophylaxis, surveillance and treatment in those deemed at risk of sepsis. these results suggest a functional role for the deleted kir2sd4 variant, previously believed non-functional in the innate immune response. in the era of novel therapeutic agents, high-dose chemotherapy and autologous stem cell transplantation (asct) remains an integral part of treatment for multiple myeloma (mm). therefore, the choice of new drug combinations for induction therapies must take into consideration the requirement to collect a suffi cient number of haematopoietic stem cells (hscs) for one or more courses of asct. lenalidomide and melphalan have been shown to impair hsc mobilisation, but induction therapies containing either thalidomide or cyclophosphamide do not have a relevant impact on the hsc collection yield. we considered the possibility that the combination of cyclophosphamide and thalidomide could have an additive impact on the hsc compartment and carried out a retrospective analysis of the outcome of peripheral blood hsc mobilisations performed in 111 mm patients. patients who had received induction therapy with ctd (oral cyclophosphamide, thalidomide, dexamethasone; n = 55) were compared with a control group of patients (n = 56) who had received vad (n = 30) or z-dex (idarubicin, dexamethasone; n = 26) during the same 4-year period. all mobilisations were performed with cyclophosphamide and g-csf. our standard collection target was 4 × 10 6 cd34 + cells/ kg, with a minimal target of 2 × 10 6 being accepted if patients failed the standard target. the total number of cd34 + cells harvested was signifi cantly lower in the ctd group (5.2 vs. 9.7 × 10 6 /kg, p = 0.002). the number of cd34 + cells harvested on the fi rst day of apheresis and per apheresis procedure were also lower in the ctd group (2.8 vs. 7.3 × 10 6 /kg, p = 0.002; 2.6 vs. 6.7 × 10 6 /kg, p = 0.002). more patients in the ctd group failed to achieve both the standard (36.4% vs. 16.1%, p = 0.021) and minimal (18.2% vs. 5.4%, p = 0.036) stem cell harvest target, despite a higher number of patients in the ctd group undergoing two or more apheresis procedures (52.8% vs. 32.1%, p = 0.012). the failure rate on the fi rst day of apheresis was also higher in the ctd group both for the standard (56.3% vs. 28.6%, p = 0.003) and the minimal target (36.7% vs. 16.1%, p = 0.041). age or number of induction therapy cycles did not have an impact on mobilisation failure in the entire cohort or the ctd group alone. these observations provide novel evidence that drugs with no previously demonstrated effect on hsc mobilisation can have a considerable negative impact when used in combination, which can result in a high rate of hsc collection failures. a. nagler (1) background: allogeneic transplantation of hematopoietic stem cells (allo-sct) from an hla-matched related (mrd) or unrelated donor (urd) is a curative option for patients (pts) with high-risk hematological disease (hrhd). in the absence of a mrd, pts have been offered investigational transplant strategies such as umbilical cord blood (ucb) or family haploidentical sct (haplo-sct). in our institution, all patients with hrhd are typed at entry; if a suitable mrd donor is missing a urd search is promptly activated. our policy is to offer an haplo-sct to adult pts lacking an mrd or urd in order to adequately treat hrhd in the ideal appropriate time according to clinical indications to allo-sct agreed in ongoing protocols for primary disease. methods: here we report the retrospective intention-to-treat (itt) analysis of alternative donor transplantation at our institution. data were obtained from institutional database. results: between jan-2004 and nov-2009, 361pts (100% of the following itt analysis; median age 48y) received indication to allo-sct according to ebmt recommendations. eightytwo pts (23%) received a transplant from a mrd; 170pts (47%) activated an urd search in the ibmdr registry; 78pts (22% of total pts, 46% of urd searching) received a urd transplant; 35pts (9.7%-20,5%) received an haplo-sct due to lacking of a suitable urd in the appropriate timing according to disease status, or absence of suitable criteria to engage an urd donor; 9 pts (2,5%-5,2%) received a ucb because lacking a suitable haplo donor. overall, 129pts received an haplo-sct (36%): 35 after ibmdr research, 94 up-front. nineteen pts died before receiving a transplant (5%), 44 (12%) are searching for a suitable donor. if we consider only pts with acute leukemia (213pts, median age 47y, range 15-72; over 50y 92pts) 47pts (22%) received a transplant from a mrd; 66pts (40%) activated an urd search; 37pts received a urd transplant (17%), 6pts (3%) a ucb, 97pts an haplo-sct (46%). eight pts died before receiving a transplant (4%), 18 (8%) are searching for a suitable donor. allo-sct was performed in complete remission in 104pts (62% alive), in persistence of disease in 83pts (20% alive). conclusion: in itt analysis, 83% of overall pts candidate received an allo-sct: 60% from an alternative donor, 23% from a mrd. the highly committed policy performed in the alternative-sct setting and the implementation of an alternative donor option are essential prerequisite to obtain these results. there was a marked variation in total numbers of transplants performed between countries ranging from 38 to 2629. the median age of hsct programs was 10 yrs (range 3-23). the total number of hsct performed per year has continued to increase and is yet to plateau. a greater proportion of transplants was allogeneic hsct (allo-hsct) compared to autologous hsct (asct) (77% vs. 23%). acute leukemia constituted the main indication for allo-hsct(37%). there was a relatively high rate of hsct for bone marrow failure (n = 1001, 17%) and hemoglobinopathies (n = 885,15%) when compared to data reported to ebmt and ibmtr. cml continued to be an indication for 8.7% of allo-hsct in 2007. the rate of unrelated donor hsct remained low, with only 2 non-umbilical cord unrelated donor transplants over the surveyed period. the use of peripheral blood stem cells varied between countries though increasingly constituted the main source of stem cells in allo-hsct. ric was used in 13.4% of allo-hsct in the the survey. asct rates continue to increase; while acute leukemia was the main indication for asct in the 1980s, overall the main indications for asct in the survey were lymphoma (45%) followed by myeloma (26%). conclusion: we present the fi rst survey of hsct activity in the em region over 23 years which refl ects the unique health conditions of this region, accounting for notable differences in transplant practices from europe and north america. annual hsct rates continue to rise. economic, logistic and other factors are likely to be responsible for disparities in activity within the region. this survey may be valuable in providing a basis for healthcare planning in the fi eld of hsct in the region. background: related haploidentical donors, as cord blood, can be alternative donor sources in stem cell transplantation (sct). severe graft-versus-host disease (gvhd), however, has interfered the progress of haploidentical stem cell transplantation (haplosct). to deal with this strong gvhd, t cell depletion has usually been used in european countries. on the other hand, based on the difference of ethnicity prone to less severe gvhd in japan, we have performed unmanipulated haplosct using myeloablative or reduced intensity preconditioning regimen for ten years. in this meeting, we will summarize our experience of ten years. , and patients over 45 years old or with comorbidities or repetitive sct (including second to fi fth sct) underwent reduced intensity preconditioning regimen consisting of flu/(ca)/bu/atg or flu/(ca)/mel/atg (haplomini, n = 146). high dose ara-c (ca) was optional to reduce tumor burden. atg (fresenius) dose was 2 mg/kg/day for four days. gvhd prophylaxis consisted of taclolimus (tac), methylprednisolone (mpsl) 2 mg/kg/day, short term mtx, and mycophenolate mofetil (mmf) 15 mg/kg/day in haplo-full, and tac, mpsl 1 mg/kg/day in haplo-mini, respectively. for elderly patients over 50 years old in haplo-mini, mmf was added. results: hematopoietic engraftment in haplosct was as rapid as that in hla-identical sct, except eight cases of graft rejection. acute gvhd (grade ii-iv) was observed in 30%. overall survival in fi ve years is 30% in haplo-full and 40% in haplo-mini, respectively. if limited to cr cases, overall survival reached over 60% in haplo-mini. there is no difference in survival rate among patients' diseases. discussion: unmanipulated haplosct is feasible and effective for refractory diseases. atg dose used in haplo-mini is rather low compared with that of european cases reported so far. although it should be too early to refer long term outcome, unmanipulated haplosct could be considered as an option to fi ght against refractory diseases. background: while chemotherapy + g-csf (g) is effective for autologous stem cell mobilization (scm) it exposes patients (pts) to risks and side effects. plerixafor (p) + g provides a safer alternative for mobilization of hematopoietic stem cells in pts with mm or nhl. to better understand the comparative effectiveness of standard and newer approaches for scm, we conducted a study aimed to: (1) determine the clinical outcomes and cost of scm with cyclophosphamide (cy) plus g (2) compare outcomes of cy + g to a clinical trial of p + g. methods: a retrospective study was conducted in all pts undergoing fi rst scm from 1/04 to 3/08 (n = 241) with cy (3 gm/m 2 ) and g (10 mcg/kg started 1 day after cy for 10 days with planned fi rst day of collection on day 11). apheresis was initiated when peripheral blood had >15 cd34 + cells/μl. positive clinical outcome (pco) was defi ned as >2 × 10 6 cd34 + cells/kg collected on planned day of collection and within 1 or 2 apheresis without a prior negative event that led to additional clinic/inpatient evaluation. the cost of drugs, apheresis, product processing, and clinical events were reported based on medicare part b physician, laboratory, and ancillary fee schedule. data on pts undergoing scm with p + g were obtained from published clinical trials in pts with myeloma (mm) or lymphoma (l) undergoing fi rst scm using g (10 mcg/kg without dose escalation) and a maximum of 4 days of p. results: among cy + g pts, 141 (61%) were males; 121 (50%) had mm, 115 (48%) had l (5-other diagnoses); and 61 (25%) pts. had prior radiation. pco was seen in 48 (20%) pts.; 23% of mm and 15.7% of l pts. median total cost of cy + g scm was $10,732 (range, 6988-30827). pco was associated with a lower cost than non-pco in the overall group, (mean, $10,371 vs. $12,870, p = 0.001), in mm pts. (mean, $10,511 vs. $12,152, p = 0.026), and in l pts (mean, $10,133 vs. $13,627, p = 0.006). assuming a similar distribution of pco in 100 pts. with mm and l, the projected per pt cost of scm would be $11,774 and $13,067 (mean, $12,421) with cy + g. projected costs of scm using p + g for 100 pts. with mm and l were $12,852 and $8986 (mean, $10,919). conclusion: scm with cy + g was associated with a lower rate of pco and higher cost than p + g, supporting front-line use of p + g for scm. future prospective studies should investigate whether scm with p + g translates into decreased resource utilization and improved quality of life for pts undergoing scm. .5x10 9 /l, platelet count (pt) >150 × 10 9 /l, and hemoglobin level (hb) >120 g/l], good partial response (gpr) [anc >1 × 10 9 /l, pt >50 × 10 9 /l, and a hb level >100 g/l] and poor partial response (ppr) [anc >0.5 × 10 9 /l, pt >20 × 10 9 /l, hb level >80 g/l and transfusion independency]. no response (nr) was defi ned as failing criteria for at least ppr. non-responding patients received a second-line therapy (hsct or androgens), a second course of ist with lg (15 mg/kg/day/ × 5 days) or tg (3.5 mg/kg/day/ × 5 days); or no treatment. subgroup analyses were conducted and differences in response were tested using the chi-square statistic test. immunoablation followed by immune reconstitution by transplantation of autologous peripheral blood stem cells is a promising approach to treat refractory or otherwise incurable s26 autoimmune diseases. it has recently been suggested that this treatment could lead to remission in some cases of the early diabetes type 1 if administered prior complete destruction of beta cells by autoimmune mechanisms. the objective of this study was to verify these fi ndings on an independent group of patients. methods: eight patients (age 19-32) with early diabetes type 1 (no more than 6 weeks from diagnosis, c-peptide positive, anti gad -antibodies positive) were qualifi ed for the treatment. the patients were subjected to 2-4 plasmaphereses to remove circulating immune complexes and then mobilized with cyclophosphamide and g-csf. leucaphereses were continued until >3,0 x 106 cd34 + cells/kg were collected. the conditioning consisted of cyclophosphamide (50 mg/kg/day on days -5, -4, -3, -2 prior to transplant) and antithymocyte globulin (atg genzyme -of 0.5 mg/kg/day given on day -5, 1.0 mg/kg/day given on days -5, -4, -3, -2 and -1). results: median follow up for the patients as of december 2009 is 10 months (range 6-19 months). no major complications were observed during the transplantation and in the posttransplantation period. all patients (8/8) became independent from exogenous insulin after the transplantation. median day of insulin withdrawal was + 24 (range + 6 to + 60). one patient resumed insulin 7 months post transplant. six out of 8 patients were given acarbose to reduce the potentially toxic infl uence of observed hyperglycemias. after the transplantation patients exhibited good control of glycemia -the average hba1c concentrations were 12.3% at the diagnosis, and 5.59%; 6.23%; 5.9% -3, 6, and 12 months after the transplantation respectively. this was correlated with increased levels of c-peptide after the transplantation. conclusion: this report independently confi rms that independence of exogenous insulin could be reached in diabetes type 1 patients following immunoablation and reconstitution of the immune system with autologous peripheral blood stem cells providing that the procedure is performed prior total destruction of beta cells in the pancreas by the autoimmune mechanisms. the use of acarbose may have potentially positive effect on the duration of remission of diabetes type 1 in the patients after the transplantation. new perspectives on the use of autologous haematopoietic stem cell transplantation in multiple sclerosis: three strategies of high-dose immunosuppressive therapy with autologous haematopoietic stem cell transplantation during the last decade high-dose immunosuppressive therapy with autologous hematopoietic stem cell transplantation (hdit + asct) has been used with increasing frequency as a therapeutic option for ms patients. we aimed to study clinical outcomes in multiple sclerosis (ms) patients after early, conventional, and salvage of hdit + asct. 155 ms patients (secondary progressive-54, primary progressive-28, progressive-relapsing-5, relapsing-remitting-68) were included in this study (mean age-33.0; male/female -63/92). beam or mini-beam conditioning regimens were used. 136 patients did not have any supportive treatment after hdit + asct; 29 patients with risk factors were administered mitoxantrone during a year after asct as a consolidation therapy. 68 patients underwent early transplantation (edss 1.5-3.0), 79 -conventional (edss 3.5-6.5), 8 -salvage transplantation (edss 7.0-8.5). median edss at base-line -4.0. the mean follow-up duration -22 months (range 6-125). neurological evaluation was performed at baseline, at discharge, at 3, 6, 9, 12 months, and every 6 months post transplant. transplantation procedure was well tolerated with no transplant-related deaths. the effi cacy analysis was performed in the group of patients who did not have consolidation treatment. out of 78 patients with the follow-up at least 9 months the following distribution of patients according to clinical response at 6 months post transplant was observed: 36 patients (46%) achieved an objective improvement of neurological symptoms; 41 patients (53%) had disease stabilization; 1 patients (1%) progression. in the patients who underwent early transplantation (n = 32) 15 (47%) patients stabilized and 17 (53%) improved. after conventional transplantation (n = 41) 22 (54%) patients stabilized, 18 (44%)-improved, and 1 (2%)-progressed. after salvage transplantation (n = 5) 4 patients were stable, and 1 improved. at long-term follow-up (median-26.5 months) out of 60 patients improvement was registered in 35 (59%) patients; stabilization -in 20 (33%) patients, and progression in 5 patients (1 patient after early; 4 -after conventional transplantation). no active, new or enlarging lesions were registered in patients without disease progression. thus, hdit + asct appears to be a safe and effective treatment for ms. further studies should be done to establish the best timing for transplantation and to evaluate consolidation therapy in patients receiving early, conventional, and salvage transplantation. in the last decade, the so-called nonmyeloablative or reduced intensity conditioning (ric) regimens for allogeneic stem cell transplantation (allo-sct) have emerged as an attractive modality to decrease allo-sct-related toxicities and nonrelapse mortality. indeed, ric allo-sct represents an attempt to harness the immune graft-versus-tumor effect while attempting to control or overcome toxicity. the work of different pioneering groups rapidly proved that this approach is feasible in several disease settings or patients' categories, and had the added benefi t of expanding the transplant option to patients who are ineligible for standard myeloablative allo-sct. currently, the use of ric allo-sct has challenged the need for high-dose conditioning regimens. unfortunately, and despite several thousands of patients receiving ric allo-sct, the true value of ric allo-sct in the management of hematological and non-hematological malignancies, especially aml is, as yet, diffi cult to delineate. currently, there are only very few, if any, prospective, randomized, or controlled trials that addressed the specifi c role of ric allo-sct versus other treatment strategies in aml. based on a large number of registry-based analyses, the ric subcommittee of the alwp has attempted over the last few years to better defi ne the role of ric allo-sct in aml patients through fi ne analysis of leukemia-free survival and overall survival balanced against treatment-related toxicity, complications, and death. a major advance was the identifi cation of the most important comorbidities that are likely to impact outcome after ric allo-sct for aml. another major achievement is the launch in 2010 of the multicenter/multinational ebmt randomized phase 3 study evaluating the role of ric allo-sct in elderly patients with aml. at present, the use of ric prior to allo-sct appears to be on the cutting-edge. the ric approach proved to be much more complex than originally thought. since the fi rst reports published in the late 90s', the ric allo-sct literature has exponentially expanded. the complexity of ric allo-sct practice is progressively deciphered and the optimism to regard ric allo-sct as a potential and promising treatment modality for many aml patients remains very high among investigators, warranting continuous and renewed clinical and therapeutic research in this area. transfusion of donor lymphocytes (dlt) for treatment of leukaemia relapse after allogeneic stem cell transplantation (sct) has been a milestone in the fi eld of immunotherapy against malignant disease. it can be regarded as the proof of principle for the graft-versus-leukemia effect. during recent years, the immunotherapy subcommittee of alwp has initiated a variety of retrospective studies to evaluate in detail the role of unmanipulated dlt in acute leukaemia. in contrast to cml and early stages of mds, dlt was less effective in the treatment of post sct relapse in highly proliferative diseases as acute leukaemia. nevertheless, when given as maintenance therapy after effective cytoreduction, dlt could induce long term remissions in selected patients in aml, both after standard and reduced intensity conditioning transplants. strategies that included dlt were more effective than treatment without the use of donor cells. in contrast, no such effects could be shown in all. these data have prompted the strategy to use donor cells already before the occurrence of overt haematological relapse, i.e in minimal residual disease, mixed or falling donor chimerism, or even prophylactically. this strategy is now widely used across european transplant centres and has shown promising results. future strategies include the combination of targeted therapies and donor cell based strategies, as well as the use of specifi c subsets of donor cells in order to augment the antileukemic effi cacy and control for side effects of dlt, such as gvhd. allogeneic stem cell transplantation (allosct) with reduced intensity conditioning (ric) is being increasingly used for acute myelogenous leukemia (aml) patients with high comorbidities not eligible for standard myeloablative conditioning. new compounds and formulations including intravenous busulfan, treosulfan and recently clofarabine have been introduced into the pre transplant conditioning regimens in an attempt to reduce transplant related toxicities while keeping or increasing the anti leukemic effect, overcoming the increased relapse rate usually associated with ric allosct. similarly, replacing bm by peripheral blood stem cell (pbsc) grafts may increase the gvl, albeit with the price of increasing toxicities, gvhd and trm. thus, the optimal conditioning regimen for adults with aml is yet unknown. in order to address these issues, the alwp of the ebmt recently performed several surveys analyzing the use of iv bu for allosct, comparing iv bu/cy to tbi/cy, comparing pbsc and bm, and is about to compare treosulfan to iv bu as conditioning regimens for adult patients with aml. in the iv bu/cy vs. tbi/cy survey that included 1479 patients, the fi ndings indicated that outcomes of allosct including engraftment, trm, rr and lfs were comparable while gvhd probability was signifi cantly lower and vod probability signifi cantly higher in adults patients with aml using iv bu/cy vs. tbi/cy conditioning, respectively. in a separate survey analyzing the use of iv bu allosct for aml the 1 year cumulative incidence of vod was found to be 8.4 + 2%. the factors associated with vod were mismatched unrelated donors and being not in remission. the pbsc vs. bm survey included 1537 patients. we observed signifi cantly higher incidence of gvhd and nrm and lower incidence of relapse rate with the use of pbsc, resulting in equivalent lfs. the treosulfan vs. iv bu survey is ongoing. in house comparison revealed similar lfs in aml patients in remission, while results were better using 4d iv bu combinations in patients with active disease. for mds the flu/treo conditioning regimen was the best. in conclusion, it is conceivable that introducing new compounds in the pre allosct conditioning regimens in combination with pre-and post-allosct targeted therapy will enable us to tailor the conditioning regimen to the specifi c disease category, reducing toxicity while improving the anti tumor activity. allogeneic hsct (allohsct) is generally considered the best consolidation option for younger patients diagnosed with an aml with high-risk cytogenetics. nonetheless, the cytogenetic high-risk category comprises different aml subtypes, and the specifi c results of allohsct for most of these cytogenetic entities are mostly unknown. moreover, several molecular lesions, such as mutations of flt3, mll or wt1 genes, identify subsets of patients with a poor-prognosis disease despite harboring a non-adverse-risk cytogenetic abnormality. therefore, the precise role of allohsct for the management of high-risk aml should be determined after a comprehensive analysis of the outcome of transplant in all these biological aml varieties with an adverse prognosis. in this context, the molecular markers sc is conducting a project to analyze the results of allohsct for diverse high-risk aml subsets, such as aml with t(6;9), aml with 3q abnormalities, and normal karyotype aml with internal tandem duplication of flt3 gene (flt3-itd three-year lfs was 28 9% and 18.9% for patients in cr1 and pif, respectively. finally, the prognostic impact of flt3-itd on the outcome of allohsct has been analyzed in a cohort of 120 patients (median age: 41, 18-60) with a normal karyotype aml. donor was an hla-identical sibling in 75% of cases, whereas conditioning regimen was a myeloablative regimen in all transplants. all patients received transplant in cr1. of note, 2-year lfs, ri, and nrm was 58.5%, 30.5%, and 13.3%, respectively, without a signifi cant difference between transplants from related and unrelated donors. in summary, the outcome of allohsct in patients with two high-risk aml subtypes such as aml with t(6;9) and flt3-itd aml showed a relatively favourable outcome, which compares favourably with reports from non-transplanted patients. on the contrary, the outcome of patients with aml and 3q26 rearrangement seems inferior, even in patients transplanted in an early phase of the disease. therefore, in order to elucidate the current role of allohsct for high-risk aml, transplant analyses should be focused on specifi c biological aml entities, since the results might differ largely among aml subtypes. finally, a careful collection of biological features at diagnosis is essential for addressing studies based on biological categories. hsct is a well-recognized option for the treatment of acute leukemia with the number of transplantations continuously growing over the last decades. however, the hsct rates vary strongly between countries. in particular, the activity in central and eastern (c&e) europe is markedly lower compared to the western part of the continent, suggesting the role of various socio-economic and geographic factors. using the human development index (hdi) as a surrogate marker we demonstrated that the socio-economic status (ses) of a country strongly correlates with the total number of hsct per population, as well as separately with sibling-hsct, unrelated-hsct and autologous-hsct in europe. we further speculated that in line with the transplant activity the results of hsct may also vary between countries or regions. direct comparison of hsct performed from sibling donors for aml in cr1 showed superimposable results obtained in c&e and western europe, however, the transplant characteristics differed with younger recipient age, longer interval from diagnosis to hsct and less frequent use of peripheral blood, tbi, t-depletion and reduced-intensity conditioning in c&e countries. subsequent analysis demonstrated that the outcome of sibling-hsct for acute leukemias in c&e europe improved over time. in particular the cumulative incidence of nrm decreased from 22 2% for patients treated between 1990-2002 to 15 3% for hsct performed between 2003-2006, despite increasing recipient age. in another analysis we demonsatrated the infl uence of the ses on outcome in europe, which, however, could not be explained by regional differences. best outcome (increased lfs and reduced ri) after myeloablative hsct from either related or unrelated hla-matched donor was observed in 8 countries with the highest hdi, while no differences could be demonstrated for the remaining 22 analyzed countries. interestingly, transplantations in countries with the highest hdi were characterized by increased recipient age and shorter interval from diagnosis to hsct. finally, we speculated that the center experience may infl uence results of hsct. using transplants with reduced-intensity conditioning (ric-hsct) as a model we analyzed results according to the team experience as defi ned by: 1) time from the fi rst allohsct and from the fi rst ric-hsct, 2) the total number of allohsct and the number of ric-hsct performed in a study period (2001) (2002) (2003) (2004) (2005) (2006) (2007) . we found that results in centers performing few ric-hsct (<15 in 7 years) were inferior compared to the remaining centers in terms of all, lfs, ri and nrm, which has been confi rmed in a multivariate model. altogether, in a series of retrospective analyses based on the ebmt alwp registry we demonstrated that both rates and outcomes of hsct for acute leukemia vary among countries and centers. both socio-economic factors and team experience infl uence results. we previously reported the ebmt experience with salvage high-dose chemotherapy (hdct) in pediatric patients with extragonadal germ-cell tumour (gct) (de giorgi u et al. -bjc 2005) . we analyzed a total of 23 children with extragonadal gct, median age 12 years (range 1-20), treated with salvage hdct with haematopoietic progenitor cell support. the gct primary location was intracranial site in nine cases, sacrococcyx in eight, retroperitoneum in four, and mediastinum in two. twenty-two patients had a nongerminomatous gct and one germinoma. nine patients received hdct in fi rst-and 14 in second-or third-relapse situation. no toxic deaths occurred. overall, 16 of 23 patients (70%) achieved a complete remission. with a median follow-up of 66 months (range 31-173 months), 10 (43%) have been continuously disease-free. of six patients who had a disease recurrence after hdct, one achieved a diseasefree status with surgical resection followed by chemotherapy and radiotherapy. in total, 11 patients (48%) were diseasefree at the time of analysis. eight of 14 patients (57%) with extracranial primary and three of nine patients (33%) with intracranial primary gct were disease-free at the time of analysis. hdct induced impressive durable remissions as salvage treatment in children with extragonadal extracranial gcts. after 7 years from this fi rst analysis (done in 2003 published in 2005), we decide to analyze the long-term results of this experience integrating these data with other from other cases from the ebmt registry previously not included, and with data from additional patients from italian registries (gitmo and aieop). high-dose chemotherapy and autologous stem cell transplantation in relapsed germ cell tumours: do we need a randomized study? p. pedrazzoli (1) nasopharyngeal carcinoma (npc) is an epstein-barr virus (ebv)-related malignancy expressing a restricted set of viral antigens. the outcome of patients with npc failing conventional radio-chemotherapy is poor, the median overall survival of patients receiving second line treatments being less than two years. hence, the need for alternative therapies capable of improving disease-free survival and associated with reduced toxicity. since 2001, we have implemented a t-cell therapy program for patients with npc failing conventional treatment. so far, we have treated 27 patients with disease resistant/relapsing after >2 lines of radio-chemotherapy in two sequential trials of cell therapy with autologous ebv-specifi c cytotoxic t-lymphocytes (ctl). in detail, ebv ctl (4 escalating doses to a maximum of 8 × 10 7 ctl/dose in the fi rst trial, or 2 doses of 3-4 × 10 8 ctl in the second trial) were administered in patients without or with preceding lymphoablative chemotherapy and recombinant human interleukin-2 (rhil-2). the proportion of patients achieving response (partial, pr, complete, cr) or stable disease (sd) lasting at least 3 months (recist criteria), as well as immunologic correlates of effective treatment, were evaluated. overall, the objective control of disease was 57%, with 6/27 patients showing complete (n = 1), partial (n = 4) and minimal (n = 1) responses, and 10/27 disease stabilization (median duration 7 months). no severe adverse events were observed after cell therapy; 4 patients showed an infl ammatory reaction at the tumor site. the use of preparative chemotherapy and increased ctl dose did not infl uence outcome. importantly, patients showing response to cell therapy showed the emergence of ebv lmp2 antigen-specifi c t-cells in their peripheral blood. ebv-specifi c ctl therapy is safe and associated with clinical benefi t in patients with advanced npc refractory to standard therapies. the use of ctl with higher specifi city for the ebv subdominant antigens expressed by the tumor, such as lmp2, at an earlier stage of disease, could further implement the strategy and ameliorate the outcome of patients with relapsing/refractory npc. this study was supported by a grant from the italian association for cancer research (airc). , and csa given orally (5 mg/kg/d). patients randomized to receive g-csf were given glycosylated g-csf from day 8 to 120 (150 microg/m 2 /d, sc). os at 6 years was 75%, it was 82% for patients with severe aa and 66% for patients with very severe aa (p = 0.001). survival decreased with increasing age from 100% (<20 years) and 92% (20-40 years) to 71% (40-60 years) and 56% (>60 years) (p < 0.001). there was no difference, overall and when stratifi ed according to age and aa severity in os (p = 0.64) and efs, defi ned by death, need for transplantation, relapse and non response as events (p = 0.36) between the study arms at 6 years ( table 1 ). the median neutrophil count was signifi cantly higher between day 30 and 240, in the g-csf group; this difference did not persist to day 360, when g-csf was stopped. there were fewer infections (36% no g-csf; 24% with g-csf; p = 0.006), and less days of hospitalization during the fi rst 90 days (p = 0.03) in the g-csf group. 44 patients died, mostly from infection (55%).there was no difference in death rates, cause of death and response rates between both groups. overall 73% of patients with and 62% without g-csf did respond to is (p = 0.49). 57 patients did not respond to fi rst-line therapy, 31 patients relapsed during the fi rst year of treatment (no difference between both groups). in conclusion, g-csf added to standard is increases neutrophil counts, and decreases rate of infections and days of hospitalization but has no impact on os, efs, remission, death and relapse rates. this has to be weighed against possibly higher risks of mds/aml, as suggested by previous studies. an hla-identical sibling (65%), 157 pts received a myeloablative regimen (78%) and 135 bone marrow (64%) with gvhd prophylaxis consisted in csa ± mtx in 154 pts (73%). during evolution, 14 pts did not engraft (7%). acute gvhd of grade >i developed in 52 patients (29%). chronic gvhd developed in 57 pts (26 extensive). after a median ±se follow-up time of 61±6 months, 64 pts died (35 infections, 18 gvhd). the 5-year os rate was 68 ± 3%. none of the common variables examined for association with os were statistically signifi cant, except for sct indications with an increasing risk from rsh, saa and thr ( figure 1 , p = 0.03). based on the 24 pairs obtained through the matching process, the 5-year os estimate after thr ( figure 2 ) was 45 ± 11% for pts with sct and 87±8% for pts without sct [hr 10.0 (95%ci 1.3 to 78.1); p = 0.01]. of note, the os among non-sct pts was signifi cantly worse (p = 0.01) in the non-matched than in the matched group, raising the question of pts over selection due to the matching process. concerning pts with saa complication, after exclusion of pts with thr and of non-sct pts with a follow-up time <6 months after saa, the 5-year os was 96±2% for pts without sct and 81±4% for pts with sct, but the 2 groups differ signifi cantly for age at saa and year of saa. conclusions: further matching processes are necessary to conclude on this large cohort of pnh pts in order to defi ne the exact place of sct in pnh, especially in the era of the eculizumab. introduction: currently peripheral blood (pb) is more commonly used as stem cell source than bone marrow (bm) in both autologous and allogeneic hematopoietic stem cell transplantation (hsct) (gratwohl, bmt, 2005) . however, pb is associated with an increased risk of chronic graft-versus-host disease (gvhd), which is a disadvantage in non-malignant diseases. a recent study of ebmt/cibmtr showed that incidence of chronic gvhd and overall mortality were higher after hsct with pb than after hsct with bm in young patients with severe aplastic anemia (saa) (schrezenmeier h, blood, 2007) . the ebmt transplant activity survey shows that the number of pbsct has increased rapidly since early 90's and exceeded the one of bmt in 1999 in family donor and in 2002 in unrelated donor (ud)-hscts. the number of pbsct has also increased in saa. we were therefore interested to review the current status of stem cell source selection in bone marrow failure ( in conclusion, pb has been increasingly used as a stem cell source in bmf despite of its higher risk of chronic gvhd. there were major differences in stem cell source distribution, regarding donor type, the global region as well as countries in regions. it may refl ect the differences in infrastructure in each center/country, donor and physicians preferences and policy of marrow donor program. clearly, recommendation for stem cell source is warranted in bmf. we conclude that 5q-syndrome can be cured by allogeneic transplantation, but additional abnormalities reduce the curability. the role of lenalidomide given before transplant will also be analysed. patients. an interim toxicity analysis was performed when the fi rst hundred patients had been included. the safety committee agreed to resume the trial. we will report the feasibility and the toxicity in both arms. an interim analysis is currently ongoing. other preliminary studies demonstrate that vtd is a highly active and relatively well tolerated regimen. the combination is used in the relapse setting, as well as fi rst line, consolidation and maintenance. in this protocol, the starting doses of velcade and thalidomide are relatively high and the duration of treatment is long. we will assess the superiority of vtd over td in the relapse setting as well as its toxicity. objectives: chronic lymphocytic leukemia (cll) is susceptible to well characterized graft-versus-leukemia effects. the leukemic clone is eradicated at the molecular level in >50% of patients. yet, up to 50% of patients have persisting disease or relapse after allogeneic hematopoietic cell transplantation (hct). the application of donor lymphocyte infusions (dli) appears to be attractive in this situation. the aim of this retrospective analysis is to study the long-term effects of dli in patients with cll. methods: data from patients with cll who received allogeneic hct between 1997 and 2008 and had received at least one dli were included. the outcome of dli was analysed in two situations. 1) "pre-emptive" dli prior to frank relapse or disease progression 2) "therapeutic" dli given after documented relapse. baseline and follow-up data were downloaded from the ebmt database. results: 217 patients fulfi lled the inclusion criteria. major characteristics were median age 52 years, a median of three prior chemotherapy regimens, 71% matched sibling donor hct, 74% reduced intensity conditioning, 85% received peripheral blood stem cells. atg or alemtuzumab was used in 37%, ex vivo t-cell depletion (tcd) in 12%, no tcd in 50% of the patients. the median follow-up after the fi rst dli was 20 months. in the cohort of 109 patients who received dli prior to relapse, 38 patients received more than one dli, 21 patients more than two dli, 11 patients more than three dli. grades ii to iv acute gvhd was reported in 9 out of 35 patients (26%) with informative data. pr or cr was documented in 10 out of 20 patients (50%). overall survival and progression-free survival 5 years after the fi rst dli was 42% (95% ci, 28% to 56%) and 22% (95% ci, 12% to 22%) respectively in this group of patients. 108 patients received dli as treatment of relapse. among these, 42 patients received more than one dli, 16 patients more than two dli, 5 patients more than three dli. the overall response rate was 38% (13 out of 34 informative patients). nine out of 50 patients (18%) experienced acute gvhd grades ii to iv. in this group the 5-year overall survival after the fi rst dli was 21% (95% ci, 9% to 33%). only 3 patients had a follow up of more than 5 years. conclusion: in patients with cll donor lymphocyte infusions appear to have only moderate long-lasting activity. further investigation to delineate factors associated with improved outcome is warranted. , 3 centres 8%, 5 centres 7.5%, 2 centres 6%, 5 centres 5%. 3 centres using 10% dmso washed cells prior to infusion although another 9 centres also washed cells in occasional patients mostly less than 10% of their patients. 43 centres added additional agents to the freezing mixture, mostly albumin (25), hydroxy ethyl starch (6), heparin (8) and tissue culture medium (5). the median amount of dmso given per centre per patient was 20 g, although the upper limit set by the centre was often considerably higher. 75% of centres did not use any delay between bags of stem cells and the median duration of infusion was 22 minutes. side effects were defi ned using nci criteria (version 3.0) and initially results were analysed by each centre. patients in centres who used washed cells or 5% dmso experienced less nausea and vomiting and 'severe other' effects, although hypotension and hypertension did not seem to be affected (p = 0.026, 0.014, 0.624 and 0.208 respectively). on a centre basis, the use of an upper limit to the amount of dmso which could be given (70 g or the amount given ≤20 g versus >20 g) did not result in any reduction of any of the groups of side effects. similarly when the amount of dmso given and the duration of dmso infusion were compared on a centre-basis, no signifi cant differences were found. further analysis will be undertaken using individual data where appropriate and the results presented. introduction: cmv infection and disease still remain serious and frequent complications after hsct. both morbidity and mortality have been reduced by prophylactic and pre emptive strategies based on biological tests and on more or less effective anti-viral drugs. however, in absence of comparative studies, there is no consensus for diagnosis, prophylaxis and treatment. then we conduct a survey to assess the current cmv management for patients less than 18 years in ebmt centres. materials and methods: in december 2009, a 40-item questionnaire about diagnostic tests, monitoring schedules, prophylactic and pre emptive strategies and treatment modalities was send to centres. defi nition of treatment failure, recourse to drug resistant cmv mutant research, practice of cell therapy and combined anti-cmv therapy were also tested. results: by the 31st december '09, we have received 35 responses from 14 countries. a second shipment will send in order to increase the number of responding centres. as expected, whatever the considered aspect i.e. monitoring, prophylactic or pre-emptive applied strategy, used drug in front or second and more line therapy etc. none centre report same procedure than another. all results will be presented and discussed during pdwp meeting in vienna. then, some proposals will be made regarding studies comparing results in different centres with "opposite" procedures. the fi nal goal of this work is to collect enough data to build consensual procedure. here we describe the results of the fi rst 87 patients. all cases were confi rmed by pcr in nasopharyngeal or bronchoalveolar lavage samples. the fi rst case was diagnosed on july-9. patients' characteristics are shown in table 1 . eight cases were considered nosocomial infections. in sct patients, the median time from transplant to infl uenza diagnosis was 588 days (7-6155). s-oiv characteristics are given in table 2 . the most frequent symptoms were: fever (86%), cough (84%), rhinorrhea (51%), odynophagia (26%), myalgia (22%), headache (11%) and dyspnea (11%). diarrhea was rare (2%). of those who presented with upper respiratory tract infection alone, 9 (14%) had progression to pneumonia. 30% had pneumonia, with no difference between sct patients (31%) and non-sct patients (28%). fifteen out 17 pneumonias in sct patients occurred in patients during the fi rst postransplant year or later but under immunosuppressive treatment. five patients were transferred to the intensive care unit (icu). four out of 5 of the most severe cases (admitted to icu or fatal course) had lymphopenia (<500/ mm 3 ) compared with 26% of those with less severe forms (p 0.010). compared with adults, children had more pneumonia (39% vs. 28%) although the difference was not statistically signifi cant (p 0.3). only one of the vaccinated patients against seasonal infl uenza were diagnosed with pneumonia compared with 41% who were not vaccinated (p .005). all patients except one were treated with oseltamivir for a median of 5 days (5-27). zanamavir was added (in combination or sequentially) in 4 cases. no major adverse events relating to anti-infl uenza treatment were reported. summary: the novel s-oiv is a serious disease in stc and oncohematological patients with a high incidence of pneumonia (26%) and signifi cant mortality (3%). diarrhea was not a frequent symptom. lymphopenia was linked to the most severe forms. vaccination for seasonal infl uenza might protect against the development of pneumonia. nevertheless, the severity of new pandemic infl uenza seems to be similar to seasonal infl uenza in this patient population. background: ebv-ptld (epstein-barr virus-related post-transplant lymphoproliferative disorder) is a rare but serious complication after hsct and number of patients at risk is increasing over time. available data do not refl ect general practice of diagnosis and treatment of this complication. objective: assessment of the current strategy of diagnosis and preemptive use of rituximab for ebv-ptld in ebmt transplant centers (tc). methods: in 2009 survey, data regarding ebv strategy from 74 participating tc were registered on specifi c forms and centrally analyzed. responses from 10 centers were excluded due to lack of specifi c strategy. results: regular monitoring for ebv after hsct is done by 47/64 (73%) tc, and in 7/64 (11%) tc is related to clinical situation. the monitoring is performed in all allohsct patients in 32/47 (68%) tc, while in remaining 22 tc in following subgroups: mud (17/22), t-depletion in vitro (8/22), t-depletion in vivo (21/22), family mismatched (14/22), cord blood transplants (16/22), and in single centers in patients with saa, ebv mismatch, or after autohsct for autoimmune disorders. quantitative ebv-dna by pcr is performed in 62/64 (97%) centers. the assay is performed in: whole blood -50/64 tc (78%), plasma -9/64 (14%), lymphocytes -4/64 (6%), serum -1 center. the test is repeated twice a week in 8/64 tc (12.5%), once a week in 39/64 (60.9%), once per 2 weeks in 8/64 (12.5%), once a month in 5/64 (7.8%) and adjusted to risk in 4/64 (6.2%) tc. the monitoring for ebv reactivation after hsct is performed for a period of: less than 3 months in 2/64 (3%), 3 months in 22/64 (34%), 6 months in 19/64 (30%), 12 months in 8/64 (12%) and adjusted to risk in 13/64 (20%). rituximab as a pre-emptive therapy for ebv-ptld is routinely administered in 51/64 (80%). the number of ebv-dna copies as an indicator for preemptive therapy with rituximab was given by 60 tc, but varied between the centers, and were based also on symptoms and signs (table) . conclusions: ebv-ptld strategy exists in most of the responding centers. differential approach regarding indications for preemptive therapy is seen between centers: rituximab is administered as preemptive therapy in 80% of participating transplant centers. objectives: the aim of this study was to analyze the clinical risk factors, donor and recipient cytokine gene polymorphisms associated with cytomegalovirus (cmv) infection within 100 days after allogeneic hematopoietic stem cell transplantation (allo-hsct). methods: we studied in a total of 203 pairs of recipients and their donors, who underwent allo-hsct at our center. we analyzed 12 single nucleotide polymorphisms (snps) in 6 pro-and anti-infl ammatory cytokines genes, tumor necrosis factor ( 92.6%of122 patients developed cmv positive antigenemia without disease, only 9 patients developed cmv disease (7 patients with pneumonia and 2 patients with enteritis). there was a higher incidence of early cmv infection in transplantation with unrelated donors (66.4% vs. 51.7%, p = 0.043) and in recipients who were cmv seropositive before transplantation(72.9% vs. 54.9%, p = 0.018). the recipient's tgf-beta1-509 and + 869 genotypes were signifi cantly associated with the incidence of early cmv infection in both the unrelated transplantation cohort and sibling transplantation cohort, but the infl uence of the donor's tgf-beta1-509 and + 869 genotypes was signifi cant only in the sibling transplantation cohort. multivariate analysis identifi ed two risk factors for early cmv infection: cmv seropositive recipients (rr: 1.712, 95%ci: 1.177-2.490, p = 0.005), recipients with tgf-beta1 + 869 c allele (rr: 2.225, 95%ci: 1.401-3.536, p = 0.001). donors with tgf-beta1-509t allele was found to be less signifi cant factor (p = 0.094). conclusion: although cmv disease has been reduced in the era of antiviral prophylaxis and preemptive therapy, our fi ndings suggest the incidence of cmv infection remains high and provide the fi rst report of relationship between genetic background of donor and recipient to the risk of cmv infection in a chinese population. hsct. toll-like receptors (tlr) are essential components of the innate immune system. c3h/hej mice that lack functional tlr4 did not develop cystitis after intravesical instillation of e. coli. individuals with the asp299gly polymorphism of the tlr4 gene showed a diminished infl ammatory responsiveness to endotoxin. because of the requirement of tlr4 in infl ammatory response we hypothesized that tlr4 asp299gly reduces the risk of bk virus-induced hc after hsct. 166 children (median age, 13 years) who underwent allogeneic bone marrow (n = 105) or peripheral blood stem cell transplantation (n = 61) in a single center and their respective donors were genotyped of tlr4 for rs4986790 (a896g, asp299gly) using taqman real-time polymerase chain reaction. the donor was hla-matched unrelated in 57% of transplants or hla-identical related in 33% of transplants. conditioning regimen was myeloablative in all cases. two forms of post-transplant immunosuppression predominated, cyclosporine a and methotrexate in 64% of transplants and cyclosporine a alone in 25% of transplants. the asp299gly polymorphism was present in 21 of the 166 patients (12.6%) and in 24 of the 166 donors (14.4%). interestingly, we found a signifi cantly reduced incidence of bk virus-induced hc in patients with the asp299gly polymorphism (0% vs. 22.8%, p = 0.009). in addition, we observed a significantly reduced incidence of bk virus-induced hc in patients who were transplanted from a donor with this specifi c polymorphism (4.2% vs. 22.5%; p = 0.05). in ten of the donor-patient pairs the polymorphism was present in both individuals and no hc was observed. the occurrence of the tlr4 asp299gly polymorphism, in either recipients or donors, had no signifi cant impact on acute and chromic gvhd, relapse rate, bacterial and fungal infectious complications, transplant related mortality, and overall survival. in conclusion, asp299gly polymorphism of the tlr4 gene in the recipient and/or the donor is associated with a signifi cant decrease of bk virus-induced hc after hsct in childhood. this study provides the fi rst evidence that the innate immune system through tlr4 signaling pathway seems to play an important role in the pathogenesis of bk virus-associated hc after hsct. design and methods: one hundred-seventeen patients, median age 52 (20-67) years, with various haematological malignancies transplanted between 1999 and 2007 entered the study. eighty-seven recipients negative for hbv surface antigen (hbsag), anti-hepatitis b core antigen antibodies (anti-hbc) and hbv-dna with hbsag negative donors were defi ned as at no risk of hbv reactivation whereas all the remaining 30 patients were defi ned as at risk. in accordance with the italian national guidelines for immunocompromised hosts, patients at risk transplanted after 2005 received lamivudine to prevent hbv reactivation from conditioning up to 12-18 months after discontinuation of all immunosuppressive drugs whereas before 2005 no prophylaxis was given. results: patients at no risk did not experience hbv reactivation/ hepatitis. among patients at risk, hbsag negative recipients from hbsag positive donors (3/3), hbsag positive recipients from negative donors (2/2) and 11/25 anti-hbc positive were treated with lamivudine. none developed hepatitis b after a median follow-up of 33 months (7-55). hepatitis developed in 3 anti-hbc positive untreated patients conditioned with a reduced intensity regimen up to 19 months after discontinuation of immunosuppression and in none of those on prophylaxis. conclusions: we observed: no risk of hepatitis b in recipients serologically negative for hbv, transplanted from hbsag neg donors; effi cacy of lamivudine in controlling hbv reactivation in both hbsag positive recipients and negative recipients from s38 hbsag positive donors; a signifi cant risk of hbv reactivation in hbsag negative/antihbc positive recipients and effi cacy of prophylactic lamivudine in this setting and the importance of prolonged prophylaxis even after the discontinuation of immunosuppressive drugs. republic -a single-centre experience p. hubacek (1,3) , d. boutolleau (2, 4) , c. deback (2, 4) , p. keslova (3) despite the improvement of infection monitoring and antiviral treatments, cytomegalovirus (cmv) infections remain an important cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant recipients (allohsct). aim was to investigate cmv resistance in paediatric and adult allohsct recipients and to clarify the cmv load in whole peripheral blood and target tissue in situation of cmv disease. between i/2002 and xii/2009, we tested 17504 whole blood samples from 427 adults (median 22/patient) and 211 children (median 32/patient) after allohsct. additional 1348 biological non blood samples were tested too.samples were tested for cmv and albumin gene quantity by rq-pcr and results were normalized to 100000 human genome equivalents. ganciclovir as fi rst-line therapy was initiated when cmv load exceeded 1000 normalized viral copies (nvcs) and switched to foscarnet or cidofovir in case of none response.clinical resistance was suspected after 2 weeks of unsuccessful well-conducted treatment.resistance was studied using ul97 and ul54 gene sequencing.in the patients deceased in consequence of cmv infection,dna quantity in the tissue was tested too. virostatic treatment was started in 142 adults (33%) and 63 (30%) children.clinical resistance was observed in 58 adults and 25 children.known genotypic cmv resistance was proved in 11 of them (5.4% of treated) detecting mutation c592g, a591v, a594v, l595s, l595f, g598s and del597-599 in ul97 and del981-982, n408k, v715m, p522s in ul54). natural polymorphisms and unknown phenotype changing were also detected in both genes. despite the virostatic treatment, symptomatic cmv disease was observed in 15 adults and 10 children. ten patients deceased in consequence of cmv infection,mainly combined with gvhd. typical pathological signs of cmv infection and antigen detection in the tissue were found in 1 patient only.despite this median of cmv quantity in mainly affected lung tissue was 9534 nvcs (range 1029-384456) while median in the whole blood was only 1065 nvcs (range 183-89072). quantifying of cmv dna is very useful in the treatment of cmv infection. unfortunately our observation suggests the limits in case of cmv disease. genotypic evidence of cmv resistance proved to be very useful in the improvement of therapeutic management of patients. further studies are required to ascertain the true nature of the novel mutations described within ul97 and ul54 same as compartmentation of cmv infection. supported by mz0fnm2005, mstm0021620813. cmv reactivation often occurs in patients after allogeneic hsct. recently, pdgfr-alpha was suggested as the cellular receptor for human cmv (soroceanu et al., nature, 2008) . pdgfr-alpha binding and activation of a downstream pi3kinase signaling pathway was essential for cmv propagation. many tkis, such as imatinib, effi ciently inhibit pdgfr-alpha at concentrations readily achievable in patients. the aim of this study was to retrospectively assess a possible link between cmv reactivation and tki therapy. . also, at 2 years, the relapse incidence was not signifi cantly associated with the type of ric regimen: 37±3%, 34±2% in the tbi-based ric and chemobased ric groups respectively (p = 0.17). finally, nrm was also comparable between both groups (21±3% and 20±2% in the tbi-based ric and chemo-based ric groups respectively; p = 0.60). despite its retrospective nature, results from this large study suggest that ric allo-sct is a valid option for aml patients not eligible for standard allo-sct. the overall outcomes (lfs, nrm and relapse) appear not to be signifi cantly different between aml patients in cr1 receiving a low dose tbi-based ric allo-sct versus those receiving a chemotherapy-based ric allo-sct using an hla-identical sibling donor. patients with all who relapse after allogeneic hct have a grim prognosis and the optimal salvage therapy remains unknown. aim of this retrospective analysis was to report the outcome of relapsed all after allogeneic hct, to identify factors which have a prognostic signifi cance and to evaluate whether any relapse treatment strategy was associated with better outcome. for this study the ebmt database was searched for: 1) adult patients with all aged >18 y at hct; 2) transplanted in remission; 3) with hematological relapse after an allogeneic hct; 4) hla identical or mud hct performed from 1995 to 2006; 5) suffi cient med-c data. 465 adult pts (median age 32 y, 18-66) met these eligibility criteria. diagnosis was 76% b-lineage all, 21% t-all, 33% ph + all and previous allograft (68% were hla identical and 32% mud) was performed in remission (cr1 65%, cr2 32%, cr3 3%). the median interval from transplant to relapse was 7 months (0.8-52). post-relapse interventions were: no further treatment in 13% pts, 43% pts received chemotherapy at a median of 5 d after relapse , 23% pts received dli at a median of 45 d post-relapse (4-344) and 20% pts underwent a second transplant at a median of 71d after relapse (7-286). 61% of ph + all pts in addition received a tki inhibitor. overall, 40% pts reached a subsequent cr. after a median follow up of 56 months (3-141), the estimated 1-, 2-and 3-year overall survival (os) was 32%, 19% and 10%. the table below shows the statistically signifi cant parameters associated with 2-year os. in the multivariate analysis, os was associated with disease status at hct (cr2 + vs. cr1 p = 0.005, hr: 0.57), time to relapse after hct (>vs< median 7 months, p<0.0001, hr: 2.27 ) and number of peripheral blood blasts at relapse (>vs<10%, p<0.0001, hr:0.49), but it was not signifi cantly infl uenced by cytogenetics (ph + vs. other), donor type (sibling vs. unrelated), stem cell source (bm vs. pb), type of conditioning (tbi vs. chemo) or type of treatment for relapse (dli or hct-based therapy vs. therapy without cell infusion). this study highlights the extremely poor outcome of all pts who relapse after allogeneic hct. better outcomes were found in patients transplanted in cr1, with late relapse after hct (>7 months) and lower tumor burden at relapse (<10% of pb blasts). these factors should be taken into consideration when discussing further therapeutic options for these patients. introduction: prognosis and survival in aml patients is associated primarily with cytogenetic abnormalities but also with molecular markers. the internal tandem duplication (itd) of the flt3 gene is one such marker associated with poor prognosis. we report here an analysis of the impact of allogeneic sct on clinical outcome of cytogenetically normal aml patients according to their flt3-itd status. patients and methods: flt3-itd mutation was analyzed by capillary electrophoresis after pcr amplifi cation of material frozen at the time of diagnosis from a total of 116/277 patients with normal karyotype entered between 1997 and 2008 into two osho studies (aml 96 and aml 2002). sct was performed after conditioning with cytoxan and 1200 cgy tbi followed by gvhd prophylaxis with cyclosporine and methotrexate. related and unrelated donor search was initiated as soon as possible and patients allocated to groups with or without a donor. a total of 46 patients were allocated to the donor group and 70 patients to the no donor group. results were analyzed as intention to treat. results: event free survival (efs) of all patients was 38% after 5 years. in patients (n = 46) allocated to receive hct from an allogeneic donor, efs was 44% compared to 33% in those allocated to the non-transplant treatment (n = 70). as previously described, detection of a flt3-itd mutation had a negative impact on efs at 5 years, which was 25% in flt3-itd positive and 46% in flt3-itd negative patients (p = 0.06). subsequently, efs was analyzed according to flt3 status and post-remission treatment. in the flt3-itd positive group, efs was inferior in patients without donor (19% at 5 years) compared to those with a donor (36% at 5 years). this difference was due to a decreased relapse incidence in the donor group (48%) compared to the no-donor group (80%). however, a difference in efs was also observed in flt3-itd negative patients in the donor (50% at 5 years) compared to the nodonor group (43% at 5 years). this difference was also associated with a reduced relapse incidence in the donor group (26%) versus the no-donor group (55%; p = 0.003). conclusion: in conclusion, allogeneic hct improves efs in aml patients with a normal karyotype by reducing relapse incidence irrespective of the flt3-itd mutation status. the median follow-up of living patients was 20 months (range, 1 to 53 months). at 3 years the overall survival (os) was 38% and progression-free survival (pfs) was 36%, while the incidence of non-relapse mortality (nrm) and relapse were 29% and 34%. we performed multivariate cox regression modelling for os and pfs while competitive event statistics were applied for nrm. in order to adjust for a potential selection bias the hct-specifi c comorbidity score, karnofsky performance status, delay between diagnosis and hct and age were forced into the multivariate model. we performed a complete case analysis based on 223 patients. the hazard ratios (hr) of a matched or a mismatched ud compared to a sibling donor were 1.1 (95% ci, 0.7 to 1.7) and 1.3 (95% ci, 0.6 to 2.0) for os and 1.3 (95% ci, 0.8 to 2.0) and 1.3 (95% ci, 0.8 to 2.1) for pfs. in interaction analyses we did not identify signifi cantly differing effects according to age and disease stage. of note, having a matched ud did not signifi cantly affect the risk of nrm (hr=0.8; 95% ci, 0.4 to 1.6). our results confi rm the current practice in high risk aml to deliver allogeneic hct from matched ud and sib. an updated analysis will be presented at the meeting. (14) ( this phase-iii randomized ebmt-intergroup trial studied the impact of a consolidating haematopoietic stem cell transplantation (autohsct) vs. wait and watch for patients with cll in binet stage a progressive, b or c, in cr, nodular pr or vgpr after fi rst or second line therapy. the primary objective was to show that autohsct increase the 5-year progression-free survival (pfs) by 30%. here we present a fi rst analysis based on 80% of expected follow-up forms. results: between november 2001 and july 2007, 223 patients were enrolled (sfgm-tc/fcllg n = 99, mrc n = 63, gcllsg n = 36, sakk n = 10, other ebmt centers n = 15). there were 74% males and 26% females. binet stages were progressive a 13%, b 67%, c 20%; 59% were in cr, and 41% in very good or nodular pr. of note, sfgm-tc/fcllg included only patients in cr. eighty three percent of the patients were enrolled in 1st, and 17% in 2nd line treatment. patients were randomized between group 1 (autohsct n = 112) and group 2 (observation n = 111) after an induction treatment which was left at the discretion of the investigators. median pfs was 26.6 months (18.3-35) in the observation group and 50.1 months (40-60.1) in the autohsct group; the 5-year pfs was 29% and 42%, respectively (p<0.001). accordingly, the 5-year relapse incidence was 54% vs. 70%; p<0.001. the cox modeling for randomization arm, binet stage, disease status, line of treatment, contributing group (country), and the interaction between randomization arm and contributing group confi rmed that autohsct signifi cantly improved pfs (hr 0.45 [0.30-0.66] p<0.001). the benefi cial effect of autohsct was stable over all contributing groups. at 5 years, the probability of os was 83% and 82% for autohsct and observation, respectively; p = 0.81. signifi cant differences in terms of non-relapse death were not observed. in conclusion, in patients with cll in fi rst or second remission, consolidating autohsct reduces the risk of progression (pfs) by more than 50%, but has no effect on overall survival. further analyses on variables affecting the outcome are underway and results from a quality of life study on both groups are awaited. improved long-term outcome after highly purifi ed peripheral blood cd34 + cell transplantation from here, we report about the long-term follow-up results of a prospective phase ii study in patients after transplantation of highly purifi ed peripheral blood cd34 + stem cells from hla-identical sibling donors for cml in 1. cp. a total of 58 pts with a median pretransplant ebmt risk score of 2 (range 1-4) have been included in this study. all patients received a myeloablative conditioning regimen with tbi, cyclophosphamid with/or without thiotepa and atg, but without any prophylactic immunosuppression post transplantation. one patient received an unmanipulated graft due to poor cd34 + donor cell mobilization, while two patients were successfully retransplanted with an unmanipulated graft from the primary donor after secondary graft failure. of the 58 pts, 56 were eligible for the application of dli, but 6 pts did not receive dli due to sustained molecular remission and complete chimerism. thirty-two pts (64%) received dli because of increasing bcr-abl transcript levels or hematologic relapse, and 18 pts (36%) as programmed t-cell add-back. the median starting dose was 0.33 (0.01 -10) x 10 6 cd3 + cells per kg with a median maximum dose 3.3 (0.17 -100) × 10 6 cd3 + cells per kg. dli alone induced a lasting reduction of median bcr-abl transcript levels (bcr-abl/gapdh ratio) of more than 3 log10 and the estimate of being in a complete molecular remission at 8 years is 76% ± 5%. 12 pts. (24%) did not respond to dli alone, but 8 of these pts. attained a cytogenetic and molecular response by imatinib (n = 9), dasatinib (n = 1) and/or interferon treatment. four patients were retransplanted with an alternative donor. the cumulative risk of grades ii-iv acute gvhd is 15% ± 5% for all study pts, and the risk of chronic gvhd is 25% ± 6%, respectively. after a median follow-up period of 88 months (range 6 -125) for all patients, the cumulative 10-year survival estimate is 91,3% ± 4%. including all therapies for molecular relapse after transplant, 50 of the 58 included patients (86%) were in molecular remission at the last time point of observation. causes of death (n = 5) were disease progression, secondary malignancy, liver failure, septicemia, and systemic capillary leak syndrome in one patient each. in conclusion, the concept of highly purifi ed pb cd34 + cell transplantation in conjunction with adoptive dli is associated with a particularly low risk of non-relapse mortality and allows induction of lasting molecular disease control in the majority of 1. cp cml. allogeneic stem cell transplantation (asct) after reducedintensity conditioning (ric) has become a reasonable treatment option for patients with advanced myelofi brosis. the role of characteristic molecular genetic abnormalities such as jak2v617f on outcome of asct is not yet elucidated. in 139 out of 162 myelofi brosis patients with known jak2v617f mutation status who received asct after ric the impact of jak2 genotype, jak2v617f allele burden and clearance of mutation after asct was evaluated. a non-signifi cant higher treatment-related mortality (31% vs. 19%, p = 0.1) and relapse incidence (30% vs. 21%, p = 0.2) was noted in patients harbouring jak2 wild-type, resulting in a decreased overall survival in a multivariate analysis (hr 2.23, p = 0.007). no signifi cant infl uence on outcome was noted for the mutated allele burden analyzed either as continous variable or after dividing in quartiles. achievement of jak2v617f negativity after asct was signifi cantly associated with a decreased incidence of relapse (hr 0.22, p = 0.04). in a landmark analysis, patients who cleared jak2 mutation level in peripheral blood 6 months after asct had a signifi cant lower risk of relapse (5% vs. 35%, p = 0.03). we conclude that jak2v617f mutated status but not allele frequency resulted in an improved survival and rapid clearance after allografting reduced the risk of relapse. early autologous stem cell transplantation in poor-risk chronic lymphocytic leukaemia. long-term results from the gcllsg cll3 trial focusing on incidence and type of secondary malignancies f. mcclanahan (1) (2), p. dreger (1) ( introduction: as previously reported, early autosct as conducted in the cll3 protocol is a feasible therapy option for younger patients (pts). purpose of the present analysis was to study the impact of fish karyotype and ighv mutational status on long-term pfs and os and the incidence and type of secondary malignancies occurring in this trial. trial design and patients: the design of the protocol has been extensively described previously. from 1996 to 2002, 216 pts were registered. as 47 cases had to be excluded due to screening failure (n = 21), withdrawn consent (n = 19) or other reasons (n = 7), 169 pts were eligible for the current analysis. male to female ratio was 5:1 and the median age at diagnosis was 51 yrs (range 27-60). results: sct was performed in 131 pts (78%) at a median time of 17 months (range 4-159) after initial diagnosis, whereas 38 pts did not proceed to sct (mobilization failure n = 14, disease progression n = 4, early death n = 3, pts preference n = 6, unknown reasons n = 11). at a median follow-up of 99 months (range 4-137), median os of all 169 pts was 10.5 yrs (10.5 yrs after sct, 6.1 yrs without sct, hr 0.26, 95% ci 0.13-0.54, p<.0001). median pfs was 6.3 yrs (6.8 yrs with sct and 4.8 yrs without, hr 0.39, 95% ci 0.23-0.67, p = 0.0007). diagnostic samples for assessment of ighv mutational status were available for 143 (85%). an unfavorable ighv rearrangement was present in 104 pts (73%), and was associated with signifi cantly worse pfs (p = .0001) and os (p = .017). fish was possible in 160 pts. whereas pfs (p <.0001) and os (p <.0001) were considerably reduced in pts with del 17p-, there were no signifi cant differences between the other subsets. altogether, 20 secondary malignancies were observed, with 6 cases of t-mds/ t-aml, translating into a 10-year incidence of 20% (95% ci 11-30%), with no signifi cant difference among pts treated with and without sct (p = 0.68). however, all cases of t-mds/ t-aml occurred after sct, yielding a 10-year incidence rate of 9% (1-18%). overall survival after secondary neoplasms was 22 months , with no difference between t-mds/aml and other malignancies. conclusions: unmutated ighv remains an adverse prognostic factor. while del 17p-is associated with a very poor outcome, autosct may eliminate the unfavorable impact of del 11q-seen with conventional therapy. secondary neoplasms are a serious problem after early autosct, but do not appear to occur more frequently than reported after sct for other diseases. reduced intensity conditioning (ric) has reduced non-relapse mortality (nrm) associated with allogeneic hsct in hodgkin lymphoma and relapse is now the commonest cause for treatment failure. however, there is little published evidence to guide management of relapse. we performed 81 ric allografts for hl (44 matched related, 37 unrelated donor) incorporating t cell depletion with alemtuzumab. 35 (43%) were primary or salvage-refractory, 47 (58%) had failed a prior autograft, and the median number of prior treatment lines was 5. patients were monitored by pet-ct and relapse defi ned by recurrence or progression of fdg-avid lesions in sites of prior disease. if fdg-avidity occurred only in new sites, relapse was confi rmed by biopsy if accessible (n = 7), otherwise an interval scan was performed at 6-8 weeks. following cyclosporine withdrawal and in the absence of gvhd, patients received dose-escalating dli for mixed chimerism (mc) or progression. the 3 year ci of nrm was 17% and of relapse was 45%. 44 patients received a total of 87 dlis (median 2, range 1-5). treatment for mc alone (n = 22) resulted in conversion to full donor in 15/20 evaluable patients, continuing improvement in 2, and 3 remained stable. only 1 of these relapsed (ci 7% at 3 years from initial dli), receiving further dlis following chemotherapy. relapse was treated in 23 patients with dli either alone (n = 13) or following debulking chemotherapy (n = 10). median maximal doses were 1 × 10 7 in unrelated and 1 × 10 8 cd3 + t cells/kg in related donor transplants. complete responses were seen in 12 and partial responses in 5 of 22 evaluable patients (response rate 77%), and did not differ signifi cantly according to donor source (10/13 mrd vs. 7/9 ud). the majority of responders developed gvhd (5 gd iii-iv acute, 4 extensive chronic). 7 cr are maintained at a median of 4.6 years from last dli (5 without prior chemotherapy), 3 died of gvhd-related complications, and 2 progressed (at 1.6 and 2.3 years). 3/5 with pr progressed. the projected 3 year os and pfs from relapse are 64% and 54% in this group. the os and current pfs for the entire cohort of 81 patients are 61% and 53% at 4 years. in conclusion, the data demonstrate favourable long term survival in this heavily pretreated hl cohort, a strikingly low relapse incidence in those receiving dli for mc, and the ability of dli guided by pet-ct to induce high response rates and durable salvage in the setting of relapse post t cell depleted transplantation. a recent update of 3 consecutive prospective trials with high-dose therapy and autograft, without or with rituximab, as primary treatment for advanced-stage follicular lymphoma shows a sizeable group of patients surviving in continuous complete remission c. tarella, m. ladetto, f. benedetti, u. vitolo, a. pulsoni, c. patti, v. callea, a. rambaldi, a. piccin, l. devizzi, m. musso, e. iannitto, p. spedini, a.m. liberati, f. ciceri, a. gallamini, f. rodeghiero, g. gini, a. de crescenzo, f. di raimondo, a. levis, t. chisesi, t. perrone, d. rota scalabrini, g. rossi, a.m. carella, g. parvis, i. majolino, r. passera, m. ruella, a. pileri, a.m ) or in the proportion of patients with systemic symptoms or with elevated serum lactate dehydrogenase. the median time from the diagnosis to asct was also comparable as well as the number of previous treatment lines and the use of rituximab before asct (49% vs. 53%). the elderly patients were less often in fi rst complete remission at the time of asct (42% vs. 53%, p = 0.003). the most common conditioning regimen used was beam (62% in the elderly vs. 58%) followed by cyclophosphamide plus total body irradiation (14% vs. 14%). non-relapse mortality (nrm) was comparable at 3 months (3.7% in the elderly vs. 2.1% in younger patients) and at 1 year (4.2% vs. 2.9%) but was higher in the elderly patients at 5 years (8.6% vs. 3.2%, p = 0.04). the most common causes of nrm were infections (53% vs. 44%). with a median follow-up of 21 months for the elderly patients and 25 months for the younger patients, the risk of relapse was 57% and 54% (n.s), respectively. progression-free survival was also comparable (38% vs. 40% at 5 years) without any plateau. overall survival was worse in elderly patients (60% vs. 69% at 5 years, p = 0.01). autologous stem cell transplantation is feasible in selected elderly patients with mcl. early nrm is low and comparable to younger patients. risk of relapse and progression-free survival are also comparable but overall survival is worse in the elderly patients. continuously increasing relapse risk indicates the need for improvements in pre-and posttransplant strategies in order to improve long-term outcome in this lymphoma type. phase ii study of radioimmunotherapy with yttrium(1) allogeneic hematopoietic cell transplantation (hct) using reduced intensity conditioning (ric) offers a potential curative therapy to patients with advanced indolent nhl. combined use of radioimmunotherapy (rit) with ric may increase anti-lymphoma activity of ric while avoiding additional toxicities. forty patients have been enrolled in a multicenter phase ii study of allogeneic hct using rit with yttrium-90-ibritumomab tiuxetan (y90-cd20, zevalin) with 0.4 mci (15 mbq)/kg on day -14 combined with ric using fl udarabine (30 mg/m² day -4 to -2) and 2 gy tbi (day 0) followed by allogeneic hct from matched related or unrelated donors. gvhd-prophylaxis consisted of cyclosporine and mycophenolate mofetil. diagnoses were follicular lymphoma (fl, n = 17), chronic lymphocytic leukemia (cll, n = 13), mantle cell lymphoma (mcl, n = 8), marginal zone lymphoma (n = 1) and immunocytoma (n = 1). median age was 56 (range, 35-68) years. pbsc grafts were either from matched related (n = 13) or matched unrelated donors (n = 27). all patients were "high risk" with refractory disease or relapse after preceding autologous hct. engraftment was rapid and sustained with no graft rejections. median time to >500 granulocytes/μl was 13 (range, 0-69) days, and to >20000 platelets/μl 4 (range, 0-69) days. in 13 patients platelets were never <20000/μl and in 6 patients granulocytes never <500/μl, illustrating the nonmyeloablative intensity of the conditioning regimen. trm in the fi rst 100 days was 10% (n = 4) and overall 40% (n = 16). no additional toxicity due to rit compared to our previous experience with the same ric as single modality was observed. incidence of grade ii-iv gvhd was 45% (ii = 3, iii = 12, iv = 3). to date, chronic gvhd occurred in 19 patients (48%, limited = 8, extensive = 11). deaths occurred due to infections = 8, gvhd = 7, relapse = 2 and cerebral bleeding = 1. 22/40 (55%) of all patients are alive with a median follow up of 672 (range, 251-1086) days, resulting in a kaplan-meier estimate 2 year survival of 51% for all, and 67% in fl, 49% in cll and 38% for mcl patients. risk factors for decreased overall survival and trm in multivariate cox regression analysis were non-fl histology (p = 0.032 and p = 0.024) and agvhd >grade 3 (p = 0.001). in conclusion, a combination of rit with ric is feasible with no additional toxicity due to rit and with stable engraftment in all patients. disease response and overall survival seems promising even in this elderly and heavily pretreated cohort of patients. allogeneic stem cell transplantation after autologous haematopoietic stem cell transplantation relapse in aggressive lymphoma patients: fi nal report of a retrospective gitmo study l. rigacci, a. bosi, b. puccini, p. corradini, l. castagna, n. cascavilla, g. milone, a. bacigalupo, r. scimé, g. specchia, a. rambaldi, p. leoni, f. ciceri, a. levis, s. guidi, b. bruno, r. oneto, r. fanin on behalf of gitmo autologous hematopoietic stem cell transplantation (ahsct) has been shown to be an effective therapy for patients (pts) with aggressive lymphoma. pts who relapse after an ahsct have a very poor prognosis. allogenic hematopoietic stem cell trasplantation (allohsct) has shown to be effective in the rescue of indolent lymphoma pts relapsed after conventional therapies. according to this data we have retrospectively analized all pts with diagnosis of aggressive lymphoma in the gitmo data-base who have performed an allohsct after an ahsct relapse. from 1995 to 2008, 181 pts were selected from the gitmo data-base. one of the principal objective in this fi nal report was the completeness of the data. data missing were: acute gvh was evaluable in 90% of pts, chronic gvh in 70% of pts, response after allogeneic transplantation in 66%, condition at the moment of allotransplant in 94%, therapy pre-allo-hsct in 77% of pts. the other characteristics were evaluable in all patients. one-hundred and one were male (56%), 160 presented a diagnosis of dlbcl. the stem cell donor was related in 115 pts, the stem cell source was peripheral blood in 151 pts and bone marrow in 30. the conditioning regimen was conventional in 55 pts and reduced intensity (ric) in 126 pts. the median time between ahsct and allo-hsct was 12 months. ninethy-seven pts (54%) obtained at least a partial remission before allo-hsct, 74 (41%) were treated with active disease and in 10 cases the data was not available. after allo 78 pts (43%) obtained a cr and 10 a pr with an orr of 49%, 69 pts (38%) showed a rapid progression of disease. ninethy-seven pts died, 46 due to disease and 51 to treatment related mortality (trm). acute graft versus host disease was recorded in 61 pts and a chronic one in 42 pts. with a median follow-up period of 16 months (4-28) from allo the os was 56% and after a median period of 12 months (6-17) from allo the pfs was 49%. in multivariate analysis the only factors which affected pfs were the status at allo and a cr after allo transplant. this conclusive retrospective analysis confi rms that the only one parameter affecting either os or pfs was the response status at the moment of all-hsct and does not confi rm that ric could permit to obtain better results in aggressive lymphoma. probably a myeloablative conditioning should be reconsidered in pts with aggressive disease because of the slow-acting graft versus lymphoma effect is overriden by the rapid growth of the tumor. patients (5%), ii in 100 (28%), iii in 80 (23%), and iv in 155 (44%). forty-nine percent of the patients had b symptoms, 7% bulky disease, and 54% extranodal involvement. karnofsky performance status < 80 was reported in 72 patients. treatment following relapse consisted on conventional chemotherapy and/ or radiotherapy in 294 patients (64%), second asct in 35 (8%), and allogeneic stem cell transplantation in 133 (29%). a significant higher proportion of patients, age < 50 years, received a second transplantation in comparison to older patients (39% vs. 16%, p =.006). after a median follow-up of 49 months (range 1-150), the os from relapse after asct was of 55% at 2 years and 32% at 5 years. in multivariate analysis, independent risk factors for os were early relapse (< 6 months), stage iv, bulky disease, poor performance status, and age > 50 at relapse. in conclusion, according to the ebmt database, most hl patients relapsing after asct were treated with chemo-radiotherapy approaches and some of them with a second transplantation. those patients in a good performance status presenting with a localized late relapse seem to be the ones showing the best prognosis. hematopoietic cell transplantation (hct) is the therapy of choice for a variety of malignancies. hct provides disease benefi t through both the high-dose conditioning regimen and an allogeneic graft versus tumor effect (gvt). however graft-versus-host disease (gvhd) still remains a major obstacle. it has been proposed that host conditioning may not only be crucial in the activation of alloreactive t cells but also determine acute gvhd organ manifestation. we wanted to investigate how the host-conditioning regimen affects the host target tissues in terms of infl ammatory cytokines and their role in donor t cell recruitment. utilizing a non invasive bioluminescence imaging (bli), and fl ow cytometry in a murine allogeneic hematopoietic cell transplantation (allo-hct) model, we compared lethally irradiated (8gy) vs. non-irradiated balb/c wild type, and balb/c rag-/-cgc-/-(dko) (h-2d) mice that received allogeneic luciferase + fvb/n t cells (h-2q). we observed that the proliferation (bli, cfse), acquisition of activation markers (cd25, cd44, cd69) and homing receptors (a4b7, aeb7, p-selectin ligand, e-selectin ligand) by alloreactive t cells occurred independently whether allogeneic recipients were conditioned or not conditioned before allo-hct. as t cell recruitment may have occurred as a result of alterations of the milieu infl ammatory cytokines in gvhd target tissues, we compared the cytokine profi le in conditioned vs. non-conditioned hosts. at days 3 and 6 after allo-hct, host tissues were analyzed for a th1/th2/th17a cytokines from the target tissues; liver, large bowel, small bowel, peripheral blood and a non target tissue: kidney. at day 3, high levels of inf-g, tnf and il-6 were detected in the balb/c wt conditioned compared to the non-conditioned host in all target tissues and most markedly in blood and the large bowel. more importantly both the balb/c rag-/-cgc-/-(dko) conditioned and non-conditioned host displayed very high levels of the infl ammatory cytokines, with balb/c wt and balb/c rag-/-cgc-/-(dko) conditioned hosts displaying higher levels. similar results with a reduced expression of the cytokines were observed at day 6 indicating that the cytokine storm peak was maybe at day 3. in summary host conditioning is not a requirement for alloreactive t cell activation rather induced infl ammatory cytokines such as tnf and inf-g are the determinant factors for effector t cell recruitment to gvhd target tissues. acute graft-versus-host disease (gvhd) after gender mismatched stem cell transplantation may be caused by female donor t cells recognizing hy minor histocompatibility antigens expressed by male recipients. hy-specifi c cd8 positive cytotoxic t lymphocytes (ctl) have been detected in peripheral blood samples collected at the onset of acute gvhd. however, it is still unclear whether these ctl reach acute gvhdaffected tissues. we validated the sensitivity and specifi city of fl uorescently labeled multimeric hla-a2 molecules containing hy peptide, i.e. hla-a2/hy dextramers, to stain cryosections prepared from skin biopsies obtained from healthy male hla-a2 positive volunteer donors. these skin explant tissues were cultured with female hy-specifi c ctl or control ha-1 specifi c ctl before cryopreservation. unlike conventional hla-a2/hy tetramers, hla-a2/hy dextramers stained hy-specifi c ctl, but not ha-1 specifi c ctl, when applied to already cryopreserved skin explant tissue. control staining of serial sections with hla-a2 dextramers containing infl uenza peptide showed no positive signal. next, the presence of hla-a2/hy dextramer positive cells was analyzed in cryopreserved skin biopsies derived from 7 male hla-a2 positive pediatric patients who developed acute gvhd of the skin after receiving a hematopoietic stem cell graft from a hla matched unrelated donor. while a few cd8 positive hla-a2/hy dextramer negative cells were detected in the skin of 4 boys who received male hematopoietic stem cells, significantly higher numbers of cd8 positive cells were detected in the skin of 3 boys who received a female graft; 50-75% of these cd8 positive cells were specifi c for hy. skin-infi ltrating hyspecifi c t cells were visualized as early as 2 weeks after sct when total peripheral blood lymphocyte counts were still low. our results underline the usefulness of this multimeric staining reagent for in situ characterization of donor-derived t cells that infi ltrate acute gvhd affected tissues. (1) granulocyte-colony stimulating factor (g-csf) is increasingly described as an immuno-modulatory agent for a diverse range of diseases and although the cytokine is usually associated with the regulation of immune responses, clear evidence exists that it can also exacerbate pathology in some settings. the clinical shift toward utilizing g-csf mobilized stem cell grafts has resulted in enhanced hematopoietic reconstitution, reduced relapse rates in advanced disease, similar levels of acute gvhd but a striking increase in chronic gvhd. the mechanisms by which stem cell transplantation (sct) promotes chronic gvhd are unclear. comparison of cytokine expression following tcr activation of splenocytes from naïve and g-csf treated b6 or balb/c donors (low and high responders respectively) showed little effect of g-csf on th1 (ifn-gamma and tnf) or th2 (il-4, il-5 and il-10) cytokine production. in contrast, il-17 production was dramatically enhanced in response to g-csf treatment in both strains but was highest in balb/c donors. the amplifi cation of il-17 production by g-csf occurred in both cd4 and cd8 conventional t cells and by using relevant knock-out mice or blocking reagents we demonstrated that this was independent of il-6, tgf-beta or il-23 signalling. however, the induction of il-17 by g-csf was completely lost in the absence of il-21 signalling. g-csf induced the production of il-21 in cd4 t cells, and this occurred independently of il-17 production itself. we next utilized multiple models of gvhd using g-csf mobilized b6 or balb/c wild-type or il-17 deficient donors, in both mhc matched and mismatched transplant settings. surprisingly, il-17 was critical for the induction of sclerodermatous chronic gvhd occurring late after transplant using either donor strain. importantly, il-17 controlled the dramatic sequestration of macrophages into skin that coincided with the fi brogenic response. this study provides a logical explanation for the propensity of allogeneic stem cell transplantation to invoke sclerodermatous gvhd and suggests a therapeutic strategy for intervention. non-hla gene polymorphisms contribute to the immune response leading to graft-versus-host disease (gvhd). we applied a systematic approach using microsattelite (ms) marker typing for a large number of immune response genes on pooled dna of japanese donors and recipients of haematopoietic stem cell transplants (hsct) to identify recipient and donor risk loci for gvhd. ms, due to their multiple alleles, are more informative than single nucleotide polymorphisms (snp). we selected 4,231 ms markers, tagging 3,093 target genes (representing the 'immunogenome') at close proximity (<100kb). we selected 922 unrelated hsct donor/recipient pairs from the japan marrow donor programme (jmdp) registry, based on clinical homogeneity (acute leukaemia, age 4-40 years, myeloablative conditioning, bone marrow source). 42.61% of pairs had a 10/10 hla match. the population was split into discovery and confi rmation cohorts with 460/462 pairs each. eight dna pools, four for each of the two independent screening steps were created using a highly accurate dna pooling method. while 4,321 ms were typed on the four pools of the 1st screening step, only markers positive here were typed on the 2nd screening pools. fisher's exact test for 2x2 (each ms allele) and 2xm chisquare tests were performed, comparing allele frequencies of recipients with gvhd grade 0-1 with gvhd grade 2-4 (donors accordingly). markers positive after both independent screening steps (p-value <0.05, same associated allele, consistent odd's ratio (or)) were genotyped for confi rmation on individual samples of all 922 pairs. the independent, 2-step pooled dna screening process has effectively reduced false-positive associations. in the fi nal analysis, 39 (recipient) and 58 (donor) ms loci remain associated with risk or protection from gvhd. (1), g. afram (2) we have evaluated the impact of nih score system in the outcome of 820 patients and studied additional prognostic factors among patients who develop cgvhd at 3 european centers. furthermore, we tried to determine the prognostic impact of the different organs involved in order to defi ne which ones must always be evaluated and which ones could be avoided in a routine examination. patients' characteristics: patients transplanted from 2000 to 2006 at 3 different institutions were analyzed; 77% received stem cells from a related donor and 23% from alternative donors. results: median follow up was 1087 days (range: 7 to 2944). 53% and 31% developed overall and extensive cgvhd, respectively. according to nih classifi cation 29%, 24% and 17% of the patients were categorized as mild, moderate and severe cgvhd, respectively. type of onset was de novo in 22%, quiescent in 27% and progressive in 12%. cumulative incidence of delayed acute gvhd was 10% while the respective value for overlapping syndrome was 17%. among patients with cgvhd, the extent of cgvhd infl uenced on survival (55% vs. 71% for extensive vs. limited cgvhd, p<0.001), as well as the development of overlapping syndrome (os 57% vs. 71% for patients who did or did not have overlapping syndrome, p = 0.01), of severe cgvhd (68%, 68% vs. 48% for patients with mild, moderate vs. severe cgvhd, p<0.001), and the type of onset (70% vs. 57% vs. 51% for de novo, quiescent and progressive cgvhd, p<0.001), while delayed acute gvhd did not infl uence on outcome. in multivariate analysis patients with severe cgvhd displayed a dismal outcome [hr = 0.31, 95%ci (0.17-0.58); p = 0,001]. the type of onset allowed to identify prognostic subgroups even among patients with severe cgvhd, so that the combination of both variables discriminate different patients subpopulations in terms of outcome [hr = 0.18, 95%ci (0.06-0.51); p = 0,001]. among targeted organs performance score at the time of cgvhd had the most signifi cant effect on survival [hr = 4.73, 95%ci (2.39-9.36), p<0.001]. also liver, gut and lung involvement adversely infl uenced on survival while eyes or mouth involvement had no infl uence on outcome. conclusion: nih scoring system plus cgvhd type of onset allows to defi ne patients subgroups in terms of survival. patients with overlapping syndrome have a poor outcome. among targeted organs, performance score was an independent prognostic factor while eyes and mouth involvement did not infl uence on outcome. chronic gvhd (cgvhd) remains the leading cause for late morbidity and mortality after allogeneic hematopoietic stem cell transplantation (hsct). the consensus conference summarized the current evidence on treatment of cgvhd and developed guidance for clinical practice. the consensus process included hsct centers within germany, austria, and switzerland participating in four meetings accompanied by two surveys on treatment of cgvhd send to all centers within the german and austrian working party on bone marrow and blood stem cell transplantation and the basel transplant center (switzerland). evidence was graded into: i (based on randomized trials), ii (based on case controlled or well designed phase ii trials), iii-1 (several case series), iii-2 (one case series), iii-3 (case reports) according to all available publications. recommendations were based on effi cacy and safety profi le: a (should always be applied), b (should generally be applied), c-1 (may be applied 1st-line), c-2 (may be applied 2nd-line), c3 (may be applied advanced line), c-4 (may only be applied in special circumstances). for 1st line treatment of cgvhd the following agents have been proposed: prednisone a i, calcineurin inhibitors (cni) c-1 ii, mmf c-1 iii. for 2nd-line treatment the following treatment options have been proposed: prednisone b iii-1, cni c-1 iii-1, mtor inhibitors c-1 iii-1, mmf c-1 iii-1, photopheresis b ii. a pulse of steroids c-2 iii-2 and rituximab c-2 iii-1 should be generally applied after 2nd-line treatment but may be used earlier in special circumstances. advanced line treatment options being proposed are: mtx c-2 iii-1, hydroxychloroquine c-2 iii-2, clofazimine c-2 iii-2, pentostatine c-2 ii, thoracoabdominal irradiation c-2 iii-2, and imatinib c-2 iii-1. additional agents in advanced line treatment are retinoids c-3 iii-2, azathioprine c-3 iii-1, and thalidomide c-3 ii. other treatment options have been rated as experimental and may only be applied in clinical trials or special circumstances: alemtuzumab c-4 iii-3, etanercept c-4 iii-3, alefacept c-4 iii-3. the low level of evidence of the proposed treatment options resulting in a low level of recommendation indicates the urgent need for further evaluation in clinical trials. moreover, the lack of indicators for response to certain agents requires a "trial and error" approach in choosing a treatment option and clinical trials for evaluation of biomarker for response are urgently needed. recently, we reported an elevation of immature cd19 + cd21-b lymphocytes in patients with active cgvhd. here, we investigated b lymphocyte subsets in cgvhd cohorts with abnormal serum immunoglobulin levels. patients and methods: seventy-fi ve patients were enrolled into our study a median of 42 months after hct. they consisted of 3 groups: 25 with cgvhd and high serum igg (>1600 mg/dl), 22 with cgvhd and low igg (<700 mg/dl) and 28 patients with resolved cgvhd and normal igg (control group). severe cgvhd was present in 64% and 45% with more than 2 organs involved in 44% and 36% in the high igg and low igg group. signifi cantly more patients in the high igg group than in the low one had autoantibodies (72% vs. 18%, p = 0.02). peripheral blood cells were analyzed by multiparameter fl ow cytometry after staining for cd19, cd21, cd27, cd10, cd38, cd95, and igd. results: while the high igg group had equal cd19 + b cell numbers, the low igg group had signifi cantly diminished ones compared to the control (400 vs. 160 vs. 400 × 10 6 /l p<0.0001). numbers of cd10high most immature b cells in the high igg, low igg and control group were 26, 7, and 7 × 10 6 /l. immature cd19 + cd21-b cell proportions were 9.2%, 16.5%, and 7.5% in the high, low igg and control group (p = 0.01). in the high igg group transitional b cells were signifi cantly increased (28 vs. 14 × 10 6 /l, p = 0.02) compared to the control. in the high igg group naive and class-switched memory b cells were equal to the control whereas in the low igg group both naïve (13 vs. 35 × 10 6 /l p = 0.02), class-switched (16 vs. 22 × 10 6 /l, p = 0.03) and non-class-switched b cells (4 vs. 10 × 10 6 /l, p<0.0001) were signifi cantly lower compared to the control. cd10-cd21low cd95high b cell proportions were the same in the high igg and control group, while they were signifi cantly elevated (23.4% vs. 9.6% p<0.0001) in the low igg group. conclusions: cgvhd patients with hypergammaglobulinemia have normal b cell numbers with elevated immature and transitional b cells but no signifi cant impairment of b cell maturation. in contrast, patients with hypogammaglobulinemia have both a signifi cant b cell defi ciency and a distortion of b cell homeostasis in the circulation. our data indicate different pathogenetic mechanisms of cgvhd leading to different clinical presentations. preliminary results of a phase ii trial of montelukast for the treatment of bronchiolitis obliterans syndrome after hsct k. williams (1) bronchiolitis obliterans syndrome (bos) after allogeneic hsct is a serious manifestation of cgvhd. current treatments yield poor and transient responses. although the pathogenesis of bos after hsct is unknown, a similar disease, bos after lung transplant is associated with elevated leukotriene levels. we present preliminary results from an irb-approved prospective, open label, phase ii trial to test the effi cacy of montelukast, a leukotriene inhibitor, for the treatment of bos after hsct. bos diagnostic criteria included: fev1<75%, fev1/vc < 0.7 or air trapping on ct and rv>120% or rv/tlc>120% in the absence of infection and presence of another cgvhd manifestation. subjects had stable or declining fev1 on stable or decreasing immunosuppression. eleven patients have enrolled. one withdrew prior to medication and 9/10 patients have reached the primary endpoint (6 months) and have continued on study medication (10 mg montelukast po nightly). study participants ranged from 15-64 years, 7/11 female, with baseline fev1 from 33 to 71% predicted, and 3/11 patients required supplemental oxygen. fev1 increased 6-10% predicted in 3 patients, remained stable in 4, and declined less than 15% in 2. comparison of patient pre-study fev1 decline to on-study fev1 values was generated using the slope of fev1 volume vs. days post-transplant. the difference in pre-and primary endpoint slope revealed: 7/9 improvement and 2/9 decline. three patients reduced immunosuppression on study with complete cessation of tacrolimus in 1, cessation of steroids in 1, and decreased tacrolimus in 1 (including 2 with stable fev1 and 1 with increase in fev1); 1 patient had a steroid increase less than 1mg/kg/day. two patients had worsening of other cgvhd manifestations, including skin fl are that resolved with local therapy (1) and gastrointestinal cgvhd fl are that improved with increased steroids and local therapy (1) . notably, after 6 months, 1 patient demonstrated fev1 increase of 14% predicted (from baseline) on tacrolimus taper after steroid cessation and 1 patient no longer required oxygen supplementation for exercise and sleep. montelukast was well-tolerated with only one grade ii attributable adverse event (insomnia) during the six-month collection period. using nih consensus criteria, improvements were also noted in 4/4 with buccal mucosa cgvhd and in 2/5 with evidence of liver disease. these fi ndings suggest that montelukast is a promising therapy for bos after allogeneic hsct. oral session 11: paediatric issues 1 o256 objective: allogeneic hematopoietic stem cell transplantation (hsct) remains the main treatment option for children with advanced primary or secondary myelodysplastic syndrome (mds). relapse rate following hsct for advanced mds ranges between 20%-50%. this analysis was performed to asses the outcome of patients treated with a second hsct (hsct2) for disease recurrence after hsct1. patients and transplant: within studies ewog-mds 98 and 2006, 73 patients with advanced mds relapsed after fi rst hsct (hsct1); a second allograft was performed in 32 of these patients. for the 32 patients with hsct2, diagnosis prior to hsct1 was primary advanced mds (n = 18), therapy-related mds (n = 9) and secondary mds after bone marrow failure disorder (n = 5). preparative regimen of hsct1 consisted of busulfan, cyclophosphamide and melphalan in 22 / 32 patients. median time to relapse was 13.8 months (0.9-77.3) after hsct1, time from relapse to hsct2 3.7 months (0.4 -14.8), and median age at hsct2 13.0 years (5.4 -25.3). twelve patients were transplanted from a matched sibling donor, 16 from a matched unrelated and 4 from an alternative family donor. in 18 cases hsct2 was performed using the same donor than in hsct1. stem cell source was peripheral blood (n = 22) or bone marrow (n = 10). conditioning regimen and gvhd prophylaxis varied widely according to the centers preferences, a tbi-based regimen was given to 12/32 patients. outcome: median follow up was 3.0 years (0.3-5.4) after hsct2. all but 2 patients engrafted. acute gvhd grade ii -iv was seen in 10 patients, chronic gvhd in 8 of 22 patients at risk. transplant related mortality occurred in 9 patients; 4 of these died from gvhd, 1 from sudden cardiac arrest 6.5 years post hsct2. a second relapse was noted in 12 patients, all of whom succumbed to their disease. there was no signifi cant difference in relapse rate according to the choice of donor (identical or different donor than in hsct1) or the occurrence of acute/ chronic gvhd. for the whole cohort of 32 patients, the probability of event free survival at 5 years was 0.35 (0.17-0.53). conclusion: we conclude that hsct2 after mds relapse is feasible and may rescue a considerable proportion of patients. reduced-intensity conditioning for children with refractory cytopenia: results of the ewog-mds study b. strahm (1) , p. bader (2) objective: refractory cytopenia (rc) is the most common subtype of childhood myelodysplastic syndrome (mds). hematopoietic stem cell transplantation (hsct) in rc following a myeloablative preparative regimen has resulted in an eventfree survival of 80% with a very low probability of relapse and a probability of therapy related mortality of 15%. this analysis was performed to asses the outcome of children transplanted for rc following a reduced intensity conditioning. patients and transplant procedure: fifty-six patients (32 male/24 female) were diagnosed with rc at a median age of 11.2 years (1.8-17.9). none of the patients had an abnormal karyotype. the median time to hsct was 0.71 years (0.12 -9.1). patients were grafted from a matched sibling donor (msd) (n = 15), an alternative family donor (n = 1) or an unrelated donor (ud) (n = 40). ud were matched 10/10 or 9/10 based on hla-a, -b, -c, -drb1 and -dqb1 molecular typing. stem cell source was bone marrow (n = 48) or peripheral blood (n = 8). all patients were prepared with thiotepa (3 × 5 mg/kg) and fl udarabine (4 × 40 mg/m 2 ). prophylaxis for graft-versus-host-disease (gvhd) was csa ± mtx, ± mmf for msd, and csa, mtx and anti-thymocyte globuline for patients transplanted from an ud. results: two patients each experienced primary or secondary graft failure. fifty-four patients reached neutrophil engraftment at a median time of 24 days (11-105). platelet engraftment was demonstrated in 48 patients at a median time of 28 days (1 -201) ; in 2 patients an additional stem cell boost was necessary. out of the four patients with graft failure three patients are alive after second hsct. the incidence of acute gvhd grade ii-iv and grade iii-iv was 23% and 5%, respectively. ten of 52 (19%) patients at risk developed chronic gvhd which was extensive in 5. one patient died of from veno-occlusive-disease with multi-organ-failure. viral infections were the most prevalent complication. fifty-four patients are alive with a median follow-up of 2.0 (0.1 -7.0) years resulting in a probability of overall survival of 0.95 and 1.0 for patients transplanted from mud and msd, respectively. conclusion: in summary the conditioning regimen with thiotepa and fl udarabine offered an excellent survival for patients rc. chronic gvhd and viral infections were the main complications. long term follow is needed to assess the expected reduction in long term sequelae. allogeneic stem cell transplantation (sct) is indicated in approximately 20-30% of children with acute lymphoblastic leukemia (all). unrelated umbilical cord blood (ucb) is an established stem cell source for sct. we retrospectively analyzed 532 children with all in complete remission (cr)1 (n = 186), cr2 (n = 238) and cr3 or advanced disease (n = 108) who received ucbt as fi rst transplant. patients were transplanted in ebmt centers from 2000-2008. median age was 6.8 years (y) (29 patients less than 1 y). the most common immunophenotype was b-cell precursor all and 17 patients had biphenotypic all. of 186 patients transplanted in cr1, 45% had poor risk cytogenetics (t4;11 or t9;22). grafts consisted of one (n = 504) or two (n = 28) units; 62% had 0-1 hla mismatches with recipients while 38% had 2-3 mismatches (antigen level for hla-a and b, allelic level for drb1). median tnc cell dose at freezing and infusion was 5.9 × 10 7 /kg and 4 × 10 7 /kg, respectively. conditioning regimen was myeloablative in 96% of cases and tbi >6gy was used in 52%. other regimens included busulphan with cyclophosphamide ± thiotepa or melphalan. atg was added in 88% of cases and gvhd prophylaxis was csa ± steroids in 75%. median follow-up was 18.5 months (3-109). cumulative incidence (ci) of neutrophil recovery, acute gvhd and trm were 82 ± 2%, 27 ± 3% and 21 ± 3% respectively. in multivariate analysis tnc infused >4 × 10 7 /kg (p = 0.001) and remission status at ucbt (p = 0.01) were associated with improved neutrophil recovery. ci of 2y relapse was 37±3% (31% for cr1, 34% for cr2, 50% for advanced). disease status at ucbt (hr = 0.36, p 0.001) and use of tbi>6gy (hr = 0.58, p = 0.01) were independently associated with lower ci of relapse. 2 y probability of leukemia-free-survival (lfs) was 38±2% (49% for cr1, 42% for cr2, 10% for advanced; p = 0.001). in multivariate analysis, disease status at ucbt was the only factor associated with improved lfs (hr = 0.32, p = 0.001). causes of death were infections or other transplantrelated events (n = 172) or disease progression (n = 95). in conclusion, in the absence of an hla identical donor, ucbt remains a valuable alternative option for children with high risk all. disease status at transplant and cell dose are the most important risk factors for outcomes. use of tbi in the conditioning regimen was associated with decreased incidence of relapse but was not associated with improved lfs. the role of minimal residual disease as a predictor of outcomes of ucbt is under investigation. treosulfan-based conditioning in children: retrospective analysis of the german and austrian experience r. beier (1) background: treosulfan (treo) is an alkylating agent with good myeloablative activity and a favourable toxicity profi le. it has been widely used in hematopoietic stem cell transplantations (hct) of high-risk adult patients. here we report on our retrospective analysis of children treated in germany and austria. patients and methods: all 25 pediatric transplant centers in germany and austria were asked to submit data on treo conditioning. nine centers reported a total of 92 transplantations, 3 autologous and 89 allogeneic. results: the median age at transplantation was 9,9 (range 0,1-20,4) years (n = 74). underlying diseases were malignancies (n = 44: 12 aml, 12 all, 6 mds, 5 ewing sarcoma, 2 neuroblastoma, 1 rhabdomyosarcoma, 1 b-all, 1 lgl, 1 biphenotypic leukemia, 1 nasopharynx carcinoma, 1 alcl, 1 nephroblastoma), immunodefi ciencies (n = 26: 8 scid, 4 cgd, 3 unclassifi ed, 3 hyper-igm, 2 interleukin-10 receptor defi ciency, 2 hlh, 1 was, 2 cvid, 1 mhc class ii defi ciency), hematologic diseases (n = 20: 8 thalassemia, 3 camt, 3 osteopetrosis, 2 sds, 1 sickle cell disease, 1 saa, 1 dkc, 1 scn), and metabolic disorders (n = 2: 1 mld, 1 mannosidosis). donors were matched sibling or matched related (n = 21), matched unrelated (n = 50), autologous (n = 3), mismatched unrelated (n = 2) or haploidentical family donors (n = 12). two patients received a combination of a cord and a haploidentical graft. treosulfan was given at a total dose of 30 g/m 2 (n = 8), 36 g/m 2 (n = 31), and 42 g/m 2 (n = 53). additional conditioning drugs included fl udarabine (n = 82), thiotepa (n = 43), melphalan (n = 22), cyclophophamide (n = 4), or tbi (n = 1, 4gy). atg (n = 37), alemtuzumab (n = 23), okt-3 (n = 11), and a combination of atg and okt3 (n = 3) were used together with csa ± mtx or mmf as gvhd-prophylaxis. eight patients experienced non relapse mortality, six of them had heavily pretreated malignancies. one engraftment failure (thalassemiahaplo) and three rejections (1 immunodefi ciency, 1 thalassemia, 1 scn) were reported. all other patients engrafted rapidly. toxicity and gvhd grades in these patients were mostly i and ii. event free survival after 3 years (data available for 65 patients) was 92% for non-malignant and 37% for malignant diseases. conclusion: treo based conditioning regimens in children were effective in terms of engraftment and survival. optimal treo dosing for children is still not well defi ned. pharmakokinetic studies in children are needed. objectives: hepatic veno-occlusive disease (vod) is a severe complication following hematopoietic stem cell transplantation (hsct), with a mortality rate of 20%-50%. the diagnosis of vod is based on clinical criteria. however, the late onset of clinical signs may delay the due treatment. studies in adults have addressed to plasminogen activator inhibitor-1 (pai-1) a possible role as marker of vod. our aim was to prospectively evaluate the role of fi brinolytic parameters to discriminate vod from other liver disorders occurring after hsct in a pediatric population. methods: we studied 161 children (males 96, females 65, mean age 7,91 ± 5,17 years) who underwent 195 hsct performed at the pediatric hematology oncology of padua university. the prevalence of vod was recorded. in 105 hsct the levels of alanine amino-transferase (alt), total bilirubin, pai-1 antigen (pai-1:ag) and activity (pai-1:act), t-pa antigen (t-pa:ag), d-dimer, pt, aptt, antithrombin, fi brinogen and platelet count were assayed before and weekly for 1 month after hsct. results: the prevalence of vod was 5.6% (11/195 hsct), and it was signifi cantly higher in patients with pre-existing risk factors for vod as compared with those without (9.7% vs. 1.9%; p<0.05). all but one patient who developed vod showed an early increase in pai-1:ag, pai-1:act, t-pa:ag and d-dimer levels, even before the clinical diagnosis of vod. the increase in fi brinolytic parameters, and in particular pai-1:ag, was statistically signifi cant in comparison with that observed both in patients without complications (pai-1:ag p<0,0001) and in those with non-vod liver disorders (pai-1:ag p<0.0001). similar fi ndings were also seen when vod patients were compared with subgroups of children with infections and/or different types of hepatic diseases complicating hsct. conclusions: our study demonstrates a role of fi brinolytic tests in the diagnosis of vod after hsct in the pediatric population. in particular, the early rise in pai-1 antigen levels may suggest an incoming vod, even in the absence of clinical signs. in addition, pai-1 antigen increase may help to discriminate vod from other hepatic complications after hsct. late onset non infectious pulmonary complications (lonipc) after allogeneic hematopoietic stem cell transplantation (hsct) such as chronic graft-versus-host disease (cgvhd) of the lung, bronchiolitis obliterans (bo), bronchiolitis obliterans organizing pneumonia (boop), sinu-bronchial syndrome and restrictive lung disease are well characterized. immunodefi ciency, with or without cgvhd, and recurrent respiratory tract infections may be a vicious circle which requires optimal supportive therapy. mucociliary dysfunction is described in chronic pediatric lung diseases like cystic fi brosis (cf) and primary ciliary diskinesia (pcd). treatment with dornase alpha as a mucolytic drug with anti-infl ammatory effect improves morbidity and mortality rate in children with various chronic lung diseases. we hypothesized that outcome of our patients with lonipc measured by mortality, lung function, inhalative therapy and systemic immunosuppressive treatment could be infl uenced by dornase alpha inhalations 2500 i.e. once daily. we analyzed 23 children with lonipc who have received an allogeneic hsct between 1994 to 2009 for malignant (n = 10) and non malignant (n = 13) diseases. we compared 12 children (study group) obtaining dornase alpha inhalations with 11 children without dornase alpha. characteristics of both groups are shown in table 1 . additional supportive care was similar in both groups. dornase alpha inhalations were well tolerated with no side effects and high compliance. children treated with dornase alpha had a lower mortality rate (17%) than in the control group (36%) and showed in the long-term follow-up an improved lung function, predominantly of the peripheral airways (mean expiratory fl ow (mef) after 25%, 50% and 75% of forced ventilation capacity), in the forced expiratory value after 1 second (fev1), in resistance (r eff) and in the intrathoracic gas volume (itgv) compared to the control group. treatment with dornase alpha reduced signifi cantly the inhalation frequency of salbutamol, fl uticason or tiotropium after 6, 12 and 24 months (4 ± 2 times per day vs. 2 ± 2, p < 0.05; vs. 2 ± 2, p < 0.05; vs. 1 ± 1, p < 0.05) compared to the control group. immunosuppressive medication was similar in both groups. our data showed that inhalation with dornase alpha in children with lonipc is a well tolerated treatment option which may improve mortality rate and lung function. additional inhalative therapy but not systemic immunosuppression could be reduced. in order to fi nd hallmark of protection we studied the functional composition and magnitude of cmv-specifi c t cell response in cmv-seropositive (cmvpos) and cmv-seronegative (cmvneg) healthy donors and compared it to children after hct. polychromatic fl ow cytometry was used for detection of cmvspecifi c immune responses. the ability of cd4 + and cd8 + tcells to produce cytokines: interferon-ã (ifng) and interleukin-2 (il-2) and to express activation marker cd40l and/or to mobilize the degranulation marker cd107a in response to cmv antigens was evaluated by intracellular cytokine staining method after in vitro stimulation. peripheral blood samples were collected from 61 patients who underwent allogeneic sct and 58 healthy donors. monitoring of viral load was performed weekly from day + 7 using quantitative rt-pcr. monitoring of cmvspecifi c t-cells begun on day + 28 and was performed weekly during the time of hospitalization and on day + 56, + 90, + 180 and + 365 after sct. we have compared functional signatures in patients controlling their pcr documented reactivations (controllers) and patients non-controlling reactivations (non-controllers). as a reference we have included healthy cmvpos and cmvneg donors. among cd8 + t-cells we found three functional signatures (ifng + , ifng + /il-2 + and ifng + /il-2 + /cd40l + ) that signifi cantly differed between controllers and non-controllers. whereas single ifng + cd8 + t-cells were the most frequent of cmv-specifi c t-cells, they were not restricted to cmv controlling individuals. we found signifi cant correlation between number of weeks with low viral load reactivations and frequency of ifng + cd8 + tcells (r = 0.4, p<0.0001). dual ifng + /il2 + cd8 + t-cells were present in majority of controllers, but they were virtually absent in non-controllers. triple ifng + /il-2 + /cd40l + positive cd8 + t-cells represented a rare subset of novel cd8 + helper phenotype. no cd4 + t-cell functional signature resolved controllers and non-controllers. we (1) ( aberrant dna methylation contributes to the malignant phenotype in cancer including myeloproliferative neoplasms and myeloid leukemia. we studied aberrant dna methylation in 14 candidate loci in blood and bone marrow samples of 87 children with juvenile myelomonocytic leukemia (jmml) and asked whether it is associated with clinical, hematologic or prognostic features of the disease. the pattern of hypermethylation allowed the categorization of jmml cases into several groups. high methylation was strongly associated with higher age and increased hemoglobin f level at diagnosis. importantly, hypermethylation at diagnosis characterized cases with signifi cantly increased probability of leukemia relapse after hsct: the 5year relapse incidence was 0.22 (0.11-0.45) in the no-methylation group but 0.69 (0.49 -0.96) in high-methylation cases (p<0.01). the predictive power of high methylation for outcome following hsct was also refl ected in a multivariate cox model which included age at diagnosis, sex, platelet count and mutational category (ras, ptpn11, cbl mutation or clinical diagnosis of nf1); here methylation was the only signifi cant prognostic factor (p = 0.013). future guidelines for intensity of graft-versushost prophylaxis in jmml will have to take the methylation status at diagnosis into account. ecfcs have recently been described as vascular progenitor cells with robust proliferative potential and vessel-forming capacity. vascular integrity depends on endothelial and pericyte functions. this study was performed to establish ideal conditions for the generation of stable vessels by ecfc/msccotransplantation. mscs were propagated as previously described. ecfcs were isolated with a novel recovery strategy and propagated under animal protein-free culture conditions with pooled human platelet lysate (phpl) replacing fetal bovine serum (fbs). ecfc long-term proliferation potential was monitored and phenotype was analyzed by fl ow-cytometry and immune-cytochemistry. genomic stability was assayed with chromosome g-banding and array-comparative genomic hybridization (array-cgh). additionally we compared telomere-length and telomerase-activity of ecfcs at different time points during culture with fl ow-fl uorescence in situ hybridization (flow-fish) and telomere repeat amplifi cation protocol-assay (trap). functionality was studied during vascular network assembly in vitro and in human vessel formation in immune-defi cient mice in vivo. a mean of four ecfc colonies/ml of peripheral blood could be recovered. the progeny of these cultures could be expanded to mean 1.5 ± 0.5 × 10 8 ecfcs within 11-25 days. consecutive analysis confi rmed ecfc purity, immune phenotype and sustained proliferation potential for >30 population doublings with preserved progenitor hierarchy. genomic stability was confi rmed by karyotyping and array-cgh. telomere-length analysis revealed longer telomeres in cord blood compared to peripheral blood-derived ecfcs and a constant shorting of chromosomal ends through passaging, which could be further confi rmed by a lack of telomerase activity. large-scale expanded ecfcs functioned to form complex vascular networks in vitro and assembled stable cd31 + /vimentin + /von willebrand factor + human vessels when transplanted together with msc in vivo under defi ned conditions. these human vessels were connected to the mouse circulation as indicated by a rich content of ter119 + murine erythrocytes. this demonstrates that functional and stable human vessels can be generated in vivo as result of ecfc/msc-cotransplantation. this procedure represents a promising tool to develop innovative experimental, diagnostic and therapeutic strategies. hematopoietic progenitor cells (hpcs) which circulate in the blood are increasingly recognized as co-regulators of the development of tumor solid tumors through their ability to support neovessel formation and the transduction of the hpcs with suicide genes offers a new modality targeted antitumor cellular therapy. further to clarify mechanisms of their tumor homing, we investigated the role of the small gtpases rac1 and 2 expressed in hematopoietic cells during tumor development in mice. mice with an inducible deletion of the rac1 gene in hematopoetic cells and/or constitutive deletion in rac2 were used. rac1 deletion was induced by polyic treatment in rac-1fl ox/fl ox mice harbouring a hematopoietic-specifi c inducible cre recombinase. mice had previously been inoculated with lewis lung carcinoma by s.c. injection. we found that in mice with hematopoietic specifi c deletion rac genes, llc tumors grew more slowly in the absence of rac genes 1 and 2 compared to wild type (wt) controls. at the same time, numbers of mononuclear cells and hpcs increased in the blood. mobilization was further increased in tumor bearing mice. in contrast, the content of cd45 + cells in tumors of mice with an induced defi ciency of rac1 and 2 in hpcs was greatly diminished as shown by immunohistochemistry. we then performed competitive homing experiments by co-injecting green and red fl uorescence-stained wt and rac1/2-/-hpcs in tumor bearing mice. this revealed a 60-70% decrease in numbers of rac1/2-/-hpcs homing to tumors compared with controls. next, we studied in vitro adhesion in parallel plate fl ow chambers to delineate specifi c adhesion defi ciencies of the rac deleted hpcs. we found decreased arrest and persitstent adhesion of rac1/2-/-hpcs on endothelial cells on sdf-1a precoated endothelial cells. this correlated with a decreased ability of these cells to migrate through gradients fo sdf-1a in transwells. moreover, analysis of fi rm adhesion on both, vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1, either alone or in the presence of sdf-1a revealed a critical role of rac1 and 2 genes in adhesion strengthening of hpcs. in conclusion, rac1 and 2 negatively regulate mobilization of hpcs in tumor bearing mice, but are critically involved in entry of hpc into tumor tissue in a process involving sdf-1a. modulation of rac activation in progenitor cells may thus be a tool to modify tumor growth and to target suicide gene delivery to tumor sites. the however, hoxa9, hoxb7, hoxc10 and hoxd8 were defi ned as good candidate markers to discriminate cb-msc and bm-msc from ussc. thus, our data suggest that the "biological fi ngerprint" based on the hox code can be used to distinguish functionally distinct stem cell populations from bone marrow and cord blood. this work was supported by a grant from the dfg ko2119/ 6-1. combined action of endothelial and mesenchymal niche cells to amplify haematopoietic progenitor expansion in a humanized system d. thaler (1) the hematopoietic stem/progenitor cell (hspc) niche is an anatomically confi ned space governing hspc proliferation, differentiation and self renewal. recent research identifi ed distinct compartments described as stromal and vascular niches. this study was initiated to directly compare mesenchymal stromal cell (msc) and endothelial colony-forming progenitor cell (ecfc) contribution to niche functions in a humanized co-culture system. mscs and ecfcs were propagated under animal protein-free conditions. autologous msc-ecfc pairs were established to avoid donor variation. purifi ed cd34 + hspcs were used as responders in a cytokine-driven (il-3, 6 ng/ml; flt-3-l, 50 ng/ml; scf, 20 ng/ml) niche cell-regulated co-culture system. different standard media were employed to compare serum-free conditions with humanized cultures supplemented with pooled human platelet lysate (phpl). primary expansion culture of hspcs with and without niche cell support was followed by colony forming cell (cfc) assays to determine maintenance of clonogenicity. liquid cultures supplemented with 10% phpl were more efficient than serum-free cultures resulting in a mean 16-84-fold increase in nucleated cell progeny within 11 days. mscs or ecfcs further amplifi ed hspc proliferation. interestingly, the supportive effect of mscs or ecfcs was constantly more pronounced in humanized phpl-supplemented compared to serum-free cultures, indicating an important role of the natural platelet-derived growth factors. the combination of mscs + ecfcs resulted in hspc proliferation with up to 567-fold nc increase. the hspc progeny was mainly comprised of differentiating myeloid cells. cfc assays revealed that phpl-supplemented liquid cultures were more effi cient than serum-free cultures resulting in a 1.2-4.5 fold cfc expansion. both msc and ecfc initiated a more than 6-fold increase of cfcs, whereas combined action of mscs + ecfcs resulted in a maximum of 23.5-fold cfc increase in phpl-supplemented cultures. the impact of human ecfc as compared to msc cotransplantation on human hspc engraftment and reconstitution in immune defi cient mice is currently under investigation. our data indicate that mscs and ecfcs are effi cient in supporting hspc proliferation and cfc amplifi cation in a humanized co-culture system. the combination of both niche cell types had no further additive effect. the humanized co-culture thus provides a novel model system for analyses of hspc-niche cell interactions. a. reinisch (1), n.a. hofmann (1) , a. ortner (1) , n. etchart (1), c. , c. dullin (2) , c. diwoky (2) osteosarcoma (os) and ewing's family tumors (eft) are the most frequent bone sarcomas in young adults. unresectable or metastatic presentations are currently characterized by an extremely severe prognosis, with a consequent great need for new therapeutic approaches. we conducted a preclinical study to investigate the potential effi cacy of cytokine-induced killer (cik) cells as adoptive immunotherapy for bone sarcomas. cik cells are a heterogeneous subset of ex-vivo expanded t lymphocytes presenting a mixed t-nk phenotype and endowed with a mhc-unrestricted antitumor activity. cik cells can be used both as autologous adoptive immunotherapy or even infused from a donor after allogeneic hct. we successfully generated cik cells from 6 patients with metastatic bone sarcomas (5 os; 1 eft). ciks were generated from pbmc, cultured for 3-4 weeks with the timed addition of ifn-a; ab anti-cd3 and il2. at the end of the culture we obtained a heterogeneous t cell population with a median of cd3 + cd56 + cells of 40% (15-45), 52% (51-55) were cd8 + and presented a high expression (>95%) of nkg2d, the receptor mediating most of ciks' antitumor activity. the median ex-vivo expansion, calculated on the cd3 + cd56 + fraction, was 50 fold (22-400). cytotoxicity experiments (n = 4) demonstrated that cik cells effi ciently killed 2 different os cell lines (86%, 85%, 81% and 68% of specifi c killing at a 40:1, 20:1, 10:1 and 5:1 effector/target ratio respectively). more striking, in selected experiments (n = 2), we could confi rm the potent killing activity of cik cells against the autologous os tumor (figure 1 ). the test was performed by staining target cells with cfse and co-culturing them with ciks at different ratios; after 5 hours data were analyzed by fl owcytometry. we confi rmed that both cell lines and autologous os cells presented a high expression of mic a/b, main ligands for the nkg2d receptor of cik cells. the tumor-specifi c cytotoxicity of cik cells was confi rmed by the absence of any signifi cant killing against non-tumoral mhc-mismatched targets. our data are the fi rst report of antitumor activity of cik cells against autologous os cells. our fi ndings are encouraging and support the designing of adoptive immunotherapy clinical trials with autologous cik cells for os patients. the observed safety across major hla-barriers suggests that also donor-derived cik cells might be considered in peculiar experimental clinical settings exploring allogeneic hct for solid tumors. m. nonn, j. knapstein, a. brunk, d. tomsitz, s.a. khan, e. distler, m. theobald, w. herr, u.f. hartwig johannes gutenberg university (mainz, de) donor lymphocyte graft engineering to separate graft-versushost (gvh) and graft-versus-leukemia (gvl) immunity is of central interest in allogeneic hematopoietic stem cell transplantation. therefore, we established a xenograft-transplantation model using nod/scid/il2r gamma chain null (nsg) mice to evaluate engraftment, residual gvh-reactivity and gvl-immunity of human leukemia reactive cd8 + t cell lines in vivo. previous studies demonstrated engraftment of resting or polyclonally stimulated human cd3 + t lymphocytes to high levels in spleen and bone marrow following adoptive transfer into nsg mice and induction of xenogeneic gvhd (x-gvhd) within 3-5 weeks depending on the applied t cell dose. the engrafted human t cells contained both cd4 and cd8 subsets in the same ratio as analyzed prior to transfer. further transfer experiments using isolated human cd4 + and cd8 + t cell subpopulations revealed long-term engraftment of cd4 + t cells and induction of xenoreactivity. cd4 + t cells isolated ex vivo from spleen from these mice demonstrated an activated and memory phenotype suggesting a xenoantigen driven response in vivo. in contrast, adoptively transferred cd8 + t cells did neither engraft nor induce x-gvhd in nsg recipients. similar results were obtained upon transfer of several leukemia-reactive cd8 + t cell lines generated by short-term mixed leukemia lymphocyte coculture of cd8 + donor lymphocytes and acute myeloid leukemia (aml) blasts. investigating different cd8 + t cell growth and differentiation promoting cytokines we found that administration of human interleukin (il)-2 or il-7 failed to improve cd8 + t cell survival and expansion in vivo, whereas co-transfer of 10% autologous, naïve cd4 + t helper cells or repetitive injections of human il-15 resulted in robust cd8 + t cell engraftment in nsg recipients. in addition to these data, fi rst results of studies obtained in a therapy model to investigate gvl-responses of hla-mismatched aml-reactive cd8 + cytotoxic t cells upon transfer into primary aml engrafted nsg mice in the presence of autologous t helper cells or il-15 will be reported. in summary, we have shown that cd8 + t cell engraftment and homeostatic proliferation in nsg mice is dependent on cd4 + t cell-mediated or, alternatively, il-15-driven signals. we conclude that our nsg transplantation model provided with these signals may be a valuable tool for evaluating gvh-and gvlreactivity of ex vivo modifi ed human donor t cell grafts in vivo. oral session 13: reduced-intensity conditioning o334 ric allo-hsct for haematological malignancies up to 70 years is feasible without extratoxicity: a retrospective study of 619 patients on behalf of the sfgm-tc p. chevallier, d. blaise, n. milpied, m. michallet, j.p. vernant, n. fegueux, m. renaud, b. rio, n. gratecos, j.y. cahn, g. socie, i. yakoub-agha, a. huynh, s. francois, j.o. bay, c. cordonnier, a. buzyn, n. contentin, e. deconinck, m the aim of this report was to assess the outcome of 619 patients aged ≥60 years who received a ric allo-sct, with a special emphasis on the comparison of the outcome of patients aged 60-65 years (y.) and patients aged >65 y. between 1998 and 2008, 619 patients aged ≥60 years with different haematological diseases were treated with a ric allo-sct, and reported to the sfgm-tc registry. patients twice allografted were excluded from this study. this series included 408 males (66%) and 211 females (34%). the median age for the whole cohort was 62 (range, 60-71) y. 369 patients (60%) were diagnosed with a myeloid malignancy. 468 patients (76%) had high risk disease features. 380 patients (61%) received a ric allo-sct from an hla-matched related donor. pbsc were used in 82% of the patients (n = 511). the conditioning regimen consisted of fludarabine and busulfan in 307 cases (50%), fludarabine and low dose tbi in 148 cases (24%). the remaining 164 patients (26%) received other socalled ric protocols. atg was used in half of cases. with a median follow-up of 23 (range, 1-125) months after allo-sct, engraftment was 96%, grade ii-iv and grade iii-iv acute gvhd occurred at a median of 31 days after allo-sct in 29% (n = 180) and 12% (n = 75) of patients, respectively. chronic gvhd was observed in 142 out of 528 evaluable patients (27%). nrm was 31% (n = 194). 2-year os was 48% (95%ci, 44-53%). in order to assess the applicability of ric allo-sct to the older age group, we next compared the outcome of patients aged from 60 to 65 y. (n = 509; median age 62 y.) and those aged >65 y. (n = 110; median 66 y.). except for age, in univariate analysis, these 2 groups were not statistically different in terms of demographic, disease or transplant characteristics. overall, the median time to anc>500/μl was 18 (range, 1-99) days in each group. the grade ii-iv acute gvhd (29% vs. 31%), the overall nrm (31% vs. 33%), the relapse (32% vs. 24%) and the deaths (54% vs. 54%) incidences, as well as the 2-year os (48.5% (95%ci, 44-53%) vs. 48% (95%ci, 38-57%)) were comparable between the younger population vs. the older one (p = ns). in a cox multivariate analysis accounting for all relevant factors, age >65 y. was not found to be a statistically signifi cant factor associated with worsened survival. despite its retrospective nature and the inherent selection biases, this data support the use of ric-allo-sct in patients up to 70 years. final results of uk multi-centre, phase ii study of campath-1h dose de-escalation prior to non-myeloablative hla-identical sibling stem cell transplantation g. orti, k. peggs, m. collin, r. clark, d. milligan, h. roddie, e. liakopoulou, c. crawley, a. clark, r. pettengel, n. chowdhry, m. roughton, j. snowden, e. morris, k. thomson, p. kottaridis, a. fielding, s. mackinnon, r. chakraverty on behalf of the uk collaborative group background: inclusion of 100mg campath-1h (cam) as part of a fludarabine/melphalan conditioning regimen is effective at preventing gvhd and reducing nrm following allogeneic sct. however, these benefi ts are offset by high rates of infection and potentially a loss of graft-versus-tumour effects. we reasoned that reductions in the dose of cam would permit improved immune reconstitution post-transplant. methods: we performed a multi-centre trial in which the total dose of cam was reduced step-wise in cohorts from 60 mg to 20 mg prior to hla-identical sibling transplantation (n = 101). eligibility criteria were patients with haematological malignancies aged 18-65, life expectancy >3 months and unsuitable for myeloablative conditioning. primary endpoints were nrm, incidence of gvhd or infection. the study received irb approval and all patients gave informed consent. the cam dose was reduced over 4 cohorts: 60 mg (group a, n = 26); 40 mg (group b, n = 24); 30 mg (group c, n = 27) and 20 mg (group d, n = 24). results: median follow up is 2.6 years. median age was 50 yrs (range 17-64). 3-yr os was 71% with no differences observed in between the groups. 3-yr nrm was 16% overall (23.1% in group a, 12.5% in b, 7.4% in c and 20.8% in d, with no significant differences between the groups). cumulative incidences of grade ii-iv acute gvhd was 3.9% in group a, 8.3% in b, 3.7% in c and 12.5% in d. 3-yr cumulative incidences of extensive chronic gvhd were 7.7%, 4.2%, 0% and 16.7% in groups a, b, c and d respectively. the incidence of infection and kinetics of cd4, cd8, total lymphocyte recovery did not differ between the groups. the lowest dose cohort (20 mg, group d) compared unfavourably to the other 3 cohorts combined (30-60mg, groups a-c), in terms of cumulative incidence of severe grade iii-iv acute or chronic, extensive gvhd (hr 4.2, 95% ci 1.1-15.6) and a trend for greater number of non-relapse deaths due to infection (hr 3.1, 95% ci 0.9-10). 2 patients died of grade iii-iv acute gvhd in group d with no deaths related to this complication in the other groups. conclusions: signifi cant dose de-escalation of cam prior to hla-identical sibling transplantation is feasible without increasing nrm, although reductions below 30mg are associated with a higher risk of severe acute and extensive chronic gvhd. in future studies, we will determine whether use of cam at a dose of 30mg for hla-identical sibling transplantation will translate into improvements in progression-free survival. comparison of bu-cy-based standard myeloablative conditioning vs. fl udarabine and busulfan (flu-bu)-based reduced-intensity conditioning m. mohty, m. labopin, g. socié, n. milpied, m. attal, d. blaise, o. ringden, p. brown, b. lorentz, m. brune, r.m. hamladji, r.f. duarte, a. nagler, v the aim of this analysis was to compare outcomes (lfs, nrm, and relapse incidence) between patients receiving the classical bu-cy myeloablative conditioning (mac) regimen vs. patients receiving a fl udarabine and busulfan (flu-bu) reduced-intensity conditioning (ric) regimen prior to allo-sct. since the use of ric in young patients is still limited, the analysis was restricted to patients aged >40 years, who were transplanted using an hla identical sibling donor for aml in cr1. in summary, in this specifi c setting of aml in cr1, lfs is not statistically different when using mac or ric allo-sct for patients older than 50 years. however, for younger patients (40-50 y. age group), the use of ric is associated with a higher relapse rate. prospective trials addressing the use of ric in patients younger than 50 years are needed. indeed, reducing toxicity without compromising disease control could be of signifi cant benefi t to many patients, but "more intensive" regimens, despite the hazard of increased toxicity, may be necessary in others. thus, the trade-off between dose intensity, toxicity, and disease control will remain to be assessed for each individual patient. long-term results of reduced-intensity conditioning with busulfan and fl udarabine ± atg prior to allogeneic hct: 10-year follow-up k. sockel, m. bornhäuser, n. kröger, d. beelen, a. fauser, r. schwerdtfeger, w. siegert, l. kraut, a. cook, t. zabelina, c. theuser, g. ehninger, j reduced intensity conditioning (ric) prior to allogeneic stem cell transplantation (hct) is being used for 10 years now. the long-term outcome of ric conditioning with respect to late relapse and late non-relapse mortality (nrm) is still subject to ongoing research. therefore, we sought to determine factors predictive for long-term nrm and overall survival (os). methods: we retrospectively analyzed data from patients who received ric based on busulfan and fl udarabine prior to allogeneic hct between march 1998 and july 2001 in six major german transplant centers. overall survival was analysed using multivariate cox regression models, while competitive event statistics were applied for nrm. results: we identifi ed 196 patients with a median age of 52 years (range, 12 to 67). the underlying hematologic diseases were: aml/mds (47%), cml (20%), cll (16%), lymphoma (10%) or other hematologic malignancies (7%). donors were matched siblings in 42%, other matched relatives in 5%, matched unrelated in 40% and unrelated with single mismatches in 12% of the patients. the incidence of acute gvhd grades ii-iv was 53%. the cumulative incidence of any episode of extensive chronic gvhd at 5 years after hct was 46%. 66 patients were alive with a median follow up of 9 years (range, 2 to 11). the 10-year os and relapse-free survival was 32% (95% ci, 21% to 35%) and 28% (95% ci, 21% to 35%). non-relapse mortality at 10 years was 30% (95% ci, 23% to 37% that, these 2 regimens produce similar 1 year os (primary endpoint). however, fba is associated with better early pfs and efs and socially acceptable cost-effectiveness ratio but worse early qol. fba is also associated with better long term disease control, whereas ftbi tends to produce lower trm and higher rejection rates. clinical data might help designing individual and optimal strategies for each candidate patient, while economical data may help hospitals to tailor their transplant program, depending on the patient population that they care for. allogeneic-related stem cell transplantation with reduced-intensity conditioning versus best standard of care in older patients with acute myeloid leukaemia in fi rst complete remission. interim analysis of a prospective multi center phase iii trial t.l. kiss (1) introduction: allogeneic transplants after reduced intensity conditioning (rict) are increasingly used in patients (pts) with acute myeloid leukemia (aml). there is however a lack of prospective data supporting this practice. we present an interim analysis focusing on safety of an ongoing phase iii multi center trial aiming at determining whether rict from an hla matched sibling donor (msd) leads to improved overall survival (os) compared to standard of care in elderly pts with aml in fi rst complete remission (cr1). patients and methods: elderly pts with de novo or secondary aml in cr1 with non favourable risk cytogenetics were enrolled if they had a potential sibling donor and assigned to the rict group or control group depending whether a msd was identifi ed. both groups were followed prospectively. in the rict group 85% of pts were conditioned with fl udarabine (150 mg/m 2 and busulphan 6.4 mg/kg iv or 8 mg/kg po). graft versus host disease (gvhd) prophylaxis consisted of ciclosporine with mycophenolate mofetil or methotrexate. the study is supported by the canadian blood and marrow transplant group. results: between 2003 and june 2009 101 pts (53 males, 48 females), median age 62 (53-74) yrs were enrolled and allocated to the rict (n = 61) or control (n = 40) group. median follow up for the rict and control group was 17 and 23 months, respectively. after a median of 2.3 months (1) (2) (3) (4) (5) (6) (7) (8) 49 pts in the rict group (80%) underwent allografting. 12 pts did not reach transplant due to non-relapse death (n = 3); relapse-related death (n = 6) or comorbidities (n = 3; infections, cardiac disease). six pts (12%) died after transplant without previous relapse, due to gvhd (n = 4), infection (n = 1) and vod (n = 1). relapse rate for patients assigned to the rict group was 34% (n = 21), for patients actually transplanted 31% (n = 15). in the control group there were 2 (5%) non-relapse deaths and 18 (45%) relapses. kaplan-meier estimates of 3 year os and pfs in the whole study group was 50% and 45% respectively, with no signifi cant difference between rict and control arms. conclusion: this early analysis indicates that rict can be performed with relatively low transplant related mortality and with a moderate relapse rate. the full potential of rict can only be assessed after inclusion of more patients, longer follow-up, and by reducing the rate of pts not reaching transplant due to early relapse. in this interim analysis, no conclusions can be drawn on the putative positive effect of rict in elderly patients with aml. t-cell depleted reduced-intensity transplantation followed by donor leukocyte infusions to promote graft-versuslymphoma activity results in excellent long-term survival in patients with multiply relapsed follicular lymphoma k. thomson (1) follicular lymphoma is an indolent disorder, which is treatable but considered incurable with chemotherapy alone. the curative potential of allogeneic transplantation using conventional myeloablative conditioning has been demonstrated, but this approach is precluded in the majority of patients with fl, because of excessive toxicity. reduced intensity conditioning regimens are therefore being explored. this study reports the outcome of 85 consecutive patients with fl transplanted using fl udarabine, melphalan and alemtuzumab. patients were heavily pretreated, having received a median of 4 lines of prior therapy, and 27% had failed previous autologous transplantation. median patient age was 45 years and 53% received stem cells from unrelated donors. with a median follow-up of 4 years, the non-relapse mortality was 15% at 4 years (8% for sibling, 21% for unrelated donor transplants), acute gvhd grade ii-iii occurred in 14%, and the incidence of extensive chronic gvhd was only 18%. relapse risk was 26%, and this was signifi cantly reduced where mixed chimerism had been converted to full donor chimerism by the use of dli (10% vs. 36%; p = 0.03). in addition, 10/13 (77%) given dli for relapse post-transplant remitted, with 9 of these responses sustained and one responding to further dli. in these patients, cr is currently ongoing at a median of 44 months (12-74) following last dli. current pfs at 4 years was 76% for the whole cohort: 90% for those with sibling donors and 63% for those with unrelated donors. the excellent long-term survival with associated low rates of gvhd and frequency and durability of dli responses makes this an extremely encouraging strategy for the treatment and potential cure of fl. we analyzed the incidence and the risk of secondary solid tumors for 17,997 adult recipients (5,598 related bone marrow s58 (bm) recipients, 3,747 related peripheral blood (pb) recipients, 6,530 unrelated bm recipients, 2,093 unrelated cord blood (cb) recipients objectives: allogeneic hematopoietic stem cell transplantation (hsct) can severely compromise patients' health related quality of life (hrqol), but there is lack of evidencebased data in this area. this is the first report to provide a longitudinal assessment of hrqol in transplanted thalassemia children and their families also using a parent proxy-report evaluation. hrqol scores in children and parents were prospectively evaluated as well as parent healthcare satisfaction. methods: fifty-eight thalassemia patients from middle eastern countries were evaluated at baseline. twenty-eight children (median age 9.5 years) were transplanted from an hlamatched donor (25 sibling, 2 unrelated and 1 haploidentical) in 2 italian hsct centers. the pedsql 4.0 generic core scales were administered to patients and their parents both before and after (3, 6, 18 months) transplantation. parent healthcare satisfaction was assessed using the pedsql healthcare satisfaction generic module. these questionnaires were also administered to 30 conventionally treated thalassemia patients and their parents, all coming from the same geographical area. change from baseline at 3, 6 and 18 months was assessed using the wilcox signed rank test. results: two-year overall survival, thalassemia-free survival, mortality and rejection were 89%, 79%, 11% and 14%, respectively. the cumulative incidence of acute and chronic gvhd was 35% and 18%, respectively. eighteen months after transplantation, total pedsqol scores were signifi cantly higher than baseline ratings, both in child ( + 8.3, p = 0.05) and parent reports ( + 13.4, p<0.01) (figure 1 ). while physical, emotional and social functioning scores generally tended to decrease after 3 months in comparison with baseline values, they all returned to higher levels at 18 months after transplantation. final pedsqol scores were also signifi cantly higher than those obtained for conventionally treated patients (p<0.05). conclusion: this is the fi rst longitudinal study showing a trend towards a better hrqol in thalassemia children at 18 months after transplantation compared to pretreatment levels and conventionally treated patients. also parent satisfaction with cure and doctor-family communication was high. these data could help to relieve some of the uncertainty surrounding the diffi cult choice of transplantation in a chronic disease like thalassemia. allogeneic stem cell transplantation (sct) is potentially curative therapy for patients (pts) with acute leukemia and mds. sct is associated with substantial mortality during the fi rst 2 years after sct due to relapse and non-relapse mortality (nrm) whereas after 2 years survival curves often reach a plateau. the pattern of late events was reported in myeloablative conditioning (mac) but is not well defi ned in the reduced intensity (ric) setting. to explore late outcomes we analyzed sct results in a cohort of 401 pts with aml/ mds and all. pts meeting standard eligibility criteria were given mac (bucy or cy/tbi)). pts considered at excessive risk for nrm were given reduced toxicity conditioning (rtc) such as fl udarabine (f) with high-dose busulfan (bu) or treosulfan or a ric regimen of f and reduced bu or melphalan. the 5-year overall survival (os) was 35% (95ci, 30-41) and was similar with the various regimens. we identifi ed 115 pts with aml/mds (n = 96) or all (n = 19) who were alive and leukemia-free 2 years after sct from related (n = 70) or unrelated donors (n = 45), using ric (n = 34), rtc (n = 37) or mac (n = 44). median age was 51 (17-72). at the 2-year time-point, 41 pts had a history of acute gvhd and 55 had active chronic gvhd which still required immune suppressive therapy (ist) in 48. the probability of remaining alive for the next 5 years was 80% (95ci, 70-90). it was 76%, 85%, and 83%, after ric, rtc and mac, respectively (p = ns). there were 16 deaths beyond 2 years; 9 due to relapse and 7 due to nrm. nrm included 3 deaths due to solid cancers. there were 4 additional secondary cancers in pts currently alive. two pts died of myocardial infarction and two of chronic gvhd. in all, the cumulative incidence of late nrm and relapse mortality was 9% (4-20) and 11% (6-22), respectively. advanced age (>50) was the most signifi cant predicting factor for shortened os; 64% and 96% in the older and younger groups, respectively (p = 0.005 results: recipients with mbl levels <1000 ng/ml were at increased risk to suffer from one or more major infections (p = 0.002) during entire follow-up. infectious susceptibility was increased after neutrophil recovery, particularly until 24 months (hazard ratio (hr) 3.4) with sustained effects afterwards (hr 2.9) (figure 1 ). in multivariate analysis mbl serum concentrations <1000 ng/ml were independently associated with major infections after neutrophil recovery (p = 0.009) when corrected for recipient age at transplantation, sex, diagnosis, history of total body irradiation, history of splenic irradiation or splenectomy, acute or chronic graftversus-host disease, and duration of immunosuppression. in subgroup analysis occurrence of severe herpes virus infections in particular was associated with signifi cantly lower mbl levels (p = 0.02). conclusion: our fi ndings indicate that low mbl levels may predict markedly increased susceptibility to severe infections with sustained effects even late after allogeneic hsct. determinations of mbl status should therefore be included into pretransplantation risk assessment. introduction: aim of this study was to evaluate frequency and risk factors of both secondary myelodysplastic syndromes/acute leukemias (smds/al) and solid tumors in lymphoma patients treated with the high-dose (hd) squential (hds) chemotherapy with peripheral blood progenitor cell (pbpc) autograft. patients and methods: data have been collected on 1,347 lymphoma patients treated at 11 centers associated to gitil ( figure 1 ). patients received either the original or modifi ed hds regimens, as shown in figure 2 . pbpc were collected after hdcyclophosphamide (cy), and, in a subgroup, after a 2nd round of mobilization with hd-ara-c. to reduce the incidence of acute and chronic graft versus host disease (gvhd) anti-t-cell globulins (atg) have been incorporated into the preparative regimen for allogeneic stem cell transplantation (sct) from alternate donors by many centers. different atg preparations are available and little is known about the optimal dosing. therefore we conducted this retrospective registry study utilizing specifi c questionnaires to participating centers. chronic myelogenous leukemia (cml) in chronic phase has been selected as underlying disease in order to have a rather homogenous patient population. a total of 1359 patients (pts) have been analyzed. 534 pts had received no atg, 288 atg-fresenius, 122 thymoglobuline® (genzyme), 261 other in vivo t-cell depletion, mainly campath, and 154 had received in-and ex-vivo t-cell depletion, utilizing thymoglobuline® for in-vivo depletion. a cumulative dose of less than 40 mg/kg atg-fresenius or less than 10 mg/kg thymoglobuline® has been defi ned as low-dose. the median follow-up for surviving pts is 62 months (range: with no statistically difference in the different pts groups. only the use of atg-fresenius and thymoglobuline® proved to be an independent positive prognostic factor for overall survival in multivariate analysis incorporating the ebmt risk score. this was due to decreased treatment related mortality. however, any of the analyzed t-cell depletion strategies increased the risk of relapse, which did not translate into overall survival, since relapse after allo sct is manageable in cml pts. when also analyzing the dosing of atg, the use of high dose atg-fresenius was associated with the best long-term overall survival of about 70%. when comparing high dose fresenius versus all others the use of high dose fresenius had the same impact on overall survival as the ebmt risk score, indicating that the use of high dose fresenius is an independent positive prognostic factor. similar effects were not seen with high-dose thymoglobuline®. interestingly, the positive effects of atg only became obvious after 4 months after transplant suggesting no protection against acute gvhd but protection against mortality from chronic gvhd. although unrelated allogeneic sct in chronic phase cml is nowadays a rather rare indication these data nevertheless prove benefi cial effects of in vivo t-cell depletion and also emphasize, that the different preparations are not interchangeable and that the dosing is of great importance. skewed t-cell receptor repertoires in very long-term survivors after allogeneic haematopoietic stem cell transplants a. rovó (1), c. arber (1), p. fisch (2), u. siegler (1) disturbed immune reconstitution and late auto-immune like phenomena have been observed after hsct and described as "late altered immunity" in very long-term survivors. in a previous cross-sectional study on 44 long-term survivors after allogeneic hsct (>10 years) and their respective donor, we demonstrated that recipients with cgvhd and a female donor had signifi cant telomere shortening of the hematopoietic cells, and subtle signs of altered late organ function as compared to their respective donor (baerlocher et al., blood, 2009 ). we were therefore interested to learn more about these differences and analyzed in the same cohort the t cell receptor beta (tcrb) repertoire diversity by spectratyping of peripheral blood t lymphocytes. we compared by visual analysis the gaussian like distribution of the peaks from 23 vb subfamilies. pattern of each vb subfamily was classifi ed as normal or skewed. normal refers to >19 vb subfamilies with a gaussian like distribution, abnormal to >5 vb subfamilies with a non gaussian pattern. the overall complexity of the tcrb repertoire (total number of peaks of all 23 vb subfamilies) from the recipients was compared with the respective donor. the median age at hsct and at time of the study was 22.5 (6-50) and 44 (27-62) years for the recipients; 23.5 (11-45) and 45 (31-61) for the donors. the median time interval between hsct and tcrb repertoire analysis was 20 years (12-25). during follow-up 4/10 analyzed recipients had cgvhd. the median number of skewed vb subfamilies was 8.5 (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) for recipients, and 2 (0-8) for donors (p = 0.008). the median overall complexity was 149 (101-170) for recipients and 159 (138-183) for donors (p = 0.041). 8/10 analyzed recipients showed an abnormal pattern of the tcrb repertoire. all but two donors had a normal repertoire (p = 0.001); one of them suffered of 2 neoplasms after donation one patient with an abnormal tcrb repertoire ( figure 1 , upn498) had still extensive, severe cgvhd and showed the shortest telomere on the leukocytes and t-lymphocytes: 3.7 and 3.6 kb respectively, versus median 6.5 (5.4-8.1) and 5.7kb (4.8-7.4) respectively. these data indicate that t cell repertoire diversity in long term survivors after allogeneic hsct is abnormal and differs from the repertoire of their respective donors. the cause of this skewed t cell repertoire remains open but gives a formal basis for the clinically observed late altered immunity. the clinical consequences thereof need to be evaluated in the future. a second allogeneic sct was given to 119 patients (median time from sct1 11 mo, from relapse 1.8 mo), using sic (43 %) or ric (57% background: center specifi c outcome data are being discussed as useful tools for patients, the insurance industry, and politicians to assess quality of specifi c centers and to guide decision making processes. hematopoietic stem cell transplantation (hsct) requires signifi cant infrastructure and is costly. hsct therefore qualifi es as a target for such an exercise. methods: we made use of the comprehensive database of the swiss blood stem cell transplant group (sbst) to evaluate center specifi c mortality rates. nine centers reported a total of 4717 hsct, 1427 allogeneic (30.3%), 3290 autologous (69.7%) in 3808 patients between 1997 and 2008 to treat acute or chronic leukemia (29%), lymphoid neoplasia (53%), non malignant disorders (3%) or solid tumors (15%). because of mandatory reporting by legal requirement these include data on all hsct performed in switzerland over this period. data were analyzed separately for recipients of allogeneic and autologous hsct for survival and transplant related mortality (trm) at day 100 and at 10 years. results: overall survival at 10 years was 49 + 4% after allogeneic and 40 + 3% after autologous transplantation. there were signifi cant differences among centers in unadjusted analyses of survival and trm. these differences became absent or marginal, when results were adjusted for disease, year of transplant and the ebmt risk score (incorporating patient age, disease stage, time interval between diagnosis and transplantation, and for allogeneic transplants donor type, and donor recipient gender combination) in multivariate analysis. conclusions: these data indicate comparable quality among centers in switzerland. they furthermore demonstrate that comparison of crude center specifi c outcome data without adjustment for patient risk factors may be highly misleading. mandatory data collection and outcome analysis including all cases within a comprehensive quality management system may serve as a model for other cost intensive therapies to ascertain quality. . contrary, the more relevant negative aspect is the increase of workload (38%). the major modifi cation produced by the qms is to recognize the critical situations (51%) and to report in proper forms the nca is useful also to resolve them (36%). also, 67% think that internal audits are helpful to check the activities. the last question aimed to understand the relevance of qms: 58% agree that qms is part of daily activity and only for 2% "there is too much to do". the collected data show that the quality culture is, today, part of the work background and this result is recognized by 70% of lab staff, 47% of nurses and 62% of physicians. in general, the perception of qms is good but there are still areas to be improved, particularly regarding the management and resolution of critical situations. it is clear that to study the perception of qms is a tool to verify his effi cacy and continuously improve it, to make clear the diffi culties and fi nd appropriate corrective actions. so, qt plans to repeat the survey at least once at year in order to build a complete evaluation's system for work environment' quality and staff satisfaction. background: the liver is the current site for pancreatic islet transplantation (tx) but has many drawbacks due to immunological and non-immunological factors as well as important technical limitations. we asked whether pancreatic islets could be engrafted in the bone marrow (bm), an easily accessible and widely distributed transplant site that may lack the limitations seen in the liver. methods: pancreatic islets were implanted into the bm of diabetic c57bl/6 mice. islet survival, function and morphology were evaluated in comparison with the liver site. moreover a phase i/ii open label pilot study to assess the feasibility and safety of bm as alternative site for islet tx was started in patients with type 1 diabetes mellitus. results: syngeneic islets engrafted effi ciently in the bm of c57bl/6 mice rendered diabetic by streptozocin (stz) treatment. for over a year post-tx these animals showed parameters of glucose metabolism that were similar to those of non-diabetic mice. islets in bm had a higher probability to reach euglycemia than islets in liver (2.4 fold increase, p = 0.02), showed a compact morphology with a conserved ratio between alpha and beta cells, and affected bone structure only very marginally. islets in bm did not compromise hematopoietic activity, even when it was strongly induced in response to a bm aplasia-inducing infection with lymphocytic choriomeningitis virus. in humans, 3 diabetic patients received an intra bm islet infusion at the level of iliac crest without any adverse effects. in all recipients islets engrafted as demonstrated by the presence insulin producing cell in addition, preferential pairing facilitated by introduction of an extra disulfi de bond in the constant regions of the tcr chains can increase cell surface expression of the transferred tcr and decrease formation of mixed tcr dimers. another strategy based on the fact that tcrs differ in their capacity to compete for cell surface expression, is to select recipient t cells with weak competitor phenotypes. both weak and strong competitor phenotype virus-specifi c t cells were used to assess improvement of ha-1-tcr cell surface expression using the different strategies. the most marked improvement in ha-1-tcr expression and functionality was observed after tcr transfer of a cysteine modifi ed and codon optimized ha-1-tcr resulting in 70% and 35% of ha-1 tetramer positive weak and strong competitor t cells, respectively. this resulted in effi cient recognition of primary leukemic cells that endogenously process and present ha-1, independent of whether the recipient t cells were strong or weak competitor t cells. furthermore, results demonstrate that next to increased ha-1-specifi c reactivity, neoreactivity after ha-1-tcr gene transfer was dramatically reduced by cysteine modifi cation of the ha1-tcr. based on these results, cysteine modifi ed and codon optimized ha-1-tcrs will be used for the planned phase i/ii clinical trial to treat patients with relapsed hematological malignancies. reactivation of latent cytomegalovirus (cmv) infection is a frequent complication in cmv-seropositive patients after allogeneic hematopoietic stem cell transplantation (hsct). the s66 cmv-related morbidity and mortality are particularly high, if the donor is cmv-seronegative and thus, no cmv-specifi c memory t cells are being transferred from donor to recipient during transplantation. grafting non-reactive t cells of cmvseronegative donors by virus-antigen specifi c t cell receptors (tcr) may be an effi cient means to transfer cmv-specifi c t cell function into allogeneic hsct recipients. in this study, we have reprogrammed t cells of cmv-seronegative donors with human tcr recognizing the immunodominant hla-a*0201-binding cmvpp65 peptide epitope 495-503. to overcome the limitations of retroviral tcr gene transduction that hamper clinical translation, we used in vitro transcribed rna encoding cmv-specifi c tcr for electroporation of non-reactive t cells. this procedure resulted in transient surface expression of the introduced tcr for at least 3 days as demonstrated on both cd4 + and cd8 + t cells by specifi c tcrvb chain and hla tetramer staining analyses. the cmvpp65 tcr rna-transfected t cells showed hla-a*0201-restricted ifn-g secretion and cytolysis against cmvpp65 495-503 peptide-pulsed target cells and against human fi broblasts upon cmv infection. we also observed that tcr rna transfection of cd4 + t cells turned them into potent cmvpp65/hla-a*0201-specifi c t helper cells. this was demonstrated by co-incubating them with immature dendritic cells (dc), which resulted in maturation of dc only in the presence of the cmvpp65 epitope. furthermore, we transfected pure naïve and memory cd8 + t cell subsets isolated from peripheral blood of cmv-seronegative donors. although 90% of naïve cd8 + t cells were cmvpp65/hla-a*0201 tetramer positive after electroporation, they mediated only marginal lysis toward cmv-infected fi broblasts. in contrast, memory cd8 + t cells showed strong cytotoxicity against cmv-infected fi broblasts for at least 3 days upon tcr rna transfection. in summary, our data demonstrate that non-reactive human t cells can be easily redirected with cmvpp65 tcr rna, thereby gaining cmv-specifi c t cell effector function for a considerable time period. we therefore believe that cmvpp65 tcr rna has the potential to be further developed as a therapeutic "off-the-shelf" reagent for cmv-seropositive patients who undergo allogeneic hsct from cmv-seronegative donors. functional cytotoxic t lymphocytes emerge in patients with ph + acute lymphoblastic leukemia under imatinib therapy and may be exploited for adoptive t-cell therapy c. quadrelli (1) an effective specifi c immune response against adult all has so far been reported only in the allogeneic stem cell transplant setting. we have recently demonstrated that bm-resident, ifng-producing, p190bcr-abl specifi c autologous t cells may occur in ph + all patients upon treatment with high dose im, and that the presence of these cells correlates with lower minimal residual disease (mrd) values and better clinical outcome. on the basis of these preliminary data, we proceeded to assess for the presence of cytotoxic t-cells among the bm resident, bcr-abl-specifi c t-cells, and characterized the cytotoxic t-cell populations identifi ed, with the aim of optimizing a protocol for the ex-vivo expansion of p190bcr-abl-specifi c cytotoxic t lymphocytes (ctls) to be employed in protocols of t-cell therapy to control minimal residual disease after chemotherapy or hsct. p190bcr-abl-specifi c ctls were identifi ed by co-colturing bm mononuclear cells (bmmc) of ph + lla patients with bcr-abl peptide-pulsed autologous dendritic cells; the peptides employed derived from the complete spanning of p190bcr-abl fusion region. the p190bcr-abl-stimulated t cell lines generated with this protocol were mainly cd8 + /cd3 + or cd4 + /cd3 + cells with effector memory phenotype, that exerted a specifi c lytic activity against bcr-abl fusion protein. in detail, we observed a p190bcr-abl specifi c lytic activity >100 lu10/106 in 7 of the 10 patients tested (median lysis against p190-derived peptidepulsed targets: 1355 lu10/106). in 3 of the patients, for whom autologous leukaemia blasts were available, we could demonstrate a strong leukemia-directed lytic activity (median lysis: 10000 lu10/106). the lysis directed towards autologous blasts was mainly mediated by cd8 + t-cells, and was hla class irestricted. lytic activity towards autologous, mock-pulsed pha blasts, or allogenic ph-blasts was low/absent. our fi ndings demonstrated that p190bcr-abl-specifi c t cell responses may emerge in patients with ph + all under im treatment, and may be stimulated and expanded ex-vivo. whether these p190bcr-abl-specifi c t cells may have a possible therapeutic role in ph + all patients, and may be expanded from healthy donors to be employed in protocols of adoptive cell therapy after hsct remains to be addressed in future studies. background: a positive selection of leukemia associated antigen (laa)-specifi c t cells would be highly desirable to amplify the gvl effect and to decrease the risk of gvhd. wilms tumor gene 1 (wt1) and the receptor for hyaluronic acid mediated motility (rhamm) are laas recognized by cd8 + t lymphocytes. streptamers constitute a novel multimer technology to select specifi c cd8 + t cells, available at good manufacturing production (gmp) level. material and methods: both tetramers and streptamers were used to detect the frequency of hla-a2 restricted cd8 + t cells in the naïve peripheral blood (pb) from both healthy donors (hds) and aml patients. rhamm-and wt1-specifi c cd8 + t cells were further characterized for the expression of cd27, cd28, cd45ra and ccr7. in the next step, laa-specifi c cells were positive selected by macs columns after labeling with streptamers and thereafter immunophenotyped. moreover, mixed lymphocyte peptide cultures (mlpcs) were performed to enrich wt1 specifi c t cells derived from the pb of hds. rhamm and wt1 specifi c cells were subjected to carboxylfl uorescein succimidyl ester (cfse) based proliferation and cytotoxicity assays. results: 21 of 40 hds showed naïve laa specifi c t cell frequencies of 0.5 to 1.6% of all cd8 + t cells. in aml patients in complete remission signifi cantly higher frequencies of laaspecifi c t cells than in patients at diagnosis could be detected, up to 6.0% of all cd8 + t cells. these cells revealed to be cd8 + cd27-cd28 + cd45ra + ccr7-effector t cells in fl ow cytometry. after positive selection by macs columns a purity of 20-87% could be achieved for laa-specifi c t cells. after a maximum of three rounds of mlpc, only a frequency of 2-5% could be achieved, thus demonstrating the power of the streptamer technology. both tetramer-and streptamer-based selections yielded similar amounts of laa-specifi c t cells. after positive selection, these t cells preserved an effector t cell phenotype and showed active proliferation in cfse staining. streptamerselected t cells showed a trend towards higher cd28 expression thus indicating a more activated t cell status. conclusion: in summary, the streptamer technology allows to select highly pure fractions of rhamm-and wt1-specifi c effector t cells with proliferative and cytotoxic properties. in analogy to dlis specifi c for viral antigens such as cmvpp65, production of leukemia specifi c dlis is feasible on a gmp level. further leukemia antigens are currently evaluated by our group. delicate balance between regulation and stimulation at the antigen presenting cell site determines the likelihood of successful priming and survival of antigen-specifi c t-cells from the naïve donor repertoire i. jedema, t.s. lam, m. van de meent, c. hoogstraten, j.h.f. falkenburg leiden university medical center (leiden, nl) although the in-vitro induction of tumor-and pathogen-specifi c t cells from the naïve donor repertoire has been shown to be feasible, the robustness of the procedure remains limited, hampering large scale clinical application. in this study, we investigated the role of individual parameters like the frequency of antigen-specifi c precursor t cells (tprec) and regulatory t cells (treg), the number of antigen presenting cells (apc) used as stimulator cells, and the number of targeted antigens (ag) on the ability to prime, enrich and expand ag-specifi c t cells from primary immune responses in-vitro. therefore, we developed an in-vitro model system allowing the monitoring of ag-specifi c activation and proliferation of individual naïve donor t cells in the fi rst 14 days of the immune response using pkh labeling, cd137 counterstaining and quantitative fl owcytometric analysis. in this model we exposed naïve tprec to allogeneic apc in different responder/stimulator (r/s) ratios in the presence of different numbers of innocent bystander cells. optimal t cell activation was seen at specifi c tprec/s ratios between 1/1 and 1/5, irrespective of the number of innocent bystander cells. lowering the number of stimulator cells per tprec resulted in incomplete activation and proliferation, but more importantly, exposure to an excess of stimulator cells resulted in induction of activation-induced cell death (aicd) of the antigen-specifi c tprec. next, we investigated the role of treg in the induction phase of the immune response. treg were like tprec attracted to the site of the apc and their activation further increased their inhibitory potential. especially when they were at a numeric advantage, treg were capable of impairing antigen-specifi c tprec priming. increasing the number of apc in the induction phase of the immune response could overcome this negative regulation since the number of treg per apc is diminished. however, at low tprec frequencies, in case of single peptide responses, this will lead to aicd of the antigen-specifi c tprec. this may be prevented by increasing the number of antigenspecifi c tprec by simultaneous targeting of multiple antigens. in conclusion, the in-vitro generation of antigen-specifi c primary immune responses can only be successfully and reproducibly performed by creating an optimal balance at the priming site of the immune response (e.g. the apc) between the number of negative regulators (treg) and responding cells (tprec). fistulas are an invalidating, often diffi cult to treat, complication of patients with crohn's disease (cd), a disabling, chronic, relapsing infl ammatory enteropathy caused by dysregulation of the immune tolerance towards intestinal bacteria in genetically susceptible individuals. mesenchymal stromal cells (mscs) represent a promising tool in approaches of regenerative medicine. we investigated the feasibility, safety and effi cacy of local injection of autologous bone marrow (bm)-derived mscs for refractory cd fi stulas. mscs were isolated and expanded ex vivo in the presence of platelet lysate from bm of 12 patients (7 males, median age 33 yrs, range 16-59). patients received intrafi stular injection of mscs scheduled every 4 weeks (median 4 infusions) and were monitored at time of each injection, and 1, 3, 6, 12 months after the last treatment. the cytokine profi le of mscs and their ability to infl uence apoptosis of mucosal t cells obtained from involved and uninvolved colonic areas were also analyzed. msc expansion was successful in all patients and no adverse event was recorded during and up to 12 months after treatment. intrafi stular injection of mscs was effective in inducing sustained closure of fi stulas, with the appearance of regenerative tissue along the tracks. in particular, 7 patients (70%) benefi ted from complete and sustained healing of fi stula tracks, while three had partial response. all patients showed a signifi cant reduction of both cd activity index (pre-and posttreatment median values: 294 sd 49 and 99 sd 32 at 6 months after the last infusion; p < 0.001) and perianal disease activity index (pre-and post-treatment median values: 13.0 sd 2.2 and 4.5 sd 2.4 at 6 months after the last infusion; p < 0.001) reaching disease remission usually after the second procedure. the immunephenotype of circulating t lymphocytes showed progressive increase of the number of cd4 + cd25bright foxp3 + cells, which became signifi cant (p<0.01) after the second procedure and remained stable up to 6 months after the last infusion. no modifi cation of serum cytokines was observed at any time point. mscs caused a sort of block of the rates of both apoptotic and living cells when incubated with t lymphocytes from diseased mucosa, whilst critically increased the apoptotic rate when incubated with t lymphocytes from apparently healthy mucosa. local injection of autologous bm-derived mscs appeared feasible, safe and successful in treating fi stulas associated with cd. we recently showed that nk cells require two signals to initiate lysis; the fi rst primes (s1) and a second triggers (s2). we found nk resistant cell lines which express s1 but lack s2 and that resting nk cells stimulated with these cells retain the primed state even after cryopreservation. these are termed tumour activated nk cells (tank). we are conducting a clinical trial in patients with aml in cr or pr with less than 25% blasts who have exhausted conventional treatment options. tank are generated from a haploidentical family donor by overnight incubation of nk cells with lysed ctv-1 cells. tank are then purifi ed from the lysate and released at a single dose of 10 6 nk/kg with a t cell contamination of <10 4 /kg and cryopreserved. pre-infusion conditioning -fludarabine (25mg/m 2 /day) for 3 days plus 2gy tbi on day 4. eleven patients have been enrolled to date, 6 of whom have been treated. patient characteristics: pt01 -57 yr female; previous autologous transplant, treated in cr3. pt02 -71 yr male; in pr after 5-azacytidine. pt03 -50 yr male; previous allogeneic transplant, treated in cr3. pt04 -71 yr male; treated during relapsing disease and circulating blasts. pt05 -73 yr female treated in cr1 following one course of induction chemotherapy. pt06 -67 yr male; treated in cr2. results: no infusional toxicity. all patients suffered a degree of bone marrow suppression needing in-patient supportive care. aplasia ranged from 3 weeks up to 45 days. pt01 remains in cr at month + 16; pt02 achieved and remained in cr until month + 11, relapsed and treated with second tank infusion; pt03 prolonged and severe pancytopaenia requiring cd34selected stem cell rescue, achieved and remained in cr until month + 11.5; currently undergoing re-induction chemotherapy. pt04 cleared peripheral blasts for 2 months; now with progressive disease. pt05 in cr, 3 months post infusion; pt06 is non-evaluable at present. extremely prolonged nk chimerism (up to + 6 months) has been seen in all patients in the absence most cord blood transplants (cbt) are performed using cord blood units mismatched with the recipient at one or two hla loci. it is not known whether the locus at which mismatches occur infl uences outcomes. we examined the effect of locusspecifi c mismatches on outcomes of 1305 hla-mismatched (one mismatch, n = 626, two mismatches, n = 679) single cbt performed for haematological malignancies at ebmt centres from 1994 to 2008. two digit typing for hla-a and hla-b and 4 digit typing for hla-drb1 was used. patients were transplanted for all (n = 563), aml (n = 411), mds (n = 166), chronic leukaemia (n = 87), lymphoma (n = 22) or myeloma (n = 10). among the 626 patients who received a cord with one hla mismatch, the mismatch occurred at hla-a (n = 191), hla-b (n = 247) or hla-drb1(n = 188). transplant-related mortality (trm) for this group at 2 years was 37±2% and was 35±4%, 35±3% and 42±4% for hla-a, -b and -drb1 mismatches respectively. on multivariate analysis the mismatched locus had no infl uence on trm when adjusted for age, year of transplant, stage of disease, cmv status and cell dose. there was a trend for less relapse for drb1 mismatches (21±3% vs. 29±3%, p = 0.051) but this did not lead to a difference in disease-free survival (dfs) between groups (38±4% vs. 39±3%, p = ns). neutrophil engraftment and acute graft-versus-host disease (gvhd) were unaffected by locus of hla mismatch. for the 679 patients who received a cord with two hla mismatches, mismatch combinations included a + b (n = 250), b + drb1 (n = 220), a + drb1 (n = 122) or two mismatches each at either a (n = 20), b (n = 34) or drb1 (n = 33). trm for this group was 42±2% at 2 years and was 41±3% (a + b), 41±4% (b + drb1), 39±5% (a + drb1), 55±12% (a + a), 42±9% (b + b) and 58±9% (drb1 + drb1) by mismatch group. the loci at which hla mismatches occurred did not infl uence trm on multivariate analysis, nor when grouped according to mhc class (class i only vs. class ii±i). however, double drb1 mismatch was associated with more grade ii-iv acute gvhd (50±8% vs. 32±2%, rr = 1.77, p = 0.03). in addition, on multivariate analysis a + b mismatches were associated with improved probability of dfs (34±3% vs. 30±2%, hr = 0.80, p = 0.03). a cell dose cut-point could not be defi ned in either population. in summary, for cbt with one hla mismatch, the locus at which mismatch occurs does not infl uence outcomes. however, for cbt with two hla mismatches, it seems that two mismatches at class i (hla-a and -b) are preferable to other mismatch combinations. matched unrelated donor is associated with lower relapse than matched related donor in reduced-intensity conditioning transplantation: implications for graft-versus-malignancy effect v. ho, h. kim, j. aldridge, g. kao, d. liney, j. koreth, p. armand, c. cutler, j. antin, r. soiffer, e. alyea dana-farber cancer institute (boston, us) as success of reduced intensity conditioning (ric) stem cell transplantation (sct) relies on graft-vs-malignancy (gvm) effect, the extent of minor hla antigen disparity in matched related donors (mrd) vs. matched unrelated donors (mud) could impact clinical outcomes. while convention dictates that mrd is preferred over mud, does this wisdom hold in ric sct? we conducted a retrospective review of 477 (279 mud, 198 mrd) ric sct performed at our institution from 9/01 through 12/08. all received uniform ric conditioning with busulfex (3.2-6.4 mg/kg) and fludarabine (120 mg/m 2 ). gvhd prophylaxis was mostly tacrolimus/mini-methotrexate ± sirolimus (98%). sc source was pbsc (97%), marrow(3%). all donors were 10/10 allele matched at hla a, b, c, drb1, dqb1. the mud and mrd cohorts were balanced in demographics, disease (mds/aml 44% vs. 45%), high disease risk (81% vs. 82%), prior sct (30% vs. 33%), and gvhd prophylaxis. median cd34 + dose was higher in mud (8.7 vs. 7.5 × 10 6 cd34 + /kg, p = 0.003). cumulative incidence of grade ii-iv acute gvhd and 2-yr chronic gvhd were similar, 21% vs. 16% (p = 0.21), and 50% vs. 47% (p = 0.17) in mud and mrd, respectively. there was no difference in transplant related mortality (trm), time to neutrophil or platelet engraftment, or % achieving over 90% donor chimerism at days + 30 and + 100. cumulative incidence of relapse was 52% for mud, 67% for mrd, p = 0.003. with a median follow-up of 25 and 28 months, 2-yr progression free survival (pfs) was 41% for mud and 29% for mrd, p = 0.004 ( figure 1 ). 2-yr overall survival (os) was 57% for mud, 49% for mrd (p = 0.35). improvement in pfs did not translate to os benefi t because many relapses were salvaged with second sct or other therapy. in competing risks regression, mrd was associated with increased risk for relapse compared to mud (hr 1.45, p = 0.004), but no difference for trm. in cox regression, mrd, prior sct, high disease risk, and mds/ aml diagnosis were associated with inferior pfs (table 1) . donor age, cd34 + dose, and year of sct were not associated with worse survival. in ric sct, mud is associated with signifi cant improvement in relapse and pfs compared to mrd, without increased gvhd or trm. this improvement is not related to younger donor age or higher cd34 + count, suggesting that hla minor antigen disparity in mud sct may mediate stronger gvm. while further investigation is warranted, these results could have great implications for the future selection of donors for ric sct. tolerance of g-csf-administration was different in the two groups: 12/171 (7,01%) sib donors stated, that they only poorly had tolerated g-csf administration, 4/106 (3,77%) mud donors said that their tolerance to g-csf had been poor. 3/171 (1,75%) sibs indicated, that the infl uence of pbsc donation to their health status had been negative, while 2/106 (1,88%) unrelated donors said that there had been a negative infl uence of pbsc-donation on their health status. 5/171 (2,92%) sibs described their current health status as "bad", while none of the 106 muds did so. the most serious adverse effects that occurred after donation were one case of hodgkin's disease in one of the related donors and one case of acute lymphatic leukaemia in an unrelated donor. furthermore, one case of squamous cell lung carcinoma occurred in a related donor and one case of mamma carcinoma in an unrelated donor. in addition to these malignancies the following health problems could be noted in the donors: subdural haematoma a few days after donation: 1 sib, 0 mud. sarcoidosis: 1 sib, 0 mud. cardiac disorder: 2 sibs; 0 mud. essential hypertension: 4 sibs, 4 muds. discus hernia: 0 sib, 4 muds. hypothyreosis: 0 sib, 1 mud. depression: 1 sib, 1 mud. tinnitus: 0 sib, 2 muds. the presented data show that serious health problems after donation may occur and therefore global collection of these data is necessary in order to calculate the actual risks of pbsc donation in advance. evaluation, consent and care of related donors and recipients at haematopoietic stem cell transplant centres in the united states: practice patterns and potential for confl ict of interest p. anderlini (1), t. pedersen (2), d. confer (2) background: a real or perceived confl ict-of-interest may arise in the situation where the same physician is responsible for the care of two individuals whose care is interdependent. in hematopoietic stem cell transplantation (hsct) from unrelated donors facilitated by the national marrow donor program, donors and recipients are under the care of separate physicians in order to provide unbiased care and eliminate the potential for a confl ictof-interest. however, the practice patterns of evaluation and care for related donors and recipients at centers performing hsct are unknown. material and methods: a practice pattern survey of transplant centers in the united states reporting to the cibmtr was conducted by the donor health and safety committee between december 2007 and july 2008. the survey was administered as an online survey tool sent to the center medical directors. the survey focused primarily on determining the type of provider involved in medical clearance, informed consent and medical management of the donor and the providers relationship to the recipient. results: of 222 centers surveyed, 98 evaluable responses were received (40%). the median number of related donor transplants per year at responding institutions was 15 (range: 1-400); the median total number of transplants per year was 70 (5-600). as shown in the table, transplant physicians in greater than 70% of centers were involved in overlapping care of the donor and the recipient during the donor evaluation, clearance and collection phases. these patterns were similar between transplant teams caring for adult or pediatric donors and recipients. conclusion: among responding centers, it appears that medical management of recipients and their related donors by the same provider is common, and may not be viewed as a potential confl ict-of-interest. whether this potential confl ict-of-interest affects donor care is unclear, and deserves further investigation. long-term follow-up and risk factors for outcomes after related hla-identical cord blood transplantation for patients with malignancies. an analysis on behalf of eurocord-ebmt a.l. herr (1) , n. kabbara (1), p. teira (1), c. bonfi rm (2) outcomes after related hla identical bone marrow or cord blood transplantation (cbt) in children have been reported to be comparable (nejm 2000) . in order to analyze risk factors for outcomes, we studied 149 patients with malignancies who had a fi rst single unit related hla identical unmanipulated cbt and were reported to eurocord-ebmt. cbt were performed since 1990, in 25 countries and 68 centers. median fu was 6.7 years (range 7 months to 17 years). nearly all patients were children, median age being 5 years. acute leukemia (al) was the main diagnosis (n = 109) followed by myelodysplasia (n = 17), chronic myeloid leukemia (n = 12), solid tumors (n = 6), lymphomas (n = 3) and hemophagocytic lymphohistiocytosis (n = 2). according to ibmtr criteria, 39 patients had low, 71 intermediate and 39 high risk diseases. all cb donors were siblings except 2: one 2nd degree relative and one child. cb units contained a median of 5.1 × 10 7 /kg total nucleated cells (tnc) at collection and 4.1 × 10 7 /kg tnc after thawing. the conditioning regimen was myeloablative for 144 patients, 50% including tbi. the most frequent gvhd prophylaxis regimen was cyclosporine alone. methotrexate (mtx) was used for 22 patients. the cumulative incidence (ci) of neutrophil recovery was 89% at d + 60 with 135 patients achieving a neutrophil count of 0.5 × 10 9 /l after a median time of 24 days. infused tnc ≥ 4.1 × 10 7 /kg (p = 0.002) and the lack of mtx in gvhd prophylaxis (p = 0.002) were independently associated with improved neutrophil recovery. ci of acute and chronic gvhd was 11% at d + 100 and 10% at 2 years, respectively. ci of non-relapse mortality was 9% and of relapse was 46% at 5 years. remission status (p = 0.04) and the use of tbi-containing regimens (p = 0.03) were independently associated with a lower relapse rate in al patients. for cbt performed in year <2000 vs. ≥2000, disease-free survival at 5 years was 34% vs. 55% (p = 0.009), and overall survival (os) at 5 years was 44% vs. 66% (p = 0.01). transplant year ≥2000 (p = 0.005), low or intermediate risk disease status at cbt (p = 0.008) and infused tnc ≥ 4.1 × 10 7 /kg (p = 0.04) were independently associated with improved os. five-year os in al patients was associated with remission status (p = 0.001): 77% for patients in cr1, 50% in cr2, 32% in cr3 and 21% in advanced phase of the disease. improving results over time and the low toxicity of related hla-identical cbt in malignancies support banking and use of family cb units with suffi cient cell dose. in the past, the outcome of adolescents with acute lymphoblastic leukaemia (all) in 2nd complete remission (cr) given hsct s72 prophylaxis was carried out with atg or okt3. primary engraftment occurred in 85%. after reconditioning, fi nal engraftment was achieved in 98%. gvhd grade 0-1 occurred in 86%. 12% had grade ii, 4% had grade iii. chronic gvhd occurred in 4 patients. no gvhd related mortality was observed. median follow up was 5.9 years. efs at 1 year was 56% (cr1), 51% (cr2) and 50% (≥cr3); efs at 5 years was 49% (cr1), 46% (cr2) and 27% (≥cr3). median survival of patients with active disease was 0.2 years. relapse rates at 1 year were 0.32 (cr1), 0.47 (cr2) and 0.46 (≥cr3); relapse rates at 5 years were 0.32 (cr1), 0.47 (cr2) and 0.64 (≥cr3). trm at 1 year was 14%, causes of death remained viral and fungal infections, no ebv lpd occurred. conclusions: positive selection of progenitor cells or depletion of t and b cells can minimize acute and chronic gvhd in both matched unrelated and mismatched related transplantations and may prevent gvhd related mortality. lethal infections occurred in 14% of the patients probably due to the delayed recovery of t cells. despite profound depletion of donor t cells, relapse rates were acceptable and remained on a stable level after the fi rst year in patients with complete remission. thus, absence of gvhd may not necessarily be associated with high relapse rates in childhood all. apart from t cells, other effector cells like nk cells are likely to exert gvl effects and may contribute to the favorable efs of our patients. fanconi anemia (fa) is a rare disorder characterized by progressive bone marrow failure, congenital abnormalities and a predisposition to cancer. bmt is the only treatment able to restore normal hematopoiesis in this disease. here, we update the brazilian experience using only cyclophosphamide 60 mg/ kg (cy60) as a preparative regimen for the treatment of pts with fa. objective: analyze the outcome of 70 pts with fa submitted to a related bmt using cy60 mg/kg. material and methods: period: 07/1999-07/2009; gender: 32f/38m age: 5-34 ys (m: 10,5). type of donors: matched siblings donors (msd): 60 pts; other related donors (ord): 10 pts. 68 pts were in aplastic phase while 2 had mds. all pts received a preparative regimen with cy60 mg/kg and gvhd prophylaxis with cyclosporine and methotrexate. statistical analysis was performed using spss. overall survival (os) was estimated using the kaplan-meier method. multivariate analysis was performed by cox method. results: 60 pts are alive between 135-3530 days (m: 1663) after bmt. os: 85,5% at 5 ys. pts transplanted below the age of 10 (37pts) had an os of 97,1% at 5 ys. there was no statistical difference between transplants performed in curitiba(56pts) and other bmt centers (86,1% × 83,2%). 2 pts died before d + 28 and were not evaluable for engraftment. only one pt had primary graft failure and he is alive and well 5 ys after the 3rd transplant. late rejection occurred in 6 pts (2 pts with mds, 2 pts with ord, 2 pts with msd) at a median of 234 days after bmt. 2/6 pts are alive and well after a 2nd bmt. cumulative incidence of rejection at 1 y was 10%. pts receiving > 25 transfusions had a lower survival when compared to those less transfused (91% × 62% p = 0,03). mucositis grade ii-iii occurred in the majority of pts;13/67 pts developed acute-gvhd grade ii-iv has been reported to be worse than that of children. in order to evaluate whether in recent years the outcome of adolescents has become more similar to that of children, we studied paediatric patients (age < 18 years at transplantation) reported to the aieop-hsct group registry who received allogeneic hsct for all in 2nd cr in a paediatric institution between 01/1990 and 12/2007. a total of 400 patients (64% males and 36% females) were anallyzed. 339 patients were children (<14 years old), whereas the remaining 61 patients where adolescents (14-18 years old). median age at hsct was 8.3 and 16.1 years, for children and adolescents, respectively. a mfd was employed in 50% of children and in 58% of adolescents; the remaining patients having received a mud hsct. conditioning regimen included tbi in 90% of patients, in most cases associated with thiotepa and cyclophosphamide. cyclosporine (csa) alone was used for gvhd prophylaxis in 88% of patients transplanted from a mfd, whereas the combination of csa, mtx and anti-thymocyte globulin (atg, 3.75 mg/kg from day -4 to day -2) was employed in 77% of patients given mud hsct. no difference between children and adolescents was observed for patient-and transplant-related variables, with the exception of the infused cell dose which was greater in children. as of october 2009, the probability of developing grade ii-iv, grade iii-iv acute gvhd or chronic gvhd was 54%, 19% and 32% for children, and 49%, 14% and 43% for adolescents (p = ns). eight-year probability of efs,trm and ri were 53%, 22% and 25% for children and 55%, 23% and 22% for adolescents, respectively (p = ns). univariate analysis showed that s3 + s4 bfm classes at relapse (p<0.0001), recipient male gender (p = 0.01), mud hsct (p = 0.02), grade 0 or iv acute gvhd (p<0.0001) were poor-risk factors for efs. only bfm class at relapse and acute gvhd (grade 0 or iv) maintained their prognostic signifi cance in multivariate analysis. adolescents and children <14 years of age, treated in paediatric institutions, had the same outcome. multivariate analysis confi rmed the gvl favourable effect in patients with grade i-iii acute gvhd.results obtained with mud hsct were comparable to those achieved with mfd hsct. adolescents should be referred for transplantation to treatment teams that have experience in the management of childhood all. long-term survival and relapse rate after transplantation of highly t and b cell-depleted stem cells from alternative donors in paediatric patients with acute lymphatic leukaemia p. lang (1) we present long term results in 68 children with all who received highly t and b cell depleted stem cells from matched unrelated (n = 17) or full haplotype mismatched related donors (n = 51) at our institution. our aim was to minimize gvhd and to avoid ebv lpd in both matched and mismatched transplantations. remission status was: cr1, n = 18; cr2, n = 23; ≥cr3, n = 12; non remission or second transplant, n = 15. graft manipulation was carried out with microbeads and the clinimacs tm device (indirect depletion of t and b cells with cd34 + positive selection (n = 50); or direct depletion with anti-cd3/anti-cd19 coated microbeads (n = 18)). t and b cell counts were reduced for 4-5 log with median numbers of residual t cells of 15 000/kg bw (cd34 + selection) and 49 000/kg bw (cd3/19 depletion). no pharmacological immune suppression was given after positive selection, whereas patients with cd3/19 depletion received mmf. the conditioning regimens were either tbi or bu based (n = 50) or a toxicity reduced protocol (flud, tt, mel) was used (n = 18). rejection more than 500 candidate donors were evaluated at our institution (median age 43 y, r 16-79 y; 185 donors ≥50 y; male 283). 460 donors were evaluated with marrow smear and cytogenetic test, 328/460 were eligible for donation (4 bone marrow harvest, 324 peripheral blood gcsf mobilization), 10 proceeded to stem cell donation without marrow evaluation. 393 out of 460 marrow were morphologically normal (85,43%), 58 (12,63% -median age 54 y, 31 donors >50 y) presented abnormalities, 9 (1,96%) were not evaluable. we registered 19 cases of plasma cells hyperplasia (10 not eligible to donation) 7 (1,52%, 5 donors > 50 y) presented abnormalities, 1 was not evaluable. we registered 5 cases of chromosome y deletion (4 not eligible to donation), 1 of pericentric inversion of chromosome 3 (p22;q29 additional follow-up will give us more important information on safety of hsc donation procedure. o373 impact of kir-ligand mismatches and kir genotypes on the outcome of patients receiving unrelated donor stem cell transplantation for lymphoid malignancies m. morelli (1), a. bermema (1) all pts received rabbit anti-thymocyte globulin for in-vivo t cell depletion. nineteen donor/recipient pairs were matched at molecular level for the hla-a, b, c, drb1, dqb1 loci and 30 were mismatched (27 at class i, 6 at class ii). the majority of pts had chemosensitive disease (n = 38, 78%). the median follow-up was 24 months (range 3-65). we studied kir genotypes using the kir-typing kit (miltenyi biotec) and hla-cw kir ligand using high-resolution molecular techniques. results: twenty-one pts were c1/c2 heterozygous, 16 were c1/c1 and 12 were c2/c2 homozygous. in the combination patient c1 absent/donor kir2dl2/kir2dl3 present (n = 12, 25%), we observed no trm and only 3 cases of acute gvhd (agvhd) as compared to other combinations (n = 37) in which 3 cases of trm and 11 cases of agvhd were observed. this combination was associated with a trend of better cumulative incidence of trm, os, and pfs os: 61% vs 67%) or in the recipient (n = 36, 74%) showed no signifi cant association with a better os or pfs. sibling donors (n = 8), haploidentical cd3/cd19 depleted grafts (n = 3), cd34 + selected graft with okt3 (1) hlh (5), leukocyte adhesion defi ciency (4), chronic granulomatous disease (3), severe immune dysregulation (3), other pid (15) mfd (13) hla matched 7-10/10). 16 of 45 were cords (7-10/10 hla matched). follow up is 2-59 months (median 16 months) 13 children died: 6/40 in the treosulfan/fl udarabine group, 7/30 in the treosulfan/cyclophosphamide group -hlh disease day-1, graft rejection and cmv, infection (4), gvhd, gvhd +infection, gvhd + cerebral infarcts, cerebral haemorrhage, pneumonitis, vod and mds/ aml. skin toxicity was common. vod occurred in 2 children in combination with cyclophosphamide and both had enterovirus in the gut. 2 patients rejected (mhc ii defi ciency successfully retransplanted autoimmunity after bmt in primary immunodefi ciency diseases: single-centre report of 184 children had aml (10 in cr1 at high risk, 10 in ≥cr2 and 2 in relapse), 5 had all (4 in cr1; 1 in relapse) and 1 had high grade nhl in relapse. conditioning was: 8gy single fraction tbi, thiotepa (4 mg/kg × 2), fl udarabine (40 mg/m² × 5), cyclophosphamide (35 mg/kg × 2) no gvhd developed in 24/26 patients, 2 developed ≥ grade ii gvhd. ten patients died (3 vod, 2 fungal pneumonia, 1 bacterial sepsis, 1 cns aspergillosis, 1 systemic toxoplasmosis, 1 adenoviral infection, 1 mof). cd4 and cd8 counts reached, respectively, 50/μl medianly on days 34 (range 19-63 days) and 24 (range 15-87)of cmv reactivation were signifi cantly fewer than after our "standard haplo" transplants. in kir ligand-mismatched transplants, speed of nk cell reconstitution/maturation and size of donor vs. recipient alloreactive nk cell repertoires were preserved. in conclusion, in the setting of haploidentical transplantation infusion of tregs makes administration of a high dose of t cells feasible for the fi rst time donor nkt cells and outcome following hla-identical peripheral blood stem cell transplantation d. nachbaur oral session 15: myelodysplasia / regulatory issues and quality management o350 monosomal karyotype, higher blast count and ex vivo t-cell depletion predict poor outcome after allogeneic stem cell transplantation for mds/saml patients with chromosome 7 abnormalities m. van gelder, j. schetelig, l. volin, j. maertens, g. socié, e. petersen, h. thomssen, a. biezen, r. brand, t. de witte, n background: high-risk mds/saml patients with a chromosome 7 abnormality have a poor prognosis with conventional therapies. these patients usually proceed to allogeneic sct (allo-sct). data on the effect of this approach are scarce. objective. description of outcome and identifi cation of risk factors of allosct in mds/saml patients with a chromosome 7 abnormality. methods: from the ebmt database 277 patients with mds/saml having any chromosome 7 abnormalities (median age 45 years) who underwent fi rst allosct between 1981 and november 2006 were identifi ed. stem cells from related donors were transplanted in 191 patients (177 hla-identical) while 85 received unrelated grafts. bone marrow was used as stem cell source in 148 patients. standard conditioning was applied in 222 patients. sixty-three patients fulfi lled the criterion for complex cytogenetic abnormalities (ca). a monosomal karyotype (mk), defi ned as at least one autosomal monosomy and at least one other chromosomal abnormality (breems da et al., jco 2008; 26:4791) , was present in 68 patients, of whom 24 did not have ca. results: median follow-up (fu) of patients alive at last fu was 5 years (range 0-18 years). estimate 5-year overall (os), and relapse-free (rfs) survival was 28 ± 6% and 26 ± 6% respectively. the relapse rate at 3, 12 and 60 months was 9 ± 3%, 29 ± 5% and 39 ± 6% resp. non-relapse mortality at 3, 12 and 60 months was 21 ± 5%, 36 ± 6% and 40 ± 6% resp. in multivariate models including age, mk, ca, % blasts at allosct, donor type, sex match, conditioning regimen, t-cell depletion and year of allosct three factors remained statistically signifi cant predictors for poor outcome: mk, ≥5% blasts at allosct and ex vivo tcd (adjusted hr for os resp. 1.65 [1.15 -2. 35 95%ci], 1.67 [1.14 -2. 46 95%ci] and 1.83 [1.21 -2.76 95% ci] resp.). when patients with ex vivo tcd were excluded, 5-year os in the remaining 97 mds/saml patients with any chromosome 7 abnormality and <5% blasts at allosct with or without mk was 8 ± 14% and 53 ± 12% resp. and for the 91 patients with ≥5% blasts 5-year os was 9 ± 12% and 29 ± 11% resp (see figure) . conclusion: this large study on outcome of allosct for patients with mds/saml and any chromosome 7 abnormality shows that long-term survival can be achieved in patients without the poor risk factors mk, ≥5% blasts at allosct and ex vivo tcd. for patients with mk new approaches that tackle the high and early relapse rate are warranted. bone marrow fi brosis on outcomes of patients with mds/saml undergoing allogeneic stem cell transplantation n. kröger, t. zabelina, a. van biezen, r. brand, t. bone marrow fi brosis has an important impact on the prognosis of patients with mds. we evaluate the impact of bone marrow fi brosis in 721 patients who underwent allogeneic hematopoietic stem cell transplantation (hsct) for mds/aml and were reported to the ebmt. no fi brosis was noted in 483 pts, mild or moderate fi brosis in 199 pts and severe fi brosis in 39 pts. diagnosis in the none, mild/moderate and severe fi brosis group were ra/rars (36%, 39% and 30%), raeb (43%,43% and 48%) and raeb-t (21%, 18% and 20%). stem cell source were from related (63%, 63% and 62%) or unrelated (36%, 36% and 38%) donors. leukocyte engraftment (>1.0 × 10 6 /l) was observed after a median of 16, 17 and 19,5 days for the days for the none, mild/moderate and severe fi brosis group, respectively (p = 0.002). there was a trend for more graft failure in severe fi brosis group (10%, p = 0.07). in a multivariate analysis bone marrow fi brosis did not infl uence non-relapse mortality, but severe fi brosis signifi cantly infl uenced risk of relapse (hr 5.7, p = 0.02), resulting in a reduced disease-free (hr 5.9, p = 0.01) and overall survival (hr 7.5, p = 0.006). other factors for reduced survival were advanced disease at transplant (hr 14.9, p = 0.005), non-cr before transplant (15.8, p = 0.001) and hla-mismatch (hr 5.3, p = 0.02). in summary, apart of the disease status, no complete remission at transplant, and hla mismatch transplantation, severe bone marrow fi brosis is an independent prognostic factor for disease-free and overall survival for mds patients undergoing allogeneic stem cell transplantation. management and outcome of myelodysplastic syndrome (mds) and secondary acute myeloid leukaemia relapsing after allogeneic stem cell transplantation -a retrospective analysis on 888 patients by the mds subcommittee of the ebmt chronic leukaemia working pa c. schmid, a. van biezen, r. brand, j. finke, l. volin, a. vitek, d. selleslag, d. heim, p. van dem borne, a. ganser, m. mohty, m. boogaerts, t. de witte, n the aim of this study was to obtain reliable data on management and outcome of relapse after allogeneic sct for mds and saml in adults. median age of 888 patients was 51 years. patients (pts.) had been transplanted for ra/rars (8%), raeb (19%), raeb-t (13%) and saml (60%), either previously untreated (20%), in cr (44%), relapse/progression (13%) or primarily refractory (24%). standard (sic) and reduced intensity (ric) transplants were performed in 59% and 41%, donors were id siblings (58%), mud or mm relatives (42%), and 5 syngeneic twins. median time from sct to relapse was 6 mo . median follow up from relapse of 188 survivors was 6.7 mo. overall survival (os) at 1, 2 and 4 years was 24%, 16% and 10%. in a cox regression model, the mds subtype at time of transplant (ra/rars vs. raeb [hr 1, 524, ci 1, 390, ci 0, 071 vs. saml hr = 2, 209, ci 1, 114, p<0.001) , and the interval from sct to relapse (1st vs. 2nd [hr = 0, 770, ci 0, 969] vs. 3rd [hr = 0, 609, ci 0, 768] vs. 4th quartile [hr = 0,404, ci: 0,318-0,513], p<0.001), were associated with survival. in 342 pts., data on the management of relapse were available. 223 pts had received dli. among them, median time from sct to relapse was 6.5 mo, median time from relapse to dli1 was 1 mo. 149, 42 and 32 patients received 1, 2, and 3 dli. median os from relapse was 7.6 mo. again, remission duration after sct (p<.001) and less advanced disease at time of sct (p = .038) were associated with better os. constitutional variability in genes involved in innate immunity (irf-3, hamp, ptx3) and in cell proliferation (atbf1 and nfat5) infl uences disease free survival after allogeneic stem cell transplantation b. martín-antonio, i. alvarez, f. márquez-malaver, p. trujillo, m. carmona, j. falantes, i. espigado, á. urbano-ispizua hospital universitario virgen del rocío (seville, es) genes involved in innate immunity and in regulation of cell proliferation may play an important role in infections and modulating the intensity of the infl ammatory response after allogeneic stem cell transplantation (allo-sct). thus, genetic variability in donor and recipient in these genes might be an important factor infl uencing the outcome of allo-sct. we have studied the potential infl uence of 15 single nucleotide polymorphisms (snps) in donor and recipient in genes of innate immunity (irf-3, hamp, ptx3, hbd2) and regulation of cell proliferation (atbf1, nfat5, akt2, nm1, cd151, tcirg1, sh3kbp1) on clinical outcomes after allo-sct, specifi cally on the incidence of acute gvhd, transplant related mortality (trm), relapse, and disease free survival (dfs). study population consisted of 106 donor-patient pairs undergoing hla identical sibling allo-sct in a single institution. patient median age was 38 years (range, 5-66). 42% of the patients were in advanced phase of disease. cumulative incidence for acute gvhd, trm, relapse and dfs was computed with the cmprsk package and with kaplan-meier. patient irf3 rs2304205 aa, donor atbf1 rs719327 aa and patient akt2 rs12460555 cc dominant genotypes, were associated with a higher incidence of relapse (p = 0.02, p = 0.04 and p = 0.002, respectively) and lower dfs (p = 0.02, p = 0.04 and p = 0.009, respectively). all of them retained signifi cance at multivariate analysis. variant rs719327 aa in atbf1 showed the same prognostic values when present in donor or in patient (relapse: 55% vs. 34% and dfs: 26% vs. 46%). when it was present both in donor and patient, the differences were more prominent (relapse: 69% vs. 33%, p = 0.003 and dfs: 15% vs. 45%, p = 0.01). donor hamp rs7251342 ag genotype and donor nfat5 rs6499244 aa dominant genotype were associated with a lower and a higher incidence of trm (p = 0.007 and p = 0.02, respectively) infl uencing in a higher and lower dfs (p = 0.04, p = 0.02, respectively). they retained its signifi cance at multivariate analysis. nfat5 is necessary for optimal t cell development and rs6499244 aa variant showed the highest mrna expression. interestingly, none of the 25 patients with a donor carrying ptx3 rs18040680 aa recessive genotype had trm but showed a higher tendency in the incidence of relapse (61%). in conclusion, genetic variability in innate immunity and in cell proliferation has a strong infl uence on the clinical outcome of allo-sct, which might be important when choosing allo-sct protocols. the factors to predict adverse events occurred within 30 days of post-peripheral blood stem cell donation at family donors y. kodera, s. kim, k. nagafuji, m. hino, k. miyamura, r. suzuki, a. tamakoshi, m . fukushima on behalf of the japan society for hematopoietic cell transplantationbackground: it has become obvious that certain adverse events might occurre at peripheral blood stem cell (pbsc) donors during or after the donation process. it is therefor crucial that certain factors which can predict the occurrence of such adverse events at normal donor are cralifi ed to prevent the adverse events. the jshct has continued the nation-wide consecutive follow up of pbsc family donors and in this project, the day 30 report of post donation was included. this time, we present the outcome of the day 30 report analysis. materials and methods: among 3,264 pbsc family donors who were consecutively pre-registered to jshct donor center between april 2000 and march 2005, 2,873 day 30 reports were obtained and subjected for analysis. the relationship between family donors' 1) gender, 2) age, 3) body-weight, 4) daily and 5) total dose of granulocyte-colony stimulating factor (g-csf), 6) current and 7) past health conditions and the occurrence of 1) thrombocytopenia, 2) prolongation of hospitalized period, 3) any adverse events (bone pain, fatigue, head ache, insomunia, anorexia, nausea, vomiting, splenomegary) as well as the mobilized cd34 + cell numbers were statistically examined. results: risk factors for thrombocytopenia were higher total dose of g-csf and older age. risk factors for prolongation of hospitalized period were higher total dose of g-csf, any present illness, older age and low body-weight. the risk factor for bone pain was higher body-weight. the risk factors for fatigue were female and lower body-weight. the risk factors for insomnia were older age and female and the risk factors for anorexia were female and low body-weight. predictive factors for lower cd34 + cell mobilization/donor's body-weight were older age and higher total g-csf administration, and predictive factors for higher cd34 + cell mobilization were male and younger age. discussion: it was revealed that certain adverse events which occurred within 30 days of post-donation and cd34 + cell numbers to be mobilized could be predicted by utilizing the basic information of donors and of pbsc mobilization/harvest process. to predict them, to notify them to donors and to prepare for possible events will contribute to keep pbsc donor's safety and trust. background and aim: allogeneic hematopoietic stem cell transplantation (hsct) has become an established therapy and the numbers of such procedures increase year by year. the risk for while 17/66 pts developed chronic-gvhd (extensive: 11 pts). in the multivariate analysis only acute-gvhd grade ii-iv and extensive chronic-gvhd were associated with a lower os. 3 pts developed oral squamous cell carcinoma (scc) between 3-5 ys after bmt (all had c-gvhd) and 1pt died. 10pts died between 12-2034 days after bmt. major causes of death were related to rejection or gvhd. trm at 100 days: 4% and at 1 y: 8%. conclusions: this regimen was well tolerated and had an excellent survival especially for pts below the age of 10. long term follow up is still needed to determine the incidence of cancer in this group of pts. survey of outcomes of unrelated cord blood transplant in patients with haemoglobinopathies: a retrospective study on behalf of cibmtr, nycb and eurocord a. ruggeri (1) allogeneic hematopoietic cell transplantation (hct) from hla-identical donors is curative in patients with hemoglobinopathies. in order to extend the availability of hct, unrelated cord blood transplantation (ucbt) has been investigated as an alternative. between 1996 and 2009, 51 patients receiving a single ucbt for hemoglobinopathy were reported to the eurocord and cibmtr registries. thirty-four had thalassemia major (thal) and 17 had sickle cell disease (scd). all thal patients were transfusion-dependent with a median time from diagnosis to ucbt of 26 months and 12 of 17 patients with scd had a history of stroke. median age at ucbt was 5 years and median follow-up was 2 years. cord units were matched at 6 of 6 (15%), 5 of 6 (34%) and 4 of 6 (51%) hla loci (antigen-level for class1, allele-level for class 2). median infused cell dose was 4.6 × 10 7 /kg (1.5-13). thirty-nine patients received a myeloablative conditioning regimen with busulfan (bu) and cyclophosphamide (n = 33) or bu with other agents (n = 6). reduced intensity conditioning regimens (ric) (n = 12) were fl udarabine-based with bu < 8 mg/kg or melphalan < 150 mg/m². cumulative incidence (ci) of neutrophil recovery at 60d was 72±6%, with 35 of 51 patients reaching neutrophil recovery at a median time of 22 days. higher cell dose (>4.6 × 10 7 /kg) (80% vs. 54%, p = 0.004) and ucbt performed after 2004 (75% vs. 61%, p = 0.001) were associated with improved neutrophil recovery. day-100 chimerism analysis was available for 47 patients: 19 patients had complete donor chimerism, 4 mixed chimerism and 24 autologous recovery. among 16 patients who did not achieve neutrophil recovery, 5 had available data on subsequent treatment. two had an autologous back-up and one received a second ucbt which engrafted. ci of grade ii-iv acute-graft versus-host disease (gvhd) was 23% and 9 patients had chronic gvhd. the 2-year probability of overall survival was 77%. thirty-eight patients (22 thal, 16 scd) are alive, 16 with full donor chimerism (8 thal, 8 scd). of the 13 deaths, 5 occurred prior to day-100, mainly due to infections. no deaths due to gvhd were reported. ten of 12 patients who received ric are alive, 4 with donor engraftment. despite the small number of subjects, these results show a particularly high risk of graft failure after ucbt for hemoglobinopathy. tnc dose plays a key role in engraftment. novel approaches modifying the conditioning regimen and/or increasing cell dose in prospective clinical trials are needed. graft rejection and second haematopoietic cell transplantation in patients with thalassaemia major s. santarone, e. di bartolomeo, p. bavaro, p. di carlo, p. olioso, g. papalinetti, p. di bartolomeo bmt center (pescara, it) graft rejection for patients with thalassemia major (tm) receiving haematopoietic cell transplantation (hct) includes early graft failure (gf) (no evidence of engraftment), late gf (lost of engraftment), and recurrence of tm. we examined our series of 126 consecutive patients aged 1 to 29 years (median 10 years) with tm who were transplanted either from hla-identical related (n = 123) or unrelated (n = 3) donor. conditioning regimen consisted of busulfan (bu) and cyclophosphamide (cy). graftversus-host disease (gvhd) prophylaxis included cyclosporine (csa) and methotrexate (mtx). all patients received bone marrow (bm) cells. the 10-years overall cumulative incidence of graft rejection was 9% + 0.07. the impact of pre-transplant patient risk factors (age, gender, number of transfusions, ferritin, splenectomy, ast, alt, hepatomegaly, hbv and hcv infection, liver fi brosis, cmv serology), donor characteristics (age, heterozygous state) and hct regimens (dose of bu, gvhd prophylaxis, bm dose) was studied in univariate analysis. no factor was univariately associated with signifi cantly increased probability of graft rejection. eleven events occurred: 4 early gf, 2 late gf, and 5 recurrences of tm. patients with either early or late gf underwent an urgent second hct, whereas 3 of 5 patients with recurrence of tm choose to be submitted to second hct. at all, 9 patients received a second hct from the same donor. their median age was 14 years (4-19). conditioning regimen for second hct was immunosuppressive for 5 patients (atg in addition to fl udarabine (flu) or cy) and myeloablative for 4 patients (bucy in 1 and thiotepa (th), treosulphan (treo) and flu in 3). gvhd prophylaxis consisted of csa alone for 5 patients and csa /mtx for 4 patients. six patients were given bm cells and 3 received peripheral blood stem cells. results: 2 patients died from hct related causes (heart failure at day 29 and acute gvhd at day 83, respectively). seven patients (78%) are living. one patient had no sign of engraftment and is now living with recurrence of tm following a third hct with double unrelated cord blood cells. tm recurred in an other patient at 3 months post-second hct. this patient is now living with transfusion therapy. five patients (56%), including the 3 patients transplanted with th-treo-flu regimen, are cured with a median follow up of 3 years (0,2-21). this study shows that the incidence of graft rejection following hct for patients with tm is low and second transplant is successful in an high proportion of patients. treosulfan is a bi-functional alkylating agent which causes less vod than busulphan and does not require anticonvulsant prophylaxis and drug monitoring. we report the use of treosulfan in 70 children undergoing hct for primary immunodeficiency (pid) at two supraregional transplant centres in the uk. children received 42 g/m² or 36 g/m² treosulfan in combination with either cyclophosphamide 200 mg/kg (30) or fl udarabine autoimmunity is a complication after haematopoietic stem cell transplantation in patients with primary immunodefi ciencies (pid) that sometime occurs when bmt is performed from alternative donors with extensive stem cell manipulation. the fact that only purifi ed stem cells (cd34 positive cells) are injected could lead to a slower and incomplete post-transplant immunological reconstitution. since the majority of children treated in our unit received haploidentical cd34 positively selected grafts, we retrospectively analysed the population of pid patients grafted in our institution between 1990 and 2008. of the 114 affected by severe combined immunodefi ciency (scid), 70 received an haploidentical transplant, while the others received a matched unrelated or a hla-identical transplant. 94 patients were affected by inborn errors; 15 received an haploidentical transplant, the others received a matched unrelated or a hla-identical transplant.we analysed the incidence of autoimmunity in these two groups of patients. out of 184 children, 17 experienced autoimmune symptoms after hsct. among this group, 11 had a diagnosis of scid, 2 of leaky scid, 2 of omenn's syndrome, one of chediak-higashi and one of wiskott-aldrich syndrome. autoimmune symptoms included autoimmune haemolytic anaemia (8), autoimmune hypotiroidism (6 patients), cutaneous autoimmunity (2), vasculitis (1) and chronic bronchoreactivity. all patients except for 2 received a myeloablative conditioning therapy. 10 of them received an haploidentical bmt and 7 a mud or a hla-identical bmt. no correlation was found analysing onset of autoimmunity and split chimerism in all groups of patients nor we could fi nd a correlation with immunsuppressive therapy. in conclusion we found a low incidence of autoimmunity despite the fact that the majority of our patients received a cd34 positive selected graft with an even distribution of the complication independently from the type of donor or stem cell manipulation procedure. introduction: there is still a discussion about the best bm harvesting method to obtain enough progenitor cells during the bone marrow (bm) harvesting procedure. the technique used in most bmt centers is the multiple aspiration of a small (2 ml) amount of bm from the iliac crest, sternum or tibia. this is proposed to minimize the dilution with peripheral blood. others are using few large amount aspirations. this procedure was not evaluated in children so far. to fi nd out possible differences in graft composition between this two methods and to evaluate the feasibility in children we initiated the following prospective study. patients and methods: 20 bm harvestings were done in 18 patients (median age 8,78, (2,48 -16,6 y), f 5, m 13). all patients were transplanted in our bm transplantation unit. there were 7 bm harvests for autologous bmt and 13 for allogeneic bmt. the autologous donors were median age 6,93 (2,5-17 y), f 1, m 5), the allogeneic donors were median age 19,75 (6,45-50 y), f 8, m 5). the amount of harvested bm was median 900 ml (440-2380 ml) and median 37 ml/kg bw of the recipient (20-55 ml/kg). the method a was defi ned as an aspiration of 2 ml bone marrow at most before the position of the needle was changed. the method b was defi ned as an aspiration of 100 ml at least before the location of the needle was changed. the bm was collected and analyzed among others for mnc, cd 34 positive progenitor cells, cfu, and t-cells. two operators carried out the bm harvest. one began with method a and changed in the second part to method b. the second operateur carried out the procedure in the opposite way. both operators collected the same amounts for each harvesting method so long that the amounts of bm harvested in one method was identically with the second. results: we found no statistically signifi cant difference between method a and b regarding the content of mnc, cd3 + t-cells, and cfu (mnc/ml 824572 a versus 725000 b wilcoxon test p = 0.7; mnc/kg 3.1 10e07 a versus 2.9 10e07, p = 0.3; cd3/ml 162500 a versus 300000, p = 0.3; cfu/10e05 mnc 1678 a versus 1315 b, p = 0.09), but a clinically not relevant but statistically signifi cant difference between cd34 positive cells (cd34/kg 2.62 a versus 2.09 b, p = 0.045). all transplanted patients showed a regular engraftment. conclusion: bm harvest by large amount few punctures method (b) is as suffi cient as the common used small amount frequent punctures method (a), and could be therefore used equally. haploidentical transplantation, with extensive t cell depletion to prevent gvhd, is associated with a high incidence of infectionrelated deaths. the key challenge is to improve immune recovery with allogeneic donor t cells without triggering gvhd. as t regulatory cells (tregs) controlled gvhd in preclinical studies, background: natural killer t (nkt) cells represent a small but signifi cant lymphocyte population which have been demonstrated to play a regulatory role in autoimmune disease as well as in gvhd/gvl effects. methods: the number of cd3 + cd56 + nkt cells in the graft was retrospectively correlated with clinical outcome of fi fty-eight patients receiving a fi rst allogeneic peripheral blood stem cell transplant (myeloablative conditioning, n = 31; reduced-intensity conditioning, n = 27) from their hla-identical sibling donors between dec 2004 and jul 2009. results: a median number of 0.15 (range, 0.01-0.57) × 10 8 /kg cd3 + cd56 + nkt cells was transplanted with the graft. nkt cells within the graft signifi cantly correlated with the number of cd3 + t cells, cd56 + nk cells, and mnc (p<0.05). overall survival for the entire cohort at two years was 55% (41%-69%, 95% ci) with no difference between pts. receiving a high (>0.15 × 10 8 /kg) or low (<0.15 × 10 8 /kg) nkt cells. the cumulative relapse incidence for the entire cohort was 40%, with a trend for a lower relapse incidence in pts. receiving a low vs. high nkt cell dose (32% vs. 47%). the nonrelapse mortality was 19% showing a trend for a lower nrm in patients receiving a high nkt cell dose (12% vs. 27%). the cumulative incidence of agvhd ii-iv was 38% for the entire cohort. the incidence of agvhd ii-iv was lower in pts. receiving a high nkt cell dose (30% vs. 45%). the cumulative incidence of cgvhd in pts. at risk was 48%, with no difference between pts. receiving low or high nkt cell numbers. by multivariate cox analysis including cd3, nk, nkt, treg, cd34 cell number, recipient age, and time interval between diagnosis and sct as numerical variables, and risk category by the underlying disease (standard risk vs. high risk), sex mismatch (male recipient/female donor vs. other combinations), and myeloablative vs. reducedintensity conditioning as categorical variables the most powerful predictive parameters for survival were standard vs. high-risk disease (p = 0.01), a high nk (p = 0.02) and a low nkt cell dose (p = 0.05). the most powerful predictive parameters for relapse were high risk disease (p = 0.01), a low nk (p = 0.02) and a high nkt cell dose (p = 0.08). discussion: these data indicate that in the setting of peripheral blood stem cell transplantation from hla-identical sibling donors a higher number of nkt cells in the graft might increase the risk of relapse by lowering the incidence of acute gvhd. objectives: adoptive transfer of nk cells with or without previous allogeneic progenitor cell transplantation may represent a promising therapeutic option in patients with hematological or oncological diseases. different nk cell isolation strategies have been pursued. here, two different methods were compared with respect to recovery, immunophenotype and cytotoxic capacity of purifi ed nk cells. methods: nk cells of healthy donors were isolated from a lrs chamber of a platelet apheresis. cells were enriched by (i) cd56 positive selection of t cell (cd3) depleted lymphocyte fractions (method i, mi) or (ii) depletion of non-nk cells (untouched protocol, method ii, mii) by magnet-activated cell sorting (macs). recovery of nk cells obtained by m i or ii were compared. nk cells derived by mi were activated by stimulation with interleukin (il)-2 (100u/ml) or il-15 (10ng/ml) overnight and analyzed for cytotoxic activity. for expansion of nk cells cd2/cd335 antibody coated macsibeads (miltenyi) were used as artifi cial stimulators in il-2 supplemented media (500u/ml) in a two weeks culture. finally, immunophenotype of effector cells and killing of target cells were assessed. results: median nk cell recovery was 35.9% (n = 4) and independent of strategy of enrichment (mi versus mii). overnight activation of nk cells led to a 2-fold enhanced killing of k562 cells. nk cell expansion led to a 100-fold increase in numbers of functional active nk cells. expanded nk cells showed characteristics of activated nk cells as indicated by the induced expression of nkp44 (cd336). surface expression of tac-tile (cd96), a marker for certain activated immune cells, was found with cd96 antibody th-111 elevated on bead-expanded nk cells in comparison to nk cells cultured only in il-2 conditioned media, which may enhance reactivity of expanded nk cells against the ligand of cd96, namely polio virus receptor expressing tumours. conclusion: t cell depletion and cd56 positive selection led to an acceptable recovery of enriched nk cells that could be successfully activated by il-2 or il-15. the described nk cell isolation and activation protocol can easily been transferred into a gmp laboratory. for clinical application, further expansion of isolated nk cells under gmp conditions may be preferable. nk cells enriched, expanded and activated by these means may be an adoptive cellular therapeutic option that in addition may be improved by combination with tumour antigen specifi c antibodies. s76 o386 a randomized trial comparing cd34 enriched grafts versus cd3/cd19 depleted grafts in partial t-cell depleted allogeneic stem cell transplantation n. schaap, d. eissens, f. preijers, a. van der meer, a. schattenberg, h. dolstra, w. van der velden, t. de witte, m. smits, n.m.a. blijlevens radboud university medical centre (nijmegen, nl) we initiated a randomized trial in aml, all and mds patients (pts) treated with partial t cell depleted allogeneic sct using immunomagnetic cd34 selection versus cd3/cd19 depletion. a benefi cial effect was hypothesized on long term engraftment and dfs using cd3/cd19 depletion. residual cell populations in the graft after cd3/cd19 depletion may play a protective role during early phase after sct and generate an increased gvl effect. from may 2006 until may 2009 pts with aml, all and mds were stratifi ed for diagnosis and randomized. median age of the pts was 46 and 45 yrs, respectively. stem cells were obtained after g-csf (10 ug/kg). t cell add back to a fi xed dose of 5 × 10e5 cd3 + t cells/kg bw was standard in all pts. pts without significant (> grade 2) acute and/or chronic gvhd were eligible for prophylactic dli. in this interim analysis we determined immune reconstitution of t cells, b cells and nk cells using fl ow-cytometry and functional essays and evaluated clinical outcome. reconstitution: nk cells are completely maintained in cd3/ cd19 depleted transplants. b cell depletion is equally effective. cd34 + cells are signifi cantly higher in the cd3/cd19 depleted grafts (median 4.3 versus 6.5 × 10e6/kg, p<0.05). all pts engrafted and the time to engraftment was not different. in the fi rst 3 months after sct, the immune reconstitution of lymphocytes, especially cd4 t cells, nk and nk-t cells was faster in the cd3/cd19 group. fourteen days after sct the cytotoxic nk and regulatory nk subsets were already present in the cd3/cd19 group whereas the cd34 group needed 2 months to full recovery. the cytolytic response against a leukemia target was stronger in the cd3/cd19 group (p = 0.02). nk receptor expression of nkg2a (inhibitory) was strongly decreased whereas expression of nkg2c (activating) was enhanced. clinical outcome; acute gvhd > grade 2 was 17% in both groups. extensive chronic gvhd was 13% (cd34) vs. 18% (cd3/cd19. one year trm, rr, lfs and survival using cd34 selection and cd3.cd19 depletion were 5% vs. 39% (p = 0.066), 36% vs. 39% (p = 0.385), 64% vs. 35% (p = 0.063), 74 vs. 41% (p = 0.016), respectively. conclusion: compared to cd34 selection, cd3/cd19 depletion resulted in a faster reconstitution of cd4 cells,nk-t and nk cells. however survival was signifi cantly better in pts transplanted with cd34 selected grafts. due to a loss of power in favor of our hypothesis (15% increase in overall survival) using statistical simulation models, this trial was stopped. (1) introduction: in haploidentical hematopoietic stem cell transplantation (hsct), the infusion of donor lymphocytes transduced to express the herpes simplex virus thymidine kinase (hsv-tk) suicide gene allows to control gvhd, to mediate gvl, and to rapidly provide an effective and polyclonal anti-infective t cell repertoire (ciceri and bonini et al., lancet oncology, 2009 ). even though their engraftment is necessary to achieve these effects, hsv-tk + cells represent the minority of lymphocytes circulating in treated patients. therefore, we investigated the putative role of hsv-tk + cells in promoting thymic activity and t cell development from graft progenitors. methods: thymic function was assessed in adult patient who underwent haploidentical hsct and infusion of suicide genemodifi ed donor t cells for hematologic malignancies, after validating the methods in healthy pediatric and adult controls. single joint t cell receptor excision circles (sjtrec) were quantifi ed by qpcr in peripheral blood mononuclear cells (pbmcs) and purifi ed t cells, and the proportion of cd31 + recent thymic emigrants (rtes) in cd4 + naïve t cells was measured with immunophenotype analysis in pbmcs. thymic output was correlated with thymic volume, assessed by computed tomography (ct) scans. t cell receptor repertoire was assessed by vbeta spectratyping. the relative contribution of hsv-tk + and hsv-tk-donor t cells to post-transplantation anti-host alloreactivity was studied by mixed lymphocyte cultures. results: at the moment of t cell immune reconstitution (defi ned as cd3 + cells > 100/μl peripheral blood), the cd4 + naïve t cell subset was almost entirely comprised by cd31 + rtes, and this percentage remained high, as compared to age, also in subsequent months. in informative patients, rte frequency before hsct and before hsv-tk + cell infusion was in line with agerelated healthy controls, suggesting a direct role of the infused cells in mediating the effect. accordingly, in treated patients ct scans documented an increase in thymic volume following hsv-tk + cell add-backs. finally, low absolute sjtrec counts could be detected, in line with the documentation of extensive peripheral proliferation and low rte absolute numbers. conclusions: these data show that after the infusion of suicide gene-modifi ed t cells a renewal of thymic activity takes place, which contributes to the recovery of the peripheral t cell repertoire. elevated pretransplant serum ferritin levels have been associated with an increased susceptibility for opportunistic infections and increased incidence of morbidity and mortality after allogeneic hct. we studied in 81 patients who underwent myeloablative allogeneic hct for acute myeloid leukemia pre-and posttransplant serum ferritin levels and correlated the serum ferritin levels with the tlr9 expression and the cellular immune reconstitution 3 and 12 months post transplant. further, we studied in vitro-experiments in kasumi 1 cells the tlr1, tlr2, tlr3, tlr5, tlr7, tlr9 and tlr10 expression after overwhelming iron exposure. the average pretransplant serum ferritin level was 1245 μg/ml (mean) in all aml-patients (mean 1100 μg/l for patients with aml in 1.cr and mean 1820μg/l for patients with aml > 1.cr). post transplant serum ferritin level increased up to 2080 μg/ml (mean) for all aml patients (mean 1290 mg/l for aml in 1.cr and mean 2350 μg/l for patients with aml > 1.cr). the application of 300 ng iron to acute leukaemia cell lines sd1, and kasumi-1 cells increased signifi cantly tlr1,tlr2, tlr3, tlr5, tlr7 and tlr9 expression in relation to the housekeeping gene abl measured by real-time rt-pcr. in kasumi-1 cells tlr1 increased up to 50,6% (p = 0.014) tlr2 35.5% (p = 0.046), tlr3 57,8% (p = 0.006), tlr5 62.9% (p = 0.005), tlr7 46.2% (p = 0.02), tlr9 44.2% (p = 0.026) and tlr10 54,7% (p = 0.07) compared to untreated kasumi 1 cells. further, patients with elevated post transplant ferritin level > 2000 μg/l had an increased tlr9 expression in mononuclear cells (tlr9/abl quotient 6485 versus 4543; p<0.05) 3 months post transplant. the number of cytotoxic t cells cd4 + cd8 + in patients with elevated serum ferritin level was signifi cantly elevated after transplant (mean 189 versus 95 cells/μl 12 months post transplant, p = 0.034), whereas no differences were found in the number of cd3 + cd4 + t helper cells, b19 + cells, nk cells. these results indicate that elevated ferritin levels might activate the innate immune system by increasing tlr expression. this might be of importance since we recently showed that increased tlr9 expression was associated with adverse impact on nonrelapse mortality in the transplant setting. further exaggerated tlr9 expression has been discussed to induce overwhelming immune responses as sirs or ards. more studies are definitely necessary to evaluate the role of elevated iron overload on the innate immune system. clinical scale generation of functional human natural killer cells from umbilical cord blood cd34-positive cells for immunotherapy j. spanholtz, m. tordoir, c. trilsbeek, j. paardekooper, t. de witte, n. schaap, f. preijers, h. dolstra umc st.radboud (nijmegen, nl) immunotherapy based on natural killer (nk) cell infusions is a potential adjuvant treatment for many cancers. we established an extremely effi cient cytokine-based culture system for ex vivo expansion of nk cells from hematopoietic stem and progenitor cells from umbilical cord blood (ucb). systematic refi nement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. the use of gbgm culture media in a clinical applicable protocol allows the ex vivo expansion and differentiation of cd34 + cells to more than 4-logs into cd56 + cd3-nk cells with very high purity. these ex vivo-generated cd56 + cell products contain nk cell subsets expressing cd94/nkg2a and kir as well express high levels of activating nkg2d and ncrs. functional analysis showed that cd56 + cell products containing alloreactive nk cells effi ciently kill myeloid leukemia and melanoma tumor cells as well as primary acute myeloid leukemia (aml) cells. we have currently translated the protocol to clinical scale production using a closed cell culture bioprocess ( figure 1 ). cd34 + selection from cryopreserved ucb samples (n = 9) using the clinimacs device was optimized and the selection resulted in a cd34 + enrichment with a mean number of 2,2 ± 1,6 × 10 6 cells and a purity of 71 ± 14 % cd34 + cells. validation experiments using these cells showed, that the generation of suffi cient numbers of nk cells without contaminating t-cells or bcells under a closed system process is feasible. the cell cultures using bags show a mean expansion of 1273 ± 506 fold (range 759-1770 fold, which generated 8,6 × 10 8 -1,9 × 10 9 nk cells from cord blood-derived cd34 + cells within maximal 6 weeks of culture. the mean purity of the nk cell product was 71 ± 9 % (range 63-80%) of the total cells in the bag cultures. in order to improve the purity of the product, we include bioreactors during the differentiation culture process. using this modifi cation we were able to synchronize the differentiation process in small (plate) and large scale (bag) experiments, which allows the generation of large scale nk scale products with a purity of more than 95% cd56 + cells devoid of t-and b cells (figure 2a and b) . these nk cell products will be used for immunotherapy in elderly aml patients. this study is a phase i dose escalation study in a series of 12 aml patients who have successfully achieved cr (<5% blasts in the bone marrow) after standard intensive chemotherapy. key: cord-005480-yg7salqt authors: nan title: oral sessions and working party date: 2008-03-26 journal: bone marrow transplant doi: 10.1038/bmt.2008.30 sha: doc_id: 5480 cord_uid: yg7salqt nan basic science award: o100 mt1-mmp and reck inversely regulate haematopoietic progenitor cell egress a. avigdor* (1) , y. vagima (2) , p. goichberg (2) , o. kollet (2) , s. shivtiel (2) , m. tesio (2) , a. kalinkovich (2) , i. petit (2) , o. perl (1) , e. rosenthal (1) , i. resnick (3), i. hardan (1) , a. nagler (1) , t. lapidot (2) (1)the chaim sheba medical center (tel-hashomer, il) ; (2) the weizmann institute of science (rehovot, il); (3)hadassah medical center (jerusalem, il) hematopoietic progenitor cell release to the circulation is the outcome of signals provided by cytokines, chemokines, adhesion molecules, and proteases. yet, the mechanisms of progenitor cell egress during g-csf mobilization are not fully understood. membrane type-1 metalloproteinase (mt1-mmp) and its endogenous inhibitor, reck, are established key regulators of tumor cell motility. we detected higher mt1-mmp and lower reck expression on circulating human cd34+ progenitors and maturing leukocytes as compared to immature bone-marrow (bm) cells. mt1-mmp expression was more prominent on cd34+ cells obtained from pb of g-csftreated healthy donors whereas reck was barely detected. g-csf mobilization in nod/scid mice, previously engrafted with human cells, increased mt1-mmp and decreased reck expression on human progenitors and maturing leukocytes, in a pi3k/akt1-dependent manner, resulting in elevated mt1-mmp activity. blocking mt1-mmp function impaired g-csf mobilization, while reck neutralization promoted egress of human cd34+ progenitors. targeting mt1-mmp expression by sirna or blocking its function reduced the in-vitro sdf-1 induced migration of human progenitors via matrigel and impaired the bm homing capacity of transplanted human progenitors in nod/scid mice. in accordance, neutralization of reck function facilitated the migration of human bm cd34+ cells in vitro. furthermore, following g-csf mobilization, we also observed a reduction of cd44 on human cd34+ progenitors in the bm of chimeric mice. this was accompanied by accumulation of cd44 cleaved products of molecular weights, expected for mt1-mmp activity, in the bm supernatants. blocking mt1-mmp function in chimeric mice resulted in less cleavage of cd44 upon g-csf mobilization, whereas in the absence of a mobilizing signal, increasing mt1-mmp activity by reck ab injection facilitated cd44 proteolysis on the bm cells. finally, mt1-mmp expression correlated with the number of cd34+ cells, collected on the first apheresis day in consecutive healthy donors and patients mobilized with g-csf. in conclusion, our results indicate that g-csf inversely regulates mt1-mmp and reck expression on cd34+ progenitors, resulting in net increase in mt1-mmp activity. mt1-mmp proteolysis of cd44 diminishes progenitor adhesion to bm components, leading to cell egress. these previously undefined cell autonomous changes in the course of g-csf treatment might serve as target for new approaches to improve mobilization. morbidity and mortality associated with treatment-related organ toxicity is a major factor limiting success of allogeneic hematopoietic stem cell transplantation (hsct). conservative therapeutic strategies have been ineffective in part, resulting in high rates of progression or complete organ failure and death. over the last decades, solid organ transplantation (sot) has been increasingly used for the treatment of terminal organ failure in hsct recipients. to date, information regarding the use of sot as treatment attempts in patients after hsct is limited. as well the risk factors accounting for the necessity of sot after hsct as well as the incidence and outcome of this therapy are not well defined. a questionnaire survey was carried out within ebmt centres. 107 centres participated in this survey, covering allogeneic hsct between 1984 and 2005 . 31 cases of sot were identified. in more detail, 13 liver-, 8 kidney-, 9 lung-, and one heart transplantations were performed in 25 different centres. indications for liver transplantation were infections leading to cirrhosis (n=4), sinusoidal obstruction syndrome (n=3), and gvhd of the liver (n=2). rejection of the transplanted liver or terminal organ failure occurred in one patient respectively. other complications after liver transplantation were infections (n=2), bleedings (n=2) and kidney failure (n=2). most kidney transplantations were performed because of chronic kidney failure due to drug toxicity (n=6). transplant rejection and/ or kidney failure did not occur. interestingly, two of the kidney donors were also stem cell donors for the transplant recipient. lung transplantation was performed in all cases because of bronchiolitis obliterans and/ or gvhd which led to respiratory failure. rejection occurred in 4 patients and in one patient terminal transplant failure occurred. other major complications in the lung transplant recipients were kidney failure (n=3) and infections (n=2). heart transplantation was performed in one patient because of pre-terminal heart insufficiency due to drug treatment. in summary, very few sot for terminal organ failures were performed. the overall survival of patients receiving an organ graft after hsct was 72.5% at 5 years with a median follow up time of 23 months. complications after sot, like infections and organ rejection were frequent, but manageable. we conclude that sot offers a viable therapeutic option for patients who develop terminal organ failure after hsct. influence of immunisation timing on the response to conjugate-pneumococcal vaccine after allogeneic stem cell transplant: final results of the ebmt idwp01 trial c. cordonnier* (1) , m. labopin (2) , v. chesnel (2) , p. ribaud background: pneumococcal infections are causes of death after sct. the efficacy of the polysaccharide 23-valent vaccine (ppv23) is limited before 6 mo after sct, or if graftversus-host disease (gvhd) . previous studies with the wyeth heptavalent conjugate prevnar® vaccine (pcv7), using different schedules after allogeneic sct, showed a response around 65-85 % of the patients. however, the optimal timing of vaccination is yet not defined, and pneumococcal infection may occur early in the first months after sct. our objective was to show that the response to early (e) (3 mo) immunization is not inferior to a late (l) (9 mo) immunization. methods: patients ≥ 5 year old and at 3 months after allogeneic myeloablative sct were randomized to receive 3 doses of pcv7 at 1 month interval, followed by a ppv23 6 months later, from 3 (e) or from 9 (l) months after transplant. the primary endpoint was the % of responders (≥ .15 µg/ml of each of the 7 pcv7 serotypes) 1 month after the 3rd dose of pcv7 (s3). ab levels were blindly measured by elisa. all patients were followed until 24 months after transplant, or until death, whichever occurred first. results: 158 patients were randomized: 75 in the early (e), and 83 in the late (l) group. most patients were adults with acute leukemia, transplanted from an hla-identical donor. 114 patients were evaluable for the primary endpoint (e: 57, l: 57). the response rate was respectively 79% vs 82% at s3, and not inferior in the early, when compared to the late group (90% ci; 8.6) . however, at 24 months, significantly less e patients were still protected when compared to l patients (26/44; 59% s 35/42; 83%, p=.013). in the lack of difference in the response between groups, the 2 groups were pooled to analyse the impact of transplant characteristics on the percentage of responders at s3. donor age (> 36y) and chronic gvhd were the only factors impairing the response in the multivariate analysis. conclusion: the ab response 1 month after 3 doses of pcv7 after allogeneic sct is about 80% and non inferior when started at 3 than at 9 months. we therefore recommend starting immunization at 3 months to offer an earlier protection. however, the e vaccination offers a significantly lower protection at 2 years, suggesting the need for a boost during the second year after e immunization. the authors are grateful to the safety committee: d. engelhard, p. reusser, and p. reinert depletion of the autoreactive immunological memory followed by autologous haemopoietic stem cell transplantation in patients with refractory sle induces long-term remissions through de novo generation of a juvenile and tolerant immune system t. alexander (1) , a. thiel (2) , g. massenkeil (1) , a. sattler (1) , s. kohler (2) , h. mei (2) , h. radtke (1) , g.r. burmester (1) , a. radbruch (2) , r. arnold (1) , f. hiepe* (1) (1)charité -universitätsmedizin berlin (berlin, de) ; (2) german arthritis research center (berlin, de) clinical trials have indicated that immunoablation followed by autologous haemopoietic stem cell transplantation (asct) has the potential to induce clinical remission in patients with refractory systemic lupus erythematosus (sle). to elucidate the mechanisms mediating the beneficial long-term clinical responses, we investigated the immune reconstitution in sle patients receiving asct as part of a monocentric phase i/ii clinical trial. seven patients with sle were evaluated during a long-term follow-up (median follow-up period 60 months) who were immunoablated with cyclophosphamide and rabbit antithymocyte globulin, followed by transplantation of purified autologous cd34+ haemopoietic stem cells. previous failure of conventional immunosuppression, including cyclophosphamide, had been an inclusion criterion. humoral immunity was evaluated, peripheral t and b lymphocytes were immunophenotyped and frequencies of t lymphocytes specific for distinct antigens of interest were assessed after short-term stimulation ex vivo. in all patients clinical and serological remission was observed, accompanied by disappearance of anti-dsdna autoantibodies and protective antibodies from serum. one patient developed a relapse 18 months after asct. in the responding patients, cd31+ cd45ra+ cd4+ t cells, i.e. recent thymic emigrants, recurred with a doubling in absolute counts compared to agematched healthy controls until 4-yr post-transplant (p=0.014). absolute numbers of cd4+ foxp3+ regulatory t cells (tregs) normalised after asct and tcr repertoires cd4+ t cells displayed a broad clonal diversity as compared to the pretransplant status. early after asct, often high frequencies of virus-specific effector t cells were detected. autoreactive t cells specific for nucleosomes or smd1 were not detectable. a normal b cell compartment developed within 12 months after therapy, as compared to the pre-existing b cell deficiencies, which had included naive (igd+) b cell lymphopenia (p=0.031), relative predominance of memory (igd-) b cells (p=0.016) and expansion of cd27high cd20plasma blasts. our data demonstrate that the long-term therapy-free clinical remissions observed in sle patients after complete immunoablation and asct are accompanied by a loss of immunological memory and a fundamental reset of the immune system. depletion of the autoreactive memory and reactivation of thymic education probably are the basis for regeneration of self-tolerance and clinical remission. m. themeli* (1), l. petrikkos (2) , m. waterhouse (2) , h. bertz (2) , n. zoumbos (1) , j. finke (2) , a. spyridonidis (1) (1)university of patras medical school (rion-patras, gr); (2)freiburg university medical center (freiburg, de) we previously demonstrated frequent genomic alterations measured by microsatellite instability (msi) in non-neoplastic epithelial tissues of pts who underwent allogeneic hematopoietic cell transplantation (hct) but not in pts after autologous hct (blood 2006; 107:3389) . confirmation in larger independent patient cohort and in an in vitro system was needed. 176 buccal samples from 71 unselected pts obtained 30-3722 days (median 322) after allogeneic hct were analysed for msi. control subjects (16 healthy and 15 pts after auto-hct, 47 samples) were negative for msi. msi was observed in 37 (52%) allo-transplanted pts. msi+ pts were significantly older than msi-patients (median age 60y vs 48y, p<0.05). by using logistic regression analysis we found that the relative risk for msi was 2-fold higher in pts who experienced extensive chronic gvhd as compared to pts with no gvhd. although the median follow up in msi+ pts was significantly lower than in msi-(336 vs 669 days, p<0.05), secondary malignancy (5 skin-and 1 adeno-ca but none in the oral cavity) was diagnosed in 5 (14%) of the msi+ pts) and only in 1 (3%) msi-pt (p<0.05). other clinical features were not significantly different between msi+ and msi-pts. in an vitro mutation analysis model we tested the hypothesis that an alloantigenic stimulus is substantially involved in the mutation process. briefly, keratinocyte (hacat) cells were transfected with a plasmid vector carrying a neomycin selectable marker, a hygromycin resistance (hygr) sequence and a (ca)13 repeat. in this system, dna slippage mutations become detectable after hygromycin treatment as hygr+ colonies. the mutant fraction was expressed as the number of hygr+ colonies corrected for relative cell survival. untreated cells served as controls. treatment of stably transfected hacat cells with tnfa (25-100ng/ml, 24h), tgfb (5-20ng/ml, 24 h) and supernatant from a mixed lymphocyte culture (mlc, 24h) didn't cause any detectable induction of genomic instability (gi). treatment with h202 (20-40µì, 1-24 hours) resulted in a time and dose dependent gi induction (max 3.5 fold). cocultivation of hacat cells with stimulated lymphocytes from mlc resulted in a 3.9 fold induction of the mutant fraction. in conclusion, our in vivo and in vitro data indicate that "alloantigenic reactions may induce genomic instability in the allotransplanted pts which might predispose them to secondary neoplasia. the ebmt risk score predicts outcome after allogeneic hsct in all haematological disease categories and is independent of stem cell source or conditioning intensity a. gratwohl*, m. stern, j. apperley, t. de witte, j. passweg, v. rocha, a. sureda, r. brand, d. niederwieser information on factors associated with outcome after allogeneic hsct is a prerequisite for risk adapted strategies. five key factors form the basis of the ebmt risk score: stage of the disease (early 0, intermediate 1, advanced 2), age of the patient (< 20 y 0, 20-40 y 1, > 40 y 2), time interval from diagnosis to transplant (< 1y 0, > 1 y 1), histocompatibility (hla-id sibling 0, others 1) and donor recipient gender combination (other 0, female donor for male recipient 1). they were identified and validated in several independent series of cml patients, but not yet in other diseases. we examined 53140 patients, 34 y of age (median, 0-77y range), 58.7% male with an allogeneic hsct for aml (15126; 28.6%), all (10756; 20.2%), cml 12321; 23.2%), mds (4112; 7.7%), mps (1184; 2.2%), lymphoma (4165; 7.8%), myeloma (1329; 2.5%) or saa (4057; 7.6%) between 1980 and 2005. donor was a hla id sibling in 78.2%, other donor in 21.8%. stem cell source was bone marrow in 64.6%, peripheral blood in 34.9% and cord blood in 0.5%.conditioning was standard in 86.5%, reduced in 13.5%. each risk factor was tested individually by multivariate analysis and confirmed as cumulative dose response risk in all subcategories with two exceptions: stage was not applicable in saa, time interval was not applicable in patients in 1st cr. cumulative incidence of transplant related mortality (trm) at 5 years increased with the risk score from 15.4% (score 0, 3500 pts) to 22.1% (score 1, 10428 pts), 27.6% (score 2, 13677 pts), 32.4% (score 3, 11729 pts), 37.9% (score 4, 8159 pts), 42.6% (score 5, 4843 pts) and 49.7% (score 6/7, 1250 pts). after stratification by risk score, underlying disease had only a minor impact on the rate of trm. inside the risk score categories, trm improved significantly during the period of observation (rr 1980 (rr -1989 rr 1990 rr -1999 rr since 2000 0.50 ). absolute trm rates declined less markedly (1980-89 36%; 1990-1999 31%; since 2000 27%) due to a shift towards higher risk patients in more recent years. the ebmt risk score separated risk categories in all diseases, for all donor types, for all stem cell sources and for patients with reduced or standard conditioning. these data show that risk categories for outcome after allogeneic hsct can be defined. they can be integrated into risk assessment algorithms and form the basis for individualised risk adapted strategies when transplant and non transplant strategies are available as treatment options. working party solid tumours 114 reduced-intensity allogeneic transplantation for breast cancer d. blaise* (1) , a. gonçalves (1) , s. fürst (1) , j.o. bay (2) , c. faucher (1) , m. michallet (3) , j.m. boiron (4), j.y. cahn (5) , n. gratecos (6) , m. mohty (1) , c. chabannon (1) , g. gravis (1) , b. esterni (1) , j.m. extra (1) , p. viens (1) (1)institut paoli-calmettes (marseille, fr) ; (2) centre jean perrin (clermont ferrand, fr) ; (3)chu edouard herriot (lyon, fr) ; (4)chu haut lévèque (bordeaux, fr); (5)chu jean minjoz (besançon, fr); (6)chu cimiez (nice, fr) we initially treated 18 pts with allo sct for advanced metastatic breast cancer. all pts (age: 45 (27-57)) underwent asct after the same reduced intensity conditioning (ric) (fludarabine (150mg/m2), busulfan (8mg/kg) and thymoglobulin (2,5mg/kg) or tli (1 cgy)) from a hlaidentical sibling (bm: 22%; pbsc: 78%) followed by csa. prior to asct a median of 3 lines of treatment (1-7) were administered over a period of 1452 days . nine pts underwent autologous sct at a median time of 1130 days (88-3012) prior to asct. all pts were measurable and had a median of 2 metastatic sites (1-4) (liver:72%, bone:50%, lung:22% and brain:11%): according to recist criteria, 13 (72%) and 5 (28%) pts had progressive (pd) and stable disease (sd) respectively. none of the 18 pts died from trm. two of them achieved partial remission (pr) at 60 and 150 days respectively (objective response (or): 18% (0-36). all pts but 1 eventually progressed and died from disease (2 year overall survival (os): 22%(9-45)). results are dramatically different in regards to disease status at time of transplant. while outcome is uniformly poor for pts with pd, patients with sd achieved a 40% (0-80) or rate for a 50% (19-80) os at 2 years with 3 (60%) patients surviving more than 2 years (640+, 834-and 1246-), which is significantly different from pts with pd. we established that ric-asct can be safely performed in brc pts, whereas highly pd pts do not benefit from this approach. we run a second trial in less advanced disease to confirm encouraging results (present accrual: 15 pts). all 33 patients will be presented. however it seems that curability in brc will be achieved only in pts in the initial disease phase: target population would need a careful selection on individual prognosis factors indicating their poor short term poor outcome: this represents the ultimate goal for future investigations. supported in part by a grant from the french ministry of health (phrc 2000; phrc 2003 ) and a special grant (pole areca) from the association pour la recherché contre le cancer (arc) we have utilized autografting to achieve maximum tumor reduction before proceeding to non-myeloablative allografting. this strategy could provide the benefit of a conventional allograft, but with reductions in the typical acute toxicities and associated mortality of myeloablative conditionings. between september 1997 and april 2004, we enrolled 17 patients with metastatic breast carcinoma. median age was 41 years. at the time of autografting, the patients had received a median of 3 (range, 2-5) previous chemotherapy lines; 14 patients had received hormone therapy, and seven patients had undergone radiotherapy on bone lesions. the primary endpoint of this study was the decrease of non-relapse mortality (nrm) from the current 20-35% noted after myeloablative allografting. patients received autografting at a median of 53 months (range, 14-152) from the diagnosis of breast cancer. no patient died after transplant. one patient who had been in complete remission and two who had been in partial remission before autografting remained in complete or partial remission. no non-relapse mortality was noted in the first 100 days after non-myeloablative allografting. thirteen patients achieved full chimerism. five patients (29%) developed grade ii-iii acute gvhd, while six patients developed chronic gvhd (five patients with extensive disease) and needed intensive immunosuppressive therapy. we have recently reported a subsequent patient transplanted from her hla-identical sister. disappearance of liver, adrenal, mediastinal, pleural, and diffuse nodes and bone metastases, observed simultaneously with clinical chronic gvhc 5 months after non-myeloablative allografting, suggested a profound graft-versus-tumor effect. renal cell carcinoma (rcc) has recently been identified as being a target for gvt effect. since 1999 there has been a number of publications describing gvt effects in patients with rcc undergoing mostly reduced intensity transplantation. at the nhlbi, patients have been conditioned with cyclophosphamide (60mg/kg x 2) and fludarabine (25mg/m² x 5) then transplanted with a g-csf mobilized blood stem cell allograft from their hla identical or single antigen mismatched related donor. twenty-nine of 74 patients have had disease regression consistent with a gvt effect (39.2 % cumulative incidence of a complete response + partial response). a better understanding of the immune cells and their target antigens that mediate tumor regression could potentially lead to the development develop more effective hct approaches for solid tumors. recently, t-cells with in vitro tumor cytotoxicity patterns consistent with recognition of minor histocompatibility antigens and tumor restricted antigens have been identified in some responding patients. the identification of tumor restricted antigens targeted by donor immune cells could lead to the development of transplant approaches that enhance gvt effects while avoiding gvhd through tumor vaccination or the adoptive infusion of in vitro expanded donor t cells with tumor antigen specificity. we detected rcc-reactive cd8+ t-cells by elispot analysis in the blood of several responding patients with metastatic rcc following hct that were absent before transplantation. we successfully generated donor cd8+ t-cell clones from lymphocytes obtained from these patients that have direct cytotoxicity against the patient's rcc cells. in one responding patient, cytotoxic t-lymphocytes and t-cell clones with rcc-specific tumor cytotoxicity were isolated from the blood after transplantation. utilizing cdna expression cloning, we identified an hla-a11-restricted 10-mer peptide (named ct-rcc-1) to be the target antigen of these rcc-specific tcells (takahashi y, harashima n. et al-j clin invest 2008 in press) ct-rcc-1-specific t-cells were detected by tetramer analysis in the patient's blood after tumor regression but not before hct. tetramer analysis of 8 hla-a11+ rcc transplant recipients showed ct-rcc reactive t-cells expanded significantly in all 3 responders in contrast to the 5 non-responders, where only 1/5 showed an increase in ct-rcc-1 reactive t-cells. the genes encoding the ct-rcc antigen were found to be derived from a human endogenous retrovirus (herv)-e previously unknown to be expressed in any human tissues; this herv-e was found to be expressed in the majority of rcc tumor lines and fresh rcc tissue but not in normal kidney cells or other normal tissues. this is the first solid tumor antigen identified using allogeneic t-cells from a patient undergoing hct. these data suggest this herv-e is transcriptionally active in rcc, encoding an immunogenic antigen that is over-expressed in rcc which could be a potential target for cellular immunity. update of the results of high-dose chemotherapy as primary or salvage therapy in germ cell tumours g. rosti* (1) , u. de giorgi (1) , p. pedrazzoli (2) , m. bregni (3) ( cisplatin-containing regimens cure nearly 80% of patients with advanced germ cell tumors. hdct has been extensively used in the last 20 years with somehow controversial results. the ebmt it-94 study on relapsing good-risk patients has not shown any difference in overall survival comparing four courses of standard second line therapy versus one late intensification single shot approach, even if patients achieving cr did significantly better if randomized in the high-dose arm. a phase iii us trial in patients with poor prognosis, treated upfront, even if not showing an advantage for hdct, has shown a significant difference for those with unsatisfactory marker decline. an input of the possible role of hdct in relapsing/refractory patients came from the retrospective data of the indiana university of tandem hdct with carboplatin and etoposide in a large series of consecutive men with metastatic testicular cancer that had progressed after receiving cisplatin-containing combination chemotherapy. this study shows 70% and 50% four-year disease-free survival in patients who received hdct as second-line or third-line or later therapy, respectively. as it is a retrospective review, one may argue that the results are biased by patient selection. this does not seem to be the case, however, as even patients with very poor prognosis achieved long-term disease-free survival -50% of survivors were classified high-risk by the igcccg classification and 45% had platinum-refractory disease. it is important to note that all patients in this series received peripheral-blood progenitors as sources of hematopoietic stem cells. this strategy allowed a rapid engraftment, thereby permitting the administration of two courses of high-dose in addition, peripheral-blood progenitors were enriched for cd34+ hematopoietic cells, a procedure which may have a role in eliminating possible cancer cells from the graft. we believe that, on the basis of the robust data provided by einhorn and colleagues, a well-designed randomized trial of hdct versus conventional-dose chemotherapy should be performed in patients with poorprognostic clinical features who relapse after initial chemotherapy. at present, there should be no debate on the use of tandem-hdct in patients with cisplatin-refractory germ-cell tumors and those who have failed second-line therapy. gitmo and igg (italian group for germ cell cancer) are planning a network of centres in italy to refer such patients for the tandem hdct. autologous stem cell transplantation international multiple sclerosis trial (astims, eudract number 2007-000064-24, supported by ebmt; www.astims.org) is now a multicenter, prospective randomized phase ii study. the primary endpoint is the number of new t2 lesions on mri. the investigational treatment comprises mobilization with cy and g-csf and conditioning with beam followed by asct and atg compared to 6 monthly i.v. pulses of mitoxantrone at 20 mg followed by 1gr of methylprednisolone. at the moment 18 patients have been enrolled (january 2008): 4 in barcelona, 3 in genova, 3 in modena, 3 in florence, 2 in chieti, 2 in reggio calabria and 1 in bergamo. the astims trial is therefore still going on, with the aim to arrive within 2 years from now at the new target of 30 enrolled cases. in the meantime, more than 400 are the ms treated in the world with ahsct and a few phase i/ii are running with the aim to identify the clinical characteristics of the patients who can really take advantage from the procedure or to evaluate the efficacy of low intensity conditioning regimens. the study of all the 58 cases treated in italy in the last 10 years with the same regimen (beam and atg), same inclusion criteria and followed by the same neurohematological teams involved in the prospective study supported by gitmo, showed that patients with a relapsing remitting clinical course respond significantly better than secondary progressive cases to ahsct, indicating the population of patients who have to be selected in the future for the design of prospective studies with a clinical endpoint. the astis trial j.m. van laar* (1) , d. farge (2) , a. tyndall (3) , o. astis investigators (4) (1) newcastle university (newcastle, uk); (2) hopital st louis (paris, fr); (3)basel university hospital (basel, ch); (4)jcuh (middlesbrough, uk) background: high dose immunosuppressive therapy (hdit) and hematopoietic stem cell transplantation (hsct) is a novel treatment for patients with severe systemic sclerosis (ssc) . previous studies showed durable responses in two thirds of patients up to 7 yrs after hsct (1) . this treatment modality is now further investigated through the astis-trial (autologous stem cell transplantation international scleroderma trial), a prospective, controlled, randomized trial to compare safety and efficacy of hdit + hsct versus monthly i.v. cyclophosphamide in ssc patients at risk of major organ failure or early mortality. objectives: to evaluate whether hdit + hsct is superior over conventional treatment in terms of safety and efficacy in ssc patients, and to assess potential predictive factors of response. methods: ssc patients with early active diffuse disease with or without major organ involvement are eligible. ssc patients randomized to the transplant arm undergo mobilization with cyclophosphamide 2x2 g/m², conditioning with cyclophosphamide 200 mg/kg, rbatg 7.5 mg/kg, followed by reinfusion of cd34+ selected autologous hsct. those randomized to the control arm are treated with 12x monthly i.v. bolus cyclophosphamide 750 mg/m². the primary endpoint is event-free survival, defined as survival until death or development of major organ failure during 2 years follow-up. progression-free survival is the main secondary endpoint. results: one hundred eleven ssc patients have been enrolled in 25 centers per january 2008: 43 male, 68 female, mean age 43 yrs, mean modified rodnan skin score 26, mean disease duration 1,8 yr, mean vc 81%, mean dlco 59%. sixty-one patients were randomized to the transplant arm, 50 to the control arm. no unexpected toxicities have yet been observed in either arm with a median follow-up of 36 months (range . grade 3,4 toxicities occurred in 15/43 transplant patients and in 13/48 controls (p=0.42). atg-related toxicity led to its discontinuation in 12/35 transplant patients. two fatalities in the transplant arm were categorised as probably treatment-related. conclusion: the ongoing astis trial has enrolled 111 patients sofar. treatment-related mortality and number of patients with serious adverse events of stem cell transplantation are lower than previously reported in registry analyses. references: 1. van laar jm, farge d, tyndall a, on behalf of the ebmt/eular scleroderma study group. the astis-trial, hope on the horizon. ann rheum dis 2005;64:1515. standard nih or eurolupus cyclophosphamide (cy) protocols and mycophenolate mofetil (mmf) as induction therapy in severe bilag a sle is still associated with 20 % failure, 50% relapse and 10% to 15 % death at 10 years in the absence of a single standard treatment worldwide for refractory sle, phase i-ii studies analysed the use of: a) rituximab (anti cd20 mab) in more than 1 000 patients showing complete to partial early response around 100% with relapse in 50 to 60% of the cases; b) autologous hematopoietic stem cell transplantation (hsct) since 1997 under the auspices of the joined ebmt-eular working party, reporting durable remission with reduced or no immunosuppressive drug requirement in 66%, one-third of whom later relapsed to some degree with a 74 ± 7% (n= 62/79) overall survival at 5 years for sle among the 863 hsct procedures registered: in 2007 in the ebmt data base. the north american, mostly single centre experience showed higher rates of remission with also some relapses. maintenance immunosuppression after induction of remission may decrease the return of disease activity. this was the basis of the ebmt approved astil trial: a prospective randomized open, multicenter, phase ii b study to compare the efficacy of autologous hsct with rituximab as remission induction, followed by mmf (2 g /day) as maintenance in both arms for severe sle patients with disease duration ≤ 5 years since the diagnosis and sustained or relapsed active bilag a sle. this analysed describes the outcome of pediatric patients receiving hematopoietic stem cell transplantation (hsct) to treat severe refractory autoimmune cytopenias. the registry of the ebmt contains data on 16 patients receiving 19 transplants. patients had autoimmune haemolytic anemia (7), evans's syndrome (7) , immune thrombocytopenia (3), pure red cell aplasia (1) and autoimmune lymphroliferative synrome (1) . 15 patients were males with a median age at diagnosis of 4 years (range 0.3-16 years) and a median age at transplant of 7.8 years (2-17 years) . the median disease duration prior the transplant was 41 months (range 2-115 months) and all patients failed multiple prior treatments. transplant were autologous for 7 and allogeneic for 12 patients, 6 of these transplanted from an hla identical donor, 2 from a family mismatched donor and 4 from a matched unrelated donor. one patient received 2 autologous transplant while another patient received an allogeneic transplant because a relapse after the first autologous transplant . the stem cell source was mobilized pbsc in 1 transplant, bone marrow in 6 and cord blood in 2 patients. the graft was t depleted in 4 of 7 recipients of autotransplant and 3 of 12 allotransplant recipient. the conditioning regimen used were heterogeneous. 3 patients died of treatment related mortality, 2 in the allo and 1 in the autologous group for a trm of 14 % eights patients had a complete and continous response after the transplantation although 1 of these died for secondary malignancy. 3 patients relapsed after the procedure (1 in the allo and 2 in the autologous group) and one of these died for disease progression. 3 patients were not evaluable for response. the present analysis has some limitations because treatment protocols, mobilization and conditioning regimen were heterogenous and doesn't allows a detailed analysis of these factor moreover these preliminary data suggest that autologous and allogeneic hsct may induce response in half of patients with severe autoimmune cytopenia of long duration unresponsive to several therapeutic options. 124b long-term follow-up of autologous stem cell transplantation for juvenile idiopathic arthritis n.m. wulffraat wilhelmina children's hospital (utrecht, nl) the majority of children with juvenile idiopathic arthritis can nowadays be treated adequately. however despite the use of combinations of antirheumatic drugs, corticosteroids and the newer so called biologicals (blocking the tnf, interleukin 1 or interleukin 6 pathways) a proportion of children with arthritis remain resistant also to these therapies and suffer from a very severe, debilitating and potentially fatal disease. for such children autologous stem cell transplantation (asct) is successfully performed since 1997. here we describe the long term outcome of the initial cohort of children with resistant juvenile idiopathic arthritis, treated with asct. the initial cohort of children was treated with a conditioning regimen containing cyclophosphamide, anti thymocyte globulins and low dose total body irradiation. overall favourable responses were seen, with a drug free remission rate of 50-55 %. in the more recent years late relapses were noted with lower percentages for drug free long term outcome. special emphasis is given on 2 cases showing very late relapses, occurring after 7 and 9 years. the observed relapses are often less severe compared to the situation before sct and can be treated successfully with conventional drugs in the majority of cases. more recently, asct was performed in 4 jia children with a fludarabin containing regimen in stead of low dose tbi. with a 4 to 5year follow up, these 4 patients are all in drug free full remission. allogeneic transplant with an hla matched family donor was reported in 2 jia cases. follow up of 1 and 3 year is sofar show clinical disease remission and tapering of medition. in conclusion, given the favourable long term outcome, sct remains a valuable treatment option for children with drug resistant jia. s7 126 multipotent mesenchymal stromal cells in the treatment of autoimmune diseases a. tyndall* (1) , f. dazzi (2) multipotent mesenchymal stromal cells ( msc) isolated from the bone marrow and other sites are currently being studied to determine their potential role in the pathogenesis and/ or management of autoimmune diseases. in vitro studies have shown that they exhibit a dose dependent antiproliferative effect on t and b lymphocytes, dendritic cells, natural killer cells and various b cell tumour lines, an effect which is both cell contact and soluble factor dependent. these soluble factors include tgf beta, il-10, indoleamine 2,3 dioxygenase, il-1ra, and hla-g among others. anti proliferative and immunomodulatory mechanisms are probably multiple and most likely due to the induction of arrest of the cell cycle in g0/g1. a plethora of phenotypic definitions and experimental conditions accounts for some of the variation in in vitro phenomena being reported previous assumptions that msc are immunoprivileged have been challenged by recent animal data in non immunosuppressed hosts. animal models of autoimmune disease and tissue injury (ischemic kidney, chemically induced lung fibrosis and liver toxicity) have mostly shown a positive clinical response, with some early warning signs in a melanoma model concerning tumour surveillance. a limited number of patients suffering from acute graft versus host disease have been treated with msc as well as sporadic case reports and small uncontrolled series in multiple sclerosis and crohns disease. prospective phase i trials are starting in multiple sclerosis and crohns disease and being considered in inflammatory rheumatic diseases. an international interdisciplinary data base has been developed to exploit the collective experience. sirolimus is associated with veno-occlusive disease of the liver after myeloablative transplantation c. cutler*, k. stevenson, h. kim, p. richardson, v. ho, c. revta, r. ebert, d. warren, j. koreth, p. armand, e. alyea, r. soiffer, j. antin dana-farber cancer institute (boston, us) veno-occlusive disease of the liver (vod) is an uncommon but important cause of mortality after allogeneic transplantation. to determine if use of the immunosuppressive mtor inhibitor, sirolimus, is a risk factor for vod, we performed a retrospective review of vod incidence and risk factors at our institution since 2000, when we began using sirolimus. methods: review of electronic medical records of all transplant patients undergoing tbi-based myeloablative transplantation with adult stem cell donors was performed. results: 510 patients transplanted between 1/2000 and 5/2007 were identified and stratified by sirolimus use (260 exposed, 250 unexposed). sirolimus patients received sirolimus/tacrolimus ± methotrexate; all others received tacrolimus/methotrexate as gvhd prophylaxis. there were no differences in the age, gender, donor-recipient gender match or diagnoses between cohorts. sirolimus patients were more likely to have unrelated or mismatched donors, were more likely to have received pbsc (p<0.001 for both) and were less likely to have gr. ii-iv acute gvhd (26 vs. 38%, p=0.004) in comparison with non-sirolimus patients. the incidence of vod in the sirolimus group was 15% and was 6.4% in the unexposed (rr 2.3, p=0.003), but vod occurred later among sirolimus patients (22 vs. 15 days, p=0.12) . among mrd recipients, the rr was 2.6 (12.2 vs. 4.6%, p=0.06). when adjusted for age, gender match, stem cell source, hla match (mrd vs. urd/mismatch), and transplant risk (standard vs. high), sirolimus use remained a significant risk for vod (adjusted or 2.54, p=0.006). cause-specific mortality related to vod was similar in sirolimus and non-sirolimus patients. despite the increase in vod, treatment-related mortality was similar in all sirolimus and non-sirolimus patients and among mrd sirolimus and non-sirolimus patients at 1 year (19 vs. 21%, and 12 vs. 13%, both p=ns). in addition, there was a trend towards increased overall survival (os) for all sirolimus patients (3 yr os 54 vs. 49%, p=ns) and for mrd sirolimus patients (3 yr os 67 vs. 53%, p=0.08). in a cox regression model, age > 50 (p=0.001), donor match (p= 0.01) and vod (p<0.001) but not sirolimus use (p=0.14) were significantly associated with overall survival. conclusions: sirolimus is associated with vod after tbibased myeloablative transplantation. despite this association, transplant outcomes appear equivalent or better than standard tacrolimus/methotrexate based immunosuppression. physical health can be compromised in very long-term survivors after hsct compared to their respective donors but not mental health: a paired analysis t. daikeler, a. rovo*, m. stern, j. halter, j.d. studt, a. buser, d. heim, j. rischewski, m. medinger, a. tyndall, a. gratwohl, a. tichelli university hospital (basel, ch) with the improvement of prognosis, health status and functional well being of long term survivors after hsct become an important issue. we performed a cross-sectional prospective study on 44 long-term survivors and their respective sibling donors at a median follow up of 17.5 years (range 11-26) after hsct. the median age of the recipients and donors at time of the study was 44.3 (24-63) and 43.4 years (22-61) respectively. both recipients and donors were seen on the same day for evaluation. the short form-36® (sf-36) health survey, which provides a generic health status measurement through 36 items assessing 8 concepts of health was used. three of the items measure physical health (pf, rp, bp), two measure both physical and mental health (gh, vt), and three measure mental health (sf, re, mh). in addition there are two summary scores for physical (pcs) and mental (msc) health. for statistical analysis norm-based scoring (nbs) was applied, where 50 is the mean score, and 10 the standard deviation of a defined general population. paired analysis between donors and recipients were performed for detecting differences. all scored items of recipients as well as of the donors were within the range of one standard deviation of the norm-based population. all scores concerning physical well being except one, (rp), were statistically lower in the recipients than in their donors. in contrast, there was no difference in scores concerning mental well being. this is confirmed by the summary measurements of physical health (pcs) with 52,8 in the recipients and 57,1 in the donors , (p=0.001) and mental health (mcs) with 50,8 versus 52,9 (p=0.831) . physical health (pcs) was lowest in patients with severe chronic gvhd compared to their donors (47,2 versus 57,2) (p=0.05), age older than 25 years at hsct 50,7 versus 56,2 (p=0.024), older than 42 years at the last control 52,2 versus 55,63 (p=0.05) and for female patients 51,8 versus 57,7 (p=0.024) . none of the factors had a statistical impact on mental heath status (mcs, p>0.05). in summary, quality of life of long term survivors after hsct measured with the sf-36 questionnaire is still within the normal variation of the general population. however, when compared to their respective donors, the physical health status is significantly compromised in the recipients. severe gvhd, older age and female gender are associated with an inferior physical health status. a retrospective analysis of sexual function, fertility and endocrine status in male long-term survivors of allografts for haematological malignancy i.h. gabriel*, r. szydlo, m. klammer, r. patterson, n. swan, n. salooja, e. olavarria, e. kanfer, d. marin, a. rahemtulla, j.f. apperely imperial college healthcare nhs trust, hammersmith hospital (london, uk) steady improvements in the outcome of allogeneic stem cell transplantation (allo-sct) have resulted in significant numbers of long-term survivors and an increasing focus on factors impacting quality of life (qol). post transplant infertility and sexual dysfunction are two such factors. using a questionnaire we audited 150 male survivors of allografting for haematological malignancies at our centre. 81 men (54%) responded. the median age at sct was 37.5 yrs. the median time from transplant was 6.9 yrs and 97% were >3 years from sct (63% >5 years). 76% had returned to full pre-sct sexual activity, however, a number of problems in sexual function were reported. 49% complained of new persistent erectile dysfunction (ed) (normal prevalence 2-20%). ed affected men of all ages (40%, 47% and 51% at < 30 years, 30-40 years and >40 years respectively). ed was seen in 52% of recipients of tbi vs 22% of those who had not received tbi. 30% experienced penile glans dryness, previously unreported which appeared to be closely associated with chronic gvhd(p 0.019). in addition urethral constriction, phimosis, genitourinary infection and peyronies's disease all complicated sct. 48% of men reported reduced libido with 37% of ed patients reporting normal libido. 13% and 17% of men suffered premature or painful ejaculation respectively. 43% of male survivors described a negative impact of infertility on themselves or their partners and 9% had utilised assisted fertility/donor insemination. other problems included dyspareunia, inability to use condoms, sicca syndrome, and poor body image. factors forming barriers to new, or worsening existing, relationships were most prominent in younger patients. compared to values pre-transplant fsh was raised in 100% by 6 months post-transplant and remained high at 5 yrs (p=0.003), indicating long-term damage to sertoli cells. lh was significantly elevated at 12 months in 15% compared to baseline (p=0.005) and normalised by 5 yrs suggesting leydig cell recovery. testosterone levels were normal in 98% of men at 12 months. lack of physician continuity, language, presence of visiting fellows/students and the impression that physicians are only concerned with managing malignancy were barriers discussing these issues at routine follow-up appointments. a high prevalence of sexual dysfunction exists post-sct. increased awareness of these complications and their effects on patients and their families would permit prompt and appropriate management. secondary malignancies in recipients of allografts for chronic myeloid leukaemia in chronic phase using hlamatched sibling or volunteer donors i.h. gabriel*, s. avery, r. szydlo, n. salooja, e. olavarria, e. kanfer, m. klammer, a. rahemtulla, j. goldman, j.f. apperley imperial college healthcare nhs trust, hammersmith hospital (london, uk) during the last decade imatinib has replaced allogeneic sct as first line treatment for chronic myeloid leukaemia (cml) and second generation tyrosine kinase inhibitors now compete with allografting as second line therapy. however over the same period there have been steady improvements in the outcome of allografting and therapeutic choices are increasingly complex. improved information regarding the long-term side effects of both chemotherapy and allografting might help inform decision-making. we now update the incidence of secondary malignancy in 481 consecutive patients of median age 33.9 years, who underwent allografts for cml in first chronic phase (291 from sibling and 184 from unrelated volunteer donors). the median follow-up is 13.9 yrs. all patients received cyclophosphamide with either total body irradiation (tbi) (n=474) or busulphan (n=7). gvhd prophylaxis was cyclosporin, methotrexate, ± t-cell depletion. the probability of developing a secondary malignancy post allo-sct was determined by the method of cumulative incidence (ci). the overall incidence in this group was 5.95% with 27 patients developing 29 new tumours. 31% were diagnosed within 5 years, 62% within 15yrs, 72% within 20 yrs. men and women were equally susceptible to secondary malignancies. the overall incidence of developing a new malignancy in our cohort is 5.95% which is higher than in an age-matched population. involved sites included: skin (5 including 2 melanoma, 2 squamous cell and 1 basal cell carcinoma), breast (4), high grade b-lymphoma, (4), tongue (3), colo-rectal (2), cervix (2), osteosarcoma (2), testis (1), bladder (1), mucoepidermoid (1), oesophagus (1), lung (1), penile shaft (1), and mds (1). 2 patients had 2 malignancies. pre-bmt treatment consisted of hydroxyurea alone in 10, busulphan in 2 and interferon in 1 and some combination of these in 9 others. the ci of secondary neoplasia increased with time reaching 1.7%, 3.3%, 6.3% and 14.2% at 5, 10, 15,and 20 yrs respectively. 13 patients have died, 10 directly attributable to the secondary tumour. as all patients had the same genetic abnormality and received only minimal prior cytotoxic therapy, this increase in neoplasia is likely to be due to sct. these data highlight the need for close follow up and screening. prospective evaluation of oxygenation index and noninvasive-ventilation in patients suffering from acute lung injury after allogeneic transplantation m. wermke*, s. schiemanck, g. höffken, g. ehninger, m. bornhäuser, t. illmer university hospital (dresden, de) objectives: respiratory dysfunction is a major cause of allogeneic transplant-related-mortality (trm). little is known about how to recognize and treat acute lung injury (ali) in this setting. we present the first prospective randomized clinical trial evaluating the use of non-invasive-ventilation (niv) for treatment of ali in allogeneic transplantation. methods: all patients (n= 530) undergoing allogeneic transplantation at a single center were investigated from 2001 to 2005. oxygenation-index (pao2/fio2) was monitored twice daily from the beginning of the conditioning regimen. patients meeting criteria for ali were randomized to receive either oxygen or intermittent niv with positive end-expiratory pressure. patients not responding to assigned therapy were allowed either to switch from oxygen to niv or proceeded with treatment on intensive care unit (icu). results: of 90 eligible individuals, 44 were randomized to receive oxygen and 42 to niv, 4 patients withdrew consent. oxygen-, niv-and control-(440 patients without ali) group were well balanced regarding known risk factors for trm. only tbi was significantly more frequent in patients with ali. patients with ali showed significantly shortened short-and long-term overall survival (os) when compared to controls (100-day-os 65 vs. 85%; median-os 7 vs. 22 months). oxygenation index was not only a major adverse prognostic factor; it also suggested ali long before clinical parameters raised suspicion. of 42 patients randomized to niv only 10 (24%) did not respond and had to be transferred to icu. in contrast 18 of 44 patients (41%) assigned to oxygen did not improve. of these, 17 switched to niv but only 12 patients had to be transferred to icu. thereafter, there was no significant difference between both groups in need for intubation (oxygen: 11 patients, niv: 7 patients). niv did not lead to improved survival in our study (100-day-os niv: 61%, oxygen: 68%; median-os niv: 6 months, oxygen: 7 months). survival of intubated patients was poor, as only 1 of 18 intubated patients survived for more than 100 days. conclusion: oxygenation-index is an easily measurable early indicator of ali and poor survival in allogeneic transplantation. niv seems to reduce the need for icu and consecutive intubation in patients not responding to oxygen. we were not able to show significant survival benefits for niv-patients, presumably because patients not responding to oxygen were allowed to switch to niv. what to do when the first allogeneic stem cell transplantation fails? m. kedmi, i.b. resnick, b. gesundeheidt , l. drey, s. samuel, r. or, m.y. shapira* hadassah -hebrew university medical center (jerusalem, il) the failure of allogeneic stem cell transplant (allo-sct) is usually cumbersome, we have retrospectively evaluated our experience in a 2nd allo-sct. patients: out of 1533 allo-transplantees, 145 patients (93 males, 52 females, median age 20.7y(8m-68.4y)) underwent 2 or more allo-sct. the indications for the 1st transplant were acute leukemia (93), chronic leukemia (17), lymphoma (3), other malignancies (3) and non malignant (29). the 1st to 2nd interval was 18d to 13.25y (median 98d). the most frequent indications for the 2nd sct were basic disease (45), rejection (23) and engraftment failure (15). the 2nd sct conditioning was radiation based (59)or chemotherapy based in the others and was myeloablative or reduced intensity (ric) in 64 and 81 respectively. in 89 of the scts the original donor was used. 2nd donor matching was full in 92 transplants (family-84, unrelated-8) or mismatched in 53 transplants (51 and 2, family and unrelated donor respectively). graft source for the 2nd sct was bm (65) and pbsc (80). 38 of the grafts were t cell depleted. gvhd prophylaxis was given in 31 2nd procedures. the median survival from 2nd sct was 70d. despite the low rate of gvhd prophylaxis used only 51 and 16 of the patients developed agvhd and cgvhd respectively. 29/145 patients (20%) transplanted survived a year after the 2nd sct (figure 1). trm was 66% including sepsis, liver and renal failure, neurologic toxicity, rejection and gvhd. factors indicating higher chance for survival were non malignant disease, longer time between procedures (more then 1y), hla matching and the use of ric (figure 2). age at transplantation, the indication for transplantation (relapse vs. others), the development of acute gvhd, radiation based vs. chemotherapy based conditioning, gvhd prophylaxis (either pharmacological or t cell depletion) or graft source were not shown to indicate better or worse prognosis. with a median follow up of 4.5y, 25 patients (17.2%) are still alive, out of which 18 are disease free. conclusion: although highly toxic, a 2nd allo-sct may be beneficial to some patients and lead to long term survival. it seems that the patients whom will gain the most out of 2nd allo-sct are those who had a longer period between scts, have a matched donor or have non malignant diseases although 14 patients with malignant diseases (some with refractory disease) survived at least a year from the 2nd procedure while in cr. it is also noteworthy that the use of ric improved outcome. s10 o136 treatment of donor graft failure with autologous or allogeneic stem cell boost or a second allogeneic transplantation based on chimerism testing a. shimoni*, n. shem-tov, a. rand, e. ribakovsky, r. yerushalmi, i. hardan, a. nagler chaim sheba medical center (tel-hashomer, il) donor graft failure (gf) is a life-threatening complication of allogeneic stem cell transplantation (sct). we performed this analysis to determine the rate and outcome of gf in the era of modern sct. we retrospectively reviewed data of 491 scts from hla-matched siblings (n=284), matched-unrelated (mud, n=180) or alternative donors (mismatched-related, haplo-identical and cord-blood, n=27) performed in a single institution since 1/2001. gf was diagnosed in 25 patients (pts), cumulative incidence (ci) 5.2% (95%ci 3.5-7.6). gf was determined when anc had not reached 0.5 x 10*9/l by day 21 (primary gf, n=21) or when anc decreased irreversibly after engraftment (secondary gf, n=4). ci of gf was 2.5%, 6.8% and 23.4% after sct from siblings, mud or alternative donors, respectively (p<0.001) but was similar following myeloablative or reduced-intensity conditioning (5.7% and 4.6%, respectively). pts with a predominant donor population in chimerism testing were given donor cell boost with no additional conditioning (n=10). pts with a predominant host population were given autologous back-up cells (n=8) or a second sct from a different donor (sibling-1, haplo-3, mud-1) with nonmyeloablative conditioning. 18 pts survived > 1 week after second graft infusion and are evaluable for engraftment. 16 pts engrafted within a median of 10 days (range, 5-15). the probability and pace of engraftment was similar in the different approaches. 11 pts (44%) were able to be discharged home and 14 died; 2 early after diagnosis of gf with no intervention, 5 within one week of second graft infusion and prior to engraftment, 2 with no engraftment and 5 early after engraftment from infection (n=3), organ failure (n=1) and gvhd (n=1). with a median follow-up of 19 months (range, 3-68), 6 are alive and 5 additional pts died (relapse-3, gvhd-1, infection-1). the projected 2-year survival for all pts was 23% (95%ci 5-41). interestingly, 4 pts given autologous cells had donor cell recovery, 1 had spontaneous autologous reconstitution within 3 weeks, 2 died within 2 months (gvhd-1, infection-1) with persistent donor cells and 1 remained complete donor until she relapsed 2 years later. in conclusion, treatment of gf with a chimerism directed method can salvage a subset of pts with gf. reserving autologous and/or donor backup cells or an alternative donor is advisable in pts at high-risk of gf. the observation of allogeneic recovery after autologous boost is intriguing and of unknown mechanism. at home autologous stem cell transplantation for haematological malignancies: the role of preparative regimens f. fernández-avilés*, m. rovira, c. martínez, a. gaya, c. gallego, a. hernando, s. segura, l. garcía, j. güell, m. valverde, e. carreras, e. montserrat hospital clínic (barcelona, es) background: the aim of this study was to investigate the impact of the most commonly used regimens on the engraftment, toxicity and readmission rate after autologous stem cell transplantation (asct) in patients managed at home. patients and methods: at home asct (since day +1) was offered to all patients with a good performance status, a travelling time to the hospital of less than 60 minutes, and a caregiver available 24 h per day. in all patients the preparative regimen was administered at the hospital. patients with lymphoma were treated with intensified beac or beam (mg/m 2 ) (bcnu 300, etoposide 1600, cytarabine 800, and cyclophosphamide (cy) 6000 or melphalan 140), patients with myeloma received melphalan 200 mg/m 2 and patients with leukaemia total body irradiation (tbi) 12 gy and cy 120 mg/kg. all patients received the same supportive care, including prophylactic i.v. ceftriaxone once daily. indications for re-admission to the hospital were: patient's or caregiver's desire; uncontrolled nausea, vomiting or diarrhea; mucositis requiring total parenteral nutrition or i.v. morphics; persistent fever; hemodynamic instability, pneumonia, or cardiac and/or respiratory distress. results: seventy-five patients were included in this study. forty-five received beac (n=10) or beam (n=35) (n=45, group a), 19 melphalan (group b) and 11 tbi-cy (group c). recovery (days) of granulocyte count above 0.5x109/l (group a: 11 (9-22), b: 12 (10-26) and c: 12 (10-22)) was significantly faster for patients from group a (a vs. b, p=0.02; a vs. c, p=0.05). fever occurred in 87%, 47% and 73% of patients in the groups a, b and c, respectively (a vs. b, p=0.003). the median (range) days with fever were 2 (1-11), 1 (1-4) and 1 (1) (2) (3) (4) (5) for a, b and c groups, respectively (a vs. b, p=0.01; a vs. c, p=0.05 and who mucositis grade upper to 2 was observed in 40%, 5% and 27% (a vs. b, p=0.006). in group a, 9 (20%) patients needed re-admission by pneumonia (n=4) and persistent fever (n=5), as compared to only 1 (3.3%) because of persistent fever in the rest of patients (p=0.04). conclusions: despite a faster granulocyte recovery, patients managed at home after beac-beam presented a higher incidence and duration of febrile neutropenia, severe mucositis, and high rate of hospital re-admission, especially when compared with patients receiving melphalan. based on this information we have established a more frequent and strict follow-up of patients having received beac-beam and managed at home. outcome of allogeneic stem cell transplantation in first remission for philadelphia chromosome-positive acute lymphoblastic leukaemia following three schedules of imatinib-based chemotherapy b. wassmann, n. goekbuget, h. pfeifer, d.w. beelen, j. dengler, n. kröger, m. stelljes, k. kolbe, w. bethge, m. bornhäuser, h.-j. kolb, m. lübbert, m. stadler, h. serve, d. hoelzer, o.g background: allogeneic sct in cr1 is considered the best curative treatment option in philadelphia chromosome-positive acute lymphoblastic leukaemia (ph+all) pts., but relapse and transplant related mortality (trm) remain significant limitations. although imatinib (im) is considered standard element of front-line therapy, the impact of different treatment schedules is not known. objective: to evaluate overall survival (os), trm and relapse risk (rr) in ph+all pts. following allogeneic sct in cr1 (n=108) after three different im-based chemotherapy regimens. patients: in a prospective, multicenter gmall study, three successive pt. cohorts received im according to one of three schedules: in cohort a (n=43) im was administered alternating with chemotherapy beginning immediately after induction phase ii (ipii)(med. age 44(23-62)yrs.). in cohort b (n=72)im was started after completion of induction phase i (ipi) and given concomitantly with chemotherapy throughout ipii, 1st consolidation (cons.i) and up to yrs.). pts. in cohort c (n=41) received front-line im starting after a 5 day prephase and continued parallel with ip i+ii, cons.i up to yrs.). results: the proportion of pts. transplanted in cr1 was 33(77%), 49(68%) and 26 (64%) in cohorts a, b and c, respectively. median follow-up since allogeneic sct was 11 (0.8-68) , 24 (0.3-51)and 9 (1-22) mo.. median os was 11 mo., 37 mo. and not reached. estimated os at 22 and 48 mo. was 39%, 57% and 71% and 33% and 47%, respectively. trm (35/108; 32%) was mainly due to infections (n=12; 34%) or gvhd (n=11; 31%). 29/35 (82%) trm deaths occurred in pts. aged >35 yrs.. trm at 3 and 12 mo. in pts. aged ≤ 35 yrs.(n=32) was 7% and 17% compared with 21% and 36% in pts. aged >35 yrs.(n=76)(p=0.05). rr was significantly lower in pts. with low or undetectable pretransplant bcr-abl levels (33% at 66 mo. vs. 100% at 18 mo., p=0.0002). conclusion: all tested schedules enable allogeneic sct in a high proportion of pts.. there was a trend towards superior os in cohort c with no significant difference in trm in the 3 cohorts. age and pretransplant mrd levels had the greatest impact on treatment outcome: trm was significantly higher in pts. >35 yrs. both low and negative pretransplant bcr-abl levels were predictive of a low probability of relapse, whereas high levels were associated with a 60% relapse incidence within the 1st year. the impact of posttransplant im on relapse risk remains to be determined. impact of flt3-itd on the outcome of hla-identical stem cell transplant for adult aml with normal cytogenetics: substantial probability of leukaemia-free survival despite increased relapse risk. a retrospective analysis of the ebmt-alwp s. brunet *, m. labopin, a. gratwohl, a. buzyn, j. harousseau, j. jouet, g. socié, a. rambaldi, m. mothy, g. cook, j. sierra, v the prognosis of patients with acute myeloid leukemia (aml) and flt3 internal tandem duplication (flt3-itd) is poor. it is unclear whether this mutation has impact on the outcome of allogeneic stem cell transplantation (allo-sct) for aml in patients with normal cytogenetics (nc). different cooperative groups have obtained controversial results. for this reason, registry data including large numbers of patients are of interest. we analysed the predictive value of flt3-itd on relapse incidence (ri), non-relapse mortality (nrm) and leukemia-free survival (lfs) in patients with aml in 1st cr with nc who underwent a myeloablative allo-sct from hla-identical siblings and were reported to the ebmt. the series included 131 patients, 89 negative itd/flt3 and 42 (32%) flt3/itd positive reported between 2001 and 2007 with a median follow up time of 16 and 10 months, respectively. no significant differences were observed between the two groups regarding gender, median age (38 vs 43 yrs), fab classification, number of induction courses to achieve cr, interval diagnosis to transplant, conditioning regimen, in vitro t-cell depletion, female donor to male recipient and cmv serostatus. in contrast, patients with flt3-itd+ had higher leukocyte counts (wbc) at diagnosis (16 vs 67.9 x10 9 /l, p=0.001) and more frequently peripheral blood was the source of stem cells (43% vs 67%, respectively). univariate (table1) and multivariate analyses demonstrated the adverse impact of flt3-itd for lfs (hr 0.29, p=0.01) and relapse incidence (hr=3.17, p=0.008). it is remarkable, however, 59% of flt3/itd positive patients transplanted for aml in first cr remain alive and disease-free at 2 years. other independent prognostic factors for lfs and relapse were: more than one induction course to achieve first cr. for relapse, higher wbc (hr=2.9, p=0.05) was also a significant prognostic factor. in summary, this analysis shows an adverse impact of flt3-itd on transplantation outcome. despite this finding 59% of flt3/itd positive aml patients in first cr and normal karyotipe remain disease-free at 2 years, a proportion that seems higher than with other treatment options. it is becoming clear that a higher lymphocyte count one month after allogeneic stem cell transplantation (sct) is associated with better transplant outcome in patients transplanted from an hla-identical sibling. however, a predictive role of the day 30 post-transplant absolute lymphocyte count (lc30) in unrelated transplants is not defined. we studied the relationship between lymphocyte counts and other engraftment parameters on outcome in 102 patients with myeloid leukemia (54 acute myeloid leukemia, 38 chronic myeloid leukemia, and 10 myelodysplastic syndrome) receiving myeloablative sct from an hla-a, -b and -dr matched unrelated donor at karolinska university hospital, stockholm from 1996-2006. median recipient age was 37 years (range 0.5-58), 22 patients (22%) were under 18 years. conditioning consisted of cyclophosphamide with busulphan (n=61) or total body irradiation (n=41). bone marrow (bm) was given to 44 patients and mobilized unmanipulated peripheral blood stem cells (pbsc) to 58. median stem cell dose was 6.8 x 10 6 /kg (0. 2-56.4 ). sixty-three patients (62%) received g-csf post-graft. immunosuppression used post graft was cyclosporine with four doses of methotrexate in 97 patients and other treatments in 5. overall survival at 5 years was 61% and relapse-free survival was 56%. the incidence of agvhd grades ii-iv was 62% in patients with an lc30 of <0.2x10 9 /l, 33% if the lc30 was 0.2-1.0 x10 9 /l and 25% in patients with an lc30 >1.0 x10 9 /l (p=0.008). transplant related mortality (trm) was 34% in patients with an lc30 <0.2x10 9 versus 19% (lc30 of 0.2-1.0 x10 9 ) and 0% (lc30 >1.0 x10 9 )(p<0.001). survival was significantly higher in 17 patients with an lc30 >1.0x10 9 /l, compared to 67 patients with an lc30 0.2-1.0 x10 9 /l, and 18 patients with <0.2x10 9 /l (91% vs. 60%, vs. 36% p=0.02 and 0.001 respectively). when analyzed as a continuous variable in multivariate analysis, a higher lc30 was associated with a lower incidence of acute gvhd grades ii-iv, improved survival, less relapse and higher relapse-free survival (see figure) . these results indicate that the lc30 is a robust prognostic factor for transplant outcome in matched unrelated as well as matched related sct for myeloid malignancies receiving either bm or pbsc with or without irradiation conditioning. further research to identify the transplant conditions leading to prompt lymphocyte recovery might lead to global improvements in sct outcome in unrelated sct. s12 o141 gitmo survey on the outcome of 2333 acute myeloid leukemia patients receiving autologous stem cell transplantation in the old and recent era: final results of the multivariate analysis a. olivieri*, b. bruno, g. meloni, m. falda, a. rambaldi, w. arcese, e. alessandrino, r. scime', r. lemoli, m. cimminiello, a. bacigalupo, a. bosi on behalf of gruppo italiano trapianto midollo osseo (gitmo) gitmo registry data files from 2333 acute myeloid leukemia (aml) adults, autotransplanted from 1985 to 2004, have been evaluated to assess the outcome according to age, conditioning and stem cell source; 2032 patients received one autologous stem cell transplantation (asct); 94 two or more asct; 207 allogeneic transplant after failure of asct. patients were categorized in 2 cohorts basing on the transplant era (before or after 1998); the two cohorts were significantly different as regard: age distribution (33% pts older >55 yrs transplanted after 1998 vs 10% pts >55 yrs before 1998); less pts <30 yrs received asct after 1998 (13% versus 28%); preparative regimens: tbi or bu-cy were less common in the recent era; status of disease: 92% received asct in 1rst or 2nd cr before 1998, compared to 90% after 1998; stem cell source: more pts (75%) received pbsc after 1998 versus 16% before 1998. with a minimum 8 yrs followup, os did not differ in the two cohorts: 37% in the 1111 patients autotransplanted after 1998 vs 36% in the 1220 autotransplanted before 1998. as a preliminary analysis suggested a possible role of pbsc in reducing the overall nrm after 1998 (from 14% to 11% after 1998), we made a multivariate analysis to evaluate the main variables (age, stem cell source, transplant era, disease status at transplant and type of conditioning) influencing the outcome, in terms of os, nrm, dfs and relapse incidence. age>55 yrs significantly worsened nrm (1,76 hr), os (1, 76) and dfs (1,5 hr) regardless the transplant era; asct performed in advanced disease (>2nd cr) was associated with a 2,9 times increased risk of relapse and nrm. stem cell source did not significantly influence os and nrm, but bm source, instead of pbsc, was associated with significantly reduced risk (0,79 hr; p=0.012) of relapse. finally tbi regimens were associated with increased nrm compared to bu-cy (1,6 hr). some conclusions can be drawn from this survey: 1-the advanced age (>55 yrs) remains an adverse factor both for nrm, os and for lfs; 2-these data definitely show that pbsc are associated with increased risk of relapse after asct, without major impact on os; 3-regimens including tbi are not recommended being associated with an increased nrm without a significant reduction of relapse. the main efforts in the future should be aimed to reduce the relapse incidence probably by designing new conditioning regimens (and by targeting the minimal residual disease post-transplant) and by a cautious use of pbsc in patients receiving a short consolidation. up-front allogeneic stem cell transplantation as part of induction therapy in newly-diagnosed high-risk acute myeloid leukaemia -an update of a prospective phase ii trial u. platzbecker*, m. füssel, m. schaich, t. illmer, b. mohr, j. schetelig, a. kiani, c. theuser, c. thiede, g. ehninger, m. bornhäuser university hospital (dresden, de) poor-risk cytogenetic aberrations, bad response to the first cycle of induction chemotherapy (ic) or the presence of an flt-3 receptor mutation define high-risk aml (hr-aml) and result in an increased risk of failure of long-term disease control. as a matter of fact, only a minority of this patient group proceed to hsct due to treatment failure or death from infectious complications during ic. therefore, there is substantial need to improve the outcome of these patients with hr-aml. we report results of an ongoing prospective trial evaluating an "early" hsct applied during induction-chemotherapy induced aplasia in forty (n=40) newly-diagnosed hr-aml patients with a median age of 50 years (17-68). a median of 12 days (range 6-34) after the first (n=18) or second (n=22) cycle of ic patients received a reduced-intensity regimen that was based on fludarabine combined with either busulfan (n=4) or melphalan (n=36) followed by allogeneic g-csf mobilized peripheral blood stem cells (pbsc) from related (n=12) or unrelated (n=28) donors. twenty-six patients were not in complete remission before conditioning therapy was started with a median marrow blast count of 20 % (range 6-85). patients with unrelated grafts received antithymocyte globulin and gvhd prophylaxis was performed with cyclosporine a only in all patients. all patients engrafted and went into remission. acute gvhd grade ii-iv occurred in 35 % and extensive chronic gvhd in 30% of patients. with a median follow-up of 17 months (range 1-91) the probability of overall and disease-free survival at 24 months is 68%. early allogeneic hsct as part of primary induction therapy seems to be an effective strategy in high-risk aml patients. comparison of up-front versus minimal residual disease triggered imatinib after stem cell transplantation for philadelphia chromosome-positive acute lymphoblastic leukaemia: interim results of a randomised phase iii study b. wassmann*, h. pfeifer, w. bethge, m. bornhäuser, j. dengler, m. stadler, d. beelen, n. basara, r. schwerdtfeger, k. schäfer-eckart, l. uharek, h. serve, d. hoelzer, o.g. ottmann on behalf of the gmall study group background: detection of minimal residual disease (mrd) following allogeneic sct for philadelphia chromosomepositive acute lymphoblastic leukemia (ph+all) is highly predictive of evolving relapse. we have previously shown that only about 50% of pts. with ph+all who convert to mrd positivity after allogeneic sct achieve renewed pcr negativity in response to interventional imatinib (im), started a median of 4 mo. after sct. failure to rapidly achieve a complete molecular response was almost invariably associated with hematologic relapse. we hypothesized that earlier initiation of im in the setting of a lower leukemic cell burden would increase response rates and improve dfs. objective: to determine whether the earliest possible initiation of im after sct is superior to delayed im triggered by reappearance of bcr-abl transcripts with respect to feasibility, tolerability and duration of molecular and hematologic remission. patients: six wks. after allogeneic sct pts. were randomly assigned to receive im either up-front (cohort 1)(n=17) or subsequent to detection of bcr-abl positivity as determined by real time quantitative and/or nested rtpcr (cohort 2)(n=17). target dose of im was 600 mg, but a lower dose of 400 mg was permitted if deemed necessary because of tolerability. im was scheduled for a total duration of one year of pcr negativity. results: to date, 34 patients have been enrolled. of the 17 pts. randomized to each cohort, 15 each underwent sct in first complete remission (cr1), 2 each in cr2. im was started in 13/17 pts. in the up-front im and 8/17 in the mrd-triggered cohort, with most pts. receiving 400 mg im (10/13 pts. and 5/8 pts., respectively). med. time from sct to start of im was 45 (cohort a) and 82 days (cohort b). after a med. follow-up of 260 (29-985) and 281 (31-1016) days in cohorts a and b, respectively, none of the 30 pts. transplanted in cr1 and 1 of 4 with sct in cr2 relapsed. three pts. (cohort a) died in cr, only 1 one of whom had actually received im. im was discontinued prematurely in 5/13 pts. in the im up-front arm and 3/8 in the mrd-triggered im cohort, due mostly to gastrointestinal toxicity and gvhd. conclusions: with rigorous monitoring of mrd, both schedules of post-sct im (up-front versus mrd-triggered) are associated with a remarkably low relapse rate, although follow-up is still short. tolerability of im is poorer than generally experienced in non-transplanted patients. the routine use of im after sct is a promising strategy to improve outcome of pts. with ph+all. we compared reduced intensity conditioning (ric, n=488) with myeloablative conditioning (mac, n=1596) in patients with aml undergoing hematopoietic stem cell transplantation (hsct), using hla-a, -b and -drb1 identical unrelated donors, transplanted between 1999 to 2005 and reported to the ebmt. in the ric group, median age was higher, transplant was performed more recently, time from diagnosis to transplant was longer, t-cell depletion was less commonly used, and more patients received pbsc vs. bone marrow (p<0.0001). in patients below 50 years of age in cr1, transplant-related mortality (trm), relapse and leukemia-free survival (lfs) were similar in the two groups. in patients in cr2-3 below 50 years of age, 2-year probability of relapse was 31% in the mac group, compared to 51% in the ric patients (p=0.006). in these patients, trm was 36% vs. 26%, and lfs was 44% vs. 36% at two years in the two groups, respectively (ns). in patients with aml advanced disease below 50 years of age, trm, relapse and lfs did not differ significantly between ric and mac patients. at two years, lfs was 22% with mac vs. 12% using ric (p=0.19). in patients above 50 years of age in cr1 and advanced disease, relapse was not statistically different in the two groups. in cr2-3, mac patients above 50 years had a probability of relapse of 18% vs. 47% for ric patients (p=0.009). there were no statistically significant differences in trm or lfs, using mac or ric in patients above 50 years. to conclude, using ric as an alternative to mac in patients with aml undergoing transplants with matched unrelated donors resulted in: no significant difference in trm in patients above and below 50 years of age, significantly increased risk of relapse in patients treated with ric in cr2-3. lfs was similar using the two conditioning regimens. higher incidence of relapse with peripheral blood as source of stem cells in adult patients with acute myelocytic leukaemia autografted in first remission n.c. gorin (1) in the past 30 years the modalities of autologous stem cell transplantation (asct) in first remission (cr1) for patients with acute myelocytic leukaemia (aml) have evolved: the age limit for asct has been gradually extended up to 70 years . total body irradiation (tbi) pretransplant has declined in favour of chemotherapy (ct). leukapheresis products (pb) have replaced bone marrow (bm) as source of stem cells. we were interested in evaluating the potential impact of these modifications on the outcome post asct. a total of 7648 asct (2947 bm, 4701 pb) were reported from january 1985 to december 2006. 79% of all pb transplants were done after 1994. therefore, we compared bm versus pb in patients patients transplanted after 1994: 1226 patients received bm and 4605 pb. the median follow up in the two groups were 46 and 16 (1-161) months respectively. patients receiving pb were older (48y vs 42,p<0.0001), had more aml of the m5,6,7 categories (p<0.0001) and received less tbi (15% vs 30%, p<0.0001). by multivariate analyses, age was a significant factor with lower trm, higher relapse incidence (ri) and lower lfs above 45 years. failure to reach cr within 40 days was associated with a higher (ri) and a lower lfs. the use of pb as compared to bm significantly resulted in a higher ri (51 ± 1% vs 43±2%, p=0.003), and a lower lfs (44±1% vs 50±2%,p=0.04) with only a trend for a slight reduction in trm (9±1% vs 12±1%, p=0.1 ). tbi was not a significant factor for outcome. we finally focused on good risk patients (cr within the first 40 days): the population consisted of 844 patients grafted with pb and 326 with bm. patients receiving pb were older (p<0.0001), and received less tbi ( p<0.0001). again in this good risk population the ri was significantly higher with pb (48 ± 2% vs 35±3%, p<0.01), with a trend for a worse lfs (47±2% vs 57±3%,p=0.1). we conclude that the shift to pb as a source of stem cells in the past 15 years has resulted in increasing the relapse incidence possibly through mobilisation of leukemic cells and /or insufficient purging of the autograft. s14 experimental stem cell transplantation/ stem cell research o146 co-transplantation of placental derived mesenchymal stromal cells produces superior engraftment of umbilical cord blood compared to double unit umbilical cord blood transplantation s. hiwase, p. dyson, s. young, b. to, i. lewis* imvs (adelaide, au) double-unit umbilical cord blood transplantation (ucbt) has been shown to overcome some of the limitations of ucbt, particularly in adult recipients. whether this simply reflects a cell dose effect has not been established. co-transplantation of mesenchymal stromal cells (mscs) has also been suggested as a means of enhancing engraftment and may be appropriate in patients where two suitably matched cords cannot be identified. in this study we have directly compared engraftment rates of double-unit ucbt with msc cotransplantation. mscs were obtained from placental tissue by enzymatic digestion and isolated by plastic adherence. placental mscs demonstrated fibroblastic morphology, immunophenotype and differentiation potential similar to bone marrow derived mscs. in a nod/scid mouse model 4 groups of mice were compared: group 1 received 5 x 10 4 cd34+ cells from a single cord unit (u1); group 2 received 5 x 10 4 cd34+ cells from u1 + 4 x 10 4 mscs; group 3 received 2.5 x 10 4 cd34+ cells from u1 + 2.5 x 10 4 cd34+ cells from u2; group 4 received 2.5 x 10 4 cd34+ cells from u1 + 2.5 x 10 4 cd34+ cells from u2 + 4 x 10 4 mscs. in 4 independent experiments mean engraftment rates were: group 1 28%, group 2 47%, group 3 24%, and group 4 44%. hence msc co-transplantation produced superior engraftment when compared with either single unit (p=0.05) or double unit transplantation (p=0.04). combining results demonstrated the superiority of msc co-transplantation with mice receiving mscs showing mean engraftment of 45% compared to mice who did not receive mscs having engraftment of 25% (p=0.005). transplantation of double ucbt did not improve engraftment when compared with a single ucbt of equivalent dose. additionally, the quality of engraftment was enhanced with msc co-transplantation producing superior engraftment of cd34+ cells. in conclusion, at equivalent cell dose single and double ucbt lead to similar engraftment, suggesting the enhanced engraftment seen with double ucbt reflects a cell dose effect. msc co-transplantation enhances engraftment of both single and double unit cords and may be a potential strategy to be explored in the clinic. infusion of allogeneic mesenchymal stromal cells can delay but not prevent gvhd after murine transplantation m. kambouris *, b. turner, l. sinfield, h. cullup, d. hart, k. atkinson, a. rice mater medical research institute (south brisbane, au) multipotent, mesenchymal stromal cells (msc) are emerging as a means of immunosuppression for patients with steroid refractory graft-versus-host disease (gvhd). despite clinical use, pre-clinical data is still lacking. we established an in vivo model using msc to control gvhd to determine their mode of immunosuppression. we showed in a mixed lymphocyte reaction that msc are highly immunosuppressive and significantly reduce t cell proliferation. we then examined the effects of donor-derived intraperitoneally (ip) or intravenously (iv) injected msc on gvhd in a myeloablative conditioned, full mismatched model of haematopoietic stem cell transplantation (hsct) [ubi-gfp/bl6(h-2b)->balb/c (h-2d)]. 4x10 5 donor-derived msc/mouse were injected 4hrs pre or 24hrs post hsct then mice were monitored daily for gvhd. only mice given msc ip 24hrs post hsct showed a signficant delay in death from gvhd, where median survival was increased by 10 days (day 7 vs day 17, p<0.001, (fig 1) . we then investigated if msc delivered ip pre or post hsct altered cytokine-driven trafficking of cellular effectors known to play a role in gvhd via timed sacrifice. control mice (hsct and no msc or msc and no hsct) were also sacrificed daily for 6 days following hsct. we found that msc given post-hsct enter an environment of significantly increased activated dendritic cells (dc) in the spleen ( fig 2) and at day 3 and day 6 post hsct, mice given msc pre hsct had increased levels of activated splenic dc compared to mice treated with msc 24hrs later. at day 3, we also saw more ifn-gamma in spleen washings in mice treated pre hsct compared to controls. we then determined the role of msc in gvhd control in a minor mismatch model [ubi-gfp/bl6 (h-2b)->balb.b (h-2b)]. msc were given on day 1, 7 or 14; or to mice with established gvhd (>25% weight loss with other surrogate gvhd markers more than 14 days post hsct). mice given msc therapy after gvhd onset showed no increase in survival compared to controls. however, msc administered prophylactically at day 1 (4x10 5 /mouse) or day 7 (1x10 6 /mouse) but not at day 14, showed significantly increased survival compared to controls suggesting that the timing of msc administration is important for their impact on gvhd. in summary, ip injection of msc influences gvhd and survival and may have in vivo influence on activated dc. elucidation of the mechanism by which msc control gvhd may ultimately lead to wider application of their use to assist hsct. s15 o148 immune reconstitution after cord blood transplantation by direct intrabone injection of cells a.m. raiola*, a. ibatici, v. pinto, a. kunkl, f. guialn, f. gualandi, d. occhini, a. dominietto, c. di grazia, s. bregante, t. lamparelli, m. mikulska , g. piaggio, m. podestà, f. frassoni, m. van lint, a. bacigalupo s. martino's hospital (genoa, it) introduction: cord blood transplantation (cbt) has been increasingly used to treat hematological malignancies. since march 2006 we have been investigating a pilot study of cbt in adults by injecting cb cells directly into the bone to overcome the cell dose barrier. pre-clinical transplant models have shown that intrabone (ibm) injection reduces the incidence of gvhd in mhc disparate donor/recipient pairs, suggesting that the way of transplant may affect the immune reconstitution. we have analysed immune reconstitution (t/b/nk cells) at different time-point after ibm-cbt in adults recipients. materials & methods: thirty-three consecutive patients with advanced haematological malignancies a single-unit graft cbt. median age was 35 years (18-62) and follow-up was 8 months (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) for 27 patients survivors longer than 30 days after cbt. median cell dose was 2.6 tnc/kg (range, 1.5-4). donor/recipient hla matching was highly disparate: 5/6 in 27%, 4/6 in 66%, 3/6 in 7%. all but 5 pts received a myeloablative conditioning regimen (tbi 1200cgy in 26/28 pts). gvhd prophylaxis consisted of csa, mmf and pretransplant atg. immunophenotype on peripheral-blood lymphocyte subsets was assessed on day +30, +60 +100, 6 months and 1 yr after cbt. results: early death occurred in 7 pts. all 26 were evaluable and engrafted 100% donor. overall survival was 50% and relapse-related death was 15% (5 pts). median time to neutrophil and platelet recovery was 23 and 40 days, respectively. grade ii acute gvhd was seen in 7 cases (27%) and grade iii in 1 case (4%). moderate/extensive chronic gvhd developed in 6/26 (23%). on day +30, median cd4+ and cd8+ count were as low as 11 and 7 cells/mmc, respectively and remained below normal values up to 6 months. at 1 year, cd4+ and cd8+ count normalized (397 and 250 cells/mmc). nk values reached values as high as 381 and 465 cells/mmc on day +30 and +60, then remained stable up to 1 yr. b cells were extremely low up to day+60, peaked to 445 on day +100 and slighty expanded over time. conclusion: data from our series showed that immune reconstitution is as delayed as in patient who received cb grafts intravenously, with prolonged t-cell lymphopenia and bcells spike since day+100 (komanduri, blood 2007). we also confirmed early nk expansion which could be due to the high donor/recipient pair hla disparity associated with the low cd4+ count. if this may have an impact on the relatively low relapse incidence observed in advanced patients warrants further studies. the effect of mesenchymal stem cells in a rat model of experimental arthritis is dose-and time-dependent e. yannaki (1) , a. papadopoulou* (1), m. yiangou (2) , e. athanasiou (1) , i. batsis (1) , a. athanasiadou (1) , a. xagorari (1) , a. anagnostopoulos (1) , a. fassas (1) (1)george papanicolaou hospital (thessaloniki, gr); (2)aristotle university (thessaloniki, gr) mesenchymal stem cells (mscs) have recently generated a great deal of excitement, due to their potential of differentiation into a broad spectrum of tissues and immune regulation by non-mhc-restriction. the latter capacity has resulted in an effective control of gvhd in several studies; however, the role of mscs in autoimmune diseases has not been extensively investigated in animal models. we aimed to explore the effect of rat mscs on cultured fibrobiast-like synoviocytes (fls) and t-cells from the spleen after adjuvant arthritis induction (aa) as well as their in vivo immunomodulatory potential in a rat model of aa resembling human rheumatoid arthritis. culture of aa-fls in the presence of supernatant from syngeneic msc culture when compared to fls in the absence of supernatant, reduced the aa-fls proliferation (p=0.00004) as well as the proliferation of cona-stimulated aa t-cells (p=0.04). coculture of activated t-cells with syngeneic mscs produced stronger inhibition (p=0.004) in a dose-dependent manner. the inhibitory effect of allogeneic mscs in activated aa t-cells was even stronger and similarly dose-dependent, either by secreted agents (p=0.017) or by cell to cell contact (p=0.00017). low doses of mscs (0.5-5x10 5 cell/recipient) administered iv, intrasplenic or intrabone marrow, at single or multiple infusions, didn't produce significant differences in disease scores of msctreated as compared to control rats. on the other hand, repeated, higher dose (6x10 6 cell/rat), iv infusions of syngeneic or allogeneic y+mscs to female recipients, before the onset of aa (d4 and d9 post aa), resulted in significantly lower arthritic scores with maintenance of a rather normal joint architecture with mild focal synovial hyperplasia/pannus formation and reduced abnormal chondropiasia or bone erosion as compared to control rats. in contrast, symptom worsening occurred when the same cell dose was injected after arthritis onset (d13 and d20 post aa). at the time of sacrifice (d30 post aa), no y+mscs were detected in the spleen or in cultured fls from synovial membrane, by pcr or fish, suggesting that the observed benefit was not due to mscs homing to the target tissues or that migration of mscs may have happened earlier. our data suggest mscs as a potential new therapeutic approach for autoimmune arthritis. however, the benefit of infused mscs seems to be dose-and time-dependent and further studies are required before the results can be clinically translated. objectives: mesenchymal stem cells (msc) suppress alloantigen-induced proliferation, interferon-gamma (ifngamma) production and cytolytic killing in vitro and infusion of third-party msc appears a promising therapy for acute gvhd. however, little is known about the specificity of immunosuppression by msc and in particular the effect on cell-mediated immunity to infectious pathogens. we have studied the effect of msc on virus-specific t-cell responses. results: peripheral blood mononuclear cells (pbmc) from 6 normal donors were stimulated for 5 days with autologous lymphoblastoid cell lines (lcl) (ebv), pp65 peptides (cmv), an adenoviral vector ad5f35 (ad) or allogeneic pbmc (allo), in the presence or absence of third-party msc (msc/pbmc ratio 1:10). msc significantly suppressed proliferation in response to allo (mean 61% suppression, p=0.003), but had less effect on the response to ebv (mean 42% suppression, p=0.016) and no suppression of the response to cmv or ad. msc had no effect on expansion of ebv and cmv pentamerspecific t cells after stimulation with their cognate antigen. elispot assays demonstrated that msc significantly inhibited ifn-gamma production in response to allo (mean sfc/10 5 cells 1125 ±274 without msc, 263±49 with msc) and to a smaller extent to ebv (3181±548 vs 2147±387), but not to cmv (2535±374 vs 2532±311). established ebvspecific cytotoxic t-cells (ebv-ctl) from 5 donors were stimulated with autologous lcl with or without msc for 5 days. the percentage of ebv-pentamer specific ctl was not affected by the presence of msc (mean 1.59% vs 1.52%). msc did not inhibit ifn-gamma production by ebv-ctl (6039±1644 vs 5885±1608). furthermore, cytolytic killing of lcl by ebv-ctl was not suppressed by msc (mean specific lysis 47±3.6% vs 52±4.7% at e:t ratio 30:1). finally, we studied anti-cmv immunity in 2 patiens who received msc for acute gvhd of the gut evolving into chronic gvhd, with a good transient response. pbmc from these patients showed persistence of pp65-pentamer positive t-cells and retained ifn-gamma response to cmv post msc infusion (sfc 69/10 5 pbmc pre-msc, 297 at 1 month and 231 at 3 months). conclusion: msc have little effect on t-cell responses to cmv, ebv and ad, which contrasts to their strong immunosuppressive effects on alloreactive t cells. these data have major implications for immunotherapy of gvhd with msc and suggest that the effector functions of virus-specific t-cells may be retained after msc infusion. intracerebral transplantation of human mesenchymal stem cells after hypoxic-ischaemic brain damage in rat h. huang* (1), j. fan (1), x. lai (1) , q. yu (2) , w. ding (1) , y. wang (2) (1)zhejiang university school of medicine (hangzhou, cn); (2)zhejiang chinese medical university (hangzhou, cn) mesenchymals stem cells (mscs) have dramatic potential of proliferation and multiple differentiation, and recently, neural transdifferentiation of mscs provoke extensive interest. our study was designed to explore the migration, differentiation and the therapeutic benefit of hmscs after hypoxic-ischemic brain damage in rats. hmscs were prelabeled with bromodeoxyuridine (brdu) for 72h before transplantation. animal models of hypoxic-ischemic brain damage (hibd) were built in one month old wistar rats, and three days after hypoxia-ischemia, hibd rats in hmscs-treated group (n=18) received intracerebral transplantation of 5±10 5 hmscs, while hibd rats received pbs of the same volume in control group (n=18). in sham-operated group (n=6) and hibd group (n=6) which just had hypoxic-ischemic brain damage but did not receive any treatment. all of the groups did not receive any immunosuppression agents. behavior tests (alternative electro-stimulus y-maze, by single blind method) indicated that pace memory capacity of rats in hmscs-treated group is significantly better at 4 weeks post-transplantation. he straining also showed that the damage caused by hypoxicischemic had lager pathological changes with bulks of neural cells necrosis and neuropil cavitations formation, even disappearance of normal architecture in cortex and hippocampus, however, it would be significantly improved in pathology in hmscs-treated group. hmscs stayed around the local area of transplantation in three days after transplantation, then migrated mainly along the ventricular system. four weeks later, hmscs were observed to migrate to the parenchyma and distribute throughout the cerebra. three days after transplantation, some hmscs expressed gfap in the local area of transplantation, and a few of cells expressed neurofilament (nf) near blood vessel. four weeks after transplantation, brdu and gfap co-express cells increased which mainly distributed in the local area of transplantation, the cortex, hippocampus and ependymal layer. nestin positive cells did not be detected at any time point. conclusion: after being transplanted intracerebrally, hmscs could migrate to the lesion area, express nf and gfap which indicated their differentiation into mature neurons and neurogliocytes, and promote tissue repair and functional recovery of hibd. furthermore, these results suggested that hmscs could be a promising treatment for hypoxic-ischemic brain damage. identification and enrichment of human bone marrow derived mesenchymal stem cells by monoclonal antibody, zuc3 x. lai, h. huang*, l. huang, j. cao, f. zeng, j. zheng zhejiang uninversity school of medicine (hangzhou, cn) human mesenchymal stem cells (mscs) could be well isolated and expanded from bone marrow and have been widely studied, however, there is no specifically definitive marker of mscs till now. in our previous study, a novel murine monoclonal antibody (mcab) zuc3 was produced by hybridoma technology, which was specifically reactive with human mscs, while showed negative cross-reactivity when screened against a variety of human tissues. flow cytometric analysis showed that zuc3 antigen expression by cultured mscs and mononuclear cells derived from bone marrow were 91.31±2.92% and 0.96±0.28% respectively, and western blotting demonstrated the molecular mass of antigen was about 33kd. zuc3 antigen positive and negative cells were separated from bone marrow mononuclear cells by immunomagnetic activated cell sorting, and plated respectively in human mscs medium consisting of 10% fbs, lg-dmem. the purity of the recovered fractions for zuc3 by macs was 76.82±6.32% by flow cytometry. the positive cells have adhered to culture flask in vitro, and the quantity of adhered cells that had fibroblast-like morphology increased and proliferated during primary expansion period, while the negative cells were observed as round shape cells without any proliferation. it was demonstrated that zuc3 antigen positive cells continued growth with spindle-shape, expansion in long-term culture. phenotype of zuc3 antigen positive cells was analyzed by flow cytometry, the culture-expanded positive cells were uniformly positive for cd29, cd44, cd105, cd106, and lack typical hematopoietic antigens such as cd14, cd34, cd45, hla-dr, which demonstrated that zuc3 positive cells sorted from bone marrow mononuclear cells were mscs. with proper medium, the zucs antigen positive cells could be successfully induced to differentiate into adipocytes, osteoblasts, and neuro-like cells which were positive of neuron markers such as nestin, nse and nf-m. conclusion: zuc3 mcab was a specific surface marker against human mscs for cell sorting. the zuc3 antigen positive cells separated from bone marrow mononuclear cells had potential capacity of high proliferation and multiple differentiation. everolimus enhances the immunomodulatory properties of cd271 positive selected human mesenchymal stem cells r. racila*, w. melchinger, j. finke, r. marks medical hospital university of freiburg (freiburg, de) immunomodulatory properties of human mesenchymal stem cells (msc) suggest their use as a potent cellular therapeutic agent for acute graft versus host disease in allogeneic hematopoietic stem cell transplantation (hct). everolimus is a mtor inhibitor which is currently being tested for gvhd prophylaxis, but its effect on mscs is not yet understood. since mtor inhibition by everolimus might interfere with protein biosynthesis, we tested whether mscs maintain their immunomodulatory properties in the presence of everolimus. in order to obtain a homogenous msc population, positive selection of cd271+ cells from bone marrow aspirates from volunteers using anti-cd271 antibody coated magnetic beads were used. the one-step procedure resulted in an enrichment of cd271highcd73+cd45lowcd34-msc with copurification of cd271lowcd73-cd45high hematopoietic cells. expansion culture resulted in a pure cd271lowcd45lowcd73highcd105highcd34low msc population. we assessed the immunomodulatory properties of cd271lowcd45lowcd73highcd105highcd34low mscs when cocultured with autologues and allogeneic t cells stimulated with anti-cd2/cd3 antibodies or third party peripheral mononuclear cells. the presence of msc in allogeneic t cell cultures did not result in a proliferative t cell response. moreover, stimulation of allogeneic msc/t cell cultures with anti-cd2/cd3 antibodies resulted in suppression of t cell proliferation as determined by thymidine incorporation. a 65-70% reduction of maximal t cell proliferation was seen when a ratio of 1:10 (msc to t cell) was used. in order to examine the effect of mtor inhibition onto the immunomodulatory properties of msc, we incubated the msc for 4 hours with 10 nm everolimus. after several washes the everolimus treated and irradiated msc were used for coculture (ratio 1:10) with stimulated allogeneic t cells. interestingly, everolimus treated mscs showed enhanced suppressive properties and a 85-90% reduction of maximal t cell proliferation. we conclude that everolimus enhances the immunomodulatory properties of cd271 positive selected human mscs and the clinical use of this mtor inhibitor might result in significant immunsupressive activity in allogeneic hct. fibrosis regression after autologous haematopoietic stem cell transplantation in systemic sclerosis l. vija, f. verrecchia, o. verola, a. de raignac, d. sibon, l. michel , d. farge st louis hospital (paris, fr) background: significant regression of clinical skin sclerosis, as assessed by repeated measure of the rodnan modified skin sclore (mrss), has been shown after autologous stem cell transplantation (abmt) in diffuse scleroderma (ssc) patients. objectives: to analyse wether 1) the clinical and histological extent of skin fibrosis can regress after hsct and 2) extra cellular matrix organization and pro-fibrotic signals elicited by tgf-b in ssc fibroblasts derived from skin biopsies vary according to the modified rodnan skin score (mrss) before and after hsct . methods: 38 ssc patients underwent 75 skin biopsies with simultaneous measure of mrss before or after treatment by immunosuppressive drugs with or without autologous hsct. the histological presence and distribution of sclerosis was assessed as : 0 = no fibrosis, + = light fibrosis, ++ = moderate fibrosis, +++ = extensive fibrosis within the papillary (pd), the superficial reticular dermis (srd), the median reticular dermis (mrd) and the deep reticular dermis (drd). human fibroblasts were established by explanting skin punch biopsy (ssc patients or healthy controls) in dulbecco's modified eagle's medium and cells were used for experiments between passages 3 and 8. immunoblotting analyses with anti-phospho smad3 (mayo clinic, rochester, mn), anti-smad3 (clinisciences, ca), and anti-bactin antibodies (santa cruz biotech, ca) and rt pcr for col1a1, col1a2 and pai-1 have been performed. results: double blind optic microscopy analysis of the biopsies seriate sections on standard matrix stains allowed to define 3 histological subgroups : 9 with grade 1 weak fibrosis, 24 with grade 2 moderate fibrosis and 42 with grade 3 severe fibrosis with significant correlation (p< 0.0001) between the grades of fibrosis and the mrss. in skin fibroblast cultures, smad3 phosphorylation levels, representative of tgf-b receptor activity, increased in parallel with the mrss. when compared to pretransplant values, a significant regression of the degree of fibrosis was observed both in the papillary and in the reticular dermis after hsct (n=7), correlated with a decreased mrss. conclusion: in ssc patients, we confirm that the histological extent of skin fibrosis correlates closely with the mrss and further demonstrate that both parameters regress after treatment by hsct. the extent of tgf-b signaling activation in ssc skin fibroblasts appears to parallel the severity of disease. we report here an update of the phase 2 multicenter, prospective study of high-dose immunosuppressive therapy and autologous haematopoietic cell transplantation in 21 nonprimary progressive ms patients showing high disease activity on the basis of both brain magnetic resonance imaging (mri) and sustained clinical deterioration despite conventional therapies. the treatment consisted of stem cell mobilization with cyclophosphamide (4 g/m²) and filgrastim, and conditioning with bcnu (1,3-bis(2-chloroethyl)-1-nitrosourea), cytosine arabinoside, etoposide, and melphalan (beam) followed by antithymocyte globulin (atg, 10 mg/kg) and infusion of unmanipulated peripheral blood stem cells (pbscs). the median follow-up is now 66 months (range 21-105). confirmed progression-free survival was evaluated according to edss disability score as the probability of being alive without clinical progression as compared to baseline. progression-free survival is 58,16% at 8,5 years after hsct (figure 1). seven patients achieved a sustained improvement in their edss score, while 6 patients remained stable and 8 worsened of at least 0,5 points in edss score. disease progression was not associated to clinical relapse. only one patient showed a clinical relapse at +52 months, spontaneously remitted without deterioration of the edss score.since transplantation patients were treated only with symptomatic therapy. hsct was capable to induce sustained progression and treatment-free survival in a large cohort of rapidly deteriorating ms patient with a modest transplantrelated toxicity. experimental animal data and single case reports have documented that allogeneic haematopoetic stem cell transplantation (hsct) potentially alters the course of severe autoimmune disease (aid). experience with allogeneic hsct for these indications is still limited. we therefore undertook a retrospective analysis of the ebmt database to identify patients, which received allogeneic hsct for aid. we identified 31 patients (pts), who underwent 34 allogeneic hsct for aid between 1984 and 2007. follow up was requested by a questionnaire, sent to the referring physicians, asking for diagnosis, transplant procedure, complications, disease status and last follow up. the completed questionnaire was available at the time of abstract submission for 15 pts. median age at transplantation for all pts was 20.07 (0.36-59.3) years; (16 male/ 14 female/ 1 not known). sixteen hsct were performed for non-haematological aid and 18 for haematological aid. three pts had two hsct for aid. eight pts had a previous autologous hsct. mean follow up time was 37.1 months (iqr 6. 2-85.2) . responses were complete for 12 pts (35%), partial for 7 (20%), no change for 6 (17%) and unknown for 9 pts (26%). complete or partial response rate was independent of underlying disease; (haematological vs. non-haematological aid) (p=0.5), gender (p=1.0) or age (p=0.69). cyclophosphamide based conditioning was associated with a better response rate (p=0,066), while atg had no influence on the response. overall mortality was 35%. three patients (9%) died of progression of aid, eight patients (25%) died of treatment related reasons. the median followup time of non-survivors (11 pts) was 5.2 months (range 2. 1-28.4 ) and of survivors (20 pts) 67.8 months (range 28. 2-86.1) . kaplan maier curves displayed similar survival rates for haematological and non-haematological aid. best survival was observed for twin donors (3 pts all survived) and matched donors (related or unrelated). tbi conditioning showed a trend to a higher mortality (p=0.09). in conclusion allogeneic hsct has the potential to induce complete remission in refractory aid. the high mortality rate limits its general use. for selected patients allogeneic hsct based on cyclophosphamide conditioning from a matched donor could be an option and should therefore be prospectively evaluated. 37 patients with intractable form of ms were included in the phase ii clinical trial involving the high dose chemotherapy with autologous peripheral blood progenitor cell (pbpc) support between 1998 and 2006. 33 patients underwent high dose conditioning beam. t cell depletion in vitro was performed on 20 grafts depending on number of harvested progenitors and available resources. 13 patients with not purged graft received atg 4mg/kg i.v. d+1, d+2 after pbpct. in 3 patients pbpc mobilization failed (cyclophosphamide 4g/m 2 + g-csf). one patient refused transplantation after improvement in disability following mobilization. median follow-up is 60 months (12 -108). median edss (expanded disability status scale) of grafted patients at the time of inclusion was 6.5 (5.0-8.5), median edss of grafted patients at last follow up was 7,0 (6.0-10.0). two patients died 31 and 58 months resp. after transplantation because of progression of ms and the cause not related to transplantation, respectively. two patients were lost for follow up. there was no treatment related mortality. at last follow up, the significant improvement (by 1.0 point and more on edss) remains in 1 patient, stabilisation of the disease occured in 23 patients (70%), 9 patients gained disability significantly (by 1.0 point and more on edss). in 3 patients occured transient significant neurological improvement lasting 12-30 months. the development of disability between the group grafted with in vitro purged graft and the group with atg i.v. was not significant. cumulative survival without significant deterioration was 80% in the group with purged grafts and 64,3% in the group without purging in vitro (p=0,178). among 4 mobilized but not transplanted patients 1 improved by 1.5 point on edss, 3 patients worsened by 1.0 point. three serious adverse events related to transplantation were observed: respiratory failure after the onset of mucositis in the patient with severe pontomesncephalic impairment, sepsis with respiratory failure following bilateral pulmonary hemorrhage, both patients needed temporary artificial ventilation and recovered. bleeding related to the inhibitor of fviii occured in 1 patient, remmission has been achieved after the immunosuppressive therapy. as majority of patients with otherwise intractable ms at least stabilized in their disability, we consider the results to be promising and requiring confirmation in a randomized trial involving also less handicapped patients. long-term results of a gitmo retrospective study on haematopoietic stem cell transplantation for paroxysmal nocturnal haemoglobinuria s. santarone*, e. di bartolomeo, a. bacigalupo, e. tagliaferri, a. iori, a. risitano, s. tamiazzo, f. papineschi, a. rambaldi, a. spagnoli, e. angelucci, p. di bartolomeo on behalf of the gitmo allogeneic haematopoietic stem cell transplantation (hsct) may cure paroxysmal nocturnal hemoglobinuria (pnh). in this study we report the results of allogeneic hsct in 26 patients (16 males and 10 females) affected by pnh who were transplanted between july 1988 and may 2007. the median age at time of hsct was 32 years (22-60). the median time from diagnosis to hsct was 33 months (3-208). all patients had received various treatments before hsct including steroids, immunosuppressive drugs and growth factors. twenty-one patients were transfusion-dependent. the median number of packed red blood cells and platelet concentrates received before hsct was 30 (4-500) and 33 (6-86) respectively. the median peripheral hematological counts at transplant were: polymorphonucleates (pmn) 1780 (20-10400) x10 9 /l, hemoglobin 8,7 g/dl (4.6-11), platelets (plt) 79 (6-355) x10 9 /l. thirteen patients developed thromboembolic episodes before hsct. twenty-four patients were transplanted from hla identical sibling and 2 from matched unrelated donors. the donor's median age was 33 years (20-59). the conditioning regimen was myeloablative for 15 patients (busulfan and cyclophosphamide), whereas 11 patients received a reduced intensity conditioning including fludarabine, cyclophosphamide, melphalan and total body irradiation. as graft-versus-host disease (gvhd) prophylaxis, 11 patients received cyclosporine (csa) alone and 13 were given csa and short course methotrexate. two patients received t-cell depleted marrow cells. twenty patients were given bone marrow cells (median nucleated cells 4.0 (2,2-7,5) x10 8 /kg) and 6 received peripheral blood stem cells (median cd34+ cells 3.5 (1.7-8.4) x10 6 /kg). twenty-five patients engrafted with a median time of 17 (10-38) days to reach >0.5 x10 9 /l pmn and 26 days to reach >50 x10 9 /l plt. the probability of developing grade ii-iv acute gvhd and extensive chronic gvhd was 42% and 16% respectively. the transplant related mortality at 6 months was 34%. causes of death were infection in 4 patients, acute gvhd in 1, chronic gvhd in 2, multi-organ failure in 1 and ebv-related disease in 1. as of december 2007, 16 patients are alive with complete hematological recovery and no evidence of pnh at a median follow-up of 109 months (8-212). the 10-year kaplan-meier probability of disease-free survival for the 24 patients transplanted from related donor is 70%. no patient developed thromboembolic disease following hsct. this study confirms that hsct is a curative treatment for the majority of patients with pnh. subcutaneous alemtuzumab is safe and effective for treatment of global or single-lineage immune-mediated marrow failure: a pilot study from the working party aplastic anaemia (wpsaa) a. risitano*, l. marando, c. selleri, b. serio, e. seneca, a. camera, l. catalano, g. scalia, l. del vecchio, a. iori, s. maury, a. bacigalupo, g. sociè, a. tichelli, j. marsh, h. schrezenmeier, j. passweg, b . rotoli on behalf of the wpsaa acquired marrow failure syndromes may globally affect all hematopoietic lineages, as in aplastic anemia (aa), or may selectively involve single lineages, as in pure red cell aplasia (prca) or in agranulocytosis (agr). standard treatment for these conditions includes immunosuppression (is), because of their common cellular immune-mediated pathophysiology. here we report a phase ii/iii pilot study with alemtuzumab (mabcampath®) (ale) followed by low-dose cyclosporine a (csa) as alternative is regimen in 24 patients suffering from marrow failure (13 aa, 7 prca and 4 agr). all patients received subcutaneous ale as escalating dose in consecutive days (3-10-30-30-[30] mg, total dose 103 for aa and 73 for prca and agr), premedicated by betamethasone, clorpheniramine and paracetamol. six patients received one or more additional courses as a result of relapse, so a total of 32 courses were administered. all patients started oral low dose csa (1 mg/kg) on day 7. an intensive anti-infectious prophylaxis has been exploited, which included oral valgancyclovir and cotrimoxazol as anti-cmv and anti-p. carinii agents, respectively. all patients completed the treatment with no relevant injection-related side effect (with exception of fever in some cases), nor significant clinical or laboratory abnormality. a complete lympho-ablation was observed in all patients within 2-3 days, which persisted for several weeks; transient worsening of neutropenia and/or thrombocytopenia were observed in some patients. at a median follow-up of 9 months, infectious events were irrelevant: in cumulative 184 patient-months, 2 hsv and 1 flu have been recorded, all resolving quickly. no cmv reactivation was demonstrated. immune reconstitution was delayed up to several months, especially affecting the cd4+ compartment. patients with adequate follow up (more than 3 months) were assessed for treatment efficacy: 8 aa patients showed 2 cr, 4 pr, 2 nr. in the 5 prcas, there were 4 cr and 1 nr; 3 out of 3 agrs obtained cr. among responding patients, 2/3 saas, 3/4 prcas and 1/3 agr experienced relapses, which were successfully treated by additional course of ale. in conclusion, subcutaneous ale is a feasible and safe is regimen for patients suffering from immune-mediated marrow failure syndromes. preliminary results suggest excellent response rate, and support efficacy even in case of relapse; such favorable risk-to-benefit ratio predicts for this regimen a leading position in the future is strategies. stable mixed chimerism has been described as being beneficial in patients with aplastic anemia after stem cell transplantation, as these patients were protected from severe graft versus host disease and from rejection, the major drivers of mortality in these patients. patients with marrow failure syndrome do not need full donor chimerism as they do not benefit from a graft-versus-malignancy effect. here we describe a protocol resulting in high rate of stable mixed s20 chimerism in patients conditioned with cyclophosphamide + atg and transplanted with peripheral stem cells depleted by campath with add-back of t-lymphocytes post-transplant at different dose levels, according to donor-recipient relationship. we included 10 patients in this protocol, median age was 35,5 (15-58) years, 5 were male, donors were identical siblings in 7, mismatched related in 1 matched unrelated in 1 and mismatched unrelated in 1, respectively. stem cell dose was 11.5 (5.4-40.2 ) cd34 x 10 6 /kg. time to neutrophil engraftment was 15 (8-17) days and to platelet engraftment 14 (8-24) days. median follow-up of surviving patients is 627 days (13-2533) acute grade ii gvhd was observed in 1 patient (with the mismatched unrelated donor), this patient died subsequently, while all other patients remain alive. there is mild chronic gvhd in 1, none of the patients lost the graft. chimerism analysis showed full donor chimerism in 2, transient mixed chimerism (defined as mixed chimerism developing into full donor chimerism) in 1, and stable mixed chimerism in 6, limited to mononuclear cells in 1 and observed in the granulocyte and mononuclear compartment in 5. mixed chimerism remained stable for up to 4 years. a protocol of conditioning with cyclophosphamide and atg and gvhd prophylaxis with t-cell depletion using campath and t-cell addback may induce stable mixed chimerism in a large proportion of patients with aplastic anemia with low gvhd and graft failure risks. i. resnick* (1), a. maschan (2), m. shapira (1), p. trakhman (2), e. skorobogatova (2), d. balashov (2), m. aker (1), r. or (1) (1 introduction: ten years ago we introduced a fludarabine (flu) based conditioning for hematopoietic stem cell (hsc) and umbilical cord blood transplantation [1, 2] for fanconi anemia (fa). this approach dramatically improved results of fa treatment. following initial case reports and small series [3, 4] , two large studies were published: by an italian group and of european experience [5, 6] . aim: here we present an update of our two centers' experience of hsc transplantation for fa. methods: thirty patients (age 3.2 to 31, median 10.3 years) underwent allogeneic hsc for treatment of fa. used conditioning protocols were flu based in 19: flu 150 or 180 mg/m 2 + cyclophosphamide (cy) 20 mg/kg or busulfan (bu) 4 mg/kg or both + antithymocyte globulin (atg) atgam 90 mg/kg or fresenius 40mg/kg. an alternative conditioning (11 patients) was cy-tai/tli or bu8cy40. follow up till december 1, 2007 was 3 to 112 months. results: in the group of patients who got flu based protocols disease free survival (dfs) is 84% vs 27% in pre-fludarabine protocols; p=0.006; or=14.2 (2.5-82.1). difference of dfs in patients transplanted from matched family donor (92%, n=12) vs matched unrelated donor (71%, n=7) was insignificant. causes of death were acute graft-vs-host disease (gvhd) grade ≥2 (n=6) in combination with sepsis (n=3), multiorgan failure (n=4), liver veno-occlusive disease (n=3), bleeding (n=2), chronic extensive gvhd (n=2), secondary malignancy (n=1). there was no clear advantage in using a combination of cy and bu compared with single agent protocols (dfs 3 out of 4 patients). three patients suffered a rejection and underwent a 2nd transplant (2 are alive and well). all surviving patients are disease free and in an excellent clinical condition. conclusion: combination of fludarabine with atg and low dose cy and/or bu is safe, demonstrates low rejection rates and is well tolerated by fa patients. lower doses of flu (150 mg/kg) can be safely used without a pronounced risk of rejection. references: 1.kapelushnik j et al. bone marrow transplant. 1997; 20:1109 . 2.aker m et al. j pediatr hematol oncol. 1999 21:237. 3.bitan m et al. biol blood marrow transplant. 2006; 12:712. 4.maschan aa et al. bone marrow transplant. 2004; 34:305. 5.gluckman e et al. biol blood marrow transplant. 2007; 13:1073 . 6.locatelli f et al. haematologica 2007 92 (10) results: cb recipients were more likely to have advanced leukemia at conditioning (relapse or induction failure, cb vs. bm = 47% vs. 31% in aml patients, and cb vs. bm = 28% vs. 21% in all patients, p<0.0001, p=0.087, respectively). the proportion of all with philadelphia chromosome abnormality was higher (38% to 25 %, p=0.001) among cb recipients. human leucocyte antigen (hla) was serologically mismatched in 93% of patients with aml and 93% of patients with all among cb recipients, while hla a, b, and dr were all matched genotipically for bm recipients. in controlled comparisons using multivariate analyses, among patients with aml, higher rate of treatment-related mortality (trm) (hazard ratio [hr]=1.51, 95% confidence interval [ci], 1.11-2.05, p=0.008) was observed in recipients of cb, which contributed to decreased overall survival (hr=1.45, 95%ci, 1.14-1.84, p=0.003). relapse rate did not differ between the two groups of aml patients (hr=1.27, 95%ci, 0.91-1.79, p=0.16). in patients with all, the relapse rate was higher with marginal significance among cb recipients (hr=1.45, 95%ci, 0.98-2.14, p=0.064). there was no significant difference between these groups for trm (hr=1.36, 95%ci, 0.94-1.95, p=0.10) or overall mortality (hr=1.25, 95%ci, 0.94-1.67, p=0.12) for patients with all. there was no increase in the incidence of acute graft versus host disease in cb recipients among patients with either aml or all despite hla disparity. conclusions: matched or mismatched cb is a favorable alternative stem cell source for patients without a suitable donor. we found different outcomes between patients with aml and all, which indicates the importance of disease-specific analyses in alternative donor studies. decreasing trm is required to improve the outcome for cb recipients, particularly for patients with aml. . the original minneapolis conditioning regimen was used in 106 (96%) pts and modified in 6 (4 or 6 gy tbi: 4 pts; atg : 2). characteristics of the grafts: a single unit was infused in 77 pts (69%), two in 35 (31%). hla compatibility was 6/6 in 6 pts, 5/6 in 36, 4/6 in 60, ≤ 3/6 in 6; 43 pts were abo matched. infused nucleated cells (nc) was 3.1x107/kg (1-9): 2.9 x 107/kg in single units and 3.7 x 107/kg in double units. csa and mmf were used for gvhd prophylaxis in all pts. results: neutrophils recovery was 85±4% at a median of 19 days (0-48) ; 14% pts experienced autologous recovery; 14% had mixed and 72% full donor chimerism at d+100. univariate analysis indicated the low weight, previous transplantation, double units and hla compatibility as significant factors for neutrophil recovery; however multivariate analysis did not find any significant factor. acute gvhd was observed in 34±5% of pts: 21, 12 and 5 pts had grade ii, iii or iv agvhd respectively and chronic gvhd in 16%. non relapse mortality was 12±3% at 6 months; relapse: 22±5%; overall survival: 72±5%. causes of death were relapse in 17 pts, gvhd in 2 pts, venocclusive disease and multiorgan failure in 5, infections in 4 and other toxicity in 3. dfs at 6 and 24 months were 68±5% and 65±5%, respectively. multivariate analysis identified 3 independent risk factors: hla disparity(0+1 vs 2+3), cell dose (<3.1x 107/kg) and age (>44y). this assessment of ucbt after nma confirms the good results of the minneapolis group (brunstein et al. blood 2007) . few events were observed between 6 and 24 mo and dfs remains high and ucbt is a good option in absence of other source of stem cells. background: cord blood transplants (cbt) are associated with delayed or failed engraftment in a significant proportion of patients. we hypothesized that direct intrabone (i.b.) transplant of cord blood cells could improve hematologic recovery. methods: unrelated cb cells were selected for 37 consecutive patients (25 patients. 4/6, 11 patients 5/6 hla and one pt. 3/6 antigen matched). median transplant cell dose was 2.5 x10 7 /kg (range 1.4 -4.2). cb cells were concentrated in 4 syringes of 5 ml each and infused in the supero-posterior iliac crest (spic) (11 patients bilaterally; 26 patients monolaterllay) under rapid general anesthesia (10 min. with propofol). patients' median age was 40 years (18-63); 30 had acute leukaemia (21 with refractory or relapsed disease and 6 high risk first remission leukemia); 2 chronic myeloid leukemia in advanced phase; 2 refractory hodgkin's disease; 2 lnh in advanced phase and 1 aplastic anemia/mds. most patients (n=28) were prepared with conventional tbicyclophosphamide. results: the infusion of cells i.b. in spic was uneventful. six patients are not evaluable because they died of multiorgan failure within 14 days from transplant. the patient with saa/mds did not engrafted and was re-transplanted. all the other patients surviving more than 14 days engrafted (100%). median for pmn engraftment (>0.5x10 9 /l) was day 25 (14-44), whereas for platelets (>20x10 9 /l) it was day 40 (range 22-64). four patients died of infection; one patients died of ptld on day +140. four patients relapsed and died. twenty-two patients are alive in remission at a median follow up of 9 months (range 2-21). from day +30 full donor chimerism was documented in cd3, bone marrow cells and progenitor cells from both the injected and non-injected spic; from day +30, cfc progenitors reached the values of normal individuals in bilateral sites documenting the colonization of the hematopoietc system and possibly an improvement of seeding efficiency. only 4 patients experienced acute gvhd grade ii and 2 grade i; 4 patients have moderate chronic gvhd and one patient extensive cgvhd. conclusion: direct intra-bone transplant of cb cells overcomes the problem of graft failure even when low numbers of hla mismatched cb cells are transplanted. low incidence/severity of acute gvhd was observed. nearly all patients for whom a cb unit was searched were able to undergo cbt. this approach may change the policy of hemopoietic cell transplants. background: the impact of donor-recipient abo matching on outcomes after allogeneic stem cell transplantation (sct) has been a matter of controversy. objective: to evaluate whether abo matching has a significant influence on overall survival(os) in patients(pts) receiving sct for hematologic malignancies. methods: we conducted an individual patient data-based meta-analysis using a pooled dataset extracted from 7 centers(ctrs) not included in previous pooled analyses. we analyzed pts who had received bone marrow or peripheral blood transplantation for hematological malignancies. the primary endpoint was os, comparing pts receiving an abo matched (ma) graft with those receiving a major (mj), minor (mi), or bidirectional abo mismatched (bi) graft using a multivariate (mv) cox model. in addition, os and mortality within 100 days were analyzed with adjustment for confounders. results: a total of 1208 sct, including 697 ma, 202 mj, 228 mi, and 81 bi cases, were analyzed. they included 709 related sct (rsct) and 184 unrelated sct (ursct) from western ctrs; 214 rsct and 101 ursct from asian ctrs. the median age of recipients was 39 years (range,1-69). the probabilities of os [95% confidence interval(ci)] at 5 years among ma, mj, mi, and bi groups were 48% (44-52), 48% (40-56), 45% (38-51), and 37% (26-49) with a median followup of 37 months (range,3-268). overall, adverse impact on os was observed for bi group [hazard ratios (hr) adjusted by age and sex:mj 1.01 (0.80-1.27), mi 1.21 (0.99-1.50), and bi 1.38 (1.01-1.87)]. among recipients of rsct, we observed no significant difference in os between the ma group and any other group. in contrast, mi and bi groups among ursct recipients experienced worse os. sources of heterogeneity were pts who received bone marrow; who had acute leukemia; who underwent transplant at asian ctrs. in mv analysis of os and early mortality adjusted for confounders, mi and bi groups showed inferior os among the subset of ursct, and an increased risk of early mortality was observed only among mj group regardless of donor type [hr (95%ci): mj 1.50 (1.09-2.05), mi 1.23( 0.90-1.70), and bi 1.21( 0.75-1.93)]. conclusion: our meta-analysis demonstrates an overall adverse association between bi transplants and survival, largely accounted for by adverse impact of minor or bidirectional abo mismatching on os in ursct. abo mismatching appeared to have little or no impact on os in rsct. larger studies including ursct of various ethnic backgrounds should be performed. intra-bone marrow injection of umbilical cord blood: no impact on rate of haematopoietic recovery c. brunstein, j. barker, d. weisdorf, t. defor, j. wagner university of minnesota (minneapolis, us) neutrophil engraftment after umbilical cord blood (ucb) transplantation is slower than that observed after adult peripheral blood or marrow transplantation.in order to augment engraftment, we evaluated the safety and potential efficacy of ucb intra-bone marrow injection(ibmi). it was hypothesized that direct ibmi to the marrow microenvironment would reduce hematopoietic stem cells (hsc) loss and improve homing. based on our prior experience with two partially hla matched ucb units, the median time to neutrophil engraftment was 23 days (r:15-41). trial success required: 1) absence of severe adverse events within 48 hours of ibmi, and 2) a 5-day reduction in days to neutrophil recovery (i.e., median anc>500/mcl by day 18). based on these measures of success, 29 patients were planned. all received cyclophosphamide 120mg/kg, fludarabine 75mg/m²/day and total body irradiation 1320 cgy with mycophenolate mofetil and cyclosporine a immunoprophylaxis. all pts received 2 ucb units randomly assigned to either iv infusion or ibmi. the ibmi unit was volume reduced to ~20ml with ~10ml infused into each posterior superior iliac crest under local anesthesia at the pts bedside. ten pts were evaluable (2 were disqualified due to inability to volume reduce the ibmi unit and ibmi unit was not randomized). median recipient age was 35 years (20-44) and median weight 73.6 kg (56-92). seven had acute leukemia and 3 had non-hodgkin's lymphoma. median infused cell doses for the iv and ibmi units were 2.0x10 7 tnc/kg (1.2-3.0) and 1.8x10 7 tnc/kg (1.4-3.4), respectively. pts received two 6/6 (n=1), two 5/6 (n=2) or two 4/6 (n=7) hla matched units. no adverse events were reported with the ibmi procedure. nine of 10 engrafted. median time to neutrophil and platelet recovery (>50,000/mcl) was 21 (17-49) and 69(30-272)days, respectively. complete chimerism was observed with one unit engrafting long term (ibmi unit in 4 and iv unit in 5). seven of 8 evaluable pts had acute graft-versus-host disease (grade ii in 5 and grade iii in 2). with a median follow up of 10 months, the probability of survival is 47%(95%ci,14-80%) at 1 year. based on this interim analysis, the study was prematurely discontinued based on the fact that the predetermined required rate of neutrophil recovery (median 18days) was unlikely with high degree of certainty. while technically easy and safe, neither neutrophil nor platelet recovery after ibmi were not improved to warrant additional patient accrual. j. fernandes*, v. rocha, d. setubal, m. bierings, m.a. champagne, r. pasquini, g. socié, primary graft failure (pgf) is a fatal complication after allogeneic hematopoietic stem cell transplantation (hsct). unrelated donor cord blood transplantation (ucbt) is frequently reported as having a delayed neutrophil recovery and the incidence of pgf varies from 10-25%. second transplants for pgf have been associated with a high treatment related mortality (trm) and a poor outcome, with an overall survival (os) varying from 0 to 30%. we retrospectively analyzed 35 patients who received a second ucbt following pgf after a first ucbt for hematological diseases, between 1995 and 2007. were defined as pgf patients that did not achieved a neutrophil count >500x10 6 /l until 60 days after ucbt or that received a second transplant for pgf in this period. patients having relapsed in the first 100 days after ucbt were excluded. median age was 11.3 years (range, 1-51). diagnoses were: acute leukemia (n=17), myelodysplasia (n=4) and bone marrow failure syndromes (n=14 -2=aplastic anemia and 12=fanconi anemia). patients received either one (n=31) or two (n=4) unrelated cord blood units matched 3-6/6 (hla a and b low resolution and drb1 allelic typing) as donors for the first transplant. median time between first and second transplants was 55 days (22-116) and median follow-up after the second transplant was 18 months. concerning the second transplants, 3 patients received mismatched unrelated bone marrow grafts, 7 haploidentical related grafts, 15 single ucb and 10 double ucb grafts. t-cell depletion was used in 8 cases. conditioning regimen was of reduced intensity in the majority of cases, the most frequent being fludarabine-based regimens. low-dose tbi was administered to 10 patients and serotherapy (alg/atg/other moab) was used in 16 patients. median time to neutrophil recovery was 19 days (10-47) and 19 of 35 patients engrafted (4 of 15 receiving a single cb unit, 7 of 10 receiving double cb units and 7 of 10 other alternative donors). chimerism analysis for these patients at engraftment showed: 16=complete donor and 3=mixed chimerism. twelve patients (34%) developed acute graft-versus-host disease (gvdh) grades ii-iv and chronic gvhd was observed in 5 of the 18 evaluable patients (28%). at 1 year, trm was 39%±1% and os was 41%±8%. in conclusion, these results suggest that second transplants in this set of extremely high risk patients are effective and should be considered early following primary graft failure after ucbt. umbilical cord blood (ucb) is increasingly used as a source of hematopoietic stem cells (hsc) for transplantation in patients (pts) with leukemia. initial reports suggested that ucb transplantation was associated with a 20-30% leukemia freesurvival (lfs). therefore, we evaluated the outcomes in 184 consecutive patients transplanted with ucb after a myeloablative therapy between 1995 and 2007 for the treatment of acute myeloid leukemia (aml,n=81), acute lymphoblastic leukemia (all,n=84), chronic myeloid leukemia (cml,n=13), and myelodysplastic syndrome (mds,n=6). conditioning consisted of either cyclophosphamide (cy) 120mg/kg, fludarabine (flu) 75mg/m²/day and total body irradiation (tbi) 1320 cgy with mycophenolate mofetil (mmf) and cyclosporine a (csa) immunoprophylaxis or the same cy/tbi with equine anti-thymocyte globulin (atg) 90mg/kg and methylprednisolone (mp) immunoprophylaxis. the median age was 16yrs (r: 0.5-52), median weight 57.3kg (r: 8-148). most were male (57%), cytomegalovirus seropositive (51%). grafts were composed of two units in 45% and hla mismatched at one (41%) or two (51%) antigens. the median infused cell doses were 3.4 x 10 7 nucleated cells/kg (r: 0.9-14), 3.9 x 10 5 cd34 cells/kg (r: 0.4-35), 1.2 x 10 7 cd3 cells/kg (r: 0.1-3.2). pts were classified as standard risk (acute leukemia in cr1-2 or cml in first chronic phase, n=138) or high risk (n=46). median follow-up for survivors is 2.8 years (r: 0.7-9.2). engraftment occurred in 164 patients (90% (95%ci, 86-94%). incidence of grades ii-iv and iii-iv acute and chronic gvhd was 43%(95%ci,36-50),14% (95%ci, (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) and 15% (95%ci, 10-20%), respectively. incidence of treatment-related mortality at 2 years and relapse at 4 years was 27% (95%ci, 21-33) and 26% (95%ci, 19-33), respectively. lfs was 43%(95%ci,35-51) and overall survival was 45%(95%ci,37-53) at 4 years. in multivariate analysis, disease risk group was the only risk factor for poor lfs (rr 2.0;95%ci,1.3-3.1;p<.01) and not year of transplant, pt age and weight, nc and cd34 cell dose, treatment regimen, gvhd, cmv serostatus and hla disparity. clearly, lfs after ucb transplantation, despite hla mismatch, represents a valuable alternative to bone marrow and peripheral blood especially for pts requiring urgent transplant or lacking an hla matched unrelated adult volunteer donor. double umbilical cord blood transplantation using reduced-intensity conditioning: a single-centre experience double umbilical cord blood transplantation (ducbt) reduces the time to engraftment and as a result may reduce associated transplant-related mortality. in this report, we describe our experience with ducbt after reduced intensity conditioning. methods: 51 subjects were treated on 2 sequential protocols between 2003-2007. all patients received fludarabine (30 mg/m²/d x 6), rabbit atg (1.5 mg/kg/d x 4) and melphalan (100 mg/m² x 1) as conditioning. gvhd prophylaxis began on day-3 and was administered through day 100 and then tapered. cyclosporine/mycophenolate mofetil (cy/mmf) was given to the first 21 patients, and sirolimus/tacrolimus (siro/tac) was given to the remaining 30 patients. all patients received 4/6 or better allele level hla-matched cord units and a supportive care regimen of antiviral, antibacterial and antifungal prophylaxis. g-csf was administered from day+5 through engraftment. results: the median age was 49 yrs (19-67) and 30 patients (59%) were male. malignant diagnoses were similar although there was a higher proportion of advanced lymphoid malignancy in the siro/tac group. subjects received a combined total of 4.4 x 10 6 tnc/kg and 1.9 x 10 5 cd34 cells/kg, without cohort differences. engraftment kinetics did not differ based on gvhd prophylaxis, with neutrophil engraftment (anc of 500) at a median of 21 days (13-70), and platelet engraftment (20 000/microl) at a median of 42 days (21-185). 5 patients experienced graft loss between days 35 and 102 (3 cy/mmf, 2 siro/tac). grade ii-iv acute gvhd occurred in 20% (33% cy/mmf, 10% siro/tac, p = 0.05). chronic gvhd occurred in 24%, without cohort differences. 100-day trm was 12%, and the risk of relapse at 1 year was 19% (cy/mmf 5%, siro/tac 32%, p = 0.03). atypical and viral infections were common, and ebv-ptld occurred in 5 patients. with a median follow-up of 20 months, relapse-free and overall survival at 1 year are 59 and 74%, without cohort differences. causes of death included infection(6), relapse(5), ebv-ptld(4), and other(4). conclusions: dubct following reduced intensity conditioning results in early engraftment and low 100-day treatment related mortality. the use of siro/tac gvhd prophylaxis is associated with less gvhd than cy/mmf prophylaxis. this approach may lead to a higher relapse incidence, although this may be explained by differences in subject baseline characteristics. dubct should be considered for all adults in whom a suitable adult donor does not exist. (1) rna encoding the wilms tumor protein (wt1) is overexpressed in the vast majority of patients with acute myeloid leukemia (aml) and peptide vaccination studies have demonstrated that wt1 is a target for active specific immunotherapy. preclinical data from our laboratory and that of hans stauss have shown that wt1 mrna-electroporated dendritic cells (dc) stimulate wt1-specific t cells in vitro (van driessche a et al. leukemia 2005; 19:1863 -1871 . therefore, we started a phase i/ii dose escalation trial in which patients with aml received intradermal injections with wt1 rnaloaded dc. feasibility, safety, immunogenicity and antileukemic activity of the vaccine were investigated. high risk aml patients (all but one of them in complete remission) underwent a leukapheresis and monocytes were isolated using cd14 immuno-magnetic beads by clinimacs. dc were generated in 6-day cultures in clinical-grade medium supplemented with serum, gm-csf and il-4 and matured with pge2 and tnf-alpha. keyhole limpet hemocyanin (klh) was added during maturation as a cd4+ t-helper antigen. mature dc were harvested, electroporated with wt1 mrna and used as vaccines. eight patients received four biweekly dc vaccines. a delayed-type hypersensitivity (dth) test was performed 2 weeks following the last vaccination. patients were monitored for minimal residual disease (mrd) by analyzing wt1 rna expression in peripheral blood by qrt-pcr. before and after the vaccination cycle, peripheral blood was collected for immunomonitoring purposes. there was successful dc generation and vaccine production in all patients selected. no serious adverse events or toxicity was seen. a decrease in wt1 rna expression was observed during the course of the vaccination in 4/6 patients who had an increased wt1 mrna level in peripheral blood at the start of dc vaccination. a vaccine-specific immune response was demonstrated in 8/8 patients by dth both to klh as well as to wt1. preliminary data from immunomonitoring in pre-and post-vaccination t cell samples from 4 patients show a mixed t helper (th)1/th2 response towards the klh and the wt1 protein following vaccination. we conclude that vaccination of aml patients with wt1 rnaloaded dc is feasible and safe. furthermore, the vaccine elicits anti-vaccine t-cell responses in vivo and a decrease in wt1 rna expression levels was observed during mrd monitoring in some vaccinated patients, strongly suggestive of antileukemic activity of the dc administered. reduced-intensity allogeneic stem cell transplantation using related haploidentical donors for relapsed or refractory lymphomas: evidence of anti-tumour activity in poor prognosis disease p. corradini*, c. carniti, a. raganato, a. vendramin, l. farina, v. montefusco, r. milani, m. milanesi, p. longoni, l. gandola, c. lombardo, a. dodero istituto nazionale dei tumori (milan, it) haploidentical stem cell transplantation (haplo-sct) has disclosed new possibilities for the treatment of hematologic malignancies in patients (pts) who do not have a hla compatible sibling or a fully matched unrelated donor. the role of haplo-sct in relapsed lymphomas is, at present, unknown. we devised a new strategy potentially useful also for pts without nk alloreactive donors (nk alloreactivity in lymphomas is untested). we report the results of a phase i-ii trial evaluating early add-backs of cd8-depleted donor lymphocytes (dlis) (from 1x10 4 up to 5x10 4 cells/kg from day+45 to day +105 at monthly intervals) after a reduced intensity conditioning (ric) regimen in 22 pts with relapsed lymphomas. ex-vivo and in-vivo t-cell depletion were carried out by cd34+ cell selection and alemtuzumab, respectively. histologies were non-hodgkin's lymphomas (nhl, n=13) [chronic lymphocytic leukemia (cll), n=5, aggressive nhl (hg-nhl), n=7 and follicular cell lymphoma (fcl), n=1] and hodgkin's disease (hd, n=9). the median age was 33 years (range, 15-65). pts received a median number of 4 lines and 86% failed autograft. at median follow-up of 29 months (range, 3-60 months), 10 pts (44%) were alive (n=7 in durable cr) and 12 died of disease (n=8) or non-relapse mortality (n=4). two-year os and pfs of the whole group were 44% (95% ci, 22%-64% ) and 33% (95% ci, 12%-56%), respectively. the main factors influencing 2-year os were age (≤ 45 years: os of 51%) and chemosensitivity of disease at time of sct (75% versus 25% for chemosensitive versus chemorefractory, p<0.02). before dlis, only 2 of 22 pts (9%) developed acute gvhd. cd8-depletion, perfomed using an immunomagnetic method, reduced the content of cd8+ tcells by a median value of 3.3 log (1.7-4). a total of 36 cd8depleted dlis were administered to 16 pts. three pts (18%) developed acute gvhd grade ii-iv following dlis. eight pts receiving cd8-depleted dlis never experienced a relapse. we observed a statistically significant expansion of cd4+ and cd19+, but not of cd8+ and nk cells, at day +120 following cd8-depleted dlis. eight pts had measurable trec/µg at 1 year after sct. spectratyping of t cells demonstrated a polyclonal vß repertoire: pre-transplant 97%, post-transplant 70%. escalated doses of cd8-depleted dlis increase the level of cd4+ in the first 4 months after haplo-sct and are feasible. this is the first report of long-term remissions in lymphomas after ric haplo-sct. k. perruccio*, f. topini, a. tosti, a. carotti, t. aloisi, f. aversa, m. f. after haploidentical stem cell transplantation, immune recovery is slow due to decaying thymic function and extensive t-cell depletion of the graft which is needed to prevent graft-versus-host disease (gvhd). consequently, infectious related mortality is about 30-40%. to address this problem, we investigated the efficacy of adoptive immunotherapy after photodynamic purging of alloreactive t cells (theralux tm , kiadis pharma, amsterdam, the netherlands) in preventing gvhd and improving immune s25 reconstitution. optimized protocol conditions provided 3,260 ± 450 (mean ± sd)-fold allodepletion, full retention of tregulatory cells, and preservation of pathogen-specific t-cell responses (against aspergillus, candida, cytomegalovirus (cmv), adenovirus (adv), herpes simplex virus (hsv), varicella zoster virus (vzv), toxoplasma antigens). here we present the preliminary results of a clinical trial. escalating doses of photodynamically allodeleted donor t cells, i.e., 1.25 x 10 5 /kg, 2.5 x 10 5 /kg, 5 x 10 5 /kg and 1 x 10 6 /kg, were infused into groups of haploidentical transplant recipients. only 1 patient developed grade iii agvhd at the 1 x 10 6 /kg cell dose and responded to immune suppressive treatment. immune assessment analyses revealed that infusion of cell doses equal or greater than 5 x 10 5 /kg are associated with significant reconstitution of t-cell counts and appearance of pathogen-specific t-cell responses. three weeks after infusion, cd4+ and cd8+ t cells were 124 ± 54/cmm and 327 ± 42/cmm (versus 11 ± 4/cmm and 8 ± 4/cmm respectively, in patients receiving t-cell doses below 5 x 10 5 /kg, p = 0.0007). aspergillus, candida, cmv, adv, hsv, vzv, toxoplasmaspecific cd4+ and cd8+ t-cell responses had recovered to frequencies within the normal ranges while they were absent in patients who received t cell doses under 5 x 10 5 /kg (p = 0.0002). in conclusion, this study demonstrates the feasibility, safety and preliminary indications of efficacy of adoptive immunotherapy after photodynamic purging of alloreactive t cells in recipients of haploidentical stem cell transplantation. a larger study will evaluate the impact of these t-cell infusions on transplant related mortality and disease free survival. leukemic stem cells (lsc) are a self-renewing subpopulation of malignant cells which is thought to play a central role in the pathogenesis of acute leukemia. lsc are likely contributing to both disease initiation and relapse and therefore represent an important therapeutic target. resistance of lcs to standard chemotherapy agents is an important consideration for the development of new therapies. in this study, we asked whether lsc of acute myeloid leukemia (aml) can be recognized and eliminated by the anti-tumor function of natural killer (nk) cells. using cd45dimcd34+cd38-as a phenotypic characteristic of the lsc population in aml, we purified these cells from peripheral blood of patients with de novo aml and examined their sensitivity to alloreactive nk cells. for this purpose, the recently described nk cell lines with single kir specificities and mismatched with respect to hla-class i allotype of target leukemic cells (diermayr et al, blood 2007 online) were employed. using the hematopoietic colony forming unit (cfu) assay in methylcellulose, we demonstrated that purified lsc gave rise to hematopoietic colonies which were maintained upon a serial passage. when lsc were preincubated with alloreactive nk cells the growth of aml cfu was significantly reduced. next, we demonstrated that cd45dimcd34+cd38-aml cells do not express the cell surface ligands, mic and ulbp, for the major nk cell activating receptor nkg2d. incubation of purified cd45dimcd34+cd38-cells with histone deacetylase (hdac) inhibitor valproic acid (va) for 2 days, resulted in an up to 2fold upregulation of mic or ulbp ligands in 8/12 tested aml samples. the va treatment had no impact on aml cfu formation in the absence of nk cells, but abrogated the clonogenic growth when applied together with alloreactive nk cells. importantly, the effect of va was limited to lsc since normal hematopoietic stem cells were unaffected, both with respect to expression of nkg2d ligands as well as the colonyforming efficiency. taken together, our results indicate that aml lsc are susceptible to nk cell-mediated cytotoxicity and that expression of nkg2d ligands makes lsc more accessible to nk cells. we propose that adoptive transfer of alloreactive nk cells in combination with pharmacological use of hdac inhibitors merit evaluation as novel approaches to prevent relapses of aml with nk cell immunotherapy. these approaches are now evaluated in an in vivo model of human aml transplanted into nod/scid mice. survival in patients receiving a transplant from an hlamismatched donor (at 10/10 alleles) can be significantly improved by selecting a donor mismatched for hla-dpb1 b. shaw* (1, 2) , n. mayor (2) recipient/donor hla matching is an important determinant of outcome in transplantation using volunteer unrelated donor (vud). there is evidence that matching for hla-a, -b, -c, -drb1, -dqb1 results in a beneficial outcome. the impact of matching for hla-dpb1 is more controversial and few studies have considered the additive effect of --dpb1. we investigated the outcome, dependent on the degree of hla matching at 12 alleles, in 488 recipients of vud transplants for leukaemia. the patients had aml (188), all (157) and cml (143). the majority of patients had t cell depletion as gvhd prophylaxis. 318 patient/donor pairs were matched for 10/10 alleles. of these, 89 were matched in addition for dpb1. 118 pairs had a single allele mismatch (9/10). of these, 30 were matched and 88 were mismatched for dpb1. 52 pairs had multiple mismatches (≥ 2), in 14 dpb1 was matched. we found a significant difference in overall survival when comparing these groups (fig 1, p=0 .006). survival was not significantly different in the 10/10 matched transplants dependant on dpb1 matching (p=0.13). however, there was a significant difference in survival dependant on dpb1 matching within the hla mismatched group (n=170) (4 years: 39% dpb1 mismatched compared 21% dpb1 matched, p=0.008). in particular, in pairs with a single hla mismatch at -a, -b, -c, -drb1, -dqb1, the presence of a dpb1 mismatch improved overall survival (4 years: 43%) compared to dpb1 match (25%, p=0.05). in multivariate analysis including disease, stage, patient/donor age, patient cmv status and gender, the significant survival benefit of dpb1 matching persisted (or 0.502; 95% ci 0.32, 0.78; p=0.002). this effect appeared to be mediated both by a decrease in relapse in the dpb1 mismatched pairs (4 yrs: 47% compared to 87% in dpb1 matched pairs, p=0.004) and a decrease in trm (4 yrs: 37% compared to 64% in dpb1 matched pairs, p=0.006). no differences in acute or chronic gvhd were seen. we conclude that in patients who receive hla-mismatched grafts (at hla-a, -b, -c, -drb1, -dqb1), a donor who is dpb1 mismatched in addition results in a superior outcome compared to one who is dpb1 matched. as physicians frequently have a choice between mismatched donors, we believe that selection by dpb1 within this group will be possible and beneficial. additionally, dpb1 is the only hla allele for which matching status impacts on disease relapse. we speculate that this may suggest a difference in the functional mechanisms of dpb1. the cxcl16/cxcr6 chemokine pathway specifically targets alloreactive t-cells to the bone marrow of mice with leukaemia r. van der voort, v. verweij, f. maas, j. vos, m. philippens, t. de witte, h. dolstra* umc st radboud (nijmegen, nl) allogeneic stem cell transplantation is an effective treatment for patients with leukemia. this therapeutic effectiveness is largely attributed to the graft-versus-tumor (gvt) response during which alloreactive donor t cells eradicate minor histocompatibility antigen (miha)-expressing tumor cells in the bone marrow (bm). unfortunately, subsets of alloreactive t cells recognize miha in healthy tissues, such as the gut, skin and liver, resulting in graft-versus-host disease (gvhd). thus, there is a strong need to separate gvt responses from gvhd. an appealing strategy to achieve this goal would be to increase the migration of alloreactive t cells to the tumorcontaining bm and/or block the infiltration of gvhd-prone organs. although the homing receptors for migration into the gut and skin are extensively studied, those involved in t cell trafficking to the bm are largely unknown. here, we characterized the pathway that alloreactive t cells exploit to infiltrate leukemia-containing bm. t cells were isolated from bm and spleen (control organ) from mice with or without acute myeloid leukemia (aml) that had previously received an allogeneic bm-transplant (bmt). in addition, we used naïve mice as controls. the expression of activation markers and a panel of 14 putative homing receptors was determined by flow cytometry, and the level of several chemokines by real-time pcr and elisa. migratory behavior was analyzed by transwell migration assays and flow chamber assays. where bm of naïve and bmt mice contained few t cells, amlcontaining bm was heavily infiltrated with cd4+ and cd8+ t cells with an effector/effector memory phenotype. interestingly, bm of mice with leukemia predominantly recruited cd4+ and cd8+ t cells expressing the chemokine receptor cxcr6. in contrast, the number of cxcr6+ t cells in bm from naïve or bmt mice without aml, and in all spleens was generally low. furthermore, the ligand for cxcr6, cxcl16, was expressed by activated macrophages and dendritic cells and its expression level increased in bm of mice with aml as compared to controls. finally, we show that cxcr6+ t cells are strongly attracted by soluble cxcl16 and show increased binding to the endothelial adhesion molecule vcam-1 upon stimulation with cxcl16. in conclusion, our data suggest that the cxcl16/cxcr6 axis is a novel pathway that specifically regulates the recruitment of alloreactive t cells into leukemia-containing bm. the pd-1/pd-l1 co-inhibitory pathway is involved in functional impairment of alloreactive cd8+ t-cell responses against leukaemia w. norde (1) , f. maas (1) , h. fredrix (1) , a. schattenberg (1), m. kester (2) , f. falkenburg (2) , r. van der voort (1) donor lymphocyte infusion (dli) following allogeneic stem cell transplantation (sct) is a potent treatment for hematological malignancies. the effectiveness is attributed to the graftversus-tumor (gvt) response, during which donor cd8+ t cells become activated by minor histocompatibility antigens (miha). consequently, these alloreactive cd8+ t cells clonally expand, acquire effector functions and kill mihapositive malignant cells. after the response, most cd8+ t cells die through apoptosis, while a small population remains to form a pool of long-lived memory cells. unfortunately, tumor relapses still occur despite the presence of miha-specific memory t cells in the patient for many years, suggesting that these memory t cells become impaired over time contributing to tumor evasion. recently, a crucial role for the co-inhibitory pd-1/pd-l pathway in inhibiting the function of virus-specific cd8+ t cells was demonstrated in chronic viral infections. here, we investigated the role of the pd-1/pd-l pathway on the functionality of miha-specific cd8+ t cells. initially, we analyzed expression of activation and co-signaling molecules on miha-specific ctl following restimulation with irradiated miha-positive ebv-lcl plus il-2. we observed that pd-1 expression was upregulated on miha-specific ctl within 24 hours after stimulation. in addition, we analyzed miha-specific cd8+ t cells from leukemia patients after dli. we observed increased levels of pd-1 on miha tetramer-positive cd8+ t cells, compared to tetramer-negative cd8+ t cell subsets in the same patient. knowing that sustained expression of pd-1 is associated with diminished proliferative capacity of virusspecific cd8+ t cells, we determined whether blocking the ligation of pd-1 could increase the capacity of miha-specific cd8+ t cells to proliferate in vitro. interestingly, we revealed that the addition of anti-pd-l1 antibody augments the proliferation of miha-specific cd8+ memory t cells. notably, addition of peptide alone or in combination with an irrelevant antibody did not induce proliferation of these cells. finally, we observed that primary leukemia cells and various hematological cancer cell lines express the pd-1-ligand pd-l1 following stimulation with ifng. these data suggest that pd-1/pd-l1 interactions in the tumor environment may impair miha-specific cd8+ t cell function, and intervening with this co-inhibitory pathway may result in improved gvt immunity after allogeneic sct. the graft versus-leukemia-effect in b-cell chronic lymphocytic leukemia (b-cll) appears to be less efficient compared to myeloid leukemias. since the aberrant coexpression of cd5 in association with cd19 is a hallmark of b-cll, we asked if both molecules might serve as a combinatorial t-cell target. b-cll cells as third party in mixed-lymphocyte reactions (mlr) induced t-cell anergy. we used recombinant proteins comprising a single chain (sc) fvcd5 antibody (ab) fused to human interleukin (il)-2 via the hinge region of human immunoglobulin (ig)g1 in conjunction with a bispecific s27 cd3x19 ab (bsab; clone okt3xhd37) to saturate cd19 and cd5 binding sites on b-cll cells. consistent with the absent immunogenic potential, b-cll cells pretreated with scfvcd5 ab, native il-2 and cd3x19 bsab were not recognized by allogeneic resting t cells. in contrast, b-cll coated with the dimeric scfvcd5-il-2 and cd3x19 bsab induced profound stimulator cell dependent allogeneic and autologous t-cell proliferation. in addition, t cells from fresh peripheral blood mononuclear cell samples from b-cll patients (n=4) pretreated with dimeric scfvcd5-il-2 and cultured in the presence of cd3x19 bsab expanded by 2-3 log within 7 days. notably, addition of soluble exogenous il-2 to both types of culture remained inferior compared to the cd5-targeted il-2 delivery. t-cell proliferation and expansion also resulted in cell-mediated cytotoxicity. in conclusion, a dual targeting approach using aberrantly expressed tumor cell surface molecules for membrane delivery of dimeric il-2 molecules using a fusion cytokine in conjunction with a bispecific antibody as shown for b-cll represents a strategy to reverse tumor-induced t-cell anergy. hurler's syndrome (hs), the most severe form of mucopolysaccharidosis type-i causes progressive deterioration of the central nervous system and death in childhood. allogeneic-stem cell transplantation (sct) before the age of two years halts disease progression and prolongs life. graft-failure and mixed-chimerism (40-50%) limit the success of sct for hs. unrelated-cord blood transplants (ucbt) are suggested to be a good alternative option for bone marrow, however, little is known about risk factors for outcomes after ucbt for this disease. we have analyzed 93 hs children that received an ucbt from 1995 to 2007 and were reported to eurocord or transplanted at duke university. median age at ucbt was 1,3 (0,2-4) yrs, and median follow up was 24 (3-140) mths. the donor was hlaidentical (hla-a and b by low resolution and hla-drb1 by high-resolution) in 13 cases (16%) and incompatible in 67 cases (84%: most with 1 (54%) and 2(26%) hla disparities). the median nucleated cell dose/kg and cd34+/kg at infusion were respectively 7.2 (2-22)x10 7 and 2,3 (0,5-17)x10 5 . with the exception of 5 patients, all received a busulfan/cyclophosphamide (+fludarabine: 6) regimen. all patients received atg or campath (4). results: median days to neutrophil and platelet recovery were 22 (10-46) and 35 (13-82) days, respectively. mixedchimerism was found in only 4%. all patients had normal enzyme levels after engraftment. in multivariate analyses for neutrophil recovery, a cd34±dose of >2.3x10 5 /kg (hr=2.0; p= 0.015) was associated with increased probability of recovery. acute-gvhd (grade ii-iv) was observed in 27%, while chronic-gvhd was seen in 10% at 2 years. two years overall survival (os) and disease/event free survival were 78% and 70%, respectively. for 2 years os, time from diagnosis to ucbt more than 6 months was associated with increased risk of death (6% for those children transplanted earlier and 30% for those transplanted later: p=0,04). in conclusion, outcomes following ucbt for hurler syndrome are encouraging. ucb appears to be a good alternative allogeneic stem cell source to transplant children with hs. earlier transplantation and higher cell dose are associated with better outcomes after ucbt for hs patients. introduction: transplant for mpsih has been associated with high rates of morbidity, mortality and graft failure. some of the difficulty of the transplant reflects pre-transplant co-morbidities including significant cardiac dysfunction, respiratory including upper airway compromise and visceral organ enlargement. in recent pharmacological enzyme replacement therapy (ert) has been available that will ameliorate many of these comorbidities. manchester protocol. we give 12 weekly doses of ert prior to conditioning and continue until donor cell engraftment. patients and methods. we have performed 71 transplants in 54 patients in manchester. 19 of these patients have received ert with transplant. in 15 of these 20 there has been full intensity conditioning with pk guided oral (hd) busulfan (40mgs/m²/dose, n=7) or iv busulfan with pk monitoring but not adjustment. results: of these 15 patients 7 received oral busulfan and the most recent 8 have received iv busulfan. of these 15 patients 14 are alive with full donor cell engraftment. one patient experienced primary graft failure following a cord transplant and was successfully transplanted with a second cord and despite achieving full donor chimerism died of adenovirus 18 months after the graft. donor source for these 15 patients was mud cord in 9, other mud in 2 (pbsc donations) and matched family donors in 4 (pbsc in 2). efs survival for this group is therefore 93% with a median follow up of 21 months. our engrafted survival rates for previous patients treated on previous protocols are 68%, including second grafts to maintain donor chimerism. 4 previous patients receiving ert and reduced intesity grafts experienced 3 rejections. administered busulfan was significantly higher in those receiving hd busulfan -they received a mean of 26mgs/kg compared with the previous dose of 20mgs/kg -(p<0.001, students t-test). discussion: manchester is the largest centre for mps transplants in europe and this is the largest collection of patients treated with both ert and sct. there were concerns that pre-transplant ert would increase rates of graft rejection. these data refute those concerns. we believe that the improving results reflect reduced patient co-morbidity after ert, improved delivery of full intensity chemotherapy with busulfan and better donor matching including use of unrelated cord blood as a donor source. s28 o180 haematopoeitic stem cell transplantation for chronic granulomatous disease -a single-centre experience e. soncini* (1), m. slatter (1) , l. jones (1), s. hughes (1), t. flood (1) , d. barge (2) , g. spickett (2) chronic granulomatous disease (cgd) is a primary immunodeficiency affecting phagocytes; mutations in genes coding for subunits of nicotinamide adenine dinucleotide phosphate cause failure of oxygen metabolite generation, resulting in impaired microbial killing, susceptibility to bacterial & fungal infections and lung & gut inflammation. antibacterial & antifungal prophylaxis with cotrimoxazole & itraconazole improve short and medium term survival but do not cure cgd; patient quality of life is burdened by frequent hospitalizations, recurrent diarrhoea, inflammatory lung damage, failure to thrive and a 50% risk of death by the third decade of life. haematopoietic stem cell transplantation (hsct) can cure the disease. we report our single centre experience. between 1998 and 2007, 20 patients underwent hsct for cgd in newcastle. a retrospective analysis of medical records to november 2007 reviewed age at hsct, donor type, hla matching, conditioning, immuno-reconstitution, significant post-hsct complications, growth, lung function and outcome. 15 received bu/cy, 3 flu/melph, 1 flu/bu, 1 bu/mel conditioning. 10 had sibling, 10 unrelated donors. 17 had 10/10 hla-match, 2 9/10 hla-match (a, c mismatch), 1 8/10 hla-match (dr & dq, hvg direction only). 15 received marrow, 3 pbsc and 2 cord blood transplants. 18 of 20 (90%) patients were alive with functioning neutrophils with follow up 0.25-9.25 years. 3/4 with significant inflammatory lung disease had an improvement in lung function. all 9 patients with colitis had resolution of symptoms, with impressive catch-up growth. 1 patient had cytopenias secondary to post hsct-cmv infection, there were no other significant infections, only 2 had significant gvhd, 1 liver that resolved with treatment and 1 gut gvhd that resolved with prolonged steroid treatment, with resulting osteoporosis and avascular hip necrosis. 2 patients died, both of complications relating to active fungal infection. 13/18 survivors are off prophylactic medication with excellent life quality. hsct is curative for patients with cgdcomplications are less frequent and outcome better in those without pre-existing infection or inflammation. hsct should be considered early in the treatment of cgd, particularly if a suitable donor is available. long-term outcome and quality of life after haematopoetic stem cell transplantation in osteopetrosis: a single-centre experience d. moshous (1) background: malignant infantile osteopetrosis (miop) is caused by defective osteoclast function. impaired bone resorption is leading to increased bone density resulting in progressive marrow failure and extramedullary haematopoiesis. the abnormal bone remodelling generates also compression of cranial nerves, especially the optical nerve, causing various degrees of visual impairment up to complete blindness, starting early in life. some patients present also neurological deterioration which is not clearly explained by the pathophysiology of miop. the genetic basis of op is heterogeneous, until now four genes with autosomal recessive inheritance have been identified: tcirg1, clcn7, ostm1 and rank-l, but there seem to be also autosomal dominant forms. in the absence of treatment, children with miop die early in life often because of bleeding or infection. the only curative treatment available consists in haematopoetic stem cell transplantation (hsct). patients and methods: we report on 40 hsct which were performed in 35 patients (17 females and 18 males) in our unit between 1984 and 2007. stem cell sources were bone marrow, peripheral blood stem cells and one unrelated cord blood unit. the donors were haploidentical related donors (26/40), hla-identical siblings (9/40), related matched donors (2/40), mud (1/40) and unrelated cord blood (1/40). the majority of patients received hsct before the age of three months (16 days to 26 months), with a mean age at first transplant of 5,5 months. two patients have been transplanted at an older age, at 3 years 4 months and 8 years 5 months respectively. conditioning consisted mostly in busulfan and cyclophosphamid based regimens, in 5 cases fludarabine was added, atg was used in 12 cases. the injected cell number varied from 0,2 -19,4 x 109 nucleated cells/kg, corresponding to 2,3 -73,2 x 106 cd 34+ cells. outcome: for two patients the follow up is still too short to conclude. out of 33 evaluable patients 16 survived (overall survival: 49%). the causes of death were interstitial pneumonia in 4 patients, veno-occlusive disease in 2 patients, multiorgane failure in 2 patients, rejection in 4 patients, transplanted related toxicity in 3 patients, bleeding in 1 and infection in 1 patient. survival was better in the setting of hlamatched hsct 8/12 (66,7%) versus hla-mismatched hsct 8/21 (38%. three patients received 2, one patient 3 hsct. detailed data especially on long-term neurological development will be provided. (2) ). there was no graft rejection; mixed donor chimerism occurred in 36%. the incidence of an acute gvhd ≥ ii was 19 %, that of a chronic gvhd ii 11 % with no difference between mrd and mud. cgvhd was associated with more than expected mri changes 2 years after hsct (p < 0.05). moreover, patients with unexpected disease progression often displayed poor immunologic reconstitution and late viral problems. 14 of 18 pts. (= 78 %) in group ii/iii remained neurologically stable with good quality of life; 12 of these continued to visit a regular school. all pts. in group i deteriorated after hsct; however, 4 out of 9 pts. stabilized and full dementia could be prevented. conlusion: the above results confirm the role of a favorable disease stage for hsct outcome, as already shown (c. peters, blood, 2004) . in addition, the data demonstrate the importance of transplant-associated factors (e.g. cgvhd). in our view, the superior neurologic outcome after mrd-bmt allows a less restrictive indication for hsct in more advanced ccald patients. j.-s. kühl* (1), g. strauß (1), b. weschke (1), w. köhler first successful bone marrow transplantation for x-linked chronic granulomatous disease using a sibling donor selected after preimplantation female sexing and hlamatching j. reichenbach* (1), h. van de velde (2), m. de rycke (2), c. staessen (2) , p. platteau (2), p. baetens (2), t. güngör (1), (1), i. libaers (2) (1 objective: allogeneic hematopoietic stem cell transplantation (hsct) from an hla-identical donor is currently the only proven curative treatment for chronic granulomatous disease (cgd). hsct with alternative donors is associated with higher morbidity and mortality. the objective was therefore to perform in vitro fertilization (ivf) and preimplantation hlamatching combined with female sexing for hsct in x-linked cgd. methods: ivf followed by preimplantation genetic diagnosis (pgd) was used to identify a female hla-genoidentical embryo in a family who needed a suitable donor for their boy affected with cgd. two pgd cycles were performed. results: in the second pgd cycle two hla-genoidentical female embryos were transferred and a pregnancy was obtained. the mother had a normal delivery of a healthy girl at term. conventional hsct had to be performed, due to insufficient cell numbers in the cord blood source, at age 12 months of the donor and age 5 8/12 years of the recipient and resulted in complete stable donor chimerism and immunological reconstitution up to 17 months post hsct. conclusion: hsct after ivf and combined female sexing and hla matching offers a new therapeutic option for patients with x-linked primary immunodeficiency such as cgd needing hsct but lacking an hla-genoidentical donor. a multicentric comparative analysis of outcomes of hla identical related cord blood and bone marrow transplantation in patients with beta-thalassemia or sickle cell disease n. kabbara* (1), f. locatelli (2) , v. rocha (1) most patients with beta thalassemia major (tm) or sickle cell disease (scd) can be cured by hematopoietic stem cell transplantation (hsct) from either cord blood (cb) or bone marrow (bm). one advantage of cb is the absence of risk associated with donation. in order to compare outcomes after hsct with cb or bm, we studied 388 patients with tm or scd who received hla identical sibling cb (n=72) or bm (n=316) allografts between 1994 and 2005. in order to avoid center and period effect, only centers that performed both types of hsct during the same period were included. we compared the incidence of hematopoietic recovery, acute and chronic graft-versus-host disease (gvhd), disease-free survival (dfs) and overall survival (os) after cb and bm transplantation. compared to bm, cb recipients were significantly younger (median of 6.2 y versus 7.2 y), smaller (19 kg vs 24 kg), and were transplanted more recently (in 2001 vs 1998) . the bm group consisted of 127 (40%) scd and 189 (60%) tm patients, and the cb group, 26 (36%) scd and 46 (64%) tm patients. the indications for transplantation in scd were not statistically different between cb and bm groups. more tm patients belonging to pesaro ii-iii risk classes received bm (65%) compared to cb (36%) (p=0.004). there were also differences in the conditioning regimen (more frequent use of atg/alg in the bm group and of fludarabine and thiotepa in the cb group) and gvhd prophylaxis (more methotrexate-containing therapy in the bm group compared to the cb group). in addition, the nucleated cell content was 10 times higher in bm compared to cb. the table below shows the non-adjusted univariate analysis for outcomes for all patients according to the stem cell source used. in tm patients, the 5 year-dfs rates were 87% and 83% for bm and cb recipients, respectively, and in scd patients, 92% and 85%, respectively. in a multivariate analysis adjusted for age and type of hemoglobinopathy, dfs was not statistically different between cb and bm recipients (rr=1.4, p=0.34). in conclusion, patients with tm or scd had excellent outcomes after hsct whether they received stem cells of cb or of bm from an hla identical sibling. these results strongly suggest that cb transplantation from hla identical siblings should be pursued when possible to avoid the discomfort and risks of a bone marrow harvest. s30 o185 communication biases during informed consent for unrelated bone marrow transplantation in adult thalassaemia patients g. caocci* (1), s. pisu (2) (1), g. la nasa (2) (1)r. binaghi hospital -asl8 (cagliari, it); (2)cagliari university (cagliari, it); (3)gimema data center (rome, it); (4)oxford university (oxford, uk) although there are numerous guidelines to evidence-based medicine, few explain how to build the information into patient oriented decision-making. several factors may hamper physician-patient communication and compromise the informed consent process. in an attempt to eliminate some of the barriers to understanding, we investigated the main factors of communication between physicians and adult thalassemia patients transplanted from an unrelated donor twenty-five transplanted thalassemia patients and 12 physicians were given a questionnaire to investigate the communicated, perceived and recalled risk for mortality and graft-versus-host disease (gvhd), the acceptable risk percentage, besides the motivation and external influences underlying the choice of undergoing hsct. the risks of dying or gvhd perceived by the patients were significantly lower than those communicated by the physicians (p=.002, p<.001, respectively). also the perception of severe gvhd as a life-threatening condition was much lower (p=.004). younger patients perceived a significantly higher risk of dying than older patients (p=.003). females perceived a significantly higher risk of developing gvhd or dying than males (p<.05 in both cases). the median percentage communicated for the risk of dying was significantly higher than the percentage remembered by the patients (30% vs 20%; p=.003). the median percentage considered to be acceptable for the risk of dying was significantly higher in the patients compared to the physicians (30% vs 20%; p=.008). hence, the balance between the risks and the benefits of the transplantation procedure had not been fully understood by the patients. therefore, it is important that physicians make every effort to overcome communication bias, heuristics and distorted processes of remembering or understanding that could possibly influence the informed consent and decisionmaking process. in adults with aml (n=24578), 43% received an hla identical family hsct, 35% an autograft, 14% an hla matched unrelated hsct, 8% an hla mismatched hsct(3% haplo, 4% hla unrelated and 1% cord blood). in adults with all (n=10932), 44% received an hla identical family hsct, 23% an autograft, 21% an hla matched unrelated hsct, 12% an hla mismatched hsct (4% haplo, 6% hla unrelated and 2% cord blood). for adults in first cr with all or aml given a hla identical sibling donor, 5-years overall survival (os) was 57%; it was 49 % for those given an autograft and 49% for those patients given a hla matched unrelated transplant. for adults with aml given an unrelated cord blood or a haplo-transplant in first cr, overall survival at 2 years were 62% and 48% respectively. many studies have been performed and published on behalf of the alwp with the collaboration of many ebmt centres. thanks to the ebmt centres, physicians and data managers, we have been able to collect specific information for those studies. therefore, in 2007, 5 studies and one editorial have been published in the most important journals in the field of medicine, hematology and transplantation, 13 retrospectives and 2 prospective studies are on going and 3 of 5 abstracts were oral presentation at the last ash meeting. moreover, the establishment of 5 subcommittees within the alwp, in the different fields of transplantation has brought enthusiasm among participants and increased the number of participants to up to 50 in the alwp meetings. we would like to thank w arcese, b labar and o ottman for their input as heads of alternative donor, developing centres and phi+all subcommittees, respectively. they have accomplished two years as subcommittee's heads. we would like to welcome a nagler, s giebel and j esteve as new heads of alternative donor, developing centres and molecular markers subcommittees, respectively. i take also this opportunity to thank m mohty and c schmid for their activity in the alwp. we hope that the enthusiasm inside the alwp can motivate young physicians and more ebmt centres to join us with their ideas and input to perform important studies for the ebmt community and importantly to continue improving the field of transplantation for patients in need. the main objective of this new subcommittee is the analysis of the impact of main molecular markers on the outcome of stem-cell transplantation. several molecular lesions described in recent years are contributing to a biological characterization of acute leukemia and provide relevant prognostic information. in this regard, mutations of flt-3, nucleophosmin (npm1) or cebpa genes, frequently found in the large subgroup of aml with normal karyotype, arise as the most relevant prognostic factors in this subset of patients. nonetheless, the precise role of different transplant modalities for each of these aml categories remains to be defined. thus, a recent analysis performed within the alwp confirmed a higher risk of relapse associated with flt-3 itd after myeloablative allotransplant transplant from an hla-identical sibling, although the procedure seemed to benefit a significant proportion of flt-3 itd aml patients transplanted in first cr, according to the survival plateau observed. nonetheless, the optimal transplant policy in these molecularly-defined high-risk patients is not firmly established. the sc is also aimed to investigate the results of transplant in patients with rare subtypes, such as aml with translocation t(6;9)/dek-can or aml with 3q26/evi1 rearrangement. given the low frequency of these entities, only those studies performed in large cooperative groups have the potential to elucidate the role of different transplant strategies for these specific subtypes. another objective of the sc is the design of biologically oriented studies analysis on the outcome of transplant in adult all, which might help to clarify the controversial role of transplant in early phases of the disease. thus, specific studies for wellrecognized prognostic categories of adult all such as mllrearranged, hypodiploid varieties or results of transplant among different t-all subtypes can be sponsored by the alwp. finally, the emergence of molecularly targeted therapy such as tyrosine kinase inhibitors in philadelphia positive all or conjugated anticd33 monoclonal antibody in aml are changing the natural history of the disease and, therefore, might modify indications and results of transplant in patients treated with these agents. reduced-intensity conditioning m. mohty* institut paoli-calmettes (nantes, fr) in the last decade, the so-called non-myeloablative or reduced-intensity conditioning (ric) regimens for allogeneic stem cell transplantation (allo-sct) have emerged as an attractive modality to decrease allo-sct-related toxicities. indeed, ric allo-sct represents an attempt to harness the well-documented immune graft-versus-tumor (gvt) effect while attempting to overcome toxicity. the work of different pioneering groups rapidly proved that this approach is feasible in several disease settings or patients' categories, and had the added benefit of expanding the transplant option to patients who are ineligible for myeloablative allo-sct. unfortunately, and despite several thousands of patients receiving ric allo-sct reported to international registries, the true value of ric allo-sct in the management of haematological malignancies is, as yet, difficult to delineate. several reasons can help understanding these difficulties. a consistent definition of "non-myeloablative" or 'ric' regimens is still lacking. the different ric regimens comprise a continuum that overlaps with standard myeloablative regimens. where in theory, reduction of the "inflammatory" component of the conditioning as in ric regimens may lead to the reduction of the incidence of gvhd, gvhd remains a matter of concern after ric allo-sct, raising questions about the need for continuous immunosuppression and its corollary of long-term infectious complications. also, the notion or definition of "ineligibility" for conventional standard allo-sct is not clearly defined. the goal of the ric subcommittee within the acute leukemia working party of ebmt is to address through retrospective and prospective studies, the specific role of ric allo-sct in net health outcomes that should include, in a specific disease setting, an analysis of disease-free survival and overall survival balanced against treatment-related toxicity, quality of life, complications and death. transfusion of donor lymphocytes (dlt) for treatment of leukaemia relapse after allogeneic stem cell transplantation (sct) has been a milestone in the field of immunotherapy against malignant disease. it can be regarded as the proof of principle for the graft-versus-leukemia effect. however, in contrast to the impressive results obtained in cml, results in acute leukemias have been inferior, although there is a small subgroup of patients that obviously responded. the immunotherapy subcommittee of alwp has set the goal to study the role of cellular immunotherapy more in detail with respect to different diseases as well as distinct variants of allogeneic sct. hence, a large retrospective analysis of dlt in relapsed aml has been performed, and similar investigations have been started in all. further, the role of dlt after haploidentical and ric transplants is in the focus of ongoing studies. finally, the conditions and the results of prophylactic or preemptive dlt, given after sct to chimeric patients in cr or in a minimal residual disease status, are currently investigated. use of hsct for patients with acute leukemia is increasing in europe. therefore many ebmt centers have been established in all countries, mainly in the new eu countries. although the number of transplants in eastern countries is growing and the results are improving, the scientific activity appears still insufficient. enhancement of the scientific collaboration in eastern europe is a principle goal of the developing centres subcommittee. to reach this goal it is planned to create a panel of young investigators representing major centres from the region. this panel is expected to discuss current clinical practice as well as to share laboratory experience. on this background it will be able to propose and coordinate multicentre studies including: 1) prospective clinical trials, 2) retrospective analyses, 3) biological studies on hsct for acute leukaemia. other goals of this subcommittee is to evaluate outcomes of hsct performed in eastern countries as well as to develop studies and methods for analyzing the role of center effect on the hsct outcomes. with these objectives two studies have been performed. in the first one, we have analyzed 640 patients (aml, 459; all, 181) treated with hla matched related donor in first complete remission in 10 countries, from1990 to 2006. with this study we have observed that results of mrd-hsct for acute leukemia in eastern europe improved over time, as a consequence of decreased nrm. another study performed was the update of the lancet paper which has demonstrated a center effect in major ebmt centers. we have updated this study and we were able to show a persistent centre effect but improvement of lfs of hla identical hsct for adults with aml in first cr over the period 1987-2005. other study proposals have been discussed such as the use of economic and social markers in order to evaluate transplant outcomes in the different eu countries in collaboration with yearly survey of a gratwohl. currently, we can find an alternative donor for almost all patients without an hla identical sibling donor, such as hla matched unrelated, haploidentical and cord blood unrelated donor. the objective of this subcommittee is to study the feasibility of those different strategies and their outcomes and compare their results. with the active collaboration with eurocord, we are able to perform studies of unrelated cord blood transplantation. a recent study in collaboration with eurocord, that will be presented during this meeting, has shown the impact of kir ligand mismatching on decreasing relapse and improving lfs in ucbt recipients with acute leukemia. also comparative studies of outcomes after ucbt and haplo have been performed in children and adults. one of the main objectives of this subcommittee is to improve the hla data of the database and therefore be able to study the impact of specific hla alleles on outcomes of hsct recipients, such as the impact of permissive hla-dpb1 alleles. with the collaboration with the immunobiology wp and donor registries, we hope to improve the data of hla high resolution typing of unrelated donors. other projects are i) to study the influence of gene polymorphisms with outcomes of alternative hsct ii) to compare various conditioning regimens in this setting; iii) to target therapy with the novel drugs preand post-alternative transplants and iv) to compare immune reconstitution after mud vs. haplo vs. cbt. update of the registration study v. rocha*, e. polge, m. arat, v. koza, h. wandt, t. ruutu, a. ferrant, g. milone, p. defabritis, w. arcese, l. verdonck, a. bosi, b. allione, a.l. herr, f. pinto, c. arrais, s. nabhan, g. socie, g. dini, e. gluckman, m the registration study is a joint prospective, multi-center, non randomized trial of ebmt al and pds wps and eurocord. primary aim is to evaluate the contribution of different strategies of treatment (matched sibling donor;msd and an alternative hsct) for adults with al. secondary aim are to compare, in a first step, policy of each centre, feasibility of each transplant modality, time to transplant. in a second step, to compare in a intent to treat analysis lfs, rr and trm according to different transplants strategies. the date of the registration of the patients is considered as the date of hla typing test. for each patient, a specific questionnaire has to be completed specifying the initial strategies and each change in those strategies : at registration, after 3-6 months after the resgitration and , each 3 months during the first year after hsct, and twice a year for the following 2 years. the intention to treat will be defined, for each patient by the choice of the treatment planned and reported by the each center and the real modality performed according to the last option, and donor availability. between december 2003 and december 2006, 727 adults, enrolled by 34 ebmt centrers, were registered for the study, 498 had aml, 216 all and 13 biphenotipic al. hla typing was performed for 448 patients at diagnosis, 179 after first cr, 43 in primary refractory la, and 53 in more advanced phase (18 in cr2 or more and 35 in relapse). a msd was found for 283 patients. the choice registered for the remaining patients was: 215 alternative donor (search for ud, cb, or haplo); autologous hsct (88 pts). chemotherapy alone (141 pts).the registration of patients has been closed on december 2006.the follow up of patients will proceed for 3 years. the final analysis will be performed on december 2009. a randomised phase iii study comparing conventional chemotherapy to low dose total body irradiation-based conditioning and haematopoietic cell transplantation from unrelated donors as consolidation therapy for elderly (>60y) patients with aml in first complete remission d. niederwieser* on behalf of osho /fhcrc/hovon sakk/austrian/french and alwp-ebmt study objectives: efficacy of allogeneic related and unrelated hematopoietic cell transplantation (hct) after reduced intensity conditioning as a consolidation treatment for patients with aml in complete remission or refractory anemia with excess of blasts (raeb) in comparison with a non-transplant approach. patients with a matched sibling or with an unrelated donor, who have entered cr1, will be eligible for randomization in a 2 (transplantation):1(non-transplantation) fashion. patients without a donor will receive post-remission therapy without transplantation. blaise, f. frassoni, m. kuenz, b. rio, n. russel, p. rebulla, g. sanz, j. garcia, j. cornelissen, c. navarette, d. niederwieser, a. nagler, g. socié, a. sureda, v umbilical cord blood (ucb) cells from unrelated donors have several advantages as compared to other sources of stem cells for allogeneic use, such as prompt availability, decreased risk of graft-versus-host disease and easy collection with little risk to the mother or to the newborn. ucb has been shown to contain sufficient progenitor cells to provide durable engraftment. however, because of low infused cell doses, single ucb transplantation (ucbt) in larger children and in adults may be associated with delayed engraftment and increased risk of graft failure. transplantation of double cord blood units (ducbt) represents a strategy to overcome this limitation. the results of both pediatric and adult ducbt studies suggest that this approach is safe, and is associated with higher engraftment rates and improved transplant outcome, in both the nonmyeloablative and myeloablative settings, as compared to single ucbt. some preliminary data also indicates a reduction in relapse with double ucb transplantation. in this view, we will discuss a proposal of a randomized trial comparing single versus double ucbt for patients with hematological malignancies. a writing committee has been established to discuss all the aspects of this protocol that will be presented during the alwp session. overall the leukaemia free survivals (lfs) at five years for patients autografted in first (cr1) and second (cr2) remission have been respectively 49 and 36% in adults and 66 and 49% in children, with some improvement in outcome after 1994. recent prospective trials from single institutions or national groups have confirmed a lfs of 50% at 5 years in adult patients autografted in cr1. several randomized studies in the pre "high dose ara-c" era had shown superior outcomes of asct over conventional chemotherapy. the only randomized study using hdara-c in the chemotherapy arm (us intergroup study,p cassileth nejm 1998, m slovak blood 2000) indeed has shown better results for asct in good risk patients by cytogenetics, better results for hdara-c in intermediate risk patients and better results for genoidentical allogeneic transplants in the high risk group. numerous retrospective studies using the ebmt registry have shown equivalence for lfs and overall survival (os) when comparing asct to allogeneic transplantation with unrelated donors and/or allogeneic transplants with reduced intensity conditioning (ric), with a higher relapse incidence (ri) for asct but a reduced transplant related mortality (trm). despite this situation, the annual reports of ebmt activities indicate a clear drop in asct for aml associated to a steady increase of allogeneic transplant activity, using ric and all cell sources including cord blood. the practice of asct itself has changed over the years: less than 15% of the patients still receive marrow as a source of stem cells and mobilization of stem cells in peripheral blood has become the s33 standard. total body irradiation (tbi) which has been shown to have the highest anti leukemic effect is used in only 15% of the patients for conditioning. in vivo purging consisting of chemotherapy consolidation courses given before mobilisation is not standardized, and higher cd34+ cell yields have been shown to contain leukemic cells and to be associated with a higher relapse incidence (n feller, leukemia 2003) . a recent retrospective survey of the awlp-ebmt (presented at this meeting) indeed indicates that the number of consolidation courses given before autografting with peripheral blood influences the outcome. in patients autografted with marrow, peripheral blood early (interval cr1-transplant<80 days) and peripheral blood later (>80 days) , the lfs at 3 years have been respectively 52,46 and 36%. the source of stem cells and the interval from cr1 to transplant have been significant in multivariate analyses. another recent retrospective survey from alwp-ebmt (nc gorin, jco, 2008 in press) has compared asct to allogeneic stem cell transplantation in patients with core binding factor mutations (inv 16 and t(8;21)) and shown equivalence in outcome for patients transplanted in cr1 with lfs around 70% at 3 years. overall the data accumulate to confirm previous and already old observations that asct remains an interesting alternative therapeutical approach for the treatment of : -older patients -patients considered for ric and/or unrelated donors transplants, with bm,pb or cord blood -chemosensitive aml (so called good risk patients) including thoses reaching cr1 rapidly (rapid remitters) and those carrying a cbf mutation. since pb has replaced almost totally pb as source of stem cells, for practicality reasons, all effort should be made at increasing in vivo purging aggressively, with, as much as feasible, minimal residual disease monitoring by molecular biology. there is still a need for randomized studies in good and intermediate risk patients, while high risk patients should be allografted whenever possible. in the largest prospective mutlicentre study initiated by the ebmt and comparing filgrastim-mobilized peripheral blood progenitor cell (pbpc) to bone marrow, patients transplanted with pbpc developed more often chronic gvhd. however outcomes, which where overall and disease free survival, relapse and transplant related mortality were similar in both cohorts. at the time of first publication we were aware that long term observation was needed to ultimately determine the role of both source of stem cells. indeed, outcome differences due to difference in the composition of the graft might become apparent only years after transplantation. we therefore planed a follow-up study in order to asses the long term outcomes, general health status, social integration as well as the occurrence of malignant and non-malignant late effects in both treatment groups. the patient accrual of the initial study took place between february 1995 and september 1999, including 350 patients transplanted for leukaemia from their hla-identical sibling. in september 2007 we sent out a questionnaire to all centres which had participated to the study. the questions included information on survival, cause of death, relapse, secondary malignancies, chronic gvhd and its treatment, the occurrence of non-malignant late effects such as hypothyroidism, bronchiolitis obliterans, sicca syndrome, blood counts at last follow-up as well as question on general health status and social integration. so far, 20 (48%) from the 42 centres responded, including 105 of 212 patients (49%) alive at 3-year follow-up. ninety-eight of the 105 patients reported patients are alive, and 7 have died after the 3rd year of follow-up (gvhd n=4, relapse n=2, other n=1). chronic gvhd is still present in 23 patients (22%), secondary malignancies occurred in 4 cases (5%), non-malignant complications in 24 (22%). the median karnofski score as a measure of general health was > 90% (range 70-100). eightyeight (83%) patients have returned to work or to school. during the late effect working party meeting 2008 in florence, italy, we will provide preliminary data on the longterm outcome, and compare both patients groups, patients transplanted with pbpc to those who had received bm. this is retrospective multicenter analysis on the incidence and risk factors of cardiovascular events after allogeneic hematopoietic stem cell transplantation (hct), in 548 long term survivors treated in 10 ebmt transplant centers. these patients received hct between 1990 and 1995 and surviving ≥1 year after transplantation. the median age of the patients at last follow-up or at time of a vascular event was 35 years (range 3-72 years), and the median follow-up time 9 years (1-16 years). twenty (3.6%) out of 548 patients developed an arterial event in at least one arterial territory. the cumulative incidence of first arterial event 15 years after hct was 6% (95% ci, 3%-10%). the cumulative incidence for patients with a high global cardiovascular risk score, including arterial hypertension, diabetes, dyslipidemia, physical inactivity and smoking was 17%, as compared to 3% in those with a low risk score. in univariate analysis, older age at last follow-up, all cardiovascular risk factors taken individually, and acute gvhd were associated with a higher risk of a cardiovascular accident. in multivariate analysis age older than 30 years at last follow-up, and patients with a high global cardiovascular risk score had a 6.4-fold and 9.8-fold increase, respectively of the relative risk for an arterial event after hct. thus, long term survivors after allogeneic hct are likely to develop cardiovascular risk factors, and present an increased risk for premature cardiovascular accidents after allogeneic hct. update on the phase ii/iii study of the incidence and outcome of vod with the prophylactic use of defibrotide in paediatric stem cell transplantation (vod-df study) s. corbacioglu* university children's hospital (ulm, de) along with graft versus host disease (gvhd) and cytomegalovirus (cmv) infection, veno-occlusive disease (vod) is one of the most frequently encountered serious complications after stem cell transplantation. the currently reported overall incidence of vod ranges from 5% to more than 60% in children who have undergone stem cell transplantation. defibrotide (df) is a polydisperse oligonucleotide derived from porcine intestinal mucosa with antithrombotic and protective properties on the microvasculature but minimal hemorrhagic risk. in large, multicenter, international phase i/ii trials targeting patients with severe vod df has emerged as a promising therapy for vod. the vod-df study is a prospective international multicentre phase ii/iii study with the aim to assess the beneficial effect of defibrotide on the incidence, morbidity and mortality of vod in children at high risk to develop vod after myeloablative stem cell transplantation (nih trial number: nct00272948, eudract number:2004-000592-33) . the study, co-sponsored by gentium and the ebmt, is open for recruitment since january 2006 with currently 27 participating centres in 10 countries. the prospective recruitment period is 3 years with a sample size of 270 patients. the current recruitment status as of january 2008 is 220 patients with an average recruitment rate of 10 patients per month. in november 2007 the first data safety monitoring board (dsmb) meeting evaluated safety data and mortality rates of the first 120 study patients who completed 30 days of follow-up. as conclusion of this meeting the types of adverse events described were considered typical for this patient population with no unexpected toxicities. no significant difference was observed in the number of adverse events between the prophylaxis arm compared to the control arm. therefore it was unanimously concluded that there were no safety considerations of concern arising from the defibrotide-treated patients on the prophylactic arm at the present time. for the determination of the finale sample size an interim analysis will be performed at 240 patients. the recruitment of this sample size should be completed by early this year. in the meantime the following centres either completed or will complete the initiation process in order to join the study: ankara, antalya, istanbul, and prague. the registration study is a joint prospective, multi-center, non-randomized trial of ebmt al and pds wps in cooperation with the eurocord. primary aim is to evaluate the contribution of different strategies of treatment (msd and alternative hsct) for children with al. secondary aim are to compare, in a first step, policy of each centre, feasibility of each transplant modality, time to transplant. in a second step lfs, according to different transplants strategies, rr, trm will be compared. the date of the registration of the patients is considered as the date of hla typing test. for each step, a specific questionnaire has to be completed: at registration, after 3-6 months, at hsct; each 3 months during the first year after hsct, and twice a year for the following 2 years, followup forms will be completed. the intention to treat will be defined, for each patient by the choice of the treatment reported to be planned by the centre in each questionnaire, the real modality performed according to the last option, and donor availability. between december 2003 and november 2007, 271 children, enrolled by 24 ebmt centres, were registered for the study. children had all (at diagnosis or in cr1, 94 patients; at first relapse or in cr 2, 73 patients; 12 in more advanced disease), or aml (at diagnosis or in cr1, 71 patients; with refractory disease, at first relapse or in more advanced phase, 17 patients). a msd was found for 83 children, the choice registered for the remaining 183 children was: chemotherapy alone (45 pts it is estimated that every year in europe about 500-600 children become donors of hematopoietic progenitor cells for siblings undergoing allogeneic stem cell transplantation. the aim of the study is the analysis of donor safety and occurrence of early side effects related to bone marrow (bm) or peripheral blood stem cells (pbsc) collection in pediatric siblings qualified to be a donor. the study is based on questionnaires send to transplant centers. following endpoints are analyzed: complications during anesthesia, complications related to bm collection, complications of catheter placement, complications related to apheresis, number of nights spent in hospital and psychological issues in donor immediately after stem cell collection and during one year follow-up. since january 2007, 112 donors (median age 9 years, range 11 months -18 years) were registered. the source of stem cells was pbsc and bone marrow, in 43 and 69 patients, respectively. preliminary data indicate that the rate of complications is very low, thus the procedure is safe for donors. due to low patients accrual, retrospective donors registration over a period of 1 year before the start of the study is being proposed. the 6th meeting of the ebmt pediatric diseases working party will be held june 2-4, 2008 in poznan (poland) and for the first time will be accompanied by the meeting of ebmt pediatric nurses. the second announcement with detailed information concerning preliminary scientific programme, call for abstracts, registration, hotel accommodation, travel, important dates etc. is available on the meeting web page www.bokiz.pl/ebmt-pds-wp-2008, which is also accessible using the link from the ebmt pds wp web page. physicians sessions (monday and tuesday, june 2-3), nurses sessions (tuesday, june 3), and joint session (wednesday, june 4) will create an excellent opportunity to summarize recent progress in the field of hsct from pediatric point of view, to initiate new ideas, and to improve an understanding and collaboration between nurses and physicians involved in pediatric hsct. apart from that the meeting will be a unique platform for initiation new trials, interaction, and cooperation. more than 30 outstanding speakers will present various aspects of topics of the meetings. however, organizers expect also a lot of interesting original oral as well as poster presentations, and an exciting and stimulating discussion. therefore, all participants are invited and encouraged to submit their own experimental and clinical results for presentation during the meetings. the deadline for abstract submission is monday, march 17, 2008. early registration is possible until monday, march 31, 2008. twenty grants from the ebmt covering registration fees and local accommodations will be awarded by the scientific committee on a competitive basis to physicians or nurses (information concerning application for grants is available on the meeting web page). pharmaceutical companies, manufactures of technical equipment and software as well as publishers are invited to display their products at the industrial exhibition which will part of the meeting. detailed information about poznan, including information how to get to poznan, is available on the web page www.city.poznan.pl. it is a real honor and a great pleasure to invite everybody interested and involved in pediatric hsct to poznan to attend the 6th meeting of the ebmt pediatric diseases working party and 1st meeting of the ebmt pediatric nurses, poznan (poland), june 2-4, 2008. 235 why is important to built a paediatric nurse group inside pds wp? s. calza* (1), v. van de crommert (2) (1)children's hospital (genoa, it); (2) transplantation group initiated a prospective, randomized, parallel-group, open-label phase iii, multicenter study, comparing vtd (arm a) with td (arm b) for mm patients progressing or relapsing after autologous transplantation. inclusion criteria were: patients in first relapse after at least one autologous transplantation, including those who may have received velcade or thalidomide before transplant. exclusion criteria: subjects with severe neuropathy or non secretory mm. 366 patients will participate (183 in each arm). primary study end point was time to progression. secondary end points included toxicity, response rate, eventfree survival and overall survival. treatment was scheduled as follows: velcade 1.3 mg/m² will be given as an i.v bolus on days 1, 4, 8 and 11 followed by a 10-day rest period (days 12 to 21) for 8 cycles (6 months) and then on days 1, 8, 15, 22 followed by a 20-day rest period (days 23 to 42) for 4 cycles (6 months). in both arms, thalidomide will be given at 200 mg/day per os for one year and dexamethasone 40 mg/day per os four days every three weeks for one year. thrombosis prophylaxis was strongly recommended as well as valacyclovir prophylaxis (in arm a) against reactivation of varicelle zoster virus. patients reaching remission could proceed to a new stem cell harvest. however, transplantation, either autologous or allogeneic, could only be performed after achieving the one year treatment. response was assessed by ebmt criteria, with additional category of ncr. adverse events are graded by the nci-ctcae, version 3.0. as of january 15, 2008, 118 patients entered the study. 79 in france (ifm 2005-04 study), 10 in italy, 9 in germany, 8 in switzerland (a sakk study), 5 in belgium, 3 in austria and the czech republic, 1 in the uk. 118 are assessable: 74 males, 44 females; median age: 60 yrs (range35-72), number of autologous transplant: one: 38, two: 80. of these patients, 60 were randomly assigned to receive vtd and 58 to receive td. treatment was discontinued in 11 patients. an interim toxicity analysis was planned to be performed when the first hundred patients had been included. this interim analysis is ongoing (january 2008). other preliminary studies demonstrate that vtd is a highly active and relatively well tolerated regimen. the combination is used in the relapse setting, as well as first line, consolidation and maintenance. in this protocol, the starting doses of velcade and thalidomide are relatively high and the duration of treatment is long. we will assess the superiority of vtd over td in the relapse setting. protocol eu-dract number: 2005-001628-35. tyrosine kinase inhibitors (tkis) are the accepted first line treatment of choice for the majority of patients with chronic myeloid leukaemia (cml) although allogeneic stem cell transplantation (sct) remains the only potentially curative procedure. although second generation tkis have shown promising results, in young patients resistant or intolerant to imatinib and with a low ebmt score allogeneic sct is still considered the best second line therapy. however, despite its curative potential, a significant proportion of patients relapse following allogeneic sct. the gold standard for the treatment of relapse following allogeneic sct is the infusion of lymphocytes from the transplant donor (dli). however, dli may result in significant complications including graft versus host disease and bone marrow aplasia and using escalating doses protocols, responses may be protracted. there is consequently a need for an alternative treatment for patients that relapse following sct, that is both efficacious, faster acting, easier to administer and safer. dasatinib has been shown to be effective in treating patients that are resistant or intolerant to imatinib and as a result constitutes a good candidate treatment option for imatinib intolerant or resistant patients that relapse following sct. the cml sub-committee has launched a prospective phase ii to investigate the efficacy of dasatinib in this setting. this study will concentrate on patients 18 years of age with ph+ cml whose disease has relapsed after transplantation from an hla-identical sibling or an hla-matched unrelated donor and have not responded to withdrawal of immunosuppressive treatment where this is possible. enrolled subjects will be commenced on dasatinib 100mg qid and receive treatment for 12 months. continuation of treatment beyond 12 months will be at the investigator's discretion. the primary end-point will be complete molecular remission at 12 months secondary end-points will include complete cytogenetic remission rates, overall survival and proportion of patients requiring dli. donor lymphocyte infusions will be administered to all patients in whom dasatinib has been discontinued indefinitely or there is evidence of disease progression during dasatinib therapy or there is evidence of disease relapse after initial response tom dasatinib. the study will open in the summer of 2008 and will aim to recruit 50 patients in 3 years. interested ebmt centres are invited to participate. (11) allogeneic hematopoietic stem cell transplantation (hsct) as first line therapy for patients with chronic myeloid leukaemia (cml) has been replaced by imatinib. the role as second line therapy in patients who failed imatinib treatment is a matter of debate. second generation tyrosine kinase inhibitors (tki) have already proven their efficacy in this setting. early transplant related mor¬tality of allogeneic hsct is considered to be too high. however, transplant outcome of young cml patients with a low risk for transplant related mortality has not been analyzed recently. method: in order to better counsel patients with a hlaidentical sibling confronted with this situation we performed a retrospective analysis of transplants reported to the ebmt be¬tween 2002 and 2005. we selected for patients who had a low risk (ebmt-) score for transplant related mortality and who were transplanted from an hla identical sibling. we analysed the outcome of those only who were transplanted in 1st chronic phase and who received best current treatment, defined as standard conditioning, no t-cell depletion and bone marrow as stem cell source. results: 214 patients (8% of all 2737 patients transplanted for cml in this time period) with a median follow up of 12 months (0-60 months) who fulfilled these criteria were identified. they were 46% males and 54% females with a medium age of 31 years (range 6 to 59 years). 21% (46 patients) were less than 20 years old and 20% were above the age of 40. the time interval from diagnosis to transplantation was less than 1 year in 86% of patients. about one third each had an ebmt risk score 0, 1 or 2. data were obtained from 81 teams in 33 countries. the probability of survival at 5 years in a competing risk model was 88% (95% c.i. 83-93) with a cumulative incidence of death without relapse of 10% at 14 months and no additional death from transplant related mortality thereafter until 60 months of follow-up ( figure 1 ). 5-years post hsct, 27% of patients were estimated to be alive after relapse (and hence the relapse free survival was 61%). conclusions: these results show the current transplant outcome which is achievable by selecting only patients with a low risk for transplant related mortality. in this context, the data shown is valid even without information on pre-and/or posttransplant therapy. allogeneic hsct is a valuable option as second line therapy after imatinib failure for cml patients with a low transplantation risk. t prolymphocytic leukemia (t-pll) is a rare, aggressive neoplasia of t lymphoid lineage which is characterized by poor survival of less than one year. incidental reports suggest that both autologous and allogeneic hematopoietic stem cell transplantation (hsct) might be effective in this disease. a comprehensive retrospective analysis of 44 patients registered in the ebmt database will be a subject of independent presentation at this meeting. however, given the limitations of conventionally collected registry data (dubious follow-up information and extreme heterogeneity), and realizing the impossibility of performing an international formal prospective trial under the current regulatory framework, we developed a new concept of two complementary projects: the first is called "ebmt prospective observational audit on allogeneic and autologous transplantation in t-pll" and means that transplant centers will be encouraged to register their patients with t-pll very timely with the ebmt, followed by mandatory submission of ebmt medb and follow-up forms. the second is the "ebmt/eln frame of orientation for allogeneic and autologous transplantation in t-pll". its purpose is mainly to avoid transplants in situations where they are very unlikely to be successful and to avoid excess heterogeneity of eventual transplants performed, thereby facilitating scientific analysis. this expert opinion-based framework covers criteria for the diagnosis of t-pll, transplant eligibility, pre-transplant remission induction strategies, remission requirements, timing of hsct, donor compatibility criteria, conditioning, gvhd prophylaxis, and mrd monitoring. with these two complementary components it should be possible to largely improve the usual quality of registry-based data and to generate scientifically sound knowledge on hsct in an orphan disease such as t-pll. primary plasma cell leukaemia (pcl) is a rare variant of plasma cell dyscrasia, associated with poor prognosis, median survival 8-12 months, significantly shorter than for multiple myeloma. treatment with alkylating agent therapy is ineffective though polychemotherapy may offer modest improvement in survival. autologous transplantation is now widely used in the treatment of pcl and this report summarises the european blood and marrow transplant (ebmt) experience. a retrospective study was carried out of 20844 patients with common type myeloma (58% igg, 21% iga and 19% light chain) and 272 patients with pcl undergoing first autologous transplant between 1980 and 2006. all patients were reported using med-a (limited data set) or med-b (extensive data set) forms and included in the study regardless of availability of complete data. comparisons used the chi-squared test for categorical data and mann-whitney test for continuous data. overall survival and progression-free survival were calculated using the kaplan-meier method and comparisons made using the log-rank test. relapse/progression and death without relapse or progression were computed by the proper non-parametric estimator for outcomes with competing risks and compared by the gray test. there was no significant difference in age, gender, calcium or albumin at presentation. haemoglobin was significantly lower (9g/dl v 11g/dl, p=0.000), creatinine significantly higher (122 micro mol/l v 92 micro mol/l, p=0.000) and b2 microglobulin significantly higher in the pcl group. there was no difference in graft type or use of total body irradiation but the pcl group was transplanted closer to diagnosis (6.0 v 7.7 months, p=0.000). while no significant difference in engraftment, pcl patients were more likely than myeloma patients to enter cr post-transplant. overall survival for pcl patients was greatly inferior to myeloma patients -25.7 (ci 19.5-31.9, p=0.000) v 62.3 months (ci 60.4-64.3), attributable to response of short duration and increased relapse-related mortality. this is the largest reported study of pcl patients and suggests improved outcome with use of autologous transplantation. it is however dispiriting to note that outcome is greatly inferior to that in myeloma despite likely pre-selection for fitness of the pcl group. there is urgent need for collaborative study of alternative approaches including highly effective induction with novel agents and optimal stem cell transplant strategy. 356 myeloma patients from 22 centres that had undergone hla typing were included in the trial. study inclusion was at the time of conditioning for first autologous transplant at the achievement of a response status of at least stable disease after vad-like induction treatment of previously untreated patients. patients with an hla-identical sibling were allocated to the auto+allo (aual)-arm (n=108) and patients without a matched sibling donor to the auto (au)-arm (n=248); single or tandem autografting was optional. conditioning for asct was melphalan 200 mg/m 2 , and for allo ric was fludarabine 30 mg/m 2 x 3 plus tbi 2 gy. the accrual period was from february 2001 to february 2005. the two treatment groups were well matched for the standard prognostic parameters such as beta-2-microglobulin, karyotype, gender, mm subtype, stage, albumin, creatinin, calcium and response status at transplantation. median age at transplantation was significantly higher in the au-arm (57 vs 53 years). the cr rate was 43% in the aual-arm and 40% in the au-arm (p=0.49). cumulative 24 months non-relapse-mortality was 11% in the aual-and 4% in the au-arm (p=0.05). at 3 years after transplantation, there was no significant difference between the treatment arms with respect to os (aual 67%, au 70%), rfs (aual 46%, au 46%) or relapse rate (aual 43%, au 48%).. however, looking at the os curve for all patients in the aual-arm, a survival plateau on the 60%-level seems to be emerging from 3 years and onwards. we conclude that no significant differences in outcome was observed in this early analysis, but longer follow-up is warranted before any definite conclusions can be drawn. updated results will be presented. allogeneic stem cell transplantation (sct) can cure patients with mds or aml. the major disadvantage of allogeneic stem cell transplantation is the high treatment related mortality. recently the introduction of dose-reduced conditiong followed by allogeneic stem cell transplantation has lowered the treatment related mortality in comparison to standard conditioning, but a prospective comparison between both approaches is lacking. the subcommittee mds of the clwp launched a multicenter, prospective phase iii-study comparing dose-reduced versus standard conditioning followed by allogeneic stem cell transplantation from related or unrelated donors in patients with mds or secondary aml. the primary endpoint is treatment related mortality at one year. the hypothesis is that a dose-reduced conditioning will reduce the non-relapse mortality from 40 % to 20 % at one year after allogeneic stem cell transplantation. a total of 160 patients is needed to achieve this goal. patients should have mds or saml (less than 20% blasts) and should be eligible for standard and dose-reduced conditioning and aged 18 -60 years if donor is a hla-matched unrelated donor (hla-a, hla-b, hla-drb1 and hla-dqb1) (one mismatch allowed)and aged 18 -65 years if donor is a hla-matched related donor ((hla-a, hla-b, hla-drb1 and hla-dqb1) (one anti¬gen-mismatch allowed). the patient will be randomised between a dose reduced conditioning (arm b) and a standard conditioning (arm a). the standard conditioning (arm a) consisted of busulfan (16 mg/kg bw orally or 12.8 mg/kg intravenously) and cyclophosphamide (120 mg/kg) and reduced conditioning consisted busulfan (8 mg/kg bw orally or 6.4 mg/kg intravenously) in combination with fludarabine (150 mg/m²). so far the protocol is activated in germany, the netherlands, russia and italy. detailed protocol as well as recruitment will be presented. a randomised trial of rabbit anti-thymocyte globulin, given on day+7 after alternative donor transplants a. bacigalupo*, f. ciceri, p. di bartolomeo, t. lamparelli, g. milone on behalf of the gitmo background: transplant mortality (trm) can be predicted by using laboratory values, on day+7 after an allogeneic hemopoietic stem cell transplant (hsct) (bone marrow transpl.2003; 32: 205) : increased mortality is mainly, but not exclusively, due to increased acute graft versus host disease (gvhd). in a pilot study, a low dose of rabbit anti-thymocyte globulin (atg) given on day+7 reduced gvhd and trm. aim of the study: the aim of this study was to test in a propsective randomized trial, whether intervention on day+7 in patients with a high risk score would reduced the risk of trm and gvhd. patients: eligible were 170 patients undergoing hsct from family hla mismatched (n=25), or unrelated donors (n=145). all patients received atg 3.75 mg/kg x2 pre-transplant, were stratified for intermediate and high risk day+7 score, and were randomized to receive an additional dose of ratg (1,25 mg/kg day +7 and day+9) (n=84) or no additional treatment (n=86) (control arm). the two groups were balanced for age, disease phase, day+7 score, and donor/recipient sex mismatch. results: the predictive value of day+7 score on trm was confirmed in the control arm (18% vs 42% for intermediate and high risk patients, p=0.03), whereas in the day+7 atg arm , there was no difference (29% vs 29%, p=1.0). trm was overall reduced from 35% in the control arm to 29% in the atg day+7 arm (p=0.37) : the difference was more pronounced in patients with early disease and high risk on day 7 (35% vs 15% p=0.08), and in hsct with female donors in male recipients (65% vs 20%, p=0.02). acute gvhd grade iii-iv was reduced overall from 16% to 6% (p=0.04), and chronic gvhd was reduced 32% to 14% (p=0.02). conclusions: we confirm that patients with different risk of trm can be identified on day+7 after hsct: in patients at greater risk of trm, the administration of atg on day+7 reduces gvhd and trm. additional intensified supportive care (including anti-infectious treatment ) may further reduce trm in patients with a high risk score on day+7. our group reported a strong association of polymorphisms (snps) within the antibacterial defense receptor nod2/card15 with severe gvhd following allogeneic sct. however, functional studies explaining these effects were so far missing. therefore, we analysed gastrointestinal biopsies from 11 controls, 6 patients (pts) prior to sct and 56 pts following sct. biopsies post sct were obtained at the time of first symptoms indicating gastrointestinal gvhd. slides were evaluated microscopically for occurrence of apoptotic cells, loss of crypts and infiltration with lymphocytes, eosinophils and neutrophils. in addition, immunohistochemical analyses for cd4, cd8, cd68, mib1 and cd25 expression were performed. all pts and donors were typed for presence or absence of nod2/card15 snps. semiquantitative histological results were compared with clinical parameters such as stage of gvhd, use of corticosteroids and nod2/card15 status. comparison of controls with pts post-transplant showed a significant increase of apoptotic cells / crypt loss associated with enhanced lymphocyte and neutrophil infiltration. whereas the number of cd8 cells in the lamina propria significantly increased after sct, cd4 cell numbers were strongly diminished. within post-transplant biopsies, loss of crypts (score 0.5 in gvhd 0-1, 1.2 in gvhd 2 and 1.3 in gvhd 3/4 ), changes in neutrophil infiltrates (score 0.3 in gvhd 0-1, 0.8 in gvhd 2 and 0.1 in gvhd 3/4 ) and reduction of cd4 infiltrates (score 1.20 in gvhd 0-1, 0.85 in gvhd 2 and 0.39 in gvhd 3/4) were clearly correlated with stages of gastrointestinal gvhd, whereas cd8 cells showed an increase and cd25 positive cells were unchanged in pts with severe gvhd. presence of nod2/card15 snps themselves resulted in a significant reduction of neutrophils (p 0.04) and cd4 cells (p 0.006) but had no impact on further parameters. this effect could not be explained indirectly by more severe gvhd in pts with nod2/card15 snps and was confirmed in multivariate analysis. our data indicate for the first time functional changes in gastrointestinal biopsies from pts after allogeneic sct in relation to the nod2/card15 genotype. the observed reduction of neutrophils and cd4 cells may result from a reduced expression of chemokines attracting these cells to inflammatory sites, as il-8 production is strongly regulated by nod2/card15 dependent activation of nf-kappab. presence of cd4 cells and neutrophils may be required to prevent dysregulated inflammation. introduction: the nih staging and response criteria offer for the first time criteria for standardized diagnosis and staging of severity as well as evaluation of physical functioning and quality of life (qol) of chronic graft-versus-host disease (cgvhd). we present the interim analysis of a prospective germany multicenter validation study on the nih staging criteria in cgvhd. methods: 102 patients (median age 45 years, range 18-67) after allogeneic hematopoetic stem cell transplantation (hsct) for hematologic malignancies were evaluated according to the nih criteria based cgvhd activity assessment, the lee cgvhd-symptom-scale, fact-bmt, human activity profile (hap), sf 36, berliner social support scale (bsss), 24 item adjective measure (24-am), hospital anxiety and depression scale (hads) and the nccn-distress-thermometer. enrolment occurred between day 100 and 1 year after hsct or in the presence of active cgvhd without time limit. follow-up surveys were conducted at 1, 2, 3, 5, 8, 12 and 18 months after baseline survey. at all time points disease status, co-morbidities and medication were documented. results: sixty five patients had cgvhd (mild n=18, moderate n=26, severe n=21) while 37 patients did not have cgvhd. the comparison of the severity grading (mild-moderatesevere) of the physician and severity grading of the patient revealed a high correlation (p<0.01, r=.66), while the comparison of 10 point scale of patient and physician revealed differences between patient and physician in the range of 3-5 points (physician), where patients graded more severity compared to physicians. the cgvhd nih consensus grading correlated inversely with fact physical well being (r=.41, p <0.001). the hap maximum activity score correlated inversely with severity of cgvhd (p<0.05, r=.35). the cgvhd symptom scale summary score correlated with physician severity grading (r=.66, p<.0001), the fact-g score (r=.66, p<0.0001), mental health (r=.56), energy and vitality (r=.5) and hap maximum score (r=.37, p < 0.001). beside fact physical and functional well being the hap maximum score was independent of other aspects of qol. discussion: the results demonstrate, that severity of cgvhd as assessed by the nih consensus grading correlates with impairment of physical well being as well as daily activities and qol. since the cgvhd symptom scale covers severity of cgvhd and aspects of qol it should be applied together with the hap in clinical routine. naturally occurring regulatory t cells (tregs) have been reported to play an important role in modulating graft-versushost disease (gvhd), a major complication after allogeneic haematopoietic stem cell transplantation. despite striking findings in animal models supporting the therapeutic use of tregs in gvhd, the data from human studies is limited and their mechanism of action remains elusive. in this study, treg modulation of cd8+ lymphocyte induced in situ graft-versushost reactions (gvhr) was evaluated using a unique in vitro human gvhd model. tregs were defined as cd4+cd25hifoxp3+ and isolated from buffy coat of healthy blood donors using robosep following rosettesep enrichment of cd4+ cells (stemcell technology). isolated tregs were expanded in vitro with anti-cd3cd28 mab coated dynabeads (invitrogen) prior to use. the alloreactive immune reactions were set up by co-culturing "donor" cd8+ lymphocytes with hla unmatched allogeneic "recipient" monocyte derived dcs (mo-dc) in the absence or presence of "donor"-derived tregs (1:4 ratio for treg: cd8+ lymphocytes) for 7-8 days. following magnetic depletion of mo-dc and tregs, allo-antigen stimulated "donor" cd8+ lymphocytes were co-cultured for 3 days with "recipient" skin tissue. the severity of histopathological changes in skin tissue was scored as grades i-iv according to the lerner gvhd grading system. in 4 out of 4 experiments the presence of tregs significantly reduced the severity of skin gvhr from grade iii to grade i. the levels of ifng, tnfa and il-5 cytokines in the supernatants from the primary co-culture of allogeneic mo-dc and cd8+ lymphocytes were significantly reduced in the presence of tregs (ifng: 2179pg/ml vs 40.41pg/ml, p<0.0001; tnfa: 128.2 vs 19.55, p=0.0001 and il-5: 1084.7 vs 11.52, p=0.0006). following allogeneic mo-dc stimulation there was a 5.5 and 7.6 fold reduction in the percentage of cd8+ lymphocytes expressing the activation marker cd69+ (13.2 vs 2.4, p=0.026) and intracellular ifng (18.2 vs 2.5, p=0.029) in the presence of tregs. cd8+ lymphocyte proliferation measured by 3h-thymidine incorporation and cfse dilution was found to be markedly suppressed (67%-96% inhibition) in the presence of tregs in alloreactions. further investigations are underway to explore the mechanisms and characterise the modulation of gvhr by tregs in an allo-antigen specific setting. donor-recipient hla class i ligands and kir-haplotype a are associated with severe acute graft-versus-host disease in unrelated haematopoietic stem cell transplantation for beta-thalassaemia r. littera (1) killer immunoglobulin-like receptors (kirs) regulate the activity of human natural killer cells, mainly through recognition of hla class i molecules. two broad haplotypes of kir genes have been defined. the a haplotype is characterised by a single activating kir gene (2ds4), whereas the b haplotype is characterised by two or more activating kir genes (2ds1, 2ds2, 2ds3, 2ds5 and 3ds1). many studies have investigated the impact of kirs and their ligands on hematopoietic stem cell transplantation (hsct) in patients affected by acute myeloid or acute lymphoblastic leukemia. however, the results of these studies remain controversial. allogeneic hsct in talassemia patients offers an ideal study model since this cohort of patients is not biased by the variability of conditioning regimens and the different clinical and immunologic characteristics of patients transplanted for oncohematologic disorders. we studied 66 thalassemia patients transplanted from an unrelated donor. the conditioning regimen was the same in all patients. donor and recipient pairs were typed for the hla-a, b, cw, drb1, drb3, drb4, drb5, dqa1, dqb1 and dpb1 loci using high resolution molecular typing techniques. kir genes were typed using kir-gene-specific primers. out of 66 transplanted patients, 52 are alive and well (disease-free survival 78.7%), 8 rejected and 6 died. twentytwo patients (22/66 -33.3%) developed acute graft-versushost disease (agvhd). in 6 of these patients agvhd was grade iii-iv. patients who were heterozygous for hla-cw groups 1 (hla-cwasn80) and 2 (hla-cwlys80) had a higher risk of developing acute gvhd than c1/c1 or c2/c2 homozygotes (14/29 vs 8/37; rr=3.3; 95% ci: 1.63-9.76; p=.03). conversely, 7/8 patients who rejected were c1/c1 or c2/c2 homozygotes (rr=6.5; 95% ci = 0.75 -56.54; p=.06 when compared with heterozygotes). these findings confirm the results of a previous study performed on a cohort of 45 thalassemia patients. in the present study, 5 of the 6 patients (83.3%) with severe grade iii-iv agvhd had been transplanted from donors who were homozygous for kir haplotype a (rr=23.4; 95% ci: 1.19-457.96; p=.008). in conclusion, it would seem that c1/c2 heterozygosity associated with donor homozygosity for the a haplotype is likely to favour donor alloreactivity and thereby increase the risk of severe gvhd. analyses of these genetic markers may help modulate conditioning regimens and the intensity of gvhd prophylaxis in patients undergoing unrelated hsct. interleukin-13 (il-13) is an immunoregulatory cytokine secreted predominantly by activated t-helper 2 (th2) cells. it suppresses the cytotoxic action of macrophages, inhibits the production of pro-inflammatory cytokines and is a central mediator of allergic inflammation. a single nucleotide polymorphism (snp) exists within exon 4 of the il13 gene at position +2044. the a allele of this snp and high il-13 levels have been linked with several inflammatory conditions and il-13 mixed lymphocyte culture (mlc) levels have been associated with graft-versus-host disease (gvhd) following haematopoietic stem cell transplantation (hsct). consequently the roles of the il13 +2044 snp and il-13 mlc levels in hsct were examined in this investigation. polymorphism studies were carried out on a cohort of 923 hsct recipients and donors from 7 transplant centres across europe. il13 genotyping was performed using pcr and rflp analysis. il-13 levels were measured in a cohort of 91 mlc supernatants using a cytometric bead array and correlated with gvh reaction (gvhr) grades from an in vitro model of gvhd. in all statistical analyses p values <0.05 were regarded as being significant. multivariant analysis of the whole hsct cohort demonstrated that the il13 +2044 a allele was significantly associated with the development of both acute (grades iii-iv) and chronic gvhd (p=0.028 and p=0.026 respectively). these associations remained significant when the cohort was stratified for transplant type and conditioning regimen. significant associations were also observed in a subset of patients diagnosed with cml; in hla-matched siblings possession of the il13 +2044 a allele was linked with a decreased susceptibility to chronic gvhd, whereas in mud transplants possession of the a allele was a risk for chronic gvhd. depending on the subset analysis, several clinical factors were also significantly associated with gvhd. analysis of the mlc data demonstrated that il-13 levels increased with gvhr grade, with significantly higher levels being observed in mlc supernatants with gvhr grades iii-iv (p=0.015). to our knowledge this is the first investigation examining the roles of both il-13 mlc levels and the +2044 snp in hsct. the findings are encouraging, indicating that il-13 may be involved in the immunopathology of gvhd. consequently, il-13 levels, as well as snp analysis could provide key pretransplant information on gvhd prognosis and be potential novel targets for post-hsct gvhd therapy. allogeneic hematopoietic stem cell transplantation (allo-hsct) is a curative treatment for hematologic malignancies, but the application is slimited due to major complications, such as severe graft versus host disease (gvhd). diagnosis of acute gvhd is based on clinical features and biopsies, a proteomic pattern specific for agvhd has been published and evaluated blindly on 902 samples collected from 168 patients undergoing allo-hsct at mhh between 2005 and 2007. the majority of the patients included were transplanted for hematological malignancies (n=158), 10 for hematopoietic failure syndromes (saa, n=6; pnh n=1; omf, n=3). forty-five patients were treated with dose-reduced conditioning regimens; gvhd-prophylaxis consisted of cyclosporin (csa) plus methotrexate (mtx) or mycophenolic acid (mmf), and antibodies respectively. most patients were transplanted from matched unrelated donors (mud, n=100),63 received stem cells from matched related (mrd, sib, 1 syngeneic), 3 from haplo-identical related, and 2 from mismatched related donors. based on the positivity of the agvhd pattern we initiated a pilot trial of pre-emptive therapy, treating patients upon pattern positivity with 1mg prednisone/kg bw and compared the outcome of the treated group to those patients who did not receive pre-emptive therapy. in 2005 90 patients were transplanted and screened for agvhd at mhh, but not pre-emptively treated. forty one patients developed agvhd (45%), 22 (24%) had agvhd >ii and were treated with standard protocols. eight patients developed agvhd grade iii or iv (8/22=36%) and 6 of these died. between april 2006 and june 2007 78 patients were transplanted at mhh. in 30 patients the agvhd proteomic pattern showed a clear correlation for gvhd >ii. twenty six (33%) of 78 transplanted patients had agvhd >ii, 12 received pre-emptive therapy, while 14 were treated upon clinical signs of agvhd according to standard treatment protocols.two patients in the preemptive treatment group (2/26=7.9%; 2 of 12 pre-emptively treated:16 %) had agvhd iii or iv and died (2/26=7.9%; 2/12: 16%). of the 14 standard therapy patients 6 developed agvhd grade iii or iv and to date 3 of these have died (6/26 =23% or 6/14 of the standard therapy group=42%). thus, taken together our results indicate that pre-emptive treatment may decrease the severity of agvhd and probably leads to a better overall survival. donor-derived t cells emigrating from the graft after solid organ transplantation have been shown to promote immunological tolerance thereby improving long-term graft survival. however, donor t cells are also able to induce lifethreatening allo-reactive graft-versus-host disease (gvhd) in the transplant recipient. the exact mechanism by which donor t cells influences this delicate balance between tolerogenicity and allo-reactivity has not been elucidated. we observed two liver transplant patients with severe gvhd who developed a donor t-cell chimerism in peripheral blood and bone marrow up to 100%, which is far above those of previously described cases. this enabled us to isolate donorderived t cells in sufficient numbers to allow for a detailed analysis of phenotypic and functional features ex vivo. we found that the donor t cells died by apoptosis over time without any evidence of rejection by host t cells. interestingly, the host-versus-donor reactivity appeared to be selectively impaired, as anti-viral t cells were still detectable in the host repertoire. these results indicate that graft t cells are able to specifically eliminate donor-reactive t cells from the host repertoire thereby preventing donor cells from subsequent rejection. since substantial donor t-cell chimerism persisted in both patients beyond resolution of gvhd, we investigated potential mechanisms of immunotolerance. we observed that the recovery from gvhd was not accompanied by an expansion of immunosuppressive cd4/cd25/foxp3-positive t cells in peripheral blood. however, we obtained formal evidence that host-reactive donor t cells were controlled by an alternative negative regulatory pathway, executed by the immunoinhibitory receptor programmed death-1 (pd-1) and its ligand pd-l1. we did not only find an exceptionally high level of pd-1 expression on host-reactive donor t cells ex vivo, but also discovered that blocking pd-l1 on host cells significantly enhanced anti-host reactivity by donor cd8 t cells in vitro. we thus suggest the interference with the pd1/pd-l1 pathway as a novel therapeutic strategy to control host-reactive donor t cells in solid organ transplant-associated gvhd. our observations might be also of relevance for other clinical scenarios of misdirected allo-reactivity, such as graft rejection as well as severe gvhd after allogeneic hematopoietic stemcell transplantation. (2) purpose: to determine risk factors of outcomes after umbilical cord blood transplantation (ucbt) for patients with advanced lymphoid malignancies. patients and methods: we evaluated 104 adult patients (median age, 41 years) who underwent unrelated donor ucbt for lymphoid malignancies. ucb grafts were 2 antigen hla mismatched in 61%, and were composed of one (n=78) or two (n=26) units, with a median cell dose of 2.5x10 7 nucleated cells/kg and 1.05x10 5 cd34 cells/kg. diagnoses were non-hodgkin lymphoma (nhl, n=62), hodgkin lymphoma (hl, n=29), and chronic lymphocytic leukemia (cll, n=13), with 85% having advanced disease and 60% having failed a prior autologous transplant. sixty-four percent of patients received a reduced-intensity conditioning regimen and 44% low-dose total body irradiation (tbi). median follow-up was 14 months. results: cumulative incidence (ci) of neutrophil engraftment was 85% by day 60, with greater engraftment in recipients of higher cd34+/kg cell dose (93% vs. 78%, p=0.0001). ci of non-relapse-related mortality (nrm) was 28% at 1 year, with a lower risk in patients treated with low-dose tbi (13% vs. 47%, p=0.007). ci of relapse or progression was 31% at 1 year, with a lower risk in recipients of double unit ucbt (9% vs. 38%, p=0.02), and those with chemosensitive disease (19% vs. 40%, p=0 .01) and indolent nhl (19% vs. 35%, p=0.04) . the probability of progression-free survival (pfs) was 41% at 1 year, with higher survival in those with indolent nhl (61% vs. 34%, p=0.04), chemosensitive disease (55% vs. 31%, p=0.004) and who received low-dose tbi (58% vs. 27%, p=0.002). conclusion: ucbt is a viable treatment for adults with advanced lymphoid malignancies. diagnosis of indolent lymphoma, chemosensitive diseases, and use of low-dose tbi and were factors associated with significantly better outcome. positron emission tomography scan performed before reduced-intensity conditioning allogeneic stem cell transplantation has a prognostic value in patients with relapsed and chemosensitive hodgkin's lymphoma or aggressive non-hodgkin lymphoma a. dodero* (1), r. crocchiolo (2) (1) ( positron emission tomography (pet) scan using 18fluorodeoxyglucose [18f-fdg] has a recognised prognostic value in patients (pts) with hodgkin lymphoma (hl) or aggressive non-hodgkin lymphoma (hg-nhl) receiving chemotherapy or autologous stem cell transplantation (sct). thus, we retrospectively assessed the prognostic role of pet scan before reduced-intensity conditioning allogeneic sct. between 2000 and 2007, 82 consecutive pts with hg-nhl or hl, responding to salvage therapy, were evaluated with a pet scan before allografting. presence (pet-pos) or absence (pet-neg) of abnormal 18f-fdg uptake was correlated to progression-free survival (pfs) and overall survival (os).interpretation of pet scan was obtained with visual assessment alone by a nuclear medicine physician (evaluation of maximal suv in pet-pos cases is ongoing). median age of pts was 36 years (range, 17 -68 years). histologic subtypes included: 38 hg-nhl [b phenotype (n=25), t phenotype (n=12), other (n=1)] and 44 hl. fortyseven pts (57%) were allografted from a hla-identical sibling donor, 16 from a haploidentical donor and 19 from an unrelated donor. sixty-eight pts (83%) failed autograft, the median number of prior regimens was 3 (range, 1-6). pet scans were performed at a median of 30 days prior to allograft: 41 out of 82 pts were pet-pos [hg-nhl (n=18), hl (n=23)] whereas 41 were pet-neg [hg-nhl (n=20), hl (n=21)]. pts with pet-pos or pet-neg scans were well balanced in terms of diagnosis, previous treatments, and type of donor. at a median follow-up of 30 months (range, 6 -86 months), 54 pts are alive and 28 died [toxicity n=12, disease n=16]. overall, the estimated 3-year pfs in pts with pet-neg or pet-pos scans were 68% (95% ci, 49% -81%) versus 30% (95% ci, 15% -47%), respectively (p<0.003). for hg-nhl pts, the estimated 3-year pfs was 70% for pet-neg as compared to 41% for pet-pos (p<0.02) whereas for hl pts, the estimated 3-year pfs was 68% as compared to 17%, respectively (p=0.05). a statistically significant higher cumulative risk of relapse was observed in pts with pet-pos scan before allograft as compared to the pet neg scan (53% versus 21%, p< 0.022). the estimated 3-year os in pts with neg or pos pet scans were 77% (95% ci; 60% -87%) versus 41% (95% ci; 24%-57%), respectively (p< 0.002). our study shows a better pfs and os for pts being pet neg before allografting. pet scan should be incorporated in pretransplant work-up to validate our findings prospectively. l. rigacci*, a. bosi, b. puccini, p. corradini, l. castagna, n. cascavilla, g. milone, a. bacigalupo, r. scimè, g. specchia, a. rambaldi, p. leoni, f. ciceri, a. levis, s. guidi, b. bruno, r diffuse large b cell lymphoma (dlbcl) is the most common lymphoid malignancy in adults. autologous hematopoietic stem cell transplantation (ahsct) has been shown to be an effective therapy for patients with dlbcl who relapsed after complete remission (cr). patients who relapse after an ahsct have a very poor prognosis and usually can not be cured with standard or high dose chemotherapy. allogeneic hematopoietic stem cell transplantation (allohsct) has shown to be effective in the rescue of lymphoma patients relapsed after conventional or high dose therapies. according to this data we have analysed all patients with diagnosis of dlbcl who have performed an allohsct from 2000 to 2005 after an ahsct relapse. seventy-five patients were selected from the data-base, 46 were male (61%), 71 presented a diagnosis of dlbcl and 4 were anaplastic large cell lymphoma. the stem cell donor was related in 61 patients (81%) and unrelated in 14 patients (19%). the stem cell source was peripheral blood in 61 cases and bone marrow in 14 cases. the conditioning regimen was conventional in 24 patients and reduced intensity in 51 patients. the median time between ahsct and allohsct was 13 months (range 1-68 months). twenty-five patients (33%) performed allohsct after the obtainment of at least a partial remission or a controlled disease, 36 (48%) were treated with active disease and in 14 cases the data was not available. after allohsct 75% of patients obtained a response (cr or pr) and did not have evidence of disease, 25% of patients did not respond and progressed. the treatment related mortality (trm) was 32%, 14 out 24 (58%) patients died in conventional regimen and 10 out 51 (20%) in reduced intensity arm. acute graft versus host disease (agvhd) was observed in 28 patients (grade iii-iv in 8 patients), and chronic (cgvhd) in 65%. after a median follow-up of 58 months from the diagnosis (range 11-196 months) and a median follow-up of 9 months after allohsct (range 0-82 months) the overall survival was 45%. the overall survival was significantly higher in patients treated with reduced intensity conditioning in comparison with patients treated with conventional conditioning (p:0.001). this retrospective study confirms that allo-transplant it is feasible and it could be really effective in a poor prognosis group of patients. moreover the use of reduced intensity conditioning improves these results. (2) median time from diagnosis to mud-sct was 25 months (range, 3 -205). 64% of the patients had failed previous autologous transplant (asct), and 25% were transplanted with chemorefractory disease. peripheral blood was the source of hematopoietic stem cells in 70% and reduced intensity conditioning regimens (ric) were used in 52% of the cases. after a median follow up for living patients of 35 months, the estimated 3-year non-relapse mortality (nrm), relapse rate (rr), progression free survival (pfs) and overall survival (os) for the whole series were 32%, 38%, 30% and 38.5%, respectively. grade ii-iv acute graft-versus-hostdisease developed in 32% of patients. patients selected for ric protocols were older (median age of 44 years vs 38 years, p = 0.02) and more heavily pre-treated; 75% had failed autograft compared with 53% in the conventional conditioning(cc) group (p = 0.01). despite these unfavorable factors, nrm for patients receiving ric was significantly lower than observed in patients treated with a conventional regimen:23% vs 41% at 3 years (p = 0.02). however, this advantage was offset by an increased rr in patients undergoing ric-mud (3-yr rr: 46% vs 30%, p = 0.2), resulting in a very similar pfs and os for both types of conditioning regimens. the prognostic factor with highest impact on pfs was refractory disease at transplantation (rr = 1.8; 95%ci 1.1 -3.1, p = 0.02). patients transplanted with chemosensitive disease had a 3 yr pfs of 35% irrespective to the conditioning regimen applied, whereas patients transplanted with chemorefractory disease had a 3 year pfs of 16% only. in conclusion, mud sct provides a true chance for cure for select patients with dlbc lymphoma who failed conventional therapies, particularity if transplanted with cemosensitive disease. ric, knowm to be associated with a reduced nrm rate, should be especially considered in patients with chemosensitive patients, whereas cc might be the preferred option for patients with more aggressive disease. prospective evaluation of 18f-fluorodeoxyglucose (fdg) positron emission tomography as a predictor of residual disease and subsequent relapse in patients with diffuse large cell ymphoma and hodgkin's lymphoma undergoing hdc and asct s. akhtar*, a. al-sugair, y. al kadhi, a. al-zahrani, m. abdelsalam, s. bazarbashi, d. ajarim, i. maghfoor king faisal specialist hosp. & res. centre (riyadh, sa) background: there is emerging data indicating poor outcome in diffuse large cell ymphoma (dlcl) and hodgkins lymphoma (hl) patients with positive 18f-fluorodeoxyglucose (fdg) positron emission tomography (pet) before high dose chemotherapy (hdc) and autologous stem cell transplant (asct). we initiated this prospective trial to evaluate the impact of pet as a predictor of post hdc residual disease and relapse in patients with dlcl and hl undergoing hdc asct. patients and methods: from july 2005 to june 2007, 115 patients with relapsed or refractory dlcl and hl were enrolled as a potential candidate for hdc asct. 43 patients did not have hdc asct due to progression (10), refusal (4), noncompliance (5) or other reasons (24). all eligible patients received eshap as salvage chemotherapy, responding dlcl or responding / stable hl patients received same chemotherapy as mobilization regimen for stem cell collection followed by beam as hdc. prior to the initiation of eshap, each patient had ct scan + other radiological studies if needed and pet ( ct-1 and pet-1), after 2-3 cycles of salvage chemotherapy / prior to hdc asct (ct-2 and pet-2) and approximately 100 days post hdc asct or earlier if clinically indicated (ct-3 and pet-3). 72 patients had hdc asct and 49 of them had both pet-2 and pet-3 available at the time of this analysis. results: patients characteristics are male: 28, female: 21, dlcl: 9 and hl: 37. relapsed 21 and refractory 28. median age at asct is 32 years (16 to 62). median follow-up from asct is 12 months. 27 patients were pet-2 negative prior to hdc asct. at the time of this evaluation, of these 27 patients, 4 (15 %) had an event, event free survival (efs) 85%. 22 patients were pet-2 positive prior to hdc asct, 10 (46 %) had an event, efs 54%. efs for pet-2 negative vs pet-2 positive has p value of 0.027. using kaplan-meier method, positive pet-2 has 53% probability of an event vs 16% for a negative pet scan (p=0.005). efs for ct-2 negative vs ct-2 positive patients is 83% vs 68%, p = 0.466. kaplan-meier for ct-2 positive showed 36 % probability of event vs 18% for ct-2 negative, p=0.198. conclusions: prior to hdc asct, positive pet scan indicates high risk of residual disease or progression. many of these patients are likely to suffer from treatment failure. these patients are potential candidate for more aggressive and experimental therapies. for many patient with pre-transplant negative pet, post transplant pet scan can be omitted. radioimmunotherapy with haemopoietic stem cell transplantation for treatment of malignant non-hodgkin lymphoma: multicentre study on thirty patients a. mele (1) (7), g. console (7), n. cascavilla (2) , p. scalzulli (2) /kilograms (range 2.55-21.6). all patients engrafted. the median number of red blood cell and platelet transfusion were 4 (1-7) and 6 (1-8), respectively. the median time to platelet and neutrophil counts higher than 20x10 9 /l and 0.5x10 9 /l were 14 (range, 9-35 days) and 10 days (range, 8-14) , respectively. mucosites occurred in all patients (grade iii and iv in 13 and 4 cases). febrile neutropenia occurred in 80% of cases. six pneumonitis and 8 blood stream infections were documented. one patient developed an atrial fibrillation. twenty-one of 30 patients were valuable for 90-day response. the 90-day overall response was 86% with 72% of cr. four early deaths before day-90 occurred: 1 case for septic shock (day +6), 1 for viral encephalites (day +60) and 2 for progression disease (day + 30, 65). the kaplan-meyer estimated treatment related mortality (trm) is 9%. seven cases (1 cr, 4 pr and 2 not response) progressed at a median follow-up of 95 days post hst (range, 60-300). twenty-four of 30 patients are alive at a median follow-up of 175 days post hst (range, 6-590). six patients died (20%): 3 for progression, 1 in cr for ards (day + 230) and 2 for trm. sixteen (53%), 5 (17%) and 2 of 30 patients are alive in cr, pr and progression, respectively. one case is not valuable for response (day+15). we analyzed the characteristics of 14 alive patients in cr: 9 had aggressive lymphoma and 13 were at least in pr before hst (p=ns). the kaplan-meyer estimated 2y-efs is 73%. conclusion. the use of rit plus transplant induces 70% of or (53% cr) with sustained engraftment, an acceptable extra-haematological toxicity and a rapid immunological recovery in patients who failed to achieve cr after first line chemotherapy. the power of this program needs to be assessed in a larger series of patients. hdt/asct has been planned either in front-line pts with high prognostic score/bulky mass/stage iii-iv or at relapse. first line therapy were mainly abvd/mine for hd and acvbp/chop ± rituximab for nhl. fdg-pet was performed after 1-5 chemotherapy cure. mine and dhap were the most frequent salvage chemotherapy used for refractory front-line therapy pts. the conditioning regimen was mainly bicnu-etoposide-aracytine-melphalan. 46 pts (53%) were pre-hdt/asct pet negative and 41 positive (47%). 8/41 pre-hdt/asct positive pts (19.5%) converted to negative by additional cross chemotherapy. after hdt/asct, 22/33 others pre-hdt/asct positive pts (67%) converted to post-hdt/asct pet negative. one negative pre-hdt/asct pet converted to positive. residual disease of positive pts was mainly treated by local radiation. after a median follow-up of 3.2 years (range 0.4-8.4) after pre-hdt/asct pet, 29 pts relapsed, 21 dead (with 18 of 29 relapses), 3 remained resistant disease. survival was measured from pre-hdt/asct pet to death (overall survival os) or relapse/death (event-free survival efs) with censoring time at the time of last follow-up. median os and efs for the two groups were not reached. estimated 3 years os was 80% and 73%, respectively for pre-hdt/asct pet negative and positive pts. estimated 3 years efs was 74% and 61% respectively for pre-hdt/asct pet negative and positive groups. a positive fdg-pet after induction chemotherapy is highly predictive of poor survival in hd and nhl pts but an additional risk-adapted treatment strategies before hdt/asct by salvage cross chemotherapy and after hdt/asct by targeted radiation may improve pre-hdt/asct pet positive pts outcome. donor lymphocyte transfusions (dlt) after allogeneic stem cell transplantation have been shown to be very effective in treatment of recurrent mylogenous leukemia but displayed limited use in chronic lymphocytic leukemia (cll) and highly malignant non-hodgkin lymphoma (nhl). here we studied whether bi20 (fbta05), a novel trifunctional bispecific antibody targeting cd20 on lymphoma cells and cd3 on t cells could induce graft-versus-leukemia / lymphoma responses in combination with dlt or mobilized peripheral blood stem cells (pbsct) after allogeneic transplantation in these diseases. six patients (3 cases each with p53 mutated cll, and highgrade nhl) refractory to standard therapy were treated with escalating doses of bi20 (range 10 -2000 µg) followed by dlt or sct. in 4 out of 6 patients, a prompt, but transient clinical and hematological response was observed. side effects (fever, chills and bone pain) were tolerable and appeared at lower dose levels in cll (>40 µg) than in high grade nhl (>200 µg). the cytokine profile was characterized by transient increases of il-6, il-8 and il-10. neither human anti-mouse antibodies (hamas) nor graft-versus-host disease (gvhd) developed allowing repeated treatment courses. in summary, the trifunctional antibody bi20 induced prompt antitumor responses in extensively pretreated, p53 mutated, alemtuzumab and rituximab refractory patients indicating its therapeutic potential. d. stachel*, k. kirby, l. corey, m. boeckh fred hutchinson cancer research center (seattle, us) background: although cmv viral load is a predictor of cmv disease in hct recipients, regulatory agencies presently do not accept it as primary endpoint for studies that evaluate new therapeutics. the aim of this study was to examine whether cmv viral load predicts transplant-related mortality (trm) or overall survival (os) in the era of preemptive therapy. methods: 2896 consecutive patients following a first hct at fhcrc between 1995 and 2005 were analyzed; 1481 of them were cmv seropositive. patients underwent weekly testing for cmv viremia by culture and quantitative pp65 antigenemia (ag) during the first 100 days after hct. preemptive antiviral therapy was given for any pp 65 ag. using univariate and multivariable cox models, we analyzed the association of initial, mean, peak cmv load, and cmv area under the curve (auc) for their association with os and trm at 1 year after hct. viral load parameters were analyzed in quartiles. pp65 ag results are expressed as cells/200,000 cells. results: trm occurred in 25.2% of all patients and 27.8% of seropositive recipients. in a model that included cmv seropositive recipients, the adjusted hazard ratios (hr) for the upper quartile of initial viral load (ag > 5), peak viral load (ag >10) and auc (ag>100) were 1.6 (95% ci 1.2-2.1), 2.2 (95% ci 1.6-3.0), and 1.7 (95% ci 1.3-2.3), respectively. the statistical models were adjusted for recipient and donor age, hla match, donor relationship, year of hct, acute/chronic gvhd and postengraftment neutropenia (to account for the toxicity associated with preemptive therapy); additional factors evaluated (but not significant in univariate analysis) were race, donor cmv serostatus, cell source, type and intensity of conditioning, t cell depletion, risk of the underlying disease, and gvhd prophylaxis. similar models that included all patients and those that used os as endpoint also showed significant associations for first and peak viral load and the auc. results are presently being validated in a separate cohort that underwent pcr surveillance. conclusion: initial and peak viral load as well as the viral auc are independently associated with trm and os in allogeneic hct recipients receiving preemptive antiviral therapy. the peak viral load showed the strongest association with trm and os. these data further support the use of parameters of viral dynamics as primary endpoints for studies that evaluate immune augmentation or drug prevention strategies. objectives: to evaluate the effect of immunoglobulin (ivig) and cytomegalovirus-hyperimmune immunoglobulin (cmv-ivig) prophylaxis in patients undergoing hematopoietic stem cell transplantation (hsct). methods: systematic review and meta-analysis of randomized controlled trials comparing systemic ivig or cmv-ivig with placebo or no intervention for prophylaxis in patients undergoing hsct. the cochrane library, medline, conference proceedings and references were searched until 2007. the primary outcome was all-cause-mortality at end of follow-up. two reviewers independently appraised the quality of the trials and extracted data. relative risks (rr) with 95% confidence intervals were estimated and pooled. results: twenty trials fulfilled inclusion criteria, 12 assessed ivig and 8 assessed cmv-ivig. for ivig vs. control there was no difference in all-cause mortality, rr 0.99; 0.88-1.12, 8 trials, fig 1. results were similar when only patients following allogeneic hsct were assessed (3 trials), when mortality was assessed at day 100-200 (5 trials) or after 2 years (3 trials), and when high-dose ivig was used (3 trials). trials' methodological quality did not impact results. overall, there was a reduction in the number episodes of interstitial pneumonitis (ip), rr 0.64; 0.45-0.89, 7 trials, although the reduction in cmv infections was not statistically significant, rr 0.84; 0.66-1.07, 6 trials. there was no difference in clinically or microbiologically documented infections, rr 1.00; 0.90-1.10 and rr 1.00; 0.88-1.15, respectively, 7 trials both). there was no difference with regard to acute graft vs. host disease (gvhd), rr 0.93; 0.83-1.04, 7 trials. veno-occlusive disease (vod) was significantly more frequent with ivig, rr 2.73; 1.11-6.71, 4 trials, fig 2, an open-label randomised study of oral valganciclovir versus intravenous ganciclovir for pre-emptive therapy of cytomegalovirus infection after allogeneic stem cell transplantation l. volin* (1), l. barkholt (2) cytomegalovirus (cmv) remains a leading cause of infectious complications after allogeneic stem cell transplantation (sct) and without preventive measures patients with cmv infection have a significant risk to develope cmv disease. a preemptive treatment approach with iv-ganciclovir or ivfoscarnet is used to restrict the use of potentially toxic drugs for patients at risk for cmv disease. recently, valgan-ciclovir, an oral prodrug of ganciclovir, has become available. the nordic bmt group started a study with the aim of demonstrating that treatment outcome of the first cmv dnaemia after sct with oral valganciclovir is not inferior to that obtained with iv-ganciclovir. the primary study endpoint was achievement of quantitative cmv pcr (qpcr) negativity on day 28 of treatment or earlier. qpcr was carried out weekly for the first three months after sct, and if positive, patients were randomized to receive either oral valganciclovir 900 mg twice a day or ganciclovir iv 5 mg/kg every 12 h for 14 days (adjusted according to renal function). qpcr was studied twice a week. if qpcr was negative on day 13-14 of treatment, the treatment was discontinued, and if negativity was not achieved but the copy number was decreasing, maintenance treatment once daily was administered for another 14 days. thereafter the treatment was carried out according to local policy. of the 96 patients receiving an allogeneic sct, 74 patients were treated for a hematological malignancy and 22 for a solid tumor. the patients in the poand iv-groups did not differ by recipient/donor cmv serostatus, age (median 55 vs 51 years), diagnosis, donor (sibling/ unrelated), or the time of pcr positivity (day +40 and +38 after sct, respectively). the median cmv dna copy number/ml at diagnosis was 2525 (200-160000) in the pogroup, and 1425 (200-41900) in the iv-group. the maximum copy number/ml was 5031 (300-160000), and 2750 (200-41900), respectively. the incidence of agvhd ≥ grade 2 was 30% in the po-group and 19% in the iv-group. the primary endpoint of the study was achieved in 30/49 (61%) of the popatients and 25/47 (53%) of the iv-patients. during the study three patients with a solid tumor de-veloped cmv disease, two in the po-group and one in the iv-group. oral valganciclovir therapy seems not to be inferior to iv-ganciclovir therapy in the preemptive treatment of the first cmv dnaemia after allogeneic sct, and the practical advantages of oral treatment are obvious. genetic variations in the nod2/card15 gene are associated with bacteraemia and sepsis after allogeneic stem cell transplantation m. grube, d. veith, g. rogler, j. brenmoehl, h. bremm, j. hahn, r. andreesen, e. holler university hospital regensburg (regensburg, de) purpose: single nucleotide polymorphisms (snps) of the nod/card15 gene resulting in a diminished nuclear factor-kappab (nf-kappab) response to bacterial cell wall products have been recently associated with an increased incidence of crohn`s disease as well as transplant related mortality and gvhd following allogeneic transplantation. in addition, a recent study from our group revealed nod2/card15 variants as independent predictors of death from septic shock in nonhematological patients. therefore, we now analysed the direct interaction of nod2/card15 variants with febrile bacteremia and sepsis in patients receiving allogeneic sct. experimental design: we retrospectively analyzed 139 donor/recipient pairs for single nucleotide polymorphisms (snps) of the nod/card15 gene (snp8, 12 and 13). bacteremia was determined by bacterial growth in the blood culture. septic syndrome was determined by classical criteria. results: 94/139 (68%) patients had unmutated snp`s (wild type group), in 45/139 (32%) patients either the recipient or the donor had any mutated snp (r or d any mutation). 68/139 (49%) patients showed febrile bacteremia, whereas 17/68 (25%) developed lethal sepsis syndrome. the cumulative incidence of bacteremia was 48% in the wilde type group and 59% in patients with any mutation (r or d any mutation). the cumulative incidence of lethal sepsis syndrome was 5% in the wilde type group and 39% in the patients with any mutation (r or d any mutation) (p< 0,001). in a more detailed analysis of individual donor and recipient snps, presence of snp8 in the recipient and any snp in the donor (either snp8, 12 or 13) were signifikant risk factors whereas the highest cumulative incidence of sepsis was found if the donor had a snp13 mutation (p < 0,001). since bacteremia/sepsis syndrome even occurred in patients without gvhd or prior to onset of gvhd our data argue against an induction of bacteremia/sepsis syndrome secondary to gvhd. in line with this view, a multivariate analysis including gvhd showed snp`s of the nod/card15 gene as independent risk factors for lethal sepsis syndrome (p<0.01). conclusion: our results suggest that defective signalling of nod2/card15 either in epithelia (recipient) or monocytes/macrophages (donor) may be directly involved in a diminished antibacterial defense. translocation of bacteria may be an important step in subsequent sct related complications. infection control interventions for cancer patients: efficacy evaluation through systematic review and metaanalysis m. paul*, a. schlesinger, a. gafter-gvili, l. leibovici rabin medical center; beilinson campus (petah-tikva, il) background: currently used infection control measures for hematological cancer patients are frequently applied with an unclear evidence basis resulting in a heterogeneous application of these interventions. methods: systematic review and meta-analysis. included were all prospective comparative studies assessing the effects of non-pharmacological interventions applied for infection prevention and control among cancer patients following chemotherapy. interventions were classified according to the transmission modality targeted by the intervention; airborne, contact or endogenous flora. the primary outcome assessed for all interventions was 100-day all-cause mortality. relative risks (rr) with 95% confidence intervals are reported. results: we identified 23 studies assessing protective environment (pe) including high particulate air filtration (hepa) ± laminar airflow (13 randomized trials, 12 hematological cancer, 9 for hsct), 10 studies comparing outpatient vs. inpatient management for hsct (all nonrandomized) and 5 miscellaneous studies assessing diet, footwear, gowns or specific room design (3 randomized trials, 2 for hsct). pe resulted in a remarkable reduction in allcause mortality at day 100: rr 0.79, 0.73-0.87 overall and rr 0.78, 0.66-0.92 in adequately randomized trials alone. the rr was 0.60, 0.50-0.72 for 30-day mortality and 0.86, 0.81-0.91 for the longest follow-up, up to 5 years. similar survival benefit was observed for allogeneic hsct patients and other patients. significant reductions were observed for all infection-related outcomes, except mold infections (table) . when isolating the effects of the different transmission modalities it became apparent that endogenous suppression using antibacterial (usually combined with antifungal) prophylaxis was the major contributor to the beneficial effect of pe ( figure) . outpatient hsct (3 allogeneic and 7 autologous) was non-inferior to inpatient treatment, rr 0.81, 0.46-1.45 for mortality; rr 0.93, 0.67-1.29 for infections; and rr 0.35, 0.15-0.80 for bacteremia. conclusions: protective isolation offers an overall significant benefit to the patient, since the assessment of all-cause mortality encompasses both infection, cancer and treatmentrelated outcomes. the additional value of hepa and strict contact isolation over antibiotic prophylaxis is unclear and probably depends on the local prevalence of mold infections. outpatient hsct appears safe and should be explored in randomized trials. m. dettenkofer*, r. babikir, h. bertz, a.f. widmer, w.v. kern, e. meyer, p for surveillance of nosocomial bloodstream infections (bsi) and pneumonia during neutropenia in adult patients undergoing bone marrow transplantation (bmt) or peripheral blood-stem cell transplantation (pbsct), an ongoing multicenter surveillance project was initiated by the german national reference centre for surveillance of nosocomial infections in 2000 (onko-kiss). methods: nosocomial infections are identified using cdc definitions for laboratory-confirmed bsi and modified criteria for pneumonia in neutropenic patients [for detailed information see : cid 2005; 40: 926-31, or in german language: http://www.nrz-hygiene.de/surveillance/onko.htm]. results: over the 60-month period from july 2002 up to june 2007 26 centres participated. altogether 4,909 patients with 72,449 neutropenic days were investigated. of these, 2,873 (59%) had undergone allogeneic and 2,036 (41%) autologous bmt or pbsct. the mean length of neutropenia was 14.8 days (9.2 d after autologous and 18.7 d after allogeneic transplantation). in total, 827 bloodstream infections and 403 cases of pneumonia were identified. site-specific incidence densities are shown in the table. there was a trend to lower incidence densities over the five years reported. following allogeneic transplantation, 17.7 bsi/100 patients and 10.4 cases of pneumonia/100 patients occurred whereas following autologous transplantation 15.7 cases of bsi/100 patients and 5.1 cases of pneumonia/100 patients were observed. the main pathogens associated with bsi were coagulase-negative staphylococci (51%). conclusions: the ongoing onko-kiss project adds to the improvement of quality of care in hct-patients by providing sound reference data on the occurrence of bsi and pneumonia during neutropenia. since 2006, surveillance is extended to neutropenic patients with acute leukemia to allow participation for centres not performing hct. t-cell-mediated immunity is an essential host factor in the control of hcmv latency in patients undergoing an allohematopoietic-stem-cell transplantation. our aims were to identify patterns of hcmv-specific immune responses associated with multiple or prolonged reactivations. we analyzed 116 recipients during the course of infection/reactivation and latency. the cd8+t-cell responses were weekly determined using hla-class i tetramers together with extended phenotypic analyses. our results showed that recipients from unrelated donors were more susceptible to multiple reactivations and that the donor hcmv serologic status influenced the occurrence of prolonged reactivations. we found that the lack of hcmv-specific t-cells during the first episode of reactivation was associated with multiple further reactivations. the sequential phenotypic follow up showed that patients with uncontrolled reactivations were unable to develop hcmv-specific t-cells of the late differentiation phenotype cd45ra+cd27-cd28-. our data indicate that the longitudinal evaluation of cd27 and cd45ra expression within the tetramer positive subset could help to identify patients developing a protective immune response. the evaluation of hcmv-specific immune responses during the first episode of reactivation, together with extended phenotypes could improve immune monitoring, especially in recipients from unrelated donors and other situations at risk of uncontrolled viral reactivation. invasive aspergillosis in allogeneic stem cell transplant recipients from alternative donor: incidence, risk factors and outcome a.m. raiola*, m. mikulska , b. bruno, m.t. van lint, f. gualandi, d. occhini, a. dominietto, c. di grazia, s. bregante, a. ibatici, t. lamparelli, e. furfaro, f. frassoni, c. viscoli, a. bacigalupo s. martino's hospital (genoa, it) introduction: invasive aspergillosis (ia) remains an important complication with high morbidity and mortality in patients undergoing haematopoietic stem cells transplant (hsct) according the donor type. materials and methods: we determined, with retrospective analysis, the incidence, risk factors for ia and outcome in 306 patients who received hsct from unrelated donor (mud), family mismatched donor, or cord blood between january 1999 and december 2006 in our unit. the diagnosis of ia was documented as proven or probable according to the 2002 european organisation for research and treatment of cancer eortc/niaid international consensus. we have also considered proven ia the presence of fungal invasion on autopsy. results: a total of 306 patients were included in the study, with a median follow-up of 297 days after hsct, (range 1-2709 days) (types of hsct: matched unrelated 60%, mismatched related 23%, mismatched unrelated 11%, cord blood 6%). there were 8 cases of proven and 37 probable ia, with prevalence of 14,7%. the median time to onset of was day + 53 (range: 4-449), with 30 (66%) cases diagnosed in neutopenia (before take o secondary neutropenia). there were 7 cases diagnosed on autopsy as a proven disease while antemortem they were classified as probable (2) or possible (4); no clinical suspicion was present in 1 patient. the diagnosis of probable ia was made by galactomannan platelia test in 29 patients and by sputum culture in 4. in 3 patients both criteria were present. in 29 cases ia was present within lungs only, whereas 14 patients developed disseminated ia. multivariate analysis identified the following risk factors for ia: late take of neutrophil cells (p=0.001) and steroid therapy (p=0.004). among 45 patients with ia, 34 died and 30 deaths were related to the mould infection. mortality was 76% (p<0.001). multivariate analysis, among patients with ia, for overall survival identified the following risk factors: atg use in conditioning regimen (p=0.046) steroid therapy, relapse, iga and cholinesterase at diagnosis of ia (p=0.009, 0.03, 0.041 and <0.001, respectively). conclusions: the prevalence if ia remains high among alternative hsct recipients, with few risk factors for ia. clinical and radiological presentation is highly aspecific and invasive procedures are rarely feasible. about concern survival of ia immuno deficiency at diagnosis seems to be important. henze (2) minimal residual disease (mrd) quantified prior to allogeneic sct has been shown to predict outcome in children with relapsed all in retrospective meta analysis. based on these results intense discussions were started as to whether transplant procedures should be adapted according to the mrd levels pre transplant. within the all-rez bfm group we have started a prospective trial evaluating the impact of pre-transplant mrd load in a well defined group of children who received their transplant in second or subsequent remission. between march 1999 and july 2005, 91 children with relapsed all treated according to the protocols all-rez bfm 96 or 2002 and receiving allogeneic sct in 2nd (n=77) or 3rd cr (n=14) have been enrolled. mrd quantification was performed within 40 days prior to sct by real time pcr using t-cell receptor and immunoglobulin gene rearrangements as clone-specific targets with at least 1 marker with a sensitivity of 10-4. probability of event free survival (pefs) and cumulative incidence of relapse (cir) in 45 patients with mrd ≥10-4 was 0.27 (±0.07) and 0.57(±0.08) compared with 0.60 (±0.08) and 0.13 (±0.06) in 46 patients with mrd <10-4 (p [efs, log-rank]=0.004; p [cir, gray] <0.001). clinical and therapeutical parameters were equally distributed between both subgroups. the difference in pefs and cir was more prominent in intermediate risk patients s2 (n=35) compared to high risk patients s3/s4/cr3 (n=56). thus, s2 patients with mrd ≥10-4 (n=14) showed pefs of 0.20 (±0.12) and cir of 0.73 (±0.15), where as patients who entered transplantation with mrd <10-4 (n = 21) had a pefs of 0.68 (±0.12) and a cir of 0.09 (±0.09); (p[efs] =0.020; p[cir] <0.001). high risk patients s3/4/cr3 who entered transplantation with a mrd load of <10-4 (n=25) showed a pefs and cri of 0.53 (±0.11) and 0.18 (±0.08), respectively. in contrast, pefs and cri were 0.30 (±0.09) and 0.50 se (±0.09) in patients who entered transplant with higher mrd load of > mrd-4. multivariate cox regression analysis revealed mrd as the only independent parameter predictive for efs (p=0.006). mrd prior to allogeneic sct proves to be the most important risk factor for outcome post transplantation. new strategies with modified sct procedures including conditioning regimen, graft manipulation, and gvhd prophylaxis and or post transplant intervention strategies are warranted and will be performed to improve prognosis in patients with a high risk of relapse. acute lymphoblastic leukemia (all) remains the most common indication for unrelated donor bone marrow transplantation in children. there have been major advances in conditioning regimes; supportive care and availability of donors, all of which have made transplant a viable treatment option for patients with relapsed or high-risk disease. we retrospectively analyzed data from 371 consecutive allogeneic transplants for all performed at the royal bristol hospital for children from october 1987 until september 2007. 371 transplants were performed in 357 patients with all. disease status at the time of transplant was complete remission(cr)-1, n=72; cr-2, n=227, cr-3, n=59 , children not in complete remission n=13.(see table 1 ) all patients were pre-treated on the current mrc-ukall protocols for the time period. eighty-six patients received stem cells from identical siblings or relatives. matched unrelated donors were used in 171 children, mismatched unrelated donors in 93, haplo-identical family donors were used in 21 children. included in these totals are 7 umbilical cord blood donations. cyclophosphamide and total body irradiation was used as conditioning therapy with the exception of children under the age of two and children receiving second transplants. ciclosporin a was used for graft verses host disease (gvhd) prophylaxis. short course methotrexate was given to mismatched and sibling donors. in the unrelated group t-cell depletion was effected using campath monoclonal antibodies. the majority of patients died due to disease relapse. the incidence of chronic graft verses host disease was low with correspondingly high karnofsky scores. this study offers a unique opportunity to analyze data from a large number of patients consistently pretreated according to united kingdom national leukaemia protocols and with a uniform approach to transplant conditioning, gvhd prophylaxis and supportive care over a 20 year period. background: intravenous bu has been used in the two dutch pediatric stem cell transplantation centers in various myeloablative regimens with different targets for the area under the curve (auc) and different dosing regimens of bu (once or four times daily). we retrospectively analyzed the association between bu exposure expressed as auc and clinical outcome. methods: all children, transplanted between 2001-2006, receiving intravenous bu as part of a myeloablative regimen, were included. patients were separated into quintiles based on the total auc of four days of treatment. the association with the primary endpoints death, graft-failure or relapse, efs and the secondary endpoints (acute graft-versus-host disease grade 2-4 (agvhd), veno-occlusive disease (vod) and mucositis grade 3-4), were tested using uni-and multivariate cox regression analysis. the lowest auc group was used as index group. results: 102 patients were included (46 malignant indications; 56 non-malignant indications). median age at transplantation was 3.1 years (range 0.2 to 21 years). the overall efs and survival were 68% and 72% respectively. in multivariate analyses a total auc between 72.5-80 mg*h/l was associated with highest efs (85%, p=0.029) and survival (90%, p=0.021). a lower auc bu was significantly associated with higher incidence of relapse or graft failure, whereas in the highest auc-percentile a high incidence of treatment related mortality (trm) was seen. covariates were hla-disparity and age. hla-mismatched donors and older children showed a significantly lower efs, the latter mainly due to a higher trm. agvhd occurred in 16%, vod in 22% and mucositis in 12% of patients. these side effects significantly correlated with a high auc bu in combination with the addition of melphalan to the conditioning regimen. the once daily dosing versus fourtimes daily did not influence any of the results. in conclusion a total auc of bu between 72.5-80 mg*h/l is associated with highest efs in malignant and non-malignant disease. a lower auc correlated with more relapse/graft failure especially in hla-mismatched donors. a higher auc was associated with more trm negatively influencing efs especially in older children. the occurrence of severe agvhd, vod and severe mucositis was mainly related to combination of bu and melphalan. dosing of busulfan in children by tdm to a target of 72.5-80 mg*h/l might improve efs. mechanical ventilation in a recent cohort of children after allogeneic haematopoietic stem cell transplantation: risk factors and outcome s. van gestel*, c. bollen, m. bierings, j. boelens, n. wulffraat, h. van vught university medical center utrecht (utrecht, nl) introduction: in previous studies, median intensive care unit (icu) mortality in children after hematopoietic stem cell transplantation (hsct) was 74% (range 25% -91%). it has been suggested that icu mortality decreased over time, but we could not confirm this in a recent meta-regression analysis (van gestel et al, submitted) . conclusions on mortality trends were limited, since recent outcome data from children requiring mechanical ventilation after hsct were scarce. objective: to assess risk factors for and outcome of mechanical ventilation in a recent cohort of children after allogeneic hsct. design: retrospective chart review. setting: a pediatric icu and hsct centre in a university hospital in the netherlands. patients and methods: all children who received an allogeneic hsct between january 1999 and april 2007 were included. patients who required endotracheal mechanical ventilation for more than 24 hours were identified. potential risk factors for the requirement of mechanical ventilation and for icu mortality were recorded. uni-and multivariable analyses were done to identify risk factors. results: 175 hscts were performed in 150 patients. thirtyfive patients (23% of hsct recipients) received mechanical ventilation on 38 occasions. none of the potential risk factors was significantly associated with icu admission. there was a trend towards an increased probability of icu admission over the years. this suggests that transplantations were carried out in sicker patients over time. sixteen admissions resulted in death on the icu, giving a case fatality rate of 42%. icu mortality was mainly associated with (persistence of) multiple organ failure on the first days of admission. none of the pre-admission transplantation characteristics significantly influenced icu mortality. based on their pediatric risk of mortality (prism) scores, our patients had a higher acuity of illness on icu admission than patients in previous studies. six-month survival in patients discharged from the icu was 82%. conclusion: we found a significantly lower icu mortality in a recent cohort of children after allogeneic hsct compared to previous studies, even though our patients were sicker. this can most probably be explained by improvements in transplantation medicine and icu treatment strategies. results from our study are promising, but need to be confirmed in other, preferably multi-centre, studies. early and comprehensive vaccination coverage in paediatric recipients of related and unrelated stem cell transplantation following coadministration of the pneumoccoccal conjugate vaccine prevenar™ and the hexavalent combination vaccine infanrix hexa™ r. meisel (1) children undergoing allogeneic hematopoietic stem cell transplantion (ahsct) lose protective immunity to vaccinepreventable disease and thus are at significant risk for lifethreatening infections. however, important issues on reimmunization after pediatric ahsct remain controversial due to the lack of prospective clinical trials. in the prospective multicenter vaccination trial ikast (nct 00169728) a total of 77 pediatric allohsct recipients (median age 8.3 (1.4-17.0) years) were therefore immunized with a primary series of three monthly doses of the hexavalent tetanus, diphtheria, pertussis, poliomyelitis, haemophilus influenzae type b and hepatitis b combination vaccine infanrix hexa (6vcv; glaxosmithkline pharma) along with the heptavalent pneumococcal conjugate vaccine prevenar (pcv7; wyeth pharma). vaccination was started at 6 months after transplantation, irrespective of immunosuppressive therapy or gvhd, with a subsequent booster dose at 18 months. immunogenicity was analysed by assessment of antibody concentrations and adverse events were prospectively collected. prior to immunization only 12.8% and 8.2% of patients (pts) exhibited protective antibodies towards pcv7 and 6vcv vaccine antigens, respectively. as a result of highly significant increases in mean antibody concentrations (p<0.001) protection to all pcv7 and 6vcv antigens was achieved in 85.1% and 89.8% of pts after primary immunization within the 1st year after allohsct, and this was independent of patient age, conditioning regimen, stem cell source, and most importantly, donor type (related vs. unrelated) and in vivo t cell depletion (all p-values>0.05). nine months later, prior to booster vaccination, 63.8% and 83.7% of pts retained protective antibody concentrations. however, mean antibody concentrations had dropped by a factor of 1.9-4.8 (p<0.001) except for pneumococcal serotype 14, thus underlining the need for subsequent vaccination. following booster immunization, antibody concentrations increased 2.4-19.1 fold (p<0.001) indicating robust memory responses, and 91.5% and 100% of pts achieved protective antibody levels against all pcv7 and 6vcv antigens. vaccination was well tolerated with no vaccine-related saes. our data show that early immunization of pediatric ahsct recipients according to our simple revaccination schedule is safe and provides early and comprehensive vaccination coverage during the first 2 years following ahsct from both related and unrelated donors. treosulfan-containing regimens achieve high rates of engraftment associated with low transplant morbidity and mortality in children with non-malignant disease and significant co-morbidities b.f. greystoke* (1), s. bonanomi (2) (1), p. naik (1), k. rao (2) , n. goulden (1) , p. amrolia (2) , r.f. wynn (1) , p.a. veys (2) (1)pendlebury children's hospital (manchester, uk); (2)great ormond street hospital (london, uk) treosulfan is an alkylating agent with both immunosuppressive and myeloablative properties, which has recently been introduced as a conditioning agent in allogeneic and autologous haemopoietic stem cell transplantation (hsct). early studies have been performed principally in adult patients with malignant disease in whom the drug appears to be well tolerated with a low incidence of regimen-related toxicity. we report the use of treosulfan in 32 consecutive children undergoing sct for non-malignant disease: immunodeficiency (n=18), metabolic disease (n=9), osteopetrosis (n=4) and other (n=1). patients received a total treosulfan dose of 36 or 42gms/m²/patient given in 3 divided doses on successive days. a range of other conditioning agents and serotherapy was administered to patients who underwent family donor sct (n=11), or unrelated donor sct (n=21). bone marrow (n=17), peripheral blood (n=9) and cord blood (n=5) were used as a stem cell source and in one patient a combination of marrow and cord blood from the same sibling donor was used. one patient (3%) died of early transplant-related complications. transplant morbidity was limited and there was no vod other than in this patient. mucositis was mild and many patients did not require parenteral nutrition. nappy rash with ulceration was noted in many patients. twenty-eight patients (87.5%) established donor cell engraftment which was full in 24 (85.7%). in 25 patients (78%) there was stable donor engraftment, including sufficient lineage specific chimerism, to correct the underlying condition. four patients required additional transplant procedures to maintain adequate donor-derived haemopoiesis, and two patients are under consideration for further hsct. twentyseven patients survive with a median follow up of 417 days. there were 4 late deaths due to progression of the underlying disease, gvhd or infection. treosulfan based conditioning regimens achieve excellent engraftment with reduced regimen-related toxicity and in children with non-malignant disease at high risk for both regimen-related toxicity and graft failure. preliminary results indicate that in a haploidentical setting cd3/cd19-depleted grafts may be advantageous regarding engraftment and immunreconstitution. since effector cell with potential antileukemic activity are cotransfused, such grafts may be suited in particular for patients with insufficient remission. nk cells have been shown both in vitro and in vivo to mediate positive effects regarding engraftment, gvhd, immunreconstitution and relapse and are an important part of the cd3/19 depleted grafts. we analyzed nk activity in 30 cd3/19 depleted grafts, which was low compared to fresh isolated pmncs. several cytokines and cytokine combinations for activation of the grafts were tested and the strongest enhancement in nk activity could be obtained with il-15. we therefore developed a protocol for the overnight incubation of the grafts according to gmp. stimulated grafts were transplanted seven times either at day 0 or as an immunotherapeutical approach after transplantation and were well tolerated. median cell counts for infused cells/kg bodyweight were 46x10 6 cd56+/3-, 13x10 3 cd3+, 9x10 3 cd19+, 88x10 6 cd14+ and 3,1x10 6 cd34+ cells. no occurrence of agvhd was seen in these patients after infusion of the cells. enhanced nk activity after stimulation could be demonstrated in vitro against k562 or primary leukemic blasts. nk cells showed increased proliferation capacity in brdu assay and [3h]-thymidine incorporation assays, especially after additional stimulation with il-2 while t cells showed only a moderately enhanced proliferation against third party stimulators or with okt3. therefore activation of haploidentical t cell depleted grafts with il-15 is a promising tool to enhance the activity of infused nk cells and should be further investigated in clinical trials. background: development of an effective strategy for patients with epmd. methods: 328 patients (pts) were registered. median age is 16.2 years (yrs) (0.4-49). primary site was extremity in 98 pts and axial/other in 217 pts (45% in the pelvis). metastatic spread was bone marrow (bm) only in 33 pts, bone only in 137 pts and bone & bm in 105 pts, other (mainly lymph nodes) metastatic sites in 33 pts. tumour volume was above 200 ml in 200 pts. six vide induction cycles were completed by 88%. local treatment included surgery when possible and/or radiotherapy (rx) as indicated. recommended hdt was busulphan (bu) 600mg/m² and melphalan (mel) 140 mg/m² with pscr. median follow up is 3 years (range, 0-7.5). results: partial remission or better was achieved after cycle 6 in 76%. the overall survival at 3 years is 32%±3. in patients with bm/bone metastasis, significantly favourable univariate factors in the unselected cohort at diagnosis (dx) were age < 14 yrs (event free survival at 3 yrs (efs) 39%, p<0.001), metastatic site (efs: bm only 47%, bone only 22%, bm+bone 15%, p=0.003), single bone lesions only (efs 37%, p=0.004), primary tumour site (efs: extremities 33%, chest/spine/hn 27%, abdomen pelvic 22%, p=0.035) and tumour volume of <200ml (efs 44%, p<0.001). ). multivariate analysis identified four major risk factors at dx: primary tumour volume >200ml p<0.001 (rr 2) and > 5 bone metastases p=0.055 (rr 1.7), age above 14 p=0.012 (rr 1.6), bm metastasis p=0.042 (rr=1.4). for pts receiving bumel it is noteworthy that 46pts of <14a and epmd achieved an efs of 46% in comparison to older counterparts >14a (efs 23%, p=0.007). conclusion: further strategy refinement and validation of hdt appears necessary within investigational , ideally randomised studies. allogeneic haematopoietic cell transplantation for chronic lymphocytic leukaemia with 17p deletion: a retrospective ebmt analysis j. schetelig*, a. van biezen, r. brand, d. caballero, r. martino, m. itala, j. garcía-marco, l. volin, n. schmitz, r. schwerdtfeger, a. ganser, f. onida, b. mohr, s. stilgenbauer, m. bornhäuser, t. de witte, p. dreger on behalf of the chronic leukemia working party, ebmt purpose: patients with advanced chronic lymphocytic leukaemia (cll) and 17p deletion have a very poor prognosis even after intensive chemotherapy. while allogeneic hematopoietic cell transplantation (hct) has the potential to cure patients with advanced cll it is not known whether this holds true for patients with 17p deletion. patients and methods: patients with 17p-cll who had received hct were identified by an ebmt-based survey. baseline data were downloaded from the ebmt database. additional information on the course of the disease, the cytogenetic diagnosis and last follow up was collected by a questionnaire. data were analysed as of february 2007. results: 56 patients were identified. twelve patients with autologous hct, haplo-identical donors or the detection of 17p-after hct were excluded from further analysis. 44 patients had received an allogeneic hct between march 1995 and july 2006 from a matched sibling donor (n=24) or an alternativ donor (n=20). the median age at hct was 54 years. the diagnosis of deletion 17p-was made by fish in 82% and by conventional banding in 18% of patients. the median interval between first diagnosis and detection of 17pwas 2.4 years and the median interval between detection of 17p-and hct was 0.5 years. patients had received a median of 3 chemotherapy regimens, including fludarabine in 98% of patients. at hct, 53% of patients were in remission. reduced intensity conditioning was applied in 89% of patients. 93% of the patients received peripheral blood stem cells. gvhd prophylaxis was performed heterogeneously. one patient experienced primary graft failure. acute gvhd grades ii to iv occurred in 44% of patients and extensive chronic gvhd in 46% of patients. after a median follow-up of 23 months (range, 2 to 90 months) of 24 patients who are alive, 18 were in complete remission, 4 in partial remission and 2 patients had progressive disease at last follow up. 4-year overall survival and progression-free survival was 47% (95% ci, 29% to 65%) and 38% (95% ci, 20% to 56%). the cumulative incidence of relapse at 4 years was 35% (95% ci, 12% to 57%). no additional relapse occurred in six patients with a follow-up between 4 and 7.5 years. conclusion: allogeneic hct has the potential to induce longterm disease-free survival in selected patients with advanced 17p-cll. given the otherwise very dismal outcome of this disease, prospective studies on allogeneic hct earlier in the course of 17p-cll seem warranted. separating patients with myelofibrosis with myeloid metaplasia into risk groups for allogeneic haematopoietic cell transplantation -results of a german multicentre analysis f. collenbusch, r. schwerdtfeger, m. schleuning, c. schmid, j. finke, m. stadler, m. bornhaeuser, d. messerer-schmid, h objective: we aimed to derive risk factors for allogeneic hematopoietic cell transplantation (allohct) in patients with s53 myelofibrosis with myeloid metaplasia (mmm) from retrospective analysis. patients: between 1999 and 2006 97 patients (pts) from 15 german centers, median (md) age 51 (19-66), were grafted from 41 related and 56 unrelated donors. 74 pts had primary, 23 secondary mmm. at allohct 32 pts were dupriez score 0, 37 score 1 and 28 score 2, 49 needed red cell transfusions. chromosomal and molecular analysis was available from 75 and 51 pts resp.: 51 pts had favourable, 24 unfavourable cytogenetics, 31 pts were jak2 mutated. conditioning was of standard, intermediate and reduced intensity in 27, 57 and 13 cases. 28 pts received bm, 69 pbsc. 5 boosts and 16 donor lymphocyte transfusions were given for 16 relapses, 2 incomplete chimerisms and one graft failure. results: at a md follow up of 985 (100 -2630) days probability of overall and relapse free survival (os and rfs) was 62 and 39%. neither time from diagnosis to transplant, age, bsymptoms, marrow fibrosis, splenomegaly, jak2, hemoglobin <10 g/dl, platelets <100/nl, ldh, comorbidity (cci or hct-ci), conditioning, type of donor nor graft source were predictive for os. log rank test showed a trend towards lower survival in cases with dupriez score ≥1 (p=0.06) and transfusion dependence (p=0.1). solely circulating blasts >1% (p=0.016), monocytes >1/nl (p=0.008) and cytogenetics (p=0.009) were predictive for survival after allohct. cox regression analysis within pretransplant variables revealed cytogenetics (p=0.016) and monocytes (p=0.036) as independent factor for os. for rfs only cytogenetics retained independence (p=0.003). combining cytogenetics, monocytes, blasts and transfusion dependence to a risk score from 0 to 4 allowed to discriminate between good (0-1), intermediate (2) (3) and high risk (4) pts for os (83, 45, 0%, p=0.0008) and rfs (68, 34, 0%, p=0.0013). following allohct pts with limited cgvhd had a projected os and rfs of 84% and 81% as opposed to 43% rfs for no and 20% rfs for extensive cgvhd, resp. (p=0.018). 7 pts treated with cellular immunotherapy achieved a partial and 7 a complete response. conclusions: pts with mmm can be scored as low, intermediate and high risk for allohct according to pretransplant disease characteristics. evidence for a potent graft-versus-mmm-effect suggests exploitation of adjuvant cellular immunotherapy to improve current results. syngeneic bmt for cml in chronic phase: update of the seattle results a. fefer*, j. radich, t. gooley, l. holmberg, m.e. flowers, s. pavletic, r. storb, f. appelbaum fred hutchinson cancer research center (seattle, us) to determine the efficacy of syngeneic bmt as treatment for chronic phase cml (cml-cp), we reviewed the results of all patients (pts) we transplanted between 1976 and 2000, before the gleevec era. of 34 pts, 5 died within 4 months (mos)due largely to pulmonary problems. of the 29 pts who went into a complete cytogenetic remission (cr), 15 relapsed at a median time of 2 years after bmt, but 14 remained in cr for up to 31 (median,19) years. all 9 pts tested were negative by pcr when last tested at 2-23 (median,13) years. thus, enduring cytogenetic and molecular cr's were achieved with syngeneic bmt, in the absence of an allogeneic graft vs leukemia (gvl) effect. at 20 years, the estimated probability of relapse, overall survival and relapse-free survival was 46%, 42% and 28%,respectively. to assess the influence, if any, of the conditioning regimen on the relapse rate, we subdivided the 34 pts into 3 groups transplanted in 3 time periods with 3 different conditioning regimens, as follows: group i (n=14), 1976-81, dimethylbusulfan (dmb) 5mg/kg iv, cyclophosphamide (cy) 60mg/kg/day x2 and total body irradiation (tbi) 10gy at a single exposure; group ii (n=10), 1982-88, cy 60mg/kg/d x2 & tbi 2gy/day x6; group iii (n=10), 1989-2000, busulfan (bu) 2mg/kg/day x4 po, cy 30mg/kg/day x2 and tbi 2gy/day x6. the median time from diagnosis to bmt for the 3 groups was 11, 3 & 5 months, respectively. relapses occurred in 5 pts in group i at a median of 4 years, 8 pts in group ii at a median of 1 eayr, and 2 pts in group iii at 1 and 4 years. thus, the relapse rate with the cy/tbi regimen was significantly higher than that for the regimens containing dmb or bu (hazard rate (hr) of relapse for group ii vs i was 4.17, p=0.01, and for group ii vs iii, hr 4.80, p=0.05). however, cy/tbi was less toxic, with no early bmt-related deaths in contrast to 2/14 and 3/10 deaths in groups i and iii. finally, we compared the risk of relapse in groups i and iii combined i.e.twins conditioned with dmb/bu-containing regimens, with that of 366 cml-cp pts who underwent bmt from allogeneic matched siblings after cy/tbi at our center. the risk of relapse is quite similar (hr=1.06, p=0.87). these results suggest that either the dmb or bu can eradicate a number of cml cells similar to that destroyed by an allogeneic gvl effect and/or that the dmb or bu can somehow induce a gvl effect exerted by syngeneic cells. we have previously shown that levels of the polycomb group (pcg) gene bmi-1 rna were significantly higher in advanced phase than in chronic phase (cp) chronic myeloid leukaemia (cml). in addition, in patients treated with hu and ifn-a, low bmi-1 expression was associated with an improved overall survival (os) (blood 2007). here, we investigated whether bmi-1 and other previously established prognostic genes (cd7, pr-3 and ela-2) are implicated in the prognosis of cml in the context of allogeneic stem cell transplantation (allo-sct). we studied 84 cp-cml patients who received allo-sct from hla-identical related donors. cd7, pr-3, ela-2 and bmi-1 expression was assessed by q-rt/pcr in the recipients pbmcs collected before allo-sct. the median expression level for each gene was used to segregate the patients into 2 groups (low: gene expression <median, and high: >median). the median fu post-allo-sct was 9.9 (range, 1.7-23.9) years. the median ebmt-gratwohl score was 3. none of the 4 tested genes showed any significant association with engraftment or with graft rejection. cd7, pr-3 and ela-2 expression was not associated with os. however, in contrast to our previous findings in the non-allo-sct setting, patients displaying a high bmi-1 expression level prior to allo-sct had significantly better os than those with low expression (p=0.005). when bmi-1 was included in a multivariate survival model and adjusted for the other prognostic variables, a high expression was found to be an independent marker associated with better survival (rr=2.72, 95%ci;1.1-6.9;p=0.034). given the impact of bmi-1 expression level on os, without a significant association with relapse, and since neither bmi-1, nor the other genes showed any significant association with leukemia-free survival, we assessed their impact on trm. there was a striking and significant association between acute gvhd and bmi-1 expression, not only in overall incidence (low bmi-1: grade 0-1 (n=21), grade 2 (n=10), grade 3-4 (n=9); high bmi-1: grade 0-1 (n=32), grade 2 (n=9), grade 3-4 (n=1); p=0.005), but also in cumulative incidence at day 100 (48% vs. 24%, p=0.016). in multivariate analysis, a low bmi-1 expression level was associated with an increased risk of grade 2-4 acute gvhd (rr=2.85, 95%ci; 1.3-6.4; p=0.011). these results suggest that bmi-1 can serve as a biomarker for predicting outcome in cp-cml patients receiving allo-sct. such measurement allows for tailored therapeutic intervention, including informed recommendation for allo-sct in patients failing tyrosinekinase inhibitors. haematopoietic stem cell transplantation in tprolymphocytic leukaemia: a retrospective ebmt analysis w. wiktor-jedrzejczak*, r. brand, a. van biesen, e. carreras, v. leblond, g. cook, m. ethell, a. nagler, t. ruutu, m.l. brune, j. schetelig, t.m. de witte, p. dreger on behalf of the clwp ebmt t prolymphocytic leukemia (t-pll) is a rare, aggressive neoplasia of t lymphoid lineage which is characterized by poor survival of less than one year. incidental reports suggest that both autologous and allogeneic hematopoietic stem cell transplantation (hsct) might be effective in this disease. however, no larger series on the efficacy of hsct in t-pll has been reported to date. therefore the purpose of the present study was to analyze the outcome of transplants for t-pll registered at the ebmt database. results: eleven patients who had undergone autologous transplantation (auto-sct) and 33 patients who had undergone allogeneic transplantation (allo-sct) were identified from the database and verified as t-pll. in the auto-sct group, there were 9 males and 2 females, with a median age of 59 (37-64) years. 5 patients were transplanted within the 1st year of diagnosis, and the remainder in later phases. 2 patients received tbi in their conditioning, while 8 received chemotherapy only. engraftment and recovery was prompt. there was one case of non-relapse death (nrm). 5 patients relapsed within the first 2 post-transplant years, translating into a median event-free survival (efs) of 14 months and a median overall survival (os) of 22 months. a single patient had prolonged disease control until relapse after 53 months.in the allo-sct group (20 males/13 females), median age was 51 (24-71) years; 18 patients (55%) had been transplanted within the 1st year of diagnosis. status at allo-sct was complete or partial remission in 18 patients (55%), and more advanced disease or unknown in the remainder. 14 patients (42%) received reduced intensity conditioning. donors were hla-identical siblings in 16 patients (48%), matched unrelated donors in 16 patients, and a mismatched unrelated donor in one patient. engraftment was documented in 29 of 30 patients (97%) with information available. 2-year nrm was 25%. 10 of 11 relapses observed occurred during the first post-transplant year, mainly in patients transplanted in advanced disease, translating into biphasic survival curves with initial steep decline (median efs 7 months, median os 11 months) but still 32% efs at 24 months suggesting sustained disease control in a subset of patients. conclusions: these data indicate that allo-sct may provide effective disease control in selected patients with t-pll. prospective investigation of allo-st in eligible patients with this otherwise fatal disorder seems to be warranted. high-dose chemotherapy with autologous stem-cell support versus standard-dose chemotherapy: metaanalysis of individual patient data from 15 randomised adjuvant breast cancer trials t. demirer*, n.n. ueno, m. bregni, d with autologous haematopoietic stem cell transplantation (hsct) for the treatment of primary poor risk breast cancer (bc) patients (pts) has lost favour over recent years in the oncology community. this is mainly due to the worrisome high mortality and morbidity of the procedure, and the lack of a clear survival benefit in early randomized studies. recently reported trials have demonstrated that hdc with hsct could still have a role in selected subgroup of pts, namely pts with ≥ 10 positive axillary lymph nodes (ln) and in pts with her-2 negative tumours. aim of this study is to re-evaluate toxicity and efficacy of hdc with hsct in a large cohort of pts receiving hdc in italy between january 1, 1990 and december 31, 2005. 1294 bc pts receiving hdc for poor risk bc were identified in the gitmo registry. in 1183 patients with >3 ln, a thorough data set including biological characteristics, toxicity and follow up was available. median age was 46 years (24-66), 62% of pts were pre menopausal at treatment, 71% had an endocrine responsive tumours and 43% had a her-2+ tumour. median number of positive axillary ln was 15 (4-63), with 23% of pts having ≥ 20 ln+. 73% of pts received alkylating agents-based hdc as a single procedure while 27% received epirubicin or mitoxantrone-containing hdc, usually within a multi-transplant program. transplant related mortality (trm) at 100 days was 0.7%, while late cardiac and secondary tumour related mortality were around 1% overall. with a median follow up of 74 months, median disease free survival (dfs) and overall survival (os) in the entire population were 6.5 and 7.5 year, respectively. exploratory subgroup analysis demonstrated that os was significantly better in endocrine responsive tumours (p=0.0000), while menopausal or her-2 status did not affect survival. in 85 poor prognosis pts with er, pgr and her-2 negative tumours (median ln+ = 18), median os was 110 months. median os was significantly better (p=0.0000) in patients receiving multiple transplant procedures, this effect being particularly evident in the ≥ 10 ln+ population. in conclusion, our retrospective analysis suggests that hdc with hsct has lower trm than expected and high efficacy in well defined subgroups of patients. multiple transplants seem more active than single hdc procedures. this analysis could be useful in selecting well defined patient populations in which to re-address the role of hdc as adjuvant treatment. long-term follow-up of metastatic renal cancer patients undergoing reduced-intensity allografting m. bregni*, m. bernardi, p. servida, a. pescarollo, r. crocchiolo, e. treppiedi, f. ciceri, j. peccatori scientific institute san raffaele (milan, it) objective: stem cell transplantation from a hla-compatible sibling donor has been advocated as adoptive immunotherapy for cytokine-resistant, metastatic renal cancer (rcc). however, the recent introduction of several targeted therapy compounds has reduced the interest in this therapeutic strategy. we have reanalyzed our transplant series with the aim to detect long-term benefit form allografting. methods: from february 1999 to may 2005, 25 patients with cytokine-refractory rcc received a reduced-intensity allograft from an hla-id sibling donor. median age was 53 years; most (24) had clear-cell histology. median number of previous treatments was 1 (0-3). median days from diagnosis to allograft were 822. all patients received a thiotepa, fludarabine, and cyclophosphamide conditioning regimen, and a cyclosporine-based gvhd prophylaxis. six patients received dli at escalating doses for progressing or nonresponding disease. results: one-year-os was 48% (95% ci: 28-68), and 3y-os was 20% (95% ci: 4-36). at a median observation time of 65 months, 5 patients are alive, one in cr, one in vgpr, and three with disease. we have analyzed the correlation of the following variables with survival: pre-transplant disease status, age at transplant, time from diagnosis to transplant, total infused cells, infused cd34+ cells, infused cd3+ cells, bm chimerism at +30, post-transplant disease status at +30 and +90, best response, day of best response, csa withdrawal day, infusion of dli, occurrence of gvhd. at univariate analysis, numbers of cd34+/kg infused cells, best response, and disease status at +90 significantly correlated with survival. at multivariate analysis, only disease status at +90 retained statistical significance (p=0.002), while cd34+ cells/kg retained marginal significance (p=0.059). conclusion: twenty percent of cytokine-refractory rcc patients are alive at a median 65 (40-72) months after allografting. all these patients have received >5x10 6 cd34+ cells/kg, and had stable or responding disease at +90 after transplant. three patients are receiving sorafenib, and two are in cr/pr without further therapies. reduced-intensity allografting is able to induce long-term disease remission in a fraction of relapsed rcc patients. it is unknown if relapse or pd after targeted therapy will be susceptible to allograft-mediated gvt effect. the place of allografting in the treatment of metastatic rcc, alone or in combination with targeted therapies, needs reappraisal. haploidentical family donors represent the ideal solution to offer to every patient with high risk leukemia the potential cure of marrow stem cell transplantation. extensive application of haploidentical transplantation (haplo-sct) is limited by high rate of late transplant related mortality (trm) and relapse associated with the delayed immune reconstitution secondary to the procedures for severe graft-vs-host-disease (gvhd) prevention. in a haplo-sct phase i-ii multicenter, open, no-randomized, trial sponsored by molmed spa, we infused donor lymphocytes genetically engineered to express the suicide gene herpes simplex thymidine kinase (tk-dli) to induce early immune reconstitution, while selectively controlling gvhd. between september 2002 and september 2007, 51 patients (pts) -median age 48-with high-risk hematologic malignancies were enrolled, 29 out of 51 pts were in complete remission (cr). after myeloablative conditioning regimen, 48 pts received a median 13x10 6 /kg cd34+ and 1.0x10 4 /kg cd3+ (median time to engraftment: 2 weeks). no immune reconstitution were observed in absence of tk-dli. twenty-seven pts received tk-dli: 22 pts obtained prompt immune reconstitution with cd3+>100/mcl at day+75 (median) from haplo-sct and day+23 from tk-dli. eleven pts developed gvhd (10 acute gvhd grade i-iv and 1 chronic gvhd) that was always abrogated by the suicide gene induction. the 3-year trm was 26%, with last infectious event at day 166 post transplant, for pts treated with tk-cells, and 69% for pts who didn't receive tk-cells (p<0.0001). immune reconstitution obtained with tk-cells infusion correlated with: 1. rapid development of a wide t-cell repertoire, 2. detection of high frequencies of t-cells specific for opportunistic pathogens, 3. abatement of the incidence of infectious adverse events (ae) and serious ae. the 3 years lfs was 45% for pts who achieved immune reconstitution and 9% for pts who failed immune reconstitution (p:<0.0001). this strategy is feasible and effective in providing immune reconstitution in haplo t-cell-depleted setting. in uni-and multi-variate analysis both status at transplant and immune reconstitution are significant risk factor. infusion of tk-cells could significantly extend the application of haplo-sct. a randomized phase iii study comparing tk-dli versus any t cell repletion strategy after haplo-hsct in high risk acute leukemia is now starting. ie-1 and pp65 specific peptide pools for the generation and expansion of cmv-specific donor t-cells after haploidentical stem cell transplantation: feasibility and first clinical experiences s. ganepola* (1), p. reinke (2) , c. gentilini (1) , m. hammer (2) , m. schmidt-hieber (1), c. tietze-bürger (1), k. freyberg (1), d. volk (2) , e. thiel (1) , l. uharek (1) (1)hematology/oncology/transfusionmedicine (berlin, de); (2)charite berlin campus mitte (berlin, de) for immunocompromised patients, rapid reconstitution of cytotoxic t cell function is mandatory for the control of human cytomegalovirus (cmv) infection and disease. the cmvassociated proteins pp65 and ie-1 are important components of a relevant cmv-directed immune response. here we report the first clinical experiences concerning feasibility and safety with a novel method for the generation of cmv-specific t-cells by stimulation with a peptide-pool containing pp65 and ie-1. material and methods: after apherisis of 1-1.2x10 9 pbmc from 4 healthy donors (2 igg positive, 2 igg negative) and 2 patients with proven cmv disease, cells were further processed under gmp-conditions. cells were stimulated with peptide pools representing the pp65 (ul83) and ie-1 (ul123) proteins and ifng producing cells were selected with the cytokine-capture-assay (miltenyibiotec) and further expanded in cell-culture conditions on a 24-well-plate. after performance of intern and extern quality controls, cells were freshly retransfused and/or kryoconserved in defined portions for further redonations. results: in 5/6 rounds we could expand cmv-specific t-cells. expansion-rates ranged from 1.2 fold to 71.1 fold (median 9.4) of the initial cell count. in a median time of 15 days (range 14-23 days), cell counts expanded from 1.2 fold up to 71.1 fold (median 9.4). facs-analysis at the end of the culture revealed that in median 80% of the cells were cd8+ (range 2.5-92.3%) and, respectively, 20% were cd4+ (range 7.2-89.6%). in cytotoxicity assays, a lysis of 50-83% (median 53%) of lcl-line cells could be achieved. so far, two patients with therapy refractory cmv disease received in vivo expanded polyvalent t-cells against multiple (ie-1/pp65) cmv epitopes. no serious adverse events occurred during the first days after administration and cmv antigenemia was decreasing in one patient. however, since both patients finally died from a septical multi-organ failure, we were not able to assess the long lasting impact of the manoeuvre on the control of cmv-disease. conclusion: selection and subsequent expansion of cmvspecific t-cells is possible with this method which allows an up to 70-fold expansion of cd8+ cells. the use of pp65 and ie-1 specific peptide pools for the prevention or treatment of cmv infection is feasible and could be of superior effectiveness as compared to approaches directed against a single cmv epitope. lmp2-specific tcr gene therapy for hodgkin's lymphoma d.p. hart, s. thomas, s. xue, h.j. stauss, e.c. morris* university college london (london, uk) ebv-positive hodgkin lymphoma typically demonstrates latency ii antigen expression, characterised by loss of most ebv antigens except for the latent membrane protein (lmp) 1 and 2 and the ebna-1 protein. reed sternberg cells expressing lmp2 can be a target for antigen-specific immunotherapy, but lmp2-specific autologous ctl for adoptive immunotherapy are difficult to generate due to poor immunogenicity. t cell receptor (tcr) gene transfer using retroviral vectors containing the tcr alpha and beta chain genes can reproducibly redirect the antigen specificity of a t cell population. the aim of this study was to generate a retroviral tcr construct suitable for the rapid and efficient production of lmp2-specific ctl. retrovirally introduced tcrs compete with endogenous tcrs for cd3 molecules required for assembly of the tcr complex. this competition may limit surface expression of the introduced tcr resulting in a transduced t cell with poor functional avidity. in an attempt to generate a 'highly competitive' lmp2-tcr the following modifications were made to the retroviral vector construct: i) nucleotide sequences were codon-optimised for efficient translation in human cells; ii) the constant region of each tcr chain was altered to contain murine sequences to enhance cd3 binding; and iii) the tcr alpha and beta chain genes were linked by a self-cleaving 2a sequence. the unmodified hla-a2-restricted lmp2-specific tcr was poorly expressed in primary human t cells (up to 2.5% of viable cd3+ t cells, as detected by facs analysis using monoclonal anti-vbeta13 antibodies), suggesting that it competed inefficiently with endogenous tcr chains for cell surface expression. however, retroviral transfer of the modified lmp2-tcr into human t cells improved lmp2 tcr expression to 55-65% cd3+ t cells. the transduced cells bound hla-a2/lmp2 pentamer, showed peptide-specific ifngamma and il2 production and killed target cells displaying the lmp2 peptide. importantly, expression of the introduced lmp2-tcr suppressed expression of almost the entire repertoire of endogenous tcr combinations, including 'mis-paired' tcrs. 'mis-paired' tcrs contain an introduced alpha chain paired with an endogenous beta chain and vice versa. the antigen specificity of such mispaired tcrs is unknown and could be auto or allo-reactive. modified tcr sequences, producing 'dominant' tcrs may improve the efficacy and reduce the potential risks of tcr gene therapy a novel form of adoptive immunotherapy. allogeneic stem cell transplantation in patient with major histocompatibility complex class ii immunodeficiency: a single-centre experience h. al-mousa*, z. al-shammari, a. al-ghonaium, h. al-dhekri, s. al-muhsen, r. arnaout, a. al-seraihy, a. al-jefri, a. al-ahmari, m. ayas, h. el-solh king faisal specialist hospital (riyadh, sa) background: major histocompatibility complex class ii (mhc ii) deficiency is a rare combined immunodeficiency disease. allogeneic bone marrow transplantation (bmt) is considered the only available curative treatment. survival rate post bmt is lower than other forms of primary immunodeficiencies. these differences were observed for both bmt performed with matched and non-matched donors. patients and methods: between june 1994 and august 2007, thirty two children with mhc ii deficiency underwent thirty seven bmt procedures at king faisal specialist hospital and research centre, riyadh, saudia arabia. five patients required second bmt trial. median age at bmt was 27 months (range, 1-129 months). the source of stem cells was unmanipulated marrows from hla-genoidentical siblings in 22 pts, hla-phenotypically identical related donors in 9 pts and umbilical cord for 1 patient. conditioning was with one of 4 regimens, regimen a (19 pts): busulfan (bu), cyclophosphamide (cy) and etopside (vp-16), regimen b (3 pts): bu/cy and anti-thymocyte globulins (atg), regimen c (2 pts): bu/cy/vp-16 and atg and regimen d (13 pts): fludarabine, melphalan and atg. median cd34 dose was 8.3 x 10 6 /kg (range, 1.5-20.7 x 10 6 /kg). graft-versus-host disease (gvhd) prophylaxis consisted of cyclosporine (csa) and methorexate (mtx) in 21 pts, csa alone in 15 pts and csa and steroid in 1 patient. results: 24 pts had adequate immune reconstitution and sustained engraftment (assessed by short tandom repeats) ranged from 24-100% for lymphoid line and 5-100% for myeloid line. seven pts (4 pts were post second transplant) died secondary to sepsis, multiorgan failure and primary disease. acute gvhd was seen in 20 pts and 4 pts developed chronic gvhd. the overall disease free survival rate was 75% with a median follow up of 5.3 years (range 0.5-10.2 years). low survival rate (20%) was seen in patients who underwent second bmt. conclusion: bone marrow transplantation (bmt) can cure the disease, provided it is performed before complications leading to severe organ failure develop. second bmt is associated with high rate of mortality. further studies and long term follow up are required to determine the appropriate conditioning regimen. to analyze the effects of cd34-trail+ cells on tumor vasculature, tumorbearing mice were perfused with sulfo-biotin and tumor endotelial cells (tec) were then revealed by horseradish peroxidase (hrp)-conjugated streptavidin. as compared with cd34-mock-or soluble (s)trail-treated mice, a 24-hour treatment with cd34-trail+ cells significantly (p ≤.001) reduced microvessel density (1850 ± 1139 vs 2227 ± 915 vs 757 ± 562 vessel per 1 x 10 5 tumor cells, respectively) and increased the thickness of the vessell wall (3.7 ± 1 µm vs 3.4 ± 1 µm vs 6 ± 1 µm, respectively), suggesting that cd34-trail+ cells induce an early vascular disruption leading to a progressive disintegration of the vascular bed. confocal microscopic imaging of tumor sections double-stained with anti-cd31 and anti-trail-r2 showed that this receptor was expressed by 8 -12% of large tumor vessels. interestingly, upon treatment with cd34-trail+ cells, but not strail, tunel staining revealed an extensive apoptosis of tec. forty-eight hours following injection of cd34-trail+ cells, a 21-fold increase of apoptotic index was detected, which was associated with extensive necrotic areas (20% to 25% of tissue section). these data show that: (i) tumor homing of cd34-trail+ cells induces extensive vascular damage, hemorrhagic necrosis and tumor destruction; (ii) the antitumor effect of cd34-trail+ cells is mediated by both indirect vascular-disrupting mechanisms and direct tumor cell killing. targeting of a therapeutic suicide gene to human alloreactive memory t-cells with stem-cell features requires il-7 and il-15 a. bondanza* (1), l. hambach (2) in a phase ii clinical trial investigating the prophylactic infusion of suicide gene-modified donor t cells after haploidentical hemopoietic cell transplantation (haplo-hct), we observed a rapid and effective immune reconstitution. after activation with anti-cd3 antibodies, t cells were modified with a retroviral vector (rv) encoding for the herpes simplex thymidine kinase (tk). tk+ cells displayed an effector memory (em) phenotype (cd45ra-cd62l-, cd28±cd27+, il-2±ifn-g+). when needed, graft-versus-host disease (gvhd) was controlled upon administration of the prodrug ganciclovir (gcv). the graft-versus-leukemia (gvl) effect was substantial in patients transplanted in remission, but failed to cure patients in relapse. genetic modification with rv is limited to memory t cells. em tk+ cells have a reduced alloreactivity. central memory (cm) t cells (cd45ra-cd62l+, cd28+cd27+, il-2+ifn-g±) share many characteristics with stem cells, namely the ability to selfrenew and to differentiate into effector cells. recently, it has been proposed that alloreactivity may be confined to memory t cells with stem-cell features. since alloreactivity is the common ground of both graft-versus-host disease (gvhd) and the gvl effect, crucial to the success of the strategy is the suicide gene-modification of cells with such properties. we found that addition of cd28 costimulation on cell-sized beads and the use of homeostatic cytokines, such as il-7 and il-15, generates central memory (cm) tk+ cells. cm tk+ cells were highly alloreactive, both in vitro and in vivo in a humanized animal model of gvhd based on the grafting of human skin onto nod/scid mice. gcv administration abrogated gvhd. stimulation of cm, but not of em tk+ cells with autologous dendritic cells pulsed with hla2-restricted peptides from the minor histocompatibility alloantigen (mhag) ha-1 or h-y efficiently induced mhag-specific t cells that lysed natural ligand expressing hla-a2+ targets. a fraction of mhag-specific tk+ cells expressed il-7ra. only il-7ra+ mhag-specific tk+ cells could self-renew and differentiate into effector cells. when infused in nod/scid mice harboring human mhag+hla-a2+ leukemia, tk+ mhag-specific t cells significantly delayed disease progression. altogether, these data suggest that targeting of a suicide gene to human alloreactive memory t cells with stem-cell features requires il-7 and il-15 and warrant their use in the clinic for a safe and powerful gvl effect. loss of foxp3 expression after in vitro expansion of human cd4+cd25+cd127-regulatory t-cells p. hoffmann* (1), t.j. boeld (1) , r. eder (1) , j. huehn (2) , s. floess (2) in animal models the adoptive transfer of donor-type cd4+cd25+ regulatory t cells (treg) protects from graftversus-host disease (gvhd) after allogeneic stem cell transplantation (sct), as shown by us and several other groups. exploring this strategy in human sct, we currently perform a first phase i clinical trial using freshly isolated treg. for future trials requiring large cell numbers for repetitive treatments, we described in vitro culture conditions that permit a more than 3-log polyclonal expansion of treg (blood 104:895; 2004) . a highly enriched starting population proved to be crucial for the generation of pure treg cell products, a criterion fulfilled by the naïve, cd45ra+ subpopulation of cd4+cd25high t cells (ra+ treg) (blood 108:4260; 2006 ). an alternative isolation strategy for treg relies on the exclusion of cd127+ cells, as activated cd4+cd25+ conventional t cells express high levels of cd127 while cd4+cd25+ treg show no or only weak expression. for a direct comparison of the two isolation protocols, we sorted cd4+cd25+cd127low/neg t cells (cd127-treg) and ra+ treg from the same leukapheresis products and analyzed the cells after 2 and 3 weeks of expansion. whereas both populations were > 94 % foxp3+ upon isolation, only ra+ treg maintained foxp3 expression throughout the expansion period (93 % [range: 78 to 97 %; n=11] after 2 and 87 % [range: 71 to 97 %; n=9] after 3 weeks). in contrast, cd127-treg cultures contained only 82 % (range: 56 to 96 %; n=11) foxp3+ cells after 2 weeks and highly variable and significantly lower numbers of foxp3+ cells than ra+ treg cultures after 3 weeks (57 % [range: 18 to 93 %; n=9; p=0.006]). further analysis identified cd45ra-foxp3+ memory-type cells within the cd127-treg starting population as a major source of foxp3-as well as il-2-and ifngamma-producing cells emerging during in vitro culture. in addition, we analyzed the dna methylation status of a defined region within the foxp3 locus termed tsdr (t reg-specific demethylation region), which has been shown to be demethylated exclusively in natural t reg cells (baron et al., eji 37:2378; 2007) . upon isolation, ra+ and cd127-treg showed complete demethylation of the tsdr, but only ra+ treg maintained this demethylated status throughout the entire culture period. based on these findings, we suggest that expansion of naive cd45ra+cd4+cd25high t cells represents the most promising strategy for adoptive treg cell therapies. several eortc aml trials were based on a 2x2 factorial design. in elderly patients, aml-13 trial was set-up to assess the value of g-csf during/after induction, and of infusional vs non-infusional mini-ice as consolidation. in the eortc-gimema aml10 study (patients ≤ 60 years old), comparing 3 anthracyclines in induction and consolidation, the value of allosct vs autosct has been assessed based on intent-totreat analysis. the "donor vs no donor" comparison was performed, overall, and according to cytogenetic subgroups and age. in the eortc-gimema aml12 (age ≤ 60 yrs) study the accrual for the 1st question (hd-ara-c vs sd-ara-c in induction) had to be expanded, due to insufficient number of patients randomized for the 2nd one (il-2 vs no il-2 after autosct). new statistical considerations were set-up for the 1st question as the trial expanded to 2000 patients. 302 statistical planning of clinical trials: an emphasis on sample size determination a. latouche* ebmt (paris, fr) an essential step when planning a trial is the calculation of the sample size or the number of patients to recruit to detect a relevant effect with sufficient power. patients enrolled in a clinical trial may experience exclusive failure causes, which defines a competing risk setting. the main decision regards the relevant quantity to assess the treatment effect: either cause-specific hazard or cumulative incidence function. the analysis should then be performed according to the method used for sample size computation, if one does not want an under-powered trial. from practical point of view, the practitioner has different options to plan a study accounting for competing risks. in this work, we thus compare these approaches. in medical applications multi-state models arise in attempting to understand complex disease or treatment processes. in these models individuals can move among a finite number of states defined by specific conditions of health, often including death. they are natural extensions of the traditional survival models in which transitions between multiple states are considered. multi-state models give more insight into the disease-recovery process because they take into account the occurrence intermediate events. in this talk i discuss some of the advantages (and disadvantages) of multi-state models and their application to bone-marrow transplantation. long-term follow-up of hcv infected patients; updated results of the ebmt prospective study p. ljungman*, a. locasciulli, a. békássy, l. brinch, i. espigado, a. ferrant, i. franklin, v. gomez-garcia de soria, j. o'riordan, m. rovira, p. shaw, h many long-term survivors after sct are infected with hcv. it has been reported that a high proportion of these patients develop late occurring liver cirrhosis. treatment options include interferon with or without the addition of ribavirin but the knowledge about these options' effectiveness is limited. therefore in 1992, the idwp of the ebmt initiated a prospective study of hcv infected sct patients having survived at least 6 months after sct. the intention is to every five years ask for follow-up on living patients. 247 patients were included in the study cohort between 1992 and 1997. this is the 3rd report of this cohort. due to limited follow-up data, 49 patients have been excluded. thus, the report is based on 198 patients with a median follow-up of 16 years (0.52 -32.8). the kaplan-meier estimate of survival is 73.9%. 43 patients have died of whom 7 have been reported to have died from liver associated complications including one after liver transplantation. two additional patients are alive after liver transplantation. 79 patients have been treated with interferon with or without the addition of ribavirin or with ribavirin alone. 37/79 patients became hcv pcr negative of whom 3 have relapsed with positive hcv pcr. the side effect profile was similar to other patient populations treated for hcv. conclusion: in this prospectively followed cohort, the prognosis of chronic hcv infection was quite favourable. treatment is feasible and associated with acceptable efficacy and toxicity. current status of the apbmt registry y. kodera*, a. yoshimi, r. suzuki, y. atsuta, l.l. chan, a. li, p.-l. tan, w.y.k. hwang, t.v. binh, t.j. chiou, a. ghavamzadeh, l. dao-pei, p.the data was submitted through the national registries in my, jp, and tw. in other countries/regions, the data was collected either by apbmt data center or by regional coordinators. the total number of hsct has been steadily increasing in most of the countries/regions and most dramatically in cn and in ir. the annual number of hsct of 2005 reported was 4,598 (data of 7 countries/region), which was doubled in these 10 years; of those, 65% were allogeneic and 35% were autologous. the number of allogeneic sct has been steadily increasing year after year; meanwhile the number of autologous sct has been recently stable. the distribution of related and unrelated donors (ud) differed widely among the countries (ud= 0% [vn] to 62% [jp] in 2005) . ud-sct is increasing consistently in cn, jp and hk, but not in other countries/regions. interestingly, cord blood appeared to be a common stem cell source in asia, accounting for 35% of ud-hsct (11% in cn to 100% in ir/vn, data of 2005). unrelated donor peripheral blood stem cell transplantation is not common in asian countries/regions except for china. in summary, this simple survey provided us basic information on current situations and trends of hsct in asia, which may be helpful to advance the patient registry. recently apbmt developed an electric data collecting system called trump and the group also agreed to share the common basic survey items with ebmt and cibmtr (med-a/ted) and join the global transplant activity survey under the umbrella of world-wide group for blood and marrow transplantation (wbmt). these international collaborations may facilitate future advances of the hsct. background: ebv associated ptld is a serious complication after sct.several risk factors may increase the risk of ptld. analysis of ebv dna viral load has been suggested to be useful for monitoring and used as the basis for preemptive therapy with rituximab with the aim to reduce the risk for ptld. methods: patients who underwent sct from 030101 until 070515 were included in this analysis. ebv serological mismatch between donor and recipient, primary ebv infection, cord blood grafts, and diagnosis of lymphoma were regarded as risk factors and used to split the patients into a high risk and a standard risk group. before 050701 (early group), patients were tested on clinical suspicion of ebv associated symptomatic infection while after 050701 (recent group) high risk patients were monitored by quantitative pcr while standard risk patients were to be sampled on clinical suspicion of symptomatic ebv infection only. 274 patients were analyzed; 150 patients in the early and 124 in the recent group. in the early group 53 were classified as high and 97 as standard risk. in the recent group, 41 were defined as high and 83 as standard risk. the ebv viral load was measured in serum by a taqman pcr technique. after 050701, rituximab was to be given in high risk patients at an ebv dna level of >10.000 copies/ml. in the early group and in standard risk patients of the recent group, rituximab was given on suspicion of ebv symptomatic infection. results: a total of 971 blood samples were analyzed for ebv dna. more samples (median 4 in the early and 6 in the recent group) were taken in the high risk compared to the standard risk group (median 2 and 3 samples, respectively). in the early group, 25/53 (47%) high risk patients compared to 31/97(32%) standard risk patients had ebv detected at least once. the corresponding numbers in the recent group were 14/41(34%) in the high risk compared to 11/83 (13%) in the standard risk group. in the early group, 13/150 (8.6%) received rituximab. while 11/124 (8.9%) in the recent cohort was given rituximab. high risk patients received more often rituximab than standard risk patients in both the early and recent cohorts (data not shown). in the early group 6 (4%) developed ptld of whom 4 died (2.6%) while in the recent group, 4/124 (3.2%) developed ptld of whom 2 died (1.6%). conclusions: a strategy of targeted monitoring to high risk patients can be safely utilized with a low risk for development and fatal outcome of ptld. a retrospective ebmt survey on the use of cidofovir for bk-related haemorrhagic cystitis after allogeneic haematopoietic stem cell transplant s. cesaro* (1), y. koc (2) bk virus has been recently associated to post-engraftment hc. cidofovir (cdv) is often used for bk-related hemorrhagic cystitis (bk-hc) treatment although data on safety and efficacy are scarce for this indication. we collected retrospectively the experience of cdv for bk-hc among the ebmt centres. 61 episodes of bk-hc in 60 patients from 16 centres were recorded. all patients but 5 had been transplanted for a malignant disease. median age at haematopoietic stem cell transplant (hsct) was 21 yrs (range 2-64) and 23/60 patients were children or adolescent (age < 18 yrs). the source of stem cell was pb in 52%, bm in 29%, and cb in 19% whilst the type of donor was sibling in 30%, unrelated donor in 57% and other related donor in 13%. myeloablative regimen + tbi was used in 74% of patients. bk-hc occurred after a median of 41 days from hsct (range 3-577) but only 8 episodes were diagnosed after day + 100. hc was scored as follows: 7 grade i, 16 grade ii, 28 grade iii, 10 grade iv, and lasted in median 33 days (range 5-749). concurrent morbidity was represented by fever in 30%, hypertension in 26%, vod in 7% and ttp in 3% of episodes. most of patients received hyperhydration plus rc and plt transfusion during hc whilst only 51 of episodes were treated with bladder irrigation through vesical catheter. cdv was started a median of 6.5 days after hc (range -22 before to 135 days after hc) and was administered for a median of 3 doses (range 1-15). the most frequent schedule was 3-5 mg/kg/weekly or fortnightly with probenecid (used in 70% of all treatments). 6 patients received intravesical cdv via catheter. cdv toxicity was reported in 20% of episodes and was limited to kidney: 3 grade i, 4 grade ii, 2 grade iii, 3 grade iv. complete resolution or clinical improvement of hc was reported in 35 (57%) and 8 (13%) of episodes, respectively. although the datum is not available for all episodes cdv treatment was associated to negativization of bk viremia in 69% (27/39) and of bk viruria in 37.5% (9/24) of episodes. we conclude the cdv therapy is a potential useful option for the treatment of bk-hc with limited moderate or severe kidney toxicity. prospective data are needed to define the best timing and schedule of treatment and the predictive factors for clinical and virological response. single-centre prognosis analysis and validation of the seattle, french and strasbourg prognosis indexes of invasive aspergillosis in adult patients with haematological malignancies or after haematopoietic stem cell transplantation r. parody*, r. martino, f. sanchez, j.l. piñana, j. sierra sant pau hospital (barcelona, es) in this retrospective study we analyzed the outcomes of 117 adults hematological patients who had a history of proven (n:23), probable (n:61) and possible (n:33) invasive aspergillosis (ia) between january 1995 and june 2007, at santa creu i sant pau's hospital of barcelona. forty-one patients (35%) were recipients of an allogeneic hematopoietic stem cell transplantation (allohsct). the main goal of the study was to analyze the prognosis factors predicting the outcome of 4-month aspergillosis free survival (afs) and overall survival (os), in order to compare the results with previously published prognosis indexes. analysis was performed in all patients and separately in allohsct and non-allohsct patient cohort. a total of sixty seven patients (57%, 30 recipients of allohsct)) died in the first 4 months after ia diagnosis [median days: 20 (range1-117)]. invasive aspergillosis was identified as the main cause of death in 51 patients (43%, 23 recipients of allohsct) [median days: 16 (range ]. according with autopsy findings, 27 cases (11 possible and 16 probable) could be recategorized as proven cases. diagnosis of ia at or before 2000 had a negative impact in both 4-month afs and 4-month os. in all patients and allo-hsct patients, 4 variables (excluded the year of diagnosis) decreased 4-month afs in multivariate analysis: (i) disseminated ia and monocytopenia <0.1 x 109/l, respectively (ii) impairment of one organ, (iii) impairment of 2 or more organs and (iv) high-doses steroids and an alternative donor, respectively. a risk model for progression was generated for each group, according with the presence of 0-1 factors,( 82 and 100% afs), 2-3 factors, (27 and 45% afs) or 4 factors, (0% afs)[p<0.001]. in base of similar results previously published our prognosis index model could be validated with both the french and seattle models for allo-hsct recipients and with the strasbourg model for hematological patients. one-year microbiological survey with molecular typing method of pseudomonas aeruginosa in a bmt unit b. bartolozzi* (1), r. fanci (1) pseudomonas aeruginosa is one of the most common nosocomial pathogens in intensive care and in oncohematological units and it still represents an important cause of morbidity and mortality. molecular epidemiological survey is a very important tool to understand the nosocomial pattern of each hospital and to identify outbreaks. from may 2006 to june 2007 we performed a "real time" aflp (amplified fragment-length polymorphism) monitoring of all p.aeruginosa strains isolated in patients within the bmt unit of florence. the patients admitted in the unit during this period were 99 (70 autologous transplants, 15 unrelated allogeneic transplants and 14 identical sibling allogeneic transplants. p.aeruginosa was responsible for ten bloodstream infections (bsis) and of two microbiologically documented infections without bacteremia. the aflp analysis showed the persistence within the unit of two clones of p.aeruginosa. one was responsible of two outbreak episodes in patients allocated in the same single room. the first episode occurred in may-october 2006 involving 4 patients; a team consisting in the bmt head physician, the bmt head nurse, the infection control officer and the infection control physician was actively involved in a strict bacteriological surveillance that allowed to isolate the same strain from a irgasan soap sample. for this reason, hyperclorination of the water network, sink tap changing, water filters installation and weekly soap monitoring were performed. nevertheless the same clone was isolated after several months in a water sample, showing the inefficacy of the control measures taken. in may-june 2007 the same p.aeruginosa clone was isolated in three infected patients and in a shower tap sample. hydraulic works inside the room were performed and until now no other case of p.aeruginosa was observed. the other persistent clone was responsible of contamination of 4 sample of irgasan soap taken from the ward cloakroom (collected from november 2006 until april 2007) and of a bsi in a patient (march 2007) . our results showed that p.aeruginosa is largely and consistently found in environment which may represent an important source of infection, difficult to eradicate. moreover, punctual microbiological surveillance and molecular typing methods are essential to early detect nosocomial outbreaks, to identify p.aeruginosa reservoir and to guide in decision making in order to understand and possibly stop the epidemic chain. donor lymphocyte infusion (dli) following t cell depleted allogeneic stem cell transplantation (sct) is an effective treatment for relapsed hematological malignancies. dli often results in a profound graft versus leukemia (gvl) effect with relatively limited graft-versus-host disease (gvhd). both gvl reactivity and gvhd are likely to be mediated by donor derived t cells recognizing polymorphic minor histocompatibity antigens (mhag) on patient cells. it has been hypothesized that t cells recognizing mhags expressed on non-hematopoietic cells are responsible for gvhd, whereas t cells recognizing hematopoiesis restricted mhags may be selectively involved in gvl reactivity. to analyze the diversity of alloreactive t cells involved in gvl and gvhd we analyzed the immune response in a patient responding to dli following hla-matched sct for aml. six weeks after dli, gvhd limited to skin and mouth developed coinciding with a rapid and sustained conversion to 100% donor chimerism. we clonally isolated and characterized activated hla-dr expressing t cells at the onset of gvhd (10% of the circulating t cells). in total 133 cd8+ t cells clones and 241 cd4+ t cell clones were tested for alloreactivity as defined by cytotoxicity or ifng production upon recognition of patient derived ebv-lcl and not donor derived ebv-lcl. 49% of the cd8+ t cell clones and 20% of the cd4+ t cell clones were identified as alloreactive t cell clones. next, recognition of patient skin-fibroblasts as a target for gvhd was determined both in the absence or presence of ifng to mimic an inflammatory environment. none of the t cell clones recognized unmanipulated fibroblasts. only after co-culture with ifng, 28% of the alloreactive cd8+ t cell clones but none of the cd4+ t cell clones reacted with patient fibroblast. by blocking studies, panel studies and t cell receptor-vb analysis the diversity of this immune response was determined. 32 and 25 different reactivities were found for the cd8+ t cells and cd4+ t cells, respectively. in conclusion, isolation of activated t cells identified a very polyclonal immune response directed against multiple mhags in all hlaclass i and hla-class ii alleles. despite the polyclonality of this immune response, reactivity of the t cell clones was relatively hematopoiesis specific, resulting in 100% donor chimerism, persistent complete remission and only moderate gvhd limited to the skin. since t cell recognition of fibroblasts was observed only upon co-culture with ifng, this may reflect a secondary reactivity induced by the ongoing gvl effect. background and aims: chronic graft-versus-host disease (cgvhd) is a recognized cause of genital complications in the vulva and vagina in female patients (pts) after allogeneic stem cell transplantation. however, clinical features and consequences of genital cgvhd are poorly described in the literature. in a retrospective study, we assess prevalence, symptomatology and effect on sexual life of genital cgvhd. in a prospective study, we try to prevent progression and sequelae of genital cgvhd. here we report early results from these ongoing studies. patients and methods: all women allografted 1996-2005 in the western region of sweden are asked to participate in a study comprising (i) structured anamnesis and validated questionnaires for the identification of depression and sexual dysfunction; (ii) gynecological examination; (iii) biopsies for pathological examination. prospective pts are seen by the gynecologists up to 3 years post-transplant. clinical findings of genital cgvhd is a dry, thin, painful mucosa, inflammation, lichenoid bands, vulvar synechiae and vaginal stenosis. systemic and local estrogens do not resolve those signs. histology findings were those of chronic inflammation, lichenoid reaction and fibrosis. results: fifty-eight consecutive pts with 1-10 yrs of follow-up were approached and so far 30 women have been examined. median age was 46 (26-71) yrs) and follow-up post-transplant was 5.5 (1-10) yrs. fifteen pts (50%) had a clinically diagnostic cgvhd, and using pathology examination (n=13) the diagnosis was confirmed (n=6) or suspected (n=4). another 12 of the 30 pts had signs suggestive of cgvhd and histology was confirmatory in one and suspect in 6 cases. two of 30 pts had vulvar synechiae and 10 (33%) had vaginal adhesions. in the prospective study, 19 pts have been included and the follow-up is now 12 (3-24) months. after 12 months of follow-up (n=14) clinically cgvhd had been diagnosed or suspected in 8 cases. local corticosteroid or takrolimus cream was commenced in 4/19 cases. female sexual distress scale intervention performed as sceduled revealed sexual dysfunction at at least once in 23 (46% of all) and beck depression inventory indicated depression 18 (37%). conclusions: genital signs and symptoms, including vaginal stenosis, are common features of cgvhd and are associated with sexual dysfunction and depression. gynecological surveillance and early intervention may reduce the risk of severe sequelae after genital cgvhd. langerhans cell chimerism early after t-cell depleted allogeneic haematopoietic stem cell transplantation k. schneiker*, t. schmitt, a. konur, j. hemmerling, k. bender, e. von stebut, a. hadian, k. kolbe, c. huber, w. herr, r.g. meyer university clinic mainz (mainz, de) skin is the most frequently affected organ in acute graft versus host disease (gvhd). data from murine studies suggest that the interaction of residing host epidermal langerhans cells (lc) and donor t cells is crucial for the initiation of acute gvhd. in an ongoing clinical protocol applying alemtuzumab-based t cell depleted (tcd) allogeneic stem cell transplantation (sct) we observed acute skin gvhd occurring very early after transplantation despite of low t cell counts in the peripheral blood (meyer, blood 2007; 109:374) . we therefore intended to analyze the lc chimerism in these patients. up to now, lc-chimerism analysis in humans has been performed by the detection of the sex-chromosomes restricting it to sex-mismatched donor / recipient pairs. in our patient-population, this would limit the analysis to less than 1/3 of the patients. consequently, we aimed at a method to isolate lc from small skin samples with high purity for a sensitive str-based chimerism analysis of general applicability. epidermal skin layers were prepared from 6 mm punch biopsies by digestion with dispase i. they were further split and used for both immunofluorescent staining and digestion with trypsin to generate a single cell suspension and cd1a/mhc-class ii-positive lc were subsequently sorted by flow cytometry. the density of lc on day + 20 after hsct was much lower compared to before transplantation or to that of healthy individuals. but still, lc could be purified in all 10 analyzed patients. the isolated lc numbers ranged from 10 to >1000. we confirmed that the purity was exceeding 93.5% in 5 patients in a facs-reanalysis, thereby reproducing the findings with skin of healthy individuals. applying two alternative str-based protocols, we obtained reliable results for lc chimerism in 8 of 10 patients and could detect signals with as few as 35 isolated cells. in 2 patients, the majority of isolated lc was of donor origin whereas the other 6 patients had predominantly host lc. after day +50 post hsct, 2 further patients showed only a few remaining lc of host origin. in summary, we have established a sensitive method that enables the chimerism analysis on highly purified lc independent of sex-mismatched donor / recipient pairs. our results on a few patients' samples cannot yet be related to clinical events. but the method allows the investigation of lc´s chimerism and potentially of other tissue-resident antigen presenting cells to study their impact on gvhd in humans. we and others have data on the activation of coagulation and fibrinolysis during and after allogeneic stem cell transplantation (sct), which may have pathogenic implications in the subsequent recovery. we characterised the outcome including graft versus host disease (gvhd) in association with adaptive mechanisms of anti/coagulation after allogeneic sct in 30 patients with a hematological malignancy. they were given myeloablative conditioning with cyclophosphamide and total body irradiation. 19 patients received the transplant from a sibling and 11 from an unrelated donor. gvhd prophylaxis consisted of cyclosporine were "unresponsive, non-progressive". the prognostic features, including cytogenetics, were similar in both groups. 70% of the patients responded to the first hdt (cr/ncr 8%, pr 48%, mr 13%). 37 patients were given a second transplant (26 "auto", 11 "allo"). 41% who received a second "auto" up-graded their response (cr 9%, pr 14%, mr 18%) while 42% who underwent "allo-ric" increased their response (cr 28%, pr 14%). median survival of the whole series was 3 years. patients progressing while on therapy had a shorter survival than the "no-change" group (median 2 yrs vs not reached, p=0.00002). finally, the 50 "non-responsive, nonprogressors" patients had similar survival than the 716 with chemosensitive disease intensified with hdt. conclusions: 1) hdt in patients with primary refractory mm results in a low cr rate, 2) patients progressing while on initial therapy have a short survival despite the intensive approach and 3) patients with "non-responding, non-progressive" disease have similar survival than chemosensitive patients. whether this good outcome is due to the impact of hdt or to the natural history of a more indolent disease remains to be further investigated. background: primary amyloidosis (al) responds poorly to conventional therapy and has poor prognosis. autologous stem cell transplantation (asct) results in a significant response rate although the procedure is hampered by a high transplant-related morbidity and mortality. aim. to analyze the outcome of a series of 34 patients with al who underwent asct at a single institution during a 10years period. patients and methods: thirty four patients (16 m, 18 f; median age 54 years, range: 33-66) who received an asct between november 1997 and september 2007 were included. fourteen patients had received previous therapy and 20 were newly diagnosed. in 71% of the patients the light chain was of lambda type. the median number of involved organs was 2 (range, 1-4) including kidney (26 patients), heart (19), liver (11) , peripheral nerve (9), autonomic system (5) and gastrointestinal tract (5). thirty-eight percent of the patients had more than 2 organs involved. all patients were mobilized with g-csf alone and the intensive regimen consisted of mel-200 in 23 patients and mel-140 in 11 patients. the median interval between diagnosis and asct was 9 months. results: the overall transplant-related mortality (trm) was 27%. there was a trend towards a higher trm in patients transplanted from november 97 -april 02 (n=14, trm: 42%) as compared to those transplanted from june 02 -sept 07 (n= 20, trm 15%) (p= 0.07). in the 26 patients who underwent asct before nov. 2006 (minimum follow-up of 1 year) the response and survival on an intent-to-treat basis were as follow: cr (28%), pr (12%), no response (26%), early death (34%).organ response was observed in 11 patients (42%). the median response duration and the overall survival were 50 and 59 months, respectively. favorable prognostic features for survival were: cardiac septum <14 mm (p= 0.03), normal beta 2m (p=0.04) and normal nt-probnp (p= 0.006). updated results on the overall series of 34 patients will be presented at the meeting. conclusion: 1) asct in al results in significant cr rate with prolonged response duration an overall survival and 2) although trm is high, there is a trend towards a lower trm over the years this most likely reflecting both a better patient´s selection and general management. outpatient-based peripheral blood stem cell transplantation for patients with multiple myeloma a. ghavamzadeh, m. khani*, a. karimi, k. alimoghadam, a. manokian, r. maheri, m. asadi, f. afshar, a. shamshiry hematology, oncology and bmt resarch center (tehran, ir) intrduction: the aim of this study was to explore the feasibility and safety and cost-benefit of performing asct on an outpatient basis. material and methods:total of 86 patients affected by mm and in complete remission (cr) or partial remission (pr) were selected to receive asct on an out-patient or in-patient basis . in the in-patient group 31, 12 patients received 200mg/m² and 140mg/m² melphalan as conditioning regiment respectively . in out-patient group 12 patients received 140mg/m2 and 30 patients recived 200mg/m 2 melphalan. in out-patient group all the patients were programmed to go home the day after asct and to be rehospitalized in the case of febrile neutropenia or other sever toxicities. we used caregiver, general physician, staff nurse as an out-patient and visit team and also unequipped routine house of the patients during neutropenia. results :median ages were 50±7.5 years, median hospital stay were 28, 6.5 days in in-patient and out-patient respectively. there were not significant difference between these groups in aphresis days ,granulocyte colony stimulating factor(gcsf) requirement for mobilization and mononoclear cell (mnc) or cluster of differentiation(cd)34 + cell parameters (p<0.1), but statistically significant reduction in total cost and hospitalization (p<0.017).there were also significant reduction (p<0.001) in parentral antibiotic,blood product requirement and need for total parentral nutrition. conclusion :many different authores have explored the feasibility of autografting patient on an outpatient basis. the ease of administration of hdm as well as the lack of excessive extramedullary toxicity, including nausea and vomiting renders patients with mm more suitable for outpatient management, in the present study, we describe an outpatient program based on management of the patient in his/her house during aplastic phase . our results clearly indicate that such a proceder is feasible and safe in a patient population with a median age of 55 years. with an accessible caregiver, the most frequent cause of readmission in other study were febrile neutropenia and sever mucositis need tpn. in particular, it is worth nothing that transplant cost, requirement for blood product and hospital stay decreased significantly in this method of transplantation. treatment of multiple myeloma with sequential autologous and low dose total body irradiation based allogeneic unrelated sct c. pfrepper*, t. lange, r. krahl, m. cross, h.-k. al ali, w. pönisch, n. basara, d. niederwieser university hospital of leipzig (leipzig, de) purpose: the limited availability of related donors poses an important limitation in the treatment of multiple myeloma (mm). the use of unrelated donors would alleviate this problem. furthermore, outcome after unrelated transplants following reduced intensity conditioning (ric) has been shown to be superior in other diseases including cll and aml, due to increased graft-versus malignancy reaction. in this study, the feasibility of autologous followed by allogeneic unrelated ric sct was tested and the results compared to autologous followed by related allogeneic ric sct and allogeneic ric sct after relapsing following autologous sct. patients and methods: we report the outcome of 39 mm patients with a median age of 55 (range 33-64) years treated with high dose chemotherapy (melphalan 200mg/m² on day -3) followed by autologous sct. subsequent ric-sct was performed using fludarabine (30mg/m²/d from days -3 to -1) and tbi (2 gy on day 0) followed by cyclosporine (6.25 mg/kg twice daily from day -1) and mycophenolate mofetil (15 mg/kg daily from day 0). eleven patients received an unrelated graft (group 1) and 18 a related graft (group 2) within median 5 (range 2-8) months from the autologous hct. ten further patients (group 3) underwent either related or unrelated sct 13 (range 2-42) months after insufficient response following autologous transplantation and additional chemotherapy. results: durable engraftment was obtained in 97% of all patients. overall survival (os) at 2 years was 62±15%, 82±10% and 67±16% (p=0.089) in groups 1, 2 and 3, respectively at a median follow up of 23 (range 2-66) months. nine patients (23%) were in cr, 15 patients (38%) in pr, 3 patients (8%) had progressive disease after allogeneic sct. however; progression free survival (pfs) was 55±15%, 20±10% and 11±10% in the three groups, respectively (p=0.014). non relapse mortality (trm) was not different between the three groups, but relapse incidence was lowest in patients after unrelated transplantation. conclusion: we conclude that autologous sct followed by low dose tbi based sct from matched unrelated donors provides rapid and sustained engraftment for mm patients comparable to that of related donors. our limited data suggest a tendency to higher pfs following unrelated compared to related sct. these results provide the basis for a phase iii study designed to compare auto-allo unrelated to auto-auto sct. background: a significant proportion of patients with multiple myeloma have a long-lasting response after autologous stemcell transplantation (asct). however, others relapse relatively quickly. the aim of this study was to determine if the presence of monoclonal plasma cells (mpc), detected by flow cytometry, in the apheresis product, and in pre-and posttransplant bone marrow samples predicts a shorter time to progression (ttp). patients and methods: we included patients diagnosed with multiple myeloma and treated with high dose therapy followed by asct between november 1, 1998 and february 1, 2007. in all casas, flow cytometric analysis was performed in the apheresis products and in bone marrow samples, both prior and after asct. the variables evaluated as possible prognostic factors were: age, sex, type of monoclonal component, durie-salmon stage, iss stage, presence of mpc (cd138+/cd38+/ cd19-/cd45-or cd45dim) in apheresis products, and in bone marrow samples (30 days before, and 100 days after asct), and accomplishment of a complete response prior or after asct. results: 55 patients were included: 21 male, 34 female. median age at asct was 58 years (range, 30-69 years). twenty-nine patients had progressed. on univariate analysis, age .14); p<0.023], iss stage [or 7.61 (2.14-27.14); p=0.08], mpc in apheresis products (p=0.005), mpc in pretransplantation bone marrow (p=0.007), mpc in postransplantation bone marrow (p=0.06), and the achievement of complete response prior and after asct (p=0.01) had a negative impact on time to progression. on multivariate analysis, the presence of mpc in apheresis [or 3.1 (1.24-2.65), p=0.01] and the presence of mpc in pretransplantation bone marrow (or 14.2, p=0 .012) were identified as independent predictors of a shorter ttp. conclusions: the presence of mpc in apheresis products and in pretransplantation bone marrow were identified as independent predictors of shorter ttp. both parameters can identify a group of patients with multiple myeloma which could benefit from most aggresive conditioning regimens or additional maintenance therapies. long-term follow-up of patients with systemic al amyloidosis treated with high-dose melphalan and autologous stem cell transplantation after induction and mobilisation chemotherapy s. o. schonland*, t. bochtler, m. hansberg, a. mangatter, j.b. perz, a.d. ho, h. goldschmidt, u. hegenbart university of heidelberg (heidelberg, de) introduction: longterm survival in al amyloidosis (al) patients (pts) has been shown recently after high-dose melphalan (hdm) (sanchorawala, blood 2007) in 21% of the patients. however, no advantage of hdm compared to conventional chemotherapy has been observed in a randomized multicenter trial (jaccard, nejm 2007) . this was probably due to high transplantation related mortality (trm) and a substantially longer time to the start of chemotherapy in the hdm group. whether treatment intensification using induction and mobilization chemotherapy prior hdm can improve results has not yet been definitively shown. methods: we have updated 23 pts with al (perz, bjh 2004) who received vincristine/adriamycine/dexamethasone as induction and additional mobilization chemotherapy (mostly cyclophosphamide based) prior to hdm in our center from 1998 until 2004. inclusion criteria were age < 70 years, nyha stage < iii and a who performance status < 3. median age was 57 years; median number of involved organs was 3. fifteen and 13 pts had symptomatic renal or cardiac involvement, respectively (on dialysis at hdm, n=4). results: the median overall survival (os) has not been reached at a median follow up of 66 months post hdm (figure). trm was 9%. a hematological response (hr) to induction and mobilization chemotherapy occurred in 47% (14% of pts achieved a complete remission (cr)). hr and cr rate increased to 84% and 67% after hdm, respectively. median time to cr from hdm was 4 months. two patients had already an organ response (or) after induction and mobilization chemotherapy. or was observed in 68% and the median time to or was 11 months post hdm (range 0-40 months). pts with cr have an estimated os of 90% after 6 years; for pts not reaching cr the median os is 48 months (p=0.002). hematological relapse or progression occurred in about half of the patients; however 6 pts (26%) are still in first cr with a sustained or and a median follow-up of almost 7 years. conclusion: we observed a very high cr and or rate using this intensive treatment approach. patients with cr after hdm have an excellent survival. therefore, the role of induction therapy in al, e.g. with bortezomib or lenalidomide, should be further investigated in randomized trials in experienced centers. the main goal of treatment in al remains achievement and maintenance of a cr of the underlying plasma cell disorder. the results of reduced-intensity conditioning allogeneic stem cell transplantation (ric allo-sct) for multiple myeloma (mm) are still under considerable debate. while ebmt data did not support the universal use of ric for mm allografts, the italian randomized multicenter study suggested that in newly diagnosed myeloma, survival in recipients of a hematopoietic stem-cell autograft followed by ric allo-sct from an hlaidentical sibling is superior to that in recipients of tandem stem-cell autografts. the aim of this multicenter retrospective national study was to identify prognostic factors for outcome of high-risk patients with mm after allo-sct prepared by ric. data from 219 patients (median age 52 years, range 27-66), who received grafts from a sibling (n=197) or unrelated donor (n=22) were analyzed. at time of transplant, only 37 patients (17%) received ric allo-sct in cr or vgpr, while 134 patients (61%) were transplanted in pr. 48 patients were transplanted either in stable disease (n=15) or were in refractory/progressive disease (n=33). all patients have received at least one autologous transplant prior to ric allo-sct. the graft source was pbscs in the majority of patients (n=183). 21% of the patients received the seattle fludarabine and low dose tbi ric regimen, while 53% of patients received fludarabine, busulfan and atg. 32 patients (15%) died of transplant-related complications. the incidences of grade 2-4 acute gvhd and extensive chronic gvhd were 37% and 20% respectively. at 3 years, overall and progression free survivals (os, pfs) were 41% (95%ci, 34-49) and 19% (95%ci, 14-27) respectively. disease status (cr, pr, sd vs. progressive) was significantly associated with overall survival (p=0.0002; fig. below) . in multivariate analysis, disease status at time of ric allo-sct, was the strongest parameter associated with an improved os and pfs (p=0.005 and p=0.004 respectively). despite its obvious caveats, the relatively low trm observed in this series, suggest that there is still space to investigate ric allo-sct for mm. however, ric allo-sct appears to result in a durable response only if it is applied early in the disease history, especially when patients are still chemosensitive. since the latter results are also expected to be further improved with the systematic and early use of maintenance therapies (bortezomib and/or lenalidomide) after ric allo-sct, randomized or quasirandomized prospective studies are still warranted. the role of reduced-intensity conditioning (ric) allogeneic stem cell transplantation (allo-sct) for adult patients with acute lymphoblastic leukaemia (all) is still under debate. all encompasses a group of chemosensitive diseases, raising concerns that significant reduction of the intensity of the preparative regimen may have a negative impact on leukemic control. in this multicenter retrospective study, the outcome of 601 adult (age at transplant >45 y.) patients with all who underwent transplantation in complete remission (cr) with an hla-identical sibling donor, were analyzed according to 2 types of conditioning: ric in 97 patients, and standard myeloablative conditioning (mac) (or high-dose) in 504 patients. both groups were comparable in terms of gender, cr status (cr1 and cr2), interval from diagnosis to allo-sct, and r/d cmv serostatus. patients in the ric group were older (median 56 y. vs. 50y in the mac group;p<0.0001). most of the patients in the mac group received high dose tbi (80%), while the majority of the ric regimens included either low-dose tbi or were atg+chemotherapy-based regimens. the majority of patients (88%) from the ric group received pbscs. in the mac group, the stem cell source consisted of bone marrow in 42% of patients. with a median follow-up of 13 months (range, , the incidences of grade ii-iv and grade iii-iv acute gvhd were: 35%, 14%, and 28%, 10% in the mac and ric groups respectively (p=ns). the cumulative incidence of nonrelapse mortality at 2 years (nrm) was 32% (mac) vs. 22% (ric) (p=0.04). the cumulative incidence of relapse at 2 years was 30% (mac) vs. 42% (ric) (p=0.0007). however, the latter differences did not translate into any significant difference in term of leukemia-free survival (lfs) at 2 years: 38% (mac) vs. 37% (ric) (p=0.42). in multivariate analysis for lfs, the status at transplant was the only factor associated with an improved lfs (p<0.0001, rr=0.55, 95%ci, 0.42-0.72). the results of this study suggest that ric regimens may reduce nrm rate after allo-sct for adult all when compared to standard mac regimens, but with a higher risk of disease relapse and no impact on lfs. the latter represent promising findings, since patients who received ric are likely to have serious comorbidities, which led the transplantation center to choose ric, and surely most of these patients would not have received a standard allo-sct in most institutions. therefore, ric allo-sct for adult all (>45 y.) may represent a valid therapeutic option when a conventional standard conditioning is not possible, warranting further prospective investigations. reduced-intensity conditioning allogeneic stem cell transplantation for patients with acute myeloid leukaemia: long-term results of a "donor" versus "no donor" comparison m. mohty *, h. de lavallade, j. el-cheikh, p. ladaique, c. faucher, s. fürst, n. vey, d. coso, a.m. stoppa, j.a. gastaut, c. chabannon, d. blaise institut paoli-calmettes (marseille, fr) the issue of possible higher relapse rates after reducedintensity conditioning (ric) allogeneic stem cell transplantation (allo-sct) is still under considerable debate. this report describes the updated long term results (initial publication, leukemia 2005) of 95 consecutive acute myeloid leukaemia (aml) patients, diagnosed between 1999 and 2003 in a single centre. using a genetic randomization through a "donor" versus "no donor" comparison, our aim was to assess the true benefit of ric-allo-sct for adult aml. in this series, 35 patients (37%; "donor" group) had an "identified" hlaidentical sibling donor, while the remaining 60 patients had no hla-matched related donor ("no donor" group). as per institutional policy, hla-mud were not considered during the study period. no significant differences in patients or aml features were found between the two groups. in the "donor" group, 25 patients (71%; median age, 51 (range, 26-60)) could actually proceed to the ric-allo-sct. the current median follow-up is 60 months. in an "intention-to-treat" analysis, the km estimate of leukemia-free survival (lfs) was significantly higher in the "donor" group as compared to the "no donor" group (p=0.003; 60% versus 23% at 7 years). when restricting the analysis to patients who could actually receive the ric-allo-sct (median follow-up, 40 months from time of allo-sct), the difference in lfs was also significant between this group of 25 patients ("transplant" group) and the remaining 70 patients ("no transplant" group; p=0.0002; 72% versus 24% at 7 years). no major toxicities were encountered during ric administration (fludarabine, busulfan and atg), and only 3 patients died from toxicity, for a cumulative incidence of trm of 12% (95%ci, 3-32%) at last follow-up. this relatively low trm translated towards a significantly higher overall survival (os) in the "transplant" group as compared to the "no transplant" group (p=0.0003). in the "intention-to-treat" analysis, os was still significantly higher in the "donor" group as compared to the "no donor" group (p=0.003; fig. below) . after controlling for all relevant factors, in the multivariate analysis, only actual performance of ric-allo-sct (p=0.0005; rr=4.1; 95%ci, 1.8-9.1), was significantly predictive of an improved lfs. based on these long term results, we conclude that if a matched related donor is identified, ric-allo-sct should be proposed since it represents a valid and potentially curative option for aml patients not eligible for standard myeloablative allo-sct. graft failure after reduced-intensity conditioning b. hertenstein*, e. dammann, r. brand, a. van biezen, d. niederwieser, t. de witte, t reduced intensity conditioning (ric) regimens use doses of chemotherapy and/or radiotherapy which per se do not eradicate the malignant cells but which should be nevertheless sufficient to allow sustained engraftment. the incidence of graft failure might therefore be higher in ric and may be dependent on the regimen used. to evaluate the incidence of graft failure and the potential risk factors in patients transplanted after ric, data files of such transplantations were selected from the clwp data base. in all records chemotherapeutic drugs and dosages as well as tbi dosages were individually checked and only records meeting the ebmt definition of ric were included. the analysis was further restricted to patients surviving > 28 days. 1720 ric transplants were identified. a chemotherapeutic ric regimen was used in 1066 patients (flu/bu 571, flu/mel 296, flu/cy 60), tbi only in 97 and a combination of chemotherapy and tbi in 557 patients. stable engraftment occurred in 1640 patients (95.3%), no engraftment in 45 patients (2.6%) and a graft loss in 35 patients (2.0%). survival for patients with graft failure was 76% at 100 days and 38% at one year. 29 patients received a second transplant and survival at one year was 60% for these patients. graft failure was lowest in patients with myeloma (1.6%) and highest in patients with mds/mps (8.7%). with the use of bone marrow as graft source graft failure was much higher than with peripheral blood stem cells (11.8% vs. 3.7%) . graft failure occurred in 3.4% of transplants from hla-identical siblings, in 6.6% from matched unrelated donors and in 12.3% from mismatched unrelated donors. there was no difference between the different conditioning regimens, neither between chemotherapy alone, tbi or combined regimens (3.4% vs. 2.3% vs. 4.4%) nor between the major chemotherapy regimens themselves (flu/bu 1.9%, flu/mel 2.6%, flu/cy 4.3% for hla-id siblings). abo match and donor-recipient sex mismatch had no impact on graft failure. in multivariate analysis underlying disease and graft source were significantly correlated with graft failure. in summary the incidence of graft failure after ric is low. mds/mps as underlying disease and the use of bone marrow as graft but not the type of the conditioning regimen were risk factors for graft failure. (2), h. esperou (2) , m. attal (2) , n. milpied (2) , b. lioure (2) , p. bordigoni (2) , i. yackoub-agha (2) , j. bourhis (2) , b. rio (2) , e. deconninck (2) , m. renaud (2) , n. raus (1) , d. blaise (2) (1)hôpital edouard herriot (lyon, fr); (2)société française de greffe de moelle et de thérapie cellulaire (saint-denis, fr) this report updates a retrospective study from sfgm-tc registry concerning 1108 patients who underwent allogeneic hematopoeitic stem cell transplantation (hsct) after reduced intensity conditioning (ric) from hla identical siblings (84%) and unrelated donors (16%) for hematological malignancies. at time of conditioning, 442 patients were in cr, 337 in pr, 107 in stable disease (sd) and 222 in progressive disease (pd). as conditioning, 255 patients received fludarabine and tbi (2 grays), 465 patients fludarabine, busulfan and atg and 388 patients other regimens. after transplant, 336 patients (30%) developed an acute gvhd grade ii (grade ii: 178, iii: 80 and iv: 78). a chronic gvhd was present in 388 patients (35%) (185 limited and 203 extensive). with a median followup of 30 months, the 3 and 5-year probability of overall survival (os) were 43.5% (40-47) and 32%(29-35) respectively and the 3 and 5-year probability of event-free survival (efs) were 35%(31-39) and 28% (24.5-31) respectively. the trm at 1 year, 2 years and 3 years was 15% (13-17), 18% (15.5-21) and 20% (17-23). a mixture model, gfcure with splus statistical package determined the percentages of long-term survivors and its adequacy was verified graphically. the probability to be a long-survivor was 24% (17.5-32.5) (fig.1 ) and to be a long event-free survivor was 23% (19-28) (fig. 2) . the multivariate analysis has tested recipient and donor age, disease status pre-transplant, number of transplants before rict, hsc source, sex matching, hla matching, cmv status and abo compatibility. the only factor which had a significant impact on long-term survival after rict was the disease status just prior conditioning: pr versus cr: hr: 3.63 [1.14-9.18] p<0.001 and pd versus cr: hr: 4.35 [2.22-8.51] p<0.0001. in conclusion, these updated data demonstrate that allogeneic hsct after ric was able to possibly cure 23% of patients with haematological malignancies and the most important factor to take into account remains to be in cr pre-transplant. the haematopoietic cell transplantation-specific comorbidity index predicts survival and non-relapse mortality in lymphoma and myeloma patients receiving a ric allograft l. farina* (1), b. bruno (2) the allogeneic hematopoietic cell transplantation-specific comorbidity index (hct-ci) has been recently developed to identify patients at high risk of morbidity and mortality after an allogeneic stem cell transplant (allosct). reduced-intensity conditioning (ric) regimens have decreased non-relapse mortality (nrm) in heavily pre-treated patients. we performed a retrospective study to assess whether comorbidities, according to hct-ci, may influence the outcome of lymphoma and multiple myeloma patients undergoing a ric allosct. between 2000 and 2007, 197 patients received a ric allosct from a hla identical sibling (n=123) or unrelated (n=74) donor in three italian transplant units. median age at transplant was 52 years (range, 17-69) and 41% of the patients were 55 or older. diseases included non hodgkin's lymphoma (n=103), multiple myeloma (n=68) and hodgkin's lymphoma (n=26). median number of previous treatments was 3 (range, 0-8). disease risk according to kahl et al. (blood 2007) was low, intermediate and high in 29%, 42% and 29% of patients respectively. patients with hct-ci of 0, 1-2 and ≥ 3 were 64 (32%), 61 (31%) and 72 (37%), respectively. variables included in multivariate analysis were age (< 55 or ≥ 55), disease risk (low, intermediate and high), number of previous lines of therapy (≤ 2 and >2), hct-ci (0, 1-2 and ≥ 3), karnofsky perfomance status (ps, >80% and ≤ 80%). oneyear os and pfs were 87%, 60%, 60% and 91%, 75%, 70% in patients with hct-ci of 0, 1-2 and ≥ 3 respectively. cumulative incidence of nrm was 6%, 22%, 25% at 1 year and 6%, 24% and 27% at 2 years, whereas relapse mortality was 5%, 20%, 17% at 1 year and 7%, 27% and 23% at 2 years. by multivariate analysis only karnofsky ps (p=0.00051) and hct-ci (p=0.0038) were correlated with os, whereas pfs was influenced by disease risk category (p=0.013), number of previous therapies (p=0.038), and both karnofsky ps (p=0.031) and hct-ci (p=0.0009). interestingly hct-ci was the only significant factor that could predict nrm (p=0.034) while karnofsky ps failed to show a significant correlation (p=0.1). of note, age (≥ 55) was not statistically significant either in os, or pfs or nrm. although the data need to be confirmed in prospective trials, these results showed that hct-ci may be a useful tool to predict os, nrm and also pfs after ric allosct in lymphoma and myeloma patients. in the clinical setting hct-ci and not age should be used for patient risk assessment. venous thromboembolism (te) occurs as a consequence of genetic and environmental factors. important genetic risk factors are deficiencies of natural anticoagulants, antithrombin-iii (at-iii), protein c (pc) and ps, and genetic mutations of factor v leiden (fvl) and prothrombin (pth a20210). thrombotic complications after hct are usually catheter-related thrombosis (crt), pulmonary te (pte) and deep vein thrombosis (dvt). our aim in this study was to evaluate the effects of the deficiencies of atiii, pc and ps, and the gene mutations of fvl and pth on the incidence of the development of te complications and liver sinusoidal obstruction syndrome (sos) at the early or late period post-hct. in our center, pre-transplant work-up includes routine thrombophilia tests. between apr 1999-jan 2007 260 patients (m/f: 145/115, median age: 34 years) admitted to our transplantation center were retrospectively analyzed for the relation of the presence of thrombophilia and the frequencies of the occurrence of a te complications and liver sos. all but 6 patients (n=254) had an hla identical sibling donor. the ratios of the detection of the plasma activation level below 50 % for pc, ps and at-iii were 5.8 % (12/206), 15.7 % (32/204) and 25.4 % (66/260), respectively. gene mutations were studied in 198 patients prior to transplant and the frequencies of gene mutations were detected in 11.6 % patients (n=23) either fvl (n=14) or pth (n=9) gene. none of the patients had both mutations, fvl and pth. at the peri-or posttransplantation period we observed venous te in 24 patients (17 crt, 2 pe and 5 dvt), liver sos in 23 patients and myocard infarction at the early period in only one patient. in 4 out of 12 patients with low pc activity crt occurred, and liver sos was observed in only one patient. in 6 out of 24 patients with genetic mutation developed a te complication, 5 crt and 1 pte. crt occurred in 4 of 14 patients with fvl mutation, while 1 crt and 1 pte among 9 patients with pth mutation were observed. liver sos was seen in only one patient with pth heterozygote positive. we found the presence of low pc level or genetic mutation increased the frequency of the development of te (or: 6.9 and 3.7, respectively), but no effect on the liver sos. in conclusion, the use of thromboprophylaxis at peri-transplant period in patients with genetic mutations is still a controversial topic; which should be elucidated with controlled studies. iron overload (io) is an adverse prognostic factor in patients who undergo allogeneic haematopoietic stem cell transplantation (hsct) for thalassemia and appears to play a similar role in patients with other hematological disorders. estimation of io is primarily based on serum ferritin, however many confounding factors particularly in hsct recipients may result in frequent ferritin overestimation. aim of the study was to quantify io by uperconducting quantum interference device (squid) after hsct and evaluate the impact on infections and gvhd; additionally the feasibility of iron-depletion has been investigated in this pilot study we evaluated io in 89 consecutive adult patients who received hsct from a matched sibling (n=66) or a matched unrelated donor (n=23). primary diagnosis included aml/mds in 54% of cases. assessment of io after hsct included serum ferritin and in those with hyperferritinemia (> 1000 ng/ml), liver iron concentration (lic) was evaluated by squid magnetic susceptometry. io assessment was performed in patients in remission at a median time of 698 days after hsct (range 48-5239 days). median serum ferritin was 821 ng/ml (range 18-11110).thirtynine of 89 (44%) patients had serum ferritin level >1000 ng/ml; lic (microgfe/g liver wet-weight) evaluated by squid was available for 35/39 patients with elevated ferritin values. overall, 26 patients (29%) had moderate (lic levels 1000-2000 microg/gww) to severe (lic levels>2000 microg/gww) io; median lic values were 1419 microg/gww (range 1030-3253). nine patients (10%) had normal lic values (lic<400) despite high ferritin. patients with lic>1000 received a median of 41 packed red blood cells before study evaluation (range 7-79). the rates of bacteremias, invasive fungal disease and chronic gvhd were higher among patients with lic>1000 as compared to patients with lic<1000 (24%, 11% and 41% vs 16%, 5% and 25% respectively; p=ns). eighteen of the 26 patients with lic levels > 1000 were treated by regular phlebotomy. for 15 of these the phlebotomy program is still ongoing, while 2 patients completed the program with ferritin normalization; one patient was switched to deferasirox due to poor tolerance. in 5 cases phlebotomy was considered contraindicated; 3 patients who relapsed did not receive any treatment. our preliminary data show that one third of hsct recipients may present moderate/severe io as assessed by squid, and iron-depletion resulted feasible in 73% of these subjects. a trend for higher rates of infectious complications and chronic gvhd was observed in patients with io. despite recent advances, mortality rates after allogeneic hematopoietic stem cell transplantation (hsct) remains high and cannot be accurately predicted. several reports from the seattle group suggested the use of comorbidity indexes (ci) to provide valid and reliable scoring of pre-transplant comorbidities that predicted non-relapse mortality (nrm) and overall survival [blood 2004 (cci) , 2005 (hct-ci) & ann int med 2006 (pam)). however, whether such indexes could be used by other groups, and if one index better predicts survival than another, is yet unknown. hct-ci and pam required grading according to pulmonary function tests (pft), which were lacking for a majority of our patients. we thus designed a modified hct-ci and a corrected pam, without pft. survival curves were estimated using kaplan-meier method. cumulative incidences (ci) of non-relapse mortality were analyzed, with relapse treated as competing event. the likelihood ratio statistics in proportional hazards models was computed for each index, with its associated p-value, as a measure of association between the comorbidity indexes collapsed into risk groups and the outcomes. discriminative performance of comorbidity indexes was then evaluated by the c-index. standard errors of c-indexes were computed from that of somers' dxy rank correlation coefficient. p-values for comparison of c-indexes were obtained using a nonparametric bootstrap procedure. we thus retrospectively studied 286 patients (169 male, 117 female) who received their first allogeneic hsct at saint louis hospital between april 2004 and december 2006. the median age was 31 years (4-64), 149 patients (52%) were transplanted from a matched related donor and 248 patients received an hsct for intermediate or high-risk diseases (86%). using cci, 25% of our patients had indexes of 1 or more; median reduced hct-ci was 1; and median corrected pam score was 24. the discriminative properties of the 3 ci were rather low in our patient population with c-index of 0.515, 0.499 and 0.582 for the cci, reduced hct-ci and corrected pam indexes, respectively. comparison of patients-and transplant-characteristics between our and seattle group's cohorts however revealed significant differences with more children, more cord blood hsct and hsct for fanconi anemia in saint louis. finally, direct comparison of scoring items and multivariate analysis revealed that age, unmatched related donor and hepatic disease were associated with nrm in our cohort. primary graft failure occurs in 10-20% of unrelated cord blood transplantation (ucbt) patients. its occurrence is associated with increased risk of death due to relapse (when associated with autologous recovery) or complications of pancytopenia. salvage transplant is possible in patients with absence of neutrophil recovery (anr): clinical tools are required to promptly identify these patients at risk. we conducted this retrospective study to identify risk factors for anr. from 1996 to 2007, 98 consecutive children underwent a first ucbt in our center. anr was defined as absolute neutrophil count ≤ 100/microl in patients alive without disease on day +42. patients for whom a salvage transplant conditioning regimen was initiated before day +42 were excluded from this analysis (n=4, 2 of them are late survivors). ninety one (91) children were eligible for analysis. median time for neutrophil recovery (≥ 500/microl) was 27 days (range, 0 to 51). anr occurred in 7/91 (7.7%) and was confirmed by bone marrow biopsy. of these 7 patients, 2 underwent a 2nd transplant, one of which succeeded. five (86%) died between day +48 and +72 of transplant complications that occurred at a median of 38 days (range 29-70) of their first transplant. in 4 of them, complications were related to persistent neutropenia and/or lymphopenia. our current strategy of waiting until day +42 for a 2nd transplant decision carries a significant risk of mortality in patients with anr. when analyzed for demographics and graft characteristics, the group with anr did not differ significantly from the group with neutrophil recovery. the specificity and sensitivity for anr prediction of white blood cell (wbc) < 200 /microl at different time points is as shown in table 1. wbc < 200 /microl on day +28 and day +35 carries a risk of day +42 anr of 58% and 100%, respectively. the negative predictive value of wbc < 200 /microl on day +35 is 100%. therefore, in order to reduce the risk of mortality associated with anr, we now proceed to 2nd transplant evaluation for children with wbc < 200 /microl by day +28, and initiate the therapy on day +35 if wbc counts remains < 200 /microl. purpose: the aim of this retrospective study was to determine whether pre-transplant iron overload assessed by serum ferritin level predict the non-relapse mortality (nrm) and overall survival (os) rate after haematopoietic cell transplantation (hsct). patients and methods: 283 patients with hematological disorders underwent myeloablative or reduced-intensity conditioning followed by the first hsct from related or unrelated donors at keio bmt program from 1995 to 2005. 35 patients were excluded from this analysis because ferritin and crp levels were not available or measured at the time of transplant. demographic data and information on comorbidities was obtained from the keio bmt database. comorbidity was scored according to the hct-specific comorbidity index (hct-ci) proposed by the seattle group without any modifications. results: median value of ferritin was 448 ng/ml(range 26-3010 ng/ml). the percentage of patients with ferritin 193 ng/ml did not vary significantly among diseases. in all but 7 patients crp were within normal range at the time of measuring ferritin. there was a strong relationship between pretransplant ferritin and os. the 5-year os for patients was 76.2% in the first quartile (ferritin 0-193 ng/ml) (q1), 58.6% in q2 (194-448 ng/ml), 50.7% in q3 (449-1060 ng/ml), and 43.6% in q4 (more than 1061ng/ml) (p=0.001). 5-year os was also significantly higher for those with ferritin level more than 1000 ng/ml compared with those with ferritin equal or less than 1000 ng/ml (61.8% vs 43.8% p=0.004). the 5-year nrm was also significantly associated with pretransplant serum ferritin level. the 5-year nrm was 12.2% for patients with ferritin more than 1000 ng/ml, and 28.8% for those with ferritin equal or less than 1000 ng/ml (p=0.004). the mean score of hct-ci was 1.3 for those in q1, 1.6 in q2, 1.6 in q3, and 2.5 in q4. the mean score of hct-ci was significantly higher in patients with hyperferritinmia (ferritin more than 1000 ng/ml) than those without (2.5 vs 1.5 p=0.001). conclusion: these results suggested that pretransplant level of ferritin could be a marker of predicting nrm, os after hct and thus useful for patient counseling before hct. whether the addition of ferritin level into hct-ci scoring system give a better prediction of posttransplant nrm and os needs further investigation. increased incidence of renal impairment after radioimmunotherapy as part of the conditioning regimen before allogeneic stem cell transplantation t. zenz* (1), r. schlenk (1) intensifying the conditioning regimen with radioimmunotherapy (rit) is feasable for high risk leukemias and mds patients. while additional radiation exposure by rit is greatest in the bone marrow, significant exposure of the kidney occurs and an increased frequency of bmt nephropathy has been observed in patients receiving rit using the rhenium188 labelled anti-cd66 antibody. in order to obtain a precise picture of the incidence of renal impairment we compared two large cohorts receiving allogeneic stem cell transplantation (sct) with or without the use of rit from our center. between 1998 and 2004, 261 patients with a median follow-up of 58 months received hla-identical or mismatched allogeneic sct. of these 123 received an intensified conditioning regimen with a rhenium188 (n=87) or yttrium90 labelled (n=36) anti-cd66 antibody. the cohorts were similar with respect to sex, stem cell source and use of nephrotoxic medication. there was an increased proportion of patients with aml, all, and haploidentical donors in the group receiving rit (p<0.01), while the conventional group had more patients with cml, tbi in the conditioning regimen, cyclosporine use and incidence of gvhd (p<0.01). the clinical characteristics were documented for 5 years post sct. we defined relevant renal insufficiency as a creatinine level above 200 µmol/l and the failure to reach creatinine values below 150 to exclude intermittent drug toxicities. the incidence of renal failure was 13% in patients with conventional conditioning regimens and 24% in patients receiving rit (p=0.02). the competing risk analysis showed a significant increase in the cumulative incidence of nephropathy (p=0.01). even though the dosimetry showed decreased renal doses in patients with y90 this did not lead to a decreased incidence of renal impairment. other factors as tbi, cyclosporine use, donor source or gvhd did not influence the development of renal failure. in a multivariate model including rit, csa, donor source, gvhd and the combination of csa and rit, the combination of rit and csa was the only significant risk factor (p=0.01). in conclusion, rit lead to an increased frequency of continuous renal impairment. the combination of cyclosporine with rit appeared detrimental while tbi (with renal shielding) did not lead to an increased incidence of renal failure. strategies to decrease the incidence of renal impairment may include nephroprotection with ace inhibitors or alternative immunosuppression. prospective evaluation of oral mucositis in allogeneic stem cell transplant recipients receiving conventional myeloablative conditioning or reduced-toxicity conditioning with treosulfan and fludarabine h. uotinen*, l. volin, e. juvonen, a. nihtinen, t. ruutu helsinki university central hospital (helsinki, fi) oral mucositis (om) is a frequent and often severe complication of allogeneic stem cell transplantation (sct). the aim of this study was to evaluate the incidence and duration of severe (who grade iii-iv) and ulcerative (grade ii-iv) om in patients receiving different conditioning regimens. between sept 2005 and nov 2007, 131 patients were prospectively observed daily from the start of conditioning until hospital discharge. conventional (convent.) myeloablative (ma) conditioning (cy/tbi n=82, bucy n=10) was given to 92 patients (34 aml, 21 all, 7 mds, 7 cml, 7 mm, 6 mf, 10 others) and treosulfan (treo)-based conditioning to 39 (13 mds, 12 aml, 9 mm, 5 others) patients. treo-based conditioning consisted of treo 3x14 g/m² (n=28) or 3x12 g/m² (n=11) and fludararabine (fld) 150 mg/m². treo-fld conditioning is regarded as ma at least at the treo dose level of 3x14g/m² but with reduced non-hematological toxicity. treobased conditioning was given to patients not eligible for convent. full intensity conditioning because of age, comorbidity or heavy preceding cytotoxic treatment. there were no significant differences in the om results with the two treo dose levels used. the median age of the patients was 48 (18-62) years in the convent. ma group and 57 (19-65) years in the treo group. in the convent. ma group the donor was sibling in 40 cases and unrelated in 52 cases. in the treo group the donor was sibling in 17 cases and unrelated in 22 cases. the incidence of severe om was 40 % in the convent. ma group and 3 % in the treo group (p< 0.005). severe om episodes had a mean duration of 4 (1-26) days. the incidence of ulcerative om was 72 % in the convent. ma group and 33 % in the treo group (p<0.005). the ulcerative om episodes had a median duration of 6 (1-34) and 3 (range 1-8) days (p=0.003), respectively. om reached maximum who grade on day 13 in the convent. ma group and on day 16 in the treo group after the start of conditioning. the median duration of hospitalisation from the sct was 24 (17-52) days in patients with ulcerative om vs. 21 (range 17-43) days in patients without (p=0.110). in conclusion, conditioning with treosulfan and fludarabine caused significantly less severe and ulcerative mucositis than conventional ma regimens, and the duration of ulcerative mucositis was shorter. this supports the role of treo-fld conditioning for allogeneic transplantation especially in the treatment of patients with increased risk of toxic complications. patients receiving cbt had significantly slow neutrophil and platelet recovery in multivariate analysis. the incidences of acute and chronic gvhd were not significantly different. unrelated bmt showed better trm (25% versus 38% at 1 year, p<0.01), relapse (15% versus 26% at 3 years, p<0.01) and dfs (57% versus 29% at 3 years, p<0.01) results compared with cbt. next, we analyzed 498 bmt (n=370) and cbt (n=128) recipients who have taken total body irradiation (tbi; >8gy) containing myeloablative regimen and calcineurin inhibitors (cyclosporine or tacrolimus) plus methotraxate (mtx) for gvhd prophylaxis without history of prior transplants. in this subpopulation, multivariate analysis revealed no significant difference between bmt and cbt in dfs (57% and 51% at 3 years; hazard ratio (hr):1.34; 95% confidence interval (ci): 0.89-2.01; p=0.16). no statistically difference was also seen in trm (25% at 1 year after bmt and 23% at 1 year after cbt; hr: 0.90; 95%ci: 0.58-1.39; p=0.64). however, the cumulative incidence of relapse was significantly lower in bmt than in cbt (16% and 21% at 3 years; hr: 1.90; 95%ci: 1.12-3.22; p=0.017). the current japanese registration data in mds showed overall results of unrelated bmt were better than those of unrelated cbt by competing risk regression models. these data also suggest that unrelated cbt could be safely and effectively used as same as unrelated bmt when adequate transplant procedures are selected. comparative studies have shown that cb transplant is characterized by a lower risk of gvhd. cb units have been reported to contain tregs, but minimal data are available on these cells. aim of this study was to compare the suppressive functions of tregs expanded from cb units with those expanded from the peripheral blood (pb) of patients who have undergone an allogeneic sct. tregs were purified from mononuclear cells obtained from cb units or pb using the cd4+cd25+ regulatory t-cell isolation kit (miltenyi biotec) and expanded for 6 days in 96-well u-bottom plates coated with anti-cd3 and anti-cd28 moabs plus il-2. to assess the suppressive functions, expanded tregs from cb units or pb were seeded with naïve autologous effector t cells stimulated with allogeneic dendritic cells (dc) pulsed with apoptotic leukemic blasts, then incubated with [3h]-thymidine and counted in a beta-counter. suppressor activity was measured as [3h]-thymidine incorporation in the presence or absence of tregs. the proportion and the immunophenotypic analysis of tregs present in the cb units (n = 9) -in terms of expression of surface cd4, cd25, cd62l, cytoplasmic ctla-4 and foxp3was comparable to those obtained from the pb of allografted patients (n = 9). in addition, tregs from cb units and from the pb of these patients showed an equivalent expansion capacity [mean fold increase (range), cb units 8.6 (1.8-24); pb patients 9.1 (1.5-16.5) ]. on the contrary, preliminary data show that tregs expanded from cb units (n = 4) exert a higher suppressive function on the proliferative reaction of t cells stimulated by allogeneic dc compared to tregs expanded from the pb of allografted patients (n = 2) [mean fold reduction (range), cb units 9.01 (2.66-15.08); pb patients 3.83 (1.49-6.17) ]. moreover, immunofluorescence analysis demonstrated that tregs expanded from cb units (n=2) are highly positive for cytoplasmic il-10 (mean 82%, range 65-99) and negative for ifn-gamma. these results indicate that tregs contained in cb units exert a potent suppressive function in mixed lymphocyte reaction culture assays and offer further insights into the understanding of the biology of cb transplant. double negative tregs are reduced in allo-transplanted patients developing graft-versus-host disease b. serio* (1), z. mciver (1), a. risitano (2) , c. selleri (2) , j. maciejeski (1) (1)cleveland clinic (cleveland, us); (2) federico ii university of naples (naples, it) during development of a healthy immune system, central tolerance is induced in the thymus by the negative selection of t lymphocytes that have a high affinity for self antigens. after bmt, central tolerance may be impaired by thymic involution and conditioning regimens resulting in dysregulated alloreactivity. various subpopulations of regulatory t cells including the tregs or suppressor t cells with cd4+cd25+foxp3+ phenotype (foxp3 tregs), nk t-cells and the cd3+cd4-cd8-cd56-a/atcr+ t regs (double negative [dn] tregs) had led to the mechanisms of peripheral tolerance. we investigated behavior of dn following human allogeneic hsct with regard to the occurrence of graft versus host disease (gvhd) and restoration of t cell receptor (tcr) repertoire. a cohort of 40 patients was investigated; 16 underwent matched unrelated hsct and 24 received matched sibling grafts. frequency of dn and tcr repertoire of cd4 and cd8 cells was measured serially and at the time of diagnosis of gvhd by flow cytometry. our patient population demonstrated skewing of tcr repertoire and we identified a very strong and linear relationship between total number of vâ family expansions and the grade of gvhd (grade 1-4, n=28, p=.005); this relationship held true when considering separately both the cd4 and cd8 compartments (p=.009 and p=.023, respectively). the median frequency of dn tregs was calculated to be 0.54% of the total cd3+ t-cell population (range 0.04 -3.11). by using the total number of vâ family expansions as a gauge of alloreactivity, we observed a reduced number of dn tregs in those individuals that developed 4 or more vâ family expansions (n=25, mean 1.16 vs 0.56 cell/ìl, p=.03). in addition we also noted a significant difference in both the mean percentage and mean absolute values of dn tregs for those individuals that developed gvhd (grade >2) when compared to those that did not (n=29, 0.40 vs 1.32%, p=.004, and 0.8 vs 2.6 cells/ml, p=.024 respectively). the size of dn treg compartment inversely correlated to the grade of gvhd (grade 1-4) as both percentage and absolute value (n=29, p<.001, and p=.019 respectively). in conclusion we found that vb family expansion are associated with degree of alloreactvity and gvhd. in addition we show that dn tregs are reduced after bmt suggesting their important role in peripheral tolerance and alloreactivity. this work contributes to better define the regulatory role of dn and to develop future therapeutic applications. patients with myelofibrosis. a study of the mds subcommittee of the chronic leukaemia working party of the de witte on behalf of the mds subcommittee of the chronic leukemia working party of the european group for blood and marrow transplantation hla disparities were 4/6 match (n=49), 5/6 (n=10), and 6/6 (n=1). , respectively. twenty-one patients received cyclosporine, and 39 patients had tacrolimus alone as gvhd prophylaxis from day c1. neutrophil engraftment was achieved in 86% (median day; 20). cumulative incidence of pir, grade 2-4 and grade 3-4 acute gvhd were 60, 45 and 31%, respectively. estimated 2-years survival was 47% (95% ci: 34-68%) sierra hospital de la santa creu i sant pau (barcelona, es) background: neurological complications (nc) of allogeneic hematopoietic stem cell transplantation (allo-hsct), involving central (cns) and/or peripheral nervous system (pns) are common and remain life-threatening in most cases. their incidence and characteristics after allo-hsct have not been well defined we excluded patients with nc due to cns relapse of their malignancies as well as encephalopaty in the context of pre-mortem multi-organ failure cns complications included seizures in 7 cases, 5 non-focal encephalopaties, 5 meningoencephalitis and 5 strokes or hemorrhages. pns complications consisted of 4 cases of neuralgia/neuritis and 4 cases of demyelinating axonal neuropathies (1 guillain-barré syndrome) piñana is supported by grants isciii (cm06/00139) o404 haematopoietic stem cell transplantation for advanced primary mds in children: results of the ewog mds study group b. strahm* (1) ); (5)wilhelmina children´s hospital and methotrexate, and additional methylprednisolone in case of a sibling donor. the median clinical follow-up time for the patients was 37 months (3-46 months). several anti/coagulation activities associated with endothelial cell activation were serially assessed up to 3 months: prothrombin fragments 1+2 (f1+2), thrombin time (tt), fviii:c, activated protein c-protein c inhibitor (apc-pci) complex and protein c act (pc). during conditioning (on day-2) as an early sign of thrombin generation, f1+2 and apc-pci complex increased 2-3 fold. after engraftment, fviii:c increased steadily to reach its high maxi-mum on day +24 (273 % ±104 %, median±sd, p<0.001). interestingly, pc rose (189 % ± 63 %) in parallel with fviii:c with a 5-fold individual variability. after the engraftment fviii:c and pc were highly interrelated. gvhd developed in 8 patients and was predicted by early low pc activity (<90%) during conditioning (p=0.007) (or=16.7). gvhd was also associated with elevated level of f1+2 (> 0.7 nmol/l) (p=0.014) and was predicted by short tt (<15 s) (p=0.004) (or= 33.1) after the transplantation (on day +10). no patient whose f 1+2 was < 0.7 nmol/l (n=11) developed gvhd. elevated level of f 1+2 (>1.5 nmol/l) after the transplantation associated with non-relapse mortality. 3 patients with the highest thrombin generation after sct all died (7-23 months). in con-clusion, early up-regulation of thrombin generation and down-regulation of pc associated with the appearance of gvhd. after allogeneic sct procedure there is an intimate relation between endothelium regulated coagulation and development of gvhd, suggestive of a new therapeutic target. final results from a phetema study l. rosiñol* (1) , j.j. lahuerta (2) , a. sureda (3), j. de la rubia (4), j. , m. hernández-garcía (6), b. hernández-ruíz (7), j.a. , j.l. bello (9), d. carrera (10), m.j. peñarrubia (11) , e. abella (12) , a. león (13), c. poderós (14) , j.c. j. besalduch (16) , r. , i. p. ribas (19), j. san miguel (8) , j. bladé (1) (1)hospital clinic (barcelona, es) ; (2) background: two randomized trials showed that tandem transplant result in a significantly longer efs and os in patients failing to achieve complete remission (cr) or near-cr with a single transplant. however, other studies failed to show survival benefit from a second transplant but there was no survival plateau. promising results have been reported using dose-reduced intensity conditioning (allo-ric), especially after debulky with an autologous transplant.aim: to investigate the efficacy in terms of response upgrading and survival from a second transplant intensification in patients with chemosensitive disease who failed to achieve cr or near-cr with a first transplant. patients and methods: patients diagnosed with mm from oct 1999 to dec 2004 younger than 70 years received 6 courses of vbmcp/vbad and responding patients were intensified with busulphan/melphalan or mel-200 followed by stem cell support. patients not achieving cr or near-cr were planned to undergo a second transplant (second auto with cvbcyclophosphamide, etoposide and bcnu -or mel-200 intensification or an allo-ric with fludarabine/mel-140 conditioning, if sibling donor available. results: eighty-five patients received a second autologous transplant while 26 underwent an allo-ric. the cr rate was significantly higher with allo-ric (35% vs. 10%, p= 0.02). there was a trend towards a higher trm with the allogeneic procedure (5% vs. 15%, p=0.09). after a median follow-up of 51 months for alive patients, there were no significant differences in efs (48 mos. vs. nt reached) and os (80 mos vs. not reached for autologous tandem vs. auto/allo-ric). however, there is a plateau in the "allo-ric" group beyond 3 years of the second procedure not observed in the autologous arm. a final update will be presented. conclusions:1) an allo-ric transplant after an autologous procedure results in a significantly higher cr rate than a second autologous transplant, and 2) although we found no significant differences in survival between the two transplant modalities, there is a plateau in the allogeneic group. (2) background: it has been assumed that patients with primary refractory myeloma benefit from early high-dose therapy/stem cell support (hdt. however, in the reported series patients with "unresponsive-progressive disease" vs those "nonresponding-non progressing" were not analyzed separately aim: response and survival after early hdt in the two populations of truly primary refractory multiple myeloma (i.e., patients with progressive disease versus those with "no change" or "stable disease" while receiving the initial therapy). patients and methods: from oct 1999 to dec 2004, 829 patients with mm received 6 cycles of vbmcp/vbad and at least one transplant. 81 of the 829 patients were refractory to vbmcp/vbad. these resistant patients were scheduled to receive a tandem transplant, the first with bu-12/mel-140 or mel-200 and the second "autologous" with cvb (ciclophosphamide(etoposide/bcnu) or mel-200 or "allo-ric" (if donor available) with fluda/mel-140. response and progression were defined by the ebmt criteria. results: 31 of the 81 primary refractory patients had progressive disease under the initial chemotherapy while 50 allogeneic stem cell transplantation (sct) is the only curative treatment approach in patients with myelofibrosis. the major limitation is the high treatment related mortality, which exclude mainly elderly patient from this treatment procedure. to determine the toxicity and efficacy of a dose-reduced conditioning regimen, consisting of busulfan (10 mg/kg), fludarabine (180 mg/m²) and anti-thymocyte globulin (atg fresenius: 3x 10 mg/kg for related and 3 x 20 mg/kg for unrelated sct) followed by allogeneic sct, we performed a prospective multicenter trial in elderly patients with myelofibrosis in 18 centers in three countries. from 2002 to 2006, 104 patients with a median age of 55 years (r.,32-68) were included and 102 were evaluable for outcome. risk profile was low risk with constitutional symptoms (18%), intermediate risk (n= 58%) and high risk (n=19%). all but 3 patients received peripheral blood stem cells as graft source either from related (n=31) or unrelated donor (n=69). all but one (1%) patient showed leukocyte and platelet engraftment after a median of 18 and 21 days, respectively. the median duration of leukocyte aplasia was 9 days (r., 3-21). acute graft-versus host disease (gvhd) grade ii o iv occurred in 19% and severe agvhd iii/iv in 7%, while chronic gvhd was seen in 32% of the patients. non-relapse mortality at 1year was 17% (95% ci: 11-27%) and significantly lower for patients younger than 50 years of age (4% vs 24%, p<0.001) and for patients with low risk vs intermediate/high risk disease (9% vs 26%, p= 0.07), while a higher nrm was seen for patients transplanted from hla mismatched donors (66 vs 17%, p= 0.006). the cumulative incidence of relapse at 3 year was 25% (95%ci: 15-43%) and influenced by time from diagnosis to sct of less or more than 24 months (18 vs 40%, p=0.05). patients with splenectomy had higher incidence of relapse (60 vs 18%, p=0.003). the estimated 3 year overall and event-free survival was 73 and 58%, respectively. the overall survival was influenced by age less than 50 years (92% vs 59%, p=0.009 and low vs intermediate/high risk (94% vs 60%, p=0.03 and hla mismatch (39 vs 70%, p= 0.02), while no impact on survival was seen for cytogenetic abnormalities, jak2 mutation status and donor (related vs unrelated) these results of a prospective multicenter study show excellent outcome of a busulafan/fludarabine based reduced conditioning regimen followed by allogeneic stem cell transplantation in patients with myelofibrosis. graft rejection after stem cell transplantation following reduced-intensity conditioning is influenced by the underlying disease, the donor type, disease stage and the cd3 content of the graft g.-n. franke*, a. mikolajewska, s. leiblein, h.-k. al-ali, e. hennig, w. pönisch, d. niederwieser, t. lange university of leipzig (leipzig, de) objectives: stem cell transplantation (sct) after reduced intensity conditioning (ric) is routinely used as a curative approach for older and medical impaired patients with haematological malignancies. in contrast to sct with conventional conditioning, graft rejection (gr) remains an important issue. we analyzed patients with sct after 2 gy total body irradiation (tbi) with or without fludarabine (flu) conditioning to identify risk factors for gr. patients and methods: 330 patients with a median age of 58 (range 17 -74) years, underwent allogeneic sct (bm=11, pbsc=319) from a related (n=115) or unrelated (n=215) donor for aml (n=121), cml (n=34), nhl/mm (n=102), mds/mps (n=38) or other diseases (n=35). conditioning regimen consisted of 2 gy tbi at day 0 and flu 30 mg/m2 from -4 to day -2 (n=305) followed by cyclosporin a and mycophenolate mofetil. results: 28 (8.5%) patients developed primary (n=23) or secondary graft failure (n=5) defined as a t-cell chimerism of <10% donor cells. in univariate analysis and also in multivariate analysis, diagnosis of cml (p=0.011), unrelated donor (p=0.029), early disease stage prior to sct (p=0.048) and low cd3 cells in the graft (p=0.002) were identified as independent predictors for gr. the relative risk (rr) to experience gr was 4.8, 4.2, 3.3 and 1.8, respectively. even in a subgroup analysis with pbsc recipients only, cml, unrelated donor and disease stage were associated with higher risk of gr. furthermore, a lower cd3 count increases the risk of gr by a rr 1.5 (p=0.06) in the multivariate model (median 3.2x108/kg bw). an increase in cd3 cells was not associated with increased incidence of acute gvhd grad ii-iv until day 100 (p=0.935). in contrast, the cd34-content of the graft had no impact on gr either as categorical (>median vs. <median) or as continuous variable (p=0.454). conclusion: in contrast to given risk factors for graft rejection, the t-cell content of the graft can be individually requested in every patient. since higher cd3 cells in the graft reduces the risk of gr without subsequent acute gvhd in our cohort of patients, we recommend the administration of cd3 cells >3.2x108/kg bw especially in patients with cml, unrelated donor and early disease stage in ric-sct. unrelated cord blood transplantation for adult patients with acute myeloid leukaemia/myelodysplastic syndrome using a reduced-intensity conditioning regimen consisting of fludarabine, melphalan and total body irradiation k. masuoka*, k. ishiwata, m. tsuji, s. takagi, h. yamamoto, d. katoh, y. matsuhashi, s. seo, n. matsuno, n. uchida, a. wake, s. miyakoshi, s. taniguchi toranomon hospital (tokyo, jp) objectives: to evaluate the efficacy of unrelated cord blood transplantation (ucbt) in a reduced intensity (ri) conditioning regimen consisting of fludarabine (125mg/m²), melphalan (80mg/m²) and total body irradiation (400 cgy), we analyzed retrospectively the results of 60 adult patients with acute myeloid leukemia (aml)/ myelodysplastic syndrome (mds) in our hospital. patients and methods: we reviewed medical records of 60 patients with aml/mds who had received single cord blood unit between november 2003 and july 2007 at toranomon acute leukaemia 2 / myelodysplasia o397 myeloablative allogeneic stem cell transplantation with unrelated donors for high-risk acute myeloid leukaemia: no increase in relapse with alemtuzumab-depleted grafts p. kottaridis* (1), k. thomson (2) the role of t cell depletion in myeloablative allogeneic stem cell transplantation for acute myeloid leukemia (aml) remains uncertain. dose intensification may compensate for any potential loss of graft-versus-leukemia effect, and the use of t cell replete grafts is associated with significant morbidity and mortality, particularly when using unrelated donors. this study describes the results in 44 patients with high-risk aml transplanted with an alemtuzumab-containing myeloablative regimen, using unrelated donors. median age at transplant was 34 years and 20 patients (45%) had donors mismatched at 1-3 hla loci. all patients were high-risk for relapse, as defined by induction failure, adverse cytogenetics, >cr1, or secondary disease (4 preceding myelodysplastic syndrome [mds] , 3 therapy-related). two patients had untreated relapse at the time of transplant, and 9 of the remaining 42 (21%) had refractory disease. the conditioning regimen was cyclophosphamide 60mg/kg for 2 days, fludarabine 30mg/m² for 3 days and total body irradiation (tbi) 14.4gy in 8 fractions over 4 days. stem cell source was peripheral blood in 37 and bone marrow in 7, and graft-versus-host disease (gvhd) prophylaxis was with 20mg alemtuzumab added to the stem cells prior to infusion and cyclosporin 3mg/kg. median follow-up for surviving patients was 36 months. estimated event-free survival (efs) was 60% at 1yr and 55% at 4 years. acute gvhd grade ii occurred in 11% (no grade iii-iv) and extensive chronic gvhd in 23%. non-relapse mortality (nrm) was 19% at 4 years with no events beyond 10 months post-transplant, and estimated relapse risk was 27% at 4 years with no events beyond 15 months. for efs and relapse risk, the only significant variable was chemosensitivity pre-transplant, with inferior efs (65% at 4 years for chemosensitive patients vs 27% for chemorefractory, p=0.026) and worse relapse risk (15% at 4 years for chemosensitive patients vs 55% for chemorefractory, p=0.010) in those not in complete remission at transplant. there was no impact on nrm or relapse risk depending on the presence of hla mismatch, or of significant gvhd (>grade i acute, or any chronic gvhd). use of this regimen therefore permits the successful transplantation of patients with high-risk disease and hla-matched/mismatched unrelated donors, with minimal acute gvhd, relatively low nrm and no evidence of an excessive relapse rate, particularly in those in complete remission at transplant. haematopoietic stem cell transplants (hsct) using unrelated donors (ud) has become an important and viable option in the treatment of acute leukaemia (al). we have previously shown an increased risk of relapse with hla-dpb1 matching and independently, with nod2/card15 genotype. in light of these data, we have analysed a larger ud-hsct cohort in order to establish the impact on outcome when both variables are considered. hla and nod2/card15 typing was performed on 304 al ud-hsct pairs. transplants were between 1996 and 2005. diagnoses were all (47%) and aml (53%). 67% of the cohort were 10/10 hla matched and 16% were also hla-dpb1 matched. myeloablative conditioning regimens were used in 74% of transplants. 82% of conditioning protocols included t-cell depletion. bone marrow was used in 72% of transplants, the remaining 28% using peripheral blood stem cells. two forms of post-transplant immunosuppression predominated, cyclosporine a and methotrexate (47%) and cyclosporine a alone (38%). based on our previous data, the cohort was grouped according to their relapse risk, group 1 (dpb1 matched; nod2/card15 snp, n=24), group 2 (dpb1 matched; nod2/card15 wild-type (wt) or dpb1 mismatched; nod2/card15 snp, n=112) and group 3 (dpb1 mismatched; nod2/card15 wt, n=168). disease relapse differed significantly between the three groups (1 year: group 1 68%, group 2 48%, group 3 30%, p=0.006). this finding persisted in multivariate analysis where being in either group 2 or 3 was protective towards relapse as compared to group 1 (rr 0.321; p=0.001 and rr 0.478; p=0.031 respectively). in group 1 (high risk), this resulted in decreased overall survival (os) (33% vs 54% in group 3, rr 0.617; p=0.080). the best os was seen in group 3 (low risk) where in addition to low relapse, there was increased acute and chronic graft-versus-host disease (gvhd) (p=0.0019 and p=0.002 respectively). in this cohort, limited cgvhd was s74 associated with reduced relapse (p=0.01) and better os (p<0.0001). in accordance with our theory, being hla-dpb1 matched and nod2/card15 snp predicts for the worst outcome with significantly increased relapse and reduced os. the ideal pairing is hla-dpb1 mismatched and nod2/card15 wt. these data suggest that prospectively typing al patients for both variables will allow the prediction of transplant outcome and will allow the effects of being independently hla-dpb1 matched or nod2/card15 snp to be offset by intelligently selecting a suitable, less precarious donor. 8 (t8) is the most common chromosomal abnormality in aml. the prognostic impact of t8 as a sole aberration in aml remains unclear, conferring either an intermediate or a poor prognosis. indeed, patients with t8 occurring with other cytogenetics abnormalities seem to have the prognosis conferred by the accompanying aberration. the aim of this study was to describe the results of allogeneic transplantation in a large series of aml patients exhibiting isolated or associated t8 and to compare outcome with intermediate risk aml patients exhibiting normal caryotype and receiving allo-hsct. 182 aml patients were identified (males n=100; females n=82) with isolated (n=136) or associated (n=46, favourable group n=8, intermediate n=30; high-risk n=7; unknown n=1) t8, allografted with an hla identical sibling (n=113) or an unrelated donor (n=69) between 1990 and 2007 and reported to the ebmt. median age was 37 years (range: 17-68). median interval between allo-hsct and diagnosis was 165 days (range: 71-946). the proportion of patients in cr1, cr2/cr3 or active disease was 59%, 13% and 28%. myeloablative and reduced intensity conditionings were performed in 148 and 34 patients respectively. gvhd prophylaxis consisted of csa/methotrexate or csa/mmf in 49%. engraftment was observed in 171 patients (95%). grade ii and iii/iv acute gvhd occurred in 21% and 11%. chronic gvhd developed in 45%. with a median follow-up of 48 months (range: 3-180), 5-year lfs, relapse rate and nrm were 45%, 30% and 25%. status at transplant (cr vs others) (p<0.0001, hr 0.3, 95% ci: 0.2-0.46), female sexe (p=0.03, hr 1.61, 95% ci: 1.05-2.48) and hla sibling donor (p=0.02, hr 1.64, 95% ci: 1.08-2.49) were significant predictors for better lfs. 5-year lfs was similar between aml patients with isolated or associated t8 (41% vs 55%, p=0.11). whenconsidering only patients allografted in cr1, 5-year lfs was similar between isolated t8 aml patients (55%, n= 82, median follow-up 51 months), associated t8 aml patients (55%, n=26, median follow-up 51 months) and aml patients with normal caryotype (58%, n=1782, median follow-up 36 months). we conclude that allogeneic transplantation in first linetherapy is a valid therapeutic option in patients exhibiting isolated or associated t8. isolated or associated t8 do not confer bad prognosis and aml patients exhibiting such aberrations have to be considered as intermediate risk patients as they likely have the same outcome of aml patients with normal karyotype allografted in cr1. regimen intensity in acute myelogenous leukaemia: addition of 400cgy total body irradiation to a myeloablative fludarabine/busulphan/thymoglobulin allogeneic transplant regimen reduces relapse without increasing transplant-related mortality j. russell* (1), w. irish (2) , l. savoie (1) some attempts to intensify myeloablative stem cell transplant (sct) conditioning protocols for acute myelogenous leukaemia (aml) have reduced relapse only at the expense of increased transplant-related mortality (trm). a regimen of intravenous busulphan, fludarabine and thymoglobulin has been well tolerated but followed by a substantial relapse rate. we report the results of a study to enhance the antileukaemic effect of this protocol by adding a low dose of total body irradiation (tbi). 179 patients were treated between 1999 and 2006 with fludarabine 50mg/m 2 daily x 5 and intravenous busulphan 3.2 mg/kg daily x 4. 88 had additional total body irradiation (tbi) 200cgy x 2 on day -1 or 0. graft-versus-host disease (gvhd) prophylaxis was cyclosporine a, methotrexate and thymoglobulin (genzyme) 4.5 mg/kg total dose. median age was 46 (range 18-66) years in tbi recipients, 42 (16-65) for the non-tbi group. there was no difference in the proportions of sct with good risk (gr, cr1 or cr2) recipients (65% vs 56%), alternative (unrelated or mismatched related) donors (49% vs 40%) cmv +ve donor +/or recipient (60% vs 68%), female donors to male recipients (18% vs 19%) and high risk (hr) recipient cytogenetics (20% vs 14%) between the tbi and no tbi groups respectively. more tbi recipients received blood cells (91% vs 74%, p = 0.001) and consequently higher cd34+ cell doses (median 5.9x10 6 /kg, range 0.75-17.69 vs median 4.310 6 /kg, range 0.64-23.87, p <0.0001). follow-up of survivors was 12-83 months (median 31) for tbi recipients and 13-100 months (median 79) for those not given tbi. there was no difference in incidence of acute gvhd grade ii-iv at 23% vs 16±4%, acute gvhd grade iii-iv at 9% vs 9% and chronic gvhd at 55% vs 64% with and without tbi respectively. outcomes at 3 years are shown in the table: after adjusting for all the above risk factors the cox proportional hazard ratio for relapse was 0.32 (95% ci 0.17-0.6) in favour of tbi (p = 0.004). there was no effect of gvhd on relapse. the impact of tbi on relapse without affecting trm resulted in a significantly decreased risk of mortality with a hazard ratio of 0.56 (0.33-0.93, p = 0.03). the hazard ratio for disease-free survival was 0.46 (0.28-0.77, p = 0.003). this regimen allows some intensification by adding a low dose of tbi without an effect on trm but a reduction in relapse, confirming the importance of regimen intensity in transplantation for aml. s75 o401 long-term survival in patients suffering from aml with a complex aberrant karyotype after early allogeneic stem cell transplantation using the flamsa-ric regimen: results from a prospective phase ii trial c. schmid* (1), m. schleuning (2) introduction: patients suffering from acute myeloid leukemia (aml) with a complex aberrant karyotype (i.e. ≥ 3 cytogenetic aberrations) usually show poor response to chemotherapy and have a grim prognosis. allogeneic stem cell transplanttaion (allosct) is the treatment of choice, although the unfavorable prognostic value of a complex karyotype was preserved in most studies. in contrast, in the flamsa-ric pilot trial, a post-hoc subgroup analysis revealed similar results for patients with a complex karyotype as compared to more favorable cytogenetic subgroups (schmid, schleuning et al, jco 2005) . therefore, a prospective phase ii trial for patients with complex karyotype aml was initiated within the german aml cooperative group, to evaluate the role of allosct, performed as early as possible after diagnosis. the flamsa-ric preparative regimen contained the sequence of intensive cytoreductive chemotherapy (flamsa), followed three days later by reduced intensity conditioning (ric; 4gy tbi; cyclophosphamide, atg). patients and methods: 20 patients from 4 centers (median age: 50,1, range 20-63 years), with a median of 7 (range: 3-15) cytogenetic aberrations were included. median time from diagnosis to transplantation was 91 days. eight patiens had received one, 12 had received two courses of conventional chemotherapy. stage at start of flamsa-ric was cr1 in 7, first cytogenetic relapse in 1, and primary induction failure in 12 patients, including 8 with persistent disease after high-dose arac. donors were hla-identical siblings in 9, matched unrelated donors in 8, and 1-ag-mismatched unrelated donors in 3 cases. results: nineteen patients engraftet (median day: +18), one died in aplasia. 17 patients achieved molecular cr, 1 regenerated with blasts, and one had cytogenetically persistent disease. agvhd developed in 13 patients, but reached > grade ii only in 3. five patients developed cgvhd. relapse occurred in 2 patients. after a median follow up among survivors of 23 (range: 3-33) months, 8 patients have died from leukemia (n=3) or treatment-related causes (n=5). overall survival at one and two years from transplantation is 74% and 58%, the corresponding leukemia free survival is 69% and 58%. conclusion: to our knowledge, this is the first trial specifically addressing patients with a complex aberrant karyotype. although the numbers are small, the results suggest a promising activity of early allosct in this otherwise very unfavorable subgroup of patients with aml. the iron (fe) chelator, desferrioxamine (dfo), has been shown to have both antiproliferative and apoptotic effects in tumor cells. fe depletion results in g1/s cycle arrest and cell apoptosis and dfo acts as hypoxia-mimetic agent by accumulating the hypoxia-inducible factor-1 alpha protein which regulates the cellular response to hypoxia by cessation of growth. in this retrospective study we evaluated the effect of dfo administration and iron overload (iro) in relapse incidence in 127 consecutive patients (pts), allografted for malignant diseases (myeloid: 76, lymphoid: 51). the disease phase was early in 45, intermediate in 38 and advanced in 44 pts. thirty received non ablative and 97 ablative conditioning. peripheral blood (116) or bone marrow (11) grafts were donated from 101 siblings, 23 matched unrelated and 3 haploidentical donors. graft vs host disease (gvhd) prophylaxis was consisted of cyclosporine or prograf plus methotrexate. non-responders or relapsed pts within 2 months post allotransplant, were excluded. according to our center policy, pts with established engraftment and iro, as evidenced by ferritin levels>2000ng/ml, elevated liver enzymes or/and liver mri indicating hemosiderosis, receive dfo. among 95/127pts with iro, 31 were treated with dfo (50mg/kg x 5days/week, iv or sc for 2 months at least). the 5year relapse rate (rr) was significantly lower in pts treated with dfo than in non-treated pts (22% vs. 53%, p=0,003) and this benefit was restricted to myeloid malignancies only. in a multivariate analysis we examined dfo, chronic and acute gvhd, disease phase, graft source, type of donor, origin of malignancy and preparative regimen as risk factors for relapse. dfo administration retained its significance (p=0,02) while the absence of cgvhd and the advanced disease phase were significant factors for relapse (p<0,03). in order to explore whether iro affects the relapse incidence we separately examined the fe-overloaded pts who received no chelation (64) vs the non-fe-overloaded pts (32). the 5-year rr for untreated pts was 60% vs 33% for those with ferritin values <2000ng/ml (p=0,04). iro remained as significant risk factor for relapse in the multivariate analysis (p=0,03). this is the first clinical study to investigate the role of dfo in relapse incidence post allotransplant suggesting a possible role for dfo therapy post-transplant. prospective studies are needed to clarify if dfo may have a role in relapse prevention. equivalent disease-free survival results after cbt and bmt from unrelated donor using tbi containing myeloablative regimen and calsinulin inhibitors plus mtx methods in patients with mds in japan: multivariate analysis by competing risk regression models s. takahashi*, t. yamaguchi, m. monna-ooiwa, s. taniguchi, h. akiyama, t. morii, y. nagari, y. takaue, s. okamoto, k. miyamura, h. sao, t. nagamura, s. kato, t. kawase, y. the result of single institutional analysis in japan has shown cord blood transplantation (cbt) is promising in adults with hematologic malignancies including myelodysplastic syndrome (mds) and is comparable with bone marrow transplantation (bmt) from unrelated or related donors. we evaluated safety and efficacy of both bmt and cbt from unrelated donors using the data of mds patients within the japan marrow donor program and the japan cord blood bank network registry database and related those to biological and procedural factors. clinical data of 965 patients with mds including transformed acute myelogenous leukemia who received unrelated bmt (n=532) or unrelated cbt (n=433) between 1993 and 2006 were collected. the median periods of follow-up for survivors were 21 months for bmt and 12 months for cbt. we analyzed the hematopoietic recovery, incidences of graft-versus-host disease (gvhd), risks of transplant-related mortality (trm), relapse and disease-free survival (dfs) using competing risk regression models.advanced primary myelodysplastic syndrome (mds) represents a subgroup of childhood mds characterized by bone marrow (bm) dysplasia and an increased blast count in peripheral blood (pb) and/or bm. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative treatment. here we report the results of 105 patients (pts) (71 males/34 females) enrolled in the prospective ewog mds study. according to the highest who type prior to hsct pts were classified as raeb (59), raeb-t (30) and mdr-aml (16). median age at diagnosis was 10.6 yrs (1.0-18.2) and the median time from diagnosis of advanced mds to hsct was 4.1 mo (1.0-31.2) . cytogenetics revealed monosomy 7 in 37 pts, a complex karyotype in 11 pts, other abnormalities in 22 pts, no abnormalities in 31 pts and was unknown in 4 pts. intensive chemotherapy was given to 31 pts prior to hsct. 43 children were transplanted from an hla matched family donor (mfd), while the remaining 62 were given an allograft from a matched or 1 ag mismatched unrelated donor (ud). the preparative regimen included busulfan, cyclophosphamid and melphalan in all cases. stem cell source was unmanipulated bm, pb and cord blood in 73, 30 and 2 pts, respectively. cyclosporine-a alone was used as graft versus host disease (gvhd) prophylaxis in most children transplanted from a mfd, while the majority of pts transplanted from an ud were given cs-a, mtx and anti-thymocyte globulin. with a median follow up of 3.1 yrs (0.1-10.2) 62 out of 105 pts are alive and disease free, 19 experienced relapse, and 24 died of transplant related complications. the 5-year probability of event-free survival (efs) is 0.56 [0.46-0.66] while the 5-year cumulative incidence of transplant-related mortality (trm) and disease recurrence are 0.23 [0.16-0.33] and 0.21 [0.14-0.32], respectively. the cumulative incidence of grade ii-iv gvhd is 0.47 [0.37-0.57]. there is no significant difference in efs, trm and disease recurrence according to donor, mds subtype, karyotype or the treatment applied prior to hsct. age at hsct >12 yrs, an interval from diagnosis of advanced mds to hsct ≥ 4 mo and the presence of gvhd contribute to a significant increase in trm. these results indicate that a large proportion of children with advanced mds can be rescued by allogeneic hsct. trm remains a main cause of treatment failure especially for children older than 12 yrs. for these patients new strategies to reduce trm will have to be defined. introduction: patients with a history of an allogeneic haematopoietic stem cell transplant(hsct)have an increasing risk to develop a secondary cancer.the association of the hla system to cancer is well known.sibling donors share the same hla alleles, and the question is,if they have an increased tumour incidence compared to the general population.this is of concern because the possibility of a tumour induction as a result of mobilisation with g-csf has been discussed. methods: all hla-identical sibling pairs with an hsct from 1974 to 2001 for a malignant disease and living in switzerland were evaluated. general data of the donors were taken from the patient's charts of the transplant unit.donors were contacted per call or mail and asked about their current health status.in case of a malignant cancer,information on date of diagnosis,localisation,treatment was obtained.data were compared with the age-,and sex adapted cancer incidence rate of the swiss association of cancer registries(sacr).the same information was retrieved for the patients. results: 318 pairs were identified,in 291(92%)the donors (142 men(m),149 women(w))could be contacted. median observation time was 13.8 years(y)(range 5-32y).146 donors were <50y,89 between 50-59y,29 between 60-64y,20 between 65-69 y and 7 >70y old, hence 85% were <60y old.seventeen (6%)donors,12 bone marrow and 5 peripheral blood donors,had developed a total of 18 cancer of 9 different localisations (mamma,prostate,skin,bone marrow, colon,bronchus,stomach,bladder,orl).according to the incidence rate of the sacr,3.3 tumours in m and 6.8 in w would have been expected,3 (m) (rr0.91) and 4 tumours (w) (rr 0.84) were found in donors between 0-49y.in the age category 50-69 y,4.5 tumours in m and 4.8 in w were expected and 5(rr1.11)respective 6(rr 1.23)observed.in the subgroup of men between 60-64y 1.07 tumour were expected and 4 s77 observed (p<0.02).no donor >70y developed a tumour.4 of the 291 donors had died,3 from the tumour,1 of cardiac disease.in 12 patients a secondary tumour was diagnosed post hsct. conclusion: we observed a definite number of donors with a malignancy after hsc donation. absolute numbers were similar to the numbers of tumours in their transplanted siblings, observed versus expected rates were similar to an age and sex matched population except in male donors between 60-64 y by now. data from this single centre cohort remain yet inconclusive but underline the need for international collaborative donor follow up. the number of transplants is consistently increasing for aml, all, and lymphoid malignancy in all countries/regions except for vietnam. meanwhile, the number of hsct for nonmalignant diseases such as aplastic anemia and hemoglobinopathy has been stable in all countries except for iran, in which country it is recently increasing. the proportion of hemoglobinopathy in all hsct differed widely among countries; 0% in japan and china, 2% in singapore, 3% in taiwan, 5% in hong kong, 10% in malaysia, 13% in vietnam, 22% in iran, (all hsct until 2006, only 2006 in taiwan) . the trends changed quite differently among countries and regions over time for other diseases, most strikingly in cml. in japan, the number of hsct for cml had consistently increased until 2000 (n=307 in 2000) and then rapidly decreased due to introduction of imatinib (n=98 in 2005). in contrast, it is dramatically increasing in china (n=8 in 2000 china (n=8 in to n=98 in 2005 and stable in other countries. the number of hsct for mds and multiple myeloma is consistently increasing only in japan (and iran for myeloma), but not in other countries. hsct for solid tumors had been commonly performed only in japan, but the number has decreased since 1997 (n=337 in 1997, n=163 in 2005) . in summary, hsct is developing in most of the asian countries/regions. however, disease indications and trends differed widely among them probably due to different economics situations, health service systems, and availability of donors and agents such as imatinib for cml. short-and long-term side effects in the healthy donors of allogeneic haemopoietic peripheral cells mobilised with lenograstim: a single-centre experience m. martino*, g. console, e. massara, e. spiniello, i. callea, f. gatto, g. messina, t. moscato, r. fedele, g. irrera, p. iacopino u.o. ematologia con trapianto (reggio calabria, it) healthy allogeneic donors, who were mobilized with lenograstim and underwent hemopoietic peripheral cells (hpc-a) collection at our institution, were enrolled in a shortand long-term surveillance protocol for a 10 year period. to date, 171 donors have been assessed with a median followup of 55 months (2 -145) ; for 71 subjects, the follow-up is > 60 months and for 20 subjects is > 96 months. healthy donors received lenograstim at a median dose of 9.9 µg/kg (range 7-15). bone pain was reported as the most common adverse event (70.2 % of donors). common associated symptoms included fatigue (16.4 %), fevers (5 % ), headache (26.3 %), nausea (12.9%), insomnia (16.4 %). spleen size increased in 4.1 % of donors (> 2 cm exceeding the marginal cost at physical examination ). all donors experienced side effect resolution within 2-4 days of lenograstim discontinuation. leukocyte mean peak values were 48 x 109/l and the nadir of platelet counts reached mean values of 93 x 109/l and; however, such a decrease was not complicated by bleeding manifestations. the hemoglobin concentration decreased slightly but not significantly. leukocyte and platelet counts returned to normal values in about one week. no vascular disorders and cardiac disease occurred. long-term observation included adverse events in donors after 30 days from hpc-a mobilization and any neoplastic or not disease developed any time post donation. 4 donors showed persistent, slight leucocytopenia until the second month, with recovery in the fourth month of follow-up. 18 donors showed an ast and alt result 2.5 times the upper limit of normal until the second months of follow-up. transit ischemic attack occurred in 1 donor, (39 months post after donation). 1 autoimmune event has been reported 28 months post-g-csf (anckylosing spondylitis); 1 donor with a history of chronic obstructive pulmonary disease developed a secondary polyglobulia (50 months post-g-csf); 1 donor developed a gastric tumor, 19 months post-donation. no hematological malignancy was observed. in conclusion, our main findings are that the primary toxicity of g-csf administration is bone pain and that no cardiovascular events was related to the donation. with a median follow-up near to 5 years, no hematological disease was observed in our cohort of donors. effect of the search of an unrelated stem cell donor in patients with high-risk leukaemia: prospective, singlecentre study on intention to treat analysis a.p. iori* (1), v. valle (1) allogeneic stem cell transplant (hsct) plays a major role in the treatment of acute leukemia (al) patients with high risk (hr) features at diagnosis or in ≥ 2nd complete remission (cr). between 1995 and 2007, 164 patients -median age 12 years (1 -59) -with hr al followed at our center and lacking a family hla compatible donor were addressed to a search of hsc donor through the bmww registry and cord blood banks. the aim of this prospective study was to assess the effect of the search on the outcome of patients with hr al on an intention to treat analysis. forty-three patients started the s78 search in i cr, 84 in ii cr, 4 in >ii cr, 33 in relapse. fifty-five % of the 131 patients who entered into the study in cr showed a disease relapse at a median of 4 (1-20) and 2 (1) (2) (3) (4) (5) (6) (7) (8) months from the start of the search for patients in i-ii cr and > ii cr, respectively. sixty-nine patients (42%) underwent an hsct, 32% in a more advanced phase compared to the start of the search. nineteen of the 33 patients (57%) who started the search in relapse obtained a cr and underwent an hsct. globally, 45% of patients failed to undergo a transplant because of lost eligibility due to disease progression. for the entire population, the 12 year survival probability and dfs were 19%. disease progression was the major cause of death. when the variables affecting outcome parameters were analyzed in univariate analysis, the occurrence of relapse during the search period and the transplant procedure affected os (p=0.001) and dfs (p<0.0001). a more advanced phase of the disease at the start of the search and no transplantation were the factors negatively affected the relapse (p=0.01, p< 0.0001). for patients who underwent an hsct, the factors which negatively affected os and dfs were a relapse during the search and a more advanced disease phase at transplant. both these factors plus the disease phase at the start of the search affected the relapse for transplanted patients. in conclusion, by decreasing the length of the search (4 months for patients in i-ii cr and 2 months for patients with a more advanced phase of the disease) the risk of relapse can be reduced, thus increasing the possibility of carrying out a transplant and the transplant success. moreover, starting the search in relapse we may obtain the cr while waiting for the hsct. therefore, during the search of an hsc donor the "timing" of the transplant must be considered the major strategic factor for patients with hr al. the goal of this prospective study was to evaluate the impact of donor's and recipient's genetic polymorphisms regarding components of innate immunity on outcome of allohsct. 102 consecutive patients with hematological malignancies, aged 32(18-58)y, treated with allohsct from either sibling (n=34) or matched unrelated (n=68) donors were included. the conditioning was myeloablative. gvhd prophylaxis consisted of csa, mtx ± atg. donors and recipients were tested for single nucleotide polymorphisms(snp)8,12,13 of the nod2/card15 gene, tlr4(299), tlr4(399), tlr5(stop codon c1174t) and il23r(11209026), as well as kir genotype. in addition, immune reconstitution was studied. os rate at 2y was significantly lower in allohsct with at least one activating kir mismatch compared to transplants with full compatibility (62%vs.86%, p=.01). in particular, the presence activating killer immunoglobulin-like receptors (kir) in the donor with its absence in the recipient (d+r-) was associated with decreased rates of os (60%vs.78%, p=.01) and dfs (58%vs.82%, p=.005), as well as increased nrm (27%vs.7%, p=.01). kir2ds1 and kir3ds1 d+r-mismatches resulted in increased risk of grade ii-iv acute gvhd, while kir2ds3 and kir2ds2 d+r-mismatches were associated with increased risk of chronic gvhd. d+r-activating kir mismatches correlated with increased cd8+/cd4+ t cell ratio up to day +100. in all cases of incompatibility regarding kir2ds1, kir2ds2 and kir3ds1, t cells with expression of respective receptors could be detected up to 360 days after allohsct. the presence of snp8 of the nod2/card15 gene in the recipient was associated with decreased probability of os (20%vs.70%, p=.005) and dfs (20%vs.70%, p=.01) as well as increased nrm (60%vs.17%, p=.01) and grade iii-iv acute gvhd (67%vs.8%, p=0.02). in a multivariate analysis adjusted for other potential risk factors, increasing number of d+r-activating kir mismatches as a continues variable appeared to independently influence os, dfs, nrm, grade ii-iv acute gvhd, and chronic gvhd. recipient snp8 of nod2/card15 was predictive for os, dfs, nrm, grade iii-iv acute gvhd, and chronic gvhd. snps of tlr and il23r genes had no impact on outcome. conclusions: both activating kir d+r-mismatches and recipient snp8 of nod2/card15 appear to enhance alloreactivity and independently influence survival after allohsct. evaluation of these polymorphisms may contribute to better donor selection and optimization of the allohct procedure. the interpretation of the role of hla-dpb1 in unrelated haematopoietic stem cell transplantation is subject to discussion. we have investigated the role of hla-dpb1 allele matching on haematopoietic stem cell transplantation (hsct) outcomes in 161 recipients who were hla -a, b, c, drb1, dqb1 matched with their unrelated donors at the allelic level (10/10). additionally, we analysed the association of polymorphic amino acids mismatches of dpb1 molecule with hsct endpoints, and the permissiveness concept of zino and colleagues (zino et al., 2004) . dpb1 allele mismatches were significantly associated with an increased incidence of acute graft-versus-host disease (agvhd), transplant-related mortality (trm) and worse overall survival (os). we observed that the mismatch at amino acid position 69 increased the risk for both agvhd and trm. risk factors for agvhd also included mismatches at positions 8, 9, 35, 76 and 84 . this is, to our knowledge, the first report of an in vivo effect of single amino acid mismatches on hsct outcomes. in our study, grouping of allelic mismatches into permissive and nonpermissive categories and their association with transplantation endpoints proved relevant for trm but not for other clinical endpoints. g.f. torelli*, r. maggio, n. peragine, m.s. de propris, b. lucarelli, m.g. mascolo, m. screnci, s. salvatori, l. malandruccolo, a.p. iori, a. guarini, r. foà sapienza university (rome, it) studies in mouse models of stem cell transplant (sct) have shown that the infusion of culture-expanded regulatory t cells (tregs) can be effective in preventing and suppressing gvhd, while still allowing a gvl effect. cord blood (cb) stem cells are now broadly used in the unrelated sct setting and key: cord-004675-n8mlxe7p authors: nan title: 2019 cis annual meeting: immune deficiency & dysregulation north american conference date: 2019-02-26 journal: j clin immunol doi: 10.1007/s10875-019-00597-5 sha: doc_id: 4675 cord_uid: n8mlxe7p nan a 34 y.o. female was referred to our clinic with a history of multilineage cytopenias/evans syndrome, a history of idiopathic thrombocytopenic purpura, hemolytic anemia, chronic neutropenia, lymphopenia, and hypogammaglobulinemia treated with ivig. our patient was healthy until she was 8 years old; at that time, she developed joint pain, rash, and bruising. she was found to have evans syndrome with idiopathic thrombocytopenic purpura (itp), neutropenia, and lymphopenia. she was initially diagnosed with lupus and was given steroids. her bone marrow biopsy did not conclude myelokathesis. when she was 15 years old, she remained thrombopenic and was started on high dose of immunoglobulin replacement therapy. in 2012 (29 years old), she developed polyarthritis in her upper and lower extremities. in 2013 (30 years old), she had a severe nosebleed, for which she was admitted and treated with amicar twice; her platelets were found to be 2,000 k/ul. she received rituximab weekly for 4 weeks resulting in an increase of platelet count to 90-100k/ul. she recently (march 2017) had a splenectomy to remove her large spleen, and since then, her platelets have rebounded to 400-500k/ul. in 2015, she was placed on long-term immunoglobulin replacement therapy after being hospitalized for bilateral pneumonia for 5 nights requiring iv antibiotics for treatment. in 2017, she developed and was treated for another pneumonia. her family history is characterized by multiple members with autoimmune multilineage cytopenia as well as autoimmune diseases such as multiple sclerosis (mother), thyroiditis and enteropathy. on physical examination, she did not present with any warts and the remainder of her physical examination being unremarkable, except for her scar from her splenectomy and a cervical lymphadenopathy. immunologic evaluations showed igg 601 mg/dl, iga <5 mg/dl, and igm 208 mg/dl. cbc with differential and lymphocyte screen were as follows (cell/mm3): wbc 12.3 x103, hemoglobin 10.2 g/dl, platelets 503 x 103; 3 % neutrophils (anc: 300), 82% lymphocytes, 10% monocytes, 0% eosinophils; absolute total t-cell number was 8884 (750-2500 cells/mcl), cd4+ t-cells 6554 (480-1700cells/mcl), cd8+ t-cells 2185 (180-1000cells/mcl), natural killer cells 206 (135-525 cells/ mcl), and absolute number of b cells was 996 (75-375 cells/ mcl). she came to our clinic with her sister, who also had multilineage cytopenia and hypogammaglobulinemia, treated with monthly ivig; and her nephew whom had neutropenia. based on this family presentation all three underwent whole exome sequencing (wes). the patient, the patients sister and the patients nephew were all found to have a variant on cxcr4 (frameshift mutation on chromosome 2, p.val324fs; refnt: tca; altnt: t). as an important note, the patient had a bone marrow biopsy, which did not conclude myelokathesis. in summary, our patient with trilineage cytopenia and hypogammaglobulinemia, without any warts or myelokathexis, had whim syndrome (warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis), which was discovered by studying her wes. with the identification of her specific diagnosis, this allowed us to discuss the potential future indication of plerifaxor (antagonist of the alpha chemokine receptor cxcr4). and equally important, we discussed family planning and future pregnancies given that the mutation is autosomal-dominant. (4) submission id#555017 taha al-shaikhly, mbchb 1 , kathleen mohan, arnp 2 , matthew basiaga, do, msce 3 introduction: complement component-3 (c3) is shared by the classical, lectin and alternative complement activation pathways. c3, a major opsonin, facilitates phagocytosis of encapsulated microorganisms. inherited c3 deficiency is rare and is associated with increased risk of bacterial infections. subjects with connective tissue diseases (ctd) and c3 nephritic factors can have low and occasionally undetectable c3 levels, yet they are at an underappreciated infectious risk. we hypothesize that excessive c3 consumption in secondary complement deficiency disorders (scd) is associated with higher risk of bacterial infections similar to primary complement deficiency disorders (pcd). objectives: to compare the rate of bacterial infections between pcd and scd patients and evaluate the association between c3 level and bacterial infection risk. methods: we performed a retrospective cohort study. subjects with an undetectable complement activity (ch50) or any of the complement components measured at seattle childrens hospital from 2002-2018 were included in our study. we recorded the number of infections, observation periods, diagnosis (pcd, scd and its underlying etiology), lowest complement component levels, and the immunosuppressive agents used. the date of birth, and date of lowest c3 level were considered as start points to calculate the observation periods for pcd and scd subjects respectively. infections requiring hospitalization or parenteral antibiotics were categorized as serious bacterial infections (sbis). descriptive analyses were performed to determine medians and ranges for continuous variables. differences in rates of bacterial infection were assessed using the chi-square and kruskal-wallis tests when appropriate. among subjects with ctds, we treated every c3 measurement as a single observation (n=1,197) and studied the association between c3 concentration and the 30-day odds of having a sbi. multivariable logistic regression was performed to determine infection risk based on c3 level while controlling for contributing factors. results: we identified 14 subjects with pcd, and 52 subjects with scd. scd consisted of three subgroups (ctd-related (n=44), nephritic factor-related (n=2), and infection-related (n=6)). collectively, ctd subjects had a lower median rate of sbi compared to pcd subjects (p = 0.004). subjects with ctd and c3 level <40 have higher rate of bacterial infection (of any severity) (p = 0.002) and of sbi (p = 0.004) when compared to ctd subjects with c3 >=40 at the beginning of observation period ( figure 1 ). while controlling for immunosuppression level 1 pediatric resident, baystate medical center 2 faculty advisor, baystate medical center introduction: zap70 codes for a 619-amino acid enzyme, zap70, a member of the syk-protein tyrosine kinase family that plays an important role in t cell development and activation. zap70 is phosphorylated at tyrosine kinase residues upon t cell receptor (tcr) stimulation resulting in tcr-mediated signal transduction with src family kinases. zap70 deficiency results in a rare t+b+ nk+ severe combined immunodeficiency (scid). we report a novel compound heterozygous mutation in zap70 leading to presumed absent zap70 function in an infant with a normal trec newborn screen and scid. case description: the patient is a term, fully immunized female, born to non-consanguineous parents who was hospitalized for rsv bronchiolitis at 2 mo. at 4 mo she developed an erythematous, papular rash on her face and extremities, nonresponsive to topical antifungal therapy. at 6 mo she was re-hospitalized with rsv bronchiolitis and subsequently treated with multiple courses of antibiotics for presumed bacterial pneumonia followed by albuterol and oral steroids for possible reactive airways disease. during this course of treatment, her rash resolved. at 8 mo she presented with failure to thrive (wt <0.1% for age), multifocal pneumonia and respiratory failure requiring intubation. bronchial alveolar lavage confirmed pneumocystis jiroveci pneumonia prompting an immune evaluation. total immunoglobulins were normal for age, however antibody titers to tetanus, diphtheria and streptococcus pneumoniae were absent. lymphocyte enumeration revealed elevated cd4 t cells and markedly diminished cd8 t cells, normal b and nk cells. t cell proliferation to mitogens (pha, pwm) and antigens (candida, tetanus) was absent, however t cells proliferated normally to stimulation with pma and ionomycin. trec number was normal by newborn screening, but was 2 std deviations below the mean and would have resulted in a positive screen upon repeat. invitae 18 gene scid panel revealed two variants of unknown significance, c.109c>g (p.arg37gly) leading to substitution of arg with gly and c.1529_1532dupgcat (p.ile511metfs*65) resulting in a premature translational stop signal expected to disrupt the last 109 amino acids of zap70 protein. parental sequencing revealed these variants to be on opposite chromosomes. the patient was successfully treated for pjp pneumonia and has since successfully engrafted a 9/10 matched unrelated donor stem cell transplant. discussion: we report a novel compound heterozygous mutation in zap70 which we presume led to t+ b+ nk+ scid. our patients clinical presentation of failure to thrive, recurrent lower respiratory tract infections, dermatologic findings and pjp pneumonia are consistent with previously reported cases of zap70 scid. her paucity of cd8 t cells, abundance of cd4 t cells and absent proliferation to mitogens are also consistent with previously described cases of zap70. normal proliferation of t cells when bypassing the tcr by stimulating cells with ionomycin and pma confirms a defect in the tcr. we believe this is the second documented case of missed scid by newborn screen in ma since the implementation of trec screening in 2008. pediatric resident (pgy iii), goryeb children's hospital 2 attending physician, pediatric and adult asthma, allergy and immunology, llc introduction: acute otitis media (aom) is one of the most common reasons for antibiotic use in early childhood. we explored the challenges when aom fails traditional therapies and immunologic evaluation does not identify a commonly described immunodeficiency. case description: an eighteen-month-old male presented with 12 episodes of aom and recurrent purulent otorrhea requiring intravenous antibiotics. laboratory evaluation revealed a normal cbc, normal immunoglobulins (igg 588, iga 76, igm 63, ige 12) and igg subclasses. lymphocyte subset panel was normal. initial responses to dtap and prevnar boosters were normal, however, there was rapid decline to tetanus and pneumococcal antibody titers. a sub optimal response to haemophilus influenza type b vaccine was noted. although vaccinated twice for mmr, he never mounted mumps specific igg. mitogen response to pha was normal with decreased responses to cona and pokeweed and no detectable tetanus nor candida responses. further investigation revealed decreased non-class and class switched memory b-cells. the patient was recently vaccinated to pcv23 and at the present time has protective titers. discussion: it has been previously suggested that decreased memory b cells may contribute to decreased antibody responses to select vaccine antigens resulting in recurrent aom in children. our case supports the need to investigate beyond typical immunologic screening for immunodeficiencies. introduction: dna mismatch repair (mmr) system corrects replication errors in newly synthesized dna, and prevent recombination between dna sequences when they were not identical (1) . msh6 is a part of mmr genes, (2) (3) (4) . case: a ten-year-old girl presented with fever, brown spots on her skin, hair loss, recurrent pulmonary infections, arthritis on the left hand and right ankle. she has also been followed up with nf ( figure 1 ). there was a first-degree cousin marriage between her parents. physical examination revealed findings of pneumonia and nf. anti-nuclear antibody, anti-ndna, anti-dsdna, anti-histone, anti ro52 and anti-nucleosome antibodies were positive. in her immunologic assessment showed low igg and iga levels associated with high igm level ( table 1 ). the coexistence of nf, hyper igm syndrome, sle, were considered in the patient. intravenous ig (400 mg/kg, every 3 weeks) treatment was started due to hypogammaglobinemia. the frame shift mutation in exon 2 of the msh6 gene was detected in the boztug's laboratory. in the follow up period, she admitted at 11 years old with back pain. a mass in the left paravertebral area, related to the spinal canal and neural foramina, was detected at the l4-l5 levels in spinal mri. the lymphadenopathy around the liver and hilum and the left parietal bone lesions were developed within two months despite surgical excision of primary mass ( figure 2 ). as a result of pet examination; suvmax was found to be around 6.5 in the mass lesion in the paravertebral region and suvmax values did not exceed 2.5 in other lymphadenopathy and masses. atypical cellular infiltration suggesting neoplastic events, which were including small-medium size atypical pleomorphic mononuclear cells and t cells. since all these formations did not indicate definite cancer, chemotherapy was not started. interestingly, although chemotherapy was not given, progression stopped, and partial spontaneous regression was observed. discussion: the effect of msh6 mutations on patients may significantly vary with the inheritance pattern (2) . leukemias or lymphomas are not common in heterozygote mmr gene defects (5, 6) . however, homozygote mutations in mmr genes show a different pattern. wimmer and etzler proposed the new term constitutional mismatch repair-deficiency syndrome (cmmr-d) for patients who have a homozygous mutation in mmr (3) . cmmr-d characterized by development of childhood cancers, mainly hematological malignancies and/or brain tumors, as well as early-onset colorectal cancers, and neurofibromatosis type 1 (3) . bi-allelic germline mutations in any of the mmr genes in which msh6 is involved increases hematological malignancies by 15% (7, 8) . msh6 mutation has been associated with many cancers since its identification. leukemia, lymphoma, colorectal cancer, endometrial cancer, brain tumors are some of these cancer types (2) (3) (4) 9) . msh6 deficiency is an important disease that can affect different systems at the same time. there is a high risk of malignancy in the cases and therefore they must be closely monitored. this case has also shown that atypical lymphoproliferation may occur in msh6 homozygous mutant cases. (normal rage: 842-1943) background: advances in inborn errors of human immunity have supported the discovery of new syndromes that are marked by striking features of autoimmunity and immune dysregulation often associated with cytopenias, lymphoproliferation, and a predisposition to reticuloendothelial malignancies leading to evaluation with hematologists/oncologists. moreover, hematologists/oncologists have also seen an increasing use of effector cell-based therapies, checkpoint inhibitors, immunomodulatory and targeted therapies resulting in autoimmunity and hyperinflammatory complications. a working knowledge of clinical immunology could help practicing hematologists/oncologists in the identification and management of these conditions. objectives: to support the advancement of aspho members and the field by facilitating education regarding the best practices in diagnosis and management of immunological disorders. to create a platform for the development of collaborative clinical research in patients with hematological/oncological manifestations of immunological disorders or those requiring hematopoietic stem cell transplantation for a underlying immunological disorder. design/methods the aspho clinical immunology sig was initiated based on collaboration with the clinical immunology society (cis). aspho members who are pediatric hematology/ oncology clinicians, clinical researchers, and trainees are eligible to participate. we have established a steering committee with representatives from across the united states and canada with diverse clinical and research expertise. through regular teleconferences and annual in-person meetings, we have developed a platform to provide our members with a network of immunology resources to ensure a strong foundation of knowledge and tools to conduct clinical care and research pertaining to the diagnosis, evaluation, and treatment of patients with immunological disorders. results we currently support over 50 members within our online community. several educational initiatives have been successfully launched. we have submitted an invited review to pediatric blood and cancer which provides a case-based review of primary immune regulatory disorders. we hosted the first immunology for hematology oncology practice (i-hop) cased-based webinar series. this series features case-based discussions of patients with primary immunodeficiency disorders presented by fellow trainees and mentored by senior clinicians. we will also be hosting an aspho webinar focusing on the laboratory evaluation of primary immunodeficiencies and immune dysregulation syndromes. we have also begun the process of laying the groundwork for clinical research initiatives. conclusion: the aspho clinical immunology sig seeks to serve as a collaborative resource for pediatric hematology/oncology clinicians and researchers. through the development of educational and research initiatives, we envision improving the care of patients with immunological disorders that are often managed by pediatric hematologists/oncologists. moreover, we hope to broaden our understanding and application of clinical immunology within pediatric hematology/oncology. we hope that this successful initiative will serve as a blueprint for the development of future collaborations with other specialty societies and patient groups. autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced) is a rare autosomal recessive disease caused by aire gene mutations. clinical diagnosis is established by the presence of at least two components of the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism, and addisons disease. in europe, the classic presentation is widely recognized and nonendocrine autoimmune manifestations are rarely reported. a recent study of 35 american apeced patients demonstrated a more heterologous presentation, with many nonendocrine manifestations including urticarial eruption, hepatitis, gastritis, intestinal dysfunction, pneumonitis and sjogrens-like syndrome, all uncommon in european reports. within the american cohort, 80% of patients developed a mean of three non-triad manifestations before reaching the classic triad. finding of aire mutations and high-titer antiifn-autoantibodies is seen in both european and american cohorts. we present the case of two siblings, who demonstrate an apeced-like phenotype with both classical and atypical features. they share the same heterozygous c132+1_132+3delinsct aire mutation. the older, an eight-year-old boy, with history of prematurity, bronchopulmonary dysplasia and onychomadesis in infancy, came to medical attention at 16 months of age due to failure to thrive (ftt), in addition to fevers and urticarial rash lasting months after his mmr vaccine. the fevers resolved with anakinra, which was discontinued two years later due to pneumonia. from age 2-4 he developed an alps negative lymphadenopathy which self-resolved. lung issues include chronic cough, initially treated as asthma but with poor bronchodilator response, and frequent lung infections, including 1-2 pneumonias per year. at age five evaluation for ftt revealed growth hormone deficiency. two years later he was diagnosed with primary addisons disease. chronic abdominal discomfort, bloating, cyclical constipation/diarrhea, recurrent rashes, dystrophic nails, and sicca symptoms are also present. his sister, age five, shows ftt, but no growth hormone deficiency. at age one, she too developed a fever and rash syndrome lasting 3 months. severe gerd and constipation started in infancy and are ongoing. at age three she developed a transaminitis, initially diagnosed as ebv, but later thought to be autoimmune hepatitis. she has frequent viral respiratory infections, and pneumonia at age two. she has had a chronic cough, with poor bronchodilator response, for most of her life. evaluation of seizure at age three showed normal brain activity. brain mri revealed partial agenesis of the corpus callosum and microgyria. her brother has similar mri findings. both children have had developmental motor delay and poor tone. brain dysgenesis and neurodevelopmental delay has not previously been described in apeced. although there were both typical and atypical symptoms, the history in combination with genetic findings led to further investigation of an apeced-like syndrome. autoantibody testing confirmed high-titer antiifn-autoantibody typical of apeced in both children and hightiter bpifb1 autoantibodies found almost exclusively in apeced pneumonitis in the brother. whole exome sequencing and copy number variation analyses are underway to further evaluate the patients condition. this case demonstrates the importance of clinical presentation in the evaluation of genetic results and in the guidance of therapeutic management. (12) submission id#570047 rationale: infants with low t cell receptor excision circles (trec) born in queens, nassau, and suffolk counties are referred to our center for further evaluation. this study elucidates the demographic and laboratory characteristics of referred infants with transient or persistent idiopathic t cell lymphopenia (tcl) without clearly identified genetic or acquired etiology. methods: a retrospective analysis was performed from september 2010 (when trec screening started) through the end of december 2017. descriptive statistics were calculated for demographic and laboratory characteristics. t-test or mann-whitney tests were used to compare laboratory variables. pearson or spearman tests were used to determine correlation between initial trec levels and t cell counts. by definition, the cd3+, cd4+, and cd8+ populations of transient tcl patients normalize by age 1 year. results: eighteen infants with transient and 17 with persistent tcl were identified. males comprised 61.1% of the transient and 47.1% of the persistent tcl cohorts. whites comprised 11.1% of the transient and 35.3% of the persistent tcl cohorts. the mean initial trec levels did not differ between the transient and persistent cohorts (67.7 vs. 78.5 trecs/l of blood, p = 0.56). mean initial absolute counts of cd3+ (2149 vs. 1300 cells/l, p <0.0001), cd4+ (1462 vs. 922 cells/l, p <0.0001), and median initial absolute counts of cd8+ (524 vs. 309 cells/l, p = 0.0075), were higher for transient vs persistent cohorts. initial trec level did not correlate with initial cd3+, cd4+, or cd8+ absolute counts. the median age of resolution for the transient cohort was 121.5 days (range 23-244). the absolute cd3+, cd4+, or cd8+ counts rarely exceeded the reported median values for age, and remained closer or below the 5th percentile for age up to 1000 days of life. the majority of both transient and persistent tcl patients demonstrated unremarkable lymphocyte proliferation to mitogens. conclusion: our centers transient tcl cohort appears to be predominantly male and non-white, whereas the persistent tcl cohort is more evenly distributed by sex but still predominantly non-white. the transient cohort had lower initial trec levels, but higher initial t cell counts. both cohorts appear to have relatively intact in vitro function. introduction: primary immune deficiency disease (pidd) is typically considered a pediatric illness, although advances in treatment and diagnosis are changing this paradigm. currently, data on pidd in older patients are very limited. objectives: to characterize the prevalence of pidd among older individuals using a patient database maintained by the consortium of independent immunology clinics (ciic), comprised of 17 specialty immunology outpatient practices in the us. methods: patients with pidd were identified in the ciic database using icd-10 codes d80, d.80. 3, d80.4, d80.5, d80.6, d81.1, d81.2, d82.0, d82.3, and d83 .0. a total of 235 records from 11 geographically-diverse clinics were identified and characterized by age, gender, and pidd diagnosis. results: of the 235 pidd patients in the ciic registry, 73 (31%) were between 60-87 years of age (see figure) . within this age group, most patients were female (n=56, 77%). the most common diagnoses among patients >60 years of age included common variable immunodeficiency with predominant abnormalities of b-cell numbers and function (d83.0; n=41, 56%) and antibody deficiency with near normal immunoglobulins (d80. 6; n=14, 19%) . in comparison, the registry included 36 (15%) patients aged 0-19 years; this age group was predominantly male (n=23; 64%). the most common icd-10 codes within the younger cohort were relatively evenly distributed between hereditary hypogammaglobulinemia (d80.0), antibody deficiency with near normal immunoglobulins (d80. 6) , and common variable immunodeficiency with predominant abnormalities of b-cell numbers and function (d83.0). conclusions: our data suggest that pidd in patients over age 60 may be more prevalent than previously reported. additional research is needed to corroborate these findings, further characterize the nature of pidd in this population, and determine whether there are unique diagnostic and treatment considerations within this demographic. introduction/background: increased susceptibility to invasive infections with neisseria has been well documented in patients with deficiency of terminal complement proteins. the molecular attack complex is constructed with complement components c5 to c9. a deficiency in complement c6 has been described previously in both african american and south african populations. complement c6 deficiency is inherited in a co-dominant pattern, with multiple known mutations. we present a case of a 19-year-old, previously healthy male, who presented with invasive n. meningitides infection. he was found to have a novel mutation noted on genetic sequencing of the complement c6 gene. objective: we present the case of a 19-year-old, previously healthy male, who presented with invasive n. meningitides infection. on genetic sequencing, he was found to have three mutations of the complement c6 gene. two of which have been described previously, and a third novel mutation. methods: a 19-year-old male with no known history presented to us with a 3-hour history of emesis. he was found to be febrile, and quickly decompensated, developing septic shock. blood cultures were drawn, and within 12 hours grew n. meningitides. he was treated with broad spectrum antibiotics upon arrival, and subsequently narrowed to ceftriaxone. his hospital course was complicated by disseminated intravascular coagulation, as well as acute tubular necrosis, leading to endstage renal disease for which he is listed for kidney transplant. results: on immunodeficiency evaluation, he was noted to have an undetectable ch50 (<13, reference range 31-60). complement levels returned with c6 of 10.8 (reference range 28-69) and c1r of 41.5% (reference range 61-102%). complement c6 function screen returned at 0% (reference range 40.7-169%). all other complement levels were within normal limits. genetic sequencing showed the patient to be compound heterozygous for two of known four variants which have been reported to recur in african patients with complement c6 deficiency. this included c.821del and c.1879del, which are predicted to result in frameshift and premature protein termination. he was also found to be heterozygous for sequence c1202g>a, which results in amino acid substitution p.arg401lys. this variant is rare, with one large database reporting it in 6 of 276000 alleles, and not in a homozygous state. it has not been reported in a case of c6 complement deficiency previously. conclusions: we present the case of a previously healthy 19-year-old male with invasive meningococcal disease. he is compound heterozygous for two mutations that have been associated with total complement c6 deficiency; however, he was found to have subtotal c6 deficiency. furthermore, he has a third novel mutation of the complement c6 gene. further investigation is warranted on the significance of this finding and impact on relevance to possible kidney transplant. background: measuring the function of the classical pathway of complement activation is useful in several disease states, including complement deficiency, autoimmune conditions such as systemic lupus erythematosus and certain forms of nephritis. the original method for assessing classical pathway activity was the haemolytic ch50 method, but this assay can be time consuming and has reagent stability issues due to the use of sheep red blood cells. there can also be high lab-to-lab variability due to differences in the protocols used. here we report the assay characteristics of an automated, commercial, liposome-based assay to measure ch50 activity. we also compare the results obtained using the traditional haemolytic method with the automated, liposome-based method used on the spaplus turbidimetric analyser. methods: a linearity study was performed based on clsi guideline ep06-a. the linear range of the spaplus ch50 liposome assay was established by analysis of a series of sample dilutions and evaluation of results against pre-defined goals for recovery and %cv. precision was assessed based on clsi guideline ep05-a2 over 21 days. 4 samples with different ch50 activities (23.7-65.1 u/ml) were run in duplicate, with two runs per day using 3 reagent lots and 3 different analysers. interference analysis was performed by spiking haemoglobin, bilirubin, chyle, ascorbic acid or saline (as a control) into samples before measuring the ch50 activity. for the assay comparison study, sera from 125 routine patient samples were used. samples were collected from chulalongkorn hospital, faculty of medicine, chulalongkorn university, thailand. ch50 classical pathway activity was assessed using a haemolytic method and also using the liposome based ch50 assay for use on the spaplus turbidimetric analyser (the binding site ltd., birmingham, uk). c3 protein concentrations were also available for 116 of these samples. results: the liposome ch50 assay gives a linear response over the range 11.8-95.5 u/ml, covering the measuring range of the assay (12.0-95.0 u/ml) at the standard analyser dilution (neat). the within run, between run and between day %cvs were all 5.4%. the total %cv was 6.8% in all 4 samples. minimal interference was observed with the four common interferents tested. a significant correlation was observed between the two ch50 methods (p<0.0001, r=0.66, y=1.1xâ±0.1), with 90.4% agreement between the methods in determining whether patients were above or below the lower limit of the assay normal range. the 12 individuals in disagreement had normal ch50 results using the haemolytic method, and low ch50 values in the liposome assay. of these, c3 values were available for 10/12, and 5 had c3 concentrations below the lower limit of the assay normal range. conclusion: the liposome ch50 assay for use on the spaplus analyser has passed assay development guidelines based on those set out by the clsi for linearity, precision and interference, and there is a strong correlation between this automated assay and the haemolytic ch50 method used here. five additional patients with low c3 concentrations were defined as having a low ch50 using the spaplus liposome method compared to the haemolytic method. (18) submission id#580179 background/aims: rotavirus vaccine is a live viral vaccine that is part of the routine u.s. childhood immunization schedule. live viral vaccines administered to infants of mothers who received biologic medications during pregnancy can potentially cause vaccine-associated disease. infant death from disseminated mycobacterial infection after vaccination with bacille calmette-guerin (bcg) in infants whose mothers received infliximab during pregnancy has been reported. it is currently recommended that infants born to women who received biologic therapy during pregnancy not receive live viral vaccines, however there is a paucity of information regarding adverse events from live viral vaccines. we report two infants, born to mothers receiving infliximab during pregnancy, who tolerated the complete series of rotavirus vaccine. methods: two infants who received rotavirus vaccine and whose mothers received infliximab (monoclonal antibody against tumor necrosis factor alpha which blocks the inflammatory response) during pregnancy were identified and their charts were reviewed. each mothers chart was assessed for timing of the biologic doses during pregnancy and concurrent immunosuppressant therapy. results: the mother of the first infant had crohn's disease and received infliximab every 6 weeks throughout her pregnancy (final infusion at approximately 35 weeks estimated gestational age [ega] ). she did not take additional immunosuppressive drugs throughout her pregnancy. the infant was born at 39 weeks ega. the infant received rotavirus vaccine at 2, 4, and 6 months of age. the infant did not have coexisting medical conditions or recorded hospitalizations during the first year of life. there were no side effects from rotavirus vaccine documented during well child examinations. the childs growth was normal during the first year of life. the mother of the second infant also had crohn's disease and received infliximab infusions every six weeks during pregnancy until 27 weeks ega. additionally, she took mesalamine (anti-inflammatory) daily. the infant was born at 33 weeks ega. the baby had a brief and uncomplicated neonatal intensive care unit stay. she did not have medical conditions diagnosed at the time of birth, or in the first year of life. the child received rotavirus vaccination at 2, 4, and 6 months of chronological age, and the infant did not experience documented adverse reactions. the child presented to the emergency department twice in the first year of life: once for thrush at 10 months of age and once for viral gastroenteritis at 11 months of age. the childs growth curve was unremarkable. conclusions: we report two infants, whose mothers received infliximab during pregnancy, who safely tolerated the 3-dose series of rotavirus vaccination. neither infant in this case series suffered from minor or severe adverse events as a direct consequence of receiving rotavirus vaccine. this suggests that administration of rotavirus vaccine may be safe in infants whose mothers received biologic therapy. introduction: combined immunodeficiencies (cids) can arise from partial loss of function variants in recognized scid genes, which can lead to relative lymphopenia with poorly functioning and oligoclonal t cells. cids have been most commonly associated with variants of the rag genes, but other genes are also implicated. clinical symptoms may be less severe, and the onset generally is delayed, compared to typical scid presentations. case report: a 28-year-old female presented with a history of recurrent and progressively worsening infections involving multiple microorganisms and organs, starting in infancy and requiring frequent hospitalizations. bacterial or viral infections included rhinosinusitis, otitis media, herpetic stomatitis, dental abscesses, pneumonias, pulmonary mycobacterial abscesses, cmv hepatitis, urinary tract infections, dermal abscesses, and groin hidradenitis. fungal and yeast infections included cryptococcal meningitis, oral thrush, dermatophytosis of the face, osteomyelitis of a finger, and onychomycosis. laboratory tests in 2018 showed: mildly low t cell counts (791/ul) with a reversed ratio of cd4/cd8 t cells (0.22); almost absent b cells (2/ul) ; and low nk cell counts (19/ul). cd4+ t cells were mostly of the memory phenotype (87%). t cell development showed low counts of th17 cells. t-cell stimulation tests demonstrated poor proliferation responses (<30%) to concanavalin a, tetanus toxoid, and candida albicans, with near-normal responses to pokeweed (>13%) and pha (>84%). she had low ig levels (iga 72, igm 23, ige <2), except for igg (872mg /ml; due to replacement since early childhood). limited genetic evaluation at age 9 showed a heterozygous variant in the rag1 gene (g.36595918t>c, c.1064t>c, p.met355thr; nm_000448.2). discussion: loss of function variants in rag1 or rag2 genes are known to cause a t-b-nk+ type scid. more than 100 missense variants have been reported for rag1, with disease-associated variants predominantly in zinc binding regions. the rag1 missense variant in our patient also lies within the zinc binding region (amino acids 354-383). the variant is rare (mean allele frequency 0.0001521 in gnomad) and has been identified in at least one other individual with scid (t-, b cell-, nk+). although classified as a variant of unknown significance, occurrence in at least two individuals with deficiencies of t and b cells-within a functionally important rag1 domainsupports an interpretation that the variant may be pathogenic. most patients with cid with rag variants are either homozygous for a poorly functional allele or have one nonunfucitonal and a second, poorly functional allele. we detected only a single potentially pathogenic allele. our patient has decreased nk cells in addition to t and b cell defects. further genetic studies including whole exome sequencing, are planned to identify further variants in rag1 or other relevant genes. rationale: infants with low t cell receptor excision circles (trec) born in queens, nassau, and suffolk counties in new york were referred to northwell health for further evaluation after abnormal newborn screens. the demographic and immune parameters of infants with transient t cell lymphopenia (ttcl) without clearly identified genetic or acquired etiology are described. tcl is considered transient if the lymphopenia resolves by 12 months of age. similar data from the following infants with low lymphocytes (fill) program of the united states immunodeficiency network (usidnet) are presented. methods: a retrospective analysis of two separate patient cohorts with ttcl are described. cohorts include patients referred to a single center, northwell health, in ny from september 2010 to december 2017 and at usidnet using data tracked by fill from june 2011 to july 2018. results: out of 1,234 referrals at northwell, 18 infants with ttcl were identified. infants were predominantly male (61.1%) and non-caucasian (89.9%). out of 71 fill participants, 9 infants with ttcl were identified. infants were predominantly male (55.6%) and non-caucasian (55.6%). initial laboratory parameters for the northwell versus fill cohorts are summarized: a) median trec levels: 54.5 vs. 47.0 trec/l of blood; b) median absolute cd3+ count: 2135 vs. 1166 cells/l; c) median cd4+ count: 1460 vs. 777.0 cells/l; d) median absolute cd8+ count: 524.5 vs. 440.0 cells/l. initial naã¯ve cd4+ t cell information was available for 0 northwell and 5 fill infants (median 52%). mitogen proliferation studies were performed in 10 (55.6%) northwell and 6 (66.7%) fill infants with 90% of these northwell and 50% of these fill infants demonstrating normal proliferation. genetic testing, such as targeted genetic panels or chromosomal microarrays (cma), was performed in 5 northwell and 0 fill infants. no genetic or chromosomal aberrations were identified. whole exome sequencing (wes) was not performed in either cohort. 11 of 18 (61.1%) northwell and 7 of 9 (77.8%) fill infants did not receive the initial rotavirus vaccine. no fill infants were vaccinated but no adverse effects were reported in 5 of 18 (27.8%) northwell infants who received the first rotavirus dose. of these, 3 of 5 (60.0%) had normal mitogen proliferation while 1 (20.0%) had decreased proliferation to phytohemagglutinin. conclusions: identifying biomarkers for ttcl and developing evidencebased guidelines for the diagnosis and management of ttcl are important knowledge gaps. this descriptive study is limited by small sample size and the constraints of registry-based research. although there appear to be differences between these cohorts, our findings suggest that ttcl may disproportionately affect different segments of the population. ttcl infants with normal mitogen proliferation may be able to tolerate rotavirus vaccination. thus, routinely checking proliferation studies in all ttcl infants may help risk stratify these patients and minimize vaccinerelated adverse events. currently, there is insufficient evidence to recommend more extensive genetic testing such as genetic panels, cma, or wes. systematically collecting information about patient characteristics and outcomes, as well as encouraging increased participation in registries such as fill, may help address these shortcomings. background: systemic lupus erythematosus (sle) is a chronic, inflammatory disease that affects multiple organs. the measurement of anti-dsdna antibodies (abs) is a gold standard serological test used in the diagnosis and monitoring of sle, with higher serum levels associated with worse prognosis. however, not all anti-dsdna abs are pathogenic, and some patients have consistently high levels with low disease activity. one mechanism suggested for the pathogenicity of these antibodies is complement activation. here we describe an assay to measure the c1q binding activities of anti-dsdna abs in sle patients. materials & methods: the concentration of anti-dsdna abs was determined using the quantalite dsdna elisa kit (inova) as per the manufacturers instructions. in order to determine the c1q binding capacity of bound abs, samples were added to the pre-coated plate and incubated. bound anti-dsdna ab/c1q complexes were then detected using a biotinylated anti-c1q antibody (570 ng/ml) and streptavidin peroxidase (1 mg/ml). normal reference ranges were developed in serum samples from healthy controls, and upper limits of these normal ranges were used as cut-offs. the dsdna abs and c1q binding capacity of bound abs was then assessed in 49 sle patients, and compared to other markers and the sle disease activity index (sledai) score. results are displayed as absorbance at 450nm (au). results and conclusions: the 95th percentile ranges for anti-dsdna abs (0.068-0.137 au) and c1q binding activities (0.207-0.313 au) were developed from the measurements generated in 17 healthy serum samples. sle patients with an increased anti dsdna ab concentration (>0.137 au) were then separated into those with low (<0.313 au) and high (>0.313 au) c1q binding activities. patients whose dsdna abs had high c1q binding activity were found to have significantly higher sledai scores (mean 6.70 vs 3.19) . serum c1q concentration, serum dsdna abs (measured by another method) and serum c3 and c4 concentrations were not significantly different between the two groups. this assay suggests that dsdna abs from sle patients differ in their ability to bind complement, and that high complement binding activity of these antibodies may be linked to a more active form of disease. x-linked lymphoproliferative (xlp) is a primary immunodeficiency, caused by signaling lymphocyte activation molecule (slam)-associated protein (sap) deficiency. patients with xlp have severe immune dysregulation, usually triggered by ebv infection, leading to fulminant infectious mononucleosis, dysgammaglobulinemia and lymphoproliferation. without hematopoietic stem cell transplant (hsct) fatality is reportedly 100% by age 40. we report the natural history of xlp in a patient, and describe the lessons learned. our patient was healthy and developed normally until 6-years of age, when he developed progressive respiratory symptoms. lung biopsy revealed mature lymphoplasmacytic infiltrate in the alveolar septa, consistent with lymphoid interstitial pneumonia (lip). he received corticosteroids and cyclophosphamide with significant improvement. at age 12, he developed severe infectious mononucleosis (fever, hepatosplenomegaly, lymphadenopathy, lymphocytosis). he had a protracted clinical course, but eventually recovered and seroconverted to a typical convalescent pattern. he subsequently developed hypogammaglobulinemia, and was started on intravenous immunoglobulin (ivig). during the same year, his 10-year-old brother developed lip, and subsequently hemophagocytic lymphohistocytosis (hlh) and died within 4 months from overwhelming candidiasis. unfortunately, his youngest brother (age 7) then developed lip and died 2 months later from a massive gastrointestinal bleed. both siblings were treated with corticosteroids and cyclophosphamide; they did not have detectable ebv infection. at age 13 years, our patient experienced recurrent strokes and was found to have biopsy-proven cns vasculitis. he was treated with interferon-and recovered with residual left sided weakness, but was lost to follow-up. he continued on ivig, with no other immunomodulatory agents for several decades. he had progressive lung disease and recurrent seizures controlled with anti-epileptics. at age 43, he developed sudden vision change, headache and right-sided weakness, followed by a seizure. mri of the brain revealed small bilateral areas of acute infarction suggestive of a central embolic event, however, no primary thrombus was identified. he did not receive any immunosuppression but was anti-coagulated. eventually he was discharged home with resolution of weakness to his baseline. the patient was referred to our clinic after discharge and we re-evaluated him after 31 years. immune profiles at the time showed therapeutic igg troughs, low/undetectable igm/a/e, normal t/b/nk-cell counts, normal spontaneous, but decreased antibody-dependent nk cytotoxicity, 0% sap protein expression (on cd3+cd8+, cd3-cd56+ and cd3+ cd56+ cells), and deletion on the x chromosome encompassing the sh2d1a gene which encodes sap. his mother was a carrier of the same deletion. his functional status excluded the option of hsct. a year later, he had rapid deterioration with recurrent lung infections, liver failure, and thrombocytopenia. bone marrow biopsy revealed hodgkins lymphoma. he declined chemotherapy and died few days after diagnosis. our case represents a rare patient with xlp surviving to the fifth decade without hsct, particularly having experienced mononucleosis and non-ebv related cns vasculitis. our patient survived decades longer than his brothers (who most likely shared the same genetic defect) without evidence of somatic reversion (0% sap expression in cd3+cd8+) to explain his milder clinical phenotype. this case may help in understanding the natural history of xlp, and confirms that prognosis remains poor without hsct. haematology and oncology, chu de quã©bec ctla-4 is a major negative regulator of immune responses, and ctla-4 haploinsufficiency has been identified as a monogenic cause of primary immunodeficiency in patients presenting with a common variable immunodeficiency (cvid) phenotype with autoimmunity. here we present the case of pb, a 40-year-old man who had been followed by the immunology service of our center for 17 years. a diagnosis of cvid had first been made when the patient presented with atypical transverse myelitis, low immunoglobulin levels, and lymphopenia. over the years, his clinical picture was dominated by various forms of autoimmunity, namely inflammatory demyelinating disorder of the central nervous system, autoimmune haemolytic anemia, immune thrombocytopenia, cryptogenic organizing pneumonia, rheumatoidlike polyarthritis, chronic liver transaminitis with biopsy-proven moderate fibrosis, and lymphocytic colitis with malabsorption. immunoglobulin replacement therapy was started at diagnosis, and autoimmunity was sequentially treated with methotrexate, interferon beta 1-a, cyclophosphamide, mycophenolate mofetil, rituximab, and finally a combination of low-dose prednisone and sirolimus, with stabilization of his neurological condition, the most debilitating complication of his immune dysregulation syndrome. bone marrow transplant had been offered, but declined by the patient due to perceived good quality of life compared to transplant-associated risks. the patient was later referred to our hematology ward in july of 2018 for septic shock complicating febrile neutropenia, which was part of a twomonth, gradual-onset pancytopenia. the diagnosis of immune-mediated aplastic anemia soon became apparent, as demonstrated by a bone marrow biopsy performed in a peripheral center two days prior to admission. the underlying pneumonia and thereafter biopsy-induced staphylococcus aureus iliac osteomyelitis and soft-tissue abscess were treated with broad-spectrum antibiotics as well as multiple surgical interventions. the patient was started on eltrombopag, high-dose corticosteroids and cyclosporin a, the latter promptly switched to tacrolimus due to liver enzymes disturbances, all of which resulted in no significant hematologic response despite over seven weeks of treatment (with concurrent treatment of complicating infection, upper gastrointestinal bleeding, and intensive-care-unite myopathy). during that time, genetic confirmation of ctla-4 haploinsufficiency was received, and the patient was thereafter started on abatacept on day 48 of current hospitalization. administration of equine anti-thymocyte was initially foregone because of perceived infectious risk in the setting of poor iliac wound healing and superimposed adenovirus viremia; however, given the lack of response, it was given on days 52 through 54 of hospitalization. haematologic response began on day 67 of hospitalization with a steady rise in alllineage myelopoiesis up to a complete neutrophil response, platelet near-complete response as well as resolution of transfusion needs by day 101. while waiting for a well-matched bone marrow donor, isolated platelet decrease was observed and attributed to multiple factors, including low-grade thrombotic microangiopathy, inflammatory consumption and drug-related thrombocytopenia, but the patient remained well. to our knowledge, our patients presentation is one of the most severe manifestation of ctla-4 haploinsufficiency to have responded to targeted therapy with abatacept, as a bridge to hematopoietic stem cell transplantation, with resolution of both immune and infectious complications, showing that genetic diagnosis is helpful in optimizing the management of presumed cvid patients. hospital 12 de octubre health research institute (i+12), madrid, spain, dept. of immunology, university hospital 12 octubre. madrid. spain background: xlf/cernnunos deficiency is a rare primary immunodeficiency classified within the dna repair defects. these patients present severe growth retardation, microcephaly, lymphopenia and increased cellular sensitivity to ionizing radiation. here, we describe two unrelated cases with the same nonsense mutation in the nhej1 gene showing significant differences in clinical presentation and immunological profile but a similar dna repair defect. methods: missense nhej1 mutation was identified by targeted next-generation sequencing with an in-house designed panel of 192 genes. for foci experiments, primary skin fibroblasts were irradiated with ionizing irradiation (137cs) or treated with 20mm etoposide for 1 hour. after irradiation, the cells were seeded at a density of 1x104 cells/ml in t75 flasks in triplicate. to evaluate cell sensitivity to gamma-ir (1 and 3 gy),adherent cells were trypsinized and counted 11 days later. pbmcs from patient and healthy controls were irradiated with 10gy, fixed and stained for cd3, cd19 and phospho-histone h2ax. mean fluorescence intensities (mfi) of gamma-h2ax were evaluated on gated cd3+ lymphocytes. results:we report two patients harboring the same homozygous mutation in cernunnos/xlf/nhej1 gene. strikingly, their clinical phenotype ranges from severe combined immunodeficiency to isolated thrombocytopenia followed until escolar age (table 1) . they harbour the same c.169c>t mutation in nhej1 gene but different immunologic features (table 2) . p2 presented with mild t lymphopenia, hypersensitivity and nhej repair defect, typical for patients with xlf/nhej1 defects. on the other hand, p1 presented a more severe phenotype (t-b-) , however hypersensitivity and nhej repair defect was similar to p2.of note, p2 has survived into the first decade of live. both patients are alive and well after hsct. discussion: usually the repair defect in these disorders is assessed by immunofluorescence assays of irradiation-induced gamma-h2ax foci using skin fibroblasts. a high throughput, sensitive and reliable assay to quantify gamma-h2ax foci in pbmcs isolated from blood samples would be a valuable tool to diagnose these patients and perform hsct early. flow cytometry (fc) can be applied as a rapid diagnostic tool for dna repair disorders. patients with the same homozygous mutation (p.r178x) in nhej1 gene have been previously reported. two patients died at 1.5 and 4 years while another of the patients is already 8 years old and is alive (without hsct). however,none of these patients presented severe t lymphopenia as it has been observed in our first patient. conclusions: the assignment of a timely and accurate diagnosis is of paramount importance in the management of patients with defects in dna repair. in the era of nbs an abnormal trec assay should be followed by ngs approach as cernunnos deficiency may present early in life as scid,as other rs-scid defects. since genetic diagnosis takes time,functional radiosensitivity assays in peripheral blood may lead to the correct diagnosis and avoid exposure to alkylating agents during the conditioning regimen prior to genetic diagnosis. it would also be helpful in cancer patients to individualize and to guide the dosing of ionizing radiation (ir) and/or genotoxic agents to avoid accumulation of cells with genomic instability that could accelerate cancer development. figure 1 ). her skin lesions also significantly improved after starting the medication ( figure 2 ). her hospitalizations were complicated by fluid overload and hypertension. both fluid overload and hypertension resolved prior to discharge. she remains on 2mg prednisone daily, cetirizine, ranitidine, cromolyn and benadryl and hydroxyzine prn. to our knowledge, this is the youngest patient successfully treated with midostaurin and she is doing very well on therapy with no apparent side effects. she has had resolution of many of her systemic mastocytosis symptoms including skin lesions, axillary mass and improvement in her diarrhea and growth as well as objective improvements in her tryptase levels. case report: a two-year-old male presented to the hospital with a painful, non-pruritic facial and groin rash. the rash started one week prior to presentation. he had no associated fevers. his history was remarkable for failure to thrive (ftt) and chronic bilateral leg pain with antalgic gait. over the preceding months, he had been diagnosed with hand-foot-mouth disease and varicella. he had also had recurrent cervical lymphadenopathy (lad) for greater than one year requiring incision and drainage. gram stain and gomori methenamine-silver nitrate stain (gms) were negative and pathology showed only acute and chronic inflammation with areas of necrosis. his family history was negative for autoimmune disease or immunodeficiency. infectious exposure history was significant for an incarcerated father with unknown tuberculosis status and history of living in a shelter. on physical examination, the patient was well appearing with multiple erythematous papules, with superficial erosions and scabbing on the face (figure 1 ), lower abdomen, genital area, buttocks and proximal lower extremities. he had large, firm, non-tender submandibular lymph nodes. he also had small palpable axillary and inguinal lymph nodes bilaterally. his laboratory workup revealed normal white blood cell and platelet counts, but microcytic anemia, an erythrocyte sedimentation rate of 140 mm/hr, and c-reactive protein of 7.5 mg/dl. full body magnetic resonance imaging (mri) revealed bilateral cervical, supraclavicular, right hilar and inguinal lymphadenopathy and a patchy right upper lobe consolidation with at least one small area of cavitation ( figure 2 ) and an adjacent smaller area of ring enhancement. it also revealed three small nonspecific hypodense foci within the right lobe of the liver and borderline splenomegaly. given these findings, there was concern for granulomatous diseases. the patient underwent a liver biopsy ( figure 3 ) which showed non-specific evidence of necrotizing granulomatous disease. microbiological cultures and stains for bacteria, acid-fast bacilli and fungi were negative. his infectious work-up was negative for hsv, tuberculosis, hiv, syphilis, histoplasmosis, and toxoplasmosis. superficial bacterial cultures from the face and groin grew mixed gram positive and negative organisms, including methicillin-susceptible staphylococcus aureus (mssa). his immunologic workup revealed borderline elevated iga and igg with normal igm, normal t,b, nk-cell counts and pneumococcal and tetanus titers. a dihydrorhodamine (dhr) flow cytometric test was positive, consistent with a diagnosis of chronic granulomatous disease (cgd). genetic testing confirmed x-linked disease. he was treated with acyclovir and ceftriaxone with resolution of his rash. conclusion: we present a case of a two-year-old male with newly diagnosed x-linked cgd. though he had been seen by multiple healthcare providers for recurrent lymphadenopathy over the preceding year, he had no other history of recurrent viral or bacterial infections or significant family history that might implicate a primary immunodeficiency. at time of presentation, he had diffuse rash which could have caused his palpable lymphadenopathy on exam. a high index of suspicion for cgd in the setting of recurrent lad and ftt prompted sending the dhr, which led to the diagnosis. chronic granulomatous disease (cgd) is an inherited primary immunodeficiency (pid) which results in both inflammatory response dysregulation and an increase in susceptibility to certain bacterial and fungal infections. without curative treatment such as a bone marrow transplant, it remains a chronic disease with daily medication management, intermittent treatment and life-long surveillance. in general, chronic disease involves physical, psychological and social effects which can affect the patients quality of life. although some research has been done on how pid affects quality of life, there is little research in the united states about how cgd affects patients quality of life. to examine the effect of cgd on patients quality of life, as a part of a voluntary research protocol examining the natural history of immune deficiencies, we administered the who qol-bref instrument to adult cgd patients enrolled on a nih irb approved protocol and seen in the infectious disease clinic at the national institutes of health (nih) over a five-month period. the who qol-bref is comprised of 26 items, which measure the following broad domains: physical health, psychological health, social relationships and environment. each item is rated on 5point likert scale. it has been validated cross culturally and has been widely field tested. the survey was interview administered to 35 patients (23 males, 12 females) with genetically confirmed cgd. the age range was 18 -60 years old (mean age 37.6 years) with a distribution of 57 % x-linked cgd and 43% autosomal recessive cgd. results have been obtained and will be presented. rationale: common variable immunodeficiency (cvid) is the most common primary immunodeficiency with an estimated prevalence of 1:25,000. we aimed to analyze the clinical presentations and their associated comorbidities amongst cvid patients in usa. methods: data on 1,546 cvid patients reported in the united states immunodeficiency network (usidnet) from 1992 to 2018 were analyzed based on clinical, immunological and genetic factors. univariate analysis with spearman rank coefficients was done to analyze correlations between disease outcomes. observed survival was estimated using the kaplan-meier method. results: among the 1546 patients, 908 (58.7%) were female and 638 (41.3%) were male. median age at diagnosis was 29 years [mean (sd), 30.1 (20.2); range, 0-82; iqr, 12-47] with median age of onset of 14 years (mean (sd), 20. 3 (19.2) ; range, 0-81; iqr, . females showed a longer delay in diagnosis (9.5 vs. 6.6 years, p=0.006). higher body mass index (bmi) linearly correlated with the age of diagnosis (r= 0.46). in survival analysis, a 5-year delay in age at diagnosis increased the risk of death by 7.4% (hr: 1.07, 95% ci: 0.98-1.18, p=0.14). conclusions: our study suggests a longer delay in diagnosis in female subjects and a strong association with diagnosis of cvid in patients with higher bmi. females may have a longer period without symptoms leading to a diagnostic delay. gender-based and disparities-based inquiry into these trends may need additional study. the physical well-being of those with primary immunodeficiency (pi) and the physical maladies of those with pi are well-documented. since the 1950s, advances in identification and treatment of pi has for many led to lives where the physical infections of these groups of diseases are manageable. however, not as well understood are the emotional and mental health aspects of living with pi. as part of a larger survey project the idf 2017 national patient survey, this study aims to quantify any potential mental health issues or challenges faced by adults with pi. our hypothesis-those with pi, suffer from statistically higher rates of depression when compared to the u.s. general population. the 2017 idf national patient survey was a nationally distributed, unincentivized, mail-based survey of 4,500 persons in the idf patient database identified as being either adults with pi or the parent/caretaker of a child with pi. the questionnaire comprised approximately 44 main questions about pi as well as the validated sf-12v2, brief fatigue inventory and the patient health questionnaire-2 (phq-2) instruments. additional questions asked about current use of prescription medications for anxiety, depression, stress and pain. for the purpose of this study, only adult respondents with pi are included as the basis for analysis. the two-item patient health questionnaire (phq-2) meets the criteria for general screening of depression suggested by the u.s. preventive services task force. scored on a scale of 0-6, a score of three or higher is suggested as the cut-point for depressive screening. according to a 2014 ahrq study that utilized meps data, 2,139 of the 23,770 (9%) respondents scored three or greater. in our survey 211 of the 925 (23%) adults scored three or greater (2 <.05.) overall, those in our survey scored lower on the sf-12v2 mcs scale when compared to the u.s. population (44.3 v.50.0, p<.05) . further, adults with pi who scored three or higher on the phq-2 had an average mcs of 31.8. those who met the phq threshold in our survey were also more likely to report moderate to severe limitations in normal activities as a result of emotional problems than those that fell below the threshold (74% versus 13%, p <.05). not surprisingly, those that met the phq threshold reported much higher use of prescription medications for anxiety, depression, stress (69% versus 33% below threshold, p <.05) as well as a higher reported use of prescription pain medications (33% versus 17% below threshold, p <.05). though moderate to severe fatigue was reported by 68% of those below threshold, 99% of those with phq scores at threshold reported experiencing moderate to severe fatigue (p <.05). health care providers should consider including the phq-2 in the overall health assessments of their patients with pi. those scoring three or higher should be referred to the appropriate professional for further evaluation. (lek et al., 2016) . the w623l is a semi-conservative amino acid substitution, which may impact secondary protein structure. in-silico analyses supported a deleterious effect, located within the sh2 domain, which is a critical functional domain (chandesris et al., 2012; koskela et al., 2012) . it was thus determined that this variant is likely pathogenic. the patients prophylactic treatment was optimized with tmp-smx (800mg-160mg) twice daily for prevention of infections. she was also started on hibiclens (chlorhexidine) baths once per week. she was referred to pulmonology for optimization of pulmonary health in the setting of bronchiectasis and mild decline in dlco. she was advised to followup on a yearly basis to the primary immunodeficiency clinic to assess for recurrent infections and for changes in pulmonary health. finally, targeted testing and clinical evaluation of both of the patients parents was recommended to determine if w623l was inherited or arose de novo. the pathogenic role of the w623l missense change would be further supported if it had occurred de novo or if it segregates with the disease in the family. uploaded file(s) uploads pulmonary function testing results.pdf j clin immunol (2019) 39 (suppl 1):s1-s151 s20 introduction: lipopolysaccharide-responsive and beige-like anchor protein (lrba) deficiency is a rare autosomal recessive disease of the immune systems characterized by hypogammaglobulinemia and decreased ctla4 expression on t regulatory cell (t regs) due to defective intracellular trafficking of ctla4. previous in vitro study has shown a significant increase of ctla4 expression on lrba deficient t cells after overnight culture with chloroquine, an older anti-malarial agent. this effect is likely due to increasing lysosomal ph. however, there is no evidence of such effect in human subjects after administration of weight appropriate doses anti-malarial agents. we are presenting a set of siblings with lrba deficiency who had ctla4 expression measured before and four weeks after starting hydroxychloroquine. case reports: case 1 is a 14-year-old east-indian boy with autoimmune thyroiditis, type 1 diabetes mellitus (dm), short stature, autoimmune cytopenias, and lymphadenopathy. he was referred to immunology clinic at 9 years of age for suspicion of autoimmune lymphoproliferative disorder. primary immunodeficiency genetic panel was sent which revealed a homozygous mutation in lrba gene (c.6480_6481del). this novel variant resulted in a frameshift and created a premature stop codon 18 amino acids downstream from this location which may lead to absent or abnormal protein. lung ct scan showed interstitial lung disease. lung biopsy showed interstitial nodular and diffuse lymphoid proliferation. this diagnosis led to the testing of his sister (case 2) given her history of autoimmune illnesses and the family history of consanguinity. case 2 is a now 13-year-old girl with type 1 dm, autoimmune thyroiditis, lymphadenopathy, psoriatic arthritis, and seizures. her lung imaging showed pulmonary nodules without interstitial lung disease. both cases received hydroxychloroquine while waiting for insurance approval of abatacept. ctla4 expression on tregs was measured prior to and four weeks after starting hydroxychloroquine treatment. at baseline, 8.6% of case 1s cd4 cells were treg (foxp3+ve, cd25hi) and 51.4% of them expressed ctla-4 (in contrast to 94.1% tregs in the healthy control) with mean fluorescence intensity (mfi) of 335. this ratio and mfi did not change after 4 weeks of hydroxychloroquine treatment (6 mg/kg/day). soluble interleukin-2 receptor levels were measured: case 1 had a baseline level of 8510 pg/ml, which decreased to 2228 pg/ml after 4 weeks of hydroxychloroquine treatment. for case 2: 8.4% of her cd4+ t cells were found to be foxp3+cd25hi and 36.1% of these tregs expressed ctla-4. this ratio increased by 7% after one month of hydroxychloroquine. increase in mfi was also noted from 298 to 386. case 2 had a drop in soluble interleukin-2 receptor level from 1265 pg/ml to 950pg/ml after treatment. conclusion: in contrast to the previous in vitro assays, we did not find a significant increase in ctla4 expression on t regulatory cells in vivo after 4 weeks of 6mg/kg/day hydroxychloroquine. interestingly, soluble il-2 receptor levels improved dramatically with hydroxychloroquine. (36) submission id#592574 human nf-kappab2 defect results in defective intrinsic b-cell differentiation, function and class switching introduction/background: autosomal dominant heterozygous mutations in nfkb2 (encoding for the protein nf-kb2) have been identified in the etiology of a form of primary immunodeficiency disorder that presents with hypogammaglobulinemia, defects in b-cell maturation, endocrinopathy, and autoimmune manifestations. in humans, the effects of altered nf-kb2 and mechanisms of immune system impairment have not been fully delineated. objectives: to understand the mechanism of the antibody deficiency in patients with hypomorphic mutations in nfkb2 (c.2564dela; p.lys855serfs*7) by evaluating b-lymphocyte proliferation, differentiation, function, and gene expression. methods: immunophenotyping of primary b-cells from subjects with mutant nfkb2 was completed by flow cytometry. proliferation of b-cells was assessed by cfse stimulation of primary cd19+ b-cells from healthy and nfkb2 mutant subjects. differentiation of healthy and affected naã¯ve b-cells (cd27-cd38-) into plasmablasts (cd27+cd38+) following stimulation was assessed by flow cytometry. the supernatant from these cells were assayed for iga, igg and igm production by elisa. to study the defect in class-switch recombination, naã¯ve b-cells and ebvtransformed b-cells from affected and healthy individuals were stimulated and expression of the aicda gene was quantified by qpcr. in parallel experiments, ebv b-cells from wildtype and nfnb2 mutant individuals were stimulated and aid (activationinduced cytidine deaminase) protein levels were determined by western blot. results: patients with hypomorphic mutations in nfkb2 (c.2564dela) had low memory b-cell (cd19+ cd27+ igd-igm+) and class-switched memory b-cell (cd19+ cd27+ igd-igm-) numbers. in vitro, primary bcells from these patients demonstrated a 50% reduction in proliferation and cell division in response to cd40l and il-10 (p =0.01). compared to healthy naã¯ve b-cells, mutant naã¯ve b-cells had a significant reduction in plasmablast differentiation (p = 0.002) and secreted significantly lower levels of immunoglobulins in response to cd40l and il-21 stimulation. mutant naã¯ve b-cells and mutant ebv b-cells failed to increase aicda expression and aid protein levels in response to cd40l and il-21 stimulation. conclusions: our studies demonstrate that a hypomorphic nfkb2 mutation in humans affects intrinsic b-cell proliferation and differentiation. the mutation impairs transcription of the aicda gene that encodes aid, a key protein involved in b-cell class-switch recombination. the nfkb2 gene defect also impairs immunoglobulin production, as seen in common variable immunodeficiency-like cases. these studies provide unique translational insights into physiological activities of nf-kb2 in downstream immunologic outputs in humans, expanding those suggested by experimental observations in mice. background: few studies have evaluated the quality of life (qol) and patient reported outcomes of primary immunodeficiency disease (pidd) patients, and no studies have assessed medical provider perceptions of their pidd patients qol, neurocognition, physical well-being and psychosocial health. understanding provider beliefs regarding patient reported outcomes is essential to improving clinical management of pidds. here we report our pidd medical provider survey results. methods: providers were contacted via email with the assistance of the clinical immunology society. participants completed adult and/or pediatric-based likert scale survey questions via a secure online survey service. in addition to demographic information, survey questions assessed provider perceptions of patients overall qol and their impression of the impact of disease or its associated treatment on mental health, physical well-being, neurocognition, social relationships and school/work performance. clinicians were expected to make their assessments based on their pidd patient cohort as a whole rather than on specific diagnoses or patients. given the small sample size, a p-value < 0.1 was considered statistically significant; repeated measures anova and paired t-test analyses were used. results: study participants (n=58) were primarily from the united states (64%), born between 1965-1979 (44%) , and trained in allergy/ immunology (77%). 85% of survey takers practiced within an academic center, 52% were female and 95% cared for children with 42% of providers concurrently caring for adults. there was a statistically significant difference (p=0.07) in the perceived overall qol of pediatric versus adult pidd patients with 41% of providers feeling as though their pediatric patients had a good qol while only 25% believed their adult patients had a good qol. clinicians believed adult pidd individuals had more difficulties related to associated co-morbidities rather than their actual pidd compared to pediatric pidd patients (p=0.046). providers felt that the neurocognition and school performance of children were more often negatively affected by a pidd than the neurocognition and work performance of immunodeficient adults (p=0.1). clinicians believe children with pidd more frequently had difficulties related to their concentration than memory (p<0.01). 96% of those who care for pidd adults believe their patients work performance or daily mental functioning is at times negatively impacted. anxiety symptoms and social relationships were viewed as being more negatively impacted by a pidd diagnosis or treatment than anger or depressive symptoms in both children and adults (p<0.01). 38% of pediatric clinicians feel their pidd patients experience anxiety symptoms often or almost always. of physical health parameters, energy, rather than mobility or pain, was deemed to be more deleteriously influenced by an immunodeficiency in adult and pediatric patients (p<0.01). conclusions: our results show that medical providers perceive the overall qol of pediatric pidd patients to be superior to that of adults with pidd, but most clinicians feel a diagnosis or associated treatment regimen for pidd can negatively impact the physical well-being, psychosocial health, school/work performance and neurocognition of both children and adults. [cbm] complex is a critical signalling adaptor that regulates lymphocyte activation, proliferation, survival, and metabolism. primary immunodeficiencies affecting each component (termed cbmopathies) result in broad clinical manifestations ranging from severe combined immunodeficiency (scid) to lymphoproliferation. we present the laboratory and clinical findings of two canadian first nations patients found to be homozygous for the same novel card11 mutation (c.2509c>t; p.r837*). results: we have identified an 8-month-old boy who presented with a severe case of entero/rhinovirus bronchiolitis with interstitial lung disease and a 17-year-old boy with a history of severe pulmonary infections (including pjp), chronic sinusitis, candidiasis, invasive bacteremia, and severe ileo-colitis and oral ulceration requiring total colectomy. both patients possessed absent tregs, absent memory b cells, and hypogammaglobulinemia. however, only the 8-month-old had poor t cell proliferation to pha, cona, and cd3. both patients were found to be homozygous for the same novel variant of card11 (c.2509c>t; p.r837*). the mutation rendered card11 protein expression unstable and it was undetectable by immunoblot. to confirm card11 deficiency, we stimulated patient b cells with phorbol 12-myristate 13 acetate (pma) and ionomycin across a time-course and immunoblotted for various signalling proteins in both the nf-b (ikk/, ib, p65) and mapk (mek1/2, mkk4, jnk1/2, erk1/2) pathways as well as various cleavage substrates of the malt1 paracaspase (relb, cyld, bcl10, hoil1). nf-b and jnk activation were completely absent and malt paracapase activity was lost, but surprisingly, mkk4 (which acts upstream of jnk) was intact. furthermore, co-immunoprecipitation experiments revealed that card11 was required for optimal malt1 association with bcl10 in response to stimulation. conclusions: these two cases highlight the crucial role of card11 in regulating lymphocyte development, function, and humoral responses. in addition, we have identified the oldest known living individual with card11 deficiency and he presented uniquely with inflammatory gastrointestinal disease in addition to scid, further adding to the spectrum of phenotypes associated with card11-related primary immunodeficiencies. abstract: the usidnet registry began in 1992 with an niaid contract with the immune deficiency foundation, which continues today. it aims to provide a resource for clinical and lab research through enrollment of known immunodeficiency patients into a national registry, the usidnet. nih is a major national and international referral center for clinical trials on inborn errors of immunity, or primary immunodeficiency diseases. it is a mechanism for depositing nih data into usidnet. a registry of patient information may help us understand how many people have each disease. the information may improve how we diagnose and treat these conditions. the patient registry is designed to obtain longitudinal data on a large number of patients with primary immunodeficiency diseases who come to nih to participate in research. the data is collected from the nih electronic medical record system, cris and is deposited into a secure registry with restricted and monitored access. all medical information is anonymized for patient privacy. department of biochemistry, emory university, atlanta, ga oas1 is an intracellular sensor for dsrna that generates the second messenger 2'-5'-oligoadenylate to activate rnase-l as a means of antiviral defense. we describe four patients with a complex early-onset autoinflammatory and immunodeficiency disease caused by heterozygous de novo oas1 mutations. patients presented early in life with lung inflammation including pulmonary alveolar proteinosis and interstitial lung disease. they had febrile flares with dermatitis specifically with macular, pustule and bullous features often progressing to ulceration. infants had episodes of bloody diarrhea in 3 patients (assoc. with villous blunting and cryptitis in two patients and oesophagitis in one patient). immunoglobulin igm, igg, and iga levels were low while t cell, b cell, and nk cell numbers were generally in the normal range. exome sequencing identified de novo heterozygous oas1 missense mutations in all patients. one patient had a heterozygous de novo oas1 mutation p.ala76val, with mutant oas1 protein being expressed in ex vivo generated t cell blasts. in sorted primary patient monocytes and b cells, oas1 p.ala76val was associated with spontaneous rna degradation and apoptosis as determined by rna chip technology and flow cytometry, respectively, while t cells were not affected. monocytes displayed disturbed terminal differentiation and functioning as indicated by reduced gm-csf-r expression and signaling. b-cells display reduced class-switch-recombination. proliferation of allogeneic t-cells was reduced in response to sorted oas1 mutated monocytes and b-cells. activation of interferon response genes in pbmcs was detected. two further unrelated patients had a heterozygous de novo oas1 mutation p.cys109tyr, which appeared to compromise protein stability in transformed patient fibroblasts and when transfected. cells transfected with this mutant protein had reduced 2-5 oligoadenylate synthesis compared to wild type transfected cells. immortalized fibroblast lines demonstrated higher levels of inflammatory cytokines and spontaneous cleavage of rnas. a 4th patient with the clinical phenotype had a heterozygous de novo oas1 variant p.val121gly, but has yet to have formal validation of the variant. three patients underwent hematopoietic stem cell transplants in an effort to control their diarrhea and skin inflammation. one patient died with ongoing chronic graft versus host disease, while the two others (p.ala76val, cys109tyr) are alive and reasonably well with a followup of 0.5-7 years. the untransplanted patient died as a result of respiratory failure. in summary, patients with de novo heterozygous oas1 mutations have chronic ongoing inflammation of multiple organs. this is at least in part due to spontaneous rna cleavage, apoptosis and production of inflammatory cytokines and type i interferons. this defines a new category of autoinflammatory disorder. introduction: increased susceptibility to infections is the most common complication of chronic granulomatous disease (cgd). hemophagocytic lymphohistiocytosis (hlh) is a severe disorder resulting from hyperinflammation and hypercytokinemia that can lead to multi-organ system dysfunction (1) characterized by certain criteria: fever, splenomegaly, cytopenias, hypofibrinogenemia or hypertriglyceridemia, hyperferritinemia, increased soluble cd25/il-2ra, evidence of hemophagocytosis, or decreased/absent nk cell cytotoxicity (2) . secondary hlh occurs infrequently but often is preceded by smoldering infection in cgd (3, 4, 5) . we present a case of hlh in a 38-day old male, the youngest reported case with cgd. case: a 38-day old male with previously diagnosed x-linked cgd, due to known family history, presented with fevers. initial evaluation was unrevealing including chest x-ray, urinalysis, and blood and csf cultures. he was admitted and treated empirically with cefepime. ct demonstrated multiple multifocal nodules of the lungs and spleen. after lung nodule biopsy was performed, antimicrobial therapy was broadened to iv meropenem, voriconazole, and micafungin. despite this, he continued to have fever and developed new onset tachycardia, respiratory distress, and lactic acidosis. further decompensation with vasoactive refractory shock was treated with vasopressors and stress dose hydrocortisone. additional laboratory evaluation revealed rising liver enzymes (ast 1670u/l, alt 307u/l), cytopenias (hemoglobin 7 g/dl, anc 90/ul, platelets 96,000/ul), and coagulopathy (fibrinogen 93-135mg/dl). splenomegaly was present on abdominal ultrasound. a diagnosis of evolving hlh was considered and dexamethasone was administered. within 24 hours of clinical decompensation, the patient died of multiorgan failure. subsequent blood cultures returned with gram-negative rods (and ultimately burkholderia cepacia). autopsy confirmed hemophagocytosis within the bone marrow. no mutations were found in genes associated with primary hlh. discussion: patients with cgd are susceptible to infectious complications and auto-inflammation most commonly involving the lungs, gi, and gu systems (6, 7) . patients with cgd can be at increased risk of hyperinflammatory syndromes secondary to infections and chronic inflammation. as shown in the included case, hlh can present in infancy and can be deadly. early consideration and directed treatment of hlh is imperative, even in the setting of sepsis malignant proliferation of gamma-delta t cells include hepatosplenic t-cell lymphoma (hstl), primary cutaneous t-cell lymphoma and t-cell large granular lymphocytic leukemia (t-lgl). the former two have often been associated with splenomegaly and cytopenias. however, reactive proliferation of gamma-delta t cells in spleen mimicking malignancy has only been reported once and has a significant risk of misdiagnosis. a 30-year-old female presented with two years of unintentional weight loss, persistent leukopenia and thrombocytopenia, with leucocytes around 1-2 x 10^9/l and platelets around 100 x 10^9/ l. she also had associated macrocytic anemia (hemoglobin=10-11g/dl) with laboratory evidence of dat (direct anti-globin test) negative hemolysis. physical examination and computed tomography (ct) imaging showed splenomegaly. there was no hepatomegaly or lymphadenopathy. serum liver function test, auto-immune studies, hemolysis and hereditary diseases workup, viral and bacterial serologies were all normal or negative, except for mild hyperbilirubinemia and ldh elevation. bone marrow examination performed four months prior to the splenectomy revealed mildly hypocellular marrow (50%) with trilineage hematopoiesis. flow cytometric analysis and cytogenetics of the bone marrow aspirate and peripheral blood were normal except for small population of large granular lymphocyte and mild low absolute b cell counts in peripheral blood. a laparoscopic splenectomy was performed for diagnostic and therapeutic purposes due to patients worsening luq pain. there was no other treatment given prior to surgery. 24 hours postsplenectomy her leucocytes increased to 13.1 and platelets to 247. her three-month post-splenectomy wbc count and platelet count was 8.9 and 391, respectively. hemoglobin also improved to 14.9. pathology showed red pulp expansion by small lymphocytes (fig. 1 ) and subsequent ihc (immunohistochemistry) was positive for cd3 ( fig. 2) , cd2, cd7, tia-1 and negative for cd8, cd5 and cd56. cd4 was difficult to interpret. eber was negative. flow cytometry ( fig. 3) showed increased gamma-delta t-cell population (20%) with positive cd3, cd2 and cd 7 and negative cd 5, cd4 and cd 8. molecular studies by pcr didnt reveal any t-cell receptor gamma or beta gene rearrangement. cytogenetics was negative for isochromosome 7q or any other abnormalities. she was symptom free at 6 months from her splenectomy. the morphology and immuno-phenotype of these gamma-delta t cells show significant overlap with the malignant cells seen in hstl and t-lgl, such as loss or downregulation of cd5, cd4 and cd8. awareness of this reactive condition is necessary to prevent making a wrong diagnosis of a malignant disease with a potentially benign, spontaneously resolving disease. additional studies of similar cases is needed in order to establish more definitive criterion to separate benign from malignant processes and delineate the role of gamma-delta t cells. uploaded file(s) uploads fig 3. flow cytomtery.pptx background: sex steroids in the human thymic environment influence aire expression as well as interactions with its partners, i.e. genes coding for aire interactors. here we investigated the effects of sex steroids on these interactions during minipuberty the surge of sex hormones that occur along the first six months of life -and up to 18 months of life. we employed a network-based approach for investigating aire-interactors gene-gene relationships and how abundantly co-expressed thymic mirnas covariate with those genes. aire-interactors networks allowed the measuring of gender-related differences in gene-gene expression correlation disclosing relevant differences between minipuberty groups. methods: total rna was extracted from thymic surgical explants obtained from male (m) and female (f) infants -aged 0-6 months (groups mm and mf, for minipuberty) and 7-18 months (group nm and nf, for nonpuberty) and used in dna microarray assays. gene coexpression network (gcn) analyses were performed for aire and its interactors and for mirna-gene coexpression analysis. the set of genes coding for the aire-targeted proteins was previously identified in tecs by abramson et al. (cell 140:123-35, 2010) . aire-interactors networks were obtained for all groups (link strength cut-off for gene-gene > |0.80| and for mirnagene < -0.80). aire expression in mtecs was quantified by immunohistochemistry. these methodologies are described in moreira-filho et al. (sci rep 8:13169, 2018) . results: the mm x mf networks comparison showed that 16 abundantly expressed mirnas are interacting with the different aire interactor genes in both networks. it is interesting to note that network topology were more similar between nm and nf groups, although aire interacts with only one distinct mirna in each network (mir-150-5p in the nm group or mir-7977 in the nf group). conversely, in the non-puberty networks the sets of mirnas and their interacting genes are distinct for each network. immunohistochemistry analysis revealed a higher percentage of mtec aire positive cells in the minipuberty groups: i.e. there is a significant difference between mm x nm (p = 0.0006) and between mf x nf (p = 0.0060). conclusions minipuberty and genomic mechanisms shape thymic sexual dimorphism along the first 6 months of life. this process does not involve changes in aire expression between genders, but differences in the interactions of aire with its partners that persist throughout the non-puberty period, probably regulated by mirnas and also by genetic and epigenetic factors. introduction: neutrophils are presumed to defend against aspergillus species by releasing reactive oxygen species (ros) and neutrophil extracellular traps (nets) to degrade fungal hyphae. triazole antifungals synergistically enhance neutrophil mediated hyphal degradation. patients with cgd are particularly susceptible to aspergillus species likely due to their inability to create ros and nets, and in severe cases may not be amenable to antifungal therapy alone. objective: we present a case of severe disseminated aspergillosis in a patient with cgd in whom gt served as an important adjunct to antifungal therapy and bridge to transplant. results: a 6-year-old boy with known cgd, lost to follow up and nonadherent to prophylaxis, presented acutely with right-sided hemiparesis. neuroimaging revealed an embolic left middle cerebral artery infarction and cardiac magnetic resonance imaging showed extensive vegetations involving both right and left ventricles and atria, with an ejection fraction of 28%. the patient was admitted to intensive care, started on liposomal amphotericin b, meropenem and vancomycin, and underwent debulking of the intracardiac masses on post admission day (pad) 1. operative findings showed severe constrictive pericarditis with multiple abscesses and intracardiac vegetations. thorough debridement of the vegetations was undertaken, however some deep seated abscesses in the myocardium were not amenable. operative cultures were positive for aspergillus fumigatus. clinical status remained precarious, with ongoing requirement for inotropic and ventilator support. antimicrobial therapy was refined to voriconazole, with amphotericin b remaining on board until therapeutic levels of voriconazole were achieved. as effective neutrophils are integral in the immune response against aspergillus, the decision was made to start granulocyte transfusions to aid in clinical stabilization prior to hsct. interferon gamma infusions were not administered because of the risks of adverse effects and potentially increasing transplant rejection. gts were started on pad 6, at a dose of approximately 1x10^10 granulocytes, three times a week. the patient tolerated the infusions well, with no allergic or inflammatory response. neutrophil oxidative burst measured one hour post infusion showed 23.9% mean fluorescent intensity, compared to a baseline of 0% ( figure 1 ). clinical improvement was seen, with inotrope cessation on pad 12 and extubation to bipap on pad 41. human leukocyte antigen (hla) allosensitizaton was tested on pad 12, 6 days after the first gt, with no evidence of hla antibodies. a total of 28 gts were given over 3 months, prior to proceeding to a 10/10 hla matched related donor transplant (pad 69), with two transfusions given before neutrophil engraftment (anc 500) on day +14. the patient is now stable 13 months post transplant, with no evidence of graft rejection. he remains on chronic suppressive antifungal therapy, to continue until full lymphoid reconstitution. conclusion: gt may be a useful adjunct to antifungal therapy in patients with impaired neutrophil function with severe invasive aspergillosis, and potentially provide a life sustaining bridge to hsct. methods: subjects were enrolled in irb protocol 00051692 for rvt-802. rvt-802 was implanted into the quadriceps with immunosuppression. results: subject 1 was normal at 22q11.2 but had hypocalcemia, an asd, pda, and abnormal ears. the subject received a cord blood transplant mismatched at hla-b and hla-c alleles at age 3 months. subsequently mild graft-versus-host disease (gvhd) developed and was treated with antithymocyte globulin, steroids and cyclosporine. donor t cells developed in low numbers. twelve years later, the subject developed epstein barr virus lymphoma and suffered two relapses. while in remission, subject 1 received unmatched rvt-802. two weeks after rvt-802 implantation, the subject developed an adenovirus infection resulting in skin and gut gvhd, presumably from activation of the cord blood t cells. subject 1 was treated with corticosteroids, cyclosporine, cidofovir and infliximab. four years post rvt-802, subject 1 is healthy with 609 genetically recipient t cells/mm3 and 40% naã¯ve cd4 t cells. subject 2 was normal at 22q11.2 but had an asd, pda, hypoparathyroidism, and no t cells at birth. his genetic defect is unknown. subject 2 was treated with a ric myeloablative, allogenic, unrelated, 10/10 cord blood transplant, and a subsequent myeloablative, unrelated 9/10 cord blood transplant. hematopoietic chimerism was established without t cell development. rvt-802 expressed the one allele in the recipient that was not expressed by the second cord donor. the post-thymic transplant course included immune thrombocytopenia requiring rituximab and splenectomy and generalized adenopathy for 3 years but no gvhd. he failed weaning of immunoglobulin replacement. three years post rvt-802, he has 930 cd3, 750 cd4, and 105 cd8 t cells/mm3. he is active in school. subject 3 had absent trecs on newborn screening with 7 cd3+ t cells/ mm.3 a single mutation in foxn1 was identified; she has sparse scalp hair. subject 3 received a 9/10 matched unrelated umbilical cord transplant. the post-transplant course was complicated by significant morbidity, and no naã¯ve t cell development. rvt-802 expressed the one allele in the recipient that was not in the cord blood donor. the subject did not develop gvhd, is healthy and at 9 months has 98 naã¯ve cd4+ t cells. she had resolution of longstanding norovirus and sapovirus gastroenteritis. conclusion: rvt-802 can improve t cell immunity after poor or failed correction with allogeneic hematopoietic transplants. in subject 1, gvhd post rvt-802 was related to an acute viral infection; cord t cells attacked hla mismatches in the recipient. subjects 2 and 3 were given rvt-802 matched to recipient alleles that were not expressed in the hematopoietic donor. we hypothesize that thymocytes developing in rvt-802, if strongly reactive to the recipient-mismatched allele, are deleted by the bonemarrow-donor dendritic cells (that acquire recipient mhc from the recipient-allele-matched thymic epithelial cells) thereby preventing gvhd. rationale: ctla4 haploinsufficiency is an autosomal dominant immune dysregulation syndrome characterized by variable phenotypes. here we present a young woman diagnosed with evans syndrome and lymphoproliferation as a child, found to have a novel ctla4 variant as a young adult, and who developed hypogammaglobulinemia and a bacterial endocarditis while stabilized on ctla-4 replacement therapy. methods: sequencing of 207 genes, including ctla4, in primary immunodeficiency panel. results: our patient was diagnosed with evans syndrome at age 2 with manifestations of anemia and thrombocytopenia recalcitrant to treatment over many years with steroids, cyclosporine, and vincristine. bone marrow biopsy reportedly showed normal trilineage maturation and her symptoms responded for a short time to splenectomy at age 14. symptoms recurred at age 16 when she was also found to have pulmonary reticular opacities, prominent lymph nodes, and elevated b cells. repeat bone marrow and lymph node biopsies at that time were unrevealing. minor responses to treatment with ivig, rituximab, mycophenolate mofetil and gcsf were noted. at age 17, she developed varicella-related encephalitis shortly after vaccination. with a strong suspicion of an immune dysregulation syndrome, immune evaluation revealed normal immunoglobulins with good vaccine responses, elevated b cell numbers, normal t cell numbers, and normal mitogen proliferation. ctla4 sequencing revealed a mutation in exon 2 [c.420c>a, p.tyr140*] causing a premature translational stop signal, which was consistent with previously reported cases of ctla4 haploinsufficiency. she was started on rapamycin initially for her cytopenias but was then transitioned successfully to abatacept with almost complete resolution of her anemia, neutropenia, and pulmonary opacities. after 6 months of stable control, she developed a precipitous drop in her platelets and was eventually diagnosed with streptococcus viridans endocarditis of her native mitral valve. this responded to antimicrobial therapy, but eventually needed surgical intervention due to ongoing insufficiency. around this time, she was also found to be newly hypogammaglobulinemic, necessitating ongoing igg supplementation therapy. during successful replacement of her mitral valve with a biosynthetic prosthesis, it was noted that her aortic valve also had evidence of previous disease, implicating a prior endocarditis as part of her clinical syndrome as well. conclusions: in this patient, the presentation of recalcitrant cytopenias, lymphadenopathy, elevated b cells, vaccine-induced viral infections and lung findings precipitated concern for immune dysregulation syndromes and allowed for identification of a novel deleterious ctla4 mutation. in addition to previously reported clinical findings, our patient presents with the first reported case of repeated endocarditis in the setting of ctla4 insufficiency disease. given the finding in this patient of prior (unrecognized) disease, regularly screening patients with ctla4 insufficiency for evidence of cardiac affectation may be prudent. clinical research nurse, johns hopkins university background: the relationship between elevated serum alpha fetoprotein (afp) concentration and age, mortality, genotype and neurologic outcome in ataxia telangiectasia (a-t) patients has remained inconclusive over the past decades, leaving afp as a useful marker for disease diagnosis without further clinical significance. objective: to examine the relationship between afp levels and age, mortality, genotype and neurologic outcome using a data set larger than any prior study. methods: we retrospectively collected data on 280 a-t patients at johns hopkins medical center (0-34 years of age) with both classical (predicted protein null) and variant a-t. this included 459 serum afp measurements (179 serial levels in 50 a-t patients, max observations 9 per patient). mixed model compound symmetry covariance was used for statistical analysis to examine the effect of age at visit on afp levels. subgroup analysis by mutation type, mortality, feeding/swallowing scores as a surrogate for neurologic function, x-ray induced in vitro chromosomal breakage and serum transaminase levels were similarly analyzed. results: significant association between age and afp level was found such that for every 1 year increase in age, afp level increases 20 ng/ml (p<0.0001). subgroup analysis by mutation type found that the 12 patients with missense mutations showed a negative linear relationship be-tween log afp levels and age (r= -0.10, p=0.03). we found greater afp levels in patients who subsequently died, after controlling for age (least square mean afp level in log scale 0.67 greater in deceased patients versus living patients, p=0.002). we found a significant decline in feeding score by 0.18 units (score range 0-5) per 100 ng/ml afp increase (p=0.05) after adjusting for age. there was no significant relationship between afp levels and serum transaminase levels. conclusion: afp increases with age in a-t patients, though this may not apply to patients with missense mutations. there is a statistically significant increase in mortality and worsened swallowing scores with increasing afp levels, but this remains to be proven clinically significant. here we present a pediatric hae patient who had recurrent abdominal attacks in which constipation, secondary to the adhd medication dexmethylphenidate (focalin), appears to be a trigger. of importance, this is the first pediatric patient with hae to be described as having safely undergone a capsule endoscopy for direct visualization of the gastrointestinal tract. this was done to decrease the risks associated with the more invasive procedure of traditional endoscopy and colonoscopy. case presentation: the patient was an 8-year-old male with hereditary angioedema who presented with 1 day history of diffuse abdominal pain and nausea. in the ed, patient was in no acute distress. abdominal ultrasound showed severe circumferential thickening of the wall of multiple bowel loops and a large amount of simple ascites. x-ray revealed stool in the colon. he was admitted for pain control and hydration. in the next year, he visited the ed five more times for exacerbations of angioedema of his hand, penis, and bowel. each time, he presented he had underlying abdominal pain and constipation. he was seen by gastroenterology and had a workup that was negative for helicobacter pylori, parasites, and other gastrointestinal infections. to further evaluate his abdominal pain, capsule endoscopy was performed and well tolerated. during an admission in january 2016 he received a full inpatient bowel cleanout, after which, his angioedema finally improved. of note, he was diagnosed with adhd and started on dexmethylphenidate (focalin) just prior to this period of recurrent angioedema attacks, and he did not have attacks during the summer months when he was off the medication. discussion: abdominal pain is a common complaint in pediatric hospitals, and further workup consists of endoscopy and colonoscopy. this may be easily accomplished in the general population, however, in patients with hae, these procedures carry greater risk and may be avoided, leading to delayed diagnosis and treatment (2, 4) . a newer and less commonly used alternative for direct visualization of the gastrointestinal tract is capsule endoscopy. some benefits are that it does not require sedation, is less invasive, and is less likely to be irritating to the mucosa (3). additionally, since psychological stress may be a trigger for angioedema attacks, the decreased stress associated with a noninvasive procedure such as capsule endoscopy, makes it safer to use (1) . limitations of capsule endoscopy include dependence on battery life and its inability to biopsy or administer therapy if needed (3) . hereditary angioedema treatment consists primarily of avoiding triggers and managing acute episodes. in this first case of hae in a pediatric patient where capsule endoscopy was used, the procedure was well tolerated without any complications. recognizing constipation as a trigger and capsule endoscopy as a safe method of direct visualization of the gastrointestinal tract will help others to control and decrease the severity of their hae attacks as well. a 45 year old male with past medical history of common variable immune deficiency (cvid) and related autoimmune complications, including granulomatous-lymphocytic interstitial lung disease (glild), hepatosplenomegaly, leukopenia, and thrombocytopenia tolerated monthly subcutaneous immunoglobulin replacement as outpatient for several years with infrequent infectious complications. four months ago, he was found to have elevated liver enzymes on routine chemistry. a liver biopsy two months later showed pathology consistent with nodular regenerative hyperplasia (nrh) without overt cirrhosis. a hepatic venous pressure gradient (hvpg) of 21 mmhg was found, consistent with portal hypertension. his hepatitis viral markers were negative, he did not drink, and portal venogram was negative for thrombosis. in early october, the patient was admitted to the hospital with anasarca and tense ascites. he underwent a diagnostic and therapeutic large volume paracentesis and was also found to have spontaneous bacterial peritonitis (sbp) and bacteremia with group b streptococcus. the patients course was complicated by polymicrobial peritonitis, vre bacteremia, fungemia, variceal hemorrhage, hepatic encephalopathy, and hepatorenal syndrome. his hepatic complications from portal hypertension were out of proportion to his liver parenchymal disease. transjugular intrahepatic portosystemic shunt (tips) was considered to alleviate portal hypertension but was not feasible due to his degree of encephalopathy. immunosuppressants such as high dose steroids were given while in the hospital with plans to start rituximab to treat patients glild after he had recovered from the acute infections. unfortunately, after two months in the hospital, the patient succumbed to sepsis and progressive liver failure. this case emphasizes the importance of systematic screening and continued vigilance for hepatic complications in patients of cvid as studies have shown that nrh of the liver is present in more than 80% of cvid patients who undergo a liver biopsy (pmid: 23219764). a cross-sectional study of patients with primary hypogammaglobulinemia and hepatic dysfunction found that histological findings of nrh were present in 84% of cvid patients and was associated with portal hypertension in 75% of cases (pmid: 17998147). another study estimated the minimal prevalence of nrh in cvid patients as 12% (pmid: 18647320), stating that this was likely a gross underestimate as nrh may also be present in patients with normal liver function tests that are not routinely biopsied. therefore, liver enzyme levels may not anticipate the severity of liver involvement. there is currently no treatment for cvid-related liver disease. other causes of non-cirrhotic portal hypertension, including hepatic veno-occlusive disease and budd-chiari syndrome should be ruled out or treated in cvid patients presenting with hepatic disease. in the case of hepatic nrh in cvid patients, early detection could lead to earlier interventions (such as tips prior to hepatic encephalopathy), to mitigate complications. we describe the application of epigenetic quantification of t regulatory (treg) cells in addition to cd3+, cd4+, cd8+ t cells, b cells, nk cells, monocytes and neutrophils from as little as 50 î¼l of fresh, frozen or dried blood. the method yields identical results to flow cytometry from fresh blood samples of a healthy donor cohort, with the advantage of being more sensitive and precise with limited amount of blood and minimal sample preparation (sci transl med 2018). we have used this method 1) to immunophenotype patients with early onset immune regulatory disorders (pird) and primary immune deficiency (pid), and 2) to evaluate cell subsets reconstitution early after hematopoietic stem cell transplantation (hsct). patients with immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) and ipex-like pird were evaluated by analyzing the treg-specific demethylated region (tsdr) of the foxp3 locus in the total of cd3+ t-cells. despite the dysfunctional foxp3 mutated protein, ipex patients exhibited elevated treg/cd3+ cell ratios which seemed to correlate with disease severity. in contrast, most of the patients with ipex-like symptoms without foxp3 mutations exhibited decreased treg/cd3+ cell ratios -in line with the possible central pathogenic role of treg function and number in pird. using epigenetic quantification of cd3+/b-and nk cells, 23 out of 24 confirmed scid and xla cases were correctly identified within a cohort of 250 newborn dried blood spot (dbs) samples (96% sensitivity, 100% specificity). the method identified one delayed onset scid as well as a xla case that were missed by combined trec/krec testing. epigenetic immune cell quantification missed one scid case with maternal engraftment that was identified by combined trec/krec testing. abnormally elevated treg/cd3+ ratio was also detected in a dbs from a newborn who was subsequently confirmed to be affected with ipex syndrome. when applied to serial blood samples during engraftment and reconstitution post-hsct, the epigenetic method allowed identification of the different blood cell subsets, including treg cells, at earlier time points than flow cytometry according to current clinical practice. this opens the way to a better understanding of the correlation between early immune reconstitution events and graft vs. host disease or viral reactivation, earlier than with the current methods, in different types of hsct. these studies underscore the suitability of epigenetic immune cell quantification for accurately measuring multiple immune cell types from limited blood sample sources. we propose this method as uniquely suitable for novel molecular diagnostic applications in settings with limited fresh blood sample or limited cell number, at the point of care as well as for newborn screening. we evaluated a 5-year-old male with hyperpyrexia, hypertrichosis, conical hypodontia, and a history of illnesses concerning for nemodeficiency syndrome. starting at six months of age, he suffered recurrent episodes of acute otitis media (non-typeable hib and actinobacter iwolffli), pneumonia, and rsv bronchiolitis. whole exome sequencing demonstrated a de novo heterozygous c.1259g>a (p.r420q) mutation in the eda-receptor (edar) gene not present in the parental dna. his physical exam findings and mutation were consistent with hypohidrotic ectodermal dysplasia (hed), a rare genetic condition characterized by abnormal development of skin, teeth, hair, and sweat glands. hed is caused by defects in the ectodysplasin-a (eda)-nfkb signaling pathway but is not typically associated with immune deficiency. consistent with this, immunophenotyping showed normal sub-populations of t-, b-, and nk-cells. immunoglobulin and complement levels were quantitatively appropriate. he had normal mitogen-induced lymphocyte proliferation and normal antibody response to pneumococcal vaccination. nk-cell studies demonstrated robust cytotoxicity. however, nasal mucosa biopsy showed diffuse squamous metaplasia and the absence of ciliated epithelial cells. we hypothesize that recurrent infections in our patient arose from impaired mucociliary clearance due to a ciliary defect. this case raises the possible association between edar variants and ciliary dysfunction. it also underscores the importance of evaluating the immune status of hed patients with recurrent infections which could mimic nemo-deficiency and have broad implications about clinical management. the rapid pace of new gene discovery and phenotype expansion for primary immunodeficiency diseases (pidds) creates challenges for genetic testing and variant interpretation. whereas well-described clinical case reports in published literature have traditionally served as the source of phenotypic data used for variant interpretation, for pidds the causal variants are often private to the patients family and thus the sole source of phenotypic information for a novel genetic variant is frequently the history provided by the clinician on the test requisition form. taking into account such heterogeneous information during variant interpretation requires establishing objective criteria for its inclusion as part of the variant interpretation process. to this end, we adapted our laboratorys preexisting, evidence-based variant classification framework, called sherloc, by developing point-based criteria for the inclusion of clinical information such as a patients phenotype, familial segregation patterns, and whether the variant is inherited or de novo in the patient. as part of this process, we defined clinical criteria for 154 pidd genes. here, we illustrate the application of this method and the importance of integrating clinical information into variant interpretation. between april 2017 and october 2018, our commercial diagnostic laboratory performed 4057 immunological genetic tests, and information about the patients clinical history was provided in 2849 (70%) of these orders. restricting our analysis to just the 154 genes for which case report information is currently used in variant interpretation, these tests revealed 3868 variants, 370 (10%) of which were classified as pathogenic or likely pathogenic (p/lp). information from case report descriptions, segregation patterns, and de novo status were applied for 32%,15% and 4% of p/lp variants, respectively. in 37 (10%) cases, the clinical information provided by the clinician on the test requisition form was used as evidence in the classification of the patients variant as p/lp. ten variants were initially classified as being of uncertain significance and reclassified following receipt of further clinical information or testing of additional relatives. in addition, 35 suspicious variants of uncertain significance were identified in which one or two additional patient case reports would allow for reclassification from uncertain significance to p/lp. these data illustrate the importance of providing good quality clinical information to the genetic testing laboratory both at the time of sample submission and following the receipt of genetic test results. background: cartilage-hair hypoplasia (chh) is a skeletal dysplasia with combined immunodeficiency, variable clinical course and increased risk of malignancy, mostly non-hodgkin lymphoma and basal cell carcinoma. there is a paucity of long-term follow-up data, as well as knowledge on prognostic factors in chh. objective: we conducted a prospective cohort study in finnish patients with chh to describe clinical course and analyze risk factors for adverse outcomes. methods: we recruited 80 finnish patients with chh in 1985-1991 and performed clinical follow-up in 2011-2015. we obtained health information from finnish national medical databases (covering time period of 1969-2016), the finnish cancer registry and the cause-of-death registry of the statistics finland and analyzed all patients' health records. standardized mortality ratios (smrs) were calculated based on the population data. primary outcomes included immunodeficiencyrelated death (from infections, respiratory diseases or malignancies), the development of lymphoma and the development of skin cancer. results: the study cohort included 35 males and 45 females. median age at recruitment was 14.6 yrs (range 2 weeks -49.6 yrs) and median duration of follow-up for the surviving patients was 29.2 yrs (range 25.6 -31.0 yrs). half of the patients (46/80, 57%) had no symptoms of immunodeficiency, while 15 (19%) and 19 (24%) patients manifested symptoms of humoral or combined immunodeficiency respectively, including six cases of late-onset immunodeficiency. in a significant proportion of patients (17/79, 22%), clinical features of immunodeficiency progressed over time. of the 15 patients with non-skin cancer, eight had no preceding symptoms of immunodeficiency. altogether 20 patients had deceased (smr=7.0, 95% confidence interval (ci)=4. [3] [4] [5] [6] [7] [8] [9] [10] [11] including deaths due to pneumonia (n=4), malignancy (n=7, smr=10, 95%ci=4.1-21) and lung disease (n=4, smr=46, 95%ci=9. . malignancy was diagnosed in 21/80 (31%) patients, mostly lymphoma (n=9) and skin cancer (n=15). severe short stature at birth (compared to normal, smr/smr ratio=5. 4, , symptoms of combined immunodeficiency (compared to asymptomatic, smr=19 (95%ci=8.0-36) vs smr=4.8 (95%ci=2. 3-8.9 ), hirschsprung disease (odds ratio (or) 7.2, 95%ci=1.04-55), pneumonia in the first year of life or recurrently in adulthood (or=7. 6/19, , and autoimmunity (or=39, 95%ci=3.5-430) in adulthood associated with early mortality. in addition, recurrent pneumonia in childhood was associated with the development of lymphoma, while warts and actinic keratosis were associated with the development of skin cancer. birth length standard deviation score correlated significantly with the age at the diagnosis of first malignancy (p=0.0029), lymphoma (p=0.011) and skin cancer (p=0.014), demonstrating that patients with shorter birth length developed malignancies at an earlier age. conclusions: patients with chh have high mortality due to infections and malignancies, but also from lung disease. some subjects present with late-onset immunodeficiency or malignancy without preceding symptoms of immune defect, warranting careful follow-up and screening for cancer even in asymptomatic patients. we provide clinicians with the risk factors for adverse outcomes to assist in management decisions. autoimmune lymphoproliferative syndromes (alps and related disorders) are characterized by insufficient apoptosis due to defects in the fas apoptosis pathway. fadd deficiency (omim 602457) is an autosomal recessive disorder resulting from a mutation in fas-associated protein with death domain (fadd), the adaptor protein involved in fas signaling to caspases 8 and 10. we present a case of fadd deficiency identified by whole exome sequencing with a novel genetic mutation we describe two brothers with recurrent febrile episodes accompanied by seizures and respiratory compromise. the older sibling initially presented with status epilepticus following the measles mumps rubella vaccination later experiencing similar episodes until his demise at 18 months of age. the younger sibling, who is unvaccinated, presented at 14 months with fever, rash, vomiting, and diarrhea. he developed status epilepticus with respiratory depression that required intubation. he also had enlarged cervical lymph nodes that regressed with antibiotics and steroids. he recovered from that episode but subsequently had a series of similar illnesses with fevers, altered mental status and seizures. with the exception of elevated hhv6 igg, extensive infectious workup up in all instances was negative. previously described fadd deficiency patients demonstrate an alps like phenotype with increased circulating double negative t cells, lymphocyte apoptosis defects, elevated fas ligand and il10, encephalopathy, functional asplenism but no splenomegaly or lymphadenopathy. our patients clinical and laboratory findings were similar. he had normal igg and iga, decreased igm, and lack of isohemagglutinins. absolute cd3+ count is elevated, with elevated percent of cd3+ tcr+ cd4-cd8-. normal mitogen and antigen t lymphocyte stimulation, but with defect in pokeweed induced b cell proliferation. fas ligand and il10 level are increased (see table 1 ). no hepatosplenomegaly, but howell jolly bodies were detected in peripheral blood indicating functional hyposplenism. whole-exome sequencing revealed two different genetic alterations in the fadd gene: a maternally inherited nonsense mutation predicted to severely truncate the protein and a paternally inherited missense mutation in codon 105. although this paternal mutation has not been described as pathogenic, a different variant in same nucleotide of fadd has been associated with fadd deficiency (reference1). there are very few cases in the literature of fadd deficiency patients and the overall prognosis is poor compared to classical alps patients, as these patients are at significant risk of deadly sepsis from encapsulated organisms or death from neurologic complications. of the fadd deficiency patients described in the literature, several died prior to 5 years old. while pneumococcal prophylaxis may reduce the risk of sepsis, hematopoietic stem cell transplant has been reported for patients with fadd deficiency (reference2), and is being considered for our patient. rationale: hcuvp is a patient product-introduction program that provides cuvitruâ® (immune globulin subcutaneous [human], 20% solution [ig20gly]) free of charge for the first 4 infusions to eligible patients with primary immunodeficiency disease (pid). using patient data from this ongoing program, our analysis described the clinical characteristics and infusion parameters of pediatric and adolescent patients who were initiated on ig20gly through hcuvp. methods: hcuvp eligibility criteria were: patients aged 2 years old, with a primary icd-10-cm code verifying diagnosis of pid, and no current or prior use of ig20gly at program initiation. data from patients who received the first ig20gly infusion between january 1, 2017, and september 1, 2017 were included. data from patients receiving infusions after october 31, 2017 were censored. descriptive statistics were calculated for patients demographic and clinical characteristics and prescribed and actual infusion characteristics by age group (<18 years and 18 years). results: in total, 817 patients who completed all 4 infusions were included in the analysis, of whom 97 were aged <18 years. among those who previously received immunoglobulin (ig) therapy, a greater percentage of patients aged <18 years were treated with intravenous ig therapy (n=46; 73%) compared with adult patients (n=222; 62%) before initiating ig20gly. nine patients aged <18 years were treatment naã¯ve. the mean infusion volume per site was lower among patients aged <18 years (25 years: 17.9 ml; 611 years: 26.4 ml; and 1217 years: 34.6 ml) than among patients aged 18 years (1864 years: 38.5 ml and 65 years: 38.9 ml). however, the mean infusion rate per site was similar between patients aged <18 years ( xmen disease (x-linked immunodeficency with magnesium defect, epstein-barr virus infection and neoplasia) is a primary immune deficiency caused by mutations in magt1 and characterized by chronic infection with epstein-barr virus (ebv), ebv-driven lymphoma, cd4 t-cell lymphopenia, and dysgammaglobulinemia. magt1 gene codifies to magt1 protein, a mg2+-selective transporter, expressed in the human immune system, specifically in the spleen and the thymus. functional studies have established the key role of magt1 in t cells and natural killer (nk) cell activation. upon cd4+ t-cell receptor stimulation, magt1 mediates a transient mg2+ influx that is necessary for phospholipase c gamma 1 (plcy1) activation, which drives ca2+ rise and downstream signaling. this mg2+ influx also regulates cytotoxic functions of nk and cd8 t cells through nkgd2, reason why these patients have impaired cytolytic responses against ebv. eleven male xmen patients have been described. we present the case of a 1-year old hispanic infant with a pathogenic variant in magt1 gene that clinically manifested with early pneumocystis jirovecii and cytomegalovirus (cmv) interstitial pneumonia, and ebv chronic infection with good response to intravenous immunoglobulins supplementation without hematopoietic stem cell transplantation or gene therapy. laboratory study highlights low levels of nkg2d ligands. the objective of this case report is to broaden the spectrum of clinical presentation of xmen disease, that manifests initially as a combined immune deficiency (cid) and evolved with a favorable course of the disease with intravenous immunoglobulins supplementation therapy and chemoprophylaxis with trimethoprim-sulfamethoxazole. introduction: lysinuric protein intolerance (lpi) is a recessively inherited disorder of the cationic amino acids transporter subunit y+lat1 caused by variants in the slc7a7 gene. the disease is characterized by protein-rich food intolerance has a heterogeneous presentation. the clinical findings are a result of depletion of lysine, ornithine, and arginine. symptoms can include hyperammonemia, failure to thrive, protein aversion, neurologic disease, and lung disease. there is also evidence that inflammatory manifestations are mediated through upregulation of nfb, il1, and tnf that occur independent of intracellular arginine levels and can lead to lifethreatening episodes of hemophagocytic lymphohistiocytosis (hlh). case presentation: a 17-year-old male presented with history of anxiety, depression, eating disorder, delayed puberty and complex partial seizures. due to poor nutrition and failure to thrive, a gastrostomy tube was placed. following commencement of enteral feeds, he presented with altered mental status, bilateral mydriasis, hyperreflexia, and agitation which lead to a picu admission. ammonia peaked as high as 181 î¼mol/l and episodes ceased with cessation of enteral feedings. prior to enteral feeds, he had been self-restricting protein in his diet. biochemical testing was consistent with lpi and illumina next-generation sequencing revealed compound heterozygous variants in slc7a7 (p.s396lfs*122 and p.e465dfs*54). hyperammonemia resolved quickly with cessation of protein intake and high rate dextrose infusion without the need for ammonia scavenging agents. he was subsequently started on proteinrestricted enteral feeds. at diagnosis he did not have any respiratory symptoms, ct scan of chest showed patchy areas of groundglass opacification that was suggestive of early pulmonary alveolar proteinosis (pap). bronchoalveolar lavage demonstrated foamy, cloudy pink fluid and elevated bronchioalveolar macrophages on cell differential. his clinical course and slc7a7 genotype led to suspicion for smoldering hlh. the findings of elevated ferritin, hypertriglyceridemia, decreased fibrinogen, splenomegaly, elevated il-2 receptor, decreased nk cell function, along with hemophagocytosis on bone marrow biopsy confirmed the diagnosis. because of his pap and hlh, in addition to dietary modifications, a trial of il-1 beta inhibition (anakinra) at 3 mg/kg/day was initiated. follow up ct scan of chest 2 months after initiation of anakinra showed complete resolution of pulmonary groundglass opacifications and pap. bone marrow evaluation showed continued hemophagocytosis in spite of the normalization in ferritin, soluble il-2 receptor, nk function, and triglycerides levels. overall, he is significantly improved on daily anakinra and no longer meets criteria for hlh or pap. discussion: recent data has shown in y+lat1 models that thp-1 macrophages and a549 airway epithelial cells upregulate il1 and tnf regardless of intracellular arginine content. this suggests that inflammatory manifestations may continue independent of dietary modifications. we present a 17 year old patient with newly diagnosed lpi who was treated dietary modification and anti-il1 therapy resulting in resolution of hlh and pap. more research is needed to see if long-term il1 blockade that can consistently control both the immunologic and pulmonary manifestations of lpi and positively impact morbidity and mortality. learning objective: recognize that symptoms of bartonella endocarditis and associated complications can share features of certain immunocompromising conditions. case description: an 8-year-old caucasian boy with history of repaired pulmonary atresia and aortic root dilation was diagnosed with pancytopenia and splenomegaly during a brief hospitalization for atypical pneumonia. pancytopenia persisted, splenomegaly worsened, and five months after presentation, he developed hypertension and renal insufficiency. he was diagnosed with hypocomplementemic, diffuse sclerosing and crescentic glomerulonephritis and was started on mycophenolate mofetil with improvement in kidney function and stabilization of cytopenias. as part of a comprehensive immune work-up, alps (autoimmune lymphoproliferative syndrome) panel was sent and demonstrated elevated double-negative t (dnt) cells with 3 out of 4 positive immunologic criteria for alps. neither targeted sequencing for alps and alpslike disorders nor whole exome sequencing revealed pathogenic mutations. by age 10, the patient remained on mycophenolate, but developed failure to thrive, with weight dropping from 37th percentile to less than 3rd percentile. he was hospitalized again for low-grade fever, increased work of breathing, left shoulder pain and fatigue and was found to have right lower lobe pneumonia. pancytopenia worsened, and he was started on cefepime and azithromycin without improvement in symptoms. echocardiogram revealed vegetations in his pulmonary conduit and bilateral branch pulmonary arteries, but multiple blood cultures were negative. upon further history, the patient reported contact with kittens. bartonella henselae titers and polymerase chain reaction (pcr) from blood were sent and were both positive. he completed a 2-week course of gentamicin, 1-month course of ceftriaxone, and was transitioned to doxycycline and rifabutin. after initiating antimicrobial therapy, his weight and energy significantly improved, his blood bartonella pcr became negative, and his splenomegaly resolved. approximately one year later, the patient underwent pulmonary artery conduit replacement and bartonella pcr testing of the tissue specimen was positive. he has had sustained weight increase, resolution of hypocomplementemia and splenomegaly, decrease in dnt cell frequency from >2% to 0.9%, and improvement though not resolution of cytopenias. he currently remains on doxycycline and rifabutin and continues treatment with mycophenolate. discussion: alps is characterized by defective lymphocyte apoptosis and clinical features such as lymphadenopathy, splenomegaly, hepatomegaly, cytopenias, and glomerulonephritis. the hallmark laboratory finding is expansion of dnts. our patient met criteria for a probable alps diagnosis based on the presence of both required criteria (chronic splenomegaly and elevated dnt cells) and secondary additional criteria (typical immunologic findings noted on alps panel). pediatric cases of bartonella henselae endocarditis have been associated with splenomegaly, cytopenias, and glomerulonephritis which mimic many features of monogenic immune dysregulatory disorders. the diagnosis of bartonella endocarditis in our patient therefore raises the question of whether his immunosuppression predisposed him to infection or if his entire clinical presentation can be explained by bartonella endocarditis. physicians taking care of patients with immune dysregulatory disorders should consider bartonella endocarditis in the differential diagnosis of onset or exacerbations of immune dysregulation. rationale: while fever is considered a sign of infection, many individuals with primary immunodeficiency (pi) anecdotally report a lower than normal average body temperature. on immune deficiency foundation (idf) friends and idf pi connect research forum online, pi patients report a diminished fever response even when other signs of infection are present. there is limited knowledge about the average body temperature in persons with pi. however, the implications of missing an infection in those with pi is well established. methods: study investigators partnered with patient investigators to design a prospective cohort study to determine whether body temperature differed between persons living with and without pi. three hundred fifty adults with pi were recruited from idf and one adult household member without pi was also recruited. mckesson digital oral thermometers (model 01-413bgm) were provided and used to record temperatures in all participants three times a day for five consecutive days. descriptive statistics were calculated. median body temperatures were compared between the two cohorts at each time point using mann-whitney test. results: data from 254 households were used for analysis (72.6% participation rate). the pi population was largely female (85.8%) with a median age of 49 years and largely caucasian population (97.6%). the non-pi population was largely male (66.9%) with a median age of 53 years and largely caucasian population (92.9%). pi diagnoses included cvid (74.8%), hypogammaglobulinemia (12.6%), igg subclass deficiency (4.7%), selective iga deficiency (3.1%), specific antibody deficiency (3.1%), agammaglobulinemia (0.4%), chronic granulomatous disease (0.4%), combined immunodeficiency (0.4%), and complement deficiency (0.4%). a total of 123 individuals with pi (48.4%) reported a lower than normal non-sick body temperature, while 108 individuals with pi (42.5%) reported a normal (between 97â°f -99â°f) non-sick body temperature. a total of 172 individuals with pi (67.7%) reported absence of fever with infection, while 50 individuals (19.7%) reported a normal fever response with infection. the median body temperature was significantly higher for the pi patients in the morning, but not evening or bedtime, reading in 4 of the 5 days (monday: pi = 97.5â°f vs. non-pi = 97.2â°f, p = 0.0291; tuesday: pi = 97.4â°f vs. non-pi = 97.2â°f, p = 0.0020; wednesday: pi = 97.5â°f vs. non-pi = 97.2â°f, p = 0.0009; thursday: pi = 97.4â°f vs. non-pi = 97.2â°f, p = 0.0575; friday: pi = 97.4â°f vs. non-pi = 97.2â°f, p= 0.0008). conclusions: despite the limitations of this non-clinical study, individuals with pi are knowledgeable about their conditions and can offer unique insights and direction to researchers. this study demonstrates that collaboration with patient advocacy groups may facilitate patient-centered and patient-driven research with high participation among the target population. introduction: familial mediterranean fever (fmf) is a hereditary condition characterized by recurrent episodes of painful inflammation caused by mutations in the pyrin (mefv) gene. alterations in the mefv gene affect pyrin production leading to recurrent fevers and painful inflammation in the peritoneum, synovium, and pleura. amyloidosis may also develop as a complication. arabic, turkish, armenian, and sephardic jewish populations are most commonly affected. homozygosity for mefv mutations are associated with a more severe course. there is a paucity of information regarding pediatric fmf in the literature. case: we present a case of a 2-year-old male with minor speech delay diagnosed with compound heterozygous fmf. patient was initially referred due to recurrent fevers and infections. at 4 months of age, he was hospitalized with septic shock requiring intubation secondary to adenovirus. at 5 months of age, the patient began to have recurrent fevers every 3 to 4 weeks, leading to multiple blood draws and courses of antibiotics prior to referral. at 11, 12, and 22 months of age, he developed three separate episodes of febrile seizures. a total of 10-15 lifetime episodes of acute otitis media occurred prior to bilateral myringotomy tube placement. four episodes of streptococcus pyogenes pharyngitis confirmed by throat culture preceded tonsillectomy. no oral ulcers, joint pain, or abdominal pain were reported. no other infections such as pneumonia, sinusitis, uti, non-viral gastroenteritis, fungal infections, or skin infections were reported. both parents are ashkenazi jewish and a maternal history of early miscarriage was noted. family history was negative for immunodeficiency, malignancy, and autoimmunity. the patients vital signs and physical exam were unremarkable. serology indicated leukocytosis of 18.53 k/l with elevated monocytes of 1390 cells/l, elevated eosinophils of 1200 cells/l, and slightly elevated cd8 t cell count of 2653 cells/l. neutrophil, cd4 t cell, b cell, nk cell enumeration, and immunoglobulin panel were normal for age. tetanus, diphtheria, rubella, streptococcus pneumoniae, and haemophilus influenzae b titers were protective. genetic analysis identified that the patient was compound heterozygous for the e148q and v726 mutations in the mefv gene. family was instructed to keep a fever diary. colchicine 0.6mg once a day was given initially, then increased to 1.2mg once a day for inadequate response. loose stools were observed while patient was maintaining a lactose free diet so he was switched to colchicine 0.6mg bid with resolution of loose stools. apart from two occasions when his colchicine dose was missed, the patient remained afebrile at his follow up visits. conclusion: we present a pediatric case of compound heterozygous fmf (e148q and v726 mefv mutations) in an otherwise healthy 2-year-old male of ashkenazi jewish background, initially symptomatic at 5 months of age. individuals who are compound heterozygous for the e148q and a second mevf mutation are generally symptomatic, although severity cannot be predicted. additional pediatric research on symptomatic heterozygous and compound heterozygous fmf is recommended. natural killer (nk) cells are innate lymphocytes that play a key role in defense against virally-infected cells and in tumor surveillance. nk cells can be divided in two subsets. the majority of nk cells in peripheral blood expressed intermediate levels of cd56 and are referred to as cd56(dim). these nk cells are responsible for nk cell cytotoxicity. a minor population of nk cells express very high expression of cd56 and are referred to as cd56(bright). these nk cells are responsible for cytokine production and are precursors to cd56(dim) nk cells. a few immunodeficiencies have been described in which there are abnormal nk cell subsets, such as autosomal dominant gata2 deficiency where cd56(bright) nk cells are absent and irf8 where there is a paucity of cd56(dim) nk cells and relative expansion of cd56(bright) nk cells. here we present a patient with an absence in cd56(bright) nk cells secondary to cd27 deficiency. our patient is a 6-year-old african american female born to non-consanguineous parents. the patients past medical history is significant for chronic lung disease secondary to prematurity, recurrent acute otitis media, failure to thrive and congenital hypothyroidism. family history is significant for an older sister that presented at age 3 with ebv-associated hodgkin lymphoma whose treatment was complicated by chronic activated ebv infection and who ultimately underwent hematopoietic stem cell transplantation (hsct). our patient presented with pancytopenia, fever, lymphadenopathy and splenomegaly. she was found to have ebv viremia with greater than 550,000 copies in whole blood by pcr. she was treated with two doses of rituximab followed by etoposide and dexamethasone as a bridge to hsct. whole exome sequencing demonstrated a homozygous mutation in cd27. cd27 is a member of the tumor necrosis factor receptor family and influences the function of t cells, b cells and nk cells. in nk cells, cd27 is primarily expressed in cd56(bright) nk cells. cd27 deficiency is an autosomal recessive disorder associated with persistent symptomatic ebv viremia, including ebv-driven hemophagocytosis and lymphoma, hypogammaglobulonemia and specific antibody deficiency. our patients immune evaluation prior to initiation of chemotherapy and immunosuppression was notable for very elevated igg, iga and igm. despite hypergammaglobulonemia patient had only 3 out of 11 protective titers against streptococcus pneumoniae. the patient had pan-lymphopenia with appropriate percentages of lymphocyte subsets. assessment of her b cell subsets showed a slight increase in the percentage of transitional b cells/plasmablast and a nearly complete absence of cd27-expressing b cells. her nk cell phenotyping demonstrated a complete loss of cd56(bright) nk cells with reduced nk cell cytotoxicity, comparable to what has been previously reported in patients with gata2 deficiency. previous reports of patients with cd27 deficiency denote normal nk cell numbers with normal to moderately reduced nk cell cytotoxicity, however, cd27 deficiency causing a specific loss of the cd56(bright) nk cell subset has not been previously reported. cd27 deficiency should be consider in patients with ebv driven disease and abnormal nk cell studies. introduction/background: the transcription factor ikaros is encoded by the ikzf1 gene and plays a crucial role in lymphopoiesis. somatic, and more recently also germline mutations of ikzf1 are associated with a hematologic malignancies, most notably b-cell precursor acute lymphoblastic leukemia. germline mutation in ikzf1 was first reported as a monogenic cause of human disease characterized by marrow failure and immune deficiency in a single neonate in 2012. subsequently, mutations leading to haploinsufficiency were discovered to underlie a proportion of patients with cvid and low b cell numbers, and dominant-negative mutations have been observed to cause more severe combined immune deficiency phenotypes. at this time, there is very little known regarding allogeneic hematopoietic cell transplantation (hct) outcomes for patients with severe dominant-negative ikzf1 mutations. concerningly, ikaros deficiency has been observed to have a negative impact on graft versus host disease in mouse models. objective: to describe allogeneic stem cell transplant outcomes in patients with the dominant-negative ikaros mutation. methods: we collected transplant data from 4 patients who underwent allogeneic hct at transplant centers around the world. results: patients underwent allogeneic hct using a variety of conditioning regimens. patients received bone marrow (n=3) or cord blood (n=1) grafts from an hla-matched sibling donor (n=1) or single allele hlamismatched unrelated donor (n=3). neutrophil engraftment occurred between day +12 and +51 post-transplant. platelet engraftment occurred between day +8 and +167 except in one patient who did not have return of normal platelet counts due to underlying liver dysfunction. all patients were documented to have greater than 99% whole blood donor chimerism at a median of 28 days (range 12-51 days) following transplant and maintained >95% donor chimerism until last follow-up. only one patient developed grade ii acute gvhd. no patients developed chronic gvhd. one patient died approximately 1 year post transplant related to cryptosporidium cholangitis which existed prior to hct. at the most recent follow up of the 3 surviving patients (range: 0.99-7.2y), ivig had been discontinued, antimicrobial prophylaxis had been stopped, and patients had received routine vaccinations. they all had excellent performance status. conclusions: allogeneic hct may be a safe option to consider for patients with dominant-negative ikaros mutation as there does not appear to be an increased risk of death or gvhd. moreover, 3-out-of-4 of the transplanted patients are alive and well and show no features of the disease. however, because of the limited number of patients evaluated and the retrospective nature of this analysis, our data do not allow firm conclusions to be made, and further studies will be needed to evaluate outcomes in larger cohorts. introduction: when evaluating patients with t-cell lymphopenia, we often are concerned about defects in lymphocyte production and function, especially in the setting of frequent infections. here we outline a case demonstrating t-cell lymphopenia due to increased loss, which should be considered in the differential diagnosis. case report: we report a 13-year-old male who initially presented with recurrent, right-sided pneumonias requiring frequent hospital admissions including severe episodes necessitating intensive care unit admission. his work up for the pneumonias included a bronchoscopy revealing normal anatomy with minimal inflammation, and a chest ct with mild peribronchial wall thickening. as his pulmonary disease progressed, he developed a persistent, productive cough with expectorated mucous plugs that were plastic-like in appearance. while his pulmonary symptoms responded to steroids, his mucous plug production persisted. sputum cultures were intermittently positive, isolating cryptococcus neoformans and aspergillus niger. he underwent vats and wedge biopsy, concerning for recurrent aspiration. an immunologic evaluation initially demonstrated normal t-and b-cell counts, but serial evaluation of his lymphocyte population demonstrated low cd4+ cells (ranging 151-367 cells/cumm), and low normal cd8 cells (ranging 101-177 cells/cumm) with normal b-and nk-cell numbers. further t-cell evaluation revealed normal ratios of naive and memory p o p u l a t i o n s ( c d 4 c d 4 5 r a + 6 1 % , c d 4 c d 4 5 r o + 3 9 % , cd8cd45ra+ 74%, cd8cd45ro 33%), normal trec (7768 copies per 10^6 cd3 cells) and normal thymic emigrants (cd4cd31cd45ra+ : 158, normal 150-1500), indicative of sufficient thymopoiesis. mitogen and antigen stimulation assays demonstrated normal responses to phytohemagglutin, concanavalin a, and pokeweed mitogen, with a low lymphocyte response to candida. he had normal quantitative immunologlobulins, normal diphtheria, tetanus and streptococcus pneumonia titers. his dihydrorhodamine flow cytometry and fish for chromosome 21q11.2 deletion were negative. given normal function and thymic output, his immunologic profile was concerning for t-cell loss. our patient was registered with the undiagnosed disease network, and had a second review of his lung biopsy, concerning for plastic bronchitis. subsequent lymphatic imaging demonstrated abnormal lymphatics within the bilateral clavicular space, right greater than left, with questionable partial thoracic duct, explaining his unilateral symptoms. he was diagnosed with plastic bronchitis secondary to abnormal lymphatic drainage, with lymphatic fluid filling his airways and secondary t-cell loss. discussion: plastic bronchitis is a rare and potentially fatal disorder, seen commonly after the fontan procedure for congenital heart disease. this process has resulted in t-cell loss into the airway and subsequent t-cell lymphopenia. in patients with fontan-related protein losing enteropathy, multiple immune abnormalities have been described including reduced immunoglobulins, lymphopenia, and selective cd4 lymphocyte deficiency. similar findings have been reported in patients with lymphatic malformations. although the impact of t-cell loss on adaptive immunity is not entirely known, there is no indication of increased risk for atypical infections. given his normal mitogen assay, our patient did not start prophylactic antibiotics. he continues to have symptomatic episodes with lymphopenia, but has had no opportunistic infections, and remains stable with an aggressive pulmonary regimen. we conclude by reiterating the importance of considering t-cell loss in patients presenting with lymphopenia, particularly with evidence of normal thymopoeisis and t-cell function. introduction: granulomatous disease (gd) has been described with a variable incidence (8.0-22.0%) in patients with common variable immunodeficiency (cvid). an increase in malignancies has been reported in cvid patient cohorts, particularly for lymphoma, reported in 1.6-8.2% of the cvid patients depending on the cohorts. prior analysis of a cohort of 436 cvid patients included 59 patients with gd (gd+). in these, there was a suggestion of more cases of lymphoma (12.5%) when compared to cases without (gd-) (5.0%) although the difference was not statistically significant (p=.07). objectives: compare the frequency of lymphoma in gd+ and gd-patients in the cvid patient cohort from the usidnet registry. methods: we submitted a query to the usidnet registry requesting deidentified data for patients with the diagnosis of cvid, through august 2018. statistical analysis was performed on spss, with comparisons done with pearson chi-square or fisher's exact test, depending on the sample sizes, using an alpha level of .05. results: a cohort of 1395 cvid patients from the usidnet registry was analyzed. ninety-one patients (6.5%) were gd+. overall, 152 patients (10.9%) had a malignancy diagnosis, 47 of these (3.4%) with lymphoma. lymphoma was present in 6/91 gd+ patients (6.6%) versus 41/1304 gdpatients (3.1%) (p=.12). overall malignancy was present in 15/91 gd+ (16.5%) versus 137/1304 (10.5%) (p=.08). discussion: in the cohort of 1395 cvid patients from the usidnet registry, we found a frequency of lymphoma of 3.4%, which is in the range of previously described cohorts. the frequency of lymphoma was 6.6% in patients with gd, higher than the 3.1% frequency for gd-patients, but these differences were not statistically significant. our identified frequency of lymphoma in gd+ patients was lower than the one previously identified in the 436 cvid patient cohort, but with similar proportional differences between gd+ and gd-patients. despite no statistical significance, the frequency of lymphoma, as shown here and elsewhere, was higher in cvid patients gd+ than gd-in both studies, with no full understanding of this increased risk of lymphoma. expanding this analysis to larger groups of cvid patients may help to confirm, or deny a more robust association, which may have a meaningful impact in the outcomes of this particular population. introduction: patients with refractory pericarditis have been treated with intravenous immunoglobulin (ivig) or interleukin 1 receptor antagonist (anakinra) with limited and transient benefit. separate or combined therapy with subcutaneous immunoglobulin (scig) and interleukin (il) 1 inhibitor (rilonacept) for refractory pericarditis in a cohort of patients has not been previously described. case descriptions: 4 patients were referred for recurrent pericarditis refractory to traditional therapies at ages ranging from 16 to 54 years. they all had multiple serious sequelae of their pericarditis and abnormal immune parameters including hypogammaglobulinemia, poor responses to vaccines, poor mitogen induced lymphocyte proliferation, and/or b cell lymphopenia. the patients had varied past medical histories and associated conditions. patients were started on ig, with some initiated on ivig, though all were transitioned to hyaluronidase-facilitated scig (hyqvia). patients were then started on either anakinra or rilonacept with 3 patients continuing on rilonacept and 1 remaining on anakinra. all patients had complete or near complete resolution of their pericarditis on dual therapy for greater than 1 year. the markedly elevated il1 prior to therapy seen in all of the patients normalized post-therapy. some patients had elevated il6 prior to therapy that also improved post-therapy. 1 patient who has also been diagnosed with familial mediterranean fever (fmf) has stopped both therapies for greater than 1 year with no further episodes of her pericarditis. discussion: 4 patients with recurrent refractory pericarditis and signs of immunodeficiency and autoinflammatory disease on laboratory testing responded to dual therapy with hyqvia and rilonacept or anakinra resulting in resolution of pericarditis. inflammasome and immune abnormalities may be implicated or associated with recurrent pericarditis and may respond to targeted therapies. chief, laboratory of clinical immunology and microbiology, idgs, dir, niaid, nih, bethesda, md, usa hypomorphic recombination activating gene 1 (rag1) mutations result in residual t-and b-cell development in both humans and mice and have been found in patients presenting with delayed-onset combined immune deficiency with granulomas and/or autoimmunity (cid-g/ai). recent studies have shed light on how hypomorphic rag1 mutations alter the primary repertoire of t and b cells, but less is known about their effect on immune dysregulation in targeted organs. in order to investigate the role of these mutations in determining intestinal disease, we set out to evaluate gut immunity and microbiota interplay in rag1 mutant hypomorphic mice. we evaluated two mouse models carrying homozygous rag1 mutations (r972q and r972w), corresponding to human mutations (r975q and r975w, respectively) described in patients with cid-g/ai. both mutations fall in the coding flanksensitive region of the rag1 c-terminal domain. on the basis of aminoacid properties and in vitro studies, the r972q mutation has demonstrated a moderate effect on rag1 protein stability while the r972w mutation resulted highly disruptive. analysis of intestinal pathology in rag1 mutant mice (niaid animal protocol lcim 6e) revealed different degrees of spontaneous colitis, with the most severe inflammatory infiltrate observed in mice carrying the most disruptive mutation, r972w. colonic inflammation was characterized by crypt elongation, epithelial hyperplasia, and an abundant inflammatory infiltrate extending to the colonic lamina propria, with occasional crypt abscesses. a significant increase in activated cd44hicd62lcd4+ t cells expressing the gut homing receptor 47 was observed in mesenteric lymph nodes (mlns) of both mutant strains, and was especially prominent in r972w mutant mice. additionally, the proportion of mln cd4+ t regulatory (treg) cells was increased in both mouse models. finally, mln of mutant mice contained a high number of myeloid cells (cd11b+ ) along with a decreased number of b220+ b cells, and these abnormalities were also more prominent in r972w than in r972q mice. in summary, we have shown that rag1 mutant hypomorphic mice present with different degrees of inflammatory bowel disease, with the mouse model carrying the most disruptive mutation presenting with the most severe phenotype. we are currently performing studies to evaluate the impact of rag1 mutations on microbiome composition and diversity in these mouse models of cid-g/ai. background: hypogammaglobulinemia or low serum immunoglobulin g (igg) levels either inherited (primary) or acquired (secondary) is associa t e d w i t h i n c r e a s e d i n f e c t i o n r a t e s . p r i m a r y ( 1â°) hypogammaglobluinemia can be caused by many primary immune deficiencies (pid) including combined variable immune deficiency (cvid), while secondary (2â°) hypogammaglobluinemia can be caused by many acquired conditions such as lymphomas, leukemias, or chemotherapies and other immunosuppressive drugs. immunoglobulin replacement therapy (irt) has been the mainstay of treatment in patients with hypogammaglobulinemia by reducing infection through replenishing the quantitative igg. there are other applications of ig therapy such as in autoimmune diseases, where the mechanism of action is thought to be ig mediated immunomodulation. innate immune cells have shown to be involved in such mechanism, but whether irt modulates adaptive immune cells in patients with hypogammaglobulinemia is not well known. hypothesis: irt has an immunomodulatory effect on t-cell function and proliferation in patients with hypogammaglobulinemia. methods: blood from thirty patients with 1â°(n=12) or 2â°(n=18) hypogammaglobulinemia recruited from the immunodeficiency clinic at the ottawa hospital was drawn for peripheral blood mononuclear cell (pbmc) isolation, before starting irt and minimum 8 weeks after starting irt. data regarding igg level, number and type of infections after receiving irt was collected. pbmcs were analyzed using flow cytometry for quantitation of t-cell subset. cultured and anti-cd3/cd28 stimulated pbmc were also analyzed for extracellular and intracellular cytokine production, measured by e l i s a a n d f l o w c y t o m e t r y, r e s p e c t i v e l y. c o m b i n e d cytomegalovirus, epstein-barr virus and influenza virus (cef) peptides were used to study specific t-cell responses. anti-cd3/ cd28 stimulated pbmc were used for celltrace t-cell proliferation a s s a y s . d a t a w a s g r o u p e d b a s e d o n n a t u r e o f hypogammaglobulinemia i.e. 1â°or 2â°. results were compared between before and after irt using wilcoxon matched-pairs signed rank test. results: irt was not found to significantly alter proportion of treg, cd4+, or cd8+ t-cell populations or activation state as measured by cd45ra/r0 expression. however, irt was found to significantly increase expression of intracellular ifn-y in cd4+ and cd8+ t-cells post-cd3/cd28 stimulation in 2â°(p = 0.007), but not in 1â°h ypogammaglobulinemia patients. there was no change in extracellular il-10 and il-17 cytokine production in both groups. in contrast, cd8+ tcells in 1â°hypogammaglobulinemia patients showed significantly higher expression of intracellular ifn-y and tnf-a post-cef viral peptide stimulation (p = 0.027). cd3+ and cd8+ t-cell proliferation after cd3/cd28 stimulation was found to be decreased after irt for both groups (p = 0.025 & p = 0.049). conclusions: our results suggest that irt can alter cd4+ and cd8+ t-cell function with differential effect in patients with 1â°o r 2â°hypogammaglobulinemia in addition to replenishing serum igg level. more experiments assessing cytotoxicity of t-cells will be conducted to further study t-cell subset function as well as bcell function. these laboratory results will be analyzed for association with clinical outcomes. uploaded file(s) uploads background: severe congenital neutropenia (scn) is a rare immunodeficiency disorder characterised by the extremely low absolute neutrophils count (anc) less than 0.5x109/l. the clinical feature of scn is recurrent bacterial infections and the patients the risk of leukemia development. the incidence of scn is estimated to be 1 in 200 000 individuals. mutations in more than 20 genes have been described causing scn and it is either recessive, dominant or x-linked inheritance. case presentation: we described an 11 years old malaysian girl who presented with recurrent abscesses over the whole part of the body, recurrent oral candidiasis, growth failure and recurrent pneumonias since 4 months old. she also had history of a few episodes of acute tonsillitis, chronic suppurative otitis media and herpes zoster infections. throughout her age, she had persistent neutropenia less than 0.5x109/l but in few occasions, her anc elevated up to more than 1.0x109/l . she was treated as autoimmune neutropenia, respectively due to few positive results of autoimmunity workout such as antinuclear antibodies (ana) and double stranded dna (dsdna) but eventually later to be negative. later at the age 9 years old, whole exome sequencing was performed and confirmed by sanger s e q u e n c i n g , f o u n d a h e t e r o z y g o u s v a r i a n t i n e l a n e gene(c.640g>t; p.gly214ter), an autosomal dominant which was described to cause scn. both parents do not carry this mutation, hence, it is a de novo mutation. currently, she had few on and off recurrent infections. despite that, she is relatively well and on prophylaxis antibiotic. conclusion: to our knowledge, we report for the first time a malaysian girl with scn, with confirmed mutational analysis of the elane gene. the delayed diagnosis might be due to the insufficient awareness of the phenotypic presentation of this rare disease. moreover, the genetic analysis is not available in malaysia and need to be done outside of the country. this case demonstrates the importance of the genetic analysis which may help in improving the diagnosis and management of the patient. (69) submission id#600360 professor of paediatrics and immunology, university college london; great ormond street hospital nhs trust; orchard therapeutics, london, uk background: ada-scid is a rare genetic disorder which causes severe combined immunodeficiency. historically, ada-scid has been treated using enzyme replacement therapy (ert) followed by allogeneic hematopoietic stem cell (hsc) transplant (hsct) from a matched related donor (mrd) or, if none is identified, a non-mrd (matched/mismatched unrelated or mismatched related donor). we developed a self-inactivating lentiviral vector (lv), in which a codon optimized human ada cdna is driven by the short form of the elongation factor-1alpha (efs) promoter (efs-ada lv). the drug product (otl-101), composed of autologous hscs transduced ex vivo with the efs-ada lv, was evaluated in a prospective, historically-controlled phase i/ii clinical trial in ada-scid pediatric subjects. we report safety and efficacy at 24 months in 20 ada-scid subjects treated with lentiviral gene therapy (gt) compared to a historical cohort of 26 ada-scid patients treated with hsct. methods: twenty subjects (9 male, 11 female; 4 mo 4.3 yrs) were treated with gt. autologous cd34+ hscs were isolated from bone marrow and pre-stimulated with cytokines before transduction with efs-ada lv. busulfan was administered at a single dose (4 mg/kg) prior to infusion of otl-101. the control group included 26 patients (0.2 mo 9.8 yrs) treated with allogeneic hsct (mrds n=12, non-mrds n=14) at great ormond street hospital, uk (n=16) or duke university childrens hospital, usa (n=10) between 20002016. results: at 24 months, overall survival (os) and event-free survival (evfs), defined as survival in the absence of ert reinstitution or rescue allogeneic hsct) were statistically significantly higher in the gt group compared with the hsct group (table) . successful engraftment of genetically modified hsc was observed in all gt subjects at 6 months, which persisted over 24 months, based on vector gene marking in granulocytes (median 0.085 copies/cell [range 0.04-2.50] at 24 months) and peripheral blood mononuclear cells (median 0.843 copies/cell [range 0.13-1.86] at 24 months), and was associated with increased red blood cell ada enzyme activity and metabolic detoxification from deoxyadenosine nucleotides. over 24 months, none of the gt subjects required peg-ada ert reinstitution and 90% were able to stop receiving immunoglobin replacement therapy (igrt), whereas 38% hsct patients required rescue hsct or reinstitution of peg-ada ert, and 52% were able to stop receiving igrt (table) . nine subjects in the gt group experienced a serious adverse event (sae), most frequently infections and gastrointestinal events; only one was considered treatment-related. in the gt group, there were no events of autoimmunity during the study. due to the autologous nature of the product, there was no incidence of graft vs host disease (gvhd) in the gt group; whereas 5 patients in the hsct group experienced acute gvhd and 3 experienced chronic gvhd events, one of whom died. conclusions: treatment with lentiviral gt for ada-scid is well tolerated and has a favorable benefit-risk profile at 24 months based on sustained gene correction and restoration of immune function, as well as improved os and evfs compared with hsct (mrd or non-mrd) at 24 months. background: ada-scid is a rare genetic disorder that causes severe combined immunodeficiency, with minimal or absent b cell function. prior to, and often after, treatment with allogeneic hematopoietic stem cell (hsc) transplant (hsct) or autologous ex vivo hsc gene therapy (gt), patients are managed with enzyme replacement therapy (ert) and immunoglobulin (ig) replacement therapy (igrt). we evaluated a gt treatment with autologous hscs transduced ex vivo with a self-inactivating lentiviral vector (lv), in which a codon optimized human ada cdna is driven by an internal short form of the elongation factor-1alpha (efs) promoter ("efs-ada lv"). at 24 months follow-up, 20 pediatric ada-scid subjects treated with gt were compared to a historical cohort of 26 ada-scid patients treated with hsct. here, we report on b cell reconstitution in these cohorts. methods: twenty subjects (9 male, 11 female) aged 4 mo -4.3 yrs received gt. autologous cd34+ hscs were isolated from bone marrow and pre-stimulated with cytokines before transduction with efs-ada lv. genetically modified cells were administered after conditioning with single dose busulfan (4 mg/ kg). the control group included 26 patients aged 0.2 mo to 9.8 yrs treated with hsct at great ormond street hospital (uk) (n=16) or duke university children's hospital (us) (n=10) between 2000 -2016. the hsct patients received an allogeneic transplant from matched related donors (mrds) (n=12) or non-mrds (n=14). subjects continued to receive igrt post-gt until a clinical decision was made to stop, factoring in b cell reconstitution, general medical condition and seasonal infections. results: by month 12, in the gt group, 45% had stopped treatment with igrt compared to 38% in the hsct group overall. by months 18 and 24, higher proportions of gt-treated subjects had stopped igrt (70% and 90%, respectively) compared with mrd hsct patients (55% and 70%, respectively) and non-mrd hsct patients (42% at both timepoints) (table) . in the gt group, vector gene marking was detectable in peripheral blood mononuclear cells within 3 months and persisted at 24 months post-infusion (median 0.843 copies/cell [range 0.13-1.86]), suggesting successful gene modification. as evidence of b cell reconstitution, iga and igm levels in peripheral blood sera more than doubled by 18 months, from 18.5 mg/dl (range 8 to 95) to 48.0 mg/dl (range 20 to 110) and 32.5 mg/dl (range 16 to 107) to 69.0 mg/dl (range 20 to 180), respectively. additionally, antibody response following tetanus vaccination, was evaluated in 3 subjects. all 3 subjects mounted a protective response to the vaccine (median antibody response 3.2 iu/ml [range 0.1 to 3.5]), based on a normal threshold of 0.01 iu/ml (hammarlund clin infect dis 2016) and a laboratory reference range (0.10 to 2.9 iu/ml). conclusions: gt with autologous hscs transduced ex vivo with efs-ada lv resulted in b cell reconstitution, as evidenced by doubled iga and igm production at 18 months, cessation of igrt in 90% of patients by 24 months, and protective specific antibody responses to tetanus vaccine in patients that were evaluated. background: x-linked chronic granulomatous disease (xcgd) results from mutations in cybb encoding the gp91phox subunit of phagocyte nadph-oxidase. attempts to treat xcgd with gene therapy (gt) using transduced autologous hematopoietic stem cells (hsc) transduced ex vivo with a gammaretroviral vector have met with limited efficacy due to transient engraftment of gene corrected hscs, gene silencing, and vector insertion-mediated activation of oncogenes leading to myelodysplasia. we developed a novel self-inactivating (sin) lentiviral vector (g1xcgd lv) with a chimeric cathepsin g/cfes myeloid-specific promoter driving gp91phox expression from a codon optimized cdna. following transplant of g1xcgd lv ex vivo transduced autologous hscs into busulfanconditioned xcgd patients, there was long-term restoration of oxidase activity in peripheral blood polymorphonuclear neutrophils (pmn) at 12 months in 6 of 9 severely affected xcgd patients without evidence of genotoxicity. here we present data about the multiple assays used to assess quality and quantity of restoration of pmn oxidase activity. methods: similar trials of gt with g1xcgd lv were initiated in the uk (n=3, plus 1 compassionate use patient) and usa (n=5). all patients had histories of inflammatory disease and severe, persistent infections (some non-responsive to conventional therapy at time of gt). g-csf plus plerixafor-mobilized cd34+ hscs were transduced with ex vivo g1xcgdlv. subjects received myeloablative conditioning with singleagent busulfan, targeted to net area-under-the-curve of 70,000 ng/ml*hr. freshly prepared or cryopreserved quality-tested genetically-modified hsc, manufactured on-site, were administered intravenously. pmn oxidase activity post-gt was assessed by p-nitroblue tetrazolium (nbt) reduction, dihydrorhodamine (dhr) flow cytometry assay, and quantitative ferricytochrome c assay (ferric) measurement of superoxide generation. results: we report results for 7 patients (aged 2-27 years) with 1-2.5 years of follow-up; two additional patients were treated but died within three months of gt from complications deemed related to pre-existing diseaserelated co-morbidities (severe pulmonary disease and anti-platelet antibodies). within 1 month post-gt, oxidase (+) pmn were present in peripheral blood based on nbt testing and dhr flow cytometry. expression of the corrective transgene was confirmed by flow cytometry using antibody detection of gp91phox. quantitative biochemical measurements of oxidase activity were also confirmed in some samples using the ferric assay, demonstrating quantitative levels of superoxide production per corrected cell that were within the normal range. functional testing of oxidase burst activity using dhr fluorescent assays was applied serially to follow levels of corrected pmn where oxidase activity per corrected cell also were in the normal range. all patients had >15% pmn dhr+ within one month, which remained stable for most patients over the follow-up period ( figure) . follow-up demonstrated sustained stable persistence of 12-46% oxidase burst positive neutrophils in 6 of 7 surviving subjects at 12 months, with restoration to clinically beneficial levels (defined as 10% of pmn being dhr+) in these patients as of december 2018. conclusion: these results demonstrate corrected pmn function within 1 month in x-cgd patients treated with autologous gt. pmn oxidase activity was sustained at levels which restore biochemical function and provide clinically beneficial levels of immunity for 12 months in 6/7 patients. the formulation for igsc 20% was developed based on the knowledge acquired from the formulation of grifols currently licensed 10% immune globulin (human), gamunexâ®-c; however, the protein concentration was increased from 10% to 20% to facilitate efficient subcutaneous administration. gamunex-c has an extensive record of safety and tolerability when administered intravenously and subcutaneously for greater than 15 years in diverse patient populations. the igsc 20% manufacturing process employs the same purification steps as gamunex-c and was demonstrated to be robust and to provide an igg product with the required potency, purity, and quality. the formulation excipient characteristics and compatibility with the drug product have been well established. glycine has been an excipient of intramuscular immune globulin (human) for fifty years and intravenous immune globulin (igiv) for over twenty years. the igsc 20% formulation has low buffering capacity, and a low ph was selected to achieve a product with low aggregates, low fragments and viscosity suitable for subcutaneous administration. to improve visual clarity, the igsc 20% formulation contains a small amount of polysorbate 80 (ps80), which is widely used in biopharmaceutical products. subcutaneous administration of the igsc 20% formulation has been well tolerated in clinical studies. objectives: the goal was to provide the pid population with a new 20% immunoglobulin liquid product for subcutaneous administration (igsc 20%). methods: igsc 20% is manufactured using the current manufacturing process for gamunex-c, followed by an additional concentration step so that the product can be formulated at a higher protein concentration. igsc 20% and gamunex-c batches were produced at full industrial scale and then subjected to a series of analytical testing including assessment of purity, composition and neutralizing activity. results: the igsc 20% and gamunex-c manufacturing processes and formulations have preserved the igg integrity, molecular characteristics and potency. the manufacturing processes have eliminated lipids, alcohols, and acetate and coagulation factor impurities, including fxia, which were undetectable by either specific or global methods. the igsc 20% and gamunex-c batches were 100% gamma globulin by agarose membrane electrophoresis, and have a subclass distribution similar to normal plasma and acceptable specific antibody content. igsc 20% was shown to be primarily monomer plus dimer igg (99â±1%) with minimal aggregate or fragment, which confirms that appropriately gentle processing conditions were used during the concentration of 10% igg solutions to 20% igg. conclusions: igsc 20% is a highly concentrated igg solution with characteristics comparable to gamunex-c, but with twice the igg concentration in order to facilitate subcutaneous administration with reduced volumes and shorter infusion times. analytical testing demonstrates suitable potency, purity, and neutralizing activity for a number of specific antigens. funding: this study was funded and conducted by grifols, a manufacturer of 20% immunoglobulin for subcutaneous administration. disclosure: all authors are employees of grifols. frequent respiratory tract infections and seizures cause recurrent hospitalizations in these children and are typically considered a result of neurological impairment and poor airway clearance. evaluation of these patients for immunodeficiency is not a common clinical practice. here we report combined immune deficiency in 2 patients with mds and recurrent respiratory tract infections. case presentation case 1: a boy with mds was initially referred at age 2 months for an abnormal newborn screen with low t cell receptor excision circles (trec) for severe combined immunodeficiency (scid). initial evaluation revealed moderate cd3+ and cd4+ t cell lymphopenia (figure 1). initial immunoglobulins levels were normal. he was placed on antiseizure medications. he later developed recurrent and severe respiratory tract infections starting in infancy. at 12 months of age, he developed hypogammaglobulinemia ( figure 2 ). in addition, t cell counts progressively decreased and stayed around 600 cells/ul. immunoglobulin replacement therapy started at 18 months of age. hospitalizations due to respiratory tract infections significantly decreased. case 2: a 3-year-old boy with mds had recurrent bacterial and viral respiratory infections which required numerous hospitalizations including intensive care unit stays. newborn screening for scid was negative. he had been on anti-seizure medications. immunologic evaluation at 3 years of age revealed low total cd3+ cells and cd8+ t cells (cd3+: 1284cells/ul[normal range 1400-3700cells/ul], cd8+:278cells/ ul[normal range 490-1300cells/ul]), hypogammaglobinemia (igg: 252mg/dl[normal range 453-916mg/dl]), and non-protective igg levels to tetanus, varicella and pneumococcus serotypes. immunoglobulin replacement therapy started at 3 years of age which resulted in reduced frequency and severity of respiratory infections, and improved quality of life. discussions: t cell lymphopenia and hypogammaglobulinemia were seen in both our cases of miller-dieker syndrome. to our knowledge, immune deficiency has never been reported in mds. one of our cases suggests that low t cell counts may start as early as at birth and may be detected by newborn screening. hypogammaglobulinemia may be primary or secondary due to antiepileptics. both children had reduced frequency and severity of respiratory infections and improved quality of life after immunoglobulin replacement highlighting the importance of screening and early management of immunodeficiency. conclusion: miller-dieker syndrome is likely another syndromic primary immune deficiency disorder. a high index of suspicion with early screening and management of immunodeficiency may be beneficial for children with miller-dieker syndrome. uploaded file(s) uploads this prospective, multi-center, open-label study assessed the pharmacokinetic (pk), safety, and tolerability of immune globulin subcutaneous (human), 20% caprylate/chromatography purified (igsc 20%) in subjects with primary immunodeficiency (pi). the objectives were to determine a weekly subcutaneous (sc) dose of igsc 20% that is noninferior to the intravenous (iv) dose of immune globulin injection (human), 10% caprylate/chromatography purified (igiv-c 10%) and to determine the steady state trough igg levels after igsc 20% and igiv-c 10% infusions. there were 3 possible phases. if not on a qualifying igg regimen at enrollment, subjects (n=44) were required to enter the run-in phase, receiving igiv-c 10% to achieve steady-state before entering the iv phase to determine steady-state area-under-the-curve (auc) of iv infusions. subjects with a qualifying igiv-c 10% regimen (300-800 mg/kg) (n=9) directly entered the iv phase for steady-state iv pk assessments. upon completion of the iv pk assessments subjects entered the sc phase, receiving weekly doses of igsc 20% for up to24 weeks, with steady-state auc determined at the 13th dose. igsc 20% was not associated with any reports of serious local infusion site reactions (isrs). the majority of local isrs were mild-to-moderate. igsc 20% (at a dose conversion factor of 1.37) provided equivalent exposure to igiv-c 10% as assessed by steady-state auc0-7 days, with 33% higher mean igg trough values, lower fluctuations in igg concentrations and the flexibility of at home administration. igsc 20% was well tolerated with a safety profile comparable to igiv-c 10%. clinicaltrials.gov identifier: nct02604810 disclosure: kecia courtney, elsa mondou, and jiang lin are employees of grifols, a manufacturer of igsc 20%. grifols is the sponsor of this study. background: in 2014 two reports described the deficiency of adenosine deaminase 2 (dada2) as early-onset lacunar strokes, intermittent fevers, livedoid rash, and early onset polyarteritis nodosa (pan). since these first reports, the clinical spectrum has dramatically expanded to include antibody deficiency, liver disease, vasculopathy, pure red cell aplasia, cytopenias, and lymphoproliferative disease. methods: forty-two patients were enrolled in an irb approved study at the nih. sequencing of ada2, the gene encoding adenosine deaminase 2 (ada2), was performed in all patients. information was obtained by chart review of all clinical, serologic, and radiographic testing. results: all 42 patients had germline biallelic loss of function mutations in ada2, leading to absent or significantly decreased protein expression and function of ada2. the cohort comprises 20 females (48%) and 22 males (52%). there were 6 sibling pairs and 2 families with 3 affected individuals. twenty-seven patients had a history of at least one ischemic stroke and 6 experienced a hemorrhagic stroke. the average age at the time of first stroke is 5.6 years (range 4 months -24 years), and the average number of strokes is 3 (range 1-11). no new strokes have occurred in patients on anti-tnf therapy. skin manifestations occurred in 86% of patients and include livedo (74%), cutaneous vasculitis resembling pan (64%), and raynauds (19%). hepatomegaly (43%) and splenomegaly (55%) were also notable. portal hypertension was observed in 6 (14%) patients, with 1 patient requiring a spleno-renal shunt for a massive variceal bleed. abdominal mra revealed arteritis and aneurysm in 7/13 patients evaluated; 3 patients developed bowel necrosis. peripheral vasculopathy was seen in 3 patients, with one requiring amputation of gangrenous digits. the most common immune abnormality seen in this cohort is hypogammaglobulinemia (62%); 20 patients have low igg, 20 patients have low igm, and 14 patients have low iga. ten of these patients are on immunoglobulin replacement. specific antibody responses to vaccines were inadequate in 5/16 patients challenged. lymphocyte phenotyping revealed decreased class-switched memory b cells in 23/32 patients (72%) tested. however, there was no relationship between absolute number of class switched memory b cells and hypogammaglobulinemia or infection frequency. hematologic abnormalities include transfusion depended anemia (7%), neutropenia (7%), lymphopenia (5%), and thrombocytopenia (2%). seven patients developed pancytopenia, 1 presented with pure red cell aplasia, and 1 developed aplastic anemia. three patients have undergone bone marrow transplant, with two of those patients requiring a second transplant for graft failure. conclusions: the spectrum of dada2 has expanded from strokes, intermittent fever, and cutaneous manifestations to include portal and systemic hypertension, immune deficiency, cytopenias, vascular abnormalities, and bone marrow failure. while initiation of anti-tnf therapy improves inflammatory markers, and no new strokes have occurred while on therapy, cytopenias do not seem to improve. bone marrow transplantation should be considered in patients with findings of bone marrow failure, although transplant of our patients has been complicated by immune mediated neutropenia. disease manifestations are heterogenous, making a comprehensive evaluation critical to our understanding of this disease. given the increase in neonatal diagnosis of athymia, clinical care is provided by the referring medical centers prior to rvt-802 implantation and patients return to the referring centers earlier after rvt-802. this creates the need for clear, concise guidelines for the care of these patients. primary goals of pre-transplantation clinical care are (1) management of pre-existing medical needs such as feeding difficulties, airway obstruction, congenital cardiac defects and developmental disabilities; (2) management of symptoms related to oligoclonal recipient t cell expansion (autologous gvhd/atypical complete digeorge anomaly) and (3) prevention of infections. most deaths in the pre and early post-transplantation period are secondary to pre-existing infections. necessary surgical and medical procedures (ie cardiac surgery, hearing aids) should not be delayed. for the first 6 to 9 months after rvt802, patients have profoundly low naã¯ve t cell numbers and may require immunosuppression to prevent rejection of rvt-802 by oligoclonal recipient t cells. immunosuppression needs to be closely monitored and titrated for desired effect while minimizing side effects such as renal toxicity, electrolyte abnormalities and hypertension. t cell counts should be performed every 3 months and are used to guide weaning of immunosuppression. most patients with successful transplants develop greater than 100/mm3 naã¯ve t cells by 12 months post rvt-802. infection prevention, clinical stability and optimal nutrition are critical for lasting engraftment. clinical guidelines have been developed to address immunosuppression, management of autologous gvhd symptoms (gut, skin and liver), preservation of renal function, and developmental considerations. after the development of naã¯ve t cells, patients should continue to be monitored regularly by an immunologist. patients may develop autoimmune complications such as thyroid disease and transient cytopenias. while risk of complications related to viral infections is greatly decreased after development of naã¯ve t cells, patients with comorbidities (central venous access device dependence, tracheostomy, chronic lung disease) continue to require complex care from multidisciplinary teams. medical conditions associated with athymia but not alleviated by thymus transplantation, such as hypoparathyroidism or cardiac defects, may require lifelong medical care. lastly, patients must be evaluated for readiness for killed and live vaccines. transplant outcomes are influenced by the clinical condition at the time of rvt-802 implantation and optimization of immunosuppression, nutrition and clinical stability in the first 9 months following rvt-802. clinical care that maintains a well-nourished, clinically stable, infection free patient yields the best chance for successful t cell development. guidance documents supporting these goals ensure patients are best prepared to receive rvt-802 and develop long lasting thymic function. hemophagocytic lymphohistiocytosis (hlh) is a life-threatening disease of immune dysregulation characterized by unchecked inflammatory responses leading to end-organ dysfunction. primary hlh results from inherited mutations that impair capacity for immune regulation whereas secondary hlh arises from inappropriate response to an immune stimulus such as infection, malignancy or autoimmunity. we report a 9-monthold male who presented with symptoms of hlh as an initial manifestation of congenital disorder of glycosylation (cdg) due to mutations in the gene component of oligomeric golgi complex 4 (cog4) resulting in cog4-cdg (cdg-iij). a 9-month-old male with history of mild motor delay presented with 3 days of fever, vomiting, and diarrhea. initial evaluation identified highly elevated ferritin and triglycerides, transaminitis, coagulopathy, and hyperammonemia. he subsequently developed generalized seizures. liver and bone marrow biopsies demonstrated erythrophagocytosis consistent with hlh. immunologic evaluation was notable for mild hypogammaglobulinemia, neutropenia, thrombocytopenia, and anemia. serum cd25 levels and nk functional studies were later found to be normal. the patient was initially treated with ammonia-scavenger therapy and fresh frozen plasma (ffp) for coagulopathy with subsequent intravenous immunoglobulin and dexamethasone several days later. within 24 hours after starting ffp, the patients ferritin level declined sharply. hyperammonemia and transaminitis also resolved, and his fever curve improved. additional immunosuppression was considered, but not initiated due to the patients ongoing clinical improvement. over the next 3 months, the patient experienced two further acute episodes of fever, liver dysfunction, coagulopathy, and sepsis physiology. the second episode was successfully treated with ffp, though no clear infectious trigger was identified. the third episode occurred 4 days after routine vaccinations. the patient had prolonged hypotension requiring ionotropic support that resolved after receiving daily ffp, and hypoxia with pleural effusions that resolved after a single treatment with protein c concentrate. as the patient had met 5/8 clinical diagnostic criteria for hlh, but also had a history of hyperammonemia, he underwent concurrent biochemical and genetic evaluation for both primary hlh and inborn errors of metabolism. whole exome sequencing identified compound heterozygous mutations in cog4, part of an oligomeric protein complex involved in golgi apparatus structure and function. cog4 mutations have previously been reported in two patients with autosomal recessive cog4-cdg (cdg-iij), who were described to have similar clinical symptoms of hypotonia, seizures, coagulopathy, and liver dysfunction, as well as recurrent infections. subsequent immune phenotyping while the patient was healthy was notable for slightly low numbers of nk cells, but normal cd107a mobilization and perforin/granzyme b expression in vitro. our patient represents a novel presentation of cdg due to cog4 defect with associated immune dysfunction manifesting as recurrent episodes of inflammatory crisis with features of hlh. cdg and inborn errors of metabolism should be considered during diagnostic evaluation for patients with hlh symptoms, as cdg patients may develop acute episodes of severe inflammation, in the absence of cellular regulatory defects, for which ffp and protein c concentrate may have therapeutic value. of the 14 deaths with identifiable causes, 10 (71%) were related to infections. the rate of death per person-year was 0.044. the most common autoimmunity-related complication was sweets syndrome, seen in 29 patients (39%) with anti-ifn-g autoantibodies. sixteen of those patients (55%) had recurring sweets syndrome. additionally, 14 patients (19%) developed lymphatic obstruction, which continued to recur in 12 patients (86%). seven patients (9%) in this study did not have anti-ifn-g autoantibodies. the median [iqr] age of autoantibody-negative patients was 38 [27, 54] years and 3 patients (43%) were female. none of the autoantibody-negative patients developed new infections during follow-up. at the end of the follow-up period, none of the patients had active/progressive disease and 2 patients (29%) had died. conclusions: ninety-one percent of hiv uninfected thai patients with disseminated ntm infection with or without other opportunistic infections had detectable anti-ifn-g autoantibodies. about one third of patients with autoantibodies to ifn-g had recurrent infections during follow-up. after approximately 7 years of follow-up, 55% of patients with anti-ifn-g autoantibodies had inactive disease following multi-drug antibiotic therapy while 8% had active/progressive disease and 24% had died. patients with anti-ifn-g autoantibodies are at risk for recurrent infections and autoimmunity-related complications. therefore, longterm follow-up is recommended. life-long secondary antibiotic prophylaxis may be required to prevent recurrence of infection in the setting of persistent anti-ifn-g autoantibodies. the study of early t cell development in patients with severe t cell immunodeficiencies is challenging because of the rarity of these diseases, the difficulty to obtain hematopoietic stem cells (hscs), and limitations in the assays to assess in vitro differentiation of hscs to mature t cells. we recently developed a serum-free system that allows faithful analysis of sequential steps of t cell differentiation. in this system, artificial thymic organoids (atos) are generated, based on the 3d aggregation and culture of a delta-like canonical notch ligand 4 (dll4)-expressing stromal cell line (ms5-dll4) with cd34+ cells isolated from bone marrow (bm) samples of normal donors (nd). in this project, we set out to evaluate the possibility of using the ato system to study t cell differentiation in patients carrying t cell defects, in order to define the exact steps of t cell development affected by different genetic defects. using the ato system, we studied in vitro t cell differentiation from cd34+ cells obtained from patients carrying defects that are intrinsic to hematopoietic cells (rag1, rag2, ak2, il2rg) or that affect thymus development (digeorge syndrome, dgs). the ak2-deficient patient showed a markedly decreased viability in cd34+ cells and a very early defect in t cell development, already at the pro-t cell stage. this defect was very similar to that observed in a patient carrying a null il2rg mutation who was reported to show autologous reconstitution after unconditioned haploidentical hsc transplantation. in contrast, cd34+ cells from a patient carrying a missense il2rg mutation and with a leaky scid phenotype were capable of differentiating into mature t cells in vitro, although with 100-fold decreased efficiency as compared to normal donors (nd). interestingly, in the patient carrying the null il2rg mutation, we noticed very few cells that could reach full maturation, with an absolute number of cd3+ tcrab+ cells around 1000-times less than in nd. at variance with pro-t cells (that failed to express the gc protein), these mature t cells did express normal levels of gc, suggesting that they may have derived from residual cd34+ cells from the bm donor. in addition, cd34+ cells from the patients carrying rag1 and rag2 hypomorphic mutations were able to differentiate to cd4+cd8+ double positive cells, but not to cd3+tcrab+ cells. finally, the dgs patient showed a completely normal in vitro t cell differentiation, confirming that t cell deficiency reflected thymic abnormalities. in summary, our data show that the ato system could be extremely useful in determining whether the lack of t cells in patients with unknown gene defects reflect hematopoietic or thymic intrinsic problems, and may therefore provide critical evidence in deciding whether hsc or thymus transplantation is warranted, even without knowing the actual gene defect. introduction: ataxia-telangiectasia (at) is an autosomal recessive disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene, which aids in detection and repair of dna damage. at is characterized by progressive cerebellar ataxia, oculomotor apraxia, choreoathetosis, conjunctival telangiectasias, variable degrees of t-cell lymphopenia (tcl) and immune compromise. patients are at an increased risk for malignancy, particularly leukemia and lymphoma, and are unusually sensitive to ionizing radiation. with the advent of trecbased newborn screening (nbs) for scid, at patients are being recognized with asymptomatic tcl in early infancy. objectives: we present an older child with at and chronic granulomatous lesions and discuss how this may be avoided in individuals with at diagnosed following abnormal nbs. case report: a 12 y/o male was born at term following an uncomplicated twin pregnancy and delivery, prior to institution of scid nbs. he demonstrated mild gross motor and speech delay as an infant and was diagnosed with at at age 3. he had received all routine immunizations, including live vaccinations. he developed granulomatous skin lesions at age 1, initially small papules on his cheeks and ears, which subsequently formed large disfiguring plaques on sun-exposed areascheeks, arms and hands (fig 1) . following an extensive workup, his lesions were found to be secondary to a mutated vaccine-strain rubella (ra27/3) based on 739bp genotyping, previously described in other immunocompromised individuals [perelygina/sullivan et al. jaci 2016] . his lesions have been refractory to multiple treatments including nitazoxanide. he is currently on daily oral and topical steroids, tmp/smx and ivig. retrieval of his nbs for trec determination revealed that he would have screened positive [mallot/puck et al. j clin immunol 2013] . when first measured at age 3, cd3 t-cells were low, 443/ul, with cd4 227/ul and cd8 140/ul. b and nk cell numbers were normal. since april 2017, 4 cases of at were seen at ucsf in infants with non-scid tcl on nbs. these 3 males and 1 female were all born at term and discharged from well-infant nurseries. at was diagnosed at 2-7 months of age. their initial trecs ranged from 5-12/ul (normal with perkinelmer enlite kit >18), and all had low t-cells on initial flow cytometry (242-1612 cd3/ul, ref range>2500) with decreased cd4 (146-1178/ul) and cd8 (87-403/ul) t-cells; however naã¯ve t-cells were present, ruling out typical scid and raising concern for non-scid tcl. three infants also demonstrated low b-cells (<20-77/ul), while nk cells were normal in all. two are currently receiving ivig, one of whom is also on tmp/smx. all have avoided not only rotavirus but also mmr and varicella live vaccinations. conclusions: at is now often diagnosed in infants with low trecs on scid nbs, prior to neurologic manifestations. benefits of early diagnosis include avoidance of live vaccines, including mmr, which led to the debilitating granulomas in our older patient. additionally, patients receive prompt immunologic monitoring and treatment, avoidance of unnecessary radiation, specialty referrals and family genetic counseling. while there is no cure for at, ongoing research may bring neuroprotective treatments in the future. introduction: subcutaneous immune globulin 20%, ig20gly, was well tolerated in the phase 2/3 north american study in patients with primary immunodeficiency diseases (pidd). here we assess comorbidities, use of concomitant medications, infusion parameters, and tolerability in advanced age patients (60 y) treated with ig20gly in the north american study. methods: patients aged 2 years with pidd received weekly ig20gly infusions at volumes 60 ml/site and rates 60 ml/h/site for~1.3 years in the north american study (nct01218438). the medical history at baseline, medical conditions that were ongoing (defined as comorbid events), use of concomitant medications, adverse events (aes), tolerability, and infusion parameters were assessed by age: in advanced age patients (60 y; n=14), adult (16<60 y; n=39), and pediatric/adolescent patients (<16 y; n=21). results: the mean number of medical history events at baseline was higher in advanced age patients (28.7 events/patient; 402 events in 14 patients) versus adult (16.8 events/patient; 657 events in 39 patients), and pediatric/adolescent patients (6.5 events/patient; 137 events in 21 patients). of these, the medical conditions that were ongoing at baseline (comorbid events) were also higher in the advanced age patients (20.9 events/patient; 292 events in 14 patients) versus adult (12.4 events/ patient; 482 events in 39 patients), and pediatric/adolescent patients (3.4 events/patient; 71 events in 21 patients). in the advanced age patients, neurological comorbidities (51 events) were the most common, followed by those related to eyes, ears, nose, and throat (49 events), gastrointestinal (43 events), and musculoskeletal comorbidities (43 events). concomitant medications were given to treat a preexisting condition in all patients in the advanced age group (225 medications in 14 patients). despite the higher mean number of comorbid conditions, infusion parameters in the advanced age patients were comparable to those in the adult age group. median maximum infusion rates and infusion volumes/site were comparable in the advanced age patients (60 ml/h/site; 47.5 ml/site) and adults (60 ml/h/site; 44 ml/site); lower infusion rates and volumes/site were reported in the pediatric/adolescent patients ( . larger infusion volumes and faster infusion rates were not associated with increases in causally related local aes in the advanced age group, consistent with the trends seen in the pediatric/ adolescent and adult patients. conclusions: despite the higher mean number of comorbidities in advanced age patients with pidd, ig20gly was infused at relatively high rates and volumes and was well tolerated. introduction: hyqvia (ighy; immunoglobulin infusion 10% with recombinant human hyaluronidase [rhuph20]) is an immunoglobulin (ig) replacement therapy approved for patients with primary immunodeficiency diseases (pidd) that allows larger infusion volumes, up to 600 ml/site, and has improved ig bioavailability compared with conventional subcutaneous ig products. a post-authorization safety study is being conducted in the united states to acquire long-term safety data on ighy and to assess prescribed administration regimens in routine clinical practice. infusion characteristics and treatment-related adverse events from an interim analysis are reported here. methods: patients aged 16 years with pidd receiving ighy were included in this ongoing, prospective, non-interventional, open-label, uncontrolled, multicenter study. as a part of routine clinical practice, patients are treated with ighy according to standard medical care and their treatment regimen is at the discretion of the treating physician. adverse events (aes) are collected from enrollment to study completion/discontinuation using a subject diary and assessed at every study visit (every 3 months or standard practice). aes are assessed based on seriousness, severity, and causal relatedness to ighy. the presence of anti-rhuph20 antibody is evaluated on a voluntary basis. treatment preferences for various attributes of ig therapy were assessed annually using a treatment preference questionnaire. results: a total of 175 patients were enrolled at 26 us study sites (data cutoff date: august 21, 2017). infusions were self-administered at home (56%) or at the clinical site (44%) most commonly using 4-week infusion intervals (56.6%). the mean maximum ig infusion rate was 302.8 ml/h and the mean ig dose was 418 mg/kg bodyweight/4weeks. the mean number of infusion sites used for administration was 1.9 and mean infusion duration was 2.8 hours. most infusions (97.3%) were administered without a rate reduction, interruption, or discontinuation due to aes. there were no serious aes (saes) related to ighy. sixteen patients experienced a causally related non-serious local ae (9.1%; 0.43 events/patient-year, 0.07 events per infusion) and 25 patients experienced a causally related non-serious systemic ae (14.3%, 0.88 events/patient year, 0.14 events per infusion). seven of 113 patients who were tested for anti-rhuph20 antibody had 1 positive binding antibody test to rhuph20 (titer 1:160; maximum titer 1:10240 at enrollment, 1:5120 during the study); no neutralizing rhuph20 antibodies were detected. of the patients who responded to the treatment preference questionnaire at the end of year 1, the majority (38/52 [73.1%]) preferred to receive their ig therapy at home; 21.2% (11/52) preferred the doctors office; 3 patients preferred treatment at the hospital, had no preference, or indicated other. almost all patients (51/52 [98.1%]) indicated a preference to continue treatment with ighy. conclusion: this interim analysis of 175 patients with pidd treated with ighy in routine clinical practice supports previous observations that ighy is a well-tolerated and preferred therapy with no reports of treatment-related saes or neutralizing anti-rhuph20 antibodies. background: cartilage hair hypoplasia (chh) is an autosomal recessive chondrodysplasia associated with variable immunodeficiency. pathogenic defects in rmrp, encoding the untranslated rna subunit of ribonucleoprotein endoribonuclease complex (rmrp), result in reduced mrna and rrna cleavage. rmrp c.70a>g is the most common variant, increased in finnish and amish populations. while cellular immunodeficiency is associated with increased morbidity and mortality, there is no established correlation between clinical and immunological phenotype. lymphocyte radiosensitivity has not been described. case: a full-term amish female infant had low trec copies on newborn scid screen. flow cytometry at 3 months-old demonstrated severe t and b cell lymphopenia (cd3+t-cells 413 cells/mcl, range: 2,300-6,500 cells/mcl; cd19+b-cells 214 cells/mcl, range: 600-3,000 cells/mcl) with normal nk quantitation (cd16/56+ 340 cells/mcl, range: 100-1,300 cells/mcl) and cd4+ memory t-cell expansion (33.2%) relative to the naã¯ve subset (67.0%). t-cell functional mitogen responses were normal. she was diagnosed with chh with homozygous rmrp c.70a>g mutation. lymphocyte subset (t, b and nk cells) radiosensitivity was evaluated by flow cytometric analysis of phosphorylated (p) atm, smc1 and gamma-h2ax after low-dose (2gy) irradiation. an increase in gamma-h2ax level was observed in a subset of non-irradiated t cells (17.66% v. 1.36% gamma-h2ax+) and nk cells (23.07% v. 1.04% gamma-h2ax+) in the patient, suggestive of a constitutive defect in dna repair. the relative distribution of t, b and nk cells expressing patm, psmc1 and gamma-h2ax at 1 hour postirradiation (ir) was not significantly different from the experimental healthy control (ehc) or pediatric reference range (prr). however, the kinetics of dephosphorylation at 24 hours post-ir was altered with residual gamma-h2ax expression in a subset of the patients t cells (delta 3.84%, mode ratio mean fluorescence intensity (mfi)=2.58; ehc: delta 0.10%, mode ratio mfi=1.39; prr: delta 2.16%, mode ratio mfi=2.42). a similar finding was observed in a subset of patient b-cells for gamma-h2ax (delta 11.35%, mode ratio mfi=1.48; ehc: delta 0.82%, mode ratio mfi=0.86; prr: delta 1.95%, mode ratio mfi=1.19). the frequency of the patient's lymphocytes with residual gamma-h2ax persistence at 24h post-ir was prominent, with 8.29% t-cells demonstrating persistence of gamma-h2ax (compared to 0.82% in the ehc, and 2.60% in the prr), and 18.02% b-cells gamma-h2ax+ (compared to 1.80% in the ehc, and 2.96% in the prr). there has been lack of follow-up, but verbal report suggests no significant immunological or infectious concerns at 1 year of age. discussion: lymphocyte radiosensitivity is a novel finding in chh with t and b cell lymphopenia. the ability of rmrp to associate with telomerase reverse transcriptase (tert) and function as an rna-dependent rna polymerase, yielding distinct silencing rna sequences, may underlie radiosensitivity in rmrp mutants. systematic characterization of lymphocyte radiosensitivity and immunological phenotype could provide useful information on whether this could serve as a biomarker for the magnitude or complexity of immunodeficiency. assessment of radiosensitivity has implications in conditioning regimen selection for patients requiring allogeneic hematopoietic cell transplantation. we recommend lymphocyte radiosensitivity assessment in chh infants identified by nbs scid and chh patients with significant immunodeficiency and/or malignancy. novel primary immunodeficiency with lymphoproliferative disease due to biallelic defects in nckap1l background: three children from 2 non-consanguineous families and different ethnic backgrounds developed lymphoproliferative disease by 2 years of age. they also had recurrent infections, including pneumonia and bronchiectasis, otitis media, and skin pustules. immune phenotyping revealed low cd4+ t cell percentages, an accumulation of memory-like cd8+ t cells, impaired t cell proliferation, and low total nk cell numbers. methods: the affected individuals, unaffected parents, and other unaffected family members underwent exome sequencing. results: all 3 affected cases had rare and bioinformatically damaging biallelic variants, with appropriate familial segregation, in nckap1l, which encodes hem1. hem1 is an essential component of the wave2 regulatory complex (wrc). immunoblotting confirmed destabilization of the wrc in all patients. immunofluorescence microscopy demonstrated defective f-actin and wave2 localization to immune synapses in nk cells. significant abnormalities were identified in patient lymphocyte and neutrophil migration and morphology, consistent with altered wrc-mediated cytoskeletal dynamics. all patients exhibited impaired inside-out integrin activation. knockdown of hem1 produced deficient proliferative responses and mtorc2-mediated akt activation in control t cells. conclusions: the immunologic and clinical phenotype in the affected individuals recapitulates the phenotype observed in hem1-deficient mice. biallelic defects in nckap1l therefore result in a novel human primary immunodeficiency disease characterized by lymphoproliferation and susceptibility to infections. background: concurrent existence/significance of immunodeficiency with new onset lymphoproliferative disease remains understudied. just two studies to date have evaluated the prevalence of hypogammaglobulinemia in chronic lymphocytic leukemia (cll) and neither studied prevalence and impact of ige deficiency on outcomes in cll [1, 2] . therefore, the objective of this study was to examine the prevalence of hypogammaglobulinemia, examining all isotypes, in newly diagnosed cll patients and to test the hypothesis that patients with hypogammaglobulinemia have a distinct clinical profile and outcome. methods: using the banked sera of 150 newly diagnosed, treatmentnaã¯ve, cll adult patients from the lymphoma molecular epidemiology resource (l-mer), ig (igg, iga, igm and ige) levels were measured. the l-mer was initiated as an observational cohort study of prospectively enrolled newly diagnosed lymphoma patients evaluated at the mayo clinic (rochester, mn) and the university of iowa (iowa city, ia) [3] . igg/a/m levels were measured using immunoturbidimetric assay whereas the ige level was determined using electrochemiluminescence immunoassay. the associations between ig deficiencies and clinical factors were evaluated with wilcoxon rank sum and chi-squared (fishers exact, where appropriate) tests. cox regression models were used to assess the effects of clinical variables on overall survival (os). time was calculated from biopsy to death due to any cause; patients still alive were censored at last contact. all tests were two-sided and assessed for significance at the 5% level using sas v9.4 (sas institute, cary, nc). results: the mean age (sd) of the selected cll cohort was 63.8 (11.0) years with a male predominance (69.3%). 96.2% of the patients were white. with a median follow-up of five years, there were 50 deaths. hypogammaglobulinemia in newly diagnosed, treatmentnaã¯ve cll was common in our cohort with 88 (58.7%) patients having a measurable isotype deficiency. the most common ig deficiency was igm (44.0%, 95% ci 35.9-52.3%), followed by igg (34.7%, 95% ci 27.1-42.9%), ige (16.7%, 95% ci 11.1-23.6%) and iga (12.0%, 95% ci 7.3-18.3%). multiple deficiencies in the same patient were common ( figure 1 ). iga and ige deficiency were associated with higher rai stages (grading system for cll) at presentation (p<0.01 and 0.04 respectively) as well as with higher white blood cell counts at presentation (p=0.02 and 0.01 respectively). a higher proportion of iga deficient patients needed second treatment during follow-up (61% compared to 36%, p=0.04). when comparing predictors of overall survival, higher rai stage [3-4 vs 0, hazard ratio (hr) 2.43, 95% ci 1.08-5.46, p=0.03] and age (hr 1.08, 95% ci 1.05-1.12, p<0.01) correlated with worse overall survival. individual immunoglobulin deficiencies did not correlate with overall survival. conclusions: a significant proportion of treatment-naã¯ve patients with cll have underlying ig deficiencies-both in isolation and a combination of different isotypes. a deficiency of iga or ige was associated with severe disease at presentation. the underlying relationship between these two immunologic disorders deserves further study. background: patients with primary immunodeficiency (pid) have an increased risk of developing autoimmune diseases, including rheumatoid arthritis (ra). management of these patients is challenging as immunomodulators can further increase their risk for infections. additionally, patients with ra that undergo therapy with drug modifying antirheumatic drugs (dmards) may develop a secondary immunodeficiency. there are few studies reviewing the characteristics of patients with a pid who later develop ra, and no studies have been reported comparing these patients to those who develop an immunodeficiency after starting dmard therapy for ra. methods: 65 patients were identified as having inflammatory arthritis and a concomitant immunodeficiency (id) at our institution between 1/1/2000-10/03/2017 using icd-9 and 10 codes. manual chart review was performed to confirm and identify the timing of diagnosis of these disorders. patients were excluded if either there was no definitive diagnosis of id or ra (clinically diagnosed by a practicing allergist/immunologist and meeting acr 2010 criteria for ra with a score of 6 or higher, respectively), or rituximab was administered prior to diagnosis of id . clinical symptoms, treatment, and laboratory data were extracted. fishers exact test was used to compare the categorical variables between the groups; ttest was used to compare the continuous variables. results: 10 patients met the inclusion criteria. 5 patients were diagnosed with an id and developed ra later in life (group 1), and 5 patients were diagnosed with ra and subsequently developed a clinically significant id (group 2). the mean ages of diagnosis of id and ra in group 1 patients were 32.0 years (sd â± 26.9) and 42.6 years (sd â± 19.0), respectively. in group 2, the mean age of diagnosis of ra was 37.8 (sd â± 14.2), compared to 54.8 years (sd â± 12.7) for the diagnosis of id. most patients in both groups were female (60% in group 1 and 80% in group 2). all patients in both groups had a humoral id, including common variable immunodeficiency (cvid) (40% of group 1 patients), specific antibody deficiency (sad) (20% of group 1 and 60% of group 2 patients), and hypogammaglobulinemia (20% of group 1 and 40% of group 2 patients). all patients in group 2 were seropositive for rheumatoid factor (rf) or anti-cyclic citrullinated peptide (anti-ccp), whereas only 20% of patients in group 1 were positive for rf or anti-ccp (table 1 ). most patients in both groups were treated with immunoglobulin replacement therapy. treatment of ra in both groups was similar, but combination dmard therapy was not used in group 1 patients in contrast to group 2 patients. conclusions: our study indicates that even though clinical characteristics and management are similar in patients with coexisting id and ra, rf and anti-ccp are usually negative in patients who develop ra after id, possibly due to impaired antibody production in immunodeficient patients. assistant professor of allergy and immunology, arkansas children's hospital, university of arkansas medical sciences introduction/background: complement deficiencies are relatively rare, comprising less than 1% of primary immunodeficiencies. they are associated with increased risk for infections with encapsulated organisms and autoimmunity. of all complement deficiencies, the rarest are defects in the alternative complement pathway. properdin deficiency is the most commonly described alternative pathway deficiency, with factor b and factor d deficiency more rarely described. fewer than 5 patients with factor d deficiency have been reported with all reported cases being children of consanguineous parents who succumbed to meningococcal sepsis. objectives: to describe a case of factor d deficiency associated with recurrent respiratory infections with streptococcus pneumoniae pneumonia with associated lung abscess and empyema. methods: retrospective chart review was conducted. laboratory investigations included lymphocyte immunophenotyping by flow cytometry, lymphocyte proliferation to mitogen, quantitative serum immunoglobulins, vaccine titers, complement assays and functional evaluation, and genetic evaluation by next generation sequencing. results: a 2 year old marshallese male was transferred from an outside hospital to our facility for further evaluation of worsening pneumonia and was found to have right-sided pleural effusion and pulmonary abscess in the right lower lobe. the abscess was drained and was found to be positive for streptococcus pneumoniae via polymerase chain reaction. he improved after chest tube placement and treatment with intravenous antibiotics. his medical history was significant for recurrent acute otitis media and prior hospitalization out-of-state for pneumonia with empyema secondary to streptococcus pneumoniae, which required chest tube placement and admission to the pediatric intensive care unit at 18 months of age. immunologic work up revealed age-appropriate lymphocyte subpopulations, lymphocyte proliferative responses to mitogens, quantitative immunoglobulin levels, pneumococcal/tetanus/diphtheria titers, and ch50 complement assay. ah50 complement assay was decreased to 44 units/ml. complement testing was repeated -with normal ch50 and ah50 of 0 units/ml. further evaluation revealed normal levels of factors b, h, i and properdin. factor d level was 0.12 mcg/ml, and factor d function was decreased to 2 units/ml, indicating a diagnosis of factor d deficiency. sequencing of the cfd gene revealed a previously undescribed homozygous deletion (c.721_723del and p.lys241del). the parents were not agreeable to personally undergoing genetic evaluation to determine if this was a de novo mutation. the patient was managed with pneumococcal and meningococcal immunizations, prophylactic amoxicillin and intravenous gamma globulin (ivig) without any further infections. unfortunately, after two ivig infusions, he was lost to follow up. conclusion: factor d deficiency is an extremely rare alternative complement pathway deficiency, described in less than 5 patients. all infections described thus far have been secondary to neisseria meningitidis. this case represents not only a novel mutation in the cfd gene leading to factor d deficiency, but also the first description of a patient with factor d deficiency developing invasive infection secondary to streptococcus pneumoniae. background: viral infections are a significant cause of morbidity and mortality in patients with primary immunodeficiency disorders and following hematopoietic stem cell transplantation. adoptive immunotherapy using virus specific t-cells (vsts) has been shown to prevent and treat viral infections in immunocompromised hosts. human parainfluenza virus-3 (hpiv3) is a common cause of severe respiratory illness in immunocompromised patients and has no approved antiviral therapies and has not previously been used as a target for t cell therapeutics. introduction: we previously reported that fatigue is increased in common variable immunodeficiency (cvid). however, in previous studies, fatigue was not defined using validated tools. our aim from this study is to identify the prevalence of patient-reported fatigue, using validated questionnaires, and determine the factors predisposing to fatigue in cvid methods: data from cvid who responded to the idf 2017 patient national survey a were analyzed. fatigue was measured using the brief fatigue inventory (bfi) questionnaire, which includes seven items to identify fatigue, and measure fatigue severity. a total of 555 patients with cvid and responses to bfi were enrolled. demographics, co-morbidities, immunoglobulin replacement therapy (iggrt) route and dose, co-morbidities, infections, depression, quality of life (qol) (using the sf-12v2) and disability were compared between fatigued and non-fatigued. logistic regression was used to identify the significant variables. ebv reactivation without ptld, treated with rituximab. alive and well. j clin immunol (2019) 39 (suppl 1):s1-s151 s58 granulomas are the most significant day-to-day problem for cvid patient management. currently, there are limited options for their treatment and the optimal therapy is unknown. in case reports and small series, infliximab has been reported effective while others found it useless. we here describe a 26yo white male referred for monthly ivig in august 2016. at age 1, he developed large areas of erythematous polymorphic plaques in his cheeks, arms and legs. a skin biopsy showed tuberculoid granulomas negative for bacteria, baar and fungi, with infiltrating cd4+ lymphocytes. a prolonged course of steroids did not improve his skin. he also had multiple pneumonias and bronchiectasis, and oral candidiasis. he received all vaccines, including bcg with no complications. with low immunoglobulins and a poor response to pneumococcal polysaccharides and tetanus toxoid he was diagnosed as cvid and placed on ivig at 7yo with excellent infectious control since then. at age 8, his skin lesions persisted and deepened to the bone on his left leg. broad spectrum antibiotics for 3 months were unsuccessful. at 16yo to 18yo, skin grafts were performed on his arms, legs and both cheeks. two ulcers persisted on his left leg until august 2018 that increased in size, deepened and became erythematous and extremely painful (fig. 1) . in september, two new ulcers appeared on his right cheek and right gluteus, respectively. one week later a third ulcer was found on his left calf. on september 28th, infliximab 5mg/kg (300mg) was administered. on the second infliximab dose, october 12th, the pain was completely gone and all ulcers were shrinking, and those ones in the cheek, gluteus and calf almost completely resolved. by the third dose, on november 23rd the ulcers in his right leg were almost closed (fig. 2) . infliximab 300mg treatment continues every 8 weeks. lab test remained unchanged from 2016 till 2018, when his wounds got worsened. (table 1 ) granulomatous disease in cvid is a challenge. both b and t cell directed therapies are encouraged. we add a new case of an infliximab responsive patient to others already reported. over 20 genes have been reported to cause monogenic cvid. a 4 year old girl presented with recurrent pneumonias and a diagnosis of cvid. the parents sought a second opinion. born at 33 weeks gestational age, she was "always smaller and sicker than her friends," and in the prior 8 months she had 3 episodes of pneumonia with fever to 104f requiring emergency department treatment. two of these were associated with rsv and metapneumovirus, respectively. laboratory evaluation confirmed low levels of igg (326 mg/dl) iga (7) and igm (6) congenital tuberculosis (ctb) is a rare disease most often associated with maternal genitourinary (gu) tuberculosis (tb) or disseminated tb. due to infertility caused by gu tb, ctb is rarely reported even in endemic countries. infants can acquire tb hematogenously via the placenta or umbilical vein or by fetal aspiration of infected amniotic fluid. presenting symptoms include respiratory distress, fever, hepatosplenomegaly, poor feeding, lethargy, and low birth weight. we report a premature female infant conceived via in vitro fertilization (ivf), who was born to indian immigrant parents at 29 weeks of gestation due to preterm premature rupture of membranes. maternal history was significant for pulmonary tb at 9 years of age. she denied abdominal or gu symptoms. infants nicu course was complicated by opacifications in the right lung and leukocytosis with neutrophil predominance, identified during evaluation of frequent apnea and bradycardia episodes at 1 month of age. clinical improvement was noted after treatment with vancomycin, amikacin and piperacillin-tazobactam; however, leukocytosis of unknown etiology persisted. at 1.5 months of age she was discharged to inpatient rehabilitation. at 3 months of age, she was readmitted for fever and respiratory distress. during this admission, an immune evaluation was undertaken due to persistence of symptoms along with unresolved leukocytosis with a peak of 58,000 cells/l with neutrophilia to 42,850 cells/l, and chest ct evidence of progressive multifocal lung disease worse in the right upper lobe despite empiric treatment with broadspectrum antibiotics. infectious work-up was negative, including acid-fast bacilli testing from bronchoalveolar lavage. due to the pronounced and persistent leukocytosis and neutrophilia, a primary immune defect was suspected. immune evaluation included: normal immunoglobulins (ig) g, a, and e, elevated igm, vaccine-specific antibody titers protective to diphtheria and 9 of 13 streptococcus pneumonia strains, mildly elevated t and b cells, a normal flow cytometry for dihydrorhodamine, myeloperoxidase stain and glucose-6-phosphate dehydrogenase level, as well as a peripheral smear with no giant azurophilic granules. her primary immunodeficiency genetic panel was unrevealing. she underwent lung biopsy via video-assisted thoracoscopic surgery, which showed noncaseating granulomas and eventual growth of multi-drug-resistant mycobacterium tuberculosis (mtb). upon treatment with an appropriately adjusted anti-tuberculosis regimen, she showed rapid clinical and laboratory improvement. endometrial samples obtained from mother showed gu tb, confirming the diagnosis of ctb. the slow-growing nature of mtb that resulted in delayed diagnosis, along with the presence of non-caseating granulomas and persistent neutrophilia, prompted an immune work up that was completely normal. this case demonstrates the importance of considering ctb in the differential diagnosis of an infant presenting with severe lung infection, persistent neutrophilia, suboptimal response to broad-spectrum antibiotics and relevant epidemiologic risk factors. furthermore, in the setting of appropriate parental exposures and infertility prompting the use of ivf, maintaining a high level of suspicion of ctb can aid in earlier diagnosis of affected neonates. 15-year-old caucasian male who initially presented with recurrent otitis media, persistent hsm, lad, and hypogammaglobinemia (igg <170 mg/dl) at 2 years of age. he was diagnosed with common variable immunodeficiency (cvid) and chronic arthritis when he was 6 and 9 years of age, respectively. subsequently, he developed hepatitis and recurrent pneumonia with mycobacterium avium complex (mac). his arthritis partially responded to anti-tumor necrosis factor (tnf) agents and tofacitinib, but did not respond to anti-interleukin-6 treatment. a combination of anti-tnf inhibitor, tofacitinib, and low dose prednisone was required to control his arthritis. hypogammaglobulinemia (igg <110 mg/dl), recurrent otitis media, pneumonia, crohn's disease, celiac disease, lad and failure to thrive at 2 years of age with more recent development of hsm. he required only immunoglobulin replacement therapy. case#3 is a 9-year-old caucasian male, the half-brother of case#2, who initially presented with recurrent pleural effusion and bilateral pulmonary infiltrates, hsm, lad, abdominal distension and ascites at 7 years of age. a transbronchial lung biopsy revealed chronic eosinophilic pneumonitis. liver biopsy showed increased eosinophils in the sinusoids with diffuse enlargement of hepatocytes, but without hepatitis. colon biopsy revealed minimal colonic eo-sinophilia. his pulmonary infiltrates and pleural effusion responded to prednisone, and he has not required additional treatment for past 1.5 years. conclusions: the clinical manifestations of the same genetic variant may be variable and unpredictable even in the same family. stat3 gof syndrome should be considered in children with multisystem autoimmune diseases, lad, hsm and low switched memory b cells regardless of presence of hypogammaglobulinemia or history of recurrent infections. background: patients with primary immune deficiencies characterized by severe t lymphopenia and/or poor t cell function and patients posthematopoietic cell transplantation are at high risk of severe viral infections. antiviral medications are expensive, not always effective and associated with significant toxicity and/or long-term side effects. as such, there has been increasing interest in the use of donor-derived or thirdparty virus-specific t cells (vsts), and several studies have demonstrated efficacy of vsts generated using various manufacture strategies. however, in depth immunologic and metabolic characterization of vsts has not been reported, limiting correlative investigations into efficacy. methods: ebv-vsts were generated from apheresis t cells collected from healthy donors using three methods: (1) stimulation and expansion with hla-matched ebv-lymphoblastoid cell lines (lcls) purchased from astarte biologics or sigma-aldrich over a period of 4 weeks, (2) stimulation with ebv peptivator from miltenyi followed by expansion over 9-12 days with different cytokines, and (3) stimulation with ebv peptivator followed by isolation of activated cells using the ifn-gamma capture system from miltenyi. immunophenotyping by flow cytometry was performed using the miltneyi macsquant analyzer. the nanostring ncounter system was used to measure gene expression for metabolic pathway analysis, and the agilent seahorse xf cell mito stress test system was used to measure mitochondrial respiration. results: ebv-vsts generated using lcls or peptivator plus il-15 both resulted in a high percentage of cd8 t cells skewed to the effector memory and terminal effector memory phenotype with high expression of the exhaustion markers pd-1, tim-3, and lag-3. conversely, ebv-vsts generated using peptivator plus il-4 and il-7 and the ifn-gamma capture system resulted in a mixed cd4 and cd8 t cell population with a high number of central memory t cells and lower percentage of cells positive for pd-1, tim-3, and lag-3. stimulation with peptivator followed by expansion with il-2 resulted in an intermediate immunophenotype. nanostring results demonstrated upregulation of the glycolytic pathway in ebv-vsts stimulated with peptivator followed by expansion with il-2 or il-15 compared to ebv-vsts generated using the other manufacture approaches. the seahorse mito stress test demonstrated that the peptivator plus il-2 ebv-vsts had a significantly lower spare respiratory capacity than other ebv-vsts and a low extracellular acidification rate despite upregulation of the glycolytic pathway. the peptivator plus il-4 and il-7 ebv-vsts had the highest basal oxygen consumption rate, atp-linked respiration, and extracellular acidification rate. conclusions: manufacture of ebv-vsts using the various approaches currently employed clinically results in t cell pools with different immunophenotypes and different metabolic profiles. ebv-vsts stimulated with peptivator followed by expansion in il-4 and il-7 and ebv-vsts isolated using the ifn-gamma capture system have immunophenotypes and metabolic phenotypes suggestive of potential greater in vivo persistence, whereas ebv-vsts expanded in il-2 and il-15 have characteristics correlated with increased effector function. however, these vsts are more likely to be short-lived and to have impaired metabolic fitness. these phenotypes will enable better correlation with clinical results and suggest combinatorial approaches depending on clinical indication. introduction: majority of patients with primary immunodeficiencies (pid) require life-long replacement therapy with immunoglobulins (ig) to prevent severe infections and irreversible complications. in addition to safety and efficacy, tolerability and convenience of administration of ig products are essential factors for patients. a new 16.5% ig preparation octanorm (octapharma, lachen; tradename cutaquigâ® in north america) has been developed for subcutaneous administration (scig) derived from the established manufacturing process of octapharmas intravenous ig (ivig) brand octagamâ®. objectives: primary outcome was assessment of the efficacy of octanorm in preventing serious bacterial infections. main secondary endpoints included (among others) evaluation of tolerability and safety of octanorm, the number and rate of other infections, number of days missed at work, and use of antibiotics. methods: a prospective, open-label, non-controlled, single-arm phase 3 study involving 25 adult patients with pid was conducted at 5 russian centers. patients treated with at least 4 infusions of ivig prior to enrollment and with igg trough levels 5.0 g/l underwent an 8-week wash-in/wash-out period followed by a 24week efficacy period. during the study, patients received weekly administrations of octanorm at the same monthly dose as during previous ivig treatment (monthly ivig dose divided by 4 for weekly dose). in total, each patient received 32 scig infusions. results: twenty-four patients completed the study. one patient terminated early (after infusion 7, during wash-in/wash-out phase; personal reasons). mean age was 35.24 years (range 18-64 years). fifteen patients (60%) were female and 10 patients (40%) male. no serious bacterial infections were recorded. during the efficacy period a total of 26 non-serious infections was observed in 14 patients. seventeen infections in 11 patients were of mild and 9 infections in 5 patients of moderate intensity. the infection rate per person-year was 2.37. in total 25 patients received 775 infusions of study drug. the average dose of cutaquigâ® was 0.11 g/kg/week. during the entire study, 59 systemic adverse events were reported (including 34 infections). three of these systemic adverse events were rated as related to study drug, all were non-serious. there was no serious or significant adverse event nor was there an adverse event leading to withdrawal. infusion site reactions were reported for 15% of infusions. serum igg trough levels were nearly constant during the efficacy period. median igg trough levels were 8.15 g/l at screening, 9.52 g/l at the end of wash-in/wash-out period and 10.71 g/l at the termination visit. one patient had a trough level 5g/l at 2 visits during the efficacy period and the dosing was subsequently adjusted for this patient. during the primary treatment period 10 patients (41.7%) used antibiotics in 19 treatment episodes (total of 229 treatment days; range 5-76 days) and 3 patients had 4 absences from work or school due to infections (total of 14 days of absence). conclusion: this study demonstrated that the new subcutaneous human normal immunoglobulin 16.5% is well tolerated, safe and effective in adult patients with pid. background: children with chronic granulomatous disease (cgd) are at high risk for fungal infections (especially with aspergillus species) and these infections usually have contiguous site involvement. most patients have pulmonary presentation. infective endocarditis and fungal osteomyelitis of skull are distinctly unusual. we report one such case. case: a 6-year-old boy, born out of a non-consanguineous marriage, presented with soft tissue swellings of skull for 2 months. his past history was significant with an episode of pneumonia at 1 year and recurrent soft tissue swellings all over the body since 1â½ years of age. on examination he was wasted, had signs of micronutrient deficiency, rickets, pallor, cervical lymphadenopathy and two abscesses, 12x4 cm on right temporo-parietal region and 4x3 cm over left frontal region. he was also found to have hyperdynamic precordium with an ejection systolic murmur. investigations revealed hemoglobin 85g/l; platelet count 7.34x109/l; total leukocyte count 13x109/l(n60/l23/m13/e1); elevated c-reactive protein( 244 mg/l) and a raised erythrocyte sedimentation rate(104 mm 1sthr). chest x ray revealed cardiomegaly (cardiothoracic ratio 67%) and 2d echocardiography showed vegetation of 6x3 mm over the anterior mitral leaflet suggestive of infective endocarditis. blood and urine cultures were sterile. culture from pus over the temporo-parietal abscess showed growth of aspergillus fumigatus. human immunodeficiency virus serology was non-reactive. immunoglobulin profile revealed elevated igg 21.20g/l (5.40-16.10g/l) and iga 5.66 g/l(0.5-2.4g/l); igm was 1.63 g/l(0.50-1.8g/l). in view of strong suspicion of cgd, nitroblue tetrazolium dye reduction test (nbt) was carried out-it revealed no reduction and dihydrorhodamine (dhr) assay showed a low stimulation index (4.34). flow cytometry for gp 47 phox and gp 67 phox was normal and dhr of mother did not reveal x linked carrier state. contrast enhanced computerized tomography (cect) of head showed osteomyelitis of the calvarial bones. contrast enhanced magnetic resonance imaging (cemri) brain showed heterogeneously enchancing soft tissue lesion in the scalp at right fronto-parietal region and left frontal region with underlying bony destruction suggestive of osteomyelitis. he was given intravenous antimicrobials (ceftriaxone, gentamycin, cloxacillin, voriconazole). after 6 weeks of therapy, he showed resolution of findings on mri brain and a repeat 2d echocardiography showed significant decrease in size of mitral leaflet vegetation. conclusion: this case highlights a rare presentation of cgd with infective endocarditis and skull osteomyelitis due to aspergillus fumigatus. to the best of our knowledge, this has not been reported previously. background: genetic defect in il12r1 affect cellular immunity, underlie mendelian susceptibility to mycobacterial disease (msmd) and inflammatory bowel disease (ibd) through different pathways. we present for the first time a patient with il-12r1 deficiency from a consanguine family with two different phenotypes. initially diagnosed as crohn's disease prior to the msmd diagnosis. method and material:patient was referred to the clinical immunology and allergy clinic at the at alzahra university hospital for immunological and genetic evaluation . blood samples from patient, his family and healthy donor controls were collected upon informed consent. in this study, we investigated effect of il12r1 mutation in il-12/ifnaxis by evaluation of patients whole blood cell response to il-12 and ifn-, il-12r1 expression in pbmcs and t cell blasts. also wholeexome sequencing has been performed. result and discussion: a 26 years old male from consanguine family , with history of right sub-axillary bcg lymphadenitis, recurrent mouth ulcers , chronic diarrhea in childhood and appendectomy at age of 5 was investigated. based on his clinical presentation abdominal pain, significant weight loss, chronic and bloody diarrhea , endoscopic and pathological findings treatment for crohn's disease (cd) was started at the age of seven . unfortunately, protracted patient's symptoms ends up to resection of his colon and colostomy two years later. he was presented with multi focal osteomyelitis at the age of 13 . although no bacteria was detected in pcr and tissue culture of the bone biopsy and the patient was not responded to antibacterials , he had a dramatic response to empirical anti mycobacterial treatment and his severe bone pain and lesions were healed. even though the bone manifestations were completely controlled, he continuously was under treatment for his gastrointestinal symptoms. genetic analysis was confirmed segregation of homozygous mutation in 3splice site of exon 15 in il-12r1. expression of gene was completely abolished in pbmcs of patient and the surface expression of il12rb1 was not detectable in t cell derived pbmcs of the patient compared to healthy control. furthermore, did not response to il12 stimulation since we could not detect increase of inf-after stimulation with il12 and bcg. our patient received bcg vaccination at birth and had bcg lymphadenitis as an infant, cd and mycobacterial multifocal osteomyelitis as a child. furthermore there are some evidences which indicate the role of atypical mycobacterial infections as a trigger for cd. conclusion: we reported for the first time contemporary msmd and ibd in 26 years old patient, who had impaired il-12 signaling and abolished il12 r1 expression in pbmcs and t cell blast. however, mycobacterial osteomyelitis is a typical phenotype of msmd patients with deficiency in ifn-r1 or stat, there were no mycobacterial osteomyelitis reported in il-12r1 deficient patients. background: advanced genetic studies help explain the occurrence of many undiagnosed, rare conditions. recently, nbas variants were identified as a causative basis of recurrent liver failure in infants (infantile liver failure syndrome 2, ilfs2). the nbas (neuroblastoma amplified sequence) gene encodes a protein involved in golgi to er retrograde transport. nbas functions seem to be broad and loss of function variants in nbas have been associated with multisystem manifestations. case report: a 5y 9m old chilean male presented to the er with a three day history of vomiting, diarrhea and one day of fever (38.3â°f). on examination he was pale, lethargic, and tachycardic. a chemistry profile revealed markedly elevated liver enzymes, increased bilirubin, and coagulopathy, consistent with the acute hepatic failure (alt 6291, ast >4000, total bilirubin 3.49 (2.82 db), ggt 52, and inr of 2.1). he was hospitalized, given vitamin k, and kept on intravenous fluids, ursodiol, and antipyretics. his liver function improved significantly within 5 days of admission (alt was down to 980, ast 45, total bilirubin 1.62). work-up of possible etiologies including autoimmunity and infectious hepatitis was negative. liver sonogram was normal, but liver biopsy was consistent with acute hepatitis with some necrosis. urine organic acid and plasma amino acid screens were not consistent with any inherited metabolic disorders. his parents recalled two previous episodes of liver failure at ages 3 and 4 years. both were preceded with a mild febrile illness and non-specific symptoms including fever, coughing, vomiting, diarrhea, lethargy, and decreased po intake. these subsequently were followed by jaundice and marked elevation of liver enzymes. flu a and adenovirus were identified as causes of febrile illnesses of the two previous episodes. for this admission, adenovirus was found in the respiratory secretions and a mild ebv viremia was also detected. genetic evaluation in chile was reportedly normal. after a literature review we obtained sequencing of nbas which revealed two variants: c.2827g>t,p.glu943* and nbas c.2951t>g, p.iie984ser. both variants have been previously reported in patients with an infantile onset, recurrent liver failure syndrome. his other clinical features include developmental and speech delays, failure to thrive, and facial dysmorphism. he also has a history of recurrent ear infections and has had 3 sets of tympanostomy tubes. further testing was limited due to the lack of insurance coverage. conclusion: nbas deficiency is a newly described syndrome of recurrent acute liver failure that occurs early in life. once individuals have survived to adulthood they do not seem to develop liver failure with illness. typically, liver crisis is triggered by a common childhood febrile illness. the mechanism of disease is thought to be thermal instability of hepatocytes which improves over time in most cases. however, although spontaneous recovery can occur following the crises, each episode can be fatal or result in permanent liver damage required liver transplantation. increased awareness of this disease will lead to the early establishment of the diagnosis. appropriate and timely management of fever at the onset of illness can significantly improve outcome in this potentially fatal disease. associate prof., infectious diseases and tropical medicine research center, isfahan university of medical sciences, isfahan, iran background: pre-eclampsia, a pregnancy-specific complication, has been shown to be associated with cytomegalovirus (cmv) infection. cmv specific t-cell response plays the major role in cmv infection or disease .we explored whether a change in cmv-specific cell-mediated immunity (cmi) is related to the development of preeclampsia. method: cmv-specific cmi was assessed using cmv-quantiferon (qf-cmv) assay in serum from 35 women with pre-eclampsia as well as 35 normal pregnancy controls retrospectively. participants were matched for gestational age individually. proportion of reactive results, mean value of interferon-level produced in mitogen and antigen tubes were compared between the cases and controls via chi-square, wilcoxon rank-sum tests, respectively. odds ratio (or) and confidence interval (ci) were calculated as well. result: no significant differences observed between demographic characteristics of the case and control groups. the qf-cmv assay turned reactive (qf-cmv [+]) in 22 of 35 of patients (63%) vs. 32 of 35 controls (91.4%) (p = 0.004). women with pre-eclampsia had lower mean ifn-levels in antigen tube (1.57 â± 1.79) compared with normal pregnancy controls (2.40 â± 2.21) (p = 0.028). there was no statistically significant differences in this value of mitogen tube between cases (3.53 â± 1.67) and controls (3.53 â± 1.67) (p = 0.209). women with suppressed cmv-cmi were 6.3 times more likely to manifest pre-eclampsia (or= 6.30, 95% ci: 1.60-24.7). this result even strengthened after adjustment for age, gestational age and gravidity (or = 12.86, 95% ci: 2.68-61.6). conclusion: our finding support an association between suppressed cmv specific cmi and pre-eclampsia. introduction: the triad of susceptibility to infections, auto-inflammation, and cancer in a patients personal and family history are always suggestive of an underlying primary immunodeficiency; however, in some cases the diagnosis might be delayed for years. furthermore, the results of immunological and inflammatory evaluation can also be affected by ongoing immunomodulatory therapy initiated by different specialists upon clinical diagnosis. objective: to describe a unique presentation of auto-inflammatory disease with combined immunodeficiency in an adult patient. case presentation: we report here the case of a 64 year old male, who had a long history of infections including recurrent sino-pulmonary bacterial infections starting during childhood, osteomyelitis at 7 years of age, recurrent tonsillitis requiring tonsillectomy at 21 years of age, recurrent cellulitis, an episode of prostatitis with septicaemia, as well as recurrent varicella zoster and warts. the patient was also diagnosed with sclerosing mesentheritis, and reynauds phenomenon, recurrent oral ulcers, arthritis, uveitis, autoimmune thyroiditis, lung fibrosis and suffered repeated episodes of abdominal pain. furthermore, there is a family history of early childhood death, multiple soft tissue cancers, crohns disease, and autoimmune thyroiditis. upon physical examination, the patient had multiple telangiectasia, baseline erythroderma, and flushing. immunological evaluation showed lymphopenia with significant reduction in both circulating b and t cells, however, assessment of humoral immunity revealed low igg and decreased igm with normal iga levels. at the time of the evaluation he had been on low dose daily prednisone (7.5mg), colchicine, and methotrexate as immuno-modifying therapy. genetic evaluation revealed a heterozygous mutation in nod2 as well as compound heterozygous mutations in the mefv gene. discussion: mutations in nod2 have been described in association with blau syndrome a multisystem auto-inflammatory syndrome which may explain many of the features experienced by our patient. to our surprise next generation sequencing revealed a second aberration in the mefv gene which causes familiar mediterranean fever, another multisystem auto-inflammatory disease, which might lead to the phenotype observed in the patient. conclusion: this is the first report of genetic lesions in two different genes leading to a severe course of auto inflammation. monogenic autoinflammatory syndromes (mais) are a diverse group of disorders characterized by primary over-activation of the innate immune system. induction of the inflammasome complex by innate immune sensors and increased production of il-1b are implicated in the pathogenesis of mais. macrophage activation syndrome (mas) is a life-threatening illness defined by acute hyper-inflammation and unopposed cytokine release. it is considered an acquired condition secondary to infection, rheumatoid disease or malignancy. the early therapeutic use of il-1b inhibition has profoundly improved the prognosis mas. it has recently been shown that increased free il-18 levels in the blood are causatively linked to the development of mas. significant overlap in clinical presentation and laboratory markers between patients with mais and mas led us to explore the role of free il-18 and therapeutic use of il-1b inhibition in a patient with cdc42 mutation. here, we report the case of an 18 months-old female who presented with hydrops fetalis in utero, and later developed failure-to-thrive, splenomegaly, anemia, thrombocytopenia, arthralgias, rashes, frequent febrile episodes and mild facial dysmorphism along with massive increase in crp, esr and ferritin. whole exome sequencing (wes) identified a heterogenous likely pathogenic de novo variant in cell division control protein 42 homolog (cdc42) c.563g>a (p.c188y). cdc42 encodes a small rho family gtpase that regulates multiple signaling pathways controlling cell polarity, migration, endocytosis and cell cycle progression. single allele mutations in the cdc42 gene were recently reported to cause takenouchi-kosaki syndrome manifesting with growth retardation, developmental delay, facial dysmorphism, and thrombocytopenia however systemic autoinflammation has not been described. cdc42 closely interacts with the wiskott-aldrich syndrome protein but little is known about the mechanism underlying immune abnormalities associated with cdc42 mutations. our patient had an inflammamosopathy-like syndrome. because of significant clinical overlap to mas, we measured il-6, il-18, free il-18 and il-18 binding protein, all of which were significantly increased. this increase in free il-18 heightened her risk of developing mas. her il1b level was normal, but an increase in il-1b is hardly ever detectable in the serum despite playing a critical role in this type of inflammation. indeed, chronic il-1b excess in the tissues promotes systemic inflammation and is associated with chronically elevated crp and esr. with this rationale we started the il-1 receptor antagonist anakinra. within 48 hours from starting anakinra, the parents observed an increase in appetite, resolution of arthralgias and improved mobility. over the course of the following weeks, fever, anemia, thrombocytopenia and rash disappeared, the spleen massively decreased in size and the patient started to meet developmental milestones. crp, esr eventually normalized while ferritin and free il-18 are still trending down. conclusions: significant increase in free il-18 and extremely encouraging clinical response to therapy with anakinra in a patient with novel cdc42 mutation suggests a link between mas and defects in cdc42. elucidating the mechanism of inflammasome activation and the drivers of il-18 increase in mas and mais more broadly may shed light on novel therapeutic targets like the use of human recombinant il-18 binding protein. j clin immunol (2019) 39 (suppl 1):s1-s151 s69 maintenance; smarcal1 is enriched in cells that maintain telomeres via the alternative lengthening of telomeres pathway and smarcal1decifient cells demonstrate telomere instability with replication fork collapse and increased telomere-associated dna damage. [1, 2] telomere analysis of 4 siod patients, including one patient who received a hematopoietic stem cell transplant (hsct) 20 years prior, as well as 5 heterozygous family members revealed significantly shorter telomeres in siod patients compared to heterozygous family members and compared to agematched, healthy controls. methods: peripheral blood mononuclear cells were isolated using a ficoll-hypaque density gradient, cryopreserved, then sent to repeat diagnostics in north vancouver, bc. telomere length measurements were performed at a single-cell level using flow-fluorescence in situ hybridization as previously described. [3] telomere length was measured in total lymphocytes, naive and memory enriched t cells, b cells, and nk cells and compared to reference samples from age-matched, healthy individuals. results: compared to age-matched healthy controls, three siod individuals had mean telomere lengths (mtls) less than the 1st percentile for age across all lymphocyte subsets (total lymphocytes, b cells, nk cells, naã¯ve and memory t cells). in comparison, three unaffected family members had normal mtls (10th percentile< x <90th percentile) across all subsets, and two unaffected family members had low mtls (1st< x <10th percentile) in some subsets. the siod individual who received a matched-sibling hsct 20 years prior, had normal mtl in nk cells (10th < x <90th percentile) but low mtls (1st< x <10th percentile) for all other subsets. conclusions: these data show that siod patients have significantly impaired telomere lengths across multiple lymphocyte lineages and support a limiting role for smarcal1 deficiency in telomere maintenance. in comparison, unaffected family members, heterozygous for smarcal1 mutations, have mean telomere lengths that are normal or slightly low for age. this suggests that abnormally short telomeres are seen in individuals with homozygous but not heterozygous smarcal1 mutations. for the individual who received a hsct, we do not have pre and post-hsct telomere data, but these results support obtaining pre and post-hsct telomere length analysis in future cases. abnormally short telomeres have been linked to widespread perturbation of gene expression. [4] we hypothesize that smarcal1 deficiency, by the effect of stalled forks and shortened telomeres, leads to perturbation in the transcriptome of affected tissues. shortened telomeres may explain the reduced hematopoietic bone marrow production in siod, as bone marrow failure is a cardinal feature of dyskeratosis congenita, a disorder of impaired telomere maintenance. future studies to investigate the role of telomere maintenance in siod include measurement of telomerase activity in polyclonally activated t cells and transcriptome analysis using rna-seq background: yellow fever is a potentially fatal disease for which only supportive treatment is available. vaccination is the primary strategy for prevention of this disease and the vaccine is extremely effective, but there are a few specific populations where it is contraindicated. regarding iga deficiency (the most frequent primary immunodeficiency), current recommendations in the literature are controversial. there are no specific studies in this disease, so case series addressing the safety or possible adverse events after vaccination are essential for decisionmaking during epidemic scenarios, as experienced in brazil in the last years. in this context, this study aimed to describe adverse events after the use of the yellow fever vaccine in iga deficient patients. method: a retrospective cross-sectional study was conducted including iga deficient patients followed at a specialized pediatric outpatient clinic between 2017 and 2018. all patients had at least one year of follow-up. immunoglobulin levels, antibody response to vaccines and lymphocyte subset count were evaluated to exclude other immunodeficiencies or the presence of abnormalities that could contraindicate vaccination. demographic data, the presence of infections and comorbidities, use of immunosuppressive medication and adverse events after vaccine administration of the vaccine were described. results: thirty-eight patients with iga deficiency were included in the study and 18 received the vaccine. vaccinated patients had a mean age at the time of the study of 13.7 years (sd â± 3.5y). six out of the 18 presented comorbidities: thyroiditis (n=3), type 1 diabetes mellitus (n=1), celiac disease (n=1) and juvenile rheumatoid arthritis (n=1). all patients were atopic and only one had recurrent infections in the last year despite the use of antibiotic prophylaxis. all 18 patients had normal igg and igm levels for their age, positive vaccine responses for measles, rubella and mumps, and age-appropriate lymphocyte subset count. after 6 months of observation, no immediate or late adverse events were reported. among the 20 non-vaccinated patients, only one had a formal contraindication (systemic erythematosus lupus using immunosuppressive therapy). five out of the 20 non-vaccinated patients reported being afraid of receiving the vaccine, 7 still intended to receive it and for other 7 patients data regarding vaccination was unavailable. conclusion: despite the small number of patients, the absence of adverse events in this case series suggests that immunization with yellow fever vaccine may be safe in iga deficient patients, excluded other contraindications. more studies are essential to confirm the safety and help the decision-making process regarding the vaccine administration for iga deficient patients, especially in this yellow fever outbreak scenario. introduction/backround: immunodeficiency, centromeric instability, and facial anomalies syndrome (icf) is a rare group of autosomal recessive disorders involving the triad of hypogammaglobulinemia, centromeric instability, and facial anomalies. the majority of patients have hypo-or agammaglobulinemia, but t cell defects have also been reported. we present the case of a child with icf-2 who presented with nk deficiency and ultimately developed an ebv-driven malignancy and was successfully treated with bone marrow transplant. methods: whole exome sequencing and nk cell function via 51-cr cytotoxicity assay and phenotyping via flow cytometry were performed at baylor college of medicine and texas childrens hospital. centromeric banding studies were performed at university of pittsburgh medical center. results: the female patient presented at 3 months of age with cmv pneumonitis and persistent cmv viremia requiring treatment followed by prophylaxis with valgancyclovir. she initially had hypogammaglobulinemia and low t, b, and nk cells; she had normal trecs, lymphocyte mitogen proliferation responses and zap 70, mhci and mhcii expression. the hypogammaglobulinemia and t-and b-cell lymphopenia resolved within 9 months after initial presentation as she clinically improved from her cmv infection. she was found to have nk cell deficiency on three separate commercially tested samples. whole exome sequencing revealed a homozygous variant in zbtb24 indicative of icf-2 syndrome that was confirmed with sanger sequencing (c.1492_1493del, p.q498vfs). repeat nk cell studies confirmed impaired function, and phenotyping showed an increase in cd56-bright and a decrease in cd16-positive cells, suggesting either impaired transition from immature to mature nk cells or impaired survival of mature cells. her karyotype and centromeric banding studies were normal, as were centromeric instability studies. she later developed a memory b-cell defect and presented at 34 months of age with persistent fever, respiratory distress, loss of vaccine titers, hypogammaglobulinemia and low b and t cells. she was found to have ebv viremia and an eber-positive diffuse large b-cell lymphoma in her right lung. due to tenuous clinical status, she received rituximab for treatment of ebv prior to definitive lymphoma diagnosis. she was treated with chemotherapy per protocol anhl1131, group b (pre-phase with cop, courses 1 and 2 with copadm, and courses 3 and 4 with cym) and her course was complicated by seizures attributed to methotrexate toxicity. she ultimately underwent reduced intensity conditioning with hydroxyurea, alemtuzumab, fludarabine, mephalan, and thiotepa followed by a cd-34 selected, hla-matched, unrelated donor peripheral blood stem cell transplant. her early post-transplant course was complicated by adeno-, ebv, and cmv viremia, all successfully treated with antivirals and a donor lymphocyte infusion. she is now greater than 8 months posttransplant, off immunosuppression with 100% donor engraftment, no evidence of organ toxicity or gvhd, and with excellent immune reconstitution. conclusions: this is the first reported case of impaired nk cell function and phenotype and ebv-driven malignancy in a patient with icf-2. this case expands the phenotype of icf-2 and suggests that early bone marrow transplant should be considered in these children. it also demonstrates a novel requirement for zbtb24 in human nk cell maturation and function. rationale: common variable immunodeficiency (cvid) is a disorder that affects the production of immunoglobulins and is associated with development of autoimmunity. multiple mutations have been described that are associated with cvid, but plcg2 mutations have only been described in patients with phospholipase c gamma 2 (plc2) associated antibody deficiency and immune dysregulation (plaid) and autoinflammatory plc2 associated antibody deficiency and immune dysregulation (aplaid). we present a case of a 44 y/o male cvid patient with recurrent upper respiratory tract infections, steroid-dependent autoimmune thrombocytopenia, low b cell count, hepatosplenomegaly, and restrictive lung disease. he was found with a variant of unknown significance at the plcg2 gene. in contrast to plaid our patient does not exhibit cold urticaria. method: case presentation of a cvid patient followed in our clinics. patients chart and previous laboratories were reviewed. sequence analysis and deletion/duplication cvid panel testing was performed using invitaeâ© discussion: genetic testing has revolutionized the diagnosis of immune deficiencies, but variants of unknown significance are being increasingly reported. in this case, a variant of uncertain significance was identified which replaces threonine for alanine at codon 829 of the plcg2 protein. this codon is located at the sh3 domain, which is part of a region that provides auto-inhibitory enzymatic functions. plaid mutations have been identified in sh2 domain, but it has been known that both sh2 and sh3 domains facilitate plcg association with other proteins. studies with deletion of plcg2 gene have shown functional abnormalities in b cells, natural killer cells and mast cells. to our knowledge, there has not been any previous report of a cvid patient with a variant mutation at the sh3 domain of the plcg2 gene without being diagnosed as plaid or aplaid. our patient has immunodeficiency, recurrent upper respiratory tract infections, steroid-dependent recurrent autoimmune thrombocytopenia, rheumatoid arthritis, hepatosplenomegaly, early-osteoporosis and restrictive lung disease. he does not have cold urticaria as seen in plaid, but exhibits autoimmunity not observed in aplaid. conclusion: conclusion: plcg2 is an important protein in the pathway of b cell development. a novel mutation in the sh3 domain of the plcg2 gene may be associated with the cvid phenotype of low b cells and autoimmunity. this could lead to a gain-of-function mutation as seen in plaid but without early-onset cold urticaria. functional studies are required to confirm the significance of this mutation. primary (or familial) hemophagocytic lymphohistiocytosis (hlh) is a rare, life-threatening hyper-inflammatory disease affecting mainly young children. it is caused by mutations in genes involved in the granule-dependent cytotoxic pathway, and is characterized by extreme inflammation and massive tissue infiltration by activated t cells and macrophages. to this day, hematopoietic stem cell transplantation is the only available curative treatment with a transplantrelated mortality of 30%. thus, the development of new, more efficient anti-inflammatory treatments would be a significant advancement in the treatment of hlh. here, we hypothesize that combination therapies targeting both jak-dependent and independent cytokines will be more effective than either one alone to reduce the lifethreatening symptoms induced by this pathology. using a perforin-deficient mouse model of hlh, we first compared the effect of targeting individual cytokines with blocking antibodies on the progression of the disease. we show that blocking ifng and il-18, but not il-6, significantly reduces the severity of hlh. targeting the jak-stat signalling pathway with ruxolitinib, a specific inhibitor of jak1 and jak2, downstream of ifng and il-6, but not il-18, is similarly beneficial. more importantly, combination therapies using ruxolitinib and blocking antibodies to either ifng or il-18 show synergistic effects, further mitigating the progression of the disease. these results suggest that jak-dependent and independent cytokines drive the pathogenicity of hlh in perforin-deficient mice. it further supports that ruxolitinib, although effective in reducing the symptoms of hlh, should be used in combination with anti-ifng and/or anti-il-18 antibodies to prevent hlh progression. this is particular relevant since the former were recently approved for the treatment of hlh while the latter (il-18 binding proteins) are in clinical trials for il-18-dependent macrophage activation syndromes. despite the increased risk of opportunistic lung infection in patients with severe t cell dysfunction (e.g. cd40l deficiency) and/or severe cd4 t cell lymphopenia, we are not aware of any reports of disseminated pneumocystis jiroveci infection in non-human immunodeficiency virus (hiv) patients with primary immunodeficiency (pid). we report the first case, to our knowledge, of disseminated pjp in a patient with cvid like/ctla4 haploinsufficiency. he had been diagnosed with common variable immunodeficiency (cvid) in 2009, approximately eight years prior to being referred to us, and was on intravenous immunoglobulin (ivig). he was also diagnosed with multilineage evans syndrome in 2015. his medical history was also significant for potential granulomatous lymphocytic interstitial lung disease (glild) (lung biopsy in the remote past with interstitial disease), significant splenomegaly (29.4 cm), severe portal hypertension, nodular liver disease (likely nodular regenerative hyperplasia) complicated by anasarca, history of chronic diarrhea (potential enteropathy), lymphadenopathy s/p biopsy with nodular lymphoid hyperplasia, and a history of multiple pneumonias. in 2017, he had developed disseminated pjp with lung, liver, and bone involvement. the t2 vertebra pjp invasion was confirmed with a bone biopsy; gomori methenamine silver staining and pcr were performed and concluded pjp. he was treated with trimethoprim sulfamethoxazole (tmp-smx) and steroids, then was continued on tmp-smx prophylaxis. due to his liver damage and his chronic neutropenia, tmp-smx was replaced by atovaquone as a secondary prophylaxis for pjp. his laboratory studies were significant for an absolute neutrophil count of 1.54 k/ul, absolute lymphocyte count of 0.61 k/ul, hemoglobin of 12.7 g/dl, platelets of 78 k/ul, total bilirubin of 2 . t-cell receptor beta chain repertoire analysis showed an oligoclonal distribution. severe combined immunodeficiency panel through ambry genetic testing was negative as was genetic testing for cd40l deficiency. given his complex clinical history, whole exome sequencing was obtained and detected an autosomal dominant heterozygous missense mutation (c.436g>a) implicated in ctla-4 haploinsufficiency and previously reported by schwab et al. our patient is currently undergoing therapy with abatacept (ctla-4 fusion protein), which has been reported to improve glild, splenomegaly and enteropathy in patients with ctla-4 haploinsufficiency. he is improving on this regimen. he has met with the stem cell transplant team, but at this point of time, due to his abnormal lung function, his liver damage and his significant splenomegaly, he is not a good candidate. defects in the nf-b signaling pathway are implicated in the pathogenesis of several primary immune deficiencies in humans. the clinical features of these conditions vary significantly, reflecting the complexity of the pathway, and its broad role in innate and adaptive immune responses, and the development and differentiation of lymphoid organs. here we report the first case of a human pid caused by a homozygous mutation in nfkbid in a 30 year-old male. he was the second child of consanguineous parents, and was diagnosed with possible cvid at the age of 16, after recurrent episodes of pneumococcal pneumonia. however the clinical features have evolved over time; he developed severe ebv infection at age 18, causing hepatitis and pancreatitis. at the age of 20, he presented with an anca-negative systemic vasculitis, manifesting as pulmonary haemorrhage, and acute necrotizing pauci-immune glomerulonephritis. pulsed methylprednisolone and cyclophosphamide induced an initial remission, however, relapse a year later led to end-stage renal failure. he is now dialysis-dependent, and due to the underlying pid, and chronic cmv viraemia, is not a candidate for renal transplantation. genomic dna was subjected to whole-exome sequencing. variants were filtered using a model of autosomal-recessive inheritance and functional analysis of primary cells was performed. we identified a novel, homozygous, single-base deletion resulting in a frame-shift, and premature stop in nfkbid. nfkbid encodes ibns, a non-classical inhibitor of nf-b signaling. at diagnosis the patient had reduced levels of igg2, iga and igm, elevated ige, with absent humoral immune responses to pneumococcal polysaccharide vaccine, and an intact response to tetanus. lymphocyte numbers were initially within normal reference ranges, albeit with an increased proportion of cd4+:cd8+ t cells. however, over time there has been a significant reduction in b cells and cd8+ t cells. cd4+ t cells demonstrated a skewing towards a central memory phenotype (cd45ro+/ccr7+), and cd4 t cell proliferative responses to pha were comparable to a healthy control. functional analysis of primary cells from the proband revealed a complete absence of bns protein expression, dysregulated nf-b signaling, and elevated pro-inflammatory cytokine production. the patient is currently receiving a trial of targeted therapy to modulate the aberrant immune responses. this novel pid highlights the importance of regulation of nf-b signalling, in orchestrating an appropriate immune response, maintenance of self-tolerance, and protection against viral pathogens. primary immunodeficiency diseases (pid) are a heterogeneous group of conditions with variable clinical features that are frequently associated with significant diagnostic delay. accurate diagnosis has significant therapeutic benefit and may lead to personalized therapies. we established the immunology flagship of melbourne genomics health alliance in australia to determine the clinical utility of genomic sequencing for diagnosis and management of individuals with suspected and confirmed cases of pid. 198 adults and children with suspected or confirmed pid (n=153), autoinflammatory disease (n=33) and hereditary angioedema (hae, n=11) were recruited to the melbourne genomics immunology flagship. whole-exome sequencing (wes) was performed, with targeted gene analysis. variant curation and reporting was performed according to the american council of medical genetics guidelines. overall, wes was diagnostic in 15% (30/198), confirming a preexisting diagnosis in 7% (14/198), and offering a new or more specific diagnosis in 8% (16/198). variants of uncertain significance were identified in a further 28 patients (14%) in genes known to be associated with their clinical diagnosis, that warrant further functional validation. in the hae group, diagnosis was confirmed in only 5 patients (45%), suggesting that wes may not be the appropriate technique for genetic diagnosis in this condition. a higher diagnostic rate was observed for autoinflammatory disorders (20%; 8/40) compared to pid (12%; 18/146). of those who received a diagnosis, immediate changes to patient management and treatment occurred for 17/29 patients (59%), including hsct for 3 and specific targeted therapy for 11 (38%) individuals. we have demonstrated the utility of wes for accurate diagnosis of complex immune diseases, with the potential to change diagnoses, guide therapeutic intervention and provide opportunities for genetic counseling. further longitudinal analysis will determine clinical outcomes and health economic implications of genomic sequencing for diagnosis and management of immunological conditions in australia. at birth he had neonatal asphyxia and cerebral palsy. at 4 years old he had presented involuntary movements, left paresis, bilateral horizontal nystagmus. at 8 years of age, he had a right nasal obstruction. it was resected by otorhinolist and informed by biopsy: inflammatory polyp and chronic sinusitis. he has had 3 pneumonias, sinusitis and diarrhea. at the age of 13 years, the ataxia telangiectasia was confirmed by sequencing with pcr (62 exons, 91711 bp) of the atm gene: transition g> a, nucleotide position 2250, codon 750, affecting splicing. alpha fetoprotein 572-606.90 u/ml. brain mri, say cerebellar atrophy. he had igg 685 mg / dl -734 mg / dl, iga 0.00 mg / dl, <1 mg / dl, igm 268 mg / dl -315 mg / dl, ige 0.10 -<1 iu / ml. subclasses of igg: igg3: 0.05 g / dl, igg4: 0.04 gr/dl, low. igg anti hepatitis b 6,22. no seroconversion. hiv negative tcd3 + lymphocytes: 32,40%, = 553 cells / mm3, ltcd4 +: 23,78% = 413,21 cel / mm3, ltcd8 +: 7,69% = 133,5 cells / mm3, cd4 / cd8: 3.09. for all of the above, common variable immunodeficiency was diagnosed. he receives human immunoglobulin. at 16, i arrived at this hospital due to fever, respiratory distress and lymphadenopathy in the neck. ct showed ganglionic conglomerate on right side neck. lymph node biopsy: strong tumors with cd20 and bcl2, weak and moderate diffuse pax-5; negativity with cd68, cd3 and cd10, and a cell proliferation index with ki67 of 50%, diagnosis: diffuse large b cell lymphoma. treated with rituximab and chemotherapy. lymphoma completely remitted. conclusion: the association ataxia telangiectasia and lymphoma is frequent. by contrast, cvid and ataxia telangiectasia are extraordinarily rare. introduction: chronic granulomatous disease (cgd) is a primary immunodeficiency wherein affected patients are susceptible recurrent infections caused by specific bacteria and fungi as a result of defective nadph activity. additionally, inflammatory complications involving the bowel and lungs can cause significant morbidity. currently the only proven permanent cure to cgd remains hematopoietic stem cell transplant. case: a 25-year-old patient was diagnosed in infancy with x-linked cgd. at age 5yrs he received a nonmyeloablative peripheral blood stem cell transplant from his 10/10 non-carrier sister as previously reported (nejm 344:881, 2001) . conditioning was cyclophosphamide (60 mg/kg) on d-6 and d-7; daily fludarabine (25mg/m2) on d-5 through d-1; antithymocyte globulin at 40mg/kg on d-5 through d-2. posttransplant immunosuppression consisted of cyclosporine on d-4 through d+100. he received 7.8x106 cd34+ peripheral blood stem cells which were t-cell depleted with 1x105 add back of cd3+ cells on day 0. after 10 days of neutropenia (anc <500) there were signs of engraftment. per protocol, he received donor peripheral-blood lymphocytes containing 2.0x106 cd3+ cells/kg on d+ 30 after transplantation. since donor t cells constituted less than 60 percent of his circulating cd3+ t cells and he had no graft versus-host disease, he received 1.0â¬107 cd3+ cells/kg on d+60. after the discontinuation of cyclosporine, he received a total of three donor-lymphocyte infusions (1.0â¬107 cd3+ cells/kg) at 90-day intervals achieving 100% t cell and myeloid engraftment at 26 months post-transplant with no acute nor chronic gvhd. at last follow-up 6 years post-transplant (2004) he had 100% and 98% lymphoid and myeloid peripheral chimerisms, respectively. the patient and family declined further periodic followup. then, in october 2018 he presented with malaise, cough and fevers. he eventually was found to have a large consolidation and a bal grew burkholderia cepacia. his dhr showed 12% activity and peripheral blood myeloid and lymphoid chimerisms were 12% and 60%, respectively. discussion: this late graft failure following peripheral blood transplant occurred following a conditioning regimen which is not the current standard for transplant in cgd. in the case series in which this patients transplant is reported (nejm 2001), another patients myeloid chimerism fell to 15% by 3 years post-transplant, remaining stable at that level of chimerism without any serious infections over regular periodic follow up to the present time. current regimens typically include busulfan to enhance engraftment and prevent graft failure. this case reinforces the need for prolonged monitoring of primary immune deficiency patients after transplantation. introduction: with the introduction of severe combined immunodeficiency (scid) newborn screen (nbs) in the state of kansas in 2017, a case of complete digeorge syndrome (dgs) was discovered in an infant born to a diabetic mother with atypical features. this is the first dgs case diagnosed after adding the scid nbs, which emphasizes the need to establish scid nbs in all 50 states. case presentation: the female infant was born via spontaneous vaginal delivery at 39 1/7 weeks to a 31 year old g1 now p1 mother. maternal history was significant for chronic hypertension, obesity, insulin dependent type 2 diabetes, anxiety, depression, and scoliosis. the infant was noted to have a left sided abdominal wall defect and hernia, imaging identifying left renal agenesis, and was initially suspicious for vater syndrome. fortunately, the infant's scid nbs revealed low t cell receptor excision circles (trecs). her initial white blood cell count was 14.1 with an absolute lymphocyte count of 2.679 k/ul. ebv pcr, cmv pcr, and hiv studies were negative. chest imaging discovered absent thymus, abnormal vertebrae with only 10 ribs on the right and 12 ribs on the left, and abnormally formed thoracic vertebrae (t7). echocardiogram detected an atrial septal defect measuring 0.32 cm, possible pfo versus secundum asd. endocrinology was consulted for management of labile calcium and phosphorus levels. fish was negative for 22q11.2 deletion. microarray r evealed a variant of unknown signif icance arr[grch37]2p11.2(86285942_86506132)x3. sequence analysis of combined and severe immune deficiency genes showed a variant of uncertain significance c.544c>a (p.leu182met). management and outcome: additional evaluation included: cd3 67ul (1700-3600ul), cd4 51ul (1700-2800ul), cd8 19ul (800-1200ul), cd45ra 14 cells/ul (1100-5200cells/ul), normal cd 19, and cd 16/ 56, normal immunoglobulin g level, and normal dihydrorhodamine assay. skeletal survey, ct abdomen and chest, and hla typing were performed in preparation for thymic transplant. discussion: patients with complete dgs, a form of scid found in less than 1 percent of patients with 22qds, have absent thymus and a t cell count <3 standard deviations below normal for age (typically <50 naã¯ve cd3+ t cells/mm3). in a large series of patients with complete dgs, only 52 percent had an identifiable 22q11.2 deletion [1] . infants of a diabetic mother have various genetic and syndromic associations including diabetic embryopathy. [2] despite the importance of immunological aspects in pregnancy, few studies have reported on the cellular immune modifications of diabetic embryopathy. diabetes during pregnancy may affect the development of the thymus and thus maturation of the immune system in the offspring. [3] the recent addition of a trec assay to newborn screening can identify such a subset of infants with atypical presentations. scid nbs uses an assay for trecs, a biomarker of t cell development. [4] [5] [6] this initial presentation now places the immunologist in the role of "first responder" with regard to diagnosis and management of these patients, who may present with atypical features. newer genetic and molecular techniques now allow for earlier identification of immune defects in such disorders with life-long clinical concerns. [7] references: introduction/background: goods syndrome is a rare cause of combined b-and t-cell immunodeficiency occurring in association with a thymoma. affected patents are susceptible to bacterial, fungal, viral, and opportunistic infections. an association with autoimmunity has also been reported. current knowledge of goods syndrome is primarily limited to case reports and small series. objectives: to examine the spectrum of clinical and laboratory features of a major cohort of goods syndrome patients in the us. methods: we conducted a retrospective analysis of patients with goods syndrome in the usidnet registry and the mount sinai hospital (msh) cohort. r e s u l t s : we i d e n t i f i e d 2 0 p a t i e n t s w i t h t h y m o m a a n d hypogammaglobulinemia (usidnet, n=11; msh, n=9; median age: 60 years; female: 45%), representing data from 151 patient years. the median age at diagnosis of thymoma and hypogammaglobulinemia were 52 years (range 31-85), and 50.5 years (range 28-86), respectively. two patients were deceased (at age 65 and 70 years, cause unspecified). all patients had low igg (median 313mg/dl, range 47-699). iga and igm were reduced in 90% and 45% of patients, respectively. low cd19+ b cells (median 0.5/mm^3, range 0-28) were reported in all available records. the absence of cd19+ b cells was observed up to 21 years postthymectomy. a wide range of additional laboratory abnormalities were identified: low cd4+ t cells (n=5), low cd8+ t cells (n=2), low cd4/ cd8 ratio (n=6), low nk cells (n=6), and absent peripheral eosinophils (n=8). the most common sites of infections were lower respiratory (70%), upper respiratory (55%), and gastrointestinal (35%). in addition, sepsis (15%), meningoencephalitis (5%), osteomyelitis (5%), and urinary tract infection (5%) were also observed. identifiable infectious agents included: bacteria (35%), virus (35%), fungus (25%), parasites (10%), and protozoa (5%), with opportunistic infections recorded in 25% of patients. opportunistic infections were significantly associated with absolute cd4 lymphopenia (p=0.048, fishers exact test). enterovirus was identified as a previously unreported cause of meningoencephalitis in this population. autoimmune manifestations were reported in 45% of patients, with a higher prevalence of inflammatory colitis (20%) than previously reported. hashimoto thyroiditis, fibromyositis, and bronchiolitis obliterans organizing pneumonia (n=1 each) were identified as previously unreported autoimmune/inflammatory conditions in this population. a case of alopecia areata was also observed. additionally, bronchiectasis was recorded in 20% of patients. all patients were initiated on immunoglobulin replacement, with antibiotics prophylaxis in 20%, and immunosuppressive medications employed in 10% of patients post diagnosis of immunodeficiency. conclusion: goods syndrome is a combined immunodeficiency, with a wide range of autoimmunity in a subset of patients. we expanded upon the spectrum of associated infectious and inflammatory complications through a major us cohort. persistent immune dysregulation was observed up to 2 decades post-thymectomy. introduction: primary immunodeficiencies (pids) constitute a large group of rare disorders that affect the immune systems function. some pid patients develop autoimmunity in addition to having increased susceptibility to infections due to their impaired immunity [917] . (1) case presentation/ management: a 43 year old caucasian female with history of bipolar disorder, factor v leiden deficiency, anti thrombin 3 deficiency, pulmonary embolism, endometriosis, and seasonal allergies was evaluated for chronic granulomatous disease (cgd) in 2007. the main symptoms were inflammatory breast lesions necessitating 4 surgeries on the right breast, and back, facial, genital, ocular, mouth, and scalp sores. biopsy with cultures of the wounds was positive for corynebacterium, coagulase-negative staphylococcus, enterococcus, bacteroides species, and provatella. neutrophil oxidative burst was ordered by the infectious disease specialist and showed normal and abnormal neutrophil populations, a finding consistent with cgd carrier. patient was started on interferon gamma-1b after failing multiple courses of antibiotics. her symptoms were well controlled on interferon gamma-1b 100mcg/0.5ml sq every other day, trimethoprim 100mg tab (2tabs in am and 1 tab in pm), cefixime 400mg once daily, and topical mupirocin as needed except for her recurrent genital ulcers. cgd can be rarely associated with oral ulcers however there is a limited literature describing associated genital ulcers. according to the international study group diagnostic criteria published in 1990 (2), the patient was diagnosed by a rheumatologist as having behcets disease (bd). there are no pathognomonic laboratory tests in bd; as a result, the diagnosis is made clinically. patient failed a trial of colchicine and was later started on cyclosporine, which resulted in decrease of her mouth and genital ulcers. discussion: bd is a rare disease mostly seen along the silk road. the prevalence has been reported as 0.12 (usa) to 370 (in a single village, northern turkey) for 100 000 inhabitants. (3) cgd is a primary immunodeficiency caused by defects in any of the five subunits of the nadph oxidase complex responsible for the respiratory burst in phagocytic leukocytes. patients with cgd are at increased risk of life-threatening infections with catalase-positive bacteria and fungi, and inflammatory complications such as cgd colitis. (4) reports of cgd female carriers with discoid lupus erythematosus, photosensitivity rashes, and other autoimmune phenomena have been published [48, 49] (4) . to the best of our knowledge, this is the first case to report bd in an affected cgd carrier. the treatment of inflammatory disease in patients with cgd poses a difficult balance between therapeutic immunosuppression and the increased risk of severe infection. (5) . high dose intravenous immunoglobulin, and targeted therapies such as ctla4-ig for t cell mediated pathologies, rituximab for b-cell mediated pathologies, and anti-tnf for ibd, may be preferable over the broad immunosuppressive activity of glucocorticoids. in addition, emerging evidence suggests that hematopoietic stem cell transplantation has indication for cases that have been difficult to control using immunosuppression. (1) given all that, our case emphasizes the need to maintain suspicion for autoimmune disorders / immune dysregulation in patients with pid. introduction: cd40-ligand deficiency is an x-linked combined immunodeficiency, characterized by susceptibility to infection, often with associated neutropenia, malignancy, and autoimmunity. central nervous system (cns) manifestations are less commonly reported than respiratory or gastrointestinal complications, but are most often attributed to infection. herein we describe a challenging case of gradual onset episodic memory loss, confusion, and unilateral hemiplegia in a young male with cd40ligand deficiency. case presentation: the patient is a 13-year-old male with cd40-ligand deficiency on immunoglobulin replacement therapy presenting with recurrent, episodic altered mental status (ams) and gradual neurocognitive decline. initial neurologic symptoms began at age 11 years, and included fever, nausea, and eyelid fluttering. initial comprehensive infectious workup at this time, including blood and urine cultures, lyme antibody, serum pcr for hsv, cmv, ebv, respiratory viral pcr including atypical viruses, csf studies including culture, lyme eia, pcrs for enterov i r u s , v z v, e b v, c m v, h s v 1 / 2 w e r e u n r e v e a l i n g . electroencephalogram (eeg) and mri displayed generalized slowing and global atrophy, respectively. definitive diagnosis was not made. the patient continued to decline with worsening developmental delay and memory loss. one year later, at age 12 years, he had a recurrent episode of ams with repeat negative infectious workup including blood and urine cultures, respiratory virus pcr including atypical viruses, csf culture including acid fast bacillus and fungi, cryptococcal antigen, viral encephalitis panel by pcr, and serum pcr for ebv and hhv-6. eeg at this time showed left hemispheric epileptogenic potential, consistent with seizure activity. his presentation, at age 13 years, was notable for right-sided hemiplegia with facial numbness, dysarthria, nausea, and fever. he was found to have anello virus on pcr of csf, abnormal left temporal region on eeg, and global atrophy with stable, diffuse generalized volume loss on mri. he was diagnosed with occult anello virus-induced encephalitis with hemiplegic migraine and discharged on valproate. discussion: here we present the first reported case of anello virus detected by pcr in a cd40-ligand deficient male with neurocognitive manifestations, attributed primarily to hemiplegic migraine. given the anello virus prevalence and relatively avirulent character, it is presumed to be unlikely culprit for encephalitis; however, the significance of this finding is as yet unknown. this case highlights diagnostic challenges in immunodeficiency: infection may go undetected by standard diagnostic techniques; however, the significance of infections identified with advanced techniques may not yet be understood. background: henoch-shã¶nlein purpura (hsp) is an iga-mediated small vessel vasculitis that presents with a tetrad of abdominal pain, arthritis, glomerulonephritis, and purpura. hsp is typically a selflimiting disease of childhood following a viral illness. there is no universal treatment for patients with chronic or recurrent hsp. we report a chronic refractory case of hsp that was successfully treated with a tumor necrosis factor inhibitor (tnfi), etanercept. etanercept functions as recombinant protein that consists of a tnf-alpha receptor ligand-binding region that links to the fc portion of human igg. it is currently approved for use in 5 diseases: juvenile rheumatoid arthritis, rheumatoid arthritis, ankylosing spondylitis, plaque psoriasis, psoriatic arthritis. tnfi are categorized into two broad categories, recombinant receptors (etanercept) and neutralizing antibodies (ex. infliximab and adalimumab). there have been prior case reports of hsp associated with tnfi agents during the treatment of other autoimmune conditions in the adult population. to our knowledge, there have been 3 prior etanercept related hsp reports, one report associated with adalimumab, and one with infliximab. however, there has been no prior report of etanercept use successfully treating chronic refractory hsp. case presentation: a 16-year-old native american male with 3 year history of chronic hsp, hla-b27 positive, and enthesitis related arthritis who was initially treated with steroids, sulfasalazine and methotrexate for symptoms of joint pain and purpura. his iga level was 545 mg/dl prior to therapy. despite treatment for one month of steroids, eight months of sulfasalazine exclusively and eight months of methotrexate and sulfasalazine, he continued to have persistent purpura on bilateral extremities without improvement. he was subsequently initiated on etanercept 50mg weekly and methotrexate was discontinued. approximately one month later, his rash significantly improved. his rash and joint pain recurs when he misses a dose of etanercept. punch biopsies were taken 3 months after initiation of etanercept. the biopsies of a lesion from his left arm showed early leukocytoclastic vasculitis and from his left leg showed weak granular deposition of iga, igm and c3 within vessel walls. there is controversy whether this is a true iga vasculitis. however, we believe that his clinical presentation and the deposition of iga and c3 within blood vessel walls seen on biopsy correlates with chronic henoch-shã¶nlein purpura. conclusion: there is no standard treatment of chronic hsp, but there are reports of benefit with nsaid and corticosteroids. per our literature review, there are no prior reports of etanercept use in the treatment of chronic hsp. tnf inhibitor, etanercept should be considered as a treatment for chronic refractory hsp in the pediatric population as it has showed rapid resolution of purpura in this case report. further studies of etanercept in the treatment of chronic hsp should be conducted given the controversial literature of anti-tnf ab induced hsp during the treatment of other autoimmune diseases. although clinical manifestations of iron overload appear to be quite uncommon in patients who are heterozygous carriers of hfa mutation, we present cases that appear to suggest an increased risk non allergic rhino-sinusitis. case report: we present a 66 year old gentleman with perennial colored rhinorrhea, with facial pressure and tenderness, constant post nasal drip, dry cough and bilateral congestion that had been going on for the past several years. he also had a frequent urge to clear his throat and had frequent episodes of sore throat despite having no history of gerd or lpr. he reported to have multiple sinus infections every year that would progress to pneumonia and eventually require long courses of oral antibiotics. all started in his 40s intensified in the recent past. he had 3 other siblings; one died in his 40s due to liver complications of hh and had a carrier sister and brother with a hx of sino nasal problems exactly similar to the patients. his exam was remarkable for bilateral narrowed nasal passages and moderate edema of the mucosa. his rhinolaryngoscopy showed significant edema and purulent drainage, most notably from bilateral middle meati. his skin test was negative. his cbc showed a wbc count of 6.7/ml with 2% eosinophils and his immunoglobulin panel showed an iga of 236 mg/dl, igg of 1190 mg/dl and ige of 31 mg/dl. patient was placed on alkalol sinus rinses and azelastine nasal spray, which he reported to work pretty well. he left for costa rica and is expected to return back with his siblings to a&i clinic in the coming months. discussion: hh is one of the most common inherited disorders in people of northern european descent with an incidence of 1:200 and carrier rate of 1:10.. most affected hh patients are homozygous for the mutation designated c282y at the hfe gene located at the 6th chromosome. unlike hereditary hemochromatosis, clinical manifestations of iron overload appear to be quite uncommon in patients who are heterozygous carriers. hh patients are at risk for a number of infections with bacteria whose virulence is increased in the presence of excess tissue iron. hh is also a risk factor for acute fulminant frs . here the mechanism is postulated to be due to quantitative or qualitative neutrophil defects as this condition is mostly seen in patients with dm, aplastic anemia, and can happen in patients undergoing antineoplastic chemotherapy. no known increased susceptibility for infections through either mechanism is postulated for patients with the heterozygous carrier state. here we present 3 hh carrier patients who present with recurrent rhinosinusitis with no allergen sensitizations and normal ige levels. since most fungal immunity is at the tissue level and is cytokine driven, it can be speculated that increased tissue levels of iron might interfere with mechanisms of innate immunity. chief, human immunological diseases section, laboratory of clinical immunology and microbiology, niaid, nih, bethesda, md background: dedicator of cytokinesis 8 (dock8) mutations are associated with a combined immunodeficiency disorder marked by atopic features, infectious susceptibility with a striking preponderance of cutaneous viral disease, and a risk for the development of malignancy including lymphoma. almost all cases can be diagnosed by documentation of the loss of dock8 protein expression. methods: we describe a 22-year-old male with a diagnosis of pre-b cell acute lymphoblastic leukemia (all) followed by epstein-barr virus (ebv) associated diffuse large b cell lymphoma (dlbcl). compound heterozygous mutations in dock8 were documented following the completion of whole exome sequencing (wes). the pathogenicity of the variants was assessed. flow cytometric quantification of intracellular dock8 protein was completed. dock8 protein function was assessed by evaluating the morphology of patient lymphocytes when migrating in a 3d collagen matrix. results: a concern for a primary immunodeficiency was raised due to a history of recurrent otitis media which began at 12 months of age. by 4 years of age, numerous warts were noted on his fingers; however, they were transient for a duration of only 2 years. no atopic features were appreciated. at 15 years of age, a diagnosis of pre-b cell all was made. during all therapy, infectious complications were severe including an intestinal perforation, osteomyelitis, and sepsis. at 22 years of age, still in an ongoing remission from his all, an incidental finding of a lung nodule led to a diagnosis of ebv-associated dlbcl. during therapy, however, infectious complications were again severe including a soft tissue infection and sepsis. wes was performed and compound heterozygous mutations in dock8 (c.1128_1132del and c.4474-1g>c) were documented. flow cytometric quantification of intracellular dock8 protein was normal when compared to a normal control. nevertheless, additional functional assessment of dock8 protein was completed. when migrating through a 3d collagen matrix, 45% of the patient lymphocytes studied demonstrated abnormal elongation (stretch ratio > 8 defined by length/width) compared with 10% of lymphocytes from a normal control. he is being evaluated for hematopoietic stem cell transplant. conclusion: autosomal recessive mutations in dock8 are a rare cause of a combined immunodeficiency marked by atopic features, infectious susceptibility with a striking preponderance of cutaneous viral disease, and a risk for the development of malignancy including lymphoma. here, pre-b cell all followed by the development of a subsequent malignant neoplasm (ebv-associated dlbcl) led to the discovery of dock8 deficiency. hence, as our case underscores, for rare instances of high clinical suspicion despite normal dock8 protein expression, additional functional testing is crucial to make a definitive diagnosis and plan treatment. understanding the spectrum of dock8 mutants and their phenotypes will improve our understanding of dock8 deficiency. background: autosomal dominant hyperimmunoglobulin e syndrome (ad-hies) is a rare primary immunodeficiency caused by heterozygous loss-of-function mutations in the signal transducer and activator of transcription 3 (stat3) gene. ad-hies classically characterized by recurrent cold staphylococcal abscesses, pneumonia, eczema, and an elevation of ige level. other additional clinical manifestations of hies have been recognized including skeletal dysplasia (scoliosis, pathologic fractures, delayed dental deciduation), pneumatoceles, coronary-artery aneurysms, brain lesions, and chiari malformations. objective: to describe a unique case of abdominal abscesses in a patient with ad-hies. method: a 22-year-old female with known ad-hies (c.1144 c>t (p.arg382trp)) and a complicated history of early pneumococcal pneumonia and meningococcemia resulting in bilateral amputation below the knees along with loss of several digits, presented for evaluation of skin infection. she had a history of recurrent staphylococcal skin abscesses and presented with inability to use her prostheses due to pain from inflammation around her amputation sites. she underwent imaging and was found to have bilateral extremity abscesses with an associated osteomyelitis of her l tibia (which was found to be mrsa after incision and drainage). while receiving intravenous antibiotics for her osteomyelitis, she developed intractable abdominal pain. imaging showed a thick-walled, multi-septated, paranephric abscess as well as several smaller abscesses scattered throughout her abdomen. she underwent multiple drain placements and drainage of retroperitoneal fluid collections via interventional radiology (ir). purulent fluid from the abdominal abscess drainage grew mrsa. the patient continued to have re-accumulation of abscesses despite multiple drainages. repeat imaging noted increased paranephric abscesses which were not communicating with drains. given lack of response to several ir-placed abdominal drains and to 6 weeks of intravenous antibiotics, she had an open surgical washout with minimal improvement. hospital course was further complicated by development of a left lower lung lobe consolidation and sub-segmental pulmonary embolism necessitating treatment with heparin. finally, after several weeks of escalating antimicrobial therapy and with additional drain placements, the retroperitoneal abscesses started to recede. repeat abdominal imaging several months later while asymptomatic revealed slow but continuing resolution of the abscesses. conclusion: the present case raises awareness of an unusual location for infection in a patient with ad-hies. although the majority of complications of ad-hies are sinopulmonary and skin infections, recalcitrant intra-abdominal abscesses should be considered in the differential of infections in hies. introduction/background: the recent epidemiologic studies have revealed that primary immunodeficiencies (pids) are more common than previously thought. however, there are very few data on epidemiology of pids in korea. objectives: we attempted to estimate the pid epidemiology and disease burden in korea and provide the background information for pid registry for future. methods: to review the previously reported scientific studies, pubmed, koreanmed, google scholar were searched. any studies on pids reported in scientific journal (korean or international) from january 2001 to november 2018 were searched. both korean and english reports were searched. diagnosis for pid was categorized from group i to group xi according to 2017 iuis phenotypic classification. study period was divided into two periods: period 1 from 2001 to 2005 and period 2 from 2006 to 2018, because there was a multicenter study to estimate pid epidemiology from 2001 to 2005. in addition, the number of pid patients and the cost for care were estimated among patients who requested reimbursement to health insurance review and assessment service (hira) korea for one year in 2017. results: a total of 334 pid patients were identified in 75 reports. one hundred and ninety-nine patients (20 reports) and 135 patients (55 reports) were found in period 1 and period 2, respectively. the pids were reported in 11 patients for immunodeficiencies affecting cellular and humoral immunity, 23 patients for combined immunodeficiency with associated or syndromic features, 143 patients for predominantly antibody deficiencies, 33 patients for diseases of immune dysregulation, 113 patients for congenital defects of phagocyte, 1 patient for defects in intrinsic and innate immunity, 4 patients for auto-inflammatory disorders, 6 patients for complement deficiencies, and none for phenocopies of pid. from hira reimbursement data, the number of pid patients were 42 for combined immunodeficiency, 486 for predominantly antibody deficiency, 47 for common variable immunodeficiency, 135 for functional defect of neutrophils, 238 for immunodeficiency associated with other major defects, 272 for other immunodeficiencies. a total of 1,220 pid patients were treated for 14,316 days and $3,351,678 was reimbursed in 2017. conclusions: we performed a systematic review on published studies for pid in medical journals and national open data system of hira to estimate the pid disease burden for the first time in korea. to obtain more information on true pid epidemiology and disease burden in korea, a national multicenter study for pid registry is required in the future. micro-thrombocytopenia is one of the most serious challenges for wiskott-aldrich syndrome (was) and x-linked thrombocytopenia (xlt) patients. thrombocytopenia leads to severe, potentially life-threatening, bleeding episodes, which require frequent transfusions and account for 23% of deaths in patients experiencing was mutations. the gold standard treatment for was patients is hematopoietic stem cell transplantation (hsct) from an hla-identical donor but more recently a number of gene therapy (gt) trials in europe and usa showed promising results. in particular, it has been shown that was patients receiving lentiviral mediated gt, consisting of autologous cd34+ cells transduced with lentiviral vector encoding the human was gene under the control of the endogenous promoter, in combination with a reduced intensity conditioning regimen, have a significant increase in platelet (plt) counts. even though plt counts do not reach normal levels, treated patients decreased the severity and frequency of bleedings. here, in a cohort of 4 xlt and 16 was patients, fifteen treated with gt, the plt phenotype and function were analyzed by electron microscopy, flow cytometry and proteomic profile. the aim of the project is to assess the presence of plt defects in was untreated patients and the impact of gt treatment on the correction of plt behavior. we demonstrate that plts of untreated was patients have reduced size and abnormal ultrastructure along with hyperactivated phenotype at steady state, showing increased expression of cd62p, activated iib3 integrin and cd40l; conversely, activation response to agonist and aggregation capacity are both decreased. analyzing plt samples isolated from treated patients, we found that gt restores plt size and ultrastructure very early after treatment and reduces the hyperactivated phenotype proportionally to was protein (wasp) expression and follow-up length. plts isolated from gt treated patients showed a normal activation response to agonists and restored aggregation capacity in 5 out of 7 analysed patients. by proteomics, various protein pathways were found downregulated in untreated plt samples, mainly involving cytoskeletal-rearrangement proteins, integrins, signal transduction molecules, vesicles-transport proteins; additionally, decreased metabolic capacity were observed. these results are in line with the functional defects observed in plts in terms of activation and aggregation. conversely, the expression of protein-pathways found downregulated in untreated patients is comparable to healthy controls in gt-treated plt samples, reflecting the amelioration of plt phenotype and function. overall, our study highlights the coexistence of multiple defects in the activation and aggregation responses occurring in was patient plts in absence of wasp. gt was able to normalize the plt proteomic profile followed by consequent restoration of plt ultrastructure and phenotype, suggesting gt is responsible for the observed reduction of bleeding episodes in treated patients. introduction: pik3cd is an autosomal dominant genetic disorder of the immune system that results in persistent activation of pi3k. signaling through pi3k is essential for immune cell regulation of metabolism, migration, proliferation and differentiation, leading patient to present with lymphadenopathy, immunodeficiency and senescent t cells. the mutated protein causes t cells to over activate and mature too quickly leading to their death, this over activation also blocks the maturation of b cells. case presentation: a 51-year-old female with a childhood history of failure to thrive, asthma, chronic rhinitis and common variable immunodeficiency on intravenous immunoglobulin replacement, was seen in immunology clinic to establish care. she reported frequent episodes of pneumonia and bronchitis in her childhood. her family history was significant for family members with leukopenia, but no diagnosed immunodeficiency. patient had 1 son who did not report symptoms concerning for immunodeficiency. physical exam was within normal limits with no lymphadenopathy. laboratory examinations exhibited normal iga (185 mg/dl), igg (800 mg/dl), and igm (100 mg/dl). while flow cytometry showed normal absolute cd3 687 (570-2400 cells/ul), cd4 (540 cells/ul), nk cells (151 cells/ul), cd19 (179 cells/ul), cd45ra (160 cells/ul), cd45ro (311 cells/ul), cd2 (757 cells/ul), and hla-dr (173 cells/ul), nonswitched memory cells (9 cell/ul) and class-switched memory cells: (15 cells/ul). (4-62 cells/ul). vaccine response was not pursued as patient had been on ivig. genetic testing was pursued, and revealed a mutation in pik3cd gene, specifically a mutation in the c.2320g>a; p.val774met variant (rs370932461). this mutation though seen in databases, is not currently reported in medical literature as associated with this condition. based on these, ct chest was ordered to screen for bronchiectasis, adenopathy and lymphoma. ct showed no cardiopulmonary disease or adenopathy, but did show an incidental adrenal mass which is now being worked up. while the pattern of inheritance of this mutation is autosomal dominant, her son is asymptomatic and testing of her son has not been pursued, though it was advised for her cousins given history of leukopenia. patient has continued on igg replacement therapy. conclusion: recent publication by the clinical immunology society suggests consideration for next generation sequencing when it can affect future family planning or it has treatment and prognostic implications. this case highlights all aspects of the importance of genetic testing as part of the diagnosis of cvid, since it can affect progeny, it offers the possibility of treatment with immune modulating agents and has implications on screening, since patients are at increased risk for malignancies. background: abnormal v(d) j recombination activity in patients with mutations in the recombination-activating genes 1 and 2 (rag1/2) results in markedly reduced usage of distal vand j genes at the t cell receptor alpha (tra) locus. mucosa-associated invariant t (mait) cells express a semi-invariant t cell receptor containing the distal trav1-2 gene. mait cells can be identified by flow cytometry using a mab directed against valpha 7.2, which recognizes the product of the trav1-2 gene. by performing high throughput sequencing (hts) of tra rearrangements and flow cytometry, we have confirmed lack of t cells using distal valpha genes in patients with known rag mutations. we now report that flow cytometry with mab against valpha 7.2 successfully identified rag deficiency in two patients with an atypical presentation. methods: tra rearrangements were analyzed by hts using gdna from sorted t cell subsets from rag-mutated patients and healthy donors. distal valpha usage was measured in whole blood by flow cytometric analysis with an anti-valpha 7.2 antibody. rag mutations were detected by sanger sequencing. patients were enrolled in niaid protocol 18-i-0041. results: hts of tra rearrangements revealed lack of distal trav and traj gene usage in patients with rag1/2 mutations. the presence of circulating mait cells in controls and patients with known rag1/2 mutations and various clinical phenotypes was analyzed by flow cytometry using mab against valpha 7.2. we found a virtual lack of valpha 7.2 expression in rag mutated patients (<0.5%) compared to controls (2-8%) . we used the valpha 7.2 assay to test two patients with unknown immunodeficiency manifesting as skin granulomas and autoimmune cytopenia, and found nearly absent expression (0.14% and 0.08%). targeted sequencing of rag1/2 revealed that both patients were compound heterozygous for rag1 mutations: p.r112h/p.c328y and p.r410w/p.r507q, respectively. conclusions: patients with mutations in rag1/2 demonstrate a skewing of their tcralpha repertoire. the reduction in recombinase activity in these patients does not allow for rearrangements of the most distal valpha segments. rapid identification of patients lacking valpha 7.2+ t cells by flow cytometry may prompt sanger sequencing and identification of rag1/2 mutations in a matter of days. this assay represents a simple but powerful tool to reduce the cost and time associated with other analysis methods. acknowledgements: supported by dir/niaid/nih. director, centro de inmunologã­a clã­nica dra.bezrodnik y equipo introduction: the fate of effector t cells is strongly dependent on the expression of bcl-6 or blimp-1, which are inhibited reciprocally through a complex signaling pathway. several studies have shown that bcl-6 is a key transcription factor for differentiation towards the follicular helper t cells (tfh) lineage able to collaborate with b lymphocytes (bl). on the contrary, the transcription factor blimp-1 is highly expressed in t lymphocytes th1, th2 and treg, thus regulating the differentiation towards tfh. materials and methods: whole fresh blood and peripheral mononuclear cells from a patient with homozygous mutation in stat5b were analysed by flow cytometry. analysis of ctfh (cd4+cd45ra-cxcr5+), ctfh1 (cxcr3+), ctfh17 (ccr6+), ctfh2 (cxcr3-ccr6-), naã¯ve bl (lb igm+igd+cd27-), memory (mbl) (lb igm+ igd-cd27+), switched (mbl-sw) (igd-igm-) and plasmablast (pbc) (cd27+cd38++) cells was performed. immunoglobulins were measured in serum. results: the patient with stat5b deficiency showed increased values of ctfh (38%) (healthy donors p10-p90: 7,9-17,8 %) that presented an activated phenotype (icos+ and pd-1+) with a skewed to a th17 profile (ccr6+), consistent with her hipergammaglobulinemia and the marked and sustained increase in the switched mbl and pbc subpopulations in peripheral blood over the years. discusion: this immunological phenotype described in the patient with stat5b deficiency could explain in part the pathophysiology of the autoimmune disorders. this patient (as well as the other two patients with mutations in stat5b previously described by our group), have had chronic hypergammaglobulinemia, autoantibodies and consequently autoimmune processes (psoriasis, hypothyroidism, eczema, alopecia and celiac disease, among others). we believe that the link between this clinical symptomatology and the molecular defect relies in the fact that the absence of stat5b promotes a greater expression of bcl-6, which generates a bias towards the production of ctfh cells, that give rise to a greater activation of lb, generation of lbm and plasma cells (dysregulation in the cg), events that manifest as hypergammaglobulinemia and autoimmunity. in summary, we provide promising evidence of the mechanisms that lead to autoimmunity in this type of patients that could also be a consequence of the defect in the regulation of gc, highlighting the crucial role of stat5b in the humoral immune response and maintenance of the tolerance of the immune system. background/introduction: the term primary immunodeficiencies (pid) encompasses a phenotypically and genetically diverse group of conditions. genetic testing for these conditions can guide treatment, reduce morbidity and mortality, allow for genetic counseling, and identification of additional at-risk family members. however, this testing can be complicated by a number of factors, including pseudogenes, high homology, methodology limitations, and the heterogeneous nature of pids. methods: mayo clinic laboratories launched their first set of nine pid next generation sequencing (ngs) tests approximately one year ago. these tests include one single gene assay for gata2 deficiency and eight targeted next generation sequencing panels for: atypical hemolytic uremic syndrome (ahus), autoinflammatory disorders, b-cell disorders, monogenic irritable bowel disease (ibd), phagocytic defects, severe combined immunodeficiencies (scid), and severe or cyclic neutropenia. herein we summarize our first year of experience with these ngs tests, with a focus on the eight targeted panel tests. results: from march 2018 through november 2018 we performed testing for 341 cases. our highest volume of tests was for the ahus panel (127/341 cases, 41%). a variant was reported in 76/341 cases (22.29%). these variants included variants of uncertain significance, likely pathogenic variants and pathogenic variants. the indication with the highest percentage of cases where a variant was reported was scid (9/13 cases, 69.23%). the number of cases that were considered solved, where the genotype likely explains the patients phenotype, varied widely by indication. twenty cases were found to have a pathogenic or likely pathogenic variant or variants; however 2/20 cases were heterozygotes for an autosomal recessive condition and were not considered solved cases. the panel with the highest percentage of solved cases is our scid panel (4/13 cases, 30.77%). conversely, we have yet to solve an autoinflammatory, irritable bowel disease, or telomere defects case; however 20% of cases in each of those three panels have had a variant of uncertain significance reported. we hypothesize that one of the reasons for the low detection rate for these three panels is inappropriate test orders. we are also actively looking for ways to update all 8 panels to increase detection rates and clinical utility, for example expanding the gene list of our ibd panel, including large deletion/duplication detection, and including ncf1, a difficult gene to capture by ngs, on the phagocytic panel. finally, we present the molecular findings from a number of interesting cases that were solved using our targeted ngs panels. conclusions: the launch of our pid ngs tests in march of 2018 has allowed us to aid patients by confirming diagnoses and providing molecular diagnoses that will enable more accurate genetic counseling and risk assessment. we have also uncovered areas for improvement, both on the clinical side: provider education is important to enable better identification of patients who can benefit from molecular genetic testing for pids, and on the laboratory side: introduction of more expanded panels and additional methodologies. the progressive decrease of red blood cells, platelets or neutrophils via a self-directed immune process is jointly termed as autoimmune cytopenias. while autoimmune cytopenias, including autoimmune hemolytic anemia (aiha), immune thrombocytopenic purpura (itp), and autoimmune neutropenia (an), are a common presentation of autoimmunity in the general population, they are particularly frequent and can appear as the first sign in patients with primary immunodeficiencies (pids). possible causes of cytopenia in pids comprise mainly immune dysregulation, bone marrow failure (bmf) and myelodysplasia. our goal is to investigate possible immune mediated mechanisms underlying chronic cytopenia in children in order to achieve an early diagnosis and consequently offer timely and appropriate therapy. we selected 24 patients affected by chronic cytopenia, evaluated with immunophenotyping by flow-cytometry; data were subjected to multivariate analysis by principal component analysis (pca). next generation sequencing (ngs) analysis of genes frequently implicated in pids was performed. among the patients, 5 were affected by bone marrow failure, of which 2 were diagnosed with fanconi anemia and severe congenital neutropenia; 12 were affected by immune-mediated cytopenia and 7 by idiopathic cytopenia. the immunephenotyping showed a typical pattern of cd8 t cell subpopulations expression in patients compared with healthy donors with an increase of naã¯ve t cells and a reduction of central memory (cm) and effector memory (em) t cells levels. we observed a decrease in total b cells, b switched and b memory cells and an increase in cd21low cells. pca showed an overlap between groups, however it revealed a peculiar trend of some single patient, suggesting the pathway involved in immune defect. preliminary results from ngs studies revealed genetic variations in genes previously associated with pids in 10 out of 11 patients investigated. in particular we identify one patient with a mutation in fas, one with a mutation in aire and one with a mutation in ikaros. concerning the remaining patients further studies are ongoing to validate the pathogenicity of the genetic variations. pca is a very effective tool to analyze several parameters at the same time, highlighting patients whose phenotype shows the main peculiarities. the presence of specific lymphocyte subpopulation patterns can be important indicators of immune-mediated cytopenias and helpful signs of specific pids that should promptly be investigated with genetic analysis. the rapid of discovery of novel, monogenic primary immunodeficiencies has been made possible by the broad availability of clinical whole exome sequencing (wes). however, clinical wes has major shortcomings that should be understood by practicing immunologists. focusing on the 2017 iuis list of~330 monogenic primary immunodeficiency genes, we show here limitations in coverage that could significantly impact clinical interpretation. on the agilent whole exome capture kit, the most common wes platform, there are a number of genes with exons that are poorly covered. specifically, there are at least 94 genes with less than 100% exonic coverage, 26 with less than 99% coverage and 5 with less than 90% coverage (e.g. ikbkb, ncf1, taci, unc93b1 and tbx1). beyond this challenging technical issue, there are more subtle issues as well. these include the presence of pseudogenes in at least 17 of our genes (e.g. ak2, c1qbp, cd46, cftr, cr2, msn, ncf1, ncstn, ikbkg, nhp2, pms2, pten, rnaseh2c, rps, sbds and was), which can make accurate sequencing very challenging. finally, there are many known causative intronic (e.g. btk, ctla-4, wasp) and copy number variant mutations (e.g. rag1 and xiap) as well as large deletions (e.g. dock8) that we cannot expect to be optimally covered using wes. this list of genes requires consideration even with a negative exome and may require additional approaches including whole genome sequencing, sanger sequencing, cnv arrays and/or long-read ngs sequencing. wes is a powerful genomic diagnostic tool, but to avoid missing key diagnostic insights using these alternative approaches may be critical when certain genes are in the differential diagnosis. going forward, as pid phenotypes continue to broaden, these issues remain fundamentally important even if these genes are not obviously implicated in a given clinical phenotype. more physicians are utilizing targeted genetic panels to reach a definitive diagnosis for their patients with immunodeficiency. however, this increase in testing also has led to the discovery of many more variants of uncertain significance (vus) in the genes tested. these findings can often leave the patient and the physician with more questions than answers. we present a patient with recurrent infections found to have multiple variants of uncertain significance in several genes associated with primary immunodeficiency. a 13-year-old female who was diagnosed with crohns disease at age 9 after intestinal perforation and jejunal resection experienced two discrete episodes of epstein barr virus (ebv) meningoencephalitis and septic shock. the first episode was diagnosed when patient had fever and altered mental status and occurred prior to her crohns disease diagnosis and the second episode was complicated with altered mental status, disseminated intravascular coagulation (dic) and hypotension requiring picu admission. aside from these two major infections, the family denied any other infections requiring antibiotics in the last 5 years and reported a remote history of repeated streptococcal pharyngitis that have not recurred. immunology was consulted at the time of the second episode of meningoencephalitis and work up was mainly unremarkable with normal immunoglobulins, adequate vaccine response to hib, tetanus, diphtheria, rubella, measles and pneumococcus (18 out of 22 protective titers). she had normal t cell numbers with slightly decreased natural killer numbers for age. neutrophil studies showed normal dihydrorhodamine (dhr) analysis, glucose-6-phosphate dehydrogenase levels and myeloperoxidase (mpo) stain. commercial testing of her toll like receptors (1) (2) (3) (4) (5) (6) (7) (8) showed normal function. invitae primary immunodeficiency panel demonstrated a heterozygous variant in nod2 (c2.104c>t; p.arg702trp) as well as heterozygous variants of uncertain significance in il7r (c.662g>t; p.ser221ile) and tlr3 (c.889c>g; p.leu297val). the patients nod2 variant is known to be associated with an increased risk for crohns disease. even with our patients presentation with recurrent severe viral infections and ibd, it is not immediately clear how these genetic results explain the pathology. innate immune defects probably contribute to her presentation and it is currently unclear if and how the combination of multiple genetic variants has left her immunologically vulnerable. we use this case to demonstrate that even when genetic testing does not elucidate a clearcut diagnosis of primary immunodeficiency, it can still provide helpful insight into a patients underlying immune phenotype. introduction: xiap deficiency is a rare primary immune deficiency characterized by hemophagocytic lymphohistiocytosis, recurrent fever and inflammatory syndromes, inflammatory bowel disease, hypogammaglobulinemia, recurrent infections, and other manifestations. loss of xiap results in abnormal tnf receptor signaling and nlrp3 inflammasome actvity which leads to dysregulated production of il-1beta and il-18. we hypothesized that suppressing the nlrp3 inflammasome with either targeted deletion or pharmacologic inhibition would suppress abnormal production and secretion of inflammatory il-1beta and il-18. methods: bone marrow derived macrophages (bmdms) from control, xiap-deficient, and xiap and nlrp3 double knock-out mice were derived with 1 week of culture in l929-cell conditioned media. bmdms were stimulated with a variety of tlr agonists or tnf-alpha, with or without a variety of inhibitors including the nlrp3 inhibitor mcc950, the cathepsin b inhibitor ca-074, and quercetin, which is a natural flavonoid (antioxidant) found in many fruits and vegetables, and available as a nutritional supplement. il-1beta, il-18, and tnf-alpha were measured in supernatants by elisa, and cell death was evaluated by flow cytometry using pi exclusion. results: as expected, bmdms from xiap deficient mice had markedly increased tlr-agonist-or tnf-alpha-induced il-1beta production compared to normal bmdms. genetic deletion of nlrp3 and the pretreatment of cells with the nlrp3 inhibitor mcc950 greatly reduced abnormal il-1beta production; residual production of il-1beta could be inhibited by caspase-8 inhibition. pre-treatment of cells with the cathepsin b inhibitor ca-074 also decreased cytokine production but was toxic at higher concentrations. quercetin reliably abrogated il-1beta, and also il-18. quercetin was found to inhibit priming of the nlrp3 inflammasome (decreased upregulation of pro-il1beta and nlrp3) and also decreased tnf-alpha secretion following tlr agonist stimulation. conclusion: quercetin suppresses the nlrp3 inflammasome and may be a promising therapeutic option for patients with xiap deficiency. it prevents il-1beta and il-18 secretion. it is a particularly appealing option given that it is a naturally occurring antioxidant, has a great safety profile, and is readily available as a nutritional supplement. human studies are needed. recently, single cell rna sequencing (scrnaseq) analysis in mice has disclosed an unexpected complexity of thymic stromal cells, and medullary thymic epithelial cells (mtecs) in particular. however, the developmental origin, hierarchy, and function of these subpopulations remain ill-defined. moreover, although cortical tecs (ctecs) are thought to represent a more homogeneous population, their characterization has been largely restricted to the adult thymus. we have previously shown that impaired lymphostromal cross-talk in the thymus of patients with combined immunodeficiency (and of corresponding mouse models) is associated with abnormalities of thymic architecture and tec maturation. here, we sought to compare tec distribution and gene expression in wild-type (wt) and in mice carrying rag1 hypomorphic mutations observed in patients with combined immune deficiency and immune dysregulation. methods: multi-color flow cytometry and scrnaseq were used to analyze composition and distribution of ctec and mtec subpopulations in wt and rag1 mutant mice at various weeks of age (niaid animal protocol: lcim-6e). results: we observed that rag1 mutant mice have an excess of ctecs, and that their mtec compartment is predominantly represented by cells with high levels of mhc class ii (mhc-ii) expression, recapitulating the phenotype of neonatal wt thymi. while mhc-iihi mtecs are thought to represent a minor fraction of mtecs in adult wt mice and include mature aire+ cells, a relative abundance of mhc-iihi mtecs is observed also at neonatal age, where they are thought to represent immature mtecs. to define more precisely tec maturation, we performed scrnaseq on sorted cd45-epcam+ cells, and identified 8 and 10 distinct clusters of tecs in wt and rag1 mutant mice, respectively. a large proportion of cells in rag1 mutant mice could be ascribed to the ctec compartment, confirming our previous flow cytometry and histopathology results. furthermore, scrnaseq analysis also disclosed a different distribution of mtec subsets in wt and rag1 mutant mice. to address the hypothesis that this difference in ctec and mtec abundance and subset distribution may reflect different maturation stages in tec development in wt and rag1 mutant mice, we will perform lineage tracing and transplantation experiments, and we will also extend tec scrnaseq analysis to wt and mutant mice of embryonic and neonatal age. in parallel, to evaluate the contribution of thymocyte maturation in shaping the stromal populations, scrnaseq will be performed on thymocytes. conclusions: we have further refined the complexity of tecs, and shown that impaired development of t cells in combined immune deficiency (as exemplified by rag1 mutant mice) has profound effects on the composition and maturation of tecs and may thus contribute to abnormalities of immune tolerance that are often associated with these conditions. the advent of next-generation sequencing (ngs), with the development of whole-exome sequencing (wes) in particular, has allowed the identification of unknown genetic lesions for many diseases and the implementation of specific therapeutic strategies. primary immunodeficiencies (pids) are a group of rare diseases which have benefited from ngs, with the discovery and molecular characterization of previously genetically undefined diseases and the identification of novel molecules involved in the regulation of the immune system. pids are often associated with autoimmune disease due to the dysregulation of the immune system as a whole. the clinical phenotypes are heterogeneous and often overlapping. while a monogenic cause of disease has been identified in a most subsets of patients, the recent application of whole-genome sequencing has found that a polygenic cause is likely. our aim is to investigate the genetic background of patients with immunedysregulations and autoimmunity and to evaluate the possible pathogenicity of the identified gene variants through extensive functional studies. we select 19 patients with sign of immunedysregulation and autoimmunity, extended immunophenotyping and next-generation sequencing (ngs) analysis of 50 genes frequently implicated in pids was performed. in six of them we identify a single gene as responsible of the clinical feature. in particular, we identify two patients with gain of function mutation in stat3, one patient with a mutation in ctla4, one patient with an activating pik3cd mutation, one with a rag1 mutation and one with a fas mutation. in most of them variants in multiple genes have been detected. interestingly, we find that some genes are recurrently mutated in more then one patient such as was, dock8, casp10, casp8, nfatc2 and fcgr3a. further studies are ongoing to validate the effect of the variations identified. our results strongly suggest that the old hypothesis, based on a single gene mutation as a cause of illness, should be revised in favor of the concept that "is the sum that causes the effect" and that a different point of view on pids now seems inevitable. physician, omni allergy, immunology, and asthma introduction/background: immunoglobulin replacement therapy (igrt) may be optimized to reduce the severity and incidence of infections and potentially delay or abrogate the development of pulmonary complications of primary immune deficiencies. pulmonary complications including bronchiectasis are common in common variable immune deficiency (cvid) and contribute significantly to morbidity and mortality in these patients. it remains unclear whether continued obstructive bronchial changes are a result of repeated respiratory infections, associated inflammation and immune dysregulation, or simply lung-damage that is irreversible by the time therapy is initiated. it has also been suggested that under-treatment in addition to the diagnostic delay may contribute to the development of bronchiectasis in patients with pid. lower serum igg levels with any given dose of immunoglobulin replacement therapy have been demonstrated in patients with bronchiectasis compared to those pid patients without this complication. in addition, earlier studies have shown that greater doses of ig (600 mg/kg/ month) may reduce the frequency and duration of infections and help prevent or slow progression of chronic lung disease. objective: to evaluate the prevalence of bronchiectasis in a cohort of patients with a diagnosis of cvid and identify associated ig dosing patterns and clinical outcomes. methods: data were analyzed from the ideal (immunoglobulin, diagnosis, evaluation, and key learnings) patient registry. this is a prospective, longitudinal registry study of patients receiving ig replacement therapy in the home or ambulatory infusion suite with one national home infusion provider. nursing and pharmacy standard of care forms were collected, and dose, infection rate, and prevalence of bronchiectasis were evaluated in patients with a diagnosis of cvid (icd-10 codes: d83.9, d83.1) results: there were 310 patients in the registry with cvid, 14 (4.5%) of which bronchiectasis was also observed. seventy-nine percent (n=246) of the study population was female, and 50% (n=7) of the cases of bronchiectasis were observed in females. the mean age of the patients with concurrent bronchiectasis was 65â±15.8 at start of care compared to 57â±15.8 in those without this observed bronchial obstruction. most bronchiectasis patients (n=11) received igrt subcutaneously every week with a mean dose of 123.8â±22.8 mg/kg/wk. the mean dose of ig in the 3 remaining patients receiving ig intravenously was 506.8â±82.0 mg/kg/month. the average annual rate of infection in ivig and scig patients with bronchiectasis was 1.6â±1.0 and 2.2â±1.3, respectively, however many were serious bacterial infections. at time of analysis, 7 of the bronchiectasis patients remained active in the registry and 7 had withdrawn. reasons for withdrawal included stopping igrt due to the following: patient decision (n=3), physician decision (n=1) insurance change (n=1), and patient expired (n=2). conclusions: there were 14 documented cases of bronchiectasis in our cohort of cvid registry patients, and dosing patterns aligned with standard doses despite the presence of bronchial obstruction. further studies are necessary to assess evolution of lung damage with respect to ig dosing in patients with cvid. background: activated phosphoinositide 3-kinase syndrome type 1 (apds1) is a combined immunodeficiency resulting from gain-offunction (gof) mutations in pik3cd, the gene encoding the catalytic subunit of phosphoinositide 3-kinase (pi3k). this form of pid is characterized by recurrent respiratory tract infections, susceptibility to herpes virus infections, impaired antibody responses, lymphoproliferation and autoimmunity. previous studies showed that patients with apds1 have b cell defects that contribute to the clinical phenotype. furthermore, these patients display t cell abnormalities, including increased numbers of memory t cells and t follicular helper cells (tfh), reduction of naã¯ve t cells and impaired t regulatory cell (treg) function. whether these t cell abnormalities are also associated with perturbations of t cell repertoire in unknown. objective: we aimed to investigate the effects of increased pi3k signaling on the t-cell repertoire of patients with apds. methods: high throughput sequencing was used to study composition and diversity of t-cell receptor (tra) and t-cell receptor (trb) repertoire in sorted treg, tfh, conventional cd4+ (tconv), and cd8+ t cells from 4 patients with pik3cd gof mutations and healthy controls. results: treg cells of patients with apds1 show restriction of tra and trb repertoire diversity, and increased clonality. no repertoire restriction was detected in tfh, tconv, and cd8+ t cells from the same patients. however, the trb repertoire of treg and cd8+ cells was enriched for the presence of hydrophobic amino acids in position 6 and 7 of the cdr3, a biomarker of self-reactivity. conclusion: these data demonstrate that the t-cell repertoire of patients with apds1 is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition. furthermore, our result support the notion that the pi3k pathway is a key regulator of treg cell development and homeostasis in humans. j clin immunol (2019) 39 (suppl 1):s1-s151 s87 (4), iii. predominantly antibody deficiencies (2), i. immunodeficiencies affecting cellular and humoral immunity (1), vii. auto-inflammatory disorders (2), ix. phenocopies of pid (1) . two non related cases of ataxia-telangiectasia and one case of schimke syndrome (smarcal1 compound heterozygous mutation) were diagnosed in the last year. we observed a wide range of age (we evaluate adult and pediatric population) with a male:female ratio close to 1: immunodeficiency, immune dysregulation, and systemic autoimmunity. clinical diagnosis of these disorders is complicated by overlapping phenotypes. in april 2017, a 207-gene next generation sequencing (ngs) panel inclusive of copy number variation analysis was launched by a commercial laboratory to facilitate clinical diagnosis of primary immunodeficiency (pid), monogenic autoimmunity and autoinflammatory disorders. we assessed the outcomes of genetic testing utilizing this panel on a cohort of pediatric patients with immunohematologic phenotypes evaluated at our tertiary care center during an 18-month period (5/1/17-10/31/18). eligible subjects were evaluated by at least two of three providers from a multidisciplinary pediatric hematology-immunology team, including a hematology physician, immunology physician and a geneticist or genetic counselor. twenty-three patients met inclusion criteria; 20 (87%) were caucasian, 12 (52%) were male with an average age of 11.7 years. the two most common phenotypic diagnoses included cytopenias, single-or multilineage (leukopenia, neutropenia, anemia, thrombocytopenia) primarily attributed to autoimmune causes or hypogammaglobulinemia. five (22%) were given a definitive genetic diagnosis as a result of panel testing, though in two of these cases, the causative mutations were listed as variants of uncertain significance (vus). diagnoses included common variable immunodeficiency due to a pathogenic variant in nfkb2, stat3 multiorgan autoimmunity due to gain-of-function mutation, and familial cold autoinflammatory syndrome due to a pathogenic mutation in nlrp12. biallelic dnmt3b vus were found in a patient whose phenotype and further laboratory studies (including karyotype) were consistent with immunodeficiency-centromeric instability, facial anomalies syndrome. further, a stat3 vus was identified in a patient with multiorgan autoimmunity and his father with hypothyroidism; studies from an outside research laboratory were consistent with gain-of-function with this variant (private communication). an additional three patients had vus identified that were suspected to be related to their phenotype, prompting eligibility for research studies. four (17%) patients had increased risk alleles in nod2, conferring an increased risk of crohns disease. three (13%) patients had pathogenic or likely pathogenic carrier findings warranting genetic counseling. in addition, 47 vus (an average of 2 per patient) thought to be unrelated to phenotype were identified, necessitating further investigation and counseling. the use of an ngs panel in a cohort of pediatric patients with immunohematologic disorders led to a definitive diagnosis in 22% of previously undiagnosed patients and prompted further research investigation in several more. genetic testing also led to the identification of clinically significant carrier findings, risk alleles and 47 vus unrelated to phenotype, necessitating genetic counseling. our experience illustrates the value of genetic testing for diagnosis of immunohematologic disorders, and the importance of multidisciplinary care, including genetic counseling, for the proper evaluation and management of these patients. background: allogeneic hematopoietic cell transplantation (allohct) is curative for primary immune deficiencies (pid). however, many patients lack a fully-matched unaffected sibling, or may have an unknown underlying genetic defect, rendering it undesirable to use related donors. many pid patients have significant comorbidities at the time they are referred to allohct, precluding the use of myeloablative conditioning. the use of alternative donors with reduced-intensity conditioning (ric) has historically led to increased rates of graft failure, graft-versus-host disease (gvhd), and transplant-related mortality (trm). posttransplantation cyclophosphamide (ptcy) as gvhd prophylaxis immunomodulates the graft through the preferential sparing of regulatory t cells and hematopoietic stem cells from its cytotoxic effects, thus allowing for robust donor engraftment that overcomes the hla barrier while effectively preventing severe acute and chronic gvhd. we report the outcomes of two institutions using a ric allohct regimen with alternative donors and ptcy in patients with pid. design: we transplanted 35 pid patients (table 1) using alternative donors and ric, either serotherapy-free (n=21) or alemtuzumab-based (n=14). all patients received ptcy for gvhd prophylaxis on days +3 and +4, either alone (n=3), or combined with mycophenolate mofetil and either sirolimus (n=21) or tacrolimus (n=11). donors included haploidentical family members (n=16), matched unrelated (n=15), and mismatched unrelated (n=4). stem cell source was t cell-replete bone marrow (n=33) or peripheral blood stem cells (n=2). results: the median follow-up is 17 months (range 0.5-8 years). at 17 months, overall survival is 91%, and event-free survival (defined as alive without graft failure) is 83%. the median days of neutrophil and platelet engraftment are 17 (range 14-42) and 28 (range 15-110), respectively. there were 10 patients who developed acute gvhd, grade 1 (n=5) or grade 2 (n=5), and there were no cases of grade 3 or 4 agvhd. seven of eight patients treated with systemic corticosteroids responded, and one was corticosteroid-dependent, then responded to second-line therapy. one patient developed skin-only chronic gvhd, which responded to corticosteroids and puva light therapy. five patients developed graft failure, either primary (n=1) or secondary (n=4), and four were successfully re-transplanted and remain engrafted. one patient with secondary graft failure had autologous recovery and has not required a second allohct given some durable infection control gained during initial engraftment. there were three deaths prior to day 180 due to infection, and one death at 1.5 years secondary to presumed overdose. in ongoing follow-up of engrafted survivors (n=30), evidence of phenotype reversal has been demonstrated in all patients, with complete or ongoing resolution of some or all of their underlying disease manifestations, including infection, transfusion-dependence, autoimmunity, malignancy, and/or immune dysregulation. discussion: we have observed high rates of engraftment, low rates and severity of acute and chronic gvhd, and low trm in 35 patients with pid transplanted using alternative donors, ric, and ptcy-based gvhd prophylaxis. ric allohct with ptcy shows promise for curing pid, and its use minimizes toxicity and widely expands the donor pool, thus allowing us to offer this curative therapy to many more patients with pid. chronic granulomatous disease (cgd) is a primary immune disorder that involves mutations in the nicotinamide adenine dinucleotides (nadph) oxidase complex (deffert, cachat, & krause, 2014) . two-third of cgd cases are caused by loss-of-function mutations in the cybb gene that encodes the gp91pox subunit of the nadph. the increased in patients' life expectancy thanks to progress in diagnosis and management has underlined the burden of inflammatory manifestations occurring independently of infectious agents (dunogue et al., 2017; marciano et al., 2018) . cgd patients develop inflammatory granulomatous disorders, notably colitis, as a consequence of a dysregulated inflammasome activation. the treatment of inflammatory manifestations remains challenging, as it can be associated with an increased risk of infections. thus, understanding the pathophysiological mechanism of auto-inflammation in cgd could help improve the therapeutic arsenal for the management of these manifestations. to reveal the precise pathophysiological mechanism of auto-inflammation in cgd, we have developed a cellular model that reproduces the cgd phenotype in phagocytic cell. through crispr-cas9 gene-editing we generated a thp-1 c e l l l i n e h a r b o r i n g t h e p r e v i o u s l y d e s c r i b e d mu t a t i o n c.90_92delccginsggt (p.tyr30ter) in the cybb gene responsible for gp91phox knock-out by early termination of translation. this cell line recapitulates the phenotype of cgd phagocytes: (i) decreased h2o2 production (ii) and enhanced inflammatory responses after pma stimulation as evidenced by increased il-1, il-6 and tnfa secretion levels (kuijpers & lutter, 2012) . these features were rescued by complementation through lentiviral transduction of a wild type cybb gene. this new model will help us to investigate the auto-inflammation reported in cgd patients and also to propose new therapeutic targets of inflammatory manifestations in this disorder. interleukin-1 (il-1) driven responses. children with irak-4 deficiency are predisposed to recurrent and invasive infections secondary to streptococcus pneumoniae, staphylococcus aureus and other pyogenic bacteria with high mortality rates in early childhood. the frequency and severity of infections is thought to decrease with age due to the acquisition of humoral immunity and immunologic memory, however due to the rarity of the disease, the natural history of this condition beyond early childhood is not well described. objectives: we present three unrelated irak-4 deficient patients with persistent chronic rhinosinusitis with nasal polyposis that developed in childhood. cases: patient 1 is a 15 y/o male with compound heterozygous mutations in irak4 (p.g75afs*14/c.717-1g>t) with a history of recurrent s. pneumoniae osteomyelitis (left hip at age 9 and left knee at age 10) and c. septicum sepsis at age 9 following acute bowel perforation. additionally, he experienced recurrent aom during infancy and recurrent uti since age 9. despite prophylactic antibiotics and ivig, he has had recurrent polymicrobial (mrsa, s. pneumoniae, h. influenzae, p. aeruginosa, a. fumigatus) rhinosinusitis with nasal polyposis since age 4 refractory to medical management requiring surgical intervention and prolonged courses of iv antibiotics. patient 2 is an 11 y/o female with homozygous deletions (exons 10-12) in irak4 with a history of ruptured appendicitis complicated by pseudomonas abscess and bacteremia at age 2, culturenegative sepsis with septic arthritis and osteomyelitis of the right leg at age 3, and septic shock secondary to mssa bacteremia complicated by rhabdomyolysis and dic at age 5. she has a history of chronic rhinosinusitis, and despite ivig and prophylactic antibiotics, she developed polymicrobial (h. influenzae, b. fragilis) rhinosinusitis with associated nasal polyposis pending surgical management. patient 3 is a 10 y/o female with homozygous mutations in irak4 (q293x/q293x on exon 8) with a history of s. pneumoniae meningitis at 3 months, m. catarrhalis epiglottitis and neck cellulitis at 4 months, rsv bronchiolitis at 6 months, enterococcus bacteremia at 8 months, s. pneumoniae sepsis at age 2 and streptococcus lymphadenitis at age 9. despite ivig and prophylactic antibiotics, she developed recurrent polymicrobial (h. influenzae, b. fragilis, mssa, v. cholera, p. aeruginosa, a. fumigatus) rhinosinusitis refractory to medical management requiring surgical intervention and iv antibiotics. conclusions: in our centers experience, irak-4 deficient patients continue to suffer from infectious complications, most prominently recurrent polymicrobial sinus infections beyond early childhood. the consistent presence of sinonasal polyps in these children is unusual, as it is not typically found in uncomplicated pediatric chronic rhinosinusitis. these infections have occurred despite antimicrobial prophylaxis and ivig, highlighting the role of irak-4 in sinopulmonary epithelium. additionally, the infectious organisms identified in our patient cohort are not commonly associated with irak-4 deficiency. further study of chronic rhinosinusitis and nasal polyposis in a larger cohort of irak-4 deficient patients and other innate immunodeficiencies may help identify pathways for targeted treatment of these patients. introduction: chronic granulomatous disease (cgd) is an inherited phagocytic defect associated with inability to clear catalase positive organisms. infections in patients with cgd are severe and recalcitrant. commonest infections are pulmonary followed by soft tissue infections and suppurative lymphadenitis. osteomyelitis is an uncommon infection in patients with cgd. it poses several diagnostic and therapeutic challenge. we herein report our experience of osteomyelitis in cgd over the last 10 years. material and methods: review of records was carried out to describe the profile of osteomyelitis in cohort of patients with cgd at pediatric immunodeficiency clinic, advanced pediatrics centre, postgraduate institute of medical education and research, chandigarh, india. the diagnosis of cgd was based on nitroblue tetrazolium dye reduction test (nbt) and dihydrorhodamine reduction (dhr) assay. results: of the 63 patients with cgd, 8 (12.7%) had osteomyelitis (6 males and 2 females; age range 1-10 years). most patients had their first episode of serious infection in early childhood (mean age: 1.5 years). stimulation index (si) of dhr assay ranged from 1 to 4.58. mutational analysis was done in 5/8 patients (3 x-linked; 2 autosomal recessive). site of involvement was variable ribs-4; vertebrae-2; radius-1; skull-2; tibia-1. aspergillus fumigatus was the most common isolate (62%; 5/8); others had aspergillus flavus, aspergillus terreus and serratia marcescens each. all 4 patients with rib osteomyelitis had concurrent pneumonia, and fungus was isolated in all of them (aspergillus fumigatus-2, aspergillus flavus-1, zygomyces spp.-1). antifungals (intravenous amphotericin b) were given for a duration of 4-6 weeks and were followed by oral voriconazole in therapeutic doses for 3 to 6 months in majority of them. debridement and resection of ribs was required in one patient, while other patients were managed conservatively. out of 8 patients, 2 (25%) succumbed to pneumonia and respiratory failure. conclusion: osteomyelitis in the context of cgd is usually caused by aspergillus spp. involvement of ribs and vertebra usually occurs with the contiguous spread of infection from the lungs. therapy often requires prolonged duration of anti-microbials, and may require surgical debridement in addition to it. a 29-year-old woman with history of hypogammaglobulinemia and acute liver failure a 29-year-old woman with a 7-month history of nausea, vomiting, and abdominal pain was admitted to an outside hospital with new onset of jaundice and anasarca. liver biopsy was thought most consistent with alcoholic steatohepatitis, and she was discharged with counseling on alcohol cessation and medical management of liver disease. she presented to our facility for a second opinion. over the following days, she developed further rise in direct hyperbilirubinemia up to 19.2 mg/dl, new coagulopathy with an inr 2.06 and hypoalbuminemia to 1.7 mg/ dl in the absence of ongoing alcohol consumption. liver sonography revealed course echotexture and patent vessels. pcrs directed against multiple hepatotropic viruses were negative and copper studies were normal. due to a history of moderate alcohol consumption, she was started on high-dose corticosteroids due to a presumptive diagnosis of alcoholic hepatitis. additional history raised concern for a possible primary immunodeficiency, including idiopathic thrombocytopenic purpura at 11 years of age, multiple episodes of sinusitis treated with antibiotics and sinus surgery, one episode of suspected bacterial pneumonia, and one hospitalization for influenza a during which she developed neutropenia. in her 20s, she developed refractory genital warts, prompting infectious diseases evaluation. initial immune evaluation had revealed low immunoglobulins (iga <7 mg/dl, igg 198 mg/dl, igm 13 mg/dl) with very low responses to tetanus and diphtheria, despite a recent booster dose, and b and t cell lymphopenia (cd19+ 89 cells/î¼l, cd3+ 567 cells/î¼l, cd4+ 345v, cd 8+ 244 cells/î¼l, cd16/56+ 236 cells/î¼l); antigen and mitogen proliferation were not assessed. intravenous immunoglobulin replacement was initiated but discontinued by the patient due to infusion-related adverse effects, and she was lost to follow up until she presented with liver failure. both parents were deceased from cardiovascular disease in their 40s and she had no siblings. she had limited knowledge of family history but no known immune diseases. due to suspicion for genetic etiology of immune disorder and liver disease, we performed next-generation sequencing of a panel of over 200 genes implicated in primary immune deficiencies. patient was heterozygous for a nucleotide substation (c.1752+1g>a) within a splice site at the exon 16/intron 16 boundary of the nfkb1 gene. during the hospitalization, immunoglobulin replacement and trimethoprim-sulfamethoxazole prophylaxis were initiated. an attempt was made to refer the patient for additional immunological evaluation and transplantation evaluation but unfortunately, she developed worsening liver failure and multiple complications, including extended-spectrum beta-lactamase (esbl)-producing e. coli bacteremia, hypotension requiring vasopressors and extensive bowel ischemia, and died in the hospital. in summary, this case highlights both the risk of diagnostic delay in adult patients presenting with a primary immune deficiency and potential for genetic testing to clarify the diagnosis. while the particular genetic change has not been described, other splice site and predicted loss-offunction mutations have been reported as pathogenic in this gene, which have been implicated in autosomal dominant common variable immunodeficiency. this case further expands on the genetic causes and spectrum of disease associated with changes in the nfkb1 gene. introduction: malnutrition and micronutrient deficiency are underrecognized causes of acquired immunodeficiency in adults, and may occur even in patients with high body mass index (bmi). methods: a 46-year-old woman with a medical history significant for one remote urinary tract infection presented to the emergency department after sudden onset of severe right flank pain. the pain was accompanied by urinary frequency and not relieved by ibuprofen; she denied fevers or chills. she was diagnosed with pyelonephritis and discharged on ciprofloxacin, which was later changed to trimethoprim-sulfamethoxazole after her culture grew resistant e. coli. her pain continued despite treatment, prompting her to return to the hospital three days later. upon presentation, she was afebrile with blood pressure of 128/88 mmhg and heart rate of 86 bpm. her body mass index was 32.4 kg/m^2. her physical exam was otherwise notable for right costovertebral angle tenderness. laboratory studies revealed a leukocyte count of 14,300/ul with 83% neutrophils; alkaline phosphatase of 146 units/l and albumin of 2.7 g/dl, but otherwise normal liver function tests; normal lactic acid; and urinalysis with 3,000 wbc/hpf, 40 rbc/hpf, moderate bacteria, and the presence of wbc clumps. ct scan of the abdomen and pelvis demonstrated an obstructing 13 mm right renal stone with hydronephrosis and a right renal abscess contiguous with a right-sided hepatic abscess measuring 7.8 x 6.0 x 7.5 cm. she was treated with ceftriaxone and metronidazole, and underwent imaging-guided drainage of the abscesses. abscess cultures again grew resistant e. coli. she was discharged from the hospital with drains in place and a plan to continue trimethoprim-sulfamethoxazole until definitive management of her nephrolithiasis with ureteroscopy and lithotripsy. discussion: there remained the question of how an ostensibly immunocompetent patient had developed such severe intraabdominal infection with little systemic inflammatory response (e.g. no fever and only mild leukocytosis). a hiv antibody screen was negative. on further interview, she described a 200lb intentional weight loss over the preceding 2 years, accomplished by dietary restriction to less than 600 calories per day. nutritional assays revealed prealbumin, vitamin c, and vitamin b6 levels below the threshold of detection. she had low-normal b12 and b1. out of concern for an acquired immunodeficiency resulting from malnutrition with micronutrient deficiency, balanced nutrition was discussed with the patient who agreed to liberalize her diet. background: the past decade has brought dozens of new mendelian disorders of immunity. yet, the genetic contribution(s) to diverse disorders of the immune system remain largely unelucidated. the majority of research participants referred to the national institute of allergy and infectious diseases (niaid) for what may be a mendelian disorder evade molecular diagnosis. making progress in this area requires a coordinated, systematic, and transparent approach to clinical genomics research which leverages the unique environment at the national institutes of health clinical center (nih cc). methods/design: this study is designed to systematically apply exome sequencing and related technologies with clinical grade interpretation and reporting to niaid research participants at the nih cc under a single protocol in order to facilitate research and clinical genetics care across niaid. we are recruiting approximately 1000 participants per year from approximately 35 intramural clinical investigators. we generate genomic data, collect standardized phenotyping and report clinical interpretation in the medical record, all while providing linked genetic counseling. results: to date, we consented 1287 participants, we sent out 1058 samples for exome sequencing and 183 samples underwent copy number variant analysis. we have completed analysis for 359 families (502 individuals) and finalized and resulted 177 cases. here we present a case series illustrating some of our findings. case 1: a 10year-old female was referred to niaid for neonatal onset multisystem inflammatory disease (nomid). developmental delay and mild intellectual disability were appreciated on clinical evaluation. exome sequencing detected a mosaic novel likely pathogenic variant in nlrp3. chromosomal microarray analysis (cma) showed 㣠5 mb interstitial deletion of chromosome 12 previously associated with developmental delay and intellectual disability. case 2: a 10year-old ukrainian male was referred to niaid for the clinical diagnosis of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced). exome sequencing and cma did not detect pathogenic variants in aire, but did find a de novo variant in fam111b. defects in fam111b are associated with poikiloderma with tendon contractures, myopathy, and pulmonary fibrosis (poiktmp). the clinical features of the patient were consistent with poikmp. case 3: a 63-year-old man had a history of brain, liver and kidney nocardiosis, disseminated mac infection, prostate cancer and lymphoma. family history was significant for prostate cancer. exome sequencing showed a heterozygous pathogenic variant in brca2, associated with susceptibility to breast-ovarian, male breast, pancreatic and prostate cancer. conclusion: this case series illustrates that multiple diagnoses, unexpected diagnoses, secondary genomic findings, and data sharing helped identify variants in candidate genes. process standardization supports data integrity and efficiency while accommodating the need for investigator flexibility and providing tailored patient care. rationale: activated pi3 kinase delta syndrome (apds) is a primary immunodeficiency caused by dominant mutations that increase activity of phosphoinositide-3-kinase (pi3k). the catalytic subunit p110 is mainly expressed in cells of the hematopoietic system, primarily lymphocytes and myeloid cells, and mutations affect both b-and t-cells. we sought to further evaluate the role of the t-cell receptor (tcr) repertoire in immune dysregulation and the pathogenesis of autoimmunity and lymphoproliferation in patients with apds. methods: we evaluated the tcr repertoire in the peripheral blood in 3 patients with pik3cd mutations and compared these to the peripheral tcr repertoire in 26 patients with common variable immunodeficiency (cvid) and 50 healthy controls to investigate the role of the tcr in disease. the tcr repertoire in affected tissue of 2 patients with pik3cd mutations was also evaluated (tissue included lymph nodes for both patients, in addition to gastrointestinal tract and lung tissue in one patient). a fixed number of tcrs were subsampled (35,000 for blood and 5,000 for tissue) and diversity was calculated using the gini and shannon indexes. results: using the shannon and gini diversity indexes, the tcr repertoire in patients with pik3cd mutations had less diversity/ increased clonality as compared to healthy controls and those with cvid ( figure 1 ). for the two apds patients with biopsy tissue available for analysis, the diversity of the tcrs in tissue was increased as compared to the peripheral blood tcr repertoire ( figure 2 ). conclusions: pi3k plays an important role in the development and function of both b-and t-cells. patients with apds were found to have decreased tcr repertoire diversity in the circulating t-cell compartment compared to healthy controls and other cvid patients. the increased tcr diversity in the affected tissues compared to peripheral blood implicates the pi3k/akt signaling pathway with t-cell trafficking and tissue immune homeostasis, and suggests this pathway may play a role in the development of inflammatory and lymphoproliferative complications in these patients. gain-of-function mutations in pi3kd result in a human primary immunodeficiency, named apds (activated pi3k-delta syndrome), characterized by lymphopenia, lymphoproliferation, respiratory infections and inefficient responses to vaccination. however, what promotes these immune disturbances at the cellular and molecular level remains unknown. we have recently published a mouse model that recapitulates major features of this disease and used this model and patient samples to probe how hyperactive pi3kd fosters aberrant humoral immunity. we found that mutant pi3kd alters the intrinsic function of t and b cells, leading to icos-independent increases in t follicular helper (tfh) and germinal center (gc) b cells, disorganized gcs, and poor class-switched antigen-specific responses to immunization. these phenotypes were associated with increased phosphorylation of akt and s6 in t and b cells, and lower threshold of activation, with altered regulation of foxo1 and bcl2 family members. moreover, b cells showed enhanced responsiveness and proliferation to both antigens and innate stimuli, accompanied by reduced cell death. strikingly, aberrant responses were accompanied by increased reactivity to gut bacteria, and a broad increase in autoantibodies that were dependent on commensal microbial stimulation, as demonstrated by striking reduction of self-reactivity upon antibiotic treatment in mutant mice. we now have further examined b cell function in these mice and demonstrate that altered foxo1 plays a major role in disruption of both b and t cell function. we further provide evidence for altered activation of metabolic pathways in b cells, compared to wt cells, that may contribute to the dysregulated b cell reactivity. our findings suggest that proper pi3kd regulation is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome. this research was supported in part by the intramural research program of the nih, nhgri and niaid. autoimmune cytopenias are seen in a significant proportion of patients with immunodeficiencies affecting antibody production. previous b-cell maturation studies using fluorescence-activated cell sorting (facs) have associated various phenotypes of primary immunodeficiency diseases affecting antibody production with differing levels of b-cell differentiation. in this study we analyzed the peripheral b-cell compartment of 84 patients with a hypogammaglobulinemia and >1% b-cells with and without a history of autoimmune cytopenias. b-cells were isolated from peripheral blood using monoclonal anti-cd19 and these cells were gated to identify the proportion of memory b cell (cd19+cd27+ ), igm+ memory b (cd27+igm+), marginal zone b-cells (igm+ igd+), isotype-switched memory b-cells (cd27+igm-igd-) and transitional cells (igmhicd38hi). pid patients with a history of aic had decreased proportions of total cd27+ b-cell (11.6% vs 25.6%; p=0.0003) and igm memory b cells (8.3% vs 18.4%; p = 0.0018). conversely, the proportion of marginal zone b-cells was increased in this group (82.0% vs 66.5%; p = 0.0043). consistent with previous reporting, the proportion of isotype-switched memory b-cells was significantly lower in the aic group (0.75% vs 2.8%; p = 0.0003). statistically significant inter-group difference was not seen within the transitional b-cell subset. our data suggest that maturation arrest of marginal zone (cd27+igm+ igd+) b-cells may be implicated in the development of autoimmune cytopenias in humoral immunodeficiency. (159) submission id#601984 taissa de matos. kasahara 1 , sudhir gupta, md 2 1 phd student, state university of rio de janeiro and university of californis irvine 2 professor, university of california at irvine, irvine, ca, usa introduction/background: common variable immunodeficiency (cvid) is the most frequent form of primary hypogammaglobulinemia with decreased serum igg and iga levels and variable levels of igm in adults. in addition to decreased serum immunoglobulins, 25-30% of cvid patients present autoimmune manifestations. the mechanisms that lead to a breakdown of selftolerance in cvid are not completely understood. however some differences in b and t cells subsets and autoreactive b and t cells can be detected. elevated expression of surface igd and downregulation of igm receptor are hallmarks of anergic naã¯ve b cells that contain autoreactive receptors in human peripheral blood. moreover, memory b cells that have class switched to igd and present an igd+igm-phenotype are also highly reactive to self-antigens in healthy individuals. the role of these autoreactive naã¯ve and memory b cells in the immunopathogenesis of cvid has not been evaluated. here we investigated the frequency of cd27-and cd27+ b cells expressing igd and igm in peripheral blood of cvid patients. methods: peripheral blood mononuclear cells (pbmc) from cvid patients (n=29) and health subjects (n=32) were separated by ficollhypaque and incubated with anti-human cd19-percp, cd27-fitc, igd-bv510 and igm-apc to identify different subsets of b cells by flow cytometry. cd19+cd27-igd+igm-and cd19+cd27-igd+ igm+ b cells were sorted, loaded with cfse and cultured with cpg and ant-cd40 for 5 days to evaluate the proliferation. results: among the compartment of cd27-b cells, cvid patients showed an increased frequency of igd+igm+ cells and a lower frequency of igd-igm-cells as compared to control group. no differences were observed in the frequency of igd+igm-cells in cd27-b cells between cvid patients and controls. in contrast, in the compartment of cd27+ b cells, cvid patients showed an increased frequency of igd+igm-, igd+ igm+ and igd-igm+ cells and a lower frequency of igd-igm-cells when compared to health subjects. when the patients were divided in two groups based on autoimmune manifestations, the group with autoimmune disease showed an increased frequency of igd+igm+ and igd-igm+ cells in cd27-b cells when compared to the control groups. both patient groups showed an increased frequency of igd+igm-, igd+igm+ and igd-igm+ cells and a lower frequency of igd-igm-cells when compared to health subjects. regarding the proliferation, naã¯ve b cells from cvid patients showed a reduced proliferative capacity in response to in vitro stimulation as compared with naã¯ve b cells from health subjects. conclusion: our results suggest that the increase of cd27+igd+igm-b cells can be related to the susceptibility of autoimmunity in cvid patients. introduction: immunoglobulin g4-related disease (igg4-rd) is a group of immune-mediated conditions where tissues are affected with dense lymphoplasmacytic infiltrations with a predominance of igg4-positive plasma cells and storiform fibrosis, usually in the setting of elevated serum concentrations of igg4. common presentations include autoimmune pancreatitis, sclerosing cholangitis, retroperitoneal fibrosis, salivary gland disease, and orbital disease, among others. symptoms of asthma or allergy are present in approximately 40 percent of patients and they typically exhibit a good initial therapeutic response to glucocorticoids. case presentation: a 61-year-old female with a history of gastroparesis, cutaneous lupus erythematosus and suspected autoimmune pancreatitis was referred to allergy/immunology clinic for evaluation of elevated igg4. she reported a 15-year history of recurrent abdominal pain attributed to recurrent pancreatitis based on previous mild lipase elevations. prior endoscopic ultrasound (eus) of the pancreas revealed edema. there was concern for gallstone pancreatitis but ercp followed by cholecystectomy, biliary and pancreatic sphincterotomy had no change in her symptoms. in 2016, she was noted to have a positive ana and high serum igg4, per patient (values from osh records could not be obtained). symptoms improved with a course of steroids, hence suspicion for autoimmune pancreatitis. in 2018 she developed a rash on her arms and face. biopsies of the affected areas revealed cutaneous lupus erythematosus on the arms and a basal cell carcinoma on the face, which was excised. ana was only 1:80 at that time. at the visit, she complained of severe allergic rhinitis, joint pains, as well as a malar rash, which responded to intermittent courses of prednisone by prior providers. laboratories obtained at initial visit were significant for thrombocytopenia (135 thou/cu mm), positive lupus anticoagulant (56 sec) and elevated igg4 (95 mg/dl; normal range 4-86 mg/dl). c3, c4, c1q, ana, anti-double stranded dna, anti-smith antibodies, antiphospholipid panel, upep and spep were all unremarkable. ct chest and abdomen were also normal. given the patient's history of cutaneous lupus erythematosus, plaquenil was started as a steroid sparing agent. eus of the pancreas with possible biopsy was ordered in an attempt to obtain a histopathologic diagnosis of igg4-rd. conclusion: this case exhibits the association between elevated igg4, pancreatitis of unknown origin, allergic rhinitis, and cutaneous lupus erythematosus, highlighting the value of identifying a pathologic connection between seemingly unrelated disorders in patients with elevated igg4, as they may be manifestations of igg4-rd. in order to make the diagnosis, histopathologic findings showcasing lymphoplasmacytic tissue infiltration consisting mainly of igg4-positive plasma cells and small lymphocytes is essential. the majority of patients respond to glucocorticoids, and while the duration of response is variable, most patients flare during or after glucocorticoids are tapered, as noted in this patient. rituximab has been shown to be effective in some patients and will be considered in this patient if symptoms persist. (161) submission id#602042 rationale: pnp deficiency is an autosomal recessive disorder due to defective purine metabolism leading to severe combined immunodeficiency (scid) and neurological deterioration. newborn screening utilizing t-cell receptor excision circle (trec) assay can detect affected patients before complications arise. herein, we describe an infant initially identified by newborn screening with pnp deficiency and congenital cmv, a previously unreported presentation. methods: cmv quantitative pcr (qpcr) was performed by nebraska medicine, pnp enzyme activity by duke and genetic sequencing by invitae. results: a small for gestational age (sga) male infant was reported to have an abnormal trec assay on day of life (dol) 7. he was hospitalized for further evaluation. initial studies revealed profound lymphopenia, normal lymphocyte proliferation to mitogens and no evidence of maternal engraftment. additionally on dol 10, he had cmv viremia and viruria; thus with sga, failed unilateral hearing screen and head ultrasound with bilateral parenchymal calcifications, congenital cmv was suspected. pnp enzyme activity was abnormal. cmv treatment was initiated with ganciclovir on dol 10. foscarnet was added on dol 13. cmv qpcr levels decreased below the limit of detection by dol 30. genetic testing found a pathogenic homozygous mutation in pnp (c.286-18g>a). the infant has a 10/10 hla-matched, unaffected, cmv positive sibling and will proceed to hematopoietic stem cell transplantation. conclusions: to our knowledge, this is the first reported case of pnp deficiency identified through newborn screening. this novel case of congenital cmv and pnp deficiency highlights the importance of cmv screening and need for treatment strategies for congenital cmv in scid. despite a dramatic increase in the use of next generation sequencing over the last decade, the majority of the more than 50 million identified human genomic variants do not have well-established clinical implications. progress is being made on this complex challenge through multiple approaches, including data sharing. to maximize our understanding of genomic data, platforms that enable effective and responsible data-sharing are essential. this means that genotypic and phenotypic data must be findable, accessible, interoperable, and reusable under conditions that are ethical and transparent. to highlight innovations in data-sharing and their potential to advance discovery, we present three data-sharing mechanisms. for each platform, we will present a case highlighting its key functionality and discuss opportunities and challenges that may arise as each platform is scaled up. (1.) genomic research integration system (gris) is a collaborationengendering web application that facilitates the identification of genetic variants associated with rare immunological disorders. users can access integrated and standardized phenotypic and genomic data that is analyzable within the platform. gris enables systematic and automated capturing, and links patient data from disconnected systems and paperbased records. standardized annotations allow for the comparison of data from different clinical studies. the main goal of this tool is discoverability of other affected individuals enrolled in separate protocols within the niaid intramural research program. this internal database was used to find a second family with a rare variant in a candidate gene. (2.) the genomic ascertainment cohort (tgac) is a resource that aims to improve our understanding of the phenotypic consequence of genetic variation by providing access to aggregate, de-identified genomic data from large nih intramural and related cohorts. participants have provided informed consent to be re-contacted for additional phenotyping in the future. the main goal of this tool is to enable further study of the clinical consequence of variants in a large, unbiased cohort of patients ascertained for many indications. this database was used to investigate findings in participants with previously published pathogenic variants in genes associated with primary immune deficiency based on medical record review. (3.) clingen is dedicated to building an authoritative central resource that defines the clinical relevance of genes and variants for precision medicine and research. through the sharing of genetic and health data, clingen seeks to answer whether a given gene is associated with a disease (clinical validity)?; whether a given variant is causative (pathogenicity)?; and whether the information is actionable (clinical utility)? this resource is meant to convene disease-and gene-specific expert groups to curate the medical literature on mendelian disease to better define gene-disease and variant-disease relationships using many lines of evidence. this resource was used to clarify clinical validity of disease-gene assertions. together these efforts help create a clinical research ecosystem that maximizes the value of clinical research data and ultimately improves patient care. this research was supported by the intramural research program of the nih, niaid. introduction: according to the population reference bureau, the number of elderly americans, defined as age 65 and older, is projected to more than double from 46 million to 98 million by 2060, rising from 15% to 24% of the total population. the impact of immunodeficiency in this important segment of the population remains understudied. methods: the usidnet registry was queried to obtain demographic, clinical data of elderly patients defined as age 65 and older. descriptive analyses were performed on the data. results: 373 participants (7.2%) were eligible out of 5176 total registry participants. the median age of the cohort was 70 years and predominantly female (74.7%) and white (78.0%) with a median bmi of 26.6 â± 6.6.the majority (81.8%) of subjects were living. humoral deficiencies comprised the majority of diagnoses (94.6%), with common variable immune deficiency being the most frequent (76.9%). of the remaining non-humoral diagnoses, immune dysregulation (1.3%) and immunodeficiency with myelodysplasia (1.1%) were the most frequent. the majority (79.1%) of subjects reported having received immunoglobulin replacement therapy (igrt) at some point, with 51.7% reporting via iv route. of the 1275 infections that occurred in this cohort, sinopulmonary infections were the most commonly reported, specifically sinusitis (18.5%), pneumonia (13.8%), upper respiratory infection (6.7%), and otitis media (5.5%). in this cohort, 107 autoimmune, 49 cardiovascular, and 11 granulomatous complications were reported . the number of patients with malignancy was 89, with some patients diagnosed with multiple malignant disorders. of the reported malignancies, the majority (69.9%) were solid tumors. conclusions: compared to the age-matched non-immunodeficiency united states population, this cohort had more females 74.7% (usidnet) versus 56.0% (us population) and fewer whites 78.0% (usidnet) vs 86.0% (us population. humoral immunodeficiencies, specifically cvid, were most common diagnoses, similar to other age groups of immunodeficiency patients. majority of these patients have received igrt, with approximately half via iv route. this cohort reported living with a variety of non-infectious complications, including autoimmunity and malignancies. more research which specifically focuses on elderly patients with immunodeficiency is needed. clinical microbiologist and infectious disease physician, university of calgary x-linked agammaglobulinemia (xla) is a primary immunodeficiency caused by mutations in the bruton tyrosine kinase gene which leads to b cell maturation failure and defective antibody production. this puts patients at risk of recurrent sinopulmonary infections, gastrointestinal infections, and recurrent skin infections including infections caused by helicobacter sp. helicobacter sp are gram negative bacilli commonly found in the gastrointestinal tract of various animals. helicobacter sp. have been linked with gastritis most notably helicobacter pylori causing gastric ulcers in humans. helicobacter sp. has been found in rare cases to cause disseminated infections including pyodermic gangrenosum and cellulitis notably in patients with agammaglobulinemia. infections caused by helicobacter bilis are challenging to diagnosis due to difficulties with culturing the pathogen as well as poor guidelines for antimicrobial management. case report: the patient was diagnosed with x-linked agammaglobulinemia at the age of 16 months with a history of recurrent sinusitis and was started on ivig q3weeks. despite regular ivig, he developed bronchiectasis. at 11 years of age in 2013, he developed a chronic rash around his left knee resembling erythema nodosum. by 2014, he had developed a left knee effusion associated with left sided calf pain. his knee pain was found to improve during courses of ciprofloxacin to treat recurrent lung infections. given case report data of h. pylori causing erythema nodosum in patients with agammaglobulinemia, he was treated empirically for an h. pylori infections with no improvement. in 2015 he was found to have progressive cellulitis with pyomyositis of the left leg. a skin biopsy of a calf nodule was found to be culture negative but 16s pcr was positive for h. bilis. he was started on treatment with ertapenem and levofloxacin with subsequent resolution of his rash. his left ankle pain progressed and by late 2015 and was found to have possible osteomyelitis of the left ankle on mri. in 2016 he was found to be bacteremic with h bilis. due to progressive symptoms with significant impact on function and rising inflammatory markers despite 12 months of antimicrobial treatment, doxycycline and flagyl were added leading to clinical improvement and normalization of his inflammatory markers. he was continued on oral doxycycline and flagyl for 12 months for a chronic osteomyelitis. discussion: h. bilis is a slow growing pathogen which is challenging to culture in the laboratory often requiring special agar plates and prolonged incubation. in patients with agammaglobulinemia and associated chronic skin infections or erythema nodosuma, h bilis should be suspected as a possible pathogen. due to challenges with culturing, 16s pcr or amplification of the 16s ribosomal subunit should be considered to try to identify the pathogen. there are poorly delineated clinical antimicrobial breakpoints to help guide therapy with minimal evidence. case reports suggest prolonged therapy with aminoglycosides and penicillin. other studies have successfully treated patients with a carbapenem, azithromycin and levofloxacin. in the absence of sensitivity data, prolonged treatment (12months) should be considered with a combination of antimicrobials. patients should be followed closely as recurrent infections are not uncommon. chief, human immunological diseases section, laboratory of clinical immunology and microbiology, niaid, nih, bethesda, md introduction: dock8 deficiency is a combined immunodeficiency characterized by eczema, recurrent sinopulmonary infections, viral skin infections, malignancy and early mortality. in recent years, liver disease and vasculopathy have been increasingly recognized as a complication of dock8 deficiency. we clinically characterized our cohort of dock8 deficient patients, with a specific focus on these newly identified areas of disease involvement. methods: chart reviews were performed on patients seen at nih with genetic and clinical diagnosis of dock8 deficiency. patients were all enrolled on irb approved niaid protocols. results: we identified 52 patients from 40 families with dock8 deficiency in our nih cohort, ranging in age from 6-44 years. of the 40 families, 17 had homozygous mutations. of the 52 patients, food allergy was diagnosed in 31 (60%), eczema in 49 (94%), and asthma in 30 (58%). chronic or recurrent viral skin infections were seen in 49/52 (94%). chronic ebv viremia by pcr positivity was seen in 18/46 patients (39%); only 2 patients were known to be ebv immune without viremia. cmv viremia was infrequent. sinopulmonary infections were common, with bronchiectasis occurring in 23 /50 (46%) with available imaging. liver disease was diagnosed in 14 (27%), with 7 having biliary tract abnormalities on imaging and stool positive for cryptosporidia; most patients with cryptosporidia were without diarrhea. the incidence of cryptosporidia is likely under-represented due to more recent availability of sensitive assays for cryptosporidia detection. other liver abnormalities included fatty liver, metastatic disease from malignancy and medication related hepatitis. vasculopathy, predominantly of the aorta and cerebral arteries, was diagnosed in 7, with patients in the last 5 years being prospectively imaged. autoimmunity was rare (5%) including autoimmune cytopenias and hypothyroidism. 36 of 50 with follow-up are alive (70%) with age range 6-44 years. of the 36 living patients, 28 (78%) have had a hsct. causes of deaths include malignancy (6), infection (1) , and hsct complications (7) . long-term follow-up of patients with hsct (up to 6 years) has revealed resolution of the infection susceptibility and eczema, no new cancers, and stabilization of vasculopathy. conclusions: in addition to the well described manifestations of dock8 deficiency including eczema, allergy, recurrent sinopulmonary infections, skin viral infections and malignancy, our cohort revealed a relatively high incidence of liver disease, frequently associated with stool positivity for cryptosporidia, as well as vasculopathy. both of these clinical manifestations should be considered during preparation for hsct as they may affect management through transplant. autoimmunity has likely been over-estimated in prior descriptions of dock8 deficiency. long-term follow-up after hsct is needed to determine the prognosis from the vasculopathy, liver disease, and malignancy risk. (166) submission id#604115 yasuhiro yamazaki 1 , stefano volpi 2 , luigi d. notarangelo 1 introduction/background: extl3 (exostosin like glycosyltransferase 3) is an exostosin family member which initiates heparan sulfate (hs) chain biosynthesis and elongation. we have reported homozygous extl3 hypomorphic mutation (r339w) as a cause of immunoosseous-dysplasia syndrome. fourteen patients who have extl3 homozygous mutation were reported so far. eight of them manifested t cell lymphopenia, and 5 presented with severe combined immunodeficiency (scid) or omenn syndrome. using patient-derived induced pluripotent stem cells (ipscs) as a model, we have previously reported that extl3 mutations affect differentiation to thymic epithelial progenitor cells as well as expansion of hematopoietic progenitor cells. consistent with the latter, previous studies have suggested that mutations in other genes involved in hs biosynthesis affect hematopoietic stem cell (hsc) differentiation. however, the exact mechanisms by which extl3 mutations affect hematopoiesis are not known. objectives: we tried to clarify gene expression difference in hscs derived from wild-type, extl3 hypomorphic and extl3 knock-out (ko) human ipscs. methods: the control bj ipsc line was engineered by crispr/cas9 gene targeting. extl3 ko ipscs were obtained which carried compound heterozygous extl3 mutations (c.1003_1004inst; c.1005_1006insgatattt). hsc differentiation was induced using the stemdiff hematopoietic kit (stemcell technologies). bulk rna from each ips cells and each differentiated cd34+cd43+cd45+ was analyzed by rna sequencing. results: as compared to control ipscs, patient-derived cells showed slightly lower capacity to generate cd34+cd43+cd45+ cells. on the other hand, extl3 ko cells showed no differentiation into cd34+ cd43+cd45+ cells. gene set enrichment analysis showed enriched expression of genes involved in hematopoietic progenitor cell differentiation, regulation of hemopoiesis, and positive regulation of hemopoiesis in both control and patient-derived cd34+cd43+cd45+ cells compared to parental ipscs. moreover, these gene sets were more abundantly enriched in control than in patient-derived cd34+cd43+cd45+ cells. the gene set of response to type i interferon was significantly enriched in control versus patient-derived cd34+cd43+cd45+ cells. conclusions: these results confirm that extl3 plays an important role for hsc homeostasis in human cells. because type 1 interferons play a role in hsc proliferation, the decreased type i interferon signature may account for the reduced number of hscs that we have previously reported upon in vitro differentiation of extl3-mutated versus control-derived ipscs. this study was supported by the division of intramural research, niaid, nih, under protocol 16-i-n139. a case of autoinflammatory syndrome with osteoporosis and specific antibody deficiency autoinflammatory syndromes are inherited disorders with an exaggerated inflammatory response with no specific trigger. the clinical phenotypes of variants of autoinflammatory syndromes may overlap. we report a case of a 13 year old male with prior diagnosis of specific antibody deficiency, periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis (pfapa) syndrome, arthralgia and moderate atopic dermatitis. he was diagnosed at 3 years of age with specific antibody deficiency based on persistently low pneumococcal titers against repeat immunizations. due to recurrent infections, he was placed on immunoglobulin replacement therapy (igrt) at 8 years of age. igrt was discontinued at 13 years of age due to full resolution in infections and patient demonstrated robust response to immunizations. patient had lifelong history of recurrent fevers (every 5 weeks) associated with pharyngitis and aphthous ulcers consistent with diagnosis of pfapa. as he became older these episodes became less frequent. last episode of fever was over a year ago. the father had similar symptoms of recurrent fevers and oral ulcers as a child but currently remains asymptomatic. paternal grandfather died of kidney disease. patient has been generally in good health until recent year with intermittent abdominal pain, arthralgia and several long bone fractures with no history of prior trauma. a bone density scan revealed osteopenia and osteoporosis with a z score of -2.2 of lumbar spine, -4.0 of left femoral neck, -3.1 of left hip. given history of familial autoinflammatory disease, and antibody deficiency genetic testing was obtained which identified a pathogenic heterozygous variant of taci and mefv c.2082g>a (p.met694lle). taci mutation has been linked to antibody deficiency syndromes. genetic study for family members is pending. the mefv gene is associated with autosomal recessive familial mediterranean fever (fmf) and has been reported in autosomal dominant fmf as well. fmf is characterized by recurrent episodes of fever associated with serositis, arthralgia, and arthritis. patients with fmf have elevation in acute phase reactants during attacks with most returning to normal levels during the episode-free periods. multiple studies have shown that patient with fmf have lower bone mineral density and zscores than the general population. inflammation in fmf is thought to be mediated by several different cytokines (il-1, il-2, il-6, il-7, il-8, il-11, il-15 and tnf-). these same cytokines play a role in osteoclast activity and bone resorption. it has been suggested chronic inflammation during acute attacks and subclinical inflammation during the disease-free period lead to bone loss and osteoporosis. regular use of colchicine, the main treatment for fmf, may slow down osteoporosis. beside careful monitoring of clinical and laboratory phenotype, genetic evaluation is an important step in distinguishing between overlapping entities and can prevent complication and promote targeted intervention. a 5 year old previously healthy boy was referred for periodic fever/ pfapa and mosquito bite hypersensitivity. eight weeks earlier he developed fever to 104f, mouth sores and exudative tonsillitis; a rapid strep screen was negative. one week later he developed moderate cervical lymphadenopathy and had a positive ebv early antigen antibody.. one month later he had several severe local reactions to mosquito bites. each manifested 6-8 cm of erythema and induration with a 1+ cm bullae which left an ulcer after rupture and healed with a hypopigmented scar. the bites were accompanied by fever to 104f for 4 days. one febrile episode was treated with low dose prednisolone for presumed pfapa, and the fever resolved within hours. his past history was positive for nasal allergy and mild asthma. his parents are not related: mom is of european-indonesian and dad european-african (creole ancestry. testing prior to this visit showed normal igg, iga and igm, elevated ige (12,000 u/l) and normal cbc. lymphocyte subsets revealed cd3+ 23% (1538/mcl), cd4+ 17% (1109/mcl), cd8+ 6% (363/ mcl), cd19+ 9% (587/mcl), nk cells 67% (4435/mcl). on examination he appeared well with height at 86th%ile and weight at 58th%ile. there was no lymphadenopathy, hepatosplenomegaly or inflammed skin lesions; there was a 1cm round scar on the right plantar surface at the site of a prior mosquito bite. laboratory studies confirmed nk lymphocytosis 64% (5459/mcl) and elevated ige (29,600 u/l). lymphoproliferation to mitogens, cd3/cd28, cmvand hsv were normal, but absent to tetanus and candida antigens. ebv antibodies reflected past infection (vca-igg+, vca-igm-, ebna+); quantitative ebv pcr was >5,000,000 copies/ml whole blood. nk cytotoxicity and cd107a expression were decreased. bone marrow nk analysis suggested conality. the patient was diagnosed with "hypersensitivity to mosquito bites with ebv-associated t-/ nk lymphoproliferation." this disorder represents a subset of chronic active ebv (caebv) that is rarely seen outside of east asia. the lack of organomegaly or lymphadenopathy with hyper-ige and nk lymphocytosis and decreased nk function support the likelihood that nk cells are the target of ebv infection in this patient. this diagnosis may be a precursor to hemophagocytosis, liver necrosis or lymphoma/leukemia, and the only curative treatment is bone marrow transplantation. the patient's sister is a 10/10 hla match. she is seropositive for past ebv infection, and she has no history of extreme reactions to mosquito bites. genetic mutations that cause familial hemophagocytic lymphohistiocytosis have not been reported in caebv, and to the best of our knowledge familial cases of this disorder have not been identified. the response to bmt in this patient is pending. introduction/background: a number of case reports have described symptomatic hypogammaglobulinemia following administration of anti-epileptic drugs (aeds), specifically lamotrigine, carbamazepine, and levetiracetam. the mechanism by which symptomatic hypogammaglobulinemia develops is unclear. we evaluated the prevalence and the clinical significance of hypogammaglobulinemia associated with use of these aeds. objectives: our aim was to characterize the prevalence of aed-induced hypogammaglobulinemia, identify specific aeds associated with hypogammaglobulinemia, and characterize the timeline to development of hypogammaglobulinemia after initiation of therapy. methods: a retrospective, multicenter, electronic medical record review spanning 18 years identified patients with hypogammaglobulinemia who were on aed therapy (lamotrigine, carbamazepine, or levetiracetam). patients were excluded if they had a pre-existing primary immunodeficiency (pid), malignancy, protein-losing enteropathy, or significant proteinuria. patients on chronic immunosuppressive therapy, those without laboratory criteria for hypogammaglobulinemia, or those on one of the aeds for less than one month were also excluded. results: of the 316 cases reviewed, 5 patients met our inclusion criteria. the median age was 35; 80% were adults, 80% were female, and 80% were white. lamotrigine was implicated in 3/5 of the cases, carbamazepine in 2/5, and levetiracetam in 1/5. tetanus and pneumococcal titers were available for 4/5 patients. of those patients, 3/4 had protective titers to both per report with responses to >70% of the serotypes. only one patient reported severe, recurrent infections while the remaining four had little to no symptoms. interestingly, the patient with severe infections did have protective titers. of the five laboratory proven hypogammaglobulinemia patients, one died of an infection, two have continued on the medication due to refractory seizures responsive only to these medications, and two are currently being tapered off of their aed. conclusion: while it appears that aed-induced hypogammaglobulinemia is quite rare, it should be considered in a patient without other secondary causes of hypogammaglobulinemia on aed therapy. many antiepileptics downregulate nfkb signaling suggestive that patients who develop symptomatic hypogammaglobulinemia may have hypomorphic mutations in the nfkb signaling pathway. (170) submission id#604503 autoimmune lymphoproliferative syndrome (alps) results from defective apoptosis of lymphocytes mediated through the fas/fas ligand (fasl) pathway. the hallmark lab finding is an expansion of t cells that express the alpha/beta t cell receptor, but lack both cd4 and cd8 (double negative t cells) in the setting of normal or elevated lymphocyte counts. patients present with chronic, nonmalignant, noninfectious lymphadenopathy or splenomegaly. for definitive diagnosis, patients need to have (1) a pathogenic mutation in fas, fas ligand or caspase 10 or (2) a defective fas-induced lymphocyte apoptosis. we describe a probable case of alps with heterozygous mutation in fas c.287a>g(p.his96arg), a variant that has not been previously reported (his lymphocyte apoptosis assay is pending). unique to this case is the patients castleman disease-like features on pathology. a 15 year-old male referred from hematology clinic presented with an 8 year history of chronic lymphadenopathy, splenomegaly, anemia, and no underlying diagnosis. malignancy had previously been excluded by bone marrow aspirate and biopsy 8 years prior. however, he had a right sided lymph node that had increased in size for the past 4 months. he was otherwise asymptomatic. a lymph node biopsy 7 years prior was reportedly normal. his exam demonstrated significant bilateral lymphadenopathy, greater on right, with an approximately 8 x 6 cm mobile right neck mass. he had splenomegaly palpated 7 cm down and across to midline. he was therefore admitted for excisional lymph node biopsy to evaluate for possible malignancy and labs were sent to evaluate for alps. labs were supportive of alps. he had elevated t cell receptor alpha beta double negative t cells (tcr a/b dntcs) in blood (10.5%). b12 level was elevated (>1000 pg/ml). plasma soluble fasl level was elevated (5517 pg/ml). interleukin-10 (il-10) and il-18 levels were elevated (88 and 909 pg/ml respectively). he had multilineage cytopenias: anemia with hgb of 9.5 g/dl and neutropenia (absolute neutrophil count of 1380 k/ul). he had hypergammaglobulinemia with an igg level of 2010 mg/dl. broad infectious work-up was negative, including hiv, quantiferon, cocci, bartonella, toxoplasma, coxiella burnetii, ebv pcr and, cmv igm. lymph node biopsy showed no evidence of malignancy. immunostains and flow cytometry showed the presence of expanded tcr a/b dntcs in the lymph node, consistent with alps. interestingly, lymph node histology showed morphologic features typical of plasma cell variant castleman disease. numerous castlemanlike follicles showed typical regressive changes with onion-skinning morphology. paracortical hyperplasia with sheets of plasma cells was noted. there was negative staining for hhv8 (a well-known cause of plasma cell variant castleman disease). the diagnosis of idiopathic multicentric hhv8-negative castleman disease was excluded by definition in the setting of alps, per evidence-based consensus criteria published in 2017. in addition, our patient did not show any symptoms typically associated with it, such as fever, night sweats, weight loss, weakness or fatigue. should his fas-induced lymphocyte apoptosis be defective (in 2 separate assays), this would confirm his alps-fas diagnosis and we would start the patient on sirolimus. head of immunology unit, children' s hospital ricardo gutierrez introduction: slc46a1 gene encodes the proto-couple folate transporter (pcft), which supports intestinal folate uptake, and participates in folate transport into the central nervous system. slc46a1 mutations cause pcft defects, resulting in low folate levels in serum and cerebrospinal fluid. hereditary folate malabsorption (hfm) is a rare, autosomal recessive disorder with pcft deficiency resulting in cerebral folate deficiency. most of the patients present megaloblastic anaemia, moderate pancytopenia in the first few months of life, failure to thrive, diarrhoea and/or later onset neurological symptoms including seizures and developmental delay. i m m u n o d e f i c i e n c y i n h f m c a n m a n i f e s t i t s e l f w i t h hypogammaglobulinemia with normal t-cell function. b-cell precursor compartment seems to be particularly vulnerable to folate deficiency in some hfm patients. this immunodeficiency can be restored with specific treatment with folic acid. aim: to describe a female patient with a homozygous pathological variation in the slc46a1 gene. results: a 17 months old girl, born of non-consanguineous parents. she started at 3 months old with diarrhoea due to rotavirus, low weight and bicytopenia with normal bone marrow aspiration. she presented low levels of folic acid 1.5ng/ml (nv 3.1-20.5 ng/ml) at first thought due to secondary to malnutrition. treatment with folic acid supplementation was administrated, improving platelets counts. at 5 months old she presented steatorrhea with severe perianal panniculitis which required surgical treatment. no germs were rescued after a skin biopsy. moreover, she suffered from a respiratory infection due to picornavirus with two episodes of pneumothorax which required intensive care. at that moment ivig treatment was administered due to hypogammaglobulinemia and clinical severity. chronic diarrhoea worsened with bloody depositions. three rectal ulcers were found in the gut biopsy. bowel inflammatory disease was suspected and mesalazine administration was started with weight improvement. furthermore, at 10 months old she presented 3 status epilepticus, with pathological eeg and normal mri; one of them related to a cmv infection, successfully treated. in the immunological evaluation igg and iga were low with normal igm and igd. the protein-antibody response was not evaluated. she presented normal lymphocyte and t cells extended populations, t cells proliferation assay, dhr, treg cells, complement, cd107a expression, alpha-fetoprotein, without autoantibodies a molecular panel testing was done by ngs and a homozygous variant in slc46a1 gene was found, causing impaired intestinal folate absorption. conclusion: hfm should be considered in the diagnosis of patients with cytopenias and hypogammaglobulinemia in order to provide specific treatment. hfm has wide clinical manifestations, not only with megaloblastic anaemia and neurological impairment but also with gastrointestinal and skin manifestations. with folate treatment, clinical and immunological defects can be normalized. introduction: multifocal epithelial hyperplasia (meh), or hecks disease, is a rare, benign infection of the mucosa caused by human papilloma virus (hpv). clinically, meh manifests as numerous painless, soft, sessile papules or plaques, and typically occurs in the labial, lingual, and buccal mucosa. meh lesions are usually associated with hpv types 13 and 32, and seen more commonly in patients of caribbean or central/south american descent. prior studies in adults have shown that tumor necrosis factor alpha (tnf) promotes hpv, and may influence duration of hpv infection. case: we present a five-year-old full term male of haitian descent referred for assessment of multiple flesh colored, papular lesions on the buccal and labial mucosa that had persisted and quantitatively increased over one year, although some lesions regressed. he had no pain or difficulty eating. medical history significant for one seizure; negative for infection. no family history of infection, immunodeficiency, consanguinity, or miscarriage. head and neck examination failed to reveal cervical lymphadenopathy, masses, or hypertrophy in the salivary glands. intraoral examination revealed multiple papular nodules, mostly flat although some were corrugated. the greatest concentration was noted on the lower left labial surface extending to the mucosal vermillion interface, not involving the vermillion or commissure region. lesions extended into the mandibular vestibule and the left buccal mucosa. no other lesions were noted on extremities, genitalia, or any other visualized mucosal surface. based on history and exam, he was diagnosed with meh. white blood cell count, neutrophils, lymphocytes, cd4 and cd8 t cell, b cell, nk cell enumeration, and immunoglobulin panel were normal for age. tetanus and streptococcus pneumoniae titers were protective. cytomegalovirus igg and igm were negative. epstein-barr virus igg was positive, igm and early antigen ab negative. serology was significant for elevated tnf (84 pg/ml; reference range <22pg/ml) while interferon gamma and interleukins 1, 2, 4, 5, 6, 8, 10, 12, 13 , and 17 were normal, as was il-2 receptor cd25. one month after the initial visit, lesions were stable and unchanged. nine-valent hpv vaccination was considered, but not administered. conclusions: meh is a rare but benign disease caused by hpv. awareness of the disease and its course is important to prevent unnecessary expanded immunodeficiency work-up and possible procedures to eliminate lesions. although mucosal immunity can be site specific, especially with hpv, our understanding of t-cell cytokine and chemokine responses to hpv in cervical and laryngeal lesions may be instructive. the mechanism which allows hpv persistence in meh is not characterized, but it likely is due to increased viral persistence and an inability for the host immune response to successfully induce viral latency and successful containment. elevated tnf levels, with normal levels of il-2, il-6, il-8, il-10, may correlate with decreased clearance of hpv and prolonged duration of meh. it remains unclear if viral persistence is the cause of, or the sequela of, increased tnf. longitudinal monitoring of cytokine (tnf, il-2, il-6, il-8, il-10) and chemokine (ccl17, ccl18, ccl19, ccl20, ccl21, and ccl22) serum concentrations may be useful biomarkers for disease resolution. introduction: autosomal dominant hyper ige (jobs) syndrome is a rare primary immunodeficiency characterized by eczema and sinopulmonary infections as well as musculoskeletal and vascular complications. as in all chronic illnesses, patient education is an ongoing need. in the rare disease population, patient education is especially important as patients must be able to explain their unique healthcare concerns in a variety of medical settings. we focused on ad-hies, due to our relatively large cohort of patients, the frequent lack of classic signs of illness often impairing diagnosis of severe infection, and the diverse nonimmunologic clinical features of this disease. objectives: we aimed to increase understanding of the clinical manifestations of ad-hies to promote earlier recognition of symptoms and to increase self-efficacy for symptom management in the adult hies population. methods: adult patients were asked to participate in a patient education project. demographic information was collected from participants. they also completed a 12-item multiple choice test about symptom recognition in ad-hies and promis self-efficacy for managing symptoms, an 8item validated survey. then, patient education handouts that focused on pulmonary symptoms, eczema, bone health, and cardiovascular complications were reviewed with the participant. six weeks later, participants were asked to repeat the 12-item test and the self-efficacy survey. the demographic information, test, and self-efficacy were collected anonymously. results: 33 participants provided demographic information, completed the test and the self-efficacy survey. of the 33 participants, 15 were male and 17 were female. participants ranged in age from 18 to 66 years. 22/33 (67%) reported looking for information about ad-hies using search engines and most patients (91%) report that they have been given information about ad-hies from a doctor. 19/33 (58%) participants identified pulmonary symptoms as the symptom that concerns them most and 10/33 (30%) participants identified more than one symptom of concern. 25 participants returned the second test and second survey. the mean test score increased from 9.08 to 10.28 with 23/25 participants achieving a score of 9/12 or higher. the self-efficacy scores were unchanged with a mean score of 50.08 before reviewing the patient education handouts and 50.13 after. conclusions: participant feedback to this project was generally positive. ad-hies patients are seeking information and an educational intervention can improve their understanding of disease. self-efficacy results were mixed and unchanged overall, but suggest that ad-hies patients manage symptoms as well as other patients with chronic illnesses. patient education should continue at each encounter. this project can be expanded to include more topics, pediatric patients, and other rare disease populations. funded by the nci contract no. introduction: bcl11b plays an important role in the development and maintenance of the immune system and the central nervous system. expression of bcl11b represses nk and myeloid factors while inducing t cell lineage genes in thymocytes at the dn2 stage. conditional loss of bcl11b expression in murine thymocytes leads to t cell deficiency while complete knockout of bcl11b was fatal within a few days of birth. recently, specific heterozygous bcl11b mutations have been reported in 11 individuals with global development delay. however, only 2 of these cases, both carrying heterozygous missense variants, had low trec values with 4 other cases having frequent infections. little is known regarding the impact of bcl11b on human nk and t cell function. methods: we identified a novel heterozygous truncating mutation in bcl11b in an infant who was first detected by trec newborn screening. she subsequently developed severe autoimmune hemolytic anemia at the age of 3 months. we used standard immunoblotting and flow cytometry methods to assess protein expression and the impact of this bcl11b mutant on t cell and nk cell development and function. results: the patient has a novel single base-pair deletion in the bcl11b gene, which is predicted to produce a truncated protein with the loss of 3 of 6 zinc finger domains in bcl11b. immunoblotting of t cell blast lysates revealed a reduced bcl11b expression in the patient consistent with the heterozygous defect in bcl11b but also generated a novel band with a smaller molecular weight that we postulate represents the truncated protein product. while mitogen responses to cona and pha were normal, both cd4+ and cd8+ t cell counts were decreased, especially cd4+ naã¯ve and cd4+cd31+ naã¯ve t cells, suggesting reduced thymic output. the function of th1 cells was skewed with reduced il-2 production but increased ifn levels after pma and ionomycin stimulation. moreover, t regulatory cell counts were below normal range. nk cell counts were normal but these were mostly cd56bright nk cells. of the few cd56dim nk cells that presented, approximately half did not express cd16, the fc receptor for adcc. perforin was only present in cd16 expressing nk cells. as such, anti-cd16 stimulation understandably led to low but not defective nk cell degranulation. function after stimulation with k562 cells was normal when controlled for nk cell counts. conclusion: we report a novel bcl11b truncating mutation with a leaky scid phenotype that manifested with t-cell lymphopenia and autoimmunity. lowered thymic-derived naã¯ve t and regulatory t cells, skewed th1 cytokine response, and incomplete nk cell development suggests that bcl11b is important for the development and differentiation of multiple lymphocyte lineages. introduction: chronic diarrhea is one of the most common gastrointestinal complaints in patients with common variable immune deficiency (cvid) and can lead to life-threatening complications such as malabsorption and malnutrition. chronic diarrhea in cvid could be caused by infections, an inflammatory bowel disease-like picture, as well as malignancy. giardia lamblia is one of the most common parasites causing diarrhea in cvid (up to 40%), and can be refractory in these patients, leading to villous atrophy, weight loss, and failure to thrive. case report: a 41-year-old female with a history of cvid presents with chronic diarrhea and significant weight loss. her cvid was diagnosed by hypogammaglobulinemia (low levels of igg, igm, and iga), inadequate responses to protein and polysaccharide-based vaccines, decreased memory b cells (cd19+cd27+ 0.5%), and recurrent sinopulmonary infections. she was started on immune globulin replacement therapy and had significant improvement in her rate of infections. four years before her presentation to our center, she developed chronic, severe diarrhea. work up revealed giardia lamblia infection on endoscopy and colonoscopy. biopsy showed intraepithelial lymphocytes, villous blunting, and atrophic gastritis with rare plasma cells concerning for non-infectious enteropathy related to her cvid, in addition to the high burden of giardia organisms. she was initially treated with metronidazole for several weeks. however, her diarrhea did not improve, and she developed significant peripheral neuropathy leading to lower extremity weakness and limited mobility. her diarrhea persisted and was associated with approximately a 20-pound weight loss. repeat endoscopy and colonoscopy two years later showed persistent high burden giardiasis of the small intestine, as well as reactive lymphocytic infiltrates and atrophic gastritis. she was treated with nitazoxanide but continued to have diarrhea, and her stool continued to show trophozoites. given the significant inflammation and the lack of response to multiple antiparasitic agents, she was referred to our center for further evaluation. she was started on oral budesonide (9 mg daily) and oral immune globulin (20 grams weekly for 12 weeks). with this regimen, she had significant improvement in her diarrhea with a 10-pound weight gain. repeat colonoscopy showed considerable improvement in inflammation and resolution of her giardia infection, though her stool antigen continues to be positive. conclusions: persistent diarrhea in our patient is most likely due to a combination of cvid enteropathy and giardiasis. a prolonged course of metronidazole and later nitazoxanide did not control her diarrhea and led to significant side effects. switching to an immunomodulatory approach significantly decreased the inflammation in her bowel and may even have helped to reduce the burden of giardia in the gut. targeting both underlying bowel inflammation as well as active infection in cvid patients with chronic diarrhea might be needed to control symptoms. introduction: sphingosine-1-phosphate (s1p) is a lipid chemoattractant that is critical for lymphocyte egress from lymphoid organs. following a s1p concentration gradient maintained by s1p lyase ubiquitously expressed in tissues, lymphocytes within lymphoid organs are drawn to efferent lymph and blood unless their s1p receptor is internalized or downregulated. owing to diminished degradation of not only s1p, but also other sphingoid bases, deleterious mutations in sgpl1 (encoding s1p lyase) perturb sphingolipid catabolism in numerous tissues. correspondingly, human s1p lyase deficiency results in multiorgan dysfunction including kidney, skin, endocrine gland, and neurologic impairment alongside expected lymphopenia. although severe t cell lymphopenia (<300 cells/microliter) rivaling that of severe combined immunodeficiency (scid) can be seen in patients with s1p lyase deficiency, no such patients have been identified by newborn screening of t cell receptor excision circle (trec) counts, which are a surrogate measure of effective t cell production. herein, we describe an infant boy with an undetectable trec count at birth who was found to have two novel, biallelic sgpl1 mutations resulting in s1p lyase deficiency. case description: a 1-day-old boy with a preceding history of fetal hydrops is born at a gestational age of 36 weeks and presents with renal failure, anasarca, and respiratory failure. trec analysis of a dried blood spot obtained at 24 hours of life reveals zero copies/microliter. subsequent peripheral blood studies show profound lymphopenia, with diminished cd3+ t (129/microliter; 96 cd4+, 27 cd8+), cd19+ b (130/microliter), and cd16/56+ natural killer (124/microliter) cell counts. recent thymic emigrants are reduced (11.3% of cd4+ t cells are cd45ra+cd31+), as is the ratio of naã¯ve-to-memory cd4+ t cells (63% cd45ra+, 37% cd45ro+). expedited whole genome sequencing identifies two novel variants in sgpl1 a paternally inherited splice site variant (c.1566+2t>c) predicted to impact a canonical splice donor site, and a maternally inherited missense change (c.854g>a; p.cys285tyr) located in a well-established functional domain of s1p. in addition to nephrotic syndrome and lymphopenia, the patient displays evidence of adrenal insufficiency and has increased plasma levels of sphingoid bases and ceramides. before further analyses could be pursued, the infant dies at 40 days of age due to ongoing complications of renal failure and eventual cardiorespiratory failure. summary: we report the first case of s1p lyase deficiency identified by newborn trec screening for scid. as sgpl1 is not included in most commercially-available, scid-tailored gene panels, s1p lyase deficiency would be missed by conventional genetic testing. therefore, analysis for variants in sgpl1 should be considered in neonates with low-to-undetectable trec counts, nephrotic syndrome, and other suggestive sequelae. w a r t s , hypogammaglobulinemia, recurrent infections, and myelokathexis) is a rare autosomal dominant primary immunodeficiency. it is caused by a defect in the gene encoding the chemokine receptor cxcr4. this receptor, along with the associated ligand cxcl12, regulates leukocyte migration. we present the case of a 40-year-old female, who presented after she self-identified the signature signs of whim syndrome in herself and multiple family members. objectives: we present the case of a 40-year-old female who presented with a history of recurrent warts, leukopenia of unknown cause, and recurrent infections as a child. as a child, she experienced multiple ear and sinus infections, along recurrent warts on her upper and lower extremities that have persisted to this day. furthermore, during a routine examination when she was 14-years-old, she had a complete blood count drawn significant for leukopenia. no further workup was undertaken at that time. when continued leukopenia was noted at the age of 30, referral to a hematologist and a bone marrow biopsy was completed. bone marrow was significant for myelokathexis with borderline hypercellular marrow for patient age (80% cellularity), and normal cell line quantity. a trial of neupoegen was undertaken, without significant improvement. her family history is significant for father and brother with both leukopenia and recurrent warts. results: genetic analysis showed a heterozygous pathogenic variant in the cxcr4 gene, c.1012_1015dup (p.ser339phe fs*6). recent complete blood count was significant for a total wbc count of 1.0 k/ul, with a differential consisting of 30% neutrophils and 57% lymphocytes. lymphocyte subsets were significant for quantitatively low cd3+, cd8+ and cd19+ subsets, with normal numbers of cd4+ and nk cells. immunoglobulin levels revealed an igg of 835 mg/dl, iga of 145 mg/ dl, and igm of 54 mg/dl; igg anti-diphtheria and tetanus titers were protective, however, none of the 23 s. pneumoniae serotype titers were > 1.3 ug/ml. mitogen (pha, cona and pwm) and antigen (candida and tetanus) stimulation of lymphocytes were normal for all stimuli. conclusions: we present the case of a 40-year-old female with a history of recurrent infections, warts, and myelokathexis. on genetic analysis, she is noted to have a pathogenic mutation of the cxcr4 gene. the substitution of a phenylalanine for a serine decreases one of the seven serine phosphorylation sites in the carboxy tail of the molecule that occurs upon binding to its ligand, cxcl12 (sdf1). additionally, the variation generates a premature stop condon terminating the remainder of the carboxy terminal amino acids including ser346-7, known to have a role in carboxy terminial beta-arrestin binding. failure to generate adequate beta-arrestin binding sites leads to prolonged cxcr4 cxcl12 interaction resulting in myelokathexis. background: lacking protective antibodies, patients with primary antibody deficiencies (pad) suffer from frequent respiratory infections leading to chronic pulmonary damage. macrolides prophylaxis has been proven effective to successfully manage chronic lung diseases as cystic fibrosis, bronchiectasis, copd. we conducted a trial to evaluate the efficacy and safety of orally low-dose azithromycin prophylaxis when added to the usual care in pad patients. methods: a 3-year, phase ii, prospective, multicenter, randomized, double-blind, placebo-controlled trial on pad patients (age 18-74 years) with chronic infection-related pulmonary disease. patients received azithromycin 250 mg or placebo once daily three-times a week for 24 months. the primary outcome was the decrease of annual episodes of respiratory exacerbations. secondary endpoints included: time to the first exacerbation, number of hospitalizations, additional doses of antibiotics, health related quality of life measures, and safety. results: forty-four patients received azithromycin and 45 patients received placebo. the mean number of exacerbations was 3â·6 per patientyear (95%ci 2â·5-4â·7) in the azithromycin arm, and 5â·2 (95%ci 4â·1-6â·4) in the placebo arm (p=0â·02). in the azithromycin group the hr for having an acute exacerbation was 0â·5 (95%ci 0,3-0â·9, p=0,03) and the hr for hospitalization was 0.5 (95%ci 0,2-1â·1) (p=0â·04). the rate of additional antibiotic treatment per patient-year was 2â·3 (95%ci 2â·1-3â·4) in the intervention and 3â·6 (95%ci 2â·9-4â·3) in placebo groups (p=0â·004). improvement in hrqofl was observed in intervention group. azithromycins safety prole was comparable with placebo. conclusion: in pad with respiratory exacerbation, azithromycin prophylaxis led to reduction of exacerbation episodes, of additional courses of antibiotics, and of risk of hospitalization. given the deleterious effects of respiratory diseases adding azithromycin to pad treatment should be considered as a valuable option. background: the autosomal-dominant hyper-ige syndrome (hies), is a primary immunodeficiency caused by mutations in signal transducer and activator of transcription 3 (stat3) that leads to defective th17 immunity. adverse reactions following 23-valent pneumococcal polysaccharide vaccine (ppsv23) have been reported in 75% of stat3-hies patients, including severe local reactions that appear to be specific to this vaccine. case report: we present the case of a six-year-old girl, second child of nonconsanguineous healthy parents, that developed an extensive inflammatory skin reaction at the vaccination site following a single dose of ppsv23. the vaccine was prescribed due to history of recurrent respiratory tract infections and an incomplete vaccine calendar with no previously administered pneumococcal vaccines. the reaction began after 2 hours with local erythema and edema at vaccination site, expanding in 48 hours to a phlyctenular lesion with no well-defined borders. within the first 3 weeks, it progressively evolved to a deep necrotic lesion that required surgical debridement. the subsequent skin defect required surgical repair with a split-thickness skin graft from her right thigh as the donor site. the complete wound healing process took about 5 months, leaving a large scar ( figure) . the patient had a longstanding history of recurrent infections with multiple hospitalizations including severe neonatal pneumonia that required respiratory support, a colon perforation with secondary peritonitis and septic shock that required a hemicolectomy at 8 months of age, recurrent oral candidiasis, recurrent pneumonias of different lobes, recurrent acute otitis media, a cervical phlegmon, three episodes of dental abscess and multiple kidney abscesses due to gram-negative bacteria treated with intravenous antibiotics and surgical drainage. family history is notable for an older sibling that died due to sudden infant death syndrome. the patients mother has large and wide nose suggestive of stat3-hies phenotype, but no history of infections. immunological work up showed mild eosinophilia (850 cells/ mm3), elevated ige (1850 mg/dl), normal igg, iga, igm and lymphocyte subsets (cd3, cd4, cd8, cd16, cd56). peripheral th17 cells were markedly decreased (0.6% vs. 3.7% of normal control). specific pneumococcal antibodies evaluated 1 month after psv23 revealed 5/10 serotypes in protective levels. high resolution thorax ct showed multilobar bronchiectasis. echocardiogram and total spine x-rays were normal. stat3-hies was suspected with a national institutes of health score of 40. a novel heterozygous missense variant in stat3 affecting the src homology 2 (sh2) domain (p.lys591glu) was found by next-generation panel sequencing. a variant in the same position (p.lys591met) has been previously reported in a hies patient (clinvar). currently, she is on monthly ivig and prophylactic antibiotics (cotrimoxazole, azithromycin and fluconazole). conclusions: the case presented raises awareness on the risk of severe local adverse reactions to ppsv23 in stat3-hies patients. the etiology of such reactions is unclear and warrants further study. the benefits and risks of immunizing stat3-hies patients with ppsv23 should be weighed carefully by medical providers. abstract (max 500 words) introduction: dock8 deficiency is a rare primary immunodeficiency characterized by susceptibility to viral infections, atopic eczema, defective t-cell activation and th17 differentiation, impaired eosinophil homeostasis and dysregulation of ige. to date, there are no reported cases from malaysia. objective: we aimed to describe the clinical, immunological profile and mutational analysis of three siblings of consanguineous parents, presented with hyper-ige and lymphopenia between the years 1998 and 2012, which were solved by mutational analysis of the second and third siblings. methods: clinical data and investigation results were collated from the medical record. scoring of the symptoms and physical examination findings using nih score was performed. t, b, nk lymphocyte subsets and serum igg, iga, igm, total ige quantification, lymphocyte proliferation test and pneumococcal specific antibody response were performed. mutational analyses were performed in freiburg, germany. result: three siblings presented at different time points over a 20-year span with raised ige levels, recurrent infections, eczema, hypereosinophilia and bronchiectasis. the nih scores for hyper-ige syndrome (hies) ranged from 39 54. we also documented two serious infections in the siblings, which were disseminated cryptococcus neoformans and salmonella sp. immunological results showed t-cell lymphopenia, defective t-cell proliferation, decreased igm, raised ige, hyper-eosinophilia and defective pneumococcal antibody responses present but not in all 3 siblings. we identified a large deletion in dock8 starting from exon 30-48 in 2 of the siblings from mutational analysis performed. we will proceed with next generation sequencing and dock8 protein assay in malaysia to further characterize the defect. conclusion: our on-going study is the first description of dock8 in a family from malaysia. the diagnosis of dock8 should be suspected in cases with raised ige levels, recurrent infections and lymphopenia, despite no warts infection in the history. this study emphasized the importance of international research collaboration and networking in solving complicated cases. the index patient presented at the age of 4 years with increased susceptibility to lower airway and gastrointestinal infections (hospital admissions 5x/year until puberty). she suffered from mumps and varicella disease despite immunization, as well as from recurrent local, partially destructive hsv infections. she was diagnosed with common variable immunodeficiency (cvid) at age 13 and started on immunoglobulin replacement therapy. following a hypoglycemic seizure at age 20, the patient was diagnosed with isolated acth insufficiency with secondary adrenal insufficiency requiring hormone substitution. during and following her first pregnancy at age 25, she suffered from recurrent bronchopneumonias including pneumocystis jirovecii infection, resulting in bronchiectases documented on chest ct at age 30. currently, chronic lung disease is severely limiting her quality of life (table 1) . her daughter was noticed to be hypogammaglobulinemic soon after birth and failed to develop antibody responses to inactivated vaccines. she was started on immunoglobulin replacement therapy. she has not suffered from severe lower airway infections, but developed alopecia totalis at age 10 and nail dystrophy. w h o l e e x o m e s e q u e n c i n g r e v e a l e d a h e t e r o z y g o u s c.2553_2554insacccgag (p.lys855profster33, nm_001077494) mutation in exon 22 of nfkb2 in both mother and daughter. this monoallelic loss-of function frameshift mutation was not found in gnomad, gvs washington or clinvar databases. as previously published, a monoallelic mutation in this c-terminal domain leads to impaired phosphorylation and subsequent reduced nuclear translocation of the nfkb2/p52 active form. pediatricians and internal specialists need to be aware of the combination of hypogammaglobulinemia, acth deficiency, immune dysregulation and ectodermal dysplasia which is unusual for cvid -possibly indicating nfkb2 deficiency. this clinical syndrome may overlap with symptoms and signs found in both apeced/ aire (ar) and eda-id/nfkbia (ad) deficiencies. besides ig and hormone replacement therapy, curative treatment with hematopoietic stem cell transplantation is a therapeutic option for patients with nfkb2 deficiency, although the experience is limited. table 1 introduction: the modes of immunoglobulin (ig) administration for primary immunodeficiency diseases (pidd) differ in pharmacokinetics, infusion parameters, and tolerability. during 3 consecutive clinical studies, a cohort of 30 patients with pidd experienced all 3 modes of administration with the same ig 10% product in sequence from intravenous (iv) to subcutaneous (sc), then to hyaluronidase-facilitated sc (ighy), providing a unique opportunity to assess each administration modality within the same patient cohort treated and observed at the same sites. here we report the rates of infections stratified by igg trough levels, and the rates of adverse events (aes) with the 3 modes of ig administration (ivig, scig, ighy) within this patient cohort. design and methods: this analysis included patients with pidd aged 4 years who participated in 3 clinical studies: in study 1 (nct00546871) patients received ivig 10% every 34 weeks followed by weekly scig 10%; in study 2 (nct00814320), patients were treated with ighy every 34 weeks; in study 3 (nct01175313; extension of study 2), patients continued with the same ighy dose. to assess a potential association between the administration route at comparable igg trough levels and the infection rate, igg trough levels were categorized as 500 <700mg/ dl, 700<900mg/dl, 900<1100mg/dl, 1100<1300 mg/dl, 1300 <1500 mg/dl and 1500 mg/dl. periods where patients had trough levels within these strata were assessed, and the infection frequency was calculated. the time periods for this analysis were 3 months for ivig and 12 months each for ighy and scig 10% (2.25 years) treatments. in order to account for differences in the frequency of administration, rates of systemic and local aes were assessed as aes/patient-year for each mode of therapy. results: for igg trough levels of <1500 mg/dl, the associated annual infection rates were lower or similar for ighy than scig ( the treatment involves the control of infections and immune dysregulation with chemotherapeutic regimens followed by definitive treatment with hematopoietic stem cell transplant (hsct). aim: to describe a female patient with a pathogenic variation in stx11 with normal cd107a expression. results: she was a 2 years old female, the 5th daughter of nonconsanguineous parents, without relevant personal or family records. she was admitted due to a prolonged febrile syndrome, lymphoproliferation, pancytopenia and hepatitis, with hhv6 rescued in bone marrow and blood. gancyclovir treatment started with good response. she was admitted one month later with similar clinical symptoms with relapsed hhv6 infection. furthermore, hemophagocytosis was found in the bone marrow and evaluation of nk cell cytotoxicity demonstrated slightly reduced cytotoxic activity. functional studies for primary fhl were performed: perforin expression and cd107a surface expression were normal. she fulfilled criteria of fhl, and treatment with gancyclovir and steroids was administered. despite this treatment, she persisted with activated macrophagic parameters, and started with hlh2014 treatment protocol. she improved the clinical symptoms and laboratory parameters, but persisted with hhv6 low viremia. three months later, when immunosupression was decreased, she was readmitted with similar clinical manifestations and added neurological symptoms (facial paralysis, abnormal movements and sleep tendency). cerebral spinal fluid was pathological with hhv6 positive rescue. immunosupresive treatment was adjusted, but hhv6 copies in blood increased markedly. foscarnet treatment was administered and immunosupression was suspended for 2 days in order to control viral infection. unfortunately the patient died 6 days later. although specific functional tests were normal, sequencing of stx11 gene by ngs revealed a homozygous variation in c.581_584deltgcc, which is a previously reported mutation responsible for fhl. conclusion: despite the fact that cd107a was normal, the strong clinical and laboratory results must keep the fhl diagnosis in mind and intensive treatment should be early administered; in order to give the patient the opportunity to achieve the curative treatment. objectives: to report and characterize the clinical course of a patient with apeced and specific antibody deficiency. methods: retrospective chart review was performed. the patient was enrolled in niaid irb-approved protocol 11-i-0187. results: the patient is a 13 year-old-girl with apeced caused by homozygous aire c.967_979del13, who manifested cmc, hypoparathyroidism, adrenal insufficiency, sjogrens-like syndrome, autoimmune hepatitis, intestinal dysfunction and autoimmune pneumonitis. she suffered from recurrent sinusitis and severe pneumonias requiring hospitalization and administration of intravenous antibiotics several times per year. at age 9, she presented to our institution with fever and cough, a computed tomography (ct) of the chest revealed bilateral pulmonary infiltrates and bronchiectasis. bronchoscopy showed mucopurulent secretions in the bilateral lower lobes with culture of the bronchoalveolar lavage fluid growing streptococcus pneumoniae. further evaluation for an underlying disorder such as primary ciliary dyskinesia and cystic fibrosis including exome sequencing and sweat chloride testing was unrevealing. quantitative immunoglobulins were normal. despite prior vaccination, specific antibody testing showed negative rubeola igg and protective levels (> 1.3 mcg/ml) to only 3 of 23 pneumococcal serotypes. lymphocyte enumeration showed normal b cell subsets. as approximately 15% of apeced patients may experience asplenia, splenic ultrasound was performed confirming the presence of a 7 cm spleen and peripheral blood smear did not reveal howell-jolly bodies. serotyping of the s. pneumoniae isolate confirmed serotype 33f, which is part of the 23-valent vaccine. follow up vaccine challenge with the 23 valent pneumococcal polysaccharide vaccine showed an inadequate response. hence, she was started on monthly immunoglobulin replacement and over the following 4 years she has experienced a single methicillin sensitive staphylococcus aureus pneumonia. she has missed very few school days and other parameters including linear growth have improved, she is now along the fifth percentile for height and along the tenth percentile for weight. although she continues to experience intermittent cough she remains active participating in sports without limitation. conclusions: we report the evaluation, treatment and outcome of a patient with apeced complicated by autoimmune pneumonitis and specific antibody deficiency. as infectious susceptibility of apeced classically pertains to the signature infectious disease, cmc, patients with invasive or recurrent infections should be evaluated for underlying immune deficiency. investigation should include assessment for asplenia, quantitative immunoglobulins and specific antibodies with response to antigens. in patients with predominate respiratory symptoms, autoimmune pneumonitis should be evaluated given the near 40% prevalence of pneumonitis observed in american apeced patients. acknowledgements: supported by dir/niaid/nih introduction: autoinflammatory diseases are genetically heterogeneous disorders of innate immunity characterized by recurrent fever, rash, and/ or serositis, which generally are considered distinct from autoimmune diseases. we report a case of a patient with lupus-like disease and a mutation of nucleotide-binding oligomerization domain-containing protein 2 (nod2 r702w, yao syndrome) suggestive of an overlap between autoinflammatory and autoimmunity processes. case presentation: a 72-year-old man was evaluated for recurrent pleural effusions, morning stiffness, erythematous rashes, and fever up to 103â°c. history was notable for hashimotos thyroiditis and multiple admissions for presumed pneumonia with recurrent bilateral lung infiltrates and pleural effusions. transbronchial biopsy showed nonspecific pneumonitis and organizing pneumonia. antinuclear and anti-dsdna antibodies were positive. he received prednisone for presumed lupus pneumonitis leading to improvement. prednisone was tapered and hydroxychloroquine was started, but his fevers, pleuritic pain and pleural effusion reoccurred. genetic testing revealed a nod2 sequent variant (r702w) associated with autoinflammatory disease. hydroxychloroquine was stopped and colchicine was added to his regimen, allowing prednisone to be tapered without recurrence of symptoms. further immunological testing revealed increased signaling through the type i interferon receptor (interferon signature). conclusion: although this patient had several clinical (serositis, arthralgia) and immunological (antinuclear and anti-dsdna antibodies, interferon signature) manifestations of lupus, his clinical presentation also was consistent with yao syndrome. in retrospect, he had been having recurrent inflammatory symptoms for many years. recent studies in both mice and humans suggest that inflammasome activation and il-1 production are involved in the pathogenesis of lupus. this case provides further support for the idea that lupus and hashimotos thyroiditis, prototypical autoimmune diseases, may have overlapping autoinflammatory features. background: the implementation of severe combined immunodeficiency (scid) newborn screening by trec assay has played a pivotal role in identifying these patients early in life. the screen has also led to the identification of infants with other immunologic abnormalities, of which the clinical implications have been unclear and there are limited data on their outcomes. objective: to review immunologic and genetic outcomes of infants referred to an immunology service of a tertiary care center with abnormal newborn scid screens. methods: we retrospectively reviewed charts of infants with positive scid screen from july 2014 to november 2018. we excluded patients who had positive screen at <36 weeks corrected gestational age. we classified outcomes into 3 groups including scid, non-scid t-cell lymphopenia (nscid-tcl) and normal t-cell count. idiopathic t-cell lymphopenia was defined as nscid-tcl (cd3+ < 2,500 cells/mcl) with negative chromosome microarray and negative whole exome sequencing/or genetic panel (either genedxâ® scid panel or invitaeâ® primary immunodeficiency panel). results: of 119 infants, 78% were male, 56% were caucasian, and 37% were african-american. fifty-four % and 46% of infants were identified by illinois and missouri screens, respectively. the mean age at initial evaluation was 22 days (4-122 days). 69% of infants had a normal tcell count (n=80) or normal repeat newborn screen (n=2), 25% had nscid-tcl, including mild (cd3+ 1,500-2,500 cells/mcl, n=20) and moderate (cd3+ 300-1,500 cells/mcl, n=10) tcl, and 6% had scid (n=5), leaky scid (n=1) or complete digeorge (n=1). genetic etiologies of nscid-tcl included 22q11 deletion (n=4), trisomy 21 (n=1), and mutations of tbx1 (n=2), foxn1 (n=1), and cd3e (n=1). three of these infants had novel variants at the time of diagnosis. secondary causes of tcl were identified in 1 infant (thoracic infantile fibrosarcoma). one infant had idiopathic tcl. eighteen infants with nscid-tcl were followed clinically without complete genetic testing performed. for scid, mutations were found in jak3 (n=2), ada (n=1), il2rg (n=1), and rag1 (n=1). the patient with leaky scid had negative whole exome sequencing. all patients with scid and leaky scid underwent hematopoietic stem cell transplantation at a median age of 5 weeks (3 weeks -4 months), with successful engraftment in all but 1 patient. of 19 idiopathic and nscid-tcl cases followed clinically, 12 had at least one follow-up visit at median age 5 months (2.6 months 2.2 years) and the majority had improved or stable lymphocyte count without serious infections requiring intravenous antibiotics, though 1 had a hospitalization for rsv infection. the mysm1 patient died after cord blood transplant from unclear etiology. our study had limitations. half of infants with nscid-tcl did not have a complete genetic workup, and only a fifth of patients with nscid-tcl were inpatients, potentially explaining the relatively low number of infants with secondary lymphopenia. conclusions: in our cohort, one-fourth of infants with abnormal scid screen had nscid-tcl. although the majority of nscid-tcl did well, approximately one-third of them had underlying genetic abnormalities associated with their t-cell lymphopenia. (189) submission id#606320 introduction: accumulation of intracellular adenosine and deoxyadenosine nucleotides (daxp) due to adenosine deaminase deficiency results in profound lymphopenia and severe combined immunodeficiency. left untreated this form of scid is uniformly fatal. while allogeneic hematopoietic cell transplant (hct) and autologous gene corrected stem cell therapy (gt) are potential cures for ada-scid , initiating enzyme replacement therapy (ert) immediately upon diagnosis regardless of definitive treatment is standard of care. hct and gt are not therapeutic options for all ada-scid patients and ert offers immediate therapeutic intervention for these patients leading to partial immune reconstitution, and durable survival in most patients treated. adagen (pegademase), approved by the fda in 1990 in the usa, is a pegylated bovine ada (nada) with the enzyme harvested from bovine intestines. this unsustainable production process led to the development of a recombinant enzyme source based on the bovine protein sequence and an improved pegylated linker by using succinimidyl carbamate (revcovitm-(elapegademase-lvlr). methods: a phase ii/iii clinical trial was performed at 5 us sites under institutional irb approval. eligible ada-scid subjects were stable on adagen and without complicating underlying conditions. demographics, medical history, lymphocyte counts, immunoglobulin levels, trough plasma ada activity and rbc daxp measurements were collected. patients were treated with adagen as a single, weekly im dose adjusted to achieve a trough plasma ada activity of > 15 mmol/hr/l and rbc daxp < 0.02 mmol/l (protocol target levels). once patients had achieved this level (3-9 weeks), a seven-day pk on adagen was done and the patients were transitioned to revcovi based on the formula for enzyme equivalent activity of 1mg revcovi = 150 units adagen. after 5 weeks on revcovi, trough ada and daxp were assessed and a seven-day pharmacokinetic study was conducted at week 9. patients were assessed periodically for clinical and laboratory values and evaluation of the study endpoints was done at week 21. subjects subsequently continued on revcovi and were assessed periodically. results: six patients, ages 16-37 entered the trial with initial adagen dosing at 7.7-42.9u/kg/wk (see table 1 ). adagen dosing was adjusted to target endpoints of ada trough activity (>15mmol/hr/l) and rbc daxp (<0.02 mml/l). patients transitioned to weekly revcovi using the aforementioned conversion formula at doses of 0.17-0.285 mg/kg/wk. the spectrum of clinical manifestations range from infections to autoimmunity and inflammation among patients with hypomorphic recombination gene 1 and 2 (rag1/2) pathogenic variants. auto-antibodies targeting cytokines ifn-alpha, ifn-omega and il-12 were reported in a large proportion of these patients and their occurrence often coincides with viral infections. we report the time of emergence and relative frequency of anti-cytokine antibodies in children and adults, and their persistence among patients with hypomorphic rag deficiency. antibodies were measured from plasma samples of patients by enzyme linked immunoassay (elisa). our rag cohort includes 28 patients with rag1 (n=17, 61%) and rag2 deficiency (n=11, 39%). antibodies targeting ifn-alpha (75%) were most common followed by il-12 and ifn-omega (40% each). two asymptomatic patients who were detected by newborn screening for scid and received hematopoietic stem cell transplantation had no detectable anti-cytokine antibodies. in the cohort of young children (ages 11 mo-7 years, n=9), all patients had detectable antibodies to ifn-alpha, prior history of severe viral infection and subsequently developed autoimmune cytopenias. other anti-cytokine antibodies were less common (ifn-omega 44%, il-12 33%). similarly, children between 10-18 yo age (n=9) also had high fraction of anti-ifn-alpha antibodies (89%) with prior history of infections (66%) and continued to have other anticytokine antibodies less commonly (ifn-omega 37%, il-12 62%). in the adult cohort (n=8, ages 25-39 years) the frequency of anti-ifnalpha anti-cytokine antibodies were lower (62%,) and il-12 and ifnomega (50% each) continued to persist. three adult patients had anticytokine (ifn-alpha, ifn-omega and il-12) antibodies tested at multiple timepoints and elevated titers persisted up to 4 years. our data demonstrates that anti-cytokine antibodies, especially those targeting ifn are frequent and emerge early in life in association with viral infections in patients with rag deficiency. a lower fraction of adult patients have detectable anti-cytokine antibodies, and maintain these over several years. anti-ifn-alpha may serve as a useful biomarker for identifying partial rag deficiency among young and adult patients with history of viral infections and autoimmune cytopenias. the role of these antibodies to cytokines is yet to be determined but a specific signature of these antibodies may help to identify an underlying immunodeficiency and initiate early definitive treatment with bone marrow transplantation. anti-cytokine antibodies appear to be a novel tool in evaluation of autoimmune diseases including rag deficiency. introduction: norovirus is one of the most common pathogens causing gastroenteritis in immunocompromised patients, often leading to chronic infection, causing villous atrophy, malabsorption, weight loss, organ failure, need for parenteral nutrition, and death. norovirus treatment in immunocompromised patients is challenging. oral immunoglobulin (poig) has been used to treat norovirus gastroenteritis with variable success. our aim in this study was to determine the outcomes of treating norovirus gastroenteritis in immunocompromised patients methods: electronic medical records were reviewed for patients with norovirus infection confirmed by rt-pcr since january 2012. our initial cohort was focused on patients with primary immunodeficiency (pid), lung, and liver transplant. data on demographics, immunological phenotype, treatment with poig, the number of bowel movements (bm), and virus clearance were collected. descriptive statistical methods were used to describe treatment outcomes. further analysis of patients immunophenotype, immunosuppression medications, and co-morbid illnesses is underway. results: twenty-six immunocompromised patients (27 norovirus infection episodes, as one patient had reinfection) were analyzed twelve females, age range 7 months-50 years. twelve patients had pid diagnosis (3 common variable immunodeficiency, 2 severe combined immunodeficiency, 1 x-linked agammaglobulinemia, 1 wiskott-aldrich syndrome, 1 digeorge syndrome, 1 hyper-igm, 1 stat3 gain-of-function, 1 nemo and 1 lymphopenia in a patient with trisomy 21), 13 patients were status-post liver transplant, and two patients were status-post lung transplant. 13 of26 patients were on ig replacement therapy at the time of the norovirus infection. the average number of bm/day in all patients was 8. 4 (range 2-20) . eight patients received poig (250-500 mg/kg) weekly for a duration from 1-12 weeks. three of those received additional nitazoxanide and 2 received ribavirin. 2/8 patients in the poig group were receiving total parenteral nutrition (tpn), and 4/19 on no treatment group received tpn. the average number of bm/day in poig before treatment was 9.5 (range 4-16), and 8.6 (range 2-20) in those who did not receive any treatment. 5 of 8 (62%) on poig vs. 11 of 19 (57%) in the no treatment group cleared the virus. the average number of weeks to return to baseline bm was 2.6 (range 1-7) in the poig group vs. 1.5 (range 3 days-5 weeks) in the no treatment group. 2 of 8 on poig continued to have chronic diarrhea that is still ongoing. conclusion: despite anecdotal reports suggesting successful use of poig in immunocompromised patients, our data did not show a significant decrease in stool output in patients treated with poig, compared to no treatment. however, poig led to a higher rate of virus clearance. a study with larger sample size might be warranted to identify the patients who benefit from poig in the context of norovirus infection and ensure the appropriate use of ig products, especially given the concerns for the national shortage of ig products. chief medical officer, novimmune sa primary hemophagocytic lymphohistiocytosis (phlh) is a life-threatening, immune regulatory disorder characterized by immune hyperactivation that is driven by high production of interferon (ifn)-. patients with hlh typically develop fever, splenomegaly, cytopenias and coagulopathy. until recently, there have been no fda approved treatments for hlh, and standard dexamethasone/etoposide-based treatment has not evolved significantly in 20+ years. emapalumab-lzsg (ni-0501) is a fully human, anti-ifn-monoclonal antibody that neutralizes ifn-and which was recently approved (november 2018) by the fda for the treatment of adult and pediatric (newborn and older) patients with phlh with refractory, recurrent, or progressive disease or intolerance with conventional hlh therapy. results of the pivotal trial supporting this approval are presented herein. methods: this open-label pivotal study (nct01818492) includes patients 18 years with a diagnosis of phlh and active disease. data presented were from 34 patients, of whom 27 had failed conventional hlh therapy prior to study entry. the initial emapalumab-lzsg dose was 1 mg/kg given intravenously every 3-4 days. subsequent doses could be increased up to 10 mg/kg based on the evolution of response parameters. dexamethasone was administered concomitantly at 5 to 10 mg/m 2 /day and could be tapered during the study. treatment duration was 8 weeks, with possible shortening to a minimum of 4 weeks, or extension up to the time of allogeneic hematopoietic stem cell transplantation (hsct). the primary efficacy endpoint was the overall response rate (orr) at end of treatment, assessed by pre-defined objective parameters, including normalization or at least 50% improvement from baseline of fever, splenomegaly, cytopenias, hyperferritinemia, fibrinogen, d-dimer, central nervous system (cns) abnormalities, and with no sustained worsening of scd25 serum levels. the primary analysis used an exact binomial test to evaluate the null hypothesis that orr be 40% at a one-sided 0.025 significance level. patients were eligible to enter an extension phase for follow-up after completing the main study (nct02069899). the data cut-off applied is july 20 2017. results: patient characteristics are summarized in table 1 and efficacy is summarized in table 2 . disease at study entry was consistent with the broad spectrum of phlh abnormalities. over 30% of patients had signs and/or symptoms of cns disease. orr was significantly higher than the pre-specified null hypothesis of 40%, meeting the primary endpoint. the response rate based on investigators clinical judgement was 70.6%. emapalumab-lzsg infusions were in general well tolerated, with mild to moderate infusion-related reactions reported in 27% of patients. the observed safety events (pre-hsct conditioning) mostly included hlh manifestations, infections or toxicities due to other administered drugs. infections caused by pathogens potentially favored by ifn-neutralization occurred in 1 patient during emapalumab-lzsg treatment (disseminated histoplasmosis), and resolved with appropriate treatment. no off-target effects were observed. conclusions: treatment with emapalumab-lzsg was able to control hlh activity with a favorable safety and tolerability profile in a very fragile population. the majority of patients proceeded to hsct with favorable outcomes. our results indicate that emapalumab-lzsg should be considered as a new therapeutic option in phlh thanks to its targeted mode of action. results: a total of 360 genes were differentially expressed between t cells of 22qds patients (n=13) and healthy controls (n=6) (log 2 fold change range (-2.0747, 15.6724)).when these 360 genes were tested for pathway enrichment, the top 5 pathways in t lymphocytes based on their p value included communication between innate and adaptive immune cells, cross talk between dendritic cells and natural killer cells, allograft rejection signaling, dendritic cell maturation, and b cell receptor signaling. the top 10 biological processes with differential expression included 36 immune response, 31 inflammatory response, 33 apoptotic process, 12 interferon gamma mediated signaling pathway, 14 nucleosome assembly, 16 defense response to virus, 8 lipopolysaccharide mediated signaling pathway, 7 positive regulation of nf-kappa b import into nucleus, 10 type i interferon signaling pathway, and 10 neutrophil chemotaxis genes. we compared gene expression between 22qds participants with low t cell counts (n=7) and 22qds participants with normal t cell counts (n=6) and found 94 genes that were differentially expressed (q<0.05) (log2 fold change range (-4.5445, 5.1297) patient began experiencing recurrent high fevers and developed splenomegaly. elevated transaminases and concern for lymphoproliferative disease prompted a splenectomy and liver biopsy. both the spleen and liver biopsy were positive for ebv but were negative for malignancy. bone marrow biopsy was unrevealing. genetic testing identified a pathogenic variant in xiap/ birc4 (1141c>t), and the patient was treated with high dose oral steroids resulting in an improvement in symptoms. subsequently, therapy with anakinra was started and steroids were tapered. during the steroid taper, he noticed a change in the vision of his left eye consistent with naion, as well as worsening of his colitis. there was loss of the inferior visual field and fundoscopic exam was significant for left optic disc swelling. oct noted superior retinal nerve fiber layer thinning. oral steroids were restarted with improvement in optic disc swelling, but without improvement or change in vision. as of his most recent exam, the patient has persistent bilateral inferior visual field defects with segmental optic nerve atrophy typical of naion. he has continued therapy with anakinra, and subsequently tapered off of prednisone; though he remains on a physiologic dose of hydrocortisone. conclusions: this case demonstrates an unreported ocular manifestation in a patient with xiap deficiency, which clinically appeared sensitive to immunomodulation. our patient is an unusual candidate for naion due to his young age, the average age of onset being the mid to late 60s, and lack of vascular risk factors. we hypothesize that his hyper-inflammatory condition contributed to irreversible vascular damage in the optic nerve head, resulting in naion. therefore, it may be useful to consider the involvement of systemic inflammatory and immune dysregulatory conditions when treating patients with atypical naion. additionally, naion should be considered in patients with xiap deficiency and sudden unilateral vision loss. the importance of de novo mutations in causing severe sporadic immune disease is well described, yet significance of such a variation in less severe and later onset of immune disease is poorly investigated. whole exome sequencing has been a powerful tool to resolve and explain the genetic basis of novel syndromes in immune related diseases. however, proving causation can be complicated due to low number of the affected individuals. we performed whole exome sequencing in a cohort of patients with noncongenital immune defects, along with detailed cellular biochemical phenotyping. we report and describe a novel non-congenital combined immune deficiency arising from a de novo gain-offunction mutation in ikbkb(c.607g>a). this gene encodes ikk2, and activates canonical nfkb signalling. cellular and biochemical studies of the proband revealed that ikk2v203i results in enhanced nf-kb signaling, as well as t and b cell functional defects. ikk2v203 is a highly-conserved residue, and to prove causation, we generated a crispr/cas9 mouse model that carry the precise orthologous missense mutation. we show that mice and humans carrying this missense mutation exhibits remarkably similar cellular and biochemical phenotypes. dysregulation in patients. total rna isolated from cryopreserved peripheral blood mononuclear cells was reverse transcribed to generate cdna. we selected four known gata2 transcriptional targets, gata1, gata2, tal1 and zfpm1 (encoding fog1) and used droplet digital pcr to quantify transcript levels normalized to the low-expressing gene tbp1. we used samples from 9 individuals with wild-type gata2 (wt), 5 known gata2 mutation patients (mut) and two individuals suspected of gata2 deficiency but without identified mutation or allelic imbalance (unk1, unk2). transcript analysis revealed significantly decreased transcript levels of gata1, gata2 and tal1 in mut pbmcs compared to wt. most wt samples had higher zfpm1 transcripts than gata2 mutated patients however it did not reach statistical significance. strikingly, we were able to use this analysis for two individuals suspected of gata2 deficiency. in the first case (unk1) a 51 yr old female with primary lymphedema, hypogammaglobulinemia, recurrent infections and possible family history of leukemia was referred for gata2 testing. no mutation was identified however it was noted that she was homozygous across the gene preventing allelic evaluation. the second patient (unk2), a 24 yr old female, had erethemya nodosa on legs, mycobacteria kansasii and cytopenias. in each of the targets analyzed, transcript levels from unk1 were lower than the wt samples and in a similar range as the gata2 mutation samples while unk2 had a profile consistent with the wt samples. we propose the use of gata2 targets as surrogate markers in cases where a mutation is not identified and allelic expression analysis is uninformative. are often under-reported and under-recognized. we sought to further understand and evaluate the prevalence, type, and association with serum immunoglobulin e (ige) for cvid patients with atopic manifestations. methods: we performed a retrospective analysis of cvid patients with atopic manifestations in the partners healthcare cvid cohort. we evaluated baseline patient characteristics, atopic diagnoses, and serum ige levels. results: in the partners cvid cohort, the average age was 52 years old (â±17) and 64% female. 92/175 (52.6%) of patients had a diagnosis of asthma, with the majority of these diagnosed by an allergist (65%) or pulmonologist (16%). eczema/atopic dermatitis was diagnosed in 47/175 patients (26%), by either an allergist (53%) or a dermatologist (8%). allergic rhinitis was diagnosed in 50/175 (28.5%) with positive skin prick testing in 52% of these patients. food allergy was diagnosed in 5 patients (2.9%). the median cohort serum ige was 7.5 iu/ml. the median serum ige was higher in patients with 2 or more atopic complications compared to those with one or less atopic condition (9 vs. 5 iu/ml), which was statistically significant (p=0.01). conclusions: we report higher rates of atopy than previously described in other cvid cohorts. consistent with previous reports, we find a low median cohort serum ige level in cvid patients compared to the general population. however, we identify a subset of patients with a predisposition towards atopy and higher ige levels within the broader characterization of cvid, and these patients may have a more specific molecular diagnosis that leads to elevated ige and atopic conditions. whole exome sequencing is underway to further evaluate this hypothesis. whim (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is a primary immunodeficiency with autosomal dominant inheritance. in most patients, the genetic cause of the disease is a gain-offunction variant in c-x-c chemokine receptor type 4 (cxcr4) that results in arrest of neutrophil migration from the bone marrow. most patients develop hypogammaglobulinemia and early waning of antibody response with vaccination. however, the exact origin of aberrant humoral immunity in whim syndrome patients is yet to be clarified. here we describe a 4-year-old iraqi female with a heterozygous cxcr4 p.ser338ter variant, which is presented with haemophilus influenzae meningitis, history of tetralogy of fallot, early onset intermittent neutropenia, lymphopenia, recurrent bacterial and viral infections. immunologic evaluation revealed hypogammaglobulinemia, elevated igm level and a lack of protective vaccine titers after tetanus and prevnar vaccinations. a bone marrow biopsy was consistent with myelokathexis. immune phenotyping, functional studies and apoptosis assays were performed on peripheral blood cells by flow cytometry in our whim patient and controls. although we found that all lymphocyte compartments were reduced, naã¯ve cd4 t helper cells and switched memory b cells were predominantly affected. spontaneous apoptosis was most pronounced in b rather than t cell compartments in whim patients. in addition, naã¯ve b cells easily activated and died upon activation in vitro. cxcl12, a ligand of cxcr4, induced elevated t helper cell migration and increased actin polymerization in p.ser338ter mutant cells. we conclude that intrinsic b cell abnormalities, such as increased rate of apoptosis and altered activation, might be responsible for defective antibody response in whim patients. although most individuals effectively control herpesvirus infections, some suffer from unusually severe and/or recurrent infections requiring anti-viral prophylaxis. a subset of these patients possesses defects in nk cells, innate lymphocytes which recognize and lyse herpesvirus-infected cells; however, the exact genetic etiologies are rarely diagnosed. plcg2 encodes a signaling protein in nk cell and b cell receptor-mediated signaling. dominant-negative or gain-of-function mutations in plcg2 cause cold urticaria, antibody deficiency, or autoinflammation. however, loss-of-function mutations and plcg2 haploinsufficiency have never been reported in human disease. we examined 2 families with autosomal dominant nk cell immunodeficiency with mass cytometry and whole-exome sequencing to identify the cause of disease. we identified two novel heterozygous loss-of-function mutations inplcg2 that impaired nk cell function, including calcium flux, granule movement, and target killing. although expression of mutant plcg2 protein in vitro was normal, phosphorylation of both mutants was diminished. in contrast to plaid and aplaid, b cell function remained intact. plcg2+/-mice, as well as targeted crispr knock-in mice, also displayed impaired nk cell function with preserved b cell function, phenocopying human plcg2 haploinsufficiency. we report the first known cases of plcg2 haploinsufficiency, a clinically and mechanistically distinct syndrome from previously reported mutations. therefore, these families represent a novel disease, highlighting a role for plcg2 haploinsufficiency in herpesvirus-susceptible patients and expanding the spectrum of plcg2-related disease. we pursued genetic diagnosis, which identified bi-allelic frameshift mutations in the rag1 gene which had not been previously described: c.967delg (p.v323sfsx22) and c.1048_1075del128insaaaagagtg (p.v350kfsx47). taken together, his presentation suggested significant immune dysfunction had evolved since transplant leading to extensive pulmonary nontuberculous mycobacterial infection and possible bronchiolitis obliterans. he therefore will undergo a subsequent unconditioned cd34+ stem cell boost from his sister, the original donor, once he completes mycobacterium abscessus treatment. this case highlights the potential long-term immune dysfunction which may evolve after unconditioned allogeneic stem cell transplant for scid, in which full engraftment in all myeloid and lymphoid compartments is not expected. it also highlights the importance of guideline-driven follow-up of these patients to monitor for said dysfunction, to prevent serious infection and long-term sequelae. somatic hypermutation (shm) in the b cell receptor (bcr) heavy (igh) and light chain genes promotes affinity maturation and also mutation away from self-reactivity, therefore serves as an important peripheral tolerance checkpoint. as an example, unmutated bcr ighv4-34 genes give rise to antibodies that bind to i/i antigen on red blood cells (rbc) and may elicit cold agglutinin disease (cad), a variant of autoimmune hemolytic anemia (aiha). in case of healthy individuals, frequent shms in the i/i binding site of bcr ighv4-34 genes decrease rbc reactivity and cad. patients with primary immunodeficiencies (pid) paradoxically develop autoimmune diseases, including autoimmune cytopenias, especially aiha. it is unclear if impaired shm of bcr, in particular mutation away from i/i binding, is relevant in the development of rbc reactivity and consequently aiha in a pid background. our studies focus on pid patients with hypomorphic recombination activating gene (rag1 and 2), combined immunodeficiency phenotype and history of autoimmunity, in particular aiha (rag cid/ai). we detected increased frequency of unmutated ighv4-34 bcr in memory b cell repertoires of rag-cid/ai patients as well as elevated titer of unmutated ighv4-34 antibodies in the patients' plasma. lower level of shm likely reflect abnormal germinal center (gc) reaction. as rag1 and 2 heterotetramer primarily shapes the pre-immune t and b cell repertoire, we studied the interaction of follicular helper t cells (tfh) and naive b cells via in vitro co-culture experiment. interestingly, tfh cells from rag cid/ai patients exhibited highly activated phenotype with increased expression of cd40l and il-21 compared to healthy controls and were able to initiate exaggerated response (class switching and shm) of healthy donor naive b cells. on the contrary, in vitro activated naive b cells from rag cid/ai patients showed impaired proliferation, class switching and decreased level of shm with diminished induction of genes involved t cell co-stimulation (cd40, il-21r) and shm (aicda, repair enzymes) compared to healthy donor naive b cells indicating intrinsic defect in patient b cells. furthermore, b cells from rag cid/ai patients also showed increased apoptosis and accumulation of gamma-h2ax foci at steady state indicating reduced cellular fitness. these findings suggest that the development of aiha is a multifactorial process in partial rag deficiency. our studies highlight that impaired germinal center reaction is an important tolerance checkpoint with the inability of patient's b cells to respond to hyperactive tfh cells and introduce proper level of shm. hence, we propose that b cell fitness is compromised which impairs proper gc interaction, shm, including mutation away from self and sustains rbc reactivity in hypomorphic rag deficiency. introduction/background: the forkhead box n1 (foxn1) transcription factor is an essential regulator of t cell development, affecting the differentiation and expansion of thymic epithelial cells (tecs). autosomal recessive mutations in foxn1 cause a t-b+nk+ lymphocyte phenotype due to a thymic aplasia in conjunction with alopecia universalis and nail plate dystrophy resulting from keratinocyte dysregulation. this is a classic nude/scid (omim # 600838) phenotype. we report on the identification of two independent patients, identified through newborn screening with absent trecs and with a t-nk+b+ scid phenotype who presented with a t cell lymphopenia who had compound heterozygous mutations in foxn1. notably, these individuals had normal hair and nail beds. objectives: to determine whether distinct compound heterozygous mutations in foxn1 cause a novel t-nk+b+ phenotype in the absence of a classic nude presentation. neutralizing autoantibodies (autoabs) against cytokines increase the susceptibility for selected infections (e.g. anti-ifn-autoabs for nontuberculous mycobacteria and non-typhoid salmonella, anti-il-17-autoabs for mucocutaneous candidiasis and anti-gm-csf-auotabs for infections by cryptococcus, nocardiae and aspergillus spp). however, the role of anti-il-6-autoabs is less clear. il-6 is a key mediator of the acute-phase response and released early in bacterial infections. patients with impaired signaling or affected production of il-6 are at increased risk for severe bacterial infections. only three patients with high-titer and neutralizing anti-il-6-autoabs who suffered from severe infections caused by s. aureus, s. intermedius and e. coli have been described so far. to investigate the prevalence of anti-il-6-autoabs in patients with bacterial infections, we investigated a cohort of 350 patients and identified three further patients, all previously healthy, with neutralizing auotabs against il-6 who hardly developed an acute-phase response. the first patient suffered from life-threatening pneumonia caused by s. pneumonia, the second patient developed a submandibular abscess and septic arthritis caused by s. pyogenes and the third patient suffered from life-threatening pneumonia caused by s. aureus. we also discovered neutralizing anti-il-6-autoabs in two adults among a cohort of patients with autoimmune diseases (n = 564), in one adolescent among a cohort of obese individuals (n = 455) as well as in three mothers of neonates with impaired il-6 signaling. so far none of the later individuals developed a severe bacterial infection. this suggests that naturally occurring and neutralizing anti-il-6-autoabs are a risk factor for severe bacterial infections yet with incomplete penetrance. (215) submission id#606931 persistent transaminitis in copa syndrome 1 researcher, immunodeficiencies research unit, national institute of pediatrics, mexico city 2 social service intern, immunodeficiencies research unit, national institute of pediatrics 3 pediatrics resident, pediatrics hospital, 21st century national medical center, mexican institute of social security 4 researcher, data science department, mexican autonomous institute of technology 5 researcher, department of research methodology, national institute of pediatrics background: inborn errors of immunity constitute a heterogeneous group of over 400 individually rare congenital diseases that involve genes coding for proteins of the immune system, and which result in increased susceptibility to infection, inflammation, autoimmunity, allergy and cancer. the complexity of the diagnostic task, and the intrinsic biases and limitations of the human mind, can be aided by computational tools. among the available machine learning approaches, decision tree algorithms select the best node to split based on entropy and information gain; random forests build hundreds or thousands of decision trees randomly (bootstrapping), to improve accuracy and reduce overfitting. aim: to implement a machine learning-assisted clinical decision support system for the diagnosis of inborn errors of immunity (iei). methods: with a local database of patients with suspected iei, we built a decision tree using c4.5 dtc, and a random forest on python 3 (jupyter notebook, scikit, mathplotlib, pandas, numpy). the database was obtained by conducting an electronic search on medsys of patients with the term immunodeficiency in their electronic medical records, and then hand-picking cases in which an iei had been confirmed or ruled out. it consisted of 234 patients, of which 201 had been diagnosed with iei. we first split the dataset randomly into training (70%) and testing (30%) sets. the decision tree was tasked with classifying correctly pid or not. after running the algorithm in the training set, we evaluated in the testing set. the random forest classified all cases by majority vote into nine groups (0 to 8), according to the iuis pid group. next, we repeated the process on a larger scale with a dataset of 2,400 patients from usidnet. accuracy was assessed by out-of-bag (oob) error estimates. results: accuracy was greater than 95% for the local dataset (pid/ not, 9 groups), and for the usidnet dataset (9 groups). we provide a list of decision nodes and a diagnostic route with those questions that achieved a greater information gain and less entropy. this might help clinicians direct their interrogation and diagnostic approach of suspected iei patients. discussion: we built two classification models. decision trees lend themselves more easily to learning and deriving rules of thumb from their sequences. random forests are more robust and better suited for categoric (as opposed to binary) classification. we next want to develop a chatbot that will ask relevant questions in optimal sequence, and extract undiagnosed patients with suspected iei, based on statistical red flags. 13 researcher, immunodeficiencies research unit, national institute of pediatrics, mexico city dna repair defects are inborn errors of immunity that result in increased apoptosis and oncogenesis. dna ligase 4-deficient patients suffer from a wide range of clinical manifestations since early in life, including: microcephaly, dysmorphic facial features, growth failure, developmental delay, mental retardation; hip dysplasia, and other skeletal malformations; as well as a severe combined immunodeficiency, radiosensitivity and progressive bone marrow failure; or, they may present later in life with hematological neoplasias that respond catastrophically to chemo-and radiotherapy; or, they could be asymptomatic. we describe the clinical, laboratory and genetic features of five mexican patients with lig4 deficiency, together with a review of 36 other patients available in pubmed medline. four out of five of our patients are dead from lymphoma or bone marrow failure, with severe infection and massive bleeding; the fifth patient is asymptomatic despite a persistent cd4+ lymphopenia. most patients reported in the literature are microcephalic females with growth failure, sinopulmonary infections, hypogammaglobulinemia, very low b-cells, and radiosensitivity; while bone marrow failure and malignancy may develop at a later age. dysmorphic facial features, congenital hip dysplasia, chronic liver disease, gradual pancytopenia, lymphoma or leukemia, thrombocytopenia and gastrointestinal bleeding have been reported as well. most mutations are compound heterozygous, and all of them are hypomorphic, with two common truncating mutations accounting for the majority of patients. stem-cell transplantation after reduced intensity conditioning regimes may be curative. 1 department of laboratory medicine, clinical centre 2 immunology, allergy and rheumatology division, department of pediatrics, baylor college of medicine, texas children's hospital, houston,texas, usa 3 laboratory of clinical immunology and microbiology, fungal pathogenesis section, national institute of allergy and infectious diseases, 4 department of intramural research, national institute of allergy and infectious diseases (niaid), national institute of health, bethesda maryland, usa card9 deficiency is an autosomal recessive primary immunodeficiency known to underlay increased fungal infection susceptibility mostly presenting as invasive cns candida infections (in infancy or adulthood) and dermatophyte infections. more recently, a rare card9 variant (c.1434+1 g>c, leading to exon 11 skipping, card9del11) showed a significant protective association towards inflammatory bowel disease (ibd) when present in heterozygosity. at the nih we studied an 8-year-old male patient (p1) born to a non-consanguineous marriage who presented as an infant with recurrent/severe thrush, candida esophagitis, and an episode of tinea pedis; p1 also has mild hypogammaglobinemia (igg 500mg/dl at age 8y). p1s gdna was tested by whole exome sequencing and showed a card9 c.1434+1 g>c mutation in homozygous state. segregation analysis and sanger confirmation determined that both parents and p1s elder brother carried the same variant in heterozygosity, while his asymptomatic younger brother (p2) was also homozygous. as previously described, this variant caused card9 exon 11 deletion as determined in p1 and p2s pbmcs by cdna sequencing and by a lower molecular weight card9 protein by immunoblot evaluation. p1 and p2s pbmcs, as well as the heterozygous parents cells, showed a defective cytokine generation (tnf-, il-1, il-6 and gm-csf) in response to heat killed candida (hkc), but not to lps. while patients pbmcs failed to induce phospho-erk and phospho-p-38 upon hkc-stimulation but presented an intact response to pma+ionomycin; the parents cells responded normally to both stimuli. moreover, t-cell activation and proliferation was affected in response to hkc but not to pha in both patients, whereas the parents exhibited normal results under the same conditions. when hek293 cells were transiently transfected with wt or card9del11 vectors together with a trim62 plasmid (e3-ubiquitin ligase, naturally associated to card9), we confirmed that card9del11 failed to bind trim62 by immunoprecipitation. furthermore, malt1, bcl10 and trim62 were only co-precipitated by wt card9, but no by card9del11, strongly suggesting trim62 is an integral part of the card9/bcl10/malt1 -cbm-complex. in summary, herein we demonstrate that the card9del11 allele fails to bind trim62, and in turn is unable to conform a complete/functional cbm complex. our data also show that card9del11 acts in a dominant negative fashion in terms of cytokine generation (previously reported), but one wt allele seems sufficient to generate normal levels of hkcinduced p-erk and p-p-38, as well as t-cell proliferation. while decreased cytokine generation associated with card9del11 in heterozygosity has been described to be sufficient to protect towards ibd, other defective pathways are affected in homozygosity and likely necessary to confer increased susceptibility to fungal infections. altogether these results suggest that card9del11 acts through a gene dosage mechanism that can dissect pathways that associate ibd protection and fungal infection susceptibility. further work is warranted to explore card9del11 role, if any, in b-cell and t-cell biology. professor, endocrinology, university of michigan medical school background: acquired generalized lipodystrophy (agl) syndromes are a heterogeneous group of diseases characterized by selective dysfunction and loss of adipose tissue after birth. this causes ectopic lipid deposition and deficiency of the adipokine leptin, which promotes metabolic dysfunction through impaired glucose handling resulting in insulin-resistant diabetes mellitus, dyslipidemia and steatohepatitis. while the metabolic effects of altered adipokine secretion are known, the molecular mechanism is less clear. many agl cases are suspected to have an autoimmune etiology. effector and regulatory t cells, dendritic cells and macrophages reside in normal adipose tissue. t cells within adipose tissue highly express pd-1 and regulatory t cells express ctla4, which limits immune activation in the adipose tissue under normal circumstances. thus, inhibition of these immune checkpoints may hypothetically cause immune activation, leading to adipocyte dysfunction and autoimmune destruction. we have encountered two cases that raise clinical concern for this process. patient cases: patient 1 is a 16-year-old female who presented with failure to thrive at 6 months. she was diagnosed with insulin-resistant type 1 diabetes and hypertriglyceridemia at ages 2 and 4 years with progressive subcutaneous fat loss and low leptin levels culminating in a diagnosis of agl. her childhood clinical course was complicated by hypertrophic cardiomyopathy, hepatomegaly, autoimmune hemolytic anemia with massive splenomegaly and severe chronic diarrhea secondary to autoimmune enteropathy. she presented at 14 years with acute liver failure, thrombotic microangiopathy, nephrotic syndrome and progressive kidney insufficiency. evaluation for her multi-faceted autoimmune presentation identified a familial heterozygous pathogenic variant in the ctla4 gene (c.4_5insgttgg,p.ala2glyfster14). despite aggressive immune therapies, including ctla4-ig (abatacept), her kidney disease and enteropathy have progressed. patient 2 is a 55-year-old male diagnosed with localized malignant melanoma of the right neck in july 2014. he underwent excisional biopsy and regional lymph node dissection with negative margins. he relapsed in november 2017 and underwent a modified radical neck dissection with 1 lymph node positive for disease and received external beam radiation from january-february 2017. additionally, he was started on anti-pd-1 therapy with the humanized antibody drug pembrolizumab in april 2017 but discontinued the drug in february 2018 in the setting of toxicities including hypothyroidism. subsequently, he developed up to 7.5% weight loss with progressive loss of subcutaneous fat first in his face, then generalized to the rest of his body. in the ensuing months, imaging with pet-ct demonstrated loss of subcutaneous fat concurrent with elevations in alt and triglyceride levels plus a low leptin level consistent with agl. conclusion: these cases raise concern that inhibition of the immune checkpoints ctla4 and pd-1 may facilitate the development of agl. we hypothesize that these defects significantly increase t cell autoimmune activity in the adipose tissue and/or alter t cell metabolism resulting in agl. disorders of immune dysregulation should be considered in the etiology of agl. similarly, patients with either genetic or pharmacologic inhibition of immune checkpoints should be monitored for the development of agl with careful physical exam and periodic monitoring of glucose and triglyceride levels. background: rosai-dorfman disease (rdd; also known as sinus histiocytosis with massive lymphadenopathy) is a rare non-langerhans cell histiocytosis. it is characterized by proliferation and accumulation of activated histiocytes in affected tissues. classically, rdd presents with bilateral, non-tender, and often markedly enlarged cervical lymphadenopathy. case presentation: a 2-year-old female presented with a 6-week history of asymptomatic, persistent and bilaterally enlarged cervical lymph nodes. she was otherwise healthy with no significant past medical history. operative excision biopsy of the largest lymph node confirmed the diagnosis of rdd. three months following diagnosis, routine bloodwork revealed that she had developed lymphopenia (lymphocyte count 1.4 x 109/l). between 1-year and 2-and-a-half-years post-diagnosis, the patient was hospitalized and treated with intravenous antibiotics for 2 presumed episodes of osteomyelitis and 2 presumed episodes of lymphadenitis. given the recurrent presumed infections and persistent lymphopenia, the patient was referred to immunology for evaluation. she received a full immunologic work-up. lymphocyte immunophenotyping revealed low cd4 (288 cells/mm3) and low cd8 (228 cells/mm3) counts. the rest of her immunologic work-up was within normal limits. approximately 3-and-a-half-years post-diagnosis, the decision was made to initiate treatment for rdd. she was started on a 6-week tapering course of prednisone therapy. within 2-weeks of starting corticosteroid therapy, the lymphadenopathy had diminished, and by 6-weeks, the lymphopenia completely resolved. at her most recent clinic visit, she had been free of serious infections for more than 3-years, and her lymphocyte counts had remained stable and within normal limits for over one year. discussion: in the literature, immune system dysfunction has been reported in rdd, with both auto-antibodies and cellular immunodeficiency implicated. in this patient, the persistent lymphopenia and recurrent episodes of presumed infections appeared consistent with an immunodeficiency. given the known association of rdd with immunologic dysfunction, this was certainly a reasonable assumption; however, when these issues resolved following corticosteroid therapy, we questioned whether her clinical presentation could instead represent a manifestation of her underlying rdd. this case highlights the diagnostic challenge of differentiating between an infection and an rdd exacerbation. the episodes of presumed infections were considered probable but not confirmed with microbiologic or histopathologic specimens. the mechanism underlying lymphopenia in rdd is not clear but may involve decreased production, increased destruction, or sequestration of lymphocytes. to our knowledge, this has not been specifically studied in rdd in the past, however lymphopenia has been linked to lymphocyte maldistribution in other diseases. for example, studies have shown that experimentally altering either the surface of the lymphocyte or the environment through which the lymphocyte travels through can cause sequestration of lymphocytes in various lymphoid organs including lymph nodes. conclusion: we describe the case patient with rdd that developed persistent lymphopenia, and multiple episodes of presumed infections resulting in hospitalization and intravenous antibiotic therapy. her lymphopenia resolved and she had sustained remission of rdd following treatment with corticosteroids. we hypothesize that lymphocyte sequestration in enlarged lymph nodes may have resulted in lymphopenia. this, combined with recurrent rdd exacerbations that clinically resemble infections created a presentation that mimicked an immunodeficiency. background: there is an expanding spectrum of immunodeficiency phenotypes linked to dna repair defects, and some patients may not be diagnosed until adulthood. the most well recognized genetic defect linked to dna repair is in the gene, ataxia telangiectasia mutated (atm), which causes ataxia telangiectasia, characterized by combined immunodeficiency, neurodegeneration, radiation sensitivity, and ocular telangiectasias. however, there are several other dna repair defects associated with immunodeficiency, including some syndromic and severe combined immunodeficiency (scid) disorders. objective: we present the case of an adult patient with prolonged history of recurrent infections, facial abnormalities, and autoimmunity who was found to have radiosensitivity suggestive of a dna repair defect. methods: retrospective chart review, immunodeficiency evaluation, flow-based radiosensitivity assay, gene sequencing. results: a 68-year-old female was referred to our clinic due to a complex history of recurrent infections and immune dysregulation. the patient had a lifelong history of sinopulmonary infections and panhypogammaglobulinemia with low vaccine responses, leading to a diagnosis of common variable immunodeficiency (cvid), necessitating treatment with immunoglobulin replacement. clinical features were also notable for congenital dysmorphia (strabismus, thin and angular face, high arched palate, nasal septal defect, small mouth, missing dentition, clinodactyly, severe equinovarus, and scoliosis). she was subsequently diagnosed with autoimmune features of vasculitides requiring trial of cyclophosphamide, azathioprine, rituximab and belimumab, which was later discontinued due to neutropenia and worsening sinopulmonary and skin infections despite immunoglobulin replacement. in the course of our evaluation she was revealed to have severe b cell lymphopenia (1%), cd4 naã¯ve t cell lymphopenia, persistent iga and igm deficiency one-year post rituximab therapy, and elevated alpha fetoprotein (afp). radiosensitivity assay revealed decreased atm phosphorylation and elevated levels of h2ax 24-hours after low-dose (2gy) radiation in her lymphocyte subsets (t, b and nk cells) . due to the evidence of radiosensitivity and elevated afp levels, there was concern for an atm or other genetic defects in a dna repair pathway. therefore, a targeted (primary immunodeficiency genes) panel was pursued for genetic testing (207 genes, invitae, san francisco). the evaluation did not identify a variant in the atm gene but rather a variant of uncertain significance was identified in the chd7 gene, in exon 38, c.8440g>a (p.gly2814arg), which may be mosaic. this variant has not been reported in population databases. chd7 is typically associated with charge syndrome, and while this patient has some dysmorphic features, she is not typical for charge syndrome. currently, studies on copy number variation (cnv) and deep intronic variants in atm are pending. conclusion: dna repair defects may occur in adult patients with a primary diagnosis of cvid. our patient exhibits some phenotypic features of both a chd7 variant, and atm leading to possible abnormal dna damage responses (ddr). the exact cause of the immune deficiency in our case remains presently unsolved. this case highlights the relevance of both functional studies and genetic evaluation of complex cases of immune dysregulation, for improving our understanding of the phenotypic variability in these immunological disorders. background: womens health issues in patients with immunodeficiency are largely underrepresented in the literature. there are no studies assessing for fertility issues in patients with antibody deficiencies, and there are few sizable studies examining pregnancy and outcomes on progeny in the same cohort. the two largest studies of pregnancy in antibody deficiency, an idf survey and a study of the czech population, provide conflicting data about the safety of pregnancy for these patients. immunoglobulin replacement has been shown to be safe and beneficial in pregnancy for patients with cvid, however, dosing strategies are unguided. we sought to further understand these and other issues associated with fertility and pregnancy in a large cohort of patients with antibody deficiencies. methods: we performed a retrospective chart review of over 100 patients with icd9 and/or icd10 codes of cvid or another antibody deficiency from january 2005 to december 2018. inclusion criteria also comprised of having reached at least 16 years of age, the beginning of child bearing years. data collected included disease characteristics, comorbidities, laboratory values, and outcomes. this was followed by a phone survey to elucidate data regarding fertility, pregnancy, delivery complications, and outcomes of children. this study was irb approved. results: the current age of women included ranged from 16 to 88 years of age, currently being in childbearing years to being post-menopausal. forty percent of the women had been pregnant, delivering an average of 2 babies per woman who had been pregnant. fertility issues were not a prominent factor for women who never became pregnant. a majority of women who had babies (64%) did not receive a diagnosis of antibody deficiency until after their child bearing years. recurrent upper respiratory tract infections, bacterial sinusitis, and urinary tract infections during pregnancy were common even in those not yet diagnosed with antibody deficiency. immunoglobulin levels and dosing of intravenous and/or subcutaneous replacement were recorded for a subset of patients with recent pregnancies. the data re-enforced that increases in dosing are needed in the third trimester. cord blood igg levels were also recorded for baby and were the same or higher than the mothers most recent igg prior to delivery. it was rare for children of our patients to be diagnosed with antibody deficiency or a related condition, although cvid, hypogammaglobulinemia, combined immunodeficiency, lymphoma, rheumatoid arthritis, and other diagnoses were found. conclusion: this is the largest report of outcomes before, during, and after pregnancy for patients with antibody deficiencies in the united states. this report highlights the importance of closely monitoring women during pregnancy for recurrent infections regardless of whether a diagnosis of antibody deficiency is present. it also highlights that close monitoring of igg levels during pregnancy is necessary for women with antibody deficiencies. backgrounds: autoinflammatory diseases (aids) are a group of disorders with an inborn error of innate immunity, characterized by recurrent episodes of fever and inflammatory attacks. the spectrum of aids is expanding, but no data on clinical presentation and symptom variability exist for the iranian population for timely precise diagnosis. this study aims at establishing the first autoinflammatory registry of an iranian population focusing on the clinical and laboratory features that may help clinicians toward a better understanding and diagnosis of these disorders. methods: clinical and laboratory characteristics of patients who clinically and or genetically diagnosed with aids collected. we used the updated version of classification criteria from the eurofever registry for the clinical diagnosis. results: in our retrospective study, clinical and laboratory characteristics of the participants collected. mean age of disease onset, disease course manifestation, the mean duration of episodes, atypical symptoms, laboratory and imaging studies as well as complications, and response to treatment also reviewed. data resulted in 26 patients of whom 16 were male. their age ranged from 2 to 68 years. 5 out of 26 were genetically diagnosed. familial mediterranean fever (fmf) was the most common clinically and genetically approved diagnosis. there were also patients suspected of nlrp12 and nod2 mutations. age at disease onset differed variably and ranged from the neonatal period to adulthood. fever was present in all the participants and the duration of episodes was 1-10 days. the frequency of attacks was between 3 to more than 12 per year. some of the common clinical manifestations were as follows: myalgia or fatigue (77%), arthralgia and arthritis (70%), abdominal pain (65%), aphthous stomatitis (38%), chest pain (34%), chronic gastrointestinal symptoms (38%), skin lesion ranging from urticarial rash and severe nodular acne to pyoderma gangrenosum (50%), exudative and or erythematous pharyngitis (46%), consanguineous parents (42%), symptoms of a type of allergy (84%), lymphadenopathy (27%), splenomegaly (27%), increased acute phase reactant (54%), elevated liver function test (19%) . 10 out of 26 of the individuals reported positive family history and in one of the cases, a patient carrying the homozygous mutation in the mefv gene has shown no clinical manifestation. conclusion: this study highlights the most common manifestations of aids in the population of iranian origin and can be used as evidencebased clinical criteria for their diagnosis. background: the term benign ethnic neutropenia (ben) is used to describe patients of african/arabic descent with absolute neutrophil counts (ancs) less than 1500 cells/ul in the absence of other causes. historically, race has been used to support the diagnosis of ben, but self-reported race is notoriously imprecise. the duffy null phenotype (fya -/fyb-) is a known molecular cause of ben and may be a more reliable marker of ben than self-reported race. in addition, although the anc is known to be lower in patients with ben, the lower limit of ancs is poorly described. it is important to differentiate patients with ben from primary immunodeficiency diseases (pidd) and to recognize their expected anc values. methods: eligible subjects included patients less than 21 years seen at the university of michigan between january 2010-july 2018. duffy null (fya -/fyb-) patients were identified using electronic medical record search engine (emerse) software and search terms duffy and fyab. 105 potential subjects were identified; 67 patients met inclusion criteria including duffy null status and the absence of other conditions or medications, potentially impacting ancs. 251 unique healthy anc values were recorded from the 67 duffy null patients. age and sex matched controls were identified using emerse software with search terms tonsillectomy, department of anesthesiology and absolute neutrophil count. subjects with conditions or medications that might impact the anc or of african/arabic descent were excluded from the control group. asian and caucasian patients included as controls were presumed to be duffy null given that <1% of these populations are expected to be duffy null. 363 control subjects were identified; 134 met inclusion and exclusion criteria. statistical analysis was performed using two-sided two-sample t-test, anova and onesample t-test. results: the median age of the duffy null cases was 4.78 years (iqr: 1.68-11.48) with 61.2% (n=41) male and all of african or arabic descent. mean anc for duffy null patients was 1190 cells/ul (n=251, sd= 650) while mean anc for controls was 4300 cells/ul (n=134; sd=1600) with a mean difference between controls and duffy null cases of 3100 cells/ul (95% ci: 2950-3380; p=0.0001). the anc levels between duffy null individuals and controls were evaluated by five age categories (p=0.0001 for all age categories). however, there was no difference in anc levels between duffy null cases at different age categories (anova, p=0.14196). 54 (21.5%) duffy null cbcs had anc levels in the nonneutropenic range (>1500 cells/ul), 99 (39.4%) cbcs had mild neutropenia (1001-1500 cells/ul), 70 (27.9%) cbcs had moderate neutropenia (500-999 cells/ul), and 28 (11.2%) cbcs had severe neutropenia (<500 cells/ul). conclusions: although neutropenia can be associated with pidds and is often a sign of a compromised immune system, duffy null patients have a wide range of values that are often much lower than previously appreciated. the degree of neutropenia related to duffy null phenotype appears to persists throughout childhood and young adulthood. in the context of patients of african/arabic descent presenting with asymptomatic neutropenia, duffy null status should be assessed, and ben should be considered in the differential. complications, hypogammaglobulinemia and a unique characteristic of decreased susceptibility to enveloped viral infections. objective: to investigate the role of impaired host n-linked glycosylation on viral susceptibility to ebola virus. methods: to mimic the condition observed on cdg-iib patients, we tested in vitro three proprietary iminosugars (emergentbiosolutionsâ©), uv4b, uv001, and uv00128, which act as competitive inhibitors of -glucosidase i and ii. their ability to inhibit the trimming of n-glycans was compared to known n-glycans modifiers as castanospermine, tunicamycin, as well as the bacterial enzyme peptide-nglycosidase f (pngase-f). ebola virus envelope protein gp1 was chosen as a prototype glycoprotein, as it is heavily glycosylated with 15 nglycosylation sites. hek 293t cells were seeded at 1x10^5 cells/well in 12 well plate. after 18h, cells were transfected with pflag-ebolavirus gp1 by coupling with effecteneâ®. after 24h, cells were treated with the inhibitors and harvested 24h after treatment. trimming of n-glycans was evaluated via molecular weight assessment by western-blot. results: all three inhibitors had comparable effectiveness in inhibiting trimming of nglycans from ebola gp1 glycoprotein compared to castanospermine. a greater molecular weight shift was seen with tunicamycin and pngase f as expected. conclusions: chemical inhibition of the n-linked glycosylation pathway was successfully achieved using three new mogs inhibitors. this approach merits further investigation on potential applications on antiviral therapies. investigator, laboratory of human genetics of infectious diseases, necker branch, inserm u1163, necker enfants malades hospital, paris, france 5 head, immunodeficiencies research unit, national institute of pediatrics stat1 gof mutations are associated with infections, autoimmunity and inflammatory manifestations; the rosacea is one of the manifestations described in this disease, however, the etiology rosacea is not clearly established. the characteristics of rosacea are not described in stat gof in the different clinical series. we describe the different characteristics of rosacea in a family with 8 affected members with stat1 gof. a family with eight members with stat1 gof mutation were diagnosed through a first affected member affected with tuberculosis and onychomycosis. seven members more had a clinical history of mycobacterial, viral and fungus infections and autoimmunity disease, in all the seven, was documented the same mutation stat1gof. in six of these adults patients, we documented rosacea, it started after adolescence, it was localized in the face and/or eyes, was progressive and not ameliorated with medical treatment and caused nose deformity. rosacea has been described previously as a unique manifestation, and the etiology is not clear, an autoimmune hypothesis has been proposed. the fact that is present in patients with stat1 gof could suggest that have effectively an autoimmune component. physicians face the patients with rosacea must look for other manifestation presents in stat1 gof mutations. genetic studies in rosacea patients could evidence an new gene defect. introduction: homozygous mutations causing loss of function of the transcription factor forkhead-box n1 (foxn1) underlie autosomal recessive severe combined immunodeficiency with congenital alopecia and nail dystrophy (nude scid). affected humans, like the scid mouse, have small or absent thymus, absent or severely diminished t cells, alopecia, and nail dystrophy. infants with nude scid have had neonatal lymphopenia and severe, life-threatening infections. studies of heterozygous carriers of foxn1 mutations are limited, some having been reported with no phenotype or mild disease manifestations, such as nail dystrophy without lymphopenia or recurrent infections. objective: we describe six infants, including two brothers, with t-cell lymphopenia (tcl) following abnormal california newborn screens (nbs) for scid. each had a single heterozygous variant in foxn1. case reports: six infants (3 female, 3 male) were referred for evaluation after abnormal california nbs for scid (table 1) , with t-cell receptor excision circle (trec) counts from undetectable to 12 (normal >18). all infants were well at the time of initial evaluation. five infants with absolute cd3 t cell counts >400 cells/ul and cd4 t cell counts >250 cells/ul began evaluation as outpatients on home isolation. patient 5, with undetectable trecs, cd3 t cell count 78, and cd4 t cell count 65 was urgently admitted for inpatient evaluation and management and immediately started on antimicrobial prophylaxis. patient 5 further evaluation was significant for lymphocyte proliferation to mitogens that was initially normal but waned with time, prompting treatment with a paternal haploidentical hematopoietic cell transplant at 6 months of age. patients 3 and 5 developed neutropenia within 6 weeks of birth treated with granulocyte colony stimulating factor (gcsf). patient 3 remains well on gcsf but has had persistent growth failure under continued evaluation. patients 1, 2, 4 and 6 remain stable off antimicrobial prophylaxis, but with persistent moderate tcl. as part of an immune evaluation, patients 1 and 3-6 had gene panel testing revealing heterozygous variants in foxn1. only the variant of patient 1 (presumed shared by patient 2, his brother) was predicted to be pathogenic; patient 1 had dystrophic nails and sparse hair most evident after 2 years of age, features shared by his mother and his brother, patient 2. the other patients lack the clinical features of the previously described phenotype of nude scid. their heterozygous foxn1 variants are of unknown significance; the functional role of these variants in the patients clinical phenotype is unknown. conclusion: six infants with abnormal nbs for scid had lymphopenia and heterozygous variants in foxn1. for these infants, variation exists in level of tcl and presence of hair and nail findings. heterozygous variants of unknown significance in foxn1 have been uncovered in others, including infants with abnormal nbs for scid, highlighting the need for functional studies to address the possible role of each heterozygous foxn1 variant in congenital lymphopenia and neutropenia. more work is needed before attributing tcl to a novel foxn1 variant of unknown significance in the absence of family history, abnormal hair or nails, or functional evidence. remains poorly understood. we characterized the intestinal microbiome and metabolome in patients with cgd to determine if intestinal microbiome and metabolomic signatures could distinguish subpopulations of patients with cgd while using the metabolome to add a functional dimension to observed microbiome signatures. methods: clinical metadata and fecal samples were collected crosssectionally from healthy volunteers (hv; n=16) and patients with cgd (n=77). metabolomic profiling and 16s rrna (v4) sequencing was performed on fecal samples (total samples: 108; reads/sample: 15,254 to 191,415; median: 60,816) . results: samples from patients with cgd had distinct intestinal microbiome signatures and metabolomic profiles depending on genotype, presence of cgd-ibd and specific interventions (e.g. treatment with an elemental diet). notably, samples from patients with active cgd-ibd (compared to samples from patients without a history of cgd-ibd) had significantly different alpha-and betadiversities, and were enriched for enterococcus spp. signal transducer and activator of transcription 1 gain of function (stat1-gof) is a primary immunodeysregulatory disease in which a subset of patients have features of autoimmunity and autoinflammation. enteropathy with growth failure and nutrient wasting is a more common feature of immunodysregulation. ruxolitinib is a janus kinase-stat inhibitor that has been shown effective for the treatment of immunodysregulatory features in stat1-gof. our patient is a 13 year old male with stat1-gof (c.983a>g p.h328r) with severe total parenteral dependent enteropathy that led to growth failure (weight 28.5kg). treatment with ruxolitinib led to resolution of diarrhea, return of normal diet, and catch up growth. a dose of 12.5mg twice daily was initially started but was decreased to 12.5mg every morning and 10 mg every evening due to elevated transaminases and thrombocytopenia. over the following year the patient thrived gaining 7.5kg with normal every other day stools. despite weight gain, he remained stable on the same dose of ruxolitinib. as he outgrew his dose, he developed an increased frequency of upper respiratory infections (parainfluenza, coronavirus, rhinovirus). one year after initiation of ruxolitinib, he again developed profuse watery diarrhea that was norovirus positive (weight 36kg, bsa 0.9). he was placed on bowel rest and ruxolitinib was dose escalated with a goal of 15mg/m2/day. when he reached 15mg twice daily, enteropathy completely resolved but liver function tests began to rise. he gained weight and began thriving after 2 weeks of therapy. six months later, enteropathy is controlled, and transaminases have remained elevated (alt 88 iu/l, ast 73 iu/ml) but stable. the appropriate dose and pharmacokinetics for ruxolitinib for the treatment of immunodysregulatory symptoms in pediatric patients has not been thoroughly studied. the dose used was extrapolated from data on the use of ruxolitinib in pediatric myelofibrosis. a dose of 15mg/m2/day appears to provide the most benefit with tolerable adverse effects. this dose should be maintained in order to prevent recurrence of disease related manifestations. abstract clathrin-mediated endocytosis (cme) is the major endocytic pathway by which eukaryotic cells internalize cell-surface cargo proteins and extracellular molecules, thereby allowing for a broad range of biological processes, including cell signaling, nutrient and growth factor uptake, and cell fate and differentiation1. the fbar domain only proteins 1 and 2 (fcho1/fcho2) are involved in the initiation of clathrin coat pit formation. whether fcho1 and fcho2 are functionally redundant or have distinct functions is unclear. we report here the first cases of a severe immunodeficiency due to a genetic defect affecting cme. by using whole exome sequencing and genomic analysis of a targeted pid gene panel, we have identified biallelic loss-of-function fcho1 mutations in five patients from unrelated families of italian (p1), turkish (p2, p3, and p5) and algerian (p4) origin with severe t cell lymphopenia manifesting as recurrent and severe infections of bacterial, mycobacterial, viral and fungal origin. p3 developed ebv-associated diffuse large b cell lymphoma. three patients (p3-p5) died in childhood, whereas p1 and p2 are alive with full donor chimerism at 13 and 1.5 years after allogeneic hematopoietic stem cell transplantation, respectively and have cleared pre-transplant infections. patients p2, p3, and p4 carried homozygous frameshift mutations predicted to cause premature termination. western-blotting analysis of ha-or flag-tagged fcho1 constructs showed expression of truncated products in p2 and p3, whereas no protein was detected in p4, presumably due to mrna decay. p1 and p5 carried homozygous splice-site mutations at the invariant -1 and +1 positions, respectively, leading to skipping of exon 6 in p1's fcho1 cdna. qpcr analysis demonstrated differential expression of the fcho1 and fcho2 genes, with the former being predominantly expressed in lymphoid cells, whereas fcho2 was more abundantly expressed in fibroblasts and k562 cells. analysis of t cell activation in p2 (the only patient for whom pre-transplant pbmc were available) revealed reduced t cell proliferation. while tcr internalization in response to cd3 cross-linking was normal (consistent with recent evidence that tcr internalization occurs through a clathrin-independent pathway), chase experiments demonstrated that transferrin internalization was abolished in activated t cells from p2. we had previously reported that a missense mutation in tfrc, encoding transferrin receptor 1, impairs transferrin internalization and intracellular iron delivery, causing a combined immunodeficiency with defective t cell proliferation. our data identify the first form of severe immunodeficiency due to defects of clathrin-mediated endocytosis, and provide additional evidence in support of the critical role played by iron cellular metabolism in t cell function and homeostasis. natural history of anti-interferon-gamma autoantibody-associated immunodeficiency syndrome in thailand submission id#601826 centralized sequencing initiative at niaid: year 1 therefore, we set out to investigate the pneumococcal-specific responses of igg, igg2, iga and igm to prevnar13â® in igg subclass deficient (iggscd) patients in this study. pneumococcal responses were measured using the vacczyme pneumococcal capsular polysaccharide igg, igg2, iga and igm elisas (the binding site group, birmingham, uk) in control (n=10, median age 57 years, range 27-64) and iggscd patients (n=10, median age 55 years, range 25-65) recruited from the immunodeficiency unit at the karolinska university hospital iga and igm antibodies in response to pcv13 vaccination was observed 4 weeks post vaccination in iggscd patients (median, 2.5th and 97.5th percentile these median concentrations were lower than those observed in control patients (median, 2.5th and 97 pcv13 igg2 71 mg/l, 14-90 however, percentage changes between pre to post vaccination concentrations of igg, igg2 and iga in response to pcv13 in iggscd patients were not significantly different to the control patients u/ml vs 17.1 u/ml, respectively) iga 26 u/ml and pcv13 igm 39 u/ml) responders and non-responders of pcv13 igg iga and igm in response to pcv13 in iggscd patients were generally lower compared to the control population. these results support the fact that in addition to igg and igg2, measurement of iga and igm could also provide useful information for the clinician gain-of-function ikbkb mutation causes human combined immune deficiency submission id#606903 neutralizing anti-il-6-autoantibodies are a risk factor for pyogenic bacterial infections national institutes of health, national institutes of allergy and infectious diseases service of immunology and rheumatology, garrahan national pediatric hospital copa mutations impair er-golgi transport and cause hereditary autoimmune-mediated lung disease and arthritis copa syndrome: a novel autosomal dominant immune dysregulatory disease analysis of pulmonary features and treatment approaches in the copa syndrome expanding the phenotype of copa syndrome: a kindred with typical and atypical features the forest and the trees: machine learning to classify cases of suspected inborn errors of immunity using decision tree and random forest algorithms submission id#607035 card9î�11 gene dosage: from mono-allelic protection to ibd, to bi-allelic increased fungal infection susceptibility yamanaka d 3 , walkiewicz m 4 , lionakis m 3 and rosenzweig s 1 stim1 mutation associated with a syndrome of immunodeficiency and autoimmunity a novel hypomorphic mutation in stim1 results in a late-onset immunodeficiency clinical, histological and genetic characterisation of patients with tubular aggregate myopathy caused by mutations in stim1 gain-of-function mutation in stim1 (p.r304w) is associated with stormorken syndrome gain-of-function mutations in stim1 and orai1 causing tubular aggregate myopathy and stormorken syndrome stormorken syndrome caused by a p.r304w stim1 mutation: the first italian patient and a review of the literature by studying ecs-pre and ecs-post patients we were able to describe the bona-fide effect of gcs on the immune system in general, and t lymphocytes in particular. decreased lymphocyte/thymic output, as well as increased apoptotic tcell death underlies lymphopenia in ecs/chronic gcs-exposed patients. under such conditions, il-21 was significantly decreased in plasma and our in-vitro studies showed that il-21 replenishment was able to increase bcl2 (anti-apoptotic molecule) and bcl6 expression, and efficiently counteract the apoptotic effects of gcs. recombinant il-21 has been explored as a co-adjuvant treatment for multiple human cancers and may offer a treatment option for lymphopenia and its genetic counselor, co-director of personalized medicine, division of hematology/oncology/bmt and the institute for genomic medicine, nationwide childrens hospital 2 genetic counselor, division of hematology/oncology/bmt, nationwide children's hospital acknowledgments. genetic sequencing was kindly provided by drs. raif geha and janet chou at the division of immunology, allergy, rheumatology and dermatology, boston children's hospital, harvard medical school. the following grants are acknowledged: 1. rui 1.1001/cippt/812036 (usm) 2. bmbf 01 eo003 (freiburg) the authors would like to thank the director general of health of malaysia for permission to publish this scientific presentation. while severe viral infections may also be an initial presentation of primary immunodeficiency, an immune evaluation is not always obtained in this scenario. patients with xla have an increased susceptibility to severe enterovirus infections, manifesting as chronic meningoencephalitis, which can be fatal. the following case describes a patient with newly diagnosed xla presenting as suspected coxsackievirus and confirmed hhv-6 meningitis, pseudomonas meningitis and bacteremia. this may be the first reported new diagnosis of xla presenting with both severe bacterial and viral coinfection. case description: a 2 year old, partially vaccinated, hispanic male with a history of febrile seizures presented to the emergency room with fever, oliguria, watery diarrhea, lethargy, meningismus, ecthyma gangrenosum and lower abdominal pain. eight days prior to presentation, he was seen by his pediatrician for facial rash and low grade temperature, and was diagnosed with hand-foot-and mouth disease. he worsened on empiric antibiotics. he had no history of sinopulmonary infections. he did not attend daycare. his vaccines were delayed due to parental choice, and he had not received live vaccines (rotavirus, mmr or vzv). full sepsis evaluation was performed. csf demonstrated pleocytosis, and he was started on empiric antibiotics and transferred to picu. due to worsening abdominal pain, ct of the abdomen was performed, which was consistent with ruptured appendicitis and septic emboli at the lung bases. csf pcr panel was positive for hhv-6 and he was started on gancyclovir. csf and blood cultures subsequently grew pseudomonas aeruginosa. immune evaluation was performed. serum immunoglobulins were undetectable. in addition to iv antibiotics, he received 500 mg/kg ivig and lymphocyte subsets revealed profound b cell lymphopenia (0.23 %, 5 cells/ul). btk protein analysis revealed hemizygous btk pathogenic variant confirming the diagnosis of x-linked agammaglobulinemia. the hospital course was further complicated by brain abscesses and pyoventriculitis. he was treated with 3 additional doses of 500 mg/kg ivig and iv antibiotics. repeat mri of the brain nearly 4 weeks after admission demonstrated significant improvement. there was significant clinical recovery. he was discharged home at baseline neurological status. his igg level upon discharge home was 605 mg/dl with the plan to increase dose to 600 mg/kg per month with close monitoring. conclusion: both severe opportunistic bacterial infections and severe viral infections as the initial presentation of xla have been well reported in the literature. this case describes the first reported severe pseudomonas aeruginosa and hhv-6 co-infection in a newly diagnosed xla patient. this case further highlights the necessity for an increased index of suspicion of primary immunodeficiency in a patient who presents with a severe first infection, despite lack of recurrent infections. we present two patients with dock8 deficiency due to compound heterozygous variants including a copy number loss at chromosome band 9p24.3 spanning approximately .107 mb with partial deletion of the dock8 gene and a novel c.2603c>t (p.ser868leu) missense variant [chr9:379933 (grch37) nm_203447] in dock8. functional data is presented to support the pathogenicity of the missense change, along with a review of the literature on dock8 variants. the proband is a 14-year-old female with elevated serum ige, severe atopic dermatitis, mild persistent asthma, food allergies, and seasonal allergic rhinitis. she is currently healthy following haploidentical bone marrow transplant in june 2018. she has a 17-year-old brother with dock8 deficiency with the same compound heterozygous variants. the brother had later onset of symptoms and a milder presentation of intermittent asthma and seasonal allergic rhinitis. each of the parents is heterozygous for one of the two variants. we evaluated the pathogenicity of the c.2603c>t missense variant with western blots of dock8 protein expression, intracellular flow cytometry, and dock8 stretch assays. flow cytometry showed decreased dock8 protein expression and stretch assays revealed t cells that were stretched in collagen gels. notably, dock8 is a large gene containing 47 exons spanning 190 kb and it is relatively common to be a carrier of a rare missense change. in fact, gnomad has approximately 1500 individuals with rare (<0.002 frequency) missense alleles in dock8. therefore, it is important to demonstrate the potential pathogenicity of any given rare missense change, since few pathogenic missense variants in dock8 have been reported. of the 168 published dock8 variants listed in the human gene mutation database (hgmd) only 13 are missense. the majority are gross deletions, 97 of which were reported in hgmd. the remaining reported dock8 variants include 19 nonsense, 15 splicing, 13 small deletions (all frameshifting), 3 small insertions (all frameshifting), 2 small indels, and 5 gross insertions/duplications. this case demonstrates the relatively infrequent but important contribution of missense changes to pathogenic dock8 alleles. functional validation of missense alleles is critical in the complex evaluation of dock8 deficiency. background: hsct is the only known curative option currently for cd40l deficiency, an x-linked disorder. in cd40l deficiency and other x-linked immune deficiencies, there is an ongoing debate regarding the use of a carrier female sibling or mother as hsct donor. skewed lyonization despite complete donor chimerism has raised concerns for incomplete disease control post-hsct. no data exist regarding the efficacy of related female carrier as hsct donor for cd40l deficiency. we herein report outcomes of three patients with cd40l deficiency who underwent hsct using a related female carrier donor. method: retrospective review of patients who received hsct from carrier female related donor at three separate institutions. results: three patients with cd40l deficiency underwent hsct between 2016-2018. patient 1 had recurrent episodes of pneumocystis jiroveci pneumonia (pjp) despite being on bactrim and immunoglobulin replacement. patient 2 presented with pjp and severe neutropenia. patient 3 presented with acute respiratory failure from severe respiratory viral infections, cmvand had severe neutropenia requiring g-csf treatment. age at the time of hsct ranged from 0.5-15 yrs. all three underwent reduced toxicity hsct with busulfan and fludarabine-based preparatory regimens. two of them received matched sibling bone marrow hsct and one received tcr and cd19 depleted mobilized maternal pbsc haploidentical hsct. donor cd40l expression varied from 37% -67% on activated cd4 cells. immunoglobulin profile and lymphocyte subset were done in two of donors, they were within normal range for age, and none had significant infection history. no history of intermittent neutropenia or oral ulcers noted in donor and the absolute neutrophil count of the donor varied between 2500 6520 /l. donor age ranged from 3.2 yrs 48 years. cd34 dose ranged from 6.1 x 106 -23.1 x 106 cells/kg and cd3 dose ranged from 1 x 105 22.1 x 107 cd3+ cells/kg. gvhd prophylaxis consisted of csa/mmf (n=2) and tcr-a/b depletion and no csa (n=1). neutrophil engraftment ranged from 11-18 days and platelet engraftment ranged from 13 28 days. none of the patients developed acute or chronic gvhd. all three patients maintain full donor myeloid chimerism at the latest testing (9 months 18 months); t cell chimerism was 100% in one and mixed in two patients (91% at nine months, 80% at 12 months). all three patients had excellent t cell immune reconstitution; two patients came off immunoglobulin replacement 5 -11 months post hsct, whereas the 3rd patient is ivig dependent, though iga level was 25 mg/dl at nine months post-transplantation. latest evaluation, 9 18 months post-hsct, revealed 27% -63% cd40l expressing activated cd4 t cells, which correlated with donor cd40l expression and t-cell chimerism. conclusion: our data suggest that hsct utilizing x-linked carrier appears to be safe and results in durable engraftment with excellent humoral and cellular immune reconstitution in patients with cd40l deficiency. longer follow-up and data from a larger cohort is needed to make a definitive determination of safety and efficacy of utilizing female carrier as hsct donors in this disease. chief, immunology service, department of laboratory medicine, nih clinical center, bethesda, md, usa background: ikaros belongs to a hematopoietic-specific zinc-finger (zf) family of transcription factors. after dimerizing and dna binding to pericentric-heterochromatin (pc-hc) regions, ikaros is described as a central regulator of lymphocyte differentiation. somatic mutations/ deletions affecting ikaros n-terminal zf have been identified in b-acute lymphoblastic leukemia (all) patients, and germline n-terminal mutations were reported in cvid patients with progressive lack of b cells, hypogammaglobinemia, autoimmune diseases and b-all. methods: we performed targeted sequencing panel for known inborn errors of immunity disease-causing genes in a previously healthy male pediatric patient with burkitt lymphoma, followed by benign lymphoproliferation, thrombocytopenia and neutropenia. b-cells and immunoglobulin levels were normal. ikaros dna-binding, nuclear localization and protein binding were evaluated by emsa, fluorescence microscopy and immunoprecipitation. protein modeling was also performed. results: a novel heterozygous germline mutation in ikaros c-terminal zf6 dimerization domain (p.r502l) was detected in this patient. this mutant showed normal pc-hc localization but dna-binding was markedly reduced in terms of ikaros dimerization and multimerization. moreover, reduced wt-mutant binding was also detected. mutant/wt cotransfection experiments suggest a haploinsufficient defect. geometry based docking of wildtype ikaros predicted that r502 is within the homodimer interface and may abolish cation-pi interactions and destabilize the ikaros-zf6 dimerization domain. conclusion: a novel germline ikaros c-terminal mutation affecting homodimerization/multimerization and resulting in reduced dna binding to its dna consensus site was detected in a patient with burkitt lymphoma, benign lymphoproliferation and cytopenias. further studies are warranted to formally establish the casual connection between this genotype and phenotype.(210) submission id#606894 patricia pichilingue-reto, md 1 , prithvi raj, phd 2 , igor dozmorov, phd 3 , quan-zhen li, md, phd 4 , edward wakeland, phd 5 , nancy kelly, md 6 , maria teresa de la morena, md 7 , nicolai s. van oers, phd 8 methods: mice were generated by crispr/cas technology to genocopy the foxn1 compound heterozygous mutations identified in one of the human patients. thymopoiesis and hair follicle extrusion was analyzed in the various heterozygous and homozygous mutant mice. gene expression analyses of the hypoplastic and normal-sized thymii and the developing skin were performed. in addition, a structure-function analysis was performed with luciferase reporter assays using 9 distinct and previously unreported foxn1 mutations uncovered in patients who presented with low trecs. results: mice harboring compound heterozygous mutations in foxn1 that match the human patient phenocopy the t-b+nk+ scid phenotype with normal hair and nails. a functional characterization of the diverse foxn1 mutations suggests that the severity of the block in thymopoiesis depends on whether the mutations affect the dna binding or transactivation domains of foxn1. a 5-amino acid segment at the end of the dna binding domain appears to be essential for tec development. however, this segment is not required for normal keratinocyte functions in the skin and nail plate. gene expression comparisons are revealing key targets of foxn1 that suggest a dichotomy in its function in the thymus versus the skin. conclusions: novel compound heterozygous mutations in foxn1 are causal to a t-nk+b+ phenotype with normal hair shaft extrusion and nail plate extension. this differs from the classic nude/scid (omim # 600838) reported for individuals with autosomal recessive mutations in foxn1. assistant professor of medicine and pediatrics, department of allergy and immunology, uva introduction: copa syndrome is a recently described monogenic immunodysregulatory syndrome. the cop protein, encoded for by the copa gene, is expressed in all cell types and is involved in trafficking from the golgi complex to the endoplasmic reticulum (1) . the most common clinical features of copa syndrome are interstitial lung disease, pulmonary cysts or follicular bronchiolitis, pulmonary hemorrhage, arthritis, glomerular disease, and autoantibody development (2, 3) . atypical features of copa syndrome identified thus far include: extrapulmonary cysts in the liver and kidney, renal and neuroendocrine malignancies, autoimmune neurological disorders such as neuromyelitis optica, and infections, such as meningitis (4) . clinical case: we present a case of a 2 year-old male with copa syndrome (de novo heterozygous mutation in exon 9, c.715g>c; p.ala239pro) manifesting as lymphocytic interstitial pneumonitis, peripheral blood b-cell lymphocytosis, mediastinal lymphadenopathy and persistent transaminitis (alt and ast 100-400 u/l, nl ast<35 u/l, alt <55u/l) with normal bilirubin, alkaline phosphatase and pt/inr. the transaminitis was noted prior to diagnosis of copa syndrome, and has persisted despite seven months of therapy with pulse dose steroids, two cycles of rituximab and maintenance therapy with hydroxychloroquine and prednisone. he has had a normal ck and aldolase excluding muscle injury as a source of his transaminitis. a congenital cholestasis panel was normal. markers of autoimmune liver disease including ana, anti-liver kidney microsomal antibody and anti-smooth muscle were negative. serum ceruloplasmin and alpha-1-antitrypsin level were normal and celiac serologies, were negative. liver ultrasound was normal. a liver biopsy did not demonstrate inflammatory changes, hepatocyte necrosis, mononuclear cell infiltrates or fibrosis. nonspecific biopsy findings included occasional intraparenchymal neutrophils. it is unclear if these scattered neutrophils and the transaminitis are due to an early as yet unidentified autoimmune process, perhaps in response to hepatocellular stress exacerbated by the copa mutation. discussion: liver involvement has not been reported in copa syndrome. we describe a child with copa syndrome who has had chronic transaminitis with no clear alternative cause. if the phenotypic spectrum of copa syndrome involves the liver, it may limit immunomodulatory options for the treatment of this disease. background: in humans, biallelic stat1 lost-of-function (lof) mutations lead to a very low or complete absence of the wild-type (wt) protein. whereas, heterozygous mutations can lead to partial loss of function. these patients are susceptible to mycobacteria and herpes virus infections. on other hand, heterozygous gain-of-function (gof) mutations in the stat1 gene result in a hyperphosphorylated state where patients develop recurrent or persistent chronic mucocutaneous candidiasis (cmc), other cutaneous mycosis, bacterial infections, disseminated dimorphic fungal infections, viral infections and autoimmune disease. methods: in this study, we evaluated 4 novel stat1 mutations, three gof and one lof. in vitro, pbmcs from these patients were stimulated with ifn-and ifn-for 30, 60, and 120 minutes and levels of phospho-stat1 were measured by flow cytometry. the stat1 phosphorylation and activity (firefly and renilla luciferase activities) were evaluated in u3a-stat1 deficient cells transfected with a reporter plasmid (for luciferase), wt or mutant-stat1 plasmids. results: we observed higher levels of stat1 phosphorylation after two hours of stimulation from three gof mutations compared to wt. however, a lof mutation showed absent stat1 activation at baseline and in response to ifn-and ifn-. luciferase reporter assay confirmed gain of function and loss of function stat1 activity observed by flow cytometry. conclusions: using flow cytometry followed by a luciferase assay, we confirmed four novel stat1 mutations. measuring phosphorylation of stat1 by flow cytometry is sufficient to determine whether the stat1 mutation is disease causing. this assay can be translated to a clinically accessible test for stat1 related disease. background: variants in recombination-activating genes (rag) are common genetic causes of autosomal recessive forms combined immunodeficiencies (cid) ranging from severe combined immunodeficiency (scid), omenn syndrome (os), atypical scid (as) and cid with granulomas and/or autoimmunity (cid-g/ai). the clinical and immunological presentation is broad, ranging from severe infections secondary to near absence of t and b lymphocytes and hypogammaglobulinemia to the occurrence of autoimmunity with late manifestations with partly preserved immune subsets and near normal immunoglobulin levels and broad spectrum of autoantibodies. objective: we aim to estimate the incidence, clinical presentation, genetic variability and treatment outcome with geographic distribution of patients with the rag defects in populations inhabiting south, west and east slavic countries. due to shared ancestry, we also investigated our cohort for founder variants in rag1 and rag2 genes. methods: demographic, clinical and laboratory data were collected from rag deficient patients of slavic origin via chart review, retrospectively. results. based on the clinical and immunologic phenotype, our cohort of 80 patients from 66 families represented a wide spectrum of rag deficiencies, including scid (n=19), os (n=36), as (n=21) and cid-g/ai (n=4). sixty-six (82.5%) patients carried rag1 and 14 patients (17.5%) carried rag2 biallelic variants. we estimate that the minimal annual incidence of rag deficiency in slavic countries varies between 1 in 180,000 300,000 live birth and it may vary secondary to health care disparities in these regions. in our cohort, 70% of the patients carried rag1 p.k86vfs*33 (c.256_257delaa), either in homozygous (n=17, 26%) or compound heterozygous (n=29, 44%) form. the majority (77%) of patients with homozygous rag1 p.k86vfs*33 originated from vistula watershed area in central and eastern poland, and compound heterozygote cases distributed among all slavic countries except bulgaria. clinical and immunological presentation of homozygous rag1 p.k86vfs*33 cases was highly diverse suggestive of strong influence of other genetic and/or epigenetic factors in shaping the final phenotype. survival of rag deficient patients without hematopoietic stem cell transplant (hsct) (n=3, 8.8%) is poor and dramatically improved in the last decade with access to hsct and tailored conditioning regimens. conclusion: we propose that rag1 p.k86vfs*33 is a founder variant originating from the vistula watershed region in poland, which may explain a high proportion of homozygous cases from central and eastern poland and the presence of the variant in all slavs. our studies in cases with rag1 founder variants confirm that clinical and immunological phenotype only partially depend on the underlying genetic defect. hsct is becoming available for rag deficient patients in eastern europe with improving outcome. clinical immunologist, centre hospitalier universitaire de montrã©al (chum) background: acute gvhd following solid organ transplantation is a rare complication. intestinal and liver transplantation have the greatest risk of gvhd among solid organs due to high number of donor lymphocytes in these organs. prevalence of acute gvhd after liver transplantation is estimated to be around 0,1-2% and has a poor prognosis (1) . chronic neurological gvhd is a rare form of gvhd with three subtypes described: cerebral vasculitis, demyelinating disease and immune mediated encephalitis. acute neurological gvhd has no clear definition and is still considered a controversial entity. case presentation: a 63 year-old male underwent cadaveric liver transplantation for alcoholic cirrhosis and hepatocellular carcinoma. the donor was a 70 year-old man who died from anoxic brain injury. the receiver was induced with basiliximab and then put on prednisone, azathioprine and tacrolimus. he was readmitted 10 weeks later for myalgia, headache, fever and neutropenia. clinical state initially improved with empiric antibiotics. he then developed a skin eruption, colitis and dic. the latter was thought to be tacrolimus-induced. he was switched to cyclosporine. skin and rectosigmoid biopsies were compatible with acute gvhd. he received basiliximab and ivig and developed a refractory convulsive state. csf analysis showed elevated proteins and slight pleocytosis. cerebral mri showed non-specific white matter lesions and conventional angiography was normal. chimerism on peripheral blood was 0% but was 45% donor on csf. with the presence of chimerism on csf, evidence of cutaneous and digestive gvhd and no infectious cause, neurological gvhd was considered the most likely diagnosis. brain biopsy showed non specific change including neuropil spongiosis, microglial activation and reactive gliosis; but no signs of vasculitis or demyelinating disease. he was treated with atg, highdose systemic corticosteroids, cyclosporine, ivig and intrathecal methotrexate and corticosteroids. csf pleocytosis, proteins and chimerism improved with treatment (45% to 2% donor). no improvement was noted regarding his neurological state and he developed pancytopenia. he was then transfer to palliative care and died shortly after (4 month and a half after liver transplant). discussion: to our knowledge, there is only one prior case published of neurological gvhd following liver transplantation (2) . both patients were old, had hepatocellular carcinoma and had at least one hla match. age >50 year, hepatocellular carcinoma and shared hla antigen are known risk factors for gvhd following liver transplantation (1). our patient had only one hla match with the donor. this case is intriguing as there was a great discrepancy between blood and csf chimerism. acute neurological gvhd following transplantation is a real complication. it must be taken into consideration in patients with neurological involvement after transplant, even solid organ transplantations. introduction: hyper-igm syndrome are rare. although no data are available on the frequency of activation-induced cytidine deaminase (aid) deficiency, this disorder is estimated to affect less than 1:1,000,000 individuals. by the year 2012, 110 cases worldwide (1) with such mutation have been described. we describe a patient with hyper igm by mutation in the aicda gene. case report: mvv, 5-year-old boy, born to consanguineous parents, was referred with recurrent pneumonia, which started shortly after discontinuation of breastfeeding at 6 months old. repetitive otitis evolved with bilateral tympanic and partial hearing loss. he was submitted to adenoidectomy without improvement. immunological evaluation showed normal numbers of b and t cells with cd3+ (1290/mm3, 65%), cd4+ (547/mm3, 28%), and cd8+ (259/mm3, 13%). immunoglobulin concentrations were: igg = 138mg/dl (p97). treatment with intravenous immunoglobulin and prophylactic antibiotic was initiated and he had no infections during the follow up except for one episode of sinusitis. at 10 years of age, molecular evaluation was performed and a mutation in homozygosity in the aicda gene (omim * 605257) at position chr12: 8.757.821 was found, confirming the clinical suspicion. conclusion: the role of aid in the immunoglobulin class-switch recombination (csr) and somatic hypermutation (shm) have not been fully elucidated. summarizing within the shm and csr processes, aicda mutation can induce dna lesions in directed sequences in the s and v regions required for dna cleavage. recurrent infections and consanguinity raised the suspicion of inborn errors of immunity in this patient. the literature described late diagnosis as in the second or even the third decade of life. it was suggested that high levels of igm antibodies may provide effective defense, at least, against some infectious agents. it is important to emphasize that the impossibility to obtain genetic diagnosis did not prevent to introduce therapy. * aicda: activation induced cytidine deaminase gene patients with chronic granulomatous disease (cgd) are at risk for recurring infections and non-infectious inflammation, reduced quality of life and life expectancy. conventional treatment with life-long anti-bacterial and antifungal prophylaxis prolongs lifespan but does not eliminate the lifelong risk of infection and inflammation. allogenic stem cell transplantation is currently the only curative option for this disease. although sct with reduced intensity conditioning has improved treatment-related mortality and efficacy, it remains a matter of debate whether all patients with cgd benefit from sct, whether pre-existing infections and non-infectious inflammation are risk factors and at what age sct should be performed. we compared patients with cgd on conventional treatment with those after stem cell transplantation for their prognosis and evaluated potential risk factors for stem cell transplantation outcome followed up in six european centers. frequency of infections, inflammatory complications, hospitalizations, operations and immunomodulative/immunosuppressive therapy, height and weight were compared in patients on conventional treatment /before stem cell transplantation versus patients after sct. correlation between transplantation outcome and patient characteristics or medical history was tested. 105 patients were recruited, 55 on ct, 50 after stem cell transplantation. before/without transplantation 98% of patients suffered from at least one infection, 84,8% from inflammatory complications. patients on conventional treatment developed infection/inflammation/ hospitalization/surgery at a median of 2,28 (range [0,29-21,82] , iqr 2,79) per year, versus 9 (range , iqr 8,5) in the first year after stem cell transplantation but 0 (range [0-15], iqr 0,53) after the first year post stem cell transplantation. there was a significant decrease of all complications after stem cell transplantation (p < .05). growth improved significantly after stem cell transplantation (z-score weight -1,692 versus -0,846 (p.017), z-score height -1,906 versus -1,064 (p.029)). nevertheless, complications post stem cell transplantation are frequent: 88% of patients had at least one infection, 8% had severe acute gvhd, 12% chronic gvhd, 16% had graft rejection, 12% died. preexisting active mold infection increased the risk for complications after stem cell transplantation. in summary infections and non-infectious inflammation are common in patients with cgd on conventional treatment, their growth is significantly impaired. stem cell transplantation, if successful, significantly reduces the risk for infections and non-infectious inflammation. however, treatment related mortality of stem cell transplantation in patients with cgd remains considerable. introduction: development of a diverse t cell repertoire is essential for full immune recovery following definitive treatment for severe combined immunodeficiency (scid), whether by allogeneic hematopoietic cell transplantation (hct); autologous gene therapy (gt); or, in the case of adenosine deaminase deficiency, enzyme replacement therapy (ert). however, the time course and depth of diversity of t cell receptor rearrangements have been difficult to measure directly, necessitating estimates from total and naã¯ve t cell counts and from spectratyping, in which t cell receptor (tcr) beta chain diversity is estimated by the length distributions of cdna amplicons between a series of tcr beta chain variable (v-beta) segments that have productively recombined with the tcr beta-chain constant region. analysis of the actual sequences of rearranged tcrs could indicate more precisely the status of the t cell compartment of these patients, and might reveal oligoclonal expansion of dysregulated t cells, t cell insufficiency, or t cell exhaustion. objectives: we wished to ascertain whether deep sequencing of individual tcr v-beta rearrangements in peripheral blood could be performed sequentially following diagnosis and treatment of scid to differentiate satisfactory immune reconstitution from incomplete or skewed repertoire development that might require further cellular therapies. methods: equal amounts of total rna were obtained from peripheral blood of controls and scid patients pre-hct and at 100 d, 6 and 12 mo, and yearly post-treatment(s). cdna was used as template to semi-quantitatively amplify rearrangements at the tcr-beta locus (trb). raw sequences were filtered to remove pcr errors, and resulting fastq files were converted into fasta format (seqtk software, github, inc), filtered for productive rearrangement, and analyzed for v, d, and j gene composition and length (imgt highv-quest software). the vdj statistics file (past program) was used to calculate a shannon entropy (h) index to measure repertoire diversity, taking into account both abundance and richness of the overall repertoire; and a gini-simpson index of unevenness, measuring inequality in the relative representation of species in a given sample. graphical representations of repertoire diversity were generated by hierarchical tree maps of the trb repertoires (irepertoire software): each dot represents a unique sequence and the dot size corresponds to frequency of that sequence in the total sample. results: tcr v-beta sequence analysis of 3 scid patients (image) showed (top) baseline poor diversity due to pre-treatment ada deficiency followed by improvement to normal complexity (shannon h >7.0) after receiving peg-ada and autologous lentivirus gene therapy at age 3 m; (middle) increasing diversity in xscid after maternal t-depleted unconditioned hct, although b cells did not recover; and (bottom) failure of initial unconditioned maternal t-depleted hct in another xscid patient at 12 m, followed by autologous lentivirus gene therapy with subsequent improvement (shannon h increasing from 3.8 to 6) 12 months later. conclusions: tcr v-beta diversity sequence analysis provided a detailed assessment of repertoire diversity in response to cellular therapies for scid. this method could become a useful predictive tool to measure successful t cell immune reconstitution, both as early as 100 d and in the years following treatment. background: the stim1 (stromal interaction molecule 1) protein, encoded by the stim1 gene, is involved in calcium regulation in the endoplasmic and sarcoplasmic reticulum. pathogenic variants in this gene are associated with three different disorders. homozygous loss-of-function (lof) pathogenic variants in stim1 have been reported to cause autoimmune cytopenias, lymphoproliferation, enamel defects, anhydrosis, and iris hypoplasia. the first described cases had frequent mortality in early childhood due to recurrent life-threatening infections and development of kaposi sarcoma (1), while recently discovered cases have had more prolonged survival, though still with recurrent serious infections (2) . heterozygous gain-of-function (gof) pathogenic variants in stim1 have been associated with both tubular aggregate myopathy (tam) and stormorken syndrome. tam is a clinically heterogeneous progressive muscle disorder with a variable age of onset. muscle biopsy characteristically demonstrates tubular aggregates, with type ii muscle fiber atrophy (3) . stormorken syndrome has a phenotype that includes miosis, thrombocytopenia, intellectual disability, mild hypocalcemia, muscle fatigue, asplenia, and ichthyosis (4) . the thrombocytopenia has not been reported to be immune-mediated; rather it is due to abnormal platelet calcium regulation (5). we report a patient with stim1 pathogenic variant presenting with tam and immune-mediated thrombocytopenia, along with lymphoproliferative features, arthritis, and a mild immune deficiency. case: the patient is a 16-year-old with a history of congenital thrombocytopenia (platelets ranging 60,000-100,000) who presented with acute arthritis of bilateral hand joints after exposure to cold temperatures, which resolved with naproxen. he had back pain without muscle weakness, and preceding sore throat and general fatigue. labs were significant for leukocytosis and elevations in his inflammatory markers and creatine kinase. mri of his lower extremities was negative for inflammatory myositis, but did demonstrate bilateral hip and knee effusions, and significant inguinal lymphadenopathy and hyperintense linear signal changes in the mid-and distal femurs with patchy red marrow signal. abdominal ultrasound could not identify a definite spleen. bone marrow biopsy was negative for malignancy but significant for toxic granulation of neutrophils, evident of inflammation. alpha-beta double negative t cells were not elevated. interferon-gamma was mildly elevated. flow cytometry demonstrated normal t, b, and nk cell absolute counts. circulating antibodies against platelets (both igg and iga) were detected. on lymphocyte antigen and mitogen proliferation testing, he did not exhibit any proliferation when stimulated with tetanus toxoid even though he had been fully vaccinated against tetanus. muscle biopsy demonstrated large vacuoles consistent with tam on both light and electron microscopies. invitaes primary immunodeficiency panel identified a pathogenic variant in stim1 (c.910c>t; p.arg304trp), consistent with a diagnosis of autosomal dominant stim1-related conditions, including stormorken syndrome (6) . conclusion: this patient expands the phenotypic spectrum of stim1 related disease. based on previous evidence, gof pathogenic variants in stim1 are associated with tam and stormorken syndrome, while lof pathogenic variants in stim1 are associated with immune deficiency. however, our patient with a stim1 gof pathogenic variant has features of lymphoproliferation and immune dysregulation in addition to tam. stim1 gof pathogenic variants should be considered in the differential of patients with immune thrombocytopenia and lymphoproliferation. references: introduction / background: card11 is critical for protein binding upstream of nf-kb (nuclear factor kappa b) and mtorc1 (mammalian target of rapamycin complex 1) the signaling pathway involved in t-cell activation and inflammatory response. prior testing of card11 mutations demonstrated variable t-cell dysfunction. in vitro studies have demonstrated reduced interferon gamma cytokine production, interference of t-cell receptor (tcr) signaling, and th2 phenotype skew in t-cells with card11 defects. while homozygous mutation causes severe combined immunodeficiency deficiency, heterozygous card11 defect is associated with atopy by way of inappropriate th2 skewing. heterozygote atopy is characterized by eosinophilia, elevated ige, and severe dermatitis. despite multiple studies demonstrating in vivo consequences of card11 on t-cell function, little is known of the clinical significance. moreover, few studies have demonstrated the impact of card11 mutations on b-cell maturation and development, despite the recognized tcr and interleukin 2 signaling deficits. objectives: this case demonstrates a card11 defect that evolved from atopy to combined immunodeficiency requiring intravenous immunoglobulin therapy. it highlights the poorly understood effect of card11 mutation on t-cell function, and the downstream impact on b-cell quality. methods: 53-year-old male, with past medical history of t-cell lymphoma and no evidence of disease status post autologous stem cell transplant, was found to have card11 e57d missense mutation by genetic testing. consistent with previous literature regarding heterozygous card11 defects, the patient suffered from frequent asthma exacerbations, aeroallergen sensitivity, and eczema. lab work was consistently positive for elevated ige and eosinophilia. family history was positive for a son born with congenital molluscum, and multiple other children with recurrent infections. one child was also identified with card11 mutation. the patient had flow cytometry demonstrating 4% of circulating cells with atypical immunophenotyped cd3+ t-cells, and positive gene rearrangement studies. his qualitative immunoglobulin levels were significant for consistently low igm, but normal quantity igg. in the patients adulthood, he had recurrent bronchitis and pneumonia requiring hospitalization and intravenous antibiotics. given his recurrent infections, the patient underwent immunodeficiency evaluation. despite previous infection with herpes zoster, the patient did not have protective titers. additionally, the patient had received the pneumococcal conjugate vaccine once, and the pneumococcal polysaccharide vaccine four times. the most recent vaccination was one year prior to evaluation. despite repeated vaccinations, titers were unprotective. consequently, the patient was diagnosed with combined immunodeficiency, and initiated on intravenous immunoglobulin therapy. results: in summary, card11 defect is a cause of atopy, observed to become less severe with age. studies of card11 heterozygote mutations have demonstrated in vitro deficiencies in t-cell activation, likely secondary to skewed or decreased inflammatory cytokine production and tcr activation. our patient demonstrates that the variable t-cell dysfunction seen in vitro can have significant clinical implications evidenced by his inadequate vaccine response, and recurrent infections. his combined immunodeficiency poses a connection between card11 defects and, not only t-cell, but also b-cell function. conclusions: further studies are needed to determine deficits in t-cell and b-cell function in the setting of card11 defect, as this case suggests the clinical implications span further than atopy. genetic variants in the scaffold gene card11 cause disorders of the immune system. the clinical course and treatment depends on whether the card11 variant causes gain-or loss-of-function. however, lymphocyte immunophenotyping and proliferation assays in cells expressing card11 variants don't easily distinguish between gain-and loss-of-function. to address this challenge in variant interpretation, we used multiplexed genome editing in a lymphoma b cell line (tmd8) to generate cell populations expressing all possible singlenucleotide variants in the n-terminal 140 amino acids of card11. to assess function in each variant, we tracked its relative abundance over multiple conditions using dna sequencing. since card11 is required for survival of tmd8 lymphoma b-cells, cells expressing clinically identified gain-of-function variants grew faster relative to cells expressing other variants, even in the presence of upstream pathway inhibitors. upon evaluation of the relative abundance of each variant in genomic dna and mrna, we found that clinically identified loss-of-function variants were depleted in mrna, which could be attributed to alterations in splicing or to nonsensemediated decay. to address the impact of splicing, we modeled a newly-identified splice donor mutation (c.358+1g>a) found in two patients from one family diagnosed with combined immune deficiency, autoimmunity and atopy that was also observed in our screen. we show that the variant causes deletion of exon four and that card11 missing exon four exerts a dominant-negative effect leading to decreased nf-kb signaling and cell growth. these experiments demonstrate the utility of multiplexed functional assays for determining variant effect in clinically-relevant genes, which will improve diagnosis and treatment in patients. mutations in the rag1 and rag2 genes in humans cause a wide spectrum of phenotypes, ranging from severe combined immunodeficiency (scid) with lack of t and b cells to omenn syndrome (os), atypical scid (as) and combined immunodeficiency with granulomas and/or autoimmunity (cid-g/ai). here, we sought to investigate the molecular basis for phenotypic diversity presented in patients with various rag1 mutations. methods: we have recently described a novel flow-cytometrybased assay in which mouse rag1-/-pro-b cells containing an inverted gfp cassette flanked by recombination signal sequences (rss) are transduced with a retroviral vector expressing either wild-type or mutant human rag1 (hrag1). the green fluorescent protein expression directly relates to the activity of rag proteins, representing a quick and powerful tool to correlate between defective activity of hrag1 mutant and severity of the clinical phenotype. the genetic variants of hrag1 analyzed in this study were affecting the various domains of the protein: ring, zinc finger ring type domain (amino acids 168-283); nbr (amino acids 387-461); hbr (amino acids 531-763) and the core domain (amino acids 385-1011). using this sensitive assay, we tested the recombination activity of 27 human rag1 variants that have been reported in patients. results: we have demonstrated correlation between the recombination activity of the mutants and the in vivo clinical phenotype of patients. in particular, similarly low levels of recombination activity were observed in patients with scid and os, whereas patients with as and especially those with cid-g/ai carried mutations that retained significant residual levels of activity. conclusions: these data provide a framework to better understand the phenotypic heterogeneity of rag deficiency. here we report a case of a child with b. cepacia lymphadenitis, ultimately diagnosed with takayasu arteritis. takayasu arteritis is a large vessel vasculitis which may have a nonspecific clinical presentation in childhood possibly leading to difficulty in diagnosis. case: a 16-month-old female presented with two weeks of fever, respiratory distress, and lymphadenopathy, and was treated with ivig for presumed atypical kawasaki disease. imaging studies performed due to worsening respiratory distress revealed retropharyngeal abscess with bilateral cervical lymphadenopathy, culture-positive for prevotella oralis and melaninogenica, with improvement following incision and drainage and antibiotic therapy. recurrence of fever and respiratory distress prompted ct imaging of her neck significant for worsening lymphadenopathy. cultures from lymph node biopsy grew b. cepacia. following treatment, she was readmitted with respiratory distress requiring chronic steroid treatment and found to have candida albicans on bronchoalveloar lavage and necrotizing granulomatous inflammation on lung biopsy. an immunologic evaluation was notable for two normal dhr assays. cgd genetic panel was negative for pathogenic variants in cybb (p91), ncf1 (p47), cyba (p22), ncf2 (p67). testing was also notably negative for hiv pcr, bartonella pcr, cryptococcal antigen, histoplasma antigen, bal afb stain and mycobacterial cultures, cmv pcr, ebv pcr, anca, serial blood cultures, and sweat test. lymphocyte subsets were normal for age. mitogen stimulation test, myeloperoxidase antibody igg, serine protease3 igg, c4 level, lad panel, and cytokine panel were normal. autoimmune lymphoproliferative disorders (alps) panel was negative. whole exome sequencing demonstrated heterozygous mutations in cfi and jak3, not considered to be clinically relevant given the patients clinical picture and laboratory evaluation. the patient was then lost to follow-up for over a year. at the age of 3 years, the patient presented with fever and back pain. imaging revealed severe large vessel vasculitis involving the aorta and subclavian, vertebral, mesenteric, and renal arteries. she also had evidence of cardio-embolic strokes on brain mri. she had had no significant interval infections, and her immunologic evaluation remained unrevealing. in the context of her new vasculitis, evaluation for deficiency of ada2 (dada2) was negative. she was ultimately diagnosed with takayasu arteritis and has begun therapy with systemic corticosteroids, aspirin, and etanercept. conclusions: we describe a case of b. cepacia infection in a child without identified immunodeficiency, ultimately diagnosed with a large vessel vasculitis. the presence of b. cepacia infection warrants a thorough investigation. burkholderia has been previously associated with giant cell arteritis, another type of large vessel vasculitis, though causation has not been established. to our knowledge b. cepacia infection has not been associated with takayasu arteritis. christopher santaralas, valentine jadoul, jacqueline squire, john cannon, jessica trotter, susan aja, neil goldenberg, david graham, jennifer leiding background: chronic granulomatous disease (cgd) is a primary phagocytic immunodeficiency secondary to mutations in any of the components of nadph oxidase. in addition to infection susceptibility, patients with cgd can develop auto-inflammatory disease that is difficult to manage. metabolomics is the systematic study of small molecule biomarkers of the clinical phenotype of disease. we sought to investigate plasma metabolic profiles in cgd as we hypothesized that unique signatures may differentiate patients with cgd. methods: plasma collected from 15 subjects with cgd (9 x-linked, 4 p47phox-deficient, 2 p22phox-deficient) and 2 x-linked cgd carriers was analyzed using a targeted multiplex assay by liquid chromatography mass spectrometry (lc-ms) and simultaneously a profiling assay by lcms. sufficient signal was present for 34 metabolites. x-linked cgd and p47phox-deficient groups were sufficiently sized for multivariate and univariate analyses in metaboanalyst. twelve patients had a single time point of plasma metabolomics analysis and three had multiple time points, including one in whom both pre-and post-hematopoetic cell transplantation time points were assessed. post-hoc comparisons were also performed for those with, versus without, clinical comorbidities of autoinflammation. results: plasma from patients with x-linked and p47phox deficient cgd had a differential metabolomic signature at baseline. many metabolites as measured by ion intensity were present at high levels, particularly homocysteine, kyneurine, tryptophan, citric acid, carnitine, methionine, and adenosine. increased values of metabolites reduced to that of normal (compared to post hct). homocysteine levels were elevated among patients with (mean 1.5x105), versus without (mean 6.8x104), clinical comorbidities of auto-inflammation (i.e., colitis, lupus). baseline samples showed elevated kynurenine among all cgd patients, relative to historical normal controls (unmatched, separate analysis). patients with colitis had elevated citric acid levels that were higher among patients with (mean 2.1x106), versus without (mean 4.5x105), colitis irrespective of genotype. conclusions: preliminary data with a small patient subset suggest that patients with cgd have metabolomic signature distinguishable by phenotype. citric acid cycle metabolites are elevated in crohns disease and ulcerative colitis. based on our data, citric acid may too act as a biomarker for inflammatory bowel disease in cgd. analyzing a larger number of samples, across time points, will likely describe a metabolomics profile for cgd and identify biomarkers for auto-inflammation in cgd. no significant medical history in mother; paternal history is unknown and unavailable.no significant medical history in mother or father. rationale: ataxia telangiectasia is a disorder with variable phenotypes characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity, and cancer predisposition which may correspond to the degree of atm protein expression and/or radiosensitivity. we used in vitro cytometric assessment of atm, smc1 and h2ax phosphorylation to assess dna damage in response to radiation and found that two siblings with the same copy number gain in atm have variable clinical neurologic and immunologic phenotypes. methods: chart review and radiosensitivity assays using cytometric assessment of patm, psmc1, and h2ax expression after irradiation with 2gy. results: patient a is a 6 month old male identified after having low trecs on newborn screening, then found to have lymphopenia and elevated igm. he has diffuse cafã© au lait macules and no neurologic symptoms. his 9 year old sister, patient b, was being followed by neurology for several years for ataxia. she has selective iga deficiency, normal lymphocyte counts, lymphocyte proliferative responses, gammaglobulins, and vaccine specific antibodies. both patients have a 4 copy number gains in atm (exons 48-61). mother and father both have 3 copy number gains in atm and are healthy without neurologic symptoms or recurrent infections. both patient a and b have normal atm protein expression. phosphorylated atm, smc1, and h2ax was assessed in lymphocyte subsets (t, b, and nk cells) after low-dose irradiation to induce dna double-stranded breaks (dsbs). these parameters were assessed at 1 hour post-irradiation when they are expected to be maximal and at 24 hour post-irradiation, when under conditions of normal and effective dna repair, the phosphorylation state returns to baseline. patient a had abnormal patm and psmc1 but normal h2ax expression 1 hour and 24 hours after irradiation of t, b, and nk cells. patient b had normal patm, psmc1, and h2ax expression in t cells but abnormal patm and psmc1 expression in b and nk cells 1 hour after irradiation. patient b, however, had abnormal atm phosphorylation at 24 hours after irradiation of t, b, and nk cells.conclusions: our results indicate that a unique copy number gain in atm within a family can correspond to different clinical and immunologic phenotypes as well as variable degree of radiosensitivity. the persistence of h2ax at 24 hours post-irradiation and impaired phosphorylation of atm and smc1 at 1 hour post-irradiation demonstrates defects in dna dsb repair, and this is variably altered in different lymphocyte subsets. correlation between atm phosphorylation in lymphocytes with outcomes may be an area for future studies and particularly important in counseling patients regarding outcomes. antibodies have been implicated in both protection and pathology of dengue virus infections. however, much of this data is gathered from serum/plasma responses that is a cumulative of historical and ongoing infection. to precisely understand the role of antibodies with respect to the ongoing dengue virus infection, we employed the cutting edge approach of generating of human monoclonal antibodies from individual plasmablasts from peripheral blood of dengue patients that allows us to probe for answers at a single cell level. this method involves ex vivo single cell sorting of plasmablasts from peripheral blood of well-characterized dengue infected patient followed by single cell molecular cloning of immunoglobulin heavy-and light-variable regions into expression vectors containing the defined constant region followed by transient cotransfection of hek 293a cells with the heavy and light chain expression vectors made from genes arising from the same cell. thus far, using this powerful technology, for the first time in india, we have made 140 number of human monoclonals, of which 80 are specific to dengue and 14 neutralize dengue virus at various concentrations. all the neutralizing antibodies are dengue-envelope specific and bind the highly conserved fusion loop of the dengue virus envelope. together, with the ongoing comprehensive analysis of the b cell repertoire and somatic hypermutations, these studies provide a detailed understanding of the dengue-specific plasmablast cell response at a single cell level and create a platform for testing these antibodies for basic research, diagnostic, prophylatic and as well as therapeutic applications. surviving. six of the 13 (46.2%) surviving patients remain dependent on ig replacement despite robust donor chimerism of 99-100% and no active gvhd. all but two received rituximab pre-hsct. of the patients who are independent of ig replacement, only one (14.2%) received rituximab post-hsct, whereas 5/6 of the ig dependent patients received rituximab post-hsct. t cell immune profiling revealed that the absolute numbers of lymphocyte subsets, cd4+ naã¯ve t cells, and cd4+ recent thymic emigrants were not statistically different between ig independent and dependent patients ( figure 1 ). however, there was a marked decrease in the number of total b cells, the percentage of memory b cells (cd27+ b cells), and classswitched memory b cells (cd27+ igd-igm-cells) in ig dependent patients ( figure 1 ). t follicular helper (tfh) cell populations (cd4+cd45ra-cxcr5+pd1+) were evaluated in four patients and the frequency was similar to healthy controls (4.5+/-1.2 vs. 3.9+/-1.4%). the ability of the patients naã¯ve b cells to class-switch was assessed following exposure to il-21, anti-cd40 antibody, and anti-human igm, and revealed normal b cell class-switching and differentiation to plasmablasts ( figure 1) . additionally, t cell ability to provide b cell help was assessed by coincubating naã¯ve b cells with activated cd4+ t cells. this revealed comparable b cell class switching to that of healthy controls. conclusion: the high incidence of poor long-term functional b cell reconstitution following allogeneic hsct for xlp-1 could be related to the use of rituximab in the post-hsct setting rather than pre-hsct. normal tfh numbers and function, and ability of b-cells to class-switch in-vitro suggest that persistent hypogammaglobulinemia is these patients is unlikely from a b or t-cell intrinsic defect. the possibility of rituximab induced acquired lymph nodal stromal defect in these patients is being explored. further studies are needed to understand the biology of persistent hypogammaglobulinemia in xlp-1. additionally, due to the high incidence of persistent hypogammaglobulinemia, exposure of rituximab should be limited post-hsct. background: tandem mass spectrometry (ms/ms) has emerged as a primary platform for many clinical and newborn screening laboratories. the application of ms/ms mainly focuses on the quantification of accumulated small metabolites in plasma resulting from various metabolic defects. however, many disorders do not yield such metabolic markers and would benefit from the direct quantification of intracellular target proteins. unfortunately, the extremely low (e.g., pmol/l range) protein concentrations in blood cells limit their detection via ms/ms. in recent years, peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-srm) has emerged as a promising technique for the quantification of low abundance proteins in complex matrices, including dried blood spots (dbs). our lab has demonstrated that immuno-srm methods are able to reliably distinguish affected patients from the normal controls for wilson disease (wd), wiskott-aldrich syndrome (was), severe combined immunodeficiency (scid), and x-linked agammaglobulinemia (xla) (j. proteome res., 2017 and front. immunol., in press). these results demonstrate the utilization of immuno-srm as a sensitive platform for multiplexed quantification of signature peptides in the low pmol/l range. methods: several candidate peptides for each protein were selected based on uniqueness using in silico blast tools and lc-ms/ms response. monoclonal antibodies (mabs) were then generated for peptide enrichment from dbs. blood from normal controls, wd, xla, scid, and was patients was spotted onto filter paper, dried, and stored at -20â°c until use. proteins were extracted from dbs, digested with trypsin, and enriched using mabs bound to magnetic beads. the enriched peptides were then eluted and analyzed using srm mode with a waters xevo tq-xs. results/conclusions: to date, immuno-srm methods have been generated for wd, was, scid, xla, and cystinosis. preliminary data shows immuno-srm methods are able to reliably quantify target proteins using signature peptides and accurately distinguish affected patients from normal controls. analysis of signature peptides found statistically significant reduction or absence of peptide levels in affected patients compared to control groups in each case (was and btk: p = 0.0001, scid: p = 0.05). intra and inter-assay precision ranged from 11 -22% and 11 -43%, respectively, and the multiplexed assay showed a broad linear range (1.39 2000 fmol peptide) . in a blinded sample set of 42 pidd patients and 40 normal controls, immuno-srm-predicted diagnoses showed excellent agreement with clinical or genetic diagnoses. every molecularly-confirmed case of was and btk was also diagnosed by immuno-srm analysis. in addition, 62 randomly selected samples provided by the nbs laboratory of washington state were tested and peptide concentrations were found to be within normal ranges. efforts are underway to validate and incorporate peptide biomarkers for adenosine deaminase deficiency, dock8 deficiency, and ataxia telangiectasia, as well as general markers for nk cells and platelets into a single multiplexed assay. in addition, scid, was and xla samples continue to be run while we focus on reducing assay costs, time, and necessary sample input. our data herein provides proof of concept for the immuno-srm workflow to be extended to various other genetic diseases as potential multiplexed newborn screening methods.(250) submission id#617782the background: the long-term effects of glucocorticoids (gcs) on the immune system have been extensively studied in patients with different underlying conditions (e.g, malignancies or autoimmune conditions), as well as in healthy volunteers receiving short-term courses of these drugs. although these approaches provided highly relevant data, neither of them answered the unbiased/bona-fide effect of long-term gcs use on the immune system. endogenous cushing syndrome (ecs) may be caused by pituitary or ectopic acth-producing adenomas, or by tumors or hyperplasia of the adrenal cortex. patients with ecs present with different gcsdependent manifestations, including those affecting the immune system as neutrophilia and lymphopenia. when tumors are removed, most of the effects of gcs tend to progressively regress. methods: paired samples from 15 patients with ecs due to acth-producing adenomas (age range 7-16y, 8 females) were studied before (ecs-pre) and 6-12 months after tumor removal (ecs-post). extended lymphocyte phenotypes and apoptosis in different cell subsets were evaluated by flow cytometry. cytokine production (elisa) and responses, as well as their effects on cell proliferation and viability, were evaluated using cell trace violet and annexin-v staining. results: among multiple immunophenotypic changes, ecs-pre patients showed significantly reduced naã¯ve t cells and recent thymic emigrants (rte) as well as increased apoptosis in t cells when compared to themselves (ecs-post) or age matched healthy controls. moreover, significantly increased exhausted cd8 t cells were observed in ecs-pre patients. interestingly, ecs-post patients showed full cellularity recovery of t cells and rte with increased proliferation and reduced apoptosis, in addition to correction of most of the other changes evidenced. significantly lower il-21 plasma levels were also detected in ecs-pre when compared to ecspost patients. to determine the role of il-21 in an ecs-resembling condition, healthy control pbmcs were treated with gcs in-vitro and the effect of il-21 and other cytokines was tested. a significant reduction in apoptosis was observed in the il-21-treated cells that almost completely countered the pro-apoptotic effects of gcs; il-21 was also significantly more efficient than il-2, il-7, ifn-alpha and ifn-gamma in rescuing cells from apoptosis. il-21-specific upregulation of bcl2 and bcl6 expression was evidenced in these cells.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-006466-e1phpqes authors: nan title: 2018 cis annual meeting: immune deficiency & dysregulation north american conference date: 2018-04-23 journal: j clin immunol doi: 10.1007/s10875-018-0485-z sha: doc_id: 6466 cord_uid: e1phpqes nan mutations in the genes encoding proteasome subunits (psmb8, psmb4, psma3 and psmb9) have been identified as the cause of candle syndrome. these mutations lead to malfunction of the proteasome, which results in buildup of cellular waste products. it is hypothesized that dysregulation in the interferon (ifn) signaling pathway in response to this waste is the driving mechanism of the inflammatory response, and may serve as a therapeutic target in these patients. objectives: to describe a case of suspected candle syndrome successfully treated with tofacitinib. methods: retrospective chart review was conducted with respect to diagnosis, treatment and response. results: a 16-month old caucasian male was admitted to the hospital for evaluation of profound anemia. his medical history was significant for extreme prematurity (born at 22 weeks from premature labor), intraventricular hemorrhage grade iv that resulted in hydrocephalus needing ventriculoperitoneal shunting, and developmental delay. he was noted to have a hemoglobin of 6.2 g/dl during a neurosurgical evaluation for routine shunt revision. he developed hemodynamic decompensation and required hospital admission for packed red-blood cell transfusion. review of systems was remarkable for intermittent pruritic macular rash, daily temperature fluctuations (fever to hypothermia), joint pain/swelling/ stiffness of multiple sites, poor weight gain, irritability, irregular breathing, abdominal distention, and regression of gross motor milestones (no longer rolling over, sitting without support, or pulling up to stand). workup excluded infections and lymphoproliferative malignancies, and he met clinical criteria for systemic juvenile idiopathic arthritis. his initial laboratory studies showed systemic inflammation (wbc 14x 10^3/ul, hgb 6.0 g/dl, platelets 558 x10^3/uul, crp 14.2 mg/dl, sedimentation rate 70 mm/h, ferritin 2370 ng/ml). imaging studies revealed serositis with right-sided pleural and pericardial effusions. he also had myositis supported by imaging and elevation of muscle enzymes (ast 52 u/l, aldolase 30.6 u/l). the patient was started on pulse iv methylprednisolone 30mg/kg daily x3 days, followed by oral prednisolone 2mg/kg/day and anakinra 2mg/kg/day with partial improvement and he was discharged home. he was readmitted 3.5 weeks later due to concerns for macrophage activation syndrome (ferritin 12,362 ng/ml) in the setting of a gastrointestinal infection and anakinra was increased to 4.5mg/kg/day. however, he continued to have persistently elevated inflammatory markers and so the dose was increased again to 7mg/kg/day. three months after initial presentation, he had an upper respiratory and ear infection and became ill with generalized rash, increased work of breathing, and poor perfusion. anakinra was considered a treatment failure at that time. he required several doses of pulse steroids and initiation of tocilizumab 12mg/kg iv every 4weeks with improvement on systemic symptoms. methotrexate 15mg/m² weekly was added soon after for persistent arthritis and inability to wean systemic steroids. he continued to have abnormal inflammatory indices, including ferritin (1,586 ng/ml) and il-18 levels (35,588 pg/ml, normal 89-540). proband only whole exome sequencing revealed a single heterozygous mutation in the psmb4 gene (c.-9g>a), a published pathologic variant. based on this finding, the patient was started on tofacitinib 2.5mg orally twice a day with a dramatic response. laboratory markers of inflammation normalized, and he was able to walk within the first month of treatment. further genetic testing to detect an additional proteasome subunit variant, as well as functional testing on a research basis to demonstrate an interferon signature are being pursued. conclusions: this case highlights the value of early genetic studies in patients with autoinflammation so that initiation of targeted therapy is not delayed in efforts to achieve control of symptoms and evade future complications. this case also illustrates the challenges in diagnosing monogenic autoinflammatory disorders in young patients that present with recurrent fevers, generalized rash, arthritis, and systemic inflammation that mimic systemic juvenile idiopathic arthritis. our experience contributes to the understanding of janus kinase inhibition in type i interferonopathies. of his recurrent infections and etiology of myasthenia gravis. results of the ct chest are notable for a thymoma. thymectomy with biopsy reveals benign pathology with a mixture of type a and b cells. he continues to have persistent fatigue, generalized weakness, diplopia, diarrhea and recurrent respiratory infections after thymectomy. immunoglobulin and lymphocyte subset panels reveal hypogammaglobulinemia with absent b cells. endoscopy reveals villous atrophy and blunting without evidence of celiac disease, inflammatory bowel disease or infection suggesting autoimmune enteropathy. the constellation of clinical and laboratory features are consistent with good syndrome with evans syndrome, seronegative myasthenia gravis and autoimmune enteropathy. the patient is started on immunoglobulin replacement therapy and pyridostigmine with resolution of recurrent infections and improvement of fatigue, generalized weakness and diplopia. three years later his fatigue and evans syndrome recur with new onset loss of appetite and a thirty pound weight loss. repeat immunologic labs were notable for elevated cd3, borderline low cd4 and highly elevated cd8 cells with low absolute number and fraction naïve cd4 and cd8 cells suggesting worsening combined immunodeficiency with peripheral t cell expansion. a bone marrow biopsy reveals large granular lymphocytic (lgl) leukemia and he is started on methotrexate. serum antibodies targeting ifn, and il-12 are negative four years after removal of thymoma. conclusions: this case is consistent with a classic presentation of good syndrome represented by thymoma, t and b cell-mediated immunodeficiency, increased susceptibility to infections and autoimmune manifestations of evans syndrome, myasthenia gravis and autoimmune enteropathy. in this case the combination of evans syndrome, autoimmune enteropathy and lgl leukemia as malignancy further worsen prognosis and is typically not seen together in good syndrome. this case depicts well the crossroad of infection, autoimmunity and malignancy in late onset immunodeficiencies. introduction/background: dedicator of cytokinesis 8 (dock8) deficiency is a known cause of autosomal recessive hyper-ige syndrome with a combined immunodeficiency. most of the mutations in dock8 are lossof-function homozygous or compound heterozygous point mutations or deletions. dock8 deficiency has been associated with low lymphocyte counts with impaired antibody responses, as well as eosinophilia, recurrent bacterial and cutaneous viral infections, malignancies, and severe atopy. we report the case of a 47 year old man with history of hyper-ige syndrome, severe atopy, eosinophilia, and antibody deficiency, phenotypically atypical for dock8, who was noted to have two variants of unknown significance in the dock8 gene. objectives: we report the case of a 47 year old man with history of hyper-ige syndrome, severe atopy, eosinophilia, and antibody deficiency, phenotypically atypical for dock8, who was noted to have two variants of unknown significance in the dock8 gene. methods: a 47 year-old man presented to us for evaluation of known hyper-ige syndrome. he had a long history of elevated ige, peripheral eosinophilia, severe atopic dermatitis, food allergies, asthma, severe eczema since early childhood which failed to respond to methotrexate, mycophenolate, cyclosporine, and omalizmuab, but ultimately responded to intravenous immunoglobulin (ivig). his infectious history included mrsa skin infections and one episode of pneumonia, and he reported a history of fungal skin infections but the history was unclear. initial immune workup revealed eosinophilia of 2898, ige level 3540 (as high as 20,000), igg level 603, igm level 106, and iga level 124. he had no random antibodies to streptococcus pneumonia 13 serotypes, but he had protective antibodies to diphtheria and tetanus. lymphocyte subsets showed cd3 1874, cd4 1597, cd8 277, cd19 85. he had normal mitogen stimulation to pha but decreased mitogen stimulation to candida. dna testing for a stat3 mutation was negative. results: we found two missense variants of uncertain significance in the dock8 gene (1.p.v194i, nm_203447.3:c.580g>a and 2.p.l1330v,nm_203447.3:c.3988c>g ). the first variant had previously been reported in the clinvar database as a variant of uncertain significance, and the second variant had not been previously reported in the literature to our knowledge. our assay could not determine if the two dock8 variants were on the same allele or on different alleles. dock8 protein expression testing is currently pending. conclusions: our patient presented with history of elevated ige, eosinophilia, atopy, severe eczema, and cutaneous mrsa and fungal infections. he was noted to have variants of uncertain significance in the dock8 gene. homozygous or compound heterozygous pathogenic variants in dock8 are associated with an autosomal recessive hyper-ige syndrome and combined immunodeficiency with clinical features of recurrent bacterial infections, cutaneous viral infections, severe atopic disease, as well as susceptibility to malignancy. our patient does not have all the typical features of dock8 deficiency and he seems to have a less severe phenotype. notably, he does not have the cutaneous viral infections or malignancy often seen in dock8 mutation hyper-ige cases. our case demonstrates new missense mutations, which have not previously been described in the literature, possibly causing a milder phenotype of dock8 deficiency. a case of igm deficiency and adult-onset still's disease negative. previous biopsy of her cervical and thoracic lymphadenopathy was unremarkable for malignancy. during her hospitalization, serum immunoglobulins were performed, which demonstrated normal levels of igg and iga, with igm level of <10 mg/dl (reference range 40-230 mg/dl), consistent with selective igm deficiency. liver function tests revealed an elevated aspartate aminotransferase (ast) of 177 u/l and an alanine aminotransferase (alt) of 177 u/l with a total bilirubin of 1.7 mg/dl and an alkaline phosphatase of 146 u/l. her ferritin was elevated at 349 g/l. the patient fulfilled yamaguchi criteria for aosd with three major criteria of evanescent rash, intermittent fevers in a quotidian pattern, bilateral arthralgias in the hips, knees, and ankles. she also met two minor criteria of liver abnormalities and lymphadenopathy. conclusions: selective igm deficiency is an uncommon immunodeficiency disorder associated with increased risk for autoimmune disorders. the recognition of co-morbid autoimmune illnesses in an immunodeficient patient is often complicated by a paucity of examples in the literature and potential confounding of laboratory serology analysis. we report the first case of a patient with selective igm deficiency and aosd. introduction/background: anaphylaxis to protamine is an uncommon but life-threatening complication of cardiac surgery and insulin therapy. here we present a case of recurrent protamine hypersensitivity during vascular surgery. objectives 1. recognize clinical signs of protamine hypersensitivity 2. recognize recurrent hypersensitivity to protamine as a serious complication of anesthesia methods: a 63 year old man with a history of diabetes, previously on nph insulin, hypertension, hyperlipidemia, chronic smoking, and peripheral artery disease with multiple vascular interventions was admitted to undergo a right lower extremity saphenous vein graft bypass. three years earlier during a similar intervention, the patient had developed intraoperative hypotension after protamine sulfate administration. protamine was subsequently held for additional surgeries, however the patient was able to tolerate protamine with slower infusion one year later. for the current vascular surgery, the patient was pretreated the day of surgery with diphenhydramine and dexamethasone, and a test dose of protamine was infused prior to full dosing. the patient initially appeared to tolerate the full protamine dose, but quickly developed facial erythema and angioedema. due to concern for laryngeal edema he remained intubated and was transferred to the surgical intensive care unit, where he received additional diphenhydramine and dexamethasone. his symptoms resolved and he was successfully extubated the next morning. results: anaphylaxis to protamine is an uncommon but lifethreatening complication of cardiac surgery and insulin therapy. protamine sulfate is a polypeptide used widely to neutralize heparin anticoagulation during cardiac and vascular surgeries, and in nph insulin. severe anaphylactic or anaphylactoid reactions caused by injection of protamine sulfate are well documented in literature, and the product contains a black box warning for such. the pathophysiologic mechanisms underlying these reactions are not clear, but ige-mediated hypersensitivity appears to play a role in many reactions, and prior sensitization or cross-sensitization (eg, to fish) have been suggested. type b adverse drug reactions are idiosyncratic drug reactions and are often unpredictable, as in our patient who previously tolerated protamine but subsequently developed an adverse reaction. hypersensitivity reactions during anesthesia should be thoroughly studied to identify the responsible drug and minimize exposure in recurrent surgeries. conclusions: this case illustrates the potential for severe reactions even with newer protamine formulations, and highlights the unpredictable nature of type b adverse drug reactions. it is important for clinicians to exhibit awareness of the potential adverse effects of protamine sulfate in such situations. introduction/background: x-linked lymphoproliferative syndrome type 2 (xlp-2) is a rare primary immune deficiency caused by loss of function in the x-linked inhibitor of apoptosis protein (xiap). common reported manifestations include recurrent hemophagocytic lymphohistiocytosis, splenomegaly, crohns-like inflammatory bowel disease, and transient hypogammaglobulinemia without reductions in major t cell or b cell repertoires, with the exception of inkt cells and mait cells. however, with only~100 known cases worldwide, we are likely only beginning to understand the phenotypic spectrum of this disease. objectives: to describe additional manifestations of xlp-2 that expand our current understanding of its phenotype. methods: a 26 year-old male with adult-onset, treatment refractory ulcerative colitis was evaluated in the immunology clinic for a history of recurrent sinopulmonary infections, skin abscesses, and recurrent ebv and vzv infections. extensive laboratory testing was performed in the course of his evaluation, including lymphocyte immunophenotyping, lymphocyte proliferation and cytotoxicity studies, quantification of total immunoglobulin levels and specific antibody function, hiv testing, and genetic testing. results: laboratory testing was significant for persistent cd4 lymphocytopenia ranging from 380-459 cells/mcl (rr: 5031736 cells/mcl). total b cell count was normal but b cell subsets showed an elevation in the percentage of naïve b cells (range: 85. .7%), low non-switched memory b cells (range: 4.7-6.0%, rr: 7.0-23.8%), and low to low-normal switched memory b cells (range: 7.2%-8.3%, rr: 8.3-27.8%), a pattern that has been seen in some autoimmune diseases. genetic testing with a commercial immune deficiency panel (invitae corp) showed a pathogenic mutation in xiap [exon 2, c.664c>t (p.arg222*)]. this mutation has previously been reported to cause a premature stop codon and reduced xiap function. the patient was referred for hematopoietic stem cell transplant and is currently awaiting transplant with a matched unrelated donor. conclusions: xlp-2 is typically reported as having normal t cell, b cell, and nk cell counts, but the presence of persistent cd4 lymphocytopenia in this patient illustrates that this is not always the case. our patient also had abnormalities in his b cell repertoire that have not been previously reported in xlp-2. additionally, xlp-2 has been associated with crohns disease and celiac-like bowel diseases, while our case indicates that the phenotype may also include ulcerative colitis. (9) submission id#420740 a case report: enteroviral encephalitis as a consequence of partial humoral immunodeficiency in a chronic lymphocytic leukaemia patient treated with rituximab hadeil morsi, st3 immunology st3, oxford university hospitals introduction/background: enteroviral (ev) infections are prevalent and usually self limited or cause mild gastrointestinal manifestations. however , in the context of primary antibody deficiency , rare cases has been reported to develop meningoencephalitis and been linked to poor outcome with fatality or chronic course. ev meningoencephalitis is even far rare reported in the era of rising secondary humoral immunodeficiency as a consequence of b cell depleting therapy e.g. rituximab and lymphoproliferative malignancies. limited treatments for ev encephalitis are available to date, apart from intravenous immunoglobulin replacement which has variable efficiency. objectives: studying such rare cases of ev meningoencephalitis as a consequence of antibody deficiencies would help to develop guidelines for intravenous immunogobulin replacement for treating these infections to improve outcome as well as predicting patients at higher risk who should be considered for prophylactic immunoglobulin therapy. methods: herein, we report a rare case of proven enteroviral meningoencephalitis following rituximab based therapy for b-cell chronic lymphocytic leukaemia and an uneventful six months period of follow up. he was found to have persistent absent b cells six months after completing six cycles of fludarabine, cyclophosphamide and rituximab therapy. interestingly, he had partial pneumococcal igg serotypes deficiency, whilst his total igg, igm and iga were all within normal limits throughout the course of the disease. the patient was treated empirically with intravenous immunoglobulin when his subtle confusion progressed to overt behavioural changes. initially his level of consciousness continued to deteriorate and he was not communicating. results: fortunately enough, the patient did have a remarkable improvement of gcs within couple of days and a slower recovery of higher mental functions e.g. memory and calculations in the next couple of months. conclusions: early suspicion and detection of entervorial meningoencephalitis in patients at risk of secondary antibody deficiency is crucial for timely ivig replacement and better outcome. patients with haematological malignancies and those on b cell depleting immunotherapy should be screened for pneumococal igg serotypes as part of secondary immunodeficiency workup. further studies on enteroviral neurological meningo/encephalitis are required to optimise ivig therapy and prognostication. furthermore, such studies provide an important asset to reveal the underlying mechanisms for humoral/b-cell mediated protective response against ev compared to other t-cell mediated viral immunity, whilst highlighting the mechanisms of immunodeficiency in cll and immunotherapy. (10) submission id#418733 a comparison of immune reconstitution following human placenta-derived stem cells (hpdsc) with umbilical cord blood transplantation (ucbt) vs. ucbt alone in pediatric recipients with malignant and non-malignant diseases introduction/background: ucbt is a safe and effective treatment in children (geyer/cairo et. al bjh, 2011) . however, due to a limited concentration of hematopoietic progenitor cells (cd34+) in ucb, ucbt has been associated with delayed hematopoietic reconstitution and a higher incidence of engraftment failure. hpdscs contain a rich population of hpcs, are low in hla class i/ii expression and t-cells, and have regenerative, anti-inflammatory, and immunosuppressive properties (cairo et al bmt, 2015) . objectives: to determine whether ucbt + hpdsc (vs. ucbt alone) is associated with enhanced hematopoietic and immune cell reconstitution in children with malignant and non-malignant diseases. methods: immune cell reconstitution at days +100, 180, 270 and 365 was assessed in children who received ucbt with hpdscs at nymc (nct01586455, ind#14949). minimum tnc was 5 x 10^7/kg (4/6 hla match) or 3.5 x 10^7/kg (5-6/6 hla match). immune cell subset counts at these time points were compared to those from a historical population of pediatric recipients of ucbt alone (geyer/cairo et. al bjh, 2011) . results: twenty four patients 18 years were enrolled. mean age was 6 (range, 0. [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] years. malignant diseases =14, non-malignant diseases =10. fourteen patients received myeloablative conditioning (mac) and ten patients received reduced toxicity conditioning (rtc). there were no severe adverse events associated with hpdsc infusion. two patients with non-malignant disease receiving rtc using alemtuzumab experienced primary graft failure. probability of neutrophil engraftment was 91.6 %, median day 22 . of evaluable patients at day 100, the probability of platelet engraftment in neutrophil engrafted patients was 100%, median day 43.5 (20-98) . at days 30, 60, 100 and 180, mean percent donor chimerism in whole blood was 94, 98, 95, and 99%, respectively. average percent of whole blood hpdsc chimerism was 1% at day 30 and <1% at beyond day 60. one patient with malignant disease relapsed. 12 month overall survival was 83.3%. there was no significant difference in cd3, cd4, cd8, cd19 and cd56 immune cell reconstitution following ucbt + hpdsc vs. ucbt alone (image 1). conclusions: these results suggest that ucbt ± hpdsc results in similar immune cell reconstitution. a larger cohort with extended follow-up would be required to confirm these preliminary findings. supported by a grant from celgene cellular therapeutics. a decade of disseminated abscesses due to mycoplasma faucium in a patient with activated pi3k syndrome 2 (apds2) introduction/background: pik3r1 monoallelic mutations are known to be responsible for apds-like 2 syndrome, a rare form of primary immunodeficiency presenting as combined immunodeficiency or hyper-igm like phenotype. this study reports a patient carrying heterozygous pik3r1 mutation with early onset and long-term disseminated abscesses due to mycoplasma faucium in both peritoneal abscess and skin, with generalized involvement in neck and both upper extremities. objectives: we describe clinical management of retroperitoneal and skin abscesses before molecular diagnosis was available in a patient with a primary immunodeficiency. identification by rdna 16s in so-called sterile abscesses may confirm the clinical suspect of an oportunistic infection. furthermore, this study offers insight on the pik3r1 suspicion even in the absence of higm-like phenotype. methods: a 16-year-old girl with 2-year history of recurrent peritoneal effusion, which had been drained repeatedly was admitted in our institution for a 10-year history of multiple supurative cutaneous and lymph node-abscesses (fig1&2a-c). she had prior diagnosis of agammaglobulinemia under standard subcutaneous immunoglobulin replacement therapy and subcutaneous interferon-gamma treatment. on physical examination at the age of 16 years, she was stunted (weight and height below the 3rd percentile), with facial, arm skin abscesses and right fistulized axillary lymphadenopaties, 5-6cm hepatomegaly and giant splenomegaly results: she had her first immunological work up at the age of 6 years during one isolated episode of knee arthritis and first episode of skin abscesses. serum immunoglobulins revealed panhypogammaglobulinemia (igg<7.3 mg/kg, iga <5.8 mg/kg, igm <4.3 mg/kg) with low b cell count. on her back, there was a 10x15cm, elastic, neither painful nor tender mass. after proper assessment by ct scan and mri she had her retroperitoneal abscess drained percutaneously, and healed with sclerotherapy (percutaneous alcohol and polidocanol instilation) by the interventional radiologist ( fig 2b) . analysis of drained pus as well as pus of skin abscesses was made by 16s rdna pcr, having coincidence of 99.9% with mycoplasma faucium . combination antibiotic therapy (doxycycline and ciprofloxacin) was started with favourable response. unfortunately skin abscesses then relapsed. t-cell phenotype only showed t-cell lymphopenia with senescent (tem & temra expansion) phenotype. whole exome sequencing revealed a heterozygous mutation, previously reported (c.1425+1g>t) conclusions: in summary, this report emphasizes the suspicion of a combined immunodeficiency in the presence of multiple abscesses by mycoplasma, the usefulness of rdna 16s in order to achieve proper objectives: we describe a 15-year-old male patient with novel heterozygous mutation of ep300 gene; his first manifestations were initially characterized by infections, cytopenia and hypogammaglobulinemia suggesting a common variable immunodeficiency (cvid), but later on, persisting lymphopenia was suggestive of a combined immunodeficiency. methods: the patient was born to unrelated healthy italian parents at 34 weeks gestation with adequate weight for gestational age. shortly after birth, he underwent several surgical procedures due to interventricular defect, aortic coarctation, double outlet right ventricle, open botallis duct, and gastroesophageal reflux. at the age of four, he came to our attention due to stomatitis. clinical examination revealed dysmorphisms (microcephaly, wide forehead, sparse eyebrows, high nasal root, low-hanging columella, thick lips, micrognathia), splenomegaly (spleen diameter 11.6 cm at abdominal ultrasound), and severe developmental delay. in the course of the infectious episode, blood tests showed leukopenia associated with neutropenia (white blood cells 2.210/mm3; neutrophils 20/ mm3) and thrombocytopenia (platelets 1.000/mm3). analysis of bone marrow aspirate revealed normal differentiation of both myeloid and erythroid lineages. treatment with high doses immunoglobulin resulted in increase of platelet counts (up to 44000/mm3 after 1 month), while neutrophil counts spontaneously returned to normal when the infection resolved. however, thrombocytopenia relapsed (2000/mm3) after 2 months and intravenous high-doses of corticosteroids did not achieve normal platelets count. despite oral corticosteroid treatment started at the age of six, two episodes of autoimmune hemolytic anemia occurred. during the following six years of follow-up, the patient experienced recurrent infections (stomatitis, upper respiratory tract infections, and skin abscesses), but none of the episodes has required hospitalization. but, at the age of ten, he was admitted to the hospital because of severe cultures negative diarrhea. despite immunoglobulin replacement therapy was started at the age of fourteen, he was admitted twice due to bilateral pneumonia requiring continuous positive airway pressure and, a few months later, acute respiratory failure with evidence of mycoplasma pneumoniae and rhinovirus infections. immunological evaluation under chronic corticosteroid treatment at different time points showed persisting lymphopenia, with lymphocyte counts ranging from 350/mmc3 to 2100/mm3 , thrombocytopenia (platelets ranging from 0/mm3 to 308000/mm3), undetectable anti-diphteria and anti-tetanus toxoid antibodies, and splenomegaly. interestingly, analysis of isohemoagglutinins, revealed low titers of anti-a (1:8 ) at 4 years of age, but normal immunoglobulins (igg 685 mg/dl, iga 73 mg/dl, and igm 83 mg/dl). at the age of seven, reduced mitogen proliferation, hypogammaglobulinemia (igg 274 mg/dl; iga 17 mg/dl, igm 185 mg/dl), increased cd3+tcr+cd4cd8 t-cell counts (2.7/3.6%) and impaired fas mediated apoptosis as measured in two separate assays (table 1 ) . at the age of fourteen, evaluation of b-cell subsets showed increase of cd21locd38lo cells and reduction of switched memory b-cells. analysis of t-cell compartment unveiled a decreased proportion of cd31+ccr7+cd45ra+ recent thymic emigrants (rte) cells and ccr7+cd45ra+ naive cells, with prevalence of effector memory t-cells (ccr7-cd45ra-) ( table 1) . interferon signature gene expression showed borderline levels of ifi27 (data not shown). because of the decrease of igg and the infectious episodes ivig treatment was started at age of fourteen. a molecular investigation performed by whole exome sequencing (wes) revealed a novel heterozygous missense mutation (nm_001429. 3:c.4763t>c , p.met1588thr) in the exon 29 of the gene ep300 encoding the histone acetyltransferase (hat) protein p300. results: few immunological reports are available in rsts patients1-4. in keeping with previous data1,3,4, our patient presented with progressive b-and t-cell lymphopenia, hypogammaglobulinemia with poor antibody response but also reduced naïve t cells, evans syndrome, splenomegaly, and defective lymphocyte apoptosis with increased dnt. at the age of seven, the patient presented the features of cvid. flow cytometry revealed expansion of cd19hicd21locd38lo b cells, that is frequently associated with splenomegaly in cvid patients7, and reduced switched memory b-cell, previously reported in a rsts patient with crebbp mutation4 (fig. s1 ). lougaris et al. reported expansion of cd21locd38lo b-cells in nf-kb1 haploinsufficiency 8, and this suggests in our opinion that alterations in the nf-kb pathway due to ep300 mutations may affect b-cell differentiation. compared to healthy controls and cvid patients with predominant infectious complications, upregulation of interferon responsive genes in cvid subgroup with noninfectious complications (i.e hematologic autoimmunity, lymphoproliferation) and lymphopenia with reduced total b cells and switched-memory b cells has been demonstrated9. borderline levels of ifi27 expression under corticosteroid treatment represent a novel finding in rsts. interferon signature may identify and better characterize subgroup of rsts patients with autoimmune cytopenias and lymphopenia. at the age of fourteen, analysis of lymphocyte subsets revealed decreased total cd3+, cd4+, cd8+, and of both naïve cd4+ and cd8+ cells. elevated and persisting igm levels were also observed (fig. s2 ). according to our data, increased igm levels may be related to high proportion of terminal differentiated igm+ cells. these data (infections requiring hospitalization, immune dysregulation, lymphopenia, reduced naïve t cells, and reduced proliferation to mitogen) together with clinical history (fisher evans syndrome and lymphoproliferation) and exclusion of known syndromic immunodeficiencies, suggested a diagnosis of combined immunodeficiency10. conclusions: our case underlines the value of wes in patients with difficult phenotype-genotype correlation. no rsts typical traits were present and, prior to wes, several syndromes and immunodeficiencies were excluded. our report expands the phenotypic spectrum of ep300 mutations, thus in syndromic patients with clinical and immunological overlap between cvid and cid ruling out ep300 mutation should be advisable. furthermore, immunological work-up should be taken into consideration in rsts patients, in order to early identify immunological abnormalities that may lead to severe immune-hematological complications. introduction/background: interferon gamma receptor (ifngr1)-related disorders are rare variants of mendelian susceptibility to mycobacterial diseases. although hematopoietic stem cell transplantation (hsct) is curative, it is complicated by high rates of delayed or failed engraftment thought to be due to high concentrations of interferon (ifn)-gamma. umbilical cord blood transplantation additionally increases risk of graft failure. objectives: describe a pediatric patient with non-functional ifngr1 who successfully underwent umbilical cord blood transplantation. methods: direct clinical care of described patient with additional electronic medical record chart review. results: the patient is a 19-month-old boy of yemeni descent who initially presented with significant hepatosplenomegaly and extensive lymphadenopathy, including a large mediastinal mass. he then developed salmonella enteritidis sepsis requiring numerous antimicrobials, vasopressor support, intubation and continuous renal replacement therapy. his evaluation showed a hyperinflammatory state with elevations in ferritin, ifn-gamma, scd25, il-10, il-13, il-17, il-6 and il-8 levels. maximal ferritin and ifn-gamma levels reached 12065.4 ng/ml and 463 pg/ml (normal <5 pg/ml), respectively. flow cytometry revealed normal expression of ifngr1 and il12r but absent ifn-gammastimulated stat1 phosphorylation, suggesting defective ifngr1 signaling. genetic testing showed a previously unreported homozygous mutation in ifngr1 (c.373+2t>c) which affects a donor splice site in intron 3 and is predicted to cause absent protein function. dexamethasone and a single dose of alemtuzumab (0.2 mg/kg) were given to decrease inflammation. he then underwent allogeneic hsct using a 5/6 human leukocyte antigen matched umbilical cord unit following a reduced-toxicity conditioning regimen of alemtuzumab (0.6 mg/kg), fludarabine (180 mg/m2) and busulfan (auc 55 mg/l*h). plasma ifn-gamma was undetectable prior to starting conditioning and on the day of transplant. neutrophil engraftment occurred on day +14 with day +30 posttransplant chimerism analysis of peripheral blood myeloid cells showing the presence of donor cells only. conclusions: these early results suggest that umbilical cord blood transplantation may be feasible in patients with ifngr1-related disorders provided adequate control of inflammation is gained prior to transplant. introduction/background: introduction: btk is a cytoplasmic tyrosine kinase that activates phospholipase c2 (plc2) via phosphorylation, which ultimately leads to the activation of nfk, which is essential for b cell development and survival. mutations in btk lead to x-linked agammaglobulinemia (xla). in addition to the pleckstrin homology and tyrosine kinase domains, btk contains two src homology domains, sh2 and sh3, which are essential for btk function. we describe a novel btk mutation (c.1197a>t) resulting in v335a substitution in the sh2 domain that results in aberrant xla function with nearly normal btk protein expression. objectives: case report/results: the male proband presented with recurrent otitis media, persistent fevers and neutropenia beginning in the first year of life with an igg level of 46 mg/dl and a lack of b cells (0 cell/mm3), as demonstrated by flow cytometry. btk protein expression in monocytes, also determined by flow cytometry, was equivalent to controls. family history is significant for a maternal uncle with history of recurrent sinus infections and pneumonias with low iga and igm, low to normal ige, and an absent vaccine response. flow cytometry also showed an absence of b cells and essentially normal btk protein expression in his monocytes compared to controls. targeted high throughput sequencing of both proband and the uncle revealed a previously unreported missense mutation in exon 12, leading to the substitution of an aspartic acid residue for a valine (v335d.) the mutation is in the highly conserved sh2 domain of btk (conservation phylop conservation score 2.8.) the probands mother and his sister were shown to be carriers of the same mutation and had normal serum immunoglobulin levels and normal numbers of b cells. methods: we hypothesized that if the btk v335d mutant protein was non-functional, the female carriers of the mutation would only express wild type (wt) btk in their b cells while their monocytes would express both wt and mutant btk. to test this hypothesis cd19+ b cells and cd14+ monocytes were purified from pbmc by fluorescence activated cell sorting (facs) from the sister, cdnas were generated from the respective populations of cells, and the btk cdna was sequenced using high-throughput sequencing. results: at a read-depth greater than 16,000, cd19+ b cells demonstrated btk expression only from the wt allele (~99% wt btk) whereas both the wild type and mutant allele of btk were expressed at approximately equal levels in monocytes. conclusions: discussion: these results define a novel mutation in btk that nominally affects protein expression, but alters function. the v335a substitution is found in the d structural element of the sh2 domain that is part of a hydrophobic phosphotyrosine binding pocket. a mutation in the adjacent residue, y334s, has been shown to alter protein conformation and decrease binding affinity to plc2 by roughly 4-fold resulting in xla. our study, using a carrier harboring the c.1197 a>t mutant btk, demonstrated that in contrast to mononuclear cells, b cells only expressed the wt allele. this is consistent with the loss of function of v335a btk protein, thereby causing xla in both the proband and affected uncle. introduction/background: pyoderma gangrenosum (pg) is often associated with systemic autoimmune diseases but it has rarely been reported with common variable immune deficiency (cvid). while genetic analysis has been increasing in both disease domains, there has been little investigation into the genetic components associated with the cooccurrence of these entities. heterogeneous nfkb1 mutations have recently been identified in familial cases of cvid, though rarely have they been associated with pg. objectives: this case describes a novel nfkb1 mutation that may link both cvid and pg, and bolsters the recent identification of heterogeneous nfkb1 mutations in cvid. methods: a 24-year-old woman with a history of frequent skin infections in childhood presented with persistent, infected wounds following cholecystectomy. upon admission, she was started on broad spectrum antibiotics but continued to have fevers and leukocytosis. labs were also notable for elevated crp (340; normal (n):0.1-3) and esr (120; , with low c3 (60; n:81-145), c4 (<10; n:16-39) , and ch50 (<10; n:>74). wound cultures grew multi-drug resistant coagulase negative staphylococcus. despite broad spectrum antibiotics, the wounds failed to heal. dermatology was consulted and punch biopsy revealed a dense neutrophilic infiltrate and no identifiable pathogens, which supports the diagnosis of pg. the patient was started on high dose steroids (1mg/kg/ day) and had a rapid response with decreased skin inflammation and lesion expansion. unfortunately, the patient developed posterior reversible encephalopathy syndrome (pres) on steroids and therefore pg treatment was changed to infliximab as recommended by dermatology. further laboratory testing found that the patient also had low igg (499; n: 700-1600) and iga (34; , and a diagnosis of cvid was made given her clinical history of recurrent skin infections. genetic testing was pursued to evaluate additional pg therapy options and this revealed a heterozygous mutation in nfkb1 (c.a2415g; p.q805q), located 2 base pairs upstream of the splice donor site for exon twenty-one. while this mutation has not been previously identified as a pathogenic variant, similar mutations in this gene have been linked to autosomal dominant cvid. the patients father also carried a similar mutation but without any evident clinical phenotype. results: nfkb1 plays a crucial role in both immune and inflammatory responses. this case highlights a novel mutation in nfkb1 that has not been previously described as a disease-causing change. other mutations resulting in nfkb1 haploinsufficiency have been associated with cvid and rarely with concurrent pg, as in this case. based on the location of the mutation, it is expected that the variant causes obliteration of the normal splice site and therefore results in defective mrna that encodes p50/ p105. interestingly, studies on p50 knockout mice show decreased levels of igg, iga, and ige but not igm and our patient similarly had low levels of igg and iga but normal igm. conclusions: further studies are needed to determine if there are other links to this novel nfkb1 mutation in patients with cvid and pg. (17) submission id#412594 a rapid flow cytometric analysis of dna repair proteins reveals a radiosensitive phenotype in bcl11b deficiency associated with severe combined immunodeficiency (scid). introduction/background: we present the second report in the literature of a patient with immunodeficiency, dysmorphic features, growth retardation, and a homozygous variant in the dna ligase i (lig1) gene with associated absence of full-length lig1 protein. results: this is a now 2 year old girl who was the fourth child of parents who are first cousins. she has one healthy older brother, a second older brother who died within 12 hours of birth of meconium aspiration, and an older sister who died at six months of age of an upper respiratory illness / pneumonia after a history of congenital anemia, poor weight gain, cardiomegaly and hepatomegaly. she was born at 37 weeks and spent the first five weeks of life in a neighboring hospital neonatal intensive care unit (nicu) for hepatomegaly, mild cardiomegaly (previously identified on fetal ultrasound) and congenital anemia (requiring transfusions). her exam was and remains notable for weight, height, and head circumference below 3rd percentile, prominent forehead, hypotelorism with epicanthal folds, downslanting palpebral fissures, and low set, posteriorly rotated and prominent ears. after discharge to home from the nicu, her course was subsequently complicated by poor weight gain, chronic diarrhea beginning after her first rotavirus vaccine, and multiple deep vein thromboses. she then became critically ill at 6 months of age with respiratory failure, and was transferred to our institution for respiratory oscillator support. absolute lymphocyte count on admission to our institution was 0.3 k/μl (total wbc 4.0 k/μl, anc 2.7 k/μl) with agammaglobulinemia. she was diagnosed with and treated for pneumocystis jirovecii pneumonia, gradually weaned from oscillator to room air, and was discharged home five weeks later on 0.4 mg/kg every other week igg replacement. she has had no serious infections requiring hospitalization in the 18 months since. alc has remained persistently below 1.0 k/μl with a corresponding uniform deficiency of t, b, and nk cells and no detectable trec positive t-cells. whole exome sequencing identified homozygosity for a c.914g>a coding region variant in the lig1 gene not previously reported in the literature. a fibroblast cell line was successfully established and western blot shows an absence of full-length lig1 protein. further molecular characterization is in progress. conclusions: this second reported case provides further evidence for dna ligase i deficiency as a distinct clinical entity comprising immunodeficiency, dysmorphic features, and growth retardation. introduction/background: chronic mucocutaneous candidiasis (cmc) is associated with a heterogeneous group of primary immunodeficiencies. autosomal dominant stat1 gain-of-function (gof) mutations have been identified in up to 50% of patients with cmc. these mutations lead to impaired il17a/f t cell immunity although the underlying mechanism is unclear. there seems to be no genotype-phenotype correlation. recently, jak inhibitor therapy has been reported to improve cmc and autoimmunity in patients with stat1 gof mutation. objectives: we describe an infant with cmc associated with a novel stat1 gof mutation. results: a 7-month-old girl was referred to our immunodeficiency clinic with chronic diaper rash since 2 weeks of life, failure-to-thrive, and history of labial abscess complicated by rectolabial fistula. she was subsequently diagnosed with food protein-induced enterocolitis syndrome triggered by cows milk-based formula. laboratory evaluation revealed normal cbc with differential, lymphocyte subsets, mitogen response, immunoglobulin levels, antigen response to candida, and neutrophil oxidative burst assay. whole exome sequencing identified a de novo heterozygous variant in stat1 (c. 1627t >c, p.cys543arg) . further evaluation of this mutation revealed increased gas (gamma activation sequence) reporter activity in response to ifng stimulation suggesting that this is a gain-of-function mutation. the patient later developed significantly elevated liver enzymes while on fluconazole treatment, candida parapsilosis sepsis, granulomatous lesions in the liver, splenic lesions, intermittent thrombocytopenia and n o r m o c y t i c a n e m i a . s e p s i s a n d l i v e r l e s i o n s r e s o l v e d on amphotericin treatment but other findings, including tpn dependency persisted. we are planning to initiate a jak inhibitor therapy, ruxolitinib. conclusions: this case is a possible genotypic and phenotypic expansion of cmc due to stat1 gof. professor, the university of british columbia introduction/background: b cell cll/lymphoma 11b (bcl11b) is a zinc finger protein transcription factor with a multitude of regulatory functions in the integumentary, central nervous, cardiac, and immune systems. it is critical for t cell lineage commitment, development, differentiation, survival, and function. in addition, it also specifies the identity and function of innate-like lymphocytes, including t cells, innate lymphoid cells (ilcs), and invariant natural killer t cells (inkt). however, little is known about its function in the human immune system, especially in the context of immune disorders. objectives: to understand the immunopathogenesis of a novel p.c826y bcl11b variant. methods: research study protocols were approved by our institutional research ethics board. two members of the family were enrolled (the index patient and her father). written informed consent for genetic testing and participation was provided by the parents for the child. genetic, bioinformatic, proteomic, and biochemical analyses were performed. results: we have identified the second described case of immune disease caused by a de novo heterozygous damaging variant of bcl11b (p.c826y). this young girl presented with intellectual disability, microcephaly, severe atopy, eczema, alopecia totalis, and brittle nails. extensive clinical immunophenotyping of patient blood showed initially unremarkable b and t cell populations. however, the patient possessed abnormal rare innate-like lymphocyte populations (inkt, dn t cells). using mass cytometry (cytof), a technique capable of concurrently analyzing 40 parameters in a single cell, we were able to examine various innatelike lymphocyte populations, including t cells, ilc1-3, and nk cells. we found that the patient possessed severely compromised numbers of t cells, thus potentially implicating the p.c826y variant in t cell development and function. conclusions: the identification of decreased t cells in a patient with a p.c826y variant of bcl11b suggests that bcl11b is important for human t cell development and provides novel insights into the roles of both bcl11b and t cells in regulating atopy and autoimmunity. introduction/background: adenosine deaminase 2 deficiency caused by mutations in ada2 gene is a newly recognized disorder. it is associated with a spectrum of vascular and inflammatory phenotypes, ranging from early onset recurrent stroke to systemic vasculopathy or vasculitis. objectives: we describe a 13 year old female patient with features of early onset immune thrombocytopenia (itp), autoimmune hemolytic anemia (aiha), chronic splenomegaly and variable abdominal lymphadenopathy. she was diagnosed with evans-syndrome and treated with rituximab at 19 and 27 month of age. from 3 years of age she developed recurrent infections, hypogammaglobulinaemia with specific antibody deficiency, progressively decreasing class-switched memory b cells, and increased cd3+cd4-cd8-//t cells (4%). differential diagnosis included common variable immunodeficiency (cvid) or autoimmune lymphoproliferative syndrome and therefore a broad search for causative genetic defect was initiated. the parents are first cousins of middle-eastern origin suggesting an autosomal recessive inheritance. patient was stable on long-term mycophenolate mofetil (mmf) and immunomodulatory dose (1g/kg/ month) ivig treatment. methods: genomic dna of the patient was sequenced with next generation sequencing technology. a panel of 250 genes linked to primary immunodeficiency was analyzed. the identified variant was confirmed by sanger sequencing. results: genetic testing revealed a homozygous pathogenic mutation in the ada2 gene with one base pair duplication in exon 2 (c.144dup. p.arg49alafs*13) that creates a frame shift starting at codon arg49. the new reading frame ends at a stop codon 13 positions downstream, likely resulting in a truncated protein. plasma ada2 activity of the patient was markedly reduced (0.2 mu/ml, normal 4.8-27.2) and confirmed the diagnosis of ada2 deficiency. the parents of the patient are heterozygous carriers of the same mutation. unlike most previously reported cases, this patient had an extended phenotype with no neurological evidence of vascular pathology, however brain mri revealed two silent lacunar infarct or vasculitis related changes. we speculate whether the long-term mmf or ivig therapy might be protective against vasculitis. conclusions: ada2 deficiency may present with a wide spectrum of clinical phenotypes beyond classical vasculopathy. the diagnosis should be considered in patients with hematological autoimmune disease, splenomegaly and/or cvid like presentation. better understanding of pathophysiology of ada2 deficiency may help diagnosis and targeted treatment. professor, university of california, los angeles ca introduction/background: adenosine deaminase (ada) deficiency as a cause of severe combined immunodeficiency (scid) is distinct from other forms of scid in several ways. historically, survival and clinical outcome of infants with ada scid have been inferior compared to infants with other scid genotypes. there are multiple treatment modalities available for ada-scid, including enzyme replacement therapy (ert), allogeneic hematopoietic cell transplant (hct) and experimental autologous transplant of gene corrected cells (in recent years preceded by low dose busulfan), designated as gene therapy (gt). in addition, there is a growing body of evidence for effects of ada deficiency on non-immunologic organ systems that may contribute to the historically poorer outcomes of these infants. therefore, it is important to evaluate the cohort of patients with ada scid separately from other scid cases. objectives: to capture incidence and treatment trends and to compare outcomes following available treatments for this rare inborn error of metabolism and other forms of scid, the primary immune deficiency treatment consortium (pidtc), a network of 45 north american immunology and transplant centers, has collected standardized data for analysis. methods: 118 ada scid patients, first treated between 1977 through 2016, were enrolled from 26 centers (range 1-25 subjects/site). ada accounted for 13% of the 926 total pidtc scid patients treated during that time. patients were entered into either a retrospective protocol (pidtc 6902, n=96) or a prospective protocol starting in 2010 (pidtc 6901, n=22) . ada-scid patients who received an hct as first therapy entered either of two strata, as with other scid patients in pidtc studies, based on whether their initial presentation met definitions for typical (n=46) or leaky scid (n=6); in contrast, patients initially treated with either ert or gt were entered into a separate stratum (n=66). results: sixty-four patients (54% of all ada-scid enrollees) had ert as first therapy, but only 6 in this cohort received ert as sole therapy; 58 went on to have subsequent hct (n=30) or gt (n=28). there were various combinations of treatment cycles among these ada-scid patients. most received hct {+/-subsequent treatments} (44%), ert followed by hct {+/-subsequent treatments} (25%), or ert followed by gt {+/-subsequent treatment} (24%); several patients received multiple successive treatment modalities, representing either failure of initial treatment or planned progression from ert to cellular therapy. two-year survival has improved over time from 69% in (1977-2000 to 92% 2001-2016 (p=0.007) (figure 1 ). the survival for all other non-ada scid patients registered by pidtc over these two eras were: 78% (1977-2000) and 81% (2001-2016) . hct (either as sole therapy or after ert) accounted for >95% of cellular therapies between 1986 and 2007; in contrast, since 2007, gt was used as commonly as hct (n=27 vs. n=29, respectively). there was a trend toward better two-year survival for patients receiving gt as first cellular therapy since 2001 (100%, n=28, all after initial ert) compared to those receiving hct over the same time period (87%, n=40, either as first therapy or after ert), although this did not achieve statistical significance (p=0.073). conclusions: this study reveals the improved prognosis for patients with ada scid in recent years and the emergence of gt as a new treatment modality. further analyses are investigating the impacts of prior infection and treatment modality, including effects of conditioning, on outcomes (survival, event free survival, clinical outcomes and completeness of immune reconstitution) for ada-scid in successive eras. this study may identify optimal treatment approaches for future ada scid patients. sponsored by the pidtc, a member of the rare diseases clinical research network (rdcrn) and funded by u54 ai 082973 (niaid and ordr, ncats, nih). dbk has potential financial conflict of interest as a member of the scientific advisory board for orchard therapeutics and an inventor on intellectual property which ucla has licensed to orchard therapeutics related to gene therapy for ada scid. jp discloses that her spouse is employed at invitae, a dna sequencing company. intern, imam abdulrahman bin faisal university introduction/background: patients with diabetes mellitus are immunologically vulnerable population to develop different types of microbial infections. immunization has an important role in infection prophylaxis. in fact, vaccines containing thymus-dependent antigens protect patients with diabetes as they produce massive and complex immune response and feature immunologic memory. the recommended vaccinations for patients with diabetes mellitus are influenza vaccination yearly and pneumococcal vaccination. in observational studies, influenza vaccine has been shown to be similarly effective in adults <65 years of age with diabetes as in older patients with or without diabetes [1] . among immunocompetent elderly, vaccine efficacy of the 13-valent pneumococcal conjugate-vaccine (pcv13) was modified by dm with higher vaccine efficacy among subjects with dm [2] . the hepatitis b vaccination should be given to unvaccinated adults with diabetes mellitus who are ages 19 to 59 years. for older patients administration only after assessment of benefits and risks of acquiring hepatitis b virus (hbv). in fact, one review suggests that dm is associated with the progression of severe liver outcomes in adults with hbv [2] . on the other hand, tetanus and diphtheria vaccinations should be updated. in addition to, vaccinations such tick-borne encephalitis, meningococcal infections and other infections that put in risk diabetic patients travelling abroad. accordingly, theres a variability of vaccines that can offer a preventive method to reduce morbidity, mortality, and medical expense. in our multicenter study among eastern province saudi arabia evaluated the degree of adherence of the physicians to the immunization recommendations for adult patients with diabetes mellitus type 1 and type 2 to increased awareness of the immunization importance in diabetic patients. objectives: to increased awareness of the immunization importance in diabetic patients. in fact, patients with diabetes mellitus are immunologically vulnerable population to develop different types of microbial infections. immunization has an important role in infection prophylaxis. methods: this is a cross sectional study involving 500 adult patients with type 1 and type 2 diabetes mellitus using a questionnaire. patients will be recruited from outpatient clinics including the primary care clinics and inpatient words of king fahd hospital-al khobar and other centers in the eastern province saudi arabia. after an informed consent, baseline data will be collected. patients will be then asked if they received the recommended vaccines and who was the provider. their knowledge regarding the needed immunization will be also tested. they will then be asked about frequency of upper respiratory tract infections and pneumonia they had over the last 2years. results: we are expecting to find low adherence to the recommended immunizations by the physician. we may also find that patients who received the vaccines has low incidence of related infections. conclusions: increased awareness of the immunization importance in diabetic patients and adherence to immunization as part of standard care of adult patients with diabetes mellitus that offer a preventive method to reduce hospitalizations, mortality, and medical expense. 1-10, 11-20, and 21-30 bases. repeatability and reproducibility of the assays were 0.994 and 0.998, respectively. 99.5% of the target regions were covered with over 15x sequencing depth. we showed that the assays had 0.748 sensitivity to detect single-exon deldups and 1.000 sensitivity to detect copy number aberrations covering two or more exons. using acmg guidelines for variant classification a diagnosis was established in 20% patients that were sent for comprehensive panel analysis. conclusions: conclusions: our results demonstrate the analytic validity of the developed tests and show that the technology is well-suited for clinical diagnostics of inherited eye disorders. it also demonstrated a cost-effective diagnostic tool to simultaneously diagnose various types of mutations from snvs to copy number variations. introduction/background: loss of function (lof) and null mutations in orai1 and stim1 cause a rare autosomal recessive immunodeficiency by abolishing calcium release-activated calcium (crac) channel function and store-operated ca2+ entry. the clinical presentation is characterized by scid-like disease, dental enamel defects, muscular hypotonia and anhidrotic ectodermal dysplasia. objectives: here we present the outcome of calcium assessments performed on lymphocytes from an adult patient with unusual infections and a purported novel single pathogenic variant in orai1. methods: the ca2+ response in lymphocytes following activation by varying concentrations of non-cross-linked anti-cd3 was assessed by flow cytometry. results: the patient was a 47 year old female with a history of seasonal and medication allergies and recurrent sinus infections, who had recently developed an acute infection of her right first metatarsal joint. cultures from the joint space grew neisseria gonorrhea and atypical mycobacteria at two different time points. hiv screening was negative. follow-up testing with the mantoux test and quantiferon gold suggested that she also had latent tuberculosis infection, for which she was started on rifampicin therapy. immunologic evaluation revealed normal complete blood count and differential, normal t, b and nk cell counts, normal immunoglobulin g, a, and m levels, as well as normal responses to polysaccharide vaccines and normal t cell proliferative responses to mitogen and antigen stimulation. however, given the identification of atypical organisms from joint fluid cultures as well as latent tuberculosis (despite the lack of significant risk factors), genetic testing was recommended by her local physicians to rule out an underlying primary immunodeficiency. an invitae primary immunodeficiency panel identified a single novel pathogenic variant in orai1. she was then referred to our institution for further evaluation. so far, individuals identified as heterozygous for lof or null mutations in either orai1 or stim1 have lacked any phenotype associated with crac channelopathy. however, there have been reports of abnormalities in calcium response in parents of a number of these patients, who are heterozygous for the disease-causing mutation. to examine the functional effect of the observed mutation in our patient, her freshly isolated pbmcs were loaded with indo-1 and the ca2+ response of her lymphocytes were assessed by flow cytometry. this showed a dose-dependent decrease in the patients t cell response to non-cross-linked anti-cd3 in comparison to the normal control, i.e. there was a clear decrease in the patients ca2+ response at 1 microgram/ml in comparison to the normal control, but the decrease was rectified upon stimulation with 5 microgram/ml or more of anti-cd3. conclusions: these findings provide functional support for the identification of a new pathogenic mutation in orai1. nevertheless, it is not yet clear if the mutation has any mechanistic role in the patients recent clinical presentation. introduction/background: patient 1: 10 years old boy, born to nonconsanguineous parents, with hypothesis of autoimmune encephalitis (vasculitis), which was not characterized. csf has already been routed to barcelona 2 times and to vienna 1 time. refractory epilepsy remains (uses 5 drugs yet). he had continuous fever during the entire hospitalization. he had adhd (took ritalin for 2 years) and central auditory processing deficit. one year ago he began to have fever for 3 days, improving later but evolving with bilateral otitis, predominantly on the left ear, accompanied by sinusitis. soon after that he began presenting epilepsy that worsened severely. he was interned and took acyclovir ev, associated with hydantoinate (had stevens-johnson by the drug, suspending and improving quickly). in march had uti + amo, several infections requiring meropenem + vanco, among others. received flebogamma 500 mg/ day/5 days, twice; he also received weekly rituximab for a few months. in use of carbamazepine, clobazan, phenobarbital, levotiracetam and vigabatrin 3 times a day. personal antecedents: allergic rhinitis, bronchial asthma, recurrent otitis media, iga deficiency. patient 2: male, 9 years old, born to non-consanguineous parents, with history of repeated infections in the upper respiratory tract from one year of age with prolonged dry cough and sore throat in all episodes, treated with dexamethasone without improvement, followed by antibiotics with resolution of the condition. at age five, in february 2012, he had a new episode of sore throat and cough that lasted three months, improving spontaneously thereafter. in june 2012, new episode of sore throat with elevated fever and whitish plaques in the tonsils, being prescribed benzetacil, without improvement in three days. he returned to the same emergency room, the antibiotic was replaced by zinnat (axetil-cefuroxime), but within the hospital he began to convulsionate, entering into an epileptic crisis, being hospitalized for 33 days in this service and being transferred to another hospital specialized in pediatrics, intensely investigated and treated with partial improvement of the condition. he remained in coma, not walking and talking for some months, recovering slowly with physical therapy and speech therapy. of relevant exams have: reduced iga, but before it was normal (probably induced by anticonvulsants); full-body magnetic resonance imaging demonstrates generalized lymphadenomegaly and hepatosplenomegaly; pet-ct showing signs of hypoperfusion in temporal (right), occipital and cerebellum regions (suggestive of hypoperfusion -vasculitis?) my first impression was of possible mevalonate kinase (mvk) or autoimmune lymphoproliferative syndrome (alps) deficiency. with these tests described above, i believe that the first diagnostic suspicion is that the epilepsy was triggered by hemophagocytosis in the central nervous system and consequent extremely severe epilepsy, triggered by ebv infection. objectives: to compare the clinical and genetic similarities and differences of both patients. methods: both patients wer submitted to whole exome sequencing looking for the genetic alterations associated to the disease of these patients. results: wes of patient 1 showed an allelic variant in the gene of rai1 (c.4625g.a; p.arg1542gln) possibly pathogenic and that could be related to the clinical features of the patient. wes of patient 2 showed an allelic variant in the gene of cd27 (c.281g>a; p.arg94his) heterozygous and of uncertain significance. another allelic variant was found in the gene of btk (c.707g>a; p.arg236gln) classified as of uncertain significance and hemizygous (as btk is in chromosome x). the expression of both proteins evaluated by flow cytometry is normal, decreasing but not abolishing the possibility of pathogenicity. conclusions: the similarity of the clinical presentations is striking, but the genetic alterations are totally different, leading to the presentation of this abstract. (inf-) have been associated with adult onset immunodeficiency in patients of asian origin. pathogens that cause infections in these patients include mycobacterium avium-intracellularae (mai), non-typhoidal salmonella, cytomegalovirus, penicillium marneffei, and varicella zoster virus. methods: chart review of one patient results: we present a thirty-two-year-old filipino female, with sjogrens syndrome, penicillin and vancomycin allergy, and shellfish allergy suffering from recurrent mai spinal osteomyelitis. after three months of conservative management of back pain, mri showed an abscess at l4/l5 and l5/ s1 vertebrae, which was diagnosed as acid fast bacilli on biopsy. she was treated for mycobacterium tuberculosis with rifampin, isoniazid, pyrizinamide, and ethambutal with subsequent change in antibiotic therapy after six weeks once cultures grew mai. mri showed spread of abscess to l3-s2 vertebrae. three months into treatment, she was found to have a new abscess at a different spinal site, and antibiotics were again changed. two months later, she had recurrence of disease with multiple large iliopsoas abscesses, cutaneous fistulas, insufficiency fractures of the sacrum bilaterally, and osteonecrosis of the l4 vertebra, requiring extensive surgical debridement. medications were adjusted and she was referred to immunology eight months after initial presentation to infectious disease. laboratories were notable for elevated igg (2580 mg/dl) and iga (474 mg/dl) and decreased cd4 (425 cell/ul) and cd16/56 (49 cell/ul) cells. serum electrophoresis showed low albumin, elevated gamma fraction, and polyclonal gammopathy. specific antibody titers, lymphocyte proliferation assay, ch50, and immunofixation were within normal limits. cytokine panel was significant for elevated il-2 receptor cd25 (3860 pg/ml), il-6(15 pg/ml), and il-13 (12 pg/ml), and normal tnf, inf, il-1, il-2, il-4, il-5, il-8, il-10, il-12, and il-17. further serologic testing was positive for autoantibodies to inf-. patient continues treatment with iv antibiotics and is awaiting enrollment in a rituximab trial. conclusions: autoantibodies to inf-should be considered in patients of asian origin presenting with adult onset immunodeficiency, particularly those with severe or recurrent infection with mai. a high level of suspicion is required to make this diagnosis: failure to consider this disease entity leads to delay in diagnosis with potentially significant consequences for the patient. allergy/immunology, university of south florida at johns hopkins all childrens hospital introduction/background: autoimmune and inflammatory conditions are common in cvid. these have been associated with increased morbidity and mortality. objectives: we sought to further understand and evaluate the prevalence of autoimmune and rheumatologic manifestations in patients with common variable immunodeficiency (cvid). methods: we performed a retrospective analysis of cvid patients with rheumatologic/autoimmune complications in the partners healthcare cvid cohort. we evaluated baseline patient characteristics as well as autoimmune and rheumatologic complications in this cohort of patients. results: in the partners cvid cohort, 120/210 (57%) had autoimmune or rheumatologic disease. autoimmune cytopenias were reported in 37/210 (18%) patients, including coombs positive autoimmune hemolytic anemia (n=18), idiopathic thrombocytopenic purpura (n=30), and autoimmune neutropenia (n=10). autoimmune thyroid disease was reported in 43/212 (20%) patients, including hypothyroidism (n=25) and hashimotos thyroiditis (n=18). inflammatory arthritis was present in 35/210 (17%), most commonly seronegative rheumatoid arthritis (ra) (n=18), followed by inflammatory arthritis (n=8), seropositive ra (+rf or +ccp antibody) (n =6), psoriatic arthritis (n=2), and juvenile idiopathic arthritis (n=1). systemic autoantibody disease was diagnosed in 26 patients (12%), with diagnoses including vasculitis (n=7), systemic lupus erythematosus (n= 6), polymyalgia rheumatica (n=4), antiphospholipid syndrome (n=3), mixed connective tissue disease (n=2), crest/ scleroderma (n=2), myositis (n=2), sjogrens syndrome (n=2), and discoid lupus erythematosus (n=1). inflammatory neuropathy was diagnosed in 21 patients, with small fiber polyneuropathy (n=10), uveitis (n=3), myasthenia gravis (n=2), bells palsy (n=2), and multiple sclerosis (n=1). autoimmune skin conditions were diagnosed in 24 patients with diagnoses including psoriasis (n=13), alopecia (n=8), and vitiligo (n=3). while the mean igm was higher in the patients with autoimmune/rheumatologic manifestations than in other cvid patients (83 vs 57mg/dl), this difference did not reach statistical significance (p=0.15). conclusions: autoimmune and rheumatologic complications are present in over half of patients with cvid. increased vigilance for autoimmune and rheumatologic complications is important as survival outcome are worse in cvid patients with non-infectious complications as previously described. further evaluation of these patients to understand the mechanism of immune dysregulation is essential, as this may promote targeted therapies and improve clinical outcomes. introduction/background: immunoglobulin concentrates have been successfully used for decades to treat patients with primary or secondary immunodeficiency disorders. this treatment has substantially decreased the frequency of life-threatening infections in these patients. octanorm is a newly developed maltose-formulated subcutaneous immune globulin (human) 16.5% liquid for the treatment of patients with primary immune deficiency (pid) and secondary immune deficiency (sid). objectives: biochemical and physico-chemical properties were investigated. methods: molecular size distribution of monomers, dimers, polymers and fragments were determined (according to european pharmacopeia (ep) monograph 8.0) by size exclusion chromatography (sec). igg and igg subclass concentrations were quantified by respective nephelometric methods. functionality of the igg was demonstrated by measurement of fc function, opsonophagocytosis and fc gamma receptor binding assays. dynamic light scattering measurement and size exclusion chromatography were used to characterize the integrity of the igg molecule. measurement of potential procoagulant activity was done by natem and tga (fxia-like activity). the capacity of the octanorm manufacturing process to robustly inactivate/remove pathogens was investigated in spiking experiments with prions and viruses. results: octanorm contains more than 96 % of human igg and is characterized by an especially low content of polymers and aggregates, low viscosity, low isoagglutinin titres, low iga and igm contents with a broad spectrum of antibodies against infectious agents. it has a distribution of immunoglobulin g subclasses closely proportional to that in native human plasma. in the final product, potential procoagulant activity is not detectable. functionality and physico-chemical properties of the igg molecules were demonstrated by state-of-the-art methods. virus safety of octanorm is obtained via a combination of three validated orthogonal methods as part of the manufacturing process: cold-ethanol fractionation, solvent/ detergent (s/d) and ph 4 treatment. a substantial depletion of prions during the manufacturing process was demonstrated. conclusions: octanorm is a state-of-the-art subcutaneous immunoglobulin. based on the excellent stability the intended shelf life of octanorm is 24 months stored at +2°c to +8°c protected from light. within its total shelf life the product can be stored at room temperature up to +25°c for up to six months. efficacy and very good tolerability of this new subcutaneous normal immune globulin 16.5% were shown in a clinical phase iii study performed in 18 centers in north america and europe. professor, sapienza university of rome introduction/background: primary antibody deficiencies (pad) are characterized by defective ig production resulting in high susceptibility to bacterial infections, especially caused by s. pneumoniae and h. influenzae. there is a limited evidence on the rate of microbial airway epithelial colonization and on the role of bacterial carriage on the development of recurrent respiratory tracts infections in such populations. objectives: the aim of this study was to investigate the prevalence of s. pneumoniae and haemophilus influenzae colonization in pad adults in italy and its clinical and immunological correlates. methods: nasopharyngeal and oropharyngeal swabs were obtained from 93 cvid and 16 patients with idiopathic primary hypogammaglobulinemia (iph) over 18 years of age and under ig replacement treatment during the period october 2016-april 2017. presence of s. pneumoniae and h. influenzae was investigated using conventional cultural methods and rt pcr. s. pneumoniae isolates were serotyped by the quellung reaction; capsular type of h. influenzae isolates was determined by pcr. the pattern of associations between the two species and potential risk factors were investigated. respiratory infections rate was recorded over 12 months of follow up. results: among cvid prevalence of carriage assessed by traditional culture was 11% and 27% for s. pneumoniae and h. influenzae, respectively. rt pcr allowed to identify a higher rate of carriage of s. pneumoniae and h. influenzae compared to standard culture. cvid and iph had not different rate of pneumococcal colonization, whereas cvid had higher rate of h. influenzae carriage identified by a culture methods and rt pcr. no synergistic association between s. pneumoniae and h. influenzae colonization was observed. among cvid, s pneumonia and h. influenzae carriage were associated to low iga and igm levels. cvid under antibiotic prophylaxis did not have an increased prevalence of carriage. no association was found between carrier status detected by culture and having chronic lung disease, bronchiectasis or the rate of infections during the follow up. rt pcr identified merely the association between igm levels and h. influenzae carriage. antibiotic resistance from isolated stains was also assessed. conclusions: this is the first study assessing the prevalence of s. pneumonia and h. influenzae carriage in cvid. objectives: to discuss clinical challenges in a subject with congenital hair hypoplasia detected on newborn screening with preserved t-cell mitogen responses but declining naive t-cell counts. parents are jehovah's witnesses, which adds complexity to clinical decisionmaking. methods: immune evaluation included complete blood count, lymphocyte subsets, flow cytometry to define t-cell subsets, immune globulins, repeat trec assay from peripheral blood, human immunodeficiency virus (hiv) and cytomegalovirus (cmv) dna pcr, screen for maternal engraftment, chromosome microarray analysis, lymphocyte proliferation to mitogens, t-cell proliferation to interleukins, t-cell receptor v-beta diversity analysis by spectratyping, and thymic ultrasound. results: repeat trec assay from peripheral blood on day 17 confirmed undetectable trecs. cd4+ thymic emigrants were low at 147 (reference 733-3181 cells/ul). naïve cd4+ and cd8+ t cell compartments declined as did naïve cd4+ t cells from 48 to 17 cells/ul and naïve cd8+ t cells from 33 to 5 cells/ul. lymphocyte proliferation to mitogens was preserved except for the cd19+ response to pokeweed mitogen. interleukin proliferation of cd45+ lymphocytes was slightly decreased after stimulation with anti-cd28 (14%, normal >38%). however, t-cell proliferation with other interleukins (il-2, anti-cd3+il-2, anticd28 as %cd3) was preserved. the t cell receptor repertoire had intermediate diversity. antibody replacement therapy was prescribed for declining igg with no history of severe infections except for rhinovirus prior to discharge from the nicu at 8 weeks, during which she required continuous positive airway pressure (cpap) therapy. the viral load for cmv and hiv dna were undetectable. breastfeeding was discontinued early because the mothers cmv serology revealed positive cmv igg. the child has had intermittent anemia, which is common in patients with chh. [2] tests for autoimmune hemolytic anemia were not performed because the patient required ivig from an early age. the parents of the child are jehovahs witnesses, complicating requests for blood transfusions. conclusions: this case highlights challenges in clinical decision making for a newborn with chh identified on nbs. t-cell function is preserved, but declining naïve t cell counts and absent trecs lead us to consider hematopoietic stem cell transplant (hsct). indication and timing of elective hsct is unclear and may depend on the natural progression of disease, including infections and anemia division of immunology and allergy, the hospital for sick children introduction/background: cartilage-hair hypoplasia (chh), caused by mutations in the ribonuclease mitochondrial rna-processing (rmrp) gene, is associated with diverse immune abnormalities including combined immune deficiency (cid). most patients with chh are managed with supportive measurements, while few have received allogeneic hematopoietic stem cell transplantations (hsct). the progression of the immune abnormalities and the impact of hsct in patients with chh and cid have not been well characterized. objectives: to characterize the progression of the immune abnormalities and the impact of hsct in patients with chh and cid methods: the clinical and laboratory findings of 2 siblings diagnosed in infancy with chh and cid due to the common 70 a>g mutation in rmrp, including the effects of hsct performed in 1 of them, were compared. results: both patients suffered from recurrent respiratory infections at early age with reduced t cells numbers and responses. patient #1 immune function continued to deteriorate leading to hsct from an hla-matched sibling at 4.5 years of age. the patient suffered acute and chronic graft versus host disease of the skin with residual mild joint contractures and scleroderma-like skin changes. seven years after hsct patient #1 has normal immune function. immune evaluations of patient #2 in the first years of life indicated mild improvement. the patient did not have a suitable related hsct donor and the family elected to continue with supportive care. at 7 years of age, patient #2 is clinically well and thriving with persistent t cell abnormalities. conclusions: close monitoring of immune function in early life for patients with chh and cid as well as the availability of suitable donors assists in determining management, including hsct introduction/background: leukocyte adhesion deficiency (lad) represents a group of distinct inherited disorders, which inhibit the normal extravasation of neutrophils and their recruitment to sites of infection or inflammation. objectives presentation of case: the patient is a girl of 8 years old with no history of primary inmunological disease (pid) in family members, including a healthy brother (currently 12 years old). she received all the immunization schedule until 1 year old (according to peruvian schedule). she had several admission to the hospital since newborn; the 1st hospital admission was at 15 days of life with diagnosis of: sepsis, pneumony, onphalitis (leukocytes count: 70 000), she had 3 more hospital admission and the leukocytes count were always above 25000. in summary, she presented other infections besides the ones admitted at hospital like: 2 episodes of sepsis, 1 episode of pneumony, 5 episodes of cellulitis (left eye, left elbow, right thigh and vaginal (2), 27 episodes of otitis, 7 episodes of tonsillitis, 6 episodes of diarrhea, 5 episodes of rhinoadenoiditis, 5 episodes of sinusitis, 3 episodes of gingivitis and 1 episode of whooping cough. she received different antibiotics for treatment, even broad-spectrum as vancomycin, meropenem and cefepime. the diagnosis of leukocyte adhesion deficiency (lad) was made at 1 year 9 months by clinical features like delaying in separation of umbilical cord, recurrent infections and persistent leukocytosis>25000. flow citometry was taken at 3 years old, resulting cd11b/cd18: 0,21%. results: discussionf: or the severe phenotype, in which leukocytes express <1% of normal levels of cd18, death occurs at an early age because of severe infection unless patients receive bone marrow transplant. however, it does not happened with her so we suspected in a possible reversion in lad1 so we took a second flow citometry at 5 years old and the results were: total leukocyte: 27870; linfocyte: 6777 cd11b+/cd18+: 0,04%; granulocyte:17438 cd11b+/cd18+: 1,41% and the conclusion was c3 receptor (cd11b/cd18) absent in linfocyte, monocyte and granulocyte. molecular study was taken at 7 years old, resulting a homozygous substitution c.562c>t identified in exon 5, causing a nonsense mutation: p.arg188, confirming lad1 diagnosis. she is, currently, receiving profilactic antibiotic and antimicotic which has reduced considerably recurrent infections. it seems that our patient may have a mixed phenotype due to a clinical expression, which may not require hematopoietic cell transplantation (hct) despite features of the severe type. conclusions conclusion: we conclude that the clinical evolution of this patient is unsual because she has severe lad1, she has not transplanted yet, profilaxis treatment has improved to decrease frecuency of infections, she exceeded life expectancy and last flow citometry confirmed that lad1 was not reverted. , ph.d. 5 , luigi d. notarangelo, md 6 , troy r. torgerson, m.d. ph.d. 7 , ph.d. 8 , hans d. ochs, m.d. 9 , m.d., ph.d. 10 introduction/background: patients with x-linked hyper-igm syndrome (x-higm) due to cd40 ligand (cd40l) deficiency often present with low blood neutrophil counts. however, even when not neutropenic and despite immunoglobulin (ig) replacement therapy, cd40l-deficient patients are susceptible to life-threatening infections by opportunistic pathogens, suggesting impaired function of phagocytes, and requiring novel therapeutic approaches. objectives: to analyze whether peripheral neutrophils from cd40l-deficient patients display functional defects and to explore the in vitro effects of recombinant human interferon (rhifn)-on such cells. methods: we investigated the microbicidal activity, respiratory burst and transcriptome profile of neutrophils from cd40l-deficient patients. in addition, we evaluated whether the lack of cd40l in mice also affects neutrophil responses. results: neutrophils from cd40l-deficient patients exhibited defective respiratory burst and microbicidal activity which were significantly improved in vitro by rhifn-. similar to humans with cd40l deficiency, cd40l-deficient mice were found to have defective neutrophil responses. moreover, neutrophils from cd40l-deficient patients showed reduced cd16 protein expression and a dysregulated transcriptome profile suggestive of impaired differentiation. conclusions: our data suggest a non-redundant role of cd40l-cd40 interaction in neutrophil development and function that could be improved in vitro by rhifn-, indicating a potential novel therapeutic application for this cytokine. methods: in this study, through the use of 23 healthy livers, paired peritumoural tissues (pt) and intratumoural tissues (it) from 236 hcc patients. results: increased expression of cd96 on nk cells was observed in intratumoral but not peritumoral regions, along with increased expression of its ligand cd155 and a poor prognosis. human cd96+ nk cells exhibited functional exhaustion, showing decreased ifn-and tnf-productions, impaired cytolysis in response to in vitro stimulation, and high gene expression of il-10 and tgf-1 with low expression of t-bet, il-15, perforin and granzyme b by global transcriptomic analysis of sorted cd96+ and cd96-nk cells. blocking tgf-1 specifically inhibited cd96 expression and reversed the dysfunction of nk cells. in addition, we compared other two receptors, cd226 and tigit, which share common ligand cd155 with cd96, and found cd96 plays a more important role in nk exhaustion. conclusions: these findings indicate that human cd96+ nk cells have features of functional exhaustion, suggesting that cd96-cd155 blockade has the potential to restore immunity against liver tumors by reversing nk cell exhaustion. introduction/background: mutations in ncf1 (encoding protein p47phox of the nadph oxidase) result in an autosomal recessive form of chronic granulomatous disease (cgd), a rare genetic disease with impaired phagocyte production of reactive oxygen species and recurrent infections. diagnosis of p47phox cgd is based on abnormal dihydrorhodamine assay and absence of p47phox protein by immunoblotting; however, these assays fail to diagnose carriers of ncf1 mutations. instead, carrier status is inferred after the birth of a child with p47phox cgd. furthermore, identification of the specific genetic defect in patients with p47phox cgd is complicated by two highly conserved (>98%) pseudogenes. the ncf1 gene has a gtgt at the start of exon 2, while the pseudogenes (ncf1b and ncf1c) delete one gt (gt). in p47phox cgd, the most common mutation in ncf1 is gt causing c.75_76delgt; p.tyr26fsx26. sequence homology between the wild type gene and pseudogenes precludes using standard sanger sequencing to identify specific mutations in ncf1. objectives: to identify phenotypic and genotypic differences that facilitate the diagnosis of patients and carriers with p47phox cgd. methods: expression of p47phox in neutrophils is determined by fixing and permeabilizing whole blood with intraprep, and then incubating with either anti-p47phox antibody or its corresponding isotype. alexafluor 488-conjugated secondary antibody is used to detect the target antigen. expression of p47phox is based on the mean fluorescence intensity of cells within the neutrophil population gated using forward and side light scatter. differential expression of p47phox by flow cytometry is validated using quantitative immunoblotting. to screen for the gt mutation, a droplet digital polymerase chain reaction (ddpcr) with two distinct probes recognizing either the wild-type gtgt sequence or the gt sequence was used to quantitate the ratio of gtgt vs. gt copies. a second ddpcr reaction established copy number by comparing one probe for an invariant region of ncf1/ncf1b/ncf1c and a second probe for the single-copy telomerase reverse transcriptase gene, tert. the results of these two assays were combined to determine the total number of gtgtcontaining and gt-containing ncf1 copies. results: analysis of p47phox expression in permeabilized neutrophils determined that neutrophils from p47phox cgd patients had negligible p47phox expression; neutrophils from p47phox cgd carriers exhibited~60% of p47phox expression compared to healthy volunteers independent of the mutation in ncf1. of all p47phox cgd patients tested by ddpcr, 83.2% (109/ 131) exhibited 0 copies of gtgt, 6.9% (9/131) exhibited 1 copy of gtgt (compound heterozygotes with 1 non-gt mutation), and 9.9% (13/131) exhibited 2 copies of gtgt (two non-gt mutations). moreover, ddpcr can identify the carriers among kindreds within the gt p47phox cgd families. unexpectedly, among normal subjects tested, only 78.8% exhibited the expected 2 copies of gtgt per 6 total ncf1/ncf1b/ncf1c copies, designated 2/6; a significant number exhibited more than two copies of gtgt (14.6% with 3/6, 1.9% with 4/6); others exhibited ncf1/ncf1b/ncf1c copy number variation (2.8% with 2/7 and 1.9% with 2/5). conclusions: flow cytometric analysis of neutrophil intracellular p47phox staining provides a quick method to identify patients and carriers of p47phox cgd. droplet digital pcr can be used to identify patients and carriers of p47phox gt, the most common mutation in p47phox cgd copy number variation is observed at the ncf1 locus among normal subjects tested. introduction/background: defects in the cd3 subunits of the tcr/cd3 complex account for a small percentage of the scid presentations. cd3 deficiencies are characterized by profound and t-cells lymphocytopenia, and normal numbers of b-and natural killer (nk) cells in the peripheral blood (t-b+nk+ scid). thymocyte development from double negative stage to double positive stage results arrested. affected individuals typically present with severe and opportunistic infections in the early infancy. objectives: to evaluate possible underlying immunodeficiency disorder in the setting of chronic ebv infection. methods: here we report our experience of a patient with an atypical presentation of cd3 deficiency. results: a 7-year-old girl previously healthy, first generation americanborn of gambia immigrants was referred to our clinic for further evaluation of chronic ebv infection. one year before presentation, she developed bilateral parotid enlargement, cervical, axillar, and hilar lymphadenopathy, bronchiectasis, and pulmonary nodules. relevant previous studies included: ebv viremia (pcr quant 63,000 copies), ebv igg and ea positive whereas igm and ebna were negative. peripheral blood phenotyping was not suggestive for malignant process and cervical lymph node biopsy was consistent with a reactive process without evidence of a clonal lymphoproliferative disorder and bal studies positive for ebv only. hiv and tb both negative. she was otherwise thriving with no history of recurrent infections and family history was negative for consanguinity or immunodeficiency. our evaluation revealed: normal blood counts, ebv pcr quant 206,677 copies, normal immunoglobulin levels and vaccine titers. she had a normal lymphocyte subsets, but skewed cd45 ra/ro consistent with low thymic output (very low cd4 naïve t cells (5.8%), low cd31+ naïve t cells (36.7%), and elevated temra (42.6%)), poor mitogen, and antigen responses. all these findings were suggestive of a scid like phenotype; therefore, scid next generation sequencing panel was pursued, and showed a novel homozygous splice site mutation (c.55 +1 g>t) in the cd3 gene. conclusions: a homozygous mutation in the cd3 gene might not necessarily imply profound t-cell lymphopenia. though this patient did not present with classic clinical course, and immune findings; her inability to clear ebv with persistent significant viremia does support a t-cell immune deficiency. she remains at risk for ebv-associated lymphoproliferative disorder, infections, and autoimmunity. bmt will be a curative treatment option. however, it is difficult to predict evolution of clinical phenotype given the atypical presentation. this case illustrates the importance of contextual interpretation of clinical findings, laboratory data, and genetic analysis for treatment approach. introduction/background: pol is multi-subunit polymerase that includes both pole1 with catalytic activity and additional pole2, 3 and 4. this holoenzyme plays a key role in proofreading damaged dna and is required for proper dna replication in proliferating cells, such as lymphocytes. germline mutations are linked to rare cause of primary immunodeficiencies whereas somatic mutations are described in colon cancer. primary immunodeficiencies are reported pole-1 deficiency in members of a large consanguineous french kindred and a palestinian female with fils (facial dysmorphism, immunodeficiency, livedo, and short stature). all reported cases with pole1 deficiency have homozygous intronic splice site variant (c.4444+3a>g) that result in a deletion of exon 34 which lead to subsequent frame shift (from p.s1483v onwards) and a premature stop codon at position 1561; this transcript results in a degraded product. the proportion of the pole1 transcript in t lymphoblasts is significantly lower (10%) in patients then carriers or healthy individuals. objectives: hereby we describe the clinical progression and treatment challeneges of the palestinian female with pole-1 deficiency secondary to homozygous g.g4444+3 a>g substitution. results: initially patient presented with viral and recurrent ear infections and cmv viremia. with age, patient had less episodes of infections even with intermittent pause in immunoglobulin replacement therapy (igrt), however had multiple admissions for fever of unknown origin with negative cultures but increased ferritin level (3300 ng/ml) and low platelet count (46,000 count/ml). autoimmune and inflammatory complications were not reported among the french kindred. her skin also worsened with poor wound healing and scarring throughout that hinders igrt via subcutaneous route. furthermore, igrt with intravenous administration has resulted in symptoms of aseptic meningitis likely related to the underlying inflammatory state. in addition, patient shows decline in immune dysfunction. initially, she had normal immunoglobulin g (igg) however by 5 years of age, she developed hypogammaglobulinemia with low igm unlike in the french family with patients. also, pneumococcal, diptheria, tetanus titers were non-protective off of immunoglobulin replacement therapy (igrt). beyond low switched memory b cells, patient also developed low b cell by 5 yo age. t cell dysfunction continued to decline from decreased lymphocyte proliferation to antigens at 2 yo age to fully absent lymphocyte proliferation to antigens and mitogens. naïve cd4 and cd8 compartments continue to be preserved. currently management challenges include treatment strategies for thrombocytopenia, inflammation and progressive skin disease that complicates the proper selection of route for optimal igrt. conclusions: beyond progression of immunological decline, our patient developed inflammatory phenotype with age. the progression of her immunological decline may be related to further decrease in the proportion of the wild type pole1 transcript and yet to be examined. overall, clinical follow up is essential in patients with pole-1 deficiency as phenotype can change with age and may pose new challenges. longitudinal follow up studies are needed to uncover the potential role of germline pole1 and pole2 pathogenic variants in cancer susceptibility. introduction/background: x-linked hyper-igm syndrome (xhigm) is one type of primary immunodeficiency diseases, resulting from defects in the cd40 ligand/cd40 signaling pathways. objectives: here, we retrospectively reviewed clinical, laboratory and genetic characteristic of xhigm in chinese population, thus further improving diagnosis and treatment for xhigm. methods: we collected and analyzed 47 chinese patients, who were diagnosed and followed up in hospitals affiliated to shanghai jiao tong university school of medicine from 1999 to 2016. targeted gene capture combined with next-generation sequencing technology and sanger sequencing were used to find out related gene mutation. results: the median onset age of these patients was 7 months (range: 20 days66 months). thirty-six percent of them had positive family histories, with a shorter diagnosis lag. the most common symptoms were recurrent sinopulmonary infections (40 patients, 85%), neutropenia (22 patients, 47%), protracted diarrhea (21 patients, 45%), and oral ulcer (19 patients, 40%). ten patients had bcgitis. six patients received hematopoietic stem cell transplantations and four of them had immune reconstructions and clinical remissions. twenty-seven unique mutations in cd40l gene were identified in these 47 patients, with 18 novel mutations. conclusions: to our knowledge, this report provides the largest cohort of patients with xhigm in china. mutation analysis is an important tool for xhigm diagnosis. infants with scid are asymptomatic at birth and unless prompt diagnosis of the disease is made they may horrifically be vaccinated. simultaneous appearance of two live vaccine associated infections in one person is rarely reported. objectives: in this study we present two infants with scid, who received bcg and oral polio vaccines early in life before the diagnosis of immune deficiency was made. both patients developed localized and disseminated infections originating from the bcg vaccine (bcgitis and bcgiosis, respectively) and in addition were diagnosed with chronic fecal secretion of vaccine-derived polio virus (vdpv); alarmingly, in both cases, the vdpv underwent reverse mutation of attenuated sites to the neurovirulent genotype. the rarity of concomitant infection from two live vaccines in one recipient, together with the multiple complexities originating from these infections in immunodeficient infants, led us to report these cases and to inquire the pathogenesis that underlies this unique condition. methods: immunological evaluation:: cell surface markers of peripheral blood mononuclear cells (pbmcs) were measured by immunofluorescent staining and flow cytometry serum concentrations of immunoglobulins were measured using nephelometry. quantitative analysis of the tcr v repertoire was performed by means of flow cytometry. quantification of t cell receptor excision circles (trecs) was determined by real-time quantitative (rq)-pcr. genetic analysis: genetic diagnosis of scid was made for patient 1 by direct sanger sequencing of candidate genes and retrieval of mutation in rag2 , and for patient 2 by wes (whole exome sequencing), followed by validation of the dna cross-link repair 1c (dclre1c) mutation using sanger sequencing. poliovirus detection and characterization: stool samples were collected monthly and transported to the national poliovirus laboratory located at israel central virology laboratory (icvl) for polio detection and characterization. results: in both patients, immunological workup revealed undetectable serum iga and igm levels with normal igg levels (table 1) , lymphocyte immune-phenotyping using flow cytometry revealed complete absence of t and b cells with presence of nk cells (table 2) . trecs, a dna marker of naive t cells and thymic output, were absent in both patients. the diagnosis of scid was made. we initiated prophylactic antibiotic (trimethoprim-sulfamethoxazole) and anti-fungal (fluconazole) treatment, as well as monthly intravenous immunoglobulin (ivig) infusions. genetic workup: the t-b-nk+ scid phenotype in patient 1 led us to search for a mutation in the rag complex genes. indeed, a sanger sequencing of the rag2 gene, revealed a g104t homozygous mutation which predicts an amino acid substitution from glycine to valine in position 35 (fig.1 ). for patient 2, who had a similar t-b-nk+ scid phenotype, we identified a homozygous mutation in the dclre1c gene (del.4bp, c.817-cttt) (fig. 2) , using wes. the genetic evaluation confirmed the diagnosis of scid due to rag2 deficiency in patient 1 and artemis deficiency in patient 2. clinical course: during hospitalization patient 1 developed disseminated bcg related disease with skeletal lesions involving the phalanx, tibia and maxillary bones, as well as involvement of the spleen, liver and pancreas. anti-tubercular therapy with isoniazid, rifampicin, ethambutol and ciprofloxacin was initiated. due to lack of response, empirical trial of g-csf (granulocyte colony-stimulating factor), in order to enhance macrophage activity . the patient showed good response to this combination therapy. patient 2 developed a palpable rigid mass on her left shoulder with surrounded redness at the site of the bcg vaccine at the age of 5 months. the clinical diagnosis of bcgitis was established and triple antitubercular therapy with isoniazid, rifampicin and ethambutol was initiated with good response. throughout their hospitalization, both infants suffered from intermittent diarrhea. pcr for enterovirus was performed and detected the presence of type 3 and type 2 vaccine-derived poliovirus (vdpv) in patient 1 and 2, respectively. during the follow-up, stool samples collection revealed accumulation of several polio virus mutations and some of the neuro-virulence attenuation sites were reverted to the neuro-virulent genotype .fortunately, both patients did not show any signs of flaccid paralysis. precautionary measures of isolation were taken to prevent spread of the vdpv. eventually, both patients underwent allogeneic bone marrow transplantation (bmt): patient 1 had bmt without pre-conditioning, from a matched sibling donor. due to engraftment failure, a second bmt was repeated, this time successfully. on follow up examination her t cell repertoire showed a normal tcr v polyclonality and trec was detected, indicating the emergence of new t cells. due to ongoing low immunoglobulin levels this patient is still on regular ivig-infusions and prophylactic antibiotic treatment, as well as isoniazid. currently, she is well, her bcgitis is not active and her stool specimens are negative for polio. patient 2 underwent an urgent haplo-identical bmt with alpha-beta t cell depletion without pre-conditioning, due to her unstable medical condition. flow cytometry analysis six months post bmt revealed lymphopenia of 7.7% (1334/mm³) with low mature t cells (cd3 32%) and absent b cells. trecs were barely detected. microsatellite analysis as a marker for engraftment revealed a stable donor chimerism of 10%. the patient is still on immunosuppressive therapy doing well, her bcgitis is not active and her stool specimens are negative for polio. conclusions: these cases highlight the importance of early recognition of scid by neonatal screening or thorough family anamnesis, and the need to further defer the timing of administration of live vaccines. introduction/background: measurement of b cells, b cell subsets and specific antibodies produced in response to vaccination are key tests used to investigate immune system function. specific antibody production may indicate b cell functionality. objectives: in this study the correlation between the different b cell subsets and antibody responses to pneumovax® in an immunocompromised population was investigated. methods: b cell subsets were assessed by flow cytometry and pneumococcal responses measured using the vacczyme pneumococcal capsular polysaccharide (pcp) igg, iga and igm elisas (the binding site group, birmingham, uk) in 39 primary immunodeficiency patients (pid) vaccinated with pneumovax®. lower limits of normal were defined as follows: b cells: 6.6%; naive b cells: 65.6%; non-switched memory b cells: 7.4%; switched memory b cells: 7.2%; pcp igg: 50 mg/l; pcp iga: 125 u/ml; and pcp igm: 140 u/ml. results: the correlation coefficients between percentage of b cells/b cell subsets and igg, iga or igm pneumovax® responses ranged from -0.61 to 0.39. the percentage of the cohort achieving a normal b cell and normal igg, iga or igm response to pneumovax® was 45%, 29% and 40% respectively. b-cell responses were measureable in the remaining patients but they did not produce normal concentrations of pcp igg, iga or igm. further stratification of patients who achieved normal percentage of switched or un-switched b cells but who failed to achieve normal igg, iga or igm responses to pneumovax® were 32%, 55% and 50%, respectively, for un-switched and 22%, 47% and 33%, respectively, for switched b cells. conclusions: the combined measurement of b cells and response to vaccination are required to provide a detailed insight into these disorders. introduction/background: this is a 2-year-old boy of african american heritage with multiple congenital malformations who was diagnosed with very early-onset inflammatory bowel disease, which proved to be resistant to treatment. subsequent testing revealed a heterozygous mutation in exon 1 of ctla4. this variant has not been previously reported in the literature in individuals with ctla4-related disease. heterozygous mutations in ctla4 cause a disease of immune dysregulation. clinical presentation is variable and may be characterized by enteropathy, hypogammaglobulinemia, granulomatous lymphocytic interstitial lung disease, lymphocytic organ infiltration in non-lymphoid organs, autoimmune cytopenias, and recurrent infections. early onset colitis has been reported with ctla4-related disease and is unique to our patient's initial clinical presentation. objectives: this is a 2-year-old boy with cloacal exstrophy of the urinary bladder, omphalocele, imperforate anus, polydactyly, and sacral agenesis who was diagnosed with very early-onset inflammatory bowel disease at six months of age. he underwent cloacal exstrophy closure, omphalocele repair, and colostomy placement in the first week of life. at six months of age, he presented with dark tarry stools. upper endoscopy and colonoscopy revealed polyps, ileitis, and colitis. he was p-anca positive and started on sulfasalazine. unfortunately, he continued to have symptoms suggestive of active colitis, prompting a change to prednisone and azathioprine. despite therapy, his colitis persisted leading to chronic bloody diarrhea and growth failure. initial immune evaluation consisted of a normal complete blood count, serum immunoglobulin, lymphocyte subsets, and neutrophil oxidase burst assay. foxp3 analysis by flow cytometry showed a moderately elevated percentage of foxp3+cd25+ cells in the cd4+ t cell population, but the regulatory t cell immunophenotype was normal. because suspicion was high for a monogenic immunologic disease to explain his symptoms, genetic sequencing was performed. a candidate gene panel was sequenced by next generation sequencing, and a heterozygous mutation in exon 1 of ctla4 (c.23g>a; p.arg8gln) was found. this variant has not been previously reported but is predicted to be pathogenic in exac and polyphen databases. results: the patients diagnosis of ctla-4 haploinsufficiency associated with very early-onset inflammatory bowel disease has provided opportunity for targeted treatment of his specific molecular defect. given his poor response to treatment thus far, the patient will be started on abatacept. abatacept is fda approved for the treatment of rheumatoid arthritis but has been used successfully for the treatment of disease-related manifestations of ctla-4 haploinsufficiency. abatacept is a ctla-4 fusion protein formed by the igg1 fc region linked with the extracellular domain of ctla-4; it replaces the defective protein in ctla-4 haploinsufficiency. in addition, given other manifestations of ctla-4 haploinsufficiency including lymphoproliferative disease in non-lymphoid organs, particularly the brain and lung, we have initiated further evaluation of these organs to evaluate for disease-specific manifestations. conclusions: the protein cytotoxic t lymphocyte antigen-4 (ctla-4) is an essential negative regulator of t cells. heterozygous mutations in ctla4 cause a disease of immune dysregulation. clinical presentation is variable and may be characterized by enteropathy, hypogammaglobulinemia, granulomatous lymphocytic interstitial lung disease, lymphocytic organ infiltration in non-lymphoid organs, autoimmune cytopenias, and recurrent infections. inflammatory bowel disease may be associated with certain variants in ctla4, but the literature remains limited, both by number of papers published as well as by ethnic subsets studied. clinicians who are presented with children who have early-onset colitis, and particularly inflammatory bowel disease that is difficult to treat, should consider possible genetic abnormalities, such as ctla-4 haploinsufficiency, as these can impact therapeutic decision-making and outcomes. introduction/background: dock8 deficiency is an autosomal recessive combined immunodeficiency syndrome associated with recurrent infections, eczema and other atopic diseases. the infections are usually viral, bacterial and fungal resulting in predominantly cutaneous and sinopulmonary manifestations. homozygous or compound heterozygous deletions or mutations in the dock8 gene (9p24) lead to abnormal cytoskeletal organization and impaired function of dendritic cells and lymphocytes. an aniline derivative belonging to the group of synthetic sulfones, dapsone has been employed in the treatment of chronic skin diseases characterized by an accumulation of neutrophils and eosinophils. methods: chart review of one patient results: we present a four-year-old male with severe eczema, persistent asthma, allergic rhinitis, as well as peanut and egg allergy suffering from recurrent skin abscesses and prurigo nodularis. abscesses began at age 18 months and required prolonged courses of antibiotics, eight in total prior to presentation. other infectious history included otitis media and lymphadenitis. there was no history of pneumonia or other severe infections. skin abscesses responded to oral antibiotics, but recurred shortly after completing extended courses of treatment. laboratory results including quantitative immunoglobulins, specific antibody titers, myeloperoxidase staining, neutrophil oxidative burst and complement were within normal limits. laboratories were notable for elevated ige (10080 iu/ml) and eosinophilia (1200 eosinophils/microl). lymphocyte immunophenotype was significant for mild elevations in cd3 and cd4. dock8 genetic sequencing by genedx revealed a heterozygous missense mutation in exon 17 (c.1979 c>a, amino acid change p.ala660asp). abscess cultures grew methicillin sensitive staphylococcus aureus (mssa) and enterococcus faecalis. mssa was sensitive to ampicillin/sulbactam, cefazolin, gentamycin, moxifloxacin, oxacillin, rifampin, tetracycline, and vancomycin. isolate was resistant to ciprofloxacin, clindamycin, erythromycin, levofloxacin, penicillin, and trimethoprim/ sulfamethoxazole. the patient was initially treated with emollients, mupirocin washes, topical steroids, anti-histamines, bleach baths, and cephalexin three times a day. he improved clinically but was unable to tolerate cephalexin for more than ten days secondary to abdominal pain. cephalexin, with addition of probiotic, was attempted several months later and again had to be discontinued because of abdominal pain and vomiting. due to limited antibiotic options, dapsone was started after ruling out glucose-6-phosphate dehydrogenase deficiency. dapsone was initiated at a dose of 1.5mg/kg, and the patient was monitored weekly for hemolytic anemia. after two weeks of treatment, anemia was noted and the dose was decreased to 1mg/kg. he has continued on dapsone 1mg/kg once a day, with significant improvement in abscess number and severity. he has not required other systemic antimicrobials since starting dapsone. conclusions: dapsone may be considered as a treatment option for children with heterozygous dock8 mutation and recurrent abscesses, particularly those requiring prophylaxis, long term treatment and lacking antibiotic options. introduction/background: autosomal dominant hyper-ige syndrome (ad-hies) is a rare complicated primary immunodeficiency disease (pid). signal transducer and activator of transcription 3 (stat3) gene mutation is found to cause ad-hies. tlr7/9 signaling plays multiple roles in b cell proliferation, activation, class-switch recombination, and cytokine and antibody production. however, little is known about b cell response to tlr7/9 agonist in patients with ad-hies. objectives: here, we aim to study the response of b cells from ad-hies patients to the tlr7/9 agonist. methods: pbmcs were isolated from peripheral blood of 5 ad-hies patients and 10 age matched healthy controls. pbmcs were stimulated with tlr7 and tlr9 agonist (r848 and cpg odn 2006, respectively), then b cells were analyzed for proliferation, the expression of certain surface markers (cd40, cd80 and cd86), intracellular immunoglobulin levels (igm and igg) and intracellular cytokine levels (il-6 and il-10) by flow cytometry. results: in response to tlr7/9 agonist, proliferative capabilities of b cells were reduced in ad-hies patients compared with those in age-matched healthy controls. besides, defective costimulatory molecule cd80 expression was observed in b cells from ad-hies patients. furthermore, significantly lower igm and igg levels, and il-10 production was detected in b cells from ad-hies patients. however, there was no significant difference in b cell apoptosis between ad-hies patients and healthy controls. conclusions: these data demonstrated that stat3 gene mutations in ad-hies patients contributed to impaired b cell tlr7/9 signaling, and further affected b cell proliferation, activation, cytokine secretion and antibody production. introduction/background: antibody function is most commonly measured by a rise in antibody titers in response to antigen introduced by vaccination or natural infection. specific antibody deficiency is defined as normal serum levels of immunoglobulins with reduced or absent antibody response to antigens, often after administration of the pneumococcal vaccine polyvalent (pneumovax® 23). a paucity of information exists about measurement of antigen-antibody binding, or avidity, as a measure of antibody function, including persons with recurrent sinopulmonary infections who have normal response to immunization with pneumococcal vaccine polyvalent. objectives: the aims of this study are to identify and evaluate children with recurrent sinopulmonary infections who had appropriate rise in pneumococcal antibody titers following immunization with pneumococcal vaccine polyvalent but low response by avidity, and to assess response with igg replacement therapy in these patients. methods: a retrospective chart review involved eight children with recurrent sinopulmonary infections with discordant pneumococcal antibody and avidity results following vaccination with pneumococcal vaccine polyvalent. these eight children subsequently received igg replacement therapy. results: the mean age of subjects was 9.75 (range 2 -15) years. the mean number of serotypes with a normal antibody response (>1.3 ug/ml) among 8 children following immunization with pneumococcal vaccine polyvalent was 18.1 (range 12-22) of 23 serotypes while the mean number of serotypes with a normal avidity response (1.0) was 4.4 (range 2-7) of 23 serotypes. igg replacement was administered subcutaneously in 8 children. the mean igg level was 720 mg/dl. local reactions were all mild and observed in 4/8 (50%) children. no serious adverse events were reported. all 8 children experienced a marked reduction in respiratory illnesses while on igg replacement therapy. conclusions: discordance between pneumococcal antibody titers and pneumococcal avidity titers was identified in eight children with recurrent respiratory illnesses. in children with recurrent sinopulmonary infections despite normal antibody response to pneumococcal vaccine polyvalent, measurement of pneumococcal avidity may identify patients with poor pneumococcal antibody function. igg replacement in these children was well tolerated and associated with a decrease number of respiratory infections. introduction/background: exome sequencing (es) is a powerful genomic tool that can be used to identify novel molecular causes of disorders with multiple etiologies. immunologic disorders are clinically and genetically heterogeneous, and therefore present unique diagnostic challenges both in the clinic and in the laboratory. objectives: the objective of this study was to assess the utility of es for determining the genetic etiology of immunological disorders and describe diagnostic yield and outcomes of exome sequencing (es) for patients with immunologic disorders and immunologic phenotypes. methods: a retrospective review was performed of 315 individuals referred for clinical es for primary abnormalities of the immune system and individuals with additional phenotypes where multiple immunologic features were reported as part of the clinical picture, as determined during internal curation. analysis of clinical es data was performed by boardcertified clinical geneticists and all variants reported were confirmed by a secondary methodology. positive es outcomes required a pathogenic or likely pathogenic variant in a gene with autosomal dominant or x-linked inheritance, or compound heterozygous or homozygous pathogenic or likely pathogenic variants in a gene with recessive inheritance. results: the most common clinical indications for es in this cohort were hypogammaglobulinemia (16%), neutropenia (14%), immune dysregulation (10%), lymphopenia (9%), and combined immunodeficiency (8%). the gender distribution was 59% male (n=185) and 41% female (n=130); 76% of cases were pediatric (<18 years, n=240) and 24% were adult (>=18 years, n=75). positive results were reported in 83 cases (26%), comparable to the overall diagnostic yield of es at our laboratory (retterer et al., 2016) . this included 20/90 (22%) of cases submitted as proband-only, 47/178 (26%) submitted as a trio, and 16/47 (34%) submitted as duo, quad or alternative family structure. diagnostic results spanned 70 different genes and recurrently reported genes with identified pathogenic variants included flg (n=10), rag1 (n=5), sbds (n=4), lrba (n=3), stat1 (n=3), and pik3cd (n=3). variants possibly associated with the phenotype, but not considered diagnostic, were reported in 58% of cases (n=183), while 13% of cases (n=41) had reportable findings in a candidate gene. conclusions: these results support that exome sequencing for individuals with immunologic-based phenotypes has similar diagnostic utility as the overall rate for clinical es. immunologic es cases with trio family structures have a higher diagnostic yield than proband-only cases, as inheritance information improves confidence in classification of variants as pathogenic or likely pathogenic. genetic heterogeneity, as demonstrated by the large number of distinct genes represented in this cohort of diagnostic es cases and rapid candidate gene discovery make es a valuable tool for genetic diagnosis in patients with immunological disorders. introduction/background: vaccination response to the 23-valent polysaccharide vaccine (ppsv23) is often used in the diagnosis of common variable immunodeficiency (cvid). unfortunately, ppsv23 titers are often difficult to interpret and many cvid patients are started on igg replacement therapy (igrt) before adequate evaluation. unlike ppsv23 titers, the enzyme-linked immunosorbent spot assay (elispot) is independent of igrt and can provide an ex vivo functional measurement of specific antibody production on the b cell level. objectives: develop and test an elispot assay to better determine vaccination response to ppsv23 compared to ppsv23 titers in cvid patients on igrt, healthy controls, and igrt patients without immunodeficiency. methods: an elispot assay was successfully optimized and used to evaluate the ppsv23-specific b cell response in 8 healthy adult controls. elispots were performed on day 1, and day 7 (when plasmablasts are best evaluated). ppsv23 titers were measured on day 1, day 7, and day 30. for igrt patients, flow cytometry for b cell subpopulations will be performed to further validate the assay. results: normal controls demonstrated a significant increase in ppsv23 antibody spot forming units (sfu) between day 1 and 7 after ppsv23 vaccination. ppsv23 titers showed generally robust initial titers at day 1, and no significant change at day 7, with day 30 results pending. conclusions: here we optimized an elispot assay that functionally measures the specific antibody response to ppsv23 in normal controls with ppsv23 titer results pending. we are actively recruiting patients on igrt (both with cvid and without immunodeficiency) for comparison. we hope to validate our assay as a useful alternative to ppsv23 titers that may be particularly useful when patients are on igrt. previous studies have demonstrated bal may be a sensitive diagnostic method for treatment failures of clinically diagnosed pneumonias, even if performed under treatment with empiric antibiotics, and can lead to a culture-directed change in antimicrobial therapy in the majority of cases. however, it has also been reported that at least in one piddchronic granulomatous disease (cgd)the diagnostic yield of bal was inferior to that of other diagnostic methods (marciano et al., 2015) . further information on the diagnostic yield of bal and other invasive procedures to obtain a specific organism diagnosis in pidd patients with suspected pulmonary infection is needed. objectives: to characterize the yield of diagnostic procedures used in pidd patients with pneumonia or other suspected pulmonary infections at the ucsf benioff childrens hospital. methods: we screened our database of pidd patients (encompassing patients seen from september 1, 1998 to september 1, 2017 cared for by the pediatric immunology service at the university of california, san francisco to identify patients with history of at least 1 bal or other invasive diagnostic procedure (fna or open lung biopsy) for etiologic diagnosis of suspected pulmonary infection. if multiple bals were performed during a single episode of illness, only the first was used for this analysis. results: we identified 22 pidd patients with history of at least one bal or other invasive diagnostic procedure, for a total of 55 events. most procedures (n=53) were performed at our institution, with 2 documented in outside hospital records. patient diagnoses included cgd (11), nemo deficiency (4), cvid (3), stat3-deficient ad-hies (2), dock8 deficiency (1) , and mhc class ii deficiency (1) . of 49 bals, 24/49 (49%) grew a predominant organism, but only 18/49 (37%) were positive for an organism believed to be causative by providers and/or for which the overall result of the bronchoscopy affected antimicrobial treatment. bal yield was highest in patients with a clinical and/or radiologic diagnosis of pneumonia (13/27, 48%). yield was poor (3/16, 19%) in minimally or chronically symptomatic patients referred for bal for interval changes on chest ct (i.e. suspected fungal infection). our nemo deficiency cohort had the highest rate of positive organism isolation by bal (6/11, 66%) for diagnosis of pulmonary infection. in our cgd cohort, 11/ 26 (42.3%) bals grew a causative organism. lung biopsy yielded positive organism isolation in 2/3 cases (2/2 in cgd, burkholderia cepacia and nocardia cyriacigeorgica; 0/1 in cvid) and fna in 1/2 cases of cgd (aspergillus fumigatus). fna or biopsy was done concurrently or after bal in 5 cases; in 4/5 (80%), fna or biopsy, but not bal, was positive for a causative organism. conclusions: at our institution, bal overall had a 37% rate of causative organism isolation in pidd patients, but had up to a 50% rate of organism isolation in those with clinical and/or features of pneumonia. our rate of causative organism isolation was slightly higher than in previous reports. however, in specific instances, biopsy was still required to make a definitive diagnosis. bal may have limitations in certain populations of pidd patients, such as in cgd, but it may be a reasonable starting point in the diagnosis of pneumonia or worsening pulmonary disease in pidd. prospective research is needed to evaluate whether fna or lung biopsy, though more invasive, could result in overall shorter time to institution of appropriate directed therapy and shorter hospitalizations for specific pidd patients. introduction/background: prior to the introduction of newborn screening, cases of severe combined immunodeficiency often presented with severe or disseminated infections. herein, we report an infant from india who presented for evaluation and treatment of a periorbital mass, presumed to be malignant. however, he was found to have disseminated bacille calmette guerin (bcg), as well as multiple other infections, and was eventually diagnosed with x-linked scid. results: a 4-month old boy was born to unrelated parents in india. he initially presented with growing periorbital mass and fever for two weeks. pet scan showed hypermetabolic areas in the bone marrow, spleen, mesenteric lymph nodes, and left shoulder, along with the periorbital mass. biopsy of the mass revealed numerous b lymphocytes without malignant transformation. there was no evidence for malignancy on bone marrow biopsy, though numerous granulomas and a significant decrease in t lymphocytes were seen. peripheral blood flow cytometry showed a complete lack of t cells. the bone marrow biopsy was reexamined, and innumerous acid-fast bacilli were found. cultures grew mycobacterium bovis, and he was diagnosed with disseminated bcg disease. genotyping revealed a novel splice site variant in il2rg, consistent with x-linked scid. further evaluation revealed multiple other infections. these included extended-spectrum beta lactamase-produce e. coli bacteremia, human metapneumovirus, cytomegalovirus, and pneumocystic jiroveci. he was also tested for vaccine-associated poliovirus because he had received the oral polio vaccine, but this was negative. four-drug therapy was started for the bcg, and the periorbital lesion completely resolved within several weeks. additionally, he was treated with intravenous ribavirin for the human metapneumovirus, and sulfamethoxazole-trimethoprim for the p. jiroveci. although the cmv was initially treated with ganciclovir, the virus eventually developed resistance and required treatment with foscarnet. due to his many infections, he underwent haploidentical stem cell transplant plus donor lymphocyte infusion from mom. this transplant failed to engraft, but a matched unrelated donor was eventually found, and our patient received a second bone marrow transplant. he currently is showing signs of engraftment, and is continuing to be treated for his multiple infections. conclusions: disseminated mycobacterial disease due to the bacille calmette-guérin (bcg) vaccination has been noted in cases of xlinked scid previously, often consisting of lymphatic, skin, and pulmonary manifestations. however, there is a paucity of published cases presenting with multiple other co-infections. nor are there reports of disseminated bcg presenting as a localized mass. this case highlights the unique considerations when evaluating a patient with immunodeficiency from another country, where vaccination practices and epidemiology differ. specifically, unusual presentations of infections or masses may warrant investigation for severe immunodeficiency. prototypic t-b+nk+ immune phenotype is caused by mutations in the iil7ra gene. the il7 signaling has an important role during t-cell development in the thymus, contributing to cell proliferation and survival. in addition, in mouse models, the rearrangement of the t cell receptor genes, specifically the gamma locus (trg), has been shown to be regulated by il7 signaling. similar to other scid phenotypes, patients with il7ra deficiency are predisposed to acquire opportunistic infections early in life and to display poor outcome and death, unless their immune system is restored by mean of hematopoietic stem cell transplantation (hsct). while most patients with il7ra mutations have full il7ra deficiency resulted in a severe t cell depletion, some have a partial deficiency with residual cells or leaky phenotype, or even present symptoms later in life. objectives: here we report two non-related infants detected by the israeli national newborn screening program for scid. despite having similar il7ra missense mutation (f40l) they displayed distinct clinical and immunological course, resulted in a completely different treatment approaches; observation in one patient with an unusual recovery, and hsct in the other patient methods: patient lymphocytes were examined for subset counts, thymic output (via excision circles), t cell receptor repertoire diversity (tcrb) and il7ra expression and function. the pathogenic il7ra mutation was found by whole exome sequencing (wes). high throughput immunesequencing was performed to characterize the trg repertoire. results: we established patients' diagnosis by validating the pathogenic il7ra mutation, and showing profoundly impaired t cell immune work up and abnormal il7ra expression and function, determined by stat5 phosphorylation assay. all these measurements improved over time for the patient with less severe clinical presentation, while remained low in the patient with the severe phenotype. characterization of their trg immune repertoire using high throughput immune-sequencing revealed restriction of t cell receptor repertoires of both patients upon their initial diagnosis, compared to healthy controls. however, skewed usage of variable (v) gene segments and abnormalities of the cdr3 length distribution were more prominent in the patient with the severe phenotype. conclusions: these studies illustrate the gap that exists in our understanding of other non-genetic parameters that may influence disease course and severity in patients harboring a similar genetic defect. furthermore, the results reinforce the role of il-7 signaling not only in cell proliferation but also in trg rearrangements introduction/background: dock8 immunodeficiency syndrome is primary immunodeficiency disease caused by loos of function mutations in the dock8 gene, which was known to play a critical role in the survival, proliferation and function of several types of immune system, especially lymphocyte. dock8 immunodeficiency syndrome is the most common cause of autosomal recessive hyper-immunoglobulin e syndromes (hies) and mainly expressed as recurrent infections and severe allergic disease affecting the skin. in addition, autoimmune features including systemic lupus erythematosus, hemolytic anemia or idiopathic thrombocytopenic purpura may be presented in dock8 immunodeficiency syndrome. objectives: we report a case of 16-month-old boy diagnosed with dock8 immunodeficiency syndrome, which was initially expressed as sle without recurrent skin infections. methods: a child with atopic dermatitis was admitted to another hospital because of fever lasting more than 3 days accompanied by swelling of the hands and foot. he developed whole body edema, perioral purpura and oliguria. complete blood count was normal and blood urea nitrogen , creatinine and albumin levels were normal on his laboratory findings. however, c-reactive protein level was high at 9.85 mg/dl, coagulation parameters were abnormal (prothrombin time 14.8 sec; activated partial thromboplastin time 67.0 sec, d-dimer 5.82 ug/ml). so he was transferred to our hospital for further examination and treatment on the 5th day of fever. additionally, ulceration of the tonsil and maculopapular rashes on the abdomen and both legs were observed in physical examination. we suspected a meningococcal infection and administered antibiotics. however, no bacterial isolates were identified in the blood and csf culture test. fever persisted despite the administration of antibiotics. we checked immunoglobulin level, complement level and autoantibodies based on fever of unknown origin. immunoglobulin g, m and a were normal, complement fractions c3, c4, and ch50 were low at 80.0 mg/ dl, 6.7 mg/dl and 13.3 u/ml, respectively. antinuclear antibodies were positive at 1:128 with homogenous fluorescence. anti-ds dna antibody was positive at 84.4. the tests for anti-ssa, anti-ssb, anti-ribonucleoprotein, anti-scleroderma 70, and anti jo1 antibodies were all negative. he developed leukopenia and thrombocytopenia over time. results: he was treated with steroid satisfied diagnostic criteria for systemic lupus erythematosus (sle) and fever subsided. he was confirmed lupus nephritis by renal biopsy later. because of onset of sle at the young age, we performed diagnostic whole exome sequencing and multiplex ligation-dependent probe amplification assays. conclusions: he was confirmed dock8 gene deletion (a deletion on one allele and point mutation on the other allele). he is preparing for hematopoietic stem cell transplantation due to autoimmunity and nonreversible parenchymal organ damage form infections although he has not yet experienced a life-threatening infection. introduction/background: during immunological investigation, it is important to distinguish those individuals who may hav e hypogammaglobulinemia (hypo) without fulfilling the criteria for the severe antibody deficiency common variable immunodeficiency (cvid) e.g. unspecified hypogammaglobulinemia from those with cvid. objectives: since low igg3 concentrations may support a diagnosis of cvid, we sought to investigate whether the measurement of additional igg subclass antibodies (iggsc) may provide further discrimination between patients with cvid and those with hypo. methods: iggsc concentrations were measured in serum samples from cvid patients (n=15, 1:1.3 m:f, median age 41.5 years, range 20-78) and hypo patients (n=19-21, 1:1.3 m:f, median age 41.5 years, range 20-78). results: cvid patients had lower median iggsc concentrations for all iggsc: igg1 290mg/dl (range 46-478) vs 365mg/dl (range 174-601); igg2 -88mg/dl (range 8-190) vs 116mg/dl (range 13-546); igg3 -15mg/dl (range 7-58) vs 30mg/dl (range 15-62); igg4 6mg/dl (range 0.2-25) vs 11mg/dl (range 0.31-108). this was significantly lower for igg1 and igg4 (p=0.04 and 0.01 respectively). a higher percentage of cvid patients had iggsc below the lower limit of the normal range compared to hypo patients: igg1 (80 vs 57.9%); igg2 (100 vs 90.5%); igg3 (60 vs 28.6%); igg4 (26.7 vs 10.5%). 100% of cvid patients had low concentrations of 2 or more iggsc vs only 68.5% of hypo patients (p=0.02). 5.2% and 26.3% of hypo patients had low levels of 0 or 1 iggsc, respectively. conclusions: igg subclass measurements may have some utility in distinguishing cvid patients from hypogammaglobulinemia patients. introduction/background: the thymus is often removed during cardiac surgery for repair of congenital heart disease, but the extent of tissue removed varies between procedures and surgeons. previous studies have shown decreased t cell counts after thymectomy but there is limited data on the effect of thymectomy on t cell receptor excision circle (trec) levels and infection risk. objectives: to determine the effect of partial and complete thymectomy during cardiac repair surgery on trec levels and infection risk. methods: a retrospective study of electronic medical records was performed on children who received cardiac surgery before age one at new york presbyterian/morgan stanley childrens hospital between 7/1/2013 and 3/31/2017. patients with heart transplant or primary immunodeficiency were excluded. data was recorded on trec levels (abnormal trec < 200 copies/μl on new york state newborn screen), number of positive cultures, viral pcr panels, and infiltrates on chest x-ray. patients were followed for a minimum of six months after cardiac surgery. study was irb approved. results: cardiac surgery was performed on 256 patients and data was available for 133 patients. of patients included, 65 had a partial and 68 had a complete thymectomy. trec levels after surgery were recorded for 79 patients. only 4% of patients had an abnormal trec level on newborn screen. there was no difference between partial and complete thymectomy on risk of abnormal trec (p = 0.58) and mean total number of infections at 6 months (p =0.42). conclusions: thymectomy rarely causes low trec levels in children undergoing cardiac surgery. complete thymectomy does not significantly increase infection rates in these children compared to partial thymectomy. these findings are possibly due to presence of ectopic thymic tissue or thymic regeneration and are reassuring for children undergoing complex cardiac surgeries. however, long-term follow-up of these children will be necessary to determine residual function of the thymus and clinical response. introduction/background: new sequencing techniques have revolutionized the identification of the molecular basis of primary immunodeficiency disorders (pid), not only by establishing a gene-based diagnosis, but also by facilitating defect-specific treatment strategies, improving quality of life and survival, and allowing factual genetic counseling. because these techniques are generally not available for physicians and their patients residing in developing countries, collaboration with overseas laboratories has been explored as a possible, albeit cumbersome, strategy. objectives: we sought to determine whether blood collected by guthrie cards could be shipped across continents by regular airmail to a cliaapproved laboratory for confirmatory testing. methods: blood was collected and blotted onto the filter paper of guthrie cards by completely filling three circles. we enrolled 20 male patients with presumptive x-linked agammaglobulinemia (xla) cared for at the vietnam national children's hospital, their mothers and several sisters for carrier analysis. dbs were stored at room temperature until ready to be shipped together, using an appropriately sized envelope, to a cliacertified laboratory in the us for sanger sequencing. the protocol for sanger sequencing was modified to account for the reduced quantity of gdna extracted from dbs. results: high-quality gdna could be extracted from every specimen. btk mutations were identified in 17 of 20 patients studied, confirming the diagnosis of xla in 85% of the study cohort. type and location of the mutations were similar to those reported in previous reviews. the mean age when xla was suspected clinically was 4.6 years, similar to that reported by western countries. two of 15 mothers, each with an affected boy, had a normal btk sequence, suggesting gonadal mosaicism. conclusions: dbs collected on guthrie cards can be shipped inexpensively by airmail across continents, providing sufficient high-quality gdna for sanger sequencing overseas. using this method of collecting gdna we were able to confirm the diagnosis of xla in 17 of 20 vietnamese patients with the clinical diagnosis of agammaglobulinemia. introduction/background: znf341 is a positive regulator of stat3 expression. it has recently been described that nonsense mutations in znf341 account for the stat3-like phenotype in four autosomalrecessive kindred. patients presented with reduced stat3 expression and diminished th17 cell numbers, in absence of stat3 mutations. objectives: here, we decribed a turkish case having nonsense mutation in znf341 developed dual malignancy in 2 years. results: a 26-year-old female patient presented with severe eczema, recurrent cold skin abscesses, herpetic skin lesions, sinopulmonary infection, otitis media and hearing loss. the parents are cousins. two younger sisters of the index case had eczema and recurrent skin infections since their infancy period. physical examination of the patient revealed severe eczema, high palate, micrognathia, maxillary hypoplasia and hearing loss. laboratory findings showed reversed cd4/cd8 ratio, high serum ige level (13.916 u / l) and low igg2 level (186 mg / dl). the patient was diagnosed as hyper ige syndrome and ivig therapy (400 mg / kg, every 3 weeks) was initiated because of igg subgroup deficiency and recurrent sinopulmonary infections. at the age of 32, a polypoid mass filling the left nasal cavity was detected in her examination. paranasal sinus ct revealed a mass obliterating the left nasal cavity, left ethmoid sinus and frontal sinus. immunohistochemical stains showed a small round cell malignant tumor in the nasal cavity. she was treated with chemoradiotherapy successfully. a homozygous nonsense mutation has been detected at exon 8 in the znf341 gene (c.1156c> t) (kindly provided by grimbacher's lab) very recently. she developed papillary thyroid carcinoma two years after completing the cancer therapy. conclusions: the relationship between znf341 defect and cancer development is unknown. the development of a second malignancy in this patient for a short time of completing the therapy might imply us a tendency for malignancy in znf 341 patients. additionally, the likelihood of increased radiosensitivity in these patients should be taken into consideration. introduction/background: prometic 10% igiv contains purified igg, 95% as monomer; with a distribution of igg subclasses proportional to that in native human plasma. we report the interim results from a phase 3 trial in the usa of prometic 10% igiv in adults and children with pidd. objectives: this was a phase 3, single-arm, open-label, multicenter trial to evaluate the safety, tolerability, and efficacy of prometic 10% igiv in adults and children with pidd. methods: adults and pediatric subjects with pidd on a stable dose of igg replacement therapy (200-900 mg/kg) for at least 3 months with serum igg trough levels > 500 mg/dl were included. subjects received prometic 10% igiv every 3 to 4 weeks for approximately 1 year at the same dose and schedule as their previous igg replacement therapy. results: an interim analysis was conducted when data were available on 50 adult subjects who received at least one dose of prometic 10% igiv (total of 461 infusions), with 15 subjects receiving at least 6 months of treatment (exposure = 27.72 subject years). at this time, pediatric exposure was only 4.29 subject years. there were no serious bacterial infections (sbis) reported, and rate/yr of infections other than sbis was 2.49 which was comparable to rate while on commercial product (2.80) all subjects achieved an igg trough level > 500 mg/dl. there were no deaths, and no subject had a study drug-related serious adverse event or an adverse event that resulted in permanent discontinuation of study drug. a total of 145 adverse reactions (ar) (0.315/infusion) occurred in 32 subjects (64.0%), with 92 infusions (20.0%) associated with an ar. most infusions (97.0%) were completed without a rate reduction. most ars were mild or moderate in severity, with 6 severe ars (0.013/infusion) occurring in 3 subjects (6.0%). the most frequent ars were headache (20.0% subjects or 0.056/infusion) and fatigue (14.0% subjects or 0.017/infusion). conclusions: in adults treated with prometic 10% igiv, there were no sbis and infusions were well tolerated. director, sean n. parker center for allergy and asthma research at stanford naddisy foundation, professor of medicine and pediatrics, stanford university introduction/background: desensitization to food allergies is being studied in clinical trials using oral immunotherapy (oit). there are limited data regarding the immune changes associated with successful oit. epigenetics involves heritable changes in gene function without modification of the underlying dna sequence. this is mediated by methylation, histone modification, or changes in microrna. objectives: to study methylation changes in the loci of four key genes of immune cells involved in allergy, interleukin 4 (il-4), interferon gamma (ifn-g), forkhead box protein 3 (foxp3), and interleukin 10 (il-10), comparing baseline to post-oit. methods: we completed a phase 2, randomized, placebo-controlled, multi-food oit trial using omalizumab, an anti-ige biologic, to facilitate desensitization for 48 multi-food allergic individuals. double-blind, placebo-controlled food challenges (dbpcfcs) to multiple foods were conducted at entry and after 36 weeks of treatment, the primary endpoint. omalizumab (n=36) or placebo (n=12) was administered for 16 weeks, with oit for 2-5 foods starting 8 weeks after the beginning of omalizumab or placebo. after 36 weeks (28 weeks of oit), participants underwent dbpcfcs to their offending foods. treatment failures (n=11) were offered open-label omalizumab. pyrosequencing of bisulfite treated genomic dna purified from pbmcs from each participant at baseline and post-oit was undertaken to investigate changes in methylation. results: forty-four participants achieved successful desensitization, defined as passing dbpcfcs to 2 or more foods following oit. we found that the -48 cpg site in the il-4 promoter region is hypermethylated over time during successful multi-food oit (fdr-adjusted p < 0.001 by wilcoxon signed-rank test). the median % of methylation at baseline was 81.6 (interquartile range 2.1%) and was 84.5 post oit (interquartile range 2.05%). there were no statistically significant (with a significance level of fdr adjusted p value of 0.001) changes in the il-10, foxp3, or ifn-g loci in the cpg sites we studied. conclusions: these preliminary results suggest that one immune mechanism involved in successful desensitization may involve suppression of th2 function by hypermethylation of il-4 in immune cells in the peripheral blood. introduction/background: atopic dermatitis (ad) is a common chronic inflammatory skin disorder afflicting from infancy to adults with itching, scratching, and lichenification. objectives: we investigated the effects of esculetin from fraxinus rhynchophylla on atopic skin inflammation. methods: for induction of atopic skin inflammation, we exposed the ears of female balb/c mice with house dust mite (dermatophagoides farinae extract, dfe) and 2,4-dinitrochlorobenzene (dncb) during 4 weeks. results: oral administration of esculetin reduced dfe/dncbinduced atopic skin inflammation symptoms based on ears swelling and scratch numbers. the immunoglobulin (ig) e, igg2a, and histamine levels in serum were decreased and inflammatory cell infiltration in skin tissue was reduced by the esculetin. it suppressed th1, th2 and th17 responses by inhibiting the production of inflammatory cytokines such as tumor necrosis factor (tnf)-, interferon (ifn)-, interleukin (il)-4, il-13, il-31 and il-17 in the ear tissue. further, we investigated the effects of escueltin on activated keratinocytes, one of the most representative cells for studying pathogenesis of acute and chronic atopic skin inflammation. as results, esculetin suppressed gene expression of th1, th2 and th17 cytokines and activation of nuclear factor-b and signal transducer and activator of transcription 1 in tnf-/ifn-stimulated keratinocytes. conclusions: taken together, the results imply that esculetin attenuated atopic skin inflammation, suggesting that esculetin might be a potential therapeutic candidate for the treatment of ad. introduction/background: autoimmunity is often seen in common variable immune deficiency (cvid) with immune thrombocytopenic purpura (itp) being the most frequent manifestation at a prevalence of 8-14% in cvid patients. in such patients, itp is often recognized and treated long before cvid, the implications of which are unknown. primary itp is a clinicopathologic diagnosis which includes an evaluation of other conditions that may mimic it including cvid. currently, it is unknown how frequently cvid is evaluated during the diagnostic workup of itp and what percentage of those patient actually have cvid. objectives: the two main objectives of this study were to determine the number of itp patients that had an igg level checked during their clinical course and if the globulin fraction can be used as a marker for hypogammaglobulinemia in itp patients at the time of diagnosis. methods: a retrospective chart review was undertaken at a large academic medical center of patients with a new diagnosis of itp between january 2009 and january 2017. igg levels were collected and globulin fractions were calculated as the difference between serum total protein and albumin within 30 days of the initial itp diagnosis. results: six hundred and twenty-three patients were found to have a new diagnosis of itp in the given timeframe. of these, only 61 (9.79%) had igg levels checked at any point during their clinical course. twelve of the 61 (19.7%) had hypogammaglobulinemia with only 3 of the 12 (25%) having a formal immunologic follow-up evaluation. two were diagnosed with a primary immunodeficiency (1 cvid and 1 ctla4 deficiency). globulin fractions were calculated on 52 patients at the time of itp diagnosis. mean calculated globulin fraction in hypogammaglobulinemic patients was 2.64 (range 2.0 3.7), versus 3.92 (range 2.4 7.5) in patients without hypogammaglobulinemia (p=0.0004). conclusions: the diagnosis of cvid is often delayed from the onset of symptoms which can include autoimmune conditions such as itp. our data indicate that clinicians do not routinely check igg levels at the time of itp diagnosis which should be considered standard of care based on the current guidelines. our data suggest that although calculated globulin fractions were significantly lower in hypogammaglobulinemic patients, the variability was substantial and hypogammaglobulinemic patients would be missed using this as an indicator of low immunoglobulins. future directives include a prospective study using igg levels checked at the time of itp diagnosis, with formal evaluation for cvid in any hypogammaglobulinemic patients to evaluate the true prevalence of cvid among itp patients. introduction/background: autoimmune lymphoproliferative syndrome (alps) is a disorder characterized by immune dysregulation due to the rupture of lymphocyte homeostasis, which occurs as a result of mutations in the apoptotic pathway mediated by fas. this disease is sometimes misdiagnosed due to its variable phenotypic expression and the overlapping of symptoms with many other hematological and immunological disorders. (1) a patient diagnosed with evans-fisher syndrome (sef) was referred to the laboratory for immunity studies. this is a patient who from infancy had multiple admissions for severe sepsis, with severe anemia and severe thrombocytopenia. in the evaluation of the history of the disease by the work team, a series of clinical data was observed that suggested the possibility of the patient presenting an alps, so it was decided to incorporate as part of the study, the quantification of the cells t cd3 + cd4-cd8-(double negative t cells or dnt) that express tcr +. the differentiation pathways of dnt tcr + and the role of fas in this process are not clear, some authors hypothesize that they may be represented by direct descendants of chronically activated positive simple t cells, with deregulated co-receptors, in a state of differentiation in which they were destined to perish by fas-mediated apoptosis. (1) (2) (3) (4) the population of tcr + dnt cells required for diagnosis must be derived from the particular study of each laboratory in their populations, but several working groups agree that the pathological limit values are 1,5% in total lymphocytes or 2,5% in cd3 + lymphocytes. (4) evidence shows that dnt alps cells are not simply accumulated in senescent withdrawal; they and their precursors remain active and proliferate under the influence of activation signals. (5) although numerous genetic deficiencies lead to lymphoproliferation of t cells, only that caused by a defective fas pathway is dominated by negative double t cells. (2) in clinical immunology, the distinction between cd45ra + and cd45ro + cells is particularly useful for determining the state of the "naive" cell compartment in relation to its thymic origin. primary immunodeficiency disorders are characterized by a decrease / absence of thymus performance, and often involve a decrease in cd45ra + t lymphocytes. scientific evidence suggests the important role of the "naive" subpopulation in the origin and maintenance of self-reactive effectors in the periphery. (5) regulatory t cells are able to effectively control autoreactive t cells, especially when negative thymic selection is defective. its differentiation and function is controlled by foxp3. the decrease or absence of regulatory t cells leads to autoimmune diseases, specifically those mediated by cd4 + t cells, and to lymphoproliferation characterized by multiorgan inflammation and other autoimmune disorders. (4, 5) objectives general: establish the diagnosis of a pediatric patient with suspected primary immunodeficiency due to dysregulation, an autoimmune lymphoproliferative syndrome (alps). specific: evaluate the tcr + dnt population, relevant in the diagnosis of alps. quantify cell populations that exhibit markers of activation, differentiation and regulation of the relevant t cells in the study of this disease. methods: monoclonal antibodies (mab) cd4fitc / cd8pe / cd3pc5 triple labeled (beckman coulter), tcrapc, cd4pe, cd45rapercp, cd45rofitc, cd8fitc, cd45rope, cd4apc, cd25f, cd56pe, cd19percp, cd3pe, cd5pe, cd45percp, cd4fitc, cd38fitc were used , hla dr and the cd4fitc / cd25pe / foxp3apc regulatory t cell kit, all from miltenyi biotec. the trial included the use of a healthy control. both samples were processed in unison to ensure reproducibility. the samples were acquired in a beckman coulter gallios cytometer, with the use of the kaluza program, version 1.2. the analysis strategy included the formation of overlapping "gate" windows for the quantification of dnt tcr + and regulatory t cells. results: the patient studied showed 11.60% of tcr + negative double t cells, in relation to 1.45% of the healthy control. the presence of this population of t cells in patients diagnosed with evans syndrome, even in the absence of lymphoproliferation, is consistent with alps. (5) (graph 1) the presence of increased tcr + dnt and lymphoproliferationexpressed as lymphadenopathies and splenomegaly of noninfectious or malignant cause, of at least 6 months of evolution; plus the typical immunohistological findings found in the patient's lymph node biopsy (paracortical hyperplasia), the presence of autoimmune cytopenias (hemolytic anemia and thrombocytopenia) and hypergammaglubulinemia, led to the "probable diagnosis" of alps in the patient studied. the accumulation of dnt in the lymph nodes and other peripheral organs is accompanied by qualitative changes in the composition of the t cell repertoire, so that the immunophenotype performed included markers of activation, differentiation and regulation. the evaluation of the activation on t lymphocytes and nk cells showed that there was a slight decrease in the expression of the receptor for il-2, cd 25. the cd19 + cd25 + population was expressed in greater percentalthough discretely-in the patient than in the healthy control, which was directly related to the presence of hypergammaglobulinemia, elevated iga and the presence of autoantibodies. it was also highlighted by the high expression of the cd19 + cd5 + autoreactive population in the patient studied (11.73% vs 0.39%). in order to know the impact on cell differentiation, the subpopulations of effector and memory cells for cd4 + and cd8 + t lymphocytes were evaluated by combining the cd45ra and cd45ro isoforms. cd45ra is expressed in naive t lymphocytes. particularly, the cd4 + cd45ra + population has an essential function as a suppression inducer, and it is diminished in the patient studied, in relation to the control. (29.11% vs. 62.75%, respectively) the same behavior was shown by the cd4 + cd45ro + memory and effector cells, 53.38% vs73.37%. in the cd4 + population itself, it was possible to confirm the presence of a clone that expressed both receptors (cd45ra and cd45ro), probably a temra population (terminally differentiated effector memory cells). when comparing the results, it was unexpectedly found that there was a slight decrease between patient and control (2.70% vs 4.0%). as the characteristic phenotype of this cell population could not be corroborated, conclusive assessments could not be made. the combination of cd45 isoforms was also used to study cd8 + t lymphocytes, but similar behavior between patient and healthy control was observed. (the lab results are shown in table 1 ) the analysis of the regulation of the immune response showed a decrease in the cd4 + cd25 + population and the expression of the foxp3 transcription factor in the patient with respect to the control. (11.16% vs. 6.30%, 0.00% vs. 1.05%, respectively) (graph 2) conclusions 1. the markedly high quantification of cd4-cd8-tcr + t lymphocytes allowed to define the "probable diagnosis" of autoimmune lymphoproliferative syndrome in the patient under study. 2. the increased activation of cd19 + cells and the presence of the cd19 + cd5 + self-reactive population, largely responsible for autoimmunity and lymphoproliferation in the alps, could be confirmed. 3. the decrease in the cd4 + cd45ra + t-cell suppressor-inducing population evidenced corroborated its involvement in this disorder by primary immunodeficiency, in relation to the thymus dysfunction that originates and maintains self-reactive effectors in the periphery. 4. the decrease in the expression of the foxp3 transcription factor observed, points towards a low regulation of the response that leads to autoimmunity and to lymphoproliferation, specifically mediated by cd4 + t cells. allergy and immunology arnp, nicklaus children's hospital 6 allergy and immunology division director, nicklaus children's hospital introduction/background: specific antibody deficiency syndrome is characterized by a weak antibody response to bacterial polysaccharide antigens when no other immune system abnormalities can be found. low titers to pneumococcal vaccine have become one of the most frequently recognized immune abnormalities in pediatric patients with recurrent sinopulmonary infections. nonetheless, insufficient data and lack of consistent testing of the response to pneumococcal polysaccharides continues to affect the optimal diagnosis and management of this specific antibody deficiency. objectives: to characterize the pre-and post-immunization igg antibody trend for each specific serotype included in the pneumococcal 13-valent conjugate vaccine (pcv13), as well as others that are routinely tested, in a cohort of pediatric patients with recurrent sinopulmonary infections. secondarily, to understand differences in the immune response to the vaccine booster between age groups. methods: this retrospective review identified 182 patients with recurrent sinopulmonary infections. in this cohort, 131 required an immune workup, and 99 were found to have low pneumococcal titers needing a pcv13 vaccine booster. baseline pneumococcal serotype-specific antibody titers at initial visit and 6 weeks after the vaccine booster were obtained. patients were categorized by age: 2 years, 3-5, 6-10, and 11-18. an adequate response to the pneumococcal conjugate vaccine was deemed to be a 4-fold increase over baseline and/or a post-immunization titer of 1.3 μg/ml or greater. results: overall, pcv13 booster provided a significant improvement in the number of protective titers, increasing from 3.6 (95% ci: 3.2-3.9) serotypes at baseline to 11.1 (95% ci: 10.7-11.5) serotypes at 6 weeks (p < 0.001). this increase correlated with improved clinical outcomes81% showed no signs of recurrent infection after the first booster and 94% after a second dose. all those who did not improve clinically suffered from co-morbidities (genetic abnormalities and rheumatologic diseases). post-immunization antibody concentrations were significantly higher than at baseline for all serotypes (p < 0.05) and only 8, 9n, and 12f did not exhibit a greater than 4-fold increase (p > 0.05) 6 weeks following the booster. across age groups, only 1, 7f, and 9v showed pre-immunization differences in titers. there were no differences between ages in post-immunization titer levels for all serotypes. similarly, all age groups had a comparable number of baseline titers (p = 0.63) and at follow-up (p = 0.10). conclusions: in pediatric patients with recurrent sinopulmonary infections, an additional pneumococcal booster proved to be effective in the protection of these children from further infections. the pcv13 booster substantially increased titer levels and concentrations, and significantly improved clinical outcomes, independent of age. this investigation has provided us with a better understanding of the response after booster vaccination, and its role in the protection of patients from recurrent sinopulmonary infections. further studies are needed to elucidate whether a fifth dose of pcv13 should be optional as part of the vaccination schedule of patients with recurrent sinopulmonary infections. introduction/background: many primary immunodeficiencies (pids) share overlapping presentations, complicating clinical diagnosis. due to the ability to include many genes in one assay and the rapid turnaround time that these panels allow, expanded next-generation sequencing (ngs) panels are valuable in facilitating the diagnosis of patients with pids. objectives: we aimed to determine the clinical utility of an expanded ngs panel for the genetic diagnosis of patients with suspected pids. methods: we performed a retrospective analysis of the clinical utility of a 207-gene pid ngs panel used in a clinical diagnostic laboratory. from april to october 2017, 260 panels were ordered for patients with suspected or known pids. results: seventy-four pathogenic or likely pathogenic (p/lp) variants were identified in 63 (24.2%) patients. eight (10.8%) of the p/lp variants were copy number variations (6 deletions, 2 duplications). fifty-two patients (20%) had 1 p/lp variant, and 11 patients (4.2%) had 2 p/lp variants. of positive patients, 31.7% (n = 20) were heterozygous carriers for autosomal recessive conditions where a second variant was not identified. twenty-two patients had p/lp heterozygous variants in genes with autosomal recessive and autosomal dominant inheritance patterns, in which the positive findings may or may not explain the patient's phenotype. for example, 13 variants in this category were heterozygous variants in tnfrsf13b (taci). genetic diagnoses were established or likely in 30% of patients with p/lp variants (7.3% of all patients). five patients were heterozygous for a single p/lp variant and a variant of unknown significance in the same autosomal recessive gene. four patients in whom a genetic diagnosis was determined were also heterozygous carriers for a second, unrelated condition. one patient was found to have two distinct genetic diagnoses. variants of uncertain significance (vus) were identified in most (94.2%) patients. the average turnaround time from test requisition to return of results was 18 days. in total, 63% percent of genetic diagnoses were for conditions that are treatable with hematopoietic cell transplantation. conclusions: these results illustrate the utility of broad ngs panels for the diagnosis of patients with pids. introduction/background: severe combined immunodeficiency (scid) is a life-threatening immune deficiency manifest by extreme susceptibility to infection. early diagnosis and definitive treatment with either hematopoietic cell transplant (hct) or, in select cases, gene therapy (gt) has been shown to significantly improve survival. newborn screening for scid has allowed for the opportunity to promptly identify these patients before significant infections occur. at ucsf, newly diagnosed infants with scid are admitted to the hospital for management and remain in isolation for definitive treatment and until adequate immune reconstitution occurs. previous work by our group demonstrated up to 60% of these parents experience psychosocial trauma manifested by depression and post-traumatic stress disorder (ptsd). the psychosocial challenges that contribute to depression and ptsd in these parents have not been qualitatively described. objectives: to understand the range of experiences and feelings of parents/caregivers of infants with scid diagnosed by newborn screening throughout their prolonged hospitalization and isolation in order to better support patients, parents, and their families. methods: voluntary participation was elicited from parents of children with scid who were status post hct/gt for one year or longer. semi-structured, in-person interviews lasting approximately 45-60 minutes were conducted with 11 parents; interviews were recorded and transcribed. parents were asked to discuss their experiences from first notification of an abnormal screening result through discharge after hct or gt. emerging themes were identified from the transcribed interviews. results: we interviewed 6 mothers and 5 fathers of 7 infants with scid. six infants received hct, while one underwent gt for ada-scid. all children were alive and well at the times when interviews were conducted. overall, once admitted, parents reported feeling well supported by the medical team and support staff. however parents identified a number of stressful events. uniformly reported key stressors included: receiving the first phone call regarding their childs abnormal newborn screening results, preparing for hct, coping with prolonged isolation, and transitioning from hospital isolation to care with ongoing isolation at home. other challenges described reflected the additional stressors of caring for a newborn, including coping with postpartum depression. overall, we identified three major themes encompassing the challenges faced by parents of hospitalized scid patients: (i) loss of normalcy and control over multiple aspects of life; (ii) prolonged waiting periods (especially the wait between diagnosis and hct and between hct and evidence of t cell engraftment); and (iii) perceived lack of guidance on realistic expectations during the hospital stay . parents sought and relied on peer support from other scid parents to learn about coping with the nuances of daily life as a parent of an infant with scid. conclusions: we identified multiple psychosocial stressors and challenges uniquely faced by parents and caregivers of infants diagnosed with scid by nbs. parents described barriers to caring for their own physical and mental health, which can be especially harmful to parents experiencing postpartum depression. recognizing these challenges allowed for the identification of opportunities to improve both healthcare delivery to their children and institutional support for future families affected by scid (table i) . emphasis should be placed on providing parents with scid-specific resources as early as the time of diagnosis, connecting parents with scid support networks, and facilitating access to psychosocial and mental health services for caregivers introduction/background: chronic granulomatous disease (cgd) is caused by a genetic defect that impairs phagocyte function. this disease results in recurrent infections and granuloma formation. rarely do patients develop cutaneous symptoms, unless associated with autoimmune disorders such as systemic erythematous lupus. previously described cutaneous findings include granulomas, abscesses, photosensitivity, malar rash, discoid lupus, vasculitis, and rarely vesicular rashes. here we describe two term infants diagnosed with x-linked cgd who present, in addition to frequent infection, with a unique papulopustular skin rash initially diagnosed as non-classic appearing eczema refractory to usual eczema treatment and antibiotics. objectives: to characterize cutaneous findings in x-linked cgd and emphasize the importance of considering further work in patients who present with similar rashes in conjunction with concerning features for primary immunodeficiency. methods: each infant was diagnosed with cgd based on abnormal dhr testing and/or genetic evaluation. after obtaining consent from both families, we have documented photographs of the development of rash in two newly diagnosed infants with cgd. one infant underwent cutaneous biopsy with resulting pathologic evaluation. results: our first patient presented at 8 months of age with episodic fever with chronic leukocytosis, iron deficiency anemia, thrombocytosis, elevated inflammatory markers, proctocolitis with elevated calprotectin, and rash. egd with flexible sigmoidoscopy showed no signs of inflammatory bowel disease, the left colon had macroscopically raised erythematous lesions but was microscopically normal with no signs of active colitis. he was initially diagnosed with fpies and eczema in the setting of diagnosis of various infections (otitis, upper respiratory illness). the skin rash was described as non-pruritic, generalized pink-purple papulopustular lesions with a pink base, most prominent on upper and lower extremities, mostly sparing the trunk. skin biopsy histopathology revealed an essentially unremarkable appearing epidermis and within the dermis, there was a superficial perivascular lymphohistiocytic infiltrate. eosinophils and plasma cells were not abundant. the histologic changes were thought to be non-specific, but could be seen in a drug reaction or urticaria. a viral exanthem could also demonstrate these changes. giemsa stain and a stain for mast cell tryptase revealed normal numbers of mast cells in the biopsy specimen. he was not on any oral medications at the time and did not respond to treatment with topical moisturizers, oral antihistamines, topical steroids, or any standard for eczema care. he had a maternal uncle who passed away in infancy from infection. heightened clinical suspicion for primary immunodeficiency led to obtaining a neutrophil respiratory burst assay which was consistent with cgd and genetic testing was positive for x-linked cgd. pathogenic mutation nucleic acid change was c. 469c>t; hemizygous; amino acid alteration was p.157arg>stp within the cybb gene locus. he had no other features of auto-immune disease including negative ana obtained later in his clinical course and his colitis diagnosed as cgd-associated colitis. his rash did not respond to systemic treatments for his colitis, oral antibiotics, and during hsct. our second patient presented at 14 months of age with persistent fevers, inguinal lymphadenopathy, leukocytosis, elevated inflammatory markers, non-bloody diarrhea and rash. there was a family history of autoimmune gi disease. he presented to the hospital with fever of unknown origin with concern for infection, atypical kawasakis, and drug rash. his rash was described as a blotchy, pink, papular rash most prominent on the upper arms but also present on lower arms, legs, and chest, faint on face. there is some induration and thickness to the rash in some areas (confluent on the arms with more erythema and induration) and more faint pink papules (scattered on legs) elsewhere. a biopsy of his inguinal lymph node showed granulomatous lymphadenitis with neutrophilic abscess formation, and culture was positive for serratia marcescens. neutrophil respiratory burst assay was consistent with cgd and genetic testing was positive for x-linked cgd, mutation with the cybb gene. pathogenic nucleic acid change was c. 141+5g>a; hemizygous; amino acid alteration was p. deletion of exon 2. the rash remained unchanged with treatment for infection, initiation of antifungal and bacterial prophylaxis, along with topical steroid therapy. conclusions: in patients who present with frequent infections and who have unusual cutaneous findings that do not fit with common infant rashes, consideration of work up for cgd should be pursued. specifically, generalized papulopustular rash with a negative skin biopsy can be misdiagnosed as atopic dermatitis, and in the right clinical context cgd should be considered. introduction/background: chronic lung disease in common variable immunodeficiency (cvid) is heterogeneous, and it is a leading cause of morbidity and mortality in this population. currently, the diagnosis and monitoring of chronic lung disease in cvid rely on radiographic findings, biopsy and/or pulmonary function tests. fractional exhaled nitric oxide (feno) is a noninvasive biomarker of airway inflammation, and it has been widely utilized to aid the in-office diagnosis, characterization, and management of inflammatory airway disease, such as asthma. however, exhaled nitric oxide in cvid patients with chronic pulmonary complications has not been examined. objectives: we aimed to determine fractional exhaled nitric oxide levels in cvid patients. methods: we measured exhaled nitric oxide in cvid patients with or without chronic lung disease. results: feno measurements were obtained in 13 cvid patients (mean age: 53, range 34-68). five patients had no known lung disease; 8 patients had chronic lung disease (bronchiectasis, n=3; lung granulomas, n=4; hepatopulmonary syndrome, n=1). four patients were on inhaled steroid (bronchiectasis, n=3; lung granulomas, n=1), 2 patients were on systemic steroid (lung granulomas, n=1; hepatopulmonary syndrome, n=1), and 1 patient was on oral budesonide (no known lung disease). feno was elevated (> 25 parts per billion [ppb]) in 9 of 13 patients, 6 of whom had known chronic lung disease. patients with granulomatous lung disease had higher feno (mean 50.8 ppb, range 41-59 ppb) compared to patients without known lung disease (mean 31 ppb, range 23-38 ppb, p=0.005), patients with bronchiectasis (mean 18.3 ppb, range 8-25, p=0.005), and the patient with hepatopulmonary syndrome (18 ppb). feno levels remained elevated in granulomatous lung disease in patients with inhaled or systemic steroid use. conclusions: this is the first report of feno measurements in cvid patients. feno is elevated in a subgroup of cvid patients, and it may differ according to the underlying lung pathology. further investigation is warranted to determine the utility of feno in the diagnosis and management of chronic lung disease in cvid. professor of pediatrics, university of british columbia introduction/background: whole exome sequencing (wes) has revolutionized the discovery, diagnosis, and treatment of primary immune deficiency diseases (pids), a group of disorders with high genetic and phenotypic heterogeneity. current bioinformatic analysis approaches for wes rely heavily on population databases to exclude variants present in the populations represented by these databases. this approach may confound the ability to detect true disease causing variants, and conversely, flag variants that are absent from population databases only by virtue of originating from minority populations. ultimately, the pathogenicity of novel variants can only be mechanistically established through rigorous biochemical and functional validation. objectives: to evaluate the pathogenicity of a novel homozygous variant in caspase recruitment domain family member 11 (card11) (c.1798g>t, p.c600y), in a patient with features of combined immunodeficiency (cid). methods: research study protocols were approved by our institutional review board. six members of one family (the affected child, healthy siblings and their parents) were enrolled. written informed consent for genetic testing and participation was provided by the parents for their children. genetic, bioinformatic, biochemical and immunological investigations were performed. results: targeted sanger sequencing confirmed the presence of the homozygous card11 variant c.1798g>t, p.c600y in the index patient (and subsequently in one apparently healthy sister). each parent was found to be a heterozygous carrier of the variant. nfkb activation in vitro was found to be normal for both the index patient and sister, and was indistinguishable from unaffected family members. conclusions: although multiple in silico tools predicted the c.1798g>t, p.c600y card11 variant to be pathogenic, the patients b and t cells did not have aberrant nfkb activation in vitro, as would be predicted given the central role of card11 in nfkb activation. this practical experience highlights how imperative it is to functionally characterize novel variants found by wes, even when variants are predicted to be damaging. chief, clinical immunology -faculdade de medicina do abc introduction/background: gata2 is a zinc finger transcription factor essential for embryonic and definitive hematopoiesis. heterozygous mutations leading to gata2 deficiency were first described in 2011. the age of clinical presentation ranges from early childhood to late adulthood, with most of them in adolescence to early adulthood. patients present clinical findings as monocytopenia, nontuberculous mycobacterial infections, myelodisplasia, viral (mainly hpv and ebv), fungal and bacterial infections. patiens may arise with many phenopytes, showing how complex is the effect of this transcription factor. objectives: we report a patient with gata2 mutation. methods: report: a 34 year old female patient was referred due to a mycobacterial non-tuberculosis pneumonia. she was a healthy child until puberty. at the age of 15 she presented genital herpes and recalcitrant vulvo-vaginal warts (hpv). at 18, she complained of lower back pain with no diagnosis for months, medication was unnefective, and she developed ischemic stroke. hemiparesia was mild and temporary. endocarditis with no agent identified was also diagnosed. therapy with acetilsalicilic acid was mantained until the age of 28. five years later, vaginal hpv spread through vulve and anal region evolving to neoplasia. she was submitted to surgery, radio and chemotherapy until october 2016. one year later, profound cytopenias led to hospitalization. during that time, she had cough and fatigue and pulmonary tuberculosis was diagnosed. despite therapy with rifampin, isoniazid and pyrazinamid, there was no improvement. bronchoalveolar lavage was positive for mycobacterium avium. main immunological evaluation showed: monocytes 162 (6%), t cells /mm3; cd4+: 28 (1,5%), cd8+: 74 (4%); b cells 2/mm3 (0,1%); nk: 9/mm3 (0,5%); normal immunoglobulin levels; incomplete response to pneumococcus serotypes; igm and igg positive for cmv; negative serologies for hiv, ebv and htlv1/2. molecular analysis identified an heterozygous mutation c.1061c>tp.(thr354met) in gene gata2. results: we report a patient with gata2 mutation. the same gata2 mutation was previously described in national institute of health nih-usa (n=5) and in australia (n=3). conclusions: it took almost 20 years for diagnosis suspicion. gynecologists should be warned about this diagnosis in order to improve patients prognosis. early diagnosis is crucial with adequate prophylaxis, prompt treatment of infection, surveillance of malignancy, and, moreover, family screening and genetic counseling. this is the first report of this mutation in latin america. introduction/background: severe combined immunodeficiency (scid) due to adenosine deaminase (ada) deficiency was the first human monogenic disease to be approached with gene therapy, and ongoing research advances over 25 years led to the approval by the european medicines agency of a stem cell gene therapy product for its treatment using a gammaretroviral vector (gv), strimvelis®. despite the high success rate using gvs for ada gene transfer without vector-related complications, the development of leukoproliferative complications from the use of gvs in gene therapy of other disorders led us to develop lentiviral vectors (lvs) to deliver the corrective ada sequence/cdna (corrigan-curay et al, mol ther 2012; modlich et al, mol ther 2009.). lvs, typically derived from hiv-1 and devoid of all viral genes, can be produced in a self-inactivating (sin) configuration, in which the viral long-terminal repeat enhancers are absent, eliminating the major identified cause of insertional oncogenesis due to gvs. we developed a lv (efs-ada) that carries a normal human ada cdna (codon-optimized) and demonstrated in pre-clinical studies its efficacy to transfer and express the ada protein, with evidence of significantly decreased potential for insertional mutagenesis compared to gammaretroviral vectors using the immortalization (ivim) assay and in murine bone marrow transplant models (carbonaro et al, mol ther, 2014) . this new lv was evaluated for safety and efficacy in parallel clinical trials of gene therapy for ada scid performed at sites in the u.s (university of california, los angeles and the national institutes of health {nih} clinical center) and u.k. (university college london/great ormond street hospital) enrolling subjects between 2012 and 2016. we report here results from the 20 patients treated in the u.s. between 2013-2016. objectives: this is a prospective, non-randomized phase i/ii clinical study to assess the safety and efficacy of efs-ada lentiviral vector stem cell gene therapy in ada-scid subjects older than 1 month. methods: 20 subjects with ada-scid were enrolled, screened to document eligibility and underwent bone marrow harvest (15-20 cc/kg). bone marrow was processed to isolate cd34+ cells, which were pre-stimulated by overnight culture in serum-free medium containing c-kit ligand, flt-3 ligand, and tpo followed by culture with the efs-ada lentiviral vector overnight. subjects received a single dose of busulfan (4 mg/kg) iv for reduced intensity conditioning. cells were removed from culture, washed, formulated and administered by iv infusion at least 24 hours after busulfan administration. enzyme replacement therapy (ert) with pegylated bovine ada (peg-ada) was continued for one month post-transplant and then stopped. subjects were followed over 24 months to assess safety and efficacy end-points. results: with 17-54 months follow-up, overall survival is 100%. eventfree survival has also been 100%, as all subjects are alive, remain off peg-ada ert, and none have required a second transplant. successful engraftment of gene-corrected cells was observed in all subjects at 6 months, and persisted over the 24 months of observation, based on vector gene marking in granulocytes and peripheral blood mononuclear cells (pbmcs), changes from baseline in rbc ada enzyme activity, and levels of metabolic detoxification of deoxyadenosine nucleotides. immune reconstitution was observed in all subjects and was sustained over the two years of observation, based on improvement of peripheral blood absolute lymphocyte counts and lymphocyte subsets (t, b and nk cells, and naïve cd4+ t cells). eighteen of 20 patients have been able to stop receiving immunoglobulin replacement therapy. subjects who had routine infections all recovered with standard of care treatment, and there were no severe or opportunistic infections. conclusions: conclusions: gene therapy using the efs-ada lv has a favorable safety profile and was efficacious in this trial. a current followon trial at ucla is using a cryopreserved formulation of the cell product and pharmacokinetic-adjusted busulfan dosing, sponsored by the california institute for regenerative medicine (clin2-09339) and orchard therapeutics. acknowledgements: this study was supported by research grants from the national institutes of health (u01 ai100801; 2p01 hl073104-01; nhlbi gtrp rsas #1101 and 1129) and the california institute for regenerative medicine (cl1-00505; fa1-00613); support from the ucla david geffen school of medicine human gene and cell therapy program and the ucla eli & edythe broad center of regenerative medicine and stem cell research; and funding from the nhgri intramural program. dbk has potential financial conflict of interest as a member of the scientific advisory board for orchard therapeutics and as an inventor on intellectual property which ucla has licensed to orchard therapeutics related to gene therapy for ada scid. introduction/background: x-linked severe combined immune deficiency (scid-xl) is a rare monogenic primary immunodeficiency disorder (pid) where male infants are born without an adaptive and innate immune system. it is a life-threatening disease due to patients inability to fight viral and bacterial infections. scid-xl is driven by any of the two hundred known pathogenic mutations in the interleukin 2 gamma receptor (il2rg) gene, which function is required for proper development of t, b and nk cells. the most effective treatment for scid-xl, if performed in the first few months of life, is allogeneic hematopoietic stem cell transplantation (allo-hsct). this treatment is limited by absence of match donors, incomplete immune reconstitution, graft versus host disease and the need for long-term immunosuppression. an alternative curative treatment for scid-xl would be genome editing-based gene therapy by ex vivo genome correction of the patients long-term hematopoietic stem cells (lt-hscs) prior to autologous stem cell transplantation (auto-sct). objectives: here we report a proof-of-concept genome editing-based approach for correcting scid-xl disease. methods: using a crispr/cas9-raav6 platform, we deliver a fulllength codon optimized il2rg complementary dna (cdna) at the endogenous start site in cd34+ hematopoietic stem and progenitor cells (hspcs). results: using an optimized genome editing protocol we achieved >80% genome editing as early as 48h and a median of 45% genome targeting while retaining >80% viability and promoting greater than 70% ex vivo expansion of cd34+ hspcs from healthy donors. we demonstrate that our approach retains proper il2rg signaling function in t-cells derived from healthy male donors and rescues the lymphopoietic defect from a patients derived mobilized cd34+ hspcs both in vitro and in vivo. we further show robust in vivo primary human engraftment potential and multi-lineage hematopoietic reconstitution of il2rg gene targeted hspcs: a median of 26% (bone marrow), 47% (spleen) and 37% (liver) of il2rg genome targeted engrafted cells was achieved at four months following primary transplantation into nsg mice and a median of 9.5% -20% was detected at 8 months from secondary transplants of il2rg targeted hspcs, thus achieving clinical levels of editing of lt-hscs. lastly, our observation of (1) an intact hematopoiesis derived from il2rg targeted cd34+ hspcs combined with (2) a normal karyotype analysis and (3) our deep analysis of potential off-target activity that showed only 2 off-target sites of no known functional significant with < 0.3% frequency of off-target activity presents strong evidence for the safety of our genome editing approach. conclusions: in sum, this pre-clinical study provides specificity, toxicity and efficacy data supportive of continued development of genome editing to treat scid-xl. objectives: to determine the risk of gvhd in msd hsct for scid patients compared to matched related donor (mrd). methods: retrospective cohort study comparing msd with mrd and the outcome of gvhd in all scid patients who underwent hsct between 1993 and 2013. all statistical analysis was done using ibm spss statistic software. results: 145 scid patients underwent 152 hsct, 82 (54%) received gvhd prophylaxis. gvhd occurred in 48 (31.5%); 20/48 (42%) had gvhd prophylaxis compared to 28/48 (58%) that did not, p value = 0.022. acute gvhd occurred at a higher rate in msd 22/120 (18.3%) compared to mrd 2/32 (6.2%) p value = 0.095. we analyzed outcome also according to period of hsct. first periods was 1993 to 2003; 48 hsct, msd: 43, mrd: 5; all had gvhd prophylaxis and there was no difference in gvhd. the second period was 2004 to 2013:104 hsct, 77 had msd and 27 had mrd, gvhd prophylaxis was used in 22.1% in msd and 63% in mrd, p value = 0.000. gvhd was significantly higher in the msd (40.2%) compared to mrd (18.5%) odds ratio of 2.9 (95ci 1.01 to 8.66) p value = 0.041. conclusions: gvhd prophylaxis in msd transplant may have a role to be considered in scid patients. introduction/background: xiap deficiency (also known as x-linked lymphoproliferative disease type 2 xlp2; mim: 300635) is an x-linked primary immunodeficiency associated with mutations in the gene encoding the x-linked inhibitor of apoptosis (xiap; mim: 300079). the pathophysiology is characterized by immune dysregulation, usually triggered by epstein-barr virus (ebv) infection. primary ebv infection is followed by hemophagocytic lymphohistiocytosis (hlh) with high grade persistent fever, splenomegaly, hematologic cytopenias and hepatitis. most patients die during this acute phase, and those who survive usually evolve with hypogammaglobulinemia, recurrent infections, cytopenias, inflammatory bowel disease (ibd) and low counts of inkt cells. dysbiosis of intestinal microbiota is believed to fuel ibd and possibly contribute to the initiation and/or perpetuation of the disease. experimental studies have provided solid evidence to support a role for the indigenous gut microbiota in the pathogenesis of autoimmune diseases, thereby raising the possibility that an altered gut microbiota is an environmental risk factor for xiap disease. objectives: in this study, we sought to investigate the identity and abundance of the bacteria in gut microbial communities in a 28 years old male patient with xiap deficiency. case presentation: at 15 years of age, the patient presented with positive serology of active ebv infection, hlh, severe hepatitis, encephalitis and myocarditis. after recovery, the patient evolved well with few manifestations for several years. approximately 3 years ago, the patient showed slow progression of hypogammaglobulinemia predisposing him to infections of the upper and lower respiratory system that required intravenous immunoglobulin replacement. immunological evaluation revealed reduction (but not absence) of inkt cells. at that time, the patient presented with intermittent diarrhea and abdominal pain that became more frequent and severe. after evaluation as ibd, the patients had been treated but without much improvement of diarrhea or resolution of his pain. after approximately 18 months, the patient presented noted pain and fistulas lesions at the scrotal and gluteal regions. the exact causes of ibd in xiap deficiency are not known, but the abnormal activation of the mucosal immune system due to exaggerated response to the commensal bacteria associated to the dysregulation of nod1 and nod 2 signaling might play an important role in the development and maintenance of the inflammatory status. methods: the gut bacterial composition was assessed by targeted metagenomic from the patients stool sample, collected before initiation of ibd antibiotics therapy. the 16s rrna amplified and its sequences were analyzed using a bioinformatic pipeline based on mothur software. we determined the bacterial community composition using 100,000 filtered reads using illumina miseq platform. results: according to the ezbiocloud database, the obtained dataset included 548 operational taxonomic units at 3% dissimilarity, distributed among the following groups: bacteroidetes (67.3%) with 37.5% of b. dorei, 18,1% b. vulgates, and 15.3% b. fragilis, firmicutes (24.8%), proteobacteria (7.7%), bacteria_uc (0.06%), actinobacteria (0.04%). conclusions: increased abundance of bacteroides species including b. dorei and b. vulgatus have been implicated in inflammation in several gut diseases such as ulcerative colitis, irritable bowel disease, and celiac disease. although this experience is limited to a single patient, the results of the present study suggest an association between altered gut microbiota and the pathogenesis of ibd in xiap disease and may be of relevance to the future development of novel therapeutic strategies for xiap deficiency. (77) submission id#427718 hematopoietic stem cell transplantation in patients with primary immune regulatory disorders: a primary immune deficiency treatment consortium (pidtc) and inborn errors working party (iewp) study introduction/background: primary immune regulatory disorder (pird) is a newly recognized group of immune-mediated diseases with prominent features of autoimmunity, autoinflammation, and non-malignant lymphoproliferation in addition to immunodeficiency. the clinical manifestations of pirds are frequently difficult to manage and hematopoietic cell transplantation (hct) can be considered as a treatment option, often in those with the most severe disease. we sought to aggregate data from patients who have undergone hct for genetically defined pird or features of immune dysregulation. objectives: we sought to aggregate data from patients with pird who have undergone hct in order to determine the quantity of patients, clinical manifestations, indication and hct, and overall outcome. methods: a questionnaire based survey was sent to all primary immunodeficiency treatment consortium sites and 3 hct referral centers in europe to determine the quantity and characteristics of patients with pirds who have undergone hct. the survey captured clinical manifestations, timing and indication of hct, strategy of hct, and outcomes from 1982-2017. results: 224 patients from 34 centers (31 in north america and 3 in europe) were included with either known genetic defects or considered to have immune dysregulation regardless of gene defect. known genetic defects were identified in 170 subjects, while 54 had symptoms of immune dysregulation but lacked a genetic diagnosis. the mean age of onset of disease was 2 years (range 0-20 years). clinical manifestations included gastrointestinal disorders (69%), failure to thrive (67%), dermatitis (51%), hematologic cytopenias (49%), and lymphoproliferative disease (39%). recurrent infections (66%), immunodeficiency (63%), autoimmunity (48%), and autoinflammation (38%) were also common. organ specific autoinflammation occurred most commonly in the lung (39%) and brain (17%). the median age of hct was 9 years (0-64 years). graft sources included matched unrelated donors (47%), matched related donors (20%), mismatched unrelated donors (16%) and haploidentical donors (3%). reduced or minimal intensity conditioning was used in 44% of transplants. five-year overall survival was 61% and the majority of survivors had resolution of symptoms that led to transplantation. among those patients that died, infection was the most common cause. conclusions: based on our survey data, pird patients commonly develop clinical features of autoimmunity, autoinflammation, and susceptibility to infection at a young age. hct can be successful and lead to disease resolution. however, further studies to define the appropriate patient, timing of hct, donor selection and pre-hct conditioning regimen are necessary to improve outcomes of patients with pird. introduction/background: pathogenic variants in tnfrsf13b (taci) are relatively common (found in about 1% of the population) but have been seen in about 7-10 % of cvid patients, interestingly in both homozygous and heterozygous states. recent articles suggest that the heterozygous state increases the risk of developing autoimmunity due to an effect on autoreactive b cell selection and activation. objectives 1) illustrate the importance of genetic testing in patients with difficulty to treat inflammatory disease 2) present a case report of inflammatory bowel disease associated with a pathogenic mutation in the tnfrsf13b gene results: 16 yo wf was diagnosed with crohns disease due to the chronic abdominal pain, vomiting, and bloody stools since 13 years of age. intestinal biopsy revealed inflammatory changes in the entire intestine with active, submucosal lymphoid hyperplasia, neutrophilic cryptitis with focal areas of crypathic damage, and submucosal epithelioid granulomas more predominantly seen in the colon and rectum. she was started on immunosuppressant medications but developed anaphylactic reaction to both infliximab and adalimumab. she was then treated with azathioprine, mesalamine, methotrexate, and oral budesonide. despite these medications, she continued to have frequent relapses, 4-5 episodes a year and required periodic systemic corticosteroid bursts. other biologics, vedolizumab and ustekinumab, were also tried without success, and she subsequently underwent a colectomy. her postoperative course was complicated by ards, poor abdominal wound healing, and sepsis. due to her complicated clinical course, immune work up was performed which revealed a normal cbc and lymphocyte subpopulations, but hypogammaglobulinemia with low isohemagglutinin titers and specific antibody levels. comprehensive genetic testing ruled out chronic granulomatous disease and other known primary immunodeficiencies but revealed a rare missense mutation in tnfrsf13b (taci). this variant, c310t (p.cys104arg) (rs34557412, exac 0.5%) is likely pathogenic. this heterozygous variant has been seen in both cvid cases and unaffected relatives but significantly more common among cvid patients. moreover, the studies on b cells of these relatives showed impaired function. increased number of autoreactive b cells were also found in the bone marrow of heterozygous individuals and these cells could give a risk of developing autoimmunity. conclusions: in difficult-to-treat autoimmune diseases, identifying the underlying immune defect may aid in the treatment decision. in this case, b cell targeted treatment such as anti-cd20 monoclonal antibody could be beneficial. introduction/background: defects in immunoproteasome caused by biallelic or digenic loss-of-function mutations in proteasome catalytic subunits cause an autoinflammatory disease identified as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (candle) associated to an increased interferon type i gene signature. the proteasome maturation protein (pomp) is a chaperone for both standard and immuno-proteasome assembly and is critical for the incorporation of catalytic subunits. here, we characterize and describe pomp-related autoinflammatory immunodeficiency disease (praid) in two unrelated patients and identify the underlying genetic mechanism of disease. objectives: determine the genetic cause and mechanism of disease in two patients with pomp variants . methods: whole-exome sequencing (wes) was performed to identify a genetic cause of our patients dysregulatory syndrome. proteasome assembly and catalytic function was assessed by sds-page and native gel respectively, using patient derived cell lines. expression of interferon type i-induced genes was measured by rt-qpcr. pomp protein was identified by western blot. results: we identified two unrelated individuals with a unique syndrome characterized by neonatal onset autoinflammation, neutrophilic dermatosis, autoimmunity, and combined immune deficiency with severe systemic viral and bacterial infections. immunologic evaluation for both individuals revealed elevated immunoglobulins, low cd8+ t cell numbers and extremely low b cell counts with persistently high titers of autoantibodies and increased expression of interferon type i-induced genes. in both individuals, truncating heterozygous de novo frameshift variants in pomp were identified by wes and confirmed by sanger sequencing. most mrna transcripts with premature termination codons should undergo nonsense-mediated decay (nmd), however in both of our patients, cdna sequencing revealed these transcripts escaped nmd. the expression wildtype and truncated versions of pomp protein was further confirmed by western blot. transfection of mutant constructs into an otherwise healthy cell line recapitulated an increased interferon signature suggesting a dominant negative mechanism. conclusions: we define praid in two unrelated individuals characterized by neonatal onset immune dysregulation and combined immunodeficiency caused by truncating variants in pomp in which transcripts that escape nmd result in a truncated protein that leads to a dominant negative (i.e antimorphic) allele. to our knowledge, praid is the first inherent defect of immunity mechanistically characterized by nmd escape. introduction/background: parvovirus viremia may occur in pediatric heart transplant patients who underwent thymectomy and developed secondary t cell lymphopenia. high dose intravenous immunoglobulin (hdivig) has been used to treat parvovirus infection in these cases. objectives: we aim to review different routes of immunoglobulin treatment in pediatric cardiac transplant patients with parvovirus viremia and compare the patients immunological phenotype. methods: data from three pediatric heart transplant patients with parvovirus viremia in a tertiary care center was reviewed including t cell counts, parvovirus viral load, route, dosage, and frequency of immunoglobulin treatment. results: all three patients received hdivig. patient 1 and 2 tolerated the treatment and viremia improved. patient 3 developed recurrent aseptic meningitis from hdivig treatment and his viral load remained >1 million copies/ml. compared to the other two cases, patient 3 had a much lower t cell count that likely contributed to the persistence of viremia. to improve his quality of life and reduce healthcare costs, a facilitated subcutaneous immunoglobulin (scig) treatment option was explored. scig treatment was well tolerated and led to a dramatic decrease in parvovirus viral loads in patient 3. conclusions: most pediatric cardiac transplant patients with persistent chronic parvovirus viremia respond well to hdivig, scig may serve as an alternative treatment option in refractory cases, especially in those with severe t cell lymphopenia. severe combined immunodeficiency (scid), by detecting tcell receptor excision circles (trecs) from dried blood spots (dbs) on routine newborn screening (nbs). this has lead to improved estimates of the incidence and prevalence of scid, decreased diagnostic delay, and improved patient outcomes. preliminary studies outside the us have demonstrated that nbs can be adapted to include screening for b-cell deficient infants before symptom onset by quantifying kappa-deleting recombination excision circles (krecs) on dbs. objectives: we report results of the initial characterization of a high throughput triplex trec/krec/rnasep assay run in 9,994 samples in new york state (nys). methods: dbs from 9994 anonymous, de-identified infants were included in the current study. dna from patients with confirmed primary immunodeficiencies (n=32), including, but not limited to, x-linked agammaglobulinemia (n=11) and scid(n=13) were obtained from the centers for disease control and prevention, and clinical immunologists working with the nbs programs in massachusetts, minnesota, wisconsin, and nys, were used as positive disease controls. all dbs were extracted and processed according to current nys nbs protocols. a trec/krec/rnasep triplex assay was designed and optimized to minimize reagent use, and maximize target amplification. cycle threshold (ct) was determined, and error detection cutoffs were identified to optimize sensitivity. results: pcr efficiency, assay quantification, intra-assay reproducibility, and error detection rates all met nys nbs standards. error detection rate for the triplex trec/krec/rnase p assay is 6%, comparable to the current error detection rate of 5% for the current duplex trec/rnase p assay. samples falling into the error detection range are repeated (analysis in process) to determine a receiver operating characteristic curve. conclusions: we show that the high-throughput trec/krec/rnase p triplex assay is feasible in a large, racially and ethnically diverse population in nys. compared to the current duplex assay, this assay has favorable performance characteristics and provides additional immunologic characterization. due to assay optimization, we were able to add the krec test at no additional cost. work is underway to further characterize other assay parameters such as sensitivity and specificity, in preparation for adoption of the triplex assay as part of routine nys nbs. highly accurate wiskott-aldrich syndrome diagnosis via rapid flowbased was protein staining samuel chiang 1 , sue vergamini 2 , ammar husami 3 , marianne ifversen 4 , kejian zhang 5 , jack bleesing, md, phd 6 , yenan bryceson 7 , rebecca marsh, md 8 introduction/background: wiskott aldrich syndrome (was) is a rare xlinked hemizygous disease commonly associated with symptoms of immune deficiency. diagnosis is based on clinical parameters including thrombocytopenia and reoccurring infections, but currently does not include any disease specific marker. as the only permanent treatment for was is hematopoietic stem cell transplantation, it is imperative that a swift and accurate diagnosis be made. absent or lowered was protein (wasp) levels have been reported in sporadic was cases. however, no systemic evaluation exists to date on the accuracy of wasp quantification for was diagnosis. objectives: to determine the accuracy of wasp staining in predicting was genetic abnormalities. methods: we retrospectively evaluated results from a rapid whole blood flow cytometry based assay on a cohort of suspected was patients and compared relative wasp staining levels to was genotype. roc curves as well as accuracy calculations were generated. results: a total of 59 patients with normal and 49 patients with a genetic abnormality in was were collected. missense mutations were most common but insertions, deletions, and gross mutations were also found ( fig a) . comparing was sequencing results to whole blood wasp expression levels provided an 82.6% sensitivity and 100% specificity for a combined accuracy of 95.3% when juxtaposed against genetic sequencing. when 3 variants of unknown clinical significance (vucs) were removed, the sensitivity improved to 89.1% (fig b) . conclusions: staining for wasp is a quick, simple, and accurate assay for the prediction of genetic was defects. introduction/background: cytotoxic t lymphocyte antigen 4 (ctla4) is an inhibitory co-receptor essential for regulatory t cell (treg) function and a central regulator of t cell proliferation and expansion. ctla4 haploinsufficiency is a recently described autosomal dominant disease, in which heterozygous ctla4 mutations result in severe immune dysregulation with variable age of onset and a wide array of clinical manifestations. herein we describe atypical findings in a patient with a novel pathogenic variant in ctla4. objectives 1) to understand whether the novel ctla4 variant identified is pathogenic and 2) to describe eosinophilic gastrointestinal inflammation and exocrine pancreatic insufficiency as possible manifestations of ctla4 haploinsufficiency. methods: next generation sequencing (blueprint genetics ©) was used to identify the ctla4 variant. polyphen and sift (blueprint genetics ©) were used for in silico analysis for the prediction of the effect of this genetic variant on protein structure/function. flow cytometry was used to evaluate ctla4 expression of regulatory t cells. results: a 10-year-old boy with type 1 diabetes mellitus and autoimmune thyroiditis presented with abdominal pain, diarrhea, and weight loss. initial studies revealed markedly elevated peripheral blood eosinophils (>4000 cells/μl) and exocrine pancreatic insufficiency (<50 μg elastase per gram of stool). prominent eosinophilic inflammation was appreciated in biopsies of the stomach, duodenum, jejunum, and terminal ileum. no parasitic infection or inciting drug/food trigger was identified. additional blood studies revealed normal total quantification of t cells but with increased memory t cells (45%, cd3+cd4+cd45ro+) and decreased treg (1%, cd3+ cd4+cd25hifoxp3hi). b cell quantification and serum immunoglobulin (ig) levels were unremarkable, save a modestly elevated ige level (74 iu/ml). comprehensive next-generation sequencing of 232 genes associated with primary immune deficiency revealed a novel heterozygous missense mutation (c.457g>a, p.asp153asn) affecting the last nucleotide in the ligand binding domain (exon 2) of ctla4. subsequent analyses revealed decreased ctla4 expression in the patients t cells compared to healthy controls as well as evolving hypogammaglobulinemia. treatment consisted of methylprednisolone and parenteral nutrition followed by sirolimus and abatacept to which the patient responded favorably. conclusions: we report a 10-year-old boy with a history of type 1 diabetes mellitus and autoimmune thyroiditis presenting with hypereosinophilia, eosinophilic gastroenteritis, and exocrine pancreatic ins uff iciency as unique m anifestat ions o f ctla4 haploinsufficiency. although not previously reported in individuals with ctla4 haploinsufficiency, peripheral blood eosinophilia and eosinophilic inflammation of the gastrointestinal tract have been observed in patients receiving ipilimumab (ctla4 blocking antibody) suggesting a potential mechanism for the aforementioned findings. severe exocrine pancreatic insufficiency is a rare but observed manifestation in individuals with type 1 diabetes mellitus. whether severe exocrine pancreatic insufficiency would be expected t o o c c u r m o r e f r e q u e n t l y i n i n d i v i d u a l s w i t h c t l a 4 haploinsufficiency and type 1 diabetes mellitus is unclear; however, our reported case and surveillance of others with ctla4 haploinsufficiency could elucidate incidence and prevalence of this manifestation. introduction/background: mild asymptomatic hypogammaglobulinemia during pregnancy is a well-described phenomenon due to hemodilution. in patients with known humoral primary immunodeficiency such as common variable immunodeficiency, women require an upwards titration of their immunoglobulin replacement dose. isolated symptomatic hypogammaglobulinemia during pregnancy in a patient is not well described in the literature. objectives 1. understand the physiology of igg during pregnancy. 2. define a rare entity with a likely genetic predisposition that manifests as hypogammaglobulinemia isolated in pregnancy. 3. management of this entity with defining goals of treatment. results: 33 year old gravida 5 para 4 female presented with progressive hypogammaglobulinemia restricted to pregnancy starting with 3rd pregnancy, recurrent otitis media requiring 3 sets of myringotomy tubes, recurrent sinusitis, and streptococcal pharyngitis. her son has common variable immunodeficiency (cvid) requiring immunoglobulin replacement (igrt). prior to conception, her igg was 849 mg/dl. during 13th week of pregnancy, her igg level was 627 mg/dl. during 21 weeks of pregnancy, she complained of fatigue, and developed an episode of sinusitis that required 2 different antibiotic treatments. at that time, her igg was 567 mg/dl. she was started on subcutaneous igrt maintain igg troughs of >650 mg/dl. she remained infection-free during her pregnancy and igrt was stopped a few months after delivery with her serum igg level returning to pre-pregnancy levels. patient was initially evaluated during her 3rd pregnancy with recurrent streptococcal pharyngitis. at that time, her igg was 682 mg/dl. diphtheria, haemophilus influenza, mumps, measles and rubella titers were protective, tetanus titer was non protective with 1/14 antipneumococcal titers protective. she was vaccinated with pneumovax and tdap with development of protective titers and remained infectionfree. igrt was not given and post-delivery, her igg levels improved to 792 mg/dl. she was seen one and half years later, for prenatal counseling for her 4th pregnancy. her immune evaluation included an igg 915. she demonstrated protection to tetanus, diphtheria and streptococcal pneumoniae. at 16 weeks of gestation, she developed recurrent upper respiratory infections requiring antibiotics. her igg was 697 mg/dl. at 28 weeks of gestation, her igg was 537 mg/dl. igrt was recommended, but patient refused at that time. after delivery, igg improved to 849 mg/dl. conclusions: decreased immunoglobulin levels during pregnancy are welldescribed phenomenon which can be attributed to hemodilution of pregnancy. igg transport to fetus generally begins in the second trimester and reaches its pinnacle in the third trimester. patients with known cvid require dose adjustments (higher) during pregnancy as they can be more symptomatic during this time. here we describe a patient who has an almost normal immune evaluation except for mildly low iga during absence of pregnancy, but during pregnancy, develops recurrent infections with significantly low igg level. her family history of a son with cvid, new daughter with low igg levels like to be transient hypogammaglobulinemia of infancy (thi), another daughter with thi suggests a genetic b-cell defect that manifests as cvid with mildly low iga and hypogammaglobinemia during metabolic stress such as pregnancy. there is only one similar report of a single pregnancy of transient symptomatic hypogammaglobulinemia during pregnancy. such patients should be adequately worked up and treated during pregnancy with igrt to decrease maternal and fetal mortality. introduction/background: card11 encodes a scaffold protein in lymphocytes that links antigen receptor engagement with downstream signaling to nf-b, jnk, and mtorc1. germline mutations in card11 are known to give rise to distinct primary immune disorders in humans, including scid (null mutations), b cell expansion with nf-b and t cell anergy (benta; gain-of-function mutations), and severe atopic disease (loss-of-function, dominant interfering mutations). objectives: here we report our experience with an expanded cohort of patients harboring novel heterozygous card11 mutations that extend beyond atopy to include other immunologic phenotypes not previously associated with card11 mutations. methods: cell transfections and primary t cell assays were utilized to evaluate signaling and function of card11 variants. results: we demonstrate that in addition to severe atopy, heterozygous missense mutations in card11 associated with dominant negative activity can present with immunologic phenotypes similar to those observed in stat3lof, dock8 deficiency, common variable immune deficiency (cvid), congenital neutropenia, and immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome. evaluation of rare or novel card11 variants found in affected patients showed that dominant negative activity was largely confined to the card or coiled-coil domains, but did not always manifest in atopic disease. conclusions: these results illuminate a broader phenotypic spectrum associated with card11 mutations in humans, and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activity. introduction/background: increasing number of states have been screening for severe combined immune deficiency (scid) as part of the expanded newborn screening program for nearly a decade. in the era of newborn screening, patients with scid present often asymptomatically and are prepared for early hematopoietic stem cell transplantation (hsct). with advances in genetic testing, mutations in over 20 genes have been associated with development of the scid or leaky-scid phenotype. objectives: to present 2 unique cases of hypomorphic x-linked scid where no known pathogenic mutations were identified on initial genetic testing, the il2r gamma-chain as protein was expressed, but subsequent testing months later revealed pathogenic il2rg mutation affecting either translation or protein function. to expedite earlier identification of pathogenic il2rg mutation, we propose screening with x-inactivation studies in maternal t lymphocytes and assessing gamma-chain function by evaluating il21r signaling in select male infants with abnormal newborn screen for scid, specifically those with scid phenotype but no identified pathogenic gene mutation on initial genetic testing, and the presence of gamma-chain expression. male patient a presented during the first week of life after his newborn screen was found to be abnormal with undetectable t cell receptor excision circle (trec) count. a phenotype of t-b+nk-scid was established and the patient was sent for bone marrow transplant evaluation. genetic testing revealed a novel hemizygous missense mutation in il2rg (p.glu59gln), which was a variant of unknown significance. the patient had common gamma-chain expression by flow cytometry on b and nk cells. he underwent haploidentical hsct with his father as donor. unfortunately, his transplant was complicated by prolonged neutropenia, slow t cell reconstitution, and eventual graft failure. to review his next treatment options, it was necessary to prove that his il2rg mutation was pathogenic. male patient b presented with concern for an underlying immune deficiency after being hospitalized for peumocystis jirovecii pneumonia. his newborn screening for scid was inadequate at birth and follow up was delayed. retrospective analysis of the newborn screening card at birth confirmed absence of trecs. his phenotype was also t-b+nk-, consistent with x-linked scid. he also proceeded to hsct with a haploidentical parent donor. despite hhv-6 viremia pre-hsct which persisted posttransplant, he has had appropriate t cell engraftment. comprehensive genetic testing on whole exome level did not reveal any known mutations contributing to his phenotype. patient did have expression of gamma-chain by flow cytometry on t, b and nk cells. however, further testing revealed an il2rg 3 utr deletion of aa that, based on similar findings from a prior study can possibly lead to mrna abnormalities.1 to expedite the association of scid phenotype with x-linked disease, implying gamma-chain pathology, we obtained x-inactivation studies in maternal t-cells that showed severe skewing in both cases. furthermore, il21r signaling was impaired on b-cells in each case. the combination of these two assays proved that both patients carry a pathogenic il2rg mutation. conclusions: in the era of newborn screening for scid, we are discovering that phenotypic variability of scid patients can be very broad and caused by hypomorphic mutations in the common chain gene. exonbased genetic testing cannot exclude all variants and novel variants of unknown significance have to be evaluated by additional assays, including functional studies for causal effect. it is important to expedite early proof of association with gamma-chain pathology, especially in the era of gene therapy. we propose that in male infants with abnormal scid newborn screening and no known or previously described pathogenic mutation on genetic screen, evaluation continues for hypomorphic il2rg mutations. the probability of this process can be increased by a simple screening test for x-inactivation of maternal t lymphocytes. allergy / immunology, allergy / immunology associates inc. / case western reserve university introduction/background: igg4-related disease (igg4-rd) is an immunologic disorder with multiple clinical presentations previously thought unrelated. it is characterized by the frequent presence of tumor-like swelling of the affected organs and several histopathological findings including tissue lymphoplasmacytic infiltrates with predominantly igg4-positive plasma cells and lymphocytes, storiform fibrosis and obliterative phlebitis. humoral immunodeficiency is a term that encompasses several disease entities associated with impaired antibody production. it is suspected in patients who present with recurrent, frequently severe, sinopulmonary infections with encapsulated bacteria, which leads to evaluation of quantitative immunoglobulin levels and vaccine responsiveness. despite a few reports of primary immunodeficiency in patients with serum igg4 elevation, no adult case has been reported of igg4-rd in a patient with concomitant humoral immunodeficiency. objectives: to present a unique case with the presence of concomitant igg4-related disease and humoral immunodeficiency. methods: comprehensive chart review of our patient and all performed exams. literature review for igg4-related disease, igg4 elevation in humoral immunodeficiency and concomitance of igg4-related disease with humoral immunodeficiency. results: our patient is an 84-year-old caucasian male with relevant past medical history of chronic bronchitis who was referred to our practice after several episodes of pneumonia in the previous years, with six courses of antibiotics just in the year prior for recurrent sinopulmonary infections. blood tests revealed hypergammaglobulinemia and low level of vaccine responsiveness. chest ct showed multiple bilateral pulmonary nodules and hilar and mediastinal lymphadenopathy. sinus ct showed left maxillary sinus opacification. pulmonary function testing was normal. he later presented with left eye edema, proptosis, diplopia, and painless submandibular salivary gland enlargement. laboratory investigation showed an igg of 1730 mg/dl, igg4 of 771 mg/dl. the patient denied any history of pancreatitis or abdominal pain and abdominal ultrasound was normal. biopsy of a salivary gland was normal. mri of the left orbit was obtained, showing lacrimal gland enlargement. based on the patients recurrent infections, lack of response to tetanus immunization, and limited, non-sustained response to pneumococcal immunization, the patient was started on ivig therapy. the patient was also diagnosed with possible igg4-rd based on his salivary gland enlargement and orbital disease in association with hypereosinophilia and increased plasmablast levels. oral prednisone 40mg daily was started for four weeks, later followed by slow steroid taper (reducing 10mg every two weeks) with considerable improvement in left eye swelling and proptosis. a few months after discontinuation of the steroids, the orbital disease returned to its previous severity. left lacrimal gland biopsy confirmed igg4-related disease, with many areas showing greater than one hundred igg4-positive plasma cells per high-power field. after another course of steroids, oral prednisone was weaned to a maintenance dose of 10mg daily and the patient became asymptomatic from his ophthalmologic complaints with normalization of his ophthalmologic exam. his last checked igg was 1105 mg/dl. igg4 was still elevated at 324.8 mg/dl , but given controlled symptoms the patient was spaced to monthly ivig infusions and continued on that daily steroid dosage. conclusions: our patient, initially diagnosed with a humoral immunodeficiency, was later also diagnosed with biopsy-proven igg4-related disease, which is a novel association of this two diseases in an adult patient. previous rare reports of association between elevated serum levels of igg4 and patients with concomitant humoral immunodeficiency were in the presence of isolated igg4 elevation and not in the presence of igg4related disease. this novel association creates a therapeutic dilemma since the patient in question is hypergammaglobulinemic, yet needs ivig, which can lead to side effects such as thrombosis due to a hyperviscous state. the description of additional concomitant cases of both diseases and further understanding of their pathophysiology will be crucial to create awareness and obtain earlier diagnosis, to refine therapeutic options and design adequate treatment protocols. igm and iga anti-pneumococcal capsular polysaccharides as prognostic tool for common variable immunodeficiency: a longitudinal study. professor, sapienza university of rome introduction/background: the clinical spectrum of cvid ranges from a poorly symptomatic form to severe phenotypes characterized by high susceptibility to infections, autoimmunity, granulomatous inflammation, lymphoproliferative disorders, and malignancies. due to high prognosis heterogeneity, prognostic factors are required. objectives: with the aim to identify additional prognostic factors, we evaluated the anti-polysaccharide iga and igm responses by elisa assay in 75 cvid in a longitudinal study over a 6-year period. methods: patients were immunized at baseline with the 23-valent pneumococcal polysaccharide vaccine (pneumovax®). twenty healthy donors (hd) were also included. results: as expected, cvid patient had lower igm/iga response than hd. for cvid, four immunological phenotypes were identified by postvaccination igm and iga levels: igm and iga responders (11%), igmhigh responders (4%), igm-low responders (20%) and non-responders (61%). to simplify, we analysed igm-high group with igm and iga responders and igm-low with non-responders. during the follow up, concomitant cvid-related conditions, immunoglobulin serum levels, respiratory infections and outcome were recorded by medical files. cvid igm-low/non-responders developed more frequently respiratory, gastro enteric and autoimmune manifestation and malignancies in comparison to igm-high/igm and iga responders (respectively, pneumonia: 62% vs 25% ; chronic diarrhea: 33% vs 18%; autoimmunity 38% vs 9%). autoimmune cytopenias were not found in the igm-high/igm and iga responders group. eleven (15%) patients died during the study time. survival analysis according to the igm/iga responder status showed that the 6-years estimated survival for igm-high/igm and iga responders vs igm-low/non-responders group was respectively: 100% vs 100%, 100% vs 95%, 100% vs 92%, 100% vs 89%, 100% vs 85%, 100% vs 85%, 100% vs 82%. interesting, in our series only two deaths were due to infective complications: five were consequent to malignancies, one to autoimmune cytopenias and three to not-cvid related conditions. conclusions: in conclusion, even if patients could not raise the protective humoral level, in cvid the anti-polysaccharide iga and igm responses could represent a prognostic factor, individuating groups of patients with less immunological impairment, lower risk of comorbidities and better survival. introduction/background: gaucher disease (gd) is a rare autosomal recessive disorder characterized by a defective function of the catabolic enzyme -glucocerebrosidase (gba) leading to a progressive accumulation of its substrate-glucocerebroside (gc) -in various organs in particular in mononuclear phagocite system. hepatosplenomegaly and cytopenia represent the most common features of the disease. moreover, gd patients also show hyperinflammatory features -secondary to machrophages engorgement and actviation-hypergammaglobulinemia, and a immune-dysregulation involving b , t and nk cells. since clinical phenotpye can be subdolous, symtoms can overlap with alps, however, few data are available on specific immunity pattern in these patients. objectives: to evaluate immune-phenotype and other alps parameters in a cohort of patients with gd methods: we evaluated lymphocytes subsets, immunophenotypic and serological features of alps (dnts, tcr alfa/beta b220, b-memory cells, tregs/hla-dr ratio, il-10, il-18), and test of apoptosis in a cohort of patients with gd followed-up at igg. results: 35 patients (28 in treatment, 5 not) were studied. dnts and tcr alfa/beta b220+ resulted to be >1.5% of t-lymphocytes and > 60% in 6/32 (19%) and in 7/32 (22%), respectively. b-memory cells and t-regs/hla-dr ratio were <15% and <1 in 11/32 patients (34%). 3/32 evaluable (9%) had all these parameters concomitantly alterated. 4/19 (21%) evaluable patients were resistant to apoposis. il-18 was pathological in 26/29 (89%) patients. all patients had normal levels of il-10 and sfas. conclusions: this study shows that some patients with gd may present an immune-dysregulation pattern that can overlap with alps features. therefore, the differential diagnosis of gd should be taken into consideration by clinicians during diagnostic work-up of patients with an alpslike phenotype. introduction/background: allogeneic hematopoietic stem cell transplantation (hsct) using unrelated and haploidentical donors is complicated by increased rates of graft-versus-host disease (gvhd) and slow immune reconstitution. selective depletion of alpha/beta t lymphocytes and b cells is a recently developed method of graft manipulation that retains mature natural killer (nk) and gamma/delta t lymphocytes, both of which may exert a graft-versus-leukemia effect and protection against life-threatening infections. objectives: to describe the rate and quality of immune reconstitution, incidence of transplant-related complications, including viral reactivation and gvhd, and overall outcomes following tcr-alpha/beta-and cd19depleted hsct for hematologic malignancy in pediatric patients. methods: forty patients of median age 11.2 years (1.7-21.7) underwent hsct for acute myeloid leukemia (n=25), acute lymphoblastic leukemia (n=12), and myelodysplastic syndrome (n=3). grafts were from unrelated (n=29) and haploidentical (n=11) donors. tcr-alpha/beta and cd19 depletion was performed with the miltenyi clinicmacs plus system. median cd34+ cell dose was 10.1 x 106/kg (4.7-15), and median cd3+ cell dose was 2.3 x 107/kg (0.21-43.3). conditioning was with myeloablative busulfan or total body irradiation, cyclophosphamide, and thiotepa. twenty of 29 unrelated donor hscts and 11/11 haploidentical hscts also included antithymocyte globulin x 3. no patient received post-transplantation gvhd prophylaxis. all but 5 patients received rituximab on day +1 per protocol for recipient positive epstein barr virus (ebv) serology. results: all patients engrafted. median time to neutrophil engraftment was 13 (8-30) days, and median time to platelet engraftment was 16 (12-40) days. one patient experienced graft rejection on day +18, and twelve patients relapsed at a median of 173 (47-625) days. overall survival was 28/40 (70%) at a median of 24.2 (5.8-34) months follow-up. two (5%) patients developed grade iii or higher acute gvhd, and 2 (5%) patients developed extensive chronic gvhd. cumulative incidence of cytomegalovirus (cmv) and adenovirus reactivation were 11/40 (27.5%) and 5/40 (12.5%), respectively. nine (22.5%) patients developed bk hemorrhagic cystitis +/-viremia. ebv reactivation was not observed. median total, myeloid, t cell, and b cell donor chimerism were all 100% (ranges 50-100%, 93-100%, 41-100%, and 99-100%) at 1 year post-hsct. immune reconstitution of all cells lines was rapid ( table 1) . eighteen of 26 (69%) patients had detectable t cell receptor excision circles (trecs) by 4 months with a median trec count of 1226 (0-5713) per 10^6 cd3 t cells, and recovery of the naïve t-cell compartment was observed by 8 months in 19/26 (73%) of patients. t cell function as measured by response to pha was normal by 8 months in 15/23 (65%) patients and continued to increase steadily with time. despite rituximab on day +1 for 35/40 (87.5%) patients, there was rapid b cell reconstitution. nineteen of 23 (82.6%) patients had present switched memory b cells at 8 months, and 22/28 (78.5%) surviving patients are off immunoglobulin replacement at a median of 4 (4-18) months. conclusions: selective tcr-alpha/beta and cd19 depletion of haploidentical and unrelated grafts results in high engraftment and rapid immune reconstitution with low incidence of gvhd in children with hematologic malignancy. cd19 depletion and routine post-hsct rituximab on day +1 are effective at preventing ebv reactivation. introduction/background: hyper-igd syndrome (hids; 260920) is an autosomal recessive disorder characterized by recurrent episodes of fever associated with lymphadenopathy, arthralgia, gastrointestinal disturbance, and skin rash. the diagnostic hallmark of hids is a constitutively elevated level of serum immunoglobulin d (igd), although patients have been reported with normal igd levels. the disease is associated with mutations in the gene of mevalonate kinase. objectives: male patient, 30 years old, born to non-consanguineous parents, with a history of severe diarrhea since childhood, followed by respiratory infections and pneumonias. moreover he presented several episodes of severe abdominal pain, with intestinal obstruction since seven months old, needing surgical intervention due to acute abdomen. this picture repeated several times until 10 years of age, being submitted to new surgeries due to intestinal suboclusion. at 16 and 17 years he had gastroenteritis and then pancreatitis. 2 years ago new severe acute gastroenterocolitis with suboclusion, submitted to laparotomy and resection of a little part of the gut. he has frequent diarrheas, triggered by coffee and tea. usually evacuates 5 times a day. he was evaluated by several pediatric immunologists in childhood, who distrusted alpha heavy chain disease. he had an intense reaction to the bcg vaccine, and then did not make any more vaccines (only opv (sabin) in the campaigns). he was tonsillectomized at 2 years of age. he had measles, chickenpox, and mumps (at 5 years old). cellulitis at 7 years, repeating several times since then. he presented improvement of pneumonias but still has sinusitis and otitis (approximately 8 times a year introduction/background: pitthopkins syndrome is a rare neurological disorder caused by mutations the tcf4 gene on chromosome 18q21. clinical features include severe intellectual disability, constipation, microcephaly, and seizures. features distinguishing it from other neurodevelopmental syndromes such as rett syndrome and angelman syndrome include breathing abnormalities (either apneic episodes or hyperventilation) and atypical facial features. typical facial dysmorphism includes bitemporal narrowing, deep-set eyes, an m shaped upper lip, and widely spaced teeth. although a very rare diagnosis (slightly over 200 reported cases), it is not known to be associated with underlying humoral or cellular immunodeficiency. there is only one report of igm abnormalities described in a patient with pitt-hopkins syndrome. objectives: to present a case of pitthopkins syndrome with humoral immunodeficiency. methods: this is a case presentation of a patient with pitt-hopkins syndrome requiring immunoglobulin replacement therapy. results: 15 year old female with genetically diagnosed pitt-hopkins syndrome who presented to our office for immunological evaluation in the setting of recurrent sinopulmonary infections. she was placed on chronic antibiotics by the department of otholaryngology for approximately two years prior to presentation. she was found to be hypogammaglobulinemic (igg: 398 mg/dl, iga: 21 mg/dl, igm: 40 mg/dl), had non-protective titers to streptococcus igg antibody (6/23 titers protective greater than 1.3 mcg/ml) despite booster vaccination with pneumovax, and had borderline tetanus (0.18 iu/ml) and diphtheria titers (0.11 iu/ml). given this laboratory evaluation and her recurrent illness, immunoglobulin replacement therapy (igrt) was started. whole exome sequencing was completed to assess for any other genetic cause of her immunodeficiency. the only abnormality was her previous known pathologic variant p.q670hfsx40 c.2010_2011delga (gln670his) in exon 19 in the tcf4 gene. she continued to have infections despite therapeutic igrt. chronic antibiotic treatment was initially tapered, however needed to be reintroduced as ivig alone was not stopping her infections, despite a igg level over 1000 mg/dl. conclusions: humoral immunologic deficits are not known to be associated with pitt hopkins syndrome. there has been one case report of a patient with poliomyelitis-like syndrome following an asthma attack in a patient with pitt hopkins syndrome, which was treated with igrt and resulted in a nearly complete recovery. however, igrt was not used for reasons of underlying immunodeficiency. to our knowledge this is the first patient with pitt hopkins syndrome with persistent hypogammaglobulinemia and frequent infections requiring immunoglobulin replacement therapy. it remains unclear why the patient continued to have infections despite igg levels > 1000 mg/dl, yet with the combination of igrt and prophylactic antibiotics the patient remains healthy. introduction/background: gata2 deficiency is a rare disease that typically presents in late childhood or early adulthood with heterogeneous phenotypes including emberger syndrome. emberger syndrome is characterized by lymphedema and predisposition for myelodysplastic syndrome (mds) and acute myeloid leukemia (aml). over 75% of patients with gata2 mutations have immune deficiencies. definitive diagnosis of gata2 deficiency is made by gene sequencing, and treatment includes infection control and potentially hematopoietic stem cell transplant (hsct). objectives: the goal of this report is to contribute data to the small documented cohort of patients with gata2 deficiency to aid in diagnosis and management of this rare, heterogeneous disorder. methods: we present a case series of three siblings with identical gata2 mutations with variable phenotypes. a usidnet query resulted 51 patients with gata2 mutations. results: three siblings (12 year-old female (a), 15 year-old male twin (b), and 15 year-old female twin (c)) and their mother had congenital deafness. clinical symptoms include (a) h1n1 influenza requiring mechanical ventilation, warts, and hypogammaglobulinemia, (b) streptococcus pyogenes neck abscess, warts, acne, and mds, and (c) lymphedema and acne. all three patients had absolute monocyte count (amc) of 0 and lymphopenia without documented lymphoid cell dysfunction. a mutation on exon 5, c.1062delg, confirmed the diagnosis. all three patients were started on mycobacterial prophylaxis with azithromycin and recommended hpv vaccination. the usidnet query average age of symptom onset was 22 years, and average age at diagnosis was 31.5 years. 35.3% (18/51) of patients had a family history of gata2 deficiency, 49% (25/ 51) of patients had warts, 7.8% (4/51) had lymphedema and only 1 patient had sensorineural deafness. 38% (19/50) of patients had amc of 0. functional data was limited. conclusions: life threatening infections as well as hematologic malignancies have been reported in patients with gata2 deficiency, which can be successfully treated with hsct. to our knowledge, the gata2 mutation detected in this family has not been previously reported. the clinical presentation in these three patients was heterogeneous despite identical genotypes, and diagnosis occurred years after initial symptoms. variable phenotypes were found in the usidnet gata2 deficiency cohort as well. a high index of suspicion for the disorder and early recognition of clinical manifestations and laboratory abnormalities may aid in timely diagnosis of gata2 deficiency, with potential for improved outcomes. consulting medical advisor, immune deficiency foundation introduction/background: as individuals with antibody deficiencies age they are susceptible to developing shingles. patients with antibody deficiencies are advised not to receive live viral vaccines such as zostavax, the shingles vaccine. objectives: our survey aimed to determine the frequency of shingles and use of zostavax in common variable immunodeficiency (cvid) and hypogammaglobulinemia patients. methods: 11,533 email invitations delivered to members of the immune deficiency foundation database requesting participation in an online survey about zoster, influenza and varicella experiences. data from 881 individuals age 19 years old or older with cvid (n=760; mean age 54 years old; 83% female; 92% white non-hispanic) or hypogammaglobulinemia (n=121; mean age 54 years old; 83% female; 93% white non-hispanic) were analyzed. results: close to one fifth (18%, n=105) of adults age 50 or older with cvid or hypogammaglobulinemia (n=568) had received shingles vaccination. the majority of those who were vaccinated reported receiving zostavax once (92%, n=97), while 5% (n=5) received a booster vaccination as well. mild side effects (e.g., skin rash and muscle pain) were only reported by 18% (n=18) of vaccine recipients after receiving their first vaccination. no side effects were reported after receiving the booster vaccination and no hospitalizations were reported as a result of receiving zostavax. when comparing shingles diagnosis and shingles vaccination, of the 568 adults age 50 years old and older, 35% (n=196) had been diagnosed with shingles, and of those diagnosed 82% (n=160) did not receive zostavax. of adults age 19 years old or older, 32% (n=279) reported a shingles diagnosis. similarly, the cdc reports almost 30% people in the united states will develop shingles during their lifetime. more than half (56%, n=157) of those ever diagnosed with shingles reported experiencing shingles once, 18% (n=51) had shingles twice and 24% (n=68) had shingles three or more times. respondents with more than three shingles episodes were more likely to report their rash lasted more than two months and their blisters became infected. conclusions: almost 20% of adults with cvid or hypogammaglobulinemia reported receiving zostavax despite recommendations against vaccinations for immunodeficient individuals. however, side effects in those pi patients who received the shingles vaccine appears minimal. though it is possible these individuals were vaccinated prior to diagnosis of their pi; additional patient and physician education on live vaccines and immunodeficiency may be needed as well. the approval of the new non-live virus component varicella zoster vaccine may be of benefit to patients with pi. introduction/background: inclusion body myositis (ibm) is a rare disorder characterized as an inflammatory myopathy with endomysial inflammation and numerous red-rimmed vacuoles seen on biopsy. five cases of ibm have been described in the literature in patients with common variable immunodeficiency (cvid). objectives: to make immunologists aware of ibm as a complication of cvid, that may be incorrectly diagnosed as myositis or autoimmune neuropathy. methods: case description results: we report a 62-year-old man with common variable immunodeficiency on gammaglobulin replacement who presented complaining of progressively worsening lower extremity pain, weakness, and fatigue. he states that over the last couple of years, it has become difficult climbing and descending steps, arising from a seated position, and has begun experiencing frequent falls. his creatinine kinase level was found to be elevated at 293u/l and he continued to be lymphopenic with total lymphocyte counts ranging from 300 to 800k/ul (cd4+ t-cells 34%, cd8+ t-cells 18%, cd19+ bcells 2%). his esr ranged from 25 to 60mm/h. anti-glutamic acid decarboxy antibody (gad) was initially elevated at 1.3u/ml and rose to 2.3u/ml within 6 months suggestive of a neuropathy. based on an electromyography (emg) and a muscle biopsy, he was diagnosed with polymyositis. he was treated with high dose steroids with no improvement. his intravenous gammaglobulin dose was then increased from 45mg every 3 weeks to 45mg per day for 3 straight days every 2 weeks. 4 months later, his creatinine kinase level dropped into the normal range (<200u/l), however, he continued to complain of worsening weakness. physical exam showed decreased muscle bulk in forearms and quadriceps bilaterally, lack of a quadriceps tendon reflex, strength 4/5 in flexor digitorum profundus and 4/5 in hip flexors, and a broad-based gait. histology and electron microscopy of a repeat muscle biopsy identified rimmed muscle vacuoles as typically noted in inclusion body myositis. conclusions: inclusion body myositis is a potential rare complication of common variable immunodeficiency. it can mimic polymyositis and inflammatory demyelinating disorders. high dose steroids and ivig are of no clinical benefit in ibm, despite decreasing serum creatinine kinase levels, which may raise a false impression of a clinical benefit. objectives: in this abstract we present the case of the development of generalized cmv infection in a child with scid. girl n. at the age of 3 months entered the children's infectious clinical hospital with complaints of cough, high febrile temperature for 5 days, refusal to eat. from the anamnesis of life the girl from the 1st pregnancy, 1 birth, was born full term in 40 weeks gestation, birth weight 4640g. for 3 months of life, a bad increase in body weight was noted and at the time of admission, the weight in 3 months was 5400 g. according to the parents, the child had atopic dermatitis. from the anamnesis of the disease on 08.01.2017, the temperature rose to 38.2°c, there was a cough and a mucous discharge from the nose. then the child refused to eat, the body temperature rose to 39.2°c. at this time, the girl's mom was borne by the ari. january 14, 2017 patient was hospitalized in the hospital with a diagnosis: acute respiratory viral infection, acute rhinitis, pharyngitis, acute bronchitis, toxicosis of 1-2 degrees. acute pneumonia? atopic dermatitis, infant form. on january 15, 2017, due to the worsening of the condition associated with the increase in oxygen (o2) -dependence, the child was transferred to the department of anesthesiology and resuscitation. methods: in the general analysis of blood upon admission, leukocytes are 14.2x10 9 / l, hemoglobin is 105 g / l, platelets are 172 x 10 9 / l, esr is 3 mm / hour, stabs are 4% (abs. -0.58 x 10 9 / l), segmented -60% (abs-8.64 x 10 9 / l), lymphocytes -24% (abs 3.46 x 10 9 / l), monocytes -10% (abs -1.44 x 10 9 / l). in a biochemical study, the total protein is 51 g / l, total bilirubin is 4.7 mol / l, urea is 7.4 mmol / l, creatinine is 60 mol / l, lactate dehydrogenase is 2249 u / l, alt is 131 u / l, asat -257 e / l, crp -5.8 mg / l. radiography of the lung from 14/01/2017 -data in favor of interstitial pneumonia. the study of the acid-base ph state is 7.367, pc02 is 34.3 mmhg, po2 is 40.1 mmhg, lactate is 1.4 mmol / l. a blood test was performed using the elisa and pcr method for markers of hsv, cmv, enterovirus and toxoplasmosis. ultrasound of the abdominal cavity revealed moderate hepatomegaly, signs of thickening of bile, splenomegaly. moderate diffuse changes in the renal parenchyma (toxic-inflammatory?). the minimum amount of free fluid in the abdominal cavity. ultrasound of the brain revealed signs of subependimal microcyst on the right. according to the immunogram, a sharp decrease in cd3 + 26% (58-85%) was detected, activated t-lymphocytes (cd3 + hla-dr +) were 19.9% (3-15%), t helper / inducers (cd4 + cd8 -26.6% (30-56%) and t suppressors / cytotoxic (cd8 + cd4-) 0.5% (18-45%), a high ratio of tx / tc (cd4 + cd8 +) was detected 53.2% (0.6-2.3), cytotoxic non-t cells (cd3-cd8 +) -1,2, an increase in the number of b-lymphocytes (cd19 +) -58.9% (7-20%), natural killers (cd16 + cd56 +) -6.6% (5-25%), natural t-killers (cd3 + cd16 + cd56 +) -0.3 (0-5%), leukocyte gates (cd45 + cd14-) -99% (95 -100 %). the absolute content of t-lymphocytes was 0.15 x 10 9 / l, b -lymphocytes -0.35 x 10 9 / l. the number of thymic migrants (cd45 + cd45ra + cd31 +) was not detected (0%). according to the results of the immunogram the diagnosis is made: severe combined immunodeficiency (t-b + nk +). 01/17/2017 ct scan of the chest was diagnosed ct signs of a polysergic two-sided inflammatory process in the lungs (figure 3 ). when blood was sown for sterility on january 19, 2017, staphylococcus epidermidis was isolated in an amount of 10 3, sensitive to linezolid, gentamicin resistant to amoxicillin, amoxicillin / clavulonic acid, and ciprofloxacin. on january 19, 2017, cmv dna was detected in an amount of 7.6 × 10 6 copies / ml. results: since the arrival clarithromycin was administered at a dose of 15 mg / kg per day in 2 divided doses from 14/01/2017 to 15/01/2017. from 16/01/2017 to 17/01/2017. change of antibacterial therapy for azithromycin intravenous at a dose of 10 mg / kg per day once a day.17.01.2017 -01/18/2017 the state of the child is very severe with negative clinical and laboratory dynamics despite the ongoing therapy. antibacterial therapy was changed to meropenem in a dose of 60 mg / kg intravenously every 8 hours, linezolid at a dose of 30 mg / kg per day and oseltamivir at a dose of 6 mg / kg per day from 17/01/2017 to 20/01/2017. 19.01.2017-20.01.2017 substitution therapy with an octagam in a dose of 0.4 g / kg was intravenously dripped. the patient's condition without significant dynamics. based on the results of pcr on cmv, ganciclovir was administered at a dose of 10 mg / kg intravenously drip 2 times a day. 01/23/2017 due to a decrease in platelet count, the platelet mass is transfused and there was a rash all over the body at night, which is associated with the development of the "graft versus host" reaction (gvhr). despite the ongoing therapy, a fatal outcome occurred. the main diagnosis: primary immunodeficiency (severe combined immunodeficiency, t0 b + nk +). complications: sepsis. septic shock. spon: ards, renal failure, dis, thrombocytopenia, anemia 3. two-sided lower-lobe pneumonia. generalized cmv infection. gvhd, acute dermal form. concomitant: atopic dermatitis, infant form. conclusions: the peculiarity of the described clinical case was that the patient's first symptoms of scid developed in the first months of life and were manifested by a bad weight gain, atopic dermatitis and the development of a life-threatening generalized cytomegalovirus infection with the development of bilateral low-grade pneumonia, respiratory insufficiency and acute cutaneous gvhd form, after transfusion of unirradiated platelet mass. an expanded immunological study confirmed the diagnosis of scid. methods: this study included 5 patients (2 boys and 3 girls) aged 2 months to 5 years. the reasons for entering the hospital were manifestations of severe hepatitis (in 2 children), acute respiratory infection (2 children) and 1 patient with symptoms of infectious mononucleosis. all patients were examined according to clinical protocols and given the severity and atypicality of the course of any infectious diseases, patients underwent immunological examination of the blood and they were consulted by an immunologist. all children were diagnosed with congenital immunodeficiency. results: in 3 cases, the trigger for the realization of the immunodeficiency state was infection (e. meningoseptica + kl. pneumonia + b. pertussis; cmv; veb); in 2 patients, giant cell hepatitis occurred. in 2 patients, despite the ongoing therapy, the disease had an unfavorable (lethal) outcome (1 patient with hepatitis and 1 patient with generalized cmv infection). conclusions: thus, it should be noted that timely diagnosis of a congenital defect of the immune system and thus timely therapy will avoid adverse outcomes. interferon gamma (actimmune®) effects on severe burkholderia cepacia pneumonia in variant x-linked chronic granulomatous disease 4 professor of pediatrics, university of utah 5 associate professor of clinical pediatrics, keck school of medicine at the university of southern california 6 professor of medicine, university of utah introduction/background: interferon gamma (ifnγ; actimmune®) has been proven to significantly decrease the overall number of infections in patients with chronic granulomatous disease (cgd) when given prophylactically (nejm 324:408, 1991) . therapy with ifnγ has also been employed to treat severe overwhelming infections in some instances, such as severe aspergillosis with success (jid 613:908, 1991) . we report here, two patients with very severe burkholderia cepacia (b. cepacia) infection, one of whom was placed on a respirator for approximately two weeks and another who was on extracorporeal membrane oxygenation for an extended period of time. both were treated with ifnγ (actimmune®) in addition to appropriate antimicrobial therapy in an attempt to affect these lifethreatening infections. objectives: the objective of this presentation is to describe two very severe variant x-linked cgd patients with b. cepacia pneumonia who were treated with ifnγ. in addition, we measured both super oxide production as well as nitric oxide production in the stimulated or unstimulated phagocytes from these patients in the presence or absence of interferon gamma. methods: case histories of both patients were reviewed in respect to the severity of their infection, the time spent on a respiratory or extracorporeal membrane oxygenation, the antimicrobial therapy administered, and the clinical results following the administration of interferon gamma as adjunctive immunomodulatory treatment. a standardized neutrophil oxidative burst assay was employed using cytochrome c reduction to measure super oxide production. in addition, nitric oxide was measured in the phagoctyes of the patients after stimulation with phorbol myristate acetate (pma) in the presence or absence of ifnγ using daf-2 fluorescence dye to detect the production of intracellular nitric oxide. results: a 2-year-old male developed a left lobar pneumonia and was admitted to primary children's medical center's intensive care unit and treated with iv cefotaxime, clindamycin, later, vancomycin and azithromycin were added. ct scan revealed a left-sided pneumonia and moderate parapneumonic effusion. subsequently, the patient decompensated, was intubated, and placed on respirator therapy. broncheoalveolar lavage and blood grew b. cepacia. neutrophil dihydrorhodamine fluorescence (dhr) demonstrated an intermediate broad peak of fluorescence with a small peak of unactivated cells, while the mother's dhr showed a broad intermediate peak suggesting the carrier state of variant x-linked cgd. both the patient, a 2.5 month old younger brother, and the carrier mother were found to have a g to a splice site mutation in exon 3 of the gp91-phox gene at position c.252 confirming the diagnosis of x-linked variant cgd. after approximately 2 weeks on the respirator, ifnγ (actimmune®) therapy was instituted with significant improvement of the patient's lung function. he was taken off the respirator approximately 3 days after the ifnγ therapy was instituted. following addition of ifnγ to his pma-stimulated neutrophil, there was a 22% increase in superoxide production and a 20 fold increase in nitric oxide in his monocytes. the second patient was a 9-year-old male who presented with fever and cough and was diagnosed with right-sided middle and lower lobe pneumonia with cavitations. bronchoalveolar lavage grew b. cepacia and nocardia. following increasing ventilatory and circulatory collapse he was placed on extracorporeal membrane oxygenation and treated with 4-5 antimicrobial agents. after 9 days of such therapy, ifnγ therapy was initiated and he was weaned from ecmo after 3 days and has remained essentially healthy since then when he is on ifnγ prophylaxis. dhr revealed a broad intermediate peak in the patient and normal and intermediate peaks in the mother suggesting variant x-linked cgd in the patient and the carrier state of x-linked variant cgd in the mother. targeted sequencing revealed a g to a splice site mutation in exom 3 of the cybb gene at position c.266 confirming the diagnosis of x-linked variant cgd. following addition of ifnγ to this patient's pma stimulated phagocytes, there was a 150% increase in superoxide production and a 200% increase in monocyte nitric oxide production. conclusions: two male variant x-linked cgd patients with splice site mutations in the cybb gene and severe life-threatening b. cepacia pneumonia, one on a respirator and one on ecmo were administered ifnγ (actimmune®) and each responded dramatically within 2-3 days recovering their respiratory capacity and coming off of assisted ventilation and ecmo, and have continued to do well when on ifnγ (actimmune®) therapy. introduction/background: hyqvia is a recombinant human hyaluronidase (rhuph20)-facilitated subcutaneous immunoglobulin (ighy) 10% replacement therapy for patients with primary immunodeficiency diseases (pidd). objectives: to acquire long-term safety data on ighy, and assess prescribed treatment regimens and administration in routine clinical practice, a global postauthorization safety study (pass) is being conducted. methods: this is an ongoing prospective, non-interventional, open-label, uncontrolled, multicenter study initiated in the united states in november 2015 to assess local and systemic effects of ighy within a routine clinical setting. patients aged 16 years with pidd who have been prescribed and/ or have started ighy are eligible for enrollment. patients are followed according to standard clinical practice and their treatment regimen is at the discretion of the treating physician. the presence of anti-rhuph20 antibody titers is evaluated on a voluntary basis. results: as of august 2017, 175 patients had been enrolled at 26 us study sites. there were no serious aes which were deemed treatment related. sixteen patients experienced a causally related non-serious local ae (9.1%; 0.43 events/patient-year, 0.07 events per infusion) and 25 patients experienced a causally related non-serious systemic ae (14.3%, 0.88 events/patient year, 0.14 events per infusion). of the 113 patients with immunogenicity data, 7 had 1 positive binding antibody test to rhuph20 (titers 1:160); no neutralizing rhuph20 antibodies were detected. conclusions: this interim analysis of prospectively-collected data of ighy use in routine clinical practice indicates that ighy is well tolerated with no treatment-related saes and has not been associated with neutralizing anti-rhuph20 antibodies in patients with pidd. introduction/background: idiopathic thrombocytopenic purpura (itp) and/or hemolytic anemia accompanied by splenomegaly occurs in up to 25% of patients with common variable immunodeficiency (cvid). treatments include steroids, other immune suppressants and rituximab. however, in some that do not respond, splenectomy may be performed. while splenectomy is known to be associated with an increased risk of infections or thromboembolic events, studies in other conditions (hemolytic disorders, hereditary spherocytosis, etc), also suggest an increased risk of pulmonary arterial hypertension (pah) after this procedure. objectives: while splenectomy is known to be associated with an increased risk of infections or thromboembolic events, studies in other conditions (hemolytic disorders, hereditary spherocytosis, etc), also suggest an increased risk of pulmonary arterial hypertension (pah) after this procedure. methods: we report three cases of pah following splenectomy for cytopenias in patients with cvid. results: the first is a 40 yo female, with long standing cvid complicated by interstitial lung disease, nodular regenerative liver disease and itp post splenectomy. the second is a 48 yo man with severe bronchiectasis, cirrhosis/nodular regenerative liver disease and itp post splenectomy. the third is a 54 yo woman with cvid (taci compound) complicated by cirrhosis/nodular regenerative liver disease, lung nodules and evans syndrome. all developed severe pah requiring chronic medications. pah in these patients is best classified as multifactorial, group v. conclusions: whether due to thrombus formation, continued cytopenias, and/or vascular changes, we suggest that pah may be a long-term complication of splenectomy in complex cvid. connective tissue, skeletal and vascular abnormalities. two isoforms exist, stat3, a 770 amino acid protein, and stat3, a 722 amino acid protein produced by alternative splicing of exon 23 resulting in a frame shift and truncated protein at the c-terminus. objectives: we follow 4 patients from 2 families with stat3 mutations leading to altered c-terminal proteins. the patients have high ige but milder features of ad-hies. methods: clinical data were collected. stat3 sequencing, stat3 functional assays as well as lymphocyte phenotyping were performed. results: patient 1 is a 58 year old man diagnosed with hies as a child due to eczema, recurrent boils and high ige (5000s iu/ml). he has tortuous and dilated coronary arteries, however denies lung infections, cmc, retained teeth, scoliosis, minimal trauma fractures, or hyperextensible joints. as an adult he developed avascular necrosis of both hips. whole exome sequencing revealed a novel splice mutation, c.2144+1g>t at the end of exon 22 causing skipping of the 43 nucleotide exon as well as utilization of the stat3 alternative splice acceptor in exon 23, resulting in a 93 nucleotide deletion. the mutant stat3 protein product has a 31 amino acid, in-frame deletion encompassing both y705 and s727 phosphorylation sites. no stat3 is made from the mutant allele. lymphocyte phenotyping was unremarkable, however total stat3 protein levels were decreased in ebv transformed b cell lines and there was decreased y705 phosphorylation after stimulation. patient 2 (family 2) was healthy until diagnosed with severe, refractory coccidiodies pneumonia complicated by pneumothorax with prolonged bronchopleural fistulae at age 16 years. this led to an immune evaluation in which he was found to have elevated ige (878 iu/ml, ref 0.0-91.0 iu/ ml). he had one perianal abscess, primary teeth requiring extraction and mild scoliosis, but denies cmc, bacterial pneumonias, eczema, or minimal trauma fractures. stat3 sequencing revealed a single base insertion in the transactivation domain, c.2185_2186insc causing a frameshift in the stat3 isoform, p.r729pfsx11, occurring immediately after the s727 phoshporylation site. the deletion occurs within the alternatively spliced region of exon 23, providing an intact, wild-type stat3. stimulation with il-6 or il-21 showed reduced pstat3 at y705, and elevated pstat1which is often seen in other ad-hies patients. lymphocyte phenotyping was unremarkable and th17 cell analysis showed low-normal levels of th17 cells while ebv transformed b cells showed reduced total stat3 levels. his mother and infant sister also share this mutation -the 8 month old infant had normal ige, and intermittent rashes; his mother has high ige (1193 iu/ml), recurrent sinopulmonary infections (complicated by tobacco use) but without bronchiectasis or pneumatocele, and denies cmc with the exception of pregnancy related vaginal candidiasis. conclusions: loss of function and gain of function mutations in stat3 lead to distinct syndromes, but it appears that stat3 mutations affecting the isoform expression, such as those reported here, can also lead to immune dysregulation with incomplete features of ad-hies. these stat3 mutations will allow us to better understand the relative roles of the isoforms stat3 and stat3 in somatic and immune cell signaling. physicians and is associated with immunological defects aswell. mutationsin the kmt2d and kdm6a genes are the most common genetic changes that lead to kabuki syndrome but for many cases the genetic basis remains unknown. objectives: recognize the varied presentation for a unique immunodeficiency syndrome methods: this is a case series. results: this is a case series describing three patients with ks and their clinical presentations which predominantly involve immunodeficiency and autoimmunity. our first patient is a 31-year-old male with autoimmune hemolytic anemia (aiha) at a young age, recurrent respiratory infections and hypogammaglobinemia which led to a diagnosis of cvid at the age of six. in addition, he has dysmorphic facial features, intellectual disabilities, and short stature but it was not until his late twenties where he was found to have a missense mutation in kmt2d (p.arg5048cys) which has been described in patients with ks. the second patient is a 34-year-old female with hypogammaglobinemia, evan's syndrome, short stature, and severe complications which include granulomatous-lymphocytic interstitial lung disease, pulmonary hypertension, and chronic kidney disease. she was also found to have a missense mutation in kmt2d (p.arg5048cys) in her early thirties and passed away from complications of her disease. the third patient is a 27-year-old female with a history of low iga/igg and poor vaccine titers, aiha, neutropenia, pulmonary nodules, and developmental delay who was diagnosed with cvid in her mid-twenties. for her immunodeficiency and autoimmunity, she was treated with immunoglobulin replacement, rituximab and cyclosporine and was found to have a missense mutation in kmt2d (p.cys1471trp). this mutation was a de novo mutation in this patient and has also been reported in another patient with ks. conclusions: these cases highlight that the presentation of ks is varied and frequently includes immunodeficiency and autoimmunity in addition to the characteristic short stature and developmental delay. a diagnosis of ks remains challenging due the diversity of symptoms and disease severity, the need for genetic testing, and due to overlapping clinical presentations with other developmental conditions. thus, often times, like in these cases, the diagnosis of ks is delayed. chief medical officer, rocket pharma introduction/background: lad-i is a rare disorder of leukocyte adhesion, resulting from itgb2 gene mutations encoding for the beta-2 integrin component cd18. cd18 deficiencies prevent integrin dimerization and endothelial leukocyte adhesion, essential for extravasation and antimicrobial activity. severe lad-i (<2% of normal neutrophil [pmn] cd18 levels) is characterized by recurrent serious infections and early mortality unless treated by allogeneic hematopoietic stem cell transplant (hsct). mortality for severe lad-i was reported as 75% by age 2 in an initial 1988 multicenter retrospective study. moderate lad-i (2-30% of pmn cd18 levels) is more indolent; although most patients (pts) survive childhood with recurrent skin and mucosal surface infections; mortality by age 40 can exceed 50%. lad-i is characterized by umbilical cord complications (delayed separation and omphalitis), poor wound healing and leukocytosis. objectives: reports regarding lad-i have been published in recent decades but no recent comprehensive prognostic assessments are available. we sought an updated understanding of severe lad-i with emphasis on prognosis in the absence of hsct, hsct outcomes and association of cd18 expression with clinical features. methods: we created a database of all published lad-i cases via pubmed searches and review of available references. results: three hundred twenty-three lad-i cases were reported between 1975-2017 in 107 publications (68 case-reports; largest series n=36). the nations reporting the most cases were iran (n=65), usa (n=50), and india (n=45); the highest number of publications were from us centers (25). 113 pts were considered to have severe lad-i, 63 moderate and 147 were not classified. pmn cd18 expression levels was reported for 265 cases and was <2% in 135 patients (51%) and >=2% in 130 pts. four pts with cd18 >2% were considered to have severe lad-i (cd18% range 2.4 17.3). gender was noted for 282 pts; 148 (52%) were male. age at presentation was reported for 146 cases. for 63 pts with cd18<2%, median presentation was age 1 m (range 0.03-18m); for 62 pts with cd18 >=2%, median presentation was age 6m (range 0.03-192m). infection details and cd18% were available for 154 (48%) cases. the most frequent infections in pts with cd18 <2% were respiratory tract (39%), sepsis (29%) and otitis media (27%) and for pts with cd18 >=2% they were periodontal (52%), otitis media (365%) and sepsis (25%). perianal skin infections and necrotic skin ulcers were noted in >10%. umbilical complications were more frequent in severe lad-i (92 of 110 pts with cd18<2% [84%] and 47 of 81 with cd18 >=2% [58%; p = 0.0002]). for severe lad-i pts with 2 years of follow-up (or death prior to 2y), there was correlation between absence of umbilical complications and survival to 24 m (p < 0.001). wbcs were reported in 143 cases (median 45 x 109/l; range 10 150 x 109/l). there were limited correlations between cd18 expression and wbc (r < 0.1) and between cd18 and cd11 expression (r < 0.5). mutation analyses were reported in 139 cases with >20 gene locations noted and mutations on exons 5, 6 and 7 accounting for 44% of specified cases. in 18 cases, cd18 expression was >30%; in 8 of 12 cases where cd11 expression was noted, at least one cd11 moiety was reported as <2%. we sought to understand whether prognosis for severe lad-i in the absence of hsct is similar to the initially-reported 25% survival to age 2. there were 66 severe lad-i cases (per investigator assessment or cd18 <2%) for whom survival to 2 years was reported, 40 of whom died prior to age 2 (61% mortality). mortality was similar for the subset of 43 cases reported since 2000 (56%, 24 deaths). early mortality was substantially lower in patients with cd18 >=2% and the majority of pts with cd18 >4% survived to adulthood. outcomes for 101 pts who received hsct were consistent with recent series; phenotypic correction was reported in 83% of pts with hla-matched sibling donors. mortality was 19% overall (11% for hlamatched sibling recipients). for 22 pts receiving haploidentical hsct there was 32% mortality and 55% received 1 subsequent hsct. conclusions: severe lad-i remains a life-threatening condition with limited 2-year survival in the absence of allogeneic hsct. umbilical complications and granulocytosis are frequent early manifestations; respiratory tract, ear, sepsis, oral and skin infections are common. hsct is potentially curative; transplant-mortality and other complications are frequent, especially in haploidentical recipients. diverse itgb2 mutations result in lad-i, and genetic evaluation may be valuable for diagnosis and prognosis. rapid identification of pts with potential lad-i (unusual or severe infections in infancy, granulocytosis and umbilical complications) is essential to enable referral to centers with disease expertise. objectives: to determine the phosophorylation of stat3 in ad-hies patients with known stat3 mutations. methods: peripheral blood mononuclear cells (pbmcs) were collected from ad-hies patients under irb-approved institutional research protocols. pbmcs were then stimulated with il-6 or il-21 for 15, 30 and 60 minutes. cells were surface stained for cd3, cd4, cd8, cd56 and cd19. they were then fixed, permeabilized and stained with anti-y705-stat3 to evaluate for phosphorylation of stat3 at position y705. cells were then washed and data acquired using flow cytometry. results: pbmcs from one ad-hies patient with a stat3 sh2 domain mutation (p.y657c) demonstrated normal y705 phosphorylation after il-6 stimulation. pbmcs from another ad-hies patient with a dbd mutation (p.h437y) exhibited highly reduced stat3 phosphorylation after il-6 stimulation and absent phosphorylation after il-21 stimulation. conclusions: these findings highlight that, in the context of ad-hies, the domain location of the stat3 mutation does not predict stat3 phosphorylation potential following stimulation and challenges the current paradigm. (106) submission id#426423 cheng sun 1 1 associate professor, institute of immunology introduction/background: as the predominant lymphocyte subset in the liver, natural killer (nk) cells have been shown to be highly correlated with the outcomes of patients with hepatocellular carcinoma (hcc). previously, we reported that nk cells were decreased and functional deficiency in hcc. however, the mechanism underline remains unknown. objectives: in this study, through the use of 23 healthy livers, paired peritumoural tissues (pt) and intratumoural tissues (it) from 236 hcc patients, methods: we have evaluated the expression of cd160 and its co-ligand receptor btla on hepatic cd8+ t cells and nk cells. results: decreased expression of cd160 on nk cells was observed in intratumoural but not pt regions, along with nk cell dysfunction, poor prognosis and tumour metastasis. human cd160+ nk cells exhibited functional activated, high capacity of ifn-secretion and nk mediated immunity by global transcriptomic analysis of sorted cd160+ and cd160-hepatic nk cells. blocking tgf-1 specifically reversed the ifn-production of cd160+ nk cells. in addition, this decreased cd160 expression is predominantly on cd56bright nk cells. conclusions: these findings indicate that cd160 expression reduction contributes to nk cell exhaustion and tumour immune escape, suggesting that cd160 has the therapeutic potential for fighting liver cancer. introduction/background: the low number of circulating lymphocytes in the blood is a marker for cellular immunodeficiency in young children. ethnicity also affects the lymphocyte count and ethnicity-specific lymphocyte norms have been used in many countries. this study analyzed the lymphocyte counts in a large cohort of infants and young children from the arabian peninsula. objectives 1-to define the normal lymphocyte counts in arab children 2-to define the possible cutoff lymphocyte count that define lymphopenia. methods: this is a cross-sectional analysis of the lymphocyte counts in 11,237 arab children. the age groups were: 1 day, 1-6 months, 6-12 months, 1-2 years, 2-3 years, 3-4 years, 4-5 years, and 5-6 years, 47% females. we analyzed the first blood count performed during their visit to the abu dhabi seha ambulatory healthcare services between april 2008 and october 2013. the median, 10th percentile and 90th percentile counts were calculated. the 10th percentile lymphocyte count was used to define lymphopenia. the kolmogorov-smirnov test, a non-parametric test, was used to compare lymphocyte counts between groups. statistical significance was defined by a two-tailed p<0.05. results: the median counts were higher during infancy. the variability (disparity) of the counts (reference intervals) progressively decreased from birth to 6 years of life. the 10th percentile lymphocyte counts were relatively constant from birth to 2 years (2.5-3.0 x109/l) and from 2 to 6 years (1.4-1.8 x109/l), table 1 . the lymphocyte counts were similar in boys and girls. the lymphocyte counts were compared to those from five other studies. conclusions: arab children have lower lymphocyte counts (10th percentile) than children in the united states, brazil and south africa, but their counts are similar to children in china and uganda. our study results support the development and use of ethnicity-specific lymphocyte count standards. the implication of our results is that using these lower cutoff values for lymphopenia will prevent a large number of arab children from having unnecessarily investigated for immunodeficiency. introduction/background: chronic granulomatous disease (cgd) is a rare phagocytic defect caused by mutations in the nadph oxidase 2 system leading to reduced or absent reactive oxygen species production. in addition to specific infectious susceptibility, patients with cgd are predisposed to hyperinflammation in response to infectious agents, autoimmunity, colitis, and other forms of autoinflammation. cgd patients with mutations in p47phox are at increased risk of diabetes and cardiovascular disease. hyperinflammatory and auto-inflammation responses are often difficult to predict and manage. intracellular adhesion molecule-1 (icam-1) and e-selectin are endothelial adhesion markers that facilitate the adhesion and transendothelial migration of leukocytes and elevations in these markers have been associated with cardiovascular disease, glomerular injury, and thrombotic events. expression of adhesion molecules is induced by pro-inflammatory cytokines and are associated with a hyperinflammatory state. objectives: to determine if e-selectin and icam-1 are elevated in patients with cgd and to determine of endothelial adhesion markers could serve as a biomarker of inflammatory disease in cgd patients. methods: thirty-eight pediatric and adult subjects with cgd (14 xlinked (xl-cgd), 21 p47phox deficient and 1 p22phox deficient, and 2 x-linked cgd carriers were enrolled. e-selectin (pg/ml) and icam-1 (ng/ml) were measured from the plasma of patients via sandwich elisa. results: all 38 cgd patients had histories of severe infection, active infection, chronic colitis, or other autoimmune disease at time of evaluation. nine p47phox deficient cgd patients had history of diabetes and/or early onset cardiovascular disease. one subject with x-linked cgd had undergone hematopoietic stem cell transplantation (hsct). plasma levels of e-selectin were significantly elevated above healthy controls (median 19,648 pg/ml) in subjects with xl-cgd (median 45,990 pg/ ml, p<0.001), p47phox deficient cgd (median 23,707 pg/ml, p=0.0017) and p22phox deficient cgd (80,108 pg/ml). plasma levels of icam-1 were also elevated above healthy controls in subjects with xl-cgd (median 553.2ng/ml), p47phox deficient cgd (median 241.7ng/ml), and p22phox deficient cgd (383.1ng/ml), although none were statistically significant. plasma quantities of e-selectin and icam-1 increased further in those p47phox deficient patients with diabetes and/or cardiovascular disease (median e-selectin 37,143 pg/ml, icam-1 356.7 ng/ml) but neither reached statistical significance. e-selectin and icam-1 quantities in female carriers of xl-cgd and in 1 xl-cgd cured by hsct were similar to values found in healthy controls. conclusions: immune dysregulatory features and hyperinflammation in cgd can be difficult to predict and manage. the endothelial adhesion markers e-selectin and icam-1 are elevated in patients with xl-cgd and p47phox deficient cgd that worsens with presence of early onset cardiovascular diseases and resolves post-hsct. elevations in e-selectin and icam-1 in the serum of cgd patients may serve as surrogate markers of inflammation and suggest a chronic endotheliopathy in cgd patients. introduction/background: the phenotypic presentation of ctla4 haploinsufficiency was only recently described. the management of these patients in the medical literature is limited to anecdotal case reports. we aimed to detail our experience with short and long term immunomodulatory therapy in treating autoimmune cytopenias in the background of ctla4 impairment. objectives: we aimed to assess the efficacy of mtor inhibitors in treating autoimmune cytopenias in patients with ctla4 haploinsufficiency. methods: we retrospectively identified 7 patients with proven ctla4 mutations and documented refractory autoimmune cytopenias while receiving care at nih clinical center (from july 2011 to august 2017). the complete (cr) and partial (pr) clinical response was assessed after six weeks of treatment and defined as hgb >10 g/dl, platelets >100k/ul and hgb >8g/ dl, platelets >50k/ul, respectively without transfusion requirement. results: all analyzed patients failed or exhibited disease recurrence on at least one prior medical therapy, including: corticosteroids, rituximab, romiplostim and eltrombopag. the initial response rate to evrolimus and/or sirolimus was 71% (3 cr and 2pr). one of the partial responders had recurrence of idiopathic thrombocytopenic purpura at four months while on rapalogs. overall, we used mtor inhibitors in 22 patients for a total of 37 patient years to treatm multiple modalities. the top three most common recorded adverse events were: clostridium difficile colitis (n=9, in 4 patients), lipid abnormalities (n =7, 3 patients required treatment) and bacterial pneumonia (n=6, in 4 patients). conclusions: our limited retrospective data suggests that mtor inhibitors might be efficacious in the treatment of autoimmune cytopenias in ctla4 haploinsufficent patients. further prospective studies are required to assess safety and efficacy of mtor inhibitors in this patient population. association with frequent upper respiratory tract infections and pharyngitis. from 2 years of age she continued to have recurrent febrile episodes in the absence of infection, with fever of 39-40 degrees celsius on a monthly basis, normally lasting 1-2 days. she also developed episodes of urticaria, temporally unrelated to her febrile episodes. the rash was noted to be induced by exposure to the cold, and would generally last 1-3 days with some clinical response to antihistamines and naproxen. her clinical picture then progressed and she developed joint pain and swelling, particularly affecting her hands and feet. over the following years the patient continued to have recurrent episodes of urticaria as well as progressive joint involvement and limitation. there was some initial improvement with naproxen and prednisone. her symptoms were refractory to subsequent treatment with methotrexate, leflunomide, infliximab and tocilizumab and she remained corticodependent. immunological work up including lymphocyte phenotype, immunoglobulins, vaccine responses to both protein and live vaccines and ch50 were normal. there was no evidence of raised inflammatory markers with esr 3-11mm/h, crp <0.2mg/l and ferritin 39ug/l. autoimmune workup including ana, ena, anti-dna, anca and c3 and c4 was normal. the initial differential diagnoses included systemic juvenile arthritis, periodic fever syndrome or cryopyrin associated periodic syndromes (caps). the patient therefore had a periodic fever gene panel that identified a heterozygous mutation in exon 3 of nlrp12, c.466c>t (p.arg156trp). whilst mutations in nlrp12 are known to be associated with familial cold autoinflammatory syndrome, this was reported as a variant of unknown significance. we therefore proceeded with functional in vitro testing to demonstrate pathogenicity. the patients monocytes showed increased secretion of il1b upon stimulation with lps as compared to healthy donors. when tested in a luciferase reporter assay, the mutated nlrp12 partially lost the capacity to inhibit the nfkb pathway. overall these in vitro studies show that this nlrp12 mutation results in defective regulation of the inflammatory response. the patient was commenced on anti-il1 therapy with canakinumab, with no clinical improvement so was therefore discontinued. we report a case of familial cold autoinflammatory syndrome due to a mutation in exon 3 of nlrp12, that to this point has remained refractory to multiple treatment modalities. functional testing was able to demonstrate mutation causality and defective regulation of the inflammatory response. objectives: authors aim to evaluate the spectrum of clinical phenotypes associated with nemo hypomorphic mutations within the usidnet registry. methods: investigators obtained demographic, laboratory, and clinical data on patients with a defect in nemo within the usidnet registry. results: there were 19 male patients within the usidnet registry with a diagnosis of eda-id attributed to nemo hypomorphic mutation. of these, 9 were associated with a known variant in nemo (e391x, f312l, m407v), and 4 were associated with previously unreported variants (d113v, e287del, c.1-16g>c). for 6 patients, a mutation was not specified. most reported having an affected family member (n=14, 74%). median age of symptom onset and diagnosis were 1 year (iqr 0.5-2y) and 2 years (iqr 1-10y), respectively. median age at most recent visit was 11 years (iqr 9-16y). infections, additional clinical features, treatments, and outcomes are summarized in table 1 . skin manifestations (n=13, 68%) and pulmonary complaints (n=10, 53%) were common, with eczema (n=11, 58%) and asthma being most prevalent (n=5, 26%). gastrointestinal conditions (n=9, 47%) were also frequently reported, and included non-specific diarrheal illness, enteropathy, colitis, enteritis, and inflammatory bowel disease. neurologic features (n=8, 42%), including seizures, hearing defect, peripheral neuropathy, and encephalopathy, were unexpectedly common, suggesting a previously unrecognized disease association. conclusions: we observed that allergic diseases, including asthma and eczema, were common in patients with nemo mutation. notably, varied neurologic features were more prevalent than previously reported. this study highlights the potential of cross-institutional registry analysis to deepen our understanding of extremely rare genetic diseases. coordinated effort across institutions is required to better characterize the spectrum of clinical phenotypes associated with hypomorphic mutations in nemo. mycobacterial lysate (ml) and purified protein (ppd) in the diagnosis of patients with mendelian susceptibility to mycobacterial disease (msmd) elimination of this infection depends mainly on the success of the interaction between macrophages and infected t lymphocytes. patients with mendelian susceptibility to mycobacterial disease (msmd) present severe and recurrent infections due to impaired signaling of the ifn/il-12 axis. objectives: our aim was to evaluated the ifn/il-12 axis of patients with clinical history suggestive of msmd using mycobacterial lysate (ml) and purified protein (ppd) by elisa assay. methods: samples of patients (n=43) with a clinical history suggestive of msmd arrived in our laboratory. for the diagnosis, 3 ml of blood diluted (1:2) in rpmi 1640 culture medium supplemented were used. the samples were distributed in two different plates, one used in the dosage of il12 (24 h) and the other in the ifn dosage (48 h). thus, they were stimulated with ml (2 ug/ml for the 24 h plate and 10 ug/ml for the 48 h plate) and with ppd (10 ug/ml for the 24 h plate and 2 ug/ml for plate of 48 h). at the same time, in half of the wells stimulated with lm and ppd, the cytokine ifn (1000 iu/ml) or il12p40 (2 ng/ml) were added in the plate. next, the plates were incubated at 37°c and 5% co² and the supernatant were collected and quantified by the elisa assay. the results were evaluated by the statistical mann whitney u test. results: six of the 43 patients presented alterations in the evaluation of the ifn/il-12 axis. the age of the diagnosis of male patients ranged from 2 to 16 years. the only two female patients diagnosed were 36 and 40 years old. the clinical history was heterogeneous: 4 had lymph node hyperplasia, 2 pneumonia, 1 colitis, 1 herpes zozter, another had a urinary tract infection and 1 bcgitis. the pathogens isolated were the m. tuberculosis, m. abscessus, m. gordonae, m. genavense and m. konsossi species found in 5 different patients. statistical analysis of the ifn-/il-12 axis evaluation by elisa was performed.the results of the dosage of ifn were significant in samples with lm and lm plus il-12 (***). similar results were observed in samples treated with ppd and ppd plus il-12 (**) when compared with healthy controls. in the il-12 dosage, statistical difference was observed in the samples with lm (***) and with lm plus ifn (*). in samples stimulated with ppd, the results did not show statistical differences (ns.) but ppd + ifn (*) were significantly different. conclusions: six patients were diagnosed by the evaluation the il-12/ ifn axis. the use of micobacterial lysate (ml) showed reliable results to the diagnosis of patients with il12/ifn pathway defects. the genetics diagnosis will be performed. introduction/background: invasive infections due to mycobacterial species are a feared complication in patients with t-cell deficiency or phagocyte disorders, and treatment is frequently complicated by antimicrobial resistance. cd4+ th1 t-cell immunity is known to be critical to antimycobacterial defense; accordingly, adoptive t-cell immunotherapy with mycobacteria-specific t-cells (mst) may be a beneficial therapy for combating these infections. objectives: to determine if ex vivo expansion of t-cells targeting common mycobacterial antigens is feasible from healthy donors, and whether the same antigens are recognized by patients with primary immunodeficiency (pid) and invasive mycobacterial infections. methods: peripheral blood mononuclear cells (pbmc) from healthy donors were pulsed with overlapping 15-mer peptide libraries encompassing five mycobacterial antigens (ag85b, ppe68, esat-6, cpf10, adk) and expanded for 10 days with cytokines il-4 and il-7. expanded msts were tested for specificity against the targeted antigens via ifn-g elispot, multiplex cytokine analysis, and flow cytometry. pbmcs from pid patients with invasive mycobacterial infections were similarly tested for presence of t-cells recognizing the tested mycobacterial antigens. a minimum of 20 spots per 1x105 cells above negative control was considered specific on elispot. results: ten healthy donors and eight patients with pid were tested. specificity against 1-5 mycobacterial antigens (median 3) was confirmed in all ten healthy donors, with a mean 6.6-fold cellular expansion during the 10-day culture. msts were predominantly cd4+ t-cells (mean/sd: 55 +/-6%), with both central memory (mean/sd 17.7 +/ -6.4%) and effector memory (mean/sd 77.4 +/-7.6%) populations. there was no clear difference in antigen specificity between bcg immunized (n=4) and bcg naïve (n=6) healthy donors. six of 8 pid patients had no detectable immunity to tested mycobacterial antigens. one patient with combined immunodeficiency has a low detectable specificity to ag85b (mean 47 spot forming colonies[sfc]), and a patient with nfkb1 haploinsufficiency mounted a response against ag85b (mean 388 sfc) and ppe68 (mean 40 sfc). conclusions: mycobacteria-specific t-cells can be rapidly expanded from healthy donors utilizing a protocol that could easily be translated to a good manufacturing practices facility. the majority of tested pid patients lacked immunity to the targeted antigens. adoptive immunotherapy with msts derived from third-party healthy donors may be a beneficial adjunctive therapy for pid patients with invasive mycobacterial infections. is an innate immune deficiency, primarily affecting the phagocytic compartment, and presenting with a diverse phenotypic spectrum ranging from severe childhood infections to monogenic inflammatory bowel disease. dihydrorhodamine (dhr) flow cytometry is the standard diagnostic test for cgd, and correlates with nadph oxidase activity. while there may be partial genotype correlation with the dhr flow pattern, in several patients, there is no correlation. objectives: in such patients, assessment by flow cytometric evaluation of nadph oxidase-specific (nox) proteins provides a convenient and rapid means of genetic triage (table) . methods: we performed dhr flow cytometry and nox flow cytometry on granulocytes and monocytes of cgd patients. results: phenotypic and laboratory patient data shown in table. *p9 had decreased p22phox (% and mfi) monocytes, but not in granulocytes. all other siblings (p6, p7, and p8), and mother (p10) had relatively higher p22phox (%) monocytes, but still lower than the healthy control. p7 had normal %p22phox monocytes, comparable to control. however, p6-p8, and p10 had normal p22phox (mfi) in monocytes and granulocytes. p6-9, and p10 have normal %gp91phox+ granulocytes, while p6and p9 have modestly decreased %gp91phox in monocytes. p6-9, and p10 have all moderately decreased gp91phox protein (mfi) in granulocytes and monocytes compared to healthy control, with a single population for protein expression. p6, p9 and p10 have bimodal populations for gp91phox in monocytes but not in granulocytes, with a larger positive population, and a much smaller negative population. the data from p6-9 suggest that the amount of gp91phox does not necessarily correlate with neutrophil oxidative burst, as measured by dhr. also, not all cybb variants affect p22phox protein expression, though both proteins are membrane-bound. the cyba vus in p8 does not appear to have affected p22phox protein expression in either monocytes or granulocytes. conclusions: the atypical clinical presentation of some cgd patients can make genotype-phenotype correlation with dhr flow data challenging. genetic testing, while necessary, can take several weeks. however, nadph-oxidase specific-protein flow assessment offers a rapid alternative to identification of the underlying genetic defect, and can be utilized as a reflex test to an abnormal dhr flow. further, it can provide insight into correlation between oxidative burst relative to protein expression in granulocytes and monocytes. head, division of immunology and allergy, the hospital for sick children introduction/background: adenosine deaminase (ada) is a ubiquitous enzyme important for purine metabolism. few studies have indicated that ada deficiency, in addition to causing profound lymphopenia and susceptibility to infections, is associated with neutrophils abnormalities. objectives: determine whether ada deficiency directly affects neutrophils and what are the mechanisms involved. methods: peripheral blood (pb) and bone marrow (bm) from 2.5 weeks old ada-deficient (ada-/-) mice that closely recapitulate the phenotype observed in ada-deficient patients, as well as ada+/-littermates were used to study neutrophils development and function. some experiments were supplemented with 6-week old ada-/-mice, maintained until 4 weeks of age with ada enzyme replacement, and littermates. results: the number of neutrophils in pb of ada-/-mice at 2.5 and 6 weeks of age was similar to ada+/-mice. the function of pb neutrophils from ada-/-mice, determined by oxidative burst, was also normal. the percentage of lin-/c-kit+/sca1+ hematopoietic progenitor cells in bm demonstrated significant reduction in ada-/-mice compared to littermates (0.93±0.23% and 1.21±0.33%, respectively, p=0.013). moreover, expansion of bm isolated from ada-/-mice in methylcellulose resulted in significantly less cd11b+/ly-6g+ neutrophils compared to healthy controls (13.6±8.4% compared to 25.3±10.3%, respectively, p=0.003). proliferation of bm cells, determined by brdu incorporation into cells dna, was higher in ada-/-mice than in littermates, possibly contributing to the normal neutrophil numbers in pb of ada-/-mice. conclusions: ada deficiency directly affects neutrophil development. further studies will help understand the significance of these effects and potential therapies for ada-deficient patients. objectives: we present here the clinico-pathologic features of a 10-yearold female with a heterozygous fancd2 gene deletion/mutation with evidence of cellular and humoral immune dysregulation. methods: we evaluated the patient using standard immunology anatomic, cellular, and biochemical functional assays. results: the patient has multiple dysmorphias including total anomalous venous return (repaired), mesomelia, absent ear canal, radial ray dysplasia, and short stature. her medical history is significant for an episode of pneumococcal sepsis despite adequate vaccination. whole exome sequencing demonstrated deletion of exons 2-17 and a pathologic mutation (c.2444g>a, p.arg815gln). repeated blood samples and immunophenotyping demonstrated severe lymphopenia. there were markedly low cd4+ t-cell counts with a low cd4:cd8 ratio (0.42). changes in the composition of the b-cell population included: significantly diminished absolute total b-cells, elevated immature cells, low levels of transitional cells, and undetectable advanced b-cell populations. there was no immunogenic response to pcv-13 or varicella/tetanus/ diphtheria vaccination. the nk-cell count was unaffected and demonstrated normal spontaneous and stimulated cytotoxic response. bone marrow analysis demonstrated hypocellularity without dysplasia. conclusions: we report here a pediatric patient with a novel fancd2 deletion/mutation presenting with severe lymphopenia in two cell compartments (b and t cells) and susceptibility to invasive bacterial infection. the findings are suggestive of combined immune deficiency. the cellular immune profile suggests that fancd2 may be involved in the transition of immature b and t cells to mature cells, a process that requires substantial dna recombination. additional genetic and biochemical evaluation is needed to further characterize this rare clinical finding. introduction/background: familial hemophagocytic lymphohistiocytosis type 2 (hlh) is a rare fatal condition due to a mutation in the pfr1 gene on chromosome 10q21-22 inherited in an autosomal recessive pattern which results in overactivation of the immune system. symptoms usually manifest before 1 year of age. a 17-year-old previously healthy male presented with acute onset of bilateral lower extremity pain and weakness, with subsequent inability to ambulate over a 10-day period. neurological exam demonstrated decreased lower extremity power, sensation, absent patellar and achilles reflexes, and a wide-based gait. objectives: not applicable methods: not applicable results: laboratory data was significant for neutropenia and thrombocytopenia, elevated levels of ferritin and serum cd25, and a positive ebv pcr. patient was initially treated with ivig for possible ebv-driven guillain-barre syndrome with some improvement in neurologic symptoms as well as thrombocytopenia. a mass of retroperitoneal lymph nodes were noted on spinal mri. testing was negative for alps and bone marrow biopsy was negative for leukemia/lymphoma. further work-up revealed absent perforin expression in cytotoxic cells and normal sap protein expression on staining, with poor nk function. genetic testing revealed a pathogenic mutation in the pfr1 gene with 1 additional variant of unknown significance. the patient was treated with rituximab for persistent ebvand returned with worsening lower extremity weakness. on mri, focal enhancements were found in the brain as well as worsening mass compression of the lumbosacral nerve roots. nerve root biopsy showed histiocytes with hemophagocytosis and a dense lymphohistiocytic infiltrate which stained positive for cd3, cd2, and granzyme b, with some loss of cd5 and no perforin. bone marrow biopsy was negative for hemophagocytes. conclusions: the patient was diagnosed with worsening familial hlh with cns involvement. hlh-directed chemotherapy (dexamethasone, cyclosporine, etoposide, intra-ommaya methotrexate/hydrocortisone) was started and hsct was performed. familial hlh is a rare and often lethal disorder that generally presents at a very young age. the acute onset and severity of presentation as occurred in this previously healthy adolescent is uncommon. familial hlh should be considered even in older patients with unexplained overactivation of the immune system. is the most profound form of primary immune deficiency, and is usually fatal in the first year of life without treatment. newborn screening for scid using quantitative analysis of t-cell receptor excision circles (trecs), has become the accepted method to facilitate early diagnosis and treatment in most of the united states, as scid babies typically do not make trecs. ikbkb deficiency is a rare form of autosomal recessive scid found in the northern cree first nations people of canada, where t cells develop normally but are non-functional. trec analysis is expected to be normal in ikbkb scid, and does not identify these cases. objectives: the objective of our study was to determine the feasibility of targeted genetic newborn testing for ikbkb deficiency. methods: we implemented a pilot project of prospective targeted genetic testing for the previously described homozygous ikbkb mutation (c.1292dupg) in newborns from 2 small northern manitoba communities. between 2013 and 2017, dna was extracted from dried blood spots of 724 newborns, and targeted sanger sequencing of the mutationharbouring ikbkb exon 13 was performed. results: all 724 infants born in the 2 selected communities underwent testing. fifty-five infants (7.6%, or 1/13.5) were found to be heterozygous carriers. one affected infant was identified, and underwent hematopoietic stem cell transplant before onset of infections. our findings are consistent with the predicted homozygosity for this mutation (1/13.5 x 1/13.5 x 1/4 = 1/729 births). conclusions: we demonstrated that targeted newborn testing for ikbkb deficiency was feasible, and provided the first prospective estimate of the ikbkb mutation carrier frequency in select manitoba northern cree first nations populations. we suggest that if we are to capture all babies with scid in manitoba, future newborn screening should be universal and include both trecs and direct mutation testing for population-specific mutations, including the first nations ikbkb mutation. high throughput analysis for trecs and targeted mutations will be introduced for universal newborn screening in manitoba. introduction/background: immunoglobulin class-switch recombination (csr) and somatic hypermutations (shm) are prerequisites of antibody and immunoglobulin receptor maturation and diversity within the adaptive immune system. the mismatch repair (mmr) machinery, consisting of homologues of mutsa, mutla, and mutsb (msh2/msh6, mlh1/pms2, and msh2/msh3, respectively) and other enzymes, is involved in csr, e.g. as backup of nonhomologous end-joining repair of activation-induced cytidine deaminaseinduced dna mismatches, and furthermore, in addition to errorprone polymerases, in the repair of shm-induced dna breaks. in line, a varying degree of antibody deficiency, from iga or selective igg subclass deficiency, to common variable immunodeficiency and hyper-igm syndrome have been shown in small numbers of patients with constitutional mmr deficiency (cmmrd) in addition to the known severe cancer predisposition due to genomic instability of patients with biallelic loss-of function mutations in one the mmr components. objectives: to elucidate the clinical relevance of primary immunodeficiency (pid) in cmmrd, we collected history and laboratory data of a novel cohort of 14 consecutive patients from 14 families with homozygous mutations in pms2 (n=7), msh6 (n=5), and mlh1 (n=2) reported to the consortium care for cmmrd (c4cmmrd) between 2014 and 2017, most of whom manifested with typical malignancies during childhood. methods: retrospective chart review according to a specific questionnaire and extended routine immunological analyses were performed with irb approval from the medical university of graz, austria. results: none of the presented patients fulfilled any classical or extended clinical warning signs of pid (infections, immune dysregulation, inflammation). furthermore, analyzing multiple specific laboratory parameters of the humoral and cellular immune system, we could not detect a uniform pattern of abnormalities. importantly, our data do not confirm previous suggestive evidence of iga or igg subclass deficiency, a specific antibody formation, or a b memory cell maturation defect. results of next generation sequencingbased detection of impaired class switch recombination and somatic hypermutations are pending. the t cell subsets and receptor repertoires were unaffected. together, neither clinical nor laboratory parameters were suggestive of pid in the present series of novel cmmrd patients. conclusions: we conclude that patients with cmmrd do not generally show a clinically relevant pid that could facilitate early diagnosis. on the contrary, these data support the prospect of potentially successful immune therapy of malignancies in the context of cmmrd. introduction/background: ada-2 deficiency is immune dysregulation diseases caused by an autosomal recessive mutation on cecr1 gene characterized by polyarteritis nodosa, childhood-onset, early-onset recurrent ischemic stroke and fever. objectives: report a new mutation on the cecr1 gene resulting in ada-2 deficient children. methods: female, 6-year-old, presented history of multiple ischemic strokes at one-year-old associated with recurrent fever and livedo racemosa. she has no siblings and parents are not consanguineous. results: the laboratory evaluation shows red cell=4.91x106/ml, hemoglobin=12.4g/dl, leucocytes=6.1x103/ml, neutrophils=4.3x103/ ml, lymphocytes=1.2x103/ml and plateletes=217 x103/ml. ige<25ui/ml, igg=467mg/dl, igm=13.8mg/dl and iga=21.3mg/dl. subsets lymphocytes shows cd3+=81.2%, cd4/cd8=1.41, cd19+ =10% and cd16/56=8.8%. due the clinical history, we performed cecr1 gene sequence homozygous substitution located at position -2 of acceptor splice site intron 6, c.847-2 g>a. this is a high conservative region with no alteration along phylogenetic studies and predicted to be pathogenic. to confirm the functional alteration, the ada-2 activity was tested in dried plasma spot showing 0.0 mu/g protein confirm the gene loss of function and the mutation pathogenicity. conclusions: the authors presented a novel mutation of cecr1 gene, the first one described in splice site causing gene loss of function and confirmed by extremely reduced ada2 activity. introduction/background: severe combined immunodeficiency (scid) is the most severe form of primary immunodeficiency characterized by severe, life threatening infections during early infancy. scid is a medical emergency associated with significant mortality if hematopoietic stem cell transplantations is not instituted early in the course of the disease. scid is a genetically heterogeneous disease caused by mutations in more than 30 different genes. different genes are implicated in different ethnic populations and geographical locales depending on the rates of consanguinity and endogamy in these populations. however, following the institution of newborn screening of scid in almost all state of the us and the widespread use of next generation sequencing in primary immunodeficiency diseases ar-scid due to mutations in rag1 and rag2 genes are found to be more prevalent than reported earlier. objectives: we performed a retrospective analysis of scid cases diagnosed at our centre and referred to us from other centres to determine the clinical, immunological and genetic basis of the disease in these cases. genetic variants both recurrent and novel were analysed in detail. methods: fifty six (56) of the 70 suspected patients met the esid diagnostic criteria. the clinical features, immunological defects and the gene sequencing results of these patients were analysed. gene sequencing was performed at the our centre and other collaborative centres at dept of pediatrics and adolescent medicine, queen mary hospital, hong kong, national defense medical college, saitama japan, kazusa dna research centre, chiba, japan and duke medical university centre, usa. mutations were detected in 36 of the 56 patients. mutations were classified as recurrent or novel after checking different databases such as exome aggregation consortium (exac), human gene mutation database (hgmd) and other relevant scid databases. the effect of novel, previously unreported mutations was determined using in-silico prediction tools such as sift and polyphen2. functional studies were also performed in few cases to determine the effect of novel mutations. results: mutations were detected in 36 patients. mutations were more common in genes causing autosomal recessive form of scid than the x-linked variant. mutations were detected in il2rg gene in 8 patients followed by mutation in the rag1 gene in 7 patients, dclre1c in 6 patients and rag 2 in 5 patients. mutations were also detected in ada gene (4 patients, 6 mutations), il7r (3 patients), stim1, pnp and nhej1 (1 patient each). nine novel mutations were detected. three in il2rg gene, 2 in rag1, 2 in ada and one each in the nhej1 and il7r gene. conclusions: autosomal recessive form of scid was more common in our cohort compared to x-linked form of the disease. mutations in rag1 and rag2 genes were the commonest (12 patients) followed by mutation in il2rg gene (8 patients). nine novel mutations in 5 different pid genes were detected in our cohort of scid patients introduction/background: rasgrp1 is a guanine-nucleotide exchange factor which phosphorylates ras-gdp to the activated form ras-gtp in response to t-cell receptor stimulation, resulting in ras activation. mutations in the gene coding for rasgrp1 have been recently described in four patients with profound t-cell deficiency, resulting in recurrent bacterial and viral infections, autoimmunity and malignancy. here we describe a two-year-old male presenting with recurrent sino-pulmonary infections, found to have two variants in rasgrp1, one not previously described. objectives 1. to describe a case of combined immunodeficiency with two pathogenic compound heterozygous rasgrp1 mutations. 2. to compare the clinical phenotype of rasgrp1 deficiency of our patient with that of previously described cases. 3. to argue for early hematopoietic stem cell transplantation in view of the increased susceptibility to epstein barr virus (ebv) induced lymphoma in patients with rasgrp1 deficiency. methods: a two-year-old male was referred to seattle children's immunology clinic for recurrent otitis media and two episodes of pneumonia. the diagnosis of combined immunodeficiency was considered based on a profound t-cell deficiency during immune evaluation and he was started on azithromycin and tmp/smx prophylaxis. at age 3 ½ years he was hospitalized with a bladder outlet obstruction and found to have two abdominal masses. biopsies were obtained and he was diagnosed with an ebv driven b cell lymphoproliferative disorder. in addition, his csf and bone marrow were considered positive based on pcr and staining, respectively. treatment with cyclophosphamide, prednisone, rituximab, and intrathecal methotrexate was initiated. due to poor csf ebv clearance, intrathecal therapy was escalated to rituximab. results: initial laboratory evaluation showed elevated igg (2290 mg/dl), normal number of cd19 b-lymphocytes, and adequate response to tetanus, prevnar-13 and varicella vaccine. b-cell phenotyping showed elevated immature/transitional b-cells. a profound t-cell defect was identified with cd4 t-cell lymphopenia (361/mm3), elevated cd8 t-cells (2074/mm3), inverted cd4/cd8 ratio (0.3) and absent proliferation in response to mitogens (pha, anti-cd3) and antigen (tetanus). forty-three percent of peripheral blood t-cells were / positive. t-cell phenotyping revealed decreased cd4 and cd8 naïve t-cells with elevated proportion of cd8+ t-effector memory t-cells. cervical lymph node and retroperitoneal mass biopsies showed atypical lymphoproliferation without malignant transformation. exome sequencing revealed two variants in rasgrp1. the first variant (c.1428+1g>a) is located at a splice site predicting an unstable transcript targeted for degradation. the second variant (c.1780c>t) is a novel mutation resulting in a stop codon. conclusions: recurrent sino-pulmonary infections are often a presentation of antibody deficiency. in this case, further investigation showed a profound t-cell defect, resembling that reported in 2 patients with homozygous nonsense mutations in the catalytic domain of rasgrp1 and 2 patients with homozygous insertion mutations leading to a premature stop codon at the bzip domain. our patient had biallelic mutations in rasgrp1 downstream of the catalytic domain, both leading to unstable transcripts. he developed an ebv induced atypical lymphoproliferative disorder, a complication reported in one rasgrp1 deficient patient whose disease progressed to b cell lymphoma and unsuccessful hsct. another patient developed ebv induced lymphoproliferative disorder after hsct for ebv-positive hodgkin lymphoma. two additional patients presented with recurrent infections, developed b-cell lymphoma and one was successfully transplanted. we are preparing the patient for hsct after chemotherapy for his lymphoproliferative disease to correct the underlying immune defect given that these patients are at high risk of developing lymphoma following ebv infection. introduction/background: immunodeficiency-centromeric instabilityfacial anomaly is a group of rare genetic disorders typically involving agammaglobulinemia. type four is caused by variants in the hells gene. the five patients previously reported with icf4 have fit the phenotype of agammaglobulinemia. here we report a patient with novel phenotype including neutropenia and neuroblastoma. objectives: describe a unique presentation of immunodeficiencycentromeric-instability-facial anomaly syndrome 4 to further expand our understanding of this disease. methods: retrospective chart review results: six-month old male was transferred to our tertiary care facility for ongoing chronic respiratory infection, chronic diarrhea, and failure to thrive. his past medical history was significant for 31-week prematurity due to rupture of membranes requiring a two month nicu stay for bronchopulmonary disease. upon discharge, he was bottle feeding and on room air. he had recurrent congestion for three months with two courses of antibiotics and one and a half weeks of diarrhea leading up to admission for difficulty breathing. he was found to have multiple infections including rhinovirus and parainfluenza virus on nasal wash, pjp pneumonia, norovirus, and pseudomonal cellulitis of his nose causing significant destruction. although previous laboratory studies revealed a normal absolute neutrophil count (anc), his anc quickly dropped to 170 cells/ul. his igg, iga, and igm were undetectable. while his total b cell count (451 cells/ul) was normal, he lacked any switched memory b cells. he had near normal total t cell count (cd3 2579 cells/ul) and cd4 count (2373 cells/ul) and a markedly decreased cd8 count (181 cells/ul) and poor proliferative response to low concentrations of phytohaemagglutinin and pokeweed mitogen. a 0.9cm x 1.6cm x 1.9cm paraspinal mass was found on chest ct, which was subsequently characterized as mibg-avid with a curie score 1 neuroblastoma. metastatic evaluation including bone marrow aspirate and biopsy was negative for malignancy. however, marked granulocytic hypoplasia and maturation arrest were present suggesting severe congenital neutropenia or, less likely, immune-mediated. whole exome sequencing detected homozygous variant of unknown significance in the hells gene (p.m223t). he was treated with intravenous immunoglobulin and g-csf with clinical and laboratory improvement. his neuroblastoma was initially observed, then subsequently removed due to a >50% increase in size. pathology confirmed mycn non-amplified favorable histology. he remains in remission 4 months after resection. he is currently awaiting bone marrow transplant for his immunodeficiency. conclusions: the significance of this case report is the novel presentation of icf4. neutropenia and malignancies have been reported in immunodeficiency-centromeric-instability-facial anomaly syndrome 2 (icf2) but not icf4. this case report thus expands upon the clinical picture of icf4 patients to include neutropenia and malignancies, and further describes the immunodeficiency. associate professor, university of south florida -johns hopkins all childrens hospital introduction/background: identification of newborns with severe combined immunodeficiency using state wide newborn screening (nbs) began in florida in 2012. abnormal results require extensive confirmatory diagnostic testing and prophylactic antimicrobial medications are needed to effectively evaluate and treat the infant. several barriers have been identified within government-sponsored health insurance programs that impede delivery of these evaluations and medications, often resulting in delays and/or inpatient hospitalization in order to provide timely and appropriate care. objectives: determine the cost differential between the initial evaluation and treatment of a scid patient detected by newborn screening in the inpatient versus outpatient setting. methods: the cost utilization of inpatient versus outpatient management of newly identified scid patients from nbs were analysed to include the cost of confirmatory testing and initiation of prophylaxis within the inpatient versus outpatient setting. laboratory tests included assessment of t cell immunity with quantitative and functional assessment, immunoglobulin measurement, genetic testing for scid variants, evaluation for maternal engraftment, and hla typing. medications included ig supplementation, pentamidine, fluconazole, and acyclovir. we compared the actual cost of inpatient stay and inpatient evaluation versus the approximate cost that would have been accrued if the patient were not admitted to the hospital. results: from 2012-2016, 4 infants with government-sponsored health insurance had abnormal nbs and were confirmed to have scid after evaluation at our institution. all 4 infants were admitted into the hospital for initial evaluation and initiation of appropriate medications for an average of 7 days. total average cost of medication administration for 7 days was $1,623, total cost of laboratory testing was $12,297, and average inpatient stay averaged $22,158 per patient. conversely, the cost that would have been accrued in the outpatient setting for medication would have been $1,097 for 7 days. laboratory testing costs would be no different as an outpatient. in total, the cost for inpatient evaluation was $36,708 versus $13,389 as an outpatient. conclusions: standard laboratory assessments and medications are necessary for infants identified with scid by population based nbs. despite government sponsoring of the florida nbs program, unnecessary barriers exist by government sponsored insurers that lead to a delay appropriate care. inpatient admission alleviates these barriers, but significantly increases cost. we advocate that standard ambulatory scid outpatient evaluation and initial treatment be authorized in children identified with scid through nbs without delay. patients with cd3g mutations reveal a role for human cd3g in treg diversity and suppressive function. introduction/background: integrity of the tcr/cd3 complex is crucial for positive and negative selection of t cells in the thymus, and for effector and regulatory functions of peripheral t lymphocytes. genetic defects that reduce, but do not abrogate tcr signaling, are associated with a variable degree of immune deficiency and immune dysregulation. in particular, while cd3d, cd3e, and cd3z gene defects in humans present mainly with severe immune deficiency, cd3g mutations lead to milder phenotypes, mainly characterized by autoimmunity. however, the role of cd3, encoded by cd3g, in establishing and maintaining immune tolerance has not been elucidated. objectives: we aimed to investigate abnormalities of treg cell repertoire and function in patients with genetic defects in cd3g with evidence of clinical autoimmunity. methods: high throughput sequencing (hts) was used to study composition and diversity of the t cell receptor (trb) repertoire in treg, conventional cd4+ (tconv), and cd8+ cells from 6 patients with cd3g mutations and in healthy controls. treg function was assessed by studying their ability to suppress proliferation of tconv cells. results: treg cells of patients with cd3g defects had reduced diversity, increased clonality, and reduced suppressive function. the trb repertoire of tconv cells from patients with cd3g deficiency was enriched for hydrophobic amino acids at position 6 and 7 of the cdr3, a biomarker of self-reactivity. overlap between treg and tconv cell repertoires was observed in cd3g mutated patients. conclusions: the treg and tconv cell repertoire of patients with cd3g mutations is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition. introduction/background: a common concern with b cell-depleting therapies is their potential effect on humoral immunity. although there have been reports of prolonged hypogammaglobulinemia in adult patients receiving rituximab, little is know about this phenomenon in children. objectives: we sought to assess humoral immunity in children receiving rituximab and determine risk factors leading to low immunoglobulin levels and infections. methods: we conducted a retrospective study on all pediatric patients ( 18 years) who received rituximab for the first time between january 2014 to december 2016 in a single tertiary pediatric hospital. charts were reviewed and data was collected prior to rituximab treatment and at 6, 12, and > 12 months after treatment. patients who received rituximab after hematopoietic cell transplantation (hct) or for a malignancy and those with an underlying primary immune deficiency (pid) at the time of treatment were excluded. results: in total, 106 patients received rituximab during the study period. of those, 38 were excluded (hct: n=31, lymphoma: n=6, pid: n=1). sixtyeight patients were eligible. indications for rituximab treatment were renal disease (n=23), neurologic disease (n=18), hematologic disease (n=12), rheumatologic disease (n=10), ebv control (n=3), other (n=1). one patient who died from autoimmune encephalitis 8 days after rituximab was excluded from the follow-up study. at any time after rituximab treatment, low igg was present in 24/62 (38.7%), low iga in 6/61 (9.8%), and low igm in 36/61 (59.0%) of patients. over a year after their last rituximab dose, 12/29 (41.4%) of patients still had low b cell counts for age, and 10/10 (100%) had low memory b cell counts (cd27+ among cd19+ cells: mean value = 2.9% +/-1.9% sd). hospitalisation for infection was required in 13/67 (19.4%) patients in the year following rituximab treatment, which was associated with having either low igg (33.3% vs 15.2%, p=0.03) or low iga (50.0% vs 18.2%, p=0.04), but not with low igm levels (19.4% vs 24.0%, p=0.33). also, receiving a treatment with more than one rituximab cycle was a risk factor for low igg (63.0% vs 20.0%, p=0.0003). conclusions: hypogammaglobulinemia following rituximab treatment was frequent, and the presence of low igg and iga were associated with a higher risk of serious infection in this context. introduction/background: subcutaneous immunoglobulin (scig) replacement therapy for patients with primary immunodeficiency (pid) is usually administered once a week. however, a variety of dosing regimens can be used to provide flexibility for patients. objectives: we used pharmacokinetic (pk) analysis to evaluate the pk characteristics of weekly and biweekly (once every 2 weeks) scig administration in patients with pid. methods: this pk substudy was part of a prospective, open-label, phase 4 study (nct02711228) in patients with pid treated with igpro20 (hizentra®, csl behring, bern, switzerland). a noncompartmental analysis of serum igg concentrations was used to calculate pk parameters and compare pk outcomes on weekly and biweekly dosing. results: of the 17 patients included in the pk substudy, 15 provided samples for both weekly and biweekly regimens. the dose-adjusted area under the concentration-time curve was comparable for both treatment regimens: 0.24 and 0.25 (h*g/l)/mg for the weekly and biweekly regimens, respectively. the igg clearance was also similar, being 4.41 for the weekly and 4.14 ml/h for the biweekly regimen. median peak igg concentrations occurred later with the biweekly regimen (3.02 days) compared to 2.00 days for the weekly regimen. igg trough levels were close for both treatment regimens, with arithmetic means slightly lower for biweekly than for weekly regimens, at 10.13 vs. 10.21 g/l respectively. the minimum igg concentrations within a dosing interval were also comparable, with arithmetic means of 9.60 and 9.83 g/l for the weekly and biweekly treatment regimens, respectively. conclusions: biweekly and weekly hizentra® administration at the same total monthly igg doses resulted in similar igg exposures. pharmacokinetics, efficacy, tolerability and safety of a new subcutaneous human immunoglobulin 16.5% in primary immune deficiency introduction/background: patients with primary immune deficiencies (pid) require life-long replacement therapy with immunoglobulins (ig) to prevent severe infections and irreversible complications. in addition to safety and efficacy, tolerability and convenience of administration of ig products are essential factors in patient acceptance. a new 16.5% ig preparation (octapharma, lachen) was developed for subcutaneous administration (scig) derived from the established manufacturing process of octapharmas intravenous ig (ivig) brand octagam®. objectives: primary outcome was to assess efficacy of a new 16.5% subcutaneous human immunoglobulin preparation in preventing serious bacterial infections. secondary endpoints included evaluating tolerability and safety, determining the pk profile, the number and rate of other infections and changes in quality of life measurements. methods: a prospective, open-label, single-arm phase 3 study involving 61 patients was conducted at 18 centers in north america and europe. pid patients who were stable on ivig treatment for at least 6 months and with igg trough levels 5.0 g/l underwent a 12-week wash-in/wash-out period consisting of weekly scig doses 1.5 times the previous ivig dose (based on published conversion rates for marketed scig products), followed by a 52-week efficacy period (64 scig infusions in total). 22 of 61 patients enrolled had complete pharmacokinetic assessments at different time points: before the switch from ivig to scig (pkiv), after the wash-in/wash-out phase (pksc1) and at week 16 of the efficacy period (pksc2). results: 61 patients (age: 2-73 years; mean age 32.2 years; 54.1% female) receiving a total of 3,497 scig infusions (0.135 g/kg/week in young children (2 years and <5 years of age) and 0.185 g/kg/week in adults; average overall: 0.175 g/kg) were included in the full analysis sets. no serious bacterial infections were recorded. among the 188 other infections observed during the efficacy period only one infection was graded as severe (bronchiolitis due to rsv virus), which led to hospitalization (2 days). all other infections were mild (72.3%) or moderate (27.1%) in intensity. infection rate per person-year was 3.43. of the 233 reported adverse events, only 14 were assessed as being related to the study drug; all of these events were non-serious. five non-study drug related serious adverse events were reported in 4 patients (6.6%). serum igg trough levels were nearly constant during the study with a minimum trough level of 6.1 g/l and mean trough plasma concentrations of 11.4 ± 3.3 g/l and 11.6 ± 3.4 g/l for pksc1 and pksc2. median igg trough levels after scig treatment were 1.9 to 2.6 g/l higher compared to ivig treatment prior to enrollment. a dosing conversion factor (dcf) of 1.37 was determined by auc (area under the curve) measurements, allowing dose adjustment to achieve bioequivalence between ivig and scig dosing. improved quality of life measurements, utilizing sf-36v2, were observed in both physical and mental health parameters when compared from the first to last scig infusion. conclusions: this study demonstrated that the new subcutaneous human normal immunoglobulin 16.5% is well tolerated, safe and effective in patients with pid. introduction/background: atopic dermatitis (ad) is a chronic, relapsing, inflammatory skin disorder with associated pruritus that affects 11 percent of children in the united states. severe atopic dermatitis refractory to conventional therapy can be concerning for an underlying immunodeficiency, especially in infants. increased incidence of hypogammaglobulinemia has been associated severe ad. a handful of cases describe a correlation with transient hypogammaglobulinemia of infancy (thi), but a thorough immunological evaluation is often missing to further understand this relationship between ad and characterization of thi. objectives: define various phenotypes of thi with their clinical presentation and laboratory findings. compare thi vs. thi associated with severe a.d. methods: a case series of six patients was conducted at a single academic center from 2/1/2014 to 12/1/2017 for patients with severe atopic dermatitis with low igg levels. all available immunological laboratory data were retrospectively collected during this time period. descriptive statistical analysis was utilized for data comparison. results: of the six patients, four had no infectious history. of the remaining two, one had recurrent skin abscesses associated with his poorly controlled atopic dermatitis requiring oral antibiotics and one patient had two episodes of staphylococcus aureus superinfection of the eczema. at the time of presentation, the mean age was 7 months with mean igg of 164 mg/dl and mean ige of 2,273 ku/l. iga and igm were within normal age cut offs. all patients had normal protein and polysaccharide specific antibody titers after completion of vaccination series. mean cd19+ count was 2,200/ul with normal cd3+, cd4+, cd8+ and cd1656+ cell counts. three patients were tested for lymphocyte mitogen proliferation and complement function which were normal. two patients, who were tested, had normal phagocyte work up. mean age of igg improvement to igg> 217 mg/dl, was 10 months. patients had total protein and albumin levels with mean 5.1 ku/l and 3.5 ku/l, respectively which eventually normalized. four patients improved with skin care and dietary modification to hypoallergenic formula. one patient improved with extensively hydrolyzed formula and three patients improved with amino acid formula. one patient improved with aggressive skin care. one patient, who was noncompliant with dietary recommendations and aggressive skin care, did not improve. conclusions: one prospective study described an increased incidence of hypogammaglobulinemia in patients with atopic dermatitis compared to controls regardless of the severity. no further prospective studies have characterized hypogammaglobulinemia in this population. according to the primary immunodeficiency practice parameters, children with thi often present with frequent viral and bacterial respiratory illnesses, low igg levels and normal vaccine responses. in one study, the period of hypogammaglobulinemia spontaneously corrects to normal by mean age of 27 months with all patients reaching normal levels by 59 months. these patients may have low igm or iga and decreased t or b cells, which eventually normalize. management is often with antibiotic prophylaxis and if refractory to prophylaxis or unable to tolerate, igg administration (igrt) based on severity of symptoms is recommended. we describe a less severe variant of thi associated with severe atopic dermatitis and characterized by very transiently decreased igg levels with earlier resolution than typical thi, normal igm and iga levels, normal specific antibody levels and normal t, b and nk cells. these patients typically do not manifest recurrent viral or bacterial respiratory illnesses. they also do not require antibiotic prophylaxis or igrt. severe atopic dermatitis maybe a positive prognostic indicator for patients with thi and is associated with an earlier self-resolution of hypogammaglobulinemia, lack of typical infections, normal iga, igm and normal t and b cell numbers. a possible mechanism for thi associated with severe ad may be transdermal loss of protein which was supported with mildly low total protein and albumin levels rather than immaturity of the immune system in patients with true thi. introduction/background: in recent years it was found that heterozygous mutation in pik3cd gene produces an autosomal dominant primary immunodeficiency characterized by onset of recurrent sinopulmonary and other infections in early childhood with defects in both b-and t-cell populations and a special susceptibility to uncontrolled viral infections. many patients develop chronic lymphoproliferation and there is also an increased susceptibility to b-cell lymphomas. here we present a patient assumed as a common variable immunodeficiency with heterozygous mutation in pik3cd gene. objectives: to describe a case of a patient assumed as a common variable immunodeficiency with heterozygous mutation in pik3cd gene. results: 60 year old woman with personal history of severe and recurrent upper and lower respiratory infections, chronic pulmonary disease with bilateral bronchiectasis, chronic diarrhea without diagnosis, mild osteopenia, focal lesion in right hepatic lobe, atopic dermatitis and anemia. she was following up in other center and in 1996 she was diagnosis with common variable immunodeficiency (cvid) and started treatment with intravenous immunoglobulin (ivig), but she referred low adherence to it. she did not referred history of lymphoproliferation nor significant viral infections. she has a daughter with spherocytosis who required esplenectomy and also had bronchiectasis and cvid, she deceased at 28 years old because pulmonary infection. other daughter and 2 sons referred healthy. in our first immunologic studies we found severe hypogammaglobulinemia (igg 428 mg%, no dosable iga and igm) with absent of b cells in peripheral blood. we started with high doses of ivig (800 mg/k/month) and antibiotic prophilaxis with improvement of the functional respiratory test and without new infections. we are planning colonoscopy to study her chronic diarrhea. thinking that her clinical picture could be other than cvid we order a genetic study. a nextera exome capture and next generation sequence with illumina hiseq was made and an heterozygous mutation in pik3cd gene (chr1:9.775.746, p.pro97ala) was found. family and functional studies are still pending. conclusions: due that clinical presentations of primary immunodeficiencies are becoming more complex, its diagnosis is a challenge for immunologist now a days. studies with next generation sequence is a very useful tool in indefinite cases, especially when more than one member in the family are involved. chief, laboratory of clinical immunology and microbiology, national institute of allergy and infectious diseases, national institutes of health introduction/background: heterozygous gain-of-function mutations in pik3cd as well as heterozygous pik3r1 mutations that affect interaction of p85 with p110 lead to constitutive hyperactivation of the pi3k pathway and cause activated pi3k delta syndrome type 1 or type 2 (apds1, apds2), respectively. we describe a female with apds2 with short stature, diffuse lymphadenopathy, recurrent upper respiratory tract infections, elevated igm, persistent ebvand cmv viremia and disseminated toxoplasmosis who gave birth to a genetically affected daughter with severe congenital toxoplasmosis. objectives: to characterize the molecular and cellular defects underlying severe toxoplasmosis in this family methods: investigation of the molecular basis of the disease was performed through whole exome sequencing, and results were validated by sanger sequencing. functionality of the pi3k pathway was assessed by analyzing akt and s6 phosphorylation in freshly isolate b cells with and without stimulation with anti-igm. an excisional lymph node biopsy from the affected mother was stained with anti-pd1 antibody to detect t follicular helper cells, and with igm and igg specific antibodies to analyze the proportion of isotype-specific b and plasma cells results: whole exome sequencing of maternal dna with targeted analysis of 362 pid genes identified a heterozygous mutation at an essential donor splice site of pik3r1 (nm_181523.2:c.1425+1g> a). sanger sequencing confirmed the presence of this mutation in both the mother and her child. functional studies on b cells freshly isolated from both patients confirmed an increase in baseline akt and s6 phosphorylation, suggesting constitutive activation of the pi3k-mtor signaling pathway. a lymph node biopsied from the mother contained numerous pd-1+ tfh cells and igm+ plasma cells conclusions: toxoplasma gondii is an obligate intracellular parasite that is usually only symptomatic in immunocompromised hosts. severe toxoplasmosis has been reported in the following primary immunodeficiencies: cd40 ligand deficiency, tap deficiency, cvid, nfkb2 deficiency, and immunodeficiency due to anti-ifn-autoantibodies. importantly, toxoplasma infection was previously reported in a 9month-old infant with apds1, and ocular involvement has been described in a 36-year-old patient with apds2. this, however, is the first report of systemic and severe congenital toxoplamosis in a mother and child with apds. to evade innate host defenses, t. gondii induces the activation of the pi3k/akt signaling pathway, reducing intracellular reactive oxygen species and creating an intracellular environment that is hospitable to parasite survival and proliferation. therefore, we postulate that the pik3r1 mutation may lead to a hospitable cellular environment for toxoplasmosis replication. this work was partially supported by the division of intramural research, niaid, nih (protocol # 05-i-0213). additional authors of this work are: ottavia delmonte and kerry dobbs, from the laboratory of clinical immunology and microbiology, niaid, nih. introduction/background: aplaid (autoinflammation and plc-gamma-2-associated antibody deficiency and immune dysregulation) is a term that was proposed for the newly discovered autoinflammatory condition resulting from a pathogenic missense variant, ser707tyr, in the cterminal sh2 (csh2) domain of plc-gamma-2 in order to distinguish it from plaid, a distinct clinical entity that results from intragenic deletions of portions of the csh2 domain of the same protein. plc-gamma-2 is a phosphodiesterase that is predominantly expressed in hematopoietic cell lines and acts on pip2 to produce ip3 and dag in the pkc and ras/raf/ erk pathways. the only formally published case of aplaid described a father-daughter pair with an autoinflammatory clinical syndrome affecting the skin, mucosa, eyes, pulmonary and gastrointestinal systems. objectives: to describe the aplaid phenotype resulting from a novel genetic variant of the phosphodiesterase plc-gamma-2 in two unrelated families methods: clinical review and case presentation results: we report a 33-year-old female with a long-standing history of recurrent pneumonia, cellulitis and cystitis with obstructive lung disease characterized as bronchiolitis and dynamic airway collapse, with negative alpha-1-antitrypsin testing. she had a history of childhood onset granuloma annulare and pressure-induced urticaria, as well as episcleritis. immune testing revealed low igm (37 mg/dl), elevated baff levels and a low percentage of cd27+ memory b-cells, prompting sequencing of plcg2, which identified a c.3422t>a, p.met1141lys variant in the calcium binding c2 domain. her 15-month-old daughter, who has a history of bullous skin lesions, failure to thrive, febrile episodes and recurrent respiratory infections was likewise found to have this plcg2 variant. like her mother, the daughter also has low igm and elevated baff levels. similar symptoms of recurrent sinopulmonary infections and hypogammaglobulinemia, have been described in an unrelated family that shares this same plcg2 variant in a doctoral thesis by rozmus. this work also describes functional analysis, in which this particular variant of plc-gamma-2 was demonstrated to have dysregulated plcgamma-2 activity leading to aberrant intracellular calcium signaling and increased apoptosis of immature b-cell subsets. conclusions: we describe our experience in evaluation and treatment of this family with a previously undiagnosed disorder. together, these new cases add to the expanding body of knowledge regarding plc-gamma-2 and its importance for the development of autoinflammatory and primary immunodeficiency conditions. (133) submission id#427357 present a case.non-celiac gluten sensitivity is an emerging entity with symptoms similar to celiac disease, but without positivity in specific diagnostic tests. it is considered more common than celiac disease patients with sigad have a greater risk of concomitant autoimmune disorders than health individuals. sigad was previously to be associated with celiac disease, but not usually in non -celiac gluten sensitive. objectives: describir a case ataxia non celiac gluten sensitivity, methods: clinic case description. results: he patient has negative serology test for gluten, but clinically respond as celiac disease. conclusions: all patient has negative serology test for gluten, but clinically respond as celiac disease, could has non-celiac disease, this case described here ,it suggest than this entity should to thought before than the celiac disease, since not-celiac disease is it more common. associate professor, federal university of rio de janeiro introduction/background: primary cutaneous actinomycosis is a rare condition caused by gram-positive filamentous bacteria and generally occur after traumatic inoculation. objectives: to report an unusual etiology of skin lesions in a patient under anti-tnf-alpha therapy. methods: we report the case of a 30-years-old female receiving conventional doses of adalimumab for ankylosing spondylitis who presented, two weeks before referral for dermatological assessment, with an erythematous nodule with purulent discharge on right pretibial region and pruritic erythematous plaques with scaling and peripheral pustules in the trunk and nose tip. results: a fungal etiology was suspected and scraping specimens from several cutaneous lesions were submitted to direct microscopic examination and culture, which were negative for fungi. cutaneous biopsy of the pretibial lesion was sent for histopathology and microbial cultures. adalimumab was interrupted and empirical therapy with oral terbinafine (250 mg/day) was started with close clinical follow-up. a folliculitis reaction pattern without granulomas was observed during histopathological examination and gram-positive cocci were isolated from biopsy sample and further identified as saccharopolyspora sp. by molecular typing. sulfamethoxazole+trimethoprim was added and discontinued after one week due to a cutaneous rash. terbinafine (250 mg/day, p.o) was used for 12 months with complete clearing of all skin lesions and very good tolerability. spondyloarthritis signs and symptoms were unremarkable and no anti-inflammatory or immunomodulating treatment was necessary. conclusions: increased risk of skin actinomycotic infections in patients under anti-tnf therapy is not consistently reported in the literature. nevertheless, they should be included as a differential diagnosis of atypical skin lesions in individuals under anti-tnf therapy. where we aim to determine the prevalence, incidence, characteristics, treatment and outcomes of primary immunodeficiency disease (pid) patients in qatar. pids are rare heterogeneous disorders of the immune system that result in an increased susceptibility to infection, immune dysregulation and occasionally, to cancer. objectives: determine the range of pids with important epidemiological data in qatar after analyzing the database and creating a registry. methods: this is a retrospective study of pid patients followed at hamad medical corporation from 1989 to 2017 using medical records. all patients who were diagnosed with a pid irrespective of age were included. patients were classified according to the international union of immunological societies expert committee on pid. the data is captured under 5 sectionsa) patient demographics including age, gender, ethnicity b) clinical presentation, c) immunodeficiency profile including age at diagnosis, type of immunodeficiency, family history d) treatment modality and e) lab/genetic data. results: we registered 150 patients (60 females and 90 males) over a span of four years. mean age at onset, diagnosis and diagnostic delay were 2.6, 4.6 and 2 years respectively. majority of the patients were arabs 77% followed by people from the asian subcontinent 12%. antibody deficiency was seen in 32%, immune dysregulation 28%, well defined immunodeficiency (at, hige, digeorge,wiskott aldrich) 25% and t/b cell cid 12%. rare diagnoses (ipex, msmd) were recorded whereas no cases of toll like receptor and complement deficiency were seen. consanguinity rate was (n = 97, % = 65) and first degree cousin marriage (n = 38, % = 40). family history was positive in 50% (n= 75) of the patients. maximum diagnostic delay was seen in scid (33% >3 months) and agammaglobulinemia (66% >3 months).during the patients life, infection was the most common presenting complaint (47%) followed by sinopulmonary disease (16%) and gi-tract manifestations (7%). the most common infections were pneumonia (46 %), otitis media and conjunctivitis (42 % each) followed by failure to thrive (30%) and sepsis (21%).microbial isolates particularly seen as causative agents of infections were p.aeruginosa and salmonella (14%) each, mrsa and e.coli (9%) each. a genetic defect was confirmed in 57% of ataxic telangiectasia and 65% of scid patients. active infections were treated and prophylactic antibiotics were prescribed in 76 cases (51%). prophylactic antibiotics were prescribed to 34% of patients with immune dysregulation and to 25% of well-defined syndromes. out of 18 patients who had hsct, 72% had successful transplants. ivig was given to 42% of the total pid patients with humoral immunodeficiency patients receiving the most (45%). one patient had gene therapy and two required interferon gamma treatment for msmd. mortality rate at 1st year of life was 4% whereas total mortality rate was 12%, excluding cases that passed away before pid was diagnosed. conclusions: the estimated prevalence of pid in qatar is found to be 6 per 100000. over the years, physicians have become increasingly aware of pid and survival rate has improved. initiation of newborn screening for scid and agammaglobulinemia will lead to earlier diagnosis and initiation of therapy with better outcomes. introduction/background: agammaglobulinemia is typically associated with a near absence of b cells secondary to a developmental block in bone marrow, and, usually but not always, manifests in early life. most of such patients are males with x-linked agammaglobulinemia (btk deficiency). females with agammaglobulinemia, of either autosomal recessive or dominant inheritance, are rare. objectives: we present and discuss the differential diagnosis for the conflicting clinical and immunological phenotypes of two adult females who present with infections, cytopenias, and agammaglobulinemia with low to normal b cell count. methods: retrospective chart review of clinical and laboratory data results case 1: a 22-year-old female who, at age 17 years, had evans syndrome (autoimmune thrombocytopenia and hemolytic anemia) that resolved after steroid and high-dose gammaglobulin treatment. at age 21 years, immunologic workups revealed a complete absence of serum immunoglobulins (igg, iga, igm) and low b cells 43 (3%) (normal range 100-500). however, three years prior, patient had detectable igm (218 mg/ dl). b cell subset analysis showed an expansion of cd19hi21lo b cells (30%, normal 0.2-8.6%), with a marked decrease in switched memory b cells (0.2%, normal 7-61%). additional immunophenotyping revealed a reduced frequency of naïve cd4+ (3.6%, normal >50%), cd8+(42%, normal >50%) t cells, and normal lymphocyte proliferative responses to mitogens and anti-cd3, anti-cd3/anti-cd28, and anti-cd3/il-2. case 2: a 30-year-old female with recurrent upper respiratory tract infection, undetectable igg, iga, igm and ige, and no response to vaccinations (tetanus and pneumococcal). thrombocytopenia (38x103 count/microliter) was noted once during her thirdtrimester pregnancy without evidence of pre-eclampsia. postpartum cd4+ t cell count was low (442 cell/microliter, normal 500-1400 cell//microliter). immunophenotyping revealed normal b cell count with reduced frequency of naïve cd4+ (17.6%, normal >50 %) and cd8+ (18%, normal >50 %) t cells. conclusions: we report two adult females who present in early adulthood with recurrent infections and cytopenias. both had agammaglobulinemia and decreased naïve t cells suggestive of late-onset combined immunodeficiency (locid). the presence of peripheral b cells makes autosomal recessive defects in b cell receptor signaling (lamda5, iga, igb, igm, blnk) less likely. differential diagnosis of locid with cytopenias includes ctla-4 haploinsufficiency, gain-of-function pik3cd mutations, ikaros defects and autosomal recessive rag deficiency. both patients remain at risk for developing autoimmune complications. a molecular diagnosis will facilitate targeted therapy for the underlying defect. professor, director of the laboratory of childhood immunology, ku leuven introduction/background: complement factor properdin (cfp) is a soluble glycoprotein which has a unique known role as a positive regulator of the alternative complement pathway by binding and stabilizing the inherently labile c3/c5 convertase enzymes. mutations in the cfp gene lead to aberrant protein expression or to expression of a dysfunctional protein which results in high susceptibility to pyogenic infections especially neisseria meningitidis. properdin-deficient individuals are at greater risk of fulminant meningococcal disease, with mortality rates as high as 75%. objectives: case report: a 5 year old belgium boy from nonconsanguineous parents presented with achronic purulent cough and recurrent infections of upper and lower airways. he suffered from a severe pneumonia at the age of 5. ct thorax showed bronchiectasis of the right middle lobe and left lower lobe. in addition, he had recurrent acute otitis media since the age of 1 year old. immunological work-up showed an absent ap50 activity, with normal ch50, c3 and c4. results: genetic and functional analysis: sanger sequencing of cfp gene identified a hemizygous c.961t>g, p.y321g (cadd score 8.081, msc_cadd 0.102) mutation in the patient and his mother. properdin elisa (hycult biotech) showed absent and 50% of the normal healthy control value of serum properdin concentrations in patient and the healthy mother, respectively. conclusions: conclusion: we diagnosed properdin deficiency in a 5y old boy presenting recurrent lower and upper respiratory tract infections. ap50 testing, added to ch50, should therefore be part of initial workup for patients with recurrent severe respiratory tract infections. indeed early diagnosis allows for appropriate prolonged antibiotic prophylaxis and immunization to reduce the risk of fatal meningococcal disease, to reduce or prevent organ damage and to allow for genetic counselling in the family. 2-4, 5-7, 8-12 and 13-18) and child depression inventory (cdi) were implemented to both children and parents in addition to sociodemographic data form; beck depression inventory (bdi), beck anxiety inventory and zarit caregiver burden scale were implemented only to parents. results: the depression inventory parent and child form values of the patient group with primary immunodeficiency were significantly higher than the control group (p=0,004 and p=0,032). according to beck depression and anxiety inventories, it was seen that the depression and anxiety inventory scores of the parents of the patient group were higher than the control group (p=0,009 and p=0,022 respectively). it was determined that the cdi parent form scores of the patients with hospitalization history were statistically higher than the patients without hospitalization history (5,4±0,5 and 4,8±0,6; p=0,009). bdi scores were significantly higher in the group which received ivig (p=0,024). while the quality of life of the patients compared to their parents was perceived as worse than the healthy children in all dimensions (p=0,003 p<0,0001 and p<0,0001), the quality of life of the children was worse only in psychosocial and total quality of life fields (p<0,0001 p=0,002 and p=0,158). it was seen that quality of life of children physical health scores of only the patients with hospitalization history were statistically significantly lower (15,5±1,9 and 14,3±2,3; p=0,04) . while no statistically significant difference was found in terms of quality of life of the child scores between the groups which received and did not receive ivig replacement treatment, psychosocial and total qualify of life scores of parents were statistically significantly lower in the group which received ivig replacement treatment (p=0,017 p=0,033). zarit care giver burden scale scores were similar in patient and control groups. although both groups were on the limits of mild to moderate caregiving burden, it was seen that the scores of the group which received ivig were significantly higher than the group which did not receive ivig (p=0,026). conclusions: as a conclusion, we think that it will be appropriate to inform and monitor the entire family in relation to psychosocial difficulties and care giving burden they may experience in time as well as the medical aspects of the disease in order to develop a holistic approach to children with primary immunodeficiency. introduction/background: chronic lung disease is the most common complications of cvid, affecting 30-60% of patients. it includes bronchiectasis affecting 50% of patients and granulomatous lymphocytic interstitial lung disease (glild) in 10 to 55%. both are associated with an increased morbidity and mortality. pulmonary functional studies and ct scans have been proposed as screening procedures for lung involvement in cvid. the definitive diagnosis of glild is established histologically, but it is too invasive in patients with radiological abnormalities and no symptoms. objectives: we aim to describe the pulmonary complications of our cohort of patients with cvid comparing them with subjects affected with other types of hypogammaglobulinemia. methods: we reviewed all clinical records of the patients with a diagnosis of hypogammaglobulinemia of any cause until december 2016. we looked at all pulmonary function tests (pft), 6-minute walk tests and ct scans performed. we classified patients according to the ct scan pulmonary disease pattern. we compared the demographic data and pulmonary characteristics of each group and intended to describe similarities and differences between them. results: we collected 34 patients and 70 ct scans from 26 patients. patients were grouped for further analysis as follows: no ct performed: 8; normal ct: 9; bronchiectasis: 9; glild: 7 and bronchiectasis + glild: 1. eight patients (24%) had no ct including six with cvid, one with x linked lymphoprolipherative disease (xlp) and one had a syndromic deletion in chromosome 11. the median age of symptom onset was 23.5yo, with a median delay to diagnosis of 1 year. nine subjects (26%) had ct scans with no chronic disease, six had cvid, 2 rituximab induced hypogammaglobulinemia (rih) and 1 mgus with hypogammaglobulinemia (mguswh). the median age of symptom onset was 58 years old with a median delay to diagnosis of 2 years. nine patients (26%) presented persistent bronchiectasis, 7 with cvid, 1 with an igg3 deficiency (sigg3d) and 1 with xlp. median age at symptoms onset was 14yo and median delay to diagnosis 13 years. seven patients (21%) had glild; all afected with cvid. median symptoms onset was 30 years old, and median delay to diagnosis was 10 years. one patient (3%) was included in the bronchiectasis and glild group. she was referred with a diagnosis of rih, in the context of a pulmonary malt lymphoma. however, her ct did not show a typical ct lung lymphoma pattern, lymphocyte monoclonality was never shown and she had a reduced pre-treatment low gamma globulin concentration and a history of respiratory infections since adolescence, we believed her diagnosis is cvid. pft with an obstructive pattern identified patients with bronchiectasis (p<0.01) and patients with glild had a significantly lower basal and post 6 minute walk o2 saturation (p<0.01, n=22) conclusions: using a systematic approach, we identified that roughly 50% of patients with hypogammaglobulinemia have chronic pulmonary ct abnormalities. half of them had bronchiectasis, that were associated to hypogammaglobulinemia of any cause, a longer disease course and a reduction in fvc and fev1 with a shorter distance reached in the 6 minute walk test. the other half had glild, spirometries in this group were useless, but o2 saturation was significantly lower, basal and after the 6 minute walk test. we found a high frequency of pulmonary disease in our cohort and a disease progression study is now in place. hies) is a primary immunodeficiency characterized by eczema, sinopulmonary infections, and musculoskeletal and vascular abnormalities. care of patients with this disease is largely supportive with the use of prophylactic antibiotics and topical eczema therapies. the use of replacement immunoglobulin is increasing. hematopoietic stem cell transplant (hsct) is being considered more frequently, but many questions remain regarding which patients should undergo transplant. as pulmonary complications are a leading cause of morbidity and mortality in this disease, and potentially improved with replacement immunoglobulin and hsct, we sought to examine more closely the patients with more frequent pulmonary hospitalizations and structural lung disease. objectives: to determine the rates of pulmonary complications in our large cohort of ad-hies patients, and examine the relationship between immunologic markers and pulmonary disease. methods: we retrospectively reviewed the records of 110 ad-hies patients seen more than one time at nih between 2010 and 2016. there were 36 pediatric patients under the age of 18 years. we reviewed the number of and cause for hospitalizations, and reviewed radiology reports for structural lung disease including bronchiectasis and pneumatoceles. we correlated these findings with specific antibody responses and lymphocyte phenotyping, such as the number of memory b lymphocytes. results: the patients range in age from 2 years to 66 years, with a median age of 23 years. 106 patients had chest cts, with 42 percent having bronchiectasis, and 41 percent having cavitary lesions. 31 percent of patients had both. these abnormalities were more prevalent in patients with low memory b cells. 31 percent of patients receive replacement immunoglobulin. in patients not receiving replacement ig, 42 percent had appropriate response to pneumococcal vaccine. during this time period, there were 190 hospitalizations, the majority of which were associated with pulmonary infections. the rate of overall hospitalization was higher in the group with low memory b cells (p=0.006), but there was no difference associated with age. conclusions: immunologic abnormalities may assist in determining the long-term prognosis of patients with ad-hies, and can be considered in management plans such as the use of immune globulin and consideration for hsct. introduction/background: purine nucleoside phosphorylase (pnp) deficiency is an autosomal recessive disorder affecting the purine salvage pathway causing a (severe) combined immunodeficiency disorder, autoimmunity, and neurological symptoms. patients typically present in the first few years of life with frequent infections and a failure to thrive. decreased plasma levels of uric acid are suggestive, while neutropenia is a rare complication. prognosis is generally poor with few patients making it to adulthood without treatment. treatment options are limited to general supportive care and hematopoietic cell transplantation. methods: we present the cases of three patients, one sister and two brothers, from a consanguineous french canadian family. they presented in early adulthood with nearly identical histories of repeated pneumonias and chronic rhinosinusitis. one patient had necessitated filgrastim to treat a parainfectious neutropenia. they did not report any neurological abnormalities or symptoms of autoimmunity. results: a primary immune deficiency was suspected. initial evaluation showed normal immunoglobulin levels, a lack of response to vaccination, positive ebv ebna iggs, and auto-antibodies. the patients were severely lymphopenic with reduced numbers of t, b and nk cells ( l: 300, cd3+81%, cd4+71%, cd8+5%, cd19+12%, cd3-cd56+7%). in particular, they had a severe depletion of naive t-helper recent thymic emigrant cells (cd4+cd45ra+cd31+2%). additional studies revealed increased urinary inosine, guanosine, deoxyinosine, and deoxyguanosine, severely reduced erythrocyte pnp activity (3% residual activity), and a normal uric acid level. a homozygous, missense mutation, c.769 c>g (p.his257asp) was found and described as pathogenic in silico. the diagnosis of pnp deficiency was made; tmp-smx prophylaxis and ivigs were started. conclusions: theses cases are atypical for a number of reasons. the first is the relatively benign immunological course, the lack of neurological symptoms, and the advanced age at the time of the diagnosis, possibly due to residual enzymatic activity. in addition, pnp deficiency has been classically described as causing a marked reduction of t cells with a relative sparing of b cells. however, b cell lymphopenia have been reported, particularly with latter presentations. while this specific mutation had been identified once previously in the literature, as a compound heterozygote, these are the first confirmed cases of homozygotes. the pathogenicity of the mutation was not only suggested in silico, it was confirmed biochemically. in addition, in vitro functional studies have demonstrated the importance of his257 for the binding of pnp to its substrate, particularly in the formation of the early transition state. indeed, pnp (p.his257asp) has been shown to have a greatly reduced affinity for its substrates and catalytic activity. further study is needed to identify the optimal management for these patients. introduction/background: whole exome sequencing has become an integral part of diagnosis and treatment of rare immunodeficiency diseases. one of these diseases is tetratricopeptide repeat domain 7a(ttc7a) mutations; a rare disorder associated with multiple intestinal atresias and severe combined immunodeficiency. histologic assessment of organ biopsies of patients with ttc7a deficiency suggest it may play a role in multiple organs as it is expressed in the cytoplasma of intestinal, thymus and pancreatic cells. many patients with ttc7a deficiency have moderate to severe combined immunodeficiency. nine patients described in the literature with ttc7a mutations have been treated with hematopoietic stem cell transplant, mostly at a young age. objectives: this case report describes a rare presentation of an adolescent with ttc7a deficiency, her clinical presentation, immune evaluation and treatment with eventual referral to bone marrow transplant at the age of 16. methods: retrospective chart review under irb approval was performed. whole exome sequencing performed at baylor texas childrens hospital and mutations were confirmed by sanger sequencing. extensive immune and nk cell phenotyping were performed by flow cytometry and nk cell function was tested using standard cr51 release cytotoxicity assays. results: a 13 year old female was referred to immunology for severe refractory warts, history of severe diarrhea with epithelial dysplasia but no atresia, failure to thrive requiring parenteral nutrition until 6 years old and multiple infections with staphylococcus aureus and candida. at 16 years of age, she developed rapidly worsening pulmonary function that improved with monthly pulse methylprednisone. her immune phenotype demonstrated a combined immunodeficiency including severely low cd4, cd8, b and nk cells. she had no igd-cd27+ memory b cells and undetectable igg, iga and igm, isohemagglutinins and vaccine titers to diphtheria and tetanus. her mitogen proliferation response was low and she had abnormal tcr v-beta repertoire. she was referred to the texas childrens hospital center for human immunobiology for the nk cell evaluation and reasearch clinic (near) the patient was found to have impaired nk cell lytic function and terminal maturation. this was demonstrated by the decreased frequency of cells that matured to the cd56dim subset and was accompanied by decreased expression of the lytic effector molecule perforin and the fc receptor cd16. this phenotype was conserved when we isolated cd34+ hematopoietic precursors and performed nk cell differentiation in vitro. whole exome sequencing demonstrated a compound heterozygous mutation in the ttc7a gene, including a c.1001+3_1001+6aagt mutation, which has been previously described as a pathologic founder mutation in the french canadian population. the second mutation is a previously unreported c.211g>a (p.e71k), a variant of unknown significance. mutations were confirmed by sanger sequencing, and parents are carriers of the mutations. she was referred for hematopoietic stem cell transplantation with a 10/10 unrelated donor option. conclusions: ttc7a deficiency is a genetic mutation associated with high morbidity and mortality leading to gut atresia and dysfunction in combination with severe immunodysfunction. we have described a unique case of ttc7a deficiency with a novel mutation which is associated with intestinal epithelial dysplasia with no atresias and a late presentation and evolution of combined immune deficiency. nk cell assessments show significantly impaired terminal maturation suggesting a critical role for ttc7a in human nk cell development. introduction/background: ras-associated autoimmune leukoproliferative disorder (rald) is a rare condition with significant overlap of clinical and laboratory findings with malignant disorders, notably juvenile myelomonocytic leukemia (jmml) and chronic myelomonocytic leukemia (cmml), but with much better prognosis without specific therapy. we report a case of rald in a 4month-old male infant originally suspected to have jmml. results a 4-month-old male infant, born to an unrelated ethiopian father and turkish mother, presented with splenomegaly and leukocytosis (26,000/l), monocytosis (7,800/l), and thrombocytopenia (14,000/l); hemoglobin was 10 gm/dl and fetal hemoglobin concentration 1.9%. family history indicated early death of three paternal uncles, two at age 6-7 months and one at age 10 years. jmml was suspected and bone m a r r o w a sp i r a t i o n w a s p e r fo r m e d ; h ow e v e r, r e s u l t s o f immunophenotypic testing with flow cytometry analysis and cytogenetic testing with fish did not support this diagnosis. patient had a normal b12 level. serum igg (1884.8 mg/dl [nl 694.0 -1618.0 mg/dl]) and igm (139.0 mg/dl [nl 35.0 -102.0 mg/dl] levels were elevated and iga was normal. lymphocyte subset analysis showed 37% cd3 [nl 51-74%], 20% cd4 [nl 34-53%], 51% cd19 [nl 17-37%] positive cells with normal percentages of cd8 and cd16/cd56 cells; there were no cd4-/ cd8-t cells detected. given the findings of leukocytosis, monocytosis, splenomegaly, hypergammaglobulinemia, and a dearth of cd4-/cd8-t cells, the patient appeared to meet criteria for rald. mutation testing with next generation sequencing showed an nras missense mutation [c.37g>t; pgly13cys] in 46% of t cells and 48% of myeloid cells. the presence of the nras mutation, in the absence of other typical jmml findings or rasopathy syndrome features, supported the diagnosis of rald. there was no therapeutic intervention and, at one year of age, the patient was reported to remain clinically well; he relocated to ethiopia with his parents. conclusions rald is a nonmalignant clinical syndrome originally classified as a subtype of autoimmune lymphoproliferative syndrome (alps) but subsequently distinguished to be a separate entity due to lack of double negative t-cells, a mutation in fas/fasl/caspase-10, or consistently elevated serum b12 levels. though also present in 25% of jmml patients, a somatic mutation in ras signaling protein (kras or nras), which controls b-cell tolerance and production of autoantibodies, is present in all reported cases of rald. while all previously reported cases of rald had somatic kras or nras mutations, our patient's nras mutation was present in more than 40% of t cells and myeloid cells, suggesting a heterozygous germline mutation; this has not previously been reported in rald and needs to be further confirmed (145) the primary objective of this study was to evaluate hct outcomes in patients with was who underwent hct since 2005 in north america. we hypothesized that survival after hct from alternative donors such as cord blood has improved compared to published results, but that overall survival would be superior in patients who undergoing hct at a young age. methods: patients were enrolled on pidtc protocol 6904, a multicenter retrospective natural history study of patients treated for was in north america since 1990. clinical features, disease status, hct type and post-hct outcomes were analyzed in 129 patients who underwent hct at 29 pidtc centers between 2005-2015. descriptive statistics such as median and range for continuous variables and counts and percentages for categorical variables were used to summarize characteristics of the study population. in addition, kaplan-meier curves were used for estimating survival probabilities results: diagnosis of was was confirmed by an expert review panel for eligibility. mutation in the was gene was available for 118 patients, including nonsense (n=27, 23%), frameshift (n=32, 27%), missense (n=31, 26%), splicing (n=20, 17%), gross deletion (n=5, 4%), in-frame deletion (n=2, 2%), pr complex (indel) (n=1, 1%). donor types included matched sibling (n=22, 17%), unrelated adult volunteer bone marrow or peripheral blood stem cells (n=66, 51%), umbilical cord blood (n=39, 30%) and other related donors (1 each, phenotypically matched related, haploidentical) . median age at time of hct was 13.6 months (range, 2.1 260.5 months). the vast majority of patients received busulfan containing conditioning regimens (87%), with some receiving other myeloablative (2%) or reduced intensity regimens (11%). with a median follow-up of 4.6 years, overall survival was excellent with 1-year and 5-year survival probabilities of 92% (95% ci, 86-96%) and 91% (95% ci, 84-95%), respectively. survival was similar at 1 year for recipients of hct from matched sibling (95%, 95% ci, 72-99%), matched unrelated donor (92%, 95% ci, 82-97%) or umbilical cord blood (90%,95% ci, 75-96% see figure) . importantly we confirmed that survival at 1 year was better in patients who were <5 years old (n=118) compared to those who were 5 years old (n=11) at the time of hct (95% versus 62%, respectively p=0.032). this difference persisted when only unrelated donor recipients were analyzed (97% vs 57%, p=0.036). overall the percentage of patients in our study who underwent hct at a young age was high (91%) compared to the literature (84% in moratto et al, 2011, blood) . the rate of second hct was only 4% (n=5). cumulative incidence of acute grade 2-4 at 100 days, acute grade 3-4 at 100 days and chronic graft-versushost disease at 1 year were 26% (95% ci 18-34%), 15% (95% ci 9-22%) and 14% (95% ci 8-21), respectively. conclusions: outcome of hct for was since 2005 shows excellent overall survival for all donor types, including umbilical cord blood with very low rates of second hct. importantly, hct at a younger age (<5 years old) continued to be associated with superior survival supporting the provision of hct earlier in the course of the disease. further analysis of the complete cohort is planned to determine whether age at hct has decreased in the modern era compared to pre-2005 and to analyze factors associated with platelet and immune reconstitution, donor chimerism and autoimmunity. introduction/background: comel-netherton syndrome is a rare disease hallmarked by congenital ichthyosis, atopy, trichorrhexis invaginata (bamboo hair) and, within the past 10 years, has been defined as a primary immunodeficiency. specific, rare genetic polymorphisms (c.230t>a, pl66h) in the fc receptor iiia (fcgr3a) on natural killer (nk) cells have been shown to decrease nk cell function. patients with these defects present with susceptibility to severe and recurrent viral infections, however, not all fcgr3a mutations result in this clinical phenotype. here we report on a child with a mixed genotype, presenting with congenital ichthyosis, atopy and recurrent bacterial and viral infections. methods: immunophenotyping of lymphocyte subpopulations were evaluated by flow cytometry. genomic dna was sequenced using next generation sequencing. all detected variants were then sequenced using sanger sequencing. cd16 dual epitope assay and evaluation of nk cell maturation was also performed. intravenous immunoglobulin replacement therapy was initiated. results: our patient presented at 9 months of age for evaluation of food allergy. she has congenital ichthyotic erythroderma with pruritic atopic dermatitis-like skin eruptions and a history of hypernatremic dehydration immediately following birth, thin and easily broken hair and a history of failure to gain weight with appropriate catch-up growth following formula fortification. she has had multiple episodes of acute otitis media and recurrent upper respiratory tract infections associated with wheezing. t-, b-and nk cell quantitation by flow cytometry was normal, including naïve and memory b cells. immunoglobulins and vaccine antibody levels to haemophilus influenza type b, tetanus and streptococcus pneumoniae were normal. lymphocyte proliferation to mitogens was normal. natural nk cell cytotoxicity was initially decreased, however, subsequent cytotoxicity testing performed at 13 months of age was normal. whole exome sequencing revealed a homozygous mutation in spink5 (c.795-11a>g) and a homozygous missense mutation in the first immunoglobulin domain of fcgr3a (c.305t>a), both variants of unknown significance and under additional investigation. this homozygous mutation in spink5 was not detected in the patients healthy, unaffected sibling. conclusions: this is a case of an infant with a clinical phenotype consistent with comel-netherton syndrome, however genotyping has revealed homozygous mutations in spink5 and fcgr3a, suggestive of a possible mixed genotype. a recent large study investigating the use of whole exome sequencing to identify variants implicated in primary immunodeficiency found that in 11% of families, more than 1 gene contributed to the immunodeficiency phenotype. it should therefore be kept in mind that variability in an individuals clinical phenotype may be attributable to the presence of a mixed genotype. introduction/background: cartilage-hair hypoplasia (chh) is a rare autosomal recessive disease caused by mutations in the rmrp gene and can manifest with scid. allogeneic hct is a curative therapeutic option for scid associated with chh. bordon et al. reported an overall survival of 62% (10/16 patients) following hct with a predominantly myeloablative conditioning regimen with busulfan and cyclophosphamide. data on outcomes of hct using a ric regimen are limited. objectives: we herein report our experience with allogeneic ric hct for scid associated with chh. methods: we reviewed records of all patients who underwent allogeneic hct for scid associated with chh at our institution, with a ric regimen containing alemtuzumab, fludarabine and melphalan. results: five patients (3 male, 2 female) underwent allogeneic ric hct for chh at median age of 8 months (range, 4 months 4 years). all patients had biallelic mutations in the rmrp gene, and met pidtc criteria for scid, prior to hct. two patients were diagnosed by newborn screening for scid. one patient received serotherapy only (rituximab 375 mg/m2 daily x 3 days, anti-thymocyte globulin 1.5 mg/kg daily x 4 days) conditioning for initial hct but developed graft failure and subsequently received a ric regimen for second hct. all patients received a ric regimen consisting of alemtuzumab 1 mg/kg over 5 days(n=4) or 3mg/kg over 4 days (n=1) and fludarabine 5 mg/kg (weight <10 kg, n=4) or 150 mg/m2 over 5 days (n=1), and single dose of melphalan 4.7 mg/ kg(weight <10 kg, n=4) or 70 mg/2(n=1, dose reduced by 50% for preexisting sclerosing cholangitis with grade 3 liver fibrosis). patients received matched (n=3) or 1-2 allele mismatched (n=2) bone marrow grafts and all but 2 grafts were from unrelated donors. all patients received cyclosporine and steroids for gvh prophylaxis. all patients engrafted with full donor chimerism. three patients developed mixed chimerism, but continue to maintain donor t cell chimerism > 90%. two patients developed vod of the liver (one patient developed mild vod whereas the patient with pre-existing sclerosing cholangitis developed severe vod). one patient developed grade 2 skin gvhd and one patient developed limited chronic skin gvhd. none of the patients developed liver or gi-gvhd. all patients remain alive at a median follow up of 4 years (range 11 months-11 years) with good t-cell and b-cell immune reconstitution. conclusions: our experience suggests that allogeneic ric hct with alemtuzumab, fludarabine and melphalan for scid associated with chh is curative, offers durable t-cell engraftment, low gvhd along with excellent survival and might be preferable over a myeloablative conditioning regimen, to further limit toxicity in young infants, especially in the era of newborn screening. refractory thrombocytopenia in a patient with wiskott-aldrich syndrome despite hematopoietic stem cell transplantation: eltrombopag as a therapeutic option. mauricio chaparro-alzogaray 1 , marcela estupiñan-peñaloza 1 , gisela barros-garcía 2 , oscar correa-jimenez 3 1 pediatric hematologist/oncologist, hsct unit, fundación homi hospital de la misericordia 2 pediatric hematologist/oncologist, fundación homi hospital de la misericordia 3 pediatrics resident, universidad nacional de colombia introduction/background: wiskott-aldrich syndrome (was) is an xlinked disorder characterized by: immunodeficiency, eczema and hemorrhage due to thrombocytopenia [1] . hematopoietic stem cell transplantation is the treatment of choice, with an overall survival of approximately 90% regardless of the source of the stem cells [2] . eltrombopag has been used as a pre-transplant stabilization therapy [3] . in our knowledge, there are no reports of its use for the management of post-transplant autoimmune cytopenias in was. objectives: to present a clinical case in which the complexity of diagnosis and management of wiskott-aldrich syndrome is highlighted, as well as the usefulness of eltrombopag in post-transplant persistent thrombocytopenia. methods: clinical case presentation and review of literature. results: clinical case: 9-month-old infant with a history of thrombocytopenia identified at the third day of life (positive serology and viral load for cmv), bacteremia due to serratia marcescens at 4 months, intermittent diarrhea from 4 months, multiple platelet transfusions, ivig cycles, and ambulatory management with corticosteroids; who consulted the er for a 5-day of bloody diarrheic stools, generalized petechiae and fever. he was irritable with generalized petechiae in the lower extremities, diaper area dermatitis with signs of superinfection. admission cbc: wbc: 20180/mm3, neutrophils 330/mm3, lymphocytes 17250/mm3, monocytes 2360/mm3, hb 11.2 g/dl, ht 34.1% mcv 80.2fl, mch 26.3 g/dl, mcmh 32.8%, platelets 15000/mm3 mpv 8 fl. bone marrow aspiration was performed that ruled out proliferative syndrome. immunological profile with normal immunoglobulins and flow cytometry showed t lymphocytosis with cd4/ cd8 inversed ratio, direct coombs was positive. he progressed to refractory thrombocytopenia, with intracranial bleeding and transfusion platelet requirement every 12h. molecular diagnosis of wiskott-aldrich syndrome was made and it was decided to carry out an allogeneic transplant of unrelated umbilical cord. he showed adequate response, 100% chimerism; however, he persisted with important cytopenias, without response to management with ivig and corticosteroids, so that on day +68 he restarted eltrombopag that he received prior to transplantation. from day +97 he received rituximab for 6 weeks. he continued with eltrombopag 10 months post-transplant. currently without cytopenia and without complications, he completed the post-transplant year without additional complications. conclusions: was represents a great diagnostic challenge in pediatric clinical practice. despite the therapeutic option of transplantation of hematopoietic stem cells, patients may persist with different complications, within these; autoimmune cytopenias will require additional therapies. eltrombopag, in addition to being used as a pretransplant transient measure, is useful for the management of post-transplant persistent thrombocytopenia in these cases. references: 1. immunol allergy clin north am. 2010; 30 (2) methods: retrospective study of medical records in two centers of immunology results: in our center we have 1205 p with pid, with made retrospective study of medical records, up today we have registered 1155p (96%). according to this record, these diseases are distributed in the following way: predominantly antibody disorders: 890p (77%), predominantly t cell deficiencies:88p (7,6%), phagocytic disorders:41p (3.5%), complement deficiencies:26p(2.2), other well pid:53p(4.5), autoimmune and immune dysregulation syndromes:8p(0.6%), unclassified immunodeficien cies:57p(4.9%). predominantly antibody deficiencies are the most common pid, which comprise more than half of our all p. these group is represented by: specific iga deficiency (sad):332p, specific igg deficiency:164p, transient hipogammaglobulinemia:206p, common variable immunodeficiency (cvid):96p, agammaglobulinemia linked x:33p, agammaglobulinemia unknown causes:8p, secondary hipogamma globulinemia:23p, selective igm deficiency:1p, subclasses deficiency:10p, cd40l deficiency:7p, hyper igm unknown causes:32p, other hyogammaglobulinemia:3p. among them, selective iga is the most common pid.in our cohort of unclassified immunodeficiency, we have 25p with auto inflammatory syndrome, such as family mediterranean fever, hyper igd syndrome and candle like-syndrome and 6 p with nk deficiency. in all our pid 244p, are under replacement gammaglobulin (gg)treatment, 171p use intravenous gg and 71p use subcutaneous gg conclusions: the lasid registry model represents a powerful tool to improve health policies, showing that are under diagnosed and should receive more attention. more data are needed to define the exact prevalence of pid to avoid underestimation of these diseases due to under reporting. as different reports in different countries, in our centers predominantly antibody deficiencies are the most prevalent. although the number of patient diagnosed with pid, is growing. many physicians still know little about these disorders. introduction/background: a number of anticonvulsant medications have been shown to cause hypogammaglobulinemia. lamotrigine is a phenyltriazine anticonvulsant medication that is approved to treat seizure disorders and bipolar disorder. objectives: we describe a patient who developed hypogammaglobulinemia secondary to lamotrigine use. methods: we performed a chart review and case-based literature review. results: a-27-year old female presented to immunology clinic for evaluation of panhypogammglobulinemia. she was initially evaluated by general internal medicine for lightheadedness and fatigue and was found to have serum igg of 382 mg/dl (767-1590 mg/dl), igm 26 mg/dl (37-286 mg/dl) and iga 33 mg/dl (61-356 mg/dl). the patient reported a history of recurrent sinus infections occurring around twice per year. she reported resolution with oral antibiotics. she denied any history of pneumonia and was never hospitalized for treatment of infection. she did report increased number of recurrent upper respiratory infections; around 4-5 per year for the last several months. she denied any family history of primary immune deficiency. she was never prescribed corticosteroids or immuno-modulators. her past medical history was significant for bipolar disorder type 2 for which she was prescribed lamotrigine 100 mg oral twice daily seven months prior to her initial visit. the medication was prescribed prior to the onset of recurrent sinus infections. she had protective post-vaccination titers against tetanus, diphtheria, and acellular pertussis and non-protective pre-vaccination pneumococcal titers. post vaccination pneumococcal titers were not obtained due to the cost of the pneumonia vaccine. the patient was started on prophylactic azithromycin three times weekly. lamotrigine was discontinued and the patient switched to lurasidone due to concern for anticonvulsant-induced panhypogammaglobulinemia. her serum immunoglobulin levels increased after 2.5 months off of lamotrigine (igg 416 mg/dl, igm 46 mg/dl, iga 34 mg/dl), and she is currently asymptomatic without further infections. conclusions: lamotrigine is likely responsible for the reversible hypogammaglobulinemia in this patient. serial immunoglobulin levels should be checked in all patients who experience recurrent sinopulmonary infections while on lamotrigine. in two separate reports, lamotrigine induced hypogammaglobulinemia began within 7 months and 13 months of starting therapy respectively. in another study, hypogammoglubinemia was reported in 28% of 74 patients taking lamotrigine. further studies are needed to accurately describe onset and frequency of hypogammgeobulinemia in these patients. currently, it is unclear whether post vaccine titers are protective in patients with lamotrigine induced hypogammaglobulinemia. introduction/background: the association of vaccine strain rubella virus with cutaneous and sometimes visceral granulomatous disease has been reported previously in 19 patients with various primary immunodeficiency disorders (pids). the majority (14/19) of these pid patients with rubella positive granulomas had dna repair disorders, namely ataxia telangiectasia (at) (n=9) or nijmegen breakage syndrome (nbs) (n=3) or rag1 (n=1) and rag2 (n=1) deficiency. objectives: to support this line of inquiry, we provide additional descriptive data on the previously reported nbs patients as well as additional previously unreported patients with rubella virus induced cutaneous granulomas and dna repair disorders as well as additional previously unreported pid patients with rubella virus induced cutaneous granulomas. methods: we provide in-depth descriptive data on the previously reported nbs patients as well as 5 additional previously unreported patients with rubella virus induced cutaneous granulomas and dna repair disorders including at (n=4) and dna ligase 4 deficiency (n=1). we also provide in-depth descriptive data on 4 additional previously unreported pid patients with rubella virus induced cutaneous granulomas, including cartilage-hair hypoplasia (n=1), mhc class ii deficiency (n=1), whim syndrome (n=1), and coronin-1a deficiency (n=1). results: the median age of the patients is 10.5 years (range 3-33). the majority are females (83%). cutaneous granulomas have been documented in all cases while visceral granulomas (spleen and liver) were observed in 3 cases. t cell and b cell lymphopenia as well as hypogammaglobulinemia or impaired antibody formation were present in most patients. all patients had received rubella virus vaccine. the median age at presentation of cutaneous granulomas was 84 months (range 14-377). the median duration of time elapsed from vaccination to the development of cutaneous granulomas was 19 months (range 12-152). the diagnosis of rubella was made by pcr in 83% of patients and by immunohistochemistry in the remainder. one patient was confirmed to have vaccine strain rubella virus. hematopoietic cell transplantation was reported in three patients. rubella associated complications did not contribute to death among those patients who died (16%). conclusions: of the now 28 cases, 19 (67%) share the diagnosis of a dna repair disorder and confirm that chronic rubella virus infection is associated with cutaneous granuloma formation. analysis of patients with dna repair disorders and other pids with this complication will help clarify determinants of rubella pathogenesis, identify specific immune defects resulting in chronic infection and may lead to defect-specific therapies. introduction/background: introduction/background: hizentra® is a 20% liquid igg product approved for subcutaneous administration in adults and children greater than two years of age who have primary immunodeficiency disease (pidd). limited information on use of hizentra® is available for children who have received hematopoietic stem cell transplantation (hsct). objectives: objectives: the aims of this study are to determine the safety and efficacy of hizentra® in pediatric patients post hsct, and to characterize reasons for switch from intravenous igg (ivig) to subcutaneous (scig) delivery following hsct. methods: methods: a retrospective chart review involved 13 pidd infants and children (mean age 6.3 [range 3.1 to 14.5] months) status post hsct who received hizentra®. ten patients received hizentra® by pump administration, and 3 patients by manual push. results: results: hizentra® administered weekly to 13 children included an average of 144 infusions (range 14-416). the mean dose was 722 mg/kg/4 weeks. the mean igg level was 889 mg/dl while on hizentra® in 11 patients compared to a mean trough igg level of 563 mg/dl in 6 patients during immunoglobulin administration prior to hizentra® of most children. four patients naïve to igg therapy were started on hizentra®. average infusion time was 4.7 (range 3-15) minutes for manual entry and 71 (range 60-90) minutes for pump entry, and the average number of infusion sites was 1.9 (range 1 to 2). local reactions were mild and observed in 3/13 (23.1%) children. ten patients had no local reactions. no serious adverse events were reported. the rate of serious bacterial infections (sbi) was 0.12 per patient-year while receiving hizentra®, similar to reported efficacy studies. the reasons for switch from ivig to scig in 13 patients (some patients had multiple reasons) were improved igg serum levels and physician desire for steady state serum igg levels (n=6), loss/lack of venous access (n=2), patient/caregiver preference (n=7), home site of care preference (n=4), and physician preference for scig (n=5). conclusions: conclusions: hizentra® is a safe and effective option in children who have received hsct. reasons for switch from ivig to scig included improved serum igg levels, desire for steady state serum igg levels, and patient/caregiver preference. consulting medical advisor, immune deficiency foundation introduction/background: it is currently unknown how many persons with primary immunodeficiency (pi) receive the seasonal influenza vaccination, and what proportion becomes ill. additionally, the effect of immunoglobulin (ig) replacement therapy on the frequency of influenza diagnosis and severity of symptoms is not known. objectives: the current study sought to measure the prevalence of seasonal influenza vaccination and diagnosis among persons with pi, specifically those with antibody deficiencies: x-linked agammaglobulinemia (xla), common variable immunodeficiency (cvid) and hypogammaglobulinemia. methods: 11,533 email invitations were delivered to members of the immune deficiency foundation database requesting participation in an online survey regarding their zoster, influenza and varicella vaccination experiences. data from 1009 persons with xla (total n=41; children age<18; n=19; adults age>17, n=22; 100% male; 81% white non-hispanic), cvid (total n=824; children age<18, n=57; adults age>17, n=767; 79% female; 92% white non-hispanic), and hypogammaglobulinemia (total n=144; children age<18, n=23; adults age>17, n=121; 77% female; 92% white non-hispanic) were analyzed for the 2016-2017 influenza season. results: overall, 74% (n=749) of the sample received a seasonal influenza vaccination during the 2016-2017 influenza season. persons with xla were less likely to receive an influenza vaccination (54%, n=22), than persons with cvid (76%, n=628) and hypogammaglobulinemia (69%, n=99). a fifth of respondents (21% overall, 34% of children and 19% of adults) who attend school and/or work stayed home at some time to avoid seasonal influenza. though the majority (81%, n=821) were not diagnosed with influenza, when stratified by age, children were twice as likely to be diagnosed than adults. of the 99 children sampled, 30% (n=30) were diagnosed with seasonal flu compared to 15% (n=139) of the 910 adults that were sampled. the role of replacement ig therapy in protection against flu can best be examined in the 41 patients with xla since these patients cannot make specific antibodies to vaccine and only 54% (n=22) received the vaccine. although 59% (n=24) of the individuals with xla were exposed to flu, only 10% (n=4) were diagnosed with influenza; two of whom were not receiving ig therapy at time of diagnosis. concerning influenza severity, individuals receiving ig therapy at the time of flu diagnosis tended to have modestly milder symptoms than those not receiving ig treatment (e.g., less likely to report sore throat (78% versus 92%, p<.05)), but the sample size is too low to draw firm conclusions. conclusions: a high proportion of antibody deficient persons received a seasonal influenza vaccination for the 2016-2017 influenza season. in addition to vaccination, many individuals attempted to avoid influenza infection by remaining home from school and/or work. there was a suggestion that ig replacement therapy may partially protect xla patients from symptomatic flu, although the role of cell mediated immunity in protection against flu is not clear. a prospective study could determine if ig replacement therapy partially protects against clinical flu symptoms in xla and cvid patients using two groups those that receive the current flu vaccine vs. those that do not receive the flu vaccine. introduction/background: fanconi anemia (fa) is an autosomal recesive or x-linked genetic disorder, characterized by cytopenia or bone marrow failure, this will lead to a severe anemia, neutropenia and thrombocytopenia, requiring frequent interventions with diversified therapies, including hematopoietic stem cell transplant (hsct). a complete immune reconstitution is requiere for a success transplant. one of the main upcoming events asociated after the hsct, is the secundary immunodeficiency, which is asociated with a significant morbility and mortality in the patients. to rich a successful alogenenic-hsct, is impending a complete reconstitution of the t cells immunity, for this it is crucial the presence of factors like; thymic activity of the hsct recipient, biological features of the allograft (eg, degree of histocompatibility, number and type of infused donor t cells) and preparative regimens. objectives: describe the kinetics of the immune reconstitution in fa patients after alogenic-hsct, as well as the infections associated during this process and other comorbilities. methods: we decribed the lympocyte population (t, b and nk) in pb measured by flow cytometry in fa patients on days +90, +120, +150, +180, +210 and +360 post alogenic hsct from a match related donor. the conditioning was base on fludarabine 150mg/m2, cyclophosphamide 10mg/kg and antithymocyte globulin rabbit 20mg/kg. infectious-disease survillance for virus was determined by the quantification by dna pcr-rt for cmv, ebv, adenovirus, as well as the presence of galactomannan and candida sp antibodies, or isolation of fungus or bacterial culturing in the cases of infectious disease of known or uknown aetiology. results: the lymphocyte population mesurement was performed in a total of five fa patients who undergo to an allogenic hsct, all of them received stem cells from a match related donor and the source was bone marrow. successful engraftment was observed in all 5 patients, there were no deaths reported. after the hsct was performed, the kinetics of recovering for the distinct lymphocytes subsets was the the following: nk cells (cd16+cd56+) were the first to recover, followed by cd+8 t cells, b lymphocyte and finally cd+4 t cells (figure 1 ) all of them rich normal values and remain stable. three out of five patients presented infectious disease: two of them were cmv positive, one patient has a concurrent detection of adenovirus, and in the other was detected aspergillus, in both of them it was presented at day +90. the third one developed acute gvhd which progressed to a chronic gvhd, at day +153 was diagnosed on him listeria monocytogenes meningitis, at that momento he has just cd+16 +cd56 and cd8+ reconstituded. there is no new infectous disease detected in any of the patients after they reconstituted cd4+ t cells. conclusions: complete immune reconstitution is the decisive for the presence of several morbilities and mortalities, mainly because of oportunistic infecctions and gvhd. we show that the kinetics of recovery of the different populations of lymphocytes follows those patterns also described for patients with other hematological malignancies: early recovery of nk cells, followed by effector cytotoxic t cells and b cells, and finally, cd4+ t-helper cells. the utility of post-transplant monitoring of pb-lymphocyte subsets for improved follow-up of patients undergoing bmt and prevent opportunistic infections. introduction/background: early detection of primary immunodeficiency diseases (pids) before serious infection is of utmost importance and a question of patient survival. one of the main organs involved in pids is skin with mucocutaneous manifestations being one of the common complaints in pids. the presenting skin symptoms may serve as an essential element in early diagnosis of pids. objectives: this study aims to determine characteristics, frequency, nature and incidence of skin manifestation in pid patients seen in qatar. methods: this retrospective study was conducted at hamad medical corporation the only tertiary hospital in qatar from january 2009 to july 2017.the subjects included were pid patients < 14 years old, who had dermatological complaints. information collected included dermatological diagnosis, gender, age at onset of signs and symptoms, age at definite diagnosis, family history of related disease, any lab results such as pathology or viral studies obtained from the clinical medical record. patients were diagnosed and classified according to clinical and laboratory criteria by the international union of immunological societies primary immunodeficiency committee. results: a total of 109 patients were studied and skin/mucocutaneous manifestations were found in 65 patients (59%) with male to female ratio of 1.5:1. age at onset of skin manifestations ranged from 0 to 12 years. skin manifestations were divided in two categories according to presentation before pid diagnosis (primary manifestations n=32, 47%) and during the disease period (secondary manifestations n=35, 53%). the type of pids predominantly having primary manifestation were scid (n=5, 15%) cvid and cgd (n=4, 12%, each), xla, at and griscelli syndrome (n=3, 9%, each).various manifestations which were atypical in their presentation and persistently found in patients who did not respond to any effective treatment (n=13) led to the basis of clinical immunological study and diagnosis including rare diagnosis such as ipex. whereas secondary manifestations were primarily reported in scid (n=9, 25%) cgd, at and digeorge syndrome (n=5, 14%, each). the nature of skin manifestations varied in both groups, primary manifestations notably had 41% cutaneous infections comprising of thrush (30%) stomatitis (17%) viral rashes (26%) bacterial rashes (13%) impetigo (8%) and fungal rash (4%). eczema/atopic dermatitis were 31% and other minor miscellaneous skin alteration cases found were apthous mouth ulcer (5) erythroderma (4) gvhd like rash (1) alopecia (1) psoriasis (1) and scleroderma (1) . secondary manifestations were identified as 53% cutaneous infections 20% eczema and infantile seborrheic dermatitis (1) pruritus (1) gvhd and alopecia (1) each. the causative microorganisms were confirmed in 44% of the cases via lab cultures whereas common infections such as chicken pox and herpes were validated through clinical symptoms. none of the cases were biopsied. overall, highest skin infection was seen in ataxia telangiectasia (11). other pids with prominent cutaneous manifestations included cgd (9), digeorge syndrome (5), hyper ige syndrome (4) and scid (rag 1 or 2, 4 cases). more than two types of skin manifestations were found in 47% patients over the due course of their illness. conclusions: an awareness of various cutaneous and skin disorders associated with pid which are persistent and unresponsive to treatments among dermatologist and family physicians is crucial to raise suspicion for early detection, timely management and prevention of complications. introduction/background: ataxic telangiectasia (at) is a rare neurodegenerative autosomal recessive disease associated with immunodeficiency, poor coordination and disability. ataxia telangiectasia has a diverse clinical heterogeneity which may often lead to an incorrect diagnosis or late detection of the disorder resulting in exposing the patient to unnecessary radiation from various sources. objectives: we present a female child from the asian subcontinent that was seen for the first time with ulcerative skin lesions, failure to thrive and marked lymphopenia. methods: a search of the pubmed database was carried out, using different combination of the terms "ataxia", "telangiectasia", typical , atypical and "presentation" results: four-year-old pakistani girl, product of first degree nuptial presented with generalized vesicular rashes mainly on abdomen and scalp which turned into ulcers with residual hypo pigmented lesions and diarrhea. she had short stature, failure to thrive (weight and height < 5th percentile) and no previous infection or family history of primary immunodeficiency diseases (pid). mild delay in milestones was observed especially in speech. immunizations were up-to-date including bcg. on examination she had mild ocular telangiectasia but no cutaneous involvement. investigations revealed lymphopenia (1.6) and eosinophilia. workup for diarrhea including duodenoscopy revealing subtotal villous atrophy however the biomarkers for celiac disease were negative. immune system workup showed high igg levels, normal iga, ige and igm level. igg subclasses: low igg 2, and 3, antibody titers to h.influenza and pneumococcus vaccine were relatively low. lymphocyte subsets showed low cd4,cd19 and high nk cells (40%), low naïve t cells (2%) and inverted cd4/cd8 ratio.t cell function with post pha was 18.7% but had normal response to cd3. alfa feto protein was requested due to continued low cd4 count and the level was found to be high: 260.9 iu/ml. next-generation sequencing (ngs) showed homozygous mutation for trp1750fs in chromosome 11 of atm gene confirmed by whole exome sequencing. immediate treatment with ivig was started leading to mark improvement of skin lesions, diarrhea and weight gain. conclusions: the index of suspicion of at should be highlighted when deciphering lymphocyte subset. currently there is no neonatal screening for pid diseases and next generation sequences in qatar. once implemented it may prove beneficial in discovering cases from early infancy and reaching upon a definite diagnosis. introduction/background: chronic granulomatous disease (cgd) is a genetic disorder of the nadph oxidase complex in phagocytes which results in impaired production of microbicidal reactive oxygen species that can lead to recurrent life-threatening bacterial and fungal infections. the majority of affected patients in the united states have a x-linked defect in the cybb that encodes gp91phox protein followed by an autosomal recessive defect in the ncf1 gene that encodes p47phox . x-linked female carriers show 2 populations of neutrophils and following lyonization, and severe skewing of x-chromosome inactivation can get cgd type infections. a recent study has shown that the lower dihydrorhodamine (dhr) percent in female carriers predicts a higher infection risk but carrier state on its own predicts autoimmunity results: a 39-year-old female presented for an evaluation of recurrent fevers. she had a history of several lobar pneumonias in childhood. at age 32, she developed erythematous bumps on her abdomen that were initially non-bothersome but later became painful. she then developed recurrent fevers, chills, and rapid weight gain. biopsy of the skin lesions showed subcutaneous panniculitis-like t cell lymphoma. she was treated with chemotherapy and found to be in complete remission. she had no previous family history of recurrent infections. at the completion of her chemotherapy, her 1-year-old son became septic in the hospital and was then diagnosed with cgd from mutated cybb gene. post chemotherapy, the patient had a prolonged course with an atypical lung infection that required bronchoscopy and video thorascopic surgery, however no bacteria was ever identified. she did improve after a long course of ivantifungals. evaluation for immunodeficiency was done after she had a recurrence of low grade fevers, cough, and fatigue. dhr flow cytometry with phorbol myristate acetate (pma) was done which showed 3.6% neutrophil oxidative burst activity after stimulation, consistent with a highly skewed lyonization pattern in x-linked cgd. conclusions: this case demonstrates an interesting lesson in a woman with recurrent infections since childhood whose clinical picture was confounded by her history of malignancy. the abnormal lung infections prior to malignancy were alarming and gave cause to do additional immunology testing. however, it begs the questions, if she did not have a son, would she have been diagnosed with severe x-lined carrier disease. introduction/background: measurement of the specific antibody response following vaccine challenge provides clinicians with a better understanding of the adaptive immune response in individuals undergoing immunological evaluation. objectives: we hypothesised that after classification of primary immunodeficiency (pid) patients based on vaccine response (vr), further division using igg subclass (iggsc) 1-3 measurements may identify additional patients with abnormal b cell function and patients with different frequencies of infection at presentation. methods: vrs and serum iggsc concentrations were quantified using the vacczyme anti-pneumococcal polysaccharide (pcp) igg elisa and human iggsc liquid reagent kits (the binding site group limited, uk) in 23 pid patients (1:1.3 m:f, median age 41.5 years, range 20-78). all patients were immunised with pneumovax®23 (sanofi pasteur msd). the lower limits of published normal iggsc ranges were used as cut-off values (igg1 3.2 g/l, igg2 1.2g/l and igg3 0.2g/l). pcp concentrations equal to or less than 70 mg/l post-vaccination was considered an abnormal response. results: agreement between vr and iggsc measurements was 48% (p=1.00). the frequency of respiratory tract infections at presentation among pid patients with a normal pcp igg vr was 45% and 55% among patients with an abnormal vr. subsequently, four separate groups could be identified by including iggsc measurements. the frequencies of infections at presentation were for the vr+/iggsc+ group (n=4) 36% vs. 48% for the vr+/iggsc-group (n=10); and 33% for the vr-/iggsc+ group (n=2) vs. 63% for vr-/iggsc-individuals (n=7). conclusions: these results confirm that vr and iggsc measurements are independent serum biomarkers of humoral immunity. taken together the vr and iggsc results provide more detailed information about the immune status that may influence diagnosis, treatment and monitoring decisions. introduction/background: hizentra® is a 20% liquid igg licensed for subcutaneous administration in adults and children greater than two years of age who have primary immunodeficiency disease (pidd). subcutaneous immunoglobulin (scig) use in autoimmune conditions is reported. stiff person syndrome (sps), a rare neurologic disorder characterized by fluctuating muscle spasms and rigidity, is mediated by autoantibodies to glutamic acid decarboxylase (gad). symptoms of sps have been shown to improve after administration of intravenous immunoglobulin (ivig) however, there is a paucity of information regarding use of scig in sps. objectives: the aim of this study is to describe the use of hizentra® in patients with sps, including indications for scig, clinical characteristics of patients, and clinical and laboratory response to scig. methods: a multicenter retrospective chart review examined 2 patients with stiff person syndrome treated with hizentra®. results: sps was diagnosed in 2 patients at 12 and 25 years. both patients were started on ivig, steroids, and rituximab prior to initiation of weekly hizentra® (ages 15 and 27 years). the average dose of hizentra was 125mg/ kg weekly. the average igg level while receiving ivig was 1,164.5 mg/dl, similar to the average igg level while on hizentra was 1,250 mg/dl. the number of administration sites ranged from 2-4, and duration of infusions ranged from 60-120 minutes. serum anti-gad antibody levels prior to hizentra® were 845 u/ml and 134 u/ml respectively. anti-gad antibody level during treatment with hizentra (available for one patient) was >5,000 u/ml, and 132,240 u/ml following discontinuation of hizentra®. one of the reasons for switching to hizentra® was lack of response while on ivig. the average number of hizentra infusions were 138. patients reported improvement in spasticity related to sps while on hizentra®, and one patient had improvement in seizures. one patient discontinued hizentra® in favor of intravenous immunoglobulin (ivig) due to physician preference. the most common side effects were local reactions including pain, pruritus, and redness. no serious adverse events were reported. conclusions: hizentra® was associated with improved symptoms in sps in both patients including decreased spasticity, and improved seizure frequency in one patient. serum anti-gad levels did not decrease following administration of hizentra. hizentra® was well tolerated in patients with sps, with most side effects reported as mild. hizentra® may be considered as an alternative to ivig treatment in patients with sps. introduction/background: combined immunodeficiency (cid) has been associated with a spectrum of secondary gastrointestinal manifestations, including infectious and non-infectious causes. abatacept, a soluble ctla4-igg1 fusion protein targeting t cell activation, has demonstrated benefit in the treatment of autoinflammatory manifestations in patients with cid due to underlying heterozygous germline mutations in ctla4 and lrba. objectives: herein, we report two patients with cid characterized by low immunoglobulin levels, low class-switched memory b cell counts, and low peripheral naïve cd4+ t cell counts. both patients suffered from severe and persistent diarrhea complicated by protein-wasting and malnutrition, ultimately diagnosed as biopsy-consistent autoimmune enteropathy. the clinical history of patient 1 was also notable for granulomatous lung disease and autoimmune cytopenias. methods: a t-regulatory cell disorder was considered in both cases, however, ctla4 and lrba were found to be unaffected by whole exome sequencing (patient 1) and/or flow cytometry (patient 2). due to progressive worsening of the autoimmune enteropathy despite management with chronic steroids (both patients), combination rituximab/mycophenolate mofetil (patient 1), and total parenteral nutrition (tpn) complicated by recurrent infections (patient 2), abatacept was trialed at 125 mg sc weekly. results: in both patients, the start of abatacept therapy produced a dramatic improvement as measured by decreased stool frequency, improved weight gain, and decreased protein-wasting. patient 1 has been maintained on this monotherapy for over one year, with improvement in the gastrointestinal as well as pulmonary autoinflammatory complications. the only documented adverse event has been hepatitis b reactivation, managed with tenofovir and abatacept continuation. patient 2 has required azathioprine/abatacept combination therapy for clinical stabilization and is without a significant adverse event to date. conclusions: we conclude that abatacept may be a relatively safe and effective therapeutic in the management of severe autoimmune enteropathy in the background of cid, even when used outside of the classical clinical context of ctla4 or lrba haploinsufficiency. objectives: as increasing the outbreak of allergic diseases, study for treatments comes to the force. in this research, we aimed to assess the effects of sg-sp1, a derivative of gallic acid, on mast cell-mediated allergic inflammation using various animal and in vitro models. methods: ovalbumin-induced systemic anaphylaxis and immunoglobulin e (ige)-induced passive cutaneous anaphylaxis are standard animal models for immediate-type hypersensitivity. oral administration of sg-sp1 hindered the allergic symptoms in both animal models. these inhibitions were deeply related to the reductions of histamine and interleukin-4. results: sg-sp1 reduced degranulation of mast cells and expression of inflammatory cytokines in a dose dependent manner. sg-sp1 showed better anti-allergic effects compared to gallic acid and dexamethasone. down regulations of intracellular calcium level and nuclear factor-b activation by sg-sp1 were causative of the reduction of allergic mediators. to anticipate the exact target of sg-sp1, phosphorylation of proteins involved in mast cell signalling was assessed. sg-sp1 suppressed the activations from lyn and was aggregated with high affinity ige receptor (fcri). conclusions: from these results, we assured that sg-sp1 directly interact with fcri. all together, we propose that sg-sp1 might be a therapeutic candidate for allergic disorders. (162) submission id#425361 systematic assessment of pain in patients with primary immune deficiency using validated pain questionnaires: a prospective study. introduction/background: the number of primary immunodeficiency (pid) patients is rising dramatically because of good medical care as well as increased awareness. around 1 in 1200 live births are affected. more than 300 disorders have been discovered so far and this number is expected to rise in the coming years. as with any other chronic illness, pid patients are also prone to acute and chronic pains. chronic pain is a big challenge and lack of understanding of the etiology and underlying mechanisms limit our ability to diagnose or treat it effectively. objectives: to systematically assess chronic pain in patients with pid using validated questionnaires and to try to understand the underlying mechanisms of neuropathic versus non neuropathic pain. methods: short-form mcgill pain questionnaire (sf-mpq) is a recognized way to ascertain different pain characteristics as well as severity. a validated arabic version of the sf-mpq was used to prospectively assess chronic pain in patients with pid. furthermore, a validated arabic version of the neuropathic pain questionnaire-short form (npq-sf) was also used to assess neuropathic pain and to differentiate it from nonneuropathic pain. a total of 27 patients with pid were included. results: males: females were 33.3%: 66.6% respectively. mean age was 29.73 years. commonest diagnosis was combined immune deficiency in 37% of the patients followed by common variable immune deficiency which was 25.9 %. chronic pain was found in 64% of the patients that participated in the study. 50% of the patients who complained of pain found it to be tiring and exhausting. 35% each had aching or heavy or sharp pain. 28% had cramping pain, 25% had tender pain and 21% characterized their pain as throbbing or burning. 18% felt shooting pain, 14% had splitting pain and 7% had gnawing pain. 28% found it to be frightening and 17% described it as being sickening in itself. the commonest pain complaint was abdominal pain in 14% of the patients followed by headache 11 % and chest pain 11%. pain attributed to neuropathy was present in about 17.8% of the study population. most patients described that they had been experiencing pain for at least 2-3 years. conclusions: this is the first international study to understand the prevalence, duration and severity of chronic pain among pid patients. a significant number of patients reported ongoing pain. this is the first time any kind of pain has been studied systematically in the patients with primary immune deficiency. treating pain should have a major impact on improving the patients quality of life. introduction/background: primary immunodeficiency (pidd) patients, particularly those with severe t cell defects, are at increased risk of infections. bone marrow transplant also contributes to significant t cell lymphocytopenia, which can be associated with similar risks. several prophylactic measures, which vary per institution, are placed on these patients in order to prevent infections. we report on a likely outbreak of rapid growing nontuberculous mycobacteria (ntm) at our institution, with a suspected association to tap water objectives: to recognize nontuberculous mycobacteria as a threat to patients with immunodeficiency disorders methods: case series of consecutive patients admitted to one institution with similar infections results: in a period of 16 months, 4/2016 to 8/2017, 5 patients admitted to our institution were found to have rapid growing ntm species on central line culture or bronchoalveolar fluid. these cultures were obtained due to fever and symptoms of systemic infection. per institutional practices no peripheral blood samples were obtained at the time of initial culture. all had significant t cell dysfunction: wiskott-aldrich syndrome (1), an undefined combined immunodeficiency (1) , and three patients post bone marrow transplantation (bmt), one 6 months (173 days) post an unrelated cord blood transplant for farber syndrome, one 120 days out of a matched related transplant for epstein barr virus (ebv)-associated lymphoproliferative disorder, and one patient with high risk neuroblastoma 106 days out of his second tandem autologous transplant. both allogeneic transplant recipients had received serotherapy. all species were rapid growing, identified as mycobacterium mucogenicum (n=3), immunogenum (n=1), or abscessus-chelonae complex (n=1). the patients had not shared rooms, caregivers, invasive procedures, or medications from the same batch. three (60%) cases met cdc criteria for hospital acquired infections (hai). all patients were in general ward rooms, and were on standard precautions given diagnosis had not been established (n=2) or they were considered outside the typical window of strict bmt precautions (n=3). ntm species were subsequently isolated from hospital tap water. this has resulted in a significant increase in infection precautions, including the use of sterile water only for cares, as well as bottled water for drinking (with no use of ice machines), for all patients being evaluated for pidd or with significant lymphocytopenia. conclusions: ntm are a threat to patients with pidd. tap water is a potential source of mycobacterial infections in pidd patients. minimizing exposure risk to water sources containing ntm is very important in this population. patients with concern for pidd or significant t cell lymphocytopenia should take steps to avoid ntm exposures, including the use of sterile water for cares, and bottled water for drinking. introduction/background: purine nucleoside phosphorylase (pnp) deficiency is a rare autosomal recessive condition leading to severe combined immunodeficiency and neurologic impairment, typically presenting in early childhood. the condition is progressive and typically fatal in the first or second decade of life without hematopoietic stem cell transplantation (hsct). objectives: to illustrate the clinical and laboratory presentation of pnp deficiency with a novel mutation in the pnp gene via a case study. results: case: a four year old female of hispanic descent, with a past medical history of spastic diplegia, initially presented with chronic nasal congestion, recurrent sinusitis, and cough. laboratory studies were significant for an alc 500 cells/mcl (2000-8000 cells/mcl). she was lost to follow-up for two years, before returning to medical attention for pneumonia. evaluation of lymphocyte subsets at age 6 years revealed cd3+ 145 cells/mcl (1200-2600 cells/mcl), cd4+ 119 cells/mcl (650-1500 cells/ mcl), cd8+ 28 cells/mcl (370-1100 cells/mcl), and cd19+ 44 cells/mcl (270-860 cells/mcl). t cell mitogen stimulation to pha measured by flow cytometry was 30% of control. pnp activity was nearly absent and urine guanosine and inosine were significantly elevated. gene sequencing revealed a homozygous c.655a>g mutation in the pnp gene. prophylactic antimicrobials were started. despite strong recommendation for hsct, the patient was again lost to follow up until age 10 years, at which time she was found to have progressive lymphopenia, bronchiectasis, and developmental delay. t cell mitogen stimulation to pha measured by thymidine uptake assay was 9% of control and pnp functional activity was undetectable. the patient underwent a 9/10 matched unrelated hsct six years after her initial presentation. one year post-transplant, the patient continues on immunoglobulin replacement therapy. her course was complicated by cutaneous gvhd, treated successfully with topical corticosteroids. conclusions: this case study illustrates the progressive nature of pnp deficiency in the first decade of life. our patient is notable in that she survived without significant medical intervention to the age of 10 years. her presentation at age 4 years was not unlike those previously reported in the literature, with muscle spasticity, ataxia, and recurring bacterial infections. to the authors knowledge, this case reports a novel mutation in the pnp gene. introduction/background: severe congenital neutropenia (scn) is a primary immunodeficiency disease characterized by early onset recurrent infections, persistent severe neutropenia and congenital genetic defect. severe idiopathic neutropenia (sin) is a rare disease defined by persistent severe neutropenia, in the absence of an identifiable etiology. objectives: here, we aim to find out clinical, laboratory, genetic characteristic and remission status in children with scn and sin in chinese population. methods: in this study, we enrolled 39 chinese children who experienced severe neutropenia longer than 6 months without any virus infection or auto-immune antibodies from june 2008 to july 2017 in hospitals affiliated to shanghai jiao tong university school of medicine. their clinical, laboratory and molecular characteristics were analyzed and the patients were followed up to observe their remission status. targeted gene capture combined with next-generation sequencing technology was used to find out related gene mutation. results: patients in this study had a mean age of 29.21±27.94 months. molecular analysis revealed that 7 patients had associated mutations of scn, including elane and g6pc3. among 26 patients with continuous follow-up, one died for unknown reason. ten patients have recovered from sin (r-sin) with mean neutropenia duration of 19.40±10.91 months. scn patients had more frequent infection (5.86±1.57 times per year) than sin (3.95±1.05 times per year, p=0.008) and r-sin patients (p=0.005, 3.89 ±1.27 times per year). scn patients had significantly higher count of anc and monocytes than sin (p=0.015) and r-sin patients (p=0.029). however, there was no difference in anc and monocytes counts between sin and r-sin patients. bone marrow examinations demonstrated a myeloid maturation arrest at the myelocyte-metamyelocyte stage in scn patients, while most of sin and r-sin patients were normal. conclusions: our study indicated that, patients with mild infection, lower anc, monocytes count and normal bone marrow are likely to be sin. whereas others with relatively more severe infection, higher anc, monocytes count and maturation arrest in bone marrow are inclined to be scn. introduction/background: patients with qualitative and/or quantitative defects of humoral immunity often require immunoglobulin (igg) replacement therapy (igrt). usual starting doses range between 400-600 mg/kg and the dose is adjusted as needed depending on the patient. there is a paucity of information about whether and how extreme bmi (obese or underweight) and route of administration may affect a patients rate of infections. objectives: the objective of the study is to determine whether rate of infection is associated with bmi, dose or route of administration in patients receiving igrt. methods: this is a retrospective chart review from december2000-october 2017. we included patients between the age of 1 month and 100 years old who were evaluated initially and had at least one follow up evaluation. data reviewed included the route of administration, dose, infection history, igg serum levels, height and weight to calculate bmi, gender, age and diagnosis requiring immunoglobulin replacement therapy. participants were excluded if the type of immunodeficiency is unknown or if the participant had incomplete data for the requested data fields. the number of infections between visits was modeled using poisson regression as a function of dose, route of administration and the bmi with the log of the follow up interval as an exposure offset. results: eighty-five patients were eligible, and preliminary results for 50 patients are presented here. the mean infection rate per 100 weeks of follow up was 1.9 in obese patients, 3.0 in overweight patients and 1.5 in underweight/normal weight patients adjusted for route of administration; however these rates do not differ significantly from one another. the mean infection rate per 100 weeks of follow up differed by administration route; 1.4 infections for ivig versus 3.1 infections for scig when adjusting for bmi (p<0.0131). the mean infection rate per 100 weeks of follow up was not associated with dosage. conclusions: overweight patients may experience more infections than obese or underweight patients, regardless of administration route. ivig patients may have a lower rate of infections compared to scig patients regardless of bmi. work is ongoing to complete analyses for the remainder of the eligible patient population. (167) submission id#421931 introduction/background: primary immunodeficiencies (pids) are a rare group of genetic disorders associated with a tendency to infectious diseases and an increased incidence of cancer. there is no data regarding the prevalence of malignancies in patients with pid in turkey. objectives: in this study, we aimed to evaluate patients who diagnosed as pids and developed malignancies, and calculate the estimated frequency of malignancies associated with pids in turkey. methods: forty five patients who were diagnosed with malignancy in the follow-up period of pid at the four tertiary immunology clinics between 1992 and 2017 years were included in the study. the data were obtained retrospectively from the hospital records and the database of esid online patient registry. results: the prevalence of malignancies in patients with pid was found as 1.05% (45/4285). the male to female ratio of the patients was 29/16, the median age was 13.5 years (minimum: 1.5, maximum: 57) and the median age at which patients get diagnosed with malignancy was 9 years (minimum: 1.5, maximum: 51) . there was no cancer history in their family members. the most common type of pid which associated with malignancy was ataxia telangiectasia (n=15, 33.3%). non-hodgkin lymphoma was the most common malignancy (n = 25, 55.5%) in our group (table 1) . ebv quantitative pcr was positive in 6 lymphoma cases (17.6%). the median number of x-rays and ct scans in patients with at and bloom syndrome before malignancy developed were 5 (minimum: 1, maximum: 24) and 1 (minimum: 0, maximum: 3), respectively. two cases had dual malignancies (papillary thyroid cancer and anal adenocancer). twenty cases were treated with chemotherapy, 7 cases with hematopoietic stem cell transplantation, 5 cases with radiotherapy, and 5 cases with surgical treatment, treatment information of 17 patients was not reached. remission was detected in 16 cases, while resistance to therapy in 2 cases and recurrences in two patients were observed. four patients are still on chemotherapy. twenty cases died. conclusions: the tendency of malignancy in patients with pids is due to the deficiency in the immune response that lead to failed surveillance against oncogenic viruses, premalignant/malignant cells, or both. lymphoid malignancies are the most common malignancies associated with pids. pids-associated malignancy incidence has increased in recent years because of that improved survival of the patients. this study is the largest cohort investigating the association of malignancy in patients with pid in turkey. additionally, we first reported tendency to malignancy in a patient with znf 341 deficiency. introduction/background: next-generation sequencing (ngs) has become an integral tool in the evaluation of primary immunodeficiency disorders (pid). we describe a patient with a previously described pathogenic foxp3 variant who met clinical and laboratory criteria for cvid. objectives: describe a patient with a previously described pathogenic foxp3 variant who met clinical and laboratory criteria for cvid. results: case description: an 8-year-old male born premature at 32 weeks presented with a history of recurrent infections. family history was negative for immunodeficiency. the patient developed recurrent acute otitis media beginning at 1 year of age, three episodes of pneumonia beginning at 3 years of age, and recurrent sinus infections requiring treatment on average four times a year beginning at 5 years of age. initial immunologic evaluation at age 8 was notable for: igg 305 mg/dl (reference 673-1734 mg/dl), igm 15 mg/dl (reference 47-311 mg/dl), iga 173 mg/dl (reference 41-368 mg/dl), ige 166 iu/ml (reference 0-90 iu/ml). lymphocyte subpopulations were normal. specific responses to vaccines showed: protective antibody titers to diphtheria, but not to tetanus or pneumococcal antigens. he did not respond to booster vaccination and was started on ivig with significantly reduced frequency of infections. at age 10, while on ivig, he developed oral ulcers (biopsy consistent with ulcerative eosinophilic granuloma), abdominal pain, and recurrent arthralgias involving ankles, elbows, hips and the sacroiliac joint. magnetic resonance imaging (mri) was consistent with sacroilitis. subsequent imaging was consistent with chronic relapsing osteomyelitis (crmo). gastrointestinal biopsies showed severe active chronic pangastritis with antralized oxyntic gastric mucosa with enterochromaffin cell hyperplasia; suggestive of autoimmune gastritis. plasma cells were present throughout the gastrointestinal tract. ngs (bcm-ngs, baylor miraca genetics laboratory, houston tx) identified a hemizygous pathogenic missense variant, c.1190g>a (p.r397q) in the x-linked foxp3 gene, that has been reported previously in patients with immunodysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome. flow cytometry studies showed a decreased percentage of treg cells of total cd4 expressing cells, 2.6% (reference 4.2-9.9%), but normal foxp3 expression within these cells, 68% of treg cells with foxp3 expression (reference 55-81%). interestingly, treg cell subset phenotyping obtained at the same time showed a normal percentage of natural tregs, 3.2% cd4 t cells (reference 0.7-4.0%), as well as normal percentage of naive tregs, 5.6% cd4 t cells (reference 0.6-9.0%). up to this point, he had not had any signs of diabetes, thyroid disease or frank enteropathy involving the small or large intestine. conclusions: we report a pathogenic foxp3 variant, occurring in a patient with a cvid-like phenotype, autoimmune gastritis, and an association with crmo. this case demonstrates the increasing utility of ngs, which can profoundly impact prognostic and therapeutic considerations. (169) submission id#413429 the natural history of patients with profound combined immunodeficiency (pcid): interims analysis of an international prospective multicenter study. immunodeficiency in this patient group. so far, 48 patients were transplanted after enrolment, overall 19 patients died (9 in the hsct group, 10 in the non-hsct group). analysis of the hsct decisions revealed the divergent decisions in patients with similar disease burden, favoring an ongoing prospective matched pair analysis of patients with similar disease severity with or without transplantation. so far, neither the genetic diagnosis nor simple measurements of t-cell immunity emerged as good predictors of disease evolution. conclusions: the p-cid study for the first time defines and characterizes a group of patients with non-scid t-cell deficiencies from a therapeutic perspective. since genetic and simple t-cell parameters provide limited guidance, prospective data from this study will be an important resource for guiding the difficult hsct decisions in p-cid patients. (170) submission id#419587 the plasma contact system and its role in common variable immunodeficiency: an explorative study. introduction/background: a growing body of evidence suggests that the contact system is involved in the activation of various vascular and immunological pathways and acts as an interface to help regulate allergic reactions, coagulation, complement, innate immunity and inflammation. as demonstrated in mice experiments, contact activation and high molecular weight kininogen (hk)-derived peptides increased homing of t and b lymphocytes into lymph nodes, which suggests an important area of research for understanding the contact systems role, specifically fxii, in immune-mediated inflammation and immune dysregulation. this novel mechanism prompted further inquiry into its role of various human disease states characterized by inflammation. plasma hk cleavage has been proposed as a useful and minimally invasive biomarker in various inflammatory disease states. this pathway has not been explored in cvid, in which inflammatory complications are found in one-third of patients with an unidentified genetic cause. characterizing the contact system biomarkers in cvid patients could elucidate a role in pathogenesis. objectives: assess the presence of contact activation at baseline in sera from cvid patients with and without inflammatory complications compared to healthy controls. methods: cvid patients were recruited in the outpatient setting and the measurement of cleaved plasma hk (chk) levels was determined by western blot analysis, under reducing conditions, with quantitation of total and chk bands using an odyssey imaging system (licor). a one-way anova test for differences among the 3 studied groups will be applied. c1 inhibitor levels, c3 and c4 levels and high-sensitivity crp were also measured as comparable biomarkers for inflammation. results: to date, 9 cvid patients were studied, 7 with and 2 without inflammatory complications. repeated determinations of cleaved hk% (chk%) revealed an average of 1.20% (range: 0.46-2.66%) in cvid patients with inflammatory complications and those without complications averaged 1.07% (range: 0.79-1.35%). healthy controls had an average chk of 1.15% (n=10, range: 0.60-2.10%). conclusions: cleaved kininogen detected in the sera of cvid patients was found at similar levels compared to healthy controls (chk<5%). findings suggest that systemic activation of the contact system might be absent in cvid, however, future considerations include developing detection methods for local tissue activation. (171) submission id#428729 the underlying primary immunodeficiencies and lung diseases, and low cd3 and cd4 counts are associated with recurrent pneumonia in hiv negative lymphopenia patients. 1 1 clinical fellow, yale university school of medicine introduction/background: lymphopenia can be considered as primary or acquired immunodeficiency. lymphopenia is associated with a considerable increase in susceptibility to infections and the treatment focus for lymphopenia is mainly consists of prophylaxis and treatment of opportunistic infections. aids is the most well known cause of acquired lymphopenia and the cd4 count serves as an effective surrogate marker for disease progression and guideline for prophylaxis for hiv positive lymphopenia patients (hplp). hiv negative lymphopenia patients (hnlp) have been following the same prophylaxis guideline for hplp patients. however, it is unclear whether same prophylaxis guideline will be appropriate for both groups since the underlying immune mechanisms are different between these two groups. objectives: we aimed to define the optimal treatment and prophylaxis guideline for hnlp. in this study, we compared the clinical phenotypes and absolute counts of lymphocyte subsets between hnlp with recurrent pneumonia (pna), recurrent upper respiratory infection (uri) and no pulmonary infection. methods: electronic medical records of hnlp (n=29) seen at an academic immunology clinic between the year of 2012 and 2017 were reviewed retrospectively. lymphopenia was defined as absolute cd3 count less than 1000/μl. the age, absolute counts of cd3, cd4, cd8, cd19, nkt and nk cell counts, history of antibiotic or antiviral prophylaxis use, autoimmune disease, lung disease, immunosuppressive therapy use, hypogammaglobinemia and ivig therapy use were compared between patients with recurrent pna (n=8), recurrent uri (n=13) and no pulmonary infection (n=8). results: this study showed that patients with recurrent pna had significantly lower absolute cd3, cd4, nk cell counts and age compared to the patients with recurrent uri ( table 1) . none of the clinical phenotype was significantly different between these two groups. when we compared the patients with recurrent pna vs no infection, all lymphocyte subset counts and age were similar between these two groups except the frequency of underlying lung disease which was significantly higher in the recurrent pna group (table 1) . lastly, we grouped the recurrent pna and uri groups together as the pulmonary infection group. when we compared the pulmonary infection group with the no infection group, lymphocyte subset counts were not significantly different, however, infection group showed significantly higher incidence of pid and the trend of lower rate of is therapy use than the no infection group (table 1) . conclusions: the initial study has several limitations. this was a retrospective study from a single clinic and patient population was limited to 29 patients. despite these limitations, we believe that this study provides valuable messages regarding prophylaxis guideline for pulmonary infection in nhlp; the underlying pids including icl and cvid, lung diseases particularly bronchiectasis and the absolute cd3 and cd4 counts less than 500/μl and 300/ μl, respectively, are associated with recurrent pna and the patients with these risk factors likely will benefit from antibiotic prophylaxis. in addition, patients with acquired lymphopenia due to chronic use of is therapies without underlying pid or lung disease less likely develop pulmonary infection despite of low cd3 and cd4 counts. further investigations are crucial to elucidate the clinical significance of our initial observation by increasing the patient population and analysis of detailed immunologic and genetic profile of these patients which will likely reveal immunological markers and genes that are involved in the pathogenesis of both primary and hiv negative acquired lymphopenia. director, neuro-oncology program, division of hematology/oncology, ucsf benioff children's hospital oakland, ca introduction/background: gata2 is a hematopoietic transcription factor, required for the development and maintenance of a healthy stem cell pool. heterozygous mutation of gata2 has been associated with different but overlapping syndromes affecting both myeloid and lymphoid cell lines including aplastic anemia, myelodysplastic syndrome, acute myeloid leukemia, pulmonary alveolar proteinosis and lymphedema. it is also associated with immunodeficiency and susceptibility to mycobacterial, fungal, and viral infections. hematopoietic stem cell transplantation (hsct) is the only available cure which should ideally occur before patients develop neoplasia, severe infections or lung disease. objectives -to describe the clinical presentation of a genetically confirmed case of gata2 mutation -to describe the characteristic hematological and immunological profile of patients with gata2 mutation -to emphasize the importance of high index of suspicion and early diagnosis in improving outcome with hsct methods: case: a 17-year-old female presented with prolonged fever, fatigue, nonspecific rash, and unremarkable clinical exam. laboratory evaluation was significant for mild pancytopenia with profound monocytopenia. bone marrow analysis was remarkable for hypocellularity without evidence of dysplasia or malignancy. peripheral blood flow cytometry showed decreased t-and b-cells and absent nk cells. results: the constellation of pancytopenia, marked monocytopenia and absent nk cells, were suggestive of gata2 deficiency. sanger gene sequencing of gata2 revealed a heterozygous nonsense mutation (p.arg337*). conclusions: mutation of gata2 is the underlying defect in overlapping clinical syndromes and is associated with immunodeficiency and malignant predisposition. incidence of organ dysfunction, infections and neoplasia increases with age. confirming the diagnosis during the phase of marrow hypocellularity/monocytopenia and pursuing hsct prior to malignant transformation may improve patient outcome. introduction/background: reduction in child mortality has kept pace with improved immunization and nutritional status. however, there are children with severe infections who have no identifiable reason. the search for children with pid was initiated in a tertiary care referral hospital from a region with no documentation of the prevalence of this disorder, but with a high rate of consanguinity. financial constraints, poor availability of laboratory facilities or therapeutic options locally, limited awareness among pediatricians and vast distances between premier centres were major obstacles. objectives to identify children with primary immune deficiency and study the spectrum of pid in north kerala to prevent infectious and other complications in these children to provide curative therapy whenever feasible methods 1. acquisition of knowledge and skills to diagnose and manage pids 2. establishment of an immune deficiency clinic 3. liaison with centres across the country offering diagnostic tests and stem cell transplant 4. formation of project team and submission of a project to the central government 5. improvement of diagnostic facilities at home institution results: 180 children with recurrent, severe or persistent infections or with two or more esid warning signs were screened for pid. severe combined immune deficiency was diagnosed in 3 children, wiskott -aldrich syndrome in 5 children, chronic granulomatous disease in 6 children and x linked agammaglobulinemia in 11 children and leucocyte adhesion deficiency in 3 children. prophylaxis with ivig was initiated in 22 children and stem cell transplant was done for 8 children. conclusions: in a country with resource constraints, limited awareness among health care providers and vast distances, it is possible to make a difference to the lives of families of children with pid by networking across centers with expertise in immunological and molecular genetic diagnostic methods and life -saving therapeutic modalities like stem cell transplantation. introduction/background: complete thymic aplasia is a rare cause of scid, and requires thymic transplantation for curative treatment. because thymic transplantation is not widely available, there can be significant delay between diagnosis and curative therapy placing the infant at risk of invasive life threatening infections. objectives: describe modalities of therapy for adenoviremia in a patient with scid due to thymic aplasia. methods: chart review was performed. treatment including hlapartially matched third party cytotoxic t cell therapy and matched related hematopoietic cell transplant were assessed for treatment of life threatening adenoviremia in a scid patient with thymic aplasia. we identified an infant with a mutation in tbox transcription factor 1 (tbx1) (c.1176_1195dup20) by way of state newborn screening for scid. she had severe t cell lymphopenia and abnormal t cell function. at 7 months age, she developed adenoviremia with associated fulminant hepatitis, and an initial viral load of 5 million copies/ml. despite prolonged therapy with cidofovir, viral load increased to as high as 264 million copies/ml. treatment with two infusions of partially hlamatched third-party cytotoxic t cells specific to adenovirus (5/10 and 4/10 hla matched, with hla class ii-mediated antiviral restrictions) led to partial clinical improvement without viral clearance. due to continued severe adenovirus-related hepatic dysfunction, unmanipulated bone marrow from an hla-identical sibling was infused without conditioning (10 million cd34+ cells/kg and 5.2x107 cd3+ cells/kg), at 9 months of age. after an initial surge in adenoviral loads attributed to massive viral lysis, the degree of viremia progressively declined and was <1,000 copies/ml within 7 weeks of marrow infusion. antiviral tcell activity against adenovirus was detected at low level in peripheral blood via ifn elispot at 3 weeks post-marrow infusion. she subsequently developed acute cutaneous and hepatic gvhd responsive to tacrolimus and steroids without recrudescence of viral illness. conclusions: delay in curative therapy in scid substantially increases risk of invasive life threatening infections. one strategy of allogeneic hct can be to eradicate severe infection in scid by providing the necessary t cell directed therapy against infectious agents. antigen-specific partial immune reconstitution can be achieved with hct in patients with thymic aplasia but concern regarding the development of full immunologic t cell diversity in athymic patients remains. senior technician, sanquin bloodsupply amsterdam introduction/background: chronic granulomatous disease (cgd), an inherited disorder of granulocyte function caused by a failure of intracellular superoxide production, normally presents as severe recurrent bacterial and fungal infections in the first years of life. the majority of affected individuals are diagnosed before the age of 2 years, although patients may remain undiagnosed until adulthood despite the early onset of the symptoms objectives: investigation of fungal infection in adult cgd patients methods: nbt and dhr 123 for detection of cgd and molecular analysis for detection of type of mutation in cgd and fungal characterization. results: we report here the detection of causative fungal infections in 2 adult patients with cgd. in the first patients we found paecilomyces formosus infection in an adult patient (18 years old female) with undiagnosed cgd who was referred to the shahid beheshti university hospital (tehran, iran) complaining of cough, dyspnea and fever for 5 weeks prior to admission. microscopic analysis revealed branching solitary phialide with ellipsoidal conidia with long chain arrangement and many chlamydospores which mostly resemble botryotrichum species but during subcultures on potato dextrose agar (pda) and sabouraud dextrose agar (sda), phialides typical of paecilomyces species appeared. typically, paecilomyces spp. rarely cause infections in humans and if these fungi are detected in blood urine or cerebrospinal fluid cultures they are considered as contaminants. the second patient was a 26-year-old man was referred to the hospital with weight loss, fever, hepatosplenomegaly and coughing. he had previously been diagnosed with lymphoadenopathy in the neck at age 8 and prescribed antituberculosis treatment. bal and serum galactomannan tests were negative. low, subnormal levels of ros were produced following stimulation of purified neutrophils with the phorbol ester pma. genomic dna was extracted and gene scan was used to determine the ratio between the number of exon 2 sequences of neutrophil cytosolic factor 1 (ncf1) gene, which encodes p47phox, and the number of -ncf1 exon 2 sequences. in addition, the fungal culture was disrupted with glass beads and dna was extracted. the dna sequence results were blasted using the ncbi genebank database, which showed 99% similarity to an aspergillus terreus isolate in the gene bank fungal library with accession no 1168. conclusions: thus, despite its current relative rarity in older patients, the presence of fungal infection is changing our understanding of the diagnosis, management and outcome of cgd. greater appreciation of the potential of fungal infection in older cgd patients is important in regions, such as iran, where tuberculosis is endemic and that sarcoidosis and cgd are considered as differential diagnosis. the demonstration of the successful patient-orientated treatment after using sequencing to confirm cgd and to identify the presence of the specific infectious agent emphasises the importance of adopting this approach across the region. introduction/background: primary immunodeficiencies (pi) represent a heterogeneous group of over 350 genetically distinct disorders which interrupt normal host-defense mechanisms and predispose to significant morbidity and mortality. presently, we are only able to screen for severe t-cell deficiencies at birth; however, the most common forms of pi often go undetected for years leading to adverse patient outcomes and excessive healthcare spending. given the relatively high incidence of pi in the general population, informatic measures could be useful for determining individual risk for pi and facilitating earlier, correct diagnosis and appropriate treatment across the spectrum of pi. objectives: the purpose of this study was to test the jeffrey modell foundations spirit (software for primary immunodeficiency recognition intervention and tracking) analyzer on the texas childrens health plan. major aims were to identify individuals with medium-high risk for pi, assess the clinical characteristics of at risk patients and determine if risk identification led to diagnosis of pi over a 12 month period. methods: after removing all known pi diagnosed patients from the database, 185,892 individual texas childrens health plan enrollees were screened for risk of pi with the spirit analyzer using relevant, weighted icd9 and icd10 codes. patient characteristics are shown in table 1 . following identification of medium-high risk (mhr) individuals, letters were sent to their primary care physicians to alert them of patient risk. a second analysis of the mhr individuals was performed 12 months later. detailed chart reviews were conducted on 769 mhr individuals to further assess clinical features of this group. the study was approved by the baylor college of medicine institutional review board (study h-38501). results: of the original cohort, 2188 (1.2%) were identified as mhr for pi. from that group, 1068 (0.6%) were accessible for analysis and 769 (0.4%) had electronic health records for review. in the 12 months following the first analysis, 43 (0.02%) were diagnosed with a pi. (figure 1 ) another 61 patients had concerning diagnoses coded warranting further investigation. (figure 2 ) concerning diagnoses included: cellulitis(18), abscess (14), recurrent otitis media(11), recurrent sinusitis (5), osteomyelitis (2), and mastoiditis (1) among others. in total 104 patients had a pi diagnosis or a history concerning for pi (0.06% of main cohort; 13.5% of mhr cohort). conclusions: the spirit analyzer is effective at identifying persons at risk for pi and facilitates diagnosis. potential mhr yield by the analyzer is over 10%. these patients are treatable and will benefit from targeted intervention once identified. the analyzer can also highlight concerning conditions worthy of additional assessment. future work should focus on longitudinal healthcare outcomes for patients diagnosed with pi via spirt and physician perspectives on the utility of the tool. introduction/background: genetic variants in card11 contribute to several diseases caused by dysregulation of the adaptive immune system. dominant-negative, loss-of-function and gain-of-function variants in card11 variants lead to primary immunodeficiency disease. however, primary immunodeficiency disease caused by card11 gain-of-function variants often progresses to b-cell malignancy. the clinical course and treatment options depend on the type of card11 mutation. unfortunately lymphocyte immunophenotyping and traditional proliferation assays can't distinguish the variant effect or predict the likelihood of malignancy. objectives: to use multiplexed functional assays for determining variant effect to distinguish between dominant-negative, loss-and gain-offunction effects in card11. our ultimate goal is to generate a variant function map that can be used to guide diagnoses and treatment of immune dysregulation caused by mutations in card11. methods: we co-delivered crispr/cas9 ribonucleoprotein complexes with libraries of single-stranded oligonucleotide repair templates thus generating lymphoma cell populations containing all possible singlenucleotide variants (~2400 different protein coding changes) in the nterminal 140 amino acids of card11. following culture with and without bcr pathway inhibitors, we used next generation sequencing to quantify variant abundance before and after culture, both with and without bcr pathway inhibitors. results: due a requirement for card11 in these lymphoma cells, those with dominant-negative and loss-of-function card11 variants grow more slowly, whereas those with gain-of-function variants grow faster in the presence b cell receptor pathway inhibitors. by tracking the relative abundance of each variant in the population by next generation sequencing over multiple conditions, we determined the functional effect of each. we assessed the functional effects of thousands of card11 variants in parallel. this enabled us to confirm several previously reported gain-offunction and dominant-negative variants in card11, as well as identify several additional novel variants. finally, we evaluated previously undescribed dominant-negative, loss-of-function and gain-of-function variants during differentiation of primary human b cells and during nf-b signaling. conclusions: the results of our experiments demonstrate the utility of multiplexed functional assays for determining variant effect in proteins where distinguishing between dominant-negative, loss-and gain-offunction effects are required to guide diagnoses and treatment. introduction/background: primary immune-deficiencies (pids) are a heterogeneous group of nearly 300 monogenic inborn errors of immunity. in recent years, whole exome sequencing (wes) became a valuable diagnostic approach for the identification of molecular defects in patients with clinical manifestations suggestive of pid. this approach provides a definitive diagnosis and may help in genetic counseling, prenatal diagnosis and pre-symptomatic identification of patients with a potentially lethal disease. the diagnostic yield using wes was found to be~25% in rare mendelian disorders and~40% in pids. we present here a markedly higher yield, of 65%, in pids with a high percentage of consanguinity (65% in this study). objectives: using the whole exome sequencing (wes) approach in 55 israeli pid patients with high percentage of consanguinity to identify the genetic causes underlying their diseases for better diagnosis and clinical management. methods: wes was performed on genomic dna obtained from wbc of 55 immune deficient patients with potential genetic causes. the sequencing data was analyzed bioinformatically. each of the discovered mutations was validated and the familial segregation was confirmed, using sanger sequencing. results: the 55 probands (32 males and 23 females) ranged in age from 2 weeks to 26 years with a mean of 4.4 years. of them, 20 patients are jewish (36%), 34 palestinians (62%) and 1 (2%) is from a greek ethnicity (cyprus). based on their clinical and immunologic phenotype at the time of initial evaluation, patients were assigned to one of seven pid groups: (i) humoral immunodeficiency 5 patients (9%); (ii) combined immunodeficiency (cid) 16 (29%); (iii) cid with syndromic features 10 (18%); (iv) scid 9 (16%); (v) congenital defects of phagocytes 6 (11%); (vi) immune dysregulation 7 (13%) and (vii) phenocopy of pid 2 (4%). we identified 67 mutated alleles, all in coding regions, that are highly likely to be causative in 36 of the 55 patients, achieving a 65% molecular diagnostic yield. among the 36 patients, the mode of inheritance in 28 patients (78%) is autosomal recessive and in 3 is compound heterozygote (8%). four patients (11%) harbor a non-inherited mutation on one allele, either de novo or somatic. the inheritance of the mutation in one patient (3%) is x-linked. the high rate of bi-allelic inheritance (93% of the alleles) is mainly due to the high frequency (65%) of consanguinity among the studied cohort. twenty eight mutated genes were identified in this study. of them, 6 were found to be novel in causing an inherited disease in man. interestingly, some genetic defects in known genes were found in patients with atypical phenotypes. in 23 patients (42% of the total number of patients and 64% of the wes diagnosed) the discovery of the genetic cause led to a change of therapy, towards a more targeted and personalized one. the revised treatments included bone marrow transplantation, conditioning protocols, reduced intensity of immune suppression, and prevention of unnecessary treatments due to their possible deleterious outcome. conclusions: except of being a useful tool for diagnosing and deciphering novel or atypical forms of pids, our wes study demonstrates an immediate and powerful impact on patient therapy in pids. early intervention with bone marrow transplant or gene therapy is critical and best when the infant in uninfected. newborn screening for scid and t-cell lymphopenia has been implemented in 45 states. the screening test is performed from dried blood spots collected at birth and involves pcr quantification of circular dna byproducts of t-cell receptor gene rearrangement, t-cell receptor excision circles (trec). trecs are generated during t-cell maturation in the thymus and are indicative of naïve t-cell output. assays and protocols to measure trecs vary by state and there is no standardized guideline at this time. washington state is unique in that it is one of few states where all newborns undergo two or more independent newborn screens, the first by which igrt treatment helps in these patients is unreported. here we present the first case of an mbl deficient patient with other complicating conditions (low igg and multiple cf polymorphisms, ciliary dyskinesia) who did not show improvement on igrt, thus refuting current literature. results: a 28-year-old caucasian male patient presented due to recalcitrant warts of the hands and feet. he had childhood otitis media status post tympanoplasty, mild molluscum contagiosum, and eczema. also, history of migraines with a negative brain mri. a trial of weekly pegylated interferon alpha 80mcg for his warts was discontinued due to significant neutropenia and depression. physical exam was notable for bulky skin colored warts on the lateral and dorsal fingers and dorsal hands including periungual skin. clusters of verrucae were noted on the feet. initial laboratory testing was notable for mild hypogammaglobulinemia with protective specific antibodies. lymphocyte subset analysis revealed a predominantly t cell lymphopenia with decreased naïve and memory cd4 and cd8 subsets, normal absolute numbers of b cells but with low memory subsets, and normal numbers and function of nk cells. whole exome sequencing ultimately revealed homozygous r169q mutations in the cecr1, a mutation previously described as causing ada2 deficiency. ada2 activity was about 40%. conclusions: ada2 deficiency is a relatively newly defined genetic defect, with a clinical phenotype that continues to evolve with newly diagnosed cases. our patient had not had evidence of vasculitis or stroke, but had recalcitrant warts with lymphopenia as his primary presentation. approach to therapy for those without vasculitis or significant cytopenias remains unknown. introduction/background: whole exome sequencing has added greatly to our library of primary immune deficiencies. however, interpretation of findings is not always straightforward. clinicians need to understand the limitations of this diagnostic tool and be familiar with the next steps in order to achieve a diagnosis. objectives: a 7 year old male presents for evaluation of bronchiectasis of bilateral lower lobes and poor growth. past medical history was significant for gerd with poor weight gain during infancy and childhood, oral thrush, recurrent respiratory infections and bilateral partial hearing loss. the bal showed abundant neutrophils and was positive for strep pneumoniae and haemophilus influenza. methods: serum immunoglobulins were normal with an elevated iga (369) and mildly elevated ige (361). tetanus igg was protective (0.4 iu/ml) but post-vaccine responses to the pneumococcal polysaccharide vaccine were poor with protective titers achieved to 3/14 serotypes. lymphocyte phenotyping was remarkable for a complete absence of b and nk cells. cd4:cd8 ratio was inverted (0.25) and the majority of the cd45+ t cells were memory phenotype. mitogen proliferation was essentially normal. due to the absence of b cells and despite normal total serum immunoglobulins, the patient was started on sc ig weekly and antibiotic prophylaxis with trimethoprim-sulfamethoxazole and fluconazole. clinically he demonstrated good weight gain and a reduction in cough and respiratory symptoms. results: whole exome sequencing was performed. a single base mutation in ada1 (c.320 t>c) was identified in one allele. this mutation (p.l107p) is known to be deleterious and when homozygous is associated with typical scid. however, this mutation was not present on the other allele. secondary analysis looking for large deletions and duplications failed to identify any abnormality in sequence in the normal allele. but the expression of this allele was markedly diminished causing us to suspect that its function might be impaired. functional testing was performed and demonstrated that there was no ada activity in the patients red blood cells, thus confirming a diagnosis of combined immune deficiency due to ada deficiency. conclusions: null mutations in ada result in an absence of ada function with profound cd3 cell lymphopenia and dysfunction. clinically affected infants have typical scid. hypomorphic mutations may lead to partial function of ada with more variable immune defects. interestingly, most patients with ada deficiency have neurologic complications, frequently hearing loss. we believe that most likely this patient has had some degree of spontaneous reversion of the mutation in the normal allele leading to a less severe phenotype. as reported previously in a case involving a mutation in il2rg chain, normal function was not restored and the patient remains with a combined immune deficiency. this case highlights an important limitation of whole exome sequencing and the need for confirming the impact of a genetic defect with a functional assay. introduction/background: the autosomal dominant hyper-ige-syndrome (ad-hies) is a rare primary immunodeficiency and multisystem disorder resulting from heterozygous loss-of-function mutations in the stat3 gene. ad-hies is characterized by skeletal dysplasia, recurrent pulmonary and skin infections (e.g. staphylococcal abscesses, eczematoid dermatitis) due to an increased susceptibility to bacteria and fungi. since many patients do rather well on anti-infective prophylaxis and supportive care, and early case reports suggested no benefit of allogeneic hematopoetic stem cell transplantation (hsct), ad-hies patients are rarely referred for hsct. the literature still contains only a handful of patients who underwent hsct. all these patients had experienced severe disease related complications before hsct and consequently the benefit of hsct was reported to be variable. objectives: currently, the general consensus is to only consider patients with severe pulmonary disease for hsct. it could however be postulated that transplanting patients earlier in their disease course and correcting their immunodeficiency before permanent organ damage due to infectious complications has occurred may extend life-expectancy and improve the quality of life of ad-hies patients. methods: we report on a 15 year old female ad-hies-patient who presented with bronchiectases following recurrent pneumonias, one pneumatocele, and normal pulmonary function tests. her past medical history also included recurrent skin infections, serious haemoptysis, pathological fractures and atopic eczema. considering that lung infections are the major life-limiting complication in ad-hies and are potentially positively influenced by hsct, our patient had enquired about a transplant. after extensive discussion of the pros and cons and obtaining full informed consent from her and her family, she underwent an elective hsct. she received bone marrow from an hla-matched sibling donor after a reduced-intensity conditioning consisting of alemtuzumab (2x0,2mg/kg), treosulfan (3x14g/ m2), fludarabine (5x30mg/m2) and thiotepa (2x5mg/kg), and graftversus-host disease (gvhd) prophylaxis with cyclosporine a (csa) and mycophenolate mofetil (mmf). results: the peri-transplant course was complicated by acute lower gastrointestinal tract bleeding and renal failure of unknown origin. continued kidney function impairment led to early tapering and discontinuation of csa on day +132 after hsct in the absence of any acute gvhd. neutrophils and platelets engrafted on days +15 and +26 respectively. she is currently on day +150, free of gvhd or infection, exhibiting full donor chimerism and recovered kidney function. in view of the preexisting pulmonary damage she currently remains on antibiotic prophylaxis and inhalation therapy. conclusions: this ad-hies patient who underwent hsct with few pre-hsct disease complications and relatively little permanent organ damage may add to our understanding of whether early hsct will lead to improvement of quality of life and possibly increased life-expectancy in ad-hies patients. it remains to be elucidated whether her rather uncommon peri-hsct complications are connected to her underlying disease. future research should be directed at identifying ad-hies patients at high risk of severe pulmonary complications early, so these could be referred for timely hsct. objectives: this is a case study of a patient who had refractory ibd symptoms and recurrent infections who was found to have xiap deficiency and mefv variant mutations. methods: a 37 year old male presented at age 16 with recalcitrant ibd unresponsive to multiple medications including steroids, mesalamine, azathioprine, infliximab, adalimumab, methotrexate, and vedolibumab. in addition, he developed severe infections from a combination of immune dysregulation and immunosuppressant medications. currently he is on ustekinumab but still has severe abdominal symptoms. patient does not have a history of hemophagocytic lymphohistiocytosis (hlh). family history was significant for ibd in both his mother and sister. he has had persistent lymphopenia which ranged between 210 to 838 cells/ mm3. t/b/nk panel showed decreased cd3 t cells (596 cells/mm3) with normal cd4, cd8, and cd4/cd8 ratios. b and nk cells were normal in quantity. t cell antigen/mitogen assays showed normal response to all mitogens (pha, pwm, con a) and most antigens (tetanus, candida, hsv, vzv, adv) but low response to cmv antigen. igg was elevated at 2,750, but iga, igm, and ige were normal. his ebv dna pcr is negative. results: exome sequencing revealed a novel xiap hemizygous variant at position c.693g>a (p.=?) of unknown significance and a mefv heterozygous variant. functional testing performed at medical college of wisconsin showed no expression of xiap on lymphocytes and a defect in nod2 pathway. given the xiap deficiency, bone marrow transplant was discussed as an option for refractory ibd and prevention of hlh. rituximab was also offered to decrease the possibility of hlh. currently the patient is in the decision making process of both treatment options. conclusions: genetic evaluation with clinical exome in early onset refractory ibd, family history of ibd, and recurrent infections demonstrated a novel mutation in the xiap gene with possible contribution of mefv variant as well. the absence of xiap protein expression and abnormal functional assay of the nod2 pathway confirm the pathogenicity of the mutation. identification of this genetic variant will help guide future therapeutic options and prognosis for this patient. introduction/background: x-linked agammaglobulinemia (xla) is a primary immunodeficiency disease caused by mutations of bruton tyrosine kinase (btk), which is essential in b cell maturation. xla is typically associated with bacterial infections of upper and lower respiratory systems, enteritis and increased risk for malignancies. objectives: to present the evolution and treatment of a complicated flexispira (helicobacter bilis) infection with delayed diagnosis in an xla patient. methods: clinical and laboratory features of a patient with xla evaluated at the national institutes of health. results: a 29-year-old male patient with xla presented for initial evaluation of indurated lower extremity and torso lesions. he was diagnosed with xla at age 2 years secondary to bacterial sepsis pneumonia and empyema. after starting ivig at age 2, he was well without significant infections except for recurrent otitis media. at 12 years of age, he developed right leg edema below the knee, which progressed to patchy skin thickening and discoloration. a tissue biopsy at 15 years of age revealed marked fibrous thickening of the subcutaneous septum with diffuse infiltrate of eosinophils with negative cultures for bacterial, fungal, and mycobacterial infections and thought to be consistent with eosinophilic fasciitis. mri demonstrated infiltration in the superficial and deep muscle compartments with fibrosis. symptoms persisted despite empirical treatment with iv antibiotics and steroids. at 16 years of age his left leg became involved with similar findings, while under treatment including multiple immunosuppressants (dapsone, methotrexate, tacrolimus, hydroxychloroquine, iv cyclophosphamide, remicade, cytoxan, and enbrel) targeting eosinophilic fasciitis. at age 28, he developed ulcerations over the left shin and ankle and was diagnosed with chronic multifocal osteomyelitis based on mri findings. physical exam was notable for bilateral leg swelling below the knees with woody appearance and induration, hyperpigmentation, and tenderness. indurated and nodular lesions without discoloration were noted above the waist line, on right forearm and above right nipple. skin biopsies of right lower extremity and right forearm were positive for numerous spirochetal-like organisms with warthin-starry stain. treatment was initiated with meropenem and gentamicin. gentamicin was discontinued due to vestibular ototoxicity six weeks later and doxycycline was added. initial response was followed by worsening of symptoms and intolerance to treatment, which led to the addition of tigecycline and azithromycin. with continued progression, seven months into initial evaluation, chloramphenicol and nitazoxanide were added to the regimen. due to the persistence of flexispira organisms on skin biopsy warthin-starry stains, fresh frozen plasma (ffp) was introduced four months later (11 months after initial evaluation) as an adjunctive treatment. cultures performed at the cdc were negative; however, pcr and sequencing resulted in identification of helicobacter bilis. 6 units of ffp was infused weekly for over three years, then reduced to every two weeks, with goal igm levels > 40mg/dl. subsequently, labs including cytopenias, inflammatory markers, and immunoglobulins improved as well as a negative warthin-starry stain. symptoms have improved with almost complete resolution of findings with only residual small areas of discoloration over both lower extremities. conclusions: xla immune deficiency is associated with flexispira (helicobacter bilis) infections, with typical appearance of discoloration and induration, which may evolve to osteomyelitis due to delayed treatment. although typically observed over the lower extremities, immunosuppressive treatment may lead to further expansion above the waist line. approach to therapy with weekly to bimonthly ffp infusions in addition to antibacterial treatment has proven to be beneficial in controlling the infection. higher igm levels resulting from the ffp may also provide antibacterial effects. introduction/background: x-linked severe combined immunodeficiency (scid) is a well described primary immunodeficiency associated with mutations in the common gamma chain. patients with x-linked scid classically present with profoundly low or absent t cells and nk cells with a variable number of b cells. the lymphocytes that are present typically have a proliferation index <10% control when stimulated with mitogens and antigens. patients must undergo corrective therapy with bone marrow transplant (bmt) or gene therapy to avoid the life-threatening infections that are associated with the nearly absent adaptive immune system. objectives: a 2-week-old boy presented to childrens national immunology clinic for initial evaluation of critical result on newborn screen. methods: targeted partial exome sequencing was performed on a 7-dayold patient who was picked up via trec assay on the maryland newborn screen. flow cytometry was completed at childrens national and proliferation studies completed at cincinnati childrens hospital diagnostic immunology lab. results: flow cytometry revealed markedly decreased lymphocytes with nearly absent cd8+ t cells and low cd4+ t cells with a r e l a t i v e i n c r e a s e i n c d 4 + c d 4 5 r o t c e l l s ( r a t i o cd45ra:cd45ro: 18%:12%). the b and nk cells were within the reference range for age. mitogen proliferation studies showed a mild decrease to pha and normal responses to pokeweed and cona (35587 cpm, 40924 cpm, and 82651 cpm, respectively). partial exome sequencing revealed a hemizygous nonsense substitution in il2rg (c.982c>t, p.r328) . maternal engraftment accounted for 3% of the t cells. the patient was started on prophylaxis with ivig, bactrim, and fluconazole with the plan to proceed with bone marrow transplant. as patient approached bmt maternal engraftment became absent in whole blood and repeat proliferation studies revealed normalization of the response to pha (stim index) with continued normal responses to cona and pokeweed. the patients flow cytometry values and ratios remained unchanged. patient completed a reduced intensity preparative regimen of busulfan, fludarabine and alemtuzumab prior to receiving his 8/8 matched unrelated donor bone marrow transplant. conclusions: it remains to be determined why initial proliferation studies showed >10% function with improvement over time in a patient with a well-described genetic mutation causing scid (187) director, division of intramural research, nih/niaid introduction/background: the advent of next-generation sequencing (ngs) has led to a proliferation of newly discovered genetic diseases and expanded phenotypes of known immunodeficiencies. availability of specific gene panels or whole exome sequencing (wes) with targeted analysis based on broad phenotypes, coupled with clinicians increasing awareness has led to higher utilization of ngs. published reports from high throughput sequencing labs indicate exome analysis identifies causative mutations in only 20-30% of probands. results: we have seen several patients who underwent high quality ngs in whom causative mutations were not identified. two separate families with multigenerational histories of leukemia, aplastic anemia, myelodysplastic syndrome, and cytopenias suggestive of gata2 deficiency had myeloid gene panel screens at commercial labs without causative mutations identified. targeted gata2 sequencing in the first family identified a novel change in gata2, c.1017g>t, p.l339l. cdna analysis demonstrated this synonymous variant resulted in aberrant splicing leading to a frameshift and premature termination. the wes bioinformatics pipeline failed to recognize the splice mutation. in the second family, pcr amplification spanning the 2 terminal exons revealed a shortened pcr product with a 426 base deletion fully encompassing the penultimate exon and leading to a 42 amino acid in-frame deletion. the deletion spanned all capture probes for the exon resulting in only the wild-type allele being captured and sequenced. additionally, capture kits targeting only coding regions of genes fail to capture deep intronic mutations such as those seen in gata2 (hsu, 2013) or in the 5 untranslated region of ikbkg, encoding nemo, (mooster, 2014; hsu, submitted) . lastly, even with good capture and sequencing, the presence of pseudogenes may confound downstream sequence alignment as seen in ncf1, encoding p47phox preventing recognition of disease causing mutations. conclusions: with ngs becoming more widely available as a clinical diagnostic tool, it is important to remember that wes results, unlike many laboratory tests, are not binary. inadequate bioinformatics pipelines, deletions, intronic or untranslated mutations, and pseudogenes can all mask the presence of causative mutations. targeted panel captures or analysis will miss novel genes. astute clinicians need to recognize the limitations of the current technology and pursue alternate assays when suspicions warrant. a cell based assay for the detection of autoantibodies to il-17 in human serum. matt phillips, phd 1 , vijaya knight, md, phd 2 1 senior scientist, national jewish health 2 director of immunology and complement adx labs, national jewish health introduction/background: patients with chronic candida infections are typically deficient in some aspect of il-17 signaling. one mechanism, which has recently come to light, is through the production of il-17 autoantibodies, particularly in patients who already suffer from specific autoimmune diseases. objectives: our objectives are to develop a diagnostic assay to accurately and easily detect il-17 autoantibodies in patient serum. methods: we developed a cell-based reporter assay using hek blue il-17 cells (invivogen) to detect the ability of patient serum to block il-17 receptor signaling. once stimulated with il-17, the cells secrete alkaline phosphatase (ap) into the surrounding media which is detected by invivogen hek blue media and an absorbance reader. addition of serum containing blocking antibodies inhibits secretion of ap. results: we were able to demonstrate, using a single patients serum with known il-17 autoantibodies, the inhibition of il-17 signaling in hek blue il-17 reporter cells. we further characterized the sensitivity of our assay with a commercially available anti-il-17 monoclonal and found it to be sensitive to between 1.4 x 10-6 m and 6.7 x 10-7 m. conclusions: loss of il-17 signaling can lead to problematic immune deficiencies including difficulty in clearing extracellular pathogens such as candida. some people who have an immune deficiency in the il-17 pathway may have developed autoantibodies to il-17 and thus have difficulty generating an appropriate immune response. we have developed a relatively low maintenance, cost effective, and simple test for detecting il-17 autoantibodies in human serum. alternations in repertoire of t and b cell subsets in patients with partial recombination activating gene (rag) deficiency with autoimmunity and history of viral infections introduction/background: patients with partial deficiency of recombination-activating genes 1 or 2 (rag1/2) can present with a wide spectrum of primary immunodeficiencies including combined immunodeficiency with granuloma and/or autoimmunity (cid-g/ai). prior case reports have highlighted alterations in b and t cell compartments; however comprehensive characterization of t and b cell receptor repertoires of lymphocyte subsets regarding diversity and autoreactivity has not been reported. objectives: defects in v(d)j recombination due to rag deficiency results in a skewed t and b cell repertoire that may be further modified by viral infections and promote inflammatory or autoimmune phenotype. methods: peripheral t and b cell compartments were sorted from two patients with combined immunodeficiency secondary to hypomorphic rag1 and rag2 mutations. b cells were stimulated with cd40l, cpg and il-21 to transition to antibody secreting cells (ascs), mimicking viral infection. repertoire analysis and single cell cloning of bcr heavy and light chain variable regions from sorted b cell populations has been performed. repertoire of t cell subsets (treg and follicular helper) were also examined results: we noted skewing towards proximal j usage in all b cell compartments (mature naïve, marginal zone, cd21-/low and memory) of two rag deficient patients compared to healthy controls. b cell clones with v4-34 v genes with low rate of somatic hypermutation expanded during b cell development. after in vitro stimulation mature naïve b cells from rag deficient patient were capable of transitioning to antibody secreting cells and enriched for polyreactivity to dsdna, insulin, lps and ifn cytokine (25 to 36%) compared to healthy control (5 to 14%). in connection to altered b cell compartments, restricted repertoire of regulatory t cells and an expanded and skewed follicular helper t subset were detected. conclusions: our data indicate that patients with partial rag deficiency have skewed t and b cell subsets that can further be altered towards antibody secretion and polyreactivity after stimulation such as viral infections. chief of the division of allergy and immunology, division of allergy immunology, the childrens hospital of philadelphia, philadelphia, pa usa introduction/background: the clinical features of 22q11.2 deletion syndrome include virtually every organ of the body. t cell lymphopenia, as a consequence of thymic hypoplasia, is the most commonly described immunologic feature and is most prominent in childhood. later in life, t cell exhaustion may be seen and secondary deficiencies of antibody function have been described in patients with 22q11.2 deletion syndrome. objectives: the role of deletion breakpoints in determining 22q11.2 deletion syndrome immunophenotype is unknown. in this study, we examined the effect of 22q11.2 deletions with and without tbx1 on lymphocyte counts. methods: lymphocyte counts were compared between 52 total 22q11.2 patients with tbx1-containing deletion (a-b, a-c, a-d deletions), and a total of 8 patients with tbx1-noncontaining deletion (b-d, c-d, d-e, d-f deletions). lymphocyte counts of patients with 22q11.2 deletions were compared to a set of 6 patients with a 22q11.2 duplication including the tbx1 locus. lymphocyte subset counts for each group were analyzed by t-test. results: cd3 counts were significantly lower in the tbx1-deleted cohort compared to the other two cohorts (mean 2169 cells/mm3 in tbx1 deleted cohort, 3709 cd3 cells/mm3 in the non-tbx1 deleted cohort, and 3657 in the duplication cohort, p<0.01 for all). similarly, cd4 counts were lower in the tbx1-deleted patients compared to the other two cohorts. there were no significant differences in cd8, cd19, and nk cell counts between the three cohorts. conclusions: these represent the first data to examine t cell counts in 22q11.2 deletion syndrome patients with different breakpoints. our data highlights an important role for tbx1 or other genes in the a-b region in regulating t cell production. paracoccidioidomycosis associated with a heterozygous stat4 mutation and impaired ifn-immunity introduction/background: mutations in genes affecting ifn-immunity have contributed to understand the essential role of this cytokine in the protection against intracellular bacteria and fungi. however, inborn errors in stat4, which controls il-12 responses, have not yet been reported. objectives: to determine the underlying genetic defect in a family with a history of paracoccidioidomycosis (pcm) disease. methods: genetic analysis was performed by whole-exome sequencing and sanger sequencing. stat4 phosphorylation and translocation from the cytosol to the nucleus, as well as ifnrelease by patient lymphocytes were assessed. the effect on stat4 function was evaluated by site-directed mutagenesis using a lymphoblastoid b cell line (b-lcl) and u3a cells. microbicidal activity of patient monocytes/macrophages was also analyzed. results: a heterozygous missense mutation, c.1952 a>t (p.e651v) in stat4 was identified in the index patient and her father. patients and fathers lymphocytes showed reduced stat4 phosphorylation and nuclear translocation as well as impaired ifn-production. in accordance, b-lcl and u3a cells carrying the stat4 mutant displayed reduced stat4 phosphorylation. patient's and father's pbmcs and macrophages (alone or in the presence of t cells) displayed impaired fungicidal activity compared with those from healthy controls that improved in the presence of recombinant human (rh) ifn-, but not rhil-12. conclusions: our data suggest autosomal dominant stat4 deficiency as a novel inborn error of il-12-dependent ifn-immunity associated with susceptibility to pcm disease. profound b cell lymphopenia in gof-stat1 that improves post ruxolitinib associate professor, university of south florida -johns hopkins all childrens hospital introduction/background: subjects with gain of function signal transducer and activator of transcription (gof-stat1) mutations have a variable clinical phenotype including combined immunodeficiency (cid). ruxolitinib, a janus kinase 1/2 inhibitor has been successful at treating immune dysregulation in subjects with gof-stat1. two subjects with profound b cell and/or t cell lymphopenia as a major manifestation are described, one of which was successfully treated with ruxolitinib. objectives: to discuss gof-stat1 mutations, their effect on the immune system, and the potential benefit of ruxolitinib in these subjects. methods: retrospective chart review was performed. results: subject 1 (c.494a>g) is a 21 year old male with a history of recurrent shingles, chronic mucocutaneous candidiasis (cmc), pneumocysitis jiroveccii pneumonia, varicella zoster meningitis, severe enteropathy, cerebral aneurysm and lymphoproliferation, and autoimmune hypothyroidism. he has profound lymphopenia predominantly affecting t and b cells (cd3+ 456 cells/ul, cd4+ 104 cells/ul, cd8+ 296 cells/ul, cd56+ 42 cells/ul, cd19+ 0 cells/ul). subject 2 (c. 1053g>t) is a 12 year old male with a history of severe cmc, recurrent pneumonia, enteropathy, and autoimmune thyroiditis. he had severe b cell lymphopenia (cd3+ 2168 cells/ul, cd4+ 1461 cells/ul, cd8+ 589 cells/ul, cd56+ 47 cells/ul, cd19+ 24 cells/ul). treatment with ruxolitinib 12.5mg bid led to clinical improvement of enteropathy and increased b cell counts in subject 2 (cd19+ 124 cells/ul). ruxolitinib has not yet been initiated in subject 1. both subjects were treated with anti-microbial prophylaxis and immunoglobulin supplementation. conclusions: combined immunodeficiency with variable degrees of b and t cell lymphopenia and hypogammaglobulinemia can be profound in subjects with gof-stat1 mutation. despite proper anti-microbial prophylaxis, this immunodeficiency can lead to severe infections. in addition to treating the autoimmune and immune dysregulatory features, treatment with ruxolitinib can improve the cid present and potentially reduce infectious susceptibility. igg4-related disease (igg4-rd), its common mimickers and response to anti-il5-(reslizumab) treatment rachel eisenberg, md 1 , arye rubinstein, md-phd 2 1 fellow in allergy and immunology, montefiore medical center 2 chief division of allergy and immunology, albert einstein college of medicine and montefiore hospital, bronx, ny, usa introduction/background: we describe a complicated case of igg4 related disease (igg4-rd) both in its presentation and novel treatment objectives: to review the common mimickers of igg4-related diseases which often lead to delayed diagnosis and treatment. to discuss novel therapeutic treatments for igg4-related disease results: a 70-year-old woman with a history of thyroid disease, sicca symptoms, lipodystrophy, relapsing parotid enlargement, asthma and erdheim chester syndrome initially presented with recurrent bacterial and fungal sinusitis despite multiple sinus surgeries. immunologic workup was notable for lymphopenia of 300/ml, cd4 count of 132 (677-1401 cells/ul normal range) and elevated igg4 of 511 (4.0-86.0 normal range). imaging was notable for nasal septal perforations and hypoplastic maxillary sinuses. there was high suspicion for igg4 disease however the patient was lost to follow up during which time she developed cachexia, eosinophilic pleural effusions (80% eosinophils), lung mass and a parotid mass with predominant t cell infiltrate misdiagnosed as follicular lymphoma. features consistent with igg4-rd included >50% ratio of igg4/igg and a predominant t cell infiltrate a biopsied lung mass showed igg4 plasma cell >50/hpf also consistent with igg4-rd. bone marrow biopsy was within normal limits. the patient was treated with rituximab, an effective treatment for igg4-rd. on treatment her igg4 levels normalized, however she developed recurrent large eosinophilic lung effusions requiring repeat drainage. fractional exhaled no (feno) was elevated to 86ppb. she was started on reslizumab at a dose of 5mg/ kg resulting in marked improvement in her respiratory status along with normalization of peripheral eosinophilia and reduction of feno to the normal level of 12ppb. conclusions: igg4-rd is a fibro-inflammatory condition which can affect any organ system and is diagnosed via tissue histology showing igg4 positive plasma cells and a typical morphologic pattern. this case outlines the common mimickers of igg4-rd often leading to a delayed diagnosis. before the final diagnosis of ig4-rd was made by us, the patient carried multiple diagnoses including: thyroid disease, recurrent parotid enlargement and seronegative sjogrens. these diagnoses in hindsight may have been mikuliczs syndrome, kuttners tumor and/or riedels thyroiditis which are common manifestations of igg4-rd. chronic sinusitis, atopic diseases, peripheral eosinophilia and destructive osseous lesions as noted in our patient are seen in up to 40% of patients with igg4-rd. destructive bony lesions and eosinophilia can mimic granulomatous polyangiitis, which was ruled out in our patient. her cachectic appearance and diagnosis of lipodystrophy can be explained by destruction of osseous tissue in the craniofacial skeleton which was later confirmed on imaging. lymphoid inflammatory infiltrates are commonly seen in igg4-rd and are can be misdiagnosed as a follicular lymphoma as in our case. a novelty in this case is the successful treatment with reslizumab targeted at the eosinophilic component of the disease. on reslizumab our patients asthma was for the first time controlled, pleuritis improved, fractional exhaled no (feno) normalized and her cachexia is improving. treatment with reslizumab should be considered in patients with igg4-rd who manifest with eosinophilic respiratory disease. introduction/background: ikbkb deficiency (c.1292dupg in exon 13) is a rare autosomal recessive form of severe combined immune deficiency (scid) originally described in canadian infants of northern cree descent. ikbkb scid is characterized by normal lymphocyte development, but impaired t-cell activation, along with innate immune defects. objectives: to report the clinical presentation, immunologic phenotype, and outcomes for patients with confirmed or suspected ikbkb scid due to this founder mutation. methods: we retrospectively reviewed hospital records dating back to 1973 of patients with confirmed homozygous ikbkb mutations, as well as patients suspected to be affected due to their clinical presentation and family relations to molecularly confirmed cases. results: fifteen patients were included. they presented early in life (average age 2 months) with invasive and disseminated viral, bacterial, mycobacterial, and fungal infections. patients had concurrent and multiple infectious organisms, with a notable predilection for candida, gram negative organisms, and mycobacteria. four patients in our cohort received bcg vaccination at birth, resulting in fatal disseminated m. bovis infection in all, and 2 additional patients succumbed to atypical mycobacterial infection following hematopoietic stem cell transplant (hsct). one newborn was identified in 2016 through new initiatives for targeted newborn screening for the mutation. immunologic features at presentation included normal to elevated lymphocyte counts with normal to elevated cd3, cd4, and cd8 t cells. when tested, response to pha varied from absent (1/10), to low (4/10), to normal (5/10). most patients had hypogammaglobulinemia, most often of the igg isotype (10/14). six had assessment of trec levels, and all had values above thresholds for screening programs. eight patients died before they could undergo hsct, and 6 received transplants in the setting of ongoing severe, lifethreatening infections. only 1 patient underwent hsct prior to the onset of infection. in our cohort, there are only 2 long-term survivors. conclusions: ikbkb deficiency is a severe form of scid with early onset of invasive and disseminated multi-organism infection. the immunologic phenotype is characterized by normal to elevated lymphocyte numbers which do not meet pidtc criteria for scid, variable (and sometimes normal) mitogen response, normal trec levels, and low igg levels. the disease is universally fatal without hsct, however, conclusions regarding efficacy and long-term outcomes of hsct are uncertain given the small sample size. immune -dysregulation mimicking systemic lupus erythematosus in a patient with lysinuric protein intolerance introduction/background: lysinuric protein intolerance (lpi) is an inherited aminoaciduria caused by defective amino acid transport in epithelial cells of the intestine and kidney due to bi-allelic, pathogenic variants in slc7a7. the clinical phenotype of lpi includes failure to thrive and multi-system disease including hematologic, neurologic, pulmonary and renal manifestations. individual presentations are extremely variable, often leading to misdiagnosis or delayed diagnosis. here we describe a patient that presented with suspected immunodeficiency in the setting of early-onset systemic lupus erythematosus (sle), including renal involvement, who was subsequently diagnosed with lpi post-mortem. objectives: describe a clinical a patient with lysinuric protein intolerance that presented as early-onset systemic lupus erythematosus (sle), including renal involvement and primary immunodeficiency. methods: after informed consent was obtained, dna samples were obtained from the proband and his parents. trio whole exome sequencing was performed to identify a cause of early onset autoinflammation resembling systemic lupus erythematosus. results: the male proband had a history of failure to thrive starting at 12 months of age, recurrent bacterial otitis media, and one episode of severe bacterial pneumonia requiring hospitalization. he presented at 30 months of age with multifocal pneumonia, anemia (hgb 8.2 mg/dl) and mild thrombocytopenia. initial laboratory studies revealed low albumin (2.8 mg/dl), elevated ldh (718), and mild hepatomegaly. renal and liver function testing was initially normal. immunologic evaluation for suspected primary immune deficiency showed normal immunoglobulin titers, low c3 (60) and low c4 (6.3). lymphocyte phenotyping revealed low b cell counts (0.6% of total lymphocytes) with t cells and nk cells within the normal range. despite antibiotic therapy, the patient worsened, developing fevers, a generalized erythematous rash, edema and nephrotic syndrome with oliguria. renal biopsy uncovered glomeruli with accentuated, global thickening and diffuse, peripheral capillary loops, as well as focal spiculated defects, there was endothelial swelling and other signs of acute damage including epithelial flattening, adluminal irregularity and extensive intraluminal proteinaceous detritus. endocapillary proliferative lesions, extracapillary crecents or tubular atrophy was not observed. immunofluorescence studies were positive for c3, igg and c1q granular deposits, mainly at the mesangium, interpreted as lupus nephropathy. endothelial swelling and massive, subepithelial electron-dense deposits with spike formation from the basement membrane were noted on electron microscopy, mimicking a stage ii membranous pattern of injury. autoantibodies included ana (1:320), anti-dsdna, smith, ssa and rnp were positive in agreement with the diagnosis of sle. immunosuppressive therapy with high dose iv corticosteroids and cyclophosphamide was initiated. despite this, the patient developed pancytopenia, elevated ferritin levels, increased triglycerides, and low fibrinogen. bone marrow biopsy displayed erythrocyte phagocytosis by macrophages, confirming a diagnosis of hemophagocytic lymphohistiocytosis (hlh). the patient subsequently died despite aggressive immunosuppression with high dose methylprednisolone and high dose iv immunoglobulin and dialysis. samples were collected from the deceased patient and his parents for research whole exome sequencing. trio analysis identified compound heterozygous missense variants in slc7a7. ammonia levels were not evaluated during the patients hospitalization. conclusions: lysinuric protein intolerance is a severe metabolic disorder that can present with protean systemic features including primary i m m u n o d e f i c i e n c y. i m p a i r e d l y m p h o c y t e f u n c t i o n , hypocomplementemia, immune-mediated glomerulonephritis, autoantibodies, and hlh are known complications of lpi. exactly how ineffective amino acid transport triggers these systemic inflammatory features is not yet understood. lpi should be considered in the differential diagnosis of early-onset sle, particularly in the absence of response to immunosuppressive therapy. director, national institute of immunohaematology introduction/background: chronic granulomatous disease (cgd) is a primary immunodeciency disorder with recurrent pyogenic infections and granulomatous inflammation resulting from loss of phagocyte superoxide production. mutations in any one of the five structural genes of the nicotinamide adenine dinucleotide phosphate (nadph) oxidase complex viz. cybb and cyba encoding for membrane bound gp91phox and p22phox; and ncf1, ncf2, and ncf4 encoding for cytosolic components p47phox, p67phox, and p40phox respectively, have been found to cause cgd. the relative incidence of these gene defects varies significantly depending on the ethnic background of the population. identification of molecular defect is important for patient management as well as for prenatal diagnosis in the affected families. the present study was aimed at studying the pattern of underlying genetic defects in a cohort of indian patients affected with cgd. objectives 1.to identify the underlying genetic defect in patients with chronic granulomatous disease in india 2. clinical,immunological and molecular characterisation 3. to utilise this information for genetic counselling and prenatal diagnosis of the affected families methods: eighty-seven (n=87) patients with abnormal nbt and dhr were included in this study. in case of male patients, mothers were first screened for carrier status to rule out x-linked cgd (xl-cgd). those patients where mother is not showing mosaic pattern were suspected for autosomal recessive cgd (ar-cgd) and were screened for the ratio of ncf1 gene to pseudo ncf1 gene by genescan analysis. additionally, evaluation of nadph oxidase components expression by flow cytometry also helped us to determine the underlined genetic defect and it is validated by dna sequencing of respective genes. results: eighty-seven patients were molecularly characterized to identify disease causing mutation which includes: 12 novel mutations in cybb, 1 in cyba, 2 in ncf2 gene in our cohort. 29.8% (n=26) of the patients belonged to xl-cgd. 58.6% (n=51) of the patients are suspected to have ncf1 gene defect among which; homozygous delgt mutation was identified in 39 patients. 6.8% and 4.5% patients showed abnormal p22phox and p67phox expression suggesting defect in cyba gene and ncf2 gene respectively. spectrum of mutations involve: 41% of delgt mutations, 15% nonsense, 14% missense, 10% deletion, 2% insertion, 14% other than homozygous delgt mutations. male to female ratio is 1.87:1. consanguinity is noted in 30% of the patients. conclusions: despite the male predominance ar-cgd is more common (70%) as compare to xl-cgd (30%) in this cohort of indian patients, which is distinct from the western data. 17% are the novel mutations suggesting, a wide heterogeneity in the nature of mutations in indian cgd patients. flow cytometric evaluation of nadph oxidase component is used as a secondary screening test to identify cgd sub-group. molecular characterisation of cgd genes was not only used in the confirmation of diagnosis but also in genetic counselling and pre-natal diagnosis in affected families. novel nlrc4 gain-of-function mutation presenting with neonatal enterocolitis and autoinflammation, with positive clinical response to rapamycin and anakinra. senior investigator, viral immunology section, national institute of neurological disorders and stroke 8 senior investigator, translational neuroradiology section, national institute of neurological disorders and stroke 9 staff clinician, neuroimmunology clinic, national institute of neurological disorders and stroke 1 0 staff clinician, laboratory of clinical immunology and microbiolology, niaid, nih introduction/background: cytotoxic t-lymphocyte antigen-4 (ctla-4) is an essential negative regulator of the immune response and its function is critical for immune homeostasis. uni-allelic mutation in the ctla4 gene leading to reduced function or expression of ctla-4, termed ctla-4 haploinsufficiency, can lead to systemic immune dysregulation with wide spread clinical disease, but with variable clinical penetrance. the neurological manifestations of ctla-4 haploinsufficiency are not known. objectives: to perform detailed phenotyping of the neurological manifestations of ctla-4 haploinsufficiency. methods: a retrospective review and prospective collection of clinical, imaging, cerebral spinal fluid, and pathological specimens was performed in a cohort of genetically confirmed patients (n=50) with ctla4 mutations who are followed at the national institutes of health. neurological symptoms and exams were collected on patient visits and from historical records. the data collected included 289 brain mris and 53 spinal cord mris that were visually inspected for evidence of inflammation. cerebral spinal fluid values were obtained from 14 patients including flow cytometry in 10 patients. pathological tissue from brain biopsies of inflammatory lesions was examined from 10 patients. results: central nervous system (cns) inflammation was found in 14/50 (28%) of the cohort. common clinical manifestations from the 14 patients with cns inflammation were headaches 13/14 and seizure 8/14. focal deficits were rare. mri findings included contrast enhancing neuroinflammatory lesions in the brain 14/14, brainstem/cerebellum, and spinal cord 8/12. figure a -c show representative inflammatory lesions. lesions were multifocal in 13/14 patients and 12/14 had recurrent inflammatory lesions on longitudinal follow up. lesions were, at times, extremely large, 5/14 with a lesion > 25 cm^3. leptomeningeal enhancement (lme) was seen in 10/13 patients and clearly preceded intraparenchymal lesion development in 3 patients. figure d shows a site of lme (green chevron) that develops into an intraparenchymal lesion (yellow chevron), mris are separated by 11 days. spinal fluid analysis showed a lymphocytic pleocytosis (mean 32 cells/mm^3) with the presence of oligoclonal bands in 6 patients. pathological features included a mixed cellular infiltrate, predominantly lymphocytes or plasma cells, with little evidence of demyelination or necrosis (figure e). conclusions: the neurological manifestations of ctla-4 haploinsufficiency include recurrent and, at times, severe neuroinflammation. however, even large lesions and lesions in eloquent anatomical locations had little to no focal clinical defects resulting in a striking clinical-radiological dissociation. future studies into the mechanisms of cns-related disease may reveal important information related to peripheral and central immune system functioning. conditioning with anti-cd45 immunotoxin in a mouse model of hypomorphic rag1 deficiency allows complete reconstitution of the immune system with lack of toxicity enrica calzoni, md 1 , cristina corsino, technician 2 , marita bosticardo, phd 3 , yasuhiro yamazaki, md phd 1 , hsin-hui yu, md phd 4 , lisa ott de bruin, md 5 , john manis, md 6 , rahul palchaudhuri, phd 7 , david scadden, md 8 , luigi d. notarangelo, md 9 treated mice and 80% in cd45-sap/200 treated mice. at sacrifice, in the bm we observed strong selective advantage for donor b cells at all developmental stages, but in particular in the most mature subsets, in both cd45-sap and cd45-sap/200 treated mice, rescuing the block in development at pre-b cell stage found in untreated f971l mice. donor engraftment in bm hsc reached levels around 70% and 80% in cd45-sap and cd45-sap/200 treated mice, respectively. donor chimerism in t and b cells in the spleen was also higher than 90% in both cd45-sap and cd45-sap/200 groups. in the thymus, full donor chimerism was achieved in both cd45-sap and cd45-sap/200 treated mice, starting at the dn4 stage and persisting at dp, sp4 and sp8. importantly, in both treatment groups, t cell development was corrected both in terms of subset distribution and absolute numbers to levels comparable to those of wt mice. finally, the thymic epithelial cell compartment was also fully reconstituted, with a normal number, distribution and maturation of both cortical and medullary thymic epithelial cells. conclusions: in conclusion, we show here that conditioning with cd45-sap immunotoxin, alone or in combination with 200rads tbi, is safe and leads to full reconstitution of the immune system in rag1 hypomorphic mice, suggesting that this conditioning regimen should be considered for testing in clinical setting. introduction/background: foxp3 is a key transcription factor for the maintenance of immune tolerance. foxp3 mutations result in dysfunction of foxp3+ regulatory t cells (tregs) causing immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome, a severe early onset autoimmune disease, which can be fatal if not promptly diagnosed and treated. our recent international study analyzing the longterm outcome in 96 i.e. patients of the two currently available treatments, pharmacological immune suppression and allogeneic hematopoietic stem cell (hsc) transplantation, showed poor long-term disease-free survival or overall survival limitations, respectively (barzaghi f. et al, jaci, 2017) . ipex syndrome is a good candidate for gene therapy as it has been demonstrated that reconstitution of wild-type treg cells can control the disease. however, foxp3 expression is highly regulated, and its safe and physiological expression in treg and teffector (teff) cells is challenging. lentiviral-mediated (lv) foxp3 gene transfer successfully converts ipex patients-derived cd4+ t cells into treg-like cells (cd4lv-foxp3 t cells) with stable suppressive capacity (passerini l. et al, sci transl med, 2013) . these ex vivo converted tregs are ideal as a short term cell-based therapy for ipex patients, but this approach does not reestablish regulated foxp3 expression in teff cells, that also likely contribute to the ipex pathology. thus, we are further characterizing cd4lv-foxp3 t cells and, at the same time, developing gene editing strategies for ipex, whereby autologous t cells or hscs are genetically modified or corrected, respectively, and reinfused into the patients. objectives: to provide more effective treatments for ipex patients, we are i) optimizing lv-foxp3 gene transfer in t cells to be suitable for clinical use, and ii) establishing a novel foxp3 gene editing in hscs and testing both approaches in preclinical models. methods: lv-foxp3 gene transfer can be obtained in cd4+ t cells activated polyclonally or in an antigen-specific manner. the vector construct is bidirectional, foxp3 expression is under the ef1a-promoter and the truncated form of ngfr, used as marker gene, is under cmv promoter. foxp3 gene editing is performed using a combination of crispr/cas9, a chemically modified sgrna targeting foxp3, and an aav6 packaged homologous donor dna template and the efficacy and safety of the resulting construct is tested in different cell types in vitro and in humanized mice. results: we demonstrate that cd4lv-foxp3 t cells can successfully be generated specific to different antigens. this result opens to new potential clinical benefit of cd4lv-foxp3 t cells with more safe and specific regulatory effect than polyclonal cd4lv-foxp3 t cells. we are currently adapting the protocol to optimal in vitro production for clinical use and assessing dose, survival and efficacy of the cd4lv-foxp3 t cells using different in vivo models. due to the wide distribution of identified mutations throughout the foxp3 gene, we have designed a gene editing strategy that uses homology directed repair to insert the coding sequence of the foxp3 gene at the start codon of the endogenous mutated foxp3 gene. this strategy permits regulated expression of the inserted wild-type, functional foxp3 protein in patient cells independent of the location of the downstream mutation. using this site-specific gene knock-in, we find that the system effectively targets expression of foxp3 in different cell types, namely tregs, teff cells and primary human cord blood-or bone marrow-derived hscs. gene editing of normal donor and ipex tregs and teff cells allowed us to test for regulated gene expression and for establishment of normal treg suppressor function and t cell proliferation upon activation. additionally, preliminary results demonstrate that gene edited hscs can be transplanted into nsg mice for long-term reconstitution. conclusions: our results show the feasibility of different gene therapy approaches for ipex syndrome. in addition, they suggest that cd4lv-foxp3 t cells, either polyclonal or antigen-specific, could be applied not only in ipex but also in immune mediated diseases of different origins. the results from the foxp3 gene editing support the use of crispr/ cas9 to treat ipex syndrome patients with autologous edited hscs. this gene editing approach may also be applied to treat other pediatric monogenic blood and immune disorders. human pi3kgamma deficiency with humoral defects and lymphocytic infiltration of barrier tissues andrew takeda, bs 1 , william comrie, phd 2 , yu zhang, phd 3 , paul tyler, bs 4 , koneti rao, md 5 , carrie l. lucas, phd 6 4 graduate student, yale university 5 clinician, niaid, nih 6 assistant professor of immunobiology, yale university introduction/background: the phosphatidylinositol 3-kinase (pi3k) signaling pathways play a key role in transducing signals from a diverse array of stimuli by producing the pip3 second messenger. class ib pi3k is primarily activated by g protein-coupled receptors (gpcrs), and this class is comprised of the p110gamma catalytic subunit in complex with the p84 or p101 regulatory subunit. in contrast to the class ia pi3k subunits, inherited mutations in the genes encoding the class ib subunits have not been described. objectives: given the leukocyte-restricted expression pattern of p110gamma, we hypothesized that mutations affecting this kinase may be found in cohorts of patients with rare immunodeficiency disorders. our objective was to identify such mutations and determine molecular, biochemical, and cellular derangements in patients with mutated p110gamma. methods: we used whole-exome sequencing of families to identify inherited gene mutations and determined the mechanistic basis of disease using biochemical assays to assess effects on protein function and cellbased assays to define functional defects with disease relevance. results: we identified a patient (here called a.1) harboring compound heterozygous mutations in pik3cg, the gene encoding p110gamma, who presented in early life with autoimmune cytopenias and eczema and, at the age of 9 years, developed cryptogenic organizing pneumonia and prominent t cell infiltration of the lungs. she also has a history of skin infections, lymphadenopathy/splenomegaly, eosinophilia, defective antibody production, and more recently, lymphocytic colitis. she inherited a frameshift pik3cg mutation from her mother and a missense mutation resulting in an r1021p amino acid substitution from her father. expression of p110gamma protein was lost, and stability of its p101 binding partner was reduced. despite defective t cell signaling responses to chemokines (i.e., gpcr stimulation), chemotaxis of patient t cell blasts in vitro was normal. intriguingly, the frequency of peripheral blood treg cells was low in patient a.1, and her cd4 t cells more frequently expressed the tissue-homing cxcr3 chemokine receptor. consistently, serum levels of cxcr3 ligands were elevated in patient a.1. moreover, we found augmented inflammatory cytokine production from m1-polarized macrophages differentiated from patient a.1 monocytes or from thp1 cells treated with p110gamma inhibitor or stably expressing pik3cg shrna. conclusions: we report the first human with loss of pi3kgamma activity and present her clinical presentation with notable t cell infiltration of barrier tissues. based on our analyses, we propose that loss of p110gamma activity in humans causes t cell-intrinsic effects of reduced tregs and increased tissue-homing propensity and the t cell-extrinsic effect of augmented inflammatory responses in macrophages. together, these consequences of p110gamma deficiency drive aberrant accumulation of t cells in lung and gut. introduction/background: pulmonary disease is a frequent complication across many primary immunodeficiencies (pidds), however its impact on the quality of life (qol) in pidds is not well characterized. objectives: to ascertain the types of infectious and non-infectious pulmonary complications occurring in pidds and to determine how these complications affect qol. methods: we analyzed the pulmonary complications, disability descriptions, and clinical status of 3610 subjects with pidds in the usidnet registry using descriptive statistics. karnofsky or lansky performance indices (n=1267) and promis29 qol data (n=120) were also analyzed. the t-test/mann-whitney test and chi square test were utilized to compare continuous and categorical variables, respectively. results: infectious pulmonary disease was reported in a majority of subjects (52.2%), most commonly pneumonia (42.3%) and bronchitis (17.9%). non-infectious pulmonary disease was reported in 30.8% of all subjects, most commonly asthma/reactive airway disease (21.7%), bronchiectasis (7.0%) and interstitial lung disease (4.2%). pulmonary insufficiency was listed as a cause of disability in 4.3% of all subjects with pidds, with highest rates of this disability in subjects with immune dysregulation (15.6%). lower karnofsky/lansky performance scores were observed in subjects with pneumonia, lung abscess, bronchiectasis, interstitial lung disease, and emphysema/copd as compared to without these disorders (p<0.001). promis29 qol metrics were largely similar among subjects with and without pulmonary disease, although physical function scores were significantly worse in those with copd/emphysema (mean= 37.5 +/-5.1) as compared to without (mean= 43.6 +/-8.4, p =0.007). promis29 physical function scores were also worse in subjects with non-infectious pulmonary disease (mean =40.3 +/-6.81) compared to those with infectious pulmonary disease only (mean =45.2 +/-8.53, p=0.046). a significantly greater percentage of patients with a history of copd/emphysema (24.0% vs. 8.95%) or interstitial lung disease (16.7% vs. 8.9%) were deceased as compared to those without a history of these disorders (p<0.0001). conclusions: both infectious and non-infectious pulmonary disorders cause significant morbidity in pidds and are associated with higher mortality in this population. infectious and non-infectious pulmonary complications were often associated with worse karnofsky/lansky scores while there was limited impact on promis29 qol measures. latin-american consensus on the management of patients with severe combined immunodeficiency, part 1: supportive measures during the time from diagnosis to definitive treatment. juan carlos bustamante ogando 1 , armando partida-gaytán 2 , francisco espinosa rosales 3 , lasid "consensus on scid" study group 4 1 pediatric allergy and clinical immunology specialist, clinical immunologist and researcher at primary immunodeficiency research unit, national institute of pediatrics 2 pediatric allergy and clinical immunology specialist, researcher at primary immunodeficiency research unit, national institute of pediatrics 3 pediatric allergy and clinical immunology specialist, president, fundación mexicana para niñas y niños con inmunodeficiencias primarias (fumeni) result in a majority of patients with late diagnosis, more comorbidities and reduced access to curative treatments. the interventions during such period are vital to keeping optimum health status to improve the probability of success of curative therapies. many interventions are not supported by clinical trials, are based mainly on clinical experience, and there are no clinical guidelines to standardize such treatments. objectives: to generate a consensus on the supportive care of patients with scid, from the diagnosis until a curative treatment is given, under a latin-american perspective taking into account particular challenges for our region. methods: in a first step, we gathered available information about scid diagnostic and therapeutic guidelines from two sources: a) literature search and b) personal communications with pid experts from europe and usa. next, we developed an expert consensus through a modified delphi technique (electronic and anonymous). we used google® forms® to gather the information and microsoft office excel® for the analysis of agreement through kappa coefficient and rounds concordance through repeated measures analysis of variance (anova). results: we gathered an expert panel of 34 subjects from 6 latin-american countries (argentina, brazil, chile, costa rica, mexico, and peru) including the primary centers caring for scid patients. we generated a document with 123 agreed diagnostic and therapeutic interventions grouped in 8 topic-domains (i.e. protective and isolation methods to decrease the risk of infections, antimicrobial prophylaxis, immunoglobulin treatment, immunizations, nutritional aspects, antimicrobial treatment, blood derivatives use, routine laboratory workup, imaging and other studies, conventional multidisciplinary approach). we also included 38 nonagreed interventions, but where relevant arguments are shared, to allow for particular clinical scenario decisions. conclusions: this is the first document of its type, and it intends to standardize clinical care of latin-american patients with scid, reduce disease burden and ultimately improve health outcomes. we see this effort as a starting point for the continuous improvement of our professional care to such patients and is intended to help as a tool not only for immunologists but for primary care physicians and other specialists involved in scid patient's care. this work will hopefully be published during 2018 as a lasid collaborative work, and it will help as a guide for clinicians caring for scid patients not only in latin america but in other world regions. also in the future, this consensus may be improved by collaboration from immunologists worldwide. no significant difference in hospitalizations one year before vs. year after treatment for prophylactic antibiotics (p=0.85) or igrt (p=0.07). baseline igg was higher in prophylactic antibiotics vs. igrt (770.6 vs. 914.4 mg/dl, p=0.03) . sex, severity of sad, igg subclasses deficiency, and lymphocyte counts were not significantly different between treatment groups. conclusions: prophylactic antibiotics are not inferior to igrt in preventing infections in some sad patients. while, clearly some patients with sad will need igrt, our date indicate that larger prospective studies are needed to identify patients who will benefit most from igrt vs prophylactic antibiotics alone. richard and barbara schiffrin presidents distinguished professor of microbiology and director, institute for immunology, university of pennsylvania introduction/background: t cell thymic development is dependent on signals received via the pre-tcr complex and we here report the first case of pre-tcr alpha (ptcra) autosomal recessive t cell immunodeficiency in an infant with a positive scid newborn screen (nbs). objectives: we sought to uncover the mechanistic links between ptcra mutations and immune dysfunction. methods: the patient was tracked clinically, with serial clinical immunophenotyping and t cell function testing. in addition, we performed deep immunophenotyping with mass cytometry and single cell rna sequencing to delineate the molecular circuitry underlying her immune phenotype. results: the patient presented with t cell lymphopenia and impaired response to mitogen stimulation. hsct was considered, but she did not meet clinical criteria and remained healthy, so she was watched closely on prophylaxis while awaiting genetic testing. response to serial mitogen stimulation remained between~20-100%, response to serial cd3/cd28 activation was normal and tcrv spectratyping was normal. whole exome sequencing revealed two mutations in ptcra. no prior human cases of ptcra deficiency have been published, but a mouse model bears a striking resemblance to this case (fehling et al, nature, 1995) , with elevated t cells and decreased t cells. her mitogen stimulation responses became persistently normal around 2 years of age with stable t cell lymphopenia, elevated t cells and normal switched memory b cells. anti-fungal prophylaxis was halted, and she remained on atovaquone alone with persistent t lymphopenia. she was able to mount an antibody response to rabies vaccine at 2.5 years and was weaned off scig replacement and is planned to initiate vaccination. deep immunoprofiling with mass cytometry (cytof) demonstrated a unique immunophenotype. single cell rna sequencing confirmed normal cd4 and cd8 tcr clonotypic diversity but increased clonotype diversity in t cells and increased and transcript levels. in addition, cd4 naïve, cd4 memory and cd8 naive t cells demonstrated both increased numbers of expressed genes and transcriptomic diversity, with altered cytoskeletal and tcr proximal signaling pathways across t cell subsets versus control. this may reflect a peripheral role for ptcra, a durable imprint of thymic signaling events mediated by ptcra, evidence of homeostatic proliferation or a combination of the above. conclusions: scid nbs led to identification of homozygous variants in ptcra causing a novel t cell immunodeficiency characterized by t cell lymphopenia, altered proximal tcr and cytoskeletal signaling and increased number of altered t cells. we will continue to pursue the mechanism of these mutations by developing ipsc and studying their t cell differentiation capacity in vitro, as well as further defining her immunometabolic phenotype. rag1 hypomorphic mouse mutants show partial preservation of thymocyte development but peculiar abnormalities of thymic epithelial cell phenotype introduction/background: the recombination-activating gene (rag) 1 and rag2 proteins are essential for v(d)j recombination. in the absence of these proteins, the development of b and t cells is blocked at early progenitor stages, resulting in severe combined immunodeficiency (scid). hypomorphic mutations in rag1, allowing residual activity, result in delayed-onset combined immunodeficiency with residual development of t and b lymphocytes, associated with autoimmunity and/or granulomas (cid-g/ai). objectives: to study in details the effect of rag1 hypomorphic mutations at the early stages of t cell development, we have generated 3 mouse models carrying mutations described in patients with cid-g/ai (r972q, r972w, f971l) (niaid animal protocol: lcim 6e). methods: we performed an extensive evaluation of the thymic phenotype in the 3 mouse models. results: the number of total thymocytes was found to be drastically reduced in all three models. however, two of these mouse models (r972q and f971l) retained a significant level of rag1 activity, and resulted in the development of mature t cells in the thymus, while the mouse model carrying the r972w mutation had minimal rag1 activity and presented a phenotype more similar to that of complete rag1 knockout mice. in r972w mice, almost all thymocytes were blocked at the double negative (dn)3 stage and there were virtually no mature t cells, as found in rag1 ko mice. on the other hand, r972q and f971l mice presented double positive (dp) and single positive (sp)4 and sp8 cells. the cross talk between t cells and thymic epithelial cells (tec) in the thymus is fundamental for the development and maturation of both types of cells. in rag1-/-mice, and consequently in the absence of mature t cells, tecs cannot complete their maturation, and the mtec subset is virtually absent. these results were also observed in the r972w mouse model. instead, in r972q and f971l mice, the residual rag1 gene activity allows development of a reduced number of mature t cells. although the number of tecs was markedly reduced in r972q and f971l mice, ctecs and mtecs were both present, but with an excess of ctecs. furthermore, mtecs were predominantly mhc-iihigh (mtechi), and only a minority of mtecs were mhc-iilow (mteclo) cells, the opposite of what found in adult wt mice. finally, mtechi cells from rag1 mutant mice were found to express aire to levels and frequencies comparable to those of wild-type (wt) mice. conclusions: our results show that tec in mouse models carrying rag1 hypomorphic mutations are affected both in terms of absolute numbers and in terms of subset distribution and maturation state. to further investigate the functional consequences of impaired cross-talk between thymocytes and tecs in rag1 mutant mice, we have performed rnaseq in sp4 and tecs sorted from r972q, f971l and wt mice. analysis of the gene expression profile in tec may thus provide novel insights in the mechanisms that govern normal and pathologic thymic t cell development. vedolizumab for autoimmune enteropathy in primary immunodeficiency: a case series of outcomes introduction/background: gastrointestinal complications are common in patients with primary immunodeficiency. infections are the leading cause, but autoimmune enteropathies including inflammatory bowel disease (ibd)-like colitis, sprue-like enteropathy, and nodular lymphoid hyperplasia (nlh) have been recognized in a subset of these patients. to date, there is no established treatment for these noninfectious disorders. vedolizumab is a humanized monoclonal antibody that binds to the alpha-4 beta-7 integrin, inhibiting the migration of memory tlymphocytes across the endothelium into inflamed gastrointestinal parenchymal tissue. it is fda approved as first-line therapy for inflammatory bowel disease. the safety and efficacy of treating autoimmune enteropathy with vedolizumab in patients with concurrent primary immunodeficiency (pid) has not previously been reviewed. objectives: to review the outcomes of a series of patients with hypogammaglobulinemia and autoimmune enteropathy following vedolizumab therapy methods: 7 patients (3 male, 4 female) at mount sinai with enteric biopsies demonstrating inflammatory enteropathy with t cell infiltrates have been treated with vedolizumab. results: five of the seven patients completed induction therapy. one patient was recently started on therapy. therapy was aborted in one patient who developed acute hepatitis during induction. another developed severe cytomegalovirus enteropathy, prompting discontinuation. two patients discontinued therapy due to response failure. at present, two patients remain on therapy at 12 months with symptomatic improvement. conclusions: vedolizumab was effective in 2 cases, but had no benefit or deleterious side effects in 4 subjects. its effectiveness in another patient is presently under investigation. introduction/background: ataxia telangiectasia (at) is an immunodeficiency most often associated with t cell abnormalities and abnormalities in serum immunoglobulin levels, primarily iga. there is a subset of patients with a hyper-igm phenotype, some with cutaneous granulomas, which may reflect a distinct clinical phenotype. a 5 yearold female presented for evaluation of concern for immunodeficiency because of frequent illnesses, presumed to be viral. she was found to have an ataxic gait, some speech delay, mild ocular and ear pinna telangiectasia, and an ulcerative rash on the left upper and right lower extremity. initial blood work showed elevated -fetoprotein levels (50 ng/ml), elevated serum igm (719 mg/dl), low igg (<75 mg/dl), and iga (0.9 mg/dl). objectives: to determine if the atm mutations in this patient are associated with perturbations in the frequencies, distributions and functions of b and t cell subsets which account for the observed phenotype. methods: next generation sequencing was used to identify the mutations in the atm gene. b and t cells were purified from the patients peripheral blood by positive selection. intracellular staining for foxp3 and t-bet was performed. b cells were activated in the presence of polyclonal f(ab)2 rabbit anti-human igm, multimeric, soluble, recombinant-human cd40l, gardiquimod (tlr7 agonist), or cpg (tlr9 agonist). the treg suppression assay was carried out by co-culturing cd4+cd25hicd127lo tregs and cd4+cd25cd127+ responder t cells at a 1:1 ratio in the presence of beads loaded with anti-cd2, anti-cd3, and anti-cd28 for 4.5 days. results: next generation sequencing revealed two pathogenic mutations in the atm gene, a novel mutation creating a premature stop codon [c.237dela,(p.lys79asnfs370)], and a nonsense mutation [c.3372c>g, (p.tyr124ter)]. proliferative responses of pbmc to mitogens (pha, cona, pwm) were reduced to roughly half of the control responses; the response to tetanus was normal whereas the response to c. albicans was absent. serum cytokine analyses demonstrated elevations in levels of tnf (14.5 pg/ml) and il-10 (7 pg/ml); levels of ifn, il-2, il-5, il-6, and il-12 were below the limits of detection. b cell abnormalities included markedly increased percentages of cd38locd21lo cells (40%) expressing t-bet and fas. activation of these cd21/low b cells through the b cell receptor, tlr7 and tlr9, and cd40 was decreased in response to all of the stimuli as evidenced by a lower percentage of b cells expressing the activation markers cd69 and cd86 relative to healthy control samples. the frequency of unswitched cd27+igd+ memory b cells was also increased (54%). among the naive b cells, the proportion of cd19+ cd27cd21cd10+igmhi transitional b cells that newly emigrated from the bone marrow (bm) was found to be diminished to 1.1% of the naive b cell compartment. in the t cell compartment, there was a decreased frequency of total cd3+ cells but normal absolute numbers of cd3+cd4+ t cells. there was also a decreased proportion of naive cd3+cd4+cd45rocd62l+ t cells and a striking increase in the cd3+cd4+cd45ro+ memory t cells (90%). this appeared to be largely attributed to the increased proportion of cd3+cd4+cd45ro+cd62l effector memory t cells (63%). the circulating t follicular receptor (ctfh) cell frequency in the patient was 5-fold higher (19%) than the average for healthy donors but icos expression levels were normal. treg frequency was decreased but suppressive capacity was not impaired. conclusions: the mutations in atm described here add to the growing understanding of the heterogeneity in degree and complex nature of the immunodeficiency seen in patients with at. these mutations resulted in perturbations in frequencies and distributions of normal and atypical b and t cell subsets, which can explain some immunologic aspects of the clinical phenotype in this patient. the immunophenotype seen here may also differentiate at patients with granulomas from those without cutaneous lesions. supported in part by grifols, the joanne siegel memorial fund, the dreizessen fund (to ewg), grants from niams t32 ar007107-41 (km) and niaid r01 ai071087 (em). introduction/background: a 10-month-old male presented with pancytopenia, b cell deficiency and developmental delay. he was born at 36 weeks with weight of 2.3kg. he was severely anemic with a hb of 7.4, and transfused on day 2 of life. he received hep b and bcg vaccines without complications. a month later he had a hb of 3.6 with a febrile illness. a bone marrow aspiration performed at 45 days, showed dyserythropoiesis without hemophagocytosis, and normal numbers of precursors. t and b cells were decreased. further evaluation with repeat bone marrow showed decrease in all 3 cell lineages. exome sequencing of the family showed homozygous variant in mysm1 (c.899_902delp. (lys300arg/s*11) omim: *612176) in the patient. both parents and hla-matched sister, were heterozygous for the same variant in mysm1. treatment consistent of replacement immunoglobulin, packed rbcs, and g-csf. there was no history of recurrent viral or severe bacterial infections except for 2-3 episodes of urinary tract infections, which were treated with antibiotics. physical exam revealed low set ears, sunken and wide set eyes, depressed nasal bridge, mild micrognathia, frontal bossing, and 1 cm x 1 cm cafeau-lait spot noted behind left knee. he was pancytopenic with a wbc count ranging 600/mcl to 6300/mcl, and anc ranging from 10/mcl to 4500/mcl (on intermittent g-csf). hb = 7.7 gm/dl requiring transfusions every 3-4 weeks, and platelet count was 91,000/mcl. b cell deficiency was confirmed with total b cell count of 36 cells/mcl. b-cell maturation was essentially normal. t cell counts were normal with ageappropriate distribution of naïve and memory t cells, and t cell function. there was normal t cell receptor repertoire diversity. objectives: to assess defect in dna repair using a flow cytometry-based assay in a patient with mysm1 deficiency. methods: patients with mysm1 deficiency are reported to have increased genomic instability. deb testing, and telomere length analysis revealed normal results. defects in the dna repair pathway were assessed using a flow cytometry-based assay measuring phosphorylation of atm (patm), smc1 (psmc1) and h2ax (gh2ax) without irradiation, or 1h or 24h after low-dose (2gy) radiation using a cs137 source. the analysis was performed in t, b and nk cells. results: the patient had higher patm and psmc1 in t cells compared to the experimental controls (hc) at 1h post-irradiation. also, the amount of gh2ax in nk cells was significantly higher than hc at 1h post-irradiation. interestingly, the patients b cells showed approximately 12% of b cells with constitutive gh2ax even without irradiation, and this subset increased slightly to 16% at 1h after irradiation. the mfi (amount) of gh2ax also increased at this time-point. at 24h post-irradiation, there was normal dephosphorylation in healthy control lymphocyte subsets. however, the patients t cells did not de-phosphorylate completely and showed higher residual patm, and psmc1 in both t and b cells. also, both t and b cells, at 24h, demonstrated a small subset of t cells (1%) with constitutive gh2ax without irradiation, which increased to 4% after irradiation. there was also an increase in gh2ax mfi in the irradiated sample. in b cells, 6% showed constitutive gh2ax without irradiation at 24h, and this increased to 37% after irradiation, with a corresponding increase in mfi. conclusions: in summary, this rapid flow analysis revealed defects in the dna repair pathway, including higher patm, psmc1 and h2ax phosphorylation in t, b and nk cells at 1h post-irradiation. at 24h, only t cells showed a residual subset with patm expression. but, psmc1, a downstream target of atm, revealed higher levels in t, b and nk cells at 24h post-irradiation. this assay, which allows lineage-specific analysis, permitted dissection of dna repair defects, in individual lymphocyte subsets revealing heterogeneity within the cell subset to radiation susceptibility. the practical benefit of this rapid multi-parameter flow assay is selection of appropriate conditioning regimen for hematopoietic transplantation, as was the case with this patient. this has significant practical implications for treatment of patients with radiosensitive immunodeficiencies. ctla-4 haploinsufficiency-associated inflammation can occur independently of t-cell hyperproliferation introduction/background: cd28 and ctla4 provide opposing proliferative signals to t cells. we identified an 18-year-old female subject (s1) with heterozygous deletions of cd28 and ctla4 and multi-organ inflammatory disease characterized by a lack of t cell infiltrates in affected organs. inflammatory disease was remarkably responsive to s1 ctla4-ig therapy. objectives: our goal was to characterize the immunologic consequences of combined deletion of cd28 and ctla4, specifically assessing t cell proliferation and treg function in comparison to patients with alps5associated ctla4 haploinsufficiency. we further sought to explain how this s1s inflammatory diseases could occur without a pathologic t cell infiltrate and why they were amenable to ctla4-ig therapy. methods: we performed phenotypic analyses of subject t cells and innate lymphoid cells (ilcs). we functionally characterized subject t cells. we created serum cytokine profiles. we stained and analyzed tissue biopsies. results: cd28 and ctla4 expression on s1 t cells were half that of control t cells. s1 t cells were hypoproliferative. s1 tregs were scarce and lacked suppressive function similarly to alps5 tregs. s1 tregs could suppress autologous t responder cells, likely due to their poor proliferative capacity. s1 colonic biopsies featured significantly fewer infiltrating intraepithelial lymphoid cells than biopsies from an alps5 patients. unlike alps5 patients whose colonic gland infiltrates were overwhelmingly t cells, s1 intraepithelial lymphoid cells were neither t cells nor b cells, suggesting the presence of ilcs. indeed, a greatly expanded population of type 3 innate lymphoid cells (ilc3) and prototypical ilc3 cytokines were identified in s1 peripheral blood. ilc3 frequency and cytokine levels decreased in response to treatment with ctla4-ig, corresponding with marked improvement in enterocolitis, hepatitis and pericarditis. conclusions: we report a novel genetic syndrome of combined cd28/ctla4 deletion and describe the immunolopathologic correlates of this disease. dual ctla4-and cd28-haploinsufficiency results in a phenotype of multi-organ inflammatory disease characterized by ilc3 expansion in the setting of t-cell hypoproliferation and quantitative and qualitative treg defects. our patients clinical response to ctla4-ig parallels published mouse studies and suggests the existence of additional stimulatory b7 receptor(s) preferentially expressed on ilc3s over conventional t-cell populations. diagnosis of radiosensitivity and dna repair defect in dna ligase iv deficiency with a rapid flow cytometry assay. introduction/background: dna ligase 4 deficiency (lig4-scid) is one of several monogenic defects affecting dna repair, and causing lymphopenia (t-b-nk+) and a radiosensitive scid (rs-scid) phenotype. the assignment of a timely diagnosis is vital in the management of patients with rs-scid. laboratory assessment of radiosensitivity is laborious, and utilizes fibroblasts (non-hematopoietic) or lymphoblastoid cell lines, and can take several weeks to months for results. objectives: we demonstrate for the first time, the application of a flow cytometric-based kinetic analysis of phosphorylated h2ax (h2ax) in lymphocyte subsets, especially nk cells, for the diagnostic assessment of lig4-scid. methods: simultaneous measurement of multiple dna repair markers phosphorylated (p) atm, smc1 and h2ax (h2ax) was performed by flow cytometry to assess dna repair defects in a 3-year-old korean female. the patient was evaluated for recurrent fevers, chronic respiratory tract infections, chronic diarrhea, and rash. genetic testing revealed compound heterozygous variants (nm_001098268, c.1341g>t, p.trp447cys and nm_001098268, c.1103a>t, p.asp368val) in lig4. functional assessment (phosphorylation) was measured in t and nk cells (b cells were absent), before irradiation (background control), or after low-dose (2gy) irradiation (1 and 24 hours). results: we observed maximal h2ax generation at 1 hour post-irradiation, with progressive dephosphorylation at 24 hours post-irradiation in healthy controls. the patient showed normal frequencies (%) of t cells and nk cells positive for h2ax (95.62% and 99.40% respectively); (controls (n=2) t cells = 99.29% and 99%; nk cells = 99.6% and 99.11%), but increased intracellular levels (mean fluorescence intensity, mfi) of h2ax (t cells = 50.67 and nk cells = 52.41) compared to controls (t cells = 24.86 and 19.58; nk cells = 41.33 and 35.03) at 1 hour post-irradiation. however, more importantly, at 24 hours post irradiation there was a lack of dephosphorylation in a substantial proportion of lymphocytes (64% of t cells and 99% of nk cells) compared to healthy controls (t cells= 1.75% and 1.51%; nk cells = 9.27% and 17.83%). further, while there was dephosphorylation of h2ax at 24h in patient lymphocytes as compared to 1h, the amount, as measured by mfi, remained elevated at 24h (t cells = 8.97, nk cells =7.57) compared to controls (t cells =1.71 and 2.25; nk cells = 3.18 and 2.75). the data from patm and psmc1 were uninformative for the evaluation of lig4-scid. conclusions: flow-based kinetic analysis of h2ax is a useful marker for the diagnosis of lig4-scid, and can be performed with a small amount (5cc) of blood, and provides a result in 3-4 days, facilitating rapid assessment of radiosensitivity in this condition. human plcg2 haploinsufficiency results in nk cell immunodeficiency and herpesvirus susceptibility objectives: we aimed to investigate the cause of disease in three patients from two kindreds with recurrent or severe herpesvirus infections and nk cell dysfunction. methods: we used exome sequencing and mass cytometry (cytof), as well as traditional immunologic techniques, to investigate the genetic causes, immune cell subpopulations/signaling, and nk cell function of these patients. we additionally used mouse models, crispr cell lines and in vitro assays to assess the role of plcg2 haploinsufficiency in disease. results: kindred a consisted of two patients presenting with hsv1 susceptibility and autoimmunity. kindred b consisted of one patient with severe cmv myocarditis and adenoviral hepatitis. both kindreds were evaluated for nk cell function and showed reductions in target killing in spite of normal cytotoxic granule degranulation against the same target. microscopy analysis suggested that granule mobility was reduced in at least one kindred. cytof revealed reductions in plcg2 phosphorylation after receptor crosslinking in the nk cells of both kindreds. kindred a also presented with a reduction in naïve b cells without perturbations in immunoglobulin output, b cell memory formation or class switching. trio whole exome sequencing was performed and revealed rare heterozygous plcg2 mutations in both kindreds. functional analysis, as well as mouse and crispr models, support a functional haploinsufficiency as a cause for nkd in these patients. conclusions: heterozygous loss-of-function point mutations in plcg2 have not been previously investigated as a cause of nk cell deficiency or recurrent herpesvirus infection. thus, these patients represent a novel immunodeficiency involving plcg2 haploinsufficiency, nk cell dysfunction, and herpesvirus susceptibility. hypomorphic rag1 mutations alter the pre-immune repertoire at early stages of lymphoid development introduction/background: human rag deficiency is associated with a spectrum of clinical phenotypes. while the most severe forms of rag deficiency manifest with severe combined immune deficiency or omenn syndrome since the first weeks of life, more recently patients have been identified who present to medical attention at a much older age predominantly with symptoms of autoimmunity and/or inflammation. many of the mutations associated with this atypical syndrome are found in the c-terminal domain (ctd) of the rag1 gene and allow residual development of t and b cells. these patients have an abnormal peripheral t and b cell repertoire, but how this is affected by abnormalities in the composition of the pre-immune repertoire vs. antigen-mediated selection and homeostatic proliferation in the periphery is unknown. objectives: in order to investigate whether mouse models with hypomorphic mutations in the rag1 ctd recapitulate the phenotype observed in patients with cid-g/ai, and to study how these mutations affect repertoire composition, cell selection and survival during t and b cell development, we generated three mouse models carrying homozygous rag1 mutations (f971l, r972q, and r972w), corresponding to human mutations (f974l, r975q, r975w) previously reported in patients with late-onset combined immune deficiency with granuloma and/ or autoimmunity (cid-g/ai). methods: mice were generated using crispr/cas9 mediated gene editing. t and b cell development, including apoptosis was studied by flow cytometry. immunoglobulins in naïve mice, baff levels and specific antibody responses were measured by elisa. serum igm autoantibodies were measured using a microarray (utsw). analysis of t cell receptor (trb) repertoire in several sorted t cell populations and immunoglobulin heavy chain (igh) repertoire in pre-b cells was performed by adaptive biotechnologies. in order to be able to detect both dj and vdj rearrangements, pro-b cells and spleen b cells were sequenced using high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (htgts-rep-seq). analysis of vk-jk rearrangements in pre-b cells was performed by pcr amplification. results: immunological characterization showed partial development of t and b lymphocytes, with persistence of naïve cells, preserved serum immunoglobulin, but impaired antibody responses and presence of autoantibodies, thereby recapitulating the phenotype seen in patients with cid-g/ai. by using high throughput sequencing, we identified marked skewing of igh v and trb v gene usage in early progenitors, with a bias for productive rearrangements after selection occurred, and increased apoptosis of b cell progenitors. this suggested that more alleles remained in germline configuration. moreover, in the rearranged igh loci, the distal v gene segments were preferentially rearranged already at the earliest stages of b cell development, a finding that has not been previously reported. in addition, rearrangement at the igh locus was impaired, and polyreactive igm antibodies were detected. conclusions: in conclusion, this study demonstrates that hypomorphic rag1 mutations reported in cid-g/ai cause abnormalities of the primary b and t cell repertoire. these changes may affect survival and selection of t and b cells, and thereby contribute to the immune dysregulation often seen in patients with cid-g/ai. senior clinician, nih/niaid/lcim introduction/background: autosomal dominant hyper ige syndrome (ad-hies) is a primary immunodeficiency due to loss of function stat3 mutations. disease manifestations include recurrent skin and pulmonary infections, eczema, mucocutaneous candidiasis, as well as tion and in vitro studies demonstrated that the enhanced signaling could be controlled by ruxolitinib, an approved jak1/2 inhibitor. informed by these experimental data, the patients were treated with ruxolitinib with remarkable improvement in a variety of clinical end-points, including hematological profiles and growth parameters. conclusions: this characterization of a human jak1 gain-of-function mutation expands our current understanding of the role of jak1 in eosinophil biology, hematopoiesis and immune function. chronic granulomatous disease, ornithine transcarbamylase deficiency and x-inactivation is a primary immune deficiency characterized by defects in the nadph oxidase enzyme complex resulting in a susceptibility to a narrow spectrum of bacteria and fungi. mutations in cybb encoding gp91phox and located at xp21.1 are associated with the most common form of cgd. deletions and rearrangements in this region are associated with other genetic diseases such as mcleod syndrome (xk), retinitis pigmentosa (rpgr), duchenes muscular dystrophy (dmd), ornithine transcarbamylase deficiency (otc), and x-linked mental retardation (tspan7). patient phenotype depends on the extent and position of the deletion, creating a "contiguous x-chromosome gene deletion syndrome. objectives: we report a four-year-old female, who presented with symptoms of x-linked cgd and otc deficiency with a large, contiguous multi-gene deletion on the x-chromosome. methods: we report a four-year-old female, who presented with symptoms of x-linked cgd and otc deficiency with a large, contiguous multi-gene deletion on the x-chromosome. the patient is the second born of a set of non-identical triplets from a clomiphene assisted pregnancy at 35 weeks gestation. (weight at birth: 5lbs 2oz). after a 7 day stay in the nicu, and cpap for one day she was discharged home. by the second month, she began having problems gaining weight and had persistent vomiting. at 3 months, she was admitted with a pneumonia diagnosed as methicillin resistant staphylococcus aureus by lung biopsy. during that hospitalization, the mother noted an enlarging lesion on the infants left hand which was biopsy proven serratia marscecens osteomyelitis and was treated with intravenous cefepime for 6 weeks. at this point a dihydrorhodamine assay (dhr) showed only 15.1% positive cells (nl 80-100%) and she was diagnosed as a carrier of x-linked cgd. bactrim was initiated for antibacterial prophylaxis but it was discontinued due to recurrent diarrhea; she was unable to tolerate antifungal prophylaxis as well due to liver function abnormalities. she continued having frequent vomiting episodes, irritability, failure to thrive and development delay. ulcerations in esophagus were confirmed by egd and colonoscopy, probably related to persistent emesis. diarrhea was unresolved. at 30 months of life she was admitted with acute encephalitis, a serum ammonia level of 218 and elevated urine orotic acid. results: given the demonstrated carrier status for cybb and apparent otc deficiency, comparative genomic hybridization was performed, revealing a deletion from xp21.1 to xp11.4 this 3.9mb loss includes 18 genes that are known to cause disease. since the diagnosis, the patient has been on reduced protein diet, resolving her diarrhea, she has subsequently grown and is on 5th percentile for weight and height. her dhr is now 28% positive. at 3 years she was diagnosed with cone rod dystrophy. currently she is on bactrim for cgd prophylaxis and l-citrulline for the otc deficiency. she continues to gain weight, and has shown great improvement in her development delay and no new cgd-related infections have recurred. conclusions: this case reminds us that x-linked carriers with large deletions may be symptomatic and genetic analysis to determine other affected genes can be important for medical management. introduction/background: wiskott-aldrich syndrome (was) is a rare and severe x-linked disorder with variable clinical phenotypes correlating with the type of mutations in the was gene. the long-term prognosis of this syndrome is generally poor, with hematopoietic stem cell transplantation (hsct) remaining the only curative choice. the syndrome is poorly characterized in china. objectives: we retrospectively reviewed patients with was referred to our hospital from 2004 to 2016, and summarize their clinical manifestations and genetic features. methods: sixty-four children suspected to be was from 62 unrelated families were enrolled in this study. the clinical data of children were reviewed in the present study. distribution of lymphocyte subsets from peripheral blood and was protein (wasp) expression in peripheral blood mononuclear cells was examined by ow cytometry (fcm). wasp mutations were identified by direct sequencing of pcramplified genomic dna results: among 725 patients with primary immunodeficiency diseases (pid), 40 (5.52%) were finally diagnosed as was with gene identified. the mean time of diagnosis was 6.25 months (range, 0.4-9.63). the common onset clinical manifestation was diarrhea, and most patients had recurrent upper respiratory tract infection, otitis media, pneumonia, and skin abscess. one patient had nephrotic syndrome and no patient with malignancy. all patients had classical was phenotype with was clinical scores 3-5. total 36 mutations in wasp were identified, including 14 novel mutations. six patients received hsct, five survived, with one died because of gvhd. compared with the other 24 patients without wasp mutations, was patients had lower numbers of cd4+ t cells and b cells, and higher eos and ige level. there was a negative association between the number of b cells and the was clinical scores. conclusions: in china, diagnosis of was has improved over the last decade, although a much higher number of cases had been expected. establishing more diagnostic centers dedicated to the care of pid will facilitate early, correct diagnosis and better care of was in china. regulatory cells (tregs) are precisely quantified by measuring demethylation of the treg-specific-region of foxp3. more recently, we have identified highly cell type-specific dna regions of demethylation for further cell populations. objectives: this novel technology allows the implementation of differential immune phenotyping into the newborn screening procedure. here, we aimed at epigenetically quantify multiple immune cell types in different biological samples including dried blood spots and samples from patients with pid with immundysregulation, where currently no approach is available. methods: using cell type-specific demethylation sites, we developed epigenetic qpcrs for quantification of t-, treg, b-, nk-, monocyte and granulocyte cell population. epigenetic qpcr is applicable for relative and absolute quantification in whole blood and dried blood spots using isolated, bisulfite converted dna. results: we demonstrated >95% concordance with flow cytometric analyses of the same fresh blood samples from healthy subjects. we have validated the method in 30 children with symptoms of immundysregulation resulting from monogenic defects, including for example foxp3, cd25, stat1, ctla4, and leading to treg/teffector cell imbalance where treg deficiency has been difficult to assess by flow cytometry. furthermore, we tested 250 dried blood spot (guthrie card) samples from healthy newborns and 30 patients with diverse pids and correctly identified 29/30 pid patients, indicating that this method holds promise for newborn screening. conclusions: the method we established for immune cell quantification based on epigenetic cell type-specific markers, is feasible and reliable in biological samples either fresh, frozen or archived, including dried blood spot. the analysis of further immune cell types introduces an innovative opportunity to diagnose a variety of pids and immunodysregulatory disorders as early as newborn screening. pancytopenia and immunodeficiency with mds in an infant due to samd9l mutation we present our data on behalf of the pcid study consortium of the inborn errors working party worth profound combined immunodeficiencies (p-cid) are inherited diseases with impaired t-cell function leading to infections, immune dysregulation or malignancies. genetic, immunologic and clinical heterogeneity make patient specific decisions on indication and timing of hematopoietic stem cell transplantation (hsct) difficult. objectives: since 2011 the pcid study recruits non-transplanted p-cid patients aged 1-16 years to prospectively compare natural histories of age and severity-matched patients with or without subsequent transplantation and to determine whether immunological and/or clinical parameters may be predictive for outcome methods: our prospective/retrospective international observational multicenter study recruits pediatric p-cid patients to identify biomarkers and clinical parameters that are predictive of outcome. results: so far >130 growth hormone deficiency comprised the majority of neuroendocrine cases (65%, 11/16). other notable neurologic diagnoses included headache (17%, 211/1227), seizure (5%, 62/1227), cerebrovascular accident (2%, 25/1227), and neurologic tumors (0.4%, 5/1227). in addition to somatic neurologic conditions, many cvid patients (37.7%, 462/1227) had reported diagnoses of depression, anxiety, and post-traumatic stress disorder. conclusions: our findings suggest that neurologic diagnoses are more common in cvid patients than previously recognized. patients with neurologic autoimmune disease appear to have a more severe phenotype with earlier age at symptom onset. many cvid patients had depression, anxiety clinical outcomes of human herpesvirus 6 reactivation after hematopoietic stem cell transplantation prognostic factors and outcome of epsteinbarr virus dnaemia in high-risk recipients of allogeneic stem cell transplantation treated with preemptive rituximab cytomegalovirus in hematopoietic stem cell transplant recipients daratumumab controls life-threatening post-hsct autoimmune haemolytic anaemia objectives: we present a 17 month old pancytopenic male who was diagnosed with myelodysplastic syndrome (mds) with monosomy 7 due to samd9l mutation. methods: whole exome sequencing was performed on a male, who presented at 8 months of age with findings concerning for a bone marrow failure (bmf) syndrome despite a normal bmf genetic panel. results: the patient presented at 8 months of age, with severe pancytopenia, fevers, e. coli bacteremia, pancolitis, and echtyma gangrenosum. bone marrow showed severe aplasia with occasional macrophages.he was treated with antibiotics, as well as steroids, etoposide and cyclosporine for presumed hemophagocytic lymphohistiocytosis (hlh). a bone marrow failure and hlh genetic panels were normal conclusions: this is one of the first reported cases of samd9l mutations causing mds since its initial discovery earlier this year. samd9l mutation should be considered in patients who present with pancytopenia and monosomy 7 mds. as new defects continue to be identified, further evaluation outside of typical bmf panels may be relevant primary immune deficiency disease in patients over age 60: an analysis from a proprietary immunology patient registry roger ucla school of medicine 2 director objectives: to characterize the prevalence of pidd among older individuals using a patient database maintained by the consortium of independent immunology clinics (ciic), comprised of 17 specialty immunology outpatient practices in the us. methods: patients with pidd were identified in the ciic database using icd-10 codes d80 conclusions: our data suggest that pidd in patients over age 60 may be more prevalent than previously reported introduction/background: the patient is a 6-month-old boy, born at 35+4 weeks gestation to non-consanguineous parents of italian origin. he was admitted to the intensive care unit at 12 days of age with profuse bloody diarrhoea, weight loss, severe metabolic acidosis and acute renal failure. he had a rapid respiratory deterioration necessitating intubation, ventilation and inotropic support. the patient developed features of macrophage activation syndrome with: (i) prolonged fever > 38.5°c, (ii) hepatosplenomegaly, (iii) bicytopenia (anaemia and thrombocytopaenia), (iv) hypertriglyceridemia, (v) high ferritin (14,7000) and (vi) haemophagocytosis on bone marrow smear. he also presented with three interesting features (i) a macular erythematous rash that slowly resolved and was replaced by reticulo-livedoid rash, (ii) a marked hypereosinophilia and (iii) no significant elevation of hladr/cd8+ t cells on lymphocyte immunophenotyping (7-15%). the patient underwent rectal biopsy which confirmed the presence of eosinophils, but without significant inflammation or architectural changes. stool microscopy showed presence of partially necrotic intestinal epithelial cells. immune work up demonstrated global t lymphopaenia without balanced subpopulation and eliminated a familial hemophagocytic lymphohistiocytosis (normal perforin and cd107a expression on cd8+ t-cell and nk cells). circulating foxp3+ cd25+cd127lowcd4+ t cells were within normal range. a dihydrorhodamine reduction assay was normal. 9 chief, laboratory of clinical immunology and microbiology, national institute of allergy and infectious diseases, national institutes of health introduction/background: hematopoietic stem cell transplantation (hsct) has been used for the treatment of hematologic malignancies and primary immunodeficiencies (pid), for several decades with increasing efficacy. however, toxicity related to conditioning regimens based on the use of chemotherapy and/or irradiation to ensure engraftment of donor cells, remains a significant problem. recently, an alternative, potentially low-toxicity, approach has been proposed, which makes use of an immunotoxin targeting cd45-expressing cells, which include hsc and more mature leukocytes. this approach is particularly attractive for leaky forms of severe combined immune deficiency (scid), with residual production of dysfunctional t and/or b cells, such as atypical forms of rag deficiency. objectives: we have developed a mouse model carrying a hypomorphic mutation in the rag1 gene (p.f971l) resulting in a combined immunodeficiency with signs of autoimmunity, recapitulating the phenotype seen in patients. methods: using this model, we have tested the efficacy of conditioning with an anti-cd45 immunotoxin (cd45-sap) alone or in combination with low irradiation (200rads; cd45-sap/200), and compared these regimens to a myeloablative dose of irradiation (800rads) [niaid protocol lcim 6e]. following conditioning, the mice were transplanted with wild-type (wt) bone marrow (bm) lineage-negative cells and followed over time to evaluate immune reconstitution. results: conditioning with cd45-sap alone or with cd45-sap/200 led to a consistent engraftment of donor t and b cells in the peripheral blood (pb) of f971l that increased overtime, reaching 90% donor chimerism at 16 weeks. myeloid (cd11b+) and nk cells in pb of f971l mice also showed high level of donor engraftment that remained stable at around 70% in cd45-sap around 24 hours of life and the second after one week. there is no data on the utility of one versus two screens for scid screening. here we present our data evaluating whether patients with scid or t-cell lymphopenia were identified on the first or second trec screen. objectives 1. determine the benefit of a second trec screen in identifying scid and t-cell lymphopenia at birth. 2. examine outcomes of trec screening in washington state since implementation. methods: results of scid newborn screening performed in washington state between january 2014 and june 2017 were reviewed retrospectively with the staff of the washington department of health laboratory where the trec assay is performed. trec thresholds (copies/μl) were defined as follows: absent (20), low (21-60), borderline (61-80) and normal (>80). all trec assays were run with a beta-actin control to assure sample adequacy. a screen is considered abnormal if there is one low/ absent trec or two borderline trec. newborns with abnormal trec screening have follow-up diagnostic testing consisting of lymphocyte flow cytometry to evaluate numbers of naïve and mature t cells, b cells, and nk cells, performed at seattle childrens hospital. results: a total of 68 positive trec screens were found in washington state between january 2014 and june 2017. five patients who did not have diagnostic flow cytometry testing were excluded from the analysis (one protocol deviation, one lost to follow-up and three who died before testing could be performed). the first screen was abnormal in forty three patients, while the second screen was abnormal in 16 patients. five patients had an abnormal third or fourth trec drawn for other newborn screen follow-up. three patients with scid were identified, all with abnormal values on first screen. fortyfive patients with t-cell lymphopenia were identified; 30 from the first screen and 11 from the second screen. there was one patient with mhc ii deficiency was missed by both first and second screens because she did not have t-cell lymphopenia. the false positive rate with the first screen was 21% versus 28% with subsequent screens. the false positive rate dropped to 3% with two abnormal trec. the positive predictive value of scid or t-cell lymphopenia with the first abnormal trec was 82% versus 96% with two abnormal trec. average age of collection among infants with a positive screen was 30.5 hours for the 1st nbs and 11.5 days for the 2nd nbs. live viral vaccines were postponed in three patients who had an abnormal secondary screen (one with idiopathic t-cell lymphopenia, one with ectrodactyly-ectodermal dysplasia-clefting (eec) syndrome and one with 22q11.2 deletion). one of these was started on pjp prophylaxis. conclusions: the practice of obtaining a second nbs from all newborns in washington state has led to increased identification of patients with tcell lymphopenia but did not result in identification of additional patients with scid. the false positive rate of the first and subsequent newborn screens was similar and decreased in patients with two abnormal trec. interventions including delaying live viral vaccines and pjp prophylaxis were instituted in patients who had a normal initial trec but abnormal secondary screen and documented t cell lymphopenia. two trec screens in all newborns can result in identification of additional patients with t-cell lymphopenia who may require intervention and additional follow-up. it is not yet clear whether the cost of a second mandatory scid newborn screen is balanced by the additional sensitivity gained by this approach. introduction/background: mannose-binding lectin (mbl) is a multimeric lectin that recognizes a wide array of pathogens independently of specific antibody, initiates the lectin pathway of the complement system, and acts as a proinflammatory mediator. mbl deficiency is reported to increase the frequency of infections in patients with impaired immune systems or cystic fibrosis (cf) patients. mbl replacement is experimental and unavailable. immunoglobulin replacement therapy (igrt) is controversial in the treatment of mbl deficiency; however, there are no reports of its efficacy or role in this condition. we describe a cf carrier patient with mbl deficiency, ciliary dyskinesia and mildly low igg who did not respond to igrt. objectives 1. understand when mbl deficiency can be symptomatic. 2. define the relationship between mbl deficiency and cf. 3. describe the treatment options of mbl deficiency. 4. define the efficacy of igrt and mbl deficiency. methods: a single case report. results: 11 year old girl with mbl deficiency, cf carrier state with polymorphisms (2752-26 a>g cf variant with 7t/7t and m470v polymorphism) and ciliary dyskinesia (diagnosed by ciliary biopsy) who initially presented at eight years of age with recurrent sinusitis, recurrent otitis media status post tympanostomy tube placement, reactive airway disease and tracheomalacia. she had nine episodes of recurrent sinusitis with six negative sinus cultures and one positive for pseudomonas aeruginosa. she required a total of nine courses of antibiotics. labs showed several mbl levels <50 ng/ml on three occasions, normal ch50, ah50, igg 577-590 mg/dl (low normal for age), normal iga, igm, t cells, b cells, nk cells, and robust specific antibody titers. despite adequate pulmonary hygiene including nebulized levalbuterol, budesonide, dornase alfa, ipratropium, hypertonic saline and compression vest twice daily, she continued to have recurrent bronchitis and cough. in addition, even with sinus rinses and intranasal corticosteroid, she continued to have 6-8 episodes of sinusitis yearly. for this reason, she underwent bilateral total ethmoidectomies and maxillary antrostomies with modest reduction in frequency of sinus infections and symptoms. however after two years, her bacterial sinusitis recurred. four episodes were positive for methicillin staphylococcus aureus or pseudomonas aeruginosa. this did not improve significantly with a trial of intranasal mupirocin. due to increased frequency of bacterial sinusitis refractory to traditional therapy, she was started on subcutaneous igrt dosed approximately 350 mg/kg/month dosing maintaining igg troughs around 700 mg/dl. after a six month trial, she did not have improvement in the frequency of sinusitis, bronchitis and otitis media. she required 3-4 courses of antibiotics for bacterial upper respiratory tract infections and igrt was stopped. prophylactic antibiotics and repeat sinus surgery were instituted. conclusions: majority of the patients with low/deficient mbl levels do not manifest significant symptomology due to the redundancy of the innate immunity. the increased susceptibility to infections is thought to be due to additional factors that compromise other components of the immune system. specifically in cf patients, mbl deficiency is associated with earlier colonization with pseudomonas, more rapid decline in lung function and earlier death secondary to end-stage lung disease. there are no reports of combined mbl and cf carrier symptomatic patients similar to this patient. there are no validated age-corrected values for mbl levels in pediatric patients. the clinical relevance of these levels to infection frequency, severity, or treatment of mbl deficiency remains to be proven. it has been proposed that <100 ng/ml is considered deficient in children. mbl therapy is still experimental and not commercially available. management when provided for severe or frequent infections includes prompt treatment with antibiotics, prophylactic antibiotics, appropriate vaccinations, and a trial of igrt. there are no reported cases describing the efficacy of igrt in mbl deficiency, and the mechanism subsequent genetic analysis identified the presence of a de-novo heterozygous mutation in the nucleotide binding domain of nlrc4 (c.1021g>c, p.val341leu). a mutation involving this amino-acid position has already been described but with a different substitution pattern in a boy and his father who presented with mas (p.val341ala) (1) . in order to prove the causality of this mutation, our team generated thp-1 cell lines expressing the two different mutations through gene-editing with the crispr system. in this system the mutation p.val341leu, as well as the p.val341ala mutation, were responsible for spontaneous activation of caspase1 , as evidenced by flica assay. the patient was treated with iv methylprednisone 2mg/kg/day. he continued to have progression of his inflammatory state, and was therefore commenced on anakinra. following confirmation of mutation, he was started on rapamycin, reasoning that (i) through autophagy induction, rapamycin could potentiate the action of anakinra (2, 3) and (ii) through mtor inhibition counteract the effect of il-18 on t-cells (4). with a combinatory therapy of anakinra up to 15mg/kg/day and ramapycin (with trough levels of 10-15 ng/l), the patient showed a marked clinical improvement, allowing weaning of steroids and establishment of enteral feeds. ferritin levels reduced to 800-1000 ng/ml. we observed a significant decrease in il-18 plasmatic level following treatment initiation (pre-vs post-treatment levels of 82844 pg/ml and 10055 pg/ml, respectively). we report a novel nlrc4 gain-of-function mutation, presenting with neonatal enterocolitis and autoinflammation with improvement under combinatory therapy of anakinra and rapamycin. to our knowledge this is the first case to report the use of rapamycin in this disease, with what appears to be encouraging results. further studies are required to elucidate the potential role of rapamycin in the management other inflammasome disorders. introduction/background: allogeneic hematopoietic stem cell transplantation (hct) is currently standard treatment for patients with severe combined immunodeficiency (scid), with 2-year overall survival >90% for typical scid (heimall blood 2017). previous studies revealed that poor clinical outcomes correlated with poor long-term t cell reconstitution, including low cd4 t cell counts and low naïve cd45+ t cell counts (pai nejm 2014) . we hypothesized that t cells developing in a poorly reconstituted immunologic environment would show features of chronically activated t cells with increased expression of inhibitory co-receptors. we further hypothesized that the intensity of the conditioning regimen would correlate with the expression of inhibitory co-receptors. objectives: to characterize the t cell phenotype of scid patients at >2 years post-hct and to investigate the impact of conditioning regimen on the quality of t cell reconstitution and the expression of inhibitory coreceptors methods: we analyzed 34 scid patients 3-28 yrs (median 14 yrs) after hct. we excluded from the analysis patients with chronic graft-versushost disease (gvhd), chronic dna viral infections or patients who had received donor lymphocyte infusion or boost in the 6 months prior to study. scid genotypes (n) included il2rg/jak3 (20), rag1/rag2/ dclre1c (5), ada (2), il7r (1) and other/unidentified (4). nine patients had received a reduced intensity (ric) or myeloablative (mac) conditioning regimen, while 25 had received either no conditioning or immunosuppression only (none/is). poor t cell reconstitution was defined as cd4 t cell counts below 500 cells/mm3 (21 patients). t cell phenotype, including expression of inhibitory co-receptors, was assessed by flow cytometry. results: compared to patients with cd4 counts above 500 cells/mm3, patients with low cd4 counts had low naïve cd45ra+ccr7+ t cells (p=0.0004) and high cd45ra-ccr7-t effector memory (tem) cells (p=0.02), low numbers of naïve thymic cd45ra+ cd31+ t cells, low numbers of trecs and a less diverse t cell repertoire (p<0.0001). additionally, they had an increased frequency of cd8 t cells expressing pd1 (8% vs 3%), ctla4 (5% vs 1.5%), cd160 (25% vs 5%, p=0.01), and 2b4 (63% vs 29%, p=0.0001) inhibitory co-receptors. increased inhibitory receptor expression was associated with a differentiation profile skewed toward a tem phenotype, reduced t cell diversity, increased markers of t cell activation, and the development of a highly exhausted cd39high pd-1high t cell population. more importantly, a fraction of ccr7+ cd45ra+ cd8 t cells expressed pd1 (6% vs 1%, p=0.03) and 2b4 (27% vs 4%, p=0.01) in patients with low cd4 counts, suggesting that some naïve t cells of poorly reconstituted patients were chronically activated. inhibitory receptor expression did not increase with hla disparities between the donor and recipient, a history of gvhd after transplant or the infection status of the patient prior to transplant. however, inhibitory co-receptor expression was correlated with conditioning regimen, with increased frequency of 2b4+ cd8 t cells in unconditioned patients (54% vs 28% in none/is and ric/mac patients respectively, p=0.01). conversely, ric/mac conditioning was associated with higher naïve cd8 t cell numbers (p=0.01), higher naïve thymic cd45ra+ cd31+ t cell numbers (p=0.001), a more diverse t cell repertoire (p=0.001) and low expression of inhibitory co-receptors. ric/mac conditioning was also associated with improved naïve t cell generation and limited expression of inhibitory receptors in il2rg/jak3 patients (23% vs 50% for 2b4, 1% vs 11% for cd160 in ric/mac versus none/is il2rg/ jak3 patients respectively), a genotype permissive to t cell engraftment. conclusions: collectively, our results suggest that the expression of inhibitory co-receptors may be a biomarker of poor t cell reconstitution in transplanted scid patients. further, we propose that lack of conditioning limits t cell reconstitution, which correlates with increased expression of inhibitory receptors on circulating cd8 t cells. antibodies targeting inhibitory receptors are now available in clinical trials to treat cancer and viral infections. it will be necessary to evaluate the relationship between inhibitory receptor expression, t cell function and clinical outcome, to see if a selected group of scid patients could benefit from these immunotherapies. wallace chair, chief of allergy immunology, children's hospital of philadelphia introduction/background: prophylactic antibiotics (abx) and immunoglobulin replacement (igrt) are commonly used to treat specific antibody deficiency (sad), but the optimal therapy is not established. objectives: to compared outcomes (number of infections and hospitalizations) in sad treated with igrt vs. prophylactic antibiotics. methods: two-center, retrospective chart review of sad patients from jan 2012-may 2017. we excluded patients with hypogammaglobinemia and/or other immunodeficiency diagnosis. characteristics and treatment were reported, rates of infections/hospitalizations among treatment groups were compared using linear regression model. results: 78 sad patients included. mean age was 18 years, 54% were females. 22 (28.8%) received prophylactic antibiotics, 45 (57.6%) received igrt, 11 (14.1%) did not receive any specific treatment. number of infections decreased from 8.2 (year before treatment) to 2.0 (year after treatment) in prophylactic antibiotics group (p=0.008), and from 7.2 to 2.3 in igrt group (p<0.001). various musculoskeletal and vascular abnormalities. little is known of gynecologic-obstetric complications, but with improved therapies women are living longer making reproductive health more pertinent. objectives: to learn more about obstetric and gynecological health in women with stat3 loss of function. methods: we prospectively interviewed and retrospectively reviewed medical records of adult women with ad-hies evaluated at the nih between 2000-2017. results: of 61 patients aged 18-66 years (mean 35 years), 47 women were interviewed, and chart reviews were performed on 14. age of menarche in our cohort was consistent with the national average (13.05 vs. 12.5 years). five of 30 patients (16.7%) reported having worsening lung symptoms with menstruation, and 14 of 34 participants (41%) reported worsening eczema during menstruation. with regard to routine health maintenance, 21 of 30 women reported having regular cervical cytology testing; 7 (23%) reported an abnormal result. of these 7, 5 had hpv that responded to treatment or was hpv only not requiring treatment; 2 had reactive changes due to yeast and 1 ascus that was subsequently normal. mastitis and breast abscesses occurred in 14 women. eleven women reported vulvar cysts/abscesses requiring drainage. nine women had progestin-releasing iuds placed, in some to suppress menses-associated vulvar eczema/abscess flares; no infectious complications were reported from progestin iud use. over 30% of women chose not to conceive given underlying disease. of 16 women with pregnancies, 15 women had 26 live births, 6 of 16 (38%) had miscarriages, and 3 of 16 (19%) experienced recurrent pregnancy loss. three women whose pulmonary symptoms worsened during pregnancy were diagnosed with progression of parenchymal lung disease post-partum. one woman experienced worsening of skin manifestations. other reported postpartum complications included one wound infection (after caesarean) and one hemorrhage leading to hysterectomy. conclusions: as women with ad-hies are living longer, significant infectious and disease-related exacerbations related to both menstruation and pregnancy were observed in this patient population. it is important to focus on maintenance of their gynecologic and obstetric health and carefully monitor for these morbidities. finally, while these women may choose to attempt pregnancy, the risk of recurrent pregnancy loss and worsening disease warrants discussion. rna sequencing identifies aichi virus 1 as the cause of chronic infection with lymphoproliferation in a patient with x-linked agammaglobulinemia objectives: we here present an xla patient with a complicated course in whom we detected aichi virus 1 (aiv1). methods: case report: the patient was diagnosed with xla at age 3 years, based on agammaglobulinemia with absent b cells and a known pathogenic mutation in btk (c82c>t, p.r28c). he had been suffering from recurrent respiratory and gastrointestinal infections since the age of 2 months. he was started on immunoglobulin (ig) substitution, which resulted in complete control of infections with igg trough levels at 8 g/l. however, at 6 years of age he developed unexplained fever, refractory temporal epilepsy, hepatitis, progressive nephromegaly with chronic renal failure, splenomegaly, episodic diarrhea and growth failure. ultrasound identified multiple focal lesions in the liver, spleen and kidney. a liver biopsy showed severe chronic hepatitis with initial perisinusoidal fibrosis. serial kidney biopsies showed variable oligoclonal cytotoxic t cell infiltrates, suggestive of chronic viral infection. standard diagnostics failed to reveal a pathogen in blood or stool samples and on biopsies. results: we finally resorted to rna sequencing on a kidney biopsy sample. this technique identified aiv1, a kobuvirus of the family picornaviridae, with a high read number. subsequently pcr confirmed the presence of aiv1 in both liver and spleen. results for cerebrospinal fluid, blood and feces are pending. conclusions: aiv1 is a picornavirus responsible for self-limiting gastroenteritis in humans. confirmatory pcrs on blood, csf and stool samples are ongoing. however, given the unequivocal result of the rna sequencing and confirmatory pcrs in other affected organs, we believe that the complications in this xla patient can be explained by aiv1 chronic infection. these findings confirm the potential of next generation sequencing techniques to identify infectious agents in patients with primary immunodeficiency. characterization and successful treatment of a novel autosomal dominant immune dysregulatory syndrome caused by a jak1 gain-offunction mutation. introduction/background: janus kinase 1 (jak1) plays an essential, nonredundant role in the jak/stat signaling cascade, a key pathway in the control of hematopoiesis and immune function. significant progress has been made in elucidating the role of jak1, but gaps in our knowledge still persist. to date, somatic gain-of-function mutations in jak1 have been linked to t-cell acute lymphoblastic leukemia. objectives: to understand and treat human jak1 gain-of-function mutations. methods: research study protocols were approved by our institutional review board. four members of the family (the affected children and their parents) were enrolled. written informed consent for genetic testing and participation was provided by the parents for their children. genetic, bioinformatic, biochemical and immunological investigations were performed. results: we describe the first known patients carrying a germ-line gainof-function mutation in jak1. the clinical phenotype includes severe atopic dermatitis, markedly elevated peripheral blood eosinophil counts with eosinophilic infiltration of the liver and gastrointestinal tract, hepatosplenomegaly, autoimmunity, and failure to thrive. functional analysis established the gain of function phenotype caused by the mutaintroduction/background: herpesviridae infection after hsct for hematologic malignancies, specifically cytomegalovirus (cmv), epstein-barr virus (ebv) and human herpes virus-6 (hhv-6), have been associated with various outcomes including post transplant lymphoproliferative disorder (ptld), graft-versus-host disease (gvhd) and mortality (1) (2) (3) . patients with primary immunodeficiency may have different incidence and outcomes with respect to herpesviridae given their differences in age at transplant, conditioning choices and underlying disease susceptibility to this viral family. objectives: the objective of this study was to describe the incidence and outcomes of cmv, ebv and hhv-6 post hsct for the primary immunodeficiency population. methods: a single center retrospective chart review of primary immunodeficiency registry patients (research ethics board protocol no. 1000005598) who received hsct from january 2000 december 2016 was undertaken. patients who received gene therapy were excluded. antiviral prophylaxis was given according to institutional protocol. demographic and clinical data were collated and analyzed with microsoft excel . the primary outcome was incidence and time to dnaemia for cmv, ebv and hhv-6. results: sixty one patients who underwent hsct for primary immunodeficiency from january 2000-december 2016 were reviewed. diagnoses are noted in table 1 . the average age of transplant was 18.0 months (range 1.9-93.8). 51 transplant recipients received busulfan and cyclophosphamide conditioning (2 with anti-thymocyte globulin (atg), 2 with alemtuzumab added), 3 transplants received busulfan, fludarabine and either atg or alemtuzumab, and 1 received atg alone. six transplants were unconditioned. 70.5% (43/61) of patients developed gvhd requiring systemic immune suppression. the overall incidence of cmv, ebv and hhv-6 post hsct for primary immunodeficiency was 9.8% (6/61), 59.0% (36/61) and 24.6% (15/61) respectively. in those with severe or profound combined immune deficiency the incidence of cmv was 7.0% (3/43), ebv 53.5% (23/43), and hhv-6 18.6% (8/43). in the 6 patients with chronic granulomatous disease, cmv incidence was 17% (1/6), and ebv and hhv-6 were both 50% (3/6). in recipients with pre-transplant negative pcr for ebv, cmv and hhv-6 (r-), time to dnaemia post transplant is seen in figure 1 . ptld was seen in 2 patients with rag1 and il2r, both of whom were d+r-for ebv status with ebv dnaemia, and one of whom died attributed to ptld. two year mortality for all patients was 23% (14/61), with mortality 24.4% (11/45) for those with either cmv, ebv or hhv-6 dnaemia vs 18.8% (3/16) in those without (p=0.64). disseminated cmv prior to transplant was attributed to cause of death for one case. conclusions: cmv incidence was rare, likely from screening for cmv negative hsct donors. cmv, ebv and hhv-6 dnaemia was not associated with differences in mortality in this cohort. ebv incidence was common, and ptld incidence was similar to previously published outcomes for hsct for other disease (2) . the advent of cytotoxic t cell therapy for ebv may help abrogate this risk. professor, senior physician, ulm university medical center, pediatrics, germany introduction/background: new-onset aiha occurs in 2-6% of pediatric patients post-hsct. incomplete immune recovery may predispose to immune dysregulation following hsct including autoimmune cytopenias. although prednisolone or other immunosuppressive drugs control most episodes, some patients respond incompletely to first or second line therapies including rituximab. objectives: we describe an innovative therapy for post-bmt aiha refractory to proteasome inhibition. three patients responded to anti-cd38 antibody (daratumumab) therapy after failing treatment with bortezomib. methods: we retrospectively evaluated data from three patients treated with daratumumab for post-transplant aiha. patients 2 and 3 were treated according to the positive response reported for patient 1. results: aiha occurred between 4-9 months following hsct. daratumumab was curative in 2 patients, the third one had only transient response and relapsed 5 months after this treatment had been initiated. following daratumumab patients no longer required any prbc transfusions. conclusions: in potentially life-threatening aiha in the context of hsct daratumumab may be an effective rescue therapy in combination with rituximab. early post-natal thymus development is strictly dependent on the level of foxn1 expression in tec these infants present severe t cell lymphopenia early in life, but in most cases their immune system gradually normalizes. however, the role of foxn1 haploinsufficiency in causing this phenotype is unclear. objectives: to analyze t cell development and tec phenotype in nu/+ mice of various age, in order to investigate whether foxn1 haploinsufficiency in mice results in impaired thymic development early in life, followed by progressive normalization, thereby recapitulating the human phenotype. methods: we analyzed 3 groups of nu/+ and +/+ littermates, divided by age: 1 day, 4-7 days, 3 weeks. the number of etp and the distribution of cortical and medullary tecs (ctecs, mtecs) were analyzed by flow cytometry. maturation of mtecs was further assessed by staining for mhc-ii and aire. real-time pcr was used to analyze the expression of ccl25, cxcl12, dll4, and scf, four key foxn1 target genes. the study was performed in accordance to niaid animal protocol lcim 6e. results: at 1 day and 4-7 days of life, nu/+ mice showed a dramatic reduction of etps, both in terms of frequency and absolute numbers, as compared to +/+ mice. however, by 3 weeks of age, the frequency and count of etps were comparable in nu/+ and +/+ mice. a slight but significant reduction in the frequency and absolute count of mtecs was observed in all three groups of nu/+ mice. additionally, the ratio between mtec expressing high levels of mhcii (mtechi) and those expressing low levels of mhcii (mteclo) was always higher in +/+ mice as compared to nu/+ mice. moreover, mtechi cells in nu/+ mice expressed lower levels of aire, the gene crucial for thymic negative selection of autoreactive t cells. finally, as compared to wild-type littermates, nu/+ mice showed reduced thymic expression of ccl25, cxcl12, dll4, and scf at day 1. reduced expression of ccl25 persisted at day 5, but normalized at 3 weeks. conclusions: these data indicate that foxn1 haploinsufficiency in mice leads to impaired thymic colonization by etps and abnormalities of tec differentiation and maturation early in life, followed by progressive normalization, thereby recapitulating what observed in newborns with heterozygous foxn1 mutations. these observations have important implications for the management of these infants, who should be monitored closely without rushing to definitive treatment for scid. introduction/background: foxn1 is the master regulator gene for the development and maturation of the thymic epithelial cells (tecs). by inducing the expression of chemokine receptors such as ccl25 and cxcl12, foxn1 also allows the migration of early thymic progenitor (etp) cells from the bone marrow. lack of foxn1 leads to the nude (nu)/severe combined immunodeficiency (scid) phenotype in humans and mice. recently, a number of newborns have been identified with low t cell receptor excision circles (trecs) at birth, associated with heterozygous foxn1 mutations. these infants present severe t cell lymphopenia early in life, but in most cases their immune system gradually normalizes. however, the role of foxn1 haploinsufficiency in causing this phenotype is unclear. objectives: to analyze t cell development and tec phenotype in nu/+ mice of various age, in order to investigate whether foxn1 haploinsufficiency in mice results in impaired thymic development early in life, followed by progressive normalization, thereby recapitulating the human phenotype. methods: we analyzed 3 groups of nu/+ and +/+ littermates, divided by age: 1 day, 4-7 days, 3 weeks. the number of etp and the distribution of cortical and medullary tecs (ctecs, mtecs) were analyzed by flow cytometry. maturation of mtecs was further assessed by staining for mhc-ii and aire. real-time pcr was used to analyze the expression of ccl25, cxcl12, dll4, and scf, four key foxn1 target genes. the study was performed in accordance to niaid animal protocol lcim 6e. results: at 1 day and 4-7 days of life, nu/+ mice showed a dramatic reduction of etps, both in terms of frequency and absolute numbers, as compared to +/+ mice. however, by 3 weeks of age, the frequency and count of etps were comparable in nu/+ and +/+ mice. a slight but significant reduction in the frequency and absolute count of mtecs was observed in all three groups of nu/+ mice. additionally, the ratio between mtec expressing high levels of mhcii (mtechi) and those expressing low levels of mhcii (mteclo) was always higher in +/+ mice as compared to nu/+ mice. moreover, mtechi cells in nu/+ mice expressed lower levels of aire, the gene crucial for thymic negative selection of autoreactive t cells. finally, as compared to wild-type littermates, nu/+ mice showed reduced thymic expression of ccl25, cxcl12, dll4, and scf at day 1. reduced expression of ccl25 persisted at day 5, but normalized at 3 weeks. conclusions: these data indicate that foxn1 haploinsufficiency in mice leads to impaired thymic colonization by etps and abnormalities of tec differentiation and maturation early in life, followed by progressive normalization, thereby recapitulating what observed in newborns with heterozygous foxn1 mutations. these observations have important implications for the management of these infants, who should be monitored closely without rushing to definitive treatment for scid. introduction/background: immunoglobulin g4rd is an immunemediated disease most commonly seen in middle-aged and older men. clinical features include autoimmune pancreatitis, salivary gland disease, orbital disease and retroperitoneal fibrosis. pathologic features include a lymphoplasmacytic infiltrate enriched in igg4-positive plasma cells and fibrosis in a storiform pattern. laboratory evaluation usually reveals an elevated serum igg4 concentration, and glucocorticoids are often used as treatment in early stages of disease. however, disease may recur off of steroids, and prolonged illness or steroid non-responsive disease is usually treated with rituximab. objectives: the objective of this case presentation is to discuss a presentation of a igg4rd is a young adult male. results: 16 year old male adopted from thailand, initially presented to hospital with five days of intermittent abdominal pain, night sweats and worsening fatigue. ct abdomen and pelvis revealed bronchiectasis in lung bases, hepatic masses and soft tissue infiltration in the porta hepatis extending into the liver, pulmonary nodules (read as possible metastases), bilateral renal masses, retroperitoneal nodes and ileocolic intussusception secondary to possible lymphoma. one year prior to presentation he developed new onset bilateral cervical lymphadenopathy. biopsy revealed a heterogenous lymphoid population and lymphoma was ruled out. he had a repeat lymph node biopsies during the admission, which was all negative for malignant cells, and was compatible with reactive lymphoid tissue with plasmacytosis. immunophenotype showed 59% lymphocytes with normal cd4/cd8 ratio, and 8% cd4-,cd8-, tcr ++ t cells. immunohistochemical stain of a lymph node fine needle biopsy showed a mixture of cd20+ b and cd3+ t cells, abundant cd138+, igg+ plasma cells(pcs) , with some igg+ pcs(ct of the neck and chest was completed for possible staging and revealed cervical lymphadenopathy, nasal polyposis, a soft tissue mass vs. enlarged lateral rectus muscle (left orbit), and mediastinal lymphadenopathy. a diagnosis of autoimmune lymphoproliferative syndrome (alps) was presumed by the hematologic/oncologic team given his elevated igg (6,682 mg/dl) and elevated vitamin b12 (>2000) with multiorgan involvement. alps panel revealed a mutation in faslg (allele 1, c.-2c>t) which is a variant of uncertain clinical significance. alps criteria/scorewas + for 1/4 criteria (cd3+cd25+/hla dr ratio < 1.0). he had been referred to the nih for further management of alps before initial presentation to our office. immunologic work up revealed immunoglobulins of igg: 6,600 mg/dl, iga: 83 mg/dl, igm: 71 mg/dl; 13/23 protective streptococcal titers (> 1.3 mcg/ml); hib: 0.46 mg/l; negative quantiferon gold, and hiv-serology. at that point igg4rd vs. castleman disease was suspected instead of alps. further work up showed a normal il-6 level, and human herpes virus 6 was also negative. igg subsets showed an elevated igg1 (2670 mg/dl), igg2 (1450 mg/dl), igg3 (355 mg/dl) and normal igg4 (20.9 mg/dl). excisional lymph node biopsy was recommended to rule out castleman syndrome or igg4rd. a left salivary gland was removed and showed mainly igg4 positive pcs with no multicentric lymphocytic infiltrates. dilution of the patients serum to determine if there was a prozone affect that minimized the igg4 level showed an elevated igg4 level of 2700.9 mg/dl. this is in the 1st percentile of all cases of igg4rd in terms of serum igg4 concentration. he was diagnosed with igg4rd, a form previously called mikulicz disease, which is comprised of lacrimal and parotid gland enlargement. rituximab was given initially because of the severity of his disease. after two cycles he showed decreased lymphadenopathy, notable weight gain and marked decrease in fatigue. conclusions: igg4rd is a rare and complex immunologically based disease process rarely seen in children or adolescents. patients with igg4rd often undiagnosed at initial evaluation. normal serum igg4 levels are seen in 40% of patients with igg4rd and thus a normal igg4 level should not be used as a biomarker to make the diagnosis of igg4rd or in treating this disease. furthermore, the possibility of having a prozone affect in measuring igg4 needs to be considered and evaluated. meticulous correlation of clinical, pathologic, and imaging findings is required to make the diagnosis. modelling human immune deficiency from novel missense mutations with orthologous heterozygous mutations engineered in mice by crispr/cas9 introduction/background: next generation sequencing has resulted in substantial progress in identification of mendelian immune deficiency syndromes. in some cases, however, putative causal mutations occur in single kindreds, or even individual patients. under these circumstances, functional analysis of patient derived cells combined with in vitro analysis of genetically manipulated cell lines can provide additional evidence in support of genetic causation, but this might not be conclusive. objectives: understanding how genetic defects result in complex syndromes of immune deficiency and immune dysregulation can be impossible to achieve in vitro. one method for overcoming these obstacles is to generate accurate mouse models of human immune deficiency methods: mouse models of human immune deficiency are a valuable tool in which the murine genome is engineered to introduce a mutation orthologous to that discovered in the patient. we have applied this strategy to elucidate causation and mechanism of immunological defect in several mutations affecting the nf-kb pathway. results: so far, defects in both canonical and non-canonical pathways of nf-kb activation have been shown to cause immune deficiency, often associated with immune dysregulation. we describe a known defects and novel putative defect identified in the canonical nf-kb pathway conclusions: crispr-cas9 mouse models can be used to elucidate mechanism of disease and provide compelling evidence that mutations are causative. introduction/background: primary immune deficiencies (pid) with or without immundysregulation are rare diseases resulting from monogenetic aberrations leading to either infections or autoimmune manifestations or both. early diagnosis and treatment are crucial for reducing morbidity and mortality. beside genetic diagnosis not commonly yet performed, current standard methods for early diagnosis are dependent on either fresh samples or limited to certain cell types. to overcome those limitations, especially in newborn screening, a novel technology of methylation-based qpcr can be applied. among the known epigenetic modifications, dna demethylation is the most stable and genomic loci with highly cell type specific demethylation sites can be identified. differential methylation can be measured from blood samples of limited availability, or with suboptimal storage. we previously showed that thymic-derived t population, and determine whether there are unique diagnostic and treatment considerations within this demographic. whitney goulstone 1 , elizabeth tough 2 1 executive director, canadian immunodeficiencies patient organization 2 lpn, alberta health services introduction/background: primary immunodeficiency diseases (pids) represent a significant collection of immune system disorders that increase susceptibility to infection, which in some cases are serious or life-threatening. patients with pid often require immunoglobulin g (igg, commonly referred to as ig) replacement therapy to prevent infections and associated comorbidities. pid treatment, in addition to symptoms and associated social and emotional impacts, has a significant impact on patients quality of life (qol). information available on the real-world diagnosis, management, and outcomes of the canadian pid patient population is limited. objectives: to better understand diagnosis and treatment of canadian patients with pid, we surveyed canadian patients with pid associated with the canadian immunodeficiencies patient organization (cipo) the primary goal of the survey was to gain insight into the canadian pid patient population with regards to demographics, diagnosis, treatment, including regimes, qol, and communication and support . methods: the authors conducted a cross-sectional survey to measure health-related qol in a cohort of patients with pid. eligible participants were identified through the canadian immunodeficiencies patient organization (cipo). the questionnaire consisted of 61 questions that covered patient-reported outcomes including diagnosis, qol, treatment regimes, and communication. results: surveys were returned by 149 patients with pid. participants conveyed significant impact on qol including personal, occupational, financial, emotional, and social impacts as a result of pid symptoms, risks, and treatment logistics, limitations, and side effects. the most common diagnoses were related to b-lymphocyte disorders (60.4%). common treatments include intravenous immunoglobulin (ivig; in hospital) and subcutaneous immunoglobulin (scig; at home), in addition to antibiotics and antifungals as required. respondents reported feeling average before treatment on a scale of 0-10 (mean 5.46 ± 2.23) with increased health after treatment (mean 6.66 ± 2.32). respondents felt current treatment was convenient (mean 8.22 ± 1.94) and were comfortable with self-infusions (mean 6.96 ± 3.46). conclusions: patients with pid are not uncommon in the canadian community, and in these patients pid is associated with a significant impairment in qol. experiences range with regards to a particular treatments advantages and disadvantages, cost, travel, and convenience. respondents hope to achieve improved qol through the following solutions: better treatment, improved infusions, gene modification, more research and clinical trials, a cure, and education and outreach. improved financial, medical, and social supports were also requested. introduction/background: severe combined immunodeficiency (scid), the most severe form of t cell immunodeficiency, is detectable through quantification of t cell receptor excision circles (trecs) in dried blood spots (dbs) obtained at birth. for many professional, humanitarian and financial reasons, newborn screening (nbs) for scid is warranted. implementation of this screening test is highly important where high frequency of consanguinity is known to exist. objectives: since october 2015, israel has conducted national scid nbs. this important, life-saving screening test is available at no cost for every newborn in israel. methods: herein, we describe two years results of the israeli scid newborn screening (nbs) program. validation includes cbc, lymphocyte subsets, trec (in a different method), t cell receptor (tcr) repertoire and response to mitogenic stimulation. whole exome sequence (wes) for genetic detection, and next generation sequencing (ngs) to demonstrate tcr clonality are used as well, in some unsolved cases. results: of 396,159 births screened, 719 (0.18%) had abnormal trec in their first screen (448 terms, 271 pre-terms), 69 (0.016%) had a repeated abnormal trec also in their second screen and were referred for a validation process. fourteen scid patients were diagnosed, so far, through the nbs program in its first years, revealing an incidence of 1:28,000 births in the israeli population. consanguine marriages and muslim ethnic origin were found to be a risk factor in affected newborns, and a founder effect was detected for both il7r and dclre1c deficiency scid. other diagnoses were made as follows: 8 cases were found to have t cell lymphopenia; 16 cases were diagnosed with syndromes; 4 cases were found to have secondary t cell lymphopenia; 10 cases were pre-term infants and 18 cases were considered as false positive with normal evaluation. lymphocyte subset analysis and trec quantification in the peripheral blood appear to be sufficient for confirmation of typical and leaky scid and ruling out false positive results. detection of secondary targets (infants with non-scid lymphopenia) did not significantly affect the management or outcomes of these infants in our cohort. we could also report several perspectives regarding t cell development in non immunodeficient newborns that emerged from the accumulated data. conclusions: already in a short term, trec nbs in israel has achieved early diagnosis of scid and other conditions with t-cell lymphopenia, facilitating management and optimizing outcomes. this program has also enabled gaining insights on t cell development in health babies. (hct) . surprisingly, 76% of these infections were acquired during the interval between confirmation of the scid diagnosis and hct (heimall, blood 2017). to investigate further, in late 2017 we surveyed pre-hct management practices for scid patients at pidtc centers. 51 physicians representing 43 north american centers responded, including 18 immunologists, 25 transplant specialists and 7 who identified as both. 33% of centers lacked a standard procedure for the management of infants with a positive nbs result. to confirm the scid diagnosis, basic t, b and nk cell flow cytometry was performed by most. testing for naïve t cells was generally included, but testing of lymphocyte mitogen proliferation was inconsistent. specialists were notified of a patients positive nbs test at median age 7.5 days (2-30 days) , and management of patients as scid was started at median age 9 days (0-90 days). when unconditioned hct was anticipated, 72% of respondents began planning as soon as possible after diagnosis, while 20% awaited genetic testing results before hct. respondants consistently implemented pre-hct prophylaxis with trimethoprim/sulfamethoxazole (84%), fluconazole (80%) and immunoglobulin infusions (96%), although timing of initiation varied. palivizumab was used by 26%. there was little consensus regarding viral monitoring. although most physicians screened for cmv by blood pcr (85%), only about half routinely screened for ebvor adenovirus. while 44% of physicians started prophylaxis against double-stranded dna viruses in all patients, 46% did so only in selected situations, such as active genital hsv at the time of delivery, or when the mother was cmv-seropositive. although all centers used only acyclovir as antiviral prophylaxis, doses and timing varied widely: from 25-90 mg/kg/day, divided into 2 or 3 doses, continued in most centers until immune reconstitution. finally, 84% of physicians recommended that cmvseropositive mothers stop breast-feeding. there was no consensus on where patients should reside prior to hct. hospital or home were favored equally, although need for a reliable family was indicated by 88% as a criterion for home-based management. for hospitalized patients, 62% of centers required patients to be in a reverseisolation/positive-pressure room and over half required staff to wear gown, gloves, and mask. with regard to visitors, 75% required parents/ relatives to perform hand hygiene. approximately 75% did not require a gown, gloves, or mask for visitors. finally, there was no consensus on allowing siblings or friends to visit, but the majority permitted grandparents or other adult relatives. this survey revealed wide variability in the diagnostic pathway, viral surveillance, and isolation practices for scid patients, although pretransplant prophylaxis with immunoglobulin, fluconazole, and trimethoprim/sulfamethoxazole were utilized consistently. we conclude there is considerable opportunity to develop diagnostic and pre-hct management pathways for scid. prospective tracking of management practices could reveal which are important for avoiding pre-hct infections. evidence-based practice guidance is needed to maximize the potential to bring each scid patient identified via nbs to hct infection-free. ikaros/ikzf1 is an essential transcription factor expressed throughout hematopoiesis. in humans, somatic mutations in ikzf1 are linked to b-cell acute lymphoblastic leukemia (all), and germline heterozygous haploinsufficient mutations cause common variable immunodeficiency-like disorder with incomplete penetrance. herein, we report seven unrelated patients with an earlyonset novel combined immunodeficiency associated with de-novo, fully-penetrant, germline heterozygous dominant negative mutations affecting amino acid n159 in ikzf1 dna binding domain. patients presented with different infections, but pneumocystis jirovecii pneumonia was common to all. one patient developed a t-cell all. additional findings included decreased b-cells, neutrophils, eosinophils and myeloid dendritic cells as well as t-cell and monocyte dysfunction. t-cells exhibited a profound naïve/recent thymic emigrant/ t-helper 0 phenotype and were unable to evolve into effector memory cells; monocytes failed to respond to different stimuli or facilitate t-cell activation. this new defect expands the spectrum of human ikzf1-associated immunodeficiency diseases from haploinsufficient to dominant negative. the beta subunit of the il-2 receptor (il2rb; cd122) is essential for il-2 and il-15 mediated signal transduction in a variety of hematopoietic cell types including t and nk cells. here we report the clinical and immunologic phenotypes of two siblings born to consanguineous parents harboring a variant in il2rb, resulting in autoimmunity, with lymphoproliferation of cd8+ t and cd56hi nk cells, and decreased regulatory t cell frequency. the proband presented with inflammatory enteropathy, failure to thrive, and disseminated cmv infection at 2 months of age, and later developed atopy, lymphocytic interstitial pneumonitis, and red blood cell autoantibodies. his younger sister presented with severe autoimmune hemolytic anemia and cmv viremia at 2 months of age, and later developed lymphocytic interstitial pneumonitis. whole exome sequencing and chromosomal microarray studies revealed a homozygous deletion within the highly conserved wsxws motif of the extracellular domain of il2rb (c.665_673delcctggagcc, p.pro222_ser224del). the deletion results in reduced il2rb expression (cell surface and intracellular) in t and nk cells. the functional consequences of the defect include complete impairment of stat5 phosphorylation through the il-2 receptor but only partial impairment through the il-15 receptor, as well as a compensatory increase in serum il-2 and il-15 levels (>100 pg/ml). cd8+ t cell proliferation responses to in vitro t cell receptor (tcr) stimulation were reduced compared to an age-matched, healthy control, but partially rescued by supraphysiologic levels of il-2 and il-15. despite reduced cd8+ t cell proliferation responses, the proband displayed a t cell population skewed toward cd8+ t cells, with an oligoclonal expansion of effector memory cells. arguing against effects of pervasive cmv infection alone, the sister displayed similar phenotypic and functional abnormalities at birth, prior to her cmv infection. our data suggest that the identified hypomorphic mutation in il2rb results in a survival advantage for those cd8+ t and cd56hi nk cells most sensitive to the exuberant serum il-2 and il-15 levels produced in response to the defect. these surviving cd8+ t and cd56hi nk cells have great lymphoproliferative potential, leading to multisystem autoimmunity and inflammatory complications. therefore, we describe il2rb deficiency as a novel primary immunodeficiency disease with prominent immune dysregulation and selective cd8+ t and cd56hi nk cell lymphoproliferation. key: cord-009567-osstpum6 authors: nan title: abstracts oral date: 2008-04-23 journal: am j transplant doi: 10.1111/j.1600-6143.2008.02254.x sha: doc_id: 9567 cord_uid: osstpum6 nan abstracts evl (target 3-8ng/ml), aza or mmf with standard (sd) or reduced (rd) csa. data at 6 months post-tx is presented here. results. the proportion of patients with cmv events, including serious adverse events and laboratory evidence of cmv infection, has remained low and broadly similar following introduction of cc administration of evl and use of concomitant rd-csa. cmv syndrome and cmv organ involvement occurred in <3% of recipients receiving evl in each of the studies. the highest rates of all cmv parameters occurred with mmf + sd-csa or aza + sd-csa. conclusion. the low incidence of cmv infection and cmv-related events observed in heart tx patients receiving fd-evl and sd-csa as de novo immunosuppression has been maintained in newer regimens of cc-evl with rd-csa. cmv syndrome and cmv organ involvement are rare with evl-based immunosuppression after cardiac tx. the low rate of cmv infections in evl-treated patients may contribute to improved long-term outcome and a lower incidence of cav; confirmatory data are awaited. fibronectin-α 4 β 1 interactions enhance p38 mapk phosphorylation and metalloproteinase-9 expression in cold liver ischemia/reperfusion injury. sergio duarte, 1 xiu-da shen, 1 takashi hamada, 1 constantino fondevila, 1 ronald busuttil, 1 ana j. coito. 1 1 the dumont ucla transplant center. expression of endothelial fibronectin (fn) is an early event in liver ischemia/reperfusion (i/r) injury. we have recently shown that cs1 peptide facilitated blockade of fn-α4β1 leukocyte interactions regulates metalloproteinase-9 (mmp-9) expression in steatotic orthotopic liver transplants (olt). this study tests the function of the cs1 peptide therapy upon mmp-9, and further dissects putative mechanisms, in an alternate model of cold ischemia/reperfusion (i/r) injury. methods and results: cs1 peptides were administrated through the portal vein of sprage-dawley (sd) rat livers before and after 24 h cold storage (500 µg/rat). sd recipients of olts received an additional dose of cs1 peptides 1h post-olt. cs1 therapy significantly increased the 14d olt survival rate (100% vs. 50%, n=8/gr, p<0.005). cs1 peptides reduced sgot levels (u/l) at 6h (1413±420 vs. 2866±864 p<0.008) and 24h (1350±142 vs. 4000±1358, p<0.006) post-olt. cs1 treated olts showed good preservation of lobular architecture, contrasting with severe necrosis and sinusoidal congestion in controls. moreover, cs1 treated livers were characterized by a profound decrease in t (31±3 vs. 64±8, p<0.0002), nk (19±2 vs. 41±3, p<0 .0003) and ed1 (21±7 vs. 57±16, p<0.008) cells as early as 6h after i/r. neutrophils, as indicated by mpo activity (0.48±0.02 vs. 3.18±0.94, p<0.02) were depressed by cs1 therapy. this correlated with decreased mrna expression of tnf-α (0.032±0.04 vs. 0.83±0.26, p<0.005) and cycloxygenase-2 (0.8±0.1 vs. 2.2±0. 6, p<0.05) . leukocyte transmigration is dependent upon adhesive and focal matrix degradation mechanisms. mmp-9, which is inducible and expressed by infiltrating leukocytes, was profoundly depressed in 6h cs-1 peptide treated olts, at both mrna (0.1±0.1 vs. 0.5±0.2, p<0.03) and protein (0.15±0.07 vs. 0.7±0.14, p<0.03) levels. moreover, mmp-9 activity evaluated by zymography was reduced by ∼3-fold in the cs-1 group. interestingly, phosphorylation of mitogen-activated protein (map) kinase p38 (0.05±0.01 vs 0.14±0.06, p<0.03) was selectively downregulated in the 6h cs-1 treated olts. in conclusion, this work supports a broad regulatory role for fn-α4β1 interactions on mmp-9 expression by leukocytes, likely mediated through activation of p38 mapkinase, and matrix pathological breakdown associated with leukocyte infiltration. this data provides the rationale for the development of novel therapeutic approaches in cold liver i/r injury. ischemia reperfusion injury is the most common cause of acute kidney injury in both native and allograft kidneys. the pathogenic mechanisms of renal ischemia reperfusion injury include changes at the level of the microvasculature as well as the tubules. within the microvasculature, leukocyte-endothelial interactions likely play a role and contribute to the well-established microvasculature dysfunction (e.g., endothelial permeability) and alterations in endothelial-leukocyte interaction occur during ischemic acute kidney injury. our previous studies have demonstrated that t cells modulate renal ischemia reperfusion injury in a murine model, we hypothesized that t cells could mediate changes in renal vascular permeability during ischemia reperfusion injury. we performed a 30 min bilateral renal ischemia followed by reperfusion in c57bl6 wild type mice and in t cell deficient (nu/nu) mice with or without t-cell adoptive transfer from their wild type littermates and evaluated rvp by evans blue dye extravasations (ebde). the time course studies of rvp showed marked increases in renal ebde within the early 3-6 hrs after ischemia. cd3 positive pan t-cells but neither cd4 nor cd8 t cells were found to infiltrate into post ischemic kidney within 6 hrs, and comparison was made with other leukocytes using immunohistochemistry technique. gene microarray analysis demonstrated that the gene for tnf-α (tnfaip1), a potent mediator of microvascular permeability as well as a t cell product, was increased in the kidney early after ischemia. tnf-α, ifn-γ and il-4 protein by an intracellular cytokine staining technique was found increased early in peripheral circulating t cells and later in renal t cells after renal ischemia. the rise in rvp was significantly attenuated in t cell deficient mice nu/nu at 6 hrs compared to the wild type littermates and this attenuated rvp in t cell deficient mice was restored after adoptive transfer of splenic t cells from wild type littermates into these nude mice. these data demonstrate that t cells traffic early into post ischemic kidney, produce tnf-α and other cytokines that can increase rvp, and directly participate in the increased rvp during acute ischemia reperfusion injury. t cell-endothelial interactions are a likely mechanisms underlying the pathophysiologic role of t cells in renal ischemia reperfusion injury . background: ischemia/reperfusion injury (iri) is a major cause of organ dysfunction after intestinal injury and transplantation. the interaction between the innate and adaptive immune systems has become a major area of focus in this field. our preliminary work showed that mice undergoing intestinal iri experienced worse survival and tissue injury in conjunction with increased infiltration of pmn and cd3+ cells as compared to sham mice. the purpose of this study was to investigate the role of the t cell in intestinal iri through the use of genetically deficient mice. methods: under anesthesia, male c57bl6 wild-type mice (wt) and cd4 knockout mice (cd4ko) underwent 100 min of warm intestinal iri by clamping of the sma. separate survival and analysis groups were performed. intestinal tissue was harvested at 4h and 24h. tissue was analyzed by histology, cd3 immunostaining, myeloperoxidase activity (mpo), and semi-quantitative pcr for several cytokines/chemokines. results: wt had significantly worse survival compared to cd4ko (30% vs. 100%, p=0.002), and worse histopathological injury mostly involving mucosal sloughing. wt had higher mpo activity than cd4ko (1.9±0.88 vs. 0.70±0.73 at 4h, p=0.059, and 1.5±0.05 vs. 0.22±0.065 at 24h, p=0.004) and increased cd3+ cell infiltration. there was also increased mrna production of cytokines/chemokines in wt vs. cd4ko at both timepoints; specifically, the data with respect to b-actin at 24h was: il-2 (0. conclusion: this study demonstrates for the first time in the intestine that cd4+ t cells are important mediators of iri. in the absence of cd4+ t cells, better outcomes were associated with less pmn infiltration implying a link between the innate and adaptive immune systems. an alteration in chemotactic signaling potentially effected by cd4+ t cells may play a role in this process. these results confirm the important function of cd4+ t cells in intestinal iri and justify further investigation into their complex role in this process. messenger rna assessment in urine sediments from renal transplant patients is rapidly evolving as a non-invasive diagnostic tool. t cells, macrophages, and often b cells are present in rejecting kidney grafts. we questioned whether urinary mrna levels of markers representing these cell types ((regulatory) t cells: cd3ε, foxp3, cd25, tgf-ß; macrophages: cd68, s100a9; b cells: cd20) are associated with rejection. materials in our institute 520 urine samples a year from over 100 renal transplant patients are being collected for mrna quantitation purposes. urine sediments are pelleted and stored in rna-preserving solution. we initially investigated the effect of incubation time of urine on mrna integrity. next, in a case-control study 17 urine samples taken at time of biopsy-proven rejection were compared to 17 samples taken during stable graft function. median time of sampling (55 days versus 43 days posttansplant) did not significantly differ between groups. rna (0.90 ± 1.18 µg) was extracted using rneasy spin columns. message of the markers mentioned above was quantified with q-pcr and normalized. results incubation of urine for 7h or longer at room temperature after acquisition resulted in a 75% decrease in 18s rrna signals (p<0.05). however, storage of urine for up to 24h at 4ºc did not result in decreased mrna expression. in the case-control study, all markers tested showed the highest expression levels in urine rejection samples. when corrected for multiple comparisons, only tgf-ß (26-fold, p=0.007) and foxp3 (21-fold, p=0.002) expression was significantly increased in rejection samples compared to non-rejection samples. tgf-ß levels highly correlated with foxp3 levels (r = 0.90, p<0.00001), suggesting a mechanistic relationship in vivo between the two molecules. conclusion rna integrity in urine samples stored at 4ºc is maintained for at least 24h. detection of increased tgf-ß and foxp3 message in urine from patients with a kidney transplant represents a means for indicating occurrence of graft rejection. this implies that mrna urinalysis renders a suitable molecular tool for non-invasive patient monitoring in clinical practice. the data furthermore suggest that rejection is associated with tgf-ß-mediated immune mechanisms in which regulatory t cells play a role. the significance of b cell and plasma cell infiltration in renal allografts remains controversial. we previously established that transcript sets associated with ifng effects or t cells reflect the inflammatory burden in renal allografts. in the present study, we identified b cell associated transcripts (bats) and immunoglobulin transcripts (igts), reflecting b cell and plasma cell infiltration, respectively. using microarrays, we analyzed bat and igt expression in relationship to histologic lesions, diagnosis, and renal function in 177 renal allograft biopsies. immunostaining confirmed that bat and igt expression was associated with b cells and plasma cells in the graft. expression of bats and igts was increased in biopsies with rejection (1.2 ± 0.2, 1.6 ± 0.9) compared to non-rejection (1.1 ± 0.2, 1.1 ± 0.5), but was not different between t cell (1.3 ± 0.3, 1.5 ± 1.1) and antibody mediated rejection (1.2 ± 0.2, 1.5 ± 0.9) and also occurred in some non-rejecting biopsies (recurrent gn). bat and igt scores correlated strongly with time post transplant (fig1), which was best modeled as a dichotomous relationship: biopsies ≤ 5 months did not express bats or igts above the level of control kidneys. other inflammatory markers were not time dependent. in biopsies ≥ 5 months, bat and igt expression correlated with interstitial inflammation, tubular atrophy, and interstitial fibrosis. in a multiple regression analysis, only time post transplant and interstitial inflammation were independently related to bat and igt scores. when correcting for time post transplant, bat and igt scores did not correlate with renal function at the time of biopsy or future function 6 months post biopsy. bats and particularly igts are a time dependent feature of injured and inflamed renal allografts. corrected for the effect of time, bats and igts do not correlate with outcomes, indicating that b cell and plasma cell infiltrates have no specific role for the mechanism of injury independent of the inflammatory burden. their accumulation in late allografts may indicate emergence of specialized lymphoid compartments in tissues with long standing low level inflammation. pearson correlation coefficient between pbts expression and renal function pbts egfr at %change in egfr from biopsy 6 months after biopsy baseline to biopsy biopsy to 6 months after qcats -0.17* -0.16* -0.19* -0.05 grits -0.23** -0.25** -0.20** -0.10 irit_d1 -0.02 -0.01 -0.09 0.13 irit_d3 -0.51** -0.29** -0.36** 0.16* irit_d5 -0.28** -0.22** -0.19* -0.02 kt2 0.27** 0.13 0.22** -0.02 * p<0.05, ** p<0.01 thus the pbts have prognostic value. surprisingly, the transcripts in the biopsy with greatest prognostic value for future gfr or recovery of gfr were not related to rejection (cytotoxic t cell or ifng associated) but to the degree of injury response. we propose that the common pathway linking various injuries (immune and non immune) to gfr is through the degree of injury response these events induce in the epithelium, particularly those in the irit_d3 set. early rejection from anamnestic response in offspring-to-mother or husband-to-wife kidney transplant. kwan tae park, 1 song chul kim, 1 duck jong han. 1 1 surgery, asan medical center, seoul, korea. accelerated rejection can be developed in the immediate post-transplant period resulting from anamnestic response due to the exposure to fetal hla antigen during the previous pregnancy in case of offspring-to-mother or husband-to-wife. however, accelerated rejections in these groups have been rarely reported. 81 cases of offspring-to-mother (offspring group) and 53 cases of husband-to-wife (spouse group), who has been sensitized to spouse via their children, underwent kidney transplants from january of 1997 to august of 2007 at our institution and retrospectively reviewed. control group was female kidney recipients transplanted at the same period from living related donors other than offspring and from living unrelated donors other than husband. acute rejection (ar) rate within 3 months after transplant were 19.7% (16/81) in offspring group and 18.8% (10/53) in spouse group. the ar rates were not different between the two groups, however they were significantly higher than control group in both groups, namely 10.2% (24/235) for offspring control and 13.5% (10/74) for spouse control. the mean onset of ar was 7.5 day (0-29) and it was significantly later than that of control groups (16.5 day, p<0.05) and 54% of ar were accelerated rejections within 7 days after transplant. proportion of acute humoral rejection was significantly higher in both groups than control group (37.5% vs 8.3% in offspring group, 60% vs 10% in spouse group). most of the ar was successfully reversed by steroid pulse and/ or plasmapheresis, ivig, rituximab. any risk factors for the ar such as number of pregnancy, preoperative cdc cross matching, pra, immunosuppressant and antibody induction couldn't be identified. serum creatinine level in ar patients were higher than ar free patients by postoperative 1 month, but last follow up creatinine level didn't show any statistical difference (1.43 mg/dl in ar vs 1.14mg/dl in ar free). 5 year graft/patient survival rate were not different between ar and ar free groups and study and control groups. risk of higher rejection rate accompanied with higher accelerated and humoral rejection was identified in female kidney recipients from offspring or spouse donor in comparison with control group. considering the anamnestic response of this cohort of female recipients, more prudent preoperative screening and careful immediate postoperative immune monitoring are required for the avoidance of rejection and impaired graft survival. kaplan-meier survival curves showed that ∆upc > 0.2 were associated with decreased patient and allograft survival. these data show for the first time that regardless of the histopathology, ∆upc > 0.2 one month after rejection is associated with poor patient/allograft outcomes. from histology to microarrays the histopathological hallmark of t cell mediated rejection (tcmr), is interstitial infiltration. assessing infiltration by histology is arbitrary, limited in reproducibility, and has never been assessed against independent standards. we recently reported that a set of cytotoxic t cell-associated transcripts (qcats) could quantitatively assess the t cell burden in tissue. objective of the present study was to re-examine the current diagnostic criteria in relationship to the qcats. in 129 renal allograft biopsies, we assessed how histology predicts the qcat burden. an independent diagnostic threshold for qcats was established in control samples. applying this qcat threshold revealed current diagnostic criteria for histology to be flawed; the threshold for interstitial infiltration is too high (100% specificity, 37% sensitivity), the types of infiltration to be considered (i.e. i-banff) are wrongly defined, and tubulitis is not increasing diagnostic accuracy. changing the criteria by lowering the histological threshold for cortical infiltration from 25% to 10%, taking into account all interstitial cellular infiltration (= i-total) and ignoring tubulitis increased sensitivity to 91% with a decreased specificity of 50%. but, this includes biopsies having interstitial infiltration without qcats, indicating that histology might not discriminate between 'active' and 'inactive' infiltration. both the refined histological and the qcat threshold had prognostic value in terms of future renal allograft function. we propose a refined histological scoring system that better predicts the active t cell burden and outcome than the current banff criteria for renal allograft rejection. there has been an increasing and well justified interest regarding the long term renal consequences of kidney donation. the collective evidence suggests, however, that kidney donors enjoy a normal life span and their lifetime risk of esrd may not be different from non-kidney donors. assessing kidney function utilizing serum creatinine is not without limitations. therefore, to better assess kidney function, 5 yeas ago, we began a large effort to measure gfr using the plasma disappearance of iohexol and first void urinary albumin/creatinine ratio (acr) in randomly selected kidney donors who donated at our institution. results: we have performed over 3600 donor uninephrectomies since the inception of our program in 1963. of these, 242 donors underwent iohexol gfr at our general clinical research center. microalbuminuria is defined as acr between 30-300mg/g and macroalbuminuria as acr>300mg/g. the mean age was 53.0±9.6 years, 11.9±8.8 years have elapsed since donation, hemoglobin was 13.7±1.23g/l, systolic blood pressure (sbp) was 121.7±14.7mmhg and diastolic blood pressure (dbp) was 73.2±9.1mmhg. 82.6% of donors had a gfr greater than 60ml/min/1.73m 2 and 17.4% had a gfr between 30-60ml/min/1.73m 2 . the detailed renal profile of these donors is shown in the table below. multivariate analysis that adjusted for age, gender, ethnicity, time from donation, sbp, dbp and body mass index (bmi) identified age at donation, female gender and bmi as independent predictors for gfr<60ml/min/1.73m 2 and time from donation and systolic blood pressure as independent predictors for micro and macroalbuminuria. conclusion: this is the largest effort describing measured gfr in previous kidney donors. it is reassuring to find out that the majorities have a preserved gfr and only a minority has albuminuria. the risk factors for reduced gfr and albuminuria are analogous to what has been described in the general population. cystatin c levels and long-term donor health markers cystatin-c <1 (n=65) psychosocial and physical health of lkds following donation is of utmost importance. unfortunately, this issue has been neglected to a large extent. we sought to examine the current practices regarding psychosocial follow-up of lkds after surgery across transplant (tx) centers. we conducted a 68-question online survey regarding this practice and issues related to lkd. the survey was e-mailed via listservs of 2 professional tx societies. several questions allowed for more than one response. characteristics of the 69 tx centers that participated in the survey are found in table 1 . only 38.3% actively follow donors regarding mental health, substance abuse, or quality of life issues after donation. the professionals most often involved are social workers (72%) and nurse coordinators (69%). the majority of follow-up is conducted via phone (83.3%); though appointments (69.4%) and questionnaires (11.1%) are also utilized. 65% initiate follow-up with donors 1-4 weeks post-operatively, whereas only 27% of programs maintain contact at both 3-6 months and 12 months. 83.3% offer post-operative psychosocial support; 62% only under certain circumstances. this support is provided by social workers (90%), psychologists (30%), and/or psychiatrists (28%). support is available indefinitely in 68% of programs. 30.5% assume the costs of post-operative medical and psychosocial care indefinitely; 50.8% for a specified period of time. 40.7% bill the recipient's insurance and 15.2% bill the donor's insurance. 83.1% of centers accept donors without health insurance and only 5.1% purchase insurance on behalf of donors to cover post-operative health care needs. the results of this survey clearly demonstrate that post-operative psychosocial follow-up of lkds is uncommon and that current practices are widely variable. without routine follow-up of donors, tx centers are less likely to capture post-operative psychosocial issues that may result from organ donation. standardized post-operative follow-up of lkds should become a mandatory part of their care, which will require increased support from health care policy makers. the role of hepatitis c and race in patient and graft survival in combined kidney and liver transplantation. dilip moonka, 1 ravi k. parasuraman, 2 kim a. brown, 1 alissa kapke, 3 dean y. kim. 4 1 gastroenterology, henry ford health systems, detroit, mi; 2 nephrology, henry ford health systems, detroit, mi; 3 biostatistics, henry ford health systems, detroit, mi; 4 transplant institute, henry ford health systems, detroit, mi. the influence of hepatitis c (hcv) and race are not well understood in combined kidney-liver transplant (klt). hcv has a negative impact on patient and graft survival in liver recipients whereas african-american (aa) race is a negative prognostic factor in kidney recipients. aim: to determine the influence of hcv and aa race on patient and graft survival in klt. methods: the unos public use database was used to identify 2296 patients undergoing klt who had known hcv ab status. 13% were aa and 68% were non-hispanic white (nhw). 39.5% were hcv ab positive. groups were assessed for patient and graft survival. results: there is a significant gradient in patient survival and both kidney and liver survival from nhw patients without hcv to aa patients with hcv (table) . the patient survival at five years drops from 72% to 54% along this gradient. there is also a 14% drop in liver and an 18% drop in kidney graft survival. on pairwise testing, the difference in patient survival at 5 yr between all patients without hcv and those hcv ab positive drops from 72% to 60% (p=0.0003). the difference in 5 yr survival between all nhw and aa patients (regardless of hcv status) drops from 68% to 57% (p=0.221). on multivariate analysis, hcv and the combination of hcv and aa race remains associated with diminished survival but race alone does not. conclusions: patients with hcv who undergo a klt transplant are at increased risk for poor overall survival and poor survival of kidney and liver grafts. this effect appears even more pronounced in aa patients. this knowledge is critical to individual centers in assessing risk and benefit from klt in these groups and these groups represent an opportunity for improved interventions. kidney: pediatrics background: pediatric renal transplant recipients have excellent short-term outcomes but long-term success is compromised by complications of chronic immunosuppressive medications and chronic allograft nephropathy. studies show that calcineurin inhibitors and steroids can be individually avoided in pediatric renal transplantation. building on that experience we designed this study to optimize short and long-term renal allograft function with minimal chronic immunosuppression using a steroid-free, calcineurininhibitor withdrawal protocol in low risk pediatric renal transplant recipients. methods: unsensitized pediatric recipients of a first living donor kidney transplant received 2 doses of campath-1h ® (0.3 mg/kg), 1 day pre-and post-transplant. subjects received tacrolimus and mmf immediately post-transplant until week 8-12 when they underwent protocol renal biopsy and were changed to sirolimus and mmf if rejection free. the planned 35 subjects have been enrolled; this report describes the clinical outcomes of the 23 with 1 year of follow up. results: the mean subject age is 12.9 yrs; 59.3% are female and 70.4% caucasian. at transplant, 16/23 were cmv seronegative and 16/23 were ebv seronegative. protocol therapy was discontinued in eight subjects due to: rejection (3), mouth ulcers (2), leukopenia (1) , unrelated (2). clinical acute rejection (ar) occurred in 4 subjects (17%) and 2 had subclinical ar; ar was cellular rejection in 5 subjects, and humoral in 1 at 4 days post-transplant who had an undetected positive crossmatch to class ii hla. there were two graft losses, one due to recurrent fsgs and one due to medication non-adherence. there were no cases of ptld and no deaths. leukopenia occurred in 11 subjects (47.8%). there were 9 infections of which 7 were urinary tract infections (34.7%) and 2 were pneumonia (8.7%). conclusions: minimization of immunosuppression using a steroid-free, calcineurinwithdrawal protocol in low risk pediatric renal transplant recipients appears to be well tolerated with acceptable rates of clinical ar and no serious infections 12 months after transplantation. male; 57% white). substantial increases in bmiz were observed within the first 6 mo.; no changes were seen from 6 to 48 mo. baseline bmiz category (<-2.0, -2.0 to +2.0, >+2.0) influenced the pattern of change. subjects with low bmiz (<-2.0) at baseline experienced the greatest increases in bmiz, but overweight was rare; increases tended to result in a bmiz in the healthy range. those with high bmiz (>+2.0) at baseline demonstrated no significant change in bmiz post-tx. younger age at tx (highest risk for those 2 to 5 y. at tx) more remote date of tx, and baseline bmi between the 25 th and 75 th percentiles were significant independent risk factors for unhealthy weight gain both at 12 mo. and persisting at 48 mo. post-tx. weight gains occur early after tx and tend to persist. counseling focused on prevention of weight gain should be a routine part of post-tx care, with the most intense efforts concentrated on the highest risk patients: young, healthy weight children. avoidance of early weight gains may have an important impact on bmi over the long term. referral. lindsey a. pote, 1 jennifer trofe, 1 erin h. wade, 1 jorge baluarte, 3 alden doyle, 2 simin goral, 2 karen warburton, 2 robert grossman, 2 jo ann palmer, 3 roy d. bloom. 2 1 pharmacy, hosp of the univ of penn; 2 nephrology, univ of penn; 3 transplant surgery, children's hospital of philadelphia. intro: few data exist on outcomes in pediatric kidney recipients who transition to an adult transplant program. purpose: to examine outcomes in pediatric kidney recipients who transition to an adult transplant program and to identify characteristics associated with non-adherence related graft loss. methods: retrospective, single center analysis of 41 pediatric kidney recipients who transitioned to an adult program. results: for the cohort overall, transition to the adult program occurred a mean of 82 ± 8.7 months following transplantation. mean serum creatinine at transition was 2.6±3.2 mg/dl and mean patient age 21.2 ± 0.3 years. 61% of patients received living donor kidneys. within 31±3.5 months of transition, 13 (31.5%) of patients experienced graft loss. causes of graft loss included admitted non-adherence (n=6), recurrent disease (n=3), chronic progressive graft dysfunction (n=3) and bk nephropathy (n=1). graft loss occurred a mean of 16±7 months post transition for non-adherent patients and 20.5± 3 months for adherent patients (p=ns). the characteristics of the cohort is shown in the table according to whether or not patients had documented non-adherence related graft loss. conclusions: 1) graft loss commonly occurs within 3 years following transition and is attributable to both patient non-adherence and late referral by the pediatric transplant program, 2) non-adherence related graft loss was more common in males, 3) factors unassociated with non-adherence include ethnicity, prior transplantation, age at transplant, & duration of dialysis, 4) the development of collaborative pediatric-toadult transition clinics may enhance adherence and lead to improved graft outcomes in this population. this 2-year, prospective randomized pilot study compares the effect of conversion to sirolimus (srl) vs. continued mycophenolate meofetil (mmf) in patients with chronic allograft nephropathy (can), on histological progression. we present 2-year data on safety and renal function. participants >1 year post-transplant with can (banff ≥ci1, ct1) on tacrolimus, mmf and prednisone were randomized to continue mmf or convert to srl (target 8-12 ng/ ml). tac dose was minimized (target 3-5 g/l), and renal biopsy performed at baseline, 1, 2 years. 3-month interval monitoring included adverse event (ae) reporting, egfr (schwartz) and immunosuppressant levels. 16/20 (mmf=10, srl=10) have completed 2-year follow-up. baseline gender, ethnicity, previous acute rejection episodes, can grade, proteinuria (upcr) were similar. srl had lower baseline egfr (79±17 vs. 109±49 ml/min/1.73m 2 , p=0.22). 285 aes were reported (mmf=117, srl=168), 43 serious ae (sae: mmf=25, srl=28). common saes were similar: dehydration & elevated creatinine (30), gastroenteritis (10) and rejection (ar). 2 episodes (1 patient) of ar occurred in mmf group and 5 (2 patients) in srl group, all attributable to non-adherence. there were no sustained change in electrolytes, hg and total wbc counts in the 1 st 2 year, except neutrophil counts were lower in the srl group (24 mo: mean 4.7 vs. 2.8, p<0.05). platelets were significantly lower at 3 months (srl) but not thereafter (24 month: mean 271±71 vs. 196±61 x10e9). cholesterol and tg increased early (p<0.05), but only cholesterol persisted after 2 years (mean 5.2 vs. 3.7 mmol/l, p<0.05). in srl group only, upcr increased over 2 years (∆upcr 94±87 mg/mmol, p<0.05). this was not associated with hypoalbuminemia at 2 years. egfr was lower in srl vs. mmf after 18 months (p<0.01), but the rate of change in egfr from baseline was similar between groups (-28±23 vs. -30±39 ml/min/1.73m 2 , p=ns). serious adverse events did not differ significantly between the srl and mmf groups. sustained srl treatment was associated with mild neutropenia, hypercholesterolemia and proteinuria after 2 years. egfr was reduced at baseline compared with mmf, and both groups had similar rates of decline in gfr over time. allograft recipients. maarten naesens, 1 oscar salvatierra, 1 li li, 1 minnie sarwal. 1 1 department of pediatrics, stanford university school of medicine, stanford, ca. background: in contrast to adult kidney recipients, little is known about the long-term evolution of tacrolimus pharmacokinetics in pediatric kidney transplant recipients. methods: one-hundred five pediatric recipients of a kidney allograft, all treated with a corticosteroid-free immunosuppressive protocol, were included. the evolution of tacrolimus doses and exposure was recorded at 3, 6, 9, 12, 18 and 24 months after transplantation, as well as all pre-dose trough levels (c 0 ; n=9376) obtained in the first 2 years after transplantation. results: dose-corrected tacrolimus exposure (c 0 /dose/kg) increased in the first 2 years after kidney transplantation in pediatric recipients (table 1, figure 1 ). this decrease in dose requirement by time was only significant in children older than 5 years at the time of transplantation ( figure 1 ). in addition, the younger patients had significantly higher dose requirements compared to older recipients, which translated in marked underexposure in 72-79% of patients <12 years of age in the first days after transplantation. conclusion: pediatric kidney transplant recipients exhibit maturation of tacrolimus pharmacokinetics with time after transplantation. this can not be explained by differences in corticosteroid use, as all patients were treated with a corticosteroid-free protocol. the higher dose requirements for younger recipients and the absence of tacrolimus maturation in the youngest recipients suggest that age-dependent changes in tacrolimus intestinal first-pass effect, metabolism or distribution play a role. whether age-specific tacrolimus dosing algorithms will improve outcome needs further study. determinants of dose-corrected pre-dose trough levels (c0/dose/kg) aih may present as acute hepatitis in 25% of patients (pts) and result in alf. early administration of corticosteroids may obviate the need for liver transplantation (lt), but features which identify aih in pts with alf have not been determined. aim: to identify clinical and histological features which distinguish alf due to aih. methods: 197/1033 pts in the alf study group registry had no evidence of viral, metabolic, vascular, and drug/toxic liver injury. all had alf defined by acute disease, encephalopathy, and coagulopathy. based upon admission clinical features, 52/197 had probable aih, and 145 were considered "indeterminate." liver biopsies (lbx) available from 56 pts were reviewed by a blinded expert hepatopathologist. clinicopathologic correlations were analyzed retrospectively. results: all lbx available from pts with suspected aih had classical histologic features of aih. moreover, 23/46 (50%) lbx from pts with indeterminate alf also had aih features: extensive necrosis (100%), fibrosis (84%), cirrhosis (22%), interface hepatitis (75%), and plasma cell-rich inflammation (69%). of all 33 lbx with aih features, centrilobular lesions associated with acute, severe aih (am j surg path 2004;28:471) were frequent: plasma cellrich central venulitis (97%), exclusive centrilobular necroinflammation (19%), and pericentral dilatation/congestion (46%). clinical characteristics and outcomes of the 75 pts with aih based upon laboratory and histologic findings differed significantly from pts whose alf remained indeterminate: aih pts were older (44 v 38 y), predominantly female (75 v 56%), and had longer jaundice-encephalopathy interval (20 v 12 d) , lower alt (660 v 1949 u/l), higher globulins (3.7 v 2.7 g/dl), lower creatinine (1.7 v 2.3 mg/ dl), and a higher prevalence of ana (68 v 24%) and asma (51 v 29%) (all p<.01). although spontaneous survival did not differ, more aih pts underwent lt (63 v 34%), and more survived 1 month after enrollment (75 v 59%; p<.0001). conclusions: using histologic criteria to classify pts with indeterminate alf, aih accounted for at least 7% of the alf study group registry, and represented 50% of indeterminate alf. lbx should be performed in all patients with alf of obscure etiology, in particular to identify centrilobular necroinflammatory lesions, which appear to be specific indicators of acute and severe aih. (u-01 58369 from niddk). (2002) (2003) (2004) (2005) (2006) . mikel gastaca, 1 miguel montejo, 1 lluis castells, 2 antonio rafecas, 3 antonio rimola, 4 ramon barcena, 5 federico pulido, 6 magdalena salcedo, 7 martin prieto, 8 manuel de la mata, 9 jose r. fernandez, 1 jose m. miro, 4 the spanish lt in hiv-infected patients working group. 1 hospital de cruces, bilbao, spain; 2 hospital vall d´hebron, barcelona, spain; barcelona, spain; univ. of barcelona, barcelona, spain; 5 hospital ramon y cajal, madrid, spain; 6 hospital 12 de octubre, madrid, spain; 7 hospital gregorio marañon, madrid, spain; 8 hospital la fe, valencia, spain; 9 hospital univ. reina sofia, cordoba, spain. background and aim: we report on the preliminary results of the prospective multicenter spanish study in hiv-1 infected patients who underwent olt. methods: the prospective multicenter spanish study fipse-olt-hiv 05-gesida 45-05 was initiated in january 2002. inclusion criteria follows the rules previously described in the spanish consensus document. hiv-infected patients transplanted between january 2002 and december 2006 were included in this study. results: 89 olt were consecutively performed in 85 hiv-1 infected patients in the period of the study. median (iqr) follow-up is 16 (8-29) months. median (iqr) age was 42 years (39-46), 74% of the recipients were male and former drug abuse was the most common hiv-1 risk factor (78%). 83% of the patients were transplanted due to hcv-related cirrhosis and 6% due to hbv cirrhosis. median (iqr) meld score was 14 (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) . pre-olt median (iqr) cd4 cell count was 288 (165-486) cells/mm3 and 64%patients had undetectable plasma hiv viral load. immunosuppression was based on tacrolimus in 70% of the patients. haart was re-started in a median (iqr) time of 10 (5-21) days after olt and was based on efavirenz in 48% of the cases. acute rejection occurred in 38 patients (43%). twenty patients died (22.5%) mainly due to hcv recurrence (8%). antiviral therapy with peg-interferon plus ribavirin was initiated in 18 patients obtaining a sustained viral response in 6 of them (33%). patient survival (95% confidence intervals) at 1, 2, 3 and 4 years was 90% (81-95%), 74% (60-84%), 67% (52-79%) and 67% (52-79%), respectively. conclusion: for selected hiv-1 infected patients under haart therapy, olt is a safe and effective procedure at mid-term. as in the hiv-negative population, hcv recurrence is the mayor cause of concern and response to antiviral therapy is still disappointing. hepatitis c virus and objectives: describe the 12-year experience of a university hospital treating chronic hepatitis c relapse, histologically diagnosed after liver transplant. material & methods: from september 1991 through july 2006, all the patients subject to liver transplant with histological diagnostics of chronic hepatitis relapse were submitted to antiviral treatment. treatment was suspended if there was a severe adverse reaction to the drugs used, non-adherence, severe rejection or no response after at least 12 months. hcv positive serology patients, alt increase (1,5x nl) and hepatic biopsy with metavir rating showing structural 1 and/or periseptal or parenchymatous portal inflammatory activity /= 2 were included. from 1995 to 2003, all patients were treated with conventional interferon and ribavirine, regardless of the genotype. from october 2003 on, patients with genotype 1 were treated with ribavirine and pegylated interferon and genotype 3 were treated with conventional interferon and ribavirine. results: 47 patients were treated during this time period, thirty-seven male (78.7%). their average age was 48. 51.1% were genotype 1, 25.5% were genotype 3. average time between transplant and beginning of treatment was 30 months. average treatment was 16 (4-36) months. 74.5% received conventional interferon, 6.4%, pegylated alpha 2a and 19.1%, pegylated alpha 2b. 48.9% had one or more cellular rejection episodes before vhc relapse diagnostics and were treated with corticosteroids. sustained virological response (svr) occurred in 48.8%. three patients had a virological response at the end of treatment (evr) and have not completed six months post treatment. out of 21 patients with svr, 20.9% were genotype 3, 9.3% genotype 1 and 18.6% were not genotyped. considering genotypes, svr was detected in 16.7% with genotype 1, in 75% with genotype 3. srv was detected when we examined hepatic biopsy, in 85.7% of the f1/f2 patients (18 out of 21), 9.5% of the f3 patients (2), 4.8% of the f4 patients (1) . survival period of 5 years for svr patients was 94% (20 out of 21) and 68.2% for patient without svr (p = 0.03 -kaplan-meier). five-year survival was 79.2% for patients with genotype 1 and 100% for patients with genotype 3. conclusions: our results show it is possible to get good five-year survival rates for treated patients. however, handling adverse reactions and long term treatment still pose difficulties in pursuing better svr rates. the impact of alcoholic liver disease (ald) and background: although liver transplant survival benefit has been shown to be associated with meld score, disease-specific analyses have not been reported. we evaluated, using srtr data, the effect of ald and/or hcv infection on transplant waitlist and post-transplant mortality, and survival benefit of deceased donor liver transplants. methods: 38,889 patients age ≥18, who were listed between sept 2001 and dec 2006, and followed until dec 31, 2006, were classified into 4 cells according to hcv or ald status: hcv+, ald-: 12,322; hcv+, ald+: 4,220; hcv-, ald+: 6,581; hcv-, ald-: 15,776). cox regression was used to estimate waiting list mortality and post-transplant mortality separately. survival benefit, which encompasses both pre-and post-transplant events, was assessed using sequential stratification; an extension of cox regression which matches transplant recipients by meld and organ procurement organization to patients on active on the waitlist at the time of transplant. results: hcv significantly (p=0.0001) increased waitlist mortality, with a covariate-adjusted hazard ratio (hr) for hcv+ vs. hcv-of hr=1.19 (p=0.0001). the impact of hcv+ was greater among ald+ candidates (hr=1.36; p<0.0001), but was also significant among ald-candidates (hr=1.11; p=0.02). the contrast between ald+ and ald-waitlist mortality was only significant among hcv+ candidates (hr=1.14; p=0.006). post-transplant mortality was significantly higher among hcv+ vs. hcv-recipients (hr=1.26; p=.0009); there was no difference between ald+ vs. ald-recipients. survival benefit of liver transplantation was significantly lower among hcv+ compared to hcv-recipients with meld 9-29, but significantly higher for hcv+ recipients with meld scores of ≥30. a diagnosis of ald did not influence the survival benefit of transplantation at any meld score. conclusion: despite higher waitlist mortality, hcv+ recipients had significantly lower liver transplant survival benefit than hcv-recipients, within categories defined by meld score. in contrast, transplant survival benefit was not influenced by ald. transplanted for hbv-related cirrhosis. giuseppe tisone, 1 daniele di paolo, 1 ilaria lenci, 1 laura tariciotti, 1 andrea monaco, 1 manuele berlanda, 1 linda de luca, 1 giuseppe iaria, 1 alessandro anselmo, 1 irene bellini, 1 mario angelico. 1 1 hepatic surgery and transplantation, university of rome tor vergata, rome, italy. background and aim: post-transplant active immunization with hbsag vaccine is a potential prophylaxis strategy against hbv-recurrence after liver tranplantation due to hbv-related disease. previous studies showed conflicting results using standard vaccines, whereas the use of the new adjuvant 3-deacylated monophosphoryl-lipid-a (mpl) significantly increased patient's immunization rate. we investigated the efficacy of a long-term (12 months) accelerated (monthly doses) vaccination schedule using the mpl-adjuvanted vaccine administered with and without concomitant hbig. methods: 18 patients (m/f:13/5) transplanted for hbv-related cirrhosis 73±38 months earlier were recruited. all were hbsag and hbv dna negative in serum and cccdna negative in liver tissue; 5 (27.7%) were co-infected with hcv and 5 (27.7%) with hdv. study protocol consisted of 12 consecutive monthly intramuscular vaccine doses (hbsag 20mg plus mpl 50mg) given together with lamivudine (100 mg/daily). each of the initial 6 doses (first cycle) was administered within 7 days after 2000 iu hbig i.v. infusion, while the last 6 doses were given after complete hbig withdrawal (second cycle). hbsab titre was determined before each vaccine dose and during the follow-up. all patients were maintaiened on low-level immunosuppression. preliminary results: all patients completed first vaccination cycle; 14 (77.7%) patients received 12 adjuvanted vaccine doses (first and second cycles) and were monitored during 3 months follow-up after vaccination end. no side effects occurred, nor evidence of hbv recurrence. at the end of first cycle all patients achieved an anti-hbs titre >100 iu/l (mean 343±209 iu/l) and 3 (16.6%) a titre greater than 500 iu/l. at the end of follow-up 8/14 (57.1%) and 4/14 (28.5%) had an anti-hbs titre greater than 100 (mean 411±599 ui/l) and 500 iu/l, respectively. conclusions: nine months after hbig withdrawal more than half of the patients reached and maintained a protective anti-hbs titre (>100 iu/l). this intensive schedule using the mpl-adjuvanted vaccine, given in combination with hbig and lamivudine, seems to be more effective than previous hbv vaccination protocols, although a longer follow-up is needed to assess its final effectiveness. heterologous immunity via alloimmune responses to hepatitis c virus replication after liver transplantation. hideki ohdan, 1 masahiro ohira, 1 yuka tanaka, 1 kohei ishiyama, 1 nobuhiko hiraga, 2 michio imamura, 2 kazuaki chayama, 2 toshimasa asahara. 1 1 surgery, hiroshima university, hiroshima, japan; 2 internal medicine, hiroshima university, hiroshima, japan. the immunosuppressive environment in liver transplantation (lt) recipients infected with hcv is believed to be associated with the progression of hcv reinfection. in the present study, we simultaneously monitored hcv levels and alloimmune status in hcv-infected lt recipients. for evaluating the immune status of 33 hcv-infected lt recipients, we employed a mixed lymphocyte reaction (mlr) assay using a cfselabeling technique. the kinetics of the stimulation index of anti-donor reactive cd4 + t cells clearly mirrored that of the hcv rna titer, and a significant reverse correlation was observed between the 2 parameters (r 2 = 0.67). we did not observe a similar relationship between the stimulation index of an anti-third party cd4 + t cells and the hcv rna titer (r 2 = 0.03). one possible explanation for this phenomenon might be that cytokines outputted by t cells responding to allostimulation display the anti-hcv activity either directly or indirectly through the activation of bystander immunocytes. to investigate such possibilities, we performed a transwell culture assay comprising a mlr culture in the upper chamber and genomic hcv replicon cell culture in the lower chamber to mimic the anatomical features of the interaction between hcvinfected hepatocytes and alloreactive t cells that infiltrate the portal area in the liver. when one-way mlr was performed using one-halpoidentical combination in the upper chamber, hcv replication was significantly suppressed in the lower chamber containing hcv replicon cells. hcv replication was further suppressed when mlr was carried out with a complete allogeneic combination. this inhibiting effect was dependent on their ifn-γ secreting activity. in addition, a similar result was obtained when ifn-γ-secreting nk/nkt cells stimulated with il-2 were cultured in the upper chamber. in conclusion, there was a close relationship between the anti-donor and the anti-hcv immune status in the lt recipients infected with hcv. cytokines such as il-2 and ifn-γ may be produced in response to allostimulation, and even these cytokines do not cause graft rejection, display anti-hcv activity. the elucidation of such a possible heterologous immunity via alloimmune responses to hcv replication might lead to the establishment of a novel method to prevent the progression of hcv reinfection in hcv-infected lt recipients. hepatitis c e2 region diversity after liver transplantation: a report from the hepatitis c iii trial. juan f. gallegos-orozco, 1 hugo e. vargas, 1 georges netto, 2 gary l. davis, 3 , and 7.3% are morbidly obese . the goal of this study was to quantify the effect of bmi on access to transplantation (att), likelihood of receiving a transplantation (lrt), turndown rates (tr), and survival benefit (sb). methods: att was defined as either registering for the deceased donor waiting list or receiving a live donor transplant, and was analyzed based on the usrds registry using logistic regression. lrt was based on time spent in active status on the waiting list before receiving a transplant, and was analyzed based on the unos waiting list dataset using cox proportional hazards time-to-event analysis. tr was defined as the relative likelihood of being turned down for an organ offer by a provider other than the patient, and was analyzed based on the unos organ turndown dataset using negative binomial regression. sb was defined as survival after kidney transplantation versus survival on the deceased donor waiting list, and was analyzed based on the unos waiting list dataset using a time-dependent cox survival benefit model. all models were adjusted for known factors influencing those outcomes. lgbp lsg lsg mean follow-up (months) 15.4 (range 3-24) 9 (range 3-18) 12.5 (12, 13) patients > 9 months since operation (n) 6/7 4/6 2/2 mean % ewl at > 9 months 61 (range 41-75) 33 (range 24-40) 62 (50, 73) transplant candidate at > 3 months 7/7 5/6 2/2 underwent transplant 0/7 1/6 1/2 complications developed in two patients (both with cirrhosis) and there was no mortality. mean follow-up was 12.3 months, and mean ewl at 9 months or later was 61% (esrd), 33% (cirrhosis) and 61.5% (esld). obesity associated comorbidities were improved or resolved in all patients. serum albumin and other nutritional parameters 9 months or later after surgery were similar to preoperative levels in all 3 groups. at most recent follow-up, 14 out of 15 patients (82%) have reached our institution's body mass index (bmi) limit for transplantation and are awaiting transplant; one patient with esld has undergone a successful lung transplant and one patient with cirrhosis has undergone a successful liver transplant. factors that contribute to inequitable access to the transplantation network (unos/ optn) include socioeconomic status, geographic location and delayed referral. the goal of the meld allocation system was to assign priority to the sickest candidate and assure equitable access to liver transplantation (lt). the meld has been validated as a reliable marker for liver disease severity. at the time of referral to the transplant center or listing a high meld (hm) is an indirect measure of delayed referral. therefore, the aim of this study is to identify factors associated with a hm at the time of listing for lt. method: using the unos database, we identified all adult candidates listed for lt from 2002 to 2006. patients who received meld upgrade (i.e. hcc, fhf) and those listed for multi-organ transplant were excluded. the data collected included demographics, insurance payor, diagnosis, and meld score. meld score at time of listing was categorized as low (< 20) and high (>20) and insurance type as private, medicaid and government (excluding medicaid) . results: during the study period 15,323 candidates were added to the lt waiting list. of these, there were 10,302 (67.2%) males with a median age of 53 (range 18 -83) . caucasians were 11,511 (75.1%), hispanic (11.6%), african americans (8.9%) and the rest (4.4%). the underlying liver disease was hepatitis c (31.9%), alcohol (15.1%), alcohol/hepatitis c (7.2%), pbc/ psc (9.9%), cryptogenic (7.5%) and others (28.4%). age, gender and liver disease etiology were not associated with a hm at listing. a hm was associated with ethnicity: aa (49.5%), hispanic (44.2%), caucasian (36.6%)[p<0.001] and insurance type: medicaid (46.3%), government (38.8%) and private (36.5%)[p<0.001].there was no strong interaction between ethnicity/race and insurance in combination as predictors of hm i.e. both were independent. conclusion: (1) aa and lt candidates with medicaid insurance are more likely to have a hm score at initial listing (2) hispanics had the lowest rate of private insurance compared to caucasians with the highest rate. the above results suggest that type of insurance and ethnicity are independently associated with a hm (i.e. sicker patients) at listing. since a hm score is associated with increased mortality, implementation of strategies that result in timely and equitable access to the transplantation network regardless of the insurance type or ethnicity of the candidate(s) should be a priority. marked increase in the use of inactive status on the kidney transplant waiting list. kim nguyen, 1 valarie ashby, 1, 2 further wait time accrued only after active status was restored. on 11/5/03, the optn implemented a change in policy that provides for the accrual of waiting time during the entire interval that wl candidates are designated as s7. methods: we investigated the impact of this policy change on the patterns of s7 designation, using the srtr/optn database. initial and subsequent status on the wl were determined for 130,986 candidates placed on the ki tx wl from 11/5/00-11/4/06. the probability of becoming active (including receiving a living donor tx) on the wl was calculated for those who were s7 at listing before and after the policy change. results: table 1 shows trends in the use of s7 before and after the policy change. of the 1,396 candidates listed as s7 before the policy change, 76% became active within 6 months and 83% within 1 year of wl. for the 11,248 candidates listed as s7 after the policy change, the corresponding figures are 48% and 60% respectively. figure 1 demonstrates that, since the implementation of the new policy, the % of patients initially wl in s7 at most us tx centers has increased. conclusion: since the implementation of this policy, the number and % of pts wl as s7 at most ki tx centers, and the duration of s7 for those initially wl as s7 has increased dramatically. the implications of this practice on access to ki tx and survival warrants further investigation. can abstracts (11%) patients were retransplanted, and 13 (9%) patients had panel reactive antibody >30. the mean cumulative thymoglobulin dose administered was 5.7 mg/kg, and the primary maintenance immunosuppression started prior to discharge was a sirolimuscontaining regimen (59%). at the end of the follow-up period, 98 (65%) patients had functioning grafts and 52 (35%) patients experienced graft loss. of the 52 patients with graft loss, 15 (10%) patients experienced graft loss secondary to death. no pertinent differences were identified between groups. the graft survivals for african american recipients with african american or caucasian donors were 95% vs. 91% at 1 year and 83% vs. 72% at year 2. conclusions: our study results suggest that donor race does not adversely affect graft outcomes in african american cadaveric kidney recipients with modern immunosuppression. donor racial differences may not play a primary role in the inferior graft outcomes of african american cadaveric kidney recipients. cardiac evaluation before kidney transplantation: are we screening too often or not enough? krista l. lentine, 1 m. a. schnitzler, 1 j. j. snyder, 2 d. c. brennan, 3 p. hauptman, 1 p. r. salvalaggio, 1 b. kasiske. 4 evaluation for ischemic heart disease (ihd) is a common but non-standardized practice before kidney transplant. we retrospectively studied pre-transplant cardiac evaluation (ce) practices among a national sample of renal allograft recipients. methods: we examined usrds data for medicare beneficiaries transplanted in 1991-2004 with part a and b benefits from dialysis initiation through transplant. clinical traits defining "high" expected ihd risk were defined as diabetes, prior ihd, or >2 other coronary disease risk factors. pre-transplant ce were identified by billing claims for noninvasive stress tests and angiography. we quantified individuals with claims for coronary revascularization procedures between ce and transplant, and abstracted post-transplant acute myocardial infarction (ami) events from claims and death records. results: among 27,786 eligible patients, 46.3% (65.4% of high-risk and 20.4% of lower-risk) underwent ce before transplant. overall, 9.5% of patients who received ce also received pre-transplant revascularization, including only 0.3% of lower-risk patients studied by ce ( table a) . the adjusted odds of transplant without ce (higher or for no ce, table b ) increased sharply with younger age and shorter dialysis duration. increased likelihood of transplant without ce also correlated with black race, female sex, and certain geographic regions. post-transplant ami rates in patients transplanted without ce allow assessment of whether ce was appropriately deferred in those who indeed face low ihd-risk after transplant. among patients transplanted without ce, the 3-yr incidence of post-transplant ami was 3% and 10% in lower and high-clinical risk groups, respectively, but varied by clinical traits within these groups. in lower-risk patients transplanted without ce, blacks patients faced increased ami-risk compared to whites (adjusted hr 1.52, 95% ci 1.16-2.00). conclusions: observed ce practices demonstrate a low yield of pre-transplant revascularization but also raise concern for socio-demographic barriers to evaluation access. women who become pregnant in the first two years after kidney transplantation have a higher risk of graft loss. nadia zalunardo, 1 olwyn johnston, 1 caren l. rose, 1 john s. gill. 1 1 division of nephrology, university of british columbia, vancouver, bc, canada. existing information regarding the risks of pregnancy is derived from voluntary data sources. using medicare claims files from the usrds (1990 usrds ( -2003 , we examined pregnancies (defined by presence of an inpatient icd-9 billing code for pregnancy or pregnancy-related complication) in the first 3 years after kidney transplantation (ktx) among women aged 15-45 years whose primary insurance payor was medicare (n=16,195) . patients were followed until graft failure, death or december 31, 2003. there were 530 pregnancies identified in 483 women during the first 3 post-transplant years. women who became pregnant during the 1st or 2nd post-transplant year had a shorter time to graft loss than women who became pregnant during the 3rd year (figure). to minimize the survivor bias among women who became pregnant during the 3 rd posttransplant year, we determined the association between the timing of pregnancy and graft survival among the subset of women (n=455) who had graft survival of at least two years using a cox multivariate regression adjusted for age, race, cause of esrd, donor source, calendar year of transplant, dialysis vintage, maintenance immunosuppression, and gfr at 6 months after ktx. pregnancy in the 1 st year (hr 2.01, 95% ci 1.35 -2.99), and second year (hr 1.79, 95% ci 1.23 to 2.62) was associated with an increased risk of graft loss compared to pregnancy during the third post-transplant year. we concluded that women who are able to wait should be counseled to become pregnant after the second post transplant year. the aging donor and recipient populations have led to new challenges in simultaneous kidney-pancreas transplantation (skpt). the purpose of this study was to retrospectively review our single center experience in skpt with respect to extended (ex) donor (d) and recipient (r) criteria. methods: over a 65 month period, we performed 83 skpts with enteric drainage (79 portal venous drainage). ex ds were defined as age <10 (n=4), >45 (n=12, mean age 50.2 yrs), or donation after cardiac death (dcd, n=4). all dcd donors were managed with extracorporeal support. ex rs were defined as age >50 (n=20, mean age 55.8 yrs) or those with a pretransplant serum c-peptide level >2.0 ng/ml (n=7, mean 5.7 ng/ml). all rs received depleting antibody induction (57 ratg, 26 alemtuzumab) with tacrolimus, mmf, and tapered steroids (39 steroid-free). results: a total of 20 ds (24%) and 23 rs (28%) met the above ex criteria. median waiting time was 10 months, mean pancreas preservation was 17 hours, and median length of stay was 10 days. with a mean follow-up (f/u) of 28 months, patient (pt) (95% ex d vs 94% non-ex d), kidney (90% ex d vs 89% non-ex d) and pancreas graft survival (85% ex d vs 81% non-ex d) rates were similar between d groups (all p=ns). the incidences of delayed kidney graft function (5% in each group) and early pancreas graft loss due to thrombosis (5% ex d vs 8% non-ex d) were also comparable between d groups. with regard to r groups, pt (87% vs 97%, p=0.13) and kidney (87% vs 92%, p=ns) graft survival (gs) rates were slightly lower in the ex r group compared to the non-ex r group, respectively. however, death-censored kidney gs rates (100% ex r vs 95% non-ex r) were comparable between groups. uncensored pancreas gs rates (83% ex r vs 82% non-ex r) were similar. the incidences of acute rejection, surgical complications, infection, and other morbidity were comparable regardless of d or r group. at 1 year (or latest) follow-up, renal and pancreas functional parameters were similar between d groups. however, the ex r group demonstrated slightly compromised renal and pancreas allograft function and a greater need for oral hypoglycemic agents. conclusion: intermediate-term outcomes in skpt from selected ex ds or rs have comparable outcomes, although ex r criteria may represent a risk factor for pt survival and functional outcomes. conclusion: spk recipients with functioning pancreas grafts have significantly improved kidney and patient survival compared to ld ka and dd ka. however, early pancreas graft failure results in kidney and patient survival rates similar to ka recipients. spk provides optimal outcomes for patients with dm1, but long-term risk associated with early pancreas loss may be a consideration when selecting spk vs ka transplantation. the impact of long-term metabolic control on renal allograft and patient survival in type 1 diabetes. christian morath, 1 martin zeier, 1 bernd dohler, 2 jan schmidt, 3 peter p. nawroth, 4 gerhard opelz. 2 1 nephrology, university of heidelberg; 2 transplantation immunology, university of heidelberg; 3 transplantation surgery, university of heidelberg; 4 endocrinology, university of heidelberg. it is a matter of debate whether pancreas allografts independently contribute to renal transplant and patient survival in type 1 diabetics who received a simultaneous pancreas kidney transplant (spk). using the data of the collaborative transplant study (cts), we studied type 1 diabetic recipients of deceased donor kidneys (ddk), living donor kidneys (ldk), or spk performed during two time periods: 1984 to 1990 and 1991 to 2000 . we analyzed graft and patient survival rates for a maximum of 18 years. ddk recipients showed inferior graft and patient survival compared to ldk and spk recipients in both time periods. ldk recipients had a superior graft survival rate initially, but the survival rate of kidneys in spk recipients reached the level of ldk toward the end of the follow up period. the results of patient survival paralleled those of kidney graft survival: an early advantage of ldk as compared to spk faded away during follow up. multivariate analysis, in which pretransplant cardiovascular risk assessment was appropriately considered, showed that patient survival of spk recipients was superior to that of ldk recipients beyond the tenth year after transplantation (hazard ratio hr = 0.55, p = 0.005). this was reflected by a lower cumulative cardiovascular death rate in recipients of spk (37.0 %) compared to recipients of ddk (45.8 %) or ldk (49.3 %) . the early survival benefit of ldk compared to spk is lost during long-term follow up, probably related to improved glycemic control in spk recipients. proposal for a grading rejection schema in pancreas allograft biopsies from a multidisciplinary panel of pathologists, surgeons and nephrologists. 1.normal 2.indeterminate septal inflammation that appears active but the overall features do not fulfill the criteria for mild cell mediated acute rejection. active septal inflammation involving septal structures and/or focal acinar inflammation. -grade ii / moderate acute cell mediated rejection multifocal (but not confluent or diffuse) acinar inflammation with spotty acinar cell injury and drop-out and/or minimal intimal arteritis -grade iii / severe acute cell mediated rejection diffuse, (widespread, extensive) acinar inflammation with focal or diffuse multicellular /confluent acinar cell necrosis.and/or moderate or severe intimal arteritis and/or transmural inflammation -necrotizing arteritis chronic active cell-mediated rejection. chronic allograft arteriopathy (arterial intimal fibrosis with mononuclear cell infiltration in fibrosis, formation of neo-intima) 4.antibody mediated rejection =c4d positivity + confirmed donor specific antibodies + graft dysfunction -hyperacute rejection -accelerated antibody mediated rejection severe, fulminant form of antibody mediated rejection with morphological similarities to hyperacute rejection but occurring later (within hours or days of transplantation). -acute antibody mediated rejection specify percentage of biopsy surface with interacinar capillaries positive for c4d. 5.chronic allograft rejection/graft sclerosis stage i (mild graft sclerosis) <30% of the core surface stage ii (moderate graft sclerosis) 30-60% of the core surface. stage iii (severe graft sclerosis) >60% of the core surface 6. other histological diagnosis e.g. cmv pancreatitis, ptld, etc. a simple, reproducible, clinically relevant and internationally accepted schema for grading rejection should improve the level of diagnostic accuracy and positively affect patient care. rank test). similar results were obtained when death was included as a cause of graft loss (data not shown). conclusion: par is associated with worse long term kidney graft survival than kar. it is likely that patients that experience par within the first year have also experienced undiagnosed sub-clinical kar. this adds associative data to the argument that the likelihood of concordance between par and kar is high and therefore, the need for increased monitoring of kidney function after par is warranted. the most efficacious immunosuppressive (immuno) regimen for spkt is debated, and information on the best regimen to prevent amr is particularly scarce. we performed a retrospective comparative cohort study that included 136 spkt patients (pts) transplanted in 2002-2005, who received maintenance immuno with tac, mmf and steroids. two groups were compared: pts who received induction with alemtuzumab (alem) (n=97) and pts who were induced with basiliximab (basilmab) (n=39). donor and recipient characteristics were similar. kt acute cellular rejection (acr) was more frequent with basilmab (2-yr 12.8% vs 3.1%, p=0.04), but the incidence of biopsy-proven kt amr was similar (2-yr 18% with basilmab vs 13.8% with alem, p=ns). no differences in prevalence were detected considering early amr (<90th day) or late amr (>90th day) separately. multivariate analyses showed that the only significant risk factor for amr was female gender (adjusted hr 2.97, p=0.016). the biopsy-proven kt rejection was associated with clinical pt rejection in 13 out of the 21 amr cases (62%), without differences between the groups. post-rejection kt graft survival was similar in both groups (2-yr basilmab/alem 94.7/91.2%), but deathcensored kt survival was lower with alem (100/91.2%, p=0.05). the predominant cause of kt loss in pts induced with basilmab was death-with-function (n=3), while in pts induced with alem acute or chronic amr was the most common cause of kt attrition (n=5). pancreas survival was similar between both groups. more pt losses due to ar occurred in alem-treated group than in the basilmab group (4 vs 1) . nearly all early episodes resolved with treatment and were not associated with worse graft survival. conversely, late amr episodes carried a worse prognosis, with decreased 2-yr kt (61.5% vs 96.2%, p<0.0001) and pt graft survival (47.7 vs 92.3%, p<0.0001). late amr was associated with graft loss in multivariate cox models (kidney loss hr 3.34, p=0.05 and pancreas hr=3.22, p=0.049) . amr is common in spkt recipients. acr is better prevented with alem than with basilmab, but no relevant difference is found between alem and basilmab in amr. late (as opposed to early) amr episodes are associated with significant reduction in spkt survival rates despite treatment. preventive and/or different treatment strategies are required to address late amr in spkt. intraoperative fluorescence imaging (ifi) in pancreas transplantation (pt) to determine vascular patency and allograft perfusion. edmund q. sanchez, 1 srinath chinnakotla, 1 marlon f. levy, 1 robert m. goldstein, 1 goran b. klintmalm. 1 1 baylor regional transplant institute, dallas/ft. worth, tx. thrombotic complications of pt are well known. we report ifi using the spy device to assess immediate vascular patency and allograft perfusion. our controlled experience is presented here. methods: pts were imaged intraoperatively using the spy device under our irb approved study. indocyanine green solution (2.5 mg/ml) was injected into central venous catheters after completion of the vascular anastomoses of whole pancreaticoduodenal allografts. the pancreas transplants were performed in the retroperitoneal portal and enteric drained technique described by boggi, et al. imaging of the allograft vasculature, perfusion of the pancreas, and perfusion of the duodenal segments was performed and recorded intraoperatively. all video sequences were archived for later review. results: ifi on 7 pancreas transplants (6 simultaneous pancreas-kidney and 1 pancreas after kidney) was performed and video sequences were recorded. all pancreas allografts demonstrated intraoperative vascular patency and complete pancreatic and duodenal perfusion. there were no side effects seen. all pancreas transplants had immediate graft function. one patient was re-explored on postop day 0 due to persistent acidosis and hypotension. repeat ifi demonstrated vascular patency and perfusion, despite an ischemic external physical appearance. there were no vascular complications that required reoperation, nor were there any graft thromboses. characteristic slow venous outflow was seen in each case. conclusion: ifi with the spy device is a simple and effective method to determine immediate patency and perfusion of the whole pancreaticoduodenal allograft. its use is beneficial in re-exploration to rule out infarcted pancreas allografts. further development in quantification of vascular flow rates and allograft perfusion indices by this device is abstracts in progress. once these are established, this information will be studied in a controlled fashion, and will be compared with clinical outcomes. we have demonstrated safety and developed a protocol for using the spy device in ifi of pancreas transplants. tolerance/immune deviation i tolerance without immunosuppression: exploiting epigenetic regulation as a new approach to achieving donor-specific allograft tolerance. liqing wang, 1 ran tao, 1 joel a. friedlander, 1 wayne w. hancock. 1 1 pathology & immunology, children's hospital of philadelphia & upenn, philadelphia, pa. given toxicities of all current immunosuppressive agents, plus complications of malignancy, infection and chronic rejection, maintaining the search for new approaches to tolerance induction is essential. while weaning of immunosuppression is fraught with risks and costimulation blockade has not yielded the expected boon, use of a broad histone deacetylase inhibitor (hdaci), such as trichostatin a (tsa) or suberoylanilide hydroxamic acid (saha), promotes foxp3 acetylation of foxp3 and binding to target genes, leading to enhanced treg suppressive function, and 2 wks of therapy with hdaci and low-dose rapamycin can induce allograft tolerance. we now show that in addition to acetylation, modulation of a second major epigenetic mechanism, dna methylation, has salutary effects, such that combined use of an hdaci and a dna methyltransferase inhibitor (dnmti, e.g. 5-aza-2 deoxycytidine) has potent effects. microarray analysis of the effects of hdaci on tregs showed down-regulation of multiple genes associated with dna methylation, including dnmt, methyl-cpgbinding domain and associated proteins, leading us to test the effects of dnmti use on treg function. dnmti administration decreased foxp3 gene methylation, enhanced treg gene expression, and increased treg suppression in vitro, and led to a dose-dependent prolongation of cardiac allograft survival (balb/c->c57bl/6) in vivo (p<0.01). the combination of hdaci and lower doses of dnmti, administered for just 2 wks, led to permanent engraftment (>100 d, p<0.001), and the permanent acceptance of second donor allografts but acute rejection of third-party (cba) cardiac allografts indicated induction of donor-specific tolerance post-epigenetic therapy. analysis of long-surviving cardiac allografts showed a minor infiltrate consisting primarily of foxp3+ tregs and an absence of chronic rejection (transplant arteriosclerosis, myocardial fibrosis), whereas combined epigenetic therapy led to only minor prolongation of allograft survival in treg-depleted recipients. in summary, brief use of 2 clinically approved agents (an hdaci plus an dnmti), can synergistically enhance treg function and induce tregdependent donor-specific allograft tolerance without use of any immunosuppression. we conclude that insights gained through epigenetic targeting may provide completely new approaches to the taming and regulation of otherwise powerful immune responses post-transplantation. a novel epigenetic approach to generate mouse and human cd4 + cd25 + foxp3 + regulatory t cells. girdhari lal, 1 nan zhang, 1 william van der touw, 1 yaozhong ding, 1 jonathan s. bromberg. 1 1 gene and cell medicine, mount sinai school of medicine, new york city, ny. background: constitutive foxp3 expression is required for stable and suppressive cd4 + cd25 + regulatory t cells (treg) . we previously showed that demethylation of an upstream cpg island of the foxp3 promoter is characteristic of stable and suppressive treg. using dna methyltransferase inhibitors, we present a novel method to generate antigen-specific treg from mouse and human cd4 + cd25 -t cells. methods: naïve cd4 + cd25 -t cells and natural treg (ntreg) were purified from wild type or foxp3-gfp transgenic mice. human naïve cd4 + cd25 -t cells were purified from pbmc. t cells were cultured with irradiated antigen presenting cells in the presence of il-2, anti-cd3ε mab, tgfβ or the dna methyltransferase inhibitor 5-aza-2'-deoxycytidine (zdcyd), which demethylates the upstream promoter. foxp3 expression was determined by flow cytometry and qrt-pcr. results: naïve t cells cultured under stimulatory conditions with zdcyd express increased foxp3 mrna (30 fold) and intracellular foxp3 protein (35% of cells vs 2% without zdcyd). foxp3 expression is synergistically enhanced with tgfβ (76 ± 4.6% vs 38.8 ± 5.3% zdcyd alone or 44.6 ± 1.4% tgfβ alone), similar to ntreg (92.4 ± 2.5%). tgfβ in combination with other dna methyltransferase inhibitors (procainamide, hydralazine, rg108) also induces foxp3. zdcyd plus tgfβ induced treg stably express foxp3 and similar surface markers and cytokine mrna as ntreg. zdcyd plus tgfβ induced treg suppress proliferation of cd4 + cd25 -t cells, prevent cd4 + cd25 -cd45rb hi induced colitis in scid mice, and enhance islet allograft survival. zdcyd plus tgfβ induce foxp3 expression in antigen-specific wild type or t cell receptor transgenic cd4 + t cells stimulated with alloantigen or peptides, and in naïve cd8 + cd25 -t cells. in vivo administration of zdcyd preferentially preserves thymic cd4 + cd25 + foxp3 + treg generation. zdcyd alone or zdcyd plus tgfb induce strong foxp3 expression in human cd4 + cd25 -t cells compared to tgfβ alone. zdcyd plus tgfβ induced human treg suppress the proliferation of naïve cd4 + cd25 -t cells, whereas tgfβ-induced treg do not. conclusion: dna methyltransferase inhibitors induce stable and suppressive foxp3 + treg from peripheral cd4 + cd25 -t cells. these finding have important implications for understanding t cell development and differentiation, and provide a clinically applicable technique for manipulating epigenetic regulation for the generation of treg for tolerance. macrophages driven to a novel state of activation can promote tolerance. katharina kronenberg, 1 seiichiro inoue, 1 james a. hutchinson, 2 beate g. brem-exner, 2 gudrun e. koehl, 1 hans j. schlitt, 1 fred fandrich, 2 edward k. geissler. 1 1 surgery, university of regensburg, regensburg, germany; 2 surgery, university hospital schleswig holstein, kiel, germany. background: the use of immunomodulatory cells in organ transplantation (tx) is a promising tolerance-induction strategy. here we describe a novel macrophage population capable of enriching t regulatory cells (treg) and promoting tolerance. methods: balb/c bone marrow, spleen and blood mononuclear cells were cultured with m-csf for 5 days, with a 24 hr pulse of ifn-γ on day 4; the resultant adherent cells are referred to as ifnγ-induced monocyte-derived cells (ifnγ-mdc) . residual lymphocytes from these cultures were recultured with the adherent ifnγ-mdc for an additional 3 days. ifnγ-mdc were phenotyped by facs and immunomodulation of lymphocytes was determined by cell counting and facs analysis for tregs. mouse heterotopic heart tx was used for in vivo testing. results: ifnγ-mdc highly express cd11b/c, f4/80 and pd-l1. compared to classically-activated (m1) macrophages, ifnγ-mdc express less cd40/cd86, but higher cd11c. ifnγ-mdc profoundly delete activated, but not resting, lymphocytes (>60%), with cell contact (via transwell system) and caspases (via inhibitors) being essential. most interestingly, ifnγ-mdc highly enrich lymphocytes for cd4 + cd25 high foxp3 + treg (41±2%; n=27). the same, or higher, proportions of treg developed when ifnγ-mdc were produced from macs-purified cd11b + and cd4 + cells (1:1) . ifnγ-mdc culture supernatant showed increased il-10 (elisa) vs. monocyte control cultures (183±42 pg/ml vs. <30 pg/ml, respectively; n=6), suggesting il-10 as one potential mediator. ifnγ receptor signaling and cd40 interactions are required for treg enrichment, as confirmed using ifnγ receptor and cd40 knock-out mice (c57bl/6 mice). ifnγ-mdc derived from cd11b + cells and lymphocytes of ova-transgenic otii mice showed enrichment of treg by 10-fold with high-affinity ova peptide (323-339) vs. treg levels using a low-affinity ova peptide (ova 323-334), suggesting ag-specific treg cells can be enriched with ifnγ-mdc. finally, a single post-heart tx (d+1) i.v. injection of 5x10 6 donor (c3h)-derived ifnγ-mdc prolonged allograft survival in balb/c recipients from 8.5±0.8d in controls (n=5) to 21.4±8.3d (n=11; p=0.001). conclusions: ifnγ-mdc are a novel macrophage population capable of deleting activated t cells and highly enriching remaining lymphocytes for tregs. therefore, ifnγ-mdc could be useful in a clinical setting for promoting transplant tolerance. plasmacytoid dc therapeutic potential is independent of ido induction due to elevated dap12 expression. tina l. sumpter, 1 bridget l. colvin, 1 zhiliang wang, 1 andrew l. mellor, 3 angus w. thomson. 1,2 1 starzl transplantation institute, dept. of surgery, university of pittsburgh, pittsburgh, pa; 2 immunology, university of pittsburgh, pittsburgh, pa; 3 immunotherapy center, medical college of georgia, augusta, ga. optimizing the mechanisms of pdc tolerogenicity may facilitate their use in the development of cell-based tolerance induction strategies. pdc may undermine t cell function via inducible expression of ido, a tryptophan catabolizer that enhances t cell apoptosis. ido is negatively regulated by dap12. in some systems, loss of dap12 correlates with immunostimulatory activation of pdc, while in others, its absence correlates with enhancement of pdc tolerogenicity through induction of ido. in these studies, we characterized the ability of wt and ido-deficient pdc to attenuate murine heart allograft rejection in order to define the contribution of ido and its regulation by dap12 relative to the immunoregulatory potential of pdc. methods: pdc and control myeloid (m) dc were generated from bm cultures of wt or ido ko c57bl/6 (b6; h2 b ) mice. wt mdc, pdc or ido ko pdc were pulsed with alloag (balb/c; h2 d ), stimulated with cpg, and then injected i.v. into syngeneic (b6) recipients 7d before heart transplant. donor ag-specific activation of t cells was analyzed by mlr and elisa. dap12 expression was evaluated by rt-pcr and abrogated with sirna. results: administration of ido ko pdc 7d before transplant prolonged graft survival beyond that seen with control mdc, but not to the extent seen with wt pdc, suggesting involvement of ido. pdc cultures from ido ko mice exhibited a more mature phenotype than their wt counterparts with reduced expression of co-regulatory molecules (e.g., icosl). although ido ko pdc induced enhanced t cell alloactivation compared to wt pdc, use of the ido inhibitor 1-mt indicated that wt pdc do not produce functional ido. further analyses showed wt pdc exhibited high levels of dap12 mrna expression. silencing dap12 had no effect on the ability of wt pdc to activate allogeneic t cell proliferation, but significantly enhanced ifnγ secretion. addition of 1-mt to mlr with dap12-silenced pdc increased t cell proliferative responses to alloag, verifying ido induction in dap12-silenced pdc. conclusions: these data underscore the prophylactic potential of pdc for cell-based therapy that may be independent of ido, due, in part, to elevated dap12 expression. additionally, our data support a role for dap12 in both immunostimulation and immunoregulation by pdc. rapamycin preferentially blocks the expansion of potentially tolerogenic plasmacytoid dendritic cells in vivo. heth r. turnquist, 1 angus w. thomson. 1, 2 1 starzl transpl inst and dept of surgery; 2 dept of immunol, univ of pittsburgh sch of med, pittsburgh, pa. rapamycin (rapa), a 'tolerance-sparing' immunosuppressant with anti-proliferative properties, inhibits myeloid (m) dendritic cell (dc) differentiation/maturation in vitro. rapa decreases cd11c + dcs in the mouse spleen and suppresses the expansion of total cd11c + dc following administration of the dc growth factor, fms-like tyrosine kinase 3 ligand (flt3l). however, the influence of rapa on plasmacytoid (p) dc,the principal type-1 ifn producers in the body, known to regulate innate and adaptive immune responses, and reported to promote experimental transplant tolerance, has not been evaluated. methods: dc were propagated from bone marrow for 10 days (d) in flt3l, in the absence or presence of a clinically-relevant dose of rapa (10 ng/ml). in addition, dcs were mobilized in c57bl/10 mice by i.p. flt3l (10 mg/d for 10 d; d1-10). untreated and flt3l-treated groups also received rapa (0.5 mg/kg/d i.p.; d3-10). on d11, dc were isolated by density gradient centrifugation and/or cd11c + positive selection. mdc (cd11c + cd11b + ) and pdc (cd11c lo cd11b -b220 + ) were identified by flow cytometry. results: rapa suppressed pdc and mdc generation in vitro; rapa-dc-treated cultures had only 22% of the pdc and 40% of the mdc found in control conditions. in normal mice, both mdc (54% of control) and pdc (24.5%) numbers were reduced significantly by rapa administration. rapa, when given concurrently with flt3l, blunted the typical profound expansion of mdc. specifically, flt3l and rapa-treated mice displayed a 46±8x increase over control (steady state) numbers compared to a 145±39x increase in mice treated with flt3l alone. flt3l-induced expansion of pdc was to a much greater extent impacted by rapa, as absolute numbers of pdc increased only 6.5±2.2x over control numbers compared to 51±3x increase in absolute splenic pdc in flt3l-treated mice. conclusion: these data identify rapa as a selective suppressor of pdc generation, as described for corticosteroids. this has significant implications, given the use of rapa following organ transplantation, and the suggested importance of secondary lymphoid tissue pdc for promotion of transplant tolerance mediated by treg. due attention to these disparate effects of rapa on regulating cell populations will be necessary to optimize therapeutic regimens for safe promotion of tolerance. the liver is tolerated better than other transplanted organs. this may reflect the inherent tolerogenicity of liver dendritic cells (dc) and interactions with foxp3 + regulatory t cells (treg). recent studies have shown that cd103 expression on dc correlates with induction of foxp3 + treg, and can be enhanced in the presence of transforming growth factor-β (tgf-β) and retinoic acid (ra), both of which are produced in the liver. this study evaluates cd103 expression on liver plasmacytoid (p) and myeloid (m) dc and the potential of liver dc subsets to induce tregs. methods: pdc and mdc were magnetically isolated from livers and spleens of c57bl/10 mice and co-cultured with cfse-labeled allogeneic (balb/c) cd4 + cd25 -t cells. after 4 or 5d of co-culture, t cells were assessed for cfse dilution and intracellular foxp3 expression. rhtgf-β and either all trans or cis-ra were added to co-cultures. cd103 expression was evaluated by flow cytometry. results: cd103 expression was elevated on liver pdc and mdc compared to spleen pdc or mdc, with higher expression on liver mdc. pdc from the liver and the spleen were poor inducers of foxp3 in cd4 + cd25 -t cells compared to mdc. foxp3 induction was enhanced with tgf-β when splenic pdc were used as stimulators. however, when liver pdc were used as stimulators, tgf-β had no effect on foxp3 induction. ra (either all trans or cis) enhanced induction of foxp3 + cells in cd4 + cd25 -t cells in the presence of tgf-β when splenic pdc but not liver pdc were used as stimulators. tgf-β and tgf-β with ra also enhanced foxp3 induction in cd4 + cd25 -t cells when either spleen or liver mdc were used as stimulators, though splenic mdc were superior inducers of foxp3. conclusions: many studies have focused on naturally-occurring foxp3 + tregs in systemic tolerance. our data show that liver mdc and pdc are poor inducers of foxp3 in t cells, even in the presence of tgf-β and ra. these data suggest that the inherent tolerogenicity of liver dc subsets may be independent of treg induction or that liver dc interact with treg through alternative mechanisms. background: t cell-mediated immune rejection occurs in organ transplantation. in addition to mhc-tcr signaling, t cell activation requires costimulation from antigen presenting cells. b7 molecules (cd80/cd86) and cd40 are critical costimulatory molecules in t cell activation. insufficient or lack of costimulation results in inactivation or tolerance. we hypothesized that blocking the costimulation pathway using small interfering rna (sirna) expression vector can prolong allogeneic heart graft survival. method: vectors that express hairpin sirnas specifically targeting cd40 and cd80 were prepared. recipients (balb/c mice) were treated with cd40 and/or cd80 sirna vectors, 3 and 7 days prior to heart transplantation. control groups were injected with a blank vector and sham treatment (pbs). after sirna treatment, a fully mhcmismatched (balb/c to c57/bl6) heart transplantation was performed. result: allogeneic heart graft survival (>100days) was approximately 70% in the mice treated simultaneously with cd40 and cd80 sirna vectors. in contrast, allogenic hearts transplanted into recipients treated with blank vector and pbs stopped beating within 10 days. hearts transplanted into cd40 or cd80 sirna vector-treated recipients had an increased graft survival time compared to negative control groups, but did not survive longer than 40 days. real time pcr and flow cytometric analysis showed an upregulation of foxp3 expression in spleen lymphocytes and a concurrent downregulation of cd40 and cd80 expression in splenic dendritic cells of sirnatreated mice. an mlr, using splenic dendritic cells (dcs) isolated from tolerant recipients, showed a significantly lower t cell proliferation capacity in cd40-and cd80-sirna vector-treated mice, compared to control groups. tolerant dcs from cd40-and cd80-treated recipients promoted cd4+cd25+foxp3+ regulatory t cell differentiation. finally, tissue histopathology demonstrated an overall reduction in lymphocyte interstitium infiltration, vascular obstruction, and edema in mice treated with cd40 and cd80 sirna vectors. conclusion: this study demonstrates that the simultaneous silencing of cd40 and cd80 genes has synergistic effects in preventing allograft rejection, and may therefore have therapeutic potential in clinical transplantation. costimulation blockade (cob) of the cd28/b7 pathway has long been suggested as a means of attaining indefinite, well-tolerated, antigen-specific prophylaxis from allograft rejection. although effective in rodent models, cd28/b7 blockade is not alone sufficient to prevent rejection in more robust primate models. the relative presence of allocrossreactive memory t cells is one mechanism of cob resistant rejection. lfa3-ig is an approved agent for the treatment of psoriasis, shown clinically to deplete tem cells in psoriatic lesions. we hypothesize that lfa3-ig specifically targets cob resistance cells, including heterologous alloreactive t cell memory, while avoiding interference of peripheral mechanisms that foster cob-mediated allograft acceptance. rhesus monkeys underwent mhc mismatched renal allotransplantation. ctla4-ig was given 20 mg/ kg iv on days -1, 0,3,7,14,21,28 and 10 mg/kg iv on days 35,42,49,56. lfa3-ig, (0.3 mg/kg iv) was given on day -1,0,3,7 then weekly (8 wks). sirolimus was given orally (1mg/kg) days 0-90 and donor specific transfusion (7 ml/kg iv) on day -1. animals were followed by polychromatic flow-cytometry to quantify t cell subsets. lfa3-ig synergized with ctla4-ig successfully preventing renal allograft rejection in this model and leading to indefinite survival in 2/5 animals (table) . this enhanced survival was associated with a significant reduction in tem cells in animals treated with lfa3-ig compared to animals without lfa3-ig treatment (figure). lfa3-ig withdrawal led to tem re-population and rejection. this therapy regimen, all clinically available agents, promotes cob-mediated graft acceptance without the use of calcineurin inhibitors, steroids, or gross t cell depletion. the aim of this study was to investigate the immunological pathways that regulate t cell dependant allograft responses in the hope of finding novel immunomodulatory compounds. islet (and skin) allografts were transplanted into full mhc-mismatched wild type (wt) and baff-transgenic (baff-tg) recipient mice. allograft function/ rejection was monitored by blood glucose levels and/or confirmed by nephrectomy. histology, splenocyte populations & t cell functions were analyzed. the b cell activation factor from the tnf family (baff) is critical for b cell survival. t cells express baff receptors suggesting that baff may also play a role in t cell responses. contrary to expectations, we found that baff-tg mice accepted a full mhc-mismatched islet allograft for >100 days. in addition, baff-tg mice also showed delayed skin graft rejection. this was due to a t cell intrinsic change in allo-responsiveness as shown by a failure of purified baff-tg t cells to reject an allograft in adoptive transfer experiments. however, baff-tg t cells were not anergic per se as they proliferated normally to mitogens in vitro and to antigenic challenge in vivo. intriguingly, baff-tg mice harbored an increased frequency of t regulatory cells in the periphery as compared to wt mice. elimination of cd25 + t cells restored normal allograft rejection to baff-tg mice, demonstrating that the increased number of treg cells were responsible for their altered allo-immunity. in a second approach, baff-tg t cells depleted of cd4 + cd25 + t cells were adoptively transferred to transplanted rag recipients, and in this case the baff-tg cd4 + cd25 -t cells rejected their allograft. proliferation assays showed that excessive baff was not promoting treg expansion by driving proliferation in the periphery. adoptive transfer of gfp expressing syngeneic splenocytes did not exhibit enhanced survival in the presence of excessive baff. however, analysis of treg cells in the thymus of baff-tg mice showed an increased intrathymic frequency. together these results demonstrate that baff-tg mice harbor an expanded number of treg cells that prevent th1-type immune responses including allograft rejection. furthermore, we demonstrate that manipulating baff levels may provide a means to generate immunosuppression free dominant allograft tolerance. we have previously reported the outcome of pig-to-baboon thymokidney transplants from galt-ko miniature swine using an immunosuppressive regimen designed to facilitate the induction of tolerance. although the results were superior to results using hdaf thymokidneys, there was a high rate of early post-operative complications. we investigated whether the elimination of steroids and whole body irritation (wbi) from the treatment protocol would decrease the complication rate in the perioperative period. methods: 7 baboons received thymokidney transplants from galt-ko miniature swine. the immunosuppressive regimen was based on the previously published protocol, but eliminated steroids and substituted rituximab for wbi. it consisted of thymectomy day -21, splenectomy day 0, atg, rituximab, mmf and anti-cd154 mab. renal function was assessed by serum creatinine levels. evidence for baboon thymopoiesis in the pig thymus was assessed by facs and immunohistochemistry. results: one animal died due to a drug reaction at pod 18 with normal renal function and no evidence of infection. the remaining 6 baboons showed no signs of rejection although igm deposition was observed, likely due to preformed non-gal nab. there were no deaths due to rejection. although early infectious complications were not seen, late complications leading to mortality were still observed, including systemic cmv infection (pod 28), pleural effusions (pod 40, 57) , ards with pulmonary hemorrhage (pod 49, 81) and acute myocardial infarction (pod 83). two animals showed evidence for early thymopoiesis in the pig thymus by facs and immunohistochemistry. cd4+/cd8+ thymocytes were seen in the thymic grafts, indicating t cell development was supported by the pig thymus. the average survival of the steroid-free group was 51 days including the animal with the drug reaction and 56 days excluding it, compared to 34.6 days in the regimen that included steroids/wbi. conclusions: a steroid-free and wbi free regimen designed for the induction of tolerance across the pig-to-baboon xenogeneic barrier had fewer early post-operative complications than previous regimens. greater than 50-day average survival of recipients of life-supporting xenogeneic thymokidneys was observed without rejection. this regimen also appears to permit baboon thymopoiesis in the pig thymus. purpose: the effects of human decay accelerating factor (hdaf) addition to the galactosyl transferase knock-out (galt-ko) background in transgenic pig kidney xenotransplantation in baboons has not been previously studied. methods: baboon recipients of galt-ko (n=4) or galt-ko/hdaf (n=2) pig kidneys received flolan, heparin and/or aspirin. steroids (s), cobra venom factor (v), atg (a), mmf (m), and/or a low-dose cd154 blockade (c) regimen were given. results: pre-transplant (tx) anti-non-gal antibody (ab) titers were inconsistently associated with early galt-ko kidney xenograft failure. high d-dimer levels 4 hours post-tx were closely associated with early galt-ko graft loss but not when assessing levels of c3a, btg, or increased cd62p expression on circulating platelets. regimens lacking mmf and/or cd154 blockade elicited by day 7-14 large anti-donor non-gal ab responses . post-operative creatinine levels for the galt-ko/hdaf recipients were lower along with superior graft survival compared to galt-ko; d-dimer, anti-donor non-gal ab, c3a, btg, and f1+2 measurements are in progress for the galt-ko/hdaf recipients. galt-ko/hdaf v,a,m,s,c >10* tbp 239 1.9; 1.5 * graft survival at time of abstract submission as graft still functioning at time of abstract submission; tbp --to be processed ! --anuric; non-life supporting, but appeared viable, pink, and well-perfused until day 7 conclusion: early failure of vascularized galt-ko organs is closely associated with activation of coagulation pathways; whether this is triggered primarily by anti-non-gal ab or by other mechanisms is not addressed by our study. preliminary results using galt-ko/hdaf pig kidneys show promise in attenuating or possibly preventing these and/or other such mechanisms. the established efficacy of cd154-based costimulation blockade with mmf to prevent induced anti-non-gal antibody elaboration is confirmed by our preliminary findings. recovery of cardiac function after pig-to-primate orthotopic heart transplant. christopher g. a. mcgregor, 1 william r. davies, 1 keiji oi, 1 henry d. tazelaar, 1 randall c. walker, 1 krishnaswamy chandrasekaran, 1 guerard w. byrne. 1 1 mayo clinic, rochester, mn. xenotransplantation has been proposed as a method to alleviate the shortage of donor organs. we report survival of up to 8 weeks of three orthotopic transplants after pig-tobaboon cardiac xenotransplantation. pig-to-baboon orthotopic transplantation was performed using an adult baboon recipient and cd46 transgenic pig. the recipients were treated with an α-gal polymer to block the effects of anti-gal antibody and with atg induction and a tacrolimus and sirolimus based maintenance immunosuppression. heart function was continuously monitored by intramyocardial electrocardiography and every 3 -4 days by serial echocardiography. standard hematological, serum chemistry and cardiac troponin levels were monitored every 2 -3 days. the animals survived 34, 40, and 57 days in a healthy condition. mortality was a result of pneumonitis, respiratory failure and bowel infarction respectively. one recipient (survival 40 days) showed impaired perioperative left ventricular function with an ejection fraction of 25% on echo. over the next two weeks, the ejection fraction in this animal returned to normal (60%) and troponin levels normalized. this study shows the longest survival of orthotopic cardiac xenografts to date with recovery of impaired heart function after early ischemia reperfusion injury. this significant improvement in ventricular function suggests that the normal reparative processes appear to function across the xenotransplantation barrier, supporting the potential clinical potential of cardiac xenotransplantation. purpose: to determine whether the galactosyl transferase gene knock-out (galt-ko) protects pig lungs from hyperacute lung rejection (halr) by human blood. the effects of adding human decay accelerating factor (hdaf) to the galt-ko background will also be examined for the first time. methods: heparinized fresh human blood was used to perfuse galt-ko pig lungs (n=10). lung function was assessed by flow, pulmonary vascular resistance (pvr), oxygen transfer, and tracheal edema using pre-defined survival endpoints. historical wild-type (wt) pig lungs (n=15) provide context for analysis. additionally, two pig lungs expressing galt-ko/hdaf were recently examined, one of which received treatment with xigris. results: median lung survival in galt-ko lungs was 114 minutes (range 15min to >240min) vs. 10 minutes in wt lungs (range 4min to 36min, p= 0.01); 2/2 galt-ko/hdaf pig lungs survived >240 min. pvr at 5 minutes for galt-ko lungs was 62±35mmhg-min/l vs.wt lungs (308±178 mmhg-min/l at 5 minutes) vs. 23mmhgmin/l for each galt-ko/hdaf lung. complement activation (∆c3a) at 15 minutes was significantly lower with galt-ko (∆c3a 133±243 ng/ml) than wt lungs (vs. 2080±1332 ng/ml, p=0.039). galt-ko was also associated with reduced platelet activation: only 4±4% of circulating platelets express cd62p at 15 min. vs. 16±13% in the wt group (p=0.05); ∆βtg at 15 min [223±151 iu/ml (galt-ko) vs. 1196±1186 iu/ ml (wt)], and less thrombin formation (∆ f1+2) at 15 minutes (galt-ko: 5±7.9 nm vs. wt: 22±39 nm). ∆c3a, % of circulating platelets expressing cd62p, ∆βtg, and ∆ f1+2 measurements are in progress for galt-ko/hdaf. platelet sequestration was delayed as mean % of the initial platelets remaining in the galt-ko lung perfusate was 52±19% at 15min vs. 27±9% in the wt lung group (p=0.02). neutrophil sequestration was also diminished in galt-ko (58±15% residual) vs. wt lung (5±3% residual, p=0.004). conclusion: galt-ko pig lungs are significantly protected from several facets of halr: complement and coagulation cascade activation, neutrophil sequestration, and platelet activation are all partially attenuated by this modification alone. preliminary results suggest that galt-ko/hdaf lungs offer even further protection. disseminated intravascular coagulation is associated with tissue factor expression on recipient platelets and monocytes. chih che lin, 1, 3, 4 mohamed ezzelarab, 1 corin torres, 1 david ayares, 2 anthony dorling, 3 david k. c. cooper. 1 1 thomas e. starzl transplantation institute, university of pittsburgh, pittsburgh, pa; 2 revivicor inc., blacksburg, va; 3 department of immunology, imperial college london, hammersmith hospital, london, united kingdom; 4 department of surgery, chang gung memorial hospital, kaohsiung, taiwan. purpose acute humoral xenograft rejection (ahxr), frequently associated with disseminated intravascular coagulation (dic), remains a challenge in pig-to-primate xenotransplantation (tx). a previous in vitro study showed that recipient platelets and monocytes were induced to express tissue factor (tf) after incubation with porcine endothelial cells, and we speculated this may contribute to thrombosis. the present study investigated whether circulating (extragraft) monocytes and platelets express tf and whether this relates to the development of dic after pig tx. methods baboons (n=7) received an aortic patch or kidney from either a wild-type (wt) or α1,3galactosyltransferase gene-knockout (gtko) pig, with or without immunosuppressive therapy. baboon monocytes and platelets were isolated from blood before and after tx. surface tf phenotype was determined by flow cytometry. functional tf activity was determined by clotting time assays after mixing monocytes and platelets with recalcified factor vii (fvii)-deficient plasma with or without added fvii. before tx, monocytes and platelets did not express tf or display tf-dependent procoagulant activity. after gtko aortic patch tx, the baboons were euthanized by day 35 without developing dic. however, by day 28, circulating platelets (but not monocytes) expressed tf and promoted tf-dependent clotting in recalcified plasma. in the absence of immunosuppression, wt kidneys survived <1 day before the onset of dic, at which time tf activity was detected on both platelets and monocytes. after gtko kidney tx in immunosuppressed baboons, platelets expressed tf as early as day 3. in contrast, monocytes began to express tf only at the onset of dic. this study links expression of tf on recipient monocytes and platelets with the development of dic. expression on platelets appeared to predict the subsequent development of dic, whereas that on monocytes was associated with the onset of dic. whilst our observations do not establish a causal relationship, they provide the basis for further study. international human xenotransplantation inventory. antonino sgroi, 1 leo buhler, 1 megan sykes, 2 luc noel. 3 1 surgical research unit, department of surgery, geneva university hospital, geneva, switzerland; 2 transplantation biology research center, massachusetts general hospital, boston, ma; 3 world health organization, geneva, switzerland. background: xenotransplantation carries inherent risks of infectious disease transmission to the recipient and even to society at large, and should only be carried out with tight regulation and oversight. a collaboration between the international xenotransplantation association, the university hospital geneva and the world health organization has established an international inventory (www.humanxenotransplant. org) aiming to collect basic data on all types of xenotransplantation practices on humans that are currently ongoing or have been recently performed. methods: we collected information using publications in scientific journals, presentations at international congresses, internet-based information, and declarations of ixa members. an electronic questionnaire is available on the website www. humanxenotransplant.org, which can be filled out and sent to the office in geneva. results: we identified a total of 16 recent or current human applications of xenotransplantation, eight were currently ongoing and one will start soon. the source animal was: pig (n=7), sheep (n=3), calf (n=1), rabbit (n=2), blue shark (n=1), hamster (n=1), and unknown (n=1). all trials transplanted xenogeneic cells, i.e. islets of langerhans (n= 6), hepatocytes (n=1), kidney cells (n=1), chromaffin cells (n=1), embryonic stem cells (n=2), fetal (n=4) and adult cells (n=1) of various organs. the treatments were performed in 10 different countries, 7 in europe, 2 in russia, 2 in asia, 2 mexico, 1 in usa and 1 in africa. six countries had no national regulation on xenotransplantation. conclusion: several clinical applications of cell xenotransplantation are ongoing around the world, often without any clear governmental regulation. this information should be used to inform national health authorities, health care staff and the public, with the objective of encouraging good practices, with internationally harmonized guidelines and regulation of xenotransplantation. cells from α1,3-galactosyltransferase gene-knockout pigs additionally transgenic for human membrane cofactor protein demonstrate resistance to human complement-mediated cytotoxicity. hidetaka hara, 1 cassandra long, 1 mohamed ezzelarab, 1 peter yeh, 1 carol phelps, 2 david ayares, 2 david k. c. cooper. 1 1 surgery, thomas e. starzl transplantation institute, university of pittsburgh, pittsburgh, pa; 2 revivicor, inc., blacksburg, va. purpose: (1) to compare the antibody binding and complement-mediated cytotoxicity (cdc) of human sera to pig peripheral blood mononuclear cells (pbmc) and porcine aortic endothelial cells (paec) from (i) wild-type (wt), (ii) α1,3-galactosyltransferase gene-knockout (gtko), (iii) human membrane cofactor protein transgenic (mcp) and (iv) gtko/mcp pigs. (2) to investigate the effect on binding and cdc of human sera following activation of paec. methods: pooled human serum was tested by flow cytometry for binding of igm and igg (5% serum concentration) and cdc (25% serum concentration) to pbmc and paec from wt, gtko, mcp, and gtko/mcp pigs. paec from all 4 pig types were activated by ifn-γ, and again tested for antibody binding and cdc. results: there was higher binding of igm and igg to wt and mcp than to gtko and gtko/mcp pbmc and paec, but there were no differences in binding (i) between wt and mcp cells or (ii) between gtko and gtko/mcp cells. cdc of wt pbmc and paec was significantly greater than of gtko, mcp, and gtko/mcp pbmc (wt 73%; gtko 37%; mcp 30%; gtko/mcp 4%) and paec (wt 51%; gtko 18%; mcp 1%; gtko/mcp 0%) (both p<0.05). importantly, there was no lysis of gtko/ mcp paec. after activation of paec, although cdc was increased against wt and gtko paec, mcp and gtko/mcp paec demonstrated significant resistance to lysis (wt 73%; gtko 41%; mcp 5%; gtko/mcp 0%). conclusions: (1) cdc of all 3 types of genetically-engineered pbmc and paec was significantly lower than of wt pbmc and paec, with lysis of gtko/mcp paec being significantly lower than to gtko or mcp cells. (2) paec from both mcp and gtko/mcp showed resistance to lysis even after activation of the cells. (3) organs from gtko/mcp pigs should provide considerable protection against cdc. role for cd47-sirpα signaling in human t cell proliferation in response to stimulation with porcine antigen presenting cells. hiroyuki tahara, 1 hideki ohdan, 1 kentaro ide, 1 toshimasa asahara. 1 1 department of surgery, hiroshima university, hiroshima, japan. we have previously proven that genetic induction of human cd47 on porcine cells provides inhibitory signaling to signal regulatory protein(sirp) α on human macrophages; this provides a novel approach to preventing macrophage-mediated xenograft rejection. a recent report indicated that the similar cd47-sirp system negatively regulates the functions of both t cells and antigen presenting cells(apcs) in humans. we hypothesize that the interspecies incompatibility of cd47 may also act as an additional barrier of t cell-mediated xenograft rejection. we have analyzed the frequency and proliferative activity of human t cells responding to either porcine or allogenic apcs by using in vitro mixed lymphocyte reaction (mlr) assay with a cfse-labeling technique. irradiated stimulator porcine or human peripheral blood mononuclear cells(pbmcs) were cultured with cfse-labeled responder human pbmcs. by fcm analysis, the number of division precursors was extrapolated from the number of daughter cells of each division and from the mitotic events, and precursor frequencies in cd4 + and cd8 + t cell subsets. using these values, stimulation index(si) was calculated. the frequencies of alloreactive and xenoreactive cd4 + t cell precursors were almost identical, 4.5±0.8% and 4.4±0.9%, respectively (n=10-12, for each type of precursor). however, the si of xenoreactive cd4 + t cells was significantly higher than that of alloreactive cd4 + t cells, indicating a stronger reaction by a single xenoreactive cd4 + t cell. in the presence of human cd47-fc(containing the extracellular domain of human cd47 fused to the fc portion of human ig, 10µg/ml), the si of xenoreactive cd4 + t cells was significantly reduced to a level similar to that of alloreactive cd4 + t cells (n=12, p<0.05), although the frequencies of their precursors were absolutely uninfluenced. the frequencies of alloreactive and xenoreactive cd8 + t cell precursors were also identical, i.e. 3.5±0.3%, 3.9±0.9%, respectively (n=10-12). the si of both alloreactive and xenoreactive cd8 + t cells did not differ: however, that of xenoreactive cd8 + t cells was significantly suppressed in the presence of human cd47-fc (n=12, p<0.05). these results suggest that the t cell responses to porcine cells are stronger than those to allogeneic cells because of the interspecies incompatibility of cd47. moreover, genetic manipulation of porcine apcs to induce human cd47 expression might attenuate human t cell-mediated xenograft rejection. reactivation of human cytomegalovirus (hcmv) is a potential risk following the clinical application of pig-to-human xenotransplantation. since little is known about undesirable side-effects arising from hcmv infection of porcine organs, we investigated the capabilities of various hcmv strains to infect porcine endothelial cells (pec) and putative immunological consequences thereof. pec from different anatomical origins were incubated with the hcmv laboratory strains ad169 and tb40/e or a clinical isolate at a multiplicity of infection (moi) ranging from 0.1 to 100. viral replication kinetics, evolution of cytopathology, and lytic end points were analyzed. consequences of pec infection on human nk cell activation were evaluated using xenostimulation assay assessing nk cell ifng secretion and cytotoxicity. infection was evident and a maximum percentage of infected cells was reached at a moi of 10. hcmv replicated in all tested pec types, with a fraction of infected cells ranging from 1% to 50%. ad169 infection of 2a2 (microvascular ec) resulted in cytopathic effect (cpe) development by 3 dpi and in lysis of 70% of the cells at 7 dpi. contrary, no cpe was observed in ped (aortic ec) up to 15 dpi. infection of 2a2 with tb40/e was non-lytic and resulted in accumulation of both extra-and intracellular virus. virus titers reached a maximum at 5 dpi, peaking at levels 20-fold higher than residual input virus. we then determined whether infected pec supported a complete replication cycle and produced viral progeny. after 5 dpi pec supernatants and lysates revealed the production of significant amounts of virus. preliminary results showed that coculture of human nk cells and infected pec resulted in increased nk killing and ifng production. altogether, these findings provide evidence that pec are permissive and support the complete productive replication cycle of hcmv. however, cpe are cell-type and strain dependent. moreover, pec infection leads to modification of the xenogeneic cytotoxicity mediated by human nk cells. our findings allow a better estimation of the potential role of hcmv cross-species infection following xenotransplantation and may be crucial to guide future clinical trials. chronic rejection/injury: innate and adaptive immunity i testing for donor-specificity of antibodies post-transplantation increases the predictability of chronic rejection. mikki ozawa, 1 arundhati panigrahi, 2 paul i. terasaki, 3 narindar mehra. 2 1 one lambda, inc., canoga park, ca; new delhi, india; 3 terasaki foundation laboratory, los angeles, ca. purpose: post-transplant hla antibodies have been shown to be linked to chronic graft failure, yet some recipients do well in the presence of antibodies. we investigated whether testing for donor-specificity of antibodies improves the predictive value of antibodies in regards to chronic rejection. methods: in a prospective study of 400 renal transplant recipients, post-transplant sera were tested for hla and mica antibodies, and positive sera were tested by single antigen beads to determine whether the antibodies were donor-specific. one year after testing, biopsy and clinical data on the graft status were collected. patients who did not have follow-up or died with function were excluded from analysis. results: of the 350 patients with follow-up and abdr typing info, 102 (29%) were found to have antibodies. sixteen of those had donor-specific a, b, or dr antibodies (dsa), while 26 had non-donor specific ab's (ndsa). sixty patients had only dp, dq, cw, or mica antibodies, and were grouped into "donor-specificity unknown", as these typing were not available at the time of this report. table 1 summarizes the antibody groups and transplant outcome one year later. interestingly, the mean serum creatinine values at the time of antibody testing were very similar among the groups, regardless of the presence of antibody or the type (ranged 1.34 to 1.73mg/dl). one year later, however, a striking 63% of dsa patients had either returned to hemodialysis, were regrafted, had died, or had biopsy-proven chronic rejection, compared to only 23% in ndsa patients and 13% in those who were negative (dsa vs. neg, p=0.00009). mica antibodies also showed significant association with graft failure or can (p=0.03), and donor mica typing is underway. conclusion: in this prospective study of 350 renal patients, dsa detected posttransplantation were highly correlated with chronic rejection. inclusion of specificity analysis in post-transplant antibody monitoring would significantly improve the predictability of chronic rejection. donor cd4 t cells within solid organ transplants contribute to graft rejection. thet su win, 1 sylvia rehakova, 1 margaret negus, 1 kourosh saeb-parsy, 1 martin goddard, 2 eleanor bolton, 1 andrew bradley, 1 gavin pettigrew. 1 1 university of cambridge; 2 papworth hospital, united kingdom. this study examines the contribution of donor cd4 t cells within heart allografts to the development of graft vasculopathy, by providing help to recipient b cells for generating effector autoantibody responses. b6 mice were transplanted with mhc class ii-disparate bm12 hearts. the allo-and auto-antibody responses were quantified, and their contribution to allograft vasculopathy assessed by incorporating b cell deficient mice as recipients. the role of donor cd4 t cells in providing help for autoantibody was examined by removing cd4 t cells from donor hearts before transplantation by three approaches: treating with anti-cd4 mab, administering 1300gy lethal irradiation, and using bm12 donors that are genetically deficient in t cells. the contribution of donor cd4 t cells to autoanitbody development was further assessed by challenge with bm12 cd4 t cells two weeks prior to transplantation. bm12 heart grafts developed progressive vasculopathy and were rejected slowly (mst=95 days, n=17). the contribution of antibody-mediated effector mechanisms was confirmed by histopathological evidence (fibrinoid necrosis and vascular proliferation), by complement c4d staining, and by a reduction in the severity of vasculopathy and long-term graft survival in b cell deficient recipients. surprisingly, no alloantibody was detected, but recipients instead developed autoantibody. the autoantibody response was completely dependent upon the provision of help from donor cd4 t cells; it was abrogated by transplanting cd4 t cell deficient hearts. to further confirm an effector role for autoantibody in vasculopathy development, b6 mice were primed for humoral autoimmunity, but not for alloimmunity, by injecting with highly purified bm12 cd4 t cells prior to transplantation. heart grafts were rejected more rapidly (mst=29 days, n=4, p<0.0001), and demonstrated severe vasculopathy. finally, bm-chimeric recipients that lacked mhc class ii expression only on b cells did not develop autoantibody, suggesting that cognate interaction between the donor cd4 t cells and mhc class ii of recipient b cells is crucial for post-transplant humoral autoimmunity. our results demonstrate the novel finding that help for autoantibody production is provided by graft-versus-host cognate recognition of recipient b cell mhc class ii by donor cd4 t cells. this autoantibody contributes to the development of vasculopathy independently of alloantibody. allograft rejection. gang chen, 1 huiling wu, 1 jainlin yin, 1 josette m. eris, 1 steven j. chadban. 1 1 collaborative transplantation research group/renal medicine, university of sydney/rpah, sydney, nsw, australia. toll like receptors (tlrs) are innate immune receptors that play an important role in innate immunity but also provide a link between innate and adaptive immunity. myd88 is a key tlr signal adaptor. a recent clinical study reported that activation of innate immunity in heart-transplantation recipients through tlr4 contributes to the development of chronic rejection after cardiac transplantation. in this study, we aimed to determine whether tlr4-myd88 signaling is required for the development of chronic allograft damage using tlr4 -/and myd88 -/mice in a murine model of chronic cardiac allograft rejection. methods: cardiac transplants were performed: b6.c-h-2 bm12 (b6.h-2 bm12 ) hearts to wt, myd88 -/and tlr4 -/mice (all c57bl/6-h-2 b -single mhc class ii mismatch) as allografts (n = 6-8/group) and c57bl/6 to c57bl/6 isografts (n = 5). blood and tissue were harvested at day 49 after transplantation. cell infiltration, fibrosis and vasculopathy were assessed histologically (grade 0 -5). cd4 + foxp3 + cells in the blood and spleen were measured by flow cytometry analysis. results: all hearts remained pulsatile until sacrifice. histology of tlr4 -/and myd88 -/recipients showed protection from leukocyte infiltration (1.2±1.1&1.0±1.1vs 3.2±1.4, p < 0.01), fibrosis (0.75±1.0& 0.6±1.2 vs 2.6±2.0, p < 0.05) and vasculopathy (0.28±0.6 & 0.04±0.1 vs 1.9±2.2) at day 49 post transplantation compared to wt recipients. by facs, the ratio of cd4 + foxp3 + regulatory t (treg) cells to total cd4 + cells at day 49 post transplantation in spleen was significantly increased in tlr4 -/and myd88 -/recipients versus wt allograft and isograft mice (18.1±0.2 & 19±0.9% vs 12.9 ±1.1 & 11.8±0.2 %, p <0.01). conclusion: absence of tlr4 and myd88 signaling reduces chronic allograft damage in a murine model of chronic cardiac rejection. one mechanism of protection may be enhancement of regulatory t cell function. promotion of treg generation via the tlr4/ myd88 pathway may be one important consequence of cross-talk between innate and adaptive immunity. pathway: implication in transplant arteriosclerosis. thibaut quillard, 1 stéphanie coupel, 1 flora coulon, 1 juliette fitau, 1 maria-cristina cuturi, 1 elise chiffoleau, 1 béatrice charreau. 1 1 inserm u643, itert, nantes, france. endothelial cell (ec) activation, injury and apoptosis are the key events associated with transplant arteriosclerosis (ta) and chronic allograft rejection (cr). notch is a major signalling pathway controlling vascular function and injury. nonetheless, the involvement of notch, at endothelial level, in both ta initiation and progression remains unknown. using a fully mhc mismatched rat cardiac allograft model of cr, we found that ta at day100 correlates with a strong decrease in both expression and activity of the notch pathway in transplant as compared to tolerant and syngeneic controls. the present study investigates the contribution of notch activity to ec survival. in this purpose, a recombinant adenovirus, encoding the notch2 intracellular domain (nicd) and the reporter gfp cdnas was constructed and used to maintain notch activity in cultured primary human arterial ec. our results demonstrate that nicd protects ec from cell death induced by tnfα in the presence of chx or by anoïkis as measured by annexinv and dna content staining. nicd mediates cytoprotection through the regulation of key-apoptosis molecules. using an apoptosis-dedicated qpcr array, we found that 14 out of a panel of 88 apoptosis-related genes were significantly regulated. nicd upregulated bcl2, bcl2a1and bcl-xl (2.5-, 8.2-and 2.8-fold compared to noninfected cells, respectively). in addition, pro-apoptotic genes bim, drp1, bok and cd40 were markedly downregulated in transduced cells (6.7-, 11.4-, 3.9-and 14 .5-fold decrease, respectively). western blotting analysis confirmed the induction of protective molecules (bcl2 and bcl-xl) and the inhibition of pro-apoptotic proteins (bad, bak and bax). consistent with these data, nicd conducted phosphorylation of akt as well as a reduction of pten expression, suggesting that the cytoprotective activity of nicd may be mediated by recruitment of the pi3k survival pathway. to conclude, our findings indicate that ta correlates with a decreased notch signaling in transplant and that activation of the notch pathway in vascular ec prevents apoptosis by promoting protective gene expression and survival pathway. these data suggest that controlling notch activity may prevent ec dysfunction associated with ta and cr. donor age intensifies the early immune response. christian denecke, 1 xupeng ge, 1 irene kim, 1 daman bedi, 1 anne weiland, 1 anke jurisch, 1 steven mcguire, 1 johann pratschke, 2 stefan g. tullius. 1 1 div. of transplant surg, bwh, transplant surg res lab, boston; 2 dept. of surgery, charite berlin, germany. increasing numbers of organs from elderly donors are currently utilized for transplantation. advanced donor age may not only be associated with physiological impairments but also with a modified immune response of the recipient. we hypothesized a more potent early immune response following the transplantation of elderly donor organs and we analyzed the immune response in a mouse heart transplant model. young b6 mice received heart allografts from 3, 12 and 18mths old bm12 donors. the recipients immune response and intragraft changes were analyzed. elderly, non-manipulated hearts contained overall significantly elevated frequencies of cd4 + and cd8 + t-cells and dcs (cd11c + ) (18mths vs. 3mths: cd4 + : 2.77% vs. 1.35%, cd8 + : 3.90% vs 1.71%, cd11c + : 46.1% vs.11.8%, p<0.05). following engraftment of 18mths old heart grafts numbers of activated dc's (cd11c + i-a b+ and cd11c + cd40 + ) had significantly increased in recipient spleens (day 14:p<0.05). in parallel, frequencies of effector/memory phenotype t-cells (cd4 + cd44 high cd62l low and cd8 + cd44 high cd62l low ) were significantly elevated with increasing age (3 vs. 12 vs, 18mths: cd8 + cd44 high cd62l low :6.7% vs. 8.4% vs. 12.2%, respectively, p< 0.01). in addition, tregs (cd4 + cd25 + foxp3 + ) were also elevated (3 vs. 12 vs, 18mths: 2.9%vs.9.6%vs.11%,p<0.05) t-cell alloreactivity, as measured by ifnγ-production, increased with donor age (3mths vs. 12mths vs.18mths: 24.4±1.5 vs 74.4±21.9 vs. 73.2±7.8 ifnγ-producing spots/5x10 6 cells, p<0.05). mixed lymphocyte reaction (mlr) at day 14 revealed a gradual increase in splenocyte proliferation with advancing donor age (p=0.0002) indicating an enhanced immunogenicity of older organs. immunohistochemical staining confirmed augmented cd4 + and cd8 + t-cell infiltrates and an intense ki67 positivity of gics in 18mths old heart grafts, emphasizing an intensified immune response towards organs from old donors. in summary, old native heart transplants contain an overall higher number of passenger leukocytes contributing to an increased immunogenicity of these organs. after transplantation, a more potent dc and t-cell activation and intragraft t-cell infiltration was observed in elderly organs. nox and chronic kidney allograft interstitial fibrosis. shannon reese, 1 madhu adulla, 1 surmeet bedi, 1 jose torrealba, 1 deb hullett, 1 arjang djamali. 1 1 nephrology, uw madison, madison, wi. to determine the role of oxidative stress (os) in kidney allograft fibrosis, we are developing strategies that would decrease the generation of reactive oxygen species (ros) instead of using ros scavengers, the standard approach to antioxidant therapy so far. we therefore started to examine the expression of nadph oxidase (nox) subunits (nox2, nox4, p22 and p47phox) and their distribution in human and rat kidney allografts with chronic interstitial fibrosis (iftanos). using double-staining immunofluorescent studies in human allografts (n=16) we showed that nox2 was present in injured tubules costained with αsma ( figure 1.1) similarly, interstitial macrophages (cd68 + -nox2 + ) and myofibroblasts (αsma + -nox2 + ) but not cd3 + t cells or s100a4 + fibroblasts expressed high nox2 levels, suggesting that nox is involved in the pathogenesis of allograft fibrosis via epithelial-tomesenchymal transition (emt), macrophage and myofibroblasts activation. we then examined the coexpression of nox2, nox4 and p22phox in normal and transplant kidneys with iftanos and showed that these molecules were upregulated in the latter group and that nox2 and nox4 were associated with p22phox expression. these results were confirmed in the fisher to lewis rat kidney transplant model (figure 1 .2). immunoblot analyses at 3 weeks and 6 months showed that nox4 and nox2 levels were increased in the allogeneic (n=7) compared to syngeneic transplants (n=3). greater nox levels were composite tissue allotransplantation (cta) is a recently introduced option for limb replacement and reconstruction of tissue defects. as other allografts, a cta grafts can undergo immune-mediated rejection, and standardized criteria are required for characterizing and reporting severity and types of rejection. this manuscript documents the conclusions of a symposium on cta rejection held at the ninth banff conference on allograft pathology in la coruna, spain on june 26, 2007, and proposes a working classification scheme, the banff cta-07, for the categorization of cta rejection. this classification was derived from public international consensus discussions regarding all published scoring systems for cta rejection. given the current limited clinical experience in cta, a formal histological classification was established for acute skin rejection with the understanding that other types of rejection involving other tissues will be developed with periodic review of this emerging field. it was agreed that the defining features to diagnose acute skin rejection would include inflammatory cell infiltration with epidermal and/or adnexal structure involvement, epithelial apoptosis, dyskeratosis, and necrosis, and that severity of rejection will be graded under five categories. this classification refines proposed schemas, represents international consensus on this topic, and establishes a working collective classification system for cta reporting. the role of skin biopsies in diagnosing clinical rejection in hand composite tissue allotransplantation (cta). christina l. kaufman, 1, 4 ruben n. gonzales, 1 kadiyala v. ravindra, 3, 4 brenda w. blair, 1 joesph f. buell, 3, 4 warren c. breidenbach. 1, 2, 4 1 christine m. kleinert institute, louisville, ky; 2 kleinert kutz and associates, louisville, ky; 3 jewish hospital transplant center, louisville, ky; 4 surgery, university of louisville, louisville, ky. aim: traditionally allograft biopsy has dictated treatment of allograft rejection. we hypothesize that histological grade of the biopsy may over diagnose rejection in cta hand cta is unique in that the organ is external and early signs of rejection can be viewed directly. skin is the first tissue in the cta graft to show rejection. methods: for this study, rejection by defined by requirement of treatment. these episodes were compared with histological grade, and hand appearance. we reviewed 127 skin biopsies taken during 9, 7 and 1 years of follow up. results: three observations surfaced. first, a rejection grading scale for cta is still evolving. a review of the literature showed at least four different criteria are in use. the criteria used to grade biopsies at our center changed over the 9 year follow up. bias towards calling higher grades of rejection, and more aggressive treatment occurred in the first two patients. a re-read of random biopsies taken from the first two patients showed a down-grade of rejection to grade ii or i in five cases. secondly, concomitant cmv infection in the last patient prevented the aggressive treatment of rejection. despite high grade histology, the swelling, rash and redness responded to topical immunosuppression. systemic treatment was not necessary. analysis indicated that swelling and/or rash, and the percentage of skin involvement seemed to be more informative markers of existing (or resolving) alloreactivity than was histology. the biopsies taken from the graft shown below showed a grade iii histology. thirdly, changes in hand function were not associated with changes in biopsy grade. conclusion: the histologic grade of skin biopsies from hand allografts appears to over estimate rejection. alterations in immunosuppression shoud be based on appearance of the allograft, and clinical course as much as on the histologic grade of the biopsy. expression of molecular mechanisms of lymphozyte trafficking correlates closely with skin rejection in human hand transplantation. theresa hautz, 1 bettina zelger, 2 gerald brandacher, 1 hans g. mueller, 3 andrew w. p. lee, 4 raimund margreiter, 1 stefan schneeberger. 1 1 dept. of general and transplant surgery, innsbruck medical university, austria; 2 dept. of pathology, innsbruck medical university, austria; 3 dept. of dermatology, innsbruck medical university, austria; 4 div. of plastic surgery, university of pittsburgh. introduction: to understand in greater depth the molecular mechanisms involved in skin rejection in hand transplantation, we investigated 8 key molecular markers of lymphocyte trafficking, cellular rejection and antibody mediated rejection in human hand transplantation. methods: a total of 130 skin biopsies taken from three bilateral hand transplants were assessed by h&e histology (grades as per previously a published classification 0-4b) as well as immunohistochemistry using antibodies for following markers: lymphocyte function-associated antigen (lfa)-1 = cd11a, intercellular adhesion molecule (icam)-1 = cd54, selectin e = cd62e, selectin p = cd62p, ve-cadherin = cd144, human leukocyte antigen (hla) ii (dp, dq, dr), psoriasin = s100a7 and c4d. levels of expression were assessed (0, +, ++, +++) and read in the light correlated with the rejection as well as time after transplantation. results: rrejection ranged between grade 0 and 4a with an average score of 0.9. in healthy skin, none of the markers investigated was consistently up-regulated. upon rejection, cd54, cd62e and cd62p staining in endothelial cells was significantly increased. expression of cd62e and cd62p correlated well with severity of rejection. the majority of infiltrating lymphocytes stained positive for cd11a. interestingly, also kerationcytes were highly positive for cd11a at the onset of rejection. cd144 was detected on endothelial cells, but its occurrence did not correlate with rejection. psoriasin expression was observed in keratinocytes in a basal and focal pattern and correlated well with rejection. for c4d, no consistent staining pattern was observed indicating that antibody mediated rejection did not play a role in these patients. conclusion: molecular markers involved in lymphocyte trafficking are up-regulated upon skin rejection after hand transplantation and represent promising target for prophylaxis and treatment of rejection in composite tissue allotransplantation. investigation of the immunomodulatory phenotype of infiltrating lymphozytes in skin rejection of human hand allografts. theresa hautz, 1 gerald brandacher, 1 bettina zelger, 2 hans g. mueller, 3 andrew w. p. lee, 4 raimund margreiter, 1 stefan schneeberger. 1 1 dept. of general and transplant surgery, innsbruck medical university, innsbruck, austria; 2 dept. of pathology, innsbruck medical university, austria; 3 dept. of dermatology, innsbruck medical university, austria; 4 div. of plastic surgery, university of pittsburgh. introduction: skin rejection has complicated the postoperative course in human hand transplantation. to better define the characteristics of the lymphozytic infiltrate human hand transplant biopsies have been investigated for expression of foxp3 and indoleamine 2,3-dioxygenase (ido), a key regulatory enzyme to induce t-lymphocyte unresponsiveness. methods: a total of 104 skin biopsies taken from three bilateral hand transplant recipients during the first 6 years after transplantation were assessed by h&e histology (graded as per previously published classification 0-4b) as well as immunohistochemistry for ido and foxp3. levels of expression were assessed (0, +, ++, +++) and interpreted in the light of clinical courses, time after transplantation, severity of rejection as well as markers for lymphozyte migration. results: overall, rejection ranged between grade 0 and 4a with an average score of 0.94. ido was found constitutively expressed in the endothelium independent of rejection. ido expression in the cellular infiltrate was significantly increased upon and correlated well with severity of rejection (rejection grade 1, 0.91+/-0.85: rejection grade 3, 2.30+/-0.82 p<0.001). foxp3 positive t-cells were mainly found in severe rejection (rejection grade 1, 0.14+/-0.35: rejection grade 3, 0.75+/-0.75 p=0.019). ido expression correlated well with foxp3 expression, although the overall staining intensity for foxp3 was lower. a strong tendency towards higher expression of ido as well as foxp3 towards later time-points after transplantation was observed (year 1 -foxp3 0.07+/-0.26, ido 0.75+ /-0.92 [n=68] , year 3 -foxp3 0.45+/-0.74, , year 5 -fox 0.25+/-0.45, ). expression of ido correlated closely with expression of e-selectin, p-selectin, icam1 and lfa-1. conclusion: characteristics of the cellular infiltrate indicate a strong tendency towards self limitation of the alloimmune response towards the skin with both time after transplantation as well as severity of rejection in human hand transplantation. further studies are warranted to clarify the clinical relevance of these findings. prospective analysis of the immunologic profile of a hand transplant recipient in the first year. kadiyala v. ravindra, 1 warren c. breidenbach, 2 joseph buell, 1 suzanne t. ildstad. 3 1 department of surgery, university of louisville, louisville, ky; 2 kleinert, kutz, and associates, louisville, ky; 3 institute for cellular therapeutics, university of louisville, louisville, ky. introduction: a major obstacle to the wider application of hand transplantation is the long term complications associated with immunosuppression. minimization of immunosuppression is an important goal in all transplant recipients. currently there are no accurate tools to evaluate the immunological responsiveness which might help tailor the level of immunosuppression for an individual patient. the response of recipient lymphocytes to pha, candida, and alloantigen may represent laboratory tool towards this end. it has been reported that these 3 responses are hierarchical with response to alloantigen being the first to be lost, followed by candida and finally pha. a 54 year old male received a proximal forearm transplant in november 2006. immunosuppression included induction with a single 30 mg dose of alemtuzumab and maintenance with tacrolimus and mycophenolate mofetil. the patient developed an episode of cytomegalovirus infection followed by acute rejection after reduction of his immunosuppression during the 3 rd post-operative month. these were successfully treated with ganciclovir and topical tacrolimus & steroids respectively. blood samples were drawn at selected time points, and subjected to phenotyping of lymphocyte subsets and immune monitoring for circulating peripheral blood regulatory t cells (t reg ) and proliferative responses to phytohemagglutinin (pha), candida, and alloantigen. results: alemtuzumab induction resulted in profound lymphopenia. at week 2 and 1 month, the response to pha was intact (stimulation index 110 and 24 respectively), but response to alloantigen and candida suppressed (si <3). a similar immunologic profile persisted up through 6 months. at 1 year, the pha and candida responses are robust (si 69 and 38 respectively), but alloresponses have not returned. current immunosuppression consists of tacrolimus (6-9 ng/ml) and mycophenolate mofetil (500 mg b.i.d.). there is no gross evidence of acute or chronic rejection. conclusions: induction with alemtuzumab alters the recovery of immune response: recovery to candida was delayed beyond 6 months and to alloantigens beyond a year. in light of this, further reduction of immunosuppression may be contemplated in future hand transplants without the risk of rejection. composite tissue allotransplantation has achieved significant clinical advances despite an adequate preclinical model to study technical and immunosuppressive strategies. we have developed a non-human primate model of facial composite tissue allografts (cta). unilateral lower hemi-facial cta (bone, muscle, skin) transplants were performed between mismatched cynomolgus monkeys. immunosuppression consisted of 28 days of continuous iv tacrolimus monotherapy followed by tapered daily im doses. six animals received prophylactic gancyclovir. all animals had serial transplant biopsies. ten transplants have been performed, with one loss secondary to line infection on day 27 without evidence of rejection. two ctas had evidence of chronic rejection (day 56, 99); with development of alloantibody after 30 days. five ctas had prolonged survival (day 60, 86, 108, 150, 177) , but developed ptlds resulting in experimental endpoints. all animals had clinically normal grafts, but 3 animals showed histological evidence of mild rejection not treated with any additional immunosuppressive therapy. ptld tumors were analyzed using short tandem repeats (str) to define donor or recipient origin. str analysis demonstrated donor origin of 4 ptld tumors and recipient origin in 1 animal. none of the ptld animals had clinical evidence of rejection of skin, bone, or muscle. ebv was not detected in the serum of 2 tested animals, and ganciclovir therapy had no effect on the development of tumors. tacrolimus levels of ptld animals were higher than animals with rejected grafts (45 vs. 34 ng/ml; p=0.02). two additional animals have healthy grafts (day 24+, 52+) without evidence of rejection or ptld, and have been converted to rapamycin after day 28. we have developed a preclinical model for facial cta transplantation that achieves prolonged graft survivals with tacrolimus monotherapy. the high incidence of ptld tumors of donor origin represents an outcome similar to bone marrow transplantation in contrast to its rarity in solid organ transplantation. our findings are a cautionary note regarding ctas that include vascularized bone elements. immunosuppressive protocol modifications have been made in an effort to decrease the incidence of these donor-derived ptlds. heterotopic heart transplantation: the united states experience. jama jahanyar, 1 tarek a. sibai, 1 matthias loebe, 1 michael m. koerner, 1 guillermo torre-amione, 1 george p. noon. 1 1 dept. of surgery, baylor college of medicine, houston, tx. heterotopic heart transplantation (hht) is utilized in patients (pts) who do not qualify for standard orthotopic heart transplantation (oht). specific indications include refractory pulmonary hypertension and a donor-recipient size mismatch. the objective of this study was to analyze the unos database and compare outcomes of hht to oht. the unos database with more than 58000 pts undergoing thoracic organ transplantation in the u.s. between 1987 and 2007 was reviewed (based on optn data as of may 1, 2007) . primary endpoint of this study was overall survival and subgroup survival [pts with transpulmonary gradient (tpg)>15, ischemic (icm) and dilated cardiomyopathy (dcm)]. secondary endpoint was assessment of pretransplant criteria. exclusion criteria were retransplantation and missing transplant dates. of 41379 who underwent oht and 178 who underwent hht, 32361 and 111 respectively, were enrolled in this study. [5] [6] [7] [8] [9] [10] survival after oht is superior to hht. this survival benefit however, disappears in pts with a tpg>15. overall the survival after hht is superior to the reported survival in pts who undergo lvad implantation as destination treatment (rematch-trial/1year survival of 52%). thus in selected pts, especially those with elevated tpgs, hht should be considered a viable option with overall good results. ventricular assist device as a bridge to heart transplantation in children: can we afford it? william t. mahle, glen ianucci, robert n. vincent, kirk r. kanter. sibley heart center cardiology, emory university school of medicine, atlanta, ga. ventricular assist devices (vads) allow children with severe heart failure to be bridged to successful heart transplantation (ht). vads are being used with increasing frequency in the pediatric population and newer devices allow even young infants to be supported. vad implantation and maintenance, however, is quite expensive and the cost-effectiveness of vad use in adults has been questioned. to date, an economic analysis of vad support in children has not been undertaken. methods: we used pediatric health information system (phis), an administrative database of the child health corporation of america (a consortium of children's hospitals in north america), to determine the costs related to vad use in children. data on subjects <18 yrs of age from 2002-2007 were reviewed. hospital charges were converted to costs based on hospital-specific cost-to-charge ratios. projected survival for subjects who were successfully bridged to ht was derived from published data. costutility was expressed as cost per quality-adjusted life years (qalys) saved, expressed in 2006 us dollars. all future costs and benefits were discounted at 3%. results: the median age at implantation from the phis database was 11.2 years, range 2 days to 17 yrs. the mean hospital cost per patient was $522,489. estimated survival to heart transplantation was 77% and estimated successful explantation without transplantation was 4%. the calculated cost-utility for vad as a bridge to transplantation was $123,232 /qaly saved. if one assumes that the children who survived to vad explantation would otherwise have a high risk of hospital death (65%) without vad support, then the calculated cost-utility would be $117,235/ qaly saved. even if abstracts survival to transplantation exceeds 90%, vad implantation does not achieve a favorable cost-utility ratio. vad support in pediatric heart only becomes cost-effective if recovery and explantation can be achieved in over 22% of subjects. conclusions: vad supports serves as an effective bridge to heart transplantation in children. however, the cost-utility of this strategy is above the generally accepted threshold for cost-effectiveness ($100,000/qaly). in a setting of limited healthcare resources vad as a bridge to transplantation may not be justified. purpose: heart transplantation (tx) in patients with hla sensitization presents challenges in organ allocation. virtual crossmatch (vxm), in which recipient hla antibodies, identified by labscreen pra beads, are compared to the prospective donor hla-type, could increase the use of allografts from distant donors (dd). accuracy of vxm and outcomes of this approach in heart tx are not known. methods and materials: to increase time-efficiency at the time of organ allocation, crossmatch testing is frequently initiated on pre-set trays which contain sera of multiple prospective sensitized recipients. we used results from these studies to determine expected accuracy of vxm. we assessed outcomes of allocation algorithm implemented in 2001 in sensitized patients. conventional prospective crossmatch was done when allografts were procured from local donors (ld), while vxm was utilized when allografts were offered from dd. there were 257 direct t-cell ahg crossmatch tests done with sera of 12 potential allograft recipients who had preformed hla-antibodies of known class i hla specificities. as shown in table 1, the positive and negative predictive values (ppv, npv) of vxm were 92% and 79%. table 2 shows outcomes in 30 sensitized patients who were eligible for vxm approach. 14 received allografts from ld with negative prospective crossmatch while 16 (53%) received allografts from dd with negative vxm. three dd patients had a positive retrospective crossmatch -npv of vxm was 81% in this cohort. vxm has high negative and positive predictive values, accurately predicting results of standard direct crossmatch in most patients. vxm allows use of allografts from dd and is likely to improve organ allocation in the disadvantaged group of sensitized patients. single center experience with new heart allocation system implemented by united network of organ sharing. biljana pavlovic-surjancev, 1 nilamkumar patel, 1 linda dusek, 1 james sinacore, 1 jennifer johnson, 1 cassie bessert, 1 alain heroux. 1 1 heart failure/heart transplant program, loyola university medical center, maywood, il. purpose: in july 2006, united network of organ sharing (unos) implemented a new allocation system for adult heart transplant (tx) candidates with the following sequence: local status 1a→local status 1b→zone a status 1a→zone a status 1b→local status 2. the purpose of this study is to evaluate the impact of new system on heart tx patients at a single center in region 7. methods: patients transplanted during 1 year (y) prior to new system (7-11-05 to 7-11-06, group 1, n=19) and 1 y following new system (7-12-06 to 7-12-07,group 2, n=26) were compared for unos status at the time of transplant, waiting time, ischemia time, length of the hospital stay (los) before and after transplant, donor age, procurement-team travel distance and cost. results: new system significantly decreased median waiting time, but increased median ischemia time without affecting short-term survival: 1 patient died in each group. number of transplants increased 37% in group 2 mostly due to increased number of status 1a patients supported by iabp without change in number of status 1b and 2 patients. median pre-transplant los increased 16-fold in group 2 (p=0.072), whereas mean pre-transplant los increased by 5 days. in group 2, thirteen patients had donor heart procured in zone a and thirteen patients had donor heart procured locally, whereas in group 1, all donor hearts were procured locally. median procurement-team travel distance increased 15-fold and median travel cost 10-fold in group 2 (p<0.05). clinical outcomes associated with simultaneous heart-kidney transplantation. tariq shah, 1, 3, 4, 5 suphamai bunnapradist, 2 jagbir gill, 2 steven k. takemoto. 1 the figure below indicates recipients of shk transplants (open symbols) had lower rates of rejection when compared to heart (square) or kidney (diamond) recipients. survival rates were initially higher for kidney recipients with rates for shk similar to heart recipients. the hazard ratio (hr) for graft loss was higher for shk recipients compared to kidney, but lower when compared to heart recipients. the lower hazard for shk in heart allografts might be attributed to risk associated with pretransplant dialysis (hr=4.09, 3.81-4.39, p<0.001) . approximately 10% (2,122) of cardiac transplant recipients initiated dialysis prior to transplantation. the differing rates of shk rejection observed in the heart and kidney analytic files might be attributed to susceptibility or treatment of heart and kidney allografts, rejection monitoring or reporting. conclusion: retrospective examination of data provided to the optn indicates simultaneous heart-kidney transplantation seems to be effective for cardiac transplant candidates who require dialysis. abstract# 123 objective: krp203, a novel structural analog of fty720, has been documented to display 5-fold greater selectivity of agonism for sphingosine-1-phosphate (s1p) type 1 (s1p 1 ) receptor compared with s1p 3 receptor. clinical trial has shown that fty720 produced dose-limiting toxicity-bradycardia, due to its effect on s1p 3 receptor. we have tested the effect of krp203 on the survival of islet allografts either alone or combined with local delivery of cd4 + cd25 + foxp3 + t regulatory (treg) cells. previous studies using knockout mice have documented a key role for the integrin cd103 in promoting allograft rejection. these data are consistent with a critical role for cd103 expressing cells in this process. however, a direct test of this hypothesis has proven problematic due to the lack of mabs that efficiently deplete cd103 + cells in wild type hosts. to circumvent this problem, we conjugated the non-depleting anti-cd103 mab, m290, to the toxin, saporin (sap), to produce an immunotoxin (m290-sap) that selectively depletes cd103-expressing cells in vivo. treatment of naive mice with m290-sap selectively depleted cd103 + cd8 + cells and dramatically reduced the overall frequency of cd8 + lymphocytes in diverse compartment including intestinal intraepithelial lymphcyte,spleen and mesenteric lymph node. m290-sap also depleted cd103 + dendritic cells (cd11c + ) and t regulatory cells (tregs, cd4 + cd25 + ) in the above compartments. in the thymus, m290-sap depleted cd103-expressing cells in both cd4 -cd8and cd4 -cd8 + subpopulations, both of which express cd103, leading to a dramatic reduction in the number of thymocytes(77.08 ± 5.74 ×10 6 vs. 2.71 ± 0.82 ×10 6 , p<0.0001, in control vs. treated respectively). we next assessed the effect of m290-sap in a fully allogeneic islet transplantation model (balb/c→c57bl/6). m290-sap produced long-term (lt) graft survival (>100d, n=9,vs. untreated median survival time 13d, n=8 ). unconjugated m290 or isotype control (igg-sap) did not significantly prolong islet allograft survival. graft histology showed little, if any, lymphocyte infiltration surrouding islet transplants in lt mice. in contrast, intense lymphocyte infiltration with disruption of islet morphology was observed in untreated mice. pretreatment of donor islets with m290-sap did not significantly prolong allograft survival indicating that the immunosuppressive effect of m290-sap resides at the level of host. interestingly, we found a 3-4 fold increase in both percentage and number of foxp3 + cd4 + cd25 + cells in the spleen and draining lymph node from lt mice, though it remains to be determined whether such cells account for graft acceptance in m290-sap treated recipients. in summary, these data document that depletion of cd103 expressing cells promotes long-term islet allograft survival. these findings point to a novel strategy for therapeutic intervention in islet allograft rejection. pancreatic objective: to engineer pancreatic islets in a rapid and efficient manner with a novel form of fasl protein chimeric with core streptavidin and test the efficacy of engineered islets for long-term survival in allogeneic hosts. methods: balb/c pancreatic islets were engineered first by cell surface modification with biotin followed by the display of a chimeric form of fasl protein that consists of extracellular domain of fasl fused c-terminus with core streptavidin (sa-fasl). sa-fasl-engineered islets were transplanted into streptozotocin diabetic c57bl/6 mice under transient cover of rapamycin. unmodified islets or those engineered with streptavidin protein (sa) served as controls. results: all the islets showed effective engineering with sa-fasl, which persisted on the surface of islets for weeks in vitro as assessed by confocal microscopy. all the islets (n=23) engineered with sa-fasl survived over the observation period of 100-400 days without detectable signs of rejection. in marked contrast, all the unmodified (n=9) and sa-engineered (n=14) islets underwent acute rejection within 40 days. the observed tolerance was localized to the engineered islets as unmodified second set of islets transplanted under contralateral kidney of long-term (>90 days) graft recipients were rejected in a normal tempo (mst=17 ± 9 days) without any effect on the survival of primary islets. conclusions: engineering pancreatic islets with exogenous immunomodulatory molecules, such as sa-fasl, in a rapid (∼ 2 hrs) and efficient (100% of targeted islets) manner represents a novel means of immunomodulation with considerable therapeutic potential for the treatment of type 1 diabetes. supported in parts by nih (r21 dk61333, r01 ai47864, r21 ai057903, r21 hl080108), jdrf (1-2001-328) the ability of embryonic stem (es) cells to form cells and tissues from all three germ layers can be exploited for the generation of cells that can be used to treat diseases. in particular, successful generation of hematopoietic cells from es cells could provide safer and less immunogenic cells than bone marrow cells, that require severe host preconditioning, when transplanted across mhc barriers. in the past, it has been difficult to derive hematopoietic cells from es cells. it has now become clear that this was due to the lack of self-renewal properties by these newly developed progenitor cells. here, we exploited the self-renewal properties of ectopically expressed hoxb4, a homeobox transcription factor, to generate hematopoietic progenitor cells (hpcs) that successfully induce high level mixed chimerism and long-term engraftment in recipient mice. hoxb4-transduced 129svj es cells (h2 b ) were allowed to form embryoid bodies. these were dismantled after 5 days and the cells treated with a cocktail of hematopoietic cytokines. by day 26, es cells had formed hpcs. these newly generated hpcs were cd45+, cd34+, cd117+ but poorly expressed mhc class i molecules and no class ii. the hpcs fully restored splenic architecture in rag2 -/-γ c -/immunodeficient mice, abstracts comparable to bone marrow. additionally, hpc-derived newly generated t cells were able to mount a peptide-specific response to lymphocytic choriomeningitis virus (lcmv) and specifically secreted il-2 and ifn-γ upon cd3 stimulation. further, hpc-derived antigen presenting cells (apcs) in chimeric mice efficiently presented viral antigen to wild type (wt) t cells. in syngeneic recipient mice, hpcs engrafted and formed more robust t and b cell populations. the majority of the hpc-derived cells were however, gr-1 + , suggesting a bias towards myeloid cells by the hoxb4. interestingly, these cells successfully engrafted in allogenic mrl (h2 k ) and balb/c (h2 d ) recipients without the need for immunosuyprresion. this ability to form mixed chimerism across mhc barriers is a consequence of their lack of mhc, cd80 and cd86 expression. our results demonstrate for the first time that leukocytes derived from es cells ectopically expressing hoxb4 are immunologically functional and escape immunological rejection when transplanted across mhc barriers allowing the induction of mixed chimerism. normalization the de is derived from the anterior segment of the primitive streak which corresponds to the early and mid-gastrula organizer during early embryo development, from which many of the major visceral organs, including the liver, pancreas, lung, thyroid and intestines are derived. es cells were cultured in a serum-free medium containing activin a and bfgf for 6-8 days, the differentiated cells developed into an epithelial monolayer yielding more than 70% of cxcr4 expressing cells. cxcr4 has been reported as a cell surface marker of the definitive endoderm. molecularly, the differentiated es cells express typical definitive endodermal genes in particular, foxa2, sox-17, gsc, and hnf4α. the cxcr4 + definitive endodermal cells were further purified using immunomagnetic bead separation to more than 99% purity in order to eliminate teratoma-forming cells. to study their engraftment and regenerative capacity, these newly differentiated cells were intravenously infused into a mouse with carbon tetrachloride-induced liver injury. harvested livers from these animals showed large de-derived cells positive for albumin suggesting that they were de novo generated hepatocytes. a second cell type was ck-19 expressing, suggesting that the engrafted cells also differentiated into cholangiocytes. therefore, we further transplanted these de cells into a factor viii null mouse and asked whether these cells could correct factor viii activity. plasma factor viii activity (coamatic assay) fully normalized to that of wild type mice and has remained stable over 60 days. these data suggest that es cell-derived de progenitor cells can restore factor viii activity in hemophilia a mouse model, presumably through protein production in de novo generated hepatocytes. more importantly, none of these animals developed teratomas. thus, es-cell derived cells can potentially be coaxed to form cellular transplants with curative capabilities. objective: we have established a novel approach, protex, to rapidly and efficiently engineer primary cells, tissues, or organs to display on their surface exogenous proteins of interest for immunomodulation. this approach involves generation of chimeric proteins with core streptavidin, biotinylation of cells, and the transient display of chimeric proteins on the cell surface. in this study, we displayed a chimeric form of fasl (sa-fasl) on the surface of bone marrow cells and tested the efficacy of these cells to establish mixed chimerism in allogeneic hosts under nonmyeloablative conditions. methods: balb/c bone marrow cells were engineered with sa-fasl and 30 million of these cells were transplanted into c57bl/6 mice subjected to various doses of total body irradiation 2 days earlier. a short course of rapamycin was used to enhance the tolerogenic effect of sa-fasl. bone marrow cell recipients were typed for multilineage chimerism at various times post-transplantation and tested for donor-specific tolerance using skin grafts. results: all the animals (n=8) treated with 300 cgy total body irradiation and transplanted with sa-fasl-engineered donor cells showed significant levels of chimerism (10-60%) on day 14 post-transplantation that showed a steady increase overtime and reached to 40-90% on day 60 post-transplantation. in marked contrast, none of the control animals (n=11) receiving bone marrow cells and a short course of rapamycin showed detectable chimerism. chimerism was multilineage and associated with donor specific tolerance since chimeric animals accepted donor, but rejected third party skin grafts. conclusions: engineering bone marrow cells in a rapid (∼2 hrs) and efficient (100% targeted cells) manner with exogenous proteins having immunoregulatory functions provides a new and effective means of immunomodulation to establish mixed allogeneic chimerism under nonmyeloablative conditions with significant potential in clinical bone marrow transplantation. funded in parts by nih (r21 dk61333, r01 ai47864, r21 ai057903, r21 hl080108), jdrf (1-2001-328) , ada , and aha fellowship 0725348b. chronic allograft dysfunction is still a major clinical problem in organ transplantation. morphologically it is characterized by changes suggestive of an alloantibody mediated mechanism such as glomerulopathy and vasculopathy or by non-specific changes such as interstitial fibrosis and tubular atrophy. alloantigen dependent as well as -independent factors contribute to the pathogenesis of these changes. in this study we analysed allospecific t cells from the peripheral blood of kidney transplant patients under different immunosuppressive protocols with or without chronic allograft dysfunction. 107 renal allograft recipients of our renal transplant clinic were screened at least six months after transplantation. all patients were on a calcineurin-based immunosuppressive protocol consisting of cyclosporine (csa)/mycophenolate mofetil (mmf)/steroid, tacrolimus (tac)/mmf/steroid, or csa/steroid. patients had to be mismatched for one or more of the five candidate hla-dr antigens for which synthetic peptides were available (dr1, dr2, dr3, dr4, and dr7). patients with biopsy proven chronic allograft nephropathy (can)with an elevated serum creatinine level of ≥1.6 mg/dl were compared with patients with stable allograft function (serum creatinine <1.6 mg/dl). t cell lines were generated from peripheral blood lymphocytes of renal transplant recipients against donor-derived hla-dr peptides presented by self apc. t cell lines generated from patients with can produced significantly more ifn-γ, while those generated from stable patients produced il-10 associated with a low proliferation index in response to the donor-derived mismatched hla-dr allopeptide in vitro. moreover, significantly more cd4+cd25+foxp3+ t cells were found in stable patients on tac and mmf as compared to patients on csa and mmf. interestingly, a higher gene expression of cd4, cd25, ctla-4, foxp3, and il-10 was observed in those patients. taken together a th1 (ifn-γ) alloimmune response is deleterious and promotes chronic graft damage, while a th2 (il-10) response seems to be associated with a lower incidence of chronic allograft nephropathy. an immunosuppression based on tac and mmf seems to favour cd4+cd25+fosp3+ t cells and to allow long-term engraftment with stable renal function. cyclosporin induces epithelial to mesenchymal transition in renal grafts. the expression of epithelial to mesenchymal transition (emt) markers is a reliable predictor of the progression towards interstitial fibrosis and tubular atrophy of the renal grafts. in vitro experiments suggest that calcineurin inhibitors (cni) can induce emt of tubular epithelial cells. although no evidence was ever provided in vivo, this suggests that emt could be involved in the pathogenesis of renal fibrosis induced by cni. we have previously reported the results of a prospective randomized trial comparing the elimination at month 3 of either cyclosporine (csa, n=54) or mycophenolate (mmf, n=54) from a triple drug regimen in 108 de novo renal transplant patients. all of them had 2 systematic graft biopsies at months 3 and 12 post engraftment. in the leftover material, we retrospectively detected in tubular cells and by immuno-histochemistry the expression of two validated markers of emt: the de novo expression of vimentin (vim) and the cytoplasmic translocation of b-catenin (cat). we were able to measure the emt score at both months 3 and 12 in a total of 68 patients (34 in each group). in the csa group, the vim and cat scores had progressed between 3 and 12 months from 1. calcineurin inhibitors (cni) are efficacious but nephrotoxic immunosuppressives. arteriolar hyalinosis is one of the characteristic histological correlates of this toxicity. yet, time course of this lesion, its reversibility, dose-dependency and the discrimination between drug-related effects, diabetic and hypertensive vasculopathy remain unclear. aim of this study was to evaluate the prevalence and time course of cni-related vascular changes after renal transplantation (tx) in protocol (pbx) as well as in indication biopsies (ibx) and to correlate this to the cni blood levels, blood pressure and diabetes. from 491 patients, a total number of 1239 pbx, taken at 6 weeks (n=380), 3 (n=420) and 6 months (n=439) after tx and 360 ibx were classified according to banff-criteria. assessement of cni toxicity included: isometric vacuolisation of tubules (isovac), intimal arteriolar hyalinosis (iah) and nodular arteriolar hyalinosis (nah) and vacuolisation of small vessel smooth muscle cells (vsm). 96% of patients received either cyclosporine or tacrolimus. in pbx, isovac was present in 15%, 17% and 15% of patients at 6 weeks, 3 and 6 months post-tx, respectively; iah in 11%, 9% and 10%; nah in 5%, 5% and 7%; vsm in 19%, 19% and 15%. in late ibx (>2 years post-tx) the prevalence of the analyzed parameters apart from isovac (9%) was markedly higher: 42% for iah, 22% for nah and 38% for vsm. in pbx, vsm, iah and nah were associated with each other but not significantly dependenct on blood pressure, rejection episodes or diabetes. through levels of cnis were not different between patients with and without vascular hyalinosis. in patients with vsm and iah in late ibx one third had these lesions already present in earlier biopsies. conclusion: the prevalence of presumed morphological signs for vascular cni-toxicity in pbx is low and constant and apparently, not associated with cni blood levels, hypertension or diabetes. in contrast, in ibx later than two years after transplantation, prevalence of vascular cni-toxicity signs is much higher. this emphasises that cni reduction protocols should be regarded within the first six months after transplantation, when vascular changes are still marginal. further elucidation of precursor lesions in pbx would help to find out patients at risk for cni-induced vascular changes. donor introduction: achieving donor specific tolerance has been the goal of the transplant community. success has been reported with donor stem cell transfusion in animal studies and prevention of chronic rejection in cardiac allograft recipients in the clinic. this report details the results of the initial phase of a study in humans. methods: a prospective phase i/ii fda approved pilot protocol was initiated to evaluate the effects of donor graft facilitating cell (fc)/stem cell infusion in kidney transplant recipients. conditioning was performed with 200 cgy of total body irradiation. bone marrow processed to remove gvhd-producing cells but retain cd8 + /tcr -fc and stem cells was infused 24 hours post-operatively. the dosage of stem cells was limited by the t cell dosage. the starting dose was 1 x 10 5 t cells. this was increased in steps of 2 x 10 5 per patient. as the study spans a 7-year period, the immunosuppression changed: 3 patients received cyclosporine (cya), mmf and prednisone; 3 were induced with basiliximab and maintained on cya(2)/fk(1), cellcept and prednisone; and 3 received alemtuzumab induction and maintenance with fk and mmf. all patients underwent tolerance testing and immunoprofiling studies. results: of the 9 patients, 6 received live donor kidney transplants. one graft was lost from arterial thrombosis on day 2. delayed graft function was seen in 3 patients. good long-term graft function was seen in the other 8 patients. acute rejection was noted only in 1 and infectious complications (cmv-1, histoplasma-1) in 2 patients. two patients died with functioning grafts -one at 4 years from lung cancer and another from complications from diabetes at 6 years. six patients are alive with functioning grafts at mean follow-up of 4.3 years with a mean creatinine of 1.3 mg/dl. no gvhd was detected in any of the patients. macrochimerism was not detected in any of the patients at any point. notably, in spite of the fact that durable engraftment was not yet achieved, none of the patients were sensitized as a result nor did they experience immunologic sequelae. conclusions: in our patients there were no untoward sequelae related to either the conditioning regimen or marrow infusion. the incidence of acute rejection even on longterm follow up has been low. we currently propose to reduce the immunosuppression further in these patients. a pilot study was performed to evaluate whether immune cell depletion with alemtuzumab would permit post-transplant weaning of maintenance immunosuppression in well-matched renal transplant recipients. patients received alemtuzumab 30 mg intravenously on the day of the transplant and the subsequent 2 days while sirolimus and tacrolimus were started on day 1. tacrolimus was discontinued at day 60 in all patients. extensive immune monitoring was performed at 1 year. at current follow-up (22 to 34 months), all patients are alive with a functioning graft (median mdrd gfr=46 ml/ min). one patient experienced clinical and biopsy-proven rejection at 9 months. all other patients remain on sirolimus monotherapy. four patients have been weaned to 1 mg of sirolimus daily as their sole immunosuppressive agent, with resulting blood levels of 3-4 ng/ml. these 4 patients have no evidence of donor-specific alloantibody, are unresponsive or hyporesponsive to donor cells by the cytokine kinetics test, and have a regulator phenotype to soluble donor antigens by trans-vivo dth. flow cytometry of peripheral blood demonstrated increased foxp3 expression in the cd3+cd4+ population (p=0.002). naive b cells (cd19/cd27neg) cells increased in 9 of 10 patients (p=0.02) and memory b cells increased in all 10 recipients (p=0.0005) when comparing pretransplant to 1 year timepoints. other than the patient with rejection, 12-month protocol biopsies did not show any evidence of rejection, although 2 of 9 showed focal c4d positivity and 1 diffuse positivity. these 3 patients also had evidence of alloantibody by luminex xmap testing. in conclusion, the cytokine kinetics test, alloantibody testing, and trans-vivo dth assay abstracts results correlated with clinical evolution of patients who successfully weaned both tacrolimus and sirolimus without rejection or alloantibody. the flow cytometry findings described occurred regardless of clinical evolution and may represent alterations of the immune system inherent in the treatment protocol independent of individual patient responses to the graft. however, the functional assays of cytokine kinetics assay and trans-vivo dth may be of potential use to correlate with the clinical immune status of the kidney transplant recipient. we have previously reported the short-term results of alemtuzumab (campath-1h) pre-conditioning with tacrolimus monotherapy and subsequent spaced weaning in living donor kidney transplantation (ldkt). we report here our 5 year experience. methods: we performed 411 consecutive unselected ldkt (donor kidneys were removed laparoscopically) from 12/11/2002 to 11/26/2007 using 30 mg (0.5mg/kg) alemtuzumab and tacrolimus monotherapy. at 6 months post-transplant and every 2 to 6 months interval, we used clinical data (including elisa antibody titers, cylex t-cell activation assay, and identification of donor specific antibodies) to wean tacrolimus when possible (bid-->qd-->qod-->tiw-->biw-->qwk). the recipients included 5 hiv+, 35 pediatric recipients, and 60 re-transplants. the mean follow up was 843.1+478.7 days. results: actuarial recipient survivals at 1-, 2-, 3-years were 98.4%, 95.6%, and 92.7%, respectively. graft survivals at 1-, 2-, 3-years were 97.6%, 90.4%, and 85.4%, respectively. the mean creatinine (mg/dl) at 1-, 2-, 3-years were 1.46+0.62, 1.56+1.08, and 1.54+0.89, respectively. the mean gfr (ml/min/1.73m 2 ) at 1-, 2-, 3-years were 73.0+28.5, 71.8+28.8, and 68.9+28.6, respectively. the cumulative incidence of acute cellular rejection (acr) at 6-, 12-, 18-, 24-, 30-, 36-, 42-, and >42 conclusions: in the current era low risk patients infrequently have ar, and have excellent short and long term graft survival without the use of depleting antibodies. given the increased costs of these drugs, the indications for using depleting antibodies in low risk ktrs of scd kidneys should be further clarified. kidney transplantation prolongs survival in hepatitis c virus-positive (hcv+) patients with end-stage renal disease (esrd). however, the effects of induction therapy and chronic immunosuppression are unknown on the course of hcv infection and potential for cirrhosis in renal transplantation (rtx) recipients. we have retrospectively assessed parameters of liver function, child-pugh (cp) and meld scores in hcv+ esrd patients who received induction therapy with t-cell depletion (group 1: thymoglobulin, n=30) or an il-2 inhibitor (group 2: basiliximab, n=46). pre-rtx liver biopsies were similar in group 1 and 2. patients were followed for a mean of 826 days (range 47 to 2679 days) following rtx and received tacrolimus, mycophenolate mofetil, and sometimes steroids post-transplant. overall graft survival was 84% in group 1 and 85% in group 2 (p > 0.05). data were analyzed pre-rtx, at 30 and 365 days and at time of last follow-up. serum ast, alt, platelets, inr, albumin and bilirubin did not change following rtx in either group. cp scores in group 1 were not significantly changed after rtx (5.5 ± 0.9 to 5.7 ± 0.9 at last follow-up, p=0.53). group 1 patients on steroid-free protocols (n=8) demonstrated declining cp scores from 6.0 ± 1.0 to 5.3 ± 0.5 at last follow-up that were not statistically significant (p=0.31). group 1 patients on steroids showed opposite trends of cp: 5.3 ± 0.6 pre-rtx vs 5.8 ± 1.1 at last follow-up that similarly did not reach statistical significance (p=0.18). group 2 cp scores declined from 5.8 ± 0.8 to 5.4 ± 0.9 at last follow-up (p=0.04). there was no difference between cp or meld scores at any point between the groups. as expected, meld scores improved significantly following rtx and remained low up until the final visit (p=0.001); this was attributed to the drop in serum creatinine post-rtx. neither the use of thymoglobulin or basiliximab resulted in acute hepatitis resurgence or the development of cirrhosis post-transplant. we have not identified any association between choice of induction agent or maintenance immunosuppression regimens, including steroid withdrawal, with impaired hepatic function or progression to liver cirrhosis in hcv+ rtx patients. t cell depletion was well-tolerated by hcv+ rtx patients and resulted in good graft outcomes. interestingly, cp scores declined after renal transplantation in the basiliximab induction group. kidney: complications i prevalence background: a few years ago we observed an expansion of blood gd t cells following cytomegalovirus (cmv) infection in kidney transplant recipient (ktr). we recently demonstrated that these cells share a strong reactivity against cmv infected cells and tumor epithelial cells in vitro. an implication of gd t cells in the immune surveillance against cancer has been demonstrated in mouse and strongly suggested in human. we tested here the hypothesis of a protective role of cmv-induced gd t cells against neoplasia in ktr through: 1/ a longitudinal case / control (ktr with cancer / ktr without cancer) study where gd t cell percentages were determined before and after cancer diagnostic (n=63), 2/ a retrospective follow-up of 131 ktr for 8.23 years looking for risk factors for malignancy. results:the median of gd t cell percentage in patients with malignancies was significantly lower when compared to control patients 18, 12 and 6 months before the diagnostic of the cancer (p<0.005). using a conditional logistic model, we determined that patients with a gd t cell percentage above 4 % were protected from cancer (p<0.008). a significant association between increase of the vd2 neg gd t cell subset and lower cancer occurrence was only retrieved in the ktr who experienced pre-or post-graft cmv infection. finally, using univariate and multivariable analysis, absence of pre-or post-graft cmv infection in ktr was associated with a risk of cancer 5.69 times more elevated (p=0.006). this study reveals an unexpected protective role of cmv against cancer in ktr most probably via the expansion of gd t cells cross-reactive against cmvinfected and tumor cells. background: viral infection (vi) is a morbidity factor in transplant recipients (tx pts). induction therapy (ind-rx) is a known risk factor for vi. although cam is thought to be a more potent ind-rx than zen, we have previously shown similar cmv infection rates in each. we have also shown that cmv-tc analyzed by cytokine flow cytometery (cfc) are consistently detectable in cmv sero(+), but not sero(-) individuals. cmv-tc(-) was associated with persistence of cmv infection in tx pts. here, we report on the effect of ind-rx on cmv-tc in kidney tx pts. methods: 58 pre-tx samples from 39 cmv-sero(+) pts and 62 post-tx samples from 44 pts were submitted for cmv tc-cfc. whole blood was incubated with a pooled overlapping peptide mixture consisting of 138 peptides from cmv pp65 and brefeldin a at 37 degrees for 6 hours and room temperature overnight. ifnγ+cd8+ cells were enumerated by cfc and results were expressed as ifnγ+cd8+ cell%. results >0.2% were considered as (+ infection associated graft loss during the entire study period is shown in figure1. infections contributing to renal allograft loss increased significantly from 1990 to 2006. this may be due to increase use of both induction agents and potent maintenance regimens. this is an important cause for poor long-term graft outcome despite decreasing rejection rates and a balance has to be maintained between prevention of rejection and avoidence of infection. serum creatinine (scr) at procurement was 101±51 µmol/l. the incidence of donor hypertension, diabetes, and death from cerebrovascular origin was 31%, 15%, and 55% respectively. multivariate analysis showed that the only clinical parameters associated with a low egfr were donor scr and donor hypertension. nyberg or pessione scores were not significantly associated with a low egfr. regarding d0 biopsies, univariate analysis showed that % of sclerotic glomeruli (sg, p=0.02), arteriolar hyalinosis (p=0.03), mean remuzzi score (p=0.03) and mean cadi score (p=0.04) were all significantly associated with a low egfr. a logistic regression showed that an integrated score including: i) donor scr (±150 µmol/l), ii) hypertension, and iii) sg (±10%) had the highest performance in predicting a low egfr at 1 yr compared to clinical or histological parameters alone. using this composite score, the adjusted or for the prediction of a low 1-yr egfr ranged from 1 if none of the 3 factors were present, to 7.1 (if sg >10% was associated with one of the 2 clinical factors), and to 27.5 (if the 3 factors were present, p=0.0003). conclusion: this study highlights that d0 biopsies are useful to predict graft outcome particularly in md population, and may perform better than clinical scores alone. in this population, a simple and routinely applicable integrated scoring strongly predicts a poor graft outcome, which may allow an optimized allocation of marginal donors. prospective kidney transplantation from small pediatric donors is increasingly being utilized as a means to optimize the organ supply, however the single most common specified reason for the discard of pediatric kidneys is vascular damage, such as shortening of the suprarenal aorta or injury to the renal artery orifices, which often precludes en bloc transplantation (ebk). at our center, damaged kidneys were salvaged by transplantation as singles (sk background: in an effort to maximize the number of recipients transplanted per donor, transplant centers in our donor service area (dsa) voted to preferentially allocate local and imported en-bloc pediatric donor renal allografts to centers willing to transplant two individuals with single allografts. after 1 year of implementing this policy into action, we report on our initial experience. methods: from july 2006 to june 2007 we reviewed our experience with 12 adult single allograft recipients of pediatric donors less than 60 months of age. there were no exclusions based on age or size with exclusion criteria consisting of donor age < 2 months and single allograft size < 5 cm. all but 1 recipient received rabbit anti-thymocyte globulin induction, tacrolimus, mycophenolate mofetil, and rapid steroid withdrawal. results from this cohort were compared to 86 consecutive recipients of adult single allografts from standard criteria donors with the same immunosuppression protocol used as historical controls. results: 12 pediatric single allografts with median donor age of 24 months (range 8-58) were transplanted into 12 adults with median age 46 years (range 24-67). showed that non-white race was associated with increased risk of death (p<0.01). this effect of race was attenuated when ltx center was taken into account [b] . finally, with the addition of meld in the model, the effect of race on waitlist mortality all but disappeared [c] . conclusions: on the surface, minority patients may appear to have higher mortality on ltx waitlist compared to caucasian counterparts. however, this association is predominantly a result of minority patients having a higher meld score, although ltx center-specific mortality may contribute. these data suggest that waitlist outcome may be improved by optimizing referral of minority patients. background: in cirrhotic patients awaiting liver transplantation (lt), low serum sodium (na) predicts short term pre-lt mortality, independently of meld. incorporation of na into meld has been recommended to improve prognostic accuracy (meld-na; gastroenterology 130:1652 gastroenterology 130: , 2006 ). however, short term interventions such as water restriction that improve na may have little effect on prognosis. hypothesis: the lowest level of serum sodium in the preceding 30 (na30), 90 (na90) or 180 days (na180) may be better than the current serum sodium (nac) for predicting pre-lt cirrhotic mortality. methods: we reviewed electronic records of 764 cirrhotic veterans referred for consideration of lt, 2/28/02-6/30/07. date of most recent na at referral was chosen as time zero for determining nac, na30, na90, and na180 and for assessing subsequent survival. findings: within 90 days, 94 patients died pre-lt (12%) and 27 underwent lt (3%). na at all time points was associated strongly (p<.001) with prelt death (censored at lt). areas under receiver operating characteristic curves (aurocs) for na30, na90 and na180 as predictors of 90d prelt mortality (mean±se) were .808±.026, .807±.026 and .783±.025, respectively, compared to .714±.032 for nac (all p<.05 vs. nac). on multivariable logistic regression analysis, meld and na90 were independent predictors of 90d prelt mortality, with best discrimination given by the following model: meld-na90 = meld + (135-na90)*1.04 with value of na90 capped at 135. aurocs for meld-na90, meld-na, and meld were .875±.027, .865±.025 and .838±.027, respectively. findings were similar when patients with hcc at referral (n=155) were excluded (aurocs .884±.025, .876±.026 and .840±.031, respectively), and when 90 day survival endpoint was changed from "death censored at lt" to "death or lt" (auroc's .890±.021, .882±.022, and .855±.026, respectively). conclusion: short term improvement in na may mask true prelt mortality risk. the lowest na in the preceding 90 days is a better prognostic indicator than current sodium. substitution of meld-na90 for meld would permit more accurate "sickest first" organ allocation, while at the same time allowing prelt correction of na without loss of priority. prospective validation of meld-na90, in comparison to meld-na and meld, is warranted. hyponatremia does not affect survival following liver transplantation. byung cheol yun, 1 w. ray kim, 1 y. s. lim, 1 joanne t. benson, 1 walter k. kremers, 1 terry m. therneau. 1 1 gastroenterology and hepatology, mayo clinic college of medicine, rochester, mn. background: hyponatremia is a common yet important complication of cirrhosis. serum sodium (na) has been found to be an important predictor of survival in patients with cirrhosis. models incorporating na have been proposed for liver allocation. concerns have been raised, however, that liver transplantation (ltx) in hyponatremic patients will adversely affect the outcome. in this work, we assessed the effect of pre-ltx na on the short term survival following ltx. methods: patient-level data on all waitlist registrants in the us for 2005 and 2006 were obtained from the organ procurement and transplantation network. demographic, clinical and laboratory data at the time of ltx and outcomes following ltx were extracted. the relationship between na pre-ltx and survival post-ltx was analyzed using multivariable regression analyses. results: there were 7411 primary transplants that met the inclusion criteria between 2005 and 2006. the median na in meq/l at the time of ltx was 136 (interquartile range, iqr: 132-139). there were 1255 patients who had a na ≤130 meq/l. the mean meld score was 22.5 (sd 9.2). median follow up was 279 (iqr: 155-371) days. the overall 30-and 90-day survival post-olt was 96% and 93%, respectively. in a multivariable logistic regression model, meld was associated with 1.04-fold increase in 90-day mortality (95 confidence interval: 1.03-1.05), while na did not have impact on survival (hr=0.99, 95% ci: 0.98-1.02). the figure represents the risk of 90-day mortality according to na after adjustment for meld, which clearly shows absence of mortality increase over a wide range of na. conclusions: hyponatremia at the time of ltx has no detrimental impact on short term patient survival following ltx. although these data do not address morbidity (e.g., central pontine myelinolysis), there is no evidence that incorporation of na in organ allocation will lead to diminished survival. objective. this study examined the relationship between meld at liver transplantation (lt) and post-lt quality of life (qol). methods. adult lt recipients (n = 247) at two centers completed the sf-36 and transplant symptom frequency questionnaire (tsfq) 1-year post-lt. high sf-36 scores indicate better qol; high tsfq scores indicate more symptomatology. clinical (lab) meld at lt, demographic characteristics, presence of ascites, encephalopathy, and variceal bleeding pre-lt, current employment status, presence of co-morbid medical conditions, and bmi were collected from medical records. results. primary lt indication was viral hepatitis (57%), cholestatic liver disease (17%), or hepatocellular disease (27%), and 63% had ascites, 51% encephalopathy, and 29% gastroesophageal bleeding. mean meld at lt was 20±9. there was almost no correlation between meld and sf-36 physical (r = 0.11) and mental (r = 0.001) functioning. statistically significant yet weak correlations were found between meld and physical functioning (r = -0.15) and role functioning -physical (r = -0.15). meld was not significantly correlated with any other sf-36 scales (r's -0.11 to -0.01). meld was not significantly correlated with any tsfq domains: affective distress (r = 0.08), neurocognitive symptoms (r = 0.05), gastrointestinal distress (r = -0.02), physical appearance changes (r = 0.09), appetite and weight changes (r = 0.09), and miscellaneous symptoms (r = 0.08). older age (ß = -0.28), female sex (ß = -0.26), viral hepatitis (ß = 0.67) or cholestatic disease (ß = 0.45), higher bmi (ß = -0.62), and >1 medical co-morbidity (ß = 0.57) were significant predictors of lower qol as measured by the sf-36 (adj r 2 = 0.12, f = 4.4, p < 0.001). older age (ß = 0.27), female sex (ß = 0.29), higher bmi (ß = 0.71), history of variceal bleeding (ß = 0.19), and >1 medical co-morbidity (ß = 0.17) were predictive of more symptoms on tsfq (adj r 2 = 0.10, f = 4.1, p < 0.001). meld was not predictive of qol. conclusions. higher disease severity, as measured by meld, at lt does not portend a worse qol outcome for patients 1-yr after transplantation. other pre-lt indicators of decompensation also do not predict post-lt qol. post-lt qol is affected more by other variables, including age, sex, bmi, and medical co-morbidities. introduction: racial disparities in access to cadaveric renal allografts have been well described for renal transplantation. however, little is known about differences in orthotopic liver transplantation (olt) rates for patients of minority racial groups following listing. the purpose of the current study was to determine if there is difference in rate of transplantation among racial groups and to examine the potential reasons for the disparity. methods: the united network for organ sharing (unos) database was obtained. data was extracted for adult olts greater than 18 years of age performed from 2/2002-12/2005. transplants for which recipient race or model for end-stage liver disease (meld) score at listing were unknown and patients active on the list were excluded. rates of transplantation as well as differences in reasons for de-listing (transplantation, death/deterioration, and improvement) were examined. in an effort to examine only patients with chronic liver disease, further analysis was performed excluding patients with acute fulminant liver failure and retransplants. results: the database contained complete meld and race information on 17,916 olts. seventy-four percent of patients were caucasian, 12% hispanic, 9% african-american, and 4% were asian. as seen in table 1 , laboratory meld score at removal differed between racial groups. examining pair-wise comparisons of the three minority groups to caucasians, only hispanics differed in reason for delisting (table 1) . subgroup analysis excluding acute hepatic failure patients and retransplants showed similar results with hispanic patients being more likely to die/deteriorate as compared to other racial groups (31% deaths vs. 24% deaths for caucasians), and being less likely to receive a transplant (69% of hispanics vs. 75% of caucasians, p<0.001). conclusion: hispanic patients, although listed with higher meld scores, are transplanted less often than caucasian patients and are more likely to die/deteriorate while awaiting olt. reasons for this discrepancy are unclear and merit further attention. background: since the implementation of the model for end-stage liver disease (meld) for liver allocation, an increasing number of candidates with renal insufficiency have undergone orthotopic liver transplantation (olt). since candidates with renal insufficiency have higher post-transplant morbidity and mortality, meld-based allocation may be shifting some waiting list mortality to the post-transplant period in these candidates. the objective of this study was to evaluate the survival benefit among candidates with renal insufficiency who underwent olt. methods: scientific registry of transplant recipients data for adult candidates age ≥18 initially listed for olt between 9/1/01 and 12/31/2006 (n=38,899) were analyzed. the effect of serum creatinine on the survival benefit (contrast between waiting list and post-transplant mortality) was assessed by sequential stratification, an extension of cox regression. each recipient was matched with candidates active on the waiting list in the same organ procurement organization with the same meld score. results: for meld scores 12-40, the survival benefit of olt significantly decreased as serum creatinine increased. among candidates transplanted at meld 12-14, the 23% with serum creatinine >1.1 mg/dl (23%) experienced no significant survival benefit ( figure) . candidates transplanted at meld ≥15 experienced significant olt benefit irrespective of serum creatinine level. conclusions: comparing two patients with meld ≥12, the patient with higher creatinine experiences significantly less survival benefit from liver transplantation. almost onequarter of patients transplanted at meld 12-14 experienced no survival benefit from olt based on 5 years of follow-up. therefore, more careful assessment of candidates is required in order to maximize the survival benefit gained by the wait-listed end-stage liver disease population as a whole. liver: living donors and parial grafts i assessment introduction: consideration of the risks and benefits of a procedure are critical in medical decision making. however, relatively little is known about risk tolerance amongst donors and transplant professionals in live-donor liver transplantation (ldlt). we conducted confidential semi-structured interviews in a convenience sample of donors, non-donors (individuals who had been assessed for donation but did not donate) and transplant team members. in addition to examining issues surrounding decision making for ldlt donation, we sought to assess the tolerance of participants, above which they would no longer contemplate donation, for a number of potential outcomes following ldlt. the outcomes that participants were asked to consider included their tolerance for risk of donor death, risk of serious donor complication, as well as risk of recipient death following transplantation. the interviews were conducted sequentially, data was coded quantitatively, and the study terminated once saturation was reached. (pre, weeks 1, 4, 12, 26 and 52 post-donation) . ambivalence detected by staff or described by donor was recorded. donor and recipient characteristics were examined and compared between ambivalent and non-ambivalent groups. results: staff identified and self identifed ambivalent donors were not equivalent. staff assessments indicated 20 ambivalent donors (16 male, 4 female). 18 donors self-identified as ambivalent (11 male, 7 female); 7 donors were on both lists (5 male, 2 female). the combinations of brother to brothers and sons to fathers were the most common pairs among ambivalent donors and more common than in total donor cohort. recipient diagnosis of alcohol or hepatitis c related liver disease was more common in ambivalent donors. ambivalent donors were more likely to be college educated and to express significant religious affiliations than the total rhl donor group. all but 1 ambivalent donor indicated that they would donate again on the 1 year qol survey. conclusions: ambivalence about rhl donation is present in approximately 20% of candidates who complete donation. staff-identified and self-identified groups showed only 20% overlap; however, both groups showed similar characteristics. brother-tobrother and son-to-father pairings and recipients with perceived self-induced liver failure were more common in both groups compared to total donor cohort. ambivalent donors had more education and stronger religious or spiritual identification than the entire cohort. only 1 donor indicated persistent doubt about donation. these results suggest that expressed or perceived donor candidate ambivalence may represent a process of careful consideration and should not be used sole basis for donor disqualification. the impact of donor age on recipient outcome for adult right-lobe living donor liver transplantation (rldlt) is unclear. aim: to analyze the effect of donor age on recipient outcome following rdldt. methods: since 2000 we have performed 226 rldlt (mean donor age 37 years, range 18-60 years), including 20 donors age 55 years or older. we analyzed the effects of donor age, as a continuous or categorical (< 54 vs > 55years) variable, on recipient outcome. recipient outcome measures included biochemical markers of hepatocytes injury (ast, alt) and graft function (inr, bilirubin), postoperative infections, bleeding, biliary complications, acute cellular rejection, as well as patient and graft survival. analyses were carried out stratified for higher recipient meld scores (< vs. >25), recipient age (< vs. > 60 years), and hepatitis c virus (hcv) infection (presence vs. absence). results: 5-year patient and graft survival after rldlt was 80% and 78%, respectively. donor age as a continuous variable was associated with increased ast (p=0.04) and alt (p= 0.022) release after transplantation, while no effect was observed on inr or bilirubin. rldlt using donors above 55 years of age resulted in an increased incidence of biliary strictures (20% vs. 6%, p= 0.028), postoperative cholangitis (25% vs. 9%, p= 0.023). no effect of donor age was found for the following recipient outcome measures: the number of bile ducts supplying the graft, type of biliary reconstruction required; rejection, hemorrhage, pulmonary or urinary tract infections, renal failure, or length of hospital stay. 5-year patient survival was identical for patients receiving grafts from donors below or above 55 years of age (81% vs 77%, p= 0.86). similarly, 5-year graft survival was comparable for young and old grafts (78% vs 77%, p= 0.71). recipient age (< vs > 60 years), recipient meld score (< vs >25), or hepatits c status of the recipient did not impact on the effect of age on patient or graft survival. conclusion: in this single center series of 226 rldlt, the use of selected older donors did not impair graft and patient survival, but was associated with an increased rate of biliary strictures. background: biliary stricture rate after living donor liver transplant (ldlt) in adults remains relatively high in comparison to the stricture rate after adult cadaveric liver transplant or ldlt in pediatric patients. the etiology or risk factors for biliary stricture development at present time are uncertain. purpose: to determine the risk factors for biliary stricture after right lobe (rl) ldlt. methods: from 5/99 to 12/07, 109 ldlt procedures were performed in 109 adult recipients. eleven patients were excluded from analysis due to <90 days follow up or need for retransplant. the following data was prospectively collected: 1. demographics, 2. acuity of illness, 3. number of bile ducts, 4. type of biliary reconstruction, 5. graft to recipient weight ratio, 6. hemodynamic parameters, 7. outcomes. these parameters were compared in patients with and without strictures. results: mean follow-up for 98 patients is 1642 days (range: 120-3052). 8 of 109 patients died during the follow-up range and 3 required whole liver re-transplants. 33 patients (34%) developed a biliary strictures during the follow-up period. comparison of risk factors in patients with and without strictures revealed the following results: mean meld >1 bile duct grwr* < 1. neither meld score, number of bile ducts or type of biliary reconstruction appear to be contributing factors to the development of bile duct stricture following rl ldlt. the biliary stricture rate was related to the volume of transplanted liver and post transplant graft recovery. therefore, the development of biliary strictures in some patients may represent yet another feature of small-for-size syndrome. background: ox40 and cd154 can be expressed by both foxp3+ tregs and activated t effector cells. however, the question as to how ox40 and cd154 function, individually or collectively, in regulating such functionally different t cell subsets in transplant models remains poorly understood. in some models, blocking cd154 costimulation is remarkably effective in prolonging graft survival, but targeting cd154 alone rarely creates tolerance. but the role of ox40 in regulating the cd154 blockade induced tolerance is completely unknown. in the present study we critically examined the role of ox40 in the activation of cd154 deficient t effector cells as well as in the regulatory function of foxp3+ tregs. we also examined the effect of ox40 on the induction of new foxp3+ tregs/th17 cells from activated cd154 deficient t effector cells. the impact of ox40 in the induction of allograft tolerance was examined using an islet transplant model. we found that cd154 deficient foxp3+ tregs constitutively expressed ox40 on the cell surface, but the cd154 deficient t effector cells did not. however, when the t effector cells were sorted and stimulated in vitro, ox40 expression could be abstracts readily induced on the t effector cells. to further examine how ox40 regulates such functionally different t cell subsets, we found that ox40 delivers potent costimulatory signals to t effector cells, which prevent the induction of new foxp3+ tregs from activated t effector cells but promote their differentiation to th1 cells but not th17 cells. surprisingly, ox40 costimulation to cd154 deficient foxp3+ tregs completely inhibited their regulatory functions. in an islet transplant model, we showed that cd154 deficient mice can reject the dba/2 islet allografts, but blocking ox40 costimulation readily induced donor specific tolerance (mst>150 days), and this tolerant status was critically dependent on the induction of foxp3+ tregs. in contrast, treatment of cd154 deficient recipients with a agonist anti-ox40 mab precipitate rapid islet allograft rejection, suggesting that ox40 costimulation is critically important in the induction of transplant tolerance. conclusions: our data suggest that ox40 is a costimulatory molecule to t effector cells but a powerful negative regulator for foxp3+ tregs. thus, a key role for ox40 in the induction of transplant tolerance is the control of t cell mediated regulation. background: foxp3 is a winged-helix family transcription factor that is the master regulator for the development and function of regulatory t cells (treg). we investigated the molecular mechanisms important for regulation of foxp3 expression, and defined the structure of the active foxp3 promoter in cd4 + t cell lineages. methods: purified cd4 + cd25 -foxp3 -gfp -t cells (naïve) and cd4 + cd25 + foxp3 + gfp + treg were cultured with antigen presenting cells in the presence of il-2, anti-cd3ε mab, tgfβ or the dna methyltransferase inhibitor 5-aza-2'-deoxycytidine (zdcyd). foxp3 promoter structure and activity were monitored with methylation-specific pcr, disulfite-sequencing, chromatin immunoprecipitation (chip) assays, electrophoretic mobility shift assay (emsa) and luciferase promoter assay. the foxp3 promoter has an upstream cpg island ∼5kb from the transcriptional start site. disulfite-sequencing and methylation-specific pcr analysis showed that this region is heavily methylated in naïve cd4 + t cells and tgfβ induced peripheral treg, but demethylated in thymic derived natural treg (ntreg). chip analysis showed that the methylated cpg island is bound specifically by the dna methyltransferases 1 and 3b. zdcyd causes demethylation of the cpg island, and in combination with tgfβ, synergistically induces foxp3 expression. chip assays for acetylated histone 3 and sp1, both markers of gene activation, showed that the cpg island is acetylated and bound by sp1 in ntreg and zdcyd plus tgfβ induced treg, but not in activated cd4 + t cells or tgfβ induced treg. emsa likewise shows the cpg island binds sp1. in contrast to the upstream promoter, the structure of the first intronic promoter differs markedly between ntreg and tgfβ induced treg, but is not affected by zdcyd. the upstream cpg island also possesses enhancer activity that is repressed by dna methyltransferases. zdcyd plus tgfβ induced treg have stable foxp3 expression and enhanced suppressive functions in vitro and in vivo. conclusion: these results demonstrate that ntreg and tgfβ induced treg are genetically distinguished from each other by the epigenetic structure of a unique upstream cpg island of the foxp3 promoter. the function of this region is regulated by dna methylation and histone acetylation. zdcyd demethylates the promoter, leading to enhanced and stable expression of foxp3 and suppressor activity, similar to ntreg. this has important implications for biology, and generating treg for tolerance. chemokine background: trafficking of lymphocytes through lymphatics to secondary lymphoid organs is crucial for immune responses. we previously showed that regulatory t cell (treg) function required trafficking from the inflammatory graft site to the local draining lymph node (dln). since the mechanisms that regulate migration through afferent lymphatics are poorly understood, we explored the role of chemokine receptors on treg for afferent lymphatic migration in an islet transplantation model. methods: islets were transplanted from balb/c mice into foxp3 gfp c57bl/6 mice. treg from wild type, ccr2 -/-, ccr4 -/-, ccr5 -/-, or ccr7 -/-c57bl/6 mice were isolated, labeled with red dye pkh26, and transferred intravenously, or locally into the islet allograft. treg migration to islet grafts and dln was determined by flow cytometry and immunohistochemistry. endogenous foxp3 gfp+ treg and transferred pkh26 labeled treg were sorted from the islet grafts and the dln, and chemokine receptor and sphingosine 1-phosphate receptor (s1p1) expression were determined by rt-pcr. islet allograft survival was determined by measurement of blood glucose. results: freshly isolated treg expressed s1p1 and the chemokine receptors ccr2, ccr4, ccr5, and ccr7. endogenous treg, and both intravenously and locally transferred treg, that were recovered from islet allografts and dln expressed similar levels of ccr2 and ccr5. ccr4 was expressed preferentially on islet migrating treg, while s1p1 and ccr7 were expressed preferentially in dln migrating treg. locally transferred treg migrated to the dln, but ccr7 -/-treg were not able to migrate to the dln. ccr2 -/and ccr5 -/-treg were impaired in their ability to migrate to the dln. this suggested that these three chemokine receptors all regulated treg entry into afferent lymphatics and migration from the graft to the dln. in contrast, ccr4 -/-treg migrated normally from the islet to the dln. importantly, ccr2 -/-, ccr5 -/and ccr7 -/-, but not ccr4 -/-treg, were impaired in their ability to prolong islet allograft survival when transferred locally in the islet allograft. conclusion: treg migrate from the inflammatory site of the allograft to draining secondary lymphoid tissue through afferent lymphatics. this process depends on ccr2, ccr5, and ccr7; and is crucial for full treg function in vivo. these results demonstrate a novel role for sequential migration from the graft to the dln in treg function and suppression. epigenetic regulation of gene expression provides a major, and especially beyond oncology, largely unexplored means to regulate host immune cell functions. our ongoing analysis of histone deacetylase (hdac) expression by foxp3+ naturally occurring murine regulatory t (treg) cells showed tcr-activated tregs had 5-6 fold more hdac6 mrna than corresponding resting treg or non-treg cells. in various cell types, hdac6 deacetylates alpha-tubulin, cortactin, and hsp90, abrogates formation of the aggresome, and blocks the unfolded protein response, though nothing is known regarding these pathways in tregs. we found that an hdac6-specific inhibitor, tubacin (but not the control compound, niltubacin), increased treg suppressive function in vitro (p<0.01), in association with increased expression of ctla, il-10, gitr, pd-1 and other treg-associated genes (p<0.05), and increased treg foxp3 protein (though not mrna) expression. tubacin enhanced the conversion of cd4+cd25-cells into cd4+ foxp3+ treg in vitro, and globally decreased cytokine production, with the exception of il-10 and il-17 mrna. comparable and dose-dependent effects were seen using the hsp90 inhibitor, geldanamycin, suggesting that the effects of hdac6 inhibition were mediated, at least in part, by blocking the chaperone effect of hsp90. use of tubacin in vivo significantly decreased the severity of colitis in two murine inflammatory bowel disease models (p<0.05), dextran sodium sulfate-induced colitis and the cd4+cd62lhigh adoptive transfer model of colitis, as assessed by standard clinical and histologic criteria. in addition, 14 days combined use of tubacin and a subtherapeutic dosage of rapamycin led to significantly prolonged cardiac allograft survival (balb/c->c57bl/6) compared to use of either agent alone (p<0.05). our data show that use of the first known small molecule inhibitor of one specific hdac has important therapeutic effects, including enhancing the production and suppressive function of tregs. while ongoing studies are directed towards unraveling the interactions of hdac6-dependent pathways and treg functions, the current data indicate the importance of understanding the functions of hdacs to the development of entirely new ways to regulate host immune responses. dendritic cells supply paracrine il-2 for treg cell functional activity. regulatory cd4+cd25+ t cells (tregs) are important for the maintenance of immune tolerance, and immunotherapy with tregs is being explored for organ and cell transplantation. treg development, expansion and function depend on il-2. because tregs do not make il-2, they must obtain il-2 from another cell. although cd4+ teffectors are a logical candidate, the identity of the paracrine source of il-2 for tregs is not substantiated. we explored whether dendritic cells (dcs) could serve as the paracrine source of il-2 for treg and rd6, a cd4+cd25+ regulatory hybridoma. using four dimensional live cell imaging we demonstrate that treg and rd6 cells establish tight contact with dcs, and cd25 is localized at these contacts. using the il-2 elispot and real-time rt-pcr we found that splenic dcs and the jawsii dc cell line constitutively make il-2. lps and cpg increases dc production of il-2. co-culture with jawsii dc cell line significantly upregulates cd25 expression on alloreactive do11.10 tregs and rd6 cells, but not on do11.10 cd4+ teffector cells. tregs and rd6 cells are functionally suppressive after activation by wild type but not il-2 knock-out allogeneic dcs, and anti-cd25 inhibits the function of treg and rd6 cells in a dose response fashion. in contrast, wild type and il-2 knock-out dcs are equally able to activate alloreactive cd4+cd25-cells. supplemental il-2 at high (1000 u/ml) but not low doses (100 u/ml) restores the function of alloreactive tregs and rd6 that were activated by il-2 ko dcs. these data indicate that treg cells acquire il-2 from dendritic cells for their gain of function and validate dendritic cells as a paracrine source of il-2 for treg. introduction: previously, it has been demonstrated that foxp3, a gene required for the development and function of regulatory t cells, was highly expressed in the graft during cardiac rejection, suggesting infiltration of regulatory t cells in the transplanted organ during an allogeneic response. in this study, we investigated whether graftinfiltrating t cells expanded from rejecting human cardiac allografts exhibit immune regulatory activities. methods: graft-infiltrating lymphocytes (gils) cultured from endomyocardial biopsies (emb; n=13) with histological signs of acute cellular rejection were expanded in the presence of donor-antigens in il-2/il-15-enriched medium for 2-3 weeks. flow cytometry was used to analyze the expression of cd3, cd4, cd8 and foxp3. to analyze the immune regulatory function, we performed mlrs with peripheral blood mononuclear cells (pbmc) of the patients and irradiated donor or third party spleen cells in the absence and presence of gils (ratio 5:1). results: of the cd3 + gils, 9% (median; range: 1-21%) stained positive for foxp3. this foxp3 expression was detected in both cd4 + and cd8 + t-cell population (median: 8% and 9%, respectively). functional analysis demonstrated that gils suppressed the antidonor proliferation of responder t cells (range % inhibition: 55-81%). interestingly, this suppression was predominantly achieved by cd8 + gils: depletion of cd8 + cells from the gils population diminished the inhibitory effect, whereas addition of solely cd8 + gils to the mlr abundantly suppressed the anti-donor response (range % inhibition: 62-77%). in contrast, gils did not inhibit the proliferation of t cells stimulated with third-party antigens. the figure below depicts a representative example. graft-infiltrating lymphocytes expanded from rejecting cardiac allograft exhibit donor-specific immune suppressive activities. these results suggest that during acute cellular rejection, graft-infiltrating lymphocytes not only consist of graft-destructing effector t cells, but may also comprise immune regulatory cells of the cd8 + phenotype. the context: it has been previously suggested that a liver allograft is immunoprotective and able to decrease the rate of rejection of a donor-specific allograft of another organ. it has been recently proposed that allografts other than the liver may also be immunoprotective. objective: the aim of this analysis was to examine one year rejection rate and the incidence of rejection free survival of all combined transplants in the collective us experience to gain insight to any possible protective effect of one organ for another. methods: the united network of organ sharing (unos) provided de-identified patientlevel data. analysis included all recipients transplanted between january 1, 1994 and october 6, 2005 who were 18 years or older (except intestinal transplants). rejection at one year was defined as treatment for one or more episodes of rejection. results: analysis included a total of 83,424 patients who received either one, or combined, simultaneous or sequential, organ transplants in all possible combinations. results are summarized in figure 1 (one-year organ allograft rejection rate). the collected data demonstrate that the rejection rate of donor-specific organ allografts which accompanied primary liver, kidney, and heart transplants was significantly lower in combined transplants as compared to that of the primary allograft transplanted alone. this was not true, however, for intestinal and pancreatic allografts where protection for the accompanying organ was not observed. we further demonstrate that transplantation of two organs of the same type (double kidneys or double lungs), i.e. increase in antigen load, also leads to decreased rates of rejection of the allografted organs. conclusions: in combined simultaneous transplants, the heart, liver, and kidney allografts appear themselves to be protected, and to protect the other organ from rejection. increased antigenic load of identical antigens in case of double lung and double kidney transplants appears to also offer immunologic protection against rejection, perhaps by different mechanisms. background: a2all (9-center adult-to-adult living donor liver transplantation cohort study) has identified risk factors for mortality after aaldlt, including center experience. the aim of this study was to determine if a2all findings are reflected in the national experience. methods: aaldlt at a2all (n=681) and non-a2all centers (n=1598) from 1/1/98 to 8/31/07 in the scientific registry of transplant recipients database were analyzed. cox regression models adjusted for recipient and donor characteristics were fitted to test associations with mortality risk after aaldlt, including center type (a2all vs. non-a2all) and case number (for each aaldlt at each center). results: aaldlt were performed at 9 a2all and 63 non-a2all centers. there was no significant difference in overall mortality risk between a2all and non-a2all centers. significant predictors of death (both groups combined) included donor age (hazard ratio (hr)=1.14 per 10 years, p=0.003), recipient age (hr=1.25 per 10 years, p<0.001), diagnosis of hcv (hr=1.22, p=0.04) or hcc (hr=1.99, p<0.001), and earlier center experience (aaldlt case number ≤15, hr=1.58, p<0.001). there was no significant effect of transplant year after adjusting for experience. cold ischemia time >4.5 hours was associated with higher mortality (hr=1.83, p=0.004); this effect was similar in a2all and non-a2all centers. there were no significant interactions between center type and any predictor except center experience ( figure) . compared to later experience, earlier center experience was associated with significantly higher mortality risk in both a2all (hr=2.24, p<0.0001) and non-a2all centers (hr=1.38, p<0.004). survival during early experience was significantly worse at a2all vs. non-a2all centers (hr=1.42, p=0.024), but survival in later experience was similar. conclusions: after the first 15 cases, aaldlt survival was similar at a2all and non-a2all centers, and similar significant mortality risk factors were identified, including center experience. these analyses support the generalization of findings from a2all centers to others performing aaldlt. abstract# 173 rejection with hemodynamic compromise (hc) and chronic allograft vasculopathy (cav) impact survival in pediatric heart transplantation (phtx). we showed that high pro-inflammatory / lower regulatory cytokine gene polymorphism (gp) profile increased the risk for acute rejection. in this analysis, we assessed the effect of genetic factors on hc and cav. methods: 406 phtx with clinical and gp data for cytokines (tnf-α a-308g; inf-γ t+874a; il-10 g-1082a, c-819t, c-592a ; il-4 c-590t; il-5 t-746c; il-6 g-174c), growth factors (tgfβ-1 t+869c, c+915g; vegf a-2578c, c-460t, g+405c), effector molecules (fas a-670g; fasl c-843t) and pharmacogenomics (abcb1 c3435t, g2677t/a) were analyzed regarding hc and cav. results: adjusting for recipient black race and age with cox regression models, we identified the following risk factors: il-10 high was associated with lower rates of hc. low th1 (inf-γ, tnf-α) with high th2 (il-4, il-5) cytokine gp profiles were protective for hc in combination with il-10 high. carriers of fas high experienced higher rates for hc and cav and high fas-fasl combination doubled the relative risk for cav. abcb1 3435cc/2677gg genotypes were also associated with lower rates of hc (table 1) . conclusion: in this large multi-center study gps with higher regulatory profiles and increased drug transport were associated with a lower incidence of hc. a genetic proapoptotic profile might contribute to the pathogenesis of cav. sponsorship: this work was supported by 5p50 hl 074 732-03 from the national heart lung and blood institute, national institutes of health. it has recently been reported that cd1d-restricted nkt cells that express invariant tcr (inkt cells) play an important role in the production of autoantibodies through the interaction with b-1 cells. this observation prompted us to investigate the possible role of inkt cells in the production of antibodies (abs) against transplant-related antigens, such as abo blood group carbohydrates and histocompatibility complex allopeptides, in a mouse model. we have previously demonstrated that b cells with receptors for blood group a carbohydrates were found exclusively in a cd11b + cd5 + b-1 subpopulation of mice, resembling humans with blood group o or b. immunization with human blood group a red blood cells (a-rbcs) elicited the extensive production of anti-a igm and igg. furthermore, the number of b-1 cells with receptors for a carbohydrates increased in the peritoneal cavity. in cd1d -/and vα14 -/-balb/c mice, which lack inkt cells, such elicited production of anti-a igm was not observed, even after immunization with human a-rbcs. however, class ii -/-balb/c mice, which lack cd4 + t cells but maintain normal levels of inkt cells, exhibited levels of anti-a igm production comparable to those in wild-type (wt) balb/c mice. moreover, anti-a igg production was absent in cd1d -/-balb/c mice even after the immunization, indicating that although inkt cells crucially contribute to anti-a igm production and igg class switching, helper t cells do not. notably, the proportion of b-1 cells in the livers of cd1d -/-balb/c mice was significantly reduced (2.66 ± 0.43%, n = 4) when compared to that in wt mice (4.76 ± 1.93%, n = 4). we next immunized cd1d -/and wt balb/c mice twice with 2 ×10 6 allogeneic b6 mouse thymocytes, and thereafter detected the anti-b6 (allopeptides) abs by flow cytometry. in the cd1d -/mice, anti-b6 igm production was comparable to that of wt mice, and igg class switching also occurred normally. these findings indicated that inkt cells play a pivotal role in the production of abs specific for blood group carbohydrate determinants that are believed to be t cell independent, but are not required in the production of abs for allopeptides that are believed to be t cell dependent. the depletion of inkt cells or the suppression of their function might constitute a novel approach for preventing antibody-mediated rejection in abo-incompatible transplantation, or in xenotransplantation, which involves similar carbohydrate antigens. background static cold storage (cs) is the most widely used organ preservation method for deceased donor kidney grafts. retrospective analyses have indicated that preservation by hypothermic machine perfusion (mp) may lead to improved outcome after renal transplantation. however, there is a lack of sufficiently powered prospective studies to test the presumed superiority of mp. in an international prospective randomized controlled trial we enrolled kidney pairs of 336 consecutive deceased donors and randomly assigned one organ to mp and the contralateral kidney to cs preservation. follow-up was directed at all 672 recipients of these grafts. the primary endpoint was delayed graft function (dgf). mp significantly reduced the risk of dgf (or 0.63; p=0.02) and more than halved the incidence of primary non-function after transplantation, when compared to cs (2.1 vs. 4.8%; p=0.04). furthermore, mp significantly reduced the risk of graft failure in the first 6 months post-transplant (hr 0.46; p=0.05). in recipients who developed dgf, 6-month graft survival was better if their transplanted kidney was machine perfused (87 vs. 76%; p=0.05). hypothermic machine perfusion reduces the risk of delayed graft function, primary non-function, and graft failure in deceased donor kidney transplantation when compared to static cold storage. furthermore, mp alleviates the deleterious effect of dgf on graft survival. we investigated the trafficking of cells after skin and heart transplantation in a dynamic fashion through the use of in vivo microscopy. antigen presenting cells were followed using mhc-cl-ii-gfp and cd11c-gfp transgenic mice. vascularized and non vascularized skin grafts as well as heart transplants were used in syngeneic as well as allogeneic settings. after syngeneic non-vascularized skin transplantation, we observed an early and massive cellular infiltration of host cells into the graft as early as 3 hours post-transplant with a gradual accumulation in the dermis. the accumulation of host-derived cells was accelerated after graft vascularization at day 4/5 post transplantation. this graft infiltration by recipient cells was more pronounced with vascularized skin grafts, and to a higher degree in heart transplants. recipient cells similarly infiltrated allogeneic grafts early on and in larger numbers than for syngeneic grafts by day 4/5 post-transplantation. when visualizing mhc-cl-ii-gfp recipient cells in a syngeneic skin transplant, recipient dcs invaded the graft early on and, by 3 weeks post transplant gradually replaced graft dcs in the dermis (dermal dcs) and the epidermis (langerhans cells). donor dcs could still be seen in the graft up to 8 days post transplant. however, virtually all donor langerhans cells were eventually replaced by recipient ones in a concentric fashion suggesting that the new langerhans cells originate from the recipient skin adjacent to the graft and not from centrally-derived precursor cells. the vascular endothelium of a syngeneic transplant was partially replaced by recipient vascular endothelial cells in a centripetal fashion with more recipient-derived vascular endothelium present at the periphery of the graft and more donor-derived endothelium remaining in the center of the graft. therefore, the graft can be seen as a "chimera" of cells from donor and recipient origin. the presence of recipient endothelial vascular cells and dcs within the graft may be important for maintaining the indirect response thought to be responsible for chronic rejection. objective: maturation resistance and tolerogenicity can be conferred on dendritic cells (dc), -crucial regulators of t cells, by exposure to rapamycin (rapa), a tolerance-sparing immunosuppressant. the mechanisms underlying this acquired unresponsiveness, typified by diminished responses to toll-like receptor (tlr) or cd40 ligation, have not been identified. thus, our objective was to elucidate a molecular basis for rapa-induced dc maturation resistance. methods: rapa administration was used to condition splenic dc in vivo and bone-marrow derived dc in vitro. dc maturation was monitored by assessment of co-stimulatory molecule expression, cytokine production, and t cell allostimulatory capacity. to identify negative regulators of maturation, microarray analysis and quantitative rt-pcr was completed, and findings confirmed via western and flow cytometric analyses. results: in vitro or in vivo exposure of myeloid dc to rapa elicited de novo production of il-1β by otherwise immature dc (cd86 lo ). interestingly, dc il-1β production, acting in an autocrine/paracrine fashion, promoted dc overexpression of the il-1 receptor(r) family member, st2l, and enhanced its surface expression. st2l is the receptor for il-33, an il-1 family member, and has also been implicated as a negative regulator of tlr signaling. consistent with this regulatory function, il-1β-induced st2l expression suppressed the responsiveness of rapa-conditioned dc to tlr or cd40 ligation. conclusion: rapa causes de novo production of il-1β by immature dc, upregulating st2l, and establishing a barrier to dc maturation following exposure to tlr or cd40 ligation. as such this work identifies a novel mechanism by which a clinically-important immunosuppressant impedes the capacity of dc to mature and consequently stimulate effector/adaptive t cell responses. these findings are particularly relevant to the potential use of rapa-conditioned dc as "negative" cellular vaccines to block alloag-specific responses, as exposure to endogenous and exogenous inflammatory stimuli can induce dc maturation and negate the tolerogenic properties of immature dc. exosomes are nanovesicles (50-100nm) released to the extracellular milieu by different cell types. exosomes secreted by dendritic cells (dcs) and other apcs express mhc ag, adhesion molecules and costimulatory molecules oriented on the membrane surface with their binding domains facing outwards. thus, exosomes released by graft-infiltrating leukocytes (gils) could function as "ag-presenting vesicles" or as vehicles to transfer alloag between recipient's apcs during elicitation of t-cell allo-immunity. aims: to test if (i) gils activate anti-donor t-cells in secondary lymphoid organs by releasing exosomes with alloag into systemic circulation; or (ii) gils that traffic to the spleen as passenger leukocytes use exosomes as a local mechanism to transfer alloag to recipient's dcs. methods: exosomes were isolated from supernatants of bm-derived [c57bl/6(b6), ia b ] dcs pulsed with the balb/c iea 52-68 allopeptide and purified by ultra-filtration and ultra-centrifugation on a 30%sucrose/d 2 o gradient. we used pkh67 + exosomes and cd45.1 congenic b6 mice for traffic studies, heart (heterotopic) and skin transplantation models (balb/c→b6, thy1.2 + ), and cfse-labeled 1h3.1 tcrtg cd4 t-cells (thy1.1 + ) specific for ia b (b6) loaded with iea 52-68 (balb/c). dcs were genetically engineered to release exosomes expressing green fluorescent protein (gfp). we have previously shown that blood-borne exosomes carrying balb/c alloag are reprocessed by different subsets of splenic dcs for presentation to indirect pathway 1h3.1 cd4 t-cells. here, we demonstrated that although gils of cardiac and skin allografts release exosomes ex vivo, they did not secrete enough concentrations of exosomes with alloag into circulation to stimulate donor-reactive t-cells in lymphoid organs. instead, our findings indicate that migrating dcs (generated in vitro or isolated from gils), once homed in the spleen, they transfer exosomes expressing gfp and carrying allopeptides to spleen-resident dcs of the recipient, identified by the congenic marker cd45.1. conclusion: exchange of exosomes between dcs in lymphoid organs might be a mechanism by which passenger leukocytes transfer alloag to recipient's apcs in secondary lymphoid organs. t cell activation is critical in initiating adaptive immunity, and pkcθ, a novel member of the pkc family, mediates non-redundant functions in the t cell receptor; however, its role in the mediation of allograft rejection remains unclear. this study is aimed at investigating whether alloimmune response can be alleviated by a deficiency of the pkcθ molecule, and whether transgenic expression of anti-apoptotic bclmethods. wild-type (wt) cardiac allografts were transplanted into pkcθ -/mice, with or without sub-therapeutic anti-cd154 mab. purified pkcθ -/or pkcθ -/-/ bcl-x l t cells were adoptively transferred into rag2 -/mice engrafted with cardiac allografts. lymphocyte proliferation assays were performed (cfse). nf-kb activation was assessed by bioluminescence imaging (bli) using luciferase transgenic mice under the control of a nf-kb promoter. results. the cardiac allografts were rejected in a delayed fashion in pkcθ -/mice with increased nf-kb activation; however, sub-therapeutic anti-cd154 mab (that normally delays rejection of cardiac allograft) induced long-term survival of cardiac allografts. the cardiac allografts were permanently accepted in rag2 -/mice with adoptive transfer of pkcθ -/-t cells, and the rejection can be elicited by transfer of pkcθ -/-/ bcl-x l t cells. in a lymphocyte proliferation assay, pkcθ -/-t cells displayed greatly reduced proliferation. in response to cd3 and cd28 stimulation, pkcθ -/-t cells underwent accelerated apoptosis and reduced th1, th17, and treg subsets compared to the wt t cells. bcl-x l restored the survival of the pkcθ -/-t cells. conclusions. the results suggest that pkcθ mediates the alloimmune response. bcl-x l transgene prevents pkcθ -/-t cell apoptosis and re-elicits allograft rejection. tolerogenic dendritic cells (dc) are immature, maturation-resistant(mr) or alternatively-activated dc that express mhc molecules and low levels or absent costimulatory signals. although mrdc administration has successfully prolonged allograft survival in murine models, the mechanism of action in vivo remains unknown. aim: to test in vivo if the down-regulation of the anti-donor response induced by donor-derived tolerogenic dc is due to: (i) direct interaction of the tolerogenic dc with donor-reactive t cells or (ii) by reprocessing of the tolerogenic dc into alloantigen (alloag) by recipient apc for interaction with indirect pathway t cells. methods: dc were generated in vitro by culturing balb/c bone marrow cells for 6-8 days in medium with gm-csf + il-4 supplemented with 10nm 1α,25-(0h) 2 vitamin d 3 (vd 3 ). we used a model of heterotopic vascularized allogeneic heart transplantation [balb/c into c57bl/6 (b6)] and cd4 t cells from 1h3.1 tcrtg mice that recognize b6 ia b loaded with the balb/c allopeptide ieα 52-68 (indirect pathway) . results: we demonstrated that vd 3 renders dc maturation resistant (vd 3 -mrdc) as vd 3 -mrdc fail to up-regulate co-stimulatory molecule expression, release il-12p70, or stimulate allo-responsive t cells after challenge with potent dc-maturation stimuli. adoptive transfer (i.v.) of balb/c vd 3 -mrdc (day -7) significantly prolonged survival of balb/c heart grafts in b6 mice in the absence of immunosuppressive therapy. interestingly, we found that in vivo, balb/c vd 3 -mrdc induced proliferation of indirect pathway 1h3.1 cd4 t cells in the spleens of b6 recipient mice, indicating that reprocessing of the balb/c dc by host (b6) apc does occur. proliferation of 1h3.1 cd4 t cells in response to balb/c vd 3 -mrdc resulted in defective activation (cd62l high , cd69 low ) of 1h3.1 t cells, leading to their peripheral deletion and outgrowth of cd4 + foxp3 + treg cells. reprocessing of balb/c vd 3 -mrdc was performed by recipient splenic cd11c high cd8α neg dc, and donor alloag continued to be presented through the indirect pathway for 3 days after donor dc administration. conclusion: these results suggest that dc-based therapies downregulate t cell allo-immunity and prolong allograft survival, at least in part, through reprocessing of the tolerogenic dc into alloag by recipient apc. early introduction: alloreactive memory t cells are present in all transplant recipients due to prior direct sensitization or heterologous immunity. these cells are known to circumvent tolerance induction and/or prevent indefinite graft survival in several models, but mechanistic details of their function are unknown. the goal of this study was to test the hypothesis that cd8 memory t cells initiate alloreocognition and express effector functions within hours of reperfusion. methods: syngeneic or a/j (h-2 a ) hearts were transplanted into wt c57bl/6 (h-2 b ), cd4-/-, cd8-/-, or rag1-/-recipients. rna and protein were prepared from total graft homogenates and analyzed by qrt-pcr and elisa. rag1-/-mice received 1x10 6 wt or ifng-/-2c cells and were used as recipients 10 weeks after reconstitution. donor-specific cd8 memory cells were purified from wt spleens 8 weeks after a/j skin grafting, and donor-specific effector cd4 cells were purified from spleens of cd90.1 mice 8 days after a/j heart transplantation. flow cytometry was used to quantify graft infiltrates. results: allografts contained elevated levels of ifng and cxcl9 mrna at 24, 48 and 72 hrs post-transplant vs. isografts. detectable cxcl9 protein was produced in allografts from wt and cd4-/-recipients but not in isografts or allografts from cd8-/-or rag1-/recipients. treatment with ctla4-ig and mr1 failed to reduce cxcl9 production. reconstitution of rag1-/-mice with ifng sufficient or deficient 2c tcr transgenic cd8 cells indicated that early allospecific cxcl9 production absolutely requires ifng made by recipient cd8 cells. although donor-specific ifng production was undetectable in splenocytes until day 4-5 post transplant, graft-infiltrating cd44 hi cd62l lo cd8 t cells were present as early as 24 hrs post-transplant. in adoptive transfer studies, effector-memory cd8 t cells reconstituted early allospecific cxcl9 production in cd8-/-mice. lastly, primed cd4 t cells adoptively transferred at day 2 post-transplant readily infiltrated allografts in control but not cd8 depleted recipients. conclusions: cd8 memory t cells infiltrate allografts rapidly post-transplant, produce ifng, and propogate an inflammatory environment which optimizes recruitment of primed effector t cells. successful neutralization of this early allorecognition pathway should provide valuable adjunctive therapy to improve graft function and survival. background: allogeneic t cell stimulation requires not only antigen-specific signals but also costimulatory signals, most importantly between cd80/86 on the antigen presenting cell (apc) and cd28 and ctla4 on the t cell. engagement of the t cell receptor without costimulation can lead to anergy and the induction of regulatory t cells (tregs). t cell activation is also controlled by expression of the tryptophan-catabolising enzyme indoleamine 2,3-dioxygenase (ido). depletion of this essential amino acid, and/or the production of tryptophan metabolites inhibits t cell proliferation. methods: a genetic approach to confer tolerogenic properties on murine dendritic cells (dcs) has been explored using lentiviral vectors, based on the equine infectious anaemia virus. firstly, an intracellular method that prevents costimulation has been developed: a fusion protein consisting of ctla4 and kdel [an endoplasmic reticulum (er) retention signal] is expressed in dcs. the ctla4-kdel binds to cd80/86 in the er and prevents expression of these proteins on the dc surface. a second approach uses an elevated expression of the ido enzyme by transduced dcs. results: ctla4-kdel-or ido-transduced dcs were unable to induce allogeneic t cell proliferation. however, using two-stage dc:t cell co-culture assays, it was shown that ctla4-kdel-, but not ido-transduced dcs, can induce donor-specific t cell anergy in vitro and in vivo. tolerance to both the direct and indirect pathways was shown using ctla4-kdel-transduced dcs. linked suppression was mediated by the generation of donor-specific tregs. ido-transduced dcs did not generate tregs. furthermore, it was shown separately that dcs expressing ido whilst lacking cd80/86 expression for potential ligation by ctla4 (although ctla4-cd80/86 ligation upregulates ido, it downregulates t cell activation) failed to generate or even sustain foxp3+ treg populations. the ability of the transduced dcs to induce tolerance to allografts was assessed in a complete mismatch and cbk→cba (indirect pathway) corneal graft model. these results support a clinical strategy to induce treg-mediated, donorspecific transplantation tolerance using ctla4-kdel-, rather than ido-expressing dcs. indirect cd4 t cells that recognise processed alloantigen on recipient apc can provide help to alloreactive cytotoxic cd8 t cells that recognise intact mhc i alloantigen on donor apc, but exactly how such 'un-linked' help is provided is not clear. the respective abilities of direct and indirect pathway cd4 t cells to provide help for cytotoxic cd8 alloimmunity were examined in a mouse model of heart graft rejection in which the recipients contain only monoclonal helper cd4 t cells, specific for self-restricted h-y antigen (female b6 mar/rag1 -/mice). mice were additionally reconstituted with 10 6 b6 cd8 t cells, and then challenged with female balb/c (no cd4 t cell help), or male balb/c (indirect pathway help), or male b6xbalb/c f1 hearts (direct pathway help) . un-reconstituted mar/rag1 -/mice lack effector b and cd8 t lymphocytes, and consequently all heart grafts survived indefinitely. in contrast, reconstituted mar/ rag1 -/mice rejected male f1 grafts rapidly (mst 8d), whereas female balb/c grafts survived indefinitely, confirming a cd4-dependent effector role for the transferred cd8 t cells. cd4 t cell help through the indirect pathway, although sufficient to elicit graft rejection, was less efficient than direct pathway help, because male balb/c grafts were rejected more slowly than the f1 grafts (mst 12d, p<0.001). we next considered whether indirect pathway cd4 t cells provide help through recognition of mhc ii complexes on the surface of alloreactive cd8 t cells, in analogous fashion to the cognate interaction between b and t lymphocytes. in support, reconstitution of mar/rag1 -/recipients with instead, mhc ii-deficient cd8 t cells, resulted in slower rejection of male balb/c hearts (mst 21d, p<0.01), whereas male f1 grafts, that still permit provision of linked help, were rejected at the same tempo. most tellingly, mar/rag1 -/mice that received simultaneously a female balb/c heart and male b6 apc (to activate mar cd4 t cells) rejected their grafts rapidly when reconstituted with male cd8 t cells (mst 9d). in contrast, grafts survived indefinitely when female cd8 t cells were transferred. flow cytometric analysis of mitogenstimulated cd8 t cells revealed surface mhc ii expression. indirect allorecognition can provide help for generating cytotoxic alloimmunity, but not as effectively as through the direct pathway. indirect pathway help is potentiated by linkage through recognition of cd8 mhc ii. purpose: tolerogenic properties of dendritic cells (dc) are supported and preserved by conditioning with the immunosuppressant rapamycin (rapa). the ability of rapaconditioned, recipient-derived dc pulsed with alloantigen (alloag) to suppress both direct and indirect alloag-specific t cells in the absence of immunosuppression has been demonstrated in a murine allograft model. dc can acquire intact mhc from cells or cell lysates. however, the ability of alloag-pulsed rapa-dc to immunomodulate directly-reactive alloag-specific t cells has not been formally demonstrated. methods: dc were generated from c57bl/6 (b6; h2 b ) bone marrow cells in gm-csf and il-4. rapa was added to indicated cultures (rapa-dc) beginning on day (d) 2. on d7, cd11c + bead-purified rapa-dc or non-treated control dc (ctr-dc) were incubated with balb/c (h2 d ) splenocyte lysates ("alloag pulsing"). following incubation, the dc were harvested and the level of donor and recipient mhc molecules on cd11c + cells determined by flow cytometry and immunofluorescent imaging. surface levels of cd86, b7-h1 (programmed death ligand-1; pd-l1), and fas-l were compared. pulsed-dc were also incubated with cd8 + t cells from rag -/-2c mice for 3d. 2c cd8 + cells express t cell receptors specific for h2-l d , a mhc class i molecule of balb/c. following incubation, 2c cell proliferation and apoptosis were both assessed. results: ctr-and rapa-dc presented detectable levels of directly-transferred mhc class i and ii on their surface after incubation with allogeneic balb/c cell lysate. donor mhc presented by "pulsed" recipient dc stimulated directly-reactive, alloag-specific 2c t cell proliferation. however, only rapa-dc induced apoptosis in the overwhelming majority of these cells responding via the direct pathway. induction of apoptosis correlated with an increased level of surface fas-l on rapa-dc and their comparatively low level of cd86 relative to pd-l1. conclusions: rapa-conditioned dc can present intact mhc molecules acquired from lysates of allogeneic splenocytes and concurrently induce apoptosis of directlyreactive alloag-specific cd8 + t cells. as such, we provide mechanistic insight into a mechanisms by which alloag-pulsed, recipient-derived rapa-dc may facilitate allograft tolerance. background: t regs actively regulate alloimmune responses and promote transplant tolerance. atg, a widely used induction therapy in organ transplantation, depletes peripheral t cells but may preferentially spare t regs . sirolimus is thought to expand natural t regs . b7 t cell costimulatory blockade inhibits effector t cell (t eff ) expansion and may promote regulation. we investigated the effect of combining mouse atg (matg), ctla4ig and sirolimus on stringent skin allograft survival, and studied the mechanisms by determining t reg /t eff balance in vivo using a unique model (abm-tcrtg-foxp3/gfp reporter mouse conclusion:this is the first report to establish that t cell depletion with matg combined with ctla4ig and sirolimus synergize to prolong stringent fully allogeneic skin allograft survival by promoting regulation and tipping the t reg /t eff balance by both preserving t regs and facilitating generation of new t regs by a conversion mechanism. these results provide the rationale for translating such a novel therapeutic combination to promote regulation and tolerance in primates and human organ transplantation. expansion of cynomolgus cd4+cd25+foxp3+ regulatory t cells using low dose anti-thymocyte globulin. to test low dose atg in vivo, 2 mg/kg (10% of depleting dose) was administered thrice (day 0,1 and 2) to a naïve monkey and to a monkey that was treated concurrently with sirolimus (trough 15-20 ng/ml) after heart transplantation. in the naive monkey, lowdose atg led to expansion of cd4+cd25+foxp3+ tregs in peripheral blood (baseline 0.57%, day 7=4.09%, day 12=2.7%) and in lymph nodes (baseline 2.47%, day 7=9.35%, day 12=7.08%) without causing t cell depletion. similarly, in the transplanted monkey peripheral blood tregs expanded from 1.4% at baseline to 4.64% on day 6. low dose atg is not only able to expand tregs ex vivo by proliferation of natural cd4+cd25+ cells, but can equally induce tregs in vivo without lymphodepletion. these findings provide the rationale for development of tolerance inducing strategies based on enhancing regulatory mechanisms in human transplant recipients. immunological background⁄aim: previously, we have shown that combination of human anti-cd40 mab, 4d11 and tactolimus exerts additive immunosuppressive effect and markedly prolongs renal allograft survival in cynomolgus monkeys. in this study, we further evaluated the immunological aspects among these transplant recipients. method: kidney transplantations were performed across mhc mismatched cynomolgus monkeys. transplant recipient was given either no-treatment, tacrolimus (1 mg⁄kg⁄day, po), 4d11 (5 mg⁄kg, iv) or tacrolimus+4d11 (n=3⁄group). peripheral lymphocyte population, mlr and serum anti-donor antibody levels and graft histology were assessed. results: mean graft survival for no-treatment, tacrolimus, 4d11 and tacrolimus+4d11 treatment groups was 6.0±1.0, 27.7±3.2, 120.3±90.6 and 213.7±28.0 days, respectively. peripheral cd20 + cells partially declined in both 4d11 alone and 4d11+ tacrolimus given animals at the early post-operation period, although the numbers recovered thereafter. cd4 + and cd8 + cells were unaffected. cd4 + effector memory population was reduced by addition of tacrolimus to 4d11 (fig. 1a ). mlr against donor and 3rd party antigens were suppressed in both 4d11 and tacrolimus+4d11 groups (fig. 1b) . addition of tacrolimus further reduced graft cd4 + , cd8 + and cd20 + cellular infiltration (fig. 1c ). anti-donor antibodies were detected in sera during the treatment course of 4d11; however, they did not develop under the tacrolimus+4d11 treatment. graft c4d deposition correlated with serum anti-donor antibody levels. the 4d11 inhibits both cellular and humoral responses against donor antigens. addition of tacrolimus strengthens these immunosuppressive effects of 4d11, leading to further prolongation of graft survival. objectives: allogeneic islet transplantation offers the potential for cure from diabetes. application of this therapy, however, is limited by immunologic mechanisms requiring medical therapy to prevent rejection of the islets. costimulatory blockade of the cd28/ cd80/cd86 and the cd40/cd154 pathways has shown promise in ameliorating the immune response to allow engraftment and function of islets. we have evaluated a new drug regimen consisting of induction therapy with 3a8, a murine anti-cd40 antibody, and basiliximab and maintenance treatment with ctla4ig and sirolimus in diabetic rhesus macaques which received allogeneic islets. methods: allogeneic rhesus macaque islets (14,100 ie/kg ± 1,802) were transplanted intraportally into diabetic rhesus macaques (n=4) under the following immunosuppressive regimen: short term administration of anti-il-2 receptor (basiliximab) and anti-cd40 (3a8), with maintenance immunosuppression using sirolimus for 134 days and abatacept (ctla4ig) for long term therapy. weekly peripheral blood flow cytometric and cmv viral load monitoring was performed. results: recipients treated with this immunosuppressive regimen had immediate return to normoglycemia following islet transplant. the graft survival in the first three animals was 141, 283 and 297 days. the fourth animal continues to exhibit good glycemic control at his current post-operative day 30. each of these animals had monthly intravenous glucose tolerance tests with monitoring of blood glucoses and c-peptides with further evidence of glycemic response and c-peptide generation. flow cytometry confirms cd40 blockade during the administration of 3a8 and return of cd40 after cessation of therapy. the treatment was well tolerated with minimal evidence of cmv reactivation and no evidence of thrombocytopenia or thromboembolism. conclusions: these preliminary results indicate that cd28/cd40 costimulatory-based immunosuppressive regimens can protect allogeneic islets from rejection. furthermore, 3a8 appears to adequately block cd40 to facilitate this engraftment and function as demonstrated by flow cytometry. iwami, 1,2 qi zhang, 1 osamu aramaki, 1 nozomu shirasugi, 1 katsuya nonomura, 2 masanori niimi. 1 1 surgery, teikyo university, tokyo, japan; 2 renal and genitourinary surgery, hokkaido university, sapporo, japan. many studies have shown immunosuppressive effects of dietary intake of fish oil containing eicosapentaenoic acid (epa) in various models such as autoimmune diseases and transplantation. however, its mechanisms remain uncertain. furthermore, there have been no studies examining the effect of purified epa. here we determined the ability of purified epa to inhibit alloimmune response in mouse cardiac transplantation model. methods: cba recipients (h-2 k ) were given single injection of purified epa intraperitoneally on the same day as transplantation of a heart from c57bl/10 donors (h-2 b ). mixed leukocyte reaction (mlr) assay and enzyme linked immunosorbent assay (elisa) were also performed to evaluate the effect of purified epa on cell proliferation and cytokine production. to determine the presence of regulatory cells, adoptive transfer study was conducted. results: untreated cba recipients rejected c57bl/10 cardiac allografts with median survival time (mst), 8 days. in contrast, cba recipients treated with purified epa (1.0g/kg) had significant prolongation of allograft survival (mst, >100 days). cba recipients treated with 0.1g/kg purified epa eventually rejected allografts (mst, 13 days). in mlr assay, treatment with 1.0 g/kg purified epa suppressed alloproliferation of splenocytes in the recipients. the treatment also inhibited production of il-2, il-12 and ifng by the splenocytes in the recipients. when splenocytes were harvested from the recipients treated with 1.0g/kg purified epa 50 days after cardiac allografting and were adoptively transferred into naïve secondary recipients, the adoptive transfer induced significant prolongation of cardiac allograft in nave secondary recipients (mst >30 days, compared to that in the recipients with adoptive transfer of naïve splenocytes, mst, 10 days). conclusions: purified epa induced significantly prolonged survival of fully mismatched cardiac allografts, and generated regulatory cells. background: chronic allograft nephropathy (can), the most common cause of late kidney allograft failure, is not effectively prevented by the current regimens. activation of extracellular signal-regulated kinases 1/2 (erk1/2) mediating intracellular signal transduction from various growth factor stimuli is required for tgf-β production, which plays a key role in the development of can. hence, the therapeutic potential of disruption of erk1/2 signaling to prevent can was examined in an experimental model. methods: kidney donors from c57bl/6j mice (h-2 b ) were transplanted to bilaterally nephrectomized balb/c recipient mice (h-2 d ). the recipients were treated with ci1040 (mek-erk1/2 inhibitor) or vehicle after 14 days post-transplantation for 28 days. can was evaluated with the banff 97 working classification. results: all six allografts receiving ci1040 treatment were survived, while two out of seven grafts were lost in vehicle-treated group. at the end of experiment, the function of grafts in ci1040 treated recipients had been maintained, indicated by lower levels of serum creatinine and bun (30±6 µm and 22±8 mm, n=6) as compared to those (94±39 µm and 56± 25 mm, n=5) in vehicle group (creatinine, p=0.0015; bun, p=0.0054). pathological evaluation indicated that ci1040 reduced can, reflected by a lower can score in ci1040-treated group (3.93, n=4 ) as compared to that (9.96, n=5) in vehicle controls (p=0.0234). further examinations showed that ci1040 treatment resulted in inhibition of phosphorylation of erk1/2 and reduction of tgf-β levels in grafts. in vitro ci1040 potently suppressed not only growth factors-stimulated erk1/2 activation and tgf-β biosynthesis in renal tubular epithelial cells, but also attenuated alloantigenstimulated t cell proliferation. conclusion: our data suggest that interference of erk1/2 signaling with pharmacological agent (i.e. ci1040) has therapeutic potential to prevent can in kidney transplantation. objective: this is the first study to investigate the role of a novel jak3 and sykinhibitor, r348, in the prevention of obliterative airway disease (oad), the major obstacle after lung transplantation. methods: trachea from brown-norway (bn) donors were heterotopically transplanted in the greater omentum of lewis (lew) rats. recipients were treated for 28 days with r348 (10, 20, 40, or 80 mg/kg), rapamycin (0.75 or 3 mg/kg), or left untreated. allografts were recovered and processed for histological evaluation determining degree of luminal obliteration, percentage of respiratory epithelial coverage, and mononuclear cell infiltration. donor reactive (igg) antibodies from the recipient's serum were determined using flow cytometry. results: r348 at 20, 40, and 80 mg/kg significantly inhibited luminal obliteration in a dose dependent manner (69±20%, 20±13%, 15±7%; p=0.003 vs. no medication). rapamycin in both concentrations significantly inhibited luminal obliteration (37±15%, 11±6%; p<0.001 vs. no medication) similarly to r348 at 40 and 80 mg/kg. r348 at 40 and 80 mg/kg significantly preserved respiratory epithelium compared to r348 at 10 and 20 mg/kg (49±35%, 76±27% vs. 0±0, 3±7%; p=0.004) and was superior to rapamycin in epithelial preservation (49±35%, 76±27% vs. 27±17%, 36±15%; p=0.01). all r348 and rapamycin-treated recipients expressed decreased numbers of peritracheal mononuclear cells in a dose dependent manner (p<0.0001). r348 20, 40 , and 80 mg/ kg treated recipients had significantly reduced igg levels versus untreated recipients (381±136, 371±61, 230±56 vs. 853±207; p<0.03). all r348 treated recipient thymus and spleen weights were significantly lower compared to the untreated group (p=0.001). bun, cr, and cholesterol levels were unaffected in r348 treated recipients. conclusion: r348 potentially exhibits its inhibitory effect by preserving the respiratory epithelium, rather than by rapamycin's mechanism of reduced smooth muscle cell (smc) proliferation. r348 occupies a beneficial pharmacokinetic profile, lacks nephrotoxic and atherogenic properties, and provides a favorable alternative to rapamycin in the treatment of chronic rejection in lung transplant recipients. genz-29155 is a novel, oral immune-modulatory agent identified in a high-throughput screen designed to find inhibitors of tnfα-induced apoptosis. the molecular target of the compound remains under investigation but is likely downstream of the tnfα cell surface receptor. in vitro studies have shown genz-29155 to be an effective inhibitor of the tnfα-triggered caspase cascade but not anti-cd3 or fas-mediated apoptosis, and thus may act by inducing allograft resistance to immune attack rather than suppressing the alloimmune response per se. it has been shown to synergize with sirolimus in murine heterotopic cardiac allotransplant models. in order to test this promising new agent in a more clinically relevant model of solid organ transplantation, we studied genz-29155 in a mismatched nhp (rhesus macaque) renal transplant model. genz-29155 (n=3) was administered (3mg/kg, iv, days 0-14) with sirolimus (1mg/kg, po, days 0-30). five control animals received only sirolimus and vehicle (1mg/kg, po, days 0-30). all animals were followed serially by polychromatic flow cytometry to determine the relative and absolute number of cd4+ and cd8+ t cell subsets. time to allograft rejection, the primary end point, was determined by a significant rise in serum creatinine and bun, as identified with biweekly monitoring. after diagnosis of rejection, allografts were removed for histological and transcriptional studies, along with splenocytes for immune function assays. in this pilot study, prolongation of rejection-free survival was significantly improved with genz-29155 and sirolimus combined vs. sirolimus alone (42.67 days vs. 17.20 days, respectively, p = 0.05). given these initial results, we have initiated a larger study (n=12) to optimize the dose and duration of genz-29155. five animals, transplanted within the past month remain alive and well in this study. further investigation of this agent will allow us to better understand the benefit of inhibiting tnfα-mediated apoptotic effects in both cellular alloimmune response and allograft injury in solid organ transplantation. targeting purpose: allospecific t memory cell responses are present in transplant recipients from exposure to cross-reacting antigens. we have previously reported that lfa-1 inhibition suppresses primary cd8-dependent rejection responses which are not controlled by any conventional immunosuppressive strategy. these studies were conducted to analyze the efficacy of this anti-lfa-1 ab for control of cd8-dependent responses in sensitized hosts. methods: fvb/n (h-2 q ) donor hepatocytes were transplanted into c57bl/6 (h-2 b ) or cd4 ko (h-2 b ) recipients. memory responses were analyzed by retransplantation with a second fvb/n allogeneic hepatocyte transplant. cohorts of mice were treated with anti-lfa-1 mab and observed for hepatocyte survival or magnitude of cd8 + t cell mediated allospecific cytolytic activity. results: the untreated secondary cd4 ko and c57bl/6 recipients rejected hepatocyte allografts with enhanced kinetics in comparison to the primary graft (mst= day 10 vs day 14, and mst= day 7 vs. day 10, respectively; p < 0.05). anti-lfa-1 mab treated cd4 ko recipients demonstrated delayed rejection (mst= day 35 vs day 10; p=0.001) compared to secondary rejection in untreated cd4 ko hosts. anti-lfa-1 mab treatment did not delay rejection in sensitized c57bl/6 recipients (mst= day 7) but did significantly reduce the in vivo allospecific cytotoxic effector function in c57bl/6 secondary recipients (20.6±4.1%; p=0.001) as compared to untreated controls (97±1.3%). the residual cytotoxicity observed in anti-lfa-1 mab treated c57bl/6 recipients is comparable to the in vivo cytotoxicity of cd8-depleted c57bl/6 secondary recipients (12.1±8.9%) and is likely mediated by alloantibody. in fact, the level of allospecific cytotoxicity in anti-lfa-1 mab treated sensitized c57bl/6 recipients correlated with the amount of alloantibody present in recipient serum. conclusion: in conclusion, treatment with anti-lfa-1 mab delayed (cd4-independent) cd8-dependent rejection in sensitized recipients but did not delay rejection in sensitized cd4-sufficient c57bl/6 recipients. despite the efficacy of treatment with anti-lfa-1 mab to significantly reduce the in vivo allospecific cytotoxic effector function in sensitized c57bl/6 mice this strategy did not delay rejection. this is likely due to alloantibody mediated rejection in sensitized c57bl/6 (but not cd4 ko recipients) which is not suppressed by treatment with anti-lfa-1 mab. abstract# 194 cytomegalovirus (cmv) represents a major cause of infectious complications after transplantation. recently, chronic infections with lcmv, hiv or hcv were shown to be associated with functionally anergic t-cells characterized by high expression of the programmed death (pd)-1 molecule. this study was carried out to characterize functional exhaustion of cmv-specific cd4 t-cells as determinant of impaired cmv-control and to elucidate whether the pd-1 pathway may be operative in active cmv-infection after renal transplantation. cmv specific cd4 t cells from 13 controls, 31 hemodialysis patients, and 51 renal transplant patients were quantified using flow cytometry and analysed for their expression of pd-1 and cytokines ifnγ and il2. cmv specific proliferation was analysed by cfda-se dilution. in viremic transplant-recipients, a significantly higher proportion of cmv-specific cd4 t-cells were pd-1 positive (median 40.9%) as compared to non-viremic transplant patients (8.8%), dialysis-patients (8.8%) or controls (3.1%, p<0.0001). in line with functional impairment, pd-1 positive t-cells produced significantly less ifnγ per single cell as compared to pd-1 negative t-cells (mean fluorescence intensity 129.4±52.6 versus 256.6±96.1, p<0.0001). moreover, unlike controls or non-viremic patients, the majority of cmv-specific t-cells from viremic patients showed a long-term loss of il-2 production. interestingly, functional anergy of pd-1 positive cmv-specific cd4 t-cells was reversible in that antibody-mediated blockade of pd-1 signaling with its ligands pd-l1/-l2 led to a 10fold increase in cmvspecific proliferation. in conclusion, expression of pd-1 defines a reversible defect of cmv-specific cd4 t-cells, and blocking pd-1 signaling may provide a potential target for enhancing the function of exhausted t-cells in chronic cmv-infection. differential background: some patients with cmv disease may be simultaneously infected with multiple viral strains. it is unknown if different strains clear differently with the commencement of antiviral therapy. we assessed response to antiviral therapy in patients with simultaneous co-infection with multiple strains of cmv. methods: pcr-based strain typing of cmv was performed using the glycoprotein b gene of cmv (gb1-4) in a cohort of organ transplant recipients with cmv disease. from this, 89 patients were identified that had simultaneous infection with ≥ 2 cmv strains. quantitative assessment of each of the strain types was performed at regular intervals after starting antiviral therapy. results: the different types of multi-strain infections were gb1+gb2 (11/89, 12%), gb1+gb3 (22/89,25%), gb1+ gb4 (7/89, 8%), gb2+gb3 (10/89, 11%), gb2+gb4 (6/89, 7%) and gb3+gb4 (8/89, 9%). 25/89 (28%) were simultaneously infected with 3 or 4 different genotypes. within individual patients, there was trend for gb1 cmv load (4.27log genomes) to be lower than the other genotypes (p=0.05-0.07) at the onset of disease. decay kinetics for all genotypes showed a bisphasic response with a 1 st phase decline of ∼0.7 days and a 2 nd phase of ∼3days. 1 st phase delines were fastest for gb1 (p=0.0001 vs gb2 and 4) while gb4 decline was slower than gb3 during the 1 st phase. 2 nd phase declines were similar between gb1 and 3 (3days and 2.9 days) but were slower for gb2 and 4 (3.45 days; p=0.01). there was a significant correlation between 1 st phase decline and log decline from baseline by day 21 (r=0.37; p=0.002). relative fitness calculations revealed complex fitness dynamics between genotypes although gb3 was always less fit than gb1, 2 and 4, and gb4 and 2 were always less fit than gb1. conclusion: in patients with cmv disease who have simultaneous coinfection with multiple strains, the 1 st and 2 nd phase declines in gb1 are significantly slower than either gb2 or 4 and have a lower log decline from baseline by day 21. these data indicate that either a significant fitness difference exists between cmv strains or that antiviral control of replication may be linked to cmv gb genotype and should aid our understanding of treatment success and failure. one introduction: parvovirus b19 (pvb19) is a single-stranded dna virus that was first reported to affect transplant (tx) recipients around 20 years ago. in the kidney tx setting pvb19 has been reported to cause anemia and proteinuria. reported incidence in a general kidney transplant cohort has been reported to be between 0%-12% and as high as 38% in an anemic kidney tx population. here we report our incidence of pvb19 infection over a 12 year span. patients and methods: all records of kidney tx recipients from 1996 until 2007 were reviewed for the presence of pvb19 infection. there were 834 kidney tx performed during this period. diagnosis of pvb19 infection was made either by detection of pvb19 via pcr in a blood/tissue sample or by detection of virus on renal tissue by immunostain. in patients found to have pvb19 infection; presence of anemia, proteinuria, concurrent infection and acute rejection rates were examined. response to treatment with ivig was also evaluated. results: incidence of infection was 2.4% as 20 patients were found to have evidence of infection. average time from tx to diagnosis of infection was 21.6 months (range 3 days-86 months). average creatinine at diagnosis was 2.32 mg/dl. anemia was present in 85% of patients with an average hematocrit of 30.8%. proteinuria was present in 45% of patients with evidence of pvb19 infection. co-infection was noted in 5 patients (4 cmv, 1ebv) and acute rejection was noted in 30% of individuals within 3 months of diagnosis. collapsing glomerulopathy (cg) was present in 5 patients and they all had subsequent graft loss at an average of 6 months after diagnosis. 4 of the 5 patients with cg had proteinuria along with anemia and were caucasian. 90% (18/20) of all patients with evidence of pvb19 infection received ivig and cleared their infection. one of the remaining 2 pts without ivig spontaneously cleared their virus. conclusion: although the incidence of pvb19 infection in our kidney tx cohort was very low, its presence portends an unfavorable outcome. the presence of cg associated with pvb19 is an especially devastating lesion with very poor outcomes. response to treatment with ivig and reduction of immunosuppression is variable. based on our data it seems reasonable to screen all tx patients with unexplained anemia and concurrent proteinuria as early detection of pvb19 may be crucial. background: prior to transplant, screening for latent tuberculosis (ltbi) by tuberculin skin test (tst) is recommended. the accuracy of tst in end stage renal disease however may be limited. the quantiferon®-tb gold assay (qft) detects interferon-δ produced by peripheral blood t-cells in response to tb specific antigens and may be more accurate for diagnosis of ltbi. methods: this prospective single center study compared the tst to qft for the diagnosis of ltbi in a cohort of adult patients listed or undergoing workup for renal transplantation. all patients had both tst and qft performed. additional data collected included demographics, tb risk history and chest x-ray results. based on demographic and radiographic findings, patients were classified as high or low risk for ltbi. a positive tst was defined as ≥5 mm and positive qft as ≥0.35 iu/ml. results: a total of 45 patients were enrolled. complete data was available for 38 subjects (5 did not return to have tst read). the mean age was 50.4 +/-12.2 years with 21 (55.3%) males and 17 (44.7%) females. the most common etiologies of renal diseases were diabetes (52.6%) and glomerulonephritis (26.3%). most subjects (35 of 38) were on renal replacement therapy (hemodialysis in 73.7% and peritoneal dialysis in 18.4%). twenty (52.6%) subjects had received bcg and 10 (26.3%) were born in or lived in a country in with tb prevalence rate > 20/100000 population. fifteen (39.5%) subjects were considered to be at high-risk for ltbi. overall 5 (13.2%) had a positive tst and 3 (7.9%) had a positive qft. the qft was indeterminate in 1 subject due to a low mitogen response. agreement between the tests was 92% (k=0.72, p<0.0001). in low-risk subjects (n=23) the tst was negative in all and the qft was negative in 22 and indeterminate in 1. in clinically high-risk subjects, 5 (33%) had a positive tst and 3 (20%) had a positive qft. the 2 subjects with discordant results, both from tb endemic countries, had both completed treatment for ltbi 4 years prior and remained tst positive, but were qft negative. in renal transplant candidates, the tst and qft are comparable for the diagnosis of ltbi. the qft has the advantage of being completed in a single visit and in our cohort indeterminate results were uncommon. optimal utilization of htlv i/ii positive organs -a nationwide survey. objective: we recently presented data from the unos database that demonstrated no significant difference in graft or patient survival between htlv i /ii (+) and (-) liver recipients. several organ procurement organizations (opo) including our own, do not offer htlv i /ii positive organs while many others find it difficult to place them. despite this, the number of htlv (+) organs is increasing with 31 utilized in 2006 alone. this prompted us to evaluate the practical difficulties in placing these organs so as to improve utilization of these "high risk" life saving organs. medthod: a telephone/email survey of all the 65 opos in usa was done over a 2 month period from october to november 2007. results: of the 65 opos, 42 responded. all screen patients for htlv i/ii with elisa. 38 centers confirm with repeat elisa, 27 confirm with western blot and 6 centers do not pursue further. 36 of the 42 centers offer the htlv i/ii positive organs. there were a total of 231 positive donors in the past 5 years of which organs from 109 donors (47.2%) were placed. 27 centers offer all the organs while 15 offer one or more organs selectively based on accepting centers. none have been able to place the pancreas. only liver and kidney were commonly accepted. several centers noted a high false positive rate. based on the unos regional analysis data, 43% of the organs are utilized in ny state alone. many opos did not know which particular centers accept these organs and consequently spend a lot of time and effort in order to place them. a majority wanted to have a list of transplant centers that accept these organs. conclusions: htlv i/ii organs are being underutilized. moreover, our prior analysis of unos data shows that these life saving organs are shared more nationally than loco-regionally which is associated with a poorer outcome. increased knowledge of successful htlv (+) donation and the centers that are willing to utilize these organs in the appropriate setting will help expand the donor pool and decrease mortality on the waiting list. increasing traditional two-drug chronic immunosuppression (is) used in organ transplantation (tx) is associated with development of ebv-driven complications because of impairment of anti-viral cd8 + t cell surveillance. since the long-term impact of alemtuzumab preconditioning combined with tacrolimus monotherapy on ebv immunity after tx has not been studied, here we aim to analyze the frequency and function of peripheral blood ebv-specific cd8 + t cells. thirteen ebv + stable kidney transplant (ktx) recipients and 12 ebv + healthy controls were recruited to this cross-sectional study. all patients received alemtuzumab preconditioning, followed by tacrolimus monotherapy. blood samples were collected at least 1 year post-tx to allow immune reconstitution. the ebv-specific cd8 + t cell phenotype and function were screened by flow cytometry and ifng elispot assay. hla-a201 restricted ebv-lytic (bmlf1) and latent (lmp2a) peptides were used to generate tetramer (tmr) probes, and for functional screening in elispot. circulating cd8 + t cells from ktx patients had recovered by 1 year, and were comparable to those of healthy controls (28.6%±12.9 vs 23% ±6.6, p=0.2). moreover, the memory distribution and the frequency of ebv-specific cd8 + t cells detected in patients and controls (bmlf1-specific: 0.56%±0.96 vs 0.88%±1.08, p=0.46, and lmp2specific: 0.05%±0.06 vs 0.24%±0.40 p=0.09) were similar. in contrast, the frequency of functional type-1 (ifn-g producing) ebv-specific cd8 + t cells was significantly lower in ktx patients than in healthy controls (bmlf1: 47±24 spots/10 5 cd8 t cells vs 164±134 p=0.06, and lmp2: 9±8 vs 64±54 p=0.03). accordingly, on average, only 8-12% of circulating ebv-specific cd8 + t cells from ktx patients produced ifn-g, while 20-30% of effector cells were functional in healthy controls. addition of il-2 (20iu/ml) during the elispot assay reversed the hypo-responsiveness of type-1 (ifn-g) ebv-specific cd8 + t in patients (range 3-8 fold increase), suggesting that these effector cells were anergic. these results support the notion that alemtuzumab-induced lymphocyte depletion followed by tacrolimus monotherapy renders ebv-specific cd8 + t cells anergic in vivo, a state that can be readily reversed by cytokines such as il-2, which are commonly released during immune activation. background: the epidemiology of the transmission of cytomegalovirus (cmv) from organ donors to recipients is not completely understood. we studied donor to recipient transmission patterns by analyzing viral genomic variants through the use of cmv glycoprotein b (gb) genotyping by real-time pcr. polymorphisms in gb ul55 allow discrimination of 4 distinct genomic variants (gb 1-4). methods: organ transplant recipient pairs or triplets were included in the study if: a) they had cmv infection, b) they received an organ from a cmv seropositive donor, and c) there was at least one other recipient from the same donor that also developed cmv infection. genotyping (gb 1-4) was performed by quantitative real-time pcr on stored blood samples. clinical charts were reviewed to evaluate the clinical characteristics and outcome of cmv infection. results: of the 78 cmv seropositive donors screened, 21 were multiple organ donors for which 2 or more of their recipients developed cmv infection. the total number of recipients from these 21 donors was 86 (median of 4 recipients per donor). of these recipients, 47 (55%) had cmv infection (16 recipient pairs and 5 recipient triplets). the prevalence of genotypes was gb1 (n=22; 47%), gb2 (n=11; 23%), gb3 (n=4; 9%), gb4 (n=0). mixed infection with two concurrent genotypes was present in 10 patients (21%). overall concordance between cmv gb genotype in recipient pairs was 62.5% (10/16). if both recipients were cmv seronegative (d+/r-) the gb concordance in recipients was 67% (2/3 pairs). gb concordance was 70% (7/10 pairs) if one of the recipients was seronegative and the other seropositive. concordance was 33% (1/3 pair) if both recipients were seropositive. concordance between genotypes was seen in 2/5 (40%) recipients triplets. in seropositive recipients with cmv viremia, the origin of the cmv strain was thought to be donor derived in 11/16 (69%) and of reactivation of the recipients own virus in 5/16 (31%) of the cases. no difference in clinical outcome or organ tropism was seen between genotypes. based on an analysis of strain concordance within recipients from common donors, transmission patterns of cmv can be assessed. in d+/r+ transplant patients, donor strain superinfection accounts for the approximately two-thirds of cmv infection. backgroud: although map kinases have been implicated in the pathophysiology of liver iri, their functional significance in the mechanism of tlr4 mediated pro-inflammatory immune regulation, remains to be elucidated. methods: map kinase activation in a murine model of liver warm iri (90 min. ischemia, 6 h reperfusion) was determined by western blots. chemical inhibitors of erk (u0126, 10 µm in vitro or 160 mg/kg in vivo), jnk (sp600125, 50 µm or 30 mg/kg), and p38 (sb203580, 10 µm, 100 mg/ kg) map kinases were utilized in vitro in primary bm-derived macrophage cultures stimulated with lps (10 ng/ml); or in vivo in liver pro-inflammatory immune responses induced by lps (1 µg/mouse, i.p.) or iri. results: erk and jnk, but not p38, map kinase activation were readily detected in liver iri. in primary macrophage cultures, lps induced pro-and anti-inflammatory genes, including tnf-α, il-1β, il-6, il-10, inos and cxcl10. erk inhibitor mainly suppressed il-1β and il-10 (80% and 90% resp), whereas jnk inhibitor suppressed the majority of genes. in lps-induced liver inflammation, erk inhibitor suppressed il-6, il-1β, inos and il-10 by >50%, but failed to affect tnf-α/cxcl10. jnk inhibitor, on the other hand, preferentially inhibited pro-inflammatory genes, but marginaly affected il-10 (<30%). and produced comparable suppression of pro-/anti-inflammatory genes (figure1). interestingly, tnf-α was the least responsive gene subjected to map kinase regulation. conclusion: erk and jnk map kinase activation: 1/ are required for tlr4 activationinduced pro-inflammatory gene induction; 2/ play critical role in the development of ir-mediated liver immune response/tissue injury. background: the jak/stat signaling is one of the major pathways for cytokine signal transduction. the signal transducer and activator of transcription 1 (stat1) is mainly activated by ifn-α/ß/ifn-γ. the activation of stat1 by ifn-γ has been implicated in hepatic inflammation. we have shown that activation of toll-like receptor (tlr) 4 complex initiates pro-inflammatory response leading to liver ischemia/reperfusion abstracts injury (iri). indeed, tlr4 signaling in vitro activates stat1, which in turn triggers production of type-1 ifn-dependent cxcl10 (ip-10). this study was designed to analyze the cross-talk between stat1 and the map kinase (erk) downstream of jak/ stat signaling pathways. methods & results: we used a mouse liver model of partial warm ischemia (90 min), followed by reperfusion (6 h) . first, we employed stat1 ko (n=17) and control wt (n=16) mice. the hepatocellular damage, as measured by salt levels (iu/l), was significantly decreased in 10/17 stat1 ko mice (p<0.005); the remaining 7/17 of stat1 ko showed salt levels comparable with wt. hence, we distinguished two groups of stat1 "protected" vs. "nonprotected" ko recipients. histology revealed minimal sinusoidal congestion without edema/vacuolization or necrosis in stat1 ko "protected" group. the induction of mrna coding for tnfα/ il-6 was higher in stat1 ko "nonprotected" livers. the expression of cxcl10, the product of stat1 activation downstream of tlr4 in type i ifn pathway, was profoundly and selectively depressed in livers from stat1 ko "protected" mice, as compared to iri susceptible livers. similarly, western blot-assisted phospho-erk expression was up regulated selectively in the stat1 ko "protected" group. in the second series, c57bl/6 mice were treated 1 h prior to liver ischemic insult with jak-2 inhibitor (tyrphostin ag490; 40 mg/kg, i.p.; n=5), or vehicle (n=5). the hepatocellular damage, as measured by salt levels (iu/l), and histology was significantly decreased in ag490 group, as compared with controls (mean = 12770 vs. 28770; p<0.05). the disruption of jak/stat signaling by inhibiting jak2 uniformly ameliorates the inflammatory immune response in liver iri. however, the blockage of stat1 alone is insufficient to reproducibly exert cytoprotection. as jak2 is upstream of stat1 as well as upstream of map kinase (erk), this study highlights the role of both signaling pathways in hepatic iri. purpose: tlr4 is required for maximal ischemic injury of the heart, liver, lung, and kidney. to better understand the mechanisms of tlr4 action, we investigated a murine model of ischemic kidney disease and examined endothelial tlr4 expression. methods: 1. animal ischemia reperfusion injury(iri): the right kidney of wildtype(wt) c57bl/10, or tlr4-deficient(ko) c57bl/10scn mice was removed. the left pedicle was clamped for 23 min, followed by 4 hr reperfusion. sham animals were controls. 2. bone-marrow chimera: four groups of bm chimeras were created: wt→wt; ko→wt; wt→ko; ko→ko (8 recipients/grp). 8 wks later, chimerism was confirmed by tail and blood genotyping, and mice subjected to renal iri. 3. tlr4 mrna detected by dig-labeled antisense; tlr4 on endothelium by anti-tlr4 and anti-cd31. 4. total genome mrna expression on ischemic vs. sham wt mice, ischemic vs. sham ko mice(4 mice/grp) determined using affymetrix mouse genome 430.2 genechips followed by genesifter analysis. quantitative real-time rt-pcr confirmed candidate genes. 5. ms1 endothelial cells were treated with h 2 o 2 (500um) for 30 min, cultured for 4 hr and then expression of tlr4 and endothelial genes determined. results: tlr4-deficient mice had less renal injury as assessed by pathology and function. radiation chimeras showed that radioresistant parenchymal cells and radiosensitive leukocytes were both required for maximal injury. immunohistology and in situ hybridization identified endothelia in the outer medulla as a major tlr4 expressing cell type at 4 hr post-reperfusion. genechip analysis revealed a panel of cytokine, chemokines, proinflammatory and cell-cycle related genes that were differentially expressed on iri versus sham kidneys. real-time rt-pcr confirmed that the following pro-inflammatory endothelial genes increased after ischemia in wildtype but not tlr4 ko mice: tlr4, pentraxin related gene(ptx3), and endothelial cell-specific molecule 1(esm1). real-time rt-pcr showed that in vitro h 2 o 2 treatment of endothelial cells induced expression of tlr4(6.6-fold), ptx3(4.6-fold), and esm1(69-fold). we found that endothelia in the outer medulla increase their expression of tlr4 after ischemia, and that the endothelial genes ptx3 and esm1 increase only in wt ischemic kidneys. we also found that h 2 o 2 , which mimics reactive oxygen species generated during iri, directly increases these same endothelial genes in vitro. nkg2d is an activating receptor expressed on nk cells and cd8 + t cells. ligands for nkg2d including rae-1, mult-1 and h60 in mouse, may be upregulated by tissues in response to stress. a study in mouse macrophages described upregulation of rae-1 in response to stimulation through tlr4 by lps. we have shown that tlr4 mediates kidney ischemia reperfusion injury (iri) and also observed rae-1 upregulation in iri kidneys. we now determined whether: 1) kidney iri could induce the expression of nkg2d ligands: 2) expression of nkg2d ligands is tlr4 dependent; 3). bone marrow (bm) derived cells or parenchymal kidney cells express nkg2d ligands. methods. kidney-ischemia was induced in tlr4 -/-, myd88 -/and wt mice for 22 min. blood and tissue were harvested at days 1, 3 and 5. primary cultures of mouse tubular cells (tecs) were also subjected to ischaemia (mineral oil overlay for 1 hr) or tlr4 activation (lps stimulation). bm chimeric mice were generated by transplanting bm into irradiated recipient mice before iri was induced. results. rae-1 mrna level in iri kidney was increased from day 1 to day 5, peaking at day 3 compared to sham operated kidney (14-48 fold increase, p < 0.001) measured by real time pcr. rae-1 protein was detected in renal tubular cells from ischemic kidney but not from shamoperated control by flow cytometry. mult-1 mrna expression was also increased from day 1 to day 5 (15-51 fold increase, p <0.005). both rae-1 and mult-1 mrna levels were reduced in tlr4 -/and myd88 -/-iri kidneys (2-8 fold reduction) versus wt controls (p < 0.01). tlr4 -/and myd88 -/primary cultured tecs submitted to iri in vitro also showed less rae1 and mult-1 mrna expression than wt controls (p < 0.01). lps stimulated rae-1 and mult-1 expression in wt but not in tlr4 -/-tecs. tlr4 -/mice bearing wt hemopoietic cells had significantly lower kidney rae-1 and mult-1 mrna expression after iri versus wt mice with tlr4 -/-bm (p < 0.05). conclusion. kidney iri causes rae-1 and mult-1 expression and kidney parenchymal cells are the dominant source. tecs can be stimulated to express rae-1 and mult-1 by ischemia or lps via the tlr4 pathway in vitro and deficiencies in this pathway provide protection against iri and rae-1 and mult-1 expression in vitro and in vivo. thus, kidney iri causes upregulation of nkg2d ligands by parenchymal kidney cells via tlr4. background: intestinal ischemia/reperfusion injury (iri) is a major clinical problem. although toll-like receptor 4 (tlr4) has been implicated as a potential link between the innate and adaptive immunity, little is known on its role in intestinal iri. our preliminary research in intestinal iri has shown that, compared to sham controls, wt mice had decreased survival, worse tissue injury/apoptosis, increased pmn infiltration, increased cd3+ cell infiltration, and increased production of tlr4, chemokines, and adhesion molecules. here we used tlr4ko mice to further investigate the role of tlr4 in intestinal iri and its effects on cytokine/chemokine programs and apoptotic signaling. methods: c57bl10 wt and tlr4ko mice underwent 100 min of total jejunoileal warm iri by clamping of the sma. separate survival and analysis groups were performed. intestinal tissue was harvested at 4h and 24h. tissue analysis included histopathology, cd3 immunostaining, myeloperoxidase (mpo) activity, rt-pcr for chemokines/ cytokines, and western blots for apoptotic and ho-1 protein expression. results: tlr4ko had superior survival compared to wt (100% vs. 33%, p<0.02). on histopathology tlr4ko had near normal-appearing villous architecture, while in contrast, wt showed mucosal erosions and villous congestion/hemorrhage. tlr4ko had reduced cd3+ cell infiltration as compared to wt (3.9±0.7 vs. 6.2±1.4 per hpf at 4h, p<0.001; and 4.8±1.1 vs. 7.0±2.3 per hpf at 24h, p<0.02). early mpo activity was also reduced in tlr4ko (0.11±0.06 vs. 0.88±0.5 u/g at 4h, p<0.005). rt-pcr analysis demonstrated decreased production of mrna for ip-10, mcp-1, rantes, and ifn-γ and increased production of il-13 in tlr4ko. there was decreased protein expression of caspase-3 and increased expression of bcl-2 and ho-1 in tlr4ko mice. conclusion: the genetic absence of tlr4 exerts protection against intestinal iri, demonstrating for the first time that tlr4 is required for intestinal iri. the absence of tlr4 signaling reduces iri through reduced neutrophil and t cell chemotaxis, and up-regulation of protective molecules. these results support data that tlr4 is a mechanistic link between the innate and adaptive immunity, implicating tlr4 as a potential therapeutic target for the prevention of intestinal iri. it is well known that liver steatosis increases hepatic vulnerability to ischemia/ reperfusion (i/r) injury as part of the transplantation process. endotoxin (lps) is thought to be a major contributing factor to the pathogensis of i/r. during portal occlusion, lps is translocated across the mesenteric tissue barrier into the portal circulation, and is delivered as a large bolus to the liver at the point of reperfusion. at this time, lps is mainly recognized by toll-like receptor 4 (tlr4). this leads to downstream signaling and the production of proinflammatory products that ultimately lead to cellular inflammation, necrosis, and apoptosis. it is well known that steatotic livers are highly sensitive to endotoxin as compared to their lean counterparts post-i/r, and we have previously seen that monoclonal antibody blockade of endotoxin dramatically improves animal survival after i/r. therefore, we propose the novel hypothesis that tlr4 signaling is a major contributor to cellular damage after steatotic hepatic i/r. to test this hypothesis, we subjected male 4-week-old c57bl/10j (control) or c57bl/10scn (tlr4 deficient, tlr4ko) mice to a high-fat diet (hfd) for four weeks. then, we subjected the animals to 35 minutes of total hepatic ischemia and 1 or 24 hours of reperfusion. there was a dramatic improvement in animal survival in the hfd tlr4ko animals versus control hfd animals at 24 hours (74% vs. 31% in control hfd animals, p<0.05). there was significantly more liver necrosis (as measured by a grading scale from 0-3) in the control hfd animals as compared to the tlr4ko hfd animals (1.4±0.3 in control vs. 0.4±0.2 in tlr4ko, p<0.05). in addition, we see significant increases in the message level of the proinflammatory cytokines il-6, il-12, and ifn-γ at one hour in the control hfd animals that is abrogated dramatically in the tlr4ko hfd animals. we do not see these dramatic changes in the control animals fed a normal diet. despite the significant increases in inflammation in the control hfd animals versus the tlr4ko hfd and normal diet control animals, we do not see changes in the tlr4 message level or endotoxin boluses, implying an increased sensitivity in the absence of an increased number of receptors. tlr4 is a critical molecule in the pathogenesis of steatotic liver ischemia/reperfusion, and represents a potential therapeutic target for expansion of the donor pool. the background: neutrophils are considered crucial effector cells in the pathophysiology of organ ischemia and reperfusion injury (iri). particularly, neutrophil elastase (ne) accounts for a substantial portion of the neutrophil function. this study was designed to explore the role of, and mechanism by which ne exerts its function in a mouse model of liver warm iri. methods: partial warm ischemia was produced in the left and middle hepatic lobes of c57bl/6 mice for 90 min, followed by 6-24h of reperfusion. mice were treated with ne inhibitor (nei; 2mg/kg p.o.; gw311616a; n=6) or control (n=6) at 60 min prior to the ischemia insult. after 6h or 24h of reperfusion, sast/salt levels and intrahepatic neutrophil accumulation (myeloperoxidase [mpo] activity) were assessed. the pro-inflammatory cytokine (tnf-α, il-6), chemokine (cxcl-1, cxcl-2, ip-10) and toll-like receptor (tlr) 4 gene expression profiles were screened by rt-pcr. liver samples were collected for histological grading, and detection of neutrophil infiltration by the naphtol as-d chloroacetate esterase stains. results: nei treatment significantly reduced sast/salt levels, as compared with controls (17695±3413 vs 10990±2411; p<0.05 / 33650±3793 vs 13510±6809; p<0.01 at 6 h, and 11778±3113 / 14483±3972 vs 3565±957 / 4810±1063; p<0.01 at 24h). the expression of pro-inflammatory cytokines, and chemokines was significantly reduced in the nei treatment group (tnf-α in 6h; p<0.05, il-6 in 6h and 24h; p<0.01 and p<0.05, cxcl-1 in 24h; p<0.01, cxcl-2 in 6h and 24h; p<0.01 and p<0.05, ip-10 in 24h; p<0.01). the mpo activity (u/g) was also significantly reduced following nei treatment (14.1±7.7 vs 4.0±1.2; p<0.05). tlr4 expression was selectively diminished in nei pretreated livers (6h; p<0.01). histological examination of liver sections has revealed that unlike in controls, nei treatment markedly reduced edema, diminished centrilobular ballooning/sinusoidal congestion, ameliorated hepatocellular necrosis, and decreased local neutrophil infiltration. conclusion: the inhibition of ne ameliorated hepatocellular damage, reduced local inflammatory responses, and neutrophil activity/infiltration in a stringent mouse liver model of warm iri. interestingly, it also downregulated the innate tlr4 signaling. this study documents the previously unrecognized ne -tlr4 cross talk, and implies neutrophil elastase in the signal transduction pathway instrumental for liver iri. lymphocyte lymphocytes are involved in the early pathogenesis of ischemia-reperfusion injury (iri) in kidney; however, their role during healing is unknown. this has direct clinical consequence since lymphocyte-targeting agents are currently administered to prevent rejection during recovery from iri in renal transplants. c57bl/6 mice underwent unilateral clamping of renal pedicle for 45 min, followed by reperfusion, and were sacrificed at day 10. mice were treated with saline (c), methylprednisolone (pred) or mycophenolate mofetil (mmf) i.p. daily from day 2 until sacrifice (n=12/group). lymphocytes were isolated from the kidneys, counted and stained with monoclonal antibodies. kidney damage (% damaged tubules) and proliferation (ki67 assay) were assessed. flow cytometry analysis demonstrated increased numbers of tcrβ + cd4 + and tcrβ + cd8 + t and tcrβ -nk1.1 + nk, but not cd19 + b cells at day 10 in the ischemic (ir) kidneys compared to contralateral. regulatory t cells, tcrβ + cd4 + cd25 + foxp3 + , and t cell subsets tcrβ + cd8 + cd122 + and tcrβ + nk1.1 + also increased. moderate tubular damage in cortex, severe injury in outer medulla and increased proliferation in both compartments characterized the repair phase. pred improved histological damage in ir kidneys, while mmf worsened it. proliferative index correlated with histology in outer medulla. pred reduced the total counts and activation of tcrβ + cd4 + and tcrβ + cd8 + t cells in ir kidneys, and increased the percentage of tcrβ + cd8 + cd122 + among total tcrβ + cd8 + t cells. mmf reduced all lymphocyte subsets, decreased the percentage of tcrβ + cd4 + cd25 + foxp3 + among total tcrβ + cd4 + t cells, and lowered il-10 tissue levels. il-6 and platelet derived growth factor-bb protein levels were also decreased in ir kidneys from mmf-treated mice. in conclusion, specific trafficking and phenotypic changes of kidney-infiltrating lymphocytes occur during recovery from renal iri, and lymphocyte-targeting agents, pred and mmf, alter tubular cell structure, proliferation, and inflammatory response in the repair phase. background: ho-1 plays an important cytoprotective role in a variety of organ injury models. we have shown that ho-1 exhibits potent cytoprotective effects against liver i/r injury. this study explores the function and mechanism of ho-1 in liver i/r injury by using sirna that suppress ho-1 expression both in vitro and in vivo. methods: using a partial liver warm ischemia model, c57bl/6 wide-type (wt) mice (n=6/gr) were injected with ho-1 sirna/nonspecific control sirna (2 mg/kg, i.v. at day -1) or ad-ho-1/ad-β-gal (2.5x10 9 pfu, i.v. at day -2). sham control wt underwent the same procedures, but without vascular occlusion. mice were sacrificed at 6 h of reperfusion; liver tissue and blood samples were collected for future analysis. in in vitro studies, ypen-1 endothelium cells were transfected with ho-1 sirna (100 nm) or ad-ho-1/ ad-β-gal. results: ho-1 sirna treated mice showed significantly increased sgot levels (iu/l), as compared with nonspecific control sirna or ad-ho-1 (7593±2859 vs. 3104±1777 and 211.5±40, respectively; p<0.05). these correlated with histologic suzuki's grading of liver i/r injury, with ho-1 sirna showing significant edema, sinusoidal congestion/cytoplasmic vacuolization, and severe hepatocellular necrosis; nonspecific control sirna showed moderate edema, sinusoidal congestion/cytoplasmic vacuolization. in contrast, ad-ho-1 revealed only minimal sinusoidal congestion without edema or necrosis. ho-1 sirna significantly increased local neutrophil accumulation and caspase-3 activity, and increased the frequency of apoptotic cells (31.8±5.5 vs. 18.5±5.4 and 3.5±2.2, respectively; p<0.005), as compared with nonspecific control sirna or ad-ho-1. both ypen-1 endothelium cells and wt mice treated with ho-1 sirna revealed markedly increased caspase-3 activity and reduced ho-1 expression. in contrast, ad-ho-1 significantly decreased caspase-3 activity and increased ho-1 and anti-apoptotic bcl-2/bcl-xl expression. conclusion: this study provides evidence that ho-1 exerts cytoprotection against i/r injury by regulating liver apoptosis and inhibiting caspase-3 activation pathway. organ specific sirna is not only a powerful tool to study local gene function, but it may also provide novel therapeutic application in transplant recipients. islet cell transplantation has recently emerged as one the most promising therapeutic approaches for diabetic patients to improve glycometabolic control. one major problem of the procedure is the requirement of an immunosuppression protocol capable of counteract both auto and allo-immune response. recent data suggest that anti-thymoglobulin (atg) can halt efficiently the mounting of an alloresponse and the recurrence of autoimmunity in nod mice by expanding antigen specific t-regulatory cells. we retrospectively reviewed our casuistry type 1 diabetic kidney-transplanted patients who underwent islet transplantation using an immunosuppressive protocol based on atg or daclizumab (as induction treatment) plus cyclosporine and mmf as maintenance therapy. 45 patients underwent islet after kidney transplantation in our center. thirty-four patients received a time course of atg as induction (125 mg per day for 7-10 days), (number of islet infused=499,596±28,780), and 9 patients received daclizumab at induction (1 mg/kg every 2 weeks for 10 weeks); (number of islets=486,108±70,644). no major adverse events were recorded in our center; no malignancies or infections outbreaks were evident. patients in the atg induction group showed a better islet survival rate compared to daclizumab (p=0.01), according to c-peptide>1ng/ml. a sustained and prolonged c-peptide secretion was evident in the atg group; while in the daclizumab group only 1 patient was functioning at 1 year. interestingly, in the atg, which reached a longer follow-up, we cannot observe a loss of beta cell mass according to our metabolic test, suggesting the preservation of islet mass. in conclusion, atg can provide a good protection towards both allo and auto immune response. the next step will be to use atg in a calcineurin free protocol, allowing a better expansion of t-regs. introduction: despite consistent achievement of insulin-independence, recent data indicate that the long term success of islets transplants using the edmonton protocol is <10% at 5 years. the cause of the nearly universal late islet allograft failure remains unknown but hypotheses include: allo or autoimmune injury, marginal mass exhaustion, hepatic site related dysfunction, and immunosuppression toxicity. we examined the series of islet transplants performed at our institution and noted marked differences in the outcome and complications in ia and iak groups. our results may provide insight as to the cause of chronic islet loss. methods: thirty-one islet infusions were administered to ia (n=9) and iak (n=8) type-1 diabetics between 9/2001-11/2007. ia and iak had similar demographics and transplanted islet mass (13,838 vs 13,411 ieq/kg). ia received edmonton like immunosuppression with zenapax induction and cni/srl, whereas iak patients received zenapax and: cni/mmf/pred (6), cni/mmf (1), cni/srl (1)). results: insulin-independence was achieved in all but 2 patients who completed therapy (2 others withdrew). compared with ia, iak exhibited better glycemic control (6-month mean hba1c 6.5 vs 6.1, stimulated c-peptide 2.3 vs 6.0), and improved islet survival; all ia eventually failed and only 1/8 were insulin free for >2-years, whereas only 3/7 iak grafts have failed with 4 exhibiting continued robust function at >38,>38,>46, and >58 months with 3/4 fully insulin independent. in addition, all ia patients demonstrated immune sensitization post graft failure versus 0/8 iak. all 9 ia developed mouth ulcers versus only 1/8 iak. conclusions: ia and iak exhibit striking differences in outcome and complications. the absence of mouth ulcers and lack of sensitization in iak may relate to steroid use and continued immunosuppression for the kidney graft, respectively. the superior outcome of the iak cohort may be a result of differences between the two groups including the use of maintenance steroids, prior exposure to thymo or the chronically immunosuppressed state of the iak recipient. perhaps the most interesting correlation with outcome is the absence of the anti-proliferative agent sirolimus in the iak group. our results provide clues to the cause of chronic islet transplant failure and may lead to novel approaches to avoid it. islet background: although islet transplantation has become an option for treatment of type 1 diabetes, all currently used immunosuppressive protocols have significant renal and islet toxicity. we describe a novel immunosuppressive protocol using sirolimus and the anti-lfa 1 antibody efalizumab that permits prolonged islet allograft survival without the need for steroids or calcineurin inhibitors (ci). methods: between february and august 2007, 4 consecutive type 1 diabetic patients with hypoglycemic unawareness and normal renal function received allogeneic pancreatic islet transplants. induction immunosuppression consisted of 2 doses of thymoglobulin given on pre-transplant days -2 and -1, efalizumab (5mg/kg sq/week starting on d -1), and sirolimus. maintenance immunosuppression consisted of sirolimus and efalizumab. results: all patients achieved insulin independence after single islet infusions (mean ieq/kg=8,905). three of 4 remain insulin independent 4 or more months after transplant (table 1) . patient 1 resumed low dose insulin (approximately 15% of original dose) 6 weeks after transplant and is awaiting a second islet infusion. her blood glucose control is markedly improved and she has not experienced any hypoglycemic episodes. all patients show persistent c-peptide secretion and have stable renal function. side effects due to efalizumab were limited to transient irritation at the injection site. conclusions: thymoglobulin induction followed by sirolimus and efalizumab maintenance is well tolerated and allows prolonged islet allograft survival. this protocol is the first ci/steroid free islet regimen resulting in insulin independence with a single donor islet infusion. by eliminating ci, this protocol minimizes renal and islet toxicity and may thus improve long-term islet survival and function. long . clinical and metabolic profiles were assessed every 3 months for18 months. results: si-exn group was on this drug for median time of 171 days pre-si. both groups were similar except for duration from post-completion to graft dysfunction (648±34 vs 221±58 in si-exn and si-c group respectively, p<0.05) and duration of graft dysfunction before si (664±83 vs 326±93, p=0.03). si-c and si-exn groups received mean of 8713±2123 and 5613±485 ieq/kg, respectively (ns). only 3/5 of si-c patients achieved insulin independence for 303, 403 and >1670 days after si. all subjects in si-exn group achieved insulin independence for more than 443, 621, 641, 654 days. at 18 months insulin independence was 20% in si-c and 100% in si-exn group. comparing pre and post-si, si-exn group had significantly lower a1c at 3-18 months and lower auc glucagon at 6 and 15 months (p<0.05). auc c-peptide in si-exn group was significantly higher than si-c at 3 and 9 months. intravenous glucose tolerance test showed significantly increased acute insulin responses to glucose at 3-18 months in si-exn and at 3 and 6 months in si-c group. si-exn group had more acute c-peptide response to glucose than si-c at 3 and 6 month (p<0.05). acute exenatide administration during intravenous glucose tolerance test at 15 month in si-exn revealed significantly increased acute insulin and c-peptide response to glucose which indicates improved first phase insulin release. conclusion: supplemental islet infusions under exenatide lead to insulin independence, restore first phase insulin secretion and result in long term insulin independence. exenatide this prospective phase 1/2 trial aimed to demonstrate safety and reproducibility of allogeneic islet transplantation (tx) in type 1 diabetic (t1dm) patients and implement a strategy to achieve and maintain insulin-independence with minimal islets. ten c-peptide negative t1dm subjects with hypoglycemic unawareness received 1-3 intraportal allogeneic islet tx. four subjects (group 1) received the edmonton immunosuppression regimen (daclizumab, sirolimus, tacrolimus). the next 6 subjects (group 2) received etanercept, exenatide and the edmonton regimen. we followed all subjects for 15 months after the first tx. the primary efficacy end point was insulin independence. secondary endpoints were hba1c, fructosamine, ogtt, mixed meal test, glucagon stimulation test, ivgtt and hypoglycemia. to study the effect of exenatide, we compared frequently sampled ivgtt, c-peptide, proinsulin, amylin and glucagon with and without exenatide. two self-limiting bleeds occurred in 18 infusions. all subjects became insulin independent. group 1 received a mean total number of islets (ein) of 1,460,080±418,330 in 2 (n=2) or 3 (n=2) tx, whereas group 2 became insulin independent after 1 tx (537,495±190,968 ein, p=0.028). all group 1 subjects remained insulin free through the 15-month follow-up. two group 2 subjects resumed insulin: one after immunosuppression reduction during an infectious complication, the other with severe gastroparesis and exenatide intolerance. hba1c reached normal range in both groups (6.5±0.6 at baseline to 5.6±0.5 after 2-3 tx in group 1 vs. 7.8±1.1 to 5.8±0.3 after 1 tx in group 2). baseline hba1c was significantly higher in group 2 than group 1 (p=0.046). pre-and post-tx hypo scores were 841.8±1217.9 and 0 in group 1 vs 812.8±941.7 and 33.5±50.0 in group 2. glucagon levels decreased significantly in all subjects. in group 2, the decrease in glucagon levels after challenge tests with and without exenatide was 18.7 fold more significant after exenatide in all subjects ( background istx has been investigated as treatment for type 1 dm. however, the hope this approach would result in long-term freedom from exogenous insulin has failed in practice. techniques for isolating islets have advanced and with availability of new is agents, strategies can now be developed specifically for istx that will provide greater immunologic protection w/o diabetogenic side effects. rejection/ vascularization still remain major limitations for success of istx. we hypothesize that bm is an accessible, immunologic privileged space with natural well-developed vasculature and may be a suitable site for istx. method wistar rats were used as donors/ recipients. dm was induced by iv-streptozocin. rats who had morning glc levels > 300 mg/dl on two separate occasions were used as recipients. ptx was performed as normal rodent standard. islets were isolated from pancreas by distending pancreatic duct with liberase. islets were separated on a discontinuous histopaque density gradient, further purified, counted, divided into aliquots transplanted into different sites (liver, bm). bw, glc and c-pep were measured in all groups before/after tx. background: in february 2007, the islet community was notified of a possible bovine product contamination in the collagenase enzyme (liberase hi) used for human islet isolations. to eliminate the potential hazard of bovine spongiform encephalopathy, we successfully adapted our human islet processing procedure to utilize a different gmp collagenase and neutral protease (nordmark/serva). here we describe what we consider the most important factors for achieving reproducible and clinically useable islet isolations. methods/results: a standard isolation protocol involving controlled enzymatic digestion followed by density gradient purification was used. seventeen donor pancreata were processed and ten were ultimately used for clinical transplantation. eight of these successful isolations were performed using the nordmark/serva enzymes (table 1) . the following factors were identified as being important for ensuring successful islet yields: 1) donor age and size: male donors 17-45 years old who were tall (>180cm) and heavy (>100kg) conclusions: incorporation of several important modifications into our existing islet isolation protocol has allowed us to routinely obtain high quality islet isolations using an alternative, bovine product-free gmp enzyme. (n=187)], and in 121 pts immuno was cni-free. tac-treated pts were younger, more sensitized and more frequently re-kt. dgf was more frequent in pts receiving cni-free immuno. as a group, these pts were older, more frequently received a kt from a donor after cardiac death and less frequently from a living donor. acute cellular rejection (acr) was diagnosed in 41 pts (6.8%), 13 occurred before day 90th (e-acr), and 28 after this date (l-acr). c4d-positive amr was diagnosed in 128 pts (21.2%), of which 58 were early (e-amr) and 70 were late episodes (l-amr rates of acute rejection (ar) and background: an increasing number of hs patients are being transplanted using desensitization protocols. these patients are considered high risk for ar, particularly antibody mediated rejection (amr). here we examined our ar rates and treatment outcomes for our hs kidney transplant (kt) recipients using our most current desensitization strategies. methods: between june 2004 (when rituximab was introduced to our desensitization and treatment protocols) to july 2007, 130 hs kt patients were transplanted using combinations of high-dose ivig, rituximab, and plasmapheresis (pp) for desensitization. we examined the overall ar, c4d-cell-mediated (cmr), and c4d+amr rates. amr episodes were treated with steroids, high-dose ivig, and rituximab. refractory and rapidly progressive amr was treated with pp. treatment outcomes and differences in ar rates between deceased (dd) and living donors (ld) was examined. recent reports suggest that treatment with the monoclonal anti-cd20 antibody, rituximab (rtx) may improve renal graft survival in antibody mediated rejection (amr); however optimal dosing for this indication is unknown. we examined the efficacy of a single low dose (500mg) of rtx for treatment of refractory amr in order to limit significant infective complications associated with conventional dosing regimens (375mg/m 2 ). rtx was used in seven consecutive patients who had refractory amr as judged by ongoing biopsy evidence of amr after 4 weeks of standard therapy consisting of pulse methylprednisolone and plasma exchange with low dose ivig (100mg/kg) (pe/ivig). all patients received tacrolimus and mycophenolate mofetil. amr was defined as 1) characteristic histology on biopsy (banff criteria), 2) graft dysfunction and 3) presence of a donor specific anti-hla antibody (dsab). b-cell counts (cd19) and serum creatinine (scr) were monitored. pe/ivig was ceased in all cases after rtx dosing. the average follow-up since rtx dosing is 16.8 months (range 5.3-28.9 mths). all patients still have functioning grafts (100% 12 month graft and patient survival), with current mean scr levels (156±17µmol/l) significantly lower than mean peak rejection levels (514±355µmol/l) p=0.05. cd19 counts fell to zero and remained <5x10 6 /l for >6 months in all patients. dsabs remain detectable by luminex flow beads in all patients despite stabilization of scr. two of 7 patients developed an infective complication post rtx dosing. one patient developed bacterial pneumonia requiring hospital admission whilst a second patient developed cmv viraemia and later bk nephropathy which has not led to significant graft dysfunction. hence, infectious complications are far less than those reported for multiple standard dose regimens in similar patient groups. large clinical trials are required to confirm the efficacy of rtx in treating amr as well as optimal dosing. standard dosing is based on oncology treatment regimens which are likely to be excessive for amr in patients already markedly immunosuppressed. our data suggests single low-dose rtx for refractory amr results in excellent patient and graft survival and leads to low rates of serious infective complications in the short-term. renal the demographic and clinical data were similar between +cxm and negative cxm groups. the rejection rate was significantly higher and the length of stay was significantly longer in +cxm group as shown in the table. the ra survival rates were 8%, 7% and 6% lower at 1, 3 and 5 years post transplant respectively among + cxm recipients. however, this did not reach significance mostly due to small sample size (p=0.11). the inferior ra outcome is seen during the initial few years after transplantation that disappears after 9 years. in clkt, pre-transplant +cxm can result in higher ra rejection rate and longer length of stay in hospital. la may not always confer immunological protection to ra in + cxm clkt and its true burden may be under recognized since cxm is not routinely performed in all clkt. study of antibody specificities or la volume to determine the effect of +cxm on ra outcome is essential. skpt in patients with positive cdc b-cell and/or flow cytometry crossmatch is associated with high amr rate despite low dose ivig and r-atg/alemtuzumab induction. pancreas graft survival is inferior in patients with positive crossmatch while kidney graft and patient survival are similar to that of negative crossmatch recipients. majority of amr can be reversed with treatment but further long-term follow-up is needed to determine the impact on graft survival. ten the first european phase iii trial with tacrolimus in the early 90´s clearly showed advantages for tacrolimus in terms of acute rejection (ar) vs the original cyclosporin formulation. however, no advantages were seen in survival rates. the present investigator-initiated, observational follow-up study collected data on patients included in the original cohort of 448 patients who participated in this large study and who were randomised to a triple immunosuppressive regimen consisting of tacrolimus, azathioprine and steroids (tac/aza/ster, n= 303) or cyclosporine, aza and steroids (csa/aza/ster, n=145). efficacy and safety parameters assessed at follow-up included: acute rejection; patient and graft survival; renal function, vital signs, basic lab results and immunosuppressive regimen for the patients 10 years after completion of the original study. results: currently, data are available from 75% of the patients. all assessments were conducted following the intent-to-treat principles. more patients in the tac than in the csa group remained on the randomised treatment. while kaplan-meier (k-m) estimates for patient survival at year 11 show comparable (72% tac vs 72%csa) results, k-m estimates for graft survival demonstrate an advantage for the tacrolimus cohort (51% tac vs 46% csa). graft half-life estimates (gjertson and terasaki method) yielded overall results of 14.2 years (tac) and 11.4 years (cs). in both treatment groups, ar was associated with inferior long-term results. for patients with ar, half-life estimates were 11.1 and 7.8 years for tac and csa, respectively, while in patients without ar half-lives were 19.2 (tac) and 15.8 (cs) years. mean serum creatinine after 10 years was significantly lower in the tac cohort vs the csa group (1.65mg/dl tac vs 2.01mg/ dl csa, p<0.05). mean glomerular filtration rates (mdrd4 estimate) were 48.7 ml/ min in tac and 41.9 ml/min in cs. after ten years of follow-up, the mean (c-0) levels were 7.6 mg/ml (tac) and 120 mg/ml (cs). conclusion: analysis of this long term follow-up of a large study confirms the benefits of a tacrolimus-based therapy. it also confirms the concept that freedom from early acute rejection is associated with superior long term results in renal fuction. therefore, long term renal allograft function is remarkably good in tac patients. cyclophosphamide a novel therapy for ivig/plex-resistant antibody-mediated rejection. (c)). moreover subgroup analyses within the different groups were made to identify potential differences. groups were analyzed for survival, graft rejection and graftvasculopathy (cad). kaplan-meier analysis was used and log-rank test was performed to detect differences. results: a total of 81 transplants (8.9%) were blood group compatible. the majority (n=32) were 0a transplants (40%) followed by 0b (n=19; 23%), aab (n=17; 21%), bab (n=7; 9%) and 0ab (n=6; 7%). overall survival comparison showed no significant difference in long-term survival (10-year) recent data has shown the utility of urinary biomarkers to predict delayed graft function early following renal transplantation, but their ability to predict longer-term allograft function is less clear. we evaluated whether urinary biomarkers measured in the early post-transplant period would predict allograft function at 6 and 12 months after renal transplantation. urinary biomarkers including n-acetyl-β-d-glucosaminidase (nag), kidney injury molecule-1 (kim-1) and matrix metalloproteinase-9 (mmp-9) were measured in 20 patients at hours 0, 6, 12, 24 and days 2 and 3 following renal transplantation. glomerular filtration rate (gfr) at 6 and 12 months were calculated using the mdrd equation. all 20 patients had functional allografts at 6 months and 2 patients lost allografts after 6 months. levels of individual biomarkers at different post-transplant time points were correlated with 6 and 12 month gfr using spearman's correlation (rho) and are shown in the urinary nag levels at post-transplant hours 0, 6, 24 and days 2 and 3 showed significant negative correlation with 6-month gfr. nag levels at hour 24 and days 2 and 3 maintained the significant negative correlation with 12-month gfr. urinary kim-1 levels at the 24 hour point showed significant negative correlation with 6-month gfr and kim-1 levels at hour 24 and day 2 displayed significant negative correlation with 12-month gfr. urinary mmp-9 levels at no time points had significant correlation with either 6-month or 12-month gfr. urinary biomarkers particularly nag shows promise as a tool in the early prediction of longer-term allograft function following renal transplantation. in kidney disease chemokines are involved in the recruitment of leukocytes causing kidney damage and progression to endstage renal failure. similar mechanisms are proposed for chronic allograft failure in renal transplant recipients. the chemokine attracted leukocytes produce proinflammatory and profibrotic cytokines contributing to fibroblast proliferation, matrix production and tubular atrophy. the role of chemokines in the acute post-ischemic tubular necrosis early after renal transplantation and their impact on long term allograft outcome has not been investigated yet. methods 30 patients (23m, 7f, mean age 49.9yrs) with dgf (with a median number of 6.4 dialysis sessions) developed biopsy proven acute tubular necrosis during the first three weeks after transplantation. the biopsies were studied by immunostaining for ki67, ccr1, ccr2, rantes, mcp-1, cd20 and cd68, respectively. the patients were followed for a mean time of 4.2yrs. none of them developed a rejection episode in the follow up time. after follow up the mean serum creatinine was 2.4mg/dl (0.8-6. the goal of a tx is for the recip to achieve long-term survival (surv.), with continued graft function, equivalent to the gen. population. we studied subsequent outcome in 2202, 10-yr kidney tx survivors (tx 1963-1997) ; 62% living donor (ld); 87% 1 st tx; 41% female; 28% type 1 diabetes; mean age at tx (±se), 33±15 yrs. actuarial 25-yr surv. is shown in table 1 ; ld patient, graft, and death-censored graft surv. is signif. ↑vs. deceased donor (p<.0001). mean creatinine (± se) at 10 yrs was 1.7±.8; 15 yrs, 1.7±.9; 20 yrs, 1.5±1; 25 yrs, 1.6±1. the 2 major causes of late graft loss (gl) were death with function (dwf) and chronic rejection (can). by multivariate analysis, risk factors for gl after 10 yrs were age ≥50 yrs at tx; type 1 diabetes, retx, hla mm ≥3; ≥1 acute rejection episode, and pretx cardiac, peripheral vascular, or liver disease (p<.05). the most common cause of dwf was cardiovascular disease (cvd); however, >20% deaths were due to malignancy. the use of kidneys from donors with a positive serology for hcv into hcv(+) recipients still remains controversial. in 1990, our units adopted this policy, subsequently modified in 1993, so these kidneys were limited to recipients with a positive rna hcv before transplantation. the aim of the present analysis was to review the long-term safety of our policy. since january 1990 to february 2007, 474 hcv(+) patients with a negative hbsag and not treated with interferon received a kidney transplant in our units. 313 patients were transplanted from an hcv(-) donor(group 1) and 161 from an hcv(+) donor(group 2). median follow-up time was 65(ir 24-117) months. remarkably, group 2 showed a significantly higher donor age (46.6±13.6 versus 41.1±18.8 years;p<0.0001) and recipient age (50.4±13.1 versus 44.9±14.6 years;p<0.0001), as well as a worse hla compatibiliy than group 1. immunesuppressive therapy did not significantly differ between the two groups. group 2 notably, only 5 patients died because of a liver disease (3 in group 1 versus 2 in group 2) and only 2 patients developed a hepatocarcinoma (both in group 1). in summary, no significant differences were observed in hcv(+) recipients according to hcv serology of the donor, in terms of death censored graf survival, patient survival and evolution of liver disease. therefore, our experience clearly demonstrates that the use of kidneys from hcv(+) donors into hcv(+) recipients is a safe strategy in the long-term and a wise way of using these kidneys, that otherwise would be lost at a moment of shortage. introduction: deceased donor kidneys are allocated to adult candidates in the u.s. primarily by waiting time and hla similarity. we investigated two alternative allocation systems, lyft-scd and lyft-dy, that prioritize offers of kidneys to adults based in part on lyft. methods: based on characteristics of each candidate and donor, lyft was calculated as the difference between expected median years of life for the candidate with that kidney donor transplant (tx) versus without a kidney. years on dialysis were weighted at 0.8 of years with a functioning graft. the relative risk of graft failure for each donor was calculated from donor factors using a cox model and the donor profile index (dpi), the percentile score of that risk among kidneys used in 2003 (100%=greatest risk of graft failure). using actual candidates and deceased donors and acceptance patterns from 2003, we modeled allocation with the kidney-pancreas simulated allocation model (kpsam). kpsam sequentially offers kidneys to candidates according to user-specified allocation rules, and models the probability for kidney placement and post-tx survival. the lyft-scd model allocated scd kidneys by lyft and ecd kidneys by dialysis years (dy). the lyft-dy model allocated organs according to the formula [.8lyft(1-dpi)]+[dy(.8dpi+.2)]+[4pra/100] to adult kidney tx candidates. results: the table compares a year's worth of kidney-alone recipient characteristics, lifespan after tx and additional lifespan (v. dialysis) due to tx among all kidney (including spk) recipients, and the donor/recipient age correlation (r) under current national kidney allocation, allocation using lyft for scd, and allocation using lyft-dy within each donation service area. conclusions: allocation systems incorporating lyft have the potential to increase the number of years of life attainable from tx and are being considered for the allocation of kidneys within the u.s. introduction : organ shortage for transplantation has led to the use of marginal kidneys, which has been associated with poorer but still acceptable results. in our center, the transplantation of two very marginal kidneys into a single recipient was used as a strategy to increase the number of organs available. the present study reports renal function and survival of dual-kidney transplants (dkt) performed at our institution. methods : from october 1999 to june 2007, 392 transplants were performed at our center, from which 63 were dkt. kidney selection for a dkt was based on refusal of the kidneys for single kidney transplantation (skt), donor's age ≥ 60 years and ≥15% of glomerulosclerosis on pre-implantation biopsy. the calculated creatinine clearance (crcl, ml/min), graft and recipient survival from dkt were compared to those of skt from ecd (based on unos criteria, n=66) and ideal donors (id, aged 10 to 39 y, n= 63) over a seven-year period. results : dkt donors were significantly older than the two other groups (dkt: 69 year old, ecd: 62, id: 24). dual transplants were offered to older recipients by design (60, 50, 44). delayed graft function was more prevalent for dkt and ecd than with id (27%, 29%, 11%). twelve, 36 and 84-month crcl were similar for dkt and ecd but lower than id (twelve months: 58, 59, 81; 36 months: 54, 60, 82; 84 months: 62, 51, 86, respectively). patient survival, actuarial graft survival and actuarial graft survival censored for death were similar between the 3 groups. conclusion : to our knowledge, this study reports short and long-term outcome of the largest cohort of dkt performed at a single institution. it shows that dual-kidney transplantation is a more complex intervention with more risks of surgical and post-op complications (data not shown). however, with adequate surgical expertise, dkt patients can expect long-term results comparable or even better than ecd. in our center, the use of dkt has increased the number of transplantations by 19% for the period of study. it allowed older patients to receive a graft within shorter delays. moreover, it permitted a more judicious allocation of a resource that is still limited. an results: very few differences resulting from donornet were seen. for regionally exported kidneys, cold ischemia time (cit) was 22.9h before donornet (bd) and 23.3h after donornet (ad); 6.4% of regionally exported kidneys had cit>36h bd, as compared with 9.0% ad. for nationally exported kidneys, cit was 27.3h bd and 26.8h ad; 17.9% of nationally exported kidneys had cit>36h bd, as compared with 16.1% ad. the same hucs of exported kidneys bd were also hucs ad. of the top 10 centers utilizing exported kidneys bd, only one was not within the top 10 centers utilizing exported kidneys ad. when we looked at cit for kidneys exported to hucs, again there was little difference before or after donornet, with a trend to possibly longer cit for hucs. for kidneys regionally exported to hucs, cold ischemia time (cit) was 23.1h before donornet (bd) and 23.4h after donornet (ad); 7% of regionally exported kidneys had cit>36h bd, as compared with 10.4% ad. for kidneys nationally exported to hucs, cit was 27.2h bd and 27.4h ad; 17.5% of nationally exported kidneys had cit>36h bd, as compared with 16.8% ad. conclusion: the first 6 months of national implementation of donornet were not successful in decreasing cit for regionally or nationally exported kidneys. furthermore, the centers to which a high proportion of these kidneys were exported did not change as a result of donornet, and for these centers allocation efficiency is no better and might even be worse than before. machine cold machine preservation has been shown to improve outcome following transplantation of deceased heart-beating donor kidneys. to determine whether cold machine preservation also improves outcome of kidneys donated after cardiac death (dcd) we undertook a multicentre randomized controlled trial in the uk comparing machine perfusion with simple cold storage. one kidney from each dcd donor was randomized to machine perfusion (organ recovery systems lifeport device with kps-1 solution), the other to perfusion with uw (viaspan) solution followed by simple cold storage. the primary outcome measure was the need for dialysis in the first 7 days (delayed graft function, dgf); secondary outcome measures included gfr at 1 week (mdrd technique); 3 and 12 month graft and patient survival. at the time of writing, one week outcome data are available for all patients. a sequential design was used with the data inspected after 60 transplants and then every 20. all recipients received basiliximab, mycophenolate sodium, tacrolimus and prednisolone. the significance of the initial results has been tested using paired t tests and mcnemar's exact tests. recruitment was stopped after 46 donors when the unadjusted sequential analysis concluded that there was no difference in the incidence of dgf. 91 of the 92 kidneys were transplanted; one was not used for anatomical reasons. one kidney suffered primary non function; it had been machine preserved. the results shown below represent intention to treat. introduction: split liver transplantation has been adopted as an alternative to expand the organ donor pool. while the adult/child split liver (a/csl) in which an extended right graft (erg) is transplanted into an adult and a left lateral segment (lls), transplanted into a child, has gained a wide acceptance within the transplantation community, adult/ adult split liver (a/asl) in which the liver is split into a full right (fr) and full left (fl) is still performed very rarely. we analyzed the experience at our center with split liver grafts over a period of 10 years. material and methods: between october 1997 and october 2007 we performed a total of 676 liver transplants in 625 recipients (370/328 children and 306/297 adults). among the 593 (310 children and 283 adults) recipients of a primary isolated liver transplant, 281 (247 children and 34 adult) were transplanted with a a/c or a/a sl. the recipients of a whole size graft (249 adults and 63 children) were used as a control. results: adults: 34 patients received a split liver graft (26 erg graft and 8 fl/fr grafts) and 249 a whole liver graft. overall the incidence of biliary complications using a split liver graft was 29% and with a whole liver graft 15% (p= 0,0506).for the recipients of a split liver graft 1 and 5 years patient and graft survival was 91%/88% and 88%/85%. recipients of a whole liver graft had a 1 and 5 years patient / graft survival of 85%/84% and 80%/78% respectively. children: 247 children received a split graft (230 lls, 12 erg and 5 fl) and 63 a whole size graft. biliary complications occurred in 42% of the recipients of a split liver graft and 5% of the recipients of a whole size graft (p < 0,0001). among the recipients of a split graft 1 and 5 years patient / graft survival was 91%/84% and 87%/80% respectively. the recipient of a whole size graft had a 1 and 5 years patient / graft survival of 84%/78% and 83%/74%. conclusion: incidence of biliary complications is significantly higher using a split graft. at a high volume center, use of split liver grafts reached comparable and even better results of a whole liver graft in terms of patient and graft survival. thus, the use of split grafts should be strongly encouraged to expand the donor pool. a groups of primary ldlt and ddlt recipients were identified by matching diagnosis, meld score, and recipient age. cost data, acquired from the hospital financial database and inflation adjusted and clinical outcomes were compared between the 2 groups. background: liver transplant recipients with (gw/rw) < 0.8% are thought to have a higher incidence of post-operative complications, including small for size syndrome (sfss), and overall inferior outcomes. we analyzed a cohort of partial liver graft recipients and compared those with gw/rw < 0.8% to those with gw/rw > 0.8%. results: between 1999 and 2007, 107 adult patients underwent partial graft liver transplant. seventy-six grafts were from live donors (ldlt) and the remaining 31 were from deceased donor split-liver transplants (slt). of these, 22 patients had gw/ rw < 0.8% (12 ldlt, 10 slt), and 85 had gw/rw > 0.8% (64 ldlt, 21 slt). median follow-up was 46.1 months and did not differ between the two groups. baseline demographics including donor and recipient age, and meld at the time of transplant also did not differ between groups (table) . three-month and 1-year graft survival for the two groups was comparable. three recipients with gw/rw < 0.8% developed sfss (13.6%); all three had inflow modification with splenic artery ligation (sal), and 1 of the 3 grafts was lost at one-year. eight recipients with gw/rw > 0.8% developed sfss (9.4%) (p = ns); six underwent sal and none had graft loss at one-year. there was a trend toward increased hepatic artery thrombosis with the smaller grafts that did not reach statistical significance (p = 0.10). the incidence of other surgical complications was similar between the two groups (table) . the diagnosis of rejection in post-transplant kidney disease depends largely on the assessment of biopsy pathology, which suffers from arbitrary scoring, subjectivity, and sampling variance. with microarray gene expression data from 186 biopsies for cause, we used predictive analysis of microarrays (pam) to build a rejection classifier. because of the imperfect gold standard, the classifier cannot and should not have extremely high predictive accuracy. the goal was to see if microarrays could detect a robust rejection signature despite this uncertainty, and to identify the top genes distinguishing rejecting from non-rejecting biopsies. by examining discrepancies between pathology diagnoses and pam predictions, and using clinical follow-up information, we combine the strengths of each method to produce a more accurate diagnostic system. biopsies with rejection plus clinical episodes (fig. 1 ) have higher positive predictive value than those lacking episodes. the majority of episodes occur in the top part of fig.1 , indicating that a valid rejection signature is being detected. of the exceptions, most had been treated with steroids before the biopsy, suppressing their gene expression patterns. samples called borderline rejection separated into two distinct high/low probability groups using pam, with all the cases classified as clinical rejection episodes occurring in the high probability group. of the 39 genes chosen by the classifier, 36 had previously been annotated in our studies of mouse kidney graft rejection. gzma, gzmb, and prf1 ranked 26th, 32nd, and 38th respectively. the top five genes were: cxcl11, gbp1, cxcl9, indo, and cxcl10, all previously annotated in rejecting mouse kidneys as interferon-γ induced genes. in summary, gene expression-based classifiers, combined with histopathology, promise more accurate diagnostic assessment of the state of kidney transplants at the time of biopsy. moreover our data driven classifier independently identifies the genes previously annotated in experimental studies. influence of donor background: renal-cell associated tlr4 activation was found to be important in mediating ischemia reperfusion injury to murine kidneys. we hypothesized that genetic variations within the tlr4 gene of the kidney donor affects the rate of delayed graft function (dgf). methods: we genotyped the functional tlr4 polymorphisms (snps) d299g (rs4986790) and t399i (rs4986791) in 268 kidney donors from 2 centers and correlated them with the occurrence of dgf. dgf was defined as the need of dialysis within 7 days post-transplant or less than 25% drop in creatinine within the first 24 hours after transplant. in a sample of 67 patients from one center, we analysed intragraft hmgb-1 (high mobility group box protein-1) gene expression in pre-and post-implantation biopsies. statistical analyses were performed using the spss statistical package. results: both tlr4 snps were in hardy-weinberg equilibrium, and were combined for further analysis. recipients of a tlr4 mutated kidney showed a significant lower rate of dgf (p=0.005), which was persistent after correction for known donor-derived risk factors (donor age, ecd vs. dcd-kidney, cold ischemia time, p=0.007, hr 4.42, cl 1.51-12.94). additionally, we confirmed the presence of a possible tlr4 ligand, hmgb-1, in pre-and post-implantation biopsies. we also confirmed the presence of a functional tlr4 receptor in human proximal tubular cells which expressed mcp-1, il1-β, il-6 and tnf-α after specific tlr4 agonist treatment. conclusion: human renal tubular epithelial cells express tlr4 and a functional tlr4 snp in donor kidney is associated with lower rate of delayed graft function. background: methylation of promoter cpg islands has been associated with gene silencing and demonstrated to lead to chromosomal instability. we postulate that differences in methylation patterns observed in tissues ranging from normal to cirrhosis to hcc may lead to the discovery of direct precipitating events that contribute to tumorigenesis in hcv-hcc patients. methods: dna from 20 hcv-hcc tumors and corresponding non-tumor hcv cirrhotic tissues as well as 20 independent hcv cirrhotic tissues and 20 normal liver tissues were bisulfite treated and hybridized to the illumina goldengate methylation beadarray cancer panel i for interrogating cpg sites in the promoter regions of 808 distinct genes. for each cpg site, a summary statistic representing "percent methylated" was estimated. for each cpg site, a jonckheere-terpstra test was applied to identify whether there was a significant monotonic trend in percent methylated across the independent normal, cirrhotic, and hcc tissues. in addition, the paired hcc and non-tumor cirrhotic tissues were analyzed using a paired t-test. the q-value method for estimating gene-wise false discovery rates (fdr) was used to adjust for multiple comparisons; cpg sites with an fdr<0.05 were considered significant. to identify if serological responses to allogenic non-hla renal compartmentspecific antigens can be detected after renal transplantation (txp). methods: 36 paired pre-and post-transplant (mean time 25 months) serum samples from 18 pediatric kidney allograft recipients were used for identification of de novo non-hla antibody formation assessed using invitrogen protoarray (v3), measuring signal post-pre. probes from the protoarray and cdna microarray platforms were re-annotated to current ncbi entrez gene identifiers using ailun. 34 cdna arrays (gse:3931) from 7 normal kidney regions (inner and outer cortex, inner and outer medulla, papillary tips, renal pelvis and glomeruli; see table) were analyzed for compartment-specific genes by sam, and the highest-ranked genes in each compartment (ks two-sample test p < 0.001) and were ranked against each individual patient's numerical antibody response across the 5057 proteins on the protoarray. results: in 83% of patients (15/18), kidney-specific serological responses against the renal pelvis were the first to be detected, followed by responses against renal cortex. control gene expression data sets from other solid organs (gse: 1133; lung, heart, pancreas) did not demonstrate immune-response enrichment. the presence of these antibodies was not associated with donor specific hla class i or ii antibody levels. conclusion: we demonstrated de novo formation of circulating non-hla non-abo antibodies after txp, irrespective of hla antibodies, are detected against kidneyspecific protein targets. as these antibodies are not seen against other solid organs, the response is likely allogeneic. the renal pelvis and renal cortex have the highest immunogenic potential. we conclude that urinary cell mrna profiles that include levels of mrna encoding tubular proteins and mrna encoding proteins implicated in emt offers a noninvasive means ascertaining renal allograft status with respect to presence or absence of can. functional functional annotation of these genes identified cell cycle pathway and carboxylic acid metabolism pathway as potentially relevant for both datasets. altogether, these data suggest that as compared with recipients requiring on-going immunosuppression, operationally tolerant liver recipients exhibit traits of immunological quiescence and activation of natural killer related pathways. liver and kidney tolerant states appear to be fundamentally different biological processes, although some common pathways could be relevant to both settings. genetic introduction: both normal and fibrotic renal allografts develop a large number of persistent changes in pro-inflammatory gene transcripts early after transplantation (1) . the aim of the current study was to determine if this "transplant effect" is attenuated by immunosuppression, fibrosis or hla-match. methods: we studied 41 histologically normal implantation biopsies (t0) and 45 12m protocol biopsies (t12) from living donor kidney transplant recipients who had no identifiable post-transplant complication (no acute rejection, polyoma virus, etc). patients were stratified into 4 distinct clinicopathologic groups. the first three groups had normal histology at both t0 and t12 and included: 1) hla identical-tac treated (n=10); 2) non-hla identical-tac (n=19); 3) non-hla identical srl (tac-free, n=8). a fourth group was histologically normal at t0 but developed mild fibrosis by t12 (n=7). mrna expression from these biopsies was measured using the u133plus2.0 microarray and custom taqman low density arrays (tlda). data for each group was tested using a generalized linear model and changes common to each dataset identified (p<0.05). results: in addition to transcript changes specific for each clinicopathologic condition, a large number of transcripts (n=1152) were altered in all groups. 68% (n=778) had higher expression in all comparisons at t12 versus t0, including fibronectin and collagen iv. 32% (n=374) of transcripts were considered down-regulated, including interleukin 6 and 1β. gene ontology and signaling pathway analyses showed many of the transcripts to be involved in pro-inflammatory and immune response signaling pathways (ex. tlr, il-10/-2, etc). custom tldas were used to study several genes considered not detected by microarray. this included tgf-β1, cd-3e/-28/-74 and vegf, all of which were significantly up-regulated in multiple comparisons. conclusions: persistent changes in pro-inflammatory transcripts occur in all renal transplants. this "transplant effect" cannot be explained by calcineurin-inhibitors (occurs in tac-free immunosuppression), increased alloreactivity (similar in hla identical/ non-identical) or fibrosis. a likely cause is persistent changes from ir injury. the role of the "transplant effect" in long-term allograft survival is unclear, but it likely interacts with other forms of graft injury. background: a critical 26 gene-set for acute renal rejection (ar) diagnosis in peripheral blood has been previously identified by our group using cross-platform hybridization of 91 unique peripherla blood samples with matched biopsy diagnosis, with ar (n=39) and without ar (sta; n=52), with cross-hybridization across 3 human microarray platforms: 30k cdna lymphochip, 44k agilent and 54k affymetrix hu133plus. in this study we proposed to verify and validate this gene-set for ar diagnosis and prediction by peripheral blood pcr-based analysis. method: 57/91 peripheral blood samples from the microarray gene-set were selected for 384-well format multiplex abi-taqman qpcrv validation of ar diagnosis across the 26 gene-set. an independant set of 66 new, time and immunosuppression matched, peripheral blood samples (30sta, 36ar) were used as a test-set for blinded prediction of ar diagnosis using qpcr across the most informative 8 genes. 42 sequential samples from 14 ar patients (at ar, 1-3 months prior to, and after ar) were also examined by qpcr for these 8 genes for blinded ar prediction, prior to biopsy proven ar. prediction models were generated using logistic regression analysis and p<0.05 considered significant. result: in the validation set of 57 samples, 8/26 genes were highly significant for confirming peripheral blood-ar diagnosis (p<0.03). in the test group of 66 samples, ar was predicted with a very high level of sensitivity and specificity (ppv and npv> 90%). in the group of 14 ar patients with sequential samples pre-and post-ar, expression for these 8 genes were significantly elevated in the pre-ar samples (vs. sta, p<0.01) but were not different between pre-ar and ar values. conclusion: a highly specific and sensitive biomarker panel of 8 genes has been derived and tested by cross-platform microarray analysis. thsi gene-set has now been validated and further verified by multiplex pcr, for ar diagnosis and prediction, even 3 months prior to the rejection event. utility of this gene-set should be further validated in real-time by serial longitudinal peripheral blood testing, for more senstive and specific minimally invasive monitoring for graft rejection. tolerance/immune deviation ii background: our previous studies showed that regulatory t cells (treg) execute suppressive function in both the inflammatory site of the allograft and the draining lymph node (dln) to suppress allograft rejection. we explored how treg influenced the migration and function of antigen specific effector t cells and dendritic cells (dc) at these two sites in an islet transplant model. methods: treg were sorted from wild type or ccr7 -/-c57bl/6 mice, labeled with pkh26, and transferred directly to islet allografts (balb/c into c57bl/6). effector cd4 t cells (te) from alloantigen specific t cell receptor transgenic mice were labeled with csfe and transferred intravenously. treg and te migration to islet grafts and dln were analyzed with flow cytometry and immunohistochemistry. chemokine expression was determined by rt-pcr. cx3cr1 gfp mice, in which dc are internally labeled with gfp, served as islet donors, and donor dc migration was tracked with fluorescence microscopy. results: during priming and rejection, islet allografts expressed a panel of inflammatory chemokines shortly after transplantation that recruited te to the islets. te accumulated and proliferated in both the islet allograft and the dln. concomitantly, donor derived dc migrated from the islet to the dln as early as 1 day after transplantation. local transfer of treg into the islet allograft inhibited islet chemokine expression, inhibited donor dc migration in an il-10 and tgfb dependent fashion, and reduced te migration to and proliferation in the graft. the transferred treg also migrated from the islet allograft to the dln, and directly inhibited te accumulation and proliferation in the dln, and migration of te to the islet. ccr7 -/-treg, which could not migrate to the dln but were retained within the graft, and were far less effective than wild type treg in inhibiting te migration and proliferation into both the islets and dln. conclusions: treg migrate to the islet and suppress parenchymal cell chemokine expression, dc migration, and te responses in the islet allograft. treg also migrate to the dln to suppress te proliferation and migration. treg trafficking from the allograft to the dln is crucial for suppressive function in order to target the separate effector mechanisms. these results demonstrate novel and important functions for migration in treg induced suppression and graft survival. tregs cd4+foxp3+ regulatory t cells (tregs) play an important role in transplant tolerance, yet basic aspects of treg biology including the mechanisms involved in their induction remain unclear. α-cd45rb is a potent tolerogenic agent that induces a 2x increase in number of tregs in wt mice, even in the absence of allo-antigen. here we use foxp3red fluorescent protein reporter knock-in mice to study treg homeostasis and identify the mechanisms by which α-cd45rb induces tregs. cfse-stained highly sort-abstracts purified foxp3+ or foxp3-cells were adoptively transferred into fully replete naive wt congenic mice. whereas only 5-10% of transferred foxp3-cells undergo homeostatic proliferation (hp) over 10d, 50-70% of foxp3+ cells proliferate -a surprisingly high rate of hp. moreover, treatment with α-cd45rb markedly enhanced hp by transferred foxp3+ cells, resulting in a 10x increase in cell number. α-cd45rb induced de novo foxp3 expression in 13-25% of transferred foxp3-cells (many of which subsequently underwent hp). thus, α-cd45rb induces both conversion of foxp3-to foxp3+ cells and promotes hp in foxp3+ cells in the absence of exogenous antigen. we then addressed the signals controlling basal hp of tregs and both hp and conversion mediated by α-cd45rb. cfse-stained congenic cd4+ cells adoptively transferred into naive wt mice were untreated, or received csa, α-cd45rb, or both and foxp3+ and foxp3-cd4 cells were assessed on d10. calcineurin inhibition greatly reduced basal hp of transferred foxp3+ cells compared to untreated mice. moreover, csa completely abrogated increased treg hp induced by α-cd45rb, although conversion still occurred. next we assessed the role of tgfβ. we found that blocking tgfβ signaling with α-tgfβ shortened allograft survival, but had no effect on basal treg hp, or on treg hp or conversion by α-cd45rb. finally, although exogenous antigen is not required, basal and α-cd45rb-induced hp may still require recognition of self-ag bytregs. indeed, preliminary results reveal that when cfse-labeled cd4+ cells are transferred into mhcii ko mice, basal hp by tregs is reduced and not restored by α-cd45rb. thus, α-cd45rb alters normal controls regulating hp by treg. moreover, both basal and α-cd45rb-mediated hp requires intact calcineurin activity and tcr signaling, but not tgfβ. understanding the signals controlling hp in treg may reveal new therapeutic targets for tolerance induction. we report the first demonstration of a tolerance-inducing strategy based on posttransplant administration of allo-ag-specific treg (aastreg) and rapamycin (rapa), in a fully allogeneic, unmanipulated mouse heart transplant model. enrichment of naturally-occurring aastreg represented the first step to counterbalance the high frequency of alloreactive t cells. selection was achieved by co-incubation of freshly-isolated cd4 + cd25 + t cells with donor bone marrow-derived dendritic cells (dc). the source of stimulatory factors necessary to sustain treg proliferation was the supernatant of cd4 + cd25 -t cells co-cultured with allogeneic dc (mlrsup). use of mature (cd86 high ) dc favored extensive expansion of foxp3cells capable of il-17 production (t h 17). co-culture of treg with immature dc in the presence of mlrsup rendered a t cell population with regulatory phenotype: foxp3 + , gitr + , ctla-4 + , ccr7 + , cd62l -. these cells inhibited in vitro effector t cell proliferation at 1:20 and 1:40 treg:t cell ratios. the suppressive activity was ag-specific; no significant inhibition was evident when effector t cells were stimulated by third party dc. aastreg were then tested for their ability to induce transplant tolerance in a mouse heterotopic heart allograft model (balb/c to c57bl/10; unmanipulated). rapa was used: i) to inhibit effector t cell proliferation, while sparing treg activity and thus enhancing the in vivo treg:effector t cell ratio; ii) to promote resolution of the inflammatory state and preserve the susceptibility of conventional t cells to treg suppression. graft recipients received rapa (1mg/kg/d; d0-9) and 2x10 6 aastreg i.v. on d7. in comparison to the untreated group (median survival time, mst=11d; n=14), rapa alone extended mst to 30 d (n=7), with no long-term graft survival. under cover of rapa, aastreg exerted a profound tolerogenic effect: >80% recipients exhibited longterm graft survival (mst>150d; n=6). this effect was stronger than polyclonal treg administration: 40% long-term survivors (mst>50d; n=5). moreover, the tolerogenic effect was ag-specific, as aastreg selected against third party dc (c3h/hej) did not prolong graft survival in comparison to the rapa-only control group. these results indicate the feasibility and therapeutic potential of ag-specific tolerogenic cell therapy based on post-transplant administration of selected aastreg. direct intravenous immunoglobulins (ivig) is an effective treatment for t-cell mediated graft rejection and autoimmune diseases. ivig treatment is associated with rapid clinical improvements without the side effects of global immunosuppression. this prompted us to ask whether ivig might enhance directly the suppressive function cd4+cd25+foxp3+ regulatory t cells in vitro and in vivo. in vitro, mouse cba/ca (h2 k ) total cd4 + or cd4 + cd25responder cells were stimulated with c57bl/10 (h2 b ) splenocytes, and human cd4 + or cd4 + cd25cells were stimulated with allogeneic antigen presenting cells. in vivo, 1*10 5 total cd4 + or cd4 + cd25 -t cells of cba/ca mice were adoptively transferred into cba/rag1 -/mice, and one day later transplanted with a tail skingraft of c57bl/10 mice. ivig was administered i.v. on day 1,3,7,10 and 14. human serum albumin (hsa) was used as a control. binding of ivig and activation status of foxp3 + cd4 + t cells were determined. in vivo, only when total cd4+ t cells, but not cd25-t cells, were adoptively transferred, ivig protected against t-cell mediated rejection of the fully mismatched skingraft (mst: ivig >100 vs hsa 16 days, p<0.01). ivig binds to 16±2% of foxp3 + t cells, while binding to foxp3 -t cells was minimal. this binding was partially fcγ receptor mediated. furthermore, ivig treatment resulted in activation of cd25 + t cells as detected by increased expression of phosphorylated zap70/syk, which could be abrogated by specific tyrosine kinase inhibition. in vitro, ivig inhibited the alloproliferative response of murine cd4 + t cells by 75±25%, but after depletion of cd25 + t cells, this inhibition decreased to 17±12% (n=6, p<0.01). similar results were found in human mlr, where depletion of cd25+ t cells resulted in twofold reduction in ivig mediated suppression. significantly, incubation of human cd4 + cd25 + with ivig enhanced their ability to suppress allogeneic t-cell proliferation (ivig 63±8% vs hsa 37±10% of inhibition, n=5, p<0.05) . our results identify a novel pathway through which ivig treatment induces direct functional activation of both mouse and human regulatory t cells. immediate binding and activation of regulatory t cells is one of the mechanisms in the immunomodulatory repertoire of ivig, which allows rapid inhibition of allogeneic responses, and therefore can be a valuable tool after organ transplantation. generation foxp3 is a dna-binding protein that is necessary for regulatory t cell (treg) function, though recent data indicate that detection of foxp3 mrna or protein alone does not necessarily indicate whether the associated foxp3+ cell is functional or not. to that end, our analysis of foxp3 sequence showed the presence of an rxxr motif, conserved between mice and humans, that is located 12 amino acids (aa) from the carboxy terminus of foxp3 and is a potential recognition sequence for cleavage by proprotein convertase enzymes. we found several proprotein convertases are expressed by naturally occurring tregs, with further upregulation upon tcr activation. we generated an antibody against the 12 aa carboxyl peptide of foxp3, and by western blotting detected a peptide of about 1.3-kda, consistent with a 12-aa long carboxy-terminal foxp3 cleavage product. both cleaved and uncleaved forms of foxp3 were resolved by sds-page, and the cleaved form was found only in the dna-bound fraction. the requirement of proteolytic cleavage, at the intact tetrabasic rxxr motif (414rkkr417), for full treg function was shown by abolishing the motif through mutagenesis, followed by retroviral expression of mutants, and analysis of proteolytic cleavage by western blotting. assays of treg function by cells expressing either long-or short-foxp3 mutants showed short-foxp3 (missing the carboxy-terminal 12-aa) suppressed teff cell proliferation significantly more effectively than wt foxp3 or an engineered and cleavage-resistant mutant, termed long-foxp3 (p<0.01). moreover, adoptive transfer of cells expressing short-foxp3 prevented experimental colitis more effectively than wt-foxp3 (p<0.01), and both were more effective than cells expressing long-foxp3. animals that received cells expressing short-foxp3 gained weight and were protected from disease. thus, function of foxp3 is regulated at a post-translational level by proteolytic cleavage and the short form of foxp3 represents the active form. our data shows proteolytic cleavage of foxp3 is key to the generation of functional tregs, with enzymes(s) of the proprotein convertase family likely playing an important role in this process and providing an extra and hitherto unrecognized important level of regulation for foxp3. blocking since tgf-β is secreted in a latent form and active tgf-β is rapidly cleared from circulation. in order to make long-lasting active form of tgf-β, the mutant tgf-β was fused to a human igg fc component. the secreted human mutant tgf-β/fc (hmtgf β /fc) fusion protein stained with both anti-human tgf-β and anti-human igg antibodies, confirming the cytokine and isotype specificity of tgf-β moiety and fc domain. moreover, in vitro bioassay, hmtgf-β/fc inhibited il-4 dependent ht-2 cell proliferation in a dose dependent manner and had a circulating half-life of 36 hours in mice. in vitro, anti-cd3 and anti-cd28 triggered cd4+ or cd8+ t cell proliferation, measured by 3h-thymidine uptake, could be synergistically inhibited by tgf-β and rapamycin. at the same time, the two reagents when added together could synergistically promote the induction of cd4 + foxp3 + t cells from peripheral naïve cd4 + foxp3 -t cells. in a pancreas islet transplantation model in which the fully mhc mismatched dba/2 islets were transplanted into c57 bl/6 mice rendered diabetic by streptozotocin, mean survival time of islet was 19 days in control recipients (n=6). administration of muttgf-β/fc resulted in a delayed islet allograft rejection (mst; 38 days, n=5). treatment with rapamycin prolonged islet grafts survival in 63% of recipients. in contrast, combined treatment with tgf-β/fc and rapamycin by four doses produced indefinite islet allograft survival in 83% cases. we have produced the long-lasting active hmtgf-β/fc fusion protein. the tgf-β/fc is synergistic with rapamycin to convert naïve cd4+foxp3-t cells into cd4+foxp3+ tregs and promote long-term islet allograft engraftment. the the recognition of microbial motifs by the innate immune system leading to the stimulation of adaptive immune responses may explain the relationship between infections and susceptibility to graft rejection. a number of infectious agents have been reported to prevent the induction of allograft tolerance, however, none of these can reverse established tolerance, a situation that is highly relevant to clinical transpalntation. we here report that established allograft tolerance (induced with anti-cd154 + donorspecific transfusion) can be reversed in a cardiac transplantation mouse model following infection with listeria monocytogenes. this reversal of tolerance was dependent on the presence of both cd4+ and cd8+ cells, as well as of the tlr/il-1/il-8 adaptor molecule, myd88. we hypothesized that the reversal of tolerance requires that alloreactive conventional t cells (tconv) escape dominant regulation by pre-existing tregs. these alloreactive tconv can then proliferate resulting in an increased ratio of tconv:tregs favoring the reversal of established tolerance. indeed, we observed that tolerant grafts harbored low numbers of infiltrating cd4 + and cd8 + t cells (1-1.4 x 10 4 /heart), with 10-43% of the infiltrating cd4 + cells co-expressing foxp3 (n=10). following the reversal of tolerance and allograft rejection, a 7-10 fold increase in cd4 + foxp3and cd8 + foxp3 -tconv was observed in the graft, but no increase in the foxp3 + subset. in contrast, we observed no significant changes in the splenic t cell subsets, raising the possibility that the escape from tregs occurs locally within the graft. infection of tolerant il-6-or ifnar1-deficient recipients with listeria monocytogenes failed to reverse tolerance, consistent with a hypothesis that the initial escape of tconv from regulation may depend on il-6, while their proliferation may be type i ifn-dependent. in summary, the conditions for the reversal of tolerance is more stringent than the prevention of tolerance, and requires myd88-signalling and the presence of both cd4 + cells and cd8 + cells, as well as il-6 and type i ifn signaling. these studies point to the potential impact of bacterial infections on established tolerance, and to novel strategies to monitor and facilitate the maintenance of tolerance in the clinic. th17 our laboratory has identified kα1 tubulin, as an epithelial autoantigen to which immune response occurs following human ltx. further, we have shown a strong correlation between the presence of antibodies (abs) to kα1 tubulin and development of chronic rejection ie bronchiolitis obliterans syndrome (bos). goal of our study is to test the hypothesis that epithelial damage due to allo immunity results in remodeling exposes otherwise cryptic self antigens including kα 1 tubulin and collagen type v (coll v) leading to cellular immune reactivity and ab production against these auto-antigens which may play a role in the pathogenesis of bos. 10 patients who developed anti-hla ab and 10 patients who had no detectable anti-donor hla abs post-ltx by luminex assay were analyzed for development of abs to kα1 tubulin and coll v using elisa method developed in our laboratory. the elisa for kα1 tubulin used recombinant protein (1µg/ml) purified on affinity resin. the elisa for collagen v uses commercially available human collagen v (1µg/ml). elispot assay for ifnγ and il-10 were performed for 5 bos-and 5 bos+ patient pbls (at the time of bos diagnosis), after 3 stimulations with kα1 tubulin protein. the levels of anti-tubulin abs and anti-coll v abs were increased significantly in bos+ ltx recipients with anti-hla compared to those with no anti-hla, table 1 (p<0.05). . surprisingly, both cd4 and cd8 subsets induced long-term survival of secondary test grafts (>100 days). however, only the cd4 + t-reg prevented tvs (ni=19+5, 21+6% of vessels), as compared to cd8 + t-reg (ni=40+9 in 50+15% of vessels). the cytokine profile indicated a dominant il-10 response. allo-antibody analysis showed up-regulation il-4/il-12 dependent igg1 and igg2c. conclusion: allochimeric protein-therapy and csa treatment generate t-reg that prolong graft survival, but only cd4 + t-reg generated from allochimeric protein-therapy prevent tvs. this cd4 + t-reg may be responsible for long-term graft maintenance through its unique ability to control anti-inflammatory and alloantibody responses. immunodominant h60 background: minor histocompatibility ags (mihas) are self peptides, derived from proteolytic processing of normal cellular proteins. miha incompatibility can induce t cell response to dominant mihas and facilitates expansion of ctls and graft rejection. however, the contribution of ctls recognizing these mihas in solid organ transplantation has not been fully evaluated. methods: balb.b (h-2 b ) donor hearts were transplanted heterotopically to c57bl/6 (h-2 b ) recipients. in vivo alloreactive cd8 t cells were monitored with peptide/mhc multimers. α-lfa-1 mab was given to recipients to prevent rejection. various numbers of h60-specific cd8 t cells or cd8 t cells lacking h60 specificity were adoptively transferred into balb.b graft bearing b6.scid recipients. results: 50% of transplantation recipients developed acute rejection and showed markedly increased numbers of h60-specific cd8 t cells at day 8-12 in spleen. abundant cd8 infiltration was found and selective infiltration of h60-specific cd8 t cells in the graft was confirmed with flow cytometry and in situ tetramer staining. α-lfa-1 mab treatment prevented acute rejection (mst>100) and allogeneic t cell expansion. it also profoundly attenuated neointimal hyperplasia ( graft-bearing b6.scid mice. we found that the degree of acute rejection positively correlated with the number of h60-specific cd8 t cells transferred. however, transferred h60-specific cd8 t cells did not cause chronic rejection. interestingly, greater numbers of cd8 t cells with h4 immunodominance were found in spleen, blood and graft at 50 days after adoptive transfer of cd8 lacking h60 specificity compared to h60-specific cd8 t cell transfer. conclusion: h60 responses dominate other miha immune responses during acute rejection after balb.b to b6 cardiac allograft. we confirmed a role of immunodominant h60 specific cd8 t cells as pathological effector t cells in acute rejection but not in chronic rejection. maintaining a h60 response may be beneficial in allotolerance to suppress miha responses that could induce chronic rejection in long-term grafts. chronic lung allograft rejection, bronchiolitis obliterans syndrome (bos), affects up to 60% of transplant survivors 5 years post-ltx. human neutrophil peptides (hnp 1-3/ α defensins), have been identified in the lavage fluids from patients with chronic rejection by proteomics. goals of our study were to determine the role of anti-hla abs and defensins in bos and to define the interactions between α-1antitrypsin (aat), and defensins in regulating inflammation leading to epithelial cell proliferation and bos pathogenesis. bal and serum samples (post-ltx and pre bos) from 21 bos+, 15 bos-patients and 12 normals were analyzed by elisa for α defensins (hnp1-3), human β defensin2 (hbd2) and aat. small airway epithelial cells (saec) were treated with hnp1 or 2, with or without equimolar aat or with anti-hla abs and analyzed for levels of hbd2 production (elisa), cytokine and chemokine (luminex), and cell surface adhesion molecules (icam, vcam) by facs. bal and serum from bos+ patients had high levels of hnp1-3 and hbd2 compared to bos-recipients or normal sera (p<0.05) ( table 1 ). there was also a significant decrease in aat levels in bos+ compared to bos-or normal serum (p=0.01) ( table 1) . saec produced human β defensins following anti-hla ab stimulation or by hnp treatment (table 2) . there was increase in adhesion molecules (icam, vcam, 2 folds) cytokines {il-6, il-1r α (2 folds), il-1 β, il-13(20 folds)}, il-10(2 folds decrease), chemokines (il-8, mcp1, 2-4 folds increase) and growth factors (egf and vegf, 2.5 folds increase) in response to treatment with hnp1 or hnp2 compared to untreated saec, that was inhibited by aat. increased defensins and decreased aat levels were seen in lavage and serum of ltx recipients with bos. anti-hla abs further stimulate defensin production by saec. we conclude that chronic stimulation of epithelial cells both by defensins and anti-hla abs can lead to increased growth factor production contributing to the pathogenesis of bos. under tubular atrophy/interstitial fibrosis (ta/if) remains a major cause of late kidney allograft loss and epithelial mesenchymal transformation (emt) is now appreciated as a key feature in this process. we hypothesize that macrophages play a direct role in the development of ta/if by creating a profibrotic microenvironment and stimulating emt within the allograft. in human kidney allografts with ta/if (n=128), macrophage infiltration detected by immunostaining was significantly upregulated (mean score 2.8±0.5) compared to biopsies without corresponding pathological changes from recipients with stable function (n=51; 2.0±0.1; p<0.0001) and the extent of this staining also correlated to the magnitude of graft dysfunction (p<0.0001). rt-pcr in ta/if grafts also showed marked upregulation for emt markers αsma (3.0±1.1-fold; p<0.05), s100a4 (8.7±2.7-fold; p<0.05), and vimentin (4.2±2.3-fold; p<0.01), compared to stable function allografts. to explore the macrophage-emt relationship, we co-cultured freshly isolated human monocytes over a primary culture of human proximal tubular epithelial cells (ptecs) using culture inserts allowing media exchange but forbidding contact between the two cell populations. by rt-pcr, co-cultured epithelial cells showed significant downregulation of bmp7 (0.45±0.09-fold; p<0.001), a negative regulator of emt, and epithelial marker e-cadherin (0.001±0.001-fold; p<0.01), with marked upregulation of emt genes s100a4 (2.43±0.40-fold; p<0.001) and αsma (5.31±2.23-fold; p<0.05) compared to ptecs cultured in media alone. investigating the mechanism of monocyte driven emt, we evaluated the effects of am80, a synthetic retinoid that also inhibits il-6 and vegf signaling. am80 markedly reversed the transcriptional profile with reduction of emt markers s100a4 (0.34±0.10-fold; p<0.01), αsma (0.29±0.06-fold; p<0.05) and vimentin (0.45±0.13-fold; p<0.01), and a simultaneous increase in expression of bmp7 (151.7±53.9-fold; p<0.01), thus reversing the pro-emt milieu. these results indicate that human monocytes induce transcription of emt related genes in ptecs, even in the absence of physical contact. moreover, this phenomenon appears to be mediated in part by il-6, vegf, and other pathways mediated by the retinoic acid receptor. thus, in addition to their typical immune functions, macrophages support a milieu within allografts that promotes ta/ if in humans. further investigation into this novel pro-ta/if pathway could ameliorate chronic injury and improve graft survival. old donor kidneys are more likely to develop long-term graft failure, especially if they are exposed to stresses (e.g. rejection). this limited ability of old tissue to withstand stress may be due to its reduced capacity of replication and regeneration. telomere shortening determines lifespan and regenerative capacity. we showed that telomere shortening occurs in old human kidneys. late-generation terc ko mice with critically short telomeres have a reduced lifespan, show an accelerated aging phenotype and are an ideal model to resemble the situation in old human donors. this study addressed the question whether iri causes greater damage in kidneys from late-generation terc ko mice. we studied terc wildtype (wt), early-(g1) and late-generation (g4) terc ko mice 1 day (wt:n=7; g1:n=8; g4:n=8), 3 (wt:n=8; g1:n=8; g4:n=5), 7 (wt:n=6; g1:n=8; g4:n=8) and 30 days (wt:n=8; g1:n=9; g4:n=6) after iri. acute tubular necrosis was found mainly on days 1 and 3 and was significantly higher in terc ko g4 kidneys. tubular atrophy (ta) and intersitial fibrosis (if), reflecting chronic damage, were first detected at day 7 and increased in all mice with a significantly higher extent of ta/if in terc ko g4 kidneys at day 30 ( fig.) . the cell cycle inhibitor p21, a downstream mediator of senescence induced by telomere shortening, was significantly upregulated in all groups after iri. p21 levels were highest in terc ko g4 kidneys at day 30 ( fig.) . proliferative capacity, as measured by ki-67 immunostainings at day 30, was significantly lower in tubular, glomerular and interstitial cells of kidneys from terc ko g4 mice. we show that late-generation terc ko mice with critically short telomeres have a greater susceptibility towards acute injury and develop more chronic renal damage. this is likely due to the reduced capacity of these kidney to proliferate and thereby to regenerate. our data strongly suggest a pathogenetic role of telomere shortening, an important senescence mechanism, for the development of long-term graft failure. results: belatacept treatment had no effect on the number of peripheral blood treg cells and t cell suppression assays. the percentage of foxp3 cells was significantly elevated in rejecting kidney allografts in belatacept-treated patients compared to cnitreated patients. conclusions: following chronic belatacept therapy, the number and function of peripheral blood treg cells are maintained in kidney transplant patients. belatacept enhances the treg population in the allograft in patients with acute rejection. therefore, our data suggest that co-stimulation blockade with belatacept does not affect treg homeostasis. the increased number of treg cells in rejecting allografts in belatacepttreated patients may provide a novel mechanism whereby belatacept can mitigate the severity of acute rejection and improve graft survival. the steady-state auc of n-desmethyl-aeb071 was minor in comparison to aeb071 and similar between the patient groups: 200 ± 90 ng.h/ml in transplantation vs 393 ± 220 ng.h/ml in psoriasis (p=0.08). demographic covariates: in the first week posttransplant with patients receiving fixed-dose aeb071, intersubject variability for c0 was 78% and for auc was 49%. aucs were similar in men vs women (8350 ± 4163 vs 10784 ± 5369, p=0.26). age, which ranged from 18-64 years, did not influence auc based on regression analysis (r 2 = 0.005, p=0.65). there was a borderline-significant negative correlation between weight (range, 51-110 kg) and auc (p = 0.07); however, its clinical relevance was low in that it could explain <9% of the variability in auc (r 2 = 0.086). there was a significant positive correlation between aeb071 c0 and auc (r 2 = 0.724, p<0.001). conclusions: (1) in the first week posttransplant, patients achieved aeb071 blood levels anticipated for this regimen. (2) there was notable intersubject pharmacokinetic variability at this time but it was not attributable to standard demographic factors such as sex, age, or weight. (3) a good correlation was noted between c0 and auc suggesting that c0 might serve as a marker for total drug exposure. a we studied which is protocol would be best after rapid discontinuation of p. between 9/01 and 4/06, 440 1st and 2nd kidney tx recips were randomized: csa-mmf (n=151) vs. high tac-low srl (n=149) vs. low tac-hi srl (n=140) (for tac and srl levels, high = 8-12, low = 3-7). all received thymoglobulin (tmg) 5 doses, and p for 5 days; tmg was continued in dgf. min f/u = 1 yr; mean = 42±19 mos. there was no diff between groups in recip age, gender, ethnicity, prim dis, donor source, % retx, pra; or in donor age, gender, ethnicity. there was no signif diff. between groups (intention to treat) in actuarial patient, graft, death-censored (dc) graft, acute rejection (ar), or biopsy-proven chronic rejection rates (cr), serum cr level or calculated (mdrd) gfr or in studied side effects. cr(sd) 1.6(.7);1.7(.9);1.8(1.3); 1.9(2);1.6(.7) 1.5(.4);1.7(1);2(2.5); 1.7(1.1);1.6(.6) 1.5(.5);1.5(.7); 1.6(.6);1.6(.6);1.7(.7) mdrd gfr(sd) 63(24),58 (21),60(26), 61(26),62 (20) 64 (20),65(24),61(27), 56(23),63 (28) 65 (20) the most common cause of graft loss was death with function (csa 40%, high tac 35%, low tac 57%). the majority of recips in each group remained p-free (csa 72%, high tac 86%, low tac 74%) but a large % were not on the medications which they were randomized to (csa 16%, high tac 51%, low tac 42%). we found a signif ↑ of new onset diabetes (nodm) (p=.03) in the tac-srl groups: csa 2%, high tac 11%, low tac 5%. there was a trend towards more use of lipd lowering medications in the tac/srl groups. in summary, all 3 is protocols were effective; with min f/u 1 yr for each group, patient and graft survival and ar rates are similar. we found an increased nodm and possibly hyperlipidemia in the tac/srl groups. purpose: we present preliminary 2-year results from a worldwide trial comparing 2 srl regimens with tac+mmf. methods: renal transplant recipients (n=451) were randomly assigned to the following treatment regimens: group 1: srl (8-15 ng/ml, then 12-20 ng/ml after week 13) + tac (6-15 ng/ml) with elimination at 13 weeks (n=155); group 2: srl (10-15 ng/ ml through week 26, 8-15 ng/ml thereafter) + mmf (up to 2 gm/day) (n=155); or group 3: tac (8-15 ng/ml through week 26, 5-15 ng/ml thereafter) + mmf (up to 2 gm/day) (n=141). all patients received corticosteroids and daclizumab. in june 2006, group 2 was terminated (after all patients were accrued) because of increased acute rejection (ar) rates. results: demographic characteristics were similar between groups except for more females in group 3. patient and graft survival were also similar among groups (see table) . biopsy-confirmed ar (bcar) was significantly greater in group 2 compared with groups 1 and 3, p<0.001, and most occurred in the first 6 months posttransplant with preponderance during the first 3 months. subtherapeutic srl trough concentrations were reported in a large number of rejectors in group 2. most of the rejections in group 1 occurred within the first 3 months before tac was eliminated. all ars were mild to moderate; grade ii ars were proportionally greater in group 3. mean nankivell gfr was numerically higher in group 2. preliminary results at 2 years show excellent patient and graft survival and similar renal function among treatment groups, despite higher ar rates in group 2. early adequate exposure to sirolimus is mandatory to achieve desired (low bcar rates) results. the goal of rapid discontinuation of prednisone (rdp) after kidney transplantation is to minimize prednisone-related side effects without increasing acute rejection (ar) rates or decreasing long-term graft survival. to date, studies have shown that rdp is associated with decreased prednisone (p)-related sided effects, and randomized trials of rdp (vs. long-term p) have shown little or no ↑ in ar rates. however, concern remains that long-term graft survival will be worse with rdp protocols. we studied t½ (the time it takes for ½ of the grafts surviving at 1 year to subsequently fail) for 1st tx recipients treated with rpd (1999-2007) (n = 652) (antibody, cni, antimetabolite [mmf or srl] , and rdp) vs. 1st tx historical controls (1995) (1996) (1997) (1998) (1999) (2000) (n = 388) treated with a protocol of antibody, cni, antimetabolite [mmf] , and long-term p. table 1 shows characteristiscs of the 2 groups. pra; rdp were more likely to get a living unrelated donor transplant. t ½ is shown separately for living (ld) and deceased (dd) donor transplants in table 2 . there was no significant difference in t½ between groups. we conclude that, compared to historical controls, rapid discontinuation of prednisone can be done without any detrimental impact on long-term outcome. a prospective, randomized study to confirm this observation is necessary. successful 1a) . we enrolled 152 patients (table 1) with an average follow-up of 19.7 + 11.7 months. subjects 19 -65 years old included primary and re-transplants, with primary endpoints of renal function and can. follow-up averaged 16.6 + 9.5 months in 47 patients withdrawn from ci (26 from group 3, 21 from group 4). we found no increased rejection after ci discontinuation and that both ratg induction dosing regimen and ci discontinuation significantly impacted renal function and the development of can. we successfully discontinued both calcineurin inhibitors and steroids with either single or divided-dose ratg induction, and single-dose ratg induction independently associated with improved renal function and reduced can. study demographics single-dose ratg (6 mg/kg x 1) divided-dose ratg (1.5 mg/kg x 4) group 1 (n = 38) group 3 (n = 38) group 2 (n = 37) group 4 (n = 39) age 45.7 ± 11.9 45. in subset analysis, we also find an enhanced negative impact of srl when combined with steroids (str) in patients without dgf. conclusion: this data supports previous findings that srl may be associated with a sustained survival disadvantage apparent early post transplant, and that this effect appears exacerbated when combined with dgf or str. several potential explanations for this effect may include srl-associated prolonged early graft dysfunction, hyperlipidemia or proteinuria. however, patients initiating srl after discharge may not evidence this survival disadvantage, and this question was not addressed here.these findings may be of particular importance to centers employing combined srl and str therapy, as well as to define the best mode of support during recovery from dgf. prospective randomized studies would be necessary to evaluate these. table 1 shows cai in both groups from 1 to 5 years. in slr group the doses of cin were significantly lower from one through 5 years (p=0.05). cai due to interstitial fibrosis/ tubular atrophy was significantly lower in slr group at 5 years. our data shows that 5 year patient and graft survival, graft function, bpar and scar were comparable between mmf and slr groups despite lower prevelance of interstitial fibrosis/tubular atrophy in slr group. registry analyses suggest that tac/mpa immunosuppression is associated with superior kidney graft survival vs. tac/srl. large single-center experience may assist in clarifying these findings, by examining outcomes related to specific utilization practice. we retrospectively examined the outcomes of 529 consecutive first renal transplants (55% deceased donor, 45% living donor) at a single center, treated with tac/srl or tac/mpa. graft and patient survival, acute rejection rates, and 1yr egfr were analyzed by era of transplant (2000-2002 vs. 2003-2006) . changes in tac/srl utilization between eras included elimination of the srl loading dose and a reduction in tac target trough concentrations. summary. compared to tac, srl+cya was associated with a 55% decreased risk of skin ca that was statistically significant. although srl+cya was associated with a 32% decreased risk of de novo solid ca, it did not reach statistical significance. this reduced risk may not reflect the unique aspects of srl itself, but may be a reflection of practice pattern, patient selection or center effect. the backgrounds: a number of studies have observed increase of malignancies following renal transplantation. however, the incidence and the site of malignancies were quite different by the follow-up times, the era and the region. methods: we reviewed the records of 694 renal transplant recipients in our institute between 1970 and 2007 and recorded the incidence and types of de novo malignancies. they were divided into two groups by immunosuppressive era; azathioprine (aza) era (1970.4-1982.3: n=172) and calcineurin inhibitor (cni) era (1982.4-: n=522) . results: a total of 57 (27 in aza era and 30 in cni era) kidney recipients out of 694 developed 60 malignancies. the tumors included 10 gi-tract cancers, 9 liver cancers, 12 skin cancers, 4 tongue cancers, 6 breast cancers, 6 renal cell carcinomas, 2 thyroid cancers, 5 leukemia, 3 lymphoma, one lung cancer, one uterus cancer and one kaposi's sarcoma (ks). the average interval between transplantation and development of malignancy was 134±86 (8-340) months. mortality was high in liver cancer (89%) and leukemia (100%). cumulative incidence of malignancies of all 694 recipients in 5, 10, 20, 30 years were 2.3%, 4.5%, 10.7% and 14.3%, respectively. graft-loss censored cumulative incidence, which was calculated to see the incidence among graft survivors under continuing immunosuppression, of all recipients in 5, 10, 20, 30 years were 2.8%,6.3%, 16.3% and 26.2%. that of 5 ,10 and 20 years in cni era was 3.0%, 6.8% and 13.9%, while that in aza era was 1.8%, 4.9% and 19.5%, showing early higher incidence in cni era outstripped by aza era by 12 years. site of malignancy in cni era occurring within 3 years, which was never observed in aza era, was focused on liver, leukemia (including atl), ks and ptld. discussions:our results demonstrated that recent potent immunosuppressive regimen shortened the interval between transplantation and viral-related malignancies. however, long-term incidence of whole malignancies has been decreasing by minimizing chronic immunosuppression in our institute. the impact of transplant center practice on the association between pulsatile perfusion and delayed graft function. jagbir gill, 1 david gjertson, 1 suphamai bunnapradist, 1 michael cecka. 1 1 ucla, la, ca. the use of pulsatile perfusion (pp) is increasing in the us, but practice varies widely among transplant (tx) centers. we describe the variability of pp use and its impact on post transplant outcomes. methods: we identified all cadaveric kidney tx from 2000-2005 using optn/unos data. the cohort was stratified by tx center pp use as follows: low pp centers (0-10% pp use), med pp centers (10-30% pp use), and high pp centers (>30% pp use). donor characteristics and the incidence of dgf were compared between and within each strata. results: pp was used by 97 % of centers, however most centers used pp <10% of the time (70.7%). compared to low and med pp use centers, kidneys pumped in high pp centers were from donors that were younger, had a lower mean terminal serum creatinine, and had a lower incidence of cva and hypertension. the overall incidence of dgf was lowest in the high pp centers (13.3%), compared to the med (24.7%) and low pp (24.8%) centers. the rates of dgf within each strata (high, med, and low pp centers) for tx performed using pp versus cold storage (cs) are outlined in the table below . within each strata, the rate of dgf did not differ between tx performed with and without pp. in ecd transplants, pp was associated with lower rates of dgf across all center groups. however, the impact of pp on scd and dcd transplants was less significant and varied across centers by pp use. hla sensitized patients (20-100%) in our dsa are now transplanted at a rate of 40%, which is significantly higher (p = 0.02) than the 23% rate when ua weren't entered into unet. furthermore, of 9 kidneys imported into our dsa and allocated to the dsawide renal candidate list (since ua entry started), 63% (5/9) were transplanted into sensitized candidates (85% to 27%), each of whom had a negative flow or ahg t cell igg crossmatch. conclusion. the higher transplantation rate for hla sensitized patients in our dsa shows that virtual a, b, & c crossmatching yields a dsa-wide ranked list of sensitized candidates likely to have a negative final class i (t cell) crossmatch and be transplanted. the data also lend support to the notion that sharing kidneys across dsa boundaries for hla sensitized candidates, based on a negative virtual hla class i crossmatch, has merit. predicting introduction: predicting graft outcome after renal transplantation based on donor histological features has remained elusive and is subject to institutional variability. we propose a pre-transplant donor path scoring system that reliably predicts graft outcome regardless of recipient ® characteristics. methods: we retrospective analyzed 286 imported cadaveric renal transplants which were initially rejected by other centers due to donor parameters between 1/00-6/05. all kidneys were re-biopsied at our center prior to implantation. morphometric analysis performed consisted of measuring glomerular-size arterioles, interlobular, and arcuate/ interlobar arteries and wall to lumen ratio (wlr) was calculated; calculating % glomerulosclerosis (gs); the presence of arteriolar hyalinosis (ah), scar, periglomerular fibrosis (pfg), and acute tubular necrosis in the biopsies. the patients were followed for a mean of 33 months. multivariate cox analysis was done to evaluate the predictive value of these path variables to graft outcome.results: ah, gs>15%, wlr>0.5, pgf, and scar were found to independently predict graft outcome. the unos board of directors has approved the change from using panel reactive antibodies (pra) to calculated pra (cpra). the cpra is a formulated pra based upon the frequency of the specificities of hla antigens found in the donor pool and is expected to standardize the degree of patient sensitization. donor organs expressing unacceptable antigens will not be offered to a recipient with donor (hla) antigen specific antibodies (dsa). highly sensitive, single antigen bead and solid phase assays (flow pra and luminex) are used to identify these hla antibodies (abs). each transplant center can determine the criteria used for identifying an unacceptable antigen, for example, based upon dsa titer or the fluorescence intensity (fi) coming from the donor-specific single antigen bead. it is unclear whether abs identified by these techniques are clinically relevant for organ allocation. we retrospectively evaluated flow-pra, flow cytometry crossmatching (fcxm), hla ab specificities and titers of 300 pre-transplant (tx) sera from transplant recipients of deceased renal allograft donors transplanted following a negative cytotoxic-anti-human globulin crossmatch. the two year graft survival of 91% for the recipients (44/54, 81%) with low-titer (≤ 1:16) donor specific hla ab and a negative (-) fcxm was significantly better when compared to the 60% two year graft survival of the 19% (10/54) of recipients presenting with (+) dsa but a (+) fcxm (p< 0.001). recipients (55/110, 50%) with non-donor abstracts specific hla abs (high or low titer) and a (-) fcxm also experienced a better two year graft survival of 89% compared to the 74% for the other 50% of recipients with (+) fcxms (p < 0.001). these data suggest that in the presence of donor-specific or non-donor-specific hla abs you can not predict the crossmatch outcome without actually performing the crossmatch which will then influence donor organ allocation and graft survival outcome. in the face of low-titer dsa and a (-) fcxm recipients experienced excellent graft outcome when compared to recipients with (+) fcxms. therefore, donor organ allocation based on cpra (ab specificity and unacceptable ags) utilizing highly sensitive single antigen bead and solid phase luminex assays may disadvantage recipients (no donor crossmatch) who could otherwise be successfully transplanted. aim: to assess vegfr2 expression in hcc and the adjacent benign cirrhotic parenchyma and its correlation with tumor differentiation, vascular invasion, and tumor morphologic parameters. background: hcc is a highly vascular tumor in which angiogenesis is mediated in part by vegf. vegf is highly expressed in hcc and mediates its effects through multiple receptors including vegfr2. the tyrosine kinase inhibitor sorafenib inactivates the vegfr2 receptor and exhibits anti-tumor effects in hcc. clinical significance of vegfr2 expression with respect to tumor parameters has not been evaluated. patients and methods: immunohistochemical staining for vegfr2 was performed in hcc and corresponding adjacent cirrhotic liver from 79 patients undergoing liver transplant. 57 patients had hcc within mc. stains were scored by estimating the % of positive surface area in veins, arteries, and sinusoidal lining cells. data are presented as median [p25, p75]; wilcoxon signed rank and wilcoxon rank sum tests were used. results: vegfr2 levels in hcc were significantly correlated to levels in adjacent non-tumorous liver. higher levels of vegfr2 in non-tumorous liver were associated with higher levels in hcc from the same patient. vegfr2 levels were significantly higher in hcc compared to adjacent areas (p<0.05). vegfr2 levels in hcc were not significantly different between patients who fell within mc and those beyond mc. however, vegfr2 levels were significantly higher in the adjacent arteries of nontumorous liver (15.8 [0, 60] vs. 0 [0, 35]; p=0.03) in those patients with hcc beyond mc. subjects with moderate or poor differentiation had significantly higher levels of vegfr2 in sinusoids and veins of hcc and in the sinusoids of adjacent non-tumorous liver. there was no correlation with vascular invasion. conclusions: elevated vegfr2 in hcc correlates with elevated vegfr2 in adjacent cirrhosis, suggesting that high expressing hcc arise in a high vegfr2 expression, pro-angiogenic environment. moreover, higher vegfr2 expression in background cirrhosis correlates with advanced hcc (beyond mc), suggesting that anti-angiogenic agents may prevent tumor formation or progression in cirrhotic patients. this novel concept warrants further study. . we further attempted to find a subgroup of patients combining tumor size, number of nodules and various levels of afp which together would correlate strongly with pdiff tumors. however, the distribution was erratic and even in extreme outliers (>4 nodules, > 8 cm in maximum size, with or without high afp), where transplantation is currently not indicated according to any current expanded tumor inclusion criteria, the incidence of pdiff did not exceed 42%. conclusions: pdiff is most highly associated with tumors that exceed the milan criteria and the expanded criteria currently used for liver transplantation. thus, tumor biopsy would not appear to be of benefit except perhaps in those patients with a high afp level. however, if tumor criteria are further extended in the future, tumor biopsy may be justified in those with higher tumor burdens. < 2 cm) ), 2,511 (85.7%) at ls=t2 (1 tumor < 5cm or 3 tumors ≤ 2 cm), and 111 (3.8%) with ls>2. results: overall survival at 36 months was 76.5%, with significant differences seen for listing stage (ls 1= 82.2%, ls 2=75.9% ls>2=73.5%, p=0.0395) and by histologic stage as determined by pathology (p<.0001). survival for patients with histologic stage 4b hcc was only 29% at 36 months, versus 83% for stage 1. patients with tumors greater than 3cm both by listing stage and by pathology fared poorly compared to those with smaller tumors (p=0. 0.002). patients listed who received ablation treatment (at) preoperatively and who had their tumors "down-staged" had better results (p= 0.0061). we observed no difference in at types (tace vs rfa). those with micro or macrovascular invasion had lower survival rates, at 66.8% and 30.5%, respectively (p <.0001). afp >500 continues to be a significant predictor of lower post-transplant survival, with a survival rate of 57.9% at 36-months (p<.0001). conclusions: we conclude that listing and histologic tumor size, presence of micro/ macrovascular invasion, and high afp are associated with poorer lt results. at is emerging as potentially effective treatment for improving lt outcomes for hcc recipients. introduction: so far, milan criteria are used to select patients with hepatocellular carcinoma (hcc) for liver transplantation (lt). herein we compare prognostic markers in patients with pretreatment by transarterial chemoembolization (tace) to a second cohort transplanted without tace pretreatment. patients and methods: between september 1997 and october 2007, 134 patients with hcc underwent lt at our institution. eighty-two patients were pretreated by repeatedly performed tace whereas in 52 patients none or other forms of pretreatment had been used (non-tace group). tace was performed using lipiodol and mitomycin. every 6 weeks tace was repeated until transplantation. tumor response was assessed by ct scans (6-week intervals). results: sixty-seven percent of the patients transplanted after tace pretreatment exceeded the milan criteria compared to 37 % in non-tace patients. the proportion of recurrence-free patients was comparable in the tace and non-tace group (77.32 % and 78.45 %, respectively). in the univariate analysis grading, angioinvasion and progress-free tace were significant predictors for recurrence after lt in patients with tace pretreatment. progress-free tace was the only significant predictor of recurrence in the multivariate analysis. in the non-tace group t classification, number of nodules, grading, angioinvasion, milan criteria and underlying disease (hcv versus all other diseases) were significant. after tace pretreatment freedom from recurrence was 92.3 % in patients with stable disease or regress but only 38.4 % in patients with progress during tace. conclusions: tace pretreatment is capable of selecting a biological entity of tumors which differs significantly from untreated tumors. this statement is deduced from the remarkable differences of predictors for tumor recurrence in patients with and without tace pretreatment. moreover, tace patients can be separated into two groups: those who experienced tumor progress during repeatedly performed tace and those who did not. stable disease during the continued tace before transplantation resulted in remarkably low tumor recurrence. liver with a median follow-up of 30 months, there was no statistical difference in the 5 year overall survival in the m (76%) and m+ (69%) groups (p=0.40). the 5-year disease free survival was significantly higher in the m (80%) vs. m+ (61%) groups (p=0.02). when stratifying for ucsf criteria tumors, the 5-year disease free survival was milan (80%) vs. ucsf (72%) vs. beyond ucsf (54%) groups (p=0.01). univariate analysis demonstrated the following factors to be associated with disease recurrence: intermediate waiting time 2-4 months (p=0.01), preoperative tace (p=0.002) or resection (p=0.04), more than 3 tumors (p=0.03), and max tumor size over 5cm (p=0.03). multivariate analysis controlling for age, gender, and waiting time demonstrated that preoperative resection hr2.78 (95%ci 1.1-7.2), more than 3 tumors hr2.3 (95%ci 1.1-4.7) and max tumor size greater than 5cm hr2.68 (95%ci 1.2-5.9) were independently associated with disease recurrence. conclusions: the overall survival after liver transplantation is excellent in the m and m+ groups, far exceeding survival rates that can be obtained via any other modality. the current unos hcc algorithm should be reconsidered, to allow extra listing points for selected patients with hcc that exceed both the milan and ucsf criteria. postoperative use of intense insulin therapy in liver transplant recipients. lama m. hsaiky, 1 iman e. bajjoka, 1 dhaval patel, 1 marwan s. abouljoud. 1 1 transplant institute, henry ford hospital, detroit, mi. hyperglycemia and insulin resistance are common post liver transplant, even in patients with no history of diabetes. in the general surgical intensive care unit (sicu) population, the use of intensive control of blood glucose has recently been shown to reduce both morbidity and mortality. thus far, limited data exist in the liver transplant population. purpose: assess the impact of intensive insulin therapy to maintain blood glucose at or below 110 mg/dl immediately post liver transplantation in the surgical intensive care unit (sicu). methods: a retrospective evaluation of liver transplant recipients who received two different insulin protocols in the sicu was performed. prior to january 2003, patients were assigned to receive sliding scale insulin therapy (ssit) to maintain blood glucose (bg) less than 180 mg/dl. the intensive insulin therapy (iit) was implemented in august 2005 with a goal bg level between 80-110mg/dl. the following data was analyzed: bg ranges, need for mechanical ventilation, blood transfusions, infection rate in the sicu, rejection episodes and patient survival. results: a total of 97 liver transplant patients were evaluated; of which 47 patients were in the ssit group and the other 50 in the iit group. demographic characteristics were comparable between the two groups. in the iit, 24% of the bg readings were maintained at < 110 mg/dl versus 5% in the ssit. the incidence of hypoglycemia (bg <60mg/dl) was less than 1% in both groups. the need for mechanical ventilation was 3.0 days vs. 2.3 days and the overall number of blood transfusion was on an average of 5.0 vs. 2 units (p<0.05) in the ssit vs. iit group, respectively. iit also reduced overall sicu infection rate by 15% (p=0.01). the rate of acute cellular rejection at 3 months post transplant was less in the iit, 14% vs. 25% in the ssit group (p=0.02). moreover, mortality during hospital stay was reduced from 5% in the ssit to 2% in the iit group. conclusions: the use of intense insulin therapy immediately post liver transplantation has resulted in reducing infection rate and rejection episodes and a trend for reduced morbidity and mortality among post surgical liver transplant recipients without the adverse effects of hypoglycemia. medical epidemiology of patients surviving ten years after liver transplantation. kerri a. simo, 1 stephanie e. sereika, 1 david a. gerber. 1 1 abdominal transplantation division, department of general surgery, university of north carolina, chapel hill, nc. background: as the population of long term survivors of liver transplantation (olt) grows, their medical epidemiology has become increasingly important. the goals of this study were to define a collective profile of liver transplant recipients ≥ 10 years post olt and to compare their co-morbidities with those of the general population. in 2005, the national health survey reported that 22% of the us population had hypertension, 14.4% had diabetes/impaired fasting glucose, and 16.8% had chronic kidney disease. methods: a retrospective review of a prospectively collected database of 125 adult patients who underwent olt at a single transplant center from september 30, 1991 to october 1, 1997 was performed. inclusion criteria consisted of survival ≥10 years post olt with >1 year follow up. results: seventy-one patients met inclusion criteria. ninety percent of patients had ≥10 years follow up. the mean age at transplant was 43 (range 18-67). the mean calculated meld score was 22 (range 9-62, median=22). indications for olt were hcv(32%), alcohol(28%), cryptogenic(18%), autoimmune(10%), hbv(7%), psc(7%), pbc(7%), and other(10%). seven patients required retransplant during the first 10 years. an additional 38 patients underwent other operations: 10 arterial or biliary revisions, 13 hernia repairs and 19 non-transplant related (7 abdominal). during analysis, the following medical co-morbidities were found: 51 patients(72%) had hypertension (41 new onset), 20(28%) diabetes (10 new onset), 33(46%) renal insufficiency and 8 renal failure (28 new onset), 10(14%) cardiovascular disease (all new onset). nine patients (13%) were diagnosed with de novo cancer. medications for chronic health problems included 18 patients on diabetic medications, 46 on antihypertensives and 18 on lipid lowering agents. initial immunosuppression consisted of 100% on steroids, 61% on cyclosporine, 40% on tacrolimus, 24% on mycophenolate mofetil (mmf) and 23% on azathioprine. immunosuppression at 10 years consisted of 42% on cyclosporine, 41% on tacrolimus, 18% on steroids, 13% on mmf, 8% on sirolimus, 1% on mycophenolate sodium, 1% on azathioprine. no patients were on triple therapy, 29 were on dual therapy, and 42 were on monotherapy. summary: patients alive 10 years post olt have a significantly higher incidence of hypertension, diabetes, and renal disease than the general population. this study supports conscientious medical follow up to ensure continued meaningful survival. liver transplantation in the morbidly obese (bmi>40). c. quintini, 1 l. kauzman, 1 k. hashimoto, 1 p. ding, 1 t. doago uso', 1 n. sopko, 1 j. rosenblum, 1 f. aucejo, 1 c. winans, 1 d. kelly, 1 b. eghtesad, 1 d. vogt, 1 j. j. fung, 1 c. miller. 1 1 general surgery -liver transplant service, cleveland clinic oh. morbid obesity (mo) is a problem seen with increasing frequency among candidates for olt. mo is considered a contraindication for olt in some centers without clear evidence to support such a practice. our aim is to describe outcomes for olt in patients with a bmi>40kg/m 2 in a single center. methods. between 1/2004 and 4/2007, 364 olts were performed in 347 patients. 20/347 patients with a bmi>40 were compared to all other olt patients with a bmi< 40. we analyzed patient and graft survival, operative time, blood transfusion requirements, and post operative events (icu and overall length of stay los, surgical complications and infections). we also analyzed the post transplant weight records of our study group at 1-3 and 6 months. results. results are summarized in table 1. outcomes of olt in the morbidly obese are no worse than those of other patients undergoing olt. bmi is often artificially elevated in end-stage cirrhotic due to severe fluid retention. these patients exhibit rapid weight loss following transplantation that is likely due to extra-cellular fluid loss from the improved homeostatic milieu provided by the new liver and possible improvement in renal function. the presence of obstructive cad was not associated with increased peri-operative morbidity or mortality. these patients experienced similar patient and graft survival irrespective of the degree of cad. significant unmodified cad should not represent an absolute contraindication to liver transplantation. olt was also examined. we found that using a trj of 3.0 m/s as a cut-off created two age-matched groups with significantly different survival curves at one year (p = 0.006) with a relative risk of mortality of 3.98 for trj ≥3.0 m/s. this study underscores the importance of screening tte for the presence of pph in the evaluation of patients for olt, suggesting that clinically significant pph may be present at lower trj than typically prompt right heart catheterization. some of the limitations of the study are the variability of the time from pre-olt echo to transplant, and the assumption of rap = 5 without assessment of ivc size. we are using these data as a framework for further prospective trials to better assess the clinical applications of the findings. hyperlipidemia has been shown to predict faster chronic kidney disease (ckd) progression over the long term in lung allograft recipients. it is unknown whether disordered lipid metabolism may also aggravate the early loss of renal function often seen in this patient population in the immediate post-operative period. we studied 230 lung allograft recipients transplanted between january 1997 and december 2003. pertinent demographic and clinical variables were recorded at baseline and one month post-transplant, including creatinine levels and fasting lipid panels. logistic regression models were created to investigate an independent association between lipid levels and change in renal function by one month post-transplant. mean +/-sd baseline creatinine was 0.8 +/-0.2 mg/dl and low density lipoprotein (ldl) was 110 +/-35 mg/dl, the latter remaining unchanged at one month. in contrast, by one month post-transplant the mean creatinine level of survivors increased significantly to 1.2 +/-0.6 mg/dl (p < 0.001), with a overall mean increase of 52% above baseline. the highest quartile of patients that fared the worst experienced a rise in creatinine > 72% above baseline. on univariate analysis, there was a strong trend toward those with one month ldl values in the highest quartile (i.e., >140 mg/dl) having an increased odds of experiencing a rise in creatinine by one month > 72% above baseline (or 1.8, p=0.09). after controlling for age, gender, pre-transplant creatinine, bmi, and the presence of diabetes prior to transplant an ldl value in the highest quartile by one month post-transplant was the only variable independently predictive of a rise in creatinine > 72% above baseline at one month (or 2.5, p=0.05). in summary, hyperlipidemia occurring early post-lung transplant predicts faster loss of renal function soon after surgery. though speculative, given the known beneficial effects of lipid lowering agents such as statins on the rate of ckd progression, perhaps timely initiation of these medications after surgery may also attenuate the early decline in renal function often observed in this patient population. the risk for acute cellular rejection (acr) following lung transplantation (ltx) is still a problem despite heavy multidrug immunosuppression. induction therapy with potent t-cell depleting agents have facilitated the implementation of minimal post-transplant immunosuppression.the impact of this protocol on the activation of proinflammatory cytokines and effector molecules that affect the cellular rejection process is not well determined. in this study, we evaluated the relationship between upregulation of t-cell and macrophage-dependent inflammatory cytokines detected by molecular methods and the clinical status of ltx patients. we studied 28 ltx patients who received anti-lymphocytic induction therapy(thymoglobulin n=8 or campath-1h n=20) followed by maintenance immunosuppression with tacrolimus and low-dose steroids. we analyzed 137 bronchoalveolar lavage (bal) mrna samples (3-8 per ltx patient) by real-time pcr for gzmb, ifn-γ, il-15, mcp-1, rantes, tnf-α, and gapdh (control). the abi prism 7700 sds and 7500 fast real-time pcr systems were used and data were analyzed by the dct and 2-ddct methods. we determined the relationship of categorical outcomes (rejection-no rejection) with continuous variables (number of cycles) by anova. early acr that occurred within the first 60 days post-ltx was associated with an increase (>3-6 fold) of macrophage-specific mrna (mcp-1, rantes, tnf-α). 7/8 thymo-treated ltx patients experienced early acr while only 5/20 campath-1h-treated patients had early acr (p<0.005). in contrast, late acr that occurred greater than 60 days post-ltx was associated with a significant upregulation (4-64 fold) of t-cell dependent mrna (gzmb and ifn-γ) in addition to increases of rantes and mcp-1 mrna. overall, ltx patients who experienced early or late acr (n=20) exhibited higher mrna levels for the above mediators compared to stable patients (n=8). our data indicated that in early post-t-cell depletion, the acr phenotype was characterized mainly by macrophage activation. in contrast, greater than 3 months post-ltx with the recovery of cd8+ t-cells, the acr phenotype was associated with cytotoxic t-cell activation. furthermore, sensitive molecular methods may detect the activation of pro-inflammatory mediators within the allograft prior to the diagnosis of rejection by transbronchial biopsies and may impact optimal patient management. antibody-mediated rejection (amr) is defined by the presence of donor-specific alloantibodies, markers of complement activation and the clinical phenotype of organ dysfunction. compared to renal and cardiac allografts, very few amr cases are documented in lung transplantation (ltx). methods. we report here on seventeen ltx patients exhibiting: 1) donor-specific hla antibodies (dsa); 2) linear, continuous subendothelial c4d deposition in lung allograft; 3) lung allograft dysfunction. the presence and specificity of dsa were determined by elisa and/or luminex. c4d deposition was assessed by immunohistochemistry in transbronchial biopsy paraffin blocks. allograft dysfunction was considered when either biopsy-proven acute cellular rejection (acr, ≥ ishlt a2) or bronchiolitis obliterans (bos) were diagnosed. the average detection of dsa occurred in the first year post-ltx, on 283±218 postoperative day (pod), range 14 to 734 days. thirteen (76%) of amr patients were females. an anamnestic humoral response was encountered in 4 cases (one pregnancyrelated and three re-transplant patients), while de novo dsa were detected in 14 cases (82%). the percent-reactive antibody (pra) was lower in de novo cases (24%) when compared to memory response (71%, p<0.05). anti-class i dsa were found in 7 cases, anti-class ii in 5 cases, while 5 patients exhibited both class i and class ii dsa. specific vascular c4d deposition was detected in 16 (94%) patients. lung allograft dysfunction was considered in 14 (82%) cases, while three patients with dsa and specific c4d deposition fulfilled the criteria of sub-clinical amr. in patients where plasma-exchange/ ivig were applied, antibody titers dropped from 1:32 to 1:4 or 1:2 in two cases; in a third case, antibody titer remained high post-pheresis (1:512) and the graft failed. conclusions: both anti-class i and anti-class ii, low pra or high pra, pre-formed or de novo dsa can be detrimental for lung allograft. the presence of donor-specific alloantibodies, vascular c4d deposition and allograft dysfunction shows that amr criteria can also be met in ltx. rationale: recently, inhaled cyclosporine has been shown to reduce mortality and bos. previously, we demonstrated that apically-dosed cyclosporine poorly transmigrated differentiated human airway epithelial cells in vitro (using a transwell system). only ∼5% of the apically deposited cyclosporine passed through the epithelial layer whereas most of the drug accumulated within the epithelium. thus inhaled cyclosporine may not be capable of inactivating airway allogeneic t cells since it may not reach the airway wall in high concentration. in this study, we hypothesized that inhaled cyclosporine alters airway epithelial signaling cascades that may ultimately result in reduced inflammatory cell recruitment to the lung. methods: human tracheobronchial epithelial (htbe) cells from healthy donors were grown in ali media to confluence at an air-liquid interface in millicells. differentiated htbes were treated on their apical surface with cyclosporine concentrations of 1000 and 10,000 ng/ml or vehicle for 24hr to mimic the effects of inhaled cycolsporine. the basilar and apical compartments (n=8-12 for each) were then assayed for cytokine secretion (il8, il6, il1, il-12p40, tnf, eotaxin, mcp-1, gm-csf, rantes and egf) by luminex assays in pg/ml. results: 10,000ng /ml of cyclosporine markedly blunted the basilar secretion of il-6 (250 ±142 vs 71± 24, p= 0.002), rantes (22±8 vs 9±3, p=0.003), gm-csf (21±16 vs 3±3, p=0.008) and mcp-1 (181±112 vs 28±19, p=0.002). il8 and eotaxin were unaffected; tnfα, il1β and il12p40 were undetectable. at 1000ng/ml cyclosporine, cytokine secretion was decreased to a lesser extent. a similar pattern of diminished cytokine secretion was seen in the apical compartment of this system. last, apical, but not basal, egf secretion was augmented by cyclosporine (2231±817 vs 1114±614 pg/ml, p=0.02). conclusion: cyclosporine decreased the secretion of critical cytokines and chemokines from human airway epithelial cells. these mediators are known to enhance mononuclear and t cell recruitment in a large variety of animal models of disease as well as in clinical studies. inhaled cyclosporine may work by reducing cell recruitment. this hypothesis will require in vivo testing. funded by: cf foundation. background: cytomegalovirus (cmv), human herpes virus -6 and -7 (hhv-6 and -7) are β-herpesviruses that commonly reactivate and have been proposed to trigger acute rejection and chronic allograft injury. the role of these viruses in the development of bos after lung transplantation remains unclear. we assessed the contribution of β-herpesvirus infection in the allograft by a prospective molecular assessment of serial broncho-alveolar lavage (bal) samples in lung transplant recipients. methods: quantitative real-time pcr of bal samples were performed for cmv, hhv-6 and hhv-7 in a prospective cohort of lung transplant recipients. a time-dependent cox regression analysis was used to correlate the risk of bos and acute rejection in patients with and without β-herpesviruses infection. results: 93 patients were included in the study over a period of 3 years. a total of 581 samples from bal were obtained (median 6 per patient). 61/93 patients (66%) had at least one positive result for one of the β-herpesviruses: 48 patients (52%) for cmv, 19 patients (20%) for hhv-6, and 19 patients (20%) for hhv-7. median time to detection was 152 days (range 8-839) for cmv, 309 days (range 28-928) for hhv-6, and 286 days (range 17-928) for hhv-7. median peak viral load was 3,419 copies/ml (range 102-41,600,000) for cmv, 258 copies/ml (range 106-131,075) for hhv-6, and 665 copies/ml (range 103-66,100) for hhv-7. acute rejection (≥ grade 2) occurred in 46% and bos (≥ stage 2) in 19%. in the time dependent cox regression model, the relative risk of bos or acute rejection was not increased in patients with cmv, hhv-6, or hhv-7 reactivation. for example, the hazard ratio of cmv and bos was 1.04 (95% ci 0.62-1.73, p=0.9) and for cmv and acute rejection was 0.81 (95% ci 0.55-1.20, p=0.3). in many of the patients, β-herpesvirus reactivation occurred after the acute rejection episode likely reflecting augmented immunosuppression. abstract# 317 mannose binding lectin in this large cohort of lung transplant recipients, local reactivation of cmv, hhv-6 and hhv-7 in the allograft was very common. however, despite high viral loads in many patients, infection was not significantly associated with the development of acute rejection or bos. introduction: the role of chemokine receptors in regulating donor-specific responses to allografts is poorly understood. cd4 + cd25 + t cells regulate alloreactive cd4 t cell responses and acute rejection of single class ii mhc-disparate cardiac allografts in c57bl/6 mice. ccr5 is expressed by a small proportion of cd4 + cd25 + t cells but the requirement for these cells in regulating alloreactive t cell responses remains poorly understood. the goal of this study was to investigate the role of ccr5 + t regulatory cells in acute rejection of single class ii mhc-disparate cardiac allografts. methods: wild-type c57bl/6 (h-2 b ) and b6.ccr5 -/received heterotopically transplanted b6.h-2 bm12 mice heart grafts. the presence of cd4 + foxp3 + t cells in the recipient spleen and in heart allografts was determined by flow cytometry. foxp3 mrna expression in the heart grafts was analyzed by qrt-pcr. donor-specific cd4 + t cells producing ifn-g or il-4 in allograft recipient spleens were enumerated by elispot assay. cell sorted naïve wild-type cd4 + cd25 + t cells and cd4 + cd25 -t cells were adoptively transferred to wild-type and ccr5 -/mice before the cardiac transplant. results: in wild-type recipients >80% b6.h-2 bm12 cardiac grafts survived more than 100 days whereas ccr5 -/recipients rejected the allografts within 24 days (18 days mean survival) with intense cd4 + t cell infiltration in the graft. donor-reactive ifn-g and il-4 producing cd4 + t cell numbers were increased 2-fold in the spleens of ccr5 -/vs. wild-type recipients at day 7 post-transplant and in contrast to wild-type recipients these numbers were sustained for at least 7 more days. allograft infiltrating cd4 + cd25 + foxp3 + cells and intra-graft foxp3 mrna expression were clearly present in allografts from wild-type recipients and were virtually absent in allografts from ccr5 -/recipients. transfer of purified wild-type cd4 + cd25 + t cells to ccr5-deficient mice resulted in the long-term survival of 60% of b6.h-2 bm12 cardiac allografts. conclusion: ccr5 + regulatory t cells control the magnitude and function of the alloreactive t cell immune response to single class ii mhc-disparate cardiac allografts. profile that were distinct from those of cd4+cd25+ tregs, naïve cd4+cd25-t-cells, and activated cd4+ t-cells. furthermore, the cd4+ converted dn t-cells were highly potent in suppressing antigen specific alloimmune responses in vitro. in this study, we further characterized and test the functional potential of the converted dn t-cells in vivo. we showed that the converted dn t-cells retained a stable phenotype after re-stimulation in vitro and in vivo. il-2 was capable of breaking the anergic status and reserving the suppressive function of dn t-cells. in an immunocompetent mhc completely mismatched islet transplant model, the transfer of 13 x 10 6 dn t-cells (converted from cd4+cd25-t-cells of naïve c57bl/6 mice by co-culture with mature dba/2 dc plus ril-15 in mlr for 6 days) resulted in a statistically significant prolongation of alloantigen specific dba/2 strain, but not third party c3h strain, islet allograft survival in c57bl/6 recipients in comparison with that of untreated control group. as il-2 was capable of breaking the anergic status and reserving the suppressive function of dn t-cells, we added il-2/fc, a long-lasting form of il-2, and low dose rapamycin with dn t-cells in a mhc mismatched skin allograft model. the single transfer of 5 x 10 6 dn t-cells plus 28 days il-2/fc and low dose rapamycin treatment significantly prolong dba/2 skin allograft survival in c57bl/6 recipients in comparison with untreated group (mst 71 days vs. 11 days, p=0.0049) and il-2/fc plus rapamycin treated group mst (71 days vs. 34 days, p=0.0091). the results of using ex vivo cd4+ t-cells converted dn t-cells in skin and islet transplantation models support the concept and the feasibility of potentially utilizing this novel cell-based therapeutic approach clinically for the prevention of allograft rejection. jessamyn bagley, 1 jonathan g. godwin, 1 joren madsen, 2 john iacomini. 1 introduction: it has been suggested that natural killer (nk) cells are critical mediators that connect the innate and adaptive immune response. cytokine production by nk cells contributes to the polarization of immune responses to t helper 1, and nk cells express co-stimulatory molecules that may affect t cell proliferation. recent work has shown that nk cells are involved in the chronic rejection of parental cardiac grafts by f1 recipients. we hypothesized that given the role of nk cells in t cell activation and proliferation, nk cells may play a role in the development and function of t regulatory cells (treg) which control alloreactive responses. methods: nk, cd4+ t cells and cd4+cd25+ treg were purified from the spleens of c57bl/6j mice using macs bead separation followed by fluorescence activated cell sorting. naïve cd4+cd25-t cells from c57bl/6j mice (h-2b) were placed in culture with tgf-beta in the presence or absence of nk cells, and the development of treg was monitored by assessing foxp3 expression by intracellular cytokine staining and flow cytometry. in addition, the effect of nk cells on the function of treg was measured by elispot assay. results: following 4 days of culture with tgf-β and anti-cd3 antibody, cd4+cd25-t cells acquire foxp3 expression. the addition of activated nk cells to cultures with cd4+cd25-t cells and tgf-β prevented the acquisition of foxp3 expression by cd4 cells. tregs induced by stimulation of cd4+cd25-t cells with anti-cd3 in the presence of tgf-β are capable of suppressing the production of il-2, ifn-γ and il-4 by cd4 t cells in response to fully allogeneic balb/c stimulators. however, in the presence of activated nk cells, induced tregs fail to function, and production of il-2, ifn-γ and il-4 by cd4 t cells is restored. these experiments were repeated with natural cd4+cd25+ tregs isolated directly from mice without induction, and found that the ability of natural treg to suppress a cd4 t cell response to alloantigen was impaired in the presence of activated nk cells. conclusions: the presence of activated nk cells in culture can prevent the development of induced treg. furthermore, the presence of activated nk cells interferes with the function of mature treg in culture and allows a productive cytokine response by cd4 effector cells in response to alloantigen. results: both the syngeneic b6 cells and allogeneic dba/2 cells survived nicely in the rag-/-il-2rg-/-mice. however, the allogeneic dba/2 cells, but not the syngeneic b6 cells, were readily killed by the nk cells in the rag-/-mice. however, both rag-/-and rag-/-il-2rg-/-mice accepted the dba/2 skin allograft long term without any sign of rejection (>100 days). thus, nk cells by themselves, though cytolytic to dba/2 cells, fail to reject the dba/2 skin allografts. to test the hypothesis that the activation status of nk cells may dictate their alloreactive potential, we treated the rag-/-mice with il-5/il-15ra complex to maximally stimulate the nk cells in vivo. we found that il-15 is remarkably potent in stimulating nk cells in vivo; and nk cells stimulated by il-15 express an activated phenotype and are surprisingly potent in mediating acute skin allograft rejection in the absence of any adaptive immune cells, as il-15 treated rag-/-, but not the rag-/-il-2rg-/-mice, readily rejected the dba/2 skin allografts (mst=18 days). nk cell-mediated graft rejection doesn't show features of memory responses. and suggests that the fate of the allografts may depend on the activation status of nk cells and the availability of nk stimulating cytokines. background: t-bet is a transcription factor that promotes th1 development. both t-bet and the cytokines ifng and il-4 have been implicated as negative regulators of th17. in contrast, il-6 promotes th17 development. il-17 production is associated with granulocytic pathologies in several disease states. hence, this study assessed the relationship between t-bet, ifng, il-4 and il-6 in th17 induction and granulocytic infiltration of cardiac allografts. (ifng-/-) mice were transplanted with balb/c cardiac allografts. recipients were left untreated, depleted of cd4+ or cd8+ cells, treated with anti-cd40l mab, and/or treated with neutralizing anti-il-4 or anti-il-6 mab. graft histology was assessed and primed donor-reactive th responses were quantified by elispot. intragraft expression of il-17 and the th17 transcription factor rorgt were quantified by real-time rt-pcr. results: wt and t-bet-/-mice rejected their allografts at a similar tempo but with distinct pathologies: allografts in t-bet-/-recipients were heavily infiltrated with granulocytes while graft infiltrating cells in wt recipients were primarily mononuclear. while th1 and th17 responses were readily detectable in t-bet-/-recipients, th1 dominated the response in wt mice and th17 were not detectable. depletion of cd4+ cells prolonged graft survival in wt, but not in t-bet-/-recipients suggesting that cd8+ cells mediated rejection independent of cd4+ help in t-bet-/-mice. cd8+ cells were the source of il-17 in t-bet-/-recipients. anti-cd40l therapy promoted long-term allograft survival in wt recipients, but not t-bet-/-mice unless cd8+ cells were depleted. additionally, anti-cd40l therapy inhibited th1 responses in both wt and t-bet-/-recipients, but not the cd8+ th17 response in t-bet-/-mice. eliminating ifng and il-4 failed to induce il-17 production, while neutralizing il-6 reduced the th17 response in t-bet-/-mice. conclusions: while cd4+ th17 have been described in detail, cd8+ th17 have received less attention. in t-bet-/-allograft recipients, cd8+ th17 emerge independent of cd4+ help. cd8+ th17 are resistant to anti-cd40l therapy and are associated with granulocyte infiltration of the graft. these data implicate t-bet, as opposed to ifng and il-4, as a negative regulator of the th17 response while il-6 is required for cd8+ th17 induction. nan zhang, 1 bernd schroppel, 1 girdari lal, 1 jordi c. ochando, 1 jonathan s. bromberg. 1 1 background: cd4 + cd25 + foxp3 + regulatory t cells (treg) are important in suppressing immunity to prolong allograft survival. treg migration and its effects on suppressive activity are poorly understood. we determined treg migration patterns and effector function in an islet allograft model. ccr2 -/-, ccr4 -/-, ccr5 -/-, ccr7 -/-, l-selectin (cd62l) -/-, and fucosyltransferase (fuct) iv-vii -/-c57bl/6 mice were used to generate treg. treg were transferred intravenously, or locally to islet allografts, following islet transplantation (balb/c into c57bl/6). transferred treg were labeled with red dye pkh26 and migration to islet grafts, draining lns (dln), peripheral lns and spleen were tracked with fluorescence microscopy and flow cytometry. islet allograft survival was determined by measurement of blood glucose. results: treg expressed p-selectin ligand, cd62l, and a panel of chemokine receptors similar to other t cell subsets. intravenously transferred wild type treg migrated to both islet grafts and dln, and prolonged allograft survival. cd62l -/or ccr7 -/-treg, which migrated to islets but not dlns, prolonged allograft survival as potently as wild type treg. fuct iv-vii -/-treg, which lack e-selectin and p-selectin ligands, migrated to dln, but not islets, and did not prolong graft survival. similarly, ccr2 -/-, ccr4 -/-, and ccr5 -/-treg migrated to dln, but not islets, and did not prolong graft survival. when locally transferred to the islet graft, treg also migrated from the allograft to the dln, and prolonged graft survival even longer than after intravenous transfer. locally transferred ccr2 -/-, ccr5 -/-, or ccr7 -/-treg were not able to migrate from the islet to the dln, and were impaired in their ability to prolong islet survival. conclusion: treg migration to allografts is essential for their suppressive function; migration to lymphoid tissues alone is not sufficient to prolong graft survival. treg migration from the islet allografts to the dlns, via afferent lymphatics, is also required for optimal suppressive function and graft survival. the sequential migration from the site of inflammation and then to dlns is necessary for treg to execute fully their suppressive program. these results demonstrate a novel and important aspect of migration in treg suppression and tolerance. abstracts failure is unknown. methods:40 patients with iothalamate gfr <40ml/min (n=34) or on dialysis (n=6) at the time of liver transplant evaluation had undergone a percutanous ct guided renal biopsy. prior to the biopsy an inr ≤1.5 and platelet count ≥50,000/ ml were achieved in the majority of cases. all patients were monitored overnight for complications. candidates were listed for slk if pathology showed ≥ 40% glomerulosclerosis (gs) or ≥30% interstitial fibrosis (if). results:13 patients were eligible for slk and 27 for lta.creatinine was higher in slk candidates but not clinically different. background -it is well known that delayed graft function (dgf) is costly for those who received a renal transplant. however, the true cost of dgf is unknown. methods -we estimated the cost of dgf for adult cadaveric renal recipients in the usrds 1995-2004 who had medicare as their primary payer. those included were restricted to single organ recipients as well as those who had no previous transplants. cost was defined as the accumulated average cost per day for everyone with a functioning graft on that day. we examined the total cost of dgf, cost associated with dialysis, and non-dialysis cost for all patients combined and separately by donor type; standard criteria donors (scd), expanded criteria donors (ecd), and non-heart beating donors (dcd) as well as time to graft failure or no graft failure. background: based on adverse outcomes during the first year post-transplant, the optn membership and professional standards committee (mpsc) peer-review process flags transplant programs for further review using one of two methods. programs performing at least 10 transplants during at 2.5 year cohort are flagged based on the comparison of observed to expected event counts (death or graft failure) and the corresponding p-value (<0.05). programs performing 9 or fewer transplants are flagged if they have any adverse events. this leads to different flagging rates for programs of different sizes. the p-value has low sensitivity to identify poorly performing programs with a "moderate" number of transplants (10 to 20) whereas flagging every event results in a high false positive rate for "small" centers with <10 transplants. during the july 2007 review, 27% of "small" centers and 7% of centers with 10+ transplants were flagged for review (all organs). the scientific registry of transplant recipients (srtr) has developed alternative approaches for flagging centers with more consistent flagging rates. methods: the new method would allow for different choices of sensitivity (rr) and specificity (p-value) for different purposes and would flag program if either {p-value < .05 (a different value could be chosen)} or {observed / expected > rr}. the resulting false negative rate is less than 50% for centers with the given rr. this approach avoids an arbitrary definition of small versus large programs, and has sensitivity (or power) > 50% to flag programs with the selected rr, regardless of size. results: the following discussion: this approach gives a more balanced distribution of flagging across programs of different sizes and has sensitivity >50% for transplant programs of all sizes. with the choice of rr=2.5, the overall number of centers flagged would be nearly unchanged from current methods. center performance ratings are of increasing importance to the transplant community with the introduction of the cms final rule. ongoing debate exists regarding how much center ratings are directly a reflection of quality of care or whether ratings can be substantially influenced by exogenous factors. the study examined data from the national srtr database from 2000-2007. centers' semi-annual graft and patient survival were calculated along with a comparison with expected outcomes adjusted for covariates used by the srtr. centers meeting three criteria for poor performance were categorized within each cohort. patient characteristics at each center were compared with performance evaluations. overall, half of transplant centers met criteria for low performance in at least one of the semi-annual intervals. several center factors investigated were not significantly associated with the likelihood to meet low performance criteria including the proportion of older, obese and privately insured patients. in contrast, centers with higher levels of ecd transplants (most ecds=70% vs least ecds=40%), african american recipients (most aas=75% vs least aas=35%) and patients with low albumin level (lowest albumin =60% vs highest albumin =32%) were more likely to meet low performance criteria. approximately half the centers that initially met criteria for low performance no longer met criteria when excluding ecd transplants and african american recipients from the performance assessment. conclusions given the extreme implications of performance ratings for transplant centers including possible loss of funding to centers with low performance, it is critical that we recognize potential weaknesses and biases of performance ratings. our results are important towards understanding factors related to performance ratings and raising questions as to whether risk adjustment techniques are adequate for fair transplant center performance evaluations. rather than only risk adjustment, stratified evaluations may be a partial solution to remove disincentives to performing higher risk transplants. it is also important to recognize factors not associated with low performance such that centers do not unnecessarily limit access to groups based on perceived deleterious impact on ratings. model. olaf boenisch, 1 takaya muramatsu, 1 francesca d'addio, 1 robert padera, 1 hideo yagita, 2 nader najafian. 1 1 transplantation research center, brigham and woman's hospital and children's hospital, boston, ma; 2 immunology, juntendo university of medicine, tokyo, japan. t cell immunoglobulin and mucin domain (tim)-3, a molecule expressed on terminally differentiated th1 cells, is an important regulator of th1 autoimmunity and in induction of transplantation tolerance. its functions in alloimmune responses during acute and chronic rejection are unknown. tim 3-23, a novel blocking antibody of tim-3, was administered to recipients in various donor-recipient strain combinations on days 0 (500ug), 2, 4, 6, 8 and 10 (250ug) after transplantation. the frequency of ifng-, il-4-, and granzyme b-producing splenocytes was measured by elispot. these data establish the regulatory functions of tim-3 in alloimmune responses in solid organ transplantation models. the inhibitory actions may be secondary to modulation of effector or regulatory t cells and appear to dominate in conditions of low levels of t cell activation, due to a restricted degree of allogeneic mismatch or absence of cd28 costimulation. the i-r injury in transplanted kidney is a major cause of dgf, an event associated with an increased risk of acute rejection. adaptative immunity was suggested to play a role in the pathogenesis of renal i-r injury, although the influence of the th1/th2 bias in this scenario is still debated. thus, the aim of the present study was to evaluate the features of t cell response during i-r injury at the peripheral and tissue level in renal graft recipients with dgf. the mrna levels of specific th1 (t-bet) and th2 (gata-3) transcription factors were evaluated in circulating lymphomonocyte of kidney transplant recipients with early graft function (egf) (n=10) and dgf (n=10), before (t0) and 24 hours after transplantation (t24) by real time pcr. infiltrating lymphocytes were characterized in graft biopsies of patients with dgf (n=40) and in a control group of patients with tubular damage by acute cni toxicity (n=10) by immunohistochemistry. in addition, we evaluated the th1/th2 bias at the renal level in a pig model of i-r injury. t-bet/gata-3 mrna ratio was similar in the 2 groups of patients at t0. at t24 the dgf group presented a significantly higher increase of t-bet/gata-3 ratio compared with the egf group (798±346 vs 288±147% of t0, p<0.001). moving to the tissue level, dgf patients presented a number of interstitial cd4 + (8.0±5.1 vs 2.6±2.1, p=0.04) and cd8 + (10.8±6.7 vs 4±2, p=0.02) t cells significantly higher compared to the control group, while no significant differences were observed in cd20 + cells number between the two groups. also at the tissue level the ratio between t-bet + and gata-3 + cells was significantly higher in the dgf compared with the control group (3.5±1.8 vs 1.1±0.9, respectively, p=0.005). to confirm that these changes were due to i-r, we investigated the presence of t-bet + and gata-3 + cells in a pig model of i-r injury. interestingly, the ratio was significantly increased after 24 hours of reperfusion (basal 2.5± 0.9 vs 24 hours 8.1±3.6; p=0.02). in conclusion, our results suggest that kidney transplant recipients with dgf present a bias toward a th1-driven immune response both at the peripheral and at the tissue level. this event, due to the i-r process, as suggested by the animal model, may represent a link between dgf and acute graft rejection. as expected a strong negative association between duration of dialysis and patient survival was seen in caucasian recipients. in contrast the relationship between patient survival and duration of dialysis was u shaped in minorities with the worst patient survivals seen among preemptive transplants and those patients with over 60 months of dialysis. the difference in outcomes between caucasians and minorities could be related to the biologic differences in the effect of dialysis on subsequent transplantation or to a differential selection bias introduced by duration of dialysis in the two populations. anti-carbohydrate natural antibodies. lorenzo benatuil, 1 jonathan g. godwin, 1 shamik gosh, 1 john iacomini. 1 1 transplantation research center, boston, ma. background. we constructed immunoglobulin gene knock-in mice lacking expression of the αgal epitope that carry rearranged vh and vl genes encoding antibodies specific for the carbohydrate antigen galα1-3galβ1-4glcnac-r (αgal). here, we describe two novel populations of b cells in the spleen of these m86vhvlgt 0 mice and their role in the production of αgal specific antibodies. methods. m86vhvlgt 0 mice were sacrificed and single cell suspensions from spleen, bone marrow, peripheral lymph nodes and peritoneal cavity were prepared and stained with different antibodies and with fluorescently labeled αgal-bsa for flow cytometric analysis. using multiparameter cell sorting, we purified marginal (mz) and follicular (fo) zone b cells, in addition to two novel splenic b cell populations. cells were adoptively transferred into b cell deficient µmt/gt 0 double knock-out mice. serum samples were collected, and production of αgal specific igm antibodies was assessed by elisa. to investigate these b cell tolerance mechanisms, we employed mice with naturally occurring antibodies (abs) against human blood group a carbohydrates in their sera and possessing b cells with receptors for blood group a determinants. b cells with receptors for a carbohydrates in mice belong to the cd5 + b-1 subset, with phenotypic properties similar to those of human b cells. when these cells were temporarily eliminated by injecting synthetic a carbohydrates, subsequent treatment with cyclosporin a or tacrolimus, which blocks b-1 cell differentiation, completely inhibited the reappearance of b cells with receptors for a carbohydrates in mice. it is probable that calcineurin inhibitors used for preventing t cell-mediated rejection simultaneously suppress b-1 cell differentiation. however, despite a very limited dose of calcineurin inhibitors, the b cell tolerance toward blood group a antigens was persistently maintained in the blood group a-to-o liver transplant recipient. b cell tolerance after abo-incompatible transplantation might be a consequence of presentation of blood group carbohydrate antigens by cells in the engrafted liver. immune fluorescence staining of the human liver reveals that blood group antigens are predominantly expressed on the liver sinusoidal endothelial cells (lsecs). we have previously proven that lsecs, which constitutively express fas-l and pdl-1 have the capacity to tolerize alloreactive t cells. taken together, we hypothesize that blood group antigen-reactive b cells are also tolerized through the interaction with the lsecs. in order to address this possibility, we used α-1,3galactosyltransferase-deficient mice. when the α-gal-expressing lsecs isolated from wild-type mice were adoptively transferred via the portal vein into the splenectomized congeneic α-gal-deficient mice, these mice lost the ability to produce anti-α-gal abs (n = 4). this finding suggests that the lsecs expressing blood group carbohydrates play a pivotal role in the tolerization of newly developed b cells specific for the corresponding carbohydrate antigens after abo-incompatible liver transplantation. success in kidney transplantation has resulted from control of t-cell-mediated acute rejection. however, little has been done to improve the fate of patients who possess pretransplant donor-specific antibody (dsa), and no proven therapies exist to specifically prevent dsa formation post-transplant. while methods to detect and characterize dsas are clearly useful, the b-cell subsets that produce dsas or more importantly sustain their production are poorly characterized. this study set out to investigate the fundamental mechanisms determining the formation of donor-specific memory b cells and plasma cells in novel mouse models of dsa formation. we have developed two complementary and novel systems to track the phenotypic and functional properties of polyclonal donor-specific b-cells. the first system involves allosensitization between normal c57bl/6 and balb/c mice. we used advanced flow cytometric methods to track donor-specific b cells with h-2k b or h-2k d tetramers. tetramers are comprised of four identical h-2 molecules bound to a fluorescently labeled steptavidin molecule that is able to bind the donor-specific b-cell antigen receptor (surface immunoglobulin). in our second system, we use donor mice that constitutively express full-length membranebound chicken ovalbumin (mova) protein in all tissues. analogously, ova specific-b cells can be analyzed flow cytometrically with the use fluorescently-labeled ovalbumin. both systems allow us to identify donor-specific memory b cells (7aad -cd4 -cd8 -ova/ tetramer + igd -b220 + cd138 -) and plasma cells (7aad -cd4 -cd8 -ova/tetramer + igd -fb220 lo cd138 + ) during skin graft rejection (day 14 post transplant). we were also able to track the development of a stable memory cell population in the bone marrow and spleen for > 90 days cells.for the ova system quantitative elisa and elisot measurements for serum dsa and dsa-secreting cells, respectively, correlated with the development of donor-specific memory b cells. using these assays we will be evaluate polyclonal donor-specific b memory subsets under multiple conditions of immunity and tolerance and for the first time, characterize their functional properties and migration patterns. these experiments will provide new information on the basic biology of the memory b-cell response to allografts in mice and facilitate the development of these methods in sensitized patients that may lead to critical therapeutic opportunities for dsa production. in the tnf-related b-lymphocyte survival factor, blys/baff, is critical for primary follicular (fo) and marginal zone (mz) b-cell survival. in vivo neutralization of blys/ baff, using a newly developed monoclonal antibody, depletes the fo and mz b-cell compartments. here, we hypothesized that targeting b-lymphocytes, via the blys/baff pathway, could promote humoral tolerance to islet allografts. cohorts of stz-diabetic c57bl/6 mice were transplanted with isolated islets from balb/c donors. treatment with anti-blys/baff alone (100mcg/mse x2 doses+15mcg/wk/mse) did not protect islet allografts from acute rejection (mst=14d; n=4) . on the other hand, a 14-day course of rapamycin (1mg/kg) prevented acute allograft rejection (mst=35d; n=9). when rapamycin was combined with the anti-blys regimen, islet allograft survival was markedly prolonged (mst>150d; n=4). importantly, islet allograft survival was coincident with the absence of detectable serum alloantibodies and blunted donor specific t-cell responses. following treatment with anti-blys, b-lymphocyte compartment reconstitution was detectable at 30 days following treatment and was characterized by stringent selection at the transitional→fo b-cell tolerance checkpoint. overall, these data indicate that the blys/baff pathway may be a logical target of immunomotherapy for the achievement of humoral transplantation tolerance. reza tavana background: highly sensitized patients' ability to be transplanted is severely compromised because of high level of antibodies against various hla antigens. interleukin-21 is a type i cytokine that signals through a receptor composed of the il-21r and the common cytokine receptor -chain ( c ). it is produced by t-cells and has been shown to be the contributing factor in terminal differentiation of memory b-cell to anti-body producing b-cell and plasma cells. objective: we evaluated the effect of il-21 co-stimulation on antibody production capacity of b-cells and also measured the expression of il-21r on b lymphocytes of highly sensitized patients compared to non-sensitized patients on the kidney transplant list. methods: patients with a pra level >20% (sensitized) were compared to non-sensitized patients from the transplant waiting list. after consent was obtained, peripheral blood was taken from patients before initiating hemodialysis. leukocytes were labelled with anti-cd19, anti-il-21 antibodies and paraffin fixed after rbc lysis. il-21r expression was measured by flow-cytometry over facscan machine on the same day. the expression of the il-21r is significantly higher on b-lymphocytes of highly sensitized patients (hsp) compared with non sensitized patients (nsp) (fig 1.p<0.05 ). in-vitro co-stimulation of isolated peripheral b-cell with il-21 and anti-cd40 results in higher igg1 production compared with anti-cd-40 or il-21 stimulation alone (fig 2) . conclusions: il-21 is an important cytokine in b-lymphocyte stimulation and increases igg1 production. il-21 receptor on b-lymphocytes is up-regulated in sensitized kidney transplant recipients. il-21 to il-21r interaction between t and b-lymphocytes may be an important pathway in antibody production in highly sensitized renal transplant recipients. we created mixed bone marrow chimeras in irradiated µmt (b cell-deficient) recipients by transplantation of syngeneic bone marrow from µmt, wildtype (wt) and mhcko (lack mhc i & ii expression on all cells) mice. µmt+mhcko chimeras lack expression of mhc i & ii on b cells but not other professional apcs such as dcs hence antigen presentation specifically by b cells is disrupted. allograft rejection and development of alloreactive memory t cells in µmt+mhcko chimeras was compared to µmt+wt chimeras that had intact antigen presentation by both b cells and apcs. skin allograft rejection was comparable between µmt+mhcko and µmt+ wt chimeras (mst = 17 and 16 days, respectively, p = 0.6, n = 5/grp). however, heart allograft rejection was significantly delayed in the µmt+mhcko chimeras compared to µmt+ wt chimeras (mst = 23 and 15 days, respectively, p=0.03, n = 5/ grp). development of alloreactive memory t cells was assessed in chimeras at 8 weeks after skin allograft rejection by quantitation of antigen-specific ifnγ producing t cells. alloreactive cd4 and cd8 memory t cells were significantly fewer in µmt+ mhcko chimeras (7-fold fewer cd4, p = 0.02, and 5-fold fewer cd8, p = 0.003, n = 5/grp) than in µmt+ wt chimeras. these results show that the disruption of antigen presentation by b cells significantly delays heart but not skin allograft rejection. development of alloreactive cd4 and cd8 memory t cells is significantly impaired in the absence of antigen presentation by b cells. conclusions: antigen presentation by b cells accelerates heart allograft rejection and leads to development of alloreactive memory t cells. these findings emphasize antibodyindependent functions of b cells in promoting alloimmune responses and highlight the need for b-cell targeted therapies to improve long-term allograft survival. purpose: to test whether depletion of cd20+ b cells at the time of engraftment alters the prevalence of anti-donor alloantibody (ab)or severity of cav in the context of therapeutic immunosuppression with (csa) or high dose cd154 inhibition. methods: forty-five mlr-mismatched heterotopic cardiac cynomolgus allograft recipients were treated with high dose anti-cd154 monotherapy (αcd154; n=14, 5 with atg induction) or αcd154 with additional anti-cd20 therapy (rituximab 20mg/ kg q wk for 4 weeks: αcd154 + αcd20; n=18, 11 with atg). thirteen other animals received therapeutic csa (target trough >500ng/ml), five of which received additional αcd20. graft survival was censored at 90 days. ab was deemed (+) if present (by flow cytometry) around time of explant. results: 12 animals died with beating grafts, mainly with atg-associated lung pathology or infectious etiologies and are excluded from this analysis. 3 animals that had sustained αcd154 levels <100µg/ml after protocol day 30 were considered "subtherapeutic" for this reagent and also excluded from further consideration. graft survival with αcd154 + αcd20 (median >90d) and proportion of grafts surviving to 90 days (6/6) was significantly increased relative to αcd154 alone (median 43d, p=0.007; 4/14 >90d). graft survival with csa + αcd20 (median >90d) and proportion of grafts surviving to 90 days (4/4) was increased relative to csa alone (median 66d, 3/6 >90d). with therapeutic αcd154 (trough level >100 µg/ml until graft explant), 12/13 (92%) developed ab vs. 1/6 with αcd154 + αcd20 (17%) (p=0.006). average cav score for the αcd154group was 2.44 ±0.44 vs. 0.7 ± 0.8 in the αcd154 + αcd20 group (p=0.006 using unpaired t test). preliminary cav scoring suggests that added αcd20 inhibited cav (scores ranging 0.0-0.1) relative to csa alone (median 2.1; range 1.5-2.4); ab analysis is in progress for these groups. conclusions: using αcd20 is associated with significant attenuation of cav when used with either "therapeutic" αcd154 or csa. our findings demonstrate for the first time that αcd20 reduces the severity of cav in conjunction with both conventional immunosuppression and costimulation pathway blockade. mechanisms include inhibition of ab production, and perhaps others that remain to be defined. subsaturating concentrations of anti-hla antibody ( sensitized recipients having donor specific anti-hla abs have been successfully transplanted following a conditioning regimen employing plasmapheresis with or without pooled human immunoglobulin. in vitro studies have shown that exposure of ecs to subsaturating concentrations (ssc) of anti-hla ab (priming) followed by subsequent exposure to high concentrations (hc) of anti-hla induces the expression of protective genes, bcl2 and ho-1 and confers protection to complement mediated lysis of ecs. however, the molecular events following priming with exposure to ssc of anti-hla ab still remains undefined. to determine the priming events and to define the kinetics of protective gene expression, we exposed human aortic ecs to ssc of anti-hla ab for 72 hrs and re-exposed them to saturating concentrations of anti-hla abs. ecs were collected at 24, 48, and 72 hrs after exposure to ssc as well as 24 hrs after exposure to hc. expression profile of signaling intermediates (mapk, wnt, nf-kb, hedgehog, pi3kinase, stress pathway, tnf family) in the ecs were analyzed by gene array. analysis of the bcl2 and ho-1 expression showed no significant increase in expression following exposure to ssc of w6/32 (priming) or control ab alone at any of the time points ( background/aims: microcirculation disturbance, endothelial injury and cytokine overproduction are implicated in the pathophysiology of hepatic ischemia reperfusion injury (iri). thrombomodulin (tm) is a membrane-bound endothelial thrombin receptor that accelerates thrombin-catalyzed protein c activation, inhibits thrombin-induced fibrin formation, and also regulates inflammation. in the present study, we investigated the effects of recombinant human soluble tm (rhstm) on hepatic iri in the rat. methods: wister hannover rat was used for preparing ischemia/reperfusion model. hepatic iri was induced by subjecting 70 % area of the rat liver to 90 minutes of ischemia followed by 6 h of reperfusion. the rats were randomly assigned to a group receiving an intravenous injection of rhstm (3 mg/kg body weight) and to a group treated with saline 60 minutes prior to the beginning of reperfusion. sinusoidal endothelial cells (secs) and kuppfer cells (kcs) were isolated using centrifugal elutriation. the plasma levels and the concentration in cultured cell supernatant of il-6 and tnfα were measured by specific enzyme immunoassays. results: the plasma alt, ast and hyaluronic acid levels were significantly decreased, and the histological damage of the liver was attenuated in rhstm-treated rats as compared to control rats. using laser doppler flow-meter we found that rhstm treatment improved hepatic microcirculation. the intrasinusoidal fibrin deposition, injury of secs and liver dysfunction during hepatic iri were weaker in rhstm-treated rats than in control rats. tm activity in secs was significantly recovered, and plasma il-6 and tnfα were significant decreased in rhstm-treated rats as compared to control rats. further, il-6 and tnfα production in isolated kcs was also significant decreased in rhstm-treated rats as compared to control rat . conclusion: the present results suggest that rhstm is useful for the prevention of secs damage and kcs activation induced by iri. our present study also suggests that disturbance of hepatic microcirculation is induced in part by intrasinusoidal microthrombus formation and by locally released inflammatory cytokines from kcs. protective effects of preservation solution including activated protein c in small-for-size liver transplantation in rats. background: small-for-size liver graft is a serious obstacle of partial orthotopic liver transplantation (olt). however, various therapeutic strategies including surgical innovations, pharmacological agents and gene therapies to protect small-for-size liver graft have not yet been developed. aims: activated protein c (apc) is known to have cell protective properties via its anti-inflammatory and anti-apoptotic activities. this study aimed to examine the cytoprotective effects of preservation solution containing apc on olt using smallfor-size rat liver graft (20% partial liver). methods: liver grafts were assigned to two groups: in the control group, the grafts were flushed and stored in histidine-tryptophan-ketoglutarare (htk) solution alone for 6 h; in the apc group, in htk solution containing apc for 6 h. results: the apc group significantly increased 7-day graft survival from 60% to 100%, decreased levels of transaminase, and improved histological features of hepatic iri compared to the control group. myeloperoxidase activity demonstrated that the apc group markedly suppressed the infiltrations of neutrophil. hepatic expressions of tumor necrosis factor-α and il-6 of the apc group were remarkably decreased. the apc group significantly reduced serum hyaluronic acid levels, indicating attenuated sinusoidal endothelial cell injury. moreover, the apc group markedly increased hepatic levels of nitric oxide caused by upregulated endothelial nitric oxide synthesis (nos) together with downregulated inducible nos, and decreased hepatic levels of endothelin-1. finally, hepatocellular apoptosis of the apc group was remarkably suppressed by downregulated hepatic caspase-8 and caspase-3 activities. conclusions: preservation solution containing apc inhibited pro-inflammatory cytokine synthesis, which leads to hepatocellular apoptosis and liver injury. one of the cytoprotective effects of the apc treatment was to upregulate hepatic enos, followed by increased expression of hepatic no, and to decrease expression of hepatic et-1, resulting in the prevention of microcirculatory disturbance. preservation solution containing apc is a potential novel and safe product for small-for-size liver transplantation to improve liver graft function and animal survival. deletion of cd39 on natural killer cells attenuates hepatic ischemia/ reperfusion injury. living donor liver transplantation (ldlt) has emerged as a solution to ease organ shortage in orthotopic liver transplantation. however, ldlt is often complicated by small-for-size liver graft that is highly susceptible to injury and shows decreased liver regeneration. suppression of liver regeneration in small-for-size grafts correlates with impaired priming as a result of limited nf-κb activation and decreased production of the priming cytokines tnf and il-6. we have shown that the hepatoprotective protein a20 promotes liver regeneration, partly through blockade of the cyclin dependent kinase inhibitor p21. however the impact of a20 expression in livers on il-6 production and/ or signaling were still unknown. in this study we demonstrate that secretion of il-6 (elisa) following treatment with lps or tnf was moderately lower in hepatocytes transduced with a recombinant a20 adenovirus (rad) as compared to non-transduced or rad.β-galactosidase transduced cells. this indicates that il-6 production in hepatocytes is not solely nf-κb dependent (not totally blocked by the nf-κb inhibitor a20). despite similar or lower il-6 levels, il-6 signaling as evaluated by phosphorylation of stat3 (western blot; wb) was enhanced in a20 expressing hepatocytes. this was confirmed in experiments showing that a20 increases stat-3 phosphorylation in response to exogenous human il-6. accordingly, hepatocyte proliferation was significantly higher in rad.a20 transduced hepatocytes as opposed to controls. the pro-proliferative function of a20 mapped to the 7zn domain. since the balance of il-6 signaling in hepatocytes is finely regulated through a negative feed-back loop provided by the il-6/stat-3 dependent induction of suppressor of cytokine signal-3 (socs3), we investigated whether a20 affects il-6 signaling by modulating socs3 expression. our results indicate that a20 indeed decreased il-6 mediated upregulation of socs3 (wb). this later result was confirmed in vivo. improved regeneration and survival in a20 treated livers correlated with a substantial decrease in socs3 levels before and 24 hours following extended (78%) liver resection in mice. these results suggest that a20 enhances priming of hepatocytes by il-6 likely through down-regulation of socs3. this added to the effect of a20 on p21 would further enhance its pro-proliferative function in hepatocytes to benefit survival and function of small-for-size liver grafts. background: we have previously demonstrated that silencing inflammatory, apoptosis and complement genes can prevent ischemia-reperfusion (i/r) injury occurring in heart transplantation. however, the method for efficiently delivering sirna into donor organ has not been established. this study was designed to develop a new method to induce gene silencing by coronal artery infusing with sirna solution for prevention i/r injury in heart transplantation. methods: multiple sirnas that specifically target tnfa, caspase 8, and c5a receptor (c5ar) genes were generated and selected. sirna protection of donor organs was evaluated in a rat heart transplantation model. heart grafts from lawis rats were infused with sirna solution via coronal artery and preserved in hkt solution at 4° c for 18 hrs, and subsequently transplanted into syngeneic lawis rats. cardiac functions were assessed by heart beating rate. gene expression at mrna level was determined by qpcr. the i/r injury was assessed by immunohistochemistry. results: after donor heart perfusion with sirna solution, sirna was found to enter the myocardial cells, indicated by the fluorescence emitted from the dye labeled sirna. the levels of tnfa, caspase 8 and c5ar genes were significantly up-regulated in the grafts after ex vivo preservation for 18 hrs. these up-regulated tnfa, caspase 8, and c5ar genes were significantly knocked down by sirna infusion. using a sirna infused organ as a donor, the graft survival was significantly prolonged in heart transplantation. while sirna solution-treated heart grafts retained strong heartbeat up to the end point of observation (>10 days), the control grafts lost function within 3 days. in addition, an improved cardiac function was observed in the graft preserved in sirna solution. the protection of graft by sirna solution is associated with prevention of i/r injury. sirna solution-treated organs exhibited almost normal histological structures as well as less neutrophil and lymphocyte infiltration, compared with control solution-treated organs. conclusions: this study developed a novel ex vivo sirna delivery system using coronal artery infusion, which can effectively silencing genes in donor hearts and prevent cardiac i/r injury. background: ischemia/reperfusion (i/r) insult is a prime factor leading to liver dysfunction. apoptosis plays key role in the early graft loss following orthotopic liver transplantation (olt). bcl-xl has been showed to exert an anti-apoptotic function both in vitro and in vivo. this study was designed to evaluate potential cytoprotective effects and mechanisms for bcl-xl in liver i/r injury by ad-bcl-xl gene transfer. methods: a mouse model of partial 90 min warm hepatic ischemia followed by 6 h of reperfusion was used. balb/c wide-type (wt) mice (n=6/gr) were injected with ad-bcl-xl or adβ-gal reporter gene (2.5x10 9 pfu, i.p. at day -2). sham control wt mice underwent the same procedure, but without vascular occlusion. mice were sacrificed after 6 h of reperfusion; liver tissue and blood samples were collected for future analysis. results: ad-bcl-xl treated mice showed significantly lower sgot levels (iu/l), as compared with ad-β-gal or wt controls (509±361 vs. 2819±706, and 2212±841, respectively; p<0.005). these correlated with histologic suzukis grading of hepatic i/r injury, with wt/ad-β-gal controls showing significant edema, sinusoidal congestion/cytoplasmic vacuolization, and severe hepatocellular necrosis. in contrast, wt mice treated with ad-bcl-xl revealed minimal sinusoidal congestion without edema/vacuolization or necrosis. ad-bcl-xl gene transfer significantly reduced local neutrophil accumulation and apoptosis (3.8±2.9 of tunel+ cells in ad-bcl-xl vs. 21.5±5.3 and 23.5±6.5 of tunel+ cells in wt or ad-β-gal treated mice; p<0.01). unlike in controls, intragraft expression of mrna coding for tnf-α, e-selectin/icam-1, and ip-10/mcp-1 remained depressed in the ad-bcl-xl group. ad-bcl-xl gene transfer markedly depressed the activation of nf-κb, caspase-3, and increased ho-1, a20, and bcl-2/bcl-xl expression, as compared with wt/ad-β-gal controls. conclusion: this study demonstrates that inhibition of nf-κb activation contributes to the cytoprtective effects after bcl-xl gene transfer in hepatic i/r injury. the induction of anti-oxidant ho-1 and anti-apoptotic a20, bcl-2 by bcl-xl gene transfer exerts synergistic cytoprotective effect against antigen-independent hepatic inflammatory injury induced by i/r. hepatocyte background: primary graft non-function (pnf) affects survival and function of renal allografts. pnf, secondary to the ischemic and inflammatory injury in the peri-transplant period, leads to acute tubular necrosis and predisposes to acute rejection. defining new preconditioning regimens to reduce pnf are desirable. a20 is part of a negative antiinflammatory loop aimed at inhibiting nf-κb in renal proximal tubular epithelial cells (rptec). hepatocyte growth factor (hgf) is a pleiotropic growth factor upregulated in acute kidney injury and acute rejection likely to modulate inflammation and promote repair. in the present study we evaluated the effect of hgf on rptec and hypothesized that some of its protective functions may relate to the upregulation of a20. methods and results: treatment of rptec with hgf (50ng/ml) led to a 2.4±0.8 (n=4; p=0.04) fold increase in a20 mrna (real time-pcr), which translated into a significant 3.0±1.5 fold increase (n=5; p=0.04) in a20 protein by 6h, as shown by western blot (wb). two lines of evidence suggested that upregulation of a20 by hgf was nf-κbindependent. hgf did not degrade iκbα in rptec (wb) nor upregulated the nf-κb dependent molecule icam-1, as shown by flow cytometry analysis (facs). further, a20 was still upregulated in rptec expressing the nf-κb inhibitor iκbα, both at the mrna and protein levels. upregulation of a20 by hgf protected rptec from a subsequent inflammatory insult, here mimicked by the addition of tnf. pretreatment of rptec with hgf for 6 hours blunted tnf-induced (10u and 25u/ml) upregulation of icam-1, as analyzed by facs. conclusion: to our knowledge this is the first demonstration that a20 could be upregulated in rptec, in a non-nf-κb dependent manner. further studies are carried out to elucidate the transcription factors involved in hgf-induced upregulation of a20. from a clinical standpoint, these results highlight the unique ability of hgf to protect rptec from inflammation by inducing the anti-inflammatory protein a20, remarkably without triggering other pro-inflammatory signals. we propose that hgf-based therapies could serve in preconditioning regimens to prevent ischemia/reperfusion injury and reduce pnf and acute rejection in renal transplantation. background. liver ischemia reperfusion injury (iri) is one of the main causes of graft dysfunction and rejection in liver transplantation. it has been documented that iri is associated with inflammatory and complement pathway activation. this study was designed to investigate the efficacy of small interfering rna expression vector (shrna) targeting tnf-α and complement 3 (c3) genes in the protection of mouse liver iri. methods. shrna expression vectors were constructed for tnf-α and c3 genes. mice received shrna by hydrodynamic injection prior to iri, which consisted of interrupting blood supply to the left lateral and median lobes of the liver for 45 minutes followed by reperfusion. iri was evaluated using liver histopathology, as well as levels of serum alanine transferase (alt) and aspartate transaminase (ast). neutrophil accumulation was determined by a myeloperoxidase (mpo) assay. lipid peroxidation was assessed by malondialdehyde (mda) levels. realtime pcr was used to test gene silencing efficacy in vitro and in vivo. result. we demonstrated that iri is associated with an increase in tnf-α and c3 mrna levels in liver tissue 6 hours after reperfusion. shrna-treatment effectively down-regulated tnf-α and c3 expression in iri livers. in comparison with vehicle control, the serum levels of alt (9843.31 ±2610.66u/l vs 1960.00 ±1361.98 u/l) and ast (7188.25 ±3295.99u/l vs 1405.13 ± 774.65u/l), were significantly reduced in mice treated with tnf-α and c3 shrna. additionally, the neutrophil accumulation and lipid peroxidase-mediated tissue injury, detected by mpo and mda respectively, were improved after shrna treatment. tissue histopathology showed an overall reduction of injury area in shrna-treated mice. conclusion. this is the first demonstration that liver iri can be prevented through gene silencing of inflammatory genes and complement genes, showing potential for shrna-based clinical therapy. kidney -acute rejection: antibody-mediated rejection the (2) in the first three days after transplantation, a temporary decrease in dsa was observed in all amr cases, and all of them quickly rebounded thereafter; (3) c4d can be detected very early (can be seen on day 1, 89% in day 4 protocol biopsies, frequently in the absence graft dysfunction, and 100% in index biopsies); (4) the pathologic changes observed in sequential biopsies were c4d deposition followed by acute tubular injury, then interstitial inflammation and peritubular capillary margination seen in index biopsies; (5) pure amr occurred early (100% at day 3), usually evolving into mixed amr with accompanying cellular rejection (87% in index biopsies); (6) most recipients (87%) had initial graft function before developing amr; (7) background: antibody-mediated rejection (amr) has been recognized as a major problem in abo-incompatible (abo-i) renal transplantation (rtx). however, little is known about the long-term impact of amr in the abo-i renal transplant setting, especially after the introduction of a tacrolimus (fk)-based immunosuppressive regimen. the aim of this study was to assess the long-term impact of amr on the clinical and pathological outcomes in abo-i rtx. methods: fifty-eight patients who underwent abo-i rtx at our institution between march 1999 and december 2004 under an fk-based immunosuppressive regimen were enrolled in this study. protocol biopsies were performed regardless of renal function at one month and one year after rtx. fifty-six of the 58 patients received the biopsy at one month and 43 of 56 patients underwent biopsy at one year posttransplant. amr was diagnosed by morphological features based on the banff '05 update and other characteristic findings for amr previously reported, such as mesangiolysis, interstitial hemorrhage, and cortical infarction. we evaluated graft survival, incidence of chronic rejection characterized by transplant glomerulopathy (tgp) at one year posttransplant, and renal function using serum creatinine at three years posttransplant according to the incidence of amr at one month posttransplant. the overall graft survival rate at 3, 5, and 8 years after rtx was 93%, 87%, and 63%, respectively. the incidence of amr at one month was 34% (19/56). the graft survival rate of the patients with amr was significantly lower than that of the patients without amr (p<0.01, 3 years: 79% vs 100%, 8 years: 38% vs 94%). the incidence of tgp in the patients with amr was significantly higher than that of the patients without amr (p<0.001, 57% vs 7%). the serum creatinine concentration at three years after rtx was significantly higher in the patients with amr than in those without amr (p<0.001, 1.95 mg/dl vs 1.32 mg/dl). in this study, we revealed that amr in abo-i rtx is associated with not only graft loss but also the progression of chronic renal impairment, functionally as well as pathologically, even after the introduction of an fk-based immunosuppressive regimen. further studies are needed to establish a more effective immunosuppressive regimen, such as rituximab induction therapy; against amr in abo-i rtx. conclusions: de novo dsa in ar is an independent predictor of graft loss and its degree of influence is comparable to other established risk factors (aa race, dgf, increased baseline creatinine). additional studies are warranted to: 1) confirm the predictive ability of dsa and 2) determine whether reduction/eliminattion of dsa will allow improvements in graft survival. was performed in all the recipients before and six months after lrkt. graft biopsies were performed as well within and after six months of the transplantation (tx). all the data of 87 recipients were collected prospectively during the period of follow-up. humoral rejection rate, donor specificity, and time of appearance of the de novo abs were retrospectively studied. results among the 87 lrkt recipients, 47 (54%) showed negative/negative results, 15 (17%) showed positive/positive results, 12 (14%) showed positive/negative results, and 13 (15%) showed negative/positive results (de novo abs) in the pre-/post-transplant flow-pra analysis. among the 13 cases with de novo abs, 5 (38%) had donorspecific abs (dsa) and the remaining 8 (62%) had non-donor specific abs (ndsa) as determined by lab single antigen analysis. four of the five recipients (80%) with dsa showed evidence of both vascular and humoral rejection in the graft biopsies performed within 6 months of the transplantation, while one of the eight recipients (13%) with ndsa showed evidence of cellular rejection during the same period. a 5-year graft survival rate of the recipients with de novo abs was 69%, compared with 96%, 88% and 93% in other groups without de novo abs (p=0.009). conclusions lrkt recipients with developing de novo abs has much higher incidence of humoral rejection and worse prognosis, especially those with donor-specific de novo abs. cautious monitoring for the appearance of anti-hla antibodies should be adopted after transplantation, even in patients without anti-hla ab prior to the transplantation. despite significantly higher response than the 4 males w/o amr (p<0.03), the other 5 females did not experience amr. conclusions: 1) cfc is a novel assay to measure allo/ do cd3-cell responses, assess the degree of sensitization, and predict amr in hs, 2) allo/do cd3-cell numbers are elevated in many hs, but not nc, 3) hs w/ high(+) cfc are at increased risk for amr and may need additional pre-tx desensitization, 4) allo/do reactivity are higher in hs females, which may explain their higher rate of amr, 5) cfc cut off levels for amr prediction may be higher in females than males, 6) monitoring hs using the cfc pre-and post-desensitization may help determine the efficacy of desensitization and risk for amr. were treated with plasmapheresis, ivig and rituximab, and pts with l-amr received ivig and rituximab. the 2-year gs post-amr in pts with e-amr and focal c4d staining was 33% vs. 50% in pts with diffuse staining; while cases of l-amr with focal c4d deposition had a gs of 55% vs. 60% in cases with l-amr and diffuse staining. the number of cases with focal staining was low, and the numerically evident differences were not statistically significant (log-rank p=ns). notably, when losses due to death with a functional graft were censored, post-amr gs was significantly lower in pts with e-amr and focal staining than in their counterparts with diffuse c4d deposition (41% vs. 67%, log-rank p=0.04). in this retrospective single center study, focally positive c4d amr carries a worse prognosis than previously thought, and causes a significant reduction of gs. whether any degree of c4d staining in the context of kt dysfunction should be treated as amr remains a pending question. association time of biopsy was 29.1±43.0 mo after kt. however, 172 cases were biopsied in the 1st year posttransplant. the extent of c4d staining was graded as <10% (0), 10-50% (1), and ≥50% (2) of ptc, and the intensity was graded as none (0), light (1), and strong (2) staining. these findings demonstrate the significant discordance between detection of dsa and c4d, which is a relatively specific histological evidence of ab-mediated injury. this factor should be taken into account when clinical decisions for treatment of patients with either c4d or dsa positivity are made. the observed discrepancy could be partially due to the technique used for staining of the biopsy specimens, inability to detect anti-hla dsa with the available technology, or non-hla dsa. long term follow up data are needed to evaluate the impact of these markers on graft outcomes. introduction epithelial to mesenchymal transition (emt) is a potential mechanism of tissue fibrogenesis. in a previous study, we had reported that the early expression of emt markers was associated with the progression of renal grafts towards interstitial fibrosis and tubular atrophy (if/ta). here, we report the long-term follow-up of this cohort, paying a special attention to the evolution of graft function. patients and methods 83 patients engrafted with a kidney from a cadaveric (n=63) or a living (n=20) donor, and in whom sequential protocol biopsies had been performed at 3 and 12 months, were included. the phenotype of epithelial cells was studied at three months according to the expression of vimentin (an intermediate filament normally expressed by fibroblast-like cells) and to the cellular localization of β-catenin. grafts in which these two markers were abnormally expressed by more than 10% of tubular cells were considered as emt+ grafts. serum creatinine and creatinine clearance (estimated abstracts by gault and cockcroft index) were collected from 12 to 24 months post-transplant and compared according to the emt status of the graft. results multivariate analysis demonstrated that the early expression of emt markers was an independent risk factor of the progression of graft fibrosis between 3 and 12 months. more importantly, these early phenotypic changes were associated with a progressive and sustained deterioration of the graft function : emt+ patients had a statistically higher serum creatinine from twelve months after transplantation, and a significantly lower creatinine clearance from 18 months after transplantation (emt+ 49.4±4.5 ml/min vs emt-61.1±2.3 ml/min, p=0.01). the difference was persistent at 24 months. conclusion the expression of emt markers by tubular epithelial cells at an early time point post-transplant (three months) is highly suggestive of an ongoing fibrogenic process, and has repercussions on the long-term graft function. therefore, these epithelial phenotypic changes are relevant and promising biomarkers for an early detection of if/ta. we recently reported that 73% of transplant glomerulopathy (tg) has evidence of alloantibody-mediated injury in biopsies for cause. we found that 1/3 of tg is c4d+ab+ and 1/3 is c4d-ab+ suggesting that c4d staining is not sensitive enough to detect all biopsies with antibody-mediated injury. we aimed to develop a new laboratory test to detect biopsies with antibody-mediated rejection (abmr) which are missed by c4d. using affymetrix microarrays, we analyzed gene expression in 173 renal allograft biopsies for cause. we previously reported that both abmr and t cell-mediated rejection (tcmr) biopsies show increased expression of transcript sets associated with cytotoxic-t cells (cats) and gamma-interferon effects (grits) compared to biopsies without rejection (p<0.05). however, abmr biopsies were discriminated by a selective increased expression of 25 "endothelial cell-associated transcripts" (endats). these genes included established endothelial markers such as vwf, pecam1, sele, cd34, and cadherin 5, which are involved in endothelial-cell activation. hierarchical clustering of 82 biopsies with ab+ using endats identified a group of c4d-ab+ biopsies (n=20) clustered with c4d+ ab+ biopsies (n=18). thus 25% of biopsies with antibody (20 of 82) had increased endat-scores despite being negative for c4d. these c4d-ab+ biopsies with high endats, had higher scores for cats and grits, increased incidence/severity of tg, tubular atrophy/interstitial fibrosis, and worse future graft function (p<0.05), but similar incidence of tcmr or borderline lesions, in comparison to c4d-ab+ biopsies with no increase in endats. the c4d-ab+ cases with endothelial activation show extensive inflammation in the allograft, as measured by the gene sets, which is similar to c4d+ abmr. there are a significant number of cases with alloantibody and no c4d that show increased expression of endothelial genes. thus the transcriptomics detects deteriorating c4d-allografts with ongoing alloantibody mediated injury. we conclude that increased expression of endothelial genes provides a new feature of abmr, and can be used as a new diagnostic test to detect and treat c4d-abmr. probabilistic ( 001) . the bayesian network model was analyzed and, interestingly, cd86 was critically related to tg, suggesting a b-cell mediated process. tg was also predicted by upregulation of ccl2, ccl3, ccl5, cxcl9, il-8, il-10, and icam gene expression. ten percent of the samples were excluded randomly from the initial model, and subsequently used for cross validation. in the validation analysis, the model effectively predicted tg (auc of 0.92, 90% ppv) and sf (auc of 0.866, 83% ppv). this study provides a compelling and clinically relevant example of the combination of quantitative gene expression with probabilistic bayesian modeling to predict renal allograft pathology. potentially important molecular pathways associated with transplant glomerulopathy were also identified. the application of this integrated approach has broad implications in the field of transplant diagnostics and interpretation of large data sets. 1, 6, 12, 24, 36, 48 and 60 months respectively. the daily dose and blood levels of tac were significantly lower in tac/slr group compared to tac/mmf group. renal function is shown. despite lower prevelance of cai in tac/slr group long-term graft function and patient and graft survival are comparable between tac/mmf and tac/slr groups. objective: the aim of this study was to determine if ethinicity impacts graft outcomes in kidney transplant patients converted to sirolimus (srl) and either maintained on calcineurin inhibitors (ci) or mycophenolate (mmf) with steroids. methods: this was a retrospective analysis of all kidney transplants converted to srl and transplanted from 7/91 to 4/07. patients were divided into 4 groups: group 1: aas converted to srl + continued on ci; group 2: non-aas converted to srl + continued on ci; group 3: aas converted to srl + continued on mmf; group 4: non-aas converted to srl + continued on mmf. pediatrics and multiorgan transplants were excluded. results: a total of 257 patients were included (61% aa). demographics, baseline immunosuppression, and reason for srl conversion were similar between groups. table 1 displays characteristics and outcomes. patients converted to srl+ci regimens had higher rates of acute rejection before srl conversion (p<0.02), but equal rates after conversion. development of proteinuria was similar across groups. figure 1 displays the graft survival rates for each group. aa patients converted to srl+mmf tended to have poorer outcomes compared to aa patients converted to srl+ci. non-aa patients converted to srl+mmf tended to have better graft outcomes compared to non-aa patients coverted to srl+ci, although this did not reach statistical significance(p=0.186). conclusion: aas converted to srl may benefit from continued ci, while non-aas converted to srl appear to have better outcomes with mmf. further prospective studies are warranted to confirm these findings. aa srl+ci (n=113) there are no large registry studies evaluating the correlation of allograft failure for recipients of kidneys from the same deceased donor. we examined outcomes in such recipient pairs using data from the united states renal data system. methods: we studied the correlation of graft failure events within 19,461 pairs of same-donor recipients transplanted during 1995 through 2003. analyses were limited to patients with functioning grafts 3 months post-transplant (tx) and adjusted for known donor, recipient, and tx management factors. we estimated odds ratios to measure the increased risk for 1, 2, and 3-year graft failure and death-censored graft failure when the contralateral kidney had such an event. we also evaluated the effect of recipient pairs transplanted at the same center vs different centers. results: there is a strong correlation in outcomes for 2 recipients with the same donor (table) . the correlation was stronger within pairs transplanted at the same center than for those transplanted at different centers. differences in the correlation of graft failures within pairs transplanted at the same versus separate centers diminished over 2 and were absent by 3 years post-tx. results for death-censored allograft failure were similar. conclusion: unmeasured donor factors contribute significantly to the correlated graft failure outcomes in paired recipients of deceased donor kidneys and need further study. kidney tx outcomes in the first year may be affected by differences in management between transplant centers, more so than in subsequent years post-tx. 1 odds ratio of death-censored graft failure and graft failure in recipients of a donor pair, given that the outcome occurred in the recipient of the contralateral kidney, with both recipients being transplanted at the same (s) center or at different (d) centers. all odds ratios significant with p<0.001. the deterioration of kidney allograft function (dekaf) study is a nih-funded multicenter observational study of late allograft (ktx) loss. the study examines two cohorts: a "long-term cohort" (ltc) of prevalent ktx with scr < 2.0 mg/dl with deterioration of function (25% increase in scr or proteinuria) and a "prospective cohort" (pc) of incident ktx developing a persistent >25% increase in scr. we examined the pathologic features of the first renal biopsy (bx) obtained for new onset deterioration in each cohort. all bx were read and scored centrally using a modified banff schema. on average, bx were obtained at 6 (pc) and 75 (ltc) months post-tx. mean scr was similar, but more patients (pts) in ltc had proteinuria. moderate to severe interstitial fibrosis and tubular atrophy (ta) were more prevalent in ltc than pc (33 vs 13% ci score ≥2; 33 vs 9% for ta). the rate of vascular sclerosis >25% was similar (10.87% pc vs 15.46% ltc); however, hyaline arteriolar sclerosis >25% was more common in the ltc (30 vs 4.35%). interstitial inflammation (i) and tubulitis (t) scores were similar in both cohorts. however, more pts in ltc had peritubular capillary infiltrates (>5 cells) and evidence of tx glomerulopathy. while rates of interstitial inflammation and tubulitis sufficient to warrant diagnosis of acute rejection are similar in the prospective and long-term cohorts, long term cohort pts had more proteinuria, interstitial fibrosis, tubular atrophy, hyaline arteriolar sclerosis, and transplant glomerulopathy. analyses of histologic findings and renal outcome are ongoing. the term chronic allograft nephropathy (can) has been abolished by the last banff meeting report (am j transplant, 2007) and 2 categories have been introduced for chronic changes: chronic active t cell-mediated rejection and chronic active humoral rejection (cahr). aim of the study was to review all cases of can diagnosed in the last 4 years and to identify immunohistochemical markers of chronic rejection. a cohort of 79 cad pts with biopsy-proven can was analyzed. each case was reviewed and assigned into 3 groups according to banff 2005 criteria: chronic rejection (cr), chronic calcineurin toxicity (cnit) or chronic lesions not otherwise specified (nos). cd4+, cd8+, cd20+, cd68+ cells and c4d deposits were assessed by immunohistochemistry. twenty-eight pts were classified as cnit,34 as cr,19 of which were cahr, and 17 as nos. serum creatinine and 24h proteinuria at renal biopsy, extent of interstitial fibrosis and glomerulosclerosis were not significantly different among 3 groups (table1).the number of cd4 + cells was higher at ti level in cr compared to cnit (table1;*p=.05). cd8 + cells were higher at ti and g level in cr compared to cnit (table1;*p=.05). ti and g cd20 + cells were not different among the 3 groups (table1). the number of g cd68 + cells was increased in cr compared to cni and nos (table1;*p=.05). no significant difference in cd20, cd4, cd8, cd68 expression was found at ti and g level between c4d + and c4dcases of cr. cd68, cd8, cd4 but not cd20 expression at ti level correlated with ti fibrosis (r 2 =.105, .077, .156, respectively, p<.05) at the univariate analysis. only ti cd4 + cells independently correlated with fibrosis at multiple regression analysis. in conclusion, our data suggest that: morbid obesity limits access to kidney transplantation and predicts adverse transplant outcomes. there are limited data on the safety and efficacy of gastric bypass (gb) as a weight reduction therapy among transplant candidates and recipients. methods: we examined usrds registry data to identify medicare-insured kidney transplant candidates and recipients with billing claims for gb procedures. gb were categorized according to occurrence before listing, on the waitlist, or after transplant. we studied the clinical characteristics of gb-treated patients, and subsequent outcomes including progression from listing to transplant and 30-day mortality. usrds surveys bmi data at dialysis start, waitlist entry, transplant, and transplant anniversaries.we computed changes between most recently reported body mass index (bmi) values preceding and following gb, when available. results: we identified 72 transplant candidates treated with gb before listing, 29 who underwent gb on the waitlist, and 87 gb cases after transplant. patients treated with gb were most commonly female, white race, and without diabetic or hypertensive renal failure (table) . 30-day mortality after gb, calculable for listed and transplanted patients, was 3.4% and 3.4%, respectively. 1 transplant recipient experienced graft failure within 30 days of gb. of 29 patients treated with gb on the waitlist, 20 proceeded to transplant. post-gb weight loss was detected for 60% with gb pre-listing, 70% with gb on the waitlist, and 90.7% with gb after transplant. among patients listed for transplant in the same era and bmi >35 at first dialysis who were not treated with gb, 22% had lost weight between dialysis start and listing. conclusions: gb has been performed in small numbers of kidney transplant candidates and recipients, and is followed by weight-loss in the majority of cases. peri-operative mortality is comparable to reports in patients without kidney disease. gb warrants prospective study as a strategy for reducing complications of obesity in esrd. introduction: the present study investigated the incidence of posttransplant diabetes mellitus (ptdm) and calculated the risk of developing ptdm under a tacrolimus and mycophenolate mofetil (mmf)-based immunosuppression based on clinical characteristics, tacrolimus-pharmacokinetics, and genetic polymorphisms related to tacrolimus-pharmacokinetics, cytokines and diabetes mellitus. methods: seventy-one non-diabetic adult kidney recipients (male 37, female 34) were studied. patients with continuous high plasma glucose levels, over 6.5mg/dl of hemoglobin a1c, or requiring insulin and/or oral anti-diabetic agents for more than 3 months after transplantation at 1-year after transplantation were diagnosed as having ptdm. fifteen genomic polymorphisms were assessed. results: one year after transplantation, 21 recipients (29.6%) developed ptdm. positive risk factors were age (p=0.028) and body mass index (p=0.048). there were no significant differences in acute rejection rate, total steroid doses, tacrolimuspharmacokinetics or its related to genetic polymorphisms between the two groups. the frequencies of ptdm were significantly higher in patients with adiponectin t45g tt genotype than in those with the g allele (p=0.034), and in patients with glucocorticoid receptor (nr3c1) bcl i cc genotype than in those with the g allele (p=0.004). conclusions: the incidence of ptdm at 1-yr after transplantation was 29.3% in our cohort. elder or obese patients were risky for the development of ptdm. the presence of the adiponectin t45g tt or nr3c1 bcl i cc genotype may also be risk factors for ptdm, suggesting that insulin and glucocorticoid sensitivity-related genes are associated with the development of ptdm. analysis of these genotypes is a possible method of predicting a patient's risk for developing ptdm and would be a valuable asset in selecting appropriate immunosuppressive regimens for individuals. pharmacokinetics persistent hyperparathyroidism (hpt) with hypercalcemia is common after renal transplantation. studies have shown that treatment with cinacalcet corrects hypercalcemia and lowers pth levels in these patients. so far cinacalcet's steadystate pharmacokinetics and their correlation with pharmacodynamics (pk/pd) have only been studied in hemodialysis patients, but not in renal transplant recipients with persistent hpt. to gain further insight into cinacalcet's effects on calcium-phosphate homeostasis, we determined its steady-state pharmacokinetics and pharmacodynamic effects in these patients. in a prospective, single center, open label study we examined the effect of a 2-week treatment with 30 mg and subsequent 2-week treatment with 60 mg cinacalcet daily on calcium-phosphate homeostasis over 24 hours and determined the steady-state pharmacokinetics of cinacalcet in stable renal allograft recipients. the urinary calcium excretion was determined in timed urine samples. median auc 0-24 was 784.8 ng*h/ml and c max was 68.5 ng/ml for 60 mg cinacalcet which is higher, and oral clearance (cl/f) was 76.9 l/h which is lower in renal transplant recipients compared to previously published data of hemodialysis patients (50 mg cinacalcet auc 0-24 179, c max 17.2, cl/f 279). we also observed a non-proportional increase of auc 0-24 after doubling of the cinacalcet dose. the once daily administration of cinacalcet dose-dependently reduced ipth and serum calcium. cinacalcet and parathyroid hormone (pth) concentrations showed an inverse correlation and were fitted to a simple emax model (e max =80 % reduction vs. baseline, ec 50 =13 ng/ml). the 8-hour fractional urinary excretion of calcium was increased after 60 mg cinacalcet (baseline 0.85±0.17 %, 30 mg 1.53±0.35 %, 60 mg 1.92±0.37 %). renal function remained stable. cinacalcet's higher and non-proportional increase of auc 0-24 in transplant recipients compared to hemodialysis patients evokes the possibility of a pharmacokinetic interaction with concomitant cyclosporine treatment. cinacalcet effectively corrected the biochemical abnormalities of persistent hpt. the transient calciuria could potentially favor nephrocalcinosis and reduce bone mineral density, suggesting that higher doses of cinacalcet need to be used with caution in renal transplant recipients with severe persistent hyperparathyroidism. screening for proteinuria in the kidney transplant clinic. bryce a. kiberd, 1 romuald panek. 1 1 dalhousie university, halifax, ns, canada. proteinuria is a predictor of progression in kidney disease. it is not clear whether measuring albuminuria will have greater clinical utility over measurement by dipstick or total proteinuria in kidney transplant recipients. there has also been a trend away from using 24 hour collections to using spot urine albumin/creatinine (ac) and protein/creatinine (pc) ratios. we compare the prevalence of proteinuria estimated by dipstick, ac and pc in prevalent patients (>6 months post transplant) in the kidney transplant clinic. significant albuminuria defined as ≥30 mg/g was present in 52% (211/403). albuminuria was seen in 29% (70/245) with negative and 67% (34/51) with trace dipstick proteinuria. significant predictors of albuminuria in a mulitvariate logistic analysis were egfr (or 0.977 per ml/min/1.73m 2 , 95% ci 0.965-0.989 p=0.001), diastolic bp (or 1.028 per mmhg, 95% ci 1.008-1.048 p=0.007), and mmf use (or 0.50, 95% ci 0.32-0.77 p=0.002). macroalbuminuria (ac>299 mg/g) was seen in 17.4% (70/403) and significant predictors in a mulivariate logistic analysis were lower egfr and higher systolic bp. sirolimus use was associated with more macroalbuminuria and mmf use with less macroalbuminuria. in a subset of patients followed for >2 years prior gfr loss was considerably greater (p=0.021) in patients with albuminuria (-0.88 ml/min/1.73m 2 /year) compared to those without (-0.15 ml/min/1.73m 2 /year). however other measures of proteinuria were also significantly (p for trend) associated with prior gfr loss (ml/min/1.73m 2 /year) as shown in the <0.13 (n=185) 0.13-0.49 (n=109) >0.50 (n=80) ∆ egfr/year -0.33 -0.26 -1.40 0.02 * a pc cut point of 0.129 g/g had a sensitivity and specificity for albuminuria > 30 mg/g of 87% and 88% respectively (c=0.92, 95% ci 0.89-0.95), and a pc cut point of 0.49 g/g had a sensitivity and specificity for macroalbuminuria >300 mg/g of 100% and 94% respectively (c=0.98, 95% ci 0.97-0.99). ac may be more sensitive and therefore have more clinical utility than other measures of proteinuria for progression. however prospective follow up of renal function change and cv outcomes is required. serum creatinine is a crude marker of gfr in renal transplant recipients and changes in gfr are frequently not accompanied by commensurate changes in serum creatinine concentration. serum cystatin c and estimates of gfr (egfr) based on cystatin c have been shown to be more accurate than serum creatinine and creatinine-based egfr in renal transplant recipients. the purpose of this study was to determine whether the filler, lebricon and rule cystatin c-based egfr equations were better able to detect changes in true gfr than the mdrd and cockcroft gault creatinine-based egfr equations. we performed two measures of 99m tc-dtpa gfr, serum creatinine and serum cystatin c on each of 183 stable renal transplant recipients at least 6 months apart. we calculated and compared the percent annual change in the measured gfr and the estimated gfr using the various gfr estimation equations. we also determined the sensitivity, specificity, positive predictive value and negative predictive value of each prediction equation for the detection of decline in measured gfr. results are presented below: the cystatin c and creatinine-based egfr equations all demonstrated poor sensitivity and diagnostic performance to detect a decline in gfr. novel equations derived and validated in the transplant population are needed to accurately assess kidney function over time. background: hypercalcemia, hypophosphatemia and renal phosphate wasting are common after kidney transplantation and are related to persistent hyperparathyroidism and hyperphosphatoninism. animal data suggest that these alterations in mineral metabolism may contribute to nephrocalcinosis and progressive graft dysfunction. supporting clinical data are limited. aim: to test the hypothesis that nephrocalcinosis is highly prevalent in the early posttransplant period and is related to a disturbed mineral metabolism. methods: biomarkers of mineral metabolism (including albumin-corrected serum calcium [ca c ], serum phosphorus [p], biointact pth, calcidiol, calcitriol and alkaline phosphatase) and renal calcium and phosphorus excretion parameters were prospectively assessed in 201 renal transplant recipients (62% male, mean age 55 ± 14 yrs) at the time of their 3-month protocol biopsy. these protocol biopsies were screened for the presence of microcalcifications. intratubular, interstitial and/or cytoplasmatic microcalcifications were observed in 30.4% of biopsies. calcifications were more prevalent in recipients of a living related donor as compared to cadaveric donor. high serum ca c levels, high serum pth levels, a high urinary ca×p product and high fractional excretion of p and low serum p levels were significantly associated with renal microcalcifications (see figure below). microcalcifications were not related to the fractional excretion of ca, use of diuretics, immunosuppressive regimen, serum alkaline phosphatase level and history of delayed graft function. the extent of microcalcifications correlated significantly with the severity of mineral metabolism disturbances. conclusion: our data demonstrate that nephrocalcinosis is highly prevalent in the early posttransplant period and suggest that a disordered mineral metabolism is implicated in its pathogenesis. polymorphism in abcb1, the gene encoding for p-glycoprotein, predicts recovery of graft function early after kidney transplantation. the pharmacokinetics of cyclosporine (csa) is characterized by wide inter-individual variability. this might be particularly relevant in the early post-transplant period, due to the detrimental effects of the drug on the kidney. p-glycoprotein (p-gp), the product of the abcb1 gene, plays a key role in the distribution of csa at cellular level. single nucleotide polymorphisms (snps) of abcb1 might potentially influence the response of patients to csa. in particular, the snp in position 3435 of the exon 26, despite its silent nature, has been recently associated with altered specificity for its ligand, such as csa (kimchi-sarfaty et al, science 2007, 315:525) . whether p-gp pharmacogenetics would help to guide csa treatment early post-transplant remains ill defined. we sought to evaluate the effects of the snps in the exon 26 on the rate of recovery of graft function, as estimated gfr early postoperatively, in 150 kidney transplant patients given csa as part of their immunosuppressive regimen. the frequency of dgf (as need for post-operative dialysis) among the different abcb1 genotypes was also estimated. of the 150 kidney transplant recipients, 35% had the abcb1 wild type (c/c) genotype in exon 26, 40% were heterozygous (c/t) and 25% were homozygous (t/t) for the polymorphic variant in position 3435. gfr values were significantly lower in patients carrying one of the two mutant alleles than in the wild type ( figure) . the frequency of dgf was 15%, 28% and 30% in patients with the cc, ct and tt genotypes, respectively. these findings demonstrate that in patients carrying the ct or tt mutant alleles in exon 26 of the abcb1 gene and given csa, the recovery of graft function is less prompt, and the risk to develop dgf higher than in wild-type cc genotype. pre-transplant screening for abcb1 polymorphism would help to identify patients who may safely receive csa early post kidney transplantation. cigarette cigarette smoking has shown to reduce graft and patient survival in renal transplant patients. however, whether it could directly produce allograft disfunction has not been investigated. the aim of this study was to assess the smoking influence on renal graft function. we studied a cohort of 827 adult renal transplant patients, transplanted from jan/96 to dec/05 and followed until dec/06. smoking habits were recordered at the time of transplant (never, former, current smoker). during summer of 2007, a telephonical survey allowed us to obtain complete information about smoking habits in 642 patients (aged 47.6 ±13, 65% male): status (never, former, current), years of habit, years of quit, and number of cigarette smoked per day. number of "pack-years" was calculated. renal function was measure by inverse serum creatinine at 3 rd month and then annually. time to decline a thirty percent in inverse serum creatinine was registered. patients were divided in two groups: those who always smoked during all transplant period (smokers, n=94) and those who did not (n=546 renal insufficiency occurs frequently after extrarenal transplantation as a result of acute tubular necrosis at transplantation, high blood pressure, and cni toxicity. we performed 56 renal biopsies in 54 patients after heart (4), lung (20) , liver (22), bone marrow (9), and cornea (1) transplantation since 2000. the time from transplantation to biopsy was 49±53 months in general and was longest after liver (60±61 months) and shortest after bone marrow transplantation (37±43 months). the histologic changes were: tubular atrophy/interstitial fibrosis of ≥20% in 52% of biopsies (heart biopsies 75%, lung 70%, liver 46%, bone marrow 22%); acute tubular changes in 54% (heart 25%, lung 65%, liver 46%, bone marrow 56%); arteriolar hyalinosis in 41% (heart 75%, lung 65%, liver 23%, bone marrow 22%); arterionephrosclerosis in 41% (heart 50%, lung 50%, liver 36%, bone marrow 22%); glomerular sclerosis of ≥20% in 21% (heart 50%, lung 25%, liver 18%, bone marrow 22%); glomerulonephritis in 13% (heart 25%, liver 18%, bone marrow 22%; that means iga-nephropathy after heart and liver, immune complex nephritis twice after liver, mpgn after liver, and membranous glomerulonephritis and minimal changes after bone marrow transplantation); thrombotic microangiopathy in 7% (lung 10%, liver 5%, bone marrow 11%); finally one case of polyoma nephritis after lung transplantation. among 1702 heart and lung transplantations, 13 patients needed kidney transplantation (0.8%) after 96±40 months; and among 2221 liver transplantations, 11 needed kidney transplantation (0.5%) after 112±65 months (sign. later, p=001). conclusion: as expected, most histologic changes were those of cni toxicity and hypertension. surprising is the high number of glomerulonephritis under immunosuppression. thrombotic microangiopathy without the typical clinical signs were interpreted as cni-related toxicity and seems to occur more often after extrarenal than after renal transplantation. patients with heart and lung transplantation reach end-stage renal failure more often and earlier than patients with liver transplantation. the context: living kidney transplantation, a superior therapy to deceased donor kidney transplantation, is underutilized. states have enacted legislation and the federal government has launched initiatives to compensate living organ donors, but the effect of policy on improving living kidney donation rates in the united states is unknown. objective: to determine whether public policies are associated with changes in living kidney donation rates in the continental u.s. design, setting, and study subjects: series of cross-sectional analyses using records of state legislatures in 48 continental states and living kidney donation rates from the united network for organ sharing. main outcome measures: living kidney donation rate during each year from 1988-2006 and change in donation rates before and after legislation enactment in each state and launch of federal initiatives. results: from january 1990 through december 2005, 28 states enacted legislation for living donors (24 mandating paid leave, 8 tax deductions, 3 unpaid leave, 2 encouraging paid leave). few states (n=5) enacted legislation prior to 1999. there was a steady increase in the mean living kidney donation rate in the continental u.s during the study period (mean (standard deviation) annual increase in donations 1.5 (0.04) donations per 1,000,000 population). in analyses accounting for length of time state legislation had been enacted, the types of legislation enacted, and the incidence and prevalence of esrd in each state, there was a slightly (but not statistically significantly) greater average annual increase in donations after compared to before state legislation enactment (annual increase in donations per 1,000,000 population [95% confidence interval ( introduction: accurate and precise renal function assessment is essential in the evaluation of prospective kidney donors. while direct measurement of gfr is the "gold standard", it is not widely available. moreover, creatinine (scr)-based estimation equations are suboptimal to assess kidney function in this setting. ct scans are increasingly being used to study renovascular anatomy in donors and has replaced angiographic exams in many institutions. 3d imaging reconstruction allows for kidney volumes (kv) measurements which have been shown to highly correlate with measured gfr in this population. thus, the purpose of this study was to develop a model to estimate measured gfr that not only incorporates scr and demographic data but also kv as measured by 3d ct scans. methods: 244 individuals who underwent donor evaluation were identified. an automated segmentation algorithm was used to measure renal parenchymal volume from preoperative abdominal cts. patient demographics and scr values were obtained from the medical records. gfr (normalized for bsa) was measured by i 125 iothalamate renal clearances (igfr). an analysis of covariance model was created to correlate measured igfr with kv, patient age, sex, race, weight, height and scr. pearson's correlation coefficient was calculated for each variable. results: kv (p<0.001), age (p<0.001), scr (p<0.001) and weight (p<0.001) significantly correlated with igfr. sex (0.60), race (0.90) and height (0.76) were not statistically significant. the new fitted regression model is: kv-egfr (ml/min/1.73 m 2 ) = 70.77 -(0.444*age) + (0.366*weight) + (0.200*volume) -(37.317*scr). we then compared the performance of the kv-egfr model to the re-expressed mdrd equation using calibrated scr assay. the r 2 was 0.61 vs 0.50, respectively; signed median % difference was +1.8% vs -12.7%, respectively (% difference b/w estimated gfr and igfr); and accuracy within 30% (% of estimated gfr values that fall within 30% of igfr) of 96.3% vs 89.8%, respectively. finally, the kv-egfr model was closer to igfr (in absolute values) than the mdrd eq. in 177/244 (70.5%) cases vs 72/244 (29.5%) cases, respectively. conclusions: kidney volumes highly correlate with igfr and the proposed gfr estimation model outperforms the mdrd equation in potential living kidney donors. the kv-egfr model could be used to estimate donor gfr in lieu of i 125 iothalamate gfr which is less clinically available. for donor selection, current reports identify unsuspected renal pathology by time 0-biopsy. aim: to explore whether the findings at time 0-renal biopsy (bx) correlates with pre-donation clinical data including renal function. methods: kt databases from 2 institutions were reviewed. time 0-renal bx are routinely performed from the upper pole during back-table and evaluated by 2 nephropathologist for interstitial fibrosis (if), tubular atrophy (ta), arteriolar hyalinosis (ah), mesangial increase (mi), and glomerulosclerosis (gs). pre-donation data gathered from the donors were demography, body weight, bmi, systolic/diastolic bp, scr, proteinuria, and egfr by levey equation clinical data is summarized in the table. and gs showed no correlation. multivariate analysis failed to sustain the significant associations found on bivariate analysis, most likely due to a low event/parameter relation. conclusions: a significant correlation was observed between time 0-bx findings and clinical pre-donation parameters. whether these histological findings at the time of kidney donation represent a higher burden/risk for the remaining kidney ought to be evaluated during follow-up. in an era where living donation is increasing, we should advise a closer surveillance of these donors in order to modify risk factors that participate in kidney damage progression. predictors of poor early graft function following laparoscopic donor nephrectomy (ldn). matthew cooper, 1 abdolreza haririan, 1 stephen jacobs, 1 michael phelan, 1 benjamin philosophe, 1 stephen bartlett, 1 joseph nogueira. 1 1 dept of surgery, urology, and medicine, university of maryland, baltimore, md. ldn has become the standard of care in many transplant centers. poor early graft function remains an important complication. we conducted a retrospective study to evaluate the risk factors for slow or delayed graft function following ldn methods: donor and recipient records from the first 1000 ldn were reviewed (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) . results: slow graft function (sgf) was defined as cr>3.0mg/dl at pod5 and dgf as the need for dialysis within the first week following transplantation. donor variables examined included age, sex, race, bmi, egfr, and number of renal arteries. recipient variables included age, sex, race, bmi, prior tx, pre-tx dm, history of smoking and drug usage. additional variables evaluated included degree of relationship (lurt), hla mismatch, antilymphocyte induction, de novo cni usage, r v. l nephrectomy, wit, total or time, and performance of simultaneous deceased donor pancreas tx (splk). univariate analysis was performed with significance defined as p<0.05. significant variables were then included in the multivariate analysis. background: a kidney exchange program is the logistic solution for patients with positive cross match (x + ) or abo incompatible donors. a major problem in all kidney transplantation programs is the sensitized recipient. we analyzed the success rate for immunized recipients in the dutch kidney exchange program. methods: from january 2004 till december 2007 242 donor-recipient pairs were registered. there were 117 couples in the x + group while 125 pairs were abo incompatible. in the x + group the median pra was 46% (2-100%). to create new combinations a match program was run 16 times (every 3 months) with a median of 46 (16-66) participating couples. allocation criteria included bloodtype (first identical, then compatible), hla match probability within the actual exchange donor pool to ensure that highly sensitized recipients have the best chance to receive a kidney, and the waittime on dialysis. cross matches between new donor and recipient were performed centrally in our reference laboratory with cdc-tests. results: after 16 match runs, we found matching couples for 83/117 (71%) x + pairs, for 21/87 (24%) abo incompatible pairs with o recipients and for 29/38 (76%) abo incompatible pairs with non-o recipients. median pra of the 83 recipients in the x + group was 42% (2-100%). after 3 match runs chances for success became small. the overall success rate for abo incompatible and x + pairs in the dutch kidney exchange program after 4 years is 55%. however, the success rate for immunized patients in the x + group is significantly higher (71%) as our match program gives priority to those recipients with the smallest chance of finding a compatible donor in each match run. thus our kidney exchange program is especially suited for immunized patients. although paired kidney donation (pkd) program is an established method to overcome incompatibilities between kidney donor-recipient pairs (drp), significant proportion of the incompatible drp participating in such program could remain unmatched. domino-kidney transplantation (kt) in which altruistic living non-directed donor kidney (lndk) is offered to a pool of incompatible drp, and is used to initiate a chain of pkd transplants, could provide more opportunities of kidney transplantation to drp in pkd program. we introduce our experience of multicenter domino kt for the last 7 years sixteen hospitals participated in the domino-kidney transplantation between february, 2001 and july, 2007. 181 domino-kidney transplants were performed with 69 domino-kt chains initiated by altruistic lndk. 2-pair chains were 43, 3-pair chains 14, 4-pair chains 9, 5-pair chains 2, and 7-pair chains 1. the development of a multi-regional kidney paired donation program. using an optimization matching algorithm the new england program for kidney exchange (nepke) efficiently matches incompatible donor/recipient pairs. in 2006, the mid-atlantic paired exchange program (mapep) and other individual centers began sharing data with nepke. this is a report of the success of two regional programs working together to increase the probability of participants in both programs finding a compatible match. methods: incompatible pairs and non-directed donors (ndd) are referred to nepke through transplant centers. donor and recipient abo, hla and recipient hla antibody screening are entered into the computer database. utilizing the optimization program, searches for compatible matches are conducted every 30 to 45 days. the program identifies potential 2 and 3-way matches, ndd chains, and list exchange chains. following the determination of compatibility nepke notifies transplant centers involved of the potential match and centers accept or decline the offer. transplant centers notify their pairs and preliminary crossmatches are performed. a conference call is scheduled to coordinate simultaneous donor nephrectomies and recipient transplants. surgeons speak prior to incision to ensure simultaneous donation. results: from july 2005 to december 2007, 186 pairs and 10 ndds have entered nepke. during this time frame over one thousand possible matches were identified, with the majority of these matches involving the same pairs in multiple matches. after "optimizing" and eliminating multiple matches 13 two-way exchanges; 15 three-way exchanges; 7 three-way list chain exchanges; and 25 ndd chain matches were offered to transplant centers as possible matches. most common reason offers were declined include: positive crossmatch (21.6%); donor factors (20%), and recipient inactive or transplanted (20%). four offers occurred in mapep alone pairs, 47 in nepke, and 14 offers were cross-regional. three matches are pending for 9 additional transplants. one previous and one pending transplant are the result of cross-regional exchanges. five transplants performed and 6 pending involve ndd chains. one pending match involves a list exchange chain. conclusion: using a computerized optimization algorithm to match 2 and 3 way exchanges, ndd and list exchange chains has lead to a substantial increase in the number of kpd matches and transplants performed. cross-regional coordination is feasible and expands the number of transplants performed beyond the ability of individual exchange programs. as living donors become an increasingly important source of life-saving organs, there is growing concern about the lack of comprehensive research on donor outcomes. in addition to possible long term consequences, there is a risk that donors will experience complications during or following surgery. of the 13,000 living kidney donors in 2005-2006, none died during surgery, and 0.5% (n=68) needed blood transfusions during surgery. in the six weeks following donation, 3.9% (n=512) had at least one serious adverse event (sae): 1.7% (n=220) needed readmission following initial discharge, 0.6% (n=72) needed an interventional procedure, 0.5% (n=61) needed re-operation, 0.3% (n=41) had vascular complications, and 2.1% (n=274) had other complications. when all saes were considered in combination, the rate was 3.9%. because over 50% of ldr forms were submitted by transplant centers fewer than 6 weeks post-donation, all complication rates should be considered minimum estimates. one donor was reported to have died from donation-related causes within 6 weeks of donation. the number of living donor kidney transplants performed by a transplant center in 2005 -2006 ranged from 1 to 362 transplants. additionally, the risk of donor complications is not equal across transplant centers. for example, there was a significant correlation between the number of living donor kidney transplants performed at a transplant center and the percentage of that center's patients who were readmitted within 6 weeks of donation, with greater donor volume associated with a lower rate of readmission. living kidney donation is relatively safe, but prospective donors should be made aware that there is a non-trivial risk (3.9%) of short-term complications post-donation. as with many other major surgical procedures, complication rates are lower, on average, at institutions that perform a larger number of these procedures. we sought to determine if intensive screening improves detection of polyomaviral reactivation in asymptomatic patients and pre-emptive stepwise modification can improve outcome of polyomaviral nephropathy (pvn). methods:this is a prospective single center study. we randomly assigned de novo kt (cluster randomization) to intensive screening (is:n=648) and routine care (rc: n=260) for the detection of decoy cells. is was initiated at week-4 of kt and rc at the time of increase in serum creatinine. this was complemented with urine and blood nucleic acid testing. all patients had biopsies performed for detection of polomya nephritis (pvn). both groups were treated with pre-specified stepwise modification of it based on cell cytology and viremia (step 1: decrease dose of cellcept by 50%, step 2: decrease dose of tacrolimus by 50%, or switch to sirolimus therapy, step 3: discontinue cellcept). primary outcome included persistence of decoy cells/viremia following each step in modification of it every three months and secondary outcomes included acute rejection, graft function and graft loss. results: polyomaviral reactivation developed in 11.7% in is group and 40% had pvn without changes in serum creatinine. the estimated cumulative rate of primary outcome in the is versus rc groups, 3-months (51% vs. 79%) relative risk (rr), 0.47;95%ci,0.20-1.08;p=0.051); 6-months (20%vs.52%) (rr=0.67; 95% ci,0.38-1.20; p=0.009); and 12-months (2%vs.33%) (rr=0.88; 95% ci, 0.41-1.43; p=0.003). secondary outcomes: despite similar degrees of step-wise is modification, rate of acute rejection non-significant (p=0.25), but 25% of patients in rc loss the graft vs no graft loss in is group. patients who continue to remain on tacrolimus vs. those who were switched to sirolimus therapy had persistent viremia (or=10.25; 95%ci, 1.2-92.0; p=0.003). conclusion: is for poloymaviral reactivation allows early detection of pvn in the presence of stable graft function. stepwise modification in it resulted in early resolution of decoy cells and viremia in both groups, albeit slowly in rc group, and it did not prevent the graft loss in rc group. background: dna sequencing of the bk viral (bkv) genome non-coding control region (nccr) from individual patient isolates demonstrate divergent sequence and alterations in the arrangement of modularly conserved sequence blocks (p-q-r-s) ranging in size from 39 to 68 base pairs. aim: primary aim to molecularly clone and analyze patient-derived bk virus nccr sequence variants. our secondary aim is to determine if reporter gene constructs of patient-derived nccr variants differ in their promoter activity upon transfection into a mammalian cell line (vero) and a human primary tubular epithelial cell line. methods: bkv dna was amplified and sequenced from blood and urine samples of 18 renal transplant recipients. via sequence alignment, unique nccrs were determined. these sequences were pcr amplified and cloned into reporter plasmids containing the renilla luciferase gene. promoter activity was measured via luminometer 24 hours after transfection into vero cells and human tubular epithelial cells. results: variation in naturally occurring bkv nccr promoter regions exist as single basepair insertions and deletions, and insertions or deletions of partial sequence blocks (p-q-r-s). single basepair substitutions were most commonly seen (46% of analyzed samples). promoter activity within vero cells ranged from 188% to 39% as compared to nccr activity of an archetypal strain (wwb). low promoter activity (<50%) was seen in isolates with duplications of the p block and large deletions of the r block. conclusion: these sequence blocks are rich in regulatory elements and control the expression of both bkv structural and regulatory genes. variation in these cisacting eukaryotic transcriptional promoter binding sites corresponds to differential promoter activity in naturally occurring bkv isolates. current plans include repeating the promoter activity studies in human primary tubular epithelial cells. humoral and cellular immunity to polyomavirus bk large t and vp1 antigens after pediatric kidney transplantation. polyomavirus bk-associated nephropathy (bkvn) has emerged as a cause of graft failure after kidney transplantation (ktx). in a cohort of 37 pediatric renal recipients undergoing prospective three-monthly monitoring for bk by blood and urine q-pcr, we evaluated antibody response, measured by enzyme immunoassay using bk vlp, and cellular immune response, reported as frequency of ifnγ-secreting cells in a elispot assay after 9-day stimulation with bkv large t (lt) and vp1 peptides. we could not observe any influence of recipient pre-transplant bkv-specific igg or t-cell levels, which were generally low, on bkv infection after allografting. after transplantation, both specific igg levels, and frequency of bkv-specific t cells increased according to the degree of viral exposure. in detail, bkv-seropositive patients who never reactivate the virus (group1, n=10) did not show significant increase in igg levels (from a median od of 0.37 at month +1 to 0.39 at the end of follow-up), while patients with urinary shedding alone (group 2, n=14) or with viremia (group 3, n=13) increased from a median od of 0.3 to 1.2 (p=0.09), and 0.26 to 2.8 (p>0.0005). in the case of cellular immunity, vp1-specific t-cells increased in the three groups. conversely, lt-specific t-cells, which were high (median 71 sfu/10 5 cells) and remained unchanged throughout the follow-up period in group 1 patients, had a significant increase in recipients belonging to both group 2 and 3. interestingly, patients with urinary shedding who do not progress to viremia show a median 3-fold increase in lt-specific t-cell levels at peak viruria, compared to no increase observed in patients who develop viremia. the latter group mount a significant response to lt only after therapeutic reduction of immunosuppression. at peak viruria, viremic patients already show a 8-fold rise in specific igg compared to the 1.5 increase observed in group 2 recipients. our data suggest that inability to reach protective levels of bkv lt-directed t cells, rather than specific igg, predispose ktx recipients to bkv replication. introduction:the antibody response to human bkv virus (bkv) is incompletely characterized. antibody responses to the vp-1 protein have been detected in kidney transplant patients, but it is not known if these have virus neutralizing activity. methods:recombinant bk, jc, and sv40 virus like particles were used to produce a panel of 20 monoclonal antibodies. these antibodies were characterized for isotype and for ability to bind the respective antigens in elisa assays. to test neutralizing activity, bkv gardner strain (atcc# vr837) viral particles were incubated with the corresponding antibodies for 2 hours at 37 degrees c, and used to infect wi 38 cells. bkv infection was monitored by quantitative real time pcr using primers directed against the vp-1 gene. neutralizing activity was defined as greater than 95% inhibition of viral yield. results:the monoclonal antibodies were of the igg 2a or igg 2b class with the exception of one igg 1 and one igm antibody. all 12 anti-bkv monoclonal antibodies bound to bkv capsids in-vitro in elisa assays. this binding affinity was species specific, as only 1 antibody showed weak binding activity to jcv and sv40 capsids. neutralization of infectious bk virus was shown for 10/12 antibodies. denaturation of capsid proteins indicated that the monoclonal antibodies recognized primarily conformational epitopes, with only monoclonal antibody appearing to have a linear component. paucity of linear epitopes was further suggested by lack of reactivity of sera from bkv seropositive subjects in elisa assays based on genotype-specific short peptide sequences derived from the bkv vp-1 loop region. four monoclonal antibodies each generated from jcv capsids and sv40 capsids did not show any bkv neutralizing activity. conclusions: bkv vp-1 protein capsids contain species specific conformational epitopes which can elicit virus neutralizing and non-neutralizing antibody responses. measurement of these antibodies in renal transplant recipients may have diagnostic and prognostic applications. bkv specific monoclonal antibodies deserve further study as potential therapy of acute infections in the viremic phase. impact of immunosuppression reduction in bk viremic patients: 3 year follow-up. s. kuppachi, 1 a. guasch, 1 c. p. larsen, 1 k. e. kokko. 1 1 transplant center, emory university, atlanta, ga. background: development of bk nephropathy is a risk factor for allograft loss. bk viremia (bkv) precedes the development of bk nephropathy. it has been reported with 1-year follow-up that reduction of immunosuppression leads to control of bkv. here, we report our 3-year follow-up experience of bkv patients that were identified by a prospective screening protocol and managed by sequential reduction of immunosuppression. methods: all kidney or kidney-pancreas transplant recipients at emory university between 5/03-5/04 were screened prospectively for bkv by real time pcr during follow-up visits (1-6, 9, 12, 24, 36) . patients with a bk viral load of greater than 10,000 copies/ml blood received a kidney biopsy to screen for bk virus nephropathy by immunohistochemistry. bkv without nephropathy resulted in reduced immunosuppression by a 50% reduction in mycophenolate dose. bkv with nephropathy resulted in discontinuation of mycophenolate. all identified bk patients were monitored every 2-4 weeks until viral load was below 10,000 copies/ml. immunosuppression was further reduced if viral loads failed to decrease. results: 135 recipients were followed over a 36-month period. 24 patients had received simultaneous kidney and pancreas, 2 a liver and kidney and the rest kidney alone. 27 of 135 (20%) patients developed bkv within a year from time of transplantation. average time to diagnosis of bkv was 4.7 months. average dose of mycophenolate at 3 years was 1.3 g/d in the bkv negative population as compared to 0.56 g/d in the bkv positive population. average 12 hour trough blood level of tacrolimus at 3 years was 9.1 ng/ml in the bkv negative population as compared to 7.4 ng/ml in the bkv positive population. survival rates for both patient and organ were comparable at 3 years in bk viremic vs bk negative patients (100% vs 92%) and (88% vs 83%) respectively. while bk viremia is a historic risk factor for organ loss, prospective monitoring and reduction of immunosuppression is associated with comparable 3 year patient and organ survival to patients that never develop bk viremia. background: there are currently no bk virus (bkv) specific therapies available for clinical use. this study evaluates viral large t antigen as a potential target for drug development, since (a) this is a key molecule that participates in several different stages of viral replication, (b) has no homologous human protein, and (c) offers multiple functional domains for chemical binding, particularly the atp binding site, the dna binding site, the hexamerization surfaces, and the hinge region. methods: virtual screening and protein modeling techniques were applied to bkv large t antigen using a model developed from the known crystal structure of sv40 large t antigen. two different structural states of large t antigen (monomer and dimer structure) in three different states (with the nucleotide pocket empty, with bound adp and bound atp), were evaluated for a total of 6 large t antigen receptor conformations. results: a computational solvent mapping analysis of small molecular probes allowed identification of multiple functional sites, which represent potential drug binding pockets on the large t antigen molecule. it was possible to classify 1200 molecular conformations centered on the atp binding site, hexamerization surface and hinge region of the viral protein. we docked 1209 medium sized fragments (<100 da) to confirm the results obtained by computational solvent mapping, and further characterize chemical properties of the atp binding site. in another approach, known chemical structures of hsp90 and rho-kinase inhibitors were used to search compound databases and obtain a subset of 8294 compounds (mean size of 350 da) capable of docking large t antigen. cross-referencing the top solutions obtained by energy ranking, we were able to identify 50 compounds that bind large t antigen in all 6 conformational states. a subset of 5 compounds simultaneously binds the atp binding site, hexamerization surface and hinge region of bkv large t antigen. conclusions: virtual screening and three dimensional homology modeling technology has allowed us to identify compounds that can bind multiple sites on the large t antigen. these compounds are predicted to preferentially inhibit viral replication without the toxicity expected from simultaneous inhibition of host cell kinases. bk virus (bkv), a human polyomavirus, causes bkv nephritis, which often leads to graft loss after renal transplantation. currently, the only efficient therapy against bkv nephritis appears to be a reduction/change of immunosuppressive agents, and this may increase the inherent risk of rejection. since human renal proximal tubular epithelial cells (hrptec) represent a main natural target of bkv nephropathy, the analysis of bkv infection of hrptec is likely to provide necessary additional insight into bkv biology and contribute to the development of strategies for treatment of bkv nephritis. here we report the ability of 3-hydroxy-3-methyl-glutaryl coenzyme a (hmg-coa) reductase inhibitor pravastatin, which is routinely used to treat hypercholesterolemia, to repress bk virus entry pathways in hrptec and, correspondently, prevent bkv infection. the percentage of hrptec infected with bkv was assessed by immunofluorescent analysis in the absence and presence of pravastatin. both, the percentage of bkv infected cells and the intensity of bkv infection, assessed by western blotting using antibodies against large t antigen, were significantly decreased in hrptec treated with pravastatin. it is likely, that pravastatin's inhibitory effect is explained by depletion of caveolin-1, a critical element of caveolae. we demonstrate that bkv enters hrptec by caveolarmediated endocytosis and disruption of caveolin 1 mrna and protein inhibits bkv infection of hrptec. we provide evidence that pravastatin dramatically decreased caveolin-1 expression in hrptec and interfered with internalization of labeled bkv particles. our data suggest that pravastatin, acting via depletion of caveolin-1, prevented caveolar-dependent bkv internalization and repressed bkv infection of hrptec. our data represent the first report of inhibitory action of statins upon bkv infection. results: 74.6% of patients were caucasian, 12.2% hispanic, 8.9% african-american and 4.4% asian. overall 1-and 3-yr patient survivals were 85% and 77%. stratified by race, only african-american survivals differed from caucasian (3-year survivals of 71% vs. 77%; figure 1 ). compared to caucasians, african-american patients were younger (48yrs ±11.5 vs. 52yrs ±9.9), were more likely to be status 1 (10.3% vs. 5.1%), to have a serum creatinine ≥1.5mg/dl (37% vs. 31%), to receive an multi-organ transplant (8% vs. 5%), to have fulminant hepatic failure (12% vs. 7%), to have a higher meld score (22.9 ± 10.4 vs. 20.1 ±9.4) , to be in the icu (16% vs. 11%), and to be ventilated pre-transplant (8% vs. 5%); all p-values <0.001. after adjustment for each of these variables, race was still an independent predictor of mortality (p=0.01, hr: 1.22, ci: 1.048-1.425). multivariate analysis of the african-american group (n=1481) alone revealed that a bmi greater than 40 (p=0.001, hr: 2.5, ci: 1.49-4.17), a creatinine greater than 1.5 mg/dl (p=0.002, hr: 1.6, ci: 1.18-2.10), and icu admission pretransplant (p=0.018, hr: 1.5, ci: 1.07-2.17) are independent predictors of mortality in this subset of patients. conclusion: in the current meld era, race is still a predictor of worse outcomes, even after adjustment for multiple clinical variables. further work is necessary to elucidate why this disparity exists. the purpose of this study was to analyze the effects of successive pregnancies in female liver transplant recipients on newborn and maternal outcomes. data were collected from the national transplantation pregnancy registry via questionnaires, phone interviews and hospital records. analyses for linear trends (proportions and continuous variables) were done by chi square and least squares regression. there were 217 outcomes of 213 pregnancies, including twins. of the 125 liver recipients who had a first pregnancy, 61 had between one and four subsequent pregnancies. there were no significant differences in the variables analyzed as noted in the conclusions: successive pregnancies in liver transplant recipients are not associated with adverse fetal outcomes and/or increased maternal graft loss. female liver recipients with excellent allograft function without significant recurrent disease or chronic rejection who wish to have more than one pregnancy should not be discouraged to conceive. recorded and categorized in a blinded fashion. univariate, multivariate and survival analyses were performed. over a 4.5-year period (2002) (2003) (2004) (2005) (2006) (2007) , 148 olt recipients were randomized to receive a cca with biliary stent (n=76) or cca alone (n=72). patients with hepatic artery thrombosis (n=8; 5.7%) were excluded. there was no significant difference in demographic or graft-related variables. the mean age at transplant was 55 years, 82% were male, the mean meld was 21 and 23% had hepatitis c. the mean donor age was 46 years, 59% of donors were male, and the mean cold ischemia time was 8.7hrs. the only complication related to the biliary stent was one occlusion. the rate of overall bc in the stented patients was 24.7% vs. 32.8% in non-stented patients, (p=ns). however, stented patients had significantly less bc in the first 60-days post-olt (6.8% vs. 21.0%, p<0.02) and significantly less anastomotic leaks (2.4% vs. 9.6%, p<0.05). over the year following olt, stented patients also required less biliary therapeutic interventions (mean 1.8 vs. 0.9 interventions/patient, p<0.04) and fewer readmissions (mean 2.8 vs. 1.6 readmissions/patient, p<0.01). we also observed improved late graft survival (>6mo) in the stented group. intraoperative stenting of the cca at olt does not appear to reduce the long-term rate of bc but it decreases the incidence of biliary leaks and significantly improves many facets of early patient management. is background: long-term outcomes after retransplantation of the liver (re-olt) is inferior compared to primary olt. however, the survival benefit of re-olt based on model for end stage liver disease (meld) is not known. a single-center analysis of 421 adult patients who underwent re-olt between february 1984 to february 2007 was performed. survival benefits, at a given meld score, were calculated by comparing re-olt survival at 3 months to expected 3-month survival without retransplantation results: of 421 pts, 380 underwent re-olt, and 41 received 3 transplants. the figure shows re-olt survival benefit at any meld score with increased significance in patients with meld scores > 18. although meld scores 30-40 predicted the highest mortality after re-olt, they also demonstrated survival benefit. multivariate cox regression identified cold ischemia time >10 hrs (rr 1.8, p 0.001), meld 30-40 (rr 2.0, p 0.04), time from first olt (8 days -1 year, rr 1.8, p 0.02) and third transplant (rr 1.7, p 0.03) as independent predictors for mortality following re-olt. conclusions: re-olt should be considered in all patients even with low meld scores. although patients with high meld scores (30-40) exhibit poor survival outcomes, a survival benefit is achieved. survival benefit that considers both probability of death without re-olt and expected survival with re-olt, should be used for selection of retransplantation candidates. this study analyzed posttransplant complications, meld score, and donor ages and their effect on length of stay (los). methods: this irb approved retrospective review of our prospectively maintained database included 1427 liver transplant recipients transplanted between 1999-2006. los <14 days, 14-30d, and >30d were analyzed. primary analysis to look at los, meld, and donor age was performed using wilcoxon two-sample test. complication groups were analyzed using logistic regression and odds ratio estimates were determined. kaplan-meier for patient survival for the los groups was performed. univariate analysis: allograft dysfunction, vascular complication of liver allograft, intra-abdominal (other than liver), biliary, cardiac, pulmonary, neurologic, sepsis, renal, and endocrine were statistically significant (p<0.001) using fisher exact test. multivariate analysis: using logistic regression, significant complications were analyzed to determine the complications linked with los >14d and >30d. analysis of 1427 liver transplants demonstrated increasing los with increasing meld in each donor age group (50-60, 61-70) p<0.0001. for donor age >70, this relationship was not significant (p=0.2206). multivariate analysis using logistic regression determined odds ratio estimates for each complication resulting in los >14 days and >30 days. los >14 days and los >30 days were associated with different complications (as shown above). allograft dysfunction (including pnf) and renal complications were not significant factors in multivariate analysis. patient survival significantly decreased (p<0.001) with increased los >14 days. introduction: some patients with primary biliary cirrhosis (pbc) may require longterm corticosteroid (cs) therapy following liver transplantation (olt) due to recurrent inflammation in the graft. our center has attempted to minimize cs use in all of our olt recipients. we reviewed our experience in this cohort of patients to determine 1) patient outcome including recurrent disease and 2) long-term requirement for cs use in pbc patients. methods: from 1988 to 2006, 1,102 olts were performed in 1,032 adults at the university of colorado of which 70 patients (6.8 %) with pbc received 74 allografts. recurrence was defined by characteristic histologic changes on biopsy. bivariate and multivariate analyses were used to evaluate predictors of cs withdrawal. 13 potential predictors of cs discontinuation were considered: age, gender, bmi, race, presence of inflammatory bowel disease (ibd), type of graft (cadaver or living donor (ld), recurrence of aih, warm ischemia time, follow up time (time since transplant), and immunosuppressant (is). results: overall survival at 5 years was 85%. the 1, 5 and 10 year recurrence-free survival was 90, 72, and 54%, respectively. disease recurred in 18 patients (25.7%). of these 18 patients, none received a second transplant because of recurrent disease. cs was withdrawn in 73% of patients at time of review. independent predictors of cs discontinuation are age (>median) (p = 0.0029) and ld graft type (p=0.0018). conversely, cyclosporine (csa) (p=0.0012), female gender (p=0.0197), and bmi > 31 (p=0.0306) were negatively associated with cs withdraw. interestingly, cs withdrawal did not influence pbc recurrence. conclusions: 1) long-term outcomes in pbc patients are favorable and disease recurrence can be managed medically without abstracts re-transplantation. 2) using an aggressive cs minimization approach, almost 3/4 of the patients were cs-free at the time of last follow-up. 3) increasing age and ld grafts were associated with successful cs withdraw; while csa, female gender, and increasing bmi were associated with unsuccessful cs withdraw. incidence cholestatic disease (cd), either chronic rejection or recurrent primary sclerosing cholangitis (psc), post liver transplantation (lt) occurs in 5-35% of psc grafts. the study objectives were to evaluate the incidence and long-term outcome of cd. from 1985 to 2006, 141 grafts in 125 consecutive psc patients and 85 grafts in 79 concurrent alcoholic liver disease (ald) patients were compared. cd was diagnosed by biliary imaging and/or histology. median follow-up was 57 months. the groups were similar including meld score and cold ischemic time; however, roux-en-y biliary anastomosis was more common in the psc group (95% vs. 13%, p<0.01). the psc group had more cmv hepatitis (22% vs. 6%, p<0.01) and acute rejection (60% vs. 30%, p<0.01), and fewer biliary anastomotic strictures (7% vs. 16%, p<0.01). cd occurred in 38 psc grafts and 7 ald grafts (p<0.01). the incidence of cd was greater in the psc group (p=0.02, fig. 1 ). no significant risk factors for cd were identified. in the psc group the graft survival was lower in the cd group (p=0.04, fig. 2) ; however, patient survival at 10 and 15 years was not effected by cd: 73% and 67% vs 75% and 60% because the re-lt rate was greater in this group (26% vs. 6%, p<0.01). at 15 years, patient survival in the psc group was better than for ald (64% vs 22%, p<0.01). long-term outcome post lt for psc is good and better than for ald; however, cd continues to develop beyond the first 5 years with a prevalence of 43% at 15 years. graft loss in patients with cd is high, but with re-tx, patient survival is similar to non-cd patients. further studies to distinguish chronic rejection vs. recurrent psc may provide insight into the prevention of cd following lt for psc. discussion: our analysis showed that patients with aih have a worse long term survival compared to pbc and psc after ddlt. this may be explained by possibly more advanced disease at the time of presentation or more septic complications due to pretransplant salvage therapy with immunosuppressants. also,pbc had the worst relative outcome after ldlt in this group-possibly explained by the older age of the recipients. overall, ldlt offered better outcomes than ddlt in terms of survival in patients with aih,pbc and psc. this study highlights an important and previously unvisited aspect of transplantation for autoimmune and cholestatic liver diseases. inflammatory bowel disease course in patients transplanted for primary sclerosing cholangitis. ariana wallack, 1 joel s. levine, 2 lisa forman. 2 1 internal medicine, univ. of colorado hsc, aurora, co; 2 gastroenterology and hepatology, univ. of colorado hsc, aurora, co. the natural history of ibd following liver transplant (lt) for psc is unknown. prior studies have not shown factors consistently associated with disease activity, but were limited by small sample sizes. the aim of the study is to describe our experience with ibd post-lt in patients with psc and determine factors predictive of disease activity. a survey was mailed to 115 liver recipients transplanted for psc between 1988 and 2006 asking about medications, ibd activity and quality of life after lt. responses were linked to our lt database. results 88 (77%) recipients responded. 76% were male with a median of 82 (7-215) months since transplant. 93% were caucasian with a median transplant age of 46 (16-71) years. 16% developed recurrent psc post-lt. 74% had a diagnosis of ibd (63% uc). immunosuppression included tacrolimus in 84% and cyclosporine in 11%. 71% experienced at least one episode of acute rejection. 73% rated their quality of health 4-5 on a scale of 1-5. there was no significant difference in demographic variables between ibd and non-ibd cohorts. 66% of respondents had ibd pre-lt. of the 30 patients without pre-existing ibd, 7 developed ibd post-lt. of the de novo ibd cohort, 43% were male, median transplant age was 45 years (39-57), and 86% were caucasian. ibd developed in 71% at more than 3 years post-lt. there was no statistical difference between the de novo ibd cohort and those with pre-existing ibd except for ibd type (57% uc vs 88%, p=0.008). significantly more patients were not on any ibd medications post-lt compared to pre-lt (75% vs 91%, p=0.02). fewer post-lt patients were on aminosalicylates (63% vs 79%, p=0.03) and prednisone (14% vs 31%, p=0.03) compared to pre-lt. post-lt recipients developed fewer ibd flares requiring hospitalization (27% vs 45%, p=0.004). 38% reported improvement in ibd activity post-l. 23% and 33% reported no change and worsening activity, respectively. there was no significant difference between reported disease activity and immunosuppression, cmv status, rejection rate, recurrent psc, transplant age, gender or race. background: symptomatic cmv continues to be a significant problem. the costs associated with its development remain high without an optimal method for prevention. the aim of this study was to measure the pharmacoeconomic impact of implementing an abbreviated pre-emptive monitoring strategy versus valganciclovir (vgc) prophylaxis in a large teaching hospital. methods: costs for this analysis were based on a societal perspective, including drug, personnel, hospital, outpatient infusion and monitoring costs related to resources needed to perform pre-emptive monitoring and treatment of cmv related events. time per hr costs were nurse/data coordinator $45, physician $200, and pharmd $52. cmv pcr cost was $203. vgc cost per mg was calculated for prophylaxis, treatment and 30 days of consolidation as needed based on an awp ($0.09 per mg). estimated cost of cmv syndrome was based on cost of picc line placement, drug, personnel and supply costs based on 21 days of therapy and was estimated to be $11,885.50. inpatient admission cost per day were $3727. results: a total of 119 patients were included in this analysis. baseline and transplant demographics were well matched. table 1 displays the total direct and indirect costs accumulated for each group. cmv syndrome occurred in three patients for each group. there was no cmv disease in either group. 27% of patients in the pre-emptive group had dnaemia, but only 4 patients required oral anti-viral therapy. provider time and lab monitoring costs were significantly higher in the preemptive group, while direct medication cost was significantly higher in the prophylactic group. conclusions: frequency of disease severity and outcomes were equal in each group. although the overall costs between strategies is equivocal, allocation of resources to provide pre-emptive monitoring places the burden of disease prevention on the health care system versus the patient necessitating further abbreviation of these strategies. we propose a non-simultaneous form of kidney paired donation that starts with a living, non-directed donor (lnd). a paired donation matching algorithm was developed to allow for lnds to start potentially never-ending altruistic donor (nead) chains in addition to closed loops of 2-, 3-and 4-way exchanges. results: in july 2007, a lnd from michigan traveled 1500 miles to donate a kidney to a woman whom he had never met in arizona. the following week, the arizona recipient's husband donated one of his kidneys to a 32-year-old woman in ohio. the following month, the mother of this recipient traveled to a city 3 hours away to donate her kidney to a patient whose incompatible donor simultaneosly gave a kidney to the fourth patient in the chain. the incompatible donor for this fourth recipient is now slated to give her kidney to a patient in maryland. over the past 9 months, 60 transplant programs have partnered to perform 10 paired donation transplants. demonstrating the advantage of nead chains over classic paired donation, 8 of 10 transplants resulted from altruistic donor chains. conclusion: in order to fully realize the potential of the above approach, one must be willing to supplant two prevalent ideas: 1) that kidneys from altruistic donors should be given to the top candidate on the deceased donor waiting list, and 2) that paired exchanges must be done simultaneously. while there are certain pitfalls to using chains of donors as opposed to traditional "swaps" (i.e. the possibility that a donor could renege, or the accumulation of type ab donors who are not likely to be able to begin another chain), this proposed paradigm shift could result in a very significant increase in both the number and quality of paired donation kidney transplants. purpose: medical literature and national best practices correlate families' understanding of brain death with organ donation rates. analysis of hospital data demonstrated variability in family communication. although surgical residents frequently interact with family members of potential donors and play a critical role in the donation process, they receive no formal cst. we sought to determine if pre-training residents improved performances in cst involving explanation and notification of brain death to family members and aided efforts to achieve the national donation rate goal of 75%. methods: in collaboration with a regional organ procurement organization (opo), an educational model for end of life cst was developed. 17 surgical residents were divided into 2 groups. the first group (n=9) attended a 3-hour didactic session at the opo that included role-playing exercises explaining brain death to families. opo staff, trained to serve as family role-players and skills station coaches, debriefed residents after each simulation. the second group (n=8) received no specialized training. six weeks later both resident groups participated in formal videotaped family communication simulations. independent observers (hospital faculty and senior opo staff), blinded to training, evaluated residents' communication skills using a 12-parameter assessment tool. all residents reviewed their videotaped performances and evaluations, then repeated the simulations after six months. results: during the study period, the pre-trained resident group assessment scores increased by 25% (p=0.0001), while the untrained group increased by 39% (p<.0001). while evidence of improvement existed in both groups, pre-trained residents consistently scored higher when compared to untrained (80% vs. 71%, p=0.037). during this same time period, donation rates in the surgical intensive care unit (sicu) increased by 21%. conclusion: our educational model demonstrated effective training in communication skills during end of life discussions. although a direct relationship cannot be established, donation rates in the sicu increased after resident training. incorporation of this training program into resident education has the potential to improve donation rates and increase the number of organs available for transplantation. program. saverio mirarchi, 1 graeme n. forrest, 1 benjamin philosophe. 2 1 medicine, university of maryland, baltimore, md; 2 surgery, university of maryland, baltimore, md. objective: to improve the efficiency of care for solid organ transplant patients by having internists work in conjunction with transplant surgeons to manage transplant patients admitted to the hospital more than 30 days post organ transplant. this includes patients who have undergone kidney, pancreas, and liver transplants which our center transplants over 300 of these organs/year. in 2002, the organ transplant program was divided into a surgical transplant service (sts) and medical transplant hospitalist service (mths). the mths consists of four full time physicians, one part time physician, and one nurse practictioner with daily rounds from a transplant surgeon on a weekly rotating schedule. methods: we analyzed our data from the past five years since the inception of the mths at a large university medical center. the length of stay (los) for the mths and sts were compared to data from the previous combined program as well as data from the university health consortium (uhc). in addition, we looked at the cost of care to determine if there were any savings related to reduced los and adjusted for the combined salary of the mths. results: in the review period, the total admissions for both the mths and sts averaged 1450 admissions/year. over the past five years the mths showed a major decrease in the los index, defined as the ratio between the observed los and the expected los based on uhc data. this index dropped from 1.27 to 0.91 for patients on the mths. there was also a parallel drop in the sts los index from 1.53 to 1.05. this was associated with a significant cost savings for the hospital. on average, the program has realized an average savings of approximately $1,000,000 per year. when accounting for the combined salary for the mths group which is 625,000 dollars/year, the total savings over the 5 year period this amounts to $1.9 million. conclusion: the creation of a mths working within an organ transplant program at a large university center has been able to demonstrate a major decrease in los for transplant patients which has resulted in decreased costs and improved efficiency. this has also allowed more focused care for a complex medical population. we believe that our program can serve as a model for similar programs at other busy transplant sites. with increasing demand for kidney transplants(tx), more patients are opting to travel outside the us to obtain transplantation. we describe the characteristics and outcomes of 32 kidney tx recipients followed at our center who traveled abroad for a kidney tx between 1995 and 2007. methods: data were obtained via chart review. we compared demographics to all tx recipients at our center during the same period and compared post-tx outcomes to a cohort of patients transplanted at our center matched for age, race, tx year, dialysis time, prior tx, and donor type. median follow-up time was 487 days (range 31-3056). results: demographics are outlined in table. patients transplanted abroad were more likely to be asian and had shorter dialysis times. most patients were transplanted in china (44%) followed by iran (16%), the philippines (13%), and india (9%). living unrelated tx were most common. all patients were discharged on a calcineurin inhibitor, pred, and either mmf (90%), aza (7%), or rapamycin (3%). 7 patients received induction. only 4 patients received cmv prophylaxis. the median duration of hospitalization was 15 days. the median time post-tx to initial visit at our center was 35 days. 4 patients required urgent admission to hospital, 3 of whom lost their grafts. 17 patients ( graft survival at 1-year was 96% for both "tourists" and matched recipients at our center. median time to graft loss was 500 (31-2832) days. mean scr and rejection 1-year post tx was not significantly different between recipients transplanted abroad and at our center. conclusion: compared to patients transplanted locally, patients transplanted abroad had similar graft survival and renal function post tx, but had a high incidence of infectious complications. [background] the primary benefits anticipated following successful induction of allograft tolerance are avoidance of the complications of long-term immunosuppression and prevention of chronic rejection. we have previously reported successful induction of renal allograft tolerance in recipients of hla mismatched combined kidney and bone marrow transplantation (ckbmt). no evidence of chronic rejection has been observed in these recipients after immunosuppression-free periods of 1 to 4 years. in the current study, we have evaluated the longer-term economic impact of this approach. [method] the conditioning regimen for ckbmt included cyclophosphamide, thymic irradiation, anti-cd2 mab, and a calcineurin inhibitor, which was discontinued after 9-12 months. 18 stable renal transplant recipients receiving ongoing triple or double drug immunosuppressive therapy with compatible follow up times were compared with the four tolerant recipients. tolerant patients and stable patients on maintenance immunosuppression were also comparable with respect to their age, their original disease and donor-recipient histocompatibility. [results] the perioperative charges for ckbmt were approximately $90,000 higher than those for conventional living donor kidney transplantation. after 9-12 months, continuing medications in ckbmt recipients included only occasional over-thecounter analgesics. in contrast, the stable conventionally treated recipients were taking an average of 8 pills daily. these included treatments required for de novo diabetes (11%), hypertension (67%), hyper lipidemia (22%) and gastro-intestinal symptoms/ prophylaxis (78%) in addition to their maintenance immunosuppression. these needs resulted in annual maintenance charges of over $15,000/year/allograft recipient and do not include unmeasured costs related to issues such as quality of life or absences from employment. [conclusion] even with this admittedly expensive approach to tolerance induction, the overall medical costs for conventional kidney transplantation with ongoing immunosuppression and treatment for complications will exceed the cost of tolerance after approximately 5 years. increasing african american donation rates in a midwest metropolitan community. susan gunderson, 1 susan mau larson, 1 david m. radosevich, 2 clarence jones, 3 bill tendle, 3 tiffany scott. 1 1 lifesource, st. paul, mn; 2 transplant information services, university of minnesota, minneapolis, mn; 3 southside community health services, minneapolis, mn. purpose: african americans are underrepresented in deceased organ donation in this +2 million community. historically minimal educational outreach had occurred and this study was designed to understand the community's disposition toward donation and to increase support for donation. methods: television, newspaper, and radio advertising aired over a 24 month period beginning in 2004 using the nationally produced donate life-african american campaign as the primary intervention. other components included related faith-based and community outreach. the opo partnered with a minority focused community health clinic to survey african americans pre-and post-intervention. a mailed, selfadministered survey was sent to a sample drawn from organizational lists with a high likelihood of including african american households. for the evaluation, the sample was separated into 1) community members and 2) church members exposed to faithbased campaigns. results: african americans in this community have a sophisticated understanding of the importance of donation (average 12 of 15 knowledge questions scored correctly) in pre-intervention survey. in both community and church groups media exposure and donation knowledge increased after the media campaign (p<0.001 and p=0.004 respectively). similarly, donor designation rates for the entire population on the drivers licenses increased (33.0% versus 40.1%, p=0.119). among all african americans the propensity to donate was, however, unchanged following the campaign. propensity to donate increased (p=0.034) in the community sample whereas there was a reduced propensity to donation (p=0.092) in the church sample. during the study time period the opo also experienced significant increases in donation authorization rates among african-americans, increasing from 37% to 75%. summary: a media based campaign combined with grassroots outreach is an effective tool to increase knowledge and awareness. partnership between the opo and community and faith based leadership was a significant positive byproduct of the project and should be included in future outreach efforts. background: it has been hypothesized that the clinical benefits of anti-thymocyte globulin (atg) induction therapy do not completely result from immunodepletion, but may also result from induction of immunoregulatory t-cells (treg). in this prospective, controlled study we investigated the effect of atg-induction therapy on the frequency and phenotype of peripheral cd4 + foxp3 + cd127 -/low t-cells in kidney transplant patients. methods: after transplantation, 16 patients received atg-induction therapy (thymoglobulin ® ) and triple therapy consisting of tacrolimus, mmf and steroids. the control group (n=18) received triple therapy only. by flow cytometry, t-cells were analyzed for markers associated with immune regulation: cd25, foxp3 and cd127. within the foxp3 + t-cell population, the cd45ro (memory) and ccr7 (homing receptor) markers were characterized. results: pre-transplant levels of cd4 + foxp3 + cd127 -/low t-cells in all patients were 3-17% (median 6%) of cd4 + t-cells. one wk post atg induction therapy, no measurable numbers of treg were present. at 12 wks post atg-induction therapy, a higher proportion of the first detectable t-cells expressed foxp3 compared to the control-group (atg vs. control-group; 7 vs. 4%, median, respectively, p=0.03) and then returned to pre-transplant levels at 26 wks. this increased proportion of cd4 + foxp3 + cd127 -/ low t-cells resulted in a significantly higher foxp3 + /foxp3 neg ratio than in the controlgroup at 12 wks; 0.074 vs. 0.046, median, p=0.02) . at 26 wks, we found a decline in the proportion of naive foxp3 + treg (cd45ro neg ccr7 + pre-transplant vs. post-transplant; 21 vs. 9%, median, p=0.02) which was associated with a rise in the proportion of memory foxp3 + treg (cd45ro + pre-transplant vs. 26 wks post-transplant; 65 vs. 82%, p=0.02). moreover, the proportion of memory t-cells exceeded that in the control-group at 26 wks (atg vs. control-group; 82% vs. 67%, median, p=0.05) which was mainly due to an increase in the proportion of effector memory foxp3 + treg (cd45ro + ccr7 neg ). conclusion: after atg-induced immune cell depletion, a shift towards cd4 + foxp3 + cd127 -/low peripheral regulatory t-cells with the memory phenotype was measured in kidney transplant patients. this finding suggests that atg treatment triggers the generation of de novo peripheral regulatory t-cells by homeostatic proliferation. introduction in a prospective study, we investigated whether donor-specific regulatory cd4 + cd25 bright+ foxp3 + t cells develop in kidney transplant patients. methods we analyzed the percentage and function of peripheral regulatory cd4 + cd25 bright+ foxp3 + t cells of 51 patients before, 3, 6 and 12 months after kidney transplantation. the immune regulatory capacities of cd4 + cd25 bright+ foxp3 + t cells were assessed by their depletion from pbmc and reconstitution to cd25 neg/dim responder t cells at a 1:10 ratio in the mlr. in the first year after transplantation, peripheral cd4 + cd25 bright+ foxp3 + t cells decreased from 8,7% ± 3,0 pre-transplantation to 6,1% ± 1,8 at 12 months (p<0.001). while mlr reactivity to 3 rd party-ag (3 rd p) significantly improved (p<0.05), the reactivity against donor antigens remained low. functional analysis demonstrated potent donor-specific regulatory activities by cd4 + cd25 bright+ foxp3 + t cells after transplantation. depletion of cd4 + cd25 bright+ foxp3 + t cells from pbmc resulted into increased proliferation upon stimulation by donor antigens (p<0.01). upon reconstitution, the capacity of cd4 + cd25 bright+ foxp3 + t cells to control the proliferation of anti-donor reactive cd25 neg/dim t cells increased over time: from 58% (median) pre-transplant to 85% post-transplant at month 12 (p<0.01). moreover, the anti-donor regulatory activities by the cd4 + cd25 bright+ foxp3 + t cells were significantly more vigorous than those controlling 3 rd p-ag stimulated responder t cells (65%, p<0.05). the generation of potent donor-specific regulatory cd4 + cd25 bright+ foxp3 + t cells in the periphery of kidney transplant patients prevents the development of adequate alloreactivity. conclusions: these results indicated that anti-donor responses could be detected even in a significant proportion of "stable" long-term hla identical kidney transplant recipients. speculatively this may be the cause of late graft failures in this group of patients. expression klotho is a gene almost exclusively expressed in renal distal tubules and loss of expression is associated with accelerated aging. in various models of acute and chronic renal injury, and with normal aging, renal klotho expression has been found to decrease. increased donor age is associated with poorer long term allograft function. we hypothesized that klotho mrna expression in renal implant biopsies would correlate with donor kidney quality, as determined by donor chronologic age and peri-transplant renal injury. our recent unsupervised microarray analysis of 87 renal implant biopsies revealed a continuum of organ quality across all samples ranging from the best living donor (ld) kidneys at one end to the worst performing deceased donor (dd) kidneys at the other (am j transpl 2007, nov 16,epub). three predominant groups were identified of ld, dd1 kidneys with low risk (9.5%) of delayed graft function (dgf) and dd2 kidneys with high risk (35%) of dgf (p < 0.05). analysis of klotho gene expression among these groups also revealed a spectrum of expression from highest levels in ld to lowest in dd2 kidneys, especially those who developed dgf. differences were highly significant among ld vs dd kidneys (p < 0.001), although donor age was not different between these groups. klotho transcript levels were significantly different among kidneys that developed dgf compared to those with immediate graft function (igf) (p < 0.05). in conclusion, reduced klotho gene expression is associated with grafts at risk of poorer function and appears to be independent of donor age. decreases in klotho expression likely reflect other factors impacting the renal tissue, which may impact potential for repair and ultimately allograft function. anti renal allograft rejection episodes produce a stereotypical response in the allograft characterized by infiltration by cytotoxic t lymphocytes (ctl), potent ifng response by donor and recipient cells, and decreased transcripts associated with the epithelium. we previously identified pathogenesis based transcript sets (pbts) that reflect the disturbance in rejecting allografts (qcats -ctl, grits -ifng response, kts -decreased function, ajt 7:2712 , 2007 . we hypothesized that anti-rejection treatment would reverse the transcriptome changes of rejection more than histopathologic lesions. using microarray analysis we measured expression of these pbts in 18 antibody mediated rejection (abmr)(11 untreated, 7 treated) and 31 t-cell mediated rejection (tcmr) (25 untreated, 6 treated) biopsies, normalized to nephrectomy samples. histopathology scoring of the primary rejection lesions were analyzed; interstitial inflammation(i), tubulitis(t), intimal arteritis(v), and glomerulitis(g) (fig 1a) . of these, only i and t differentiated between abmr and treated abmr (p<0.05) yet none differentiated between tcmr and treated tcmr. pbts on the other hand differentiated treated from untreated for both abmr (p<0.04) and tcmr (p<0.005) with all 3 pbts (fig 1b/c) . the changes in transcript expression in treated cases were dramatic. particularly impressive was the consistent correction of the disturbances in pbt expression despite the heterogeneous treatment following abmr episodes. unlike abmr treatment, tcmr treatment always included steroids which may contribute to the more pronounced changes compared to abmr. the time of treatment before the biopsy, which also varied within the groups, did not affect the changes in pbt expression since values in treated cases approached those of nephrectomy samples. thus, histologic rejection lesions persist following anti-rejection treatment whereas transcript disturbances of rejection are greatly reduced compared to untreated cases. this study suggests that assessment of anti-rejection treatment is more sensitively monitored by transcript expression than histopathology. blood deciphering the mechanisms of tolerance and chronic immune-mediated rejection remains a major goal in transplantation. data in rodents suggests that toll-like-receptors (tlr), regulators of innate immune responses, play a role in determining graft outcome. however, few studies have focused on tlr in human kidney transplant recipients. we addressed this issue by analyzing the peripheral blood (n = 75) and graft biopsies (n = 26) of renal transplant patients and healthy volunteers. we analyzed, for the first time, the expression of tlr4 in pbmc from kidney recipients with contrasted situations: operational tolerance and chronic immune-mediated rejection (banff 2005), compared to patients with normal histology and stable graft function, non transplant patients with renal failure and healthy volunteers. we found that myd88 and tlr4 were significantly contrasted in the pbmc, and in particular in monocytes, of patients with chronic immune-mediated rejection vs. operational tolerance. chronic rejection patients had significantly increased tlr4 and myd88 compared to operationally tolerant patients, who resembled healthy volunteers and non transplant patients with renal failure. interestingly, analysis of tlr4 transcripts in graft biopsies from patients with normal histology or chronic immune-mediated rejection reflected the blood findings, with a significant increase of tlr4 in chronic immune-mediated rejection. thus, we provide data to support a link between tlr4 expression and long-term graft outcome. the role of tlr and their endogenous ligands in mediating allograft rejection or acceptance therefore warrants further investigation and could give rise to new strategies of therapeutic intervention. our results suggest that peripheral blood tlr4 shows potential as a biomarker of chronic immune-mediated rejection and that measuring blood tlr4 levels may help to identify patients experiencing chronic rejection who require a biopsy. moreover, our data suggest that absence of tlr signaling may be a feature of operational tolerance to kidney grafts. identification of immune identification of immunological tolerance is an important prerequisite in order to establish an individually-tailored approach to the post-transplant management of allograft recipients. it will also provide new insight into the mechanism underlying the balance between tolerance and rejection. here we present data from a multi-centre study aimed at identifying tolerance to renal allografts. we have collected samples from five selected groups of renal transplant recipients: drug-free tolerant patients that were functionally stable despite remaining immunosuppression-free for more than one year; functionally stable patients on minimal immunosuppression (<10 mg/ day prednisone); stable patients maintained with calcineurin inhibitors (cni); stable patients maintained on cni-free immunosuppression regimen; and patients showing signs of chronic rejection. a group of age and sex matched healthy volunteers was also included as control. several biomarkers and bioassays, were combined to provide an immunological 'fingerprint' of the tolerant state. immunophenotype showed a selective expansion of peripheral blood b and nk lymphocytes in drug-free tolerant patients. this group of patients was also characterized by the absence of anti-donor specific antibodies. the differential expression of several immune relevant genes and a high ratio of foxp3/ α-1,2-mannosidase expression in these patients was observed. tcr landscape analysis highlighted differences between the vβ repertoires of drug-free tolerant recipients and chronic rejection patients. additionally, direct pathway donor-specific hyporesponsiveness by ifnγ elispot and lack of indirect pathway anti-donor responses assessed by trans-vivo dth were detected in drug-free patients. the diagnostic capabilities of the combined results of several of the above mentioned biomarkers and bioassays are as follows: specificity 0.964, sensitivity of 0.933 and a positive predictive value of 82.4%. these biomarkers could be used to inform drug weaning protocols of kidney transplant recipients. bile acid aspiration stimulates lung allograft immunity. bile acids detected in the broncho alveolar lavage (bal) as a marker of aspiration has been associated in a dose dependent fashion to earlier development of bronchiolitis obliterans syndrome. we sought to study the relationship between bile acids and active immune molecules as detected in the bal. methods: bal collected prospectively from lung transplant recipients at routine surveillance bronchoscopies were assayed for bile acids. samples were then assayed by luminex for cytokines , and by elisa for pulmonary collectins (sp-a, sp-d). results were analyzed according to levels of bile acids as per roc testing for accuracy for bronchiolitis obliterans syndrome diagnosis (high levels ≥3.5 µmol/l). results: we prospectively examined 120 lung transplant recipients and a total of 271 bal samples were collected. in 114 no bile acids were detected, low levels were present in 110, and high levels were detected in 47 samples. samples with high bile acids had significantly greater innate (tnf-a, il-1b, il-12, il-10, il-6) and adaptive (ifn-g, il-2) cytokines as well as greater chemokines (mcp-1, il-8) compared to the other samples. in contrast pulmonary collectins sp-a and sp-d were significantly reduced in samples with high bile acids. the figures shows the median and interquartile range for each molecule according to bile acid levels. conclusion: bile acids detected in the bal as markers of aspiration stimulate the lung allograft immunity in a dose dependent fashion. in particular high levels of bile acids are associated with an impaired lung specific innate defense system provided by the pulmonary collectins and with a broncho-alveolar district "cytokine storm". tolerance/immune deviation iii the results of using ex vivo cd4+ t-cells converted dn t-cells in nod mouse models support the concept and the feasibility of potentially utilizing this novel cell-based therapeutic approach clinically for the treatment of autoimmune type i diabetes. horng-ren yang, 1 gouping jiang, 1 john j. fung, 1 shiguang qian, 1 lina lu. 1 1 immunology and general surgery, cleveland clinic, cleveland, oh. liver transplant tolerance was recognized by spontaneous acceptance of liver allograft in many species. the underlying mechanism remains unclear. interestingly, although liver allografts are accepted, hepatocyte transplants in the same combination are promptly rejected, indicating a crucial role of liver tissue cells in immune suppression. we have demonstrated a profound t cell inhibitory activity of hepatic stellate cells (hpsc), which are known to participating in repairing and fibrosis during liver injury. addition of activated (a) hpsc, but not quiescent hpsc, significantly inhibited allo-dc induced-t cell proliferative responses (mlr) in a dose dependent manner, which was associated with enhanced t cell apoptosis (tunel). neutralization of b7-h1 by anti-b7-h1 mab significantly reduced the hpsc-induced t cell apoptosis and reversed the inhibition of t cell proliferation, suggesting a key role of b7-h1. to evaluate this in vivo, balb/c islets (300) were co-transplanted with 3 x 10 5 activated hpsc (b6) into stz-induced diabetic b6 recipients. co-transplant with hpsc effectively protects islet allografts from rejection. this was associated with reduction of graft infiltrating t cells and enhancement of apoptotic activity. co-transplant with hpsc from b7-h1 -/livers markedly lost their islet graft protective capacity, associated with less apoptosis of infiltrating cells. to determine the subsets of apoptotic t cells, t cells were isolated from spleen, draining lymph nodes, and grafts for phenotype and function analyses. on pod 8, a marked reduction of graft infiltrating cd4 + (30.7 %) and cd8 + t cells (31.3 %) was seen in hpsc co-transplant group, as compared to islets alone, which was further progressed thereafter. cd8 t cells dropped ∼7 folds on pod 140. cd4 + / cd8 + ratio was increased from 0.5 at the early pod to 2.0 in the long-term survival grafts. adoptive transfer of cfse-labeled des transgenic t cells was used to track the response and fate of antigen-specific cd8 + t cells. the results showed active division of des + cells within the allograft in both the islet only and hpsc co-transplantation groups. however, accumulation of these des + cells was significantly lesser in the hpsc co-transplantation group as compared to that in the islet only group (1.9×10 3 vs. 10×10 3 cells per graft). these findings suggest that hpsc induce antigen-specific cd8 + t cell death, and may not inhibit their activation. donor-specific memory t cells are potent mediators of allograft rejection due to their ability to proliferate and give rise to cytotoxic and inflammatory cytokine-secreting effectors within hours of stimulation. furthermore, memory t cells have been shown to be relatively resistant to the effects of many tolerance-induction protocols, including blockade of the cd28 and cd154 pathways. while seminal studies have shown that donor-reactive memory cells, generated through pre-sensitization with donor tissue or infection with pathogens with cross-reactive epitopes, contribute to costimulation blockade-resistant rejection of fully mhc disparate allografts, the role of memory t cells specific for minor antigens in costimulation blockade-resistant rejection has not been well studied. we addressed the ability of memory t cells specific for a single donorderived class i epitope to mediate this process. tcr transgenic t cells (ot-i) specific for siinfekl/kb were adoptively transferred into naive b6 recipients, which were then infected with ovalbumin-(siinfekl) expressing listeria monocytogenes (lm-ova). at memory, mice received a skin graft expressing ovalbumin (mova), and therefore the siinfekl epitope. results showed that the transferred ot-i t cells proliferated in response to lm-ova, but not in response to a control infection with wild-type listeria (lm). at day 9, ot-i t cells comprised ∼15% of the total cd8+ t cell compartment. during memory, the siinfekl-specific cells comprised ∼1-2% of the total cd8+ t cell compartment. engraftment of mova skin on lm-ova memory recipients resulted in rejection with accelerated kinetics relative to lm infected controls (mst=13d vs 20d). following treatment with ctla-4 ig and anti-cd154, 9/10 lm-ova infected recipients experienced costimulation blockade-resistant rejection, while lm-infected controls went onto long-term graft survival (p<0.001). lm-ova-infected recipients also resisted the engraftment of mova-expressing donor bone marrow following a tolerance induction protocol containing busulfan, ctla-4 ig, and anti-cd154. these results suggest that memory t cells specific for a single surrogate minor antigen are sufficient to induce costimulation blockade-resistant rejection, and support the feasibility of using this model to study the specific requirements for donor-reactive memory t cell activation and tolerance induction during transplantation. the during an immune response, cd4 helper t cells can be instructed by non-antigenspecific signals to differentiate into functionally distinct subsets with mutually exclusive patterns of cytokine production. we find that ikaros, a zinc finger transcription factor required for lymphocyte development, is crucial for the development of polarized t helper subsets. in the absence of ikaros dna binding activity, cd4 t cells induced to undergo th2 differentiation in vitro or in vivo produce high levels of il-4, but fail to silence expression of ifn-gamma and il-17, cytokines that contribute to inflammatory disease processes such as autoimmunity and organ transplant rejection. similarly, ikaros is required for repression of il-4 and il-17 expression by th1 cells, and inhibition of ifn-gamma and il-2 production by th17 cells. our results show that ikaros controls the expression of transcription factors such as gata-3, c-maf, stat-6, and runx3, and in polarized th2 cells, ikaros inhibits ifn-gamma gene expression through direct repression of the t-bet locus. these studies place ikaros, a dna binding protein previously recognized only as a regulator of lymphocyte development, as a master regulator of peripheral t cell differentiation and function. introduction as the fastest growing subpopulation seeking organ transplantation is >65 yrs of age, it will be imperative to discern how aging impacts the acquisition of transplantation tolerance. prior work has demonstrated that viral infections induce the development of alloreactive t cells, which impede the induction of transplantation tolerance. in this study, we tested the hypothesis that aging alters host defense against viruses leading to the development of cross-reactive t cells, which impair transplantation tolerance induction. we first examined how aging modifies the function of plasmacytoid dcs (pdcs), as ifnα production by pdcs is essential for control of viral infections. using both in vitro and in vivo murine systems, we found that aged pdcs produced lower levels of ifnα in response to tlr9 activation with cpg sequences or herpes simplex (hsv)-2 virus (elisa). aged mice (18-20 mths) failed to the clear this virus as effectively as young (2-4 mths) mice. this was associated with increased liver inflammation, the release of systemic th17-skewing cytokines, il-6 and il-21, and augmented splenic il-17 levels in aged mice (elisa). prior to transplantation, unmanipulated aged mice (b6 or cba background) produced more donor-specific il-17 effector-memory t cells as compared to unmanipulated young mice (elispot). furthermore, mlr assays demonstrated that aged memory cd4+ t cells from unmanipulated mice produced significantly more il-17 in response to donor antigen compared to young t cells (elisa). to determine if aged recipients manifest an altered response to therapies that prolong allograft survival, aged and young b6 mice received balb/c skin allografts and perioperative anti-cd45 and anti-cd154. we found that aged mice rejected their allografts significantly faster (median survival 20 days) than young mice (mst, 76 days, p <0.01). similar results were noted when we employed perioperative treatment with anti-cd154 + dst and when we altered the donor-recipient (b6 to cba) strain combination. pre-treating aged mice with an anti-il-17 mab improved the efficacy of the graft-prolonging therapy compared to aged mice that received control mab (p = 0.04). conclusion our results suggest that impaired control of viral infections with aging leads to the generation of alloreactive il-17 producing t cells that impair therapies that may induce transplantation tolerance. prevention of type 1 diabetes by thymus genetic modification with protective mhc class ii molecules. jesus r. paez-cortez, 1 michela donnarumma, 1 chaorui tian, 1 john iacomini. 1 1 transplantation research center, boston, ma. introduction. susceptibility to type 1 diabetes is determined by multiple genetic factors, among the strongest of which is the inheritance of at-risk genes that lead to disease development. here we examined whether diabetes can be prevented by providing protective mhc class ii genes through directly infecting the thymus of diabetes prone nod mice. methods. after direct exposition of the thymus, lentiviruses encoding control (phage-cmv-dsred-ires-zsgreen-w) or protective mhc class ii iaβ d (phage-cmv-iaβ d -ires-zsgreen-w) genes were injected in a single thymic lobe of 3 to 4 week old female euglycemic nod mice. gene expression was determined by microscopic examination at different time points after injection and blood glucose levels were monitored weekly to examine whether this approach prevents the development of diabetes. the presence of diabetogenic cd8 + t cells were detected by flow cytometry via tetramer staining of splenocytes. pancreatic islet integrity and insulin production was assessed by immunohistochemistry. results. viral gene expression was observed exclusively in thymic epithelial cells beginning at 5 days post-injection in both groups. nod mice injected with control lentivirus developed diabetes by 22 weeks post injection, similar to non-treated animals (18-20 weeks). in contrast, phage-cmv-iaβ d -ires-zsgreen-w injected animals remained normoglycemic at 12 months post-injection. diabetogenic cd8 + t cell population were not detected in splenocytes of iaβ d injected animals using mhc class i tetramers. islet integrity and insulin production was preserved in the treated group, in marked contrast to controls, which exhibited characteristic lymphocytic infiltration of islet cells and low insulin storage. conclusions. thymic genetic modification with protective a mhc class ii iaβ d molecule can be used to prevent diabetes in nod mice. central deletion of diabetogenic t cell populations may be involved in prevention of autoimmunity in these animals. role of invariant nkt cells in liver sinusoidal endothelial cell-induced immunosuppression of t cells with indirect allospecificity. masayuki shishida, hideki ohdan, yuka tanaka, masataka banshodani, yuka igarashi, toshimasa asahara. surgery, hiroshima university, hiroshima, japan. we have reported that liver sinusoidal endothelial cells (lsecs) endocytose portally injected allogeneic splenocytes and can negatively regulate t cells with indirect allospecificity via the fas/fasl pathway. as a result of in vitro transmigration across the lsecs from balb/c mice treated with a portal injection (pi) of b6 mhc class iideficient (c2d) splenocytes, the naive balb/c cd4 + t cells lost their responsiveness to the stimulus of balb/c splenic antigen presenting cells (apcs) that endocytosed the donor-type alloantigens. however, they maintained a normal response to the stimulus of balb/c apcs that endocytosed third-party c3h alloantigens. in the present study, we examined whether invariant nkt (inkt) cells influence the ability of lsecs to endocytose irradiated allogeneic cells. balb/c wild-type (wt) mice or balb/c cd1d-deficient (cd1d -/-) mice that lacked inkt cells were portally injected with 30×10 6 irradiated b6 c2d splenocytes labeled with pkh-26. only 3.56 ± 3.19% lsecs endocytosed the labeled splenocytes in the balb/c cd1d -/mice at 12 h after pi, whereas 16.82 ± 2.69% lsecs endocytosed the labeled splenocytes in the wt control mice (p < 0.001, n = 4 each). when balb/c wt mice intraperitoneally received 4 µg α-galcer, before pi of b6 c2d splenocytes, the expression of mhc class ii on lsecs and endocytic activity of lsecs were enhanced. thus, we found that the endocytic activity of lsecs was regulated by the inkt cells. intraportal adoptive transfer of lsecs isolated from balb/c wt mice, treated with a pi of b6 c2d splenocytes, into balb/c mice significantly prolonged the survival of subsequently transplanted heart allografts (n = 4) as compared to the adoptive transfer of lsecs isolated from balb/c cd1d -/mice, treated similarly, into the balb/c mice (n = 4). however, intraportal adoptive transfer of lsecs isolated from balb/c wt mice, which received 4 µg α-galcer intraperitoneally prior to pi of b6 c2d splenocytes, into balb/c mice did not result in a further prolonging of the effect (n = 4). these findings indicate that inkt cells are required for such lsec-induced immunosuppression of t cells with indirect allospecificity; however, α-galcer-induced activation of inkt cells does not promote such suppressive effects on these t cells. in conclusion, naive inkt cells play a pivotal role in the lsec-induced immunosuppression of t cells with indirect allospecificity. notwithstanding the considerable amounts of in-vitro data supporting the nonimmunogenicity and immunomodulatory effects of mesenchymal stem cells (msc), scanty and conflicting data are available on their in-vivo immunomodulatory capacities. in this study we formally investigated whether msc had immunomodulatory properties in solid organ transplantation, using a semi-allogeneic heterotopic heart transplant mouse model, and studied the underlying mechanism(s). msc, isolated from bone marrow by adherence, were depleted from cd45+cd11b+ cells before injection. bone marrow-induced hematopoietic mixed chimerism is the most robust mechanism for the induction of transplantation tolerance. however, immunogenicity of bone marrow cells requires harsh immunosuppressive regimens that can lead to severe sideeffects including death. here, we examined whether embryonic stem (es) cells can be successfully coaxed to form hematopoietic progenitor cells (hpc) which potentially could be less immunogenic than bone marrow cells. here, we transduced es cells with hoxb4, a hematopoietic transcription factor that confers self-renewal properties to hematopoietic cells and differentiated them into hematopoietic cells. transduced cells had a 100-1000fold greater proliferation capacity than controls. at the end of the differentiation procedure, most cultures were >80 % cd45 + . hpcs were purified using immunomagnetic bead separation. the separated cells were further characterized for leukocyte markers and showed a high percentage of cd34, cd117, cd31 and low class i, but no class ii expression. further, they poorly express co-stimulatory molecules such as cd80 and cd86. when transplanted in rag2 -/γ c _/_ mice, hpcs fully reconstituted bone marrow, forming multi-lineage hematopoietic cells. to now determine whether these cells engraft in allogenic recipients, the cells were transplanted in syngeneic and allogenic mrl mice. all transplanted animals became chimeric (n>30), reaching 20-30 % after 28 days. thereafter donor cells declined as a result of out-competition by resident bone marrow cells. this pattern was identical in both syngeneic and allogenic recipients. unexpectedly, allogenic chimeric mice became tolerant to donor-type cardiac allografts as monitored over 100 days. grafts showed no mononuclear cell infiltration or signs of chronic rejection. interestingly, the t cells in tolerant animals showed responses to mrl alloantigen similar to that of controls, suggesting that our protocol was likely non-deletional, but could involve regulatory t cells. indeed, when stained for cd25 + foxp3 + cells, the allografts showed a high percentage of these cells, but not in controls, confirming our hypothesis. thus, these data show for the first time the potential of es-derived hpcs to regulate engraftment of allografts, providing an alternative approach for the induction of transplantation tolerance. we had previously shown that a20 is part of the regulatory atheroprotective response of endothelial (ec) and smooth muscle (smc) cells to injury. a20 is a nf-κb dependent gene with potent anti-inflammatory effects in ec and smc, through blockade of nf-κb. a20 also serves an anti-proliferative function in smc and opposite anti-apoptotic or pro-apoptotic functions in ec and neointimal smc. based on these functions, a20 would be a good candidate to prevent transplant arteriosclerosis (ta) and chronic rejection in vascularized organ grafts. this is supported by a20 expression in ec and smc correlating with the absence of ta in rat kidney allografts and long-term functioning human kidney allografts. fully mismatched c57bl/6 (h2 b ) and balb/c (h2 d ), were used as donors and recipients of an aortic to carotid allograft. in this combination, ta lesions start at 4 weeks and become occlusive by 8 weeks when left without immunosuppression. a20 expression in the graft was achieved by recombinant adenoviral (rad) mediated gene transfer prior to retrieval. control mice were infused with saline or control rad beta-galactosidase. the grafts were harvested at 4 weeks and analyzed for ta lesions by measuring intima to media ratios (i/m) and for markers of inflammation and of the immune response by immunohistochemistry. a20 expressing vessels were significantly protected from intimal hyperplasia with i/m reaching 0.4± 0.09 as compared to saline (1.62±0.17) and beta-gal (1.86±0.06) treated vessels. this effect of a20 did not associate with a decrease in infiltrating cd3, cd4 or cd8 t cells in a20 vessels as compared to controls. a possible modification of the phenotype of these t cells (t-regs vs. effector t cells) is being explored. rather, protection from ta correlated with increased expression of endothelial and inducible nitric oxide synthases (nos) in ec and smc of a20 expressing vessels, suggesting that this effect was, at least in part, related to increased in situ production of nitric oxide. in conclusion, we present the first direct evidence that expression in the vessel wall of the anti-inflammatory and atheroprotective protein a20 prevents transplant arteriosclerosis through a mechanism implicating increased expression of nos. carbon monoxide inhalation reverses established chronic allograft nephropathy through the no pathway. g. faleo, a. nakao, j. kohmoto, r. sugimoto, k. tomiyama, a. ikeda, m. a. nalesnik, d. b. stolz, n. murase. thomas e starzl transplantation institute, university of pittsburgh, pittsburgh, pa. chronic allograft nephropathy (can) is the most common cause of graft loss; however an established therapeutic strategy in preventing/treating can is not yet available. we have previously shown that carbon monoxide (co) effectively inhibits can development. here, we examine the mechanisms of co in overturning can through vascular endothelial cell protection. methods: orthotopic kidney transplantation (ktx) was performed in lewis to binephrectomized bn rats under brief tacrolimus (0.5 mg/kg, d0-6, im). by d60 after ktx, bn developed can with decreased creatinine clearance (ccr, 0.58 ±0.1 ml/min), significant proteinuria (92.8 ±30.5 mg/24h), and increased banff scores for intimal arteritis, interstitial fibrosis, and tubular atrophy. recipients were then treated with inhaled co 20 ppm from d60 to d150. results: inhaled co effectively reversed the severity of can and markedly improved renal function at d90 (table) and recipient survival (>150d vs. 81d air control). co treatment resulted in reduced cytokine mrna levels (tnf-α, ifn-γ) and improved banff scores compared to untreated controls. in untreated allografts, cd31 expression on peritubular capillaries (ptc) was markedly diminished, while co-treated grafts showed normal cd31 expression, suggesting significant improvement in maintaining ptc integrity with co. interestingly, enos and inos protein expression was significantly upregulated in untreated grafts, while it was maintained at steady levels in co-treated grafts. immunohistochemistry revealed enos expression on vascular endothelial cells while inos on infiltrates. further, serum nitrate/nitrite levels were significantly higher in untreated than in co-treated recipients. elevated levels of mda, a marker for oxidative stress, in air control group were accordingly reduced in co-treated grafts. renal cortical blood flow data showed a better perfusion in co-treated group at 90d (69. chronic allograft vasculopathy (cav) is a component of chronic rejection and a major cause of graft loss. non-immunologic factors and indirect allorecognition participate in the pathogenesis of cav. new therapies with tolerogenic dendritic cells (dcs) are based on in situ-delivery of alloag to "quiescent" dcs of the recipient's lymphoid organs via apoptotic cells, vesicles or particles. we have shown that the ability of apoptotic cells to deliver alloag and an inhibitory signal to dcs down-regulates the indirect alloresponse and prolongs allograft survival in mice. aims: to test if targeting of recipient's dcs in situ with donor apoptotic cells ameliorates cav by down-regulating indirect pathway allo-immunity. methods: we performed functional aortic (abdominal) transplantation in mice [balb/c→c57bl/6 (b6)]. b6 mice were injected i.v. with 10 7 balb/c uvb-induced early apoptotic splenocytes (d-7). sixty days later, grafts were evaluated in sections with h&e, vangieson's (elastic fibers) and masson's (collagen) techniques. cfse-labeled 1h3.1 tcrtg cd4 t-cells specific for ia b (b6) loaded with ieα 52-68 (balb/c) were used to evaluate the indirect pathway t-cell response. results: pkh67 + balb/c apoptotic cells injected (i.v.) in b6 mice were captured by splenic cd8and cd8α + dcs, but not plasmacytoid dcs. splenic dcs with apoptotic cells remained quiescent in vivo (mhc-i/ii lo , cd80/86 lo , icosl + , pdl-1/2 + ) and were unable to up-regulate mhc-ii and cd86 upon culture with gm-csf. injection of donor apoptotic cells induced defective activation and deletion of indirect pathway 1h3.1 cd4 t-cells. therapy with donor apoptotic splenocytes reduced intimal thickness in aortic allografts (100±21 vs.196±28mm in controls; p<0.001 ) and proliferation of α-smooth muscle cells and collagen deposition. the effect of apoptotic cells was allospecific, superior than that of cells alive, and depended on the physical properties of apoptotic cells, since necrotic cells did not achieve the effect. treatment with donor apoptotic cells decreased significantly the indirect pathway t-cell response (assessed by elispot for ifn-γ) and reduced the level of circulating alloab. conclusion: in situ-targeting of recipient's dcs with (early) apoptotic cells carrying donor alloag is a novel approach to prevent cav by down-regulating the indirect pathway alloresponse. the antifibrotic agent, pirfenidone, has direct inhibitory effects on t cell activation, proliferation, and cytokine and chemokine production, leading to suppression of host alloresponses. gary a. visner, 1 fengzhi liu, 1 hanzhong liu, 1 liqing wang, 2 wayne w. hancock. 2 1 medicine, children's hospital boston, boston, ma; 2 pathology, children's hospital of philadelphia, philadelphia, pa. there is an urgent need to develop new therapies effective against the fibrotic and other complications of chronic allograft rejection. while pirfenidone (pfd) is an established anti-fibrotic agent, we previously showed that pfd treatment reduced acute rejection in a rat lung transplant model suggesting that it might have direct immune modulating properties. accordingly, in this study, we tested the effects of pfd on t cell responses. we first evaluated whether pfd alters t cell proliferation and cytokine release in response to t cell receptor (tcr) activation in vitro. since pfd can inhibit tgf-β by mononuclear cell fractions, we also examined whether pfd affects the suppressive effects of regulatory t cells (cd4+cd25+). the effects of pfd on alloantigen-induced t cell proliferation in vivo were then assessed by adoptive transfer of cfse-labeled t cells across a parent->f1 mhc mismatch, as well as by using a murine heterotopic cardiac allograft model (balb/c->c57bl/6). pfd was found to significantly inhibit tcr-stimulated cd4+ t proliferation in vitro (p<0.01), whereas cd8+ t cell proliferation was not significantly affected. while the beneficial effects of pfd were not associated with increased cd4+ t cell apoptosis, pfd use inhibited tcr-induced production of multiple cytokines and chemokines, including . interestingly, there was no change on tgf-β production by purified t cells, and pfd also had no effect on the suppressive properties of naturally occurring regulatory t cells. similar to the in vitro studies, pfd inhibited allo-antigen-induced t cell proliferation in vivo (parent->f1 model), and showed synergistic effects with low dose rapamycin in this model. lastly, though pfd alone did not affect the tempo of acute cardiac allograft rejection across a full mhc mismatch, use of pfd plus a subtherapeutic regimen of rapamycin significantly prolonged allograft survival (p<0.05), decreased mononuclear cell infiltration and prevented development to chronic rejection, including arteriosclerosis and myocardial fibrosis. we conclude that pfd may be an important new agent in transplantation, with particular relevance to combating chronic rejection by inhibiting both fibroproliferative and alloimmune responses. we have previously reported studies in miniature swine showing that transplantation (tx) of prevascularized donor islets as part of composite islet-kidney (i-k) reversed diabetic hyperglycemia across fully allogeneic barriers, while free islets did not. in order to test the potential clinical applicability of this strategy, we have extended it to a fully allogeneic nonhuman primate model. methods: two diabetic baboons received composite iks and one diabetic baboon received free islets across fully allogeneic barriers. (1) i-k preparation in donors: two i-ks were prepared by isolating islets from 60% partial pancreatectomies and injecting them under the autologous renal capsule, allowing for vascularization before allogeneic tx. these i-ks were harvested at days 203 and 102 for allogeneic ik tx. (2) induction of insulin-dependent diabetes (iddm) and allogeneic i-k or free islet tx: all recipients received streptozotocin at 2000 mg/m 2 x (body surface area). iddm was induced successfully in two animals, while one animal required total pancreatectomy to induce iddm. after confirming iddm by fasting blood sugar (fbs) >300mg/dl for 3 consecutive days, either i-ks or free islets were transplanted (single donor to each recipient) with atg (day -3) followed by mmf and low-dose tacrolimus. free islets were injected into the liver through the ileocolic vein. islet function was assessed by fbs and renal function was assessed by serum creatinine. immunologic status was examined by cml/mlr assays. results: all three recipients had strong ctl/mlr responses to donors pretx, indicative of a fully allogeneic combination. fbs decreased immediately after i-k tx and no insulin therapy was required throughout the experimental period (days 225 and 279). bs levels averaged 82.2+/-15.6 mg/dl in the first baboon and 125.4+/-42.7 mg/dl in the second. normal creatinine levels (<1.0mg/dl) were maintained by these life-supporting ik grafts. in contrast, the recipient of allogeneic free islets had unstable bs levels and required insulin from day 60 (bs 400 at day 60) conclusions: life-supporting i-ks from single donors achieved glucose regulation without insulin therapy and maintained normal renal function. these results demonstrate the feasibility of composite i-k tx in a non-human primate allogeneic model, with possible clinical applicability for the cure of diabetic nephropathy. donor cell infusion without immunosuppression as a novel therapy for induction of donor-specific tolerance in islet cell transplantation. xunrong luo, 1 kathryn pothoven, 1 derrick mccarthy, 2 matthew degutes, 2 aaron martin, 2 xiaomin zhang, 3 guliang xia, 3 dixon kaufman, 3 stephen miller. 2 1 medicine, northwestern university; 2 microbiology and immunology, northwestern university; 3 surgery, northwestern university, chicago, il. background in autoimmune models, peptide-pulsed splenic antigen presenting cells that are chemically fixed with ethylcarbodiimide (ecdi) have been used as a powerful and safe method to induce antigen specific t cell tolerance. ecdi fixed donor cells for allo-antigen specific transplant tolerance has not been well studied. material and methods c57bl/6 mice were rendered diabetic by stz. kidney subcapsular allogeneic islet transplant was performed 10 days after diabetes stabilization (day 0). 1x10 8 ecditreated donor splenocytes were injected i.v. either once (day -7) or twice (day -7 and day +1). animals were analyzed for graft outcome. results control mice receiving islet graft alone rejected the graft between day 11 to day 18 (mst = 14 days, n=8). mice receiving one dose of ecdi-treated donor splenocytes (on day -7) showed similar graft survival as controls (mst = 15 days, n=5). in contrast, mice receiving 2 doses of ecdi-treated donor splenocytes (on day -7 and day +1) showed significant prolongation of graft survival with 72.7% functional grafts at day 60 (n=11), and some remained functional >100 days. this protection is donor-specific as mice receiving ecdi-treated sjl cells rejected balb/c islet grafts as controls (mst = 18 days, n=4). immunohistochemistry of protected grafts showed positive insulin staining within well-defined islet architecture. peri-islet infiltrates were composed of cd4+, cd8+, and cd11c+ cells, with occasional foxp3+ cells. anti-donor antibody production (igg1,g2a,b, g3) was completely abolished in long-term graft survivers. this tolerance was undisturbed by anti-cd25 antibody treatment during maintenance stage, but could not be established if treatment was given around the time of the first donor cell infusion. in addition, lack of pd-l1 also impaired tolerance induction evidenced by using pd-l1 -/as recpients. conclusion 1. multiple infusions of ecdi-treated donor splenocytes significantly prolonged allo-graft survival in the islet transplant model. 2. the protective effect is donor-specific and is dependent on regulatory t cells as well as the pd-l1 signaling pathway. therapy with ecdi-treated donor cells may emerge to be a novel and potent agent for induction of donor-specific transplant tolerance. mice. rebecca stokes, 1 k. cheng, 1 c. scott, 1 w. hawthorne, 2 p. o'connell, 2 j. e. gunton. 1 1 garvan institute, darlinghurst, nsw, australia; 2 nptu, westmead, nsw, australia. the aim was to investigate the effects of increasing hif-1α protein in human islets upon islet-transplant outcomes. hif-1α is a transcription factor which co-ordinates a program of cellular responses to stressors including hypoxia. in other cell-types hif-1α improves survival following hypoxic-challenge. hif-1α functions as a heterodimer with arnt which we have shown to be important for normal β-cell function (1) . islet transplantation subjects islets to hypoxia. it is thought up to 70% of islets die within 1 week of transplantation and this is at least partly due to hypoxia. the role of hif-1α in β-cell function is unknown. we hypothesized that increasing levels of the protective factor hif-1α in islets before transplantation would improve survival and engraftment and thus improve islet transplant outcomes. isolated human pancreatic islets from 9 separate donors were cultured overnight in control media, or media supplemented with desferrioxamine (dfo), a small molecular stimulator of hif-1α protein. islets were transplanted into diabetic scid mice. there were 3 transplant groups, with mice receiving: 1.supra-physiological-mass transplant of 2000 control-cultured ieq (islet equivalents) 2.minimal-mass-transplant of 600 control-cultured ieq, or 3.minimal-mass-transplant of 600 ieq cultured with dfo. for each human donor, at least 1 of each of the 3 transplant groups was performed to avoid the confounder of inter-donor variability. recipients of 2000 control ieq cured in 42% of cases. minimal-mass-transplantation was ineffective: 600 ieq cured 0% mice at 28-days. however, minimal-mass-transplant of dfo treated islets had 53% success (p<0.001 vs group 2 and p=ns vs 2000-control-ieq). blood glucose levels were markedly improved in the 600 dfo treated group compared to 600 ieq control group (p<0.0001) and equivalent to 2000 control ieq transplants (p=ns). this data demonstrates increasing hif-1α in human islets prior to transplantation markedly improves islet transplant outcomes. hif-1α and dfo may have a therapeutic role in human islet transplantation. long-term disappearance of neovascularization of transplanted islets. eba hathout, nathaniel chan, annie tan, john chrisler, john hough, naoaki sakata, john mace, ricardo peverini, richard chinnock, lawrence sowers, andre obenaus. loma linda university, loma linda, ca. we recently reported an in vivo time-line for neovascularization of transplanted islets using dynamic contrast enhanced (dce) magnetic resonance imaging (mri) over a 14-day period. however, vascularization of transplanted islets must be maintained for extended periods to provide long-term function. in this dataset, we investigated whether vascularization was maintained in transplanted feridex-labeled syngeneic murine subcapsular islets (400 ieq per kidney) using dce imaging on an 11.7t mr scanner and subsequent immunohistochemistry over 180 days. sub-capsular transplants could be visualized at post-transplant days 3 and 14 using t2 weighted imaging. however, the islets could not be seen on mri at post-transplant day 180. injection of the contrast agent gadolinium (gd)-dtpa for dce at 3, 14 and 28 days showed increased signal in the transplant area. at 180 days, there was no change in signal intensity after contrast injection during dce. immunohistochemistry confirmed mri and dce findings. these results suggest that islet neovascularization occurs early after transplantation but is likely not maintained for the 180-day duration of our experiments. this work was supported by nih/niddk grant # 1r01dk077541. a) t2 imaging at day 3 clearly identifies iron-labeled islets (arrows) in the subcapsular region. no iron-labeled islets are observed at day 180 (arrows). b) dce imaging for neovascularization of transplanted islets in the subcapsular region demonstrates a temporal decline in signal intensity. oleanolic acid, a natural triterpenoid, significantly improves islet survival and function following transplantation. n. angaswamy, 1 d. saini, 1 s. ramachandran, 1 n. benshoff, 1 w. liu, 1 n. desai, 1 w. chapman, 1 t. mohanakumar. 1, 2 1 surg, wusm; 2 path & immunol, washington univ sch med, st. louis, mo. oleanolic acid (oa), a triterpenoid in medicinal herbs, is an integral part of normal human diet. oa has anti-oxidant, anti-inflammatory properties (inhibits inos & cox2) & lowers plasma glucose levels. we hypothesis that these properties of oa will prevent early islet cell loss following transplantation & also benefit long term function of allograft. c57bl/6 mice, made diabetic by streptozotocin (200mg/kg) were transplanted with 500 balb/c islets (isolated by collagenase digestion) under kidney capsule. oa (0.5mg/day) was administered i.p. in 100 µl of pbs (with 6m dmso) or pbs-dmso as vehicle control daily from day -1 onwards. blood glucose was monitored daily. immunohistochemical analyses of grafts were performed for cd4 & cd8 markers. cellular immune responses to donor antigens & cytokines produced by cells and in sera were measured using elispot & luminex assays. effect of oa on function of transplant with suboptimal dose of islets (100-250) was also analyzed. optimal dose of islets (500) transplanted into diabetic bl/6 mice administered with oa significantly reduced time taken to reverse diabetes following transplantation (<2±1 vs 4±2 days, p=0.003). further, oa treatment reversed diabetes even with suboptimal dose (200) of islets while untreated animals did not achieve normoglycemia. as expected, control diabetic mice rejected on 6±2 days whereas, oa administration alone prolonged islet allograft survival to 23±3 days (p<0001). oa treatment resulted in >3 fold increase in serum kc, il-10 & vegf (p<0.0003) & 2 fold decrease in mcp-1, ip-10 & il-4 (p<0.005) in luminex assay. stimulation of splenocytes from oa treated mice with donor balb/c cells resulted in significantly reduced ifng (4.5 fold), il-4 (3.5), il-2 (2.3) & il-17 (4). in addition, proliferation in mlr was also reduced 2.5 fold. immunohistochemical analysis of grafts showed significant reduction in cellular infiltration in oa treated animals with reduction in both cd4 and cd8 t cells. daily administration of oa markedly improved islet engraftment & function with reversal of diabetes even when suboptimal dose of islet were transplanted. further, oa treatment allowed significant long term survival of allograft with no other immunosuppression. we demonstrate that prevention of inflammatory signaling cascades by oa resulted in marked reduction of cellular infiltration into graft allowing long term function of allograft. endoplasmic reticulum stress may be an important cause of cell loss after human islet isolation. soon hyang park, 1 michel tremblay, 2 steven paraskevas. 1 1 surgery, mcgill university health center, montreal, qc, canada; 2 mcgill cancer center, mcgill university, montreal, qc, canada. purpose: to evaluate the presence of endoplasmic reticulum (er) stress, induced by conditions to which islets are subjected during isolation (ischemia, nutrient deprivation, thermal stress and cytokine release) in human islets and to determine if this leads to the unfolded protein response (upr), which could alter cell survival. methods: human islets were purified from cadaveric pancreata by collagenase dissociation and continuous density gradient purification. islet preparations were cultured in serum-free medium and sampled at the end of isolation and daily thereafter. total mrna was purified and gene expression evaluated by rt-pcr. activity in upr signaling pathways was evaluated by immunoblot. apoptosis was measured by a caspase-3 activity assay. representative trends observed in >5 isolations are described. results: following isolation, a rapid increase in upr signaling was observed in the perk and ire-1 modules of the upr. these include the phosphorylation of perk target eif2? and splicing of mrna for the transcription factor xbp-1. these changes occurred concurrently with a rapid spike in jnk activity and a rise in expression of the upr target gene chop. after these signals peaked, caspase-3 activity increased with time (apoptotic cells), as did expression of er chaperone bip (surviving cells). conclusion: we consistently observed upr activation in human islets. er stress and the upr may be one important and unrecognized cause of apoptosis in this context. current investigations focus on upr modification and determination of a causal relationship with apoptotic cell death. immunosuppression and the risk of renal transplant failure due to recurrent glomerulonephritis. atul mulay, 1 carl van walraven, 1 greg knoll. 1 1 the ottawa hospital, ottawa, on, canada. glomerulonephritis (gn) is the most common cause of end-stage renal disease among those who undergo kidney transplantation. recurrent gn is a major cause of kidney transplant failure. immunosuppressive medication is used to treat gn in the native kidney prior to the development of end-stage renal disease but the impact of different immunosuppression on recurrent gn post-transplantation is unknown. we used the united states renal data system to determine the association of routine post-transplantation immunosuppressant use with time to renal allograft failure due to recurrent gn. immunosuppressants were treated as time-varying covariates. the study-cohort included patients with kidney failure due to gn who received first kidney transplant between 1990 and 2003. the study cohort included 41,272 patients with a median follow-up of 51 months. ten-year overall graft survival (including death as graft loss) and death-censored graft survival was 56.2% and 70.5% respectively. use of cyclosporine (hazard ratio 0.93; 95% ci 0.62-1.41), tacrolimus (hazard ratio 1.03; 95% ci 0.67-1.60), azathioprine (hazard ratio 0.88; 95% ci 0.68-1.13) or mycophenolate mofetil (hazard ratio 1.10; 95% ci 0.85-1.40) was not associated with risk of graft failure due to recurrent gn after adjusting for important covariates. there was no difference of recurrent gn causing graft failure between cyclosporine and tacrolimus (p=0.4) or between azathioprine and mycophenolate mofetil (p=0.1). however, change in any immunosuppressant during follow-up was independently associated with graft loss due to recurrence (hr 1.31, 95% ci 1.07-1.60, p=0.01).when we restricted the analysis to patients who had no change in immunosuppression during follow-up we again found no association between any of the immunosuppressive medications and the risk of graft loss due to recurrent gn. despite the increased use of tacrolimus, cyclosporine and mycophenolate mofetil to treat gn in native kidney disease, the use of these medications following kidney transplantation had no impact on the risk of graft loss due to recurrent gn. glomerulosclerosis. junichiro sageshima, 1 gaetano ciancio, 1 alessia fornoni, 1 linda chen, 1 carolyn abitbol, 1 jayanthi chandar, 1 warren kupin, 1 giselle guerra, 1 david roth, 1 sherry shariatmadar, 1 gaston zilleruelo, 1 george w. burke iii. 1 1 university of miami miller school of medicine, miami, fl. background: disease recurrence is a major obstacle of kidney transplant for focal segmental glomerulosclerosis (fsgs). anti-cd20 antibody (rituximab) has been used for nephrotic syndrome of native kidney. the significant reduction of proteinuria in transplant recipients with fsgs recurrence was also reported after rituximab use for posttransplant lymphoma. we hypothesized that rituximab induction could alter the posttransplant course of fsgs recipients, particularly in those patients with rapid progression to end-stage renal disease who are higher risk of recurrence. methods: we compared the outcome of transplants for primary fsgs treated with and without rituximab. from jan. 2000 to dec. 2003 received renal allografts along with our "standard" immunosuppressive protocol, consisting of tacrolimus, mycophenolate, corticosteroids, antithymocyte globulin and/or daclizubab. from jan. 2004 to dec. 2007 received rituximab in addition to the "standard" immunosuppression. posttransplant proteinuria was treated with plasmapheresis (pp) and maintenance angiotensin blockade (ab). results: there was no adverse event related to rituximab infusion. the overall incidence of posttransplant proteinuria was significantly lower in recipients with rituximab induction (p < 0.05). four recipients treated with "standard" immunosuppression developed massive proteinuria (u-protein/creat. > 10) immediately following transplantation; they responded poorly to pp and ab. four other recipients had moderate proteinuria. in contrast to this, of the 18 patients induced with rituximab, only 2 had massive proteinuria and 4 had mild to moderate proteinuria which responded well to pp and ab. with a median follow-up of 26 months, there was no significant difference of graft survival between 2 groups (2-year survival: 81% without rituximab vs. 84% with rituximab). a half of the graft loss was related to non-compliance. conclusion: while the mechanism of action is unclear, our observation indicates that rituximab induction may decrease the incidence and severity of recurrence of fsgs following kidney transplantation. a larger-scale study is desirable to confirm this observation. mesangial chimerism in recurrent iga nephropathy. geoffrey talmon, 1 dylan miller. 1 1 department of pathology and laboratory medicine, mayo clinic, rochester, mn. background iga nephropathy (in) is the most common primary glomerulonephritis and nearly 50% of patients who undergo a renal transplant for in recur. data support that bone abstracts marrow-derived cells are capable differentiating into various mesenchymal cells within the kidney. the extent to which this phenomenon versus proliferation of resident mesenchymal cells is involved in populating mesangium is not well understood. the mesangial injury and/or hypercellularity seen in in provides a robust in vivo model for determining if this phenomenon is prevalent in human kidneys. design follow-up biopsies from male patients receiving female renal allografts for in that showed recurrent disease were selected. fluorescent in-situ hybridization and immunofluorescent staining was performed on unstained slides from the paraffinembedded tissue for smooth muscle actin, x, and y chromosome centromeres. cells within nonsclerotic glomeruli with triple positivity (y+) were assumed to be mesangial cells derived from the recipient. results four cases of recurrent in with nonsclerotic glomeruli were obtained, each displaying at least minimal mesangial proliferation by light microscopy (one "minimal", two "mild", one "moderate"). mesangial cells with y chromosome centromeric material were observed in each case (100%). between 6 and 34 mesangial cells were present in each glomerulus (mean 16.45) with no to three y+ cells seen (mean 1.8). these accounted for between 8% and 16% of mesangial cells in individual glomeruli. the ratio of y+ to total mesangial cells in each case ranged from 1:7.8. to 1:11.3 (mean 1:9.35 ). the case exhibiting minimal mesangial hypercellularity had a ratio of 1:9.6, those with mild had ratios of 1:7.8 and 1.8.7, and that with moderate 1:11.3. conclusions recipient-derived mesangial cells make up a fraction of the population of glomerular cells in renal allografts affected by recurrent in. although the number of cases is small, the number of recipient-derived cells does not seem to be directly related to the degree of mesangial hypercellularity seen by light microscopy. the consistent presence of these "colonizing" cells in patients with recurrent in does, however, suggest that there may be a role for targeted therapy directed against circulating recipient cells. in the mid 1970's, patients developing esrd secondary to systemic lupus (sle) were deemed to be poor transplant candidates because of concern for early recurrent lupus nephritis (rln) leading to allograft loss. subsequently, rln was considered an unusual complication of kidney transplantation, occurring in <4% of allografts. however, over the last decade, several reports have shown the frequency of rln to range from 8-30%. we sought to determine the frequency of rln at our center and to identify any clinical variables associated with rln. between 6/1977 between 6/ -11/2005 allografts in 166 patients with esrd due to sle functioned for more than 90 days after engraftment. immunosuppression consisted of azathioprine (aza), or cyclosporine (csa) and aza, or mycophenolate (mmf) and csa, or tacrolimus and mmf depending on the date of transplant. all received steroids. proteinuria was defined as 2+ on dipstick or urine protein/creatinine ratio >0.5. medical charts were reviewed. we found pathologic evidence of rln in 18 (24%) of 75 patients who underwent biopsy due to allograft dysfunction or proteinuria, comprising 10% of all patients transplanted for sle. characteristics of these patients are shown below: randomized studies have shown little or no increase in ar in kidney tx recips on p-free is; but concern remains about long-term outcome. we present 8-yr f/u of a protocol incorporating rapid (<6 days) discontinuation of p, with now over 1000 patients transplanted using this protocol. between 1/1999 between 1/ and 10/2007 between 1/ , 1003 adult tx recips were treated with thymoglobulin (5 doses)(extended in dgf), p (5 days), a cni, and either mmf or srl. of these, 658 were ld (260 lurd); 345 dd. of the 1003, 41% were female;88% white; mean recipient age was 47± 14 years and mean donor age was 39.7± 13.2 years. diabetes was present in 34.3% of the recipients and 12.5% of the transplants were retransplants. the peak pra was >10% in 20% of recipients; 14% had tx pra >10%. table 1 shows actuarial survival rates. graft survival rates were significantly better in ld vs dd transplants (p=0.002) and and acute rejection rates lower (p=0.04). compared to national data from srtr, overall outcomes were not significantly different. with mean follow-up of 3.5 years, a total of 110 (11%) recipients have died -the most common cause being cerebrovascular accident (14%) followed by malignancy (9%). there were only 5 (4.5%) patient deaths due to cardiac causes. of 195 (19.4%) graft losses, 86 (44%) were from dwf (death with function); 43 (10%) from cr/can. renal function has been stable with mean serum cr of 1.7 mg/dl at 3 and 5 years posttransplant with cretinine clearance of 63 and 60 respectively. at 5 years posttx, compared to ppretransplant values, recipients showed a 9.6% increase in weight, a 3% decrease in serum cholesterol, and a 10.8% decrease in serum lipid values. 84% of the kidney recips remain p-free; the most common reason for restarting p was acute rejection (ar). conclusion: short-term data suggests kidney tx recips do well with rapid discontinuation of p. our intermediate-term data suggests that patient and graft survival rates remain good and renal function remains stable. ongoing long-term follow-up is necessary. background. since 4/02 our program has employed a steroid-free, rapamycin and neoral maintenance immunosuppression regimen for kidney transplant recipients. prior to that time recipients were treated with prednisone, mmf and neoral. we noted a significant reduction in acute cellular rejection (acr) after implementing this regimen. this retrospective analysis was performed to examine the impact, if any, on the incidence, character, and outcomes of early ahr. results. this study includes 1611 consecutive kidney recipients transplanted between 1/99 and 12/06. there were 717 recipients in the prednisone, mmf, and neoral era (grp 1) and 894 in the steroid-free, rapamycin and neoral era (grp 2). recipient age, gender, african-american race, ab and dr mismatch, and frequency of pra>10% was not statistically significantly different between the 2 groups. however, 43% of grp 1 pts vs 63% of grp 2 pts received a living donor kidney due to a more recent volume increase in this procedure (p<0.001). there were a total 722 kidney biopsies in 466 pts performed in the first 6 months post-transplant; 478 in 287 pts when excluding pre-perfusion biopsies. nineteen percent (54/287) of these showed >50% peritubular capillary (ptc) c4d deposition. comparison of grp 1 to grp 2 demonstrated the following: 1) the incidence of clinical acute rejection in the first 6 months was 12.7% (91/717) in grp 1 and 5.6% (50/894) in grp 2 (p<0.001), 2) the overall incidence of a c4d+ biopsy was similar in the 2 groups ( conclusions. despite a significant reduction in the incidence of acute rejection using our newer, steroid-free immunosuppression protocol, there has been no reduction in the incidence of early (0-6 months) ahr evidenced by c4d+ kidney biopsy. however, the percentage of ahr unassociated with acr has significantly increased. the poor graft survival in pts with early ahr has not improved with our newer immunosuppression regimen. conclusions: four risk factors for ar were identified in the rdp study population: retx, aa race, age 18-50 (vs. >50) and pra >50 (vs. <50). four risk factors for gl were identified: pre-tx t1 dm, ar, dgf and dd tx (when ar and dgf omitted). these risk factors for ar and gl are the same as we observed in prednisone-containing protocols. additionally, many of these factors are not modifiable. identification of high risk groups allows for individualization of is. increasing lds and utilization of is protocols to decrease or minimize dgf and ar are goals for improving graft outcome. effect with the advent of more potent immunosuppression hla matching has been deemphasized in the allocation of deceased donor kidneys due to the limited impact on acute rejection and graft survival. an unforeseen consequence of poorer matching could be an increase in sensitization of patients in need for a repeat transplant. our study examined candidates listed in the us from 1988-2007 from the srtr database that were re-listed following loss of a primary kidney transplant (n=19,827). the primary outcome of the analysis was change in pra from the listing prior to recipient's initial transplant to the subsequent listing. absolute change in peak and current pra levels were examined in general linear models as well as the proportion of patients with a rise in pra level in a logistic model. results hla(a,b,dr)-matching in the primary transplant was strongly associated with change in pra level (p<.001, figure 1 ). among recipients with 6-hla mm, over 50% had a rise in pra at re-listing as compared to 25% of 0-hla mm recipients. younger recipient and donor age, males, deceased donor transplants and african american recipients were also significantly associated with elevation in pra. in addition, the effect was apparent stratified by primary donor type. while there might be a limited impact of hla matching on graft survival, many patients might be negatively impacted from poor hla matching from their first transplant when needing a second transplant. as high pra is one of the strongest risk factors for not getting transplanted, this should be taken into account when evaluating the impact of hla matching in kidney transplantation. this might be particularly important in younger patients and in patients with a long life expectancy in general because of the high likelihood of needing a second transplant during their lifetime. the were about 25% less likely to die or lose an ecd kidney from a donor aged 50-59 and 70% more likely with kidneys from donors older than 69. a similar trend was seen with older recipients, but the risks seemed to be lower. logistic regression indicates recipients older than age 65 were 5 times more likely to have a donor older than age 70 than recipients younger than 50, hypertension and creatinine >1.5 were less likely in older donor kidneys. interestingly, the use of these kidneys relative to the total number of ecd kidneys has decreased since 2002 (odds ratio=0.63, 0.53-0.76, p<0.001). conclusion: an increase in the supply of kidneys might be achieved with increased utilization from deceased donors older than age 70. outcomes were similar to those from donors age 60-69 in recipients that were older than age 50. outcomes , but approximates non-dcd survival thereafter. dcd listing for retransplantation and graft failure progressed continuously over 180 days versus 20 days in non-dcd. when retransplanted, dcd recipients waited longer and received higher risk allografts (p=0.039) more often from another region. more dcd recipients remain waiting for retransplantation with fewer removed for death, clinical deterioration, or improvement. conclusions: dcd utilization is impeded by early outcomes and a temporally different failure pattern that limits access to retransplantation. allocation policy that recognizes these limitations and increases access to retransplantaton is necessary for expansion of this donor population. orthotopic liver transplantation with allografts from dcd donors. roberto c. lopez-solis, 1 background: in the current era of liver transplantation, organ shortage continues to be a significant problem. the use of extended criteria allografts from donation after cardiac death (dcd) donors to increase transplantation rates is widely practiced. this study is a review of one of the largest single center experiences utilizing dcd donors in the world with a follow-up of almost 15 years. methods: from 03/01/1993 to 10/31/2007, 3,431 liver transplants were performed at our institution, 146 (4.2%) of which were liver allografts from dcd donors. patient and donor demographics, recipient and graft survival, and the incidence of primary non function, hepatic artery thrombosis, retransplantation, and bile duct complications were analyzed for this subset of recipients. results: kaplan meier analysis showed a 1-and 5-year patient and graft survival of 81% and 71%, and 71% and 59%, respectively. the mean age of recipients was 52 ± 10 years with an average meld score of 18.7 ± 9.3 (range, 6 to 40), and there were 101 male patients (69%). donor mean age was 36 ± 16 years and cold ischemia time was 646 ± 173 minutes. one hundred and three patients (70.5%) are alive and 24 (16.4%) underwent retransplantation. the incidence of primary non-function was 11.6% (17 patients) and hepatic artery thrombosis was 4.1% (6 subjects the fda warns against using sirolimus (srl) in liver transplants, reporting increased hepatic artery thrombosis (hat), excess mortality and graft loss when srl is used as initial immunosuppression (is) with calcineurin inhibitors. we report the largest experience to date of patients with srl used as initial is, assessing hepatic artery complications and survival outcomes. materials and method all 1554 olt pts from 1998-2007 were reviewed. those using srl as initial is were identified, and the remaining olt pts from that time period were used as controls. ultrasound assessed graft vascular status and any issues were verified by angiogram. . there were no significant difference in demographics variables, lt indication or pre-lt meld score between the two gr. mean follow-up was 6.8±3.9 months. all the enrolled pts were treated with abstracts an initial dose of cs of 2mg/kg/day, to target 100ng/ml for the first 10 days. pts were randomized on day 10 into one of the two following gr on a 2:1 basis. ev gr: initial dose of ev was 2 mg/day, to reach blood level of 8 ng/ml. the dose was increased on day 30, when cs was discontinued, in order to reach an ev blood level between 10 and 12 ng/ml. cs gr: after the 10 th post-operative day the dose of cs was adjusted to a target level of 250 ng/ml until day 30, then to 200 ng/ml until the end of month 6. all pts received basiliximab induction on day 0 and 5 after lt. pts were weaned off prednisone by 5 weeks. pt survival at 6 and 12 months was similar in the ev and cs gr (95.2% and 86.6% vs 87.5% and 87.5% respectively; p= ns). causes of death were sepsis (1), hcv recurrence (1), pulmonary embolism (1) in the ev gr, and sepsis (1), rupture of splenic artery aneurysm (1) the overall incidence of infection episodes was comparable between two gr (5.9% ev gr vs 12.5% cs gr; p=ns). cholesterol but not triglycerides increased in the ev gr compared with the cs gr (p<.05); ev dose reduction decreased such parameters without the need for statin implementation. conclusion ev monotherapy in de novo lt showed similar patient survival and incidence of morbidity compared to a cs immunosuppressive protocol. the primary endpoint was achieved inasmuch as renal function was statistically better in the ev gr. background: sir is a potent immunosuppressive agent that inhibits t-cell activation and proliferation. in lt recipients, sir has primarily been used as a renal-sparing agent, but its toxicity and tolerability in this population has not been well defined. aims: to identify the adverse effects and predictors of discontinuation of sir in lt recipients. methods: records from 327 adult lt recipients transplanted between 1/2000 and 12/2006 were reviewed. reasons for starting and discontinuing sir were captured, as were all significant adverse effects and laboratory abnormalities. factors predicting sir discontinuation in univariate analysis were further analyzed by multivariable logistic regression (mlr). results: mean age of the study group was 50 ± 11 years, and 75% were male. underlying liver disease was hcv ± alcohol in 56%, 24% had hepatocellular carcinoma, and 14% received living donor grafts. calcineurin inhibitors (cni) were started post-operatively in 91% (85% tacrolimus/15% cyclosporine), with or without mycophenolate and prednisone. 179 patients (54%) started sir a median of 15 days (iqr: 4-138) post-lt primarily for renal insufficiency (76%) or cni neurotoxicity (7%). sir was overlapped with tacrolimus and cyclosporine in 65% and 16%, respectively. prior to starting sir, total cholesterol was 139 ± 69 mg/dl, ldl-cholesterol 90 ± 49, triglycerides 193 ± 126. peak lipids after sir were 263 ± 97, 138 ± 81, and 475 ± 456 mg/dl, respectively, despite lipid-lowering therapy. serum creatinine was 2.05 ± 1.04 and 1.08 ± 0.5 mg/dl before and after sir, respectively. before sir, 70% of patients had no proteinuria, but only 36% had no proteinuria after sir. high range proteinuria (>300mg/dl) was noted in 3% before and 16% after sir. finally, sir was discontinued in total of 81 (45%) patients, for indications of cytopenias (20%), hyperlipidemia (19%), mouth ulcers (11%), sepsis (7.5%), skin reactions (7.5%), nephrotic syndrome (6%), gi intolerance (4%), pneumonitis/boop (2.5%), myopathy (2.5%), and combinations of above (20%). mlr failed to identify any pretreatment predictors of discontinuation. conclusions: immunosuppression with sir improves azotemia at the expense of considerable hematologic, metabolic, dermatologic, renal, pulmonary and muscle toxicity. considering the high incidence of proteinuria after sir treatment, the use of sir as a less nephrotoxic agent must be re-considered. and to identify the most effective protocol. peripheral blood was obtained from 4 ebvseronegative and 4 ebv-seropositive pediatric heart (h) tx patients. lcl vs dc-based methods were compared as follows: (i) lcl (ii) lcl + il-12 (iii) type-1 polarized dc (treated with il1-b, tnf-a, il-6 and ifn-g) loaded with mhc class i-restricted ebv-peptide pool (dc/pep.) and (iv) dc/pep. + il-12. the ebv-specific cd8 + t cell phenotype and function were screened using flow cytometry, ifng elispot and cytotoxicity assays. the yields and the functional activities of in vitro co-cultures differed based on the induction method employed, and on the ebv status of the patients tested. for the ebv-seropositive pediatric htx patients, all four methods resulted in the successful expansion of functional type-1 ebv-specific cd8 + t cells, suggesting that memory cd8 + t cell are readily reactivated in vitro. for the ebv-seronegative pediatric tx patients however, only the lcl + il-12 approach resulted in significant augmentation of type-1 ebv-specific ctls that were competent to secrete ifn-g (400±50/10 5 cells) and to kill (300±170 lu/10 7 cells) ebv + targets. we found that il-27 secreted by lcl (and not by dc) was critical in triggering expression of il-12rb2 on naive cd8 + t cells, and rendering these cells responsive to il-12p70. further addition of exogenous il-12p70 (which is generally not produced by lcl) proved to be essential for effective type-1 priming. however, blocking il-27 during ebv-priming has abolished il-12rb2 expression and subsequent ifng production. these results demonstrate that the inducible expression of il-12rb2 on naïve cd8+ t cells was dependent on il-27, and support the critical early role of ebv infected b cells in the in vivo priming of naïve precursors into potent ebv type-1 cd8 + t cell in children. serial ebv load monitoring in pediatric heart transplant patients (phtx) has identified a group of asymptomatic children that exhibit persistently high ebv loads in peripheral blood (≥16,000 copies/ml on at least 50% samples over a period of at least 6 months). these patients have a high rate of progression to late ptld. our goal is to characterize the deficiency of ebv-specific cd8 + t-cell immunity that allows this state to occur and be maintained. twenty-one stable ebv + phtx patients were categorized as follows: group 1 (n=6) no detectable viral load; group 2 (n=12) low viral load (≤16,000 copies/ ml); group 3 (n=4) high viral load (≥16,000 copies/ml). twelve healthy subjects were recruited as controls. flow cytometric analysis with hla-a2 ebv-tetramer (tmr) probes in conjunction with mabs against memory/activation markers was performed on peripheral blood cd8 + t cells, and their ebv-specific ifn-γ production was measured by elispot. ebv-"latent" specific cd8 + t cells in g2 patients were mostly cd62l + / cd45ro + (central memory) and expressed heterogeneous levels of pd-1 and high cd127 (il-7 receptor α), the ebv-lytic-specific cd8 + t cells were more frequent, and biased toward cd62l -/cd45ro + (effector memory) and cd62l -/cd45ra + (stable effector memory), corresponding to terminally differentiated memory compartments. this cell population also expressed heterogeneous levels of pd-1 and down-regulated cd127. in contrast, both ebv-lytic and -latent specific tmr + cd8 + t cells from g3 patients were homogeneously cd62l + /cd45ro + (effector memory), cd38 + and cd127 -, suggestive of "recently activated" phenotype. interestingly, although patients in groups g2 and g3 had high frequencies of ebv-specific tmr + cd8 + t cells (g2 0.65%±1.5, g3 1.9%±3.5, p=0.3), only g2 patients exhibited a direct correlation between tmr + cd8 + t cells and ebv-specific ifn-γ production. these results demonstrate that different levels of chronic ebv-antigenic pressure trigger significant differences in the phenotypic and functional features of ebv-specific cd8 + t cells from phtx, suggesting that the immunologic characterization of high ebv load carrier state is a combined "activated" phenotype with "exhausted" function of ebv-specific cd8 + t cells. ebv-encoded lmp1 indirectly activates the jak/stat pathway through induction of ifnγ. abstracts evidence of ptld. 15 children were enrolled at 5 sites. mean age at transplant 7.1 years, mean time to ptld 43 months . organs transplanted were lung 6, heart 5, kidney 4. all ptld were of b cell origin and expressed cd20 and all were ebv positive. histology was: polymorphic 11, monomorphic 3, hodgkins-like 1. 7 patients received 4 doses, 7 patients 8 doses and 1 patient 7 doses. treatment was associated with minimal side effects in 12, mild-moderate infusion related reactions in 2 and moderate reaction in 1. no patient had treatment discontinued because of side effects. twelve patients (80%) showed complete response after 4-8 doses, 2 had progressive disease and one had stable disease. at 24 months, 10 (67%) were alive with one graft loss (kidney) and none with residual disease. at latest follow-up (mean 60.4 months, 42-82), 10 remain alive with 1 further graft loss (lung) and no ptld. the 5 deaths occurred between 2.5 and 13 months and were associated with progressive disease (2), chronic rejection (2), and complications of elective surgery (1) . conclusions: these findings support prior registry data and suggest that rituximab without chemotherapy is a successful second line treatment in approximately two-thirds of children with refractory ptld. cancer after organ transplantation in france. jean michel rebibou, 1 fabienne pessione, 1 francois aubin, 1 bernard loty. 1 1 agence de la biomedecine, france. cancer prevention appears as a major challenge in transplantation. estimating cancer frequency is a major step toward designing prevention policy. we report on patterns of cancer incidence among 47000 transplantations registered in the french data base. ). the risk of lung cancer is higher for recipients of a thoracic organ and the risk of kidney cancer appeared higher for kidney recipients. ten year cumulative incidence was 8.6% for all cancer and transplantation types, 1.7% for nhl. multivariate analysis demonstrated that cancer risk increased with recipient age (p<10 -3 ), 10 year cumulative incidence was 15% for recipients older than 60 year. it was higher in male (p<10 -3 ) and in thoracic organ recipients when compared with kidney recipients (p<10 -3 ). cancer incidence did not vary according to the transplantation period (1995 ( -1999 ( vs 2000 ( -2005 .95 p=0.34) and nhl risk was significantly lower during the period 2000-2005 (rr=0.8, p=0.02). this work does not report any increase in cancer incidence among transplant recipients while cancer incidence increased in the general population. the observed decrease of nhl risk is of particular interest. the both costimulatory and co-inhibitory signals are delivered by b7 ligands through the cd28 family of receptors on t lymphocytes determining the ultimate immune responses. although b7-h4, a recently discovered member of the b7 family, is known to negatively regulate t cell immunity in autoimmunity and cancer, its role in transplantation rejection and tolerance has not been established. to study its role in physiologic rejection processes, we first treated b6 wt recipients of balb/c hearts with a blocking mab against b7-h4 or isotype igg control and found no difference in graft survival (mst 7 days, n=8 vs. 7, n=10). however, b7-h4 blockade resulted in accelerated allograft rejection in cd28 deficient b6 recipients (mst 9.5 vs. 19, n=8, p=0.003) , indicating that b7-h4 signaling can mediate negative regulation in the absence of cd28 costimulation. we next studied b7-1/b7-2 double deficient (dko) b6 recipients of balb/c heart allografts, as these mice are truly independent of cd28/ctla-4:b7 signals. while cardiac allografts were accepted in control dko recipients (mst>100, n=5), blocking b7-h4 precipitated rejection (mst 45, n=5, p=0 .01) demonstrating non-redundant functions of these two negative pathways. based on these results, we next evaluated the role of b7-h4 in acquired transplantation tolerance by blocking cd28:b7 using ctla4-ig. b7-h4 blockade abrogated prolongation of allograft survival by ctla4-ig (250 µg on day 2) in the fully mhc-mismatched cardiac allograft model (mst 13 vs. 46.5, n=8, p=0.0002) . we conclude that the novel b7-h4 molecule can regulate alloimmune responses independent of an intact cd28/ctla-4:b7 costimulatory pathway. the interplay between positive (stimulatory) and negative (regulatory) costimulatory signals is an important determinant of the outcome of the alloimmune response and could be exploited to induce tolerance. specific in a model of mhc-mismatched kidney allograft in the rat, treatment with anti-cd28 antibodies induced a form of tolerance independent of treg cells but associated with a two-fold accumulation of mdsc in the blood. to further characterize these cells, we analyzed their phenotype and mechanism of action by flow cytometry, western blotting and suppression assays. mdsc expressed cd80 and cd86, nkrp-1, cd172a (sirpa), cd11a, cd11b, his48 and for a fraction of them cd4 but did not express mhc class ii molecules. mdsc dose-dependently suppressed the proliferation of t cells in mlr and after stimulation with anti-cd3 + anti-cd28 antibodies. although detected in blood, bone marrow, spleen and lymph nodes, mdsc were only suppressive in blood and bone marrow. this suppression was lost after physical separation from the responding t cells by semi-permeable membranes in transwell assays, as well as after addition of l-nmma, a selective inhibitor of inducible nitric oxide synthase (inos), suggesting a role for no in the suppression. western blot analyses revealed that inos was expressed only after contact between mdsc and activated cd4 + cd25effector t cells and to a much lesser extent after contact with activated cd4 + cd25 high t reg cells. mdsc affected the viability of stimulated cfse-labeled effector t cells by blocking their proliferation but not their activation. in contrast, mdsc did not block the response of t reg cells stimulated by anti-cd3 + anti-cd28 antibodies. this selective suppression of effector but not of regulatory t cells was confirmed by cytokine profile analyses. in vivo, the expression of inos was higher in the blood of tolerant recipients, as well as in the graft, as compared with isografted recipients. in addition, the injection in stable tolerant animals of aminoguanidine, which inhibits inos, induced graft rejection within 3 weeks. in conclusion, these results suggest that mdsc, accumulated in the blood of tolerant recipients of kidney allografts, release high levels of no after contact with activated effector t cells and specifically control their proliferative response. liver non-parenchymal components inhibit dendritic cell differentiation and maturation. ching-chun hsieh, 1 horng-ren yang, 1 guoping jiang, 1 john j. fung, 1 shigunag qian, 1 lina lu. 1 1 immunology and general surgery, cleveland clinic, cleveland, oh. the inherent tolerogenicity of liver allografts could be due to comparatively large numbers of potentially tolerogeneic antigen presenting cells, in particular dendritic cells (dc). it is not clear whether the unique antigen presenting function of liver dc is intrinsic, or is altered by the microenvironmental factors within the liver. in the present study, we investigated the effect of hepatic stallet cells (hpsc), a unique tissue cells in the liver which were actively expanded in the liver allografts, on generation and function of bone marrow derived dc (bm dc). we hypothesize that liver hpsc may modulate immune response via inhibition of liver dendritic cells (dc) which are known of bone marrow (bm) origin. in this study, dc were propagated from b6 bm cells with gm-csf for 5 days. irradiated hpsc isolated from b6 mouse liver were added at the beginning into the culture at hpsc : bmdc progenitor ratio of 1:20. the differentiation, maturation and function of propagated dc were determined by characterizing their surface molecule expression and functions of instructing t cell activation / differentiation. the results showed that addition of hpsc markedly blocked the differentiation of dc from bm precursors (most cells remained in cd11b + cd11cprecursors stages). the incidence of cd11c + cells was 7% vs. 41.6% in normal bm dc culture (without hpsc). the presence of hpsc also prevented maturation of cd11c + dc, as evidenced by low expression of cd40, cd80 and cd86, but high of b7-h1. the inhibitory effect appeared to be mediated by soluble factor (s) produced by hpsc, since addition of the hpsc culture supernatant or transwell culture provided comparable inhibitory activity. culture of allogeneic cd4 + t cells with hpsc-dc elicited poor proliferative response in a 3d mlr assay, with low il-2 and ifn-γ production. three color staining of t cells stimulated by hpsc-dc showed that cd25 + foxp3 + t cells were preferentially expanded, suggesting that hpsc-dc were capable of inducting treg. in contrast, bm dc induced vigorous cd4 + t cells proliferation, most of activated cd4 + t cells were cd25 + foxp3associated with high levels of ifnγ and il-2 in the culture, indicating induction of th1 cells. in conclusion, liver tissue cells, such as hpsc markedly inhibit dc differentiation and maturation, suggesting that the tolerogenic property of liver dc may not be intrinsic, but is altered by the microenvironmental factors in the liver. . allograft rejection was also significantly accelerated when bm12 hearts were transplanted into cd28ko recipient (mst 14 days). to investigate the mechanisms of these findings, lymphocytes harvested at day 10 post-tx from spleens and regional lymph nodes were assessed by flow cytometry for phenotypic differentiation of the activation status, regulatory t cell markers and effector cell generation. the ratio of effector to regulatory cells was 1.4: 3.8: 5.2 in the wt, cd28ko and b7dko recipients, respectively. cytokine production was evaluated by elispot using donor specific antigen stimulation of recipient splenocytes. the mean ifn-γ production was 173 spots per 500,000 splenocytes in wt, 206 in cd28ko and 702 in b7dko. this study for the first time demonstrates the paradoxical role of b7-cd28 co-stimulation in fully allogeneic and class ii mismatched grafts and proposes a possible mechanism through the re-alignment of the effector to regulatory t cell ratio in favor of a highly alloreactive immune response. the major concern regarding kidney donation has been whether the occurrence of hyperfiltration, on the background of increasing prevalence of hypertension with aging and the decline in glomerular filtration rate (gfr) noted in some people as they get older, may put donors at a higher risk for progressive kidney disease. we have previously reported on donors > 20 years after donation and found them to incur no excessive risk of hypertension or kidney disease. herein, we report on renal and non-renal outcomes on the world's largest experience of kidney donors to date (n=302) who donated more than 35 years ago. methods: kidney donors were asked to fill out a survey detailing their medical history since donation and obtain a physical exam and laboratory testing by their local physician. results are expressed as mean±standard deviation (sd the graph below depicts the cross-sectional distribution of last serum creatinine available 35 years or more after donation regardless whether the donor is presently alive or not though the majority is from currently alive donors. conclusion: in the longest follow-up of kidney donors to date, this data indicates that 4-5 decades of living with one kidney has no serious adverse renal effects and a prevalence of hypertension that is probably similar to the general population considering the age of these donors. centers ) . before and after adjustment for comorbidity, 19 (7.8%) and 18 (7.5%) centers, respectively, met all criteria for review, but only 16 met criteria for review both before and after comorbidity adjustment. we conclude that failure to adjust for pre-existing recipient comorbidity results in grossly inaccurate estimation of expected gf per ktx center, more often resulting in expected gf that is too low. using data that are not adjusted for comorbidity to judge the quality of tx programs could encourage denial of access to high-risk patients. introduction: the purpose of our study was to examine temporal trends in the regionalization patterns and center volume-outcomes relationship for lung transplantation in the united states over the past decade. a retrospective analysis of all adult single-organ lung transplants included in the scientific registry of transplant recipients for three consecutive time periods between 1997 and 2006 was performed. for each time period, lung transplant centers were divided into three groups based on each center's annual volume of the procedure (low-volume group = 1-17 procedures per year, medium-volume group = 18-30 procedures per year, high-volume group = greater than 30 procedures per year). oneyear observed-to-expected patient death ratios were then calculated and compared for each group in each time period. a temporal analysis of the percentage of transplants being performed relative to center volume was also performed. statistical comparisons were made using chi square testing. results: a total of 12,603 lung transplant procedures were included in the analysis. in period 1, there was not a significant difference in the one-year observed-to-expected patient death ratio of low-volume lung transplant centers when compared to high-volume centers (ratio 1.15 for low-volume centers vs. 0.79 for high-volume centers, p = 0.07). by period 3, however, a significant relationship between center volume and outcomes had emerged (ratio 1.30 for low-volume centers vs. 0.79 for high-volume centers, p = 0.006). over this same time period, the percentage of lung transplants within the united states that are performed at low-volume centers has decreased significantly (from 43.3% of all lung transplants in period 1 to 22.7% in period 3, p < 0.0001), while the percentage being performed at high-volume centers has increased significantly (from 20.5% of all lung transplants in period 1 to 51.3% in period 3, p<0.0001). conclusions: a significant relationship between center volume and patient outcomes has emerged for lung transplantation over the past decade. at the same time, the percentage of these procedures being performed at high-volume centers has increased. these findings suggest that regionalization patterns for a given procedure may be influenced by the presence or absence of a volume-outcomes relationship for that procedure. liver transplantation (lt) has emerged as one of the few curative treatment modalities for patients with hepatocellular carcinoma (hcc). however, the increase in the incidence of hcc recurrence due to immunosuppressants administered after lt is a serious issue. we have recently proposed a novel strategy of adjuvant immunotherapy for preventing the recurrence of hcc after lt: intravenous administration of il-2-stimulated natural killer (nk) cells extracted from donor liver graft to liver transplant recipients. since the immunosuppressive regimen currently used after lt reduces the adaptive immune components but well maintains the innate components of cellular immunity, the augmentation of nk cells response might be a promising immunotherapeutic approach. we confirmed that the il-2-stimulated donor liver nk cells exhibited a significantly high level of trail and showed vigorous cytotoxicity against an hcc cell line without cytotoxicity against normal cells. after obtaining approval from the ethical committee of our institute, we successfully applied this therapy to 13 cirrhotic patients with hcc from january 2006. the average number of nk cells that had been administered to lt recipients at 4 days after lt was 304.2 ± 225.5 × 10 6 cells/body. the lt recipients were categorized as follows: (1) based on the milan criteria, 8 recipients met the criteria while 5 did not, and (2) based on the tnm stage, 2 recipients were categorized as pathological tnm stage i; 6, stage ii; 4, stage iii; and 1, stage iv. in our institute, the 2-year recurrence-free survival rates of the lt recipients treated with and without this therapy were 100% and 71.3%, respectively. kinetic studies revealed that in the early postoperative period, the peripheral blood obtained from the treated lt recipients exhibited a significant improvement in cytotoxicity against hcc cell line as compared to the untreated lt recipients (p < 0.01). furthermore, flow cytometric analyses revealed that the frequency of trail + nk cells increased remarkably in the peripheral blood of the treated lt recipients (p < 0.05). in conclusion, the administration of il-2-stimulated donor liver nk cells contributes to the promotion of host anticancer activity and has the potential to regulate hcc recurrence after lt. abstract# 500 vegf, a well-established angiogenesis factor, is expressed within allografts at high levels in association with acute and chronic rejection. in previous studies, we have reported that vegf possesses potent proinflammatory properties in part via its ability to mediate leukocyte trafficking into allografts. recently, we discovered that vegf mediates cd4+ and cd8+ t cell migration via interaction with its receptor kdr (also called vegfr2). blockade of t cell kdr significantly inhibits transendothelial migration. these observations suggest that kdr may be a novel t cell receptor for allogeneic lymphocyte recruitment. here, we first examined the expression of kdr on peripheral human cd4+ and cd8+ t cells by facs analysis. we found that ≤ 1% of circulating t cells express kdr, and its expression was at low levels on individual unactivated t cells. further, we found that the expression of kdr on t cells increased markedly following activation with mitogen and following interactions with activated allogeneic endothelial cells. induced expression of kdr on cd4+ and cd8+ t cells was at a similar level as that observed on endothelial cells. therefore, kdr appears to be selectively expressed on t cells, that traffick into allografts. to test the pathophysiological significance of these observations, we analyzed the expression of kdr in a total of 19 cardiac, and 5 renal, human allograft biopsies. we correlated the expression of kdr with cd3+ t cell infiltrates; and by double immunofluorescence staining, we determined co-expression of kdr on individual cd3+ t cell infiltrates. in cardiac allografts we found that kdr was expressed throughout the endomyocardium, and was most notable on endothelial cells in all biopsies examined. by grid counting of 3-4 areas of each biopsy, we found that the mean number of cd3+ t cell infiltrates ranged from 4 to 79 cells/hpf (x600 mag.). by double staining, we noted that kdr was expressed on 29 ± 4 % (mean±sem) of these cd3+ t cell infiltrates. similarly, we found that kdr was co-expressed on cd3+ t cells within renal allografts. while infiltrates were more focal, again 30 ± 2 % (mean±sem) of graft infiltrating t cells expressed kdr. collectively, these observations for the first time identify kdr as a novel receptor on allogeneic t cells. we suggest that intragraft vegf may interact with t cell kdr to facilitate homing and recruitment of allospecific lymphocytes into allografts. interaction of infiltrating cd8 + t cells and tissue cells in tolerant liver allografts: using tcr transgene approach. guoping jiang, 1 qiwei zhang, 1 horng-ren yang, 1 kathleen brown, 1 john j. fung, 1 lina lu, 1 shiguang qian. 1 1 immunology and general surgery, cleveland clinic, cleveland, oh. the liver allografts are accepted without requirement of immunosuppression in mice. the underlying mechanisms are not completely understood. we hypothesized that it resulted from an abortive t cell response within the liver due to hyporesponsiveness or apoptosis. to test this, we examined the activation and fate of allo-ag specific cd8 + t cells following liver transplants (ltx) compared with heart transplants (htx) that were acutely rejected. following transplantation [b10 (h2 b )→c3h (h2 k )], cfse labeled cd8 + t cells (10x10 6 ) from des tcr tg mice (h2k b specific tcr) were adoptively transferred into recipients. animals were sacrificed two days following des t cell administration for analyses of t cells in the grafts or draining lymph nodes (d-ln). host cd45 + leukocytes were quickly infiltrated following ltx. among cd3 population ∼48% were cd4 + and ∼52% cd8 + . cd8 + t cells were further increased thereafter in grafts and d-ln, associated with high inf-g production. cd8 + t cells in the liver grafts rapidly reduced to 36% by pod 14, and to 12% by pod 40. however, the incidence of cd4 + t cells remained high. cfse dilution assay and elispot showed an active division of des + t cells in liver allografts either on pod 7 or 40. these ag-specific cd8 + t cells functioned well evidenced by ifn-g production in response to allo-ag. however, compared to htx, the accumulation of des + cells in grafts was significantly lower in ltx [9.6% (17x10 4 /heart), and 0.59% (6.14×10 4 /liver)] on pod 7, and further dropped to 0.14% (3.14×10 4 /liver) on pod 40. the expended cohorts of adoptively transferred cells followed by their elimination suggested elimination of ag-specific cd8 + cells. to examine the role of liver environment, graft cd45non-parenchymal cells (npc) were isolated tested fro regulatory effect on des + t cell response. liver allografts showed significantly expansion of cd45 -npc, which were donor mhc class i + (h2b + ), b7-h1 + , trail + and low for cd40 and cd86. these cells did not inhibit des + t cell proliferation in response to b10 spleen stimulation, but significantly enhanced their death, which was dependent on b7-h1/trail. this was confirmed by using cd45 -npc from b7h1 -/or trail -/mice. in conclusion, activated t cells in liver grafts may stimulate tissue cells to express inhibitory and death inducing molecules, resulting in t cell death and graft acceptance. foxp3 + graft-infiltrating lymphocytes (gil) have been detected in the rejecting allografts of transplant patients. foxp3 is a marker for regulatory t cells (t reg ). published reports suggest that human foxp3 can be upregulated following tcr stimulation without induction of regulatory function. to investigate whether foxp3 + gil during acute rejection are t reg , we analyzed the phenotype of gil harvested from acutely-rejected non-human primate kidney allografts. methods: renal allografts with histologically-confirmed acute rejection were harvested at the time of necropsy from macaques that had undergone experimental transplantation. following digestion of kidney fragments using collagenase, gil were isolated using lymphocyte-separation media and cryopreserved. axillary lymph nodes (ax ln) were also isolated either at the time of necropsy or by biopsy. for seb-stimulated lymphocytes, ax ln cells were stimulated for 5 days in the presence of 25 ng/ml seb for 5 -6 days. to perform intracellular interferon-gamma (ifng) analysis, cells were stimulated with pma and ionomycin in the presence of brefeldin a for 5 -6 hours at 37°c. samples were prepared for flow cytometry by first staining for extracellular antigens. cells were then fixed and permablized (kit from ebiosciences) prior to intracellular staining for foxp3, ki67 and ifng. results: the percentage of foxp3 + in cd3 + gil was similar to that found in ax ln ( table 1 ). the majority of these cells were cd4 + . while 80% of the cd3 + /cd4 + /foxp3 + population of both ax ln and gil were cd25 + , a significantly higher frequency of cd39 + and ki67 + cells were found in gil (p < 0.01; student's t-test). simlar levels of cd39 and ki67 expression were found in seb stimulated lymphocytes. unlike the seb-stimulated lymphocytes, few ifng -producing cells were demonstrated following pma/ionomycin stimulation of gil. conclusion: our current data indicates that the majority of foxp3 + gil from acutely rejecting renal allografts are recently activated cd4 t cells that lack effector function. this suggests that foxp3 t cells within rejecting allografts may indeed be t reg . findings in mouse models of transplantation often fail to translate well in humans. three variables may account for the discrepancy: (1) evolutionary divergence between mice and humans, (2) influence of infection history on alloimmunity, and (3) use of highly inbred strains of laboratory mice. here, we investigated whether the use of inbred mouse strains skews the rejection phenotypes and their response to treatment due to decreased genetic diversity and/or fixation of undesirable genetic loci, known as inbreeding depression. we examined heterotopic cardiac allograft survival in outbred and inbred mouse populations in the presence or absence of immunosuppression. in the absence of immunosuppression, heart transplantation within or between outbred stocks of mice (n = 35) resulted in three distinct rejection phenotypes that resemble accelerated (1 -4 days), acute (8 -25 days), and chronic rejection (> 75 days), respectively. in contrast, all fully allogeneic grafts transplanted between inbred mice (n = 12) were rejected acutely (7 -13 days) as were historical controls (n > 50). the accelerated phenotype, present in 29% of outbred to outbred transplantations, was characterized by extensive hemorrhagic necrosis of the heart with thrombosis, neutrophil margination and neutrophilic arteritis, and did not correlate with donor:recipient mhc ii disparity. immunosuppression with t cell costimulation blockade did not prevent accelerated rejection (incidence = 27% in the treated group, n = 26) but did convert the acute rejection phenotype into longterm allograft survival in all groups studied. the same accelerated phenotype was observed if transplantation was performed from outbred to inbred mice (incidence = 39%; n = 23) but could not be duplicated if inbred to outbred transplantation was performed (n = 20). finally, c3 depletion with cobra venom factor abrogated the accelerated rejection phenotype in outbred to outbred transplantations (n = 16), suggesting a role for complement in the pathogenesis of this phenotype. in summary, our data (1) indicate that the use of outbred mouse stocks may uncover clinically-relevant rejection phenotypes not observed in inbred mouse strains, and (2) underscore the importance of the donor background in determining the phenotype of rejection. outbred mouse stocks may provide a platform to uncover mhc-unlinked genetic loci that play an important role in the outcome of solid organ transplantation. th17 are limited in their ability to reject allografts. elderly recipients represent the most rapidly growing segment of patients on the waiting list. however, little is known about age-dependent alterations of the immune response in organ transplantation. we examined age dependent t-cell functions in a transgenic mouse transplant model. effector t-cell phenotype, -function, cytokine production and regulatory t-cell function were analyzed in 3 and 18mths old b6 mice. in an in vivo transplant model, bl/6 nude mice were reconstituted with 2x10 6 young or old transgenic alloantigen-specific cd4 + tcells and engrafted with bm12 skin grafts. t-cell phenotype and cytokine secretion were sequentially analyzed in all lymphatic compartments. splenocytes of naïve old b6 mice contained significantly higher frequencies of t-cells with an effector/memory phenotype (cd4 + cd44 high cd62l low and cd8 + cd44h igh cd62l low; p<0.005). in vitro proliferation and ifnγ-production were significantly reduced in aged mice indicating an impaired t-cell response with increasing age as assessed by mlr (p<0.005) and elispot (p<0.001). in parallel, regulatory functions remained age-independent as alloantigen-specific cd4 + cd25 + foxp3 + t-cells isolated from sensitized old mice demonstrated a dose-dependent well preserved suppressor function. next, we tested the age-dependent alloantigen -specific cd4 + t-cell function in a transgenic skin transplant model: age did not significantly impact rejection kinetics (young vs. old: 10.1 vs. 14.3days, n.s.) however, t-cell migration and activation were significantly different: fewer numbers of activated cd4 + cd25 + and effector/memory phenotype t-cells (cd4 + cd44 high cd62l low ) were found in recipient spleens (p<0.05) and draining lymph nodes (dln) (p<0.05) after transfer of old t-cells. chemokine receptor staining revealed less cxcr3 + and ccr7 + t-cells in dln following the transfer of old t-cells (total cell numbers x104: cxcr3 + : 10.9±4.2 vs.0.95 ±0.2, ccr7 + : 4.7±1.0 vs. 0.35±0.2, p<0.05). this was paralleled by reduced intragraft t-cell infiltration as observed by immunohistochemistry. in summary, native elderly mice showed an increased frequency of effector memory t-cells but an overall impaired t-cell response. regulatory -t-cell function remained preserved. in vivo allospecific cd4+ t-cell activation and migration was impaired in elderly transplant recipients. the background: the sensitized transplant recipients may undergo an "accelerated" form of rejection, which is mediated by t cell-dependent mechanisms. these patients often experience increased rate of early rejection episodes, which are difficult to control with currently used immunosuppressive agents. methods: in our model of cardiac graft rejection in sensitized recipients, b6 mice are first challenged with b/c skin, followed 40-60 days later by b/c heart transplant (htx). unlike in naive hosts, htx rejection and alloreactive cd8 activation in this model are cd154 blockade-resistant. we first performed systemic analysis at the intragraft transcriptional level by microarray to identify disparities in local immune responses in naive vs. sensitized hosts. aiming to improve the efficacy of costimulation blockade in the sensitization settings, we then determined the role of cd4 t cells in costimulation blockade-resistant alloimmune response by using cd4 depleting (gk1.5) vs. cd4 blocking (yts177) ab, in conjunction with cd154 blockade (mr1). results: htx harvested from groups of naïve (day 4-6), sensitized (day 2-4), control ig or mr1 ab treated mice (n=3/gr) were subjected to microarray analysis. mr1 treatment suppressed htx expression of proinflammatory genes (il-1β, il-6, tnf-α), and t cell-targeted chemokines (rantes, mig, cxcl10) early after htx in naïve, but not sensitized recipients. five groups of sensitized mice treated at the time of htx with: (1) 5). ctl activation was determined by facs phenotyping at day 10 and 30. the simultaneous blockade of cd154 costimulation and cd4 help, but not a single blockade with mr1 ab or anti-cd4 ab, was required to inhibit peripheral alloreactive cd8 activation in sensitized mice. additionally, cd8 activation in the absence of cd4 help showed defective cytotoxic molecule profile, with suppressed perforin but upregulated granzyme b expression at the graft site. conclusion: cd154 blockade-resistant cd8 activation is critically dependent on cd4 t cells. this study provides novel immunological basis to study the potential synergy between adjunctive cd4 and cd154 targeted therapies to control accelerated graft rejection in sensitized hosts. mediators. g. einecke, l. g. hidalgo, p. f. halloran. department of medicine, university of alberta, edmonton, canada. the hallmark of t cell mediated rejection (tcmr) are interstitial inflammation and tubulitis. the mechanisms of tubulitis and epithelial deterioration during tcmr are unknown. we previously showed that tubulitis in mouse allografts is independent of cytotoxic molecules (gzma/b, prf) and is preceded by molecular changes with loss of epithelial genes, reflecting epithelial dedifferentiation. human tcmr is associated with loss of the same epithelial genes and re-expression of embryonic pathways (wnt, notch). we hypothesized that tcmr is mediated through soluble factors released by effector t cells or macrophages in the interstitium, and that supernatants of effector t cells would simulate these changes in epithelial cultures. we established an in vitro model in which cultured primary human renal epithelial cells are incubated with supernatants from effector t cell/monocyte co-cultures. the transcript changes in this model, analyzed by microarrays, closely simulated those in human and mouse tcmr (fig1), with loss of epithelial transcripts, activation of wnt/ notch pathways, and increased expression of ifng-inducible and injury-related transcripts previously defined in our mouse model. some of these changes were reproduced by incubation of epithelial cells with ifng or tgfb. the in vitro model identified additional epithelial transcript changes not previously identified in vivo (not affected by ifng or ischemic injury, not expressed in t cells, macrophages, b cells, or nk cells). expression of these transcripts (n = 305) was highly altered in human tcmr compared to nonrejecting biopsies of 177 human renal allograft biopsies and distinguished tcmr from antibody-mediated rejection in a hierarchical cluster analysis. thus we have established an in vitro model that closely simulates the epithelial events during human tcmr and confirms that these changes are independent of direct contact with inflammatory cells, supporting the hypothesis that interstitial effector t cells mediate allograft deterioration by soluble mediators. together with previous mouse and human data these results provide the first in vitro model of the epithelial consequences of tcmr. objective. c4 split product deposition to hla antigen-coated microparticles ([c4d] flowpra) was previously shown to be a specific marker of c4d-positive antibodymediated rejection (amr). the objective of this study was to assess the predictive value of [c4d]flowpra reactivity in a cohort of non-biopsied patients with stable graft function during the first year. methods. a total of 133 kidney transplant recipients were enrolled (inclusion criteria: functioning graft at 12 months; prospective collection of sera taken before and at 1-3, 6, and 12 months after transplantation). included patients were serially screened for humoral panel reactivity applying [igg] and [c4d]flowpra screening. results. fifty-four of the included 133 recipients had stable graft function within the first year and were not subjected to diagnostic renal biopsy. in this particular patient group, detection of complement-fixing hla reactivity tended to be less frequent than in the 79 patients with biopsied graft dysfunction (≥10% [c4d]flowpra before transplantation: 9% vs. 18% of recipients, p=0.2; ≥10% [c4d]flowpra after transplantation: 11% vs 24%, p=0.06). in line with our previous results, within the group of biopsied patients, pre-and/or post-tx [c4d]flowpra reactivity was tightly associated with the immunohistochemical detection of peritubular capillary c4d deposition (p=0.005) reflecting ongoing amr. remarkably, in initially stable patients, detectable [c4d] flowpra reactivity was not associated with inferior long-term outcomes. within this patient group, recipients with and without (pre-and/or post-transplant) c4d-fixing anti-hla reactivity did not differ with respect to 4 yr allograft survival (p=0.4), 4 yr serum creatinine levels (1.5 vs. 1.5 mg/dl; p=0.7), and proteinuria at 4 yrs (0.2 vs. 0.18 g/24h; p=0.8). similar results were obtained for a comparison of [igg]flowpra positive vs. negative subjects. conclusion. our data suggest that a considerable number of patients with initially stable graft function may have excellent long-term graft function despite serologically detectable levels of (complement-fixing) alloreactivity. for these antibody-positive recipients, a potential role of graft accommodation can be speculated. the immunoproteasome subunit beta 10 as a novel peripheral blood and intragraft biomarker of chronic antibody mediated allograft rejection in clinical transplantation. joanna ashton-chess, 1 in an attempt to identify non-invasive biomarkers of specific histological scarring, we compared publicly available gene sets derived from microarray studies of human renal transplant biopsies published in the literature with our own microarray data derived from studies of rat heart allografts. in this way we identified an immunoproteasome subunit (proteasome subunit beta 10 -psmb10) as a potentially interesting candidate. psmb10 is one of three members of the immunoproteasome that are induced by abstracts interferon gamma. messenger rna profiling in renal transplant biopsies (n = 52) with normal histology, interstitial fibrosis and tubular atrophy, calcineurin inhibitor toxicity, transplant glomerulopathy or chronic antibody-mediated rejection (banff 2005) revealed psmb10 to be strongly and significantly increased in chronic antibody mediated rejection vs. the other three histological diagnoses. receiver operator characteristic (roc) curve analysis showed that psmb10 mrna could diagnose chronic antibody-mediated rejection with an auc of 0.99, a sensitivity of 1.0 and a specificity of 0.95. moreover, psmb10 mrna was significantly increased in the pbmc (n = 24) of patients with chronic antibody-mediated rejection compared to those with normal histology. roc analyses revealed an impressive auc of 1.0 with all patients being correctly classified. similar results were also observed in a rat allograft model where psmb10 was significantly increased at day 100 post transplantation in both the heart allograft and the pbmc of animals presenting chronic transplant vasculopathy vs. syngeneic grafts. moreover, inhibition of the proteasome by administration of velcade® at 0.1 mg/kg every other day for the first 20 days post transplantation significantly and dose-dependently prolonged allograft survival (mst 31.7 days in velcade-treated vs. 6.3 days in untreated animals).together our data point towards psmb10 as a blood and intragraft biomarker of chronic antibody-mediated rejection as well as a potential therapeutic target. furthermore, our results suggest that using a threshold of psmb10 in the blood could help in guiding the decision to biopsy in the clinic. baff monitoring after b-cell depletion therapy for acute renal transplant rejection. valeriya zarkhin, 1 snehal mohile, 1 li li, 1 jonathan martin, 1 minnie sarwal. 1 1 pediatrics, stanford university, stanford, ca. introduction: the objective of this study was to investigate the interaction between b-cell activation factor of the tnf family (baff) level and circulating b-cell repopulation in pediatric patients with acute kidney transplant rejection treated with the b-cell-depleting agent rituximab. methods: 10 pediatric patients (3-23 yrs) with biopsy proven b-cell positive ar were treated with steroids and rituximab (4 x 375 mg/m 2 /dose/week). all patients were followed up for 12 months. peripheral blood cd19 cells and donor specific antibodies (dsa) were monitored monthly. serum level of baff was measured by elisa at ar, 1, 3, 6, and 12 months post-ar treatment and correlated with clinical outcomes. results: complete depletion of circulating and intragraft b-cells was observed with rituximab, with improvement in ar grade in all patients. the median time of peripheral b-cells repopulation was 5 months (range 3-12 months, fig. 1a) . no correlation was found between pre-treatment peripheral b-cell number and the b-cell repopulation time (r=0.59, p=0.09). baff levels rose significantly with b-cell depletion with maximum values at 3 months post-treatment (7.5 fold increase, p=0.0001) and returned to pre-treatment levels, with b-cell recovery, at 12 months (fig. 1b) . serum baff levels correlated positively with b-cell depletion >6 months (r=0.91, p=0.004, fig1b). a lack of depletion of dsa i, but not dsa ii correlated with higher baff levels (r=0.99, p=0.007). the timing of b-cell repopulation and depletion of dsa i may be dependant on serum baff level. anti-baff treatment may be considered in addition to rituximab or standard immunosuppressive treatment protocols in patients with persistent and/or antibody mediated rejection. the background: the development of donor specific hla antibodies (dsa) post-transplant has been associated with graft failure. we have shown in a longitudinal study that increases in dsa may precede rejection by months. this retrospective analysis evaluates changes in maintenance immunosuppression (mi) and appearance of dsa in stable transplant recipients. methods: sera from stable renal transplant recipients were collected at 4-6 month intervals and tested for the presence of dsa. the types and doses of immunosuppression were correlated with the appearance of dsa. two hundred eighty stable renal transplant recipients who received either a deceased or a living donor kidney were monitored post-transplantation for the development of dsa. patients have been followed for 1 to 7 years and had a minimum of 4 serum samples analyzed. all recipients received anti-lymphocyte induction therapy. maintenance immunosuppression (mi) consisted of a calcineurin inhibitor, prednisolone, and mycophenolic acid. hla single antigen beads analyzed in the luminex instrument were used to establish donor specificity of the antibodies. a chart review was undertaken to determine the doses of the mi posttransplantation. all mi was managed by the transplant team and changed according to clinical indications without regard to dsa. results: of the 280 patients monitored 37 developed dsa post-transplantation with a functioning graft. dsa was against hla-class ii antigens in 26 of 37 (70%); class i antigens in 8 of 37 (22%); and against both class i and ii antigens in 3 of 37 (8%). dsa against class ii was against dq in all except one case. in the majority of the recipients the appearance of dsa was preceded with dose reduction of the mi, either calcineurin inhibitor or mycophenolic acid or both. conclusions: our data show that dsa developed predominantly against hla-class ii antigens and that the appearance of dsa was often preceded with reduction of one or more of the mi. this data shows the importance of monitoring dsa with mi decreases in a stable allograft recipient. antibody production and antigen presentation are directly inhibited by mycophenolate mofetil. anat r. tambur, 1 joe leventhal, 1 nancy d. herrera, 1 joshua miller. 1 1 division of organ transplantation, northwestern university, chicago, il. immunosuppressive medications are primarily designed to target t cell proliferation. mycophenolate mofetil (mmf) exerts its effect by inhibiting de-novo synthesis of guanine, a dna building block. we, and others, have previously shown that mpa (active metabolite of mmf) affects the differentiation of monocytes into dendritic cells (dc). we further demonstrated that cell-surface receptors associated with antigen up-take and antigen-processing and presentation (cd83 and cd205) are down regulated when cells are matured in the presence of mpa. this phenotype translated into a decreased uptake of alloantigens and reduced stimulation of t cells. we concluded that mmf inhibits also cell functions requiring mrna synthesis. we now present data regarding the role of mpa in maturation and function of b-lineage cells. pbmcs from 10 subjects were cultured in the presence of cpg, il-2, il-10 and cd40l for 5 days to induce memory b and plasma cell maturation in-vitro. cultures were performed in the presence or absence of mpa (50 ugr/ul) for the length of the incubation period. in-vitro stimulation of b cells increased the memory population (cd19+ cd27+) from 2.8 +/-1.1% to 7 +/-2%. similarly, plasma cells were increased from 4.4 +/-1.3% to 6.4 +/-1.8%. the addition of mpa to the culture inhibited stimulation for both memory and plasma cells (4.7 +/-3% p=0.01; and 4.7 +/-2.3% p=ns, respectively). we have further analyzed the effects of mpa on antibody secretion using an elisa (measuring soluble antibodies) as well as a b-cell elispot assay (assessing the number of b cells that produce antibodies). while an expected increase in od values was observed for stimulated samples compared with non-stimulated samples, a significant decrease was observed when stimulation occurred in the presence of mpa. nonstimulated cells: 0.249 +/-0.28; stimulated cells: 0.391+/-0.33; mpa treated stimulated cells: 0.279+/-0.43; p<0.0005). the number of antibody producing cells was also significantly lowered when cultures were done in the presence of mpa (a mean of 106 cells were counted for the stimulated cells compared with 1-2 cells for non-stimulated and mpa-treated-stimulated cells). to our knowledge this is the first time where in-vitro experimental data document the inhibitory effect of mpa on b lineage cells and antibody secretion, although clinically known for some time. these results confirm our previous observations regarding the effects of mmf on non-proliferating immune cells. a non-allogeneic stimulus triggers the production of de novo hla and mica antibodies. luis e. morales-buenrostro, 1,2 lluvia a. marino-vazquez, 1 anh nguyen, 3 paul i. terasaki, 2 josefina alberu. 1 1 nephrology and transplantation., instituto nacional de ciencias medicas y nutricion salvador zubiran, mexico city, df, mexico; 2 terasaki foundation laboratory, los angeles, ca; 3 one lambda inc., canoga park, ca. background: in a previous study, we found that healthy people developed hla abs after immunization against hepatitis b virus. the aim of this prospective study was to establish if stimulation with influenza vaccine is capable of triggering the production of hla and mica abs. methods: we determined the presence of hla and mica abs (de novo and preformed abs) in 3 groups of patients vaccinated against influenza: a) 42 healthy adults, b) 40 esrd patients, and c) 25 tr. additionally, we followed 22 healthy unvaccinated people without exposure to sensitizing factor: d) control group. sera samples were collected at baseline (pre influenza shot), at 1 week, and monthly up 6 months after immunization. hla abs were assessed with labscreen single antigen beads for luminex. all samples of each patient were tested simultaneously. a luminescence value higher than 500 was considered positive only if it was 3 times the baseline value. we analyzed the data using chi square, one way anova test, and logistic regression. results: the table shows the types of abs in each group. interestingly, we found preformed abs across all four groups, including the control group (which is free of any known sensitizing factors). the proportion of de novo abs was higher in the group b and c. multivariate analysis shows that the only independent factor associated with development of de novo abs was the presence of preformed abs. we observed a nonspecific immunologic response triggered by external stimulus and was not necessarily associated to the vaccine in people previously sensitized. a introduction. decisions about the minimization and ultimate withdrawal of immunosuppression (is) would be facilitated by the identification of biomarkers associated with operational tolerance (ot). methods. as part of an itn/nih supported study tolerant kidney transplant recipients (off all is for > 1yr with stable function, n=22) were compared to recipients with stable function on is (sis, n=34), recipients with chronic allograft nephropathy (can, n=20), and healthy volunteers (hv, n=18). pbmc, whole blood total rna, and urine samples from each group were examined using flow cytometry, microarrays, and rt-pcr respectively. results. analysis of microarrays revealed significantly higher expression of b cell differentiation genes in tolerant recipients compared to the sis and can groups. consistent with this finding, tolerant recipients also displayed higher numbers of naïve b cells in peripheral blood and increased expression of cd20 in urine relative to the sis and can groups. no differences in treg or genes associated with regulatory cells were observed in tolerant recipients relative to other groups. these analyses failed to demonstrate significant differences between tolerant recipients and hvs although support vector machine learning methods suggested potential differences in a number of genes including nfat and calcineurin. finally, relative to tolerant patients, those with can showed decreased numbers of t and nk cells and expressed lower levels of genes associated with immune cell activation in peripheral blood. conclusions. differences in b cell numbers may be useful in identifying tolerant renal transplant recipients or those predisposed to developing tolerance and could potentially provide insights into the mechanisms of tolerance. erythrocyte development of antibodies (abs) to mismatched donor hla antigens has been associated with acute and chronic rejection. complement activation, and c4d deposition, has been correlated with humoral rejection of allografts. however, utility of c4d staining in ltx has been controversial. a recent study (arthritis rheum. 2004 nov; 50(11) :3596-604) shows a strong correlation between erythrocyte bound c4d (e-c4d) in diagnosis and monitoring of sle. goal of our study is to determine the utility of measuring e-c4d in the diagnosis of humoral rejection following human ltx. 26 ltx recipients were analyzed post-ltx for e-c4d using facs of rbcs incubated with anti-c4d (quidel) followed by fitc-goat anti-mouse.10 normals were also analyzed. the serum was analyzed for development of anti-hla abs by solid phase assays and for the presence of autoabs to kα1 tubulin and collagenv (elisa). biopsies from 11 patients were stained immunohistochemically for c3d deposition. summary of results are presented in table 1 . infection=2/10 ar=0/10 % e-c4d in normals-10.35%; mfi in normals-5.46; mfi=mean fluorescence intensity; ar=acute rejection; dsa=donor specific abs 16 out of 26 patients show significant increase in the % bound e-c4d (p<0.05) as compared to controls.11/16 had anti-hla and 13/16 had autoabs which were significantly different from those with low e-c4d (p= 0.005). staining of the biopsies showed c3d deposition in 6 recipients with increased e-c4d. all 4 patients with acute humoral rejection had elevated e-c4d. we conclude that there is a significant correlation between increase in % e-c4d in ltx recipients and development of abs to either hla antigens or auto-antigens during the post-ltx period. biopsies from patients with increased e-c4d showed deposition of c3d in the allografts. preliminary data suggest that measurement of e-c4d using a non-invasive method of flow cytometry may be of value in monitoring ltx patients for humoral rejection. innate immunity: chemokines, cytokines innate immunity is emerging as an important initiator and modulator of the adaptive immune response. in the setting of transplantation, ischemia-reperfusion injury and tissue trauma appear to potentiate the alloimmune response. one of the mechanisms through which the innate immune system modulates an adaptive immune response is dendritic cells (dc). in this study, we examined dc activation in a mouse model of skin transplantation by monitoring the expression of mhc ii and p40 chain of the proinflammatory cytokine il-12. the p40 expression was detected in live cells using the yet40 reporter mouse, in which a transgene for yellow fluorescent protein (yfp) was placed downstream of the endogenous il-12p40 gene, thus faithfully "reporting" p40 expression. skins from c57bl/6 or balb/c donors were grafted on the dorsal thorax of c57bl/6.yet40 mice. draining and non-draining lymph nodes (ln) were harvested at 24 hours and examined for yfp expression by fluorescent microscopy. a marked increase in the numbers of yfp-expressing dc was observed in draining ln in both syngeneic and allogeneic graft recipients. surprisingly, yfp-expressing dc also increased in nondraining ln when compared to non-transplanted controls. similarly, dc in draining and non-draining ln showed higher mhc ii expression than those in non-transplanted controls. upregulation of mhc ii was highest at 24 hours and decreased significantly by 48-72 hours. to assess whether dc activation in non-draining ln was functionally significant, we monitored the activation of adoptively transferred ovalbumin-specific ot-ii tcr transgenic t cells in response to footpad antigen challenge in mice with or without a syngeneic skin transplant on the contralateral upper thorax. otii t cells in transplanted animals proliferated approximately 5-to10-fold better and a higher percentage of the otii cells produced il-2 than those from non-transplanted animals. thus, local surgical trauma results in a widespread, time-limited, functional activation of dc that appears to act as a partial adjuvant for t cell responses. together, these data suggest that surgical trauma may incite a systemic barrier to transplantation via the activation of dc and therapeutic interventions that reduce the surgical trauma and dc activation may help to improve survival of transplanted grafts. tolerance of cardiac allografts: studies with cx3cr1-deficient mice. takaya murayama, 1 katsunori tanaka, 1 takuya ueno, 1 mollie jurewics, 1 guleria indira, 1 fiorina paolo, 1 paez jesus, 1 smith n. rex, 2 sayegh mohamed, 1 reza abdi. 1 1 transplantation research center, renal division, brigham and women's hospital, harvard medical school, boston, ma; 2 department of pathology, massachusetts general hospital, harvard medical school, boston, ma. although donor/tissue dendritic cells (ddc) have long been known to play a key role in mounting alloimmune responses, however, their generation, trafficking and role in tolerance have not rigorously examined. we have used b6.fvb-tg (itgax-dtr/ egfp) 57 mice which has a gfp linked to the cd11c promoter. using these mice as the donors of heart allograft transplantation provided us a unique model to study ddc posttransplantation. our trafficking data indicate there is a rapid migration of ddc into spleen (3 hours post transplantation) but not lymph nodes and that ddc were unexpectedly detected in the spleen of the recipients long after rejection of heart allografts suggesting ddc could escape from the immunosurveillance of host immune system. our data also show that ddc proliferate in the lymphoid tissue of the recipients and co-express class ii molecule of the recipients. we then show that cx3cr1 pathway regulates generation abstracts of heart tissue dc constitutively. as compared to wt hearts, cx3cr1 -/hearts contain lower number of dc and transplanting cx3cr1 -/donor hearts into wt balb/c mice led to significant prolongation of allograft survival without immunosuppression (mst of 8 vs. 17 days, respectively). increasing cx3cr1 -/heart dc by implanting donors with flt3-producing hybridoma cells has restored the time of rejection. unexpectedly, induction of long-term survival with anti-cd154 blockade (mr1) and ctla-4 ig (but not low dose rapamycine) was abrogated when cx3cr1 -/hearts were used as donors, with concomitant lesser tregs in the cx3cr1 -/heart allografts as compared to wt. furthermore, co-transplanting hearts from wt and cx3cr1 -/into the same recipient treated with mr1 resulted in significant prolongation of cx3cr1 -/heart allograft survival. depleting the ddc of heart donors prior to transplantation with diphtheria toxin also worsened markedly chronic rejection in the recipients at day 100 post-transplantation. our data indicate that, in contrast to the widely accepted dogma, the presence of donor dc in graft tissue is not only central to allograft rejection but also is necessary for the induction and maintenance of peripheral tolerance. local c5a interaction with c5ar on dcs modulates dc function, subsequently up-regulating allospecific t cell responses. qi peng, ke li, steven h. sacks, wuding zhou. mrc centre for transplantation, department of nephrology and transplantation, king's college london, guy's campus, london, united kingdom. the innate system of immunity plays an important role in ischemia-reperfusion injury and allograft rejection. the early stages of inflammatory processes are accompanied by complement activation. one biological consequence of this activation is the release of potent inflammatory anaphylatoxins, c3a and c5a, which have been reported to regulate a range of inflammatory responses. we previously reported that dcs express c3ar and c5ar, and c3a-c3ar interaction has a positive impact on murine bm dcs, in terms of activation phenotype and capacity for ag uptake and allostimulation. however, the role of c5a in modulating dc function remains unclear. the aim of this study is to investigate the role of local c5ar signalling in modulating murine bm dc function and subsequent regulation of the allospecific t cell response. we first evaluated if c5a-c5ar interaction could result from local expression of factors. our results showed that c5ar mrna was detected in wt dcs at different stage of dc culture by rt-pcr, and c5a was detected by elisa in the culture supernatants from different stages of dc culture. we next determined if c5a-c5ar interaction modulates dc function in allospecific t cell stimulation in vitro and in vivo. we found that bm dcs cultured from c5ar-/-mice or treated with c5ar antagonist (c5ara, w54011) exhibited a less activated phenotype (producing significantly less il-12 and more il-10, in response to lps stimulation); both c5ar-/-and antagonist-treated dcs (lps stimulated) showed reduced capacity to stimulate naïve alloreactive t cells, as measured by ifn-γ production and thymidine uptake. as regards interaction in vivo, following i.p. administration of the c5ara-treated dcs into allogeneic mice for 10 days, ex vivo mixed lymphocyte reaction showed that cd4+ t cells from those recipients have reduced thymidine uptake, but increased il-4 production compared to that with untreated dcs. conversely, dcs treated with c5ar agonist (c5a) exhibited a more activated phenotype (producing more il-12 and less il-10) and were more potent in allospecific t cell stimulation. our findings demonstrate that murine bm dcs can express c5ar and c5a can be generated locally; c5a-c5ar interaction up-regulates murine bmdc activation and their allostimulatory capacity. thus, targeting c5a-mediated signal may be able to prevent allograft injury. role of tnfα in early chemokine production and leukocyte infiltration into heart allografts. daisuke ishii, 1 austin d. schenck, 1 robert l. fairchild. 1 1 immunology, glickman urological and kidney institute., cleveland clinic, cleveland, oh. objectives: the acute phase cytokines il-6 and tnfα are produced early during inflammatory processes, including wound healing and ischemia/reperfusion. the goal of this study was to investigate the role of these cytokines in the induction of early chemokine production and leukocyte infiltration into heart allografts. methods: c57bl/6 (h-2b), balb/c (h-2d), and balb/c.il-6-/-mice received vascularized syngeneic or complete mhc mismatched, a/j (h-2a), cardiac grafts. grafts were retrieved at different time points and total rna and tissue protein were prepared and analyzed by quantitative rt/pcr and elisa to test expression levels of tnf-α, il-6, cxcl1/kc, cxcl2/mip-2, and ccl2/mcp-1 in the grafts. anti-tnfα mab (500 ug) was given at the time of transplantation with or without anti-cd154 mab (200 ug on days 1 and 2). infiltration of cd4+, cd8+ t cells, neutrophils and macrophages was assessed by flow cytometry and immunohistochemistry. donor-reactive t cell priming to ifn-g producing cells in the recipient spleen was measured by elispot. results: expression of tnfα and il-6 mrna reached an initial peak at 3 hrs post-transplant and a second peak at 9-12 hrs with equivalent levels in both iso-and allo-grafts. the neutrophil and macrophage chemoattractants cxcl1/kc, cxcl2/ mip-2 and ccl2/mcp-1 reached peak levels at 9 hrs post-transplant in both sets of grafts and then declined to background levels. il-6 deficiency in the recipient or the cardiac allograft did not prolong allograft survival. in untreated mice, heart allografts were rejected at 8.6 ± 0.6 days after transplantation. anti-tnfα mab decreased neutrophil and macrophage chemoattractant levels 50% at 9 hrs post-transplant and subsequent neutrophil, macrophage and cd8+ cell infiltration into the allografts as well as extended graft survival to 14.1 ± 0.8 days. anti-tnfα mab also decreased the number of donor-reactive ifn-g producing cd8 t cells almost 90% on day 7 post-transplant. whereas anti-cd154 mab prolonged survival to day 21, administration of anti-tnfα and anti-cd154 mab delayed rejection to day 32 and resulted in the long-term (> 80 days) survival of 40% of the heart allografts. conclusions; these data indicate that anti-tnfα antibodies can delay donorreactive cd8 t cell priming and leukocyte infiltration into heart allografts rejection. as a conjunctive therapy, tnfa antibodies can promote long-term survival of the allografts. introduction recent evidence indicates that inflammation impairs immune regulation, yet the mechanisms behind this effect are not clear, in particular in regards to transplantation tolerance. in this study, we investigated the role of inflammatory cytokines, il-6 and tnfα, in transplantation tolerance induction. we first examined the impact of il-6 + tnfα on in vitro t cell alloimmune responses. t cells that were stimulated by allogeneic apcs in conditioned media harvested from lps-activated dcs proliferated more than apc-stimulated t cells that were cultured in conditioned media derived from non-lps-activated dcs. the ability of cd4 and cd8 t cells to respond to allogeneic apcs in the lps-activated conditioned media was significantly impaired by the addition of either anti-il-6 or anti-tnfα mabs. the addition of both mabs further diminshed t cell proliferation, indicating that il-6 and tnfα synergize to augment in vitro t cell allostimulation. in support of these findings, we noted reduced t cell proliferation during the mlr when t cells were cultured in conditioned media derived from either il-6-/-or tnfα-/-lpsactivated dcs. furthermore, these diminished responses was restored by the addition of recombinant il-6 or tnfα, respectively. to examine the in vivo implications of these findings, we employed a murine skin allograft model, with recipients that were either b6 wild type, il-6-/-or tnfα-/-. these groups were transplanted with a balb/c skin graft and treated with (or without) perioperative costimulatory blockade (ctla4 ig and anti-cd154). in the absence of immune modulation, all groups rejected balb/c skin allografts at a similar tempo (<12 days). in the presence of costimulatory blockade, il-6-/-recipients (median survival time, mst = 22 days, p =0.05) rejected their allografts at a slower tempo compared to wt (mst = 16 days) or tnfα-/-recipients (mst = 18 days). however, this response in il-6-/-recipients was further delayed by administering a tnfα inhibiting mab (mst = 60 days), indicating that synergy between il-6 and tnfα occurs in vivo and prevents the ability of costimulatory blockade to delay the onset of allograft rejection. conclusions we conclude that synergy between il-6 and tnfα augments t cell alloimmune responses and impairs the effects of costimulatory blockade to delay allograft rejection. abstract# 529 background: calcineurin inhibitors (cni) are involved in the development of post transplant diabetes mellitus (ptdm). changes in insulin secretion and sensitivity are central mechanisms involved in the development of ptdm. in addition alterations in endothelial function seem to be involved. the present study investigated the effect of cni's on these factors. methods: in a predefined sub-study of a previously published randomized trial it was aimed to compare the effect of cni treatment (n=27) with complete cni-avoidance (n=27) on insulin secretion and sensitivity as well as endothelial function. an oral glucose tolerance test and endothelial function investigation with laser doppler flowmetry was performed in 44 patients, 10 weeks and 12 months following transplantation. results: insulin sensitivity differed already 10 weeks posttransplant and was significantly better after 12 months in patients never treated with cni drugs (p=0.043). endothelial function was significantly correlated with insulin sensitivity (n=27, r 2 =0.22, p=0.013) at 10 weeks posttransplant, but not after 12 months (p=0.54). insulin secretion tended to be higher in cni treated patients both at week 10 and month 12 (p=0.068). conclusions: findings in the present study indicate that long-term cni treatment reduce insulin sensitivity which was associated with impaired endothelial function. in response to this peripheral insulin resistance a tendency towards a compensatory increase in insulin secretion was seen. these effects combined may indicate a future risk for premature cardiovascular disease in cni treated renal transplant recipients, but this hypothesis needs further study. group(n) ktx ptx(kptx) ecd re-tx pra(>20%) race(aa) low immunologic risk alem (113) overall pt, ktx, and ptx survival are 96, 92, and 86% at 16 months median follow-up. actuarial survival rates, initial length of stay, delayed graft function, steroid free rates, major infection, and incidence of ptld (1 ratg pt) were similar for alem and ratg groups, but treated acute rejection (ar) occurred in 17(15%) alem pts compared to 29(27%) ratg pts (p=0.03) and biopsy proven rejection (bpar) in 13(12%) alem pts compared to 23(21%) ratg pts (p=0.04). only 1 alem bpar has occurred after 12 months. total daily mmf doses were similar for alem and ratg groups at 3 months (1567±457mg vs 1670±508mg). neupogen use was greater in the alem group, 26(23%), than in the ratg group (14(13%), p=0.03). excluding pak, chronic allograft nephropathy (can) was observed in 19(17%) alem pts and 27(25%) ratg pts. (p=0.14). conclusions: alem and ratg induction both provide excellent 1 and 2 yr pt, ktx, and ptx survival. alem is associated with lower acute rejection rates and perhaps less can, but requires increased neupogen administration to help maintain mmf dosing. thymoglobulin dosing intensity and density: effects on induction efficacy ruth-ann m. lee, 1 adele h. rike, 1 background: alemtuzumab (campath 1h) has been used as induction therapy for kidney transplant recipients with acute rejection rates reported by us and others of 7 to 20% at one year. the histologic type of rejection and the time frame for occurrence after treatment with alemtuzumab have not been well established. this study is a retrospective single center review of acute rejection episodes of kidney transplant recipients treated with alemtuzumab induction with respect to the kinetics and histologic patterns of acute allograft rejection. methods: from 11/01/03 to 10/31/06, 416 kidney transplants were done meeting the inclusion criteria for this review. all patients had negative t and b cell flow cytometric crossmatches and received induction therapy with alemtuzumab 30mg iv intra-operatively, methylprednisolone 500 -750 mg during the first 24 hours, and were then maintained on tacrolimus (target level 5-7) and mycophenolate mofetil without steroids. all episodes of biopsy-proven acute rejection (ar) were reviewed. patients in pre-transplant desensitization protocols or with documented non-adherence with medications were excluded from the analysis. results: a total of 44 of 416 patients (10.6%) experienced ar during the study period, with a mean follow up of 28 months (range 14-48 months). of the ar episodes, 19 (43%) occurred within the first 3 months post-transplant, 13 (30%) occurred between 3-6 months, 5 (11%) occurred between 6-9 months, 1 (2%) occurred between 9-12 months, and 6 (14%) occurred more than one year post-transplant. of the rejection episodes within the first 3 months post-transplant, 9/19 occurred within the first 30 days. histologic analysis showed that 9/19 rejection episodes (47%) within the first 3 months included an antibody mediated component (6/9 within the first 30 days.) in contrast, 3/25 rejection episodes (12%) which occurred greater than 3 months post-transplant were antibody mediated. of the 12 patients with antibody mediated rejection, only 3 patients had panel reactive antibody (pra) levels > 25% at the time of transplant. conclusion: this large experience with alemtuzumab induction therapy with a steroidfree maintenance protocol, demonstrates that the majority of rejection episodes occur within the first 6 months post-transplant, with the largest fraction in the first 3 months. a significant number of early rejection episodes are antibody mediated and occur in unsensitized recipients. in a randomized, international, multicenter study, comparing the use of thymoglobulin (tmg) and basiliximab (bas) in recipients at high risk for delayed graft function (dgf) or rejection, tmg was associated with less acute rejection (15.6 % vs 25.5%, p=0.02) and a lower triple endpoint (rejection, death or graft loss, 20.6% tmg vs 33.6% bas, p=0.02) but not a significantly lower quadruple endpoint including dgf. the purpose of this study was to compare the efficacy of tmg and bas for induction stratified by donor source: standard criteria donor (scd), extended criteria donor (ecd) or donor with hypertension (htn). methods: retrospective review of data collected in the original randomized trial. data-capture limitations necessitated defining ecd as donor age > 60 or donor age between 50 and 60 with both a donor history of htn and donor renal insufficiency (history of atn or creatinine above 2.5 mg/dl during the 24 hours prior to organ recovery/start of cold ischemia time). results: 75 recipients received ecd kidneys [tmg n=40 (28.4%), bas n=35 (25.6%), p=ns]. outcomes are presented below. there were no differences in the rates of dgf between the groups examined. conclusion: standard and non-htn donor recipients had a tremendous benefit of tmg compared to bas with less acute rejection and death. contrary to the perceived niche of tmg in ecd recipients, tmg has its most beneficial effect in scd recipients and recipients of donors without htn at risk for acute rejection or dgf. evaluation we have changed our immnosuppressive protocol in abo-incompatible kidney transplantations and attempted to determine whether the changes in agents have resulted in better outcomes. we used tacrolimus(fk), mycophenolate mofetil(mmf) and methylprednisolone(mp) in immunologically high risk patients between 2000 and 2007. moreover, we performed splenectomy at the time of the transplant surgery in 117 patients (group 1) with aboincompatibilities between 2000 ansd 2004, and administered rituximab as an alternative to splenectomy in 38 patients(group 2) with abo-incompatibilities between 2005 and 2007. in this study, we compared the graft survival rates as well as the incidence of acute rejection in these two treatment eras. the graft survival rate at one year was 94% in group 1 and 97% in group 2 (p=ns). the graft was lost in one case of the 38 cases of group 2 due to insufficient doses of the immunosuppressive drugs. the incidence rate of acute rejection was 15% (18/117) in group 1, and 11% (4/38) in group 2 (p<0.01). there were no significant differences in serum creatinine level one year after transplanatation between two groups(1.5±0.3 in group 1 vs.1.4±0.4mg/dl in group 2). no serious adverse events associated with rituximab or splenectomty were encountered in either groups. in abo-incomaptible kidney transplantation, rituximab under fk/mmf combination as an alternative to splenectomy seems to yield an excellent result in terms of the incidence rate of acute rejection. introduction: the choice of immunosuppression in the elderly kidney transplant recipient remains unclear. the objective of this study was to compare outcomes with different t cell-depleting induction agents in the elderly. method: all solitary kidney transplant recipients over the age of 60 years that received induction therapy with either alemtuzumab or thymoglobulin from 2002 to 2007 were included in this unos analysis. overall graft survival, the risk of graft loss, and the risk of rejection were compared using kaplan meier, cox proportional hazards, and logistic regression, respectively. results: patients receiving alemtuzumab had a significantly lower 3-year graft survival (70.8%) than patients receiving thymoglobulin (79.3%), p=0.02) (figure) after adjusting for other risk factors, alemtuzumab had a higher risk of graft loss compared to patients given purpose. the aim of this prospective randomized study was the comparison of efficacy and incidence of adverse events in two induction therapy regimens (atg versus basiliximab) in patients receiving a dual immunosuppression. methods. 120 recipients of first or second deceased donor kidney transplants were prospectively randomized to receive either atg (fresenius) or basiliximab (novartis) as induction therapy. dual immnosuppression consisted of tacrolimus (astellas) and methylprednisolone. cmv prophylaxis was not applied on a regular basis. statistical analysis was performed with fisher's exact or chi-squared test, anova or mann-whitney u test, kaplan meier curves and log-rank test. results. patient characteristics of populations treated with atg versus basiliximab were similar concerning average age (48 years), gender and dialysis time prior to transplantation (78 vs. 88 months). average donor age and cold ischemia were also comparable (43 vs. 41 years and 833 vs. 852 minutes). the actuarial 5-year patient survival for the atg subpopulation is 91,7 % in comparison to 85 % in the basiliximab group (n.s.). analyzing graft survival after 5 years, rates of 88,3 % in atg patients compared to 75 % in the basiliximab group can be observed (n.s.). the incidence of acute rejection episodes was similar in both groups (atg: n=20 vs. basiliximab: n=16). 18 (30%) patients in the atg group and 20 (33,3%) in the basiliximab group showed a delayed graft function. serum creatinine was not significantly different at 1 and 5 years (atg: 1,4±0,6 mg/dl and 1,4±0,8 mg/dl vs. basiliximab: 1,3±0,7 mg/dl and 1,5±0,9 mg/dl). patients in the atg group had a higher rate of cmv infections (n=13 vs. n=3; p=0,05), whereas patients treated with basiliximab had significantly more hematological complications like anaemia, leukopenia and thrombocytopenia. conclusion. comparing induction therapy with atg and basiliximab, our data shows similar patient and graft survival rates with slightly better results in the atg group. patients treated with atg had a higher rate of cmv infections but less hematological complications. predicting cardiovascular events (cvd) and the varied effects of immunosuppressive medication on cvd risk factors requires an understanding of how traditional and nontraditional risk factors impact cvd after kidney transplantation (ktx). single-center studies have generally lacked statistical power and generalizability. registry studies have lacked sufficient data on cvd risk factors. the port project is creating a multicenter international database of ktx recipients with the primary objective of developing risk prediction models for post-transplant cvd. as a preliminary data assessment, an analysis was done on 19,669 ktx from 1990-2007 from 11 transplant centers representing 5 european centers, 4 north american centers, and 2 centers from asia/oceania. all data were extracted from preexisting databases at each individual transplant center and processed into the consolidated port database. 1,131 major adverse cardiac events (mace) were identified, defined as non-fatal or fatal myocardial infarction, cardiac abstracts arrest, and sudden death. the mace-free survival curves by participating center are shown in the figure. in this preliminary analysis, the overall one-year cumulative incidence of mace was 2.2%, ranging from 0.2% to 4.4% across centers; and the five-year cumulative incidence was 5.8%, ranging from 0.6% to 13.5%. the prevalence of diabetes pre-transplant varied from 10% to 47% across centers. in cox proportional hazards models adjusted for age, gender, race, donor type, transplant center, and reported history of diabetes, hypertension (htn), and ami, patients with a reported history of ami had a 112% increased risk (79%-152%, p<0.0001) for mace. for the final analysis, the definition of mace will be expanded to include major revascularization events. the final port database will be used to develop and validate an equation to predict mace and other health outcomes of interest, after accounting for differential cvd risk factors internationally. abstracts to center. the most commonly used bp medications were beta-blockers, followed by ccbs, and both were used with the same frequency at 1 and 6 months. in contrast, the percentage of patients on an acei or arb more than doubled between 1 and 6 months, suggesting reluctance to use these agents early after tx (a practice not necessarily evidence-based); however, more than 70% of subjects did not receive acei/arb therapy even at 6 months. fewer than half of all patients received aspirin, including only 60% with dm and/or cvd. similarly, only half with dm and/or cvd received a statin at 1 and 6 months. these data indicate current management of ktx recipients fails to utilize optimal cvd risk reduction measures in a timely fashion, perhaps missing an opportunity to reduce long-term morbidity and mortality from cvd in this at-risk population. background: cardiovascular (cv) risk reduction has been a primary reason for pursuing early corticosteroid withdrawal (ecswd). to date, actual cardiovascular event data (cve) (rather than cv risk) has not been reported for ecswd. therefore, we analyzed and compared actual cv events (cve) and cv-related survival in ecswd (≤7 days) and chronic corticosteroid (ccs) pts. methods: cve and heart failure (hf) data were prospectively collected. cve were defined as sudden death, myocardial infarction, angina, and cerebrovascular accident/ transient ischemic attack. hf events were defined as pulmonary edema or hf diagnosis. conclusions: rtx recipients receiving ecswd experienced: 1) fewer cve and 2) a trend toward overall better pt survival. these differences in cve and pt survival do not present until at least 3 yrs ptx. and therefore require long term followup to become evident. abstracts immunohistochemistry was performed for cd4 + and cd8 + cells. results were compared with those of the patients on maintenance is (gr-is n=29) and the liver tissue from normal subjects (gr-normal n=11). results: the follow-up time in gr-tol was longer than that in gr-is.(gr-tol and gr-is: 121m and 52m, p<0.01) in gr-tol, typical features of neither acute nor chronic rejection were observed following banff criteria. the extent of graft fibrosis in gr-tol, however, was greater, than those in gr-is and gr-normal (gr-tol, gr-is and gr-normal ; 1.6, 0.9 and 0 (ishak's modified staging )(gr-tol vs. gr-is, gr-is vs.gr-normal p<0.01). each number of cd4 + and cd8 + cells in graft infiltrates was increased in gr-tol, compared with that in gr-normal, but equivalent with that in gr-is (cd4 + gr-tol, gr-is and gr-normal ;14.6,10.6 and 5.3 cells/field, gr-tol vs.gr-normal p<0.05, gr-tol vs.gr-is ns / cd8 + gr-tol, gr-is and gr-normal; 27.6, 26.3 and 10.5 cells/field gr-tol vs.gr-normal p<0.01, gr-tol vs.gr-is ns). conclusions+discussion : in tolerant graft after pediatric living-donor ltx, neither acute nor chronic rejection was observed, but fibrosis developed. because of the similar extents of cd4 + and cd8 + cells infiltrates and different follow up time between tolerant and immunosuppressed patients, it remains questionable whether fibrosis in tolerant graft is antigen-dependent. serial protocol biopsy before and after starting weaning is will detect fibrosis early, and observing whether reintroduction of maintenance is reverses fibrosis in that case will answer this question. operational tolerance may not always guarantee intact graft morphology. development of "operational tolerance" after pediatric liver background : in the setting of our pediatric living-donor liver transplantation (ltx), 15% of all the patients (significantly higher proportion, compared with those of other transplant centers) achieved complete withdrawal of immunosuppression (is), which is reffered to as "operational tolerance". nonetheless, some patients encountered rejection while they were undergoing weaning from is. it is,therefore,essential to identify and characterize the differences that will enable patients in these two distinct populations to be distinguished reliably. methods: the study groups consisted of group tolerance(gr-tol) in which 88 patients are successfully weaned off from is, and group rejection (gr-rej) in which 22 patients experienced clinically evident rejection during or after weaning process. the correlation between the clinical outcome (success or failure of weaning is) and following parameters was assessed ; donor/recipient age, donor/recipient gender, abo compatibility, hla mismatch, graft size, early (<1month) rejection episode and initial immunosuppression.results: there was no difference between gr-tol and gr-rej with respect to donor/recipient age (gr-tol and gr-rej;32y and 32y ns/35m and 26m ns), or donor/recipient gender (gr-tol and gr-rej (female);60% and 50% ns/60% and 73% ns). abo compatibility did not differ between the two groups(gr-tol and gr-rej (i dentical:compatible:incompatible);63%:27%:10%and 55%:27%:18% ns).the presence of hla-b mismatch was more frequent in gr-tol than that in gr-rej (gr-tol and gr-rej;95% and 76% p<0.05 ), while the presence of hla-a or dr mismatch did not affect success or failure of weaning is(hla-a gr-tol and gr-rej;73% and 76% ns, hla-dr ;83% and 82% ns). graft size did not differ between the two groups(gbwr gr-tol and gr-rej;2.9% and 3.2% ns). the patients in gr-rej experienced early rejection more frequently than those in gr-tol(gr-tol and gr-rej;17% and 50% p<0.05). mean trough level of tacrolimus within 7 days after ltx was compatible between the two groups (gr-tol and gr-rej;11ng/ml and 11ng/ml ns). conclusions: development of operational tolerance after pediatric ltx was associated with the absence of early rejection and the presence of hla-b mismatch between donors and recipients. auxiliary partial orthotopic liver transplantation (apolt) in children with fulminant hepatic failure. patients with fulminant liver failure (fhf) who undergo auxiliary partial orthotopic liver transplantation (apolt) have a chance to come off immunosuprresion (isp) when the native liver regenerates. it may be most beneficial for children with fhf; however, the literature regarding its use in children has been limited. from the beginning of the pediatric liver transplant program at our institution, 31 patients underwent liver transplantation for fhf. of those, 7 received apolt and the remaining standard liver transplantation (olt). seven children (age 8months to 8 years) who received apot (apolt group) were compared to matched control group of 11 patients (olt group). since apolt was offered routinely at out instituting since 2005, 6 of apolt cases were done since 2005. in apolt group, either left lateral segment or left lobe graft was used. recipients left lobe was removed in all cases. in olt group, 7 received whole liver graft and 4 received partial liver graft. all native livers showed submassive to massive necrosis at the time of transplant in pathology. all children (100%) in apolt group are currently alive with a median follow up of 761 days (range 116-4139 days) where 8 (72%) patients are alive in olt group (median follow up 1724 days). six of 7 children in apolt group (87%) showed native liver regeneration. first four apolt recipients (57%) are currently off isp with fully regenerated native liver. two of those patients developed complete atrophy of the graft liver, one underwent graft removal due to sepsis caused by severe rejection. one remaining patient who is off isp is displaying progressive atrophy of the transplant liver. incidence of acute rejection was 57% (4/7) in apolt group vs 30% (3/10) in olt group. other postoperative complications included hepatic artery thrombosis (hat) (n=1), bile leak (n=1), bilary structure (n=1) and bowel obstruction (n=1) in apolt group, hat (n=1), bile leak (n=2), bowel perforation (n=2), chylothorax (n=1) and aplastic anemia (n=2). median posttransplant length of stay was 15 days in apolt and 20days in olt group. conclusions: apolt was safely performed in children with fhf. significant proportion of recipients displayed native liver regeneration and came off immunosuppression. natural killer cell dysfunction in pediatric acute liver failure. nada yazigi, 1 greg tiao, 1 alexandra filipovich, 2 john bucuvalas. 1 in pediatric patients, indeterminate acute liver failure (alf) accounts for ∼50% of all cases, and carries a particularly poor prognosis without transplantation. evidence exists to suggest that acute liver failure may reflect a disproportionate immune response to a common stimulus. nk cells comprise a central component of the innate immune system. we hypothesized that nk cell dysfunction (innate or secondary to an antigenic insult) plays a pathologic role in indeterminate alf. we reviewed peripheral nk cell function in a series of 15 consecutive children cared for at cincinnati children's hospital, who met criteria for indeterminate alf as defined by the pediatric alf study group. peripheral blood testing was carried for nk cell number, cytolytic function and perforin and granzyme activity as part of our clinical alf protocol. seven of fifteen patients had nk cell dysfunction. only the severity of cholestasis was statistically higher in the nk cell dysfunction group. there was no statistical difference between the 2 groups with respect to age, inr, or peripheral blood cell counts. 3 of seven patients with nk cell dysfunction died in contrast to none of eight in the normal nk cell group. of the 3 patients with nk cell dysfunction who eventually received a liver transplant, 2 had severe early recurrence of chronic hepatitis in the graft at a year follow up. those outcomes are in sharp contrast to the group with no nk cell dysfunction where 7 patients needed transplantation, but all had no complications both on short or long term follow up. we documented nk cell dysfunction at the time of alf diagnosis in 7/15 pediatric patients with indeterminate alf. this subgroup of patients was found to have higher: mortality, risk of infections, as well as recurrent disease in the graft. our findings suggest that nk cell dysfunction is involved in the pathogenesis of indeterminate alf. as such, it could therefore be a prime target for therapeutic intervention, with goals to rescue the patient from liver failure and /or to improve post-transplantation outcomes. background hrs is a reversible renal failure which occurs in pts with advanced liver disease and portal hypertension and is characterized by a marked decrease in gfr and rpf in absence of other identifiable causes. vasodilation theory is currently the most accepted hypothesis to explain the pathogenesis. in decompensated cirrhotics, probability of developing hrs is 8-20%/yr and increases to 40% at 5 yrs. ideal treatment is ltx. however, there is an urgent need for effective alternative tx to increase surv chances for pts until ltx can be performed. interventions that have shown some promise are vasoconstrictors in splanchnic circulation and tips. main objective was to compare efficacy of two different regimens (albumin/terlipressin resp hes/terlipressin both w/ wo midotrine) against tips whereas grf was considered as primary efficacy endpoint. pts/tx dx of hrs was based on criteria, as proposed by international ascites club. only pts with esld on the waiting list for ltx were eligible to be enrolled. pts were assigned to tx arms and randomized w/wo midotrine. volume/vasoconstrictor tx lasted for 10d, mitodrine was continued; follow up for 90d. results iia (albumin/terlipressin); iib (hes/terlipressin); iic (tips) discussion combination of volume expansion/vasoconstriction improved effectively gfr in pts with hrs. use of albumin shows no advantage compared to (cost effective) hes. although a marked improvement was observed during iv-treatment, renal fct deteriorated upon treatment withdrawal whereas pts with continued mitodrine showed superior long term outcome. we analyze our single institution experience to quantify the long-term incidence of renal failure based on month 3 gfr compared to subsequent determinations. methods: this is an irb approved retrospective review of the prospectively maintained database of lt recipients. exclusion: patients on renal replacement therapy (rrt) at time of transplant, combined liver kidney, fulminant hepatic failure, and <1-year follow-up. gfrs (i 125 iothalamate glofil method) were measured at initial evaluation (ie), month 3 (m3), year 1 (y1), 2 (y2), 5 (y5), 10 (y10), and 15 (y15). patients were grouped by gfr >80 (g1), 60-80 (g2), and <60 (g3). ie and m3 gfr were used as starting points for longitudinal analyses. paired data analysis for ie, m3, y5 and y10 was also performed. renal failure was defined as gfr <30, received kidney transplant, on dialysis, or on kidney transplant list. results: 592 liver transplant patients were reviewed between 1985 and 1999. paired glofil data was available for the y5 and y10 analysis in 114 patients. m3 gfr correlated more with long-term renal function (p<0.0024). g1 demonstrated largest reductions in gfr over time. g2 and g3, when corrected for patients that got kidney transplantation and rrt, demonstrated progressive reduction in gfr. g3, g2, and g1 were statistically significant (p<0.001 wilcoxon two-sample test). this study clearly demonstrates progressive decline in gfr continuing out to 15 years after liver transplantation. m3 gfr correlates better with long-term renal function compared to ie gfr (not truly reflective of renal function at time of lt). if m3 gfr <60, data showed a high rate of renal failure in our paired data analysis by y5 (p<0.0024). by correcting for patients with renal failure, the previously reported stability in gfr between y1 and y5 is not seen. analysis of grouping demonstrates that g1 patients at m3 have lower incidence of renal failure >10 years after lt. g2 and g3 patients will be at higher risk for renal failure each year after transplantation. introduction: calcineurin inhibitors have demonstrated efficacy in liver transplantation. however, they have a potential to impair renal function. delayed tacrolimus (tac) administration may reduce the risk of renal dysfunction. methods: a prospective study included liver transplant pts randomised to delayed introduction of tac (day 5) + daclizumab (dac) (group a) or to immediate tac administration (group b). in both groups tac t0 was 10-20 ng/ml until week 4 and 5-15 ng/ml thereafter. mmf was given at 2 g/d for 2 months, and corticosteroids (cs) at standard doses. pts with a serum creatinine (scr) > 180 µmol/l at 12 hours (h12) were excluded. the primary endpoint was the rate of pts with a mean scr > 130 mmol/l at month 6. month 24 results are presented. results: 207 pts were randomised. baseline characteristics were similar. at month 6, mean tac t0 was 9.6 (group a) and 11.2 ng/ml (group b median follow-up post-tx was 8 years (5-21). most frequent tx indications were alcoholic (34%) and hcv (10%) cirrhosis. amdrd glomerular filtration rate (gfr) was < 60 ml/min/1,73m 2 in 11% (bt), 46% (1m), 48% (1y) and 55% (5y) of the patients. changes in gfr were then compared according to the immunosuppressive protocol: -group "cni+mmf" = a calcineurin inhibitor (cni) + mycophenolate mofetil (mmf). -group "cni" = a cni without mmf. in this group, some patients received only cni and some cni + azathioprine. there was no difference between those 2 sub-groups, neither on rf nor on cni doses. all those patients were thus pooled. in both groups, gfr decreased from bt: -14% in "cni+mmf" vs -25% in "cni" at 1m (p=0.06), -14% vs -29% at 1y (p=0.03), and -12% vs -33% at 5y (p=0.02). although their mean gfr bt was lower (86 vs 97 ml/min/1.73m 2 , p=0.0002), the decrease in rf in "cni+mmf" patients was less severe. nearly 50% of the patients had renal insufficiency in the 5 years following liver tx. the reduction in the gfr is less pronounced in patients treated with mmf even if they were significantly more at risk bt. except at 1m, there was no difference in cni doses between the 2 groups, suggesting that the sustained lower decrease in rf observed in "cni+mmf" may not be only explained by a cni dose reduction. acute rejection is a complex biologic process involving multiple cell types, cytokines and chemokines/chemokine receptors. we hypothesized that an mrna panel that included genes implicated in the anti-allograft response would distinguish allografts undergoing acute rejection from normal allografts with a high degree of accuracy. we tested this hypothesis by measuring levels of urinary cell mrna and peripheral blood cell mrna for cell surface proteins cd3, cd20, cd25, cd103, and ctla4; chemokines/ chemokine receptors ip10, mig, cxcr3; cytotoxic attack molecules granzyme b(gb) and perforin, and immunoregulators foxp3, tgf-beta1 and il-10. gene specific primer pairs and probes were used in pre-amplification enhanced real time quantitative pcr assays to measure mrna and transcripts for 18s rrna. for each cell source, we used logistic regression to identify a linear function of up to 5 log-transformed measures that would distinguish biopsies of ar patients from those of stable transplant patients. our study demonstrates that molecular signatures developed using urinary cell levels of just 5 genes (signature 1, urinary cell levels of mrna for ctla4, foxp3, gb, cd3, and mig), or a combination of 3 urinary and blood cell levels (signature 3, urinary cell level of ctla4 mrna and peripheral blood cell levels of cd103 and ctla4) differentiate ar from stable biopsies with 100% sensitivity and 100% specificity. blood cell levels alone are also informative, but less so. we conclude that molecular signatures, developed from noninvasively ascertained mrna profiles of urinary cells/ peripheral blood cells, predict acute rejection with extraordinary accuracy. clinical trials to validate the predictive value of these signatures are worthy of pursuit. we have described the association of cd20+ b cell infiltrates in renal transplant (tx) biopsies (bxs) with acute cellular rejection (acr) and tx dysfunction (dysfx). we have also found metabolically active plasma cells (pcs) staining for s6 ribosomal protein (s6rp) within these txs. herein, we report the significance of cd138+ pcs in rejection (rj) and evaluate the impact of cd20, cd138, and s6rp on long term tx fx by calculated creatinine clearance (crcl). we studied 46 tx bxs from 32 pediatric (ped) patients (pts) who were bxed for suspicion of rj from nov 2001 to nov 2004. pts were given daclizumab and maintained on prednisone, mycophenolate mofetil, tacrolimus or cyclosporine. immunohistochemical staining and quantification for cd20, cd138, s6rp, and c4d were performed under 500x light microscopy. bxs were classified by modified banff 97 criteria. crcl was followed 2 yr post-bx. cd138+ pcs were associated with c4d-negative acr (p=0.0002) but not antibody mediated rj (amr, p=0.82). roc analysis confirmed >5 cd138+ cells/hpf strongly associated with acr, yielding 89% sensitivity, 83% specificity, correctly classifying 84% and comprising total roc area 0.92 (95% ci 0.82, 1). higher cd138 counts at bx correlated with worse tx fx (fig 1) . a univariate regression model showed that cd20, cd138, s6rp and time were associated with a decline in tx fx at bx. multivariate model showed that cd20, cd138, and time had the main effects on crcl decline, with s6rp dropping out. all patients regardless of rj status had a 16 ml/min/1.73 m 2 crcl decline exerted by time (p=0.004). pts with cd20 had an additional sustained 19 ml/min/1.73 m 2 crcl decline seen 2yrs post-bx (p=0.03). pts with cd138 also had 19 ml/min/1.73 m 2 crcl decline at bx (p=0.03), but there was an interaction between time and cd138 that negated a sustained effect (p=0.04). this study identifies a numerical threshold of >5 cd138 cells/hpf that is associated with acr and tx dysfx. infiltrating cd20 cells had the greatest, sustained effect on tx dysfx. we conclude that cells of the b lineage, particularly cd20, play a key, but undefined, role in acr. intragraft there is now evidence that foxp3+ cells are not indicators of tolerance, since foxp3 is also increased during acute rejection. however, it is unknown whether foxp3+ cells are present during chronic antibody mediated rejection. moreover, the relative balance of regulatory, effector and cytotoxic pathways in chronic vs. acute injury has yet to be explored. here we addressed this issue. intragraft regulatory, effector and cytotoxic transcriptional profiles were analysed within renal transplant biopsies (n = 43) classified (banff 2005) as displaying normal histology, chronic calcineurin inhibitor toxicity (cnitox), chronic antibody mediated rejection (camr) and acute cellular rejection (acr). granzyme b, tbet and foxp3 mrna were measured by quantitative pcr and foxp3 -positive cells were additionally quantified in graft biopsies by immunohistochemistry. distinguishing mrna profiles were analyzed in the peripheral blood (n = 26). our data show that foxp3 mrna is increased not only in acr (p<0.0001) but also in camr (p<0.05). expression of foxp3 mrna correlated tightly with the density of foxp3 protein-positive cells by immunohistochemistry (spearman r = 0.71; p < 0.0001); foxp3+ cells were found in aggregates and within tubules. moreover, graft cytotoxic, effector and regulatory pathways were all found to be active in chronic as well as acute graft injury. significant increases in granzymze b, tbet and foxp3 mrna were observed in camr, cni-tox and acr compared to normal histology (p<0.0001, p<0.001 or p<0.05). however, differences in the relative contribution of each pathway were evident, with significant accumulation of foxp3 mrna predominating in acr and granzyme b predominating in camr. thus, camr can be distinguished from both acr and cni-tox by an unfavorable intragraft granzyme b/foxp3 mrna ratio (p< 0.05). interestingly, this ratio was reversed in the blood, suggesting different migratory patterns for regulatory and cytotoxic cells between the blood and the graft.our data thus confirm that intragraft and peripheral blood foxp3 accumulation is also a feature of camr of kidney grafts. moreover, camr can be distinguished from other graft injury types based on its intragraft or blood cytoxicity/regulatory profile. survival of solid organ grafts depends on life long immunosuppression which results in increased rates of infection and malignancy. induction of tolerance to allograft would represent the optimal solution for controlling both chronic rejection and side effects of immunosuppression. we previously showed that operational tolerance after kidney transplantation could occur in some patient. here, the potential of high throughput microarray technology allowed us to study the peripheral blood gene expression profile associated to operational tolerance and chronic rejection in a cohort of human kidney graft recipients (n=26). microarrays were used to compare the gene expression profile of pbmc from patients with chronic rejection and drug-free operationally tolerant recipients. results have been treated using a classical statistical and a non-statistical analysis based on the identification of key leader genes associated respectively to chronic rejection and operational tolerance, either as those mostly changing their expression or having the strongest interconnections. 343 differentially expressed genes were identified between operational tolerant patients and patients with chronic rejection. abstracts defined as missing > 10% of prescribed doses on mam and mems, and >2 sd among consecutive blood serum levels. results: participants were 28 transplant patients (m = 14.11 +3.38 years old, 75% male, 71.4% caucasian). on the mam, 67.9% of the patients acknowledged some non-adherence but minimized how many doses they missed. using mems technology, 90% had some non-adherence and specifically, 23.8% of the participants missed doses and only 56% of their doses were taken within the allowable time frame. using > 2sd criteria for blood serum levels, 42% of the participants were considered non-adherent. non-adherence worsened with years since transplant. more missed (r = .59, p = .001) and late doses (r = .59, p = 0.04) on the mam and >2 sd among blood serum levels (r = .83, p = .001) was associated with higher incidence of acute rejections. adherence data was examined for patients with documented acute rejections (n = 12). sensitivity and specificity of each detection method was also examined. the mam and sd detection methods each identified non-adherence in 67% of the patients with acute rejections; mems did not identify any additional non-adherent patients. only 25% of the patients with acute rejections were identified consistently by all three adherence detection methods; all patients with acute rejections were identified by at least one method. discussion: non-adherence worsening with time since transplant and was associated with acute rejections. since no single method of detecting adherence identified all the patients with acute rejections, multi-method adherence assessments should be used to accurately capture patients who are non-adherent. non na is a leading cause of allograft loss and results from multiple factors. locus of control (loc) and beliefs regarding health have been associated with adherence in other populations. 51 randomly chosen ktr's were interviewed using a confidential questionnaire administered by an outside investigator that included questions regarding loc, health beliefs and self-efficacy. the population was 55% female, 92% black, 15% hispanic, 71% deceased donor kidney, 47% diabetic, 49% greater than high school education, 36% employed, 30% married or cohabiting, 63% income <20k per year, 65% insured by medicaid. mean age 47.8±13.5 yrs, time on dialysis 72.8±51 mos, months since transplant 23.9±14.3, total meds 7.3±3.0. by pearson correlation, non-adherence (na), defined as "having missed doses of immunosuppression over the preceding 3 months", was not correlated with race, gender, income, type of insurance, age, marital status, type of txp, mos on dialysis, time since transplant, or number of medications. na was correlated with higher education level (r=0.4, p=0.006), current employment (r=0.3, p=0.05), and knowledge of most recent creatinine value (r=0.35, p=0.015). na was associated with concerns regarding prednisone (long term effects, dependency) r=0.33, p=0.018, feelings of greater personal control over illness (r=0.36, p=0.011), and inversely correlated with powerful others loc (feeling that one's health is dependent on other people), r=-0.31, p=0.029 and belief in the necessity of medication for maintenance of transplant health, r=-0.3, p=0.031. we conclude, in our population of inner-city patients: 1. na is not associated with standard demographic factors including income, race and gender. 2. contrary to findings in other populations, na is associated with higher education and current employment. 3. na is associated with knowledge about creatinine value, concern regarding long-term effects of prednisone and disbelief in the importance of transplant medications. 4. na is associated with feelings of personal control and feeling that powerful others (e.g. health care providers) are not of high importance in the outcome of illness. 5. education programs designed to address na in this population should be targeted towards altering negative beliefs regarding medications and stress the importance of partnering with the transplant team for optimal long-term outcome. purpose: the present study aimed to prospectively examine the relationships among nonadherence, health-related quality of life (hrqol), and family factors in adolescent kidney, liver, and heart transplant recipients. method: 68 adolescent transplant recipients aged 11 to 20 years (m = 15.8, sd = 2.5; 44% female; 57% kidney, 25% liver, 18% heart) and their parents participated. at baseline and 18-month follow-up assessments, adolescents and their parents independently completed phone interviews assessing self-/proxy-reported medication adherence, hrqol, and family cohesion and conflict. medical record reviews were conducted to obtain current medications, immunosuppressant drug assays, and clinical outcomes in the past year (i.e., rejection episodes, hospitalizations, graft loss). results: at baseline, adolescents classified as nonadherent based on self-report and tacrolimus standard deviation (sd) reported significantly lower general health perceptions (f(3, 64) = 3.63, p < .05), self-esteem (f = 4.58, p < .01), mental health (f = 3.09, p < .05), and behavior hrqol (f = 2.75, p < .05) compared to adolescents classified as adherent. similarly, parents of adolescents classified as nonadherent reported significantly lower physical functioning (f = 3.18, p < .05), self-esteem (f = 5.85, p < .01), and behavior hrqol (f = 2.82, p < .05) for their adolescents. family conflict was correlated with adolescent report of behavior (r = -.49, p < .01), physical functioning (r = -.33, p < .01), self-esteem (r = -.42, p < .01), and mental health hrqol (r = -.34, p < .01). family conflict was correlated with parent report of behavior (r = -.46, p < .01) and physical functioning (r = .24, p < .05). improvement and deterioration in hrqol from baseline to 18-month follow-up is currently being examined. it is expected that increased family conflict and decreased medication adherence will be associated with deteriorations in hrqol. the interrelationships between medication adherence, family conflict, and hrqol domains such as self-esteem and mental health suggest that interventions targeting these domains may result in improvements in medication adherence behavior. the use of cam in general is associated with non-disclosure by patients to physicians. 51 randomly chosen ktrs were interviewed using a confidential questionnaire administered by an outside investigator, including questions on cam usage, whether it was doctor-recommended, and whether the patient disclosed use. cam was defined as ingestion of herbal or other preparations, use of mind-body techniques or manipulation of the body for healing by someone not an allopathic medical provider. use of vitamins and spirituality were excluded. the population was 55% female, 92% black, 15% hispanic, 71% deceased donor kidney, 47% diabetic, 49% > high school education, 36% employed, 30% married or cohabiting, 63% income <20k per year, 65% insured by medicaid. mean age 47.8±13.5 yrs, time on dialysis 72.8±51 mos, months since transplant 23.9±14.3, total meds 7.3±3.0. 45% of patients (n=23) used cam. by pearson r, cam use was correlated with na to immunosuppressants, p= 0.041, r= 0.4, blood sugar-lowering medications, p= 0.003, r= 0.57, and cholesterol lowering medications, p= 0.0001, r=0.84, worries about long-term effects of medicines, p=0.02, r= 0.324, belief that doctors place too much trust in medication, p=0.032, r=0.3, and that natural remedies are safer than medicines, p= 0.034, r=0.3. cam use was inversely related to belief that health depends on allopathic medicines, p=0.014, r= -0.341, medicines protect from worsening disease, p=0.048, r= -0.28, and that following doctors orders is the best way to stay healthy, p= 0.04, r= -0.29, and that having a kidney transplant makes them feel happy, p= 0.005, r= -0.39. we conclude, in our population of inner-city patients: 1. use of cam is correlated with medication non-adherence. 2. patients who use cam are more worried about long term effects of medication, believe that natural remedies are safer than medications and that doctors place too much trust in medication. 3. patients who use cam do not believe that their health depends on allopathic medication, that medicines protect from worsening disease, or that following a doctor's orders is the best way to maintain optimal health. 4. patients who use cam are less happy with their kidney transplant. 5. disussing cam use and motivation for use is important in the transplant clinic and may alert the provider to possible risk for non-adherence. by multivariate cox analysis the risk of tg related to the presence of hla-iiab death censored graft loss occurred in 4.6% of patients without tg and in 38.4% of patients with tg (p<0.0001) hla-iiab are associated with higher risk of tg and reduced graft survival. furthermore, the risk of tg and its prognosis relate to the level of hla-iiab quantitated in a solid phase assay term survival of cardiac allografts in wild-type mice by alloantigen (alloag)-specific foxp3 + cd4 + cd25 + natural regulatory t (nt reg ) cells. guliang xia, 1 jie he methods: fresh naive cd4 + cd25 + nt reg were isolated from congeneic b6.pl mice via automacs and enriched for alloag specificity by in vitro culture with either anti-cd3/ cd28-coated dynabeads (d0-11), then donor bone marrow-derived dendritic cells 8% (d+16) for dc/beads-expanded nt reg , while total fold of expansion of nt reg remained similar (24.8∼30.2 for beads/dc-or 5.2∼7.1 for dc/beads-expansion) regardless of the presence or absence of tgf-β. introducing ra (100 nm) into bead/dc-based, tgf-β/ il-2-conditioned culture resulted in marginal improvement with 28.9% (d+18) nt reg being foxp3 + . in mlr assays, nt reg expanded with tgf-β/il-2 exerted more potent suppression than cells conditioned with il-2 alone. in vivo, beads/dc-expanded, tgf-β/ il-2-conditioned nt reg synergized with transient host t cell-depletion (anti-thy1.2 mab i.p. 50 µg at d-3 & 25 µg on d+2) in c57bl/6 mice to suppress balb/c heart allograft rejection with 57.1% (n=7) and 100% (n=10) allografts surviving over 100 days when 4x10 7 or 8x10 7 cells/mouse were injected immediately post-transplant, respectively. anti-thy1.2 treatment alone led to only 18.8% long-term survival. infused nt reg survived long-term (5.8 % circulating t cells (8x10 7 cell dose) or 1.2 % (4x10 7 cell dose) at d+7 post-transplant) and expressed high level foxp3 (40∼60%) in vivo. long-term surviving allografts showed characteristics of 'acquired immune privilege' with cellular infiltrates that were foxp3 + , tgf-β + , il-10 + and indoleamine 2,3-dioxygenase (ido) + , although signs of mild to moderate chronic rejection were still evident conclusion: t-bet deficiency results in up-regulation of il-17 expression in addition to th2 associated cytokines resulting in acceleration of chronic rejection despite profound deficiency of ifn-γ. t-bet deficiency may contribute to the alloimmune responses independent of ifn-γ by in situ hybridization, tir8 mrna level was higher (p<0.05) in cortical tubuli, glomeruli, perivascular and peritubular areas of kidney grafts at 1, 3 and 6-7 d post-tx, than in naive kidneys. to assess how local expression of tir8 affects the outcome of kidney grafts, we transplanted tir8 -/-b6x129 kidneys into dba/2 mice. most (83%) recipients of tir8 -/-kidneys rejected their grafts with a median survival of 9.5 d (n=12, p<0.05 vs wt) and had more severe graft dysfunction (bun levels) at day 1, 3 and 6-7 days post-tx, than recipients of a wt allograft (p<0.05) opticept trial: efficacy and safety of monitored mmf in combination with cni in renal transplantation at 12 months. r trough-based dose adjustments were made in the mmf cc arms. antibody induction and/or corticosteroids were administered according to center practice. primary endpoints were the proportion of patients with treatment failure (biopsy-proven acute rejection [bpar], graft loss, death), and mean percent change in calculated glomerular filtration rate (gfr; nankivell equation) at 12 months. safety endpoints were incidences of adverse events (aes) and serious aes baseline characteristics did not differ among treatment groups with living donors accounting for approximately 50% of grafts. 82% received tacrolimus (tac) and 18% cyclosporine (cya): cni doses and levels were significantly lower in group a. mmf doses were greater in cya-treated subjects in all groups cya treated patients and in group a (p=0.08); stability of renal function over time was greatest in group a. despite higher mmf doses in group a (p<0.001) at most time points, significantly fewer mmf withdrawals occurred in group a vs. groups b and c. conclusions: a concentration-controlled mmf and reduced level cni regimen is not inferior to that of fixed-dose mmf and standard-dose cni as regards bpar and other end points. this regimen facilitated higher mmf dosing without an overall increase in adverse effects, and with a trend toward preservation of kidney function versus standard-dose cni regimens comparison at one year of interstitial fibrosis (if) by automatic quantification in renal transplant recipients with cyclosporine (csa) discontinuation and sirolimus (srl) introduction introduction: we previsouly reported the clinical results of a multicentric study showing that csa conversion to srl at week (w) 12 is associated with a significant improvement in renal function. using routine renal biopsy (rb) performed at w52 during this study routine rb was performed at w52. for each rb, a section was imaged using a colour video camera and analyzed by a program of colour segmentation which automatically extracts green colour areas characteristic of if. results were expressed as percentage of if and grade according to banff classification. results: male donor gender was associated with higher if (30±2% vs. 23±2%, p =0.03). if was numericaly higher in patients who had experienced acute rejection (33±4%, n = 18 vs. 26±1%, n=99, p=0.06) there was a positive correlation between renal function and the percentage of if on rb (p=0.004). despite significant improvement of renal function at w52 in the srl group intent to treat (n=117) mean if (%) grade i (%) grade ii (%) grade iii (%) sirolimus (n=58) conclusion: despite significant improvement in renal function after csa to srl conversion at 3 months, we found no difference of if on rb at w52. the observed improvement of renal function may be due to a hemodynamic effect. a longer delay may be necessary to observe histological improvement. the higher if score than the one previously reported by others may be explained by the use of expanded criteria donors abstract# 528 effects of cni or mmf withdrawal on carotid intima media thickness in renal transplant recipients methods: we included 119 stable renal transplant patients on cni-based immunosuppression, including steroids (10 mg/d) and mmf (2g/d), who were randomized to mmf-withdrawal (group a: csa-auc 3000 ng*h/ml) or cni-withdrawal (group b: auc-mpa 75 µg*h/ml). patients were treated for traditional risk factors according to stringent predefined targets. ambulatory bloodpressure (abpm), lipids, estimated creatinine clearance (mdrd) and imt were measured at baseline and after 12 months. results: groups were comparable with respect to demographic characteristics, immunological profile, renal function, systolic and diastolic bloodpressure and lipids. mean duration of follow-up was 17.4±5.5 months. only 1 patient (1.3%) in group b and 3 patients (3.8%) in group c experienced acute rejection despite adequate exposure (p=0.31). imt did not change final renal function outcomes from the spare-the-nephron (stn) trial: mycophenolate mofetil (mmf)/sirolimus (srl) maintenance therapy and cni withdrawal in renal transplant recipients purpose: to compare the effect on renal function of maintenance immunosuppression with mmf and srl to that of mmf and a cni in renal allograft recipients. methods: in a 2-year open-label, prospective, randomized, controlled, multicenter study, 305 subjects maintained on mmf and a cni were randomized 30-180 days posttransplantation to either mmf (1-1.5 g bid) plus srl (2-10 mg followed by ≥ 2 mg/ day results: outcomes of the first 123 subjects receiving mmf/srl and 126 receiving mmf/cni (tac, n=98; csa, n=28) completing 1 year of follow-up will be reported here. final outcomes of all 305 subjects will be presented at the congress. mean time from transplant to randomization in both groups was 117 days. groups were similar at baseline for all reported renal function endpoints after 12 months of therapy, maintenance immunosuppression with mmf/ srl after cni withdrawal appears to preserve renal function when compared with a mmf/cni-containing regimen improved outcomes after de novo renal transplantation: 2-year results from the symphony study. h. ekberg, 1 h. tedesco-silva frei, 10 y. vanrenterghem, 11 p. daloze, 12 p. halloran at 2 years, the rate of uncensored graft loss was lowest in patients receiving tacrolimus (8% vs 10-13% in other groups; kaplan-meier estimates). gfr at the end of the core study was slightly better in the follow-up itt patients (65-70ml/ min) than in the core study itt patients (57-65ml/min), suggesting inclusion of betterperforming patients in the follow-up. renal function was generally stable over year 2. a slight improvement in gfr in the sirolimus group (+1.6ml/min) was observed, whereas the tacrolimus group still had superior gfr (69 vs 65-67ml/min in other groups). conclusions: in follow-up patients, renal function was stable during the second year and gfr differences were less marked than at 1 year a prospective randomized study of alemtuzumab vs rabbit anti-thymocyte globulin induction in kidney and pancreas transplantation gautreaux, 1 s. iskandar, 4 p. adams, 2 r. stratta. 1 1 surgery; 2 medicine; 3 pharmacy alemtuzumab (alem) and rabbit anti-thymocyte globulin (ratg) are the most commonly used t-cell depleting induction agents in kidney (k) and pancreas (p) transplantation (tx) expanded criteria donors (ecd) were included. results: between 2/1/05 and 8/24/07 225 pts enrolled and 222 pts were transplanted. of 222 pts, 180(81%) had ktx alone, 38(17%) kptx, and 4(2%) paktx. of 180 ktx alone, 152(84%) were deceased donor, and 61(34%) were ecds. recipient age, race, re-tx abstract# 535 purpose: to determine the impact of alginduction on long-term outcomes post-renal tx. methods: between 01/85 and 01/86, 123 consecutive adult pts received a deceased donor renal tx at a single institution results: the incidence of acute rejection was lower in gr.2 (28% vs. 75%, p<0.0001). the incidence of cmv infection was 10% in gr.1 and 18% in gr.2 (p=ns). the overall incidence of cancer was 22 abstract# 538 single-dose induction with rabbit anti-thymocyte globulin (ratg) safely improves renal allograft function and reduces chronic allograft nephropathy 1 clifford miles, 1 gerald groggel, 1 lucile wrenshall. 1 1 divisions of transplantation and nephrology we conducted a prospective, randomized trial in renal transplant recipients comparing two dosing protocols [single dose (6mg/kg) vs. divided doses (1.5mg/kg for 4 doses)] of rabbit anti-thymocyte globulin (ratg; thymoglobulin®). we present herein the results of the first 142 patients throughout the first 6 months post-transplantation, recipients of kidneys from non-marginal deceased donors derived the greatest benefit in renal function (egfr) from the single-dose regimen (p = 0.006). the incidence of chronic allograft nephropathy (can) was also lower in the single-dose group, in both clinically-indicated and protocol biopsies combined (p = 0.045) and in 12-month protocol biopsies alone high risk (race, pra) 8 (11%) in multivariable regression, allograft failure strongly predicted increased risk of subsequent cve. among listed candidates, receipt of a transplant was associated with significant time adjusted for baseline factors, cve after transplant predicted increased risk of subsequent mortality: hr 5.15 (ci 4.53-5.85) after is microalbuminuria post-renal transplantation is related to inflammation and cardiovascular risk our objective was to define the relationship between microalbuminuria and these risk factors in stable rtr. methods: over one year, we identified 223 stable rtr who were at least 2 months post-transplant and provided 3 successive urine albumin-to-creatinine ratio (acr) measurements, excluding those with recent illness and overt proteinuria. microalbuminuria was defined as averaged acr ≥ 2.0 in men and 2.8 in women (cda 2003). framingham-based traditional as well as novel cardiovascular risk factors associated with microalbuminuria were determined by univariate (p < 0.20), followed by stepwise backwards elimination (p >0.05) multivariate logistic regression analysis microalbuminuria did not correlate with prior acute rejection, delayed graft function, or any specific antihypertensive or immunosuppressive agents. conclusions: post-transplant microalbuminuria is highly prevalent and is associated with elevated crp, elevated bp, and smoking. its relationship to these other factors suggests that it reflects an inflammatory state in otherwise stable patients and thus may indicate graft and patient health the first year after kidney transplantation (tx) is associated with increased mortality relative to dialysis. early post-tx deaths are often cardiovascular (cv) and frequently occur after the first week post-tx. ctnt is a sensitive and specific maker of myocardial injury. in this study we investigated whether ctnt relates to early post-tx survival. methods: 396 patients received kidney tx from 9/2004 to 12/2006, 74% from living donors. ctnt was measured during the pre-tx workup and periodically while on the tx waiting list. 261 patients (64%) had a dobutamine stress echo (dse) and 103 (26%) had a coronary angiogram. the combined end point of the study was death or major cardiac events. survival was censored for graft loss. results: mean age was 51+12, 59% males. pre-tx ctnt level was elevated (>0.01 ng/ ml) in 56% of patients other dse derived parameters did not relate significantly to survival. ctnt further stratified the risk associated with other variables. thus, among patients with ef<55%, 2 year survival was 97%, 88% and 60% (p=0.0003) in patients with ctnt <0.01, 0.01-0.03 and >0.03, respectively. similarly, these ctnt ranges stratified risk in patients with low albumin conclusion: an elevated pre-tx ctnt is a strong and independent predictor of reduced early post-tx survival. ctnt allows stratification of risk in patients who have other risk factors such as low ef, low serum albumin and dialysis>2 years. in all patients, independent of any other variables, a normal ctnt was an excellent predictor (97%) of survival abstract# 545 validation of framingham risk assessment by actual cardiovascular event data in renal transplant recipients alloway, 2 michael cardi, 3 gautham mogilishetty, 2 shazad safdar excellent outcome after liver transplantation in children with cystic fibrosis some studies have reported benefits of liver transplantation (lt) in cf patients, but large outcome studies are not available. we report the outcomes of a large cohort of cf patients undergoing lt. methods: pre and post-lt patient characteristics, post-lt morbidity and mortality, and patient and graft survival were 2702 patients age < 18yr) received a 1 st isolated lt. 62 cf patients were listed for 1 st lt, neither waitlist deaths nor the probability of death from time of listing was different from non-cf. 42 (1.6%) cf patients underwent lt with an average followup of 3yrs (0-10yrs) average peld: 5.4 (86.5% had a peld < 10), median age: 11.9 yrs (0.7 -17.5) graft survival in cf patients was 85.0%, 81.2%, and 81.2% at 1, 3, and 5 yrs compared to 85.6%, 80.7%, and 78.1%. rejection rates were not different (50.6% cf vs 56.4% non-cf @ 5 yrs with 50% of these patients requiring dialysis. standardized height and weight scores showed no improvement over 2 years followup in the cf patients (height z -1.4 at tx to -1.4 at 2 yrs., weight z -1.2 to -1.5), but tended to improve in the non-cf group in addition, death rates from time of listing are not increased compared to non-cf patients. these data support lt as a treatment for cf liver disease, but studies investigating the lack of growth improvement and increased renal complications in these patients may further improve outcomes. abstracts full cni group. conclusion: compared to full cni, low cni/mmf a) allows renal function to recover in patients with impaired renal function at the time of ltx and b) preserves long term renal function. cni sparing in combination with mmf may become cellular islet autoimmunity influences clinical outcome of islet cell transplantation methods: twenty-one t1d patients received cultured islet cell grafts prepared from multiple donors and transplanted under anti-thymocyte globulin (atg) induction and tacrolimus plus mycophenolate mofetil (mmf) maintenance immunosuppression. immunity against auto-and alloantigens was measured before and during one year after transplantation. cellular auto-and alloreactivity was assessed by lymphocyte stimulation tests against autoantigens and cytotoxic t lymphocyte precursor assays, respectively. humoral reactivity was measured by auto-and alloantibodies. clinical outcome parameters remained blinded until their correlation with immunological parameters. results: all patients showed significant improvement of metabolic control and 13 out of 21 became insulin-independent. multivariate analyses showed that presence of cellular autoimmunity before and after transplantation was associated with delayed insulinindependence (p=0.001 and p=0.01, respectively) and lower circulating c-peptide levels during the first year after transplantation (p=0.002 and p=0.02, respectively). 7/8 patients without pre-existent t-cell autoreactivity became insulin-independent, versus 0/4 patients reactive to both islet autoantigens gad and ia-2 before transplantation. autoantibody levels and cellular alloreactivity were not associated with outcome. conclusions: cellular islet-specific autoimmunity affects clinical outcome of islet cell transplantation under atg-tacrolimus-mmf immunosuppression bmp-7 is downregulated & tgfβ1 to bmp-7 ratio favors emt during acute rejection of human renal allografts allospecific cd154+ t-cells predict rejection risk and measure immunosuppressive effect after abdominal organ transplantation in 100 recipients methods: allospecific cd154+t-cells were measured in <24 hours with polychromatic flow cytometry to identify rejectors (who had experienced acute cellular rejection within 60 days post-transplantation) in single mixed leukocyte responses (mlr) from 100 cross-sectional recipients-88 children with liver or intestine allografts, and 12 adults with renal allografts. where possible, results were correlated with proliferative alloresponses measured by cfse-dye dilution (n=45), allograft biopsies (n=46), and expression of ctla4, a negative t-cell costimulator, which antagonizes cd154-mediated effects (n=52). results: in the first 33 children, logistic regression identified donor-specific, memory cd154+ t-cytotoxic cells (tc) as enhanced among rejectors, compared with non-rejectors (408±231 vs 90±36 per 10,000 cells, p=0.003), relatively drug-resistant (r with drug levels =-0.5, p=ns), with greatest sensitivity/specificity (>93%) for rejectors noninvasively developed molecular signatures accurately predict acute rejection of human renal allografts greater emotional well-being (sf-36) and felt that their transplant interfered significantly less with various aspects of their life (iirs). conclusions: findings highlight the potential utility of assessing attachment style in transplant populations cni sparing in de novo renal transplantation: 3-year results from the symphony study one background: single center non-randomized results with steroid avoidance have shown patient and graft benefits. methods: 130 unsensitized, primary kidney recipients, 0-21 yrs of age, were enrolled from 12 us transplant programs (2004)(2005)(2006), in a prospective 1:1 randomized multicenter study of steroid-free (sf) vs. steroid-based (sb) immunosuppression with matched demographics. 24.1 % of sf and 27.5% of sb were african americans and 15.5% of sf vs. 8.7% of sb had esrd from fsgs. sf patients received extended (6 mo) vs. standard (2 mo) daclizumab induction in the sb group. patients in both arms received tacrolimus and mmf maintenance. protocol biopsies were performed at 0, 6, 12 and 24 mo, and for renal dysfunction. primary end-points were differences for standardized height scores and biopsy proven acute rejection (bpar) at 1 year. results at 1 year: 60 sf and 70 sb patients were enrolled; 10 sf and 16 sb were 0-5 yrs of age. patient survival was 100% in both arms. graft survival was similar (96.7% in sf vs. 98.6% in sb). intent to treat median delta height sds scores from baseline for different age groups were: 0.58 for sf and 0.48 for sb in the 0-5 yr old (p=0.80); 0.34 for sf and 0.34 for sb in the 6-12 yr old (p=0.86 protection of liver ischemia reperfusion injury by silencing of tnf-α and complement 3 genes. roberto hernandez-alejandro, 1 xusheng zhang, 2 dong chen, 2 xiufen zheng, 2 hongtao sun, 2 weihua liu, 2 marianne beduhn, 2 aminah shunnar, 2 motohiko suzuki, 2 norihiko kubo, 2 bertha garcia, 2 anthony jevnikar, 1,2 living kidney donation is rapidly increasing worldwide to offer a partial (?) solution for the numerous esrd wait-listed pts. in spite of properly followed guideline criteria conclusion: dcd donors are a viable source of liver allografts for transplantation. patients who receive dcd livers have outcomes comparable to subjects who receive grafts from brain dead donors. use of dcd livers from donors over 60 years of age is accompanied by a higher incidence of retransplantation and biliary complications. background: hypothermic machine perfusion (hmp) is in its infancy in liver transplantation (ltx). potential benefits include diminished reperfusion injury and improved early function. methods: the study was designed as a phase 1 trial of liver hmp. exclusion criteria included: multiple organ recipients, meld>35, icu patients, and patients >65 years of age. donor livers >65 years, biopsy with >30% macrosteatosis and dcd were also ineligible for hmp. seventeen patients were enrolled transplanted with livers that underwent hmp for 4-7 hours using dual centrifugal perfusion with vasosol solution at 4-6°c. patient, operative and early outcome variables were recorded. we compared outcomes to 17 matched cold stored (cs) controls from the same era. results: all 17 hmp grafts functioned immediately by usual clinical criteria with intraoperative bile production. results are summarized in table 1 . synergy between il-6 and tnfα promotes t cell alloreactivty and impairs the graft-prolonging effects of costimulatory blockade. hua shen, 1 bethany m. tesar, 1 wendy e. walker, 1 daniel r. goldstein. 1 1 internal medicine, yale university, new haven, ct. a novel role of th17 cells in allograft rejection and vasculopathy. francesca d'addio, 1 jesus paez-cortez, 1 m. javeed ansari, 1 laurie glimcher, 2 john iacomini, 1 mohamed sayegh, 1 xueli yuan. 1 1 transplantation research center, renal division, brigham and women's hospital, boston, ma; 2 harvard school of public health, boston, ma. introduction: transcription factor t-bet plays a crucial role in th1/th2 development. here, we investigated the role of t-bet in th17 differentiation and function of th17 cytokines in allograft rejection using an mhc class ii mismatched model of cardiac allograft vasculopathy. methods/results: cardiac allografts from bm12 mice were transplanted into wild-type as well as t-bet and ifn-γ deficient c57bl/6 recipients. t-bet-/-mice showed significantly accelerated allograft rejection (mst=14.75±0.96 days). however, as previously reported, all ifn-γ-/-and majority of the c57bl/6 mice accepted grafts for greater than 60 days. upon in vitro stimulation of recipient splenocytes by irradiated donor cells, t-bet-/-and inf-γ-/-lymphocytes produced significantly less inf-γ and more th2 cytokines. interestingly, production of the proinflammatory cytokines il-17 and il-6 was significantly higher in t-bet-/-(337±94.4 and 339±10.5 pg/ml) than c57bl/6 (135.1±68.3, 75.3±25.9pg/ml, p=0.0399 and 0.0001 compared to t-bet-/-) and inf-γ-/-(95.8±29.8, 20844.5 pg/ml, p=0.0135 and 0.0093 compared to t-bet-/-) mice. in vivo administration of il-17 neutralizing antibody (mab421) significantly prolonged survival of bm12 hearts (mst>30 days, p<0.01 compared to the 15.3±2.6 days of the control igg group) in t-bet-/-mice. immunofluorescence staining of bm12 hearts harvested from t-bet-/-recipients indicated that both cd4 and cd8 infiltrating lymphocytes produced il-17. however, t-bet-cd4 double knockout mice did not reject bm12 heart grafts, nor did the grafts exhibit chronic vasculopathy. in contrast, t-bet-cd8 double knockout mice rejected (mst:18.9±3.2 days). splenocytes from t-bet-cd4 knockouts produced significant lower il-6 (12.9±3.8) and il-17 (7.9±4.6 pg/ml) than observed in t-bet knockouts (940±176.3 and 210.2±73.4 pg/ml) and t-bet-cd8 knockouts (1240.5±215.6 and 113.1±61.2 pg/ml respectively) recipients when re-stimulated with donor cells, while there was no significant difference in inf-γ production. induction in the elderly transplant recipient: an analysis of the optn/ unos database. suphamai bunnapradist, 1 steven takemoto, 2 jagbir gill, 1 tariq shah. 2 1 medicine-nephrology, ucla, la, ca; 2 medicine-nephrology, national institute of transplantation, la, ca.we examined the incidence and mortality implications of cerebrovascular events (cve) after kidney transplant. we also compared variations in risk on the transplant waitlist and after allograft failure. methods: we used registry data from the us renal data system to retrospectively investigate ischemic stroke (is), hemorrhagic stroke (hs) and transient ischemic attacks (tia) among 29,614 adults who received kidney transplants in 1995-2002 with medicare as primary payer. patients with prior indications of cve in the registry were excluded. we ascertained events from billing claims, and estimated incidence of first events by the product-limit method. at-risk time was censored at: loss of medicare, 3yr transplant anniversary, non-cve death or end of study (12/31/2002). cox regression was used to identify independent correlates of cve, and to examine cve events as time-dependent mortality predictors. we estimated cve incidence after graft failure among patients without cve diagnoses prior to graft loss (n=2,599), and amongthe association between hyperuricemia at six months after kidney transplantation and the development of new cardiovascular disease, many studies have previously reported safe withdrawal of prednisone (pw) late after kidney transplantation (ktx). to determine the best immunosuppression regimen during the pw, we performed a prospective trial with stable ktx patients randomized to either csa or 's' based regimen. methods: all patients received antibody induction therapy at the time of rtx and maintained on csa, p and cellcept®. patients excluded if they had >1 acute rejection, >1 gm/d proteinuria or serum creatinine >2.5 mg/dl. 161 patients were enrolled and data presented for 141 patients with >52 weeks follow-up (f/u) with mean f/u of 112.2±29.2 weeks. no differences observed in baseline characteristics in both groups. all patients then randomized to either csa (n=69) or 's' (n=72) and cellcept® converted to equivalent dose of ms. csa dosed by c2 level (2-hour) with goal level of 600 ng/ml. sirolimus target level was 8 ng/ml. results: 5 patients withdrew from study, 12 patients on s returned to csa regimen because of side effects. 5 patients in the csa group and 1 patient in 's' group had ar (3 of them due to drug non-compliance). death censored graft survival was 100%. mean csa drug level acheived was 664±188 ng/ml and 's' drug level was 8.2±1 ng/ml. no significant differences noted in hematological values or bp measurements. csa ( purpose: the clinical significance of c4d positiviity in patients with acute rejection is well defined but its significance in stable graft function is undetermined. this study was performed to evaluate the clinical outcome of protocol biopsy-proven c4d positive renal transplants with stable graft function in the early posttransplantation period. methods: 151 renal allograft biopsies were included. protocol biopsies (n=79) were performed from stable allografts on day 14 posttransplantation, and indication biopsies (n=72) were performed from dysfunctioning allografts. incidence of c4d positivity was compared between protocol and indication biopsies. clinical characteristics, biopsy findings, graft function, acute rejection episodes, and graft survival rates were compared between the c4d-positive and c4d-negative grafts in each group. results: c4d deposition in protocol biopsies was detected in 4 of 79 biopsies (5.1%), whereas 9.7% (7 of 72 biopsies) in indication biopsies. the histological findings of c4d-positive protocol biopsies were minimal inflammation of tubulointerstitium. on the other hand, those of c4d-positive indication biopsies were various including acute humoral rejection, acute cellular rejection, acute tubular necrosis and calcineurin inhibitor toxicity. in the protocol biopsy group, graft function during 1 year after biopsy, acute rejection rate, and cumulative graft survival did not differ between the c4d-positive and c4d-negative grafts. all c4d-positive allografts maintained stable graft function without any antirejection therapy. in the indication biopsy group, graft function during 1 year after biopsy and acute rejection rate did not differ between the c4d-positive and c4d-negative grafts. however the cumulative graft survival rate was worse in the c4d-positive grafts than the c4d-negative ones (p=0.008). conclusion: c4d positivity associated with allograft dysfunction indicates a poor graft outcome. however, c4d-positive allografts with stable graft function in the early posttransplantation period take an indolent course. are methods: this was a retrospective single centre study reviewing all the adult patients who had a kidney transplant biopsy between april 2003 and october 2006 at guy's hospital. results: 45 patients had diffuse (>50%) c4d staining out of 228 who had kidney transplant biopsies in this time and had been followed up within the centre. of these 45 patients, 20 also had dsa prior or at the time of biopsy. the fall in egfr in this group a year post biopsy was greater than those with diffuse staining for c4d but no dsa. the mean change in egfr from the day of biopsy at a year was -3.1ml/min/1.73m2 (+/-12.1) in those with dsa compared with +17.7 ml/min/1.73m2 (+/-23.2) for those with c4d but without dsa. the changes in egfr from the pre-biopsy baseline at one year showed a fall in egfr in both groups but this was greater in those with dsa (-21.5 compared with -9.6 ml/min/1.73m2 ).of 179 patients who never had diffuse or focal c4d staining on biopsy, only 81 had had dsa tested. of these only 8 (10%) had a positive dsa result. from these eight, four had features of rejection and four did not. one person in each of these groups is dialysis dependant and one person in the rejection group has egfr <15 ml/min/1.73m2. although small numbers, this outcome appears to be worse than that of c4d negative patients with no dsa but features of rejection who in fact showed an improvement in egfr from the day of biopsy by 14.4 m l/min/1.73m2 or an improvement from their pre-biopsy baseline of 1.3 m l/min/1.73m2 at one year. conclusion: dsa is of additional value in evaluating risk of graft failure. this appears to be of value in those with and without diffuse c4d staining on biopsy. utility of post-transplantation flow cytometry crossmatching in predicting graft outcomes. michelle willicombe, 1 graham shirling, 2 ray fernando, 2 henry stephens, 2 paul sweny, 1 peter j. dupont. 1 1 department of renal medicine, royal free hospital, london, united kingdom; 2 histocompatibility laboratories, anthony nolan trust, london, united kingdom. de novo development of donor-specific anti-hla antibodies after renal transplantation may be associated with increased rejection and decreased graft survival. flow-cytometry crossmatches (fcxm) have been suggested as method of screening for development of donor-specific hla antibodies post-transplantation, but interpretation of crossmatch results can be confounded by antibodies directed against antigens other than hla. we assessed the impact of developing a positive fcxm post-transplantation on clinical outcomes in a cohort of live donor renal allograft recipients. methods: 29 patients were studied. 23/29 (79%) received tacrolimus-based and 6/29 (21%) ciclosporin-based immunosuppression. median follow-up was 24 months. all patients had negative complement-dependent cytotoxic (cdc) t cell crossmatches pretransplantation. 4/8 (50%) in the group with a positive fcxm had an acute rejection episode in the first 12 months compared with 10/21 (41%) in the group with a negative fcxm (p=ns). graft function at 12 months was not different between the groups (positive fcxm -median creatinine 137mmol/l; negative fcxm -median creatinine 141mmol/l; p=ns). 2/8 grafts (25%) were lost within the first year in the positive fcxm group compared with 1/21 (5%) in the group with negative post-transplant fcxm (p=ns). the development of a positive fcxm post-transplantation alone is not predictive of adverse clinical outcomes. this may be explained by the poor correlation between a positive fcxm and the presence of antibody directed against mismatched donor hla antigens. surveillance for development of donor-specific anti-hla antibodies after transplantation may be best performed using high-resolution bead technologies rather than fcxm. use of the fcxm alone, without establishing antibody profiles, is of limited predictive value. based upon the amount of antibody (ab) measured by the titer of donor specific hla antibodies, dsa, or the fluorescence intensity (fi) of the donor specific single antigen bead. it is unclear whether abs indentified by sensitive single antigen bead and solid phase assays (flow pra and luminex) correlate with and are predictive of a clinically relevant end-point (a + fcxm). we evaluated the pra, dsa, bead specific ag fi and fcxm reactivity of pre-transplant (pre-tx) sera from 219 recipients of a deceased donor renal allograft to determine whether amount of ab (measured by fi) predicts a (+) fcxm. patients with a (+) dsa, a (+) fcxm and pre-tx class i pra ≥ 80% (n = 103, mean pra of 91 ± 6%) when compared to patients with pre-tx pra < 80% (n = 58, mean pra 53 ± 21%) had comparable mean fis (7,407 ± 4,305 vs 7,483 ± 5,380) , fi ranges (1,000 -20,243 vs 1,552 -17,153 ) and median fis (5,681 vs 5,670). the class ii comparisons were of the same pattern. surprisingly, patients with a (+) dsa, a (-) fcxm and pre-tx class i pra ≥ 80% (n = 22, mean 90 ± 6%) compared to patients with pre-tx pra < 80% (n = 36, mean pra 50 ± 18%) had comparable mean fis (6,758 ± 4,841 vs 6,412 ± 3,844) , fi ranges (1,656 -16,596 vs 2,162 -11,637) we have recently showed that pre-treatment of the donor with epo causes a substantial reduction of the dysfunction and injury associated with the transplantation of kidneys recovered after cardiac death. 5-aminoisoquinolinone (5-aiq) a potent water soluble parp inhibitor has proven to reduce renal ischemia-reperfusion (i/r) injury. the aim of our study was to determine the effects in the graft and in the receptor of the pre-treatment of the donor with epo and treatment of the recipient with 5-aiq, in a porcine model of dcd kidney transplantation. material/methods:24 landrace pigs were killed by lethal injection; their kidneys were subjected to 30 min of warm ischemic time (wit) and then transplanted after 24 h of cold storage in celsior. in the pre-treated group, donors received a single dose of epo (1000 iu/kg) 30 min before cardiac arrest. in the treated group, recipients received a continuous dose of 5-aiq (5mg/kg/h) 10 minutes before reperfusion and maintained during 60 minutes. blood, urine and renal tissue samples were collected at the end of the experiment for biochemical, histological and immunohistochemistry (pars, inos and cox-2) evaluation. data analysis performed with graph pad prism statistical package; p<0.05 considered statistically significant. results:transplantation of kidneys from dcd resulted in: a significant rise of the levels of creatinine, n-acetil-b-d-glucosaminidase, glutathione-s-transferase, ast, ldh, alt, fractional excretion of na+, interleucin 1 and 6, malondialdehyde levels and myeloperoxidase activity (p<0.05); a significant reduction in urine flow and creatinine clearance, disturbances in the histological and imunohistochemistry pattern. administration of epo before ischemia and 5-aiq before reperfusion reduced significantly the biochemical (p<0.01), histological and imunohistochemical evidence of glomerular dysfunction and tubular injury. they also reduced systemic injury, inflammatory response and oxidative stress. conclusions:pre-treatment of the donor with epo and treatment of the recipient with 5-aiq causes a substantial reduction of the dysfunction and injury associated with the transplantation of kidneys recovered after cardiac death. in the hmp group perfusate ast levels strongly correlated with recipient peak ast by linear regression (p<0.001). conclusions: hmp of liver grafts provides safe and reliable preservation in our pilot series. perfusate ast may allow pretransplant prediction of reperfusion injury. a larger randomized trial will be necessary to demonstrate the magnitude of benefits of hmp over cs in ltx.purpose: liver discard rates have increased in a large, urban organ procurement organization from 8 % to 24 %. the reason is likely the result of increased transplant surgeon willingness to consider organs close to the margin of clinical acceptability. however, the costs associated with recovering these organs are high if the liver is discarded. we endeavored to determine whether a model based on pre-recovery data could predict liver discard introduction: we report our 6 years experience with the use of campath-1h (c1h) in adult liver transplantation. from december 2001 until july 2007 we administered c1h induction with low dose maintenance tacrolimus immunosuppression to 166 adult recipients of a liver allograft. most common primary diseases were laennec (n=60), cryptogenic cirrhosis (n=31) and autoimmune: psc (n=17), pbc (n=16) and aih (n=14). the first dose of c1h was administered immediately before (n=74) or after (n=92) the transplant procedure. follow up was until september, 2007. results: five year patient and graft survival was 90% and 85% respectively. there were 14 deaths due to stroke (n=2), chronic rejection (n=2), failure to thrive/pneumonia (n=3), sepsis (n=2), hepatic artery thrombosis, hcc, prostate cancer, graft lymphoma and non-compliance (one each). seven patients were retransplanted, for primary non function (n=2), portal vein thrombosis (n=1), hepatic artery thrombosis (n=1), hepatitis b (n=1) and chronic rejection (n=2). thirty six patients had biopsy proven rejection episodes: mild (n=28), moderate (n=9) or severe (n=3). the average tacrolimus 12 hour trough levels were 6.6, 5.2 ng/ml and 2.78 ng/ml for the 1rst, 3 nd and 5th year post-transplantation, respectively. there was no significant difference in the outcome of the transplant so far, between patients that received c1h before or after the transplant procedure. immunosuppression-related complications included a). opportunistic infections: most common were herpes zoster (n=19), cmv (n=3), and herpes simplex (n=3), b). neoplasms: skin cancer (n=3), kaposi sarcoma (n=1), lymphoma (n=2) and c). nephrotoxicity: five patients received a kidney graft for diabetic nephropathy (n=2), nephrotic syndrome (n=1) and calcineurin nephrotoxicity (n=2). conclusion: the use of c1h induction with half the usual dose of tacrolimus is an effective regimen in adult liver transplantation. the timing of c1h administration does not seem to affect the clinical outcome so far. a. david mayer, 1 james m. neuberger. 1 1 the liver unit, queen elizabeth hospital, birmingham, united kingdom. introduction: in the prospective respect study, primary liver transplant patients were randomised to 1 of 3 groups: a) standard-dose tacrolimus (target trough level > 10 ng/ml) for the 1 st month; b) 2 g mycophenolate mofetil (mmf) iv until at least day 5, 2 g po thereafter + reduced-dose tacrolimus (target trough level ≤ 8 ng/ml); and c) mmf as in group b + reduced-dose tacrolimus introduced on day 5 (target trough level ≤ 8 ng/ml) + daclizumab on days 1 and 7. steroids were given in all groups according to local centre protocol. results at 1 year showed that 2 g mmf + delayed and reduced tacrolimus + daclizumab is associated with significantly less impairment of renal function compared with standard treatment. here we present the results of the per protocol (pp) population. methods: the pp population, which was defined prior to the sub-group analysis, consisted of patients from the full analysis set who had no inclusion/exclusion criteria violation, had at least one creatinine clearance (crcl) value beyond 6 months, were treated according to the protocol and did not receive any prohibited medication during the first 14 days and for less than 1 week at any time during the study. a composite endpoint comprising freedom from renal dysfunction (≥ 20% decrease from baseline in calculated crcl), acute rejection, graft loss or death was also investigated. results: the full analysis set included 181, 168 and 168 patients, whereas the pp population only included 69, 67 and 67 patients in groups a, b and c, respectively. the mean difference in calculated crcl from baseline to 1 year was significantly smaller in group c compared with group a (-10.7 ml/min vs -22.0 ml/min, p = 0.038), but was not significantly different between groups a and b (-20.50 ml/min). the incidences of death (n = 2, 0, 1) and graft loss (n = 1, 0, 0) were similar in all 3 groups. the incidence of the composite endpoint at 1 year was in both the full analysis set and the pp population significantly lower in group c compared with group a (pp population: 70% vs 93%, p < 0.0001), but was not significantly different between groups a and b (85%). the pp analysis confirms the results from the full analysis set that 2 g mmf + delayed and reduced tacrolimus + daclizumab is associated with less impairment of renal function compared with standard treatment with no negative effect on death and graft loss. aims: post transplant lymphoproliferative disorder (ptld) is a serious complication of solid organ transplantation that is closely associated with epstein barr virus (ebv) infection. ebv + ptld lymphomas express several latent viral genes including latent membrane protein 1 (lmp1), a proven oncogene that is essential for human b cell transformation. lmp1 is able to activate erk, jnk, p38, nfκb and pi3k. the aim of this study is to determine whether lmp1 isolated from ptld tumors differs in signaling ability from lmp1 derived from the b.95 strain of ebv, originally isolated from a patient with infectious mononucleosis. methods: lmp1 variants isolated from a panel of ebv + ptld-associated b cell lines were cloned and sequenced. inducible chimeric constructs containing the lmp1 c-terminus and ngfr transmembrane domain were created for each tumor variant and expressed in the burkitts b lymphoma cell line bl41. lmp1 signaling in bl41 clones was induced by crosslinking of ngfr. activation of p38, erk, akt and jnk was assayed by western blotting (wb) with phospho-specific antibodies. nfκb activation was assayed by wb for iκb and cfos induction was analyzed by wb and the transam cfos binding assay. results: all three tumor variants of lmp1, as well as the b.95 lmp1 isoform, were able to induce p38 activation within 30min of ngfr crosslinking while akt and jnk were activated within 10min. all variants showed similar ability to activate nfκb. however, tumor lmp1 variants induced prolonged erk activation (up to 3hrs) while the b.95 lmp1 variant induced a transient response. cfos is induced only during the sustained phase of erk activation. indeed, the tumor variants of lmp1, but not b.95 lmp1, were able to induce cfos protein. similarly, cfos binding to the ap1 consensus site was only observed in tumor lmp1-induced nuclear lysates. two mutations in the c-terminus-aa212 (s vs g) and aa366 (t vs s) -are conserved in the tumor variants lmp1 compared to b.95 lmp1. point mutation of either of these amino acids from the b.95 to tumor variant version allowed for sustained activation of erk and subsequent cfos induction and binding to the ap1 site. conclusion: tumor-derived lmp1 has enhanced ability to induce the cfos oncogene and this property can be localized to two amino acids in the c terminus. these findings suggest that these specific amino acid residues of lmp1 are important in determining whether ebv infection is benign or results in ptld. the absence of interferon regulatory factor-3 (irf-3) confers protection against the liver ischemia and reperfusion injury through an il-10 independent pathway. elizabeth r. benjamin, 1 xiu-da shen, 1 feng gao, 1 yuan zhai, 1 genhong cheng, 1 ronald w. busuttil, 1 jerzy w. kupiec-weglinski. 1 1 surgery, dumont-ucla transplant center, los angeles, ca. toll-like receptor 4 (tlr4) mediated liver reperfusion damage after warm ischemia requires signaling through the myd88-independent, irf3-dependent pathway with cxcl-10 (ip-10) playing a central role in the injury development. studies using cxcl-10 ko mice have shown that these mice are protected through an il-10 dependent mechanism. we chose to investigate irf3, upstream of cxcl-10, to further characterize its role in the injury progression, and to better understand the involvement of il-10 in this pathway. methods: we used irf3 ko mice and their wt counterparts in a model of partial hepatic warm ischemia with 1, 2, and 6h of tissue reperfusion (n=2 ko, wt at 1 and 6h; n=3 ko, wt at 2h). wt bone marrow derived macrophages were generated and stimulated with lps to determine the kinetics of il-10 production. tissue was analyzed for histology and mrna levels were measured by qpcr. results: kinetic studies showed peak il-10 production at 1 and 2h post-reperfusion (pr). on pathology, irf3 ko mouse livers were protected from ir injury both early pr, at 1 and 2h, and at 6hrs pr when compared to wt. consistent with these data, il-6 mrna induction was decreased in irf3 ko, as compared with wt at 1, 2, and 6h. although il-10 induction was maintained in the cxcl-10 ko mice, the irf3 ko mice showed decreased levels of il-10 at 1h pr. by 2h, il-10 levels were normalized to wt. conclusion: irf3 ko mice are protected from liver ir injury with evidence of this protection as early as 1h pr. although cxcl-10 ko mice are protected from ir injury with maintained il-10 expression, the absence of the upstream molecule, irf3, confers protection in an il-10 independent manner. these data suggest a novel mechanism of ir injury mediated by irf3 in the liver. background: bone marrow (bm) transplantation may induce donor-specific tolerance to prevent rejection of allogeneic solid organs while maintaining immunity against infections and tumors. currently allogeneic bm transplantation is limited by donor t cell mediated graft-versus-host disease (gvhd), as well as a variable requirement for recipient marrow ablation and high numbers of donor bm cells. furthermore, sustained macro-chimerism has not yet been easily or predictably achieved in partially ablated patients or large animals. while rejection of allografts is mediated primarily by recipient t cells, recent studies have demonstrated the capacity of nk cells to reject allogeneic bm and to prevent long-term mixed chimerism. thus, nk cells represent a barrier to long term bm engraftment even with t cell tolerance. we have previously identified a novel type of regulatory t (treg) cell with a "double negative" (dn) phenotype (tcrab + cd3 + cd4 -cd8 -). dn-treg cells can effectively suppress anti-donor t and b cell responses and prolong graft survival in allo-and xenotransplantation models. we therefore tested the capacity of dn-treg to alter nk cell function. methods: c57bl/6 bm cells were i.v. injected into sub-lethally-irradiated (6.5 gy) cb6f1 (h-2b/d) in a "parent to f1" model, or into allo-disparate balb/c mice. bm cells were co-transplanted with various numbers of c57bl/6 dn-treg cells or cd4 + or cd8 + t cells as controls. recipient spleen cells were collected 7 days after to detect donor progenitors in a colony-forming-unit (cfu) assay. mice then received cardiac (n=8) or skin transplants (n=8) to confirm tolerance. we found that donor-derived dn-treg cells suppress nk cell-mediated allogeneic bm graft rejection in both "parent-to-f1" and fully mhcmismatched bm transplantation models. adoptive transfer of dn-treg cells with donor bm cells promoted the establishment of stable mixed chimerism and donor specific tolerance to bm donor cardiac and skin grafts (mst>160 days), without inducing gvhd in sub-lethally irradiated mice. perforin deficient dn-treg cells were unable to efficiently inhibit nk cell function, and donor bm did not engraft. these results demonstrate a potential approach to control innate immune responses and promote allogeneic bm engraftment and donor specific tolerance through the use of dn-treg cells.framingham risk score (frs) predicts cardiovascular (cv) risk in the general population, but may underestimate cv risk in kidney transplant (txp) patients (pts). frs has not previously been validated by prospective cardiovascular event (cve) data collection in kidney txp pts. the purpose of this study was to validate frs with actual observed cve data in kidney txp pts. methods: cve data was collected at routine intervals in our kidney txp pts and entered in a cardiovascular risk database. frs was calculated from baseline to 7 yrs posttransplant (ptx) individual frs factors of age, sex, smoking, diabetes mellitus (dm), high-density lipoprotein (hdl), total cholesterol (tc), and blood pressure (bp) were evaluated for their ability to predict acutal cve occurring after kidney txp. pts with coronary artery disease (cad) were excluded from the frs analysis. cve were defined as sudden death, myocardial infarction, angina, and cerebrovascular accident/transient ischemic attack. frs factors were evaluated by cox proportional hazards in univariate (uva) and multivariate (mva) models. rho kinase (rok) modulates calcium sensitivity of vascular smooth muscle cells and contributes to the regulation of peripheral vascular tone in man. in essential hypertension increased rok-activity contributes to the generation of vascular resistance. arterial hypertension is a common complication in renal transplant recipients. in this study we were interested in the role of rok for systemic hemodynamics in hypertensive renal transplant recipients (tx). we tested the specific inhibitor of rok fasudil. 10 tx and 10 matched control subjects (c) received either fasudil (1600 g/min) or placebo over a period of 30 minutes intravenously. peripheral blood pressure and heart rate were recorded every 5 min over a total of 120 minutes. measurements for pulse wave analysis (sphygmocor vt) were performed every 10 minutes during this period. statistics by anova for repeated measurements.compared to placebo fasudil significantly reduced peripheral mean arterial pressure p<0 .05; figure 1) and increased heart rate (+ 6.31.1 bpm, p<0.001) in tx but not in c. likewise, central systolic pressure(p=0.006; (figure 2), augmented pressure and augmentation index were decreased in tx only.we conclude that acute inhibition of rok by fasudil consistently and effectively lowers blood pressure in tx with a calcineurin inhibitor-based immunosuppression. interestingly, rok-inhibition also reduces central blood pressure and arterial stiffness in these patients. improvement of both these parameters has been linked to a reduction in cardiovascular morbidity and mortality in large trials. hence rok inhibition might prove beneficial for the treatment of hypertension in renal transplant recipients. use death with function causes half of late ktx failure, and cardiovascular disease (cvd) is the most common cause of death. chronic kidney disease (ckd) is a cvd risk equivalent, justifying aggressive risk reduction with blood pressure (bp) control, statins, aspirin, and use of angiotensin converting inhibitors (acei) and angiotensin receptor blockers (arb). dekaf is an nih-sponsored prospective observational study examining causes of ktx failure at 7 transplant centers in the us and canada, with current enrollment of over 1800 subjects. we examined the use of cardioprotective medications among patients transplanted after 10/1/05 with at least 6 mos follow-up, focusing on subgroups with preexisting diabetes (dm) and/or cvd. we conducted a retrospective cohort study to asses the prevalence and the predictors for the development of hyperuricemia at 6 months after kidney transplantation and the association between hyperuricemia and clinical outcomes including patient and graft survival, new cardiovascular events and chronic allograft nephropathy (can). adult patients who underwent kidney transplantation at mount sinai medical center between 1.1.2001-12.30.2004 were included. patients who died or lost the allograft within 6 months after transplantation were excluded from analysis. of the 307 patients with a functioning allograft at 6 months after transplantation, 163 patients (53%) had normal uric levels and 144 patients (47%) had hyperuricemia. after age, race, sex adjustment, receiving a cadaveric kidney, having an egfr<50 ml/min, and taking diuretics and cyclosporine were associated with a higher odds ratio of hyperuricemia. over a mean of 4.3 years of follow-up, 81 patients had one, or more, of the pooled outcomes; 40 had new cardiovascular events, 41 developed biopsy-proven can, 4 patients died, and 20 had graft failure. kaplan-meier survival curves demonstrated that the pooled outcomes of events occurred more frequently in hyperuricemic patients (figure, p < 0.001). due to association between low egfr and hyperuricemia, we analyzed the clinical outcomes in patients with low and normal egfr. while 44.7% of hyperuricemic patients with an egfr<50 ml/min had one of the pooled outcomes, it was 20.8% in patients with normal uric acid levels (p=0.038). among patients with an egfr ≥50 ml/min, 19.4% of normouricemic and 25% of hyperuricemic patients had one of the events. these results suggests an important association between hyperuricemia at 6 months after transplantation and the new cardiovascular events, biopsy-proven can, and graft loss in kidney transplant recipients with decreased allograft function. background: long-term survival after liver transplantation (lt) is now the rule rather than the exception. hence, assessment of outcomes for children after lt must consider not only the quantity, but also the quality, of life years survived and restored. aim: to examine key hrqol themes after pediatric lt raised by both recipients and their parent proxies, with evaluation by time (1-3 yrs, 3-5 yrs, 5-10 yrs, and >10 yrs) since lt. methods: semi-structured 1:1 item generation interviews were conducted in person with children (c) and parents (p) at time of ambulatory lt follow-up at 4 pediatric lt programs in canada and uk. all interviews were audio-taped, transcribed verbatim, and subjected to content analysis utilizing qsr nvivo 2.0 software for hrqol related theme generation. the participants interviewed were part of a larger research program aimed at developing a disease-specific instrument to assess hrqol for children after lt. results: data representing 91 (47% male) pediatric lt recipients was obtained from a total of 146 (62 c, 84 p) item generation interviews. median recipient age at lt was 2.4 (range, 0.05 to 17) yrs, for primary indications including biliary atresia (45%), fulminant liver failure (17.6%), metabolic liver disease (4,4%), malignancy (8.8%) and others (24.2%). median patient age at time of interview was 9.9 (range, 1.1 to 18.8) yrs. themes emerging at all time points post-lt included infection risks, limitations on physical activities, side effects from immunosuppression meds, educational supports, and ongoing bloodwork. themes identified within the medium (1-5 yrs) follow-up included worries about rejection episodes, need for future re-transplantation, school absenteeism, and altered sibling and family dynamics. the impact of living with a surgical scar was a more frequent theme with recipients >3 yrs from lt. as time from lt increased to >10 yrs, themes suggest a focus on normalization and health promoting behaviours, along with expressed desires to be like healthy peers. conclusions: unique hrqol themes emerged from item generation interviews not captured by currently available generic hrqol tools. hrqol themes identified after pediatric lt suggest the importance of considering time trajectories from lt, and a focus on elements of 'everyday life' apart from lt. the shortage of cadaveric donors has led many transplant centers to expand their criteria for accepting life-saving organs. utilization of donation after cardiac death (dcd) donors has been estimated to increase the number of cadaveric donors. we report our experience with a recently established dcd program at a pediatric hospital and the outcome with the transplanted grafts. methods: in 2005 a protocol for dcd was established at a free standing pediatric hospital. from 2005 to 2006 all patients undergoing withdrawal of care were evaluated for dcd. patients meeting criteria for dcd underwent withdrawal by the critical care team and organ retrieval was initiated if asystole was reached in less than 60 minutes. in addition, one dcd liver was imported and was included in the liver results. results: during the 2 year study period 7 patients (25% of total donors) underwent dcd resulting in 14 organs (11 kidneys and 3 livers) transplanted. the 7 cases had a mean donor age of 9 yrs (range 3-16), wit of 22 min (9-55), time sbp < 60 of 11 min (7-15), and time from asystole to aortic flush of 8 min (6) (7) (8) (9) (10) (11) (12) . four kidneys were transplanted locally with cit 5-14 hrs, no dgf, and one month creat 1.2-1.7. the remaining 7 kidneys were exported for transplant. four livers were transplanted locally with donor age 7 mth-16 yrs, wit 12-28 min, recipient age 14 mth-61 yrs, cit 6-15 hrs, ast peak 190-3630, ast day 7 33-44, inr peak 1.4-1.8, inr day 7 1.0-1.1, total bili one month 0.3-1.0, and graft survival 1.5-2.9 yrs. there were no vascular or biliary complications. conclusions: a protocol for dcd at a pediatric hospital increased the number of pediatric donors by 25%. liver and kidney grafts from pediatric dcd donors demonstrated excellent graft function and survival. liver retransplantation in children. a 21 year single centre experience. christophe bourdeaux, andrea brunati, magda janssen, jean-bernard otte, etienne sokal, raymond reding. pediatric liver transplant program, université catholique de louvain, saint-luc university clinics, brussels, belgium. when graft failure occurs in liver recipients, secondary transplantation represents the only chance of long-term survival. in such instance however, several surgical and immunological aspects should be carefully considered, with respect to their impact on final outcome.in the present study, the epidemiology and outcome of graft loss following primary pediatric liver transplantation (lt) were analysed, with the hypothesis that early retransplantation (relt) might be associated with lower immunologial risks when compared to late relt. between march 1984 and december 2005, 745 liver grafts were transplanted to 638 children at saint-luc university hospital, brussels. among them, a total of 90 children (14%) underwent 107 relt, and were categorized into two groups (early relt, n=58; late relt, n=32), according to the interval between both transplant procedures (< or > 30 days).ten-year patient survival rate was 85% in recipients with a single lt, versus 61% in recipients requiring relt (p=0.001). ten-year patient survival rates were 59% and 66% for early and late relt, respectively (p=0.423), the corresponding graft survival rates being 51% and 63% (p=0.231). along the successive eras, the rate of relt decreased from 17% to 10%, whereas progressive improvement of outcome post-relt was observed. no recurrence of chronic rejection (cr) was observed after relt for cr (0/19). two children developed a positive cross-match at relt (2/10, 20%), both retransplanted lately for cr secondary to immunosuppression withdrawal following a post-transplant lymphoproliferative disease.in summary, the current need for relt has been decreasing over years, with a parallel improvement of its outcome. the results presented could not evidence better results for early relt when compared to late relt. the latter did not seem to be associated with higher immunological risk, except for children with immunosuppression withdrawal following the first graft. the background: a serum conjugated bilirubin greater than 100 umol/l (cb100), in neonates who receive parenteral nutrition (pn), has been demonstrated to be a predictor of end-stage liver disease requiring transplantation. given the recent interest in the role of omega-6 lipids in the development of parenteral nutrition associated liver disease (pnald), we sought to examine in a multiple variable model the role of days of maximal lipid (>2.5 g/kg/day), in the development of this outcome. method: between 2003 and 2004, data were collected prospectively on all neonates undergoing an abdominal surgical procedure. univariate logistic regression models for the prediction of cb100 were developed with the following predictors: gestational age, percentile weight, percent predicted small bowel and colonic length, resection of the ileocecal valve, presence of a stoma, post-operative enteral tolerance, number of septic episodes, days of pn amino acid > 2.5g/kg/day, days of pn lipid > 2.5g/kg/day, and total days of pn. univariate predictors significant at the 0.2 level were entered into a backward stepwise multiple variable logistic regression. results: 152 infants received pn post-operatively, and 22 developed cb100. predictors that met criteria for consideration in the multiple variable model were: age (p=0.079), weight (p=0.059), small bowel length (p=0.001), presence of a stoma (p=0.163), proportion of enteral feeds post-operatively (p=0.049), days of pn amino acid > 2.5g/ kg/day (p=0.00), days of lipid > 2.5 g/kg/day (p=0.00), and total days of pn (p=0.00).the final multiple variable model which had a negative predictive value of 97.6% and positive predictive value of 52.6% is presented in the table below. our model suggests a key role of pn lipids and intercurrent septic events in the development of cb100 from pnald. these data may provide targets, such as careful line care, reduction in maximal lipid dose, or the use of alternate lipids such as omega-3 fatty acids, to prevent cb100 an identified marker for the need of subsequent liver transplantation in infants with pnald. terminal renal failure occurs in more than 10 % of liver transplant recipients after 10 years. we have previously shown that, beside renal toxicity of calcineurin inhibitors, renal lesions may be related to diabetes, arterial hypertension, accumulation of hydroxyethylstarch (elhoes), and the etiology of the liver disease. we made the hypothesis that these lesions may be already present at the time of liver transplantation (lt), a finding that could lead to adapt the perioperative management. this work investigated prospectively whether renal histopathological lesions were present before lt by performing systematically a renal biopsy by endovenous route in 60 candidates to lt with end-stage liver disease. these patients were 58 ± 10 years old, 46 males ; 10/60 had a diabetes, and 21 an arterial hypertension ; the liver disease was related to alcohol in 32 cases, hcv in 12 cases, hbv in 5 cases, and to a cholestatic disease in 7 cases. at the time of the pre-lt workup, the biochemical parameters were : child score10 ± 2, meld score 18 ± 4, prothrombin rate 50 ± 12, creatinin serum level 90 ± 6 umol/l, proteinuria 0.12 ± 0.04 g/24h. severe side effects related to the procedure were limited to 2 cases of macroscopic hematuria, lasting less than 24 hours. in 10 cases, the material obtained during the procedure did not allow the histological analysis. among the 50 samples available, 21 were considered as normal ; in 29 cases, lesions related to mesangial iga glomerulonephritis (14 cases), diabetic glomerulosclerosis (12 cases), elhoes accumulation (5 cases), thrombotic microangiopathy (1 case) were found, often associated ; in 5 cases, the lesions were severe and lead to combined kidney/liver transplantation in 2 cases. in conclusion, significant renal lesions are detectable in more than 50 % of the candidates to lt. interestingly, histological findings often combined lesions related to the liver disease and to an associated cause (diabetes, previous treatment by elhoes or interferon). results of histological analysis could help to decide either to perform a combined renal/liver transplantation, to adapt the immunosuppressive regimen, or to abandon the lt project. . because serum creatinine is one of the components of meld, liver candidates with renal insufficiency have been transplanted in increasing numbers, with some candidates receiving a kidney along with the liver transplant. we aimed to compare the liver graft outcomes for liver alone (lta) transplants with those from combined liver-kidney transplants (clkt). a propensity score analysis was used to reduce the impact of selection bias in the comparison of outcomes in the two groups. methods. demographics, clinical factors and outcomes on lta and clkt recipients from 3/1/02 to 12/31/06 (n=10,388) obtained from the optn database were used for the analysis. univariate post-transplant survival rates were estimated using kaplan-meier survival, and multivariable post-transplant outcomes were analyzed using a cox regression model with and without stratification by categories of the propensity score. the propensity score (probability of receiving a clkt) for each recipient was estimated using a logistic regression model. several donor and recipient factors were included in both the cox and logistic regression models. in this cohort, liver graft outcomes for clkt were significantly better than those for lta based on the multivariable analysis. the results were similar, although slightly less significant, when the model was adjusted for propensity. the superior outcomes of clkt may be due to unobserved differences between these groups of recipients, reflecting data not currently captured by the optn. effect of liver the tgfβ1 to bmp-7 ratio was higher in the ar group (median ratio: 1635) compared to recipients with stable graft function & normal biopsy (median ratio: 438, p=0.0002).our observations that bmp-7 is specifically down-regulated during an episode of ar and that the balance between tgfβ1 & bmp-7 is in favor of emt advance a mechanism for the deleterious impact of ar on the long-term outcome of human renal allografts. the non-statistical bioinformatic approach identified 68 leader genes which define the highest interaction genes derived from the 343 sam-gene list. an interaction map between the genes identified has been calculated. this network is formed around 2 majors clusters: a network of interleukins and a network of signal transduction which allow us the identification of key genes such as bank1, a negative modulator of cd40mediated akt activation, thereby preventing hyperactive b cell response in blood from patients with operational tolerance and il7r, a specific marker absent on potentially regulatory cd4 + cd25 +high t cells in blood from patients with chronic rejection. we have identified by a non-statistical analysis of the peripheral blood gene expression in human kidney recipients a cluster of genes which are strongly interconnected and which could be a starting point for further analysis of the molecular mechanisms of kidney graft operational tolerance and chronic rejection. fecal algorithm based on multiparameter mixed lymphocyte reaction assay for tailoring maintenance immunosuppressants after living donor liver transplantation. yuka tanaka, hideki ohdan, toshimasa asahara. department of surgery, hiroshima university, hiroshima, japan.background: no reliable immunological parameters exist for identifying liver allograft recipients in whom immunosuppressants can be safely withdrawn. for minimizing maintenance immunosuppressants, we established an algorithm determining anti-donor alloreactivity based on multiparameter mixed lymphocyte reaction (mlr) assay, wherein the number and phenotype of alloreactive precursorscan be quantified. we enrolled 68 adults undergoing living donor liver transplantation (lt). the initial immunosuppressive regimen comprised tacrolimus/cyclosporine and methylprednisolone, which were gradually tapered off by 6 months after lt. thereafter, therapeutic adjustments were determined by a policy of slow tapering off in the case of normal liver function. mlr assay was performed at 6 month intervals to monitor immune status. in this assay, cfse-labeled pbmcs from recipients were used as responders. irradiated donor and third-party pbmcs were used as stimulators. after coculture, the responder cells were stained with cd4 or cd8 mabs along with cd25 mab, followed by fcm analyses. the proliferation and cd25 expression of cd4 + and cd8 + t cell subsets in response to anti-donor and anti-third-party stimuli were analyzed; the immune status of lt patients was categorized as hypo-response, norm-response, or hyper-response for cd4 + t cells and as hyper-response for cd8 + t cells. of the 68 patients, 33 had normal liver function at >6 months after lt. we examined the fluctuation of immunosuppressants at 6 months after mlr in these patients. in patients whose immune status was categorized as hyper-response for cd4 + or cd8 + t cells (n=4), immunosuppressants had to be increased. in patients with norm-response immune status (n=7), immunosuppressant tapering was abandoned. immunosuppressant therapy was successfully tapered off in patients with hypo-response immune status (n=22). of 10 patients with hypo-response immune status at >3 years after lt, immunosuppressants were completely discontinued in 3. in these "operational tolerance" patients, the precursor frequency of anti-donor cd4 + t cells (mean=5.2±1.9%) was not reduced compared to that in non-tolerance patients, suggesting that donor-specific immune tolerance is maintained via inhibitory/ suppressive mechanisms rather than via clonal deletion. conclusion: multiparameter mlr assay can provide a clinically validated rule predicting the success of tailoring/weaning immunosuppression. psychological factors associated with non-adherence among adolescents before and after kidney transplant. nataliya zelikovsky. 1 1 dept. of pediatrics, div. of nephrology, the children's hospital of philadelphia, philadelphia, pa.purpose: little is known about psychosocial risk factors for poor adherence among pediatric transplant patients. identification of variables that impact illness management can guide targeted interventions to improve adherence. methods: a longitudinal study was conducted to determine whether quality of life (pedsql), family functioning (fad), and parent adjustment (pip) would predict adherence in adolescent transplant patients. psychological questionnaires were administered prior to and 12 months after the transplant. medical adherence measure (mam), a semi-structured interview was used to assess adherence. adherence was calculated as % missed and % late doses of those prescribed. results: 60 patients (m =14.32 years +2.18, 75% male, 63% caucasian) and their parents were evaluated at the time of listing for kidney transplant. the rate of non-adherence prior to transplant was high, with 90% of patients reporting some degree of nonadherence. of these patients, 37% missed and 28% took late > 10% of prescribed doses. on the quality of life measure, behavior issues were associated with missed (r=-.42, p=.01) and late doses (r=-.39, p<.05), and mental health issues were associated with late doses (r=-.38, p<.05). adolescent reports of problems in affective responsiveness among family members was associated with missed (r=.33, p=.04) and late (r=.51, p=.001) doses. missed doses were also associated with mother reports of difficulties with overall family functioning (r=.34, p<.05), communication (r=.41, p<.01) and role definitions (r=.33, p<.05) among family members. 49 of the families were re-evaluated one year after the kidney transplant. 65% had been on dialysis prior to transplant and 42% received living-related transplants. 58% of the patients reported some degree non-adherence post-transplant, and using more stringent criteria, 17% of patients reported missing and 27% reported taking late >10% of prescribed doses. worse quality of life such as limitations due to emotional problems (r=-.32, p<.05), behavioral problems (r=-.39, p<.01), and difficulties with family cohesion (r=-.32, p<.05) was related to worse adherence. discussion: adolescent quality of life in behavioral and emotional domains, and family functioning play a significant role in adherence both before and after transplant. programs to improve adherence among transplant patients should incorporate psychosocial supports and behavioral interventions to improve adjustment of patients and families. kidney transplantation leads to marked improvements in health, yet transplant (tx) recipients often have difficulty with sexual functioning, which can affect quality of life. specific sexual concerns of tx recipients remain under investigated. the purposes of this study were to 1) further establish the psychometric properties of the sexual concerns questionnaire (scq) including reliability and preliminary construct validity and 2) identify the sexual concerns of kidney tx recipients. the scq was answered by 390 kidney tx recipients who rated each item on a 0 (not at all) to 5 (extremely) scale. a cronbach's alpha correlation coefficient was calculated to determine the reliability of the scq. an alpha value of .83 was calculated for the questionnaire indicating it was reliable. exploratory factor analysis (efa) was performed to establish preliminary construct validity of the scq. as a result from the efa, 14 items were dropped and a 5 factor structure was accepted. examples of items and responses include question 1: "how difficult is it for your vagina to get or stay wet or moist?" (for women) and "how difficult is it for you to get or keep an erection?" (for men) and question 2 "how comfortable are you talking about sexual concerns with your doctors and nurses?"participants were also asked to indicate how important their sexuality was to them on a 0 (not at all) to 6 (extremely) rating scale. twenty-six percent of participants rated their sexuality as quite a bit important, 26% rated their sexuality as very important, and 20% rated their sexuality as extremely important. the findings provide evidence of a reliable questionnaire with evidence for preliminary construct validity. they also indicate that sexuality is an important issue for a majority of kidney tx recipients. a cross-sectional study of fatigue before and after liver transplantation. james r. rodrigue, 1 timothy antonellis, 1 p=0.78 # 1 (none) to 10 (extremely high); ♣ higher score = more fatigue, poorer sleep quality, or more mood disturbance; ¶ higher score = better qol one-third of pre-lt (32%) and post-lt (33%) patients reported severe fatigue. poor sleep quality was reported by 68% and 79% of pre-and post-lt patients, respectively. pre-lt fatigue was predicted by higher bmi (ß = 0.21) and meld (ß = 0.20), depression (ß = 0.35), and poor sleep quality (ß = 0.48), adj r 2 = 0.44, f = 10.72, p < 0.0001. post-lt fatigue was predicted by older age (ß = 0.19), tension-anxiety (ß = 0.27), anger-hostility (ß = 0.38), and poor sleep quality (ß = 0.18), adj r 2 = 0.38, f = 7.45, p < 0.0001. more fatigue was associated with lower sf-36 physical (r = -0.34) and mental (r = -0.44) qol. conclusions. fatigue and poor sleep quality are clinically significant problems for lt candidates and recipients. bmi, psychological functioning and sleep quality are modifiable variables that predict fatigue severity and should be targets of intervention when addressing fatigue symptoms. health literacy in kidney transplant recipients. elisa j. gordon, 1 michael s. wolf. 2 1 medicine, albany medical center, albany, ny; 2 medicine, northwestern university. background: in order to successfully manage the transplant long-term, kidney recipients must have a basic understanding of key transplant-related concepts and terms indicative of their condition and treatment to properly communicate with health care providers and manage their health. kidney recipients must also possess numeracy skills to enable proper medication-taking and to monitor serum creatinine levels, bodily temperature, etc. the objective of this study was to examine health literacy levels among kidney transplant recipients. methods: we surveyed 124 consecutive adult renal transplant recipients using the test of functional health literacy in adults (s-tofhla), and a modified version of the rapid estimate of adult literacy in medicine, called the "realm-transplant," which measured patients' knowledge of 69 kidney transplant-related terms that patients are expected to have familiarity with. open-ended and multiple choice questions assessed numeracy related to kidney survival. results: most kidney recipients (91%) had adequate health literacy (s-tofhla), but 81% were unfamiliar with at least 1 kidney transplant-related term (realm-t). patients who were less educated (p<0.0001), had lower income (p<0.002), and were single or without a partner (p=0.046) had significantly lower health literacy levels (s-tofhla). patients less familiar with transplant-related terms (realm-t) had less education (p<0.0001), lower income (p<0.0001), and were nonwhites (p=0.033). the five least familiar terms were: sensitization (50%), urethra (45%), trough level (41%), blood urea nitrogen (32%), and toxicity (31%). sixteen percent wanted more information about their transplant. numeracy levels varied: 21% knew the likelihood of 1-year survival; 29% knew that half of kidney recipients have problems with the transplant in the first 6 months; 86% knew the normal range of creatinine for kidney recipients; and 86% were aware of the risk of death within the first year of transplantation. conclusion: at this clinic, kidney transplant recipients generally had high levels of health literacy. however, most had difficulty recognizing frequently used transplantrelated terms, which could impede their understanding of health information and self-care management. greater efforts are needed to educate kidney recipients about transplant concepts, which may foster better self-care management, and ultimately transplant outcomes. abstracts by a single pathologist using the banff, cadi and cnit classifications. all indication biopsies with clinical acute rejection (ar; 23.3% for sf and 21.4% for sb) were excluded from this analysis. the histological and clinical parameters were assessed using multivariate generalized-estimating-equations statistical analysis. results: subclinical ar was present in 12.2% sf vs. 8.5% sb bx at 6 mo and 0% sf vs 5% sb bx at 12 mo (p=ns); borderline ar was seen in 2.4% sf vs. 8.5% sb bx at 6 mo and 10% sf vs. 5% sb bx at 12 mo (p=ns). despite the pristine condition of the kidneys at implantation, regardless of steroid exposure, there was a significant trend increase (p<0.0001) in chronic tubulo-interstitial damage; 35% of 6 mo bx and 53% of 12 mo bx demonstrated ifta; with moderate/severe changes (ifta grade 2-3) in 6.8% and 10% of 6 and 12 mo bx respectively. the prevalence of biopsies with ischemic glomerular changes (p<0.0001), tubular microcalcifications (p=0.009), vascular intimal thickening (p=0.0008) and the number of sclerosed glomeruli (p<0.0001) increased over the first year after transplantation, without any difference between the sb and sf group. a critical risk factor for ifta injury by multivariate analysis, independent of time after transplantation, was smaller recipient size. in this first ever serial histological analysis, embedded in a randomized multicenter pediatric study of steroid avoidance, we found significant progression of chronic graft injury in the first year post-transplantation in both study arms. small recipient size is the primary risk factor for tubulo-interstitial damage, likely related to vascular size discrepancies between recipient and the graft, resulting in chronic graft ischemia. in the 1-year symphony core study, a regimen with 2g mycophenolate mofetil (mmf) + low-dose tacrolimus (3-7 ng/ml) + daclizumab + steroids resulted in less acute rejections and better glomerular filtration rate (gfr) compared with 2g mmf + steroids and either standard-dose cyclosporine (csa), low-dose csa (50-100 ng/ml) + daclizumab or low-dose sirolimus (4-8 ng/ml) + daclizumab. methods: 960 patients participated in an optional follow-up of 2 years. gfr data from 79% of included patients (48% of the core itt population) were available at 3 years.here we present results in the itt population. results: at inclusion into follow-up 47%, 34% and 17% of patients received csa, tacrolimus or sirolimus, and at 3 years 37%, 31% and 14%, respectively. many follow-up patients had been switched to tacrolimus in the 1st year, including 24% of patients randomized to sirolimus. at 3 years 95% of patients were on mmf and 69% on steroids.over the 2nd and 3rd year all arms had a low rate of biopsy-proven acute rejection (bpar; 2-4%) and of graft loss (3-5%). low-dose tacrolimus remained clearly superior in terms of bpar (13% vs. 26%-38% in the other arms). uncensored 3-year graft survival was 89% with low-dose tacrolimus and low-dose csa, 87% with standarddose csa and 85% with low-dose sirolimus (p=0.19). patient survival was between 94% and 97% (p=0.52). in the four arms, the mean gfr change over the 2 nd and 3 rd year was between +1 and -3 ml/min and low-dose tacrolimus still had the highest gfr (69 vs 64-66 ml/min, p=0.15). observational follow-up results based on approximately half of the core symphony population indicate that during the 2nd and 3rd year renal function was stable, bpar and graft loss rates were low and many patients changed treatment regimen substantially. still, the itt arm with 2g mmf + low-dose tacrolimus + daclizumab + steroids was superior at 3 years with respect to renal function and graft loss but differences were less marked and statistically not significant. discovering histological evaluation of time zero donor kidney biopsies has not conclusively predicted graft outcome. we hypothesize that gene expression analysis could provide additional information to determine graft outcome in the first year of transplantation. to this end, we evaluated all 49 implantation biopsies obtained post reperfusion in 18 deceased donors (dd) and 31 living donors (ld) at our center. biopsies were evaluated and scored using banff criteria. low density real time pcr arrays were utilized to measure intragraft expression of 95 genes associated with programmed cell death, fibrosis, innate and adaptive immunity, and oxidative stress signaling. results of expression were defined as folds compared to a pool of 25 normal kidney biopsies. in dd, histological features of atn were more common (44%) than in ld grafts (6%; p<0.001), whereas arteriosclerosis was infrequent in both groups (6% and 15%, respectively), as well as the extent of glomerular sclerosis (0% and 2%). there was no association between these histological features and renal function at 1 year post transplant. not surprisingly, dd grafts displayed a pattern of gene expression remarkably different from ld, including an increased expression of complement protein c3 ( background: isa247 is a novel calcineurin inhibitor (cni), developed using a pharmacodynamic approach for use in autoimmune disease and solid organ transplantation. in moderate to severe plaque psoriasis, a canadian phase iii trial has demonstrated that isa247 is efficacious with minimal changes to renal function and a european trial is presently underway comparing isa247 to cyclosporine a (csa).in renal transplantation, a phase iia study comparing isa247 to csa in stable renal transplant recipients demonstrated isa247 to be efficacious and well tolerated. a phase iib study in de novo renal transplant patients comparing isa247 to tacrolimus is ongoing and final data will be available may 2008. we hypothesize that isa247 is non-inferior to tacrolimus in terms of efficacy.methods: this is a 6 month, randomized, multicenter, open-label, concentrationcontrolled study comparing three oral isa247 dosing groups (0.4, 0.6, or 0.8 mg/kg bid) to tacrolimus in 42 north american transplant centres. all cni's were titrated to target trough concentrations. inclusion criteria included males and (non-pregnant) females between the ages of 18-65 who were receiving a first deceased or living donor renal transplant. cold ischemia times were to be ≤ 24 hours, and peak panel reactive antibodies ≤ 30%. the primary efficacy parameter of the trial is non-inferiority (in at least one dose group) in biopsy proven acute rejection (bpar) at 6 months as compared to tacrolimus. secondary objectives include: renal function; pk/pd relationships; patient and graft survival; and proportion of patients with hypertension, hyperlipidemia or new onset diabetes mellitus (nodm).results: interim data, as previously presented at the 2007 atc, demonstrated that isa247 had rejection rates similar to tacrolimus (isa247 0.4 mg/kg bid 11%, isa247 0.6 mg/kg bid 8%, isa247 0.8 mg/kg bid 3%, tacrolimus 9%) and confirmed previous results indicating an improved safety profile. 334 patients have now been enrolled between january 2006 and june 2007, with an optional extension to 12 months added to the trial. a recent approval by both fda and health canada has allowed continued use of isa247 in these patients until commercialization. the six month final results will be available for presentation at atc 2008. key: cord-022888-dnsdg04n authors: nan title: poster sessions date: 2009-08-19 journal: eur j immunol doi: 10.1002/eji.200990224 sha: doc_id: 22888 cord_uid: dnsdg04n no abtract the humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. ptx3, the prototype of long pentraxins, plays a nonredundant role in resistance against a. fumigatus lung infection. the model proposed suggests that upon binding, ptx3 facilitates recognition, phagocitosis and killing of a. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly th1-oriented adaptive response. actually, ptx3-deficient mice are highly susceptible to aspergillosis and develop th2 skewed responses; moreover, ptx3-deficient resident macrophages and neutrophils show defective conidia phagocytosis. both in vitro and in vivo defects can be rescued by the administration of recombinant ptx3, which does not show direct activity on fungal cells. finally, ptx3 alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically. in the present study, we investigated the mechanisms underlying the ptx3-mediated opsonic activity and the involvement of complement, complement receptors and fcg receptors, by in vitro studies and genetic approaches in vivo. in vitro ptx3 amplified the complement-dependent effects on a. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. accordingly, in the presence of ptx3-opsonised conidia, cd11b activation, internalization, recruitment to the phagocytic cup and cd11b-dependent phagocytosis were increased. as pentraxins interact with fcgreceptors, which in turn can control cd11b activation, the phagocytic assay was performed in the presence of fcgr blocking abs. data obtained strongly suggest that upon conidia opsonisation with ptx3, fcgriia/cd32 mediates inside-out activation of cd11b and consequently phagocytosis of c3b-opsonised conidia. in vivo phagocytosis experiments performed with c1q-and fc common gamma chain-deficient mice and complement inhibitors support in vitro data. these data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, ptx3) and the cellular arm (fcgrs, cr3). moreover, they confirm previous studies on the interaction between pentraxins and fcgrs and support the idea that pentraxins behave as predecessors of antibodies. innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. the humoral arm of the innate immunity includes soluble pattern-recognition receptors (prrs) such as collectins, ficolins, complement components and pentraxins. the prototypic long pentraxin ptx3 is rapidly produced and released by diverse cell types in response to proinflammatory signals. ptx3 binds selected microorganisms such as aspergillus fumigatus and restores protective immunity against this pathogen in ptx3-/-mice. neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. a beneficial effect of breast feeding on newborn health is highly demonstrated. this protective effect is mediated by nutrients, immunomodulatory mediators (ifn-g, tnfa, or tgf-b), innate immunity factors (soluble cd14, immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. we thus hypothesized that milk may contain ptx3. we found high concentration of ptx3 in human colostrum (47.62 ± 13.5 ng/ml at day 1 post-delivery) compare to the one found in human serum ( x 2 ng/ml). the presence of ptx3 in human colostrum seems to be due to the secretion of ptx3 by human mammary gland since we report the production of ptx3 by these cells. this prr is also found in human milk cells (hmc), mainly in leukocytes, and penetrate into newborn tissus after suckling. furthermore, human colostrum upregulated the ptx3 production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their ptx3 production after lps injection. collectively, these data demonstrate that newborn have three distinct ways of ptx3 supplying by breast feeding: (i) soluble ptx3 in colostrum (ii) hmc that can secrete ptx3 upon stimulation in the specific tissue, (iii) an increase of ptx3 production by immune cells in the presence of colostrum. thus, soluble or cell-derived ptx3 may participate to the beneficial role of breast feeding on the newborn health. a. m. baru 1 , j. stephani 2 , h. wagner 2 , t. sparwasser 1 1 twincore, institute for infection immunology, hannover, germany, 2 technical university of munich, institute for medical microbiology, immunology and hygiene, munich, germany toll-like receptors (tlrs) represent the best characterized pattern recognition receptor family in mammalian species. the family currently comprise of 10 receptors in humans (tlr 1-10) and 12 in mice (tlr 1-9, 11-13). as transmembrane receptors, tlrs are expressed on the cell surface (tlr 1, 2, 4, 5, 6, (10) (11) (12) (13) and at endosomal membranes (tlr 3, 7, 8 and 9) . toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. bacterial dna has been shown to possess immunomodulatory activity about a decade prior to the identification of cpg motifs. about 5 years later to this, toll-like receptor 9 (tlr9) was identified and shown to be the receptor for unmethylated cpg dna which is present mainly in non-vertebrate genome. studies have defined potential role of tlr9 as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. one explanation for these discrepancies could be the fact that expression of human-tlr9 (hutlr9) is restricted to b-cells and plasmacytoid dendritic cells (pdcs) whereas murine-tlr9 (mutlr9) is also expressed on conventional dendritic cells (cdcs). consequently, tlr9 ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. by employing bacterial artificial chromosome (bac) technology, we generated transgenic mice with hutlr9 (henceforth called as hut9 mouse) integration in their genome under human epigenetic control. to avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto mutlr9 knock-out background. we expect that hut9-mutlr -/mice mimic the human specific expression pattern of tlr9, i. e. exclusively in b-cells and pdcs, allowing us to investigate detailed in vivo functions of hutlr9. by studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of tlr9 ligands in human immunotherapy. the fractal analysis provides unique physical insights into the interactions between c1q and the prp protein. if one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. if this is true, then surely, this is worth exploring further. any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as alzheimers is defintely worth the effort. interleukin-12 is a heterodimeric cytokine consisting of the two subunits p35 and p40. the main inducers of il-12p40 are microbial components activating toll-like receptors with the magnitude of il-12p40 induction depending on the specific tlr engaged. differential induction of il-12p40 upon tlr stimulation correlated with striking differences in the kinetics of nfkb activation. cpg-dna strongly induces il-12p40 due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal il-12p40 promoter region and (ii) to stimulate prolonged rela activity. here we were interested in further changes in chromatin structure of the il-12p40 promoter upon tlr triggering. we did not observe a change in dna methylation, but using chormatin immunoprecipitation (chip) we were able to detect a strong increase in histone 3 and 4 acetylation in specific regions of the proximal promoter region. acetylation of h4 showed a specific distribution pattern and occured mainly in regulatory elements within the il-12p40 promoter, whereas acetylation of h3 took place over all regions analyzed. tlr tolerance has been reported to be associated with specific chromatin alterations. methylation status of lysine residue 4 on h3 turned out to be important for the inhibition of gene expression upon repeated stimulation. modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses. a. d. koepruelue 1 , w. ellmeier 1 1 medical university of vienna, institute of immunology/division of immunobiology, vienna, austria macrophages are important in innate and acquired immunity. failures are associated with inflammatory and autoimmune diseases. understanding their stimulation is the basis for therapeutic targeting. members of the tec kinase family (bmx, btk, itk, rlk and tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. mutations in btk represent the source of human x-linked agammaglobulinemia (xla). a mutation in the murine btk gene accounts for a similar syndrome, x-linked immunodeficiency (xid). although the tec family members tec, btk and bmx are expressed in monocytes/macrophages, little is known about their function there. tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or tlrs. several studies in xla or xid macrophages and in monocyte/macrophage cell lines implicated roles for tec kinases in tlr signaling and as well as other macrophage effector functions like phagocytosis. inspired by these findings, we aim to determine the role of tec kinases in bone marrowderived macrophages (bmm), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. in a comprehensive functional analysis of tlr-mediated bmm activation from mice deficient for one or more of the tec family members in vitro, we reveal which of the kinases play a role in which tlr pathway. based on the results of this analysis, we set the goal to further study how tec kinases regulate the respective signaling cascades. our study will contribute insights into the role of tec kinases in this important cell population of the innate immune system. g. lunazzi 1 , m. buxadé 1 , j. minguillón 1 , r. berga 1 , j. aramburu 1 , c. lópez-rodríguez 1 1 universitat pompeu fabra, department of experimental and health sciences (dcexs), barcelona, spain nfat5 is a transcription factor that regulates the expression of cytokines such as tnfa and lymphotoxin b in response to osmotic stress. in addition, nfat5 participates in multiple processes not linked to the response to hypertonicity. in this regards, it has been recently reported that nfat5 is required as a novel host factor that supports hiv replication in macrophages. given the established connections between nfat5, the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether nfat5 could be activated by receptors for pathogens expressed in macrophages. the activation of toll-like receptors (tlrs) is central to innate immunity. upon stimulation of tlrs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. as a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. we have analyzed whether nfat5 is expressed in primary macrophages through the activation of different toll-like receptors. likewise, we have explored whether the activity of nfat5 is induced during the response to tlrs. in addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of tlrs could interfere with the expression of nfat5. our work indicates that nfat5 is a novel transcriptional regulator acting in response to the activation of tlrs. our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. objectives: compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by nf-xb, a master switch of inflammation. recent studies implicate some tlrs in tumor development or regression, and immune escape. however, mechanisms leading to tumor growth or apoptosis induced by tlr stimulation are not fully understood. several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. we hypothesized that some tlrs can contribute to lung inflammation and tumor development in vitro and in vivo. methods: tlr expression in lung cancer was assayed by immunohistochemistry or flow cytometry. nfxb activation was determined by western blot and nuclear translocation assay after tlr stimulation. clonogenicity of stimulated cells was analyzed by colony assay. transcriptomic analysis were performed by taqman lda technology. tumor growth in vivo was analyzed in nod/scid mice after subcutaneously engraftment of human lung tumor cell lines. we have observed that primary human lung tumors express tlr3, tlr4, tlr7 and tlr8 and that stimulation of these receptors in lung tumor cell lines by poly i:c, lps, loxoribine or poly u induces nfxb activation through atypical signaling pathway, with phosphorylation of ixba without its degradation and nuclear translocation of p50 and p65 nfxb subunits. interestingly, we observed that tlr3 stimulation induces apoptosis depending of the histological type of the tumor. on the contrary tlr4, tlr7 and tlr8 stimulation induces cell survival and increases clonogenicity. this is correlated with an up-regulation of bcl-2 expression. moreover, despite a common atypical activation of nfxb, our transcriptomic analysis revealed major differences in gene modulation after triggering of tlr3, tlr4, tlr7 and tlr8. finally, in vivo tlr7 stimulation of human lung tumor cells dramatically increases tumor size and metastasis. conclusions: altogether, these data emphasize that tlr4, tlr7 or tlr8 triggering can directly favor tumor development whereas tlr3 signaling can induce tumor cell death. these data suggest that anticancer immunotherapy using tlr adjuvants should take into account the expression of these tlrs in lung tumor cells. objective: dasatinib (bms-354825) is a small molecule src/abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and philadelphia chromosome-positive acute lymphoblastic leukaemia. members of the src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. the purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (tlr) signalling, natural killer (nk) cell cytotoxicity as well as antigen-specific cd8 + and cd4 + t cell function. methods: to analyse tlr signalling in vitro murine bone marrow derived (bmd) macrophages were stimulated with the tlr 4 ligand lipopolysaccaride (lps) in the presence of dasatinib and tumour necrosis factor a (tnf-a) in the culture medium was measured. the response to tlr stimulation was also tested in vivo, dasatinib-treated mice were challenged with lps and tnf-a in the serum was quantified. in addition, the clearance of the rma-s cells, a mhc class i deficient thymoma sensitive to nk cell lysis, was analysed in mice undergoing dasatinib treatment. to investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic cd4 + and cd8 + t cells specific for ovalbumin were utilised to measure antigen specific t cell proliferation. endogenenous cd4 + and cd8 + t cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. results: we show that dasatinib impairs: 1. innate immune response; dasatinib treatment reduced the (a) production of tnf-a following tlr 4 stimulation of bmd macrophages in vitro, (b) production of tnf-a in vivo in response to lps and (c) ability of nk cells to eliminate mhc class i deficient cells in vivo . 2. adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic t cells, (b) endogenous antigen-specific helper t cell recall-responses and (c) t cell-mediated cytotoxic effector function. conclusions: these findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock. i. zanoni 1 , r. oatuni 1 , m. collini 2 , m. caccia 2 , p. castagnoli 3 , g. chirico 2 , f. granucci 1 1 university of milano-bicocca, biotechnology and bioscience, milan, italy, 2 university of milano-bicocca, physics, milan, italy, 3 singapore immunology network (sign), biomedical sciences institutes, immunos, singapore, singapore the recognition of mamps by tlrs expressed on dendritic cells (dcs) plays an essential role for the regulation of the immune responses. by recruiting different combinations of adapter proteins, individual tlrs turn on signal transduction pathways leading to the activation of different transcription factors. interleukin-2 (il-2) is one of the molecules produced by dcs shortly after stimulation with different tlr agonists. based on this observation and by analogy with the events following t-cell receptor (tcr) engagement leading to il-2 production, we hypothesized that the stimulation of tlrs on dcs might lead to activation of the ca2+/ calcineurin and nfat pathway. we found that dc stimulation with lps induces extracellular ca2+ influxes, leading to calcineurin-dependent nfat activation. the activation of this pathway was independent of tlr4 engagement, depending instead exclusively on cd14. we also found that lps-induced nfat activation in dcs was necessary for the efficient synthesis of cyclooxygenase-2 (cox-2) that, by generating prostaglandins (pgs), such as pge2, regulates different dc functions including migration and polarization of t cell responses. our findings reveal novel aspects of the molecular signaling triggered by lps in dcs and define a new role for cd14. given the essential involvement of cd14 in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via cd14 represents a major step towards the development of potential treatments with modes of action involving interference with cd14 functions. we have examined the interaction of cd55, a 80-kda glycosyl-phosphatidylinositol (gpi)-anchored membrane protein, with the monocyte signalling receptor, cd14. human monocytes were isolated from healthy adult donor's peripheral blood. this involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. the cells were labelled at saturation with anti-cd14 antibody coupled to biotin visualised by qd-525-streptavidin and anti-cd55 antibody coupled to allophycocyanin. the images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. single particle fluorescence imaging (spfi) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. the images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that lps promotes the interaction of cd55 and cd14, suggesting a new functional role of cd55 as a member of a multimeric lps receptor complex. l. lundvall 1 , r.r. schumann 1 1 charité -universitätsmedizin berlin campus mitte, institute for microbiology and hygiene, berlin, germany meningitis is a life-threatening disease mainly caused by bacteria and viruses. bacterial components such as lipopolysaccharide (lps), lipoproteins or peptidoglycan breakdown products (i. e. mdp, mesodap) stimulate pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the intracellular nod-like receptors (nlrs) for an inflammatory response. we hypothesize that a synergistic effect of tlr-induced nf-xb activation and nlr-mediated caspase-1 induction leads to an increased release of mature il-1b during bacterial meningitis in brain-derived cells. a mouse meningitis model with s. pneumoniae (d39) was established for assessing il-1b induction during this disease. the murine raw 264.7 cell line, the human astroglial u-87 mg and the murine microglial cell-line bv-2 were stimulated with the tlr4 ligand lps, the tlr2 ligand pam 2 cys, the nod2 ligand mdp, or the nod1 ligand c12-ie-dap, and, as control, atp alone or in combination. we assessed il-1b by elisa and caspase-1 and pro-il-1b expression by western blot. furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by sirna suppression of elements of the il-1b induction pathway. s. pneumoniae (d39) infected mice showed a significant increase in il-1b release after 24 hours. in vitro, an increase in il-1b levels after costimulation with lps or pam 2 cys, and mdp or c12-ie-dap was observed in a dose-dependent manner. a synergistic enhancement of il-1b by tlr-and nlr-ligands was observed in raw cells, bv-2 cells, u-87 cells and primary astrocytes. active caspase-1 (p10) was induced by mdp or c12-ie-dap, corresponding with high il-1b responses when lps or pam 2 cys was added. sirna experiments show that a knock-down of nod2 leads to a diminished il-1b release after lps-and mdp-stimulation. the precursor forms of il-1b and caspase-1 seem to be constitutively expressed in astrocytes and microglia. a synergistic enhancement between tlrs and nlrs in il-1b release in brain-derived cells was observed. so a two-step stimulation seems necessary for the release of high levels of mature il-1b by astrocytes and microglia. bacteria containing both, tlr-and nlr-ligands thus have the potential to induce high levels of il-1b which may contribute to disease pathology and may point to novel intervention strategies. j. rosenberg 1 1 toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i-like (rlrs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . in the case of tlrs most sutides to date have delineated tlr expression and function on antigen presenting cells like dendritic cellof this research. extension of the profiling and presence of tlrs on cell characterized as adaptive immune cells such as t cells is the subject of this line of research. using a cd3 and cd28 activation model system -tlr4 presence on cd4+ cells is found in mouse t cells, human t cells and jurkat cell lines. following cd3/cd28 activation for 48 hours we have identified a small but distinct populationof tlr4+ cells. further characterization indicates these cells to be cd4+cd25+ cells. further characterization of the expression and functional acitvity of the tlr4+ t cells indicates co-expression of tlr4 with md-2 indicating a functional tlr4 receptor. in addition lps activiation did not lead to upregulation of tlr4 expression in t-cells. the data indicate that tcr activation leads to tlr4 expression. the expression appears to be associated with cd4+cd25+ cells and refelecting an activated t cell phenotype which will be further characterized as perhaps related to tregs or other tcell subsets. s. m. lehmann 1 , d. kaul 1 , c. krüger 1 , f. zipp 1 , r. nitsch 2 , s. lehnardt 1 1 charité-universitätsmedizin berlin, cecilie-vogt-clinic for neurology, berlin, germany, 2 charité-universitätsmedizin berlin, institute for cell biology and neurobiology, berlin, germany the innate immune system is the first line of defense against various pathogens and requires the expression of toll-like receptors (tlrs). in macrophages, tlr7 plays a crucial role in immune responses elicited by gu-rich ssrna (i. e. ssrna40) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). these compounds were initially described to activate mouse tlr7 (and human tlr8) and are potent immune response modifiers leading to important antiviral and antitumor activities. microglia serve as the major innate immune cells in the central nervous system (cns). employing various techniques including pcr, in situ hybridization, and immunocytochemistry, we demonstrate that tlr7 is expressed in these cells. incubation of microglia with all three of the above mentioned tlr7 ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as tnf-a and il1-b in a dose-and time-dependent fashion. analysis of wild type (wt) and tlr7 knock out (ko) microglia by realbecause neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. using a proteomic approach, coronin-1 was identified as a cytosolic protein cleaved during neutrophil apoptosis. coronin-1 is an actinbinding protein that can associate with phagosomes and nadph oxidase but its involvement in apoptosis was currently unknown. in coronin-1-transfected plb985 cells, coronin-1 overexpression did not modify the kinetics of granulocyte differentiation as assessed by cd11b labeling. concerning apoptosis, increased coronin-1 expression in dmf-differentiated plb985 significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. likewise, coronin-1 significantly decreased trail-induced apoptosis with less mitochondrial depolarization, caspase-3 and caspase-9 activities, but not caspase-8 or bid truncation suggesting that coronin-1 interfered with mitochondria-related events. to validate the prosurvival role of coronin-1 in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (cf) patients were studied. circulating neutrophils from cf patients had more coronin-1 expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. this was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. in addition, inflammatory neutrophils from cf patients lungs showed an intense coronin-1 immunolabeling. we concluded that coronin-1 could constitute a potential target in resolving inflammation. p.-n. hsu 1 1 national taiwan university, graduate institute of immunology, taipei, taiwan, republic of china human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (tnf) ligand superfamily members and their receptors. many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. recent evidence indicates that the tnf-related apoptosis-inducing ligand (trail) of the tnf ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated t cells. we show in this work that trail can induce osteoclast formation from human monocytes and murine raw264.7 macrophages. we demonstrated that both cell models differentiate into osteoclast-like cells in the presence of trail in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (trap)-positive multinucleated cells and bone resorption activity. the trail-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. however, trail-induced osteoclastogenesis is dependent on activation of nf-xb, erk, and p38 map kinase. the trail-induced osteoclastogenesis was significantly inhibited by treatment with traf-6 sirna and traf-6 decoy peptide, indicating this pathway is traf-6 dependent. thus, our data demonstrate that trail induces osteoclast differentiation via direct engagement with the trail death receptor through a signaling pathway distinct from apoptosis. our results indicate that in addition to triggering apoptosis, trail induces osteoclast differentiation. it provides a novel role for trail in regulating osteoclast differentiation and in osteoimmunology. microglia are considered to be the local antigen presenting cells (apcs) of the central nervous system (cns) which are thought to play a crucial role in local reactivation of autoreactive t cells during cns autoimmunity e. g. in multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) . in this study we investigated if the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor gamma (pparg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. sustained activation of pparg both reduced microglial signalling via mhc molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules b7-h1 and b7-dc. moreover, also production of pro-inflammatory cytokines like tnf-a and il-6 was profoundly reduced if microglia were pre-treated with the pparg-agonist pioglitazone (pio). in contrast to this, the lack of pparg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that pparg might play a cell-intrinsic role in controlling microglia immunogenicity. importantly, if pparg was activated in microglia, the capacity to prime ovalbumin-specific t cells was impaired. t cells primed by pio-treated microglia produced reduced amounts of il-2 and ifn-g which could not be overcome by restimulation with acd3. this indicates that t cells primed by pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. furthermore, microglia were able to induce antigen-specific differentiation of naive cd4 t cells into t helper 17 (th17) cells, which have been associated with autoimmune pathogenicity during eae. however, if pparg was activated, microglia were no longer able to induce th17 differentiation. in conclusion, activation of pparg impairs microglial apc function leading to reduced activation of antigen-specific t cells and, in addition, inhibits the induction of th17 cells. therefore, activation of pparg in microglia is a promising approach to limit local activation of autoreactive t cells in the cns in cns-autoimmune deseases. bacterial lipopolysaccharide (lps) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. however, once exposed to lps, they become hyporesponsive to a subsequent endotoxin challenge. this phenomenon is defined as lps desensitization or tolerance. previous studies have identified some components of the biochemical pathways involved in negative modulation of lps responses. in particular, it has been shown that the il-1 receptor-related protein st2 could be implicated in lps tolerance. the natural ligand of st2 was recently identified as interleukin-33 (il-33), a new member of the il-1 family. in this study, we investigated whether il-33 triggering of st2 was able to induce lps desensitization of mouse macrophages. we found that il-33 actually enhances the lps response of macrophages and does not induce lps desensitization. we demonstrate that this il-33 enhancing effect of lps response is mediated by the st2 receptor since it is not found in st2 ko mice. the biochemical consequences of il-33 pretreatment of mouse macrophages were investigated. our results show that il-33 increases the expression of the lps receptor components myeloid differentiation protein-2 (md2), cd14 and tlr-4 and the myeloid differentiation factor 88 (myd88) adaptor molecule. in addition, il-33 pretreatment of macrophages enhances the cytokine response to tlr-2 but not to tlr-3 ligands. thus, il-33 treatment preferentially affects the myd88-dependent pathway activated by the tlr. c. klotz 1 , b. lenz 1 , r. lucius 1 , s. hartmann 1 1 humboldt-university berlin, molecular parasitology, berlin, germany chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. recently we showed that recombinant parasite cystatin (avcystatin), derived from the model parasite acanthocheilanema viteae, effectively abolished ova-induced allergic airway responsiveness in a mouse model of asthma (schnoeller et al., 2008) . the cystatin effect was blocked by the application of anti-il-10 receptor antibodies and by depletion of macrophages using clodronate liposomes. we hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive interleukin-10 (il-10). the aim of the present study was to elucidate the molecular mechanisms by which avcystatin induces il-10 in primary macrophages. in vitro experiments with peritoneal macrophages from balb/c mice confirmed specific and concentration dependent il-10 production after avcystatin stimulation. application of specific inhibitors revealed that the il-10 induction was p38 and erk dependent, and inhibitor titration indicated a higher sensitivity towards p38. western blotting experiments confirmed the phosphorylation of p38 and erk in macrophages after avcystatin stimulation. in addition, by using specific inhibitor and western blotting, we showed that avcystatin induced il-10 is also regulated by the phosphatidylinositol-3-kinase (pi3k) -proteine kinase b (akt) pathway. further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. hence, we conclude that avcystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of il-10 and possibly other regulatory markers. in general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. we screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. this screen revealed the impact of several novel kinases as well as kinases with previously established function. one of the top candidates identified to block endotoxininduced tnf-a secretion was carkl, a gene with no previously described function. subsequent biochemical analyses unequivocally revealed that carkl is a phosphotransferase protein using sedoheptulose as a phosphate acceptor and atp as a donor. sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. the product sedoheptulose-7-phosphate (s-7p) is also an intermediate metabolite of the pentose phosphate pathway (ppp) and so far was only known to be produced by condensation of ribose-5p and xylulose-5p via a transketolase reaction. to identify the molecular mechanism by which carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. so far, our data favor a model where post-stimulatory downregulation, i. e. loss of carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. disentangling the signaling pathways responsible for the rapid regulation of carkl unearthed nf-kb and p38/jnk but not erk as driving forces. counterbalancing endotoxin induced loss of carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. comparison of wild type and catalyticinactive forms of carkl unveiled that most of the effects of carkl on the inflammatory response were due to its phosphotransferase activity. expression profiling using gene chip analysis further supported the concept that carkl may represent a new key modulator of inflammatory processes. taken together, detailed analyses to study the molecular function of carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. in addition, connecting the ppp and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. in this context, the sedoheptulose kinase carkl and its product s-7p may provide a novel basis for interfering with adverse immune responses. t. bosschaerts 1 , y. morias 1 , p. de baetselier 1 , a. beschin 1 1 vib, cmim, vub brussels, brussels, belgium the development of classically activated monocytic cells (m1) is a prerequisite for effective elimination of parasites, including african trypanosomes. however, persistent m1 activation causes pathogenic damage including liver injury during infection, resulting in death of the host. we aim to identify mechanisms involved in regulation of m1 activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. methods: we have scrutinized the phenotype and cellular origin of liver m1 in trypanosoma brucei infected by facs analysis and bone marrow transfer experiments. the contribution of different signaling pathways, including myd88, ifng, il-10, ccr2 and nf-kb to the development and/or recruitment of pathogenic m1 in the liver was investigated using knock-out mice or by delivering il-10 in infected mice. results: we established that cd11b+ly6c+cd11c+ tnf and inos producing dcs (tip-dcs) represent the major m1 liver subpopulation. tip-dcs differentiated in an ifng/myd88-dependent manner from cd11b+ly6c+ inflammatory monocytes in the liver of infected mice. ccr2 promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to tip-dcs in the inflamed liver. as a consequence, ccr2 ko mice experienced reduced pathogenic symptoms. on the other hand, the absence of il-10 enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to tip-dcs, resulting in exacerbated pathogenicity and early death of the host. in addition, the therapeutic liver-specific delivery of il-10 in t.brucei infected mice efficiently limited the differentiation and maturation to tip-dcs, hereby limiting disease-associated pathogenicity. finally, the absence of the nf-kb member p50 was associated with increased tissue injury associated with increased production of pathogenic tnf and no by inflammatory monocytes, but not by tip-dcs. conclusion: our data demonstrate that nf-kb p50 and il-10 play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic m1, in particular tip-dcs. the inflammatory activity of liver m1 is controlled by il-10 and/or p50 nf-kb at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic tip-dcs, or their production of tnf and no. a. popov 1 , j. driesen 1 , z. abdullah 2 , a. niño castro 1 , t. chakraborty 3 , m. krönke 4 , o. utermöhlen 4 , c. wickenhauser 5 , j.l. schultze 1 1 limes institute, laboratory for genomics and immunoregulation, university of bonn, bonn, germany, 2 institute of molecular medicine and experimental immunology, bonn, germany, 3 institute of medical microbiology, university of giessen, giessen, germany, 4 institute for medical microbiology, immunology and hygiene, university of cologne, cologne, germany, 5 institute for pathology, university clinic leipzig, leipzig, germany dendritic cells (dc) and macrophages play an important role in pathogen sensing and antimicrobial defense. here we report on a new role for the myeloid antigen presenting cells (apc) in granulomatous infections. infection of myeloid dc and macrophages with listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine 2,3-dioxygenase, cyclooxygenase-2 and cd25 and production of prostaglandin e2 (pge 2 ) and interleukin 10. all these molecules are strictly dependent on autocrine tnf, released during infection, and are in concert suppressing t-cell responses; cd25, expressed by regulatory myeloid cells, acts as an il-2 scavenger. importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. induction of regulatory and protective phenotype in macrophages and dc require at least two signals provided by tnf and either pge 2 or tlr ligands. transcriptional changes in human macrophages, infected by mycobacterium tuberculosis, resemble the ones induced in dc during infection with l.monocytogenes. in fact, granuloma in patients with tuberculosis and listeriosis are enriched for cd25 + ido + cox-2 + regulatory myeloid cells, whereas most effector cell populations, such as t cells, b cells and nk cells, are expelled from the granuloma. of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. altogether, our studies provide strong evidence that intracellular pathogens such as m.tuberculosis and l.monocytogenes induce a specific polarization of myeloid dc and macrophages characterized by a functional preponderance of inhibitory mechanisms. we postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. on one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated t cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. the interferon-gamma (ifn-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. induction of ifn-g secretion by human t and nk cells through synergistic co-stimulation with interleukin 12 (il-12) and il-18 in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with nk or t cells in the relevant experiments. the possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. to resolve this issue, we have determined the production of ifn-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (elispot) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of il-12 and il-18 or with macrophage-colony stimulating factor (m-csf) were able to produce ifn-g when further stimulated with a combination of il-12 and il-18. in addition, naturally activated alveolar macrophages immediately secreted ifn-g upon treatment with il-12 and il-18. therefore, human macrophages in addition to lymphoid cells contribute to the ifn-g response, providing another link between the innate and acquired immune response. a. j. denzel 1 , m. rodriguez gomez 2 , m. niedermeier 2 , y. talke 2 , n. göbel 2 , k. schmidbauer 2 , m. mack 2 1 unversity hospital regensburg, internal medicine ii, regensburg, germany, 2 university hospital regensburg, regensburg, germany we have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of il-4 and il-6. activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin fc-receptors. we now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. peripheral blood basophils are markedly reduced during the first hours after restimulation. in the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to 24h, with basophils in the bone marrow binding the lowest amount of antigen. depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. our datas show that basophils are recruited to draining lymph nodes during a memory response. tnf-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including mycobacterium tuberculosis. it is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. three tnf-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against tnf-a (adalimumab and infliximab); the other is a soluble tnf receptor/fc fusion protein (etanercept) . tnf-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. we studied the effects of tnf-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. all three drugs had an inhibitory effect on ifn-g-induced phagosome maturation in pma-differentiated human thp-1 cells infected with m. bovis bcg, the avirulent m. tuberculosis h37ra strain and the virulent m. tuberculosis h37rv strain. adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (mdm) in the presence or absence of ifn-g. macrophages secreted tnf-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with ifn-g. treatment of infected macrophages with tnf-a increased maturation of mycobacteria-containing phagosomes. these results suggest a role for tnf-a in activating phagosome maturation and highlight a novel mechanism through which tnf-a blockade can affect the host innate immune response to mycobacteria. z. g. dobreva 1 , l.d. miteva 1 , s.a. stanilova 1 1 trakia university, faculty of medicine, molecular biology, immunology and genetics, stara zagora, bulgaria il-23 is a heterodimeric cytokine composed of a p19 subunit associated with the il-12/23p40 subunit. like il-12, il-23 is expressed by the activated antigenpresenting cells and both cytokines induce ifn-gamma secretion by different t cell subsets. the proper balance between il-12p40-related cytokines controls the appearance of normal th 1 and pathological th 17 mediated immune responses. in this study, we examined the dynamics of inducible il-12p40 and il-23p19 mrna expression and protein production in purified human monocytes and how jnk and p38 mapks inhibitors influenced il-12p40 and il-23 production. the cytokines' quantity determination was performed by elisa. quantitative real-time polymerase chain reaction (qrt-pcr) was performed for mrna transcripts detection. results were calculated in fold increase compared with gene expression in nonstimulated monocytes. il-23p19 gene expression was higher than those observed for il-12p40 gene at all time-points. the level of il-23p19 mrna increased after 6 th h and reaching a maximum level at 9 th h (43.4 fold for c3bgp and 22.7 fold for lps). c3bgp and lps triggered il-12p40 gene transcription were almost equal at the 3 rd h (4.4 and 4.1 fold) and at 9 th h (7.8 and 7.9 fold, respectively) after stimulation. the higher level of il-12p40 gene expression was detected at 6 th h in lps compared to c3bgp stimulated monocytes (8.1 vs. 4.9 fold). however, il-12p40 and il-23 protein production was increased in the highest level after c3bgp stimulation. the inhibition of p38 led to the statistical significant augmentation of c3bgp stimulated il-12p40 production. the inhibition of the same map kinase enhanced lps stimulated il-12p40 production without significant difference. the inhibition of jnk and p38 mapks significantly decreased c3bgp stimulated il-23 production from human monocytic cells.in summary, the present study demonstrates the different time-course and ability of c3bgp and lps to induce the expression of il-12p40 and il-23p19 mrnas in purified human monocytes. we showed that inhibition of p38 mapk down regulated il-23 and upregulated il-12p40 production in stimulated monocytes. we concluded that in human monocytes p38 map kinase activation has an opposite effect on the il-12p40 and il-23p19 expression. neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, t and b cells. for instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (modc). indeed, upon interaction with neutrophils, modc up-regulate the expression of costimulatory molecules, such as cd83, cd86 and cd40, and secrete il-12, thus acquiring the ability to induce proliferation and th1 polarization of naï ve t cells. in order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. in human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc), the latter further divided in three different subsets based on the expression of cd1c, bdca-3, and cd16. by analyzing different chronic inflammatory pathologies, such as crohn's disease, psoriasis and sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mdc) with characteristics resembling the cd16+ subset of mdc. in order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and cd16+ dc were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. here we show that neutrophils modulate different effector functions of cd16+ dc, including their survival and their ability to produce il-12p70. besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. chitin is a linear polymer of n-acetyl-d-glucosamine (glcnac) residues present in human pathogenic fungi or nematodes. chitotriosidases (cht) and acetic mammalian chitinase (amcase) have been identified as the only functional chitinases in mammalians. the expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. amcase is considered as predominant chitinases in mice while cht is regarded as major chitinases in humans. interestingly, cht is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. although, amcase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of cht in humans is virtually unknown. to evaluate the physiological function of cht we have characterised the substrate specificity of human cht and the mode of substrate cleavage by analysing chtproduced fragments of chitosan, a close but water soluble derivate of chitin. degradation products of chitosan have been investigated by gel electrophoresis and maldi-tof mass spectroscopy. moreover, the application of a computer-based model of cht activity revealed the mode of substrate cleavage. we found that cht is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. in further studies we could show that those cht-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines tnf-a, il-6, il-8 and mcp-1 involving the transcription factor nfxb. moreover, these stimulated monocytes/macrophages revealed an increase of cht expression indicating an autocrine positive feed-back regulation. our data suggest that human cht is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments. m. hasenberg 1 , s. wolke 2 , a. brakhage 2 , m. gunzer 1 1 otto-von-guericke universität, institut für molekulare und klinische immunologie, magdeburg, germany, 2 hans-knöll-institut, abteilung für molekulare und angewandte mikrobiologie, jena, germany since their discovery in 2004 nucleic extracellular traps (nets) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. recently it was found by us and others that neutrophil granulocytes release nets also upon contact to the filamentous fungus aspergillus fumigatus. in the present study we aimed to characterize this process in more detail focusing on the kinetics of net-formation as well as clarifying the responsible cell-biological mechanisms. by the use of several microscopic techniques (scanning electron microscopy, fluorescence widefield microscopy, confocal-and 2-photon microscopy) we initially demonstrated the generation of net like structures after coincubation of a. fumigatus germlings and freshly isolated murine or human pmn. the analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of nets up to 3 hours later. moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. therefore we added the nadph-oxidase inhibitor dpi to the cell coincubation and found clearly reduced net formation. by fluorescence staining of reactive oxygen species we could demonstrate that ros are released prior to net detection. interestingly, our data currently suggest that in contrast to other pathogens investigated so far, nets are not directly toxic to fungal elements. whether and how nets control growth of a. fumigatus currently remains open. to summarize our data, we found rapid net formation as a commonly observed immune response of neutrophil granulocytes contacting a. fumigatus. consistent with studies on different pathogens this mechanism seems to be ros-dependent, however not toxic for the fungus. thus, in the future we will have to clarify whether net-formation really occurs in vivo and how nets can control the outgrowth of a. fumigatus at sites of infection. production of type i interferons (ifn-i, mainly ifn-a and ifn-b) is a hallmark of innate immune responses to all classes of pathogens. when viral infection spreads to lymphoid organs, the majority of systemic ifn-i is produced by a specialized 'interferon-producing cell' (ipc) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pdc). it is unclear whether production of systemic ifn-i is generally attributable to pdc irrespective of the nature of the infecting pathogen. we have addressed this question by studying infections of mice with the intracellular bacterium listeria monocytogenes. protective innate immunity against this pathogen is weakened by ifn-i activity. in mice infected with l. monocytogenes systemic ifn-i was amplified via ifn-b, the ifn-i receptor (ifnar) and transcription factor interferon regulatory factor 7 (irf7), a molecular circuitry usually characterisitic of non-pdc producers. synthesis of serum ifn-i did not require tlr9. in contrast, in vitro differentiated pdc infected with l. monocytogenes needed tlr9 to transcribe ifn-i mrna. consistent with the assumption that pdc are not the producers of systemic ifn-i, conditional ablation of the ifn-i receptor in mice showed that most systemic ifn-i is produced by myeloid cells. furthermore, results obtained with facs-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pdc is responsible for bulk ifn-i synthesis. the amount of ifn-i produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. based on these data we propose that the engagement of pdc, the mode of ifn-i mobilization, as well as the shaping of the antimicrobial innate immune response by ifn-i differ between intracellular pathogens. t. naessens 1 , s. vander beken 1 , p. bogaert 1 , j. grooten 1 1 ghent university, biomedical molecular biology, zwijnaarde (ghent), belgium introduction: although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. recent insights in monocyte and macrophage heterogeneity render the picture even more complex. in addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. in this study we focused on the dynamics and function of resident alveolar macrophages (ram) during and after allergic bronchial inflammation. strategy: we used an ovalbumin (ova)-alum based mouse model of allergic asthma and an ova-complete freund's adjuvant (cfa) based mouse model of hypersensitivity pneumonitis, constituting a th1-driven immunological counterpart of the th2-driven experimental asthma. ram were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. as complementary approach, ram and elicited alveolar macrophages (eam) were distinguished using cd45 bone marrow chimeric mice. combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited am populations in the course of th1-and th2-driven allergic airway inflammation. results: during the acute phase of the allergic response, isolated ram and eam showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. in both types of allergic inflammation, microsphere-tagged cd45.1 + ram remained constant in cell number for the first 2 days of chronic ova-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day 4 of ova-exposure. as a consequence, following the clearance of inflammation, inflammation-experienced ram replaced the initial ram population. strikingly, in both types of allergic inflammation, this secondary ram population showed a markedly altered responsiveness to lps stimulation. this involved macrophage activation markers and nf-kb inducible inflammatory genes. however, especially genes induced by ifn-beta showed strongly increased expression in secondary ram as opposed to their near lack of induction in primary ram. this switch from an ifn-beta deficient to an ifn-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection. e. schlecker 1 , a. stojanovic 1 , a. cerwenka 1 1 german cancer research center, innate immunity, heidelberg, germany myeloid-derived suppressor cells (mdsc) are a heterogeneous population of cells that expand during cancer, inflammation and infection. these cells play a critical role in suppressing t cell responses. the exact nature and function of mdsc remain unclear. here we show that a subpopulation of mdsc (gr-1 + cd11b + f4/80 + ) isolated from rma-s tumor-bearing mice did not suppress but rather activated nk cells to produce ifn-g. additionally, nk cells eliminated this subpopulation both in vitro and in vivo. in order to identify molecules and pathways that might be involved in mdsc accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of mdsc subpopulations was performed using whole genome microarrays. understanding the reciprocal interaction of mdsc with nk cell could improve the efficiency of cancer immunotherapy. g. solinas 1 , f. marchesi 1 , m. fabbri 1 , s. schiarea 2 , c. chiabrando 2 , a. mantovani 1,3 , p. allavena 1 1 istituto clinico humanitas, rozzano, italy, 2 istituto mario negri, milano, italy, 3 università di milano, milano, italy experimental and clinical evidence has highlighted that tumor-associated macrophages (tam) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. macrophage population is generally divided into two distinct subsets: m1 and m2. m1 macrophages act as a first line of defence against pathogens whereas m2 cells participate in wound repair and maintenance of tissue integrity. in the tumour micro-environment tam interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. to investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. in selected cultures, about 50 % of the monocytes differentiated after 5 days into macrophages. the phenotype analysis of tumor-conditioned macrophages (tc-macro) demonstrated high expression of the mannose receptor, cd16, cd68 and low levels of mhc class ii. tc-macro produced il-10, il-6, tnf but not il-12, even after lps stimulation. moreover, tc-macro produced a panel of chemokines including ccl2, cxcl8, ccl17 and cxcl10. the transcriptional profile of tc-macro revealed that several genes in line with an m2 polarization are highly expressed. the nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( g 100.000 kda). il-3 and il-6 were not detectable in tumor supernatants whereas m-csf was present at low levels. by mass spectrometric techniques, we surprisingly found that the tumor-derived m-csf had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. the characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. genomic effects of glucocorticoid hormone (gc) are exerted by glucocorticoid receptor (gr)-mediated changes of gene expression. this is relatively timeconsuming process, needing hours to develop. in contrast, non-genomic effects may occur within minutes. gcs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. moreover, many cells and cell lines of haemopoetic origin are sensitive to gc-induced apoptosis. recent findings indicate, that non-genomic gc effects mediated by mitochondrial gr may have important function in generating pro-apoptotic signals. we aimed the investigation of non-genomic gc effects on in vitro cultured rbl-2h3 rat mast cell line. we demonstrate that gr nuclear translocation begins within 5 minutes and completes after 30 minutes in dx treated rbl-2h3 cells. since genomic effects occur in the nucleus through gene expression changes, we considered gc effects within 5 minutes as non-genomic. studying gc-caused apoptosis, rbl-2h3 cells proved to be gc-resistant and no mitochondrial gr translocation neither impaired mitochondrial function could be observed upon gc treatment. in further experiments we used rbl-2h3 cells sensitized with anti-dnp (dinitrophenyl) ige and dnp-conjugated bovine serum albumin was used for stimulation. 5 minutes of dx treatment inhibited ca 2+ -signaling in antigen stimulated rbl-2h3 cells in the concentration range of 100nm -10mm. moreover, 5 minutes of dx treatment altered the tyrosine phosphorylation pattern of rbl-2h3 cells. dx treatment alone caused slight increase in tyrosine phosphorylation, while dx treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. the tyrosine kinase syk plays indispensable role in regulating mast cell activation through the fc[epsilon] receptor i. our immunoprecipitation studies show, that dx treatment results in decreased syk phosphorylation in both resting and activated cells. this finding raises the possibility, that syk phosphorylation thus kinase activity may be directly or indirectly regulated by gcs via non-genomic pathway. taken together, our experiments along with the clinical experiences suggest that gcs rapidly influence mast cell activation via a non-genomic pathway, too. the elucidation of the exact signal transduction mechanisms behind rapid gc effects need further experiments. high mobility group box 1 (hmgb1) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. notably, hmgb1 is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin (1) . extracellular hmgb1 can act as a cytokine and a chemoattractant, mediating inflammatory responses. interestingly, hmgb1 exerts antibacterial functions in human adenoid and testis (2) . recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (nets), made of dna fibers, nuclear proteins (histones) and granule proteins. it has been shown that histones on nets have an anti-microbial activity (3). we asked whether hmgb1 from neutrophils is a component of nets and whether it has a function in nets. we purified human primary neutrophils from peripheral blood of healthy volunteers on ficoll gradients. to induce net formation, we stimulated cells for 40 or 120 minutes with 25 nm phorbol ester (pma), 100 ng/ml interleukin 8 (il-8), or 100 ng/ml lps. the presence of nets was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (mpo) and against a dna-histone h2a-histone h2b complex. dna was stained with hoechst. using a polyclonal antibody we found hmgb1 in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of nets induced by all stimuli. elisa assays revealed that hmgb1 is not present in the supernatants of activated neutrophils, confirming its binding to nets. in conclusion, we found that hmgb1 localizes on nets. we hypothesize that net-bound hmgb1 might exert a direct antimicrobial function, or that nets might concentrate hmgb1 locally to recruit macrophages to the site of infection. these receptors were present on the mast cell surface. incubation (37°c, 3 h) of hlmc with vegf-a, vegf-b, vegf-c, vegf-d and placental growth factor-1 induced concentration-dependent chemotaxis that was blocked by a combination of anti-vegfr-1 and anti-vegfr-2 antibodies. these data indicate that human mast cells represent both a source and a target of vegfs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors. macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as mycobacterium tuberculosis. a very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the ifn-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. these include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. we hypothesize that the very early steps of pulmonary infection directs the outcome of disease. therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. preliminary data show that m. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. cells harboring mycobacteria are found during early and late stages of infection. both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of il-18. altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. human cd1 molecules are dedicated to lipid presentation to t cells and are implicated in inflammatory and auto-immune responses. the cd1a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. our previous studies have demonstrated that ii associated with cd1a and cholesterol-dependent lipid rafts impact on cd1a surface expression and cd1a-restricted t cell response. bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated dcs acquire the ability to stimulate cd1-restricted autoreactive t cells. this mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by cd1 group i molecules. the functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by cd1a molecules could impact in such pathologies. we have thus conducted a preliminary study to understand the implication of cd1 molecules in multiple sclerosis. we first analyzed cd1 expression on monocytes from 16 ms patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. results obtained in this preliminary study demonstrate that cd1a was not expressed on ms patient monocytes, while the other members of the cd1 family were expressed. moreover ms sera and plasma induced an earlier and more rapid dendritic cell differentiation than ab sera. these preliminary results confirm our hypothesis that cd1 molecule expression is modified in ms and also reveal that serum from patients with ms modifies lipid-antigen presenting cells. further studies should contribute to define precise mechanisms involved in lipid presentation by cd1 molecules in this context. c. ohnmacht 1 , d. voehringer 1 1 ludwig-maximilians-universität munich, institute for immunology, munich, germany basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. however, their development and in vivo functions are largely unknown. here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth nippostrongylus brasiliensis. for this purpose, brdu incorporation experiments and in situ fluorescence microscopy of il-4 reporter (4get) mice as well as in vivo depletion of basophils are used to uncover their role during type 2 immune responses. our results demonstrate that under homeostatic conditions basophils have a lifespan of about 60h. n. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of n. brasiliensis in the absence of th2 cells. these results demonstrate that basophils play a crucial role as effector cells in type 2 immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. during acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. neutrophils show pro-inflammatory activity and may contribute to tissue damage. in pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. this damage may be due to excessive neutrophil activity. we here show that transgenic expression of bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. the persistence of neutrophil brain infiltrates was accompanied by high levels of il-1beta and g-csf as well as reduced levels of anti-inflammatory tgf-beta. significantly, bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. in vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. the inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. in wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. these results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. objectives: to investigate the existence of systemic inflammatory response to subchronic oral warfarin (wf) consumation in rats. methods: dark agouti (da) rats were treated with warfarin in drinking water (10 mg and 100 mg daily) for 30 days. oxidative activity (cytochemical nbt reduction) and myeloperoxidase (mpo) intracellular content of peripheral blood neutrophils, plasma levels of il-6 and tnf-a (elisa) and superoxide dismutase (sod) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. changes in prothrombin time (pt), as basic biological warfarin activity was determined as well. results: significantly increased pt was noted at the lower wf dose, with tremendous rise after the higher dose. increase of pma-stimulated neutrophil nbt reduction capacity (neutrophil priming) was noted at both wf doses, while increase in mpo intracellular content was noted at the higher wf dose solely. warfarin consumation resulted in no changes in plasma levels of il-6 and tnf-a. significant decrease in the sod activity was detected in red blood cell lysates at both wf doses, suggesting systemic oxidative activity. conclusion: increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. this work is funded by serbian ministry of science and technological development (grant 143038). objectives: although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. we have recently reported that immune complexes (ic) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. in disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with ic. here we address the consequences of ic opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type-1 macrophages, mph1), and during resolution of inflammation (type-2 macrophages, mph2). methods: mph1 / 2 were generated by culturing cd14 + human monocytes for 6 days in the presence of gm-csf or m-csf, respectively. phagocytosis by mph1 / 2 of ic opsonised and unopsonised neutrophils was assessed by flow cytometry. after phagocytosis mph1 / 2 were stimulated with lps and secreted il-6, il-8, il-10, il-12p40 and tnf were quantified by elisa. results: mph2 are relatively efficient phagocytes for apoptotic neutrophils whereas mph1 are only poorly phagocytic. opsonisation with ic leads to enhanced neutrophil uptake by both mph1 and mph2 which is specifically inhibited in the presence of a blocking mab for macrophage fcyrii. uptake of ic opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. in both macrophage subsets il-6, il-12 and tnf production is suppressed while il-10 secretion is increased. in contrast, engagement of macrophage fcyr with ic alone induces the release of pro-inflammatory cytokines. conclusion: our data demonstrate that ic opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of ic opsonisation. we suggest that ic present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. excessive production of reactive oxygen species (ros) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. on the other hand, intracellular heat shock proteins (hsp) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. in this work, correlation analysis was applied to analyze relationship between ros production and intracellular hsp70 in neutrophils of elderly people. neutrophils were isolated from peripheral blood of donors of 90 years old and older (long-livers). intracellular ros and hsp70 levels were registered by flow cytofluorimetry upon labeling with 2',7'-dichlorofluorescin diacetate (invitrogen) and anti-hsp70 antibody (brm-22, sigma), respectively. intracellular level of hsp70 was also estimated in neutrophils after heat shock (hs) performed at 43°c for 10 min. extracellular ros production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. a positive correlation was determined for intracellular ros level and zymosan-mediated extracellular ros release although the dynamics of ros release at 1-15 min time range varied within the group. the correlation was unaffected by hs of neutrophils performed for 1 min at 42°c, although this short heat treatment decreased significantly ros release. there was no correlation between basal intracellular hsp70 (hsp70 basal ) and ros level, both intracellular and extracellular. at the same time increased hsp70 level immediately after hs (hsp70 (0 min)) correlated negatively with intracellular ros (initial and after hs). the hsp70 increase value (hsp70 (0 min) -hsp70 basal ) correlated negatively also with intracellular ros and extracellular ros release in response to zymosan; and the correlation with ros level became lower when hsp70 increase was registered in 60 min after hs (hsp70 (60 min) -hsp70 basal ). thus it was found that within this age group the alteration in hsp70 induced by hs in neutrophils but not basal hsp70 itself is the parameter associated negatively with both spontaneous ros level and ros production in response to activating action of zymosan. this work is supported by istc grant #3303. d. goyeneche-patiño 1 , z. orinska 1 , f. mirghomizadeh 1 , s. bulfone-paus 1 1 forschungszentrum borstel, borstel, germany several studies have shown different roles of mast cells (mc) in innate and adaptative immune responses. in fact, crosstalk between cd8 + t cells and mc has shown to induce multiple genes implicated in the signaling of specific programs such as type 1 ifn. two novel genes, receptor transporter protein (rtp4) and virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) are ifn inducible and were found to be over-expressed in chip analysis. the aim of this study is to characterize the expression and protein production of rtp4 and viperin in mast cells after tlr ligand stimulation in mice lacking of ifnra and the adapter proteins myd88 and trif. bone marrow derived mast cells (bmmc) from wt, ifnra -/-, mydd8 -/and trif -/mice, were exposed to tlr ligands (lps, pic, cpg, p(da-dt) and new castle disease virus (ndv)) during 8 and 48 h. mrna and protein extraction were performed for further qrt-pcr and sds page analysis for rtp4 and viperin. intracellular stimulation of tlr was performed transfecting cells with nucleic acids using lipofectamine 2000. stimulation of wt cells with pic, pda-dt and ndv showed an increased expression of viperin and rtp4 in comparison to control cells (untreated). the same trend was observed for mc from the trif and myd88 knockout mice. in contrast, in the ifnra deficient mice, expression of genes and protein production was abrogated to the same levels of wt untreated cells. lipofectamine stimulation does not increase the expression/production of the genes. direct stimulation of the well recognized viral sensors tlr 3 and 9, as well as, infection of mast cells with ndv (rna virus) induce the expression of rtp4 and viperin. the findings suggest that activation of mc with the ulterior expression of genes is type i ifn dependent. in contrast, the adaptor proteins myd88 and trif and the pathways that they represent are not relevant in the expression of rtp4 and viperin. these findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of mc in innate immune responses. in recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. we have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (apcs) to activate lymphocytes; but inflammatory macrophages do not express c-myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-myc. we studied some phenomenons suggested to be myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. finally, using lentiviral vectors, we over-expressed exogenous c-myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy. g. germano 1 , e. erba 2 , r. frapolli 2 , m. d'incalci 2 , a. anselmo 1 , s. pesce 1 , p. allavena 1 , a. mantovani 1, 3 1 humanitas clinical institute, rozzano, italy, 2 mario negri institute, milan, italy, 3 institute of general pathology, university of milan, milan, italy several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (tam) as well as tumor cells, and are attractive target of novel anti-tumor therapies. trabectedin (et-743) is a natural product derived from the marine tunicate ectenascidia turbinate, it binds the minor groove of dna, affects transcriptional factor activity and blocks cell cycle. this novel anti-tumor agent is currently used in phase ii studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. we previously demonstrated that trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. we tested the effect of trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. trabectedin significantly reduces ccl2, cxcl8 and the inflammatory protein pentraxin3 (ptx3) either at transcriptional and protein level, especially after tnfa/il1b stimulation. down-regulation of ccl2, cxcl8, ptx3 and also of il6 and vegf were confirmed in primary cultures of liposarcoma. according to the previous in vitro data we now show in a mouse model , using the fibrosarcoma mnmcai , that trabectedin treatment selectively affects monocytes in the blood and bone marrow. moreover trabectedin treatment strongly reduces the number of macrophages and of cd31+ vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. overall these results suggest a possible triple role of trabectedin. besides its direct cytotoxic effect on tumor cells, trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. as the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of trabectedin makes it an attractive candidate in inflammation-associated tumors. interleukin-4 (il-4) is a key cytokine of the t helper 2 cell response. il-4 has been found to be a major regulator of immunoglobulin class switching to ige and has important functions in the regulation of allergic diseases. here, the onset of the il-4 production after birth was investigated in equine neonates. the form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. il-4 producing cells were measured in peripheral blood mononuclear cells (pbmc) of neonates and foals by flow cytometric analysis. at day 3-6 after birth, a small population of il-4 producing cells was observed in the absence of any stimuli. the il-4 + population was not detectable at 6 or 12 weeks of age. other cytokine producing cells (ifn-g, il-10) were not detected using these conditions. the stimulation of neonatal pbmc with pma and ionomycin did not alter the il-4 + cell population. phenotyping of the neonatal il-4 + cells showed that they were ige + /mhcii -/cd4cells. the occurrence of cd4 + il-4 producing cells after pma stimulation increased slowly with age and did not reach adult levels by 12 weeks after birth. magnetic cell sorting of the ige + /mhciicells identified them as basophils. previous work has shown that foals do not produce endogenous ige for at least six months of life. ige bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. here, the stimulation of neonatal pbmc with anti-ige induced the secretion of il-4 at day 5 after birth. neonatal pbmc collected before colostrum uptake did not produce il-4 in response to anti-ige. in summary, equine neonates provide a model to investigate ige mediated il-4 responses after birth. the transfer of maternal ige from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal il-4 response mediated by the mare's accumulated acquired immunity to allergens. s. schmechel 1 , d. voehringer 1 1 ludwig-maximilians-university munich, institute for immunology, munich, germany macrophages display broad phenotypic heterogeneity depending on their microenvironment. the initial inflammatory response to th1 cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a stat6-dependent manner when stimulated with the th2 cytokines il-4 or il-13. alternatively activated macrophages (aam) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. furthermore, it has been shown that aam suppress the proliferation of t cells by a yet to be determined mechanism. currently there is still very limited information about the phenotype, migration and function of aam. we began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a stat6-dependent manner. to this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and stat6-deficient mice and infected these chimeras with the helminth nippostrongylus brasiliensis to determine whether lack of stat6 in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. the highest turnover of macrophages was found in the peritoneum, irrespective of stat6 expression. no major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on stat6 expression in macrophages. we could further confirm that in vitro generated aam from wild-type but not from stat6-deficient mice have a strong inhibitory effect on t cell proliferation. we are now trying to identify the mechanism(s) by which t cell proliferation is inhibited. furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. we have previously shown that aam can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. to identify potential aam-derived eosinophil chemotactic factors we currently compare the gene expression profile of il-4 exposed macrophages from wild-type and stat6-deficient mice. candidate genes will be expressed using retroviral transfections of stat6-deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. macrophages are an essential component of leukocytes infiltration in the tumor. they are identified as tumor associated macrophages (tams). these cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. the aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines. we tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. we have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. in the study we used macrophages isolated from a total of 38 pleural effusions, including 15 malignant and 23 nonmalignant tumors. the following human malignant cell lines were tested: a549, ht29, hct116, sw60, mcf7, mda-mb231, jurkat and hl60. results: our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. the up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins. the results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to m1, and the tams isolated from the malignant effusions similar to m2. among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating ms4a4a and ms4a6a: highest trascriptional level after 18h of stimulation with 10-6m dexamethasone. ms4a murine genes are differently expressed respect to the human counterpart and only the homologs of ms4a6a (ms4a6b, 6c and 6d) have a similar regulation. finally, egfp-tagged ms4a4a, ms4a6a, and ms4a7 expressed in cho cells showed that all molecules traffic to the cell membrane. though the biological functions of these ms4a proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized ms4a members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. during inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. these signaling circuitries are even more interfaced during infections in which microbial agonists activate toll-like (tlr), rig-like (rlr) and nod-like (nlr) receptors. on the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein-1 (mcp-1)/ccl2 and the gpcr ligand fmlp or the tlr4 agonist lipopolysaccharide (lps) on monocytes. in fact, the bacterial tripeptide fmlp, but not the cytokines il-1b or ifn-g, significantly and dose-dependently synergized with ccl2 in monocyte chemotaxis. furthermore, lps rapidly induced the expression of interleukin-8/cxcl8, but not of the ccl2 receptor ccr2 in monocytic cells. in turn, the induced cxcl8 synergized with ccl2 for mononuclear cell chemotaxis and the chemotactic effect was mediated by cxcr1/cxcr2, because cxcl8 receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to ccl2 intact. these data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. objectives: in recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. most data were collected on endothelial cells and on thrombocytes. however, there are only few data on vesicles derived of neutrophilic granulocytes (pmn), and most of these investigations applied only pharmacological agents. our aim is to investigate pmn-derived cell-free particles and their possible role in bacterial killing methods: preparation of pmn and investigation of bacterial killing by our semi-automatic method was described by rada et al. (blood, 2004) . cell-free vesicles were prepared after co-incubation of human pmns with different activating agents for 20 min at 37°c with gentle shaking, followed by spinning down of pmns for 5 min, at 4°c and 500g. the supernatant was sedimented at 15000g for 10 min, 4°c, and we used the sedimented fraction for our investigations. formation of particles was followed by fluorescent and electron microscopic assays. the amount of particles was estimated with flow cytometer and by their protein content. we observed that upon co-incubation of pmns with s. aureus, opsonized by mixed human serum, pmns produce a well detectable amount of vesicles. omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. production of particles could be inhibited with diphenyl-iodonium (dpi), cytochalasin b (cb) or with azide treatment. treating pmns with dnase or withdrawing glucose during co-incubation had no effect on vesicle formation. in killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. this antibacterial activity could be inhibited by dpi, cb, azide treatment or by withdrawing glucose from the medium during the killing assay. however, treatment of the microvesicles with dnase had no effect on their antibacterial capacity. for long, cd56 has been used for the detection and identification of natural killer (nk) cells. recently, the presence of a minor subset of cd56 low cd33+ blood monocytes (mo) in healthy individuals and the increase in cd16+cd56+ blood mo in patients with inflammation has been reported. the functional activity of human cd56+ blood mo has been studied in vitro but not tested ex vivo so far. healthy people living permanently in malaria endemic areas are exposed to plasmodium infection, and we hypothesized that blood mo of these individuals could be activated and display increased cd56 expression. we tested if this phenotypic expression was associated with detectable changes in the mo anti-parasitic activity. the mo phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. myeloid cell markers included cd33 and activation markers such as hla-dr and trem-1. percentages of blood mo involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given mo phenotype and phagocytosis activity was then looked for, using spss ® and statview ® softwares. our results showed that, compared with malaria naï ve individuals, there was a 12.3 fold increase (p x 0.0001) in the total number of circulating cd56 low mo present in the blood samples of healthy malaria exposed asian individuals living in thailand. according to the density of surface antigen determined by fluorescence intensity (fi), the decrease in cd33 and the concomitant increase in hla-dr expressions indicated that in this malaria endemic area, blood mo were mature and highly activated by comparison with surface markers of mo from malaria naï ve donors. the relative levels of cd56+ blood mo were associated with the percentages of membrane-bound ifn-g present at the mo surface. in conclusion, (i)-a subset of cd33+ blood mo expressed increased levels of cd56 on mo of healthy malaria exposed individuals; (ii)-blood mo with activated (hla-dr+) and mature (trem-1+) phenotypes were present in these healthy individuals; (iii)-increased expression of hla-dr and cd56 on cd14 high mo was associated with a high phagocytosis activity. introduction: adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. for instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. the present work focuses on the effects of leptin on the reactivity of m1-and m2-polarized human macrophages. methods: monocytes were isolated from the peripheral blood by magnetic cell sorting. polarization to m1 and m2 macrophages was induced by culture in the presence of mcsf or gmcsf respectively. polarized cells were characterized by flow cytometry, stimulated with lps and response assessed by characterization of cytokine profiles via cytometric bead array (cba). results: culture of monocytes in the presence of mcsf or gmcsf induced two different phenotypes. cells cultured in the presence of gmcsf represented the m1 type and were cd14 negative but cd80 and mhcii positive and produced high levels of il-8, tnfalpha and il-6 following lps stimulation. culture in the presence of mcsf resulted in induction of the m2 phenotype. these cells were cd14 positive with intermediate expression of cd80 and mhcii expression and produced high levels of il-10, il-6 and il-8 following lps stimulation. interestingly, already baseline il-8 production was high in these cells. stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. however, if leptin was present in cultures stimulated with lps, the induction of cytokine production was significantly reduced in both, m1-and m2-polarized cells as compared to cells stimulated with lps alone. summary: whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the tlr4 ligand lps. thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens. methods: hl60 cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to pma (50 ng/ml) treatment for 48h. morphological evaluation was performed with conventional microscopy and electron microscopy. immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. a colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ros). results : besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the pma-treated fibronectinadherent hl60 cells expressed antigen receptors cd14, tlr2, tlr4 and cd68 , and displayed enhanced phagocytic activity and production of ros. expression of cd13, cd33 and cd15 was also maintained however the cells were hla-dr and cd1a negative. conclusion: we describe the enhanced ability of fibronectin-adherent hl60 cells to differentiate into macrophages in response to pma. hl60 may provide a functional model for macrophage differentiation. above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies. a. aporta 1 , n. ferrer 2 , a. gómez 2 , j. gonzalo 2 , a. arbués 2 , a. anel 1 , c. martín 2 , j. pardo 1 , apoptosis, immunity and cancer 1 university of zaragoza, molecular and cellular biochemistry and biology, zaragoza, spain, 2 university of zaragoza, mycobacterium genetics, zaragoza, spain mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. it has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. it has been proposed that m. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. however the biological relevance of this process is not clear. our group has generated so2, a m. tuberculosis phop mutant strain that was shown (perez et al 2001) to be more attenuated than the present attenuated vaccine strain bcg and conferred protective immunity against m. tuberculosis infection in mice and guinea pigs (martin et al 2006) . in the present study, we compare the time course and phenotype of cell death induced by so2, bcg and wild type m. tuberculosis on the murine macrophage cell line j774 and on bone marrow derived mouse macrophages. our results indicate that wild type m. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. of note cell death presented apoptotic features like caspase-3 activity and nuclear condensation. in order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. experiments are ongoing with macrophages derived from trl2x4 deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phop m. tuberculosis strain. objectives: vip is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. type 1 receptor for vip (vipr1) gene is highly conserved through species and, in humans, is highly polymorphic. vipr1 has been reported to be down-modulated in cells of the immune system after activation. an association of some snps with some autoimmune diseases has also been reported. in this study we have investigated the correlation between these snps and gene expression in monocytes exposed to lps. methods: monocytes from 53 blood donors were separated from pbmc and stimulated with lps. total rna was reverse transcribed and the level of vipr1 in untreated or lps-stimulated monocytes was measured by real-time rt-pcr and protein expression. protein level was measured by western blot and densitometric analysis. the kinetic of expression of vipr1 after 3, 6, 9 and 12 h of exposure to lps was firstly analysed in monocytes from five individuals. there were two kinetics: one in which a reasonable high levels ( g 50 %) of mrna was maintained trough time and a second one in which the decrease of mrna was pronounced. the experiments were repeated using monocytes from 53 donors that had been typed for the relevant vipr1 snps. the down-regulation of vipr1 correlates with the presence of a t at rs896 mapping in the 3'-end of the gene (p= 0.004). the vipr1 protein level was decreased about 40 % in monocytes of 3 subjects typed as t/t at rs896 whereas 3 subjects typed as c/c at rs896 maintained a high level of expression after 9h of lps treatment. the data show that different haplotypes of the vipr1 gene correlate with a different kinetics of gene expression in activated monocytes. a possible consequence of these data is that the anti-inflammatory properties of vip governed by the vipr1 vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases. j. oujezdska 1 , t. vavrochova 1 , d. filipp 1 , immunobiology 1 institute of molecular genetics as cr, immunobiology, prague, czech republic phagocytes which appear in early mouse development (e7.5-13.5) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, ys-derived phagocytes. in addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. this origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. the aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. here we report the temporal and spatial expression of toll-like receptors (tlrs) and cd14 in the early mouse embryo (me). facs analysis of cell suspension prepared from 10.5 day me showed that about 0.7-1 % of cells were positive for cd11b. these cells exclusively were also positive for cd14, tlr2, and cd45 antigens. using qpcr and flow cytometry we show that tlrs and other tir domain-containing signaling molecules are expressed in the embryo through embryonic days 7,5-13,5. reciprocal matings between wild type and mhcii-egfp knock-in mice revealed that while maternallyderived mhcii + macrophages are present in the embryo from early developmental stages (e7,5), embryo-derived mhcii + macrophages start to appear in the embryo around day 13. multicolor facs analysis of cd11b, cd45, cd14, f4/80, tlr2, tlr4, c-kit and mhcii surface markers revealed differential expression of tlr2 and c-kit on embryonal phagocyte subpopulations. moreover, the microarray analysis of cd11b + tlr2 + cells isolated from the e10,5 embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. these molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. these results are first to characterize the regulated expression of tlrs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. humans may be exposed to a variety of mycobacteria ranging from environmental or bcg vaccine to more pathogenic mycobacteria. only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. il12/il23-dependent ifng pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. due to the high rate of consanguineous marriages in the north african countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. in tunisia, among patients affected with primary immunodeficiencies 16 presented with disseminated bcg infection (bcg-osis). among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and 11 have a mendelien susceptibility to mycobacterial disease. using a candidate gene strategy, we have identified in 9 out of these 11 patients mutations in several ifng pathway genes, other candidate genes are being investigated for the 2 other patients. in the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including mhc and nramp1. we did focus on the study of 2 genes which are considered as important pathogen recognition receptors of the innate immune system: tlr2 is the principal mediator of macrophage activation in response to mycobacteria through nfkb pro-inflammatory signaling pathway and dc-sign is the major receptor of m. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory il-10 cytokine. using a case/household-contact cohort we did investigate polymorphisms of these 2 genes in tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of snp and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis. host inflammatory responses play a major role in granuloma formation and control of the infection. unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. ifng therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant m. tuberculosis strains. francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. research into francisella has expanded over recent years due to its designation as a potential biological warfare agent. several species of francisella exist and have varying degrees of pathogenicity. f. tularensis live vaccine strain (lvs) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. however, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. f. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of f. tularensis would decrease and antibiotic treatment may be more effective. a flow cytometric assay was developed to measure bacterial uptake. this method used a fitc labelled anti-f. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. a series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (mhs) and on ex-vivo mouse lung tissue to determine whether uptake of f. tularensis lvs could be altered. the presented data shows that several inhibitors work efficiently to reduce lvs uptake by up to 70-80 % in both the in vitro and ex vivo assays. however, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. in addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against f. tularensis. it may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã crown copyright. dstl, 2009. hsp70 are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. it is also known that extracellular hsp70 have pronounced immunomodulatory properties. to study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface hsp70 on reactive oxygen species (ros) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). neutrophils were isolated from human peripheral blood by using a standard protocol. respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. for the experiments human recombinant hsp70 (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface hsp70 were used. exogenous hsp70 was used in concentration 1-10 ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio 1:1 and 2:1 directly before the measuring. as the control we registered amplitude of oxidative burst in samples supplemented with pbs or live mouse thymocytes having no hsp70 on their surface. results demonstrating effect of exogenous hsp70 on phagocytosis-induced ros release from human peripheral blood neutrophils have been obtained. it was demonstrated marked dose-dependent inhibiting action of exogenous hsp70 on amplitude of respiratory burst. the cells expressing surface hsp70 impacted on ros production in this model similarly. the results of chemiluminescence analysis demonstrated that zymosan induced ros production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. the effect was more pronounced for increased amount of thymocytes added to the samples. thus, immunomodulatory effects of exogenous hsp70 might be caused by influence of the protein on ros release from phagocytes. we suppose that the registered effects are connected with ability of hsp70 to inhibit activity of nadp-oxidase -the key enzyme for ros production during respiratory burst. results: we recruited 28 pts, with so far five complete pathological remission, five partial responses and five no responses. no substantial changes were detectable in the number of circulating monocytes. in contrast we observed a clear expansion of cd14/cd86 and cd14/cd163 double positive subsets. this event was transient; it abated at the later time point suggesting a causal relationship to the treatment. it correlated with sensitivity to the treatment. in fact we observed that in the responder patients the expansion of the cd14/86 subset was clear in the first weeks of treatment and decreased there after. in contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. all the patients had an initial expansion of the cd14/163 subset. in the responder patients this population was still present at the time of surgery. the immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative m2 polarization. conclusion: these data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors. m. dimitrijević 1 , i. pilipović 1 , s. stanojević 1 , k. mitić 1 , k. radojević 1 , v. pešić 2 , g. leposavić 1,2 1 institute of virology, vaccines and sera "torlak", immunology research centre "branislav janković", belgrade, serbia, 2 faculty of pharmacy, university of belgrade, department of physiology, belgrade, serbia the primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (no) and hydrogen peroxide (h 2 o 2 ) production and phagocytic activity of these cells. in addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. to address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to 14-day-long propranolol treatment and measured both no and h 2 o 2 production and phagocytic activity of these cells. using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b 2 -and a 1 -adrenoceptors on macrophages was revealed. furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. in vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated no production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide h 2 o 2 production. an increase in h 2 o 2 production in the presence of the a 1 -adrenoceptor antagonist ebrantil provided support for this. chronic propranolol treatment in vivo led to increased no and h 2 o 2 production by peritoneal macrophages. furthermore, this treatment resulted in opposing effects on the expression of b 2 -and a 1 -adrenoceptors on peritoneal macrophages (a stimulatory effect on b 2 -adrenoceptors and a suppressive effect on a 1 -adrenoceptors). in conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on h objectives: monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: cd14 ++ cd16 -('classical') and cd14 + cd16 + ('pro-inflammatory') monocytes. a central monocyte function is cytokine production in response to toll-like receptor (tlr) ligation. the cd14 + cd16 + monocytes display higher tlr2 and -4 expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the cd14 ++ cd16monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following tlr2 stimulation. methods: cord blood (n=8) and peripheral-blood (n=8) mononuclear cells were stimulated in vitro for 24hrs with peptidoglycan and subsequently analysed for cd14 and cd16 and intracellular il-12p70 and tnf expression. the mann-whitney u-test was used to evaluate differences between groups. results: a significantly higher percentage of neonatal monocytes were positive for il-12p70, both unstimulated and after peptidoglycan stimulation, as compared to adults. geomfi of il-12p70 was low and similar between groups, although significantly higher in newborns after stimulation. in both newborns and adults, il-12p70 (% positive cells and geomfi) was significantly higher for cd14 + cd16 + cells than for cd14 ++ cd16cells, unstimulated and stimulated. regarding tnf, neonatal and adult monocytes did not differ in unstimulated cultures, however geomfi of tnf was significantly higher in neonatal monocytes after stimulation. whereas the tnf response following stimulation was similar between the adult monocyte subsets, in newborns the cd14 ++ cd16cells were positive for tnf to a significantly higher extent than the cd14 + cd16 + cells. in particular the tnf response to tlr2 stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. notably, in newborns the cd14 ++ cd16monocytes were positive to a higher extent for tnf following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. objective: chronic granulomatous disease (cgd) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. cgd generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating cgd may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features. we report a 16-year-old boy who admitted with complaints of seizures during the previous 2 months. there was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. the family history was unremarkable, and the parents were unconsanguineous. physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. a large frontol mass with diffuse peripheral vasogenic edema was discovered on mri. subfalcine herniation was noted secondary to mass effect. cgd was suspected and the analysis with flow cytometric dihydrorhodamine assay (dhr assay), for functional analysis of neutrophils was compatible with the diagnosis of cgd and no bimodal histogram pattern spesific for x-cgd was found in the mother and sister. after the diagnosis of cgd, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. aspergillus fumigates grew from the culture; liposomal amphotericin b and voriconazole were started; which were found to be sensitive to the cultured species. in addition, interferon-g (50 mgr/m2/day, subcutaneously every other day) was started. after 2 months, control mri showed regression of the lesion, and the anti-fungal treatment was continued for 3 months. the screening of the other family members with dhr assay demonstrated that one of his sisters had also cgd and phenotype was autosomal recessive. mutaton analysis in "hot spot" in ncf1 gene concerns the well-known gt deletion in the second exon of ncf1 gene both at the patient and his sister. results: this was an atypical clinical presentation of cgd in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. we documented a good response to the combination of ifn-g, liposomal amphotericin b and voriconazole after surgery. conclusion: cgd should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. to test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (cci) model in sprague-dawley rats. animals (n = 45) subjected to moderate brain injury were randomized, and received either 1.5 mg/kg ubiquitin or vehicle (placebo) intravenously within 5 min after cci. levels of tnf-a, il-1b, il-6, il-10 and il-1 receptor antagonist were analyzed in brain tissue using real time rt-pcr at 4 and 72 hours after treatment. immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at 24h and 7 days. data were analyzed with the mann-whitney u test and a two-tailed p x 0 .05 was considered significant. all cytokines were highly up-regulated 4 hours after cci but no differences between the groups were observed at this time point. three days after trauma the levels of il-10 were significantly lower in the ubiquitin treated animals, whereas the levels of il-6 and tnf-a were higher when compared to the placebo group. interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day 7. the infiltration of neutropils was not affected by ubiquitin treatment. here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (ptb) patients. efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (ptb) patients. there is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (tpp), total antioxidant activity (taa), malondialdehyde (mda), and oxidative stress index (osi) in ptb patients during chemotherapy with or without micronutrient supplementation. the present study was designed to assess the levels of neopterin, tpp, taa, mda, and osi during chemotherapy with (c+m) or without (c-m) micronutrient supplementation using elisa and spectrophotometric methods. thirty-eight (38) newly diagnosed ptb patients and forty non-ptb apparently healthy subjects volunteered to participate in this study. twenty of the ptb patients were on anti-tuberculosis drugs supplemented with micronutrients (c+m) while 18 were treated with anti-tuberculosis drug alone (c-m) for a period of four weeks. the levels of neopterin (p=0.02), tpp (p=0.00), osi (p = 0.00), mda (p = 0.00) were significantly raised but taa (p = 0.01) was significantly reduced in ptb patients compared with controls. the levels of mda (p = 0.04), neopterin (p=0.00) and tpp (p=0.00) were significantly reduced in c+m after two weeks of treatment compared with baseline values before commencement of treatment. the levels of tpp (p=0.00), mda (p=0.00), neopterin (p=0.02), osi (p=0.00) were significantly reduced while taa (p=0.01) was significantly raised in c+m after 4 weeks of treatment compared with the baseline concentrations. in c-m, only mda showed significant decreased after 4 weeks of treatment when compared with the baseline values. plasma level of neopterin, tpp, osi and mda declined faster in c+m than c-m. therefore, micronutrient supplementation of ptb drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. this will improve deranged macrophage activation and reduce oxidative stress indices in ptb patients. a. p. aguas 1 , e.m. cunha 1 , m.j. oliveira 1 1 icbas, university of porto, anatomy, porto, portugal the acute in vivo intake of mercury (hg) microparticles (20 nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of hg by scanning electron microscopy coupled with x-ray elemental microanalysis (sem-xem). the intracellular distribution of hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. balb/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to 5 minutes after the injection. in some mice, before the hg injection, peritoneal phagocytes were activacted by ip injection of bsa. pre-injections with a selenium (se) salt were also performed in order to study the putative modulatory role of se on hg intake by phagocytes. peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with pbs, they were cytospinned, fixed with formaldehyde, and processed for observation by sem-xem. five min after the hg injection more than half of the mouse phagocytes were positive for hg. a higher percentage (70 %) of macrophages contained the metal particles than neutrophils (55 %). phagocyte activation enhanced the number of hg particles seen inside the phagocytes. pre-injection of the peritoneal cavity of mice with se resulted in finding that more than half of the hg intracellular particles were coupled with se. subcellular topography of hg particles showed that they were presented in individual small cytoplasmic vesicles. we conclude that hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. this study was supported by a grant from fct, portugal. mast cells play central roles in allergic inflammatory reactions and innate immunity. swap-70 is a rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. in b cells and mast cells swap-70 regulates f-actin cytoskeletal rearrangements, cell polarisation and cell migration. (pearce et al., 2006; sivalenka and jessberger, 2004) . swap-70-/-bone marrow derived mast cells (bmmc) are specifically impaired in fceri-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (gross et al., 2002; sivalenka and jessberger, 2004; sivalenka et al., 2008) . crucial regulators of these processes are members of the rho family of small gtpases such as rac1 and rhoa. swap-70 interacts with rac1 in vitro and preferentially binds the active gtp-bound rac1. swap-70 supports the increase of active rac1 in vitro by a yet to be defined mechanism (shinohara et al., 2002) . in this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between swap-70 and rac1. it was found that fulllength swap-70 preferentially binds to constitutively active rac1 (rac1q61l) but not to its dominant negative form (rac1t17n). binding assays with swap-70 truncated mutants showed interaction of swap-70's n-terminus with gtpgs rac1 or rac1 depleted of guanine nucleotide, whereas swap-70 central or c-terminal regions do not bind to any form of rac1. preliminary competitive-binding assays with overlapping 18mer peptides, spanning the entire swap-70 sequence, mapped the rac1 binding site near the n-terminus of swap-70. full-length swap-70 site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of rho gtpases. elucidating the molecular interactions of swap-70 with rho gtpases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express swap-70. v. c. barbosa 1 , c. d. polli 1 , m.c. roque-barreira 1 , m.c. jamur 1 , c. oliver 1 , g. pereira-da-silva mast cells are essential cells in ige-associated immune responses. fceri crosslinking induces mast cell degranulation and release of proinflammatory mediators. we have previously shown that the lectin artinm induces mast cell activation but the mechanisms involved in this activity remain unknown. objective: the present study was undertaken to further characterize the ability of artinm to activate mast cells. methods: rbl-2h3 cells were sensitized with ige anti-tnp and stimulated with dnp 48 -hsa or artinm. artinm binding to rbl-2h3 cells was assessed by flow cytometry. mast cell degranulation was determined by measurements of released b-hexosaminidase activity. microplate binding assays were utilized to assess artinm binding to ige. to investigate fceri recognition by the lectin, western blots of cell lysates were stained with biotinylated artinm and be's antibodies specific for fceri b-subunit. intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. mcp-1 and tgf-b levels released by mast cells were measured by elisa. results: artinm binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of ige. the release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of d-mannose, suggesting that mast cell degranulation induced by artinm might be the result of interactions between the lectin crds and glycosylated components on the cell surface, like fceri or ige. indeed, it was observed that the lectin bound to ige in a dose-dependent manner and recognized the fceri b subunit in western blot analysis. exposure to artinm resulted also in phosphorylation of intracellular proteins, mcp-1 release and tgf-b production. significant increases in these activities were observed upon sensitization with ige. conclusions: these results suggest that artinm may bind to glycans of the high affinity ige receptor and/or of the ige (bound to fceri) and that such interactions would be implicated in its ability to activate and degranulate mast cells. in view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. the adhesion receptor l-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. it is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. a distinct feature is the exclusive presentation of l-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. this topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. other adhesion molecules such as p-selectin glycoprotein ligand 1 (psgl-1), b1 and b7-integrins also share this special phenotype. taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown. in this study we identified the highly conserved transmembrane domain of l-selectin to regulate microvillus receptor positioning and adhesion under flow. taking advantage of the inverse surface expression pattern of cd44 (cell body) compared to l-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. in turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. objectives: macrophages are one of the principal effector cells involved in the innate immunity response. they kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as il-1b, il-18 and tnf-a. herpes simplex virus 1 (hsv-1) is an enveloped dna virus that infects mostly oral mucosa and sensory neurons. innate immunity responses activated by hsv1 infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. il-18 and tnf-a are cytokines produced by macrophages that contain known anti-hsv properties. the objective of this study was to characterise the secretome of human primary macrophages infected with hsv1. methods: human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. macrophages were left untreated or primed with poly(i:c) (10ug/ml), a mimetic of double-stranded rna, after which cells were left uninfected or infected with hsv-1 for 18 h. after this, cell culture supernatants were collected, concentrated and proteins purified. the secreted proteins were digested into peptides, identified and quantified using itraq (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanolc-ms/ms. the raw ms/ms data was analysed using proteinpilot 2.0 software. results: in the first itraq experiment over 300 human proteins were identified in the hsv1 infected cell supernatants. from these proteins 119 had at least 3 fold increase after poly(i:c) + hsv1 infection compared to the uninfected cells. hsv1 infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: itraq labelling showed a total of 2.7 fold increase in the protein amount in the poly(i:c) + hsv1 infected cell supernatant and a 2.6 fold increase in the hsv1 infected cell supernatant when compared to the uninfected cell supernatant. amongst the upregulated proteins there were known inflammatory proteins: chemokine (c-x-c motif) ligand 10, il-6, tnf-a induced protein 6, complement factor b, galectin-1 and mxa. at present, further experiments are on-going for more detailed analysis of the hsv1 infected macrophage secretome. h. p. prakash 1,2 1 german cancer research centre, translational immunology, heidelberg, germany, 2 max planck institute for infection biology, molecular biology, berlin, germany chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, chd and copd. chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (iaps). the significance of cellular inhibitor of apoptosis protein-1 (ciap-1) and x-linked inhibitor of apoptosis proteins ( xiap) in chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap-1 and xiap knockout (ko) mice using a novel non-invasive intra-tracheal infection method. in contrast to wildtype, iap knockout mice failed to clear the infection from their lung. wildtype mice responded to infection with a strong inflammatory response in the lung. in contrast, the recruitment of monocytes and macrophages was reduced in iap ko mice compared to wildtype mice. the concentration of interferon gamma (ifn-g) was increased whereastumor necrosis factor (tnfa) was dysregulated in the lungs of infected iap ko mice compared to infected wildtype mice. ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that iaps are required for innate immune responses of these cells. our findings thus suggest a new immunoregulatory role of iaps in c.pneumonaie pulmaonry infections. methods: human monocytes were purified from venous blood of normal volunteers by ficoll density gradient centrifugation. hrgal-3 (25 mg/ml) binding to monocytes, in the presence or absence of 10mm lactose or sacarose, was assessed by flow cytometry and confocal microscopy. in transwell systems, assays were performed using hrgal3, laminin or fibronectin immobilized or not on the filters. these were added to wells containing soluble hrgal3 or rpmi and monocytes (1x10 5 ) were added into each insert. when necessary, hrgal3 was pre-incubated with 10mm lactose or sacarose. mcp-1 (100ng/ml) was used as positive control. we observed that hrgal-3 binds to the surface of human monocytes through its crd, since this interaction can be inhibited by lactose. we corroborated some data of literature that hrgal-3 is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. when we evaluated the participation of the ecm laminin and fibronectin in monocyte migration induced by hrgal-3, we observed that the association between these glycoproteins and hrgal-3 resulted in a 60 % increase in the number of migrating cells. both n-and c-terminal domains of hrgal-3 are involved in the association between laminin or fibronectin and hrgal-3, since the presence of lactose resulted in 50 % and 20 % inhibition of monocyte migration induced by the lectin, respectively conclusions: our results showed that hrgal-3 induces monocyte migration by haptotaxis, through the interactions established between both n-and c-terminal domains of the lectin and ecm glycoproteins, laminin and fibronectin. in a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. recent studies have shown that monocyte heterogeneity is conserved in humans and mice. the different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. in order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hesc).culturing of embryoid bodies for 3 weeks in the presence of bmp4,vegf and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of cd14+cd45+ cells. the sorted cd14+cd45+ cells were further cultured for 7 -10 days in the presence of m-csf and gave rise to a homogenous population of adherent mature macrophages. embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. while virtually all embryonic-derived macrophages expressed the lps-receptor cd14, m-csf receptor cd115 and the scavenger-receptor cd36, we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the cd14 and cd16 (fcgammariii) : the cd14lowcd16-and cd14+ cd16+. trancscriptional, phenotypic and functional assays suggest the alternative (m2) polarization of cd14+cd16+ embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, m2 -related gene expression).the exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. b.-s. choi 1 , p. kropf 1 1 imperial college london, immunology department, london, united kingdom the balance between t helper (th) 1 and th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized th1 or th2 responses are not sufficient to account for healing or nonhealing. we have recently shown that arginase-induced l-arginine depletion results in local suppression of antigen-specific t cell responses in nonhealing leishmaniasis. healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. moreover, supplementation with l-arginine restored t cell effector functions and resulted in reduced lesions size and parasite load. however, despite the efficient production of ifn-g by cd4 + t cells at the site of infection and despite the reduced pathology, the mice did not heal. we hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to ifn-g mediated signals. to test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. in addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (inos) to kill the intracellular parasites. vg9vd2 t lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. the phosphoantigen isopentenyl pyrophosphate (ipp) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. previous work has shown that a molecular complex homologous to mitochondrial atp synthase (ecto-f1-atpase) is expressed on many cell types and is a possible specific ligand for the vg9vd2 tcr. the present study aims at understanding the role of f1-atpase in antigen regognition. using video microscopy calcium imaging in single vg9vd2 t lymphocytes, we can now show that the t cell response to ipp requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface f1-atpase. purified f1-atpase immobilized on polystyrene beads can partly replace the need for cell-cell contact. ipp in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in t cell activation assays clearly shows that it is not recognized as such on tumors. however, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. one of these, a derivative of ipp, is barely able to stimulate vg9vd2 cells in the absence of apcs, as opposed to the non-nucleotidic antigen ipp. however it can bind stably to f1-atpase. thus the f1-atpase complex acts as a presenting structure for nucleotide phosphoantigens. altogether, our data suggest that vg9vd2 t cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. surface plasmon resonance was used to analyse the molecular interaction between tcr and f1-atpase. by using purified f1-atpase and peptides derived from vg9vd2 tcr sequences, interaction sites between f1-atpase and tcr were identified on both ligands. based on these findings a generalized model for vg9vd2 t cell activation is proposed. ligands for the cytotoxic lymphocyte activating receptor nkg2d are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by nkg2d(+) lymphocytes. a key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific t cells. a second key issue is the scope and scale of events that follow nkg2d activation in vivo. by transiently overexpressing the nkg2d ligand rae-1-beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident vgamma5vdelta1 gamma-delta t cells and langerhans cells (lc), that were swiftly followed by epithelial infiltration of unconventional alpha-beta t cells. these data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of nkg2d ligands is sufficient to provoke a series of multicomponent immunological changes. the effects on lc, which lack nkg2d and presumably respond to changes initiated by local gamma-delta t cells, are particularly interesting. ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to nkg2d ligands in vivo. to assess the scope of this response, ovalbumin was applied to the skin concomitant with rae1 induction. the primary systemic th2 response is increased by concomitant responses to a stress antigen. we will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. in addition, the many ligands available to the nkg2d receptor suggest that different ones may play unique roles. a novel nkg2d-ligand, h60c, is uniquely expressed in mouse skin. when the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of rae-1 induction. such studies may help resolve a long-standing puzzle over the pleiotropy of nkg2d ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels. a.-s. invariant natural killer t (inkt) cells are a distinct lineage of t lymphocytes that co-express a highly conserved ab t cell receptor (tcr) along with typical surface receptors for natural killer (nk) cells. these lymphocytes recognize glycolipid antigens presented by the non-classical class i molecule cd1d. inkt cells are characterized by their capacity to produce rapidly large amounts of both th1 (ifn-g, tnf) and th2 (il-4, il-13) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. converging studies in mouse models suggest that inkt cells can prevent the development of type 1 diabetes. the frequency of inkt cells is lower in non-obese diabetic mice (nod mice). manipulation of inkt cells, either by increasing their frequency or by stimulating them with agonists such as a-galcer, inhibits diabetes onset in nod mice. recently, a new population of cd4 -nk1.1 -inkt cells producing high levels of the pro-inflammatory cytokine il-17 has been identified (inkt17 cells). given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of inkt17 cells in type 1 diabetes. interestingly, nod mice exhibit a higher frequency of inkt cells producing il-17 as compared to c57bl/6 mice. this increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. as previously described in normal mice, inkt17 cells present in nod mice were mainly cd4 -nk1.1 -, express the ror-g transcription factor and il-23 receptor, both molecules being usually associated with th17 commitment. we are currently analyzing, using co-transfer experiments, whether these inkt17 cells play a beneficial, a deleterious, or any role in the development of type 1 diabetes in nod mice. j. s. dodd 1 , r. muir 1 , s.s. affendi 1 , p.j. openshaw 1 1 imperial college london, respiratory medicine, london, united kingdom natural killer t (nkt) cells are a heterogeneous population of innate t cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. upon activation with their cognate glycolipid antigen presented by cd1d molecules, activated nkt cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. consequently, nkt cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. respiratory syncytial virus (rsv) is a common cold virus of the family paramyxoviridae. it is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. it is known that cd1d-deficient mice with poor nkt cell responses have inefficient induction of cd8 t cells and reduced clearance of rsv, perhaps because of ifn-g release by activated nkt cells. we now show that activation of lung nkt cells with intranasal agalcer during rsv infection of mice boosts th2 immunity (increasing il-5 and il-10), promoting pulmonary eosinophilia and ablating cd8 t cell recruitment. by contrast, intraperitonal injection of agalcer enhances nk cell recruitment and boosts pulmonary cd8 t cell activity (as measured by cd25 expression), increasing ifn-g production in the airway and lung and inhibiting viral replication. effects on illness (as measured by weight loss) were similarly distinct: intranasal agalcer induced early (d4) weight loss independent of conventional t cells, whereas intraperitonal agalcer enhanced late (d7) weight loss by a cd8 t cell dependent mechanism. therefore, nkt cells stimulated by agalcer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. in general, neonatal conventional t cells, especially cd4 + ab t cells, are regarded as immature or t h 2 biased. vg9 + vd2 + t cells are unconventional lymphocytes: they are mhc-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (e)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (hmb-pp)) or aminobisphosphonates (e. g. zoledronate) in adults. until now, little is known on the functional reactivity of neonatal vg9 + vd2 + t cells towards these activators. because il-23 is preferentially secreted by neonatal dendritic cells (dc) upon tlr stimulation, we investigated the potential costimulatory effect of this cytokine on hmb-pp and zoledronate-treated neonatal vg9 + vd2 + t cells. herein, we observed that zoledronate induced neonatal vg9 + vd2 + t cell proliferation and ifn-g production in cord blood mononuclear cells (cbmc) cultures. other t h 1-like cytokines like tnf-a and gm-csf were also produced upon this stimulation, but less than ifn-g, while t h 2-like cytokines such as il-4 and il-5 were not induced. addition of il-23 to zoledronate selectively costimulated ifn-g production from neonatal vg9 + vd2 + t cells. furthermore, zoledronate/il-23 treatment resulted in neonatal vg9 + vd2 + t cells expressing high levels of the cytotoxic mediators perforin and granzyme a. zoledronate induced the expression of the receptor for il-23 (il-23r) and the transcription factor t-bet, which is known to be important for the production of ifn-g in gd t cells. in addition, costimulation with il-23 resulted in a further increase of t-bet expression in neonatal vg9 + vd2 + t cells. these changes in the expression of il-23r and t-bet likely contribute to the observed selective ifn-g response towards zoledronate/il-23 treatment. of note, in contrast to adult peripheral blood vg9 + vd2 + t cells, hmb-pp had no or only a minor effect on the functional reactivity of neonatal vg9 + vd2 + t cells. altogether, these observations show that neonatal vg9 + vd2 + t cells are functionally active and that this t cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when dc-derived il-23 is produced in response to microbial stimuli. the evasion of antigen presentation is a feature common to herpesviruses. one of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral e3 ligases such as hhv8 encoded k3, k5 or mhv68 encoded mk3. these viral genes represent homologues of the march family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. ubiquitination targets surface molecules to the lysosome via the multivesicular body (mvb), a structure which also has an important role in the budding of many viruses. we investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral e3 ligases manipulate them. we discovered that both the cellular march and viral e3 ligases ubiquitinate cd1 molecules. however, whereas viral molecules inhibit cd1-antigen presentation, the march molecules are essential for the recirculation and function of the long-lived and lysosome-resistant cd1 molecules. in contrast mhc class ii was only targeted by cellular and not by viral e3 ligases. furthermore cd1 molecules could be found in viral particles as a result of ubiquitination, presumably via the mvb. thus, virally expressed and cellular e3 ligases have opposite effects, despite their homology. how this is achieved is a matter of active investigation. gamma delta (gd) t cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. our previous study revealed that vd2 negative ( neg ) gd t lymphocytes isolated from transplant recipients infected by cytomegalovirus (cmv) killed both cmv-infected cells and ht29 colon cancer cells in vitro. in order to investigate the anti-tumor effects of vd2 neg clones in vivo, we generated hypodermal ht29 tumors in immunodeficient mice. concomitant injections of vd2 neg clones, in contrast to vd2 + cells, prevented the development of ht29 tumors. vd2 neg clones expressed chemokine c-c motif receptor 3 (ccr3) and migrated in vitro in response to chemokines secreted by ht29 cells, among which were the ccr3 ligands macrophage inflammatory protein (mip)-1d and monocyte chemoattractant protein (mcp)-4. more importantly, a systemic intraperitoneal (i. p.) treatment with vd2 neg clones delayed the growth of ht29 subcutaneous (s. c.) tumors. the effect of in vivo gd t cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-ccr3 antibody. gd t cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since vd2 neg clones were not able to inhibit the growth of a431 hypodermal tumors. our findings suggest that cmv-specific vd2 neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. more recently, we generated ht29 cells expressing the luciferase and realized orthotopic injection of ht29-luc cells. progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. intraepithelial lymphocytes (iel) compose large, oligoclonal, tissue-associated repertoires of non-mhc-restricted t cells that play key roles in immunosurveillance. it is commonly considered that the characteristic iel repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating iel function. however, no such molecules have been identified. here we characterise skint1, currently the only known determinant of a canonical iel compartment, that is selectively required for vg5vd1 + dendritic epidermal t cell (detc) development. we show that both peripheral and thymic skint1 expression is essential for full detc development. its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects detc, nor affects any other t cells. unexpectedly, however, skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. mouse genetic models allow powerful insight into skint1 function; for example, we demonstrate that the constitutive expression of wild-type skint1 fully restores detc development in a skint1 mutant mouse, but does not rescue normal detc function. thus, skint1 provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated t cell compartments, and how normal versus dysregulated tissues may be demarcated. marginal zone (mz) b cells are strategically localized in the mz of the spleen. since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. besides being able to rapidly secrete antibodies, mz b cells may also act as professional antigen presenting cells (apcs). they are known to express high levels of cd1d which is the presenting molecule for nkt cells which are also located in the mz. therefore we hypothesised that mz b cells may be efficient activators of nkt cells. to test this hypothesis, we used freshly sorted splenic mz b cells (cd19 + cd21 hi cd23 lo cd11c -) and splenic conventional dendritic cells (cdcs) (cd11c hi cd8a +/-cd11b +/-b220 -) from wt and cd1d -/mice as apcs for nkt cells from va14-ja18 transgenic or wt mice. the apcs were treated with agalactosylceramide (agalcer) or heat killed (hk) listeria monocytogenes or salmonella typhimurium. both mz b cells and cdcs proved to be highly efficient apcs for priming of nkt cells and induced robust proliferation. in contrast, other populations of b cells failed to activate nkt cells. we showed, using cd1d -/mice as well as blocking antibodies to icosl, that proliferation of nkt cells depends on tcr/cd1d and in case of mz b cells, also on icos/icosl interactions. importantly, apcs primed with hk bacteria were not able to induce nkt cell proliferation. interestingly, mz b cells exclusively induced production of il-4 by nkt cells. in contrast, cdcs mostly induced production of ifn-g and il-4 producing cells were scarce under these conditions. cytokine production by nkt cells proved to be independent of tcr signalling, but dependent on icos/icosl interactions when mz b cells were used as apcs, and gitr-dependent when cdcs were used. taken together, our data suggest that both mz b cells as well as cdc act as professional apcs for nkt cells. notably, the nature of apcs appears to be critical for polarization of the immune response: mz b-cell-primed nkt cells induce cytokine milieu fostering a t h 2 response, whereas cdc-primed nkt cells rather favor a t h 1 response. objectives: il-18 is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. sle and ra) and the allergic disease atopic eczema. in mice, injections of il-18 give rise to an early polyclonal isotype switched antibody response which is absent in inkt cell deficient (cd1d -/-) mice. we set out to investigate the activated b cells in il-18 injected mice and how these are regulated by inkt cells. methods: mice received daily i. p. injections of il-18 (2 mg) for 10 days and the antibody response in serum was monitored using elisa. the b cell activation in the spleen at day 14 was evaluated by flow cytometry and immunohistology. results: mice injected with il-18 developed self reactive (anti-pc and anti-dna) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. sle and atopic eczema. the antibody producing cells formed cd138 + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. surprisingly, the antibody response induced by il-18 was increased in inkt cell deficient (cd1d -/-) mice, in contrast to published data. an increased response to il-18 was also observed in ja281 -/mice, which lack the a-chain of the tcr used by inkt cells, and thus our data suggest that inkt cells inhibit antibody producing cells in il-18 induced antibody responses. further characterization of the recruitment of b cells in il-18 injected mice revealed a marked expansion of the marginal zone b cell (mzb) population in the spleen, suggesting an important role for mzbs in the il-18 induced autoreactive antibody response. mzbs are innate-type b cells that express high levels of cd1d, are prone to autoantibody production and often involved in early immune responses. the il-18 induced antibody response in mzb deficient (cd19 -/-) mice was either decreased (igg) or delayed (ige), supporting the importance of mzbs in il-18 induced antibody responses. we conclude that the role for inkt cells in il-18 induced antibody responses is to inhibit the production of autoreactive antibodes from mzbs in extrafollicular foci. objectives: amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan entamoeba histolytica. there are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ala). interestingly, only a small proportion of e. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. so far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (ifn-g). however, information is lacking about the sources of early ifn-g production as well as the amoeba antigens involved in this activation process. methods: using a recently developed c57bl/6 mouse model for ala, the contribution of natural killer t (nkt) cells for protection against amoebic disease was investigated. applying nkt cells and dendritic cells as antigen-presenting cells from various ko-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by nkt cells was analysed. results: nkt cells were found to play a key role in the defense against ala. specific activation of nkt cells by a-galactosylceramide (a-galcer) induced significant protection, whereas jalpha 18-/-and cd1d-/-mice lacking inkt as well as dnkt cells suffered from more severe abscess formation. a lipopeptidophosphoglycan, which is present in large quantities on the surfcae of e. histolytica trophozoites (ehlppg), was identified as a major amoeba antigen that activates nkt cells resulting in the production of ifn-g, but not of il-4. moreover, ifn-g production required the presentation of ehlppg by cd1d and signaling through the tlr receptor cascade in combination with a simultaneous secretion of il-12. similar to a-galcer application, treatment of mice with purified ehlppg significantly reduced the severity of ala in amoeba-infected mice. our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of e. histolytica-infected individuals do not develop amoebic disease. a few years ago, we have observed a significant expansion of circulating effector gamma delta t cells following cytomegalovirus (cmv) infection in kidney transplant recipients (ktr). these unconventional t cells display tcr dependent cytotoxicity against both cmv-infected cells and carcinoma cells. in the present study, an extensive phenotyping of gamma-delta t cells allowed us to demonstrate an over-expression of cd16 in cmv-infected individuals. cd16 is the fcgammariiia, a natural killer cell marker usually absent on conventional t cells. we found that 71.9 ± 15.9 % of gamma-delta t cells from 21 cmv-infected ktr expressed cd16, when compared with only 19.8 ± 16.7% in 11 non cmv-infected ktr (p x 0.0005). similarly, 51.9 ± 25.7 % of gamma-delta t cells from 13 cmv-seropositive blood donors expressed cd16 compared to 27.1 ± 25.1 % in 15 cmv-seronegative donors (p x 0.01). cd16+ gamma-delta t cell lines generated from cmv-infected individuals were able to produce ifn-g (a potent anti-viral cytokine) in a cd16-dependent manner when activated by cmv/igg immune complexes. this production greatly increased in the presence of il-12 and ifn-alpha, two cytokines highly produced during cmv-infection. the supernatants of gamma-delta t cells activated with agonist anti-cd16 mab inhibited cmv replication in vitro and this effect was abrogated in the presence of a blocking anti-ifn-g antibody. cmv/igg immune complexes were also able to induce the expression of the cytotoxicity marker cd107a on cd16+ gamma-delta t cell lines. cd16 is well-known to mediate antibody-dependant cellular cytotoxicity (adcc), especially in natural killer cells. accordingly, we demonstrated that cd16+ gamma delta t cell lines could make adcc against the daudi lymphoma cell line and the a431 skin carcinoma cell line pre-incubated either with rituximab (anti-cd20) or cetuximab (anti-egfr), respectively. in contrast, no addc could be observed against cmv-infected fibroblasts pre-incubated with polyclonal anti-cmv igg (cytogam), probably because cytogam weakly stained infected cells. these data reveal a new cd16-dependent anti-cmv function of gamma-delta t cells through recognition of immune complexes and secretion of ifng. moreover, they demonstrate that these cells are able to kill through adcc lymphoma and skin carcinoma cells, two tumour types frequently encountered in ktr. dendritic epidermal t cells are a prototypic population of intraepithelial gd t cells in the mouse skin. found in the basal layer of epidermis and in close contact with langerhan's cells and keratinocytes detc facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd t cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-mhc restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. by using gd t cell knock-out mice tcrd-/-, ovalbumin transgenic k5mova mice and a skin grafting model we aimed to elucidate the role of gd-detc in adaptive immune responses associated with elimination of foreign antigen presented in the skin.we show that in the absence of gd t cells in the skin there is a decrease in rejection of ovalbumin expressing skin grafts compared to wildtype mice. we show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. however frequency of primed host animals is reduced in tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting detc enhance cross presentation of classical mhc bound antigens in the skin. considering the incapability of gd t cells to recognize peptide antigens in the context of mhc we plan to dissect the relationship between detc and professional antigen presenting cells in the skin. understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional apc function in skin by detc and potentially other epithelia by intraepithelial gd t cells and can be useful in designing therapies to epithelial infections and malignancies. we demonstrate a rapid and hmb-pp-dependent crosstalk between gd t cells and autologous monocytes that resulted in the production of inflammatory mediators including il-6, ifn-g, tnf-a, osm, ccl2, cxcl8, cxcl10, and trail. moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory dcs with antigen-presenting functions. these cells expressed cd40, cd86, hla-dr, and dc-sign, and lost cd14, ccr2, ccr5, and cxcr4. addition of further microbial stimuli (lps, peptidoglycan) induced ccr7 and enabled these inflammatory dcs to trigger antigenspecific cd4 + effector ab t cells expressing ifn-g and/or il-17. importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from pd patients and translated directly to episodes of acute pd-associated bacterial peritonitis, where vg9/vd2 t cell numbers and soluble inflammatory mediators were elevated in patients infected with hmb-pp-producing pathogens. conclusion: our findings suggest a direct link between invading pathogens, microbe-responsive gd t cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. the mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of nkt cell manipulation remains unpredictable. there is growing evidence that the nkt cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of nkt cellmediated immunosuppression. we examined the differential ability of nkt cell subsets from the thymus and liver to prevent type i diabetes when transferred into prediabetic nod mice. the transfer of abtcr+dn thymocytes (a population enriched for nkt cells) has previously provided robust protection against tid development; however it has not been formally shown that nkt cells are solely responsible for the protection. our study found that while the transfer of thymic dn nkt cells can prevent tid and severe insulitis in nod mice, not all nkt cell subsets show the same tolerogenic capabilities. these findings both formally demonstrate the disease-preventing effects of nkt cell transfer in nod mice and provide further evidence that nkt cells are a functionally heterogeneous population. objective: vg9/vd2 t cells constitute a minor t cell population in human blood that expands specifically and rapidly in response to the microbial metabolite hmb-pp. our previous microarray studies showed that vg9/vd2 t cells stimulated with hmb-pp in the presence of il-21 express markers associated with a possible follicular b cell helper function. we therefore investigated in more detail whether and how hmb-pp and il-21 regulate expression of the b cell attracting chemokine cxcl13/bca-1, its receptor cxcr5, and co-stimulatory molecules involved in b cell help. purified peripheral vg9/vd2 t cells were co-cultured with autologous monocytes or b cells (as feeder cells) for up to 4 days with and without hmb-pp, in the absence or presence of il-2 or il-21, or in medium alone. cells were analysed by flow cytometry and immunofluorescence microscopy. results: high levels of cxcl13 protein were detected in co-culture supernatants only when both il-21 and hmb-pp were provided, implying an il-21-dependent and tcr-dependent expression. vg9/vd2 t cells were confirmed as producers of cxcl13 by flow cytometry and immunofluorescence. under the same conditions, activated vg9/vd2 t cells expressed cd27, cd28, cd40l, cd70, icos and ox40. in contrast, neither cxcr5 nor ccr7 changed markedly by il-21 stimulation of peripheral vg9/vd2 t cells. conclusion: our findings confirm on the protein level that stimulation of vg9/vd2 t cells with hmb-pp and il-21 induces markers typically associated with follicular b helper t (t fh ) cells. these data suggest that gd t cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. ongoing analyses of gd t cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings. y. emoto 1 , m. emoto 1 1 gunma university school of health sciences, department of laboratory sciences, maebashi, japan invariant (i) natural killer (nk)t cells become undetectable after stimulation with a-galactosylceramide (a-galcer) or interleukin (il)-12. although downmodulation of surface t cell receptor (tcr)/nkr-p1c (nk1.1) expression has been shown convincingly after a-galcer stimulation, it is unclear whether this holds true for il-12 stimulation. to determine whether failure to detect inkt cells after il-12 stimulation is caused by dissociation/internalization of tcr and/or nkr-p1c or by block of de-novo synthesis of these molecules, and to examine the role of il-12 in disappearance of inkt cells after a-galcer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mrna expression of tcr/nkr-p1c by inkt cells after stimulation with a-galcer or il-12, and influence of il-12 neutralization on down-modulation of stcr/snkr-p1c expression by inkt cells after a-galcer stimulation were examined. the s/ctcr + s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of a-galcer, which was partially prevented by il-12 neutralization. whereas s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of il-12, s/ctcr + inkt cells were only marginally affected. mrna expression of tcr/nkr-p1c remained unaffected by a-galcer or il-12 treatment, despite the down-modulation of ctcr and/or cnkr-p1c protein expression. in contrast, ctcr + cnkr-p1c + stcr -snkr-p1c -inkt cells and cnkr-p1c + snkr-p1c -inkt cells were detectable after in-vitro stimulation with a-galcer and il-12, respectively. our results indicate that tcr and nkr-p1c expression by inkt cells is differentially regulated by signaling through tcr and il-12r. they also suggest that il-12 participates, in part, in the disappearance of inkt cells after a-galcer stimulation by down-modulating not only snkr-p1c but also stcr. the fetus and infant are highly susceptible to viral infections. a number of viruses, including human cytomegalovirus (cmv), cause more severe disease in early life compared to later life. it is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd t cells are unconventional t cells that can react rapidly upon activation and show mhc-unrestricted activity. herein, we show that upon cmv infection in utero, fetal gd t cells expand and become differentiated. the response was restricted to vg9-gd t cells, irrespective of their vd chain expression. differentiated gd t cells expressed high levels of ifn-g, transcription factors t-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. in addition, congenital cmv-infection induced a highly restricted complementary-determining region 3 d1 (cdr3d1) and cdr3d2 repertoire, with a striking enrichment in a specific germline-encoded cdr3d1 sequence. differentiated gd t cells and the enriched cdr3d1 sequence were detected as early as after 21 weeks of gestation. our results indicate that functional fetal gd t cell responses can be generated during development in utero and suggest that this t cell subset could participate in anti-viral defense in early life. results: spectratyping showed only in-frame selection for vd1-jd1 and vgi-jg1.3/2.3 rearrangements in tcrgd thymocytes and to a lesser extent in tcrgd cb cells. in contrast, clear in-frame vd2-jd1 and vg9-jg1.2 selection was seen in pb tcrgd cells. detailed analysis of the cdr3 motifs revealed selection determinants in both vg9-jg1.2 (canonical length and cdr3 motif) and vd2-jd1 (minimal cdr3 length in combination with an invariant t nucleotide) rearrangements. upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve tcrgd thymocytes (˚4) and tcrgd cb cells (6-7) to tcrgd pb cells (˚10 or more). no increase was seen between cb and pb tcrgd t cells within the first year of life, suggesting that peripheral proliferation occurs later in life. our results indicate that the human peripheral tcrgd repertoire is shaped by (antigenic) selection and proliferation processes. moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. background: natural killer (nk) t cells have been implied in the regulation of disease in the non obese diabetic (nod) mouse model of type 1 diabetes (t1d). we have previously shown that transgenic expression of a cd1d-restricted, va3.2-vb9 tcr in nod mice lead to an increase in cd1d-restricted type ii nkt cells (24abnkt cells), and prevention of the development of t1d in the transgenic mice. in this study we have investigated the requirements and underlying mechanism of disease protection by type ii nkt cells in a disease transfer model. to investigate the mode of regulation by 24abnkt cells, we explored a disease transfer model into nod.scid mice using transgenic diabetogenic bdc2.5 cd4+ t cells, in the presence or absence of selected cells from 24abnkt cell transgenic mice. results: in 24ab transgenic mice a high frequency of activated transgenic nkt cells was found in the pancreas of the protected mice. in this organ, 24abnkt cells expressed a high level of cxcr3 and a low level of ccr7 and cd62l, a pattern similar to that observed in t cells homing to inflammatory tissues. adoptive transfer of cd4+ bdc2.5 t cells into nod.scid recipients rapidly induced onset of diabetes. using this model, we found that co-transfer of spleen cells from 24ab transgenic mice with bdc2.5 cd4+ cells resulted in the prevention of diabetes development. the protection from disease required a minor cd4+ subset of 24ab+ nkt cells, but was independent of cd25+ t regulatory cells. analogs of alpha galactosylceramide (a-galcer) that may modulate the strong activation of inkt and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. a new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by cd1d and recognized by inkt. the structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the o glycosidic linkeage with the ceramide by a n. in this basic structure, substitutions in the cyclohexane ring with oh in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the n linkeage conform a series of analogs that have been analyzed in their capacity to stimulate inkt cells. proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that inkt cells are specifically stimulated by some of the analogs tested. in particular, the active compound hs44, induces in vitro inkt cell expansion and ifng and il-4 secretion in a similar fashion but less potently than a-galcer. dose response assays show a bias towards a th2 profile response after recognition by nkt cells, more similar to the response induced by och. the degree of structural similarity of the cyclitol ceramides with a-galcer parallels their cellular activities. these data open the way towards the development of a new class of a-galcer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential inkt cell sphingolipid agonists that can modulate the immune response due nkt cell activation. objectives: invariant natural killer (ink) t cells represent an innate lymphocyte subset with important modulatory functions. in the presence of pathogens or tumors, inkt cells play an adjuvant function that boosts t cell immunity through cytokine secretion and dc maturation. in steady-state conditions, i.e. in the absence of pathogens, inkt cells acquire a regulatory function that promotes t cell tolerance and prevents autoimmune disease. our aim was to assess the mechanism of action of inkt cells in the steady state and, specifically, to test the hypothesis that inkt cells promote immune tolerance through modulation of dcs. methods: to assess the direct influence of regulatory inkt cells on dc maturation in resting conditions, we derived murine inkt cell lines in vitro and, after staining with agalcer-loaded cd1d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid dcs in the absence of any other maturation signals. we analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of inkt cell-modulated dcs (inkt-dcs). the cell-cell interaction with inkt cells provoked dramatic phenotypical changes on immature dcs that acquired the cardinal features of tolerogenic dcs such as intermediate levels of mhc class ii and co-stimulatory molecules expression and high secretion of il-10 with no release of pro-inflammatory cytokines. most importantly, inkt-dcs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory tr1 cells and immune tolerance in vivo. dcs, simultaneously stimulated with inkt cells and through toll-like receptor (lps) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. conclusion: it is still mysterious how inkt cells can play a dual role and either boost t cell immunity or promote immune tolerance. our results suggest that the same mechanism could underlie both inkt cell functions. in the presence of pathogen-driven maturation signals, the inkt cell-modulation of dcs favors their acquisition of a pro-inflammatory phenotype and function. on the contrary, if inkt cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature dcs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. th17-type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. the th17 immune response is characterized by the secretion of il-17a and il-17f. the il-17 locus encodes the highly conserved il-17a and il-17f cytokines that are syntenic in 44kb distance to each other. besides cd4 + th17 and nkt cells, approximately 50 % of the il17a producers are gd t-cells. like cd4 + th17 cells, il-17 producing gd tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. our findings show a difference between the il-17 production of gd t cells in the peripheral system and mucosal epithelia. mucosal gd t-cells generally do not produce th17 cytokines. in the periphery, we define novel subsets of gd t-cells that can produce either il-17 or ifn-g. combined with the well known classification of il-17 producing gd t-cells along the markers cd27 and cd122, our data point at specialized functions of the different gd t cell subsets depending on their location and origin. functional studies are currently carried out in order to address the role of the different gd t-cell subsets for th17-type immune responses in vivo. in this context, the potential redundancy of il-17a and il-17f may complicate the analysis. so far, most studies were carried out with il-17a single-deficient or il-17f single-deficient mice. to further clarify these issues, we will have to address the above mentioned findings in il-17a and il-17f double-deficient mice. several subsets of gd tregs have been described and intensively studied, but the potential regulatory role of innate t cells in controlling immune responses remains unclear. lymphocytes expressing gd tcr are involved in both innate and adaptive immune responses. vg9vgd2 t cells, which represent a major peripheral blood gd t-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.here we report that tgf-b1 and il-15 differentiate in vitro a subset of gd t lymphocytes with regulatory functions (vd2 tregs) in the presence of specific antigen stimulation. these cells express the forkhead/winged helix transcription factor (foxp3) and, similarly to ab tregs, suppress the proliferation of anti-cd3/anti-cd28 stimulated-pbmc. detailed knowledge about the phenotype and functionality of vd2 tregs will improve our understanding of the role of gd t cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-galcer) has the potential to activate invariant (i) nkt cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-galcer. here we show that treatment with a-galcer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following listeria monocytogenes infection. liver infiltration of granulocytes and g/d t cells was accelerated by a-galcer treatment. granulocyte and g/d t cell depletion exacerbated listeriosis in a-galcer-treated mice, and this effect was more pronounced in granulocyte than in g/d t cell depletion. although secretion of gm-csf and il-17 was detected among the nkt cell population in the liver and bone marrow immediately after a-galcer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mab. yet, in parallel to the numerical increase of granulocytes expressing cd11b in the liver following a-galcer treatment, numbers of cells lacking cd11b diminished in the bone marrow. in addition, respiratory burst in granulocytes was enhanced by a-galcer treatment. our results indicate that a-galcer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d t cells, into the liver. we also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. objectives: the aim of this work is to evaluate whether phenotypic and functional features of vgamma9/vdelta2 t cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in cll. methods: eighty seven previously untreated cll patients were evaluated for in vitro vgamma9/vdelta2 t cells expansion upon stimulation with zoledronic acid (za) and interleukin-2 (il-2). gammadelta t cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. the mutational status of the tumor immunoglobulin heavy chain variable region (igvh) was analyzed by dna sequencing. the activity of the mev pathway was determined by 1) the bioinformatic analysis of gene expression profiling data 2) the quantification of mev pathway metabolites. results: proliferation of gammadelta t cells was observed in 43 patients (49 %) (responders, r), whereas 44 patients (51 %) were non-responders (nr). vgamma9/vdelta2 t-cell subset distribution was well balanced in r patients, whereas effectors subsets [i. e., effector memory (tem), and terminally differentiated effector memory (temra)] were largely predominant in nr patients. temra of nr patients mainly expressed the inhibitory receptor ilt2, whereas temra of r patients had an higher expression of the costimulatory molecule nkg2d. the proliferative response of vgamma9/vdelta2 t cells was significantly associated with igvh mutational status, which is a well known prognostic factor in cll. indeed, 82 % of r patients were m, whereas 77 % of um patients were nr (p x 0.001). given this association, we evaluated the activity of the mev pathway in tumor cells of m and um patients. the pathway was more active in tumor cells of um than m patients, suggesting that the former can more easily engage gammadelta t cells and drive their differentiation into functionally exhausted t emra . given the association between the r/nr status and the igvh mutational status we also analyzed the independent prognostic impact of r/nr status in multivariate cox analysis. nr patients had a significantly shorter time to first treatment thus pointing to r/nr status as an independent prognostic factor. conclusion: these data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in cll patients. the studies reported here were undertaken to ascertain and delineate the ability of kupffer cells to regulate the response of inkt cells to biliary obstruction. methods: c57bl/6 mice were not treated or rendered kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. to clarify the factors that elicit inkt cell activity, additional mice were administered anti-il-12 p40 (clone r2-10f6; atcc) or anti-cd-1d (clone 1b1) monoclonal antibody (mab) prior to surgery. midline laparotomies were performed; the common bile duct was ligated twice and divided. sham-operated animals served as controls. blood and liver samples were collected at periodic intervals post-surgery. the hepatic lymphoid population was purified and characterized by flow cytometry. the nkt cell population was increased significantly in the livers of control, but not kupffer cell-depleted, mice at 18 hours post-bdl. the response of inkt cells was diminished in mice pretreated with mab specific for il-12p40, a component of both il-12 and il-23; pretreatment with anti-cd1d mab had no effect. il-12rb-deficient mice also exhibited a marked increase in hepatic inkt cells following bdl suggesting that il-12 was not a critical factor. this suggestion is supported by the increased expression of il-23p19 and il-12p40 (but not il-12p35) mrnas by kupffer cells purified from the livers of bdl animals. these findings imply that il-23 production by kupffer cells promotes the response of hepatic inkt cells to biliary obstruction. objectives: p-glycoprotein (pgp or abcb1) is a member of the abc family of transporter proteins which are characterized by their ability to pump molecules across membranes in an atp-dependent manner. although pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. our work primarily focuses on gd t cells that complement and regulate the activities of ab t cells, particularly in tissues. we have recently described functional subsets of gd cells based on cd27 expression. gd 27+ cells secrete interferon-g, while gd 27cells are capable of producing il-17. this study investigates the role of pgp in gd cells with specific reference to these recently-identified cd27-defined subsets. methods: pgp activity was measured based on the expulsion of rhodamine 123. cells were incubated with rho followed by a period in the presence or absence of the pgp inhibitor cyclosporine-a. cell populations were identified using monoclonal antibodies and flow cytometry. percentages of subpopulations were compared by anova, statistical results are shown as p values that were calculated using a newman-keuls multiple comparison post-hoc test. results: up to 40 % of intraepithelial lymphocytes (iels) from the small intestine are tcrgd + . of these, virtually all displayed pgp activity. indeed, pgp activity was generally higher in tcrgd + than tcrab + iels. in the thymus, pgp activity was observed in only˚2% of gd 27+ cells but not at all in gd 27cells. by contrast, in peripheral lymph nodes, mesenteric lymph nodes and peyer's patches, 40-60 % of gd 27+ cells were positive for pgp activity, although their gd 27counterparts remained largely negative (p x 0.01). conclusion: this study demonstrates that subsets of gd cells display different levels of pgp activity depending on their location in the body and their expression of the newly identified functional marker cd27. as pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd 27+ and gd 27cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. objectives: an effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. in this context, the vgamma9vdelta2 t cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. the activation of vgamma9vdelta2 t cells by aminobisphosphonate drugs such as zoledronic acid (zol) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (dcs) and b lymphocytes. the aim of this work was to evaluate the ability of activated vgamma9vdelta2 t lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. methods: peripheral mononuclear cells (pbmc) and purified vgamma9vdelta2 t cells from healthy donors were stimulated with different compounds (zol, ipp) for 24 hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and elisa tests. we showed that soluble factors released by zol-stimulated vgamma9vdelta2 t cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated vgamma9vdelta2 t cells. moreover, mcp-2 depletion by neutralizing ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. altogether, these data show a vgamma9vdelta2-mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of vgamma9vdelta2 tlymphocytes in orchestrating the immune response. conclusion: an immune modulating strategy targeting vgamma9vdelta2 t cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. objectives: the aim of this study was to analyse the activity of vg9vd2 t lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. human vg9vd2 t cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. interestingly, drugs already in clinical use, such as zoledronic acid, are able to promptly activate vg9vd2t cells through an indirect mechanism involving the block of farnesyl pyrophosphate synthase of the mevalonate cycle. the vg9vd2 t cell activation by zoledronic acid results cytokines and chemokines synthesis and cytotoxic activity. glioma are tumors arising from glia in the central nervous system. unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. methods: in order to analyse the activation of vg9vd2 t cells and their effects on the viability of glioma cells, we expanded in vitro vg9vd2t cells from pbmcs of healthy donors by using phosphoantigen stimulation and tested the ability of vg9vd2t cell lines to kill three different glioma cell lines (t70, u251, u373) by cytokinic/cytotoxic mechanism by flow cytometry. results: our results demonstrated that vg9vd2t cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. moreover, we analysed whether zoledronic acid treatment could improve the susceptibility of glioma cells to vg9vd2 t lines. we showed that zoledronic acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of vg9vd2 t lines. conclusions: altogether, our results suggest that the induction of a strong antitumor response in vitro of vg9vd2 t cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately 15 % of all cancers and represent a failure of host immunity to control chronic viral infection. natural killer t (nkt) cells are a population of regulatory t lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. using a mouse model of chronic viral infection in the skin, in which human papillomavirus (hpv) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for nkt cells in abrogating protective immunity to viral antigens in cutaneous tissue. we show that local hpv-e7 protein expression in the skin attracts a large lymphocytic infiltrate, including a population of cd1d-restricted nkt cells. this nkt infiltrate is required to maintain local hpv-e7-induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. the local suppressive environment evident in e7-expressing transplanted skin is dependent on interactions between populations of cd1d-expressing cd11c+/f480+ myeloid cells and nkt cells. removal of either donor-resident or host-infiltrating nkt cells is sufficient to break immune suppression and allow e7 graft rejection. dissecting the suppressive properties of nkt cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of nkt cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. nkt cells were expanded from total pbmcs from healthy donors by treatment with il-2 and a-galcer. expression of cd1a, cd1d and the costimulatory molecules cd86 and hla-dr, was established by flow cytometry. rna was quantified by real time-pcr. functional assays were performed by analysis of nkts cytokine production (ifn-g, il-4) and cytotoxicity against treated-idcs. results: idcs stimulated with olive pollen lipids up-regulated cd1d expression on the cell surface in comparison with control cells. in contrast cd1a expression was decreased. cd86 and hla-dr slightly increased, indicating certain grade of maturation. the amount of cd1d mrna was higher in treated cells than in control cells. by contrast, there was less transcription of cd1a, cd1b and cd1c genes than in control cells. nkt cells efficiently killed treated idcs as "in vitro" cytotoxic killing assays showed. ifn-g producing cells increased slightly in response to treated idcs compared to unstimulated cells, but the number of il-4 producing cells was not modified. similar results were obtained using monocytes as antigen-presenting cells. conclusions: idcs treated with lipidic extracts from olive pollen up-regulate the expression of cd1d on the cell surface. in addition, nkt cells are able to recognize idcs and monocytes treated with lipids from pollen, producing ifn-g and cytotoxicity. all these data suggest that nkt cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. outline: in humans, 0.5-10 % of circulating lymphocytes express a vg9vd2 t cell receptor, yet strikingly little is known about the function and properties of such unconventional t cells. we performed cdna microarrays to find vg9-enriched genes compared to conventional mhc-restricted cd8 + ab t cells, and found reciprocal enrichment of nectin-like adhesion molecules igsf4 & crtam in gd and ab t cells respectively. because igsf4 binds to crtam, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. interestingly, previous studies show that activated nk, nkt and cd8 + ab t cells express crtam, and that engagement of igsf4 on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. our data extends this to the prospect of cytolytic immunoregulatory interactions between t cells mediated by igsf4/crtam. we therefore sought to answer: 1. what is the function of igsf4/crtam on gd t cells? 2. how is the igsf4-crtam axis regulated in t cells? results and conclusions: flow cytometry showed igsf4 enrichment on resting gd t cells, with expression also detected on˚10 % of ab t cells. the properties of those cells are being examined. however, igsf4 generally correlates with markers of activation/antigen experience such as cd45ro. thus, igsf4 cells may comprise activated-yet-resting/pseudo-memory unconventional t cells and memory-effector conventional t cells. stimulating vg9 + t cells in vitro led to rapid crtam induction, resulting in the majority of cells co-expressing both igsf4 and crtam within 48 hours. however, engagement of igsf4 by crtam or vice versa is not sufficient to induce cytotoxicity, as stable cho cell transfectants expressing either molecules were not specifically lysed by pbmc in vitro, compared to efficient and parallel targeting of mica + cells. instead, our current experiments address the possibility that crtam-igsf4 may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as mica-nkg2d. this may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that crtam-igsf4 interactions are sufficient for cd8 t cells to kill gd t cells and/or vice versa. instead, crtam-igsf4 interactions may set the threshold for cytotoxic immune-surveillance responses. t cell receptor (tcr) is a multisubunit complex in which the invariant subunit cd3z is a 16 kda transmembrane protein indispensable for coupling antigen recognition by tcr to diverse signal transduction pathways. approximately 3-6 % of human peripheral blood lymphocytes express the gd tcr and the majority of these cells express the vd2 tcr variable segment associated with the vg9 segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. these compounds trigger vg9vd2 t cells without antigen presentation. in vitro stimulated vg9vd2 t cells with antigens are able to produce ifn-g and tnf-a and exert a powerful cytotoxic activity against infected cells as hiv-infected cells. however, during hiv infection a marked decrease of vg9vd2 t cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. aim of the present work was to study the mechanisms of vg9vd2 t cell anergy observed in hiv+ patients. to this aim, cd3z expression and ifn-g production by vg9vd2 t cells from hiv+ and hiv-subjects were analyzed. we show that vg9vd2 t cells from hiv-infected patients expressed lower level of cd3z compared with healthy donors. a direct correlation between cd3z expression and ifn-g production capability by vg9vd2 t cell was found. however, pkc activation by pma is able to restore cd3z expression and ifn-g production. our findings may contribute to clarify the molecular mechanisms of vg9vd2 t cell anergy found in hiv+ patients and have implication in the design of effective immune-based therapies. l. abeler-dörner 1 , m. swamy 1 , s.l. clarke 1 , a. hayday 1 1 king's college london, immunobiology, london, united kingdom gut intraepithelial lymphocytes (iel) constitute one of the largest t cell compartments in mice and in man. their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance. intestinal iel comprise many unconventional t cells including tcrgd cells and tcrab cd8aa or cd8 -cd4cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. unlike conventional t cells, iel might initiate an immune response rather than simply being late effector cells. it is therefore important to elucidate the "immunological information flow" in the gut. to this end, this project characterizes different subsets of iel and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo. in the past, it has been notoriously difficult to study iel ex vivo. to solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to 15 days. the cells are initially activated by plate-coated acd3 antibody and a cytokine cocktail and maintained further in medium containing low levels of il-2. after a resting period, the cells can be restimulated in vitro. in this new system, we studied responses of different iel subsets to stimulation via tcr, nkg2d and cytokine receptors, either alone or in coculture with epithelial cells. as readouts we monitored proliferation, cytokine secretion (ifng, il-6) and expression of activating and costimulatory molecules. reactivation in response to various stimuli could already be observed after 6 hours. the in vitro data set forms the basis for analysing iel responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. the characterization of iel responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. objective: behcet's disease (bd) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. increased expression of cd94 in bd patients suggested that nk receptors may play a pathogenic or regulatory role in the pathogenesis. considering the regulatory functions of nkg2 molecules in heterodimer with cd94, we screened the presence of these receptors on t cell subsets in bd. the expression of nkg2 a/c/d molecules on gd and cd8+ t cells were analyzed in 17 active and 9 inactive patients with bd and 21 healthy controls. expression of nkg2 molecules was evaluated on cd8+, gd t and cd56+ nk cells by using flow-cytometry. results: gd t cells were increased in patients with bd compared to controls (4.4 vs. 2.8 %, p=0.001). in addition to the increase of gd t cells, increased expression of activating nkg2c molecules was also observed on gd t cells (20 % vs. 11 %, p= 0.01). nkg2a expression on gd t cells was found to be higher than nkg2c expression in patients and controls; but nkg2a expression on the t cells was not statistically different in both groups (33.5 vs. 40 %). nkg2d receptors were present on most of the gd t cells in both groups. however these activating molecules on cd8+ cells were decreased in patients with bd compared to controls ( revlimid is a therapeutic agent used to treat myelodysplastic syndrome (mds), a group of haematological disorders characterised by ineffective haematopoiesis. the mechanism of action for revlimid is poorly understood, but there has been increasing interest in the strong association reported between mds and defects within the immunoregulatory nkt cell compartment. indeed, some studies now suggest an important outcome of revlimid treatment is the restoration of normal cytokine production by nkt cell levels and an increase in their overall numbers. we have conducted the most thorough study to date of the nkt cell compartment of mds patients treated with revlimid/ancestim and can report that mds patients had normal nkt cell levels prior to treatment, and no significant increase as a result of revlimid/ancestim treatment. furthermore, nkt cells from mds patients produced high levels of th1 and th2 cytokines when stimulated with pma/ ionomycin and the proportion of nkt cells capable of cytokine production did not increase significantly after revlimid/ancestim treatment. these are highly significant findings given the recent emphasis on nkt cells as a potential therapeutic target for mds. our study provides an extensive analysis of the impact of revlimid/ ancestim treatment on the nkt cell compartment and sheds new light on the role of nkt cells in mds and the mechanism of revlimid immunomodulation. objectives: human gd t cells are potent killers of a variety of tumour cell lines, and mice lacking gd t cells suffer from high incidence of experimentally-induced tumours. however, the molecular mechanisms mediating tumour cell recognition by gd t lymphocytes remain largely unknown. we aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd t cells for tumour cytolysis. as immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for vg9/vd2+ lymphocytes. we have screened a panel of 26 lymphoma and leukaemia cell lines using a conventional in vitro killing assay using vg9/vd2+ cells, and selected two susceptible ("target") cell lines (over 60 % death in the assay) and two vg9/vd2+ resistant ("non-target") cell lines (under 20 % death) for cdna microarray analysis. we compared the differential expression in pairs of tumour cell lines of identical origin: the burkitt's lymphoma cell lines daudi (target) vs raji (non-target), and the pre-b cell leukemia cell lines rch-acv (target) vs 697 (non-target), and validated the results by rt-qpcr quantification. results: we identified 37 commonly up-regulated and 50 commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ulbp1, ifitm1 and prame, for example, are up-regulated, whereas cd274 and clec2d are down-regulated in target cell lines. as these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. the expression of these candidate genes was studied by rt-qpcr in a broader panel of cell lines and primary biopsies. we are currently testing, in functional assays based on rna interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd t cells. the comparison between the transcriptomes of vg9/vd2+ target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd t cells. mice and humans are the only species in which phenotype and function of inkt cells have been properly described. our aims are to directly identify this cell population and to investigate cd1d, in the rat. mice and rats have very similar cd1d and inkt tcr genes, with the exception of the va14 gene segment, which is a multimember gene family in the rat. novel monoclonal antibodies with nearly identical binding capacities to mouse and rat cd1d revealed a very similar pattern of cd1d distribution, and could inhibit cytokine production after agalcer stimulation of primary cells in both species. response to agalcer was studied in five different rat strains, showing big inter strain differences. notably, ifn-g and il-4 production was 10-100 fold lower in the best responder rat strain (f344) compared to mouse (c57/bl6). since nkrp1a (rat homologue of mouse nk1.1) and tcr are not appropriate markers for rat inkt, cd1d oligomers where tested for binding to inkt-tcr transduced cells. newly generated agalcer loaded rat cd1d dimers, recognized rat inkt tcr and, although less efficiently, bound to mouse inkt tcr. however, mouse cd1d agalcer dimers did not bind to rat inkt tcr. agalcer loaded rat cd1d dimers were then used to stain primary intrahepatic lymphocytes. but, although mouse inkt cells were stained to some extent, the identification of a discrete population in the rat was not possible. the reasons behind could be: that the avidity of the dimers for the tcr is not high enough to stain primary cells and/or that the frequencies are so low that the detection by facs analysis is difficult. in order to clarify these issues we currently produce and test rat cd1d tetramers. burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. this disease is endemic in tropical regions, especially thailand and northern australia, and has a serious outcome for many infected individuals. b. pseudomallei is an intracellular bacterium and many b. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. in addition to this, no vaccine is currently available to prevent the disease. human g9d2 t cells are involved in the immune response to infection with a number of intracellular pathogens including brucella suis and mycobacterium tuberculosis. g9d2 t cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. phosphoantigens cause expansion and activation of g9d2 t cells during infection with intracellular pathogens including fransicella tularensis and m. tuberculosis. analogues of natural phosphoantigens have been developed to manipulate g9d2 t cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of hepatitis c virus. we aimed to determine in vitro whether enhancing gd t cell responses in human blood using the synthetic phosphoantigen picostim could reduce growth of intracellular b. pseudomallei in the human monocytic cell line thp-1. a significant (p x 0.01) reduction in intracellular bacterial numbers was observed (n=8) in the presence of pbmcs cultured with picostim+il-2 in comparison with pbmcs cultured with il-2 or media alone. picostim+il-2 caused significant expansion and activation of gd t cells following culture of pbmcs for 10-14 days. purified gd t cells stimulated with picostim were able to reduce intracellular b. pseudomallei numbers 100-fold. this data demonstrates that pbmcs, stimulated with the synthetic phosphoantigen picostim+il-2, reduced growth of intracellular b. pseudomallei in a gd t cell-dependent manner. objectives: vgamma9/vdelta2 (gd) t cells play a major role in innate immunity against microbes, stressed and tumor cells. they represent less than 5 % of peripheral blood lymphocytes (pbl), but can be expanded in vitro by zoledronic acid (za)-treated monocytes or dendritic cells (dc).the purposes of this study are: 1) to determine whether dc generated from multiple myeloma (mm) patients are as effective as their normal counterparts in the ability to activate gd t cells; 2) to evaluate whether gd t cells can exert immunoadjuvant activity on dc generated from mm patients and primed with tumor-specific antigens (survivin-sv); 3) to establish whether the same issues could be solved using a simplified protocol of dc generation. 1) dc were generated from cd14 + cells of healthy donors/mm patients; immaturedc on day 6 were induced to fully mature by incubation for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za. after 7 days of co-culture dc:pbl, percentages and total counts of gd t cells were determined by flow cytometry; 2) idc generated from cd14 + cells of hla-a*0201 + healthy donors/patients were pulsed with sv-peptide and stimulated for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za; after 2 rounds of autologous t cells stimulation by dc, the frequency of sv-specific cd8 + t cells was determined by svpentamers staining; 3) the same experiments were performed both with dc generated following a standard protocol and a 48h protocol (dc fast objective: depletion of or deficiency in gd t cells aggravate colitis in different animal models. additionally, reconstitution of mice with syngeneic gd t cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd t cells in intestinal inflammation. therefore, we asked whether human gd t cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or crohn's disease. hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd t cells were determined in vitro. methods: human gd t cells were isolated from whole blood of healthy donors by macs technology. the proliferation was determined by [6-3 h]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via cfse fluorescence intensity. the cytokine profile was determined by elisa from culture supernatants as well as by flow cytometry intracellularly. finally, the in vitro characteristics of gd t cells were compared to those of cd4 + cd25 + regulatory t cells (treg). human peripheral gd t cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin-2 on stimulation and proliferate poorly. while the proliferation of gd t cells and treg cells is comparable, the suppression of gd t cells on responder cell proliferation is even stronger than the suppression by treg cells though gd t cells being foxp3 negative. additionally, gd t cells are strong producers for tgf-b, particularly by the vd1 subset. conclusion: human peripheral gd t cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. their suppressive activity is even superior to treg cells and might be due to strong tgf-b secretion. for application of human gd t cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. there are previous descriptions of gamma-delta t lymphocytes (gd) from behçet's disease patients (bd) but, in most of cases, they are incomplete or contradictory. it has been suggested that nkg2d on gd is involved in bd lesions through interaction with mica molecules. furthermore gdcd8+ have been recently proposed as a new regulatory t subset (treg). objectives: to study gd phenotype in bd active (bda) (n=8) and inactive (bdna) (n=20), versus healthy controls (hc) (n=30) and patients with recurrent oral ulcerations (ru) (n=14). to determine gd cytokine profile and surface markers treg-related in bd (n=9) and hc (n=11). methods: we obtained mononuclear cells from peripheral blood (pbmc). we determined by flow cytometry: -surface expression of: gd tcr, vdelta1, vdelta2, cd8alpha, cd8beta, nkg2d, nkg2a and cd103. -intracellular expression of ctla-4, and foxp3. -intracellular expression of il-2, il-4, ifngamma, il-10 and tgfbeta after pbmc polyclonal stimulation. we used two tailed test for means comparison (mann-whitney u or student's t test). -vdelta2+ cells were significantly increased in ru. vdelta1+ and gdcd8+ lymphocytes were significantly increased in bd versus ru and hc. -the mean fluorescence intensity of nkg2d was slightly increased in gd from bda. -nkg2a expression by gdcd8+ was not different in bd versus hc. -most of gdcd8+ presented cd8alpha-alpha homodimers in bd and hc and were negative for cd103, foxp3 and ctla-4. gdcd8+ and gdcd8-subsets were (in bd and hc): -high ifngamma-producers without differences. -low il-2-producers: il2+ cells were lower in gdcd8+ than in gdcd8-. -low il-10-producers: il10+ cells were lower in gdcd8+ than in gdcd8-. -low tgfbeta-producers: tgfbeta+ cells were lower in gdcd8+ than in gdcd8--very low producers of il4 in most of cases. the hallmark in bd was the increase of gdcd8/vdelta1+. this subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to nkg2a in intraepithelial intestinal lymphocytes from celiac patients. we did not find a cytokine profile or a phenotype t-reg-related for gdcd8+, except a lower percentage of il-2+ cells than in the gdcd8-subset. gdcd8+ from bd did not show significant differences versus hc. natural killer t (nkt) cells comprise a highly heterogeneous subset of t lymphocytes that co-express a t cell receptor (tcr) and nk cells markers such as cd56 in humans. a subgroup, the invariant nkt cells (inkt), expresses the va24vb11 tcr rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. objectives: to establish a method to growth cord blood-derived nkt cells (cd3 + cd56 + ), in order to evaluate their phenotypic characteristics and the tcrvb repertoire. methods: mononuclear cells were isolated from 10 healthy umbilical cord blood samples and stimulated with ifn-g (50 ng/ml), anti-cd3 (25 ng/ml) and il-2 (500 ui/ml). these cells were cultured for 21 days and the expanded cd3 + cd56 + cells were isolated by immunomagnetic methods. surface markers were determined by flow cytometry. total rna was extracted from the purified cd3 + c56 + cell suspension using trizol ® reagent and mrna expression of twenty tcrvb gene families was measured by semiquantitative rt-pcr. statistical analyses were performed using mann-whitney u test and one-way anova, a p value of x 0,05 was considered significant. results: we could significantly expand cord blood cd3 + cd56 + nkt cells from 0,87±0,57 % to achieve an enrichment of 46,89±13,31 % (p=0,0002). table 1 shows the percentage (mean±sd,n=10) of phenotypic markers in cd3 + cd56 + cells at baseline (day 0) and after 21 days of culture. expression of mrna for the vb families studied was confirmed in each individual cell culture with a significant high expression of vb4 and vb8 families (p x 0,001). conclusion: our results show that cord blood-derived nkt cells are mainly cd8 + and cd94 + subsets, similar to peripheral blood nkt cell with a low percent of inkt cells. additionally, we confirm a diverse tcr vb repertoire with a significant expression of the vb4 and vb8 families in these cells. l. marischen 1 , d. wesch 1 , p. rosenstiel 2 , a. till 2 , d. kabelitz 1 1 institute of immunology, kiel, germany, 2 institute of clinical molecular biology, kiel, germany gd t cells account for a minority of t cells in human blood, but represent the majority of intraepithelial t cells in the intestinal tract. due to their ability to respond rapidly and in an mhc-independent fashion to particular antigens by cytokine production, gd t cells are considered as a link between innate and adaptive immunity. in addition, the expression of distinct pattern recognition receptors such as toll-like (tlr) and nod-like receptors (nlr) are characteristic for cells of the innate immune response. recent reports have demonstrated the tlr expression in human and murine gd t cells. here we provide evidence also for a gd t cell responsiveness to muramyl dipeptide (mdp), the putative ligand of the nlr family member nod2. peripheral blood mononuclear cells (pbmcs) containing gd t cells as well as freshly isolated gd t cells were stimulated via the gd t cell receptor in the absence or presence of mdp and analyzed for proliferation and ifng-production. while the proliferation of gd t cells within pbmcs was decreased, ifng-production was increased after costimulation with mdp compared to the stimulation with a non-activating dd-stereoisomer of the ligand (mdpi). the enhanced ifng production of pbmcs after costimulation was mediated mainly by gd t cells as shown by intracellular flow cytometric staining. with regard to the ifng-production after co-stimulation with mdp vs. mdpi, freshly isolated gd t cells from different healthy blood donors can be divided into responder and non-responder. responder gd t cells showed a significant increase of the ifng-production due to mdp-stimulation, whereas ifng-production was not influenced in non-responder gd t cells. in further experiments, as first approach to explain the different reactivity patterns of gd t cells, it is planned to analyze the polymorphisms of the nod2 gene in various donors. taken together, our preliminary data indicate that gd t cells are a major source of ifng-producing cells among pbmcs when challenged with specific antigens plus mdp, and support the role of gd t-cells as an important team player in the early immune response against bacteria. objectives & methods: an increasing of gamma-delta t cells during acute p. vivax infection and convalescent period has been reported. moreover, the activation of gamma-delta t cells leads to the inhibition of blood stage p. falciparum parasites in vitro. to determine the killing mechanisms of p. vivax parasites by gammadelta t cells comparing with what has been found in p. falciparum, the gamma-delta t cells were enriched by isopentenylpyrophosphate (ipp) from naïve pbmc. different number of gamma-delta t cells and normal pbmc were incubated with intact of p. vivax parasites and protein extract of p. vivax parasites, recombinant pvmsp1 19 and pvama1 proteins. gamma-delta t cells was daily determined the cytokine and granzyme intracellular releasing by flow cytometry until day 5 culturing. results: among the enriched gamma-delta t cells, the percentage of cells expressing cd69 + and cd25 + was elevated after co-culturing with intact and the proteins of p. vivax parasites. the overall gamma-delta t cells showed proliferation at day 3 after the co-cultivation. moreover, the gamma-delta t cells expressing ifngamma + and cd107a + (lysosomal associated membrane proteins: lamp-1) elevated from the first day of pbmc collection after co-culturing with the intact and p. vivax antigens. this level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition. our results showed the activation of gamma-delta t cells during p. vivax infection in vitro. this suggests that gamma-delta t cells could be stimulated by p. vivax parasites and these actively activated gamma-delta t cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. this study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. objectives: several evidence suggest that invariant nkt cells (inkt) connect innate and acquired immune system. they are able to produce both th1 and th2 cytokines after stimulation. atopic dermatitis (ad) is a chronic inflammatory skin disease. th1-like and th2-like cytokines have been implicated in the pathogenesis of ad, but there are controversial data on their role in ad. the frequency and absolute number of inkt cells in mononuclear cells (pbmcs) of peripheral blood of patients with atopic dermatitis (ad) (n=43) and healthy controls (n=13) were determined by flow cytometry using anti-cd3 and monoclonal antibody specific for the cdr3 loop of the invariant tcr a chain of inkt cells (clone:6b11). furthermore, after pma/ionomycin stimulation for 4 hours, intracellular ifng and il-4 cytokines were detected in cd4+cd8-, cd4-cd8-(dn), cd4-cd8+ and cd4+cd8+ subsets of inkt cells by five colour flow cytometry in patients with ad (n=10) and healthy controls (n=10). results: both frequency and absolute number of inkt cells were significantly lower in patients with ad (p x 0.01) compared to healthy controls. the frequency of dn subpopulation was significantly lower in ad patient (p x 0.01). there was a positive correlation between the frequency of dn cells and inkt cells both in ad patients (r=0.726 and p x 0.001) and healthy controls (r=0.693 and p x 0.001). in the intracellular ifng level there were no significant difference in any of the inkt subsets of ad patients, however the intracellular il-4 level was significantly higher in dn subpopulation of inkt cells of ad patients compared to healthy controls (p x 0.05). the frequency, the number of inkt cells and the cytokine producing capacity of the cd4/cd8 inkt subsets are different in peripheral blood obtained from ad patients compared to healthy controls. our result suggest that the dn inkt cell subset can serve as a source of il-4 that promotes the th2 differentiation in ad patients and might play a role in the pathogenesis of this disease. introduction: intrahepatic immune cells (ihic) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. aims: in the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of ihic. these cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and dnase. results: the mechanical disruption yielded viable ihic in considerably greater numbers than those obtained following enzymatic digestion. the ihic isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which b, t, natural killer (nk), nk t cells, granulocytes and macrophages were the major populations (constituting 37.5%, 16.5%, 12.1%, 7.9%, 7.9% and 7.5% of the total number of cells recovered respectively). the ihic obtained following enzymatic digestion contained markedly lower numbers of nk t cells (1.8 %) . the b, t and nk t cells among ihic isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin a and alpha-galactosylceramide respectively) and produced immunoglobulin m and interferon-gamma. conclusions: thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent ihic for various types of investigation. nature killer t cells (nkt) are a special t cell population with co-expresses nk and t cell surface markers. murine nkt cells include cd4 + nkt and cd4 -cd8 -nkt cells. nk1.1 + nkt cells may release large amounts of il-2, il-4, ifn-g and il-10 after they are activated. it has been reported that a-galactorsykeramide (a-galcer), a glycolipid, may induce proliferation of nkt cells with the role of immune regulation by stimulating mouse spleen cells. this study demonstrated that superantigen staphylococcal enterotoxin b (seb) , a kind of peptide, can activate the nkt cells with the function of immune tolerance. the response ability of seb-activating effect cells to cona, lps and il-2 had significantly decreased compared with that of normal lymphocytes. the effect cells exerted an inhibitory effect for the response of normal lymphocytes to cona and il-2. there was a significantly increase in the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cells identified from the seb-activated cells. based on the cell distribution detected in the upper part of the facs picture, expression of cd69 molecule existed in 68.95 % of the cells from large-scale selection. the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cell subsets in the giant lymphocytes were enhanced to 84.0 and 38.9 folds, respectively. under a light microscope at x400 magnification, the seb-activating lymphocytes in size were larger than not only the cona-activated cells but also the adherent macrophages with an increase of 5 fold observed under a microscope. there were a few granules seen in cytoplasm. the value of cytoplasm vs nuclei was less than 1.0 and they are non-adherent cells. the differentiation pathway of the seb-activating cd8 + and tcrvb8 + nkt cells was not relative to a nk source. they were produced directly from t cell population and were considered as a subsets of t lymphocytes. our results suggest that the superantigen seb can act on the cd8 + nkt cell and tcrvb8 + nkt cells. and the two nkt cell subsets may play a critical role in seb mediated tolerance. gd t cells in the intestinal intraepithelial compartment (gd iiel) show an intrinsic activated phenotype. we hypothesised that their t cell receptor gd (tcrgd) is implicated in the activation of gd iiel. because the tcr gd ligands in mice are not well described, monoclonal antibodies (mab) directed against the gd tcr, like the clone gl3 which binds the d subunit of tcr gd, are important tools to specifically activate gd t cells. using cytometric indo-1am measurement, we could detect calcium flux of intestinal and peripheral gd t cells from tcrd-h2begfp reporter mice. stimulation with anti-gd clone gl3 or anti-cd3 clone 2c11 elicited activation of gd t cells suggesting that tcr gd and cd3 molecules in gd t cells are functional and signalling competent. next, using elisa and cytometric bead array, we found that iiel stimulated with plate bound gl3 in vitro produced ccl4, ifng and tnfa. therefore, we were interested whether the ccl4 production of gd iiel influenced the homing of ccr5 cells such as lamina propria (lp) cd4 + foxp3 + cells (tregs). to test this, wt mice were i. p. treated with gl3 mab and lp tregs were analysed by cytometry at various time points post inoculation. we found similar frequencies of lp tregs population but a slight decrease in ccr5 + tregs. however, when we compared wt and tcrd -/mice, we found both lower percentages of total lp tregs and of lp ccr5 + tregs in tcrd -/mice compared to wt mice. in conclusion, our data suggest that intraepithelial activation of gd t cell may directly or indirectly induce changes in the iiel and lamina propria (lp) lymphocyte compartment and influence the ccr5 expression and the homeostasis of lp treg. the ability of nkt cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different nkt cell subsets. diversity in cytokine production by nkt cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation. here, we investigated cytokine production by murine nkt cell subsets in vivo under conditions where minimal manipulation of the cells was required. to this end, we examined il-4 production in g4 reporter strains in which dna encoding green fluorescent protein (gfp) was inserted into the first exon of the il-4 gene. in the absence of any manipulation gfp was expressed from the il-4 locus in populations of immature thymic nkt cells (predominantly cd4+cd44lotcrhi cells on a balb/c background, and cd4+cd44lonk1.1-on a c57bl/6 background) and some splenic nkt cells, with overall numbers of gfp+ cells in both tissues decreasing with age. after i. v. administration of the nkt cell ligand a-galactosylceramide, il-4 production was induced predominantly in cd4+ nkt cell subsets of the liver and spleen, and after i. n. administration, in cd4+ nkt cells of the airways. spontaneous and a-galcer-induced expression from the il-4 locus occurred in the absence of stat6 signalling, and did not require initial exposure to il-4 protein from other sources in the host. diversification in cytokine expression by nkt cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. interleukin-17 (il-17) plays an important role in neutrophil recruitment. herein, we investigated the role of il-17 receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (clp). methods: adult c57bl/6 (wt) and il-17 receptor gene-deficient (il-17r ko) mice were subjected to non severe (ns-clp) sepsis. intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated 6 hours after surgery. the ability of il-17 mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. the means of different treatments were compared by analysis of variance (anova), followed by bonferroni's t test and the survival rate by the mantel-cox log rank test. results: it was observed that il-17r ko mice, subjected to ns-clp sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to wt. as a consequence, the mice showed an increased mortality rate. moreover, il-17 induced neutrophil migration in vivo and in vitro. besides, we demonstrated that neutrophils harvested from il-17r ko mice already show reduced microbiocidal activity, compared with wt, suggesting a physiological role of il-17receptor signaling in the microbiocidal activity of neutrophils. furthermore, wt neutrophils treated with il-17 showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. conclusion: during ns-clp besides the importance in recruit neutrophils to focus of infection, il-17 also enhances the microbiocidal activity of neutrophils. therefore, our results demonstrated that il-17 receptor signalization plays a critical role on host protection during polymicrobial sepsis. objectives: members of the toll-interleukin-1 receptor (tir) family are important for host defense, inflammation, and immune regulation. their canonical signaling pathway involves adaptor proteins and il-1r associated kinases to activate nfxb and p38 mitogen-activated protein kinase. the il-33-induced signal transduction in mast cells is poorly understood. in this work we studied the signal transduction of il-33 in different mast cell subsets. methods: different mast cells subsets (hmc-1, human cbmcs and murine bmmcs) were stimulated with il-33. the resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-kit, perk1/2, pakt, pnfxb, p38 and pjnk). additionally, we studied the signal transduction of il-33 in il-33r transfected hek293t cells. results: we found, that a tir family member, il-33r, transactivates the receptor tyrosine kinase c-kit in mast cells and that il-33-induced cytokine production depends on c-kit transactivation. il-33r and il-1r accessory protein (il-1racp) form a physical complex with c-kit. thereby the complexation is dependent on the activity of c-kit. conclusion: these results show for the first time that the biological function of an il-1r family member is dependent on the presence of an activated receptor tyrosine kinase. furthermore, these results reveal that certain il-33-induced signaling pathways and effector functions are dependent on activated c-kit and could therefore explain the effects of il-33 in mast cells in absence of iger activation. (1) . we now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo tnfmediated programmed cell death. independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to tnf, presumably due to the participation in the fenton reaction of the fe atom present in the protoporphyrin ix ring. once exposed in vitro to free heme, a sustained c-jun n-terminal kinase (jnk) activation was observed in hepatocytes in response to tnf, an effect that promotes further free radicals production. pharmacologic or genetic (shrna) inhibition of jnk in hepatocytes avoids free radicals accumulation and caspase-3 activation, also mimicked by the anti-oxidants n-acetylcystein (nac) or butylated hydroxyanisole (bha). expression of the heme catabolyzing enzyme heme oxygnease-1 (ho-1) in hepatocytes affords protection against heme sensitization to tnf cytotoxicity. recombinant adenovirus mediated ho-1 expression in the liver suppresses tnfmediated hepatocyte apoptosis and prevents the lethal outcome of plasmodium infection in mice. in conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo tnf-mediated cytotoxic effect, critically involved in the outcome plasmodium infection. the multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. responsiveness to chemotactic ligands is mediated by g protein-coupled receptors (gpcrs) which are finely regulated by a family of cytosolic proteins, betaarrestin 1 and 2. recent evidence indicates that, in addition to playing a regulatory role in gpcr desensitization and internalization, beta-arrestins may contribute to gpcr signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. on this basis, we investigated the physiological role of beta-arrestin 2 in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the rap1 small gtpase, recently emerged as pivotal regulator of integrin function. the analysis of kc the (keratinocyte-derived chemokine) rap1 activation profile in rbl (rat basophilic leukemia) cells expressing mcxcr2 shows a bimodal kinetic, with the first peak at 30''/1' and the second at 5' after stimulation. rna interference-mediated depletion of beta-arrestin 2 specifically inhibits the occurence of the second wave of rap1 activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin 2 is involved in rap1 activation and that the oscillations in the formation of rap1-gtp are regulated by different molecular mechanisms. in order to elucidate the gefs and gaps involved in the gtpase regulation we are at present down-regulating the expression of c3g (rap1gef) and spa1 (rap1gap): preliminary results suggest that spa-1 has probably a role in the early activation peak. since this oscillatory chemokine-induced rap1 activation is present on other myeloid cell lines (hl60, 32d) and fresh pmn's we are also translating our research to these more appropriated cells. interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin 2, localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. in order to find sh3 containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of 153 different sh3 domains that revealed over 20 putative beta-arrestins putative interactors, some of which isoform specific. granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin (il)-3 and il-5 stimulate proliferation, differentiation, survival and functional activation of myeloid cells. the cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. as a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. here we present novel insights into the role of the ig-like domain of the gm-csfra in gm-csf binding. deletion of the ig-like domain abolished direct gm-csf binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. the results indicate a previously unrecognized role for the ig-like domain of gm-csfra. furthermore, we address a longstanding controversy in the field of gm-csf, il-3 and il-5 receptor biology, by performing a systematic study of the role of n-glycosylation upon on the bc, and related murine b il-3 , in ligand-binding and receptor activation. these data demonstrate definitively that n-glycosylation does not play a role in mediating ligand-binding or receptor activation. these findings clearly establish that the determined human bc structures lacking glycosylation at asn 328 are biologically relevant conformers of the human bc ectodomain. our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the gm-csf: gm-csfra: hbc receptor complex. reference: (1) micrornas (mirnas) are endogenous small non-coding rna molecules acting as key regulators of immune cell differentiation and innate immune responses. mirna-146 expression is induced by activation of the toll-like/interleukin-1 receptor pathway (tirpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. since tnfa also up-regulates the expression of mirna-146a, we decided to explore whether this mirna is involved in the regulation of apoptosis. to this end, we used the hela human epithelial cell line as a model system for tnfa signaling. following tnfa and cycloheximide (chx) treatment mirna-146a transfected cells showed significantly reduced levels of the active proapoptotic caspases 8 and 3 (casp8/3). in line with this, mirna-146a conferred enhanced protection against tnfa-induced dna fragmentation and mitochondrial potential drop-down. our results demonstrate that mirna-146a is a regulator of receptor-mediated apoptosis. similar to the tir-pathway, mirna-146a seems to be part of a negative feedback mechanism of the tnfa signaling cascade. ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by mirna-146a. furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. objectives: the aim of this study was to evaluate the role of single nucleotide polymorphisms (snps) located in il-15, il-7r a-chain and il-15r a -chain genes in hiv disease progression. methods: we studied 70 antiretroviral treated patients (progressors) and 71 long term non progressors (ltnp). we analyzed 2 snps in the il-15 gene, 5 snps in the il-7r gene and 3 snps in the il-15r gene. in univariate analysis, we found an association between the presence of at least one mutated a allele in il-15r aa 182 and a higher possibility of being ltnp ( our study suggests that genetic polymorphisms located in il-7r and il-15r genes can influence the rate of disease progression in hiv+ patients, especially when a combination of aplotypes is present. mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of cd4 and cd8 t cells homeostasis. aims: mono-adp-ribosyltransferases (arts) are gpi-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an adpribose moiety from extracellular nad+ to specific arginine residues of target proteins. in this study, we report that human tumor necrosis factor (tnf) is adpribosylated by art1, and that adp-ribosylation affects both the release of tnf from cells and its cytolytic action. methods: transcription of art1 in human leukocytes was analyzed by rt-pcr. adp-ribosylation of tnf was detected by monitoring the incorporation of adpribose from labeled nad. release of tnf from transfected hek cells was monitored by elisa. binding of tnf to tnf receptors was analyzed by biacore. tnf cytotoxicity was monitored by flow cytometry. the adp-ribosylation site on tnf was analyzed by lc/ms mass spectrometry. results: we identified art1 transcripts by rt-pcr analysis in human blood leukocytes. soluble art1, released from the surface of transfected cells by phosphatidylinositol-specific phospholipase c (pi-plc), adp-ribosylated recombinant human tnf in vitro. co-transfection of hek293 cells with art1 and tnf resulted in modification of tnf at at least 2 distinct sites, i. e. one within the tnf ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by tnfa converting enzyme (tace). analysis of modified recombinant tnf by mass spectrometry provided evidence that the tnf ectodomain is adp-ribosylated at r32, a site that has previously been implicated in binding to tnfr2. binding assays indicated that adp-ribosylation inhibited binding of tnf to its receptors. importantly, modified tnf was less potent at inducing cell death in the human t cell lymphoma line kit225 than wildtype tnf. furthermore, cell surface adp-ribosylation of hek cells co-transfected with tnf and art1 resulted in reduced release of tnf into the supernatant. conclusions: adp-ribosylation of tnf or other cell surface proteins interferes with the biology of tnf signals by at least two distinct mechanisms. adpribosylation of tnf blocks binding to its receptors, thereby inhibiting tnf-mediated cytotoxicity. additionally, adp-ribosylation of tnf or another protein on the surface of tnf-producing cells inhibits the proteolytic release of tnf. noninflammatory chronic pelvic pain syndrome : immunological study in ejaculate g. n. drannik 1 , t.v.poroshina 1 1 institut of urology amsci of ukraine, laboratory immunology, kyiv, ukraine chronic prostatitis (cp) is a disease which likely is associated with abnormalities in local immune responses. secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. the aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. methods: 23 patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) and 11 control men were assessed for slpi, tnf-alpha, il-8 and free tgf-b1 in ejaculate by elisas using stat-fax 303 plus. a 3 to 5 day sexual abstinence period was required from the subjects before semen collection. after liquefaction and centrifugation, seminal plasma samples were kept at -20 degrees c°until assayed. the materials were processed after the standard programmes for statistical analysis.the study was approved by the local ethics committee. the slpi concentration was elevated in all patients (5127.571 ± 971.731 pg/ml, p x 0.001) in seminal fluid, in comparison with the healthy control subjects. the tnf-alpha concentration was elevated in all patients in seminal fluid (125.49 ± 17.9 pg/ml; p x 0.05). the il-8 concentration was elevated in all patients (366.7± 49.5 pg/ml; p x 0.05) in seminal fluid. free tgf-b1 was present in normal seminal plasma in high concentrations (346.4 ± 29.2 pg/ml), while in ejaculates of patients with noninflammatory cp/cpps tgf-b1 concentrations were 36.2 ± 6.2 pg/ml. conclusion: ejaculate's slpi, tnf-alpha, il-8 and free tgf-b1 are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome. m. r. marrakchi 1 , e. a. elgaaeid 1 1 faculté des sciences de tunis, biology, tunisie, tunisia ulcerative colitis (uc) and crohn's disease (cd), collectively referred to the inflammatory bowel disease (ibd), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. the excessive production of either th1 or th2 cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of ibd. studies have identified a gene that encodes the nod2/card15 protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to cd. indead, it has been suggested that high rates of asca (saccharomyces cerevisiae ) in absence of panca (perinuclear anca: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of cd and that the important rise of panca was more frequent at uc . in a sample of tunisian patients, we examined the contribution of nod2/card15 gene in cd. we performed a cases /controls study upon 75 cd patients and 90 healthy controls. this study suggests that in northen tunisian population, 3020insc mutation in nod2/card15 gene is a prevalent mutation leading to the typical crohn's disease including ileal location, stricturing and penetrating clinical types and asca expression. since conflicting results were obtained on il-10 polymorphisms as risk factor for ibd, the aim of our study was also to explore anti-inflammatory il-10 cytokine genetic profile in patients with ibd. we examined the contribution of il-10 gene promoter polymorphisms (-627 and -1117) to crohn's disease (cd) phenotype, and the possible genetic epistasis between these polymorphisms and card15/nod2 gene mutations in cd presentation and location. in tunisian population, the 3020insc insertion in nod2/card15 gene is a marker of susceptibility to cd, while the a allele at position -627 in the il-10 promoter increases the risk of cd ileal location and severe disease presentation. a genetic epistasis between il-10 gene polymorphisms and card15/nod2 gene mutation was suggested. in conclusion, genetic and serologic markers might be useful in defining patien gangliosides were shown to inhibit the il-2-dependent proliferation of t-cells, implying that gangliosides interfere with one or more of the il-2-driven events. it is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of il-2 molecule by ganglioside. but gangliosides apparently can also form complexes with il-2r; such complexes influence on the signal transduction through il-2r. this effect of gangliosides may lead to the failure of this pathway. unfortunately, the biological and structural aspects of this problem are poorly understood. in this study we propose possible modes of interactions between exogenous gangliosides and il-2r subunits. in our work we use il-2-dependent cytotoxic t-cell murine line ctll-2. two different approaches for study of possible interaction types between exogenous gangliosides and il-2r subunits were applied: antibody staining of il-2r subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with 125 i-dcp-gm1 followed by immunoprecipitation of il-2r subunits. the fluorescence intensity of the antibody-labeled il-2r a-subunit substantially decreases after the treatment of cells with gangliosides. it has been shown that the fluorescent labeled cell fraction decreases by 29.5 % after cells incubation with ganglioside gm1, and by 10.7 % after incubation with gm2. labeling of the cells with antibodies to the il-2r b-subunit results in a less significant fluorescence decrease after cells incubation either with gm1 (2.8 %) or with gm2 (5.9 %). to determine the mode of this impressive masking influence of ganglioside gm1, photoaffinity cross-linking has been used. in our modification this method could identify is interaction of gm1 with subunits of il-2r occur with or without incorporation exogenous ganglioside into plasma membrane. electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of il-2r, but not for il-2r a-subunit. these results demonstrate that exogenous ganglioside gm1 can interact with a-and bsubunits of il-2r in different modes. interaction of il-2r b-subunit with ganglioside gm1 requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit. a. respa 1 , j. bukur 1 , s. purpose: loss of interferon (ifn)-g inducibility of hla class i antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. this kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. methods: phospho-specific western blot analyses were performed to verify the functionality of the different ifn-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and hla class i cell surface antigens, quantitative real time-pcr experiments to confirm the absence of jak2 and presence of pathway relevant molecules as well as, genomic pcr and chromosome typing technique to prove the deletion of jak2. results: different ifn-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong ifn-g inducibility. resistance to ifn-g treatment was associated in one melanoma cell line with the lack of jak2 expression due to a gene deletion on chromosome 9, whereas the expression and functionality of other ifn-g signal transduction components like stat1 and jak1 were not affected. jak2 blockade by two jak2-specific inhibitors resulted in reduced levels of hla class i surface antigens. conclusion: structural abnormalities of jak2 leading to a lack of jak2 expression are associated with loss of ifn-g inducibility as well as reduced constitutive hla class i surface expression. in addition the jak2 inhibition modulates the expression of the hla class i antigen processing components. of renal cell carcinomas (rccs) are associated with the size, grade, t, n, m, stage and death of rccs patients. the genotypes were compared with those of a random sample of 506 controls of the spanish population. methods: two il18 polymorphisms located on the il-18 promoter region, snps -607 a/c (rs1946518) and -137 g/c (rs187238) were genotyped using taqman snp genotyping assays. the functional il-18 gene polymorphisms studied do not influence the susceptibility to rccs in the spanish populations (il-18 -607 p=0,318. il18 -137 p=0,740) but may contribute to disease onset and aggressiveness: the genotype il-18 -607 cc genotype, which is associated with higher il-18 production, was significantly associated with higher tumor size (p=0,001), grade (p=0,030), t (p=0,001), m (p=0,012) and stage (p=0,002). the influence of the il-18 -103 gg genotype was less relevant, however was correlated with higher tumor size (p=0,036), grade (p=0,017), t (p=0,026) and stage (p=0,011). the multivariate analysis with cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas il-18 polimorphism can not be considered as independent prognosis factors. our results suggest that the polymorphism variants from the il-18 gene (il-18 -607 and -137) may be associated with an worse prognosis of rcc. high levels of il-18 production can play an important role in grow, invasion and metastasis of renal cancer. interleukin-18 (il-18) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. the most prominent biologic property of il-18 is its ability to induce the production of ifn-gamma in presence of il-12. moreover, it stimulates the expression of tnf-a and il-l, enhances the differentiation of t cells to the thl and impairs the synthesis of the anti-inflammatory cytokine il-l0. then it seems that il-18 has a crucial role in immunity against brucella infection. since the expression of il-18 can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different il-18 gene polymorph isms and brucellosis. methods: a total of 188 patients with brucellosis and 77 healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. all individuals were genotyped for six il-18 polymorphisms at positions -656, -607, 137, +113, +127 and codon 35/3 using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). the distribution of alleles for il-18 polymorphisms at position -137 g/+113t/+127c (correlated with high production of il-18), codon 35/3c and -656g/-607c (correlated with higher production of il-18) were significantly higher in the healthy controls than the patients (p=0.000022, p=0.00185 and p=0.0441, respectively). discussion: as data revealed genotypes that have correlation with higher production of il-18 are more frequent in the controls than the patients. then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of il-18 at the onset of infection and it leads to more ifngamma production and control brucella infection before appearing brucellosis. abstract withdrawn by author objectives: a dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (ibd) pathogenesis. to dissect the influence of these cytokines, specifically interferon gamma (ifng), on the inflammatory response and colitis we have created a cell specific ifng receptor 2 (ifngr2) mouse mutant. we have generated a mouse line carrying a conditional ifngr2 allele using the 2 loxp 2-flp recognition target (frt) approach. a targeting vector with 2 loxp sites flanking exons 4-6 and 2 frt sites flanking the neomycin resistance cassette was generated. after confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with flp deleter mice. cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific cre expressing mouse lines. functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total stat1 following ifng stimulation. results: successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. functional inactivation of the receptor with no stat1 activation following ifng stimulation has been demonstrated in the complete knock out mice. furthermore, flox/flox mice retain full responsiveness to ifng. breeding with lysm cre and cd4 cre mice will been completed to create a cell specific deletion of the ifng receptor in macrophages/granulocytes and t cells respectively. the specificity of this deletion will be confirmed through cell sorting and functional assays. in order to determine the influence of ifng on a specific cell type a conditional gene targeting approach has been utilised. this has allowed the generation of conditional mice mutants with deletion of ifngr2 on macrophages and t cells. this has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. moreover, the ability to cross the conditional mice with additional cre lines will enable the analysis of more cell types in the future. a. gonzalez 1 , r. carretero 2 , p. saenz-lopez 2 , j. cantón 2 , j. carretero 2 , f. ruiz-cabello 2 , l.m. torres 1 , cts-143 1 hospital universitario virgen de las nieves, departamento de ginecología, granada, spain, 2 hospital universitario virgen de las nieves, departamento de análisis clí nicos, granada, spain objetives: cervical cancer is almost associated with infection by human papillomavirus (hpv). however, only a subset of infected women will ever develop the malignancy. ifng dinucleotide (ca) repeat polymorphism is responsible for genetic differences in interferon-gamma production. our objective was to investigated the relationship between ifgn polymorphism and cervical intraepithelial neoplasia (cin) methods: we have studied nineteen women with cin and 130 normal women control. dna was extracted from blood samples, and was genotyped for functional microsatellite (ca) repeats in the first intron of ifn-gamma gene. results: heterozygosis in (ca)12 allele of ifgn is significant more frequent in healthy control than in cin patient (p=0,030) in contrast homozygosis for (ca)12 allele did not show significant differences between both population. analysis of relation between this polymorphism and the cin stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p=0,030 and p=0,045 respectively). conclusions: our study suggest that ifng (ca)12 allele may influence in cin risk and progression. ifng is associated with hpv clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. further studies of the role of ifng and other cytokines may contribute to the understanding of cin promotions and progression. introduction: macrophages play a fundamental role in controlling of brucella infection, mainly through the secretion of cytokines such ifn-y and tnf-a. interleukin-15 (il-15), a th1-related cytokine, triggers inflammatory cell recruitment and increases the expression of ifn-y. as the cytokine production is under the genetic control, we decided to investigate the association between il-15 single-nucleotide polymorphisms (snps) and susceptibility to brucellosis in iranian patients. methods: 190 patients with brucellosis and 78 healthy animal husband men who kept animals with brucellosis, were included in this study. all individuals were genotyped for il-15 (c267t, g367a, c13687a and a14035t) polymorph isms using pcr-rflp. results: at position 267, the distribution of c allele and cc genotype were significantly lower in the patients than the controls (p=0.025 and p=0.042, respectively). at position 13687, the distribution of c allele and aa genotype were significantly higher and lower in the patients than the controls, respectively (p=0.050, p=0.015). discussion: as shown the frequency of cc genotype and c allele at position 267 were higher in healthy controls than the patients. hence, our study provides evidence that presences of cc genotype and/or c allele are significantly associated with susceptibility to brucellosis. the frequency of aa genotype at position 13687 is lower in patients than the healthy controls and the frequency of c allele at position 13687 is higher in patients than the healthy controls. hence, our study provides evidence that presences of c allele and lack of aa genotype are significantly associated with susceptibility to brucellosis. objectives: increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, mcf-7 and skbr-3, as well as their ability to respond to several cytokines and to the g-1 gpr30 agonist. the mcf-7 and skbr-3 human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: il-2ra, il-2rg, il-3/il-5/gm-csfr, il-4/il-13r, il-5ra, il-6ra, il-6r (gp130), il-7ra, il-10r, tnfr i, tnfr ii, ifngra, cxcr1, cxcr2, cxcr3, cxcr4, cxcr5. cells were incubated with il-10, ifn-g, tnf-a and tnf-b (for which both cell lines displayed receptors) alone and with the gpr30 agonist. cytosolic ca 2+ concentrations [ca 2+ ] i were measured by the microfluorimetric technique. results: different cytokine receptors phenotypes emerged for the two cell lines. the less well differentiated skbr-3 cells were found positive for a larger number of receptors than the mcf-7 cells. both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. pretreatment of mcf-7 cells with il-10 reduced the calcium response to g-1, while pretreatment with ifn-g and tnf-a potentiated the calcium response to g-1. tnf-b had no effect on mcf-7 g-1 stimulation. no direct effect on basal [ca 2+ ] i stimulation could be noticed when administering the cytokines alone. in skbr-3 cells, pretreatment with il-10 or tnf-b had no effect on basal [ca 2+ ] i and did not significantly alter the calcium response to g-1, while pretreatment with ifn-g induced calcium oscillations and potentiated the calcium response to g-1. pretreatment with tnf-a produced calcium oscillations and reduced the response to g-1. conclusions: mcf-7 and skbr-3 cell lines express distinctive cytokine receptor phenotypes. furthermore, their ability to respond to cytokines in terms of modulating the gpr30 stimulation proved to be different. the susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. results: in initial analyses of the early infection phase we identified splenic pdcs expressing ifnb/yfp. furthermore, we show for the first time in vivo that these ifnb producing pdcs are not directly infected with mcmv and that not only cdcs but also cd8a -pdcs expressed gfp as a marker for mcmv infection. we observed that at early time points equal frequencies of cd8a -pdcs and cdcs were infected with mcmv, whereas after 24h p. i. the frequency of infected cdcs wins over that of the pdcs. conclusions: with this experimental system we are able to visualize the ifnb response vs. the infection status of mcmv in vivo on a single-cell level. from our initial results we can conclude that infected cells in the spleen induce ifnb production in noninfected pdcs which initiate the antiviral immune response in this organ. recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (lcmv) carrying the vesicular stomatitis virus (vsv) envelope gene (rlcmv/vsv-g). since interferon (ifn) signaling is known to be crucial for adaptive immune responses against wildtype (wt) lcmv and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rlcmv/vsv-g infection. mice lacking both, ifn type i and ii receptors generated potent virus-specific cd8 t cells and neutralizing antibody responses against rlcmv/vsv-g, even exceeding the respective responses in wild type animals. in further contrast to wt lcmv infection, ifn type i and ii signaling was dispensable for rlcmv/vsv-g control. rlcmv/vsv-g infection of rag1-deficient mice (lacking mature t and b cells) resulted in persistent levels of circulating viral rna that was solely detectable by qrt-pcr but not by classical measures of infectivity. overt viremia was only found in mice lacking both rag1 and ifn type i receptor (ar). thus, viral attenuation for vaccine use was found to considerably relax the ifn-dependence of adaptive immune responses and virus control. this redundancy of ifn and adaptive immune responses in rlcmv/vsv-g control provides a better understanding of the attenuation of this vector. it furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of cd8 t cell and antibody responses, which may have implications for vaccine development. objectives: the class of type i interferons (ifns) consist of multiple ifnas and a single ifnb subtype. although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. while in general the first ifn to be produced is ifnb, the cell types responsible for its initial production remain unclear. to assess the cellular sources of ifnb and its role for the outcome in bacterial infections we use an ifnb reporter-knockin mouse model, in which yellow fluorescent protein (yfp) is expressed from a bicistronic mrna linked by an internal ribosomal entry site (ires) to the endogenous ifnb mrna. methods: to induce a type i interferon response we intravenously injected the tlr agonists poly(i:c) and cpg 1668, respectively, or infected reporter mice or bone marrow derived macrophages (bmdms) or dendritic cells (bmdcs) with the facultative intracellular pathogen listeria monocytogenes. the spatiotemporal tracking of the ifnb response was done using facs analysis and immunofluorescent microscopy. results: after poly(i:c) injection in vivo a small subpopulation of ifnb + cd8a + dendritic cells relocalize from the red pulp via the marginal zone to the t cell areas of the spleen whereas ifnb + activated pdcs were localized in the splenic white pulp at the t/b-cell interface after cpg administration. in vitro infection of bmdms, bmdcs and flt3-l derived dcs with listeria monocytogenes resulted in a low frequency of ifnb + cells depending on the listeria strain used and multiplicity of infection with bmdms being the most potent producers of ifnb. in vivo mostly activated f4/80 + macrophages were accountable for the ifnb expression. simultanous visualization of the cellular state of infection shows that ifnb + bmdms carry a higher bacterial load then ifnbcells. the cellular detection of ifnb expression reveals a remarkably low frequency of ifnb-producing cells in response to distinct pamps or the infection with intracellular bacteria. this hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. additional data will be presented further resolving the timecourse of the ifnb response vs. the state of infection after bacterial challenge. the spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens. we investigated the role of the spleen in the generation of antigen-specific cytotoxic cd8 t cell (ctl) responses in a systemic infection with recombinant adenovirus expressing ovalbumin (adova). although adenovirus mainly infects the liver, the ctl response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific ctl response upon systemic challenge with adova. additionally, dendritic cells (dc), macrophages and an intact splenic structure were mandatory for the induction of an efficient ctl response. we detected adova specific ctl responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. furthermore, we asked how toll-like receptor (tlr) ligands influence adova-specific ctl responses. tlr ligands as "danger signals" are generally known to exert immune stimulatory effects. importantly, we observed that systemic administration of single-stranded rna (ssrna) prior to adova infection inhibited the generation of antigen-specific ctl responses in a tlr7 and type i interferon (ifn) dependent manner. ssrna injection induced the production of type i ifns as detected in sera and supernatants of splenocytes isolated from wild-type mice. experiments performed in ifnbeta reporter mice revealed that splenic plasmacytoid dcs represented the cellular source of type i ifns. additionally, splenic cd11c+ dcs purified from ssrna pretreated mice showed a reduced capacity to cross-prime ova-specific cd8 t cells upon adova challenge. in vivo pre-activation of endogenous ova-specific cd4 t cells as well as an adoptive transfer of in vitro activated transgenic ova-specific cd4 t cells prevented the ssrna mediated suppression of the ctl activity. we assume that dcs preactivated by systemic ssrna were impaired in their ability to activate naive cd8 t cells in response to an adova infection due to an impaired cd4 t cell help. taken together, we show that type i ifns cannot only stimulate, but also inhibit the induction of ctl responses in the spleen. within hours after infection many viruses induce early type i interferon (ifn) responses that can confer protection against lethal infection. modified vaccinia virus ankara (mva) is a highly attenuated vaccinia virus (vacv) strain generated by more than 500 passages in chicken embryo fibroblasts. mva stimulates systemic ifn responses, whereas vacv-induced cytokine milieus are dominated by il-12. to study the impact of virus-triggered ifn on the induction of t cell responses, we used recombinant mva-gp33 and vacv-gp33 expressing the gp33 epitope of lymphocytic choriomeningitis virus. for adoptive transfer experiments transgenic mice expressing the p14 t cell receptor recognizing the gp33 epitope were intercrossed with mice devoid of the ifn receptor (ifnar -/-) to obtain p14ifnar -/mice. upon adoptive co-transfer of p14ifnar -/and p14ifnar wt/wt t cells and subsequent challenge with mva-gp33, a massive expansion of p14ifnar wt/wt t cells was observed, whereas p14ifnar -/-t cells expanded less efficiently. in contrast, challenge with vacv-gp33 induced a rather similar expansion of ifnar competent and ifnar deficient p14 t cells. in the absence of ifnar-triggering t cell expansion was associated with reduced proliferation capacity and increased apoptosis. to study the impact of ifnar-signaling on the expansion of endogenous t cells, conditional mice with a t cell-specific ifnar-ablation were infected with mva. interestingly, in those mice the virus-specific t cells showed a reduced expansion compared to t cells of wt mice. additionally, we found that ifnar-triggering of dendritic cells but not of macrophages further supported specific t cell expansion. thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific t cells. to delineate h5n1-specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. blocking of specific signaling pathways revealed that h5n1 induced an exceptionally nf-kb dependent antiviral response. irf3 is essential part of this interferon-response of human endothelia. furthermore, we identified hmga1 as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by h5n1. finally, nfatc4 was found to be a transcriptional regulator for specifically h5n1-induced genes. we therefore describe for the first time defined signaling patterns specifically activated by h5n1 which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. objectives: to study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. methods: to dissect the contribution of the unique viral antagonist ns1, delta-ns1 influenza virus (a recombinant influenza virus lacking the ns1 gene) was compared side by side with wild type influenza virus in vivo. mice infected with influenza virus were sacrificed at different time points after infection. to study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by multiplex elisa and quantitative pcr (qpcr). to determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (dc) migration to the draining lymph nodes was quantitated in infected mice. further, functional in vitro and in vivo assays in infected animals with influenza-ova viruses were used to determine whether antigen-bearing migratory dcs were capable of priming transgenic t cells. to investigate the effect of ns1 during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including ifns, cytokines, and chemokines occurred. this burst preceded the robust dc migration and t cell activation in the lymph nodes. virus-loaded dcs appear in the lymph node starting 2 days post-infection, reached its maximum at day 4, and triggered t cell priming in vivo. a direct comparison of delta-ns1 virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the ns1 protein. we demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza ns1 protein. thus, we propose that the virally encoded ns1 protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. in addition, keratinocytes actively participate in immune response. viral double-stranded rna (dsrna) is the most important viral structure involved in activation of innate immune response. intracellular detection of dsrna triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis. here we have used subcellular proteomics to characterise dsrna-induced human keratinocytes. cells were transfected with a mimetic of dsrna, poly(i:c), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. these proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. we show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsrna stimulation. in mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. we show that cytokeratin 18 is cleaved during poly(i:c) stimulation and fragments are solely localized in mitochondria. similar degradation of cytokeratin 18 is seen in emcv-and vsv-infected keratinocytes. cytokeratin fragmentation after dsrna stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. in addition, we show that 14-3-3 proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsrna-stimulation and during viral infection. viral dsrna also induced transient phosphorylation of 14-3-3 target proteins. thus, these results suggest that 14-3-3 proteins have a regulatory role in host defence against viral infections. i. wessels 1 , d. fleischer 1 , l. rink 1 , p. uciechowski 1 1 institute of immunology, rwth aachen university hospital, aachen, germany objectives: the proinflammatory cytokine interleukin (il)-1b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. however, the molecular regulation of il-1b expression is not elucidated, yet. it is known that the il-1b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing il-1b when stimulated. b-cells which are il-1b non-producers reveal an inaccessible promoter structure. in this study the chromatin structure of the il-1b promoter and the impact of methylation on il-1b expression were examined in a cellular monocytic differentiation model. methods: promyeloid hl-60 cells were differentiated into monocytic cells after dihydroxyvitamine d 3 treatment. the monocytic phenotype was confirmed by flow cytometry. the il-1b promoter was analyzed using the chromatin accessibility by real time pcr (chart) assay. to test the influence of methylation, cells were treated with 5-aza-2-deoxycytodine (aza) and changes in il-1b expression were measured by pcr, elisa and western blot analyses. results: in contrast to undifferentiated hl-60 cells, differentiated cells displayed upregulation of cd14 antigen and acquired the ability to express il-1b. by comparing the accessibilities of il-1b promoter we detected that the il-1b promoter was not accessible in undifferentiated hl-60 cells but highly accessible in differentiated monocytic cells. the accessibilities of differentiated cells were comparable to that observed in primary monocytes. lps stimulation did not affect promoter accessibility in promyeloic and monocytic hl-60 cells, demonstrating that the chromatin remodeling of the il-1b promoter depends on differentiation but is independent of the transcriptional status of the cell. demethylation via aza led to the induction of il-1b expression in both undifferentiated and differentiated cells which could be increased after lps stimulation. conclusion: two independent mechanisms involved in the regulation of il-1b expression were found. our data indicate that the il-1b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. as a second mechanism, il-1b expression is regulated by methylation acting independent of the developmental stage of myeloid cells. a. holweg 1 , g. wetzel 1 , h. arnold 1 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany the p65 family members of interferon (ifn) inducible gtpases also known as guanylate binding proteins (gbps) are among the most abundantly induced transcripts after stimulation with ifn-g. although the stimulatory capacities of the toll like receptor ligands lps and cpg, cytokines like ifn-b and il-1b and the intracellular pathogens listeria monocytogenes and toxoplasma gondii have been described to induce their expression, the function of gbps during bacterial infections is still ill defined. here we report for the first time the massive induction of murine gbps in two independent in vivo models of pneumonia (infection with streptococcus pneumoniae and pseudomonas aeruginosa). a strong and rapid induction of mgbp2, 3, 4, 5 and 6 mrna after intratracheal infection was detected by realtime pcr analysis of bronchoalveolar lavage (bal) cells. using newly generated antibodies in western blots and fluorescence microscopy, we identified macrophages as the main producers of mgbps in bal samples. although the signaling cascade involved in the upregulation of mgbps after ifn-g stimulation has been extensively studied, the mechanisms responsible for mgbp induction upon bacterial stimuli are unclear. in this study we could show that the induction of mgbps upon infection is abrogated in ifn type i/ type ii receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. in contrast, the tlr adaptor molecule myd88 was found to be dispensable arguing for a trif-mediated interferon production subsequentially resulting in enhanced gbp expression. based on these findings our future experiments will address the functional role of gbps in innate and adaptive immune responses against extracellular bacteria. (2)). activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. clearance of replicating virus from the cns required mcmv specific cd8 + t lymphocytes whose effectors functions still remain incompletely defined (j. immunol. 2008 aug 1;181 (3)). humoral immunity appeared to also play a role in the control of mcmv infection in developing brain. infected newborn mice treated with mcmv-specific antibodies had lower viral titers in the cns, significantly less cns inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (j. virol. 2008 dec; 82 (24) ). conclusion: peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse cns followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. passive immunization of infected newborn mice reduces mcmv-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during cmv infection of developing cns. chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. tumors enhance the accumulation of myeloid derived suppressor cells (mdsc), which contribute to tumor escape, immune tolerance and immune suppression. previously, we have shown that tumor-derived inflammatory cytokines, such as il-1b in the tumor microenvironment can induce a massive accumulation of mdsc in the spleen of tumor bearing mice and induce t cell suppression. in this work, we describe a novel polymorphnuclear mdsc subpopulation -inflammatory mdsc (infmdsc) which accumulates in the bm and spleen of mice bearing inflammatory 4t1 breast cancer cells over expressing il-1b (4t1/il-1b) tumor cells, but rarely in untransfected 4t1 cells. secretion of il-1b from tumor cells is crucial for infmdsc generation and accumulation. infmdsc have the ability to suppress nk cell activity via reduction of the nk activating receptor nkg2d in vivo, and in a cell-cell contact dependent manner in vitro. inflammation up-regulates il-4ra expression on the cell surface, which correlates with tumor growth and induction of suppression on nk cells. our data suggest that tumor derived inflammation enhances a specific mdsc subset which has the ability induce suppression of nk cells, and perhaps can serve as a new chemotherapy target. objectives: lps constitutes a main target of innate immune recognition of gram-negative bacteria and other lps carrying pathogens. cytokine production after in vitro stimulation of whole blood with lps is used as an expression of individual lps reactivity. we assess genome-wide data to analysed the association to lps induced cytokine response, and replicated the top findings in two independent cohorts. materials and methods: we used 130 healthy caucasian blood donors as discovery samples, and replicated snps from affymetrix -genome-wide human snp array 5.0 having p x 10 -4 in two independent cohorts each consisting of 200 blood donors using a customized chip from illumina and real-time pcr respectively. in all the three cohorts whole blood samples was stimulated for 24h and we measured the levels of il-6, il-8, il-10, tnf-alfa and il1-ra with r&d ® assays on luminex platform.. the association analysis was performed with wald statistical test assuming an additive model. the discovery sample statistical analysis revealed 150 snps with p x 1,0*10 -4 . to identify/replicate the association of cytokine production for these 150 we reanalysed these on a 200 cohort. a combined analysis revealed 10 snps with p x 9,1*10 -5 .these results are not genome wide significant. the 10 snps showing nominal association to lps cytokine response are being analysed in a replication cohort conclusion: we find 10 nominal associated snps between lps stimulated cytokines in blood samples of healthy caucasian blood donors. the importance of these snps are to be determined in a replication cohort. adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. the physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. heat shock proteins, such as hsp60 have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive. here, we investigated the regulatory effects of hsp60 on adipocytes. for the first time we could show a hsp60-stimulated release of the proinflammatory cytokines il-6, cxcl1 and mcp-1 in a time-and concentration-dependent manner from murine 3t3-l1 adipocytes. analyses of hsp60-signalling in these adipocytes revealed that members of the mapk-family (erk1/2, p38) and the transcription factor nfkb are involved in hsp60-mediated induction of the mediators il-6, cxcl1 and mcp-1. binding-studies with fluorescence-labelled hsp60 demonstrated that the interaction of hsp60 with adipocytes exhibits basic features of a receptor-mediated binding. hsp60-binding to adipocytes was saturable and reached its maximum at 3.5 mm. binding was inhibitable only by the unlabelled ligand (52 %), but not by unrelated proteins, thereby proving the specificity of hsp60-binding. further analyses to characterize hsp60-receptor structures on adipocytes revealed the presence of toll-like receptor (tlr)4 on adipocytes. tlr4 has been found to be expressed on macrophages and to interact with hsp60, therefore suggesting tlr4 as a potential receptor candidate for hsp60 on adipocytes. in order to identify the responsible binding-epitope of hsp60 we investigated the effect of specific antibodies directed against different epitopes of the hsp60-molecule. incubation with antibodies directed against the n-terminus of hsp60 (aa1-200; 5-25 mg/ml) were capable of inhibiting the hsp60-binding to adipocytes (47-80 %) indicating that the n-terminal region of hsp60 is involved in receptor binding. our experiments demonstrate that hsp60 stimulates the release of proinflammatory adipocyte mediators. the findings implicate that the hsp60-mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. legionella pneumophila, a gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as legionnaires' disease. classically, type i ifns (ifna/b) have been associated with antiviral immunity. ifna/b signal through the ifna/b receptor (ifnar) leading to the induction of hundreds of ifn-stimulated genes (isgs), many of which have anti-microbial activities. recently, it was demonstrated that ifna/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic dna. here we show by rnai that l. pneumophila infected host cells produced ifnb dependent on irf3. we observed enhanced l. pneumophila replication in mouse macrophages lacking ifnar and human cells after irf3 knock-down, suggesting that endogenously produced ifnb activates a cell-autonomous defence against legionella. moreover, ifnb treatment restricts legionella replication in human and murine host cells. ifna/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of er markers nor fusion of the legionella-containing vacuole with the lysosome. moreover, ifna/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. we thus hypothesize the crucial involvement of antibacterially acting isgs. ongoing studies focus on the role of ifn-induced immunity-related gtpases (irgs). mesenchymal stem cells (mscs) are identified by their capacity to differentiate into connective tissue cell types. mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. several authors have reported the expression of alfa-smooth muscle actin (a-sm actin) by msc. this protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. this activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of msc. in this work, we study the presence of alfa-sm actin in human msc by flow cytometry and immunoflurescence. we also study the contractility of these cells under the effect of different cytokines. human bone marrow samples were obtained from bone marrow aspirates. bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in opti-mem culture medium with 3 % of fetal calf serum at 37°c and 5 % co2. bone marrow nonadherent cells were removed after 24 h, and culture medium was refreshed twice per week thereafter. cells grew adherent, with a fibroblastic morphology and expressed cd10, cd29, cd73, cd21 and stro-1 and were negative for cd45. intracellular staining was performed for alfa-sm actin. cell contractility was measured with the collagen gel contraction assay. alfa-smooth muscle actin was detected in almost 100 % of msc. interleukin-2, ifn-gamma and tnf (th1 cytokines) increased msc contractility, whereas il-10 (a th2 cytokine) decreased msc contractility. by immunofluorescence, we observed that il-2, ifn-gamma and tnf increased the incorporation of alfa-sm actin into the stress fibers of msc, whereas il-10 decreased that incorporation. our results suggest that th1 and th2 cytokines regulate msc physiology by acting on their contractility. aims: thapsigargin (tg) is a sesquiterpene lactone (sl) of guaianolide type isolated from the mediterranean plant thapsia garganica l. it is widely recognized as an inhibitor of sarco-endoplasmic reticulum ca 2+ -atpase leading to elevation of intracellular calcium. this activity is shared by trilobolide (tb), a sl from laser trilobum (l.) borkh. tg has been shown to possess prospective immunotherapeutic properties. it kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. the aim of our work was to investigate possible immunostimulatory potential of tg and tb. methods: the effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (pecs), and human peripheral blood mononuclear cells (hpbmcs). they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. supernatant levels of ifn-g were determined by elisa. production of no by animal pecs was assayed using griess reagent. possible contamination of the samples with lps was excluded using the chromogenic limulus amoebocyte assay. results: we have found that both tg and tb at as low doses as 40 nm and 400 nm induce ifn-g secretion in rat pecs and hpbmcs, respectively. the concentration of ifn-g produced by the highest dose of the agents (10 mm) at the 24-h culture interval reached the values of approximately 3 ng/ml and 2 ng/ml in rat pecs and hpbmcs, respectively. the increase was apparent within the interval of 2-6 h in rat pecs. the 24-h interval was optimal for accumulation of ifn-g in cultures of hpbmcs. only modestly enhanced secretion of ifn-g was observed in mouse pecs. production of ifn-g was found to depend on activation of nf-xb and map kinases p38 and erk1/2. it was not suppressed by the calcium chelating agents bapta-am and tmb-8. the tg-and tb-induced ifn-g production was associated with activation of the high-output production of no. conclusions: sesquiterpene lactones tg and tb are potent inducers of ifn-g in animal and human cells. the effect is independent of their serca inhibitory potential. underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. acknowledgements: the work was supported by the grant gacr 305/07/0061. pin1 is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated ser/thr-pro protein motifs and catalyzes the cis/trans isomerization of peptides. mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are pin1 substrates and targeting sites. recent data show that pin1 interacts with apo-bec3g (a3g). the pin1/a3g interaction results in a reduced a3g expression and a diminished a3g-mediated restriction of hiv. two single nucleotide polymorphisms (snps) in the promoter region of the pin1 gene (-842 g/c and -667 t/c) modulate pin1 expression; in particular, the -842 gc genotype or cc haplotype are associated with reduced protein levels (neurobiol. aging, 28; 69-74, 2007) . the -842 c/g and -667 t/c polymorphisms in the promoter of pin1 gene as well as pin1 protein levels were analyzed in 30 exposed seronegative individuals (esn), heterosexual partners of hiv-infected patients; 40 hiv-infected patients (hiv) and 40 healthy controls (hc). the genotype and allele distributions of the -842 snp was skewed in esn (genotype: p= 0.008; allele: p= 0.013). in particular esn showed a significantly lower frequency of the -842 gg genotype compared to hiv and hc (p=0.017 and p=0.019, respectively) and consequently a lower g allele frequency (p=0.026 and p=0.028, respectively). no significant differences were found for the -667 snp. these snps are in linkage disequilibrium and combine to form haplotypes. conclusions: our findings support the role of hiv viral replication as the most important promoter of immune activation and prove the importance of art in reducing immune activation and viral replication even in a sub-saharan african environment, where patients are exposed to an abundance of other infectious agents. our data further indicates that hiv replication rather than host genetics is the key determinator of circulating cytokine levels among the studied hiv infected participants. aims: acyclic nucleoside phosphonates (anps) are synthetic analogues of natural nucleoside monophosphates. they have proved to be outstanding antivirals inhibiting replication of both dna-viruses and retroviruses. tenofovir, 9-(r)[2-(phosphonomethoxy) propyl]adenine [(r)-pmpa] is broadly used for therapy of aids, adefovir, 9-[2-(phosphonomethoxy)ethyl]adenine (pmea) has been approved for the treatment of hepatitis b. the aim of our work was to investigate possible interactions of anps with production of cytokines and nitric oxide (no) implicated in antiviral defence mechanisms. the immunobiological effects of anps were screened in vitro using mouse resident peritoneal cells. they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. secretion of cytokines was determined after the 5-h culture by elisa. production of no was assayed at the interval of 24 h using griess reagent. approximately 300 compounds belonging to several distinct anp groups were included in the study: a) pmea derivatives, b) pmedap i. e. 9-[2-(phosphonomethoxy)ethlyl]-2,6-diaminopurine derivatives, c) 9-[2-hydroxy-3-(phosphonomethoxy)propyl]-adenine (hpmpa), and hpmpdap derivatives, d) guanidino analogues of pmpdap, e) 9-heteroalkyl substituted 2-amino-6-guanidinopurines, and f) 2-amino-3-(purin-9-yl)propanoic acid derivatives. possible presence of lps in the stock solutions of the samples was checked and excluded using the chromogenic limulus amoebocyte assay. results: approximately 30 compounds were found to activate the secretion of anti-hiv effective chemokines rantes and mip-1a and cytokines tnf-a and il-10. although these anps did not stimulate biosynthesis of no on their own, they substantially augmented production of no triggered by ifn-g. no clear-cut relationship between the chemical structure and biological effects of anps was observed. however, the most effective proved to be the n 6 -cycloalkyl derivatives of pme-dap. the effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as 2 to 5 mm. the remarkably enhanced secretion of chemokines was reached within 2-4 h of the cell culture. the effects were found to depend on activation of nf-kb. conclusions: it may be suggested that anps represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. acknowledgements. the work was supported by the grant 1m0508. host related immune factors in childhood chronic hepatitis b and change of initial profile with ifn-a treatment needs to be clarified. sixteen patients were included, and 10 million units of ifn-a treatment three times a week for 6 months was initiated. before and after treatment: percentages of the il-2 and ifn-g in cd4+ t cells were assessed to determine intracellular t helper cell 1 (th1) type cytokine expression. similarly, percentages of intracellular il-2 and ifn-g were detected to verify cytotoxic t cell 1 (tc1) type cytokine expression in cd8+ t cells. percentage of th2 and tc2 type cytokine expression, (il-4 and il-13) were determined in cd4+ and cd8+ t cells, respectively. six (50 %) of these were evaluated as having no response and the other half with partial/complete response. all patients had higher percentages of th2 cells with respect to healthy controls before treatment. tc percentages, both tc1 and tc2, were significantly different between these groups, being higher in the patient group. when values of no responder group were compared with healthy controls, il-4 expression was higher and the percentages of th1 type cells were significantly low. il-13 expression in th and tc cells decreased after treatment in the unresponsive group. intracellular cytokine profiles of treatment responders and normal controls were not different. this has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. the frequency (29,8 %) of high concentration of igg anti-oxldl antibodies in patients with hcv infection were significantly elevated in comparison to healthy subjects (6,6 % according to bibliographic data). the immune response was significant but it was not assosiated with the viral load. it is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of hcv-infection. abstract withdrawn by author t. schwandt 1 , f. juengerkes 1 , b. schumak 1 , g. gielen 1 , j. kalff 2 , p. knolle 1 , b. holzmann 3 , a. limmer 1 1 university hospital bonn, institute of molecular medicine and experimental immunology, bonn, germany, 2 university hospital bonn, department of surgery, bonn, germany, 3 department of surgery, tu munich, munich, germany bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity. here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. to mimic abdominal surgery, mice were subjected to intestinal manipulation (im). to study septic conditions, mice underwent colon ascendens stent peritonitis (casp) or received e.coli intravenously. we monitored the distribution of gut-derived bacteria by in vivo imaging (xenogen) and additional microbiological assays and determined antigen-specific ctl responses towards subsequent infection with recombinant adenoviruses (av). we detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent casp or were injected i. v. with e.coli. in addition, mice infected systemically with av lacked an antigen-specific ctl response, whereas the ctl responses of locally av infected mice were not affected. in contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to im or received e.coli by injection into the hepatic portal vein. here, the ctl response was not impaired. depletion experiments imply that kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. we suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. objectives: varicella-zoster virus (vzv) is one of the most frequent pathogens for which a vaccine is available. tropism of vzv for skin is the most obvious manifestation of vzv infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. the striking difference in the clinical outcome of infection by rush inducing circulating virulent vzv strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. therefore, we comparatively investigated the impact of vzv clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (idcs) and epithelial gd t cells. methods: skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. phenotypic changes of cutaneous dcs and monocyte-derived dcs after vzv infection were investigated by flow cytometry. the cytokine profile of vzv-infected dcs and epithelial gd t cells was determined through elisa. results: we observed that innate lymphocytes recognize dcs, which infiltrate herpes zoster lesions, after infection with vzv. strikingly, only the vaccine but not vzv clinical isolates could license the bidirectional crosstalk between idcs and gd t cells resulting in release of ifn-g and il-12, the signature cytokines of antiviral immune responses. this is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite. we describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. this result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair dc instruction in order to allow stimulation of powerful antiviral immune responses. a. jafarzadeh 1 1 rafsanjan university of medical sciences, rafsanjan, iran, islamic republic of objective: it has been reported that the caga + h. pylori strains induce more severe gastric mucosal inflammation. the aim of this study was to investigate the association of the h. pylori virulence factor caga with serum levels of il-17 and il-23 in h. pylori-infected duodenal (du) patients and h. pylori-infected asymptomatic (as) carriers. methods: totally, 45 h. pylori-infected du patients (23 patients were positive for anti-caga antibody and 22 patients were negative for anti-caga antibody), 30 h. pylori-infected as carriers (15 subjects were positive for anti-caga antibody and 15 subjects were negative for anti-caga antibody) and 15 healthy uninfected subjects (as a control group) were enrolled to study. a blood sample was obtained from all participants and the serum levels of il-17 and il-23 was measured by elisa method. the mean serum levels of il-17 in total du patients (9.28 pg/ml ± 5.48) was significantly higher than those observed in total as subjects (5.19 pg/ml ± 3.75, p x 0.001) and healthy control group (3.55 pg/ml ± 3.76, p x 0.0001). in du group, it was found that the mean serum levels of il-17 in subjects with positive test for anti-caga (10.84 pg/ml ± 5.79) was significantly higher than those observed in subjects with negative test for anti-caga (7.65 pg/ml ± 4.74; p x 0.05). the mean serum levels of il-23 in du (8.66 pg/ml ± 8.41) and as groups (7.25 pg/ml ± 5.66) was significantly higher than those found in uninfected control group (3.64 pg/ml ± 3.36, p x 0.02 and p x 0.03, respectively). however, no significant difference was observed for mean serum levels of il-23 between du and as groups. moreover, in both du and as groups the mean serum levels of il-23 was not significantly differ in subjects with positive test for anti-caga and those were negative for anti-caga antibody. the results of the present study showed higher serum concentrations of il-17 and il-23 in h. pylori-infected subjects as compared with control group. in du group the expression of il-17 influenced by the bacterial caga factor. a. aral 1,2 , a. atak 1 1 gazi university faculty of medicine, department of immunology, ankara, turkey, 2 gazi university institution of health sciences, department of immunology, ankara, turkey objective: ebv is a human herpesvirus, which infects human b lymphocytes latently and immortalizes the cells due to transformation. ebv infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (im) in adolescence. in immunodefective patients, the virus may lead to malignancies like burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. the virus can also cause latent infections. cytokine production in response to ebv infection differs according to the phase of the infection. neopterin, ifn-g and il-6 levels are elevated in acute ebv infection in vitro; but in chronic phase, particularly, il-6 elevation could not be detected. tnf-a enhances the effects of ifn-g on neopterin synthesis while neopterin enhances the secretion of tnf-a via various stimuli. ifn-g levels are elevated particularly in the acute phase of im. since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. ifn-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity. method: in order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via ebv. neopterin, ifn-g, tnf-a and il-6 levels have been measured with eia kits in culture supernatants. results: neopterin levels increased dependent on time and independent of ebv transformation. in ebv-transformated cell culture tnf-a levels increased beginning from the 48th hour and reached to maximum levels at the 1st week and decreased again at the 3rd week; however there were no significant differences between the levels among three weeks. likely tnf, ifn-g levels also increased at the 1st week and started to decrease at the 3rd week. il-6 reached to its peak at the 3rd week. conclusion: according to these results, neopterin levels, which increased dependent on time but independent of ebv transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation. v. jurisic 1 , m. jurisic 2 1 university of kragujevac, school of medicine, kragujevac, serbia, 2 university of belgrade, school of dentistry, belgrade, serbia tnf-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. we investigated tnf concentration in 43 radicular inflamed cysts and 15 odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. further, we estimated the role of cyst wall on production of tnf-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. the concentrations of tnf-alpha in the cystic fluids were measured by elisa, while proteins analyzed after separation by two-dimensional gel electrophoresis. the presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for cd3, cd20 and cd68. tnf-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. higher concentration of tnf-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (mann-whitney u-test, p x 0.05) in radicular cysts. no correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of tnf-alpha. objectives: interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. in susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. we will study prolactin role in defensive reaction of immune system to bacterial infection. as a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system. the aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery. 1) drawings blood from 20 patients and 20 healthy donors. blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. status of patient had to meet these conditions: a) the presence of systematic inflammatory response syndrome (sirs) according to standard clinical and laboratory criteria. b) positive hemoculture or determination of septic focus with demonstration of microbiologic source. 2) detection of the gene expression of prolactin and tlr-2 in cd14+ peripheral blood monocytes from patients with septic status and from healthy controls has been performed by rt-pcr at the level of mrna and flow cytometry at the level of intracellular protein results: we have found statistical significant differences (p p 0.05) in expression profile between patient and control groups. these differences were at both levels of expression, mrna and protein. conclusion: septic statuses change tlr-2 and peripheral prolactin expression in cd14+ monocytes. the function of interferon (ifn)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of mhc-class i antigens. the experiments performed here challenge this existing paradigm of i-proteasome function. we demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under ifn-induced oxidative stress. ifns up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. i-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. background: revived by the description of new functions, b cells are considered to be essential in the genesis of autoimmune diseases. in strong support of this interpretation, baff would play a key role in their physiology. however, the correlation between circulating baff levels and disease activity is not clearly established. conflicting results concerning levels of baff and b-cell repopulation after rituximab treatment have been reported. in fact, basal serum levels of baff reported in literature vary according to the antibodies (ab) used in the elisa and the mode of calculation. the aim of this study was to understand these variations. material and methods: different anti-baff abs were tested to verify whether they recognize glycosylated baff purified from u937 culture supernatant. sera from patients with autoimmune diseases were also tested. a western-blot analysis of sera was performed to evaluate anti-baff abs specificity and the best combination of anti-baff abs validated for elisa. then, different commercial baff elisa-quantification kits were compared to our "in-house" elisa. results: unexpectedly, the binding of some anti-baff ab was restricted to glycosylated baff. however, both glycosylated and non-glycosylated forms of baff were found in sera from patients with autoimmune diseases. most of the kits commercially designed to quantify baff suffer from some limitations. some sera are indeed positive with a kit and negative with another and vice versa. furthermore, there seems that rheumatoid factors (rf) interfere and correlate with the optical density of the anti-baff elisa. conflicting results concerning levels of baff and disease activity or auto-abs titers should be reconsidered in light of the glycosylation status of baff. (table-2 ). when tb household contacts and healthy controls were compared, cfp10 and esat6 seemed to be more useful than tst in tb contacts for displaying ltb (table-2) . although cfp10 spot numbers were much more than esat6 spots at the beginning and follow up period, statistically there was no difference in terms of median values(p: 0,069)( table-3 ). both esat6 and cfp10 spots were prone to decline during the follow up period. [ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by hcv. to gain insight into some features of the innate immune response activated in vivo in the context of acute hcv replication, we analysed cytokine and chemokine levels in serum samples from chronic hcv patients undergoing liver transplantation. luminex multiplex assays and elisas were used to quantitate serum levels of g 20 analytes immediately prior to liver transplantation and at sequential time points up to 90 days post-transplantation. the increase in serum hcv rna levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including ifn-gamma, tnf-alpha, ip-10, il-6 and il-10. notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary hcv infection. high-magnitude elevations in systemic type 1 ifn levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by hcv similar to that observed in in vitro studies. we suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute hcv infection. to test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (hae). the hemagglutinin of the pandemic virus a/hong kong/1/68 (h3n2) (hk) differs from its putative avian precursor by 7 amino acid substitutions. we generated the complete recombinant virus rhk and its ha variants with amino acid reversions back to the ancestral avian sequence (rhk-5aa-i62r, n81d, k92n, s193n, g144a, human (2-6); rhk-r2-l226q, s228g and rhk-7aa-i62r, n81d, k92n, s193n, g144a, l226q, s228g, avian (2-3)). among these variants, the double mutant rhk-r2 and the seven mutant (rhk-7aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions l226q and s228g.we infected hae cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. virus titers were determined in mdck cells, and concentrations of about 50 pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. many cytokines, including t-cell-attracting chemokines such as ip-10 and rantes, were induced to similar levels by different viruses. however, some mediators were induced significantly stronger by avian-like viruses rhk-r2 and rhk-7aa as compared to rhk and rhk-5aa. in particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as g-csf and il-8, shedded adhesion molecules (cd25, vcam-1, icam-1), and pro-apoptotic factors (trail). remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rhk and rhk-5aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. these data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. a total of 98 newborn infants were enrolled in the study. forty-nine newborn infants (group i), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of pct, il-10, and neutrophilic cd64 levels, procalcitonin and il-10 were measured by elisa technique while, neutrophilic cd64 by single colour flowcytometric technique. of these 49 "infected" infants, 16 had positive blood culture (subgroup ia: culture-positive sepsis), and 33 infants were diagnosed to have clinical sepsis with negative blood cultures (subgroup ib: culture-negative sepsis). another 49 newborn infants classified as control group (group ii) . results: sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for pct, il-10, cd64, and crp. il-10 had the highest sensitivity and specificity, 92 % and 84 %, respectively, using cutoff n 17.3 pg/ml. for pct, the highest sensitivity and specificity, 65 % and 60 %, respectively, were at a cutoff value of n 36.4 pg/ml. neutrophilic cd64 had maximal sensitivity and specificity of 92 % and 71 %, respectively, at cutoff value of 2.6 %. combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. we found that the best combination was il-10 and neutrophilic cd64, which together provided sensitivity and specificity of 95 % and 83 %, respectively, and npv 86 %. the combination of il-10 and crp had high sensitivity and moderate specificity, 93 % and 79 %, respectively. conclusions: il-10 and neutrophilic cd64 levels determination can be used as good tests for early detection of neonatal sepsis. procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. objectives: the assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the nod-like receptors (nlr). the nlr family members nalp3, naip5 or ipaf and the adaptor asc are involved in caspase-1 activation in response to bacterial infection, triggering the processing and secretion of il-1b and il-18. recent studies have demonstrated that tlr-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. autophagosome biogenesis and maturation requires activation of the class iii pi3k, vps34 and can be inhibited with the pi3k inhibitors wortmannin and 3 methyladenine (3ma). in contrast, activation of akt, via class i pi3k, results in inhibition of autophagy. the aim of this study was to determine the nature of this inflammasome and whether autophagy influences il-1b secretion in dendritic cells. methods: murine bone marrow-derived dendritic cells (bmdm) were treated with tlr ligands in combination with 3ma, wortmannin or an akt inhibitor. supernatants were analysed for il-1b by elisa. results: 3ma enhanced il-1b secretion by bmdc treated with the tlr3 and tlr4 ligands poly(i:c) and lps, but not with any other tlr ligands tested. similar results were obtained using wortmannin. this increase in il-1b secretion was greatly reduced in bmdc from nalp3 -/mice compared to wild type c57/bl6 controls. treatment with the akt inhibitor had no effect on lps-induced il-1b secretion by bmdc. tlr-dependent secretion of il-1a was also enhanced by treatment with 3ma. conclusions: these data demonstrate that il-1b secretion by bmdc in response to treatment with pi3k inhibitors, in combination with lps or poly(i:c), is dependent on the nalp3 inflammasome. this response is limited to tlr3 and tlr4 agonists. inhibition of akt had no effect on lps-induced il-1b production, suggesting that the effect of wortmannin and 3ma on inflammasome activation is not dependent on the inhibition of akt activation by class i pi3k. objectives: in the last few years, several evidences have shown the modulation of toll-like receptors (tlrs) by g-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of tlr2 signaling by the inflammatory lipid mediator sphingosine 1-phosphate (s1p) through receptors 1 and 2 in human monocytes-macrophages, which could explain some of the s1p anti-atherogenic effects. since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of tlr-s1p receptors crosstalk on the expression of adhesion molecules in endothelial cells. methods: for the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of tlr ligands and s1p, and later analyzed by flow cytometry. a pharmacological analysis of the s1p receptor subtype involved in the response was also performed. results: data from flow cytometry experiments revealed that icam-1 expression was increased following lps and s1p concomitant stimulation in both venous and arterial cells, suggesting that tlr4 and s1p receptors cooperate in the expression of icam-1. conversely, no cooperation was observed when tlr2 ligands were used. in order to elucidate which s1p receptor subtype was involved in the increase of icam-1 expression, we used a pharmacological approach with s1p receptor inhibitors and pertussis toxin. results showed differences between arterial and venous cells. while in arterial cells elevated icam-1 after lps and s1p challenge was significantly reduced by blocking s1p receptor 3 and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of s1p receptors 2 and 3, which suggest that s1p receptor 1 might be involved in the effect. conclusions: altogether these data demonstrate that tlr4 and s1p receptors can interact to increase adhesion molecules such as icam-1 in human endothelial cells, and the s1p receptor subtype involved in the effect differs between arterial and venous cells. 15 with ssc without pah) and a pool of 12 sera of healthy controls (hc) were tested. results: in 1 dimension immunoblot, serum igg from ssc patients, patients with ipah and hc tested individually reacted with 7-10, 4-8 and 2-5 protein bands in a human vsmc protein extract with qualitative and quantitative differences between groups, respectively. in 2 dimension immunoblot, igg of pools of patients with ipah, igg of pools of patients with ssc with or without pah, and igg of a pool of hc recognized 145±48, 127±26, 130±25 and 150 protein spots respectively. twenty one protein spots were recognized by more than 80 % of igg of pools of sera in each group of patients and not by igg of hc. twenty seven protein spots were recognized by the great majority of igg of pools of patients with a higher intensity than igg of pools of hc. identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein 1 and peroxyredoxin 6 and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise. we have identified anti-vsmc abs in the serum of patients with idiopathic and ssc-associated pah. these abs bind to cytoskeleton, oxidative stress and cell cycle antigens. objectives: this study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species. methods: this rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. results: this rat model is adequate to study the regeneration of transplanted lymph nodes. lymph node fragmentation seems to affect transplant regeneration negatively. an immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. however, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. conclusion: lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. this disease causes lifelong disability due to chronic swelling and increased risk of infections. it currently lacks an effective treatment. methods: 27 patients suffering from different diseases were enrolled in our study. 16 patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas 11 of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). blood specimens were collected before hyperbaric oxygen treatment and the serum levels of icam-1 and vcam-1 were assesed by an enzyme immunoassay (elisa). results: 11 out of the 27 patients (40,7 %) had elevated levels of the intercellular adhesion molecule. 6 out of the 16 patients suffering from bone diseases (37,5%) had raised values (mean value 400 ng/ml) whereas 5 patients out of the 11 suffering from soft tissue diseases and diabetes (45,5 %) had raised values (mean value 735,5 ng/ml). reference value for icam-1 was 130-299 ng/ml. vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. conclusions: our study revealed a high rate of patients (40 %) having increasd levels of icam-1. high icam-1 levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes (45,5 %) than in patients with bone diseases (37.5 %). mean values were found 735,5 ng/ml and 400 ng/ml accordingly. those findings verify the positive correlation between icam-1 and inflammatory diseases and tissue damage but not for vcam-1. colorectal cancer (crc) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of crc carcinoma potentially through repression of tumour angiogenesis. we investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of crc: 16 cases of ulcerative colitis (urc), 16 adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, 33 tubular or tubulo-villous adenomas with low grade dysplasia, and 33 infiltrating adenocarcinomas. immunohistoobjectives: to determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of cd18 in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (cec). methods: they were included in way randomizada double blind 20 patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (cec). 40 mg of pravastatina oral 2 hours they were administered before the procedure (group study, n=10) or placebo (group placebo, n=10), and control (group control, n=10). samples of outlying veined blood were extracted to the 24 hours. the separation of leukocytes was made in peripheral blood, to determine the expression of cd18 in such. in all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. results: 3 types of patterns were distinguished: cytoplasmic, of membrane and compound. the intensity of the expression was classified in 3 degrees: degree 0. without expression. degree 1. weak; degree 2. moderate; degree 3. intense. in the group control: most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree 0-1. the placebo group: mixed pattern, degree 1-2. group study (40 mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: degree 2-3. the percentage of cells that expressed cd 18 was greater in the group study (40 mg. oral pravastatina). the preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression cd18 answer induced by the cec; it seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases adam10 and adam17. we recently identified transmembrane chemokines, junctional adhesion molecule-a (jam-a), and members of the proteoglycan family as novel substrates for these proteases. here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble jam-a and proteoglycan ectodomains. this release is enhanced by treatment with the proinflammatory cytokines ifng and tnfa. the enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. both, constitutive and induced release required the presence of adam17 as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of adam17. these data suggest that the proinflammatory cytokines ifng and tnfa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells. to investigate the physiological relevance of this induced shedding, mice were treated systemically with ifng/tnfa leading to increased presence of soluble jam-a in the blood serum. both cytokines also stimulated jam-a release from excised murine aortas with was associated with enhanced adam17 activity in the tissue. in the presence of the adam17 inhibitor induction of jam-a release was suppressed. in cultured epithelial cell lines enhanced shedding of jam-a or proteoglycans was not associated with increased mrna or surface expression of adams but rather with increased activity of cellular adam17 as shown by means of a synthetic substrate assay. our study demonstrates that the proinflammatory cytokines ifng/ tnfa upregulate adam17-mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings. rium, and the haplotype vegf-460/ vegf+405 is associated with rcc risk ( p= 0,017), metastases ( p=0,043), nuclear grade ( p=0,05), tumor stage ( p=0,029), and tumor size (p=0,04). on the other hand, the polymorphism vegf -2578 a/c is not associated with rcc risk and clinical parameters. our results shed a new light to the knowledge on the association between vegf polymorphism and rcc risk and development. these data could help to improve our understanding of the rcc pathogenesis and disease progression. pten is a lipid phosphatase, whose substrate is phosphatidylinositol 3,4,5-trisphosphate. therefore, pten is one of the main antagonists of the pi3-kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. here we investigated the role of pten in collagen induced arthritis. we show that conditional deletion of pten under the lysm promoter (lysmcrepten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (cia). histological analysis of cia, lysmcrepten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. total anti-collagen antibodies, however, as well as anti-collagen iggs were identical in both groups. upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of il-6 as well as il-8 levels. furthermore, pten deficient macrophages and dendritic cells showed reduced induction of il-6 as well as il-12 and il-23 mrna upon stimulation with various tlr-ligands. since these cytokines play an important role in the induction of pathogenic th-17 t cells, we measured th-17 cytokines in lymph nodes after immunisation with collagen. although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of il-17 and a strong reduction of il-22 mrna in the draining lymph node of immunized lysmcrepten flox/compared to wild-type mice. one of the mechanisms through which il-10 exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. in this context, particularly important is the production of il-1ra, whose expression is induced by lps in human neutrophils and monocytes and significantly potentiated by the presence of il-10. based on our previous observation that support a direct role of il-10-activated stat3 in the enhancement of il-1ra transcription induced by lps, we plan to characterize the transcriptional activators recruited to the il-1ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of lps-treated cells to il-10. quantitative chromatin immunoprecipitation (chip) studies were employed to examine the in vivo binding of transcriptional activators to the il-1ra promoter. crosslinked nuclear lysates were immunoprecipitated 30 and 60 min after il-10 addition with different antibodies and immunoprecipitated dna was analyzed by quantitative real-time pcr for the presence of target sequence located in the il-1ra promoter. chip assays showed that the pol ii recruitment to the il-1ra promoter induced by lps is significantly increased by il-10, further strengthening the concept that the rapid enhancement of lpsinduced il-1ra gene expression by il-10 initially occurs by targeting transcriptional events. as expected, real-time pcr of anti-stat3 immunoprecipitated dna showed statistically significant levels of stat3 binding to the il-1ra promoter only in cells stimulated with lps in the presence of il-10. surprisingly, anti-p65 and anti-p50 chip assays revealed enrichment of both p65 and p50 recruitment to the il-1ra promoter when il-10 was added to lps-stimulated cells, suggesting that il-10 enhances the recruitment of nf-kb to the il-1ra promoter. interestingly, when nf-kb is recruited to this promoter in lps + il-10-treated cells, the overall nf-kb nuclear translocation (analyzed by western blot) and dna binding activity (detected by emsa analysis) were not modified with respect to cells stimulated with lps alone. the enrichment of nf-kb at the il-1ra promoter site is dependent on il-10-activated stat3, since it is greatly reduced when stat3 activation by il-10 is impaired. the molecular mechanism through which il-10-activated stat3 promotes the recruitment of nf-kb to the il-1ra promoter is currently under investigation. major components of mast cell secretory granules are proteases. we could recently report that intracellular stored mast cell-produced cytokines regulate mc protease activities and provided evidence that il-15 acts as a specific negative transcriptional regulator of mouse mast cell protease-2 (mmcp-2). we examined the mechanisms underlying the repression of mmcp-2 gene expression. our data show that the "repressor" effects of il-15 on mmcp-2 promoter activity are still operating on the mmcp-2 591 bp long minimal promoter. moreover, il-15 deficiency in mast cells causes a specific dysregulated expression of the transcription factors c/ebpb and yy1. furthermore, chromatin immunoprecipitation revealed that il-15 promoted specific reciprocal recruitment of c/ebpb but not yy1 to the mmcp-2 promoter. finally, il-15 deficient mast cells display a predominantly non-cpg methylated pattern of the mmcp-2. thus, we proposed that the expression of mmcp-2 and possibly other immunoregulatory genes may be regulated by il-15 through epigenetic modification and by balancing the content and binding of c/ ebpb and yy1 in mast cells. i. nagy 1 , k. filkor 1 , a. vörös 1 , l. kemény 2 , a. szász 1 1 bzaka, baygen, szeged, hungary, 2 university of szeged, department of dermatology and allergology, szeged, hungary micrornas (mirnas) are evolutionary conserved small non-coding rnas that act as key regulators of gene expression at post-transcriptional level by targeting mrnas for translational repression and/or degradation. mirnas have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. during the last years several studies highlighted that mirnas play critical role in the differentiation and function of the adaptive as well as innate immunity. little is known however, about the differential regulation of mirnas following the activation of pattern recognition receptors. in order to tackle this issue, we treated hacat keratinocytes with staphylococcus aureus-derived peptidoglycan (pgn) once or repeatedly, the latter mimicking persistent infection. after appropriate treatments we first analyzed the expression profile of mirnas mir-203, mir-146a and mir-155, which are known to participate in immune processes of the skin. repeated pgn-treatment significantly decreased mir-203 expression; in contrast, pgn re-stimulation had no further effect on mir-146a and mir-155 expression. next, we investigated the correlation between the expression of mir-203 and its two known direct targets: regulatory protein p63 and suppressor of cytokine signalling-3 (socs-3). although the gene-expression profile of neither p63 nor socs-3 changed, we found that the expression of mir-203 reversibly correlates with both p63 and socs-3 protein expression, a phenotype that we verified by two independent protein analysis methods (western blotting and immunofluorescent labeling). importantly, transfection of hacat cells with anti-mir-203 prior to pgn-treatment completely abolished both p63 and socs-3 down-regulation, revealing the involvement of mir-203 in pgn-induced transcriptional regulation. finally, methylation-specific pcr experiments unravelled the role of dnamethylation in regulating mir-203 expression upon pgn-treatment. taken together, our results strongly suggest that sets of mirnas may be differentially regulated during persistent infection. results: tgfb1+/-had a lower incidence and burden of benign papillomas when compared to tgfb1+/+ animals. however, more scc developed in the tgfb1+/-mice. after acute and chronic promotion, tgfb1+/-skin showed a reduced proliferative response with no increase in epidermal tgfb1 or nuclear p-smad2 compared to tgfb1+/+ mice. tpa-induced pkca activity as well as phosphorylation of specific pkc substrates in keratinocytes correlated with tgfb1 gene dosage. further, pharmacological inhibition of alk5 suppressed tpa-mediated pkca activation suggesting that physiological levels of tgfb1 are required for maximal activation of pkc-dependent mitogenic responses. even though the tpa-induced inflammatory response was greater in tgfb1+/-skin, tgfb1+/+ papillomas had more tumor infiltrating neutrophils. tpa-induced proinflammatory gene expression was sustained in tgfb1+/-skin and primary keratinocytes but it was elevated in v-ras ha -transduced tgfb1+/+ but not tgfb1+/-keratinocytes, indicating that tgfb1 switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. despite this differential proliferative and inflammatory response to tpa and enhanced papilloma formation in the tgfb1 +/+ mice, there was no increase in conversion to scc in this genotype. conclusions: tgfb1 acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate pkc activation in skin, yet retains a suppressive function for malignant conversion. background: proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (ra). in the present study we studied the relationship between survivin and urokinase (upa), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. material and methods: levels of survivin and upa were measured in the paired blood and synovial fluid samples of 132 patients with ra, using elisa and compared to controls with non-inflammatory joint diseases. the ability of upa to induce survivin and requirement of upa receptor (upar) was studied in primary synovial fibroblasts and pbmc of ra patients, human monocytic (thp-1) and fibroblast (mrc-5) cell lines employing antibodies against upar, sirna technique, and synthetic inhibitors of intracellular pathways. the ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. results: in the present material of 132 ra patients and 82 controls the levels of survivin correlated to urokinase (upa) (r=0.46), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. we found that 30/132 ra patients had high circulating levels of survivin. these patients had erosive arthritis and were characterized by high levels of upa. in vitro studies showed that upa induced survivin in leukocytes and this process was dependent on signaling through upa receptor. in turn, survivin was required for expression of upar. additionally, survivin was essential for upa production in mrc-5 and synovial fibroblasts. down-regulation of survivin with sirna was followed by significantly reducion of upa synthesis. finally, treatment with downregulation of survivin by sirna in vivo efficiently abrogated upa-induced arthritis in mice model. these findings indicate that survivin is an essential mediator of arthritogenic properties of upa regulating its synthesis in synovial fibroblasts and upar expression in leukocytes. close correlation between survivin and upa in patients with ra supports the improtance of these interaction for the pathogenesis of arthritis. upon cell activation, ubiquitously expressed inositol 1,4,5-trisphosphate 3-kinase type b (itpkb) phosphorylates inositol (1, 4, 5) trisphosphate (ins(1,4,5)p 3 ), a calcium-mobilizing second messenger with pleiotropic effects. itpkb inactivation leads to severe t cell deficiency, altered thymo-independent b cell responses and neutrophil hyperactivation. we here report that itpkb-deficient (itpkb -/-) mice also display profound alterations in mast cell development and function. indeed, while mast cell number, c-kit and fcepsilonri expression were comparable in itpkb-deficient and proficient mice, itpkb -/mast cells were almost completely devoid of granules. this phenotype could be partially reversed upon treatment with sodium cromoglycate. nevertheless, fcepsilonri or c-kit activation on mast cells led to increased ca 2+ responses and to stronger erk phosphorylations. however, itpkb -/mice displayed an attenuated sensitivity to ige-mediated passive systemic anaphylaxis, correlated to the absence of fcepsilonri-dependant histamine release and to downregulation of h1 and h2 receptor expression due to high basal histamine concentrations. production of neosynthesized mediators remained normal. finally, itpkb deficiency also severely impaired scf-induced mast cell differentiation in vitro. taken together these results demonstrate that itpkb is a key regulator of mast cell activation. itpkb antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. the large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. il-1b is an important proinflammatory cytokine produced by activated macrophages and monocytes. il-1b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase-1 to generate the mature secreted active form. caspase-1 is also synthesized as an inactive form that requires processing by the inflammasome to become active. we have used a subset of the trc lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. we were able to silence the expression of 425 genes involved in splicing with an average 5-fold coverage. after the primary screen and several rounds of phenotypic validation, we have identified 30 genes that significantly affect the production of il-1b by thp-1 cells after a 24h challenge with pfa-fixed e. coli, as measured by elisa. knockdown levels were analyzed by qrt-pcr for the most significant candidates to validate the phenotypes observed. exon array analysis are being performed to identify possible targets of the most significant splicing factor candidates obtained by the shrna screening in order to dissect their mechanism of action in the regulation of the inflammasome and il-1b secretion. tissue transglutaminase (tg2) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. we have previously described the key role of tg2 in cystic fibrosis (cf), a genetic disease characterised by chronic lung infections and inflammation. in cf, mutation on the cftr gene results in an increased tg2 expression and activity leading to functional sequestration of the anti-inflammatory pparg and increase of the classic parameters of inflammation. here we tested whether in vivo inhibition of tg2 can reverse inflammation in chronic inflammatory diseases. to assess the importance of tg2 not just in cf but in chronic inflammatory diseases in general, we injected cystamine, a potent tg2 inhibitor, in a transgenic mouse model cf and in the taz10 transgenic mice that spontaneously develop autoimmune thyroiditis. intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the cf model, where it decreased tg expression and activity. the treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional pparg. interestingly, cystamine injections could also block inflammation in the taz10 tcr transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of tg2 in generating inflammation in two very different pathologies. this work underlines the critical role of tg2 in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. we have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated dc, a-dc) display potent angiogenic activity in vivo which is mediated by the release of biologically active vegf-a. here, we investigates the molecular mechanisms leading to vegf-a secretion in lps+pge 2 stimulated a-dc. preliminary results indicate no accumulation of hif-1alpha in a-dc, therefore suggesting that vegf-a is induced by a non-classical, hif-1alpha independent pathway. in addition, we found that vegf-a secretion depends on the activation of mapk p38 but not erk1/2 or jnk. inhibitor studies, nascent transcript analysis and polimerase ii recruitment on the promoter show that the induction of vegf-a is largely due to new transcription and not to changes in mrna stability. chromatin immunoprecipitation studies aimed at the characterization of the modifications of vegf-a regulatory regions in a-dc and at the identification of transcription factors bound to vegf-a promoters are being performed. this will possibly allow the description of novel transcription factors involved in vegf-a activation in a-dc. wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. they are best known for their role in embryonic development and tissue homeostasis. deregulation of wnt signaling has been shown to promote carcinogenesis. recently we identified wnt signaling to be involved in the regulation of inflammatory processes: wnt5a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines il-12 and ifn-gamma. to gain deeper insights into wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous wnt homologs. we monitored wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by real-time pcr and measured the secretion of inflammatory cytokines by elisa. exogenous wnt5a -but not wnt3a -was able to induce cytokine expression in primary macrophages. in infection experiments wnt5a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. in contrast, addition of wnt3a reduced the expression of inflammatory mediators upon mycobacterial infection. these data corroborate our previous findings and further support the notion that tlr/nf-kappab and wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected infection of immunocompetent mammals with t. gondii induces a chronic infection of the brain. t. gondii cysts persist in neurons and escape elimination by the immune system. in immunodeficient individuals, the infection can be reactivated resulting in a lethal toxoplasma encephalitis (te). in te, the parasite is primarily controlled by infiltrating t and b cells. also brain resident cells may contribute to control of the disease and however, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic te are largely unknown. in a previous study, we could show that expression of gp130 on astrocytes in mice is critical for survival of te. in the present study, we analyzed the function of neuronal gp130 in te. after infection with t. gondii, mice lacking neuronal gp130 (synapsin-cre gp130 fl/fl ) died significantly earlier in the chronic phase of infection than control gp130 fl/fl mice. death of synapsin-cre cre gp130 fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. additionally, te of synapsin-cre gp130 fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. in vitro, apoptosis of gp130-deficient neurons was also significantly increased upon infection with t. gondii or stimulation with tnf as compared to gp130 expressing neurons. interestingly, the intracerebral parasitic burden was not increased in synapsin-cre gp130 fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function. t. objectives: persistent production of tnfa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. targeting tnfa with neutralising antibodies or tnf receptor fusion proteins is often, but not always, an effective therapy. high serum concentrations of tnfa, il-1b, il-6 and il-8 have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of il-4, il-10 and tgfb are reduced. this suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. thus, understanding the homeostatic control of tnfa by tgfb1 further may generate more effective therapies. as tnfa mrna 3' untranslated region (utr) contains an au-rich element (are), which targets mrnas for degradation, we wished to test whether tgfb1 suppresses tnfa protein production by upregulating the rnabinding protein fxr1, which can bind to tnfa mrna and inhibit translation. methods: using raw 264.7 cells and mouse bone-marrow derived macrophages stimulated with lps and tgfb1, we assessed mrna expression by q-pcr and tnfa protein expression by flow cytometry. the 3'utr of tnfa mrna was isolated and inserted into a luciferase reporter vector on a constitutive promoter. transfected raw cells were treated with lps and tgfb1 and luciferase expression was quantified. cells treated with lps and tgfb1 were also examined for fxr1 expression using pcr and western blot. following fxr1 knockdown using sirna, the influence of tgfb1 and lps on tnfa protein production was examined by flow cytometry. results: we find that while tnfa mrna expression remains constant, lps induced tnfa protein expression is suppressed by tgfb1. using the luciferase-tnf-3'utr vector we show that tgfb1 targets the 3'utr of tnfa. furthermore, tgfb1 and il-10 both upregulate fxr1 mrna and protein; and treatment with tgfb1 and lps can synergistically upregulate mrna expression, more than tgfb1 alone. following sirna inhibition of fxr1, tgfb1 can no longer inhibit lps-induced tnfa production. comtb up-regulated mmp-1 and mmp-3 secretion from saecs, nhbes and fibroblasts to a peak of 2.5 +/-0.5 ng/ml, at 72 hours. interleukin-17 augmented comtb-stimulated up-regulation of mmp-1 and mmp-3 secretion from saecs and fibroblasts in a synergistic manner. in contrast, interleukin-17 down-regulated mmp-9 secretion from saecs by 50 %. interleukin-22 up-regulated mmp-1 and mmp-3 secretion from fibroblasts but not from saecs. timp1 secretion from saecs was enhanced by interleukin-17 but there was no effect of interleukin-22. mmp up-regulation by interleukin-17 and comtb was inhibited by the pi3kinase inhibitor ly294002 and on western analysis akt (protein kinase b) was phosphorylated at 30 minutes. chemical inhibition of the p110d isoform of pi3kinase with ic87114 abrogated the il-17 and comtb driven secretion of mmp-3 from the small airway epithelial cells. chemical inhibition of the tumour suppressor phosphatase, pten (phosphatase and tensin analogue on chromosome 10) accentuated mmp-3 secretion. these inhibitory effects were confirmed with sirna. mmp-3 up-regulation was secondary to increased gene expression with promoter activity peaking 24h after stimulation. in summary, interleukin-17 and interleukin-22 drive transcription dependent mmp-1 and mmp-3 secretion from airway epithelial cells and fibroblasts. interleukin-17 also increases timp but down-regulates mmp-9 gene expression and secretion. this may contribute to the matrix degrading phenotype in tuberculosis. the pi3kinase pathway is central in interleukin-17 driven tissue destruction in the context of m. tuberculosis infection. v. delgado-maroto 1 , l.s. moreira 2 , e. gonzalez-rey 2 , m. delgado 1 1 institute of parasitology and biomedicine lopez-neyra , csic, granada, spain, 2 university of seville, sevilla, spain objectives: atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. atherosclerosis will probably become the most common cause of death within 15 years. one of the initial hallmarks of the disease is the uptake of oxldl particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. t cells undergo activation after interacting with foam cells, which process and present local antigens including oxldl, generating a t helper 1 response. cholesterol metabolism is regulated by factors such as pparg1 (proliferator activated receptor g), srb1 (a class b scavenger receptor), cd36 or abca1, that can induce cholesterol exit from the macrophage which may help to solve the lesions. expression of these factors depends on intracellular camp. adrenomedullin (am), urocortin (ucn) and vasoactive intestinal peptide (vip) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. they are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit th1 immune response. their mechanism of action involves binding to gpcr and adenylate cyclase activation with subsequent increase of camp in the cell, which is recognized as an anti-inflammatory response. methods: we investigate am/ucn/vip effect on bone marrow-derived macrophages stimulated with oxldl. we determine the levels of pparg1, srb1, cd36, and abca1. we also analyze the cholesterol metabolism of oxldl-stimulated macrophages after neuropeptides incubation using oil red o staining of lipids drops and tritium labelled cholesterol. objective: endovascular aortic repair (evar) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. however up to 60 % develop a systemic inflammatory response syndrome (sirs), resulting in prolonged convalescence. as yet there is no satisfactory explanation to this severe response. previous studies have shown a high level of il-6 in the mural thrombus lining the aneurysm. the thrombus is manipulated during the procedure, but whether or not it is the source of circulating il-6 and/or other cytokines during and after the operation is unknown. methods: quantitative analysis of the pro-inflammatory cytokines il-6, tnf-a, il-1b, il-8 and il-12, and the anti-inflammatory cytokine il-10, in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma il-23 was performed using the luminex platform. the cytokine levels were compared to the clinical response, in terms of sirs. results: evar induced the production of il-6 and il-10, and in some cases, of il-23. the maximal plasma levels of il-6 and il-10 were found at 24 hours and of il-23 between 48-72 hours. modest plasma levels of il-8 were also observed, with maximal production at various time points (4-24 hours). by contrast, production of tnf-a, il-1b and il-12 did not occur to a significant extent, while production of il-17 occurred sporadically. although our preliminary data indicate that sirs is associated with enhanced cytokine responses, the production of il-6, il-8, il-23 and il-10 also took place in patients who did not develop sirs. conclusion: evar is associated with the sequential production of il-6, il-10, il-8 and il-23, i. e. a mixed pro-and anti-inflammatory response, even in the absence of sirs; but the production seems to be exaggerated in patients developing sirs. further studies involving 165 patients are in progress, and will clarify this. we hypothesize that sirs is elicited by il-6, activated by manipulation of the mural thrombus. to reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. il-33 is a novel il-1 cytokine family member that is expressed as an intracellular precursor (pro-il-33) and is thought to be cleaved by caspase-1 to yield a mature bioactive form of the molecule (mat-il-33). to date however, evidence of cell-associated proteolytic processing and caspase-1 dependent secretion of mat-il-33 has not been reported. here we show that pro-il-33 but not mat-il-33 is released from uvb-irradiated keratinocytes. we demonstrate binding of pro-il-33 to the il-33r and also il-33r-dependent bioactivity of pro-il-33 on mast cells. we propose a previously unrecognized role for pro-il-33 as a pro-inflammatory mediator and suggest a direct link between uvb-mediated epithelial cell damage and cutaneous mast cell activation. we have previously shown that induction of er stress and tlr signalling synergistically enhance il-23 p19 mrna expression in myeloid cells, and markedly increase secretion of il-23, but not il-12, by dendritic cells. the aim of this study is to investigate the mechanism of this synergy. we examined the il-23 promoter for potential binding sites for er stress induced transcription factors and identified a putative site for chop10. chromatin immunoprecipitation (chip) assays using anti-chop10 and isotype control mab were performed using nuclear lysates from u937 cells and il-23 promoter dna measured by qpcr. chop10 binding on the il-23 promoter was detected following stimulation of u937 cells with lps or tp alone, but this was significantly enhanced when er stress and tlr stimuli were combined. il-23 promoter dna was not detectable following chip with the isotype control antibody. to confirm the role of chop10 in il-23 gene transcription, u937 cells expressing shrna's specific for chop10 or non-specific gene target were tested for their ability to express il-23 following tlr and er stress stimulation. u937 expressing three independent shrna targets for chop10 exhibited significant reductions in il-23p19 mrna (up to 87 % reduction of the response to lps+tp) compared to u937 expressing a control shrna. chop10 shrna expression did not affect the expression of other lpsresponsive genes, including il-1, il-8, ccl3 and sod2. to identify if er stress induction of il-23 mediated by chop10 expression plays a role in a more physiological setting, we examined the role of chop10 in the induction of il-23p19 gene expression following chlamydia trachomatis (ct) infection. infection of u937 cells with live but not g-irradiated ct induced expression of er stress response genes, including chop10. u937 infected with live ct exhibited increased il-23p19 mrna expression compared to u937 infected with nonviable bacteria. chop10 silencing significantly reduced the ability of live ct to induce il-23p19mrna, confirming the important role of chop10 in this response. these data suggest that er stress induction of chop10 could contribute significantly to the pathogenesis of diseases in which il-23 plays an major role, through induction of il-17 and il-22 producing cells. the clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. here, we show that gadd45b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. gadd45b expression was inducible in different in vitro and in vivo models of negative selection. strikingly, only tcr-ligating peptides resulting in negative selection induced gadd45b expression, while positively selecting ligands or dexamethasone, a tcr-independent apoptosis agonist, failed to do so. expression of gadd45b maintained a sustained activation of p38 kinase and thereby promoted tcr-mediated apoptosis. in contrast, thymocytes from gadd45b-deficient mice showed only transient p38 activation and reduced caspase activation. interestingly, we observed a switch to positive selection in gadd45b-deficient mice since a higher percentage of single positive thymocytes was found. moreover, markers of positive selection as cd5 and cd69 were elevated on gadd45b-deficient thymocytes. thus, we provide evidence that gadd45b and a resulting persistent activation of p38 constitute a novel apoptotic pathway involved in negative selection. these results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. di santo 1 1 institut pasteur, paris, france, 2 monash university, victoria, australia > the thymus represents the ''cradle'' for t cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. although il-7 is recognized as an essential factor for thymopoiesis, the nature of the thymic il-7 niche remains poorly characterized in vivo. > using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (yfp) is under control of il-7 promoter, we identify a subset of thymic epithelial cells (tecs) that co-express yfp and high levels of il7 transcripts (il-7 hi cells). il-7 hi tecs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (ccl19, ccl25, cxcl12) and cytokines (il15) that are critical for normal thymopoiesis. in the adult thymus, il-7 hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature tecs. interestingly, the frequency of il-7 hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining il-7 levels. conversely, the frequency of il-7 hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in t cell development. this augment indicates that thymocyte-tec crosstalk may condition il-7-expression by tecs. > together, our temporal-spatial analysis of il-7-expressing cells in the thymus suggests that thymic il-7 levels are dynamically regulated under distinct physiological conditions. this novel il-7 reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic il-7 expression in vivo. two lines of evidence have recently demonstrated that the pre-b cell receptor (pre-bcr) is associated with autoimmunity, through its surrogate-light-chain (slc) components l5 and vpreb. it has been shown that pre-bcrs are polyreactive for several self-antigens. the polyreactivity of the pre-bcr induces pre-bcr signaling and activation. furthermore, in human a self-reactive b cell subset was identified that co-expresses immunoglobulin light chain (ig lc) and the slc components. these vpreb + lc + b cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like sle and ra. to elucidate the contribution of pre-bcr components to the development and activation of autoreactive b cells, we have recently generated a slc transgenic (slc-tg) mouse model in which all b cells express slc proteins. slc-tg mice exhibit spontaneous igm + plasma cell development. moreover, aging slc-tg mice have elevated anti-nucleosome igm levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. nevertheless, vpreb + lc + b cells may induce pathology when self-tolerance mechanisms fail. to test this hypothesis, slc-tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) em-bcl2-tg mice with b cell-specific overexpression of the anti-apoptotic protein bcl2 and (ii) fcgriib-deficient mice. both in young slc-tg;em-bcl2-tg double tg mice and in slc-tg;fcgriib -/mice spontaneous germinal center (gc) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control em-bcl2-tg and fcgriib -/mice. in slc-tg;fcgriib -/mice, numbers of splenic igg2 plasma cells and serum igg2 levels were˚5-10 fold increased. importantly, serum from young slc-tg;em-bcl2-tg and slc-tg;fcgriib -/mice contained high titers of igg auto-antibodies in 2/3 and 6/6 cases, respectively. these values were increased when compared with control groups: 1/6 in fcgriib -/and 0/6 in bcl2-tg. finally, we found that the collagen induced arthritis (cia) was significantly enhanced in slc-tg;fcgriib -/mice, compared to fcgriib -/mice. taken together, these findings demonstrate the slc components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in ra. t cells are generated from progenitor cells that enter the thymus from bone marrow via blood. these progenitor cells once within the thymus have little selfrenewal capacity. differentiation from hematopoietic stem cells to early t lineage cells proceeds through a series of intermediate precursor populations. however, it is largely unknown to what extent these cell populations contribute to t cell development in the presence of other precursor populations and how the earliest intrathymic t cell progenitors are generated from extrathymic precursors. to assess the relative contribution of potential precursors to t lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological t cell precursor or many. we showed that, under such competitive conditions, t lineage progenitors are confined to the cd27 + cd135 + fraction of bone marrow cells. notably, t lineage reconstitution was not restricted to either cd117 hi cells, representing multipotent progenitors, or cd127 + cells, representing common lymphoid progenitors, both of which contributed to t lineage differentiation with different kinetics. in conclusion, our data suggest that multiple physiological extrathymic t cell precursors exist, which are able to compensate for the loss of depleted populations. thus, our findings may have implications for devising strategies for improved t lineage reconstitution after hematopoietic stem cell transplantation. background: previous results from our group have demonstrated ephb2 and ephb3 expression on both thymocytes and thymic epithelial cells (tecs). we used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and tec development, and how they regulate interactions between both cell types. objectives: in order to better define the importance in thymus of eph-ephrin b interactions we have analyzed the effects of the lack of ephrin b1 and/or ephrin b2, the ligands of ephb2 and ephb3 receptors. this approach is specially interesting taking into account that eph-ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. methods: for this purpose we have used cre-loxp recombination systems for deleting ephrinb1 or ephrinb2 genes specifically on either thymocytes or tecs. results: animals with ephrin b deficient thymocytes showed thymic hypocellularity and alterations on t-cell development whose severity depended on the background of the analyzed mice. in these mice only a few changes occurred in the cortical tec network. on the contrary, mice with conditioned deletions in tec, especially ephrinb1/b2 double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of cd45+ cells in the cortex, increased proportion of k5+k8+ cells and high presence of cysts. in addition, t-cell development was partially blocked at the dn cell stage. conclusion: these data reveal an autonomous and non-autonomous role for ephrinb1 and ephrinb2 in the development of both t cells and tecs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. we discuss how eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in t cell differentiation. a. rolink 1 , d. vanhecke 1 1 university of basel, developmental and molecular immunology, basel, switzerland the importance of normal t lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of t cells is defective. in order to identify molecules/genes and elucidate developmental processes that are essential for human t cell development we use a novel in vitro tool, the op9-dl1 cell culture system (1) . using this in vitro assay we obtain large numbers of human cycd3 + and cd4 + cd8 + double positive thymocytes starting from umbilical cord blood (ucb) derived cd34 + hsc. signals and molecules that are involved in t cell development are being addressed by using blocking antibodies and/or chemical inhibitors. similar as in mice we found an essential role for il-7 and notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. among the molecules that are rapidly induced in cd34 + cells upon notch signaling is cd7 followed by cd127. t cell specification is accompanied by the induction of cd1a and loss of cd34 on cd7 + cd127 + cells. these cd34 -cd1a + cd7 + cells become dependent on continuous il7 and notch signaling for sustained survival and further differentiation into cd4 + cd8ab + dp thymocytes. we found that flt3l is not essential for the differentiation of cd4 + cd8 + human thymocytes but that addition of exogenous flt3l in the co-cultures increases the number of cd34 + precursors and consequently result in higher yields of developing cd4 + cd8ab + dp thymocytes. finally few mature tcrab + t lymphocytes develop from the cycd3 + cd4 + cd8ab + dp subset in this in vitro assay suggesting that op9 stromal cells lack the required selecting mhc-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. this in vitro assay will allow us now, by using rna interference, to test additional genes for their role during human lymphoid development. from a clinical standpoint, a better understanding of the mechanisms controlling human t-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant t-cell disorders. brain derived neurotrophic factor (bdnf) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. emerging evidence also indicates an essential role for bdnf in the immune system, e.g. in the b and t cell lineages. we therefore investigated the impact of bdnf on thymocyte development using bdnf knockout (ko) mice and conditional ko mice lacking bdnf specifically in t cells. in both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. in contrast, the percentage of naturally occurring regulatory t cells and the expression of activation markers were unaltered. moreover, the lack of bdnf did not result in enhanced thymocyte apoptosis. the increase in double negative thymocytes was due to a partial block in the transition from the dn3 to dn4 stage, where bdnf and its receptor p75 are expressed as revealed by real-time pcr. the observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral t cells, their homeostatic proliferation and without compromising peripheral immune responses in general. in summary, our findings point to a critical role of t cell lineage derived bdnf in thymocyte development acting in an autocrine and/or paracrine manner. r. berga 1 , c. lópez-rodríguez 1 1 pompeu fabra university, barcelona, spain nfat5 is a transcription factor that belongs to the rel family (nf-kb and nfatc proteins). its expression in primary cells and organs is restricted to certain proliferative tissues, like activated t lymphocytes and thymocytes, where levels of nfat5 are relatively high and its subcellular distribution is predominantly nuclear. recent mouse models suggest that nfat5 participates in thymocyte development and also indicate its involvement in t cell proliferation and survival. nfat5 deficient mice present a t cell immunodeficiency consistent on lymphopenia, which is more accused for cd8 + lymphocytes. these observations are of substantial relevance as we and others have described that, in vivo, nfat5-null mice are unable to mount cd4+-and cd8 + -immune responses. data from our laboratory indicate that nfat5-null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. to selectively analyze the t-cell autonomous effects derived from the lack of nfat5 during the development of t lymphocytes, we developed mouse models that delete nfat5 at early (lck-cre + /nfat5 flox/flox ) or late (cd4-cre + /nfat5 flox/flox ) stages of thymocyte maturation and that present isotonic plasma. our work indicates that nfat5 is expressed at all stages of t cell development. in addition, analysis of mouse models that lack nfat5 at different points of t cell development indicate that it participates at early stages of the ontogeny of t cells. objectives: apoptosis mediated by the tumor suppressor molecule p53, is regarded as a major player in tumor prevention but this may not be its only role. we have investigated this by creating a mouse (m ¿ pro) lacking residues 58-88 of the proline-rich domain of p53. methods: we compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. apoptosis was measured by staining with annexin v in vitro and by detection of caspase activation in vitro and in vivo. we also compared their ability to undergo cell cycle arrest using brdu staining. tumor development was monitored in cohorts of m ¿ pro, p53 null (p53-/-) and wild type (p53+/+) mice, with or without prior irradiation. results: apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset b-cell lymphoma, but not the thymic t-cell tumors found in p53-/-mice. interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated b-cells. bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated b-cells in the bone marrow and spleen. we propose that the apoptotic function of p53 has an important role in b-cell homeostasis, which, in turn, is important for prevention of b-cell lymphomas moreover, our data suggest that the apoptotic function of p53 is not important for preventing thymic t-cell tumors. s. myrczek 1 , r. pardi 2 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany, 2 vita-salute san raffaele university school of medicine, milano, italy jab1 is the catalytic subunit of the highly conserved cop9 signalosome. this complex plays a central role in various cellular processes as proliferation and cell cycle control. jab1 regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. furthermore jab1 regulates the activity of ap1 transciption factors. to date jab1 is thought to be essential for every cell type as jab1 knock out mice are embryonic lethal and t cell development is blocked by t cell selective absence of jab1. to investigate the function of jab1 in b cells we established a mouse strain deficient for jab1 selectively in b cells. mice with floxed alleles of jab1 kindly provided by r. pardi were crossed with a mouse strain expressing the cre recombinase under control of the mb1-locus (m. reth, freiburg). ablation of jab1 expression resulted in an almost complete block of b cell development at the pro b cell stage. the absence of peripheral mature b1 and b2 cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with borrelia burgdorferi. the observed block in b cell development is rescued by over expression of the anti apoptotic protein bcl2 under the control of the m enhancer. facs analyses revealed that all b cell subtypes analyzed in the jab1-deficient, bcl2-transgenic mice are present albeit at reduced numbers compared to wild type animals. serum immunoglobulin titers are detectable and after borrelia infection specific antibodies are produced. we confirmed the absence of jab1 in sorted spleen b cells by immunoblot analysis. in summary, we show for the first time that cells are viable and functional without jab1 when apoptosis is prevented. t. nitta 1 , s. murata 2 , k. tanaka 3 , y. takahama 1 1 university of tokushima, tokushima, japan, 2 university of tokyo, tokyo, japan, 3 rinshoken, tokyo, japan how self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of t cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive t cells and thereby preventing autoimmunity is well established. a recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (ctec) and is required for the optimum generation of cd8 t cells. here we show that ctec display a thymoproteasome-specific spectrum of class i mhc-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class i mhc-restricted t cells. indeed, cd8 t cells generated in the absence of thymoproteasomes display a markedly altered tcr repertoire that is defective in both allogeneic and antiviral responses. these results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent cd8 t cells. defects in helper t cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. our understanding of the genetic control of helper t cell differentiation into specific functional subsets is still far from complete. here we present the results to date from a genome-wide enu mutation screen for mice with inherited deficits in specific helper cell subsets. these deficits were detected by multi-colour facs analysis of peripheral blood samples, and by antibody production following immunization with heat-killed b.pertussis and cgg coupled with the hapten arsonate (aba) in alum, which induce internally polarized th1 and th2 antibody responses, respectively. using this screen, a number of new mutant strains have been isolated with complete or partial loss of cd4+ t cells or functional deficits that selectively interfere with th1 or germinal centre responses. in this talk i will present data from some of the first 12 strains that have been identified including the first 5 strains where we have been able to identify the causative mutation. systematic genetic analysis of helper t cell differentiation in the resulting strains will illuminate how t cell help is correctly polarized for immunity and to avoid immunopathology. intrathymic t-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. in order to highlight the differences and similarities between fetal and adult t-cell development at the molecular level we performed a microarray study. labelled rna from facs purified fetal and adult dn1 c-kit high (etp), dn2 and dn3 thymocyte populations was hybridised to affymetrix mouse 430a-2.0 genechips. the resulting data were grouped into four distinct gene clusters: cluster i contained genes over-expressed throughout adult development and included a large proportion of transcription factors (85 out of 623 genes), illustrating a significantly different transcriptional program acting during adult differentiation. conversely, cluster ii consisted of genes that were over-expressed in fetal progenitors and included 64 signal transducers (out of 590 genes) such as acvr1, bmpr1, fzd7, chemokine receptors cx3cr1, cxcr6 and integrins a2, a4, a9, ae and av, pointing to a difference in microenvironments. genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster iii. amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. on the other hand, cluster iv was limited to genes that were homogeneously up-regulated during development. these included gata-3, tcf-1, notch-1, rag-1, rag-2 and pre-ta, which are indispensable for t cell development. interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the etp and dn2 stages, suggesting that the molecular program of t-cell development is more advanced in the early stages of fetal differentiation. discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult dn3 population, therefore implying a more committed state of fetal progenitors. finally, transcriptional signatures of each developmental stage were defined by "recursive feature elimination" with support vector machines. this approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. tcrgd+ cells and tcrab+cd8aa+ intraepithelial lymphocytes (iels) of the gut are unconventional t cells that reside in tissues and provide innate-like immune responses to "stressed-self". as these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional t cell developmental pathway based on tcr signal strength at the dn stage. the pre-t-alpha chain (pta) that pairs with tcrb to generate the pre-tcr, has two isoforms; pta a and pta b . both can form a functional pre-tcr with tcrb. ligand-independent signalling by the pre-tcr is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pta chain. pta b lacks 3 out of 4 of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. we have hypothesised that pta a and pta b permit differential signal strength through the pre-tcr at the dn stage, facilitating the divergence of the conventional and unconventional lineages of tcrab+ t cells. preliminary semi-quantitative pcr data suggest that pta a and pta b are differentially expressed in wt thymocytes at different stages of ontogeny. retroviral transduction of pta -/-e14 thymocytes with either pta a or pta b alone, followed by fetal thymic organ culture, confirmed the rescue of abt cell development by both isoforms. however the two isoforms appear to differentially regulate the kinetics of thymocyte development by 7-10 days of culture; pta b expression generates a greater percentage of tcrab+ cells while pta a expression results in the accumulation of isps. these results suggest different roles for the two isoforms of pta in the thymic development of abt cells. in order to determine the mechanism by which pta a and pta b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pta a and pta b that lack the "dimerisation residues" necessary for internalisation of the receptors. in addition, bac transgenic mice that express singly either pta a or pta b under the pta promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and t cells, and precursor of b cells can be detected and counted at day 18 of culture. if needed for further functional analyses, long-term proliferating lines and clones of progenitor t and b cells can be established at this point of "in vitro" development. hence the "in vitro" differentiation of es cells to different hematopoietic cell lineages and their progenitors can be quantified. it allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different es or ips cells. aim: increasing evidence includes wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems. although their precise functions in bone marrow and thymus are still controversial, numerous studies show that wnt signalling is able to control the proliferation of hsc and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. in the present work we analyse the effect of transient stimulation of canonical wnt pathway in the differentiation potential of lin -cd34 + cd1ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into t cells, nk cells, monocytes, conventional dendritic cells (cdc) and plasmacytoid dcs (pdcs). methods: human thymus samples from patients aged 1 month to 3 years undergoing corrective cardiac surgery were obtained and used according to the declaration of helsinki. transient b-catenin stabilization was triggered culturing purified thymic lin -cd34 + cd1precursors with recombinant wnt3a (100 ng/ml) or with licl (10 mm) for 12hr. active b-catenin, was detected by flow cytometry using anti-human active b-catenin mab (8e7) under conditions of phosphatase activity inhibition. wnt3a or licl pre-treated precursor were assayed in chimeric human-mouse ftoc, in il-15 and scf-supported cultures for generation of nk cells and in co-cultures with murine bone marrow stromal st2 cells suplemented with il-7 and flt3l. phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. expression profile of transcription factors was analysed by real-time quantitative rt-pcr . our results demonstrate that giving a boost to canonical wnt signalling triggered by transient exposure of thymic progenitors to wnt3a or licl, change their differentiation capacity enhancing nk cell production. on the contrary, wnt3a or licl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cdc, as well as reduce their capacity to differentiate into pdc lineage. as a possible mechanism for this effect we show that wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are il-7 and flt3l and modify the transcription factor profiles of cd34 + cd1thymocytes mainly increasing hes-1 and id3 expression levels. human th17 clones and circulating th17 cells showed lower susceptibility to the anti-proliferative effect of tgf-beta than th1 and th2 clones or circulating th1oriented t cells, respectively. accordingly, human th17 cells exhibited lower expression of clusterin, and higher bcl-2 expression and reduced apoptosis in the presence of tgf-beta, in comparison with th1 cells. umbilical cord blood naï ve cd161(+)cd4(+) t cells, which contain the precursors of human th17 cells, differentiated into il-17a-producing cells only in response to il-1beta plus il-23, even in serum-free cultures. tgf-beta had no effect on constitutive rorgamma t expression by umbilical cord blood cd161(+) t cells but it increased the relative proportions of cd161(+) t cells differentiating into th17 cells in response to il-1beta plus il-23, whereas under the same conditions it inhibited both t-bet expression and th1 development. these data suggest that tgf-beta is not critical for the differentiation of human th17 cells, but indirectly favors their expansion because th17 cells are poorly susceptible to its suppressive effects. m. irla 1 , w. reith 1 1 university of medecine, pathology and immunology, geneva, switzerland objectives: medullary thymic epithelial cells (mtecs) are specialized for inducing central immunological tolerance to self-antigens. to accomplish this, mtecs must adopt a mature phenotype characterized by expression of the autoimmune regulator aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. the mechanisms that control mature aire(+) mtec development in the postnatal thymus remain poorly understood. however, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and tec control not only t cell development but also the differentiation and organization of tec, a phenomenon designated as 'crosstalk'. the aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mtec, key mediators of central tolerance. we have demonstrated by generating different transgenic mouse models, that although either cd4(+) or cd8(+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mtec population requires autoantigen-specific interactions between positively selected cd4(+) thymocytes bearing autoreactive t cell receptor (tcr) and mtecs displaying cognate self-peptide-mhc class ii complexes. these interactions also involve the engagement of cd40 on mtecs by cd40l induced on the positively selected cd4(+) thymocytes. conclusion: this antigen-specific tcr-mhc class ii-mediated crosstalk between cd4(+) thymocytes and mtecs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mtec population competent for ensuring central t cell tolerance. q. qiu 1 , i. ravens 1 , g. bernhardt 1 1 hannover medical school, institute of immunology, hannover, germany cd155 is originally identified as human poliovirus receptor (pvr) and as rodent tage4, which is overexpressed in rodent colon carcinoma. cd155 is also known as necl-5, a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. cd155 expression is very low in most adult organs, but is abundant in the developing or regenerating liver. in addition, cd155 is overexpressed in transformed cells and promotes the cell cycle. thus, cd155 seems to be an oncofetal protein that functions in embryonic development and cancer progression. t-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (dn), double positive (dp) and single positive (sp) based on expression of the co-receptors cd4 and cd8; the dn subset is further subdivided into four stages (dn1-4) by differential expression of cd44 and cd25. thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. during their final maturation in the medulla, semi-mature sp thymocytes down-regulate activation markers and subsequently exit into periphery. while semimature cd4+ sp are sensitive to negative selection, it remains elusive when negative selection occurs in the cd8 lineage. here we show that the frequency of terminally matured cd8+ sp cells but not that of cd4+ sp present in thymus varies depending on age. in mice lacking expression of the adhesion receptor cd155, a selective deficiency of mature cd8+ sp thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. evidence is provided that the mature cells emigrate prematurely when cd155 is absent thus cutting short their retention time in the medulla. moreover, in unmanipulated wild type mice semi-mature cd8+ sp thymocytes are subjected to negative selection as reflected by the diverging t cell receptor repertoires present on semi-mature and mature cd8+ t cells. in cd155 deficient animals, a shift in the tcr repertoire displayed by the pool of cd8+ sp cells was found demonstrating that cd155 is involved in negative selection. in the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. the analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (passegué et al. j. exp. med. 2005 , 202, 1599 . during development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. by using complementary, quantitative analyses (brdu labelings, hoescht 33342, propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from e9 (e, gestational day) on, in yolk sac, splanchnopleura/agm, blood, liver, spleen and bone marrow. we have observed that around half of cd71 + ter119 + erythroid and cd45 + cd11b + myeloid cells are simultaneously cycling (s/g2/m) in the post-gastrulation mouse embryo (e10-12). the peak of lymphohematopoietic cell proliferation occurs at e13 in a sort of wave-like pattern. these high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. interestingly, nucleated erythroid cells suddenly go into quiescence 24-48 hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. we are also analysing the proliferation state of the first b and t lymphoid progenitors emerging at e11-12 that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. we attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. objectives: the role of cd40-cd154 interactions in t cell activation of antigen presenting cells and b cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. following an initial discovery that b lineage cells in the bone marrow (bm) as early as pro-b cells express cd40, we hypothesised a role for cd40-cd154 interactions in the control of b cell haematopoiesis. the objectives of this study were to investigate this hypothesis further. methods: flow cytometry was used to investigate cd40 expression by precursor b cells using b cell specific markers. reverse transcription of bm stromal cell rna and pcr were used to assess the presence of cd154 message and cell lineage specific mrna. irradiation and bone marrow transplantation (bmt) in both directions between cd154-/-and wt mice was used to assess potential functional contributions of stromal or haematopoietic cd154 on reconstitution of b cell numbers following depletion. we show that cd40 is expressed by pro-b cells, and these cells proliferate in response to cd40 signalling in vitro. pcr identified a source of cd154, negative for cd3eta, in the bm of wt mice showing this cd154 is not provided by activated re-circulating t cells. we have shown that when cd154 -/-mice are recipients, but not donors of bmt, b cell recovery after irradiation is significantly delayed regardless of the donor cell source. in the in vitro experiments we found that the pta gene is expressed from the dn1 (cd4 -/cd8 -/cd44 + /cd25 -) to the dp (cd4 + /cd8 + ) stage, whereas no yfp expression could be observed in the b lineage. the in vivo analysis of thymocytes confirms the appearance of yfp positive cells during t cell development from the dn1 stage on. in the bone marrow we found yfp + /b220 + and yfp + /b220populations. thus these pta expression analyses show closely similar pattern to those observed with hucd25 preta-reporter transgenic mice (gounari f. et al. 2002 , martin et al. 2003 . the bac pta reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis. gounari f et al., nat. immunol. 3, 489-496 (2002 ) martin c. h. et al., nat. immunol. 4, 866-873 (2003 . the individual functions and the reason for the tightly regulated expression of igm and igd during b cell development are poorly understood. our data show, that igd requires stronger stimuli than igm to induce b cell activation and that this silences autoreactive vdj recombination products when expressed as igd. in agreement with this, mhc and dhc, the respective heavy chains of igm and igd, differ dramatically in pre-bcr signaling, which represents the prototype of an autoreactive receptor. together with published data, our results reveal a novel role for igd and suggest that the differential expression of igm and igd is important to raise the activation threshold of mature b cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens. p. d. rymkiewicz 1 , g. klein 1 1 zmf (center for medical research), section for transplantation immunology and immunohematology, tübingen, germany thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. the core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type iv, nidogens and perlecan. a marker molecule for the conduits in the human thymus is the laminin isoform lm-332 which is synthesized by the medullary thymic epithelial cells (tecs) which tightly surround the conduits. functionally the conduits are too small to transport cells but they are able to transport small molecules x 70kda.mmp-19, a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting lm-332. in the human thymus medullary, but not cortical thymic epithelial cells strongly express mmp-19. by western blotting the zymogen and the activated form of mmp-19 can be detected in whole thymus lysates, whereas in lysates from isolated tecs mainly the activated form is present. an in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. using confocal laser scanning microscopy double immunofluorescence staining showed that lm-332 and mmp-19 can be found in close neighbourhood, but they do not exactly co-localize. why activated mmp-19 which can be secreted by medullary tecs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. two natural inhibitors of mmp-19, timp-2 and timp-3, are found in the thymic medulla, but they are not expressed and secreted by tecs. whether mmp-19 plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. to study the process of t cells differentiation in more detail, we intend to establish an inducible gene expression system (tet-on system) in primary t cells. the tet-on system comprises two retroviral vectors. the response vector contains an inducible modified minimal cmv promoter which per se is unable to induce expression of the gene of interest (goi). the second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. in this state, the transactivator enables the minimal cmv promoter to transcribe the gene of interest. therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest. to date we have tested two tet-on systems (revtet system and retro-x tet-on advanced inducible expression system) that differ in the sequence of their inducible promoters. to monitor successful transfection in retrovirus-generating phoenix cells and transduction in t cells, respectively, we have cloned the reporter gene gfp under the control of a constitutive cmv promoter, into the response vector of the revtet system. this allowed identification of transduced gfp-positive cells via facs. however, when we used a red fluorescent protein, tomato, as a surrogate goi, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. in contrast, we found comparably low leakiness when using the retro-x tet-on advanced inducible expression system. here, co-transfection of phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of 15-20 % compared to only 5 % basal rate. therefore, the retro-x tet-on advanced inducible expression system appears suitable for our studies. future experiments will aim at establishing this system in primary t cells. although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (gcs) on t-cell development, and thereby t-cell-mediated immune response, their relevance for these processes is still far from being understood. the study was undertaken to explore relevance of basal levels of gcs for t-cell differentiation/maturation. eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by tcrab/cd4/cd8 expression were examined using flow cytometry analysis. it was found that adrenal gc deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of t-cell differentiation/maturation leading to an overrepresentation of the cd4+cd8+ double positive (dp) tcrab low cells entering selection, and their cd4+cd8+ dp tcrab-immediate precedents followed by underrepresentation of the selected cd4+cd8+ dp tcrab high and the most mature cd4-cd8+ and, particularly, cd4+cd8-single positive (sp) tcrab high cells. the study suggests that withdrawal of adrenal gcs produces alteration in thymocyte selection processes that may affect diversity of functional t-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of cd4-cd8-double negative tcrab high cells. in addition, it indicates that gcs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature cd4+cd8-/cd4-cd8+ sp tcrab high cell ratio. in the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mtecs) -express a highly diverse set of tissue-restricted antigens (tras) representing essentially all tissues of the body, which is known as promiscuous gene expression (pge). this allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing t cells. the scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. thus, even lack of a single tra can result in spontaneous organ-specific autoimmunity. promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for tras. for better understanding these features, we set out to precisely define the genomic organization of this gene pool. in particular, we probed to what extent and according to which rules predefined genomic clusters of tras are transcribed in mtecs. our analysis proceeded from the bioinformatic definition of tra clusters via gene expression analysis in mtecs using whole genome arrays to the in depth analysis of selected tra clusters by rt-pcr at the population and single cell level. patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pge and adaptive immunity. although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (ar) blockade failed to show any significant effects on thymic cellularity. bearing that in mind, the present study was undertaken to explore: i) a 1 -ar expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a 1 -ar-mediated mechanisms in modulation of thymic cellularity and t-cell development. for this purpose a 1 -ar expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (cd4/ cd8/tcrab), in adult wistar rats subjected to 14-day-long treatment with a 1 -ar blocker urapidil (0.20mg/kg body weight/day s. c.). the a 1 -ar immunoreactivity was found in both thymocytes (mainly less mature cd3and cd3 low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ed1-postive cells, which comprise macrophages and dendritic cells). chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. the thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. in addition, in these rats changes in distribution of major thymocyte subsets delineated by cd4/ cd8/tcrab expression were observed. these changes comprised of an increase in the percentage of cd4+8+ tcrabthymocytes, which was accompanied by the reduction in that of cd4+8+ tcrab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive tcrab high thymocytes. compared with saline-administered controls, the percentage of cd4+8-tcrab high thymocytes in urapidil-administered rats was increased, while that of the cd4-8+ tcrab high was reduced. in addition, the percentage of cd4+ t regulatory and cd161+tcrab+ nkt cells was increased. collectively, this study clearly showed the expression of a 1 -ar on both lymphoid and nonlymphoid thymic cells, and indicated that a 1 -ar-mediated mechanisms may be implicated in modulation of multiple steps of t-cell development. we have recently shown that the thymus is a common target for mycobacterial infections. of notice, while bacterial growth is arrested in the spleen around 4 weeks post infection with mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. this observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. since t cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. methods: c57bl/6 mice infected with m. avium (10 6 cfus, iv) were sacrificed at different time points after infection (5, 16 and 25 weeks). non-infected animals were used as controls. bacterial load was assessed in the spleen and thymus and cytokines (such as ifn-g, tnf, il-10) were quantified by rt-pcr in tissues (normalized for hprt expression) or by elisa in the supernatants of cultured thymocytes and splenocytes. statistical significances were determined by anova. we observe increased levels of ifn-g in infected thymi at 16 weeks post infection. this increased expression of ifn-g is concordant with the late bacterial growth arrest within the thymus. at 24 weeks post infection this difference is still present. throughout the course of infection no significant differences are found in the expression of tnf and il-10 in this organ. in the spleen, ifn-g reaches a peak of expression earlier (5 weeks post infection) and this is accompanied by increased tnf expression. conclusion: our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. since the cytokine milieu influences t cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. such implications should next be investigated. precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening dna, but how trans-acting regulatory proteins work to establish and maintain dna loops during gene activation remains largely unexplored. lps-induced transcription of the mouse igx gene in b lymphocytes utilizes three distal enhancers and requires the transcription factor nf-xb, whose family members include rela and c-rel. using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that lps-induced igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and rna polymerase ii, the apparent molecular tie for the bases of these loops. rela and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-rel. we have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during igx gene activation. this investigation was supported by grants gm29935 and ai067906 from nih and grant i-823 from the robert a. welch foundation to wtg, and by grants hl067256 and hl61897 from nih to lst. allelic exclusion of immunoglobulin (ig) genes supports burnet's clonal selection theory. the recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the rag complex. attempts to prove the probabilistic or epigenetic nature of monoallelic v(d)j recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or x-chromosome inactive genes, ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (mostoslavsky, singh et al. 2001) . we are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each b cell progenitor, which of the ig alleles is going to be activated for rearrangement and expression. for this, we are generating b cell clones from b cell progenitors of (c57bl/6 x balb/c)f1 mice, because the original strains have ig heavy chain (igh) alotypes distinguishable by monoclonal antibodies. we are analyzing if individual heterozygous clones show biased expression of a particular igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. we have already shown, for the igh gene, a clear segregation of monoallelic expressors among b cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell b cell clones. this result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. we are currently exploring this result by the analysis of the igh silenced allele rearrangement status in sorted fractions of igm+ b cell clones. we are also testing the same epigenetic hypothesis for the ig kappa light chain gene (igk), using f1 mice in which the igk constant region from both alleles can be distinguished by antibodies. a. giniewski 1 , s. lang 1 , m. stein 1 , t. winkler 1 1 friedrich-alexander university, erlangen, germany vdj recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to rag recombinase. accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. h3ac) or methylation of h3 on lysine 4 (h3k4me2/3). previous studies in our lab revealed three regions in the intergenic part of the distal v h cluster (ivars), which are associated with high levels of active chromatin marks (h3ac and h3k4me2/3) only in pro b cells but not in pro t cells. it is also known that vdj h recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. since one ivar (ivar #3) shows promoter activity in antisense orientation, it was analysed in more detail. we found three transcription start sites by 5'rlm race at the ivar #3 element. background: t-all is a malignancy of the lymphoblast committed to the t-cell lineage with translocations between tcr genes and oncogenes as a genetic hallmark. these translocations are thought to be driven by v(d)j-recombination mechanisms. we believe that these mechanisms only partly facilitate the occurrence of tcr translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. the lmo2 locus, is thought to be accessible only during the double-negative (dn) 1 and 2 thymocyte stages based on mrna expression, implying that translocations between lmo2 and tcr genes can only occur within these stages. gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as tlx1 (hox11), which are not expressed in thymocytes, as being accessible for translocations to occur. objectives: we aimed 1) to evaluate lmo2 and tlx1 breakpoint-site accessibility during thymocyte development; 2) to determine in which stage of development there is an increased chance for lmo2 or tlx1 translocation to occur based on this accessibility. methods: dna of immunologically "healthy" sorted thymocytes was isolated using faire (formaldehyde-assisted isolation of regulatory-elements). this dna was used to quantitatively assess lmo2 accessibility during thymocyte development at both the transcription start site (tss) and negative regulatory element (nre), and within different in t-all documented breakpoint-sites of both the lmo2 and tlx1 loci. results: quantitative analysis on the tss showed a correlation with mrna expression, with the dn1 and dn2 development stages showing the highest accessibility. the nre, showed an inverse pattern of accessibility to the tss region. analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, dn1, dn2, dn3 and immature single positive (isp) stages for both lmo2 and tlx1. conclusion: our findings show that both the lmo2 and tlx1 loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to dn1 and dn2 stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. we addressed this issue, by building a model able to account for of v-ja gene rearrangements observed experimentally during thymus development of mice. we developed, based on experimental data, a numerical model on the whole tra/trd locus to estimate va and ja genes accessibility to rearrangements. the progressive opening of locus to v-j gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. furthermore, the possibility of successive secondary v-j rearrangements was introduced in the modeling. the model points out some unbalanced v-j associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the j genes, depending on their location in the locus. the model shows that 1 to 3 successive rearrangements are sufficient to explain the use of all the v and the j genes of the locus. finally, the model provides information on the kinetics of rearrangements and on the frequency of each v-j association. the model accounts for the essential features of the observed rearrangements on the tra/trd locus and may provide a reference for the repertoire of the v-j combinatorial diversity. the genetic programs of b-cell differentiation and the first dj h gene rearrangements appear in the post-gastrulation mouse embryo (e10-12), shortly after the first multipotential hematopoietic progenitors do emerge. these dj h joints represent the unselected baseline of the ig repertoires. we have undergone a systematic sequencing of embryo dj h joints obtained from normal balb/c embryos and heterozygous embryos obtained from rag2 -/mothers mated to balb/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. the embryo dj h s displayed unexpected mechanisms of diversity, including short stretches of non-templated n nucleotides in one-third of the studied sequences (in the absence of tdt expression) and frequent dj h s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. because the dna polymerase m (polm), a highly-homologous tdt member of the x dna polymerase family, showed an increased expression in the embryo, we analysed the dj h s of polm -/mouse embryos. we observed that polm was mainly responsible for introducing n nucleotides at the mouse embryo dj h joints. also, and based on its dna-dependent polymerization ability, polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. we think that, more than attempting to increase diversity, polm acts as a "connector" in the embryo, subsequently participating in the repair of rag-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. along the end of gestation, further selective pressures acting over these first v-dj h products will contribute to establish the differential neonatal ig repertoires. although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. most children with infantile bronchiolitis are under 6 months of age, and most cases are due to respiratory syncytial virus (rsv) infection, for which vaccines continue to be elusive. we now show that, compared to adults, the antibody response to rsv infection is very poor in neonatal mice and is unaffected by cd4 cell depletion. however, cd8 depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. to test the possibility that poor antibody boosting is caused by rsv-specific cd8 t cells killing rsv-infected b cells, we sorted cells from the lungs of infected neonates. viral copy number was high in neonatal b cells, but viral load in surviving b cells was unaffected by cd8 cell depletion. in addition, fas ligand (fasl) deficient gld mice responded to rsv infection in the same way as normal mice, indicating that fasl is not required for the inhibition of antibody responses. this new mechanism of regulation of b cell responses by cd8 t cells has important implications for vaccine development against neonatal infections. (2008) showed that irf8 knockout mice had significantly reduced numbers of pre-pro-b cells in marrow and a phenotype similar to agammaglobulinemia. these results prompted us to consider icsbp/irf8 as a candidate gene in the pathogenesis of defective early b cell development. therefore we decide to undertake direct sequencing of the gene encoding icsbp/irf8 in a small cohort of patients with autosomal recessive agammaglobulinemia. methods: eight patients affected by agammaglobulinemia were included in this study. all patients were under regular ig replacement therapy. informed consent was obtained from all patients. genomic dna was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. direct gene sequencing of the eight exons of icsbp/irf8 were obtained using abi prism sequencer. results: seven of the eight patients result wild type while only one patients present a synonymous snp in exon v, yet documented as rs17444416. conclusions: although recent findings indicated that irf8 function is essential for early b cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in icsbp/irf8 that may be responsible for this disorder. the hh/ptch signaling system is known to control the development and neoplastic transformation of several cell types. however, the role of hh/ptch for the differentiation of b and t lymphocytes from hematopoietic stem cells (hsc) has not been assessed so far. to analyze the function of hh/ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the ptch gene can be conditionally inactivated by virtue of the cre/loxp recombination system. we show that targeted disruption of ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated b and t lymphocytes from peripheral lymphoid organs. the developmental block occurs at the level of the common lymphoid progenitor cell (clp), which defines an early branching point of hsc differentiation and lineage commitment. in contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (cmp) appears normal. our data identify hh/ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. hence, the progression of tumors, which are initiated upon oncogenic hh/ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system. l. calderon dominguez 1 , t. boehm 1 1 max-planck institute for immunbiology, developmental immunology, freiburg, germany in many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. the ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. by analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. we demonstrate how an epithelial niche, rendered functionally inactive by disruption of the foxn1 transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the b and t cell lineages, respectively. the strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. silencing of dc-sign using lentiviral rna interference revealed its critical function for pd-l1 expression on dcs after m. tuberculosis infection. as a counterpart to expression of its ligand, we showed that cd4 and cd8 t cells from tuberculosis patients highly express pd-1 when compared to healthy uninfected individuals. in addition, analysis of pd-1 expression in lung biopsies from tuberculosis patients revealed that pd-1 is expressed on cd4 and cd8 t cells confined to lung granulomatous lesions. finally, blocking of the pd-1/pd-l1 axis using monoclonal antibodies abrogated the down-modulation of t cell proliferation and ifn-g production induced by manlam, a mycobacterial cell wall glycolipid and ligand for dc-sign. taken together, our results suggest that the pd-1/pd-l1 pathway is involved in the exhaustion of t cell responses to m. tuberculosis. the inflammatory canonical nfkb pathway is critically involved in virtually all aspects of inflammation in general. yet, the role of the alternative, non-canonical nfkb pathway in inflammation and adaptive immunity remains largely elusive. the alternative pathway is primarily mediated through the nfkappa-b inducing kinase (nik) which in turn leads to the phosphorylation and the cleavage of p100 to p52. among the receptors engaging nik is the ltbr, which is also required to form the anlage for secondary lympoid tissues (slts). due to a point mutation within nik, alymphoplasia (aly) mice do not develop slts and are highly immunodeficient. however, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of nik itself could potentially lesion the development of immune responses. to verify this notion, we generated a series of bone marrow chimeric mice (bmc) in which the absence of slts was disconnected from the hematopoietic loss of nik function. we generated mice, which lack all slts, but are equipped with a normal systemic immune system (wt 1 aly), and conversely, mice with normal slts, but lacking nik in all leukocytes (aly 1 wt). surprisingly, we discovered that nik is vital for the development of autoimmune disease, while slts (ie. lns, spleen etc.) are essentially dispensable for cell-mediated immunity. we found that nik is required for the polarization of effector t cells and that th17 and th1 cells cannot be generated in the absence of nik. preliminary data implicate the involvement of nik in a discrete and novel pathway required for the formation of cell-mediated immune responses. the family of nfat (nuclear factor of activated t-cells) transcription factors is indispensable for t cells, for example playing an important role in cytokine gene regulation. in peripheral cd4 + t cells, nfatc1 and c2 are predominantly expressed. nfatc1 is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. here we address the functional difference of the short isoform nfatc1/a, which is highly induced upon t cell activation, and the long constitutively expressed isoform nfatc1/c. as demonstrated by y2h screen and co-ips, nfatc1/c-specific c-terminus can be highly sumoylated. confocal microscopy studies revealed that upon sumoylation nfatc1/c -but not the unsumoylated nfatc1/a -translocates to promyelocytic leukemia-nuclear bodies (pml-nbs). this leads to interaction with hdacs followed by deacetylation of histones (co-ips), which in turn induces transcriptionally inactive chromatin (chip and confocal microscopy). as a consequence, multiple expression studies revealed sumoylation dependent suppression of the nfatc1 target gene interleukin-2. other lymphokines like ifng and il13 are reversely regulated. interestingly, ntreg cells which do not express il2 exerted only nfatc1/c, but no nfatc1/a expression (qrt-pcr). these findings demonstrate that the modification by sumo converts nfatc1 from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for nfatc1 function. therefore, especially ntreg cells and anergized cd4 + t cells might be regulated by the long sumoylatable isoform nfatc1/c. lnk/sh2b3 and aps/sh2b2, two members of the lnk/sh2b family of adaptor proteins, play an important role as negative regulators in b cell lymphopoiesis. they possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. mice deficient for these proteins demonstrated that lnk inhibits expansion of pro/pre-b cells while aps controls mature b-1 cell population, suggesting specific roles for these adaptors during b cell development. however, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. to address this question, we used primary and b cell lines at different stages of differentiation as our cellular system. analysis of lnk/aps expression pattern showed that lnk is expressed at all developmental stages, while aps is only detected in immature and mature cells. we then first examined the role of lnk in il-7 signalling in pre-b cells overexpression of lnk dramatically inhibits il-7-dependent growth demostrating that lnk negatively regulates il-7 pathways. furthermore, we showed that il-7 stimulation induces lnk phosphorylation and its subsequent association with important signalling effectors, notably the e3 ubiquitin ligase cbl. we next analyzed the role of aps in mature b cells by imaging and biochemical techniques. our results showed that aps colocalizes with the bcr complex after bcr triggering. interestingly, lnk is not recruted to the bcr signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. moreover, we showed, for the first time, that aps can associate, upon bcr stimulation, with the signalling molecules cbl and vav1. to address the functional implications of these interactions, we examined specific b cell responses, notably bcr trafficking and cytoskeleton remodelling. we demonstrated that overexpression of aps enhances ligand-induced endocytosis of bcr, possibly through interaction with cbl and affects the kinetics of bcr-induced cell spreading. our results therefore suggest a regulatory function of aps in bcr internalization and cytoskeleton dynamics. altogether, our findings demonstrate that lnk and aps display sequential specific regulatory roles during b cell development that are important for maintaining b cell homeostasis. signaling through the t-cell receptor (tcr)-cd3 complex is a critical event in adaptive immunity. it is still not clear how ligand binding to the tcr is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the cd3 complex. it is widely accepted that dimerization or multimerization of tcr is required for tcr triggering. in our model t-cell activation is initiated by recognition of monomeric mhc/peptide complexes on the surface of antigen presenting cells (apc). critical to tcr triggering is the movement of the t-cell across the apc. engagement of a mhc/peptide complex on the surface of the apc will change the mobility of the tcr leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for t-cell activation. furthermore, the change in mobility will lead to dislocation of the itams from the plasma membrane so that they become accessible to tyrosine kinases. to test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pmhc and a fab that recognizes an epitope tag that we express on the t-cell surface. binding of the fab to the expressed epitope tag will constrain the lateral mobility of the tcr that is engaged by the pmhc arm of the same complex. the bifunctional complexes induced activation and proliferation as well as ca influx and cytokine production in human cd4 + t-cell clones that displayed the epitope, but not in t-cells that did not display the epitope. activation required interaction of the fab with its epitope on the t-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the fab binding site, was added. these results demonstrate that a monomeric copy of a pmhc is sufficient to trigger tcr and that formation of a tcr dimer is not an obligatory step in t-cell activation. the bifunctional complex we generated may also have a great immunotherapeutic impact. exchanging the fab with a fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of t-cell responses. adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. adaptor proteins containing characteristic sh2 or sh3 domains known to mediate protein-protein interactions are key players in these processes. sly2 (sh3 domain protein expressed in lymphocytes 2) was identified as a putative adaptor protein containing a sh3 and a sam domain as well as a bipartite nls. sly2 belongs to a family of three molecules: sly1, sly2 and sash1.in humans, the sly2 gene is located on chromosome 21, in mice on chromosome 16. sly2 is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. subcellular fractionation showed that the sly2 protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.to elucidate the function of sly2 we searched for possible interaction partners by yeast two hybrid screening with a mouse t cell lymphoma library. this approach identified sin3-associated polypeptide p30 (sap30) as a putative interaction partner of sly2. sap30 is a conserved member of the sin3a-hdac corepressor complex that contains histone deacetylase 1 (hdac1) and histone deacetylase 2 (hdac2) and acts as a transcriptional repressor for a variety of genes. we confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected 293t cells. in addition, we could show a direct interaction between sly2 and hdac1. to investigate the functional impact of this molecular interaction, we performed hdac enzymatic activity assays. we were able to show that sly2 increases the activity of hdac1 in whole cell lysates and, more precisely, in nuclear extracts of 293t cells. the interaction of sly2 with sap30 and hdac1 indicates a transcriptional function of this protein. within the sin3a-hdac corepressor complex sly2 might act as a switch for the activity of hdac1. cd46-cyt1 and cyt2 are co-expressed in human t cells and undistinguishable from the cell surface. in order to determine their specific role in t cell activation, we have expressed chimeric proteins consisting of the extracellular domains of cd19 (b cell marker) fused to the transmembrane and intracellular domain of cd46-cyt1 or cyt2 in primary t cells. we show that these two isoforms differently control human t cell function. specific cyt1 coengagement controlled il-10 secretion, while cyt2 coligation inhibited ifng production. moreover, our preliminary data suggest that cd46-cyt2 inhibits the phosphorylation of several molecules known to be activated by cd46 stimulation. these data suggest that these two isoforms act as molecular switches for t cell activation, either promoting or turning off t cells. they demonstrate for the first time the distinct roles of cd46 cytoplasmic isoforms in primary human t cell activation. this also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. nck is a ubiquitously expressed adapter protein that is almost exclusively built of one sh2 domain and three sh3 domains. nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. in t cells, nck participates in different and interdependent signalling pathways linking t cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. we previously showed that nck directs the death factor fasl to the cytotoxic immunological synapse when t cells encounter putative target cells. we now performed a systematic screening for interaction partners of the four individual interaction modules of nck in primary and leukemic t cells. we precipitated putative binding partners from untreated or pervanadate-treated pha blasts, jurkat and hut78 cells with gst fusion proteins containing full length nck, the three sh3 domains or the individual sh3 and sh2 domains. binding proteins were excised from gels after staining with coomassie, silver or flamingo pink and processed by tryptic in gel digestion for mass spectrometrical analysis. as expected, we observed major differences in nck binding proteins precipitated from resting versus activated t cells. we not only verified established interactions (e. g. with the tcr signalling components slp76 and cd3epsilon, the actin-regulatory proteins wasp and wip and the nuclear protein sam68) but also identified novel nck-interacting proteins. the interaction with the actin-binding protein hip55 once more underscores the fundamental role of nck in tcr-mediated actinreorganization. the identification of the nuclear proteins sfpq/nono points to novel, yet unknown functions of nck that might be associated with the recently reported nuclear translocation/localization of nck. accordingly, employing laser scanning microscopy, we clearly detected nck within the nucleus also in human t cells. the present data highlight that nck serves versatile functions in t cells, which include the different interdependent pathways of tcr-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of nck. cytotoxic t lymphocytes (ctl) mediated killing is tightly regulated according to the strength of t cell receptor signal. killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (csmac) within the immunological synapse. although much has been learnt about the mechanisms controlling the strength of tcr signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the tcr is able to control the degree of ctl-mediated killing so finely. here we examine how the strength of tcr signal controls polarization of the secretory apparatus leading to ctl-mediated killing using tcr transgenic ot-i ctl. decreasing the tcr signal by reducing the concentration of ova peptide or using the weak agonist peptide, g4, results in a slight reduction in the number of ctl target cell conjugates formed, and the number of conjugates in which a csmac (visualized by a patch of lck-staining at the immunological synapse) was formed. tcr signals result in reduced or absent (in the case of g4) staining with psrc and perk antibodies in the immunological synapse and reduced or absent (g4) degranulation as measured by cd107a assays. the centrosome docks at the csmac of the immunological synapse even with relatively weak tcr signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. inhibitors support a role for pi3k in granule polarization. together these data demonstrate that the strength of tcr signal controls the level of ctl mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the csmac and the accumulation of psrc and perk at the synapse. the centrosome polarizes to the csmac even with relatively weak tcr signals, but granule recruitment requires a higher threshold of signaling. these findings reveal how ctl can fine tune the degree of killing in response to tcr signals at the single cell level. cytotoxic t cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. cytolytic t-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. they are capable of undergoing regulated secretion in response to a t cell receptor engagement which involves binding to a cognate mhc class i-peptide complex. the intracellular transport of lysosomal proteins from the golgi to the lysosomes is mediated by the cationindependent mannose-6-phosphate receptor which exhibits structural and functional similarity to the vps10p-receptor sortilin. sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. in the secretory pathway, sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. to explore whether sortilin has a broader functional relevance, we asked if sortilin might act as an alternative receptor for the cation-independent mannose-6-phosphate receptor in cytotoxic t cells. first, we demonstrate that sortilin is expressed in t cells. to examine its function during an adaptive immune response, we analysed sortilin-deficient cytotoxic t cells derived from a knockout mouse strain. in strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in sortilin-deficient cytotoxic t cells. whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of interferon-g was found to be decreased. the enhanced release of cytotoxic molecules from sortilin-deficient cytotoxic t cells translated into an increased cytotoxicity in vitro. thus, the deletion of sortilin imposed a specific phenotype in cytotoxic t cells which could not be compensated for by other sorting receptors. our localisation studies of sortilin in t cells were consistent with the results previously described in neuronal cells which indicated that sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the trans-golgi-network to endosomes and lysosomes. taken together, we suggest that sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. there is growing interest in the soluble splice variant of ctla-4 (sctla-4) as an immune inhibitor secreted by t cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. however, little is known of the biology of sctla-4 in immune responses. using a specific anti-human soluble ctla-4 monoclonal antibody, jmw-3b3 that selectively binds the soluble isoform but not membrane bound ctla-4, or cleaved fragments of it, we demonstrate that sctla-4 plays a vital role in regulating antigen-specific immune responses. we used antibody blockade to show that antigen-specific t cell responses are strongly enhanced upon blockade of sctla-4, secreting increased amounts of cytokines including interferon-g, il-17 and tnf-a, but lower amounts of il-10. soluble ctla-4 was also prepared from sera for use in experiments by antibody based affinity purification techniques. addition of sctla-4 induced secretion of the immunoregulatory cytokine il-10 by human pbmcs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. the immunosuppressive indoleamine 2,3 dioxygenase enzyme cascade was also initiated by sctla-4. it is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound ctla-4 it is crucial to t cell inhibition. membrane-bound ctla-4 exists as a homo-dimer on t cells but sctla-4 is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link b7 ligands on antigen presenting cells. a third important observation from this study is that sctla-4 exists both in serum and culture supernatants as a natural 46kda homo-dimer, and not as a monomer. this goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. together, these results lead us to reappraise sctla-4, concluding it to be a mediator of negative feedback, secreted as a recall regulatory t cell response to antigenic stimulus, rather than a product of resting t cells. this work also raises the possibility that where il-10 dependent regulation is most critical, boosting sctla-4 secretion by regulatory t cells could be a novel therapy for immune mediated diseases. recently, we identified a new adaptor protein, swiprosin-1/efhd-2, in lipid rafts of b cell lines that undergo apoptosis after b cell receptor (bcr) stimulation. swiprosin-1/efhd2 is expressed in immature b cells of the bone marrow, in resting and activated splenic b cells, in t cells, macrophages, mast cells and some nonlymphoid tissues. ectopic expression of swiprosin-1/efhd-2 in the immature murine b cell line wehi231 enhanced spontaneous and bcr-induced apoptosis. in contrast, shrna-mediated down-regulation of swiprosin-1/efhd-2 impaired spontaneous and bcr-elicited apoptosis, but not bcr-induced g1 cell cycle arrest. to understand how swiprosin-1/efhd2 enhances pro-apoptotic bcr signals, we analyzed whether swiprosin-1/efhd2 is involved in proximal bcr signalling. in fact, ectopic expression of swiprosin-1/efhd2 enhanced bcr-induced calcium flux in wehi231 cells, whereas shrna-mediated down-regulation of swiprosin-1/ efhd2 impaired bcr-elicited calcium signals. concomitantly, gst-pulldown experiments revealed that swiprosin-1/efhd2 interacts with phospholipase cg2 (plcg2) and with the tyrosine kinase syk (splenic tyrosine kinase), both of which are important for bcr-induced calcium flux. the interaction of plcg2 and swiprosin-1/efhd2 was further established by co-immunoprecipitation. reconstitution of bcr-elicited calcium signals through complementation of swiprosin-1/efhd2 silenced wehi231 cells with swiprosin-1/efhd-2 was inhibited by the syk inhibitor bay 61-3606. in analogy, swiprosin-1/efhd2 regulated syk activity positively. moreover, swiprosin-1/efhd2 re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of plcg2 and of syk tyrosine residue 352, which is involved in syk activation. finally, reconstitution of swiprosin-1/efhd2 knock-down cells with swiprosin-1/efhd2 mutants revealed that the n-terminal putative sh3-binding site, the first ef-hand, and to a lesser extent, the second ef-hand and the c-terminal coiled-coil domain, are important for bcr-induced calcium flux in wehi231 cells. interestingly, swiprosin-1/efhd2 re-expression in swiprosin-1/efhd2-silenced cells induced already in unstimulated cells raft partitioning of syk, plcg2 and the bcr, which was reversed after 2 min of bcr stimulation. in summary, swiprosin-1/efhd2 is an accelerator of proximal bcr signalling and acts through syk and plcg2 by assembling a syk-dependent calcium initiation complex in lipid rafts. this might be relevant for memory b cell signalling or central b cell tolerance. to test the biological relevance of cbl-b e3 ligase activity, these mice were analyzed for t cell proliferation, susceptibility to autoimmunity, in vivo t cell tolerance responses, and tc1 tumor rejection. results: when stimulated, t cells from rf mutant mice hyperproliferate compared to wild type t cells, even in the absence of cd28 co-stimulation. preliminary data also suggest that rf mutant mice are more susceptible to autoimmunity. in addition, rf/p14 mice die within hours after a second challenge with p33 peptide, indicating a severe defect in t cell tolerance induction. more importantly, cbl-b e3 ligase dead mice can spontaneously reject tc1 tumors. conclusion: cbl-b e3 ligase dead mutant mice phenocopy total body cbl-b knock out mice, thus indicating that cbl-b e3 ligase activity is indispensable for its regulatory in vivo functions. intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. to further elucidate the cellular mechanism of cbl-b mediated tumor rejection we have now generated the conditional cbl-b e3 ligase dead mutant mice to for the first time study the cbl-b ubiquitination function in a tissue specific and temporal fashion. our research is also currently focused on identifying the relevant in vivo cbl-b ubiquitination substrates. interferon alpha (ifn-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. for a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to ifn-a therapy. however, recent studies in mice have revealed that ifn-a/b also exerts effects on several host immune cells. ifn-a has been shown to enhance cd8 t cells (ctls) responses against soluble antigens in mice. this immunostimulatory activity of ifn-a results at least partly from its direct ability to induce maturation of dendritic cells. several studies have recently demonstrated that ifn-a/b also acts directly on murine ctls, inducing clonal expansion and differentiation into effector and memory cells. to date, little is known about the effects of ifn-a on human ctls. to approach this issue, magnetically sorted untouched human cd8 + cd45ro -t cells (mainly naï ve cells) were unstimulated or stimulated with human ifn-a and gene expression profiles were compared using an affymetrix human array. interestingly, ifn-a stimulation of highly purified human ctls without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (trail) and chemotaxis (ip10 and itac). in a second genome-wide array analysis, we analyzed the effects of ifn-a on human ctls responding to antigen (signal 1) and co-stimulatory signals (signal 2), provided by beads coated with anti-cd3/cd28 antibodies. gene expression patterns were compared for cells stimulated with anti-cd3/cd28 beads alone or along with ifn-a. ifn-a regulates the expression of a number of genes that promote proliferation, activation and survival of ctls, tcr stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in ctls effector functions (granzyme-b, ifn-g, trail, fasl) and chemotaxis (ip10, itac). the enhanced expression of granzyme-b, ifn-g, trail and ip10 were further confirmed at the protein levels by flow cytometry analysis and/or elisa. enhancement of granzyme-b-and trail-mediated cytolitic functions was also found by functional assays using anti-cd3-coated p815 cells and trail-sensitive caki-i cells as targets. our results show that ifn-a provides a strong signal-3 to human ctls leading to their differentiation into effector ctls. t cell activation is an important process of the adaptive immune system, which requires recognition of mhc-associated antigens by antigen presenting cells (apcs) via the t cell receptor (tcr). to induce a productive t cell response the interaction of t cells with apcs needs to be stabilized by adhesion molecules. junction adhesion molecules (jams) are a recently discovered group of immunoglobulin (ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. the third member of the jam protein family, jam-c, is highly expressed in platelets and endothelial cells, whereas expression in t cells is largely unknown. to investigate the regulation of jam-c in t lymphocytes, we determined jam-c gene expression in quiescent and activated human t cells. treatment with the polyclonal t cell activator phytohemagglutinin (pha) increased surface and total jam-c expression in t cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. by contrast, no up-regulation of jam-a in activated t cells was detectable. the highest level of jam-c up-regulation by pha was observed in cd3 + foxp3 + and cd4 + cd25 high t cells. moreover, t cell receptor activation with combined anti-cd3 and anti-cd28 stimulation induced jam-c expression in t cells. jam-c induction occurred at the mrna level suggesting a transcriptional regulatory mechanism of jam-c expression. accordingly, we studied the regulation of the human jam-c gene promoter in transiently transfected t cells. luciferase activity of a jam-c promoter gene construct with three potential consensus sites for the transcription factor nfat was markedly induced in activated t cells. finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin a and fk-506, but not with mapk inhibitors, blocked jam-c induction in activated t cells. in summary, the present data indicate that jam-c is induced in activated human t lymphocytes via a transcriptional mechanism and suggests a major regulatory function of jam-c for the t cell response. hiv-1 infection leads to immune dysfunction owing to a successive loss of the cd4 + t cell compartment. the molecular mechanisms underlying this depletion are not well-understood but may involve the viral nef protein. nef is a multifunctional accessory protein that is required for full hiv-1 virulence and the maintenance of high viral loads. nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. cd4 and mhc class i, and modulating signal transduction pathways. the latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis. in this study we identify a signaling complex assembling at the n-terminus of nef, which contains the kinases lck and pkcv. formation of this complex, termed nakc for nef-associated kinase complex, led to activation of lck, as assessed by in-vitro kinase assay, and recruitment of pkcv to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. recruitment of pkcv to membrane rafts is a hallmark of t cell activation and has been associated with activation of the nfxb transcription factor. however, contrary to our expectations, nef-mediated nakc formation did not activate nfxb. instead, it led to a strong induction of erk1/2. this correlated with a nakc-mediated increase in hiv transcription that was demonstrated by luciferase reporter assays suggesting that erk1/2 directly targets hiv transcription, possibly via induction of transcription factors. to our surprise, however, the effect of nakc on hiv transcription was found to be independent of ap-1, nfat and nfxb suggesting an alternative mechanism of nakc-mediated enhancement of hiv transcription. on the basis of our previous results we propose that nef enhances hiv transcription via removal of inhibitory factors and thus derepression of the hiv promoter. how erk1/2 is involved in this mechanism and whether nakc targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined. p. otahal 1 , t. brdicka 1 , v. horejsi 1 1 institute of molecular genetics as cr, praha, czech republic aims: c-terminal src kinase (csk) and cd45 are key regulators of src-family kinases in leukocytes. while cd45 is a transmembrane phosphatase, csk is localized mostly in cytosol. however, a fraction of csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. currently, it is accepted that sh2 domain of csk binds phosphotyrosine 317 of transmembrane adaptor protein pag and via this interaction is recruited to the cell membrane and lipid rafts. however, pag knock-out mice still have cell membrane-associated csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane csk. thus, the mechanisms of membrane targeting of csk remain unclear. to analyze the role of membrane and lipid raft targeting of csk on lymphocyte signaling we targeted csk to different membrane compartments by fusing csk with transmembrane domains of lat, lax, cd25 and n-terminal part of src kinase. methods: csk chimeras containing n-terminal membrane targeting motif and c-terminal orange fluorescent protein were cloned into retroviral vector pmxs. jurkat t cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these csk chimeras on t-cell receptor (tcr) signaling by measuring calcium flux and cd69 upregulation. the efficiency of inhibition depended on the membrane targeting motif, while lat-csk chimera completely inhibited tcr signaling and src-csk chimera inhibited the signaling only partially; lax-csk and cd25-csk chimeras showed almost no inhibition of tcr signaling despite efficient presence at the plasma membrane. conclusions: our data demonstrate that the function of csk strongly depends on its targeting to the specific areas of plasma membrane. it also strongly supports the idea that membrane compartmentalization is critical for regulation of t-cell signaling. peripheral cd8 t cell tolerance can be generated outside lymphatic tissue in the liver. however, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating t cells is unclear. here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (lsec) and not by other antigen presenting cells in the liver or spleen. uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve cd8 t cell-retention in the liver but again not in other organs. using bone-marrow chimeras and tie-2kb mice, we could show that antigen cross-presentation by lsec was both essential and sufficient to cause antigen-specific t cell-retention under non-inflammatory conditions, which was followed by cd8 t cell proliferation and expansion, but ultimately led to the development of t cell tolerance. our results show that cd8 t cell tolerance towards circulating systemic antigens is predominantly generated in the liver by lsec, which preferentially take-up and cross-present circulating proteins to cd8 t cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. these insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of cd8 t cell responses. alphapix is a rho gtpase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. it has been shown that pix proteins play roles in some immune cells, including neutrophils and t cells. in this study, we report the immune system phenotype of alphapix knockout mice. we extended alphapix expression experiments and found that whereas alphapix was specific to immune cells, its homolog betapix was expressed in a wider range of cells. mice lacking alphapix had reduced numbers of mature lymphocytes and defective immune responses. antigen receptor-directed proliferation of alphapix deficient t and b cells was also reduced, but basal migration was enhanced. accompanying these defects, formation of t-cell-b-cell conjugates and recruitment of pak and lfa-1 integrin to the immune synapse were impaired in the absence of alphapix. proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of pak and expression of git2 in both t cells and b cells. these results reveal specific roles for alphapix in the immune system and suggest that redundancy with betapix precludes a more severe immune phenotype. s. merluzzi 1 , s. parusso 1 , b. frossi 1 , g. gri 1 , c. pucillo 1 1 university of udine, dstb, udine, italy in this study, we investigated whether primary mcs could modulate the activation and proliferation of primary b cells. we performed co-culture assays using mouse splenic b cells and bone marrow-derived mcs. naï ve and activated b cells proliferation could be induced by nonsensitized mcs while an increase in b cell proliferation was observed when mcs are activated. moreover, b cell proliferation was partially abolished when mcs and b cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. using both il-6 -/-mcs and anti-il-6 receptor antibody, we demonstrated that in co-culture of primary b cells and mcs, il-6 derived from activated mcs is a key cytokine implicated in the b cell proliferation. moreover, we showed that activated mcs can influence the surface expression of costimulatory molecules as cd40 on naï ve b cells and the interaction of cd40 on b cell surface and cd40l on mcs is important for the further differentiation of b cells to plasmacells. indeed, we presented for the first time evidence that cytokines produced by activated mcs and interaction between cd40l e cd40 on mc and b cells respectively can contribute to differentiate mature b cells to iga secreting cells. in conclusion, in the present report, we showed a novel role of mcs as promoter of both the survival and activation of naive b cells and of the proliferation and further differentiation of activated b cells through soluble factors production and cell-cell contact, suggesting that mcs can contribute to the regulation of specific immune response. e. fourmentraux-neves 1 , n. bercovici 1 , a. caignard 1 1 inserm u567, paris, france inhibitory killer ig-like receptors (kir2dl1-2/3) which bind to hla-c molecules are expressed by human natural killer cells and effector memory cd8 + t cell subsets. these receptors suppress cd8+ t cell activation through recruitment of the src homology 2 domain-containing protein tyrosine phosphatase 1 (shp-1). to further analyse the yet largely unclear role of inhibitory kir receptors on cd4+t cells, kir2dl1 transfectants were obtained from a cd4 + t cell line and primary cells. the transfection of cd4 + t cells with kir2dl1 dramatically increased the t cell receptor (tcr)-induced production of il-2 independently of ligand binding, but inhibited tcr-induced activation after ligation. kir-mediated tcr activation requires intact itim motifs, involves kir2dl1-itim phosphorylation, shp-2 recruitment, zap-70 and pkc-v phosphorylation. synapses leading to activation were characterized by an increase in the recruitment of p-tyr, shp-2 and p-pkc-v but not of shp-1. in contrast, the kir2dl1/hla-cw4 interaction led to a strong synaptic accumulation of kir2dl1 and the recruitment of shp-1/2, inhibiting tcr-induced il-2 production. kir2dl1 may induce two opposite signaling outputs in cd4 + t cells, depending on whether the kir receptor is bound to its ligand. these data highlight unexpected aspects of the regulation of t cells by kir2dl1 receptors. b cell receptor (bcr) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific b cells to powerful co-stimulatory signals, such as t cell help or toll-like receptor ligands. the role of bcr binding in modulating the access to these second signals is complex and varies between stimulatory conditions. by quantitative tracking of b cell responses in vitro we can measure which signals affect b cell proliferation or differentiation, or both, and thereby establish a novel understanding of how b cells respond appropriately to different combinations of stimuli. we utilised hel-specific bcr transgenic sw hel mice to assess the effect of a specific antigen signal on b cell responses to the t-independent mitogen lipopolysaccharide (lps). the presence of antigen renders a greater proportion of cells responsive to lps stimulation and profoundly influences effector cell differentiation. antibody secreting cell formation is dramatically inhibited by hel, but we found that isotype switching to igg1 is strongly upregulated. both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. when b cells are exposed to antigen for a limited period of time, switching to igg1 still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of igg1 antibody during these conditions. the observed igg1 switching behaviour mimics that of b cells responding to lps and il-4, but is mediated by a different, stat6-independent pathway. these data are indicative of the important role specific antigen signals play in regulating b cell responses in stimulatory environments. a. quintana 1 , c. schwindling 1 , m. pasche 2 , c. junker 1 , c. kummerow 1 , u. becherer 2 , e.c. schwarz 1 , j. rettig 2 , m. hoth 1 1 saarland university, biophysics, homburg, germany, 2 saarland university, physiology, homburg, germany the adaptive immune response requires the interaction between antigen-presenting cells and t cells. this cell-cell interaction, called the immunological synapse (is), facilitates the activation of t cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of crac/orai1 channels. to allow sustained activity of crac/orai channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. objectives: the purpose of the study was to analyze local and global calcium signals in t cells and to test the hypothesis that the is controls these signals through mitochondrial positioning. methods: we used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human t helper cells and cell lines. to test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. results: following is formation, we found that mitochondria translocated to the is in a calcium-dependent way. the distance between mitochondria and the plasma membrane at the is was lower than 150 nm. following accumulation at the is, mitochondria limited calcium entry to the orai channels localized right at the is by preventing their calcium-dependent inactivation. in contrast, no calcium influx was observed at sites where no mitochondria were accumulated as orai channels were inactivated at these sites. mitochondrial positioning at the is thus induced local calcium influx at the is without the necessity to enrich orai channels at the is. mitochondria took up calcium at the is distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the is low enough to prevent calcium dependent orai inactivation and to prevent excessive calcium clearance by the calcium aptases in the plasma membrane, which could inhibit an efficient t cell activation. conclusion: mitochondria positioning at the is controls local calcium entry through orai channels. mitochondria prevent orai inactivation and excessive calcium clearance at the is to facilitate calcium-dependent t lymphocyte calcium signalling. we aimed to determine the functional correlates of cd4 + t cell tolerance and immunity in vivo. ovalbumin (ova)-specific transgenic cd4 + t cells were adoptively transferred into syngeneic mice immunized with soluble ova protein ± lipopolysaccharide (lps) by the i. v. route, and analyzed for a variety of immunological parameters over a period of 21 days. under tolerogenic conditions (ova alone), cd4 + t cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (icos, 4-1bb), from t cells activated by ova+lps. this difference in activation also translated into differing cd4 + t cell expansion and contraction kinetics in the early phase of the t cell response (days 1-6). in the late phase of the primary response (days 7-21), under immunizing conditions, the large majority of transgenic cd4 + t cells in the spleen developed into mature effectors with a prominent capacity to secrete il-2, ifn-g, and il-17a, and only few ova-specific foxp3 + regulatory t cells ( x 10 %) were observed. germinal centers were prominent and ova-specific ig of all isotypes were generated. in contrast, under tolerizing conditions, antigen-specific cd4 + t cells failed to migrate into the b cell follicles, but production of ova-specific igm was nevertheless observed. in these animals, the proportion of splenic ova-specific regulatory t cells (30 %) was substantially increased. on day 14, both groups of mice were re-challenged via the airways with ova+lps to functionally assess their immune status. in tolerized animals, the transgenic t cell population in the lung infiltrate was composed of ova-specific regulatory t cells (50 %) or t cells with a reduced capacity to secrete effector cytokines. in contrast, in immunized animals, this population almost exclusively consisted of cd4 + effector t cells with a pronounced inflammatory cytokine profile (ifn-g, il-17a). with this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. we identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. mast cells (mcs) are well-recognized as key effector cells in immunoglobulin e (ige) -associated immune responses and as prototypic regulators of innate immunity. the characteristics, importance, and molecular requirements for interactions between mast cells (mc) and cd8 t cells (tc) remain to be elucidated. using myelin/oligodendrocyte glycoprotein (mog), we demonstrated that mcs induce antigen-specific cd8 tc activation and proliferation. the antigen crosspresentation by mcs induces the secretion of interleukin-2, interferon-g and macrophage inflammatory protein-1 by cd8 tc. in vivo evidence that mcs modulate t cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (eae), the standard animal model for multiple sclerosis, in which both cd8 tc and mcs are now recognized as key players. one of the main central nervous system (cns) antigens recognized by autoreactive tc in eae is the myelin oligodendrocyte glycoprotein (mog). to investigate the in vivo-relevance of the identified mc-cd8 tc interactions, we have employed the eae as a model of organ specific autoimmune disease in wild type mice and mc-deficient w/w sh mice. wt and w/w sh mice were immunized with the mog 35-55 protein. our results provide direct evidence that mc contribute to cd8-specific priming in eae and show that the tc proliferation failure is specific for cd8 tc from mog 35-55 -immunized w/w sh mice. the role of mc-cd8 tc interaction in induction of autoimmunity will be further investigated in eae. in summary, we provide the first evidence that mcs regulate antigen-specific responses of primary cd8 tc in vitro and in vivo. our study further supports the emerging concept that mcs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding cd8 tc responses. this newly uncovered mc function might be of great biological relevance in situations where effector cd8 tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. activation of resting t cells in vitro is triggered by combined t cell receptor (tcr) and cd28 engagement and can be modulated by simultaneous ligation of various other surface receptors. although the fasl is best known for its capacity to initiate cell death in fas-bearing cells, it has recently been implicated in the regulation of t cell activation. thus, a crosstalk between the tcr and fasl is likely, but far from being biochemically elucidated. we report that fasl engagement by immobilized but not soluble fasfc fusion protein and anti-fasl antibodies blocks the activation of primary human peripheral t cells even in the presence of cd28 costimulation at the level of an early signal initiation. inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in tcr signal transduction and also with a lowered calcium response. the data presented stress the importance of the fas/fasl-system for signal initiation via the tcr/cd3complex and provide further arguments for a retrograde signaling capacity of fasl or a crucial role of fas as a costimulatory molecule. golgi network (tgn). moreover, trim specifically associates with the cytoplasmic tail of ctla-4, but not via any conventional motifs in this region. overexpression of trim augments ctla-4 surface expression, whereas down-regulation of trim expression by shrna results in disturbed ctla-4 localisation, mainly restricted to the tgn. ctla-4 vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in ctla-4 trafficking. here, we identify additional transmembrane adapter protein (trap) family members as novel binding partners and regulators of ctla-4 expression. although there is some redundancy amongst traps, our results highlight the importance of this family of proteins in ctla-4 transport to the cell surface. it is imperative to reveal the mechanisms by which ctla-4 is transported to the cell surface, given that minor changes in expression can have major effects on t-cell function and in the development of autoimmunity. natural killer t (nkt) cells are found within the liver and are known to exhibit immune regulatory function. upon recognition of glycolipids presented on cd1 molecules, nkt cells are activated and release cytokines, including ifn-g, il-2 and il-4. nkt cells are efficiently recruited to the liver via cxcr6-dependent chemotaxis toward cxcl16 and constitute a large proportion of the liver-resident lymphocytes. we have previously shown, that liver sinusoidal endothelial cells (lsec) can scavenge circulating soluble antigens, and can cross-present these antigens to naive cd8 t cells. cross-presentation leads to initial t cell activation and expansion, but ultimately these cd8 t cells are rendered tolerant. as both naive t cells and nkt cells come into close contact with lsec in the hepatic sinusoids, we investigated whether nkt cells can modulate cd8 t cell tolerisation via interaction with lsec. to this end we analysed cd1d expression on lsec and their ability to activate nkt cells by presentation of the cd1d-binding glycolipid a-galactosylceramide (agalcer). we found that lsec express functional cd1d, as agalcerpresenting-lsec were capable to induce tnf-a, il-2, il-4 and ifn-g production in nkt cells in vitro. the interaction of agalcer-presenting-lsec with nkt cells led to the upregulation of cd54 and b7-h1 on lsec. as naï ve cd8 t cell tolerisation by lsec critically depends on b7-h1, we hypothesise that hepatic nkt cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of lsec. improved antibody responses by class-switched memory b cells require enhanced signaling from their antigen receptor (bcr). however all bcr classes on naïve and antigen-experienced b cells utilize the canonical iga/igb subunit for signaling. we identified the signal amplification mechanism of the igg-and ige-bcr. for these isotypes tyrosine-based signaling is not confined to iga/igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. the phosphorylated immunoglobulin tail tyrosine recruits the adaptor grb2 in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. hence membrane-bound igg and ige not only recognize antigen but also exert bcr-intrinsic costimulation to render memory b cells less dependent on t cell help for activation. objectives: the majority of circulating human gd t cells harbor tcr containing vg9, jg1.2, and vd2 gene products. they recognize nonpeptide antigens like (e)-4hydroxy-3-methylbut-2-enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (ipp) in malignant cells. recently, we and others found out that gd t cells express a variety of costimulatory molecules including icos, and pd-1. one of the inhibitory receptors, pd-1, is a member of cd28/ctla-4 family and contains a single ig v-like domain in its extracellular region. pd-1 can bind to two b7 homologue molecules, pd-l1 and pd-l2. it has been reported that interaction of pd-1 with its ligands resulted in peripheral immune regulation and tolerance in ab t cells. in this study, we show that pd-1 is expressed on activated human gd t cells and regulates the effctor functions of gd t cells. methods: peripheral blood mononuclear cells were resuspended in yssel's medium and stimulated with 2-methyl-3-butenyl-1-pyrophosphate plus il-2 to obtain gd t cells. pd-l1+ and pd-l1-human tumor cell lines were established from cancer patients. in order to prepare anti-pd-l1 mabs, the pd-l1 extracellular domain was expressed in e.coli as inclusion bodies and refolded in the standard arginine-based buffer. mice were immunized with the refolded protein and mabs were established. to determine the function of pd-1 in gd t cells, we determined cytokine production and cell mediated tumor lysis by activated gd t cells in the presence of inhibitors of pd-1/pd-l1 interaction. results: gd t cells expressed pd-1 upon simulation with nonpeptide antigens and many tumor cell lines expressed pd-l1. we first examined whether or not the engagement of pd-1 receptor could modulate the cytotoxic activity of gd t cells. pd-l1-expressing tumor cells tempered cytotoxic activity of pd-1+ gd t cells, and cytokine production such as tnf-a was down-regulated by pd-1 engagement. in addition, inclusion of anti-pd-l1 mab reversed cytotoxic activity and cytokine production when pd-l1-expressing tumor cells were challenged by pd-1-expressing gd t cells. conclusion: pd-1 delivers inhibitory signals in gd t cells upon engagement with pd-l1. peripheral tolerance plays an important role in preventing t lymphocyte responses to self or harmless antigens. one of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and il-2 production by t cells in response to antigenic challenge. the acquisition of the anergic phenotype is an active process, with negative regulators of t cell signalling being induced. among these are the e3 ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. the e3 ubiquitin-protein ligases cbl-b, itch and grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate tcr signalling elements. our objectives were to determine the expression of cbl-b, itch and grail in antigen-specific cd4 + t cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. in order to accomplish these objectives we induced priming or tolerance of ovalbumin (ova 323-339 peptide)-specific t cells from do11.10 tcr transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with e3 ubiquitin-protein ligases expression and the ubiquitination status of the tcr signalling machinery. cbl-b, itch, grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and western blotting studies. moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using laser scanning cytometry. our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific t cells by mutant e3 ligase-, signal target-and ubiquitin-constructs. collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific t cells. s. j. keppler 1 , p. aichele 1 1 immh, university freiburg, immunology, freiburg, germany interleukin 12 (il-12) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. it was postulated recently that il-12 has a direct influence on cd 8 + t cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to tcr engagement (signal 1) and costimulation (signal 2). we analysed direct il-12 signaling to cd8 t -12 signaling exhibited normal degranulation activity, cytolytic functions, ifn-g and tnf-a production. however, cd8 t cells lacking il-12 signaling failed to up-regulate klrg1 and to down-regulate cd127 in the context of listeria but not viral infections. thus direct il-12 signaling to cd8 t cells determines the cell fate decision between short-lived effector cells (slecs) and memory precursor effector cells (mpecs), dependent on the pathogen-determined local cytokine milieu. cd8 + t lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. we could now reveal that individual cd8 + t cells are tightly controlled in their effector functions by cd152 (ctla-4). we demonstrate that signals induced by cd152 reduce the frequency of interferon-gamma (ifn-g) and granzymeb expressing cd8 + t cells. for this novel function cd152 specifically represses the transcription factor eomes, but not t-bet. a cd152 mediated induction of the inhibitory transcription factor ckrox has been ruled out. ectopic expression of eomes reversed cd152-mediated inhibition of effector molecule production. additionally, enhanced cytotoxicity of individual cd8 + t cells differentiated in the absence of cd152 signaling could be demonstrated in vivo. the novel insights that cd152-mediated signal transduction in vivo indeed alters cd8 + t cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of cd8 + t cells. objectives: atp constitutes a damage associated molecular pattern (damp) and contributes together with pathogen associated molecular patterns (pamp) to the efficient priming of the innate immune system. atp is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed p2 receptors. p2x 1-7 receptors open to non-selective ion channels, whereas p2y1, 2, 4, 6, 11-14 are g-protein coupled receptors, which bind preferentially adp, udp, utp or udp-glucose. as the role of p2 receptors in the control of b cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular atp production and release by human b cell subsets. methods: intracellular atp measurement has been performed using a bioluminescence assay while extracellular atp has been measured by hplc. storage and release of atp by b cells have been elucidated using confocal and tirf microscopy, to study vesicles distribution and dynamics near the plasma membrane. results: in both human naive and memory b cell we observed a prominent increase of atp synthesis upon tlr9 but not bcr stimulation. glycolytic pathway rather than oxidative phosphorilation was involved in atp synthesis. p2x7 antagonists inhibited both proliferation and differentiation to plasma cells of human b cells thus suggesting that atp is released in the pericellular space. labelling of resting and activated human memory b cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of atp, confirmed with subcellular fractionation on sucrose equilibrium gradients. tirf imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-ig and this event was ca(2+)-dependent. conclusion: these data provide evidence that atp is produced by b cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of atp release in human b cells. atp released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of b cells. o. ballek 1 , a. brouckova 1 , d. filipp 1 1 institute of molecular genetics as cr, laboratory of immunobiology, prague, czech republic two src family tyrosine kinases lck and fyn are critical for the proximal t-cell signaling. we have previously demonstrated that induced lck activation outside lipid rafts (lr) results in lck translocation to lr. central in this sequence of events is the rapid translocation of kinase active lck to lr, yet the mechanism underpinning this process is unknown. the main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to lr and forming immunological synapse. we have recently characterized the c-terminal yqpqp sequence as a novel cis-acting component essential for partitioning of lck to lr. here we report that the expression of the c-terminal truncate of constitutively active lck ( ¿ fqpqp) in nih3t3 cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active y505flck. comparative 2-d gel analyses followed by ms/maldi identified rack1 as a candidate protein for interaction with the c-terminal tail of lck. co-expression in nih3t3 cells of ha-tagged rack1 with either a wild type lck or constitutively active y505flck revealed a significantly enhanced complex formation between y505flck and rack1 compared to that of wtlck. ectopic expression of y505flck with its domain-inactivating mutations showed that lck-rack1 interaction depends on functional sh2, sh3 and the c-terminal tail sequence of lck. lck-rack1 interaction is readily detectable also in primary cd4 + lymph node t cells. upon their activation, only the pool of lck molecules associated with high molecular weight complexes can translocate to lipid rafts. co-purification of rack1 with these fractions further suggests that it plays a role in the translocation of lck to lr. in addition, lck and rack1 co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. moreover, the importance of interaction between activated lck and rack1 in the context of lck translocation to lr is further strengthen by the observation that rack1 is associated with elements of cytoskeleton. these results are the first to characterize rack1 as a candidate molecule involved in the regulation of lck translocation to lr through linking the c-terminal sequence of lck to cytoskeletal network. human b cells are currently not known to produce the pro-apoptotic protease granzyme b (grb) in physiological settings. we have discovered that b cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin 21 (il-21) can induce secretion of substantial amounts of grb by human b cells. grb response to viral antigens was significantly stronger in b cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. both, naï ve and memory b cells differentiated into grb-secreting cells, which featured a homogeneous cd19+cd20+cd27-cd38-igd-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. b cellderived grb was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic t cells. our findings suggest grb-secreting b cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum grb levels found in various viral diseases. further studies will elucidate whether b cell-derived grb induces cytotoxicity towards virus-infected cells or exhibits other functions. results: transfer of otii cells augments the wild type th2 response to alumova inducing large early germinal centres and massive plasma cell formation with more than 75 % of these switching to igg1. the plasma cells up-regulate cxcr4, but not cxcr3, a chemokine receptor that attracts plasma cells to inflammatory sites. oti cells respond to alumova by producing ifng, a th1-associated cytokine. when both oti and otii cells are transferred switching is diversified with plasma cells being igm (˚5%), igg2a (˚30 %), igg2b (˚30 %) or igg1 (˚30 %). in addition to cxcr4, some 70 % of these plasma cells strongly express cxcr3. the induction of cxcr3 in these plasma cells correlates with their increased expression of the transcription factor t-bet, which has been linked with igg2a switching during th1 responses. this is functionally significant for oti-dependent cxcr3 expression, as well as induction of switching to igg2a, are dependent on t-bet expression by the responding b cells, although t-bet-deficient b cells still switch to igg2b. t-bet is known to be induced in b cells exposed to ifng or tlr9 stimulation. these two hypothetical mechanisms are currently being tested in mice injected with blocking anti-ifng antibodies or mice deficient in myd88. objective: a successful immune response against malaria has to be tightly controlled. the early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. activation of t lymphocytes through tcr signalling takes place within the context of numerous other cell surface protein interactions. to prevent unnecessary activation of t cells the immune system has developed an intricate balance between positive and negative costimulatory signals. costimulatory signals determine whether antigen recognition by t lymphocytes leads to full activation or to anergy. in contrast negative costimulators expressed by t cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. recently, btla (b and t lymphocyte attenuator, cd272) was described as a novel negative costimulatory receptor. btla is predominantly expressed on t and b cells and dampens t cell activation. in this study, we analyzed the function of btla during experimental malaria infection. to study the function of btla we employed a mouse model of blood-stage malaria using p. yoelii nl infection of btla-deficient and hvem-deficient mice. p. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. immunity in this model depends on cd4+ t cells and hence the role of negative costimulators that modulate t cell function can be studied using this model. results: peak parasitemia of p.yoelii-infected btla-deficient and hvem-deficient mice was much lower compared to wild type mice. the increased immunity of btla-deficient mice depends largely on cd4+ t cells. we found that btla::hvem interaction regulates the size and the cytokine-production of the responding t cell pool. however, in contrast to the ctla-4 pathway, the manipulation of btla::hvem interaction triggers no pathology during infection. the hvem::btla interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. . so far, the contribution of actin regulatory proteins to this process remained largely unknown. here we demonstrate that the actin-bundling protein l-plastin is indispensable for segregation of lfa-1 and cd2 in the psmac of untransformed human t-cells. in marked contrast, tcr/cd3 accumulation in the csmac is not dependent on l-plastin. the relocalization of l-plastin in the immune synapse occurs within seconds of t-cell/apc contact formation and relies on actin polymerization. importantly, binding of calmodulin to l-plastin is required for the maintenance of l-plastin in the immune synapse and inhibition of calmodulin prevents psmac formation. thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein l-plastin and calmodulin. protective t cell responses are based on expansion and persistence of clones with optimal affinity for antigen. presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. therefore we hypothesized that apoptosis might play an important role during this phase of t-cell biology. here we show that the balance between the pro-apoptotic protein noxa and its antagonist mcl-1 regulates interclonal t cell competition during acute and chronic immune activation. we found p53-independent noxa gene induction and mcl-1 downregulation upon t cell activation. concomitant we observed the release of death-inducing factor bim from mcl-1, which was delayed in noxa -/cells. using ot-1 cells and altered peptide ligands we observed that the level of mcl-1 downregulation in activated t-cells depended on the antigen affinity of the t-cell receptor. since mcl-1 -/mice are embryonic lethal, noxa -/mice were used to study the functional implications of this mechanism in vivo. at a young age noxa -/mice have a normal lymphoid compartment, but accumulate effector t cells over time. upon acute influenza infection, normal levels of effector cells were generated. however, the quality of the antiviral (np366) response was impaired in these mice as many subdominant clones persisted in the effector t-cell population of noxa -/mice at the peak of infection. this increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. in a model of chronic immune activation, effector t cells rapidly accumulated in the noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. these results establish a novel role for the noxa/mcl-1 axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a darwinian selection of t-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. cytotoxic t lymphocytes (ctls) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice. here, we refer to estrogen receptor-binding fragment-associated antigen 9 (ebag9) as a negative regulator of secretory lysosome release. in gene deleted mice we show that loss of ebag9 confers ctls with enhanced cytolytic capacity, in vitro and in vivo. here, we show that ebag9, which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g2-adaptin in t cells. both interactions suggested an involvement of ebag9 in endosome-lysosome related organelle biogenesis and membrane fusion. efficiency of granzyme b sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of cathepsin d proteolytic processing. while the formation of the immunological synapse remained unaffected in ebag9-/-ctl, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in ebag9-deficient ctls. these data imply a role for ebag9 in regulating the formation of mature ctl granules and identify ebag9 as a tunable inhibitor of ctl-mediated adaptive immune response functions. finally, ebag9 defines a novel negative regulator of secretory lysosome release in ctls. thus, the elucidation of the ebag9-related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. the liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector t cells. recently, we could show that liver sinusoidal endothelial cells (lsec) inhibit activation of naive cd8 t cells by antigen-presenting dc. this regulatory effect of lsec on dc function was mhc-independent and not limited to soluble mediators, but required physical contact. interestingly, interaction with lsec led to reduced dc expression levels of cd80/86 and il-12. in addition to indirect inhibition of t cell activation by de-licensing of dc, we now detected another influence of lsec in form of direct inhibition of t cell priming. in the presence of lsec, stimulation with acd3/acd28 or pma/ionomycin could not significantly activate cd4 and cd8 t cells. thus, lsec did not only inhibit t cell priming triggered by tcr activation but also after elicitation of ca 2+ influx into the cytoplasm. furthermore, we found that ifn-g secreted by t cells in the early phase of activation is crucial for licensing the inhibitory function of lsec. taken together, these data indicate that the inhibitory effect of lsec is mediated by a machinery induced at the early phase of t cell activation, however, interferes with late events in the t cell activation cascade. we propose a model of "inducible inhibition", where on the one hand naive t cell priming is directly inhibited by lsec, and on the other hand tolerogenic priming by antigen-presenting lsec is still allowed. taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which lsec not only tolerize t cells themselves, but also inhibit the responsiveness to local activation stimuli. m. almena 1 , s. carrasco 1 , i. merida 1 1 centro nacional de biotecnología csic, inmunología y oncología, madrid, spain self-tolerance acquisition is essential for the immune system to control its own response. t cells achieve self-tolerance trough thymic selection and anergy, two processes where rasgrp1-ras-erk signal intensity is critical to determine the final cell outcome. rasgrp1 is a gef for ras that is activated in a diacylglycerol (dag)dependent manner. dag is generated by plcg after tcr stimulation and is consumed by diacylglycerol kinases (dgk). dag generation, as a result of the concerted regulation of these two enzymes, activates ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response. 1. analyze the impact that dag metabolism plays in t cell tolerance in vivo, using transgenic mice where dag generation is impaired. 2. develop a method to sense dag production and localization, in both thymocytes and peripheral t cells, and its correlation with the strength of the stimulus used. methods: we generated transgenic mice expressing a constitutively active dgk in t cell lineage. this protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after tcr stimulation. ot-i cd8 cells expressing gfp-c1 domains were used with peptide-pulsed apcs to study dag generation and dynamics by confocal microscopy. results: transgene expression was obtained in thymic and peripheral t cells. no major defects were observed in t cell subsets but analysis of peripheral t cells demonstrated important defects in t cell activation. we are currently studying thymic selection breeding our transgenic with h-y mice, in order to check if t cell populations are being properly selected. using t cell-apc conjugates with peptides with different tcr binding affinities we found a clear correlation between the strength of the stimulus, dag production and ras-mapk activation. conclusion: our data demonstrate that dag generation not only activates c1 domain containing proteins but regulates a mechanism by which t cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in t tolerance. future experiments will help to define the exact contribution of the lipid to tcr signaling pathways and to t cell homeostasis. the inducible costimulator (icos, h4, cd278), a cd28-like costimulatory molecule, has an important role in the development of efficient t cell responses. early data showed that icos costimulation produced th2 biased responses and high production of the anti-inflammatory cytokine il-10, and was essential to the development of germinal centres. however, icos can also help in the il-21-dependent differentiation of inflammatory th17 cells. these different functions could be due to differences in the apcs bound by icos-expressing t cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of icos. icos shares with cd28 a yxxm cytoplasmic motif that can bind, upon tyr phosphorylation, the regulatory p85 subunit of class ia pi-3 kinases. these can complex with one of the three 110 kda catalytic subunits (p110a, p110b, and p110d) expressed by leukocytes that generate pip 3 affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. there is also evidence that the regulatory and catalytic class ia pi3k isoforms fulfill specific functions in macrophages and lymphocytes. we have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of icos, and particularly the presence of distinct pi3 kinase isoforms. then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of icos activation. pull down of t cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of icos was carried out. proteomic and immunoblot analysis of bound proteins showed that phosphorylated icos bound the different pi3-k regulatory (p85a, p85b, p53a) and catalytic (p110a, p110b, and p110d) pi3-kinase subunits expressed by leukocytes. these data were confirmed in icos immunoprecipitates from pervanadate-activated cells. icos bound regulatory and catalytic subunits in the order p85a g p53a g g p85b and p110a g p110d g g p110b, in agreement with quantitative rt-pcr and immunochemical estimation of subunit abundance in the t cells and t cell lines used. the use of specific pi3-kinase inhibitors has confirmed the relative importance of the catalytic isoforms in icos function, including reorganization of actin cytoskeleton induced by icos ligands, or costimulation of tcr/cd3-induced secretion of il-10 and il-4. during the process of antigen recognition between t-cell and antigen-presenting cell (apc), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the immune synapse (is) reorganize, displaying a segregated localization. in this context, the translocation of the microtubule-organizing center (mtoc) is an early event that occurs during the formation of the is, bringing with it the golgi apparatus, thus providing the basis for a polarized secretion. however, the molecular mechanisms involve in the localization of the mtoc at the contact area between the t cell and the apc are not completely understood yet. we have studied the possible role of scaffolding protein akap450, a member of the a-kinase anchoring protein (akap) family that localizes at the centrosome and interacts with pka regulatory subunit and other signalling molecules, in mtoc polarization and immune synapse formation. either the overexpression of gfptagged c-terminal cg-nap/akap450 construct that acts as a dominant negative, or sirna knockdown of endogenous akap450 expression in t cells prevents the correct organization of cd3z and pkcv to the is and mtoc reorientation towards t cell-apc contact area in antigen and superantigen-dependent human models, resulting in a disorganized is; lfa-1 localization was also analyzed to assess p-smac architecture and, interestingly, confocal 3d reconstruction revealed that lfa-1 ring was not clear in the akap450-disrupted cells. moreover, akap450 was required for tcr signalling since the knock down with specific sirna and overexpression of c-terminal of akap450 decrease the phosphorylation of molecules such as lat, plcg1 and pkcv. these defective activation events as reflected in a reduction of il-2 production. together, our results underscore a key role for akap450 in the organization of the immune synapse and in the antigen-specific reorientation of the mtoc. the tcrbeta/ptalpha pre-tcr complex signals the expansion and differentiation of developing thymocytes. functional properties of the pre-tcr rely on its unique ptalpha chain, which suggests the participation of specific intracellular adaptors. in fact, we have recently identified cms, a member of the cin85/cms family of adaptors, as a ptalpha-binding protein that specifically interacted with the human ptalpha cytoplasmic domain via its sh3 domains, and to the actin cytoskeleton via its c-terminal region. we found that cms co-localized with polymerized actin in pre-tcr clusters at the pre-tcr activation site, and also in the ptalpha endocytic compartment. since actin polymerization plays a critical role in regulating signalling through the alpha/beta tcr in mature t cells, we decided to investigate the potential function of cms as a regulator of actin polymerization and pre-tcr signalling in pre-t cells. using pre-t cells expressing a mutant pre-tcr lacking the cms-binding motif in the ptalpha tail and short hairpin irna-based gene silencing, we demonstrate that binding of cms to ptalpha contributes to cytoskeleton dynamics and pre-tcr-mediated signalling in human pre-t cells. cms-deficient cells specifically showed defects in pre-tcr-induced ca2+ mobilization and cell activation involving the pi3k, nfat, plcg and erk signalling pathways, together with defects in actin polymerization and cell motility. cms therefore links cytoskeleton dynamics with the function of discrete pre-tcr signalling components, suggesting the functional implication of cms in human t-cell development in vivo. abstract withdrawn by author j objectives: most signaling pathways engaged after bcr activation have been described. however, several negative regulators of these pathways are unknown. the characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. carabin (tbc1d10c) has been recently described as a negative regulator of tcr signaling. it interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking nfat nuclear transport. moreover, carabin maintains ras protein under an inactive form, thus inhibiting ras-mapk cascade. expression of carabin is finely regulated following tcr signaling, and its knockdown (kd) enhances t cell activation. considering the important molecular similarities of antigen receptor signaling pathways in t and b cells, we studied the role of carabin in b cell. could carabin play a role of negative regulator of b cell function? methods: we studied by quantitative rt-pcr 1) the expression of carabin in different purified subsets of bone marrow and splenic mouse b cells, as well as 2) the kinetic of expression of carabin in bcr stimulated murine splenic mature b cells. 3) we then studied the phenotype of carabin kd (shrna expressing) a20 b cells after bcr stimulation. 1) the expression of carabin is significant in murine b cells, with an increase during b cell development, from bone marrow pro/preb to immature, to splenic t1 tot2 b cells and to follicular mature b cells. 2) the kinetic of expression of carabin in bcr stimulated murine mature b cells suggests a fine regulation of carabin expression. 3) bcr simulation, but not lps stimulation, of carabin kd a20 b cells shows an acceleration of ras target erk1/2 phosphorylation, without any for the phosphorylation of mapk jnk, which is not targeted by ras. conclusion: carabin is expressed in murine b cells in a developmental regulated manner, with the highest expression in mature compartment. bcr stimulation leads to a fine regulation of carabin expression in wild-type mature b cells, and to a faster activation of erk1/2 pathway in carabin kd b cells. altogether, these results strongly suggest a role of carabin as a negative regulator of b cell function toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. toll like receptor 3 (tlr3) binds doublestranded rna, a nucleic acid frequently associated with viral replication. we observed that freshly isolated human cd4 + t cells express tlr3 and respond to the well characterized synthetic tlr3 ligand polyinosinic-polycytidylic acid [poly(i:c)]. the expression of activation markers and cytokine production by cd4 + t cells upon t cell receptor (tcr) stimulation is enhanced in response to co-stimulation via tlr3. tlr3 stimulation on its own had no effect on expression of activation markers and cytokine production. to elicit the molecular basis of a potential cross-talk between tcr and poly(i:c) induced signaling, we used jurkat cells to perform luciferase assays. we observed that costimulation with poly(i:c) in comparison to tcr stimulation alone enhanced nf-kb but not nfat activation in jurkat cells. similarly to jurkat cells, tcr stimulation activated nf-kb in primary cd4 + t cells. this effect was further enhanced by additional poly(i:c) stimulation as shown by real-time-pcr and western blot analysis. on the other hand, we observed that poly(i:c) stimulation on its own activated the transcription factor interferon regulatory factor 3 (irf3) as revealed by realtime-rcr analysis of ifn b and irf7, whose transcription depends on the activity of irf3. combined tcr and poly(i:c) stimulation further enhanced the transcription of these two genes. these results indicate that tlr3 signaling modulates tcr-driven responses and vice versa both in jurkat cells and in freshly isolated cd4 + t cells. this study was supported by dfg spp 1110 "innate immunity" (ka 502/8-3). the initiation of protective t cell responses requires the recognition of mhc-bound peptides from pathogen or tumor antigens by the t cell receptor (tcr). how this signal is transmitted across the t cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. in textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the cd3e cytoplasmic domain (cd3e cd ) binds to synthetic lipid vesicles that contain acidic phospholipids. this binding is predominantly due to electrostatic interactions between basic residues of cd3e cd and acidic phospholipids. in the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. to study the interaction of the cd3e cd with the membrane in live cells, we have developed a fluorescence resonance transfer (fret) assay which measures the proximity between a fluorescent protein (tfp) attached to the c-terminus of cd3e cd and a fluorescent membrane dye (r18). with this assay, we show that the cd3e cd is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. additionally, in vitro analysis confirm functional domains for cd3e cd lipid binding and conformational change. finally, nmr spectroscopy analysis reveals key features in membrane binding dynamics of cd3e cd to lipid bicells. membrane binding by the cd3e cd could thus be subject to dynamic regulation during the engagement of the tcr and further activation of the t cell. m. xydia 1 , y. ge 1 , u. quitsch 1 , p. beckhove 1 1 german cancer research center (dkfz), heidelberg, germany in peripheral tissues and the factors affecting their proliferation. cd4+ t cell help is believed to contribute to optimal cd8+ memory expansion via cd40l on cd4+ t cells binding cd40 on dendritic cells. however, a few reports suggest that cd40l-cd40 engagement may mediate direct cell-cell contacts between cd4+ and cd8+ t cells. in this study, we investigated the importance of cd4-cd8 co-operation and cd40l-cd40 interactions for t em proliferation. methods: we isolated human cd4+ and cd8+ t em cells from peripheral blood of healthy donors by facs or macs sorting. separated or mixed cd4+ and cd8+ populations were activated in vitro using anti-cd3/cd28 beads. proliferation was measured by [ 3 h]-thymidine incorporation, in some experiments after irradiation of one t em subset and/or incubation with blocking mabs against cd40 or cd40l. furthermore, facs staining was used to assess cell-surface markers. statistical comparison was performed by student's t test. results: upon activation mixed t em populations showed a highly better proliferative response than separated cd4+ or cd8+ t em cells, demonstrating that optimal t em expansion requires direct cd4-cd8 interactions. surprisingly, not only cd8+ but also cd4+ t em cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal cd4+ t em proliferation depends on signals from cd8+ t em cells. activation induced the expression of cd40 on both populations and cd40l on subsets of cd4+ and cd8+ t cells. blocking of cd40l on cd8+ t em cells impaired significantly cd4+ t em proliferation, which confirms that the improved expansive potential of cd4+ t em cells in mixed populations depends on cd40l co-stimulation by the cd8 t em subset. conclusions: our data demonstrate for the first time that activated cd8+ t em cells deliver help to the cd4+ t em subset via cd40l-cd40 signalling and may play an important role for cd4+ t em expansion upon stimulation. the t cell surface glycoprotein cd5, a member of the scavenger receptor cysteine-rich (srcr) family of proteins, targets to the immunological synapse upon t cell binding to antigen presenting cells (apc). however, it has not been established whether this translocation is due to the binding of a ligand expressed in the apc, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control cd5 localization upon t cell:apc conjugation. we have questioned which domains of cd5 mediate the localization within the is, and for this we have expressed cd5 mutants as gfp fusion proteins in human t lymphocytes. we have also used jurkat cell lines expressing different cd5 mutants. t cells were incubated with superantigen-loaded raji b cells, and following the establishment of stable interactions between the cells, we analyzed the localization of cd5 by immunofluorescence and confocal microscopy. interestingly, our results show that the translocation of cd5 depends on sequences within the cytoplasmic domain, as a cd5 deletion mutant lacking most of the cytoplasmic tail, cd5.k384 stop , is randomly distributed through the whole cellular surface, even in sustained t-apc interactions. the cytoplasmic domain relevant to cd5 translocation was mapped within amino acids glu 418 and his 449 since the cd5.h449 stop mutant, just short of 22 aa is still able to translocate to the is, whereas cd5.e418 stop , that lacks 2 important tyrosine residues, is no longer transported to the is upon t cell: apc interactions. although these studies do not exclude a role for the extracellular domain binding to an elusive apc-expressed ligand, they suggest that a major mechanism of regulation of cd5 translocation is dependent on molecular association of a short stretch of its cytoplasmic region (glu 418 -his 449 ) to intracellular signaling effectors. however, in hodgkin's lymphoma (hl) ebna-2 is missing, but lmp-1 is still expressed. using a hl derived cell line, we have shown that the cytokine il-4 can induce lmp-1 expression in vitro and can replace ebna-2. we have investigated the molecular events for this mechanism. stat proteins bind to the palindromic ttc(n) x gaa sequence, where × is 2, 3 or 4. a high affinity stat6 binding site is spaced by 4 nucleotides. we found three potential stat binding sites in the lmp-1 promoter, which we named lrs, tr and edl1. they were spaced by 4, 3 and 2 nucleotides, respectively. electrophoretic mobility shift (emsa) experiments were performed with nuclear extracts prepared from il-4-treated or non-treated kmh2-ebv cells. dna binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (gl) epsilon promoter, which is known to contain a high affinity stat6 binding site, or lrs-stat6. a stat6 complex binding to the gl-epsilon promoter and lrs-stat6 was induced by il-4. the specificity of the stat6 complex was shown by supershift experiments with anti-stat6, but not anti-stat5 antibodies. when gl-epsilon or lrs-stat6 was used as cold competitors in a 100-fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the lrs-stat6 contains a functional stat6 binding site. oligonucleotides, corresponding to lrs in which the stat6 site had been mutated, could not compete for stat6 binding. interestingly, the unlabeled lrs-tr with 3 nucleotides as spacer could also function as competitor. however, when ttc/gaa palindrom was spaced by 2 nucleotides (lrs-edl1), it could not compete. thus, expression the transforming protein lmp-1 can be induced directly by the t cell derived cytokine il-4 in a stat6 dependent manner. it is likely that this mechanism operates in vivo as well and determines expression of the ebv encoded protein lmp-1 and thus the pathogenesis of ebv carrying hls. established knockout/ knockin mice with a fasl deletion mutant that lacks the intracellular portion (fasl ¿ intra). co-culture experiments confirmed that the truncated fasl protein is still capable of inducing apoptosis in fas-sensitive cells. preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular fasl domain does not alter the intracellular fasl localization in activated t cells. we are currently investigating signalling and proliferative capacity of b-and t-cells derived from homozygous fasl ¿ intra mice. our data point to a rather inhibitory role of fasl reverse signaling during immune responses. during an immune response numerous receptor-mediated signals delivered to t cells direct their proliferation, survival and differentiation. we are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms t cells use to process these multiple signals. previous experiments with ot-i cd8 t cells revealed that tcr affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (ji 2007 (ji . 179:2250 . furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. here we examine the same question for cd4 t cells. again we find that lower affinity peptides stimulate t cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. in contrast varying the number of dendritic cells used to stimulate cd4+ t cells primarily affect the proportion of cd4+ t cells going into division rather than affecting division time or cell death in subsequent divisions. currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules cd40, cd80 and cd86 on parameters of cd4+ t cell proliferation to inform quantitative models of the immune response under different conditions. our goal is to develop quantitative models of t cell behaviour that can accommodate information at the molecular, cellular and population level. interaction between cd40, a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as b cells, and cd40l, a member of the tumor necrosis factor family transiently expressed on activated t cells, are essential for the development of humoral adaptative immune response. various studies have shown that dual stimulation of b cell through antigen binding on bcr and cd40 leads to an enhancement of ig and cytokine production. the current dogma postulates that these 2 signals are necessary and sufficient to drive naive b cell proliferation and differentiation to ig secreting plasma cells. however, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. indeed, b cells can be activated through engagement of a variety of innate immune receptors, including toll-like receptors (tlrs). soluble cd40l is unable to induce murine b cell proliferation. however, we and others have shown that recombinant mouse cd40l (rmcd40l) can increase proliferation induced by tlr3 (poly ic) and tlr4 (lps) agonists. by contrast, we never observed any synergy between rmcd40l and tlr1/2 (pam3csk4) or tlr2/6 (pam2csk4) agonists. to go further in the study of cd40l/tlr agonist synergetic effect, we have developed trimeric synthetic molecule to mimick cd40l, named mini-cd40ls, based on a c 3 -symmetry core holding cd40-binding motif lys-gly-tyr-tyr. in surface plasmon resonance experiments, mini-cd40ls bind to immobilized human cd40 and compete with the binding of cd40l homotrimers and diplayed effector functions that matched those of the much larger recombinant cd40l homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of trypanosoma cruzi infection. as soluble cd40l, mini-cd40ls synergize tlr4 (lps), tlr3 (poly ic) and tlr7/8 (r848) agonist-induced murine b cell proliferation but no synergy was observed between mini-cd40ls and tlr 1/2 (pam3csk4), tlr 2/6 (pam2csk4) and tlr9 (odn 2395) agonists. synergy between cd40l and tlr agonist provide the ground to use such a combination as adjuvant in vaccination strategy. however, to reach this goal, evaluation of cd40l/tlr combinations on murine and human b cell activation and differentiation in antibody producing cells are under investigation. interaction of naïve cd8 + t cells with immature dendritic cells (idc) expressing self-peptides can result in their abortive activation (aa), which leads to the induction of cd8 + t cell tolerance. we have defined a phenotypic profile for cd8 + t cells undergoing such aa. these cells undergo limited proliferation which is associated with lack of ifn-g production, low cell surface expression of cd25 and cd69, and high levels of expression of cd62l and ly6c. whereas, cd8 + t cells undergoing productive activation (pa), following encounter with mature dc, form effector ctl which is evidenced by extensive t cell proliferation, high levels of ifn-g, cd25 and cd69, and loss of cd62l and ly6c expression. ly6c is a gpi-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. in this study, we show that mab-blocking of ly6c in vivo and in vitro results in pa rather than aa. we hypothesize that the interaction of ly6c, expressed on naïve cd8 + t cells, with its ligand on idcs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive cd8 + t cells. objectives: organophosphorus compounds (opcs) are commonly used in the manufacture of insecticides and pesticides. exposure to opcs is associated with neurological toxicity but the effect on the immune system remains ill-defined. in this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. methods: balb/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for 3 weeks. during the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. at the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by elisa. for in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of s. typhimurium. animal survival was followed for up to 60 days after infection. results: daily injection of paraoxon induced g 50 % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining 2 weeks of treatment. mice exposed to paraoxon exhibited g 80 % reduction in the rate of body weight gain over the treatment period in comparison with control group. at the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. to analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of s. typhimurium and followed for survival. unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with 80 % of mice surviving the infection compared to 20 % in control group. protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. conclusion: our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. this suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. t cell-dependent (td) and t cell-independent (ti) igg autoantibodies have been described in the context of the autoimmune disease systemic lupus erythematosus (sle). however, their different roles in autoimmunity are unknown. here we show that ti antigens induce anti-inflammatory igg antibodies and protect from antigen-specific immune pathology. administration of antigen-specific anti-inflammatory igg antibodies was sufficient to mediate this effect independent of the igg inhibitory receptor fcgammariib. ti but not td igg autoantibodies were further associated with inhibition of pro-inflammatory th1 and th17 cells and disease in mice deficient for fcgammariib, a spontaneous model for sle. the data suggest a novel immune regulatory function for ti immune responses through the generation of anti-inflammatory igg antibodies. objective: class i phosphoinositide 3-kinases (pi3k) constitute a family of enzymes that generate 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (tyr) kinase-associated receptors or g protein-coupled receptors (gpcr). the class i pi3k are divided into two types: class ia p85/p110 heterodimers, which are activated by tyr kinases, and the class ib p110g (p110gamma) isoform, which is activated by gpcr. although the t cell receptor (tcr) is a tyr kinase-associated receptor, previous studies showed that p110g deletion affects tcr-induced t cell stimulation. mice lacking p110g show a partial defect in t cell differentiation, activation and survival. p110g participates in signaling pathways that regulate pre-tcr dependent differentiation and cd4+/cd8+ t cell lineage commitment. in the mrl/lpr mouse model of systemic lupus erythematosus, administration of a pi3kg-specific inhibitor causes a reduction in the number of cd4+ memory t cells that mediate renal injury. similarly, pi3kg deletion in p65 pi3k transgenic mice also reduces the numbers of cd4+ memory t cells. there is therefore evidence that pi3kg has an important function in tcr-mediated t cell activation, although the mechanism by which pi3kg regulates this process is not well understood. we studied the specific role of p110g in t cell activation. methods: we studied whether the tcr activates p110g and the consequences of interfering with p110g expression or function on t cell activation. results: we found that after tcr engagement, p110g interacts with and forms a complex with ga q/11 , lck and zap70. tcr stimulation activates p110g, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. we show that tcr-stimulated p110g controls rac1 activity, f-actin polarization, and the interaction between t cells and antigen-presenting cells (apc). we show that p110g deletion affects the activation of many pathways downstream of tcr crosslinking, as well as the interaction between t cells and apc; these findings could explain the defective activation of p110g-/-t cells. our observations clarify the activation mechanism and mode of action of p110g in the control of t cell activation, confirming a crucial role for p110g in tcr-induced t cell activation. we investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic t lymphocytes (ctl). we show that cytolytic granules in ctl can be delivered to the secretory domain via two different pathways -"short" and "long". the choice between these pathways is regulated by the kinetics of early tcr signaling which depends on the strength of tcr/pmhc/co-receptor interactions. meanwhile, the molecular hardware used to deliver the granules remains the same. we conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (mtoc) and the mtoc polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of ctl cytolytic response. our findings reveal a mechanism of well-documented flexibility in t cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. objectives: in addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. nicotine was shown to suppress both cellular and humoral immune responses. previously we found that two nicotinic acetylcholine receptor (nachr) subtypes, a4b2 and a7, expressed in mouse b lymphocytes regulate their development within the bone marrow. the aim of the present study was to evaluate the roles of these two nachrs in b lymphocyte activation. methods: b lymphocytes were magnetically separated from the spleens of c57bl/6j mice. they were stained with fluorescently labeled igm-, cd40-or cd23specific antibodies in the presence/absence of unlabeled nachr subunit-specific antibodies to be examined by flow cytometry. b lymphocyte activation was studied by 3 h-thymidine incorporation upon stimulation with anti-cd40 and nachr-specific agonists or antagonists. the antibody response of mice immunized with cytochrome c with or without a7 nachr antagonist methyllicaconitine (mla) was studied by elisa. results: antibodies against a4 or b2 nachr subunits inhibited binding of igm-and cd23-specific antibodies but facilitated that of cd40-specific antibody. in contrast, antibody against a7 subunit prevented binding of anti-cd40 but not of anti-igm or anti-cd23 suggesting that a7 nachrs are located close to cd40, while a4b2 ones are close to bcr/cd23. consequently, anti-cd40-induced b lymphocyte proliferation was increased by mla much stronger than by a4b2-specific antagonist dihydro-b-erythroidine. it was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine-3. in contrast, proliferation of b lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. mice co-injected with cytochrome c and mla responded with igm antibodies faster than those injected with cytochrome c alone, while the secondary / igg responses were similar. the cd40-mediated b lymphocyte proliferation, but not the igm-igg switch or memory b cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a7 nachrs. therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.this work was supported by philip morris usa inc. and philip morris international. binding of cd4 + t h -lymphocytes to antigen presenting cells or of cd8 + cytotoxic t-lymphocytes (ctl) to their target cells lead to a tight contact between these two cells, called immunological synapse (is). formation of the is induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the is, all of which are crucial for t-cell functions such as cytokine release or target cell killing. objectives: using primary human t-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. for testing protein functions, down-regulation by rnai technology is thus an important tool. we used short interfering rnas (sirnas) to analyze the role of proteins involved in calcium influx and proliferation (stim1 and trpc3), and to analyze snare proteins which were shown to accumulate at the is and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. methods: to validate down-regulation of different mrnas quantitative rt-pcr was used. down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). results: transfection efficiency of sirnas in t-lymphocytes was about 96 %. down-regulation of stim1 was confirmed by qrt-pcr and by calcium imaging, but only for early time points following activation of cd4 + t h -lymphocytes, probably because of stability problems. to increase stability of sirnas within t-lymphocytes we used modified sirnas published by mantei et al. (eji, 2008) . we show that these sirnas down-regulate various snare proteins in ctls more efficiently than non-modified sirnas. the optimal sirna concentrations for transfection in primary human t-lymphocytes was found to be 300-600 pmol, which is lower than the concentrations reported in other cell types. conclusions: following optimization, down-regulation of mrnas by sirna is a powerful tool to investigate the role of different proteins involved in the activation of t-lymphocytes in primary human cells. chemical modifications increase the lifetime and efficiency of the sirnas in primary human t-lymphocytes. stress-inducible heat shock protein 70 (hsp70) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific cd8 + cytotoxic t-lymphocyte (ctl) and cd4 + t-helper cell (th1) responses. in this study, we investigated the behavior of t-cell subsets stimulated with endotoxin-free recombinant hsp70 with respect to proliferation, cytokine expression, cytotoxicity against allogeneic b-lymphoblastoid cell line (b-lcl) and k562 cells as well as targetindependent cytotoxicity. cd4 + cells exhibited a strong increase in proliferation after stimulation with hsp70, with rates of up to 29 %. in the presence of target cells, a 35-fold up-regulation of granzyme b mrna was observed after stimulation of cd4 + t-helper cells with hsp70 in combination with il-7, -12 and -15. the target cell-independent secretion of granzyme b by cd4 + cells was greatly augmented after stimulation with hsp70 plus il-2 or il-7, -12 and -15. in this study, we have shown that hsp70 is capable of inducing a cytotoxic response of t-helper cells in the absence of lps or any other pamps. the granzyme b secretion and the cytolytic activity of cd4 + t cells is induced in a target-independent way, whereas the cytotoxic activity of cd3 + and cd8 + t cells can be further enhanced in the presence of the target cells. our data provide novel insights into the role of extracellular hsp70 on t-cell immune response concerning the induction of target-independent t-helper cell cytotoxicity. jun n-terminal kinases (jnk) have been shown to play controversial role in regulation of cell fate. cd40, which is responsible for germinal centre formation in lymph nodes, trigger jnk activation. the role of other b cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. the aim of this study was to find out whether cd150 receptor contributes to jnk activation in mature human b cells. protein expression and phosphorylation were studied by western blot analysis. protein associations were evaluated by immunoprecipitation and gst-pull down assays. hpk1 overexpression in a model system was achieved by transfection. pjnk1/2 expression in primary hrs cells was assessed by immunohistochemistry. ligation of cd150 on resting (dense) and activated (buoyant) human tonsillar b cells lead to jnk2, but not jnk1 activation. cd40 ligation on primary tonsillar b cells also resulted in jnk2 activation. however, bcr crosslinking did not affect the level of jnk1/2 phosphorylation. cd150-mediated jnk2 activation was independent from sh2d1a/sap adaptor protein expression, and was demonstrated for all studied b-lymphoblastoid, burkitt's lymphoma and hodgkin's lymphoma (hl) cell lines of b cell origin. we were searching for serine/threonine kinase that could coprecipitate with cd150 and link this receptor with jnk pathway. using immunoprecipitation and gst-pull down assays we found that hematopoietic progenitor kinase 1 (hpk1) was associated with cd150 in primary b cells as well as in b cell lines. cd150-hpk1 association was independent from cd150 tyrosine phosphorylation and sh2d1a expression. overexpression of hpk1 in a model system significantly enhanced cd150mediated jnk2 phosphorylation. it is known that tnf family receptors such as cd30, cd40, rank trigger survival signals in hrs cells. we observed the expression of pjnk1/2 in hrs cells of primary classical hl. cd150 could be involved in sustained jnk2 activation in primary hrs cells, and this may reflect the role of cd150 receptor as well as other receptors in the regulation of hrs survival. overall, it was shown that jnk2 is activated via cd150 in primary b cells and in all studied cell lines of b cell origin. serine-threonine kinase hpk1 is involved in cd150-mediated jnk2 activation. objectives: cd5 has been shown to act as a negative regulator of tcr signaling during thymocyte development. however, the molecular mechanisms involved in this process remain elusive. one potential key molecule involved in the downmodulation of tcr signaling is c-cbl, a ubiquitin ligase that physically associates with cd5 upon tcr crosslinking in thymocytes. the objective of this study was to determine which sequences within the cytoplasmic tail of cd5 are involved in c-cbl phosphorylation and association. methods: el4 thymoma cell line was stably transfected with wild-type human cd5 or hcd5 cytoplasmic tail mutants: cd5.k384stop (maintaining only a pseudo itim); cd5.h449stop (lacking the distal s and y in the carboxy-terminal region); cd5. ¿ e418-l444stop (lacking the pseudo-itam, putative site for c-cbl association). phosphorylaton of y700 in c-cbl was analyzed, which is required for vav recruitment and c-cbl dependent degradation by the proteasome. stable clones were stimulated with anti-murine cd3 in combination or not with anti-human cd5 biotinylated antibodies and phosphorylation of c-cbl was detected by flow cytometry after intracellular staining anti-phospho c-cbl (py700) antibody. murine thymocytes were used as positive control. data was analyzed using flowjo software. unpaired two-tailed student t test was used to calculate statistical significance (p x 0.05). in murine thymocytes, co-crosslinking of cd3 with cd5 induces an increase in c-cbl phosphorylation compared to cd3 alone. analysis of the el-4 transfectants showed that mutants cd5.k384stop and cd5.h449stop lost the ability to costimulate cd3-mediated phosphorylation of c-cbl. in contrast, cd5. ¿ e418-l444stop mutant, was able to efficiently costimulate cd3-mediated c-cbl phosphorylation, similarly to the hcd5wt. our results indicate that the absence of the pseudo itam in cd5 does not interfere with c-cbl phosphorylation in response to cd3 plus cd5 crosslinkiing on the other hand, sequences present in the carboxy-terminal region of cd5 appear to be important for c-cbl phosphorylation. therefore, c-cbl phosphorylation might not require physical association with the cd5 cytosplasmic tail, but rather, may indirectly associate with cd5 through the interaction with other sh2-sh3 domain-containing molecules, that may be recruited to cd5 through its carboxy-terminal region. l. kolly 1 , s. narayan 1 , j. tschopp 2 , a. so 1 , n. busso 1 1 chuv, rheumatology, lausanne, switzerland, 2 unil, biochemistry, epalinges, switzerland apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) is an adaptor protein that is essential for the recruitment of pro-capase-1 into inflammasomes and thus plays a key role in regulating capase-1-dependent il-1b and il-18 production. despite recent evidence implicating asc in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for asc involvement in adaptive immune responses remains largely unexplored. to investigate the impact of asc on t cell activation and subsequent effector function. asc +/+ and asc -/-t cells or purified cd4 + and cd8 + t cells were activated in vitro through anti-cd3 stimulation and their proliferative potential and cytokine profiles characterized. proliferative responses by asc -/-t cells were significantly inhibited two-fold following tcr-cd3 ligation when compared to asc +/+ t cells. furthermore, cytokine analysis revealed that anti-cd3 activated asc -/-t cells predominantly displayed a more th 2 phenotype, producing more il-10 (199 vs. 692 pg/ml; asc +/+ vs. asc -/-t cells respectively; p=0.0074) and less ifn-g (15,831 vs. 6 ,921 pg/ml; asc +/+ vs. asc -/-t cells respectively; p = 0.0021). when asc +/+ and asc -/-t cells were purified into cd4 + and cd8 + t cell fractions and activated individually using anti-cd3, no inhibition in proliferation was observed amongst activated asc -/-cd4 + and cd8 + t cells. interestingly, the activated asc -/-cd4 + t cell fraction produced significantly more il-10 when compared to activated asc -/-cd8 + t cells and asc +/+ cd4 + and cd8 + t cells (asc -/-cd4 + t cells = 380 pg/ml il-10; asc -/-cd8 + t cells = undetectable il-10; asc +/+ cd4 + t cells = 11 pg/ml il-10; asc +/+ cd8 + t cells = undetectable il-10). cd4 + and cd8 + t cell mixing experiments revealed that asc -/-cd4 + t cells are able to inhibit the proliferative ability of asc -/-cd8 + t cells, asc +/+ cd4 + and cd8 + t cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated asc -/-cd4 + t cells. collectively, these results demonstrate that the absence of asc drives cd4 + t cells towards a suppressor cell phenotype, suggesting that asc might play an important role in determining the fate of cd4 + t cells. various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. in particular, e-series prostaglandins, pge 1 and pge 2 suppress some t-cell functions including proliferation, activation and cytokine production. pge 2 signals through four types of gpcrs called the ep receptors. at low concentrations, pge 2 is believed to be necessary for t cell function, whereas at higher concentrations, pge 2 inhibits t cell proliferation. these effects are largely governed by various cell specific stimuli and tissue microenvironment. objectives: to delineate, compare and contrast the effects of pge 2 and ep receptor antagonists on t cell activation. methods: flow cytometry, proliferation assays, migration assays. we have observed that pge 2 diminishes expression of early, intermediate and late t cell activation markers. in contrast, pre-treatment of cd4 + t cells with ep receptor antagonists was found to impair cell surface expression of cd71, cd69, cd25 and ox40 but not cd44. suppression of t cell proliferation by pge 2 has already been widely studied. however, blocking ep receptors in cd4 + t cells by the use of ep antagonists prior to activation surprisingly caused a defect in t cell proliferation. migration of cd4 + t cells to the chemokine sdf-1b was also found to be reduced due to pre-treatment with ep antagonists. in order to study the physiological relevance of these findings we studied the trafficking of basal and activated t cells to regional lymph nodes during inflammation in the presence and absence of ep receptor antagonists. this model revealed that the use of ep antagonists causes a reduction in the amount of cd44 + cd4 + adoptively transferred t cells in the regional lymph node following the induction of a local inflammatory response. conclusions: in our study we show for the first time that ep receptors are required for expression of activation markers and activating proliferation in murine cd4 + t cells. our results also suggest that considering pge2-mediated camp signaling in cd4 + t cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in t cell activation. objective: our objective was to investigate how ros affect the different stages of t cell activation. because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ros affect calcium signalling. the experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, t-cell activation assays and molecular biology. results: we show by direct measurement of ros that t-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. the effect of ros on calcium signalling in jurkat t-cells as well as in primary naï ve and effector cd4 + human t-cells was examined. oxidation affects several ca 2+ signalling pathways by altering the activity of ip 3 receptors, trp channels and store operated ca 2+ channels in a concentration dependent manner. interestingly, calcium signalling is differentially affected in naï ve and effector t cells. thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of ca 2+ signalling in t-lymphocytes. cysteins are the main carrier of thiol groups in proteins and we show that orai ion channels contain reactive cysteine groups that mediate ros effects on the calcium influx pathway. conclusion: ros regulate the calcium dependent t-cell activation in a complex way, affecting all three major calcium signalling pathways. by mutational analyses of the orai proteins, we are able to pinpoint molecular targets of regulation. the activation of t cells during an immune response is a crucial but tightly regulated event. to make the grade, the t cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. this enables the t cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. in this study we present data of the signaling mechanisms underlying the potent t cell inhibitory receptor cd147 (emmprin, basigin) , a member of the ig-family. previous studies reported that lymphocytes from cd147 knockout mouse possess enhanced mixed lymphocyte reactions and cd147 monoclonal antibodies can interfere with t cell activation. these observations already pointed to a negative crosstalk of cd147 signals with the t cell antigen receptor or co-stimulatory signals. consistent with these studies, we found that rna interference (rnai) with cd147 in jurkat t cells augments the secretion of the t cell growth-factor interleukin-2 (il-2) upon t cell activation. this up-regulation is at least partially due to an increased activity of the nuclear factor of activated t cells (nfat), which resulted in an enhanced il-2 promoter activity. by reconstituting the rnai-mediated knockdown with various truncated rnai-resistant forms of cd147, we identified the immunomodulatory sub-domain of cd147. supported by the gen-au program of the austrian federal ministry of science and research. mirnas play a critical role in the control of hematopoiesis. the goal of this project is to determine whether mirnas function also during the antigen-induced activation of mature b lymphocytes. therefore, we determined mirna profiles in primary splenic b cells before and after polyclonal activation with either lps (simulates t cell-independent activation) or a combination of anti-igm, anti-cd40 and il4 (simulates t cell-dependent activation). microarray assays identified about 104 mirnas in unstimulated b cells. 35 of these were downregulated and one was upregulated upon stimulation. in silico analyses with various mirna target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by mirnas during the antigen-induced activation phase of mature b cells. among these targets are bcr signalling molecules and transcription factors that control proliferation, igh class switch as well as differentiation in antibody-secreting plasma cells. one of these transcripts codes for the interferon regulatory factor-4 (irf-4). the graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. first results indicate that the expression kinetic of irf-4 transcripts differs from that observed for irf-4 protein abundance after b cell stimulation. further analysis identified the irf-4 transcript as a target whose expression is obviously fine-tuned by a mirna upon antigen stimulation. we are in the process to biochemically verify potential targets for each of the differentially regulated mirnas and determine the effect of ectopic and retrovirally mediated expression of mirnas on b cell differentiation. the work was in part supported by the izkf erlangen, the dfg graduiertenkolleg gk592 and the dfg forschergruppe for832. objective: transforming growth factor-b (tgf-b) signals through type i (tgfbri) and type ii (tgfbrii) tgf-b receptors and receptor regulated smad proteins. tgf-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. the mechanisms of resistance against tgf-b have not been fully elucidated. our aim is to describe how b cell lymphoma cells respond to tgf-b compared to normal peripheral b cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to tgf-b. methods: proliferation assays were performed on 11 different b-cell lymphoma cell lines and normal peripheral b cells to screen for tgf-b-induced effects. western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to tgf-b signaling pathways. facs analysis was used to measure tgf-b receptor surface levels. cells were treated with demethylating agents to examine changes in gene expression levels. s. manthey 1 , f. hauck 2 , i. berberich 1 , f. berberich-siebelt 2 , gk 520 -immunomodulation 1 institute for virology and immunobiology, university of wuerzburg, würzburg, germany, 2 university of wuerzburg, department of molecular pathology, würzburg, germany the transcription factor ccaat/enhancer-binding protein b (c/ebpb) can not act only as a transcriptional activator but also as a transcriptional repressor. in murine cd4+ t lymphocytes, the transcription factor is predominantly expressed in t helper 2 (th2) compared to t helper 1 (th1) cells. in contrast, by binding to the c-myc promoter(s), c/ebpb represses c-myc expression thereby arresting t cells in the g1 phase of the cell cycle. both, transactivation and repression depend on the n-terminal transactivation domain of c/ebpb. blimp-1 encoded by prdm1 is a transcription factor necessary for terminal differentiation of b cells to plasma cells. furthermore, blimp-1 is expressed in differentiated effector t cells where it is higher in th2 than th1 cells. the regulation of the blimp1 expression is not fully understood. interestingly, we found that c/ebpb can bind to the prdm1 promoter and activates blimp-1 expression in t cells. as c/ebpb is also expressed in b cells, we hypothesize that this transcription factor might as well influence the expression of blimp-1 in b cells. so far, we were able to show a similar expression profile of c/ebpb and blimp-1 in b cells using cre recombinase. moreover we found a new putative blimp-1 isoform lacking exon 2. currently, we analyze the expression of c/ebpb and blimp-1 in primary b cells and b cell lines after various stimulations. to get more insights into the function of c/ebpb in b and t cells, we are generating mice carrying a b as well as a t cell-specific deletion of c/ebpb. engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase c gamma (plcy) and plays an important role in activation of cells. by screening 53 cvid patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in b and t cells isolated from the peripheral blood of patients in the group ia of the freiburg classification as compared to non-ia patients and healthy donors (hd). ia patients are characterized by the expansion of an unusual cd21low b cell population in which calcium mobilization is strikingly lower than in other b cell subsets. common subpopulations like naï ve and mz-like b cells as well as cd4 + t cells but not transitional b cells or cd8 + t cells also revealed significantly decreased calcium peaks. the cytometric data correspond to a semiquantitative rt-pcr assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein-1a (mip-1a), and abrogated activation and proliferation, respectively. preliminary data on b cell receptor (bcr) mediated phosphorylation of plcy2 revealed constitutively high background levels in cd21low b cells of ia patients. since phosphorylation in the other b cell populations as well as calcium flux upon ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of plcy and upstream of internal store release, thus resulting in defective calcium signalling. the underlying mechanism yet remains to be elucidated and is part of our work in the future. c. balas 1 , v. courtois 1 , k. de luca 1 , r. sodoyer 1 1 sanofi pasteur, marcy l'etoile, france the presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. a potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. for instance monitoring of the expression level of cytokines, such as il-2, il-4 or il-6 could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. the analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. methods: genetic tools (primers and qpcr probes) capable of discriminating and quantifying alternatively spliced messenger rnas from il-2, il-4 and il-6. furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated. the genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract no a-136-0033-01835). conclusion: implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. in a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. parasitic helminths affect about one third of the world population. therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. from other parasitic infections it is known, that the regulatory receptor cytotoxic t lymphocyte antigen-4 (ctla-4) plays a crucial role during infections. here, we use the strongyloides ratti infection of mice as an experimental system to investigate the role of ctla-4 during nematode infections. we employed a quantitative real-time pcr (qtpcr) analysis to quantify the migrating larvae (il3) in the tissue and the released eggs and first stage larvae (l1) in the feaces. the cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mln) upon stimulation with polyclonal a-cd3 and s. ratti antigen was determined. additionally the humoral response was analysed in the primary and the secondary infection. to investigate the role of ctla-4 during the infection, a neutralysing antibody (a-ctla-4; 4f10) was administered intraperitoneally (300 mg) two hours before subcutaneous infection with s. ratti il3. the in vivo neutralisation of ctla-4-signalling by applying a-ctla-4 during s. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. we detected an increase in th2 cytokines, such as il-4 and il-5 and a reduction of the proinflammatory cytokines ifn-g and il-17. the investigation of the humoral response showed a remarked increase of the igg1-titer in the serum during secondary infection in mice that had been treated with a-ctla-4 during primary infection. furthermore, the blockade of ctla-4 resulted in a diminished worm burden as indicated by reduced release of l1 in the faeces. these results suggest that the blockade of ctla-4 during s. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. in particular the transition of the t cell cytokine profile towards a th2 response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. the strong increase of igg1 during secondary infection also reflects the induction of a potent th2 response. objectives: cd36 is a class b scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. this clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (sle). it was recently described that cd36 is highly expressed also on the marginal zone b cell subtype. we therefore set out to investigate the role of cd36 on the regulation of b cell in the setting of apoptotic cell clearance and autoimmune activation. we used a mouse model for sle where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. elisa was used to measure antibody levels and flow cytometry to study cell activation as well as cd36 expression. cd36 knock-out (ko) and wild type mice were used. results: preliminary in vivo data show a tendency for a higher antibody response towards ds-dna and the common self-antigen pc in cd36 ko compared to heterozygous mice. since reduced levels of cd36 are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison. in support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with lps is gone in cd36 ko mice. after one injection of apoptotic cells, cd36 ko b cells are activated while wild type b cells are not. after four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that cd36 on b-cells are involved in setting this threshold. conclusion: our data suggest that cd36 is involved in the early regulation of b cell response towards apoptotic cells and production of autoreactive antibodies. it does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. successful t cell immunity requires lymphocytes to be at the right time at the right place. the co-receptor cd152 acts as a major check-point of immune responses, but the mechanism by which cd152 controls peripheral t cell responses is unknown. the consequences of cd152 signaling on murine th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. the genetic and serological inactivation of cd152 in th1 cells reduced migration towards ccl4, cxcl12 and ccl19. crosslinking of cd152 together with cd3 and cd28 stimulation on activated th1 cells increased expression of the chemokine receptors ccr5 and ccr7, which in turn enhanced cell migration. sensitive liposome technology reveals that mature dendritic cells but not activated b cells were potent at inducing surface cd152 expression and cd152-mediated migration-enhancing signals. importantly, migration of cd152 positive th1 lymphocytes in in vivo experiments increased, as compared to cd152 negative counterparts, showing that indeed cd152 orchestrates specific migration of selected th1 cells to sites of inflammation and antigenic challenge in vivo. these data show that cd152 signaling does not just silence cells, but selects individual ones for migration. this novel activity of cd152 adds to the already significant role of cd152 in controlling peripheral immune responses by allowing t cells to localize correctly during infection. it also suggests that interference with cd152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. here we analyzed the role of cd152 signaling on the longevity of cd28 null t cells. using a sensitive staining method for cd152, we show that human cd4 + cd28 null and cd8 + cd28 null t cells rapidly express surface cd152. serological inactivation of cd152 using specific fab fragments or blockade of cd152 ligands using ctla4ig in cd4 + cd28 null and cd8 + cd28 null t cells reduces the number of non-apoptotic cells in a fas/fasl-dependent manner. cd152 crosslinking on activated cd28 null cells prevents activation-induced cell death (aicd) as a result of reduced caspase activity. apoptosis protection conferred by cd152 is mediated by pi3'k dependent activation of the kinase akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule bad. we show that signals triggered by cd152 act directly on activated cd28 null t lymphocytes and, due to its exclusive expression as a receptor for cd80/cd86 on cd28 null t cells, prevention of cd152 mediated signaling is likely a major target mechanism taking place during therapy with ctla4ig. objectives: cd45 is a transmembrane protein tyrosine phosphatase (ptp) expressed in all nucleated leukocytes. it activates src family kinases (sfks) by dephosphorylating inhibitory tyrosines in their c-terminal tails. in cd45 -/mouse t cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. at least in part, it is due to the activity of another transmembrane tyrosine phosphatase cd148 (ptprj, dep-1) which acts as a positive regulator of sfk in cd45 -/-b cells and macrophages and can compensate for cd45 deficiency in these cells. indeed, combined deficiency of cd45 and cd148 in mice results in defective macrophage and b cell signaling and development, a phenotype much more severe than the loss of either protein alone. naïve murine t cells do not express cd148 and its expression is increased only after activation. accordingly, no defects in t cell development and signaling in cd148 -/mice were reported so far. however, in human t cells the role of cd148 may be different since naive human t cells express cd148 at a level comparable to b cells. using cd45 -/-/cd148 -/human t cell line (jurkat-derived js-7 cells) we tested the ability of cd148 to complement cd45 deficiency in t cells. we used retroviral transduction to express human cd45 or cd148 in js-7 cells and tested their ability to reconstitute major signaling pathways. we also employed substrate trapping mutant of cd148 to identify direct substrates of this phosphatase. in agreement with previously published data, defective t cell receptor (tcr) signaling was observed in js-7 cells. expression of wild type cd45 or cd148 in js-7 cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased cd69 upregulation after tcr cross-linking. moreover, the carboxy-terminal tyrosine of lck, major t cell sfk, was hypophosphorylated in js-7 cells expressing cd148 when compared to control cells. finally, cd148 substrate trapping mutant expressed in js-7 cells interacted with lck in vivo suggesting that lck is a direct substrate of cd148 in js-7 cells. the results suggest a level of redundancy between cd45 and cd148 in human t cells not appreciated so far. during the past decades, great efforts have been made to get insights into the complex process of antigen-induced t cell activation and the underlying signal transduction pathways. the t cell antigen receptor signaling cascade is initiated by phosphorylation of itam-tyrosine residues through the t-cell specific src protein tyrosine kinase family member lck. during t cell activation, lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the itam-motifs. in order to resolve conformational changes of lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive förster resonance energy transfer (fret) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. for the live-fret imaging and biochemical assays we complemented lck-deficient jurkat t cells (jcam1.6) with the biosensors. by introducing point mutations affecting the two major regulatory tyrosines tyr 505 and tyr 394 we found a dramatic decrease and increase, respectively, of intramolecular fret efficiency compared to the wild type biosensor. these results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. thus, our biosensor is able to detect phosphorylation modifications of key residues. however, we could not detect any overall change in fret and thus conformation of membrane-associated lck molecules during t cell activation indicating that other mechanisms, presumably reorganization of localization, underlie lck regulation. furthermore, we observed a contribution of intermolecular fret, which indicated homophilic interaction of lck. indeed, by performing single molecule analysis and native 2d immunoblotting we found lck dimers and higher order oligomers. together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the t cell antigen receptor. it has been reported that mitochondria accumulate under the immunological synapse (is) in response to tcr (t cell receptor) stimulation. this process seems to be required to allow proper tcr-induced calcium influx in t cells in contact with antigen presenting cells (apcs), because mitochondria can sequester calcium and thus keep crac (ca2+ release-activated ca2+) channels open. however, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the is. thus, we speculated that other signals are involved in recruiting the organelles to the contact region between t cells and apcs. we found that the adhesion molecule lfa-1 (leukocyte function-associated antigen 1) induces localization of mitochondria at the is. this process is antigenindependent and is enhanced by the presence of chemokines in the t cell environment. however, tcr triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. our data suggest that, by recruiting mitochondria to the cell-cell contact region, lfa-1 prepares and facilitates tcr signaling. we are performing experiments to understand the signalling pathways involved in mitochondria translocation at the is. burkitt lymphoma (bl) is a high grade b cell malignancy (non-hodgkin lymphoma (nhl)) derived from germinal center b cells, that harbours a chromosomal translocation juxtaposing the protooncogene myc next to the regulatory elements of one of the immunoglobulin loci. however, the precise contribution of myc to the pathogenesis of this tumour is poorly understood. based on the definition of a distinguishing gene expression signature for the molecular burkitt lymphoma (mbl) with myc as one hallmarking signature gene (hummel et al.2006) we describe a non-viral vector based approach (vockerodt et al. 2008) to express myc in primary human gcb cells from pediatric tonsils. comparative whole genome gene expression profiling was performed in 9 independent preparations. our data reveal a global change in gene expression in lymphoma precursor cells by myc giving new insight into potential changes of the gene expression program of gcb cells on the accidental way to bl in addition as a first step the function of selected signature genes in bl is accomplished. in a representative cell line with a mbl signature and with a non-mbl signature rnai directed inhibition of elements of the cd40 signaling cascade was conducted. after activating this particular signaling cascade (cd40) we analysed respective gene expression profiles of ikks, trafs and mapk deficient cells. based on these different rnai-mediated ge-profiles a comparison between both lymphoma types is performed. first attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (markowetz et al. 2005 ). a rat thymic epithelial cell (tec) line (r-tnc.1) was established from a long-term tec culture. this line was characterized as a type of rat cortical tec with nursing activity (tnc). very little is known about molecular mechanism of the tnc/thymocyes interaction. in our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by r-tnc.1 cell line in vitro. it was found that a number of adhesion molecules, such as cd2, cd4, cd8, cd11a, cd18, cd54, cd90 was involved in these processes. objectives: a main goal of this study was to define the adhesion molecules involved in the interaction between r-tnc.1 line and activated thymocytes. methods: experiments was performed on inbred ao rats. monoclonal antibodies (mabs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of cona and il-2 activated syngeneic thymocytes and unstimulated or ifn-g stimulated r-tnc.1 cells. we found that both the adhesion (30 min and 3h) of activated thymoytes were partially blocked by mab to cd2 and cd8 molecules (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells). early adhesion was inhibited by mab to cd90, abtcr, mhc class i molecule (ifn-g stimulated r-tnc.1 cells) and cd4 molecule (ifn-g unstimulated r-tnc.1 cells). after prolonged incubation, significant inhibition was obtained using anti-mhc class i mab (ifn-g unstimulated r-tnc.1 cells). almost all mabs which were inhibitory in the binding assay were inhibitory in the engulfment assay (6h), namely mab to cd2, cd4, cd8, cd90 molecule (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells) and mhc calss i and mhc class ii molecule (ifn-g unstimulated r-tnc.1 cells). our results also suggest the involvement of cd11a/cd18 dependent -cd54 independent pathway in adhesion and cd11a/cd18 dependent -cd54 dependent pathway in emperiopolesis. the obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by r-tnc.1 cell line are tightly regulated processes in which multiple adhesion molecules are involved. the crucial roles of cytokines in shaping t cell responses have been documented in both healthy and disease conditions. interleukin-27 (il-27), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. il-27 favours naï ve cd4 t cell differentiation into th1 cells to the detriment of th17 or th2 differentiation. the il-27 receptor (il-27r) is a heterodimer composed of tccr, which confers ligand specificity, and gp130, a signal transducing chain that is utilized by several other cytokines. il-27 has been demonstrated to promote cytotoxic lymphocyte functions of mouse cd8 t cells, but the potential impact of il-27 on human cd8 t cells has not been elucidated. our goal is to investigate the impact of il-27 on human cd8 t cell functions. we used peripheral blood mononuclear cells (pbmc) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. we first assessed whether the il-27r is detectable on ex-vivo t cells using flow cytometry. we observed a greater proportion of cd8 than cd4 t cells expressing the complete surface il-27r (gp130+tccr). however, we detected high amounts of intracellular tccr in both, cd4 and cd8 t cells, but only polyclonal activation (anti-cd3) of cd8 t cells led to an actual increase of il-27r surface expression. purified cd8 t cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. il-27 activated stat1 and stat3 signalling with rapid kinetics in both cd8 and cd4 t cells, indicating the capacity of il-27 to signal through these molecules. addition of il-27 to anti-cd3 activated cd8 t cells led to a significant dose dependent increase of proliferation (as measured by cfse-based assay) and ifn-gamma and granzyme b production (determined by intracellular staining). these results demonstrate a pro-inflammatory impact of il-27 on human cd8 t cells. defects in immune regulation could result in the breakdown of immune tolerance leading to development of multiple sclerosis (ms). the pd-1/pd-l1 pathway is associated with production of the immunoregulatory cytokine il-10, the suppression of t lymphocytes proliferation by inhibition of akt phosphorylation (pakt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in ms. we analysed by flow-cytometry the surface expression of pd-l1 and pd1, as well as myelin basic protein (mbp)-stimulated il-10 production, pakt inhibition, and apoptosis (annexin v), in 50 ms patients with relapsing-remitting disease. twenty-six patients were diagnosed as being affected by acute disease (ams); 24 had a diagnosis of stable disease (sms). results showed that: 1) pd-l1 -expressing cd14+ and cd19+ cells are reduced in ams compared to sms individuals (p=0.04); and 2) pd1 expression is increased in cd4+ t cells of sms individuals and is comparable on cd8+ t cells of ams and sms patients. this is associated with a significant decrease in il-10 production by mbp-stimulated cd14+ and cd19+ cells of ams patients (p=0.03). additionally, cd8+ anexin v+ (av+) cells were diminished and cd8+ pakt+ cells were higher in ams compared to sms patients, while similar percentages of cd4+av+ and cd4+ pakt+ were observed in both groups of individuals. data herein show that the impairments of the pd-l1/pd-1 pathway seen in ams patients result in a reduced mbp-specific il-10 production by cd14+ and cd19+ cells as well as in a reduced apoptosis (annexin v) and an augmented proliferation (pakt) of mbp-specific cd8+ t. the pd1/pdl1 pathway plays an important role in the pathogenesis of multiple sclerosis. monitoring of the expression of these proteins could be a novel diagnostic tool. anti-4-1bb in cd8 cells. this difference could be due to down regulation of cd28 by activated lymphocytes and possible preferential response of cd8 cells to anti-4-1bb costimulation. moreover, increase in ifn-g concentration in costimulated cultures also may enhance the suppressive function of mscs which again could explain the inability of costimulation in proliferation recovery. likewise, reducing tgf-ß by costimulation is not sufficient to abolish suppressive effect of mscs. in overall, these results suggest that lack of costimulation expression by mscs is not the mechanism of msc suppression and other mechanisms are involved. cytotoxic t lymphocytes (ctls) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the ctl, the immunological synapse (is). t cell receptor (tcr) enrichment at the is is one of the early and key events of is formation. objectives: soluble nsf attachment receptor (snare) proteins are required in almost all fusion events in cells. in the present study we tested if the snare protein syntaxin7 (stx7) is part of the is and whether it serves as a key player of is formation and/or the fusion process itself. methods: pcr-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (tirf) were used on primary human ctls to test the function of stx7. rna interference technique was also used to down regulate stx7 expression in primary human ctls. results: we identified stx7 in ctls by pcr and immunocytochemistry. stx7 accumulates at the is after ctl/target cell contact. when stx7 function was blocked by overexpression of a dominant negative stx7 mutant (deletion of the transmembrane region), functional studies with tirf showed a reduced accumulation and fusion of lytic granules at the is. furthermore, confocal studies showed a loss of tcr accumulation at the ctl/target contact side. conclusion: these results imply that the snare protein stx7 is present at the is and moreover is required for is formation in ctls. the observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. objectives: despite the 20 years history of mouse t h 1 and t h 2 subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. we have developed mouse t h hybridomas to study these differences between polarized t h cells. the in vitro established hybridomas were first characterized as t h 0, t h 1 or t h 2 phenotypes, based on their cytokine production (il-2, ifng or il-4). a comparative analysis of t-bet, ifng and il-4 mrna levels was also done on quiescent and activated t h hybridomas. in the present study, the ca 2+response, membrane raft expression/organization, k + -and ca 2+ -ion channel expression/function and sensitivity to apoptosis (aicd) were compared in these hybridomas. expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. ion channnels were functionally analyzed by patch-clamp technique. apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, dna fragmentation) and flow cytometry. results: expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin b-staining) showed the following rank: t h 1 g t h 0 g t h 2, although the membrane cholesterol level (detected with anti-cholesterol ab, ac8) was similar in the three cells. in connection, tcr displayed stronger colocalization with rafts and appeared more polarized in t h 1 cells upon activation than in t h 2 cells. t h 1 cells produced a more sustained calcium response with higher amplitude than t h 2 cells to the same tcr-mediated triggering signal. interestingly, this does not coincide with the expression of cav1.2 and kv1.3 ion channels, major functional determinants of the sustained calcium influx. t h 2 cells expressed the highest levels of these two ion channels. there were also marked differences in their sensitivity to activation induced apoptosis (aicd) as assessed by three different markers of apoptosis. the results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these t h hybridomas may be responsible for the observed differences in their functional characteristics. objectives: bone morphogenetic proteins (bmps) belong to the tgf-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. whereas tgf-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of bmps in b lymphoid cells remains more elusive. in this study we investigated the effects of bmps on mature human b-cells. methods: b cells were isolated from peripheral blood of healthy donors using cd19-dynabeads. cd19 + isolated cells were facs sorted into cd19 + cd27naïve b or cd19 + cd27 + memory b cells. dna synthesis was measured by 3h-thymidine incorporation, immunoglobulin (ig) levels in cell supernatants were measured by elisa and phospho-protein levels were measured by western immunoblotting analysis. results: all bmps significantly suppressed anti-igm-induced proliferation of cd19 + cd27naï ve b cells, of which bmp-6 and -7 were most efficient (40 % suppression). similarly, all bmps suppressed cpg-induced proliferation of cd19 + cd27 + memory b cells by 40 -50 %. to induce differentiation, both naï ve and memory b cells were stimulated with cd40l and il-21. this increased the production of igm, iga and igg 10 -100-fold compared to medium control, whereas addition of bmps inhibited the production of all ig classes. all bmps highly induced phosphorylation of smad1/5/8 in cd19 + b cells. the mechanisms for how bmps mediate their inhibitory effects are currently being explored in more detail. conclusion: bmps have prominent inhibitory effects on anti-igm-and cpg-induced proliferation of naive and memory human b cells, respectively. they also suppress cd40l/il-21-induced production of igs in mature human b cells. s. gutenberger 1 , k. warnatz 1 1 university medical centre freiburg, freiburg, germany background: signals through the b cell receptor (bcr) and co-receptors are essential for the survival, differentiation and effector function of b cells. the stimulation of the bcr initiates several independent but interrelated signaling pathways. one important pathway leads to the activation of mitogen activated protein kinases (mapk) and especially the phosphorylation of extracellular signal-regulated kinases 1 and 2 (erk 1/2). in a subgroup of patients with common variable immunodeficiency (cvid) we have previously demonstrated intrinsic defects in the activation of b cells revealed by the insufficient cd86 upregulation and proliferation after b cell receptor (bcr) stimulation. therefore we assessed signaling pathways downstream of the bcr in order to identify defects in the activation of b cells. methods: pbmc of 20 hd and 25 cvid patients were stimulated by anti-igm. different igm expressing b cell subsets were analyzed separately for erk1/2 phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to erk1/2. to increase the signal intracellular phosphatases were inhibited by h2o2. as markers of activation and initiation of proliferation, cd86 and ki67 expression were measured after 2 days of in vitro stimulation. k. theil 1 , p. aichele 1 1 immh university freiburg, immunology, freiburg, germany type i interferons are homone-like molecules that are produced early after viral and bacterial infections. they signal via the type i interferon receptor (ifnar) and have pleiotropic effects on different cells of the immune system. their best known function is the antiviral activity. to test the direct effect of type i interferons on cd8 t cells in vivo we adoptively transferred lcmv glycoprotein specific tcr transgenic p14 cd8 t cells that are deficient in type i interferon receptor (ifnar-/-) into wild-type b6-recipient mice and compared their expansion with wild-type (wt) p14 t cells after viral infection. we could demonstrate a severe impairment in the capacity of p14 t cells lacking type i ifnr (ifnar-/-) to expand after lcmv infection. following infection of recipient mice with recombinant vaccinia virus, recombinant vsv (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing lcmv glycoprotein, p14 t cells expansion was considerably less dependent on type i ifnr expression. therefore direct type i ifn signalling is essential for cd8 t cell expansion and survival only after lcmv infection. our experiments showed that the lcmv generated cytokine milieu is responsible for the failure of expansion of ifnar-/-t cells during lcmv infection. a suitable model for elucidating the impact of the lcmv generated cytokine milieu is the transfer of p14 t cells into h8 mice. h8 mice ubiquitously express the lcmv immunodominant glycoprotein-epitope gp33-41. therefore the antigen-presentation can be uncoupled from the lcmv induced cytokine milieu when the h8 mice are infected with lcmv8.7. this is a lcmv variant that has got a point mutation in gp33-41 and consequently cannot be recognized by the p14 t cells. s. frischbutter 1 , r. baumgrass 1 1 deutsches rheuma-forschungszentrum, signal transduction, berlin, germany antigen-specific stimulation of t helper cells induces activation of the main transcription factors nfat, nf-kb and ap1 which are important for expression of cytokines such as il-2, ifng and il17. it is known that the immunosuppressive drug cyclosporin a (csa) blocks the activity of the ser/thr phosphatase calcineurin and thereby the activation of the transcription factor nfat. however, we and others observed that this drug also inhibits the activation of nf-kb. to detect targets of calcineurin within the nf-kb pathway we analyzed phosphorylation and degradation levels of different nf-kb signaling proteins in the presence of csa and other calcineurin inhibitors. we found that phosphorylation of the signaling protein bcl-10 was prolonged in cells treated with inhibitors. our data do not indicate an enhanced bcl-10 phosphorylation but rather an inhibition of bcl-10 dephosphorylation. furthermore, calcineurin and bcl-10 co-precipitated with each other. interestingly, this interaction was observed only in t cell receptor-but not in tnfa-stimulated cells. in our proposed model, we hypothesize that calcineurin interacts with the carma/bcl-10/malt1 signaling complex and dephosphorylates bcl-10 and, thus, promotes nf-kb activation. therefore, calcineurin is not only a hub for nfat but also for nf-kb activation. a. t. fulop 1 , j. lamoureux 1 , c. fortin 1 1 université de sherbrooke, medicine, sherbrooke, canada objectives: aging is accompanied by a decrease in immune functions, called immunosenescence. the exact cause is still not known. changes in t cell subpopulations, thymic involution were invoked. we have demonstrated that the signal transduction is altered with aging. in the present work we studied the negative regulatory molecules in the t cell signaling to explain the altered activation of t cells with aging leading to decreased clonal expansion. methods: 25 healthy young and elderly subjects were studied. lymphocytes were separated by fycoll-hypaque. the molecules participating in the negative control loop of lck were studied by western blot and confocal microscopy. the surface expression of ctla-4 has been studied by facscan. the translocation of the molecules in the membrane lipid rafts (mlr) was also studied by western blot. the activity of phosphatases was also determined. results: we found that the phosphorylation of pag was altered with aging explaining the decreased release of csk from mlr and the decreased lck activation. the activation of fynt was also altered. the phosphatase activity studies showed an increase in their activities with aging. the ctla-4 expression was higher after stimulation in t cells of elderly. there was differences between cd4 and cd8 t cells with aging. conclusion: these results suggest that the negative regulation is preponderant in t cells with aging on the positive activation and as such explaining the defect in t cell functions with aging. this opens new therapeutical avenues in the future. in contrast to other members of the tumour necrosis factor superfamily, fas ligand (cd95l) contains a cytosolic proline-rich domain (prd) that enables interactions with sh3 and ww domain proteins. since fasl surface expression is regulated by adam10-mediated ectodomain shedding and fasl might be subsequently released into the cytosol by regulated intramembrane proteolysis (riping) through the secretase-like enzyme sppl2a, we are interested in defining interactions involving the generated intracellular fragment of fasl. employing a monoclonal antibody directed against the intracellular domain of fasl, we observed that previously described fasl-interacting proteins of the pch family selectively bind to the full length molecule but not to n-terminal fragments (ntfs). in order to identify other sh3 domain proteins that potentially interact with the riped fasl prd, we used a sh3 domain phage display library containing all 288 sh3 domains expressed in humans. the screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. it could be demonstrated that tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the snx9 family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known fasl-interacting proteins. of note, in contrast to pch proteins, the snxs bound both ntfs and unprocessed fasl, indicating that individual interactors might influence different facets of fasl biology. in conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of fasl to the full length protein or ntfs. this more detailed glance at the fasl interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conseoptimal t cell activation requires the engagement of the t cell receptor (tcr) by the specific mhc/antigen complex and costimulatory signals as the interaction of b7 family members on antigen-presenting cells with cd28 on t cells. remarkably, whereas classical glucocorticoids (gcs) effectively suppress solely tcrtriggered t cell activation in vitro, additional cd28 co-stimulation leads to gc-resistance. in this study, we compared the non-steroidal selective glucocorticoid receptor agonist (segra), compound12, with classical gcs regarding their suppressive effect on cd28-costimulated t cells. human primary t cell subpopulations and jurkat cells were stimulated in vitro with plate-bound anti-cd3 and anti-cd28, and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. remarkably, a clearly improved inhibition of ifn-gamma secretion was observed in cd28-costimulated human memory/effector cd4+ t cells by compound12 than by classical gcs. interestingly, apoptosis and activation antigen expression were similarly regulated. improved inhibition of lymphokine secretion by compound12 was also seen after pma / ionomycin stimulation of human primary t cells and jurkat cells. when investigating the in vivo effects of compound12 and prednisolone in acute and subacute dnfb-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of t cell-dependent skin inflammation when treating before hapten challenge. in contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for compound12 than prednisolone. when evaluating possible mechanism for the increased activity of compound12 in inhibition of t cell activation we got hints for a specific inhibition of the calcineurin pathway by compound12 which was not prevented by the partial gc receptor antagonist, ru-486, in vitro. moreover, in vivo we observed less induction of il-1beta and tnf-alpha by pre-treatment with compound12 than with prednisolone. our data indicate that the non-steroidal segra, compound12, may represent a promising drug candidate for the treatment of t cell-dependent inflammatory diseases where therapy with classical gcs is hampered by t cell resistance. influenza a infection of b6 mice elicits robust cd8+ t cell responses, with virus-specific cells showing a distinct pattern of cytokine production: tnfa+ cells always express ifng; and il-2+ cells are contained entirely in the ifng+tnfa+ subset. interestingly, the co-expression of ifng and tnfa varies for different epitope specificities. almost all ifng+ pa 224 -specific cells also express tnfa, but only about half of the ifng+ np 366 -specific cells co-express tnfa. this was originally linked to the avidity of the responding population for the specific peptide/mhc complex, with the ifng+tnfa+ phenotype representing cd8+ t cells with higher avidity and a more differentiated phenotype. however, the same cytokine pattern is seen in adoptively transferred cd8+ t cells expressing a clonal tcr, implying avidity alone cannot control development of cytokine profiles. co-expression of ifng and tnfa by adoptively transferred cfse-labelled ot-i cells following infection with influenza a virus expressing ova 257-264 peptide shows a close correlation with division in vivo. early after antigen encounter (0-2 divisions) the vast majority of cells express only tnfa. after 3-4 divisions cells begin to co-express ifng and tnfa. the emergence of an ifng+tnfa-phenotype increases with subsequent divisions (4-6 divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both b cells and cd4+ t cells. titration of adoptively transferred ot-i cells, which controls the level of expansion in vivo, reveals that more cd8+ t cells develop an ifng+tnfa-phenotype with increased expansion. thus we conclude that while tcr avidity and co-stimulation can impact the differentiation of cd8+ t cells, expansion plays a very important role in the regulation of cd8+ t cell effector function. in addition to its chemo-attractant function, sdf-1a (stromal-cell derived factor-1a, cxcl12) has been described to costimulate cd4 + t cell during tcr triggering. our objective is to clarify the mechanism regulating this costimulatory activity. tcr-driven proliferation of human cd4 + t cells was increased by immobilized sdf-1a to a level similar to that obtained with the costimulatory molecule cd28. as visualized by real time confocal microscopy, t cells entering in contact with sdf-1a formed a tether and displayed an active scanning activity. since sdf-1a induced a similar activity in t cells stimulated with a sub-optimal dose of anti-cd3 mabs, it is conceivable that the sdf-1a-driven scanning may favour productive tcr engagement. to test this hypothesis, we are studying the effect of sdf-1a on tcr internalization, calcium mobilization, mapk activation and actin cytoskeleton reorganization. we are also studying the role of sdf-1a in the context of cd4 + t activation by antigen-presenting cells secreting sdf-1a. this study should help us to better define how sdf-1a modulates cd4 + t cell activation beyond its chemo-attractant function. background: propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. caffeic acid phenethyl ester (cape), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. in this study, the effect of cape on the functions of t cells, which play the major role in chronic airway inflammation of asthma, was evaluated. method: cd4 + t cells isolated from human peripheral mononuclear cells by automacs were stimulated with anti-cd3 and anti-cd28 antibodies and cape for 2 days. cytokine levels were dertermined by elisa and lymphoproliferation was analyzed by 3h-thymidine incorporation method. signaling pathway of t cells was studied by western blot. result: it was found that cape significantly inhibited ifn-g and il-5 production and lymphoproliferation in cd4 + t cells stimulated by anti-cd3/cd28. cape could inhibit nuclear factor-kb (nf-kb) activation, but not mitogen-activated protein kinase (mapk) family phosphorylation in t cells. cape could also inhibit akt phosphorylation. conclusion: these results indicated that cape inhibits cytokine production and lymphoproliferation of t cells which might be related to the nf-kb and akt signaling pathway. this study also provided a new insight into the mechanism of cape in immunology and the rationale for propolis in the treatment of allergic disorders. objective: upon activation, cd4 t cells express a variety of molecules on their surface, such as mhc-class ii, cd80, cd86, cd70, whose ligands are constitutively expressed on resting t cells. whereas these molecules are physiologically expressed on antigen presenting cells, their function on t cells is not understood. we tested the hypothesis that activated cd4 t cells might induce t cell proliferation and differentiation from cd4 resting t cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. methods: cd4 t cells from the peripheral blood of healthy donors were co-cultured with fixed activated t cells from the same donor. after 5 days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. results: cd4 memory t cells but not naive t cells proliferated in response to contact with activated t cells. these cells showed a mild activated phenotype assessed by the expression of cd25, cd30, and cd69. analysis of the cytokine profile of these cells revealed the differentiation of il-10-and ifn-g-double-producing cells in response to contact with th1 effector cells, and il-4-producing cells in response to contact with th2 effector cells. the levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-cd3/cd28 stimulation. whereas neutralization of ifn-g or il-4 during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. blocking particular receptor/ligand interactions by neutralizing antibodies against hla-dr, cd70, cd80, and cd86 could not prevent cytokine production induced by t-t cell interaction. however, simultaneous addition of all antibodies significantly inhibited cytokine production to 64-85 %. conclusion: interaction of cd4 memory t cells with activated t cells resulted in the production of the cytokines il-4, il-10, and ifn-g. given the immunomodulatory capacity of il-4 and il-10, these findings might indicate a novel potential negative feedback mechanism to control t cell-driven immunity. a. nasir 1 , s. thompson 1 , j.j. murphy 1 1 king's college london, division of immunology infection and inflammatory disease, london, united kingdom the murine bcl1 leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines il-2 and il-5 and this is characterised by a marked reduction in proliferation and production of large amounts of secreted igm. these cells were observed to express significant levels of the zinc fingercontaining protein zfp36l1 by western blot analysis. this protein is reported to act in post-transcriptional regulation of gene expression by binding to au rich elements (ares) of mrnas of certain genes and consequently promoting mrna degradation. at a cell functional level, zfp36l1 has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. cytokine-induced bcl1 differentiation was observed to be associated with downregulation of zfp36l1 protein. in an attempt to determine whether zfp36l1 downregulation was directly linked to bcl1 differentiation, a zfp36l1 shrna expressing lentivirus (psicor) was employed to knockdown zfp36l1 expression. this reagent downregulated zfp36l1 expression very effectively . shrna infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. zfp36l1 shrna infected cells also produced more secreted igm per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. these results are consistent with a role for zfp36l1 downregulation in promoting bcl1 plasmacytoid differentiation. vidual lysates of peripheral blood lymphocytes (pbl) of 32 patients with igg multiple myeloma and healthy controls were investigated for the expression of sialic acid (sa), galactose (gal) and n-acetylglucosamine (glcnac), the sugars known to specify the glycoforms of human serum igg. the degree of glycosylation and signaling status of all 32 isolated myeloma igg bcrs were correlated and compared with the glycosylation of the igg paraproteins isolated from sera of the same patients. it was shown that bcr igg in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of igg bcr is associated with higher levels of tyrosine phosphorylation (signaling activity) of the igg bcr supramolecular complex and that bcr igg and serum igg paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. the results suggest that the development of the malignant clone in mm from postswitch b cells expressing igg bcr at their surfaces to plasma cells secreting igg paraprotein may be followed by permanent glycosylation changes in the igg molecules. caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to tpen. conclusion: the signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. in light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than tms, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. although best known for its pro-apoptotic function, it seems clear now that cd95 (fas, apo-1) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. we investigated effects of fas co-ligation during tcr/cd3/cd28-triggered activation of freshly isolated human t-lymphocytes. to this end, tcr-triggered cells were incubated in presence or absence of different ligand concentrations of anti-apo1 mab, faslfc or faslstrepfc fusion proteins, or leucin zipper (lz-)cd95l. interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and t cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked tcr-induced cell proliferation without inducing cell death. the positive costimulatory effect of fas at low concentrations is associated with elevated il-2 and ifng production, upregulation of activation markers, adhesion molecules and cell-cycle regulating cdks and cyclins. in addition, we observed an increased activation of important signalling molecules including mapk and caspases. using pharmacological inhibitors, we demonstrate that fas is internalized upon ligation. we also observed an increased tcr internalisation following fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal t cell activation. in stark contrast, most fas ligands at high concentrations almost completely inhibited cell proliferation of tcr-triggered lymphocytes. in this context, crucial events associated with t cell activation, i. e. tyrosine and erk1/2 phosphorylation, the expression of various activation markers, the il-2 production and caspase activation were almost completely abrogated. these findings highlight that fas-triggering accelerates or blocks t cell activation, depending on the strength of the stimulus. in addition, we provide further evidence for an anti-apoptotic function of fasl during signal initiation in human t lymphocytes. sponsored by the dfg (sfb415) and the medical faculty kiel (to oj) it has been shown that glycosylation of cell surface proteins controls critical t cell processes, including homing, thymocytes maturation, activation, and cell death. plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic t cell activation stimulus. the galb1,3galnac-specific lectin from amaranthus leucocarpus (all) shows a differential binding pattern to murine thymocytes and peripheral cd4+ and cd8+ t cells. in addition, mitogenic stimulus increase 3-fold the all binding to cd4+ t cells. previous studies in human pbmc showed that all binds to human cd4+ t cells and all-binding increased after a mitogenic stimulus using total cell cultures as murine studies. these data suggest that all detects selectively activation-related changes in cd4+ t cell surface carbohydrate but none study has been performed to examine the all effect on human t cell activation. to examine the effect of all on human t cell activation, we analyzed the anti-cd3-dependent activation of purified cd4+ t cells from pbmc in presence or absent of all by measuring proliferation using cfda-se staining, expression of the surface activation marker cd25 and calcium influx by flow cytometry. results showed that all did not induce significantly t cell proliferation or cd25 expression, but enhanced the anti-cd3-dependent proliferation and cd25 expression of purified cd4+ t cells. analisis of calcium influx showed that all enhanced anti-cd3 dependent calcium influx. our findings indicated that all alone does not affect t cell activation but suggested that all induces a costimulatory effect on human cd4+ t cells by up-regulating t cell activation mediated by anti-cd3 stimulus, as further studies have to be performed to elucidate all-induced costimulatory effect. financed in part by papiit-unam (in214609) a. the adaptor protein lat (linker for activation of t cells) has a prominent role in the transduction of intracellular signals elicited by the tcr/cd3 complex. upon tcr engagement, lat becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. the balance between cell survival and cell death is critical for normal t cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as cd95 receptor. it has been previously demonstrated that cd95 ligation in t cells induces the proteolytic cleavage of several adaptor proteins, including gads, slp-76, slap-130 and lat. given the dual role of lat as a transducer of activation and negative signals in t cells, we have analyzed the role of the lat cleavage in t cell functions and studied the proteases responsible for this cleavage. objective: the study is designed to explore preliminarily the need of t cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of t cells, and by detecting the expressions of il-rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (hpbls). the results may lay a theoretic and experimental basis for developing the condition media qualified especially to t cell culture. methods: pbls were isolated , and cultured in different media. both immunocytochemistry staining and cell enzyme linked immunosorbent assay (celisa) were used to detect the expressions of il-1r, il-2r, il-3r, il-7r, il-12r, il-18r, cd27, cd28, cdw137( 4-1bb), cd 95(fas) and cd178 (fasl) on hpbls in different cultured time, i. e. 0d, 1d, 3d, 5d, 7d, 9d, 11d and 14d. using typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. to evaluate the cellular activity, growth situation and cell cycle of t cells, both mtt and fcm analysis were also performed separately. 1. the expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. 1) the expressions of membrane immune molecules before cultured. 2) expressions of the membrane molecules on hpbls during culturing. 5% fbs rpmi 1640 group (1640 group), il-2 group, pha group... (1) mtt assay. (2) proliferative times and growth curves of hpbls... 1. during cultured in vitro, there are expression changes of the il-1rs (il-1ra, il-2ra, il-2rg, il-3r, il-7r, il-12r, il-18r), co-stimulatory molecules (cd27, cd28, 4-1bb) and apoptosis associated molecules (fas/fasl) on hpbls in different time and cultured media. the expression patterns of the most molecules checked are similar in 1640 group, il-2 group and pha group, but the rests are different. 2. our data also suggest that the hpbls cultured in cd3mcab+cd28mcab+il2+il1a group has a great proliferative potential compared with the other groups. using this condition medium, may have a practical prospect to tumor therapy. 3. celisa will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale. f. beceren-braun 1 , r. tauber 1 1 zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany l-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. apart from its role in adhesion, l-selectin functions as a signal transduction molecule. crosslinking of l-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. in addition to process signals coming from outside of the cell, the intracellular part of l-selectin (lscyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p56lck and the ras/rac2 signalling pathway (1) followed by mitogen-activated protein kinases (2) and c-jun n-terminal kinase (1), which leads to an enhanced binding of l-selectin to soluble ligands (3). in our previous work we described an association of lscyto with isozymes of the pkc family which phosphorylate the receptor on serine residues (4). here we show that the protein phosphatase 2a inhibitor phapii is a novel direct interacting partner of the lscyto. we propose a model in which the l-selectin mediated signalling is regulated by the interaction of pkc, pp2a and phapii: phapii binds to the unphosphorylated lscyto. upon l-selectin crosslinking lscyto is phosphorylated, pha-pii dissociates and inhibits the phosphatase pp2a. in addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of l-selectin. v. heissmeyer 1 , e. glasmacher 1 1 helmholtz center munich, molecular immunology, munich, germany during self-antigen recognition, roquin dependent posttranscriptional downregulation of icos prevents t cell help to b cells and autoantibody production. the molecular mechanism by which roquin interferes with icos translation remained unclear. we have identified two critical regions in roquin. the amino-terminus is required for rna binding and can be functionally replaced by conserved sequences from its paralog mnab. the carboxy-terminus mediates p body localization and has specialized in roquin for efficient repression of icos in t cells. using knockout cells of dicer or ago1-4 genes, we prove that roquin mediated repression of icos occurs in the absence of mirisc formation. instead, roquin function required intact p bodies, and was impaired after knockdown of lsm1 and rck or expression of dominant-negative gw182. interestingly, roquin activity is blocked through induced mirisc formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses. s objectives: upon encountering their antigens, naï ve t cells are activated and driven to clonal expansion and differentiation into armed effector cells. according to the two-signal hypothesis, the induction of an optimal cd4 + t-cell immune response requires both antigen-specific and co-stimulatory signals. in contrast, stimulating naïve cd8 + t cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. thus, cd8 + t cells require additional signals for full activation and further differentiation into effector cells. methods: in this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for cd8 + t-cell activation and differentiation. naïve cd62l hi cd44 lo cd8 + t cells were sorted and stimulated by anti-cd3 and anti-cd28 antibodies. results: firstly, we show that the activation and differentiation of cd8 + t cells require il-2 provided by activated cd4 + t cells at the initial priming stage after stimulation. secondly, this critical il-2 signal is delivered through il2rbg of cd8 + cells and is independent of il-2ra. besides promoting cell proliferation, il-2 stimulation increases the amount of ifng and granzyme b produced by cd8 + t cells. conclusion: therefore, our studies demonstrate that a full cd8 + t-cell response is elicited by a critical temporal function of il-2 released from cd4 + t cells, providing mechanistic insights into the regulation of cd8 + t cell activation and differentiation. most antigenic peptides recognized by cd8 t lymphocytes are produced through degradation of intracellular proteins by the proteasome. however, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. mage-a3168-176 is a tumor antigenic peptide presented by hla-a1 and widely used for vaccination of melanoma patients. we observed that proteasome and tppii inhibitors failed to block presentation of the antigen by tumor cells. however, processing of this peptide occurred in the cytosol because tap inhibition prevented its presentation. to characterize the cytosolic peptidase producing mage-a3168-176 we setup an in vitro digestion assay using a 20-mer precursor peptide encompassing the sequence of the antigenic peptide. we observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. this production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. this inhibitor also blocked the presentation of mage-a3168-176 by tumor cells. by electroporating hla-a1 cells with a precursor peptide blocked at the c-and the n-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. one such enzyme is insulin-degrading enzyme (ide). we observed that depletion of ide abolished the capacity of a cytosolic fraction to produce the antigenic peptide. furthermore, recombinant ide was able to produce the peptide in vitro from the precursor peptide. lastly, silencing of ide with sirna reduced presentation of the peptide by tumor cells. with tppii, ide is the second example of a proteasomealternative pathway in the production of class-i restricted peptides. antigen-specific t cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. immune escape, due to impairment of hla dependent tumor epitope presentation is believed to be one major reason for this failure. to identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane mart-1/melan-a tumor antigen were exposed to two or three rounds of brief co-culture with mart-1/melan-a 26-35 specific cytotoxic t lymphocytes (ctls). immune selected melanoma cell clones, being resistant to lysis by mart-1/melan-a 26-35 ctls due to impaired epitope processing were further investigated. our results show that in addition to previously described immune evasion mechanisms like down regulation of mhc class i and mart-1 expression, the ifn-gamma independent endoplasmic reticulum associated degradation (erad) pathway is crucial for mart-1/melan-a 26-35 epitope generation. moreover, deregulation of several erad components is essentially responsible for the observed immune escape of the immune selected melanoma cells. in support, re-expression of down-regulated erad components in ctl-resistant melanoma cells completely restored immune recognition by mart-1/melan-a 26-35 ctls. thus, our studies demonstrate for the first time that erad not only plays a central role in the production of cd8 + t cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. studies of t cell responses to hen egg lysozyme suggest that several conformers of peptide-mhc class ii complexes can be generated for a single peptide epitope and that distinct cd4 t cell repertoires known as type a and type b recognise these different conformers (lovitch and unanue, immunol rev 207: 293-313, 2005) . type a t cells recognise peptide-mhc complexes generated from intact proteins after intracellular antigen processing under h2-m (dm) control, where as type b t cells respond to synthetic peptides in the absence of dm editing, but fail to respond to processed intact protein. type b t cells escape thymic deletion in mice (petersen et al., immunity 11: 453-462, 1999) , with implications for autoimmunity. so we studied whether type a and type b recognition patterns occur in t cell responses to autoantigens such as the rheumatoid arthritis (ra)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type b t cells are found in inflamed joints. lymph node cells from aggrecan-immunised balb/c mice proliferated in response to intact aggrecan and to the immunodominant peptide 84-103, whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. t cell hybridomas generated from 84-103 peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type a and type b t cell recognition. responses to staggered and alanine-substituted peptide sets showed that type a and b t cell hybridomas recognized the 84-103 epitope in the same register, consistent with this peptide epitope binding to mhc in distinct conformers. type b t cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce raassociated aggrecanases. our data suggest that inflammation generates extracellular peptides that activate type b t cells. we are also characterising human type b t cell responses as well as searching for type b t cell ligands in synovial fluid from ra patients. we propose that extracellular cartilage degradation generates ligands that induce autoreactive type b t cell responses which participate in the pathogenesis of autoimmune arthritis. a to cope with mhc i antigen presentation hcmv encodes for several post-translational strategies which have been extensively studied in transfected cells. in this study we analysed the plc in naturally hcmv-infected cells and monitored the composition of the plc throughout hcmv replication. metabolic labeling experiments revealed the absence of tapasin incorporation into the plc. in contrast, western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. tapasin mrna levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. taking advantage of a novel method, in which newly transcribed rna is selectively labeled and analysed (dölken et al, 2008), we found, after an initial induction at 8 hrs p. i., a strong inhibition of tapasin transcription at 24 hrs p. i. furthermore, also reduction of tap1 and tap2 transcription was observed contrasting to the elevated levels of erp57 and mhc i transcripts. importantly, ectopic expression of tapasin restored the incorporation of tapasin into the plc in hcmv-infected cells. the data indicate that hcmv controls mhc i antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. most peptides presented by mhc class i molecules are produced by the proteasome during degradation of intracellullar proteins. two main proteasome types have been described, differing in their content of catalytic subunits. the standard proteasome comprises catalytic subunits ß1, ß2 and ß5, which are replaced by their ifng-inducible counterparts ß1i, ß2i and ß5i in the immunoproteasome. the thymoproteasome represents a third proteasome type, where catalytic subunit ß5i is replaced by a thymus-specific subunit ß5t. the standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to ifng and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. we produced a panel of novel antibodies that recognize subunits ß1i, ß2i, ß5i and ß5 in their native form. using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. one comprises ß1,ß2 and ß5i (single intermediate proteasome), and the other comprises ß1i, ß2 and ß5i (double intermediate proteasome). we quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent 10-20 % of the total proteasome content of those tumor cells. they are also present in dendritic cells, where they represent about 50% of the proteasome content. we characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor ctl. with respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( tapasin is a multi-functional protein dedicated to mhc-i biosynthesis; it serves as a structural component in the so called mhc-i peptide loading complex (plc), as a chaperone putatively acting as an active peptide editor and mhc-i quality control mechanism, as an er retention signal for immature mhc-i, and as a chaperone stabilizing tap expression and increasing tap-performance. furthermore, tapasin has been found outside the er, where it has been suggested to regulate retrograde transport of escaped immature mhc-i back to the er from the trans-golgi compartment. the role of tapasin as an active peptide-editor has been debated and we here set out to study the effect of tapasin on binding of peptides of both high-and low-affinity to a human mhc-i allele (hla-a*0201) using protein interaction-and peptide-competition assays. specifically we wanted to in detail compare the binding of two peptides of the same affinity. at high concentrations all of the tested hla-a*0201 binding peptides (tap-transported high-affinity peptide (ttp-ha), signal-peptide of high affinity (sp-ha), tap-transported mediumaffinity peptide (ttp-ma)) induced dissociation of hla-a*0201 from tapasin, but only ttp-ha dissociated hla-a*0201 from tapasin at lower concentrations. using peptide-competition assays against ttp-ma, a peptide of lower affinity, we could show that ttp-ha, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide sp-ha. however, analysis of mhc-i peptide loading in the tapasin-negative cell line lcl-721.220-a2 showed no competitive advantage of ttp-ha compared to sp-ha supporting a role for tapasin as a selective facilitator of mhc-i peptide binding. in conclusion, we here show that peptides of different affinities dissociate hla-a*0201 from tapasin in a dose-dependent manner, and that tapasin facilitates ttp-ha, but not sp-ha replacement of a lower-affinity peptide (ttp-ma). together these data strongly suggest a role for tapasin as a selective facilitator of peptide binding to mhc-i. importantly, this study implies that criteria in addition to peptide-affinity determines whether tapasin will promote peptide binding to hla-a*0201. m. basler 1,2 , c. lauer 2 , m. groettrup 1,2 1 biotechnology institute thurgau, kreuzlingen, switzerland, 2 university of constance, division of immunology, department of biology, konstanz, germany two lmp7-dependent antigens have been described that relied on the 'structural presence' of lmp7 in the proteasome but not on the activity of lmp7. here we have investigated processing of the h-2d b -restricted uty 246-254 epitope of the male minor antigen uty reported to be lmp7-dependent. using splenocytes from lmp7 -/-, lmp2 -/and mecl-1 -/mice we found that the uty 246-254 epitope requires lmp7 and lmp2 but not mecl-1. curiously, a selective lmp2 inhibitor did not interfere with uty 246-254 presentation. objective: we investigated why the deletion but not the inhibition of lmp2 interferes with uty 246-254 presentation. we hypothesized that the 'structural' requirement for lmp2 is based on replacement of the caspase-like activity of b1 in the proteasome. methods: it was determined if t1a mutants of lmp2 and/or b1 can rescue the uty 246-254 epitope. we used a b1-selective inhibitor to determine if the inhibition of the caspase-like activity of b1 preserves the epitope. finally we determined by mass spectrometry if the uty 246-254 epitope embedded within a 25mer precursor peptide is differentially cleaved by lmp2-deficient and proficient immunoproteasomes in vitro. results: we found that t1a mutants of lmp2 and b1 rescue presentation of uty [246] [247] [248] [249] [250] [251] [252] [253] [254] . also inhibition of cells with a b1-selective inhibitor preserves uty [246] [247] [248] [249] [250] [251] [252] [253] [254] presentation. an aspartate in position 7 of the uty 246-254 sequence wmhhnmdli is preferentially used as a cleavage site by lmp2-deficient but not half as frequently by lmp2-proficient immunoproteasomes. the generation of the uty 246-254 epitope relies on the replacement of the caspase-like activity of b1 by lmp2 because the b1 activity destroys the uty 246-254 epitope. this is the first example for the 'structural' requirement of lmp2 for generation of an epitope. eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. thus we have discovered a so far unrecognized mechanism how lmp2 and perhaps also lmp7 and mecl-1 exert their function in antigen processing. a. linnemann 1 , a. musiol 2 , r. lindner 1 1 hannover medical school, cell biology, hannover, germany, 2 hannover veterinary school, graduate school for biomedical sciences, hannover, germany objectives: mhc i molecules are constitutively endocytosed and recycled to the cell surface. this process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on mhc i. in fibroblasts, mhc i is known to internalize via a clathrin-independent, arf6-regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. although this observation suggests that membrane rafts are involved in the internalization of mhc i, no evidence for an association of mhc i with membrane rafts has been found in this cell type. methods: a novel detergent extraction protocol was used to investigate the association of mhc i with membranes rafts. endocytosis of mhc i was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. for characterization of mhc i internalization pathways, dominant negative mutants of gtpases (dynamin and arf6) were overexpressed in 3t3 fibroblasts. we show that antibody-mediated oligomerization of mhc i in 3t3 fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. this change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized mhc i internalized faster and more completely and arrived at different endocytic organelles. the two mhc i internalization pathways differed in their sensitivity towards dominant negative arf6: endocytosis of oligomerized mhc i was not affected, whereas non-oligomerized mhc i endocytosed more slowly and changed its subcellular distribution. unlike transferrin receptor internalization, none of the mhc i endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoa. conclusion: we propose that mhc i switches from an arf6-regulated to a novel, arf6-independent internalization pathway in response to a change in membrane environment induced by oligomerization of mhc i. since mhc i is one of the cellular receptors for sv40 virus and since sv40 binding triggers mhc i oligomerization, this novel pathway may be involved in sv40 uptake. antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. in this study, we tested the effect of sirna-mediated knockdown of 57 rab gtpases, the key regulators of membrane trafficking, on antigen cross-presentation. twelve rab gtpases were identified to be associated with antigen cross-presentation, and among which rab3b, 3c were found to be colocalized with mhc class i molecules at perinuclear tubular structure. tracing with fluorescence protein tagged beta2-microglobulin demonstrated that the mhc class i molecules were internalized from plasma membrane to rab3b and rab3c postitive compartment. moreover, the recycling ligand transferrin was enriched in the rab3b or 3c positive vesicles. furthermore, the rab3b, 3c positive compartment were colocalizd with a fraction of rab27a at a juxtaposition of phagosomes. together these data demonstrate that rab3b and rab3c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. introduction: while the proteasome is thought to generate most of the hla peptidome, other proteases were also proposed to be significant for this process. both t cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model hla peptides. the hla-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the er, binding stability at the cell surface and retrograde uptake of the hla molecules back into the cytoplasm. objectives: the role of the proteasome in hla peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire hla peptidome. the peptidomes of both the authentic membranal and a recombinant soluble form of the hla molecules were collected for analysis at different time points after the inhibition of the proteasomes. the turnover rates of the hla-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. the hla molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and orbitrap tandem mass spectrometry. both the endogenous membranal and soluble mhc molecules were studied in parallel from the same cells. peptides were identified by their ms/ms fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide. results: a few thousands hla-peptides were identified, and for a large portion of them, the turnover rates could be defined. proteasome inhibition did not affect the complexity of the hla peptidomes or reduced significantly the amounts of membranal hla molecules. many peptides were labeled relatively rapidly with heavy leucine, indicating that the hla peptidome contains also the products of newly synthesized and rapidly degrading proteins. the source proteins of the hla peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells. the centrality of proteasomal degradation in hla-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. epstein-barr virus (ebv) is a ubiquitous y-herpesvirus, infecting over 90 % of adults worldwide. it can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for b-lymphocytes and epithelial cells. ebv persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. one such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. however, for ebv replication and dissemination to occur, it must enter the lytic phase. here, over 80 viral proteins are expressed, creating many potential antigens for presentation to cytotoxic t -lymphocytes. ebv can circumvent possible eradication by cd8+ t lymphocytes during the lytic phase by interference with antigen processing and presentation through hla class i in the infected cell. the viral proteins bnlf2a and bglf5 have been shown to achieve this by impairing peptide-loading of hla class i and inducing the degradation of mrnas encoding hla molecules, respectively. a third ebv lytic phase protein, the g-protein coupled receptor (gpcr) bilf1, has now been found to down-regulate cell surface hla class i expression (zuo et al, plos pathogens 2009). this represents a novel function for a virally-encoded gpcr. bilf1 is expressed early in the ebv lytic cycle and is localized predominantly at the cell surface. there it can interact with hla class i molecules, resulting in their internalization and lysosomal degradation. this has a profound effect on the ability of cytotoxic t-lymphocytes to recognize cells displaying antigens derived from ebv proteins. interestingly, bilf1 displays a differential effect on distinct hla class i haplotypes. furthermore, we have shown that the intracellular c-terminal tail of bilf1 is required for its effect on hla class i expression. however, the ability of the gpcr to activate intracellular signaling pathways is dispensable in this regard. thus, by reducing the cell surface expression of hla class i molecules, ebv bilf1 can hinder the recognition of virally-infected cells by cytotoxic cd8+ t lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. t lymphocytes mature in the thymus, generating a non-dangerous t cell repertoire. for the adquisition of tolerance, thymocytes suffer positive and negative selection processes. during t cell maturation, tcrs contact with different mhc-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. to obtain a non-self-reactive t cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of mhc-peptides complexes representative of the proteins that t cells will found in periphery, including tissue restricted antigens (tras)-derived peptides. in the last decade, transcription of tras in thymus has been well-reported. furthermore, the expression of many genes codifying for tras are dependent.on the expression of the autoimmune regulator (aire). aire is mainly expressed in medullar thymic epithelial eells (mtecs), which are involved in negative selection. so far no systematic study have been made to describe the peptide repertoires associated to hla molecules in the thymus. in addition, although many data of tras transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no hla ligand arising from any tra have been reported in thymus. in this report we present the results of analyzing the hla-dr-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. we describe 131 natural ligands, including two peptides derived from semenogelin-1, a tissue restricted antigen expressed mainly in the prostate, and present in semen. using qpcr we demonstrate that semg1 is transcribed in thymus from both male and female individuals. finally, we detected the semg1 mrna expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. the proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for mhc class i antigen presentation. vertebrates express two sets of catalytic subunits, constitutive (beta1, beta2, beta5) and immuno-subunits (beta1i, beta2i, beta5i). deficiency in beta5i results in profound reduction of mhc class i expression, demonstrating the significance of this subunit for efficient antigen presentation. currently, this is attributed to the specific proteolytic activity of the beta5i subunit, its role in the maturation of immunoproteasomes or both. however, re-expression of catalytically inactive beta5i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. here, we show that following infection with listeria monocytogenes induction of beta5i expression increases the cellular proteasome content in the infected organs. our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. further, mhc class i antigen presentation on beta5i-deficient cells could be restored by treatment with d3t, which increases the amount of proteasomes independent of beta5i via induction of mixed proteasomes containing beta1i, beta2i and beta5. consequently, not the lack of the specific proteolytic activity of beta5i or immunoproteasomes, but the reduced proteasome quantity in beta5i deficient cells is the major limiting factor for mhc class i cell surface expression. . we have previously shown that lc, in contrast to ddc, do not express cell surface tlr2, 4 and 5, which results in their inability to respond to both gram-positive and gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. therefore, the question remained what the role is of lc in class ii mhc-mediated activation of anti-bacterial t cells. we determined the capacity of ddc and lc to internalize and process whole bacteria and present bacterial antigens to cd4 + t cells. in vitro generated lcs and ddcs were cocultured with gfp-expressing bacteria and subsequently analysed by clsm and facs for their uptake capacities. furthermore we investigated their capacity to stimulate autologous bacteria-specific t cell lines as a measure for antigen presentation. results: we found that lc are principally able to internalize bacteria, but far less efficient than ddc. moreover, visualisation of bacterial uptake by em revealed different uptake mechanisms by lc and ddc. both in lc and ddc internalized bacteria were detected in the endosomal and lysosomal compartments of the mhcii processing route. nevertheless, presentation of bacterial antigens by lc on mhcii was inefficient compared to that of ddc, as indicated by a low capacity to activate autologous bacteria-specific cd4 + t cells. the presence of exogenous tlr3 and tlr8 ligands did not overcome the differences between lc and ddc, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific t cells is not due to the lack of tlr signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of lc. conclusion: we propose that the epidermis of the skin is an immune-privileged site where lc play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria. e. james 1 , i. bailey 1 , t. elliott 1 1 university of southampton, cancer sciences division, southampton, united kingdom regulatory t cells (tregs) play a pivotal role in the suppression of tumour specific t cell responses. depletion of tregs in balb/c mice results in a robust immunity to the normally poorly immunogenic ct26 colon carcinoma. this response is long lasting and mediated by both cd4 and cd8 t cells. importantly, the treg depleted ct26 specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (a20, c26, bcl1, renca) implying a broader repertoire of response. we have characterised one of these cross-protective antigens, gsw11, which is h2-d d restricted. analysis of the generation of gsw11 in ct26 revealed that the peptide is susceptible to over-processing by the er-resident aminopeptidase eraap. inhibition of eraap in ct26 cells substantially increased the amount of gsw11 present, observed by increased t cell responses to the tumour in vitro and hplc analysis. this increase was in spite of an overall reduction of mhc class i molecules at the cell surface. to investigate whether the increase in immunogenicity following knockdown of eraap would protect mice, we generated stable eraap knockdown (kd) ct26 and immunised balb/c mice. greater than 80 % of mice injected with eraap kd ct26 were found to reject the tumour. analysis of t cell responses revealed the presence of gsw11-specific t cells, however, these responses were small (0.5-1 %). this compared to a much larger response to ct26 (˚5 %). preliminary results indicate that the majority of the t cell responses (non-gsw11-specific) in these mice are directed toward unstable peptide/mhc complexes, possibly indicating presentation of n-terminally extended peptide antigens. this highlights manipulation of the peptide repertoire as a potent tool for the generation of t cell responses in vivo. minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of graftversus-host-disease and in the graft-versus-tumor reactivity in hla-identical stem cell transplantation [1] . the di-allelic hla-a2 restricted minor histocompatibility antigen ha-1 locus codes for the highly immunogenic ha-1 his and the non-immunogenic ha-1 arg nonapeptides, differing in one amino acid. the only difference that could explain the absence of the ha-1 arg immunogenicity was the estimated numbers of cell surface presented copies i. e. 80/cell for ha-1 his and less than 5/ cell for ha-1 arg [2] . as ha-1 his/arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the ha-1 his allele is currently used for boosting the graft-versus-tumor responses after hla matched ha-1 mismatched stem cell transplantation. to elucidate the mechanisms underlying the differential cell surface presentation of the ha-1 allelic peptides, we investigated the impact of the ha-1 his/arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of hla class i-bound peptides. therefore, proteasome-mediated digestion experiments, tap translocation analyses, and hla-dissociation assays with ha-1 his and ha-1 arg peptides were performed. moreover, the crystal structures of hla-a2 in complex with either ha-1 his , ha-1 arg or a ha-1 variant with a citrulline residue at position 3 were determined in order to obtain atomic level insights into the conformation of the hla-a2/ha-1 peptide complexes. our results exclude a role for antigen processing in preventing ha-1 arg to be presented at the cell surface and both the structural and hla-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the ha-1 arg allele in the hla-a2 peptide binding groove [3] . they provide a rationale for the lack of ha-1 arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. proteasomes play an important role in mhc class i antigen processing. exposure of cells to proinflammatory cytokines such as tnfa or ifng leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. immunoproteasomes generate many pathogen-derived cd8 t cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific cd8 t cell response. on the other hand, immunosubunit expression is not essential for development of cd8 t cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear. we observed that mice that lack the immunosubunits lmp7 (ib5) and mecl-1 (ib2) develop a variety of autoimmune responses, including a latent form of t1d (or insulin dependent diabetes mellitus, iddm), following irradiation and bone marrow reconstitution. iddm development in these mice is characterized by inflammation of the islets of langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of cd8 but not cd4 t cells. a cd8 t cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose-6-phosphatase catalytic-subunit-related protein" (igrp) mrna, was identified as an important target of the cd8 t cell response. this epitope, like many other known diabetes-associated epitopes, binds its presenting mhc class i molecule with low affinity. as t cells specific for low affinity binders most likely can escape central and peripheral tolerance while t cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind mhc class i molecules with low affinity, by a higher affine peptide species towards which t cell tolerance exists. thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune cd8 t cells that enter inflamed tissues. endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express hla-ii molecules with compact conformation and are therefore expected to stably bind autologous peptides. the role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response. to study in situ t cell responses without characterizing self-reactive t cells, we have identified natural hla-dr-associated peptides from autoimmune organs that will help finding peptide-specific t cells in situ. here we report the first analysis of hla-dr natural ligands from ex-vivo graves' disease-affected thyroid tissue. using mass spectrometry, 162 autologous peptides were identified from hla-dr-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which hla alleles were these peptides associated in vivo. all but two of the thyroglobulin peptides showed low binding with the corresponding alleles. the two peptides with relatively high binding affinity were presented in the context of dr3 and dr4. analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a lys and arg was observed for all of them, independent of the restricting allele. our data demonstrate that although the hla-dr-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to hla-dr in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. the t cell response generated following herpes simplex virus type 1 (hsv-1) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. despite this, the relative contributions of cd4 + and cd8 + t cells in hsv-1 immunity have yet to be clearly elucidated. to better understand the role of hsv-1-specific cd4 + t cells in immune control we have identified a 13 amino acid epitope derived from glycoprotein d of hsv-1. following flank infection, gd-specific cd4 + t cells were first detected in the draining brachial and axillary lymph nodes (ln) 5-days post-infection (pi), peaking at day 7 and declining thereafter. gd-specific cd4 + t cells were first recovered from the spleen, skin and dorsal root ganglia (drg) at day 6 pi and peaked at day 9. while hsv-specific t cells were first observed in the draining ln at day 5 pi, hybridoma assays showed ex vivo presentation of the gd epitope by brachial ln cells as early as 2 days pi, with peak activity 4 days pi before declining to background by day 7. however presentation of the gd epitope was much more prolonged in vivo as proliferation of transgenic gdspecific cd4 + t cells was observed up to 23 days post-infection in the brachial ln. ex vivo analyses suggest that only cd11c + cells were involved in gd antigen presentation at days 2, 5 and 15 post-infection. subdivision of dendritic cells (dcs) populations indicated that both skin-derived dcs and cd8a + dcs can present the gd antigen to cd4 + t cells at day 2 pi, whereas by day 5 pi the skin-derived dcs were the predominant population presenting the gd epitope. together these data show that following hsv-1 infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. furthermore, we present evidence that different dc populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection. complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. we also verified that the zippers did not influence the substrate "preferences" of the respective erap. our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric erap1 and erap2 unable to form dimers. this observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. conclusion: allosteric interaction of erap1/erap2 in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human er. during the biogenesis of class i molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (cnx) and its associated thiol oxidoreductase erp57. upon assembly of the heavy chain with b 2 m, the class i molecule enters a peptide loading complex (plc) that consists of the tap transporter, tapasin, the calnexin homologue calreticulin (crt) plus associated erp57. both crt and erp57 are required for efficient assembly of peptide-loaded class i molecules and their subsequent expression at the cell surface. we examined functional sites on crt and erp57 to gain insights into their mechanisms of action in class i biogenesis. for crt, its lectin function is thought to be crucial for its association with class i molecules. however, when crt mutants lacking lectin function were expressed in crt-deficient cells, they completely complemented all class i biosynthetic defects. thus polypeptide-based contacts either mediated through erp57 or directly between crt and the heavy chain are sufficient to effect the chaperone and quality control functions of crt in class i biogenesis. we also tested the notion that erp57 must be recruited by cnx or crt to function on class i molecules. we found that the rates of heavy chain disulfide formation were normal in cells lacking cnx, crt or both chaperones. furthermore, an erp57 point mutant that fails to bind to cnx or crt was just as effective as wild type erp57 in normalizing rates of disulfide formation. we conclude that erp57 does not require recruitment by cnx or crt and likely acts directly on class i heavy chains to promote disulfide formation. furthermore, in cells expressing the erp57 point mutant, class i heavy chains, crt and the tapasin-erp57 disulfide conjugate were present at normal levels in the plc, indicating that the interaction between erp57 and crt is not required for plc assembly. finally, we show that mutations that destroy the enzymatic function of erp57 have no effect on plc stability or class i surface expression, suggesting that erp57 plays a structural as opposed to catalytic role in plc function. autoimmune pancreatitis (aip) underlies 5-11 % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. a strong association of aip with the hla-drb1*0405/dqb1*0401 haplotype has been reported, but identification of the predisposing hla gene(s) has been precluded by strong linkage disequilibrium. here, we show that hla-dr*0405 transgenic ab0 nod mice suffer from aip and additional pneumonitis after sublethal irradiation and adoptive t cell transfer from syngenic donors, leading to complete pancreatic atrophy. pancreas histology is characterized by destructive infiltration of the exocrine tissue with cd4+ and cd8+ t cells, b cells and macrophages. mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. hla-dr*0405 transgenic mice (cd4+ t cell competent) develop aip even unprovoked, similar to ab0 nod mice (cd4+ t cell deficient), while hla-dr*0401, hla-dq8 or hla-dr*0405/dq8 (double-) transgenic controls all remain normal after same treatment. we conclude that hla-dr*0405 fails to protect from aip, likely due to defects in the induction of cd4+ regulatory t cells. our results identify hla-dr*0405 as a prominent risk factor for aip on the hla-drb1*0405/dqb1*0401 haplotype. this humanized mouse model should be useful to study mechanisms that underlie the hla association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human aip. s. khan 1 , c. britten 1 , h. overkleeft 2 , g. van der marel 2 , k. melief 1 , d. filippov 2 , f. ossendorp 1 1 leiden university medical center, section tumorimmunology, leiden, netherlands, 2 leiden university, biosynthesis group, leiden, netherlands objective: we have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic tlr ligands to study the impact on mhc class i and class ii antigen presentation. the potency of the vaccine was addressed by monitoring antigen presentation, priming of t-cells and tumor protection. results: our data show that this type of targeting of peptides greatly improves antigen presentation and t-cell priming compared to free peptide. vaccination of mice with the tlr-ligand peptide conjugates induced high numbers of functional cd8 and cd4 t-cells that could protect mice for aggressive melanoma. this potency relies on tlr signaling since peptide coupled to a non-functional tlr ligand was unable to support induction of specific t-cells. these data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal t-cell activation by dendritic cells, and show the potency of tlr-l peptide conjugates as a vaccine modality. y. shi 1,2 , x. hu 1,2 , a. kawanatachikawa objectives: nef protein of human immunodeficiency virus (hiv) holds some important immunodominant ctl epitopes. two overlapping 8-mer and 10-mer epitopes (rypltfgwcf (nef138-10) and rypltfgw (nef138-8)) were found to be presented by hla-a*2402 and some immune escape mutants of these two epitopes have also been found in some patients, e. g. y2f, y2w, t5c, f6l, w8r, f10r, f10y etc. or their combinations. it's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of hiv. methods: refolding method was used to attain the soluble protein pmhc. crystals are grown using hanging drop vapor diffusion method and x-ray diffraction technology is used to determine the structure. we have determined six peptide-mhc(pmhc) structures containing nef138-10 (wild type) and its four mostly common immune escape mutants (y2f, t5c, y2f&t5c, f6l), and also nef138-8 (wild type). we found that there was little difference between the nef138-10 (wild type) and nef138-10 (y2f) when they were displayed in the peptide-binding groove of mhc molecule, except water molecule distribution near the anchor residue y2 or f2. interestingly the central bulge region of the peptide was becoming very flexible for the nef138-10 (t5c) and nef138-10 (y2f&t5c), which may affect the binding of peptide and the recognition of t cell receptor. for nef138-10 (f6l), the side chain of l6 was more flexible compared to the nef138-10 (wild type). alignment of the nef138-10 and nef138-8 showed that the nef138-8 became flat and the side chain of f6 was not solvent-exposed due to shortening of the length of the peptide. conclusion: as the peptide nef138-10 was featured, while the peptide nef138-8 was featureless, so the different topology of these two epitopes indicates that they have different tcr repertoire diversity in hiv-specific responses. different immune escape mutants of nef138-10 was using different strategies to avoid the killing of host ctls, which indicates that the therapy strategy based on the cellular immune response should be diversity. for the in vivo or ex vivo activation of antigen-specific t cell responses long synthetic peptides are used to activate both cd8+ and cd4+ t cells. in this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in mhc class i. we observed a large variation in the effectiveness of activation of specific t cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. for the hla-a2 restricted cmvpp65 derived nlv epitope specific t cells were most efficiently activated by n-terminally extended variants of the minimal epitope, while the use of c-terminally extended variants resulted in a 2-3 log reduction of activation efficiency. this pattern was seen for 5/9 epitopes tested in different hla restrictions. furthermore, for all epitopes tested, extending both the c-terminus and n-terminus led to 2-5 log less efficient activation of the specific t cells, compared to the minimal peptide. exchange of the c-terminal sequence of the c-terminal extended hla-b7 restricted cmvpp65 rph peptide with the c-terminal extended nlv peptide led to the enhancement of t cell activation by the exchanged nlv peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. tap-deficient t2 cells loaded with extended nlv peptides efficiently activated nlv-specific t cells, indicating that the route of presentation was tap-independent. addition of lactacystin did not affect activation of specific t cells, illustrating that crosspre-sentation was proteasome-independent. primaquine reduced the activation of specific t cells by extended nlv peptides, but not by the minimal nlv 9-mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. these data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling mhc class i molecules. not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. therefore, a rational design of peptides is crucial for efficient activation of cd8+ t cells in approaches of vaccination, adoptive transfer and immune monitoring. antigenic peptides presented by mhc class i molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. for two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. one of them is derived from melanocytic protein gp100 and requires excision of a four-amino acid intervening segment. the other peptide is derived from protein sp110, and requires splicing in the reverse order of two segments initially separated by six amino acids. the first spliced antigenic peptide described was derived from fibroblast growth factor-5 (fgf-5) and was recognized by human cytotoxic t lymphocytes directed against kidney cancer cells. it is made of two spliced fragments, which are initially separated by a long segment of 40 amino acids. the splicing mechanism of this peptide has not been worked out. the length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. we therefore evaluated the role of the proteasome in the splicing of this peptide. we observed that the spliced fgf-5 peptide was produced in vitro after incubation of proteasomes with a 49-amino acid long precursor peptide. we evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. the results confirmed the transpeptidation model of splicing. we further compared the production of the fgf-5 spliced peptide by cells transfected with mutant constructs encoding fgf-5 proteins where the intervening segment was shortened from 40 amino acids to 30, 20 or 8 residues. we observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. finally, using the spliced gp100 peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. the polymorphic residues within the peptide binding cleft of hla class i molecules not only diversify the range of peptides presented to cytotoxic t lymphocytes but also influence the pathway of antigen presentation. in order to acquire high affinity peptides, some class i allotypes, such as hla-b*4402, are heavily dependent upon tapasin and other molecules comprising the peptide loading complex (plc). other class i molecules, like hla-b*4405, appear to largely bypass this complex but are consequently loaded suboptimally with peptide. hla-b*4402 and b*4405 are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. we have previously speculated that such tapasin-independent class i molecules may have been selected in response to viral inhibitors that target the plc, such as the human cytomegalovirus us3 protein. to address this hypothesis, us3 was stably coexpressed in b lymphoblastoid cell lines expressing hla-b*4402 or hla-b*4405. in the presence of us3, the surface expression of hla-b*4402 was substantially reduced whereas hla-b*4405 expression was relatively unaffected. although us3 was able to form complexes with both hla class i allotypes, only hla-b*4402 was retained intracellularly in an immature form whereas hla-b*4405 was transported to the cell surface. accordingly, in the presence of us3, hla-b*4405, but not hla-b*4402, constitutively presented a hla-b44 restricted alloantigen to reporter t cells, suggesting that us3 binds hla-b*4405 without interfering with peptide loading. us3 has been reported by others to bind the plc but surprisingly we have not detected such us3-plc complexes in our system. rather, in the presence of us3 we identified a pool of class i molecules distinct from the plc and only present in us3 expressing cells, implying that us3 may act independently of the plc. these findings demonstrate how hla class i polymorphism not only impacts upon the t cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation. c. massa 1 , b. seliger 1 1 martin-luther-university halle-wittenberg, institute of medical immunology, halle, germany in the attempt to optimize vaccine dc, modifications have been proposed both in the antigen loading and in the maturation protocols. for dc loading "whole antigens" are now preferred to peptides. therefore, it is important to consider not only the costimulatory properties of the vaccine dc, but also their antigen processing abilities. this is even more important since there is the trend to stimulate dc with tlr ligands combined with ifn-y in order to induce dc not only able to correctly migrate, but also secreting the bioactive il12p70. since ifn-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the dc in parallel to their costimulatory potential. for this purpose monocyte-derived dc were stimulated for 24h with the gold standard of maturation (tnfa, il1b, il6 and pge2) or a combination of ifn-g and different tlr ligands. the dc obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic pbmc, but differ for the pattern of proteases expression as evaluated by real time pcr. with the exception of the downregulation of the tripeptidyl peptidase ii (tppii), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature dc. in response to the "ifn-gcontaining" cocktails there was a similar tppii downregulation, but also the induction of many other enzymes. the cytosolic leucine aminopeptidase-3 (lap3) had a more than 20-fold increase in transcription levels, whereas the mrna expression of the aminopeptidases of the endoplasmic reticulum erap1 and erap2 and of the immunoproteasome subunits lmp2 and lmp10 was enhanced between 2 and 5-fold under these culture conditions. with regard to the different tlr ligands used in combination with ifn-g, there was a reproducible higher mrna induction in the presence of the tlr4 ligand mpla in comparison to the tlr-3 and 7/8 ligand polyi:c and r848. these data suggest that the maturation cocktail of dc may alter the peptide repertoire presented by hla class i surface antigens. it has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for t cells. however, whether cognate interactions between mast cells and class ii restricted cd4 + t cells actually occur, is still an open question. we addressed this question using peritoneal cell-derived mast cells (pcmc) as an antigen presenting cell model. our results show that in vitro treatment of pcmc with ifn-g and il-4 induced surface expression of mature mhc class ii molecules and cd86. when ifn-g/il-4 primed pcmc were used as antigen presenting cells for cd4 + t cells they induced activation of effector t cells but not of their naive counterparts as evidenced by cd69 up-regulation, induction of proliferation and cytokine production. confocal laser scanning microscopy showed that helper ot-ii t lymphocytes form with pcmc functional immunological synapses, characterized by pkcq enrichment and ifn-g polarized secretion towards the antigen-presenting mast cells. finally, upon cognate interaction with ot-ii t cells, mast cells lowered their threshold of activation via fceri. our results show that mast cells can establish cognate interactions with class ii restricted helper t cells, implying that they can actually serve as resident apc in inflamed tissues. h the vast majority of peptide ligands presented by mhc class i molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (er) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of er proteins remains little characterized. studying processing and presentation of er-targeted and cytosolic forms of proinsulin (pi), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type 1-diabetes, we found that er-targeting of this model antigen has profound effects not only on how pi is degraded, but also on regulation of its synthesis. as expected, proteasome inhibition inhibited degradation of cytosolic pi as well as presentation of the epitope insulin b15-23 to specific cd8+ t cells. in contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of er-targeted pi (pre-pi) through induction of er stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. experiments using conditions permissive for pre-pi expression showed that er-targeting modified proteolytic processing of pi for mhc class i presentation. these experiments suggested that two proteolytic pathways contribute to degradation of er-targeted pi, with their relative contribution depending on the stability of the protein. while degradation of unmodified pre-pi was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-pi for presentation, while introduction of a site for n-glycosylation had the opposite effect. these findings imply that er-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. cd8 + t cell immune response to exogenous antigens relies on cross presentation by dendritic cells (dcs) in secondary lymphoid organs. recently, in several infectious murine models, it has been shown that in addition to dc located in tissues, de novo differentiating dc participate in the protective th1 immune response. the role of de novo differentiating dc in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the apc subsets patrolling for exogenous ag. a prerequisite for cross presentation is a moderate ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to mhc molecules. this prerequisite is of special importance considering dc precursors (such as monocytes), which are not yet dcs and may take up antigen before differentiating into dcs. the objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to cd8+ t cells and whether they are able to sustain this cross presentation while differentiating into dcs. we have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate cd8+ tumor-specific t cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to cd8+ t cell clones, faure, 2009, eur j immunol 39 (2): 380-90). in the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into dcs. we have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate cd8+ t cells after their in vitro differentiation into dc, 6 days following their antigenic pulse. the delineation of apc subsets able to sustain ag cross-presentation and t cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. viral genomes contain alternative reading frames (arfs) encoding for mhc-i restricted epitopes (arf-epitope). in the siv/macaque model, ctl responses directed against arf-epitopes participate in controlling viral replication. we previously described that hiv-1 genome contains arfs within gag, pol and env genes encoding for a panel of hla-b*07 restricted epitopes. qprsdthvf (q9vf/5d) is one such epitope but its parental epitope qprsnthvf (q9vf/5n) has a significant higher frequency among hiv-1 isolates. strikingly, q9vf/5d-or q9vf/5n-specific ctls recognize apcs infected with hiv strains encoding for q9vf/5d (e. g. hiv lai ). in contrast, hiv strains (e. g. hiv nl-ad8 ) encoding for q9vf/5n do not activate ctl responses raising the possibility that q9vf/5n epitope is not presented by infected cells. we asked whether introducing mutations within q9vf might be a mean for the virus to escape ctl responses directed against this arf-encoded epitopes. we dissected the mechanism responsible for the lack of q9vf/5n mhc-i presentation. we modified hiv lai to introduce a d to n mutation in q9vf. introducing this single amino-acid mutation abrogated ctl recognition indicating that this asparagine (n) alters q9vf mhc-i presentation. we performed in vitro proteasomal digestions of 28mer peptides encompassing q9vf/5d or q9vf/5n and cleaved polypeptides were analyzed by mass spectrometry. the asparagine (n) in q9vf/5n is a preferential proteasomal cleavage site. thus suggesting that proteasome cleavages within q9vf/5n might be responsible for its lack of mhc-i presentation. we then sought in hiv-infected patients for the presence of proviruses encoding for q9vf/5d or q9vf/5n, and ctls responses directed against these epitopes. far thus, two out of three donors tested recognized the q9vf/5d peptide. we cloned and sequenced hiv-1 genomes from the three donors. surprisingly, out of 20 hiv proviral genomes isolated from pbmcs of q9vf/5d reactive donors, we could not find any virus bearing the q9vf/5d sequence. the isolated hiv sequences either encoded for q9vf/5n or had a stop codon within the epitope. in contrast, viruses encoding for q9vf/5d were isolated from pbmcs of the q9vf/ 5d nonreactive patient. altogether, our data suggest that ctls exert a selection pressure on viral arfs. hiv-1 seems to escape immune surveillance by introducing mutations altering processing of arf-derived epitopes. i. e. flesch 1 , y. wang 1 , d.c. tscharke 1 1 the australian national university, biochemistry and molecular biology, canberra, australia vaccinia virus (vacv) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. cd8 + t cells recognizing viral peptides in association with mhc class i molecules on infected cells play a crucial role in the defence of viruses. despite the large number of possible mhc class i-peptide combinations, cd8 + t cells only recognize a small number of epitopes, a phenomenon called immunodominance. using recently defined cd8 + t cell epitopes for vacv in mice, we have investigated how heterozygosity of mhc class i molecules influences immunodominance patterns in h-2 bxd f 1 mice compared with their inbred parent strains. we find that the immunogenicity of vacv peptides defined using inbred mice is variable in f 1 progeny, with some peptides being almost equally immunogenic in f 1 and inbred mice, while others elicit responses that are reduced by more than 90 % in f 1 mice. during acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in f 1 mice. in line with these findings, a multiepitope construct expressed by a recombinant vacv was less immunogenic in f 1 mice than would be predicted from its performance in parent strains. in terms of mechanism, we find evidence of altered tcr repertoires including in the case of one epitope, the loss of many diverse tcr vb clones and outgrowth of cd8 + t cells with a restricted vb usage in f 1 mice. these data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how mhc diversity can alter the range of epitopes that are immunogenic in outbred populations. objective: tlr ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (dc). therefore we aimed to study the efficacy of a tlr2 agonist, s-[2,3-bispalmitoyiloxy-(2r)-propyl]-r-cysteinyl-amido-monomethoxyl polyethylene glycol (bppcysmpeg), a synthetic derivative of the mycoplasma macrophage activating lipopeptide (malp-2), as an adjuvant for cross-priming against cellular and soluble antigens. malp-2 has been characterized as an effective mucosal adjuvant and synthesis of bppcysmpeg further improved solubility and pharmacokinetic features of the adjuvant. methods: dc isolation, in vitro and in vivo t cell stimulation, intracellular cytokine staining, in vivo cytotoxicity assays. results: systemic administration of bppcysmpeg induced maturation of cd8 + and cd8 -dc in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. in addition, administration of bppcysmpeg and cell-associated ova resulted in generation of an effective ctl response against ova in vivo in a t-helper cell-dependent manner, but independent of ifna. delivering antigenic peptides directly linked to bppcysmpeg led to superior ctl immunity as compared to giving antigens and adjuvants admixed. in contrast to other tlr ligands such as cpg, systemic activation of dc with bppcysmpeg did not result in shutdown of antigen presentation by splenic dc subsets, although cross-priming against subsequently encountered antigens was reduced. we provide evidence that bppcysmpeg stimulation of dc via tlr2/6 results in the generation of an effective ctl response and that delivering antigenic peptides linked to bppcysmpeg is a promising strategy for vaccination. while bppcysmpeg-matured dc retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after tlr-mediated dc maturation. altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation. tumor immunevasion. to verify whether the loss of erap1 expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of eraap (murine erap1) in a murine t lymphoma cell line, rma. we used a method that allows an efficient and continuous expression of mirnas that directly silence eraap and obtained several eraap-deficient rma clones with different levels of eraap expression (up to 90 % of reduction at the protein level). microsomal aminopeptidase activity and mhc class i surface expression were decreased in all clones proportionally to eraap expression. moreover, low expression of eraap affected the stability of mhc class i molecules as evaluated after acid and brefeldin a treatment. de-regulated er peptide trimming also drastically affected the tumor formation of rma cells and host survival. eraap-deficient rma clones with different levels of eraap, 50 and 10 % as compared to control rma cells, were injected s. c. in the flank of c57bl/6 syngenic mice, and analysed tumor growth. all mice injected with control rma cells developed a tumor but survived up to 45 days after injection. all mice injected with rma clone with a 50 % level of eraap expression developed a tumor and died within 23 days after injection. surprisingly, any animal injected with rma clone with a 10 % level of eraap died or showed a visible tumor. thus, knockdown of eraap expression appears differently to affect the immunogenicity of rma cells, depending on the eraap silencing level. hemophilia a is an x-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor viii (fviii). treatment consists of regular administration of fviii, but is complicated by the formation of inhibiting antibodies against fviii. both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia a. the development of inhibitory antibodies in hemophilia a patients has been shown to be a cd4 + t-cell driven process. therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of t cells against fviii in hemophilia a patients using mhc class ii tetramers. cd4+ t-cell responses of two monozygotic twins with severe hemophilia a were analyzed. one of these subjects developed a high titer inhibitor (207 bu/ml) following intensive factor viii (fviii) treatment. high dose immune tolerance therapy together with anti-cd20 therapy resulted in eradication of the inhibitor. in contrast, his twin brother developed a low titer inhibitor (2.5 bu/ml) which declined rapidly after tolerance induction. fundamental differences in the twins' antibody responses were further suggested by elevated and persistent igg4 levels in the subject with the high titer inhibitor. in order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated drb1*0701-restricted t-cell responses of the high titer inhibitor subject, using fluorescent mhc class ii tetramers loaded with 20-mer synthetic fviii peptides to stain epitope-specific cd4+ cells.cd4+ t-cells from the high-titre inhibitor subject recognized three peptides corresponding to the fviii a2 domain: fviii 405-424 , fviii 421-440 and fviii 653-672 , as well as the c1 domain peptide fviii 2093-2112 , but not any c2 domain peptides. the c1 domain peptide contains a sequence that was reported as a promiscuous t-cell epitope (jones td et al., j thromb haemost.85:123-33, 2005 ). analysis of t cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of t cells that may underlie the discordant immune responses of these twins to infused fviii. m. forloni 1 , s. albini 1 , m.z. limongi 1 , l. cifaldi 1 , d. fruci 1 1 ospedale pediatrico bambin gesù, rome, italy neuroblastoma (nb) is a pediatric tumor that derives from neural crest. the most aggressive forms are characterized by amplification of the mycn oncogene and severe reduction of hla class i expression. mycn has been claimed to hinder hla class i expression through affecting the expression of the transcription factor p50 nf-kb subunit. since in many human tumors the expression of hla class i molecules is positively co-ordinated with that of er aminopeptidases, erap1 and erap2, we wondered whether in nb cell lines mycn may impair expression of these aminopeptidases. to explore this possibility, nb cell lines that differ in mycn expression were quantified for expression of mycn, erap1, erap2 and hla class i heavy chains by western blotting and for surface hla class i expression by flow cytometry. we found that mycn negatively correlates with expression of hla class i, erap1 and erap2. this negative correlation was confirmed in a nb cell line expressing a tetracycline repressible mycn transgene. then, by the use of tnfa (a nf-kb nuclear translocation stimulator), sulfasazine and ikba mutant (two nf-kb nuclear translocation inhibitors) and knockdown of p65 nf-kb subunit, we demonstrated that nf-kb is involved in erap1 and erap2 expression in nb cell lines and that mycn does not affect nf-kb expression. furthermore, we showed that mycn and nf-kb are recruited to the promoter regions of erap1 and erap2 and that mycn affects the recruitment of nf-kb binding to these promoter regions. in conclusion, the present results indicate that an enhanced mycn level, linked or not to mycn amplification, represses erap1, erap2 and hla class i expression in nb cell lines by affecting the recruitments of nf-kb binding to their promoters. s. brosch 1 , s. tenzer 1 , h. schild 1 , e. von stebut-borschitz 1 1 uniklinik mainz, mainz, germany infection of inbred mouse strains with the intracellular protozoan parasite leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. c57bl/6 mice) or systemic disease (susceptible phenotype; balb/c mice) depending on the genetic background of an individual. healing of leishmania infections is based on th1 immunity, whereas ifng secretion of both cd4 + th1 and cd8 + tc1 cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. stimulation of antigen-specific effector t cells is driven by l. major-infected dendritic cells (dc) in an il-12-dependent manner. proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective cd8 + t-cell responses via the mhc class i. here, we analysed the role of the ifng inducible immunoproteasome for the priming of cd8 + t cells in l. major infections. using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome lpm7 (lmp7 -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with l. major mimiking natural transmission of the parasite as compared to wild type c57bl/6 mice. in addition, ex vivo co-cultures with infected dc from either lmp7 -/or wild type mice together with antigen-specific t cells from infected wild types showed no differences in tc1 cell ifng secretion and the dc restimulatory capacity of cd8 + t cells. furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble leishmania antigen; sla) cd8 + t cells, isolated from low dose infected c57bl/6 wildtype or lmp7 -/mice, were not detected. in summary, our data indicate that despite the fact that cd8 responses in l. major infections are important for disease outcome, processing of antigen and thus priming of cd8 + t cells against l. major is independent of the lmp7 subunit of the immunoproteasome. studies have defined an essential requirement for autoantigen-specific b cells as antigen presenting cells in rheumatoid arthritis. however, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by b cells are unknown. in this study we have developed a system to investigate how antigen-specific b cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. we have utilised these cells to characterise the mechanisms by which aggrecan-specific b cells could induce autoimmunity. we have constructed plasmids encoding an aggrecan-specific b cell receptor and have transfected them into the b cell line a20, generating b cell lines that specifically recognise and target aggrecan for presentation to t cells. in addition, we have established conditions for a panel of aggrecan-specific t cell hybridomas to recognise aggrecan pulsed b cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. we used inhibitors of mhc class ii transport, endosomal ph and enzymes involved in aggrecan degradation. we found that aggrecan-specific b cell lines presented the major arthritogenic cd4 + t cell epitope (84-103) from the g1 domain of aggrecan 10,000 times more efficiently than non-specific b cells and over 100 times more efficiently than the macrophage line j774. however, despite this highly efficient aggrecan capture, processing and presentation of the 84-103 epitope took at least 5 hours, comparable to the time required for presentation of aggrecan by j774. treatment of aggrecan-specific b cells with ammonium chloride to raise endosomal ph or brefeldin-a to disrupt golgi transport inhibited presentation of the 84-103 epitope, suggesting a requirement for low endosomal ph and presentation by newly synthesised mhc class ii. interestingly, aggrecan presentation by antigen-specific b cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium understanding the mechanisms of antigen processing and presentation by autoantigen-specific b cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. tapasin is an mhc-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of mhc class i molecules within the peptide loading complex (plc). class i molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions 114, 115 and 116 at the peptide binding groove. position 116 also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. the mhc class i antigen b27 is strongly associated to ankylosing spondylitis (as) and other spondyloarthropathies. hla-b27 subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the plc. tapasin also modulates b27 folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of b*2705, peptide loading is relatively independent of this chaperone. we analyzed the effect of b27 subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of b27 subtypes. the association of b27 heavy chain with tapasin was analyzed in c1r cells transfected with hla-b27 subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody pasta-1, which recognizes human tapasin. we also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the b27 peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the me1 monoclonal antibody that recognizes b27properly folded b27/peptide complexes. the formation of fully assembled b27 molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody hc10, which recognizes mhc class i free heavy chains (hc), or with me1. maturation was analyzed by pulse-chase analysis, immunoprecipitation with me1 and treatment with endoglycosidase h (endo h). hla-b27 polymorphic positions other than 116, both at the a and c/f pockets modulate tapasin binding and the optimization of the peptide cargo. the stability of the peptide repertoires critically influences the folding efficiency of b27 subtypes. from as early as the initial phases of infection, hiv is coated with complement (c) fragments and following seroconversion, the circulating virus forms immunecomplexes with igg and complement. recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (idcs) with differentially opsonized hiv. the opsonization pattern of hiv may additionally have profound consequences for the outcomes of the antigen-presenting capacity of dcs and their ability to mount an adequate immune response. in this context, we compared the impact of differential hiv-opsonization on the antigen-presenting capacity of dcs and found that c-opsonized hiv triggered ctl responses, while igg-coated virus did not. these in vitro generated ctls showed an enhanced ifn-g secretion and recognized the help independent ctl epitope slyntvatl. c-generated ctls also degranulated upon stimulation with specific hiv peptides and were able to elicit antiviral activity against hiv-infected cd4 + t cells. our results indicate that c-opsonization of hiv drives the virus towards the mhc class i pathway in dcs, thereby promoting a more efficient stimulation of naïve cd8 + t cells. this ctl-stimulating property of c could be exploited when searching for a novel approach against hiv. igg isolated from patients with high titers of anti-ccp antibodies showed a cross-reactivity with hcit peptides. vaccination experiments supported a triggering role of hcit for the development of arthritis in mice model. conclusions: diamination process is significantly increased in patients with ra while carbamylation is suppressed. production of specific antibodies against diaminated residues in ra patients may have a modulating role for the development of autoimmune arthritis. the classical pathway of mhc class i antigen presentation involves cytosolic degradation of viral proteins by the proteasome. peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (tap). previous reports have shown that viral epitopes are presented to ctl independently of tap in smaller viruses. we hypothesized that presentation of vacv by mhc class i might proceed by alternative pathways. the aim of this study was to characterize these alternative pathways in tap-deficient mice. our results show that ctl derived from c57bl/6 mice immunized with vacv, recognized tapdeficient dendritic cells infected with the virus. approximately 15 % of vacv global presentation in the context of h-2 b was independent of tap. in addition, vacv infection induced a virus-specific ctl response in mice deficient in tap. dendritic cells (dc) initiate robust ctl immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class i molecules (pmhc). two major dc subtypes have been described, cd8+ and cd8-dc, which differ in their mhci antigen presentation capacities. cd8+ dc are the major dc subset responsible for cross presentation (presentation of exogenous antigen by mhci), while cd8-dc display little cross presenting capacity. here, we examined the mhci antigen presentation pathway of cd8+ and cd8-dc in more detail. first, turnover (half-life) of total mhci at the cell surface of cd8+ and cd8-dc was determined. surprisingly, cd8+ dc exhibit rapid surface mhci turnover compared to cd8-dc (following culture in the presence of brefeldin a). following activation of dc with cpg, mhci levels at the surface of both cd8+ and cd8-dc were stabilized and no longer underwent rapid turnover. this suggests that cd8+ and cd8-dc differ in their regulation of surface mhci turnover and that this is subject to regulation by antigen-associated signals. second, we examined the ability of cd8+ and cd8-dc to generate pmhci complexes containing cross presented antigen. we utilized the model antigen ovalbumin (ova) and an antibody that can detect h-2kb loaded with the ova-derived peptide, siinfekl. cd8+ and cd8-dc isolated from ova-expressing mice (actin-ova transgenics) displayed abundant kb-siinfekl complexes at their cell surface. in contrast, in response to exogenous soluble ova protein, only the cd8+ dc, but not the cd8-dc, displayed kb-siinfekl complexes at the cell surface. similarly, when dc were pulsed with ova-coated splenocytes, kb-siinfekl complexes were only detected on the surface of cd8+, and not cd8-dc. this data further validates the role for cd8+ dc as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other dc subsets from accessing the important cross presentation pathway. objectives: the action radius of matrix metalloproteinases or mmps is not restricted to massive extracellular matrix (ecm) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. although many instances exist in which cells disintegrate, often in conjunction with induction of mmps, the intracellular mmp substrate repertoire or degradome remains relatively unexplored. the aims of the present study were to identify novel intracellular mmp targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. methods: multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to thp-1 cytosol using gelatinase b/mmp-9 as a model enzyme. in the first dimension, ion exchange chromatography separated the thp-1 proteins by their net charge and/or isoelectric point (pi) followed by cleavage of the proteins by mmp-9. in the second dimension, potential substrates were separated by molecular weight on sds-page. to evaluate the effect of proteolysis by mmp-9 on the immunogenicity of the intracellular protein pool, mice were immunized twice with thp-1 cytosol in complete freund's adjuvant. lymph node t cells were isolated and stimulated with mmp-9-cleaved or intact thp-1 cytosol. proliferation was assessed by measuring incorporated 3 hthymidine. results: 100-200 mmp-9 candidate substrates were isolated, of which 69 were identified, revealing many novel mmp-9 (candidate) substrates from the intracellular matrix (icm), such as actin, tubulin, stathmin,... about 2/3 of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. remarkably, a significantly lower t cell proliferation was observed in the presence of cleaved vs. intact cytosol. conclusion: multidimensional degradomics technology is a valuable tool for high-throughput identification of novel mmp substrates. proteolysis by mmp-9 decreased the immunogenicity of the intracellular contents, suggesting that mmps may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury. a preference for hla-a versus -b molecules; e3-19k did not detectably associate with hla-c molecules under identical conditions. this locus specificity may provide a functional advantage to ads by inactivating t-cell receptors, while avoiding activation of nk receptors. finally, we showed that residue 56 in hla-a11 and residue 93 in e3-19k are highly critical for association of both proteins. this defines a putative interaction surface between e3-19k and class i molecules. conclusions: our studies provide novel insights into the functional relationship between e3-19k and the class i antigen presentation pathway. moreover, because soluble e3-19k can differentiate between polymorphic gene products encoded in the mhc, our results may contribute to define paradigms for how class i substrate specificity is established for er retention. overall, our studies represent an important step towards a molecular understanding of the strategy evolved by ads to establish life-long persistence in host cells. objectives: the transporter associated with antigen processing (tap) belongs to the abc transporter superfamily and is a heterodimer consisting of the two subunits tap1 and tap2. tap transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (er) and is thus a key element of the mhc class i antigen processing machinery (apm). methods: target-specific tap knock downs were generated by shrna technology. the resulting transfectants were subsequently analysed regarding mhc class i surface expression using flow cytometry, whereas mrna and protein expression levels of tap1 and tap2 were analysed by rt-pcr and western blots, respectively. furthermore, the protein stabilizing effect of tap1 on tap2 was investigated in the presence of two distinct proteasome inhibitors. results: previous findings obtained with rare tap1 mutants suggested that the lack of tap1 protein expression is associated with a strong reduction of tap2 protein levels, which could be restored by tap1 gene transfer, whereas no such regulation is found vice versa. to investigate this stabilizing effect of tap1 on tap2 different shrna plasmids specifically targeting tap1 or tap2, respectively, were stably transfected into constitutively tap1 and tap2 expressing hacat keratinocytes and colo794 melanoma cells. in both cell types the shrna-mediated tap1 and tap2 inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. the knock down of tap1 caused not only an almost complete loss of tap1, but also a strong decrease of tap2 protein expression. in contrast, the tap2 knock down exhibited no influence on the tap1 expression. specific inhibition of the proteasome prevented the degradation of tap2 in the tap1 knock down variants. the results of our study emphasize that an unidirectional stabilisation of tap1 on tap2 protein expression is not restricted to rare tap1 mutants, but rather suggest a common regulatory mechanism for the tap complex. uv injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. in this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following uv exposure. therefore, the buttock skin of 27 healthy volunteers was exposed twice to 1.5 minimal erythema dose of uv. blister roofs were then collected before and 1, 4 and 10 days after uv-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. we demonstrated that, along with the rapid activation and migration of langerhans cells (lc), uv skin radiation exposure promotes the infiltration into the epidermis of a monocytic cd14 + cd36 + and of a macrophagic cd14 -cd36 + cell subsets that emerge 1 and 4 days post exposition, respectively. more importantly whereas classical cd1a hi cd207 + lc are the unique dendritic cell (dc) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal dc namely cd1a low cd207and cd1a low cd207 + that emerged 1 and 4 days post irradiation, respectively. these two distinct populations of epidermal dc (edc) differ from classical epidermal lc by their activation/maturation profile as assessed by the strong expression of cd86 and hladr. finally, 10 days post-exposure, we observed that lc represented almost the only haematopoietic cell population in the epidermis. these results suggesting that the uv-recruited edc and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis. i. bailey 1 , e. reeves 1 , t. elliott 1 , e. james 1 1 university of southampton, school of medicine, cancer sciences division, southampton, united kingdom there is accumulating evidence that cd4+ cd25+ regulatory t cells (treg) play an important role in anti-tumour immunity by preventing effective t cell responses to tumour antigens. tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. the depletion of tregs prior to challenge with the murine colorectal tumour, ct26, stimulates a robust, protective t cell response which is also protective to challenge with other tumours of different histological origins, such as b cell lymphomas and a renal cell carcinoma. this cross protection has not been seen with other tumour cell lines. we have identified a ct26-derived cross-protective antigen, gsw11, which was found to be encoded within the ectotropic murine leukaemia virus (emv-1) envelope protein, gp70. this protein has previously been shown to encode ct26-specific cd8 and cd4 antigens, implicating it as a 'hot-spot' for ct26 tumour antigens. interestingly, we have identified a truncated version of gp70 which may be responsible for generation of gsw11. expression studies have revealed increased gp70 expression in ct26 compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (gsw11) generated. the current knowledge of hla class ii antigen presentation and peptide binding is mainly based on studies of hla-dr molecules. they contain a large hydrophobic p1 pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while p4, p6 and p9 tune the peptide repertoire that can be presented by individual hla-dr alleles. the same rules and requirements do not necessarily exists for peptide binding to hla-dp molecules, however. the present study adresses this issue. we have expressed and affinity purified soluble recombinant hla-dp2 molecules from drosophila melanogaster cells and studied its binding of a number of peptides known to bind hla-dr, -dq or dp molecules. unexpectedly, the immunodominant epitope in multiple sclerosis (ms), the myelin basic protein derived peptide, mbp85-99, bound to hla-dp2 with high affinity (10-30 nm). binding studies of mbp85-99 derived peptides containing single alanine substitution at each position revealed that only three of the peptides (f91a, f92a and k93a) were affected, and only by a 20-50 fold reduction in affinity for hla-dp2. the observation that none of the substitutions resulted in a complete loss of binding to hla-dp2 indicates that 1) hla-dp2 binding to peptides does not depend on a large hydrophobic residue accomodated in p1, or 2) mbp85-99 can bind in more than one register. we will present data addressing this issue. the hla-dp peptide binding capacity was increased at neutral as compared to acidic ph, and by the presence of n-butanol, a small organic mhc loading enhancer (mle). in summary, the hla-dp2 molecule binds the immunodominant epitope in ms, mbp85-99, possibly in more than one register. additional studies are required to resolve the hla-dp2 peptide binding properties, and to determine whether expression of hla-dp2 affects the disease course in ms patients. results: depletion of mncs for cd14+ cells abrogated the tg-induced cytokine production and proliferation of cd4+ t cells, indicating a primary role for monocytes as apcs. however, the encounter of t cell with antigens presumably occurs in b cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. to mimic the conditions prevailing there, we depleted pbmcs for g 98 % of the monocytes (without significant loss of t-cells), and compared the tgelicited t-helper cell responses in the presence and absence of b cells. the tg-induced cd4+ t cell proliferation was significantly reduced in cd14/cd19-depleted mnc cultures, as compared to cultures depleted for cd14+ monocytes alone. the same applied to the production of il-2, il-6 and tnf-a. production of ifn-g and il-10 was generally not observed. our data indicate that normal b cells are capable of inducing a pro-inflammatory cytokine response in mnc-cultures, where monocytes and monocyte-derived cells are not preponderant. studies addressing the relative contributions to this cytokine production, by b cells themselves and by t cells (following antigen-presentation by b cells), are in progress. j. kyosiimire-lugemwa 1,2 , p. pala 1 , g. miiro 3 , j. todd 3 , p. kaleebu 1,2 , n. imami 2 , f. gotch 2,3 1 mrc uganda, basic science, entebbe, uganda, 2 imperial college, london, united kingdom, 3 mrc uganda, entebbe, uganda background: hiv-1-specific t-cell responses are preserved in hiv-1 infected individuals with non-progressing hiv-1 disease. "long term non progressors" (ltnps) were defined as art naï ve individuals infected with hiv-1 for g 8 years, maintaining cd4+ t-cell counts g 500, and with minimal cd4+ decline over time. we tested the hypothesis that gag-specific t-cell responses are inversely correlated to disease progression whereas nef-specific t-cell responses are not. methods: 17 art naï ve hiv-1 infected patients from the entebbe cohort in uganda were recruited and stratified by cd4+ t-cell count, cd4+ decline slopes, and time of enrolment, into 2 groups -10 ltnp and 7 rapid progressors (rp). all patients were women reflecting the patient base at the entebbe cohort. we measured plasma viral load, current cd4 t-cell count, and ifn-g, il-2 and il-4 elispot responses to pools of 22 to 34 peptides (18-mers overlapping by 10aa). peptides were based on consensus sequences of gag and nef from hiv-1 clades a1, a2 and d. medians and inter-quartile ranges were calculated and comparisons between groups were performed using the mann-whitney u test. correlations were presented using spearmann's linear correlation coefficients. results: some gag-specific ifn-g and il-4 responses were significantly higher in the ltnp than in rp (p=0.02, ifn-g responses to gaga2 pool 1; p=0.04, il-4 responses to gaga1 pool 2). il-2 responses were low and not significantly different between ltnp and rp. there was a positive correlation between il-4 responses to gaga1 pool 2 and cd4 t-cell counts (r 2 =0.502, p=0.04), but no correlation between either il-4 or ifn-g responses and viral load. cytokine responses to nef peptides were not significantly different between the ltnp and rp. conclusion: overall, gag hiv-1 specific responses were higher in ltnps than in rps confirming previous results. non-specific il-4 responses were high possibly reflecting baseline th2 responses to helminths a common environmental exposure in the study population. objectives: in human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the hla class i antigen processing machinery (apm) have been described, which were associated with a reduced antigenicity of these cells. so far, the molecular mechanisms of such defects have not been elucidated in detail. to investigate whether impaired apm component expression was due to altered transcription and associated with cell growth properties murine her2/neuand her2/neu + fibroblasts were employed. methods: using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qrt-pcr and western blot. flow cytometry was employed for determination of the distinct phases using 7-aad for dna analysis and specific antibodies directed against the proliferation marker ki-67, the m-phase specific phistone h3 as well as for the h-2l d surface antigens. in addition, chromatin immunoprecipitation (chip) experiments with an antibody directed against rna polymerase ii were performed to investigate the transcriptional levels of tapasin in her2/neuversus her2/neu + cells. results: serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the g 0 /g 1 -, s-and g 2 /m-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. tapasin mrna level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the g 0 /g 1 -phase, which continuously raised and peaked within the s-phase. however, h-2l d surface antigen expression was not altered in her2/neucells during cell cycle progression. in contrast to her2/neufibroblasts the her2/neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered h-2l d surface expression. this was confirmed by a reduced promoter activity and decreased accessibility of the rna polymerase ii to the tapasin promoter. conclusion: these findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. recent studies suggest that dendritic cells (dcs) are key players in shaping the respiratory syncytial virus (rsv) specific immune response. before, dcs within the airway epithelium were characterized as langerhans cells. in this study, in vitro counterparts of langerhans cells expressing langerin (cd207) and ccr6 were cultured from cd34+ stem cells under the influence of tgf-b (tgf-b-dcs) and compared to cd34+ derived dcs, which passed through a monocytic stage . after infection with rsv, both types of dcs generated viral rna and viral-proteins. although tgf-b-dcs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by il-4-dcs. the increased expression of viral proteins is most likely responsible for the pronounced inhibition of t-cell functions by tgf-b-dcs. since there is evidence that langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of rsv replication is characteristic of a more mature answer against rsv. the occurrence of inhibitory antibodies against exogenous factor viii (fviii) remains the major concern of fviii replacement therapy in patients with hemophilia a. initiation of the immune response implies the endocytosis of fviii by professional antigen presenting cells (apcs): b lymphocytes, dendritic cells (dcs) and macrophages (mø). the organ where the anti-fviii immune response is initiated and the type of apcs involved in this process have not been investigated. we hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where apcs interact with fviii to initiate the anti-fviii immune response. we first administered radiolabeled fviii at therapeutic doses to fviii-deficient mice. fviii was found to preferentially accumulate in the spleen and liver of the mice. levels of fviii in the spleen remained stable for up to 45 min following fviii administration, while they rapidly decreased in the liver. unlabelled fviii was then administered to fviii-deficient mice that had been splenectomized or sham operated and the anti-fviii humoral responses were compared. removal of the spleen resulted in significantly reduced levels of anti-fviii igg. using flow cytometry, fviii was found to preferentially accumulate with splenic mø than dcs and b cells. elimination of apcs by treatment of the mice with clodronate-containing liposomes prior to fviii administration resulted in a drastic reduction of the anti-fviii igg response, as compared to control mice treated with pbs-containing liposomes. taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-fviii immune response and that splenic mø have an important part in this process. the interactions between antigen presenting cells (apc) and t-lymphocytes are a relevant current issue. the area of contact between an antigen presenting cell and a t-lymphocyte is termed immunological synapse (underhill et al, 1999) . the present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. in this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. objectives: as central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be t cells. methods: autologous total human leukocyte cultures, from samples of 30 healthy blood donors were harvested at various times and centrifuged and performed as previously reported (cabral and novak, 1992, 1999) . cytopreparations of each experiment were performed. statistical analysis: regression model. results: experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p x 0,0001), anova for regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are t-cells, cd4+, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as chloroquine or brefeldin a, were added, rosettes were not formed, g) monoclonal antibodies anti-human mhc ii precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. conclusion: taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. objective: to investigate if patients with multiple sclerosis (ms), without the typical increase of antibodies in csf, are less likely to develop neutralizing antibodies (nabs) against ifnb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. methods: overall, 2252 patients were obtained from the swedish multiple sclerosis registry and the swedish nab registry, and treatment information was available for 2207 of them. for 538 of these patients hla-drb1 data was available. results: a significant correlation between lack of antibodies in csf and nab-negativity was found (p=0.02). patients without csf antibodies were to a lesser extent nab-positive when treated with the ifnb-1a preparations, whereas no differences were shown for ifnb-1b. an association between hla-drb1*11 and nab-negativity was detected (p=0.028). the known associations between hla-drb1*15 and csf-positive ms and hla-drb1*04 and csf-negative ms were confirmed. conclusion: we show for the first time that patients without antibodies in csf have a different propensity to induce nabs compared to csf-positive patients, indicating an extended immunological difference between the two ms sub-groups. hla-drb1 potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. in csf-negative patients the reaction against ifnb-1a molecules, possibly through a t-cell dependent pathway, is lower than for csf-positive patients. however, reaction against ifnb-1b, which might also be activated through a t-cell independent pathway, shows no difference in seroprevalence between the groups. abstract withdrawn by author tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using tyrosinase-specific ctls, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. there was no correlation between the level of antigen presentation and mrna expression levels for the three antigens; however, our data suggest that tyrosinase protein stability may play a major role in the high level presentation of this antigen. measurement of the half lives of these proteins revealed a hierarchy in protein stability, with mart-1 and gp100 more stable than tyrosinase. by the use of the cofactor dopa, which stabilizes the tyrosinase protein, significant decrease of hla-tyr complexes presentation was achieved. in addition to the study of antigen presentation, these tcr-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. by generating a whole igg antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (adcc). with the addition of point mutations in the fc fragment, which increased the affinity of the fc to the fc receptor, enhanced tumor cell lysis was achieved. g. schiavoni 1 , s. lorenzi 1 , f. mattei 1 , f. spadaro 1 , l. gabriele 1 1 istitituto superiore di sanità, cell biology and neurosciences, rome, italy cross-presentation is a crucial mechanism for generating cd8 t cell responses against exogenous antigens (ag), such as dead cell-derived ag, and is mainly fulfilled by dendritic cells (dc), particularly cd8a + dc. however, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector cd8 t cell responses. type i ifn are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. in particular, type i ifn have been shown to promote the cross-priming of cd8 t cells against soluble or viral antigens, partly through the stimulation of dc. in this study we evaluate the role of type i ifn to affect dc capacity to capture and cross-present apoptotic cell-derived ag. by using uv-irradiated ova-expressing eg7 thymoma line, we show that type i ifn promote the ability of cd8a + dc to capture apoptotic eg7 cells and to undergo phenotypic activation, both in vitro and in vivo. remarkably, ifn-treatment prolongs the survival of ag-bearing cd8a + dc and the persistence of apoptotic eg7-cell ag within the phagosomal dc compartment, a process that is known to facilitate the recruitment of ag into the mhc-i presentation pathway. accordingly, type i ifn-treatment increases cross-presentation of apoptotic eg7-derived ova ag by dc, as revealed by higher expression levels of siinfekl peptide in association to mhc-i molecules on cell surface of phagocytic cd8a + dc. as a result, eg7-loaded dc become competent at inducing ot-i cd8 t cell proliferation and activation both in vitro and in vivo. our data indicate that type i ifn promote the cross-presentation of apoptotic cell-derived ag by cd8a + dc and suggest that these cytokines may act as a switch signal for cross-presenting dc, thus skewing the immune response from tolerogenic to immunogenic. (2). we have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic dc subsets. using biochemical methods, we show that only cd8+ dc efficiently transfer soluble antigen to their cytosol. the amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to tlr ligands cpg, poly i:c or pam3csk4, and this correlated with enhanced cross-presentation. the increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. measurement of the proteasome activity at different times after exposure to tlr ligands revealed that tlr signalling induced transient inhibition (maximum at two hours) of the proteasome in cd8+ dc but not cd8-dc, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting dc. this correlated with formation of aggresome-like structures only in cross-presenting dc exposed to tlr ligand. by limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. to our knowledge this is the first report of a direct, immediate effect of tlr activation on proteasome activity. exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. they are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. here we explore whether exosomes of different origin can selectively target different immune cells. we compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and b cells to peripheral blood mononuclear cells. flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (imagestream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an epstein-barr virus (ebv) transformed b cell line selectively target b cells. our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. one of the hallmarks of cancer cells is the resistance to cell death. it has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. the proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. here, we investigated that dm1, which is a saponin derivative isolated the stem bark of dracaena mannii induced autophagocytosis on a549 human lung cancer cell line. methods: dm1 induced cell cytotoxicity was measured by mtt assay and propidium iodide staining on a549 cells. we examined the morphological change using optical microscope. and gfp-lc3 punctation was measured using confocal. the protein expression was measured by western blot analysis and the mrna expression was measured using reverse transcription poly chain reaction (rt-pcr). results: dm1 was showed high cytotoxicity on various cancer cell line, especially a549 cells. dm1 treated a549 cells did not show regular dna fragmentation and caspases activation. we also analyzed protein expression of apoptotic marker, but protein level didn't change. as a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. we checked lc3 and beclin-1 protein and mrna expression and inhibitory effect of autophagocytosis inhibitor. the expression level of lc3 was increased concentration and time-dependently. beclin-1 also was increased. and then, we examined gfp-lc3 punctation on a549/ gfp-lc3 stable cells using confocal. a549 cells were formed gfp punctuation after dm 1 treatment. to confirm these data, we measured cell death ratio using 3-methyladenine (3-ma), an autophagocytosis inhibitor. after 3-ma treatment, dm1 induced cell death was decreased comparing with dm1 treatment. conclusion: dm1 did not induce apoptotic cell death. but dm1 showed several characteristics of autophagic cell death. taken together, dm1 induced autophagocytosis on a549 cells. these finding indicated that therapeutic potential of dm1 by triggering autophagic cell death. s. b. rasmussen 1 , s. r. paludan 1 1 university of aarhus, department of medical microbiology and immunology, aarhus, denmark during viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (pamp)s. in the case of herpes simplex virus (hsv), detection is, among others, conducted by toll like receptor (tlr)9, which is a transmembrane receptor located in the endosomes where it detects unmethylated cpg rich dna of extracellular origin, e. g. viruses. upon binding to dna, tlr9 initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (ifn)a and ifnb being some of the most essential ones. the exact route of hsv to the endosomes and thus tlr9 recognition is not clear-cut. the endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in tlr mediated viral recognition has been drawn in to focus. we have found indications of an autophagy dependent ifn expression during hsv-1 infection. by use of an entry defect glycoprotein l and glycoprotein h deficient hsv-1, we found that tlr9 dependent ifn regulatory factor 3 activation was abrogated. in addition, inhibition by 3-methyladenine of phosphoinositol 3-kinase class iii, which is crucial in autophagosome formation, abrogates ifnb induction. these findings points to a role for autophagy in tlr9 dependent recognition. in the ongoing project we are examining how hsv-1 triggers formation of autophagosomes. especially the role of endoplasmatic reticulum stress and doublestranded rna-dependent protein kinase will receive attention. also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated. j. zovko 1 , i. berberich 1 , gk 520 -immunomodulation 1 universität würzburg institut für virologie und immunbiologie, würzburg, germany members of the bcl-2 family control the integrity of mitochondria and thereby influence survival and death of cells. most bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their c-terminal portion. murine a1 and its human homologue bfl-1 are anti-apoptotic members of the bcl-2 family. a1 is expressed in small amounts in the bone marrow and immature b cells, but in high amounts in mature b cells. thus the protein seems to be important for b cell maturation. we analyzed the function of the c-terminus of a1. unless the c-terminal ends of other bcl-2 proteins the tail of a1 does not function as a strong membrane anchor. nevertheless, the last amino acids of a1 are important for the protein. in fact, the c-terminus of a1 serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. we show that a1 undergoes proteosomal degradation controlled by its c-terminus. interestingly, binding to the proapoptotic bcl-2 factor bimel results in increased stability of a1. this is due to reduced ubiquitination of a1 after binding of bimel. we conclude that the cterminus of a1/bfl-1 serves as a docking site for e3 ubiquitin ligase(s) that control the stability of a1 by targeting the protein to the proteasomal pathway. very recently, we have identified a putative e3 ubiquitin ligase that interacted with a1 in a yeast two-hybrid screen. currently, we are trying to confirm this interaction in mammalian cells. suppressors of cytokine signaling (socs), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. here we describe novel function of socs-1 in the suppression of rosmediated apoptosis and associated mechanisms using tnf-alpha induced t cell apoptosis as a model system. both in jurkat t cells and primary splenocytes, socs-1 is induced under tnf alpha-induced apoptosis conditions. the tnf-induced apoptosis was mediated by generation of ros, and the over-expression of socs-1 significantly suppressed tnf-induced ros levels and the subsequent apoptosis. such anti-apoptotic function of socs-1 was manifest not only by the suppression of jaks acting upstream of p38 mapk, but also protection of ptps which down-regulate the tnf alpha -induced jak 1/2 activities. we further show that tnf-alphainduced ptp inactivation can be prevented by socs1 which up-regulates thioredoxin levels. finally we present data that there is a molecular interaction between thioredoxin and ptp which is formed in response to ros-generating stimuli and sustained in socs-1 overexpressing cells. the results strongly suggest that socs-1 acts as an anti-oxidant and anti-apoptotic factor to sustain t cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. aim: inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. the effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the cns by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. cxcl12/sdf-1a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. moreover, it has been described that tnf-a mediates in cxcl12 effects on primary astrocytes, and it is clear that tnf-a participates in the pathogenesis of many neurological conditions. methods: we used the astrocytoma cell line u-87, and sk-n-mc as neuroblastoma cells. detection of tnf-a mrna expression was carried out by rt-pcr. transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. we performed cotransfection experiments of nfat promoter construct with a dominant negative version of nfat (dn-nfat). neuronal death was performed by mtt and tunel assays. nfat translocation was confirmed by western-blot. p53 and fas-l expression was carried out by western-blot. results: cxcl12 induced mrna-tnf-a and transcriptional activity of tnf-a promoter in human astrocytes. this cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on nb cultures, neuronal death increased in a direct and indirect way. surprisingly, tnf-a did not induce nf-kb activation in nb cells but it induced nfat activity. nfat translocation was confirmed by western-blot. neurotoxicity was absolutely reverted in the presence of ciclosporin. we discard p53 pathway as responsible for tnf-mediated toxicity since we did not find any alteration in p53-ser46 levels in stimulated cells. in addition, we found increased fasl expression in neuroblastoma cells after 24h of tnf-a treatment. conclusions: cxcl12 induced-tnf-a promotes nfat activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of fasl expression without alter p53function/levels. s. y. demiroglu 1 , r. dressel 1 1 universitätsmedizin göttingen, zelluläre und molekulare immunologie, göttingen, germany objectives: intracellular hsp70 is part of the cellular stress response system and can inhibit specific apoptotic pathways. extracellular hsp70 on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic t lymphocytes (ctl). interestingly, hsp70 does not protect against cell death mediated by ctl. acute overexpression of hsp70 can even increase the susceptibility of target cells to ctl, which use the granule-exocytosis pathway to induce apoptosis (dressel et al. cancer res 63: 8212) . granzyme b is one of the main effector proteases of cytotoxic granules. therefore, we analyzed the effect of acute overexpression of hsp70 on granzyme b-induced apoptosis. methods: hsp70 was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (ge-tet). the effect of hsp70 induction on granzyme b-mediated apoptosis was now analyzed after delivery of granzyme b into the cytosol of the target cells by the endosomolytic activity of adenovirus type 5. results: hsp70 did not protect the melanoma cells against granzyme b-mediated apoptosis when annexin v binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-3 were evaluated. instead, we observed a moderate but significant pro-apoptotic effect of hsp70 in ge-tet cells when late apoptosis was analyzed at the nuclear level by sub g1 peak measurements (p=0.01). in contrast, a partial protection of ge-tet cells was observed after acute hsp70 overexpression when apoptosis was induced by staurosporine. conclusion: acute overexpression of hsp70 does not seem to protect tumor cells from granzyme b-induced apoptosis; it appears even to accelerate the progression of apoptosis to dna fragmentation and nuclear condensation. it is conceivable that this hsp70 effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated dnase (liu et al., blood 102:1788) . thus, granzyme b might be able to kill even those tumor cells that undergo an otherwise protective stress response. the work has been supported by the grants grk1034 and dr394/2-3. the authors thank prof. c. j. froelich (evanston, usa) for the granzyme b and dr. t. seidler (göttingen) for the adenoviruses. tumor necrosis factor (tnf) elicits its biological activities by stimulation of tnfr1 and tnfr2, both belonging to the tnf receptor superfamily. tnfr1-mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical nfkb pathway, cell death induction and map kinase signaling. in contrast tnfr2-associated signal transduction is poorly defined. earlier findings demonstrated that only membrane tnf, but not soluble tnf, properly activates tnfr2, resulting in depletion of traf2 from the cytoplasm. here we confirm that tnfr2 induced depletion of cytosolic traf2 by the use of tnfr1-and tnfr2-specific mutants of soluble and membrane tnf. corresponding with the known inhibitory role of traf2 in the alternative nfkb pathway, we show that tnfr2 induced activation of the alternative nfkb pathway. thus, we identified activation of the alternative nfkb pathway as a tnf signaling effect that can be specifically assigned to tnfr2 and membrane tnf. j. c. morales 1 , d. ruiz-magaña 1 , d. carranza 1 , c. ruiz-ruiz 1 1 universidad de granada, instituto de biopatologí a y medicina regenerativa. centro de investigación biomédica, armilla, spain different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (trail)-mediated apoptosis. epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to trail by regulating gene transcription of trail receptors and other trail pathway related-genes. in the present study we have analyzed the effect of several histone deacetylase inhibitors (hdaci), belonging to different structural families, on trail-induced apoptosis in leukemic t cell lines. moreover, we have analyzed the activity of hdaci in normal t lymphocytes. we have found that, to a greater or lesser extent, all hdaci potentiate the induction of apoptosis in leukemic t cells by enhancing the signals triggered upon trail ligation, from the activation of the most apical caspase-8. in contrast, hdaci do not sensitize primary resting or activated t lymphocytes to trail-mediated apoptosis. the analysis of the expression of several pro-and anti-apoptotic proteins involved in the trail-signalling pathway indicates that most hdaci regulate the expression of trail-r2 and c-flip in leukemic t cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. zfp36l1 is a zinc finger containing protein that is involved in post-transcriptional gene regulation. it can bind to mrnas containing adenine uridine rich (are) regions and subsequently mediate their degradation. we have previously reported a role for this protein in promotion of b cell apoptosis. one mechanism whereby zfp36l1 may mediate cell apoptosis could be by degradation of cell survival gene mrnas. the bcl-2 protein is an important cell survival protein at different stages of b cell development. bcl-2 mrna also contains are regions that could possibly be targeted by zfp36l1 protein. in the present study, we have tested the ability of zfp36l1 protein to bind to a bcl-2 mrna are probe and to degrade bcl-2 mrna. recombinant bacterially expressed zfp36l1 protein was shown to bind specifically to a bcl-2 are probe by rna electrophoretic shift assays (remsas). furthermore, remsas using cell lysates of ramos burkitt lymphoma b cells stimulated to express high levels of endogenous zfp36l1 also provided evidence that endogenous zfp36l1 in b cells could bind to the bcl-2 are. in order to examine whether zfp36l1 binding to bcl-2 are resulted in bcl-2 mrna degradation, actinomycin d rna degradation assays were carried out on murine embryonic fibroblast (mefs) cells from zfp36l1 knockout mice and wild-type mice using quantitative real-time pcr analysis. bcl-2 mrna was expressed in both wild-type and knockout mefs. the half-life of bcl-2 mrna was found to be extended in knockout mefs compared to wild-type mefs suggesting that zfp36l1 does play a role in degradation of bcl-2 mrna. overall, our data are consistent with a role for zfp36l1 in degradation of bcl-2 mrna which could be a mechanism for the reported role of this protein in induction of b cell apoptosis. the epstein-barr virus (ebv) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. its infection is associated with several human malignancies (hodgkin's lymphoma, burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein lmp1. since lmp1 is able to transform numerous cell types, it is considered as the main oncogenic protein of ebv. the principal mechanism of lmp1 function is based on mimicry of activated member of the tnf receptor super family (tnfr), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like nfkb, c-fos-jnk, pi3-kinase...involved in the regulation of cellular processes. we previously generated two unique model, a monocytic (te1) and lymphocytic (nc5) immortalized by ebv and which expressing type ii latency program. here we developed original dominant negative (dn), by generating a fusion between gfp and tes1 or tes2 (transforming effectors site) derived from the c-terminal intracellular part of lmp1, in inducible vectors. then, we generated cell lines conditionally expressing these dns. we showed these dns not only inhibit survival processes resulting to the impairment of nfkb and akt pathway but increase apoptosis in this cell lines. we demonstrated that this pro-apoptotic effect is due to i) the depletion of lmp1's specific adapters and ii) the recruitment of theses adapters by dns interestingly allowed generation of apoptotic complex involved tradd, fadd and caspase-8. using this nc5 tumorigenic model in scid mice, we showed that induction of the dn lmp1-tes2 prevent development of tumours and mouse death. these dominant negative derived from lmp1 could be used to develop therapeutic approaches in malignant diseases associated with epstein-barr virus, but also in inflammatory pathologies. recent studies indicate that suppressors of cytokine signaling (socs) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. since tumor cells often exhibit aberrant expression of socs genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of socs isoforms during dna damageinduced apoptotic response and cell cycle changes in various tumor cell types. by using tumor cell lines transduced for over-expression or knock-down of distinct socs isoforms, it is found that socs1 and socs3 differentially affect apoptosis and cell cycle changes induced by dna damaging agents in a cell type-specific manner. in t lymphocytic leukemia cell line jurkat, socs1 exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p38mapk activities, while socs3 promoted apoptosis with an increase in p38 activities. in contrast, both socs1 and socs3 display proapoptotic effect in rko colon cancer cell lines upon exposure to gamma radiation or ros-generating agents. notably, effects of socs proteins on cell cycle changes induced by dna damaging agents were rather similar in that over-expression of either socs1 or socs3 induced a slight decrease in g1 or s phase cells and a prominent increase in g2/m cells, regardless of their distinct effects on apoptosis. the analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and cdk inhibitors affecting g1/s transition and g2/m arrest induced by socs1 or socs3. socs1 promoted dna damage-induced p21 induction and g2/m cyclin b expression, while socs3 induced decrease in g1 cyclin e expression. the results suggest that socs isoforms potentially modulate growth of tumor cells exposed to dna damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. the heat shock protein 90 (hsp90) is a highly conserved a widely expressed molecular chaperone. it is known to regulate the activity of several protein kinases or the proper folding of client proteins. hsp90 has also been identified as an important regulator of cellular survival. besides these intracellular functions, extracellular hsp90 can initiate cross presentation or immune responses. apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. however the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. hsp90 is released during necrotic cell death and proinflammatory effects of extracellular hsp90 have been observed. thus, we asked whether apoptozing cells cleave hsp90 during apoptosis or how hsp90 is disposed by these cells. we induced apoptosis either in activated or resting primary human cells and analyzed the hsp90 protein content. we observed that hsp90 is degraded during apoptosis resulting in the formation of a fragment of about 50 kda. this fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. analyzing the isoforms of hsp90 (hsp90 alpha and hsp90 beta) we could show that the 55 kda fragment is formed after degradation of the alpha isoform of hsp90. further, we were able to show, that hsp90 cleavage is dependent on caspase activity and most probably mediated by calpain. analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous hsp90 and caspase inhibitors. we observed a rather proinflammatory cytokine profile, if cleavage of hsp90 was inhibited or if exogenous hsp90 was added. these results demonstrate that cleavage of hsp90 represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells. activity. mifc integrates the advantages of flow cytometry and fluorescence microscopy in one system, the imagestream. upon induction of autophagy, cytosolic lc3-i is processed to lc3-ii, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. an increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. mcf-7 gfp-lc3 cells were therefore incubated in starvation medium for 3 hours, +/-bafilomycin, which potently inhibits lysosomal activity. classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. we conclude that imagestream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. jnk seems to mediate the bcl-2/beclin-1 control of autophagy. recently, jnk was shown to be necessary for beclin-1 upregulation, and jnk-mediated phosphorylation of bcl-2 is associated with both, starvation-and ceramide-induced autophagy. the nfkappab pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. we report here the novel findings that under conditions of starvation, pharmacological inhibition of nfkappab decreased the autophagic flux in mcf-7 cells, while jnk inhibition shows an enhancing effect on autophagy induction. ingenol 3-angelate (pep005), a novel activator of protein kinase c (pkc), has been shown to induce apoptosis in acute myeloid leukemia cells. we show here, that in contrast to leukemic cells, pep005 provides a strong survival signal to resting and activated t cells. this anti-apoptotic effect was dependent upon the activation of pkcv, a pkc isoform restricted to t cells and myocytes. expression of pkcv in the acute myeloid leukemia cell line nb4 turned their response to pep005 from an increased to decreased rate of apoptosis. furthermore, our data show that pep005 inhibited t cell apoptosis through the activation of nfxb downstream of pkcv, leading to increased expression of the anti-apoptotic proteins mcl-1 and bcl-xl. we conclude that pkcv expression determines whether pkc activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. this finding may be of considerable importance for the development of pkc -targeting antileukemic therapies. the neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. several reports indicate that survival and activation of normal b lymphocytes are regulated by nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) autocrine circuits. however, the production and the role of neurotrophins were not evaluated in b lymphoma cells. diffuse large b-cell lymphoma (dlbcl) is a common and often fatal malignancy. despite major advance in the treatment (r-chop protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. we hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of dlbcl tumor cells. this hypothesis was investigated with dlbcl cell lines (su-dhl). thus, we evaluated the ability of su-dhl cells to produce neurotrophins (ngf, bdnf) and to express their receptors (p75, trka and trkb) in different cell culture conditions. our preliminary data show for the first time the production of neurotrophins by dlbcl tumoral cells whose level decreased in apoptotic conditions, in association with bad dephosphorylation suggesting its pro-apoptotic role. furthermore our results suggest that up-regulation of autocrine circuits (expression of trka known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral b cells. objectives: ataxia telangiectasia (a-t) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. this gene encodes atm, a protein kinase which has a major role in dna double strand break response. a-t patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. the susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. although atm is known to have a central role in the induction of apoptosis in response to unrepaired dna double strand breaks its role in extrinsic apoptotic pathways is unclear. this study aimed to investigate if atm has a role in fas induced apoptosis. a bank of lymphoblastoid cell lines (lcls) derived from a-t and normal individuals and tumour samples with wildtype or mutant atm were used in the study. apoptosis was induced by incubating cells with the fas activating antibody ch11 and analysed by flow cytometry. expression of the caspase 8 inhibitor cflip which inhibits fas induced apoptosis was detected by western blot. results: there was no significant difference in the susceptibility to fas induced apoptosis or cflip protein expression between atm mutant and control groups. however cells expressing high levels of cflip protein do show greater resistance to fas induced apoptosis than those with lower expression. whilst the lcls expressed both long and short forms of cflip, the tumour cells expressed only the long form. conclusion: atm mutations do not affect susceptibility to fas induced apoptosis or alter cflip protein expression in lcls or tumour cells. cflip protein levels and fas susceptibility vary greatly between individuals but this is independent of atm status. high expression of cflip protein correlates with reduced apoptosis in response to ch11 treatment but there is no clear difference in cflip expression between atm wildtype and mutant cells. labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. however, little is known about their possible role in the apoptotic cell death machinery. we report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase 8 in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. activation of caspase 8 by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases 9 and 3. diterpenoids also led to time-dependent cleavage of bid. inhibition of caspase-8 abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. in addition, pretreating cells with neutralizing antibodies to fas ligand, tumor necrosis factor receptor 1 (tnf-r1), and tumor necrosis factor (tnf)-a receptor 2 (trail) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ros). however, increased ros production was not directly involved in diterpenoid-triggered apoptosis. these results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. conclusion: cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. synthetic alkyl-lysophospholipids (alps) are a group of unnatural lipids with promising anticancer capability. a prototypic member is the ether lipid 1-o-octadecyl-2-o-methyl-rac-glycero-3-phosphocholine (et-18-och 3 ; edelfosine), which induces selective apoptosis in tumor cells through activation of fas/cd95 independent of its ligand fasl/cd95l. fas/cd95 is activated by edelfosine via its translocation in lipid rafts. in this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zvad-fmk while fasl-stimulated cells stayed mostly alive. furthermore cells could not be rescued by addition of zvad-fmk in combination with necrostatin-1, an inhibitor of death receptor-induced necrosis. fas resistant solid tumor cells overexpressing members of the anti-apoptotic bcl2-family as well as cells overexpressing the cellular regulatory protein flip went in contrast to fas stimulation to apoptosis after treatment with edelfosine. therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present dna mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. the present study focused on the potential influence of oncolitical treatment (5-fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. colo 201, sw1116, lovo, caco-2, ht-29) . in addition, expression of antigens involved in tumour proliferation and apoptosis (ki-67, pcna, p53, bcl-2) was compared with gene expression in the presence or absence of stimuli treatment. percentages of apoptotic cells were detected by using annexin v/fitc and propidium iodide double staining, while progression through cell cycle phases was evaluated by using pi staining. correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from rt-pcr with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of dna changed. data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. the results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. the effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. the purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways. the randomized studies were performed in 88 patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. lymphocytes expressions of bcl-2, bax, caspase 3, 6, 9, nfkb, parp1/89kda, tnfr1/cd120a and cd95/fas were assessed by western-blot and flow cytometry. results: before and after surgery the expression of bcl-2, bax, nfkb, parp1 was significantly lower and expression of caspases, tnfr1 as well as percentage of cd95 cells significantly higher as compared with control group. caspase 3 expression was significantly higher as compared with nfkb, parp1 and tnfr1. in comparison to the standard nutrition preoperative immunonutrition increased bcl-2 and nfkb expressions and decreased caspases and parp1 expressions. in addition, we found a significant down-regulation of bcl-2 expression after surgery, but insignificant in patients with preoperative immunonutrition. conclusion: preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. this modulatory effect of glutamine and omega-3 fatty acids has no influence on patients outcome. the capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. in this study the immunomodulatory effects of salvia mirzayanii a native plant that is widely distributed to iran was investigated. s. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p x 0.01). in flow cytometry analysis, accumulation of apoptotic cells in the sub-g1 phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. dna fragmentation analysis of these cells showed a typical dna laddering. immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p x 0.007). these results indicated the presence of immunomodulatory compounds in the extract of s. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. a new randomized, double-blind, placebo-controlled clinical trial was conducted with 54 healthy volunteers receiving either la1 (10 9 cfu/day) or placebo, during 57 days prior to uv (2 × 1.5 med). blister roofs, liquid and skin biopsies were collected 1, 4 and 10 days after uv exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in uv-induced immune response, quantification of inflammatory cytokines, a dna damage marker (p53). while a similar decrease of lc for both groups was observed on day 1 after uv exposure compared to placebo, la-1 group presented a faster increase of a new subset of epidermal dendritic cells (dc), namely early lc precursors (cd1a low cd207 -) associated with a minor recruitment of monocytes. concomitantly, inhibition of il-10 and a tendency to inhibit il-6 was observed in la-1 group compared to placebo. on day 4, la-1 group presented a greater recruitment of early lc precursors and a trend to increase cd1a low cd207 + lc precursors compared to placebo. additionally, a faster reduction of inflammatory and immunosuppressive cytokines (il-6, tnfa, il-8, and il-10) was observed in la1 group compared to placebo. we show that la1 limits uv-induced immune-suppression and skin inflammation. this contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of la1 supplementation for photoprotection at a lower dose (1 log). the thymus is one of the primary lymphoid organs and plays a central role in the immune system. it provides the essential microenvironment for proper t cell development. in the thymus, the maturation of t cells depends on many interactions between t cells and different stromal cell types, mainly composed of epithelial cells (tecs). foxn1 is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. foxn1 appears to be expressed in all epithelial cells of the early thymic rudiment starting around e11.5. previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. using the cre-loxp system, we showed that a single epithelial cell, when reverted to express foxn1 in a nude (foxn1-deficient) background, can give rise to a functionally competent thymic microenvironment. hence, we hypothesize that the epithelial progenitor cell expresses foxn1. if true, it should be possible to target this cell type by use of foxn1-promoter driven transgenes. conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as huntington's disease. considering this, we are expressing transgenic egfp variants containing either 19 or 82 glutamine residues under the control of foxn1 promoter, leading to different degrees of tec degeneration. furthermore, also under the foxn1 promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (mtz) into a cytotoxic cross-linking agent for conditional cell ablation. preliminary results describing the phenotypes of these mice will be presented. t. kamei 1,2 , y. toriumi 3 , k. kimura 4 1 european university viadrina, frankfurt (oder), germany, 2 shimane institute of health science, izumo, japan, 3 shimane university faculty of medicine, department of pediatrics, izumo, japan, 4 japan yoga niketan, yonago, japan as a method in relieving stress, yoga is popular today. many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. we examined changes of electroencephalograph (eeg) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. the subjects consisted of eight yoga instructors who had been practicing yoga for several years. a 10-minute-rest period, followed by a 15-minute yoga exercise called asana, a 15-minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a 20-minute meditation were performed. throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (fp2). blood samples were drawn before and after each exercise. nk activity and percentages of t-cell and b-cell subsets were measured. during the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in nk activity (r=0.83, p x 0.02), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of t lymphocytes (r=0.77, p x 0.05) were observed. furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of cd4 (r=0.96, p=0.0001). these findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of nk cells and t lymphocytes, mainly of cd4, within a short period of time. we conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. t regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. several recent studies suggest also that diverse populations of regulatory t cell play an important role in regulating t-helper 2 response to allergens, maintaining functional tolerance and preventing allergy. here we demonstrate that cd4 + cd25 + t regulatory (t reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (mcs) without affecting cytokine release. this effect is shown to require a cell-cell contact and depends on interaction between ox40l expressed on mcs and the constitutive expression of ox40 (members of the tumor necrosis factor [tnf] and tnf receptor family, respectively) on t reg cells. this interaction does not alter the activation of plc-g, syk and lat in ige/ag stimulated mcs upon co-incubation with t reg cells, whereas it induces a decrease in the phosphorylation levels of akt. moreover, we find that upon co-incubation with t reg cells, mcs show increased levels of camp, which is known to inhibit mcs function, as a result of ox40l signal. antagonism of camp in mcs reverses the inhibitory effects of t reg cells restoring normal ca 2+ responses and degranulation. the cross-talk between t reg cells and mcs through ox40-ox40l interaction defines a previously unrecognized mechanism controlling mcs degranulation. loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. a healthy immune system responds to antigens with a combination of polarized th1 or th2 effector cells and the induction of antigen specific foxp3+ regulatory t cells (treg). it is believed that the dominant subset determines the quality of the eventual immune response. in allergic asthma there is a clear dominance of th2 cell responses to non-pathogenic environmental antigens. recently it was shown that the specific transcription factors that characterize the th2 and treg subset, gata3 and foxp3 respectively, counter regulate each other (mantel y et al., 2008) . we hypothesize that children with allergic asthma will respond to allergens with low induction of foxp3+ tregs and high gata3+ th2 cells. in order to prove this hypothesis pbmc of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and lps. almost 100 million allergic patients are sensitized to the major birch pollen allergen bet v 1, which cross-reacts with major allergens of fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. the epitopes of bet v 1 recognized by allergic patients' ige antibodies belong to the conformational type and therefore have not been characterized in detail. here we used antibodies raised against peptides spanning the bet v 1 molecule in ige competition experiments to search for sequences which are involved in ige recognition. the strongest inhibition (i.e., g 90 %) of patients' ige binding to bet v 1 was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa 30-59 (p2) and aa 74-104 (p6) of bet v 1. cross-reactive ige epitopes between bet v 1 and related pollen allergens and plant food allergens involved primarily p2. p2 and p6 are not adjacent peptides in the bet v 1 sequence but define a surface-exposed patch on the three-dimensional structure of bet v 1. as determined by surface plasmon resonance, monoclonal antibody mab2 specific for p2 and mab12 specific for p6 showed high affinity, i. e., dissociation constants, k(d) = 8.35e-11 m and k(d) = 1.05e-9 m, respectively. interestingly, peptide-specific mabs inhibited allergic patients' ige antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. this finding indicates that the surface patch defined with anti-p2 and anti-p6 antibodies contains several ige epitopes. in summary, we have defined a surface-exposed patch on the bet v 1 allergen which seems to harbor the majority of the ige epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. background: antigen-specific th1 cells as well as tc1 cells, induced by biolistic gene transfer using plasmid dna encoding the model allergen b-galactosidase (bgal) under control of the fascin promoter (pfascin-bgal), inhibited the elicitation of systemic th2 immune responses and suppressed ige production in an experimental mouse model. moreover, protective biolistic dna vaccination with pfascin-bgal prevented th2-mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bgal protein, but led to the recruitment of th1/tc1 cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (ahr). objective: to analyze the modalities of ahr induction in mice biolistically vaccinated with pfascin-bgal. methods: balb/c mice were immunized with pfascin-bgal using the gene gun. subsequently, mice were challenged by consecutive intranasal application of bgal protein. cd4 + and cd8 + t cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-cd4 (gk1.5) or anti-cd8 (53.6.72) monoclonal antibodies. neutrophilic granulocytes were depleted by treatment of animals with either anti-gr-1 monoclonal antibody rb6-8c5 or monoclonal antibody nimp-r14. one day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (bal) was performed and the frequency of ifn-g-producing cd8 + effector t cells in the lung was determined. results: whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of cd4 + t cells, ahr was not significantly affected, implicating that the elicitation of ahr by cd8 + t cells is dissociated from the activity of neutrophils. this notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered ahr. in contrast to cd8 + t cells, cd4 + t cells induced strong neutrophilic infiltration and ahr. transfer experiments with cd4 + or cd8 + t cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of th1 and tc1 cells operative in the elicitation of airway inflammation. conclusions: robust type 1 immune responses, although highly effective in the counter-regulation of local th2-mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of ahr. respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. house dust mite (hdm) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line nci-h292. the proteolytic activity of these hdm allergens has been proposed to be involved in the activation process. the aim of this study was to compare the influence of hdm extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. in contrast to the hdm major allergen der p 1, we were able to show that the major allergen of phleum pratense, phl p 1, although sharing molecular similarities with der p 1, does not display any enzymatic activity under physiological conditions. therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified phl p 1. chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mrna was used for cdna-microarray analysis. first data show that both, hdm extract and grass pollen allergens, induce the release of il-6 and il-8 from nci-h292 cells. furthermore, stimulation with hdm extract leads to the release of tnf-a, gm-csf and ifn-g. interestingly none of these mediators was induced after stimulation with grass pollen extract or purified phl p 1. in contrast to hdm extract grass pollen allergens induce the release of mcp-1 from respiratory epithelial cells, as well as moderate levels of il-12. detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with hdm induced expression profiles will be helpful towards understanding the allergic response in general. (supported by dfg sfb tr22) results: collectively, responses to blg, but not to bsa, were observed in all groups analyzed, included healthy controls. nevertheless, 3 distinct profiles of response were obtained: children with ige mediated cma had a significant increased level of proliferation (mean±sd of stimulation index(si): 7.2±5.7) and of il-13 (mean±sd: 1157±909 pg/ml), and reduced il-10 (mean±sd of il-10-spot forming units/2x10 5 cells (sfu): 912±510), compared to healthy subjects (3.4±2.7 si, p x 0.05; 355±396 pg/ml, p x 0.05; 1272±623 sfu, for proliferation, il-13 and il-10, respectively); children with non-ige mediated cma had a significant reduction of il-13 (192±362 pg/ml), compared to ige patients (p x 0.0004) and an increased, although not statistically significant, production of ifn-g (33.7±54.6 sfu) compared to control (9.5±9.5 sfu) and to ige-allergic patients (0.6±0.8 sfu). finally, tolerant patients showed reduced il-13 (636±1048 pg/ ml, p x 0.05) and proliferation (3.9±3.5 si), compared to acute ige-cma children. interestingly, the high level of il-10 observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to blg; by contrast, il-4 was undetectable in all patients even blocking the il4-receptor. conclusion: blg-specific, immune responses can be recalled in peripheral blood of cma patients, as well as of normal and tolerant children. a th2-like response with il-13 and proliferation is dominant in ige-mediated cma patients; by contrast a th1-skewed response with ifn-g is present in non-ige-mediated allergic and in those children who outgrew ige-allergy. y. f. tang 1 , b. chua 1 , f.c. lew 1 , a. ho 1 , k. wong 1 , k.l. wong 1 , d. m. kemeny 1 1 national university of singapore, immunology programme, yong loo lin school of medicine, singapore, singapore allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. using a mouse model, this response can be divided into an induction phase, in which cd4 th2 t cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. in the lung, recruited th2 cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (ahr). previously we, and others, have shown that transfer of cd8 t cells inhibits the induction of the th2 response. here we have investigated the effect of cd8 t cells on the effector phase of the inflammatory lung response. in vitro activated ot-i cd8 t cells were transferred to ovalbumin (ova)/ alum immunized mice one day before the first of 3 airway challenges with ova. eosinophil infiltration was inhibited by transfer of cd8 + t cells (36.7 %±4.1 % to 17.6 %±2.7 %). when ifn-gamma -/-ot-i cd8 t cells were transferred, we found that the inhibitory effect on eosinophilia was reduced (39.6 %±5.1 %), suggesting an important role for cd8 t cell ifn-gamma. cd11c + cd103 + cd11blung dcs from cd8 transferred mice secreted higher levels (500pg/ml) of il-12p70 following ex-vivo stimulation as compared with animals that were not given cd8 t cells. these data show that, in addition to regulating the induction of the allergic immune response, cd8 t cells can subsequently divert the local lung environment to one that favors th1 immunity. the chain terminator drug abacavir triggers a serious hypersensitivity reaction in 8 % of patients with hiv infection. this reaction is strongly associated with hla-b*5701 and appears to be mediated by cd8+ t cells producing inflammatory cytokines. we show that cd8+t cell responses can be primed in vitro, in normal blood donors who are hla-b*5701+, but not in non-b*5701+ donors. cd8 t cells, but not cd4 t cells, are expanded by abacavir pulsed autologous apc over a13-day culture, producing ifn-gamma and tnf-alpha upon re-stimulation with apc expressing hla-b*5701. similar responses were detected in abacavirhypersensitive hlab*5701+ patients. responses were not detected using mutant apc deficient in tap or tapasin, or when normal apc were aldehyde fixed before loading with abacavir, indicating a reliance on the conventional mhc-i ag presentation. responses were exquisitely restricted to hla-b*5701 since they were undetectable using apc expressing closely related hla-b57 or b58 allotypes. responses to apc expressing mutants of hla-b*5701 demonstrated a crucial role for residue 116. isolation of peptide fractions from abacavir-loaded cells has led to the identification of specific fractions recognised by an abacavir-specific t cell line. our data suggests that abacavir forms a conjugate with an endogenous peptide that is presented by hla-b*5701 triggering cd8 t cells. we speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic mhc-i, contributing to the responses observed in abacavir naï ve individuals. the molecular mechanisms underlying altered hla-b*5701 may be relevant to the role of other disease-associated class i allotypes such as hla-b27 and b51. a. jenckel 1 , s. bulfone-paus 1 , n. föger 1 1 research center borstel, immunobiology, borstel, germany mast cells play a crucial role in acute inflammatory and allergic reactions. upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. the actin-associated protein coro-nin1a (coro1a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. recent data of our group also indicate a regulatory role of coro1a in mast cell function. coronin proteins have been described to be differentially phosphorylated in vivo. however the molecular mechanisms by which coro1a is regulated in response to physiological stimuli are still poorly characterized. here we investigated the modalities of coro1a phosphorylation during the activation of mast cells. immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of coro1a on serine residues upon antigen-specific engagement of fc-epsilon-receptors. as the phosphorylation status of coro1a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of coro1a during mast cell activation. cell fractionation experiments demonstrated that the association of coro1a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in coro1a phosphorylation. a functional correlation between coro1a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of coro1a. thus, coro1a is a downstream effector molecule of fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. allergen-specific t and b lymphocytes play an important role for the pathogenesis of asthma. t cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. recently, we have shown in a mouse model for allergic airway inflammation that b cells directly interact with t cells in the inflamed tissue and locally produce ige. to analyse t/b-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific t cells and nitrophenolspecific b cells. recipient mice are then challenged intranasally with an np-ova conjugate. this system allows to track single allergen-specific t and b cells in all stages of the immune reaction using flow cytometry and immunohistology. in addition, cells can be re-isolated by flow-sorting for in-depth analysis. using this system we could define several phases of the inflammatory reaction. t and b cells first become activated in the lung-associated lymph nodes. granulocytes can be found very early in the lung tissue and also activated t cells very rapidly emigrate to the site of inflammation. however, clusters of allergen-specific t and b cells can only be found in later stages of the reaction. as another focus, we used our in vivo system to define the role of t cell costimulatory molecules for airway inflammation. costimulatory receptors are key regulators of t cell activation and differentiation and therefore promising targets for therapeutic intervention. of special interest is the t cell-specific icos molecule which is important for t/b cooperation as well as the regulation of chronic inflammatory reactions. using icos knock-out mice we were able to delineate the specific role of icos for the different stages of airway inflammation. in particular, we analysed the impact on t cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. the integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. coronins constitute a family of evolutionary highly conserved wdrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. in mammalians seven coronin family members have been described. the high degree of sequence conservation amongst coronin family proteins suggests common features and functions. however, individual family members may also have developed additional selective and specific functions. our recent studies on coronin1a (coro1a) deficient mice have demonstrated that coro1a exhibits an inhibitory function on the cellular steady-state f-actin content, is required for chemokine-mediated functions in t cells and is involved in the maintenance of t cell homeostasis. coronin1b (coro1b) is a closely related homolog of coro1a and the two genes are co-expressed in hematopoietic cells. to address the question of functional redundancy in vivo, we have generated coro1b deficient mice and crossed them with coro1a deficient mice to obtain coro1a/coro1b double deficient mice. analysis of t lymphocytes from coro1a/coro1b double deficient mice revealed defective chemotactic responses and a severe peripheral t cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. an analysis of coronin deficient mast cells also revealed an involvement of coro1a/coro1b in the regulation of actin cytoskeletal dynamics and the function of mast cells. however, in contrast to the inhibitory effects of coro1a/1b deficiency on t cell function, mast cell degranulation and migration was enhanced in coro1a/1b double deficient mast cells. thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. initially found by serial analysis of gene expression, murine samsn1 (also known as hacs1 or sly2) is a putative adaptor and scaffold protein with a sterile-alphamotif (sam), a src homology 3 (sh3) domain and a predicted bipartite nuclear localization signal. the samsn1 gene is located on mouse chromosome 16 and encodes a well conserved protein with 364 amino acids, which is predominantly expressed in hematopoietic tissues. initial overexpression studies suggest a contribution of samsn1 in b cell activation and differentiation, however its physiological function is yet unknown. to investigate samsn1 expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time pcr. our data revealed an expression of samsn1 in all tested hematopoietic cell types. the highest expression level of samsn1 mrna was seen in mast cells compared to lower levels in macrophages, dendritic cells, cd4+ and cd8+ t cells and b cells. the other two members of the sly family of adaptor proteins -namely sly1 (hacs2) and sly3 (sash1) -were expressed only at a very low level in mast cells. the high level of samsn1 mrna expression in mast cells, together with minimal expression of other sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. to address the potential role of samsn1 in mast cell differentiation and function we are analyzing bone marrow derived mast cells from samsn1 deficient mice. initial in vitro experiments indicate normal proliferation and differentiation of samsn1 deficient mast cells. in additional studies we are now investigating the effects of samsn1 deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. analyzing the role of samsn1 in mast cells will help to define the biological function of this novel class of adaptor proteins. introduction: we showed previously that the ability of murine igg1 antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain n-linked to the antibody fc region (silva et al; j.immunol., 2008). then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic igg1 abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in b cells. objective and methods: to prove this hypothesis it was analyzed the expression of st8siai-v; st6galnac i-iv, st3gal ii -v genes quantitatively by real time-pcr in the hybridomas producer of these two types of igg1 abs. results: we observed that the expression of st3gal i, iii and v coding genes was similar in both hybridomas, while the st3gal ii and iv genes were less expressed in the hybridoma producer of non-anaphylactic igg1. in addiction, the expression levels of st8sia and st6galnac genes in the hybridoma producer of anaphylactic igg1 were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic igg1. conclusion: these data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the igg1 abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. financial support: cnpq, capes, fapesp. basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of tolllike receptors (tlrs), which have been described in basophils, has a similar effect. we found that, in contrast to other tlr agonists tested, only the doublestranded rna poly(a:u) induced the typical t h 2 cytokine and histamine production in vitro. this compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. we took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. this is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils. ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. to investigate the effect of activation of nkt cells at different allergic asthma progression, we administered balb/c mice with a-galactosylceramide (a-galcer), a stimulator for nkt cell activation, before or after ova immunization and measured the airway inflammation of that mice after 2 times of intranasal ova challenge. in our results, the total numbers of bronchoalveolar lavage (bal) cells were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. moreover, significant increased percentage and cell numbers of eosinophils in bal of mice administered with a-galcer before ova immunization was noted. il-5 and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. il-5 and eotaxin levels in the bal fluid were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. these data demonstrate that activation of nkt cells at different allergic asthma progression dictates the different outcome of asthma. in addition, the activation of nkt cells in naïve mice induces airway inflammatory responses. the potential risks of treatment with nkt cell activation on human diseases should be considered. objective: bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of ige and inflammation. histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. the transcription factor irf1 mediates the differentiation into th1 cells by activating multiple genes which are independently crucial for the development of naive t cells into th1 cells. because irf1 is expressed in many different tissues, it can be considered as a master switch factor for th1 cell differentiation. methods: here, we tested mice deficient in irf1 in the murine acute asthma model to evaluate its importance in this th2 cell-mediated disease. the protocol setup was the following: 3 sensitizations s. c. with ova, followed by 3 challenges via ova inhalation and adoptively transferred wildtype cd8+ t cells prior to initial sensitization. in our experiments, we could demonstrate that only after priming of irf1 deficient mice with the help of adoptively transferred cd8+ t cells, asthma symptoms in these mice were more severe than in wildtype controls. as an example, eosinophil infiltration into the lung was increased by 3.5 fold. likewise, ovaspecific antibodies and numbers of goblet cells (fig. 1) were also significantly higher in irf1 deficient mice. conclusion: interferon regulatory factor 1 plays a role in the severity of the development of asthma. in its absence, proinflammatory parameters in the lung are increased significantly. this effect is only visible in the presence of wildtype cd8+ t cells. mechanistically, a potential counterregulation of asthma by th1 cells is not available in irf1 knockout mice. together with our previous report that irf1 represents a susceptibility gene for allergy in the human, our data highlight irf1 as key in regulating the severity of asthma. the sensory neuropeptide substance p (sp) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (apc) activity. here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. to address this question, atopic dermatitis-like allergic dermatitis (ad) was induced in c57bl/6 mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. animals were additionally exposed to noise stress for 24hrs prior to challenge. in this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. at the same time, an increased number of apc could be observed in these areas and contacts between nerve fibers and apc were found. under the same conditions, we were able to show increased nk1-receptor and ppt1 mrna levels. accordingly, sp had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of cd11c expressing apc substets characterized by cd4 and cd8 expression in vitro. in vivo we found a stress dependent shift of cytokine mrna levels towards a th-1 cytokine profile and increased levels of il-2 mrna. further the number of cd25 + t-regulatory cells was increased in vitro. additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. proton pump inhibitors (ppis) that are the cornerstone of gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (ee) in patients with associated symptoms. the most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in ee. under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. nonetheless, a number of evidences suggested the possibility that ppis could have a direct effect in molecular pathways involved in asthma and ee: 1) the inhibitory mechanism of ppis implies alkylation of cysteine residues in gastric atpase3, 2) asthma and ee are prototypic th2 diseases in which the cytokines il-4 and il-13 play a principal role through the activation of the transcription factor stat6, and 3) we have recently demonstrated that some chloromethyl ketones can downregulate stat6 by mechanisms involving cysteine alkylation. on the theoretical basis that cysteine reactivity of ppis may affect the regulation of stat6, we analyzed its effect in the activation of stat6 by il-4 and il-13. we found that treatment of cells with ppis inhibited the ability of il-4 and il-13 to signal stat6 activation in a dose-dependent manner in multiple cell types from different origin. given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. in argentina more than three million people suffer from asthma, and the number is rising. asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. a th2 mediated immune response prevails in these patients. in the asthmatic exacerbation period, crisis (cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (i). we studied 12 asthmatic patients both at cr and i. we evaluated eosinophils, basophils and ige expressing b lymphocytes; as well as t regulatory cells (by expression of cd4 and cd25 high (treg)), that might inhibit the development of a th2 response, together with gdt cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. to evaluate the t cell response, mononuclear cells were cultured for 48 hours in the presence of m. tuberculosis (m) or s. pneumoniae (spn), or absence of them (c). then the percentage of activated cells was determined (expression of cd69 or cd25 at 48 hours). all the parameters were evaluated in peripheral blood by flow cytometry. discussion: even though the pathophysiological characteristics of asthmatic patients in periods of cr and i are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at cr and i. we might be able to detect differences if we studied cells from the lungs, the target organ. we demonstrate that murine and human lc expressed the h4r. the level of intracellular ccl2 production in human lc was reduced after stimulation with h4r agonists and basal production could be restored when h4r was blocked with the specific antagonist jnj7777120. moreover histamine and a h4r specific agonist augmented the migration of lc from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice. in conclusion, the h4r is expressed on murine and human lc and influences the immunomodulatory function and migration of these cells. these findings underline the relevance of the h4r in allergic skin diseases and encourage further exploration of the h4r as a therapeutic target in allergic skin diseases. expression data of the non-coding rna gene, prins (psoriasis susceptibility-related rna gene induced by stress) identified and characterized by our workgroup, suggests a role for prins in psoriasis susceptibility and in cellular stress response. in order to asses the function of prins, we aimed to identify genes regulated by prins and intracellular molecules interacting with this stress-induced non-coding rna. to identify prins regulated genes, we carried out a cdna microarray chip experiment on hela cells where the expression of prins was silenced. this experiment identified g1p3, an interferon-inducible anti-apoptotic gene that was down-regulated by prins silencing. g1p3 was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. to detect prins interacting proteins we applied ribonucleoprotein (rnp) purification in hacat cells. with the help of matrix-assisted laser-desorption ionization time-of-flight (maldi-tof) method we identified nucleophosmin, a protein that physically interacts with prins rna. nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. our data indicate that the non-coding prins rna forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. we suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic g1p3 which can contribute to the pathogenesis of psoriasis. atopic dermatitis (ad) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. rituximab is a monoclonal anti-cd20 antibody therapy that targets pre-b cells and mature b cells, but not plasma cells. ad is generally considered as a biphasic, with switch to initial th2 to chronic th1-predominant disease, in which rituximab may have multiple effects. objectives: to report three patients with severe ad refractory to conventional treatments and to anti-ige monoclonal treatment (omalizumab). materials and methods: three patients with severe refractory ad with high levels of serum ige that received 4 weekly intravenous infusions of rituximab at a dose 375 mg/m 2 body surface each. subsets of lymphocytes were analyzed with multiparametric-flow cytometry (facscalibur, bd) at baseline and at specific intervals after treatment. serum immunoglobulins levels were quantified by nephelometry. results: at baseline, all patients had highly elevated levels of total ige ( g 5,000; g 6,000; g 19,000 mg/dl, respectively). all patients underwent prior treatment with omalizumab for 6 months, with only partial response. then, we started rituximab therapy, resulting in a clear and complete improvement of ad eczema area and severity of skin lesions in all patients. remission of pruritus was observed from the 2 nd week after initiation of rituximab therapy up to 1 year. whereas allergen-specific ige levels were not altered, we observed a large reduction in total serum ige concentrations after initiation of therapy with rituximab. in the first treated patient (follow-up 1 year), ige levels decreased from 5,512 to 1,500 mg/dl. the other two patients are in the 3 and 1-months of the follow-up period. importantly, during follow-up no other therapies were required for ad control. conclusions: treatment with an anti-cd20 antibody led to a dramatical improvement in our series of patients with severe refractory ad. this study support further evidence on the efficacy and safety of rituximab in severe ad. we have previously demonstrated that chronic topical exposure of mice to the contact allergen dncb or to the respiratory sensitiser trimellitic anhydride (tma) preferentially activates t helper (h) 1 and th2 cells, respectively. in addition, a single application results in divergent cutaneous cytokine production and the migration of langerhans' cells (lc) with different tempos. to explore events occurring after allergen application, balb/c strain mice were exposed to a single topical dose of either 1 %dncb, 25 %tma or to vehicle alone for 0.5-6h. measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. exposure to dncb provoked rapid production of il-2 (mean=131pg/ml, n=12, p x 0.001), il-17 (mean=69pg/ml, n=12, p x 0.001) and il-1a (683pg/ml, n=12, p x 0.001) in skin compared with tma-or aoo-treated mice. in subsequent experiments, mice received an intradermal injection of 50ng/ear of murine recombinant il-2 or of the known regulator of lc migration; il-1b. interleukin-1b induced a significant loss of epidermal lc numbers, measured as a function of reduced frequency of mhc class ii positive cells within epidermal sheets, after 4 (32 %) and 16h (31 %) (n=6, p x 0.001). in contrast, il-2 or control injections were without effect. however, il-2 administration caused an increase in cutaneous il-17 production (347pg/ml, n=12, p x 0.001) compared with control injection and naï ve tissue (19 and 63 pg/ml, n=12, ns). in addition, systemic treatment with anti-il-2 antibody failed to impact on lc migration provoked by dncb (33 % reduction; n=6, p x 0.001). in parallel experiments dncb-induced lc migration was blocked by treatment with anti-tumour necrosis factor (tnf)-a antibody, another cytokine known to regulate lc migration. however, dncb-induced cutaneous il-1a (987pg/ml, n=10, p x 0.001) and il-17 (248pg/ml, n=6, p x 0.01) expression was reduced to baseline levels by anti-il-2 treatment. these data demonstrate that il-2 is not involved in the regulation of lc migration, unlike il-1b and tnf-a. however, il-2 is involved in the regulation of the production of other cutaneous cytokines provoked by dncb. therefore it is hypothesised that il-2 may influence lc and dermal dc maturation, via the expression of il-1a and il-17. allergic contact dermatitis (acd) caused by nickel ions (ni) represents the most common form of human contact hypersensitivity. along with other allergies its incidence is increasing in the us and worldwide (nhanes iii survey 2005), but the majority of molecular events underlying this kind of t-cell mediated disease are still widely unknown. to elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. by applying dige technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen ni. in the attempt to further characterize the role of a certain down regulated p38-mapk-pathway related protein (p38prp) in acd, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. our results strongly support an involvement of p38 mapk pathway in allergenspecific signaling responses. it is expected that identification of differentially allergen-regulated proteins and detailed analysis of acd-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (work supported by eu-project novel testing strategies for in-vitro-assessment of allergens, lshb-ct-2005 -018681, www.sens-it-iv.eu.) objectives: schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. in the long term, 15 % of patients develop a lymphoproliferative disorder. schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. the recent finding that treatment with il-1 receptor antagonist (il-1ra; kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine il-1b and of il-1-like cytokines in the pathogenesis of the disease. methods: two patients with recently diagnosed schnitzler's syndrome were treated with kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. blood samples were collected before and after initiation of therapy. serum cytokine levels were measured by elisa, and expression of il-1-related genes by real-time pcr on mrna from blood cd14+ monocytes. results: compared to normal controls, schniztler's monocytes had similar expression of il-18, il-18bp, and caspase-1, both before and after therapy with kineret. il-1b expression was similar to controls before therapy, and was decreased five-fold after therapy. at the serum level, neither inflammatory (il-1b, tnfa, il-12) nor anti-inflammatory cytokines (il-10, tgfb) were detected. as expected, il-1ra was only detectable after therapy. il-18 was detectable in schnitzler's sera at higher levels than in controls (23.0 vs. 10.3 pm) and decreased after therapy (13.2 pm). the circulating il-18 inhibitor il-18bp was lower than in controls and not affected by therapy. thus, free il-18 levels were increased in schnitzler's patients as compared to controls (17.6 pm vs. 7.2 pm in controls) and decreased after therapy (9.9 pm). conclusions: schnitzler's syndrome is not associated to enhanced expression of il-1-related cytokines (il-1b, il-18), nor of the il-1/il-18-converting enzyme caspase-1 in blood monocytes. however, the high circulating levels of il-18 suggest an increased activity of caspase-1, as in the case of autoinflammatory diseases. experiments are in progress to test this possibility. atopic dermatitis (ad) is a chronic relapsing allergic skin disease with a high and growing prevalence. currently around 20 % of the children in industrialized countries are affected. in most cases patients exhibit increased systemic ige-levels (so-called extrinsic form) accompanied by sensitization to allergens. while ad is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. most ad-patients show topical colonization with staphylococcus aureus indicating a defective innate immune response. as a class of pattern recognition receptors, toll-like receptors (tlrs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. all known tlrs except tlr3 signal via myd88 to induce nfxb-dependent gene transcription. tlrs are also known to be involved in the pathogenesis of autoimmune diseases. as chronic ad also has an autoimmune component, the study of myd88 signaling in ad might provide new insights into the function of tlrs. to investigate the role of tlrs in the immunopathology of allergic reactions and skin infections, we induced ad-like symptoms in c57bl/6 myd88-deficient mice by repeatedly sensitizing the mice to ovalbumin (ova) after mechanical disruption of the skin barrier by tape stripping. first results show that myd88-/-mice display reduced inflammation of the treated skin area compared to wildtype mice. immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. skin-draining lymph nodes are less enlarged in the ova-treated knockout mice compared to wildtype and differ in cellular composition. serum antibody levels determined by elisa show reduced systemic total and ova-specific igg1-titers in ova-treated myd88-/-mice compared to wildtype mice, although the nacl-treated myd88-/-control group has higher total antibody titers than the wildtype nacl control group. total ige levels are increased in the knockout mice compared to wildtype mice under both conditions. to further investigate the role of staphylococcus aureus during ad development, we will include topical application of the superantigen staphylococcal enterotoxin b (seb) or living bacteria into our analyses. following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. introdution: the skin of vertebrates is the target for over 15,000 species of hematophagous arthropods. among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. bovines present variable and heritable levels of resistance to the tick rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. in order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, nelore (bos taurus indicus) and holstein (b. t. taurus). methods: skin biopsies (6 mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype n = 4) or from non-infested contra-lateral sites. expression of mip-1a, igf-1, mcp-1 and ip-10 genes was quantified with realtime rt-pcr. sections of paraffin-embedded skin were stained with may grünwald-giemsa for differential cell counts. lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. results: as expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. however the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. inflammation of nelores contained significantly more basophils, eosinophils and mononuclear cells expressing cd3, cd4, cd8, cd21, mhc class ii, and p46 than that of holsteins. lymphocytes expressing wc1 and cd21 antigens were significantly diminished in infested skin of holsteins when compared with control skin (p x 0.05). infested skin of nelores contained significantly more message for mip-1a, igf-1, mcp-1 and ip-10 than that of holsteins. conclusions: although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. supported by cnpq, capes, fapesp, and icttd. in the last decade it has become clear that keratinocytes play an important role in the skin immune system. upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. in a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. it has been proposed (goodman et al.) that keratinocytes rather support a th2 response by interacting lymphocytes. we examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with cd3+ t cells from the same donor. during the coculture either seb or antigen were added. in all experimental approaches the addition of keratinocytes resulted in higher production of ifng by t cells. furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. again, the induction of ifng by the presence of keratinocytes resulted in a marked and significant increase of ifng production by t cells. we were able to show that il-1 plays a crucial role in the induction of ifng in t cells keratinocyte interaction. in addition blocking of lfa-1 in the co-cultures resulted in significantly reduced ifng production by t-cells underlining that icam-1/lfa-1 binding is also crucially important for ifng induction. we conclude from our study that keratinocytes rather support a th1 than a th2 local response pattern by virtue of il-1 secretion and icam-1/lfa-1 interaction. this property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a th2 like micromilieu in acute towards a th1 dominated milieu in chronic lesions. the genotyping of ccl4l gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. this work was supported by project grants of merck-serono and instituto de salud carlos iii (pi07/0329). psoriasis is an inflammatory dermatosis with 2 % prevalence among caucasians. hla-cw6 allele is the gene that confers susceptibility to psoriasis and it is placed near to tnf loci with several snp in promoter region. the most common polymorphisms are two g to a transitions in -308 and -238 positions. strong association was found between polymorphisms in the -238 region with psoriasis. in several diseases, the association with hla and clinical manifestation is different between genders, for example in spondyloarthropathy and hla-b27, and this is a question of increasing interest. the objective of this study was to identify clinical and molecular differences between male and female in brazilian psoriatic patients. sixty-nine individuals assisted at the dermatology outpatient clinic of the teaching hospital, university of campinas, with diagnosis of psoriasis of early-onset (up to 40 years of age) were selected. hla-a -b -c -dr -dq alleles and tnf-238 and -308 snp were differentiated by pcr/ssp. analyzing the total group, 21 patients (30.5 %) were male, 48 (69.5 %) were female. in the male group, the mean age at disease onset was 16,3 years. severe forms were seen in this group (psoriatic arthritis in 4 cases and erythroderma in 2). seven patients (33,3 %) had a favorable evolution of the disease, but 14 (66,4 %) developed extensive psoriasis, covering over 30 % of body surface requiring systemic treatment. the main molecular risk factor for the disease, cw*06 allele was positive in 10 cases (47,62 %), tnf 238 g/a genotype was found in 5 (23,8 %) and tnf 308 g/a in 4 (19,1 %). in the female group, the mean age at disease onset was 14,6 years, one case of psoriatic arthritis and one of erythroderma. twenty-nine (60,4 %) had a favorable evolution of the disease and 19 (39,6 %) an unfavorable evolution. cw*06 allele was positive in 26 cases (54,2 %), tnf 238 g/a genotype was presented in 16 (33,3%) and tnf 308 g/a in 8 (16,6 %). severe disease was seen in male patients. there was no difference in frequency of cw*06 allele between male and female groups, but there was a tendency of significant difference in tnf 238 g/a genotype. we found that c57bl/6 mice were more susceptible than balb/c and dba/2 mice. higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as il-6 and mrp8/14, among different inbred strains of mice. furthermore, we identified the cells which are involved in this cytokine production. as expected, histopathological analysis showed that s. aureus infection induces an influx of monocytes and granulocytes. our study shows that not only bacteria-but also host-specific differences are associated with different courses of s. aureus skin infection. aims: to investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. methods: 136 cases, which were studied retrospectively, included children (84 boys and 52 girls), aged 3-14 years, who had an anaphylactic reaction, out of the 915 that were examined in total. the statistical analysis of the data was held with the spss program. the commonest causes were proved to be food (44 %-particularly sea food and dried fruit), drugs (25 %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites (9 %-mainly caused by hymenoptera). the symptoms included mainly the presence of pruritic pomphus with erythema (76 %), and gastrointestinal symptoms (37 %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. total ige g 100 was found in 26 out of the 47 severest cases (55,4 %), in which the adequate control was held, while in their vast majority (45 out of 47) there was no previous anaphylactic reaction. on the other hand, it was proved in total, that in 53,7 % (73 cases) there was a hereditary family history of atopy, while in 52 children (38 %) there was also a personal history of asthma. finally, at a great percentage (69 %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered. conclusions: it has, therefore, been shown that 1)the anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used. 2) in particular, in many cases it is proved that there is a personal but also a family history of atopy. 3) increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. allergic contact dermatitis (acd) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. as classical type iv delayed hypersensitivity reaction this response is mediated by t-cells. since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. up to now the identification of potential allergens completely relies on animal testing, like buehler assay or guinea pig maximization test (gpmt). due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. since dendritic cells (dcs) play a pivotal role in the initiation of contact dermatitis we chose dcs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between dcs primed by allergens and t-cells in the test tube. the known allergens nickel, dinitrochlorobenzene (dncb) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on dcs derived from monocytes that display a langerhans cell (lc) type-similar phenotype. we used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. in addition to the upregulation of cd86 we could observe dose dependent upregulation of programmed death ligand 1 (pdl-1) and downregulation of the dendritic cell immunoreceptor (dcir). furthermore we observed enhanced t-cell proliferation in mixed leukocyte reactions (mlrs) applying lcs stimulated with allergens ex ante. since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour facs and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. we want to present the case of a 60 year old iraqui patient with arabic ancestors who had been suffering from psoriatic arthritis since 35 years. in march 2006 a treatment with fumaric acid esters in combination with ibuprofen was introduced. this led to the complete healing of the skin lesions. for this reason the dose could be reduced to one tablet fumaric acid esters (120 mg) every second day. in april 2008 the patient presented himself in the consult with multiple livid papules with a diameter of 3 mm in the area of the auricle. the histological examination showed an hhv-8 positive kaposi sarcoma. the differential blood cell count demonstrated a lymphocytopenia. the hiv-serology was negative. the staging examinations (chest x-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. after the diagnosis the treatment with fumaric acid esters was discontinued. over the course the livid papules showed a spontaneous complete regression. a spontaneous regression is known from the iatrogenic ks caused by immunosuppressive therapy when the immunosuppression is terminated. as our patient also showed a spontaneous regression of the kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. sarcoidosis is a multisystemic granulomatous disease with unknown etiology. although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of th1 type immune response. recently, suppressor of cytokine signaling (socs) proteins have been identified as regulators of cytokine signaling pathways. in this study we aimed to evaluate the roles of socs1, socs3 and foxp3 in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. peripheral blood (pb) and broncholaveolar lavage (bal) mononuclear (m) cells from sarcoidosis patients in remission following treatment (responders, n:4), the patients who showed recurrence or progression after treatment (non-responders, n:4) and stage i/ii sarcoidosis cases which were followed up without any treatment (untreated, n:7) were evaluated for socs1, socs3 ve foxp3 mrna expressions by taqman pcr, and also flow cytometric analysis was performed for lymphocyte markers including cd3, cd4, cd25, foxp3, cd4 + cd25 high , cd4 + foxp3 + . expression of socs3 and foxp-3 mrna in pbmcs and balmcs from responders were found to be significantly higher in comparison to other two groups . socs1 was found significantly elevated in pbmcs of responders when compared with other two groups. it was also elevated in balmcs of responders when compared with with those of untreated cases. the proportions of cd25, foxp3, cd4+cd25 high , cd4 + foxp3 + cells in pbmcs and balmcs of responders were found to be increased in comparison to nonresponders and untreated cases. our data demonstrates that socs1, socs3 and t regulatory cells may have potential roles in the control of sarcoidosis. we think that if the roles of socs1 and socs3 molecules and t regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. the inorganic dust, containing free crystalline silicon dioxide (fcs) is critical for the development of silicosis. several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the fcs in affected individuals. the role of fcs in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.we studied the changes of neopterin, as a marker for ifn-g dependent macrophage activation and circulating immune complexes (cic), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing fcs.we survey a group of 62 silicosis patients, with mild (21), moderate (23) and severe (18) silicosis, 92 coal workers, exposed to inorganic dust containing fcs (exposed), and 43 healthy workers without exposure to dust aerosol (controls).the serum quantity of neopterin and cic, containing iga(igacic), igg(iggcic) and igm(igmcic) was detected by elisa. differences between investigated groups were detected by student's t-test and a p-value less than 0.05 was considered significant.neopterin level was significantly elevated in exposed (3,2±0,8ng/ml) compared to controls (1,8±1,3ng/ml; p=0,0001). moreover, the neopterin level in exposed was similar to silicosis patients (2,9±1,6ng/ml).the levels of iggcic was significantly elevated in the exposed compared to controls (81,9±22,7au vs 64,3±16,7au p=0,0001) and to silicosis patients (69,6±20,0au p=0,002). in contrast, igmcic was significantly elevated in silicosis than in exposed (70,2±18,8au vs 62,1±24,2au; p=0,03).in comparison with exposed, significantly higher igmcic was found only in mild, but no in moderate and severe silicosis. in contrast, the level of iggcic in mild and moderate silicosis was significantly lower compared to the exposed (p=0,001 and 0,02 respectively).the obtained results showed that activation of alveolar macrophages mainly depends on the presence of fcs in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. the dynamics of cic suggest the involvement of fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing fcs. described pathologic similarities between sarcoidosis (sa) and tuberculosis (tbc) suggest m. tuberculosis antigens as caustaive agentes. it seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. so different hla haplotypes have been described as a predisposing factor to develop sarcoidosis (hla a*01, b*08, drb1*03 (these of good prognosis) *12/*13/*14/*15) or tbc (drb1*02/*05/*14/*16 and associations with dqa1*02/*03/*05) we describe two cases of two female patients from the same geographic region with mantoux and zhiel-neelsen negative tests and high levels of tnf diagnosed of tbc and sa respectively. both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the tc study (pulmonary nodules and mesenteric adenopaties). we found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. after being treated with anti-tbc drugs, the first one presented a good clinical improvement, so she was diagnosed as tbc. the second one did not improved, so she was treated with corticosteroid, with good results. therefore, she was diagnosed as sarcoidosis. after hla analysis, we noticed that the tbc patient was hla a*01, b*08 and drb1*03 (sarcoidosis good prognosis haplotype) and the patient diagnosed as sarcoidosis was hla a*01, drb1*14. as the results show, could there not be a direct relationship between the hla system and the development of sa or tbc, or in contrast, was the first patient missdiagnosed of tbc being a good prognosis sa? objectives: experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. for example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. ovalbumin (ova), house-dust-mite (hdm) and cockroach (cra) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. the objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. methods: for the induction of asthma, mice were initially sensitised by intraperitoneal injection of ova, hdm, cra or a combination of all three, followed by repeated challenge by intratracheal application of ova, hdm or cra. inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (bal) and by determination of chemokine and cytokine levels in bal and lung tissue using elisa and multiplex technology. additionally, serum levels of ige and igg antibodies were measured. airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. results: significant differences were measured in cell influx, chemokine/cytokine and total ige levels. compared to hdm and cra, ova induced an higher cell influx in the bal, hdm showed an increase of chemokines in bal and increased ige levels in serum. using a combination of all three proteins resulted in the most severe form of asthma. conclusions: to our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. the most sustained and severe form of asthma was induced by the combination model. this model offers particular advantages for evaluating existing and novel therapeutic agents. furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. the present study focused on peri-smi connective tissue capsule formation, the most frequent post-operative local complication in patients receiving smi. we investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. to this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (pbmcs) from the same patients were isolated and analyzed via facs, concentrating on t-effector cells (teff) and t-regulatory cells (tregs: cd4 + , cd25 ++ , foxp3 + ), cytokine profiles, t-cell receptor (tcr) repertoire and reactivity against human heat shock protein 60 (hhsp60). intracapsular tregs were visualized by immunohistochemistry and functionally tested in suppression assays. the cellular composition of intracapsular mononuclear cells showed a preponderance of cd4 + t-helper cells and a significant subset of tcrg/d + cells, exceeding that observed in peripheral blood. il-17, il-6, il-8, tgf-b and ifn-g production prevailed, pointing to a th17/th1 weighted immune response. furthermore, intracapsular t-cells displayed a restricted tcr a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hhsp60. importantly, numbers of intracapsular tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral tregs. our results suggest that silicone triggers a specific local immune response via activated th17/th1 cells, promoting fibrosis due to the production of profibrotic cytokines. clonal restriction of the tcr repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. in this context, hsp60 might be a prominent candidate. taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of smi. the inverse correlation between the degree of capsular fibrosis and the number of intracapsular tregs suggest that tregs may initially be able to inhibit the progress of capsular fibrosis. however, as numbers of tregs, as well as their suppressive capacity decreases over time, fibrosis develops. supported by the competence center medicine tyrol (kmt) and the lore-and-udo-saldow donation. objectives: recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. the present project deals with the standardization and further development of a modified elisa test system (silisa ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (smis) based on the protein signature adhering on the surfaces of such devices (1) . the current silisa ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. the optimization of the silisa ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. the essay was further developed with antibodies against annotated proteins that were not yet tested in the past. all experiments were carried out in a 384 well plate format for high-throughput analysis. statistical analysis has been performed using spss. the extended essay has been successfully established in the system with antibodies against seven already tested proteins, including c-reactive protein, collagen-i, collagen-iii, fibronectin, igg, c3-complement, myeloid related protein 14 and two new proteins, integrin-ß4 and fibrinogen. data from more than 100 patients have been obtained and exploited so far. the intra-and inter-assay variability of the test was reduced to less than 10 % and 16 %, respectively. patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. conclusion: applying the silisa ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. the distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. this work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. it was included at least 3 mice per group with distinct cytokine pattern: th1 (c57bl/10, c57bl/6) and th2 (balb/c). muscular injury was performed by injection of bupivacaine. both th1-dominant strains presented more areas with regenerating myofibers and macrophages at 4 dpi. regional lymph nodes showed significant increase of cellularity and relative numbers of cd3 bupivacaine-inoculated balb/c mice compared to non-inoculated matched mice at 4 dpi. balb/c mice showed increased collagen expression and decrease of mmp-9 activity associated with more mrna for tgf-b1. this study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (th1 cytokines) or myonecrosis and collagen deposition (th2 cytokines). the severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (dcm) is imputed to the stimulatory autoantibodies against the second extracellular loop (ec ii ) of the ß1-adrenergic receptor (anti-ß1ec ii ). to analyze their pathogenic impact as a single causal factor we used a human-analogous lewis rat model of dcm, where monthly subcutaneous immunization of the rats with the ß1ec ii peptide as a glutathione-s-transferase (gst) fusion protein induced production of anti-ß1ec ii and eventually dilated cardiomyopathy. in this model we isolated a ß1ec ii -specific rat monoclonal antibody (clone 13f6), and showed by elisa that it binds to the linearized ß1ec ii peptide. additionally, we confirmed with flow cytometry that 13f6 also binds the ß1ec ii in its native conformation, i. e. directly labeled circular ß1ec ii (dyl649-ß1ec ii ) peptide. moreover, we demonstrated activation of the ß1-adrenoreceptor by 13f6 using a fluorescence resonance energy transfer (fret) assay system in vitro. these data further corroborate the pathogenic role of anti-ß1ec ii antibodies in mediating dcm in this animal model, thus rendering them a potential therapeutic target. therefore, we investigated a novel anti-ß1ec ii -specific peptide-based therapy, by intravenously applying a circular ß1ec ii peptide in the dcm lewis rat model to neutralize the anti-ß1ec ii antibodies. while the peptide therapy strongly reduced the anti-ß1ec ii titers in the serum by up to 80 % and consecutively lead to clinical remission, elispot assays for the detection of ß1ec ii -specific antibody-secreting cells (asc) indicated no difference in the number of long-lived plasma cells in treated animals. in contrast, elispot and flow cytometrical analyses revealed a decrease in the number of ß1ec ii -specific memory b cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. our newly developed anti-ß1ec ii -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory b cells involved in the generation of these autoantibodies. these results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (dcm), are a frequent cause of mortality in humans with dcm being the most common reason for heart failure in young adults. infections with coxsackievirus b3 or cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). immunization with the well-characterized myhca 614-629 epitope elicits autoreactive cd4 + t cell responses that have been shown to be the major mediators of autoimmune myocarditis in balb/c mice. it is known that professional antigen presenting cells (apcs) such as dendritic cells are crucial for initiating and maintaining t helper (th) cell responses affecting the heart muscle. however, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. we therefore generated a tcr transgenic mouse harboring t cells that specifically recognize the myhca 614-629 peptide. in a first step, hybridoma cells were generated by fusing bw5147 tcra -cd8lymphoma cells with myhca 614-629 -specific th cells. tcr expression and antigen specificity was assessed by facs analysis and elispot assay. following subcloning, the variable regions of the expressed tcr were characterized by pcr-sequencing. the rearranged v(d)j regions were subcloned into tcr cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. using this novel tcr tg mouse we plan to investigate in detail the activation of myhca 614-629 -specific t cells during the process of autoimmune myocarditis. furthermore, this new tool will help to generate a high resolution analysis of the contribution of different apcs in the activation and differentiation of autoreactive th cells during inflammatory heart disease. m. relle 1 , a. schwarting 1 , p.r. galle 1 1 university medical center of the johannes gutenberg university mainz, medical clinic i, mainz, germany several mouse or rat models have been established to explore the role of proteinase 3 (pr3), in anca-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that anca alone are not sufficient to induce these diseases directly. therefore, we assessed the expression, mobilization and enzymatic activity of pr3 in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of pr3-positive neutrophils in the peripheral blood of frequently used mouse strains. neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human il-8. pr3-mrna from the murine cancer cell line wehi-274 was amplified by race-pcr and subsequently sequenced. sequence comparisons were done with dnasis software package and the blast tool of the ncbi. promoter analyses were performed with the genomatix software matinspector. we could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of furthermore, we identified an alternative pr3-promoter in the second intron of the mouse pr3 gene. this promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. the identification of an alternative pr3 transcript and its promoter indicates that the murine pr3 may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. objective: recent studies show that in vivo administration of och, a synthetic lipid that specifically activates natural killer t (nkt) cells, results in suppression of th1 mediated immune responses in autoimmune diseases. nkt cell activation depends on lipid presentation via the mhc-i like molecule cd1d on antigen presenting cells such as mature dendritic cells (mdcs) and upon activation by och nkt cells rapidly produce large amounts of th2 cytokines. the goal of this study was to investigate the effect of och and och-primed dendritic cells on atherogenesis. methods & results: ldl receptor deficient (ldlr -/-) mice were fed a western type diet and atherosclerosis was induced via collar placement around both carotid arteries. subsequently the mice were treated i. p. with och (n=13) or pbs (n=11) twice a week for seven weeks. the injections with och did not affect atherosclerotic lesion size. to improve the presentation of och to nkt cells in vivo, bone marrow-dendritic cells were maturated via tlr4 activation, in the presence/ absence of och. subsequently we transferred 1.5x10 6 mdcs (n=11) or och-primed mdcs (n=11) (3 times) to ldlr -/mice. afterwards the mice were put on a western type diet to induce atherosclerosis. vaccination with och-primed dcs resulted in a 70.6 % reduction in plaque size compared to mice treated with mdcs (p x 0.05). during the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant 23.7 % (p x 0.05) reduction in cholesterol levels in the mice treated with och-primed dcs. the number of nkt cells in blood and liver was monitored and a 2 to 3-fold increase in these cells was detected 3 days after the last treatment with och-primed dcs (p x 0.05). additionally, the nkt cells in the liver of mice treated with och-primed dcs produced more il-10. discussion: we conclude that immunotherapy using och-primed dendritic cells efficiently activates nkt cells, resulting in a th2 phenotype of the nkt cells and this leads to an efficient protection against atherosclerosis. these data indicate that immunotherapy based on ligand specific primed dcs may be a novel way to treat atherosclerosis. systemic lupus erythematosus (sle) is characterized by high serum titers of igg anti-nuclear antibodies secreted by plasma cells. however, the characteristics of the igg+ plasma cell antibody repertoire in sle has never been determined on a single cell level and little is known about the role of germinal center (gc) reactions for the development of sle autoantibodies. the igg inhibitory fcgriib knock-out mouse on the c57bl/6 background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsdna and chromatin. to characterize the igg+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed 350 igg antibodies from single isolated gc b cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type c57bl/6 and fcgriib deficient mice. igh and igl chain gene sequence analyses revealed no major differences in the ig gene usage between wild-type and autoimmune mice, but gc b cells of fcgriib were enriched for antibodies with positively charged igh cdr3 regions and anti-nuclear specificity. the overall frequency of autoantibodies was similiar between wild-type and fcgriib deficient mice. however, strongly autoreactive antibodies to dsdna and murine igg2c were isolated only from fcgriib deficient mice, but not from c57bl/6 control mice and somatic mutations contributed to their generation. in summary, our data suggest that the gc reaction plays an important role for the development of self-reactive antibodies in fcgriib deficient mice. the finding that the frequency of autoreactive antibodies is higher in gc b cells than in spleen or bone marrow plasma cells may indicate that autoreactive gc b cells are partly regulated even in the absence of fcgriib. autoantibodies against double-stranded dna (dsdna) and nucleosomes (ncs) represent a hallmark of systemic lupus erythematosus (sle). however, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. high mobility group box 1 protein (hmgb1), a nuclear dnabinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. it is not released, since apoptotic cells are immediately engulfed by phagocytes. conversely, in conditions of clearance deficiency, which is observed in a subset of patients with sle, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing ncs containing the "endogenous adjuvant" hmgb1. we investigated if hmgb1-containing ncs contribute to the breakdown of immunological tolerance against dsdna and ncs. we found that hmgb1 remains associated with ncs released from late apoptotic cells in vitro. hmgb1-ncs complexes were detected also in the blood of patients with sle. hmgb1 containing ncs from apoptotic cells induced secretion of il-b, il-6, il-10, and tnfa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (dc), respectively. cytokine release from murine macrophages was dependent on myd88 and toll-like receptor 2. neither hmgb1-free ncs from living cells nor from apoptotic hmgb1-or hmgb1/2-deficient cells induced marked cytokine production or dc activation. specific inhibition of hmgb1 activity by the antagonistic a box domain significantly reduced capacity of "apoptotic "ncs to induce tnfa and il-10 release by macrophages. immunizations with hmgb1-containing ncs from apoptotic cells induced anti-dsdna and anti-histone igg responses in non-autoimmune mice in tlr2-dependent manner. in conclusion, hmgb1 in complex with ncs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against ncs/dsdna and, hence, to the immunopathogenesis of sle. objective: apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (sle). in agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. still, little is known about how apoptotic cell-derived self-antigens activate autoreactive b cells and where this takes place. methods: injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. binding studies were performed using apoptotic cells and cho cells transfected with class a scavenger receptors (sr). repeated injections of syngeneic apoptotic cells in sr deficient and wild type mice were conducted and antibody production by autoreactive b cells was measured. autoreactivity against sr was followed in two sle prone mice strains over the development of disease and in a cohort of sle patients. an antibody against the sr was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. results: in this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. autoantibodies against sr are found before the onset of clinical symptoms in sle-prone mice, and they are also found in diagnosed sle patients. furthermore, injections of an antibody binding sr enhance the antibody production by b cells when co injected with either apoptotic cells or tnp-ficoll. conclusion: our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of sle. e. glasmacher 1 , k.p. hoefig 1 , e. kremmer 1 , v. heissmeyer 1 stretches and helps in the selection of the correct splicing borders. a allele of (r61h) creates a strong binding site for a splicing enhancer protein srp40 according to bioinformatics. our findings indicate that, the putative branch point, r61h snp and the t stretch located downstream of exon two, plays a role in the alternative splicing of bank1. finally, we believe that bank1 delta 2 protein work as a dominant negative isoform in b cell activation and antobodies production, and may antagonize the effect of the full-length protein. these properties of the delta 2 protein may contribute to the observed reduction in sle susceptibility. s. beermann 1 , r. seifert 1 , d. neumann 1 1 hannover medical school, pharmacology, hannover, germany the biological function of histamine is mediated by four different receptors, namely histamine h 1 receptor (h 1 r), h 2 r, h 3 r, and h 4 r. during an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and t cells. in these cells, histamine may modulate the synthesis of characteristic t cell cytokines such as ifng, which plays a central role in a number of autoimmune diseases. the present study was initiated to analyze the involvement of histamine on the induced production of ifng by immune cells. mouse spleen cells were stimulated in vitro by either immobilized a-cd3 antibodies or cpg-oligonucleotides (cpg-odn) in the presence or absence of histamine or 4-methylhistamine, a h 4 r-selective agonist. ifng production was evaluated by analysis of cell culture supernatants by elisa. both, histamine and 4-methylhistamine concentration-dependently reduced ifng production in splenocytes obtained from control c57bl/6 mice induced by either a-cd3 antibodies or cpg-odn. this histamine effect was completely inhibited by the h 2 r-specific antagonist famotidine, while h 4 r-, h 1 r-, and h 3 /h 4 r-selective antagonists had no or only moderate effects. interestingly, the h 4 r-selective reagent jnj7777120, which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-cd3 induced ifng synthesis, but in contrast it slightly enhanced the histamine effect. thus, at the murine h 4 r, jnj7777120 may be a partial agonist. we conclude that histamine modulates the induced production of ifng by t cells via mainly the h 2 r and, to a much lesser extend, the h 4 r. using this assay system, cells obtained from control c57bl/6 mice will be compared to those from sle-prone mrl lpr/lpr mice and the respective wild type strain mrl +/+ . i objectives: resolvins are products of omega-3 fatty acids and they exert potent anti-inflammatory properties. in this study we examined their effects on cytokine release in healthy subjects and autoimmune patients. to test the in vitro effects of 20 ng/ml resolvin e1(rve1) on the release of tgfb, il-6 and il-17 in the culture of peripheral mononuclear cells (5x10 6 /ml) stimulated by phorbol ester (pma) (1nm), and the combination of pma and ionomycine (3 mg/ml) for 72 hours. methods: mononuclear cells were prepared by ficoll-uromiro gradient centrifugation from 7 healthy subjects and from 10 patients each with sle and sjögren's syndrome (ss). level of cytokines was measured by elisa method. results: in the patients with sle (p = 0.010) and sjögren's (p=0.017) mononuclear cell stimulation by pma resulted in a reduced release of tgf b compared with controls. rve1 significantly reduced tgfb release from control mononuclear cells stimulated by either pma (p=0.041) or pma+ionomycin (p=0.021), however rve1 was ineffective at reducing tgfb release in the sle and ss patients. rve1 caused a non-significant decrease in il-6 release from control mononuclear cells, but was again ineffective in sle and ss patients. the production of il-17 was not significantly modified by rve1 in any of the groups tested. the release of tgfb by 20 ng/ml of rve1 can be significantly reduced in healthy control subjects but not in subjects with sle or ss. at the single dose of rve1 tested, il-6 and il-17 release were not significantly affected in healthy or autoimmune patients. omega-3 fatty acid derived rve1 may affect inflammation in healthy patients by reducing tgfb production but its effects on inflammation in sle and ss patients may be expected to be smaller or non-existent. in addition, the tgfb release in the pma activated mononuclear cells of sle and sjögren's patients is less than that of healthy subjects. g. e. fragoulis 1 , a.k. tsirogianni 1 , m. herrmann 2 , h. m. moutsopoulos 1 , m.n. manoussakis 1 1 university of athens, dpt pathophysiology, athens, greece, 2 university of erlangen-numberg, institute for clinical immunology, erlangen-numberg, germany objectives: altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (sle) that is thought to lead to impaired clearance of apoptotic remnants. herein, we assessed comparatively the phagocytic capacity in the peripheral blood of ss and sle patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of ss patients. methods: patients studied included 29 with primary ss (american-european criteria 2002) and 14 with sle (acr criteria 1997). age-and sex-matched healthy blood donors to the ss and sle groups (13 donors each) were also studied in all assays. the phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (gaipl et al, j autoimmunity, 2007) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (mb-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal pbmc (snec-phagocytosis). salivary gland biopsies of patients with ss with and without malt lymphoma (5 patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (tunel assay) and the presence of macrophages (cd68-staining). results: in agreement to previous studies, mb-phagocytosis was found significantly decreased in granulocytes and monocytes of sle patients (both for p=0.0001). in ss patients, defective mb-phagocytosis involved only monocytes (p x 0.0001) and significantly correlated with the presence of extraglandular manifestations (p=0.02). compared to controls, snec-phagocytosis was significantly increased in the granulocytes of sle (p x 0.0001) and of ss (p=0.001). in the salivary gland biopsies of ss patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. these lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. these phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ b-cell lymphoma. conclusion: in a manner similar to sle, ss patients appear to manifest altered phagocytic capacity. this may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. the autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (sle) patients, but clinical value of such antibodies often is not understood. the purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -adenozine deaminase (ada) in sle and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. methods: 30 healthy persons have been included in our study and 71 sle patients (66 women and 5 men) with various clinical signs (44 persons had 1 st degree of disease activity, 27 persons -2 nd degree of pathological process activity). 18 women had habitual noncarrying of pregnancy (hnp) in anamnesis. antibodies of igg class to ada (anti-ada) determined by technique of indirect elisa developed by us with the use of immobilized form of ada as an antigenic matrix. b 2glicoprotein-i-dependent antiphospholipids (aphl) of igg classes were determined using commercial "anti-phospholipid screen igg/igm" test set (orgentec diagnostica). results: at admission an anti-ada was revealed in 36,6 %, aphl of igg class -in 45,1 % sle patients. it has been noted that igg-aphl were found out in anti-adapositive patients more often and in higher antibody titer, than in anti-ada-negative sle patients (x 2 =6,4; p x 0,02). development of cytopenic syndrome was noted reliable more often in sle patients with associated presence of igg-aphl and an anti-ada in comparison with patients who has not the combinations of these antibodies in blood (x 2 =3,9; p x 0,05). the increased levels of anti-ada were revealed in 11 of 18 women with hnp, and the combination of anti-ada and aphl (9/18) was found out more often than isolated anti-ada (2/18, x 2 =6,5; p x 0,02) or isolated aphl (3/18, x 2 =4,5; p x 0,05). conclusion: taking into account the imbalance of immunoregulatory functions in sle, the further studying of autoantibodies to ada generation seems to be very promising. presence of hnp in anamnesis is the evidence of necessity of careful biochemical monitoring of aphl and anti-ada in women for the prevention of abortus fetus and administration of adequate therapy. objectives: sjögren's syndrome (ss) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). dendritic cells (dc) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. two main subsets of dc have so far been identified in human peripheral blood: myeloid dc (mdc), which can be further divided into mdc1 and mdc2, and plasmacytoid dc (pdc), also known as ifn-a/b producing cells. the pivotal role of dc in inducing and maintaining tolerance could be critical in ss as alterations among dc populations might contribute to autoimmunity. purpose of this study was to quantify mdc1, mdc2 and pdc in peripheral blood from primary ss patients by flow cytometry and compare the results with gender-and age-matched healthy controls. methods: blood samples from 31 pss patients fulfilling the american european consensus group criteria (aecc) and 28 gender-and age-matched healthy controls were collected in heparin tubes. dc populations were stained with the blood dc enumeration kit, miltenyi, according to the manufacturer's manual. cells were analyzed on a facs canto ii, bd, and data analysis was performed with flowjo software, tree star. for the statistical analysis, a two-tailed mann-whitney u test was performed using prism, graphpad. results: pss patients have significantly reduced amounts of pdc (p=0,0002) and mdc2 (p x 0,0001) in peripheral blood. conclusion: alterations in dc populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (sle) or diabetes. in ss patients, up-regulation of interferon-regulated genes has been shown previously. therefore, decreased pdc numbers in peripheral blood from pss patients might explain the fact that an increased ifn signature is found in salivary glands of pss patients, but no elevated levels of ifn-a are measured in serum. recently we reported that malignant cd5+ b cells from patients with b chronic lymphocytic leukemia (b-cll) produce granzyme b (grb) and are rapidly undergoing apoptosis in a granzyme b-dependent manner following interleukin 21 (il-21) stimulation. several autoimmune diseases have been linked to both elevated frequencies of cd5+ b cells and increased il-21 levels. we therefore hypothesized that il-21 may have similar biological effects on cd5+ b cells in autoimmune diseases. here we demonstrate that the amount of il-21 in the serum of systemic lupus erythematosus (sle) patients but not of healthy subjects highly correlated with serum levels of grb. in contrast to b cells from healthy individuals, where no baseline grb expression was found, we demonstrate that up to 14 % of cd5+ b cells in sle individuals expressed grb. in-vitro experiments revealed that il-21 was able to induce expression of grb in b cells from individuals with sle and other autoimmune diseases including psoriasis and rheumatoid arthritis. this effect was direct and was strongly enhanced by engagement of the b cell receptor or toll-like receptor 9. importantly, il-21 significantly decreased the cd5+/cd5-b cell ratio in both sle peripheral blood and healthy cord blood samples, suggesting a preferential induction of cd5+ b cell death. these results suggest that il-21-induced grb may play a regulatory role for cd5+ b cells similar to what we described earlier in b-cll cells. this is the first report uncovering an interrelation between il-21 and grb levels in sle and showing that il-21 reduces the cd5+/ cd5-b cell ratio in b cells from sle peripheral blood and healthy cord blood. endogenous il-21 may therefore play a disease-modifying role and may explain elevated grb serum levels in autoimmune diseases. further studies should evaluate the therapeutic potential of il-21 in sle and other autoimmune diseases. r. de palma 1 , e. d'aiuto 1 , s. vettori 1 , g. abbate 1 , g. valentini 1 1 second university of naples, clinical & experimental medicine, napoli, italy ssc is considered an autoimmune puzzling disease whose pathogenesis is unknown. in the last years, there have been increasing evidences that an interplay between activated t cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (ssc). we have previously shown that peripheral t cells from ssc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same t cell clonotypes found in the affected skin. here, using the same experimental approach, we found that the t cell clonotypes expanded in co-cultures are ab positive, hla-dr positive, and promote apoptosis of autologous ssc fibroblasts. we also found that, in these co-cultures, ssc fibroblasts up-regulated fas and underwent apoptosis that paired with the expression of fas ligand (fasl) on cd4+ t cells. finally, when we added a blocking anti-fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of fas/fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. it has to be reminded that the absence of fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble fas in sera of patients affected by ssc. moreover, in the co-culture supernatants we found tgf-beta, il-1beta, il-6 and il-8, cytokines known to have a role in promoting fibrosis in systemic sclerosis. taken together, these data suggest that t cell response in ssc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. indeed, fibroblasts of ssc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. however, the overall outcome of the t cell response triggered by fibroblasts in ssc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. these findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. systemic lupus erythematosus (sle) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in b cell self-tolerance as reflected by elevated serum igg levels of predominantly antinuclear antibodies (anas). serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. however, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in sle. to determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in sle, we cloned and expressed 169 igg antibodies from single facs purified cd19+cd27+cd38+cd138+ bone marrow plasma cells of 3 patients with sle and tested the recombinant monoclonal antibodies for self-reactivity. our preliminary data on the ig gene repertoire and reactivity profile of human igg+ sle bone marrow plasma cells in comparison to healthy controls will be discussed. z. amirghofran 1 , e. moazemi godarzi 1 , e. kamali sarvestani 1 , e. aflaki 1 1 shiraz university of medical sciences, shiraz, iran, islamic republic of interleukin 6 (il-6) has been shown to be related to the pathogenesis of systemic lupus erythematosus (sle). two polymorphisms in the promoter region of il-6 gene at positions -572 g/c and -174 g/c have been described that are key regulators of il-6 gene. in the present study the relationship between these two polymorphic sites and disease susceptibility in a group of iranian patients with sle was investigated using polymerase chain reaction-restriction fragment length polymorphism method. the genotype distribution and allele frequencies of il-6 gene polymorphism at -174 position showed no significant difference between sle patients and controls. at this position the frequency of gg genotype as well as g allele was higher than c allele in both patients and control groups. in contrary, both allelic and genotypic frequencies at the -572 position significantly differed in sle patients and controls. at this position gg genotype was observed in 77.9 % of patients compared to 68.9 % in the control group (p x 0.014). the frequency of -572 g allele in patients (87.3 %) was also higher than in controls (83.2 %) (p=0.034). the haplotype study showed no significant difference between patients and healthy subjects. the relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. -174 polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p x 0.04). at -572 polymorphism, a significant difference with regard to photosensitivity in male patients (p=0.04) was found. in conclusion, results of this study showed that -572 polymorphism plays an important role in susceptibility to sle and that -174 polymorphism could influence the presence of antinuclear antibodies in the patients. the eukaryotic constitutive proteasome is the main protease expressed in most tissues. recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating nf-kb activity during the intestinal inflammation. in comparison with healthy controls and patients with ulcerative colitis (uc), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with crohn's disease (cd) at both mrna and protein levels. in our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between cd and uc patients. since ifn-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the ifn-g levels are highly elevated in inflamed intestine of cd patients, induction of immunoproteasomes in cd might be mediated by this cytokine. our data with human leukemic t cell lines and primary macrophages show a significant increase in the nf-kb controlled production of proinflammatory cytokines after the ifn-g-mediated induction of immunoproteasomes in these cells. in the dss-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing nf-kb activity in the epithelial and immune cells. however, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. objectives: inflammatory bowel diseases (ibd) including crohn's disease (cd) and ulcerative colitis (uc) are characterized by unknown etiology and chronic intestinal inflammation. noninvasive serological tests to differentiate cd from uc have been searched for a long time. testing for panca together with ascas has good predictive values to identify patients with ibd.the aim of this study was to find evidences for diversity of ascas and anti-mycobacterium paratuberculosis antibodies (anti-mpt) by elisa method. in addition, to examine whether combination of these elisas is useful for distinguishing cd from uc. methods: the study population contained 161 patients with ibd (89 with cd, 41 with uc, 31 with gluten sensitive enteropathy, gse) and 33 healthy control subjects. serum asca igg, asca iga and anti-mpt antibody levels were measured by solid phase enzyme immunoassay. adsorption of 7 asca positive sera was performed by baker's yeast suspension. results: elevated level of asca igg, iga and anti-mpt was shown in cd and gse but not in uc compared to healthy controls. serum levels of asca igg, iga showed a significant positive correlation with anti-mpt antibody levels in cd. repeated adsorptions with yeast removed asca igg and iga from sera of patients, but did not change levels of anti-mpt. these results indicate the diversity of asca igg, iga and anti-mpt (accordingly their antigens) and suggest that combination of these elisa can have a role in the differential diagnostics of ibd. it is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. we have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. we show that distinct populations of t cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative t cell population (ie tcrab+ve, cd4-ve cd8b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating cd4(+) cells in inflammatory bowel disease (ibd). having previously shown in mice that gut-resident t cells express high levels of the regulator of g protein signalling-1 (rgs-1) protein, we have now found substantial over-expression (10-100 fold) of rgs-1 in human gut-derived t cells, particularly in this unconventional t cell population. furthermore, levels appear even higher in t cells derived from inflamed gut. transfection of rgs-1 decreases primary t cell responses to cxcl12 and ccl19, strongly implying that it may regulate t cell localisation. thus, rgs-1 may be a novel target for modulating t cells in ibd, consistent with which snps in rgs-1 have been associated with both coeliac disease and type 1 diabetes. mechanisms involved in the induction of oral tolerance (ot) or systemic immunization through the oral rout are still poorly understood. in our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. conversely, if they are immunized with peanut proteins prior to a challenge diet (cd) containing peanuts they develop chronic inflammation of the gut. our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. adult, female c57bl/6j mice were divided in control (c) and experimental (e) groups. c1-c3 received peanut protein immunization, animals of the control groups c4 were sham immunized, and control group c5 received ovalbumin (ova) immunization. the experimental group was immunized with peanut protein extract. before initial exposure to a 30 day peanut containing cd, the experimental group was divided into 5 groups (e1-e5). ova feeding began 7 days prior cd (e1) on day 0 (e2), 7 (e3), 14 (e4) and 21 (e5) during cd. our results show that oral exposure to a novel protein (ova) in the absence of gut inflammation (e1) leads to low levels of systemic antibody titers, comparable to tolerant animals. conversely, as off initial induction of inflammation, groups submitted to ova (ot) protocol develop increasingly higher systemic antibody (ab) titers similar to animals of the immune control group. in conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. therefore, we assessed the effect of particles on dendritic cells (dc) as key players in the manifestation of intestinal immunity.in in vitro studies we could show that ultrafine tio 2 as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived dc. this effect appears to result from enhanced cell death in immature dc but also from direct stimulation of the cells.to analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase 1 and secretion of bioactive il1-b.in our hands certain nanoparticles induced apoptosis of immature dc, as well as enhanced secretion of active il1-b. we therefore hypothesize that particles can induce the inflammasome which leads to the activation of dc.to study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying 5 % destrane sulphate sodium (dss) in the drinking water for 8 days to wildtype mice. when ultrafine nanoparticles were administered on day 6 and 7 by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. in future experiments we will also analyse the effect of different particles in the il10 -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease. m. schmohl 1 , n. schneiderhan-marra 1 , m. blum 2 , g. stein 2 , m. schmolz 2 , t. joos 1 1 nmi-natural and medical sciences institute at the university of tübingen, biochemistry, reutlingen, germany, 2 edi gmbh, reutlingen, germany the human immune system represents a highly complex system that protects the organism against diseases. there is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. epithelial layers function as a barrier against pathogens. as the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. the ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. this ability appears to be breaking down with chronic inflammatory bowel diseases (ibd) like crohn's disease and ulcerative colitis [1] . to date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. for this purpose a human organo-typic (hot) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. the human caco-2 cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of 24-well culture plates, containing whole-blood. this co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. as a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular mapk and rtk-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. this approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of inflammatory bowel diseases. inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. osteopontin (opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of ibd patients. opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental uc, indicating a controversy in this field, while its role in experimental cd remains unknown. in our study we investigated the role of opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (tnbs) colitis, a t h 1-associated model that resembles cd, and dextran sulphate sodium (dss) colitis, a t h 2-like-associated model for uc. deficiency of opn (either by antibody-mediated neutralization or use of opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that opn, and especially, the secreted isoform of opn (opn-s) is important for the initiation of acute intestinal inflammation. importantly, we discovered that opn drives il-17 production and t h 17 polarization and decreases recruitment of cd4 + cd25 + foxp3 + t regulatory (treg) cells in mesenteric lymph nodes (mlns) of mice with colitis. also, there was an effect of opn on recruitment of cd11c + dcs, which were significantly elevated in mlns of opn -/or anti-opn-treated, as compared to opn +/+ or ig-treated control mice. this finding implies that opn deficiency results in enhanced recruitment of regulatory cd11c + dcs which may mediate treg induction and protect from colitis. overall, our findings indicate that opn is proinflammatory in both types of colitis, by promoting pathogenic t h 17 and attenuating treg cell recruitment, implying also common mechanisms in the pathogenesis of cd and uc. c. shen 1,2 , g. van assche 3 , p. rutgeerts 3 , a. liston 1 , j. l. ceuppens 2 1 k.u. leuven, autoimmune & genetics lab, vib, leuven, belgium, 2 k. u. leuven, experimental immunology lab, leuven, belgium, 3 k. u. leuven, department of pathophysiology, gastroenterology section, leuven, belgium background: haptoglobin (hp) is one of the acute phase proteins synthesized during inflammation. hp-1 allele is associated with the disease behavior in crohn's disease but not in ulcerative colitis. however its role in inflammatory bowel disease has not been defined. aim: to determine whether hp modulates the immune responses in experimental colitis. methods: we induced 3 types of colitis dss (th1/th17), tnbs (th1) and oxazolone (th2) in hp ko mice. neutralizing anti-il-17 mab was injected into dss and tnbs hp ko mice. severity of colitis was evaluated by body weight, colon length and histology. th17/th1 cells were analyzed by flow cytometry. cytokines were measured by elisa or rt-pcr. 1) compared to the wt mice, hp ko mice developed much severer dss and oxa induced colitis. dss induced lethal colitis in hp ko but not in wt mice; 2) in dss but not in oxa colitis mice, il-17, ifn-g, tgf-b and il-6 were significantly increased (p x 0.01, dss vs control) in lamina propria and mesenteric lymph nodes (mln), and this is much evident in hp ko mice compared to those in the wt (p x 0.05, ko vs wt). in tnbs colitis, we found elevated il-12 and ifn-g (p x 0.01, tnbs vs control). although not significant, il-17 was also somewhat upregulated; 3) in dss colitis we observed that il-23 enhanced differentiated th17 cells in vitro, this effect could be abrogated by coculture with serum from wt but not hpko mice. furthermore, in vitro in the presence of tgf-b, il-6 and il-21, more mln-t cells from hpko mice differentiated into th17 cells; 4) anti-il-17 mab improved dss and tnbs colitis, and partially rescued hp ko mice from lethal dss colitis. in line with this, mice treated with anti-il-17 showed reduced il-6, il-17 and ifn-g in both mln and lp (p x 0.05, anti-il-17 vs control). our results reveal that hp has a protective role in the development of mucosal inflammation. in dss and tnbs colitis hp may exert its beneficial effect partially through inhibiting production of il-17, supporting further pre-clinical and clinical application of hp for treatment of crohn's disease. p. engelmann 1 , g. talabér 1 , g. süt" o 2 , p. németh 1 , t. berki 1 1 university of pécs, clinical center, department of immunology and biotechnology, pécs, hungary, 2 university of pécs, clinical center, department of immunology and rheumatology, pécs, hungary objectives: inflammatory bowel disease (ibd) resembles as an autoimmune-like disease. ibd is most common in developed countries: it is calculated that 2.2 million people in europe suffer from ibd. several hypotheses are raised in the pathogenesis of inflammatory bowel disease. one of the most favored is the dysregulation of the immune response due to failure of regulatory t cells. the most well known regulatory t cells are the cd4+cd25hi+ t (treg) cells. furthermore, other immune-regulatory cells are known such as invariant natural killer t (inkt) cells producing both th1 and th2 cytokines rapidly upon antigen (lipid) stimulation. methods: based on this hypothesis we aimed to investigate the role of various immune-regulatory t cells in human ibd. we attempt to measure the proportions of inkt cells, treg cells in peripheral blood of patients with crohn's disease (cd) and ulcerative colitis (uc) compared to normal controls. blood samples were collected from normal controls and ibd patients; then lymphocytes were labeled for inkt and treg markers with specific monoclonal antibodies and measured with flow cytometry. results: according to our results a decline in the total inkt cells of ibd patients was observed, interestingly the proportions of cd4+ and double negative (dn) inkt subgroups showed a characteristic shift among the study groups. percentages of dn and cd4+ inkt subpopulations were assessed after gating of total inkt populations. in controls we observed high percentage of dn inkt cells (74.5 ± 3.5 %, mean ± sem), while cd4+ inkt cells ratio was moderate (25.4 ± 3.5 %). in uc and cd patients we found a reduced proportion of dn inkt cells (uc: 38.0 ± 7.1 %; cd: 34.0 ± 5.2 %, mean ± sem), while the percentage of cd4+ inkt cells was elevated (uc: 62.0 ± 7.1 %; cd: 65.9 ± 5.2 %, mean ± sem) in both disease groups. proportions of foxp3+ treg cells also showed a decline in ibd patients comparing to normal controls. conclusion: this study can provide useful data about the pathogenesis of ibd and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. objectives: the aim of this project is to explore whether exosomes from tgf-b1 gene modified bone marrow-derived immature dendritic cells (md-imdc) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (ibd) in mice, the underlying mechanism was also investigated. methods: exosomes were isolated from supernatant of md-imdc transfected with tgf-b1 adenovirus (tgf-b1-exo). the t cell inhibitory function of tgf-b1-exo was determined by mixed lymphocyte reaction (mlr) in vitro. to evaluate the protective effect of tgf-b1-exo in the development of ibd, dextran sulfate sodium(dss) induced murine ibd was established and mice were treated with tgf-b1-exo. the main symptoms of ibd were observed. the inflammatory degree of colon was also evaluated by histological examination. the relative cd4 + foxp3 + treg cell numbers from spleens and mesentery lymph nodes (mlns) were analyzed by facs. results: it was demonstrated that tgf-b1-exo could inhibit the proliferation of t cells in mlr in vitro. in murine ibd model, after treated with tgf-b1-exo, the main symptoms of ibd such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. analysis of cd4 + foxp3 + regulatory t cells (treg) revealed that the relative numbers of cd4 + foxp3 + treg increased in lymphocytes from mesentery lymph nodes (mlns) of inflammatory site but not from spleens. conclusions: these results demonstrate that immunosuppressive exosomes obtained from tgf-b1 gene modified md-imdc can delay the development of ibd. this protective effect is mediated by the induction of cd4 + foxp3 + treg. tgf-b1-exo might provide a novel strategy for the therapy of ibd. results: hcv-specific cytokine expression by cd8+ t-cells was similar in the four vaccinees as observed by ifng, il-2 production-profiles. however, the killing capacity of expanded cd8+ t-cells was distinct as observed by the competence to kill ns3-peptide presenting transfectants in vitro. as depicted in figure 1 , cd8+ t-cells cells from both vac1 (cleared ) and vac2 (chronic) produced il-2 and ifng after stimulation with ns3-peptide59. however, specific killing of the peptide loaded transfectants was only observed in vac 1, who was able to clear its hcv infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers. [ figure 1 ] killing of ns3 peptide presenting cells was restricted to the vaccinee that was able to clear hcv infection. these results suggest that controlling hcv replication as initiated by this dna-prime mva-boost vaccine-protocol was partly mediated by antigen specific cd8+ t-cells. hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent cd8 t-cells. objectives: infection by hepatitis c virus (hcv) is characterized by its high tendency to chronicity, which is usually associated with a low or absent t-cell response against viral antigens. immune response specific for non-structural protein ns3 from hcv was associated with viral clearance. we have demonstrated that fusion of an antigen to the extra domain a from fibronectin (eda) targets the antigen to tlr4-expressing dendritic cells and improves its immunogenicity. thus, we tested if covalent linkage between eda and ns3 might constitute an alternative for vaccination against hcv infection. methods: recombinant plasmids expressing a secretable version of ns3 or eda-ns3 under the control of cmv promoter were prepared. recombinant ns3 and the fusion protein eda-ns3 were produced in e. coli. the recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. hhd transgenic mice (expressing the human hla-a2 molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(i:c) and anti-cd40 agonistic antibodies. elispot and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. intrahepatic expression of hcv-ns3 rna was measured after a hydrodynamic injection with a plasmid encoding hcv ns3. results: immunization of mice with the plasmids expressing eda-ns3, but not ns3 alone, induced strong t cell responses against the main hla-a2 restricted cytotoxic t cell determinants from ns3. the recombinant eda-ns3 fusion protein, but not ns3, was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of tnf-a by the thp-1 monocyte cell line. immunization of hhd mice with eda-ns3 fusion protein induced both cd4+ and cd8+ t cell responses against ns3 and, when immunized with poly(i:c) and anti-cd40 antibodies, was able to down-regulate the intrahepatic expression of hcv-ns3 rna. the recombinant eda-ns3 fusion protein may be considered for the development of prophylactic or therapeutic vaccines against hcv infection. vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of hiv infection even 25 years after virus detection. therein we propose the intra-dermal inoculation of dna vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus e2 protein encoding an artificial multi-component hiv antigen. this inoculation is followed by electroporation in order to increase dna uptake. we used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. among them, langerhans cells (cd207+cd1a+), located in the epidermis, are dendritic cell subset capable to elucidate specific cd8+ responses. the present work emphasizes molecular and cellular biodistribution of the dna vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in hiv studies. technical approach considers an intra-dermal injection of dna followed by topical electroporation of the injection sites. skin and draining ln biopsies were collected at different time points. these biopsies were used for ihc fluorescent staining in order to establish biodistribution dna-encoded antigens and co-localisation with different cell types. kinetic of antigen expression was studied by bioluminescence in vivo imaging. t cell responses were measured by ifn-g elispot assays up to 3 years after dna vaccination. we show that a dna vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of cd1a+ cells in the draining ln in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. our data suggest that electroporation after intradermal injection of dna vaccine involves langerhans cells from the epidermis that elucidate qualitative anti-hiv immune responses. this new approach that comprise new dna vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human melan-a, in combination with the immunostimulatory cytokine gm-csf, selective targeted dcs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of dcs. in mice, a single application of only low doses elicited strong and long-lasting cytotoxic t-cell responses which provided protective antiviral and antitumor immunity. furthermore, the efficient activation of human tumor-specific cd8+ t cells by mature dcs transduced with melan-a-recombinant human coronavirus 229e indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. as the application of gm-csf already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant coronavirus-based vectors expressing different immunostimulatory cytokines. thereby cytokines will be acting on t cell and dc level. to enhance t cell response interleukin 2 (il2) and interleukin 15 (il15) will be involved, and fms-like tyrosin kinase 3 ligand (flt3l) will be expressed to modulate dendritic cells. il2 is known to enhance early t cell expansion and limits t cell overshoot, whereaes il15 guarantees survival of high affinity t cells during memory phase. on the other hand flt3l enhances dc proliferation and accumulation. with these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific ctl response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches. f. demircik 1 , ag waisman 1 uniklinik mainz, 1. med, mainz, germany in murine cytomegalovirus (mcmv) infection, cytotoxic cd8 t cells and nk cells play a critical role. previously it was shown that mice deficient for b cells are more susceptible to mcmv-related disease, caused by virus reactivation. to better understand the role of b cells and antibodies in the response to mcmv, we made use of different mouse strains that lack b cells, secreted antibodies or il-10 production by the b cells. we found that for the initial t cell response to the virus b cells are important, but antibodies do not play an important role. this implicates b cells as potential important antigen presenting cells (apcs) in the activation of the virus-specific t cells. the reduced t cell response to the virus was observed whether the mice were b cell deficient from birth or if they were depleted later in life. six month after infection mice were tested for the memory cd8 t cell response. interestingly, we found that in mice that lack antibodies (mice that lack b cells all together and mice that have b cells but no secreted antibodies) maintain a rather high t cell response to viral peptides, in a level similar to the acute response 7 days after infection. we conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of t cells. finally, we tested the role of il-10 produced by b cells by conditional deletion of the il-10 gene in these cells. we found that b cell secreted il-10 has a suppressive effect on the t cell response to mcmv, as this response is elevated in these mice. we conclude that b cells are important for an efficient acute response to mcmv and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for b cells in the efficient acute and memory response to mcmv. this work is supported by the deutsche forschungsgemeinschaft grant sfb490 to aw. objectives: ebv infection leads to life-long viral persistence. although ebv infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by t cells. ebv-specific ifng-producing t cells could be demonstrated in acute and chronic infection by many researchers. recent studies in hiv and leishmania provide, however, evidence that assessing ifng alone is insufficient to assess the quantity and quality of a memory t cell response and support the crucial role of multifunctional t cells in disease control. in this study we therefore analyzed ebv-specific t cell responses in peripheral blood (pb) and bone marrow (bm). methods: paired pb and bm samples were obtained from 8 healthy virus carriers who underwent total hip arthroplasty. t cells were expanded for 10 days in the presence of il-2 and il-7 with exposure to overlapping peptide pools of latent ebna-1 and lytic bzlf-1 antigens. ebv-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, cd3, cd4, cd8, ccr7, cd45ra, il-2, tnfa, ifng and cd107a. the majority of ex vivo ebv-reactive cd4+ t cells as well as ebna-1-reactive cd8+ t cells were il-2 and tnfa-producing memory cells, the later being more frequent in bone marrow (cd4+, median, ebna-1: bm 0.69 %;pb 0.12 %; bzlf-1: bm 0.37 %;pb 0.01 %, p=0.039). after in vitro expansion a major subset of ebv-specific cd4+ and cd8+t cells displayed a differentiated effector ifng/tnfa phenotype. a comparable number of ebv-specific cd4+ and cd8+ t cells retained, however, a tnfa single, tnfa/il-2 or triple producer phenotype resembling early differentiated or multifunctional memory t cells, respectively. interestingly, both cd4+ and cd8+ t cells generated from bm revealed significantly higher cytotoxic potential. sorting of ccr7/cd45ra differentiation subsets, revealed that ebv-specific t cells were predominantly expandable from the central memory compartment. conclusion: our data shows that multicolor assessment of ifng, tnfa and il-2 delineates various subsets of ebv-specific memory t cells, which reflect the profile of a protective immune response. human adenovirus (hadv) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (allosct). reconstitution of hadv-specific t cells has been reported to be associated with sustained protection from hadv disease, but epitope specificity of these responses has not been further characterized. furthermore, the relative contribution of hadv-specific cd4 + and cd8 + t cells in the protection from hadv disease after allosct remains to be elucidated. in this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with cd154 or peptide-mhc tetramer staining, that clearance of hadv was associated with a combined hadv hexon specific cd4 + and cd8 + t cell response in both pediatric and adult allosct recipients. based on this observation, we developed a clinical grade method for the rapid generation of t cell lines with high and defined specificity for hadv hexon epitopes for adoptive immunotherapy. activation of hadv hexon-specific cd8 + and cd4 + t cells in peripheral blood with a hexon protein-spanning pool of synthetic 15-mer peptides followed by ifng-based isolation allowed rapid expansion of highly specific t cell lines from healthy adults, including donors without detectable frequencies of hadv hexon-specific t cells. the frequency of hadv-specific t cells was increased to 29-90 % in the t cell lines and the absolute numbers of both hexon-specific cd4+ and cd8+ t cells were 2 to 3 log increased compared to the starting material. detailed analysis showed that hadv-specific t cell lines recognized multiple mhc class i and ii restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of hadv infected target cells. this strategy may be used for adoptive transfer of donor-derived hadv hexon-specific cd8 + and cd4 + t cells for treatment of disseminated hadv infection after allosct. several studies showed that hbv persistance correlates with a failure of an efficient virus-specific t-cell response. induction of hbv-specific t cells by vaccination may be an innovative approach to overcome virus persistance. dna prime-recombinant adenovirus serotype 5 (ad5) boost strategy proved to be effective in stimulating t cell responses and control of viral infections. woodchuck hepatitis virus (whv) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. the efficacy of plasmid dna and ad5 vaccine vectors expressing whv core protein was first examinated in c57bl6 mice. groups of mice were immunized with a dna prime-ad5 boost regimen or with dna and ad5 alone. ad5 was injected i. m. or s. c. t cell response was evaluated by intracellular ifng staining of splenocytes stimulated in vitro with whc-derived peptide pools. anti-whc antibodies were detected by elisa. we detected cd8+ t cell responses against peptide pools 1 and 3 in spleens of dna and dna-ad5 immunized mice. however, in prime-boost group the percentage of of detected ifng+ cd8+ t cells was lower in comparison to dna group. in splenocytes of animals vaccinated with ad5 very weak cd8+ t cell response was observed. in dna vaccinated animals we determined high level of anti-whc already after second immunization. after boosting with ad5 level of antibodies did not change. those antibodies were only igg2a subclass what indicates th1 t helper type of response. ad5-immunized mice had over 3-fold lower level of anti-whc: both igg2a and igg1 subtypes were detected. the weak response induced by ad5 may be due to the low expression of whcag. in ongoing expreriments we improved the protein expression level by insertion of an intron. we currenly investigate the new construct in mice. the new peptide construct containing four m2e-peptide sequences coupled to t helper epitopes from the plasmodium falciparum cs protein and the hepatitis b virus antigen was administered together with adjuvants intranasally and subcutaneously as described (mozdzanowska et al., virology journal 2007) into various mouse strains. in contrast to its predecessor peptide, we found that vaccination induced much higher anti-m2e serum ab titers against peptide and native m2e. this correlated with a large number of m2-specific ab-secreting cells in lungs and bone marrow. moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype a/fm (h1n1), which contains a variant m2e-sequence different in 3 amino acid positions. importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza a strains x31 (h3n2) and the highly pathogenic pr/8 (h1n1) with remarkably reduced viral titers in lungs and noses of mice. in conclusion, our studies show promising results towards the further development of vaccination with m2e as a potential "universal" influenza vaccine. this research is supported by a nih t32 fellowship ca09171-32, the nih grant ai 46457 and a grant from the commonwealth of pa. l. yu 1 1 zhejiang university, zhangzhou, china interleukin-18 (il-18) is a cytokine produced by stimulated mononuclear macrophage system. in this report, 18-day-old chicken embryos were vaccined with the plasmid dna (pci-chil-18) encoding chicken interleukin-18 and the copy numbers of chil-18 in peripheral blood, spleen and bursa of fabricius at different time points post-embryonic-vaccination were detected by real-time fluorescent quantitative pcr. the polyprotein of infectious bursal disease virus (ibdv) was prepared into dna vaccine, and the dna vaccine was co-administrated with pci-chil-18 in 18-day-old chicken embryos, then boosted after two weeks, and challenged with virulent ibdv four weeks later. the results indicated that allantoic cavity vaccinated with pci-chil-18 could accelerate high concentrations of chil-18 in nonage peripheral blood, accelerate high expression of chil-18 in nonage spleen and bursa of fabricius and promote the body early immune response capacity. embryo vaccination with chil-18 could significantly enhance the nonage proliferation responses of t lymphocytes from spleen and b lymphocytes from bursa. meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent ibdv induced by dna vaccine. the results indicated that the nonage immune responsing to ibdv dna vaccine was highly enhanced by embryonic coadministration with chil-18 (p x 0.05). due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("follicular targeting"). applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (apcs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles. in in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. transcutaneously applied nanoparticles in the size range of 40nm, were capable of penetrating the epithelium and entered into human epidermal lcs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle. the use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (dc) in cd8 cell cross-priming and suggests that vaccination via the transcutaneous (tc) route may be relevant in the induction of cellular immune responses. advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. for this purpose, we developed a standard operating procedure (sop) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. in a pilot study on 12 volunteers and a phase i study on 24 volunteers vaccinated with an influenza vaccine, we found that this newly developed sop is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific cd8 cells. induction of t cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. this study proposes new perspectives for the development of vaccination strategies that triggers t cell immune responses in humans. objectives: all anti-hiv-1 neutralizing antibodies are directed toward the viral envelope glycoproteins (gp) 120 and the transmembrane protein gp41. two sites on gp120 and gp41 are attractive targets for vaccine design: the epitope in the third hypervariable region (v3) is recognized by the human monoclonal antibody 447-52d and the epitopes in membrane proximal external region (mper) were recognized by the human monoclonal antibodies 4e10 and 2f5. in order to elicit anti-hiv-1 neutralizing antibodies we have designed virus like particles (vlps) displaying either the gp120-v3 region or the gp41-mper. the vlps are based on the acyltransferase component (e2 chain) of the pyruvate dehydrogenase complex of geobacillus stearothermophilus. the e2 chain self-assembles into a 24 nm protein scaffold resembling a vlp and that contains 60 copies of e2. efficient display and refolding of the v3 and mper regions in e2 vlps are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the 5' of the gene coding for e2. the priming and boosting with a combination of vlps and specific hiv-1 envelope dna were used to immunize mice and rabbits. results: the v3-e2 and mper-e2 vlps were purified as stable 60mers from e. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. binding of 447-52d, 4e10 and 2f5 antibodies to hiv-e2 monomers was confirmed by western blot. we obtained high titers of hiv-1 gp140-specific antibodies in mice immunized with a combination of vlps plus dna (hiv-1 sf162 gp160). these antibodies generated a low (20 %-27 %) level of neutralization. moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. three doses of e2 vlps plus dna elicited a low titer of hiv-1 gp140 specific antibodies. additional rounds of immunizations in rabbits will be performed, in combination with gp160 plasmid dna, to enahance the responses to envelope and to induce neutralizing activity against these key epitopes. our results demonstrate that e2 vlps are able to display antigenic determinants of hiv and to induce high titers of hiv-1-specific antibodies. the e2 vlps represent a promising tool for a vaccine design. now a day we paid for vaccination of previous generations. as a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. the most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. we offer a way of immunity according to the world scientific theory and practice. the method is based on biochemical, electrophysiology, and biology way of correction physical status. at first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. all of it attends indemnity necessary microelements that were extracted from wild officinal herbals. we don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. in our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. that's why when we secure new physical statement in the cns program we forming new nearest and distant men health. we tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. it's completely different disturbances of physician state, which should have different therapeutic approach. the threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. we have previously shown that baxter's vero cell-derived h5n1 whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. more recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic h5n1 influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [1] . the study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a h5n1 whole virus wild-type vaccine, could induce an immune response and protect mice against h5n1 influenza virus infection. a trivalent seasonal influenza vaccine was shown to elicit anti-h5n1 antibody and t cell responses and partially protected mice against a lethal challenge with wild-type h5n1 virus. the protective efficacy of the trivalent vaccine derived mainly from the h1n1 component. moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with h5n1 virus, suggesting that antibodies are the main contributor to protection. h1n1 specific serum did not inhibit neuraminidase activity of h5n1 virus suggesting that protection was not mediated by neuraminidase n1-specific antibodies. next, we investigated the combination of the trivalent seasonal influenza vaccine and the h5n1 whole virus wild-type vaccine. a prime with the seasonal influenza vaccine followed by immunisation with the h5n1 vaccine enhanced anti-h5n1 antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the h5n1 vaccine. hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a h5n1 candidate vaccine. [1] gioia c et al. aims: to register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to 6 years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. methods: in the study we checked the vaccination history of 335 children in the first grade of primary school in the area of central and west macedonia. there were 234 greek and 101 foreign children. as fully vaccinated were considered those who had already undergone at least one dose of hib, meningococcus and pneumococcus, two doses of hav, as well as four doses of dtp-sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. in all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. results: the percentages of the compliance found, are presented in the following 1) it should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to 100%. 2) high percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media). 3) on the contrary, as far as the hepatitis a and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. 4) finally, a statistically significant difference was found relating the response to the vaccination coverage, between greeks and foreigners, but also between the greeks themselves, in relation to their educational and socioeconomic level. objective: over the past three decades, the incidence of type 1 diabetes has dramatically increased in europe and north america, inversely correlated to the decrease of infections. according to the hygiene hypothesis, pathogens may prevent the onset of the disease. om-85, a bacterial extract of both gram positive and gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect non obese diabetic (nod) mice from diabetes development. we aimed here at understanding the mechanism underlying this protection. methods: nod mice and nod-cd28 -/mice, which are devoid of natural regulatory t cells (tregs), were treated with om-85. cytokine secretion, activation and proliferation of b cells and foxp3+ tregs were monitored. as toll-like receptors (tlr) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for tlr2, tlr4 or the myd88 adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of om-85. two synthetic tlr4 agonists used as adjuvant in human (om-174-dp and om-197-mp-ac) were also tested for their capacity to protect nod mice from diabetes. the om-85-induced protection of diabetes required natural tregs, as nod-cd28 -/mice were not protected. remarkably, om-85 activated b cells and not t cells, promoting their proliferation and il-10 secretion, two phenomena that were tlr4-and myd88-dependent. om-174-dp and om-197-mp-ac two synthetic murine tlr4 agonists effectively prevented diabetes onset in nod mice, promoted the expansion of cd4 + cd25 + foxp3 + t cells and the proliferation of il-10 secreting b cells in a dose-dependent manner. conclusion: our results argue for the involvement of tlr4 signaling in the protective effect of om-85 on development of diabetes and show that two other tlr4 agonists induce proliferation of b cells and their secretion of il-10 as well as stimulation of regulatory cd4 + cd25 + foxp3 + t cells. activation of the innate immunity by tlr-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease. d. de wit 1 , a. legat 1 , s. thomas 1 , m. van mechelen 2 , p. hermand 2 , m. goldman 1 1 institute for medical immunology/université libre de bruxelles, gosselies, belgium, 2 glaxosmithkline biologicals, rixensart, belgium aminoalkyl glucosaminide 4-phosphates (agp) are lipid a mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting toll-like receptor 4 (tlr4). since natural lipid a from bacterial lipopolysaccharide (lps) depends on membrane-bound or soluble cd14 (scd14) for its tlr4 ligand activity, we investigated the involvement of both forms of cd14 in the responses elicited by crx-527, a prototypical agp. first, we found that crx-527 efficiently induces nf-kb and irf3 activation in hek cells transfected with tlr4 and md-2 genes, whereas the responses to lps required co-transfection of the gene encoding membrane-bound cd14. likewise, crx-527 efficiently induces the synthesis of nf-kb and irf-3 dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound cd14 prevents lps responses. we then observed that monocyte-derived dendritic cells (dc) which are devoid of membrane-bound cd14 respond to crx-527 but not to lps in serum-free medium. the addition of the soluble form of cd14 did not modify the levels of il12 and tnf produced by crx-527 stimulated dc but increased the levels of interferon-b (ifn-b). when scd14 was added to hek cells expressing tlr4/md-2, nf-kb activity was not modified but irf3 activity was increased in a dose-dependent manner in response to crx-527. we will further compare the responses induced by crx-527 in wild-type and cd14 deficient mice. we previously showed that the transcriptional transactivator (tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. these observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. as we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (ptat) can raise a humoral immune response against two model peptides. these two peptides, which originate from diphtheria toxin (pdt) and from toxin alpha (pt), both contain an i-ad restricted t-cell epitope but are nonetheless non-immunogenic in balb/c mice of the h-2d haplotype when injected with alum. the ptat, pdt, pt, ptatpt and ptatpdt constructs were prepared by chemical synthesis, purified by reverse phase hplc and characterized by mass-spectrometry. pdt+ptat, pt+ptat, ptatpt and ptatpdt were respectively injected twice at two weeks interval in balb/c mice and animals were bled 14 and 28 days after the second immunisation. the sera were subsequently incubated in microtiter elisa plates previously coated with pt and pdt peptides respectively in order to assess the humoral immune response. we observed a lack of antibody response for the immunizations made with the mixture of peptides (pdt+ptat and pt+ptat) but an anti-pdt and anti-pt response for the immunizations made with the two hybrid constructs (ptatpt and ptatpdt). our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect. interleukin-15 gene polymorph isms (c267t, g367a, c13687a and a14035t) and susceptibility to brucellosis in iranian patients russian federation some epidemiological and observational data suggest that farm and pets exposure [1] in early childhood may be conducive to reduced atopy. currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. as we previously reported the decreasing of intracellular ifn-g production by cbmc statistical analysis was performed using the kruskal-wallis and mann-whitney tests. results: we revealed that newborns from rural mothers (n=14) have higher amount of both nonactivated (subtype infg+/cd69-, p=0.02) and activated (subtype infg+/cd69+, p=0.028) cbmc, producing ifn-g, as compared with newborns from urban mothers (n=79) exposure to pets and the risk of allergic symptoms during the first 2 years of life intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study pc09/16 to what extent t-spot.tb could be used in the diagnosis of tuberculosis in children exposed to tb infection? s. a tb) in children, especially in bcg-vaccinated is difficult for diagnosis because of the low percentage of smear positivity (12-14 %) and clinical futures only in severe forms of disease. the purpose of the present study was to evaluate the diagnostic value of t-spot.tb (oxford immunotec, oxford, uk) compared to tuberculin skin test (tst) in children exposed to tb contact in the family. forty three children with a history for bcg vaccination/revaccination, treated in the university clinic for lung diseases in children sofia, bulgaria were enrolled in the study. the patients were divided according to age in the following groups: 5 months -3 years (n=22), 4 -7 years (n=15) and 8 -12 (n=6) tb has the highest diagnostic value in children n 4 years of age in early childhood the diagnostic value of t-spot.tb and tst does not differ cfp-10 antigen is more sensitive for detection of tb-specific t cells compared to esat-6 antigen. 4. in children with tst 5-14 mm t-spot.tb has a high diagnostic value objectives: the goal of this study is to determine the role of tlr2 and tlr4 in the development of spontaneous lupus disease by creating tlr2 or tlr4 deficient c57bl/6 lpr/lpr mice. methods: tlr2 and tlr4 deficient lupus prone mice have been generated by crossing c57/bl6-tlr2 -/-or c57/bl6-tlr4 -/-mice with c57/bl6 lpr/lpr mice which develop a moderate type of lupus related to fas deficiency. we analysed the phenotype of the disease, autoantibody production and renal injury. statistical comparisons were performed using the mann-whitney u-test. results: these mice developed a less severe disease and few immunological alterations. indeed, in tlr2 or tlr4 deficient lpr mice, glomerular igg deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsdna and anti-cardiolipin autoantibody titers were significantly reduced. however, the response against nucleosome remained unaffected, indicating a role of tlr2 or tlr4 in the production of autoantibodies directed against certain slerelated autoantigens. analysis of b cell phenotype showed a significant reduction of mz b cells, particularly in tlr4 deficient mice suggesting an important role of tlr4 in the sustained activation of these cells likely involved in autoantibody production. interestingly, the lack of tlr4 also affected the production of cytokines involved in the development of lupus disease. conclusion: our data show that deficiency in tlr4 pc14/13 expression of full length mcl-1 and its splice variant in juvenile systemic lupus erythematosus (jsle) neutrophils: differential modulation by gm-csf granulocytemacrophage colony-stimulating factor (gm-csf) can prolong neutrophil survival by increasing mcl-1, an anti-apoptotic protein. a splice variant of mcl-1 arises by removal of exon 2 and induces cell death rather than preventing it. here we investigate the expression of both the full length mcl-1 (mcl-1l) and its splice variant (mcl-1s) in jsle neutrophils compared to controls and investigate whether the addition of gm-csf changes the expression of both isoforms of mcl-1. method: neutrophils were isolated from children (diagnosed x 17 years) with jsle (n=14) and non-inflammatory conditions (control, n=14) and incubated with control serum, jsle serum alone or with jsle serum plus 20pg/ml gm-csf. quantitative real time pcr was used to assess mcl-1l and mcl-1s mrna expression (mean ± sem) following incubation in the above conditions and immediately following neutrophil isolation the ratio of mcl-1s to mcl-1l was also higher in jsle patients compared to controls (p x 0.05). the addition of gm-csf to jsle serum was associated with an increase in mcl-1l (1.66 ± 0.31) and a decrease in mcl-1s (2.56 ± 1.1) mrna expression the addition of gm-csf to jsle serum can abrogate the increased neutrophil apoptosis. alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. our results suggest that jsle neutrophils may be more apoptotic due to differential expression of mcl-1 compared to controls, with jsle neutrophils having greater expression of the pro-apoptotic isoform mcl-1s, and less anti-apoptotic full length mcl-1 cyld is a tumor suppressor gene known to play an important role in the nf-kb pathway. to analyze the function of cyld in vivo we used the cyld ex7/8 mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (scyld) to further investigate the connection between scyld overexpression in t cells and colonic inflammation, we used an adoptive transfer model of colitis. therefore naive cd4 + cyld ex7/8 t cells were transferred into rag1 -/-mice which were analysed by mini-endoscopy weekly after cell transfer. here we could demonstrate that cyld ex7/8 cd4 + t cells exhibit less capacity to induce colitis compared to control cells. consequently we investigated if regulatory t cells (t regs ) of cyld ex7/8 mice are capable to control inflammatory responses. for this purpose cd4 + cd25 + cells were co-transferred with naïve wt cd4 + t cells into rag1 -/-recipients. interestingly, rag1 -/-recipients of cyld ex7/8 t regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. our findings demonstrate that overexpression of scyld leads to a hyperresponsive t cell phenotype and higher production of inflammatory cytokines by t cells pc17/16 the role of hla complex in inflammatory bowel disease: crohn's disease and ulcerative colitis de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd). university hospital virgen arrixaca the allele frequencies of hla class i in cd and uc patients were not different to those observed in controls, although we found an increased frequency of a*03 in cd vs uc. haplotype frequencies of hla class i and ii in cd and uc were also not different to those observed in controls. however, we found increased frequencies of drb1*13, *01 and *0103 alleles, and a decreased allele frequency of drb1*15 in cd vs uc patients and controls. these data are in concordance with other previous studies suggesting that, in patients with isolated colonic cd, drb1*0103 is associated with the development of severe disease and positive association of cd with drb3*0301 and drb1*13. indeed, drb1*15 was negatively associated with cd. this allele appears to confer protection against all subgroups of cd, in all ethnic groups including japanese. however, hla-drb1*07 frequency allele, associated in unselected patients with cd in other studies, was not different in our cd and uc patients, and controls. additionally, an increased frequency in hla-drb1*04 in cd was not found in our patients in a different manner to other reported studies. on the other hand, in our uc patients, allele frequencies of drb1*15 were strongly increased with respect to cd and controls. however, the frequency of drb1*04 was decreased in uc with respect to cd and controls. in this sense, our data are agreed with other reports showing that hla-drb1*15 is associated with uc in european, north american, japanese and korean populations methods: a total of 176 children were studied, (92 boys and 84 girls), up to 17 years of age, with symptoms suspicious for epstein-barr virus infection. the elisa method was used to look for specific antibodies against the capsid of the virus vcaigg and against the nuclear antigen ebv-igm, while taking into consideration the possible increase of the vcaigg title between two serum samples. results: totally, 51 positive cases of children were found (29 %) with active infection : 28 boys (14 x 5 years of age, 12 5-10 years of age and 2 g 10 years of age) and 23 girls (10 x 5 years of age, 6 5-10 years of age and 7 g 10 years of age). pharyngitis was present in 47 children (92,2%), 39 had fever (76,5%) and 48 had lymphadenitis (94 %). the lab tests revealed leukocytosis up to 20.000 leukocytes in 29 cases (56,9 %) and leukocytosis g 20.000 in 9 cases (17,6 %). the most frequent complication documented was streptococcal superinfection in 13 children (25,5 %) and thrombocytopenia in 8 children (15,7 %). a past infection (negative ebv-igm values and positive vcaigg values) was virus infection is common among children and teenagers serum negative are mainly the children of little age and 3) there is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks general hospital of rethymno, rethymno, greece shows that the il-13ra2, previously believed to be a decoy for il-13 only, is able to transmit a signal via il-13. our results support this and may suggest that il-13/ il-13ra2 signalling causes disease in oxazolone-induced colitis. currently we are dissecting the role of single cell populations expressing il-4ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. together this data can define a role for il-13 or il-13ra2 and identify specific cell populations methods: splenic apcs exposed to enteroantigen (eag) +/-probiotics were used to stimulate cultured cd4 + cd25 -t cells to which titrated numbers of tregs were added. neutralizing antibodies against il-6 and il-1b and elisa-based cytokine analyses were used to monitor the effect of cytokines secreted in the t cell cultures. results: exposure of apcs to eag and probiotics did not influence eag-specific cd4 + cd25 -t cell proliferation. however, exposure to three of the six probiotics tested (b. bifidum bi-98, l. acidophilus ncfm tm and b. bifidum bi-504) consistently reduced regulatory activity of tregs in a cell-dose dependent manner. the tregreducing activity of probiotics was analyzed using fractionated components of the b. bifidum bi-98 strain. data indicated that bacterial cell-wall components were responsible for reducing treg activity and not components of nucleus or cytoplasm. the probiotic-induced down-regulation of treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as il-6 or il-1b. conclusion: we conclude that certain probiotic strains can modify apcs to cause reduced treg activity in an eag-specific t cell proliferation assay. this effect apparently depends on a direct apc-to-treg cell contact and not secreted cytokines. the apc/probiotics-mediated inhibitory effect on tregs may oppose antiinflammatory activities desired from probiotic therapy palmieri 1 1 'la sapienza dysregulated innate and adaptive immune responses against commensal flora lead to crohn disease (cd) and ulcerative colitis (uc), two different forms of inflammatory bowel disease (ibd), a lifelong inflammatory condition of the gastrointestinal tract methods: we analyzed 21 pediatric cd patients (13 active, 8 remission), 24 pediatric uc patients (17 active, 7 remission), and 37 age-matched non-ibd controls. nkg2d/ligand expression was evaluated by immunostaining and multiparametric facs analysis (on pbmc subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). differences between groups were analyzed with non-parametric and parametric tests; a level of p x 0.05 was considered significant. results: nkg2d expression is selectively upregulated on circulating "innate-like" t cell populations (g/d and cd3+cd56+ nkt cells), in active, but not in quiescent ibd patients; receptor upregulation correlates with disease type (observed in uc, but not in cd patients). in the same patient groups, the appearance of nkg2d ligands on circulating monocytes is also observed. the dramatic increase of nkg2d+ lymphocytes, and the strong upregulation of nkg2d ligands on both epithelial and immune components, are observed in active ibd lesions. conclusions: our observations document the dysregulated expression pattern of nkg2d/ligands on selected innate immunity populations in pediatric ibd patients, both at mucosal and systemic level pc17/26 peripheral and intestinal regulatory t cell dynamics in pediatric ibd patients is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. regulatory t cells (t reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. aims: to evaluate the frequency of regulatory t cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric ibd and non mucosal regulatory t cells were identified by immunohistochemistry; circulating regulatory t cells were analysed by immunofluorescence and facs analysis. differences were analyzed with parametric and non-parametric testsconsidered significant. results: foxp3+ t reg were significantly increased in the intestinal lesions of active ibd patients (cd or uc), and returned to normal levels in post-therapy remission phase. at variance, circulating cd4+ t reg frequency was elevated in patients affected by both forms of ibd, independently of disease activity, as it persisted in the remission phase. a selective imbalance in the frequency of t and nk subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent ibd patients. conclusions: regulatory t cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; t reg dynamics in pediatric ibd patients only partially matches previous data obtained in the adults; quiescent ibd is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically the increased expression of immunoproteasomes in the inflamed mucosa of ibd patients was shown to contribute to this pathology by enhancing nf-kb activation. due to the relation between nf-kb and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in ibd. lmp7 knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß5i and therefore are devoid of immunoproteasomes. to test our hypothesis, we employed the dss colitis model. in contrast to wild-type mice, colitis was attenuated in lmp7 knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. in addition, lmp7 knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by rt-pcr and elisa. especially nf-kb regulated genes show enhanced induction in wild-type mice unlike lmp7 knock-out mice synaptic systems gmbh, braunschweig, germany objectives: although more than 200 million people worldwide are chronically infected with hepatitis c virus (hcv) no prophylactic or therapeutic vaccines do exist to prevent or cure hcv infections. our major objective is to develop a dendritic cell (dc)-based immunotherapy enhancing virus-specific cellular immune response for treatment of hcv infections based on this approach we aim at generating adec-205 antibodies conjugated with immunodominant hcv proteins to induce hcv-specific protective immunity. methods: recombinant hcv proteins are expressed using "expression-ready-clones" containing n-terminally his-tagged hcv-core (aa 1-191) or hcv-ns3 (aa 1027-1218) sequences. protein purification is performed by metal-affinity chromatography on ni-nta-agarose hcv-specific t cell responses are monitored at different time points after immunization by facs and in vitro t cell proliferation assays. results: to obtain high amounts of recombinant ns3 and core we successfully optimized culture and protein purification conditions. briefly, ns3 was purified natively using pbs-based buffers with ph-gradient. in contrast, purification of core was performed under denaturing conditions in presence of guhcl and urea and a ph-gradient elution. moreover, optimized conditions allowing conjugation of adec-205 to recombinant hcv proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. efficient conjugation was verified by western blot analysis. after successful generation of adec-205/ hcv-protein conjugates we are currently establishing optimized vaccination conditions to induce hcv-specific immune responses pd01/2 mva-nef vaccination induces polyfunctional cd4 t-cells and increases the proliferative capacity of cd8 t-cells in hiv-1 infected individuals under haart several vaccination trials have made use of the modified vaccinia virus ankara (mva) as delivery vector. in a therapeutic vaccination trial, we demonstrated that mva expressing the hiv-1 protein nef (mva-nef) was safe in hiv-1 infected individuals under haart and immunogenic in regard to the elicitation of ifn-g mediated cd4 t-cell responses. recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of ifn-g provides limited information on the quality of antigen-specific t-cell responses. the evaluation of several functions is essential, as simultaneous production of multiple cytokines by t-cells is associated with superior control of viral replication. methods: in a retrospective setting, we simultaneously assessed the production of ifn-g, il-2 and mip-1b, the expression of the activation marker cd154 and the differentiation marker cd45ra in nef-specific cd4 and cd8 t-cell populations during the course of the vaccination trial. furthermore we applied a multi-colour cfse based proliferation assay investigating the proliferative capacity and the simultaneous expression of ifn-g, il-2 and mip-1b. results: following mva-nef vaccination, we observed a significant increase of the total nef-specific cd4 t-cell response and a significant increase of polyfunctional nef-specific cd4 t-cells, simultaneously expressing ifn-g, il-2 and cd154. using the standard ics no increase of nef-specific cd8 t-cell responses was observed. however, by the cfse based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of nef-specific cd8 t-cells following mva-nef vaccination. notebly, we observed a correlation between the increase of ifn-g, il-2 and cd154 expressing cd4 t-cells and the increase of proliferating cd8 t-cells suggesting the possibility of a causal link between the two functions. conclusions: the mva-nef vaccine is able to change the quality and quantity of the nef-specific cd4 t-cell immune response and has the potential to increase the proliferative capacity of nef-specific cd8 t-cells in hiv-1 infected subjects under haart this preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (spr) tests. this ab inhibited hiv-1 mediated membrane fusion and p24-detected replication. db81 was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant esn individuals. further characterization of the antibody and of its binding epitope is ongoing following intradermal vaccination with 25mg dna and electroporation of balb/c mice, splenocytes have been incubated with peptides representing class i and ii epitopes, and specific t cell-responses were examined by elispot-assays. the specific antibody responses have been measured by sandwich eli-sas, and neutralizing antibodies have been investigated by hi-assays. results: the vaccibody constructs have been found to be expressed and correctly folded in vitro. the in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific cd4 + and cd8 + t cells. conclusion: antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to apcs. methods: the hcv recombinant proteins rns4 (1677-1756 aa) and rns5a (2061-2302 aa) were conjugated with immunomax using the heterobifunctional reagent sulfo-smcc. balb/c and dba/2j mice were immunized intraperitoneally 2 times at a month interval with different doses (0.1 -2 mg/mouse) of the proteins without adjuvants, as conjugates with immunomax, or with complete freund's adjuvant (cfa) the other combinations were not immunogenic at given doses. it should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of ns4 protein. immunization with rns5a-immunomax conjugate and rns5 in cfa (1.4 mg/mouse) induced a similar antibody activity, but a different t-cell responses. the conjugate induced splenic accumulation of t cells specifically reacting in vitro with ns5a recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. immunization with rns5a in cfa induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. conclusion: covalent conjugates of hcv nonstructural proteins with immunomax effectively induce humoral and cell immune responses pd01/21 degree of cross-genotype reactivity of hcv-specific cd8 t cells directed against ns3 the existence of multiple hcv genotypes characterized by marked sequence differences is a challenge for immune control. the aim of this study was to compare the antiviral cd8 t cell response targeting hcv genotype 1 (gt1) and genotype 3 (gt3) as the most predominant genotypes in germany and to determine the extent of cross-genotype reactivity of specific t cells. we analyzed a cohort of patients with past or ongoing intravenous drug use (ivdu) hypothesizing that multiple exposures to different genotypes may occur. methods: 53 subjects (17 with gt1, 22 with gt3 and 14 anti-hcv-pos/hcv-rna-neg) were analyzed. hcv-specific t cells were expanded from pbmc in the presence of peptide pools covering ns3 from gt1 or gt3. individual reactive peptides and the degree of cross-reactivity between the gt1 and gt3 variants were determined by ics. complete ns3 is sequenced from all viremic patients pd01/22 anti-retroviral effects of type i interferon subtypes in vivo ifna subtypes 1, 4, 6 or 9 suppressed fvreplication in vitro, but differed greatly in their antiviral efficacy in vivo. treatment of fv-infected mice with the ifna subtypes 1, 4 or 9, but not 6 led to a significant reduction in viral loads. decreased splenic viral load after ifna1 treatment correlated with an expansion of activated fv-specific cd8 + t cells and nk cells in the spleen, whereas in ifna4-and ifna9-treated mice it exclusively correlated with the activation of nk cells. other ifna subtypes like ifna2, 5 and 11 are under investigation pd01/23 elimination of immunodominant epitopes from multispecific dna-based vaccines allows induction of cd8 t cells that have a striking anti-viral potential immunodominance limits the tcr diversity of specific, anti-viral cd8 t cell responses elicited by vaccination or infection. to prime multispecific t cell responses, we constructed dna vaccines that coexpress chimeric, multidomain antigens (with cd8 t cell-defined epitopes of the hepatitis b virus (hbv) surface (s), core (c) and polymerase (pol) proteins, and/or the ovalbumin (ova) antigen as stress protein-capturing fusion proteins. priming of mono-or multispecific, hla-a*0201-or k b -restricted cd8 t cell responses by these dna vaccines differed. k b /ova 257-264 -and k b /s 190-197 -specific cd8 t cell responses did although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening ebv-associated diseases, such as posttransplant lymphoproliferative disorders (ptld). thus, immunotherapeutic strategies using adoptively transferred ebv-specific t cells are promising. one option is the generation and expansion of cd4 + and cd8 + t lymphocytes by using ebv-specific synthetic peptides for the stimulation of pre-existing memory t cells. aim of our study was to identify a set of mhc class-ii peptides for each antigen promiscuitive peptides with high syfpeithi scores were tested for immunogenicity using an ifn-g-elispot. pbmcs of at least 16 healthy, randomly chosen blood donors were cultured for12 days in the presence of each candidate peptide. functional and phenotypic analysis of t cells of several donors was performed by multicolor flow cytometry. 48 out of 72 tested peptides could be identified as t-cell epitopes. two of them were defined as immunodominant, as more than 50 % of tested blood donors showed peptide-specific t cell responses. so far, eight of the tested peptides could be identified as mhc class-ii epitopes. furthermore, a highly immunodominant class ii peptide mix consisting of 5 peptides was selected. in conclusion, we could identify several new ebv-specific mhc class-ii epitopes which can be used for united kingdom, 3 hospital de clínicas during persistent hbv infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.in order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic hbv infections a chimeric tcr against hbsag, which aims to increase the percentage and quality of antigen-specific cd8 + t cells, was developed moreover, we pre-conditioned the liver microenvironment by injection of cpg oligodeoxynucleotides (odn) to optimize the recruitment of transferred cd8 + t cells to the liver and to overcome the tolerogenic microenvironment of the liver. we found that the il-12-exposed cd8 + t cells showed at least five-fold increase of survival rate in vivo than il-2-exposed cd8 + t cells did treatment of the recipients with cpg-odn could increase the percentage and also the total amount of transferred cd8 + t cells mainly in the liver. by in vivo brdu incorporation, we demonstrated that the higher in vivo survival rate of il-12-exposed cd8 + t cells and the effect of cpg-odn were due to the up-regulation of the proliferation of those cells. to sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific cd8 + t cells to the liver to exhibit their effector functions. the detailed mechanisms responsible for the il-12 and cpg-odn effects on the regulation hyper igm (him) and wiskott-aldrich syndrome (was) than those of corresponding controls (p x 0.01) . there was a significant elevation of t ada and ada1 activities in iga deficient patients as compared to healthy individuals (p x 0.01) . our results hypothesized that altered ada activity may be associated with altered immunity. therefore, serum ada level could be used as an indicator along with other parameters pd01/61 hiv-1 sequence evolution after dendritic cell-based immune therapy in a phase i/ii clinical trial hiv rna was extracted from plasma samples collected before the startof haart and early after vaccination when haart was terminated. rna was amplified by rt-pcr and sequenced using standard protocols. sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. hiv sequences spanning known and predicted epitopes of the relevant hla alleles from each participant were analyzed in detail. results and conclusion: immune therapy was well-tolerated and no severe adverse effects occurred. after haart termination, plasma viral load became detectable in all patients after 2-6 weeks. follwing the viral rebound a set point was reached, that was lower than the viral load before start of haart. using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract b. de keersmaecker et al.). for studying sequence evolution, complete sets of both pre-haart and post-vaccination sequences were obtained in 12 out of 17 patients. with one exception, variation in sequences of vaccine and control genes of pre-haart samples compared to samples taken early after vaccination was limited. this indicates that there was no significant impact of the immune response on virus evolution at this stage. more focussed analysis on viral sequences spanning specific hla islamic republic of newcastle disease (nd) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. the purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to nd live vaccine. one hundred and twenty, 10-day-old ross 308 broiler chickens divided randomly into 3 groups, 2 experimental and a group as control one. the first experimental group vaccinated by a monovalent nd live vaccine with cl/79 strain, and the second experimental group vaccinated by a bivalent newcastle disease and infectious bronchitis live vaccine with cl/79 and h120 strains, via the drinking water at 10 days of age at the same time, and the control group received no nd vaccine. the antibody response to vaccination was assessed using the hemagglutination inhibition (hi) test by taking blood samples three times, first the day before and the next, 7&14 days post vaccination. results indicated that, although the strain of studied nd live vaccines were the same united kingdom t cell-based ifng release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active tb from successfully treated disease or latent infection. t-cell cytokine profiles vary with in vivo antigen load in viral infections cd4 t cells from 25 patients with active tb and 28 patients with successfully treated tb were analysed for simultaneous expression of ifng and il2 at the single cell level using multi-colour flow-cytometry after 6 hours stimulation with ppd. moreover, cells were stimulated with esat-6 and cfp-10 receiver operator characteristics analysis revealed that a percentage of ifng /il2 dual positive cells x 56 % served as an accurate marker for active tb patients (specificity 100 %, sensitivity 65 %), while frequencies g 56 % were observed in treated as well as active tb patients. in conclusion, quantitation of antigen-specific t cells based on the analysis of ifng only does not allow distinction of patients with active and successfully treated disease pd03/7 necessity of postpone bcg vaccination -lesson from primary immunodeficiencies v. thon methods and results: the czech national database of primary immunodeficiencies (pid) was established in 1993 and is connected with the european database of primary immunodeficiencies (esid). the prevalence of pid in the czech republic (approximately 10 100 000 inhabitants) is 5.33 to 100 000. among these patients there are children diagnosed with severe combined immunodeficiency (scid) and chronic granulomatous disease (cgd) too. according to the czech national database of pid, 12 out of 14 children with later proved scid were immunised with bcg vaccine in the first days of life. nine of them developed disseminated and generalized bcg infections. five children with scid died. moreover, reactivation of bcg was also seen in healthy children after admission of combined vaccines with hepatitis b given at the age of twelve weeks. on the other hand, this was not the case in thousands of children of hbsag positive mothers who were vaccinated against hepatitis b after delivery in the first place and later immunized with bcg vaccine. systematic vigilance against tuberculosis (tb) and vaccination significantly lower the prevalence and risks of tb. in the czech republic, the prevalence of tb is currently 6.12 to 100 000 inhabitants. unfortunately, temporary interruption of bcg vaccination in three large districts in the period of 1986 to 1993 led into higher incidence of tb and appearance of new cases of aviary mycobacteriosis. these complications were not observed in vaccinated children. conclusion: we recommend a change of current practice of bcg vaccination considering new immunization schedule with hexavalent vaccine pd03/8 novel analogues of thalidomide inhibit cd80 expression and production of tnf-a, il-12, ifn-g, cxcl-9 this work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating enl and other diseases. supported by: cnpq, fapemig and capes, brazil. of the b cell follicle. cta1-dd augmented gc-formations, specific antibody responses and cell-mediated immunity to the t cell-dependent antigen np-cgg, but failed to do so when used together with t cell independent antigens, such as np-ficoll or np-dextran. this effect required adp-ribosyltransferase activity, as mutant cta1r7k-dd failed to exert an adjuvant effect. the adjuvant function appeared to correlate with the fdc-localization and turned out to require complement and/or complement receptors (cr) chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by elisa. we found that, in contrast to alum, plg and ps particles, chitosan induced bmdc maturation on its own, as determined by the expression of cd80 and cd86. these effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. the effect of chitosan on cytokine production was investigated using a panel of different tlr agonists in combination with chitosan particles. results show an increase in the secretion levels of il-1a and il-1b, while il-6 levels were not affected. finally we studied the role of inflammasome activation in the enhancement of il-1b production. using bmdc from nlrp3 -/-mice we examined il-1b production in response to different tlr and chitosan combinations. results show that the ability of chitosan to enhance il-1b production is dependent on nlrp3. collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan severe sepsis, induced in mice by cecal ligation and puncture (clp), led to ho-1 expression in infiltrating peritoneal leukocytes, kidney and liver. mortality rate of clp increased from 20 % in wild type (hmox1 +/+ ) mice to 87 % in ho 1 deficient (hmox1 -/-) mice. hmox1 -/-but not hmox1 +/+ mice developed end-stage multiorgan failure. mortality of hmox1 -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. clp induced a significant increase in cell-free hemoglobin free heme was found to sensitize primary hepatocytes to tnf, anti-fas antibody, h 2 o 2 or peroxynitrite mediated apoptosis. this cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine hmgb1. similarly, circulating and cytoplasmic hmgb1 was increased in hmox1 -/-relative to hmox1 +/+ mice following clp. in conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe and b-1,2-linked mannose residues elicit inhibition effect. it was found that inhibition activity of oligosaccharides increases with chain length. immunization with mannan-hsa conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. in the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. acknowledgements: this work was supported by the grant agency of slovak academy of sciences all subjects received dtwp vaccine at 4-6 years of age (booster vaccination), following the national vaccination schedule of iran. blood samples were collected before and 2-4 weeks after the vaccination. immunogenicity of the vaccines was assessed by elisa using commercial kits. results: the geometric mean titers (gmt) of the antibodies induced against diphtheria and tetanus by dtwp-local were 7.7 and 9.4 iu/ml and those of dtwp-pasteur were 8.2 and 8.6 iu/ml, respectively. there was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. the gmts of antibodies produced against pertussis were 30.2 eu/ml for dtwp-local and 47.9 eu/ml for dtwp-pasteur vaccines, respectively (p x 0.001). no significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. conclusion: immunogenicity against diphtheria and tetanus was similar for the two vaccines pd05/18 united kingdom haemorrhagic septicaemia (hs) is an acute disease of cattle and buffaloes in tropical countries, caused by pasteurella multocida serotype b:2 , a gram-negative coccobacillus. jrmt12, an aroa mutant of pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. in this work, the immune response of calves was tested after intramuscular vaccination with single dose of 10 8 cfu of jrmt12. a possible contributory role of cellular immunity against hs was investigated in vaccinated and in control calves after challenge. a lymphocyte stimulation assay was used to assess the effects of a cell-free extract (cfe) of p. multocida on peripheral blood mononuclear cells (pbmcs) isolated from calves at different times after challenge. the results were indicative of a possible immunosuppressive effect of challenge with p. multocida b:2 on calf pbmcs. the suppressive effect was further investigated by in vitro experiments. calf pbmcs obtained from normal calves were treated with cfe for 1 h before adding concanavalin-a (cona) pd06 -vaccination and immunotherapy against parasitic diseases pd06/1 evaluation of simian adenoviral vector adch63 expressing msp-1 as a candidate blood-stage malaria vaccine this successful regime incorporated a human adenovirus serotype 5 (adhu5) prime, boosted eight weeks later with a modified vaccinia virus ankara (mva) vector. adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. recent clinical trials have suggested that the simian adenoviral vector adch63 is a promising clinical candidate. we have developed vectors (of human and simian origin) and mva encoding a novel construct based on p. falciparum msp-1 and have undertaken comparative immunogenicity studies in mice. the antigen, termed 'pfm128 while asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by plasmointo carbon monoxide (co), iron and biliverdin. when infected by plasmodium hb sad mice are protected against experimental cerebral malaria (ecm), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. ho-1 expression and activity are strictly required to suppress ecm in hb sad mice, as demonstrated by functional deletion of the hmox1 locus or pharmacologic inhibition of its enzymatic activity. the protective effect of ho-1 is mediated by co, which inhibits the accumulation of protein-free heme in plasma following plasmodium infection conclusion: topical treatment of cutaneous leishmaniasis with gsno accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben gp63 expression was confirmed by sds-page and elisa using monoclonal antibody against gp63. discussion: today researchers attempt to find a suitable vaccine for leishmaniasis. although some researchers have reported proper vaccines of interest, a5-180recp is recognized only by sera collected from resistant bovines infested with all stages of r. microplus, but not by sera from similarly infested, susceptible hosts. furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with a5-180recp. our results show that reverse immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. supported by cnpq and fapesp. for the maintenance of ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. memory b-cell elispot together with ab titres might therefore prove useful as independent marker for duration of protection. objective: this study focused on establishing experimental conditions and optimizing the performance of the memory b-cell elispot assay by detection of specific memory b-cells against anti-tetanus vaccine and naturally acquired toxoplasma gondii infection as a model. methodology: twelve healthy subjects who had received the tetanus vaccine at least 6 month previously were enrolled. peripheral blood mononuclear cells (pbmcs) were isolated from each donor using cell preparation tubes (cpt). plasma was obtained after centrifugation of cpt and stored at -20°c until used for elisa. specific igg-secreting b-cells were determined by elispot assay, using tetanus toxoid (tt) and t. gondii surface antigen as model antigens. results: to optimize our assay, conditions were changed and compared to the previously established protocol. we detected low frequencies of total igg memory b-cells and tt-specific memory b-cells in all donors four seropositive and 2 seronegative donors had positive responses in elispot. no correlations were found with serum antibody titers and frequencies of memory b cells (r=0.216, p=0.641) or with t. gondii-specific b-cells conclusions: following optimization of several assay parameters, we demonstrated that the memory b cell elispot could be reliably used to determine low numbers of antigen-specific memory b-cells in individuals naturally exposed to infection or following vaccination our previous work demonstrated that il-17 also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, bfu-e, but inhibiting the growth of late stage erythroid progenitors, cfu-e, from normal murine bone marrow. we also provided in vitro evidence that at least part of its effect on cfu-e is mediated by nitric oxide (no) generation. in the present study we demonstrated that the in vivo reducing effect of il-17 on bone marrow cfu-e was prevented by co-treatment with the no synthase (nos) inhibitor, l-name, implying that this effect is mediated through nos activation. the data obtained in cultured bone marrow cells showed the ability of il-17 to upregulate the expression of mrna for both the inducible (i)nos and the constitutive, endothelial (e)nos isoform. both the nos-inducing effect of il-17 and il-17-related inhibition of cfu-e growth were dependent on p38 mapk activity, since the p38 mapk inhibitor, sb203580, markedly downregulated il-17-induced activation of nos and reversed the growth inhibitory effects of il-17 on cfu-e. the in vivo stimulating effect of il-17 on bfu-e colony growth in the bone marrow was not affected by co-treatment with the nos inhibitor, pointing to different mechanisms for il-17 effects on bfu-e and cfu-e. however, the in vivo exposure of the mice to l-name, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that no itself is important regulator of hematopoietic progenitor cell activity. overall, the data presented gave an insight into the mechanisms by which il-17 acts on bone marrow cells and also revealed a link between the il-17, no and hematopoiesis. further studies on il-17-mediated induction of both inos and enos methods: a total of 1785 blood donor samples were tested for hbsag and anti-hbc with the immunoenzymic method elisa, while simultaneously, molecular blood test (nat) was applied. the positive samples for anti-hbc were also tested for anti-hbs and anti-hbc igm. results: a total of 68 samples (3,8 %) were found anti-hbc positive conclusions: it is proven, therefore, that in some cases the levels of hbsag, following an infection from the hepatitis b virus, are probable to remain low, so that it is not possible to detect them using elisa method. in these cases anti-hbc can be the only serological marker of the infection. consequently, patients with positive anti-hbc and levels of anti-hbs x 100 iu/l are possibly not immune enough, so that they can become blood donors. that was the reason why some blood donation centers in our country, until recent years when there was no capability for nat testing of blood donors, had 100 iu/l as a limit for anti-hbs levels. however, in present days that nat testing of blood donors is used in our country, it has offered great safety and it is possible that anti-hbc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of 10 iu/l anti-hbs in all the blood units, which also goes for our study pd14/20 neonatal allo immune thrombocytopenia and igg glycosylation patterns michaelsen 1,2 1 national institute of public health in milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. the reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. recently influence of glycosylation patterns of igg on the biological activity has been realized. variation in carbohydrate structures attached to asparagine 297 can cause differences in the interaction with fc-receptors, and hence a difference in thrombocyte elimination capacity of the igg molecule. patient sera from norway and the netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate igg antibodies against the hpa-1a alloantigen and analyze the glycopeptides using mass spectrometry. the glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. so far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated n-glycans on their anti-hpa-1a iggs we monitored 12 children (9 boys and 3 girls) in ages from 3.2 to 17.2 years with average age of 8.9 years. in 11 of them all was diagnosed for the first time. 1 subject had the second relapse of all. one patient received maintenance chemotherapy, all the rest (11 subjects) induction chemotherapy. methods: leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. we valuated spontaneous and zymosan induced hemiluminescence. we used onsonised zymosan as the inductor of "respiratory paroxysm mice with a homozygous mutation in the rc3h1 gene, that encodes the zinc finger and ring finger containing protein roquin, develop severe autoimmune disease. the observed lupus-like phenotype involves follicular helper t cells, which express higher levels of icos. these cells provide inappropriate t cell help to b cells, leading to the production of autoantibodies (vinuesa et al. 2005, nature 435, 452-8). it has been shown that the half-life of icos mrna is shortened when roquin is over-expressed. such repression requires the 3'utr of icos, in which a 47bp sequence, containing a possible mir-101 binding site, was sufficient (yu et al. 2007, nature, 450, 299-303). mnab is the paralogue of roquin, and has been shown to bind to nucleic acids (siess et al. 2000, j biol chem 275, 33655-62). we demonstrate that in primary mouse t cells and embryonic fibroblasts roquin, but not mnab, inhibits translation of icos. we map critical domains in the roquin protein to icos repression using deletion-and point-mutants of roquin, as well as chimaeras that swap sequences from roquin to mnab and vice versa. addressing the mechanism of roquin mediated icos repression; we demonstrate binding of roquin to icos mrna in primary mouse t cells and in cotransfection experiments. our current work dissects the requirement of cellular rnai, the stress response pathway or p-body function by testing roquin repression of icos mrna in dicer-, tia-1-and ago2-deficient mef cells and in knockdown approaches. acknowledments: j m m-v is a recipient of a harvard real colegio complutense (rcch) grant. work in dr tsokos' lab is supported by grant phs nih r01 ai 42269. we have recently reported that 6-hydroxyl-1-methylindole-3-acetonitrile (6-hma) isolated from brassica rappa inhibit nuclear factor-kappa b (nf-xb) activity in raw 264.7 macrophages. in this report, we investigated the effect of 6-hma on dextran sulfate sodium (dss)-induced colitis model in mice. methods: we induced colitis with dss in mice and evaluated disease activity index (dai), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (mpo) accumulation. through h&e staining, histological change was observed. the expression of inducible nitric oxide synthase (inos), inhibitory kappa b-a (ixba) and nf-xb were detected by western blot and immunohistochemical staining. in-vitro system, the expressions of interleukin-8 (il-8), monocyte chemotactic protein-1 (mcp-1) in ht-29 human colon epithelial cells were measured by rt-pcr. results: in dss colitis model, the dai score and detection of mpo accumulation brevealed 6-hma significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. moreover, h&e staining also indicated 6-hma suppressed the thickness of muscle layer, edema, mucosal damages by dss. these results were related to the regulation of nf-xb activation. 6-hma attenuated the dss-induced phosphorylation and translocation of nf-xb subunit p65. in addition, this effect was accompanied with parallel blocking degradation of ixba. moreover, pretreatment of 6-hma significantly reduced the mrna levels of il-8 and mcp-1 stimulated by tumor necrosis factor-a (tnf-a) in the ht-29 cells. pretreatment of 6-hma also significantly blocked the ixba degradation and nf-xb p65 nuclear translocation stimulated by tnf-a in the ht-29 cells. these results were concurred with the effect on nf-xb activation in dssinduced colitis model. conclusions: these results for the first time demonstrated that alleviation of 6-hma mediated by regulation of nf-xb activation and suppression of chemokines in vitro and in vivo. therefore, 6-hma could be new potential therapeutic agent for inflammatory bowel disease.cd4 serves as receptor of hiv and is a self-antigen. we have previously characterized the anti-cd4 igg immune response in hiv-1-exposed, seronegative (esn) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp120-binding and that there is an epitope specificity distinct from that seen in hiv-infected patients (second cd4 domain preferred). to generate antibodies able to inhibit the infection of hiv virus trying to learn from what happen in nature in esn we used a particular immunization procedure. we immunized mice with autologous cells expressing gp120, reacted with the 2 external domains of soluble human cd4, in the absence of the target cells expressing the co-receptor ccr5. the latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the cd4-gp120 complex and trigger the membrane fusion between effector (gp120 expressing) cells and target (ccr5 expressing) cells. thus, in the absence of ccr5 we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during hiv-1 infections. a conventional protocol for the generation of monoclonal antibodies was used. db-81 (igg1, x), one of the anti-cd4 antibodies obtained, recognized preferentially cd4 complexed to gp120, as compared to cd4 alone, not competed for the gp120 binding site on cd4 and was specific for the second extracellular domain of cd4. g. röder 1 , l. geironson 2 , a. darabi 3 , m. harndahl 1 , c. schafer-nielsen 4 , k. skjödt 5 , s. buus 1 , k. paulsson 2 1 copenhagen university, institute of international health, immunology and microbiology, department of experimental immunology, copenhagen, denmark, 2 lund university, immunology bmc d14, lund, sweden, 3 lund university, rausing laboratory, division of neurosurgery, department of clinical sciences, lund, sweden, 4 schafer-n, copenhagen, denmark, 5 department of immunology & microbiology, university of southern denmark, odense, denmarkcytotoxic t-lymphocytes become activated by binding to mhc-i molecules presenting antigenic peptides. the loading of peptides onto mhc-i takes place in the er and involves different chaperones and enzymes. tapasin binds mhc-i molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported mhc-i molecules. how tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. here, we have generated the first 87 n-terminal amino acids of human tapasin, tpn , and shown that this fragment of tapasin facilitates peptide dependent folding of hla-a*0201. to further investigate the properties of tpn and tapasin, we generated multiple mouse monoclonal antibodies towards tpn and wildtype human tapasin. one clone, atpn 1-87 /80 , was found to be specific for natural human tapasin and stained cellular er localized tapasin. using peptide chip technology, the epitope of atpn /80 was demonstrated to be located on tapasin [40] [41] [42] [43] [44] , which recently was shown to be a surface-exposed loop of the tapasin structure. together, these results demonstrate that, the first 87 n-terminal amino acids of tapasin are able to facilitate peptide-binding to mhc-i, and as well, this fragment can be recombinantly expressed in e.coli and fold into a structure, which at least partially, resembles that of wild-type human tapasin. we speculate that this region of tapasin might support empty, open and receptive mhc-i peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this tapasin region might be essential for enabling peptide editing. a objectives: antigen processing and presentation through hla class i molecules is critical for an effective destruction of infected or transformed cells by cd8+ t lymphocytes. different intracellular routes governing the processing of endogenous and exogenous antigens have been described. we show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to ctls. methods: to produce genetic in-frame tat fusion proteins, dna sequence encoding for the amino acid region 301-498 of the influenza a virus nucleoprotein (np) was inserted into the expression vector ptat-ha. starting from tatnpflu recombinant protein we produce hybrid proteins, in which the hla-b*2705-restricted np-flu epitope (aa 383-391) was replaced by hla-b27 or hla-a2-restricted epitopes of ebv and hcv, respectively. cross-presentation was evaluated according to the standard 51 cr release assay and through the ifn-g production. results: using hla-b27 or hla-a2 restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the hla-b27 molecules follow a proteasome-independent pathway which is active in different cell types, whereas the hla-a2 molecules present the epitopes in a classical proteasome-dependent pathway performed by dcs. furthermore, different hla-a2 restricted epitopes can be inserted in tandem and presented to the specific ctls without interfering each other. the data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. these findings also enlighten hla-b27 as a remarkable hla-class i molecule that, differently from hla-a2, can present peptides through additional, unconventional antigen presenting routes. this could concur to an imbalance of the immunological properties of the hla-b27 molecules leading to a more effective response towards viral as well as self -antigens. objectives: although cytotoxic t cells (ctl) in human immunodeficiency virus (hiv-1)-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. ctl play a key role in limiting viral replication in infections caused by e. g. epstein-barr virus, cytomegalovirus, hepatitis c virus and hiv1. consistent patterns of immunodominant and subdominant ctl-responses have been found between individuals with the same hla-alleles in both acute and chronic infection. as the ctl-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. however, the factors determining these ctl-response hierarchies are largely unknown. while structural differences between peptide-hla class i complexes may be important for tcr-repertoire selection and clonal expansion, it is less obvious how they impact ctl-response hierarchy formation and timing. other factors may also contribute, e. g. epitope abundance at the cell surface. methods: antigen processing efficiency of ctl epitopes from the p17-gag and p24 region was determined in vitro. 25mer peptides were digested with i20s and c20 proteasomes and the fragments identified by mass spectrometry. for epitope precursor peptides generated by the proteasome, we then determined tap affinity, trimming by eraap and hla-binding affinities and analyzed patient responses by elispot. results: we show that ctl-immunodominance in regions of hiv-1 p17-and p24-gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (tap) affinity and endoplasmatic reticulum aminopeptidase (eraap)-mediated trimming, and moderately by hla affinity. proteasomal cleavage-preferences were affected by flanking and intra-epitope ctl-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting t cell response avidity and clonality. conclusion: our analyses reveal that antigen processing plays a pivotal role in determining ctl-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of ctl-epitope-based vaccines. t. f. gregers 1 , g. koster 1 , o. landsverk 1 , f. skjeldal 1 , o. bakke 1 1 university of oslo, molecular biosciences, oslo, norway mhc ii is synthesized and assembles in the er together with invariant chain (ii). ii facilitates mhc ii assembly followed by transport to the mhc ii loading compartment (miic) where peptide loading occurs. miic is multivesicular late endosomal compartments resembling conventional multivesicular bodies (mvbs) found in all cells. it is not known whether the biogenesis of miics is regulated by the same mechanisms as formation of mvbs. expression of ii induces the formation of enlarged endosomes and we have previously shown that ii modulates antigen processing and presentation. we have suggested that ii itself can act as a tethering factor involved in fusion of ii containing endosomes, and our main question is whether ii can regulate the formation of an endosomal pathway dedicated for antigen processing and mhc ii loading.in order to investigate this we use cell lines expressing ii controlled by an inducible promoter, thus being able to control the ii expression level and thereby the endosomal size. live imaging and high through put microscopy of ii expressing cells treated with inhibitors and/or specific sirnas have revealed that ii induced endosomal fusion is independent on type iii pi3 kinases and thus ptdins(3)p. this is in contrast to conventional endosomal fusion and mvb formation. thus other factors might be important for miic biogenesis. by using small rnai libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell. m. bouvier 1 , l. visvabharathy 1 , j. fu 1 1 university of illinois at chicago, microbiology and immunology, chicago, united statesobjectives: adenoviruses (ads) cause persistent infections. the e3-19k protein from ad targets class i mhc molecules for retention in the endoplasmic reticulum (er), thereby preventing the cell-surface presentation of viral peptides. this escape from immune surveillance allows ads to freely replicate in host cells. the molecular mechanism of e3-19k-mediated class i retention is mostly undefined. it is clear that further characterization of this mechanism is important to understand the susceptibility of the class i antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of ad pathogenicity. we used biophysical and cell-based approaches to examine interaction between ad type 2 e3-19k and class i molecules.results: we showed that e3-19k associates with immature (peptide-deficient) and mature (peptide-filled) hla-a11 molecules, with the mature form being more tightly associated. we also provided evidence that e3-19k does not compete with the class i assembly proteins for binding onto class i molecules. importantly, immature class i molecules sequestered by e3-19k can still bind peptides. together, these results suggest that ads have evolved to interfere with the early and late stages of the class i antigen presentation pathway. evidence was also provided that e3-19k displays an allele-and locus-specificity towards class i molecules with high-density lipoprotein (hdl) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. recent data indicate that qualitative changes of hdl including oxidative modifications and alterations of the protein cargo of hdl may alter its biological activity. here we analyzed the anti-inflammatory potential of hdl and compared it with hdl obtained from patients with end-stage renal disease (esrd), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. we demonstrate that freshly isolated, but not oxidized hdl from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (apc) such as monocytes and dendritic cells, which are regarded as the most potent apc. production of typical proinflammatory cytokines (il-12, il-6, tnf-a) were significantly suppressed by hdl after stimulation of monocytes or dendritic cells with toll-receptor ligands 2 and 4, but also with the t-celldependent stimulus cd40l (cd154) indicating an immunomodulatory effect independent of agonist neutralization by hdl. moreover, surface expression of crucial activation and costimulatory molecules like cd40, cd83, and cd86 was inhibited by freshly isolated, but not oxidized hdl. the negative regulatory effect of hdl on cytokines and surface receptors occurred at the transcription level, while hdl did not modulate the activity of the major inflammatory transcription factor nf-kb or the map kinases p38 and erk-1/2. strikingly, hdl from esrd patients not only failed to block, but rather promoted proinflammatory cytokine production and apc activation. these data identify hdl as a novel potent anti-inflammatory regulator of professional apc, which may help to dampen excessive inflammatory responses of the innate immune system. conversely, qualitative changes of hdl leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in esrd patients. objectives: cd4+ t cell abnormalities may play a role in the autoimmune pathogenesis of churg strauss syndrome (css). on one side, th2 (il-4+) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of css. on the other, th1 (ifn-g+) cells could participate in vessel wall damage and granuloma formation. in order to define this th1 / th2 balance and to identify potential t cell target antigens (ags), we analyzed circulating cd4+ t cell responses to polyclonal stimuli and to myeloperoxidase (mpo) in css and healthy subjects. methods: ifn-g and il-4 expression in peripheral blood cd3+cd4+ lymphocytes were measured in 9 ccs patients and 7 healthy subjects (hs) upon polyclonal stimulation, both by intracellular staining and by elisa. mpo-driven il-4/ifn-g production was assessed by elispot on t cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated mpo, negative control protein or hexavalent vaccine (positive control recall ags). results: upon polyclonal stimulation, higher il-4 and lower ifn-g intracellular expression were detected in cd4+ t cells from css patients, as compared to hs (il-4: 1.3±0.3 % vs. 0.50±0.21 %, p x 0.05; ifn-g: 14.2±4.5 % vs. 27.0±4.8 %, p x 0.025). similar results were obtained by elisa (il-4: 0.39±0.16 vs. 0.30±0.07 pg/ml, p x 0.05; ifn-g: 31.0±14.3 vs. 79.0±15.0 pg/ml, p x 0.05). elispot counts of hexavalent vaccine-stimulated cd4+ cells were positive for il-4 in 4/5 (80 %) css patients and in 5/5 (100 %) hs, and for ifn-g in 2/9 (22 %) css patients and 7/7 (100 %) hs. mpo stimulation determined significant ifn-g release in 5/8 (62 %) css patients, but not in hs (0/5) no il-4 response to mpo in both groups was observed. conclusion: polyclonally or recall ag-stimulated cd4+ cells from css patients show a th2-polarized cytokine profile. mpo is here first identified as a css-related ag targeted by cd4+ t cells, and responses towards it are instead th1-polarized. these data unfold one molecular target and possible pathogenic mechanisms of cd4+ t cells in css. a. voigt 1 , e. opitz 1 , k. savvatis 2 , k. klingel 3 , k. stangl 1 , u. kuckelkorn 1 , p.-m. kloetzel 1 1 charité -universitätsmedizin berlin campus mitte, berlin, germany, 2 charite -campus benjamin franklin, berlin, germany, 3 universität tübingen, tübingen, germanymurine models of coxsackievirus b3 (cvb3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. immunoproteasomes (ip) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. our previous work has established that ip expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.here, we investigated the impact of ip function in enterovirus myocarditis. mice, which are deficient in immunosubunit lmp2 of the stress-induced ip, were infected with 1x10e5 pfu cvb3 nancy strain. in concurrence to wt littermates, we observed a pronounced up-regulation of cardiac ip subunit lmp7 as early as day 4 p. i. in lmp2-deficient mice. however, lmp2-deficiency was linked to less severe myocarditis at day 8 p. i. (he stain of cardiac tissue sections: wt 1.95 ± 0.20 vs. lmp2-deficiency 0.71 ± 0.06 (grade of myocarditis; scale 0-4; p x 0.001). whereas the cardiac output (co) was reduced in wt littermates in enterovirusmyocarditis (p x 0.05), there was no difference in lmp2-deficient mice in comparison to sham-treated mice. maximal left ventricular pressure and dpdt max were impaired in acute myocarditis in wt littermates. in contrast, systolic function was not affected by cvb3 infection in lmp2-deficient mice. likewise, diastolic function was preserved in lmp2-deficient mice upon enterovirus infection. our findings of less severe myocarditis in lmp2-deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.in conclusion, this study suggests an impact of lmp2-immunosubunit function in regulatory processes of viral replication. absence of lmp2 confers host protection in enterovirus myocarditis. h. w. liao 1 , j. xu 1 , j.q. huang 1 1 sun yat-sen university, guangzhou, chinathe characteristic of the dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. the pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. in this work, we showed that dengue virus serotype 2 (den-2) strain induced apoptosis in human umbilical vein endothelial cells (huvecs). additionally, fas expression was increased on infected huvecs. trailr1 and tnfr1-2 were constantly very low whereas trailr2-4 decreased after den-2 infection. fasl was expressed at similar levels on huvecs throughout den-2 infection. the apoptotic rates in huvecs were decreased upon addition of caspase family inhibitors and activated caspase 8, caspase 3 were also observed by western blot after by den-2 infection. there were no significant changes of no in our study. we thus proposed that the fas/fasl pathway might be involved in apoptosis induced by dengue virus in vascular endothelial cells in vitro. dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. it seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. aim. to study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. materials & methods. group i. s. epidermidis was injected daily for 7 days into wis rat paw web tissue in saline containing 7.5x10 7 cells. group ii. s.epidermis was injected as in group 1 after ligation of lymphatics below the popliteal lymph node. nodes were isolated on day 8.they were weighed, the cell number was counted and cells were stained with mabs for immunohistochemical analysis. immunohistochemical pictures were analyzed by microimage program. results. group i. skin contained some mhcii cells. the popliteal lymph nodes became enlarged on the bacteria injected side. there was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors 2,23 and 3,91 respectively (p x 0.05). immunohistochemical pictures showed increase in percentage of ox62 (migrating dendritic cell), mhc ii, his48 (granulocytes), ox7 (stem cells) and cd54 (icam i) subsets in the subcapsular , follicle, paracortex and medullary areas. group ii. after ligation of afferent lymphatics the weight of nodes was not significantly increased. skin showed presence of multiple mhc ii, ed1 (macrophages) and ox62 cells. popliteal lymph nodes contained evidently less of ox62, his48 and mhcii cells than in group i (p x 0.05). summary & conclusions. afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. these observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. omega-3 fatty acids, and in particular docosapentaenoic acid (dha) and eicosapentaenoic acid (epa) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. our presentation focuses on the role of omega-3 fatty acids in the prevention and treatment of cardiovascular disease. it is based on reviewing and processing data obtained by search of scientific and medical databases. search terms used were: atheroma, atherosclerosis, cardiovascular disease (cad) ,coronary disease, antiinflammatory drugs, omega-3 fatty acids, epa, dha. we also searched epidemiological research web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega -3 fatty acids consumption. the results indicate that omega-3 fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. omega-3 fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. combined therapy with omega-3 fatty acids and statins shows a synergistic effect. methods: on ultracentrifugation of serum at density 1.24 and 202,000g, top 20 % layer contained lipoproteins only and 20-50 % layer contained lipoproteins as well as immunoglobulins. the bottom layer was shown to contain immune complexes (ic) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.both these forms of lp(a) were western blotted and probed with jacalin-hrp, anti-gal-hrp and anti apo(a)-hrp. anti-gal was prepared by affinity chromatography on guar galactomannan and complexed with lp(a) in vitro. ic formation by lp(a) was measured in terms of reduction in response in a new elisa for lp(a) involving addition of lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-hrp detection. ic formation was also shown by migration of lp(a) from free lipid layer to ic layer below in ultracentrifugation. results: anti-gal and lp(a) could be liberated from precipitated ic using specific sugar. immune complexed lp(a) in serum was found to be more o-glycosylated, larger in size and binding more anti-gal than lp(a) in free form in western blots. while ic formations within homologous free anti-gal-free lp(a) pairs were few, those within heterologous pairs were more rampant. conclusions: lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. anti-gal comprising 1 % of serum igg is rich in igg capable of complement fixation and macrophage mobilisation. present results offer a viable mechanism of lp(a)-mediated immune injury to vessel walls leading to vascular damage. even though no receptors have been detected for lp(a), unlike for ldl, the present results may explain the internalization of lp(a) in the form of lp(a) immune complexes by macrophages since the latter can phagocytose ic. extended specificity of the a-galactoside-specific anti-gal for t-antigen in lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin-1. objective: the aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the e2f2 mutation, and other affecting b cell apoptosis control, such as bcl-2 over-expression, can induce the development of ais. methods: mice: mice with both genetic abnormalities were generated in a non-susceptible c57bl/6 (b6) genetic background. e2f2-/-bcl-2tg were obtained backcrossing e2f2-/-and e2f2+/+hbcl-2tg mice. e2f2-/-bcl-2tg, e2f2-/-, e2f2+/+hbcl-2tg and control mice (e2f2+/+) were followed up to 18mo-old. serologic studies: serum samples obtained at 3, 9 and 15 month of age were test for igg and iga ana and anti-dsdna by elisa. histopathologic studies: kidney paraffin sections of 3, 9 and 15 mo-old mice were stained with hematoxylin-eosin (h&e) and masson's trichrome to identify histological changes. immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse igg, igm and iga. to evaluate b cell homeostasis, absolute number of b cell in blood, primary and secondary lymph organs were assessed by flow cytometry. in vitro proliferation was measured with [h3]-thymidine and brdu was used to assess in vivo proliferation capacity of immature b cells. results: overexpression of hbcl-2tg in b lymphocytes of e2f2-/-mice induced the production of high titres of igg and iga ana and anti-dsdna, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the iga isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. e2f2-/-bcl-2tg mice showed an altered b cell homeostasis as demonstrated in proliferation and apoptosis studies. e2f2-/-mice showed neither autoantibodies nor nephropathy. this study demonstrates that the isolated deficiency of e2f2 or the overexpression of a bcl-2 tg in the b6 genetic background do not induce an ais. when combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild ais with overproduction of iga autoantibodies. an alteration in the b cell compartment, but not in the t cell compartment, seems to be underlying the syndrome described in the present work. in different mouse models of the autoimmune disease systemic lupus erythematosus (sle) loss of toll-like receptor 9 (tlr9) abolishes the generation of antinucleosome igg2a and igg2b autoantibodies but exacerbates lupus disease. however, the tlr9-dependent tolerance mechanism is unknown. here we show that loss of tlr9 in b cells of lupus prone mice prevents the generation of protective t cell-dependent self-reactive igm and thereby enhances the development of th1 and th17 t cells. transfer of a synthesized monoclonal polyreactive igm to tlr9 deficient lupus prone mice inhibits t cell activation and abolishes development of lupus disease. thus, these results document a protective tlr9-dependent tolerance mechanism in b cells that induces the generation of self-reactive igm to prevent autoimmunity. cloning and production of polyreactive or antigen-specific igm might therefore be a powerful tool to treat autoimmunity. objectives: to investigate cytokine and autoantibody levels in serum from patients with primary sjögren's syndrome (pss), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. methods: minor salivary gland tissue was obtained from a group of patients fulfilling the revised eu-us criteria for pss (n=115) (vitali et al. 2002) . ninety-seven of 115 (84 %) patients had focal mononuclear cell infiltrates corresponding to focus score (fs) g 1 (fs+), while biopsies from 18/115 (16 %) patients lacked characteristic focal mononuclear cell infiltrates (fs-). germinal center (gc)-like lesions were determined in 27/115 (23 %) minor salivary gland biopsies. serum samples were used for cytokine and autoantibody evaluations. the mean level of unstimulated whole saliva was significantly lower in the fs+ patients compared with the fs-patients, and in the gc+ patients compared with the gc-patients (p x 0.05). interleukin (il) 17, il-1ra, il-1beta, il-12p40, il-15, macrophage inflammatory protein (mip) 1alpha, mip-1beta, eotaxin, interferon (ifn) alpha, and il-4 levels were significantly increased in the gc+ patients (n=27) compared with the gc-patients (n=70). in addition, minor differences in cytokine levels were found when comparing age groups. degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pss may represent "burned out" inflammation. no significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the fs-and fs+ subgroups. the reduced salivary flow observed in gc+ patients may be influenced by the elevated levels of il-4 found in these patients (gao et al. 2006) . increased titers of th17-associated cytokines, il-17, il-1beta and the il-23 subunit il-12p40, may indicate a higher activity of these cells in gc+ patients (nguyen et al. 2008) . differences in cytokine levels may be utilized when sub-grouping the ss patients into disease phases and may consequently have implications for treatment. objectives: c-reactive protein (crp) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine il-6. the rapid increase of crp during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of crp typically remain low in disease flares of systemic lupus erythematosus (sle), a systemic autoimmune disease. another feature of sle is the so called 'interferon (ifn) signature' which implies high levels of ifn-alpha and/or up-regulation of ifn-alpha related genes. ifn-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. since high levels of ifn-alpha coexist with a muted crp response in sle disease flares and in viral infections, we hypothesized that ifnalpha inhibits crp synthesis. methods: crp promoter activity was studied in a crp-promoter and luciferase reporter transfected human hepatoma cell-line, hepg2. production of the acute phase protein serum amyloid a (saa) and the negative acute phase protein transferrin were analysed by elisa as reference. results: the crp-promoter activity was inhibited by all ifn-alpha subtypes. mixes of type i ifns that were induced by sle-like immune complexes or virus also inhibited the crp-promoter activity. virus-induced purified leukocyte ifn-alpha had the most prominent inhibitory effect ( g 50 %) on crp promoter activity. saa synthesis was inhibited by ifn-alpha in a similar fashion as for crp promoter activity, whereas transferrin was unaffected. conclusion: our data indicates that ifn-alpha is an inhibitor of crp-promoter activity. we suggest that this could explain the muted crp response seen in sle disease exacerbations. further, i may contribute to differences in crp response between viral and bacterial infections. background: b cell activating factor of the tnf family (baff) is an essential b cell survival and maturation cytokine. mice overexpressing baff (baff tg mice) develop lupuslike autoimmunity, b cell hyperplasia, and lymphomas. autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be t cell-independent (ti) and myd88-dependent. this suggested the involvement of transmembrane activator and caml interactor (taci), which is a receptor for baff that is essential for ti immune responses and is upregulated by myd88-dependent tlr activation. we assessed the role of taci in baff-driven ti autoimmunity. methods: we tested the importance of taci in ti autoimmunity by generating baff tg bone marrow (bm) chimeras reconstituted with taci -/or taci +/+ bm then comparing their disease severity by flow cytometry, autoantibody elisa, immunofluorescence microscopy for ig deposition. results: as expected, baff tg chimeras reconstituted with taci +/+ bm produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (rhf), and ig deposition in the kidneys and salivary glands was observed. by contrast, baff tg chimeras reconstituted with taci -/-bm had greatly ameliorated levels of circulating proinflammatory autoantibodies, rhf, and ig deposition. b cell hyperplasia was greater in taci -/-1 baff tg chimeras. defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (sle). autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize t-cells after phagocytosis of apoptotic material. these processes of silent apoptotic cell clearance seem to be disturbed in sle patients. a characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). these microparticles have been recognized as mediators of intercellular communication. therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human sle. we observed an engulfment of microparticles by monocyte-derived dendritic cells. further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. however, microparticles caused a remarkable downregulation of mhc class ii molecules. further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to lps stimulated dendritic cells. finally, these dendritic cells pulsed by membrane microparticles did not cause a significant t-cell expansion. interestingly, dendritic cells obtained from sle patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the mhc class ii downregulation and a higher constitutive secretion of il-8. objectives: increased levels of il-18, an innate and inflammatory cytokine of the il-1 family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. here, expression of il-18 and other genes of the il-1/il-1r families was examined in human systemic lupus erythematosus (sle) and in mrl lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. methods: serum, urine, and monocytes were collected from 48 patients and 32 healthy controls. lymphoid (lymph-nodes, spleen, thymus, peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from mrl +/+ and lpr/lpr mice of different ages. il-18 and il-18bp were measured by elisa. gene expression was assessed by real-time pcr and expressed relative to b-actin. results: in sle, serum and urine levels of total and free il-18 are higher than in controls. serum il-18 correlates with disease activity and decreases upon remission. monocyte expression of the receptor il-18rb is increased and correlates with disease severity, while expression of tir8/sigirr (a down-regulatory receptor of the il-1r/il-18r family) is reduced. in mrl lpr/lpr mice, expression of il-18, caspase-1 and il-18rb genes precedes disease onset in lymph-nodes. in other organs, changes in il-1-related genes (il-33 and tigirr-1 up-regulation, tir8/sigirr down-regulation) occur after disease onset. free il-18 levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. conclusions: free il-18 levels correlate with autoimmune lupus both in mice and humans. free il-18 may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in sle. both in human and mouse syndromes, upregulation of il-18rb is a marker of pathology, suggesting increased il-18-dependent activation. both in mouse organs and human monocytes, tir8/sigirr expression decreases with disease, suggesting impaired control of il-18r activation. thus, il-18 may be involved in autoimmune lupus pathology, and il-18-related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. in this study we compared the epitope specificity of anti-topo i autoantibodies present in sera of dcssc, lcssc and sle patients. we have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with igg purified from patients' sera. regions of topo i selected from the library were expressed as recombinant fusion proteins and were tested with elisa and western blot. we unexpectedly found that antibodies against a fragment of topo i (fragment f4 (amino acid (aa) 451-593) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than ssc and sle. using sera of dcssc, lcssc and sle patients we showed that the pattern of recognized epitopes is different between these patient groups. fragment f4 was recognized by all patients. fragment f1 (aa 5-30) was recognized by 9 of 34 dcssc patients. fragment f8 (aa 350-400) was recognized by 4 of 8 sle patients. analysis of clinical data revealed a significant difference between the f1 negative and f1 positive groups of ssc patients in age and in the duration of the disease. according to our results the newly identified fragments f1 and f8 could represent characteristic epitopes for dcssc and sle, respectively. background: previous studies demonstrated that depletion of regulatory t cells (tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. studies performed by us and others showed that in human connective-tissue diseases a reduced number of tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. objectives: based on these observations and the fact that rapamycin (rapa) has the ability to expand tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of cd4 + t cells isolated from systemic lupus erythemaosus (sle) patients by ex vivo expansion of tregs. methods: pbmcs or peripheral cd4 + t cells from sle patients were cultured in the presence of specific stimulation with or without rapa and ril-2. by facs the initial percent of tregs and after expansion protocol were determined. in order to verify the suppressive capacity of expanded tregs, cd4 + t cells enriched in tregs were co-cultured with activated cd4 + effector t cells (teff) stained with cfse, after one week teff cells proliferation was measured by facs. additionally, cytokine and igg release in cell culture media were analyzed by multiplex and elisa, respectively. expanded cd4 + t cells anergy was also evaluated based on cbl-b, grail and foxp3 mrna by realtime rt-pcr. results: in vitro expansion of tregs was more efficient when the starting cells were cd4 + t cells. the presence of rapa during expansion protocol significantly increased the number of tregs. sle tregs cells expanded in vitro in the presence of rapa had the capacity to suppress proliferation of both sle and hd teff cells. rapa inhibits igg secretion in the pbmcs culture, inhibition dependent on tregs level. rapa during tregs expansion protocol stimulated some type of cytokines while suppressed others. rapa had the capacity to re-establish sle cd4 + t cells anergy by induction of anergy genes, grail and cbl-b. conclusions: our data show that the above described protocol permits ex vivo tregs expansion and that suppressive capacity of the expanded tregs depends on the source of both tregs and teff cells. in this study, we look for a more specific approach to remove b-1 cells through targeting p110d by shrnas strategy. methods: we used the drugs, ly294002 and wortmannin, pan-specific inhibitors against pi3ks. then we designed shrnas carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p110d. we then introduced either pan-specific inhibitors against all pi3ks or p110d-targeting shrnas into an sle-prone animal model, nzb/w f1 mice, for therapeutic purposes. the results suggested that pi3ks are not only important for the development of b-1 cells but also remain essential to maintain their population after birth. shrnas carried on lentiviral systems were designed to knock down the expression of p110d. either pan-specific inhibitors against pi3ks or p110d-targeting shrnas were introduced into the sle-prone animal model, nzb/w f1 mice. one inhibitor, ly294002, and shrnas delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-dna auto-antibodies and prolonged the life span. conclusions: our findings are promising for developing treatments for sle. moreover, knowing pi3ks are critical for the maintenance of b-1 cell populations might shed light on future treating other diseases associated with b-1 cells, such as certain melanoma, lymphoma, or leukemia. a. m. zaghlool 1 , m. alarcón-riquelme 1 , s. kozyrev 1 1 institution of genetic and pathology, uppsala university, uppsala, swedenrecently, we discovered that the bank1 gene, which plays a role in b cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution g/a (rs10516487, r61h). we identified that bank 1 gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon 2 (delta 2). the two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. to address the question of how bank1 is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the bank1 isoforms were tested in vitro. qpcr analysis revealed that, another t/c snp (rs17266594), which is in complete ld with r61h snp and located in the putative branch point, has a strong affect on the isoforms expression levels. deletion of a polypyrimidine (py) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein tia1, which bind to t objectives: cerebral ischemia is the most common presentation of antiphospholipid syndrome (aps), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (apl). we report the case of a subject with atypical movement disorder related to aps successfully treated with oral anticoagulation agents. case report: a 57-year-old woman with a previous history of recurrent foetal losses was admitted to our hospital due to cognitive dysfunction and headache. she presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. mild executive dysfunction was observed. her deep tendon reflexes were symmetric and normal. pathological reflexes were absent. biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. the immunoglobulin g (igg) isotope of anticardiolipin antibody (acl) was elevated, whereas igm isotype and anti-2gpi antibodies were undetectable. the lupus anticoagulant (la) was negative such as antinuclear antibodies (ana). no evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. brain magnetic resonance imaging (mri) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. she was diagnosed as having an atypical neurologic manifestation probably linked to aps. she was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . the therapy leads to a late, gradual improvement of symptoms that persisted at the last 1year follow-up evaluation. conclusions: antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of aps (foetal losses, livedo reticularis, thrombosis). a.-s. korganow 1 1 cnrs 9021, strasbourg, france b lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. several b cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. a few genes have been suggested to be implicated. one of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.we made the postulat that "intrinsic" abnormalities of b cells could be a common point in very quiescent patients. we choosed 18 patients, with minor clinical and/or biological manifestations of their disease, for at least 6 monthes. known of them received immunosuppressive drugs since this period. the mean sledai score was below 2. b cell surface markers expression was determined by flow cytometry. we analysed most of the already described and phenotypically distinctive b cell populations. we confirm the presence of activated b cells even in quiescent patients. we do not confirm the significant increase of a specific memory b cell compartment. above all, we described a decreased expression of the cd19 surface protein for all patients. this cd19 lower expression is associated with cd45 lower levels. it is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. these findings suggest some mechanisms in lupus genesis. objectives: tgf-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. recent work from our group has shown that tgf-beta signalling in t-cells is protective in a mouse model of colitis associated cancer. smad ubiquitin regulating factors (smurf) are ubiquitin ligases that are involved in the regulation of tgf-beta signalling. the aim of this study was to determine the function of smurf2 expression in t-cells on the pathogenesis of experimental colitis associated colon cancer. methods: we could isolate a known splice variant of smurf2 lacking an exon in the c2-domain. to analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses smurf2 in t-cells. smurf2 expression were analysed by qpcr. wild type (wt) and transgenic (tg) mice were treated once with the mutagenic agent azoxymethan (aom) followed by three cycles dextran sodiumsulfate (dss). after each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. results: smurf2 expression was upregulated by tgf-beta stimulation in t-cells and smurf2 was markedly upregulated in tumor infiltrating cd4+ lymphocytes in aom/dss treated mice. whereas wt mice suffered from severe colitis resulting in colon tumors beginning at day 42, smurf2 transgenic mice had less colitis and were significantly protected from tumor development. interestingly, t-lymphocytes overexpressing smurf2 showed an upregulation of the tgfbrii and an activation of smad2 and 3 as compared to wild-type t-lymphocytes, which were previously described as typical smurf2 targets for degradation. in addition the transfection of smurf2 and a caga-luc plasmid into cos-cells for smad3-promotor studies yielded the same effect as shown by upregulation of the smad3 activity. conclusion: although, wt-smurf has been described as a negative regulator of the tgf-beta signalling pathway, our data show surprisingly that a smurf2 splice variant upregulates the tgf-beta receptor expression and increases tgf-beta signalling effects. due to immunosuppressive effects on t-cells smurf2 has beneficial effects on mucosal inflammation and tumor development. smurf2 thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. the transcription factor stat3 has important functions in cytokine signalling in a variety of tissues. however, the role of stat3 in the intestinal epithelium is not well understood. we demonstrate that development of colonic inflammation is associated with the induction of stat3 activity in intestinal epithelial cells (iec) both in humans and in mice. studies in genetically engineered mice showed that epithelial stat3 activation in dss colitis is dependent on il-22 rather than il-6. il-22 was secreted by colonic cd11c+ cells in response to toll-like receptor stimulation. conditional knockout mice with an iec specific deletion of stat3 activity were highly susceptible to experimental colitis, indicating that epithelial stat3 regulates gut homeostasis. stat3 iec-ko mice, upon induction of colitis, showed a striking defect of epithelial restitution. gene chip analysis indicated that stat3 regulates the cellular stress response, apoptosis and pathways associated with wound healing in iec. consistently, il-22 and epithelial stat3 was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. in summary, our data suggest that intestinal epithelial stat3 activation regulates immune homeostasis in the gut by promoting il-22-dependent mucosal wound healing. stat3 seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury. map and ma infection induced an increase in both cd40 and tlr4 expression at day 3 and day 7 after infection. mycobacterial infection did not result in differential tlr2 expression as compared to uninfected cells. cd40 is involved in stimulating th1 pro-inflammatory responses, although map may interfere with cd40 signalling (1) . tlr4 signalling elicits anti-inflammatory responses, which can contribute to bacterial replication (2) .in conclusion, monocyte-derived macrophages from crohn's disease patients show an increase in cd40 and tlr4 receptor expression in response to both map and ma infection. as ma is a known human pathogen of immunocompromised hosts, this findings further support a role for map in the immunopathology of crohn's disease. objectives: for our understanding of the pathogenesis of human ibd, animal models of intestinal inflammation are indispensable. most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of ibd. the occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. to further dissect the pathophysiological mechanisms responsible for initiation and progression of ibd and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of cd8 t cells by exogenous antigen leads to colitis. methods: eight million naïve cd8 + ot-i cells, transgenic for a t-cell receptor specific for an ova-derived peptide (siinfekl) in the context of h2-kb, were transferred i. v. into b6 mice. at day 0 and 4, mice were treated intra-rectally (i. r.) with 50 % ethanol. thirty minutes later, ovalbumin (ova) or bovine serum albumine (bsa) were applicated i. r. proliferation of cfse-labelled cells was measured at day 2 after the injection of ot-i cells. the phenotype of effector cells was evaluated at day 5 by measuring ifng production and by in vivo cytotoxicity assay. based on histology and immunhistochemistry for cd8, the severity of colitis was scored. results: local application of the exogenous antigen ova but not of bsa led to antigen-specific activation and proliferation of adoptively transferred naïve ot-i cd8 + t cells. these cells differentiated into fully activated effector t cells with the capacity to secrete ifng upon re-stimulation ex vivo and possessed in vivo cytotoxicity to siinfekl-loaded target spleen cells. furthermore ova treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of cd8 + t cells. our study demonstrates that the local activation of antigen-specific cd8 t cells by exogenous antigen in the colon leads to fully activated effector t cells with the capability to promote local intestinal inflammation in non-immune-compromised b6 mice. aims: to determine the immune system response of the greek population against helicobacter pylori (hp), given the fact that hp infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc). the results of express qualitative detection method for igg and iga antibodies were studied of 535 patients, (248 male and 287 female), with age average 69,7 years of age. patients who received antibiotics and excretory medicine in the last year were excluded. anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using spss, x 2 and t-test programmes. results: in 372 patients (69,5 %,with age average 62,7 years of age) no antibodies were detected. on the contrary, in the remaining 163, 52 male and 111 female, (30,5 %, with age average 76,4 years of age), antibodies were detected. out of them, in 106 cases the results were strong positive (32 male and 74 female) and weak positive in 57 cases (20 male and 37 female). the statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, mcv and rdw). conclusions: it is proven, therefore, that: 1) helicobacter pylori infection is relatively common in the general population (30,5%).2) there is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women).3) there seems to be no correlation with anemia. it is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of helicobacter pylori infection in our country. abstract withdrawn by author m. durilova 1 , t. ulmannova 1 , k. stechova 1 , k. tesarova-flajsmanova 1 , v. stavikova 1 , j. nevoral 1 1 charles university, pediatrics, prague, czech republicobjective: was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. methods: breast milk of 20 mothers whose infants were diagnosed with allergic colitis and 20 mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. breast milk samples were collected at the time of diagnosis of allergic colitis (2-27 weeks, average 16.8 weeks of infant's age) or at the age of 12 weeks in control group. concentrations of the following cytokines were analyzed using elisa method: il-4, il-6, il-10, il-17, il-23, ifn-gamma, tgf-beta1, egf and eotaxin. man-whitney u test was used for statistical analysis, p x 0.05 was considered statistically significant.results: il-10 as the only cytokine was not detected in any of the tested samples in both groups. significant difference was seen in concentration of ifn-gamma, which was higher (p x 0.001) in breast milk of mothers whose infants were suffering from allergic colitis (range 0-8.45 pg/ml, mean 2.1 pg/ml) than in control group (range 0-3.41 pg/ml, mean 0.35 pg/ml). higher concentrations of il-4 and lower concentration of tgf-beta1 were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. conclusion: immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. the results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. supported by national project no. 8310-5. background: ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. the chemokine il-8 (murine homologs kc and mip-2) and its receptor cxcr2 are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. this study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. methods: peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated 12 h post intraperitoneal injection of thioglycollate and phenotypically (facs analysis) and functionally characterized in an in vitro chemotaxis assay. four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (dss) colitis followed by bioluminescence imaging of whole body and ex vivo organs at 2, 4, 16 and 22 h post-transfer. anti-kc antibody or its isotype control was administered at 20mg/mouse one hour before transfer followed by whole body and organ imaging 4 hours post-transfer. results: facs analysis revealed 80 % neutrophil purity, 35 % of which were cxcr2 + . in vitro, the cells migrated towards kc and this was inhibited by anti-kc. in the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of dss recipients at all time points. neutrophil recruitment to the colon was detected only in dss recipients and was inhibited by anti-kc, 4 h post cell transfer. this study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. the human gut contains more than 10 15 bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. the barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (s)iga and sigm. sigs are generated via epithelial polymeric ig receptor (pigr)-mediated transfer of iga and igm from the lamina propria to the intestinal lumen. to assess the role of sigs in colitis development, we constructed pigr knockout (ko) mice and tested them in the dextran sodium sulfate (dss) colitis model (1.5 % dss in drinking water for 1 week, followed by pure drinking water for 1 week). pigr ko mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of sigs is to prevent pathology induced by commensal microbiota. in contrast, 2 % dss was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. as previously documented by medzhitov and co-workers (rakoff-nahoum et al, cell 2004), treatment of commensal-depleted mice with the tlr4 ligand lps in drinking water protected against the lethality of 2 % dss. thus, the commensal microbiota serve two distinct roles in the dss colitis model. at dss concentration of 1.5 % they may become pathogenic and drive an intestinal inflammation. at 2 % dss commensals protect against the toxic effect of the chemical via their tlr ligands. in mice lacking sigs, due to deleted pigr, the severity of colitis induced by 1.5 % dss was greatly enhanced suggesting that one role of sigs is to prevent commensal microbiota from becoming pathogenic. ulcerative colitis (uc) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. although uc is associated with a type 2 immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. in a mouse model resembling uc, oxazolone induces il-13 production which is an important pathological factor. neutralizing il-4 or il-13 prevents or ameliorates disease significantly. as many aspects of the mechanisms involving these th2 cytokines in colitis remain undefined, we used mice deficient in il-4/il-13 or the key receptor through which they signal, il-4ra, to further dissect their role in oxazolone-induced colitis. disease was exacerbated in il-4ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. this was in contrast to il-4/il-13 double deficient mice which were protected from colitis. removing il-13 production from il-4ra -/mice, by using il-4ra/il-13 double deficient mice, reversed the susceptible phenotype to protection. together these data strongly suggest that il-13 mediates susceptibility in an il-4ra independent manor. recent evidence pc17/33 introduction: the activation of cd4 + t-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (ibd). whereas cd is associated with increased production of th1-like cytokines, the cytokines profile in chronic uc is characterized by the increased production of several th1 cytokines, such as il-5,-6 and il-13. however, the functional role of t cell transcription factors such as nuclear factor of activated t cells (nfat) in ibd is poorly understood. the aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in uc. cryosesctions of uc and cd patients were analysed by immunohistochemically methods. a significantly higher expression of nfatc2 was found in uc and cd colonic tissue compared to control specimen. transmitted to the th2-mediated oxazolone-induced colitis model, nfatc2-production is significantly increased in both diseases, too. nfatc2 deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in nfatc2 deficient mice compared to control mice, which can be observed by tunel assays, caspase3 and annexin v staining, as well as in lamina propria t cells. contrary, anti-apoptotic proteins, like bcl-2 and bcl-xl were downregulated for induction of apoptosis. this observation was associated with a reduced production of il-6, ifn-gamma, il-13 and il-17 by mucosal t lymphocytes, tested by elisa assays. further studies with the oxazoloneinduced colitis model showed that nfatc2 regulates il-23/il-17 in an indirect way. last, administration of il-6 blocked the protective effects of the nfatc2 deficiency in experimental colitis, suggesting that nfatc through il-6 signal transduction plays a direct pathogenic role in vivo. conclusion: our data define a unique regulatory role of nfatc2 in colitis by controlling mucosal t cell activation in an il-6 dependent manner. the examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. the pivotal role of micrornas in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (ibd) as well. the study of the expression of micrornas in ibd will unravel their role in this disease. in addition, micrornas by their mechanism of action, are promising new therapeutic agents or targets. a possible therapeutic application of micrornas is the introduction of novel, artificial micrornas or microrna mimics to regulate specific genes. because ibd is a heterogeneous disease in human we decided to define microrna expression in a well defined model of experimental colitis. as a result of this study we found a number of micrornas involved in different phases of experimental colitis. to study the role of mirnas in experimental colitis in mice we have used a well defined colitis model that resembles human ibd. this colitis is mediated by cd4cd45rb high t cells that are injected i. p. in scid mice. in control mice in addition to the cd4cd45rb high t cells also regulatory cd4cd45rb low t cells are transferred and no colitis develops. to study mirna expression we collected colonic tissue from the mice at 3 different time points during colitis progression. after 3 weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at 9 weeks. microrna was isolated from colons of mice in different stages of colitis progression (3, 6 and 9 weeks) and control mice that do not induce colitis (n=3 for each timepoint). from all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.the rna isolation as well as the microarray analysis has been outsourced to miltenyi biotec gmbh, bergisch galdbach, germany. we used the mirxplore tm microarrays for microrna expression profiling. from 11 micrornas that demonstrated an induction during the development of disease we selected 4 micrornas for in situ hybridization and for a proof of principle of the efficacy in the cd4cd45rb high transfer model. objective: the purpose of this clinical trial (id: nct00287677 of www.clinicaltrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (gh). methods: successfully highly active antiretroviral therapy (haart) treated hiv infected patients that failed to elicit a humoral response to tetanus toxoid (tt), or to hepatitis a (hva) or to hepatitis b (hvb) virus have been selected for the trial. growth hormone was given for 6 months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. patients have been randomized in 3 groups: group a (n=8) receiving haart+ gh (for 6 months) + tt+hva/b vaccines (at month 6 post gh adminsitration); group b (n= 6) receiving haart+gh but not vaccines; and group c (hiv control group, n=7) with haart+vaccines (at month 6) but without gh. all patients are followed up 6 months further. results: preliminary results show that an increase in thymic size was observed in gh recipients and not in controls. furthernore after 24 weeks of administaring hormone the absolute numbers of cd4 incresase from 562 ± 93 to 704 ± 112 cells per mm 3 (mean and sem; p x 0.0025). in contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the cd4 absolute numbers from 550±97 to 470±103 cells per mm 3 (p x 0.02). viral load remained undetectable in all patients. despite the increase in cd4 counts the percentage of recent thymic emigrants (as assessed by the expression of cd31) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. finally, specific responses to hepatitis a virus seem to be restored in a major proportion of patients treated with gh (group a) than in the other groups. conclusions: although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. a major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. in the course of the eradication of small-pox, freeze-dried vaccinia virus which proved to be extremely stable was used to overcome this limitation (dryvax ® ). before usage, dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. an alarming report by the who has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. each year, an overwhelming number of infections with hiv (80,000-160,000), hepatitis c virus (hcv; 2.3-4.7 million) and hepatitis b virus (hbv; 8-16 million) are thought to originate from the reuse of needles and syringes by health-care providers. in this report, we took advantage of the stability of freeze-dried vaccinia virus mva and directly applied it into the nostrils of mice without prior reconstitution. this direct mucosal application induced systemic antibody and t cell responses comparable to those achieved by intramuscular administration. importantly, mucosal application of lyophilized mva conferred protective immunity against a lethal vaccinia virus challenge. additionally, recombinant mva expressing the model antigen ovalbumin induced long-term and protective immunity against listeria monocytogenes-ova challenge. the data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized mva. methods and results: seventeen haart-treated asymptomatic hiv-1 infected patients with g 350 cd4 + t-cell counts and plasma hiv-rna levels of x 1.7 log 10 copies/ml were treated with a dc-based vaccine. the vaccine consisted of autologous mature dc electroporated seperately with either sig-tat-dc-lamp, sig-rev-dc-lamp or sig-nef-dc-lamp mrna and were each administered at a distinct anatomical site. after four monthly vaccinations haart treatment was interrupted. pbmc from 4 timepoints, before during and after vaccination, were analysed for ifn-g production (elispot), proliferation (cfse assay) and lytic capacity (fatt-ctl) in response to the antigens used in the vaccine. pbmc were screened upon ex vivo stimulation with pools of overlapping tat, rev, nef and gag peptides and ifn-g secretion was analysed using elispot ('peptide elispot'). elispot was also performed on re-stimulated t-cells with electroporated dc ('dc elispot') in vitro. responses were considered positive when the number of spot forming units per million t-cells was g 50 and when the responses were g 2 times the standard deviation above the mean of replicate negative controls (mock electroporated dc). 16/17 patients were screened with both peptide and dc elispot. an increase of responses to the vaccine-antigens after vaccination was found in both assays. based on the dc elispot data, we observed immune reactivity against tat in 4/16 patients before and 11/16 patients after vaccination. for rev, 7/16 patients showed a pre-existing rev specific response and 14/16 patients responded to rev after vaccination. nef was the most immunogenic antigen used in this vaccine with already 11/16 patients responding before and 16/16 patients responding after vaccination. for our control antigen gag, we observed an anti-gag response in 13 out of 16 patients before vaccination and 16/16 patients after vaccination. the results of the other assays correspond to the dc elispot results be it less pronounced. conclusion: therapeutic vaccination of hiv-infected patients with dc electroporated with tat, rev and nef induces and/or enhances antigen-specific t-cell responses, especially when monitored with the dc elispot. background: enterovirus 71 (ev71) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. no effective vaccine for ev71 is available yet. polysaccharide purified from ganoderma lucidum (ps-g) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of ps-g as the possible adjuvant with ev71 inactivated virus. methods: mice were immunized intraperitoneally with 10 mg inactivated virus /mouse with one of the following samples: pbs, cfa/ifa, and ps-g. each mouse received the same dose of boosters after 0, 2, and 4 weeks. blood samples were collected at 0, 21, 35, and 45 days. the total serum anti-ev71 igg level was determine by elisa, and the neutralization assay was also done. to evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. cytokines assay regarding ifn-g and il-5 from splenocytes was also measured. results: immunization with ev71/ps-g showed that the anti-ev71 igg levels were significantly increased compared with ev71 alone or ev71/cfa/ifa in balb/c mice. neutralization assays demonstrated an effective protection of ev71/ps-g group compared to ev71 alone. the splenocyte proliferation test showed that production of ifn-g significantly increased in ev71/ps-g-immunized mice compared to those of ev71 or ev71/cfa/ifa-immunized mice, indicating a th1 cells response elicited by heat-inactivated ev71 vaccine with ps-g adjuvant. conclusions: vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. the results here suggested that ps-g can be used as a novel, safe and natural vaccine adjuvant. objectives: the search for a vaccine against hepatitis c virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees. here we describe the differential modulation of cd8+ t-cell responses induced by a dna prime mva boost vaccine regimen in four individual chimpanzees and their association with viral clearance. methods: an ex vivo expansion protocol was used to map peptide specific cd8+ t-cell responses against hcv-ns3, studying induction of il-2, ifng and tnfa cytokine production as well as killing capacity. to assess the killing capacity and mhc restriction of the peptides, a non-radioactive killing assay was designed. peptides that induced both il-2 and ifng production by cd8+ t-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee mhc class i molecules. introduction: high levels of hiv-1-recognizing cd8 + cytotoxic t lymphocytes (ctl) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of hiv-1 replication, and for the prolonged survival of infected individuals. a potential immunotherapeutic strategy would be the adoptive transfer of t cells, which are reprogrammed by introduction of an hiv-specific t cell receptor (tcr). up to now, such ctl were generated by retroviral transfer of tcr-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). methods: therefore, we investigated the transfer of tcr-rna into cd8 + t cells by electroporation, and chose tcrs which were able to recognize the hla-a2 restricted hivpol-peptide iv9, or the hivgag-peptide sl9. results: t cells, reprogrammed with these receptors, released the pro-inflammatory cytokines il-2, tnf, and ifng simultaneously, and showed up-regulation of the activation marker cd25, after stimulation with peptide-loaded target cells or target cells (i. e. ebv-transformed b cell and cd4 + t cells) presenting the naturally processed epitope. furthermore, these cells maintained their ability to proliferate after stimulation. more importantly, killing assays demonstrated that the tcrreprogrammed cd8 + t cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. a comparison of our reprogrammed t cells with the parental ctl showed that the transfected t cells were only one order of magnitude lower in avidity than the parental ctl. also, the parental clone's recognition pattern of mutant peptides was preserved in tcr-rna-transfected t cells. the transfection of tcr-encoding rna into cd8 + t cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred tcrs is possible. background: dendritic cells (dcs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. the aim of this study was to investigate the capacity of splenic dcs that ingest antigen coated magnetic beads to induce hcv cellular immune responses. methods: splenocytes of flt3l pretreated balb/c mice were incubated for 3 hrs with hcv ns5-coated magnetic beads. the cells were harvested and cells that contained beads were purified by passage over a magnetic column. the isolated population contained g 80 % dcs and was used for immunization. dc expression of the maturation markers cd40, cd80 and cd86 was determined before and after ingestion of ns5-coated beads, showing a significant increase of all maturation markers induced by phagocytosis. cellular immunity was assessed using a conventional ctl assay, a cfse-t-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably ns5 expressing sp2/0 cells). results: in immunized animals, the ctl activity was increased 3-fold compared to mock immunized mice. accordingly, tumor challenge with ns5 expressing tumor cells showed a significant reduction in tumor growth. the number of cd4 + ifn-g + cells was increased g 3-fold and the number of cd4 + il-2 + increased g 5fold in the dc-ns5-bead immunization group. these results paralleled the proliferative response of splenocytes to ns5 protein obtained from immunized animals with the most significant response in the cd4+ population of dc-ns5-bead immunized animals. the use of ns5 coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. the induction of a strong th1 biased t cell response makes this approach a promising new tool in therapeutic immunization in chronically hcv infected patients. the success of anti-dec-205 antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. in their absence, anti-dec-205 induces antigen-specific tolerance rather than immunity. we hypothesize that regulatory t-cell epitopes contained in anti-dec-205 promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. this hypothesis is based on our recent discovery of a set of natural regulatory t-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory t cells. we have verified experimentally that these epitopes generate an expansion of regulatory t cells and suppress inflammatory immune responses. here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-dec-205 antibody can be developed. we screened the anti-dec-205 sequence computationally for putative hla dr4-restricted, regulatory t-cell epitopes as targets for mutations that will reduce epitope binding affinity for hla. amino acid substitutions predicted to interfere with hla binding were identified and are being incorporated into an array of anti-dec-205 antibody variants recombinantly fused to a test antigen, hiv gag. variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced gag immunogenicity, in terms of cellular and humoral responses. we predict that the modification of regulatory t-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-dec-205 as a hiv antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. supported by nih 1r21ai078800 a live oral vaccine based on human adenovirus (ad)4 has proved safe and effective in us military recruits for nearly 50 years. in these experiments, we have investigated whether replication-deficient ad4 can be an efficient potential vaccine carrier for oral vaccination. ad5 vectors were used throughout to provide a benchmark for efficacy. we generated novel ad4 and ad5 vector systems based on dna recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://adz.cf.ac.uk). egfp was inserted with a hcmv ie promoter as a model transgene. preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with ad4 than ad5. ad5 routinely infected and provided transgene expression in˚10 % of human cd4+ and cd8+ t cells. stimulation of the hcmv ie promoter post infection increased detection of egfp to 25 -30 % of cd8+ t cells present, showing that ad5 infected a surprisingly large proportion of t cells. in comparison, ad4 provided egfp expression in x 2 % of either cell type, even after t cell activation. in contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with ad4 and ad5 vectors were comparable. preferential infection of dcs is likely to be beneficial in the context of a vaccine. in vivo, we observed that following oral delivery both ad4 and ad5 induced restricted yet strong expression in the intestine. the vectors were rapidly taken up into the peyers patches, with optimal expression detected day 3 after dosing, and transgene expression being reduced below detectable levels by day 8. interestingly, when delivered together ad4 and ad5 vectors targeted the same subset of cells. together, these data show that ad4 is a viable alternative to ad5-based vaccines which may also avoid unwanted infection of activated t cells. aims: monoclonal antibodies (mabs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies (20 recently commercialized; g 300 in clinical trials). to date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. however, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. as induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mabs, in addition to blunt viral propagation, may also modulate the endogenous immune response. methods: we have developed a preclinical mouse model of fatal leukemia induced by the frcas e murine retrovirus. mice were infected with frcas e and treated, or not, with a neutralizing mab (the 667 mab). viral propagation, survival and development of immune responses were followed up for several months. results: using this model, we have shown that 667-treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. these results show that neutralizing mabs act as immunomodulatory agents capable of inducing long-term protective immunity ( g 8 months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (gros et al, 2005; gros et al, 2008; michaud et al. submitted) . although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral t-cell responses. conclusions: our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral t-cell responses. our observation might have important consequences on the design of antiviral mab-based immunotherapies. objectives: we have analyzed the potential of virus-like particles (vlps) from rabbit hemorrhagic disease virus (rhdv) as a delivery system for foreign t-cell epitopes. to accomplish this goal, we generated chimeric rhdv vlps incorporating a cd8 + t-cell epitope (siinfekl) derived from chicken ovalbumin (ova). the ova epitope was inserted in the capsid protein (vp60) of rhdv at two different locations: 1) the n-terminus, predicted to be facing to the inner core of the vlps (rhdv-vlp-2), and 2) a novel insertion site predicted to be located within an exposed loop (rhdv-vlp-306). both constructions correctly assembled into vlps and we analyzed the immunogenic potential of both chimeric rhdv vlps in vitro and in vivo. in vitro, dendritic cells (dcs) were able to process and present siinfekl peptide in the context of mhc class i from chimeric rhdv vlps for cd8 + specific recognition in a dose-and insert position-dependent manner. moreover, chimeric rhdv vlps activated dcs for tnf-alpha secretion.in vivo, mice immunized with the chimeric rhdv vlps without adjuvant were able to induce specific cellular responses mediated by cytotoxic (ctl) and memory t cells. although both chimeric rhdv vlps were able to induce specific ifn-g-producing cell priming, insertion of the siinfekl peptide at the amino-terminal position (rhdv-vlp-2) was more immunogenic than insertion at position 306 for induction of ctls and anti-viral immunity.more importantly, immunization of mice with chimeric rhdv vlps at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing ova in target organs. in addition, immunization with chimeric rhdv-vlp-2 at the highest dose tested was able to resolve vv-ova infection. conclusion: our data demonstrated that immunization with chimeric rhdv vlps was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. objectives: a major issue pertaining to use of dna vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. in this study, we describe the targeting of antigen-encoded dna directly to dendritic cells (dcs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-s-glyceryl cysteine, a known tlr-2 ligand. methods: agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to dna encoding for green fluorescent protein (gfp), influenza hemagglutinin (ha) or nucleoprotein (np). transfection efficiencies of dcs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. stimulation of t cells by np-transfected dcs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific cd8+ t cells. subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. results: electrostatic binding of the lipopeptide to dna plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the dna backbone and retard its ability to migrate towards the anode. high levels of gfp, ha or np were detected in murine spleen-derived cultured dcs following transfection with these complexes concomitant with the upregulation of surface mhc class ii molecules compared to when dna alone was used. the ability of transfected dcs to stimulate cd8+ t cells from influenza virus-infected mice was also investigated. subsequently, vaccination by lipopeptide-dna complexes resulted in the induction of higher numbers of ifn-g producing np 147-155 specific cd8+ t cells in the spleen and lymph nodes of mice compared to those that received dna alone. conclusion: altogether these results demonstrate that the use of a tlr-2 targeting lipopeptide-based system that can facilitate the delivery of dna by directly targeting and concurrently activating antigen-presenting cells, such as the dc, could prove to be advantageous in enhancing cellular responses induced by dna vaccination. the level of interferon in blood serum of non-infected mice was determined by elisa kit and by the cytopathic test in the l929cell culture after aplication of ridostine and ridostine ointment. the effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. the statistical processing of the data was carried out by the student t-criterion. ridostine was administered to patients once or twice in the case of high temperature. the clinical signs were recorded (temperature, rhinitis, headache etc). for prophylactic and treatment purposes the ridostine ointment was intranasally applied twice per day during 7 days. the effectiveness of the preparation was evaluated by clinical sings and the level of cd2+, cd4+, cd8+, cd16+ t -lymphocytes. results: ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. ridostine and ridostine ointment stimulated synthesis of interferon and phagocytic activity. ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. prophylactic intranasal application of ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of t-cell immunity. application of ointment at an early stage of disease markedly activated t-cell immunity, reduced the duration of the disease and the intoxication syndrome by 1,5-2-fold. conclusion: interferon inducer based on natural dsrna stimulates some reactions of innate immunity and resistance to influenza virus. the drugs based on dsrna show promise for treatment of influenza. objectives: the creation of gene engineering vaccines against hepatitis c virus (hcv) based on recombinant proteins is one of relevant approach. since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. the aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural ns4 and ns5a hcv proteins and immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. objectives: ifn-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. a/h5n1 influenza virus inhibits the ifn-g synthesis (mibayashi et al., 2007) and causes a decrease in cd4 + and cd8 + t-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (tumpley t. m. et al., 2000) . thus, ifn-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. an essential defect of this cytokine is its fast degradation in blood. the goal of this work was to study immunomodulating properties of an ifn-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded ifn inducers. methods: a recombinant human ifn-g analog deltaferon is distinguished by a 10 amino acid deletion at the c-end of the molecule and substitutions of amino acids in positions 129-131 (tat'kov c. i. et al., 2000) . deltaferon was i. p. administered to male non-inbred mice in doses of 2-40*10 4 iu once or twice at an interval of 48 hours, alone or in combination with double-stranded yeast rna preparation (5 mg/kg). the content of ifn-a and ifn-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by mtt-test (mosmann t., 1983) . results: when deltaferon was administered in doses of 2-20*10 4 iu alone it did not influence the content ifn-a in blood, but caused a transient increase in the level of ifn-g. the injection of the preparation in a dose of 2*10 4 iu led to a an increase in both spontaneous and conconavalin a-induced proliferation of splenocytes. the two-fold administration of deltaferon in the maximal dose increased a level of ifn-g and inhibited cell proliferative activity. the combined administration of deltaferon (2*10 4 iu) and dsrna markedly increased the level of ifn-a and enhanced splenocyte proliferation. the recombinant human ifn-g analog is able to enhance ifn-g synthesis, splenocyte proliferation and to modulate the effect of ifn inducer. these data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. by using a combined approach of routes of immunization and vaccine delivery systems such as poly-lactic acid (pla) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. we showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (id), transcutaneous (tc), subcutaneous (sc)). while id and tc route remain mostly efficient for the induction of antigen-specific cd8 responses (tetramer+ hiv-1 gag p24 cells), the quality of humoral responses (igg, iga) remained distinct between the two routes. in addition, sc route is less efficient than id and tc routes for the induction of cd8 responses after pla-p24 immunization. we have also shown that a lower antigenic charge of pla particles was needed when pla-p24 were injected using id and tc routes of immunization. currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. this unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system. b. dí az-freitas 1 , c. prego 2 , s. vicente 2 , m.j. alonso 2 , a. gonzález-fernández 1 1 university of vigo. area of immunology, department of biochemistry, genetics and immunology, vigo, spain, 2 university of santiago de compostela, nanobiofar group, department of pharmacy and pharmaceutical technology, santiago de compostela, spainobjectives: the design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. in this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis b surface antigen (rhbsag) as a model. our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. materials and methods: loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer 188, with a counter ion, tripolyphosphate, containing the free rhbsag. the immunogenicity of these polysaccharidic-based nanocarriers with 1 or 2 immunizations to balb/c female mice (4 weeks old) was assessed following intranasal or intramuscular immunizations. blood samples from the mouse maxillary vein were collected at different intervals (from day 15 to 260 post-primary immunization). specific igg antibodies levels directed against rhbsag in serum were measured by indirect elisa in miu/ml. results: chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rhbsag loading were prepared. the results of the specific igg levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. the response elicited was delayed respect to the alum based vaccine, and very interestingly, igg concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. after intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. the igg titers elicited were enough to induce full seroprotection against the disease (100 miu/ml). polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. more studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. the success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (dcs) in an environment that provides optimal immunostimulatory signals. in our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigenbackground: infection with human immunodeficiency virus type 1 (hiv-1) is characterized by dysfunction of hiv-1-specific t cells. to control the virus, antigenloaded dendritic cells (dcs) might be useful to boost and broaden hiv-specific t-cell responses. poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. we investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p24 antigen from hiv-1 and the tlr3 ligand poly i:c as a maturation factor. methods: monocyte-derived dc (mddc) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly i:c. potential maturation of dc was evaluated by flow cytometry. mddc from hiv-infected patients under highly active anti-retroviral therapy (haart) were similarly pre-incubated with p24 microcapsules containing p24 and poly i:c. these antigen loaded mddc were used to directly stimulate autologous peripheral blood lymphocytes (pbl) in elis-pot. they were also co-cultivated for 10 days with autologous pbl to evaluate the immunogenic capacity. potential expansion of specific t cells was measured by comparing elispot responses of pbl before and after coculture, using a pool of overlapping p24 peptides. intracellular staining of interferon-gamma (ifn-g), interleukin-2 (il-2) and cd107a after p24 stimulation was also performed. results: mddc from hiv(-) subjects, incubated for 24 hour microcapsules alone did not induce maturation of dc, but when poly i:c was present the dc did mature. mddc from haart treated hiv-infected individuals, cultivated with p24 containing microcapsules with poly i:c, were able to efficiently expand and broaden autologous effector memory t cells which contain and secrete ifn-g and il-2, upon p24 peptide restimulation. objectives: we aimed at investigating whether and how the distance of a cytokine from the vlp surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. methods: we inserted one, two or four ig-like domains of hcd16b between our model cytokine il-2 and the minimal gpi-anchor acceptor sequence. subsequently, we compared particle production by western blotting for p30gag, targeting of cytokines to lipid rafts and and vlp upon isopycnic membrane separation and biological activity in il-2 dependent proliferation assays of il-2 variants. results: murine il-2 attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the il-2 dependent cell line ht-2. we found that the membrane anchors comprising one and four ig-like domains (il-2::1iggpi and il-2::4iggpi) resulted in severely reduced vlp production by 293 producer cells and despite of an increased targeting of il-2::4iggpi to vlp, a reduced stimulatory capacity of producer cell crude supernatant, when compared to il-2 fused to the minimal gpi-anchor acceptor sequence of hcd16b (il-2::gpi). il-2::2iggpi, however, showed comparable particle production and biological activity in vitro when compared to il-2::gpi. furthermore, il-2 fused to 2ig::gpi showed an increased capacity to co-stimulate primary p14 tcr transgenic t-cells specific for lcmv-gp 33-41 in the context of h2-d b . conclustions: besides the minimal gpi-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to vlp. 2ig::gpi has a biological activity in vitro, which is comparable to the minimal gpi anchor. moreover, il-2::gpi displays increased co-stimulatory potential in the context of specific mhcp complexes. this work was supported by grants sfb f1816-b13 of the austrian science foundation, the austrian research promotion agency (forschungsförderungsgesellschaft) bridge grant 812079 & biomay ag, and the christian doppler laboratory for immunomodulation. a. roemhild 1 , interdisciplinary transplant laboratory 1 charite berlin, insitute of nephrology and medical immunology, berlin, germany immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. missing viral immunity, particularly against ebv, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (ptld). ptld affects 2-25 % of solid organ transplant recipients, depending on the organ transplanted. healthy individuals control ebv infection by ebv-specific cytotoxic t lymphocytes (ctls), but some patients under immunosuppression are unable to do so. in these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific t cells into the patient. currently these t cells are generated by repetitive stimulations with ebv-infected autologous lymphoblastoid cells (lcls). due to a generation time of 2-3 months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. t cell lines were generated with two different protocols. in the first protocol t cells are generated by repetitive stimulation with ebv-infected autologous lcls. the second protocol is based on stimulation with 6 different overlapping ebv peptide-pools and immunomagnetic cell isolation. expanded t cells were analysed using multicolour flow cytometry. cells were stained for diverse surface markers and intracellular cytokine production. cytotoxic capacity and specificity was determined by a calcein release assay. our group developed a new protocol for the production of ebv specific t cells, thereby shortening the generation time from 2,5 month to 16 days. t cell lines are composed of cd8 and cd4 cells with a mainly effector memory like phenotyp. after restimulation the cells produce more tnfa than ifng. depending on the generation protocol t cells specifically recognized and lysed autologous lcls alone or loaded with ebv-peptides. the detailed characterization of ebv-specific t cell lines should help to further improve the adoptive immunotherapy and its outcome. the novel, short time generation protocol did not affect phenotyp and cytokine production of the t cells. nevertheless their therapeutic potential in vivo has to be tested in further experiments. s. s. schmucker 1 , m. assenmacher 1 , a. richter 1 1 miltenyi biotec gmbh, r&d cell biology, bergisch gladbach, germanyadoptive transfer of virus-specific t cells provides a promising treatment of infection in immunocompromized patients. as expansion of virus-specific t cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific t cells from naive hosts exist. in this study we established a cell culture system for priming of highly rare naive cmv pp65-specific cd4 + and cd8 + t cells from cmv-seronegative donors in vitro.magnetically isolated naïve (cd45ro -cd25 -) cd4 + and cd8 + t cells from pbmc of cmv-seronegative donors were co-cultured with autologous mature monocytederived dc loaded with cmv pp65 peptide pool and cd3-depleted autologous pbmc as feeder cells in the presence of il-2, il-7, and il-15. already 9-13 days after primary activation pp65 495-503 /a2-tetramer + cd8 + t cells were detectable for 3 hla-a2 + blood donors. to analyze cd8 + t cells having other specificities than for the peptide pp65 495-503 as well as probably primed cd4 + t cells, we looked for the production of cytokines after a second stimulation. we found ifn-g secretion in up to 3.9% of the cd8 + t cells and up to 3.8% of the cd4 + t cells after restimulation with pp65 peptide pool, but not with either irrelevant ie-1 peptide pool or without antigen, in each of eight donors tested. for generation of t cell lines, we magnetically enriched the primed t cells according to their ifn-g secretion. subsequent cultivation for 20 days led to a 10 -100 fold expansion of pp65-specific t cells, defined by their sustained capability to produce ifn-g. evaluation of the antigen-specificity of the expanded t cells also showed upregulation of the activation markers cd154 and cd137 only if restimulated with the pp65 peptide pool. further cytokine analysis of the cells revealed co-production of ifn-g, tnf-a, and il-2, indicating the functionality of the in vitro primed and expanded t cells.in conclusion, we established a cell culture system, which enables the in vitro priming and expansion of cmv-specific cd4 + and cd8 + t cells derived from the naive compartment. this should extend the application of adoptive t cell therapy to patients for whom immune donors are not available. a. i. wolf 1 , k. mozdzanowska 1 , l. otvos 2 , j. erikson 1 1 the wistar institute, philadelphia, united states, 2 temple university, philadelphia, united statesthe influenza virus a matrix protein 2 ectodomain (m2e) sequence has remained highly conserved among various human influenza a strains and is therefore a promising target for a protective vaccine. based on previous work using a synthetic m2e-based multi-antigenic peptide vaccine (mozdzanowska at al., vaccine 2003; virology journal 2007), we generated a novel peptide and investigated its efficacy in inducing an anti-m2e antibody (ab) response and its ability to confer protection against viral challenge.objectives: cytomegalovirus (cmv) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (hsct). due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived cmv specific cd8 + t cells, have been considered. clinical data confirm a crucial role for antiviral cd8 + t cells inversely correlating with the incidence of cmv reactivation and disease. cmv specific cells have to reach protective levels in order to be effective. levels of such cells correlating with protection against cmv infection and disease have only been reported in patients expressing hla-a*0201 and hla-b*0702 previously. considering other frequent hla alleles cmv specific cd8 + t cells were monitored longitudinally in 30 hsct patients in this study to establish the cell number thresholds at which patients are protected from cmv reactivation. methods: we have correlated the pattern of different ex vivo cmv peptide specific cd8 + t cell responses (frequency analysis using tetramer staining and interferon gamma elispot analysis) with the cmv viral load (dnaemia) and clinical status in patients. different response groups were compared using the mann-whitney-u test.results: our results demonstrate that the presence of different cmv specific cd8 + t cells inversely correlates with the ability to detect of cmv reactivation in patients at different cell number thresholds. we show that the cell number thresholds for hla-a*2402/pp65 (341-349) (7.6x10 6 cells/l) and hla-b*3501/pp65 (123-131) (4.4x10 6 cells/l) specific cd8+ t cells are significantly lower than those for hla-a*0101/pp50 (245-253) (17.2x10 6 cells/l) and hla-a*0201/pp65 (495-503) (13.2x10 6 cells/l) specific cd8 + t cells in hsct recipients post transplant. this difference is also evident in healthy cmv seropositive volunteers. conclusion: these findings suggest a differing efficiency of the responses restricted by the two sets of alleles. the data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of ag specific t cells to be monitored after therapeutic strategies for cmv in hsct patients. (2,5 -50 mcg) . no adverse effects were indicated during trials (up to 25 month of observation).hiv-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after 4th immunization with 50 mcg of vichrepol.no differences were detected in cd4+ and cd8+ t cell counts and cd4+/cd8+ ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to hiv infection. the results of phase i clinical trials of vichrepol vaccine were approved by who authorized russian national control institution and transition to phase ii immunogenicity trials was recommended. objectives: to improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pix. the adenoviral nanoparticle vaccine was evaluated in the friend virus (fv) mouse model and compared to conventional adenoviral vectors. methods: adenoviral nanoparticle vectors were constructed by deletion of pix from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. for vaccination against fv, that is a retrovirus complex of friend murine leukemia virus (f-mulv) and spleen focus forming virus, we constructed fusion proteins of pix and the f-mulv surface env protein gp70 or gag. to elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid dna encoding either pixgp70 or gp70 alone. conventional adenoviral vectors were used that express full-length f-mulv env and gag. the vaccines were tested in cb6f1 hybrid mice that are highly susceptible to fv infection and develop viremia and splenomegaly after fv infection. results: vaccination of cb6f1 mice with adenoviral nanoparticles expressing fusion proteins containing gp70 resulted in protection from viremia and splenic enlargement after fv challenge that was superior to vaccination with conventional vectors. immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of f-mulv env-specific cd4 + t cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. we could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. conclusion: adenoviral nanoparticles are a useful tool for the induction of antibody and cd4 + t cell responses that are superior to conventional adenoviral vectors. this new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . antibody levels were monitored by elisa and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative rt-pcr, and cellular production of ifn-gamma was measured via flow cytometry. results: we found that animals vaccinated with caf01 exhibited higher levels of serum igg and mucosal iga than the ones which received the vaccine alone, and that they excreted 90-99 % less virus. animals that received only vaxigrip were producing ifn-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also caf01 did not show any increase in their levels of ifn-gamma. conclusion: caf01 enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. evaluation of the t-cell specific immune response is very important for global eradication of measles and rubella. peripheral blood lymphocytes (pbl) from 13 children aged 1-2 years old (6 boys and 7 girls) -group 1, and 11 children (6 boys and 5 girls) 6-7 years old -group 2 were isolated on a gradient of density before vaccination ( or revaccination) with priorix, 1 week, and 2 months after and incubated with cfse. then 2 million/ ml pbl were incubated in rpmi-1640 supplemented with 10 % fcs (the negative control), at presence of 5 mcg/ml pha (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing 5 % cî 2 at 37°c within 7 day. intensity of a fluorescence estimated on fl1 by flow cytometr facscalibur (bd biosciences, usa). cytokines production was measured in the same cultures by bioplex technology (biorad, usa). in the negative control 90 % pbl in both groups did not enter mitosis. in the positive control 90 % of cells have passed one and more mitoses. in group 1 measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in 2 months 15-25 % of lymphocytes demonstrated antigen-specific proliferation. in group 2, on the contrary, before the vaccination the most part of cells (75-80 %) has not entered division, but 20-25% of cells have passed 2 and more mitoses. in a week specific lymphocyte proliferation decreased and in 2 months it was increased up to 40-50 %. production of the interleukin (il) 6, ifn-g, tnf-a, il-4, il-1 was more informative than il-5, il-7, il-8, il-12. measles and rubella antigens induced cytokines production in pbl of immune children and did not influence on pbl of intact children. thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. objectives: broadly neutralizing human monoclonal antibodies (mab) and patients' sera recognizing functionally conserved epitopes on hiv envelope (env), such as the gp120 cd4-binding site (cd4bs), appear to be uncommon. therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. here we describe the generation of two anti-idiotype (ai) murine antibodies recognizing human anti-hiv-1 neutralizing polyclonal iggs directed against the cd4bs. the mabs were shown to react with an anti-cd4bs human neutralizing mab (b12), to elicit antibodies that recognize the gp120 molecule and an anti-hiv-1 neutralizing response in rabbits, confirming them as cd4bs mimotopes. these mabs were also used as probe to detect the expression of clonally distinct anti-gp120 antibodies in sera of hiv-infected individuals. methods: broadly neutralizing sera were collected from long-term non-progressor patients. anti-cd4bs iggs were purified and used to immunize mice for hybridoma generation. mabs reacting in elisa with the anti-cd4bs igg fraction were used to immunize rabbits. rabbit sera were then tested for anti-gp120 titer and hiv neutralizing activity by pseudovirus-based neutralization assay. sera from hiv-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the ais. serial dilution of b12 in sera from healthy hiv-negative donors were used to determine elisa sensitivity. results: two clones (p1 and p2) reacted in elisa only with the cd4bs-directed igg fraction. the clones were also recognized in elisa by b12. p1 and p2-immunized rabbit sera showed a strong anti-gp120 titer. in the pseudovirus assay the ais-immunized rabbits showed a neutralization activity against virions bearing hxb2 strain glycoproteins. in particular, 3/5 rabbits in the p1 group and 1/5 in the p2 group showed an 80 % hiv neutralization at dilutions ranging from 1:20 to 1:150. the immunoenzymatic assay used, allowed to detect a p1 and p2 reactivity in hiv-positive sera and was able to detect a b12 concentration equal to 10 ng/ml. conclusions: these data demonstrate that immunogens designed on the idiotype of broadly neutralizing abs are feasible and could help in the design of effective anti-hiv vaccines or diagnostic assays. yellow fever vaccines (17d and 17dd) are well tolerated, with a very low rate of severe adverse events (yf-sae), such as serious allergic reactions, neurotropic (yf-and) and viscerotropic (yf-avd) diseases. viral and host factors have been postulated to explain the basis of yf-sae, especially those able to modify the host immune response to the yf vaccine. however, the mechanisms underlying the occurrence of yf-sae still remain unknown. in the present investigation, we present a detailed immunological analysis of a 23-year-old us citizen female patient, who developed yf-and characterized by encephalitis associated with pancreatitis and myositis following 17d-204 vaccination. our findings highlighted that yf-and exhibited decreased expression of fc-g-r in monocytes (cd16, cd32 and cd64) along with increased levels of nkt-cells (cd3 + cd16 +/-cd56 +/-/cd3 + ratio) and activated t-cells (cd4 + and cd8 + ) and b-lymphocytes. enhanced levels of plasmatic cytokines (il-6, il-17, il-4, il-5 and il-10) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within nk-cells (inf-g + , tnf-a + and il-4 + ), cd8 + t-cells (il-4 + and il-5 + ) and b-lymphocytes (tnf-a + , il-4 + and il-10 + ). the analysis of cd4 + t-cells revealed a complex profile with increased frequency of il-12 + and ifn-g + and decreased percentage of tnf-a + , il-4 + and il-5 + cells. depressed cytokine synthesis was observed in monocytes (tnf-a + ) following in vitro antigenic stimuli. these results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of yf-sae. this is the first case report of yf-sae investigated by members of the international laboratory network for yellow fever vaccine associated adverse events. g. mester 1 , h.-g. rammensee 1 , s. stevanović 1 1 eberhard-karls-universität tübingen, department of immunology, tübingen, germany adenovirus (adv) is a widespread pathogen in humans and can persist in its hosts after infection. persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. bmt recipients, with high rates of mortality. however, the cellular immune response against adv is poorly characterised, and very few t cell epitopes have been published up to now. thus, our aim was to detect dominantly immunogenic adenoviral cd8 t cell epitopes by analysing the responses of healthy blood donors who have overcome infection. we have predicted possible cd8 t cell epitopes for the frequent mhc class i alleles a*01, a*02, and a*24 from the proteins pii (hexon), pviii, and e1a of adv strains ad2 and ad5 by using the syfpeithi software developed by our group (www.syfpeithi.de). subsequently we performed a 12-day recall stimulation of pbmcs from at least 16 healthy donors with synthetic peptide followed by ifn-g elispot screenings to identify naturally occurring t cell responses and assess their frequency in the population. tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific cd8 t cells. we could identify 27 new peptides eliciting ifn-g responses, several of which were confirmed as novel cd8 t cell epitopes. amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined hla restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. these findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro t cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. methods: sle (5 ara criteria) was diagnosed in a 40-year-old african female patient with hiv-1 (clade c) infection. good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of cd4 t-cell count x 200 cells/mm 3 . initiation of 500 mg mmf bid was associated with biological and clinical remission of sle and cd4 t-cell increase. no opportunistic infections or cancers were noted during a 3-year follow-up and the patient remained always naive to art. hiv-1-specific cd4 and cd8 t-cell responses were analyzed after 18 months of mmf by ifn-g elispot assay and polychromatic flow cytometry assessing ifn-g, tnf-a and il-2 production following stimulation with a panel of 192 hiv-1-derived optimal epitopes (9/10-mers) covering various hiv regions and a pool of 105 hiv-1-derived peptides (15-mers overlapping by 11 aminoacids) encompassing the entire gag protein. all peptides are derived from hiv-1 consensus strain iiib. results: highly polyfunctional hiv-1 specific cd4 and cd8 t-cell responses against gag were detected. 11 epitope-specific cd8 t-cell responses were identified: except for one response restricted by hla a*23 and another one by hla cw*07, all the others were restricted by hla-b alleles and mostly by b*58 (n = 5). seven out of 11 responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. ifn-g+, tnf-a+ and il-2+) regardless of the viral region and hla restriction. conclusion: strong, broad and polyfunctional hiv-1 specific cd4 and cd8 t-cell responses known to be associated with nonprogressive infection were detected during mmf treatment.we therefore suggest that mmf use in the context of sle-hiv is not detrimental to the establishment or preservation of protective hiv-1 t-cell immunity. the rabies virus was propagated in the vero cell line. virus was titrated by focus fluorescent units. virus preparations having a titer of 10 6 dl50/ml were inactivated with b-propiolactone. aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. male mice of the strain cf-1 of 12-16 g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group a was immunized with virussqualene, group b with virus-aluminium hydroxide, group c with the antigen alone, group d with saline buffer-squalene, group e with saline buffer-aluminium hydroxide and group e was inoculated with mock-infected cell culture supernatant. mice were boosted at the 7 th day. all mice were properly bled to prepare preimmune sera and hyper-immune sera. at the end of the immunization protocol the igg raised against the rabies virus was tested by an indirect elisa. results: the highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. there was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected vero cell supernatant. conclusions: the use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. supported by: concyt-46767, cofaa and cgpi-20090305. . state of vaccine-induced measles immunity was determined by means of elisa in 1-3, 4-6 and 7-9 years since revaccination with live measles vaccine (lmv) before and after tuberculosis chemoprophylaxis. statistic data were processed with t-, w-and u-criteria. results: during the first three years since lmv revaccination igg level was middle (children with negative and long-term positive mt) and high (children with conversion and hyperergic mt). in 4-6 years since lmv revaccination uninfected and long-term infected children showed a significantly decreased (p p 0.05) measles immunity and antibody level much lower (p p 0.05) than among children with mt conversion. in 7-9 years the comparison group kept decreased (p p 0.05) measles immunity, the majority (92±5.54 %) of persons had minimal protected igg level, but the observation groups were characterized by average immunity level, which was higher (p p 0.05) than in the comparison group. comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles igg level among long-time infected children on completion of chemoprophylaxis decreased (p p 0.05), the majority (83.3±4.1 %) of persons lost protected antibody level; among children with mt conversion in 1-3 years since lmv revaccination immunity state didn't change, but in further periods antibody level decreased (p p 0.05) to low values; among children with hyperergic mt igg level decreased (p p 0.05) and reached low (in 1-3 years), minimal protected (in 4-6 years) and lower than protected (in 7-9 years) values. -at the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-in remote periods since lmv revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-in countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. objectives: to evaluate the balance of ifn-gamma and il-17 producing cells in lungs during the immunotherapy of tuberculosis with the dna vaccine encoding the heat-shock protein 65 (dnahsp65). methods: balb/c female mice were infected by intra-tracheal route with 10 5 h37rv mycobacterium tuberculosis. immunotherapy with endotoxin free dnahsp65 genetic vaccine was done at days 30, 40, 50 and 60 post-infection. each dose consisted of 100 micrograms of dna vaccine in the quadriceps. intracellular cytokine staining of cd4+, cd8+ and gamma-delta t cells from lungs were determined 10 and 50 days after the end of the therapy. bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. differences of p x 0.05 were considered significant (t test). results: at day 10 after the end of the immunotherapy, dnahsp65 treated mice exhibit increased numbers of absolute cd8+ and gamma-delta t cells when compared to non-treated animals. the percentage of ifn-gamma and il-17 producing gamma-delta t cells were the same between treated and non-treated animals. in contrast, dnahsp65 treated mice showed more ifn-gamma producing cells in both cd8+ and cd4+ cell populations. at day 50 after the end of the therapy, the main observation in mice which received dnahsp65 treatment was the augment of all three populations producing ifn-gamma. although non-treated animals also increased the frequency of cd4+ and gamma-delta t cells positive for ifn-gamma, they did not increase the numbers of ifn-gamma cd8+ cells, together with a more frequency of gamma-delta t cells producing il-17. finally, the immunotherapeutic effects of dnahsp65 vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis. the results presented here indicates that cd8+ cells producing ifn-gamma and the reduction of the frequency of gamma-delta t cells secreting il-17, are the main effects of dnahsp65 immunotherapy of murine tuberculosis. furthermore, these results have important implications since they indicate the importance of an appropriate balance of il-17 and ifn-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. the detection of quantitative changes in mrna expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time pcr) screening methods. because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a reverse-transcriptase multiplex ligation-dependent probe amplification (rt-mlpa) method. rt-mlpa is a reliable, robust, low cost and user friendly technique permitting rapid mrna expression profiling of as many as 60 loci in a single reaction. genes of interest can be selected on a tailor-made basis. the assay is highly reproducible, has an extensive dynamic range of 3-5 log depending on the genes of interest, and a pcr amplification step within the rt-mlpa ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. since this assay is relatively high throughput (96-well format), requires only 100 ng rna per sample, and allows mrna profiling in direct ex vivo whole blood samples (from e. g. pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. to illustrate this, we have been able to successfully implement this assay in 5 different laboratories in sub-saharan africa. thus far we have applied rt-mlpa to characterize the human immune response to mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. a particularly useful application of the rt-mlpa is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (tb) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active tb patients. initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with tb disease. these biomarkers may provide new and relevant information that can be applied in future tb studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. preclinical m. leprae infection is a major source for leprosy transmission. therefore, early detection of individuals infected with m. leprae is crucial. however, to date there are no diagnostic tests available that can identify preclinical leprosy. such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.newly developed hla based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of m. leprae infection.using this post genomic approach, we were able to identify candidate proteins and peptides unique to m. leprae containing predicted t cell epitopes restricted via several major hla-class i and ii alleles. since the selected genes were of unknown function, their expression in m. leprae bacilli was assessed.evaluation of the immunogenicity of these m. leprae proteins in pbmc from a brazilian population showed that 5 candidate antigens induced significant ifn-g levels in m. leprae infected individuals but not in healthy controls from an endemic area.importantly, among exposed healthy controls 71 % had no detectable igm antibodies to the m. leprae specific pgl-i, but instead responded to one or more m. leprae antigen(s). to further improve the diagnostic potential of these m. leprae sequences, synthetic peptides spanning all 5 m. leprae proteins were analyzed similarly. determination of cumulative t cell responses towards 4 of these peptides that activated pbmc of leprosy patients increased the sensitivity compared to single peptides to 100 % in pb, 75 % in rx and 93 % in hhc, without compromising specificity.since diagnostic tools should be applicable in several populations regardless of the genetic background, these m. leprae antigens are also tested in populations on the african (ethiopia) and asian (nepal) continent.in addition, we have applied these antigens in a new user-friendly ucp-lf assay to detect different cytokines. this assay proved to be more sensitive than elisa for detection of ifn-g and can be easily applied in field sites. tuberculosis, an infectious disease caused by mycobacterium tuberculosis (mtb), affects millions of people. m. bovis bcg is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. paratuberculosis, an enzootic bacterial disease in ruminants, due to mycobacterium avium subsp. paratuberculosis (map), has a significant economic impact on livestock production, and moreover, map infection may be one of the microbial triggers of crohn's disease in humans. map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. cd40l, a co-stimulatory molecule preferentially expressed on activated cd4+ t cells, is the ligand of cd40. cd40-cd40l interaction induces the production of il-12 and the initiation of a th1-type immune response. several studies show that cd40l is required for the activation of macrophages and the maturation of dcs. moreover, cd40l enhances the capacity of cd8+ t cells to produce ifn-g and to lyse mtb-infected monocytes. in this study we attempt to improve existing tb and map vaccines with a recombinant bcg expressing cd40l. we have constructed the recombinant bcg strain expressing cd40l (rbcg2) by electroporation of bcg with pgfm11/signalsequenceag85b-cd40lec and an another recombinant strain with empty vector pgfm11 (rbcg1) as a control. the expression of cd40l has been evaluated by western blotting. balb/c mice were vaccinated with the recombinant bcg vaccines. bcg growth kinetics were compared by counting viable bacteria (cfu) in spleen and lungs. the immune response was evaluated by measurement of th1 type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. two months post vaccination, mice were challenged with mtb and map and protection was evaluated. preliminary results show normal persistence of the two recombinant bcgs. analysis of the immune response shows an effect of cd40l 2 weeks after vaccination but not at 4 and 8 weeks. rbcg2 seems to be more protective against paratuberculosis than rbcg1, but not against tuberculosis. another vaccination experiment is required to confirm these results. the effects of bcg-cd40l on cultured dcs in vitro will further be explored. objectives: tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. the regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. methods: using the guinea pig model of mycobacterium bovis bacillus calmette-guérin (bcg) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (ppd) of mycobacterium tuberculosis or infected with bcg or m. tuberculosis. the expression of ferritin in co-ordination with key immuno-regulatory proteins, tnfa, ifng and il-1a, was examined using real-time pcr. to determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the ppd re-stimulation studies by the addition of guinea pig specific tnfa and ifng antibodies. results: a typical pro-inflammatory immune response was observed with significant up-regulation (p x 0.05) of tnfa, ifng and il-1a after re-stimulation with ppd and mycobacteria. of interest was a trend in ferritin down-regulation after re-stimulation with ppd and bcg and this was significant (p x 0.05) after restimulation with m. tuberculosis. the down-regulation of ferritin was also affected by the addition of tnfa antibody in the ppd re-stimulation study. conclusions: ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. the data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve tnfa. these results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. this work is funded by health protection agency phd studentship award. methods: anti-cd40 mab and ag85a were chemically treated with sata and sulfo-smcc respectively, in order to produce a stable crosslinker between both proteins. crosslinking was confirmed by western blotting and cd40 binding on cd40 transfected l929 fibroblasts. the conjugates were tested in vivo in wild type and cd4 + cell-depleted mice for the induction of specific anti-ag85a serum antibodies. splenocytes were challenged ex vivo with ag85a and were examined for their ability to produce th1-related cytokines. elispot assays were performed to determine ifng production and flow cytometry was used to analyse intracellular cytokine staining for tnfa, ifng and il-2. we developed a method to successfully crosslink anti-cd40 mab to ag85a. serum antibodies against ag85a were detected after immunisation with this conjugate vaccine in both wild type and cd4 + cell-depleted mice. t cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in ifng production (elispot), when compared to mice vaccinated with ag85a alone. production of two other th1-related cytokines, tnfa and il2, was also increased in these t cells as shown by intracellular cytokine staining. conclusion: our results suggest that anti-cd40 conjugate vaccines could provide a new way to increase vaccine efficacy. this new conjugate vaccine may be able to by-pass the need for cd4 + t cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients. h. schäfer 1 , r. burger 2 1 robert koch-institute, cellular immunology, berlin, germany, 2 robert koch-institute, infectious diseases, berlin, germanyobjective: immunity against mycobacterial infections is mediated by both cd4-positive and cd8-positive t-lymphocytes. cd8-positve cells respond to peptides derived from cytosolic proteins and presented on mhc class i molecules of antigen presenting cells (apc) via the endogenous pathway. some apc however, are able to take up extracellular antigens and present peptides thereof on mhc class i molecules. this process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. to define the contribution of cross-presentation to activation of cd8+ t cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. methods: purified t lymphocytes from guinea pigs immunized with bcg or complete feund's adjuvant were labeled with the intracellular fluorescent dye cfse and incubated with ppd and/or apc for 5 days. surface phenotype and proliferation of t-lymphocytes were analyzed by flow cytometry.results: up to 30 % of lymph node t lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with ppd-pulsed macrophages. no difference was observed between bcg-(living mycobacteria) and freund's adjuvant (heat killed mycobacteria)-immunized animals. the responses of both t cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime cd4+ t-lymphocytes in vivo. similarly, the cd4-positive subpopulation should primarily respond to soluble antigens presented on mhc class ii molecules. proliferation of both the cd4+ and the cd8+ subpopulation depended on the presence of apc. stimulation oft cd8+ cells as a consequence of direct loading of peptides onto mhc class i molecules was ruled out by using mhc-class i-positive fibroblast cells instead of professional apc, which did not lead to proliferation of primed t-lymphocytes. conclusion: cross-presentation of soluble antigens to cd8-positive t cells is a highly effective means to stimulate the response of cytotoxic t cells against mycobacterial antigens even without direct contact to infected cells. therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. objectives: due to broad antigenic cross reactivity of purified protein derivatives (ppd) with bcg vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (tst) is not reliable to evaluate the specific anti-tuberculosis immune response. therefore, new tools are required to improve mycobacterim tuberculosis (tb) diagnosis and treatment, including enhanced ability to compare new treatment strategies. among different antigens early secretory antigenic target 6 (esat-6) protein is highly specific for tb complex and elicit strong t-cell response in human. in order to monitor the immune response against the pathogen, 83 iranian and afghan adults (38 patients with sputum smear and culture positive tuberculosis, 24 recovered patients during 6 months after full course of chemical treatment and 21 healthy individuals) were recruited to quantify the frequency of esat-6 and ppd specific t-cells in their peripheral blood by home made elispot ifn-gamma assay. results: considering cut off of 4 spot forming unit ( g 20 spots per million), we found detectable response to esat-6 in almost 80 % of patients with active disease. this frequency among treated patients after disease recovery was not significantly different and 77 % of these individuals had detectable esat-6 specific response even after six months completing treatment. neither of healthy individuals showed such response. t cell response against ppd was identified in 14 %, 57% and 62 % of healthy participants, active patients and healing individuals, respectively. conclusion: elispot ifn-gamma assay showed a sharp induction of th1 immune response, against esat-6, in tb patients which persists after successful treatment and full recovery. these results may show potential application of tuberculosis-specific elispot testing as a proxy measure of tb diagnosis and treatment. bcg is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. both environmental and genetic host factors as well bcg strain variability and virulence of the intruding m. tuberculosis strain have been suggested to affect the efficacy of bcg vaccination. in mouse and bovine models it has been shown that pre-exposure to mycobacterium spp. negatively affected protective efficacy of bcg. we use non-human primates (nhp) for the evaluation of new tb vaccine candidates and possible identification of immune mechanisms of protection. however, in naive rhesus macaques a variable efficacy of bcg reminiscent of the clinical situation was revealed. by meta-analysis we compared immune response parameters measured after vaccination using bcg strain danish 1331 in the context of protection measured by gross pathology evaluation after experimental infection with a constant m. tuberculsosis strain erdman. although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of bcg. most remarkably, while bcg induced levels of ifng secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of tb diagnostics for nhp, negatively affects the protective efficacy of bcg. together, these results are relevant for evaluation and interpretation of tb vaccine tests in nhp and support further research into the identification of (mechanisms of) protective immunity in the primate host. p. s. nagpal 1 , p.k. upadhyay 1 1 national institute of immunology, pdc-1, delhi, indiaobjective: study was aimed for the preparation of dry powder formulation containing live mycobacterium indicus pranii for pulmonary immunization against tuberculosis. pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. secondly, pulmonary delivery is a needle-free delivery system, long desired for vaccine delivery. dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. method: sodium alginate solution with suspended mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. aerosols so generated were entrapped in cacl 2 solution with poly-vinyl alcohol (pva) as surfactant. particle so formed collected by centrifugation and lyophilized for dry powder formulation. pva and alginate concentration varies the size, surface and shape of the particles. formulation so prepared was delivered to c57bl/6 mice directly into lung by endotracheal intubations of mice. proliferation index (pi) of spleenocytes of immunized mice was measured after in-vitro stimulation with mycobacterium tuberculosis antigen. result: 2.4 % alginate, 0.012 % pva concentration gives particles with size of 2-10 micrometer as confirmed by particle size analyzer and scanning electron microscopy. viability of the mycobacterium indicus pranii was best achieved with 5 % trehelose and 3.5 % pvp (poly vinyl pyrollidone). there was 6 fold increase in proliferation index of spleenocytes and releases 600 pico-gram of interferon gamma after 4 week of immunization. formulation also induces the activation of dendritic cells after their in-vitro incubation as shown by 10 % increase in cd86 and 10.5 % in ccr7 expression as compared to blank alginate formulation. bacterial exopolysaccharides (epss) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. epss are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. some epss contain sulphate groups as inorganic substituents. the presence of these groups contributes to the biological activity of epss, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. b100 is a constitutively sulphated eps produced by halomonas maura, a recently discovered halophilic bacterium. in preliminary experiments we found that modification of its eps by adding sulphate groups to the native polymer (thus obtaining b100s) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. at the same time we found that other epss produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. it was therefore of interest to determine whether the antiproliferative activity of b100s was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by b100s. by cell cycle analysis we determined that b100s is able to induce apoptosis in up to 80 % of jurkat and molt-4 t cell leukemias. the examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by b100s is restricted to cells of the haematopoietic lineage and that leukemic t cells are particularly sensitive to death induced by b100s, but that untransformed cells are not. a time-course of caspases activation indicated that caspase 9 is the first to be cleaved, followed by caspases 3 and 8, thus suggesting that b100s triggers apoptosis through the mitochondrial pathway. it is noteworthy that b100s also induces vigorous apoptosis in primary leukemic t cells obtained from the peripheral blood of patients. therefore, b100s may well provide a satisfactory therapeutic alternative to patients with acute t cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting dcs. polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. this layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. by using the biopolyelectrolytes dextran-sulphate and poly-l-arginine, biodegradable microcapsules can be obtained. in this study, we have chosen the lungs as a non-invasive route for vaccine delivery. as demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-l-arginine microcapsules were readily taken up by local pulmonary apcs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards apcs. microcapsule instillation also affected the pulmonary apc activation status, indicated by the emergence of an apc population expressing increased levels of mhcii, the co-stimulatory ligands cd40, cd80 and cd86, and of the inflammatory cytokines il-6, il-23 and mcp-1. using ovalbumin (ova) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. analysis of the alveolar infiltrate, cd4 t cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard th2 and th1/th17 skewing adjuvants alum and complete freund's adjuvant (cfa). in response to ova aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while alum and cfa respectively induced typical eosinophilic and neutrophilic inflammations. striking differences were also observed on the level of cd4 t cell responses. microcapsule based vaccination resulted in a marked induction of il-17 secreting th17 cells, without inducing strong th1 (cfa) or th2 (alum) responses. these differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (igg1, igg2c and ige).in conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung apcs, and possess immune stimulating activities distinct from alum and cfa. due to their capacity to generate th17 responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. pneumolysin is an important virulence factor produced by virtually all clinical isolates of streptococcus pneumoniae. the protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of cd4 + t cells and increased production of pro-inflammatory cytokines in immune cells. in this study we investigated the interaction of pneumolysin with murine dendritic cells (dc). we found that pneumolysin induced the activation of dc, reflected in the enhanced expression of the costimulatory molecules cd80, cd86 and cd40 and mhc class ii molecules. the toxin alone was found to be a poor inducer of cytokine production by dc but it did enhance the secretion of tlr agonist-induced cytokines such as il-6 and tnf-a. previous published findings have shown that pneumolysin activates peritoneal macrophages in a tlr4-dependent manner. however, we found that pneumolysin was capable of activating dc from both wildtype and tlr4-defective c3h/hej mice, by inducing cell maturation and synergising with tlr agonists to enhance cytokine secretion. importantly, we also found that pneumolysin is a strong inducer of il-1b secretion by dc, through its effects on caspase-1 processing, which is also tlr4-independent. the results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require tlr4 signalling. objectives: transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. the objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. methods: pregnant balb/c mice were supplemented with placebo (maltodextrin) or probiotics (lactobacillus paracasei ncc2461 (st11) or lactobacillus rhamnosus ncc4007 (lpr), each at 5x10 8 cfu/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. at weaning, pups were immunized with live attenuated measles vaccine (mv-s, aventis-pateur). weight evolution of pups was followed from week 1 to week 7 of life. fresh feces were collected at 3, 5 and 7 weeks of life for determination of iga levels (assessed by elisa). pups were bled 2 and 4 weeks after immunization for determination of measles-specific igg1 and igg2a antibodies. analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. results: all newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. fecal iga production increased progressively in all pups from weaning, reflecting a normal development status. nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning s-iga production in pups. lpr supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. interestingly, no significant effect was observed in the st11-fed group. finally, a modification of the microbiota composition was observed in pups of supplemented mothers. particularly, there was a significant increase in lactobacilli in pups from the lpr group as compared to controls. conclusion: this study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. moreover, these first results seem indicate that the effects are strain specific. chitosan, (1-4)-2-amino-2-deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. chitosan (chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. nanoparticles (chi-np) were prepared from 200 kda chitosan by ionotropic gel formation. bovine serum albumin (bsa) and myoglobin, human immunoglobulin g and superoxidedismutase (sod), and chicken lysozyme were fitc labeled. chi-np were preincubated with proteins at 3:1 ration and washed 3 times. after washing chi-np containing bound proteins were run by denaturating gel electrophoresis. all proteins were able to form complexes. most effective binding was shown for bsa, sod, and lysozyme. the stability of chi-np complexes with proteins was studied in vitro on macrophage cell line raw264.7 by confocal microscopy. for this chi was labeled with rhodamine and nanoparticles were coincubated with fitc labeled proteins before addition to the cells. we showed co-localization of chi and fitc for all proteins studied. these results demonstrate that chi-np form stable complexes which are internalized by macrophages. the family euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. in the present study, different aspects of the immunomodulatory activity of 24 extracts from 10 euphorbiaceaes on peripheral blood mononuclear cells (pbmc) from healthy individuals were evaluated. the pbmc were exposed to the extracts w/o phytohaemagglutinin a (pha), cycloheximide (chx) or lipopolysaccharide (lps) as agents that induce proliferation, apoptosis and cytokine production in pbmc. the lymphoproliferative activity of pbmc was evaluated by thymidine incorporation and cfse dilution assay using flow cytometry. the mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using dioc6/propidium iodide staining by flow cytometry and tnf-a secretion in the culture supernatans by elisa. we found that 15 up to 24 euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. however, only the bark extract of croton spp. insoluble in hexane:diclorometane:methanol (hdm) and the latex extracts of euphorbia cotinifolia and euphorbia tirucalli induced strong proliferation, apoptosis and also tnf-a production in pbmc. these extracts were subfractioned by sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. additionally, we started with the characterization of the specific immune effects of these subfractions on pbmc. all three subfractions induced proliferation predominantly on cd3+ cells. these effect was also observed in isolated t cells indicating that accessory cells are not necessary for the subfractions'activity. the lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates d-(+) galactose or a-methyl-mannopyranoside. these results demonstrate the presence of immunomodulatory compounds in plants from the euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. we found significant increase on lymphocytes and eosinophils populations obtained from lps + p. acnes-treated group in relation to control group.on 35 th day, we detected a significant negative correlation between eosinophils absolute number and fec. both il-5 and ige serum levels were increased on animals from group i when compared to control.the enhancement on th2 immune response pattern induced by lps and p. acnes treatment diminished drastically parasitic load. conclusion: our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. indeed, current influenza a vaccines do not generate significant immunity against serological influenza a virus subtypes and would thus be ineffective in the face of a pandemic novel variant. hence adjuvant usually needs to be added to those types of vaccines. here we show that wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. experiments performed in mice showed induction of specific cd8+ ctl cells against conserved proteins that were accompanied by the induction of ifn-g producing cd4+t cells, following single immunisation. in addition increased hi titres and higher levels of specific igg2a and igg2b antibodies were found even long periods after single vaccination with reduced doses of vaccines. consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. this promising finding on the improvement of seasonal influenza vaccines by wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase i clinical trials in humans. s. lindgren 1,2 , n. almqvist 2 , a. lönnqvist 1 , s. östman 1 , c. rask 1 , e. telemo 2 , a.e. wold 1 1 university of göteborg, department of clinical bacteriology, göteborg, sweden, 2 university of göteborg, department of rheumatology and inflammation research, göteborg, swedenobjective: dietary antigens normally evoke immunological tolerance. a prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. the gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in western societies in parallel with reduced complexity of the infantile commensal microflora. we have observed that children neonatally colonized with s. aureus in the gut seem protected against development of food allergy. here we examine whether a s. aureus toxin affects tolerogenic processing by the intestinal epithelium. methods: mice were given s. aureus enterotoxin a (sea; 0.8 mg/ml) in the drinking water for 5 days and, 3 days later, 50 mg ovalbumin per os. one hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). results: recipients of serum from sea pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with sea. this was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin-5 and interleukin-13. examination of gut sections from sea treated donor mice revealed increased density of cd8a + intraepithelial lymphocytes. our results show that sea promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes. abstract withdrawn by author to develop an efficient vaccine against cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like ompa, omcb, and pmp21, proteins of the inclusion membrane like incc, secreted proteins like cpaf, and the heat shock protein groel. cpg-dna 1826, a highly stimulatory oligonucleotide for apcs, was used as adjuvans. subcutaneous co-injection of ompa, omcb, pmp21, or groel together with cpg-dna 1826 reduced the chlamdial burden in nasally infected mice. however, symptoms like substantial loss of body weight were not influenced. in contrast, a low dose infection with cp. pneumoniae almost completely prevented the loss of body weight upon challenge. to improve the efficacy of the vaccine we used poly-dl-lactide-co-glycolide microspheres loaded with the protein ompa or pmp21 together with cpg-dna 1826. the microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same apc. intranasal but not subcutaneous vaccination of mice with ompa or pmp21 microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. pmp21 microspheres induced protective ifng-secreting cd4 + t-cells and raised pulmonary pmp21-specific iga levels in vivo. also, pmp21 microspheres caused lower il-6 serum levels upon administration than the injection of pmp21 together with cpg-dna 1826, indicating fewer side effects. objectives: staphylococcus (s.) aureus superantigens are highly potent t cell mitogens and the causative agents of toxic shock syndrome (tss) and food poisoning. most s. aureus have superantigens and patterns are highly variable. to date, the role of superantigens in bacteraemia is not well defined.to analyse whether superantigens play a role in bacteraemia, we investigated s. aureus strains and anti-superantigen antibody responses in 44 cases of s. aureus bacteraemia in iv drug users and 44 cases in nonaddicts. a rise in neutralising antibody titers indicates that superantigens are produced during infection. the study comprised 44 iv drug users with positive s. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original fintrova and finlevo trials (ruotsalainen 2006).all s. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-pcr was applied to determine the superantigen gene pattern. sera from patients were obtained at diagnosis (day 0) and four weeks thereafter (day 28). neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.results: genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (holtfreter 2007) . there were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. interestingly, the staphylococcal lineage st59 (spa-type t172, agr 1, and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p=0.01).most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. we frequently observed a rise in antibody titers during infection. surprisingly patients with st59 strains did not show any elevations in neutralising antibody levels. conclusion: s. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. this indicates that superantigens are produced during infection. however, the action of superantigens is frequently modulated by specific neutralising antibodies. this and the special behaviour of s. aureus st59 strains need further investigation. objectives: down syndrome (ds) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. to test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (tt), which is part of the dutch immunization program. methods: after booster vaccination at 4 and 9 years of age, quantitative (titer) and qualitative (avidity) tt responses were investigated in 15 and 7 ds children, respectively. samples were taken before and 3-4 weeks after vaccination. tt-specific igg and igg-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (elisa), avidity of igg 1 -anti-tt by an avidity elisa. the results were compared with reference values from the laboratory. results: at 4 years, post-vaccination anti-tt-titers were decreased (geometric mean total igg, igg 1 , igg 2 and igg 4 ). at 9 years, ds children had lower postvaccination geometric mean igg 4 anti-tt-titers only. post-vaccination igg 1 -anti-tt avidity levels were decreased in 8/15 and 4/7 ds children at four years and nine years of age, respectively. the quantitative and qualitative anti-tt-responses in both ds groups are shifted downwards compared to the reference values. although the anti-ttresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that ds children have profound and lasting difficulties with specific anti-tt antibody formation. 2 kda; 30, 6 kda; 23, 9 kda; 19, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] 6 kda and 18, [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 7 kda were expressed by a majority of examined strains independently of the associated diseases. we assume that these omps could be conservative proteins of h. pylori. conclusion: considering omps as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of h. pylori omps as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. the increasing cases of severe diarrhoea and invasive lethal infections in children caused by salmonella typhimurium are a major public health problem in mexico. the rapid dissemination of multidrug-resistant s. typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. the objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant salmonella infections was associated with a reduced in anti-salmonella immunity. a cohort of 100 families, from a mexican agricultural community with a high incidence of endemic salmonella infections, was followed prospectively for an 8-month period. sera were obtained from healthy subjects from the same community (2 months to 88 years of age). the highest incidence of salmonella-associated diarrhea, 74/1000, occurred in children under 5 years of age. the lowest incidence, 10/1000, was observed in the population aged 10 to 59. whereas serum from individuals ranging 15-70 years of age showed maximum igg1, igg3 and iga anti-s. typhimurium titres, children less than 5 years-old did not show detectable igg1 and igg3 titres and had weak igg2, iga and igm antibody levels; only their igg4 levels were comparable to those detected in adults. moreover, the levels of igg2 and igg3 antibodies were lower in adults with a diarrheal-associated episode. interestingly, s. typhimurium yuhs 07-18, a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to fc-mediated phagocytosis by human monocytes. these data contributes to define the protective immune response involved in anti-s. typhimurium immunity. diseases caused by the yeast of candida genus are a serious clinical and social problem. despite this fact, there is no effective prevention against these opportunistic pathogens yet. although c. albicans is the major cause of the mycoses (67%), the number of the multiresistant non-albicans isolates increases. c. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates. the surface mannan of candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). in our study we prepared oligosaccharides from acid-stable part of mannan c. dubliniensis by conventional acetolysis. this procedure specifically cleaves the a-1,6linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. obtained oligosaccharides were used in inhibition elisa and spr (surface plasmon resonance) measurements. the reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after 7-fold immunization with mannan-hsa conjugate injections in week intervals as well with inactivated c. dubliniensis whole z. neščáková 1 , s. bystrický 1 1 institute of chemistry, slovak academy of sciences, bratislava, slovakiaour newest approach to sub-cellular vaccine against gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified lps) as the target antigen. this is achieved by conjugation of carbohydrate to a protein carrier which secures the t cell dependent immune response. the goal of the immunization with this conjugate is to generate the effective production of memory b cells. here we prepared subcellular conjugate with the detoxified lps from vibrio cholerae strain o135 using polymer carrier and a protein. the cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. activation and differentiation of b-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. a single-platform approach based on flow cytometry and defined number of fluorospheres was used to count b cells. however in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. the protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. like pan-b cell marker cd19, expressed almost on all blood and tissue b cells, was used. here we investigate the characteristics and development of antibody (iso)types after secondary immunization with mencc or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. methods: volunteers, age 18-55 years, were immunized with mencc or received a secondary immunization with mencc or plain menc ps. blood samples were obtained before and seven time-points after immunization. igg, iga, igm, igg1, igg2 and avidity were assessed by a multiplex immunoassay. functional antibodies were determined by a serum bactericidal assay. results: high levels of antibodies were still present 5 years after primary mencc immunization. secondary immunization resulted in increased igg and sba titers after 5 to 7 days. in primed individuals, igm was still present, and this only increased following a secondary immunization with plain ps. in addition, immunization with ps induced a higher igg2 response compared to mencc immunization. discussion: secondary immune responses are quiet slow. the composition of the ig (iso)type distribution is different between mencc and plain ps and might be of influence on functional titers. although this study indicates that immunological memory was previously induced by a single mencc vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as n. meningitidis. the immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of igg, iga and igm isotypes and by phagocytic and respiratory metabolic activities of granulocytes. results: prime-boost immunization strategy resulted in enhanced production especially dlps-specific igg and igm isotype antibodies in both experimental groups (peak titers 1:3200). igm-igg isotype switch was more pronounced with o-sp. peak values of dlps specific iga isotype were signicantly lower than igg and igm ones (1:800 vs. 1:3200). flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with o-specific polysaccharide and ip final boost (stimulation index was 5. 85 fold of normal control). subcutaneous immunization gave a weaker stimulation: 1. 52 fold of normal control. the second de-oac conjugate exerted different pattern of stimulation, sc intervention was more effective. our results are indicative for immunological effectiveness of novel dlps derived glycoconjugates; thus promising further application in cholera subcellular vaccine. this work was supported by apvv 0032-06 and vega 2/7029/27 grants of the slovak grant agencies. background: the yeast candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. a long-acting, effective and safe vaccine that protects against medically important candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a-1,6-linked d-mannopyranose units and many branches composed of a-1,2 a-1,3 and/or b-1,2-linked mannopyranose units that are connected to the backbone. investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. objective: in this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, bsa) was used. methods: in order to study the immunogenicity of heptamannoside -bsa conjugate as inducer of hummoral and cell-mediated responses, balb/c mice were subcutaneously immunized without adjuvant (6mg oligosaccharide per one conjugate dose) two times in 14 days intervals and then intraperitoneally or subcutaneously boosted. cell-mediated and humoral responses were analyzed on day 14 after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using elisa. results: prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. intraperitoneal boost elicited significantly higher igg and igm levels than subcutaneous boost. immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. introduction: bulgarian immunomodulator respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. it is composed of killed bacterial bodies and lysates of six microbial species (streptococcus pneumoniae, branhamella catarrhalis, streptococcus pyogenes, haemophilus influenzae, staphylococcus aureus, klebsiella pneumoniae). the immune response in respiratory tract includes not only systemic immunity in lungs, but also balt as a part of common mucosal immune system. most animals develop balt after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. therefore, immunostimulation of balt may contribute to more efficient mucosal immunity in respiratory tract. aim: to study balt development in different terms after oral application of respivax in guinea pigs. methods: male guinea pigs (250g-350g) were treated orally with 50 mg respivax five consecutive days. after the last application, on days 3, 7, 10, 14, 28 and 42 six animals on each term were sacrificed and lungs were removed. morphological changes were evaluated on 4mm thick serial sections, stained with hemalauneosin. the populations of cd8, cd4 and b cells were identified on cryosections by using indirect peroxidase immunostaining. zio technique was used to detect intraepithelial dendritic cells (dc). results: balt was not identified in control animals. in the treated group on day 3 subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. the following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. they were composed mainly of cd8 positive cells, which number reached maximum at the end of the second week. on day 14 b cell lymphoid follicles with different size were found. lamina propria was presented by abundant lymphocyte infiltrates, composed of cd4 and cd8 positive cells. on days 28 and 42 the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. numerous intraepithelial dc's were detected in treated animals, comparing to controls in which only a few were identified. conclusion: oral administration of respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of dc and balt development. g. gupta 1 , s. majumdar 1 1 bose institute, molecular medicine, kolkata, indiavisceral leishmaniasis (vl) caused by the protozoan parasite, leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. chemokines, particularly cc chemokines, have been shown to render protection against leishmania infection. there is no clear understanding about the immunoprotective role of cxc-chemokines in vl.in the present study, the comparative potential of cxc chemokines, interferon gamma inducible protein-10 (ip-10) and interleukin-8 (il-8) in restricting leishmania donovani infection via the release of nitric oxide (no) and proinflammatory cytokines was studied in an in vitro model. no, a crucial mediator for ip-10 mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase2 (inos2) expression and was linked to the mapk signaling pathway via antagonistic regulation of p38mapk and erk1/2. further, ip-10 was also able to abrogate the survival of leishmania in an in vivo model of vl by restoration of th1 cytokines and no. thus this study strongly demonstrates that ip-10, like cc chemokines, is involved in rendering a protective response in vl via upregulation of proinflammatory mediators. african trypanosomiaisis (at), known as sleeping sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. at is caused by the protozoan trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. the dna vaccines could be the answer for this disease by being able to induce production of igg antibodies and induce of th1/th2 cytokines mediated by cd8+ t cells and activating cd4+ t helper cells. in this study, we shows that balb-c mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from trypanosoma brucei, named invariant surface glycoprotein (isg), trans-sialidase (tsa), and fosfolipase c (plc) are able to produce igg antibodies anti-trypanosoma. this immunization process was able to control the mortality level when mice were submitted to challenger assay with trypanosoma brucei brucei parasites. in mice co-infected with s. ratti and l. major (nl) neither clearance of l. major nor strongyloides infection was changed. mice co-infected with s. ratti and p. yoelii (nl) showed the same course of parasitaemia as single infected mice. these results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, s. ratti and l. major or p. yoelii. if this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. in the present study ,we evaluated tsa -encoded dna vaccine against l.major in balb/c mice. igg and ifn-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p x 0.05) following immunization and after challenge with leishmania major. il-4 values were increased in all groups, but there was no statistical difference between the groups(p g 0.05) following immunization and after challenge with leishmania major. the immunized mice with the dna vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p x 0.05) in this regard . the survival time of the immunized mice with the vaccine was significantly higher than the control groups (p x 0.05) after the challenge with leishmania major. the immunized mice had significantly lower parasite load comparing to the control mice(p x 0.05). the findings of this study indicated that the tsa -encoded dna vaccine increased the cellular response and induced protection against infection with leishmania in the mice. the tsa -encoded dna vaccine may be an excellent candidate for future vaccine developments against leishmania. there is a lot of evidence showing that bcg vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. in this study the protective effect of autoclaved leishmania major (alm) vaccine in combination of either rectal or subcutaneous bcg on susceptible balb/c mice was evaluated.one month after bcg vaccination, balb/c mice were immunized subcutaneously twice with alm+alum at 3 week intervals. three weeks after booster injection, 5×10 5 stationary phase l. major promastigotes were inoculated subcutaneously in one footpad. immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. high level of interferon gamma was induced in the spleen and significant increase in the serum ratio of igg2a/igg1was observed only in rectal immunized group. rectal immunized mice showed comparable nitric oxide production and inos induction in peritoneal macrophages .the obtained results in rectal bcg vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal bcg immunization against leishmaniasis might be due to the long-lasting induction of type 1 immunity. methods: two groups of balb/c mice were infected by l. tropica. one group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. mice were challenged by l. major in the right footpad after establishment of l. tropica infection. the immune response was evaluated at two intervals: one week and one month after challenge. single cell suspensions were prepared from draining lymph nodes of mice. cells were stimulated by phorbol myristate acetate (pma). cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. the following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (dth) to l. major challenge, production of gamma interferon (ifn-g) and interleukin 10 (il-10), and cellular expression of cd4 and cd25. results: infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of dth and ifn-g, lower levels of cd4+ lymphocytes, and higher protection against l. major challenges. conclusion: intradermal infection of l. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in balb/c mice. therefore, we propose the route of infection as an important variable in this experimental model. this factor should be considered for development of an appropriate experimental model for human l. tropica infections. objectives: many mammals exhibit a periparturient relaxation of immunity (ppri) to gastrointestinal nematode parasites culminating in increased worm burdens. it has been suggested that the extent of ppri may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. however, this effect is often confounded with increased food intake and thus increased energy levels. here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host. the nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian ppri. lactating rats, re-infected with 1,600 infective n. brasiliensis larvae on day 2 post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (me). parasite burdens were assessed by counting worms in the small intestine at day 6 post secondary infection. histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mrna levels in the mesenteric lymph nodes were carried out to assess the host immune status. results: increasing cp supply, but not increased me supply, reduced worm burdens. whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased cp supply increased mucosal mast cell numbers and levels of n. brasiliensis specific antibody (total igg, ige, igg1 and igg2a). this was independent of level of me supply. feeding regime did not affect levels of the type-2 cytokines il-4 and il-13. conclusion: this study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. this anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. this data emphasises the role of immunonutrition in combating infectious disease. protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. background: gp63 is the major surface glycoprotein of leishmania that exhibits protease activity and has an important role in the biology of the parasite. the aim of this study was cloning and expression of gp63 of l.major strain mrho/ir/75/er. methods: l.major promastigotes were grown in rpmi1640 supplemented with 10 % fcs. l.major rna extraction and cdna synthesis were carried out. gp63 gene segment was amplified by specific primers and cloned into ptz57r to construct ptz57r/gp63. the presence of gp63 into ptz57r was confirmed by pcr. then, ptz57r/gp63 was sent to determine the sequence of its nucleotides. after that the gp63 gene segment was sub-cloned into pet32 a (+) expression vector and transformed into e.coli bl21 (de3) plyss and gp63 protein was expressed in presence of 1mm iptg. objectives: the development of a vaccine against malaria caused by plasmodium falciparum is an urgent public health priority. influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. at appropriate antigen doses, seroconversion rates of 100 % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (genton et al., plos one, 2007) . the aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated. the study design was a single centre, randomized, controlled, double-blind, age deescalating trial including 50 volunteers. 10 male volunteers (18 and 45 years) for the adult group, and 40 children of both sexes (5-9 years) were enrolled. subjects received virosomal formulations containing 50 mg of ama 49-c1 (pev301t), an apical membrane antigen-1 derived synthetic phospatidylethanolamine (pe)-peptide conjugate and 10 ug of uk39 (pev302t), a circumsporozoite protein derived synthetic pe-peptide conjugate. comparator groups received the influenza vaccine inflexal v. volunteers received two injections at study days 0, and 90. results: safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. this vaccine showed a very good safety and tolerability profile in all study participants. curcumin dissolved in dmso when administered orally to p.berghei infected mice has been shown to have antimalarial activity, enabling 29 % of the treated mice to survive till 21 days after infection by which time all of the untreated mice had died. under such condition we found that bioavailability of curcumin was only 0.04 % of the amount fed and it remained in circulation in the blood only for 30 minutes post feeding. we therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to 0.4 % ( of the amount fed but it's circulation was sustained till 6 hrs post feeding. under such conditions when 200mgm of curcumin bound to 300mgm of chitosan nano particles were fed one time daily for 10 days post infection to plasmodium yoelii infected mice 100 % of mice were cured and survived atleast for 100 days without any infection and were resistant to reinfection with the same parasite. curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. we believe that it is one of the ways by which curcumin may be killing the parasite. among immune cells, nk and gamma-delta t cells are suspected to play a critical role in the early control of plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. gamma-delta vgamma9vdelta2 t cells, a non-conventional t cell subset specific of primates, are activated and expanded during primary p.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of ifn gamma. furthermore these cells inhibit in vitro growth of p.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an nk and t cell specific cytotoxic molecule has been incriminated. so far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear. to further investigate the anti-parasitic activity of gamma-delta t cells an rnai strategy based on a lentiviral vector approach was undertaken. we demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta t cells. concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta t cells. in addition, we also provide evidence that such a mechanism may occur in infected patients. altogether these data highlight a new mechanism by which gamma-delta t cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta t cells to prevent or cure malaria. the immune system has a number of mechanisms to prevent self-destructive responses. amongst these, regulatory t cells (treg) have the ability to actively suppress effector responses. many questions surround the issue of antigen specificity of treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. the purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. such a model is provided by murine autoimmune hemolytic anemia (aiha) induced by immunisation with cross-reactive rat red blood cells (rbc). mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. the re-establishment of tolerance is associated with the loss of t-cell proliferative responses, and emergence of il-10 responses, to epitopes on the dominant rbc autoantigen, anion exchanger-1 (ae-1, or band 3) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. here we show that transfer of tolerance to naï ve recipients is dependent on ido mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of 1 methyl tryptophan, an inhibitor of ido, were refractory to tolerance and developed hemolytic disease. induction of ido is therefore an important process in antigen-specific tolerance, and initiators of ido activity, including ctla-4 + regulatory t cells or soluble forms of ctla-4, may also be crucial components of this regulatory pathway. consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. objectives: it was shown alpha-fetoprotein (afp) induced immunosuppression of cell-mediated immunity in vivo. our previous work discovered afp-activated mice bone marrow hematopoietic stem cells (hscs) suppressed effector reactions of cell-mediated immunity in vitro. we investigated relationship existed between afp-induced hscs suppressor activity and immunosuppression of cell-mediated immunity during afp-produced teratocarcinoma development. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and rna interference (rnai) were used in this work. results: as a result, there was a negative correlation (r medium =-0.81) between dynamics of hscs suppressor activity elevation in spleen and inhibition of nk cells, nkt cells and cd8 + t cells cytotoxic activities ex vivo during tumor growth. besides, the inhibition of spontaneous and induced cytokines productions such as ifng, tnf-a and tnf-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as tgf-b 1 (r medium =-0.74) and il-10 (r medium =-0.65) in isolated splenic hscs ex vivo. analysis of effector cd4 + t cells in spleen showed decrease of t h 1 cells quantity and simultaneous t h 2 cells number increase during teratocarcinoma development. moreover, it were found elevated numbers of cd4 + cd25 + ctla-4 + -and cd4 + cd25 -il-10 + il-4regulatory t cells in spleen as well as increasing suppressor activity of isolated regulatory t cells ex vivo. number boost kinetics of t h 1, t h 2 and regulatory t cells were correlated (r th1 =-0.74, r th2 =0.86 and r th3 =0.80 and r tr1 =0.68) with kinetics of hscs suppressor activity level. in addition, dynamics of regulatory t cells activity were linear (r th3 =0.84 and r tr1 =0.76) to hscs suppressor activity level in spleen during tumor growth. quantities of tgf-b1-and il-10-produced hscs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. however, inhibition of afp expression by rnai caused to inhibition as immunosuppression activity of hscs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. conclusion: thus, hscs suppression activity is correlated with changes in cell-mediated immunity during endogenous afp productions by teratocarcinoma cells and may play a role in afp-mediated dysfunction of normal immunoregulation during afp-produced tumor development. syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. our studies showed that naturally aquired s.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected cba mice. while the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. beside cellular changes, we also evaluated the impact of the s.obvelata on mitogen-activated protein kinases (mapk) signaling in bone marrow cells and found that infection upregulated all three mapk families, p38, jnk and erk. additionally, s.obvelata enhanced the expression of mrna for the inducible nitric oxide synthase (inos). to evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin-17, t cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. bone marrow myeloid and erythroid progenitors from s.obvelata-infected mice displayed altered sensitivity to il-17, as compared to non-infected controls. the infection also altered the effect of il-17 on mapk activation by preventing its stimulating effect on p38 mapk. moreover, in s.obvelata-infected animals il-17 markedly down-regulated the expression of both inos and constitutive, endothelial (e)nos, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. il-17 induced no production through the activation of both inos and enos. besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. 2.-validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified mc were serially diluted from 1 ⁄2 to 1/64 with a normal serum pool. 3.-implementation as a standard method for analysis of the mc in patients with paraproteinemia. the method showed good linearity: r 2 g 0.98. the analytical range was from 1 g/l to 70 g/l. the coefficient of variation (cv) was x 10 % for [mc] n 1 g/dl, and x 20 % for [mc] x 1 g/dl. this procedure was successfully implemented to quantify the mc in 1100 serum samples between march 2008 and february, 2009. among these samples, we have quantified light chains, heavy chains, igd and biclonal paraproteinemias. conclusions: 1. we have developed a simple, reproducible and low-cost method to quantify the mc using standard analyses of serum protein electrophoresis (spe), serum albumin, and densitometric quantification of mc and albumin regions. 2. the procedure allows monitorization of the mc in patients at diagnosis, after therapy, and evaluation of complete remission. objectives: direct influence of alpha-fetoprotein (afp) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (hscs) was detected in our previous work in vitro. we investigated possible role of endogenous-produced afp in induction hscs immunosuppressor activity at tumor-bearing mice. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric elisa, methods of molecular biology and rna interference (rnai) were used in this work. results: our results demonstrated afp endogenous synthesis adduced to elevation of immunosuppression activity of hscs in bone marrow. this afp effect becomes developed from 7 day and reached plateau level after 30 day of teratocarcinoma insertion. moreover, cd34 + cd38cells showed in spleen and main lymph nodes from 15 day and achieved plateau level after 60 day of teratocarcinoma growth. however, immunosuppression activities of purified hscs from spleen and lymph nodes discovered at 22 day and had a maximum pick at 60 day of teratocarcinoma inoculation. besides, immunosuppression activity of hscs from spleen and lymph nodes was more than 1,5 times lower than in bone marrow in the same period of tumor development. isolated hscs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as tgf-b 1 , il-10, pge 2 and no. inhibition of such suppressor factors lead to levelling of hscs immunosuppression activity ex vivo. kinetic of hscs quantity and activity had significant correlation (r cell number =0.81 and r activity =0.92) with afp level dynamics in blood serum. in addition, inhibition of afp expression by rnai caused to diminishing of hscs immunosuppression activity as well as hscs appearance in spleen and lymph nodes. conclusion: therefore, afp plays role not only specific inducer of hscs immunosuppression activity but also as a factor of activated hscs penetration into spleen and lymph nodes during afp-produced tumor development. cd40-ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated cd40l is cleaved to soluble (s)cd40l. we sought to examine the levels of scd40l in platelet concentrates (pcs) having led to an acute transfusion reactions (atr), and to test for its biological effect on b-lymphocytes. we recorded 5 atr episodes that could lead to investigation of residual platelets in container. two fractions of aliquots from each pc (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; scd40l -along with other products -were assayed by quantitative elisa. levels of il8, cd62p and pdgf-ab in pc supernatants and lysates from pcs associated with atr were similar to that in controls. supernatants of pcs associated with an atr contained higher scd40l levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l . to examine if scd40l was biologically active, we stimulated purified b cells recovered from healthy blood donors and exposed those normal b cells to supernatants and cell lysates of pcs implicated in atr, or control material, and we measured il-6 secretion. the il-6 concentration was consistently below 5-10 pg/ml in pc supernatants and lysates, and unstimulated b cells did not secrete detectable levels of il-6. the addition of supernatant from atr-associated pc samples to purified b cells consistently resulted in sustained il-6 production over control (p x 0.05) at d2 after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l (p x 0.05). pre-incubation of b cells with cd40-blocking antibodies substantially abrogated il-6 secretion, unlike isotype-matched control. the partial blocking of cd40 binding on cd40 + b cells strongly suggests a potentially synergistic role in b cells for cytokines other than scd40l (under investigation) and indicates a sustained role for pc-derived scd40l. these data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of scd40l by collected/stored cpa. objectives: there is accumulating evidence for a role of natural killer (nk) cells in the antitumor response against hematological malignances. nk cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. analysis of activating receptor pathways in nk cells has revealed a dominant role for natural cytotoxic receptors (ncrs) and dnax-accessory molecule-1 (dnam-1) in the lysis of acute myeloid leukemia (aml) blasts. here, we investigate the expression of these activating receptors on nk cells from aml patients stratified by age. methods: we analyses by flow cytometry peripheral blood mononuclear cells (pbmcs) from 30 aml patients before specific anti-leukemia therapy and 47 healthy donors. all results were analyzed statistically using spss version 15. results: aml patients under 65 years showed a significant reduction in the expression of dnam-1, nkp30 and nkp46 compared with age-matched controls. both healthy individuals and aml patients older than 65 years showed a reduction of these receptors compared with young donors. in contrast, we have found that nkp44 expression was increased in some patients of aml. on the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or fab subtype. in addition, an inverse correlation in the expression of dnam-1 on nk cells and its ligand cd112 on aml blasts has been found in aml patients under 65 years. to analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy nk cells. the initial recognition of aml cells by nk cells may represent a crucial process to prevent tumor development. here we described for the first time, a decrease of dnam-1 expression on aml patients and confirm previous reports showing a significant decrease of nkp30 and nkp46 on aml-nk cells. altogether, these alterations of the major receptors involved in nk cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival. a. stelmaszczyk-emmel 1 , e. gorska 1 , u. demkow 1 , m. wasik 1 1 medical university of warsaw, department of laboratory diagnostics and clinical immunology of developmental age, warsaw, polandglucocorticosteroids are often used in leukemia treatment. their therapeutic use is limited due to several side effects. one of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. dexamethasone is used in schedule of children's all-b treatment and the response on glucocorticosteroids therapy is very important. the aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in all-b and their tendency to begin apoptosis. the study involved 10 children with all-b. bone marrow cells isolated by centrifugation on histopaque at the day of diagnosis were cultured for 2 or 48 hours with or without dexamethasone in concentration 10 -6 m. analysis of: p-gp surface expression, p-gp function (rhodamine 123 test), phi (carboxy-snarf) and apoptosis test (annexin-v and pi test) were performed with the use of flow cytometer coulter epics xl. for statistical analysis nonparametric wilcoxon test was used.the results showed that p-gp expression on lymphoblasts was 14,52 %±11,32. after 48 hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. average percentage of lymphoblasts with rhodamine 123 efflux, which characterized p-gp activity, was 2,62 %±3,15. after 2 hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine 123 (4,31 %±4,03, p=0,015). average phi in lymphoblasts was 7,78±0,19. acidification of cells incubated 2 hours with dexamethasone was seen in 10-25 % percentage of cells 17) . rest of lymphoblasts showed alkalization (phi -8,00).the percentage of lymphoblasts in early stage of apoptosis after 48 hours incubation with dexamethasone (annexin-v test) was higher than in control cells (19,34 % vs 14,13 %; p=0,01). we concluded that dexamethasone does not influence surface p-gp expression on lymphoblasts of patients with all b but significantly increases activation of this protein. functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of p-gp is not identical with its activity. moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. those effects may contribute to the treatment outcome. . however, small numbers of clonal pc can also be detected in the peripheral blood (pb) of the majority of patients and, in a minority of cases, mm is transformed into pc leukaemia (pcl). here, we describe that tumoral pc can express cd49d/cd29 integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the bm, respectively. objectives: to evaluate the activation state of cd29 on malignant pc from bm and pb, as well as its regulation. methods: pc and active integrin expression on these cells were detected with anti-cd38 and anti-cd29 (clon huts21) moab, respectively, by flow cytometry. to study the integrin activation with divalent cations and pc index proliferation (brdu+ cells), we used short-term pc cultures (18 hours). results: cd29 active form was expressed in the majority of normal and tumoral bm pc from healthy subjects (67.3±6.6, n=9) and mm patients in the early stages of the disease (32.6±7.5, n=17). in these cells, huts21 epitope was clearly upregulated by mn 2+ . in contrast, circulating pc were almost all huts21 negative, and levels did not significantly augment when these cells were exposed to mn 2+ . moreover, not only pb but also bm malignant cells from pcl patients were also huts21 negative and divalent cation refractory. it was also observed that pc from pcl patients showed an increased proliferative index (2.35±0.9 brdu+ cells, n=3) in comparison to pc from mm patients in the first stages of disease (1.3±0.2 brdu+ cells, n=5). these results suggest that the active form of cd29 must be expressed on pc to retain these cells within the bm environment. moreover, its downregulation is associated with increased numbers of circulating pc and disease progression. multipotent mesenchymal stem cells derived from (hucb) represent promising candidates for the development of future cellular therapy strategies. they are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues. in the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hmsc) starting from diagnostic samples of bone marrow aspirates and the cord blood (cb). the protocols differed in the presence of either 10 % fetal bovine serum (fbs) with and without fgf2,or 10 % human platelet lysate (hpl), 5 %hpl, (10 % fbs + 10 % hpl), (10 % fbs + 5 % hpl). we obtained a significantly better expansion with hpl, compared to cells with a selected batch of fbs and in fewer days.in the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. we compared the quality and the quantity of protein expression before and after differentiation (day 18). two bm and two cb differentially expressed spots were observed between the two groups (before and after differentiation).we noted the low pourcentage of hmsc in cb samples: in ten samples, only two made msc colonies as in bm samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red s staining, for adipogenic differentiation, the cells were stained with oil red o to visualize lipids droplets. background: inherited bone marrow failure syndromes (ibmfs) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. activation of p53 tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. we studied the presence of p53 dna (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (fa) and those with acquired aplastic anemia (aaa). subjects and methods: this is a cross sectional study that involved: 1) ten cases with fa diagnosed on the basis of dna breakage analysis, 2) ten cases with aaa, and 3) ten normal control cases. the presence of p53 dna was measured in both bone marrow and peripheral blood samples using a real-time quantitative pcr by taqman assay. results: p53 dna was demonstrated in bone marrow of 90 % of children with fa, compared to 10 % in children with aaa (p x 0.001), while, no p53 dna was seen in normal control. a positive correlation between dna breakage and presence of p53 dna was seen in bone marrow from fa (p x 0.02, r0.81). the presence of p53 tumor suppressor gene by real time pcr in bone marrow of fa may represent an early indicator of significant dna genetic alteration in those patients. key: cord-005453-4057qib7 authors: nan title: the 45th annual meeting of the european society for blood and marrow transplantation: physicians – poster session date: 2019-07-03 journal: bone marrow transplant doi: 10.1038/s41409-019-0559-4 sha: doc_id: 5453 cord_uid: 4057qib7 nan background: allogeneic hematopoietic stem cell transplantation is routinely offered to patients with high-risk or advanced all in the hopes of improving outcomes. use of truly non-myeloablative (nma) conditioning reduces toxicity in other contexts but outcome data for all patients after nma transplants is lacking. we report the outcomes of 31 patients with all transplanted using a nma conditioning without t cell depletion. methods: first transplant patients between october 2006 and june 2018 were reviewed. these were consecutive patients until 2015 then only those considered unfit for fmc conditioning as per the ukall 2014 protocol. all patients were conditioned with fludarabine 25mg/m 2 /day for 5 days and cyclophosphamide 1g/m 2 /day for 2 days. short course mtx and ciclosporin were used for gvhd prophylaxis. standard supportive care was employed. thirty-one patients with a median age of 43 (23-67) met the criteria for this case review. 30 had b-all and 10 were philadelphia chromosome positive. 24 patients (77%) had high risk disease by standard diagnostic criteria. 27 (87%) were in first complete remission (cr1). matched sibling donors were used in 13 instances with the remaining being fully matched unrelated donors. 58% of patients had a hct-ci score of 0, 32% a score of 1 or 2 with 3 patients having a score of 3 or higher. median cd34 dose was 5.3 x 10 6 /kg (0.93-34.12) with a median cd3 dose of 2.13 x 10 8 / kg (0.12-7.37) results: trm was low at 7% at 1 year and 11% at 2 and 3 years respectively. no factors included in a univariate analysis (which included age, diagnosis, disease status, hct-ci, donor type, cmv risk and cell dose) significantly impacted trm. the incidence of classical acute (a) gvhd grade 2-4 and 3-4 was 18% and 8% by day 100 and 29% and 13% by day 180 if late onset agvhd is included. 24 out of 30 eligible patients developed chronic gvhd of any stage. relapse incidence was low (22% at 3 years in all patients, 17% in cr1 patients) and was not impacted by any pre-transplant factors including positive mrd post phase 2 induction (present in 6 patients). notably, in univariate analysis relapse was significantly lower in patients who developed chronic gvhd. background: allogeneic stem cell transplantation (allosct) is the treatment of choice for many patients (pts) suffering from acute myeloid leukemia (aml). the graft vs. leukemia effect (gvl), applied by immunocompetent cells of donor origin, is the most important effector mechanism for the eradication of leukemia, the presentation of leukemic or allospecific antigens by malignant blasts is regarded as a crucial trigger for an effective allogeneic immune response. conversely, insufficient stimulatory capacity by the leukemic blasts is thought to be a relevant escape mechanism from cellular immunotherapy (allosct or donor-lymphocyte infusion (dli)). the purpose was to test, whether the ability of malignant blasts to differentiate in vitro towards dendritic cells of leukemic origin (dc leu ) is associated with response to allosct or outcome after immunotherapy (second allosct or dli) for post-transplant relapse in aml. methods: leukemic blasts were isolated from peripheral blood (pb) or bone marrow (bm) samples of aml patients before allosct (n=47) or at relapse after allosct (n=22). a panel of 6 different assays was used to generate dc leu in vitro (5 of them containing gm-csf). finally, in vitro results were correlated with clinical characteristics and outcome of patients treated with donor lymphocyte infusion and/or allosct. results: dc leu could be generated in vitro from all 69 samples. when correlating proportions of dc-subtypes generated ex vivo with clinical data, significantly higher mean proportions of dc leu in the dc-fraction were found in responders vs. non-responders to immunotherapy (76.8% vs 58.8%,p=0.006, range:13%-99%). vice versa, the chance for response to immunotherapy was significantly higher, if a dc leu /dc ratio of >=50% could be reached in vivo (p=0.004). those patientswere characterized by a longer time to relapse (p=0.04) and by a higher probability for leukemia-free survival (p=0.005). similarly, generation of higher amounts (>8%, p=0.04) of dc leu in the mnc-fraction, and generation of more mature dc (>47% cd83+, p=0.03 using the best gm-csf containing assay) were associated with a longer time to relapse in the respective patients. moreover, overall survival was improved, if >70% dc leu /dc could be generated with the best gm-csf containing assay (p=0.048). conclusions: in vitro generation of dc/dc leu from leukemic blasts obtained in active stages of aml before allosct or at relapse post transplant were associated with clinical outcome. this observation supports a role of antigen presentation by leukemic cells for an allogeneic immune response in aml. disclosure: nothing to declare background: the role of autologous hematopoetic cell transplantation (hct) in the treatment of aml is not clear. trials in the past have shown that autologous hct consolidation lowers the risk of relapse, however the magnitude of this effect is limited . autologous hct is advocated in patients with aml with lower genetic risk in cr1.many of these patients will eventually relapse and will undergo reinduction followed by allogeneic hct in cr2. methods: the aims of this study is to analyze outcome of allogeneic hct performed in cr2 comparing patients with prior consolidation by autologous hct vs. patients with chemotherapy consolidation. primary outcome is non relapse mortality (nrm) of allogeneic hct in cr2 in patients with, or without prior autologous hct in cr1. secondary outcomes include leukemia free survival (lfs), relapse rate (ri), graft versus host disease free relapse free survival (grfs), overall survival (os), and treatment related toxicities. results: 2619 adult patients reigstered with the alwp of the ebmt with de novo aml were included, receiving a first allogeneic hct in cr2, in 2000 cr2, in -2017 or without (n=2202) prior autologous hct. patient and transplant characteristics are shown in the table. patient groups were not entirely comparable, patients with prior autologous hct were younger, had less often a favorable cytogenetic profile, had more commonly donors other than matched siblings and more often received reduced intensity conditioning (ric) as compared to mac conditioning. univariate outcomes are shown in the table with slightly higher nrm risks in patients with prior autologous hct consolidation. in multivariate analysis nrm risks in patients with prior autologous hct were 1.34 (1.07-1.67), p=0.01 after adjustment for patient age, cytogenetic risk category, year of transplant, donor type, conditioning intensity, sex matching, time from diagnosis to relapse and time from relapse to allogeneic hct as compared to patients with chemotherapy consolidation. similarly, risks of events in lfs and grfs were higher with prior autologous hct, 1.17 (1.01-1.35 ), p=0.03 and 1.18 (1.03-1.35 ) p= 0.02, respectively, risk of death was also higher 1.13 (0.974-1.32) p=0.1 but this was not statistically significant. conclusions: we may conclude that some of the advantages of potentially higher anti-leukemic activity of high dose chemotherapy and autologous hct when given to patients with aml in cr1(as was shown in a randomized trial by vellenga e et al with lower relapse and higher lfs by approximately 10% but no significant differences in overall survival) may be lost by higher toxicity of allogeneic hct in cr2 in case of subsequent relapse. background: although relapse is a major cause of mortality in patients receiving allogeneic hematopoietic cell transplantation (hct) for acute leukemia, limited and conflicting data exist on extramedullary relapse (emr). we aimed to describe the incidence, risk factors, outcomes and prognosis in relapsed hct recipients. methods: we retrospectively reviewed charts of consecutive allogeneic hct recipients transplanted in our center with the indication of acute leukemia (7/1990-7/ 2018). we recorded: age, gender, disease, previous extramedullary involvement, phase at transplant, type of transplant, donor, conditioning, graft-versus-host-disease (gvhd), infections, treatment-related mortality and relapse mortality. in patients with extramedullary relapses, additional data on clinical manifestations, imaging, cerebrospinal fluid testing, histopathology and management were additionally documented. incidence of isolated emr (iemr) and bone marrow relapse (bmr) was calculated using cumulative incidence (ci) analysis, with each and treatment-related mortality considered a competing risk. results: among 554 allohct recipients followed for 1.8 (0.04-27.75 ) years, 61 (11%) patients presented with emr. the majority of emrs involved the central nervous system (cns, 56%). isolated emr was observed in 38 patients at 9.5 (1.8-67.3) months. 10-year cumulative incidence (ci) of 10.5% for iemr was associated only with pre-transplant advanced disease phase (p< 0.001). bmr was observed in 149 patients at 9 (0.3-276 months), with a 10-year ci of 34.8%. in the multivariate analysis, bmr ci was independently associated with fungal infections (p< 0.001), pre-transplant disease phase (p< 0.001) and lines of treatment (p=0.042). 10-year trm of our whole cohort was 33.2%. the majority of iemr and bmr (75% and 81%, respectively) patients received systemic treatment combined with local radiation for iemr (26%) and donor lymphocyte infusions (dlis, 16% and 28% respectively) when feasible. extensive chronic gvhd was recorded in 47% of iemr and 48% of bmr patients. outcomes were poor in iemr, with 10-year overall survival (os) of 18.3%. favorable os in iemr was associated only with sibling donors (p=0.049) and not with other factors, such as treatment with dlis or presence of chronic gvhd. similarly poor outcomes (10year os of 19.1%) were observed in bmr. favorable os was independently associated only with the diagnosis of aml (p=0.050) and absence of bacterial infections (p=0.049). in the whole cohort, both iemr and bmr were independent unfavorable predictors of os (p< 0.001) along with extensive chronic gvhd (p=0.012). conclusions: in a large population with long-term follow-up, incidence of iemr was relatively high, developed at the late post-transplant period and associated only with disease phase at transplant. furthermore, iemr and bmr conferred similarly poor outcomes despite systemic treatment or extensive chronic gvhd. these independent predictors of survival highlight the unmet clinical need of novel approaches either as maintenance or treatment to reduce extramedullary or systemic relapse post allohct for acute leukemia. disclosure: no competing financial interest. impact of t-cell depletion on outcome in patients undergoing allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia background: after a diagnosis of acute myeloid leukemia (aml) the majority of patients (pts) who achieve complete remission (cr) eventually relapse, with only approximately 30% of pts maintaining cr for 3 years or longer. late relapses (after 3 years in cr) occur rarely (6-10%) in pts receiving hsct in cr1 and late effects are followed up by routine surveillance as well as preventative measures. the purpose of this study was to investigate long-term outcomes in pts with diagnosis of aml undergoing hsct at our institution in cr1. methods: a standardized follow-up of hsct-survivors is applied at our center. we analyzed 116 adult pts with aml in cr1 consecutively transplanted between january 2004 and december 2016 at our institution. a written consent was given for the use of medical records for research. a landmark analysis was adopted for patients in cr at 2-y after hsct (ltcr -long-term cr). results: ltcr was achieved after hsct in 91/116 patients (male 55, female 36) transplanted in cr1. the median follow-up was 6 years and the median age at transplant 52 years (r 20-72). the selected donor was a family haploidentical relative in 29 cases, an hla identical relative in 21, a match unrelated donor in 39 and a cordblood in 2. in this cohort of ltcr, the 5-year overall survival was 92% (95% ci 83-96). cumulative incidence of relapseevaluated in competing risk with transplant related mortality (trm) -and trm -evaluated in competing risk with relapse -were respectively 7% (95% ci 1-23) and 2% for the cr1 cohort. the event-free-survival (efs) was 91% (95% ci 83-95). the causes of death were relapse (6/10 pts), second cancer (3/10 pts) and sepsis (1/10 pts). the 5-year incidence of dyslipidemia -defined as cholesterol >/= 200 mg/dl, and/or ldl >/= 115 mg/dl, and/or triglycerides >/= 150 mg/dl or need for specific treatment -was 24%. the 5-year incidence of osteopenia / osteoporosisdefined as t-score lower than -1 and greater than -2.5 and t-score lower than 2.5 respectively -was 38%. the 5-year incidence of second cancer was 11%: 10 nonmelanoma skin cancer, 2 lung carcinoma, 3 cervical intraepithelial neoplasm, 1 thyroid cancer, 1 gastric cancer and 1 colon cancer. the 2-year incidence of chronic moderate-severe gvhd was 27% (95% ci 13-38), with the latest diagnosis performed on day 570. of note, 4/24 pts are still on active treatment at last follow-up. conclusions: relapse incidence is low for patient that reached ltcr: patients in cr1 at transplant can obtain excellent os and efs once reached the target of ltcr. a proactive long-term follow-up and strategy of counseling are essential to keep at best quality the survival advantage offered by hsct in patients with aml in cr1. disclosure: chiara bonini has research contract with intellia therapeutics. the other authors declare that they have no conflicts of interest. background: relapse, graft-versus-host disease (gvhd) and gvhd-associated mortality are major obstacles to success of transplantation from unrelated (mud) donors in children with acute leukemia (al). negative depletion of αβ t cells and cd19+ b lymphocytes, conserves the mature donor-derived natural killer cells and γδ t cells in the graft, may improve gvhd control, immune reconstitution and prevent the relapse. we present a retrospect analyses of a cohort of pts with al in cr transplanted from mud with depletion. methods: a total of 59 children with acute leukemia (34 aml, 25 all, 21 female, 38 male, median age 8,5y) underwent allo hsct from matched unrelated donor between june 2012 and july 2017. all pts were in complete remission (cr1=34, cr2=23, cr>2=2). all pts, except one, received treosulfan-based conditioning. either melphalan (n=56) or thiophosphamide (n=2) or etoposide (n=1) were added as a second agent. fludarabine was used in all pts. two types of gvhd prophylaxis were used: type 1 (n=35): hatg 50 mg/kg and post-hsct tacro/mtx (n=30) or without prophylaxis (n=5); type 2 (n=24): thymoglobulin(ratg) 5mg/kg, rituximab 200mg/ m 2 with either bortezomib on days +2, +5 (n=21) or tacro/ mtx (n=3) . aβ t cell depletion with clinimacs was used in all cases. the median dose of cd34+ cells was 9 x10 6 / kg, aβ t cells -15 x10 3 /kg. median time of follow-up for survivors was 5,3 years (range, 2, 3 -6,5) . results: primary engraftment was achieved in 100% pts., the median time to neutrophil and platelet recovery was 15 and 14 days, respectively. all evaluable pts achieved sustained complete donor chimerism by day +30. early (100 day) mortality was 3,4% (1pt -bacterial sepsis, 1pt -adv fulminant hepatitis), 5-years overall ptrm at 4 years was 13,5% (95%ci:7-26). six late trm events were due to: viral infection in 2 pts (cmv=1, adv+cmv=1), bacterial sepsis in 2 pts and 2 pts had bacterial and viral infection, all late deaths were associated with cgvhd and prolonged corticosteroid therapy. ci of acute gvhd grades ii-iv was 36% (95% ci: 25-50), acute gvhd grades iii-iv 3,7% (95% ci :1,5-14,5) . ci of cgvhd was 27%(95%ci:18-41). regimen 2 was more effective in prevention of agvhd ii-iv in comparison with regimen 1: 8% (95% ci: 2,2-30) vs 45,7%, respectively, p=0,04. all events with acute gvhd grades iii-iv had pts with regimen 1. ratg was also effective in prevention of cgvhd: ci at 4 years after hsct was 12,5% vs. 37%, respectively, p=0,04. cumulative incidence of relapse was 25% (95%ci: 14-50) without difference between ratg and hatg. event-free survival (efs) (event=death or relapse) at 4 years was 61% (95%ci: 48-73), overall survival 59%(95% ci:47-72), there were no difference between age and diagnosis. conclusions: we confirm that the depletion of tcrαβ +/cd19+ t lymphocytes from the graft ensures high engraftment rate. transplant-related mortality is caused by infections, mostly associated with cases of chronic gvhd. gvhd prophylaxis including ratg/rituximab/ bortezomib improves gvhd control in recipients of tcrαβ+/cd19+depleted grafts in comparison to hatg/ tacro/mtx apparently without loss of anti-leukemic activity. disclosure results: at baseline, r/r all with emd and lbl were diagnosed in 7 and 11 ino patients and 5 and 6 sc patients. median (range) age of the ino and sc patients was 55.5 (20-78) and 47.0 (28-64) years, with 8/18 (44.4%) and 8/11 (72.7%) males, respectively. the rate of cr/cri was significantly higher in the ino group (12/18 [66.7%] , 95% confidence interval [ci] : 41.0-86.7) compared with sc (2/11 [18.2%], 95% ci: 2.3-51.8; p=0.0144) (table) . allogeneic hematopoietic stem cell transplantation was carried out in 6/ 18 (33.3%) ino and 2/11 (18.2%) sc patients prior to any post-study induction therapy. the pfs hazard ratio [hr] was 0.502 (97.5% ci: 0.203-1.240; p=0.0410), with median pfs of 4.4 (95% ci: 1.9-7.1) months among ino and 1.6 (95% ci: 0.8-3.7) months in sc patients. the os hr was 0.661 (97.5% ci: 0.269-1.621; p=0.1478), with median os of 5.9 (95% ci: 3.4-9.4) months in ino versus 5.5 (95% ci: 2.1-6.7) months in sc patients (figure) . all patients had adverse events (aes). serious aes occurred in 10/18 (55.6%) ino and 5/11 (45.5%) sc patients; 4 (22.2%) ino and 0 sc patients had grade 5 ae. one (1/15, 6 .7%) patient in the ino group died from veno occlusive disease. conclusions: among r/r all patients with emd and lbl, improvement in remission rates, transplant rates, and progression free survival was shown in the ino group versus the sc group. although patient numbers were small and limited the ability for a robust comparison, these results support the use of ino in patients in this difficult to treat population with r/r all and emd or lbl. background: bcr-abl-targeted tyrosine kinase inhibitors (tki) revolutionized the outcome of patients inflicted with ph+ b-all. moreover, addition of tki may be relevant strategy for ph-like all patients. methods: we hypothesized that overcoming the bm microenvironment-mediated protection of all cells from tki-mediated apoptosis may further enhance the responsiveness to tki therapy. results: in vitro treatment of bcr-abl-positive all cell lines nalm1 and nalm20) with dasatinib resulted in significant dose-dependent cell growth inhibition, with ic50 of 10-15 nm (p< 0.01). furthermore, dasatinib exhibited significant growth suppression of bcr-abl -negative all cells (nalm6 and reh), with ic50 of 250 nm and 185 nm, respectively. however, when cocultured with bone marrow stromal cells (bmscs), dasatinib-mediated effect was abrogated in both ph-and ph+ all cells. furthermore, dasatinib treatment promoted significant upregulation of chemokine receptor cxcr4, on both mrna and cell surface levels. elevated cxcr4 expression was accompanied by increased responsiveness of all cells to cxcl12 stimulation, resulting in strong and sustained phosphorylation of erk1/2 and akt and increased adhesion capacity to bmscs. therefore, dasatinib-induced upregulation of cxcr4 promotes stroma-mediated survival advantage of all cells upon tki therapy. next, in order to overcome the cxcr4-mediated stromal protection, we choose to combine dasatinib with the histone deacetylase inhibitor panobinostat, for its known ability to deplete cxcr4 in aml cells. single-agent treatment with panobinostat demonstrated significant inhibition of ph-and ph+ all cell growth at low nanomolar concentrations (p< 0.01). importantly, combination of panobinostat with dasatinib synergized (ci< 0.5), effectively overcoming the protection provided by bmscs and inducing the apoptosis of ph-and ph+ all cells, as demonstrated by phosphatidylserine externalization, mitochondrial depolarization and dna fragmentation. furthermore, combining panobinostat with dasatinib significantly reduced cxcr4 surface levels in ph-and ph+ all cells. accordingly, cxcl12mediated responses, including erk1/2 and akt activation and adhesion to bmscs were significantly reduced upon combined panobinostat/dasatinib treatment. these data indicate that panobinostat effectively suppresses both basal and dasatinib-induced cxcr4 expression and function in all cells overcoming stroma-mediated resistance to dasatinib. to determine the molecular mechanism, we performed gene and protein expression analysis. panobinostat, alone or in combination with dasatinib, significantly down-regulated the protein levels of calcineurin, a serine-threonine protein phosphatase previously implicated in t-all and b-all pathogenesis, as well as of nfatc1, a critical effector of the calcineurin signaling cascade, and nfatc1-regulated target genes. it was previously found that calcineurin signaling positively regulates cxcr4 expression in t lymphocytes. additionally, cyclosporin a (csa) decreased both basal and dasatinib-induced cxcr4 surface levels in all cells, overcoming the protection of the bmscs which result in potentiation of the cytotoxic effect of dasatininb and panobinostat. combining csa with panobinostat resulted in deeper suppression of nfatc1-regulated target genes. we thus link the effect of panobinostat with calcineurin-dependent downregulation of cxcr4, blocking the ability of the leukemic cells to respond to cxcl12mediated stromal support. conclusions: taken together, our results identify calcineurin signaling pathway as a novel target of panobinostat in all cells and indicate that hdac inhibition with panobinostat may be effective strategy for facilitating the anti-leukemic activity of tki therapy. disclosure: nothing to disclose background: the treatment of relapsed/refractory acute lymphoblastic leukemia (rr-all) remains a clinical challenge with a generally dismal prognosis. allo-sct using a sequential conditioning ("flamsa"-like regimen) has shown promising results in relapsed/refractory aml, but little is known about the efficacy of this procedure in rr-all. methods: we identified 115 adult patients (45% females; median age: 38 y; range, 18-66) with all in primary refractory phase (26%) or in relapse (74%), allografted between 2000 and 2017 from a matched sibling (31%), matched unrelated (58%) or haploidentical donor (11%) at ebmt participating centers. almost half (49%) of the patients had t-all and 23% had a positive philadelphia chromosome. six patients (5%) underwent a previous autotransplant. karnofsky score was above 90 in 52% of patients. conditioning was myeloablative (mac) with high dose tbi in 30% of patients, reduced intensity (ric) including low dose tbi in 22%, or with chemotherapy alone in 48%. in vivo t cell depletion was performed in 77 cases (69%). most patients (74%) and about half of the donors (47%) were cmv positive. 14% of patients were males who received a graft from a female donor. the median follow-up was 37 (range, 13-111) months. results: overall, 14 patients (13%) failed to engraft, 18 (16%) died within 100 days after allo-sct without relapse, and 64 (56%) could achieve complete remission. at day 100, the cumulative incidences of grade ii-iv and grade iii-iv acute gvhd were 30% and 17%, respectively. the 2year cumulative incidences of chronic and extensive chronic gvhd were 25% and 11%, respectively. the 2-year relapse incidence (ri) and non-relapse mortality (nrm) were 45% and 41%, respectively. the 2-year leukemia free survival (lfs), overall survival (os) and gvhd relapsefree survival (grfs) were 14%, 17% and 12%, respectively. in a multivariable cox analysis, karnofosky score below 90 negatively affected ri, lfs, os and grfs. also, conditioning with chemotherapy alone, compared to tbibased conditioning, negatively affected relapse rates (hr=4.13; p=0.0006), lfs (hr=2.32; p=0.004) and os (hr=2.29; p=0.006). conclusions: allo-sct using a sequential conditioning regimen is proposed by different teams in rr-all, and could be an option, especially when considering a tbibased regimen. however, the overall 2-year lfs of 14% suggests that these patients still face extremely dismal outcomes, highlighting that other therapies (e.g. bite antibodies, inotuzumab, car t cells) need to be combined prior and/or after allo-sct in order to further improve outcome. disclosure: no conflict of interest, no funding received chemotherapy courses, only 2 pts were not treated: 1 pts for the worsening of the general status and the other for invasive fungal infection. results: forty-three pts (42%) were in complete remission (cr) and negative minimal residual disease (mrd) at the time of hsct; 16 pts were in active disease (16%), and 44 (42%) showed a morphological cr with positive mrd. 41pts (40%) developed chronic graft versus-host disease (cgvhd) as followed: 23pts (22%) mild, 17pts (16%) moderate, and only 1 sever grade respectfully. only 1 patient developed cgvhd after dli. the overall leukemia free survival (lfs) time was 16 months, the absence of cgvhd (hazard ratio -hr: 5,968; p = 0,01) and the pre-hsct disease status (hr 2,353; p = 0,028) were the most important factors on lfs. all pts treated with chemo-based regimens died due to progression or infective complications. 1 patient of aza/dli group is still alive with a extramedullary relapse; 2 pts treated with bl/dli are in cr. os was better for the dli group compared to the chemotherapy group (28 vs 2 months respectfully; p < 0,001). conclusions: dli after allo-hsct has exhibited definite anti-leukemic effects in post-transplant patients. bl and aza were reported to increase dli's graft vs-leukemia (gvl) effect. although cgvhd could be the most important protective factor against the relapse but it remains the main cause of morbidity. maximising the gvl effect without putting the patient at risk of gvhd still represents an unmet need. our data show that the combination of either bl or aza with dli infusion is safe and might represent an improvement in disease control in the early phase of relapse. disclosure: nothing to declare p018 increased detection of (leukemiaspecific) adaptive and innate immune-reactive cells under treatment of amldiseased rats and one therapy-refractory aml-patient with blastmodulating, clinically approved response modifiers (pg-e2,kit-k) or +pge1 (kit-m),patent 102014014993) convert myeloid blasts into dendritic cells of leukemic origin (dc leu ). after stimulation with dc leu , antileukemic tcells can be generated ex vivo. the compounds are approved for clinical use and are therefore attractive tools for immunotherapy in myeloid leukemia. methods: dc/dcleu-culture from rats'/patients' wholeblood (wb) with kits, mixed lymphocyte culture (mlc) of tcells with kit-treated blood, functional blast-cytotoxicity and leukemia-specificity assays (csa/elispot/degranulation/intracellular cytokine-assays). in addition flowcytometric evaluations of cellular and (leukemia-specific) lymphocyte compositions were performed from rats'/pts' blood in the course of the disease. results: 1) aml-diseased rats: each 3rats were treated with "i", "k" or "m" or were untreated (controls). a significant increase of dcleu could be detected in spleen/pb in kit-(esp. m) treated compared to untreated animals without induction of blasts' proliferation (ki67positivity): a significant reduction of blasts was seen with "m" (p=0.03/ 0.0001 in spleen/pb) and "i", but not "k". successful treatment correlated with an increase of cd62l+tcells, most likely representing tmem-cells, (p=0.07) and a reduction of cd4+treg (p=0.037). 2) 6 therapy-refractory aml-patients (during the course of decitabine/ld-aractreatment): kit-m was shown to ex vivo generate dcleu, activate immunereactive cells and mediate leukemia-specific/antileukemic response. activated or leukemia-specific lymphocytes were monitored in low proportions in active stages of the disease as well as of two patients during the further course of persisting disease. one of these patients (72 yo male), was offered an individual systemic salvage-treatment (kit-m, applied as continuous infusions) for refractory leukemia. after approval from the local ethical commitee,extensive information of the patient about the experimental nature of the treatment and obtaining his written informed consent. clinically the treatment was well tolerated and the patient improved clinically. neutrophils in wbc increased from 10% to 50%, thrombocytes reached 100 g/l after 24 days. after 4 weeks of treatment, the patient was discharged in good clinical conditions. 12 days later, progression of aml was seen with high blast counts in pb and bm. the patient developed severe sepsis and died few days later. immune monitoring showed (other than before treatment and in the patients without kit-m-treatment) a continuous increase of proliferating and non-naïve tcells, nk, cikand nkt-, th17 cells, bmem-cells and dc in pb. the production of ifnɣ producing t-, cik and nkt-cells was demonstrated, suggesting an in vivo production/activation of (potentially leukemia-specific) cells. immune stimulatory effects decreased after discontinuation of therapy. conclusions: treatment of wb as well as leukemically diseased organisms with blast-modulating kits (especially gm-csf and pge1) was well tolerated and induced clinical and immunological improvement (adaptive and innate immune system), whereas low counts of (leukemiaspecific) activated immune-reactive cells were found in non-kit-treated organisms. disclosure: nothing to declare p019 long-term outcomes after allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia with non-myeloablative and myeloablative conditioning: a single-center cohort study of 438 consecutive patients 1lars klingen gjaerde, niels smedegaard andersen 1 , lone smidstrup friis 1 , brian thomas kornblit 1 , søren lykke petersen 1 , ida schjødt 1 , henrik sengeløv 1 1 rigshospitalet, copenhagen, denmark background: since 2000, we have at our institution used a non-myeloablative (nma) conditioning regimen for older (>50 years) or significantly comorbid younger patients undergoing allogeneic hematopoietic stem cell transplantation (allo-hsct) for acute myeloid leukemia (aml). we aimed to compare the long-term outcomes of nma conditioned patients with myeloablative (ma) conditioned patients. methods: we studied 220 nma and 218 ma conditioned adult (>15 years) consecutive patients receiving their first allo-hsct for aml from 2000 to 2017 at rigshospitalet. nma conditioning consisted mainly of 2 gy total body irradiation (tbi) and fludarabine 90 mg/m 2 (95% of cases). ma conditioning consisted mainly of cyclophosphamide 120 mg/ kg and either 12 gy tbi (86% of cases) or busulfan 12.8 mg/ kg (5% of cases), or fludarabine 150 mg/m 2 and treosulfan 42 mg/m 2 (6% of cases). five percent and 19% of nma and ma conditioned patients, respectively, received anti-thymocyte globulin. patients were followed until death or end-of-followup on october 31 st , 2018. cumulative incidences with 95% confidence intervals (ci) of acute graft-versus-host disease (agvhd) grade ii-iv, chronic graft-versus-host disease (cgvhd), relapse and non-relapse mortality (nrm) were calculated and compared between nma and ma conditioned patients using gray's test with death as a competing risk (or relapse when comparing nrm). overall survival (os) was estimated by the kaplan-meier method. results: nma and ma conditioned patients were comparable when regarding sex (49% and 48% female, respectively) and donor (matched related donor in 34% and 36%, respectively), but differed, as expected by indication, with regards to age (median of 60 versus 42 years, respectively) and karnofsky score (< 90 in 18% and 11%, respectively). nma conditioned patients had generally a lower aml stage at transplant (1 st complete remission in 68% versus 49% of ma conditioned patients) and a lower aml cytogenetic risk (adverse risk in 17% versus 21% of ma conditioned patients). patients were followed for a total of 2090 person-years (median follow-up in surviving patients was 6.2 years). agvhd grade ii-iv occurred less frequently in nma conditioned patients (20% [ci: 15%-26%] versus 38% [ci: 32%-45%] in ma conditioned patients, p < 0.01), while cgvhd occurred in similar rates (50% [ci: 43%-56%] in nma conditioned patients and 51% [ci: 44%-58%] in ma conditioned patients, p = 0.77). there was a trend towards a higher relapse rate in nma conditioned patients (34% [ci: 28%-40%] versus 28% [ci: 22%-34%] in ma conditioned patients, p = 0.07), and nma conditioned patients had, however not with statistical significance, lower nrm (20% [ci: 14%-25%] versus 25% in ma conditioned patients, p = 0.27). os ( figure) was comparable, with 5-year os rates of 55% (ci: 48%-62%) in nma conditioned patients and 54% (ci: 47%-61%) in ma conditioned patients. conclusions: patients with aml undergoing allo-hsct with nma conditioning at our institution were older and frailer than ma conditioned patients, but their overall survival after transplantation was comparable. this might be explained by a generally lower aml stage and cytogenetic risk at transplant in nma conditioned patients. jedlickova 1 , saskia güller 1 , rosa toenges 1 , juliane steinmann 1 , hans martin 1 , hubert serve 1 , gesine bug 1 background: allogeneic hsct is urgently indicated in patients with aml in first complete hematologic remission (chr) after intensive chemotherapy with increasing or recurrent minimal residual disease (mrd). these patients are at high risk of hematologic relapse (hr) during preparation of their transplant and hsct with active aml was found associated with poor outcome. azacitidine has recently been shown to substantially delay or even prevent hr in >50% of patients (relaza2 trial, platzbecker et al., lancet oncology 2018) . we here present the outcome of a small cohort of consecutive patients with mrd-positive aml who received low dose cytarabine (ldarac) as bridging therapy prior to hsct. methods: mrd was assessed by quantitative polymerase chain reaction (qpcr) using mutated npm1 (n=5), runx1-runx1t1 (n=2), cbfb-myh11 (n=1) or kmt2a-ptd (n=1). mrd negativity was defined as ratio of oncogene to control gene (abl1) ≤0,01% while increased or recurrent mrd required a ratio >1% (shayegi et al., blood 2013) . primary endpoint of our retrospective analysis was progression to hr (≥5% bone marrow blasts or extramedullary disease); secondary endpoints were achievement of molecular remission prior to hsct, neutropenia g4 according to ctcae, thrombocytopenia g4, anemia ≥g3, admission to hospital, os and rfs. os and rfs were calculated from the first dose of ldarac. ldarac was self-administered subcutaneously by the patients at home at a flat dose of 20mg bid over 10 days and repeated after 4 weeks if necessary. results: between 12/2015 and 10/2018, nine patients (median age 55, range, 22-68 years) with low (n=7), intermediate (n=1) or high-risk cytogenetics (n=1) according to eln criteria 2017 were treated in continuous chr for increasing (n=2) or recurrent mrd (n=7) starting at a median of 260 (range, 154-651) days after the last consolidation therapy, i.e., duration of chr was >6 months in all pts. patients received one (n=4), two (n=2) or three cycles (n=3) of ldarac prior to hsct. in three patients, neutropenia g4 occurred and one patient needed platelet transfusion. all patients were managed in the outpatient setting. in eight out of nine patients (89%), hr was successfully prevented and 3 patients (33%) even became mrd negative prior to hsct. one patient (runx1-runx1t1 positive aml) progressed to hr after one cycle of ldarac and received salvage therapy with high-dose arac and mitoxantrone (ham) prior to hsct. all patients proceeded to hsct from a matched related (n=1), unrelated (n=7) or haploidentical donor (n=1) and are still alive (median follow-up of 666 days). conditioning regimens included fludarabine (flu)/melphalan (mel)/tbi (n=5), flu/mel (n=1), flu/tbi (n=1), flu/busulfan (bu)4 (n=1) and thiotepa/bu3/flu (n=1). after hsct, only the ldarac-refractory patient relapsed, resulting in a probability of rfs of 88% at 2 years. conclusions: our data suggest that a bridging therapy with up to three cycles of ldarac prior to hsct is feasible and was associated with favorable outcomes in patients with npm1-mutated or core binding factor aml and molecular relapse >6 months after achieving a first chr. the treatment has low costs, can be administered on an outpatient basis and is very well tolerated. clinical background: allogenic hematopoietic stem cell transplant (hsct) is the only curative treatment for all the patients with aml. high risk disease qualifies for upfront hsct irrespective of the presence of matched sibling donor (msd). in the absence of msd, haploidentical stem cell transplant is easier option with success rates as high as msd in a high volume transplant centre. we present our experience from a single centre. methods: we analyzed retrospective data of aml patients who have undergone hsct at our centre between january-2013 and august-2018. for msd transplant we used fludarabine + busulfan or fludarabine + melphalan conditioning regimen, in matched unrelated donor transplant (mud) regime used was fludarabine + busulfan + atg. we followed john hopkins's protocol for haploidentical hsct. cyclosporine + methotrexate was used as gvhd prophylaxis in msd and unrelated donor group and cyclophosphamide + tacrolimus + mycophenolate was used for haploidentical post-transplant. day 100 survival, overall survival (os), incidence of gvhd and cmv reactivation was computed. results: a total of 96 aml patients underwent hsct during the study period, the basic and clinical characteristics of the study patients are presented in table 1 . conditioning regime did not have significant impact on os. survival at day 100 was 78%. the os function and relapse free survival (rfs) function did not significantly differ between msd and haploidentical transplantation (68.3% vs 60.0%; p=0.225) and (68.3% vs 75.0%; p=0.760) (graph 1). disease status at latest follow up showed that 82% were in remission and 18% had relapsed. overall one year survival and five year survival in the entire cohort was 68% and 58% respectively. the average cost of msd transplant at our centre is inr 10,00,000 (€ 10000-12000), haploidentical transplant is inr 20,00,000 (€ 25000-27000) and mud transplant is inr 32,00,000 (€ 30,000 + 10000 for stem cell procurement). conclusions: our study showed comparable outcomes in msd and haploidentical transplant with respect to day100 survival, os, and rate of gvhd. in a developing country like india where patients are not covered under state health insurance, the additional cost of procurement of stem cells in a mud transplant would add to the financial burden to the patients. haploidentical transplant is a feasible option in case of non-availability of msd, due to ease of donor availability and strong motivation from the family donor to donate the stem cells. background: allogeneic stem cell transplantation (allo-hsct) is not indicated as consolidation of first complete remission (cr1) in favorable-risk acute myeloid leukemia (aml) bearing mutations in nucleophosmin (npm1) in the absence of flt3 internal tandem duplication (flt3-itd). nevertheless, a substantial proportion of patients eventually proceed to allo-hsct beyond cr1 or for chemoresistant minimal residual disease (mrd) while in cr1, which might compromise transplantation outcomes. the study aimed at examining the characteristics and results of allo-hsct in aml cases with mutated npm1 and wild-type flt3 (npm1mut/flt3wt), with special focus on molecular monitoring of mrd following transplantation. methods: from 11/2010 until 04/2018, 16 patients (women/men, 9/7) underwent allo-hsct for npm1mut/ flt3wt aml. at transplant, median age of patients was 44.5 years (range, 35-63) , and disease phase was cr1 (n=5), cr2 (n=9), or primary refractory (n=2). among the 13 patients who were transplanted in cr and had available molecular mrd assessments, 10 had detectable mutant npm1 transcripts by real-time quantitative pcr (rq-rcr). also, 4 patients fulfilled criteria of molecular relapse (increasing levels of npm1-mutated transcripts in two successive bone marrow samples), with mutant npm1 load of 386-4,900 transcripts/10,000 abl transcripts). the conditioning regimen was myeloablative in the majority of cases (n=14) or reduced-intensity (n=2). the type of donor varied, namely hla-identical sibling (n=6), matched unrelated (n=5), haploidentical relative (n=3), or double umbilical cord blood (n=2). results: engraftment was achieved in all cases, with a median time to absolute neutrophil count >500/ul of 16 days (range, 12-29) . among the 13 patients with posttransplant monitoring of mrd by rq-pcr, 9 exhibited a stable molecular remission whereas a rising level of npm1mutated transcripts was observed in 4 cases due to either hematologic (n=3) or molecular (n=1) relapse of disease. the cumulative incidences (cin) of hematologic relapse and non-relapse mortality (nrm) were 18.75% and 25% at 12 months, respectively. no events of relapse or nrm were encountered beyond 6 months from allo-hsct. out of 3 patients with hematologic relapse post transplant, 2 died of disease whereas one achieved a stable complete remission after withdrawal of immunosuppression. at a median follow-up time of 40 months (range, 14-89), 10/16 patients continue to be alive in cr. the estimated disease-free background: cpx-351 (vyxeos®) is an advanced liposomal encapsulation of cytarabine/daunorubicin at a synergistic 5:1 molar ratio. cpx-351 is approved by the us fda and ema for the treatment of adults with newly diagnosed, therapy-related aml or aml with myelodysplasia-related changes. methods: safety data were pooled from 5 studies of cpx-351 in adults aged 18-75 years with newly diagnosed or relapsed/refractory aml. cpx-351 induction consisted of 100 units/m 2 (cytarabine 100 mg/m 2 + daunorubicin 44 mg/m 2 ) on days 1, 3 , and 5 (second induction: days 1 and 3) . cpx-351 consolidation consisted of 65 or 100 units/m 2 (varying by study) on days 1 and 3. cpx-351 was evaluated against standard-of-care controls. results: baseline characteristics were generally balanced between cpx-351 (n=375) and controls (n=236); the majority of patients were aged ≥60 years (78%; 87%) and had secondary aml (55%; 72%). controls included 7+3 (n=192) and salvage therapy with mitoxantrone/etoposide/ cytarabine (n=23), idarubicin/cytarabine (n=8), other cytarabine-based chemotherapy (n=12), and mitoxantrone/ etoposide (n=1). the treatment-emergent adverse event (teae) profile of cpx-351 100 units/m 2 was comparable to induction controls, but associated with a greater proportion of patients with teaes, grade ≥3 teaes, and serious teaes during consolidation (table) . therefore, the cpx-351 consolidation dose was reduced to 65 units/m 2 in latter studies; this dose demonstrated an improved teae profile similar to consolidation controls. the most frequent system organ class was gastrointestinal disorders for both cpx-351 and controls; a lower incidence was reported for cpx-351 (90%) versus controls (95%), with this difference driven by the lower incidence of diarrhea for cpx-351 (46%) versus controls (66%). the most frequently reported grade ≥3 teaes were febrile neutropenia (cpx-351: 62%; controls: 59%), pneumonia (16%; 13%), hypoxia (10%; 11%), and bacteremia (10%; 3%). early mortality rates, both overall and by treatment period, appeared lower with cpx-351 versus controls at day 30 and day 60 ( table) ; the majority of early deaths were attributable to teaes. conclusions: across the 5 studies comprising the cpx-351 clinical development program, cpx-351 demonstrated a safety profile comparable to conventional chemotherapy in adults with newly diagnosed or relapsed/refractory aml. background: haploidentical hematopoietic stem cell transplantation (hsct) with post-transplantation cyclophosphamide (pgcy) marked improved clinical outcome. recent studies comparing allogeneic hsct using unrelated donors versus haplo donors in patients with acute leukemia have suggested equivalent outcomes. the depletion of tcells with pgcy was subsequently applied for unrelated hsct setting for patients with unrelated donor. methods: we performed a retrospective study on 90 patients with acute leukemia in order to compare the outcome after hla haploidentical (n=30) and unrelated hsct (n=60) with pgcy. the main characteristics of patients were similar in both groups. baseline disease were: 19 aml (63%) and 11 all (37%) for haplo group and 33 aml (55%) and 27 all (45%) for unrelated group. disease state at time of haplo and unrelated-hsct were following: 19 and 50 patients in cr1 (63% and 83%) and 11 and 10 non cr1 (37% and 17%). for aml recipients mainly received thiotepa, busulfan and fludarabine and for all recipients received tbi and etoposide conditioning. all patients who received pbsc graft were treated with rabbit antithymocyte globulin (atg) on days -2 and -1. results: at the time of analysis, the os and dfs did not differ between the haplo and unrelated groups (67% vs 63%, and 63% vs 56%). incidence of severe (grade [3] [4] acute gvhd was the same in two groups (10% versus 8%). recipients of haplo-hsct transplant were statistical significance less likely to experience disease relapse (3% vs 28%) and chronic gvhd (20% vs 47,5%). however, gvhd free relapse free survival (grfs) rate was slightly higher after haplo-hsct (77% vs 64%). addition, cumulative incidence of trm rate was higher after haplo-hsct (30% vs 15%).for haplo and unrelated groups who underwent hsct in cr1, the os were 84% and 67% versus 22% and 45% for those in non cr1. for aml, the os was same in two groups (haplo 63% versus unrelated 67%). however patients with all, the os was higher in haplo group compared with unrelated group (72% versus 59%). the impact of pretransplant disease state have a more powerfull effect on survival in the haplo-hsct setting (for aml cr1 77% versus non cr1 33% and for all cr1 100% versus non cr1 0%). viral reactivations were significant concern in both groups. conclusions: our retrospective analysis suggests largerly similar os and dfs with haplo versus unrelated transplants with pgcy for acute leukemia. our data indicate that haplo-hsct results in a lower incidence relapse and of chronic gvhd and higher grfs compared with unrelated hsct. in addtion, the pretransplant disease state have the important effect on the outcomes in both groups. allo-pbsc with atg can be used safely and effective as graft source in haplo-hsct with acceptable post-transplant outcomes and replaced bm in this settings. more statistical data for transplant related characteristics will be provided at the presentation.we emphasize that use the same pgcy gvhd prophylaxis for all types of allogeneic transplant. based on our results, we recommend haplo-hsct with pgcy against unrelated transplant for patients with acute leukemia. disclosure: disclosure of conflict of interest: none. excellent efficacy and tolerability of inotuzumab ozogamicin in b-cell all relapsed after allo-hsct background: donor lymphocyte infusion (dli) could be used prophylactically to reduce relapse after allogeneic hematopoietic stem cell transplantation for very high-risk leukemia/lymphoma without effective targeted therapy. to compare the safety and efficacy of prophylactic dli for prevention of relapse after allogeneic peripheral blood stem cell transplantation from haploidentical donors (hid-sct) and matched-sibling donors (msd-sct) in patients with very high-risk acute myeloid leukemia (aml), we performed a retrospective, observational cohort study enrolled in 21 hid-sct and 13 msd-sct recipients. methods: the very high-risk features were defined as: (i) in the non-remission (nr) state prior to transplantation, including primary induction failure, relapse untreated or refractory to reinduction chemotherapy, or untreated aml evolution from mds; (ii) achieving complete remission 1 with ≥3 cycles of induction of chemotherapy; (iii) carrying tp53, dnmt3a, tet2 or flt3-itd gene mutation. the scheduled time of the prophylactic dli was +30-60 days after transplantation for msd-sct recipients and +60-90 days for hid-sct recipients. the g-csfmobilized peripheral blood stem cells were infused to the recipient at a dose of 2×10 7 cd3 + cells/kg. csa was given at 2 mg/kg b.i.d from day -3 to day +90 (hid-sct) or to day +60 (msd-sct), and then tapered at 33% per month to be discontinued on day +150-180 (hid-sct) or on day +120-150 (msd-sct) unless graft-versus-host disease (gvhd) developed. if the patients received dli before day +90 (hid-sct) or day +60 (msd-sct), csa was given 8 weeks after dli in hid group and 4 weeks in msd group at a though concentration of 150-250 ng/ml for dliassociated gvhd prophylaxis, and then tapered and discontinued within 2 weeks unless gvhd developed. if gvhd occurred before the scheduled time of prophylactic dli, it would be delayed for 8 weeks when gvhd was well controlled. results: prophylactic dli was administered at a median of 71 (34-240) days for hid-sct recipients and 53 (35-97) days for msd-sct recipients (p=0.008), and both groups displayed similar baseline characteristics except for donor's gender distribution (table 1) . grade 2-4 acute graft-versushost disease (gvhd) at 100-day post-dli was higher in hid-sct group than that in msd-sct group (59.5% vs. 30.8%, p=0.05). grade 3-4 acute gvhd (17.5% vs. 7.7%), 1-year chronic gvhd (36.6% vs. 33.2%) and severe chronic gvhd (15.3% vs. 27.3%) were similar between two groups (p>0.05). one-year non-relapse mortality was higher in hid-sct group than that in msd-sct group with marginal significance (27.9% vs. 0.0%, p=0.061). one-year relapse rate was similar between hid-sct group and msd-sct group (21.6% vs. 36.5%, p>0.05). estimated 1-year overall survival (os, 55.1% vs. 83.9%) and relapsefree survival (rfs, 50.1% vs. 74.0%) rates were both similar between hid-sct group and msd-sct group (p>0.05). in multivariate analyses, non-remission status prior to transplant, poor-risk gene mutations and donor's age ≥ 48 years predicted a higher risk of relapse after dli. nonremission status prior to transplant predicted inferior os and rfs. patient's age ≥ 40 years also predicted an inferior os. conclusions: prophylactic dli after hid-sct demonstrated similar tolerance and efficacy for reducing relapse compared to that after msd-sct for very high-risk aml. disclosure: the authors declare no conflict of interest. prognostic impact of pre-transplant tim3 levels on transplant outcome in acute leukemia patients background: t cell immunoglobulin and mucin domaincontaining protein-3 (tim3), a negative regulator of t cells, is expressed on a variety of tumors including hematological malignancies like acute myeloid leukemia (aml) and some lymphoma types in which it was shown to be associated with an adverse prognosis. the aim of this study is to identify the prognostic impact of pre-transplant tim3 levels on early and late transplant related complications as well as post-transplant relapse and survival methods: a total of 177 hematopoietic stem cell transplantation (hsct) recipients with an initial diagnosis of acute leukemia [median age: 36(16-66) years; male/ female: 111/66] were included in the study. aml was the initial diagnosis in 99 patients (55.9%), acute lymphoblastic leukemia (all) in 74 patients (41.8%), mixed phenotype acute leukemia in 3 patients (1.7%) and blastic plasmacytoid dendritic cell neoplasm in 1 patient (0.6%). soluble tim-3 levels in pre-transplant serum samples were measured with enzyme linked immunosorbent assay (elisa). results: median pre-transplant tim3 level was 955.6 (65.8-3784.4) pg/ml in the whole cohort. pre-transplant tim3 levels were significantly higher in aml patients when compared to all [1063.7(409.5-3784.4) vs 831.4 (65.8-3254.4 ); p=0.01]. tim3 levels were significantly lower in patients with abnormal cytogenetics when compared to normal karyotype (p=0.017). cytogenetic abnormalities, including mainly a complex karyotype or chromosome 8 abnormalities, were more frequent in patients with low tim3 levels (p=0.053). pre-transplant tim3 levels were significantly higher in patients who developed post-transplant viral hemorrhagic cystitis (p=0.034). a positive correlation was demonstrated between tim3 levels and acute graft versus host disease (gvhd) grade (p=0.013; r=0.299). at a median follow-up of 14.6 (0.2-160.9) months, overall survival (os) was found to be better in low-tim3 group when compared to high-tim3 group, without statistical significance (%35.2 vs % 20.4; p>0.05) ( figure 1 ). probability of os was relatively better in both aml (42.6% vs 26.7%; p>0.05) and all patients (29.5% vs 19%; p>0.05) representing low pretransplant tim3 levels in the subgroup analysis conclusions: in this study, elevated levels of pretransplant tim3 levels in aml patients were compatible with the previous reports which had underlined an increased tim3 expression on aml stem cells. the possible association of tim3 expression with cytogenetic features should be confirmed with further studies as there is no adequate data except its relationship with flt3-itd mutational status. tim-3 is also expressed on exhausted t cells in patients with viral infections, including human immunodeficiency virus, hepatitis b and hepatitis c virus. it plays an essential role in the regulation of antiviral and antitumor immune responses which may be an explanation for the increased frequency of hemorrhagic cystitis in patients with higher tim-3 levels. the adverse prognostic impact of tim3 on gvhd and os was confirmed without statistical significance which may be related to small sample size. as tim3 has a wide spectrum of action in the tumor microenvironment including stimulatory and inhibitory activities, further clues are required to define the exact role of this molecule in the clinical course of allogeneic hsct in order to develop targeted therapeutic strategies clinical trial registry: n/a disclosure: nothing to declare p029 homozygous hla-c1 is associated with increased risk of relapse after hla-matched transplantation in recipients with acute lymphoid leukemia: a japanese national registry study background: after hematopoietic stem cell transplantation (hsct), the role of natural killer (nk) cells which express killer-cells immunoglobulin-like receptors (kirs) and recognize hla-class 1 ligands is important. kir2dl1 recognizes not hla-c asp80 (c1), but hla-c lys80 (c2) and has polymorphism based on the 245 th amino acid of the transmembrane domain. low frequency of c2 and high frequency of strong kir2dl1 are characteristics observed in japanese. by using large transplant database, we reported that homozygous hla-c1 (c1/c1) recipients displayed lower relapse rates than did c1/c2 recipients after hla-matched hsct for acute myeloid leukemia (aml; hr = .79, p = .006) or chronic myeloid leukemia (cml; hr = .48, p = .025). this effect seemed to be independent of acute graft-versus-host disease (agvhd) or cytomegalovirus reactivation occurrence (arima n et al bbmt 2018) . methods: relapse rates of japanese recipients who first underwent hla-matched hsct between 1996 and 2016 for the treatment of acute lymphoid leukemia (all) were compared between c1/c1 pairs and c1/c2 pairs, using data from japanese data center for hematopoietic cell transplantation and adjusting for transplant characteristics. cord blood transplantation was excluded. multivariable competing risk regression analyses were performed to evaluate relapses and relapse-free survival (rfs) was estimated using kaplan-meier method. results: after 61 recipients who did not achieve remission or experienced graft failure and 41 recipients not-expressing c1 were excluded, resting 2779 recipients aged 0-72 years (median, 31.2 years) were analyzed. the median follow-up period for survivors was 5.0 years. there were 2447 recipients expressing c1/c1 and 332 recipients expressing c1/c2, respectively. after hla-matched hsct, c1/c1 recipients had higher relapse rates than c1/ c2 recipients (hr = 1.55, p = .003), resulting in worse rfs among c1/c1 recipients (hr = 1.27, p = .034). the frequent relapse in c1/c1 recipients than in c1/c2 was noticeable among recipients with agvhd (hr = 1.89, p = .002), those without cytomegalovirus reactivation (hr = 1.84, p = .002), and those with ph-negative all (hr = 1.88, p = .001). conclusions: kir2dl1-positive nk cells may promote graft-versus-leukemia (gvl) in c1/c1 recipients with aml or cml but suppress gvl in c1/c1 recipients with all. one interpretation is that transplant-activated nk cells impair antigen-presenting cells or deprive cytotoxic tlymphocytes of their gvl effects on all cells. this hypothesis may be explained by the fact that agvhd was necessary for the recessive relapse in c1/c1 recipients with all. furthermore, ph-positive all cells sometimes mimic aml cells in terms of their frequent myeloid antigen expression and might be directly targeted by nk cells. it would be necessary to further clarify in vitro the character of nk cell-affecting in the transplant immunity against residual leukemia cells. disclosure: authors have nothing to declare. hematopoietic stem cell transplantation with sequential conditioning for children with relapsed/refractory acute leukemia nao yoshida 1 , kazuki matsumoto 1 , daiki yamashita 1 , yiqing zhu 1 , daichi sajiki 1 , ryo maemura 1 , hirotoshi sakaguchi 1 , asahito hama 1 1 children's medical center, japanese red cross nagoya first hospital, nagoya, japan background: patients with acute leukemia who fail to achieve complete remission show a dismal prognosis even with allogeneic hematopoietic stem cell transplantation (hsct). this study evaluated whether sequential conditioning approach that is cytoreductive chemotherapy applied shortly prior to the main conditioning followed by hsct can improve prognosis in such high-risk patients. methods: we retrospectively analyzed the outcomes of 90 children (median 8, range 0-18 years old) with primary refractory (n = 11) or refractory relapsed (n = 79) acute leukemia (aml n = 43, all n = 47) who received hsct in our department between 1990 and 2016. the stem cell source was related peripheral blood (pb) in 4 patients, related bone marrow in 31, unrelated bone marrow in 40, or unrelated cord blood in 15. the grafts were hla serologically matched (n = 63) or mismatched (n = 27) with the recipient. in total, 29 patients received the sequential conditioning approach. as cytoreductive chemotherapy, fludarabine/cytarabine/idarubicin/g-csf (flag-ida) was used in 12 patients, mitoxantrone or daunorubicin/cytarabin in 10, or other regimens in 7, and 6 of them were combined with gemtuzumab ozogamicin. without waiting for hematological recovery, the patients promptly underwent hsct; therefore, the median interval between cytoreductive chemotherapy and main conditioning was 11 days. the main conditioning regimens were total body irradiation-based myeloablative (n = 22), busulfan-based myeloablative (n = 4), or reduced intensity (n = 3). results: in 90 children with relapsed/refractory acute leukemia, the 5-year overall survival (os), leukemia-free survival (lfs), cumulative incidence of relapse (ri), and transplantation-related mortality (trm) were 24%, 21%, 53%, and 26%, respectively. in multivariate analysis, the use of sequential conditioning was identified as the most favorable factor for lfs (hazard ratio [hr] 0.37; p = 0.001), although there were no differences in the outcomes according to the types of cytoreductive chemotherapy or the main conditioning regimen. hla-matched donor (hr 0.46; p = 0.005) and pb blasts-negative at the beginning of conditioning (hr 0.49; p = 0.02) were also independently associated with better lfs. with sequential conditioning, leukemia burden prior to the hsct was significantly reduced; pb blasts became undetectable at the beginning of conditioning in 66% patients given the approach, while in 38% patients without the approach (p = 0.02). notably, the outcomes in the patients without pb blasts at the beginning of conditioning who received sequential conditioning were promising; the 5-year os and lfs reached 73% and 62% and the 5-year ri and trm were 33% and 5%, respectively. conclusions: our study reveals that hsct with sequential conditioning can be an effective and tolerable treatment option for children with relapsed/refractory acute leukemia. the treatment strategies that focus on the reduction of leukemia burden immediately prior to hsct may contribute to the induction of long-term remissions in patients with high-risk acute leukemia. disclosure: this research was funded by japanese red cross, nagoya 1st. hospital research grant nfrch18-0028. use of blinatumomab to achieve remission and consolidation with haploidentical transplant with cyclophosphamide post for the treatment of children with refractory acute lymphoblastic leukemia (all) background: most of patients with all in relapse or refractory to conventional treatment have only 30% possibilities to achieve long term remission. this report refers to the therapeutic efficacy and adverse events from the blinatumomab to achieve molecular remission in patients with pre-b cd19+ which lead to haploidentical with cyclophosphamide post transplant as a consolidation. methods: a pilot study was conducted in children with refractory all preb-cd19 +. as a strategy to achieved remission blinatumomab was used at a dose 10 μg/m2 for continous infusion of 48 hours, increasing the dose to 15 μg/m2 during 28 days, patients with a mrd of < 0.002 log, after 2 cycles received an haploidentical bone marrow transplant as a consolidation, the conditioning regimen was with total body irradiation scheme at 200 cgy/day/3 days, cyclophosphamide and etoposide. receiving prophylaxis for gvhd with cyclophosphamide. results: a total of 10 patients were included, seven of them achieved complete remission after 2 cycles of blinatumomab, one with partial remission (table 1) , these seven patients, six received an haploidentic transplant achieving graft in 6 of the transplanted patients. one patient had a bone marrow relapse in the first 6 months of the follow-up and 5 patients are free of disease with a follow-up to 20 months (figure 1). as a acute complication the 10 patients presented cytokine release syndrome, during the infusion of blinatumumab 10 patients presented tachycardia (table 3 ) and the 6 patients presented agvhd after hsct (5 grade i-ii and i grade iv). conclusions: allogeneic bone marrow transplant constitutes a treatment option on those patients that relapse or become refractory to treatment, one of the major problem is basically to identify a hla-identical donor, the alternative is an haploidentical donor. the most important factor to get these results is the disease status before transplant. the use of blinatumomab has proven to be effective in achieving remission in relapse acute linfoblastic leukemia pre-b cd 19+ or refractory to treatment. characteristics nº % male 4 40% median age at diagnosis, (range), years 9. 22 (7-12) status of disease 2 o + 3 relapse 6 60% refractory to primary or salvage therapy 4 40% complete remission after blinatumomab 7 70% partial remission after blinatumomab 1 10% active disease 2 20% [[p031 table] 1. table n°1 . demographic characteristics of patients undergoing blinatumomab (n=10)] disclosure: a. olaya-vargas, r. rivera-luna, y. melchor-vidal, h. salazar-rosales, g. lopez-hernandez, n. ramirez-uribe. we wish to confirm that there are no known conflicts of interest associated with this abstact, the only financial support was provided by mexican associations that helping children wiht cancer in a few patients. sequential high-dose chemotherapy reinduction followed by myeloablative allogeneic transplant for active acute myeloid leukemias methods: at our center, 27 relapsed/refractory aml patients were transplanted during chemo-induced neutropenia after high-dose salvage chemotherapy. median age at transplant was 52 years (range 21-62). patients suffered from de novo (n= 18/27, 67%) or secondary aml (n=9/ 27, 33%). genetic risk stratification was reported using stardardized groups proposed by the european leukemia net (eln) in 2010. favorable, intermediate i and ii and adverse risk category at diagnosis was observed in 1/27 (4%), 19/27 (70%), 7/27 patients (26%) respectively. all patients had active disease at the time of sequential therapy and median marrow blast count was 25% (range 7-88%). patients received a high-dose cytarabine based (mec in 17/ 27, 63%) regimen as salvage therapy. donors were haploidentical relatives for 15/27 (56%) patients, identical siblings and matched-unrelated for 6/27 patients (22%) and 6/27 (22%), respectively. a myeloablative conditioning was used to further implement anti-leukemic effects. conditioning, thiotepa-busulfan-fludarabine in 89% patients, was started at a median of 8 days (range [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] after the last day of chemotherapy. bone marrow and peripheral blood stem cells were used as graft source in 11/27 (41%) and 16/27 (59%) patients. graft-versus-host disease (gvhd) prophylaxis and supportive care were administered accordingly to each hsct platform. results: all patients engrafted. median day of neutrophil recovery was day +16 (range 12-23). median follow-up of survivors was 31 months (range 4-125). non relapse mortality and relapse incidences (nrm, ri) were 16% and 48% at 1 year and 16% and 58% at 3 years, respectively. overall cumulative incidences of acute and chronic gvhd were 48% and 43% at day +100 and + 400. one and 3 year overall survival (os) were 58% and 34%, while 1 and 3 year event-free survival (efs) were 35% and 26%. significant better os and efs were observed in patients with favorable-intermediate i-ii versus adverse risk score (1-3 years os 64% and 50% vs 43% and 0% p=0.022; 1-3 years efs 43% and 36% vs 14% and 0% p=0.013). adverse risk had a significant impact on os (hr 3.24, p=0.030) and efs (hr 3.33, p=0 .018) by univariate analysis and on ri (sdhr 3.02, p=0.031) by fine and gray test. conclusions: though small the patient cohort, our findings suggest that sequential therapy with a myeloablative hsct is feasible in treating relapsed/refractory aml. transplant-related toxicity was low (16%) and relapse was the major treatment-failure. however, even with this approach, patients with adverse cytogenetic features have a very dismal prognosis. for these patients, the use of new drugs before hsct and/or maintenance therapy after transplant is highly encouraged to improve outcomes. disclosure: alessandro busca: honoraria from gilead sciences, merck, pfizer pharmaceuticals and jazz background: in spite of satisfactory results of overall survival (os) after allohsct in 1 st and 2 nd cr aml, relapse free survival (rfs) and graft-versus-host-disease free/relapse free survival (grfs) require further improvement. the detection of mrd is one of the factors which influence on the outcome of allohsct in aml is unclear but identification is important to improve risk-adapted relapse prophylactic treatment after allohsct. aim. to evaluate outcomes of allohcst in 1 st and 2 nd cr pediatric aml depending on the level of mrd status before myeloablative (mac) or reduced intensity conditioning regimens (ric). methods: the data of 72 children with aml in 1 st and 2 nd cr underwent allohsct between 2008 and 2018 were analyzed. median age at the moment of allohcst was 8 years old (2-18). mrd negative status had 42 (58%) patients, 30 (42%) were mrd positive by flow cytometry. mac based on busulfan (16 mg/b.w.) received 27 (37%) patients, on treosulfan -7 (10%) patients. ric based on melphalan received 20 (28%) patients, based on busulfan (8 mg/b.w.) -18 (25%) patients. patients received prophylaxis of agvhd by atg 20 (28%) or ptcy -48 (66%) patients plus csa -23 (32%) or tacrolimus ± sirolimus -43 (60%) patients that depended on source of transplant (related, unrelated or haplo donor) . results: at the median follow up 3 years in the cohort of mrd positive patients os is 66% vs 72% in mrd negative (p>0,05). rfs is 56% vs 83% accordingly (p=0,01). graft-versus-host-disease free/relapse free survival (grfs) in mrd positive patients is 37% vs 51% in mrd negative (p>0,05). os, rfs, grfs in mrd positive patients after mac is 57%, 42%, 30% vs 75%, 68%, 43% after ric accordingly (p>0,05). os, rfs, grfs in mrd negative patients after mac is 75%, 85%, 55% vs 65%, 82%, 47% after ric accordingly (p>0,05). os, rfs in mrd negative patients with/without ptcy is 82%, 86% vs 42%, 78% (p>0,05); grfs is 62% vs 28% accordingly (p=0,042). os, rfs, grfs in mrd positive patients with/without ptcy is 66%, 55%, 54% vs 66%, 57%, 27% (p>0,05). conclusions: mrd status does not statistically significant affect on os that can be related to different approaches to the treatment of relapse after allohsct. mrd positive status statistically significant decreases rfs that underline the necessity of posttransplant therapy improvement. ric vs mac in all patients in first and second remission do not show statistically significant impact on os, rfs, grfs. ptcy significantly improves grfs in mrd negative patients. disclosure: none of the authors has anything to disclose. background: with increasing overall-survival (os) of lymphoma patients, higher incidences of therapy-related clonal bone marrow diseases, such as acute myeloid leukemia (aml) and myelodysplastic syndrome (mds) are occuring. generally, the outcome is considered poor. allogeneic hematopoietic stem cell transplantation (allo hsct) often remains the only potentially curative treatment option. nonetheless, there is only little data available concerning this patient group. methods: we retrospectively collected data from 33 patients with therapy-related aml (taml) and mds (tmds) after treatment for hodgkin's lymphoma (hl; n=7 and n=2) or non-hodgkin's lymphoma (nhl; n=10 and n=14), who received an allo hsct between 2000 and 2018. median follow-up of surviving patients was 3.1 years (range 2.6 months-9.4 years). background: the prognosis of relapsed/refractory acute leukemia (r/r al) is poor and the treatment is challenging. in this setting, allogeneic stem cell transplantation (allo-sct) constitutes the only curative option although the high relapse rate and non-relapse mortality (nrm). the sequential conditioning regimen followed by allo-sct has been used for persistent disease and aims to improve disease control by intensified chemotherapy, thus conceding more time for the presumed graft-versus-leukemia effect to occur. methods: the clinical outcome of r/r al with the sequential conditioning regimen combining a chemotherapy rescue followed by ric allo-sct in our center is described. patients who underwent a sequential allo-sct from 2005 to 2017 are included. the primary endpoint was progression free survival (pfs) and overall survival (os) that were estimated by the kaplan-meier method. secondary endpoints were non-relapse mortality (nrm). background: recommendations of the 2017 european leukemia net (eln) for favorable-risk genetics (frg) acute myeloid leukemia (aml) favor consolidation over transplantation, although reviews suggest advantage of autologous stem cell transplant (asct) in event free survival. our objective was to compare the progression free survival (pfs) and overall survival (os) of normal karyotype npm1 mutated without flt3 itd or allelic ratio < 0.5 (npm1+) aml patients treated with consolidation chemotherapy alone (cc), asct or allogeneic stem cell transplant (allosct). methods: retrospective review of npm1+ frg-aml patients, treated in one institution (2008 to 2017) with the following induction regimens: cytarabine (ara-c) and vp-16 with daunorubicin (ade) or mitoxantrone (mice). consolidation regimens were ara-c with daunorubicin (ac-d), idarubicin, vp-16 and ara-c (mini-ice) or highdose ara-c (hidac). in asct, conditioning regimens were bucy or bvac and in allosct were bucy or flubu. pfs and os were calculated from the start of the last consolidation or stem cell infusion. results: a total of 39 patients were evaluated, with a median age of 53 years (y) (23-68y), 69% female, 95% with ecog performance status (ps) 0-1 and 36% with ageadjusted charlson comorbidity index (aacii) ≥2 at diagnosis. patients were treated with cc in 33% (n=13), asct in 36% (n=14) and allosct in 21% (n=12) of cases. there were no differences between groups for age, aacii, ps, leucocytes at diagnosis or extra-medullary disease. flt3-itd was more frequent in allosct group (64%) than cc (23%) or asct (8%; p=0.07). at induction, ade was used in 82% and mice in 18% of patients, with a complete remission (cr) rate of 95%. there were no differences between groups for induction regimen or cr. in cc group, consolidation regimens were 1 cycle (8%) and 2 cycles ac-d (61%) and 2 cycles mini-ice (31%). asct patients received consolidation with 1-2 cycles ac-d (78%) and 1 cycle mini-ice (22%), while allosct patients received 1-2 cycles ac-d (84%), 2 cycles hidac (8%) and no consolidation in 8%. [[p037 image] 1. figure 1 . pfs at 3y in cc, asct and allosct groups.] median follow-up was 39 months, pfs at 3y was 53% and os at 3y was 64%. pfs at 3y for asct group was superior then cc and allosct groups (61%, 51% and 44%, respectively; figure 1 ), although not statistically significant. os at 3y was statistically similar between groups, although inferior in allosct comparing to cc and asct (44%, 77% and 70%, respectively). conclusions: in this historical cohort review, although there was no advantage in os for asct in npm1+ frg aml, our data suggests that there might be a pfs improvement in asct over cc, which needs to be further addressed in prospective studies. disclosure: nothing to declare background: acute myeloid leukemia is a hematological malignant disease that motivates the persistent struggle in the scientific world to provide effective cure that can establish acceptable survival rates in this group of patients. autologous stem cell transplantation with myeloablative conditioning is still a powerful weapon that can be used against this entity methods: we have evaluated retrospectively patients with aml where autologous stem cell transplantation was performed in the period from 2000 till 2018. our group consisted of 94 patients; male patients 45 (47.8%), female patients 49 (52.2%). median age at diagnosis was 44 years (16-68). the average period from time of diagnosis to autologous sct was 7.05 months. results: in the majority of our group, we used myeloablative conditioning regimen with busulphan-cyclophosphamide, 60 patients (63.8%), in 2 patients (2.1%) we have added melphalan to bu-cy conditioning, in 22 (23.4%) patients we used beam conditioning and in the rest, 10 patients (10.6%) we used bam conditioning regimen. as auto graft we used peripheral blood stem cells (pbsc) in 78 patients (82.9%), and in 16 patients (17.1%) we used bone marrow. the main mobilising regimen for pbsc was g-csf + etoposide and it was performed in 44 patients (46.8%), and in the remaining 34 patients (36.1%) mobilising of pbsc was performed only with g-csf. the mean number od apheresis procedures done in our group was 1.55, and the mean number of collected mononuclear cells was 3.05x10 8 /kg tt. the mean time to engraftment was 12.8 days (9-23). the transplant related mortality (trm) was 2.1 %. the 5 year overall survival of our patients was 46.7 patients. the main reason for death was relapse of the primary disease(73%). 20 patients (21%)were treated with salvage chemotherapy regimen (flag-ida) because with the standard induction regimen 7+3 there was absence of adequate therapeutic response, or predominantly no complete remission was achieved. all patients were transplanted in complete remission conclusions: autologous stem cell transplantation could be an acceptable therapeutic solution for patients with aml as a consolidation therapy, where neither suitable compatible donor is available nor allogeneic stem cell transplantation could not be performed from various reasons depending on the bone marrow transplant unit disclosure: nothing to declare p040 prophylaxis dli alone may not prevent relapse of flt3-itd positive aml after allogeneic hct background: one of the most potent prognostic factors affecting outcomes in aml is the presence of cytogenetic and molecular markers which can guide the selection of post-remission therapies. recently, favorable outcomes of npm1 wt /flt3-itd neg /non-cebpa dm group after allogeneic hematopoietic cell transplantation (allo-hct) have been reported, that is similar to those of favorable risk by the eln risk classification. however, the role of allo-hct compared to consolidation chemotherapy has not yet been elucidated. methods: the data of 88 patients who were diagnosed with aml and received intensive induction therapy from 2015 march to 2017 july were included in the current study. to address the time dependence of the allo-hct, the simon and makuch method was used in the graphical representation and the mantel-byar test and andersen and gill methods for identifying risk factors for long-term survival. results: median age of the patients were 53 years (range 21-69), and 49 patients (56%) were male. npm1 mutation was detected in 14 patients (16%), and flt3-itd were none, low, and high ratio in 69 patients (78%), 9 (10%), and 10 (12%), respectively. the eln risk classification divided the patients into favorable, intermediate, and adverse risk group in 31 patients (35%), 38 (43%), and 19 (22%), respectively. npn1 and flt3-itd both negative group included 29 patients (33%). allo-hct was performed in 48 patients (55%). overall, complete response (cr) after induction therapy achieved in 63 patients (72%), and 7 patients (8%) were primary refractory disease. cr rates did not differ between npm1 wt /flt3-itd negative group (n=17/29, 58.6%) and other intermediate risk group (n=6/9, 66.7%; p=0.967) . with median follow-up duration of 12.9 months (range 1.3-39.0 months), one-year os rate were 100%, 83.5±6.9%, 56.1±12.8% in favorable, intermediate, and adverse risk group (p < 0.001). among intermediate risk group, os rate of npm1 wt /flt3-itd negative group was similar to other intermediate risk (p=0.403). allo-hct was performed in 11 patients of npm1 wt /flt3-itd negative group. one-year os rates did not differ between npm1 wt /flt3-itd negative and other for allogenic hematopoietic stem cell transplant (allo-hsct), as a strategy to prolong survival. methods: data from aml pts over 60 years, who underwent ric allo-hsct in our institution between september 2011 and september 2017, was retrospectively collected from clinical files to evaluate the overall survival (os) up to november 2018. we calculated the os using kaplan-meyer curves. results: we identified 15 pts, median age 62 y.o. (60-67) and median htc-i score 2. the median follow-up was 25 months. one patient (pt) had cml blast crisis and was on first major molecular remission. of the remaining 14 aml pts, 7 were in 1 st complete remission (cr), 4 in 2 nd cr and 1 with progressive disease (pd); the other 2 pts could be classified as mds according to 2016 who diagnostic criteria and were in cr1. donors (d) were: 3 matched unrelated (mud), 5 mismatched unrelated (mmud -9/10), 6 matched siblings and 1 haploidentical. thirteen pts were infused with peripheral blood hsc and 2 with bone marrow. conditionings were: flubcnumel in 5 unrelated donor (ud) pts and 4 siblings, flumel in 1 ud pt and 1 sibling, flubu in 2 ud pts and flutbi 2gy in 1 sibling and in the haploidentical. graft versus host disease (gvhd) prophylaxis was tacrolimus (tac) + mmf in 5 ud pts and 1 sibling, tac + mtx in 2 ud pts and cyclosporine (cya) + mmf in 1 ud pt and 5 siblings. all mmud pts had atg. ptcy was done in the haploidentical setting with tac + mmf. the median time to neutrophil and platelet engraftment for the whole cohort was 14 and 11 days, respectively. one pt with secondary engraftment failure required re-infusion of selected cd34+ cells. ten pts presented with mild acute skin gvhd. eleven pts had chronic gvhd, 2 classified as severe; 7 required systemic therapy, 5 of those beyond 1 year. the median time on immunosuppressants was 404 days. at 2 years the os was 63.5%. there were 6 deaths: 3 disease-related (2 relapses at 22 and 58 months and 1 pd at d+30), 2 infection complications (2 septic shock) and 1 to secondary neoplasia. other relevant complications were hypoxemic pneumonia in 5 pts, 1 urinary sepsis, cmv and ebv reactivation respectively in 8 and 4 pts; pulmonary and renal toxicity either in 2 pts. at end of follow-up, 9 pts were in remission, 8 without negative measurable residual disease (mrd), the other mrd negative pt died of septic shock and severe intestinal gvhd. conclusions: in this small cohort, chronic gvhd and infectious complications were major causes of morbidity but there were no treatment related deaths before d+100. pts maintaining or achieving mrd negativity after transplant had better survival. although with only 15 pts, these results suggest that allo-hsct is feasible as consolidation strategy in selected aml pts over 60 years. [[p043 image] 1. overall survival] disclosure: nothing to declare. background: hematopoietic stem cell transplantation (hsct) is the only curative option for fanconi anaemia (fa); an inherited disorder characterized by congenital anomalies, progressive bone marrow failure (bmf) and a predisposition to develop malignancies. methods: we retrospectively analysed the data of 27 consecutive patients that underwent hsct at this centre from 2001 till june 2018. the data was analysed for variables affecting the outcome in terms of overall survival (os). results: median age at diagnosis was 10 years (2-20 years). median age at transplant was 11.3 years (4-25 yrs). all patients at transplant were in aplastic phase. male to female ratio was 1.2:1. twenty-four (88.9%) patients had congenital anomalies along with bmf while 3 were phenotypically normal. twenty-three (85.2%) patients were 10/10 hla matched with siblings, 2 with parents and 2 with cousins. eleven (40.7%) patients had gender mismatch transplant. three patients had major and 6 had minor abo mismatch. background: paroxysmal nocturnal hemoglobinuria (pnh) is a rare clonal non-neoplastic hematopoietic stem cell disease whose incidence is 1.5-2.9 cases/million of individuals worldwide. disease characteristics and natural history have been mostly analyzed by multicenter, retrospective studies, with the limit of heterogeneous approaches. herein we report the incidence of severe complications and outcome in a real life setting scenario of pnh patients consecutively diagnosed and managed at our pnh referral center between january 1985 and june 2018. methods: patients received a homogeneous diagnostic and treatment approach according to the period of observation (availability of diagnostic tests and eculizumab). all patients treated with eculizumab received vaccination with conjugated anti-meningococcus acwyserotypes and, since 2016, conjugated anti-meningococcus b-serotype. in the event of any complication, patients could refer to dedicated hematology emergency rooms (er) 24 hours daily. the occurrence of renal failure and pulmonary hypertension was specifically evaluated. the renal function was studied according to the cockcroft-gault formula and the lung function was prospectively monitored by daytime-on exertion, nocturnal pulsoximetric profiles and complete spirometric tests, with dlco measurement. results: overall,48 pnh patients, median age 36 years (range 17-84), were analyzed. at diagnosis, 26 patients had classic pnh, 19 aplastic pnh and 3 an intermediate form. the cumulative incidences (ci) of thrombosis, and clonal hematologic neoplasm were 29%, and 6%, respectively. ci of pancytopenia in the 26 patients with classic pnh was 23%. one patient showed a spontaneous disappearance of the pnh clone. since 2005, eculizumab was administered in 28 patients. after eculizumab treatment 50% and 32% of patients reached hemoglobin level > 11g/dl and >8< 11g/ dl without transfusion, respectively, while 18% were nonresponsive. during eculizumab treatment no thrombotic event was observed while two severe infectious episodes (respiratory tract and urinary tract infection) were observed in only one of the 28 patients. extravascular hemolysis was demonstrated in 50% of patients. no patient showed a significant reduction of the renal function.out of 24 patients prospectively monitored for lung function no pathological alteration in any diurnal or nocturnal pulseoximetric test was observed. no patient showed obstructive or restrictive ventilatory deficiency, nor reduced dlco values. 30-years overall survival (os) was 90% (4 patients who died for non-pnh related reasons were censored at the last follow-up).a better os, even if not statistically significant,was associated to the absence of thrombotic events (90%vs70%), and the period of diagnosis (100% in 2006-2018, 91% in 1996-2005, 75% in 1985-1995) . conclusions: our study reports a better os and lower rate of severe complications in pnh compared to previous experiences. although renal failure and lung hypertension have been reported by other groups, we did not observe these complications along a prolonged follow-up. we can assume that the availability of a dedicated er service enabled an early diagnosis and prompt treatment in case of hemoglobinuria crises (reducing the risk or organ damage) or other complications. the use of eculizumab, together with improved supportive approaches, presumably accounts for the trend towards a better survival witnessed over the last decade in the management of pnh patients. disclosure: nothing to declare haploidentical and unrelated allogeneic stem cell transplantation in aplastic anemia:single center experience zafer gulbas 1 , elif birtas atesoglu 1 , meral sengezer 1 , i̇mran dora 1 , cigdem eren 1 , suat celik 1 , demet cekdemir 1 background: aplastic anemia is a syndrome of bone marrow failure characterized by peripheral pancytopenia and marrow hypoplasia. allogeneic stem cell transplantation from hlamatched sibling is performed in the firstline setting in young aplastic anemia patients and in elderly patients who are refractory to immunosuppressive treatment. but if the patient does not have a hla-matched sibling, allogeneic stem cell transplantation is performed from unrelated and haploidentical donors. in this study, we analyzaed and compared the results of aplastic anemia patients who had undergone allogeneic stem cell transplantation either from matched unrelated or haploidentical donors. methods: we collected and analyzed data of aplastic anemia patients who had undergone allogeneic stem cell transplantation from matched unrelated or haploidentical donor between 2011 and 2018. results: there were 10 patients who had received allogeneic stem cell transplantation from unrelated donors and there were 10 patients who had undergone haploidentical transplantation. but in 4 patients who had undergone haploidentical transplantation, engraftment failure had occurred and they were transplanted from different haploidentical donors fort he second time. so a total of 10 unrelated and 14 haploidentical transplants were performed. the median age of patients who had undergone unrelated transplantation was 29(16-55) and the median age of patients who had undergone unrelated transplantation was 22(19-61) . the results of the haploidentical and unrelated transplantations are shown in table 1 . the neutrophil and platelet engraftment times were significantly longer in haploidentical transplantations (p=0,006 and p=0,005, respectively). however, vod, gvhd and 100 day mortality rates were not different in the 2 groups. similarly overall survival (os) of the patients who had undergone haploidentical or unrelated transplantation were not significantly different (p=0,38) ( figure 1) . conclusions: although the number of patients are low in this study, we can conclude that urelated and haploidentical transplantation in aplastic anemia have comparable toxicity and efficacy. background: autologous stem cells transplantation (ahsct) is an effective treatment for very aggressive autoimmune diseases such as multiple sclerosis (ms). however, toxicity remains the major concern to a wide application of this approach. post transplant viral reactivations may induce severe complications and a rigorous monitoring of peripheral blood viral load for a prompt and effective therapy is required. a higher rate of ebv and cmv reactivation has been observed in patients affected by ms and conditioned with beam plus atg compared with a controlled group of lymphoma patients without atg in the conditioning regimen [1] . we report here the policy of our center about both monitoring and treatment of such side effect. methods: a series of 37 consecutive patients with ms, transplanted between 2014 and 2018 is included in this analysis. all patients were mobilized with cyclophosphamide 4g/sqm + g-csf and conditioned with beam plus rabbit atg (thymoglobulin©, 7.5mg/kg). monitoring of cmv/ebv dna on whole blood by quantitative pcr was performed after the engraftment, weekly for at least one month, then at longer intervals. pre-emptive treatment with valgancyclovir was started in case of cmv viral load ³1x10^4 copies/ml. in case of ebv assay between 1x10^4 and 1x10^5 copies/ml further determinations were performed and rituximab-based treatment was started if the viral copies exceeded 1x10 5 copies. patients received treatment in case of symptomatic disease for any value of the pcr of both viruses or ebv titer ³1x10^5 copies/ml. results: detectable dna for cmv was observed in 15/ 37 (40,5%) patients at a median time from transplant of 23 days (range 12-36) and 6/37 (16%) required pre-emptive treatment. all patients promptly responded to treatment within 2 weeks. ebv viral load was detectable in 19/37 patients (51,3%) at a median time of 22 days (range 12-52). one patient out of 19 started the treatment on first determination for high viral load (>1x10^6/ml); nine presented an ebv viral load over 1x10^4 copies/ml, three of them were treated thereafter for the persistent increase of the viral load (> 10 5 /ml). six patients spontaneously recovered the ebv reactivation. previous treatments were not predictive of any higher risk of viral reactivation. no impact on engraftment related to the reactivation was observed. conclusions: this policy shows that, despite a high rate of cmv and ebv reactivation, no grade iii-iv adverse events were observed, suggesting the key role of viral monitoring in these patients and the efficacy of the preemptive treatment. ebv reactivation at low titers should be monitored to identify those cases that could achieve a spontaneous resolution and avoid the induction of further immunosuppression by rituximab. these data confirm that patients diagnosed with ad undergoing autologous hsct need a more intense pattern of care than hematological patients. background: autoimmune diseases are chronic serious conditions that are often refractory to standard therapies. since 1996, autologous haematopoietic stem cell transplantation (hsct) has been a very promising alternative that has shown satisfactory long-term results. the aim of this study is to evaluate immune reconstitution and mortality following hsct in patients with autoimmune disease. methods: a retrospective study was conducted on patients with diagnosis of autoimmune diseases that underwent autologous hsct between july 2012 and january 2018 at a tertiary referral center in colombia, south america. descriptive statistics were used to analyze patient's demographic and clinic characteristics. results: seven patients were included, with a mean age of 37 years (range 26-57). five patients were female (71%). the indications for hsct were systemic sclerosis (n=3) , multiple sclerosis (n=2), and myasthenia gravis (n=2). the conditioning regimen administered in patients with systemic sclerosis was cyclophosphamide + human anti-t lymphocyte immunoglobulin, beam (carmustine, etoposide, cytarabine (ara-c), and melphalan) + human anti-t lymphocyte immunoglobulin in patients with multiple sclerosis, and cyclophosphamide + human anti-t lymphocyte immunoglobulin in myasthenia gravis. median time to myeloid engraftment (neutrophils>0.5×109/l) was 12 days post-transplantation, and platelet engraftment, defined as >20,000 platelets/mm 3 untransfused, was 11 days post-hsct. median time of hospitalization was 21 days (range 11-66); longer in-patient management was due to infectious complications. infectious complications included bacteremia caused by e. coli and pneumocystis pneumonia that resulted in septic shock and acute respiratory failure, respectively. evaluating t-cell immune reconstitution, none of the patients had reached normal cd4+ cell value after one year of hsct follow-up. four patients (57.1%) reached normal cd8+ cells value at 3 months post-hsct. regarding bcell immune reconstitution, 85.7% of the patients (6/7) had reached both igg and iga normal levels at one-month post-hsct, and four patients (57.1%) had achieved normal igm background: multiple sclerosis(ms) is an inflammatory disease caused by autoimmune reactivity of t cells against myelin. there is accumulating evidence of the efficacy of highdose chemotherapy followed by autologous haematopoietic stem cell transplantation(ahsct) in ms patients who failed response to standard immunotherapy, despite a variability in eligibility criteria, conditioning regimens and outcome. methods: we retrospectively reviewed ms patients submitted to ahsct in our centre (2014) (2015) (2016) (2017) (2018) . patient eligibility criteria were active relapsing remitting(rrms) or secondary-progressive ms (spms), with prior failure to treatment with disease-modifying therapies and evidence of disease activity (clinical relapse or new active lesions in magnetic resonance [mr] ). mobilization of cd34+cells to peripheral blood was performed with granulocyte colony-stimulating factor(g-csf 10μg/kg/day) and conditioning regimen according to beam protocol. the severity of ms disability was classified according to the expanded disability-status scale (edss) and ahsct toxicity was evaluated by ctcaev5.0. results: seven ms patients had undergone ahsct (4 female/3 male), with a median age at ahsct of 39.9 years (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) . median age at ms diagnosis was 31.6 years(27-36) and median time from diagnosis until ahsct was 6.8 years (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) . four patients (57.1%) had spms and 3(42.9%) had rrms, with 4(57.1%) having mr active lesions. pre-ahsct relapse rate per year was 2 (1) (2) (3) (4) . median baseline edss was 6.5(5.5-7.5). median number of previous dmts was 6 (3) (4) (5) (6) (7) (8) (9) . all patients had been treated with corticosteroids and copaxone, 5(71.4%) received rituximab, 6 (85.7%) natalizumab, 1 (14.3%) alemtuzumab, 2(28.6%) fampridine and 2(28.6%) fingolimod. all patients collected enough cd34+ cells in a single apheresis session. median number of cd34+ cells infused was 7.2±3.8x10^6/kg, for a mean dmso volume of 50.4 ±14.8 ml. median inpatient stay during ahsct was 28 days(21-47). all patients developed febrile neutropenia, one was admitted to intensive care unit due to sepsis. one patient developed an anaphylactic reaction to transfusion and another a self-limited encephalopathy. two (28.7%) patients had grade≥2 oral mucositis, and all had gastrointestinal toxicity (grade 2-3). median time until neutrophils>500/μl was 11(6-12) days, to platelets>20,000/μl was 9 days(5-12), and to engraftment was 13 days (12) (13) (14) . patients were transfused with a median of 1(0-2) unit for erythrocytes and 3(1-8) for platelets. there was no treatment-related mortality and no long term side effects have been observed so far. for a median post-ahsct follow-up of 8.7 months, no patient developed new lesions in mr, but 2(28.6%) experienced symptoms consistent with a clinical relapse, at a median time of 20.5(5-35) months, effectively rescued with corticosteroids. the absence of evidence of disease activity at 6-months was 71.4%. although there was no variation concerning edss punctuation, 4(57.1%) patients self-reported significant benefits, especially concerning limb strength and sphincter continence improving. conclusions: our real life results claim a stabilization effect of ms patients with highly active/progressive disease after ahsct, with no significant toxicity. the failure in reporting benefits in edss punctuation is probably due to a small sample size and short follow-up. more studies are needed to establish the best patient selection criteria and define the ideal time to include these patients in transplantation programs, as well as to evaluate its long term outcome. disclosure: nothing to declare. elena poponina 1 , elena butina 1 , anna yovdiy 1 , galina zaytseva 1 , natalia minaeva 1 , igor paramonov 1 background: reactivation of epstein-barr virus (ebv) represents a potentially life-threatening condition in approximately 30% of patients after allogeneic stem cell transplantation, with no specific treatment available. methods: we have previously developed a manufacturing protocol for the expansion of cytomegalovirus (cmv) and ebv-specific t cells by stimulation of g-csfmobilized stem cell grafts with defined peptide pools (gary, 2018) . this advanced therapeutic medicinal product is currently investigated in an ongoing phase i/iia trial (eudract number: 2012-004240-30) . however, the expansion of virus-specific t cells relies on a pre-existing virusspecific memory compartment in the stem cell donor. in virus-seronegative donors, no expansion can be achieved. we therefore aim to identify ebv peptide-specific t cell receptors (tcrs) that can be translated into off-the-shelf cell products for the treatment and prophylaxis of ebv infection and ebv-associated malignancies. leftover cells from five allogeneic stem cell grafts were expanded in vitro in the presence of hla-b35*01-restricted peptides (hpvgeadyfey from ebna1, eplpqgql-tay from bzlf1) associated with latent and lytic ebv infection. after expansion, single ebv-specific t cells were facs sorted using peptide-mhc (pmhc) tetramers, and individual tcr αand β-chain pairs were determined with single cell sequencing (han 2014 , penter 2018 . to confirm peptide specificity, dominant tcr pairs were transfected into a murine 58αβreporter t hybridoma cell line with nfat-driven gfp expression (siewert, 2012) . functional tcrαβ candidates were transduced into human peripheral background: lymphoid and myeloid acute leukemia are the most frequent cause of cancer related death in children. interactions between nkg2d receptor, expressed in cytotoxic immune cells, and its ligands (nkg2dl), that are upregulated in many types of tumor cells including leukemic blasts, are important for anti-tumor immune surveillance. nevertheless, tumor cells may develop immune scape strategies like ligand shedding, which reduces nkg2dl expression and may cause nkg2d receptor downregulation. engineering t lymphocytes with nkg2d car may overcome immune evasion and become an effective therapeutic strategy. methods: cd45ra -t cells were obtained by depletion of non-mobilized apheresis with cd45ra magnetic beads using clinimacs. nkg2d-car t cells were generated by lentiviral (nkg2d-41bb-cd3z) transduction of cd45ra -t cells with moi=2. the expression of nkg2d ligands was analyzed in peripheral blood or bone marrow samples from a total of 97 leukemia patients (aml=13, b-all=52 and t-all=19), at different status of the disease (diagnosis, remission, relapse/refractory), and in 10 different leukemia cell lines by qpcr and flow cytometry. cytotoxicity of nkg2d-car t cells against leukemia cells was evaluated by performing conventional-4 hours europium-tda assays. soluble nkg2dl (snkg2dl) concentration was measured in the sera of leukemia patients by elisa. to evaluate the effect of snkg2dl on nkg2d-car t cells, those were cultured in the presence or absence of different concentrations of snkg2dl for 7 days. one week later, cell proliferation and car downregulation were measured by flow cytometry using cell trace violet and nkg2d labeling, respectively. the production of ifn-g and tnf-a was measured in the supernatants by elisa. the effect on cytotoxicity was evaluated in a 2 hoursdegranulation assay by co-culturing snkg2dl pretreated nkg2d-car t cells against k562 cell line. results: nkg2d ligands were expressed in leukemia cell lines and leukemic blasts. nkg2dl expression changed with disease status with a trend to decrease at diagnosis and relapse/refractory compared to remission. nkg2d-car t cells were cytotoxic against 8/10 leukemia cell lines with a percentage of specific lysis over 50%. myeloid and t-all cell lines were more susceptible to nkg2d-car t cells (specific lysis ranging from 50-78%) compared to b-all cell lines (19-52%). physiological concentrations of snkg2dl caused an increase in nkg2d-car expression. however, supra-physiological levels of snkg2dl decreased nkg2d-car expression up to 5 times and increased cell proliferation up to 4 times. cd4+ subpopulation was more affected by downregulation, while proliferation had more impact on cd8+ subset. the effects of snkg2dl were dose-dependent and attenuated by il-2. conclusions: nkg2d-car t cells are cytotoxic against leukemia cells, specially aml and t-all, and thus could be a novel therapeutic approach for non-b leukemia, or those b-all that relapse with undetectable cd19 after cd19-car treatment. nkg2d-car expression may be downregulated only by supra-physiological levels of snkg2dl, although antitumor activity is not affected. il-2 softens the negative effects of snkg2dl inducing nkg2d expression, cell proliferation and cytokines production. the changes observed in nkg2dl surface expression at the different stages of the disease could be related to ligands release and immune escape. disclosure: nothing to declare denis-claude roy 1,2 , ines adassi 1,2 , céline leboeuf 1,2 , vibhuti p. dave 1,2 1 hôpital maisonneuve-rosemont research center, montréal, canada, 2 university of montréal, montréal, canada background: for patients with high-risk leukaemia, allogeneic haematopoietic stem cell transplantation is the only curative treatment. the presence of alloreactive t cells in the donor graft, however, leads to a high probability of developing graft-versus-host disease (gvhd) . t-cell depletion minimises the presence of gvhd-causing alloreactive cells, but often results in an increased incidence of infections and disease relapse. photodepletion treatment (pdt) can specifically deplete activated alloreactive t cells while conserving resting t cells. pdt-treated cells have been utilised after t-cell-depleted haploidentical transplant to help reduce infection and relapse. the efficacy and safety of such pdt-treated cells is currently under clinical investigation in a phase iii trial (hatcy, nct02999854; kiadis pharma). here the reactivity of pdt-treated donor t cells was assessed toward tumour-associated and viral antigenic peptides derived from wilm's tumour protein 1 (wt1p), preferentially expressed antigen in melanoma (pramep), and from cytomegalovirus and epstein-barr virus (cmv/ ebvp). methods: healthy donor (hla-a*0201) peripheral blood mononuclear cells (pbmcs) were co-cultured with irradiated pbmcs from another mismatched donor (1:1) in a 4-day mixed lymphocyte reaction. th9402, a photoactive rhodamine derivative, was added and cells were exposed to visible light to deplete the th9402-containing activated alloreactive cells. elimination of alloreactive cells post-pdt was assessed using cd25 and hla-dr as activation markers. an ex vivo expansion protocol was exploited to evaluate the impact of pdt on reactivity to tumour and viral antigenic peptides. post-pdt t cells were co-cultured with irradiated autologous monocyte-derived dendritic cells (10:1) pulsed with wt1p, pramep or cmv/ebvp. antigen-specific t cells were re-stimulated on days 7 and 14 with wt1p-or pramep-pulsed autologous pbmcs or with cmv/ebvp added directly to the culture. mhctetramer staining was performed on days 14 and 21; ifn-γ elispot was conducted on day 21. [[p057 image] 1. functional wt1-specific and viralspecific t cells can be expanded post-pdt] results: pdt resulted in a drastic decrease of cd25 and/ or hla-dr activation marker-expressing cd4+ and cd8 + t cells. pdt-treated cells showed a significant increase in background: acute lymphoblastic leukemia (all) is the most common childhood cancer and relapsed or refractory all is still difficult to treat. engineered t cells equipped with a synthetic chimeric antigen receptor (car) targeting cd19 have demonstrated remarkable efficacy to treat all. however natural killer (nk) cells are known for their target-independent cytotoxic potential without induction of cytokine release syndrome (crs) or graft-versus-hostdisease (gvhd), car-nk cells can overcome the persisting problem in the therapy with car t cells. as the use of viral vector generated car nk cells is limited by theire genotoxicity, cost and regulatory demands, we are developing an innovative protocol using non-viral sleeping beauty (sb) transposition of third party nk cells as a source to produce 'off the shelf' car-engineered cell products. methods: nk cells are isolated from peripheral blood mononuclear cells (pbmcs) using cd56 selection kits. they are successfully expanded ex vivo with il-15 cytokine stimulation under feeder-cell free conditions. after few days of expansion nk cells are electroporated using pmaxgfp. transfection efficiency and percent of living cells after electroporation is analyzed by flow cytometry. transposition based nucleofection using an sb100x mrna and a minicircles (mc) dna vector is performed at different time points after nk cell isolation. the transient mc-venus longtime expansion and the viability after sb100x based nucleofection is measured over two weeks. furthermore, α-retroviral (α-rv) cd19-car transduction of nk cells with different viral amounts (moi) is conducted and the cytotoxicity of the engineered cd19-car-nk cells against the cd19 positiv cell line supb15 is addressed. results: for an α-rv cd19-car transduction of maximal 1x10 4 nk cells we could show transduction efficiency of 68,96% for moi10. the α-rv cd19-car modified nk cells had a high killing activity against cd19 positiv supb15 cells (e:t ration 1:1 90,85%) compared to cd19 negativ k562 cell lines (e:t ration 1:1 16,42%) and the non-transduced nk cells (e:t ratio 1:1 9,03%). in first experiments with pmaxgfp vector based nucleofection, we could show an increasing efficiency of 55,9% 48h post electroporation with a only slightly decreas of living cells (21,5%) comparing to the non-electroporated nk cell viability. using sb100x mrna with mc-venus dna we electrotransfected 1x10 6 nk cells after fews days of cultivation and we reached 36,6% of transfected nk cells 24h post electroporation and a transient expression of mc-venus positive nk cells up to 54,6% efficiency with an increasing rate of live cells over 14 days after electroporation. conclusions: the sleeping beauty based nucleofection of nk cells is a very promising non-viral method to generate more easy, safer and higher amounts of genetically modified third party nk cells for therapy of all and has also a broad range of clinical applications. disclosure: winfried s. wels is an inventor on a patent describing chimeric antigen receptors with an optimized hinge region. axel schambach is an inventor on a patent describing alpharetroviral sin vectors. michael hudecek and zoltan ivics are inventors on patents related to sleeping beaut gene transfer technology. the remaining authors have nothing to disclose. background: mature immune cells from the stem cell graft are essential for the graft-versus-tumor (gvt) effect to eliminate residual malignant cells after hematopoietic stem cell transplantation (hsct), but donor cells are also involved in complications such as graft-versus-host disease (gvhd). methods: we performed a prospective study of the detailed graft composition in 102 recipients of peripheral blood stem cells (pbsc) or bone marrow (bm) in order to identify correlations to clinical outcomes, table 1. grafts were characterized with concentrations of t cells and nk cells together with a multi-color flow cytometry panel with staining for tcrαβ, tcrγδ, vδ1, vδ2, cd3, cd4, cd8, hla-dr, cd196, cd45ro, cd45ra, cd16, cd56, cd337 and cd314 for detailed immune phenotyping. cell contents in stem cell grafts were analyzed both as fractions of cd45 positive lymphocytes and as absolute concentrations converted to transplanted cells/ kg. fractions were evaluated in patients receiving both bm and pbsc (n=102), while concentrations (cells/kg) were only analyzed in patients transplanted with pbsc (n=88). table] 1. table 1] [[p059 image] 1. figure 1 ] results: we found, that patients transplanted with graft nk cell doses above the median of 27x10 6 /kg and fractions of nk cells out of lymphocytes above the median of 8.1% had significantly increased relapse-free-survival compared to patients transplanted with grafts containing nk cell doses below these values, figure 1 ; results stayed significant in multivariate analyses. relapse incidence was significantly lower in uni-and multivariate analyses in patients receiving grafts with high nk cell fractions compared with low fractions, p=0.01, with 1-year relapse rates of 8% versus 27% in patients transplanted with high versus low fractions of nk cells, p=0.01. peripheral blood concentrations of nk cells obtained from samples from 17 pbsc donors before g-csf mobilization were significantly correlated to graft concentrations-and fractions of nk cells.. analyses of graft contents of nkt cells showed that the incidence of grade ii-iv acute gvhd were significantly lower in patients background: extracorporeal photopheresis (ecp) is an immunomodulatory treatment that has shown efficacy in steroid refractory acute gvhd, but the mechanism of action is only partially understood. there is no clear relationship between the ecp-treated mononuclear cells (mnc) or lymphocyte numbers and response to ecp. the objective of the study was to analyse the relationship between the infused subpopulation cellularity and response. methods: 65 patients from 7 different centers with a total of 1008 ecp procedures were retrospectively analized. ecp procedures were performed from january-2011 to june-2017. all ecp procedures were performed with the off-line system. the response was defined as responder (complete and partial response) and non-responder. infused cell numbers for lymphocytes, monocytes and mononuclear cells (lym+mon, mnc) were calculated. for analytic purposes, the median number of cells infused per procedure until response (or until the median number of procedures until response for non-responding patients) and the cumulative number of cells infused until response (or until the median in non-responders) were calculated for all the subgroups. same procedures were performed with the number cells infused until day 30 of ecp. finally, the response and survival impact of infusing a number of cells above or below the median and in different tertiles was assessed until the median number of procedures needed to achieve a response. [[p060 image] 1. results: the median number of procedures until response was 3. we observed a trend towards a higher median number of monocytes per procedure and cumulative infused monocytes in responding patients (median number infused 19.0 vs 13.5 x10 6 /kg p=0.071, cumulative infused median number 71.5 vs 41 x10 6 /kg p=0.067) that was lost in the day 30 of treatment. there was also a trend toward higher median infused mnc until response for responders (54 vs 37 x10 6 /kg p=0.087). we observed no differences in the number of lymphocytes infused, but patients who received a number of lymphocytes per procedure over the first tertile (31 x10 6 /kg) presented higher response rates (75% vs 45%, p=0.0317). none of the other analysed parameters showed a significant impact in overall survival. conclusions: patients with acute gvhd who responded to ecp received higher numbers of monocytes and mnc in the early phase of the treatment (a median of the first 3 processes). also the patients who received higher numbers of lymphocytes in the first procedures achieved a higher response rate. these findings suggest the possibility that higher number of treated and infused cells could influence the response to ecp, but specifically designed prospective studies are need to asses this possibility. disclosure: nothing to declare background: the field of kidney transplantation has made enormous progress over the last decades towards being a standard treatment for patients with end-stage renal disease. however, administration of immunosuppressive drugs is still one of the major limitations of long-term allograft survival. therefore, strategies for induction of donorspecific tolerance are highly desirable. to this aim, a clinical phase i study with donor-derived modulated immune cells (mics) was conducted. methods: donor-derived mics were manufactured under gmp conditions. potency of mics was tested by different in vitro bio-assays. mics were administered to patients with an escalation from 1.5 x 10 6 mics/kg on day -2 (n=3, group a), to 1.5 x 10 8 mics/kg on day -2 (n=3, group b) or on day -7 (n=4, group c) before kidney transplantation accompanied by standard immunosuppressive medication post-transplantation. frequency of adverse events (ae) was assessed from day 30 until day 360 post-transplant. dynamic changes of various lymphocyte subsets in patients after mic therapy were detected by multicolor flow cytometry. donor-specific immunosuppression was assessed by measuring anti-donor antibodies and mixed lymphocyte reaction (mlr) against donor and thirdparty cells. results: in all kidney transplant recipients, we observed a median serum creatinine of 1.4 mg/dl at day 30 which remained stable until day 360 (median creatinine of 1.4 mg/ dl) without significant proteinuria. none of patients experienced rejection episode. 69 aes were observed while three aes being severe. most importantly, none of them was associated with mics transfusion. besides two infectious complications, no post-transplant positive cross match results against the donor or titers of de novo donorspecific antibodies were recorded. notably, immunosuppressive therapy could be reduced without signs of rejection in group c. after infusion, we observed a dramatic increase of cd19 + b cells up to a median of 300 cells/μl until day 30, followed by a reduction to 35 cells/μl on day 180 in group c. notably, regulatory b cells significantly increased from a median of 2% on day 30 to 20% on day 180. in parallel, the plasma il-10/tnf-α ratio increased from a median of 0.05 before cell therapy to 0.11 on day 180. after mic cell therapy recipient lymphocytes showed no or only minimal reactivity against irradiated donor pbmcs in vitro, while reactivity against 3 rd -party-donor pbmcs was not impaired. moreover, in vitro mics product demonstrated their immunomodulatory potency by inducing tolerogenic dendritic cells (tdcs) characterized by low expression of costimulatory (cd80, cd86) and maturation (cd83) as well as high expression of inhibitory marker cd103. functionally, tdcs could inhibit not only the release of ifn-γ but also the proliferation of cmv specific cd8 + t cells. moreover, mic-induced tdcs showed the capacity to inhibit donor-specific allo-reactive cd4 + and cd8 + t cell proliferation. conclusions: mic cell therapy modulates the immune system of kidney transplant recipients by increasing the ratio of regulatory b cells and facilitates the reduction of conventional immunosuppressive therapy without allograft injury or rejection episodes. therefore, mic cell therapy represents a promising strategy in transplantation medicine. we currently prepare a phase ii trial with mic cell therapy. disclosure: nothing to declare p062 genome editing of graft-derived t cells for postbackground: immunotherapy using car t cells has shown promising results to fight cancer. however, car-t cell production requires specialized infrastructure and operators, which implies high cost and centralized production. automated production of car-t cells in clinimacs prodigy device allows clinical-grade manufacturing of car t cells. methods: 100 million cd45ramemory t cells from healthy donors were cultured in texmacs supplemented with 100 iu/ml il-2. at day 0 cells were activated with t cell transact for 24h. at day one, activated cd45ramemory t cells were transduced with nkg2d-cd8tm-41bb-cd3z lentiviral vector at moi = 2. then, nkg2d-car t cells were expanded for 10-13 days. nkg2d-car t cell products were next harvested, counted and analyzed for viability, nkg2d-car expression and anti-tumor cytotoxicity. different quality tests including sterility, vector copy number, genetic stability, quantification of viral particles in the supernatant, myc/tert expression and endotoxin detection were performed. spare cells were cryopreserved either in autologous plasma and 10% dmso, m199 35% albumin and 9%dmso or hypothermosol. after 12 months, cryopreserved nkg2d-car t cell products were analyzed for viability, nkg2d-car expression and cytotoxicity. results: nkg2d-car memory t cells expanded up to 2076 ± 697 million with 77,8 ± 20% nkg2d-car expression and 76 ± 10% viability. harvested car t cells showed 90 ± 14% of specific lysis against jurkat cells and 31 ± 16% against 531mii osteosarcoma cell line. no microbiological contamination was observed in final car t cells products. vector copy number was ≤5 in all validations except for one. cgh and karyotype showed no genetic alterations. free viral particles were undetectable in the supernatants. no overexpression of myc/tert was found except for one validation. endotoxins were ≤0.25eu/ml. all cryopreserved nkg2d-car t cell products kept nkg2d-car expression one year after freezing. however, viability and cytotoxicity was best preserved using autologous plasma 10%dmso. conclusions: automated production of large-scale clinical-grade nkg2d-car t cells using clinimacs prodigy is feasible and reproducible, allowing a decentralized protocol to generate car t cells for clinical use. background: immune reconstitution (ir) is essential to control severe infections after hematopoietic stem cell transplantation (hsct). reconstitution of adaptive immunity may take up to 2 years to recover t-lymphocytes (lt). delay in early lt recovery increases the risk of relapse, viral infections and transplant related mortality. adoptive transfer of selected t cell subset with low alloreactivity potential is emerging as a strategy to improve ir. methods: depletion of cd45ra+ naive t cells, preserving cd45ro+ memory t cells could provide functional lymphocytes to protect against infection and leukemia relapse with low risk of graft versus host disease (gvhd). we present our experience with high-dose donor cd45ro + memory t cell as donor lymphocyte infusions (dli) to assess safety and outcome. a total of 58 dli of cd45ro+ after hsct was performed in cases of cmv/ebv reactivation (50%), mixed chimerism (26%), persistent lymphopenia (8,5%), graft rejection (3,5%) , relapse (3%) or to boost ir (7%). dli product was obtained performing a cd45ra depletion on donor leukapheresis product using the clinimacs® device. results: twenty-two pediatric patients, median age 11 years (range 3-18), with malignant (n=15) and nonmalignant diseases (7), received cd45ra+ (n=14), tcr alpha/beta (n=2) depleted grafts from haploidentical and cd45ra+ depleted grafts from match unrelated (n=6) and match related (n=2) donors. at a median of 97 days (range 15-462) after transplantation, patients received a total of 58 dli of cd45ro+ cells, median 2 (range 1-6), containing a median of 2.87x10 7 /kg (range 4.8x10 4 -2x10 8 /kg), cd3 +cd45ro+ 1.05x10 7 /kg (range 4.8x10 4 -1.09x10 8 /kg) and cd45ra+ cells 5 x10 2 /kg (range 0-9.8x10 4 /kg). all infusions were well-tolerated and did not develop or worsen gvhd. a total of 11/29 episodes of cmv/ebv viral reactivations decreased viral load, 4/29 cleared viral load and 5/29 showed a clinical improvement. a total of 4/5 patients with persistent lymphopenia there was a slightly increase in total lymphocyte count, but not to normal levels. prophylactic dli of cd45ro+ to boost ir increased lymphocyte count in 2 of 3 cases. none of the dli administered in cases of mixed chimerism, graft failure or relapse were effective in reverting those situations. conclusions: our preliminary data suggest that infusions of high dose cd45ro+ memory t cells are a safe adoptive immunotherapy strategy. efficacy has been observed in patients with lymphopenia and cmv/ebv reactivation, with no positive results in patients with mixed chimerism, graft failure and relapse. however, to determine the real efficacy of this strategy, prospective studies are required. disclosure: nothing to declare. background: increasing clinical trials have confirmed that chimeric antigen receptor t cells (car-t) targeting cd19 antigen (car-t-19) is a promising effective approach for the treatment of relapsed/refractory(r/r) b-cell lineage malignancies. considering cd19 is frequently expressed in large part of t(8;21) acute myeloid leukemia (aml) cells, we suppose that car-t-19 may be used as an approach to rescuing r/r t(8;21) aml patients. methods: both patients received lymphodepletion chemotherapy with decitabine 20mg/m 2 ×5d, fludarabine 30mg/m 2 ×3d and cyclophosphamide 300mg/m 2 ×3d (dac +fc). two days after chemotherapy, autologous/allogeneic cart-19 cells provided by the unicar-therapy biomedicine technology co.(shanghai, china) at a total dose of 5-10×10 6 cells per kilogram(kg) were infused dose escalation within 2 to 3 days. the research protocol was approved by the institutional review boards of the first affiliated hospital of soochow university and both patients gave written informed consent. results: both cases responded well with transient and reversible toxicities. case 1 presented with grade 1 cytokine release syndrome (crs), manifested by intermittent fever and chill from day 4 after car-t-19 infusion for half months associated with neutropenia. car-t cells expansion were observed in blood without obvious increase of cytokines. after infusion, case 1 achieved and maintained molecular complete remission (cr) for more than 10 months. case 2 presented with grade 3 crs manifested by continuous high fever, hypotension and grade 1 liver disfunction from day 1 after car-t-19 cell infusion for 1 week. obvious cytokines releasing (peak il-6 serum concentration 1774.5 pg/ml, peak crp serum concentration 367pg/ml) were detected which were associated with car-t-19 cell expansion in blood and no severe off-tumor effect was observed. after infusion, case 2 achieved hematological cr and cytogenetic cr and got 3 months disease free survival. conclusions: our report implicates that car-t-19 is a safe and promising approach to managing r/r t(8;21)aml with cd19 expression, and may provide a salvage treatment approach for all aml patients with cd19 expression and benefit a certain population with aml besides b-linage malignancies. clinical trial registry: na disclosure: nothing to declare. this work was supported by research grants from the national key r&d program of china (2016yfc0902800), national natural science foundation of china (81873443, 81270645, 81400155, 81500146) background: chimeric antigen receptor engineered t (car-t) cells have emerged as a powerful cellular therapy to treat malignant disease, which is currently revolutionizing field of cancer immunotherapy. a cryopreservation step postmanufacture is not only a logistical necessity for large scale cell manufacturing processes but also a mandatory request by regulatory authorities. in case relapse after 1 st car-t cell transplantation, a second application, maybe at a higher dose constitutes a therapeutical option. however, data concerning clinical grade car-t cell stability and functionality after months of cryopreservation have not been released by companies so far. to investigate the effect of cryopreservation on car-t cells, we performed this study. methods: different batches of cd19 car-t cells were manufactured according to gmp requirements at our institution. final car-t products were frozen at concentrations of 1 x 10 7 cells/ml (high batch) and 2 x 10 6 cells/ml (low batch) by a controlled freezing process with the biofreeze bv40 device and stored in liquid nitrogen tanks below -150°c until release. quality control tests for sterility, endotoxin and mycoplasma were performed for each batch according to european pharmacopoeia and united states pharmacopoeia guidelines. stability of cd19 car-t cells in terms of viability, recovery, transduction efficiency and functional capacity were determined by microscopy, multi-parametric flow cytometry as well as chromium-51 release tests following our sops. results: all the results of quality controls fully met the requirements of the regulatory authorities. stability results were highly robust and reproducible over time for all our gmp car-t batches. duration of cryopreservation (up to 90 days) had no negative influence on cell viability, recovery of viable cd19 car-t cells and transduction efficiency. however, the cell concentration for cryopreservation has a significant impact on the post-thawing viability (low batches vs. high batches: 96.33 ± 2.17 vs. 74.87 ± 8.68, p < 0.05) and recovery (low batches vs. high batches: 89.67 ± 6.76 vs. 74.90 ± 9.19, p < 0.05) of cryopreserved cd19 car-t cells, but not the transduction efficiency. moreover, we observed four transient side-effects of cryopreservation on the amount of cytokines released by car-t cells, the cytokine release on a per-cell basis, the multifunctionality of car-t cells and the killing capacity. of note, functional capacity of cryopreserved car-t cells after overnight resting was comparable or even enhanced for inf-γ and tnf-α release by cd4 + and cd8 + cd19 car-t cells when compared to fresh car-t cells. the multi-functionality of car-t cells could be preserved. furthermore, the killing capacity of cryopreserved cd19 car-t cells after overnight resting could reach the level of non-cryopreserved/fresh car-t cells. conclusions: cryopreservation up to 90 days has no harmful effect on transduction efficiency and functionalities of car-t cells. however, the cell number per milliliter freezing medium matters. dose over 1 x 10 7 cells/ml should be avoided. for the conduction of in vitro bio-assays to determine the function of car-t cells, an overnight resting process could mimic the situation after clinical application and eliminate the transient side-effects of cryopreservation to fully regain the functional potency of car-t cells. disclosure: nothing to declare background: dc and specific t-cells are important mediators of ctl-responses. we could already show that allogeneic donor-or autologous t-cells obtained from amlpatients can be stimulated by dc leu , resulting in a very efficient lysis of naive blasts. methods: chemokine-release (cxcl8, -9, -10, ccl2, -5, and il-12) was analysed by cytometric bead array in serum of aml/mds-pts as well as in supernatants from 5 different dc-generating-methods and correlated with pts' clinical course, dc-and t-cell-interactions as well as specific t-cell-reactions. the lytic activity of dcleu/blast -stimulated t-cells in mlc against naive blasts was quantified in a cytotoxicity assay. results: minimal differences in median chemokine-levels in pts' serum subdivided in subtypes were seen, but higher release of cxcl8, -9, -10 and lower release of ccl2 and -5 tendentially correlated with more favourable subtypes (< 50 years of age, < 80% blasts in pb). in persisting disease, a higher serum-release of ccl5 and at relapse a significantly higher ccl2-release were found compared to first diagnosis -pointing to a change of 'disease activity' on a chemokine level. whereas chemokine-levels in dc-culture supernatants compared to serum were variable, clear correlations with lateron (after stimulating t-cells with dcleu in mlc) improved antileukemic t-cell activity were seen: higher values of all chemokines in dc-culture supernatants always correlated with improved t-cells' antileukemic activity (compared to stimulation with blast-containing mnc as control) -whereas with respect to the corresponding serum values higher release of cxcl8, -9, and -10 but lower values of ccl5 and -2 correlated with higher probabilities to improve antileukemic activity of dcleu-stimulated (vs. blaststimulated) t-cells. predictive significant cut-off-values could be evaluated separating the groups compared. moreover, correlations with lateron achieved response to immunotherapy and occurrence of gvhd were seen: higher serum values of cxcl8, -9, -10 and ccl2 and lower values of ccl5 correlated with achieved response to immunotherapy. predictive cut-off-values could be evaluated separating the groups compared in 'responders' and 'non-responders'. higher levels of ccl2 and -5 but lower levels of cxcl8, -9, -10 correlated with occurrence of gvhd. conclusions: we conclude, that in aml-pts' serum higher values of cxcl8, -9, -10 and lower values of ccl5 and in part of ccl2 correlate with more favorable subtypes and improved antitumor'-reactive function. since in dcculture supernatants higher values of all chemokines correlated with improved antileukemic t-cell reactivity we conclude a change of functionality of ccl5 and -2 from an 'inflammatory' or 'tumor-promoting' to an 'antitumor'reactive function. this knowledge can contribute to develop immune-modifying strategies that promote antileukemic adaptive immune-responses. disclosure: nothing to declare background: allogeneic hematopoietic cell transplantation (allo-hct) is a curative treatment option for patients suffering from hematologic malignancies. infusion of donor lymphocytes (dlis) can induce sustained remission in case of minimal residual disease or relapse through potent graftversus-leukemia (gvl) effects, although graft-versus-host disease (gvhd) represents a common dose-limiting toxicity. as invariant natural killer t (inkt) cells are known to prevent gvhd while promoting beneficial anti-tumor effects, we investigated the role of inkt cells for successful dlis. methods: we analyzed dli samples by flow cytometry. inkt cells were identified by staining with pbs57-loaded cd1d tetramers. culture-expanded and purified dli-inkts were then tested against tumor cell lines and primary leukemia cells in an ex vivo tumor control model. tumor cell viability after coincubation with dli-inkts was measured by flow cytometry using 7-aad. results: inkt cells represent 0.05% (range 0.001-0.55%) of donor lymphocytes and can be expanded 300fold following a two-week protocol with a preferential expansion of cd4+ inkt cells. tumor cell lines such as jurkat were efficiently lysed after coincubation with dli-inkts. cd107a as a marker of degranulation was significantly upregulated on dli-inkts after stimulation by jurkat. in addition, increased concentrations of tnfα, ifn-γ, sfasl and perforin were measured after coincubation of dli-inkts with jurkat. we observed that tumor cell lysis correlated with the expression of the mhc-i-like molecule cd1d. consequently, adding a cd1d antibody to the coculture abrogated the dli-inktmediated kill of tumor cells. dli-inkts also efficiently lysed primary leukemia cells such as aml blasts: expression of cd1d on these aml blasts significantly correlated with dli-inkt-mediated tumor cell lysis (r 2 =0.7, p=0.03). conclusions: ex vivo expansion of dli-inkts and subsequent dli enrichment is an immunotherapeutic approach that could improve leukemia control and thus, prevent relapse after allo-hct without exacerbating gvhd. disclosure: nothing to declare. generation of antigen-specific cytotoxic t lymphocytes targeting wt1 using activated b cells sun ok yun 1 , kyung won baek 1 , hee young shin 1 , hyoung jin kang 1 1 seoul national university, seoul, korea, republic of background: the wilms tumor antigen 1 (wt1) is highly expressed in many malignancies including leukemia and targeting wt1 as a tumor associated antigen (taa) in cancer immunotherapy is attractive. in this study, we generated wt1-specific cytotoxic t lymphocytes to confirm if activated b cells can act as a cancer antigen presenting cell and induce ctls. methods: for the induction of ctls against wt1, activated b cells were used as an antigen presenting cells. b cells were isolated from pbmcs of normal healthy donors and activated with α-galactosylceramide (α-galcer) and nucleofected with wt1-coding plasmid dna. activated b cells were the cultured with pbmcs for 17days in vitro and harvested for assay. results: cells expanded about 3 times after 17 days of culture. we examined characteristic of wt1-specific ctls by their surface markers. wt1-specific ctls had more than 90% cd3+ marker, and ratio of cd8 to cd4 was 1.7-5.8. we also examined nkt cell markers to see if nkt cells were activated by il-15, a cytokine used in the induction of ctls, and the portion of nkt cells was about 2%. the ctls showed a decrease in naïve cell (cd62l+cd45ra +) and an increase in effector memory (cd62l+cd45ra-) and central memory (cd62l-cd45ra-) compared with non-stimulated pbmcs. subsequently, the ifn-γ elispot (enzyme-linked immunospot) assay was performed to confirm the response of the induced wt1-specific ctls to the wt1 antigen. when wt1-specific ctls encounters a target that does not have a wt1 antigen, it did not produce ifn-γ, but when it encounters a target cells loaded wt1 antigen, it responded to secrete ifn-γ. killing assays were also performed to determine the immunogenicity of induced ctls. the induced wt1 ctls was found to be killing more than 90% when the e:t ratio was 10:1 when the autologous pbmc met the target with wt1 pepmix. in addition, we found that wt1 ctls has killing activity when it encounters leukemia cell lines that express wt1 and matched hla-a*0201. conclusions: in this study, we can induce antigenspecific ctls that specifically react to wt1 using activated b cells as antigen-presenting cells. these observations confirmed that b cells activated by α-galcer can act as a taa presenting cell to induce taa specific ctls as viral antigen, such as pp65 and ie1, and consequently wt1specific ctls could be induced. moreover, ctls induced activated b cells had ability to recognize and kill the target cells expressing wt1 correctly. our results demonstrate that these in vitro expanded wt1-specific ctls using activated b cells can be a promising candidate for adoptive immunotherapy against cancer. disclosure: nothing to declare judith böhringer 1 , michael schumm 1 , christiane braun 1 , marina schmidt 1 , patrick schlegel 1 , christian seitz 1 , murat aktas 2 , georg rauser 2 , sandra karitzky 2 , peter lang 1 , rupert handgretinger 1 1 university children's hospital tübingen, tübingen, germany, 2 miltenyi biotec gmbh, bergisch gladbach, germany background: t cells with chimeric antigen receptors (cars) on their surface facilitate to target specific surface expressed antigens. research and clinical trials with cd19-car t cells show impressive remission induction rates and increased survival in heavily pretreated patients. therefore, car t cells are introduced as new potent cellular therapeutics in the clinical routine. in order to establish the manufacture of cd19-car t cells, validation runs with the fully automated clinimacs prodigy t cell transduction process have been performed using the miltenyi anti-cd19-car lentiviral vector. methods: unmobilized leukaphereses from 3 donors (2 x healthy, 1 x all) were used for the clinimacs prodigy t cell transduction process. leukocytes undergo a cd4 + / cd8 + t cell enrichment via magnetic beads, followed by stimulation with macs ® gmp t cell transact™, transduction with an anti-cd19-car lentiviral vector, expansion with il7 and il15, and final formulation to the cellular product. during and after the manufacture, facs analyses were performed as well as cytotoxicity assays after cd19-car t cell production. results: total volumes of leukaphereses were between 60 and 280 ml with 2.6 -4.0 x 10 9 total mononuclear cells. after enrichment 100x10 6 cd4 + / cd8 + t cells were transduced with anti-cd19-car lentiviral vector and were further expanded. cells were harvested on day 12. the final cell counts of the cellular products were 6.1, 7.2 and 5.0 x 10 9 mononuclear cells from two healthy volunteers and the all-patient, respectively. the transduction efficiency of the cd19-car t cells was 48.6%, 43.4% and 32.0% among viable cd3 + cells. the final count of car t cells was therefore 2.9, 3.0 and 1.5 x 10 9 cells. the final products exerted excellent cytolytic activity against cd19 + bcp-all cell line nalm-6. importantly, cd19-car t cells generated from the all patient demonstrated complete eradication of autologous blasts at 0.3 to 1 e:t ratio after 24 hours incubation. conclusions: the clinimacs prodigy t cell transduction process has been shown to run a fully-automated manufacturing process over 12 days without any deviations in a clean room environment on a single device. the user interaction was reduced to activities at only 3 days to set up the system and provide fresh medium and reagents. the transduction process yielded a high number of t cells with a high frequency of cd19-car t transduced cells. the results were comparable for both unmobilized leukaphereses from healthy donors and showed expected slightly lower results in the patient. finally, these results demonstrate that the clinimacs prodigy t cell transduction process is well suited to provide the clinical mb-cart19.1 r/r cd19+ bcm study with appropriate investigational medical products. disclosure background: allogeneic hematopoietic stem cell transplantation (allohct) is an effective strategy in the long term control of several hematologic diseases, however, patients could experience complications, as graft versus host disease (gvhd) and disease relapse. recently, the introduction of post-transplant cyclophosphamide (ptcy) allowed to significantly reduce gvhd, but disease relapse remains an important issue. donor-lymphocyte infusion (dli) is an established adoptive cell therapy for disease relapse after allohsct, but, in order to be efficient and safe, patients have to be off immunosuppression treatments and gvdh-free. here we report our data about efficacy and safety of dli infusion as treatment for disease relapse in patients who received peripheral blood stem cell transplantation (allopbsct) from hla-matched unrelated/related plus ptcy as gvdh prophylaxis in our clinical trial (nct 02300571). methods: we collected data from 13 patients, treated with ptcy (50 mg/kg/die, days +3+4), mofetil mycophenolate (mmf) and tacrolimus (t) as gvhd prophylaxis after allo-pbsct, who received dli infusions. they were treated between january 2013 and october 2018. we report data about overall response rate (orr), disease control rate (dcr), and dli-related mortality and morbidity. diagnosis were as follow: 5 had multiple myeloma, 3 had acute myeloid leukemia, 3 had acute lymphoblastic leukemia and 2 had lymphomas. all patients but one, who had chimerism loss, received dli because of disease relapse. results: median time between transplant and dli was 9 (range 3-87) months. median number of dli infusions was 2 (range 1-13). 10 patients (77%) received cyclophosphamide 300 mg/m2 preparative regimen the day before the cryopreserved dli infusions, while in the other 3 cases dli were associated with lenalidomide, ponatinib and 5-azacitidine. the overall response rate (orr) was 50%, while disease control rate (dcr) was achieved in 75%. the patient who received dli because of loss of chimerism converted it in full donor after 2 infusions. after dli treatment the incidence of acute gvhd grade i-iii was 54%, while was 46% for grade ii-iii and patients were started on short course of systemic immunosuppression treatments . none of these patients died because of dli adverse events. estimated 1-year overall survival was 77% with a limited follow-up length (6 months). conclusions: the infusion of non-manipulated lymphocytes from allogeneic donors is a valuable and safe strategy of treatment for patients relapsing after allopbsct with ptcy. ptcy showed high efficacy in gvhd prevention, allowing early discontinuation of immunosuppression drugs. because of this, we can reach the goal to transform transplant in a platform where we could add early dli infusions as a new strategy for disease control. clinical trial registry: nct 02300571 disclosure: nothing to declare p074 extracorporeal photopheresis in the treatment of refractory chronic gvhd: analysis of mononuclear cell infusion gillen oarbeascoa 1 , maria luisa lozano 2,3,4 , luisa maria guerra 5 , cristina amunarriz 6 , nuria revilla 2,3,4 , pastora iniesta 2,3,4 , cynthia acosta fleitas 5 , jose luis arroyo 6 , eva martinez revuelta 7 , andrea galego 8 , dolores hernandez-maraver 9 , mi kwon 1,10 , aurora viejo 9 , jose maria garcia gala 7 , concepcion andon saavedra 8 , jose luis diez-martin 1, 10, 11 background: extracorporeal photopheresis (ecp) is an immunomodulatory treatment that has shown efficacy in steroid refractory chronic gvhd, but the mechanism of action is only partially understood. in some studies, a correlation has been suggested between treated mononuclear cells (mnc) or lymphocytes and response to ecp. the objective of the study was to analyze the relationship between the infused cellularity and response in chronic gvhd. methods: 48 patients from 7 different centers with a total of 930 ecp procedures were retrospectively analyzed. ecp procedures were performed from january-2011 to june-2017. all ecp procedures were performed with the off-line system. the response was defined as responder (complete and partial response) and non-responder. infused cell numbers for lymphocytes, monocytes and mononuclear cells (lym+mon, mnc) were calculated. for analytic purposes, the median number of cells infused per procedure until response (or until the median number of procedures until response for non-responding patients) and the cumulative number of cells infused until response (or until the median in non-responders) were calculated for all the subgroups. same procedures were performed with the number cells infused until day 30 of ecp. finally, the response and survival impact of infusing a number of cells over or below the median and in different tertiles (t1, t2 and t3) was assessed until the median number of procedures needed to achieve a response. results: the median number of procedures until response was 3. we observed no differences in the median number of lymphocytes, monocytes or mncs infused until response or until day 30 between responding and non-responding patients. there were no differences in response if patients received lymphocytes or monocytes above or below the median number. nevertheless, patients that received a total absolute number of mncs above the median (64x108 cells) showed a trend towards a higher response rate (75% vs 61%, p=0.09). the patients that received a cumulative number of lymphocytes in the 3 first ecp procedures above the median showed improved overall survival (os) (2y os 85% vs 55%, p=0.024). patients that received a number of monocytes above the median showed a trend towards better survival (p=0.09), that was significant when the number of monocytes infused surpassed the first tertile (2y os 38% for t1, 79% for t2, 92% for t3, p=0.003). finally, the patients that received a cumulative number of mncs above the first tertile also showed improved survival (2y os 47% for t1, 74% for t2, 94% for t3, p=0.015). conclusions: there were no differences in the infused cellularity between responding and non-responding patients with chronic gvhd. at the same time, we found that except for a trend toward better response with higher mncs infused, there was no relationship between lymphocytes and monocytes with the response rate as other previous studies have suggested. however, even if there is no relationship with the response rate, the patients receiving the highest numbers of lymphocytes, monocytes and mncs in the cohort showed an improved survival, suggesting that larger quantities of cells could exhibit a protective effect. nevertheless, prospective studies that address this relationship are needed. disclosure: nothing to declare comparative analysis of the cytotoxic potential of cytokine-induced killer and natural killer cells for neuroblastoma therapy annekathrin heinze 1 , beatrice grebe 1 , eva mudry 1 , jochen früh 1 , bushra rais 1 , claudia cappel 1 , sabine hünecke 1 , eva rettinger 1 , thomas klingebiel 1 , peter bader 1 , evelyn ullrich 1,2 1 university hospital frankfurt, frankfurt, germany, 2 german cancer consortium (dktk) partner site:, frankfurt, germany background: neuroblastoma (nb) is the most common solid extracranial tumor in childhood. despite therapeutic progress, prognosis for high-risk nb is poor and innovative therapies are of medical need. therefore, we investigated the cytotoxic potential of interleukin (il)-activated natural killer (nk) cells compared to activated cytokine-induced killer (cik) cells against different human nb cell lines in vitro. methods: nk cells were isolated from peripheral blood mononuclear cells (pbmcs) using cd56 enrichment or cd3/cd19 depletion kits. they were successfully expanded ex vivo with different cytokine combinations such as il-2, il-15, il-18 and/or il-21 under feeder-cell free conditions. in contrast, cik cells were generated from pbmcs by ex vivo stimulation with interferon-γ, il-2, okt-3 and il-15. a comparative analysis of expansion rate, purity, phenotype and cytotoxic activity against different nb cell lines following different culturing protocols was performed. results: cd56 enriched nk cells showed a median expansion rate of 4.3-fold after 10 to 12 days in culture with a final frequency up to 99.0% nk cells and a median frequency of 0.5% cd3 + cd56 -t cells. in contrast, the starting cell product after cd3/cd19 depletion consisted of a median frequency of 43.5% nk cells that expanded significantly faster with 7.5-fold and also reached up to 98.6% purity without any relevant t cell contamination. cik cells expanded with a median rate of 30.8-fold and contained 3.3% nk, 84.2% t and 6.2% nk-like t cells. interestingly, nk cells, particularly after cd3/cd19, showed a significantly higher median cytotoxic capacity against nb cells depletion (46.6% for cd56 enrichment, 53.7% for cd3/cd19 depletion) compared to cik cells that induced 7.2% killing of nb cells with e:t ratio 5:1 in a 3 hours' co-incubation assay. interestingly, prolonging the ex vivo stimulation after cd3/cd19 depletion to 15 days enhanced the median expansion rate to 12.3-fold with a slightly reduced cytotoxic potential (40.9% for 11 days' ex vivo expansion, 31.1% for 15 days' ex vivo expansion, comparison of the same donors). the addition of an il21boost prior harvesting increased the expansion rate to median 12.6-fold (compared to 11.7-fold for the same donors) with an improved cytotoxicity of 51.5% (compared to 45.8%) . fortunately, all nk cell products showed a high viability and no relevant t or b cell contamination (median < 0.2%). interestingly, further optimization of the culturing procedure with use of another cell culture medium led to an improved median 24.4-fold (compared to 9.6-fold) nk cell expansion rate in 15 days, also resulting in comparable cytotoxicity of 52.5%. conclusions: nk and cik cell products may offer an innovative immune therapeutic option for patients with high-risk nb after allogenic stem cell transplantation. our study revealed that nk cells have a significantly higher cytotoxic potential to combat nb. interestingly, the use of il-15 expanded and il-21 activated nk cells developed from a cd3/19 depleted apheresis product is highly promising as additional immunotherapy in combination with haploidentical stem cell transplantation of children with nb. disclosure: nothing to declare. quantitative determination of donor allo-reactive t-cells in haploidentical donor-recipient pairs by enzymelinked immunospot (elispot) and mixed lymphocyte culture (mlc) assays background: t-cell alloreactivity is responsible not only for graft versus host disease and morbidity, associated with hematopoietic stem cell transplantation (hsct) but also for graft-versus-leukemia (gvl) activity. in this regard, monitoring and quantitation of alloreactive t-cells (allo-t) may potentially provide valuable information for individualized clinical management of transplant recipients. the aim of this study was the optimization of allo-т detection and comparison of the elispot and mlc assays. methods: allo-t were determined in 20 haploidentical donor-recipient pairs before hsct. donor mononuclear cells (mnc) served as effector cells (ec) . patient cd3depleted mnc were used as stimulatory cells (sc).the ratio ec:sc were 5:1 and 10:1. the frequency of allo-t in donor peripheral blood was tested in elispot assay and mlc. elispot provides the detection and quantitation of activated t-cells on the basis of cytokine secreted by each cell. the co-incubation time was 24 h for ifn-gamma and 48 h for il-2 detection. in mcl assay donor mnc were labeled with cfse and allo-t, proliferating in response to stimulation with alloantigens, were determined by flow cytometry on day 5. results: the median number of ifn-gamma producing allo-t per 300 000 donor mnc was 143,5 (2-1469; ec:sc ratio -5:1) and 75,0 (1-1440; ec:sc ratio -10:1). the median frequency of allo-t was 0,056% (0,00076 -0,538; ec:sc ratio -5:1) and 0,019% (0,00019 -0,632; ec:sc ratio -10:1) among lymphocytes. il-2-producing allo-t were less frequent in donor mnc in comparison with ifngamma-producing allo-t. the median number per 300 000 mnc was 7,5 (0,5-356; ec:sc ratio -5:1) and 6,0 (0-169; ec:sc ratio -10:1). the median frequency of il-2-allo-t was 0,0028% (0,0002 -0,130; ec:sc ratio -5:1) and 0,0022% (0 -0,0619; ec:sc ratio -10:1) among lymphocytes. the ec:sc ratio 10:1 is enough for stimulation of il-2 producing by mnc in elispot assay, but for optimal stimulation of ifn-gamma producing cells ec:sc ratio 5:1 is preferable. this suggests that allo-t are predominantly ifn-gamma producing cells. alloreactive proliferating t-clones were detected in mlc in 10 of 14 donor-recipient pairs on 5 day of cocultivation. median percentage of proliferating t-clones were 9,5% (2,1 -32,5; ec:sc ratio -5:1) and 7,6% (3,0 -24,1 ; ec:sc ratio -10:1) among lymphocytes. however, mlc assay only permit a qualitative analysis that confirmed the presence of alloreactive t-clones, giving no information on their frequency within the culture. results of elispot and mcl assay directly correlated. conclusions: allo-t were detected in 100,0% of assayed haploidentical donor-recipient pairs by elispot and only in 71,4% by mlc. this difference in detection is due to the fact that elispot allows to detect single cytokine secreting cell whereas mlc can reveal proliferating аllo-t clones. the analysis of allo-t in haploidentical donor-recipient pairs may provide rationale to manipulate the allo-immune response and to exploit the powerful ability of allo-t to control hematologic malignancies. disclosure: nothing to declare allogeneic mesenchymal stromal cell as rescue therapy in an infant with life-threatening respiratory syndrome due to a filamin a mutation background: cell-based therapy has gained attention in the respiratory system diseases and encouraging results are reported following mesenchymal stromal cells (mscs) administration. due to their capacity to produce and secrete a variety of paracrine factors and bioactive macromolecules, mscs became a key player in lung tissue injuries and function, reducing fibrosis, promoting the normal development of alveoli and pulmonary vessels. for the first time we used the msc infusions as rescue therapy in a pediatric patient with flna gene mutation and life-threatening respiratory syndrome. methods: a child with a new pathogenic variant of the flna gene c.7391_7403del; (p.val2464alafster5) at the age of 18 months, due to the serious and irreversible chronic respiratory failure and dismal prognosis, was treated with 4 intravenous infusions of allogeneic bone marrow (bm)-mscs at the dose of 1×10 6 mscs/kg body weight. bm-mscs were produced at "cell factory", fondazione irccs policlinico s. matteo, pavia,isolated and expanded ex vivo from healthy donor bm, following a previously reported protocol. premedication with antistaminic drug, 30 min before every infusion to avoid any potential reaction was performed. the evolution of the respiratory condition was detected. peripheral blood were collected before each msc treatment for treg and th17 monitoring. treg, defined as cd4+ cd127neg cd25+ cells expressing the forkhead box p3 (foxp3) transcription factor, and th17, defined as cd4+ cells expressing intracellular il-17, evaluation was performed by flow cytometry (facscanto; bd biosciences, san diego, ca) as previously reported, following standard procedures. results: no acute adverse events related to mscs infusion was recorded. during follow-up, patient maintained a good general condition and showed a regular growth. no systemic or respiratory infections occurred. after the second infusion, the child experienced a progressive improvement of his clinical respiratory condition, with a good adaptation to mechanical ventilation, in the absence of episodes of respiratory exacerbations. the baby maintains adequate volumes of exchange with substantial reduction of the inspiratory support. a reduction of trigger sensitivity was also obtained. thorax ct scan showed a recovery of the basal parenchyma bilaterally and the improvement of the anatomical-functional alignment and aerial penetration. after the first msc administration, an enrichment of treg and th17 percentage in peripheral blood, was observed. while, after the second msc infusion a significant increase in treg/th17 ratio was noted. conclusions: this report suggest that msc serial infusions are a promising therapy in aiding the respiratory failure, even in a pediatric patient with flna mutation. intravenous administrations of allogeneic mscs are feasible and safe without toxicity. our results suggest that to mitigate lung injury, mscs may act as regulators of treg and th17 balance. further investigations are upcoming to establish the useful of this therapeutic proposal in interstitial lung diseases in children. disclosure: nothig to declare p078 feasibility of il-15 stimulated donor nk cells manufacturing for early infusion in patients with high risk acute myeloid leukemia undergoing haploidentical transplantation background: nk cells provide a potent antitumor effect in the setting of manipulated haploidentical hematopoietic stem cell transplant (haplo-hsct). we propose a novel strategy to enhance the antitumor effect of allogeneic transplant through the infusion of nk cells stimulated with il-15 exvivo in adult high-risk acute myeloid leukemia (aml) patients undergoing unmanipulated haplo-hsct. the objective of this study was to provide efficiency and productivity data obtained in the manufactured cellular products infused. methods: selection criteria included patients with highrisk aml undergoing unmanipulated haplo-hsct. lymphoapheresis of the haploidentical donor was performed using spectra optia (terumo® bct) on days +6 and +13 after transplant. from the obtained product a double immunomagnetic cellular selection with clinimacs system (miltenyi biotec®) was performed in two steps: cd3+ depletion followed by positive cd56+ selection. the obtained an enriched cellular product of cd3-cd56+ nk cells was incubated with il-15 (10 ng/ml) between 12 and 18 hours at 37ºc and 5% co2 in gmp conditions. quality and microbiological controls were performed at the end of each manufacturing step. dxh cellular counters (beckman coulter®) and multiparametric flow cytometry were used for lymphocyte subpopulations and viability analysis (navios cytometer; beckman coulter®, conjugated monoclonal antibodies; miltenyi biotec®). the final product was infused intravenously to the patient on days +8 and +15 if manufacturing conditions were met (range of 0.5-100x10 6 nk/kg, purity ≥ 80%, viability≥ 70% and < 1x10 4 cd3+ cells/kg). if not, it was discarded. nk cell activation in the product was measured by the expression of cd25 and cd69. results: between november 2017 and april 2018, 3 patients were included in this ongoing trial. two products were manufactured for 2 of the patients, and only one for the first patient, due to transplant complications between first and second infusion. one product did not meet minimum viability criteria and was discarded. in the infused final products mean and sem of nk cell purity, recovery and viability were 83.7%±4.4, 30.9%±4 and 76.3%±17.4, respectively. log cd3+ depletion ranged between -5.48 and -6.03. median infused doses of nk cells and cd3+ cells per kg were 3.78x10 6 (2.8x10 6 -4.57x10 6 ) and 114 (87-532). complete manufacturing data of all 5 procedures are shown in table 1 background: cytokine-induced killer (cik) cells are a promising immunotherapeutic approach to combat relapse following allogeneic hematopoietic stem cell transplantation (hsct) for acute leukemia or myelodysplastic syndrome. to show safety and efficacy, a multicenter clinical study with 20 pediatric and 20 adult patients including up to eight cik cell applications with escalating doses is ongoing. methods: we favor single large scale cik cell generation with the aim to apply fresh cik cells and cryopreserve ready-for-use doses according to the study protocol in contrast to recurrent manufacturing. therefore cryopreserved cik cells were tested against freshly generated cik cells to approve equivalence. furthermore, an alternative medium supplement for cik cell culturing was investigated to avoid supply bottlenecks in ab-serum. results: fresh frozen plasma (ffp), platelet lysate (pl) and ab-serum in cik cell culture showed median expansion rates of 10-fold, whereas cultivation without medium additive resulted in significantly lower proliferation (p< 0.01). cik cell composition including t cells, nk like t cells and a minor part of nk cells was not significantly influenced by changing the medium additive. moreover, neither cytotoxicity against thp-1 cells nor cd25 expression on nk like t cells were significantly influenced by the different medium additives. for cik cell generation either ficollized peripheral blood (pb) or unstimulated leukapheresis (lp) products were utilized. with regard to repeated manufacturing within the clinical study, also cryopreserved lp and pbsc as starting material came into the focus of interest. comparing cik cell expansion rates, no significant differences for the entire cik cells and the subgroup of t cells were detected between the four starting materials. cryopreservation of cik cells had no significant effect on cik cell composition, cytotoxicity and cd25 expression on nk like t cells. a small, albeit not significant effect of cryopreservation on viability was detected, which was 86.1% before and 79.4% after freezing and thawing. conclusions: the challenge was an efficient time-, personal-and cost saving production of cik cells within the clinical study. introducing ffp enabled cik cell manufacturing for an increased patient cohort by avoiding supply bottlenecks in ab-serum. furthermore, cryopreservation allows the storage of ready-for-use cik cell doses fulfilling the demands of the clinical study. clinical trial registry: eudract number 2013-005446-11 disclosure: nothing to declare. automated generation of cd45ra depleted donor lymphocyte infusion (dli) with the clinimacs prodigy® cd45ra system 1, 2 methods: the current clinimacs cd45ra system was developed for graft engineering. up to 20x10e9 magnetically labeled cd45ra+ cells from leukapheresis products can be depleted from up to 50x10e9 white blood cells (wbc). we developed a new clinimacs prodigy® process in order to ease the procedure for routine-use, to reduce the specifications according to reported cell numbers for dli applications, and to enable the use of peripheral blood products with high amounts of red blood cells (rbc). the new system was tested by performance runs. an new fluorescent flow analysis protocol was developed. results: the resulting clinimacs prodigy pb-45ra system is an automated procedure with integrated labeling and washing steps. the new application software pb-45ra depletion enables to deplete up to 1.8x10e9 cd45ra + cells from up to 5x10e9 total wbc from peripheral blood products. a major difference of this process is the rbc removal option based on an integrated camera for cell pellet detection. the final cell product is provided in physiologic saline. verification runs with peripheral blood products (n=6 in total, n=3 with whole blood, n=3 with leukapheresis products) resulted in a mean depletion of 5.0 log (range 3.9 -5.7) for cd45ra + t cells in the cd45ra depleted product. viability of the target products was always above 96%, and mean wbc recovery was 66%. the mean process time was 3h23min (range 3h to 3h50min) without including the manual steps, i.e. tubing set installation and downstream analysis of blood products by flow cytometry. this data were in line with preceding evaluation runs (n=6), and results obtained in cooperation with an external beta test site. 3 the performance results were furthermore in line with results obtained on clinimacs plus instrument runs. for quality control of cd45ra depleted products we developed a flow cytometric analysis strategy for fast, accurate, and convenient analysis of even rare counts of remaining unwanted cells. it allows to determine naïve t cells at two different levels of subset staining. the minimum requirement for the flow cytometric analysis includes 4 colors to define viable cd3+cd45ra+ cells. for further evaluation of the naïve t cell subsets 3 additional colors are used to define viable cd3+cd45ro-cd95-cd62l+cd197+ cells. conclusions: the automated clinimacs prodigy pb-45ra system process is capable to deplete cd45ra+ cells efficiently from peripheral blood products within 4 hours. the new process is a fast, convenient, and regulatory compliant method for the preparation of ready-to-use cd45ra-depleted cell products for clinical applications. the submission to an european notified body for ce certification is an important next step. myeloablative conditioning regimen was preferred for 24 patients out of 25. gvhd prophylaxis regimens are csa +mtx: 20(%80), csa+mmf: 2(%8), csa only: 1(%4). atg was given 20 patients. despite been given gvhd prophylaxis 47(%27,1) patients out of 173 transplanted patients had gvhd features. of 47 patients, 25 had experienced steroid resistant gvhd after transplantation, including 17 (%68) grade 3 and 8 (%32) grade 4. ecp treatment was started mean 11 days after diagnosis of steroid resistant gvhd and 2 (%8) patients had complete response while 12 (%48) patients had partial and 11 (%44) patients had no response to ecp treatment on day 28. sixteen out of 25 patients had also received mesenchymal stem cell therapy as salvage therapy. only one patient had experienced hypocalcemic tetany, a complication of ecp procedure. thirteen patients had died and 12 were directly related with steroid resistant gvhd. other conditions like relapse of primary disease or pres syndrome also played role in death. conclusions: extracorporeal photopheresis is a reliable and effective second line treatment modality in steroid resistant gvhd. starting ecp sessions as soon as gvhd symptoms occur increases its effectivity. mesenchymal stem cell administration with ecp for 16 (%64) patients limits our study to reach o conclusion for efficacy of ecp itself. need for hemodialysis catheters, the prolonged sessions while adequate flow is not possible and catheter related infections are the lmitations for feasibility of ecp. disclosure: nothing to declare donor lymphocyte infusion administrations after allogeneic stem cell transplantations in pediatrics: a single center experience background: loss of chimerism is one of the major problems after allogeneic stem cell transplantation(sct). donor-lymphocyte infusions(dli) are used as a treatment after taper or stopping immunosuppression. in this study, dli experience in 20 patients with loss of chimerism after sct due to various benign and malign hematological diseases was presented. methods: between july 2015-august 2018, twenty patients, detected chimerism loss and received dli after sct were evaluated retrospectively. patients received myeloablative or reduced intensity conditioning, atg, cyclosporine a and methotrexate for gvhd prophylaxis. chimerism analyses were performed with short tandem repeat(str) method from peripheral blood. results below 95% were considered as mixed chimeric and below %5 were nonchimeric. when patients considered as mixed chimeric, immunosuppression therapy was ceased immediately and treated with dli. donor lymphocyte infusions were performed at two-week intervals with chimerism follow-up. student t, mann whitney u, ki kare tests and kaplan-meier analysis were used. results: between 1-16 ages (median 4), 7 female, 13 male patients were evaluated. the initial diagnoses were thalassemia major(10), aplastic anemia (3),all(4), aml (3) . dli initiation time was 119.65+-92.71 days after sct, total number of dli administrations were 3.5+-2.19. dose of dli was 1x10 5 -63.6x10 6 /kg (mean 20.5x10 6 /kg). nine patients' chimerism out of 20, fell below 95% at first month after transplant; 4 patients were nonchimeric, 2 of them were complet chimeric and 3 were mixed chimeric. eleven patients´chimerism were below 95% between 1-6 months after sct, 5 patients were nonchimeric and 6 were mixed chimeric. early mixed chimerism was found relevant with graft rejections (p=0.04). patients were followed up for 91-645 days. eight patients' chimerism increased after dli infusion and continued to decrease in 12 patients. after dli, acute gvhd has been seen in both group.the group with decreased chimerism after dli, dose was mean 12x10 6 ±23x10 6 /kg while the group with increased chimerism had dli dose mean 22x10 6 ±19x10 6 /kg. although the difference was not statistically significant, numerical value revealed significantly different. eventually10 patients out of 20 were mixed chimeric, 6 patients were complete chimeric and 4 were none. in thalassemic patients, 7 patients with thalassemia-trait donor were mixed chimeric, in 3 patients whose donors were normal, 2 of them were complete chimeric and one of them was nonchimeric.the difference was significant (p=0.02). the cd34 infusion doses revealed mean 6.61 ±5.06x10 6 /kg in mixed chimeric patients, 7.16±3.31x10 6 /kg in complete chimeric patients and 6.625±1.37x10 6 /kg in the patients with loss of chimerism. cd34 amount was seen high as numerical value in complete chimerics but no statistical significance was found. overall survival was 85%, disease-free survival was 25%. conclusions: we evaluated the efficacy of dl for patients with mixed chimerism in our patient group. we concluded that chimerism loss in patients with early decreased chimerism is similar to those in literature in spite if dli practices. dose and application frequency were greater in patients with increased chimerism. the small number and the heterogeneity of the patients limited our study. in this regard, studies with larger series and homogeneous groups are acquired. disclosure: nothing to declare phase i clinical trial of repeated administrations of bone-marrow derived mesenchymal stem cells in steroid-refractory chronic graft-versus-host disease patients nayoun kim 1 , young-woo jeon 2 , jae-deog jang 1 , keon-il im 1 , nak-gyun chung 2 , young-sun nam 1 , yunejin song 1 , jun-seok lee 1 , seok-goo cho (cghvd) is the most common long-term complication of allogenic hematopoietic stem cell transplantation which is associated with poor quality of life and increased risk of morbidity and mortality. currently, there is no standardized treatment available for patients who do not respond to steroids. as an alternative to immunosuppressive drugs, mesenchymal stem cells (mscs) have been used to treat and prevent steroidrefractory acute gvhd patients. these studies and reports have also provided a basis for using mscs in steroid refractory cgvhd patients. methods: to evaluate the safety and efficacy of repeatedinfusions of mscs, we enrolled ten severe steroid-refractory cgvhds patients. steroid refractory was defined as either no response to steroids lasting at least 4 weeks or progression of disease during treatment or tapering lasting at least 2 weeks. patients were intravenously administered with mscs produced from third-party bone marrow donors at a 2-week interval for a total of four doses. each dose contained 1x10 6 cells per kg body weight and all four doses consisted of mscs from the same donor and same passage. results: we enrolled ten patients (3 female/ 7 male, with a median age of 41.5(range 17-68). median of cgvhd affected organs was 3 (range 2-4) including the skin (n=4), eyes (n=8), oral cavity (n=9), lung (n=1), liver (n=2) and joints (n=6). all ten patients received their planned four doses of mscs, administering a total of 40 infusions. median time from initial cgvhd diagnosis to first msc treatment was 709 days (range 222-4413). msc infusions were well tolerated with no immediate or delayed toxicities. after 8 weeks of the first msc infusion, all ten patients showed partial response showing alleviation in clinical symptoms and increased quality of life. organ responses were seen in skin (n=2), eyes (n=5), oral cavity (n=8), liver (n=1), and joint(n=5). however, one patient died of progressive gvhd and one patient relapsed from primary disease. conclusions: repeated infusions of mscs was feasible and safe and may be an effective salvage therapy in patients with steroid-refractory cgvhd. further large-scale clinical studies with long-term follow up is needed in the future to determine the role of mscs in cgvhd. background: the majority of pregnant polish women (84%) have heard of cord blood banking. however, most doctors do not have sufficient knowledge about the possibility of using cord blood in order to respond to their potential concerns. only 16.5% of healthcare professionals were aware that cord blood could be used to treat haematological diseases. in order to make doctors aware of this issue and provide patients with the information they expect, we would like to present data on the use of cord blood stored in our blood bank for haematological and nonhaematological therapies. methods: the table presented below has been created using data from the general database of the polish stem cell bank, warsaw, poland. no data regarding umbilical cord blood data have been excluded. all patients were planned to be assessed on day 1, day 28, on discharge, 100 days after transplantation and 1, 2, 3, 4, and 5 years after transplantation, but in some cases, patients were lost from follow-up due to a persistent lack of reports from transplantation centres. results: in 32 cases, the therapeutic use of cb was transplantation (replacement of patients' own tissue); in 13 cases it was administration (infusion without destruction of patients' own tissue). thirty-three were administered as standard therapy and 13 as experimental therapy. conclusions: the survey study cited above, indicated low awareness of cord blood use among healthcare professionals. on the other hand, one study indicated that the expectations placed in cord blood banking may be unreasonable. as a private cord blood bank, we support recommendations which underline the importance of patient education. in poland, cord blood has been approved as standard therapy in approx. 80 diseases; most of them are rare, but polish law allows the use of cord blood for the siblings, parents and grandparents of a donor. additionally, 168 active and planned clinical trials throughout the world evaluate the therapeutic efficacy of cord blood in such areas as haematology, neurology, cardiology, diabetology, congenital paediatric disorders, ophthalmology, dermatology, gastroenterology, hiv infection, and the quality of life during aging. therefore, further indications may be expected in the future. background: cytokine release syndrome (crs) has been identified as a clinically significant, on target, off tumour side effect of the chimeric antigen receptor (car) t-cell therapies. it is clinically increased in interkeukin 6 and elevations in other cytokines, lactate dehydrogenase (ldh), c-reactive protein (crp), and ferritin. these side effects can include fever, fatigue, headache, encephalopathy, hypertension, tachycardia, coagulopathy, nausea, capillary leak and multi organ dysfunction. crs symptoms can appear as early as one day after infusion and can resolve quickly or last for weeks. it´s severity to be related to the disease burden prior to car t-cell therapy. methods: the bristol oncology and haematology centre will be providing car t-cell therapy to patients in early 2019. on collating from the leading consultant on the ward and fellow nursing team members it was apparent that an effective way at managing patients post car t-cell therapy side effects is to provide an educational and informative poster depicting a flow chart that will aid the practitioner to recognise and effectively treat/manage a patient with crs symptoms. results: none as of yet as this is a prospective tool ready for our first patient in early 2019 conclusions: through continuing reading and study days prior to the ward receiving its first car t-cell patient it is increasingly important that the entire multi disciplinary team recognise crs and understand the importance of early detection, careful monitoring and early intervention. background: allogeneic stem cell transplantation (allosct) is the only curative procedure for primary and secondary myelofibrosis (pmf, smf). elderly people are mainly affected, limiting the feasibility of intensive myeloablative chemotherapy regimens. the introduction of reduced-intensity conditioning (ric) made allosct feasible and effective for old patients. nevertheless, the incidence of pmf and smf is not negligible in young patients, theoretically able to tolerate also high-intensity therapy. very few data are available about the efficacy of ric-allosct in the particular setting of young-aged mf patients. methods: this study includes 56 myelofibrosis young patients (age < 55y) who received allosct between 2002 and 2016 at the university hospital hamburg/germany. four patients were previously splenectomized. patients mostly fall into intermediate risk groups according to dipss. four patients belonged to the high-risk triple-negative category (jak2/calr/mpl-). asxl1-mut was tested in 50 patients (pos: 17). in 96% graft source was pbsc, 2 patients received bmsc. only 30% of patients had a 10/10 hla-matched sibling, the others were transplanted from fully-matched (36%) or partially-matched (34%) unrelated donor. all transplants were conditioned according the ebmt protocol with busulfan (10 mg/kg po or 8 mg/kg iv), fludarabine (150 mg/m2), atlg (grafalon® neovii, germany) administered in 3 days at a dose of 20 mg/kg die for mud, 10 mg/kg die for mrd transplants, followed by cylosporina, and mycophenolate in the first 28 days. results: engraftment rate was 98%, median neutrophil engraftment time 15 days. platelet engraftment was reached by 51 patients (91%, median 19 days). four patients (7%) developed poor graft function, successfully treated with cd34+ selected pbsc-boost. after a median follow up of 8.6 years, estimated 5y-pfs and os were 68% and 82% respectively. dipss-risk and donor hla-matching resulted the only significant impacting factors on os. neither cytogenetic nor molecular abnormalities were significantly related to os. twenty-five patients (44%) experienced agvhd grade >1. c-gvhd was observed in 34 patients (65%), mostly (82%) beginning in the first 300 days after transplantation. cumulative incidence of trm was 7% at 1 year, with a plateau after the first year (5y trm = 12%). trm was observed only in patients with maximal grade (3) of marrow fibrosis. furthermore, trm never occurred in previously splenectomized patients (p=0.00), but no significant impact from splenectomy on os was observed (p=0.32). after transplant, 11 patients (20%) relapsed: 1 died without any treatment because of infection, 9 received dli (3 durable cr), 7 patients (6 after dli) underwent a second allosct, with long-term survival in 5 cases. conclusions: ric followed by allogeneic sct is a curative treatment approach for younger patients with myelofibrosis with a low nrm. the most important outcome-determining factor is donor hla-matching. interestingly, marrow high grade fibrosis showed to significantly impact trm. biological markers such as asxl1 mutation and cytogenetics, largely known as highly predictive for poor prognosis in the disease natural course, did not show any impact on survival, suggesting that patients harboring these abnormalities could get the greatest benefit from allosct. further data collection, and a prospective randomized trial are needed to confirm our conclusion. disclosure: nothing to declare p087 abstract already published. splenectomy as a risk factor for relapse after allogeneic hematopoietic stem cell transplantation in patients with myelofibrosis -retrospective cohort study background: splenectomy is a common procedure in patients (pts) with myelofibrosis (mf) performed to achieve improvement in blood cell counts and reduce b-symptoms. however, it has also been shown that splenectomy may adversely predispose to leukemic transformation in this setting. aim: to evaluate in a single-center retrospective analysis the long-term impact of pre-or post-transplant splenectomy on transplant outcome regarding overall survival and relapse risk. methods: this retrospective analysis comprises the data of 163 pts (93 male and 70 female) with primary (n=108) or secondary (n=55) mf after allo-hsct from hla-matched sibling (n=48) or unrelated (n=115) donors in our center between 1994 and 2018. the median age was 56 years (range, 22 to 75 years). a myeloablative conditioning regimen was performed in 142 pts, while 21 pts where treated with a reduced intensity conditioning. peripheral blood stem cells (n=154) or bone marrow (n=9) with a median of 7.0 x 10 6 cd34 + cells/kg bodyweight (bw) (range, 1.0 to 30) were transplanted. splenectomy was performed in 41 of 163 pts: 21 pts were splenectomized prior to and 20 pts after allo-hsct. relapse was diagnosed in 22 (14%) of 163 pts. the median duration to relapse after transplantion was 13 months (range, 3-99 months). results: the median duration of follow-up of this cohort was 28 months (range, 2-120 months), the 10-year overall survival (os) was 46%. 74 pts died, including 7 pts who relapsed and 67 pts who died of treatment related causes. the observed probability of relapse was significantly higher in splenectomized pts than in non-splenectomized pts: 37% versus 10% (relative risk (rr) 3.7, 95% ci, 1.5-9.4, p=0.007). at 10 years, the os was 50% in nonsplenectomised and 39% in splenectomised pts (p=0.35) (fig.1) . the relapse rate in splenectomised pts was independent of pre-(5 of 21 pts, 24%) or post-transplant (6 of 20 pts, 30%) treatment (rr 1.3, 95% ci, p=0.73) . conclusions: on the basis of our cohort, we could assert that pre-and post-allo-hsct splenectomy was equally and significantly associated with an increased relapse ratio in patients with mf, which also tends to negatively affect overall survival. [[p088 image] 1. figure 1 : the overall survival after allo-hsct in patients with myelofibrosis.] background: b-cell prolymphocytic leukemia (b-pll) is a very rare lymphoproliferative disorder. although allogeneic stem cell transplantation (allosct) could be a curative option, patients often do not qualify for this consolidation treatment due to an aggressive course of disease. in this case study, we report on three patients who failed ibrutinib therapy but achieved complete remission and even mrd negativity after treatment with the chimeric cd20-antibody rituximab, enabling them to undergo allosct. methods: clinical data and follow-up data were collected by chart review. results: all three patients (pt#1: male, 42 years; pt#2: female, 66 years; pt#3: female, 64 years) were referred with b-pll harboring highly complex aberrant karyotypes, including 17p abnormalities in pt#1 and pt#2. a tp53 mutation could be detected in pt#2 and pt#3. all three patients had symptomatic disease with rapidly increasing hyperleukocytosis and massive splenomegaly. two of them were treatment-naive and one relapsed after chemoimmunotherapy. all patients were put on ibrutinib 420mg. despite initial response to treatment, two patients developed progressive disease after 4 (pt#1) and 9 months (pt#2) on ibrutinib, whereas pt#3 remained in partial remission with persisting leukocytosis, precluding consolidating allosct as originally intended. in pt#1, ibrutinib was replaced by venetoclax, but without response. in order to control rapid lymphoproliferation, rituximab was added to venetoclax. grade 4 infusion reaction / tumor lysis syndrome (tls) (fever, tachycardia and hypotonia requiring intravenous vasopressors) followed each rituximab administration despite fractionating rituximab to small doses. however, continuation of rituximab (100mg/d over 10d) led to complete and durable clearance of hyperleukocytosis (from 250/nl to mrd negativity) despite venetoclax cessation. a similar pattern was observed in pt#2, who received rituximab while showing rapidly increasing leukocytosis upon ibrutinib. again, complete clearance of b-pll cells in the peripheral blood (from 148/nl to mrd negativity) occurred after initial grade 4 tls despite only modest cd20 expression on tumor cells in this patient. also, pt#3 achieved profound b-pll cell depletion (from 38/nl to a mrd rate of 1.1%) upon addition of rituximab to ibrutinib (without tls in this case). subsequently, all three patients were able to undergo allosct after conditioning with fludarabine and total body irradiation with 8 gy. pt#1 received stem cells from a hla-ident sibling donor, whereas pt#2 and pt#3 had unrelated donors (hla-ident and hla-matched respectively). with follow-up times of 17 and 11 months post-transplant, pt#1 and pt#2 are currently in ongoing mrd-negative remission. pt#1 developed an acute graft-versus-host disease (gvhd) of the liver (grade 3), nevertheless the clinical course was well controlled by immunosuppression. in pt#2 a chronic gvhd of the skin occurred. pt#3, who achieved mrd negativity after allosct, developed acute and chronic steroidrefractory gvhd of the skin and gastrointestinal tract. nine months post-transplant, gvhd deteriorated and after further complications the patient died of pneumonia 11 months post-transplant. conclusions: supplementary treatment with rituximab can induce deep remissions in patients with ibrutinibresistant, genetically poor-risk b-pll, thereby enabling them to undergo successful consolidation with allosct. a high risk of life-threatening infusion reactions / tls associated with the addition of rituximab has to be taken into account. background: there is little experience on the use of the newer targeted therapies in cll patients relapsed after allogeneic stem cell transplantation (allo-sct). against this background, we evaluate the safety and efficacy of the bcr inhibitors (bcri), ibrutinib and idelalisib, administered after allo-sct for the purpose of treating the cll relapse. methods: data from 11 cll pts who relapsed after sct, and were subsequently treated with ibrutinib (n=6), idelalisib (n=3) or both (n=2),were retrospectively collected in collaboration with the spanish group of cll (gellc) and the spanish group of stem cell transplantation (geth). results: transplant characteristics are summarized in table 1. eight patients received the bcri as the first salvage treatment after sct relapse, whereas 3 patients had received ≥2 prior lines of treatment. at the time of the onset of the bcri, 7 patients had rai 4 stage and 6 patients had a lymph node size ≥5 cm. del17p was present in 4 patients and del11q and complex karyotype in 2 patients, respectively. tp53 gene mutation was detected in 3 patients (all with del17p13). median time from sct to bcri therapy was 53.9 months, being shorter in patients treated with ibrutinib (n=8, median 51 months) than in those treated with idealisib (n=3, median 111 months). median time on ibrutinib and on idelalisib was 7.3 months (4. 1-18.8 ) and 6 months (3.0-23.4 ), respectively. the best overall response rate (orr) obtained with ibrutinib was 75% (1 cr, 4 pr, 1 pr+l) whereas it was of 40% for patients receiving idelalisib (1 cr, 1 pr+l). among the 8 patients treated with ibrutinib, 7 (87.5%) presented an adverse event (ae), being diarrhea (n=3), asthenia (n=2) and infections (n=5) the most frequent. hypertension was seen in 1 patient and none patient developed atrial fibrillation. five patients stopped ibrutinib treatment, due to toxicity (n=4) or progression (n=1). after ibrutinib discontinuation, 4 patients were newly treated with idelalisib (n=2) or venetoclax (n=2). all patients treated with idelalisib developed at least one ae, being diarrhea (n=3), pneumonitis (n=2) and neutropenia (n=2) the most common. four patients discontinued idelalisib because progression (n=3) or toxicity (n=1). venetoclax was given after idelalisib in 3 patients. although acute and/or chronic gvhd before bcri was documented in 7 (63.6%) and 5 (45.5%) patients, respectively, only one patient (treated with idelalisib) reactivated a mild chronic gvhd. none patient received infusion lymphocyte from donor after bcri and one patient underwent a second sct. with a median follow-up of 14.3 months (5.9-33.9) after the onset of the bcri treatment, 4 patients had died, all of them due to cll progression (3 richter´s transformation), whereas 5 patients remained in response (3 cr, 2 pr). the overall survival probability of the whole series at 12 months was 77.8% ±13.9%. conclusions: in our study, ibrutinib and idelalisib, administered in cll patients relapsed after sct did not increase the risk of gvhd reactivation but they show high incidence of adverse events. nevertheless, bcri offers a possibility of disease control in these patients with poor prognosis. further studies are needed to confirm these data. background: prior to the introduction of tyrosine kinase inhibitors (tki), median survival of chronic phase chronic myeloid leukemia (cp-cml) patients was approximately 60 months and the standard treatment with interferon-alpha resulted in complete cytogenetic responses in about 30% of the patients. autologous stem cell transplantation (auto-sct) was first attempted for patients in transformation in order to restore a second cp and was introduced secondarily in cp to try to prolong the response. the main rational for autografting in cp resides on the reduction of the tumor burden and the number of leukemic cells at risk of developing blastic transformation. nevertheless, auto-sct alone was not able to maintain a long-term remission. nowadays, tkis represent the state-of-the-art therapy for cml and the concept of auto-sct has only little interest while long-term follow-up and outcome in this setting are currently unknown. the aim of our study is to evaluate at a first time the longterm outcome of cml patients who received auto-sct in chronic phase, and to evaluate at a second time in a subgroup analysis, the outcome of those who received tki after having been auto-transplanted, mainly for disease progression/loss of response and/or to enhance disease response. methods: we found a total of 969 patients who received auto-sct for cp-cml in europe between years 1989 and 2004, 578 (60%) were males, median age at auto-sct was 47 years (range: 19-67), the median time between diagnosis and auto-sct was 19 months, stem cells source was peripheral blood in 84% of patients, most frequent conditioning regimen was busulfan 4mg/kg/day 4 days + 1 day of melphalan 140 mg/m² one day prior to the cells reinfusion. information about receiving tki post auto-sct was available only for 103 patients, first tki was imatinib for 89 (86%) patients, dasatinib for 8 (8%), nilotinib for 6 (5%) and ponatinib for one (1%) patient. results: after a median follow-up of 9.5 years (range: 1-27) from time of auto-sct for the whole population, the probability of overall survival (os) at 10 years was 50% (95% ci: 46-53); there was 540 (56%) patients who relapsed after a median time of 16 months after auto-sct. there was a total of 530 patients transplanted before the tki era and survived until the availability of tkis. when we performed a landmark analysis evaluating the outcome of patients who received auto-sct, survived until the tki era and received tki (n=103), the 10 years os probability of these patients from tki treatment was 70% (95% ci: 58-78). additional data requests will be sent to centers querying about prognosis, molecular responses, treatment and disease details. conclusions: we demonstrate here with these preliminary results that the introduction of tki has improved survival of cml patients. in addition, patients who received auto-sct, survived until the tki era and also received tki, had encouraging rates of long-term survival. an extensive analysis will be performed when additional data will be available and the study will be updated with more results. disclosure: nothing to declare a 35 year single center transplant experience in chronic myeloid leukemia background: allogeneic hematopoietic stem cell transplantation (hsct) has been considered for decades the only curative approach for patients with chronic myeloid leukemia (cml). in the tyrosine kinase inhibitors (tkis) era, hsct for cml has been reserved only to patients not achieving a cytogenetic remission or showing progressive disease after multiple tki treatment lines. however, a progressive improvement in the long-term survival has been obtained in the overall hsct population. the present study aimed at evaluating whether in cml patients transplanted at our center over a long time period -from 1983 to 2018 -the outcome improved over time. methods: 136 consecutive patients who underwent a transplant between 1983 and 1999 were compared to 43 patients who received the transplant between 2000 and 2018. overall survival (os), leukemia-free survival (lfs) and graft-leukemia-free survival (glfs) were estimated using the kaplan-meier method and the log-rank test was used to compare risk factors categories. results: of the 179 patients [median age 35 years (range7-66)], 148 (82.7%) were in 1 st or 2 nd chronic phase, 25 (13.9%) in accelerated phase and six (3.4%) in blast crisis. matched related donors and alternative donors (matched unrelated donors, cord blood or mismatched related donors) were used in 156 and 23 cases, respectively. as stem cell source, bone marrow was used in 142 patients, peripheral blood in 33 and umbilical cord blood in 4. tbibased conditioning regimens were used in 89 patients, while in the other 90 cases irradiation-free conditioning regimens were used. both in univariate and multivariate analysis, irradiationfree conditioning regimens (hr 1.8; 95%ci 1.1-3.0, p=.0014) and transplants performed in 1 st chronic phase (accelerate phase hr 2.1; 95%ci 1.2-3.8, p=.008 -2 nd chronic phase hr 4.9; 95%ci 2.3-10.3, p=.0001 -blast crisis hr 2.5; 95%ci 1.0-6.4, p< .05) were associated with a better os. patients transplanted before 2000 had a worse os (hr 6.5; 95%ci 2.7-15.5, p < .0001) and dfs (hr 2.2; 95%ci 1.0-4.8, p=.045). a trend for a worse glfs was observed in univariate analysis (hr 1.6; 95%ci 1.0-2.7, p=0.05), in the first period of observation. conclusions: our single center experience confirms that higher os, dfs and glfs are observed in cml patients allografted in more recent years. improvement of conditioning regimens, use of tbi-free conditioning regimens and supportive therapy, have presumably contributed to these results, together with the more recent strategy of close monitoring of minimal residual disease, and prompt use of tki or donor lymphocyte infusion in case of relapse. hsct is nowadays a safer therapeutic procedure in cml patients that should be considered timely in tki-resistant patients to avoid progression into a more advanced disease phase. disclosure: the authors declare no conflict of interest. reduced-intensity transplantation (rit) in patients with high-risk or advanced chronic lymphocytic leukemia in last 5 years: improvement of transplant outcomessingle centre experience . hct-ci ≥ 3 was in 20% of pts. source of stem cells was peripheral blood in 80% and bone marrow in 20% of pts. the median of infused cd 34+ cells was 5,4x10^6/ kg. the conditioning regimen consisted of fludarabine and melphalan (+atg in unrelated donor). gvhd prophylaxis were cyclosporine and methotrexate. results: all pts engrafted. none of 3 pts in cr before rit progressed at day +30 after rit and among 17 pts beyond cr before rit all of them achieved at least pr at day +30 after rit. 13 pts (65%) developed acute gvhd (2 pts grade iii-iv) and among 19 evaluable pts 10 (53%) of them developed chronic gvhd (6 mild, 2 moderate, 2 severe). with median follow-up 50 months (range 6-63 months) 15 pts (75%) are alive in cr. 3 pts (15%) relapsed or progressed 5, 19 and 21 months after rit and 2 of them died. last relapsed patient achieved next cr after ibrutinib. 3 pts (15%) died due to nrm. nrm till day +100 after rit was 5%. the estimated probabilities of 2-years cgrfs, pfs and os are 55%, 73% and 83%. conclusions: in spite of relatively small number of evaluated pts and retrospective type of analysis our data show that rit in pts with high-risk or advanced cll has achieved promising results (2-and 5-years pfs and os 73% and 55% resp. 83 and 57%) in recent years and these results are better than outcomes of our historical patient cohort from period 2010-2012 (2-and 5-years pfs 41% and 26% resp. os 56% and 35%, p=0.009) or ebmt published data of pts transplanted for cll in period 2000-2010 (2and 5-years pfs 46% and 35% resp. os 62% and 45%). current results of transplantation should be taken into account in our future decision-making process on indications for transplantation in pts with high-risk cll, of course also in the context of new or updated results of targeted cll treatment and its complications. disclosure methods: retrospective data and treatment outcomes were collected from the singapore childhood cancer registry (sccr). most children with cancer in singapore receive therapy at one of the two public paediatric cancer centers (kkh or nuh). a total of thirty two cases were diagnosed with cml and received treatment in either of these centers over a twenty year period (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . results: the age at diagnosis of the thirty two children ranged from 4 to 17 years (median 12.5years). six patients in the pre-tki era were treated with an upfront hsct. the remainder twenty six patients were initially started on a tki. of these 12/26 (46%) had a hsct at a median period of 22.5 months from diagnosis (range 5-43 months). the reason for hsct in ten out of the twelve children was due to high risk features i.e. accelerated/blastic phase/ no ccr/no cmr. the remaining two patients had a hsct due to parent and patient preference for attempt at upfront cure rather than the use of life-long and expensive tki therapy. non-compliance to tki therapy was a major finding in our teenage cohort. eleven of the eighteen transplants used a matched sibling donor. three patients had cord blood as their stem cell source. one patient had a single antigen mismatched related donor and three patients had a mismatched unrelated donor for their hsct. all patients except one had myeloablative conditioning with busulfan and cyclophosphamide. atg was added according to physician preference. one patient had cy/tbi conditioning because of pre-transplant lymphoid blast crisis. anti gvhd medications included cyclosporine/ methotrexate or tacrolimus and methylprednisolone in the cord transplant patients. six of the eighteen (33%) patients who had a hsct died. four died due to treatment related mortality (2 infections, 1 acute gvhd and 1 pulmonary fibrosis). one patient died due to an early relapse and one had a late relapse related mortality. for the pre-tki era, hsct related 5 and 10 year os was 83% and 67% respectively. post-tki era 5 and 10 year os was 75%. for the entire cohort, the 10 year os was 70%. conclusions: the post-tki era transplant outcomes from our two centers is comparable to leading centers in the world. outcomes for patients with mismatched unrelated donors was poor in our cohort. we recommend a haploidentical related donor transplant or an unrelated cord blood stem cell source for patients when a matched sibling or unrelated donor is not available. clinical trial registry: na disclosure: we have nothing to disclose. fludarabine, busulfan, and thiotepa may be a promising conditioning regimen for myelofibrosis patients undergoing allogeneic stem cell transplantation background: allogeneic stem-cell transplantation (sct) is a curative therapy for patients with myelofibrosis. however, recurrent disease and non-relapse mortality (nrm) are frequent causes of treatment failure. the optimal conditioning regimen for sct in this disease has not been defined. methods: we retrospectively analyzed transplantation outcomes of all adult patients given sct for myelofibrosis between 2003 and 2018 at a single large academic medical center. patients (n=59) were treated with several conditioning regimens that were grouped according to conditioning intensity. myeloablative conditioning (mac) included busulfan 12.8 mg/kg and cyclophosphamide 120 mg/kg (bucy, n=10), fludarabine and busulfan 12.8 mg/kg (flu/ bu4, n=9) and fludarabine and treosulfan 30-36 g/m 2 (flu/ treo, n=6). reduced-intensity conditioning included fludarabine and busulfan 6.4-8.0 mg/kg (flu/bu2, n=22). more recently we adopted the tbf regimen including fludarabine, busulfan 6.4-9.6 mg/kg and thiotepa 5-10 mg/ m2 (n=12). all patients were also given anti-thymocyte globulin during conditioning, irrespective of donor source. results: the median age was 59 years (interquartile range [iqr] 52-63). the majority of patients had documented splenomegaly (81%) and were not previously exposed to ruxolitinib (71%). donor type was an hla-matched sibling (41%), 10/10 (51%) or 9/10 (8%) matched unrelated donor. the dipps+ score distribution was intermediate-1 (16%), intermediate-2 (45%), or high (n=39%). the median followup was 3.2 years since the success of tyrosine kinase inhibitors (tkis), transplant-related mortality is considered too high to justify allogeneic hematopoietic stem cell transplantation (allohsct) as first-line treatment for chronic myeloid leukemia (cml) patients in chronic phase (cp). allohsct is currently considered for patients failing to at least 2 tkis or with disease in advanced phase. nevertheless, the optimal timing for transplant referral is still not well defined. methods: we performed a retrospective analysis on 23 consecutive patients with cml in cp receiving first transplants from an hla-identical sibling donor with partially t-cell depleted grafts from 1998 to 2016 at our center. partial t-cell depletion (ptd) consisted of in vitro alemtuzumab incubation of a part of the graft for infusion at day 0 while the rest, containing 100x10 6 cd3+ cells/kg was given as a t-cell add-back at day 1. donor lymphocyte infusions (dlis) were provided, in the absence of gvhd, in case of disease relapse or mixed chimerism. molecular monitoring was performed by 3-month bcr-abl1 rt-qpcr testing in peripheral blood during at least a 5-year period after hsct. thereafter, 3-month testing schedule was maintained where possible, or followed by a 6-month one. kaplan-meier method was employed to determine the probability of overall survival (os) and leukemia free survival (lfs) since allohsct. results: median age at hsct was 36 years (range, 18-58). all patients were in first cp but one who was in second cp. twelve patients were tki-naïve at hsct (1998 hsct ( -2001 , 4 patients had presented suboptimal response or/ and intolerance to imatinib (2002-2004 period) , while the last seven patients had presented suboptimal response or/ and intolerance to imatinib, dasatinib and nilotinib (2005-2016 period) . the time interval from diagnosis to transplant was < 12 months in 11/23 (48%) patients. 15 (65%) patients had an ebmt risk score of 0-2, while 8 (35%) patients of 3-4. the conditioning regimen was myeloablative for all but one patients. the stem cell source was pbsc for 22 patients and bone marrow for one. all patients engrafted. 14 patients presented molecular relapse and one patient hematological relapse with a median interval from transplant to relapse of 9 months (range, 5-70) . 17 patients received dlis (15 for relapse and 2 for mixed chimerism), while 3 patients in relapse also received tki. without prior administration of dli, 3(13%) patients presented grade ii agvhd and 2 patients moderate cgvhd. after dli, agvhd occurred in 3 and cgvhd in 4 patients. one patient died of disease progression 3 years after hsct and one of myocardial infarction 19 years after hsct. with a median follow-up of 14.4 years (range 2.3-20.6), 15-year os and lfs were 95%. at the time of the analysis 21/23 patients were alive and in major molecular response. conclusions: these results of excellent long-term survival and no transplant-related mortality suggest that ptcd improves the outcome of cp-cml patients transplanted from an identical sibling donor and they can be useful for deciding risk-adapted strategies. we believe that ptcd could allow earlier transplant referral of patients failing tkis and having an identical sibling donor. disclosure: nothing to declare single tertiary centre experience in allogeneic haematopoietic stem cell transplantation (allo-hsct) for primary and secondary myelofibrosis (mf) the only curative option for fit patients is allo-hsct. novel therapy is emerging but current recommendation is that eligible patients with life expectancy less than 5 years should be considered for allografting. methods: we retrospectively looked at the clinical features and outcomes of all allo-hsct for mf performed in our centre since 2010. results: 12 patients (10 male, 2 female) aged between 29-68 years old (median age 60) with intermediate-2 or high-risk mf as per the international prognostic scoring system (ipss) or dynamic ipps (dipps) were transplanted in our centre since 2010. 6 of them (50%) were diagnosed with pmf and the remaining 50% with secondary mf; 4 post-et and 2 post-pv mf. 2/12 of our patient group received a sibling allograft and 10/12 a matched unrelated donor allograft (70% received a 10/10 human leukocyte antigen (hla)-matched transplant, 20% a 9/10 hlamatched and 10% a 8/10 hla-matched graft). all patients received a reduced intensity conditioning (ric); 3/12 patients with fludarabine/ melphalan/ campath (fmc), 8/ 12 fludarabine/ busulphan/ atg (fbatg) and 1 fludarabine/ ara-c/ campath (flag/ campath); all received peripheral blood as source of hsc. engraftment occurred between day 13-48, with a median of d+17. one late graft rejection occurred. all patients were alive at d+100. 9 patients are currently alive; overall survival (os) is 75%. transplant related mortality (trm) was 16.6% at 1 year, 25% at 3 years. 1 patient died of graft versus host disease (gvhd) and 2 patients of septicaemia leading to multiorgan failure. acute gvhd grade ii skin occurred in 5 patients, grade iii and above in 2 patients. 5 patients have limited chronic gvhd. 2/12 patients received donor lymphocyte infusion (dli) for mixed chimerism (one of which had 2nd graft failure). out of these 2 patients 1 developed acute grade 4 gvhd and died. response rate: 6/9 alive patients i.e 66.6% exhibit no fibrosis in trephine biopsies, 1/9 alive patients had residual fibrosis but 100% donor chimerism, 1/9 alive patients had residual fibrosis with mixed donor chimerism, other patient non-assessable. conclusions: allo-hsct remains the only potentially curable option for myelofibrosis. in our centre which serves 1.1 million population, with 12 new cases per year, 12 patients were transplanted since 2010. our data suggest that close collaboration between mpn-treating haematologists and transplant physicians is required so that all suitable patients have a transplant assessment early in their disease course. novel molecular prognostic systems are likely to identify those best placed to benefit in future but this series currently supports allo-hsct survival and cure. (range, 105-4624) . dynamic international prognostic scoring system (dipss) score at the time of hct was intermediate-2 or high risk in 20 patients (95%), intermedate-1 in 1 patient. molecular evaluation was available in 14 out of 21: jak2 v617f mutation was detectable in 8 patients, mpl-w515k in 1 patient, carl in 1 patient. 4 patients were "triple negative" for driver mutations. cytogenetics information was available for 6 out of 21; among which 3 patients had complex karyotype, 1 trisomy 8 and 1 trisomy 9. 3 patients underwent splenectomy before hct. ruxolitinib was administered in 4 patients before hct. 10 (48%) patients received stem cells from an hla identical sibling, 9 (42%) from a matched unrelated donor and 2 (10%) from an haploidentical sibling. graft source was bone marrow in 7 patients (34%) and peripheral blood in 14 (66%). conditioning was myeloablative in 16 patients (76%), reduced intensity in 5 (24%). all patients engrafted. acute graft versus host disease was absent in 12 patients (57%), grade i-ii in 7 (34%), grade iii-iv in 2 (9%). in 18 evaluable patients chronic graft versus host disease was limited in 3 (17%), extensive in 4 (22%) and absent in 11 (61%). transplantrelated mortality at 180 days was 24%. main causes of death were: acute gvhd in 2 patients, chronic gvhd in 1, pancreatitis in 1, pulmonary aspergillosis in 1. relapse occurred in 7 patients and was the main cause of death in 4 of them. notably, 2 patients experienced late relapse after 6.6 and 17.6 years after hct. both of them are living while receiving ruxolitinib therapy. after a median follow up of 581 days (range, 38-11660), 10 out of 21 patients are alive. 7 of them (33%) are disease-free and 3 are living. the kaplan-meyer overall survival and disease-free survival at 10 years was 40% and 35%, respectively. conclusions: our experience confirms that hct is a valid option to achieve cure in one third of mf patients. two patients experienced very late (> 5 years) recurrence of mf. the rarity of this condition limits the amount of data and cases available for evaluation and study. life-long follow-up of all mf transplanted patients is warranted to better understand this rare event. disclosure: nothing to declare methods: а 37-years old female was diagnosed with jak2v617f-positive pmf, 46xx, ipss low risk, dipssplus intermediate -2 risk, subacute budd-chiari syndrome and portal vein thrombosis four years before allohsct. to reduce the splenomegaly and constitutional symptoms we performed pre-transplant ruxolitinib therapy 30 mg daily. after three months of therapy the patient achieved clinical improvement (eln criteria). contrast-enhanced computer tomography and magnetic resonance imaging showed enlarged intrahepatic collateral vessels and signs of portal vein thrombosis with cavernous transformation and multiple dilated collateral veins. gastroscopy documented enlarged esophageal veins. allogeneic stem cell transplantation was performed from 10/10 -hla matched unrelated donor with peripheral stem cells (6.1 x 10 9 сd34+ cells/kg). conditioning regimen consisted of fludarabine (180 mg/ m 2 ), busulfan (10 mg/kg p.o.). post-transplant cyclophosphamide was administered at 100 mg/kg at day +3, +4, and ruxolitinib 15 mg was used from d+5 till d+100 as graft versus host disease prophylaxis. results: starting d+1 the patient experienced eight episodes of ebv some of them with severe blood loss. to treat the bleeding episodes blackmore tube was placed six times with temporary effect. to place blackmore tube the patient was two times intubated and required mechanical ventilation. at d+18 leukocyte and neutrophil engraftment, full donor chimerism and molecular remission were achieved. platelet engraftment was documented only at d +42 and poor graft function was present due to cytomegalovirus reactivation (d+41) and parvovirus b19 reactivation (d+62). evb was stopped at d+95 only after two esophageal veins ligations, and two procedures of gastric veins sclerotherapy. soon (d+111) the patient achieved complete platelet recovery (more than 50x10 9 /l) and became red blood cells transfusion independent. at day + 180 complete remission was confirmed by splenomegaly resolution, regression of bone marrow fibrosis, full donor chimerism, jak2v617f-negative molecular status. cbc showed hb 130 g/l, platelets 73x10 9 /l, leucocytes 3,8x10 9 /l. ultrasound examination after transplant documented portal vein thrombosis recanalization. at day + 180 she developed mild (nih) chronic graft versus host disease with eyes and mouth involvement, which was managed with topical steroids. at d+958 after transplant the patient is alive in complete remission and has no recurrent bleedings. conclusions: splanchnic vein thrombosis can significantly complicate the course of allohsct in pmf. easy access to surgical, intensive care unit and endoscopic teams is required to make allohsct more feasible in this group of patients. disclosure all patients received treosulfan-based mac regimens, treosulfan(total dose, 36-42gms/m2) was given in combination with different conditioning drugs. the most commonly used regimen was treosulfan, fludarabine (150mgs/m2) and thiotepa(10mgs/kg) referred to as ftt that was used in 59%(n=55). serotherapy was given in 93% of patients(n=87), as either alemtuzumab or antithymocyte globulin in 82%(n=77) and 11%(n=10), respectively. post-transplant graft-versus-host disease (gvhd) prophylaxis was given in all patients, based mostly on ciclosporin. 46 patients(49%) received the transplant from identicalrelated donors, 46 patients(49%) received the transplant from matched-unrelated donors, and two patients(2%) had haploidentical transplants. 90% of the patients(n=85) were fully hla-matched. all stem cell sources were used as bone marrow in 59%(n=55), peripheral blood stem cells in 33%(n=31), and umbilical cord blood in 8%(n=8). this treosulfan-based conditioning was given as the 1 st transplant in 92%(n=85), and as the 2 nd transplant after the failure of a first procedure in 8%(n=9). two patients received treosulfan-based conditioned transplant twice. results: neutrophil engraftment and platelet engraftment occurred at a median of 13 days and 18 days respectively. chimerism was full donor in 55%(n=52), high donor in 18%(n=17), and mixed donor in 9%(n=8). gvhd developed in 43% of patients(n=40), with acute gvhd grade i/ii and grade iii/iv developed in 29%(n=27) and 2%(n=2), respectively. chronic gvhd grade i/ii and grade iii/iv developed in 13%(n=12) and 2%(n=2), respectively. all chronic gvhd were mild, limited, non-extensive, and resolved completely. none of our patients had persistent gvhd necessitating long-term systemic immunosuppression. mild vod occurred in 13%(n=12), and severe vod occurred in 2%(n=2). one of them died but was believed to be related to the underlying disease (wolman syndrome). viral reactivation occurred in 55% of patients(n=52), with cmv, ebv, and adenovirus reactivation was found in 34%, 21%, and 14%, respectively. five patients had invasive adenoviraemia that contributed to death in two of them. primary graft failure happened in two patients(2%) due to adenoviraemia. seven patients(7%) had secondary graft failure with autologous reconstitution. graft failure was significantly lower (p0.045) in the ftt group than other conditioning groups. at a median follow-up of 35 months (range, two-174 months), eleven patients(11.9%) died, with overall survival of 88.1%, and event-free survival of 80.9%. five patients died due to complications related to their original disease, while six patients died due to transplant-related causes (transplant-related mortality 6.5%). immune reconstitution in alive patients was achieved at a median of eight months. this time was significantly longer (p0.034) in ftt group. conclusions: this study demonstrates that treosulfan is a safe and effective conditioning drug that can achieve engraftment, with low rates of graft failure, transplantrelated mortality and morbidity, even if it is used twice in the same patient. disclosure: nothing to declare background: high-dose chemotherapy (hdc) followed by autologous stem cell transplantation (asct) is the treatment of choice for the patients with relapsed or high risk nhl. although the high-dose conditioning regimens commonly used in patients with non-hodgkin lymphoma (nhl) are beam (bcnu, etoposide, cytarabine, and melphalan), beac (bcnu, etoposide, cytarabine, and cyclophosphamide), survival of patients with nhl received above high-dose chemotherapy followed by asct was still unsatisfactory. methods: we prospectively evaluated the efficacy and toxicity of busulfan, etoposide, cytarabine and melphalan (bueam) including iv busulfan instead of bcnu of standard beam as a conditioning for asct in patients with nhl. the high-dose chemotherapy consisted of bu (3.2 mg/kg i.v. q.d. from day -6 to day -5), e (200 mg/m 2 i.v. b.i.d. on day -4 and day -3) a (1 g/m 2 i.v. q.d. on day -4 and day -3) and m (140 mg/m 2 i.v. q.d. on day -2) at 7 centers in korea. results: two hundred five patients were enrolled onto the study. main subgroup was diffuse large b cell lymphoma (n=104, 50.7%), t cell lymphomas (n=59, 29.8%), and nk/t cell lymphoma (n=22, 10.7%). upfront asct was performed in 160 patients (78.0%), and salvage asct in 45 patients (22.0%). the disease status of the patients before hdt/asct consisted of 133 patients (64.8%) with complete response and 72 patients (35.2%) with partial response. treatment related toxicities included nausea in 149 patients (72.7%), diarrhea in 127 patients (62.0%), anorexia in 107 patients (52.2%) and stomatitis in 97 patients (47.3%), which were grade i or ii in the majority of cases. the common grade iii toxicities were stomatitis (6.9%), diarrhea (5.9%), and anorexia (5.4%). there were no vod, and transplant-related mortality occurred in 4 patients (1.95 %), due to infection. one hundred fifty three patients (74.6%) achieved a complete response and 13 patients (6.3%) after asct, while 28 patients (13.7%) showed progressive disease. at a median follow-up duration of 38.6 months, the estimated 3-year overall survival and progression free survival for all patients was 74.5% and 56.6%, respectively. conclusions: the conditioning regimen of bueam for asct was well tolerated and seemed to be effective in patients with relapsed or high risk nhl. disclosure: none of declare background: allogeneic hematopoietic cell transplantation (hct) is potentially curative for high risk acute myeloid leukemia (aml) and myelodysplastic syndrome (mds), however both gvhd and disease relapse remain major challenges. we recently introduced a combination of posttransplant cyclophosphamide (ptcy) and atg (4.5 mg/kg) as graft-versus-host disease (gvhd) prophylaxis. the purpose of our study was to compare outcomes between ptcy/ atg and other gvhd prophylaxis regimens for high risk aml and mds. methods: we retrospectively investigated outcomes of 159 patients that underwent allogeneic hct between january 2014 and july 2017 for high risk aml (n=120, 75%) and mds (n=39, 25%). gvhd prophylaxis regimens were compared for overall survival (os), cumulative incidence of relapse (cir) and non-relapse mortality (nrm) in univariate and multivariable analysis. high risk aml was defined as secondary aml, therapy related aml, high risk cytogenetics (eln criteria) in cr1, good/ intermediate cytogenetic risk aml in cr2 and primary induction failure; high risk mds was defined as high/very high risk wpss score. results: median age of patients was 56 years (range 22-73 years). donors were matched related in 52 (33%) patients, matched unrelated in 89 (56%) patients and haploidentical in 18 (11%) patients. graft source was peripheral blood stem cells in 158 patients (99%). myeloablative conditioning was used in 54 patients (34%), reduced intensity regimens in 105 (66%) patients. ptcy combined with atg was used in 69 (43%) patients, other gvhd prophylaxis regimens were used in 90 (57%) patients. both donor and recipient were cmv negative in 18 (11%) patients. median follow-up of survivors was 29 months (range 14-56 months). univariate analysis demonstrated os of the entire cohort at 2 years was 49% (95%ci 41-57%), cir at 2 years was 22% (95%ci 16-29%) and nrm at 2 years was 32% (95%ci 25-39%). concerning gvhd prophylaxis regimen, 2-year os for ptcy/atg versus others was 46% (95%ci 33-58%) versus 51% (95%ci 40-61%) (p=0.87, figure) , 2-year cir for ptcy/atg versus other was 31% (95%ci 20-43%) versus 16% (95%ci 9-24%) (p=0.02) and 2-year nrm for ptcy versus other was 28% (95%ci 18-39%) versus 34% (95%ci 25-44%) (p=0.35). grade ii-iv acute gvhd was seen in 23% of ptcy/atg patients versus 59% using other regimens (p< 0.0001). chronic gvhd was observed in 20% of ptcy/atg patients versus 42% using other regimens (p=0.004). multivariable analysis for os confirmed that the gvhd prophylaxis regimen has no influence (p=0.19), while the predominant predictor of survival was age at hct (hr 1.03, 95%ci 1.01-1.05, p=0.01). for cir, the ptcy/atg combination had no influence compared to other gvhd prophylaxis regimens (p=0.6), while ric conditioning was the predominant predictor of relapse (hr 3.05 for ric, 95% p=0.01) . for nrm, the atg with ptcy combination demonstrated no significant difference (p=0.12), while age at hct was the predominant predictor (hr=1.04, 95%ci 1.01-1.07, p=0.02). conclusions: the ptcy/atg combination for gvhd prophylaxis has demonstrated on multivariable analysis similar os, cir and nrm with other previously used regimens at our center. a decrease in atg dose may potentially decrease the relapse rate while retaining the advantage of decreased gvhd. [ background: the combination of fludarabine with myeloablative doses of busulfan (fb4) represents a standard of care conditioning regimen before allogeneic transplantation in patients with myeloid malignancies (giralt, s.: the lancet oncology 2015). fb4 has potent antileukemic activity and is associated with low transplantrelated mortality and acute gvhd. however, early after transplantation (days 30-90), a proportion of patients may not convert to a full donor haemopoietic chimerism, particularly if anti-t lymphocyte globulin (atg) is used as gvhd prophylaxis (rambaldi a, et al.: the lancet oncology 2015) methods: we retrospective analyzed 104 patients who underwent an allogeneic stem cell transplantation after fb4 conditioning regimen at our hospital, from november 2007 to august 2018. the median age was 51 years (range 22-67) and diagnoses were aml 76%, mds 18% cml 5% mfi 1%). the disease status at transplantation was: cr1 in 59%, cr2 in 5% and active disease in 36% of patients. the stem cell source was represented by pbsc in more than 95% of cases and anti-t lymphocyte globulin (atg) was part of the conditioning regimen in more than 95% of cases at a dose of 5 mg/kg. the donor was a hla identical sibling (26%), a matched unrelated (65%) or mismatched (one allele or one antigen mismatched) unrelated, 9%. hematopoietic chimerism was molecularly evaluated by variable number of tandem repeats (vntr) on bone marrow (bm) mononuclear cells or peripheral blood (pb) t lymphocytes, purified by immunomagnetic positive selection (miltenyi, biotec). the analysis was performed at day 30, 60, 90, 180 and 360 after transplantation results: after 30, 60 and 90 days from transplantation, the proportion of patients with a full bm chimerism was 94%, 87% and 83%, respectively. at the same time points, the pb t cell chimerism was 47%, 65% and 69%. before day 100, 10 patients required the infusion of dli to treat a pending or overt hematologic relapse and 12 patients to convert the lymphoid chimerism from mixed (median 44%, range 0-76), to complete (successfully in 7 cases). after day 100, 13 additional patients required dli to treat disease relapse or progression and 8 patients to improve the chimeric status or the immune reconstitution. at 5 years, the overall survival is 64%, with a relapse and non-relapse mortality of 21 % and 11%, respectively ( figure 1 ). by uni and multivariable analysis, aml diagnosis and a mixed bm chimerism before day 100 were associated with relapse and overall survival while age > 50 was the only factor significantly associated with nrm. a mixed pb t-lymphoid chimerism before day 100 does not adversely impact on non-relapse mortality, cumulative incidence of relapse, leukemia-free and overall survival. conclusions: after fb4 and atg, a progressive increase of pb lymphoid donor chimerism develops gradually after transplantation, in most of cases without the need of dli. early mixed lymphoid chimerism does not compromise the main long-term clinical outcomes and may at least partially explain the low non-relapse mortality. an incomplete bm chimerism within the first 3 months strongly correlates with early disease progression or relapse. background: busulfan (bu) is widely used as a component of myeloablative conditioning regimen before hematopoietic stem cell transplantation (hsct) in children. bu has a narrow cumulative exposure window. the relation of bu exposure with toxicity is well established, but the link between the exposure and efs is not clear due to conflicting reports especially in pediatric patients. obtaining the ratio of bu to its metabolite i.e. metabolic ratio (mr) may serve as an indicator of bu gsh conjugating capacity of an individual, thus cumulative exposure of bu for a particular day that could be used along with auc as a marker to predict efs. the present investigation is aimed at evaluating the utility of bu mr to predict efs in children undergoing allogeneic hsct. methods: two different cohorts with children receiving bu in four times daily (qid, n=44) and once daily doses (qd, n=13) at st. justine's hospital, montreal were studied. bu and su levels were measured on day 3 of the conditioning regimen at the end of infusion (dose 9 in qid or dose 3 in qd dosing). efs was defined from the time of transplant until death, relapse, or rejection, whichever occurred first. a receiver-operator characteristic curve (roc) for bu mrs measured was plotted to show the trade-off in sensitivity vs. 1-specificity rates for efs, as the cut-off of the test was shifted from low to high. cutoff values were defined based on the youden´s j statistic (i.e. sensitivity+specificty-1). results: twenty-two males and 22 females aged from 0.1 to 19.9 years (mean±sd: 7.2 ± 5.7) from bu qid cohort had the mean mr of 5.9 (sd: 3.2). a cut off value of 4.9 in mr was chosen in roc analysis in this cohort, with better sensitivity (71 %) and specificity (70 %) for efs prediction (p=0.01, auc= 0.7 (95 % ci= 0.6-0.8). in qd cohort nine females, and four males aged between 0.4 and 15.8 years (6.7±5.1) had the mean mr of 29.3 (sd: 16.6). in roc analysis, a cut off value of 25.06 was chosen with better sensitivity (100 %) and specificity (100 %) for efs prediction (p=0.003; auc=1.0). conclusions background: treosulfan is an alkylating agent increasingly used prior to hematopoietic stem cell transplantation (hsct). the main objective of this study was to develop a population pharmacokinetic model of treosulfan in pediatric hsct recipients and to explore the effect of different covariates on treosulfan pharmacokinetics (pk). also, a limited sampling model (lsm) was developed. methods: in this multicentre study, 91 patients, receiving a dose of 10, 12 or 14 g/m 2 treosulfan a day, administered during 3 consecutive days, were enrolled. a population pharmacokinetic model was developed using nonlinear mixed effect modelling (nonmem version 7.3.0, using psn toolkit 4.7.0 and piraña version 2.9.7 as modelling environment). demographic factors, as well as laboratory parameters, were included as covariates. results: treosulfan pk was best described by a twocompartment model. a bodyweight-based allometric model improved the model more than a model incorporating body surface area (bsa). clearance (cl) and intercompartmental clearance parameters were 6.07 l/h/15.6kg (95%ci 5.46-6.68) and 2.15 l/h (95%ci 1.39-2.91). typical volumes of distribution of the central and peripheral compartments were 8.00 l/15.6kg (95%ci 6.88-9.12) and 2.05 l (95%ci 1.52-2.58). a model-based dosing table based on bodyweight is created to achieve a target exposure of 1540 mg*hr/l (table 1) , which was the median exposure of our population. estimated glomerular filtration rate (egfr) was shown to be the only parameter that significantly reduced interpatient variability in cl from 36.5% to 34.8%. a limited sampling model with 3 samples (taken at 1.5, 4 and 7 hours after start of infusion) accurately estimated pharmacokinetic parameters of treosulfan. conclusions: to the best of our knowledge, this is the largest cohort of pediatric patients treated with treosulfan used for a population pharmacokinetic study. we developed a two-compartment model with weight and egfr as covariates influencing treosulfan pk. recently we showed a relationship between treosulfan exposure and early toxicity. patients with an exposure >1650 mg*hr/l have an increased risk of developing grade 2 or higher mucositis and skin toxicity. another study in 87 pediatric patients with thalassemia major reported an association between treosulfan clearance (< 7.97 l/h/m 2 ) and poor overall survival. our model, together with the limited sampling strategy, can be used to adjust the dose, prior to or during treosulfan administration. ongoing studies conducted in different disease settings will determine if treosulfan exposure can influence patient outcome. subsequently, the optimal target exposure can then be established. background: autologous stem cell transplant (asct) is an effective treatment method for non-hodgkin lymphoma (nhl). until recently, carmustine, etoposide, cytarabine and melphalan (beam) was the most commonly used conditioning regimen. despite acceptable efficacy with beam, carmustine is associated with major pulmonary toxicity. for this reason, the aim of this study was to investigate the safety and efficacy of beb conditioning regimen for asct in nhl. methods: we conducted a prospective, multicenter, phase ii study for beb conditioning regimen for asct in nhl patients. a total of 33 patients were enrolled from 3 centers. they underwent asct with beb conditioning regimen (busulfan 3.2mg/kg for 3days, etoposide 400mg/ m 2 for 2days, bendamustine 200mg/m 2 for 2days) between 2016 and 2018. [[p111 image] 1. two year progression-free survival and overall survival.] results: the median age was 52 years (range 21-66) and 16 patients (48.5%) were men. the most common type was diffuse large b cell lymphoma (n=23, 69.7%) and more than half of patients (n=19, 57.6%) were classified as ipi score 3 or 4. eight patients (27.3%) had a history of relapse and 19 patients (57.6%) received more than 2 lines of chemotherapy before asct. most patients (n=27, 81.8%) were complete remission (cr) state at asct. a median number of 5.85x10 6 /kg cd34 cells were infused (range 2.0-18.6). all patients engrafted after a median time of 11 days (range 10-14). twelve patients (36.4%) experienced neutropenic fever and 16 patients (48.5%) had grade 3 toxicities during asct. however, no one had a documented infection, veno-occlusive disease, or treatment-related death. three months cr rate was 81.8%. during a median follow-up period of 10.2 month, 7 patients (21.2%) exhibited relapse or progression, while 1 patient (3.0%) died of the disease. the estimated 2-year pfs and os rate were 73.0% and 89.8%, respectively ( figure 1 ). conclusions: the beb conditioning regimens for asct is a feasible with tolerable toxicity in patients with nhl. disclosure: nothing to declare long-term report of total marrow or total lymphoid imrt in advanced leukemia, myeloma and lymphoma background: during the last three decades, total body irradiation (tbi) continues to play an important role in the conditioning regimens for patients undergoing stem-cell transplant (sct) for a wide variety of advanced hematological malignancies. however, tbi showed boundaries in dose limits for toxicity in allogenic and moreover in autologous stem cell transplantation. currently, the choice of conditioning regimen is based on the use of the least-toxic regimen to achieve the optimal therapeutic result. this report aims to assess the feasibility of a conditioning strategy based on high dose chemotherapy and whole-body radiotherapy focused on selective extensive tumor burden irradiation, both in allogeneic and autologous stem cell transplantation. methods: since december 2009, sixty-two patients (pts) have been irradiated by helical tomotherapy (ht) to extensive target before allogeneic or autologous transplantation. selected total marrow irradiation (tmi) schedules were planned to treat patients with high risk acute leukemia (all or aml) or multiple myeloma (mm) as a part of conditioning regimen. total lymphoid irradiation (tli) was planned for patients with refractory or relapsed (r/r) hodgkin (hd) or non-hodgkin lymphomas (nhl). results: tmi and tli allowed delivering therapeutic dose over extensive selected targets with wide reduction of toxicity to all the organs at risk (oars). the higher radiation doses rate to the oars is reduced from 30% to 70%. allogenic conditioning regimen was tli (4gy x 3fx) than fludarabine + endoxan for patients with hd (4 pts). tmi (4gy x 3fx) + fludarabine + melphalan for patients with mm (4 pts). tmi (4gy x 2 fx) + thiotepa + fludarabine + busulfan for advanced lam patients (4 pts). tmi as the boost (2-3gy) after conventional tbi was (12 gy in 6 bi-fractionated doses) by cyclophosphamide (18 pts). autologous preparation to sct consisted of tli (4gyx 3fx) followed by high-dose bendamustine and melphalan for patients older than 40 years and conventional feam (fotemustine, etoposide, cytarabine, and melphalan) for younger patients, in hd e nhl (20 pts). while tmi (4gy x 3 fx) plus melphalan was delivered for autologous sct in mm and lam (12 pts). no unexpected acute toxicity was found. in the allogenic setting, all the patients' engraftment was achieved in all patients. no acute graft versus host disease increasing was detected. within the autologous setting, only 33% developed grade 3/4 mucositis. none experienced grade 3/4 extra-hematological toxicity. outcomes of the specific disease will be reported. conclusions: the current report describes the clinical feasibility of using ht to deliver tmi or tli in the setting of autologous transplantation or during allogenic stem cell conditioning regimen, to allow all patients (old, fragile or with high tumor burden) to achieve an ablative regimen before sct. to our knowledge, this single institution experience describes data from one of the largest cohort of patients treated in europe since the development of this irradiation techniques. disclosure induction therapy in both groups of patients was based in polychemotherapy without the use of new drugs. case matching was performed according to age, clinical stage at diagnosis, and response to induction therapy. conditioning regimen consisted of iv bu at a dose of 3.2 mg/ kg once a day on days -5 to -3 followed by mel at a dose of -140 mg/m 2 on day -2 in the bumel group versus mel200 in the control group. maintenance therapy after transplant consisted of interferon and steroids in the majority of patients. results: the cut-off date for this update was june 30, 2018. after a median follow-up of 56 and 63 months in the bumel and mel200 groups respectively, 35 patients had relapsed in the bumel group and 82 patients in the control group. median pfs was 33 (95% ci, 25.4-48.3) months in the bumel and 24 (95% ci, 20.1-32.7) months in the mel200 group (p = 0.04) ( figure 1 ). in this update, 12 patients in the bumel group are in maintained response and 7 of them are in continuous cr (two with negative status for minimal residual disease) between 9 and 12 years after transplantation. ten-year os was not significantly different between both groups, being 41 (95% ci 30-58) months in the bumel and 29 (95% ci 18-47) months in the control group.transplant-related mortality was similar in both groups of patients (4% in the bumel and 2% in the mel200 group). regarding toxicity, bumel was associated with a higher incidence of mucositis and liver toxicity than the melphalan-only approach but no patient in our series developed sinusoidal occlusive syndrome and the hepatic toxicity observed was only grade i/ii. finally, no long-term side effects have been reported among bumel recipients. conclusions: this long-term follow-up analysis confirms that a therapeutic strategy including bumel as conditioning regimen beforeasct in patients with newly diagnosed mm is highly active and safe in these patients. [[p113 image] 1. figure 1 . progression free survival in the bumel (____) and control group (…… frequency of acute gvhd grade iii-iv [cc: 11%; ct: 17%; tt: 24%, p=0.057], and transplant-related mortality was higher in tt-carriers (cc:26%; ct:28%; tt:46%, p=0.02 cc&ct vs tt) . ta-tma, cmv infection/reactivation and cgvhd were also not different according to donor genotypes. fungal infections occurred more frequently as causes of death in carriers (cc: 9.7% vs. ct: 38.1% vs tt: 33.3%, p=0.022). conclusions: our results suggest that donor tgfb1 -1347c>t may exert an adverse influence on the outcome of myeloablative conditioning. our finding might be explained by the combination therapy of calcineurin and mtor inhibition in gvhd prophylaxis in myeloablative conditioning. disclosure: nothing to declare. treosulfan-based reduced intensity conditioning in hla-haploidentical transplantation using ptcy as gvhd prophylaxis in high-risk mds /aml of the elderly background: standard conditioning regimens prior to allogeneic hematopoietic stem cell transplantation (allo-hsct) are often associated with a considerable risk of severe adverse events, especially in elderly patients suffering from high-risk (hr) mds/aml. previous clinical studies have demonstrated feasibility of treosulfan-based reduced-intensity conditioning (ric) by stable engraftment, low non-relapse mortality (nrm), and favorable survival in elderly patients undergoing hla-matched related or unrelated allo-hsct (beelen et al, ash 2017 #0521). however, data for treosulfan-based conditioning in the t-cell-replete hlahaploidentical (haplo-hsct) setting in high-risk aml/mds patients are rare. here we report on the outcome of eleven patients treated with a treosulfan-based conditioning undergoing haplo-hsct using exclusively post-transplantation cyclophosphamide (ptcy) as gvhd prophylaxis. methods: eleven patients with high-risk (hr) aml (n=9)/mds (n=2) who underwent haplo-hsct using treosulfan for reduced intensity conditioning (ric) and ptcy as gvhd prophylaxis were retrospectively analyzed with respect to outcome and toxicity. all patients were >55 years old and transplanted between january 2016 and february 2018 at our institution. the majority of the patients (9/11) suffered from active disease at time of treatment initiation, only two patients presented in cr. all but one received sequential conditioning with cytoreductive chemotherapy using flamsa applied shortly prior to treosulfan-based ric (10g/m 2 over 3 days). a bone marrow graft was used in 9/11 patients. post-grafting immunosuppression consisted of cyclophosphamide, tacrolimus and mmf. national cancer institute common terminology criteria for adverse events version 3.0 were used for nonhematologic toxicity assessment starting from sequential therapy initiation or conditioning until day +30. results: median age of the entire cohort was 63 years (range: 58-71). the hct-ci was ≥2 in eight pts (median hct-ci=2, range: 0-5). no graft rejection occurred. neutrophil and platelet engraftment were achieved in 100% and 91% of the patients at a median of 20 (16-23) and 26.5 (13-30) days, respectively. acute gvhd grade ii-iv occurred in 18% of the patients, exclusively involving the skin. no one developed severe (°iii-iv) acute gvhd. no patient died prior to haplo-hsct. severe nonhematologic regimen-related toxicities (°iii-iv) occurred in 2/11 patients, predominately affecting the gastrointestinal tract. no patient suffered from ≥two iii-iv°toxicities. all patients developed fever during treatment course, four with positive blood cultures. cmv reactivated in 6/7 patients at risk. no ebv reactivation or ptld occurred. six patients had clinical and radiological signs of pneumonia (probable invasive aspergillosis) without detection of aspergillus/antigen in the bronchoalveolar lavage. ci of nrm at day +180 was 0%. four patients relapsed within the first year after haplo-hsct, with two of them dying due to relapse. at last follow-up (dec 2018) 9/11 patients were alive. with a median follow-up of 5 months (2-31) estimated 1-year os and dfs were 80% and 59%, respectively. conclusions: treosulfan-based unmanipulated hlahaploidentical allo-grafting using ptcy as gvhd prophylaxis in hr mds and aml patients aged over 55 years is safe and well tolerated resulting in stable engraftment and a favorable toxicity profile. our preliminary data further show promising outcome with low nrm, no severe acute gvhd and favorable survival offering an attractive alternative in ric for haplo-hsct of the elderly. disclosure: nothing to declare comparison of outcomes of total body irradiation (tbi) vs non-tbi conditioning regimens in acute lymphoblastic leukemia for allogeneic transplantation background: in adult patients diagnosed acute lymphoblastic leukemia (all) long-term results are poor with intensive chemotherapy. allogeneic stem stem cell transplantation is the potential treatment that provides cure for these patients. myeloablative preparation regimens include total body irradiation (tbi̇)+ cyclophosphamide(cy) and busulfan + cyclophosphamide.in adult all patients wbi/cy widely used, but the toxicity rate is higher. the aim of this study is to compare the result and effect of the tbi/cy and busulfan/cy regimens in allogenic bone marrow transplantation in all patients. methods: between 1993 -2018 there were 137 all patients who underwent transplantation using myeloablative preparation regimen with or without addition tbi in the adult bone marrow transplantation units of medipol medical faculty, istanbul university istanbul medical faculty, sisli florence nightingale hospital, atakent acıbadem hospital adult bone marrow units . we analyzed overall survival (os), progression free survival (pfs), veno occlusive disease, acute and chronic graft versus disease development rates in these patients. results: demographic characteristics of patients summarized in table -1 there was no significant difference between groups in donor age, gender, stem cell source. it was observed that the relapse rate was not statistically significant in both group.there was no statistically significant difference between the patients who underwent myeloablative regimen and myeloablative regimen with tbi in relaps,death, os, pfs. (figure-1) [[p117 image] 1. figure 1 ] in terms of transplant complications there was also no respectable difference in development of vod and acute and chronic graft versus disease but vod was more common in the group that did not use tbi (p: 0.068) ( conclusions: although there are contradictory data in the literature, in our multicentre study, it was revealed that the addition of tbi in the myeloablative preparation regimen compared with myeloablative preparation regimen alone did not have a positive or negative effect on overall survival.we think that if we can prepare a good vod prophylaxis approches, we can give up tbi in future. disclosure (n=3) . for gvhd prophylaxis, cyclosporine a was given either alone (n=21), with mmf (n=13) or with methotrexate (n=1). the graft source was bone marrow (bm) in most cases (n=31), pbsc in seven cases, matched sibling cord +bm in two cases and one matched related cord. twentyfive of the donors were family donors and ten were unrelated. twenty-nine of the donors were 10/10 hla matched, six were 9/10 mismatched and one haploidentical. four patients had engraftment failure and required a second transplant, two of them were re-transplanted with cyclophosphamide and tbi, one with fludarabine, busulfan and campath, and one with no conditioning. thirty of the 35 patients are alive (86%). four patients died of transplant complications and one died of metastatic squamous cell carcinoma. eight survivors are mixed chimeras (81%-94% donor) and are all doing well, none of them developed any gvhd. nine patients developed acute gvhd, four of them with grade 3-4. seven of these patients later developed chronic gvhd, two of them have extensive disease. conclusions: our results show a high survival rate of 86%, with a low rate of engraftment failure and reasonable rates of gvhd. only one of our patients died of late effects of hsct for fa. mixed chimerism does not seem to present a problem. we conclude that reduced intensity fludarabine based conditioning regimens are a good treatment option for patients with fanconi anemia undergoing hsct. disclosure: nothing to declare total marrow irradiation + bendamustine as reducedtoxicity myeloablative conditioning prior to allohsct for younger patients with multiple myeloma background: the prognosis of patients with multiple myeloma (mm) has improved markedly over the last two decades. despite that, allohsct remains the only treatment option with curative potential. however, its use is limited due to high incidence of non-relapse mortality (nrm) after myeloablative conditioning while insufficient efficacy of reduced-intensity regimens. we developed a new protocol characterized by reduced toxicity while preserved myeloablative potential, based on the use of total marrow irradiation (tmi) in combination with bendamustine. the aim of this study was to evaluate its safety and efficacy in a singlecenter experience. methods: between years 2013-2018, mm patients below 55 years old were offered tandem auto-allohsct as part of first-line therapy. the decision was based on individual patient preferences after detailed description of potential risks. autohsct was preceded by melphalan 200 mg/m 2 iv. the conditioning prior to allohsct consisted of tmi performed using helical tomotherapy at the dose of 4 gy/d on days -3, -2, -1 (total 12 gy) and bendamustine 140-220 mg/m2/d iv. on days -5, -4 (total 280-440 mg/m2). the immunosuppressive therapy consisted of cyclosporine + methotrexate +/-atg. peripheral blood was used as a source of stem cells. results: the analysis included 18 patients (women -9, men -9). the median follow-up was 28 (4-68) months. the median age at allohsct was 44 (26 -53) years. the disease stage before allohsct was as follows: cr-6, vgpr-4, pr-6. patients were treated with hsct from either hlamatched siblings (n=7) or unrelated donors (n=11). the interval between autohsct and allohsct was 5 (4-23) months. all patients engrafted after allohsct with median time of neutrophil and platelet recovery of 14 and 12 days, respectively. one patient (6%) experienced grade 2 acute gvhd, while there were no cases of grade 3-4 acute gvhd. the incidence of mild, moderate and severe chronic gvhd was 17%, 0% and 6%, respectively. the rate of grade 3 non-hematological toxicities was 11%. one patient died of late bacterial infection. the incidence of trm was 5%. grade 4 adverse events were not reported. disease status 3 months after allohsct was: cr-10, vgpr-5, pr-3. the probability of os and pfs after 30 months was 94% (+/-6%) and 77% (+/-12%), respectively. the incidence of progression and trm was 17% and 6%, respectively. conclusions: allohsct using tmi 12gy + bendamustine conditioning protocol is characterized by good tolerance and low risk of gvhd. it may be used for younger patients with mm as part of tandem auto-allohsct strategy. encouraging results reported in this study should be confirmed in prospective clinical trials. disclosure: nothing to declare p120 comparison between two reduced intensity conditioning regimens in patients with a myeloid malignancy: a single center experience comparing fb2 with flumel background: hematopoietic stem cell transplantation (hsct) remains the only curative option for high-risk myeloid neoplasms. the optimal reduced-intensity conditioning (ric) is still debated. methods: a single-center retrospective analysis was conducted at our institution to compare two different ric regimens in adult patients transplanted for myeloid malignancy from 2001 to 2018. a total of 137 patients were analysed, 74 of them treated with busulfan-based (fludarabine 150 mg/m 2 , busulfan 6.4 mg/kg, fb2) and 63 with melphalan-based conditioning regimen (fludarabine 150 mg/m 2 ,melphalan 140 mg/m 2 , flumel). antithymocyte globulin (atg) was administered in all patients while no one received tbi. partial in vitro t-cell depletion was performed using alemtuzumab for low risk patients. results: the two groups were well balanced with a median age of 61 and 62 years in the fb2 and flumel group, respectively, and a median follow up of 46 months. the most frequent indication for transplant in both groups was aml (59.5 and 69.8% for fb2 group and flumel group, respectively) and the stem cell source was peripheral blood in 94.6 and 96.8% of patients. more patients in the first group had near to significant worst karnosfky status (< 90) at transplant compared to second (35. 1 vs 19%, p=0 .057) and more patients received a t-partial depleted graft (54.1 vs 33.3%, p= .028). the neutrophil engraftment was significantly shorter after flumel (15 vs 18 days, p < .01). the 3-year overall survival (os) and disease-free survival (dfs) were of 43.0 and 36.5%, respectively, after fb2 and 54.9 and 52.0% after flumel, respectively, and were not significantly different (p=.41 for os and .15 for dfs), with a karnofsky >= 90 being the only factor significantly associated in univariate analysis with better os and dfs (p=.02 for both). the cumulative incidence (ci) of grade 2 to 4 acute graft-versus-host disease (agvhd) was 16.2% after fb2 and 38.3% after flumel (p< .001) and was associated in multivariate analysis with both t depletion and ric type (p< .001 and .005, respectively). the ci of chronic gvhd at 3 years was 13.9% in fb2 and 22.1% in flumel group (p=.24) . the ci of non-relapse mortality at 3 years was 18.7% after fb2 and 29.6% after flumel (p=.11). the ci of relapse at 3 years was 44.8% for the first and 18.4% for the second group (p< .001) and was associated with conditioning regimen in multivariate analysis (p=.02). no difference in 3-years gvhd-free/ relapse-free survival (grfs) was observed between the two group (25.5% for fb2 and 37.6% for flumel, p=.48). conclusions: when comparing two ric regimens for myeloid neoplasms, we observed a higher incidence of agvhd after flumel whereas no statistical difference was noted for the cgvhd occurrence. while the toxicity appears to be higher after flumel, this result is counterbalanced by a higher proportion of relapse after fb2, accounting for no difference in os, dfs and grfs between the two groups. these findings could be partially explained by a larger proportion of patients receiving a partial t-depletion after fb2 ric, but a larger trial is needed to clarify this issue. disclosure: nothing to declare. once-daily vs 4-times daily intravenous busilvex in conditioning regimen before allogeneic stem cell transplantation for patients with myeloid malignancies: safety and efficacy background: busilvex (bu) is part of standard conditioning regimen before allogeneic stem cell transplantation (asct) for patients with myeloid malignancies and usually administered as an intravenous (iv) infusion 4-times daily. this study aimed to compare the saftey and efficacy of this schedule to a once-daily iv bu. we conducted a retrospective study in adult patients (≥18 years) with myeloid malignancies who received asct from hla-identical sibling donors between january 2011 and june 2018 following iv bu-based preparative regimens. graft-versus host disease (gvhd) prophylaxis consited of cyclosporine and short course of methotrexate. intravenous bu was administered 4-times daily (0.8 mg/kg every 6 hours x12 to 16 doses) or oncedaily in a 3-hour infusion (3.2 mg/kg x 3 to 4 days) since june 2015. results: ninty-nine patients were enrolled (54 men and 45 women). median age was 35 years (range, 18-50 y). the median time from diagnosis to asct was 5 months (range, 51days -7 years). diagnosis were acute myeloid leukemia (n=79, 80%), chronic myeloid leukemia (n=8, 8%), myelodysplasic syndrome (n=6, 6%), primitive myelofibrosis (n=4, 4%) and chronic myelomonocytic leukemia (n=2, 2%). thirty-seven (37.3%) patients had ebmt-score ≥2. sixty-five (65.6%) patients were transplanted in cr1, 5 (5%) beyond cr1 and 24 (24.2%) had active disease. conditioning regimens consisted of bu/cyclophosphamide in 86 patients (86.8%), bu/fludarabine in 13 patients (13.1%). four-times daily bu was given to 58 patients (58.5%, groupe1) and once-daily bu to 41 patients (41.5%, groupe 2). stem cell source were bm in 46 patients (46.5%) and pbsc in 53 patients (53.5%). globally, patients characteristics were well balanced between the two groups. the rates of severe complications were similar between the two groups with no statistically significant differences except oral mucositis (table1). non-relapse mortality (nrm) was comparable in the two groups (21% and 17% in groups 1 and 2, respectively, p=0.65). the relapse rate was 24% and 32%, respectively (p=0.4). after a median follow-up of 2 years (range, 5days -7years), the os was not significantly different between groups 1 and 2 : 64% vs 56% (p=0.09). however, the rfs was significantly better in the groupe 1 : 62% vs 56% (p=0.03). conclusions: once-daily iv bu regimen seems to be an efficient and safe alternative to the 4-times daily protocol. however, results should be interpreted with caution because the historical comparison and lack of bu pharmacokinetics studies. disclosure background: standard therapy of the most patients with juvenile myelomonocytic leukemia (jmml) is allogeneic hematopoietic stem cell transplantation (ahsct). the choice of optimal conditioning regimen for patients with jmml is crucial as well as long-term observation. we aimed to estimate the long-term follow-up and survival rates of patients with jmml after ahsct with the help of busulfan or treosulfan-based conditioning regimens. methods: thirty eight patients with jmml underwent ahsct in 2002-2018. we compared equal groups of patients received busulfan (n=19) and treosulfan-based (n=19) conditioning regimen. m:f=28:11. median of age at hsct was 2.5 (0.6-5). donor type: hla-related 10/10 -29% (n=11), hla-related 9/10 -2.6% (n=1), hlaunrelated 10/10 -39.4% (n=15), hla-unrelated 9/10 -15.8% (n=6), and haploidentical -13.2% (n=5). stem cell source: bm -55.3% (n=21), pbsc -26.3% (n=10), ucb -10,5% (n=4), and ucb+bm -7.9% (n=3) . disease status on hsct: cr -73.7% (n=28), refractory -26.3% (n=10). results: median follow-up 15.5 months (1-129 months) . the estimated 10-year overall survival (os) probability in patients received busulfan-based conditioning was 48,8 ±13,4% in comparison with 68,0±10,8% in patients with treosulfan-based regimen (р=0,458). event-free survival (efs) was 42,1±11,3% in group with busulfan-based regimen and 57,0±11,5% in patients with treosulfanbased conditioning (р=0,224). background: post-transplant relapse remains the leading cause of treatment failure in high risk (hr) acute myeloid leukemia (aml), myelodysplastic syndrome (mds), myeloproliferative neoplasia (mpns) receiving allogeneic hematopoietic cell transplantation (allo-hct), especially for patients with relapsed or refractory aml. recently, a sequential transplant approach, as developed by the munich group, comprising of intensive cytoreductive chemotherapy flamsa (fludarabine/amsacrine/cytarabine) to decrease leukemia cell burden shortly prior to conditioning regimen, has been successfully used for high-risk (hr) aml/mds with promising results. methods: we studied 48 patients (median age 53 years, range 26 -68) with hr aml (n=38), as defined by refractory, relapsed disease, secondary leukemia, or high/ very risk disease risk index risk, and hr mds (n=10) according to ipss-r, undergoing allo-hct using the sequential transplant approach in 2 institutions between january 2009 and october 2018. the sequential transplant approach combined a cytoreductive chemotherapy, which consisted of either flamsa (n=17), flag +/-ida (fludarabine/cytarabine/granulocyte colony stimulating factor /idarubicin) (n=23), or clo-arac (clofarabine/cytarabine) (n=8), followed by reduced (ric) (n=43) or myeloablative (mac) (n=5) conditioning regimen. all patients received peripheral blood stem cell from matched related donors (n=27) matched unrelated donors (n=14), or mismatched unrelated donors (n=7). post-grafting immunosuppression consisted of calcineurin inhibitor and mycophenolate mofetil in all patients. thymoglobulin was added for gvhd prophylaxis for unrelated donor transplant. results: the median time to neutrophil > 1000/μl was 10 days (range, 9-25) . with a median follow-up of 28.2 months (range, 1.4 to 103.1 months), the kaplan-meier estimate of leukemia-free (lfs) and overall survival (os) at 5 years were 45 % (95% ci, 8-30), 46% (95% ci, 8-30), respectively. patients receiving flag or clo-arac based sequential regimen showed a trend towards more favourable overall survival (os) as compared to patients given flamsa (5 year os: 53% vs 31%; p=0.236). at 2 years, the cumulative incidences of relapse and non-relapse mortality (nrm) were 46 % (95% ci, 31-60 %) and 15 % (95% ci, 7-21 %), respectively. in multivariate analysis, the type of sequential conditioning regimen did not show any significant impact on lfs, os, nrm or relapse. conclusions: sequential transplant conditioning with flamsa, flag or clo-arac followed by allo-hct is an effective strategy in overcoming the dismal prognosis of hr aml and mds, and enabling long-term disease free survival. more studies on effective strategies such as posttransplant maintenance therapy of prophylactic donor lymphocyte infusion, are needed to further eliminate the risk of relapse, without increasing risk of treatment related toxicity. disclosure: nothing to declare optimization of the blood sampling procedure for busulfan therapeutic drug monitoring (tdm) to optimize our sampling scheme (15 minutes, 1, 2, 3, 4, 6, 8 and 10 hours after the end of a 3-hour infusion), we reduced the number of blood samples collected, reducing nursing and laboratory staff time and increasing patient convenience. this study aims to show the performance of a simplified sampling protocol which includes the first 5 samples from the original protocol. methods: individual pk parameters were retrospectively estimated using 5 samples (simplified protocol) and were compared with those obtained after 8 samples (original protocol). individual pk parameter values for a one compartment model were estimated using a maximum likelihood estimation modelling algorithm (adapt 5.0) and the statistical analysis of the results was performed (statgraphics centurion xv). results based on the approved dosage recommendations, mean (sd) initial dose was 171.1(69.3) mg. after tdm, mean (sd) calculated dose at day 1 for the remaining days (to achieve the defined target cumulative auc) was 158,5 (72,6) mg obtained from the original protocol. according to the simplified protocol the result would be 159,8(73,7) mg. the median and the mean variation of the calculated dose were 0% and 1% (0-8%) between protocols. a strong relationship between the cl of the day 1-3 obtained from the original protocol and the simplified protocol is observed (r 2 =0.9985). this high correlation is also observed for patients with busulfan t 1/2 >3h (r 2 =0.9936), a population were the reduction of sampling could be more problematic. anova test for the log cl with the factors: patient, day of busulfan and type of sampling protocol was performed. sampling protocol was determined as non-statistically significant (p = 0.7248). conclusions: results suggest that both protocols are equivalent concerning to the busulfan cl estimation and calculated auc. variation between protocols regarding the calculated dose at day 1 for the remaining days to achieve the defined target cumulative auc is considered acceptable. we verified a strong relationship between busulfan cl obtained from both protocols and sampling protocol doesn't influence cl statistically. a reduced sampling collection of 5 determinations until 4 h after the end of the infusion is shown to be sufficient for the tdm of busulfan, so this was implemented in our centre in line with published data. disclosure: nothing to declare p125 impact of anti-thymocyte globulin doses in unrelated hematopoietic stem cell transplantation for patients with myeloid neoplasm background: anti-thymocyte globulin (atg) is widely used for the prophylaxis of graft-versus-host disease (gvhd) in hematopoietic stem cell transplantation (hsct). however, there is still controversy regarding the optimal dose of atg. therefore, we analyzed the impact of atg doses in unrelated hsct for patients with myeloid neoplasm. methods: this was a retrospective multi-center study that assessed the impact of atg doses on clinical outcomes in patients with acute myeloid leukemia (aml) or myelodysplastic syndrome (mds) undergoing an unrelated hsct. the patients who received peripheral blood stem cells (pbsc) transplantation after conditioning regimens containing i.v. busulfan (bu), fludarabine and rabbit atg between 2010 and 2017 were included in this study. results: a total of 96 patents, median age 45 years, with aml (n=74) or mds (n=22) were included in our analyses. 66 patients (69%) received a myeloablative regimen (i.v. bu>6.4 mg/kg). high-atg (atg 9 mg/kg), intermediate-atg (atg 4.5-5 mg/kg) and low-atg (atg 3 mg/kg) were given in 11, 49 and 36 patients, respectively. after a median follow-up of 23 months, the cumulative incidence of extensive chronic gvhd was 9.1% in the high-atg group, 13.8% in the intermediate-atg group and 29.7% in the low-atg group (p=0.31). conclusions: our study shows that the incidence of extensive chronic gvhd was similar regardless of the doses of atg after transplantation of pbsc from unrelated donor for patients with aml or mds. however, the rate of relapsefree survival and the rate of a composite end point chronic gvhd-free and relapse-free survival were significantly higher in the intermediate dose ( methods: we retrospectively retrieved data from the electronic medical records for consecutive patients aged 65 and older, who underwent an asct for lymphoma over the last 10 years at our institution. results: forty four patients ≥ 65 years old underwent asct between 1 aug 2008 and 31 aug 2018. twenty eight of them received a reduced-dose conditioning (median 25%, range 20%-33% dose reduction). the dose was reduced for 92% of patients ≥ 70 years old and for 53% of patients aged 65-69. the outcomes of the following three groups of patients were compared: a) age ≥ 65; without dose reduction, b) age 65-69; with dose reduction and c) age ≥ 70; with dose reduction (table 1). only one patient aged 70 received full-dose conditioning. there was no significant difference between the groups in the number of previous chemotherapy cycles (median 2, range 1-3). however, significantly more patients at the age of 65-69 were in complete remission (cr) pre-transplant in both full and reduced-dose conditioning groups (a and b). no significant intergroup differences were observed in the occurrence of complications (mucositis and infections), day 30 transplantrelated mortality (trm) or engraftment day. similarly, no significant differences were found either in the 1-year progression-free survival (pfs), which was 50%, 64% and 50%, or 1-year non-relapse mortality (nrm), which was 17%, 7% and 10%, respectively for groups a, b and c. the 1-year overall survival (os) tended to be higher in group b (85%), compared to groups a (66%) and c (70%). conclusions: beam/beac conditioning dose reduction was not found to adversely affect 1-year pfs and os rates. despite the fact that 2/3 of the patients in the age group ≥ 70 underwent asct in partial remission and had dose reduction, theier achieved trm, pfs and os rates were similar to those of patients aged 65-69. beam/beac conditioning at a 75%dose may be a suitable option for patients in their seventh decade requiring asct. this strategy should be further evaluated in prospective clinical trials. background: the transplant related mortality in autologous transplants for lymphoma and multiple myeloma, reported worldwide ranges from 0-5%. from 2004-2015, the trm at our center for these two diseases was approximately 20%. we introduced changes in mobilization schedule, conditioning regimens and drug dosages to determine whether these changes affected the transplant related mortality and overall survival. methods: from april 2004-december 2015, we used beam (bcnu: 300 mg/m 2 on day -6; etoposide 200 mg/ m 2 on days -5 to -2, cytarabine 200 mg/m 2 on days -5 to -2 and melphalan 140mg/m 2 on day -1 as conditioning chemotherapy for patients admitted in transplant unit for autologous transplants in hodgkin's and non-hodgkin's lymphoma. in patients with multiple myeloma high dose melphalan (200mg/m 2 ) was used. the mobilization protocol consisted of cyclophosphamide 1.5gm/m 2 followed by gcsf 5μgm/kg twice daily till stem cell collection was completed. from january 2016, we changed the beam protocol to bendaeam with dose modifications that included: bendamustine 150mg/m 2 on days -5 and -4, cytarabine 150mg/m 2 on days -5 to -2, etoposide 150mg/m 2 on days -5 to -2 and melphalan 100mg/m 2 on day -1. for multiple myeloma melphalan was reduced to 150mg/m 2 . we used only gcsf for mobilization of stem cells, which was continued till stem cell harvest was complete. response to treatment was evaluated by comparing trm and overall survival for two time periods: 2004-2015 and from 2016 till date. results: from april 2004 till december 2015, n=78 autologous transplants were performed. the male:female ratio was 2.4:1. fifty seven patients underwent transplant for lymphomas, n=16 for multiple myeloma and n=5 for other diagnosis. median age was 23±14.6 (2-64 years). the mean mnc was 4.7 × 10 8 ± 1.7/kg. engraftment was achieved in 80% of patients. the transplant related mortality was 19.5% and overall survival was 72% (follow up: 104 months). since january 2016 till march 2018 we have performed n=18 autologous transplants of which n=17 were males. fifteen transplants were performed for lymphomas (nhl:8, hd:7) and n=3 for multiple myeloma. median age was 24±15 (20 -64 years). the mean mononuclear cell count was 5.8 x 10 8 /kg and the mean cd34 count was 3.7 x 10 6 /kg. engraftment was achieved in all patients. the transplant related mortality was 0% and the overall survival was 83% (follow up 22 months). conclusions: we were able to reduce the autologous transplant related mortality to 0% by decreasing dosages of conditioning chemotherapy and changing the mobilization protocol. long follow-up is needed to determine late mortality and late relapse in comparison to standard chemotherapy dosages disclosure: nothing to declare risk and benefit of thiotepa based conditioning followed by autologous stem cell transplantation in high risk lymphomas 36 years (16-62) . stage (ann-arbor) at diagnosis of hd/dlbcl/pcnsl: stage ie n=0/ 0/10 (0/0/91%), stage ii n=7/2/0 (47/18/0%), stage iii n=2/ 0/1 (13/0/9%), stage iv n=6/9/0 (40/82/0%). median time from diagnosis to asct hd/dlbcl/pcnsl: 25/11/ 6 month (4-72). induction treatment in hd patients was abvd, in most dlbcl patients r-chop and in pcnsl patients high dose methotrexate and cytosin arabinoside. tumor status at asct hd/dlbcl/pcnsl: complete metabolic remission (cmr) n=4/1/11 (27/9/91%) and from pcnsl patient's n=8 (73%) were in first complete remission (cr1). type of stem cell graft was periferial blood stem cell in all case. conditioning: thiotepa (250mg/ m2 on days -9 to -7, busulphan 3,2mg/kg on days -6 to -4 and cyclophosphamide 60mg/kg on days -3 to -2 plus rituximab 500mg/m2 on day -10 in dlbcl and pcnsl. median follow up from asct 711 days . tumor stage at asct was defined with computer tomography with positron emission tomography (pet-ct). results: median time of engraftment was 10 days (9-14). thiotepa caused toxicoderma appeared at 9 (24%) patients. cytomegalovirus (cmv) reactivation was seen in 3 (8%) cases with low dna content (284,454,5500 copies/ml) and responded completely to oral valgancyclovir therapy. transplantation related mortality hd/dlbcl/pcnsl n= 2/3/1 (9/27/13%), in 4 cases bacterial sepsis and one systemic mycoses and one pulmonary fibrosis. incidence of long-lasting grade iii-iv thrombocytopenia and anaemia: n=15 (40,5%) and n=3 (8%), median time of duration from transplantation 71 days (31-720) and 35 days ( background: this study evaluated the efficacy and toxicity of intravenous busulfan and thiotepa as a conditioning regimen for autologous stem cell transplantation (asct) in patients with multiple myeloma (mm). methods: we retrospectively analyzed the data of 68 patients with mm who received the intravenous busulfan and thiotepa conditioning for asct between november 2016 and april 2018 in korea. results: the median time to transplant was 5.4 months, and 66 patients (97.1%) underwent asct within 12 months of the diagnosis. the overall response rate after asct was 95.6%, including 55.9% with complete response, 22.1% with very good partial response, and 17.6% with partial response. the most common severe non-hematologic toxicity (grade 3-4) was infection (44.1%). three patients (4.4%) developed venous-occlusive disease. one patient (1.5%) died due to severe pneumonia after asct. after a median follow-up of 13.0 months, the median progression-free survival (pfs) and overall survival (os) were not reached. conclusions: in conclusion, a conditioning regimen of intravenous busulfan and thiotepa was effective and tolerable. clinical trial registry: not applicable disclosure: the authors have declared no conflicts of interest. myeloablative haploidentical bone marrow transplantation with post-transplant cyclophosphamide in paediatric patients with haematological malignancies santanu sen 1 , sameer tulpule 1 background: haploidentical transplants have been shown to be safe and effective in treating haematological malignancies in the paediatric population. we have previously reported on our experience of using reduced intensity conditioning with post transplant cyclophosphamide in haploidentical patients. we herein report our experience of using a tbi based myeloablative conditioning to treat our first 8 patients with haematological malignancies. methods: 8 patients were enrolled in the study, 5 with relapsed acute lymphoblastic leukemia (all) and 3 with relapsed/resistant acute myeloid leukemia (aml). all aml patients had genetic markers of high risk disease and all all patients had very early relapses (either on therapy or within 6 months of stopping therapy). all patients were conditioned with an identical protocol using tbi-based myeloablative preparative regimen (fludarabine 30 mg/m 2 /d × 4 d and tbi 150 cgy bid on d −4 to −1 [total dose 1200 cgy]) followed by an infusion of unmanipulated peripheral blood stem cells from a haploidentical family donor. postgraft immunosuppression consisted of cyclophosphamide 50 mg/kg/day on days 3 and 4, mycophenolate mofetil through day 35, and tacrolimus through day 180. results: median time of neutrophil and platelet engraftment was 11 and 19 days, respectively. all patients achieved sustained complete donor chimerism by day +28. acute gvhd, grades ii-iv and iii-iv, was seen in 75% and 25%, respectively. disease progression occurred in 2 patients: 8 & 10 months after transplant and there was one death due to severe fungal infection. estimated twoyear survival and relapse were 75% and 24%, respectively. 2 patients had severe bk viremia and cmv reactivation occurred in 4 patients. all patients were successfully managed with appropriate supportive and antiviral therapy. conclusions: we report good outcome with a myeloablative conditioning in haploidentical transplants with excellent engraftment and hopefully a longer life expectancy. with small number of patients, it is difficult to state whether using a myeloablative conditioning would lead to better long term outcomes in this cohort of patients with very haematological malignancies, but we certainly showed that it is possible to achieve excellent early results. disclosure: nothing to declare fludarabine in combination with melphalan and atg can be the best conditioning for hematopoietic stem cell transplant of children with hemophagocytic lymphohistiocytosis methods: in this prospective study, we analyzed the outcome of two pediatric patients with hlh who had received hsct, using reduced-intensity conditioning (ric) regimen. they received the same ric regimen based on the use of fludarabine (30 mg/m 2 /day for 5 days) in combination with melphalan (70 mg/m 2 /day for 2 days) and horse antithymocyte globulin (atg 10 mg/kg/d for 4 days). cyclosporine and methotrexate were used as graft-vs.-host disease (gvhd) prophylaxis. results: a 2 months boy with primary hlh (fhl2) was transplanted from his mother and a 4 years girl with secondary hlh was transplanted from her brother. both of donors were hla match with their recipients. they were received 6 x 10 6 /kg and 10 x 10 6 /kg cd34 + cells from the harvested peripheral blood stem cells, respectively. they achieved full neutrophil and platelet recovery. the time to neutrophil recovery was 13 and 11 days, respectively. full chimerism was achieved for both of them. in addition, they was developed grade 3 and 2 of acute gvhd, respectively. gvhd was completely controlled with prednisolone. they are alive and in complete remission without any significant complications after 36 and 14 months, respectively. conclusions: it appears that fludarabine in combination with melphalan and atg may be the best conditioning regimen for hematopoietic stem cell transplant of children with hlh. due to a few number cases of this study, a study with sufficient sample size is required. disclosure background: hematopoietic cell transplant (hct) recipients often report depression and impaired quality of life (qol) before transplant. mixed evidence suggests depression may be a risk factor for greater mortality and worse qol. inconsistent findings may be due to the fact that previous studies have not evaluated antidepressant use. the aim of the study was to compare pre-transplant patientreported physical functioning and post-transplant overall survival (os) between four groups of hct recipients: 1) non-depressed/taking antidepressant (treated depression), 2) depressed/taking antidepressant (undertreated depression), 3) depressed/not taking an antidepressant (untreated depression), and 4) not depressed/not taking an antidepressant (control). it was hypothesized that physical functioning and os would be worse among patients with untreated and undertreated depression relative to those with treated depression and controls. methods: this retrospective case-control study included patients completing depression (phq-8) and quality of life (sf-12) questionnaires at pre-transplant. analyses were conducted separately for allogeneic and autologous recipients. results: participants (n=1,797) were 58% men, mean age 57 years (19-79), 39% allogeneic recipients. regarding depression and antidepressant use, 146 (21%) allogeneic patients were characterized as having treated depression, 47 (7%) as untreated depression, 49 (7%) as undertreated depression, and 461 (65%) as controls. hierarchical linear regression models indicated that after adjusting for significant univariate factors (performance status, disease status, and regimen intensity), allogeneic patients with treated depression (b=-2.58, 95% ci=-4.63, -0.54) reported better physical functioning than patients with undertreated depression (b=-6.06, 95% ci=-9.43, -2.70) and untreated depression ) but worse physical functioning than controls (p values <0.05). cox regression models indicated depression/antidepressant usage was not associated with os among allogeneic patients (p values>0.10).among autologous patients, 195 (17.82%) were characterized as having treated depression, 83 (7.59%) as untreated depression, 77 (7.04%) as undertreated depression, and 739 (67.55%) as controls. hierarchical linear regression models indicated that after controlling for significant univariate factors (gender, performance status, diagnosis, and disease status), autologous patients with treated depression (b=-2.97, 95% ci=-4.71, -1.23) reported better physical functioning than patients with undertreated depression (b=-8.63, 95% ci=-11.23, -6.03) and untreated depression (b=-8.62, 95% ci=11.15, -6.08), but worse physical functioning than controls (p values <0.05). cox regression models showed depression/antidepressant usage was associated with os (p values <0.05), with patients with treated depression demonstrating significantly worse os than other groups (p=0.05), but this association was no longer significant in multivariate analyses controlling for diagnosis and disease status (p=0.09). conclusions: patients with untreated or undertreated depression pre-transplant may benefit from depression screening and treatment to improve physical functioning. disclosure: hslj: consultant for redhill biopharma and janssen scientific affairs p133 eltrombopag (epag) induces a high percentage of responses in patients with post allo-hsct poor graft function (pgf) and no active gvhd lourdes aguirre 1 , aitziber lizardi 1 , pilar bachiller 1 , brigida esteban 1 , carmen gonzález 1 , nagore argoitia 1 , maría araiz 1 , aranzazu aguirre 1 , anunciación urquía 1 , carlos vallejo 1 background: persistent cytopenia is a life-threating complication after hsct. several causes can lead to this situation (viruses, gvhd, drugs, etc) . a specific entity is the one called "poor graft function (pgf)", which is diagnosed in pts with ≥2 cytopenias after day +30, in the presence of donor chimerism and the absence of gvhd or relapse. pgf is more frequent after alternative allo-hscts, such a haplo-identical, mismatched, or ucb. several therapeutic approaches for pgf, with poor results, have been tested. recently, epag has been shown to improve platelet counts in the post-allo-hsct setting. in this study, we analysed the efficacy of epag in pts with post-transplant persistent cytopenias. methods: the population analyzed includes all 175 pts who underwent allo-hsct from june 2015 through may 2018 in our unit. median age was 52 years (12-69). 102 were male (58.3%) and 73 female (41.7%). baseline diseases were: 69 aml, 39 lpd, 20 all, 18 mds, 15 mpd, 9 mm, and 5 bmf. donor was unrelated in 101 (54.3%) and was family in 74 (42.3%) (including 25 haplo-identical). conditioning was ric in 95 (54.3%) and intensive in 80 (45.7%). sc source was pb in 164 (93.7%) and bm in 11 (6.3%). median followup was 24 months (6-41). epag was initiated at some point during the first 6-month post-hsct period in 12 pts (6.9% of the series) due to thrombocytopenia (< 20000/mcl) plus, at least, one other cytopenia. patients characteristics shown in table 1. epag was started at 50 mg/day and escalated each 2 weeks to 75, 125 and 150 mg/day if platelet count was < 20000/mcl. global response was considered when, after epag, the patient needed no transfusions and reached the three of the following: platelets >50000/mcl, hgb >10 g/dl, and anc >1000/mcl. epag was tapered off in responders and discontinued if no response was reached after 16 weeks. results: at epag initiation, all the 12 pts had thrombocytopenia (< 20000/mcl), 10 had anemia (hgb < 10 g/dl), and 5 had neutropenia (anc < 1000/mcl). counts pre and post and response to epag are shown in table 2. among the 8 responders, all but one (who relapsed from thrombocytopenia and died from bleeding) were alive at analysis close (87.5%). among the 4 non-responders, three pts had gvhd-associated cytopenias, and finally died from infectious complications; the other patient relapsed from her aml, reached a new cr after treatment, and is alive and well 27 months afterwards. epag was tapered off and discontinued in 6/8 pts who responded; 2/8 responders are still on epag. epag was discontinued in the 4/4 pts who did not respond. rest of treatment details shown in table 2. conclusions: 1) epag worked striking well in subjects with pgf, an otherwise a life-threatening situation for patients. 2) epag induced impressive responses in platelets, but strong bilinear and trilinear responses were also seen. 3) epag did not improve gvhd-associated cytopenias. 4) to confirm these innovative and transcendent results, we have just initiated a multicenter prospective study on the role of epag for treatment of post-hsct pgf. 1232 * five out of the six urdt were mismatched ** the donor was a woman in the six cases: three sisters and three daughters background: hepatic vod/sos with multi-organ dysfunction (mod; typically, renal or pulmonary) may be associated with >80% mortality. defibrotide is approved for treating severe hepatic vod/sos post-hsct in patients aged >1 month in the eu, and for hepatic vod/sos with renal or pulmonary dysfunction post-hsct in the us. this analysis provides an overview of the safety results from 3 studies of patients with vod/sos, with or without mod, who received defibrotide 25 mg/kg/day. methods: safety data were pooled from patients with vod/sos post-hsct treated with defibrotide in a phase 3 trial (n=102) and a phase 2, randomized dose-finding trial (n=74 receiving 25 mg/kg/day). safety data for historical controls (hc) from the phase 3 study (n=32) also are provided. reported separately, due to differences in patient population and data monitoring protocol, are aes from the expanded-access program (t-ind) in patients with vod/ sos with and without mod (n=1000 post-hsct). vod/ sos was diagnosed by baltimore criteria/biopsy for the phase 2/3 studies; diagnosis by baltimore or modified seattle criteria was permitted in the t-ind. results: median patient age at hsct for the phase 2/ 3 studies was 24.0 years, 18.0 years for the hc, and 14.0 years for the t-ind. in the phase 2/3 studies defibrotide-treated group (n=176), 169 (96.0%) experienced aes; most common (>10%) were hypotension (36.9%), diarrhea (24.4%), and multi-organ failure (21.6%). treatment-related aes were at least possibly related to defibrotide (table) . any hemorrhage (an ae of special interest) occurred in 101 patients (57.4%); most commonly epistaxis (13.6%), gastrointestinal and pulmonary alveolar hemorrhage and hematuria (8.5% each), and conjunctival hemorrhage (6.3%). all 32 hc experienced an ae; most common (>25%) were hypotension (50.0%), tachycardia (43.8%), diarrhea (37.5%), nausea (31.3%), and pyrexia, agitation, and petechiae (28.1% each). any hemorrhage occurred in 24 patients (75.0%): most common (>10%) were petechiae (28.1%); hematuria, epistaxis, and pulmonary alveolar hemorrhage (15.6% each); and lip hemorrhage (12.5%). in the t-ind (n=1000), 385/512 patients with mod (75.2%) and 324/488 patients without mod (66.4%) had an ae; other than vod/sos and mod, most commonly (>10% in either subgroup) hypotension (15.2% and 8.4%, respectively). traes occurred in 210 patients (21.0%) ( table) . any treatment-emergent hemorrhage occurred in 166 patients with mod (32.4%) and 124 patients without mod (25.4%); most commonly (>5% in either subgroup) pulmonary hemorrhage (8.2% and 4.7%, respectively) and gastrointestinal hemorrhage (5.5% and 4.3%, respectively). conclusions: the incidence and type of aes were as expected in these critically ill patients. of the pooled patients, 96% had aes; 57.4% had a hemorrhage. all hcs had an ae, with 75.0% having a hemorrhage. in the t-ind, patients with mod had higher rates of aes. support: jazz pharmaceuticals event, n(%) phase 2/3 studies (n=176) disclosure: paul g. richardson has served on advisory committees and as a consultant, and has received research funding from jazz pharmaceuticals. angela r. smith and leslie lehmann have nothing to disclose. nancy a. kernan received grants from gentium during the conduct of the study, and her research was supported by the national cancer institute of the national institutes of health under award number p30 ca008748; the content is solely the responsibility of the author and does not necessarily represent the official views of the national institutes of health. she has a research grant from jazz pharmaceuticals. robert ryan and william tappe are employees of jazz pharmaceuticals and hold stock and/or stock options in jazz pharmaceuticals plc. stephan a. grupp has served on a steering committee and as a consultant to jazz pharmaceuticals. defibrotide for treatment of adults with hepatic vod/ sos with or without multiorgan failure after hematopoietic cell transplantation: results of a systematic review/meta-analysis background: although hematopoietic cell transplantation (hct), autologous or allogeneic, is potentially curable in various hematologic malignancies, the procedure is associated with serious and potentially life-threatening complications, among them veno-occlusive disease/sinusoidal obstructive syndrome (vod/sos) of the liver. several studies, prospective or retrospective, have reported outcomes of defibrotide, when used as prophylaxis or treatment, in a mixed population of adult and pediatric patients. in this systematic review/meta-analysis, we analyze outcomes of defibrotide when specifically used for treatment of adult patients with hepatic vod/sos with or without multiorgan failure. methods: a comprehensive search of 3 large databases (medline/pubmed, cochrane and embase) on november 2, 2018 identified 642 publications. analysis was restricted only to adult patients (defined as median age older than 16 years) who received defibrotide for treatment of vod/sos and were reported in prospective or retrospective (which included ≥ 5 patients) studies published in full manuscript form. there were no limitations based on language. data were extracted in relation to benefits [complete remission (cr) rate and overall survival (os)] and harms (hemorrhage, any site or organ-specific). a total of 15 studies (prospective=6; retrospective=9) with 1437 patients met inclusion criteria. results: the median year of publication of prospective studies was 2013 (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) and for retrospective ones 2016 (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) . the prescribed starting dose of defibrotide varied among studies ranging from 6.25 mg/kg/day to 80 mg/kg/day, mostly for a 21-day course. the pooled cr rate was 39% (95%ci=28-49%) for prospective and 54% (95%ci=39-69%) for retrospective studies. the pooled day +100 os rates were 43% (95%ci=37-48%) and 65% (95% ci=53-75%) for prospective and retrospective studies, respectively. the pooled rates of hemorrhage (any site) were 15% (95%ci=2-35%) for prospective and 21% (95% ci=4-43%) for retrospective studies. when analyzing organ-specific hemorrhage, 3 prospective studies (n=1091 patients) reported pooled rates of pulmonary alveolar (pa) hemorrhage of 2% (95%ci=1-3%) and of 5% (95%ci=3-7%) for gastrointestinal (gi) hemorrhage. only one retrospective study (n=14 patients) reported an incidence of pa hemorrhage of 7% (95%ci=0-34%) and a different study (n=14 patients) reported an incidence of gi hemorrhage of 14% (95%ci=2-43%). none of the 15 studies reported cerebral hemorrhage as a complication of defibrotide therapy. conclusions: this systematic review/meta-analysis confirms the efficacy of defibrotide for treatment of vod/sos with or without multiorgan failure, yielding cr rates of 39-54% and day +100 os rates of 43-65%. the purportedly higher pooled cr and os rates observed with retrospective (vs. prospective) studies are likely due to assignment-bias inherent to observational studies. moreover, although the pooled hemorrhage (any site) rates of 15-21% is considered proportionally significant, the pooled rates of pa and gi hemorrhage were ≤ 5%, in prospective studies. clinical trial registry: not applicable disclosure: m.a.k-d: consultancy for pharmacyclics m.m: received lectures honoraria and research support from jazz pharma efficacy and safety of defibrotide in the treatment of hepatic veno-occlusive disease/sinusoidal obstruction syndrome following hematopoietic stem cell transplantation: interim results from the defifrance study background: hepatic veno-occlusive disease/sinusoidal obstruction syndrome (vod/sos) is a potentially lifethreatening complication of conditioning for hematopoietic stem cell transplant (hsct) but may occur after nontransplant chemotherapy alone. vod/sos with multi-organ dysfunction (mod) may be associated with >80% mortality with supportive care alone. diagnosis of vod/sos was traditionally based on baltimore or modified seattle criteria; however, the ebmt recently published separate diagnostic criteria for adults and children. defibrotide is approved for treating severe hepatic vod/sos post-hsct in patients aged >1 month in the eu, and for hepatic vod/sos with renal or pulmonary dysfunction post-hsct in the usa. the goal of the defifrance study, requested by the french health authorities, is to collect real-world data on safety and efficacy in a broader patient population in france, including all indications. this is the first interim analysis of the largest current evaluation of defibrotide for the treatment of vod/sos in europe. methods: defifrance is an observational, multicenter, post-marketing study that includes any patient treated with defibrotide from hsct centers in france. this interim analysis is based on all patients treated with defibrotide, including those with severe and very severe post-hsct vod/sos. vod/sos was diagnosed using traditional criteria. day+100 survival, complete remission (cr; total serum bilirubin < 2 mg/dl and resolution of mod), and safety profile are reported. results: a total of 324 patients treated with defibrotide were included retrospectively and prospectively between july 2014 and october 2018 from 36 table] disclosure: mohamad mohty: has received honoraria and research funding from jazz pharmaceuticals, delphine lebon: nothing to disclose, ann berceanu: none, charlotte jubert: has received funding from jazz pharmaceuticals, ibrahim yakoub-agha: has received honoraria from jazz pharmaceuticals, stéphane girault: none, marie detrait: has received research funding from jazz pharmaceuticals, cécile pochon: none, fanny rialland: none, virginie gandemer: none, jean-hugues dalle: has received honoraria from jazz pharmaceuticals, régis peffault de latour: has received research grant / honoraria / board from pfizer, novartis, alexion; research grant amgen; and honoraria from jazz pharmaceuticals, david michonneau: has received honoraria from jazz pharmaceuticals, myriam labopin: has received honoraria from jazz pharmaceuticals, floriane delaval: employee of jazz pharmaceuticals and holds stock and/or stock options in jazz pharmaceuticals plc, gerard michel: none, anne sirvent: none, laurence clement: none anne-lise menard: none, anne huynh: has received honoraria from jazz pharmaceuticals, virginie bouvatier: employee of jazz pharmaceuticals and holds stock and/or stock options in jazz pharmaceuticals plc, raj hanvesakul: employee of jazz pharmaceuticals and holds stock and/ or stock options in jazz pharmaceuticals plc, zakaria medeghri: employee of jazz pharmaceuticals and holds stock and/or stock options in jazz pharmaceuticals plc p137 incidence and predictors of severe cardiotoxicity in patients with severe aplastic anemia after haploidentical hematopoietic stem cell transplantation zheng-li xu 1 , lan-ping xu 1 , yuan-yuan zhang 1 , yi-fei cheng 1 , xiao-dong mo 1 , feng-rong wang 1 , yu-hong chen 1 , wei han 1 , chen-hua yan 1 , yu-qian sun 1 , ting-ting han 1 , yu wang 1 , xiao-hui zhang 1 , xiao-jun huang 1 1 peking university institute of hematology, peking university people's hospital, beijing, china background: severe cardiotoxicity after hematopoietic stem cell transplantation (hsct) is a rare but fatal complication. the aim of this study was to evaluate the frequency of severe cardiac complications and to assess the ability of various factors to predict these complications in patients with aplastic anemia after haploidentical transplantation., this is the first study evaluating the values of both clinical and imaging factors in the prediction of severe cardiotoxicity among saa patients after haploidentical transplantation. methods: a retrospective study was conducted in 216 consecutive aplastic anemia patients who received haploidentical transplantation from 2006 to 2017. all patients received a unified regimen including busulfan, cyclophosphamide (ctx) and antithymocyte globulin at our single center. results: a total of 12 (5.6%) patients developed grade iii or iv cardiac toxicity. patients with cardiotoxicity had significantly poorer overall survival (os) than those without cardiotoxicity (12.5% vs. 89.6%, p< 0.001). our multivariable model identified four independent adverse predictors of severe cardiotoxicity, including pre-transplant ecog score (≥2), abnormal st-t wave on 12-lead electrocardiogram (ecg), hyperlipemia and recalculated ctx dose (≥1.8 g/m2/d). a predictive risk model was refined as low risk (0-1 factor), intermediate risk (2 factors) and high risk (3-4 factors) . the respective incidences of severe cardiotoxicity were 50.0%, 6.0%, and 1.3% in the high-, intermediate-and low-risk groups (p< 0.001). the corresponding os rates were 49.0%, 80.4%, and 90.3% in the three groups (p< 0.001) at the last follow-up. conclusions: patients with high risk scores had the poorest outcomes and should be monitored closely. a reduced intensity conditioning might be recommended for these patients. disclosure: there are no conflicts of interest to declare. background: allogeneic stem-cell transplantation (allo-sct) is associated with significant transplant-related mortality (trm). acute renal failure (arf) is a frequent complication and usually presents early after the procedure, compromising its feasibility. the aim of this study is to analyse the incidence of arf, its risk factors and its potential impact on trm after allo-sct. methods: 422 patients were included (244 males [58%]; median age 43 years, range 16-67) treated with allo-sct consecutively between january 2001 and april 2012 in a single institution. patient characteristics are detailed in table 1. median follow-up was 1.8 years (range, 1.0-2.7). renal function was evaluated using creatinine and data was collected pre-transplant (baseline) and at the point when arf was developed after allo-sct. arf was evaluated using akin criteria, being akin-1 an increase 1.5-to 1.9-fold from baseline, akin-2 an increase 2.0-to 2.0-fold and akin-3 an increase ≥3-fold. chronic renal disease was evaluated one year after the date of arf using kdigo criteria. results: cumulative incidence of arf at 1 year was 63% (akin-1, 25%; akin-2, 27%; akin-3, 15%). in the multivariate analysis, arf (akin-1/2) was associated with: non-use of antithymocyte globulin in conditioning chemotherapy, p=0.02 (hr 2.3, 0.2 to 0.9) and development of severe agvhd, p=0.04 (hr= 1.5, 1 to 2.3). in patients with arf akin-3, the most important variables in the multivariate analysis were: use of methotrexate (mtx) plus cyclosporine vs mycophenolate mofetil plus cyclosporine as gvhd prophylaxis, p=0.009 (hr=1.9, 1.2 to 3.1); myeloablative conditioning vs reduced intensity, p=0.03 (hr=1.7, 1 to 2.8) and use of total irradiation therapy in conditioning, p=0.02 (hr=1.7, 1.1 to 2.8). trm at 1 year increased significantly according to akin: akin-1, 25%; akin 2, 35%; akin 3, 51%; p=0,003; hr=11.2. overall survival at 3 years according to akin was: akin 1, 52%, akin 2, 45% and akin 3, 29%; p=0,004 (figure 1). the incidence of chronic renal disease at 1 year after allo-sct according to arf was: no arf (8%), akin-1 (11%), akin-2 (15%) and akin-3 (16%); p=0.006. conclusions: arf is a frequent complication during the first year after allo-sct and is associated with several factors. arf akin-3 was associated with more intensive strategies received during conditioning, meanwhile akin-1/2 were related to development of gvhd. there is an association of arf (akin-1, 2 or 3) with development of chronic renal disease. background: the introduction of cellular therapies such as car-t and modalities of gvhd-prophylaxis with posttransplant/cyclophosphamide (ptcy) that increase the number of admission days have boosted the pressure of available beds in the bm-units. in this sense, our centre started an at-home allogeneic stem cell transplantation (allo-sct) program to follow aplasia from the d+1 until independent ambulatory patient. to evaluate the feasibility and safety of allosct, we compared two groups: allohsct/athome (ah-group) vs. allohsct/in-patient (ip-group). methods: we included 78 patients receiving allosct (january 2014-november 2018) in a single centre: 39 patients, ah-group and 39, ip-group. all patients received conditioning at the hospital. gvhd-prophylaxis consisted in tacrolimus (tk) plus mycophenolate (mpm) or methotrexate, or ptcy (d+3, d+4) plus tk (d+5). all patients received prophylaxis with levofloxacin, fluconazole and acyclovir. besides that, ah-group patients received prophylaxis with ceftriaxone 1g/24h iv or ertapenem 1g/ 24h iv, and aspergillus-prophylaxis with inhaled liposomal amphotericin-b or posaconazole during neutropenia. patients of ah-group since d+1 or d+6 (in ptcyprophylaxis) received a nurse visit at-home once daily. the visits by the physician were performed at the hospital and only during complication events. first-line therapy of neutropenic fever was meropenem 1 g/8h in both groups, using a portable infusion pump in ah-group. in this group, the absence of focal infection or signs of severe sepsis allowed returning home after the initiation of antibiotics. the platelets support was performed at-home and the red blood support at hospital. results: the median (range) age (years) of the series was 54 . the median follow-up of the series has been not achieved. the source of the sct was peripheral blood in all cases. we didn't find statistical differences between two groups (ah vs ip) in terms of age, diagnosis, type of donor, intensity of conditioning, gvhd-prophylaxis, toxicity (mucositis, acute renal injury, neutropenia and thrombocytopenia), agvhd, aspergilosis and trm. interestingly, a significant reduction of neutropenic fever was observed resulting the lower use of meropenem in the ah-group than ip-group. the admission median days were similar in the both groups and it represented 21-23 days the reduction in the total economic cost of the ah-group. the whole analysis of the results are detailed in table: in-patient group, conclusions: in our experience, at home allosct, including ptcy-gvhd prophylaxis, is a feasible and safe procedure reflected in similar trm and aspergillosis incidence. at-home allo-sct is associated with a significant lower risk of neutropenic fever than in-patient group, as well as a very low readmission rate. disclosure: gonzalo gutiérrez-garcía: honoraria from gilead. grant from jazz pharmaceutical and janssen. laura rosiñol: honoraria from takeda, janssen, amgen and celgene. the others author do not have any disclosures to declare. background: renal complications in sickle cell disease (scd) include episodes of acute kidney injury (aki), progressive chronic kidney disease (ckd) and hyperfiltration, defined by abnormally high glomerular filtration rates (gfrs). hematopoietic stem cell transplant (hsct) from an hla identical sibling donor is a well-established curative treatment for scd, but traditional myeloablative conditioning (mac) regimens pose risks of kidney injury due to intensive use of chemotherapeutic agents, infectious risks, and use of calcineurin inhibitors (cnis). aki and subsequent fluid overload (fo) are common in pediatric hsct with reported aki incidence of 21%-50% (kyung-nam koh et. al., 2017). we report renal outcomes in pediatric patients with scd who received hsct following a non-myeloablative conditioning (nma) regimen without cni exposure. methods: retrospective chart review describing renal outcomes in pediatric patients (18 years of age or younger) with scd (hbss) who underwent nma hsct in alberta, canada from july 2013 to february 2018. the nma regimen is illustrated in figure 1 . reported renal outcomes: 1) measured gfr (dtpa) pre-hsct, 2) aki (kdigo definition) post-hsct by reviewing all serum creatinine levels from pre-hsct to one month post-hsct, 3) %fo calculated: (max post hsct weight -baseline weight)/ baseline weight x 100 for the two first weeks post-hsct, and 4) estimated gfr (egfr) using the pediatric schwartz formula at last follow-up post-hsct, ckd defined as egfr < 60 ml/min/1.73 m 2 , mildly reduced gfr: 60-90ml/min/1.73 m 2 , and hyperfiltration: gfr ≥ 150 ml/ min/1.73 m 2 . [[p140 image] 1. results: eighteen patients (33% male, 3-18 years old at transplant) were included. most common pre-morbid events: vaso-occlusive crisis (n=17), acute chest syndrome (n=8), splenic sequestration (n=6), and cholelithiasis (n=4). median follow-up time: 27 months (range: 7 -62 months). all patients engrafted successfully with no acute or chronic gvhd. baseline measured gfrs were all > 60 ml/min/1.73 m 2 (range: 79-227) with mildly reduced gfr and hyperfiltration seen in one (5.6%) and 12 (66.7%) patients respectively. at baseline (pre-hsct), the only aki event was one transplant related aki secondary to delayed hemolytic reaction after exchange transfusion in preparation for transplant. post-hsct, there were no aki events. additionally, no substantial %fo post-hsct was observed. average %fo week one post-hsct: +0.01% (range: -4.2% -+1.0%) and week two post-hsct: +0.04% (range: -4.24% -+1.5%). post-hsct egfr remained > 90 ml/min/1.73 m 2 at last follow-up in all patients. hyperfiltration was present in 5 (27.8%) of the patients. conclusions: this is the first study describing stable kidney function in children with scd after the present nma hsct regimen with alemtuzumab/300 cgy total body irradiation (tbi) with prolonged post-hsct sirolimus. no episodes of aki or significant fluid overload were observed during the first month post-hsct, and no patient developed ckd during follow-up. further prospective studies are needed to confirm our findings and to determine if stable renal function persists during longer-term followup. disclosure: nothing to declare. lung microbiota in patients with idiopathic pneumonia syndrome (ips) after hct background: idiopathic pneumonia syndrome (ips) is a non-infectious pulmonary complication after hematopoietic cell transplantation (hct) and the etiology remains unknown. recent studies have reported that various diseases are associated with changes of microbiota. the aim of this study was to evaluate the lung microbiota in hct recipients with ips and identify microorganisms potentially associated with ips. methods: frozen bronchoalveolar lavage (bal) samples from hct recipients with ips (n=18) and research bal samples from asymptomatic hct recipients as controls (n=12) were retrospectively analyzed. all samples were negative for common viruses by quantitative pcr. sequencing libraries were made with 1ng of input dna per sample (nextera xt, illumina). samples were pooled and sequenced by hiseq 2000 to obtain 100-bp paired end data. sequence data analysis and read classification were performed with sunbeam and the quality control and read classification were performed using komplexity and kraken, which classifies bacterial, archeal, and viral genomes. we used sequence data of bronchoscope prewashes from a separate cohort as controls for environmental sources (n=24). bray-curtiss dissimilarity among samples was calculated using the vegan r packages. permanova and a two-sided wilcoxon rank sum test were used to compare between the study groups. results: bal samples started at a median of 22x10 6 raw read pairs per sample and reduced to 21x10 3 reads assignable to microbial taxa following quality control. the bacterial phyla proteobacteria and firmicutes were most abundant followed by bacteroidetes and actinobacteria in both bal and bronchoscope prewash samples. separation of bal and prewash microbiota using bray-curtiss dissimilarity plots showed that bal samples were distinguished by sequences assigned to staphylococcus, acidovorax, and bradyrhizobium species, while prewash samples were distinguished mostly by pseudomonas and elizabethkingia species, consistent with environmental sources (figure) . within bal samples, staphylococcus species were the main drivers of separation between ips cases and the controls (p=0.002, permanova, figure) . consistent with this, a linear discriminant analysis to identify taxa best distinguishing cases and controls identified staphylococcus, especially s. epidermidis, in ips cases with lactobacillus and streptococcus species in controls. we then compared relative abundances of s. epidermidis between all study groups. ips case samples were significantly enriched in s. epidermidis compared to control (p< 0.001, two-sided wilcoxon rank sum test) and prewash samples (p< 0.001). viruses were classified by category as human pathogens, non-human pathogens, and bacteriophages. torque teno viruses (ttv) was the most commonly detected virus among viruses that replicate on human cells, and there was a trend towards higher abundance in ips case samples than controls. conclusions: lung microbial sequences in hct recipients predominantly consisted of proteobacteria and firmicutes, and had considerable overlap with environmental background. patients with ips had significantly more staphylococcus sequences detected than asymptomatic hct patients. these results suggest that patients with acute lung injury post-hct show distinct patterns of lung microbiota, although heterogeneity of sample collection and processing cannot be excluded and no singular organism was uniquely associated with ips. a prospective study is required to confirm these findings and define the clinical significance of differences in abundance patterns. disclosure: nothing to declare p142 abstract withdrawn. romiplostim for the treatment of thrombocytopenia after allogeneic stem cell transplantation background: thrombocytopenia is a common complication after allogeneic stem cell transplantation (allo-hct). with variable possible causes, such as drug side effects, infections, poor graft function, graft vs host disease (gvhd) and immune mediated. the purpose of this study was to evaluate the efficacy of romiplostim, a thrombopoietin receptor agonist, in patients with prolonged thrombocytopenia with no obvious cause after allogeneic transplantation. methods: retrospective analysis of allo-hct patients who received romiplostim at a single bmt unit between november 2015 and november 2018. romiplostim was given because of prolonged (>3 weeks) thrombocytopenia (< 60,000 μl) that couldn't be explained by obvious causes such as administration of drugs (antibiotics/antivirals), infection or gvhd. all patients were in complete remission and had complete chimerism. response to romiplostim treatment was considered transfusion independence or plt>80.000/μl. results: in total, 19 patients (median 45 years, 19-67) received romiplostim. patients (10 male, 9 females) had aml (10 pts), all (8), mds (2) or hodgkin (1), received a myeloblative (busiphex-based: 16, tbi-based:1) or ric (2) conditioning and were transplanted from a sibling (5), vud (11) or haploidentical (3) donor with pbsc (16) or bm (3) . all patients revealed primary neutrophil (median 14 days, range 10-19) and >20.000/μl platelet (13 days, 7-31) engraftment. romiplostim was started at median day +104 (range 58-419) with a median dose 5 μg/kg (1) (2) (3) (4) (5) . the median platelet count before commencement of treatment with romiplostim was 24.000/μl (range 12.000-57.000) and 10 them (59%) were transfusion-dependent. in total 14/17 (82%) patients responded to romiplostim treatment. eight out of the 10 (80%) transfusion dependent patients responded to the administration of romiplostim. six out of the 7 patients (86%) who were transfusion independent at romiplostin initiation (plt median 28.000/μl, range 19.000-56.000) responded. the median duration of treatment was 74 days (15-253) and the median follow up from the commencement of romiplostim was 177 days (15-1080). three out of 17 (18%) patients experienced relapse of thrombocytopenia after discontinuation of romiplostim and re-initiation of romiplostim was commenced in all of them, of which 2 responded and 1 didn't. the administration of romiplostim was done on an external basis and was well tolerated by the patients. two patients experienced gvhd during romiplostim treatment (both patients transplanted from 7/8 unrelated donor, 25 and 42 days after initiation treatment with romiplostim). 3/19 patients interrupted romiplostim due to disease relapse. 11/19 patients receiving romiplostim are alive in complete remission and 8 died (3 due to relapse, and 5 due to trm). conclusions: we present high response rates to romiplostim in patients with prolonged thrombocytopenia after allogeneic transplantation. in this retrospective study there were no side effects from the administration of romiplostim. however, the administration of romiplostim after allo-hct should be controlled in prospective trials. disclosure we report a single-center analysis of 29 adult patients (median age 22 years, range 18-57, m/f 13/16), receiving tpo agonists for isolated severe thrombocytopenia (n=7) and spgf (n=22) after allo-hsct. primary diagnoses were aml (10), all (6), mds (7), pmf (4), mds/mpn (4), saa (4), cml (1), nhl (1) . severe pgf was defined as cytopenia in ≥ 2 lineages (platelet < 20 × 10 9 /l, anc < 0.5 × 10 9 /l, hemoglobin < 70 g/l any time after sustained engraftment), full or stable mixed donor chimerism > 90 % and no signs of relapse. median dose of romiplostim was 5 (range, 3-5) mcg/kg weekly, eltrombopag -50 (range, 50-150) mg/day. overall response (or) included cr (platelet ≥ 100 × 10 9 /l, anc ≥ 1.5 × 10 9 /l, and hemoglobin ≥ 100 g/l) and pr (platelet > 20× 10 9 /l, anc ≥ 0,5 × 10 9 /l, hemoglobin > 70 g/l). results: median time from pgf diagnosis to treatment with tpo agonists was 14 days (0-119), median treatment duration was 3 weeks (1-43). tpo agonists were well tolerated with no cases of grade iii-iv toxicity. tpo agonists were combined with rituximab (n=4), rituximab and dli (n=3) and hsc boost (n=1) in 8 (28 %) patients. a total of 14 (48 %) patients met criteria of response (cr: n=4, 14 %; pr: n=10, 34 %). combination therapy showed no difference in or compared to tpo agonists alone. or was not depended on the tpo agonist used nor the time to therapy initiation. median increase in anc in responders was 3.4 × 10 9 /l (0.8-6.0), in platelet count -48×10 9 /l (21-205). a total of 15 patients died due to relapse (n=2), gvhd iii-iv grade (n=3) and infection (n=10). two-year os from the start of tpo agonist therapy was 44 % (95 % ci, 25-62) with a significant difference between responders and non-responders: 71 % (95 % ci, 33-90) vs. 18 % (95 % ci, 3-40) (p=0,002). conclusions: this study showed promising results of tpo agonists for management of spgf. further studies are warranted to specify optimal timing and dosing regimen, predictors of response. [[p144 image] 1. two-year os in responders and nonresponders to tpo agonist therapy] disclosure: there are conflicts of interest to disclose p145 cytomegalovirus reactivation kinetics and peak titers as novel predictors of survival and relapse after allogeneic cell transplantation for hematologic malignancies saskia leserer 1 , evren bayraktar 1 , nikolaos tsachakis-mück 1 , michael koldehoff 1 , lara kasperidus 1 , esteban arrieta-bolanos 1 , mirko trilling 1 , katharina fleischhauer 1 , dietrich w. beelen 1 , amin t. turki 1 1 university hospital essen, essen, germany, background: after allogeneic hematopoietic cell transplantation (hct), human cytomegalovirus (cmv) reactivation associates with non-relapse mortality (nrm) but also with reduced relapse in patients with leukemia, as shown by numerous studies that evaluated cmv reactivation as a qualitative yes/no parameter in the first months posttransplant. we hypothesized that longitudinal quantitative assessment of cmv reactivation kinetics and virus loads might improve patient-specific clinical outcome associations. methods: this retrospective study included 705 patients with hct for hematologic malignancies treated between 01/2012 and 12/2017 at university hospital essen, germany. cmv titers were monitored weekly by quantitative pcr (qpcr); cmv reactivation was defined by a cutoff of >500 genome copies per ml. patients were included for analysis, if at least 5 measurements were available during the first 200 days after hct. in total, 11,508 samples were analyzed. subgroup analyses were performed according to the time of cmv reactivation (before/after +30d) or the cmv viremia titer (>100,000, 20,000 -100,000 and 500 -20,000 copies/ml). results: cmv reactivation was detected in 350 (median age 58 years; range 17-76 years) out of 705 patients. baseline characteristics (age, gender, underlying disease, transplant) of patients without cmv reactivation were comparable. cmv reactivation kinetics followed a gaussian normal distribution with a median first reactivation at +33d and peak titers at +47d. all except 1 patient reactivated before 100d, 40 % before +30d. overall survival (os) of the cmv reactivation group as a whole did not significantly differ from the non-reactivation group (34 vs. 38 months). however, in subgroup analyses os was significantly reduced in patients with very early (< +30d) compared to later reactivation (17 vs. 59 months, p=0.040). moreover and importantly, os was significantly reduced in patients with cmv reactivation at high titers of >100,000 copies/ml compared to those with lower titers ((10 vs. 45 months) p< 0.0001).cox regression analyses confirmed significantly reduced os for patients with cmv reactivation >100,000 copies/ml and < day +30 as compared to the other cohorts (hr 2.03, 95%ci 1.45-2.86, p< 0.0001 and hr 1.36, 95% ci 1.01-1.83, p=0.041) respectively). the nrm was consistently higher (hr 2.59; 95%ci, 1.69-3.97, p< 0.0001) for patients with cmv copies >100,000/ml. the risk of hematologic relapse was exclusively reduced in patients with a peak cmv viremia between 20,000 and 100,000 copies/ml (hr 0.55, 95% ci 0.32-0.95; p=0.033) as compared to patients without cmv reactivation. for other levels of cmv reactivation this effect was not observed. conclusions: our data showed that cmv reactivations before +30d or with high titers of >100,000 copies/ml associated with significantly reduced os, while cmv reactivations at intermediate titers between 20,000 and 100,000 copies/ml had a positive impact on relapse incidence. these findings underline the complexity of cmv reactivations after hct outcome, and support longitudinal evaluation of cmv titers and individualized quantitative kinetics models for risk assessment after hct to distinguish the advantageous from the detrimental aspects of cmv reactivation. disclosure: att has received lecture fees from jazz pharmaceuticals and travel subsidies from neovii biotech outside the submitted work. the other authors declare no competing financial interests within the submitted work. association of serum ferritin levels before start of conditioning with mortality after allosct -a prospective, non-interventional study of the ebmt transplant complication working party background: elevated serum ferritin levels occur due to iron overload or during inflammation and macrophage activation. a correlation of high serum ferritin levels with increased mortality after allosct has been suggested by several retrospective analyses as well as by two smaller prospective studies. methods: this international multicentric study aimed to study the association of ferritin serum levels before start of conditioning with allosct outcome. patients with acute leukemia, lymphoma or mds receiving a matched sibling allosct for the first time were considered for inclusion, regardless of conditioning. data were prospectively collected between 8/2014 and 2/2018. a comparison of outcomes between patients with high and low ferritin level was performed using univariate analysis and multivariate analysis using cause-specific cox model. variables included in the multivariate analyses were age, sex mismatch, diagnosis, disease status, karnofsky score, number of cd34 cells given, intensity of conditioning, type of gvhd prophylaxis, atg use, time from diagnosis to transplant, year of transplant and cmv status. results: twenty centers from 10 european countries reported data on 385 allosct recipients. patient characteristics are given in table 1 . the ferritin cut off point was determined at 1500μg/l (median of measured ferritin levels). overall survival of allosct recipients with ferritin levels above cut off measured before start of conditioning was significantly shorter ( figure 1a , univariate hr=2.3 ci=1.4-3.6 p=0.00041; multivariate hr=2.5, ci=1.5-4.1, p=0.0005). progression-free survival was also shorter ( figure 1b , univariate hr=2.1 ci=1.4-3.2 p=0.00014; multivariate hr=2.4, ci=1.6-3.8, p< 0.0001). excess mortality in the high ferritin group was due to both higher relapse incidence (univariate hr=1.7 ci=1-2.8 p=0.03; multivariate hr=2.2, ci=1.2-3.8, p=0.007) and increased non-relapse mortality (univariate hr=3.1 ci=1.5-6.3 p=0.002; multivariate hr=3.1, ci=1.5-6.4, p=0.002). non-relapse mortality was driven by significantly higher infection-related mortality in the high ferritin group (univariate hr=3.9 ci=1.6-9.7 p = 0.003; multivariate hr = 3.9, ci = 1.6-9.7 p = 0.003). acute and chronic gvhd incidence or severity were not associated to serum ferritin levels. conclusions: ferritin levels before start of conditioning can serve as routine laboratory biomarker to predict mortality after allosct. disclosure: the authors declare no confict of interest related to this study p147 prediction of reduced lung function and acute gvhd by surfactant protein d in allogeneic stem cell transplantation transplantation (hsct) and may progress to bronchiolitis obliterans that has a high mortality rate. surfactant protein d (sp-d) is an innate defense molecule involved in immune regulation at the epithelial surfaces, particularly in the lungs, and elevated levels have been associated with exacerbation of chronic obstructive pulmonary disease (copd). the aim of this study was to investigate, whether sp-d plasma levels and variants in the gene encoding sp-d may predict the development of reduced lung function after allogenic hsct. methods: we performed a population-based, singlecenter study of children (aged 6-18 years) treated with allogeneic hsct. the study consisted of 1) a prospective study of serial plasma sp-d levels and rs721917 genotypes in 55 patients during the first 6 months after hsct, and 2) a retrospective study of rs721917 genotypes within the sp-d gene in 247 patients transplanted between 1990-2017. pulmonary function tests were performed regularly as part of the clinical monitoring. results: at the day of graft infusion (day 0) sp-d levels were reduced compared to levels before start of treatment with conditioning chemotherapy, defined as baseline (615 ng/ml (quartiles 441-1132) at day 0 vs 771 ng/ml (542-1348) at baseline, p< 0.01). from day +7 sp-d levels increased and remained increased during the whole study period (771 ng/ml (542-1348) at baseline vs 1287 ng/ml (713-2549) at 6 months, p< 0.01). acute gvhd (agvhd) occurred in 25 patients, of those 17 patients with grade 2-4. high sp-d levels at day +14 were associated with the development of agvhd (1402 ng/ml (1244-2023) vs 839 ng/ml (523-1630), p< 0.01) ( fig. 1 ). the c/c genotype was associated with generally low sp-d levels and low fev1/fvc at all time intervals compared to the other genotypes, significantly 24-36 months post-hsct (p=0.02). there was no overall correlation between sp-d levels and lung function, but stratifying for genotype, high baseline sp-d levels were predictive for reduced fev1/fvc at 8-24 months in cc and tt homozygous individuals. conclusions: patients with a genotype causing low capacity for sp-d production are at increased risk of developing pulmonary impairment after hsct. in addition, our data lend support to other studies indicating that spd production may increase during inflammatory pulmonary disease, acting as a reactive, protective mechanism. further research is warranted to define the role of sp-d levels and genotypes as a prognostic tool for lung function and agvhd. [[p147 image] 1. background: allogeneic hematopoietic cell transplantation (allohct) means a long period of restricted mobility and a range of therapy related side effects on muscle function. in this context patients demonstrated a huge decline of physical capacity and muscle mass in particular, accompanied with a decrease of quality of life (qol). resistance training could maintain muscle mass but is limited by patientsb lood values (platelet-count) and well-being. whole body vibration (wbv) was shown to maintain muscle mass during bed rest and has less impact on blood pressure than conventional resistance exercises. furthermore it was also shown to be feasible in patients during high dose chemotherapy. therefore the aim of our study was to examine the effects of wbv during allohct on patients physical and functional performance as well as qol. methods: 43 patients receiving allohct were randomly allocated to either a wbv exercise group (ig) or an active control group (cg) doing stretching and mobilization. both groups exercised during the whole time of hospitalization for 5 times per week and underwent pre-, post-and followup-assessment. physical capacity was determined by maximum oxygen consumption (vo 2peak ) and maximum power (p max ) during cardiorespiratory exercise test and by maximum strength of the knee extensors and flexors (ex max , flex max ) during isokinetic strength test. functional performance was assessed by jumping height during counter movement jump (cmj) and time of chair rising test (crt) as well as power output during both tests. qol was assessed by questionnaires of the eortc. results: during allohsct vo 2peak and p max decreased in both groups but till follow-up an increase is seen in the ig (p=0.035; p=0.011). at day +180/follow-up a vo 2peak group difference is seen (p=0.034). ex max (p=0.003) and flex max (p=0.044) were only reduced in the cg during hospitalization. jumping height and power output decreased in the cg during hospitalization (p=0.005, p=0.039) and a difference between groups were seen in changes of jumping height from pre-to follow-up-assessment (p=0.033): increase in the ig and decrease in the cg. the ig showed a decrease in time from baseline to follow-up (p=0.022) in the crt and an increase of power output (p=0.009). qol decreased only in the cg during hospitalization (p=0.015) while during follow-up qol increased in both groups (ig: p=0.013; cg: p=0.037). in the cg physical functioning decreased during intervention (p=0.001) whereas an increase was seen in the ig from pre-to follow-upassessment (p=0.035). body image was significant worse in the cg compared to the ig at hospital discharge (p=0.007) as well as at follow-up measurement (p=0.030) where it got worse over time (p=0.036). conclusions: wbv was shown to maintain maximum strength, jumping performance and qol during allohct. although cardiorespiratory fitness could not be maintained by wbv during hospitalization, it seems in the follow up period till day + 180 that recovery of the cardiorespiratory system is enhanced by wbv carried out during allohst. nevertheless reasons for this changes in recovery have to be analyzed in further studies as well as treatment effects of wbv compared to conventional resistance training. disclosure: supported by a grant of the faculty of medicine and comprehensive cancer center freiburg respiratory virus infection within 1 year after of allo-sct is the significant risk factor of obstructive ventilatory disturbance kosei kageyama 1 , michiho ebihara 1 , mitsuhiro yuasa 1 , daisuke kaji 1 , aya nishida 1 , shinsuke takagi 1 , hisashi yamamoto 1 , go yamamoto 1 , yuki asano-mori 1 , naoyuki uchida 1 , atsushi wake 1 , akiko yoneyama 1 , shigeyoshi makino 1 , shuichi taniguchi 1 1 toranomon hospital, hematology, tokyo, japan, background: obstructive ventilatory disturbance (ovd) is one of the major life-threading complication at the chronic phase of allogeneic stem cell transplantation (allo-sct). bronchiolitis obliterans has been the most established etiology as a part of chronic graft-versus-host disease and major cause of late non-relapse mortality of allo-sct. but other etiologies impact on respiratory function after allo-sct and risk factor of ovd have not been well understood. methods: to address these issues, we retrospectively reviewed the medical record of 747 consecutive patients who first allo-sct at toranomon hospital between 2009 and 2017. to detect ovd, forced expiratory volume in 1 second (fev1.0) showed less than 80% of predicted in spirometry test was defined as positive. in the recipients who showed fev1.0 less than 80% in pre-transplant test, more than 20% reduction of fev1.0 was regarded as positive. nasopharyngeal swab of those who had upper respiratory tract symptoms were tested for the presence of respiratory viral antigens (adv, piv, and rsv). patients with ecog performance status of 4, had active infection at transplant were excluded from this analysis. the cases of early death or relapse before 30 days post-transplant, and the cases of graft failure were also excluded. results: the median age was 55 years (range, 16-74). underlying diseases were aml in 403, mds/mpd in 73, cml in 26, all in 82, atl in 19, hl in 12, nhl in 104, and others in 28. five hundred twenty-nine (71%) were not in remission at the time of transplant. five hundred eightythree patients (78%) were conditioned with myeloablative regimens, whereas 164 patients received reduced-intensity regimens. donor sources consisted of related peripheral blood /bone marrow (bm) (n=85), unrelated bm (153) forty-six developed ovd on median of 198 (60-804) days post-transplant. cumulative incidence of ovd was 6.4% in total population. in 490 recipients those who could spirometry, overall survival at 5 years was 73.2% in patients who developed ovd and was comparable with those who did not develop it (64.3%, p=0.486). in univariate analysis, disease status (cr/aa or noncr), recipient age (age< 55 or ≥55), prior autologous stem cell transplantation (yes or no), intensity of conditioning regimen (mac or ric), tbi dose (< 8 gy or ≥8 gy), busulfan dose (< 9.6mg/kg or ≥9.6mg/kg), donor source (cord blood or non-cord) had no impact on the incidence of ovd. patients who developed respiratory virus infection showed significantly higher incidence of ovd compared to those who did not developed it (12.4% vs 5.3%, p< 0.01). in multivariate analysis, respiratory virus infection was the only significant risk factor for the development of ovd (hr=2.43, 95% ci 1.30-4.57, p< 0.01). conclusions: respiratory virus infection within 1 year after allo-sct is the significant risk factor of ovd. disclosure: nothing to declare. background: metabolic syndrome (mets) is related to increased risk of cardiovascular disease and type-2 diabetes (dm-2) and usually seen in overweight individuals in the general population. we investigated mets and clinical risk factors two decades after hsct. methods: all male survivors treated with myeloablative allo-hsct during childhood (< 17 years) between 1980-2010 in denmark were invited to a follow-up study. mets was defined as the presence of at least three ncep atp iii criteria: fasting plasma triglyceride (tg) ≥1.7 mmol/l, high density lipoprotein (hdl) < 1.03 mmol/l or medical treatment of hyperlipidemia; fasting plasma glucose (fpg) ≥5.6 mmol/l; abdominal circumference (ac) >102 cm; bp ≥130 mmhg (systolic) / ≥85 mmhg (diastolic) or medical treatment for hypertension. patients with overt dm-2 were included into the mets group. furthermore, patients were examined for chronic graft-versus-host disease (cgvhd) by the nih-criteria at the time of follow-up and high sensitivity c-reactive protein (hscrp) was measured. the prevalence of mets was compared to a nordic reference group (hildrum et al. 2009) . results: we included 49 out of 97 eligible males (participation rate 51%) aged 18-44 years, median 29 years. median (range) follow-up was 21 (8-32) years. of these 49 males, 74% had a malignant diagnosis and 65% were treated with tbi-based conditioning. donors were matched siblings (n=21), matched relatives (n=3) or matched unrelated donors (n=25). mets was more prevalent (33%) in the young adult survivors compared to the prevalence reported for 20-39year-olds in the nordic reference (16 %). instead the prevalence was comparable to that reported for the 50-69year-olds (32%). of the components of mets, elevated tg (51%), hypertension (47%), and decreased hdl (40%) were frequent, while fpg was elevated in 16%. importantly, only 4% of those with mets had increased ac and mean bmi (23.8 kg/m 2 ) of the hsct survivors was within normal range in contrast to features of mets observed in the background population. having mets was significantly associated with tbi (rr = 7.9, 95%ci (1.1-55.3), p=0.004) as was the following single components of mets (mean in tbi group vs. mean in non-tbi group): elevated tg (2.34 mmol/l vs. 0.93 mmol/ l, p= 0.006), lower hdl (1.04 mmol/l vs. 1.38 mmol/l, p=0.001) and higher diastolic bp (80 mmhg vs. 72 mmhg, p=0.03). mets was only demonstrated in one patient who received non-tbi based conditioning. sixteen of 49 patients had cgvhd of which nine were moderate to severe cases, but cgvhd was not associated with mets. however, low-grade inflammation measured by hscrp was related to increased ac (rho=0.41, p=0.004) and tg (rho=0.34, p=0.028). conclusions: our results indicate that male long-term survivors of allo-hsct during childhood have a high risk of mets at an earlier age than the general population. the presence of mets despite normal bmi in several patients suggests unconventional etiologies like the effect of tbi and low-grade inflammation. disclosure: nothing to declare. results: this survey was completed by transplant directors (46%), transplant consultants (41%), nonconsultant grade physicians (8%), hsct clinical nurses specialists (cns) (3%) and other (2%) from 114 centres in 24 countries. 58% of the centres are adult-only, 21% paediatric-only and 21% treat adult and paediatric patients (mixed centres). 46% are located higher than 50 degrees latitude (northern countries) and 54% lower than this latitude (southern countries). at the time of the survey 84% were members of the european union (eu). measurement of serum vd is routinely performed in 47% of the centres prior and in 70% after allogeneic hsct. the main clinical indications are known osteopaenia/osteoporosis (86%), previous fracture (71%), treatment with steroids (68%), premature menopause (46%) and established menopause (32%). monitoring occurs every 3 months (39%), every 6 months (24%), once a year (18%) or at other time-points (19%). in this regard, seasonality is not taken into account in the majority of the centres (94%). local and national/international guidelines (nice) are only followed by 19% and 18% of the centres, respectively. the most common cut-off value of serum vd for commencing on replacement is 50 nmol/l (32%). northern countries tend to use values of ≥75 nmol/l whereas southern countries ≤50 nmol/l. 15% do not use cut-off values. following hsct, 83% of centres prescribe vd supplements to maintain calcium metabolism and bone health (92%), enhance immune reconstitution post-hsct (24%), gvhd prevention (17%), enhance immune-suppression to treat gvhd (10%), treat depression/fatigue (3%) and reduce relapse risk 2%. a "loading" dose is administrated in 30% (54% adult, 25% mixed and 22% paediatric), with a mean duration of 4 weeks . the median daily loading dose is 2,000 iu (286-20,000). the median "maintenance" daily dose is 800 iu (67-10,000). there are not remarkable differences between adult and paediatric centres or northern and southern countries. vd replacement is prescribed by transplant physicians (75%), family physicians (10%), endocrinologists (3%), cns (3%), others (4%) and in 5% of the centres, patients are advised to buy it over-the-counter. vd is prescribed combined with calcium carbonate in 52% and alone in 48% of the centres. it is eventually discontinued by 69% of the centres when therapeutical levels of vd are reached (69%), dexa scan returns to normal (12%) and symptomatic improvement (9%). conclusions: this survey has demonstrated discrepancies in monitoring and replacement of vd across ebmt allogeneic hsct programmes. although awareness has arisen over the last decade, there is still lack of evidence about the optimal levels of vd required for immunemodulation post-hsct. this survey emphasises the need for specific guidelines to harmonise the current management of vd deficiency in adult and paediatric hsct setting. disclosure background: the use of unmanipulated haploidentical sct (haplo-sct) with post-transplant cyclophosphamide (pt-cy) as gvhd prophylaxis has widely extended. primary and secondary graft failure are relatively uncommon complications. however, poor graft function (pgf) after haplo-sct with pt-cy has not been described thoroughly. the objective of this study is to describe characteristics, treatments and outcomes of patients with pgf after haplo-sct with pt-cy. methods: we retrospectively analyzed 132 haplo-sct with pt-cy consecutively performed between 2011 and 2017 in our centre. pgf was defined as either occurring after initial engraftment: persistent neutropenia (anc < 500/ul) with the need of at least 3 doses of g-csf and/or thrombocytopenia (platelets < 20.000/ul) with platelet transfusion dependence, with complete donor chimerism and without concurrent severe gvhd or disease relapse. results: nineteen patients were excluded from the analysis due to early mortality (death before day +30), primary graft failure (absence of neutrophil engraftment by day +28, with mixed chimerism) or secondary graft failure (development of severe cytopenias and mixed chimerism after initial achievement of neutrophil engraftment). thirty one patients (27,5%) were diagnosed with pgf. main characteristics of these patients are summarized in table 1 . twenty six patients (84%) presented with neutropenia and were treated with g-csf, while 5 patients (16%) only developed severe thrombocytopenia without neutropenia, and were treated only with platelet transfusion. twenty four patients (77,5%) had at least 1 cmv reactivation, 15 patients (48%) had 2 or more cmv reactivations and 21 patients (67%) received valganciclovir for cmv reactivation treatment. although most patients achieved adequate peripheral blood counts (pbc) with initial salvage therapy, 6 patients (19%) had persistent cytopenias in spite of g-csf, platelet transfusion, cmv reactivation resolution and myelotoxic drugs withdrawal. four of them were treated with a boost of cd34+ selected peripheral blood donor cells at a median of 170 days after . median cd34+ cells infused was 3,42 x10 6 /kg. these 4 patients achieved adequate pbc after salvage therapy and two developed gvhd. the other 2 patients were treated with increasing doses of thrombopoietin (tpo) receptor agonist (tra) eltrombopag. one patient started treatment 160 days after hsct with 25mg daily and increased dose to 125mg daily, with complete recovery of pbc 6 months after initiating tra. the second patient started treatment 110 days after hsct with 25mg daily and increased dose to 100mg daily, with complete recovery of pbc 2 months after initiating tra. twenty one patients (67%) with pgf diagnosis had long term survival. conclusions: poor graft function is a frequent complication after haplo-sct with cy-post. cmv reactivation and myelotoxic drugs could be the most relevant factors associated with development of this entity. although most patients recover pbc without specific therapies beyond g-csf and platelets transfusion, there is a small group of patients with persistent cytopenias. boost of cd34+ selected cells is effective in reverting this condition, with gvhd as main complication of this procedure. use of tra seems to be an interesting option for these patients, although more experience is needed to draw definitive conclusions. disclosure: nothing to declare. were also frequently observed. the high risk patients for anxiety (hads-a score ≥ 8) and depression (hads-d score ≥ 8) was found in 14.9% and 13.6%, respectively. 10.4% of patients was in high distress status (nccn dt score ≥ 4). we found that younger age (< 60 years) was significantly associated with poor quality of life score (fact-bmt) (p=0.001) and high risk of fatigue (p=0.008), anxiety (hads-a) (p=0.001), and depression (hads-d) (p=0.025). female sex was significantly related to lower physical well-being score and higher distress score (p= 0.046 and p=0.05, respectively). acute lymphoblast leukemia (all) survivors after allo-hct showed significantly worse quality of life score (fact-bmt) (p=0.006) and higher depression score (hads-d) (p=0.028) compared to those with other disease. chronic graft versus host disease (gvhd) and continuous immunosuppressant usage also have significant adverse impact on lower fact-bmt score (p=0.024 and p=0.033, respectively) and higher hads-d score (p=0.015 and p=0.019, respectively). but there was no significant difference in fact bmt, hads-a, hads-d, nccn dt according to donor type, conditioning intensity, anti-thymocyte globulin use, acute gvhd. smoking and alcohol drinking was continued in 7.5% and 17.9% of allo-hct survivors. 20.9% of survivors did not exercise regularly. regular health screening tests have been done only in 40 patients (59.7%). conclusions: allo-hct survivors over 2 years following allo-hct still have many physical and psychological symptoms. younger patients (< 60 years), female, all, chronic gvhd, and sustained use of immunosuppressant were significant risk factors for poor quality of life and anxiety. we need to build more active survivorship care plan after allo-hct especially for those patients. disclosure: all authors have nothing to declare. evaluation of the new ebmt criteria for the diagnosis of vod/sos in 693 consecutive transplant patients using an electronic patient record analysis system asha aggarwal 1 , nicola gray 1 , oliver lomas 1 , katalin balassa 1 , nadjoua maouche 1 , robert danby 1,2 , andy peniket 1 , grant vallance 1 background: veno-occlusive disease (vod), or sinusoidal obstruction syndrome (sos), is a recognised complication of haematopoietic stem cell transplantation. hepatic vasculature endothelial cells are damaged by conditioning chemotherapy, leading to venous occlusion and centrilobar necrosis. the ebmt criteria for diagnosis of vod are bilirubin >=34 with two of painful hepatomegaly, >5% weight gain and ascites. vod is often under-diagnosed, and as a result, treatment may be delayed. integrated electronic patient record (epr) systems are now widely used, and provide an opportunity to retrospectively audit practice to identify patients in whom vod may have been un-diagnosed or in whom treatment was delayed. in addition these systems have potential for alerting clinicians to the potential diagnosis of vod. methods: we have developed software to analyse the data downloaded from epr to identify patients in whom vod was a possible diagnosis according to the new ebmt criteria. in order to identify patients who may have had vod we first screened for patients with a bilirubin of >= 34 mmol/l (which is an absolute requirement for the clinical diagnosis of vod) within the first 50 days of transplantation. epr data was then used to assess whether patients had >5% weight gain. radiology reports were reviewed for patients who had bilirubin >= 34 mmol/l to ascertain if they revealed ascites or painful hepatomegaly. results: 652 patients underwent 693 transplant procedures (january 1st 2013 to july 31st 2018). 162 of all transplant patients (23.4%) were found to have a bilirubin of >= 34 mmol/l. 39 of 403 (9.6%) autograft patients and 123 of 249 (49.4%) allograft patients had an elevated bilirubin at this level. these 162 patients were assessed for evidence of 5% weight gain. this was the case in 30 patients overall-1% of autograft patients, 10.5% of allograft patients. seven patients (2 autograft and 5 allograft) had radiological evidence of ascites. two patients had a recording of painful hepatomegaly (both post allograft). overall our analysis identified 5 patients (0.7% overall) fulfilling the ebmt diagnostic criteria for classic early vod all of whom received defibrotide. all patients had received allogeneic transplants. we failed to identify any cases of late onset vod or any undiagnosed patients over this period. conclusions: this analysis enabled us to efficiently perform a complete audit of our practice to identify patients with vod. we would recommend using electronic patient records to retrospectively audit practice in this way. the tool that we have created for this analysis will be made freely available for public use and the details will be presented at the ebmt meeting. we now plan to extend the function of our epr system to provide alerts to clinicians when vod is a possible diagnosis and may lead to more rapid treatment of these patients. our data suggests that elevation of bilirubin and weight gain of > 5% will be the most frequently occurring criteria on which to base these alerts. disclosure: g.vallance has performed consultancy work for jazz pharmaceuticals. endothelial activation and stress index in predicting outcome of allogeneic stem cell transplantation-a retrospective cohort analysis zinaida peric 1 , tomislav taborsak 1 , nadira durakovic 1 , lana desnica 1 , alen ostojic 1 , ranka serventi-seiwerth 1 , radovan vrhovac 1 1 university hospital centre zagreb, zagreb, croatia background: endothelial dysfunction is a common pathophysiology of major complications after allo-sct, such as graft-versus-host disease, veno-occlusive disease, thrombotic microangiopathy and sepsis. endothelial activation and stress index (easix) is a simple score comprised of standard laboratory parameters (creatinine, ldh and thrombocytes) developed as a potential tool to predict allo-sct mortality by luft and colleagues. a recent validation of easix included three retrospective cohorts and showed that easix taken before start of conditioning can be used as an independent predictor of survival after allo-sct. methods: the aim of our study was to retrospectively evaluate pre-transplant easix in our cohort of consecutive patients who underwent allo-sct in the university hospital centre zagreb between 2012 and 2017. with the use of a cut-off used in the validation cohorts, we compared two groups of patients for overall survival (os) and transplantrelated mortality (trm). group comparisons were done using the log-rank test or gray test for competing risks outcomes. a multivariate analysis evaluated the association of os with relevant variables by using a cox's proportionalhazard regression model. results: our study group included 313 patients and comprised 180 males (57%) and 133 females (43%, with a median age of 48 years (range, 18 to 67 years) at the time of transplantation. the most frequent malignancies in our population were acute leukemia (196 patients, 63%) and myelodysplastic/myeloproliferative neoplasm (451 patients; 16%). the donor was an identical sibling for 106 patients (34%), matched unrelated donor for 176 patients (56%) and haploidentical for 31 patients (10%). 104 patients (33%) received a myeloablative conditioning regimen while 209 patients (67%) received a reduced-intensity conditioning regimen. with a median follow-up of 16 months (range, 12-60) for the whole study group, the os at 24 months was 60%, (95%ci 54-68) in the group of patients with low easix score and 43% (95% ci 33-56) in the group of patients with high easix score (p=0.004). this difference was mainly attributed to higher trm in the group with high easix score (32%, 95%ci 22-45 at 12 months) compared to the group with low easix score (18%, 95%ci 13-23 at 12 months) (p=0.009). in the multivariate analysis which included easix, patients' age, intensity of conditioning, diagnosis (lymphoid vs myeloid), status of the disease at transplant and type of the donor, worse os was independently associated only with older age of patients (hr 1.66; 95% ci, 1.07-2.59, p=0.02) and high easix score (hr 1.51; 95% ci, 1.01-2.24, p=0.04). conclusions: our retrospective data support previous data and suggest that easix could potentially serve as a valid tool for prediction of allo-sct outcomes. as a simple biomarker panel, easix could easily be implemented in clinical decision making in the field of allo-sct. these retrospective data need validation in a prospective study which is currently being conducted. clinical background: veno-occlusive disease (vod) is a potentially devastating complication that can occur after hematopoietic stem cell transplant (hsct) and in severe cases can lead to multi-organ failure. (mohty 2016) defibrotide has been proven to be effective to prevent and treat vod, and it is critical that clinicians are aware of how to diagnose and treat this serious complication of hsct. this study was conducted to determine if an online, simulation-based continuing medical education (cme) intervention could improve performance of hematologists/oncologists (hem/ onc) and advanced practice providers (nurse practitioners and physician assistants, apps) in the diagnosis and treatment of patients with vod. ( methods: a cme certified virtual patient simulation (vps) was made available via a website dedicated to continuous professional development. the vps consisted of 2 cases presented in a platform that allows clinicians to assess the patients and make diagnostic and therapeutic decisions supported by an extensive database of diagnostic and treatment possibilities, matching the scope and depth of actual practice. clinical decisions were analyzed using a sophisticated decision engine, and tailored clinical guidance (cg) employing up-to-date evidence-based and faculty recommendations was provided after each decision. one case was about vod and the other case was about acute myeloid leukemia (aml). decisions were collected post-cg and compared with each user's baseline (pre-cg) decisions using a 2-tailed paired t-test to determine p-values (p < .05 indicates significance). data were collected between 9/21/2017 and 11/7/2018. results: at the time of assessment, 115 hem/oncs and 409 apps had fulfilled the participation criteria for completing the vod case simulation. conclusions: this study demonstrates that vps that immersed and engaged clinicians in an authentic and practical learning experience improved evidence-based clinical decisions related to the management of vod. this vps increased the percentage of clinicians who utilized standardized criteria to diagnose vod and who ordered defibrotide and iv fluids for vod management. however, further education is needed to increase the competence and performance of clinicians, particularly apps, in these areas in order to positively impact patients. disclosure: nothing to declare. a nationwide retrospective study of hematopoietic stem cell transplantation in solid organ transplant recipients: on behalf of jshct, transplant complications working group background: the outcome of hematopoietic stem cell transplantation (hsct) in solid organ transplant remain unclear. to address this issue, we conducted a retrospective survey of the 404 japan society for hematopoietic stem cell transplantation centers. methods: to address this issue, we conducted a nationwide retrospective survey of the japan society for hematopoietic stem cell transplantation (jshct) centers. a first questionnaire was emailed to jshct centers requesting information on cases of hsct in sot recipient. patients' data about sot were collected by sending a second questionnaire to the centers with the patient. based on these reports, patients' data about hsct was identified in the japan transplant outcomes registry database by the transplant registry unified management program (trump), confirmed in 2017. results: of the 404 jshct centers, 238 responded to the survey (58.9%). 14 of the responding centers reported a total of 19 patients who had undergone sot from living donor, and subsequent hsct. they consist of three autologous hsct (auto-hsct) and 13 allogeneic hsct (allo-hsct). in auto-hsct, all patients had received liver transplant for hapatoblastoma. they achieved neutrophil engraftment at 30 days after hsct, and two of three patients were alive at one year after hsct. in allo-hsct (n=16), seven patients had received liver transplants, and nine patients had received kidney transplants. five patients received hsct from unrelated donor, and 11 patients received hsct from related donor; two donors were identical in sot. their stem cell sources were seven peripheral blood stem cell, six bone marrow, and three cord blood. all but one patients achieved neutrophil engraftment at 30 days after hsct. five-year overall survival (5yos) was 37.5%. while 5yos in patients with bone marrow failure (n=4) was 100%, that in patients with malignant disease (n=12) was 16.7%; all but one patients with malignant disease received allo-hsct in non-remission. seven of nine kidney-transplant recipients experienced dialysis, and three patients experienced renal rejection after hsct. on the contrary, no liver-transplant recipient experienced hepatic rejection. conclusions: in sot recipients, the outcome of allo-hsct for malignant disease was poor, partly due to disease status before allo-hsct. severe renal complications were common in kidney-transplant recipients, suggesting renal care with caution during and after allo-hsct. disclosure: this work was supported in part by the practical research project for allergic diseases and immunology (research technology of medical transplantation) from japan agency for medical research and development, amed. high incidence but low mortality of ebv related ptld after t-cell replete allo-peripheral blood hct with aggressive monitoring and without pre-emptive rituximab background: the aim of the study is to report the incidence and outcome of post-transplant lymphoproliferative disorder (ptld) in the setting of allogeneic peripheral blood hematopoietic stem cell transplantation (allo-hsct) combining post-transplant cyclophosphamide (ptcy) and anti-thymocyte globulin (atg) as graft versus host disease (gvhd) prophylaxis. methods: between october 2015 and may 2018, 195 adult patients diagnosed with hematological malignancies underwent a first t-cell replete allo-hsct in our center. all patients received a reduced intensity conditioning regimen with fludarabine, busulfan, and 200cgy of total body irradiation, combined with rabbit-atg, ptcy and cyclosporine (csa). ebv titres were monitored weekly by quantitative pcr in plasma samples. the cut-off value for test positivity was >600 copies of ebv dna/ml of plasma. last follow up was november 2018. median follow up for patients known to be alive was 19 months (range 5-35). results: patient information is summarized in table 1 . ebv reactivation was documented in 117 (60%) patients. median time to ebv reactivation and the diagnosis of presumed/proven (p/p)-ptld were 75 (16-326) days and 97 (54-306) days [3 (0-10) months], respectively. median time between first ebv reactivation to p/p-ptld was 21 (0-175) days. seventeen (14%) of the 117 patients developed p/p-ptld. median age was 55 years . two (12%) received mrd, 9 (53%) 10/10 mud, 1 (6%) 9/10 mud, and 5 (29%) haploidentical donor grafts. twelve (71%) were on therapeutic cyclosporine at diagnosis. pre-emptive therapy was not given to any case and only probable or proven ptld were given rituximab. treatment was based on reduction of the immunosuppression in 3 patients and with the addition of weekly rituximab 375 mg/ m 2 in 15 cases. fifteen (88%) achieved complete clinical responses with pcr negativity. two (12%) patients died secondary to ptld. conclusions: atg based conditioning is associated with increased viral reactivations. frequent ebv monitoring and pre-emptive treatment may lead to rapid disease control. further research is required to optimize monitoring and management strategies in allo-hsct recipients. disclosure: nothing to declare p160 acoustically enriched extracellular vesicles as potential markers for allogeneic hematopoietic stem cell transplantation complications hooi-ching lim 1 , robert palmason 2 , stig lenhoff 2 , thomas laurell 1 , stefan scheding 1,2 background: extracellular vesicles (evs) contain a number of condition-specific proteins, dna and rna types and might therefore be used for the early detection of posttransplant complications. however, traditional ev isolation (ultracentrifugation) is time consuming and requires large sample volumes thus making it difficult to perform longitudinal studies on larger patient cohorts. we therefore investigated whether recently-developed acoustic trapping could be applied to isolate evs from patient plasma for biomarker development. methods: plasma samples were collected from 10 consecutive patients before and up to 3 months after allogeneic hematopoietic stem cell transplantation. patients (age: 22-58 years) with high-risk or refractory/relapsed diseases were transplanted with mobilized pbsc from related (n=2) and unrelated donors (n=8) after standard conditioning. gvhd prophylaxis was cyclosporine and methotrexate. plasma samples were frozen and thawed for ev enrichment using a novel acoustofluidic-based technology (acoustic trapping). acoustic trapping uses ultrasound as a local λ/2 acoustic standing wave produced by a piezoelectric transducer over a capillary. first, 12 μm polystyrene beads are captured which serve as seeding particles. after washing, target particles (evs) are then captured ("trapped") in the acoustic field. a semi-automatic trapping device (acoutrap) was used to isolate evs from diluted plasma (1:2 in pbs). the number of evs and size distribution were analyzed by nanoparticle tracking analysis. mirna analysis was performed by qpcr.evs were enriched in duplicate from 50μl and 300μl of diluted plasma for nanoparticle tracking analysis and qpcr analysis, respectively. results: evs were successfully isolated from all plasma samples. a total of 89 plasma samples were processed. numbers of trapped evs ranged from 3.7x10 8 -5.5x10 9 before conditioning to 4.4x10 8 -1.5x10 10 per 50μl diluted plasma after transplantation. the maximum change in ev numbers in individual patients compared to pretransplantation values ranged from 2-fold to 7-fold. most patients showed slight increases in ev size after transplantation. eight of the patients showed signs of infection and received i.v. antibiotics. increased levels of evs (> 2-fold) were recorded in three patients during these episodes. furthermore, increased ev numbers were observed in a patient who required i.v. antiviral therapy for cmv reactivation. acute grade i gvhd was observed in five patients of which two had increased ev numbers (> 2-fold). one patient developed grade iv gvhd which was accompanied by a 4-fold increase in ev numbers. interestingly, progressively increasing ev numbers preceded the detection of early relapse in a pre-b all patient by three weeks. rna isolation from trapped evs yielded sufficient material for mirna profiling. here, first mirna profiling data demonstrated that mirnas were detected in ev samples (mir-103a, -23a, -30c, -142 and -451a) , and that acoustically enriched evs were not affected by hemolysis in contrast to the corresponding whole plasma samples (dcq of mir-23a and mir-451a). conclusions: acoustic trapping allows for efficient and rapid enrichment of evs from small volume plasma samples. trapped ev samples contain sufficient amounts of mirna for downstream analysis and are thus promising candidates for biomarker development in transplantation. disclosure: laurell and scheding are founders and board members of acousort ab, a lund-based biotech sme that develops particle and cell sorting methods based on ultrasound. the incidence, risk factors and outcomes of primary poor graft function after allogeneic hematopoietic stem cell transplantation fei gao 1,2 , jimin shi 1,2 , yi luo 1,2 , yamin tan 1,2 , xiaoyu lai 1,2 , jian yu 1,2 , he huang 1,2 , yanmin zhao 1, 2 background: allogeneic hematopoietic stem cell transplantation (allo-hsct) is a curative therapy for both hematologic malignancy and many other blood disease. while, primary poor graft function (pgf) is still a severe complication following hsct which lead to poor prognosis. up to now, the incidence and risk factors of pgf have not been totally revealed. methods: from january 2013 to december 2017, a total of 647 patients who received allo-hsct in our center were analyzed retrospectively. there were 9 males (47.4%) and 10 females (52.6%) with a median age of 36.21 years (21-49 years) . pgf was defined as persistent neutropenia (≤0.5×10 9 /l), thrombocytopenia (platelets≤20×10 9 /l), and/ or hemoglobin≤70 g/l after engraftment with hypocellular bone marrow and full donor chimerism, without concurrent graft-versus-host disease or disease relapse. incidence was calculated from all patients. of the 647 total patients, nineteen (2.94%) developed primary pgf. a 1:4 ratio of nested case control study using the good graft function (ggf) subjects transplanted in the same year with the same sex and age of ±5 years was carried out. results: data was analyzed by univariate and multivariate logistic regression, and univariate analysis identified disease species, the time from diagnosis to transplantation, disease states, myelofibrosis, splenomegaly, serum ferritin (sf) level, cmv infection, mononuclear and cd34+ cells in graft as potential risk factors (p <0.1) for pgf. multivariate analysis identified 3 elements as the independent risk factors (p <0.05), including cd34+ cells <5×10 5 / background: transplant survivors affected by cgvhd usually take one or more immunosuppressants, as well as prophylactic antimicrobials; use of multiple medication classes concurrently poses a risk for drug-drug interactions or amplified side-effects. the use of medications other than cgvhd-direct immunosuppressive therapies has not been well-characterized. this study aims to evaluate patterns of opioid analgesic use in a cohort of patients severely affected by cgvhd. methods: patients (n=335) with cgvhd were consecutively enrolled in a cross-sectional natural history study (nct00092235) from 10/2004-12/2016 at the nih. participants underwent a comprehensive evaluation including a detailed history and physical examination (including current medications), multidisciplinary evaluations, and laboratory and diagnostic testing. for this analysis, respondents were classified as receiving or not receiving an opioid analgesic. following the initial screening by univariate methods (n=335), multivariable logistic regression analysis (mlr) was used to identify a set of factors which could jointly impact opioid use. for mlr data were divided into a training (n=167 patients) and a validation set (n=168). results: study participants´median age was 48.5 years (19-75), 44% were female, 74% had severe cgvhd per nih scoring criteria, and 77% were currently receiving high or moderate levels of systemic immunosuppression. approximately one third (33%) were taking opioid analgesics (oa). based on the univariate screening results (p< =0.05), a set of 24 parameters was evaluated by univariate logistic regression in the 167-patient training set, and the following parameters retained their significance and were included in the mlr model: nih average score per organ, total lss, patient impression of severity, nih cgvhd severity, presence of skin erythema, karnofsky performance score (kps,) clinician's therapeutic intent, nih joint score, and with the presence of several cgvhd symptoms including rashes, mouth sores, avoidance of food, vomiting, weight loss, joint and muscle aches, joint limitation, energy loss, need for naps, fevers, anxiety. multivariable logistic regression identified kps < 77% as predictive of oa use, or 0.92, 95% ci 0.89-0.95. in the training set 56.4% of pts using opioids were correctly identified, 78.2% of those not taking opioids were identified, an overall fraction of correctly identified pts was 70.9% (95% ci 63.3 -77.7%), while in the testing set, 45.5% of those using opioids were correctly identified, and 67.9% of those not taking opioids were correctly identified, with overall 60.5% (95% ci: 52.6-68.0%) classification accuracy. conclusions: this study showed the burden of oa in this cgvhd cohort. lower kps was significantly associated with oa use, as well as self-reported symptoms and a more severe cgvhd disease, which could be of interest in the development of non-pharmaceutical interventions in this patient population. additional, prospective studies are needed to explore the indications for and effectiveness of oa in this population of survivors. disclosure: no conflict of interest to declare. rcts that tested an internet-based program and patientcentered survivorship care plans for hct survivors. patient and caregiver input is essential to inform the design and features for the mobile app platform so that it is usable and engaging for those it targets. methods: using a qualitative research design, we conducted telephone focus groups of adult patients and caregivers in the united states. adult (age >18 years at the time of study entry) hct recipients had to be at least oneyear post-hct to participate. participants had to be able to communicate in english, and could have received a hct for any diagnosis, and from any donor source or stem cell type. those who had multiple transplants were included. participants were asked to review printed and online visual presentations of the mobile app before the focus groups so they were prepared to discuss their responses to the materials during the call. focus groups were conducted to saturation, when no new qualitative content was offered. results: three focus groups were conducted with 22 total participants (20 patients, two caregivers/patient advocates). all patients received an allogeneic hct; average time since hct was 8 years (range: 2-22 years).the majority of participants were female (77.3%). participants had differing perspectives on the usefulness of the app to track follow-up appointments, lab values, and other health care plans. there was high interest in having the app tailored to meet specific needs of patients, including tracking information over time (e.g. test results, medications), and having health information available specific to their needs. to minimize duplication of information and data entry, participants recommended syncing the app with their calendars and online patient portals they already use. reasons provided for not using the app included perception that the materials repeated information already received, side effects such as graft-versus-host disease that restricted vision or motor skills, and lack of comfort with apps for some older participants. conclusions: many health technology and mobile apps are being created to improve patients' health and survivorship care. in this study, hct survivors and caregivers identified a variety of features that they would want in an app or website, in particular, features tailored to individual needs. health technologies provide an opportunity to improve survivorship care, but patients and caregivers should be engaged in the process of developing these tools to assure the technology fits their needs and will be used. given the effort required to maintain these technologies, they require testing for health benefits in rigorous clinical trials. clinical background: thanks to allogeneic stem cell transplantation (allo-hsct) patients suffering from hematologic malignancies have seen an increase in there life duration expectancy, but they are many side effets including decreasing in physical performance and in quality of life. the intensity of physical performance decrease is variable between patients, and today we did not know why. the aim of our study was first to characterize the physical performance of subjects less than 1 year following allo-hsct by the use of a cardiopulmonary exercise testing (cpet), and then to determine the predictive factors of exercise performance. methods: we did a retrospective analysis from 59 patients who had an allo-hsct at hematology department of toulouse-oncopole and cpet from 01/2015 to 09/2017. the cpet was performed using a cycle ergometer with o2 and co2 analyzer breath by breath, (masterscreen cpx carefusion, san diego, usa), a continuous 12-lead electrocardiogram, and a blood pressure monitoring. the protocol included a 2-min rest period, a 2-min warm-up of 20w pedaling followed by a 10w/min incremental phase, up to exhaustion, then a 2-min active recovery of 20w pedaling, then a 2-min passive recovery. three exercise markers were analysed: the peak of oxygen uptake (peak vo 2 ), the ve/vco 2 slope and the first ventilatory threshold (vt 1 ). data relative to conditioning regimen, short-term complications, impairment at cpet day, and physical activity since allo-hsct were gathered. results: after allo-hsct, nearly 3 over 4 patients reported fatigue, a half reported dyspnea, and 1 over 4 or more reported pain, muscular, neurological or psychological impairment. more than 60% of patients suffer from moderate or severe physical intolerance, particularly when myeloablative conditioning regimen was used. only 37% of patients followed rehabilitation sessions supervised by a physiotherapist, and non-supervised physical activity has been performed by 87% of patients. despite normal lung function tests and echocardiography findings in most patients, 80% had exercise intolerance (ei), 85% exercise deconditioning, and 55% had abnormal ventilatory efficiency. patients with moderate and severe impaired exercise capacity were significantly younger at diagnosis and at allo-hsct, such as patients with severe deconditioning conclusions: based on a retrospective study, we reported for the first time complete results from cpet and detailed clinical evaluation concerning deficiency and disability following first year after allo-hsct. these results confirm that exercise impairment is very frequent with more than a half of patients suffering from alterations of one or more of the three performance markers, despite being active. disclosure: nothing to declare demyelinating disorders: a paradigm of immunity disorders after hematopoietic stem cell transplantation background: neurologic complications are a major problem in patients who undergone hematopoietic stem cell transplantation (hsct). given the higher survival of transplanted patients, the burden of neurological complications is increasing in the last years. a significant reduction in overall survival was demonstrated in patients who developed neurological complication after hsct, irrespectively of the hematopoietic stem cell (hsc) source. neurologic disorders in transplanting setting comprise a wide variety of ethiologies including demyelinating disease, which are caused by immune and non-immune mechanisms. here, we analyzed the clinical presentation and the underlie ethiologies of patients developing hsct-related demyelinating disorders in order to give diagnostic and prognostic clues useful to manage these severe but treatable complications in the transplant setting. methods: a total of 45 patients of our department which developed neurological complications after hsct were consecutively collected and 33 (73%) of them, namely those having a diagnosis of a demyelinating disorder, were grouped and described according to the ethiologies of their neurological disorder. results: in 14/33 (42%) patients, an immune-mediated process was found, while 10/33 (30%) were diagnosed as having an infective etiology and 2/33 (6%) were supposed to have a demyelinating disorder caused by toxic exposition. a definitive etiologic diagnosis was not formulated in the remaining 7/33 (21%) patients. when patients who developed an immune-mediated demyelinating disorder (10/33) were compared to those in which a clear immune pathogenic mechanism was not detected (23/33), a higher incidence of acute graft-versus-host disease (agvhd) was detected in the former than in the latter (20% vs 8%). moreover, comparison of these two groups revealed that those with no evidence of immune-mediated process have a slight higher prevalence of t-cell depleted hsct thanthose with an immune-mediated demyelinating disorder (60% vs 50%). finally, a lymphoproliferative disorder pre-existing the hsct was detected in 5/14 (36%) patients with immune-mediated demyelinating disorder but only in 4/19 (21%) of those without evidence of immune-mediated processes. conclusions: demyelinating disorders may be responsible of near 40% of neurologic complications in the posttransplant setting and, among them, an immune-mediated process is likely to be involved in more than 40% of cases. our results suggest that the immune mechanism that underliesthe agvhd may also be involved in developing demyelinating disease in transplanted patients. it also may be possible that the lymphoproliferative disorder preexisting the hsct is a risk factor able to increase the risk to develop an immune-mediated demyelinating disorder in the post-transplanting setting. using a t-cell depleted hsct can increase the risk of immune-mediated disorders in at least a small fraction of transplanted patients. despite our results should be validated on a larger cohort of patients, we can speculate on the possible connections between the wide range of complex and still poorly defined immunity disorders which can influence the prognosis and course of transplanted patients. disclosure background: injury to the mucosal barrier and subsequent development of oral mucositis (om) is among the most common toxicities of allogeneic stem cell transplantation (sct). despite the high prevalence of om and its debilitating nature, prospective studies evaluating determinants of om are scarce. we therefore prospectively evaluated the occurrence of om following sct. risk factors for om and its implications short and long-term outcomes were assessed. methods: om was prospectively evaluated on a weekly basis in patients undergoing allogeneic hsct. the grade of om was determined based on the national cancer institute common toxicity criteria for adverse events (ctcae) scale (v. 4.0). severe om was defined as grade ii to iv. conditioning regimens were evaluated individually and according to intensity; myeloablative (mac), reduced intensity (ric) or reduced toxicity (rtc). the latter category included only patients receiving fludarabine and treosulfan at dose of 30-42 g/m2 (flu/treo). risk factors for the development of severe om were initially identified by a univariate analysis and then analyzed in a multivariate logistic regression model. association of om with peritransplant infectious complications, iv morphine consumption, hospitalization length, neutrophil engraftment, acute and chronic graft-versus-host disease (gvhd), non-relapse mortality (nrm) and overall survival were assessed in a univariate analysis. competing events were considered in analyzing engraftment, gvhd, and nrm. results: 115 patients who underwent an allogeneic sct between 2016 and 2017 were included. median follow-up was 316 days. leading indications for transplantation were acute myeloid leukemia (49%), lymphoma (16%), and myelodysplastic syndrome (15%). the majority of patients received an allograft from a matched sibling or unrelated donor (88%) and methotrexate gvhd prophylaxis (74%). the median time to om onset was 7 (interquartile range [iqr] 5-9) days. prevalence of grade ii-iv om was 60%. the median duration om was 10 [6-13] days, and iv morphine was administrated for a median of 5 [6-12] days for patients with grades iii-iv om (45%). in a univariate analysis a younger age (p=0.023), lower bmi (p=0.01), recent smoking history (p=0.08), recent antibiotics exposure (p=0.018), mac (p< 0.001), and use of methotrexate (p=0.009) were associated with an increased risk for grade ii-iv om. in a multivariable model the risk for grade ii-iv om was lower with rtc (i.e., flu/treo) vs. mac (odds ratio [or] 27.31; p< 0.001) and rtc vs ric (or 7.25; p=0.001), mycophenolate mofetil vs. methotrexate (or 3.43; p=0.027) and recent smoking (or 4.7; p=0.075). compared to lower grades, grade ii-iv om was associated with a longer hospitalization duration (median 29 days vs. 27 days; p=0.006), delayed neutrophil engraftment (median 14 vs. 12 days; p=-0.004), and more gastrointestinal related infections (10% vs. 0%; p=0.045). grade ii-iv om was not associated with increased risk of bloodstream infections, acute or chronic gvhd, non-relapse mortality, and increased mortality. conclusions: oral mucositis is prevalent among allogeneic-sct recipients. importantly, fludarabine-treosulfan, which is considered a myeloablative is associated with a markedly reduced risk for om. consequences of om include prolongation of hospitalization, delay in neutrophil engraftment, and a tendency for gastrointestinal infections, but does not increase the risk for gvhd and mortality. disclosure: nothing to declare background: the advent of recent diagnostic techniques for the assessment of iron overload (t2*-mri) and their systematic use as screening tools in the setting of secondary hemochromatosis have led to an increased awareness that focal nodular hyperplasia (fnh) represents a possible incidental finding after hematopoietic stem cell transplantation (hsct). methods: clinical and radiological features of patients undergoing hsct in a single pediatric institution have been retrospectively reviewed for fnh. in order to provide an estimate of the prevalence of fnh after hsct, we analysed all the t2*-mri scans performed during the last 5 years in our centre and recorded the number of patients with fnh (group a). in addition, data about patients incidentally diagnosed with fnh at abdominal imaging performed for different clinical indications have been collected (group b). results: eight out of 118 (7%) transplanted patients who underwent at least one t2*-mri scan from september 2013 to september 2018 were incidentally diagnosed with fnh. group b included 3 subjects with fnh incidentally found at ultrasound or non-t2* mri scans performed before 2013. overall, 11 transplanted patients (5 males, 45%), transplanted for al (9 cases) or bone marrow failure (2 cases) at a median age of 13.1±3.6 years, were diagnosed with fnh between 0.6 and 12.8 years after hsct, namely 3.2±2.6 years in group a and 7.0±4.1 years in group b. a variable degree of iron overload was demonstrated in all patient (lic: 230-340±50 microg/g; baseline serum ferritin: 685-3189 ng/ml). the potential risk factors for fnh are reported in table 1. in 8/11 patients, the radiological finding was pathognomonic; in 1/11 the diagnosis of fnh was confirmed histologically, while 2/11 subjects were labelled as "fnhlike", although a potential diagnosis of hepatic adenoma could not be ruled out. in 3/11 patients, fnh presented with an isolated lesion, while 8/11 had 2 to more than 10 hepatic nodules at diagnosis. the size of nodules at diagnosis ranged from 3 to 41 mm. in unenhanced mri scans, lesions were predominantly hyperintense on both t1-and t2weighted sequences. in dynamic studies with contrast medium, all lesions strongly enhanced during the arterial phase, with a variable degree of wash-out in the late venous scans. hepatic function tests were normal in all the enrolled patients at diagnosis of fnh. among the 9/11 patients for whom at least a follow-up scan was available, 1 presented a complete regression, 3 a reduction and 3 an increase in the size and/or number of lesions, while in 2 patients the nodules remained substantially unchanged after a mean radiological follow-up of 4.5±3.3 years. no malignant transformations were observed. conclusions: fnh represents a relatively frequent incidental finding after hsct. although a malignant transformation is rare, given the demonstrated variable evolution of the hepatic nodules, a radiological follow-up is highly recommended. disclosure: nothing to disclose. incidence and risk factors for hepatic sinusoidal obstruction syndrome after allogeneic transplantation: retrospective multicenter study of turkish hematology research and education group (threg), updated data methods: ten centers from turkey were enrolled in the study. we retrospectively evaluated the medical records of patients who were treated with allo-sct between january 2012 and december 2015. a baltimore criterion was used for assessment of hsos. four hundred twenty six (97.2%) of 438 patients who were treated with prophylaxis with defibrotide alone or one or more of the n-acetylcysteine, diuretics and heparin used defibrotide (10-25 mg/kg/day). results: the study included 1153 patients (687 males/ 466 females) with median age of 38 (15-71) years. the demographic and clinical characteristics of patients were summarized in table 1 seventy-three (84.9%) of patients with hsos were treated with defibrotide after diagnosis. the median time of starting defibrotide in these patients was 13.5 (2-29) days. thirty-seven (43%) of patients with hsos recovered completely and forty-nine (57%) of them died as a result of multi organ failure. the incidence of hsos-related mortality in allo-hsct cohort was found to be 4.2%. in univariate analysis, statistically significant associations were not found between hsos incidence and age/sex of recipient, type of conditioning regimen, stem cell source and type of gvhd prophylaxis. on the other hand donor type, engraftment status and prophylaxis for hsos were significantly associated with hsos development. hsos prophylaxis was significantly decreased hsos-associated mortality (p=0.001). conclusions: hsos still remains a serious lifethreatening complication of allo-sct. although the incidence is low, hsos is associated with increased 100-day non-relapse mortality. hsos prophylaxis especially with defibrotide, seems to reduce hsos associated mortality in high risk patients. disclosure: nothing to declare prophylaxis with defibrotide in adults at very high risk of veno-occlusive disease: results in 11 patients background: hepatic sinusoidal obstruction syndrome/ veno-occlusive disease (sos/vod) is a life threatening complication that can occur after hematopoietic stem cell transplantation (hsct). severe sos/vod rapidly evolves in multiple organ dysfunction syndrome (mods), associated with a mortality rate exceding 80%. precocity of defibrotide (df) treatment is the leading factor for efficacy. prophylactic use of df is recommended in children, but its value has not been validated in the adult population, although factors for individual risk assessment for vod are debated. we here present a real-world experience of df prophylaxis in adult patients at very high risk of sos/vod receiving allogeneic hsct. methods: from 2016 to 2018 we treated with prophylactic defibrotide and ursodeoxycholic acid (udca) 11 patients, median age 30 years (range 21-59). nine patients received allogeneic hsct for acute lymphoblastic leukemia (8 b-all and 1 t-all), one patient for severe aplastic anemia, one patient for primary myelofibrosis. they were all at high risk for sos/vod because of previous hepatotoxicity (3 patients), previous hsct (6 patients), double alkylating agent (5 patients) or previous treatment with inotuzomab ozogamicin (io; 7 patients). of the 7 patients treated with io, 6 received 2 cycles of io, and 1 received 1 cycle, with the last io dose administered a median 41.5 days before hsct (range 34-61 d). defibrotide was administered in 4 daily doses for a total dose of 25 mg/ kg per day and udca at the dose of 300 mg twice per day, starting from day -6 prior transplant. all patients received treosulfan-fludarabine based conditioning. in 5 patients thiotepa was added to the conditioning and in 2 patients a low dose 4gy tbi. gvhd prophylaxis included posttransplant cyclophosphamide, rapamycin and mycophenolate in all patients, except one patient with aplastic anemia receiving atg, rapamycin and mycophenolate. donor source was pbsc in all cases. seven patients received family haploidentical (mmrd) transplant, 1 patient a mrd transplant and 3 patients a mud transplant. results: the median duration of defibrotide therapy was 34 days (range 32-64 days). documented non-severe gastrointestinal bleeding occurred in 2 patients requiring defibrotide temporarily discontinuation, no other significant bleedings were experienced. four patients developed grade ii-iv acute gvhd and no transplant-associated thrombotic microangiopathy were diagnosed. overall, sos/vod occurred in 3/11 cases within 21 days after hsct (days 9, 10 and 13) and no late-onset sos/vod were diagnosed. sos/vod was very severe, causing mods and death in all 3 cases. all 3 patients were characterized by a common pattern of very high risk factors for sos/vod by prior hsct and salvage treatment for b-all with 2 cycles of io close to hsct. furthermore, they all received a fully myeloablative conditioning regimen with treosulfan and thiotepa and a mmrd transplant. conclusions: defibrotide prophylaxis was safe and well tolerated with no severe related complications. sos/vod occurred despite continuous df prophylaxis in 3/7 patients treated with inotuzomab ozogamicin close before undergoing 2 nd transplant. to reduce the incidence of severe vod, pre allo-hsct treatment with inotuzomab ozogamicin should prompt avoidance of other cumulative risk factors for vod, such as use of double alkylating agents. disclosure background: busulfan is the backbone of many preparative regimens administered to children undergoing allogeneic and autologous hematopoietic stem cell transplantation (hsct). among its many long-term adverse effects, busulfan can cause various degrees of pulmonary injury. although well described in adults, there are few large series exploring pulmonary toxicity of busulfan in children. we describe long-term pulmonary follow-up in a large group of children treated at a single center who had received high-dose busulfan and examine the relationship of systemic drug exposure and lung function over time. methods: all surviving children who had received highdose busulfan between 1993-2013 in the context of hsct at the schneider children´s medical center, were referred for serial pulmonary function monitoring (including spirometry, plethysmography and diffusing capacity for carbon monoxide [dlco] . pre-transplant testing was available for children who were old enough to perform the procedure. spirometry results were adjusted according to the revised global lung initiative formulas for age, gender, and height. pulmonary injury was defined as a z score below -1.96 for spirometry, or < 80% of predicted for the other parameters. busulfan levels were monitored following the second drug dose. all patients received busulfan in four daily doses. area under the curve (auc) calculations were performed by bayesian calculations. results: between 1993-2013, 263 patients aged 0-18 years were diagnosed with malignant or non-malignant diseases and treated with high-dose busulfan. of 130 shortterm survivors, 75 had at least one post-transplant pulmonary function evaluation. the mean age at treatment with busulfan was 7.9 years (range, 0.4-27 years). of these 75 children, 22 children had undergone autologous transplantation and 53 children had an allogeneic transplant. 7 of these patients eventually relapsed and 3 died. 26 children had one or more pulmonary risk factors before hsct -chest or upper abdomen radiation (13), chest wall tumors or lung metastasis (8), chest surgery (5), prior administration of pulmonary-toxic drug (3) or asthma (2). during follow-up (up to 14 years, median 5.5 years), fev1 and fvc spirometry tests both decreased significantly (p=0.002 and 0.001, respectively), while the decrease in dlco was not statistically significant. 35% of patients had abnormal pulmonary function tests and seven children had symptomatic disease which in two may have been manifestations of gvhd. interestingly, no correlation was found between busulfan auc, busulfan peak levels, the number of busulfan doses administered, the type of transplantation (autologous vs. allogeneic) or primary disease to pulmonary injury. even after censoring of children with pre-transplant pulmonary risk factors we noted a decrease of fev1 and fvc. conclusions: as in adults, pulmonary injury is observed in children treated with high-dose busulfan prior to hsct. no correlation was observed between busulfan auc and pulmonary injury. follow-up of children who receive this drug should include regular pulmonary monitoring, referral to a pulmonologist when subclinical pulmonary compromise is found, and counseling regarding measures that might prevent or ameliorate pulmonary damage. continued follow-up of this cohort of patients should inform our pretransplant patient information sessions, and the future use of busulfan in children. disclosure: nothing to declare background: transplant-associated thrombotic microangiopathy (ta-tma) is a specific complication of allogeneic hematopoietic stem cell transplantation (hsct). post-hsct tma has been attributed to the vascular endothelial damage caused by high-dose chemotherapy, calcineurin inhibitors (cnis), graft-versus-host disease (gvhd), infections. there is a little evidence published regarding the efficacy and factors influencing the outcome of withdrawal of cnis. methods: the analysis comprised a total of 54 patients, with diagnosed hematologic malignancy (aml (16), all (12), mds (4), hodgkin lymphoma (5), cml (3) and neuroblastoma (1) received allo-sct, from a matched related, unrelated or haploidentical donor between 2007 and 2018. patients were diagnosed with ta-tma based on cho criteria. the median age of patients was 23 (37 adults, 17 children). gvhd prophylaxis was performed with tacrolimus (tac) in 42, cyclosporine a(csa) in 11, combination tacrolimus+sirolimus (sir) in 12. 24 patients received atg and 26 ptcy. withdrawal of cnis was accompanied by administration of systemic steroids (21 patients) or substitution with sir after reaching levels of csa< 100 ng/ml or tac < 3 ng/ml in 13. the target concentration of sir was 3-9 ng/ml. in pediatric patients who received combination tac+sir, the tac was discontinued in one step while sir continued. median time to development tma was 31,5 days after allo-sct (range 1-408). median follow-up of surviving patients was 395 days. the primary outcome was overall survival (os) up to 2 years after development of ta-tma. results: the following significant predictors of 2-year os were identified: tac replacement with sir (p< 0,001), ptcy in prophylaxis (p< 0,001), acute gvhd (agvhd) grade 2-4 (p=0,029), previous sepsis (p=0,003), level of ldh in debut (p=0,001), combination sir+tac in prophylaxis (p=0,05), major ab0-mismatch (p=0,022), severity of cns symptoms (p< 0,001). there was no significant difference in os according to patients' age, sex, "salvage" disease status at transplantation, previous vod, viral (hhv 1,2,6, cmv, ebv) reactivations, count of cd34+ cells transfused, ldh level, shizocytes and creatinine in the debut of ta-tma. in the multivariate analysis replacement of cnis with sir (hr 0.27, 95%ci 0.08-0.96, p=0.018) and baseline ldh level (hr 1.01, 95%hr 1.00-1.01, p=0.029) were associated with survival differences. the cut off for ldh was 4xunl. agvhd grade 2-4 (hr 1.96, p=0, 081) and use of ptcy (hr 0.535, p=0.317) were not significant in the multivariate analysis (figure 1). ta-tma cases after ptcy were significantly less frequently associated with clinically significant agvhd (19% vs 68%, p< 0,001). the survival was higher after ptcy (74% vs 15%), but not significant due to sample size and other ta-tma factors. leading causes of death were: gvhd progression (11%), bacterial infection (11%), tma (17%) and other (19%) . conclusions: replacing tac by sir is an effective therapeutic strategy in a group of patients with debut of ta-tma at least after ptcy, where it is less likely to be associated with agvhd. there is a significant overlap of populations with ptcy prophylaxis and substitution with sir, thus the study is not powered to provide guidance for patients on conventional prophylaxis with ta-tma. [[p172 image] 1. disclosure: none of the authors has anything to disclose. donor-recipient ab0 mismatch effect on the allogeneic hematopoietic stem cell transplantation outcome: a single-center retrospective study background: because transmission of major histocompatibility complex and blood group system genes is independent from each other, approximately 40-50% of all allogeneic hematopoietic stem cell transplantations (allo-hsct) are realized crosswise the ab0-blood group boundary. however, due to the widespread expression of ab0 antigens on a variety of human tissues other than erythrocytes, ab0 incompatibility may have an impact on the outcome of allogeneic hsct that goes beyond the wellknown immune-hematological complications such as immediate hemolysis due to the presence of isoagglutinins and delayed hemolysis due to passenger b lymphocytes. here we aimed to assess the donor-recipient ab0 mismatch effect on the allo-hsct outcome, comprising non-relapse mortality (nrm), overall and relapse-free survival, posttransplant prc transfusion requirement, as well as relapse rate, incidence of graft-failure and acute gvhd. methods: clinical and laboratory data from 180 consecutive patients undergoing allogeneic hsct between 01/2008 and 06/2018 at the fondazione irccs ca' granda maggiore policlinico hospital in milan, italy, were retrospectively collected. kaplan meier estimates were used for the analysis of survival outcomes while nrm, relapse and acute gvhd cumulative incidences were investigated by competing risk analysis. results: the patient series included 105 ab0-match, 31 major ab0-mismatch, 34 minor ab0-mismatched and 10 bidirectionally ab0-mismatch transplants. indication for allo-hsct were mainly aml/mds (77 pts), all (34 pts) and t-nhl/ctcl (32 pts). mean overall survival for groups of patients undergoing ab0-identical, major ab0 mismatch and minor ab0 mismatch hsct were 66 months (95% ci [55 ;77]), 47 months (95% ci [28; 65) and 46 months (95% ci [31; 61]), respectively. nrm in the three groups were significantly different, with point estimates of 12%, 29% and 26% at 5 years, respectively, whereas no significant differences were observed for relapse rate and graft failure incidence. although not statistically different, incidence of acute grade iii-iv gvhd was twice as high in patients transplanted from minor ab0mismatched donors than in the ab0 identical group (16% vs 8%). following transplantation, prc transfusion requirement was significantly higher in the major ab0 mismatch then in the ab0-match transplanted patients (median 14 vs 7, p=0.01), with a marginal positive correlation between the anti-donor a/b igg titers measured prior hsct and the total number of prc transfused during the first year following transplantation. we observed only one case of prca occurring in a 50year-old 0+ woman who was transplanted from a 32-yearold male a+ hla-identical sibling using peripheral blood as the stem cell source following a myeloablative conditioning for aml in first complete remission. anti-a igg isoagglutinin titers prior to transplantation were 1:256. during the first year post transplantation, the patient required a total of 46 prc transfusions, with gradual resolution occurring only after introduction of danazole treatment. conclusions: in our patient cohort, both major and minor ab0 mismatch associated to a significantly higher nrm. major ab0 mismatch associated to a higher prc transfusion requirement. a more frequently occurring severe acute gvhd was also suggested in minor ab0-mismatch transplants. altogether, our results suggest that allo-hsct outcome may be significantly affected by ab0 blood group mismatch. disclosure: nothing to declare background: at-tma is a severe endothelial injury complication and it may involve the intestinal vasculature. intestinal tma could be fatal and missdiagnosed. clinical and pathological criteria to differentiate from intestinal gvhd are needed. the aim of this study was to analyze the incidence and histological characteristics of intestinal tma in patients diagnosed of systemic tma. methods: we analyzed the incidence of tma in 555 patients who underwent allo-hsct in our institution between january 2010-august 2018. tma diagnosis was based on ho criteria. we do a pathological review in 103 biopsies from 25 out of 52 patients in whom an endoscopy have been performed 30 days before and 60 days after the diagnosis of tma for suspicious of gvhd. review was performed by a pathologist expert in gvhd, who examined the biopsies in search of hystopathological features of gvhd, tma or viral infection. diagnosis of gastrointestinal gvhd was stablished according to mcdonald and sales criteria, while intestinal tma diagnosis was stablished by warren et al criteria. results: 52 out of 555 patients (9,4%) were diagnosed of tma. transplant characteristics and tma data of patients with systemic tma are shown in image. 47 out of 52 patients with tma (90%) had been diagnosed with prior/ simultaneous acute gvhd, 20 of them grade iii-iv, and 80% with gastrointestinal gvhd. intestinal tma have been reported only in 7 out of 25 patients (28%) at diagnosis, whereas when review based on warren criteria was performed, in 19 patients (76%) the pathologist found at least 1 of the criteria of endothelial damage and 48 % of the patients 3 or more warren criteria were founded. the most frequent features were endothelial cell swelling (n=16, 64%) and perivascular mucosal hemorrhage (n=15, 60%). review hystological features of biopsies are shown in table 3 of the image. regarding gvhd, it was found in 21 patients (84%) at diagnosis and in 23 (92%) at pathological review. with a median follow-up of 10 months (1-73) 32 patients of the 52 with systemic tma (62%) are dead. 9 of the deaths (41%) were related to tma (3 tma, 3 tma +gvhd, and 3 tma+infection). patients with 3 or more warren criteria in pathological review had poor outcome compared with patients less than 3 criteria (30% alive vs 51% at 12 months, p=0.9). conclusions: intestinal tma is a life-threatening underdiagnosed entity. only 7 patients of 25 patients were diagnosed of intestinal tma. we found that most of our patients had endothelial damage in the gastrointestinal biopsy pathological reviews. gvhd histological criteria were present in most of the patients, mainly histological grade i-ii. prognosis of these patients is poor and pathologist effords in diagnosed the entity is guarranted. disclosure: nothing to disclosure p175 strategies to reduce neutropenic fever and hospital readmission in multiple myeloma patients managed at home after autologous stem cell transplantation background: neutropenic fever (nf) is the most frequent cause of readmission in the outpatient autologous stem cell transplantation (asct) programs. in our at home model for multiple myeloma patients, we added primary prophylaxis with ceftriaxone, decreasing the incidence of fever during aplasia phase from 85% to 57.6%. the aim of this study was to analyze the addition of two strategies to reduce the non-infectious nf: withdrawal of g-csf and the addition of primary prophylaxis for engraftment syndrome with corticosteroids after asct. methods: between january 2002 and august 2018 111 myeloma patients were managed at-home since day +1 of asct. all were conditioned with mel200. all patients received prophylaxis with quinolone, fluconazole, aerolized pentamidine, low-dose acyclovir (hvs+), and ceftriaxone (since day +4). the patients were classified into 3 groups: group a (n=33; g-csf since day +7 without corticosteroid), group b (n=32; no g-csf and no corticosteroid), group c (n=46; no g-csf with prednisone 0.5 mg/kg/day since day +7 until granulocyte recovery). first-line therapy at home of nf was piperacillin-tazobactam 4.5 g/6h i.v. using a portable intermittent infusion pump. fever was an indication of immediate medical consultation and those patients presenting signs of focal infection or severe sepsis were admitted. other indications for readmission were: willingness of the patient or caregiver, uncontrolled nausea, vomiting or diarrhoea, and mucositis requiring total parenteral nutrition or i.v. morphics. results: the main characteristics of the patients and outcomes are shown in table 1. there were no differences between groups regarding age, gender, immunological subtype, response before asct, hct-ci, and cd34 cell dose infused. there were more patients with advanced disease (iss iii) in group c compared to group a (19.5% vs. 6.1%; p=0.003). the duration of neutropenia was longer in those groups that did not receive g-csf (a: 8 days, b: 11 days, c: 10 days; p< 0.01). comparing group a with group c, we observed that the incidence of nf and the readmissions rates were lower in group c (nf: 57.6% vs. 23.9%; p=0.002; relative risk reduction: 0.41, and number needed to treat 2.97; readmissions: 12.1% vs. 2.2%; p=0.07, respectively). the 10-day cumulative incidence of nf were 54.5% in group a, 40.6% in group b, and 23.9% in group c; p=0.009. the non-administration of g-csf with the addition of prophylactic corticosteroid did not modify the incidence and grade of mucositis, the first day and duration of fever, nor the number of bacterial infections documented. in the multivariate analysis, this combination (no g-csf with corticosteroid) maintained its protective effect for the development of nf and hospital readmission (or 0.07; p=0.001 and or 0.07; p=0.05, respectively). conclusions: the non-use of g-csf and the addition of prophylactic corticosteroid in mm patients managed at home after asct minimize the incidence of non-infectious fever and optimize hospital resources by reducing hospital readmissions. disclosure: nothing to declare. background: antibody titers to vaccine-preventable diseases decline during the 1-10 years after allogeneic hematopoietic stem cell transplantation (hsct) if the recipient is not revaccinated. it is therefore considered best practice to try to offer hsct recipients the same level of protection against all vaccine preventable diseases as the general population. few data in the literature are available concerning vaccine-related problems in hsct recipients. we performed a farmacovigilance evaluation in a cohort of allotransplanted patients followed in our clinic during a 1 year period. methods: from october 2017 to november 2018 we administered a list of recommended vaccines to 49 hsct recipients attending our routine out patient clinic who fulfilled the following criteria: cd4 t cells>200/μl, cd19 b cells>20/μl, anti-cd20 antibody infusion>6 months, ivig therapy>2 months, no active and severe graft-versus-host-disease (gvhd), no chemotherapy or biological therapeutic agents on going. vaccines suggested were influenza, pneumococcal conjugate (pcv13), polio (inactivated polio vaccine), diphteria, tetanus, acellular pertussis, hepatitis b, hepatitis a, haemophilus influenzae type b, meningococcal quadrivalent (mcv4), human papillomavirus, meningococcal b, measles-mumps-rubella (mmr), varicella. live vaccines (mmr and varicella) were not recommended before 2 years after hsct and in patients with chronic gvhd. all the patients were asked to take the list to the local health facilities in order to have the vaccines injected and a vaccination table arranged with the doses already received and those to receive. we checked the vaccination tables at each visit and monitored potential side effects and gvhd status at 3, 6, and 12 months after the first vaccine injection. results: twenty-nine out of 49 patients were evaluable (table 1), 16 without gvhd and 13 with chronic gvhd (5 mild, 4 moderate, 4 severe). median time after hsct was 34 months (16-240). median number of vaccines received was 8 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) . as regards patients without chronic gvhd, 2 out of 16 experienced fever after vaccine injections; 1 out of 16 developed transient mild reduction of platelet count; 1 patient reported headache and otalgia after vaccine injection, while another one transient joint pain; 1 out of 16 patients presented signs of mouth chronic gvhd (score 1 nih) and transaminase increase (grade 1 according to world health organization toxicity scale) 3 months after the first vaccine dose, so that cyclosporine dose had to be augmented. as regards patients with chronic gvhd, 4 out of 13 experienced fever after vaccine injections; 2 patients with mild chronic gvhd of the mouth presented hepatic flare two and three months after the first vaccine dose, respectively. in both cases a new increase of cyclosporin and methylprednisolone doses determined progressive normalization of liver enzymes. conclusions: these data show that vaccines were globally well tolerated in hsct recipients, even when they suffered from chronic gvhd. however, close monitoring is warranted in order to better evaluate possible vaccine side effects in this setting of patients. background: allogeneic hsct improves survival for aml patients over the age of 60 years of age when compared to chemotherapy alone. the haematopoietic stem cell transplantation comorbidity index (hct-ci) and ebmt score predict for non-relapse mortality and overall survival, yet little is known about whether qol is preserved in this patient group and whether hct-ci and other performance scores pre-bmt correlate with qol post allo-hsct. methods: we conducted a retrospective analysis of patients 60 years and older who underwent ric allo-hsct at the university hospital of wales, cardiff between september 2011 and december 2017 (n=41). hct-ci, karnofsky performance score (kps) and ebmt scores were calculated prior to transplant and qol measured using the fact bmt (version 4) questionnaire, which was completed at 3, 6 and 12 months post transplant. patients were grouped at the 3-, 6-and 12-month time points for each of the different performance indices, allowing group comparison against compound sub scores using the mann-whitney u test. results: 41 patients were included in this study, with median age 65 years (range 60-74). patient characteristics, including conditioning, donor type, pre-transplant hct-ci and kps scores are summarised in table 1. the 2 year and 5 year overall survival (os) for the patient cohort was 65.4% and 48.7% respectively. hct-ci of ≥3 vs 0 was significantly associated with poorer bmt-related qol domains at 3 months (p=0.035) and general qol domains at 6 months (p=0.025) post-transplant. while ebmt score showed no correlation with qol parameters, patients with kps of 100 vs ≤90 showed significant differences in both general (p=0.01) and bmt-related qol (p=0.04) at 3 months and in all qol domains at 6 months (symptomrelated qol p=0.05, general qol p=0.01, bmt-related qol p=0.01). importantly neither the hct-ci nor the kps pre-transplant predicted for qol at 12 months post transplant. conclusions: patient selection is key to ensuring maximum benefit from allo-hsct both in terms of overall survival but also with regards to qol and survivorship. we note that while patients with hct-ci ³3 or kps ≤90 had significantly poorer qol at 6 months post allo-hsct, qol was recovered by 12 months post transplant, with this significant difference no longer seen. our data shows that in selected aml patients over the age of 60 years with good performance status and low comorbidity index, a favourable outcome can be achieved with good qol maintained throughout the post transplant period. background: advances in allosct technology, supportive care, and use of reduced intensity conditioning regimens for older patients have led to significant improvements in longterm survival after transplant. the survivors have an elevated probability of late morbidity and mortality, including abnormalities in phosphocalcic metabolism and bone disease. rapid and progressive bone loss occurs within the first 6-12 months after transplant, and this is followed by a slow process of recovery, with bone loss persisting for 48 to 120 months. bone fractures can worsen the quality of life of allosct survivors, but the real burden of the disease is unknown. the objective of the study is to ascertain the prevalence of bone pathology and vertebral fractures early after transplant in our center. methods: this is a retrospective and observational study. forty-nine patients (25 male/24 female, median age 54y, range 19-69) that underwent allosct were included in the study in the period of 6 to 24 months after transplant (may 2016-december 2017). pre-and post-transplant risk factors associated with bone disease were recorded: age >65 years, female sex, menopause, hormone replacement therapy, previous treatment with steroids, previous fractures, weight < 40 kg, bmi < 20-25, low physical activity, low calcium intake, smoking, alcohol intake, and history of femoral fractures in parents. in all patients laboratory data (including serum calcium, 25-hydroxyvitamin d, and pth), lumbar and femoral bmd (dxa), and spinal x-ray were also evaluated. a vertebral fracture was defined as a reduction of >20% in the anterior, middle or posterior high of the vertebral body. results: we identified vertebral fractures in 12 (24%) patients. five patients had fractures prior to transplantation, and 7 patients presented "de novo" vertebral fractures following transplantation; therefore, the prevalence of "de novo" postransplant fractures was 7/49 (14%). most (85%) of these fractures were asymptomatic at the time of diagnosis. most patients (64%) with vertebral fractures had >3 pre-sct risk factors (median risk factors pre-sct 3, range 2-6), the most frequent being low calcium intake, steroid exposure, presence of previous fractures, and menopause. those patients with fractures and less than 3 risk factors pre-tph, added new risk factors after transplant, mainly steroid treatment. forty-four patients (90%) had vitamin d insufficiency (< 30ng/ml), 15 (32%) had osteopenia and 9 (18%) had osteoporosis. vitamin d insufficiency and bone disease were more frequent in women than in men (98% vs. 84% for vitamin d, 37% vs. 28% for osteopenia, 29% vs. 8% for osteoporosis, and 25% vs. 20% for vertebral fractures, respectively). conclusions: the prevalence of post-transplant bone disease and vertebral fractures in our series is high. most fractures appearing "de novo" after allosct were asymptomatic and were diagnosed by x-ray. patients who presented vertebral fractures frequently had more than 3 risk factors identified pre-sct. patients undergoing allosct should have their bone health assessed early in their treatment and, if indicated, should start preventative therapy to avoid bone loss and fractures. other measures such as physical exercise, vitamin d and calcium supplementation, and dxa and spinal x-ray at baseline and following transplantation are also highly recommended. disclosure: maría suárez-lledó received a grant from dkms-spain foundation. other authors have nothing to declare the use of g-csf in selected patients after autologous stem cell transplantation is associated with low incidence of engraftment syndrome background: the use of g-csf after autologous stem cell transplantation (asct) accelerates neutrophil recovery, however it has been related to an increased risk of engraftment syndrome (es) development in some studies. for this reason, we do not routinely prescribe g-csf after asct and we only use it in patients with significant complications (enterocolitis, severe sepsis, atrial fibrillation) after stem cell infusion. the main objective of this study is to evaluate the incidence of es in patients who receive asct for monoclonal gammopathies (mg), non-hodgkin lymphoma (nhl) and hodgkin lymphoma (hl) and receive g-csf only if needed. as secondary objectives we evaluate differences in the engraftment day as well as the length of inpatient stay. methods: we retrospectively analyzed patients with mg or lymphoma, who underwent asct conditioned with high dose melphalan (140-200 mg/m 2 ) or beam, respectively, between 2015 and 2017 in our center. specific clinical features for es according to spitzer and maiolino criteria were evaluated between 3 days before and 7 days after the engraftment. statistical analysis was performed with spss v. 15.0. results: thirty-one patients with mg and 34 patients with lymphoma were analyzed. median age at transplant was 56.8 years (48.7-65.4 ) and 41 patients (63.1%) were male. median prior lines of treatment in patients with gm or lymphoma were 1 (1-3) and 2 (1-5), respectively. table 1 shows patients´characteristics. mobilization with g-csf ± plerixafor was performed in 27 patients (36%) and chemotherapy + g-csf ± plerixafor in 38 patients (64%). median cd34 x 10 6 /kg cells infused was 3.6 (2.7-5.3). eleven patients (16.9%) received g-csf, 5 due to infection (2 enterocolitis, 1 listeriosis, 1 acute hepatitis, 1 septic shock) and 6 because of atrial fibrillation or fibrilloflutter. median time from sct to first day of g-csf was 5 days (5-7) and median time on g-csf treatment was 5 days (4-7). patients who received g-csf showed a short time to neutrophil engraftment (≥0.5x10 6 /l), 10 days vs. 13 days, p< 0.001 but longer duration of hospitalization, 18 days vs. 15 days, p =0.050. non-relapse mortality at day +30, +100 and +180 was 0%. es was diagnosed in 4 (6.2%) patients, 1 amyloidosis, 2 multiple myeloma and 1 plasmablastic lymphoma. there was not statistical difference in the incidence of es between patients who received g-csf (9.1%) and patients who did not (5.6%), p=0.533. analyzed by disease, es appeared in 1 of 6 patients who received g-csf in the lymphoma group (16.6%) but none of the 7 patients with mg that received g-csf developed it. we did not find statistical differences between patients who developed es and those who did not in age (49 years vs. 56 years, p=0. 314), length of hospitalization (19 days vs. 15 days, p=0.185) and the number of cd34 x 10 6 /kg cells infused (3.65 vs. 4.62, p=0.408) . conclusions: the use of g-csf in selected patients is associated with low incidence of es. our study confirms that the use of g-csf accelerates neutrophil recovery but it is unclear if it can increase the incidence of es, especially in patients with lymphoma. [[p180 image] 1. background: graft failure is one of the top-3 problems of allo-hsct (after gvhd and relapse). the problem of graft failure becomes more significant due to increasing number of allo-hsct with ric conditioning regimen from haploidentical and hla-mismatched unrelated donors. role of t cells in graft failure is well known. here we report an impact of t-memory cell subsets count before antithymocyte globulin (atg) administration on primary graft failure after allo-hsct. methods: sixteen patients with acute leukemia transplanted in national research center for hematology were included on this prospective study. all patients received horse atg at dose 10 mg/kg/day from day -4 to -1 before allo-hsct as gvhd prophylaxis and were balanced by other factors that could affect engraftment. detailed patients characteristics are listed in table 1 . peripheral blood samples were collected on day -4 before allo-hsct (before atg injection) in edta-tubes. flow cytometry analysis was performed on bd facs canto ii (becton dickinson, usa) to define t-memory subsets: t-naive and t-stem cell memory (tnv+scm) -cd45r0-ccr7+cd28+; t-central memory (tcm) -cd45r0+ccr7+cd28+; t-transitional memory (ttm) -cd45r0+ccr7-cd28+; t-effector memory (tem) -cd45r0+ccr7-cd28-; t-terminal effector (tte) -cd45r0-ccr7-cd28-, among cd4+ and cd8+ t-cells . sysmex xe-2100 was used to calculate absolute count of different t-cell subsets. mann-whitney u test was used for nonparametric data analysis between two groups. fisher's exact test was used for 2x2 tables. p-value less than 0.05 was considered statistically significant. results: an influence of t-memory cell subsets count before atg administration on primary graft failure is shown in figure 1 . according to our data high absolute number of cd4+ttm and cd4+tte is associated with primary graft failure. conclusions: based on these findings high absolute number of cd4+ttm and cd4+tte could be one of the prognostic factors of primary graft failure after allo-hsct. optimizing atg dose due to recipient absolute t-memory cell subsets count before atg administering may prevent graft failure and improve posttransplant results. background: upper gastrointestinal graft-versus-host disease (gi gvhd) has been an increasingly recognised entity following allogeneic stem cell transplantation (sct). budesonide, widely used in inflammatory bowel conditions, has also been found beneficial in gi gvhd. the major benefit of budesonide is attributable to its poor absorption and extensive first-pass metabolism via cytochrome p450 (cyp) 3a4, which translates to less systemic steroid-related effects. however, transplant patients are often exposed to multiple drugs, among which some agents act as cyp3a4 inhibitors and therefore can increase budesonide bioavailability and might lead to systemic toxicity. azole antifungal drugs are probably the most common concomitantly used cyp3a4 inhibitors in transplant recipients. methods: we reviewed allogeneic sct records for patients treated with oral budesonide for gi gvhd at our transplant centre between 2015 and 2018 retrospectively. the aim of the work was to assess the development of adrenal suppression with or without clinical features of iatrogenic cushing`s syndrome. the standard dose of budesonide was 3 mg three times a day. patients receiving prednisolone or other glucocorticosteroids and those with no available serum cortisol level measurements were excluded. results: our analyses identified four allogeneic sct patients in whom adrenal suppression was diagnosed with undetectable serum cortisol levels during oral budesonide treatment. of these patients two developed iatrogenic cushing`s syndrome and both patients were treated with cyp3a4 inhibitors concomitantly: 1. clarithromycin and fluconazole; 2. clarithomycin and voriconazole. the development was rapid (within 3 and 4 weeks). symptoms included morphological features such as moon face, high blood pressure, weight gain, peripheral oedema and proximal myopathy. symptoms resolved gradually following cessation of azole antifungal agents and on gradual weaning of budesonide. conclusions: although single agent budesonide treatment given for gi gvhd is rarely associated with systemic side effects, patients on azole antifungal drugs and macrolide antibiotics are at higher risk of systemic toxicity due to drug interactions. patients who are allergic to penicillin and receive macrolide-based prophylaxis can be especially vulnerable. to our knowledge the number of cases reported in literature about systemic effects of oral budesonide in transplant recipients is less than 10. our observation supports previous reports on the potential of oral budesonide to induce systemic effects. we therefore advise careful monitoring of patients treated with budesonide in combination with cyp3a4 inhibitors, including antimicrobial agents routinely used in sct. disclosure: none implemented strategies to overcome barriers in the establishment of a consolidated hematopoietic stem cell transplantation program in a developing country background: the national institute of medical sciences and nutrition "salvador zubiran" is a national health institute located in mexico city. although mexico is considered an upper-middle income country, more than 50% of the population lives in poverty without health care coverage and patients within this social stratum are referred to our institution. the first hematopoietic stem cell transplantation (hsct) in mexico was performed at our institution in 1980. from this year until 1997, hsct were sporadically performed (n=33), showing a poor overall survival (os) and high non-relapse mortality (nrm). these outcomes resulted from an unstructured hsct program, limitedresources, patient low socioeconomic status, and paucity of population-adapted procedures. in 1998, according to these results, a decision to establish a hsct program was made. therefore, in order to set up a successful hsct program, implementation of financial and medical strategies were necessary. the objectives of this study were to describe the barriers and implemented strategies for the establishment of a hsct program in mexico along with the outcomes of patients undergoing this procedure throughout the reorganization of the program. methods: this study is a health services research. barriers were detected based on the results of the hsct program from 1980-1997 (not shown). table 1 shows the financial, medical, and research strategies that were implemented for each barrier. results: from november 1998 to november 2018, 363 hsct have been performed in 322 patients at our institution. most hsct were autologous (n=213, 59%). forty one patients underwent 2 hsct. from the 322 patients, most were males (n=196, 61%) and the median age was 33.5 years (range, 15-65). the most frequent underlying diseases for auto-hsct were lymphomas (n=68, 36%), non-seminomatous germ cell tumors (n=42, 22%), and multiple myeloma (n=42, 22%). acute leukemias (n=41, 34%), aplastic anemia (n=25, 21%), and myelodysplastic syndromes (n=20, 17%) were the most frequent diagnosis for patients undergoing allo-hsct; and acute leukemia was the most frequent diagnosis for patients undergoing haploidentical hsct (n=10, 83%). acute and chronic gvhd were present in 25% (grades i-ii 89%) and 35% (limited 76%), respectively. for allo-hsct, 30, 100day, and 1-year nrm was 2.5%, 8%, and 12%, respectively; 30 and 100-day nrm in auto-hsct was 1.5%; 10year os was 63% and 56% for auto and allo-hsct, respectively. conclusions: future perspectives of the hsct program include the acquisition of funds for unrelated donors; to improve outcomes of patients undergoing haploidentical hsct, and to increase the number of in-patient rooms. we conclude that despite paucity of resources and other limitations, the implementation of financial, medical, and research strategies have shown that barriers can be effectively overcome in a developing country in order to establish a consolidated and nationally renowned hsct program, providing good outcomes for patients. disclosure: none of the authors have any conflict of interest to disclose. the effect of protective buffering on daily stress and relationship quality in dyads following hematopoietic stem cell transplantation: results from daily process methodology malgorzata sobczyk-kruszelnicka 1 , aleksandra kroemeke 2 , zuzanna kwissa-gajewska 2 , sebastian giebel 1 background: cancer-related support communication (e.g., protective buffering) may impact the risk for psychological and relationship distress in patients following hematopoietic stem cell transplantation (hsct) and their caregivers. previous studies have revealed that protective buffering (i.e., hiding one's concerns and denying one's worries) has mixed effects: is beneficial (for "protected" person), costly (especially for the person using it), or unrelated to dyadic wellbeing. there has been, however, little evidence linking dyadic protective buffering with distress using daily process methodology. we assessed (1) the relationship between daily protective buffering, and same-and next-day stress and relationship quality in patient-caregiver dyads following hsct and (2) whether similarity or complementarity in protective buffering between dyads is adaptive. methods: two hundred patients (after first autologous or allogeneic hsct) and their caregivers (spouse or another relative) independently completed measures of daily protective buffering, daily relationship quality, and daily stress for 28 consecutive evenings after patients´hospital discharge. actor-partner-interdependence model (i.e., both partners' and caregivers' reports regarding support communication and distress were studied) was used to test study hypotheses. results: for both patients and caregivers, multilevel structural equation modeling showed a significant positive relationship between daily protective buffering and sameday relationship quality. association of protective buffering with same-day stress level was negative. in next-day analyses, patient-reported protective buffering was related to patient's higher relationship quality, whereas caregiverreported protective buffering increased patient's daily stress. complementarity in protective buffering was related to higher immediate same-day relationship quality for both patients and caregivers, while benefits from similarity have delayed effects, although only in patients. conclusions: contrary to previous studies, protective buffering rather has a beneficial effect in dyads following hsct. protection of the partner and relationship against revealing negative emotions and powerlessness was not related to costs in both parties. the findings suggest that the effect of daily protective buffering in dyads following hsct depends on support timing (same-or next-day effect) and differs for both parties. patients seem to benefit the most from the similarity in protective buffering, while caregivers from complementarity. the "fit" between patient and caregiver in support communication ought to be taken into consideration in the practical approach. disclosure: nothing to declare. virus reactivation and low dose of cd34+ cell were associatied with secondary poor graft function within the first 100 days after allogeneic stem cell transplantation yuqian sun 1 , xiao-jun huang 1 background: secondary poof graft function (spgf) was defined as the secondary cytopenia after initial engraftment of hsct. it was shown to be associated with poor prognosis, however there are very few reports on the incidence, risk factors and outcomes of spgf. methods: patients who received transplantation from peking university people's hospitial during january, 2015 to december, 2015 were retrospectively reviewed if they fulfilled the following conditions: (1) diagnosed with acute leukemia or myelodysplastic syndrome; (2) received allo-sct from either matched sibling donor (msd) or haploidentical related donor (hid). pgf was defined as persistent neutropenia (≤0.5×10 9 /l), thrombocytopenia (platelets ≤20×10 9 /l), and/or hemoglobin ≤70 g/l for at least 3 consecutive days, transfusion-dependence, associated with hypoplastic-aplastic bone marrow (bm), and complete donor chimerism without concurrent graftversus-host disease (gvhd) or disease relapse. primary pgf was defined as the failure to achieve initial engraftment by days 28 after transplantation, while secondary pgf was defined as the fulfillment of the criteria after initial engraftment hsct. results: during january, 2015 to december, 2015, 564 patients who received transplantation from peking university people's hospitial were retrospectively reviewed. among the 490 patients who achieved initial engraftment, 28 patients developed spgf. the cumulative incidence of spgf on day 100 was 5.7%. the median time of secondary pgf was 54.5 (34-91) days after transplantation. low (< median) cd34+ cell dose (p=0.019, hr 3.07(95%ci, 1.207-7.813)), ebv reactivation (p=0.009, hr 3.648(95%ci, 1.382-9.629)) and cmv reactivation (p=0.003, hr 7.827 (95%ci, 2.002-30.602)) were identified as independent risk factors with spgf. there is no significant difference of pgf incidence in msd group and hid patients (p=0.44). the overall survival of patients with spgf at 1 year after transplantation was significantly poor than patients with ggf (50.5% versus 87.2%, p< 0.001). conclusions: in conclusion, spgf develop in 5.7% patients after allo-sct, especially in patients with cmv, ebv reactivation or infused with low dose of cd34+ cell. the prognosis of spgf is still poor due to lack of standard treatment. disclosure: there is no conflict of interet thiotepa with treosulfan and busulfan based conditioning are significantly more gonadotoxic than treosulfan previous studies suggest that busulfan results in long-term gonadal toxicity. no previous studies have compared gonadal toxicity outcomes after treatment with busulfan with treosulfan, a newer agent with similar marrow toxicity to busulfan but with reduced non-marrow toxcitiy. our aim was to determine whether there are differences in pubertal and fertility outcomes in paediatric patients treated with treosulfan compared with busulfan. methods: inclusion criteria were patients who had received either busulfan or treosulfan or treosulfan with thiotepa, only one hct and were aged 14 years and above in august 2018. eligible patients were reviewed in clinic as part of their routine follow-up, thus research ethical approval was not required. follice stimulating hormone, luteinising hormone, oestradiol, and pubertal history were noted. ovarian reserve was estimated in female patients by measuring serum anti-mullerian hormone (amh). male patients had serum testosterone measured and were also offered semen analysis. results: thirty-five patients met the inclusion criteria, of which twenty-five wanted to be reviewed (71%); seventeen females and eight males. mean age at hct was 13 years, mean age at review was 19 years and mean years since hct was 5 years. female patients treated with busulfan or treosulfan with thiotepa (n=14) had minimal amh and none of these patients were having regular periods. females treated with treosulfan (n=3) had normal amh and regular periods without needing hormone replacement. only four male patients opted for a semen analysis and all had significantly reduced sperm counts. conclusions: our results suggest that females treated with treosulfan have minimal (if any) reduction in ovarian reserve compared to other conditioning regimens which casue significant compromise. although this was a small study, and thus not suitable for statistical analysis, the clinical findings are marked. future studies should further investigate optimal doses of treosulfan that could be used to achieve bone marrow engraftment and limit long-term effects on fertility. disclosure background: autologous and allogenic hematopoietic stem cell transplantation (hsct) are potentially curative treatments for hematological malignancies. patients with related complications may need admission to the intensive care unit (icu) for specific therapy and organ support. mortality risk factors, supportive care and principal causes of admission in icu are described in our cohort of patients (pts). methods: we retrospectively studied 326 pts, 185 male, with a median age of 56,63 years (range: 18-73) who underwent allo-hsct in our center between july 2014 and october 2018. two hundred and twenty-seven(69,6%) pts received autologous hsct (auto-hct) and 99 (30,4%) allogenic hsct (allo-hsct); 50 from unrelated donor, 38 from identical sibling, and the remainder, mismatched related donor 11. twenty-three (7,1%) out of 326 pts were admitted in the icu in the transplant procedure admission. results: fifteen (65,2%) out of 23 pts were male with a median age of 55 years (range: 28-69). patients' baseline diseases were: multiple myeloma (34,8%), non-hodgkin´s lymphoma (26,1%), hodgkin´s lymphoma (8,7%), acute lymphoblastic leukemia (8,7%), myelodisplasic syndrome (8,7%), solid tumor (8,7) and acute myeloblastic leukemia (4,3%). fifteen (65,2%) pts received auto-hsct, 5 (21,7%) allo-hsct from unrelated donor, 2 (8,7%) allo-hsct from identical sibling, and the remainder haploidentical hsct (1) (4,3%). so, 6,6% of auto-hsct pts and 8% of allo-hsct were admitted in the icu. the median stay in the icu was 5 days (range: 1-30) and reasons for admission were: respiratory insufficiency (60,8%), septic shock (30,4%), renal insufficiency (4,3%) and multi-organic failure (4,3%). twenty-one (91.3%) pts required respiratory support with: nasal cannula or oxygen mask (c/m) (19%), non-invasive mechanical ventilation (nimv) (66,7%) and invasive mechanical ventilation (imv) (14,3%). fourteen (60%) pts needed inotropic agents for shock treatment. finally, 4 (4,5%) pts required substitutive renal therapy with hemodialysis or haemofiltration (hd/hf). eleven (47,8%) out of 23 pts died, 7 (63,6%) were male with a median age of 55 years (range: 24-64). ten of them (90,9%) needed imv and were treated with inotropic agents. all patients who required hd/hf (n=4) died. imv and treatment with inotropic agents were associated with icu mortality (or 6,5; p=0,03, or 7; p=0,008; respectively). conclusions: in our series of pts, 7,1% needed admission in the icu, presenting a mortality rate of 48% approximately. there were no differences in the prevalence of icu admission regarding hsct donor. main reason for admission was respiratory failure with imv requirement in 14,3% of pts. imv and treatment with inotropic agents were associated with icu mortality. an early identification of pts at risk of icu admission could have a beneficial impact on survival improvement disclosure: nothing to declare is there any association between thrombotic risk factors and veno-oclusive disease in childhood allogeneic hematopoietic stem cell transplantation? background: veno-oclusive disease (vod) is a major complication of hematopoietic stem cell transplantation (hsct). in some studies levels of fibrinolytic factors especially plasminogen activator inhibitor-1 (pai-1) level were found associated with vod. however, little is known about the relationship between thrombophilia risk factors and vod. in this study we aimed to investigate association of major thrombophilic gene mutations on vod in pediatric hsct patients. methods: we reviewed retrospectively 35 patients with vod who underwent hsct between 2010-2018 in ankara pediatrics and pediatric hematology-oncology training and education hospital, bone marrow transplantation unit, turkey. fifty-one patients who did not develop vod and transplanted during the study period were accepted as control group. we evaluated plasma homocysteine and lipoprotein a level, protein s and c activity and antigen levels and factor v g1691a mutation, prothrombin g20210a mutation, methylenetetrahydrofolatereductase (mthfr) c677t and a1298c mutations, plasminogen activator inhibitor-1 -675 4g/5g polymorphism before hsct. we also evaluated the patients' hospital files and noted the demographic values and complications of hsct. statistical investigations were done with spss statistics 17.0 for windows and p< 0.05 has been accepted as significant. results: there was no difference between control and vod groups as regard to age, sex, diagnosis, donor type, conditioning regimen, hsc source, and hla typing . there was no difference between the groups according to homocysteine, lipoprotein a, protein s and c activity and antigen levels. we did not find any relation between the genetic variations of thrombophilia and vod (table 1 ). in vod group there were 6 patients (17.1%) with acute graft versus host disease (agvhd) and in control group there were 7 (15.9%) patients with agvhd (p=0.046). febrile episodes were more frequent in vod group compared to the controls (respectively; n=30, 85.7% vs. n=23, 54.8%, p=0.006). 8-year overall survival was %77.1 in vod group and 100% in control group (p=0.001). disease free survival was also different between vod and control groups (respectively; 74.3% vs. 97.3%, p=0.001). conclusions: in literature there are recent studies showing higher pai-1 levels in patients with vod. however, in our study we did not find any relationship between congenital thrombophilia factors and vod. new studies with larger sample groups is necessary to better evaluate the association of congenital thrombophilia factors and vod. disclosure: nothing to declare p189 different strategies of chemotherapy-induced nausea and vomiting (cinv) prevention in hematological patients receiving an autologous hematopoietic stem cell transplantation: a single center experience ilaria cutini 1 , riccardo boncompagni 1 , chiara nozzoli 1 , antonella gozzini 1 , stefano guidi 1 , chiara innocenti 1 , massimo di gioia 1 , lorenzo tofani 1 , riccardo saccardi 1 background: despite the improvements of pharmacological control, cinv still represents a major problem in patient undergoing hematopoietic stem cell transplantation (hsct). we present here a comparison of two pharmacological strategies for preventing cinv in multiple myeloma (mm), hodgkin (hl), and non-hodgkin lymphoma (nhl) patients who received an autologous hsct in our institution. methods: from january 2015 to july 2018, we retrospectively analyzed 250 consecutive patients, median age 58 years (22-71yo) , diagnosed with mm, hl, and nhl, who underwent an autologous hsct following a melphalan 200 mg/sqm and beam/feam condition regimens, respectively. the first 122 patients received cinv prophylaxis with palonosetron i.v and dexamethasone 8 mg die (regimen a), whilst the following 128 were administered with fosaprepitant iv, ondansetron iv and dexamethasone 8 mg die (regimen b) both cinv prophylaxis was administered the day of melphalan infusion (day -1 form transplant). emesis breakthroughs were treated with alizapride and metoclopramide. nausea and vomiting were assessed through the ctcae 4.0 score system. categorical variables were compared with pearson chi-square test. results: the overall incidence of nausea was 78%, (55% grade 1, 41 % grade 2, and 4% grade 3, respectively). in regimen a was shown to be 80%, (56% grade1, 41% grade 2, and 3% grade 3, respectively) while in regimen b was 77% (54% grade 1, 41% grade 2, and 5% grade 3, respectively). pearson chi-square test did not show any differences between the 2 groups (p=0.679). the overall observed vomit was 32% (83% grade 1, 16% grade 2, and 1% grade 3). in regimen a it was (47% (84% grade 14% grade 2, and 2% grade 3), and 17% in regimen b (77% grade 1 and 23% grade 2). conditioning regimens didn't' have any significant impact on either nausea or vomit. patientsyounger then median (58 yrs), were reported to have higher incidence of both nausea, (p=0.028) not related to cinv treatment, and vomit (40% vs 24%, p=0.012). in multivariate analysis the overall incidence of nausea is related to age (younger patients have higher probability to develop nausea (or 2,282; p= 0,024) whilst the higher incidence of vomit is related to: regimen a (or 3.958; p< 0,001), previously reported nausea (or 4,506; p< 0,001), and no smoking habits (or 2,761; p=0,02). conclusions: both regimens are equally effective for nausea control however regimen b evidenced a better vomiting control. this finding is particularly relevant when the center policies include an early discharge program, therefore improving both patient's quality of life and procedure cost-effectiveness. clinical background: patients who underwent an allogeneic hematopoietic cell transplantation (hct) are challenged by medical, psychological and social complications. support groups might help hct-survivors to cope with these challenges. however, the existing literature about post-hct support groups is scarce. moreover, data on professionallyfacilitated support groups do not exist. the aim of this project was (1) to establish a professionally-facilitated support group and (2) to assess the discussed topics. methods: from 11/2013 until 6/2017 all patients who received an allogeneic hct at the adult stem cell transplantation program of the university clinic mannheim were invited to participate in a professionally-facilitated support group. additionally, spouses and life partners were invited. a theologian who is also a physician served as facilitator. he had no further function within the transplant team. the format of the group was unstructured without any rules regarding regular attendance. the facilitator did not provide topics or a curriculum. during the first year the group met every 14 days followed by a monthly schedule. from the fifth until the 39th meeting the attendance and the discussed topics were minuted by the facilitator. the content of the minutes was analysed by a combination of an inductive and a deductive approach. all participants provided their informed consent for the study. results: altogether 23 patients (female: n=10; male: n=13) and 10 spouses/life partners (female: n=9; male: n=1) participated. 13 patients (57%) and 6 spouses (60%) attended more than one meeting. among those who participated in ≥2 meetings the median time of participation was 16 months. the median count of participations was eight. 30% of the participants attended the meetings longer than one year, 9% longer than three years. there was no sex difference with respect to the frequency and the duration of participation. however, the frequency of participation decreased significantly the longer a participant was attending the meetings. during 35 group meetings the facilitator recorded 5138 thematically different contributions to the discussions divided in 37 distinct topics. these topics were grouped into 5 main categories [(a) medical topics, (b) private life and environment, (c) human relationships, (d) physical and mental condition and (e) the support group itself] and eight further categories [(1) compliance, (2) economic issues, (3) religion, (4) sexuality, (5) death and dying, (6) support and coping, (7) objectives and needs and (8) not otherwise specified issues] which could not be grouped in one of the main categories. the most frequent issues were medical topics (34%), human relationships (16%), physical and mental condition (15%), private life and environment (14%), financial issues (5%), the support group itself (4%), support and coping (4%) and objectives and needs (4%). noteworthy, death and dying (0.5%) were rare topics and sexuality was never mentioned. conclusions: to our knowledge, this is the first prospective and systematic analysis of a professionallyfacilitated support group for hct-survivors. these data might help to establish support groups and to identify psychosocial needs of patients and targets for specific support. disclosure: nothing to declare background: endothelial damage is associated with inflammatory complications that appear early after hsct, such as sinusoidal obstruction syndrome or acute gvhd. engraftment syndrome (es) is an inflammatory condition diagnosed by maiolino clinical score. potentially, es can exhibit high morbidity and mortality, especially after autologous-hsct in multiple myeloma (mm) patients since the introduction of new drugs such proteasome inhibitors and immunomodulatory drugs (imids). the objective of the present study was to evaluate if es is associated with endothelial dysfunction in patients with mm who underwent auto-hsct. methods: we included six patients with mm who received induction treatment including new drugs and consolidated their response with an autologous-hsct. we analysed comparatively the effect of incubating endothelial cells in vitro with serum samples from patients with es vs. no es. serum samples were collected before (pre), and after 5, 7, and 10 days from the transplant. an additional sample was collected at the es onset and at the discharge day (no es group). endothelial cells (hmec) in culture were exposed to media containing 20% of serum from each patient for 24h. cell growth was controlled morphologically. expression of the adhesion receptor icam-1 on the cell surface was analysed by immunofluorescence, and activation of the inflammation related p-38 mapk signalling pathway was evaluated by sds-page and western blot. results: exposure of hmec monolayers to sera from patients who developed es (onset day, n=4) resulted in an increased icam-1 expression on the cell surface, higher that the observed with sera from patients who did not develop es (discharge day, n=4) (26.4% of labelled area vs. 6.4%, respectively). in addition, in experiments with sera from patients not developing es, icam-1 expression on cells exposed to sera from day +10 was reduced with respect to the observed with sera from day +5, probably due to the corticosteroid used as a prophylaxis in our centre. this reduction was not observed in es patients. regarding phosphorylation of p-38, it was significantly higher in cells exposed to sera from es patients than in response to sera from patients who did not develop es. conclusions: the increase in the expression of the adhesion receptor icam-1 on the surface and the intracellular activation of p38mapk in endothelial cells exposed to sera from patients developing es indicates the existence of endothelial activation in association with es. interestingly, the prophylaxis of es with corticosteroid seems to be less effective in patients who developed es than in patients who did not develop this complication. these results need to be validated in a higher number of patients and modifications in additional markers of endothelial dysfunction should be investigated. disclosure: gonzalo gutiérrez-garcía: honoraria from gilead. grant from jazz pharmaceutical the other authors do not have any disclosure to comment. p192 association between uric acid levels before and after allogeneic haematopoetic stem cell transplant and transplant outcomes: a single centre experience background: uric acid (ua) is a known endogenous danger signal which activates the nod-like receptor protein (nlrp)3 inflasome.ua is released from injured cells during conditioning in allogeneic stem cell transplantation (hsct). a pre-clinical study has demonstrated that nlrp3 inflasome-mediated il-1 production regulates graft-versushost disease (gvhd). the ua role in inflammation and gvhd is unclear. there are discordant reports in the literature about a potential protective role of ua on gvhd after a hsct. methods: we performed a retrospective study to assess the association between serum ua levels pre-and posttable] 1. table 1 ] results: the characteristics of the 142 patients are shown in table 1 . median age was 52 years (range 15-69), and 80 patients (56%) were male. twenty-seven patients (19%) received low doses atg as part of gvhd prophylaxis. allopurinol was from the day before start of conditioning therapy until day 0. the median levels of ua were 4,8 mg/ dl before conditioning, 2,85 mg/dl at day 0, 3,1 mg/dl at day +7 and 3,2 mg/dl at day +14. there was no impact between the ua levels and os at any time of the hsct. ua levels at day +7 were associated with a higher ci relapse at 5 years (34% [95% ci, 20-49%] for ua level > 3,1 mg/dl, and 17% [95% ci, 8%-30%] for ua level ≤ 3,1 mg/dl [p= 0,046]). there was a trend for a higher ci of grade ii-iv agvhd for the subgroup of patients not treated with atg with ua < 4,8 mg/dl (48% vs 30%; p= 0,083) on day -8 and a higher nrm with ua < 2,85 on day 0 (50% vs 30%; p=0,080). conclusions: in our study the ua levels showed no impact on os, and only a tendency for ci of grades ii-iv agvhd grades ii-iv and nrm for the subgroup of patients not treated with atg. surprisingly, high levels of ua at day +7 of hsct were associated with a significant higher incidence of relapse. disclosure: dkms foundation, pi14/01971 (instituto carlos iii) and sgr288 (grc), generalitat de catalunya. background: veno-occlusive disease (vod) is an early, uncommon but serious complication of stem cell transplantation (sct) that is associated with high morbidity and mortality. defibrotide is the only licensed treatment for vod, and time to start of treatment (tst) affects outcomes. minor differences exist between the seattle, baltimore and classical ebmt (2016) criteria, which may trigger different start points for treatment. late onset vod (>21 days) is less recognised and we hypothesize, may have worse outcomes with longer time to diagnosis, and more limited treatment options across different healthcare systems. methods: electronic patient records from sept.2013 -oct.2018 at king´s bmt centre and pharmacy databases were reviewed, timepoint to clinical and bio-chemical manifestation of vod, diagnosis, tst, survival and longterm outcomes were analysed. results: 30 of the 532 patients(5.6%) who underwent an allogeneic sct, developed vod, including 2 paediatric cases. none of the autologous sct patients developed vod. the paediatric and autologous sct patients were not analysed any further. 28 adult patients (male=22;78.5%) developed vod at a median age of 56 years(range 26-72), of whom 21 developed < 21 days and 7 patients had late-onset vod as per ebmt criteria(range 22-93 days). 24 cases classed as severe and 4 as moderate vod. 10 patients received defibrotide at diagnosis, 7 patients within 3 days, 5 patients between 4-7 days, and 6 patients received treatment after 7 days. overall mortality for this cohort was 50%(14/28). 12/21 (57.1%) of patients with early onset vod and 2/7(28.5%) patients with late-onset vod died. of the 14 deaths, 10 died of liver failure and a further 2 patients had vod as a likely contributing factor in their deaths. 1 patient died with subarachnoid haemorrhage and 1 with relapsed disease. patients that received defibrotide after 7 days, 5/6 patients(83.3%) died, as compared to 3/5(60%) for treatments between 4-7 days, 6/17(35.2%) for treatments within 3 days. the lone surviving patient who received treatment after 7 days has severe chronic liver disease and it's complications. of the 21 patients who fit seattle criteria for early-onset vod, only 6 fit the baltimore or ebmt criteria for classical vod. 4 of these 6 patients met the baltimore criteria later than the seattle criteria were met(range = 2-9 days). conclusions: vod carries high morbidity and mortality, and beyond the known risk factors and with the caveat of limited numbers in this study, we strongly suspect this is further increased when time to definitive treatment with defibrotide is delayed, particularly beyond 7 days. nearly a quarter of cases with vod are late-onset as per classical ebmt criteria. however contrary to our hypothesis, their overall outcomes and mortality do not appear worse, with time to treatment again emerging as a strong predictive factor. conditioning treatment related factors, which play a stronger role in endothelial dysfunction in the hepato-portal circulation, may not be as much at play, perhaps for late-onset disease. uniformity in the use of diagnostic criteria, and high degree of vigilance, even beyond 21 days, leading to early treatments may improve outcomes in vod. disclosure: nothing to declare background: hsct-associated thrombotic microangiopathy (ta-tma) affects 10-30% of patients receiving an allogenic sct, with a high mortality up to 80-90% in severe cases. endothelial injury mediated by complement activation has been atribuited a major role in the pathogenesis, and blockade of c5 with eculizumab offers promising results. methods: we present our experience with 6 pediatric cases of ta-tma treated with eculizumab. the diagnosis of ta-tma was stablished attending to jodele et al criteria. clinical data were collected retrospectively from medical records. results: all cases were diagnosed between august 2016 and april 2018, with a median age of 11 years (2.5 -17) at time of diagnosis. primary disease was acute leukemia in 2 cases (1 all and 1 aml), severe aplastic anemia in 3, and primary immunodeficiency in 1. they received their first sct in all cases, 3 from mud and 3 from mmrd (cd45ra+ depleted haploidentical grafts), with mac regimen in 2 cases, and ric in 4 cases. 3 of them received calcineurin inhibitors (cyclosporine) as gvhd prophylaxis. all patients developed agvhd (grade 2 or higher in 3 cases). and 5 patients presented viral reactivation. hypertension was present in 4 cases at tma diagnosis, requiring 2 or more antihypertensive drugs in 3 of them. all patients had renal injury consisting of less-than-normal glomerular filtration rate (median of 41 (20-59)) and proteinuria, with urine protein-to-creatinine ratio higher tan 2 mg/mg in 2 cases (data not available in 2 patients). serum haptoglobin was decreased in just 2 cases at diagnosis, and schistocytes were detected in 3 patients. cutaneos signs were present in all cases, digestive symptoms in 2, neurological affection in 2, and notoriously all of them developed polyserositis. c3 and c4 were normal in all cases, with sc5b9 higher than 244 ng/ml in 2 patients and lower in 1 (data not available in 3 cases). all patients received defibrotide as treatment, and 4 cases received also rituximab, associated to therapeutical plasma exchange in 3. all of them received eculizumab, as first line in 2 cases (median of 40 days between diagnosis and eculizumab start). treatment was correctly monitorized with ch50 levels in 3 cases (not available quick enough in other 3). median number of doses needed in induction therapy was 8, and median interval between doses was 7 days. 2 patients required reduced interval and higher doses to maintain ch50 supressed. 2 patients did not respond, and died because of tma. 4 patients had hematological response, with chronic renal injury in 3 of them and resolution of acute renal failure in 1 case. nevertheless 1 patient responding to eculizumab died because of tma related complications, and 1 because of an invasive fungal infection. 2 patients are alive, with a median follow up of 6 months from treatment start. conclusions: our experience supports promising results of eculizumab based treatment for ta-tma, highlighting the importance of an early treatment and a careful therapy monitoring by ch50 supression. prospective studies are needed to achieve a better knowledge of this pathology and its treatment. disclosure: nothing to declare background: approximately 40-50% of allogeneic hematopoietic stem cell transplant (allo-hsct) are made with some sort of abo blood group system incompatibility. an hsct abo donor-recipient incompatibility implies risks of complications during the process of infusion as acute hemolytic anemia (ah), delayed graft and other later complications due to the presence of isohemaglutinins (pure red cell aplasia or passenger lymphocyte syndrome). also, abo incompatibility could impact on graft versus host disease (gvhd) incidence, and could be associated with not relapse mortality (nrm) and overall survival (os). there are not concluded evidence about the abo incompatibility impact, so the aim of this study was to identify complications and response associated with abo incompatibility in patients undergoing allogeneic hematopoietic stem cell transplantation. methods: a retrospective study was performed on patients who receive an allo-hsct between january 2014 and august 2018. two groups were performed according to the presences or not of abo incompatibility. demographic and clinical information was collected from physical and electronic medical records, and information was analyzed in spss v21 results: sixty-eight patients were enrolled in the study, 54% male, the median age was 34 years (19-61) with the following diagnoses: acute lymphoblastic leukemia 44%, acute myeloblastic leukemia 26.5%, granulocytic chronic leukemia 17.6%, myelodysplastic syndrome 4.4%, dendritic cell neoplasm 4.4%, aplastic anemia 2.9%. ninety-one percent of the patients received a transplant from an identical hla donor and 8.8% received a haploidentical transplant. fifty-two patients (76%) were abocompatibility (g1) and 16 patients (24%) had aboincompatibility (g2). none patient with aboincompatibility received a haploidentical transplant. the contrast between groups didn't show differences in fever, infections, bacterial isolation, presence and degree of acute or chronic gvhd and relapse of the disease. graft failure was 6%(g1) vs 20%(g2) (p=0.27), intermediate risk cmv serostatus 79%(g1) vs 87(g2) (p=0.35). the most relevant characteristics and complications are described in table 1. contrast analysis between g1 vs g2 showed that within the whole group there were 29 deaths (40% vs 50% respectively) (p=0.69), the overall survival 1-year was 74% vs 66% (p=0.58) with a median of 29 vs 34 months respectively; mortality associated with relapse was 68% vs 87% respectively, and mortality related with transplantation was 35% vs 13 % respectively. conclusions: abo incompatibility did not show association with complications related with the infusion, but there was a higher tendency of graft failure in the abo incompatibility group. it has no statistical significance, but it is important to expand its study. disclosure: none declared methods: retrospective data for 142 nhl patients who underwent asct between 1999 and 2017 was analysed. patients were identified using the swbmt database and data on mets was collected using paper and electronic hospital records. forty-eight patients were excluded due to loss of follow-up, inaccessible/incomplete records, or death. cause of death was not determined. the ncep-atpiii definition of mets was used. this requires ≥3 of 5 criteria to be met. a bmi of ≥30kg/m 2 and hba1c of ≥42mmol/l were used to replace central obesity and impaired fasting glucose, respectively. other criteria include triglycerides (tgs) ≥1.7mmol/l or treatment, high density lipoprotein cholesterol (hdl-c) < 1.0mmol/l (male), < 1.3mmol/l (female) or treatment and blood pressure >130mmhg systolic or >85mmhg diastolic, or treatment. results: the prevalence of mets in the cohort was 33% (n=31). eighty-two percent of patients (n=77) met one or more criterion for mets. twenty-seven percent (n=25) fulfilled only one criterion, 23% (n=22) fulfilled two criteria, 20% (n=19) three criteria, 12% (n=11) four criteria, and 1% (n=1) five criteria. the greatest prevalence of mets was in the 60+ age group, accounting for 17 out of 31 (55%) patients with mets. overall prevalence decreased with declining age ( table 1 ). the number of patients aged < 20 years was too small to make any judgement on risk. raised triglycerides was the criterion most frequently met (61/94 patients), followed by hypertension (48), raised bmi (26), low hdl-c (23) and an increased hba1c (15). conclusions: the prevalence of mets in our cohort (33%) was higher than the estimated worldwide prevalence of 25%, with the majority in the 60+ age category. this is in keeping with other post-transplant studies, which show an increase in prevalence of mets after transplantation. moreover, the overall prevalence of mets was greater in the older population, which could be associated with the cumulative effect of ageing on the decline of normal metabolic homeostatic mechanisms. background: acute renal failure (arf) is a frequent complication in the early post-allogeneic hematopoietic stem cell transplant (allohsct) period with either myeloablative (ma) or non-myeloablative (nma) conditioning regimens. the aim of this study was to compare the incidence of arf in both types of hsct and to evaluate its impact on overall survival (os) and non-relapse mortality (nrm). methods: all allosct performed in one center between 2010 and 2018 were included in this study. allohsct from cord blood and from haploidentical donors were excluded. the renal function and the incidence of the main complications after allosct from day 0 to day +90 were evaluated. arf was defined according to kdigo (kidney disease improving global outcomes) classification; the relative increase of serum creatinine levels was considered a marker of kidney damage. results: seventy-seven patients received a ma allohsct and 72 a nma allohsct. recipients of nma allohsct had a higher median age (61 years [range: 18-69] vs. 41 years , p< 0.001), higher frequency of arterial hypertension (29% vs. 6%, p< 0.001) and showed most frequently active disease at allosct (42% vs. 18%, p=0.002). in both groups the most frequent graft-versushost disease (gvhd) prophylaxis regimen was cyclosporine a and methotrexate. the median follow-up time was 2.3 years for the nma group and 3.6 years for the ma group. patients from the ma group had higher incidence of grade 3-4 mucositis (60% vs. 22%, p< 0.001) and acute gvhd of any grade (70% vs. 53%, p=0.029) than patients from the nma allohsct. the incidence of arf was similar in both groups (72% in nma and 71% in ma). in the nma group arterial hypertension (hr 2.05, p=0.018), obesity (hr 4.16, p< 0.001) and prior pneumonia (hr 3.19, < 0.001) were predisposing factors for arf by multivariate analysis, whereas any factor was identified in the ma group. arf had no impact on 2-year os in both groups (28% vs. 35% p=0.406 for the nma group and 45% vs. 40% p=0.623 for the ma group). however, worse os were observed in patients with grade 2-3 arf in the nma group (18% vs. 43%, p=0.048) and in patients with grade 3 arf in the ma group (25% vs. 59%, p=0.011). in turn, arf had no influence on nrm in the ma group but was associated with a trend for higher nrm in the nma group (59% vs. 35%, p=0.084). conclusions: arf is a frequent complication in patients receiving allohsct irrespective of the intensity of the conditioning regimen. moderate and severe arf had negative impact on os. disclosure: supported by grants from: asociación española contra el cáncer, aecc (gc16173697biga), instituto carlos iii (pi14/01971 fi), 2017-sgr288 (grc), cerca program from generalitat de catalunya, and "la caixa" foundation. treatment and risk factors of hepatic veno-occlusive disease after pediatric hematopoietic stem cell transplantation: a single-center experience barbaros sahin karagün 1 , ilgen sasmaz 2 , ali bülent antmen 1 background: defibrotide emerged as a promising treatment option for hepatic veno-occlusive disease, a significant cause of mortality in recipients of hsct. as vod diagnosis is quite difficult even with the recently introduced ebmt 2017 criteria, studies which report treatment outcomes and response to prophylaxis are required. our aim was to evaluate the efficacy of defibrotide prophylaxis in hsct recipients at our center. methods: a total of 236 transplants in 210 patients from january 2013 to july 2018 were included in this study. all patients had factors that increased the risk of vod and all received 25 mg/kg/day prophylaxis. patients' coagulation, renal and liver function test were monitored daily and all clinical findings and complaints were recorded. diagnoses were made via the ebmt 2017 vod criteria and patients who developed vod received treatment with increased df dose (40 mg/kg/day) and supportive interventions. after complete remission of vod findings, patients were returned to the prophylaxis dose. close follow-up of patients was performed until 100 days. results: in total, 17 patients developed vod (7.2%), none of the cases were severe (13 mild, 4 moderate). median age was 8.5 years and the most common clinical findings were weight increase, hepatomegaly, right upper quadrant pain and ascites development. in those with vod, treatment with 40 mg/kg/day df was initiated and average duration of treatment with this dosage was 7.4 (5-11) days. no adverse events were reported in any of the patients. conclusions: our findings are consistent with previous studies on this topic, and we believe that the use of df as a prophylactic agent for vod is beneficial for pediatric patients with risk factors. disclosure: the authors report no conflicts of interest in this work. background: several factors might influence outcome of allo-hsct. analysis of the impact of donor-receptor blood group-incompatibility have been performed in different series not always showing the same results. as a consequence, its clinical impact remains controversial. minormismatch is characterized by the ability of donor b lymphocytes to produce anti-recipient antibodies. in majormismatch cases, antibodies against donor antigens are present in the recipient. methods: 343 pts underwent allo-hsct between may 2011 and august 2018 in our center. median age was 52 years (range: 7-69). 193 pts were male (56.3%) and 150 female (43.7%). baseline diseases were: 138 aml, 76 lpd, 44 mds, 41 all, 21 mpd, 16 mm, and 7 bmf. donor was unrelated in 191, and related in 152 cases (including 36 haplo-identical). donor-recipient abo compatibility was as follows: 69 (20.1%) majormismatched (including 11 bidirectional), and 274 (79.9%) nonmajor-mismatched (including 68 minormismatched and 206 matched). donor-recipient rh compatibility was as follows: 50 (15.6%) major-mismatched, and 293 (84.4%) nonmajor-mismatched (including 34 minor-mismatched and 259 matched). the impact of donor-recipient abo and rh compatibility on transfusion needs (prbc and platelet concentrates) and survival by day +100 was analyzed. results: for the global series the median number transfusions by day +100 was: 4 (0-81) prbc and 4 (0-92) platelets concentrates. day +100 overall mortality was 9.3%. rh-incompatible and nonmajor abo incompatible cases showed no different results. however, major abomismatched cases needed more prbc transfusions (median: 6; range: 0-49) and more platelet transfusions (median: 7; range: 0-60), and had higher day +100 mortality (18.8%) (p < 0.05) (see table) . conclusions: our analysis showed: 1) donor-recipient rh-incompatibility, as well as minor aboincompatibility had no impact on prbc and platelet concentrates transfusion needs nor on 100-day mortality; 2) contrarily, donor-recipient major abo-incompatibility had a significant adverse impact on prbc and platelet concentrates transfusion needs and 100-day mortality. 3) donor-recipient rh-incompatibility and minor aboincompatibility.might be considered of marginal importance at the time to choose a potential donor. 4) donorrecipient major abo-incompatibility should probably be a factor to be considered, along with other features, to choose the best donor background: survivors of haematopoietic stem cell transplantation (hsct) are at significant risk of developing treatment-related complications, including cardiovascular risk factors such as arterial hypertension, that could eventually lead to cardiovascular disease. the aim of this study is to evaluate the incidence and risk factors of hypertension following hsct in a colombian population. methods: a retrospective cohort study was conducted to assess the incidence and risk factors of hypertension in 220 consecutive adult hsct recipients who underwent transplantation between 2009 and 2017 at a tertiary referral center in colombia, south america. blood pressure data, from two different measures, were collected at 7 time points: day of mobilization for autologous hsct and day 0 before infusion for allogeneic transplantation, day 7, and months 1, 3, 6 and 12 post-transplantation. hypertension was defined as having a systolic blood pressure >=140mmhg and/or a diastolic blood pressure >=90 mmhg. patients with history of arterial hypertension were excluded. results: one hundred and seventy-five patients were included, with a mean age of 44 years (range 15-67). ninety-one patients (52%) were male. one hundred and sixteen patients (66.3%) underwent autologous hsct and 59 (33.7%) allogeneic hsct. the most common indication for hsct was acute leukemia (26.3%), followed by non-hodgkin lymphoma (23.4%) and multiple myeloma (22.9%). twelve patients (6.9%) had medical history of type 2 diabetes mellitus (dm), 11 (6.3%) dyslipidemia, 24 (13.7%) alcohol consumption, and 25 (14.3%) tobacco smoking. only two of the patients with history of tobacco smoking were active smokers at time of transplantation. twenty-four patients (13.7%) had developed hypertension by the end of the first year post-hsct follow-up. two patients (8.3%) had systolic and diastolic, 12 (50%) had only systolic, and 10 (41.7%) had only diastolic hypertension. only one patient was hypertensive at more than two time points. the incidences of hypertension at each time point were 2.3% on day 7 post-hsct, 6.9% at first month, 9.8% at three months, 12.1% at 6 months, and 13.8% at one-year post-transplantation. allogeneic hsct (p< 0.01), therapy with calcineurin inhibitors (p< 0.01), pre-hsct fasting glucose levels (p< 0.05), acute gvhd (p< 0.05), chronic gvhd (p< 0.05), and media of diastolic blood pressure (p< 0.05) were significantly associated with the development of arterial hypertension. however, age, history of type 2 dm, history of tobacco consumption, volume of infusion, prophylactic treatment for gvhd with mycophenolate, chronic gvhd, serum creatinine level on day of hsct, and being overweight or obese at time of transplantation were not significantly associated with the development of hypertension. conclusions: arterial hypertension is a fairly common complication in hsct recipients. similar to findings reported in previous studies, association between allogeneic stem cell transplantation, therapy with calcineurin inhibitors, and acute and chronic gvhd, and post-hsct hypertension was found in the present cohort. further studies are needed to assess the link between hsct and developing long-term cardiovascular complications. disclosure: nothing to declare tramadol-based pain management of oral and esophageal mucositis in pediatric hsct recipients background: mucositis is one of the most common early hsct complications seen in about 70% transplant recipients with 20% of patients developing gr iii-iv mucositis. mucositis is characterized by painful gastrointestinal mucosa lesions impairing the solid and liquid foods intake and increased risk of infections, bleeding, and intestinal paresis. thus, it greatly decreases the quality of life of a transplant recipient. according to who recommendations, the moderate pain control in pediatric patient is based on the use of low-dose morphine. however, there are some factors such as genetic polymorphisms causing variable morphine pharmacokinetics in children, side effects, and social factors (caregivers' general unwillingness to use narcotic analgesics), which cause the need for alternative pain relief options in pediatric practice. tramadol, which has both opioid and non-opioid mechanisms of action, may be a feasible option in mild to moderate pain. it may be delivered via patient-controlled analgesia (pca), although there is no consensus on its optimal parameters in pediatric practice. methods: a total of 69 pediatric patients with a median age of 8 (range 2 to 18) years receiving an autologous or allogeneic hsct in our clinic as part of the treatment regimen for solid tumor (n=40), leukemia (n=24), acquired aplastic anemia (n=3) or inherited condition (n=2) were included. conditioning regimens were myeloablative (mac) in 54 and reduced-intensity (ric) in 15 patients. all patients had oral and/or esophageal mucositis accompanied by moderate pain. the pain severity was assessed using the scales corresponding to patient's age and varied from 3 to 6 points. the pain control was based on intravenous tramadol administration using patientcontrolled analgesia (pca) approach. the following pca parameters were used: loading dose of 0.5 mg / kg (not exceeding 25 mg), basal infusion rate of 0.25 mg / kg (not exceeding 12.5 mg), a bolus of 0.25 mg / kg (not exceeding 12.5 mg), lockout interval of 25 min. the maximal daily dose was 8 mg/kg/day. the pain control was considered adequate if a patient was satisfied or the basic and breakthrough pain score values were not higher than 3 and, accordingly. in case of inadequate pain control nsaids were added. non-responders were switched to morphine. all patients were divided into 2 groups based on conditioning regimen intensity. results: as a whole, 46% of patients did not require pain control measures escalation. the tramadol pain control rate was slightly higher for ric (n=9, 60%) compared to mac (n=23, 47%) recipients. in most cases the inadequate pain control was due to progressive mucosal lesions. the pca regimen used was characterized by very few complications. drowsiness was observed in 4 (7%) of patients, in all cases the patients also had anemia. there was only 1 (7%) patients with severe nausea requiring switching to morphine. conclusions: tramadol is an effective pain control option in transplant recipients with mild to moderate pain due to oral and esophageal mucositis without progressive mucosal lesions. the pca allows achieving a very low complication rate. therefore, this option may be considered for both mac and ric recipients. disclosure: no immune reconstitution of lymphocyte subsets after allogenic stem cell transplant (sct) and vaccination background: infectious diseases are a major cause of morbidity and mortality after allogenic stem cell transplant (sct). vaccines constitute an effective strategy to prevent infections but the optimal timing to start vaccinating is not well stablished. in order to individualize the early vaccination schedule, we studied the lymphocyte subsets involved in generating enough response to produce protective serological levels. methods: we studied retrospectively 20 patients that had undergone allogenic sct at our hospital. patient distribution -age range: 21-68 years-old; diagnosis: acute leukaemia/myelodysplastic syndrome/ chronic myeloid leukemia (16 patients), lymphoma (4 patients). analytic parameters: tcd4+, tcd8+, nk, total b and functional b lymphocyte subsets (naïve igd+cd27-, memory igd+cd27+ and igd-cd27+, and effectors cd27++cd38++). immunoglobulin levels (igg, iga, igm) and specific igg for pneumococcus, tetanus, hbv, chickenpox, measles, rubella and mumps. clinical parameters were collected from medical records. results: we distributed patients in two groups, based on the timing of lymphocyte analyses: -less than 12 months since sct (5 patients) no patient showed complete immune reconstitution, although 2 had enough t and functional b lymphocytes to generate response to vaccination. in these patients, vaccination for pneumococcus was completed and they generated sufficient protection antibody levels, despite being under immunosuppressive treatment. -more than 12 months since sct (15 patients) before the beginning of vaccination, we collected specific antibodies of 7 patients. we compared the serological status before and after sct and observed that protection against tetanus was the most frequently preserved (6 patients) and hbv the least frequent (2 patients). other than one patient treated with alemtuzumab, all patients in this group had minimum absolute count of tcd4 + (>200 cells/microl), tcd8+ (>200 cells/microl), nk (>100 cells/microl) and b cells (>100 cells/microl). we also observed presence of b effector and b memory cells, with predominance of igd-cd27+ memory cells. immunoglobulin levels were within the normal range. in this group, we registered vaccination in 13 patients. all of them were vaccinated against flu, and 11 against pneumococcus and hbv. the rest of vaccines administered were heterogeneous in type and timing. 6 patients were under immunosuppressive treatment at the time of vaccination and were able to generate enough specific antibodies for pneumococcus. conclusions: immune reconstitution was not completed 12 months after sct, although minimal immunological reconstitution was observed tcd4+ and no-switching memory b lymphocytes were the last ones to reach minimum normal values according to patient age. some patients maintain serological protection after allogenic sct. immunoglobulin levels were normal, suggesting no need for immunoglobulin administration to prevent infections. flu, pneumococcus and hbv vaccines were the most frequently administered. pneumococcus vaccination generated a much larger serological response than hbv. this seroconversion occurred in patients under immunosuppressive treatment. the analysis of lymphocyte t, nk, b total and b functional subsets could be useful when programming an early vaccination schedule after sct. completion of the vaccination schedule was heterogeneous despite giving specific indications. therefore a more rigorous supervision of the process may be required. background: the significant advances that have been achieved in the allogeneic transplantation (allohct) field, have resulted in better post-transplant outcome and therefore complications other than the graft vs. host disease (gvhd) or disease recurrence become increasingly important. the post transplant metabolic syndrome (pt-ms), which caused by several factors (i.e. immunosuppressive agents, chemo-radiotherapy, anti-viral, and biologic therapies) is a well known post transplant complication in pediatric allografted long-term survivors however, only few studies have evaluated the prevalence of the pt-ms in adults. in this retrospective study, we sought to evaluate the incidence, the risk factors and the impact of the pt-ms on the allosct outcome. methods: since 2011, 42 patients (25 males and 17 females) with adequate clinical and laboratory data and a minimum follow-up of 6 months were included in the study. their median age was 35.5 (17-62) years and after a myeloablative (n=28) or a reduced intensity (n=14) regimen they received either mobilized peripheral blood stem cells (n=34) or marrow graft (n=8), originated from full-matched siblings (n=35) or haploidentical donors (n=7). calcineurin inhibitors plus either short-term methotrexate or mycophenolate mofetil were given as gvhd prophylaxis. the diagnosis of pt-ms was based on the ncep-atpiii criteria; for patients with unknown data for abdominal circumference the body mass index (bmi) ≥25kg/m 2 was consider as a criterion for pt-ms diagnosis. the independent t-test, logistic regression analysis and logrank tests were used for the statistical analysis. results: twenty (47.6%) patients (12 males, 8 females) assessed to have pt-ms within the first 6 months following the allograft. seventeen diagnosed after the 1 st trimester post allosct and additional 3 patients after 2 nd trimester. sixteen out of 20 patients had elevated glucose and bmi>25kg/m 2 , 13/20 elevated triglycerides levels, 12/20 low hdl levels and 10/20 hypertension. four (20%) had already known history of ms before allosct (for 10 patients no data were available for ms diagnosis before allosct). interestingly, for 7/20(35%) patients who had diagnosed with pt-ms either in the 1 st or in the 2 nd trimester the syndrome was reversible and did not fulfill the criteria for pt-ms beyond 6 months post allosct. patients' gender, age, bmi, the type of conditioning regimen and gvhd co-existence evaluated as potential predisposing factors for pt-ms diagnosis. in univariate and multivariate analysis only the: bmi>25kg/m 2 and age>35 years were detected as significant risk factors (p< 0.03). the pt-ms did not affected negatively the survival or the nrm incidence post allosct conclusions: in our study, in agreement with other publications, we demonstrated that the pt-ms is not an uncommon complication post in the early post transplant period however, for a significant number of patients the syndrome was a reversible. for patients with high risk features (bmi>25kg/m 2 , age> 35 years, known history of diabetes-mellitus, dyslipidemia, hypertension) apart of close monitoring, specific diet and encouragement for adequate exercise might help to reduce the incidence and the severity of pt-ms. nevertheless, prospective and well design trials are warranted to determine the accurate incidence, severity and the impact of pt-ms on the allosct outcome. disclosure: no conflict ofinterest experience of a single center in the humanization of the hospitalization process: technology and team training impact on the qol of the patient and family maria claudia moreira 1 , marcia rejane 1 , marcia garnica 1 , andrea ribeiro 1 , paulo cesar dias 1 , ilza fellows 1 background: hematopoietic stem cell transplantation (hsct) is one of the most aggressive therapeutic modalities of internal medicine, making it a highly stressful experience for the patient and his family. the duration of hospitalization can be prolonged by several intercurrences, frequently generating anxiety in the patient and their caregiver, which may lead to confinement and reactive depression. interventions in the hospital environment, in addition to the continuous training of the multidisciplinary team, can have a positive impact in this process with improvement in the process of discharge and quality of life of the patient and his / her family. methods: the objective of this research was to evaluate the impact of a reformulation in the unit, completed in may 2018, which modified the facilities with availability of hermetic balconies in each room, with a view of an internal garden. there was also the addition of a screen in the corridor of the floor with images -technology known as videoowall, interconnected to motion sensors (kinects) that allow interaction between patients and families, besides facilitating physiotherapy and physical exercise. there was re-training of the multidisciplinary team with emphasis on the practice of humanization. the methodology consisted in the application of questionnaires of satisfaction to patients and their families during the period of hospitalization in a bone marrow transplant unit in the third quarter of 2018. the items evaluated ranged from the quality of the information provided by the medical team and nursing, to the cordiality and agility with which the patient and his patient were treated by the global team. the results were compared with a similar period of the same unit in the previous year and with the indices collected simultaneously in another unit of the same hospital (cardio-intensive). results: overall and segmental satisfaction scores in the various items surveyed were higher when compared to the previous period of the same unit and were also higher in those obtained in a high complexity unit of the same hospital, composed of patients submitted to mental and psychological stress similar to onco-hematologicos.a reports of "free speech" were also obtained anonymously, in order to guarantee the authenticity and free expression of the subjects analyzed. conclusions: the results obtained allowed the validation of the technical and professional team initiatives, bringing indicators that will allow better monitoring and support of these patients and their relatives in this difficult time of treatment. they served as an initial tool in the continuous process of humanization and stimulated the multidisciplinary team to continuously improve this process. disclosure background: pure red cell anemia (prca) is a rare complication of abo-incopatible hematopoetic stem cell transplantation characterized by anemia, reticulocytopenia and absence of erythroid precursors in patient's bone marrow. most patients with prca resolve spontaneously within months, however a small number of patients requires continued red blood cell (rbc) transfusions. the treatment of this complication is difficult and not standardized. different approaches has been used such as rituximab, donor lymphocytes, plasma exchange with different outcome. recently, a remarkable response to treatment with bortezomib has been described in a case of prca. methods: we reviewed 146 patients who received an allogeneic hematopoetic stem cell transplant (hct) between januar 2012 and august 2018 at our institution. sixty eight patients received a major abo-mismached hct. prca was defined as a completely absence of erythroid precursors on day +30 bone marrow puncture, with absence of donor red cells and the recipient requiring rbc transfusion. results: only one patient developed prca (1.5%). a 18 years old male received a myeloablative hla-matched abomismatched sibling donor transplant (brother, 12 years) for acute myeloid leukemia (aml), with t(8;21) cr1,mrd positive (runx1-runx1t1). the donor was blood type a rh positive and the patient 0 rh positive. the patient had no complication after transplant. the day +30 bone marrow puncture has shown only few erythroid precursors and day +100 puncture and biopsy no erythroid precursors, he had transfusion dependent anemia requiring a rbc transfusion every two weeks and retukulocytopenia. parvo virus and cytomegalovirus were negative. due to very high ferritin level (>4.000u/l) and increased luiver enzymes without signs of gvhd, the treatment with deferasirox has been started. the patient has achived cr1, mrd negative, and has evidence of complete chimerism. high titers of anti-a and anti-b issohemagglutinin was present.we started the treatment with rituximab 375mg/m2 weekly, 4 weeks, however without response. the pathogenesis of the prca is thought to be due to the recipients plasma cells, bortezomib, a proteasome inhibitor inducing apoptosis of plasma cells has been given s. c. 1,3mg/m2 two times weekly, for two weeks. the patient responded to the treatment two weeks later with increase in hb, which was 12,9 g/dl and increase in retikulocyte number. the patient has continued to be well at the last control. conclusions: prca aplasia is a rare but serious complication after abo-incompatible hct. bortezomib is an effective treatment for this complication if mediated by residual host isohemeagglutinins after hct and should be recommended as standard of care. clinical methods: this work is retrospective, observational, cross-sectional and analytical. it included all patients who received hsct at stem cell transplantation unit (utmo, by its spanish acronym) at solca-guayaquil, between the years 2009 -2016.we use the kaplan-meier method to analyze the survival rate between the autologous and allogeneic transplant. the information collected for this study was obtained from the database of the solcay institute and the review of the files of the patients included. results: at least, 150 patients have been undergoing to hsct between 2009-2016 years. according to the type of hsct, 42.1% received an autologous transplant and 57.9% received an allogeneic transplant, from which 79.3% were from a related donor. the main source of transplant was peripheral blood in 86.67%, followed by 12% obtained from umbilical cord blood and 1.33% by bone marrow aspiration. the most frequently reported pathologies were acute lymphoblastic leukemia (all) (34%), multiple myeloma (mm) (22%) and acute myeloid leukemia (aml) (13.33%). the overall survival was 68% (ic: 95%). the 82.53% of patients that were undergoing to autologous transplant have survive, meanwhile the patients that were undergoing allogeneic transplant only the 57.47% have survived (p< 0.05). the highest death rate occurred during the first year after hsct, and decreased considerably after that period. the main cause of mortality related to transplant (mrt) was the graft-versus-host disease (gvhd) (8%); however, the main cause of mortality in the study population (n=150) was relapse in 12.66% of the patients, presented more frequently in all. conclusions: the results showed that 68% of patients undergoing to hsct have survived. a high rate of deceased patients in this study, have died in the first year before the transplant (26.6 %%), due to relapse. the main cause of deceased in the study is not related to hsct, and was the relapse in 12% of patients, in compare the gvhd was the main cause of mrt (8%). we consider that hsct is a technique that is still under development in ecuador, but despite the short time it has been taking and the institutional and medical limitations present in the health field, has presented excellent results comparable to studies conducted in developed countries. [ background: pigmented epithelioid melanocytoma (pem, early known as 'аnimal type' melanoma) is a rare tumor with unpredictable clinical behavior and metastatic potential. pem generally has favorable prognosis. involvement of regional lymph nodes is not rare. extranodal and distant nodal metastases are extremely rare. we report about patient with fanconi anemia (fa) and pem with developed distant metastases in the early term after allogeneic hematopoietic stem cell transplantation (hsct). methods: 10-years old boy with fa was hospitalized for hsct. the blue-black painless nodulus 15х15 mm was noted on the left cheek. this lesion was observed from early childhood and during life only slightly increased in size. there were no distant and regional metastases on computerized tomography (ct) and scintigraphy with 99m tc. the nodulus and regional lymph nodes were radically removed before hsct. the resection margin was within the normal tissue. microscopically the derma and subcutaneous fat were infiltrated with epithelioid and spindle cells with total expression of s100, melana, mhb45, cyclind1. ki-67 expression level was 15-20%. histological structure was specific for pem. hsct with tcrαβ+/cd19+ graft depletion from match unrelated donor was performed. the conditioning regimen included total lymphoid irradiation 2gy, fludarabin 150 mg/ m 2 , cyclophosphamide 40 mg/kg, rabbit atg 5 mg/kg and rituximab 100 mg/m 2 . results: at +45 day after hsct was detected the tumor on the left cheek and parotid region with a histological structure identical to the primary lesion. on ct in s6 segment of the left lung was detected focus 20x20 mm with a cavity. invasive aspergillosis was suspected and empirical antifungal treatment was started. but in 15 days the lung lesion increased in size to 52x30x32 mm and penetrated in the bronchus. after bronchoscopy with biopsy, pem metastasis was histologically confirmed. moreover, the tumor on the face continued to grow. therapy with cobimetinib and vemurafenib was not effective and patient died from progression of pem on +98 day after hsct. conclusions: pem was early described as indolent tumor with rare distant metastasis and favorable prognosis. we suspect that pem may acquire an aggressive course in the absence of immunological control, especially in high immunocompromised patients after hsct. disclosure: nothing to declare p208 abstract withdrawn lidia gartcheva 1 , antoaneta mihova 1 , penka ganeva 1 , margarita guenova 1 , branimir spassov 1 background: the main objective of the study is to assess the dynamics of quantitative and qualitative changes in the parameters of the b cell population and the production of immunoglobulins in patients after autologous transplantation of hematopoietic stem cells in the course of recovery of the immune system. methods: 56 patients with hematological neoplasms undergoing autologous transplantation were included in the study: 30 women and 26 men, with an average age of 31 years. patients were diagnosed with lymphoma (n = 30), multiple myeloma (n=7), leukemia (n = 7) and solid tumors (n = 12). at the time of transplantation, 16 patients were in complete clinical remission or at least with very good partial response, 30 patients were in partial remission and 10 patients -with progression. all patients were evaluated in nine time points through 356 examinations by clinical-laboratory, flow cytometric and immunochemical methods. results: the percentage of cd19 (+) b cells reached the minimum values one month after transplantation then began to increase in the second month reaching a plateau around the mean values in the period 6-12 months after transplantation. the absolute number remained low during the entire period of observation. the amounts of igg and igm serum immunoglobulins gradually increased within the reference range throughout the entire period, while the iga level varied around the lower reference range. conclusions: implementation of an adequate humoral immune response is hampered by the reduction of circulating b cells, suppressed proliferative potential and functional deficits. restoration of b-cell function occurs over a period of 6 months to 2 years after autologous transplantation. clinical trial registry: no clinical trials disclosure: nothing to declare justyna background: allogeneic hematopoietic stem cells transplantation (allo-hsct) is a life-saving and well established therapy for wide range of diseases. however, it is still uncommon treatment for infants less than 12 months of age. the data about indications and outcome of allo-hsct in the youngest group of patients is sparse. the primary objective of this study was to assess the incidence, indications, post-hsct complications and general outcome of allo-hsct among infants not older than 12 months. latter sequelae of hsct such as physical and cognitive development were secondary aim of this study. methods: we retrospectively analyzed data of 63 patients who underwent allo-hsct before 1 year of age in department of pediatric hematology, oncology and bone marrow transplantation in wrocław during years 1999-2017. clinical and epidemiological features as well as indications for transplantation, early and late complications and general outcome were assessed. results: infants who underwent hsct in our department comprise 8.2% of all patients undergoing hsct in analyzed period of time. thirty-one (49.2%) patients received stem cells from matched unrelated donor (mud), 26 (41.3%) from mismatched (haploidentical) related donor (mmrd) and 6 (9.5%) from a sibling donor (msd). non-malignant disorders were indication for transplant in 49 (77.8%) patients and malignant diseases in 14 (22.2%) . acute graft versus host disease (agvhd) occurred in 33 (52%) infants, chronic graft versus host disease (cgvhd) in 15 (24%). majority of graft rejections were seen in infants transplanted from mmrd 7(63.6%), whereas the rest 4 (36.4%) was associated with mud. median follow-up in study cohort was 860 days, 1148 days for alive patients (range 72 days-19.5 yrs) and 72 days for those deceased (range 3 days-341 days). overall survival (os) in study cohort was 0.682 and transplant related mortality (trm) was 0.317. in children with malignancy 5 (35.2%) patients died comparing to 15 (30.6%) patients in non-malignant group respectively. main cause of death in analyzed group of infants was infection (60%). conclusions: 1. allo-hsct is rarely performed in children less than 12 months of age. 2. majority of those patients receive stem cells due to non-malignant disorder. 3. among youngest hsct recipients, haploidentical transplant are more common than in general pediatric transplant population. 4. graft rejection is a significant problem in infants transplanted from mmrd. disclosure: nothing to declare unusual non-infectious lung complication after allogeneic haematopoietic stem cell transplantation claudia lucia sossa melo 1,2 , manuel rosales 2 , francisco fernando naranjo junoy 1,2 , sara inés jiménez 1,2 , luis antonio salazar 1,2 , angela maría peña 1,2 , maría angélica chacón manosalva 3 , maria luna-gonzález 1 , claudia marcela chalela 1 , manuel ardila-báez 1 jirovecii infections, viral infections or nocardia. we describe the case of a patient with acute lymphoblastic leukemia (all) diagnosis with pap associated to a hsct and pulmonary pneumocystis. methods: a 55-year-old colombian female patient diagnosed with b-precursor all of high-risk in january 2017, positive philadelphia chromosome, positive bcr / abl in february 2017, infiltration to the central nervous system (cns), 2.53% of lymphoblasts, and karyotype without legible metaphases. refractory to induction according to the pethema protocol (vincristine, daunorubicin, prednisone, l-asparaginase) with presence of 13.9% blasts at the end of the induction. re-induction was performed with the flag-ida protocol (idarubicin, fludarabine, cytarabine) achieving complete remission, obtaining minimum residual disease (mrd) < 0.01. dasatinib was initiated by bcr / abl expression and cns involvement at the time of diagnosis. an allogeneic hsct was performed, from a male brother donor, with low intensity conditioning tt buflu and prophylaxis of graft-versus-host disease (gvhd) with tacrolimus and sirolimus. patient showed early posttransplant complications, given the reactivation of cytomegalovirus and hemorrhagic cystitis grade i due to adenovirus. late complications such us gvhd at the cutaneous level and subsequent hepatic and gastrointestinal involvement were seen too, for which immunosuppressive therapy was administered with high doses of systemic corticosteroid. results: patient was hospitalized on day +270 posttransplantation due to febrile neutropenia and respiratory symptoms, with normal chest ct, and ct of paranasal sinuses with acute pansinusitis, for which she received meropenem 1gr intravenously every 8 hours plus vancomycin 1gr intravenously every 12 hours during 14 days with symptom resolution. she remained hospitalized for cytopenias with normal bone marrow and 100% chimerism. on day +291 posttransplant she presented fever and leukocytosis, with acute respiratory failure with chest ct that showed bilateral alveolar occupation, "crazy-paving" pattern and frosted glass (see image), so diagnostic fibro-bronchoscopy was performed, reporting postoperatively for pneumocystis jirovecii. she received 21 days of trimethoprim-sulfamethoxazole, with a torpid evolution requiring mechanical ventilation and tracheostomy, persisting with hypoxemia. the report of cultures for fungi, mycobacteria, and respiratory panel of filmarray were negative. a pathology report was obtained with 30% neutrophils, as well as pas staining with acellular pink material and elevated serum ldh, with a diagnosis of secondary pap. the patient continued with poor general condition, refractory hypoxemia, high ventilatory parameters and hemodynamic instability, due to which she was not able to be a candidate for treatment with total pulmonary lavage; leading to multi-organ failure and later death. [[p211 image] 1. high resolution chest ct. sample opacification in frosted glass (a) and pattern ''crazypaving'' (b)] conclusions: the importance of considering the diagnosis of pap as a noninfectious pulmonary complication in patients with allogenic hsct despite its low incidence is recognized. disclosure: nothing to declare methods: once the project was approved by the clinical trials and ethics committee, 154 pairs of blood samples were drawn (154 from picc line and 154 from venepuncture) from 33 voluntary allo-hsct recipients who were receiving continuous infusion tacrolimus from february through august 2018. the pts had inserted a double-lumen polyurethane picc. tacrolimus was always administered through the red line, and the blood draw always performed through the purple line. all of the patients signed the informed consent. 22 were male and 11 women. median age was 55 years (30-68). 24 of the venepunctures were carried out in the arm where the picc was set, and the other 130 from the contralateral arm. a limited group of nurses performed the extractions of the samples. results: as shown in the table, tacrolimus trough levels determined in blood from venepuncture were similar to those in blood drawn through the picc (median: 10.7 vs 11.1 ng/ml). when comparing one by one in the individual patients, the differences were not significant, and changed the dosing prescription in no cases. conclusions: in our experience, there are not significant differences in tacrolimus levels draw from the picc line, compared with a peripheral site. so, in our opinion, if the line for tacrolimus infusion is properly identified and the one used for the sample draw is the alternative one, venepunctures to obtain sample from peripheral sites are not justified for tacrolimus levels measurements. background: patients undergoing a hsct may require icu admission due to transplant-related toxicities. the aim of this study was to analyse a single centre experience with hsct patients requiring icu admission and the factors affecting outcome. methods: we included all adult patients (age >=18) who had an allogeneic or autologous hsct during 2017 (d0 between 1-1-2017 to 31-12-2017) at st. george's hospital. data was retrospectively collected from patients' notes. icu outcome and 100-day survival were analysed. for those patients who were admitted to icu more than once, outcome was analysed from their last icu admission. results: 20 allograft patients were included. 14 were male, with a median age 47 years (range 23-68 years). 6 were female, with median age 61 years (range 51-67 years). diagnosis n (%) includes all 3 (15%), aml 7 (35%), acml 1 (5%), cmml 3 (15%), hl 1 (5%), mds 2 (10%), mds/mpn 1 (5%), fl 1 (5%), scd 1 (5%). sixteen (80%) patients received their first transplant, 4 (20%) received second transplant. eight (40%) patients had sibling donor, 12 patients (60%) had unrelated donor. sixteen (80%) patients had 10/10 matched donor, 2 (10%) patients had 9/ 10 matched donor, 2 (10%) patients had 8/10 matched donor. nineteen (95%) received reduced intensity conditioning (ric), one (5%) received myeloablative (ma) conditioning. majority of ric allo-hsct patients were conditioned with fludarabine, mephalan, campath (fmc). a small number were conditioned with busulfan, fludarabine and atg. the ma allo-hsct patient was conditioned with tbi, cyclophosphamide. gvhd prophylaxis was ciclosporin alone starting on day -1 with a target level of 150-250 ug/l for all ric and ciclosporin and methotrexate for the ma patients. two (10%) allograft patients were admitted to icu on three occasions. both patients were male, 58 and 68 years old. one had mmud allograft for mds/mpn. the other had 2 nd mud allograft for relapsed aml. the reasons for icu admission include sepsis, cardiac arrest and respiratory failure. the median duration of icu admission was 5 days (range 2-9). there were 2 deaths within 100 days of transplant. one patient died on day +11 during his second icu admission with multi organ failure (mof). one patient died after icu discharge on day +23 with relapsed disease, bronchopneumonia with disseminated fungal infection. icu mortality rate was 50%, and 100-day mortality rate was 10%. nineteen autologous patients were included (median age 61 (range 36-71 years)), 16 (84%) were myeloma patients who were conditioned with melphalan, 3 (16%) were lymphoma patients who were conditioned with beam. the icu admission was 0%. the 100-day mortality rate was 0%. conclusions: our centre's icu admission rate, icu mortality rate, cause of icu admission in allo-hsct patients and autologous patients is comparable to literature reports. autologous transplant is safe with no deaths and icu admissions despite an older age. the mortality rate for allo-hsct patients requiring icu admission remain high. all patients were appropriately referred to icu and there was no one who was denied icu admission. this analysis is being extended to preceding years. disclosure: nothing to declare liposomal doxorubicin for the treatment of iatrogenic kaposi sarcoma following hematopoietic stem cell transplantation background: iatrogenic kaposi's sarcoma (iks) represent a rare complication after hematopoietic stem cell transplantation (hsct), related to hhv-8 infection in hivnegative immunocompromised patients (pts). methods: we describe a case of iks occurred after an allogeneic hsct and we provide a review of the literature using pub med. results: a 70-year-old man, hiv-negative, received full hla-matched related hsct after a reduced intensity conditioning regimen for relapsed aml. gvhd prophylaxis was based on atg (fresenius 30 mg/kg), cyclosporine (cya) and methotrexate. no severe complication occurred in the first 100 days after transplant. shortly after cya withdrawal, he developed grade i acute gvhd. gvhd resolved after restarting cya. at fifth month after transplant, the patient developed several red and purple angiomatous plaque and nodules involving the skin of both lower limbs, right arm and the nose (figure 1). skin biopsy revealed multiple localizations of iks and positive hhv-8 viremia was detected in the peripheral blood. a visceral involvement was excluded. patient was treated with cya tapering and nine courses of liposomal doxorubicin 20 mg/m2 every 15 days, obtaining a negativity of hhv-8 viremia and partial response of the skin lesions. at last follow up, at 15 months after transplant, the patient was in complete remission (cr) for aml, cya-free without signs of gvhd recurrence and with his single stable residual iks lesion on his left limb, currently waiting for local radiotherapy. we found additional 18 iks published cases after hsct. most of post-hsct iks were secondary to an allogeneic-hsct (16 out of 19, 84.2%) and occurred in adult (78,9%) and male (68,4%) pts. median age at the time of iks diagnosis was 47.5 years (range 7-70). thirteen pts (68.4%) had mediterranean origin. the most frequent underlying disease was aml (47.4%). gvhd prophylaxis was primary based on calcineurin inhibitor. half of the pts developed gvhd and were treated with steroid and other immune suppressive drugs. median time between the hsct and the occurrence of iks was 8.5 months (range . cutaneous iks was the prevalent form of manifestation, however visceral involvement was reported in 7 pts (36.8%). in four cases (21.1%) an hhv-8 associated bm failure was report. immune suppression drugs tapering (36.8%) and chemotherapy (26.3%) were the most frequent actions taken after the diagnosis of iks. in most cases, liposomal doxorubicin was used as chemotherapy. cr rate was high, 63.2%, whereas progression disease occurred in 5 out 19 pts (26.3%), all of which had visceral involvement. in 3 pts (15.8%), iks was the cause of death. conclusions: withdrawn of immune suppression drugs and anthracycline based chemotherapy can represent a feasible treatment option for pts with iks after hsct. clinical background: acquired haemophilia a (aha) is an autoimmune disease caused by the spontaneous production of neutralizing immunoglobulin g (igg) autoantibodies (inhibitors) targeting endogenous fviii. treatment of these inhibitors presents additional challenges in a hematopoietic stem cell transplantation (hsct) recipient, because preservation of the graft that restores a normal hematopoiesis is critical. here we describe the management of a case of aha in an acute myeloid leukemia patient following hsct. methods: the clinical, laboratory and molecular aspects of a 56-year-old italian male who developed aha after allogenic bone marrow transplantation were collected and presented in order to show how we diagnose and manage this severe but rare complication within the special setting of hsct. results: a 56-years-old man with a flt-3 itd, npm-1, runx1-runx1t1 and cbfb-myh11 negative, not differentiated, chromosomally normal acute myeloid leukemia (aml) in third complete remission (cr) was submitted to a hematopoietic stem cell transplantation (hsct) from his haploidentical son. the conditioning regimen consisted of oncothiotepa, busulfan and fludarabine and was followed by the infusion of a t-cell depleted bone marrow graft. gvhd prophylaxis consisted of cyclosporine a (csa) and mycophenolate mophetyl (mmf). neutrophil engraftment occurred on day +24. recipient's autoimmunity was negative. at 27 months post-transplantation the patient received an antipneumococcal vaccination. fifteen days post-vaccination the patient was admitted to our in-patient ward due to general malaise, diffuse muscle and joint pains, cutaneous bleedings, oedemas, hyperchromic urines and constipation. physical examination revealed diffuse ecchymosis, swelling of deep muscles with a progressive functional disability due to hematomas and hemorrhagic suffusions of the tongue frenulum. and anti-factor viii inhibitors 6.46 bu/ml (high titers >5 bu/ml). thus, a diagnosis of acquired autoimmune haemophilia a was made and treatment with feiba combined with prednisone was started. patient's clinical conditions dramatically improved as he referred an improvement of movements and the resolution of joint and muscle pains despite the persistence of deep hematomas just after one day of treatment that had determined an increase of fviii:c value to 1.32% and an improvement of aptt to 47.06 seconds. on the following medical checks physical examination showed the progressive disappearance of deep muscle hematomas, and normal values of fviii:c. conclusions: aha is a rare but severe complication following hsct and it could appear years afterengraftment. a prompt diagnosis and an early treatment with feiba and corticosteroid are necessary to avoid life-threatening sequelae. the inclusion of the coagulation panel in the laboratory exams performed during the follow-up is advisable in order to early detect this life-threatening complication. disclosure: nothing to declare background: splanchnic thrombosis is an uncommon complication of myelofibrosis and a controindication to proceed to hematopoietic stem cell transplantation (hsct) due to the risk of additional vascular and endothelial complications. we present a patient with myelofibrosis (mf) that proceeded to hsct from an unrelated donor, despite splanchnic thrombosis unresolved after heparin treatment and unable to proceed to surgical treatment due to severe thrombocytemia. methods: a 67-year woman with mf secondary to essential thrombocythemia, with intermediate-2 score according dynamic international prognostic staging system (dipss) and with extreme splenomegaly (maximum diameter 30 cm), refractory to ruxolitinib, showed an extensive thrombosis of the portal and splenic veins, unresolved after 4-week heparin therapy, at the time of availability of an hla (8/8) and abo matched unrelated donor. she received a conditioning regimen including fludarabine and thiotepa and a gvhd prophylaxis with atg thymoglobuline, cyclosporine and methotrexate, followed by the reinfusion of 5.8x10 6 /kg cd34+ pbsc. at the time of transplant we were aware of an high risk of developing sos, on the basis of the older age of the recipient, the unrelated donor, the advanced stage of myelofibrosis and the ferritin serum level of 2.223 ng/mg. results: on day +9 after hsct sos complicated the aplasia phase, characterized by jaundice, ascites, weight gain, progressive increase in creatinine and bilirubin serum levels. an ultrasound of abdomen confirmed an unchanged thrombosis extension and the development of ascites. on day +10 the patient was categorized as very severe sos stage, according to ebmt severity criteria, because of doubling of bilirubin serum level in 48 hours and a 20% increase in comparison with her baseline weight. therefore, defibrotide was promptly started in association with diuretic therapy. the treatment was continued for 3 weeks and allowed gradual restoration of the water balance and normalization of bilirubin serum level. at the last follow-up, 6 months after hsct, the patient shows the persistence of a non-transfusion dependent anemia, platelets 80.000x10^3/ul, palpable spleen 4 cm below the rib, >95% allogeneic chimerism in the granulocytic compartment and 90% in the t lymphocyte compartment. splanchnic thrombosis is partially recanalized and replaced by collateral circles with cavernous aspects. the patient is on treatment with fondaparinux and has shown neither significant infectious episodes or acute or chronic gvhd. conclusions: we conclude that defibrotide treatment allowed to perform a successfull allogeneic transplant in a patient with mf associated with an overt picture of splanchnic thrombosis. background: hematopoietic stem cell transplantation (hsct) is associated with an increased incidence of secondary malignancies including skin cancer. squamous cell carcinoma (scc) is the most common type in patients who are receiving immunosuppressive therapy and chronic graft-versus-host disease (cgvhd) appears to be an important risk factor for its development. recent studies describe voriconazole exposure as an independent factor that may contribute to this increased risk as well. in our best knowledge, no cases of scc have been reported in pediatric allogeneic hsct to date. methods: we present a case report of a 9 year-old boy who developed a scc with high-risk features six years after undergoing hematopopoietic stem cell transplant. results: a 9 year-old boy with acute lymphoblastic leukemia (all) underwent a matched unrelated bone marrow transplant 7 years ago. he developed grade iv agvhd followed by extensive cgvhd with generalized scleroderma. he required intensive and continued immunosuppressive therapy and was on prolonged antifungal prophylaxis with voriconazole. in march 2017, he developed scc involving left temporal region that was completely excised. two months later, more lesions in scalp and nose were noted and intralesion treatment with methotrexate was started. however, an unfavorable evolution was noted and he was put on systemic treatment including cisplatin and cetuximab receiving the whole scheme from january to march 2018 and continuing only with cetuximab, ten doses in total, until may, for unaceptable and severe tubulopathy that required admission at the hospital in several ocassions. he achieved a very good partial response but progression was noted shortly in follow up. at this point, non curative therapeutic options were found and he was put on intralesion methotrexate and photodynamic theraphy in a weekly basis with palliative intention. unfortunately, tumor growth was fast and patient passed away in august 2018, fifteen months after squamous cell carcinoma diagnosis, due to tumoral progression. conclusions: 1) scc is a rare, non-previously described, secondary malignancy in children undergoing hsct. 2) high-risk features scc constitutes an aggresive disease with a median overall survival below 1 year. 3) cgvhd appears to be an important risk factor for its development. 4) voriconazole induced-photosensitivity might have played a role. 5) cisplatin based regimens +/-cetuximab are a therapeutic option in disseminated and/or high risk cases. as outcomes are unsatisfactory in these cases, alternative therapeutic options need to be explored. disclosure background: pregnancy is a rare event after allogeneic stem cell transplantation (sct) for acute leukemia. here we report, to the best of our knowledge, for the first time on a successful pregnancy after treosulfan-based conditioning. methods: a 29-year old woman was diagnosed with acute myeloid leukemia (aml) secondary to chronic myelomonocytic leukemia in july 2015. ovarian preservation was performed by leuprolide acetate depot injection prior to cytostatic chemotherapy. of note, no cryopreservation of oocytes or ovarian tissue was conducted. she received two cycles of chemotherapy consisting of idarubicine (12mg/m² on day 1-3) and cytarabine (1000mg/m² b.i.d. on days 1, 3, 5 and 7). due to secondary origin of aml sct was performed in first complete remission of aml after conditioning with treosulfan (14g/ m² days 3-5) and fludarabine (30mg/m² days 1-5). she received 4.94×10 6 cd34-positive cells per kilogram body weight from a hla-matched unrelated donor. results: follow-up bone marrow aspirates showed continuous complete remission of aml. seven months after sct she became pregnant, but decided for induced abortion. in january 2018, 25 months after hsct she became pregnant again and desired the child. medical examinations were performed monthly on an outpatient basis in stringent cooperation with the maternity clinic. the course of pregnancy was unremarkable, although she was hospitalized due to premature labor in the 34 th week of pregnancy. however, gynecological examination showed no clinical significant findings, so that section was planned and she could be discharged again. in the 38 th week of pregnancy she gave birth to a healthy girl (50cm, 2810g) by cesarean section. peripartum she developed hypoethesia of the left body half. neurological examination showed no abnormalities and she recovered immediately. there were no other postpartum complications. breastfeeding was established but additional food was necessary for a sufficient nutrition of the child. conclusions: this case of successful pregnancy following sct demonstrates that fertility can recover after treosulfan-based conditioning. however, detailed studies of ovarian function and fertility are necessary to gain more insight into the risk of premature ovarian failure. disclosure: nothing to declare. experimental stem cell transplantation p219 cd19-cart therapy before allo-hsct in children and adolescents patients who diagnosed r/r b-all with e2a-pbx1 background: b-all with e2a-pbx1 in children and adolescents is described with favourable prognosis. but there are more than 10% patients with e2a-pbx1 diagnosed as relapsed or refractory. the results of allo-hsct in children and adolescents with this group leukemia in our center was analyzed in order to understand the therapeutic effect of cd19-cart on the patients. methods: retrospective analysis, from june 1st, 2012 to july 31,2018, all children and adolescents diagnosed relapse or refractory b-all with e2a-pbx1 who received allo-hsct, total 30 cases. all patients was divided into two groups depending on whether or not accepted cd19-cart before allo-hsct. according to fcm-mrd and e2a-pbx1 level before allo-hsct, os lfs and cumulative recurrence rate were analyzed. r 3.2.0 was used as statistical analysis software. results conclusions: 1. for r/r b-all with e2a-pbx1 in children and adolescents, fcm-mrd pre-transplant hasn't obvious effect on the outcome of allo-hsct, while the level of e2a-pbx1 has obvious effect. the out come of e2a-pbx1 negative group was obviously better than positive group. 2. cd19-cart can obviously improve the os and lfs, it is mainly because of cd19-cart can makes more patients fusion to zero. 3. for r/r b-all with e2a-pbx1 in children and adolescents, if chemotherapy can't make the fusion to zero. it is suggested to accept cd19-cart therapy to make the fusion zero. it can improve the outcome of os and lfs. disclosure background: currently, hematopoietic stem cell transplantation (hsct) represents the only curative treatment for numerous hematopoietic malignancies like leukemias, immune deficiencies or metabolic diseases. cd34 serves a quality marker for stem cell grafts, which is not solely expressed on stem cells but also on a variety of progenitors. the role and the impact of these subpopulations remains unknown. we made use of our genetic barcode system to analyze the influence and contribution during reconstitution on a clonal level. methods: fluorescence activated cell sorting (facs) was used to sort hematopoietic stem and progenitor populations, namely hscs, mpps, cmps and clps, which were lentivirally transduced with our previously established bc32 barcoding system. after mixing the marked cells with bone marrow support, lethally irradiated recipient animals were and transplanted and monitored over 16 weeks. we focused on bone marrow, blood, spleen and thymus, on chosen endpoints (1w, 3w, 8w, 16w) and samples were used to analyze the contribution of the subpopulations during the reconstitution process based on fluorescent protein (fp) expression. to investigate the clonal contribution in different organs, we performed next generation sequencing (ngs) and frequencies of unique barcodes in a sample were analyzed by bioinformatical approaches. results: a maximum of 15% of cells expressed the encoded fps, which were mostly derived from the hscs and mpps. cmp-derived cells were only detected 1 week after transplantation in the myeloid compartment. cells derived from the clps were not detected at any time point. we analyzed the barcode content of the differently marked cells after next-generation-sequencing. in accordance with the facs data, the majority of the clones during the 16 weeks of observation are derived from hscs and mpps. cmp-derived clones were only contributing during the first weeks and clp-derived clones are barely detectable. we did not observe any major differences with regard to age of donor or recipient, despite the total number of clones is higher in the group, which received the "aged" graft, independently from the transduced cell population. conclusions: here we show the suitability of our highly complex multi-color barcode system to study the clonal contribution of hscs and three progenitor populations after hsct. our results will contribute to a better understanding how these different populations interact to support the establishment of a new hematopoietic system. emphasized by the variability in data of graft and recipient age, this comprehensive analysis gives rise to an impression to the necessity of personalized graft composition, by which treatment success could be influenced. disclosure: nothing to declare survival and fate of adipose derived mesenchymal stem cells in a rat brain injury model background: mesenchymal stem cells have been identified as promising candidates in the treatment of central nervous system (cns) injury through neurotrophic support and immunomodulation. adipose tissue is an attractive source of mesenchymal stromal/stem cells (ascs) for regenerative therapeutic applications because they can be harvested from autologous donors with minimally invasive methods, can be rapidly expanded ex vivo, show low immunogenicity if allogeneic, and can be used in autologous or heterologous settings. the present study examines the fate and effects of intracerebroventricularly (icv) transplanted ascs in a traumatic brain injury (tbi) model. methods: ascs were isolated from inguinal fat pad of adult wistar rats under sterile conditions and cultured according to standard procedures. ascs at passage 2 (2x10 5 cells) were seeded and transfected with sleeping beauty transposase and pt2 venus-neo r plasmids. selection with g418 antibiotic resulted in the generation of a homogeneous asc population which expressed fluorescent venus protein for several passages, phenotypic characterization showed that these cells were 99.6% double positive for cd44 and cd90 stem cell markers, verifying their mesenchymal origin. tbi was induced by stereotactic surgery under deep anaesthesia and subsequently icv transplantation of venus+ ascs was performed on adult wistar rats. normal ascs-transplanted and tbi-saline transplanted rats were used as controls. the proliferation, migration, survival and fate of transplanted ascs and their effect on injury restoration were examined six weeks post transplantation (pt). results: six weeks pt ascs expressed the fluorescence venus protein and therefore were identified in brain parenchyma. their presence into brain was also confirmed by masson trichrome staining, which revealed their collagen depositions. ascs were found in lesser numbers compared to those transplanted and exhibited no proliferative activity. ascs were found scattered distributed in brain as individual cells, and there were no aggregates of ascs or mass formation into lateral ventricles. extensive migration of ascs was mainly performed through white matter tracks in the corpus callosum and fimbria of hippocampus. six weeks pt ascs retained the characteristics of mesenchymal cells and did not differentiate into cells of neural lineage. ascs exhibited limited long-term survival, which is restricted in perivascular areas probably contributing to vascular formation. homing of ascs into peri-injured area was detected in half of the animals and achieved through the corpus callosum, as revealed by the collagen depositions, in this white matter track. transplanted ascs reduced the area of tbi cavity and did not enhance the astroglial scarring in peri-injured area. in tbi +ascs transplanted animals, the cortical injury site, showed a significantly smaller volume and lower % tissue loss compared to that of tbi+vehicle animals (1.90 ±0.38mm 3 and 12.25±2.83% respectively, versus 1.12 ±0.34mm 3 and 6.57±1.67%, p=0.015 and p=0.019 respectively). conclusions: considering the effects of ascs on inflammation and regeneration, we suggest that their transplantation after brain injury may promote host brain repair mechanisms. ascs transplantation may be beneficial in tbi, however some of its effects need careful and indepth evaluation. disclosure: nothing to declare xie-na cao 1 , yuan kong 1 , zhong-shi lyu 1,2 , qi wen 1 , min-min shi 1,2 , qian-yu sun 1 , yu-hong chen 1 , yu wang 1 , lan-ping xu 1 , xiao-hui zhang 1 , xiao-jun huang 1,2 background: poor graft function (pgf) remains a serious complication after allogeneic hematopoietic stem cell transplantation (allo-hsct). our previous work reported that abnormal bone marrow (bm) endothelial cells (ecs) were involved in the pathogenesis of pgf patients after allo-hsct (bbmt2013; bmt 2016; blood2016), but the explicit mechanism requires further clarification. autophagy is a self-degradative process responsible for the elimination of cytosolic components including proteins and damaged organelles. recent findings demonstrated that stimulation of autophagy could reduce oxidative status and angiogenic potential in ecsafter high-glucose exposure, from diabetic patients.however, little is known regarding the autophagy of bm ecs in pgf patients. therefore, the current study was performed to evaluate whether autophagy in bm ecs play a role in the pathogenesis of pgf. moreover, to investigate the effects of autophagic regulation on ecs and thereby regulating hematopoietic stem cell (hscs). methods: in the prospective case-control study, the autophagy levels were compared in bm ecs from pgf patients, and their matched good graft function (ggf) patients.the expression levels of autophagy-related markers (lc3, beclin1, and p62), and intracellular autophagosomes were detected by immunohistochemical staining, flow cytometry, western blot and transmission electron microscopy. subsequently, rapamycin (the autophagy activators) or hydroxychloroquine (hcq, the autophagy inhibitor) were administrated tothe 7-day cultivated bm ecs and human umbilical vein endothelial cells (huvecs), respectively.the autophagic vacuoleswere detected by monodansylcadaverine (mdc) staining assay. the bm ecsand huvecs were evaluated by cell counting, dii-ac-ldl and fitc-lectin-uea-1 double staining, migration, cell proliferation, and levels of reactive oxygen species (ros). to explore whether autophagy would affect the ability of bm ecs to support hscs in vitro, bm cd34+ cells from healthy donors were co-cultured with cultivated bm ecs and huvecs. colony-forming unit (cfu) and the apoptosis of co-cultured hscs were analyzed. results: the defective autophagy in bm ecs, characterized by decreased intracellular autophagosomes and autophagic vacuoles, decreased expression of lc3-ii and beclin1, and high level of p62, were observed in pgf patients compared with ggf patients. moreover, the coculture of bm cd34+ cells with bm ecs showed significant deficient cfu plating efficiency, and increased apoptosis of cd34+ cells in pgf patients. in vitro upregulation of autophagy by rapamycin quantitatively and functionally improved bm ecsand huvecs, which manifested as more dii-ac-ldl and fitc-lectin-uea-1 double stained cells, increased capacities of migration, lower levels of ros and apoptosis via regulating beclin1 pathway, whereas inhibition of autophagy by hcq aggravated the huvecs and bm ecs from pgf patients. furthermore, in vitro upregulation of autophagy by rapamycin significant improved cfu plating efficiency, and decreased apoptosis in bm hscs co-cultured with huvecs and bm ecs from pgf patients. conclusions: these findings suggest that defective autophagy in bm ecs may be involved in the pathogenesis of pgf. the effect of rapamycin in pgfpatients is potentially mediated by improving the dysfunctional bm ecsto support hscs. therefore, it would be of value to investigate whether upregulating of cytoprotective autophagy of bm ecs may ameliorate pgf, thereby providing a novel clinical intervention for pgf in the future. clinical background: heparanase (hpse) in an endoβ-glucuronidase that specifically cleaves the saccaride chains of heparan sulphate proteoglycans (hs), leading to a loss of integrity of the extracellular matrix and to release of hs-bound cytokines, chemokines, angiogenic and growth factors. hpse gene is polymorphic and includes approximately 300 snps. the combination of two snps, rs4693608 and rs4364254, are involved in the regulation of hpse expression with an inverse correlation between mrna expression and protein levels: gg-cc, gg-ct, gg-tt, ga-cc (low group) expressed high hpse concentration; ga-ct and ga-tt (median group) expressed intermediate hpse levels; aa-tt and aa-ct expressed low hpse concentration (high group). we studied hpse snps in the allogeneic stem cell transplantation (hsct) setting to evaluate a possible association with post-hsct outcomes. methods: we enrolled 228 patients submitted to hsct in our department since 2005 to 2016. for each couple recipient-donor, rs4693608 snp was genotyped using restriction fragment lenght polymorphism assay, whereas for rs4364254 snp an allele-specific polimerase chain reaction was applied. hpse genotype distribution was compared in different groups according to post-hsct outcome: graft-versus-host disease (gvhd), transplantrelated mortality (trm), overall survival (os), infectious complication and disease-free survival (dfs). statistical analysis was performed using ncss 10. results: distribution of rs4693608 snp was as follows: gg 16.7%, ga 49.8% and aa 33.5% among recipients and 17.6%, 53.3% and 29.1% among donors, respectively. hardy-weinberg equilibrium (hwe) was respected. distribution of rs4364254 snp was as follows: cc 15.1%, ct 37.8% and tt 37.8% among recipients and 15.3%, 36% and 48.7% among donors, respectively. rs4364254 snp distribution did not respect the hwe. an association was found between recipient rs4364254 snp and the cumulative incidence of agvhd among patients submitted to a reduced intensity conditioning (ric): 37.3% for tt genotype and 69% for ct or cc genotype (p=0.03). on the other hand, an association was identified between donor rs4693608/rs4364254 snps combination and the cumulative incidence of agvhd: 81.5% for low group donor, 48% for median group donor and 40.8% for high group donor (p=0.04). conversely, aa genotype for donor rs4693608 resulted independent risk factor for cgvhd de novo development (p=0.049, od 2.1) together to donor-recipient sex mismatch (female donor to male recipient vs. others: p=0.005, od 3.56) . considering cmv reactivation rate after hsct, an association was observed according to recipient rs4364254 snp: 82% for cc genotype, 63.5% for ct genotype and 57.1% for tt genotype (p=0.049). multivariate analysis confirmed recipient rs4364254 snp as independent risk factor for cmv reactivation after hsct (p=0.04, od 2.62) together with recipient cmv serostatus at transplant (positive vs. negative: p< 0.01, od 8.49). conclusions: hpse role was widely studied in the setting of inflammation, autoimmune diseases, hematological disease and tumor. however, it still remains debated the inducing or protective activity of hpse in the setting of gvhd. obviously, our results need to be confirmed in a validation cohort. clinical trial registry: na disclosure: nothing to declare novel protocol for autologous hsct in patients with high risk of complications: ambulatory chemomobilization and transplantation of fresh hematopoietic stem cells with backup storage background: autologous hematopoietic stem cell transplantation (ahsct) is standard of treatment in many patients with high risk of complications: dialysed patients, patients with heart and kidney amyloidosis or patients with systemic sclerosis. we introduced recently a novel protocol for ahsct: combination of ambulatory mobilization with very low doses of ara-c and g-csf connected with direct ahsct with fresh cells. this protocol allowed us to reduce the transplant risk in various patient groups traditionally connected with high risk of complications. in this work we summarize the experience in such high risk patients. methods: the prospectively collected database of patients after ahsct was searched for patients who underwent ahsct after chemomobilization with ara-c and transplantation with fresh cells and who fulfilled at least one study inclusion criteria: a) dependence on dialysis b) amyloidosis c) systemic sclerosis d) disqualification from transplantation at other centre due to the high risk of complications. there were together 19 patients selected for this analysis -9 with amyloidosis (6 with ≥ 2 organs involved), 9 dialysed, 2 with systemic sclerosis, 2 unfit at other centre. the database included prospectively recorded serious adverse events during the mobilization and transplantation. results: there were 20 transplantations performed in this group of patients. mortality was 0% at 100 days. all patients underwent successful ambulatory mobilization. all patients received mephalan conditioning with single infusion with median dose of 200mg/m2 (min 140, max 200). mean engraftment was 10.5 days for white blood cells and 12.7 days for plt over 20 g/l. the rate of complications was low with 7 cases of neutropenic fewer, 1 single bacterial culture with staphylococcus epidermidis without clinical signs of infection, median mucositis grade of 0.4 and without patients on parenteral nutrition. the median time of hospitalization was 19 days (min 14, max 29). conclusions: we present here novel protocol of transplantation combining chemomobilization and ahsct with fresh cells with excellent safety profile among most severely ill patients allowing for safe and efficient transplants. with this protocol we were able to overcome multiple risk factors and perform full intensity transplantation in very fragile patients. disclosure: nothing to declare single umbilical cord blood transplantation provides durable disease remission of advanced hematological malignancies in elderly patients background: although allogeneic hematopoietic stem cell transplantation (allo hsct) is potentially curative therapy in a variety of hematological malignancies, little has been reported of the outcome for elderly patients who are not in remission at transplantation. but it has been pointed out that recipient age alone can not be regarded as contraindication for allo hsct in the literature recently, supported by suitable donor, conditioning regimens and appropriate management of complications. we conducted a retrospective study of elderly patients who had advanced hematologic malignancies to elucidate the outcome of single umbilical cord blood transplantation (sucbt) in toranomon hospital kajigaya, japan. methods: we retrospectively investigated the outcomes of 19 patients aged over 65 who underwent their first ucbt from june 2013 to december 2017 in our medical center. results: diseases included acute myelogenous leukemia (n=12), myelodysplastic syndrome (n=3), adult t-cell leukemia/lymphoma (n=2), myelofibrosis (n=1) and chronic lymphocytic leukemia (n=1). the median age at transplantation was 69 years (range, 65-75) and follow-up for survivor post transplantation was 642 day (range, 391-767). all patients were not in complete remission (cr) at the time of transplantation. reduced intensity conditioning (ric) regimens were used in 10 patients. all patients received tacrolimus and mycophenolate mofetil as graftversus-host disease (gvhd) prophylaxis. all cases except 4 early death achieved neutrophil recovery at median 18 days (range, 13-28). at 1 year, overall survival (os) rate and disease free survival (dfs) were 31,6% (95% confidence interval (ci), 12.9-52.2). we performed univariate analysis to identify the factor that influenced os at 1 year, but no statistical significance was demonstrated at the age of transplantation (aged 65 to 69 vs. ≧70, 33.3% (95% ci, 10.3-58.8) vs. 42.9% (95% ci, 9.8-73.4), p=0.68). the cumulative incidence of non-relapse mortality (nrm) at 100 days was 47.4% (95% ci, 23.6-67.9%) and relapse at 1 year was 9.1% (95% ci, 0.0-24.6%). only two patients developed acute gvhd(ii-iv) and one developed severe gvhd at 49 days after transplantation. the main causes of death was infection (n=10), including sepsis (n=8) and viral encephalitis (n=2), followed by idiopathic pneumonia syndrome (n=2) and thrombotic microangiopathy (n=1) during the early phase of transplantation. in contrast, no patients died of recurrence. conclusions: although our report consisted relapsed/ refractory disease of elderly patients at the time of sucbt, durable remission and lower incidence of gvhd could be noteworthy compared with previous reports. further strategies to reduce the rate of nrm and longer duration of follow up would be warranted. disclosure background: pearson syndrome and kearns-sayre syndrome are metabolic disorders caused by a de-novo deletion in the mitochondrial dna (mtdna). allogeneic stem cell transplantation has shown to improve metabolic function in distal organs in several metabolic disorders, but bears significant morbidity and mortality, especially for patients with mitochondrial disorders. novel gene therapies may correct diseases rising from genomic dna mutations, but targeting the mitochondrial dna is complex. mitochondria are able to transfer into cells and between cells, as seen in preclinical models of mitochondrial and other metabolic disorders. here, we introduce a novel concept of mitochondrial augmentation therapy (mat) of autologous cd34+ cells in 4 children with mitochondrial deletion syndromes. methods: patients were treated under a compassionateuse program, approved by the sheba medical center irb and the israeli ministry of health. briefly, mobilization was performed using gcsf alone (n=1) or in addition to plerixafor (n=3) . cd34+ cells were isolated via miltenyi clinimacs system and co-cultured with maternal mitochondria, drawn from peripheral blood and confirmed nondeleted, for 24 hours, and re-infused to the patient without any conditioning. patients were followed for clinical and metabolic parameters. results: all four patients presented with different deletions in mitochondrial dna, and different baseline characteristics, and were treated at the age of 6.5, 7, 11 and 14 years. despite normal cbc, significant bone marrow hypocellularity was seen in 3 evaluated patients (20%, 30% and 50% cellularity at age 7, 11 and 14), which correlated with low colony forming unit capacity of patients and low yield of cd34+ mobilization in the leukapheresis product. patients received on average 2x10 6 enriched cells/kg (range, 1.1 -2.8), and the median enrichment of cd34+ cells was 135% (range, 103-162%). no infusion reactions occurred, and the only severe adverse events of this cellular therapy were leukapheresis-related anemia, hypokalemia, hypocalcemia and alkalosis, all resolved promptly with proper supplementation. follow-up duration is variable, ranging 5-22 months. we were able to show improvement in mitochondrial heteroplasmy (proportion of deleted mtdna of total mitochondrial dna) and in normal mtdna content, starting 1-5 months from cell therapy, which correlated with improved atp production in peripheral blood derived mononuclear cells. clinically, patients showed improvement in aerobic function and endurance (measured by the half-bruce protocol, sit-to-stand test and 6-minute walk test), muscle strength (hand-held dynamometry), and in quality of life, measured by the international pediatric metabolic disability scale. no metabolic crises occurred following cell infusion. conclusions: patients with deletion in mtdna have metabolic dysfunction, including poor bone marrow cellularity and function. hematopoietic stem cells in patients with mtdna deletions can be enriched with normal mitochondria, via mat, as first shown in our patients. this novel process is safe and results in increase in the normal mtdna in peripheral blood of patients, and in improved metabolic and clinical function. clinical trial registry: clinicaltrials.gov nct03384420 disclosure: moria blumkin, noa sher and natalie yivgi ohana -minovia therapeutics, employment p227 high cytotoxic efficiency of alpharetrovirally engineered cd19-specific chimeric antigen receptor natural killer cells for treatment of acute lymphoblastic leukemia stephan müller 1 , tobias bexte 1 , annekathrin heinze 1 , franziska schenk 2 , axel schambach 3 , winfried s. wels 4,5 , ute modlich 2 , evelyn ullrich 1, 5 background: autologous chimeric antigen receptormodified (car) t cells with specificity for cd19 showed potent antitumor efficacy in clinical trials regarding relapsed and refractory acute lymphoblastic leukemia (all). natural killer (nk) cells are cytotoxic lymphocytes that are capable to kill their targets in a non-specific manner and additionally do not cause gvhd. therefore, using cd19-car-nk cells exhibits several advantages, such as safety in clinical use, possible allogenic settings and the potential to also attack heterologous leukemia cells which lost cd19. previous approaches used cd19-car-nk cells pre-stimulated by feeder cells, bearing potential risks. thus, we focused on the optimization of generating cd19-car-nk cells by viral transduction under feeder-cell free conditions. methods: human nk cells were isolated from healthy donor peripheral blood mononuclear cells via cd56 negative selection. after a feeder-cell free expansion phase with interleukin 15, transductions were performed with an egfp or a cd19-car encoding vector at different multiplicities of infection (moi). to optimize gene modification different transduction enhancers (retronectin and vectofusin-1) and viral vector systems (lentiviral and alpharetroviral) were compared. finally, generated cd19-car-nk cells were tested in their ability to kill cd19positive and cd19-negative cell lines. results: nk cells transduced with a lentiviral egfp encoding vector or a lentiviral cd19-car vector using retronectin and vectofusin-1 showed similar transduction efficiencies for both transduction enhancers (egfp: retronectin moi 10: 12.9%; vectofusin-1 moi 10: 12.8%; cd19-car: retronectin moi 5: 10.7%, moi 10: 9.2%; vectofusin-1 moi 5: 11.3%, moi 10: 14.4%). the generated cd19-car-nk cells showed increased cytotoxic capacity against cd19-positive cells compared to nontransduced (nt) nk cells (72.7% vs. 23.6%, effector to target (e:t) ratio 1:1). both nk cell populations were equally efficient in killing cd19-negative cells (30.5% vs. 25.7%). alpharetroviral transduction of nk cells with an egfp encoding vector showed higher transduction rates with vectofusin-1 than with retronectin (retronectin moi 1: 5.3%, moi 5: 9.7%; vectofusin-1 moi 1: 55.3%, moi 5: 51.6%). further using vectofusin-1, similar transduction efficiencies could be achieved with an alpharetroviral cd19-car encoding vector (moi 1: 11.1%, moi 5: 49.2%, moi 10: 68.9%), outperforming the efficiencies of lentivirally generated cd19-car-nk cells in the same experiments (moi 1: 1.5%, moi 5: 8.4%, moi 10: 14.9%). additionally, alpharetroviral cd19-car-nk cells showed a higher cell killing activity against cd19-positive cells than lentiviral cd19-car-nk cells or nt-nk cells (90.5% vs. 62.5% vs. 9%, e:t ratio 1:1). interestingly, similar killing activities were achieved with an e:t ratio of 0.5:1 (88.9% vs. 58.3% vs. 10.3%) and alpharetroviral cd19-car-nk cells remained a stable cytotoxicity level at lower cell concentrations down to an e:t ratio of 0.1:1. all three nk cell populations were equally efficient in killing cd19negative cells (16.4% vs. 23.6% vs. 12.3%, e:t ratio 1:1). conclusions: cd19-car-nk cells can be successfully generated under feeder-cell free conditions using different transduction enhancers and viral vector systems. these data suggest the usage of vectofusin-1 in combination with alpharetroviral vectors to genetically modify nk cells to achieve sufficient amounts of transduced cells. these cd19-car-nk cells mediate high cytotoxicity and therefore may offer a new therapeutic option in the treatment of all. disclosure: axel schambach is an inventor on a patent describing alpharetroviral sin vectors. winfried s. wels is an inventor on a patent describing chimeric antigen receptors with an optimized hinge region. the remaining authors have nothing to disclose. graft-versus-host diseaseclinical walter spindelböck 1 , bianca huber-krassnitzer 1 , barbara uhl 1 , gregor gorkiewicz 1 , hildegard greinix 1 , christoph högenauer 1 , peter neumeister 1 background: steroid-refractory acute gastrointestinal (gi) graft-versus-host disease (agvhd) is a severe complication of allogeneic hematopoietic stem cell transplantation (allo-hsct) associated with a high mortality rate. loss of intestinal bacterial diversity is thought to be associated with severity of gi-agvhd and an impaired intestinal microbiota with reduced diversity is an independent predictor of mortality. methods: the fecal microbiota transplantation (fmt) procedures were performed according to a protocol approved by the local ethical committee (29-027ex 16/17) after obtaining informed consent. donors were healthy adult subjects screened for potential infections by serologic and microbiologic tests according to local standards. donor stool was diluted with saline and homogenized to a volume of~250 ml fecal solution for instillation into the terminal ileum and caecum via colonoscope. microbiota sequencing analysis of 16s rdna was performed before fmts and afterwards at predefined timepoints. results: we report the outcome of nine patients refractory to 3-6 lines of immunosuppressive therapies with lower gi-stage iii (n=1) or iv (n=8) agvhd following repetitive fmts from a single donor. all patients had received an allo-hsct for mds (n=3) , aml (n=4), pmf (n=1) and mm (n=1) following a reduced intensity (n=5) or mac (n=4) conditioning regimen using pbsc as stem cell source. after an onset of lower gi agvhd between 11-465 days after allo-hsct, nine patients refractory to several lines of immunosuppressive therapies received 1-6 fmts (6 patients were treated with more than 2 fmts, in 3 patients fmt was only administered once or twice) mostly in weekly intervals. five patients achieved a clinical complete response with resolved diarrhea and no gastrointestinal complaints, and four of these could be discharged without gvhd symptoms. two patients (pr, nc) were discontinued after 2 or 3 fmts in pr or nc due to concomitant infections (metapneumoviral pneumonia, cmv gastroenteritis), the 2 other non-responders succumbed to gvhdrelated infectious complications. the establishment of donors' microbiota with the emergence of new taxa, an increase in bacterial richness/diversity, and the disappearance of the "enterococcus signature" were associated with disease control and response to fmt. except the possible transmission of adenovirus by fmt in one patient, no other immediate procedure-related infections or other side effects were observed. conclusions: restoration of dysbiosis by fmt might represent a promising novel therapeutic approach for a subset of patients with refractory lower gi-agvhd. vigorous donor screening for infectious disease is mandatory. clinical background: migration of allo-activated donor effector tcells from lymphoid tissues to target organs is an important step in acute graft versus host disease (gvhd). the sphingosine-1-phosphate-1 (s1p1) receptor plays a crucial role in lymphocyte trafficking. data from animal models suggest that pharmacological modulation of the s1p1 receptor reduces gvhd and improves mortality. we investigated this mode of action by using the secondgeneration s1p1 modulator krp203 for the prophylaxis of gvhd in a pilot clinical trial in patients undergoing allogeneic hsct. methods: a multi-centric, phase 1b, prospective, open label, two-part study was conducted to evaluate the safety, tolerability and pharmacokinetics of krp203 in patients undergoing allogeneic hsct for hematological malignancies. primary endpoint was safety. initial efficacy was explored based on the incidence of gvhd, mortality and relapse. part 1 was a single arm open label study to investigate the safety of 3 mg/day krp203 added to standard of care gvhd prophylaxis (csa/mtx) in 10 patients. part 2 was a randomized two-arm open label study to compare the safety, efficacy and pk of 3 mg/day of krp203 in combination with tacrolimus/mtx to 1 mg/day of krp203 in combination with csa/mtx in 13 patients. in both parts, treatment with krp203 was initiated 10 days before hsct and continued for an additional 100 days. patients were followed up for up to 2 years. results: 23 patients were included in the study. 16 of 23 patients completed the 110-day treatment with krp203 at the assigned doses. median duration of follow-up was 264 days (range 153 to 271 days). krp203 was safe and well tolerated. 11 serious adverse events (saes) suspected to be related to krp203 were observed. macular edema (n=3) and peripheral edema (n=1) as s1p related adverse events occurred and resolved without sequelae. of note, the incidence of macular edema in hsct recipients is unknown. neutrophil engraftment was confirmed in all patients with a median of 16 days (range 12 to 45 days). 5 of 23 patients presented with grade iii or iv acute gvhd (on days 50, 56, 68, 99 and 102) . no gvhd or infection related death occurred during the first 100 days. 100-day survival was 96%, with no death occurring during krp203 treatment. 1 death occurred on study day 90 due to lymphoma relapse. a second death occurred on study day 121 due to liver gvhd. four patients died in the follow-up period due to gastrointestinal gvhd (day 265), aspiration pneumonia (day 327) and relapse (day 533 and day 877). the kaplan-meier estimate of overall survival at 1 year was 0.75. when comparing the data from the two dose groups (1 and 3 mg krp203), no major differences in safety, engraftment, gvhd rate or mortality were observed. conclusions: this clinical trial was the first to test s1p modulation in this population. our data suggest that krp203 had no negative impact on engraftment and overall, was safe, and well tolerated. based on exploratory data, when comparing to matched historical mortality data, krp203 may have favorable effects on overall survival ( figure 1 ). background: uric acid is a danger signal contributing to inflammation. relevance to allosct has been demonstrated in preclinical models: the depletion of uric acid led to improved survival and reduced gvhd (j exp med. 2013 sep 23;210(10):1899-910). results of a clinical pilot trial suggested that peri-transplant uric acid depletion reduce acute gvhd incidence (bbmt 2014 may;20(5):730-4). methods: this international multicentric study aimed to study the association of uric acid serum levels before start of conditioning with allosct outcome. patients with acute leukemia, lymphoma or mds receiving a matched sibling allosct for the first time were considered for inclusion, regardless of conditionning. data were prospectively collected between 8/2014 and 2/2018. a comparison of outcomes between patients with high and low uric acid level was performed using univariate analysis and multivariate analysis using cause-specific cox model. variables included in the multivariate analyses were age, sex mismatch, diagnosis, disease status, karnofsky score, number of cd34 cells given, intensity of conditioning, type of gvhd prophylaxis, atg use, time from diagnosis to transplant, year of transplant and cmv status. results: twenty centers from 10 european countries reported data on 385 allosct recipients. patient characteristics are given in table 1 . the uric acid cut off point was determined at 4.3mg/dl (median of measured uric acid levels). overall survival (os) and progression free survival (pfs) of allosct recipients with uric acid levels above cut off measured before start of conditioning were significantly shorter ( figure 1a , os univariate hr=2.4 ci=1.6-3.7 p< 0.001; multivariate hr=2.8, ci=1.7-4.7, p< 0.0001) ( figure 1b , pfs univariate hr=2 ci=1.1-3.7 p=0.02; multivariate hr=2.7, ci=1.4-5, p=0.003). nonrelapse mortality was significantly increased in allosct recipients with high uric acid levels prior to start of conditioning (univariate hr=2 ci=1.1-3.7 p=0.018; multivariate hr=2.65, ci=1.41-5.01, p=0.003). in addition, there was a non-significant trend towards higher acute gvhd incidence (gvhd grades ii-iv univariate hr=1.2 ci=0.8-1.9 p=0.4; multivariate hr=1.5 ci=1-2.4, p=0.08) in allosct recipients with uric acid levels above cut off before transplantation. finally, the incidence of relapse after allosct was moderately increased in the cohort with higher uric acid levels (univariate hr=1.6 ci=1-2.5 p=0.09; multivariate hr=1.59, ci=1.02-2.49, p=0.04). conclusions: high uric acid levels before start of conditioning correlate with high mortality after allosct. our results can serve as rationale for clinical trials on depletion of uric acid during allosct. results: we found significant correlation between donors' ctla-4 +49a>g polymorphism and hsct outcome. genotype aa was present in 170 donors, ag in 183 donors and 44 donors was homozygous for g allele. recipients who received graft from g allele carrier donors showed significantly increased cumulative incidence of relapse (at 24 months aa: 20.7%, ag: 23.6% and gg: 34.3%; p=0.04). on contrary, the frequency of the acute gvhd grades iii-iv and cytomegalovirus (cmv) reactivation/disease decreased according to the presence of the g allele in the donor ctla-4 genotype [agvhd: aa: 20%, ag: 12%, gg: 5%; p= 0.014; cmv: aa: 24%, ag: 16%, gg: 9%; p= 0.039]. cumulative incidence of agvhd was also markedly decreased among patients with g allele carrier donors (at 100 days aa: 19.9%, ag: 10.4%, gg: 6.4%; p=0.01). donor genotype similarly influenced hsct outcome in mud donor and mac conditioning subgroups. overall survival (os) was not different in patient subgroups according to donor genotypes [os at 24 months: aa: 55.5 ±3.8%, ag: 54.4±3.7%, gg: 49.4±7.6%; p= 0.68]. we did not find any correlation between recipients' ctla-4 +49a>g polymorphism and hsct outcome. conclusions: several ctla-4 snps have previously been described to be associated with relapse rate, incidence of agvhd and os, but results are often contradictory in the publications. in our study, ctla-4 +49a>g polymorphism of hsct donors influenced risk of relapse, agvhd, cmv and cause of death, but not overall survival. the genotyping of ctla-4 +49a>g polymorphism in donors may help in the risk assessment process and the choice of personalised therapy. disclosure: nothing to declare. background: although steroids remain first-line therapy for the treatment of acute graft versus host disease (agvhd), response rates in patients with grade iii-iv disease are poor, with no apparent improvement in survival over the past 15 years. we performed a prospective, multicenter trial to assess the efficacy and safety of the combination of ruxolitinib and etanercept as a novel approach to treat grades iii-iv sr-agvhd . methods: forty malignant hematologic disease patients with grades iii-iv sr-agvhd after allo-sct from three centers in east china were enrolled from january 2017 to june 2018. ruxolitinib was initiated at a dose of 5-10 mg bid for 2 months, and then tapered gradually for another one month. etanercept was administrated at 25mg biw for 2-8 weeks. results: the median age of patients was 25 (range 15-59) years. at day 30 after the combination treatment, the overall response rate (orr) was 90% including 30 crs (75%) and 6 prs (15%). the median time to the optimal response was 13 (range 3-34) days. the incidences of cr per organ were 95.7%, 80.8%, and 80% for skin, liver, and gut, respectively. the agvhd relapse rate was analyzed for the patients who had achieved cr or pr and survived beyond 60 days. relapses in agvhd occurred in 9.38% (3/ 32) of responsive patients. the patients who received ruxolitinib within 14 days after agvhd onset have a significant higher cr rate that those with delayed ruxolitinib therapy (96.2% vs. 42.9%, p=0.001). and the patients without gut infections have a significant higher cr rate than infected cohort (92.6% vs. 46.2%, p=0.002). by logistic regression analysis, the time from agvhd to ruxolitinib (rr=4.17, p=0.011) and gut infection (rr=3.31, p=0.031) were independent predictors for incomplete response. thirteen patients (13/40, 32.5%) suffered from at least 1 infectious episode after the start of the combination therapy, and pulmonary infectious diseases was a frequent complication (9/40, 22.5%). iii-iv cytopenia and cmvreactivation were observed in 30% and 47.5% of patients. the 1-year overall survival (os) after initiation of the combination therapy were 76.8%. the 1-year nrm and relapse incidence was 17.9% and 19.9%, respectively. patients with complete response on day 30 had significantly higher os probability than non-cr patients (1-year os: 86.1% vs 48.0%, p=0.01). compared with the historical cohort of basiliximab and etanercept for sr-agvhd in our center (n=31), no significant difference was found on the baseline. although the orr in patients treated with ruxolitinib and etanercept is identical with the historical cohort, ruxolitinib group achieved rapider remissions in liver agvhd and gut agvhd than the historical cohort (gut agvhd: 11 days vs. 17 days, p=0.026; liver agvhd: 21 days vs. 28 days, p=0.039), thus, with regard to hospital stay after agvhd onset, the ruxolitinib cohort stayed shorter (median: 18 days vs. 29 days, p=0.005) than basiliximab cohort. conclusions: combined treatment with ruxolitinib and etanercept resulted in a rapid cr to visceral agvhd and meanwhile reserve graft anti-leukemia (gvl) effect as the relapse rate of primary disease is relatively lower. the various infection complications associated with ruxolitinib merit more attention. disclosure: nothing to declare background: graft-versus-host disease (gvhd) remains one of the main life-threatening complications after allo-hsct, especially in patients with non-malignant diseases. the standard gvhd prophylaxis strategy is mostly based on the use of calcineurin inhibitors alone or in combination with other immunosuppressive (is) post-transplant cyclophosphamide (ptcy) is effective gvhd prophylaxis optiont for adult patients (pts), but has limited data in children. methods: the study aim was to evaluate ptcy as gvhd prophylaxis in pediatric pts with inherited disorders undergoing allo-hsct. 96 pts, the most of them are pediatric age (median age -3 y.o., range 7 month -30 y.o.) with different types inherited disorders (β-thalassemia -10, bone marrow failure syndromes -26, storage diseases -46, primary immunodeficiencydisorders -14) were inrolled in retrospective study. donor type was: matched/mismatched unrelated (mud/mmud) -69, matched related donor (mrd)-15, haploidentical (haplo) -12. conditioning regimen was: myeloablative (mac) -43, reduce-intensity (ric) -53. graft sourse was: bone marrow (bm) -68, peripheral blood stem cells (pbsc) -26, combintions bm +pbsc/bm+cord blood -2. ptcy 50 mg/kg days +3, +4 based gvhd prophylaxis recived 33 pts., standart gvhd prophylaxis based on calcineurin inhibitors -63 pts. results: cumulative incidence (ci) of agvhd was 48%. grade 2-4, 3-4 agvhd were 40% and 22% respectively. ptcy based gvhd prophylaxis reduced ci of agvhd (35% vs 56%, p=0,025). another reduce ci of agvhd factors were mac (31% vs 59% in ric pts group, p=0,044), mrd (13% vs 44% in haplo group vs 56% in mud/mmud group, p=0,024), bm as a transplant source (44% vs 62% in pbsc group, p=0,05). in a multivariate analysis mac (hr 2,6 95%ci 1,7-6,, p=0,02), time from diagnosis to allo-hsct less then 22 month (hr 2,6 95%ci 1,1-6,2, p=0,03) were predictive for reducing ci agvhd. for agvhd 2-4 st. significant factor increase ci was female donor both in univariate (51% vs 34%, p=0,04) and multivariate analysis (hr 0,7 95%ci 0,2-0,8, p=0,02).5 years overall survival (os) was 60%. improving os factors were: transplant age younger then 5 y.o. (80% vs 35%, p=0,000), time from diagnosis to allo-hsct less then 22 month (72% vs 38%, p=0,000), engraftment (72% vs 22%, p=0,000). in a multivariate analysis only transplant age younger then 5 y.o. (hr 3, 3 95%ci 1, (4) (5) (6) (7) (8) p=0, 006) and engraftment (hr 0,3 95%ci 0,1-0,7, p=0,005) were predictive for os. conclusions: ptcy-based gvhd prophylaxis can be effective options for reduce risk of acute gvhd. using unrelated donors, bone marrow as transplant source and mac can reduce ci of gvhd. performing allo-hscr earlier from diagnos and in earlier age can improve os patients with inherited disorders background: diarrhea is a frequent complication after allo-sct. at onset it is often difficult to differentiate gi gvhd from other causes of enterocolitis. recently, non-invasive tests, such as fecal calprotectin (fc), have been validated as markers of gut inflammation in patients with inflammatory bowel disease, but only a few studies have been published regarding its use as a diagnostic marker in gi gvhd. methods: our aim in this study was to explore the levels of fc in allo-sct recipients with new-onset diarrhea. so far we have included 43 allo-sct recipients who developed acute diarrhea ≥ stage 2-4 at a median of 75 days (range:12-328) post allo-sct. stool samples were analyzed as soon as possible after the onset of diarrhea. fc levels were determined in addition to an extensive microbiological panel for infectious enterocolitis (including norovirus pcr and c. difficile associated diarrhea). endoscopies for histologic analysis were performed according to the treating physicians' discretion (n=15). results: patients characteristics are summarized in table 1 . median follow-up for survivors was 524 days (range:151-1834). twenty-eight patients (65%) were diagnosed of gi-gvhd. the additional causes of diarrhea were: drug-related enterotoxicity (n=6), viral enteritis (n=2), food intolerance (n=2), c.jejuni-enteritis (n=1), and non-specific causes (n=4). the concentration of fc was higher in patients with gi gvhd vs. other causes of diarrhea (544μg/g +/-71 vs. 58 μg/g +/-33, p= 0.03). patients who did not develop severe enterocolitis had normal to slightly raised calprotectin at the onset of diarrhea [< 100-150 in 16 out of 19 (89%) cases], including 100% (6/6) of patients with enterotoxic drug-related diarrhea. among the 28 patients with gi-gvhd, 13 (30.2%) were later found to be steroid-resistant. as shown in figure 1 , we found a significant association between high fc (≥400μg/g) and severe-refractory gvhd (hr 5.7, p=0.01). of note, high values of fc were also found in 3 patients with severe infectious enteritis (norovirus, adenovirus and c.jejuni infections), with baseline fc>800 μg/g, respectively. overall survival was 78% (ic95%:65-91) at 12 months. hypoalbuminemia and thrombocytopenia were the only variables linked to 1-yr os in univariate analysis, regardless of the cause of enterocolitis. conclusions: in the absence of standarized (and expensive) biomarker panels for analyzing and predicting gvhd onset and outcomes, the fc test may be an useful tool in the allo-sct setting. our initial results show that fc is helpful in predicting mild causes of diarrhea and to identify patients with a high probability of developing severe (and potentially steroid-refractory) gi gvhd, although high levels are also found in severe infectious enteritis. background: there is an urgent need for effective therapy for severe acute gvhd. results of gvhd therapies beyond 6 months are rarely reported. we here report a median follow-up of 4 years. we introduced mesenchymal stromal cells as therapy for severe acute gvhd, with a dramatic response in some, but not all patients. the placenta protects the fetus from the mothers haploidentical immune system during pregnancy. we found that maternal stromal cells from the fetal membrane, so called decidua stromal cells (dscs) were more immunosuppressive than other sources of stromal cells. methods: we treated 21 patients, median 49 years of age (range 1.6-72) for severe acute gvhd. all had biopsy proven gastro-intestinal gvhd. all were steroid refractory,11 after >7days or with progression and 10 after >3 days. we used an improved protocol where dscs were thawed and infused in a buffer with 5% albumin. dscs were given at a median dose of 1.2 (0.9-2.9) x 10 6 cells/kg and 2(1-6) doses, given one week apart. viability of frozen and thawed dscs was 95% (89-100) and cell passage was 4 (2-4). results: complete resolution of gvhd was seen in 11 patients and 10 had a partial response. the cumulative incidence of chronic gvhd was 52%. six had mild, 4 moderate and one severe nih overall gvhd severity scoring. nine patients died, 3 from relapse, 1 acute gvhd and septicemia, 1 zygomycetes infection, 1 liver insufficiency, 1 cerebral hemorrhage, 1multiorgan failure and 1 chronic gvhd with obstructive bronchiolitis. four years transplant related mortalliy was 28.6% and overall survival was 57%. survival was not significantly worse (p=0.33) than 66% for all 293 patients undergoing allogeneic hematopoietic cell transplantation during the same period 2012-2015. conclusions: to conclude, dscs seems to be a promising therapy for severe acute gvhd. randomized trials are under way. disclosure: nothing to declare p237 anti-apoptotic protein bcl-2 is upregulated in graftversus-host disease stem cell transplantation (allo-hsct) with 30-80% developing either acute or chronic gvhd. recently, bcl-2 inhibitor venetoclax was approved for treatment of chronic lymphocytic leukemia. induction of apoptosis and depletion of lymphocyte subpopulations e.g. follicular b-cells or cd4 + and cd8+ t-cells led to further exploration in autoimmune disease. methods: to establish expression levels of genes in the bcl-2 pathway, low-input rna sequencing was performed on t cells isolated from non-inflamed skin and peripheral blood of hsct recipients at 5 different time points before until 1 year after transplantation. furthermore, we analyzed blood, lung, gut and skin samples of 105 patients post allo-hsct with and without previously untreated acute or chronic gvhd by rt-pcr, flow cytometry and tissue immunofluorescence. [[p237 image] 1. bcl-2 is up-regulated in t and b lymphocytes of acute and chronic gvhd lesions.] results: rna-sequencing revealed that t cells upregulated bcl-2 upon conditioning treatment (day 0) and cells of patients who later developed gvhd failed to downregulate bcl-2 after transplantation (day+14, day+100). bcl-2 protein levels were elevated in overall leukocytes and pathogenic cell subsets including monocytes, cd8+ t lymphocytes and nkt cells showed significantly higher expression of bcl-2 in peripheral blood of gvhd patients as compared to healthy controls. these results could be recapitulated in tissue samples, where disease-promoting lymphocytes (t, b, nk, nkt) were numerically expanded and expressed bcl-2 in acute and chronic gvhd skin lesions. notably, non-pathogenic cell types such as keratinocytes did not exhibit increased bcl-2 expression compared to control samples from hsct recipients and healthy donors. while bcl-2 rna expression did not depend on type of conditioning (mac vs. ric) or gvhd grade, it correlated to disease severity and was significantly elevated in biopsies of patients with steroidrefractory gvhd. conclusions: we could show exclusive upregulation of bcl-2 in gvhd-mediating cell types in peripheral blood and tissue samples affected by gvhd, correlating to gvhd severity and response to first-line therapy. thus, bcl-2 inhibition may present a novel and urgently needed targeted therapy in treatment of steroid-refractory acute and chronic gvhd. disclosure: supported by a docmed fellowship od the austrain academy of sciences background: graft-versus-host disease (gvhd) represents a major contributor to morbidity and mortality in recipients of allogeneic hematopoietic cell transplants (hct). several therapeutic strategies exist for gvhd prophylaxis and include post-transplant cyclophosphamide (ptcy) and antithymocyte globulin (atg). while several groups have described the use of ptcy in younger patients, there is a paucity of data about the efficacy of ptcy in older individuals, particularly when combined with atg. we investigated the combined effect of ptcy with atg on transplant outcomes in older patients at princess margaret cancer centre, toronto, canada. methods: this retrospective study included all patients age ≥60 who underwent allogeneic hct for any indication at our centre between december 2013 and july 2017. overall survival (os) was calculated using kaplan-meier analysis and multivariable cox proportional hazards regression. cumulative incidence of relapse (cir) and non-relapse mortality (nrm) were calculated using competing risk regression (fine and gray method). incidences of acute (agvhd) and chronic (cgvhd) were compared using the fisher's exact test. results: of 133 patients, 84 (63%) were male. median age was 65 (range 60-74) and median follow-up among survivors was 28 months (range 6-60). acute myeloid leukaemia (aml) was the most common indication for hct (57 patients, 43%), followed by myelodysplastic syndrome (37 patients, 28%) and myelofibrosis (17 patients, 13%). eightyfour (63%) patients had a matched unrelated donor, 37 (28%) had a matched related donor and 12 (9%) had a haploidentical donor. one hundred twenty-five (94%) patients received reduced intensity conditioning. sixty-two (47%) patients received ptcy combined with atg (4.5 mg/kg) while 71 (53%) received other forms of gvhd prophylaxis. os at 2 years was 46% (95% confidence interval (ci) 37-54) in the entire cohort. patients who received ptcy with atg had a superior 2-year os compared with other gvhd prophylaxis regimens ( figure 1a ): 57% (95% ci 44-69) vs. 37% (95% ci 26-49), respectively (hr=0.6, 95% ci 0.4-0.9, p=0.02). the 2-year nrm for the entire cohort was 37% (95% ci, 29-46). patients who received ptcy with atg had a lower 2-year nrm compared to those who did not ( figure 1b ): 23% (95% ci 13-34) vs. 49% (95% ci 37-60), respectively (hr=0.4, 95% ci 0.2-0.7, p=0.002). the 2-year cir in the whole group was 24% (95% ci 17-32). use of ptcy with atg was associated with a modest increase in cir at two years ( figure 1c ): 35% (95% ci 22-49) vs. 16% (95% 8-25), respectively (hr=2.1, 95% ci 1.0-4.0, p=0.04). there was a trend toward lower incidence of grade ii-iv agvhd among patients who received ptcy with atg compared to those who did not: 15% vs. 30 % (p=0.06). the incidence of grade ii-iv cgvhd was lower in individuals who received ptcy with atg compared to those who did not: 26% vs. 45% (p=0.03). conclusions: in older hct recipients, use of ptcy combined with atg is associated with improved os, lower nrm, decreased risk of both agvhd and cgvhd and a modest increase in relapse risk. therefore the ptcy with atg combination represents an effective strategy for gvhd prophylaxis in older allogeneic hct recipients. disclosure: the authors have no conflict of interest to declare. outcome of severe graft versus host disease in pediatric patients with nonmalignant diseases after allogeneic bone marrow transplantation. a single center experience irina zaidman 1 , sigal grisariu 1 , batia avni 1 , ehud even-or 1 , bella shadur 1 , adeeb nasereddin 1 , polina stepensky 1 background: hematopoietic stem cell transplantation (hsct) remained the only curative option for many nonmalignant diseases in pediatric patients. survival after hsct has improved the last few years due to significant advancement in human leukocyte antigens (hla) typing techniques, less toxic conditioning regimens and better supportive care and resulted to 90% survival and cure in some non malignant diseases. graft-versus-host disease (gvhd) remains a major complication of hsct and leading cause of morbidity and mortality. prognosis of patients with high grade gvhd is dismal and survival rate varies between 25% to 55% in pediatric patients. methods: the retrospective study included patients with non malignant diseases who underwent allogeneic hsct at hadassah medical center from 2008 to 2018. the collected data included patient´s clinical data and transplant characteristics. the study was approved by the institutional helsinki committee. results: 182 children with nonmalignant diseases underwent 194 allogeneic bone marrow transplantations in hadassah university hospital during ten years period. fifty seven patients (31%) developed agvhd grade 1-4, twenty five of them (13.7%) grade 3-4. median age was 6.34 (range 0.37-17.7), most patients were males (17 males, 8 females). 9 patients underwent bmt from fully matched family members, 6 children were transplanted from matched unrelated donors and 10 from mismatched donors. twenty one of 25 patients with severe gvhd (83%) survived. four patients (17%) died from severe gvhd and complications of immunosuppressive treatment. 3 of 4 deceased patients were transplanted from mismatched donor, in 3 of 4 cases the age of donor was advanced, 2 of 4 patients developed severe gvhd and died after second hsct. all 4 patients were refractory to different treatment modalities. three of 4 patients died in 2012 and one in 2015, it was no death from severe gvhd in 12 patients that were transplanted and developed high grade gvhd after 2015. conclusions: the results of this study show a high survival rate of 83% in pediatric patients with non malignant diseases and severe gvhd. significant risk factors for mortality in our group included mismatched donor, advanced age of donor and second transplant. trend to better survival was observed after 2015. additional multicentral studies analyzed the outcomes of agvhd in pediatric patients with nonmalignant diseases are urgently required. background: chronic graft-versus-host disease (cgvhd) is a serious late complication after allogeneic hematopoietic stem cell transplantation (allohsct) with heterogeneous presentation and still poorly understood pathophysiology including inflammation and endothelial dysfunction. factor viii (fviii) and von willebrand factor (vwf) are coagulation factors but also known indicators of endothelial dysfunction and inflammation in different settings, and therefore could serve as interesting candidate biomarkers of cgvhd. methods: since 2013 patients after allohsct were assessed by the multidisciplinary cgvhd team at the university hospital center zagreb, croatia, using established nih cgvhd-related measurements. an extensive history, physical and laboratory evaluations were performed, including fviii, vwf:ag and vwf:ac analysis. descriptive statistic and non-parametric analyses were performed. variables that showed significant univariate correlations were used in multivariate logistic regression (mlr) to identify the most predictive for fviii, vwf:ag and vwf:ac in cgvhd patients. results: 70 cgvhd patients and 41 controls (subjects after allohsct without cgvhd) were analysed. median age of cgvhd patients was 42 (9-65) years, 50% females, 91.5% underwent allohsct for hematologic malignancies, 55.7% had myeloablative conditioning and 52.9% matched related donor. median time from hsct to study was 450.5 days and from cgvhd diagnosis to study 82 days. there were no demographic neither transplant related significant differences between cgvhd patients and controls beside stem cell source (peripheral blood 71.4% vs 51.2%, p=0.041) and history of acute gvhd (70.0% vs 22.0%, p< 0.001). majority of patients had moderate (52.9%) or severe (42.6%) nih global cgvhd score, 57.2% active cgvhd by clinician´s impression. median number of organs involved by cgvhd was 3 (1-6), and the most frequently involved organs were mouth, skin and eyes (52.0% each). cgvhd patients compared to controls had higher fviii levels (median 206 (52-453)% vs 182 (51-406)%, p=0.044, reference range 50-149%) and higher vwf:ag (median 261.6 (76.6-601)% vs 203.2 (51.9-600)%, p=0.030, reference range 50-160%), while vwf: ac showed a trend toward higher levels among patients (median 253.4 (54-601)% vs 178 (48.6-601)%, p=0.084, reference range 50-150%). patients had higher ggt (p=0.002), lower anticardiolipin igg (p=0.001) and igm (p=0.003), and lower albumin (p=0.018) than controls, without differences between other laboratory parameters. univariate analysis showed that among cgvhd patients higher fviii was associated with worse karnofsky score (ks) (p=0.031) and performance score (ps) (p=0.030), higher leukocytes (p=0.031), cholesterol (p=0.003), triglycerides, ast, alt, ggt, ldh, and lower albumin. higher vwf:ag and vwf:ac in cgvhd patients were associated with worse ks and ps (p< 0.001), with more active cgvhd (p< 0.001), worse nih cgvhd liver (p=0.042; p=0.039) and nih cgvhd mouth (p=0.012; p=0.009), higher total nih score (p=0.044; p=0.005), higher number organs involved (p=0.013; p=0.003), higher esr, monocytes, ddimers, ast, alt, ggt, ldh, triglycerides, β-2-microglobulin, ferritin, total proteins, iga and lower albumin. mlr analysis showed leukocytes (p=0.018) and cholesterol (p=0.010) as the strongest predictor of fviii (r2=49.8%; p< 0.001), while strongest predictor of vwf: ac was number of organs involved by cgvhd (r2=71.7%; p=0.031). conclusions: results of this study detected high fviii and vwf levels in cgvhd patients with possible reflections to cgvhd manifestations, what needs to be further confirmed in larger longitudinal studies. disclosure: this work was supported, in part, by the unity through knowledge fund project entitled "clinical and biological factors determining severity and activity of chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation", and also, in part, by the croatian science foundation project entitled "new biomarkers for chronic graft-versus-host disease". antonela samardzic -work financed by the croatian science fondations`"young researchers`career development project -training of doctoral students" background: thrombotic microangiopathy (tma) is a severe complication of allogeneic hematopoietic cell transplantation (hct) with multisystem involvement. a few recent reports have recognized evidence of tma in the intestinal vasculature (intestinal tma/itma) of patients with graft-versus-host disease (gvhd) with or without tma. we aimed to identify patients with itma and describe histological, clinical and prognostic features. methods: we prospectively evaluated available endoscopic samples (stomach and/or colon) from consecutive adult hct recipients for previously described histopathologic signs of itma (january 2017-september 2018). systemic tma was diagnosed according to the international working group criteria. we compared findings among 3 clinical groups: gvhd/systemic tma, gvhd/no systemic tma and no gvhd/no tma. results: we studied 20 patients, 5 classified as gvhd/ systemic tma, 11 gvhd/no systemic tma and 4 no gvhd/no tma. baseline transplant characteristics (age, donor, hla matching, conditioning) did not differ significantly among groups. histological features of itma, including loss of glands, total denudation of mucosa, apoptosis and detachment of endothelial cells, intraluminal fibrin, intraluminal microthrombi and mucosal hemorrhage were found in 6 patients. previously described features of intraluminal schistocytes were not observed in our patients. interestingly, loss of glands, total denudation of mucosa, apoptosis and detachment of endothelial cells were also found in patients with gvhd and no itma, suggesting that these features are not pathognomonic of itma. among 6 itma patients, two patients were classified in the clinical group of acute gvhd/systemic tma, while the other 4 patients had clinical and histopathological features of itma and severe grade iii-iv steroid-refractory acute gvhd (3 patients) or extensive chronic gvhd (1 patient) but no evidence of systemic tma. in the majority of patients (5/6), itma occurred during the early posttransplant period at 1.7 (0.9-4) months. clinical features (gastrointestinal bleeding, diarrhea, pain, nausea) presented no differences between patients with or without itma. prognosis was poor for patients with itma who suffered from a significantly higher mortality rate of 83% compared to the rest patient population (p=0.014). with a median follow-up of 11.1 (2.1-67.5) months, 1year overall survival probability (os) was 22.2 for itma, 55% for gvhd and 60% for systemic tma. unfavorable predictive factors for os were itma (p=0.048), hla mismatched donors (p=0.008) and gastro-intestinal bleeding (p=0.021). conclusions: intestinal tma has emerged as a novel distinct entity in patients with gvhd and/or systemic tma. distinct histological features may be useful in differential diagnosis of these severe hct complications. mortality rates higher than those of systemic tma highlight the need of proper recognition of itma that needs to be further studied in terms of diagnostic and therapeutic potential. disclosure: e.g. was supported by the european hematology association clinical research grant. the remaining authors declare no competing financial interest. the beneficial effects of thrombomodulin gene polymorphisms after hematopoietic stem cell transplantation background: chronic graft-versus-host disease (cgvhd) remains the major cause of late morbidity and mortality after allogeneic blood and marrow transplantation. treatment options for cgvhd, particularly its sclerotic forms remain limited. active hedgehog (hh) signaling was shown as a therapeutic target in both mouse and human cgvhd, with limited efficacy and significant toxicities described in a published clinical trial (defilipp, 2017). methods: adult patients with steroid refractory sclerodermatous cgvhd, defined as requiring >0.5 mg/kg/day of prednisone dose equivalent (pde), or need for second-or third-line therapy beyond corticosteroids and calcineurin inhibitors or sirolimus were eligible for this open label study of vismodegib, a first generation hh pathway inhibitor. primary endpoint was failure free survival, defined as absence of non-relapse mortality, no recurrent malignancy, steroid dose at 6 months =< 0.2 mg/kg/day of pde, and no addition of new systemic treatment. vismodegib was administered orally for 6-12 months, with dose reductions at development of toxicities. peripheral blood mononuclear cells were isolated from samples collected at treatment initiation and every three months thereafter. the immune profile of circulating b cells was analyzed by flow cytometry and t helper polarization by qrt-pcr of sortpurified cd4+ t cells. results: at the time of interim analysis, 6 patients were evaluated. 3 patients completed 6 months of treatment and five patients completed 3 months of treatment. therapy was discontinued in 3 patients prior to 6 months due to treatment-related (n=2) and unrelated (n=1) side effects. most patients experienced grade 2 toxicities (muscle cramps and dysgeusia), with only a single grade 3 toxicity (weight loss). 5 patients who completed 3 months of therapy demonstrated partial response, and overall, the primary endpoint was reached in 50% (3/6) of patients. in 2 patients who discontinued vismodegib, cgvhd worsened acutely after discontinuation. correlative analysis of immune cellular subsets in peripheral blood in 4 paired samples (pre-treatment and month 3 of therapy) documented modulation of b cell subsets pathogenic in cgvhd (pregerminal center and plasmablast-like b cells) and diminished t helper 2 polarization in cd4 t cells. conclusions: overall, use of vismodegib was associated with potential clinical efficacy in sclerodermatous cgvhd with possible mechanistic evidence arising in correlative studies. while side effects were common, further studies of hh inhibition in cgvhd are warranted. future studies should employ adjusted dosing regimens, along with supportive care interventions to offset side effects, and testing of novel hh inhibitors with enhanced safety profiles. clinical background: graft-versus-host disease (gvhd) results from recognition of host antigens by donor t cells following allogeneic hematopoietic stem cell transplantation (sct). we tested the hypothesis that somatic neomutations occurring after sct from donor and/or recipient dna may trigger gvhd. methods: we longitudinally analyzed both constitutive and somatic mutations by whole exome sequencing (wes) in 2 patients who received sct from a sex-matched hlaidentical sibling for npm1 mutated acute myeloid leukemia (pt#1) and jak2 v617f mutated primary myelofibrosis (pt#2). both patients were initially refractory to alloreactivity, i.e. not displaying any signs of gvhd, even after several donor lymphocyte infusions. acute gut gvhd finally occurred after a further dli preceded by a lymphodepleting chemotherapy. in pt#2, gvhd correlated with a graft-versus-tumor effect. wes was performed on dna from recipient saliva and donor pbmcs (germline samples) and from sequential post-sct pbmcs samples on a hiseq2500 illumina with 2x100bp paired-end reads at a mean depth of coverage of 190-210x. germline and somatic mutations were determined using in-house bioinformatic pipelines (named ewok from the curie institute and smaug from the henri mondor hospital), using briefly gatk as variant caller for germline samples, and a combination of 3 variant callers for matched normaltumor pairs. we adjusted parameters to detect somatic mutations at a minimal variant allelic frequency (vaf) of 5% compared to recipient and donor germline for all variations (minimal coverage = 8x for germline and 5x for tumor sample). results: wes allowed detecting somatic driver mutations explaining aml and pmf for both patients in the initial timepoint and all these driver mutations disappeared at the following timepoints. as expected, the somatic variant rate was 10x higher in pt#1 with aml than in pt#2 with pmf at each timepoint, except for the final gvhd timepoint. indeed, at this final point, the somatic variant rate dramatically decreased by 80% as compared to previous timepoints. by subtracting variants detected pre-and post-sct from those identified at the ultimate time-point of gvhd occurrence, we created 2 sets of 5 and 7 variants respectively for each patient (keeping only variants with at least 4 reads of mutated dna). these variants can be classified in 2 categories: (i) those with only with a slight increase at time of gvhd, i.e. ≤ 2-fold compared to highest previous vaf (lrrc43, or8u1, or10g9, alpp, frg1, frg2b and lilrb3 genes), and (ii) those with a significant increase at that time, i.e. > 2-fold compared to highest previous vaf (phf2, smpd1, ercc8 and krtap9-1 genes). none of the variants or genes involved was common between the 2 patients. ontology classification of mutated genes showed the implication of some of them in cell death, regulation of map kinase activity, mrna splicing and immune system process, making them good candidates for further studies. identification of variants appearing pre-gvh and turning off at time of gvhd is ongoing to unveil putative neoantigens that could trigger the alloreactive response. conclusions: using a comprehensive, pre-and post-sct, wes of donor/recipient pairs, we identified several neomutations from donor and/or recipient dna correlating with gvh/gvt effect development. disclosure results: a total of 169 patients experienced cgvhd, and mild, moderate, and severe cgvhd were observed in 66, 67, and 36 patients, respectively. the 2-year cumulative incidence of total cgvhd was 60.5% (95% ci, 54.7-66.3%), and the 2-year cumulative incidence of moderate to severe and severe cgvhd was 36.6% (95% ci, 30.9-42.3%) and 12.7% (95% ci, 8.8-16.6%), respectively. the patients who had 3 loci mismatched had a higher 2-year cumulative incidence of total cgvhd (66.2% vs. 54.5%, p=0.025) and moderate to severe cgvhd (42.3% vs. 30.6%, p=0.028) compared to those of the patients who had 1-2 loci mismatched. the patients who had maternal donors had a higher 2-year cumulative incidence of moderate to severe cgvhd (50.8% vs. 32.7%, p=0.018) compared to that of the patients who had other donors. the patients who had grade iii to iv acute graft-versus-host (agvhd) had a higher 2-year cumulative incidence of total cgvhd (91.7% vs. 50.0%, p< 0.001) and moderate to severe cgvhd (70.8% vs. 26 .3%, p< 0.001) compared to those of the patients without agvhd. in multivariate analysis, grade iii to iv agvhd was the only independent risk factor for total cgvhd (hr=2.6, 95%ci, 1.6-4.2; p< 0.001) and moderate to severe cgvhd (hr=3.8, 95%ci, 2.1-6.7; p< 0.001). in the model excluding agvhd, maternal donor was the risk factor for moderate to severe cgvhd (hr=1.5, 95%ci, 1.1-2.3; p=0.030). conclusions: we observe that severe agvhd was the most important risk factors for cgvhd after haplo-hsct, and further interventions should be considered in these patients to prevent severe cgvhd. disclosure: none of the authors have any potential financial conflict of interest related to this manuscript. background: extracorporeal photopheresis (ecp) has been successfully used for the treatment of graft-versus-host disease (gvhd). ecp therapy might restore the balance between effector and regulatory cells which is severely impaired in gvhd. nk cells are the first lymphocyte subset to be reconstituted after allogeneic hematopoietic stem cell transplantation (allo-hsct). as an important innate immune cell population, nk cells can temporally bridge the transient period of t-cell deficiency post allo-hsct, by protection from opportunistic infections and prevention of leukemic relapse by graft-versus-leukemia (gvl) effect. nk cells not only preserve homeostasis through targeted killing of allo-reactive t cells and thereby control gvhd but also enhance inflammation by secretion of tnf-α and ifn-γ and thereby promote gvhd. therefore, we investigated here the role of nk cells in gvhd patients under ecp therapy. methods: thirty four patients with steroid-refractory/ resistant agvhd ≥ ii°and moderate to severe cgvhd received ecp therapy which performed according to the guidelines. glucksberg and nih criteria were used for clinical staging of agvhd and cgvhd under ecp therapy, respectively. the comprehensive phenotypical analysis of nk cells was evaluated by multicolor flow cytometry. nk activity in terms of killing function, cytokine release capacity and proliferation function was monitored by chromium-51 release assay, intracellular cytokine staining and cfse staining, respectively. results: five different nk cell subsets were defined based on cd56 and cd16 expression. cd56 bri nk cells displayed an immature and activation profile with high expression of cd62l and nkg2d. agvhd patients had a higher frequency of cd56 bri nk cells when compared with hds and cgvhd patients, who were characterized by significant increase of the cd56 dim cd16 + and cd56 -cd16 + nk cell subsets with high expression of differentiation markers cd11b and cd57. of note, cd56 bri cd16 -nk cells could serve as a novel predictive biomarker for the response of agvhd patients to ecp treatment. in responding agvhd patients, an increase of cd56 bri nk cells was observed already during the early ecp treatment phase, suggesting immune reconstitution. after priming of the progenitors, ecp could differentiate immature cd56 bri nk cells into mature cd56 dim nk cells with reduction of cd62l on cd56 bri nk cells. moreover, cd56 dim nk cells could further be matured through upregulation of cd57 expression by ecp. notably, ecp therapy could shift the nk cells from a cytotoxic to a regulatory phenotype within the cd56 bri nk cells. in spite the immunomodulatory effect of ecp on nk cells, nk activity could be kept intact under ecp therapy. the killing activity of nk cells was stable as confirmed by a 51 cr release assay. ecp therapy had no negative effect on the quantity and quality of cytokine release by nk cells upon k562 stimulation. especially, the polyfunctionality of nk cells was not altered significantly by the ecp therapy. conclusions: nk cells play an important role in gvhd and could serve as a predictive cell population for the clinical response to ecp therapy. in the current study, ecp influenced the differentiation, maturation and education of nk cells ameliorating gvhd without comprising the antiviral immune defense and gvl effect. disclosure: the authors declare no competing financial interests, except the following: therakos mallinckrodt gave a financial support to as and ms for the documentation of the clinical course and for the analysis of immune cells of the patients, pw has honoraria and membership on advisory boards for sanofi-aventis. abstract withdrawn. cyclosporine levels >200µg/l on day 10 post-transplant was associated with significantly reduced acute graftversus-host disease following allogeneic hematopoietic stem cell transplantation monica bianchi 1 , dominik heim 1 , claudia lengerke 1 , martina kleber 1 , dimitrios tsakiris 1 , jakob passweg 1 , alexandar tzankov 2 , michael medinger 1 background: acute graft-versus-host disease (agvhd) remains a major complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). affected patients, especially with steroid-refractory agvhd, have a very poor prognosis. prophylaxis with cyclosporine a (csa) is the backbone of gvhd prevention in most conditioning regimens. methods: in a retrospective analysis of patients treated with allo-hsct, we correlated csa levels at the day of transplantation (day 0) and day +10 with the incidence of acute and chronic gvhd. we postulate that higher target csa levels >200μg/l will result in a lower incidence rate especially of agvhd after allo-hsct. results: we assessed 660 patients with either aml n=248, lymphoma/myeloma n=127, mds/mpn n=124, all n=79, cll n=36, cml n=23, or bone marrow failure n=22. in patients with clinically relevant agvhd grade ≥2, mean csa levels was lower on day 0 and day +10 (142±88 μg/l; and 183±64 μg/l; respectively) compared to patients without agvhd (156±81 μg/l; and 207±67 μg/l; respectively; day 0: p=0.003; day +10: p=7.57 x 10 -9 ). in patients with csa level < 200 μg/l, the incidence of agvhd was significantly more frequent compared to patients with csa levels >200 μg/l [(234/356; 66%) versus 91/248 (37%); p= 1.34 x 10 -12 ]. in patients with cgvhd, there was no significant difference between csa levels < 200 μg/l (128/330) compared to csa levels >200 μg/l (96/ 233; p=0.312). the optimal csa cut-off level for the prevention (i.e. roughly 50% incidence reduction) of agvhd was >201 μg/l at day 0 and >195 μg/l at day +10 ( figure 1 ) in a competing risk analysis, time to agvhd grade ≥2 (using death of other causes as competing risk) was associated with csa levels >200 μg/l on day 0 and on day 10, unrelated donors, myeloablative conditioning (mac), and for the diagnosis lymphoma/myeloma. conclusions: our data support close monitoring with active adjustments of csa dosing to maintain therapeutic csa levels above 195 μg/l in the first 10 days of allo-hcst to reduce agvhd. disclosure: noting to declare. liposomal cyclosporine a for inhalation (l-csa-i) to treat bronchiolitis obliterans syndrome: novel formulation with therapeutic potential for patients with bos following allo-hsct noreen roth henig 1 , emilie hofstetter 2 , dominik kappeler 3 , gerhard boerner 3 background: bronchiolitis obliterans syndrome (bos) is a rapidly progressive lung disease caused by t-cell mediated inflammation that leads to blockage of bronchioles, leading to respiratory failure and death shortly after diagnosis. approximately 4% to 10% of patients who undergo allogeneic hematopoietic stem cell transplant (allo-hsct) will develop bos, with 72-100% developing bos as a respiratory form chronic graft-vs-host disease (cgvhd) in addition to other signs of cgvhd. mean time to bos diagnosis ranges from 273 to 547 days post-transplant. the histopathology of bos after allo-hsct and lung transplantation is identical. early studies of l-csa-i for the prevention of bos in lung transplant recipients demonstrated therapeutic benefit. l-csa-i is a novel, liposomal formulation of cyclosporine administered via a pari investigational eflow â nebulizer which delivers a potent immunosuppressant to the site of disease. pharmacokinetics and tolerability of l-csa-i is presented. methods: retrospective review of two clinical studies of l-csa-i (isotonic, 4mg/ml) for bos associated with lung transplantation. both studies had a control arm and results reported here are for patients who received l-csa-i. subjects received 5mg (single lung transplant) and 10mg (double lung transplant) bid via inhalation. blood samples for pharmacokinetic analysis of cyclosporine a concentrations were collected before inhalation, immediately after inhalation, and thereafter in intervals of 15, 30, 60 min and 2, 4, 8 and 12 hours. local and general tolerability of l-csa-i was investigated. results: between the two studies, 85 subjects received either 5 or 10 mg bid of l-csa-i. pharmacokinetic models predict a constant drug level in the lung. maximum serum cyclosporine a concentration after inhalation was 57.42 ± 34.26 ng/ml. trough levels for up to 2-years of daily administration was 4-5 ng/ml with no evidence of accumulation following repeated exposure. tolerability data was assessed from 1068 patient-month exposure to l-csa-i. reported symptoms were: pharyngeal soreness 1%; cough 22%; dyspnoea 7%; and wheezing 1%. no subject discontinued due to intolerability. inhalation time is on average 9-13 min. conclusions: l-csa-i provides high and constant concentrations to the airways of the lungs and the site of bos. l-csa-i is well tolerated in lung transplant patients. use of l-csa-i instead of augmentation of systemic csa reduces the total drug exposure. a multicentre phase 2 safety and exploratory efficacy trial for the treatment of bos in allo-hsct recipients is underway. disclosure background: there are a number of biomarkers that predict non-relapse mortality (nrm), graft-versus-host disease (gvhd) and relapse incidence (ri) after conventional gvhd prophylaxis based on calcineurin inhibitors with or without antithymocyte globulin. currently there is limited data whether the conventional predictive biomarkers work with posttransplantation cyclophosphamide (ptcy) prophylaxis. methods: prospective single-center study in 2015-2016 enrolled 79 adult patients with acute leukemia in cr (34% with all, 66% with aml). 26 received matched related bone marrow (bm) graft with single-agent ptcy and 53 received unrelated peripheral blood stem cell graft (pbsc) with ptcy, tacrolimus and mmf. the grafts were studied by flow cytometry (facs aria ii, antibodies by miltenyi biotec). the following populations were analyzed: cd3, cd4, cd8, cd16cd56, nkt, inkt, treg, double-positive t-cells, double-negative t-cells, tcralpha/beta, tcr v11 memory cells. the crypreserved plasma from were analysed by elisa (commercial kits by ebioscience and critical diagnostics) for vegf a soluble tnf receptor (stnfr), il-8, il-6, soluble il-2 receptor, st2, il-17 and stnfr. the above mentioned biomarkers were tested in logistic regression with roc analysis, assays with auc>0.600 were selected for analysis in fyne-gray regression with competing risks. cut off levels were determined for significant parameters. results: median follow-up was 19 months (range 12-30). in the whole group overall survival (os) was 77%, eventfree survival (efs) 73%, grade ii-iv acute gvhd 13%, moderate and severe (m&s) chronic gvhd 20%, nrm 6%, mortality in patients with gvhd 0%, ri 20%. there was no difference between bm/related and pbsc/unrelated grafts in the incidence of gvhd, nrm and ri (p>0.11). the only significant predictor of acute gvhd were low levels of il-8 level on day+7 (p= 0.0490, 0% vs 15% with the cut off 40 pg/ml). m&s chronic gvhd was predicted only by the high percentage of inkt cells in the graft (p=0.0003, 31% vs 12% with the cut off 0.03%). there was a correlation between il-8 levels and number of nk cells in the graft (p=0.02). nrm was related to infectious complications, nonetheless high levels of vegf a on day 0 (p= 0.0458, 16% vs 0% with the cut off 100 ng/ml), st2 on day+30 (p= 0.0041, 11% vs 0% % with the cut off 40 ng/ml) and low percentage of cd16+cd56-cells in the graft (p=0.0215, 22% vs 2% with the cut off 1.3%). the identified biomarkers of nrm had no association with the pre-transplant crp and ferritin levels (p>0.4). the only significant parameter for ri was the level of cd34 cells in the graft (p=0.0434). none of the identified biomarkers significantly predicted overall survival (p>0.09). conclusions: in the related and unrelated grafts with ptcy the study of biomarkers has low clinical utility due to very low gvhd-related mortality. however st2 and vegf a can predict infection-related mortality. also the study verified previous observations that high level of il-8 is associated with reduced gvhd incidence after ptcy 1 and identified the importance of nk and inkt cells in the induction of tolerance with ptcy. references background: hematopoietic cell transplantation (hct) is the only curative approach for many hematological malignancies but life-threatening toxicities, such as graft-versushost disease (gvhd) and infections, still limit its fullpotential impact on the disease. strategies for keeping allohsct more effective and safe are needed in order to reduce morbidity while improving its immunological effect to control disease relapse. post-transplant cyclophosphamide (ptcy) has been demonstrated to improve acute gvhd (agvhd) and chronic gvhd (cgvhd) control in allogeneic bone-marrow hct from identical and haploidentical donor. the use of ptcy, after peripheral blood stem cell transplantation (allopbsct) from hla-matched unrelated/related donors, has been investigated by our group in a clinical trial (nct 02300571) and preliminary results were published last year. here we report updated efficacy and safety data about the expanded cohort of patients treated with ptcy followed by tacrolimus and mycophenolate mofetil (t/mmf). methods: we analysed data about 71 consecutive patients with high-risk hematologic malignancies received allopbsct from hla-matched unrelated/related donors between march 2011 and august 2018. gvhd prophylaxis was ptcy 50 mg/kg (days +3 +4), tacrolimus from day +5 and mmf from day +5 to day +28. primary objectives were cumulative incidence of agvhd and cgvhd. secondary objectives were event-free survival (efs), cgvhd-efs, overall survival (os) and non-relapse mortality (nrm). results: patients median age at transplant was 50 (range 19-74) years. 34 (48%) patients were transplanted in first complete response (cr), 16 (23%) patients in second/third cr, the others in disease control. a median dose of 7.0 (range 2-15) x 10^6 cd34/kg was infused. primary graft failure was observed in one patient. all patients were off mmf on day +28, the median day of tacrolimus discontinuation was 112 (range 39-467). eight out of 71 (11%) patients developed agvhd, 6 (8%) of them were grade ii-iii; median day of onset was day 68 (range . no grade iv was observed. no cases of late-onset agvhd were reported. cumulative incidence of cgvhd was 8% (6/ 71), median day of onset was 162 (range 140-268). systemic treatments were required, but all patients were able to discontinue immunosuppression (is). with a median follow-up of 22 (range 4-94) months, efs was 58%, cgvhd-efs was 52% and os was 72%. non-relapse mortality (nrm) was 3% (2/71): 2 patients died because of multidrug resistant bacteria septicemia. nowadays 41 patients are alive with no evidence of disease, being continuously off is and completely reintegrated in their normal daily life activities. conclusions: the updated reported results confirm, in a larger cohort of patients with a longer follow-up, that ptcy after pbsc-hct is highly active in agvhd and cgvhd prevention with extremely limited nrm. this strategy, not only allowed earlier discontinuation of immunosuppression, but also reduced the overall time of exposure to is for most of the patients. all these features might contribute, in the future, to transform hct into a safe immunologic platform that may be combined with advanced form of cellular therapies (car-tcells), aiming to increase safely the graftversus-tumor effect. clinical methods: pediatric patients (0-21 years) with nmd undergoing unrelated hct were eligible for this single center, phase i trial. following reduced intensity conditioning, abatacept (10 mg/kg iv on days -1, +5, +14, +28) was added to standard gvhd prophylaxis (cyclosporine, mycophenolate mofetil [mmf]). patients were followed for 2 years for standard hct outcomes. [[p257 image] 1. figure 1 results: since june 2014, 10 patients have been enrolled and transplanted (table 1 , excluding #7). donor source was bone marrow in all. with median follow-up of 3.5 years, 8 of 10 patients survive without disease. initial engraftment was successful in 9, at a median of 20 and 16 days, for neutrophils and platelets respectively. one patient (5) had secondary graft rejection in the setting of viral reactivation (cmv/ebv), with successful engraftment following a 2 nd unrelated hct. in 8 engrafted patients, myeloid (cd33) chimerism was 100% at all timepoints; t-lymphoid (cd3) chimerism was mixed but reached >/=95% (figure 1 ). one patient (7) with saa had primary graft rejection in the setting of inadequate tnc dose (0.5 x 10 8 /kg) and died from marrow aplasia/infection despite 2 nd hct. a second death from wilms' tumor occurred 17 months post successful hct, in a patient (1) with dba and constitutional chromosome abnormality. except patient 7, all patients received 4 doses of abatacept, which was well tolerated, with all severe adverse events expected for a hct population. cmv and ebv reactivation occurred in 3 patients each, with resolution using standard anti-viral therapy. one patient (6) was diagnosed with ebv-driven post-transplant lymphoproliferative disease, which responded to rituximab and immune suppression withdrawal. no patients developed severe acute (grade iii-iv) or chronic gvhd (table 1) , and no patients required systemic immune suppression at >1 year. conclusions: these preliminary data suggest that abatacept can be safely added to cyclosporine and mmf gvhd prophylaxis in pediatric patients with bone marrow failure undergoing unrelated donor hct, with encouraging rates of gvhd despite half of patients having a mismatched (7/8) donor. given the higher risk of graft rejection in this non-malignant cohort, rejection (in addition to gvhd) will be a primary focus in our subsequent multi-center, phase 2 trial. clinical trial registry: clinicaltrials. gvhd and may to have be separated from from toxicity to infectious complications in the early phase after allohsct. methods: from our files we identified 65 patients which had upper gastrointestinal tract endoscopy after allohsct in with biopsies were taken from the esophagus, stomach and duodenum simultaneously. of these patients 14 were excluded because of infection, reflux disease or drug toxity and the remaining 51 patients were included in our study. we evaluated the routine stained esophageal biopsies, applied a grading scheme and compared the histological findings with those within the stomach and duodenum, the endoscopic findings and the clinical course. results: in 32 of 51 biopsy samples of the esophagus, we identified histological features of acute gvhd, ranging from vacuolar degeneration (grade 1) and single-cell apoptosis (grade 2) to the formation of clefts (grade 3) and mucosa denudation in advanced cases (grade 4), resembling epithelial lesions in acute gvhd of the skin. these findings correlated with gvhd involving the stomach and duodenum and the clinical manifestations of gvhd in other organs. endoscopically patients with gvhd revealed signs of inflammation, ranging from erythema to ulceration in the more advanced cases, sometimes reminiscent of reflux or infection. clinically these patients had abdominal discomfort ranging from inappetence to nausea, accompanied by emesis or diarrhea and weight loss. conclusions: we have shown that acute esophageal gvhd occurs after allohsct and is correlated with acute gvhd in stomach and duodenum. it could be diagnosed and graded histologically. the endoscopic findings are signs of inflammation. our results may help to establish the histological diagnosis of acute gvhd using endoscopic biopsies from the esophagus and to explain the alterations observed in the esophageal mucosa in patients after allohsct. [ background: intestinal acute graft versus host disease (agvhd) is a major thread after allogenic hematological stem cell transplantation (allohsct), with a high mortality in patients which were refractory to steroid treatment in particular. recent papers point to a correlation of histological grading of intestinal gvhd and prognosis in patient after allohsct. however a comparison with clinical scores has not been performed so far. methods: in this analysis, retrospective data from 89 patients who underwent endoscopy due to clinical signs of agvhd (day +20 to +200 after allohsct) were evaluated. of each patient least 3 biopsies from different sites of the colon which were taken simultaneously. of each biopsy series the maximum histological grad of agvhd according to the lerner scheme was obtained and compared with the glucksberg stage of the lower gastro intestinal tract (gslgi) and the overall glucksberg grade (ogg). these three grades were compared for non-relaps related mortality using the log-rank test and for sensitivity to steroid treatment applying the receiver operating characteristic for the patients who received steroid treatment. for these patients the non-relaps related mortality for responder and non-responder were calculated using also log-rank test. results: the histological grade strongly correlated with the survival (p=0.009). a statistical significant correlation was also found for the gslgi (p=0.02), whereas the ogg revealed no significant correlation (p=0.09). non-relaps related mortality was mainly related to infection or sepsis (in 32/56 patients who died). -eighty-one of the patients received steroid therapy. the sensitivity to the steroid therapy correlated with each of the three scores (p< 0.0001) but was the strongest for gslgi (area under the curve (auc) 0.829), compared to ogg (auc 0.795) and the histological score (auc 0.691). the survival of the patients, which were sensitive to steroid treatment was significantly better than those of steroid refractory patients (p=0.005). conclusions: we found that histological and clinical grading in patients after allohsct with intestinal gvhd was correlated with survival and respond to steroid treatment. histological scoring may predict survival more precisely than ogg and gslgi but did not add substantial information to the prediction of treatment response. [ emerging evidences suggest that regulatory b cells (bregs) play essential roles in inflammation, autoimmune diseases and tumors. few data exist about the role of bregs in the contest of hematopoietic allogeneic stem cell transplantation (hsct). some authors have observed that bregs from patients with chronic graft-versus-host disease (cgvhd) were less frequent and less likely to produce il-10 than bregs as compared to healthy donors or patients without cgvhd. these findings suggest that bregs may be involved in cgvhd pathogenesis. the purpose of our study was to evaluate a possible role of b cell subsets on gvhd occurrence. methods: lymphocyte subset enumeration was performed by aquios cl flow cytometer (beckman coulter), a quantitative automated analyzer that performs two diagnostic panels: tetra-1 cd45-fitc/cd4-rd1/ cd8-ecd/cd3-pc5 and tetra-2 cd45-fitc/cd56 +cd16-rd1/cd19-ecd/cd3-pc5. b cell subsets (memory, mature and transitional b cells) on peripheral blood samples were analyzed by aquios designer software, a tool for the creation of user-defined applications. panel-1 cd19-fitc/cd10-pe/cd38-ecd/cd24-pc5/cd27-pc7 and panel-2 cd19-fitc/cd5-pe/cd38-ecd/cd24-pc5/ cd20-pc7 were specifically designed by beckman coulter for our center. the flow cytometric analysis was performed as follows: in donors and patients at basal level; on graft products and in patients at days +30, +60, +90, +120 after hsct. statistical significance was assessed with prism software (graphpad) by mann withney test. p < 0.05 was considered statistically significant. results: actually we enrolled 84 patients submitted to hsct in our center from november 2017. a preliminary statistical analysis was performed on 55 patients. stem cells source was peripheral blood (pb) in 27 cases and bone marrow (bm) in the others 28. the conditioning regimen was myeloablative in 26 patients and ric in 29 patients. agvhd was diagnosed in 22 patients (40%). no associations were found between b cell subsets in donors and patients at baseline and the occurrence of agvhd. however we found a higher median percentage of transitional b cells in graft products in patients without agvhd (9.6%, 4.5-26.7) compared to patients with agvhd (6.9%, 1.5-21.1) (p=0.02, fig 1a) . in addition, patients without agvhd showed a lower median percentage of memory b cells (24.2%, range 11.6-49) in graft product as compared to patients with agvhd (34.2%, range 10.3-68.15) (p = 0.03, fig.1b ). finally in the subgroup of patients receiving pb as stem cell source we observed a higher percentage of cd3+ lymphocytes in graft product in patients with agvhd (75%; range 68-80) compared to patients without agvhd (71%; range 62-78) (p=0.01). in the monitoring of b cells reconstitution we observed that cd19+ events did not appear before day +90 after hsct and these were b transitional immature events predominantly. conclusions: our data suggest a possible protective link between transitional b cells and agvhd development. these results data need to be confirmed in a larger cohort of patients. moreover, it will be interesting to evaluate the relationship between transitional b cells at day +90 and the occurrence of cgvhd. clinical background: agvhd is a major complication of allogeneic hematopoietic stem cell transplant (hsct) and a risk factor for post-hsct mortality. the objective of this analysis is to describe patients with agvhd who had a suboptimal response to corticosteroids. methods: patients who developed ibmtr severity index ii-iv agvhd after first hsct between 1/1/14 to 6/ 30/16 were included in an ongoing chart review at centers in the united states. patients who had ever participated in a gvhd prophylaxis trial or used jak inhibitors were excluded from the study. suboptimal response to corticosteroids was defined as use of additional systemic anti-gvhd therapy, inability to taper high-dose steroids (≥1 mg/ kg) by ≥25%, or tapered corticosteroids by ≥25% but not to < 10 mg/day. results: the analysis included 64 patients with suboptimal response to corticosteroids. mean age was 55 years; 66% were male. median time from transplant to agvhd diagnosis was 33 days. at the time of maximum agvhd grade, 33% of patients were grade ii and 66% were grade iii-iv; 58% had lower gi involvement, and 59% had ≥2 organs involved. from time of diagnosis to maximum agvhd grade, 52% of patients had new organ involvement or an increase in agvhd grade. median time from diagnosis to maximum grade was 5.5 days, and was 2.5 days for patients with lower gi involvement. systemic corticosteroids were initiated on the day of diagnosis for 73% of patients. average starting daily dose was 93 mg (1.1 mg/kg) for prednisone and 136 mg (1.6 mg/kg) for methylprednisolone. steroid dose was increased for 44% of patients during follow-up; 80% were unable to taper below 10 mg/day. among patients who received additional systemic anti-gvhd therapy (n=32), 41% increased their corticosteroid dose before initiation of additional anti-gvhd therapy. median time from initiation of corticosteroids to additional therapy was 16.5 days. frequently used therapies were mycophenyalate mofetil (25%), atg (16%), extracorporeal photophoresis (13%), tocilizumab (13%), etanercept (9%), and sirolimus (9%). agvhd recurred in 31% of patients and was managed by increasing corticosteroid dose in 70% of patients. 44% had any infection within first 100 days post-hsct. forty patients (63%) required hospital readmission(s); 40% had ≥2 readmissions within 100 days post-hsct, with a mean inpatient lengthof-stay of 28 days. relapse of underlying malignancy was reported for 13 (20%) patients. two-thirds (66%, n=40) patients died at a median of 87 (interquartile range (iqr): 44-180) days from agvhd diagnosis; a higher proportion (76%) of patients with maximum grade iii-iv agvhd died at a median of 63 (iqr: 41.5-186.5) days; majority (81%) of patients with lower gi agvhd died at a median of 68.5 (iqr: 44-137) days. conclusions: a majority of patients with agvhd who had suboptimal response to systemic corticosteroids had severe and rapidly progressing disease and resulted in a high mortality rate (66%); progression was more rapid and mortality increased for patients with lower gi involvement. most patients required readmission to the hospital with extended length-of-stay. an urgent need exists for effective and tolerable therapies that quickly resolve life-threatening agvhd in early stages of disease. disclosure results: median time to onset of bo from allohct was 6.9 months (range 0.6-31.2). previous acute gvhd in 52.2% (n = 24) [grades iii-iv 29.2% (n = 7)]. in 23.9% (n = 11) cgvhd had exclusive lung involvement, while the other 35 patients (76%) had other organs affected. at diagnosis of bo, 69.6% (n = 32) were under immunosuppressive treatment. 32.6% (n=15) of patients with bo received ecp as second-line treatment. median duration of treatment was 22 months (1.5-36.8 ) and time to response 9.9 months (1.9-30.2). median of sessions was 40 (4-108). evaluation of response was based on the evolution of fev1 measurement: 26.6% (n = 4) complete response; 40% (n = 6) partial response and 20% (n = 3) stable disease. one patient did not get any response and another was not evaluable. 66.6% of patients (n = 10) could reduce immunosuppression, and in one case it was completely discontinued. there is a trend for early separation between survival curves in favor of ecp ( figure 1 ). one patient had sepsis secondary to central venous catheter infection as complication related to ecp. conclusions: ecp has emerged as a promising treatment for bo after allohct. in our experience, ecp was effective to stabilize or improve the disease in many patients and allowed to taper esteroids with minimal associated complications. however, prospective studies and longer follow-up are needed to support these findings. disclosure: nothing to declare background: the key role of il-6 signaling in acute graft vs. host disease (agvhd) and cytokine release syndrome (crs) has evoked growing use of tocilizumab, an anti-il6 receptor (il6-r) antibody, in these settings. apart from regulation of t-and b-cell differentiation, immune cells migration to inflammatory sites and t-cell recruitment, il-6 complex with il6-r through gp130 upregulates production of fibrinogen (fg) and other acute phase proteins, including c-reactive protein (crp). methods: we retrospectively analyzed data of 7 patients treated with tocilizumab (8mg/kg) due to steroid-refractory (sr) agvhd and 4 patients because of crs. median age was 37 and 47 years, respectively. seven patients were transplanted from unrelated donors (mud/mmud) and 4 from sibling donors. eight patients received myeloablative and 3 reduced intensity conditioning regimen. analyzed data included concentrations of fg, crp, an incidence of infections at tocilizumab administration and in weeks following the infusion. results: stage ii agvhd was diagnosed in 1 patient, stage iii in 4, and stage iv in 2 patients. involvement of the gastrointestinal tract (gi) was observed in 85% of cases. the median fg concentration before tocilizumab administration was 2.56 g/l (range, 1.4-7) and crp 21 mg/dl (range, 1-260) and 60% of patients had an active infection. after infusion of the antibody, we observed a decline of fg and crp levels. the median level of fg was 1.02 g/l (range, 0.46-1.7) 7 -14 days after the tocilizumab infusion with no severe bleeding complications. a median crp value was 1.82 mg/dl (range, 0.1-36) despite confirmed infectious complications. three weeks after infusion of tocilizumab fg raised to the normal range in 85% of patients (fig 1) . five patients with sr agvhd achieved a complete response, and 2 had a partial response after tocilizumab therapy. [[p265 image] 1. fibrinogen levels in gvhd patients following tocilizumab infusion.] a group treated with tocilizumab due to crs had higher initial levels of fg 5.1 g/l (range, 4.1-6.6) and crp 143 mg/ dl (range, 80-227) before administration of the drug. reduced fg and crp levels from a baseline value were also observed in this group. however, concentrations were higher than in gvhd patients: fg 2.02 g/l (range 1.2-3.4) and crp 3.2 mg/dl (range 2.7-11). in all patients, a differential diagnosis of disseminated intravascular coagulation was excluded. conclusions: 1. fibrinogen declines after tocilizumab therapy due to its cytokine-regulated production in the liver. coagulation monitoring should be performed during the first 3 weeks after administration of the antibody to avoid serious bleeding complications. 2. crp concentrations remain low despite the presence of active infections following infusion of tocilizumab. crp fails as a marker of infection during 3 weeks following the therapy. 3. tocilizumab is an effective therapy in patients with agvhd, especially with the gi involvement. disclosure: nothing to declare vanishing bile ducts after allogenic hsct: is it really gvhd? antonio grasso 1 , lorenzo d'antiga 2 , aurelio sonzogni 2 , massimo gregori 3 , alessandra maestro 3 , roberto simeone 3 , natalia maximova 3 background: evaluation of liver gvhd was historically based by elevation of bilirubin levels and by reduction and degeneration of small bile ducts on histological samples of post-transplant liver biopsy. however, there is a lack of studies that compared histological finding of ductopenia between post-autologous hsct and post-allogenic hsct. studying severity of ductopenia following allogenic hsct, we aimed to demonstrate lack of correlation between ductopenia and clinical signs of liver gvhd. methods: we retrospectively collected a series of 72 allogeneic hsct performed from 2005 to 2017 in the institute burlo garofolo. all patients undergo percutaneous liver biopsy in most cases at three months, one year and three or more years after hsct. indications for biopsy were alteration noted at 2 weekly follow-up assessments of at least one clinical or laboratory marker of liver impairment or cholestasis. ductopenia was defined by number of portal tracts with no interlobular bile duct divided by the total number (severe if the ratio was less than 0.2). clinical gvhd was defined by nih consensus criteria results: our population involved 64% males and 36% females with oncological (70%) and non-oncological underlying disease (30%). clinical signs of liver gvhd were present in 18% of the patients (n=13), 8% with contextual intestinal involvement, 8% with cutaneous and intestinal involvement. 71 patients underwent biopsy at a mean time of 110 +/-32 days after hsct, 45 patients underwent a biopsy at 12 months after hsct and 35 patients after three or more years from hsct. results of biopsies are showed in table 1. no difference in incidence of ductopenia were found between liver gvhd group and no gvhd. table] 1. table 1 : incidence of ductopenia 3-6 months, 12 months and 3 or more years after hsct in total population, gvhd group and no-gvhd group] the group that not received chemotherapy prior the hsct had an overall incidence of ductopenia of 77% (severe ductopenia of 42%) statistically significative in comparison with the oncological underlying disease group (94% of ductopenia and 64% of severe ductopenia). furthermore, a little sub-group of 16 patients extrapolated from our population received liver biopsy before hsct for diagnostic assessments: of the 10 with an oncological underlying disease 70% already showed ductopenia, while no signs of ductopenia were found in the others with a nononcological disease. conclusions: there is no correlation between incidence of gvhd and histologically finding of ductopenia on liver biopsy. ductopenia may be caused in the first place by chemotherapy treatment received before hsct and myeloablative conditioning for hsct and it's not related with gvhd. this hypothesis is strengthened by the subgroup analysis of pre-hsct biopsy. background: second and third line therapies for steroid refractory acute graft versus host disease (agvhd) after allogeneic stem cell transplantation (asct) are still lacking. ruxolitinib, a selective januskinase 1/2 inhibitor could show high efficacy in agvhd, as well as extracorporeal photopheresis (ecp). here we report a single center experience of combining both therapeutic approaches in severe steroid refractory agvhd with additional analysis of immune status of these patients to elucidate direct effects of this treatment on immune response. methods: from june 2015 to february 2017, 18 patients (77.8% male, 22.2% female, median age: 58.5 years, r: 21-73) with steroid refractory agvhd of lower gi-tract after asct were treated with ruxolitinib and extracorporeal photopheresis as third, fourth or fifth line therapy. some patients showed additional agvhd of skin (n=7), liver (n=6) or upper gi-tract (n=2). all patients had an overall grade iii (50%) or iv agvhd (50%). steroid refractoriness was defined as no improvement in 7 days or aggravation after 5 days of steroid treatment.median start of ruxolitinib or ecp was day 86.5 after asct (r: 35-257). medianduration of ruxolitinib therapy was 59.8 days (r: 14-192) with a median start dosage of 20 mg per day (2 x 10 mg; r: 10-20 mg). all patients started with 2 ecp treatments per week with an individual reduction of treatment frequency. median number of ecp treatments was 20.5 (r: 2-71) with a median frequency of ecp therapy once a week (r: 0.5-2.1). cytomegalovirus (cmv) status of all patients and immune status of ten patients (lymphocyte count with cd4 + t helper lymphocyte and regulatory t cell count) were collected previously, after four weeks of starting combined treatment and four weeks after stopping the treatment. results: one-year estimated overall survival (os) of all patients was 50% with a median estimated os of 314 days. 3 patients died because of relapse of underlying disease, one of severe therapy refractory agvhd of lower gi tract and 5 due to infection complications in agvhd refractory setting. overall response was 55.5% (complete remission rate: 44.4%, partial remission rate: 11.1%). 72.2% (n=13) of the patients had cytopenia ctc i-iii during the treatment, no grade iv cytopenia was reported. cmv reactivation during ruxolitinib occured in 66.7% of cases (n=12). tapering of steroids could be performed rapidly with a medium reduction time of 1.75 days for reducing to half of the dosage.remarkably, regulatory t cells significantly increased during combined ruxolitinib/ecp treatment compared to regulatory t cell count before treatment (p=0.02) and after stopping treatment, regulatory t cell count decreased again (p=0.02, see figure) . significant changes in whole lymphocyte count or in cd4+ t helper cell count were not observed. conclusions: treatment of severe steroid refractory agvhd with ruxolitinib plus ecp could show a high complete remission rate of 44.4% with an one year os of 50%. detecting increased regulatory t cell count during the treatment underlines its direct effects on immune response and encourages to pursue this promising therapeutic approach. [ background: due to increased immunosuppression infections remain the main cause of death followed by higher risk of relapse in patients treated for acute graft versus host disease (agvhd) after allogeneic stem cell transplant (sct). here we report a single-centre experience with extracorporeal photopheresis (ecp) for acute gvhd that was introduced in order to reduce steroid treatment. comparison of overall survival (os) for patients on ecp and patients that received standard first line therapy for agvhd was performed. methods: we retrospectively analysed 62 patients (28 %) with acute gvhd grade ii-iv treated from january 2010 to october 2018 out of total 221 allogeneic sct in that period. all patients received calcineurin inhibitors or sirolimus while receiving steroid treatment for agvhd. twenty-five patients (40 %) received ecp with steroid lowering intent. we defined response as (1) reduction of steroid dose for at least 50% from baseline while not adding another immunosuppresive agent and (2) not repeating second steroid treatment if the ecp was started after lowering of steroids to prevent agvhd flare. we checked separately patient responsive and refractory/dependent to steroids. on average patients received ecp procedure once weekly. results: tapering of immunosuppressive therapy as defined was successful in 13 (52 %) out of 25 patients in ecp group. in a group of patients without ecp 14 (37 %) patients had steroid refractory or steroid dependent agvhd compared to 11 (44 %) patients in ecp group. four (16 %) patients with steroid refractory or dependent agvhd showed improvement in ecp group compared to only one (2,7%) in non ecp group. twenty (74 %) patients died due to infectious complication and 7 (26%) due to relapse in non ecp cohort. in ecp cohort 10 (77%) patients died due to infection and 3 (23%) due to relapse. median os was 5 months in non ecp group (r., 1-99) compared to 21 months (r., 2-76) in ecp group and os of 20 % at 5 years in non ecp compared to 35 % in ecp cohort was observed. patients with agvhd treated with ecp and faster steroid tapering had longer os compared to patients without ecp (p=0,03). conclusions: ecp enables successful tapering or withdrawal of steroid therapy in many patients, even in those who are steroid refractory or steroid dependent. reduction of immunosuppression leads to reduced incidence of infection and relapse which translates into a better overall survival. background: the curative potential of allogenic stem cell transplantation is hampered by graft-versus-host disease (gvhd). pre-clinical study showed an efficacity of jak1/2 inhibitor, ruxolitinib, in treatment of steroidrefractory gvhd. methods: we reported in this monocentric retrospective study, ruxolitinib response and follow up of 44 cases of chronic gvhd (cgvhd) not improved with standard immunosuppressive therapy. complete organ response (cr) was defined as the resolution of clinical manifestations of cgvhd in a specific organ. very good response partial (vgpr) was defined as an improvement of clinical manifestations of cgvhd with more than 50% decrease of corticosteroid, while a partial response (pr) was associated with less than 50% decrease of corticosteroid. treatment failure was defined by the absence of improvement of cgvhd, deterioration of cgvhd in any organ by at least one stage, the development of cgvhd manifestations in a previously unaffected organ, and the use of any additional agents to control the disease. results: median age at transplant was 52 years (range, 19-66). 59% of patients presented an acute myeloid leukemia. donor type was sibling (n=12), unrelated (n=24) or haploidentical (n=6).two patients benefited a cord blood transplant. patients received either myeloablative (64%) or reduced intensity (36%) conditioning regimens. stem cell source was peripheral blood for 75% of patients. patients presented mild (n=11), moderate (n=21) or severe (n=12) cgvhd according to nih score. median number of regimens prior to ruxolitinib was 1 (range, 0-6), among those corticosteroids (n=36). median follow-up after ruxolitinib was 18 months (range, 4-32). overall responses rate (orr) at 1 month was 84% with 34% cr, 6% vgpr and 43% pr ( figure 1 ). 13% of patients failed at 1 month after introduction of ruxolitinib. the rate of cr increased with time : 45% at 3 months (n=38), 53% at 6 months (n=30) and 65% at 12 months (n=23). but vgpr rate was rather stable at 3 months (24%), at 6 months (33%) and at 12 months (22%) vs 6% at 1 month. among the patients under steroids, 21 (58%) patients discontinued steroids. the 12-months overall survival (os) and diseasefree survival (dfs) after ruxolitinib was 89% (80%-99%, 95% ci) and 86% (76%-96%, 95% ci), respectively. severe cytopenia (grade 3 and 4) was observed in 12 patients. after introduction of ruxolitinib, 4 patients presented bacterial infections, 3 patients presented an invasive pulmonary aspergillosis and 1 patient developped a pneumocystis. cytomegalovirus reactivation requiring preemptive treatment was observed in 7 patients. no toxicities required withdrawal of ruxolitinib. [[p269 image] 1. figure 1 and partial response to mesenchymal stem cells (msc), as second-line therapy, varies from 15% to 82% in acute gvhd patients. we report our experience using mscs to treat refractory agvhd. methods: the study was a retrospective single center study. all data were collected from patients' files. twenty patients were enrolled (age ranging from 7 months to 19 years) between april 2014 and april 2018. results: five of these patients received reduced intensity conditioning and 15 patients received myeloablative regimens before hsct. one haploidentical, 1 autologous, 1 cord blood, 14 mud, 3 msd transplantations were performed. the patients were eligible if they developed grades ii-iv agvhd. all patients were treated with standard first-line treatment with corticosteroids and at least one second-line therapy. the definition of steroid resistant agvhd considered as either no response to steroid treatment lasting at least 7 days or progression during treatment of at least one grade within the first 72 hours. prophylactic treatment with calcineurin inhibitors continued at therapeutic dose level. totally, 77 doses of mscs were infused. the median dose of msc was 1.02 × 10 6 cells per kg body weight. the median duration between the diagnosis of agvhd and initiation of mscs therapy was 16 days (range: 4-241). the received msc doses ranged from one to seven. none of our patients had severe side-effects during infusions of mscs. overall, complete response (ocr) was obtained in 10 patients, partial response in 7 patients and no response (nr) was documented in 3 patients. in our study group, the complete response rates in liver, gastrointestinal, skin agvhd were 20%, 35%, 72% respectively. four patients (20%) died in 90 days after using mscs from complications of agvhd. eleven of 20 patients (55%) were still alive with a median follow-up of 558 days (range:171-989 days) after first mscs infusion. one year estimated probability of overall survival for patients achieving ocr and partial remission/no remission in 90th day of mscs were 87.5% and 20%, respectively. conclusions: in conclusion, mscs appears to be a safe and effective treatment option for pediatric patients with steroid refractory agvhd. disclosure: nothing to declare effect of extracorporeal photopheresis on production of serum elafin in chronic graft versus host disease arun alfred 1 , charlotte burton 1 , kathryn goddard 1 , nichloas matthews 1 background: extracorporeal photopheresis (ecp) is a second line therapy for steroid refractory, dependent or intolerant chronic gvhd (cgvhd). in order to guide ecp there is an unmet need for predictive and diagnostic biomarkers. elafin is a serine-protease inhibitor primarily produced by epithelial cells, particularly keratinocytes in inflammatory skin diseases and plasma and epidermal elafin have been identified as biomarkers of skin gvhd (1, 2) . since skin cgvhd is noted for a particularly high response rate to ecp, we conducted a study to investigate whether ecp affects the production of elafin. methods: serum samples were collected from 72 cgvhd patients (39 male /33 female; age range: 25-74) and 17 age-matched healthy controls (10 male / 7 female) before ecp and at 3 month intervals up to 1 year. patients had gvhd affecting skin (61/72), mucosal membranes (16/ 72), liver (14/72), joints (8/72), gut (17/72), eye (8/72), genital (4/72), and respiratory involvement (4/72). serum elafin was assessed by elisa (r&d systems). data were analysed using graphpad prism 6. statistical tests performed include 2-tailed mann-whitney, pearson's correlation test, and 2-way anova with repeat measures, as appropriate. results: chronic gvhd patients presenting for ecp had significantly elevated serum levels of elafin (p=0.0029; median of 22ng/ml, iqr 15-45ng/ml) compared to healthy controls (median of 14 ng/ml, iqr 9.6-18ng/ml).while 85% of patients had skin involvement, only 40% had elafin levels above the iqr of healthy controls. where disease scores were available (n=25) there were no significant correlations with modified rodnans (r=0.19) or nih bsa scores (r=0.37).sub-analysis was performed by grouping cgvhd patients according to quartiles of serum elafin at pre-ecp baseline. retrospective analysis of patients after 12 months of ecp (n=66) revealed that those with serum elafin levels in the upper quartile (elafin hi ) pre-ecp (min-max: 46-117ng/ml), showed a significant reduction after 3 months of therapy (p< 0.05; mean +/-sd :72ng/ml +/-26 ng/ml vs 53ng/ml +/-23ng/ml, respectively), which was sustained up to 12 months of ecp (p< 0.0001; mean +/-sd: 37 ng/ ml +/-20ng/ml). in contrast, patients with elafin levels below the upper quartile (elafin lo ) showed no significant change (mean +/-sd: 21ng/ml +/-11ng/ml vs 26ng/ml +/-20 ng/ml, respectively). of note, pre-ecp patients with elafin below the median received significantly more corticosteroid (cs) than those above, (p< 0.05; mean +-sd: 27+/-18mg/d vs 16+/-16mg/d, respectively), which was significantly reduced after 3 months of ecp (p< 0.001; to 13+/-10 mg/d), while cs dose was not significantly changed in elafin hi patients until 6 months (p< 0.05; mean +/-sd: 16mg/d +/-16mg/d vs 6mg/d +/-5.5mg/d, respectively). conclusions: consistent with recent data, we found that serum elafin is significantly elevated in a subset of cgvhd patients compared to healthy controls, but did not correlate with skin disease scores. ecp administration was associated with a reduction in serum elafin in the elafin hi subset. further, elafin lo and elafin hi patients tolerated different rates of ecp-mediated tapering of cs immunosuppression suggests pre-ecp elafin measurements may have predictive value. references : background: allogenic hematopoietic stem cell transplantation (hsct) is a potential curative treatment for many malignant and no malignant hematologic diseases, primary immunodeficiencies and some metabolic and deposit diseases in children. graft versus host disease (gvhd) is a major cause of morbidity and a leading cause of non-relapse mortality. corticosteroids are the standard first-line systemic treatment for both acute and chronic gvhd, whereas no second line option for corticosteroid-refractory patients is standardised. ruxolitinib is a potent inhibitor of jak 1/ 2 showing significant responses in refractory gvhd patients in recent reports. methods: we present two centres experience with ruxolitinib for gvhd treatment in pediatric patients. the study was conducted in two spanish pediatric hsct centres, hospital vall d'hebron (barcelona) and hospital universitario la paz (madrid). all patients receiving ruxolitinib since the drug was available were included for retrospective analysis. results: between march 2017 and december 2018 19 pediatric patients with acute or chronic gvhd with refractoriness to corticosteroids were treated with ruxolitinib, in 22 different episodes (one patient received it in 3 different moments, and one patient received it in 2). patient's sex at birth was female in 11 and male in 8 cases. median age at hsct was 10,94 years (0,79-18,11). primary disease was malignant in 11 patients and non malignant in 8. median time of gvhd diagnosis was 47,5 days (6-525). all gvhd episodes were treated with corticosteroids as first line, with maximum doses between 1-5 mg/kg/day (the main dose used was 2 mg/kg/day, 15/22 episodes). patients received a median number of 3,5 (2-7) previous lines of treatment including steroids before starting ruxolinib; they were extracorporeal photopheresis (19/22 episodes), sirolimus (7/22), mesenchymal cells (5/22) ruxolitinib initiation was indicated for acute gut refractory/steroid dependant gvhd in 9 episodes and chronic multisystemic in 7 episodes. other indications were chronic lung (3/22 episodes), chronic skin (2/22) and acute skin gvhd (1/22) . median post-hsct time of ruxolitinib start was 300 days. doses ranged between 2,5-10 mg/12h depending on age, weight, and tolerance (hematologic and liver toxicities). average duration of treatment was 186 days (11-616). complete response (cr) rate was 18,1%, global partial response (pr) 72,7%, and no response (nr) 9% (progression in one patient and recent treatment start in other patient). mean time to maximum response was 4 weeks. treatment stop cause was cr in 2 cases, infection in 4, liver toxicity in 1. no severe side effects directly related to ruxolitinib treatment were described. conclusions: ruxolitinib has been recently introduced as second line strategy for rescuing corticosteroid-refractory gvhd in pediatric patients. while results of randomized trials are lacking, we present our experience (two centres). the main indications for starting treatment were acute gut and chronic multisystemic gvhd. most patients achieved some grade of response (partial or complete), allowing stopping or tapering corticosteroids. toxicity profile appears to be acceptable. disclosure: nothing to declare. stability of tacrolimus concentration early after allogeneic hematopoietic stem cell transplantation reduces the risk of acute gvhd background: tacrolimus is used as an immunosuppressive drug after allogeneic hematopoietic stem cell transplantation (allo-hsct). it is well known that early concentration level of tacrolimus is correlated with the risk of acute graft versus host disease (agvhd), however, whether range of standard derivation (sd) of early tacrolimus concentration after allo-hsct also affect to the risk of agvhd still remains unknown. here, we investigate the correlation between the range of sd of early tacrolimus concentration after donor hematopoietic cells engraftment and the development of agvhd. methods: we retrospectively assessed 207 patients who underwent allo-hsct in our hospital from 2010-2017. all patients received standard gvhd prophylaxis by continuous intravenous (iv) tac with starting dose of 0.02 mg/ kg/day from 1 day before allo-hsct (day -1) and iv methotrexate on day 1, 3, 6 at dose of 10 mg/m 2 , 7 mg/m 2 , 7 mg/m 2 , respectively. tac dosage was adjusted to target the serum concentration of 8-12 ng/ml until at least day 30 and then tapered. to evaluate the sd of weekly tacrolimus concentration, the range of sd of tacrolimus concentration at day1-7 (week-1), day8-14(week-2), day15-21(week-3) and day22-28(week-4) were calculated. the difference of the range of sd between the 2 groups that develop or did not develop agvhd was compared by using mann-whitney u test. multivariate analysis was performed by using multiple logistic regression analysis. patients had given written consent allowing the use of medical records for research, in accordance with the declaration of helsinki, and the institutional review board approved the study. results: there were 114 males and 93 females and the median age was 45 years (range, 16 to 68 years). the risks of disease were low-standard in 141 and high in 66 pts. the number of donors were in 6 hla-identical sibling, 13 in hla-mismatched related donor, 81 in hla-matched unrelated donor and 107 in hla-mismatched unrelated donor. thirty-seven patients developed agvhd (grade i-ii; 28, gradeiii-iv; 9 patients). as a result, the wide range of sd at week-3 significantly increased the risk of agvhd (agvhd-group; 1.178±0.8159 ng/ml, agvhd+ group; 1.447±0.7359 ng/ml, p=0.02). multivariate analysis demonstrated that narrow range of sd of tacrolimus concentration at week-3 reduce the risk of agvhd (or=4.19; 95% ci: 1.59-14.80; p=0.005). there were no correlation between gender, age, disease status, hla with the development of agvhd. conclusions: the range of sd at week-3, an engraftment phase of donor hematopoietic cells, was significantly correlated with the development of agvhd. fine tuning of early tacrolimus concentration with narrow range of sd reduces the risk of agvhd, resulting in improvement of the overall survival after allo-hsct. disclosure: nothing to declair p274 ruxolitinib treatment for steroid-refractory graftversus-host disease han-seung park 1 , je-hwan lee 1 , jung-hee lee 1 , eun-ji choi 1 , miee seol 1 , young-shin lee 1 , young-ah kang 1 , mijin jeon 1 , kyoo-hyung lee 1 background: steroid-refractory graft versus-host disease (gvhd) is one of the most lethal complications after allogeneic hematopoietic cell transplantation. recent studies have shown that ruxolitinib, a janus kinase 1/2 inhibitor, is effective in patients suffering from gvhd. here, we report a retrospective result of ruxolitinib treatment for steroid-refractory gvhd. methods: all patients had received cyclosporine and a short course of methotrexate as gvhd prophylaxis. antithymocyte globulin was added for unrelated or mismatched familial donor hct. ruxolitinib 5 mg twice daily was added to immunosuppressive treatment in patients with steroid-refractory gvhd. results: a total of 27 patients with gvhd (acute, 8, including 3 patients with donor lymphocyte infusion [dli]related; and chronic, 19) were included in the analysis. all patients had grade 3/4 acute gvhd or severe chronic gvhd at the time of ruxolitinib treatment. six (75.0%) of 8 patients with acute gvhd responded to ruxolitinib, including 3 with complete response (cr). the median time to response was 13.5 days (range, 8-25) . nineteen patients received ruxolitinib for severe chronic gvhd, with the median of 3 involved organs (range 2-5). fourteen patients (73.7%) showed response to ruxolitinib, including 4 crs. the median time to response was 24 days (range, 12-138). five responders discontinued ruxolitinib and 9 patients are still on the agent. after a median follow-up duration of 8.7 months, 5 died (2 from relapse of disease, 3 from infection). the 1-year survival probability was 70.1%. eleven of 20 responders discontinued ruxolitinib. gvhd relapsed in 3 of 11 patients at 14, 35, and 149 days after ruxolitinib discontinuation. thrombocytopenia (12/27, grade3/4; 4) was the most common adverse event of ruxolitinib. during treatment, 4 with grade 3/4 infectious adverse events occurred; 2 pneumonias, 1 brain abscess, and 1 liver abscess. conclusions: ruxolitinib treatment seems to be effective for the treatment of steroid-refractory gvhd including long-standing chronic gvhd. the agent was well tolerated and relatively safe. disclosure: nothing to declare. il6-receptor antibody tocilizumab as salvage therapy in the treatment of severe chronic gvhd after stem cell transplantation: a retrospective analysis background: severe chronic graft-versus-host disease (cgvhd) remains the most relevant factor affecting survival and long-term quality of life after allogeneic hematopoietic stem cell transplantation (hct). besides corticosteroids there is no established therapy for cgvhd and many of the used immunosuppressive agents may lead to significant toxicity incl. infectious complications. tocilizumab (an il6-receptor antibody) has shown efficacy in acute gvhd and cgvhd. we retrospectively analyzed the efficacy and safety of patients having received tocilizumab for treatment of advanced cgvhd at our center between the years 2015 and 2018. methods: 9 patients with severe steroid refractory cgvhd and a median age of 50 years (range: 21-62 yrs) having received at least two prior lines of therapy for cgvhd (range: 2-8 regimens) were treated with tocilizumab for at least one cycle (q4w, dosage: 8 mg/kg iv, maximum: 800 mg) with a median number of 4 cycles (range: . nih consensus criteria grading for cgvhd and the immunosuppressive regimen were noted at the time of the first tocilizumab administration and after 3, 6 and 12 months of therapy. all patients received additional concomitant immunosuppressive agents already given at least 4 weeks without response before start of tocilizumab. no new immunosuppression (is) was added in parallel to tocilizumab and response assessment was stopped at start of any additional new is. all patients had received peripheral stem cell allografts. gvhd prophylaxis consisted of a calcineurin inhibitor in combination with methotrexate or mycophenolate and in case of unrelated donors atg was added. 8/9 patients had quiescent onset of cgvhd, one patient developed de novo cgvhd. the median number of days between hct and onset of cgvhd was 215 (89-545). the median number of days between hct and initiation of tocilizumab therapy was 1033 (510-1749) days. at cgvhd onset, 7/9 patients had mild cgvhd and 2/9 patients had moderate cgvhd. the thrombocyte count was < 100/nl in 5/9 patients. organs involved at initiation of tocilizumab therapy were skin (100%, all grade 3), eyes (78%), mouth (67%), fascia (67%), lungs (44%) and genitals (22%). 5/9 patients are still receiving tocilizumab at the time of analysis. results: as tocilizumab was given fairly recently in most patients, 6-and 12-month follow-up was only reached in 3/ 9 patients (33%). at three-month follow-up after initiation of tocilizumab therapy, 4/9 patients (44%) showed partial remission, 2/9 patients stable disease (22%), and 2/9 patients progressive disease (22%) of cgvhd. maximal response was partial remission (56%), stable disease (11%) and progressive disease (22%). 3 patients required subsequent new immunosuppressive treatment. one patient has not yet reached 3-month follow-up. during tocilizumab therapy none of the patients suffered recurrence of underlying malignancy. two patients developed significant respiratory infection and one patient developed soft tissue infection, all requiring antibiotic treatment and pausing of tocilizumab administration, hospital admission was not required. the os and rfs was 100% with median follow up of 8.5 months (range 2-12 months). conclusions: tocilizumab appears to be a promising treatment option in advanced cgvhd but further evaluation within a phase ii trial is required. disclosure: nothing to declare background: allogeneic hematopoietic stem cell transplantation (hsct) is for many patients suffering from aml the only curative treatment option. one major complication is graft versus host disease (gvhd), caused by donor immune cells attacking healthy tissue. regulatory t cells (treg) have been getting huge attention during the past years because of their important role in maintaining immune balance. here we collected peripheral blood samples from 11 patients at different time points after hsct to investigate immune-reconstitution of treg as predictive marker for the development of gvhd. methods: we collected blood samples from 11 patients in the course of allogeneic hsct prospectively once a week from d+7 up to d+200. all patients received conditioning regimen with fludarabine and melphalan, combined with alemtuzumab for t cell depletion. 9 patients developed acute gvhd in the later course. after isolation of pbmc`s we performed facs multicolor staining of t cell and nk cells. treg were identified as cd3 + cd4 + cd25 ++ foxp3 + , nk cells were characterized as cd3 neg cd56 + cd16 + and divided in nk cell subpopulation due to their expression of cd56 dim or cd56 high . results: 1. cd52 neg t cells: all patients developing acute gvhd in the later course showed significant elevated levels of cd4 + cd52 neg t cells, especially cd52 neg treg at d+50. 2. cd52 neg treg / cd8 + cd52 + t cells: one patient not developing acute gvhd showed lots of cd52 neg treg but missed cd8 + cd52 + effector t cells. we recently showed that cd8 + cd52 neg effector t cells are of impaired effector function. these data suggest that cd52 neg treg are only of relevance combined with functional cd8 + cd52 + effector t cells in the development of agvhd. 3. t cell marker: patients without agvhd showed elevated expression of garp on treg. garp was significantly higher expressed on cd52 + treg, indicating a better suppressive capacity of cd52 + treg. this was detected throughout from d+50 until d+200. tigit and ilt3 showed a heterogeneous expression profile without significant differences between the two groups. 4. nk cells: we detected a higher ratio of cd65 ++ /cd56 dim nk-cell population in patients without. we could also show that tigit is mainly expressed on cd56 dim nk cells. conclusions: we and others showed reconstitution of cd52 neg t cell subsets after alemtuzumab mediated t cell depletion -our data on effector t cells showed an impaired effector function for cd52 neg cd4 and cd8 t cells. recently we presented data on impaired suppressive capacity of cd52 neg treg and the association with acute gvhd retrospectively (wölfinger ebmt 2018, ash 2017). here we provide prospective data on patients after the use of alemtuzumab in the context of hsct: our preliminary data suggest that the total amount of cd52 neg-treg and the ratio of cd52 neg treg to cd8 + cd52 + treg on d+50 after allogenic hsct could predict agvhd. this data may be a basis for immune monitoring of patients at d+50 to evaluate their risk for agvhd and could lead to the use of prophylactic treg dli in the context of alemtuzumab mediated t cell depletion. disclosure: medac -travel support, novartis -consultancy fee, pfizer -consultancy fee, shireconsultancy fee background: multiple factors such as disease activity and severity, therapy and/or dietary habits can cause changes in nutritional status independently or by interacting with each other. presence of malnutrition or significant weight loss in chronic gvhd (cgvhd) patients was reported in literature up to 43%. the aim of this cross-sectional study was to identify factors that affect nutritional status in cgvhd patients. methods: nutritional status in patients with cgvhd treated at the university hospital center zagreb, croatia from 2015 to 2018 was assessed. anthropometric measurements (height, body weight (bw), body mass index (bmi)) and clinical validated tool patient-generated subjective global assessment (pg-sga) (where patients were categorized as well-nourished (pg-sga a), moderately malnourished (pg-sga b) or severely malnourished (pg-sga c)) were used. all patients were evaluated according to 2005 nih criteria for cgvhd diagnosis. descriptive and correlation analysis were preformed. results: in total, 44 adult cgvhd patients were included in the study, 23 women (52.3%), median age 47 (18-65) years, with mild cgvhd in 6 (13.64%), moderate in 24 (54.55%) and severe in 13 (29.55%) patients. according to the pg-sga rating 19 (43.18 %) patients had pg-sga a, 19 (43.18 %) pg-sga b and 6 (13.64%) had pg-sga c, giving a total malnutrition or risk of malnutrition prevalence of 56.82%. the mean bmi was 23.83±4.3 kg/m 2 with correlation to pg-sga rating (r=0.446, p=0.0024). malnutrition according to the bmi (defined as bmi< 21 kg/m 2 ) was found in 8 patients (18.18%). bw changes (10% or more in 6 months) were significant in 14 patients (31.82%). according to the pg-sga assessment tool, oral symptoms reported by 31 patient (70.45%) and decreased appetite reported by 12 patients (27.27%) were associated with oral cgvhd nih score (r=0.536, p=0.0002; r=0.441, p=0.0027) but not with bw or bmi. gastrointestinal (gi) symptoms assessed with sga, were generally mild with no correlation to gi cgvhd nih score. no significant association was found between nutritional status and other nih cgvhd scores. corticosteroid therapy present in 12 (27.27%) correlated with pg-sga rating (r=0.494, p=0.0006) but not with bw, bmi or appetite changes. in 23 patients (52.7%) with altered pg-sga rating, bmi, appetite and body weight changes, dietary counseling and oral nutritional supplementation were initiated. conclusions: oral symptoms, decreased appetite and corticosteroid therapy in our cgvhd patients were associated with altered nutritional status according to the pg-sga, but not with bmi. therefore, pg-sga might be a more sensitive tool in assessment of changes of the nutritional status and detection of patients at risk of malnutrition than bmi since it includes different factors like physical examination, presence of gi symptoms and corticosteroid therapy in its scoring system. nutritional counseling and support are important in cgvhd patients especially in presence of oral symptoms. disclosure: nothing to declare. background: sclerotic skin changes are common features in chronic graft versus host disease (cgvhd). one of the most challenging aspects in the diagnosis and management of sclerodermoid cgvhd (scgvhd) is the differentiation between reversible symptoms related to active cgvhd and nonreversible symptoms related to residual permanent damage such as long-standing fibrosis. although several candidate biomarkers of cgvhd inflammatory activity have been proposed, none of them are currently validated. therefore, there is a need for the development of more quantifiable and reproducible measurements tools to guide clinical decisions. we report our experience evaluating the usefulness of high-frequency ultrasonography (hfus) plus doppler ultrasound (doppler-us) and serum fibrosis biomarkers to determine the inflammatory activity of scgvhd. methods: we report 6 patients with scgvhd. hfus plus doppler-us were performed at diagnosis of scgvhd and at different time-points after treatment initiation. serum hyaluronic acid and pro-colagen-iii were measured as fibrosis biomarkers simultaneously with hfus and doppler-us. nih cgvhd 2014 consensus conference diagnosis criteria, scoring system, and response criteria were used to assess global and organ-specific cgvhd, and to measure overall response to therapy. abnormal ultrasound findings were defined as the presence of ≥ 1 of the following: hypoechogenic dermis, dermo-epidermal junction effacement, hypoechogenicity of septa and/or hyperechogenicity of lobules in hypodermis, hypoechogenic fascia, or myositis, for hfus; and, vessels thicker than 1mm in dermis and/or hypodermis, systolic pressure >10 cm/sec, and index of vascular resistance >0.75, for doppler-us. inflammatory activity was classified as mild, moderate and severe according to the severity of doppler-us findings. results: hfsu showed abnormal findings in all patients at diagnosis with no changes except in two patients along the treatment follow-up. inflammatory activity by doppler-us was observed in 5/6 patients at diagnosis (1 mild, 3 moderate, 1 severe). four patients responded to treatment (2 complete responses, cr, and 2 partial responses, pr), one presented clinical improvement less than pr, and one, progressive disease. all patients with clinical response had also a p-rom improvement or normalization. all patients achieving a response showed normalization (n=2) or improvement (n=2) of doppler-us findings. the patient with clinical improvement less than pr and the patient with progressive disease showed persistence of inflammatory doppler-us findings. most patients had normal or light increase of pro-collagen levels at diagnosis and no significant changes were observed during follow-up. levels of hyaluronic acid tended to be very high in patients with progressive scgvhd (patients 2 and 5) and tended to decrease or normalize in those who responded to therapy (patients 1, 2, and 3). conclusions: in this exploratory study, hfsu was a reliable method for evaluating sclerotic skin changes in scgvhd. doppler-us showed a good correlation with disease activity and response to treatment. serum hyaluronic acid levels might be a biomarker of disease activity that deserves further investigation. hfsu plus doppler-us is a useful, non-invasive, repeatable device in monitoring patients suffering from scgvhd. according to our results, doppler-us may be a more sensitive parameter than hfsu in assessment inflammatory activity of scgvhd. disclosure: maría suárez-lledó received a grant from dkms-spain foundation. other authors have nothing to declare post-transplant cyclophosphamide versus antithymocyte-globulin in hla-matched unrelated and haploidentical transplantation for hematologic malignancies background: post-transplant cyclophosphamide(ptcy) and antithymocyte-globulin(atg) are the most commonly used regimens for the prophylaxis of graft-versus host disease(gvhd). we compared these two regimens in hlamatched unrelated (mud) and haploidentical transplantation for hematologic malignancies. methods: we retrospectively analyzed the consecutive adult patients with hematologic malignancies who received mud and haploidentical transplantation at chungnam national university hospital between january 2013 and january 2018. patients who received second transplantation and had refractory disease were excluded. results: this study included 45 patients with median age of 54 (range, 18-71) years: 29 (64.4%) patients received mud transplant (8 and 21 patients in ptcy and atg group, respectively), and 16 (35.6%) patients received haploidentical transplant (11 and 5 patients in ptcy and atg group). graft source was peripheral blood stem cell in all patients. median follow-up duration was 15.5 months (range, 0.5-58.0). in mud transplant, the estimated 20-months survival rate were 85.7% in ptcy vs. 80.7% in atg (p=0.835), the 20-months relapse rate were 37.5% in ptcy vs. 35.0% in atg (p=0.663), the cumulative incidence of grade 2 to 4 acute gvhd were 25.0% in ptcy vs. 22.2% in atg (p=0.706), and the estimated 20-month extensive chronic gvhd rate were 25.0% in ptcy vs. 16.7% in atg (p=0.902). in haploidentical transplant, the estimated 20-months survival rate were 55.4% in ptcy vs. 40.0% in atg (p=0.936), the 20-months relapse rate were 42.9% in ptcy vs. 33.3% in atg (p=0.328), the cumulative incidence of grade 3 to 4 acute gvhd rate were 29.9% in ptcy vs. 33.3% in atg (p=0.686), and the estimated 12 month extensive chronic gvhd rate were 25.0% in ptcy vs. 50.0% in atg (p=0.575). patients receiving ptcy had significantly longer neutrophil engraftment time than those receiving atg in haploidentical transplant [median(range); 17.0(14.0-21.0) days vs. 14.0(13.0-16.0) days, p=0.005]. conclusions: ptcy might be a good option for the prophylaxis of gvhd in hla-matched unrelated transplant as well as haplo-identical transplant. disclosure: nothing to declare early fam therapy for post allo-hsct bronchiolitis obliterans syndrome background: bronchiolitis obliterans syndrome (bos) is a potential major complication after allogeneic hematopoietic stem cell transplantation (hsct). attributed to an allo-immune reaction against the small airways, bo is considered a pulmonary manifestation of chronic gvhd. reported incidence of bos ranges from 5 to 12%, and bos-attributed mortality as high as 30%-80%. a few years ago, a new therapeutic approach with fluticasone, azithromycin, and montelukast (fam) was described (norman bc, et al. bmt 2011) . our aim was to analyze the outcomes of pts who developed bos and were precociously treated with the fam scheme. methods: all the 209 allo-hsct performed in our center from january 2015 and july 2018 were included in the analysis. baseline diseases were: 69 aml, 49 lpd, 31 mds, 28 all, 16 mpd, 10 mm, and 6 bmf. day +100 and day +365 overall mortality were 9,1% and 24,4%, respectively. rest of characteristics of the series are shown in table. fam therapy was systematically started when any patient was first diagnosed with bo. results: eleven patients (5,3%) were diagnosed with bos. at diagnosis of bos, the pts exhibited a fev1 80% of predicted (median fev1: 74%; range; 54-86%) and/or a decline >10% from pre-hsct . at day +100, 6 pts had already the syndrome. two of them died before the end of the first year: one due to invasive zygomycosis (cns plus pulmonary) and the other to baseline disease progression. at day +365, 5 more pts had bos. two more pts with bos died at 15 and 17 months post-hsct due to baseline disease progression. at the close of the analysis, 7 of the 11 pts were alive. so, with a median follow-up of 29 months (range: 8-44), mortality and bos-attributable mortality of the pts with the complication were 36,4% and 9,1%, respectively. conclusions: 1) bos is an infrequent but very severe complication of allo-hsct; 2) bos seems to be less frequent in pts with prophylactic pre-transplant ratg or post-transplant cyclophosphamide, as well as in pts undergoing transplantation with bm (compared to pbsc). 3) early diagnosis and therapy are critical to minimize the bos-attributable mortality. disclosure background: donor lymphocyte infusion (dli) is an established treatment for patients with hematological malignancies relapsed after allogeneic hematopoietic stem cell transplantation (hsct). however, it is associated with an increased risk of graft-versus-host disease (gvhd) and modest anti-tumor activity. compared to the infusion of nonmobilized lymphocytes, granulocyte colony-stimulating factor (g-csf)-primed dli might induce a stronger anti-tumor effect and reduce the risk of infusion-induced gvhd. due to the limited experience of g-csf primed dli in patients relapsed after haploidentical hsct, we conducted a retrospective study of all patients at our hospital who received dli for the relapsed hematological diseases following related hla-matched or hla-haploidentical hsct. methods: the institutional research board approved the study. we identified 94 patients with hematological malignancies receiving dli following related allo-hsct at national taiwan university hospital between 1999 and 2018 aug. the infusate was obtained from the cryopreserved specimen, which had been collected and stored at multiple aliquots at the same time as the initial haploidentical peripheral stem cell graft. patients received dli for either hematological relapse, preemptive or prophylactic treatment. univariate and multivariate analysis was performed using cox proportional hazard regression model. results: for the 61 patients following related hlamatched and the 33 patients following hla-haploidentical hsct received 119 and 72 doses of dli, respectively. in comparison, the median cd3+ cell dosage of haplo-dli is significantly lower (p = 0.0224) than that of dli from sibling donors, with median cell dosage 0.45 × 10 7 /kg (range, 0.05-12 × 10 7 /kg) and 1.1 × 10 7 /kg (range, 0.04-9.78 × 10 7 /kg), respectively. the median time to dli from initial sibling hsct and haplo-hsct was 152 days (range, 13-3357 days) and 155 days (range, 13-946 days), respectively. overall, 12 (20%) of the 61 patients following sibling hsct developed grade 2-4 acute gvhd after dli, whereas 12 (36%) of the 33 patients receiving haplo-hsct developed grade 2-4 acute gvhd after dli (p=0.1460). importantly, for patients receiving dli with cd3+ cell dosage less than 1 × 10 7 /kg, there is no difference in the risk of developing grade 2-4 acute gvhd between patients receiving dli from sibling or haplo donors ( figure 1a) . interestingly, for patients receiving dli with cd3+ cell dosage more than or equal to 1× 10 7 /kg, 4 (50%) of the 8 patients following haplo-hsct developed grade 2-4 acute gvhd after dli, significantly more than 5 (14%) of the 37 patients following sibling hsct developed grade 2-4 acute gvhd after dli ( figure 1b) . the cumulative incidence of grade 2-4 acute gvhd at day 100 after haplo-hsct and sibling hsct were 50% (95% ci: 0.13 -0.79) and 13.5 % (95% ci: 0.05-0.27), respectively ( figure 1b , p = 0.0146). [[p282 image] 1. conclusions: our study shows that the administration of g-csf mobilized dli is feasible after haploidentical hsct for relapsed hematological malignancies. however, dli with cd3+ cell dosage more than or equal to 1× 10 7 /kg in patients receiving haplo-hsct is associated with significantly higher risk of developing acute gvhd than dli from the sibling donors. disclosure: the authors declare no competing financial interests. background: the fresenius phelix is a uva irradiation device used to photoactivate mnc collected on the amicus. the system is closed, utilizing a special mnc kit and modified instrument software. the preliminary results of a phase i safety trial involving three patients (12 treatments) with chronic graft vs. host disease are presented. methods: reasons for transplantation for the patients ages 37, 61 and 62 years were: acute myelogenous leukemia, myelodysplastic syndrome, and myelodysplastic syndrome with pnh. stem cell source was peripheral blood with a 10/10 match for all. each developed chronic skin gvhd. inclusion criteria included wbc and plt counts > 1000 and 25x10 9 /l, gfr > 30 ml/min/bsa, and ast 10-120 unit/l. exclusion criteria included active gi bleeding, nyha cardiac disease greater than grade iii, and the presence of light-sensitive diseases. amicus software 4.51 and phelix software 1.0 were used. settings included: 80 ml/min max draw rate, 2000 ml fixed cycle volume, 1.25 mg/kg/min citrate infusion rate, and 12:1 acd-a ratio. venous access was peripheral or subcutaneous port. target uva dose was 1.5 j/cm 2 and 8-methoxypsoralen dose was 3.4 ml. results: the following mean + sd procedure results were obtained: 2,341 + 14 ml whole blood with acd-a drawn, 191 + 3 ml acd-a used, 691 + 127 ml saline used, 91 + 5 minutes procedure time, and 4,881 + 419 ml total blood volume. minor alarms (n=4) on the amicus and no alarms on the phelix were encountered. all 14-day aerobic and anaerobic cultures were negative and mean endotoxin levels were 0.425 + 0.1752 eu/ml. mean pre/post cbc and plasma hemoglobin levels were: 12.5/11.8 wbc, 9.9/9.5 neutrophils, 0.06/0.05 basophils, 0.21/0.15 eosinophils, 1.06/1.03 lymphocytes, 1.23/1.06 monocytes, 314/293 platelets x 10 9 /l, 40/ 37% hct, 13.3/12.2 g/dl hgb, and 26.8/23.4 mg/dl plasma hemoglobin. plasma hemoglobin delta in the product was 0.00+0.001 grams and the subject was -0.15+0.70 grams. collected product hct. mean 1.95+0.255%. yields are in the table. adverse events included one each: acute respiratory failure, respiratory failure, muscular weakness, musculoskeletal discomfort, and peripheral swelling. three of four events occurred in one patient two weeks after the study procedure. none of the adverse events were considered related to the procedure or investigational product. the patient who experienced acute respiratory failure was removed from the study because of death due to pneumonia, felt to be unrelated to the procedure. conclusions: results indicate the new closed photopheresis system is capable of collecting sufficient mnc and irradiating the cells producing high lymphocyte apoptosis, with minimal alarms and adverse reactions. (21.4%)). 10 (23.8%) of the 42 patients also had acute gvhd of the skin or liver. 37 patients (88.09%) could be treated and controlled with methyl-prednislone monotherapy, 5 patients had steroid refractory gvhd of whom 3 patients (7.14%) could be salvaged with additional drugs (infliximab:2; tacrolimus:1); 2 patients (4.77%) had refractory acute gut gvhd and could not be salvaged despite more than three lines of therapy. at the time of reporting, 26 patients (61.9%) of the 42 are alive. 11 patients died due to transplant related mortality, while 5 patients developed relapsed disease. on binary logistic regression analysis, no baseline clinical or treatment related predictor (disease indication, disease status at transplant, transplant type, graft source, type of conditioning) could be identified for developing acute gvhd of the gastrointestinal system. conclusions: acute gvhd of the gastro-intestinal system is a significant cause for morbidity in allo-hct patients at our centre. further studies are warranted in our cohort, and a prospective analysis of gut microbiome analysis, faecal multi-drug resistance organism surveillance, conditioning related toxicity and antibiotic usage is ongoing. clinical trial registry: not applicable disclosure: the authors declare no potential conflicts of interest benefits and precautions of ruxolitinib in steroidrefractory acute gvhd background: corticosteroids are the standard first-line treatment option for patients with acute graft-versus host disease (gvhd), but approximately half of patients become refractory to steroids and require second-line treatment. ruxolitinib has the potential to treat gvhd in steroidrefractory (sr) patients based on retrospective clinical data. the ongoing prospective trials are currently enrolling patients to evaluate the therapeutic potential of ruxolitinib for gvhd. methods: we analyzed retrospectively clinical experience with ruxolitinib in patients (n=15) with grade 2~4 steroid-refractory acute gvhd patients compared with the control group not receiving ruxolitinib. in addition, immune status was evaluated about 6 weeks~8 weeks after the administration of ruxolitinib using flow-cytometry. ruxolitinib was used as a third option for sr gvhd, combined with previously used immunosuppressive drugs. and steroids were gradually decreased according to the symptoms and discontinued. patients received ruxolitinib 5 mg twice daily (bid), with increase to 10 mg bid if hematologic parameters are stable and no treatmentrelated toxicities. results: fifteen patients all were assessable for response. seven patients achieved a complete response, 5 had a partial response, and 3 had no response at 8 weeks after the first ruxolitinib dose. overall response rate was 75%. three were treatment failures. most adverse effects were manageable, except infectious complications. infectious complications were occurred in about 73% patients (n = 11), resulting in two deaths. common cause of infectious events included cytomegalovirus (n =5), herpes-zoster (n=2), epstein-barr virus (n=2), fungal infection (n = 2), pneumocystis jiroveci (n = 2), bacterial infections (n = 1), and pneumonia of unknown origin (n = 1). t cell counts tended to decreased in the group with ruxolitinib compared with the control group, especially cd4 cell counts. conclusions: ruxolitinib is effective in controlling sr gvhd and can lead to clinical benefits. however, we need to be aware of the infectious complications because ruxolitinib may lead to increased risk of opportunistic infections or reactivation of latent infections. in addition, common infectious complications are presumed to involve t cell dysfunction. clinical background: graft versus host disease (gvhd), being one of most common life-threatening complication post hsct, contributes significantly to morbidity and mortality. when affecting gastrointestinal tract (gi) it is the major cause of death in early period post hsct. due to widespread tissue involvement in most patients diagnosed with gi gvhd, surgical treatment is rarely considered. methods: among 972 allo-hsct performed in department of pediatric hematology, oncology and bone marrow transplantation in wroclaw, poland during years 1996-2018, 291 (29,9%) cases were diagnosed with gi gvhd. in this study we present 3 cases (1%) which were referred to and benefit from surgical approach. results: 1. male, 4 years old underwent hsct from matched unrelated donor (mud) due to chronic myelogenous leukemia (cml) and subsequent molecular relapse succesfully treated with donor lymphocyte infusion, followed by agvhd (skin and gut involvement, grade iv). extensive immunosuppression (steroids, mycofenolate mofetile, atg, okt3) resulted in significant resolution of agvhd symptoms. however aggravating severe abdominal pain and lack of gut movement suggesting bowel obstruction. due to presence of acute abdomen patient was immediately directed for laparotomy. resection of constricted bowel segment followed by 2 subsequent laparotomies for secondary obstruction provided complete resolution of abdominal symptoms. after 16 years of follow-up patient is alive and well. 2. eleven years old male was diagnosed with skin and gut grade iv agvhd on day +84 post mud-hsct performed due to acute myelogenous leukemia (aml). he received pronlonged immunosuppressive treatment including steroids, antibodies, msc and ecp which led to resolving of skin leasions and diarhoea. nevertheless patient was suffering from severe paroxysmal abdominal pain and incidentally vomiting. ct enterography showed partial small bowel constriction. after numerous surgical consultations, eventually on day+ 503 patient underwent laparotomy with constricted bowel resection. histopatological examination of resected tissue revealed moderate gvhd. immunosuppersion was tapered to low dose of steroids with ecp. for now, 2 years post hsct patient is alive, rarely experiencing mild abdominal cramps 3. fourteen years old female developed severe abdominal pain and high volume diarhoea on day +24 post mud-hsct performed for severe anaplastic anemia (saa). despite extensive immunosuppression (steroids, anti-tnf, anti-il2 antibodies) patient condition did not improved. through consistent stomach pain, suspected subileus confirmed by ct enterography, laparotomy was performed (day+158). resection of inflamated and obstructed bowel was made. microscopic evaluation confirmed prior gvhd diagnosis therefore immunosuppression including csa and tapered doses of steroids was continued. complete resolution of abdominal symptoms was almost immediately achieved post-surgery, however 2 months after recurrent abdominal cramps were observed and are now well controlled by pain killers. conclusions: commonly gi gvhd is diffused inflammatory process. however in some cases it may be localized and may lead to partial bowel constriction. in case of severe and prolonged stomach pain, despite of partial resolving of other gvhd symptoms, ileus should be considered. ct enterography may be useful for diagnosis confirmation. in those patients, surgical intervention may improve quality of life or even be a salvage approach. disclosure: nothing to disclose is there any impact of the uric acid levels during the preand early post-graft infusion period, on the gvhd occurence and allotransplant outcome? 36.8(16.7-61.6 ) years, who underwent allogeneic stem cell transplantation (allosct) from full-matched sibling donors for acute leukemia (n=31), very severe aplastic anemia/pnh (n=5), lymphoma (n=3), myelodysplastic/ myeloproliferative syndrome (n=3) . thirty-two patients were in remission at the time of allosct (cr1: 23, cr2: 7, beyond cr2: 2). for a better and more accurate assessment of the ua levels on the agvhd incidence, unlike to the other published studies which evaluated the ua levels only at day 0, we evaluated the ua levels in different time points during the the peri-transplant period (at the conditioning regimen initiation, and at days 0, +7 and +14). because the majority of our patients developed agvhd within the 15-30 days post-transplant, we did not incorporated in the study the of ua levels beyond the +14 day. we also investigated the effect of the ua on survival and the non-relapse mortality (nrm). the vast majority of patients received allopurinol from the 1 st day of conditioning regimen till day -1. the independent t-test, kaplan-meir method and logrank test were used in the statistical analysis. results: the median ua levels were 4.2, 2.5, 2.7 and 3.2 mg/dl at days -7, 0, +7 and +14 respectively. for the statistical analysis purposes, we grouped our patients as low-ua if they had values < 3 mg/dl or high-ua if they had >3 mg/dl. this threshold was chosen based on the ua values from all the collected samples (n=175). finally 16/ 42 (38%) patients developed agvhd; 14(33%) were assessed as gr ≥ii, while 7(16%) as gr iii-iv. the incidence of the agvhd gr ≥ii was similar (ranged from 30-35%) in both groups of patients (low-ua and high-ua) and for all the estimated time points (days -7, 0, +7, +14). we noticed a better 2-years overall survival for patients with low-ua (75% vs. 63%) however without any statistical significance. ten patients succumbed to nrm causes; 6/10 deaths attributed to gvhd complications. the nrm was assessed higher in the high-ua group (38% vs. 18%) but also this difference was not statistically significant. conclusions: though our study bears the limitations of the small number of patients and the retrospective origin, at least to our knowledge is the first which evaluates the impact of ua levels at different time points in the peritransplant period, on the agvhd incidence. in our study the ua levels did not influence the incidence or the severity of agvhd. the higher nrm rates for patients with ua>3.0 mg/dl merits further evaluation. definitely, the role of ua on the allosct outcome will be clarified through well designed prospective trials. disclosure results: five male patients (12%) had genital cgvhd manifestations presented by urethral stricture in 4/5 patients and phimosis requiring surgical treatment in one patient. all five patients had simultaneously cutaneous, oral, and/or ocular cgvhd manifestations. the first patient underwent urethroplasty of bulbomembranous part of urethra with termino-terminal anastomosis and urethroplasty of penile part of urethra with buccal mucosa autograft -bmg (dorsal onlay) that resulted in significant improvement of symptoms and normal miction afterwards. biopsy of the urethra showed mononuclear infiltration in lamina propria consistent with cgvhd. biopsy of the buccal mucosa was done prior to surgery and was negative for cgvhd involvement. the second patient underwent urethrotomy due to circular strictures, but symptoms reappeared again and he is now candidate for bmg. in two patients urethral dilatation was done, and the fifth patient presented with phimosis requiring circumcision, resulted in significant improvement of symptoms. conclusions: male genital cgvhd is an underrecognized and under-reported manifestation. patients after allo-hsct need to be actively asked about their genital symptoms and sexual function, especially if they are diagnosed with other mucocutaneous or ocular cgvhd. multidisciplinary approach, early recognition and frequent follow-up is necessary for timely start of treatment. new methods, such as bmg for cgvhd patients with urethral stricture seem promising and should be further investigated. disclosure: nothing to declare. p289 abstract withdrawn. heracles: a phase ii single-arm prospective study to assess the efficacy of fecal microbiota transfer in the treatment of steroid refractory gastro-intestinal agvhd post allo-hsct background: steroid-refractory acute graft-versus-host disease (sr-agvhd) is associated with an 80% mortality rate and reduced quality of life (qol). so far, there is no approved standard of care for agvhd second-line treatment. there is an urgent need to identify effective therapy for sr-agvhd to improve patients' outcomes. fecal microbiota transfer (fmt) might be beneficial to substantially improve the prognosis. higher gut microbial diversity is strongly associated with increased survival in gvhd patients. recent studies reported promising results of sr-agvhd patients treated with fmt. further evaluation to confirm the efficacy and safety of fmt for agvhd is warranted. the ongoing phase 2 study (heracles) investigates the efficacy of allogeneic fmt in the treatment of patients with sr-agvhd. heracles was launched after the odyssee study showed promising results in the reconstruction of gut microbiota diversity after induction chemotherapy with fmt in acute myeloid leukemia patients. we expect that fmt-based biotherapeutic drugs could be effective treatments to contain sr-agvhd, and thereby reduce the risk of life-threatening complications after allogeneic hsct. methods: heracles is a single-arm, multicenter prospective trial in 5 european countries. patients aged ≥18 years-old, who underwent allogeneic hematopoietic stem cell transplantation (allo-hsct) and developed a first episode of stage 3 or 4 agvhd with gut predominance resistant to a first-line steroid therapy are eligible for inclusion. main exclusion criteria comprise the use of other second-line gvhd therapy, patients with grade iv hyperacute gvhd, late onset agvhd, and overlap chronic gvhd and agvhd after donor lymphocyte infusion. patients receive a first maat013 enema within 5 days after sr diagnosis (v1) and 2 additional ones 1 week apart (v2/ v3) from each other. maat013 is a highly-diverse, microbiome-rich enema formulation obtained from pooled, rigorously screened faeces from healthy donors, manufactured with a standardized process using the signature maat microbiome restoration biotherapeutic (mmrb) platform. at inclusion (v1), before each dosing (v2,3), and 28 days post inclusion (v4), patients' faeces and blood are collected. safety monitoring will be performed with corresponding blood analyses. exploratory measures on faeces include characterization of gut microbiota composition and evolution, impact of maat013 on metabolism, and gut inflammation. immune system phenotyping will be performed by flow cytometry on peripheral blood mononuclear cells, and by elisa assay on plasma. patients' qol will be assessed using a standard, eq-5d-5l questionnaire. the primary objective is to assess the efficacy of maat013 by evaluating complete response (cr, according to modified glucksberg criteria) and very good partial response (vgpr, defined by martin et al., bbmt, 2009) 28 days post-inclusion (primary follow-up). secondary objectives include fmt safety assessment and evaluation of fmt impact on several endpoints, such as overall, relapsefree or gvhd-free survival and chronic gvhd evaluation, as well as multi-drug resistant bacteria carriage. patients will be followed-up until1 year after inclusion. overall, 32 patients are planned to be enrolled and treated, to assess overall response rates and maat013's safety profile. results background: anti-programmed cell death protein 1 (pd1) monoclonal antibodies can be used as "bridge to" a subsequent allogeneic hematopoietic stem cell transplantation (hsct) in patients with relapsed/refractory hodgkin´s lymphoma (hl). this strategy has been reported to be effective, but a frequent onset of steroid-refractory graft versus host disease (gvhd) was also reported. we report 3 clinical cases of patients affected by hl undergoing allogeneic hsct after having been treated with nivolumab. methods: the 3 patients of 48, 18 and 42 years respectively had advanced hl and had relapsed after a previous autologous (2) or allogeneic (1) hsct. they underwent a rescue therapy with 18, 12, 16 nivolumab cycles respectively, depending on the time of partial response achievement and the availability of a donor. two patients received a thiotepa-fludarabinecyclophosphamide conditioning, atg-based prophylaxis and pb cells from unrelated donors. the third patient received bm cells from an haploidentical donor using the "baltimora" nonmieloablative platform. results: at a follow-up of 11, 12, 15 months after hsct, respectively, all patients achieved and maintained a complete remission by pet-ct scans. all the 3 patients developed acute gvhd on day +32, +54 and +107, respectively. patient 1 progressed to grade iv acute gvhd with hepatic and intestinal involvement unresponsive to first line 2 mg/kg steroid therapy and second line etanercept plus extracorporeal photopheresis (ecp). third line therapy with ruxolitinib partially controlled the gvhd. gvhd onset in patients 2 and 3 was preceeded by a prolonged fever without microbiological findings. patient 2 developed hepatic grade ii gvhd with high transaminase levels, initially responsive to steroid therapy, then it progressed to gut requiring second line therapy with etanercept. patient 3 progressed to severe chronic gvhd with skin involvement and resulted unresponsive to steroids and ecp and it was partially controlled by ruxolitinib. immune reconstitution was delayed in all 3 patients: at 6 months post transplantation cd3 levels were 80/μl, 17/ μl and 127/μl and cd4 levels were 36/μl, 16/μl and 65/ μl respectively. only patient 2, that underwent haploidentical transplant and received post-trasplant cyclophosphamide (pt-cy), is off of immunosuppressive treatment at 11 months after hsct, without evidence of gvhd and no history of infections. out of the 2 patients receiving pbsc from unrelated donors and atg prophylaxis, patient 1 developed a disseminated fusariosis on day + 180 and died of cns fusarium localization 1 year after hsct, despite targeted antifungal therapy. patient 3 had pulmonary aspergillosis, sepsis by multidrug resistant psuedomonas aeruginosa and otomastoiditis: at +15 months after hsct, he is on ruxolitinib treatment with skin clinical partial response. conclusions: this case series confirms that nivolumab as "bridge to transplant" is effective in appropriately selected patients. however, risk of acute gvhd and delayed immune reconstitution may require a careful consideration at the moment of planning the transplant. a possible advantage of pt-cy gvhd platform and haploidentical donors should be addressed in larger studies. background: acute graft-versus-host disease (agvhd) is the most important complication after an allogeneic hematopoietic stem cell transplant (hsct). no standard secondline treatment has been established for the corticosteroid refractory agvhd. the anti-tnfα agents are a good option of treatment for these patients, especially when lower gi tract is involved. methods: from april 2010 to july 2017 we reviewed the outcome of 19 patients with steroid-refractory (sr) agvhd treated with etanercept as at least, second line treatment. etanercept dose was 25 mg twice a week for the first 4 weeks, followed by 4 weekly doses. results: median age was 52 years (range 15-69 years), and 12 patients (63%) were male. fourteen patients (74%) had a non-advanced disease status at hstc. eleven patients (59%) received a myeloablative conditioning, and the stem cell source was peripheral blood in 18 patients (95%). sixteen patients (84%) were 8/8 hla matched. the characteristics of the 19 patients, their agvhd stage previous to rescue treatment with etanercept and their outcome are shown in table 1 . seventeen patients (89%) had a classic agvhd while 2 had a late-onset agvhd. etanercept was given as a 2 nd , 3 rd and 4 th line in 3 (16%), 10 (53%) and 6 (31%) patients respectively. the median doses of etanercept administered were 7 (range 1-12), and just 4 patients (20%) completed the 12 doses planned treatment, of whom 3 were alive at 38, 24 and 18 months from the onset of rescue treatment. complications during etanercept treatment were: infection (n=9 [47%]: gram negative bacilli [n=6]), grade 2-4 neutropenia (n=8) and grade 3-4 thrombocytopenia (n=6). etanercept was indicated as a rescue treatment due to: progression after 3 days of agvhd treatment (n=1), no response after 7 days of treatment (n=11), no complete remission after 14 days of treatment (n=3) and relapse due to decrease corticosteroid doses (n=4). at the end of treatment 1 patient achieved a complete response and 3 patients a partial response, all of them are alive. these 4 patients received etanercept as a 2 nd (n=3) and 3th line (n=1) treatment, all of them had lower gi agvhd without any other organ significantly involved. causes of death were: agvhd with or without infection in 15 patients (78%) and leukemia relapse in 1 patient. conclusions: although if etanercept is an option for treatment of sr agvhd in some patients, their prognostic remains poor and more effective alternative strategies are needed. a prompt initiation of etanercept as a rescue treatment for sr agvhd is crucial to improve the prognosis. (58) 5(26) background: although both cyclosporine (csa) and tacrolimus are calcineurin inhibitors, csa is more widely used in pediatric hematopoetic stem cell transplantation (hsct) as a prophylactic drug for acute graft versus host disease (agvhd). there are some clinical experience but very few data about the clinical efficacy of conversion to tacrolimus. here, we present our single center data on this arguable topic. methods: this study involves the data of 71 pediatric hsct patients in medical park göztepe hospital between 2014-2018. all 71 patients had prophylactic csa therapy and for various reasons csa was converted to tacrolimus therapy. most of the patients had this conversion due to agvhd. as steroid is the first line therapy for agvhd, conversion to tacrolimus is done concurrently at the start of steroid therapy (within 72 hours after the start of steroid). and also, patients who had any other immunosupressive therapy for agvhd are excluded. response is defined as resolution of symptoms within 7 days after conversion. results: mean age of the study population is 130 months (5-266 months), male/female ratio is 1,3 (40/31), donor types are mud 48 patients (67%), mfd 18 patients (26%), haplo 5 patients (7%) and mean conversion time is 32 days (1-142 days) . the rationales for conversion are agvhd for 40 patients, unproper csa plasma levels for 7 patients, allergic reaction for 6 patients, nephrotoxicity for 6 patients, hepatotoxicity for 5 patients, severe headache for 2 patients, high arterial blood pressure for 2 patients and one each for refractory vomiting, autoimmune thyroiditis and visual disturbance. the subgroup analysis of agvhd patients reveals that mean conversion time for agvhd is 41 days (8-142 days) and there are only 11 responders whose agvhd resolve completely (%27) after conversion. all of the patients had proper tacrolimus levels after conversion due to unproper csa levels and also patients in allegic reaction, severe headache, visiual disturbance and refractory vomiting group responded to conversion completely but only one of the 6 patients in nephrotoxicity group responded and also 3 of the 5 patients in hepatoxicity group responded. the only one patient suffered from autoimmune thyroiditis did not respond to conversion. conclusions: in this study, it is obvious that there are response to conversion for some specific adverse effects of csa and tacrolimus is a good alternative for the patients who have unproper csa levels. conversely, the high percentage (%73) of non-responders shows that it is not feasible to make a conversion to tacrolimus for acute gvhd. disclosure: nothing to declare background: capillary leak syndrome is caused by the dysfunction of the vascular endothelial cells,and is characterized by weight gain,generalized edemas,unresponsive to diuretic treatment,and hypotension.it usually develops in the first 15 days post hsct.and it is of great difficuty to distinguish from other complications which are occured post the allo-hsct. to diagnose this complication at the early stage,it is very difficulty. methods: a 34-year-old man was admitted to ningbo first hospital for its abnormal in the peripheral blood .he was diagnosed with aml-m5 by the classical morphology and immunophenotype.cytogenetic evaluation showed a normal 46, xy(20).the patient achieved cr with induction therapy including idarubicin, cytarabine and etoposide. after consolidation therapy,an allo-hsct from hla identical related dornor(33-year-old male, donorrecipient matched by high resolution hla typing at hlaa, -b, -c, drb1, and dqb1, 10/10 matches) was performed.the recipient received conditioning with busulfan, 4 mg/kg/day injection for 3 days; cyclophosphamide, 50 mg/kg/day injection for 2 days; cytarabine, 2 g/m2/day injection for 1 day; semustine, 250 mg/m2/day orally for 1day; donor peripheral blood stem cells (pbsc:mnc: 6.43×109/l, cd34+:4.05×106/l) were mobilized, pheresed and administered to the recipient. gvhd prophylaxis consisted of traditional cyclosporine, short-course methotrexate (15 mg/ m2 at day +1, 10 mg/m2 at days +3, +6, and +11) and cyclosporin a injection 5mg/kg qer day was mot reduced untill the hematopoietic reconstitute sucessfully . on day +19, complete donor chimerism was acheieved. the csa was gradually reduced and tapered.on day +150,the patients was manifested with increasing in the time and volume of the faeces, he was diagnosed with ii°gvhd (gut).the standarded does of immunosuppressive drug including methylprednisolone and cyclosporin a was administrated. the immunosuppressive drug was gradually reduced when the gvhd was controlled.on day+271,the patient felt distress and the distress was not related to with the exercise,the temperature was normal,and he did not gain weight.there was no edema in the body.laboratory test including routine blood test,c-reactive protein,procalcitonin,blood gas analysis,cmvdna,ebvdna was normal. the ct scan shows that 1the pleural is filled up with water, and could not be enlarged promptly,2there is pericardial effusion in the body.pulmonary function test shows that 1 reduced function in ventilation and diffusion fuction.the laboratory test of the pleural effusion was normal,the blast cell was not detected in the pleural effusion,the cd34+ cell count was below the dectable level,the next generation sequencing for minimal residual disease shows that there was no gene mutation .thus, post capillary leak syndrome was considered .sirolimus was adopted and taken the place of cyclosporin a,immunoglobulin was adminstrated to reduce the edema. results: taking together comprehensively,the effusion in the pleural and cardiac was absorbed well. conclusions: occurance of capillary leak syndrome is rare,there is limited data about capillary leak syndrome. comprehensively,the mechanism of cls has not been totally identified.and there is no standard treatment to treat the complication.at present,the cls of this patient was absorbed well by administrating sirolimus,closely followup is needed. disclosure: nothing to declare graft-versus-host diseasepreclinical and animal models p295 short-term krp203 and posttransplant cyclophosphamide for graft-versus-host disease prophylaxis emi yokoyama 1 , daigo hashimoto 1 , takahide ara 1 , eko hayase 1 , takanori teshima 1 1 hokkaido university faculty of medicine, hematology, sapporo, japan background: post-transplant high-dose cyclophosphamide (ptcy) in combination with other immunosuppressants such as calcineurin-inhibitors (cis) has been increasingly used as gvhd prophylaxis after hla-haploidentical or matched hematopoietic stem cell transplantation (hsct). however,cis could hamper reconstitution of regulatory t cells (tregs) and tolerance induction after hsct, facilitating us to develop novel ci-free/ptcy-based gvhd prophylaxis. in the current study, we developed a novel gvhd prophylaxis in which ptcy was combined with short-term administration of krp203, a selective agonist of sphingosine-1-phosphate receptor type 1 (s1pr1), using murine models of mhc haploidentical bone marrow transplantation (bmt). methods: b6d2f1 (h-2 b/d ) recipients were lethally irradiated and transplanted with bone marrow cells and splenocytes from allogeneic b6 (h-2 b ) donors. cy at a dose of 50 mg/kg was intraperitoneally injected into the recipients on day +3, and krp at a dose of 1.0 mg/kg was orally administrated daily from day 0 to day +4 after bmt. donor t cells in the target organs and secondary lymphoid organs were evaluated by flow cytometric analysis. plasma levels of tnf-α were determined using cytometric beads array. to evaluate graft-versus-leukemia (gvl) effects, recipient mice were intravenously injected with luciferase-transduced p815 cells (p815-luc) on day 0, and in vivo bioluminescence imagingwas conducted weekly after bmt. results: severe gvhd was developed in allogeneic recipients and all mice died by day 50 after bmt.ptcy alone at a dose of 50 mg/kg significantly ameliorated gvhd and 30 % of ptcy-treated allogeneic recipients survived. oral administration of krp203 alone enhanced contraction of donor t cells in the lymph nodes and also ameliorated gvhd as has been previously shown with multi-s1pr agonist, fingolimod. next, we tested if shortterm krp203 on days 0 to +4 added to ptcy enhances anti-gvhd effects of ptcy. we found that survivals of ptcy+krp203 group were significantly prolonged compared to those of ptcy-alone group ( figure a) . plasma levels of tnf-a, clinical gvhd scores ( figure b) , and donor t-cell infiltration into the target organs such as the gut and skin were also significantly reduced in ptcy +krp203 group compared to ptcy-alone group (figure c and d) . unlike cis, addition of krp203 to ptcy promoted treg reconstitution after bmt. finally, bioluminescence imaging demonstrated that proliferation of p815-luc injected on day 0 was significantly delayed in ptcy +krp203-treated allogeneic recipients compared to control mice transplanted only with t-cell depleted bone marrow cells, suggesting that significant gvl effects persisted in ptcy+krp203-treated recipients. conclusions: a combination of short-term krp203 and ptcy is a promising novel calcineurin-free gvhd prophylaxis in mhc-haploidentical sct. we recently showed that donor inkt cells can be expanded ex vivo and that they are able to prevent activation and proliferation of alloreactive donor t cells while promoting efficient graft-versus-leukemia effects (schmid et al. 2018 ). however, the underlying mechanisms how human inkt cells induce immune tolerance after allogeneic hct are not fully understood. methods: monocyte-derived dendritic cells (dcs) were cultured in a mixed lymphocyte reaction with mhcmismatched t cells and culture-expanded inkt cells. tcell activation and proliferation was analyzed by multiparametric flow cytometry and released cytokines were measured via multiplex analysis. transwell assays and imaging flow cytometry were performed to elucidate cellcell interactions. bead-controlled flow cytometry-based cytotoxicity assays were used to evaluate dc apoptosis. apoptotic dcs were then purified by fluorescence-activated cell sorting to investigate their tolerogenic potential to prime regulatory t cells (tregs). results: the addition of inkt cells to mixed lymphocyte reactions resulted in a significantly reduced activation and proliferation of mhc-mismatched t cells. transwell assays and imaging flow cytometry revealed a cell contactdependent mechanism between inkt cells and dcs leading to apoptosis with increasing dna fragmentation of dcs over time. interestingly, various fluorescence-activated single cell sorted inkt-cell subsets were all able to induce apoptosis of host dcs. multiplex analysis revealed that dcs triggered inkt-cell release of cytotoxic factors like perforin, granzyme b and granulysin. blocking the inktcell receptor engagement with a cd1d antibody prevented inkt-cell degranulation as well as the subsequent induction of host dc apoptosis. inhibition of cytotoxic factors also abrogated apoptosis of dcs. in turn, sorted apoptotic dcs induced tolerogenic dcs characterized by a high expression of pd-l1 in mixed lymphocyte reactions. such tolerogenic dcs promoted the expansion of cd4 + cd25 + foxp3 + tregs and prevented activation and proliferation of mhcmismatched t cells. conclusions: we propose a novel mechanism how culture-expanded human inkt cells prevent gvhd after allogeneic hct. host dc apoptosis through donor inkt cells induces a tolerogenic immunoenvironment characterized by pd-l1 high dcs and expanding donor tregs inhibiting activation and expansion of alloreactive donor t cells. our findings pave the avenue for clinical translation of adoptively transferred culture-expanded inkt cells in humans. disclosure: nothing to declare results: vip-ko mice transplanted with allogeneic tcd bm alone had increased graft rejection with lower levels of donor chimerism and 33% day 75 survival compared with 73% survival of wt recipients. transplanting tcd bm plus 3 × 10e6 donor t cells from b10.br or balb/c donors in vip kio mice led to >95% donor chimerism and significantly increased gvhd-mortality compared with wt recipients, with 0% vs 40% survival in the b10.br-->b6 model (p< 0.01), and 0% vs 73% survival in the balb/c-->b6 model (p< 0.01). donor-derived t cells in vip-ko recipients had significantly higher th1 and th17 polarization, with higher rorγt in both cd4+ (p< 0.0001) and cd8+ (p< 0.0001) t cells, and higher frequencies of ifn-γ (p< 0.001), tnf-α (p< 0.05), and il2 (p< 0.01) in cd4+ and cd8+ t cells compared to wt recipients. b10.br-->b6 second allogeneic transplantation of radiation chimeras caused lethal gvhd mortality in vip-ko-->vip-ko and wt-->vip-ko mice, but not in wt-->wt or vip-ko-->wt b6 mice, demonstrating the protective effect of vip was due to synthesis by non-hematopoietic recipient cells. immunofluorescent imaging of allo-bmt recipients showed marked up-regulation of vip in lungs post-transplant and high vip production within neurons innervating the lungs. finally, we demonstrated that short-term administration of vip (10mcg/day) from day 0 to day 10 prevented gvhdmortality in vip-ko recipients transplanted with b10.br-->b6 mhc donor bm & t cells. conclusions: the absence of vip in recipient cells led to increased graft rejection in the absence of donor t cells and increased lethal gvhd when donor t cells were transplanted, indicating vip induced post-transplant regulates allo-reactivity of host graft-rejecting lymphocytes and donor gvhd-causing t cells. the protective effect of parenteral vip administration suggests vip-mimetics represent a novel approach to prevent and treat gvhd. these data also suggest a mechanism of action for the mitigation of gvhd by alpha-1 anti-trypsin (aat) whereby aat inhibits the proteolytic inactivation of endogenous vip. disclosure: dr. waller reports personal fees and other support from cambium medical technologies, grants from celldex, personal fees from kalytera, grants and personal fees from novartis, grants and non-financial support from pharmacyclics, and equity ownership in cerus corporation and chimerix outside the submitted work. in addition, dr. waller has intellectual property related to vip signaling that has been licensed to cambium oncology in which he holds equity. low-density neutrophils expansion is associated with acute graft versus host disease in allogeneic hematopoietic stem cell transplant patients background: low-density neutrophils (ldns) are distinguished from normal-density neutrophils (ndns) by their anomalous sedimentation within the mononuclear cell fraction after density gradient centrifugation of peripheral blood (pb). by analysing ldns and ndns from g-csfstimulated donors or lymphoma patients, we have previously demonstrated that, depending on physiopathological conditions, immature cd66b + cd10 -ldns can promote t cell survival and ifn-γ production, while mature cd66b + cd10 + ldns can exert immunosuppressive proprieties. aim of this study was to establish the frequency of cd66b + cd10and/or cd66b + cd10 + ldns in pb of allogeneic hematopoietic stem cell transplant (hsct) patients throughout immune reconstitution, and verify their potential correlation with acute graft versus host disease (agvhd). methods: patients undergoing hsct in our institution between december 2015 and june 2018 were prospectively enrolled in the study upon informed consent and after institutional board approval. criteria of inclusion were age ≥ 18 years and absence of rheumatologic or viral diseases. pb samples were collected at day +21, +42, +60, +90 and +180 after hsct and any time within day +180 in case of gvhd, before first-line therapy. eight healthy donors (hds) were enrolled as control. mononuclear, polymorphonuclear, and whole blood cells were analysed by flow cytometry after cd45 vioblue, cd16 apc-cy7, cd11b pe-cy7, cd10 pe, cd66b fitc staining. cd66b + ldns were expressed as percentage of cd45 + pb mononuclear cells (pbmcs) or cd45 + whole blood cells and were further characterized based on cd10 expression. cd66b + ndns, expressed as percentage of cd45 + whole blood cells, were also analysed for cd10 staining. results: 39 patients (m/f 25/14, median age 47) were enrolled in the study. patients received hsct from hlaidentical (13) or haploidentical (6) related and from hlaidentical unrelated (20) donors. after a median time of 33 (15-95) days, 13 patients developed grade ii-iv agvhd. no patients were receiving g-csf at agvhd onset. the scheduled assessments were interrupted in agvhd patients at the beginning of first-line treatment and in 4 patients relapsed of their primary malignancy. no patients developed de novo late-acute or chronic gvhd. starting from day +21 the frequency of ldns within cd45 + pbmcs was higher in all patients as compared to hds. the 25 patients that did not develop agvhd showed a decreasing frequency of cd66b + cd10 -ldns, with a progressive increase of cd66b + cd10 + ldns, from day +21 to +180. interestingly, patients with agvhd showed a significantly higher frequency of cd66b + cd10 -ldns as compared to patients without agvhd throughout the same time lapse (i.e. from day +21 to +90) (83.15 vs 50.8, p=0.027). consistently, patients with agvhd had a significantly lower frequency of cd66b + cd10 + ldns (12.1 vs 50.05, p=0.0014). the frequency of mature cd66b + cd10 + ndns was normal in all patients since day +21. conclusions: ldns are more represented in hsct patients than in hds, with a significant expansion of the cd66b + cd10subpopulation (with a parallel decrease of the cd66b + cd10 + subpopulation) in patients with agvhd as compared to those without agvhd. according to the previously demonstrated t cell activating function of cd66b + cd10 -ldns, it is tempting to speculate that the expansion of this subpopulation may contribute to agvhd development. disclosure: nothing to declare background: acute graft-versus-host disease (agvhd) is a major complication after allogeneic hematopoietic stem cell transplantation (allo-hsct) which has negative impact on the morbidity and mortality of the patients. accumulating evidences suggest that abnormalities of foxp3+ regulatory t (treg) cells contributed to the pathogenesis of gvhd, but the underlying molecular mechanisms still remain largely unknown. methods: in this study, we enrolled all the 40 patients treated with allogeneic hsct at the institute of hematology, chinese academy of medical sciences between 2016 and 2018,as well as 10 age-matched healthy adults as control samples. the ratio of tregs in pb and bm of healthy controls (hcs) and patients with and without agvhd was determined by flow cytometry. the transcription profile between tregs from patients with or without acute gvhd was measured,the pathway enrichment analyses were performed by the kyoto encyclopedia of genes and genomes (kegg) pathway database and geneset enrichment analysis (gsea).the expression of lkb1 at transcript levels and protein levels was measured by realtime pcr and analyzed by the nanopro1000tm system. a series of functional assays in vitro were performed to assess the function and stability of tregs from patients with and without agvhd.meanwhile, to assume the affect of lkb1 on gvhd outcome, we established a murine transplant model,which recipient balb/c animals were transplanted with the same amount of mixture made by bm, cd4 +cd25-tcon cells from c57bl/6 and cd4+ foxp3 yfp+ tregs from either foxp3crelkb1f/f or foxp3cre mice. results: in this study, we demonstrated that bm had decreased frequencies of tregs, accompanied with a reversed lower ratio of tregs frequencies between bm and pb in agvhd patients. meanwhile, the number and function of tregs in bone marrow also affected hematopoietic reconstitution. futhermore,to elucidate these mechanisms which regulate tregs homeostasis, we examined the role of lkb1 on tregs in patients with agvhd and in agvhd murine model. studies demonstrated that lkb1deficient tregs lost foxp3 expression and weaken suppressor function during agvhd. transcriptional profiling and pathway analysis revealed that nf-kb signaling activation and the impairment of a wide spectrum of immunosuppressive genes in agvhd tregs. further mice experiments suggested that cns2 methylation might lead to the instability of tregs in agvhd group. transplantation with marrow grafts from foxp3crelkb1f/fmice exacerbates gvhd lethality. conclusions: these studies indicate that lkb1 is a critical homeostatic regulator for tregs during agvhd. targeting of lkb1 therefore represents a novel therapeutic strategy that promote immune tolerance to mitigates the severity of agvhd. disclosure: national program on key basic research project (973 program) role of aryl hydrocarbon receptor in intestine after allogeneic hsct in mice won-sik lee 1 , soung-min lee 1 , sj-kil seo 1 1 inje university, busan paik hospital, hemato-oncology, busan, korea, republic of background: aryl hydrocarbon receptor (ahr) is a ligandactivated transcription factor that is activated by various small molecules from the diet, microorganisms, host metabolism, and xenobiotic toxic chemicals. the function of ahr has been demonstrated as a crucial regulator in intestinal homeostasis. here, we investigated the regulatory role of ahr in intestine of recipients after allogeneic hematopoietic cell transplantation in mice. methods: wild-type (wt) b6 (h-2 b ), ido -/-(h-2 b ) and ahr -/-(h-2 b ) mice were lethally irradiated and transplanted with 5 x 10 6 tcd-bm plus 2 x 10 6 t cells from balb/c donor mice. ahr activation in colon tissue of recipients was determined by the ahr target genes cyp1a1 and cyp1b1 expression using real-time pcr. the recipient mice were monitored every other day for survival and clinical score. histopathology and pathogenic effector cytokine levels in colon tissue were analyzed for evaluating ahr function. results: we observed that cyp1a1 was constitutively expressed in the colon tissue of naïve recipient mice. although the expression levels were increased by tbi conditioning, the additive up-regulation of its levels with donor t cell alloreactivity was not observed. in contrast, cyp1b1 expression was markedly induced in the colon tissue by donor t cell alloreactivity. we further observed that the cyp1b1 expression was significantly decreased in the colon of ido-/-recipients with donor t cell alloreactivity, but cyp1a1 was not changed. ido-/-and ahr-/recipient mice showed higher histopathological score for intestinal gvhd and increasing pathogenic cytokine levels in the colon compared with wt mice. conclusions: our results demonstrate that ahr-induced target gene profiles might be differently induced in intestine by ligand dependent manner after hsct, which affect intestinal gvhd. disclosure: nothing to declare. abstract already published. abstract withdrawn. in vitro platelet activation evaluation in allogeneic hematopoetic stem cell transplanted patients in response to haemostatic stimulation and cytomegalovirus stimulation (gvhd), complication of which one of the risk factor is cmv reactivation. the resultant inflammatory platelet response during the high-risk period of gvhd after allogeneic hsct remains unknown. our study aimed to characterize spontaneous platelet activation during the 2d and 3d months after allogeneic hsct, and in response to haemostatic stimulation and cmv stimulation. methods: we compared a group of healthy volunteers to a group of allogeneic hsct patients followed between the 30 th and the 90 th days after hsct. platelet activation was determined by the platelet surface expression of cd62p and cd63 using flow cytometer after stimulation by an haemostatic agent, thrombin-receptor activating peptid (trap) and after stimulation by cmv glycoprotein b. the inflammatory response was determined by the detection of immune mediators, rantes, cd62ps, pf4, cd40l and ccl3, using the elisa technique in the stimulated platelet supernatants. results: no platelet activation or molecules release were observed after stimulation by cmv glycoprotein b in both groups. rantes and cd62ps baseline levels are spontaneously higher in allogeneic hsc patients than in healthy volunteers. platelets from allogeneic hsct patients can be activated after haemostatic stimulation and release cd62ps and rantes. in this situation, platelets release more cd62ps, rantes and pf4 than platelets from healthy volunteers. conclusions: although no platelet activation was detected in response to cmv glycoprotein b stimulation, our study revealed a chronic platelet activation condition during the 2d and 3d months after allogeneic hsct with an haemostatic inducible hyper-responsiveness. this leads to the release of molecules with immune-modulating properties involved in the pathophysiology of gvhd. as we move further away from the hsct, that phenomenon seems to gradually weaken. clinical trial registry: nct03009708, fipalloc https://clinicaltrials.gov/ct2/show/nct03009708 disclosure: nothing to declare p305 efficient process and characteristics of umbilical cordderived mesenchymal stromal cells as a feasible source for anti-inflammatory therapy background: recently, umbilical cord (uc) has become attracted source of mesenchymal stromal cells (msc), because of abundant sources and ease of collection of fetal origin without invasive process for the donor and low immunogenicity with immunosuppressive ability and tissue repair potency. objectives of this study were to explorer the efficient and safe products and to evaluate the antiinflammatory potency of uc-mscs for the application of acute graft versus host disease (gvhd). methods: informed consent was obtained from mothers planning to have cesarean sections. uc tissue was cut and once cryopreserved. the safety assessment including infections and baby's health and development were done after 6 months of birth, and performed small-scale quality test of the frozen uc. then we initiated to isolate master uc-mscs from frozen-thawed uc by an improved explant method, which was passed for quality test. the master uc-mscs were cryopreserved once and thawed and expanded until p4. product cells were cryopreserved in original serum-free cryoprotectant dba-d solution. mixed lymphocyte reaction (mlr) assay co-cultured with uc-mscs was carried out using responder mononuclear cells (mnc) stained with cfse, and proliferation and cytokine secretion were analyzed by flowcytometry. results: uc-msc cultured showed significantly higher proliferation ability compared with those from bone marrow-derived mscs, and positive for cd105, cd73, cd90, and negative for cd45, hla-dr. cd80, and cd86 were negative even in the high concentration of ifn-γ, while bm-mscs became positive for hla-dr. pd-l2 was constitutively expressed in uc-msc, while pd-l1 was induced by the addition of ifn-γ. in mlr, responder t cell proliferation triggered by allogeneic dendritic cells was inhibited efficiently by 3rd party derived uc-mscs, in which was induced ido, pge2, hgf, and tgf-β analyzed by rt-pcr, and inhibited ifn-γ and tnf-α in the supernatant by cytokine beads array. uc-mscs migrated toward the tnf-α treated mnc and increased regulatory t cells incidence in peripheral mononuclear cells by the coculture. conclusions: these results demonstrated that cryopreserved uc are feasible and efficient source of mscs and frozen-thawed uc-mscs have high anti-inflammatory background: a new protocol is under development on the amicus separator that enables the device to perform ecp procedures. the amicus separator is used with a photoactivation device, disposable kit and 8-mop to provide ecp therapy in a closed system. the objective of this study was to evaluate the safety and performance of the investigational amicus ecp system in healthy human subjects. methods: an irb-approved written informed consent was obtained from 17 subjects (12 male, 5 female). the amicus ecp system processed either 500, 2000 or 4000 ml whole blood (n ≥ 5 per arm) using double-needle access and acd-a anticoagulation at a 12:1 wb:ac ratio. after mnc collection was completed, the subject was disconnected from the device. 8-mop (3.4 ml, 20 μg/ml) was injected directly into the collected mnc product and saline (approximately 200 ml total), which was photoactivated with 1-2 j/cm 2 uva light. post photoactivation, the amicus separator reinfused the treated mncs into a transfer pack. subject laboratory and safety parameters were evaluated; in vitro evaluations were performed on subject whole blood, collected mncs, treated mncs, and reinfused cells. lymphocyte and monocyte analysis were performed on samples purified using density gradient separation and cultured for up to 3 days post treatment. results: in 17 procedures, median (range) wb processed was 2016.0 (509 -4024) ml using 172.0 (51 -333) ml of acd-a. procedure time was 93.0 (66 -119) minutes, including photoactivation. no adverse events were reported. subjects' vital signs and hematology values were unremarkable and within expected values. the wbc count of the collected mncs was 13.50 (3.3 -30. 2) x10 3 /μl, comprised of 77.10 (47.9 -87.0) % lymphocytes, 15.50 (7.0 -36.8) % monocytes and 5.70 (2.9 -25.5) % granulocytes and platelet count was 94.0 (70 -169) background: transfusion of white blood cells (wbc) causes a number of transfusion reactions and complications, for example transfusion-associated graft versus host disease (tagvht), which still does not have effective treatment and is a fatal complication of transfusions. the only effective method of preventing tagvht is irradiation of blood components with ionizing radiation (x-ray or gamma radiation). but the use of ionizing radiation sources has a number of technical and material difficulties. the emergence of pathogen reduction technologies (prt) in blood components targeted by nucleic acids has opened the possibility of using these technologies as an alternative to irradiating of blood components. several prt demonstrated effective inactivation of wbc in platelet concentrates and blood plasma. so, determination of the influence of prt based on the combined effect of riboflavin (rf) and ultraviolet (uv) on the viability and proliferating potential of lymphocytes in whole blood is important. methods: samples of whole blood were obtained in 35 healthy volunteers. each sample was divided into three unequal parts: untreated control, gamma irradiated, and treated by rf and uv prt (mirasol, terumo bct inc.). mononuclear cells (mnc) were cfse stained, viability and proliferating activity were tested at intervals of 24 hours for 3 consecutive days by flow cytometry. statistical analysis was performed with xlstat 7.0. levels of significance were calculated by mann-whitney test, expressed as p-values (p< 0,05). results: the median viability of mnc after application of both methods of treatment was over 85,0% on day 0 and decreased to day 3 -median percentage of viable mnc were 84,0% (control group), 69,0% (after gamma irradiation) and 63,0% (rf/uv prt). the median of spontaneous proliferative activity on day 3 of untreated and gamma irradiated mnc did not differ (1,8% and 2,1% respectively, p< 0,05). phytohemaglutenin (pha) induced proliferation on day 3 in gamma-irradiated samples was significantly lower in comparison with control group (4,0% and 45,0% respectively, p< 0,01). in samples treated with rf/uv, spontaneous and stimulated proliferating cells was not detected. median percentage of proliferating mnc was less than 0,2%. the use of this prt on whole blood, as well as gamma irradiation, significantly reduces the viability of lymphocytes during storage for 3 days. conclusions: inactivation of wbc using rf/uv prt is a useful and very necessary bonus for a number of reasons. in one procedure two effects are achieved: infectious and immunological safety. the use of prt on whole blood gives the potential for obtaining pathogen-reduced and immunological safety components of blood, which reduces their material cost and staff loading. the use of rf/uv system does not have such complex security requirements and difficulties in servicing as the use of sources of ionizing radiation. the results demonstrate a promising potential for using this technology as an alternative to irradiation disclosure: nothing to declare p308 influence of patients´serum after allogeneic stem cell transplantation on t cell proliferation and treg function background: acute or chronic graft versus host disease (a/ cgvhd) is one of the major complications after allogeneic hematopoietic stem cell transplantation (ahsct). application of regulatory t cells (treg) as "immunosuppressive dli" to prevent or treat gvhd is investigated in clinical trials. here we ask the question, if there could be clinical conditions (e.g. cytokines or drug effects) limiting the efficacy of this approach. to face this problem we tested the influence of patients´serum on t cell proliferation and treg function. methods: lymphocytes from healthy donors were incubated with t cell medium (90% aim v + 10% serum + il2/okt3) containing serum from healthy donors or serum derived from patients after ahsct with or without gvhd (n=10). next we evaluated the suppressive function of treg by performing treg suppression assays, also comparing serum from patients suffering from gvhd versus serum obtained from healthy donors (n=8). proliferation of cfse stained t cells was measured after 5 days. to test the effect of immunosuppressive drugs on treg we performed treg suppression assays after incubation of treg with corticosteroids or tacrolimus or the combination of both drugs. results: serum of patients with acute or chronic gvhd had a negative effect on t cell proliferation. to avoid bias tests were performed with samples from patients without or only with low levels of immunosuppressive drugs. incubation with serum of patients without gvhd or with serum of healthy individuals showed no differences in t cell proliferation. treg from healthy donors showed a stronger antiproliferative capacity when incubated with serum derived from patients with gvhd. treg previously incubated with immunosuppressive drugs showed no decreased suppressive capacity. conclusions: components of serum from gvhd patients seem to have an antiproliferative effect on t lymphocytes itself. this fact might influence the clinical course of gvhd, but should not be a limiting factor for therapeutic application of treg dli. even the systemic treatment with immunosuppressive drugs e.g. corticosteroids or calcineurin-inhibitors should not diminish the treg application. in a next step we will analyze serum components responsible for this immunosuppressive effect with multi cytokine assays and proteomic analysis. the aim of our project is to develop new strategies to avoid gvhd and to optimize clinical settings for treg dli. disclosure background: hypercalcaemia can be very severe following stem cell transplant (sct) in some osteopetrosis patients. denosumab is a fully human monoclonal antibody that binds the cytokine rankl (receptor activator of nfκb ligand), an essential factor initiating bone turnover. rankl inhibition blocks osteoclast maturation, function and survival, thus reducing bone resorption. we describe the effective management of hypercalcaemia in a patient with rank mutation osteopetrosis who received a haploidentical sct. methods: our patient was diagnosed with osteopetrosis at 2 year of age with a defect in the tnfrsf11a gene which codes for rank and received a maternal haploidentical sct aged 4 years. the patients calcium levels were monitored regularly post sct. denosumab was administered for hypercalcaemia as per laboratory reports or clinical symptoms. the drug was diluted with water for injection to make 6mg/ml solution to facilitate subcutaneous administration. results: significant hypercalcaemia emerged on day +18 with a level of 3mmol/l and treated with hyper-hydration and diuretics. this was ineffective in reducing the hypercalcaemia; therefore denosumab was initiated on day +20 post-transplant. initial dosing was determined using the only available paediatric case report at 0.13 mg/kg. a repeated larger dose of 0.19mg/kg was given 4 days later due to an inadequate response (calcium decreased from 3.9mmol/l to 3.80mmol/l). the calcium decreased to 1.93mmol/l after this dose. four weeks later a third dose was required at 0.26mg/kg as the calcium level had increased to 4.1mmol/l. the dose was further increased to 0.32mg/kg for another four doses and then further increased to 0.65mg/kg for another 3 doses and repeated every 7 weeks. normalisation, but not excessive drop in calcium was achieved with these larger doses. over the 9 month follow up post-transplant there were three admissions lasting less than 24 hours for symptoms of hypercalcaemia. these were managed with denosumab administration and hyper-hydration. the remaining doses were given in an outpatient setting. conclusions: denosumab can be safely used as a first line agent in treating post stem cell transplant hypercalcemia in patients with osteopetrosis. a dose of 0.3mg/kg is required as an initial starting dose in order to control hypercalcemia. this is a new higher dose than previously suggested by the original report. denosumab can be effective even after dilution and safely given in children weighing less than 10kg. disclosure: nothing to declare methods: the clinical, laboratory and molecular aspects of this italian male patient who developed such a complication were collected and presented in order to discuss the origin, clinical outcome and management of this very rare post-transplant event. results: a 68-years-old man affected by a high-risk chromosomally abnormal, ph1-, mll-pro-b (egil b-i) all relapsed during maintenance treatment, nonresponsive to re-induction chemotherapy, in second complete remission (ii cr) after blinatumumab treatment received a female cb transplant. according to sorror's and ebmt scores he was considered a high-risk transplant. the patient and the cb unit were sex-mismatched, shared the same blood groups and were both cmv+/ebv+. he received a tbf conditioning regimen that was followed by the infusion of 0.54x10 5 /kg cd34+ cb cells. gvhd prophylaxis consisted of rabbit atg, cyclosporine a (csa) and mycophenolate mophetyl (mmf). neutrophil engraftment occurred on day +28, whereas platelets were never >20.000/μl. on day +67 a 2 cm bulged area became apparent on the left parietal region of the skull. an echotomography showed that the lesion adhered to the bone without infiltrating it and lacked blood vessels and suggested that it may be either a site of disease relapse or an area of infection. at the same time a bone marrow (bm) aspiration showed morphological cr confirmed by immune-phenotypic studies and x-y fish a complete chimera. since the patient was still febrile no biopsy was performed, but on day +94 the axial diameter of the lesion that on a ct scan showed the same appearance revealed by the previous echo-tomography increased to 4 cm. thus, the lesion was surgically removed and histological examination showed cd33+, cd14+/-, cd163+/-, cd45/lca+/-, cd21-, cd23-, cd35-, cd207-, and s100-neoplastic cells whose phenotype suggested a granulocytic sarcoma rather than a histiocytic sarcoma. immuno-chemistry confirmed this suggestion by showing a nuclear npm1 positivity. fish studies demonstrated that these neoplastic cells were of recipient's origin. a novel bm aspiration showed cr confirmed by immune-phenotypic studies and fish revealed a complete chimera. since the patient was still pancytopenic due to anti-cmv treatment, radiotherapy with 18gy in nine fractions were given and the lesion completely resolved. conclusions: a granulocytic sarcoma of recipient's origin occurring three months after a cb transplant is a very rare and unusual event. in order to explain such a complication we suggest that granulocytic sarcoma cells were dormant but already present at the time of pro-b all diagnosis and survived not only the initial all treatment but also the cb transplant conditioning regimen. we can't exclude that immune-suppressive treatments given early post-transplant might have promoted the outgrowth of these neoplastic cell population. disclosure: nothing to declare haemoglobinopathy and inborn errors of metabolism p311 abstract already published. addition of fludarabine on to anti-thymocyte globulin, busulfan and cyclophosphamide conditioning improves outcomes in low-risk matched-related bone marrow transplantation in children with severe thalassaemia flu-atg-bucy. atg dose was 4 mg/kg in all patients except patients with splenomegaly > 3 cm from costal margin and/or sex-mismatched/maternal donor in whom atg was increased to 7 mg/kg. all patients were younger than 15 years and had no hepatomegaly (liver ≤ 2 cm from costal margin) at bmt. results: actuarial overall survival (os) in the atg-bucy and flu-atg-bucy groups is 89% and 98%, thalassemia-free survival (tfs) 75% and 93%, gvhdfree and thalassaemia-free survival (gtfs) at a median follow up of 23.5 and 10.6 months was 72.6% and 93.3% months respectively, which is a significantly improved outcome by log-rank statistics (p=0.002) in the flu-atg-bucy group. there was no significant difference between the groups in pre-transplant characteristics and posttransplant complications except for the following: median cell dose more in 2 nd group with total nucleated cell dose of 8.7 vs 6.4 x 10 8 cell/kg with p< 0.0001; csa taper started later in the new protocol (184 day vs. 152 p=0.0005); median age at bmt (7.2 vs. 4.6 years, p=0.001); number of pre-bmt transfusions (p=0.01) and ferritin at bmt (2.214 vs. 1.599 ng/ml, p=0.002) were higher in the second group; day 30 and 60 chimerisms were also significantly higher in new protocol (p=0.02 and 0.03 respectively). there was a trend towards increased incidence of veno-occulsive disease (vod) and posterior reversible encephalopathy syndrome (pres) on the second group but this difference did not reach statistical significance. conclusions: adding fludarabine and targeted dose increase of atg in the standard bucy context seems to significantly improve outcomes of thalassaemia transplants without contributing to excessive gvhd or infectious complications. this protocol can be easily administered in low resource setting without major additional costs. clinical is an acquired clonal disorder of the hemopoietic stem cells for which the only curative treatment is allogeneic hematopoietic stem cell transplantation. however, there are still few reports on the outcomes of allogeneic hematopoietic stem cell transplantation (allo-hsct) in patients with pnh compared to paroxysmal nocturnal hemoglobinuria-aplastic anemia (pnh-aa) syndrome. our study aimed to compare the outcomes of allo-hsct for pnh with pnh-aa syndrome. methods: the clinical data of 46 pnh patients received allo-hsct (pnh = 16, pnh-aa = 30) in our center from july 2007 to june 2018 were analyzed retrospectively to compare the outcomes of pnh group with pnh-aa group. the clinical data including 28 male patients and 18 female patients, the median age was 29 years (range 6-54). all patients had received various treatments before transplantation such as steroids, androgens, cyclosporine (csa), antithymocyte globulin, and growth factors. the median interval from pnh diagnosis to hsct was 6 months (range 3-240). the conditioning regimen was modified bu/cybased regimen in haploidentical donors and unrelated donors, csa, mycophenolate mofetil (mmf) and shortterm methotrexate (mtx) were administered for graftversus host disease (gvhd) prophylaxis. patients with matched sibling donors were treated with the flu/cybased regimen and csa were administered for gvhd prophylaxis. results: there were no differences of baseline between the 2 groups (p>0.05) except gender and haploidentical donors. the median values of absolute nucleated cell counts were 10.58 (3.83-13.83 ) ×10 8 /kg in the pnh group and 10.81 (3.96-33.40 ) ×10 8 /kg in the pnh-aa group (p = 0.668). the median doses of cd34 + cells infused were 5.00 (3.14-8.42)×10 6 /kg and 3.57 (1.97-6.17)×10 6 /kg (p = 0.002), respectively. all patients attained complete engraftment, no patient occurred graft failure. the median time for myeloid engraftment were 11 (range, 7-14) days in the pnh group and 12 (range, 10-26) days in the pnh-aa group (p = 0.003). the median time for platelet engraftment were 13 (range, 11-16) days and 18 (range, 12-75) days (p = 0.002), respectively. with a median follow-up of 36 (4-132) months in the pnh group and 26 (4-75) months in the pnh-aa group (p = 0.428). in pnh and pnh-aa groups the incidences of grade i-iv acute graft-versus-host disease (agvhd) were 12.50% and 33.30% (p = 0.121), grade ii-iv agvhd were 6.25% and 20.00% (p = 0.209); chronic gvhd were 12.50% and 35.67% (p = 0.274), moderatesevere chronic gvhd were 0.00% and 14.39% (p = 0.146). in haplo-hsct and msd groups the incidences of infection were 37.50% (6/16) and 33.33% (10/30) (p = 0.777). no patient occurred early death and relapse. 3-year estimated overall survival (os) of pnh and pnh-aa groups were 100.0% ± 0.0% and 85.7% ± 6.6% (p = 0.141), gvhd-free and failure-free survival (gffs) were 100.0% ± 0.0%、78.7% ± 7.7% (p = 0.067). conclusions: the preliminary results indicated that allo-hsct is a feasible choice for pnh with favorable outcomes, time for myeloid and platelet engraftment in pnh group were faster than pnh-aa group. there were no differences in os and gffs between pnh group and pnh-aa group. disclosure: no disclosure pattern of calcineurin inhibitor-associated neurotoxicity in sickle cell disease patients receiving a stem cell transplantation background: allogeneic hsct with a msd represents currently the only curative option for sickle cell disease (scd), limited by a donor availability < 20%. neurotoxicity (nt) contributes significantly to hsct-associated morbidity and mortality. calcineurin-inhibitor (cni) associated nt ranges from 4.2%-28.8% (severe nt 4%-11%). the elevated incidence of nt in scd (around 30%) might be triggered by the systemic vasculopathy of scd, with the brain being the primary target. although both cyclosporine a (csa) and tacrolimus (fk506) have a proinflammatory effect, it is more pronounced in csa. infusion modalities also might impact (10.3% after bolus injections versus 3.3% after continuous infusion). methods: in a pilot study, we compared t-cell depleted haploidentical hsct (t-haplo hsct) with msd hsct in patients (pts) with advanced stage scd, using almost identical conditioning regimens. 32 pts (3-31 years; yrs) with homozygous scd or hbs 0/+ ß-thal were treated between 2012 and 2018. nine pts received a msd bone marrow graft, 23 pts received 24 t-haplo-hsct (1 second t-haplo due to graft rejection). immunosuppression consisted of either csa (6 msd, 16 t-haplo) or fk506 (3 msd, , in combination with mycophenolate mofetil (mmf). fk506 was administered as a 20-hours continuous infusion, csa as 4-hours bolus injections; both target level adjusted (csa: 100-120 ng/ml; fk506: 5-8 ng/ ml). duration of immunosuppression was >6 months in thaplo-sct and < 6 months in msd, depending on chimerism. results: cni-related nt was observed in 36.4%, severe nt (pres, visual disturbance, aphasia) in 21.2%. nt was more prevalent in msd (n=5, 55.5%) than in t-haplo (n=7, 29.2%). the incidence of nt was identical under csa (8/22; 36.4%) and fk506 (4/11; 36.4%), however the majority of severe nt (all pres) occurred with csa. complete recovery of nt was achieved in all pts either spontaneously or after switching to fk506/everolimus or withdrawal of fk506. moreover, 66.6% of pts with nt were >18 yrs, and 91.6% >12 yrs, suggesting an increased risk with age. only 37.5% of pts with pre-existing cerebrovascular disease experienced post-hsct nt. of note, 57.1% of pts with severe nt also developed mild acute gvhd. the overall (os) and disease-free survival (dfs) with a median follow-up of 17 months in t-haplo-hsct and 22 months in msd hsct was 91% vs. 100%, respectively. conclusions: our data confirm an elevated nt risk in scd pts following allo-hsct. importantly, the incidence of nt seems to be related to age (91% of pts with nt were >10 yrs), donor source (msd 55.5% vs. t-haplo 29.2%) and type of cni inhibitor where almost all severe nt (71.4%, particularly all pres) was observed under csa. continuous infusion of fk506 vs. bolus injections of csa might have levelled concentration peaks. the nt observed with csa could be the consequence of predominantly csarelated vascular toxicity inflicting pre-damaged vessels in scd. the mechanism of action could be related to other systemic endotheliopathies such as vod, tam and agvhd, which was observed in 57.1% of pts with severe nt, compared to an overall agvhd rate of 30%. disclosure: nothing to declare background: matched-related bone marrow transplantation (bmt) may cure over 80% of low-riskchildren with severe thalassemia (st) defined as a thalassemia syndrome with inability to keep a spontaneous hemoglobin > 7 g/dl. it is well known that patient status at the time of transplant is critical in predicting transplant outcome. liver size > 2 cm is an established adverse prognostic factor in terms of transplant-related mortality and, in our own experience,a spleen size > 3 cm from costal marginis associated with increase rejection rates (blood 2017 vol. 130 no. suppl 1 1944) . optimising liver and spleen size prior to transplant is likely to improve transplant outcomes. methods: we retrospectively reviewed the effectiveness of our strategy to reduce liver and spleen size pre-transplant using hydroxyurea, super-transfusion and intensive iron chelation. we considered liver size < 2 cm and spleen size less than 3 cm below costal margins as good risk features. liver biopsies were not performed thus pesaro risk classification could not be assigned. all transplant candidates were started on hydroxyurea for a minimum of 3 months and pre-transfusion haemoglobin was maintained > 7 gm/dl while on hydroxyurea. if the child had hepatospenomegaly at enrollmentand no improvement in liver and spleen size after an adequate trial of hydroxyurea (minimum of 3 months of treatment achieving maximum dose of 50 mg/kg day or tolerable haematological toxicity, i.e. neutrophil count between 1000 and 1500/μl and/or platelet count between 100.000 and 150.000/μl) patients were given a trial of supertransfusion maintaining haemoglobin above 12 g/dl) for a minimum of 3 months prior to declaring the patient as having failed downstaging. results: out of 119 transplants across 3 collaborating centers in india, 85 patients had no hepatosplenomegaly at enrolment and hence were not actively downstaged. twelve patients were excluded due to inadequate information on their records. all of the remaining 22 patients with enlarged liver and/or spleen were downstaged to low-risk features. all patients received adequate hydroxyurea trial among which seven (32%) patients required super transfusion in addition to maximal hydroxyurea. out of the 22 patients 18 (82%) were successfully down-staged with the above strategy and proceeded to transplant as low-risk patients. among the remaining 4 (25%) patients 3 had liver > 2 cm and one had a spleen > 3 cm only. there was significant improvement in liver and spleen size from the time of enrollment to transplant (p value 0.0004 and 0.0002 respectively by wilcoxon test for paired samples -two tailed) with median duration of downstaging of 9 months (range 2-27 months). there was no significant difference in overall survival (os) and disease-free survival (dfs) by log rank test between the downstaged group and those who did not have hepatosplenomegaly at enrollment (p value 0.59 0.64 respectively). conclusions: in the majority of children with thalassaemia and high transplant risk features liver and spleen size can be reduced pre-transplant using hydroxyurea and supertransfusions thereby decreasing transplant risk. disclosure: nothing to declare p316 abstract already published. abstract withdrawn. longitudinal analysis of the effect of hematopoietic cell transplantation on ocular disease in children with mucopolysaccharidosis i shows ongoing disease progression background: corneal clouding is seen in nearly all patients with mucopolysaccharidosis-1 (mps-1) causing visual impairment. hematopoietic cell transplantation (hct) is able to stabilize disease in many organs including the brain. however, residual disease in peripheral tissues is often described. therefore, the aim of this study was to determine the long-term effect of hct on ocular disease in mps-1 patients. methods: corneal clouding (grade 0-4) and visual acuity (decimal scale) were prospectively collected from all consecutive mps-1 patients treated with hct between 2003 and 2018 at the umc utrecht. the primary outcomes of interest, the effect of time on corneal clouding and visual acuity, were analyzed using a linear mixed model. the correlation between corneal clouding and visual acuity was analyzed with pearson's rho. other parameters studied were clinical phenotype, age at time of transplantation and hematological enzyme level after transplantation. other outcomes of interest analyzed included intra-ocular pressure, refraction, and macula and lens abnormalities. [[p318 image] 1. results: 24 successfully engrafted mps-1 patients were included (92% with >95% chimerism and normal enzyme levels after hct). corneal clouding stabilized during the first years after hct, but increased rapidly beyond three years (figure 1). other predictors for increased corneal clouding were age at time of transplantation (0.74, 95%ci 0.34:1.15; p=0.0026) and clinical phenotype (-1.02, 95%ci -0.18:-1.86; p=0.0335). visual acuity also worsened significantly over time (-0.03, 95%ci -0.06:-0.007; p=0.01). corneal clouding was strongly negatively correlated with visual acuity (ρ -0.60, p = 7.12e-11). conclusions: after initial stabilization, ongoing ocular disease is seen in mps-1 patients despite successful hct. this hallmarks the shortcomings of current standard therapies. new therapies that overcome the weak spots of current therapies are necessary to improve the late outcomes of these patients. clinical trial registry: n.a. disclosure: b.t.a.v.d.b. was supported by a research grant from the sylvia toth charity foundation, the hague, the netherlands, while working on this study. the sponsors of this study are public or nonprofit organizations that support science in general. they had no role in gathering, analyzing, or interpreting the data. all authors would like to thank all parents and patients for participating in this study. all authors state they have no competitive (financial) interests in this study. background: paroxysmal nocturnal hemoglobinuria (pnh) is an acquired clonal disorder of the hemopoietic stem cells for which the only curative treatment is allogeneic hematopoietic stem cell transplantation. haploidentical donor hematopoietic stem cell transplantation (haplo-hsct) is now increasingly applied as a curative therapy for patients with hematologic diseases. however, there are still few reports on the use of haplo-hsct for the treatment of pnh. our study aimed to compare the outcomes of haplo-hsct with matched-sibling donor transplantation (msd-hsct) for pnh. methods: the clinical data of 40 pnh patients received hsct (haplo-hsct = 25, msd-hsct = 15) in our center from july 2007 to may 2018 were analyzed retrospectively to compare the outcomes of haplo-hsct group with msd-hsct group. the clinical data including 23 male patients and 17 female patients, 13 classical pnh and 27 pnh-aa syndrome, the median age was 29 years (range 6-54). all patients had received various treatments before transplantation such as steroids, androgens, cyclosporine (csa), antithymocyte globulin, and growth factors. the median interval from pnh diagnosis to sct was 6 months (range 3-240). the conditioning regimen was modified bucybased regimen in haplo-hsct group, csa, mycophenolate mofetil (mmf) and short-term methotrexate (mtx) were administered for graft-versus host disease (gvhd) prophylaxis. patients with msd-hsct were treated with the flucy-based regimen and csa were administered for gvhd prophylaxis. results: there were no differences of gender, age, patients of pnh-aa and median time from diagnosis to transplantation between the 2 groups (på 0.05). the median values of absolute nucleated cell counts were 10.74 (4.80-22.86) ×10 8 /kg in the haplo-hsct group and 12.19 (5.14-17.25) ×10 8 /kg in the msd-hsct group (p = 0.866). the median doses of cd34 + cells infused were 3.57 (0.68-7.80) ×10 6 /kg and 4.00 (3.02-8.42) ×10 6 /kg (p = 0.151), respectively. all patients attained complete engraftment, no patient occurred graft failure. the median time for myeloid engraftment were 12 (range, 9-26) days in the haplo-hsct group and 11 (range, 7-15) days in the msd-hsct group (p = 0.065). the median time for platelet engraftment were 19 (range, 11-75) days and 13 (range, 11-25) days (p = 0.027), respectively. with a median followup of 26 (4-65) months in the haplo-hsct group and 36 (4-132) months in the msd-hsct group (p = 0.294). in haplo-hsct and msd-hsct groups the incidences of grade i-iv acute graft-versus-host disease (agvhd) were 32.00% and 20.00% (p = 0.343), grade ii-iv agvhd were 16.00%、13.33% (p = 0.759). chronic gvhd were 30.69% and 24.62% (p = 0.418), moderate-severe chronic gvhd were 12.73% and 7.14% (p = 0.522). in haplo-hsct and msd groups the incidences of infection were 32.00% (8/25) and 26.67% (4/15) (p = 1.000). no patient occurred early death and relapse. 3-year estimated overall survival (os) of haplo-hsct and msd-hsct groups were 86.5% ± 7.3% and 93.3% ± 6.4% (p = 0.520), gvhd-free and failure-free survival (gffs) were 78.3% ± 8.6% and 92.9% ± 6.9% (p = 0.250). conclusions: the preliminary results indicated that haplo-hsct is a feasible choice for pnh with favorable outcomes, haplo-hsct and msd-hsct had similar therapeutic efficacy. disclosure: no disclosure p320 pres in bmt for thalassemia major in india: lower incidence and limited impact background: posterior reversible encephalopathy syndrome (pres) is a relatively common complication seen after blood or marrow transplantation (bmt) for hemoglobinopathies with a reported frequency of 10-19%. pres has also been associated with poorer survival rates. severe hemoglobinopathies are one of the most frequent indications for bmt in the developing world, particularly in india. given the risk of rejection in multiply transfused patients and the need to minimize gvhd risk, immunosuppression post-bmt for these non-malignant conditions can be particularly intense and prolonged. we sought to measure the incidence and impact of pres in developing countries. methods: we analysed 194 successive transplants for thalassemia using protocol 1 (atg-bucy+csa/mmf or csa/mtx) maintaining cyclosporine a (csa) blood levels 100-150 ng/ml for 74 patients and protocol 2 (flu-atg-bucy+csa/mtx) maintaining higher csa levels post, i.e. 150-250 ng/ml for 120 patients from fully matched donors with g-csf-primed bone marrow. for 3 patients this was the second transplant from a different matched related donor. pres was confirmed with brain ct/mri for all patients. results: all recipients who had pres had sibling donors, 5 males and 2 females. age median 7.4 (iqr 5.6-8 years). the frequency of pres was 3.6%; disease free survival for patients who had pres was 100%. pres resolved completely in all. csa was switched to mmf in 5 patients who had received mtx and were on csa only at the time of pres occurrence, while csa was stopped but mmf continued in 1 patients taking csa/mmf combination and csa was continued for 1 patient. three patients with pres had grade 2 acute gvhd, 1 had grade 1 gvhd and none developed chronic gvhd. csa levels at the time of pres were a median of 133 ng/ml (iqr: 89 to194) with 1 patient having 419 ng/ml. three patients had pres while they were thrombocytopenic. hypertension stage 2 was observed in four patients, stage 1 in one patient, one patient was not hypertensive and in one patient blood pressure values were not available. two patients were on methylprednisolone 1 and 1.3 mg/kg/day and one was on dexamethasone 10 mg/m2/day. one patient was started on csa again after the pres episode and within 2 weeks had another one while on csa (level 30 ng(ml), methylprednisolone 1.5 mg/kg/day and ruxolitinib for gvhd. protocol 2 had statistically significant improvement in disease free survival from 67% to 91% (p< 0.001) with probability of occurrence of pres increasing from 1.4% to 5.0% (p = 0.17, see figure 1 ), yet had a benign course in all patients. conclusions: not stopping immunosuppression may have been the key factor which could explain why we have better outcomes with pres than what is reported. intensifying immunosuppression pre-bmt did lead to more pres, albeit not significantly, and yet it was quite manageable. even with addition of fludarabine our pres incidence is lower than previously reported. [[p320 image] 1. background: sickle-cell diseases (scd) are a group of genetic hemoglobin disorders marked by brain vasculopathy. allogeneic hematopoietic stem cell transplantation (hsct) is a curative option able to stop vascular disease progression. diffusion-tensor imaging (dti) is a magnetic resonance imaging (mri) technique sensitive to the brownian motion of water molecules and cellular environment. this microscopic quantitative technique is able to detect white matter (wm) alterations before a conventional mri. the aim of this study was to use dti to evaluate axonal damage and structural connectivity in the brain of patients with scd submitted to hla-identical sibling allogeneic hsct. methods: sixteen scd patients with no extensive vasculopathy detected by conventional mri (11 male, age range: 9 -33 years) and 17 age-matched healthy controls (10 male, age range: 6 -31 years) participated in this prospective study. mri acquisitions were performed in a 3t scanner two times for patients (before and 1-5 years after hsct) and at a single moment for controls. from dti acquisitions, fractional anisotropy (fa), mean (md), radial (rd) and axial diffusibility (ad) were calculated in the wm of the whole brain. structural connectivity was also analyzed, based on graph theory, obtaining efficiency, length path and clustering coefficients of the brain network. an anova test was applied to analyze fa differences among controls and patients, before and after hsct. a paired two-tailed t-test was used to determine statistical significance of changes in the fa, diffusivity mean values and network parameters before and after hsct. results: mean fa was lower in patients before hsct than controls (p = 0,038) and increased after hsct being not statistically different when compared to controls (controls = 0,3504; patients before hsct = 0,3328; patients after hsct = 0,3422; post hoc dunnett's test -error 0,03; anova test). when patients were compared before and after hsct, md and rd decrease after hsct (p = 0,038 and 0,047, respectively). on the other hand, fa increased (p = 0,044). after hsct, efficiency was higher (p = 0,023) and path length index was lower (p=0,027) than at study entry (table 1) . conclusions: this study indicates that, before hsct, patients with scd present axonal damage not detectable by conventional mri, when compared to healthy controls. we also suggest that hsct is able to promote axonal recovery and reorganization. partial diffusivity recovery could be associate to a still unidentified mechanism of myelin regeneration. in the future, longer follow up and comparisons with other forms of treatment are required. background: bmt is a well-established treatment modality for haemoglobinopathies, limited by the availability of related donors. unrelated transplantation has historically shown variable outcomes driven by gvhd and toxicity, and usually restricted to 10/10 matches, but the impact of reduced toxicity conditioning regimens is yet to be known. methods: from 2011 to 2018 twenty-five consecutive unrelated bone marrow transplants were conditioned with fludarabine 160 mg/m 2 , treosulfan 42 g/m 2 , thiotepa 10 mg/ kg and atg (thymoglobulin) 11.25 mg/kg if the source of stem cells was marrow (n = 21) or ptcy if pbsc (n = 4). endogenous haemopoiesis was suppressed pretransplantation for a minimum of 8 weeks. gvhd prophylaxis was provided with ciclosporin/sirolimus and mmf. thirteen patients were transplanted for b thalassaemia major, one of a thalassaemia major and 11 sickle cell disease. the median age was 8 years (2 -19). ten patients were 10/10 matched (7 thalassemia and 3 sickle) and 15 patients had a 9/10 match (7 thalassaemia and 8 sickle). the median cell dose was 3.88 x 10 8 tnc/kg (range 1.38 -13.3) and 5.22 x 10 6 cd34+/kg (range 1.10 -27.41). the median survival was 13.4 months (0.7 -68.3). patients with thalassaemia were pesaro class i or ii (pesaro class iii patients were intensively chelated pretransplantation to return to class i or ii). patients with sickle cell disease were transplanted for stroke or recurrent vaso-oclusive crises and/or acute chest syndrome not responding to hydroxycarbamide. results: all patients engrafted and achieved evidence of donor haemopoiesis on day +28 and achieved transfusionindependence and donor haematological values, but subsequently one 9/10 patient with thalassaemia suffered secondary graft failure on day +75 after macrophage activation syndrome. median neutrophil engraftment was 13 days (range 9 to 19) and 12 days (9 -22) for 10/10 and 9/ 10 patients respectively. patient with sickle cell disease had the platelet count maintained >50 x 10 9 /l at all times. the median platelet engraftment >50 x 10 9 /l was 31 days (range 21 to 53) and 40 days (range 15 to 86) 10/10 and 9/10 patients respectively. there were three deaths, all in the 9/10 matched group: two with thalassaemia (day +257 due to idiopathic pneumonia syndrome and day +102 due to mas) and one with scd (day +43 due to ips). there were different trends of complications seen by degree of matching that did not segregate otherwise by disease. conclusions: in conclusion, unrelated bmt for haemoglobinopathies with reduced toxicity regimens is feasible. whilst gvhd caused significant morbidity during the transplant period, other alloreactive/endothelial complications (vod, macrophage activation syndrome, idiopathic pneumonia syndrome) were only seen in the 9/10 transplants. disease-free survival, dependent on transplantrelated mortality, and lack of long-term toxicity, including chronic gvhd, are determined by the degree of matching. 10/10 matched transplants have excellent long-term outcomes with no chronic gvhd >18 months and can be considered for patients without a related donor; whereas 9/ 10 transplant have significant toxicity and mortality, warranting a haploidentical approach. disclosure: no conflict. long-term safety and efficacy of lentiglobin gene therapy in patients with transfusion-dependent β-thalassemia following completion of the phase 1/2 northstar study patients with transfusion-dependent β-thalassemia (tdt) may benefit from gene therapy involving β-globin gene addition to hematopoietic stem cells (hscs) enabling production of functional hemoglobin (hb). lentiglobin gene therapy contains autologous cd34+ hscs transduced ex vivo with the bb305 lentiviral vector encoding β-globin with a t87q substitution under transcriptional control of the encoding β-globin locus control region. the safety and efficacy of lentiglobin was evaluated in adults and adolescents with tdt in the 2-year phase 1/2 northstar study (hgb-204; nct01745120). methods: patients with tdt (≥ 100 ml/kg/year of red blood cells [rbcs] or ≥ 8 rbc transfusions/year) received g-csf and plerixafor for hsc mobilization. to generate drug product (dp), cd34+ hscs were transduced with the bb305 lentiviral vector. patients underwent single-agent, myeloablative busulfan conditioning, were infused with the dp, and were followed for safety and efficacy. results: eighteen patients have been treated in the completed northstar study. as of 14 september 2018, patients had a median follow-up of 38.9 (min -max: 29.3 -48.1) months. the median age at consent was 20 (min -max: 12 -35) years including 15 patients ≥ 18 years old. patients received a median cell dose of 8.1 (min -max: 5.2 -18.1) cd34+ cells x10 6 /kg with a median dp vector copy number (vcn) of 0.7 (min -max: 0.3 -1.5) vector copies/ diploid genome. the median liver iron content (lic) at baseline was 5.7 (min -max: 0.4 -26.4) mg fe/g dw. outcomes by age and baseline iron status will be presented. the median time to neutrophil and platelet engraftment was 18.5 (min -max: 14 -30) and 39.5 (min -max: 19 -191) days, respectively. four patients had platelet engraftment ≥ day 60 and four patients had platelet counts of ≤ 100x10 9 /l at month 12. none of these patients had ≥ grade 3 bleeding events post-lentiglobin infusion. transfusion independence (ti, defined as weighted average hb ≥ 9 g/dl without rbc transfusions for ≥ 12 months) was achieved in 8/10 patients with non-β 0 /β 0 genotypes and 3/8 patients with β 0 /β 0 genotypes. in patients who achieved ti, total hb at last visit was 9.1 -14.1 g/dl. lic increased from baseline in patients who achieved ti by a median of 55.6% and 12.5% at month 12 and 24 then decreased from baseline by a median of 9.2% and 44.4% at month 36 and 48, respectively. non-hematologic grade ≥ 3 adverse events post-infusion in ≥ 3 patients included stomatitis, febrile neutropenia, pharyngeal inflammation, and irregular menstruation. there was no transplant-related mortality, vector-mediated replication competent lentivirus, or clonal dominance. two patients experienced grade 3 serious veno-occlusive liver disease (table 1) . events resolved following treatment with defibrotide and were attributed to myeloablative conditioning. conclusions: in the northstar study, 80% of patients with tdt and non-β 0 /β 0 genotypes and 38% of patients with β 0 / β 0 genotypes achieved transfusion independence. the safety profile of lentiglobin remains consistent with myeloablative busulfan conditioning. longer time to platelet engraftment was observed in some patients, but no graft failure was reported. clinical background: sickle cell disease (scd) is an inherited hemoglobin disorder associated with high morbidity and mortality. currently, allogeneic hematopoietic stem cell transplantation (hsct) is the only curative therapy for scd. transplant outcomes with thiotepa, treosulfan and fludarabine (ttf) preparative regimen are encouraging but this regimen has not been directly compared to other preparative regimens in scd. we therefore planned to compare the event free probability for death, rejection and high grade acute graft versus host disease (agvhd) between ttf and busulfan and fludarabine (bf) regimens. methods: in this retrospectively cohort study, we included all patients with scd who received allogeneic hsct at our center or who were transplanted in other centers and referred to ours for follow up before day 100. patients were transplanted between july 2007 and december 2017. we used kaplan-meier curve to estimate the event free probability for death, rejection and high grade agvhd (grades 3-4). cox regression was used to assess the impact of the preparative regimen on these outcomes. results: a total of 61 patients were included with a median age of 20 years (interquartile range [iqr]: 14-26) and a median hemoglobin of 10 g/dl (iqr: 9-10). sixtytwo percent were males. the proportion of patients who had splenectomy, stroke and acute chest syndrome was 34%, 23% and 57% respectively. all patients received peripherally collected hematopoietic stem cells from a matched sibling donor with a median stem cell dose of 6 x 10 6 /kg (iqr: 5. 1-8.8 ). most patients, 95%, received cyclosporine or tacrolimus based agvhd prophylaxis. most patients received ttf (41%) or bf (53%) preparative regimens. all patients in the bf group received atg. the median follow-up time was 44 months (range: 2-127). four patients died during the follow-up period with an os of 93% (95% confidence interval [ci]: 87%-100%) at 5 years. the os was not different (hr 1.3, p = 0.82) between the ttf (91%) and the bf (94%) regimens. the probability of high grade agvhd free survival at day 100 was 91% (95% ci: 84-99) for all patients. this probability was 85% in the ttf group and 93% in the bf group and the difference was not statistically significant (hr 2.2, p = 0.39). the rejection free survival at 12 months was 93% (95% ci: 87-100) for all patients. no patients in the ttf group rejected while the rejection free survival at 12 months for the bf group was 90%. this was not statistically significant (p = 0.07). conclusions: in patients with scd undergoing allogeneic hsct from a matched sibling donor, the ttf preparative regimen is not associated with improved os, rejection free or high grade free agvhd survival when compared to the bf preparative regimen. larger studies are needed to confirm these findings. disclosure: nothing to declare. novel strategy for haploidentical hematopoietic stem cell transplant in sickle cell disease methods: 9 consecutive patients suffering from scd who underwent hhsct between jan 2018 till date were enrolled in the study. all 9 underwent autologous backup (target dose>5x10 6 /kg) followed by pre-transplant immune suppression (ptis) 2 cycles at 3 weekly intervals using fludarabine @30mg/m2/day(d1-d5) + cyclophosphami-de@1000mg/m2/day(d1) + dexamethasone@20mg/m2/ day(d1-d5) along with hypertransfusion (target hb 11-13gm/dl), hydroxyurea (20mg/kg/day) and azathioprine (2mg/kg/day) from day -60. the graft was mobilized using gcsf@10mcg/kg/day(d1-d5) + plerixafor@0.24mg/kg s/ c on d5 6-8 hours before the pbsch. conditioning included thiotepa 10mg/kg in two divided doses (d-7), fludarabine 30mg/m2 (d-6 to d-2), cyclophosphamide 14.5 mg/kg (d-5, d-4), tbi 2gy with thymic shielding (d-1), ratg (genzyme thymoglobulin 1.5 mg/kg (d-9 to d-7). gvhd prophylaxis included ptcy 50 mg/kg/day on d3 and 4, sirolimus (target levels 10-15ng/ml) (till 9-12 months post hsct) and mmf (till d35) starting from d5. results: the median age of patient's was 7 years (range 3-22 years). before transplantation all patients had repeated episodes of one or other complication warranting a transplant, non-responsive to hydroxyurea. six had maternal donors, 2 paternal and 1 sibling. median age of the donor was 41 years (range 19-51 years). all were dsa negative with a cutoff mfi of >2000 iu. all patients received 10x10 6 /kg cd34 cells irrespective of harvested dose which ranged from (10.13-34.82 x10 6 /kg). median cd3 dose was 16.59 x10 7 /kg (range 10.44-41.9 x10 7 /kg). all patients engrafted with median time to neutrophil engraftment 13 days (range 12-15 days) and median time to platelet engraftment 13 days (range 11-16 days). median duration of hospital stay was 30 days (range 24-35 days). one patient had cytokine release syndrome needing tocilizumab. five had engraftment syndrome treated with short course of steroids. two had cmv reactivation needing treatment with ganciclovir/valganciclovir. acute gvhd grade ii was seen in one patient. till date of analysis none had features compatible with chronic gvhd. of the 9 patients, 8 are alive without sickle cell disease with lansky/ karnofsky scores of 100. at median follow up of 164 days (range 61-271) the probabilities of survival, sca-free survival, and transplant-related mortality after transplant were 88.9%, 88.9%, and 11.1%, respectively. one patient died due to mdr klebsiella sepsis after being discharged initially while he was receiving iv ganciclovir on day care basis. he had full donor chimerism. none of the patient had primary or secondary graft failure. conclusions: pre-transplant immune suppression and upfront use of plerixafor for graft mobilization decreases the risk of graft failure and graft versus host disease leading to overall better survival in hhsct for sickle cell disease. disclosure: none. combined haematopoietic stem cell transplant and enzyme replacement therapy in wolman disease: outcomes and challenges jane kinsella 1 , denise bonney 1 , helen campbell 1 , robert wynn 1 , simon jones 1 background: infantile lysosomal acid lipase deficiencymore commonly known as wolman disease -is an autosomal recessive lysosomal storage disease, characterised by storage of cholesterol esters in the liver, spleen and gastrointestinal tract. these children present under the age of 6 months and traditionally had a poor prognosis, with almost all being dead by the age of 12 months. bone marrow transplant has been used to correct disease manifestations, but limited by high procedure-related mortality with the significant co-morbidities. the survival has changed over the past few years due to pharmacological enzyme replacement therapy but still presents challenges for these patients and their clinicians. in these children haematopoietic stem cell transplant we have offered bmt with enzyme replacement therapy, in certain specific circumstances. methods: four children with wolman disease being treated with enzyme replacement therapy, limited by alloantibody, or poor venous access, received treosulfan-based, myeloablative conditioning with serotherapy followed by a matched haematopoietic stem cell transplant: two family donors, one sibling donor and one unrelated donor. results: three of the four children survived transplant. they have continued to receive enzyme replacement therapy but at reduced dose and frequency with improved tolerability. they have continues to grow and develop. growth and gastrointestinal histology is improved for children having received transplant compared to those receiving enzyme replacement alone. monitoring of peripheral blood chimerism has shown a disease-associated engraftment defect, with mixed chimerism in the 3 surviving patient. conclusions: haemopoietic stem cell transplant is a suitable treatment option in children with wolman disease in whom receiving enzyme replacement therapy is not possible because of venous access, sensitisation or cost reasons. it improves their tolerability of the enzyme treatment and allows for a reduction in enzyme dose and frequency. however, the results of engraftment are not as good as expected for a transplant with myeloablative conditioning and a matched donor. an engraftment defect has been observed in lysosomal acid lipase deficient animal models. a further understanding of this poor engraftment in children with wolman disease is required as to determine whether the risks of transplant is beneficial in these patients and for the consideration of future treatment options including gene therapy. background: thalassemia major is the most common transfusion dependent hemolytic anemia in the world. the absent or reduced production of the β-chain of hemoglobin causes severe ineffective erythropoiesis, massive erythroid hyperplasia in the bone marrow and extramedullary hematopoesis occurs. patients require regular transfusion therapy lifelong. currently, the only proven curative treatment of thalassemia is allogeneic stem cell transplantation (sct). methods: we evaluated the immune reconstitution results of 23 patients at 1 year after hematopoetic stem cell transplantation at our pediatric bone marrow transplantation center between january 2015 and december 2018. all patients were not receiving any immunosuppressive treatment at least for 3 months and they have normal lymphocyte counts, immunoglobulin levels and transfusion independent. lymphocyte subtypes and chimerism percentages and the relationship with the donor type were evaluated at 1 year of transplantation. results: ages of transplantation was ranged between 1-17 years (median: 5 years). seven (30%) of them was male. matched unrelated donor type was chosen in 7 patients while others (16 patients) were transplanted from family matched donor (matched sibling: 9 patients, matched family: 7 patients). all patients received myeloablative conditioning regimen containing busulfan/treosulfan, cyclophosphamide, thiotepa and fludarabine. follow up time was between 12-47 months (mean: 24 ± 10 months). in 9 patients, whole bone marrow product was used while peripheral stem cell harvest in remaining patients. cd3 levels were found low in only 4 patients, in normal patients mean was 59% ± 14%. cd4 levels were severely low in 18 patients while cd8 in only 1 patient. cd8 levels were increased in total 13 patients in as compensatory. cd4/cd8 ratios were very low in all patients (range: 0.2-0.7). b cells (cd19+) were low in 3 patients while immunoglobulin levels were normal. chimerism values between 55-99% (mean: 93 ± 11%). donor and product types did not differ in cd3+ lymphocyte reconstitution at 1 year (p=0.15, p=042 respectively). all patients were alive and well at 1 year after transplantation. conclusions: after 1 year of transplantation, although patients are in well condition regarding to infection frequency and transfussion dependency, it was seen that their lymphocyte subtypes reconstitution could not be achieved enough as in normal children. we can conclude that low cd4+ cell levels were an expected finding in almost all patients. so, these patients may have a tendency to suffer serious bacterial and viral infections, and close follow up be required in terms of infections as long as cd4 levels continue to be low. immunoglobulin replacement therapy did not required even in patients with low b cell levels. disclosure: nothing to declare p328 phase 2 international, multicentre trial to assess haploidentical aß t-cell depleted stem cell transplantation in patients with sickle cell disease with no available sibling donor background: sickle cell disease (scd) is an inherited disorder with an estimate of 300,000 affected newborns per year worldwide. allogeneic hematopoietic stem cell transplantation (hsct) with a matched sibling donor (msd) is currently the curative standard of care for scd patients (pts). however, msd availability is < 20%. a t-cell depleted haploidentical hsct (t-haplo-hsct) from a relative, mostly a parent, expands the donor availability while exhibiting low gvhd rates and thus could offer cure to the remaining 80% of scd patients. in a pilot study, comparing t-haplo-hsct with msd hsct in advanced stage scd, using almost identical transplant regimens for both. the overall (os) and disease-free survival (dfs) was 90% vs. 100%, respectively. methods: these results led to the design of a clinical trial to assess tcd-haplo-hsct prospectively which aims to demonstrate that a hsct from a haploidentical relative is not inferior to a msd hsct with regard to major outcome parameter. this phase 2, prospective, stratified, open-label study is targeting enrollment of 212 patients aged 1-35 years with homozygous hbs disease or heterozygous hbsc or hbs 0/+ ß-thal suffering from severe or moderate scd related complications. inclusion criteria are clinically significant scd related complications such as stroke, silent crisis, pathological angio-mri, transcranial doppler (tcd) velocity >200 cm/s, 2 or more episodes of acute chest syndrome (acs) in a lifetime, chronic transfusion dependency, transfusion-refractory allo-immunization and others. pts fulfilling inclusion criteria will be stratified according to donor availability. pts with a msd will receive a bone marrow graft, pts requiring an alternative donor will be transplanted with an aß/cd19 depleted graft from a haploidentical family donor. the conditioning regimen for both groups will be identical with the exception that antithymoglobulin (atg-neovii ® ) is given upfront in thaplo-hsct versus day -3 to -1 in msd. chemotherapy consists of thiotepa, fludarabine and treosulfan. posttransplant immunosuppression will consist of mofetil mycophenolate and tacrolimus for a duration >6 months in t-haplo-hsct and < 6 months in msd, depending on chimerism. (eudract number: 2018-002652-33) results: primary efficacy endpoint: event free survival (efs). event is defined as incidence of acute gvhd, grade iii -iv, chronic gvhd, rejection (graft failure) or death (for any reason). key secondary endpoint(s) are os, dfs, graft failure, hematological and immunological reconstitution, quality of life (qol) assessment and fertility. the primary null hypothesis is: efs of scd patients treated with t-haplo-hsct is non-relevantly inferior to efs in the msd arm. conclusions: results will help to determine if an a/ß depleted t-haplo-hsct can be considered equivalent to msd hsct with regard to dfs, adverse events and safety, in order to offer this form of cure to the majority of patients with scd. disclosure: nothing to declare hit three birds with one stone: successful stem cell transplantation from one family donor to three siblings methods: in august 2016, three thalassemic siblings were admitted to hospital for stem cell transplantation from a full match donor, their 17 years old sister. the patients' general health conditions and specific health issues due to thalassemia were checked extensively. it was decided to perform first transplant to older sister whom the disease and transplant complications are expected more intense due to prolonged transfusion and chelation therapy. the oldest daughter of family, healthy, was planned to accompany her sisters in transplantation unit so parents can take care the others and organize this period for whole family. results: the 13 years old sibling was first admitted to bone marrow transplantation unit in july 2017. the conditioning regimen was busulfan, fludarabine, cyclophosphamide and thiotepa with antithymocyteglobulin(atg) and defibrotide prophylaxis was given. the healthy donor was admitted to hospital and received g-csf for 5 continuous days before harvesting. stem cells were collected peripherally on day 0 and viable cd34 + cells were 2211/ul. patient received 5,5 x10e6/kg stem cell and the other cell products were divided into 4 parts according to other recipients´weight. no infusion problems were recorded in stem cell transfusion. gvhd prophylaxis was given with cyclosporin and methotrexate. severe sinusoidal obstruction syndrome was observed and successfully managed with supportive therapy. neutrophils were engrafted +11. day, and platelets were on day 64. full blood chimerism results were %98 in day 30, %99 in day 60 and %98 in day 180 consecutively. after 3 months from first transplant the 4 years old sister was admitted to hospital on october 2017. same conditioning with defibrotide prophylaxis and gvhd prophylaxis were given and 6,8 x10e6/kg peripherally derived and previously frost stem cell was infused without any complications. mild sinusoidal obstruction syndrome was observed and managed with supportive therapy successfully. neutrophils were engrafted +11. day, and platelets were on day 16. full blood chimerism results were %99 in day 30, %99 in day 60 and %99 in day 180 consecutively. the third transplant was performed on january 2018 with the same conditioning and prophylaxis regimen. although defibrotide was used mild sos was observed and treated with supportive therapy with success. neutrophils were engrafted +10. day, and platelets were on day 27. full blood chimerism results were %99 in day 30, %99 in day 60 and %99 in day 180 consecutively. conclusions: the patients are being followed for over a year after first transplat, neither adverse nor gvhd symptoms were observed. we presented this case for being a unique example for match family donor transplant and the first successful example from one donor to three recipients. disclosure: nothing to declare results: our female patient admitted for anemia at 3 rd month of birth and was transfused every 4-5 months from 6 th months to 3.5 years of age. since investigations directed towards hemoglobinopathies or membrane defects like hereditary spherocytosis were unremarkable, she was not transfused for 6 years after the age of 3.5-years because hemoglobin level was constant over 7 g/dl. her bm examination showed erythroid hyperplasia and feature of dyserythropoiesis with a few binucleated erythroblasts. it was decided to follow-up the patient with a diagnosis of cda ii. after the age of 10-years, the need for transfusion started again for every 4 to 5 months which led the parents of our patient to request for bone marrow transplantation, however, the diagnosis was not definite, and because of the insufficient data for the transplantations for cda ii patients, it was decided to go on to follow-up. nevertheless, after 2 years, the frequency of transfusion gradually increased to every 2-3 weeks, and bone marrow transplantation was brought into question again. at that time, genetic examination was started and sec23b gene was analyzed by direct sequencing. hsct decision from her hla 10/10 matched brother, carrying sec23b mutation in heterozygous state, was taken. in the preparation regimen, busulfan (bu) at a myeloablative weight adjusted dose (4 days), 200 mg/kg cyclophosphamide (cy) (4 days), and 30 mg/kg antithymocyte globulin (atg fresenius) were used. graftversus-host disease (gvhd) prophylaxis was with cyclosporin a started on day -1 and short-term methotrexate on day +1,+3 and +6. she was transplanted with bm with a dose of total nucleated cells=7.1x10 8 /kg and cd34=3x10 6 / kg. neutrophile and platelet engraftment were achieved at +20 and +38, respectively. indeed, grade 4 hemorrhagic cystitis due to bk virus and a moderate veno-occlusive disease prolonged platelet transfusion days which concealed the exact engraftment day of platelet. the patient was discharged on day 45 with no more need for any transfusion and followed up as a complete chimeric with no type of gvhd since then. now, she is 20 years old, under regular surveillance at our transplant centre without any symptoms. conclusions: hsct data in cda ii patients is still insufficient, however based on data from tm patients with similar treatment approaches in td cda ii patients, it is seen that the hsct is reliable and effective. disclosure: nothing to declare hematopoietic stem cells background: the prognosis after frontline therapy in b-all patients have improved due to monoclonal antibodies (cd20, cd19, cd22) and approximately 90% of patients achieve complete remission. in relapsed and refractory (r/ r) b-all and also in mrd + outcomes are relatively poor. disease-free survival (dfs) in this cohort is 10-20%. in this cohort allo-hsct is indicated and complete remission before transplantation is crucial for prognosis. conventional chemotherapy is associated with high failure rate and significant toxicity. immunotherapy with monoclonal antibodies and car-t are more promising approaches. the aim was to evaluate the efficacy (frequency of responses, os, dfs) and toxicity, especially neurotoxicity and cytokinerelease syndrome, of a bispecific monoclonal antibody blinatumomab in patients both children and adults with persistence of minimal residual disease (mrd + ) or r/r b-all as a bridge to allo-hsct. methods: this study included 120 patients with high risk b-all blinatumomab treated in 2013-2018, among them 14 pts (12%) with t(9;22), 10 (8%) with t(4;11), with mll 11(9%), 84 pts (70%) who were refractory to previous chemotherapy, 66 (30%) after allo-hsct from deferent type of donors. median age was 21 y.o. (range 5m-71y.o), 55 children 0-18 y.o. (46%) and 65 adults >18 y.o. (54%). r/r all had 63 pts (52%), mrd + -57 pts (48%), median days of follow up were 227 (18-720). blinatumomab was applied as 28-day cycles followed by a 14-day off-period before the start of the following cycle. majority pts received one cycle (n=94, 78%). in r/r all group dose was of 9 mcg/d during the first 7 days and afterwards 28mcg/d. patients with weight less than 45 kg received 5 mcg/m 2 /d and 15mkg/m 2 /d accordingly. in mrd group dose was 15 mcg/m 2 /d. results: the frequency of responses to blinatumomab was higher in mrd + pts in comparison r/r all pts (85% vs 62 % p=0.007). in mrd + pts cr mrdwas achieved in 47 pts (82.5%), 10 pts (17.5%) were mrd+ after blinatumomab. two-year os in this group was 61%. twenty pts (34%) received allo-hsct. in rr all pts cr mrdwas achieved in 30 pts (48%), 9 pts (14%) were mrd+ after blinatumomab, 24 pts (38%) had no hematological response . two-year os in r/r all was 43%. fifteen pts (24%) received allo-hsct. os in cr mrdpatients who received allo-hsct was not significantly different in comparison with patients who received blinatumomab as a monotherapy (84% vs 71%, p=0.08). no significant differences in dfs were observed at two years in cr mrdpts depending status of the disease before therapy-mrd vs r/r (66% vs 59%). of the reported adverse events, febrile fever was the most common 91pts (76%), neutropenia 43 (35%), thrombocytopenia 46 (38%), infection 32 (26%), neurotoxicity 29 (24%), cytokine-release syndrome 8 (7%). all complications were reversible. conclusions: blinatumomab is effective option in patients with high risk b-all especially in the group with mrd persistence after previous chemotherapy and facilitates effective bridging to hsct. blinatumomab therapy is generally well tolerated. disclosure here we address the transcriptional regulation of differentiated cells from human embryonic stem cells (escs) using self-assembling peptide hydrogel without stromal cells, and compare with embryoid body (eb) culture system. methods: esc differentiation was induced in eb culture system or three-dimensional (3d) hydrogel culture system. the engraftment potential of differentiated cells was evaluated by flow cytometry. cd34 + cells from mobilized peripheral mononuclear blood cells (mpbmcs) or differentiated from escs at different times (day 7, day 10, day 14) were purified by fluorescent-activated cell sorting. sorted cells were captured on medium-sized microfluidic chips using the fluidigm c1 single cell auto prep system. sequencing was performed by hiseq x ten. results: self-assembling peptide hydrogel formed a 3d scaffold for cell culture, the pore diameter of which ranged from 50 to 200 nm. compared to eb culture system, escs in 3d culture system differentiated more potently. the differentiated cells from 3d system were short-term engrafted in the nog mice, and myeloid cells, b cells and t cells could all be detected in peripheral blood after transplantation. however, the engraftment was not obtained in differentiated cells from eb culture system. we obtained and analyzed 301 escs, 554 cd34 + cells from eb culture system, 440 cd34 + cells from 3d culture system, and 218 cd34 + cells from mpbmcs. the cells were divided into 11 cluters ( figure 1a ). in both differentiation systems, the cd34 + cells from day 7 were more heterogeneous than cd34 + cells from day 10 and day 14 ( figure 1b) . however, cd34 + cells from mpbmcs were more homogeneous, probably because the differentiated cd34 + cells contained several cell lineages, including hematopoietic cells, endothelial cells and mesenchymal cells. there is transcriptional overlap between individual cd34 + cells from eb and 3d culture systems. however, we found that cluster 3, which is composed mainly of cd34 + cells from 3d at day 14 and day 10, expressed similar level of several hematopoietic regulator as hsc, such as tal1, lmo2, erg ( figure 1c ). the cluster 6, which is almost the cd34 + cells from 3d at day 7, also expressed the highest gata2 among the clusters from differentiated cells ( figure 1c) . conclusions: our study demonstrates that 3d hydrogel culture system facilitates hematopoietic specification of escs. disclosure: nothing to declare higher cd34+ cell dose increases overall survival in the setting of dual t-lymphocyte suppression with atg and ptcy in matched related and unrelated donor allosct background: there is no consensus on the cd34+ donor cell numbers required for optimal outcomes in allogeneic stem cell transplant (allosct). there is controversy on the benefits or harm in higher cell dose for allosct. this study aims to evaluate the impact of cd34+ cell dose in allosct patients receiving reduced intensity conditioning (ric) combined with anti-thymoglobulin (atg) and posttransplant cyclophosphamide (ptcy) using related (mrd) and 10/10 and 9/10 matched unrelated donors (mud). methods: this is a single-centre retrospective analysis of 140 adult patients who received allosct for hematologic malignancies between october 2015 and may 2018. all received ric using fludarabine (30mg/m 2 /day: day -5 to -2), busulfan (3.2kg/m 2 /day: day -3 and -2) and total body irradiation (200 cgy: day -1). all patients also received rabbit-atg (4.5 mg/kg: day -3 to -1), ptcy (50mg/kg/day: day +3,+4) and cyclosporine (from day +5). unmanipulated peripheral blood stem cells were infused on day 0. analyses were done using 2 thresholds: (1) an arbitrary cd34+ cell dose of 6 x10 6 /kg (as this was our target dose) and (2) cell dose according to quartiles (< 5.67, 5.67-7.98, 7.99-11.34 and ≥ 11.35 x10 6 /kg). results: median cd34+ cell dose was 7.98 x10 6 /kg. median follow up was 19 months (range 5-35). median neutrophil engraftment was 16 (range 13-31) days and platelet engraftment was 21 (range 11-83) days. a cell dose greater than 6 x10 6 /kg was associated with an increased overall survival (os) at 1 year (71.3%; 95% ci, 62.3-80.3 vs 46.7%; 95% ci 30.3-63.1; p=0.018, figure 1 ). the higher dose was also associated with shorter platelet engraftment time (p=0.016, figure 2 ). there was no significant difference in neutrophil engraftment, nonrelapse mortality (nrm), relapse free survival (rfs), grade ii-iv acute graft versus host disease (agvhd) and moderate to severe chronic graft versus host disease (cgvhd), (table 1) . analyses using quartile cell dose thresholds showed a trend towards decreased os with a cell dose of < 5.67x10^6/kg, however this was not statistically significant ( figure 1 ). higher cd34+ cell doses were associated with shorter platelet engraftment time (p=0.005, figure 2 ). there was no significant difference in neutrophil engraftment, nrm, rfs, agvhd and cgvhd (table 2) . conclusions: cd34+ cell dose greater than 6 x10 6 /kg significantly increases overall survival in the setting of ric and dual t-lymphocyte suppression with atg and ptcy in mrd and mud allohsct. further studies in a larger number of patients and longer follow up are recommended to validate these findings. disclosure methods: fifty two adult patients were included. median cd34+ cells requested for infusion were 6x10^6/kg. all patients received the same ric regimen including fludarabine (30mg/m2/day day -5 to -2), busulfan (3.2kg/m2/day day -3 and -2), and total body irradiation (200 cgy) (day -1) combined with rabbit-atg (4.5 mg/kg: day -3 to -1), ptcy (50mg/kg/day: day +3,+4), and cyclosporine. unmanipulated peripheral blood stem cells were infused. last followup was november 2018. median follow-up was 13 months (range 5-26). median cell dose count infused was 9.83 cd34+/kg. we arbitrarily divided the cohort in two groups with cd34+ dose of >8x10^6 cd34/kg as cut-off point. results: findings are summarized in figure1. the infusion of more than 8x10^6 cd34/kg dose had a significant worse impact on overall survival (os) (p=0.022), relapse-free survival (rfs) (p=0.042) and cumulative incidence of acute gvhd (p=0.000). chronic gvhd could not be compared between the two cohorts due to the different median follow-up. conclusions: the infusion of a cd34+ cell dose count higher than 8x10^6 cells/kg had a significant adverse impact in overall survival and grade ii-iv acute gvhd in the setting of ric and dual t-lymphocyte suppression with atg and ptcy for haplohsct. disclosure: nothing to declare p335 long-term thymic activity and immune-reconstitution after haplo-identical allografting with post-transplant cyclophosphamide background: the use of post-transplant cyclophosphamide (ptcy) has expanded the application of t repleted haploidentical stem cell transplantation (haplo-hsct). in this setting, to investigate thymus role in longterm clinical outcomes, evaluation of immune reconstitution kinetics was performed. methods: twenty-nine patients (median age 53) were enrolled. blood samples were collected before conditioning and at 1, 3, 6, 12, 18, 24 months after haplo-hsct. analyses of cd4 and cd8 t-cell subsets by flow-cytometry were correlated by generalized linear models with real-time pcr (rt-pcr) quantification of signal joint t-cell receptor excision dna circles (sjtrecs), specific marker of naive t-cells thymopoiesis. a) naive; b) central; c) memory; and d) revertant cd4 and cd8 t-cells were defined as follows: a) cd45ra+cd62l+; b) cd45ro +cd62l+; c) cd45ro+cd27-; and d) cd45ra+/45ro +, respectively. sjtrecs rt.pcr was performed on genomic dna (100 ng) extracted from sorted cd4 and cd8 t-cells. results: a gradual increase in absolute numbers of all cd4 and cd8 t cell subsets and of sjtrecs copies from the first month up to 2 years post-transplant was observed ( figure 1) . however, at 2 years, cd4 and cd8 t-cell levels and sjtrecs levels were lower than those observed in healthy donors. sjtrecs kinetics was associated with the increase in cd4 naive t-cells (overall, p < 0.002). this correlation suggests that most of cd4 naive t-cells derives from thymic re-education of donor precursor stem cells, whereas cd8 naive t-cells undergo peripheral expansion after thymic production. furthermore, an increase in cd4 revertant memory t-cells was also significantly correlated with sjtrecs kinetic (p 0,041). central and effector memory t-cells showed a faster thymic-independent expansion in both cd4 and cd8 tcells. interestingly, sjtrecs levels and thymic dependent immune-reconstitution were higher in a cohort of 63 patients undergoing hsct from hla identical donors (manuscript in preparation). clinical outcomes and thymic function were correlated starting at 6 months after hsct. lower thymic output was significantly associated by multivariate analysis with low pre-transplant trecs values (p 0,002 and p < 0,001 in cd4 and cd8, respectively), moderate-severe chronic graft-versus-host disease (gvhd; p < 0,001 in cd8), and age (≥50 years, p 0,006 in cd8). conclusions: the thymus, despite age-dependent involution, substantially contributes to t-cell reconstitution after haplo-hsct. chronic gvhd and older age were significantly correlated with reduced thymic function. overall, lower production of sjtrecs after haplo-hsct as compared after hla identical sibling hsct may partly be due to a higher degree of "mismatching" of mhc molecules during thymic re-education. [[p335 image] 1. figure 1 ] background: the use of allogenic hematopoietic stem cell transplantation (hsct) in the treatment of adolescents and young adults (aya) with philadelphia negative all is decreasing with the adoption of pediatric inspired protocols to treat this age group and the incorporation of minimal residual disease assessment in the routine care of all patients. previously, its use was defined mainly by disease risk features at presentation. methods: a study on 209 aya (age 14-39 years), who underwent allogenic hsct at our institute for philadelphia negative all, between february 2005 and december 2015. all the studied patients received calgb based adult chemotherapy protocol for induction, and underwent a matched related donor (mrd) transplant with cy/tbi conditioning and mtx/csa as gvhd prophylaxis. the patients were eligible for allogeneic hsct, if they have a mrd plus one or more of the following risk factors: (1) age ˃ 30 years, (2) high presenting wbc count (>30 for b-all, >100 for t-all), (3) high risk immuno-phenotyping (pro-b, pro-t, early t, and mature t), (4) bulky splenomegaly or bulky lymphadenopathy, (5) high risk cytogenetics (4;11, 1;19, low hypodiploidy/near triploidy, complex), (6) cns involvement, (7) relapsed or refractory disease at d28 of induction. in this study, we investigated the impact of those different risk factors on the long term outcome of allogeneic hsct. results: the median os of our studied patients was not reached at 12.5 years, with a median dfs of 8.3 years (figure 1 ). in a univariate analysis, relapsed or refractory disease prior to transplant was the only independent risk factor for os and dfs (p-value= 0.36, and 0.01 respectively) (figure 2 ). in addition, patients who had 3 or more risk factors (41, 19.6%) prior to transplant had a significantly lower long term outcome compared to patients, who had one (100, 47.9%) or two risk factors (68, 32.5%) with a median os of 23 months, and a median dfs of only 11 months (p-value=0.32, and 0.009 respectively) ( figure 3) . conclusions: our results show that the long term outcomes of hsct in aya with philadelphia negative all treated on an adult type chemotherapy regimen, were significantly better in patients who showed a good response to initial therapy and a limited poor prognostic factors at presentation, with worsening of dfs as the number of poor prognostic features increase. we can conclude that, using this risk score can be helpful in predicting the outcome of allogenic hsct in aya with philadelphia negative all treated with adult type chemotherapy protocol. disclosure: no conflict of interest a prospective single center survey on donor-specific anti-hla antibodies and desensitization strategy in patients undergoing an allogeneic stem cell transplant background: in the setting of hematopoietic stem cell transplantation (hsct), considering the risk of poor engraftment or graft failure (gf), the detection of antibodies (ab) directed against donor specific hla loci (dsa) represents a contraindication to proceed with the same donor, suggesting the search of other donors. in many cases, there is not sufficient time to search for alternative donors and it is necessary to plan an immunosuppressive strategy to decrease the dsa level, thus reducing the risk of gf. to date, there is no consensus on desensitization standards to manage dsas in hsct. the aim of this study was to determine the incidence of anti-hla ab and dsas in hematologic patients candidate to an allogeneic hsct, and the efficacy of our desensitization protocol. here, we present an update of the results obtained with our strategy. methods: between august 2014 and september 2018, we prospectively screened for dsa 140 consecutive patients candidates to an allogeneic hsct. anti-hla ab research was carried out using the luminex bead assay (lifecode screen and lsa i/ii-immucor). the results were expressed as mean fluorescence intensity (mfi); mfi >1000 was considered positive. in case of a mismatched related donor, a flow cytometric crossmatch test (fcxm) was performed. if the patient had dsas and only one available donor, a desensitization strategy was employed, scheduled with rituximab on day -15, single-volume plasmapheresis procedures (pp), usually on day -9 and -8, intravenous immunoglobulins on day -7, infusion of hla selected platelets for dsa absorption in case of persistent antibodies directed against class i hla antigens. the aim of this schedule was to avoid interferences with chemotherapy and anti-t-cell globulins, infused during condition regimen results: since august 2014, 140 patients have been prospectively screened. thirty-three patients (23.6%) showed anti-hla ab and 9 of them (6.4%) had dsas: 6 were treated with the desensitization strategy, applied according to the mfi score and the fcxm result, and all of them obtained an engraftment; in 2 cases, an alternative donor was selected and in 1 case the research for an alternative donor is still underway. dsa detection was performed every 7 days after hsct for the first month and 60, 180 and 365 days following hsct. neither a dsa rebound nor other complications were observed during the follow-up. conclusions: our prospective analysis underlines the high frequency of anti-hla antibodies detection in hematologic patients, confirming the necessity to routinely evaluate the presence of dsas before an allogeneic mismatched hsct. our desensitization schedules based on the combination of pp, rituximab, ivig and platelet absorption proved successful in reducing dsas. we confirm the necessity of a prospective multicenter collaboration to better define the role of dsas against each hla locus and the critical mfi cut-off level associated with a higher risk of gf. transplant and transfusion specialists should joint to define a consensus for a standard desensitization strategy. disclosure the most frequent technique used for counterbalance partial incompatible hsct is cd34+ selection that is associated with sustained engraftment and effective reduction of t cells that minimizes gvhd. on the other hand, this approach could delay immune reconstitution and increase risk of viral and fungal infection. in mud setting the use of pbsc is the procedure that most centers have recently adopted. this implies the infusion of a relevant higher number of t cells 5 to 10 times more as compared with bone marrow (bm). since in our centre most part of our patients are primary immunodeficiencies, we applied a procedure to minimize the risk of severe gvhd infusing a controlled number of cd3 positive cells. methods: we report data about 91 paediatric patients who received 92 mud hsct (1 patient received 2 hsct) between 2001 and 2018 in the bmt unit of the children's hospital of brescia. patients received conditioning, according to the european group for bone marrow transplantation (ebmt) and the european society for immunodeficiencies (esid) guidelines. cd34+ selection has been realized by a milteny column with an ideal addback of cd3 positive cells of 30x10 6 /kg. stem cell source was bm in 62 cases and pbsc in 30 cases. results: median patients age at transplant was 2 years (range 2.6 months-17 years). the mean number of infused cells were: 12x10 6 /kg cd34+ and 36x10 6 /kg cd3+ in bm product, while 20x10 6 /kg cd34+ and 39x10 6 /kg cd3 + in pbsc. mean time for engraftment was day 15 post-hsct. as concerns acute gvhd overall incidence 64.1 % (59/92) of the children presented this complication, but only 11% (7/ 59) presented gvhd grade iii and none gvhd grade iv, while chronic gvhd presented in 7.6% (6 limited, 1 extensive/92). while acute gvhd incidence and severity weren't significantly different between bm recipients and pbsc recipients, the cases of chronic gvhd were prevalently in the latters. no major infections presented in the post-transplant period and immunological reconstitution both cellular and humoral was completed by 12 months. overall survival at 10 years is 75% (23/92). the results obtained show how it is possible control severity of gvhd if an addback of a controlled number of cd3+ lymphocytes. acute gvhd wasn't severe and only few children presented with limited chronic gvhd. the method allows to graft primary immunodeficiencies patients even with pbsc without infusing too many t cells. in fact, especially in very young children, the number could be excessive and risky. nevertheless in case of an oncohaematological patient, gvl effect is preserved. disclosure background: dc is a rare genetic disorder that results from a defective telomere length maintenance and is characterized by mucocutaneous features, bone marrow failure (bmf) and a high predisposition to cancer and pulmonary fibrosis. bmf remains the major cause of mortality and the hsct is the only definitive treatment to restore hematopoiesis but is limited by a high incidence of treatmentrelated mortality. methods: a retrospective analysis of 28 patients (pts) with dc who underwent hsct at the bone marrow transplantation unit in the clinical hospital of federal university of paraná, brazil, between july-1993 and november-2017. results: 15 boys and 13 girls, with a median age of 14y (3 -30y) received a hsct from a mds (n=7), mud (n=17) or mmrd (haploidentical, n=4). 27pts received bone marrow (bm) and 1pt received a cord blood unit (cbu). the median of tnc infused was 4,76x10 8 /kg (range 2,26-10,12x10 8 /kg) and in the cbu was 6,5x10 7 / kg. two pts received a myeloablative preparatory regimen with busulfan (bu) 12mg/kg + cyclophosphamide (cy) 120mg/kg or fludarabine (flu) 150mg/m 2 + antithymocyte globulin (atg). the remaining pts received a ric regimen with cy200mg/kg (n=5), flu150mg/m 2 + cy60mg/kg + atg5mg/kg (n=17), and flu150mg/m 2 + cy30 + tbi200rads (n=4, haplo). graft versus host disease (gvhd) prophylaxis consisted of cyclosporin (csa) and methotrexate or steroids (cbu) and post-transplant cy + csa + mycophenolate mofetil in the haploidentical transplants. 26 of 27 evaluable pts engrafted with a median time to neutrophil recovery of 20 days (range:13-36 days). one patient experienced primary graft failure (haplo) while second graft failure occurred in other 3pts. all these 4pts went a second hsct and 3 survived. acute gvhd grade ii-iv occurred in 6 of 26 pts at risk. moderate to severe chronic gvhd occurred in 6 pts with 5 cases occurring in pts who had previously presented acute gvhd. overall survival (os) was 53,6% at a median follow-up of 6y. the 5y os was slightly better in msd transplants compared to the others (54,5% x 52,9% p=0,053). causes of early death include adenovirus sepsis (n=1), toxicity to preparatory regimen and sepsis (n=2), primary graft failure (n=1). 13pts remain alive between 1-16y after hsct with a median fu of 8y. among them only 1pt has developed organ involvement by the underlying disease: hepatopulmonary syndrome (hps). 7pts died due to pulmonary fibrosis (n=1), liver fibrosis(n=1), gi bleeding(n=1), hps (n=2); cgvhd and sepsis(n=1), infection (n=1), and 2pts were lost to fu. conclusions: early mortality from bmf can be reduced by hsct, but late outcomes remain a consequence of the underlying disease. long term fu is essential in order to detect late complications related to the hsct procedure or the underlying disease. disclosure: nothing to declare single intra-bone cord-blood transplantation with a treosulfan-based regimen, atg-free and sirolimusbased gvhd prophylaxis: fast hematopoietic engraftment and immune-reconstitution in 20 patients background: cord blood transplants (cbt) require less stringent hla-matching, compared to peripheral blood stem cell or bone marrow. however, cbt has been associated with delayed engraftment and immune reconstitution, especially if in vivo t-cell depletion, such as antithymoglobulin (atg), is used. methods: from 2010 to 2018, 20 patients with high-risk diseases received intra-bone infusion of unwashed single cb unit with an atg-free gvhd prophylaxis; 7 were in active disease at cbt and 8 had received prior allogeneic stem cell transplantation. median age was 44 y [range (r) . conditioning regimen was myeloablative, with treosulfan and fludarabine in all, intensified with melphalan in 15 or with 4gy tbi in 4. hla matches was 4/6, 5/6, 6/6 in 12, 5 and 3 cases, respectively. gvhd prophylaxis included sirolimus and mycophenolic acid (mmf). results: after thawing, median cd45+ cells was 1.39 x 10 7 /kg [r 0.69-5.9], median cd34+ cells 0.08 x 10 6 /kg [r 0.04-0.23], and median cd3+ cells 3.05 x 10 6 /kg [r 1.29-5.9 ]. of the 16 evaluable patients all engrafted with a sustained full donor chimerism at day 100. median time to neutrophils (16/16, anc> 500/μl for 3 consecutive days) and platelet engraftment (14/ immune-reconstitution of cbt patients (tables 1a-b ) was compared with two cohorts of patients transplanted at our center from any adult donor with (81) or without (126 patients, including post-transplant cyclophosphamide cohort) atg in association with sirolimus and mmf. profiles of immune-reconstitution at day 90 -180 -365 showed a better cd4+ recovery at any time-point in both cbt and no-atg versus atg cohort, with no statistic significant difference in the first 2 cohorts. moreover, cd4 +/cd8+ ratio at any time point was better in the cbt cohort vs the no-atg cohort. b cell recovery was faster in the cbt cohort; immunoglobulin recovery was superimposable across different platforms. focusing on late events (>180 days from cbt), 3/12 pts experienced ebv reactivation, median time 586 days [362-655] treated with rituximab, and 1 experienced late hhv6 and cmv reactivation, both solved at last visit. sirolimus was withdrawn after a median of 188 days [r 64-394]. only 1 patient developed severe chronic gvhd, solved at last visit. overall, after a median follow-up of 330 days [r 9-1311], 11 pts are alive and well. conclusions: our data confirm that intra-bone cbt without in-vivo t-cell depletion is associated with fast hematopoietic engraftment and immune-reconstitution, with very low rate of chronic gvhd and late infective events. background: a promising improvement of hematopoietic stem cell transplantation (hsct) may lie in the transplantation of high numbers of pluripotent stem cells to minimize the time span between transplantation and immunological reconstitution. hence, an ex vivo platform is needed that supports hsc proliferation before application and, at the same time, the maintenance of pluripotency by diminishing hsc differentiation into lineage-specific progenitor cells. methods: to artificially model the natural hsc niche in vitro, we used 3d bone marrow (bm)-like scaffolds made of polydimethylsiloxane (pdms). these structures are based on a human long bone cross section as a representative of the bm. human cryoconserved hscs were cultured in distinct cultivation systems for 14 days under different conditions. cell counting and facs analyses at day 14 were conducted. for characterization of the cultivated hscs, we used antibodies against cd34 alone or in combination with antibodies against cd38, cd90, cd45ra and cd49f. results: for optimization of culture conditions for human hscs, a commercially available medium was supplemented with a panel of cytokines and valproic acid. we found a significant increase in the number of cd34+ hscs by simultaneously increasing their vitality using the 3d system compared with conventional 2d culturing. a further improvement was achieved by introducing a silicon oxidecovering of the 3d pdms structures, suggesting that hydrophilic surface properties offer superior attachment for semi-adherent hscs. for a more precise characterization of the cultivated hscs, we introduced a panel of facs markers reflecting the immaturity of the amplified hsc. surprisingly, with increasing immaturity of the cultivated hsc, non-covered 3d pdms revealed to be best suited for amplification: cell number of vital immature hscs was increased after cultivation on non-covered 3d pdms compared with silicon oxide-covered 3d pdms and the 2d system. conclusions: by establishing a 3d scaffold according to the human bm, we found a platform mimicking the natural niche of human hsc which is suitable to amplify human hscs in vitro and support their vitality, pluripotency and ability for self-renewal. [[p341 image] 1. with the introduction of sion covering of 3d pdms structures the maintenance of cd34+ hscs could be further improved. despite the better conservation of cd34 + hscs by using silicon oxide-covered 3d pdms, we found that immature human hscs obviously prefer more hydrophobic conditions found on non-covered 3d pdms. disclosure: nothing to declare. abstract already published. improvements in neutrophil engraftment following changes in freezing method background: in the setting of autologous haematopoietic progenitor cell (hpc) transplants for haematological disorders, peripheral blood stem cells are routinely collected via apheresis and cryopreserved. leicester royal infirmary had been using a controlled rate freezer (crf) to cryopreserve cellular therapy products up until 2017. in 2017 a literature review of cryopreservation techniques was undertaken, since the crf required replacement. this review found consistent evidence that cryopreservation using minus 80 o c is comparable to crf, and engraftment times should not be negatively affected by changing to a more simplified method of freezing. there would also be cost saving benefits from switching from a crf to minus 80 o c freezers. methods: as a result two minus 80 o c freezers were purchased following the acceptance of a preparation process dosier (ppd) which was prepared for the human tissue authority. validation was carried out, and from january 2018 stem cell laboratory at the lri switched from the crf method to minus 80 o c for cryopreservation of cellular therapy products. briefly, cells are frozen using 10% dmso (wak-chemie) in 20g/l human albumin solution (grifols) in cyrobags (origen biomedical, inc). the cells are transferred on cold packs to the minus 80 o c freezer. cells are packaged in between stainless steel heattransfer plates. the plates are placed within a bubble wrap bag, and are placed in a rack within the minus 80 o c freezer, which allows air to circulate freely around each bag. in one plate a el-usb-tc thermocouple data logger (thermosense) is inserted between the bag and stainless steel plate, to record the freezing profile. this data is downloaded after each run. after an overnight freeze at minus 80 o c, the cells are subsequently removed from the bubble wrap and plates, and are transferred to minus 150 o c freezers the following morning. results: a total of 40 patients have had autologous stem cells frozen using this method so far this year. in addition to engraftment data for neutrophils, post-freeze trypan blue viabilities were also compared to the previous year. during 2017 a total of 51 patients, who had all their cells cryopreserved, underwent 72 collections. the post freeze median viability was 90% (75-98%). a total of median neutrophil engraftment was 12.0 days, with a median cd34 dose infused of 4.0 x 10 6 /kg. during 2018 so far, 40 patients have undergone 57 collections. median viability is 95.5% (82-98%). subsequent median neutrophil engraftment is 11 days, with a median cd34 dose infused of 4.1 x 10 6 /kg. conclusions: ongoing savings of approximately £6400 per annum have been made by changing our procedure. the benefit of changing to a simplified method of freezing has also resulted in a reduction in staff working overtime. more importantly, this simplified cryopreservation method has resulted in an improvement in neutrophil engraftment times since changing the cryopreservation technique from the previous method using crf to mechanical freezing using a minus 80 o c freezer. disclosure: nothing to declare low doses of granulocytes in the apheresis product predict a better outcome of autologous hematopoietic stem cell transplantation in multiple myeloma patients background: high-dose chemotherapy with autologous stem-cell transplantation (asct) remains the standard consolidation therapy for multiple myeloma (mm). peripheral blood stem cell collection may be contaminated with large quantities of granulocytes and its consequences on the outcome of asct are still unclear. on the other hand, the effect of performing apheresis with high levels of monoclonal component (mc) on outcome is unknown. the objective of this study was to analyze the effect of total nucleated cells (tnc) and granulocytes count (considered as contaminating components of apheresis products) as well as the influence of mc in the apheresis product on outcome of asct in mm. methods: eighty-two patients diagnosed with mm were mobilized with filgrastim 10 μg/kg/day (plus plerixafor if insufficient mobilization of cd34+ cells on day 4). apheresis collection was performed with cmnc program by spectra optia cell separator. cd34+ count was carried out according to ishage protocol (target: ≥2x10e6 cd34 +/kg). subsequent cryopreservation were performed according to the local protocol. results: the medians (range) of collected cd34+, tnc and granulocytes were 4.69x10 6 /kg (1.69-15.54), 9.35x10 8 / kg (2.16-19.66) and 13.7x10 9 /kg (1.4-90.6), respectively. the medians (range) of infused cd34+, tnc and granulocytes were 2.99x10 6 /kg (0.89-10.93), 6.46x10 8 /kg (1.93-18.60) and 8.30x10 9 /kg (1.1-86.0), respectively. a successful collection after first line therapy was performed in 94% of patients. treatment for mm was continued after carrying out apheresis in 40% of patients, as per protocol. a significant reduction of mc was observed prior to asct, indicating a further improvement in responses after apheresis (p=0.005). an optimal response (cr or vgpr) at the time of apheresis was achieved in 45% of patients and a suboptimal response (partial or minimal response) was observed in the remaining 55%. undergoing apheresis in optimal response did not result in lower number of tnc or granulocytes in the harvest. the subtype of mc did not influence on the number of tnc and granulocytes in the product of apheresis. no differences in collected tnc and granulocytes were observed when plerixafor was used as mobilizing agent. the type of chemotherapy given prior the apheresis did not have influence on the characteristics of the apheresis product. a significant improvement in overall survival (os) probability (95%ci) was observed when low tnc (< 6.46x10 8 figure 1a ). lower incidence of relapse (p=0.044) ( figure 1b ) and non-relapse mortality (p=0.040) was observed in patients who received low granulocyte count in the graft. no significant correlation was observed between the time of engraftment and the number of tnc or granulocytes infused. similarly, no increase in the frequency of the engraftment syndrome was observed when higher number of tnc or granulocytes were infused. conclusions: in our series, low doses of granulocytes in the product of apheresis predicted a better outcome of asct in mm patients. the amount of mc at the time of apheresis did not have influence in the characteristics of the harvest. efforts for avoiding contamination in grafts are important for its impact on outcome of asct in mm patients. disclosure: dkms foundation, pi14/01971 (instituto carlos iii) and sgr288 (grc), generalitat de catalunya. background: our initial experience (from 2012 to 2017 years) with hematopoietic stem cell transplantation (hsct) from mud with tcrαβ+/cd19+ graft depletion in patients with cgd showed high rate of secondary graft dysfunction, the incident of graft rejection was 29%. to improve the outcome, we hypothesized that the use of plerixafor and g-csf as additional agents in conditioning regimen would offers advantages and better outcomes. this trial was registered at www.clinicaltrials.gov (nct03547830) methods: between april 2018 and september 2018, 5 patients with cgd underwent allogeneic hscts from mached unrelated donors with tcrαβ + / cd19 + graft. the conditioning regimen included -treosulfan 42 g / m2, fludarabine 150 mg / m2, thiotepa 10 mg / kg, g-csf 50 mcg / kg and plerixafor 720 mcg / kg. all patients received rabbit atg -timoglobulin® ("genzyme europe b.v.", the netherlands) 5 mg/kg for serotherapy. in all cases, tcrab +/cd19+ graft depletion was used with the immunemagnetic method (miltenyi biotec, bergisch gladbach, germany) according to the manufacturer's instructions. stem cell source was g-csf mobilized peripheral blood stem cells in all cases. posttransplant gvhd prophylaxis was not performed. minimal follow up was 90 days after hsct. results: neutrophil and platelet engraftment occurred at 12-17 days post-hsct in all patients. 3 patients had full donor chimerism in whole blood and in 2 cases we observed predominantly donor mixed chimerism at last follow up. all patients had full donor chimerism in cd15+ compartment and mixed chimerism in cd3+ lineage, but it stable without any sign of graft dysfunction. acute gvhd is verified in 1 of 5 cases and was limited to skin grade 2. all patients are alive for periods from 3 to 8 months post-hsct with good graft function without severe clinical problems. conclusions: we presented first experience of g-csf and plerixafor addition to conditioning regimen before hsct with tcrαβ+/cd19+ graft depletion in cgd patients . we suppose, the preliminary results are encouraging, as the frequency of primary and secondary graft dysfunction in patients from this group is not observed today, there are no significant toxic complications, as well as clinically severe manifestations of gvhd. currently, the recruitment of patients is continuing, and estimation of the rates of immune reconstitution and a more detail analyses will be evaluated later. background: introduction -it is believed that aboincompatibility is of minor importance in allogeneic stem cell transplantation (allo-sct) and that the clinical outcome is equivalent to abo-compatible sct. therefore, we performed a single center retrospective study to characterize the impact of abo-incompatibility on the outcome of haploidentical stem cell transplantation (haplo-sct). methods: this analysis included 27 consecutive patients who underwent haplo-sct for various hematological malignancies at our center between october 2010 and may 2018. we used our institutional database to evaluate details and characteristics of patients and transplant outcomes. results: demographic features of the patients and donors have been summarized in table 1 . all of the patients had advanced hematological disease with a high risk of relapse (22 patients with acute leukemia). out of 24 patients, early transplant-related mortality was seen in this cohort of 5 patients. the remaining 19 patients were followed in 1 and 42 months. donor type abo group switch was observed in a median of 45 days (30-60 days) after transplant. we were not able to show any statistical difference in terms of blood group switch between minor and major abo incompatible transplant. the median red blood cell (rbc) transfusions in the first 30 days for the abo compatible and incompatible transplants were median 4 units (range, 0-11) and median 4 units (range, 3-12) (p=0.6). no statistical difference was also encountered for the rbc transfusion need for stem cell source, peripheral blood vs bone marrow. a total of 15 patients were followed up for reticulocyte engraftment. the median time for reticulocyte engraftment was 19 days (range, 15-60) for all patients. reticulocyte engraftment was tended to be faster in minor abo-mismatched group (p=0.05) than major or abo-compatible ones. nineteen patients achieved independence from rbc support after a median time of 44 days (range, 11-61 days) in abocompatible patients, 25 days (range, 7-52 days) in minor abo-incompatibilityand 34 days (range, 20-57 days) in major abo-incompatibilitygroup, respectively (p>0.5). the engraftman kinetics due to major and minor aboincompatibilitytransplants were presented in table2. pure red cell aplasia was not developed in our cohort. conclusions: the present single center study provides new evidence for the importance of the abo system for erythrocyte recovery in haploidentic stem cell transplantation. it's important to note that, randomize prospective and larger studies are warranted. disclosure: nothing to declare low dose of anti-t lymphocyte globulin protects against severe forms of graft versus host disease in patients undergoing allogenic stem cell transplantation background: graft versus host disease (gvhd) is the most important complication after allogeneic stem cell transplantation (allosct). optimal dose of different anti-tlymphocyte globulin (atg) formulations in this setting has not been established yet. the aim of this study was to analyze the impact of a low dose of atg-fresenius (atg-f) in allosct outcomes. methods: we analyzed 57 adult patients who received an allosct for hematologic malignancies from october 2012 to march 2018. the gvhd prophylaxis included a total dose of 21 mg/kg (7 mg/kg on day -3, -2 and -1) of atg-f for patients who received a graft from peripheral blood, an unrelated donor; with a mismatch, and/or were older than 55 years; associated to a calcineurin inhibitor and mycophenolate mofetil/short course-methotrexate. statistical analysis was performed using spss v.22 and r software. results: median age was 57 years (18-70) and 66.7% of patients were males. seventy-four percent of patients underwent myeloablative conditioning. the stem cell source was peripheral blood in 51 patients (89.5%), 80% were from unrelated donors (28% mismatched). seventeen (29.8%) patients had high risk cmv status (d-/r+) (see image 1b). engraftment was observed in 54 patients (94.7%). primary graft failure occurred in 3 patients (2 myelofibrosis, 1 aml). twenty (39.2%) out of 51 evaluable patients developed grade 2-4 acute gvhd. the cumulative incidence of severe agvhd and moderatesevere chronic gvhd were 8.8% (95% ci, 3.2-17.9%) and 35.2% (95% ci 22.7-47.9%), only 5 (8.7%) patients developed severe cgvhd. twenty-nine patients (56.8%) discontinued immunosuppression before the first year of transplant. the median duration of immunosuppression for patients with moderate-severe cgvhd was 488 days (207-2046). at 2 years non-relapse mortality (nrm) was 21.9% (95% ci, 12.0-33.7%). thirty-nine (68%) patients developed relevant infectious complications. two (3%) patients died within the first 30 days due to gram negative blood stream infection. eleven (19.2%) had at least two episodes of cytomegalovirus (cmv) reactivation between day 30 and 100. three (5%) patients developed cmv gastrointestinal disease, 2 (3%) had probable invasive fungal infection and 1 (1.7%), post-transplant lymphoproliferative disorder associated to epstein barr virus. with a median follow up of 28 months for alive patients , the gvhd and relapse free survival (grfs) at one year, overall survival (os) and progression free survival (pfs) at two years were 47.6% (95% ci, 42.8-52.2%), 59% (95% ci, 54.5-63.2%) and 52% (95% ci, 46.9-56.5%), respectively. the relapse incidence at two years was 26.3% (95% ci 14.7-39.4%). complete remission at transplant was associated with better long term survival (80% at 2 years, p < 0.01). hla disparity did not affect os (see image 1a). conclusions: the use of low doses of atg-f is protective against severe forms of acute and chronic gvhd in a cohort with high prevalence of unrelated donors and a high median age. this strategy showed good results in grfs, os and pfs in a population at high risk for developing gvhd or relapse. disclosure: nothing to declare performance parameters of a ngs-product for chimerism monitoring -applicable in patients after hematopoietic stem cell transplantation methods: for this purpose, samples from patients with mixed chimerism (mc) with increasing amounts of recipient dna were analyzed and compared using realtime pcr of insertions/deletions (indels), fragment analysis of short-tandem repeats (str) and ngs of indels. results: whereas real-time pcr displayed excellent sensitivity down to 0,01 % mc, but poor precision above 20 %, fragment analysis exhibited good precision with limited sensitivity (> 2,5%). in contrast, ngs chimerism demonstrated good sensitivity, with a limit of detection (lod) of 0,1 % mc, and precision throughout the whole spectrum of patient/donor mixed chimerism. the ngs chimerism product (devyser chimerism) exhibited at least three (average eight) and at least two (average 5) informative genetic markers (indels), suitable for monitoring mixed chimerism of patients with their corresponding matched unrelated (60) or related (56) donor samples. in order to establish the performance of the separate techniques for determination of mixed chimerism on retrospective patient samples, a cohort of 27 patient monitoring samples (3-7 weeks post-hsct) with low (< 5%), intermediate or high mixed chimerism (> 20 %) were included and analyzed. dna from all monitoring samples was extracted from sorted cell fractions. the results show that although all evaluated techniques are suitable for monitoring patient/donor chimerism after allogeneic hematopoietic stem cell transplantation (hsct), only the ngs chimerism product exhibits high sensitivity (lod 0,1 %) and a broad dynamic range (detection range 0,1-100%) with good precision and accuracy throughout the whole spectrum of mixed chimerism (% patient/donor). in addition, the ngs chimerism product employ 24 non-population dependent highly informative genetic markers providing stable resolution power and thus suitable for monitoring mixed chimerism. disclosure: dan hauzenberger is medical adviser at devyser ab and shareholder in devyser holding gender distribution: male -59% (n=58), female -41% (n=41). age median -5,8 years old (8 months -17). stem cell source: bone marrow -80% (n=81), peripheral blood stem cells -19% (n=19). 82 patients (83%) received 10/10 matched unrelated donors hematopoetic stem cells transplantation and 17 patients (17%) -9/10 matched unrelated donors hematopoetic stem cells transplantation. differences in the antigen blood system: single group 41% (n=40), minor 32% (n=31), major 20% (n=18), mixed 11% (n=9). age of donor: 18-25 years old -25% (n=16), 26-35 -42% (n=27), 36-45 -23% (n=15), 46 and more11% (n=7). gender differences in donor/recipient: male/female 25% (n=24), female/male 18% (n=18), one sex 57% (n=56). we also took into account the impact of gender difference and cytomegalovirus serostatus in the donor/recipient pair. results: in 10/10 group the estimated probability of overall 2-years survive was 76% and in the 9/10 group 2-years survive was 63%. the increase in donor age of 10 years reduces the 2-years survive by 9-11% (p=0,117), however, the 2-years survive from donors over 46 years old was 100%. we have found no difference between 2-years survive in transplants from donors that are compatible/ incompatible with the antigen blood system, cytomegalovirus serostatus, or the gender differences in donor/ recipient. in the study of donor-related factors, we found the negative impact of an human leucincompatibility (9/10) on the incidence of chronic gvhd -29% (p = 0.019). the combination of cytomegalovirus positive serostatus of the donor and the negative status of the recipient increases the risk of primary graft rejection up to 50%, in comparison with others (p = 0.001). conclusions: our study showed the role of genetic matching on the hla system between the patient and the unrelated donor, and the donors age value. 10/10 transplants have better outcome and lower incidence of severe a. gvhd and ch. gvhd. younger donor increases 2-years survive, but there is a significant increase in 2-years survive if the donor is over 46 years old. disclosure: nothing to declare allogeneic stem cell transplantation in chronic myelomonocytic leukemia. a single center experience nine patients (45%) relapsed with a median of 6,3 months (1-87) with different strategies at this point: in all cases we modulated immunosuppression, in 2 cases as the unique strategy, in 2 cases with donor lymphocyte infusion (dli), in 3 cases we employed hypometilating agents (hma) and in 2 cases with intensive chemotherapy, reaching cr only in two patients, one of them after dli and the other one after hma and consolidation with a second asct. eleven patients (55%) died being the relapse the main cause (64%). transplant related mortality (trm) at +100 were 5% and global trm were 15%. in the last follow-up, 9 patients (45%) are still alive, 8 (89%) in cr and 1 (11%) in relapse situation. with a median follow-up of 58 months (5-138), the event free survival (efs) were 19 months (5-32) and the overall survival (os) were 56 months (0-116). we observed advantage in terms of os in those patients that reach cr at +100 post asct and in those who develop chronic gvchd (p=0.065 and p=0.012 respectively) conclusions: asct is still the only curative option despite the high relapse rates. to reach cr at +100 post asct and the development of chronic gvhd seems that they confer advantage in terms of os. the importance of knowing the molecular profile of the entities that we consider for asct. disclosure this study documents a first experience of a cell processing lab seeking to integrate process automation technology to wash and volume reduce products which can account for the initial material source volume variability, product characteristics, and number of bags. methods: here we report the pre-clinical assessment of the lab's initial work with the lovo cell processing system for a 5 product experience over 2 days with 1 machine. this study used products intended for destruction. the workflow used parallel and sequential processing schedule. after water-bath thawing, bags were sampled, weighed to determine volume, and subsequently connected to lovo or pooled into a transfer pack and then connected to lovo. the bags were then diluted 1:1 at 50 ml/min with lovo at +4-8c using 6% hydroxyethylstarch 130/0.4 (voluven, fresenius kabi) and processed using a 3 cycle procedure. after processing the bags were weighed for volume, sampled, and stored in a 4-8c refrigerator in their lovo final product bags. samples were assessed from t=0 to t=24 hours. results: cd34+ viability and absolute counts were determined using flow cytometry. processing duration and solution volume consumed was determined by the lovo's sensors and confirmed by the operator. data is presented as a percentage relative to the post-thaw values. note, the values presented are not total process yields. the results focus on the lovo processing step. conclusions: the operators easily integrated into the software to drive the machine. the machine demonstrated it's flexibility with a wide-range of volumes, cell-inputs, and number of bags. the lovo produced products which meet our specifications in a quick and reliable manner. further work on this platform will be performed to validate and qualify this system for production use. properties. the aim of this study was to evaluate prospectively the efficacy of a fbm protocol for the prevention of om in patients undergoing a hsct. methods: all patients consecutively who underwent a hsct at our center from 201x onwards received five weekly fbm sessions with a bidiodic laser (lumix 2®, prodent, italy), which simultaneously emitted at 650nm and 910nm with a power of 89mw and energy of 4j per point. the procedure started the day before the beginning of the conditioning regimen up to the tenth day post-transplant. the laser was applied in a defocused mode on each of the mucosal surfaces (12 areas). at each session, the morphine dose, the om level (according to the who scale) and pain through a numerical rating scale (nrs) were recorded. results: 27 consecutive patients (19 male/8 female) submitted to a hsct were analyzed. the median age was 44 years (range 4-66). eighteen patients had acute leukemia, 3 myelodysplastic syndromes, 6 lymphoproliferative diseases. the median number of treatment lines before hsct was 2 (range 1-5). at transplant, 13 patients had advanced disease. the myeloablative conditioning regimen mac (thyotepa, busulphan, fludarabine) was employed in 17 patients; the same conditioning, with a reduced dose of busulphan (ric), was infused in 10 patients. seven patients (26%) had no evidence of om. the incidence of grade ii-iv om was 65% in the group of patients receiving mac and the median duration 12 days (range 3-28); grade 4 om was observed, for 1 day, in 1 patient. in the ric group the incidence of om was 50%, the median duration 11 days (range 7-16); no patient had evidence of grade iv om. in the whole population, the maximum nrs value was 4. morphine administration was required in 23 patients, due to the occurrence of non-oral complications. conclusions: in our experience, prophylaxis with fbm to prevent or reduce om was safe and effective, compared to results of previous experiences reported in the literature, which used no prevention against this complication that negatively affects the quality of life of transplanted patients. further studies on a large series of are necessary to confirm our results. disclosure: nothing to declare background: cytogenetic abnormalities are an essential part of prognostic systems in myeloid malignancies before hematopoietic stem cell transplantation (hsct), however, their role in posttransplantation prognosis is unknown. the aim of this study was to assess the prognostic impact of genetic risk stratification of aml and mds patients on posttransplantation course, which could be an additional tool in making decisions regarding preemptive therapy. methods: a retrospective analysis covering patients treated with allo-hsct between 2012 and 2018. cytogenetic studies included karyotyping (c-and gbanding) and fluorescence in situ hybridization (fish). the number of analyzed cells exceeded european cytogenetics association guidelines (for each fish at least 600 interphase nuclei were analyzed). cytogenetic risk group in aml was assessed based on the eln 2017 criteria. patients with mds were stratified into three groups; favorable (good and very good prognostic score), intermediate, and adverse (poor and very poor) prognostic score according to ipss-r 2012. interestingly, the poorest survival was in patients with monosomy of chromosome 7, which was present in 6 patients of whom 5 succumbed to refractory disease, while all patients who had deletion of long arm of chromosome 7 (del 7q)-are alive at the time of writing of this report after a median follow-up of 34 months (21-73). relapse was diagnosed in 31 patients (31%), including; 13 (42%) with adverse, 15 (48%) with intermediate and 3 (10%) with favorable cytogenetic risk. among 12 patients with a complex karyotype and/or cytogenetic evolution prior hct: 8 patients (67%)relapsed, including 6 (50%)-who died. follow-up cytogenetic studies in relapse after transplantation were performed for 24 patients; 4 of them (17%) had clonal evolutions of the original karyotype with additional abnormalities-(50% died) and 7 (29%) had new aberrations in cells without primary changes (all died). in 18 patients (18%) (8 unfavorable, 8 intermediate group) cytogenetic relapse was diagnosed by fish analysis and they were treated with azacitidine (+/-dli) achieving cr (n=9, 50%), stabilization-(n-=3, 16%), transient response (n=1), while 5 deceased). conclusions: cytogenetic studies in patients after transplantation may facilitate assessment of mortality. karyotype may undergo cytogenetic evolution after allo-hsct. patients with monosomy of chromosome 7 seem to have a particularly poor prognosis. transplanted patients are vulnerable to new cytogenetic alterations. disclosure: nothing to declare methods: the primary end-points were the rate of complete response (cr), defined as no emesis and no nausea without rescue medications, for both acute (cr-24) and delayed (cr 25-120) cinv and rate of post-transplant complications until discharge. we prospectively analyzed 61 patients undergoing autologous (85%) and allogeneic (15%) stem cell transplantation and receiving cinv prophylaxis with nepa and dexamethasone (schedules shown in fig.1 ). in our series, 30 patients (49%) were female. patients median age was 55 years (20-74). the most frequent diagnosis were myeloma (46%) and lymphoma (39%), while 15% of patients were diagnosed with aml or mds. myeloma patients received one day hd-ct with melphalan (75% mel200/25% mel140). lymphoma patients were conditioned with feam (87,5%) or tt_flu_edx (12,5%) hd-ct. busulfan-based mds-ct regimen was offered to aml/mds patients. results: the incidence of cr-24 and cr 25-120 observed was 82% (50/61) and 47,5% (29/61), respectively. more than grade 1 nausea and vomiting (according to ctae-4), was reported in 36% (22/61) and 5% (3/61) of patients, respectively. female sex was associated with an increased risk of acute (hr 19,6; p 0,037 95% ci 7.980-2.191) but not delayed (hr 1,58; p 0,06 95% ci 200-2.042) cinv. similar rate of cr24 and cr25-120 was observed in one-day hd-ct (82% and 50%) compared to mds-ct (89% and 42,5%) group (pns). median lenght of stay was 23 days (15-51). no case of cardiotoxicity and no exitus was observed. the incidence of febrile neutropenia was 61% (70% fuo; 22% sepsis; 18% pneumonia). only one patient experienced an agvhd on day +9. neutrophil (>1000/mcl) and platelet (>50000/mcl) recovery occurred in median on day 11 (9-26) and on day 15 (4-45) respectively. conclusions: nepa seems to be safe and effective in preventing acute and delayed cinv in patients receiving both one day hd-ct and mds-ct as conditioning regimen for hsct. more studies are needed to define the better 5-ht3ra and nk-1ra combination and the better schedule in transplant setting. disclosure: "nothing to declare background: vod and ta-tma represent two early endothelial complications occurring after allogeneic stem cell transplantation (sct) sharing many pre-transplant risk factors. the aim of our study is to evaluate the impact of donor graft composition, engraftment kinetics and infections on the development of these endothelial complications (ec). methods: we retrospectively reviewed 55 consecutive sct recipients at our institu-tion between january 2015 and june 2018. the median age was 48 years (range 17-65). acute leukemia was diagnosed in 33 patients (60%). complete remission was documented in 63% of patients at transplant. donor source was from hla mismatched donor in 53% and from unrelated donor in 56% of the patients. hbv positive patients were 13% of the sample. conditioning regimen was busulfan based in 63% of patients. ursodeoxycholic acid and unfractionated heparin were given to all patients as vod prophylaxis. cyclosporine was used as gvhd prophylaxis. lymphocytic subpopulation analysis (cd3+, cd4+, cd8+ and cd16+/cd56 +) and cd34+ cells count on the donor graft were performed using bd facs cantoll. all patients had routine monitoring for ebv and cmv pcr, hemolysis tests, creatinine and electrolyte panels, proteinuria, complete blood count, blood pressure and schistocytes by direct examination until day +100. the fisher's exact test was used to compare categorical variables, while continuous variables were analyzed with anova test. diagnosis of vod and ta-tma were carried out by using ebmt and cho criteria respectively. results: the incidence of very severe vod and tma was 13% (7/55) and 14,5% (8/55) respectively. cmv reactivations with viral load over 30.000 cv/ ml was 27% and 5% in patients with and without ec, respectively (p 0,041; hr 2,97 p0,0055 95%ci 1,38-6,4). the median day to neutrophils (ns) engraftment (500/ml and 1000/ml) was 14 vs 17 and 14,5 vs 18 in vod/tma group vs control group (p0,03 and 0,027, respectively). more rapid neutrophils engraftment (ns >500/ml and ns>1000/ml within 13 days) was related to a higher risk of ec with a hr of 2,67 (p 0,015; 95%ci 1,2-5,9) and 2,94 (p 0,006; 95% ci 1,4-6,3). patients with ec received a donor graft with a higher median numbers (x10e6/kg) of cd3+ and cd8+ (p>0,05) and a lower numbers (x10e6/kg) of nk cells (p>0,05). patients who received a cd8+ cells count >12x10e6/kg and nk cells count < 2,5x10e6/kg presented a relative risk of ec of 2,37 (p 0,036; 95% ci 1,06-5,3) and 2,35 (p 0,004; 95% ci 1,02-5,4), respectively. there were no differences with respect to the other analyzed variables between patients who developed vod/tma compared with those who did not. (pic_01) conclusions: cmv viremia, early neutrophils engraftment and donor nk and cd8+ cells infused are associated with the risk of vod and tma. very few studies evaluated the link between these variables and the risk of developing such two complications. it could be interesting to investigate these relationships on larger series. clinical trial registry: not applicable disclosure: nothing to declare p358 when the last hope turns out to be just as good as best: haplosct following tbf conditioning, pt cyclophosphamide and tacrolimus as gvhd prophylaxis background: hematopoietic stem cell transplantation is an effective therapy for a variety of severe hematological diseases. in last decades, haploidentical sct (haplosct) followed by ptcy as gvhd prophylaxis has been reported as a valid alternative for patients who lack an hla matched donor. we therefore analysed outcomes with this. methods: 38 patients without hla-matched donor received a haplosct between 04/13 and 08/18. thiotepa 5 mg/kg (2 days), fludarabine 50 mg/m2 (3 days) and oral busulfan 1 mg/kg/6 h (3 days,with pkd dose adjustments) was used as conditioning regiment; in patients >55 years busulfan administration was limited to two days. gvhd prophylaxis consisted of cyclophosphamide 50 mg/kg on day +3 and +4, and tacrolimus as a continuous iv infusion from day +5. all patients received pbsc as the stem cell source. outcomes analysed were overall survival (os), progression free survival (pfs); cumulative incidences (ci) of gvhd, relapse and non-relapsed mortality (nrm). results: median ages was 54 (range 25-71). 53% male and 47% female. diagnoses were: aml 20 (53%), mds 7 (18%), all 5 (13%), hl and nhl 5 (13%), and 1 mm (3%). 37% (n 14) were transplanted in early disease status, while most cases (63%) were in advanced status, including, second/third cr (29%, n 11), one (3%) in aplasia without progressive disease and 23% (n 9) had active/progression disease, 8% (n 3) had stable disease; four cases were second alosct. thus, 50% of patients had a high or very high rdri and 50% had intermediate. ebmt score was ≤ 4 was in 58% of patients (n 22) . the donor was as son/ daughter in 50%, 29% a sibling and 21% a patient's mother. median time to neutrophil (0.5x10e9/l) and platelet (>20x10e9/l) recoveries were +20 and +27 days, respectively (g-csf was not used). only one patient had a primary graft failure attributed to anti-hla donor specific antibodies. median follow up in survivor is 22 months (range 2-63). overall survival is 81±6.5% (at 12 months) and 61±9% (at 63 months). pfs is 67±8% and 52±9% respectively. the cumulative incidence of relapse was 20% and 35%, respectively, while nrm is 13% at 63 months. day +120, grade 2-4 acute gvhd were 13%, while mild/ moderate and severe chronic gvhd were 6% and 3% respectively. ebmt ≤ 4 and first alosct were the only variables to clearly impact 60-months os in univariate analysis. a combined covariate of ebmt ≤ 4-no prior alosct vs other patients showed a 60 month os 81±13% vs 38±12% (p 0.001), pfs 65±13% vs 35±11% (p 0.014) and nrm 0% vs 29% (p 0.008) but without impact on relapse 34% vs 35% (p 0.68). conclusions: haplosct with an age-adapted tbf conditioning regimen, pbsc and ptcy followed by tacrolimus, led to very encouraging results, mainly in patients with a low ebmt score and as a first alosct. although formerly considered as a last alosct strategy, we now agree that the time has come to compare this strategy with hla mud (and even elderly sibling donors) in ongoing prospective randomized multinational trials. disclosure: nothing to disclosure background: autologous hematopoietic stem cell transplantation (autosct) for acute myeloid leukaemia (aml) is increasing becoming a viable option for an increasing number of patients due to limited availability of matched sibling or unrelated donor for allogeneic hematopoietic stem cell transplantation (allosct). we examined the relevant long-term outcomes in our local patient cohort. methods: we retrospectively reviewed the data for all autosct done for aml in our centre over a 17-years period between 1 st january 2001 until 31 st dec 2017 from our electronic record. patients with acute promyelocytic leukaemia (apml) were excluded from this analysis. patients were further stratified based on the number of high risk features present; not achieving complete remission (cr) following induction chemotherapy, high presenting total white cell count (wbc > 100 x 10 6 /ml, adverse cytogenetics (example: complex cytogenetics) and adverse molecular mutations (example: flt3-itd & mll gene arrangement). outcome data including mortality (overall survival (os) and non-relapse mortality (nrm)) and morbidity (leukaemia free survival (lfs)) were recorded and analysed. results: a total of 64 patients were identified. median age at diagnosis is 34-years old. the cohort comprised of 34 males and 30 females. the overall median os and median lfs is 3.9 years and 2.2 years respectively. the nrm is 1.6% (1/64). there was no difference in the median os and median lfs for the patients achieving cr following induction chemotherapy and those not in cr following induction chemotherapy; 4.4 years versus 3.9 years (log-rank, p=0.9) and 3.4 years versus 2.1 years (log-rank, p=0.9) respectively. the median os were statistically significant for patients with zero versus one and two and more high risk features present; 10.2 years versus 3.7 years versus 2.2 years (log-rank, p=0.4) respectively. however, the median lfs were not statistically significant for these three patient cohorts; 3.6 years versus 1.9 years versus 1.4 years (log-rank, p=0.7) respectively. conclusions: in our patient cohort, autosct appeared to be a feasible option for patents with aml without matched sibling or unrelated donor available. disclosure: none to declare methods: between 2014-2018, 16 thalassemic patientsunderwent hisct. the median age of patients was5 (1-8)years with male preponderance (n=10, 62.5%). across the gender and abo mismatch transplants were done in 43.7% and 25% of patients. stem cell source was bone marrow in 5 (31%) while peripheral blood in 11 (69%) of patients. mean stem cell dose was 5.6 ± 2.9 x 10 6 cells / kg and mean volume of product was 188 ± 60.58 ml. preparative regimen included anti-thymocyte globulin, busulfan, fludarabine and cyclophosphamide.graft versus host disease (gvhd) prophylaxis comprised of posttransplant cyclophosphamide on day +3 & +4 followed by tacrolimus and mycophenolate mofetil. patients were observed for hematopoietic recovery (neutrophil and platelet engraftment) and transplant related mortality including acute and chronic gvhd for skin, gut, liverand lungs, primary and secondary graft failure and infectious complications. results: nine(56.25%) of sixteenpatients were engrafted with full donor chimerism. twelve (75%) patients belonged to pesaro class i and 4 (25%) toclass ii patients. median time to neutrophil and plateletengraftment were13(11-20) and16(12-36)days respectively. average number of packed red cell and platelet transfusions were 4.35±7.54 and 20.8 ±19.18 respectively.primary graft failure was observed in 3 (19%) and secondary graft failure was observed in 4 (25%) patients. two patients received a second dose of stem cells and they engrafted at 20and 32 days of infusion respectively.2 of 3 patients with primary graft failure died, one with sepsis (day +23) and the other because of intracranial bleeding (day +21).acute gvhdof gut and skin (grade ii-iii) was observed in 2 patients each, within first 100 days post-transplant. none of the patients had grade iv gvhd. cytomegalovirus reactivation occurred in 50% of patients, all of them received pre-emptive therapy with intravenous ganciclovir. none of them developed cmv disease. invasive fungal infection was not observed in any of the patient. culture proven bacterialinfection was documented in 62% of patients requiring intravenous antibiotics during first 100 days post-transplant.overall survival and relapse free survival were 81.25 % and 56.25% over a median follow-up of 500 (21-1757) days. conclusions: haploidentical transplant is a suitable modality for thalassemic patients lacking a full matched donor in pakistan. in view of our results, we suggest that thalassemia patients should be offered hisctas an option for cure. clinical background: gemtuzumab ozogamicin (go) is an anti-cd33 monoclonal antibody with significant activity in de novo and relapsed/refractory (r/r) acute myeloid leukemia (aml). a relevant side effect consists of hepatotoxicity and especially sinusoidal obstruction syndrome (sos). the objective of this study was to analyze tolerability of go during the induction and reinduction therapy in patients with aml, and its possible impact on subsequent hematopoietic stem cell transplantation (hsct). methods: from 2004 to 2017, 24 patients who had received go in three hospitals were collected and their medical records were retrospectively reviewed. results: fourteen patients diagnosed with de novo aml received go (3mg/m 2 ) on day +1 in combination with standard chemotherapy (idarubicin and cytarabine, 3x7 schedule) as induction therapy. hyperbilirubinemia (bilirubin >1.5 unl) was detected in 4 patients and increase of aspartate aminotransferase (ast) (>2.5 unl) in 1. twelve patients achieved complete remission (cr) and one was refractory (1 not evaluated). in the r/r setting, 10 patients diagnosed with aml (n=8), biphenotypic acute leukemia (n=1) and acute promyelocytic leukemia (n=1) received go as 3rd or subsequent rescue therapy either as monotherapy (n=4) or in combination with cytotoxic chemotherapy (n=5). prior hsct was performed in 5 patients (autologous [n=2], allogeneic [n=3] ). rescue therapy was indicated for refractoriness (n=2), relapse (n=5), partial response (n=1) or absence of donor (n=2). four patients received 3 doses of go (3 mg/m 2 ) and 3 patients, one dose. hyperbilirubinemia (>1.5 unl) was observed in 1 patient and increase in ast (>2.5 unl) in 2 patients. seven patients achieved cr, 1 was refractory, 1 obtained partial response and 1 died early during induction). thirteen patients received subsequent hsct (autologous [n=4], allogeneic [n=9]) after go therapy (10 in the de novo aml and 3 in the r/r group). the reasons for not performing hsct in the remaining 11 patients were: low cytogenetic risk (n=1), active chronic graft versus host disease (gvhd) in previous hsct (n=1), early death during treatment (n=3), relapse (n=1), severe complications in rescue treatments (n=1), and unknown (n=4). the conditioning regimen was myeloablative (n=8), non-myeloablative (n=4) and sequential (n=1), and the donors were matched sibling (n=6) or unrelated (n=3) . cyclosporine, methotrexate and thymoglobulin were administered as gvhd prophylaxis in 7 patients and cyclosporine, mycophenolate and thymoglobulin in 3. hyperbilirubinemia was observed in 2 patients belonging to the de novo aml group. death after hsct occurred in 10 patients due to infection (n=5), relapse (n=3), gvhd (n=1) and traffic accident (n=1). three patients are currently alive in remission. no sos was observed in any patient. conclusions: in both de novo and r/r aml the administration of low dose go is feasible and does not have impact on subsequent hsct outcome. although some degree of hepatotoxicity was observed, no cases of sos were observed, either before or after hsct. disclosure: supported by grants from: asociación española contra el cáncer, aecc (gc16173697biga), instituto carlos iii (pi14/01971 fi), 2017-sgr288 (grc), cerca program from generalitat de catalunya, and "la caixa" foundation. outcome of allogeneic hematopoietic stem cell transplantation in patients with benign hematological disorders saqib ansari 1 , tahir shamsi 1 , uzma zaidi 1 , saima siddiqui 1 , tasneem farzana 1 background: allogeneic hematopoietic stem cell transplantation is a potentially curative treatment modality for hematological disorders. we evaluated the outcome of patients suffering from benign hematological disorders, including aplastic anemia, fanconi's anemia and thalassemia after matched related allogeneic transplantation. methods: all patients having hematological disorders including aplastic anemia (aa), beta thalassemia (btm), fanconi's anemia (fa) and severe combined immune deficiency disorder (scid) with hla identical related donors who underwent allogeneic transplantation were included. donors were given g-csf at a dose of 10 μg/ kg/day daily for four days prior to harvest. the conditioning regimens for thalassemia included cyclophosphamide (cy) + busulfan (bu) in 21 (20%), bu + cy + thiotepa in 4 (4%) and bu + cy + antithymocyte globulin (atg) in 81(76%). conditioning regimens for aplastic anemia included, fludarabine (flu) + cy in 38 (33%), flu + atg in 28 (25%) and cy in 38 (33%), cy+ atg in 9 (8%) patients. for fanconi's anemia flu + atg in 12 (52%), flu in 2 (9%), cy + atg in 4 (17%) and flu+ cy + atg in 5 (22%). flu + atg in 2(25%) and cy given in 4 (50%) and no conditioning regimen was offered to 2 (25%) patients with scid. results: a total of 250 allogeneic transplants were performed for benign hematological disorders including aa (n=113), btm (n=106), fa (n=23) and scid (n=8) from 2011 to july 2018. median age was 7.5 years (range 0.5 -48). across the gender and abo blood group transplants were 109 (43.6%) and 80 (32%). the median time to neutrophil and platelet recovery was 13 days (range: 9-46) and 18 (range: 10-35). primary and secondary graft failure was observed in 34 (13.6%) and 29 (11.6%). overall survival in aplastic anemia (63/113, 56%), beta thalassemia (82/106, 77%),fanconi's anemia (15/23, 65%) and severe combined immune deficiency disorder (6/8, 75%). eighty four patients expired (33.6%) among them 41 patients expired within 100 days post transplant main cause of deaths included sepsis (31%), multi organ failure (6%) and gut gvhd (5%) conclusions: in developing world scenario where non malignant disorders are leading cause of morbidity and mortality. bone marrow transplantation has been successfully implemented with better long term diseases free survival and quality of life. clinical background: hematopoietic cell transplantation (hct) remains the only curative therapy for many diseases, yet transplant survivors carry an unusually high burden of morbidities, primarily because of exposure to intense chemotherapy, radiation and /or gvhd. this study aimed to evaluate the burden of chronic diseases at the end of life after allogeneic-hct and to identify the disability-adjusted life years (daly). methods: the pubmed, medline, and ovid databases were queried utilizing specific mesh terminology (post, allo stem cell, hematopoietic, bone marrow, transplantation).we collected data on the impact of the hct on the variables affecting survivor's health in all aspects .the rates of late complications were compared to the risks in the general population (united states). results: a total of 7 studies fulfilled the selection criteria totaling to 6619 patients (table 1) . median os at 5-year mark varied widely between studies from 19% to 92%. majority of the patients at 10-year mark were found to have new comorbidities thereby indicating a huge burden of late effects at the end of life, though exact dalys could not be calculated due to incomplete data. hct survivors were found to have higher risk of premature arterial disease (pad) at 6.8% compared to the general population, however gvhd or the addition of tbi to the conditioning regimen were not found to be significantly associated with pad. regarding the risk of new cancers, the cumulative incidence of their development at 5 and 10 years was 1.71, and 3.61, respectively. increased risks compared with the general population were seen for some solid cancer including cancers of the lip: p=0.02, tonsils: p=0.05, oropharynx: p< 0.01, bone: p< 0.01, soft tissue: p< 0.01, and vulva: p=0.01. with respect to mental health, depression was prevalent in (10.8%) survivors, in whom (82.5%) were still on antidepressants at the last follow-up. cognitive impairment and other psychiatric disorders were found in (2.4%) and (2.7%) survivors, respectively. the most common cause of nrm in the first 5 years was gvhd. however, after 10 years, the leading cause of death in those conditioned with mac regimen was secondary cancer, but in the ric group, new cancers and gvhd contributed equally. conclusions: hct survivors remain at risk of significant complications which lead to premature death and their burden of comorbidities at the end of life is significantly more than that of general population. background: late onset hemorrhagic cystitis (hc) is a common complication of hematopoietic stem cell transplantation (hsct) frequently associated with reactivation of bk virus (bk-hc).there is no consensus as to the best therapy for bk-hc, and many different treatments have been reported. hyper baric oxygen therapy (hbot) is used as primary or adjuvant therapy in diverse clinical situations involving hypoxic injury to tissues and has been explored as a useful tool in treating bk -hc. we report our experience with hbot in combination with non-invasive supportive care in children and adolescents suffering from bk-hc following allogeneic hsct. methods: the computerized database of schneider children´s medical center of israel was reviewed for all patients aged 0 to 21 years who underwent hsct between january 2000 and june 2018 and developed bk-hc. hbot therapy consisted of 2 hours sessions at 2 atmospheres, with patients breathing oxygen by mask. parents accompanied patients during the treatment. results: fourteen patients with a variety of underlying diseases received (hbot) for treatment of bk-hc following hsct. the initial treatment for children with bk-hc at our center prior to 2008 included continuous bladder irrigation and intravesicular instillation of various medications. beginning in 2008, we adopted a non-invasive strategy that included the administration of oral anticholinergics (oxybutinin), systemic pain management, hyperhydration and the administration of weekly cidofovir with probenecid. hbot was administered to patients who failed the above regimen. with this protocol, the average time of starting hbot dropped from 13 (prior to 2008) to 9.8 days. the median onset of hc was 33 days post hsct. all patients were receiving immunosuppressive treatment at the onset of bk-hc. all patients suffered from macrohematuria with blood clots (grade iii cystitis), 12 (92.3%) experienced severe dysuria and 11 (84.6%) urgency. bk viruria was present in all patients, and concurrent bk viremia was detected in 80% of those who were tested. patients reported symptomatic improvement at a mean of 3.6 days following the initiation of hbot. no patient experienced serious adverse effects due to hbot, but two patients required insertion of tympanic ventilation tubes. eleven of our 13 patients (84.6%) experienced complete remission of bk-hc following hbot, with an overall response rate of 92.3% (12/13 patients).eight of our patients (61.5%) eventually succumbed due to either hsct complications or disease relapse. conclusions: hyperbaric oxygen therapy is a safe, effective, non-invasive and well tolerated treatment modality for bk -hc and should be considered for first line therapy for this complication of hsct. clinical background: every year, almost one thousand cases of hematological malignancies in pediatric population are reported in peru. allogeneic hematopoietic stem cell transplantation (allo-hsct) is an alternative strategy in many of these cases. only between 20-30% of the pediatric population that requires a hsct has a compatible human leukocyte antigen (hla) donor. the remaining 70% have to access international donor registries, extending the awaiting time and conditioning the progress of the disease. allo-hsct has the potential to help children with several hematological disorders with non-compatible hla donor. hla genotypically identical sibling donors are the best option when pursuing an hsct. nevertheless, patients' alternative sources of stem cells could be obtained from an haploidentical donor like one of their parents. the haploidentical transplantation program with macs was implemented in peru in 2016 to reduce the risk of graft-versus-host disease (gvhd), support the immune system reconstitution and to expand pool of donors. it allows patients to access a treatment that is efficient and safe, as shown in the depletion of positive tcrα/β+ and b cells procedures for allo-hsct. methods: the mobilized leukapheresis products (n=19) of haploidentical healthy donor was washed to remove platelets and preparations were performed according to miltenyi's clinimacs® manual for tcrα/β+ and cd19+ cell depletion. analysis of the initial leukapheresis product and tcrα/β + and cd19+ depleted graft (target and non-target product) was performed using flow cytometer. cells were analyzed for cd3+, cd45+, 7-aad, cd20+, tcrα/β+, tcrγ/δ +, cd34+ and cd133+ with fluorochrome-labeled antibodies from miltenyi biotec using a novocyte cytometer. the results obtained with the ex vivo t-cell depleted allo-hsct procedures show an overall survival (os) over 70% with an ic95% at the end of the first year with low incidence of gvhd. macs of tcrα/β+ and cd19+ cells are effective (logp 3.9 and logp 3.3, respectively) obtaining minimum levels of depleted lymphocytes within clinical established parameters for diverse pathologies. in addition, tcrα/β+ and cd19+ cell depletion does not significantly affect hematopoietic stem cell populations such as cd34+ and cd133+ cells or tcrγ/δ+ cell population. cd34+ and tcr γ/δ cells are highly recovered (93.15% and 88.76, respectively), which contributes with a better engraftment after allo-hsct. conclusions: peruvian results oscillated within european ranges with an os over 70%. our data suggests that the macs method is an efficient, effective and safe strategy for haploidentical hsct which has a remarkable cost-benefit ratio and makes it viable in countries of the latin american region with peruvian socio-economic characteristics. evaluation of genoresistance for viral reactivation treatment has been implemented as a strategy to improve os. better results are achieved in patients after allo-hsct with the validation of these tests in peru. preliminary pharmacoeconomic evaluations allow us to establish magnetic activated cell sorting (macs) as a promissory strategy compared to other alternatives for haploidentical hsct. it is necessary to increase the number of procedures in order to confirm efficacy and safety of macs in a larger population. disclosure: all authors have no conflicts of interest. abstract already published. micro-costing study of hematopoietic stem cell transplantation in two hospital institutions from southern of brazil background: hematopoietic stem cell transplantation (hsct) is a potentially curative treatment indicated for patients with onco-hematological, hereditary and immunological diseases. considering the increase of patients indicated to the hsct and the lack of knowledge about the costs resulting from this treatment, is important to identify and detail the resources consumed in each phase of hsct and provide knowledge to the public health brazilian system. we aimed measure the total cost of related hsct, based on micro-costing study of patients assisted in two hospitals in the south region of brazil. methods: hsct costs were estimated using the timedriven activity based costing method (tdabc), which measured cost of services / products based on actual consumption of resources. we collected data from medical records of 12 patients submitted to allogeneic hsct in 2017 from public and private (philanthropic) hospitals. we interviewed professionals involved in the tcth activities, we performed chrono-analysis and, we consulted financial and administrative systems reports of hospitals. in order to compare costs according to clinical complications observed in patients, we grouped into two ranges of complexity: low/ medium and high. the study was divided into stages: hsct processes mapping; costs measurement; and analysis of results. finally, the costs were compared: by activity, by resource and by hospital. this study was financed by psid-uhs, by an agreement signed between ministry of health and moinhos de vento hospital, through adjustment term number: 04 / 2014, and approved by research ethics committees. results: from the hsct processes mapping, the following steps were defined: (i) hospitalization; (ii) conditioning; (iii) transplantation; (iv) period of aplasia; (v) engraftment; (vi) observation; (vii) pre-and medical discharge. seven patients were classified in low / medium complexity level, with hospitalization median time of 41 days and an median cost of usd 66,278.29, whereas the other five patients, classified as high complexity, presented median time of 101 days and median cost of usd 300,367.13. the hsct costs evaluation identified that steps ii and iv presented greatest cost in high complexity patients. lower complexity patients presented, in steps ii and iv, median costs of usd 44,274.52 while in higher complexity usd 100,226.26. in addition, median costs of materials and drugs were usd 13,926.02 and usd 181,094.15 in lower and higher complexity patients. conclusions: tdabc method allowed the identification of the moment when patients consume the most resources. of all the hsct stages, periods of conditioning and aplasia presented higher costs, representing 76.90% of the total hospitalization value. in these stages, higher complexity patients presented three times higher the median cost. the resources that had the greatest impact were medicines and medical materials, costing 120 times more than lower complexity patients. conclusion: this study allowed a detailed identification of the hsct costs in patients with different complexity ranges in two hospitals from southern brazil. therefore, the identification of service demand regarding the clinical complexity, allows the generation of important information for the management of the best care in the health service. disclosure background: immunodeficiency due to lrba deficiency is characterized by hypogammaglobulinemia and autoimmunity. hemolytic anemia, lymphadenopathy, autoimmune hepatitis and, above all, autoimmune enteropathy are the fundamental characteristics of these patients together with the history of recurrent invasive infections.the therapy includes immunosuppressants, endovenous immunoglobulins. bone marrow transplantation is the final therapy of these patients, especially in the most serious cases and in recent years, also on the light of increasingly targeted conditioning regimes, it is associated with ever better prognosis. methods: we present the case of caterina, an 8-year-old patient with lrba deficiency, diagnosed at 5 years of age for a history of hypogammaglobulinemia, recurring invasive, bacterial and fungal infections and a picture of autoimmunity represented by ahia, myelitis (c3-c5), autoimmune hepatitis and enteropathy in treatment with abatacept and sirolimus. it also presents leptin deficiency lipodystrophy.she presented to our observation for ostemielitis in multiple outbreaks (tibia, femur and left knee) secondary to sepsis from mrsa. broad spectrum antibiotic therapy and curettage surgery were performed during hospitalization.after 5 months of broad-spectrum antibiotic therapy (daptomycin, rifampicin, ceftaroline, cotrimoxazole, levofloxacin, dalbavancin) there was resolution of the infections.because of the seriousness of the disease it is therefore decided to subject catherine to hematopoietic stem cell transplantation. results: therefore were performed bone marrow transplant from mud after conditioning at reduced intensity, delayed in 9 days, with treosulfan, fludarabine and thiotepa. prophylaxis for gvhd was performed with atg (5mg / kg / day), sirolimus (already practiced for enteropathy) and mmf. because of the hepatic picture, we also performed prophylaxis for vod with defibrotide for 21 days. transplantation was performed by peripheral stem cells with 18 x 10^6 cd34 / kg and 30 x 10^6 cd3 / kg.the patient had always presented good general conditions with engrafment of the pmn to the d + 13 and the plt to the d + 11. the chimerism at d + 20 was 100% donor both on pmn and on pbl. prophylaxis for gvhd was changed on d + 7 by replacing the sirolimus with tacrolimus for the appearance of grade i cutaneous gvhd . conclusions: we have successfully performed bone marrow transplantation in patients with lrba deficiency. the new antibiotic molecules, used to induce infectious remission, the new low-intensity regimens, the prevention of the most fearful complications (vod) have been the key to success in such complicated case. the high number of cd34 cells infused with a controlled number of cd3 were the key then of rapid engraftment with minimal gvhd readily controlled by the immunosuppressant. disclosure background: in the absence of hla-matched related donor, allogeneic stem cell transplantation from haploidentical donors are potential alternatives for patients with hematological malignencies with an indication to allogeneic stem cell transplantation. herein, we retrospectively assessed the outcome of haplo-sct for patients with refractory hematological malignancies. methods: this analysis included 27 consecutive patients who underwent haplo-sct for various hematological malignancies at our center between october 2010 and may 2018. we used our institutional database to evaluate details and characteristics of patients and transplant outcomes. results: demographic features of the patients and donors have been summarized in table 1 . all of the patients had advanced disease with a high risk of relapse. the majority of patients underwent haplo-sct from their parents. out of 24 patients, early transplant-related mortality was seen in this cohort of 5 patients. four patients treated with second haplo-sct and recovered hematopoiesis after second transplant. the remaining 19 patients were followed in a median of 4 months. donor type abo group switch was observed in a median of 45 days (30-60 days) after transplant. the median time for engraftment was 19 days (range, 15-60) for all patients. after the first transplant, 9 patients developed acute gvhd (37.5%) with 7 patients having grade ii-iii acute gvhd. five (18.5%) had chronic gvhd, none of them with extensive manifestation. the prepative regimen was relatively well tolerated with limited regimen-related toxicity. cmv reactivation occurred in 11 patients (40.7%) during the follow-up of the study. eight patients (29.6%) relapsed after a median of 132 days post transplant (range, 45-588 days). cr was achieved in 17 (63%) patients after haplo-sct. mean estimated 5-year os and pfs are 66.7%±0.9% and 92.3%±0.7%, respectively. conclusions: given the growing data on the similarity of outcomes after hla-matched and haploidentical sct, further studies are required to determine whether factors may be more important for donor selection than hlamatching. clinical trial registry: -disclosure: nothing to declare outcome of allogeneic stem cell transplantation for hodgkin and non-hodgkin lymphoma: single center experince from turkey ayşe uysal 1 , hale bülbül 2 , nur akad soyer 2 , mahmut tobu 2 , murat tombuloglu 2 , guray saydam 2 , filiz vural 2 background: allogeneic sct (allosct) is generally optionally treatment choice for young and fit patients with relapsed/refractory lymphoma who were heavily pre-treated and after the failure of autologous stem cell transplantation (asct). relapse after asct is associated with a poor prognosis and allosct is a potentially curative therapy for lymphomas which have relapsed after asct. methods: in this study, we evaluated 19 patients with hl and nhl who had treated with allo-hsct between november 2014 and december 2018 in ege university adult hematology transplantation unit. results: patients, disease and transplant characteristics were illustrated in table. histologic subtype of nhl was evaluated as t cell lymphoma (n=8;61,5%), mantle cell (n=2;15,4%), diffuse large b-cell lymphoma (n=2;15,4%) and b-cell lymphoma, unclassifiable, with features intermediate between dlbcl and classical hodgkin lymphoma (n=1;7,7%). all histologic subtype of hl was determined as nodular sclerosing. the median number of prior treatments before allo-hsct was 3 (range, 1-4). twelve (63,2%) patients had refractory disease, 3 (15,4%) patients were in complete remission and 4 (21,1%) patients were in partial remission before allo-hsct. the median time from diagnosis to allosct was 24 (range, 8-144) months. peripheral stem cell was used for stem cell source in all of them. total body irritation plus fludarabine plus cyclophosphamide and busulfan plus cyclophosphamide were preferred most frequently for conditioning as non-myeloablative and myeloablative, respectively. neutrophil engraftment was occurred median of 17 (range, 10-21) days. graft versus host disease (gvhd) prophylaxis was applied all of them and cyclosporine plus methotrexate was preferred most frequently (n=16;84,2%). gvhd was occurred in 68,4% of them (42,1% acute gvhd, 31,6% chronic gvhd and 7,7% both). veno-occlusive disease (vod) was occurred in 2 (10,5%) patients. transplant related death was observed in 5 (26,3%) patients. overall survival (os) and disease-free-survival (dfs) were evaluated as median 9 (range, 0-45) and 7 (range, 0-45) months, respectively. analyze of os and dfs was illustrated in figure. six patients are alive without disease. after a rapid tapering of immunosuopressive therapy was underwent a therapy with ponatinibat dose of 45mg/die results: afther a month of therapy we observed a rapid decrease in minimal residual disease on molecular assessment with an mmr of p190-bcr-abl/abl non detectable confirmed by bone marrow revaluations at days + 190, +222nd +244 after the salvage therapy. the patient has not had experienced of graft-versus-host disease, ponatinib treatment was well tolerated and considered safe with easily manageable side. conclusions: maybe in the era of tyrosine kinase inhibitors (tkis), philadelphia chromosome positive acute lymphoblastic leukemia (ph+ all) it could benefit from a combined treatment between transpalnt and tkis however more studies are needed to confirm these hypotheses. disclosure: nothing to declare immunodeficiency diseases and macrophage background: although a number of patients with hiv infection and hematological disease have successfully undergone allogeneic hsct together with combination anti-retroviral therapy (cart), short and long-term outcomes remain not well known. we report the largest spanish experience of hiv-infected adult patients with high-risk hematological malignancies with allogeneic hsct. methods: we retrospectively reviewed 22 hiv-positive patients who received allogeneic hsct between 1999 and 2018 in 5 spanish centers within geth (grupo español de trasplante hematopoyético y terapia celular). results: baseline and transplant characteristics of patients are shown in table 1 . median age was 44 years and 77% of the patients were men. the most frequent underlying malignancies were non-hodgkin lymphoma (9, 41%) and aml (7, 31%). in half of the patients an hlaidentical sibling was the donor; and in the other half, an alternative donor was used. peripheral blood was used as graft source in 86% of the transplants. at the time of hsct, all patients had been receiving suppressive cart for a median of 6 years and only 2 of them showed detectable plasma hiv rna, one of them because of poor adherence to cart together with the accumulation of multiple resistance mutations; and the other patient had detectable hiv rna at low levels (< 150 copies/ml). all patients received cart throughout the transplant procedure, being temporally stopped in two patients due to significant mucositis. after a median follow-up of 65 months (8-112), 5-year overall survival (os) and event-free survival (efs) were 46%. nrm was 14% at 12 months and relapse was 24% at 24 months. grade ii-iv agvhd rate was 40%, and moderate/severe cgvhd rate was 41% at 24 months. a significant proportion of patients (68%) showed infectious complications with viral infections as the most frequent cause. two patients had invasive aspergillosis and one patient presented disseminated tuberculosis. causes of death included infections (50%), relapse (43%) and toxicity (7%). among the 6 patients who died due to infections, 3 had severe chronic gvhd and were under immunosuppressive therapy. two patients showed severe toxicity related to drug interaction with anti-retroviral therapy. all survivors except one showed undetectable hiv load at last follow-up after hsct. conclusions: allogeneic hsct is an effective therapy for high-risk hematological malignancies in patients with hiv infection, providing long-term disease free survival together to long-term hiv suppression with cart. however, drug interactions with anti-retroviral agents, occurrence of gvhd, and frequent infectious complications account for a complex procedure in this population. selected hiv-infected patients with hematological malignancies should be considered for allo-hsct when indicated, in experienced centers, with a multidisciplinary care. disclosure background: primary immunodeficiencies (pid) are rare diseases often associated with genetic defects in the immune system, predisposing individuals to recurrent infections and increased risk of allergy, autoimmunity and malignancy. allogeneic haematopoietic stem cell transplantation (hsct) has been successfully used as a curative therapy for most severe forms of pid. because pid is a genetic disease, < 25% of these children will have a healthy, human leukocyte antigen (hla) matched sibling donor available, and umbilical cord blood grafts from unrelated donors are a suitable alternative cell source. we report the results of umbilical cord blood transplantation (ucbt) performed in 112 patients with pid between 2014 and 2018 at children's hospital of fudan university in china. methods: 112 patients included chronic granulomatous disease (cgd, n=39), severe combined immunodeficiency (scid, n=23), interleukin-10 receptor-a deficiency (il-10rad, n=27), wiskott-aldrich syndrome (was, n=7), leukocyte adhesion deficiency (lad, n=5), severe congenital neutropaenia (scn, n=3) and other immunodeficiencies (n=8). all patients were assessed by clinical immunologist to confirm clinical phenotype and genetic diagnosis. median age of 112 patients was 13 months (range, 2 to 144 months), and median body weight was 8.75 kg (range, 3.2 to 36 kg). all patients received a ≤3/10 hla alleles-mismatched cord blood unit, 16 were hla fully matched, 28 were 9/10 matched, 49 were 8/10 matched and 19 were 7/10 matched. median nucleated cells of the cord blood were 14.23x10 7 /kg (range, 3.55 to 51.5 x10 7 /kg), and median cd34+ cells were 3.86 x10 5 /kg (range, 0.73 to 30.27 x10 5 /kg). results: median follow-up time was 13 months (range, 1 to 57 months), the overall survival rate at 1 year for all patients was 74.6%, and was 83.7%, 78.3% and 60% for cgd, scid and il-10rad, respectively. 27 patients died, most deaths (19/27, 70.4%) occurred in +100 days after transplantation, the main cause of death was infection (24/ 27, 88.9%). 87/112 (77.7%) patients engrafted, median time of neutrophil engraftment was 24 days (range, 11 to 60 d), and median time of platelet engraftment was 36 days (range, 9 to 59 d). the cumulative incidence of grade 3-4 acute gvhd was 8.9%, and that of chronic gvhd was 10.7%. conclusions: unrelated ucbt should be considered for pid patients without an hla -matched sibling donor. effective control of infection before and after transplantation is important for improving survival. disclosure background: dedicator of cytokinesis (dock 8) deficiency causes a combined immune deficiency characterised by recurrent bacterial infections, susceptibility to viral infection, eczema, food allergies, vasculitis and increased risk of malignancy. due to the high morbidity and mortality of the disease hsct has been increasingly offered to patients as a potentially curative therapy 1 . methods: we retrospectively reviewed the outcomes of hsct for patients with dock 8 deficiency at great north children's hospital newcastle upon tyne between 2011 and 2018 (5 in published reference). results: ten patients with dock 8 deficiency were treated with hsct (median age 7.5y range 4.5-16.8y). median duration of follow up was 4.5years (range 1.3-6.5y). there were a range of donor sources (3 msd, 3 mud and 4 tcr ab/cd19+ depleted haploidentical), conditioning regimens (6 treo-flu, 4 treo-flu-thiotepa) and serotherapy (5 alemtuzumab, 3 atg+rituximab, 2 none). one patient who received a cd3+ tcr alpha beta/cd19+ depleted haploidentical transplant received add back t-cells with caspacide molecular safety switch (bellicum pharmaceuticals). skin only agvhd occurred in 3/10 patients (1x stage 1, 2x stage 3). no patients had cgvhd. overall survival was 60% (6/10). survival was comparable regardless of donor source. all deaths occurred within 13 months of transplant. the 4 patients who died had significant burden of disease pre-transplant: 1 patient had chronic liver failure secondary to cryptosporidial sclerosing cholangitis,1 had a cirrhotic liver secondary to cryptosporidium, cerebral vasculitis, an axillary aneurysm and aortic vasculitis requiring grafting of an ascending aortic aneurysm,1 was pn dependent for failure to thrive with a history of cryptosporidium infection and 1 had candida in a bal pre-transplant. causes of death in these patients were: respiratory failure (n=1), progressive encephalopathy (n=1), multi-organ failure with septic shock and encephalopathy (n=1) and multiorgan failure and septic shock after treatment for tma (n=1). two of these patients had reactivation of cryptosporidium prior to their death. pretransplant cryptosporidium was associated with mortality (graph 1). one patient who survived had suffered from stroke pretransplant. one suffered from a basilar artery aneurysm 7 years post-transplant at 19yo. at the time of latest follow up donor chimerism was 100% in 5/6 survivors and high level mixed in the other(100% cd3, 89% cd15 and 92% cd19). conclusions: this single centre study of hsct for patients is consistent with literature indicating that hsct is a potentially curative therapy for patients with dock 8 deficiency. the increased morbidity associated with cryptosporidial infection is likely to be a consequence of overall disease burden rather than an infection specific effect. this does however highlight the improved outcomes of transplant prior to development of multiple comorbidities and suggests that hsct should be considered early. it is unclear whether the late occurrence of vascular complications after transplant were caused by a manifestation of disease which is not corrected by transplant or a result of vascular injury sustained pre-transplant. reference methods: referred infants underwent testing for: immune phenotype (ab, gd, naïve and memory t cell, b cell and nk cell numbers); functional activity of t and nk cells; maternal engraftment; adenosine deaminase (ada) and purine nucleoside phosphorylase (pnp) enzyme activity; and genetic testing. those with a confirmed diagnosis of scid underwent either allogeneic hematopoietic stem cell transplant (hct) or (if eligible) gene therapy (gt). infants identified as having ada deficiency as the etiology of their scid received enzyme replacement therapy prior to proceeding to definitive therapy. results: twenty-three (66%) infants were confirmed to have scid. three (13%) of these infants had a family history of scid but would not have been identified without nbs. in addition, one infant, born prematurely at 28 weeks, was diagnosed as having pnp deficiency only after developing infections. this infant was identified by nbs but repeat testing at 32 weeks gestation was normal likely due to support of transient t cell production from exogenous enzyme provided by red cell transfusion. twelve infants with confirmed low trecs had a non-scid diagnosis: 5 with transient lymphopenia of infancy who normalized trec, immune phenotype and function, 1 with prenatal exposure to 6-mecaptopurine (6-mp), 3 genetically confirmed with digeorge syndrome, and 3 with prolonged lymphopenia. of the three with prolonged lymphopenia, two had recurrent infections: one ultimately diagnosed with ataxia telangiectasia and one with absent trec but near normal number of t cells, normal pha but no specific antigen responses, and absent b cells who will be undergoing transplant in the near future. the third continues with absent trec, short telomeres, low numbers of a/b t cells, presence of g/d t cells, vaccine responses and freedom from infection with no identified genetic etiology. in summary, 30% of the patients referred to msk with confirmed abnormal nbs for scid have a non-scid diagnosis. there is no uniform collection of data for these infants and the threshold trigger for repeat testing varies from state to state, so the incidence of significant non-scid disorders identified will also likely vary from state to state. although our institution specific experience is biased, as most infants had confirmation of a low number of trec prior to referral, the significant number of disorders in the non-scid cohort emphasizes the importance of full evaluations and follow-up for these infants. disclosure: none of these relate to the work being presented. susan prockop -research funding mesoblast and atara biotherapeutics. nancy kernan -research funding jaz pharmaceuticals. richard o´reilly research funding and royalties atara biotherapeutics. kevin curran consulting juno pharmaceuticals, novartis. j.j. boelens avrobio, magenta, chimerix and bluebird bio background: post-transplant autoimmune cytopenia (aic) is challenging and associated with substantial morbidity and mortality. we aimed to study the cumulative incidence (ci) of post-hct autoimmune cytopenia (aic) and its predictors in a cohort of children with primary immunodeficiency (pid). methods: in this retrospective study, we included 199 children with pid who underwent their first hsct with fludarabine(f)-treosulfan(t)±thiotepa(thio) at great north children's hospital from 2007-2017. main outcomes of interest were the ci of aic and its predictors. fine-and-grey regression models were used to analyse predictors of aic, considering death as a competing event. variables included were age at transplant (< 2.5 years vs >2.5 years), gender, diagnosis (scid vs immune-dysregulatory disorders vs other pids), pre-transplant aic, pre-and posttransplant respiratory virus, donor (mfd vs mud vs mmfd/mmud vs haploidentical donor), abo incompatibility, conditioning (ft vs ftthio), serotherapy (none vs alemtuzumab 0.3-0.6mg/kg vs alemtuzumab 0.9-1.0mg/kg vs atg), stem cell source (marrow vs pbsc vs cord vs exvivo t depleted pbsc), infused stem cell doses (tnc, cd34 and cd3), agvhd (none vs any agvhd), cgvhd (none vs any cgvhd), viral infections (cmv/adenovirus/ ebv/hhv6 viraemia), chimerism (full vs mixed chimerism (wb < 95%) within first year post-hct). impact of thymopoiesis using naïve t cell recovery was studied. results: median age at transplant was 2.4 years (range, 0.11-18.3 years). primary diagnoses were scid (22%), immune-dysregulatory disorders (28%) and other pids (50%). donors were mfd (21%), mud (48%), mmfd/ mmud (16%) and haploidentical parents (15%). stem cell sources were marrow (30%), unmanipulated pbsc (38%), ex-vivo t-depleted pbsc (23%) and cb (9%). 16% received additional thiotepa and 87% had csa/mmf as gvhd prophylaxis. median duration of follow-up of survivors was 2.9 years (range 0.2 to 10.2 years). 5-year os for the entire cohort was 81%. 6-month and 1-year ci of aic were 5% and 13%. of 21 developed aic, 17 (80%) had aiha, 3 (15%) had aiha±itp and 1 (5%) had aiha ±itp±ain. median onset of aic was 6.2 months post-hct (range 0.2-12.8 months). patients were treated with a median of 3 treatment modalities (range, 1-4). one (5%) had steroid, 8 (38%) had steroid+high-dose-ivig, 8 (38%) had steroid+high-dose-ivig+rituximab, 1 (5%) had steroid +high-dose-ivig+sirolimus and 3 (14%) had steroid +high-dose-ivig+rituximab+sirolimus. the median time to resolution in 18 (85%) who achieved remission after first aic was 6.5 months (range 1.4-28.6 months). 1 had one relapse and 2 had two relapses. 2 died after development of aic (1 aspergillus pneumonia; 1 multi-organ failure). of 19 (91%) surviving patients after aic, 4 had on-going aiha at median of follow-up 2.5 years post-hct (range 0.9-4.8 years). on univariate completing-risk analysis, age at transplant >2.5years (p=0.02) and pre-transplant aic (p=0.02) were associated with higher incidence of aic (figure 1a -ic). on fine-and-grey models, only age at transplant (hr 3.08, 95%ci 1.12-8.49, p=0.03) was independently associated with aic. of 157 with complete immune reconstitution data, naïve t cells >100cells/ml at 6 months post-hct was associated lower incidence of aic (hr 0.36, 95%ci 0.15-0.91, p=0.03) (figure 1d) conclusions: younger age and thymopoiesis were associated lower incidence of aic in children with pid after hct clinical background: human heme-oxygenase-1 (ho-1) deficiency has been reported to present with tetrad of anemia, nephritis, inflammation and asplenia and is fatal if not treated. its an auto-inflammatory disorder. macrophages/ monocytes express ho-1 and are engaged in recycling of red cells. human ho-1 deficiency results in intravascular hemolysis and severe damage to the endothelial system, kidneys, and other organs. transplantation of either healthy wild type macrophages or new macrophages produced by sct from healthy donor has been proven to be curative for ho-1 deficiency in mice. in 2018, we had reported first successful allogeneic sct for human ho-1 deficiency. here we report second successful non-myeloablative msd hsct for a child with ho-1 deficiency. methods: a 9-yr-old-girl presented with complaints of fever, anemia and severe hypertension. in the past, at the age of 7 years she was admitted for high fever for 1 month and needed blood transfusion for the first time for severe anemia and had high platelets and high ferritin. she was treated as macrophage activation syndrome with prednisolone alone and later cyclosporine was added. she had short stature, abnormal facies but normal development. hemoglobin 9 g/dl, urine for haemoglobin was positive, platelets 1055, 000/ul, ferritin 2568 mcg/l and urine albumin 4+ and urine rbc 20-30/hpf. ultrasound and ct scan abdomen showed asplenia. a diagnosis of ho-1 deficiency was suspected. mutation analysis showed homozygous missense mutations in exon2 (r44x) on chromosome 22q12, which would result in the absence of the functional ho-1 protein. both parents were carriers of this mutation. we managed her over next 6-years with prednisolone, hydroxyurea and mycophenolate mofetil (mmf). however she remained steroid dependent. hla-typing confirmed her healthy unaffected 13-year-old brother to be a fully matched donor. at the age of 16 years she was taken up for msd sct after taking informed consent. she weighed just 16 kg. we conditioned her with alemtuzumab-0.8mg/kg, fludarabine-160mg/m2, cyclophospamide-29mg/kg and total body irradiation 2 gray. we infused 9 million/kg peripheral blood stem cells from her brother. graft-vs.-host disease (gvhd) prophylaxis consisted of tacrolimus & mmf. results: she tolerated procedure very well. her entire hospital stay was uneventful and lowest platelet count recorded was 30,000/ul. her neutrophils engrafted on day +13 and she was discharged on day+19. his urine albumin was nil by day+7. she had no gvhd. her chimerism on day+22 showed 97 % donor cells, on day+60 was 98% and on day+180 was 98% donor. now he is day+210 post-sct and doing well. she has no evidence of hemolysis, proteinuria, hypertension, fever. she has normal ferritin and platelets. she has gained 3 cm height and 5 kg weight in last 7 months. she had no viral reactivation and her immune recovery at 6 months post sct is good. conclusions: non-myeoablative allogeneic msd sct is a curative treatment option for human ho-1 deficiency. disclosure: nil two decades of excellent transplant survival in children with chronic granulomatous disease: a report from a supraregional immunology transplant centre in europe background: haematopoietic stem cell transplantation (hsct) confers life-long curative therapy for chronic granulomatous disease (cgd). the ability of donor-derived neutrophils to replace recipient's defective neutrophils makes hsct a superior therapy compared to conventional standard of care using antimicrobial therapy. methods: we examined the outcome of children with cgd who received a first hsct at great north children's hospital from 1998 to 2017. outcomes included overall survival (os), event-free survival (es), toxicity endpoints, autoimmune disease, long-term survival and graft function. cox proportional-hazard models were used to analyse predictors of os and es. variables included for predictor analysis were age at transplant, donor, stem cell source, stem cell doses and conditioning. results: =55 children were included in this analysis. median age of transplant was 5.3 years (range, 0.6-18.0 years). 45 (82%) had x-linked and 10 (18%) autosomal recessive cgd. twenty (36%) had matched family donor, 31 (56%) had unrelated donor and 4 (8%) had parental haploidentical donor. prior to 2007, various conditioning regimens were used, with 21 (38%) patients undergoing conditioning with pharmacokinetic guided intravenous (iv) busulfan (bu) and iv cyclophosphamide with or without serotherapy. from 2007, the conditioning regimen was switched to flu-treosulfan-alemtuzumab with gvhd prophylaxis using ciclosporin (csa) and mycophenolate mofetil (mmf) for family and unrelated donors (n=24, 44%). flu-treosulfan-thiotepa-atg-rituximab was used for cd3 tcr alpha-beta cd19 depleted haploidentical grafts (n=4, 7%). ten (20%) patients had grade ii-iv acute gvhd while 5 had (9%) had grade iii-iv acute gvhd. none had chronic gvhd. the 5-year os for the entire cohort was 89% (95% ci, 67-95%) (figure 1 ). analysis by age at transplant revealed a 5-year os of 100% for children transplanted at < = 5 years of age and 81% (95%ci, 60-92%) for the children >5 years of age (p< 0.04) (figure 2) . the os was comparable between match family donor (88%, 95% ci, 61-97%) and unrelated donor transplant (89%, 71-95%) ( figure 3 ). all four haploidentical transplants were successful. the 5-year es for the entire cohort was 77% (95% ci 62-87%). none of the variables was associated with es. all seven patients with slipping chimerism received a successful second transplant. the five deaths were all due to transplantrelated complications (2 multi-organ failures; 1 pulmonary haemorrhage; 1 graft iv acute gvhd; 1 post-transplant lymphoproliferative disease). the median age at transplant of deceased patients was 10.0 years (range 8.4 to 18 years). the 1-year and 5-year cumulative incidence of autoimmune diseases were 9% and 12% respectively. three (5%) had immune cytopenia while 3 (5%) had autoimmune endocrinopathy (2 thyroid dysfunction; 1 type 1 diabetes mellitus). the median age of long-term survivors was 14 years (range, 2 to 36 years) with the median duration of follow-up of 6.5 years (range, 0.32 to 19.5 years). there was no late death in the entire cohort. the median donor myeloid chimerism was 100% (range 23 to 100%) conclusions: despite the limitations of a single centre study, our findings confirm that hsct is a safe and longlasting curative therapy for children with cgd disclosure: none non -medical challenges in the diagnosis and transplantation of patients with primary immune deficiency: an experience from a tertiary care center in india sagar bhattad 1 , stalin ramprakash 1 , raghuram cp 1 , chetan ginigeri 1 , fulvio porta 1,2 background: primary immune deficiencies (pid) are increasingly being recognized in several parts of india. despite being diagnosed, many patients fail to receive optimal care due to financial and social constraints. methods: case records of patients diagnosed and treated (including hematopoietic stem cell transplants) for pid diseases during feb 2018 -nov 2019 at aster cmi hospital, bangalore, india were analysed. factors leading to deferred or suboptimal care were assessed in detail. results: 65 patients with various pids were diagnosed during the study period (details in table). 35 of them warranted a hematopoietic stem cell transplant (hsct) as definitive curative treatment. a total of 7 children received 9 hsct. 2 of them died while 5 of them are alive and well. 17 children (13 with severe combined immune deficiency) died before a hsct could be carried out. 12 of them were critically ill at presentation, while 5 were stable but deferred further treatment citing financial and social constraints. 11 children needing transplant continue to remain on follow-up and have not been transplanted to date (4 of them have significant financial constraints, 3 families are not convinced about the need for transplant and 4 of them are being prepared for transplant). table: (scid -severe combined immune deficiency, vodi-veno-occlusive disease with immunodeficiency, cgd -chronic granulomatous disease, hlh -hemophagolymphohistiocytosis, was -wiskott aldrich syndrome, xlt -x linked thrombocytopenia, lad-leukocyte adhesion deficiency, msmd -mendelian susceptibility to mycobacterial disease, xla -x linked agammaglobulinemia, cvid -common variable immune deficiency, apeced -autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, cmcc -chronic mucocutaneous candidiasis, at -ataxia telangiectasia). conclusions: we present our experience from a developing country and discuss non-medical factors leading to suboptimal care in children with pid. only 20% children warranting hsct could be transplanted in our cohort. among those where hsct is potentially curative 48% of children died before hsct could be offered. transplants in developing countries pose unique challenges due to the absence of government funding and/or universal insurance coverage. in addition to delay in diagnosis and critical state of patients at admission, financial and social factors significantly contributed to poor outcome. disclosure: none the outcome of hematopoietic stem cell transplantation (hsct) in pediatric patients with hemophagocytic lymphohistiocytosis (hlh) in korea methods: the korea histiocytosis working party retrospectively collected nation-wide data from the patients diagnosed with hlh and underwent allogeneic hsct between 2009 and 2017. the clinical characteristics and treatment outcomes of the patients were analyzed. results: a total of 44 patients were enrolled. there were 31 patients with fhl (4 fhl2, 26 fhl3, and 1 fhl4), 7 infection associated hlh, and 6 secondary hlh of unknown cause. all the patients were treated with hlh-2004 protocol, and 30 patients achieved complete response (cr) after treatment for 8 weeks, while 14 did not. the main reasons for receiving transplantation were fhl in 26, reactivation in 17, and refractory disease in 1. the conditioning regimens were busulfan-based in 16 patients, fludarabine-based in 4, treosulfan-based in 7, and busulfan/fludarabine-based in 17. stem cell sources used for hsct were from peripheral blood in 36 patient, cord blood in 7, and bone marrow in 1. the donor types of hsct were unrelated donor in 33 patients and related in 11 (7 matched sibling donor, 4 haploidentical donor, 1 partially matched donor). the causes of death of 7 patients were disease reactivation/ progression in 3, acute gvhd with/without vod in 3, and graft failure in 1. five year overall survival rates were 82.4%, respectively. the disease status at the time of hsct was cr in 37 patients, and non-cr in 7. the 5-year survival rate of patients who received hsct in cr was 87% and 63% for patients transplanted while in non-cr status (p=0.046). patients who received hsct using peripheral blood stem cells had a better 5-year survival rate of 86% compared to 75% of patients who received cord blood stem cells, significantly. the presence of neurologic symptoms, disease status after intial 8 week therapy, conditioning regimen, and cd 34 positive cell count did not have statistically significant impact on survival. conclusions: hsct improved the survival of patients who had familial, or relapsed, or refractory hlh in the korean nation-wide hlh registry. these results are similar to other reports in the literature. the disease status at the time of hsct and the stem cell source of the transplant were the important prognostic factors that affected the survivals of the hlh patients who underwent hsct. clinical trial registry: no registry number. disclosure: to the best of our knowledge, the named authors have no conflict of interest, financial or otherwise p382 hematopoietic cell transplantation with reduced intensity conditioning regimen using fludarabine/ busulfan and fludarabine/melphalan for primary immunodeficiency diseases background: primary immunodeficiency disease (pid) is congenital disorders of innate or acquired immune system. hematopoietic cell transplantation (hct) was a treatment option for pids with life-threatening infections or immune dysregulations. reduced intensity conditioning (ric) was increasingly used to prevent complications in hct, but optimized regimens have not been established. we performed hct for pids with ric using fludarabine and busulfan (flubu) or melphalan (flumel) according to the guidelines of european society for immunodeficiencies (esid) / european society for blood and marrow transplantation (ebmt), and assessed the efficacy and safety of these ric. methods: from april 2004 to december 2017, 42 pid patients underwent ric-hct using flubu or flumel in tmdu were analyzed retrospectively. the auc of bu was set to 30 mg*hour/l for severe combined immunodeficiency disease (scid) and 60 mg*hour/l for non-scid. overall survival (os) was analyzed. results: the median age at hct was 2.0 (0.3-21) years old (35 male and 7 female patients). flubu was used for 23 patients (7 scid, 9 combined immunodeficiency disease (cid), 3 ectodermal dysplasia (eda), and 4 severe congenital neutropenia (scn)) and flumel was used for 19 patients (8 scid, 6 cid, 4 xiap deficiency, and 1 eda). anti-thymocyte globulin was used in 8 patients of flubu group and 7 patients of flumel group. cord blood in 21 and bone marrow in 21 was used for donor sources. matched donor was used for 7 and 8 patients in flubu and flumel groups (30.4 % and 42.1 %), respectively. median follow up period was 2.2 years. two years-os of all patients, flubu group patients and flumel group patients was 79.6 %, 89.8 % and 64.1 %, respectively. neutrophil engraftment was 92.9 %, 91.3 % and 94.7 % (all patients, flubu group and flumel group). in scid, all 7 patients in flubu group achieved engraftment and survived. seven out of 8 in flumel group achieved engraftment, but 1 patient had secondary graft failure and 3 patients died. in non-scid, 14 out of 16 in flubu group achieved engraftment, but 6 patients had secondary graft failure. all 11 non-scid patients in flumel group were engrafted and survived. two and 7 patients in flubu group and flumel group suffered from severe acute graft-versus-host disease (grade iii-iv). ten patients had hemophagocytic lymphohistiocytosis (hlh). viral reactivation or infection was observed in 20 patients, and resolved in all but one patient. conclusions: the ric-hct using flubu or flumel was advantagous for neutrophil engraftment, and flubu for scid and flumel for cid with immune dysregulation may be an effective opinion. flubu regimen needs to be improved for secondary graft failure in non-scid. prevention of hlh after transplantation using dexamethasone palmitate will be considered. disclosure: nothing to declare. survival after hematopoietic stem cell transplantation with tcrαβ/cd19 graft depletion in older children with primary immunodeficiencies background: tcrαβ/cd19 depletion is a graft engineering method that proved valuable in increasing the survival rate after hematopoietic stem cell transplantation (hsct) in patients with primary immunodeficiencies (pid). decreased survival rate in older patients with pid was previously reported after transplantations with nonmanipulated grafts. methods: 148 patients with various pid (excluding classic scid) who received allogenic hsct with tcrαβ/ cd19 graft depletion from 2012 to september 2018 in our center were analyzed. the median age at hsct was 3,5 years (range 0,43-17,63). patients were divided into 3 age groups: 0-6 years -95 patients, 6-12 years -30, 12-18 years -23. 112 patients received hsct from matched unrelated, 31 -haploidentical donors, 5 -siblings. conditioning regimens with 1-2 alkylating agents and antithymocyte globulin were used in all patients. 113 patient received short courses of various posttransplant immunosuppressants. median follow up after hsct is 1,95 years (range 0,04-6,3 years). results: overall survival (os) in 148 patients was 0,85 (95% ci 0,79-0,91). we observed similar os in the younger age groups: 0,89 (95% ci 0,83-0,96) in 0-6 years and 0,89 (95% ci 0,78-1,0) in 6-12 years of age. seven of 23 patients in older group (12-18 years of age) died, the os was only 0,65 (95% ci 0,43-0,86), p=0,065. all patients from older age group who died had combined pid: 1 -wiscott-aldrich syndrome, 1 -undefined pid, 1 -il2rg deficiency, 1 -dclre1c deficiency, 1 -nijmegen breakage syndrome (nbs), 1 -kabuki syndrome, 1 -icf syndrome. the median time of death after hsct was 0,64 years (range 0,26 -1,58). six of those had transplant-related mortality (trm). five patients had hsct-associated viral infections: 2 -cmv pneumonias, 3 -adv infections (1 -fulminant hepatitis, 2 -multiorgan). interestingly, 2 of them had prolonged history of disseminated viral infections (adv), with the reduction of viral load in blood and other fluids and tissues upon treatment. one patient with kabuki syndrome after hsct developed hhv8 associated kaposi sarcoma, was successfully treated. eventually all 3 patients with reduction of viral infection and sarcoma symptoms developed multiorgan failure with some clinical and laboratory evidences of endothelium cell damage syndrome. one patient with nbs died of high grade lymphoma progression. conclusions: hsct with tcrαβ/cd19 depletion demonstrates high survival rate in 148 patients with various pid. in our group patients' age older than 12 years predisposes to decreased posttransplant survival. patients with combined pid are at higher risks of posttransplant mortality. we conclude that at least in some of our patients with prolonged history of viral infections after hsct the cause of death could be multiorgan failure due to endothelium cell damage syndrome resulting from persistent inflammation and drug toxicity effects. disclosure background: chronic granulomatous disease (cgd) is a primary immunodeficiency (pid) caused by a mutation in 1 of the 5 subunits of the nicotinamide dinucleotide phosphate (nadph) oxidase, which leads to a reduction in the microbicidal activity of phagocytic cell. starting at an early age, cgd patients suffer from severe recurrent infections, as well as inflammatory events. allogeneic hematopoietic stem cell transplantation (hsct) is a curative option for cgd in patients with insufficient benefit from supportive care and prophylactic antibiotics. we reported a series of 39 patients with cgd who underwent unrelated umbilical cord blood transplantation (ucbt) at our center. methods: in this retrospective study, we observed a series of 39 consecutive ucbt performed at our center in children with cgd between 2015 and 2018.median age at transplantation was 17 months (range, 4 to 144 months), median body weight was 10 kg (range, 6 to 34 kg). 32/39 patients received a myeloablative conditioning regimen consisting of busulfan, fludarabine, cytarabine, cyclophosphamide and g-csf, 7/39 patients received another myeloablative conditioning regimen consisting of busulfan, fludarabine, cyclophosphamide and atg. prophylaxis for graft-versus-host disease (gvhd) was tacrolimus. results: engraftment occurred in 29/39 (74.4%) patients. 10/39 (25.6%) patients occurred graft failure, all of them received a myeloablative conditioning regimen without atg. median time to neutrophil and platelet engraftment were 24 (rang, 14-46) and 36.5 (rang, 15-51) days. 22/39 (56.4%) patients developed acute gvhd, with 4/39 (10.3%) episodes of grade iii-iv agvhd. chronic gvhd occurred in 3/29 patients (10.3%). at a median follow-up of 17 months (rang, 1 to 44 months), the overall survival rate was 83.7%, and event-free survival rate was 66.7%. conclusions: unrelated ucbt should be considered as potential curative methods in children with cgd. cgd patients who used myeloablative conditioning regimen with atg shows better graft and survival. disclosure: nothing to declare background: invasive fungal infections (ifi) remain a major cause of treatment-related morbidity and mortality in aml patients. although not uncommon, the presentation of unusually severe clinical features might be indicative for an underlying immunodeficiency. caspase-associated recruitment domain 9 (card9) is recognized to have a crucial role in effective antifungal response, leading to th1 and th17 differentiation and to the initiation of the inflammatory cytokine cascade. particularly interferon-gamma (ifng) increases macrophage activity. patients with homozygous card9 mutations are known to have a significantly increased susceptibility to life-threatening systemic candidiasis. however, some sequence variants may lead to increased ifi-susceptibility even in heterozygosity, e.g. under immunosuppression. ifn-γ has been described as an additive treatment option because of its immune stimulating effect on the leukocyte immune response in a situation of immunological "blindness". methods: here, we report the case of an 11-year old male with aml m6 with a severe systemic candida tropicalis infection, unresponsive to triple-antimycotic regimen, leading to multi-organ failure. he was discovered to bear a heterozygous card9 mutation, and ifn-γ immunotherapy leaded to complete response of all disseminated infections. results: the patient developed septic fever immediately after the first chemotherapy cycle. unexpectedly, candida tropicalis was confirmed in the blood culture within 24 hours. liposomal amphotericin b (ambisome ® ) was started immediately, however candida rapidly disseminated to lungs, liver, spleen, kidneys and cns despite extended antimycotic therapy with caspofungin, voriconazole and fluconazole. the patient was splenectomized due to massive infiltration ( figure 1 ). genetic testing for mycosis predisposition revealed a heterozygous mutation in the card9 gene, inherited from the father (c.809a>t(p.glu270val)). ifn-γ treatment was started (100 μg subcutaneously, 3 times per week), leading to an almost complete response of disseminated infections. due to the severe infection, chemotherapy had to be interrupted after one course. however, bone marrow remained in complete remission for almost one year. the patient experienced altogether two relapses requiring an unrelated allogeneic and a haploidentical hsct. under combined ifn-γ and ambisome/ fluconazole prophylaxis no further mycosis was observed despite extensive and prolonged immunosuppression. conclusions: ifi in aml patients are common, however an unusual presentation in presumably immune competent individuals should raise the suspicion for immunodeficiencies. in our case, an unexpected early candida-sepsis was completely unresponsive to an adequate multi-agent treatment. while ifn-γ is used in adults as an immune stimulatory cytokine, little data are available for children. to our knowledge, this is the first case of successful ifn-γ treatment of a pediatric aml patient with disseminated candida sepsis, bearing a card9 mutation. given the elevated mortality risk for ifi, and the apparently safe and well-tolerated application of ifn-γ, an adjuvant immunotherapy might be considered. further studies are needed to define the indication and duration of this kind of adjunctive immunotherapy. moreover, considering the wide heterogeneity of genetic mutations involved in ifi-susceptibility, genome-wide expression profiling might be useful for pediatric cancer patients, as the identification of specific immune pathways might help to identify individual ifisusceptibility in order to improve the outcome of those high-risk patients. [[p385 image] 1. background: chronic granulomatous disease (cgd) is curable by allogeneic hematopoetic stem cell transplant (hsct). recent reports of haploidentical donor hsct with with post transplant cyclophosphamide (ptcy) from family donors in pediatric primary immune deficiencies have shown encouraging results. however, it has not been reported in cgd. here we describe successful haploidentical hsct in a child with cgd with myeloablative conditioning and ptcy. a 3 year-old, male child diagnosed with cgd showed oxidative activity 0.3 % by dihydrorhodamine (dhr) test. he had no matched related or unrelated donor available so underwent haploidentical hsct after taking informed consent of the parents in may 2018. donor was his 5/10 hla matched healthy elder sister (oxidative activity 55 % by dhr). he has had multiple admissions for recurrent pneumonia prior to hsct. the conditioning was with rituximab 100 mg/m2 iv on day -8, thiotepa 10 mg/ kg/dose intravenous (iv) for 1 day (day-7), busulfan 3.2 mg/kg/dose daily iv for 4 days (day -6 to -3) and fludarabine 40 mg/m2/dose daily iv for 4 days (day -6 to -3) and rabbit anti-thymoglubulin (thymoglobulin) 1.5 mg/ kg/dose daily for 3 days (day-4 to -2). peripheral blood stem cells (8 million/kg cd34+ cells) were harvested from his sister and transfused to the patient on day 0. graft vs. host disease (gvhd) prophylaxis was with ptcy 50 mg/ kg on day+3 & 4, intravenous cyclosporine from day-3 (targeting levels 150-250 ng/ml) and mmf from day+5. results: his neutrophils engrafted on day+15 and platelets on day+17. chimerism on day+30, 100 and 6 months was fully donor. he developed no acute or chronic gvhd. at 6 months his lymphocyte counts showed cd4-280/ul, cd8-670/ul, cd19-15/ul and cd16/56-120/ul. his had no viral reactivation. he is disease free and gvhd free on day+190 post hsct and is on tapering doses of cyclosporine. his dhr test showed oxidative activity of 42 % on day +100. background: primary immune deficiencies (pid) are a functional disorder of inheritance immune system that increase predisposition to infectious disease in number and severity. the incidence is 1: 10,000 birth live; its immunological dysregulation may increase the predisposition of autoimmune diseases and malignancy, the latter being more frequent (4-25%). at present, the only curative treatment is hematopoietic stem cells transplant (hsct). methods: we describe all patients transplanted with primary immune deficiencies at instituto nacional de pediatria. the conditioning regimen depended on the type donor and pathology: myeloablative (41.07%), reduced intensity (37.5%) and non myeloablative (21.43%) without modification statistically significantly in overall survival. results: a total of 71 patients were included from 1998 to january/2017. severe combined immunodeficiency (scid) is the pathology most frequently transplanted ( figure 1) . seventy three percent have molecular diagnosis, and 44.83% have cases of family pid. the most used sources were umbilical cord blood (ucb) with 42.6% and peripheral blood (39.3%), however the trend of the source of obtaining it has been modified a few years ago (figure 2) . the median graft was 14 days for ucb, 16 days for bone marrow (bm) and 12 days for peripheral blood (pb) (figure 3 ) the main complications are infectious (bacterial 48.3% and viral 39.7%) and non-infectious as pre-graft syndrome (23.1%). conclusions: the overall survival was 54.1% survival according to pathology was: 100% chediak higashi syndrome, 68% scid, 66.7% griselli syndrome, 66.7% hyper igm syndrome, 60% was, 57% cgd, 50% hemophagocytic lymphohistiocytosis. disclosure: ramírez-uribe rosa maria nideshda, salazar-rosales haydeé, olaya-vargas alberto, lópez-hernández gerardo, del campo-martínez maria de los àngeles we wish to confirm that there are no known conflicts of interest associated with this abstact, the only financial support was provided by mexican associations that helping children wiht cancer in a few patients. long-term outcome following hematopoietic stem cell transplantation of wiskott-aldrich syndrome in a single institute mamoru honda 1,2 , yukayo terashita 1 , minako sugiyama 1 , yuko cho 1 , akihiro iguchi 1 background: wiskott-aldrich syndrome (was) is an xlinked disorder of hematopoietic cells, characterized by thrombocytopenia with small platelets, eczema, and immunodeficiency. hematopoietic stem cell transplantation (hsct) is the only curative treatment, and it is recommended to be performed as soon as was is diagnosed. myeloablative conditioning before hsct is recommended because there is a high risk of development of autoimmune disease in patients with mixed chimera after hsct. however, there are few reports about late complications such as pubertal development and eruption of teeth in patients with was receiving hsct. thus, we evaluated late complications in patients with was receiving hsct at hokkaido university hospital. methods: we reviewed medical records of 8 male patients with was who received hsct between 1995 and 2017. results: mean age at hsct was 1.4 (range, 0.6-6.8) years, and median follow-up time after hsct was 13.8 (range 2.9-23.4) years. conditioning regimen in all patients comprised busulfan at 4 mg/kg for 4 days and cyclophosphamide at 60 mg/kg for 2 days or 50 mg/kg for 4 days. additionally, anti-thymocyte globulin at 2.5 mg/kg/day for 1-2 days was administered in 6 patients. engraftment, normal platelet count, and complete chimera were confirmed in all patients. no patients showed complications such as severe chronic graft-versus-host disease, autoimmune disease, short stature (≤ -2.0 sd) and second malignancy. however, high ige level was observed in 4 patients. pubertal development has been confirmed in 6 patients. lack of complete eruption of permanent teeth has been observed in 3 patients who received hsct at age of < 2 years. conclusions: although this was a small-cohort study in a single institute, complete chimera has been achieved in all patients who received hsct with busulfan-based myeloablative conditioning. however, late complications such as male infertility and incomplete eruption of permanent teeth remain major problems. disclosure: nothing to declare methods: we performed unrelated umbilical cord blood transplantation (ucbt) in 5 consecutive children with lad-i. median age of 5 children was 13 months (range, 8 to 131 months), and median body weight was 13 kg (range, 8 to 24.3 kg). all patients received myeloablative conditioning regimen consisting of busulfan, fludarabine and cytarabine. prophylaxis for graft-versus-host disease (gvhd) was tacrolimus. all patients received a ≤2 hla alleles-mismatched cord unit, 1 was hla fully matched, 3 were 9/10 matched, 1 was 8/10 matched. median nucleated cells of the cord blood were 14.23x10 7 /kg (range, 4.6 to 20.62 x10 7 /kg), and median cd34+ cells were 3.87 x10 5 / kg (range, 1.95 to 5.77 x10 5 /kg). results: all patients engrafted, median time of neutrophil engraftment was 24 days (range, 13 to 28d), and median time of platelet engraftment was 33 days (range, 32 to 56d). median follow-up time was 13 months (range, 2 to 25 months), all 5 patients were alive with continuous completely donor engraftment, and achieved complete clinical remissions. 4/5 patients developed grade ii/iii acute graft-versus-host disease (gvhd), and 1/5 patients developed chronic gvhd with skin. conclusions: it is the first successful unrelated ubct for lad-i children in china. our data shows ucbt provided excellent outcome for patients with lad-i. disclosure: nothing to declare p390 excellent outcome using 'nktm' enriched hematopoietic stem cell transplants for patients with inborn errors of immunity results: majority of patients in the 2 cohorts had significant infective co-morbidities at the time of hsct with patients in the later cohort entering hsct earlier. patients in the later cohort were sicker at hsct. final engraftment occurred in all except 1 patient who received a hla mis-matched cord blood hsct. graft failures occurred in 6 patients (2 in earlier and 4 in later cohort); 3 of these patients received unmanipulated hscts from hla mis-matched unrelated donors (1 cb, 2 bm). second hsct were with same donors in 3 and different donors in 2 patients. no grade ii to iv acute gvhd or extensive gvhd occurred. one patient (cbt) died of infections/ non-engraftment. all 9 patients in the later cohort compared to 7 of 9 patients in earlier cohort are alive, engrafted and cured. performance status were 100% in all alive patients. of the 9 patients in the later cohort, 7 (6 hla mis-matched related and 1 hla matched related donors) received 'nktm' enriched hsct. in the 6 hla mis-matched 'nktm' enriched hscts, patients received high cd34+, cd3+cd45ro+ and nk cell doses, with median of 16.88 (range, 5.1 -36.5), 113.9 (range, 3.3 -232.6) and 89.0 (range, 22 -654) x10 6 /kg, respectively. no invasive infections occurred in these patients and immune reconstitution in t, b, nk compartments were complete at 1 year after hsct with cd4 > 200 by 6 months and tcrαb > 1500 by 1 year after hsct. background: viral infections contribute to significant morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-hsct), increasing both the human and the financial cost. antiviral agents are often ineffective or/ and associated with toxicity. methods: in view of t-cell anti-viral immunotherapy in greece, we evaluated the actual cost of conventional pharmacotherapy for cmv, ebv and bkv reactivations after allo-hsct, by calculating the costs of (i) the antiviral agents, (ii) the treatment (excluding transfusions) of antiviral drug primary toxicity (e.g. graft failure, cytopenias, renal or hepatic dysfunction) and secondary toxicity (e.g. leukopenia-associated bacterial infections), iii) the treatment (excluding transfusions, ie for bk cystitis) of infectionrelated complications, iv) the transfusions due to treatmentrelated toxicity (ie cytopenias) or infection-related complications (ie, bk cystitis), v) the inpatient or outpatient daily care. notwithstanding that blood and its products, as a common natural good, are provided free in our country, the costs related to blood and platelet collection, processing, storage, laboratory testing and infusions were included in our model. results: the treatment cost of cmv, ebv and bkv reactivations/infections for the first six months post allo-hsct was evaluated in 38/51 patients who reactivated viruses and were transplanted between 10/2015-11/2016 from matched related (17/51), matched unrelated (20/51), mismatched unrelated (8/51), haploidentical (5/51) and mismatched related donors (1/51). we detected 29 cmv, 35 ebv and 18 bkv infections/reactivations in 20, 32 and 17 patients respectively, with a mean of 2±0.4 infection per patient from all three viruses (1-7/patient). of note, 22/38 patients experienced reactivations from more than one virus, requiring repeated treatments with antiviral agents and/or rituximab. the cost of antiviral agents for all cmv, ebv and bkv reactivations/infections was 78,656€, 58,504€ and 11,331€ respectively (3,146€, 2,089€ and 1,416 €/patient, respectively). the treatment cost of toxicity related to antiviral drugs and infection-related complications was 70,358€ (4,309€/patient) excluding transfusions and 676,107€ (28,171€/patient) including transfusions. in particular, the cost of transfusions for bkv hemorrhagic cystitis reached 22,751€/patient. repeated (1-5) and/or prolonged (δm 38d, range 6-150d) hospitalizations were needed, up to a total of 745 and 159 days of inpatient hospitalization and short-term outpatient treatment, respectively. hospitalizations further increased the cost of inpatient and outpatient post-transplant care by 81,640€ and 8,000€ respectively (2,634€ and 500€/patient, respectively), onthe basis of a, rather underestimating the true cost, fixed, unified hospitalization fee (60€/day and 200 €/day). conclusions: overall, in a six-month study period, the treatment of cmv, ebv and bkv infections substantially increased the cost of post-transplant care by 956,283€ (27,322€/patient). the actual cost is undoubtedly higher as the hospitalization fee for transplant recipients is largely underestimated in greece. considering not only the hematopoietic but also the solid organ transplant recipients, the financial burden of antiviral treatment for national economies is enormous. given that antiviral pharmacotherapy is often associated with suboptimal efficacy, toxicity, development of drug resistance, reactivation recurrences and repeated hospitalizations, it is expected that a one-time treatment with multi-virus-specific t cells, able to expand in vivo and provide a long-lasting protection without significant toxicity, will serve as a powerful and costeffective treatment over conventional pharmacotherapy. disclosure: nothing to declare methods: this is a single-centre retrospective analysis of 293 consecutive patients who underwent tcd allo-hscts for myeloid malignancies between january 2012-june 2016. ebv-dna was monitored frequently on whole blood samples with standardised quantitative real-time pcr. serum protein electrophoresis was routinely tested with immunoglobulin subclasses identified by immunofixation electrophoresis. histological confirmation of ptld was based on standard who diagnostic criteria ('proven'), while those without biopsy were classed as 'probable' based on clinical & radiological criteria as defined by ecil-6 guidelines. results: majority of patients had aml(n-152/293) and mds(n-107/293) with a median age of 58 years(range . median follow up of survivors was 32 months(range 4-65). majority of patients(n-220/293;75%) developed ebv-r with a median time of 79 days[inter quartile range(iqr) 27-160 days] &higher cumulative incidence with atg(n-132) versus alemtuzumab(n-161)(p< 0.001). figure-1a shows schematic representation of ebv and ptld events (cumulative incidence of 6.8%(95%ci-4.0%-10.6%) at 12 months). significantly higher peak ebv dna viral load(evl) were noted in patients with ptld(p-< 0.001). development of post-hsct mg was observed in 29%(n-85/292). roc curve identified peak blood evl>150,000 copies/ml significantly correlated with risk of developing ptld (sensitivity-70.6%,specificity-79.4%;auc-0.82,p< 0.001). based on these estimates, subgroup of patients with no ebv-r(n-72/292), peak evl < 150,000(< 150k)copies/ ml(n-165/292) & >150,000(>150k)copies/ml(n-55/292) were categorised in 6 groups along patients with/without mg accordingly (groups 1-6;figure-1b). patients with ebv-r had significantly better os [5-year os of 52% vs 35%(no ebv-r);log-rank p< 0.001],with this survival benefit mainly driven by subgroup of patients with lower evl(< 150k)(p< 0.001). ptld patients had trend towards inferior 3-year os(15% vs 54%;p-0.051). patients with mg had a significantly better os irrespective of degree of evl (group 1-3,p< 0.001).we report a 'sweet spot' of low evl & presence of mg in these patients, with a clear survival advantage compared to those with no ebv-r and/or no mprotein (group-2 5-year os 62% vs 27% in group-6; hr-0.15;95%ci:0.06-0.34;p< 0.001;figure1b). overall cumulative incidence of relapse (cir) was 28%(95%ci:23-37) and non-relapse-mortality(nrm) of 24%(95%ci:18.6-30) at 5 years. multivariate analysis(mva) revealed absence of m-protein,high evl (>150k copies/ml) or no ebv-r and absence of any gvhd as significant factors for high cir. similarly, high evl or no ebv-r, absence of m-protein and itu admission were significant predictors of high nrm. conclusions: this study adds to our understanding of role of ebv viraemia & associated mg in tcd-hscts while highlighting its significant impact on risk of ptld, os, nrm & cir. low ebv burden and development of mg is protective with significantly better survival outcomes and we recommend pre-emptive approach of using rituximab for ebv-r /ptld is best employed at higher ebv burden (e.g. >150k copies/ml dna) in high risk patients and be prospectively evaluated in future studies. clinical trial registry: n/a disclosure: nothing to declare p394 impact of early candidemia on the long-term outcome of allogeneic hematopoietic stem cell transplant in non leukemic patients: an outcome analysis on behalf of idwp background: to assess the incidence of, and risk factors for, candida infection in the first 100 days post-allogeneic hematopoietic stem cell transplantation (hsct) and the impact on long-term survival. methods: outcome analysis of 50,188 patients, 61% male, median age 46 years (range 0-80), with diagnosis of hemoglobinopathies in 3176 (6.3%), bone marrow failure in 7626 (15.2%), lymphoma in 17743 (35.4%) and myelodysplastic/myeloproliferative disesases in 21643 (43.1%) patients who underwent hsct from 2000 to 2015: 420 with candidemia by day + 100, and 49,768 without candidemia. the incidence of 100-day candidemia was estimated by using the cumulative incidence method. the univariate and multivariate risk factor analysis for 100-day candidemia was performed with the cause-specific cox regression model. the occurrence of candidemia was analyzed as a timedependent covariate. the overall survival and non-relapse mortality after day +100 were assessed in a land-mark setting, this analysis was restricted to patients surviving to day +100 post transplant. [[p393 image] 1. figure 1a -1b] results: the incidence of candidemia by day +100 was 0.85% (95% c.i. 0.77-0.93) (420/50,188) and occurred at a median of 17 days post-hsct (range -7-100). considering the candidemia within 100-day from hsct as a time dependent covariate, a higher 100-day non-relapsemortality (nrm) (hr 3.47 (2.75-4.38), p < 0.0001), and a lower 100-day overall-survival (os) (hr 3.21, 95% ci 2.67-3.85), p< 0.0001) were obtained from the cox model for patients with candidemia. factors significantly associated with candidemia occurrence in the multivariate analysis were: gender female, increased age at hsct, bone marrow failure, lymphoma or myelodysplastic/myeloproliferative diagnosis, bone marrow or cord blood stem cell source, t-cell depletion, less recent year of hsct. among patients alive at day + 100, the 5-year nrm and os with and without candidemia were 28.2% vs. 18.8%, p < 0.0001, and 50.5% vs. 60.7%, p< 0.0001, respectively, after a median follow-up of 4.3 years (95% ci 4.3-4.4) (figure 1 ). in multivariate analysis, the occurrence of a candidemia episode within day + 100 was an independent risk factor for higher nrm, hr 1.52 (1.18-1.97), p=0.0013, and lower os, hr 1.30 (1.08-1.57), p=0.0061. conclusions: despite the general improvements in prophylaxis and treatment, the occurrence of early post-hsct candidemia had a negative impact on transplant outcome as showed previously in leukemic patients. abstract already published. carbapenem-resistant enterobacteriaceae colonizationimportance in the risk of cre bacteremia and mortality in stem cell transplant (hsct) and acute leukemia patients marcia garnica 1,2 , marco a f bellizze 1 , priscila g a de jesus 1 , rafaela r c gomes 1 , filipe m akamine 1 , alan j marçal 1 , luzinete co rangel 2 , andreia assis 2 , marcia rejane valentim 2 , angelo maiolino 1 background: spread of infections due to carbapenemresistant enterobacteriaceae (cre) is a worldwide phenomenon and has been associated with high mortality and clinical complications. gut translocation is the most important portal of entry of bacteria during neutropenia, and cre gut colonization is a possible risk factor for bacteremia during neutropenia. goals: in the present study, we describe the frequencies of cre colonization and analyzed its relationship with development of cre bacteremia and mortality in two different scenarios: stem cell transplant patients (hsct) and leukemia patients. methods: prospective cohorts of hsct (from 2012 to 2017) and leukemia patients (from 2016 to 2018). hsct patients were analyzed from conditioning until discharge (pre-engraphment phase) and leukemia patients from first induction chemotherapy until last intensification. if an hsct was performed in leukemia patient the patient was censored in leukemia cohort and included in hsct cohort. all patients had rectal swabs performed weekly during hospitalization for the identification of cre colonization. patients with at least one positive swab (cre colonization group) were compared to patients with no documentation of colonization (controls). the outcomes analyzed were bacteremia due to cre, and overall mortality. results: there were 493 hsct performed during the study (408 [83%] autologous and 85 [17%] allogeneic). multiple myeloma and non-hodgkin lymphoma were the most frequent baseline diseases (n=251; 51%, and n = 88; 18%), respectively. cre colonization was documented in 10% (n=50), and it was more frequent among allogeneic hsct and leukemia patients (p< 0.001 for both). cre colonized patients had longer hospitalization (25 vs. 20 days, p< 0,001), higher frequency of cre bacteremia (6% vs. 0.2%; p=0.004), and mortality (16% vs. 2.4%, p< 0,001) compared to non-colonized hsct. negative and positive predicted values for cre bacteremia were 6% and 99%, respectively. thirty-one patients were analyzed in leukemia cohort, accounting to 92 hospitalizations (median 3 hospitalizations per patient, ranging from 1 to 6). the median age was 58 years, and 90% aml vs. 10% all. cre colonization was documented in eight (26%), with a median time from leukemia diagnosis and colonization of 93 days (9 -503 days). cre bacteremia was documented only in colonized patients (25% vs. zero; p=0,046). all eight colonized patients were submitted to other cycles of chemotherapy after colonization, in one of them cre bacteremia relapsed. conclusions: a routine surveillance of cre colonization showed colonization frequencies from 10% to 25% in hsct and leukemia patients respectively and was effective to stratify cre bacteremia risk as the predictive negative value was over 95%. colonization had association with cre bacteremia and overall mortality. efforts to minimize risks for colonization and mortality are necessary. the information of surveillance can be a tool to improve adequacy in empirical febrile neutropenia therapy in hsct and leukemia patients. disclosure: nothing to declare background: the incidence of hepatitis b virus infection is high to 6.2% in asian population, so there is more and more attention to the risk of hepatitis b virus(hbv) reactivation in the hepatitis b core antibody positive patients during chemotherapy, anti-cd20 monoclonal antibody, hsct, or other intense immunosuppressive drug therapy (isdt). hepatitis b core antibody is associated with a significant risk of hbv reactivation in patients undergoing hsct. however, there are remain uncertain that the effect of anti-hbsag antibodies in hepatitis b virus reactivation among the hepatitis b core antibody positive patients undergo hsct. we aim to investigate the role of the anti-hbs and the necessity of anti virus in hepatitis b surface antigen(hbsag) negative, hepatitis b core antibody positive patients during hsct. methods: we enrolled 791 hematological malignant patients received hsct in our center from 2008 to 2016. we classified 665 hbsag negative and undetectable hbv dna patients into 4 groups as anti-hbc(-)anti-hbs(-) (n=189), anti-hbc(-)anti-hbs(+) (n=176), anti-hbc(+) anti-hbs(-) (n=49), and anti-hbc(+)anti-hbs(+) (n=251). results: hbv reactivation was identified in 16 patients (2.4%) after hsct. there was a significant difference in hbv reactivation rate in anti-hbc(+)anti-hbs(-) (12.2%) vs anti-hbc(+)anti-hbs(+) (2.8%) (p=0.01) and anti-hbc (+)anti-hbs(-) (12.2%) vs anti-hbc(-)anti-hbs(-) (0.0%) (p=0.000), anti-hbc(+)anti-hbs(-) (12.2%) vs anti-hbc(-) anti-hbs(+) (1.7%) (p=0.004), but not among anti-hbc(+) anti-hbs(+) (2.8%) and anti-hbc(-)anti-hbs(-) (0%) and anti-hbc(-)anti-hbs(+) (1.7%). whereas there were no difference according to the donor viral profile(p=0.774). the median time of hbv reactivation in hbsag negative patients accepted hsct was 645 (455-1957) days after hsct. all of the patients with hbv reactivation have been controlled with nucleos(t)ide analogues drugs, and 5 of them achieved reverse seroconversion which detect persistent anti-hbsag antibodies in their bodies. conclusions: the anti-hbsag antibodies negative and anti-hbc positive patients have the highest risk of hbv reactivation after hsct in resolved hbv patients. the anti-hbsag antibodies play a protective role in resolved hbv patients receiving hsct. we recommend not prophylactic anti hepatitis b virus in hbsag negativity and anti-hbsag antibodies positive patients following hematopoietic stem cell transplantation. disclosure: nothing to declare methods: 2230 allo-hscts performed between 1997 and 2016 for acquired bone marrow failure (70.6%) or hemoglobinopathies (29.4%), with bm±cb (75.8%) or pb±cb+bm (24.2%) as a stem cell source were included in this retrospective registry megafile idwp ebmt study. results: demographics: the median age of recipient was 17.7 years (range: 0.1-78), and 50.8% were children. 79.0% recipients and 75.4% donors were ebv-seropositive. 67.8% had hsct from a matched family donor, 4.6% from a mismatched family donor, and 27.6% from an unrelated donor. t-cell depletion was performed in vivo in 82.2%, and ex vivo in 6.6% patients. conditioning regimen was myeloablative in 63.7%, ric in 36.3%. median follow-up was 4.7 years (95% c.i. 4.3-5.0). transplant out-comes: ebv-seropositive recipients in comparison to ebv-seronegative recipients had lower os (85.4% vs 88.4%, p=0.035), and higher nrm (10.0% vs 6.4%, p=0.018). no other significant differences were found for: ri, rfs, and acute or chronic gvhd with respect to ebv pretransplant serostatus donor and/or recipient. multi-variate analysis: ebv serostatus as a risk factor did not reach significance, while a trend towards higher risk of development of cgvhd (hr=1.31; 95%ci 0.97-1.78; p=0.081) and better survival (hr=0.78; 95%ci 0.59-1.04; p=0.087) in allo-hsct from ebv-seropositive donors. allo-hsct in ebv-seropositive recipients had a trend towards lower risk of development of cgvhd (hr=0.75; 95%ci 0.56-1.02; p=0.066). when 4 subgroups (r-/d-, r-/ d+, r+/d-, r+/d+ ebv serology) were analyzed, the ebv serostatus had no significant impact on os, rfs, ri, trm and development of acute or chronic gvhd. conclusions: allo-hsct from ebv-seropositive vs ebv-seronegative donors are at 31% higher risk of chronic gvhd in patients with non-malignant hematological disorders undergoing allo-hsct, however this difference is non-significant in multivariate analysis. disclosure: nothing to declare. results: twenty-eight (30%) pts (19 male, 9 female) were tested positive (group 1) for subtype a (n=4, 14%), b (n=18, 64%) or a (h1n1) (n=6, 21%) while 67 (70%) pts (32 male, 35 female) were negative (group 2). vaccination rate in group 1 (32%) was significantly lower compared to group 2 (51%, p=0.002). the median time after transplantation (790 vs 565 days), t-cell counts (349 vs 296/ μl), bcell counts (162 vs 135/ μl), igg-level (8,3 vs 7,6 g/ l), proportion of immunosuppressed pts (75% vs 63%), male/ female ratio was not significantly different between groups 1 and 2. within group 1 influenza subtypes were similarly distributed in vaccinated and not vaccinated pts (a 11% vs 16%, b 67% vs 63%, a (h1n1) 22% vs 21%). pts. with subtype b infection had higher levels of t-(482 vs 274/ μl) and b-cells (232 vs 108/ μl) and a longer follow up from sct (1610 vs 470 days) compared to subtype a / a (h1n1) infection but differences were not significant. conclusions: influenza could be proven in one third of all tested pts. dominance of b and a (h1n1) pdm0e9 subtype occurrence corresponded to the flu epidemic dissemination in the german population. the most important protective factor for outpatient sct recipients was influenza vaccination. disclosure: nothing to declare background: cmv infection is one of the most frequent complications after haplo. some risk factors are well known but the best strategy (prophylactic or preemptive treatment) to mitigate this complication is not still well defined. the primary endpoint in our study is to describe incidence and risk factors to develop cmv infection or disease in haplo. as secondary objective we analyzed efficacytoxicity of treatment and cmv related mortality. methods: we analyzed 60 patients who underwent haplo in our center between may 2012 and may 2018. all of them received ptcy (d+3 and d+4), tacrolimus and mycophenolate as graft versus host disease prophylaxis. a preemptive therapy based on viral load was applied. treatment was started when >1000 ui/ml of cmv were detected in one determination or >500 ui/ml in two consecutive determinations. cmv analyses were made in plasma using cobas pcr technique® and positive viral load cut-off point was 137 ui/ml. the cmv viremia was determined weekly until d+100 and then every two weeks until immune reconstitution. results: the cmv infection and disease incidence at d +100 was 61.3% (38 episodes) and 19.4% (12 episodes), respectively. cmv disease was digestive (n=8), pulmonar (n=2), neurologic (n=1) and disseminated (n=1). the median time to first cmv infection was 39.5 days (20-151). thirty-six patients had at least one episode of cmv infection: 24 of them (66.7%) had one episode, 7 (19.4%) had two episodes and 5 (13.9%) had 3 or more episodes, respectively. only pre-transplantation cmv status was significantly associated with cmv infection (p< 0.001). risk factors are shown in image 1. the median viral load in first cmv infection and disease was 8314 ui/ml (542-51158) and 24842 ui/ml (271-126279), respectively (p=0.02).the median counts of cd4 lymphocytes at d+100 in cmv infection and disease were 262/mm3 and 120/mm3, respectively (p=0.09). preemptive therapy for the first 2 episodes of cmv infections (n=48) was valganciclovir (58,3%), ganciclovir (35.4%) or foscarnet (6.3%), reaching a complete viral load clearance in 77%, with a median time to response of 19.2 days (6-34) and a median treatment duration of 21 days (2-39). grade iii-iv toxicity (mainly hematologic) was observed in 55.6% (n=28), 30% (n=17) and 16.7% (n=3), respectively. three patients had an ul54 mutation, one of them with clinical and microbiological resistance to the 3 mentioned drugs. three patients (5%) had a graft failure secondary to cmv infections. five patients (8.3%) died as consequence of cmv infection: 3 before d+100 secondary to cmv disease (2 pulmonar, 1 disseminated) and 2 after d+365 due to graft failure and infectious complications. with a median follow up of 12.5 months, overall survival at 18 months for patients who had cmv infection was 58.9% compared to 66.5% for those who had no infection (p=0.08). conclusions: a high incidence of cmv infection in haplo with ptcy was shown in our series and it contributed to mortality in 8.3% of patients. only cmv status (d-/r+ and d+/r+) was significantly associated with higher risk of infection. identification of high risk patients and new prophylactic and treatment strategies may improve these results. disclosure: nothing to declare. methods: consecutive patients admitted at the sct unit between january-18 to november-18 were reviewed. only first admission was analysed. screening consisted of rectal and perineal swap on admission and weekly until discharge. in case of detection of mdro, patients were isolated and infection control strategies were applied. results: 67 patients were analysed, median age 53 years (18-70). 65% were male (n=44). median duration of hospitalization was 30 days(16-133). 293 swabs were performed, with a median of 4 swaps/patient (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) . patient characteristics are shown in table 1 . 60 patients (90%) spiked fever in a median of 9 days after admission . 13% (n=9) had previous documented mdro colonization. median neutrophil engraftment was 15 days (95%ci 14-15), in 24% patients (n=16) of patients had a positive screen: in 8 (50%) patients at baseline and in 8 (50%) patients were detected for the first time beyond baseline screen. cumulative incidence of colonization at 7 days was 10.4% (95%ci 3.2-17.6), at 15 days 18% (95%ci 8.2-27.8), and at 30 days 19.5% (9.9-29.1%) (figure 1 ). mdro identified were: 8 with extended-spectrum beta-lactamases producing e. coli (esbl-ec), 4 multidrug-resistant pseudomonas aeruginosa (mr-ps), 3 vancomycin resistant enterococci (vre) and 1 patient with carbapenemaseproducing (cp) citrobacter freundii. 7/16 colonized patients developed mdro infection (44%): 4 patients mr-ps, site of infection was 2 urinary tract infection (uti), 1 urethritis, 1 genital ulcer. two patients were treated with ceftolozane/ tazobactam, 1 with meropenem+amikacin;2 patients esbl-ec both uti treated with meropenem; 1 patient cpcitrobacter freundii uti treated with ceftazidime/avibactam. in 75% patients (12/16) antibiotic treatment at febrile episode was guided by positive screening. no mdro related icu admission or mortality was observed. in 76% patients (n=51) background: hepatitis e virus (hev) can cause chronic infection and liver cirrhosis in immunocompromised individuals. there is limited data on hev infections in patients undergoing allogeneic hematopoietic stem cell transplantation (hsct). the aim of this study was to investigate the frequency and clinical importance of hev in a swedish cohort of hsct recipients. methods: we analyzed serum samples from 262 hsct patients (241 adults and 21 children), collected 6 months after hsct. hev igg and igm were detected by elisa (dia.pro®), hev rna by reverse transcriptase pcr, and quantification of hev rna was performed by digital pcr. in all patients, who were positive for hev-rna and/or serology at 6 months, also samples collected at the time of hsct from both the patients and their donors were analyzed. in the hev rna positive patients, additional samples were analyzed to determine the duration of viremia. three hev rna negative controls were selected for each case of hev infection, matched for age, diagnosis, conditioning regimen and donor type. results: hev rna was detected in 8/262 (3.1%) patients. in three of the patients hev rna was positive during a period of 3-8 months, and two of these patients were infected already at the time of hsct. in five patients hev-rna was positive, at a low level, only at 6 months. 11/262 (4.2%) patients had detectable hev igg and/or igm, whereof eight patients were hev rna negative. in 4/8 (50%) patients with hev infection (hev rna positive) alanine aminotransferase (alt) was > 3 upper limit of normal (uln), in 1/8 (12.5%) patients > 1.5 uln, and in 3/8 (37.5%) patients alt was normal, at 6 months after hsct. bilirubin was elevated > 1.5 uln in 1/8 (12.5%) patients, and > 3 uln in no patient at 6 months after hsct. two patients died with ongoing signs of hepatitis and hev rna detected in blood. one of them developed acute liver failure, at the time interpreted as drug toxicity, and died of multi-organ failure. the other patient died of unrelated causes. the remaining six patients had cleared the infection at 7-24 (median 8.5) months after hsct. active gvhd was present at 6 months after hsct in 5/8 (62.5%) patients with hev infection, involving the liver in 3 of these patients. corticosteroid treatment was ongoing in 6/8 (75%) patients; the mean dose during the 14 preceding days was > 0.5 mg/kg in 1/8 (12.5%) patient, 0.25-0.5 mg/ kg in 3/8 (37.5%) patients, and < 0.25 mg/kg in 4/8 (50%) patients. hev infection correlated to elevated alt > 1.5 uln, or 8.3 p=0 .02) and > 3 uln, p=0 .02) at 6 months, but not at 3 months, after hsct, compared to hev rna negative controls. conclusions: hev infection was detected in 3.1% of patients tested at 6 months after hsct and was correlated to abnormal alt. spontaneous clearance was common but one patient died in acute liver failure, where hev may have contributed. hev infection is a differential diagnosis in patients with elevated alt 6 months after hsct. disclosure: nothing to declare monitoring of t-cell responses to viral-coded antigens in pediatric patients receiving tcrαβ-depleted haplo-hsct followed by bpx-501 cell administration background: αβ t-cell-depleted haplo-hsct is an effective option for children with hematological disorders in need of an allograft. however, recovery of adaptive immunity is impaired in these patients. thus, in order to accelerate immune reconstitution, we developed a novel approach based on post-transplant infusion of a titrated number of donor t cells, transduced with the suicide gene inducible-caspase-9, ic9 (bpx-501 cells, sponsor bellicum pharmaceuticals®; nct02065869). we previously reported on immune recovery of 108 children transplanted at our institution, showing that bpx-501 cells infused after αβ t-cell-depleted haplo-hsct expand in-vivo and persist over time, contributing to fasten adaptive immunity recovery (merli, ash2017). here, we report the results of lymphoproliferation assay to viral-encoded antigens to assess tcell function in patients transplanted with this approach. methods: we evaluated 142 children, 78 male and 64 female. median age at transplant was 5.7 years (range 0. [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] . patients had either malignant (69 children) and nonmalignant (73) disorders. no patient was given any posttransplant graft-versus-host disease prophylaxis. nine children were enrolled in the phase i portion of the trial consisting of 3 cohorts receiving escalating doses of bpx-501 cells. the remaining 133 patients (phase ii portion) received the recommended dose of 1x10 6 bpx-501 cells/kg identified in phase i. bpx-501 cells were infused at a median of 17 days post-hsct (range 10-82). antigendriven activation of peripheral mononuclear cells was evaluated by lymphoproliferation assay with 3 h-thymidine pulsing at d+4 and harvesting at d+5. stimuli included pha or cmv, ebv and adv whole viral lysate. results are given as stimulation indexes (si, cpm stimulated sample/cpm unstimulated control). thresholds for positive response were arbitrarily set at si>3 for viral-encoded antigens and at si>5 for mitogenic stimulation with pha. fractions of responders are indicated in the figure. results: patients were analyzed from d+30 to d+720 post-hsct. pha responders (a) increased to 80%, while cmv (b), ebv (c) and adv (d) responders were 61%, 52% and 61% at 2 years after haplo-hsct. responses to ebv and adv antigens were slightly delayed but improved over time. responses to pha and to cmv (e,f) were analyzed in the cmv-reactivating and cmv-non reactivating groups (cmv-yes/cmv-no). significant differences in pha response were observed at d+60 and d+270. moreover, increased cmv responses were observed in cmvreactivators at d+270, d+360 and d+720, with approximately 100% of responders at d+720, as opposed to cmvnon reactivators which comprised 50% responders. neither primary disease, age nor tbi during the conditioning regimen influenced proliferative capacity of the two subgroups (not shown). conclusions: we showed a rapid recovery over time of t-cell function after αβ t-cell-depleted haplo-hsct followed by bpx-501 cells administration. when patients were grouped according to cmv reactivation (previously demonstrated as a strong driver of immune reconstitution), a significant difference in the number of responders among the patients experiencing viral reactivation was observed using the cmv lysate only but not the immunodominant pp65 protein (not shown), suggesting that other viral antigens account for increased t-cell responses. results of t-cell function after bpx administration complements the phenotypic data we already reported. clinical trial registry: nct02065869 disclosure: nothing to declare. background: cytomegalovirus (cmv) is associated with significant morbidity and mortality in allogeneic hematopoietic cell transplantation (allo-hct) patients (pts). cumulative incidence of cmv infection in high-risk patients such as cd34-selected or haploidentical hct have been reported as high as 61.8-84.5% and 53-81%, respectively. letermovir (ltv) was approved in 11/2017 for prophylaxis (ppx) in cmv-seropositive recipients (r+) of allo-hct. since 12/2017, ltv ppx was implemented at our center for both primary and secondary ppx. we report our real-world experience. methods: adult cmv r+ allo-hct pts who initiated ltv as primary and/or secondary prophylaxis were identified between 1/1/2018 and 6/30/18. cord blood transplants were excluded. the primary outcome was the incidence of clinically significant cmv infection (cmv viremia requiring preemptive treatment or cmv disease). pts were followed through 9/2018. results: 53 pts initiated ltv. 69.8% pts were at high risk for cmv reactivation and disease (primarily ex vivo t-cell depleted hct [n = 18; 34%] or haploidentical t-replete hct [n = 12; 22.6%]). the most common indication for hct was acute myeloid leukemia (n = 16; 30.1%) and the majority of patients received myeloablative conditioning (n = 34; 64.2%). 39 pts (73.5%) received ltv as primary ppx after hct, with a median day of ltv initiation of d+7 (range d+7─d+40). at ltv initiation, 34 pts had an undetectable cmv dna, and 4 had cmv < 137 iu/ml. clinically significant cmv infection requiring preemptive treatment occurred in 2 of 39 pts (5.1%). one patient was treated with valganciclovir (vgv) for persistent cmv < 137 iu/ml and received ltv as secondary ppx. a 2nd patient developed persistently detectable cmv (< 137 iu/ ml) and breakthrough cmv viremia with a mutation in ul56 at site c325yltv successfully treated with vgv. the median duration of primary ltv ppx was 116 days (54-221), with primary ppx continuing beyond 14 weeks post hct in 29 pts. the median additional follow-up in patients who discontinued ltv was 40 days (0-154), without clinically significant cmv infection to date. an additional 14pts (15 pts overall; 28.3%) received ltv as secondary ppx after cmv pre-emptive therapy. the median duration of secondary ltv ppx was 127 days (18-270), with no reactivation. ltv was not discontinued due to toxicity or intolerance in any patient. cmv outcomes are summarized in figure 1 . all-cause mortality for the 53 pts over the observational period was 11.3%. conclusions: primary ltv ppx significantly reduced cmv reactivation, and high-risk patients may benefit from extended prophylaxis. in patients who received preemptive therapy for cmv, use of secondary ppx showed no recurrent cmv reactivation. ltv is well tolerated. additional studies are needed to determine optimal ppx duration and to clarify role of secondary cmv ppx in high-risk allo-hct. the future standard of care will likely include extended primary ppx and secondary ppx and result in decreased morbidity and mortality associated with cmv. disclosure: andrew lin -nothing to declare, molly a. maloy -nothing to declare, valkal bhatt -nothing to declare, lauren derespiris -nothing to declare, meagan griffin -nothing to declare carmen lau -nothing to declare, anthony j. proli -nothing to declare, juliet barker -angiocrine bioscience , letermovir primary prophylaxis (pp) has been shown to reduce clinically significant cmv infection with a favorable safety profile. letermovir pp will improve the outcome of seropositive patients. however, patients who did not benefit from pp and experienced > 1 cmv episode (infection or disease) after hct may be candidate to secondary prophylaxis (sp). indeed half of them will have >1 recurrent episode after pre-emptive treatment (pet). letermovir is available since november 2017 as part of the french early access program for pp and sp. we report the outcome of patients who benefited from letermovir sp in the context of this program. methods: letermovir is granted, in a restrictive manner, by the french drug agency (ansm) on a case-by-case basis for prophylaxis of cmv episode, in cmv-seropositive adult allogeneic hct recipients. sp patients should have a negative baseline cmv pcr, have already experienced >1 cmv episode, in the context of a potentially harmful pet according to physicians. planned letermovir daily dose was 240 mg in case of concomitant cyclosporine and 480 mg otherwise. all patients were routinely screened by blood or plasma cmv pcr. results: between november 2017-july 2018, 57 patients received letermovir in the early access program, 22 for pp, and 35 for sp. among the 35 sp patients, 6 had previous cmv disease (gut: 5; cns: 1). mean age was 55±13 years, m/f ratio was 22/13. the sp cohort included one cord blood and 10 haplo-identical hct. main diagnoses were acute leukemia (46%) and myelodysplastic syndrome (26%). the conditioning regimen was myeloablative in 47% and included atg in 61%. based on available data (6 missing data, md), previous gvhd was present in 22 (76%) patients, and active at letermovir initiation in 17 (59%). thirty two (91%) patients were planned to receive immunosuppressants. donor's cmv serology was negative in 12/15 (80%) (20 md). at baseline, cmv pcr was detectable in 1/35 patients. letermovir was initiated a median of 195 days (iqr: 154-308) after transplant for a mean duration of 112 ± 47 days. only one (3%) patient developed cmv breakthrough. the median follow-up from letermovir initiation was 103 days. among the 57 patients exposed to letermovir prophylaxis, two patients permanently discontinued because of letermovir-related adverse events (acute gvhd and nephropathy for one, loss of appetite, pruritus, diarrhoea and weight loss for the other); two deaths occurred with no causal relationship to letermovir. data were consistent with the known safety profile of letermovir. conclusions: letermovir is or will be soonly available in most european countries for cmv prophylaxis in hct recipients. pending its routine use, letermovir used as sp was well tolerated and effective, with only 1/35 patients developing a breakthrough infection. in this high-risk population for cmv recurrence, letermovir may provide a safe bridge between pet and specific immune reconstitution, pending tapering or discontinuation of immunosuppressants. whether sp may improve survival deserves further studies. disclosure: thierry allavoine is a former employee of msd france, nathalie benard and amir guidoum are employees of msd france, marion masure is an employee of icta pm, sophie alain and catherine cordonnier have participated in advisory boards and have been members of the speaker bureau of msd. ibrahim yacoub-agha has received honoraria from msd, other authors: nothing to declare p407 real-world data on letermovir prophylaxis for cytomegalovirus reactivation after allogeneic hematopoietic cell transplantation: a single center experience patrick derigs 1 , maria-luisa schubert 1 , paul schnitzler 1 , carsten müller-tidow 1 , peter dreger 1 , michael schmitt 1 1 heidelberg university hospital, heidelberg, germany, background: reactivation of cytomegalovirus (cmv) still contributes substantially to morbidity and mortality after allogeneic hematopoietic cell transplantation (allohct). recently, letermovir became available as the first drug approved in europe for prophylaxis of cmv reactivation in seropositive patients who have undergone allohct. letermovir is neither myelo-nor nephrotoxic, and significantly reduced the incidence of cmv reactivation in a pivotal phase iii trial (nejm 2017; 377:2433) . therefore we adopted letermovir prophylaxis according to the label as standard policy in our institution: in seropositive recipients letermovir was initiated after engraftment and continued until day +100 or cmv reactivation. the aim of the present study was to investigate if the favorable trial results could be reproduced under real-world conditions. methods: the study cohort consisted of the first seropositive 35 patients who received letermovir prophylaxis at our institution (between march and august 2018). these were compared with a control cohort transplanted between august 2017 and march 2018 before the advent of letermovir. study and control cohorts were matched for cmv donor/recipient sero-status, underlying disease and donor type source of stem cells and application of atg. cmv viremia was monitored by a quantitative pcr twice a week during the inpatient period and weekly thereafter. patients reactivating cmv prior to engraftment were not considered as event in both groups. results: no major side effects of letermovir intake were observed. with altogether 5 reactivation events, the cumulative rate of cmv reactivation on day +100 was 14% (95%ci 1-45%) in the letermovir cohort and thus significantly lower than in the control group (20 events, 58% (95%ci 42-71%); hr 0.23 (0.10-0.51); p=0.0003). the median time to reactivation was 53 days for the control group and not reached for the letermovir group. the cumulative number of days on valganciclovir before d +100 was 151d for the 35 letermovir patients vs 689d for the 35 control patients. there were no hospitalizations for foscavir administration in the letermovir group compared to 5 hospitalizations in the control group. there were 2 deaths before d +100 in the letermovir group (one pd, one nrm) and 3 deaths in the control group (all pd). conclusions: this observational study confirms the safety and efficacy of letermovir for the prophylaxis of cmv reactivation in seropositive patients after allohct in a real-world setting. our results are in good concordance with the phase iii trial. although letermovir appeared to reduce the need for therapeutic valganciclovir and foscavir tremendously, larger samples with longer follow-up are needed to assess the impact of letermovir prophylaxis on non-relapse and overall mortality as well as on resource consumption. background: cmv viremia occurs in 40%-80% of cmv r + hct recipients. pet use has reduced the risk of cmv end-organ disease (eod) and associated mortality; however, pet use may lead to substantial antiviral use and healthcare resource utilization. limited real-world data are available on the outcomes of pet. therefore, we aimed to examine cmv outcomes (eod, resistance), cmv-related mortality by day (d)180 and healthcare resource utilization between pet and no-pet groups among cmv r+ recipients undergoing first hct. methods: we conducted a retrospective cohort study of adults, cmv r+ recipients of first peripheral blood or marrow allograft at mskcc identified from march 2013 through december 2017. data was extracted from electronic medical records and hct databases. cmv+ recipients were monitored weekly by quantitative pcr assay starting on d14 through d180 post hct. use of antiviral therapy for cmv viremia defined pet. high cmv risk (hr) comprised t-cell depleted (tcd) hct by cd34+-selection regardless of donor hla match or conventional hct from mismatched or haploidentical donors; low risk (lr) included conventional hct from matched related donors. cmv eod was scored by standard criteria. cmv resistance mutations were confirmed by sequencing (viracor-eurofins). length of stay (los) for hct admissions and readmissions were identified through d180. stratified analyses were performed to examine outcomes by pet use and cmv risk. background: in a phase iii randomized, double-blind, placebo-controlled study of cmv-seropositive post-hsct recipients, letermovir prophylaxis significantly reduced the incidence of clinically significant cmv infection through week 24. the objective of this research was to assess the impact of cmv prophylaxis on rates of rehospitalization in adult cmv seropositive allogeneic hsct recipients from the letermovir phase 3 clinical trial. methods: rehospitalization was recorded as an exploratory endpoint in the clinical trial at end of treatment (week14), time of primary endpoint (week24) and through an extended follow-up period (week48). cmv-related rehospitalization was assessed in the trial. prespecified analyses describe the observed rates of rehospitalization for the letermovir and placebo groups at the specified times. fine-gray cumulative incidence function(cif) regression models were used to explore the rate of all-cause, and cmv-related rehospitalization accounting for the competing risk of mortality. a multiple linear regression model was used to describe the cumulative length of stay (los) for all-cause rehospitalizations that occurred through week48 (excluding time of initial transplant stay). results: observed rates of all-cause rehospitalization were lower for the letermovir group compared to placebo at end of treatment (36.6%vs.47 conclusions: letermovir was shown to significantly reduce the rate of clinically significant cmv infection in a placebo-controlled randomized clinical trial. these analyses suggest that there is also a reduction in the rate and cumulative days of rehospitalization. this trial was not sufficiently powered to detect differences in this exploratory endpoint. nonetheless, these data provide valuable insights into the economic burden of cmv. real world data and findings from future clinical trials are needed to better understand the nature of the association between cmv and rehospitalizations. clinical methods: all consecutive patients with hematologic disorders who received hsct at our center between january 2013 and august 2018 were included. among the 278 evaluable patients, 172 received levofloxacin as antibacterial prophylaxis (group a) while 106 did not receive any fq prophylaxis (group b). baseline characteristics were similar in the two groups, except for the number of patients with advanced disease (34% in group a and 47% in group b, p 0,042). median duration of neutropenia was 16 days (range 9-44) in group a and 14 days (range 4-31) in group b. a positive rectal swab for carbapenem-resistant enterobacteriaceae (cre) was detected in 3 patients in group a and 8 patients in group b. results: overall, bsi was detected in 58 patients (20,9%), 29 (16,9%) in group a and 29 (27,4%) in group b (p=0,048). the median onset of bsi was 8 days post transplant (range 0-28), without significant differences between the two groups. in univariate analysis, fq prophylaxis (or 0,54; 95% ic 0,30-0,97) and bone marrow stem cell source (or 2,08; 95% ic 1,05-4,12) were significant factors associated with the risk of bsi. gramnegative bacteria accounted for 44,8% (n=13) of bsi in group a and 65,5% (n= 19) in group b, and gram-positive bacteria for 48,3% (n= 14) of bsi in group a versus 27,5% (n= 8) in group b, without statistically significant differences (p = 0,16). polymicrobic bsi were 6,9% (n=2) in group a and 6,9% (n=2) in group b. mdrgram negative bsi were detected in 4 patients (14%) in group a and in 6 patients (20,7%) in group b (overall, 2 cre, 7 esbl producing enterobacteriaceae and 1 mdr-pseudomonas). death attributable to bsi occurred in 6 of 58 patients (10,3%); 5 of these patients did not receive fq prophylaxis, but 2 of them had both a pre transplant kpc colonization and active disease at transplant. neither antibacterial prophylaxis (p = 0,98) nor bsi (p = 0,4) had a significant impact on overall survival (os). conclusions: the preliminary data of our study show that fq prophylaxis is associated with a reduced incidence of bsi, in particular gram-negative infections, with no impact on os. the limitations of our study may be the different group sizes and the retrospective nauture of the study. whether antibacterial prophylaxis should be avoided in the pre-engraftment period in still a matter of debate and needs to be evaluated in larger prospective studies. disclosure: nothing to disclose. gillen oarbeascoa 1 , nieves dorado 1,2 , laura solan 1,2 , rebeca bailen 1,2 , pascual balsalobre 1,2 , carolina martinez-laperche 1,2 , ismael buño 1,2 , javier anguita 1,2 , jose luis diez-martin 1,2,3 , mi kwon 1,2 1 hospital general universitario gregorio marañón, hematology, madrid, spain, 2 instituto de investigación sanitaria gregorio marañón, madrid, spain, 3 universidad complutense de madrid, madrid, spain background: incidence and outcome of invasive fungal infection (ifi) are not well characterized in the setting of peripheral blood, non-manipulated haploidentical stem cell transplantation with postransplant cyclophosphamide (haplosct). the aim of the study was to analyze incidence and risk factors of ifi in patients who underwent haplosct at our institution. methods: 132 consecutive patients who underwent peripheral blood haplosct with postransplant cyclophosphamide between 2011 and 2017 at our centre were reviewed. ifi was classified according to eortc definitions. proven and probable ifi were included. results: patients´characteristics are shown in table 1 . primary antifungal prophylaxis was performed with micafungin from day -1 until oral intake, followed by posaconazole until day +35. patients on steroid treatment for gvhd received prophylaxis with micafungin or posaconazole. 92% of patients obtained neutrophil engraftment. twenty-two episodes of ifi were observed in 20 patients: 10 proven and 12 probable, with a cumulative incidence of ifi of 17% at 500 days. most commonly isolated organism was aspergillus spp (n=5), followed by candida spp (n=4: 1 c. kruseii and 3 c. parapsilosis), and fusarium spp (n=2). isolated cases of inonotus spp, mucor spp and trichosporon ashii were observed. pulmonary involvement was the most frequent clinical presentation (n=10), followed by fungemia (n=5: 4 candidemia, 1 trichosporon ashii) and skin-pulmonary involvement (n=2). among patients with lung involvement, 10 showed probable ifi: 5 with elevated serum galactomannan and 3 positive galactomannan in bronchoalveolar lavage (bal). there were 2 patients without galactomannan, one with a positive bal culture for penicillum spp and the other with an aspergillus spp. median time to ifi diagnosis was 21 days. thirteen cases were diagnosed in the pre-engraftment period, 4 after engraftment and 5 cases after day +30. among patients with late ifi, median time to development was 220 days. all of them were associated with gvhd (3 grade iii-iv acute gvhd and 2 moderate/severe chronic gvhd). ifi outcome was favorable in 13 out of the 22 ifi. treatment was liposomal amphotericin b in 7 cases, voriconazole in 4 and combined treatment (amphotericin b and azole) in 6. there were 7 ifi related deaths, with a cumulative incidence of ifi related death of 6.4%. prior transplant (or 4.5, p< 0.01), particularly allohsct was associated to ifi development (or 8.2, p< 0.01). patients with previous allohsct presented ifi mainly from molds: 3 aspergillus, 2 fusarium, inonotus, trichosporon and mucor. there were also 2 candidemia episodes. no other factors were significantly associated to ifi occurrence. conclusions: in our experience, cumulative incidence of ifi in the setting of haplosct with posttransplant cyclophosphamide was similar than observed in previous studies in allosct. having received a previous sct, especially allosct, was the most significant factor related to ifi development. this high risk population should be closely monitored and could benefit from prophylaxis with azoles. disclosure: nothing to declare. methods: rsv infection was diagnosed in nasal wash (nw) or bronchoalveolar fluid (bal) by dfa (millipore, usa) or pcr (seeplex, seegene, kor). urti and lrti were defined according to ecil-4 guidelines. death from all causes was assessed within 90 days after rsv infection and was attributed to rsv if the patient had persistent or progressive rsv infection with respiratory failure at the time of death. neutropenia and lymphocytopenia were defined as an absolute neutrophil count (anc) < 500/ul and absolute lymphocyte count (alc) < 200/ul, respectively. results: median number of confirmed rsv infections per year was 12, ranging from 5 to 34. an outbreak of rsv was detected in 2017, possibly due to a lack of compliance with contact precautions in the unit. median patients' age was 26 years and time to rsv infection was day 80 (-11 to 1837). twenty-three patients (pts) had received an autologous transplantation (17.2%) and 111 were allogeneic hsct recipients (82,8%). median time to engraftment was 15 days, ranging from 10 to 27 days. at rsv diagnosis, 108 pts presented with urti (80.6%) and 26 with lrti (19.4) . surprisingly, around 18% of the auto hsct recipients had rsv pneumonia at diagnosis. variables significantly associated with lrti at diagnosis were mud hsct (no/ yes, or 0.42; ci95 0.20-0.89); anc < 500/ul (or 2.75; ci95 1.01-7.45); alc < 200/ul (or 3.25; ci95 1.12-9.45); and recent or pre-engraftment hsct (no/yes, or 0.38 ci9 0.14-0.98). among the 108 pts with urti at diagnosis, 19 progressed to lrti (17.6%). forty-four of the 134 pts died (32.8%) and mortality rate was significantly higher in pts with lrti in comparison with pts with urti (53.8% versus 27.8%, p=0.011). death was attributed to rsv in 11 of the 44 pts who died (25%). conclusions: autologous hsct recipients are also at risk of lrti caused by rsv. risk of rsv lrti is higher in mud hsct, infection acquired pre-engraftment or early after hsct, and low neutrophil and lymphocyte counts. continued education is necessary to sustain compliance to contact precautions in hsct units. disclosure background: measles is a life-threatening infection after allogeneic hct. due to the decreased coverage of vaccination in many countries, the disease reappears, increasing the risk of outbreaks worldwide. allogeneic hct recipients have been shown to be seropositive for measles in roughly 40-50% of the cases 3 years after transplant. however, these data were obtained before the 2000's from hct populations mainly conditioned with myeloablative (ma) regimens. our aim was to assess measles immunity before considering vaccination in a cohort of hct survivors including patients conditioned with reduced intensity (ric) or non-ma regimens. methods: allogeneic hct adult recipients who had not been vaccinated for measles since hct were routinely screened for measles immunity. measles igg titers were determined with a chemiluminescence immunoassay (liaison measles igg kit, liaison xl analyser, diasorin, italy). patients were considered to be seropositive if the igg titer was > 16.5 ua/ml. risk factors for seropositivity were analyzed. qualitative variables were described as numbers (%) and compared using the chi-2 test or fisher exact test as appropriate. quantitative variables were described as median or mean (range) and compared using the kruskall-wallis test. ors were estimated separately for factor yielding a p-value < 0.20 in the univariate analysis using logistic regression models. results: eighty-six patients, transplanted 1.5 to 38 years (mean: 13,5 years) ago, were included. the mean age was 53 years (range: 21-79), the sex ratio m/f: 0,5. the underlying diseases were acute leukemia: 49 (57%), myelodysplastic syndrome: 5 (6%), lymphoproliferative diseases: 17 (19.5%), myeloproliferative neoplasms: 11 (13%) and non-malignant diseases : 4 (4.5%). the hct was performed from an hla-identical donor in 52, an unrelated donor in 30, and a cord-blood in 4. conditioning regimen were ma in 48 (56%), ric in 20 (23%) and non-ma in 18 (21%) patients no patient had experienced measles or had received measles vaccination since transplant. fifty-seven of the 86 (66%) patients were seropositive for measles. measles seropositivity was not associated with conditioning regimen, patient age at transplant, patient age at time of assessment, donor age at transplant, lymphocyte count or gammaglobulin levels, or type of transplant (hlaid. vs others) measles vaccination before transplant or previous measles before transplant. the only parameters significantly associated to seropositivity were absence of previous gvhd (any type or severity, p=0,033 or 0,31 [0,10-0,94]), and absence of previous extensive chronic gvhd (or 0, 28 [0, 87] p0,027). conclusions: sixty-seven percent of allogeneic hct are seropositive for measles at a median of 7 years after hct before vaccination. the only risk factor strongly associated with seronegativity is extensive chronic gvhd. in patients background: cytomegalovirus (cmv) reactivation is a frequent complication after hematopoietic stem cell transplantation (hsct). extracellular vesicles (evs) have emerged as a promising new category of biological biomarkers in different scenarios, including inflammation, tissue damage, cancer and viral infections. we recently reported on the potential use of serum evs as biomarkers of agvhd (lia g. et al. leukemia (2018) 32, 765) . here, we investigated the potential correlation of cmv reactivation with plasma evs in post-transplant cyclophosphamide (ptcy) haploidentical-hsct (haplo-hsct). methods: plasma samples were collected after mononuclear cell separation at given time-points (pre-transplant, on day 0, 3, 7, 14, 21, 28, 35, 45, 60, 75 and 90 after haplo-hsct) and evs were extracted by a protamine-based precipitation method and their concentration and dimension were characterized by nano-tracking particle analysis (nanosight). after extraction, evs were analyzed by flowcytometry (guava easycyte flow cytometer) with a panel of 14 antibodies (cd44, cd138, cd146, krt18, cd120a, cd8, cd30, cd106, cd25, cd26, cd31, cd144, cd86, and cd140a). results: thirty-two patients with hematological malignancies underwent haplo-hsct between 2011 and 2015. cmv reactivation was observed in 20/32 (62,5%) and occurred at a median of 50 (range: 10-275) days after transplant. preliminary analysis (17/32 patients) showed that cd140a fluorescence (platelet-derived growth factor receptor-α or pdgfr-α), cd30 fluorescence (ki-1 antigen) and cd144 fluorescence (ve-cadherin) were associated with an increased risk of cmv reactivation (or 2.67 p=0.045; or 3.11 p=0.011; or 2.37 p=0.08), whereas cd31 (platelet endothelial cell adhesion molecule, pecam-1) concentration level was associated with a decreased risk of cmv reactivation (or 0.26, p=0.032). all these biomarkers showed a signal change before cmv reactivation (an increase with cd140a, cd30 and cd144, a reduction with cd31). (figure 1 ). conclusions: we observed a potential association of 4 evs membrane proteins with cmv reactivation: cd140a, cd30, cd144 and cd31. these proteins are crucial for endothelium and immune cells interaction. cmv can infect different cell types including endothelial cells (bentz gl. pnas 105 (14) 2008). moreover, cd140a (pdgfr-α) has been shown to function as an entry receptor for cmv expressing gh/gl/go complex (wu y. et al. plos pathog 13 (4) 2017). we plan to implement our analysis characterizing evs contents (mirnas) and will be applied to investigate other viral reactivations (e.g. epstein barr virus and human herpes virus 6). [[p414 image] 1. methods: to explore the value of cmv dna extracted from gi tissue for the diagnosis of cmv gastroenteritis, we retrospectively evaluated 71 patients, aged 17-67 (median 44.8 years) who received allo-hct from sibling(26), matched unrelated(40) or haploidentical donors(5), after receiving myeloablative (56) or reduced intensity conditioning(15). they all underwent endoscopy for gastrointestinal symptoms between 2012-2018. cmv dna from tissue samples and parallel blood samples were measured by q-pcr. positive cmv dna on the tissue was considered cmv gi infection.cmv gi disease was proven with the identification of cmv inclusion bodies or positive immunehistochemical staining using anti-cmv antibodies. results: overall, 91 endoscopic tests were performed (55 gastro-,36 colonoscopies) at a median of 73 days (iqr:145) post transplantation. symptoms included nausea, vomiting, diarrhea, abdominal pain and weight loss. cmv dna was positive in 41/91 tissue samples: median 536 copies/ml, range: 11-131x10^6. only half patients (22/41) had concurrent cmv viremia (plasma viral load>100c/ml). cmv gi infection was not correlated to the type of transplant, acute or chronic gvhd. gi cmv disease was documented by biopsy in 13 patients. cmv dna of the tissue, but not the plasma viral load, was a predictor of biopsy positivity (or: 1.6, 95%ci: 1.1-1.8, p=0.006). thirty-six out of 41 cmv dna positive patients received specific treatment for at least 10 days. symptoms resolved in 21/36 patients (60%) and the gi viral load was not a significant factor to predict cure. gi gvhd was diagnosed in 42/91 patients, among which 45%(19/42) with cmv dna positivity. median os was 453 days (95%ci: 297-608) for patients with cmv infection, similar to those without (median os: 890, 95%ci: 80-1699 days, p=ns). we studied separately endoscopies of the upper (55/91) or lower gi tract (36/91) . there was no significant relationship between cmv gastritis proven by biopsy and cmv dna levels in gastric tissue. however, the viral load of the colon was a predictor of cmv enteritis (or: 1.9, 95%ci: 1.9-3, p=0.007). the auroc of the q-pcr was 0.849 (95%ci: 0.659 to 1), the sensitivity was 85.7% and the specificity was 78.6% with a cutoff value of 370 copies/ml dna. conclusions: pathognomonic findings in the biopsy remain the gold standard for the diagnosis, especially for the upper gi tract. however, when the lower gi tract is involved, quantification of cmv viral load in the tissue may be a valuable tool to support the diagnosis. positivity of cmv dna of the gi tissue, in linearity to the cmv viremia, may guide to preemptive treatment for prevention of cmv disease . disclosure: nothing to declare background: clostridium difficile infection (cdi) is caused by cd overgrowth in antibiotic-disturbed intestinal microbiota. antibiotics targeting unselectively beneficial for t-regulatory cell formation strains of clostridiales may increase pro-inflammatory processes in the guts promoting or augmenting the development of graft vs. host disease (gvhd). the efficacy of cdi treatment has impact on the persistence of inflammation which might influence the alloreactive reactions. methods: we retrospectively and, from 2016, prospectively analyzed the data from 5 transplant centers concerning cdi occurrence, treatment efficacy, and gvhd development. the study included 77 patients with hematological malignancies who underwent allogeneic hematopoietic cell transplantation (allohct) between 2012-2018. results: median time to cdi was 14 days post-allohct with detection of both toxins a and b in 57% of cases. disturbance of intestinal microbiome was confirmed by a 59% rate of colonization with multidrug-resistant bacteria (mdrb). the cdi symptoms resolved with the negative toxins after the first line treatment in 76.6% of patients. the median time to remission and therapy duration was 8 and 10 days, respectively. fifteen therapeutic failures were observed after treatment with metronidazole (10), vancomycin (2) and a combination therapy (3) . eleven patients responded to second line treatment. thirty-seven (48%) patients died due to infections (17), relapses (10) and gvhd/infections (10). we noted recurrent cdi in 6 cases. eight patients died with active cdi. we observed occurrence or exacerbation of gvhd in 35 (44%) patients following cdi, including 25 cases with gut involvement (gi-gvhd). treatment with metronidazole and failure of the first line therapy increased the development or escalation of gi-gvhd (p= 0.03 and p< 0.001, respectively). the duration of cdi exceeding 10 days also had impact on the gi-gvhd incidence (p= 0.002). conclusions: 1. patients colonized with mdrb are at high risk of cdi. 2. high mortality due to infections and/or gvhd in patients with cdi. 3. due to lower efficacy and harmful immunomodulatory impact, metronidazole should not be the first line treatment in cdi post-hct. 4. emphasis must be put on fast cdi resolution to interrupt a vicious circle of the intestinal inflammatory processes. disclosure: nothing to declare establishing optimal preemptive cytomegalovirus therapy threshold post allogeneic hct in a patient population with high prevalence of seropositive status background: preemptive therapy (pet) for cytomegalovirus (cmv) reactivation post allogeneic hematopoietic stem cell transplantation (hct) was shown to decrease the incidence of cmv disease. however, the optimal pet threshold is unknown and there are significant toxicities associated with anti-cmv therapy. at our institution, we initiate pet at cmv dna titer above 1000 copies/ml (1560 iu/ml). our aim was to examine the efficacy of this approach including the incidence of spontaneous clearance in a population with high prevalence of cmv seropositive status. methods: after due irb approval, patients that underwent allogeneic hct were identified and records retrospectively extracted.cmv reactivation was defined as the first detectable viral titer post hct from plasma samples whereas clearance of viremia as the first date of two negative pcr values obtained at least 1 week apart. cmv monitoring was initiated post hct performed at least weekly during the first 100 days and every 2-4 weeks thereafter. a high sensitivity assay abbott realtime cmv was used with detection threshold of 20 copies/ml (31.2 iu/ml). analysis was computed using jmp v. 14.0.1 results: a. baseline characteristics: a total of 195 patients were identified and included with a median follow up of 18.1 (0.7-95.6) months. median age was 26 (14-63) years and 58% were male. indication for hct was for a malignant disorder in 77% of cases. the majority had a matched related donor (87%) and cmv igg was positive in both donor and recipient in 98% of cases. myeloablative conditioning was given to 117 (60%) and 109 (56%) received tbi. in vivo t-cell depletion was given to 76 (39%); atg in 39 (20%) and alemtuzumab in 37 (19%). b. cmv reactivation and pet: a total of 178 (91%) patients had a positive cmv pcr with median days to reactivation post hct of 17 ; 129 (66%) patients had peak cmv titer < 1000 copies/ml (low titer) whereas the remaining 49 (25%) had a peak titer ≥ 1000 copies/ml (high titer). patients with high titer were more likely to be older (p = 0.019), have malignant disease (p = 0.019), haploidentical or unrelated donor (p < 0.0001) and higher incidence of agvhd grade ii-iv (p = 0.003) as shown in the table. median peak titers for the low and high groups were 111 vs. 4638, respectively (p < 0.0001).120 (93%) patients with low titers cleared spontaneously with median time to clearance of 40 days (4-188), 1 (1%) received anti cmv therapy and the remaining died with active viremia (range 49-561 copies /ml) with active disease. one patient in the high titer group developed cmv disease. 2-year os and ci-nrm was 67.9% vs. 55.4% (p = 0.1) and 8% vs. 19.1% (p = 0.034) in the low and high titer groups, respectively. conclusions: cmv reactivation was high in this cohort however of low titer viremia in over 70%. a pet threshold of 1000 copies/ml (1560 iu/ml) appears desirable as it was associated with spontaneous clearance in almost all patients while minimizing treatment related toxicity. validation of these observations is warranted. background: the risk of pneumocystis pneumonia often warrants antifungal prophylaxis for recipients of blood and marrow or solid organ transplantation. however, complications such as myelosuppression, nephrotoxicity, and intolerance with the existing standard, trimethoprim/sulfamethoxazole (tmp/smx), may hinder or interrupt prophylaxis. rezafungin (rzf) is a novel echinocandin in development for prevention of invasive fungal disease caused by candida, aspergillus, and pneumocystis species in blood and marrow transplant patients. rzf has a favorable safety and tolerability profile and a low risk of drug-drug interactions. furthermore, the stability and pharmacokinetics of rzf allow for once-weekly dosing and broad distribution to the lung and other target organs. rzf was shown to prevent in vitro pneumocystis biofilm formation and to reduce the viability of mature biofilms. a previous prophylactic study was conducted using a broader range of rzf doses. in the current study, the efficacy of rzf was evaluated to better understand the minimum doses necessary to prevent pneumocystis growth in a mouse model. methods: c3h/hen mice were immunosuppressed (dexamethasone 4 mg/l in acidified drinking water) and then infected intranasally with p. murina (2 x 10 6 /50 μl). given the slow growth of p. murina, test agents were administered at the same time mice were inoculated to test for prophylactic efficacy. mice received intraperitoneal injections of either vehicle (control/steroid [c/s]), tmp/ smx 50/250 mg/kg/3x/week (wk), caspofungin 5 mg/kg once daily, or rzf 5 mg/kg or 0.5 mg/kg once daily, 1x, or 2x/wk. after a 6-week dosing period, mice were sacrificed and lung homogenates were processed for analysis to quantify the nuclei (trophic) and asci (cyst) forms of pneumocystis. prophylaxis efficacy was based on reduction of organism burden compared with c/s. nuclei and asci counts were log transformed and analyzed by anova; individual groups were compared by the student-newman-keuls t test. survival rates were compared using graphpad prism v5. results: all mice in the rzf groups had significantly reduced nuclei and asci burdens compared with the c/s group, and all but the lowest doses of rzf (0.5 mg/kg 1x or 2x/wk) worked as well as tmp/smx at reducing nuclei levels. similarly, all rzf groups except for the 0.5 mg/kg 1x/wk group showed reductions in asci levels comparable to that of tmp/smx. the survival rates were not statistically different between treatment groups. conclusions: rzf demonstrated potent in vivo efficacy for prophylaxis against pneumocystis in an in vivo mouse infection model at dose regimens much lower than the human equivalent phase 3 regimen. these data support the development of rzf for the prevention of invasive fungal infections including pneumocystis pneumonia. disclosure: melanie t. cushion: research funding (cidara therapeutics) taylor sandison: employee, stockholder (cidara therapeutics) alan ashbaugh: nothing to declare. yuhua ru 1,2 , ziling zhu 1,2 , yang xu 1,2 , suning chen 1,2 , xiaowen tang background: immunocompromising period following allogeneic hematopoietic stem cell transplantation (allo-hsct) may allow opportunistic pathogens to thrive and result in fatal complications. epstein-barr virus (ebv) infects more than 90% of chinese population, and its reactivation after hsct is one of the major concerns due to the increased risk of ebv diseases and post-transplant lymphoproliferative disease. with the development of infection prophylaxis and supportive care after hsct, demographic data on ebv reactivation post-hsct needs to be updated. methods: we retrospectively analyzed the data of patients who received allo-hsct between july 2011 and july 2014 in the first affiliated hospital of soochow university. quantitative pcr (q-pcr) was used to monitor ebv-dna load in peripheral blood dynamically. ganciclovir (pre-hsct) followed by acyclovir was given as viral prophylaxis. the treatment protocol for ebv reactivation consisted of tapering of immunosuppressive agents, antiviral agents (including ganciclovir and sodium phosphonatel), and rituximab for persistent positive patients. results: totally 890 cases from most of the provinces in china were enrolled (characterized in table 1 ), among whom ebv reactivation developed in 175 recipients. most reactivation events (95.4%) occurred in the first year post-hsct, with a peak of 113.8 incidence rates per 100 personyears at the second month. besides, more episodes of lateonset reactivation occurred in patients receiving grafts from haploidientical donors ( figure 1a ) . multivariate analyses revealed that the major impactors of ebv reactivation included atg as gvhd prophylaxis (p< 0.001), hlamismatched donor (p=0.001) and the appearance of chornic gvhd (p=0.042). cumulative incidence of ebv reactivation was low (2.9%) among 890 patients with no major risk factors, but increased to 11.7%, 27.3% or 41.9% with 1, 2, and 3 major risk factors, respectively ( figure 1b) . there was no statistical difference of overall survival between people with or without ebv reactivation (p=0.871). conclusions: we concluded that there are similar ebv reactivation impactors in chinese population compared to literatures, including atg use, hla-mismatched donor and the appearance of chronic gvhd. additionally, incidences of ebv reactivation increased significantly with the accumulation of risk factors. however, ebv reactivation had no impact on overall survival in current virus management protocol. disclosure: nothing to declare background: several studies have shown loss of diversity of the gut microbiome in association with significant gut injury following hematopoietic stem cell transplantation (hsct). prolonged broad spectrum antibiotic use further promotes loss of microbiome diversity and increases the risk of intestinal colonization by multi-drug-resistant (mdr) bacteria. aims of this study were to prospectively evaluate the overall changes in gut microbiome composition after hsct and differences in patients colonized by mdr bacteria and treated with carbapenems. methods: we performed a prospective observational study evaluating the gut microbiota of 20 hematological patients undergoing hsct, from admission (t0) through day +28 (t5). fecal microbiota was assessed by 16s amplicon-based sequencing. clinical, and microbiological data as well as fecal samples were collected every 7 th day from admission. results: one-hundred fecal samples were analyzed. overall, we found a progressive decrease of bacterial richness from t0 to t5, with a significant reduction of blautia, ruminococcus and dorea species, which are strictly associated with the production of short chain fatty acids (sca) (fig.1) . moreover, in the 30% (no.6) of patients who were colonized by esbl bacteria, we observed a significant reduction of clostridium spp and bifidobacterium species. as for antibiotic therapies, carbapenems were used as second line treatment of febrile neutropenia in 50% (no 9) of cases, usually associated with aminoglycosides. in patients treated with meropenem, a strong decline of blautia and ruminococcus species was observed. this finding suggests a correlation between carbapenem regimens and increase of pro-inflammatory bacterial strains in the gut. conclusions: our data support the hypothesis that loss of intestinal commensals that produce short-chain fatty acids may increase dysbiosis. moreover, for the first time we report significant and progressive alterations in the composition of blautia, ruminococcus and bifidobacterium species in patients treated with meropenem and colonized by esbl bacteria, respectively. our findings offer potential modifiable targets to reduce risk of colonization by mdr bacteria and to promote a carbapenem-sparing approach in the hsct setting. clinical background: cmv is associated with significant morbidity after allogeneic hematopoietic stem cell transplantation. strategies to prevent cmv-related complications include universal prophylaxis and preemptive therapy, more widely spread. antivirals used for cmv reactivation (cmv-r) produces major toxicities and costs. rate and characterization of cmv-r after haploidentical transplantation with post-transplant cyclophosphamide (haplo pt-cy) is scarce. our goal was to analyze cmv-r rate after haplo pt-cy, outcome, complications associated to therapy, and to identify risk factors. methods: one hundred haplo pt-cy transplants using peripheral blood as stem cell source performed between 2011 and 2016 in our center have been retrospectively reviewed. gvhd prophylaxis consisted of pt-cy 50 mg/ kg/day on days +3 and +4, mmf and csa from day +5 for all cases. cmv pcr was performed in a biweekly basis during admission for transplant and treatment, and weekly thereafter. cmv-r was considered with any cmv dna level by pcr assay above 100 copies/ml. prior four consecutive negative weekly pcrs were needed to consider a new reactivation episode. preemptive strategy was applied in all cases. data collected in relation to cmv-r included: cmv serostatus of donor/recipient (d/r), number of cmv reactivations, length of each reactivation, antiviral treatment used, need for admission to receive treatment and adverse events related to cmv reactivation and/or antiviral treatment. results: patients characteristics are summarized in table 1 . among 100 patients, 78 of them with positive cmv serology, 128 episodes of cmv-r were detected. seventysix patients (76%) had at least one cmv-r in a median of 30 days after transplant. none of them had cmv disease or die as a consequence of cmv-r. median duration was 22 days (15-37). valganciclovir or ganciclovir was used in 101 episodes (79%). foscarnet was used in 43 episodes (34%). six of the episodes occurred after initial discharge, and required re-admission for treatment, with a median length of hospitalization of 16 days (6-27). cytopenias requiring transfusion or g-csf support occurred in 36 episodes (36%) treated with ganciclovir or valganciclovir. three of them needed further cd34+ cells booster for graft rescue. mild acute renal failure and genital ulcers were found in 21 (49%) and 5 (11,6%) events treated with foscarnet, respectively. no cases of severe renal failure were observed. serological status different than negative/negative (n/n) (p 0.001) and older age (46 vs 38 years, p 0.02) were significantly associated with cmv-r. no relationship was observed with gender, disease, donor relationship, conditioning, gvhd or cells infused. more than 2 reactivations were more frequent among patients with grade ii-iv acute gvhd (agvhd) and moderate-severe chronic gvhd (cgvhd). conclusions: in our experience, rate of cmv-r after unmanipulated haplo pt-cy, using pbsc as stem cell source, is considerably high. a significant proportion of patients presented complications associated with cmv-r and its treatment. cmv serological status other than n/n and older age are associated with high risk of cmv-r. patients with grade ii-iv agvhd are at higher risk of multiple reactivations. this population could be benefited from primary prophylaxis, in order to decrease treatment´s complications, re-admissions and costs. disclosure: nothing to declare. impact of infectious events occurring during the first hundred days after hsct for hematological malignancy: a monocentric retrospective study over a five-year period marie-pierre ledoux 1 , célestine simand 1,2 , karin bilger 1 , annegret laplace 1 , bruno lioure 1 background: patients undergoing hematopoietic stem cells transplantation (hsct) for hematological malignancy often present with infectious events in the early stages of the procedure, some of which having a documented impact on the outcome of the graft. for instance, cytomegalovirus (cmv) has been shown by some authors to have a protecting effect against relapse, whose features remain to be elucidated. we conducted a retrospective monocentric study regarding the outcome in terms of graft versus host (gvhd), relapse and survival of 224 consecutive patients over a period of 5 years, whether they presented or not with an infectious event by day 100 among the following: cmv viremia, epstein-barr virus (ebv) viremia, human herpes virus 6 (hhv6) viremia, bk virus (bkv) viruria, bacterial bloodstream infection (bsi) or invasive fungal infection. results: a high proportion of cmv seropositive recipients underwent a viral reactivation of cmv by day 100 of the hsct: 81% if the donor is seronegative and 74% if the donor is seropositive. we observed that cmv wasn't associated with a lower relapse rate in our cohort, and data weren't sufficient to conclude firmly, but showed a trend towards a worse acute gvhd (hazard ratio hr 2.08, pvalue 0.06). no significant correlation was found for ebv viremia. occurring in 26% of our patients and mostly with an early timing, hhv6 strongly correlated with worse acute gvhd (hr 3.25, p-value < 0.001) but its impact on survival was not significant. bkv (27% of our patients) and bsi (46% of our patients) both correlated with poorer outcome in terms of overall survival (logrank < 0.001 and 0.014 respectively) although not significantly associated with relapse or acute gvhd. fungal infections were too rare events to draw any conclusion. conclusions: thus, contrary to many studies, we found no protection against relapse induced by cmv, although the trend for worse acute gvhd was obvious. the mechanisms behind this discordance could include early treatment, but remain to be studied. whether hhv6 is a cause rather than a consequence of acute gvhd or its treatment is debated, but the correlation is strong and the sequence of events suggests hhv6 might act as a trigger for gvhd. the association between bkv viruria and a higher mortality is in contrast with previous observations, and the lack for association with gvhd and relapse could suggest bkv is a surrogate for poor immune recovery and therefore other causes of non-relapse mortality. in addition to the direct lethal risk of bacteriemia, bsi also are a promoter of late non-relapse mortality through indirect toxicity. through the expansion of immune effectors they promote, one could assume that infectious events play a role in gvhd and gvl, and therefore have an interference with relapse. however, the association between each infectious event and outcome remains to be clarified to guide our prophylactic and therapeutic choices by a better understanding of the bright and dark sides of infectious events. disclosure background: rezafungin (rzf) is a novel echinocandin in phase 3 development for treatment of candidaemia and invasive candidiasis and for antifungal prophylaxis against invasive fungal diseases caused by candida, aspergillus, and pneumocystis in blood and marrow transplant patients. rzf is differentiated by stable, prolonged pharmacokinetics (pk) that allow for once-weekly dosing and a pk-pharmacodynamic (pd) profile correlating with efficacy. clinical in vivo evaluations of drug interaction potential were performed proactively to assess the risk of drug-drug interactions (ddis) with respect to the phase 3 dose of 400 mg once weekly and known pk exposure in healthy individuals. methods: this open-label study of 26 healthy inpatients assessed ddis between rzf (as perpetrator) and drugs known to have interactions with cyp enzymes and transporters (probe drugs): repaglinide (cyp2c8), metformin (oct/mate), rosuvastatin (bcrp/oatp), pitavastatin (oatp), caffeine (cyp1a2), efavirenz (cyp2b6), midazolam (cyp3a4), and digoxin (p-gp), as well as tacrolimus, a drug likely to be coadministered with rzf. an initial dose of rzf 600 mg was administered on the first dosing day, to approximate a steady state plasma concentration of multiple once-weekly 400-mg doses, followed by 2 once-weekly 400-mg doses on days 10 and 15. probe drug cocktails containing ≥2 drugs were administered, once before and once after rzf administration, on a schedule designed to allow for washout between doses and to limit interactions with other probe and test drugs. samples were analysed to determine respective drug concentrations in plasma (except for tacrolimus which was in whole blood) to characterize the pk profile of each analyte. area under curve (auc) and maximum concentration (c max ) were calculated from the plasma/blood concentration-time profiles by noncompartmental analysis. ln-transformed pk parameters were statistically analysed using an analysis of variance model. the ratio of geometric least squared means between each substrate drug when administered with and without rzf and corresponding 90% confidence intervals (cis) were calculated for lntransformed c max and auc. results: when rzf was given concomitantly with the probe drugs, six of nine substrates (metformin, pitavastatin, caffeine, efavirenz, midazolam, and digoxin) statistically demonstrated the absence of drug-drug interaction, as their 90% ci were all included within the default 80-125% noeffect boundary. three substrates had the upper (repaglinide and rosuvastatin) or lower (tacrolimus) bounds of their ci falling just outside of this range (figure 1 ), and these changes are considered unlikely to be clinically significant. conclusions: no meaningful pk interactions were observed between rzf and 9 drugs known to have ddis and/or likely to be coadministered with rzf. these findings provide evidence that no dose adjustment is expected when rzf is co-administered with these commonly used drugs, which stand in contrast with the ddi complications widely associated with azole antifungals. disclosure: voon ong: employee, stockholder (cidara therapeutics), michael boily: employee (altasciences), hong wong: employee (altasciences), taylor sandison: employee, stockholder (cidara therapeutics), shawn flanagan: employee, stockholder (cidara therapeutics) abstract withdrawn. background: cytomegalovirus (cmv) continues to cause morbidity following allogeneic hematopoietic stem cell transplantation (hsct). letermovir is a newly approved drug for cmv prophylaxis in cmv-seropositive allogeneic hsct recipients. however, there is a paucity of data for its efficacy in patients receiving in-vivo t-cell depletion (tcd). at weill cornell medical center, we perform in-vivo tcd with alemtuzumab for related and hla-identical unrelated transplants, and anti-thymocyte globulin for umbilical cord blood transplant supported by third party accessory cells (haplo-cord transplant).although these drugs reduce the frequency of graft-versus-host-disease (gvhd), they significantly delay t-cell immune reconstitution post hsct, and may cause higher rates of cmv reactivation. our historical rate of cmv reactivation in cmv seropositive recipients receiving high dose valacyclovir prophylaxis is approximately 35%. therefore, we implemented letermovir for cmv prophylaxis in february 2018. the primary aim of this study is to determine the incidence of cmv infection (defined as cmv viremia warranting treatment or development of end-organ disease) in tcd cmv seropositive allogeneic hsct patients who received letermovir prophylaxis. methods: this is a single center, retrospective cohort study to determine the incidence of cmv infection in adult, cmv-seropositive recipients receiving letermovir prophylaxis after in vivo tcd hsct with atg or alemtuzumab for gvhd prophylaxis. all included subjects were at least 100 days post-transplant. results: 31 allogeneic hsct transplant recipients met inclusion criteria. median age was 60 years, iqr [47, 67] and 48% were male. eight (26%) had a matched related donor, six (19%) had a matched unrelated donor, and 17 (55%) were haplo-cord transplants. their underlying malignancy and conditioning regimens are summarized in table 1 . 17 (55%) received atg and 14 (45%) received alemtuzumab for gvhd prophylaxis. median follow up time for survivors is 141 days, iqr [107, 187] . the incidence of cmv infection in the first 100 days post-transplant was 3% as only one patient reactivated with detectable cmv viremia. this same patient developed cmv pneumonitis with documented ul 56 resistance, and was successfully treated with ganciclovir. the incidence of cmv infection within the first 150 days post-transplant was 5% (1/19 patients) . six patients (19%) developed acute gvhd in the first 100 days, and one (3%) had relapse of their malignancy. five patients (16%) died within 100 days post-transplant, but none of these deaths were cmv related. background: infectious complications caused by endogenous adenovirus (adv) are common and associated with morbidity and mortality rates in patients after hematopoietic stem cell transplantation (hsct). adv infections occur in about 3% to 20% of hsct recipients, with significantly higher rates in pediatric patients. a better understanding of adenoviral-specific t-cells (advt) response in donors can serve as a basis to develop more effective strategies for antiviral therapy. methods: frequencies of cytomegalovirus (cmv)-and adv-specific t cells were determined by enzyme-linked immunospot (elispot) assays with adv5 hexon and cmvpp65 respectively in 80 health donors. we used 3x10 5 of mononuclear cells (mnc) per well in elispot assays. all donors were divided into 3 groups according to the number of spots per well (spw) as follows: high responders (hr) (≥50 spots; n=32), low responders (lr) (>10 and < 50 spots; n=39), nonresponders (nr) (≤10 spots; n=9). the average spot area of adv-and cmv-specific lymphocytes was calculated by immunospot® multiplate autocount™. cd45ra+ and cd45ro+ t-cells were generated by immunomagnetic negative selection. hla typing for class i and ii was performed by sequence specific oligonucleotides technology. statistical analysis was performed using graphpad prism v7.00 software. levels of significance were calculated by mann-whitney rank-sum test, expressed as p-values (p< 0.05). results: the median frequency per well of advts were 72 in hr group, 24 in lr group, 4 in nr group. the median spw of cmv-specific t cells in donors mnc were 147 and didn´t differ between 3 groups. antiviral activity may depend not only on the amount of advt but also on their ability to produce ifnγ. the average spot area for advt did not differ between hr, lr and nr groups and were 22,4, 23,1 and 19,1 mm 2 respectively. the median of the average spot area for anti-cmv t-lymphocytes was equal to 13,5 mm 2 . thus, the frequency of advt was lower than cmv-specific t-cells, but advt have the ability to produce more ifnγ per cell (p< 0.0001). in order to evaluate the distribution of the advt between naive and memory t cell compartments, we evaluated response to adv in preselected cd45ra+ and cd45ro+ fractions of t-cells in a group of 17 donors. the median frequency of advt in unfractionated mnc was 87; the median frequency of advt in cd45ra and cd45ro fractions were 2 and 73, respectively. the amounts of cd45ra and cd45ro tcells were normalized to their amounts in mnc. we evaluated the impact of hla-alleles on the anti-adv response of t-cells in different groups and found 2 association: hla-a*1 with hr group (p-value=0,0043; rr=1,954; 95% ci: 1,285 to 2,604) and hla-a*31 with lr group 0251; rr=1, 889; 95% ci: 1, 135 to 4, 426) . conclusions: in this study the frequency of donors with advt is 88,75% which corresponds to the reported frequency of adv-seropositive people (95%) in population of russia. advt are exclusively cd45ro-positive cells. the analysis of advt in potential hsct donors will allow to determine more accurately the amounts and functional activity of specific antiviral t-lymphocytes administered to patient and optimize antiviral therapy. disclosure: nothing to declare p429 abstract withdrawn. chemotaxis and exhaustion of γδ t cells in the allografts are associated with cmv reactivation after hematopoietic transplantation background: cytomegalovirus (cmv) reactivation and its related diseases remain the most common and serious complications in patients who underwentallogeneic hematopoietic stem cell transplantation (allohsct). we previously reported that the incidences of total and refractory cmv reactivation reached approximately 90% and 50% after haploidentical hsct. while majority of studies in the literatures focused on the adaptive cd8 + αβ t cellsand nk cells in anti-cmv immunity, increasing evidences highlighted the important role of γδt cells in this context. a progressive and prolonged expansion of vδ1 + t cells in response to cmv reactivation was observed after allohsct. the effect of vδ1 + t cells associated with cmv clearance has been reported in vitro and in vivo. in contrast to the reconstituted γδt cells post transplantation, whether the phenotypes of γδt subsets in allografts correlate to cmv reactivation in hsct recipients have not been documented. methods: the proportions and phenotypes of γδ t cells were detected inallografts those were unmanipulated g-csf-mobilized bone marrow (bm) and peripheral blood (pb) harvests from 20 donors for haplohsct. bm grafts were collected by aspiration on the fourth day of g-csf treatment (filgrastim, 5 μg/kg/day), and pb grafts were obtained on the fifth day by leukapheresis. immunophenotyping for γδ t-cell subpopulations, including the expression of cd3, cd4, cd8, tcrγδ, tcrvδ1, tcrvδ2, hla-dr, nkg2d, cxcr4, ccr5, pd1, ki67, ifnγ, tnfα, and il-17, was performed using flow cytometry. for detection of the intracellular cytokines, bm and pb grafts were pre-stimulated with 1x cell stimulation cocktail (500x, ebioscience). cmv dna in the peripheral blood of recipients was routinely monitored by quantitative pcr. the association of γδ t-cell contents in allografts with cmv reactivation in haplohsct recipients was analyzed using the mann-whitney u test and spearman test. all calculations were performed using spss 22.0 statistical software. results: we found that the proportions of total γδ t cells, and vδ1 and vδ2 subsets in both bm and pb grafts for cmv+ and cmv-recipients were comparable. neither the expression of hla-dr nor nkg2d in the allografts were significantly different in correlation to cmv reactivation after hsct. the productions of intracellular cytokines of γδ t subsets did not varied in bm and pb grafts for cmv+ and cmv-recipients. interestingly, the proportions of cxcr4+vδ1 and ccr5+vδ1 cells in bm grafts for cmv + recipients were significantly higher than those for cmvrecipients (p = 0.012 and 0.045, respectively). meanwhile, pma-stimulated ki67+vδ1 cells in bm grafts for cmv+ recipients were less than those for cmv-recipients (p = 0.039). in parallel, the concentration of pd1+vδ1 cells in pb grafts for cmv+ recipients were significantly higher than those for cmv-recipients (p = 0.023). conclusions: this study is the first to connect the chemotaxis and exhaustion of γδ t cells in grafts to the risk of cmv infection after allogeneic hsct. future studies should explore how the expressions of chemokines and exhaustion marker on the effector γδ t cells in allografts facilitate cmv replication and/or dissemination in the setting of hematopoietic transplantation. disclosure: all authors do not have conflicts of interest. this study is supported by the national natural science foundation of china (grants no.81770191 and no.81670167) results: incidence of ic was 2,9%: allo-hsct -3% (n=15), auto-hsct -2,7% (n=7). the etiology: c. parapsilosis 50%, c. albicans 27%, c. krusei 14%, candida tropicalis 5%, candida dubliniensis 4%. the most frequent underlying diseases was acute leukemia -45% (n=10). the median age was 8 y.o. [3 month -18 years] . the median day of onset of ic after allo-hsct was 63 , auto-hsct -12 [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . febrile fever was the main clinical symptom; septic syndrome develops in 32% cases. antifungal therapy was with echinocandins -23%, lipid ampho b -27%, azoles (fluconazole, voriconazole) -32%, without therapy (the early mortality) -18%. overall survival (os) at 30 days from diagnosis of invasive candidiasis was 50%. the central venous catheter (cvc) removal was the only factor significantly improved os (70% vs 33%, p=0,035). conclusions: incidence of invasive candidiasis in children after hematopoietic stem cell transplantation was 2.9%. the main etiology agent was c. parapsilosis. invasive candidiasis infections most often affect leukemia patients, developed later after allo-hsct than auto-hsct. overall survival at 30 days from the diagnosis was 50%. removing of cvc improved overall survival in children with invasive candida infections after hsct. disclosure: nothing to declare background: graft versus host disease (gvhd) and virusassociated enteropathy in allogeneic hematopoietic stem cell transplantation (allo-hsct) may cause severe quantitative and qualitative composition changes of intestinal microbiota, leading to the development of small intestinal bacterial overgrowth (sibo) on the background of immunodeficiency, which can have a negative impact on treatment effectiveness. the gold standard for diagnosis and the main criterion for sibo is the detection in the jejunum aspirate >10 5 /ml bacteria and/or the appearance of colonlike microbiota in small intestine. it is also acceptable to use an alternative non-invasive technique -hydrogen breath test, which could be especially important in patients with severe mucositis, grade iii-iv and thrombocytopenia grade iv. sibo diagnosis in the setting of the gastrointestinal tract damage and dysfunction in patients treated with allo-hsct is insufficiently studied. methods: the study included 7 patients with acute myeloid leukemia (n=3), acute lymphoblastic leukemia (n=1), myelodysplastic syndrome (n=1), non-hodgkin´s lymphoma (n=1), hurler syndrome (n=1), who underwent allo-hsct from an unrelated (n=4) and haploidentical donor (n=3) , and which were complicated by enteropathy development. in 2 cases, the enteropathy reason was a combination of intestinal gvhd and viral colitis (hhv-6), in 5 cases -viral colitis (hhv-6). all patients had esophagogastroduodenoscopy with species aspiration from descending part of the duodenum and feces collection, with further bacteria pcr identification. hydrogen breath test was performed also in which patients were treated with oral lactose 2 g/kg with subsequent hydrogen assessment after 30 and 60 minutes. the study was performed in the period from 20 to 741 days after allo-hsct. results: according to feces analysis data, colon microbiota composition significantly differed from the reference values. at the same time total bacterial mass of the duodenum was less in comparison with colon microbiota: 2e+08 (2e+7/8e+9) and 9e+10 (9e+7/3e+12), respectively (p< 0.004). quantitative composition of the duodenal microbiota was comparable to that of colon: lactobacillus spp. 4e+5 (1e+5/2e+7) > 1.5 e+5 (1e+5/4e+11), (p=0.48); bifidobacterium spp. 6e+6 (1e+05/3e+7) < 7.5 e+6 (2e+5/1e+9), (p=0.423); escherichia coli 7e+5 (1e+5/2e+6) > 5e+5 (4e+5/9e+5), (p=0.2); bacteroides fragilis group 6e+6 (0e+0/8e+9) > 5e+6 (2e+5/3e +12), (p=1.0); faecalibacterium prausnitzii 1e+6 (0e+0/ 1e+7) < 1.5 e+6 (1e+5/3e+10), (p=0.54), indicating the presence of sibo. in this case, the hydrogen breath test was completely uninformative: basal values -0.035 (0.01/0.06) ppm, hydrogen concentration in 30 minutes -0.04 (0.01/ 0.06) ppm, in 60 minutes -0.02 (0.01/0.07) ppm, which is less than in healthy volunteers. conclusions: quantitative composition of the duodenal and colon microbiota is similar in the case of intestinal gvhd and/or virus-associated enteropathy in allo-hsct patients, which may be of diagnostic value for sibo confirmation. the hydrogen breath test is an uninformative method for sibo identification in patients after allo-hsct. disclosure: nothing to declare johannes schulte 1 , patrick hundsdörfer 1 , sebastian voigt 1 background: adenovirus (adv) infections or reactivations frequently occur in the pediatric hematopoietic stem cell transplant (sct) setting and these infections contribute to increased morbidity and mortality. the nucleotide analog cidofovir might be effective in reducing adv load, however, nephrotoxicity is a considerable side effect. the new antiviral compound brincidofovir (bcv, cmx-001), a lipid-conjugate nucleotide analog with broad-spectrum antiviral activity in vitro, has been reported to be effective in cases where cidofovir treatment was unsuccessful. methods: data of eight pediatric patients undergoing sct for malignant and nonmalignant indications were analyzed. all patients were weekly monitored for adv viremia by pcr. in case two consecutive positive adv pcr results indicating a viral copy number > 1000/ml were documented, patients received a weekly dose of cidofovir. if no reduction of adv load was seen within two weeks after the commencement of treatment or side effects demanded cidofovir discontinuation, bcv was obtained through an emergency expanded access programme. results: eight pediatric patients developed adv viremia with maximum viral loads ranging between 9350 and 4430000 copies/ml. six patients had c type adv and two patients had non c type adv infections. five patients had viral co-infections: two had an additional cmv infection, one had an epstein-barr virus (ebv) and herpes simplex virus co-infection, one patient had an ebv co-infection and one patient had a bk virus co-infection. all eight patients initially received cidofovir, however, a substantial decrease in adv load could not be observed in any patient after a two-week administration course. except in one patient who had extensive intestinal graft-versus host disease (gvhd), adv infection was cleared in all patients within three weeks after the beginning of bcv treatment. in addition, all coinfections were cleared. no nephrotoxicity or other side effects were observed. conclusions: bcv was effective in all but one patient. oral bcv might not be effective in advanced upper gut gvhd, especially when applied via a gastric tube, yet this was observed in only one patient. eventually, without nephrotoxic side effects, bcv could be an useful alternative to cidofovir. disclosure: nothing to declare. background: the use of post-transplant high-dose cyclophosphamide (ptcy) has overcome the need for extensive depletion of t lymphocytes from haploidentical donor grafts, which traditionally resulted in severe and prolonged immunosuppression. however, reconstitution of cellular immunity may be delayed even after t cell replete haploidentical stem cell transplantation (haplo-sct) with ptcy. the study of the incidence and severity of viral reactivation is therefore relevant to the outcomes of haplo-sct with ptcy. methods: our study enrolled 42 patients (women/men, 19/23), who underwent t cell replete haplo-sct from 12/ 2013 to 10/2018 and achieved hematopoietic engraftment. median age at transplant was 53.5 years (range, 19-70) . the underlying disease was aml (n=17), all (n=9), mds (n=9), myelofibrosis (n=4), cml (n=2), or cll (n=1). the conditioning regimen was myeloablative (n=31), reduced-intensity (n=10) or non-myeloablative (n=1). peripheral blood was the graft source in the majority of cases (n=29) and bone marrow in the remaining (n=13). recipient/donor cytomegalovirus (cmv) serostatus was -/-(n=2), -/+ (n= 4), +/-(n=8), or +/+ (n=28). the combination of tacrolimus and mycophenolate mofetil was administered in addition to ptcy for prevention of graftversus-host disease. cmv, epstein-barr virus (ebv), and human herpesvirus-6 (hhv-6) reactivation was monitored by real-time quantitative pcr (rq-pcr) in blood twice weekly post haplo-sct. bk virus (bkv) reactivation was assessed by rq-pcr in urine and/or blood specimens in cases with symptoms suggestive of bkv-associated hemorrhagic cystitis (hc). results: with a median follow-up time of 25 months (range, , the cumulative incidences (cin) of relapse and non-relapse mortality (nrm) were 15.5% (95% ci, 6.2-28.6%) and 33.3% (95% ci, 18.9-48.2%) at 2 years, respectively. median disease-free (dfs) and overall survival (os) were 53.3% (95% ci, 39.5-71.9%) and 58.4% (95% ci, 44.6-76.5%) at 2 years, respectively. the cin of cmv reactivation/infection (>100 copies/ml) reached 75.7% (95% ci, 58.8-86.4%) at 3 months. cmv infection developed in 30 out of 40 patients who were at risk, whereas recurrent cmv reactivation was observed in 15 patients with a median number of 2 episodes (range, 2-6) per patient. the median total duration of antiviral therapy for cmv infection was 27 days (range, 14-199) . cmv disease (pneumonia) was documented in 2 patients. the cin of ebv reactivation (>1,000 copies/ml) was 50.9% (95% ci, 34.2-65.3%) at 12 months. no case of ebv-related post-transplant lymphoproliferative disorder was observed, however preemptive therapy with rituximab was required in 2 patients with rapidly increasing ebv viral load. hhv-6 reactivation (>1,000 copies/ml) was observed in 5 patients (cin, 12.3% at 6 months; 95% ci, 4.4-24.5%), with none of them requiring specific therapy. bkv-related hc occurred at a cin of 23.9% (95% ci, 12.3-37.7%) at 6 months. cystoscopy for bladder hemostasis was required in 4/11 and nephrostomy in 1/11 patients with hc. conclusions: despite preservation of non-alloreactive memory t cells, haplo-sct with ptcy is associated with substantial rates of viral reactivation (especially cmv and bkv) resulting in the need for prolonged antiviral therapy and considerable morbidity as well. therefore, strategies to prevent viral reactivation and disease are still warranted in haploidentical stem cell transplantation. disclosure: nothing to declare. background: adenovirus(adv) infections are a wellrecognised cause of morbidity and mortality in children and adults receiving an allogenic stem cell transplant(hsct).the reported incidence of adv infection is higher(6%-42%) in paediatric hsct than in adults(3-27%),but we currently lack accurate data of adv infection burden among adults.cidofovir has been extensively used as a pre-emptive anti-adenoviral therapy and is current standard of care.we present our single centre experience of adv incidence and outcomes with pre-emptive approach in adult patients receiving t-cell depleted(tcd) hscts for myeloid disorders. methods: this is a single-centre retrospective analysis of 332 consecutive hsct patients for myeloid disorders including aml, mds, mpn & aplastic anaemia between january 2012-june 2016 using atg or alemtuzumab based tcd.adv screening was performed in all patients with standardised real time quantitative pcr on weekly basis during standard risk period. figure 1a -1b] results: baseline characteristics (table1) of patients were similar across both cohorts with or without adv infection. overall 12.6%(n-42/332) patients were positive for adv dna on atleast one of the sanctuary sites(upper respiratory airway,blood,faeces,urine) and 45%(n-19/42) of these experienced disseminated infection(defined by adv in ≥2 sanctuary sites or rising adv dna copies in blood), while 2 developed typical adenoviral disease (pulmonary). among patients with disseminated infection,majority had adv in gastro-intestinal(42%),10.5% in genitourinary and 21% as both sanctuary site of infection,in addition to blood viraemia(85% of all cases).cumulative incidence of adv infection was 10.6%(95%ci:7.6-14.2%) at 12 months with median time of 108 days(iqr:19-304 days) to detect adv-dna post hsct.overall survival(os) at 5 years for whole cohort was 41%(95%ci:31-50;median os-57months) with no statistical difference between patients with disseminated adv infection vs those with none(log rank; p-0.68; fig-1a ). overall cumulative incidence of non-relapse mortality (nrm) was 23%(95%ci:18-28%) and relapse(cir) was 25.5%(95%ci:19-32%) at 5 years,but no statistical difference noted between patients with disseminated adv infection & those with none(nrm:p-0.31;cir: p-0.50;gray test).pre-emptive therapy with cidofovir (3mg/kg weekly iv infusion for 2 weeks and fortnightly thereafter until infection free) was required in 33%(14/42) of symptomatic adv infection patients and 52%(10/19) with disseminated infections.one patient required brincidofovir therapy for refractory disease,but one patient died due to severe sepsis, before adv specific therapy could be given.remaining patients were monitored and all self-recovered on cessation of immunosuppression.all patients treated with cidofovir developed renal impairment(defined by atleast >25% increase in baseline creatinine),however majority(73%) recovered their renal function near their baseline (fig-1b) . conclusions: adv infection remains a significant cause of morbidity in adult hsct patients, however pre-emptive management with cidofovir has improved os and nrm despite use of tcd conditioning.renal toxicity remains common with cidofovir but with use of intermediate doses, majority do recover their renal functions. clinical trial registry: n/a disclosure: nothing to declare background: publications on invasive fungal disease (ifd) in lymphoma patients are limited especially after allo-hsct. there are no data on outcome of allo-hsct in lymphoma patients with prior ifd. this study focuses on epidemiology of ifd before and after allo-hsct in children and adults with hodgkin's lymphoma (hl). methods: single center prospective study included 86 patients with classical r/r hl who received allo-hsct from 2002 to 2018. the median age was 27 (13-49) y.o., children (< 18 yo) -13%. allo-hsct from mud was performed in 45,4% (n=39), mrd -24,4% (n=21), mmud -15,1% (n=13), haplo -15,1% (n=13), with ric (100%) and predominantly ptcy-based gvhd prophylaxis (71%). primary antifungal prophylaxis was fluconazole in 85%, secondary -voriconazole (100%). eortc/msg 2008 criteria for diagnosis and bronchoscopy before allo-hsct in pts with ct-scan lung lesions were used. "active ifd" means ifd diagnosed just before hsct. median follow-up time was 12 months . results: incidence of ifd before allo-hsct was 12,8% (n=11). ifd prior to hsct were invasive aspergillosis (ia) with lungs involvement. antifungal therapy before allo-hsct was used in 81,8% pts with median duration -2 months. complete response to antifungal therapy was in 45,4% pts, partial response or stabilization -36,4%, and 18,2% pts had an "active ifd". after allo-hsct all pts received voriconazole as an antifungal therapy or secondary prophylaxis. cumulative incidence of relapse or progression of ia after allo-hsct was 18,2% with the median 49 day after hsct, which were successfully treated with voriconazole in post hsct period. incidence of ifd after allo-hsct for naïve patients was 17,6% (n=13/74). etiology of ifd after allo-hsct was ia -69%, invasive candidiasis (ic) -15%, mucormycosis -8% and 8% combined ifd caused by aspergillus fumigatus + rhizopus stolonifer. the median day of onset of ifd after allo-hsct was day+ 114 and was associated with post-hsct relapse of hl (p=0,04). the main site of infection were lungs (88%), the main clinical symptom -febrile fever (100%). antifungal therapy was used in all patients: voriconazole -59%, micafungin -17%, posaconazole -8%, lipid amphotericin b -8% and combination lipid amphotericin b with caspofungin -8%. overall survival (os) at 12 weeks from the diagnosis of ifd after allo-hsct was 80%. the 2-year os in children and adult with hl after allo-hsct was 73,3%. development of ifd after allo-hsct do not decrease the 2-year os rate (69,2% vs 74%, p=0,77). the impact of prior ifd on 2-year os in allo-hsct recipients was not statistically significant in all group (63,6% vs 74,7%, p=0,47) , and separately in children and adults. conclusions: incidence of ifd in children and adults with hodgkin's lymphoma before allo-hsct was 12,8%. incidence of ifd after allo-hsct in patients with hodgkin's lymphoma was 17,6%. the major etiology agents as before as after allo-hsct were aspergillus spp. ifd was a late complication after allo-hsct and associated with post-hsct relapse. despite the high incidence ifd before or after allo-hsct didn't influence the outcome in children and adults with hodgkin lymphoma. disclosure: nothing to declare our community has high cmv positive serostatus, which is a known risk for cmv infection or reactivation. we conducted a study to explore the incidence and outcome of cmv infection among post-hsct children. methods: medical records of pediatric patients (age ≤ 14 years) undergoing single allogeneic hsct from january 2014 to december 2016, at king faisal specialist hospital and research centre, riyadh, saudi arabia, were reviewed. all patients with active cmv infection or disease before and during transplant were excluded. a total of 307 patients were included in the study; 160 were female. median age at hsct was 5 years. recipient cmv serostatus was positive in 213 patients before hsct, and 232 donors were cmvpositive. the recipient-donor (r/d) serology was 63.7% r +/d+, 15.3% r+/d-, 13.7% r-/d+, and 7.3% r-/d-. indication for hsct was immune disorders 24.1%, hemoglobinopathies 20.8%, bone marrow failure 20.5%, malignant disorders 20.5%, histiocytic 8.1%, and metabolic disorders 5.9%. source of stem cells was bone marrow in 223, cord blood in 74 and peripheral blood stem cell in 10 cases. donor was matched related among 198, unrelated matched/mismatched in 74, haploidentical 24, and related with 1-antigen mismatch in 11. total body irradiation (tbi) based conditioning was used for 47 patients, while atg was used in 169 patients. results: out of a total of 307 patients, 101 patients developed cmv infection post-hsct (32.9%). incidence in female recipients was high (38.1% versus 27.2%, p-value 0.042). both recipient and donor cmv serology positive (42.5%) developed cmv infection (p-value < 0.001). however, no cmv infection in both recipient and donor negative group (r-/d-). the incidence of cmv infection post-hsct was high in patients received tbi based conditioning (22 out of 47, 46 .8%, p-value 0.027), and in haploidentical transplant with 66.7% (p-value 0.008). source of stem cells, myeloablative versus nonmyeloablative conditioning, atg use in conditioning and agvhd, did not exhibit significant association with cmv infection. in multivariable setting, when adjusted for primary indication for transplant, donor hla type, tbi based conditioning and recipient and donor cmv serology at transplant, haploidentical donor (odds ratio: 3.491, p-value: 0.041) and donor-recipient cmv sero-positivity (odds ratio: 2.228, p-value: 0.007) were found to be significant risk factors. cmv infection resolution rate was 83.2% (84). with a median follow-up time of 32.9 ±1.5 months from infusion, five-year overall survival of cmv infected group was lower (0.658±0.087) as compared to non-cmv infected (0.706±0.032, p-value: 0.285). conclusions: incidence of cmv infection post-hsct in our center is comparable to other centers. our data suggest that donor-recipient cmv positive serostatus, haploidentical donor, and use of tbi based conditioning necessitate close attention and surveillance. background: toxoplasmosis is a rare and underestimated complication following allogeneic stem cell transplantation (allo-sct) with an often fatal course. this is in part due to limited diagnostics relying mainly on imaging and detection of parasite dna by pcr. we present here eleven cases of toxoplasma disease following allo-sct. methods: we retrospectively analyzed 534 consecutive adult patients who received an allo-sct in our bone marrow transplant unit between july 2008 and july 2018. eleven (2%) of these patients were diagnosed of toxoplasma disease. the main characteristics of the patients are shown in table 1. all patients, except two cord blood, have received peripheral blood stem cells. fludarabine-based conditioning regimes were used in all patients. only the two cord blood patients received thymoglobulin in the conditioning. graft-versus-host disease (gvhd) prophylaxis consisted on tacrolimus plus mycophenolate mofetil in 5 (46%) patients and post-transplant cyclophosphamide followed by tacrolimus in 4 (36%). before the allo-sct was performed the igg/igm toxoplasma serology of the recipient and donor. we reviewed the absolute lymphocyte count (alc) and cd4 + lymphocyte count within four weeks prior to the diagnosis of toxoplasmosis, and if the patients took effective primary prophylaxis for this parasite. toxoplasma disease was defined as the presence of toxoplasma infection plus clinical, radiological or pathological evidence. toxoplasma disease was considered the main cause of death when no other major life-threatening infection or other potential fatal complication occurred immediately before death. results: median (range) age (years) of the eleven patients diagnosed with toxoplasma disease was 54 (19-69). for pancytopenia, no patient received trimethoprimsufmethoxazole (tmp-smz) but pentamidine for pneumocystis jirovecii-pneumonia (pcp) prophylaxis in 10 cases and atovaquone in one. toxoplasma serology pretransplant was positive (igg+/igm-) in ten of the eleven patients. all donors were seronegative (igg-/igm-) except two. toxoplasmosis was diagnosed a median (range) of 85 days (35-330) post allo-stc. the clinical presentations were as cerebral-encephalitis (n=4), chorioretinitis (n=3), pneumonitis (n=2) and disseminated toxoplasmosis (n=2). one case, patient and donor seronegative pre-transplant, was presented as a primary infection in form of chorioretinitis. all three patients with chorioretinitis were diagnosed after day +100 of allo-sct. at the time of toxoplasma disease, 8 of 11 (73%) of patients had an alc < 500 cells/μl and all of them with immunosuppressive therapy and corticosteroids for acute or chronic gvhd. we had cd4 + lymphocyte count only in four patients and in three of them was < 200 cells/μl. eight of the eleven (73%) patients died, with a median (range) of 21 days (9-65) since diagnosis of toxoplasmosis, and in 6 of them the toxoplasma disease was the main cause of death. conclusions: in our series, the incidence of toxoplasma disease after allo-sct is low and is related to high mortality, in accordance with what has been reported by other groups. positive pre-transplant serology and gvhd and its treatment were factors strongly related with toxoplasmosis. we encourage the use of tmp-smz instead of pentamidine for pcp-pneumonia prophylaxis in patients seropositive for toxoplasma gondii pre-transplant. clinical trial registry: data about its epidemiology in children are scarce. we retrospectively analyzed the incidence, the severity and the risk factors that contribute to the manifestation of this complication in a pediatric population. methods: during a 10-year period (january 2008 -june 2018) we performed in our center 376 allogeneic transplantations, 237 for malignant hematological diseases and 139 for non-malignant. the majority of our patients received myeloablative conditioning regimens. diagnostic criteria of hemorrhagic cystitis were the detection of the virus with pcr in urine samples and/or in blood samples, in combination with hematuria and lower urinary tract symptoms (dysuria, urinary frequency, urgency, suprapubic pain) that couldn't be attributed to any other reason. we defined the hemorrhagic cystitis as severe when one of the following factors was present: formation of clots and continuous bladder irrigation, obstructive uropathy with creatinine elevation or need for urological intervention. results: a total of 376 patients with median age 7,2 years (0,5-20) were studied. 37 children (10%, 95% ci, 7,2-13,3) manifested bk virus associated hemorrhagic cystitis with median age 12,9 years (5, [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 5) . onset of cystitis occurred at a median time of 27 days (day +-day +52) after transplantation. in 17 children cystitis was severe. the median duration of symptoms was 27 days (8-200). the median time of hospitalization for children with severe cystitis was 70 days (29-242) whereas for those who didn't manifest cystitis was 45 (25-184) . in 25 of the 37 patients we examined the presence of the virus not only in urine but also in blood samples. in 11 of them the test was positive and almost half of them (6) manifested severe cystitis. the risk factors that were examined were age, administration of antithymocyte globulin, type of disease, graft source, type of donor and the presence of acute graft versus host disease (agvhd). in multivariable analysis, independent risk factors for the manifestation of hemorrhagic cystitis were age > 6 years old (hr: 8,46, 95% ci, 2,86-25, p< 0,001), transplantation for malignant disease (hr: 2,26, 95% ci, 0,97-5,27, p=0,05) and the presence of agvhd (hr: 4,04, 95% ci, 2,01-8,1, p< 0,001). the overall survival of children with hemorrhagic cystitis was 46,3% vs 69,3% of those who didn't manifest this complication, but in multivariable analysis for survival cystitis wasn't a statistically significant risk factor. conclusions: according to our results, stem cell transplantation in children > 6 years old who suffer from a malignant disease and the presence of agvhd consist independent risk factors for the manifestation of bk virus associated hemorrhagic cystitis. the identification of the risk factors of this serious complication will contribute to better management of transplanted patients. further research through prospective trials can contribute to the better understanding of the pathophysiology of hemorrhagic cystitis and to the establishment of appropriate diagnostic and therapeutic guidelines. disclosure: nothing to declare p442 impact of natural killer cell reconstitution on outcomes in patients with early cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation background: cytomegalovirus (cmv) reactivation influences survival after allogeneic hematopoietic stem cell transplantation (sct) and induces natural killer (nk) cell expansion. we evaluated nk cell reconstitution and clinical outcomes following early cmv reactivation after sct. methods: lymphocyte subsets were measured by flow cytometry on day 100 in patients with hematologic malignancies undergoing sct between january 2009 and december 2017 at kanagawa cancer center, excluding patients with graft failure or death within 100 days. cmv reactivation was defined as initiation of preemptive cmv therapy following pp65 antigenemia surveillance. results: the subjects were 152 males and 94 females with a median age of 51 years (range: 18-69 years). the median follow-up period for survivors was 3.2 years (range: 0.8-9.6 years). there were 128 patients with acute myeloid leukemia, 64 with acute lymphoblastic leukemia, 32 with myelodysplastic syndromes, and 22 with other diseases. at transplantation, 166 patients were standard risk and 80 were high risk. myeloablative conditioning and reduced-intensity conditioning were employed in 99 and 147 patients, respectively. bone marrow transplantation, peripheral blood stem cell transplantation, and cord blood transplantation was performed in 98, 36, and 112 patients, respectively. cmv reactivation occurred in 141 patients (57%) at a median of 45 days (range: 15-93 days) after sct. grade ii-iv acute gvhd and chronic gvhd affected 89 patients (36%) and 80 patients (33%), respectively. among all patients, 5-year overall survival (os), cumulative nonrelapse mortality (nrm), and cumulative relapse (cir) rates were 66%, 10%, and 33%, respectively. in patients without cmv reactivation (cmvr-) versus patients with cmv reactivation (cmvr+), 5-year os, nrm, and cir were 79% vs. 55% (p < 0.001), 3% vs. 16% (p = 0.005), and 27% vs. 38% (p = 0.142), respectively. among all patients, the median level of cd3-cd56+ cells, cd16 +cd57cells, and cd16+cd57+ cells on day 100 was 236/μl (range: 19-2150/μl), 275/μl (9-2660/μl), and 98/μl (3-1767/μl), respectively. nk cell subsets showed no significant differences between cmvr-and cmvr+ patients. when patients were divided into low and high groups at the median level of each nk cell subset, cmvr+ patients with high cd3-cd56+, cd16+cd57-, or cd16+cd57+ cells showed significantly better 5-year os than those with low cells (72% vs. 35%, p < 0.001; 69% vs. 40%, p < 0.001; 66% vs. 45%, p = 0.001, respectively). high cd3-cd56+ cells were significantly associated with lower nrm (7% vs. 26%, p = 0.002), while high cd16+cd57-cells were significantly associated with lower cir (26% vs. 50%, p = 0.007). multivariate analysis confirmed these nk cell subsets as prognostic factors in cmvr+ patients. conclusions: nk cell reconstitution may contribute to improved transplantation outcomes in subgroups of cmvr + patients. disclosure: nothing to declare background: rezafungin (rzf) is a novel echinocandin in development for prevention of invasive fungal infections caused by candida, aspergillus, and pneumocystis spp. in patients at high risk of infection. rzf has demonstrated in vivo prophylaxis efficacy and low risk of drug-drug interactions. furthermore, the stability and pk profile of rzf allow for once-weekly dosing. rzf is also in development for treatment of candidemia and invasive candidiasis using a dosing regimen of rzf 400 mg followed by 200 mg once-weekly, which achieved >90% target attainment against candida. while lower doses might be useful to prevent candida and pneumocystis, invasive aspergillosis is a different challenge. we evaluated rzf dosing for prophylaxis against aspergillus fumigatus in blood and marrow transplant (bmt) patients using pk/pd simulations of the treatment dosing regimen. methods: a previous population pk model was refined using data from phase 1 and phase 2 trials of iv rzf (nonmem vers 7.2). stepwise forward selection (α = 0.01) and backward elimination (α = 0.001) were used to assess for relationships between interindividual pk variability and covariates, such as age, sex, bsa, albumin, liver and renal function markers, and infection status. the final model was validated by comparing model-based predictions to observed data. the model and demographic data from 100 bmt recipients at stanford medical center were used for monte carlo simulation (n=2,000) of expected rzf concentrationtime profiles in bmt patients receiving iv rzf 400 mg on week 1 followed by 200 mg weekly x 11. of the 100 patients included in the demographic dataset, 39 were female (mean values at baseline: age, 58 years [19-75 years]; weight, 69.8 kg [42-123 kg] ). the median (range) bsa in the demographic dataset was 1.84 m 2 (1.27-2.61), and albumin was 3.35 g/dl (1.8-4.43 g/dl) . free-drug concentration-time profiles were evaluated (97.4% human protein-binding) relative to the a. fumigatus minimal effective concentration required to inhibit 100% of isolates tested (mec 100 ; jmi 2014-2017 sentry international surveillance data). results: the population pk model was a linear, 4compartment model with zero order iv input. albumin, sex, infection status, and body surface area were statistically significant predictors of interindividual variability; clinical significance of these factors was not determined. the model provided precise, unbiased fits to the observed data (r 2 =0.993 observed vs individual-predicted concentrations). rzf plasma free-drug concentrations at weeks 1, 2, and 12 were above the a. fumigatus mec 100 (0.03 mg/l) for the entire dosing interval in 98.0%, 92.0%, and 90.2% of simulated patients, respectively, and in ≥99.8% for all 12 weeks based on the mec 90 (0.015 mg/l). conclusions: these data modelled from bmt patients support the rzf dosing regimen of 400 mg iv followed by 200 mg once-weekly for prophylaxis against a. fumigatus. current antifungal prophylaxis may be limited by toxicity, ddis, or patient factors such as mucositis. the pk of rzf and its spectrum, safety, tolerability, and lack of ddis may address current unmet needs in ifi prophylaxis for bmt and other immunocompromised patients. disclosure: janice brown: research funding, cidara therapeutics, elizabeth lakota: research funding, cidara therapeutics, shawn flanagan: employment, cidara therapeutics, taylor sandison: employment, cidara therapeutics, voon ong: employment, cidara therapeutics, christopher rubino: research funding, cidara therapeutics p444 is fungal prophylaxis necessary in non myeloablative peripheral blood stem cell allogeneic transplantation in the pre-engrafment period? julien vaidie 1 , jean-baptiste woillard 1 , stéphane girault 1 , marie-laure dardé 1 , arnaud jaccard 1 , daniel ajzenberg 1 , bernard bouteille 1 , pascal turlure 1 background: non myeloablative peripheral blood stem cell transplantation (pbsc), by limiting toxicity, can be proposed to elderly patients or patients with comorbidities. however, fungal infections remain a key issue that can negatively impact outcome, and increase duration and cost of hospitalization. systematic fungal prophylaxis have demonstrated benefits in outcome in the context of myeloablative conditioning but are not currently in reduced intensity conditioning allograft with pbsc. fluconazole prophylaxis is currently recommended in this situation (ecil). methods: primary objective of this retrospective study was to evaluate fungal infection incidence after allograft procedure in patients who received a non myeloablative allograft with pbsc in limoges university hospital between june 2009 and june 2018. patients received fludarabine 30 mg/m2/day between d-6 and d-2 before allograft and busulfan 3.2 mg/kg/day at d-4 and d-3. gvh prophylaxis consisted in rabbit anti-lymphocyte serum at the dose of 2.5 mg/kg at d-2 and d-1, and ciclosporin at the beginning dose of 3mg/kg per os twice a day. mycophenolate mofetil was adding for patients with hla-matched or mismatched unrelated donors. patients did not systematically receive antifungal prophylaxis during the neutropenic pre-engraftment period. when patients had fever during more than 72 hours, an empirical fungal treatment (caspofungine) was added to empirical antibiotics. as soon as neutropenic recovered and in the case of apyrexia without microbiologic documentation, antimicrobial treatments were stopped while in the case of microbiologic documentation, treatments were adjusted to germ in term of dosing and time of administration following recommendations. however, some patients received antifungal azole prophylaxis during the neutropenic pre-engraftment period in case of history of previous invasive aspergillosis (ia), or a nasal colonization by aspergillus. in post-engraftment period, posaconasole prophylaxis was administered for patients with systemic corticotherapy for acute graft-versus-host disease. results: 91 patients were evaluated (median [min-max] age of 60 [33-71] years). 20% of patients received an hlaidentical related donor, 69% an hla-matched related donor and 11% an hla-mismatched unrelated donor. the five years overall survival and survival without relapse or gvhd were 70% ic95 [59%-81%] and 62% respectively ic 95 [52%-73%]. the median time for neutrophil recovery was 15 days. 79 patients did not receive prophylaxis and only 12 patients received systematic fungal azole prophylaxis in the pre-engraftment period. two patients received an empirical treatment by caspofungine. only 1 ifi was documented during the neutropenic period : candida krusei in blood culture. in the post engraftment period, 37 patients with acute gvhd treated by corticotherapy received an antifungal prophylaxis by posaconazole and only 1 patient had a probable ia at day 100 despite prophylaxis by posaconasole. conclusions: except for patients with previous history of ifi, our results provide additional arguments against systematic fungal prophylaxis after reduced intensity conditioning with pbsc allogenic transplantation in the pre-engraftment period with a very low incidence of invasive fungal infections. in post-engrafment period, posaconazole prophylaxis is required for patient with gvhd treated by corticotherapy. disclosure methods: a simple, rapid and sensitive method using hplc with a diode-array detector (dad) was developed and validated for the quantification of letermovir in human serum using sorafenib as internal standard. after pretreating serum samples by liquid-liquid extraction with tert-butyl methyl ether, separation was achieved on a x-terra rp-18 column (dimension 150 x 2.1mm, 5μm) at 30 0 c using gradient elution with a mobile phase of 20 mm ammonium bicarbonate ph 7.8 (mobile phase solvent-a) and acetonitrile:20mm ammonium bicarbonate ph 9.2 (mobile phase solvent-b). samples were eluted at a flow rate of 0.3ml / min throughout the 20-minute run. uv wavelength mode was used, detection was at 260 nm. results: the calibration curve was linear (r > 0.99) in a concentration range of 25 -5000 ng / ml for letermovir. the hplc assay established for letermovir determination showed a high rate of accuracy and precision with an intraday variability of -9.21 to 12 % (accuracy) and 0.31 to 3.75 % (precision) and an interday variability of -2.6 to 6.9 % (accuracy) and 3.74 to 7.41 % (precision), respectively. 16 letermovir serum concentrations of 9 patients (8 male / 1 female, mean age 66.3 years) were determined in daily clinical practice. the mean concentration was 4752 ng / ml (median 4163 ng / ml, standard deviation 3925 ng / ml, range 89 -11452 ng / ml). conclusions: the newly developed hplc method is useful for the determination of letermovir concentrations. patient samples analyzed in a routine clinical setting demonstrated considerable interindividual variability. all measured concentrations were above the ec50 of letermovir. monitoring the concentration of letermovir could help to prevent over-or underexposure, especially in patients with polypharmacy which is frequent in allogeneic hematopoietic stem cell transplant recipients. disclosure background: the use of preemptive strategy (pet) has lowered the incidence of cmv disease in allo-sct to 5-10%. nonetheless the use of this strategy implies that more than 50% of seropositive patients will replicate cmv. several studies have shown that cmv replication is detrimental for patient survival although the viral load related to this bad outcome variates among studies. objective: to analyse thel impact of cmv replication in overall survival (os) in allo-hct patients. methods: to analyse the impact of cmv replication in os we perform a unicentric, retrospective study on 117 consecutive first allo-hsct patients transplanted between jan-2015 and oct-2018 with a median follow-up of 398 days (5-1266). all patients were monitored post-hct with real time pcr cobas-taqman® /cobas6800® (rtpcr) in plasma. the cut-off for inception of pet was 150 iu/ml. cmv mutations (ul54/ul97gene), were studied in plasma samples by sanger sequencing, median cmv viral load 3130 iu/ml (565-23900). results: patients (117): women/men (49%/51%), median age was 55 years (range 18-71). 101 identical allogeneic scts (86%), 16 haploidentical scts (14%). donors were related in 60 cases (51%) and 57 (49%) unrelated. progenitors source was 98% peripheral blood and 2% bone marrow. cmv status was (d+/r+) in 51%(n=60), (d+/r-) in 9%(n=11), (d-/r+) in 26% (n=31) and (d-/r-) in 11% (n=13), unknown 2 cases. positive pcrs were detected in 63 patients: one episode in 41 (65%); 2 episodes in 15(24%) and 3 to 5 episodes in 7 patients (11%). fifty-five patients (87%) received preemptive therapy. fourteen episodes (25%) were refractory/ probable refractory cmv infections (according to the criteria of chemaly r. cid 2018). a resistant mutation (ul54 gene) was detected in one patient with refractory infection patients that developed cmv infection had an inferior non-significant os at 3 years (61,3% vs 76,8% log-rank p 0,346). those patients that received pet for cmv had a significant inferior os compared with those that replicate cmv but didn't receive preemptive therapy (55,6% vs 100%, log rank p=0.04). os of patients that received pet was inferior compared with those without pet (with or without infection) (55,6% vs 79,9%, logrank p 0,05). no difference in survival was found for those patients treated pre-emptively that were refractory vs no refractory (53% vs 57,1%, log-rank p 0,51). conclusions: patients that received preemptive therapy had a significant inferior overall survival compared with those that didn´t replicate and those that replicate cmv but didn't receive preemptive therapy. this reinforce the relevance of prophylactic strategies for cmv with drugs with good safety profile like letermovir that in a randomised trial proved to decrease the need for preemptive therapy. disclosure: rafael, de la camara: has received grants from astellas, gilead, janssen, merck, novartis and pfizer clinical evaluation of stenotrophomonas maltophilia infection in allogeneic hematopoietic stem cell transplant recipients -retrospective single-center data analysis negative bacillus that causes severe infections associated with high morbidity and mortality in immunocompromised patients. the aim of our study was to determine incidence, characteristics and outcome of s. maltophilia infection in patients (pts) who underwent allogeneic hematopoietic stem cell transplantations (allo-hsct) in institute of hematology and transfusion medicine between october 2010 and november 2018. methods: we retrospectively evaluated incidence, clinical features and outcome of s. maltophilia infections in 338 consecutive patients with median age-42 years (range 19-71), who underwent allo-hsct from unrelated donors -238 (70.4%), matched sibling donors -87 (25.7%) and haploidentical donors -13 (3.9%) in our center. s. maltophilia was detected by culture-based microbiological tests. invasive infection was defined by isolation s. maltophilia from cultures in the presence of both clinical symptoms and signs of infection -blood stream infection (bsi), pneumonia with or without pulmonary haemorrhage. the only colonization status was defined as s. maltophilia culture-positive samples in the absence of infection symptoms. in vitro susceptibility tests to antibiotics were performed. results: 21 pts (6.2%) with median age-46 years (range 19-66) with s.maltophilia culture positive samples were identified. 19 (90.5%) underwent allo-hsct from unrelated donors, 1-from matched sibling donor and 1-from haploidentical donor. among them bsi developed in 7 pts (33.3%), pneumonia in 4 pts (19%) -with fulminant and fatal pulmonary hemorrhage in 3 pts (14.3%). all patients with pneumonia demonstrated bsi. positive sputum cultures were detected in 6 pts, in 4 pts hemoptysis was observed. the rest of isolated strains were identified as colonization (throat -in 3 pts, stool -in 9 pts). all 7 patients with invasive s. maltophilia infection before pathogen identification demonstrated persistent fever despite of the use of broadspectrum antibiotics (carbapenems, glycopeptides, aminoglycosides, colistin), prophylactic antifungals and antivirals. all of them received fluoroquinolone (ciprofloxacin) as a standard antibacterial prophylaxis before neutropenic fever occurred. all 3 patients (100%) with bsi, pneumonia and pulmonary hemorrhage died before engraftment (anc -0.0 g/l) -2 of them during 48-72 hours from the onset of a positive blood culture for s. maltophilia. the c-reactive protein (crp) concentration before identification of s. maltophilia invasive infection was > 26x-69x upper normal limits (unl). susceptibility to antibiotics of isolated strains from blood and sputum was respectively: 86% and 100% for ceftazidime, 100% and 100% for trimethoprim-sulfamethoxazole, 64% and 50% for levofloxacin; while 56% and 50% strains were resistant to ciprofloxacin. 1-year overal survival (os) and 2-y os for this group was 52.4 % and 47.6% respectively compared with 77.2% and 68.3% for group without s. maltophilia infection. conclusions: s. maltophilia invasive infections are associated with high morbidity and mortality in allo-hsct recipients especially in the period from conditioning therapy to engraftment. an exposure to broad-spectrum antibiotics in the treatment of neutropenic fever or confirmed bacteremia of other etiology is one of risk factors of breakthrough s. maltophilia infections. empiric therapy against s. maltophilia in selected patients in risk of such infection before pathogen identification may be lifesaving procedure. disclosure: nothing to declare. role of cmv reactivation following allogeneic stem cell transplantation in preventing relapses in patients with acute myeloid leukemia background: cytomegalovirus(cmv) reactivation is common in patients undergoing allogeneic stem cell transplantation. it has been shown recently that cmv reactivation is associated with reduced risks of relapse in patients undergoing allogeneic stem cell transplantation for aml. however the analysis of cibmtr data did not show any effect of cmv reactivation on relapse. with this background we conducted an analysis of patients suffering from aml who are undergoing allo-sct for their long term disease free survival with respect to cmv reactivation. methods: after obtaining permission from hospital medical records committee, we retrospectively analysed data from electronic medical records of patients undergoing allo-sct for aml at our center between january 2013 to august 2018. patients who underwent matched sibling, matched unrelated and partially matched allo sct were included. all patients underwent cmv monitoring with weekly pcr starting from the time of engraftment till d+100 following allo sct. value of ≥ 1000 copies/mcl was considered as cut off for initiation of treatment in matched sibling donor transplant but in unrelated donor or partially matched donor transplants, ≥ 500 copies/mcl was used as cut off for initiation of pre emptive therapy. results: total of 96 patients were included in study. median age was 28.01 ± 17.09years (2-69yrs). 60(62.5%), 30(31.3%) and 6(6.3%) patients underwent matched sibling, haplo (partially matched) and mud transplantation respectively. median follow up was 15 months(1-73months). (table 1) acute gvhd (grade2-4) was observed in 50(52.1%) of patients. cmv reactivation occurred in 70(72.9%) of patients. overall survival at last follow up was 63.5% (61/ 96patients). 17(17.7%) patients relapsed during follow up. relapse free survival at at last follow up was 61.5%. 64(90.1%) of 70 patients who had cmv reactivation didń t relapse, whereas 10(40%) of 25 patients who didn´t have cmv reactivation relapsed which was statistically strongly significant p < 0.001. (figure 1 ) similar results were seen in recently published paper from japanese society for hematopoietic cell transplantation (jshct) transplantation-related complication working group. conclusions: 1. cmv reactivation following allo sct had beneficial effect on preventing relapse in patients with aml. 2. probable immune activation resulting due to cmv reactivation may result in better graft versus leukemia effect preventing subsequent relapses. [ background: human herpesvirus 6 (hhv-6) causes lifethreating central nervous system disorders such as encephalitis after allogeneic hematopoietic stem cell transplantation (hsct). recent studies showed that cd134, a member of the tumor necrosis factor receptor superfamily, has been implicated as a specific receptor of hhv-6b, and that its expression levels in cd4-positive t cells after hsct could be related to the reactivation of hhv-6. real-time quantitative polymerase chain reaction analysis (qpcr) is the most commonly used method for detecting and evaluating hhv-6 reactivation after hsct, but more sensitive detection method is required. we recently developed a new monitoring method for hhv-6 reactivation using digital pcr (dpcr) which provides high sensitivity of detecting hhv-6 dna in clinical samples. in this prospective study, we evaluated the relationship between hhv-6 reactivation monitored by dpcr and expression of cd134 on cd4 + t cells before and after allogeneic hsct. methods: thirty-four patients who underwent allogeneic hsct for hematological diseases at keio university hospital (tokyo, japan) between january 2017 and march 2018 were consecutively enrolled into this study. peripheral blood samples of the patients were obtained before the conditioning (pre), the day of transplant (day 0), and weekly during the first month after transplantation (days 7, 14, 21, and 28) . hhv-6 viral load in plasma was quantitatively measured by dpcr. the primers and a probe of dpcr for hhv-6b were selected from immediate-early 1 (ie-1) protein transactivator region (u90). we evaluated the relationship between hhv-6 reactivation and the serial expression rates of cd134 in cd4 + t cells (cd134/cd4 ratio) measured by flow cytometry before and after hsct. results: median age of the patients was 51.5 years. onethird of patients received cord blood as a stem cell source. hhv-6 reactivation was detected in 23 patients (68%) with dpcr. a comparison of cd134/cd4 ratio between the patients with and without hhv-6 reactivation after hsct revealed that cd134/cd4 ratio was significantly higher in patients with hhv-6 reactivation than those without before conditioning ( in contrast, there was no such significant difference after transplant (days 7 to 28) . in multivariate analysis, higher cd134/cd4 ratio before conditioning (odds ratio (or) = 10.5, 95% confidence interval (ci): 1.3-85.1, p = 0.03) and stem cell source from human leukocyte antigen mismatched donor (including all cord blood transplantation cases) (or = 15.4, 95%ci: 2.0-121.0, p = 0.04) remained to be significantly associated with the incidence of hhv-6 reactivation. conclusions: higher cd134 expression rate in cd4 + t cells before hsct was associated with higher risk of hhv-6 reactivation, which could be a promising marker for predicting hhv-6 reactivation after allogeneic hsct. careful observation and monitoring may be needed in cd134 highly expressed patients. it is a subject of further research to clarify the role of cd134 + cd4 + t cell in hhv-6 reactivation. disclosure: nothing to declare. methods: criteria for the administration of ici (vistide) were grade iii-iv (clinically significant hematuria with clots) bk-related hemorrhagic cystitis after allo-hct which showed no improvement after symptomatic therapy with hyperhydration and bladder irrigation. cidofovir was diluted in 100 ml of normal saline and installed via a foley catheter which was blocked for 1 hour. not knowing the level of absorption of the drug we decided to give probenecid prophylaxis in all patients. ici was repeated weekly according to severity of symptoms. urine and plasma bkv viral loads were quantified by rq-pcr results: six patients (median 38 years, 29-51) received ici after allo-hct. patients had haematological malignancies (aml 2, all 3, mds 1), received busilfex-based myeloablative conditioning and a graft (pbsc 5, bm 1) from a 7/8 hla-matched (4pts), 8/8 (1pt) or haploidentical (1pt) donor. median time for the onset of bkv-hc after allo-hct were 53.5 days (range 32-117). all patients were under standard cyclosporine prophylaxis and none of the patients had any signs of acute gvhd at the time of onset of hc. the median pcr-bkv viral load at the onset of bkv-hc in urine and plasma were 5.75x10 8 (range 0.15x10 8 -59x10 8 ) and 141.5 (range 0-255), respectively. the median maximum pcr-bkv viral load in urine and plasma were 37.5x10 8 (range 4.7x10 8 -450x10 8 ) and 1.100 (range 0-400.000), respectively. five patients had impaired renal function (median egfr 49 ml/min, range 33-53) at first ici which was probably multifactorial. the median dose of intravesical cidofovir was 5 mg/kg (range 2.5-5 mg/kg) and a median number of 3.5 instillations (range 2-7) were given. in 5/6 cases symptoms of cystitis improved dramatically and hematuria resolved. virological response (at least 1 log reduction) was observed in all 5 cases. two patients experienced relapse of hemorrhagic cystitis and were retreated with ici which resulted in resolution of the symptoms and the hematuria. no deterioration of renal function of other systemic adverse effects were observed. after a median follow up of 190.5 days after transplantation (range 105-360), 3/6 patients are alive without cystitis symptomatology and 3 died (1 due to relapse and 2 due to trm). conclusions: in this retrospective study we propose that local therapy of bkv-hc with ici is safe and has high clinical and virological response rates. the administration of ici after allo-hct should be controlled in prospective randomized trials. disclosure: nothing to declare background: since cmv-preemptive therapy approach was implemented, cmv disease frequency is very low. however, cmv reactivation and the need of using nephrotoxic plus/less myelotoxic drugs is very frequent. in addition to the toxicity of the medications to avoid cmv disease, other potential adverse effects of cmv have been mentioned in medical literature. in this study, we wanted to estimate how recipient/donor serologic status influences the outcome of allo-hsct in our most recent series of patients. methods: the population analyzed for this report is the all 224 patients who underwent allo-hsct during the 4-year period from october 2014 september 2018 in our unit. median age at transplant was 52 years (range: 7-69). one hundred and thirty were male (58%) and 94 were female (42%). baseline diseases were: 83 aml, 51 lpd, 29 all, 29 mds, 14 mpd, 12 mm, and 6 bmf. donor was unrelated in 120 transplants (54,5%) and was family in 104 (45,5%) (including 34 haplo-identical). conditioning regimen was ric in 121 procedures (54%) and intensive in 103 (46%). stem cell source was pb in 210 (93,7%) and bm in 14 cases (6,3%). median follow-up was 23 months (range: 3-50). patient's and donor's cmv igg were positive in 188 (82,6%) and 92 (58,5%), respectively. recipient/donor serology was +/-(risk group 1) in 62 (27,7%), +/+ (risk group 2) in 126 (56,3%) , -/+ (risk group 3) in 6 (2,6%) y -/-(risk group 4) in 30 (13,4%). results: two pts underwent a second transplant before day +100 due to graft failure. overall mortalities (om) at days +100 and +365 of the rest of the series (222 pts) are shown in table. the highest risk group (recipient cmv + / donor cmv -) exhibited more than double om at day +100 and more than four times om at day +365, when compared with pts at lowest risk (recipient cmv -). those striking differences were mainly due to nrm. om for risk group ii (recipient cmv + / donor cmv +) was intermediate. conclusions: in our studied population, mainly adult patients, the combination of cmv-seropositive patient with a cmv-seronegative donor had a very clear adverse impact on hsct outcome. as a result, we considered that the election of a cmv-positive donor for a cmv-positive patient continues to be strongly advisable, whenever is possible. on the other hand, once letermovir has proved to be efficient and well-tolerated and has been licensed for prophylaxis of cmv in high risk recipients, this approach appears to be very attractive to try to avoid the adverse impact of recipient cmv-seropositivity, particularly when finally chosen donor is cmv negative. disclosure: nothing to declare an active surveillance and an early and individualized management is critical to avoid mortality from respiratory viral infections in allo-hsct recipients background: respiratory viral infections (rvis) are frequent among the general population. in transplant recipients, rvis are known to cause an important morbidity and potential mortality. for this reason and several others, as the need of preventing other pts from contagious or avoiding misdiagnosis with other infections processes, a high index of suspicion of vris is necessary. during the last few years, we have implemented an active and systematic surveillance policy orientedto early detection and management of rvis in the hsct recipients. methods: the population analyzed for this report is the 175 patients who underwent allo-hsct from january 2015 through march 2018 in our unit. median age at transplant was 51 years (range: 7-69). one hundred and four were male (59.4%) and 71 were female (40,6%). baseline diseases were: 64 aml, 38 lpd, 23 all, 19 mds, 15 mpd, 10 mm, and 6 bmf. donor was unrelated in 92 transplants (52.6%) and was family in 83 (47.4%) (including 29 haplo-identical). conditioning regimen was reduced in 91 procedures (52%) and intensive in 84 (48%).stem cell source was pb in 163 (93.1%) and bm in 12 pts (6.9%).median follow-up was 27 months (range: 8-46); at the close of the analysis, majority of the series (89.1%) had a follow-up superior to one year from hsct. a throat swab(ts) was taken from every patient with any, even minor, respiratory symptoms. the respiratorysamples were tested whith a complete pcr panel of human respiratory viruses: rhinovirus (rv), influenza a and b virus (iv-a, iv-b), parainfluenza virus (pivs 1-4), respiratory syncytial virus (rsv), metapneumovirus (mpv), coronavirus (cov), adenovirus (adv), and bocavirus (bov). results: day +100 overall mortality of the series was 8,6%. day +365 overall mortality was 25,1% (13,7% nonrelapse mortality -nrm-, and 11,4% progression/relapse mortality). causes of nrm reflected in table 1. no patients died due to rvis. from 1 st july 2016 through 30 th june 2018 (a 24-month period), 581 ts samples were obtained from 129 pts (73,7%).the median number of samples/patient was 3 (range: 1-17).a total of 162 (1-8) rvis episodes were diagnosed in 87 pts (49,7%).the median presentation of the first rvi was at the day + 226 (3-924) post-hsct. the viral distribution was: 47 rv (26.6%), 36 iv (20.3%), 33 piv (18.6%), 28 rsv (15.8%), 12 mpv (6.8%), 11 cov (6.2%), 9 adv (5.1%), and 1 bov (0.6%).there were 13 mixed (two or more viruses) rvi episodes. the temporary distribution of vri episodes is shown in figure 1 . conclusions: 1) symptomatic infections due to respiratory viruses are very frequent among the allo-hsct recipients. 2) a high level of suspicion, as well as an early and systematic screening and management policy, are critical to avoid potential attributable mortality and the nosocomial spread of rvis among the transplant recipients. 3) in our series, rhinovirus, parainfluenza and adenovirus might be detected at any moment of the year; the rest of the viruses showed a clear seasonal pattern (november to april). [[p452 image] 1. background: trimethoprim-sulfamethoxazole (tmp-smx) is the most suitable drug for prophylaxis against pneumocystis pneumonia and infections with toxoplasma after allogeneic haematopoietic stem cell transplantation (allo-hsct). allergic reactions or hypersensitivities, mainly exanthemas, occur in about 3-5 % of the patients, usually resulting in the use of alternative prophylactic drugs (e.g. pentamidine or atovaquone). it has been hypothesised that allergies might be cured with allo-hsct. methods: we conducted a retrospective chart review of patients with tmp-smx re-exposition after allo-hsct from december 2017 to september 2018. follow-up is current as of december 2018. results: six patients (f/m: 4/2, median age: 45 years, range: 25 -66 years) with a history of tmp-smx hypersensitivity prior to allo-hsct were re-exposed to tmp-smx after engraftment of a matched related (mrd, n=1) or matched unrelated (mud, n=5) donor. median time to re-exposition was 18.5 (range: 11-341) days after allo-hsct with one oral dose of tmp-smx. in four patients, tmp-smx was tolerated without any signs of hypersensitivity reactions and has been continued for a median of 231 days (range 87-315) until last followup. one patient (mud, re-exposition at d+341) experienced pruritus and erythema some hours after tablet intake. another patient (mud, re-exposition at d+18) developed an exanthema one day after re-exposition which was later diagnosed as a cutaneous gvhd. conclusions: re-exposition of tmp-smx in patients with prior hypersensitivity is feasible after allo-hsct. after successful re-exposition, patients can be treated with the best-studied drug for prophylaxis of infections with pneumocystis and toxoplasma. disclosure: nothing to declare brincidofovir for adenoviremia in paediatric hsct for primary immune deficiency background: reactivation of adenovirus is a severe complication of hsct associated with significant morbidity and mortality, particularly for children with primary immune deficiency (pid). the only drug currently licensed to treat adenovirus infection is cidofovir. brincidofovir is a lipidlinked derivative of cidofovir which has been shown to be a safe and effective alternative treatment to cidofovir. there is limited data describing the use of brincidofovir in patients undergoing hsct for primary immune deficiency. we reviewed all patients who received brincidofovir after undergoing hsct for primary immune deficiencies between 2016 and 2018 at the great north children's hospital, newcastle upon tyne, uk. results: of 78 patients transplanted for pid, 11 developed significant adenoviraemia (14%). all were treated with cidofovir initially but 6 were switched to brincidofovir because of a failure to respond or because of renal toxicity. of these, 4 resolved their adenoviraemia within 21 days of commencing treatment (figure 1). donor sources were tcr alpha/beta/cd19 depleted haplo-identical (n=4), tcr alpha/beta/cd19 depleted mmud (n=1) and 12/12 mud (n=1). patients were conditioned with treosulphan/fludarabine/thiotepa/atg/ rituximab (n=4), treosulphan/fludarabine/atg/rituximab (n=1) or treosulphan/fludarabine/alemtuzumab/ gcsf/plerixafor (n=1). occurrence of agvhd and treatment of agvhd are outlined in table 1. patient 5 died +111 days post-transplant of multi-organ failure, severe thrombotic microangiopathy and sepsis. although patient 2 initially responded to brincidofovir, reactivation occurred after cessation of treatment; severe diarrhoea precluded the reintroduction of brincidofovir and the adenoviraemia persisted with poor immune reconstitution. treatment with addback t cells was attempted however the patient died 194 days post-transplant after a cerebral haemorrhage. patient 3 had long-standing chronic diarrhoea which was thought not severe enough to warrant cessation of brincidofovir treatment. conclusions: the complete resolution of adenoviraemia in 4/6 patients who had previously failed to respond to prior therapy with cidofovir suggests that brincidofovir may be an effective treatment option for adenoviral reactivation post-hsct for pid. however, resolution of adenoviraemia is influenced by many other factors, including the adequacy of immune reconstitution, the degree of induced immune suppression and the presence of comorbidities such as gvhd. due to the small sample size it was difficult to assess the relative importance of these factors in this cohort. brincidofovir was well tolerated however its effectiveness may have been limited by poor gastrointestinal function in one patient (patient 3) and could not be used after a viral reactivation in another for the same reason. further studies of the use of brincidofovir in this specific cohort are needed to clarify the role and effectiveness of this treatment. background: there is a high prevalence of cmv seropositivity in algerian population. because of high morbidity and mortality in pts who underwent allo sct with cmv reactivation, effective surveillance and timely treatment using anti-viral therapy s required. the risk of cmv reactivation depends on the type of stem cell source, immunosuppression (is) and serological status of the donor/ recipient pair. methods: over a 24 months period (from 01/01/2016 to 31/12/2017), 254 pts underwent allo-hsct for malignant or non-malignant hematology diseases of which 246 pts are evaluated for this study. cmv reactivation was observed in 66 pts (26.82%) (aml: 33 pts, all: 8 pts, cml: 4 pts, multiple myeloma: 2 pt, nhl skin: 1 pt, primary myelofibrosis: 1 pt, aplastic anemia: 14 pts, fanconi anemia: 2 pts, β-thalassemia: 1 pt), with a median age of 30 years (4-54), sex ratio (m/f) of 1.75. allo-hsct done with sibling donors: 51 pts, haplo-identical donors: 14 pts and pheno-identical donor: 1 pt. all pts were treated by chemotherapy alone with myéloablative conditioning (mac) in 41 pts and reduced intensity (ric) in 25 pts. all pts received peripheral blood stem cells with an average rate of cd34 + cells: 7, 8.10 6 /kg (3.4-12.7). additional bone marrow graft was used in 7 pts that received a haploidentical graft without pt-cy. gvh prophylaxis associated cyclosporine (csa) and methotrexate (sibling and phenoidentical); csa-mtx-mmf or csa-mtx-cyclophosphamid (haplo-identical). before transplantation, donor/recipient pair is at high risk reactivation in 65 pts (98.4%). detection of cmv reactivation done by antigenaemia pp65 or by quantitative pcr weekly for the first 3 months and during an is treatment for acute or chronic gvhd. pre-emptive therapy is initiated by ganciclovir as soon as positivity of antigenaemia or increased viral load in pcr. results: a first reactivation occurred on average day 44 (16-83) in 66 pts (26.82%) of which 32 pts under corticotherapy for acute gvhd (27 pts), thrombotic micro-angiopathy (1 pt) and renal failure (4 pts) or due to a reinforced is for haplo-identical transplantation (5 pts). one pt with chronic gvhd presented a late reactivation 18 months after transplant. twenty pts presented a 2 nd reactivation on average day 135 (106-180) and 8 pts a 3 rd reactivation on average day 136 (130-143). pre-emptive treatment is introduced in the first episode by a viral dna polymerase inhibitor (ganciclovir: 22 pts; valganciclovir: 43 pts, foscarnet: 1 pt). the negativity of antigenaemia is observed on average at 5 days of treatment (3) (4) (5) (6) (7) . second line treatment was required in 14 pts (22%) due to resistance (12 pts), severe cytopenia (1pt) or renal failure (1 pt). the onset of severe cytopenia imposed a dose reduction (4 pts) or a therapeutic stop (6 pts) before 15 days. two pts received additional maintenance treatment for negativation delay. three pts (4.5%) died from cmv infections resistant to antiviral treatment (pneumonia: 2, colitis: 1). conclusions: cmv infection is a serious complication after allo-hsct. in the absence of vaccination, the systematic monitoring for cmv reactivation is strongly recommended for the establishment of a rapid and effective preemptive treatment. disclosure: nothing to declare p456 abstract withdrawn. results: in transplanted group, episodes of bkv reactivation occurred in 54 patients (42 %). in 26 cases only urine colonization (c) found before hsct. in this group in 10 patients (39 %) virus was transmitted from urine to the blood (b) . dysuria and/or hc were observed in 4/26 (15%) patients . all of them (100%) had urine and serum involvement. in 28 cases bkv replication was found after hsct (3 -cases detected in urine, 25 cases-bothserum and urine). dysuric syndromes and/or hc were found in 5/28 of cases (18%)-all in patients with serum and urine involvement. urinary tract was always first location of the virus. there was no case of isolated serum reactivation. the incidence of bk infection was higher in patients older than > 5 yrs (p< 0.05), transplanted from family donor (msd) (p< 0.05). mud recipients had more often both serum and urine reactivation (p< 0.05) than isolated urine involvement. sex, day of neutrophil recovery, conditioning regimen, or use of total body irradiation were not significant risk factors for bkv infection, or hc . six patients were treated with cidofovir (range 1-4 doses) with good response. there was no death due to evident bkv infection. conclusions: bkv reactivation remains one of the most frequent infectious complication in children undergoing allogeneic hsct. most of patients experienced mild infection and age < 5 years was the positive prognostic factor influencing its incidence. bkv monitoring and prompt treatment of hc resulted in excellent outcome. we observed surprisingly high rate of new bkv replication after hsct. disclosure: nothing to declare background: high-dose chemotherapy (hd-ct) and auto pbsct have been the standard therapy for multiple myeloma (mm) for more than two decades, despite a wide range of new therapeutic options. recurrent/refractory malignant lymphomas and recurrent/metastatic germ cell tumors (gct) also benefit from this intensive therapy. in comparison to allogeneic transplantation, this treatment is known for lower complication rates, e.g. infections. however previous studies have schown that treatment related toxicity may not be underestimated and depending on the conditioning regimen used. methods: we retrospectively analyzed 193 patients (225 cases) who underwent hd-ct plus auto pbsct between 2011 and 2017 in a single-center study. to anlyze the incidence of infections depending on the conditioning regimen, we formed the following categories based on the agiho: no infections, neutropenic fever, sepsis and severe sepsis. results: the median age in this analysis was 59 years; 74.2% were male. the most frequent diagnosis was mm (62.7%) receiving high dose melphalan (mel), followed by malignant lymphoma (26.7%) receiving beam (bcnu, etoposide cytarabine, melphalan) and relapsed/metastatic germ cell tumours (gct) (9.8%) receiving high dose carboplatin/etoposide (ce). 8% of all patients developed severe sepsis, 5 patients had to be ventilated and 2 patients died. sepsis was documented in 25.3% of all cases (57 cases). the majority of patients (58.2%, 131 cases) developed neutropenic fever and 8.4% (19 cases) didn´t have any infection complications. the beam conditioning regimen showed the highest tendency to result in a septic course (43.3%), followed by ce (27.3%) and mel (24.1%). the most commonly documented pathogen in 153 blood cultures was s. epidermidis (41.7%), followed by e. coli (18.5%) and s. mitis (9.9%). only in one blood culture we detected a multi-resistant pathogen (3mrgn e. coli). p. aeruginosa was detected in 4 blood cultures (2.6%), l. monocytogenes in 2 (1.3%) and s. aureus in 6 (4%). 77.8% of all patients developed diarrhea, only in 10.9% of these cases we could detect c. difficile. the conditioning regimen shows no significant effect on the incidence of c. difficile. the mean neutropenic period was 13.5 days in malignant lymphoma patients, followed by 10.8 in mm patients and 8.7 days in gct patients. the hospital discharge, calculated from the day of transplantation, was significantly different: for malignant lymphoma the mean was 20.8 days, for mm 19.6 days and for gct 14.5 days. conclusions: our data correspond to former published results by many groups. the beam regimen shows the highest infectious complication rate followed by ce and mel. the duration of neutropenia and hospital stay depends on the conditioning regimen. the type of infectious complication doesn't effect the progression free-and overall survival in our analysis. disclosure: nothing to declare. impact of donor and recipient cytomegalovirus serostatus on outcomes of unrelated allogeneic haematopoietic stem cell transplantation background: cytomegalovirus (cmv) is an important cause of morbidity and mortality in allogeneic haematopoietic stem cell transplant (hsct) patients. the aim of our study is to evaluate the outcomes of our cmv seropositive recipients who received grafts from seropositive unrelated donors (d+r+) compared with grafts from seronegative unrelated donors (d-r+). methods: this is a retrospective single center study on a series of cmv seropositive recipients who underwent hsct from unrelated donors between febuary 2012 to july 2018. a total of 96 patients were analyzed. their clinical course and laboratory results were reviewed for evidence of cmv reactivation and/or cmv disease. we defined cmv infection as detection of cmv reactivation or primary infection by antigenaemia or polymerase chain reaction (pcr) assays, but was not accompanied by signs and/or symptoms suggestive of a systemic disease. cmv disease occurred when cmv was isolated from any site in association with organspecific signs and/or symptoms. monitoring for cmv infection commenced upon engraftment (approximately day +14). peripheral blood samples were sent twice a week for cmv antigenaemia or cmv quantitative pcr. the duration of twice weekly monitoring was at least about 100 days. longer monitoring was performed in patients who experienced cmv infection after hsct. results: all patients received graft-versus-host-disease (gvhd) prophylaxis using anti-thymocyte globulin (atg) at 4.5mg/kg in addition to cyclosporin or tacrolimus. among the entire cohort of 96 patients, 72 (75%) had cmv infection, including 23 (82.1%) out of 28 patients from the d-r+ group and 49 (72%) out of 68 patients from the d+r + group. 16 patients (57.1%) from the d-r+ group and 19 patients (27.5%) from the d+r+ group had ≧2 cmv reactivation above the threshold for preemptive therapy respectively; p=0.007. 11 patients developed cmv disease, 6 (21.4%) from the d-r+ group and 5 (7.4%) from the d +r+ group. cmv resistance to both foscarnet and ganciclovir was detected in 3 patients (10.7%) from the d-r+ group but none from the d+r+ group. 2 patients died due to cmv disease, both were from d-r+ group. 2 year overall survival (os) were 60% versus 55% for d-r+ group and d+r+ group respectively; p=0.706. median survival was not reached at 2 years. 2 year non-relapse mortality (nrm) were 35% for d-r+ group and 24% for d +r+ group respectively; p=0.37. conclusions: the incidence of recurrent cmv infection was higher in the d-r+ group compared to the d+r+ group. there were no statistically significant differences between the 2 groups in terms of os and nrm. however, there was a trend towards higher nrm in the d-r+ group compared to d+r+ group. our findings suggest that for matched unrelated hsct, it may still be important to select a seropositive donor for a seropositive recipient. disclosure: none background: it´s known that some patients submitted to allogeneic stem cell transplantation (asct) could present a greater susceptibility to infection even when they are in long term complete remission or potentially cured. this fact is related to the dynamic of immunological recovery that is variable in every single patients and it is dependent from many factors: the haematological disease, the conditioning regimen, the age of patient and donor, the number of stem cell and lymphocytes infused, the anti-gvhd prophylaxis, the use of anti-thimoglobulins and others. in clinical practise we can observe patients who are potentially cured, who tapered and stopped the immunosuppressive treatment months or years ago and who are suddenly graved from opportunistic infections. the largest part of these infections is represented from varicella-zoster virus (vzv) cutaneous eruption. methods: in this report we retrospectively analysed a monocentric cohort of 38 patients submitted to asct for haematological malignancies from a median time of 30 months. all of them were free of disease. they stopped the immuno-suppressive treatment in a median time of 270 days after asct (range: 160-1642) and did not present later chronic gvhd needing treatment neither other moderate or severe chronic post transplant complications nor other diseases. prophylactic treatment with anti viral agents (acyclovir or valacyclovir) has been conducted simultaneously to immuno-suppressive treatment and for a period ranging between 1 to 6 months after its suspension. in this cohort of patients we considered the incidence of vzv eruption occurred after the suspension of the immunosuppressive treatment, and we analysed the immunological recovery in terms of lymphocytes sub-population after 6, 12, 18 and 24 months from asct. results: of these 38 patients considered, 10 developed at least one vzv manifestation. all the vzv presentation were cutaneous, we did not observe neurological, ophthalmic or visceral presentation. all the vzv manifestation occurred in patients who ended the anti-viral prophylaxis. median time of presentation was 575 days after asct (range: 174-1642) the remaining 28 patients did not present vzv manifestation nor other kind of opportunistic infection despite the absence of anti-viral prophylaxis. the analysis of lymphocyte sub-population after 6-12-18 and 24 months did not show a significant difference in b, t, t4, t8 and nk lymphocytes in the different post transplant period. conclusions: vzv reactivation seems not to be correlated with the number of the different lymphocyte subpopulations in the post transplant period. actually it is not possible to distinguish patients more suitable of vzv reactivation on the basis of lymphocyte sub-populations analysis, so anti-viral prophylaxis should be prolonged for a medium period after suspension of immuno-suppressive drugs. in absence of anti viral prophylaxis a careful clinical surveillance should be performed in order to treat early eventual vzv manifestations. disclosure background: infection and disease cytomegalovirus (cmv) are common problems in patients undergoing hematopoietic stem cell transplantation (hsct). cmv infection has a high overall seroprevalence, therefore, during the first 100days post-hsct, it is important to prevent reactivation of cmv. the international clinical recommendation is the use of ganciclovir as prophylaxis in hsct patients; however, the cost of this treatment is not accessible for our population. in this respect it has been used as an alternative valganciclovir because of its lower cost and oral administration. our study´s aim was to assess the response and safety of valganciclovir in comparison with ganciclovir to prevent viremia and cytomegalovirus disease in patients undergoing allogeneic hsct methods: a retrospective study was performed on patients who receive an hsct-allo between january 2014 and august 2018. participants were enrolled in two groups according to prophylaxis treatment: (a) ganciclovir 5mg/k once daily and (b) valganciclovir 450mg twice daily for 7 days pretransplant, at day +100; viremia was measured by pcr. demographic and clinical information was collected from medical records and furthermore analyzed in spss v21. results: sixty-eight patients were enrolled in the study, 54% male, the median age was 34 years (19-61) with the following diagnoses: acute lymphoblastic leukemia 44%, acute myeloblastic leukemia 26.5%, granulocytic chronic leukemia 17.6%, myelodysplastic syndrome 4.4%, dendritic cell neoplasia 4.4%, and aplastic anemia 2.9%. ninety-one percet of the patients received a transplant from an identical hla donor and 8.8% received a haploidentical transplant. thirty-four patients received ganciclovir (g1) and thirtyfour valganciclovir (g2). median age was 39 vs28 years (p=0.022), intermediate risk cmv 85% vs 76 (p=0.048), associated bacterial infections was 15%vs 32% (p=0.15), and fungal infections 6% vs9% respectively (p=0.5). the reactivation by cmv was presented in 21% vs 15% respectively (p=0.70). there were no significant differences in fever, bacterial isolation, dysfunction or graft failure, presence and degree of acute or chronic gvhd and relapse of the disease. the most relevant characteristics and complications are described in table 1. within the whole group there were 28 deaths, 53% in the ganciclovir group and 32% in valganciclovir group (p=0.14), overall survival 1-year was 66% vs 80% (p=0.58) respectively; in both groups 72% was associated with relapse and 28% associated with transplantation. conclusions: ganciclovir and valganciclovir were effective in preventing the reactivation of cmv, the only statistically significant difference was that the presentation of the disease appeared earlier in the valganciclovir group. no difference in toxicity between the groups was identified. disclosure: none declared background: invasive pulmonary aspergillosis (ipa) is a severe and serious complication that occurs in the immediate post-transplant period due to severe neutropenia or late usually following prolonged corticosteroid therapy during treatment of graft-versus-host disease (gvhd). the objective of this study is to analyze the epidemiological, diagnostic and evolutionary characteristics of this major complication over a period of 3 years. methods: from january 2015 to december 2017, 392 patients (pts) received an allogeneic hematopoietic stem cell transplantation (allo hsct) for malignant and nonmalignant haematological diseases. during the transplant procedure, anti-infectious prophylaxis consisted of pts isolation, digestive decontamination, fluconazole and aciclovir. secondary prophylaxis done for pts with prior history aspergillosis. during the follow-up, a standard chest x-ray is performed systematically at each control or in case of clinical signs a thoracic ct scan is requested from suspicion. the diagnosis of ipa is made according to the criteria of the eortc-msg based on the predisposing criteria of the host and clinico-radiological criteria (possible infection). galactomannan antigen and histopathology criteria are not common practice. results: a total of 29 ipa episodes (7%) were identified in 26 pts (aml: 15, aa: 5, all 3, cml 2, mm 1) of median age 36 (8-56) , sex ratio: 0.44. all of them had transplantation from a family donor (geno-identical: 22, haplo-identical: 4) with conditioning by chemotherapy alone and a graft of csp (24 pts) and peripheral stem cells-bone marrow (2 pts). all pts had at least one predisposing risk factor: antecedent of aspergillosis (3 pts), prolonged neutropenia> 10 d (5 pts), acute gvhd (8 pts), chronic gvhd (4 pts), prolonged corticosteroid therapy ≥ 0,3 mg /kg/day exceeding 21 days (12 pts). the diagnosis of api was possible on average at j172 (20-930) after appearance of clinical signs (in all cases) and evocative radiological in 25 cases (in 4 cases, the standard chest x-ray was normal). at the time of thoracic ct scan, 11 pts (37%) had characteristic signs: halo sign (6 pts), crescent sign (1 pt) and cavity (4 pts). other minor radiological signs are found in the other pts. empirical first-line antifungal therapy was started as monotherapy in 14 pts (voriconazole: 8, caspofungin: 6 pts) or in combination in 12 pts. a secondline treatment was required in 10 pts for failure after an average duration of 7 days . three pts presented a second episode after an average delay of 5 months (3) (4) (5) (6) with a favorable evolution of resumption of thetreatment. fourteen pts (54%) are alive with complete resolution after a median treatment time of 10 months (2-19). twelve pts (46%) died rapidly on average 14 days after diagnosis (ipa 11, relapse of his disease: 1) conclusions: ipa occurring after an allograft of allo-hsct is a severe complication with high mortality. it is essential, in each case, to identify the pts with risk factors, perform a thoracic ct-scan, send serum serology for apergillus galactomannan antigenand start specific treatment as soon as possible while waiting to be able to reinforce the diagnosis by direct examination or sputum or brochoalveolar lavage with aspiration. disclosure: nothing to declare background: cmv (cytomegalovirus) has a prevalence varying between 45-100%. its pathogenicity is relatively low in the general population, usually resulting in a selflimiting viral illness. in an immunosuppressed host, infection can lead to life threatening illness. disseminated cmv infection can manifest in a number of organs and is diagnosed using internationally accepted criteria. in the post solid organ and stem cell transplant (sct) setting, it is postulated that it is viral reactivation, rather than primary reinfection that leads to cmv viraemia. prevention of reactivation requires the presence of a competent immune system, mediated by t-cells. this accounts for the increased incidence in intensive and t-cell depleting sct conditioning regimens. despite improved outcomes following the introduction of cmv monitoring by pcr and pre-emptive treatments (current uk guidance), cmv pneumonitis still carries a high mortality. the use of cmv specific immunoglobulins (cmvig) for the treatment of this complication is generally not recommended post chemotherapy or sct in haematological cancers due to lack of evidence. however, cmvigs are widely used in the setting of cmv reactivation post solid organ transplants. we report the use of cmvig in patients with suspected cmv pneumonitis at a single uk centre. the aims of this retrospective study were to establish safety and review efficacy in this highly immunocompromised group of patients. methods: data was collected retrospectively on the use of cmvig in patients with haematological cancers post sct or chemotherapy alone between 2007 and 2017 at manchester royal infirmary, uk. all patients included had cmv positive pcr in blood (and or from bronchoscopy), as well as high resolution ct imaging evidence of cmv infection. the data was sourced from pharmacy database and crossreferenced with a departmental list. for each patient identified, case notes and prescriptions were sourced. data collected included patient baseline characteristics, timing of treatment, number of doses of cmvig and outcome. results: eight patients received cmvig for suspected cmv pneumonitis. seven patients were post sct and one patient was severely immunosuppressed with chemotherapy alone. median age was 40 years (range 16-68). the cmvig regimen used was 4ml/kg of cytotect ® on days 0, 2, 4 and 6, followed by 2ml/kg every four days until resolution of symptoms. there were no infusion related reactions observed. patients received a median of 4 doses of cmvig. four out of 8 patients responded to the treatment and showed full recovery but only 3 are alive and well to date. conclusions: this study shows that the use of cmvig is safe in the post-sct setting of acutely unwell patients with multi-organ failure. despite limitations of retrospective studies, there appears to be benefit for the use of cmvig in our patient population, with 50% of patients showing a full recovery from that episode. allogeneic sct plays a confounding role in the outcome of patients although the numbers in our study are small. there is clearly a need for better treatments of cmv pneumonitis. cmvig is a promising treatment but further studies are needed to identify the optimal dosing regimen and provide evidence of efficacy. disclosure: biotest-honaria p464 abstract withdrawn. background: the risk of fungal infection related to allogeneic transplantation is a well-known cause of morbidity and mortality. the main agents implicated are yeast during the neutropenic period and filamentous fungi after this period. methods: we decided to evaluate the effectiveness of a prophylactic regimen containing fluconazole since day -3. after discharge fluconazole was kept until day or 75 or switched to posaconazole in high-risk patients. patients with gvhd under steroids were kept under prophylaxis.the group of high risk patients was defined by one of the following variables: 1-non related donors 2-atg, campath or fludarabine in the conditioning 3-presence of gvhd with need of steroids above 0.5 mg/kg we have analyzed the patients submitted to allobmt during 2016 and 2017. all patients were first admitted to an isolation room with hepa filters.patients under secondary prophylaxis were excluded. breakthrough fungal infections during the first year and toxicity leading to discontinuation was evaluated. results: sixty six patients were included with 67 transplants. male/female ratio was 36/30. the age range was 0.6-64 yo with a median of 37. malignant (53) and nonmalignant (13)diagnosis were included. donor type was related (16) haploidentical (9) and non-related (42). the conditioning regimen includes atg in 21, campath in 5 and fludarabine in 41. fourteen patients were treated after discharge with fluconazole and 52 with posaconazole. three patients fluconazole were switched to micafungin for hepatic toxicity, two cases to amphotericin due to persistent fever and in one case to caspofungin for a proven fungal infection (candida parapsilosis in blood stream in day +18). after discharge and during the first year of follow-up a single case of possible fungal infection was diagnosed, in a patient with gvhd with a lung nodule. conclusions: during the neutropenic period after transplantation the main risk of fungal infection is associated with candidiasis. the greatest risk of aspergillosis occurs later and have a significant relation with gvhd. except for candida parapsilosis the main source of yeasts are the gi tract. the main source of aspergillus are aerosolized particles retained by hepa filters. in patients without a previous episode of fungal infection the main risk of filamentous fungi occurs only after discharge. we conclude that fluconazole alone or followed by posaconazole in high risk patients is a feasible and effective regimen for primary prophylaxis, in allogeneic transplantation. disclosure background: bk virus-associated hemorrhagic cystitis (bkv-hc) has emerged as a serious infection after hematopoietic stem cell transplantation (hsct). it is characterized by painful hematuria due to hemorrhagic inflammation of the urinary bladder mucosa, this causes significant morbidity, prolonged hospital care with extensive nursing requirements and increases in healthcare costs. the purpose of this study is to determine the incidence, risk factors, and duration of treatment in our center. methods: we performed a retrospective review of hsct patients at luis calvo mackenna children´s hospital in santiago, chile diagnosed with bkv-hc, from 1st january 2016 to 30th november 2018. we investigated the incidence, risk factors and duration of treatment of bkv-hc in paediatric patients undergoing hsct over a 35 months period. bkv-hc was defined as bk virus (bkv) detection in urine by pcr testing in association with clinical symptoms and hematuria grade 2 or higher. sixty-seven patients were trasplanted during this period. results: eleven patients were diagnosed with bkv-hc at our institution, only one with bk viremia. the cumulative incidence of bkv-hc in our series was 16%. all of them were treated with cidofovir. the median age at diagnosis was 9 years old (range: 3-13 y.o.). the median time from hsct to hemorrhagic cystitis (hc) was 40 days (range: 25-240 days), the median length of treatment was 9 weeks (range: 2-36). all patients received myeloablative conditioning regimens and used cyclophosphamide (100%); ten (90%) were unrelated cord blood transplant recipients and nine (81%) used antithymocyte globulin. a concomitant viral reactivation (cmv/vh6) was demonstrated in six (56%) patients. no patient died due to bkv-hc or its complications, but in the follow up three patients died, one in relapse and two of other post transplant´s complications. conclusions: bkv-hc is the result of a complex interaction between patient characteristics, donor type and conditioning regimen intensity. these patients experienced significant morbidity and prolonged treatment. in our cohort bkv-hc of all patients but one were transplanted with an unrelated umbilical cord blood unit, all of them received myeloablative conditioning regimen with cyclophosphamide and most of them received anti-thymocyte globulin. we also observed frequently co-existence of viral infections from herpes family as cmv and vh6. the main limitations of this work are its retrospective nature and it´s from a single center. more studies are necessary to better understand the epidemiology and risk factor associated with bkv-hc and the morbidities associated with its treatment. disclosure: nothing to declare how we manage hhv-6 reactivation in the posttransplant setting oscar borsani 1 , anna amelia colombo 1 , daniela caldera 1 , paolo bernasconi 1 1 university of pavia, san matteo hospital, pavia, italy background: hhv-6 encephalitis is a life-threatening complication in the post-transplant setting and it develops in about 1% of patients receiving traditional hsct. several risk factors were described. a differential diagnosis between hhv-6 encephalitis and other neurological complications is extremely important but often not-easy to achieve because of the highly heterogeneous clinical and radiological features and complexity of interpretation, especially in transplanted patients. here we described vignettes that represent and highlight distinct problems in the diagnosis and management of transplanted patients with suspected hhv-6 reactivation. methods: we collected the clinical, laboratory and radiological (electroencephalogram, brain mri and brain ct) data of transplanted patients who developed a neurological syndrome suspected for hhv-6 reactivation. hhv-6 was detected on serum and csf using rt-qpcr. results: 1) a 61-years-old patient developed a diffuse erythema and subsequent encephalitic syndrome following hsct. the brain mri revealed clear signs of limbic encephalitic and searching for hhv-6 on serum and csf revealed 150.000 copies/ml and 220.000 copies/ml respectively. an antiviral therapy was started but no clinical benefit was achieved. 2) a 59-years-old patient developed a typical neurological syndrome without brain mri findings of encephalitis and with no evidence of skin involvement. the lumbar puncture and csf analysis showed a total of 49.200 hhv-6 dna copies/ml. antiviral therapy with ganciclovir and foscarnet was promptly started with clinical improvement and a drastically reduction of hhv-6 dna on both csf and serum. a new brain mri revealed an acute limbic encephalitis. 3) a slight neurological syndrome consisting of confusion and amnesia developed in a 49-years-old-patient. brain mri findings were compatible with a wernicke syndrome, but no improvement of neurologic symptoms were obtained with thiamine supplementation. csf analysis did not revealed hhv-6 dna, which was detected at low copies number on serum analysis. a second brain mri was conclusive for limbic encephalitis, so an antiviral therapy with foscavir was started and radiological but not clinical improvement was noted. the patient died after few days. 4) in the last case we present a 52-years-old patient who developed a clinical picture of encephalopathy (i.e. amnesia, ataxia, drowsiness, weakness, depression) with rapid progression to coma after seventy-eight days from hsct. a brain mri showed a slight contrast enhancement in parietal-occipital regions. during the recovery phase from conditioning-induced cytopenia, an increasing in serum hhv-6 dna was detected. searching for hhv-6 dna on donor's follicles showed a chromosomally integrated hhv-6 (cihhv-6). cyclosporin a (csa) was interrupted and neurological improvement was observed in the following hours: a diagnosis of pres was made. conclusions: hhv-6 encephalitis should be suspected in transplanted patients with a clinical syndrome of encephalopathy. pcr detection of hhv-6 dna in csf associated with either typical brain mri abnormalities or a clinical diagnosis of nonspecific encephalopathy must lead to the urgent initiation of systemic antiviral treatment. if an increase of both serum hhv-6 dna and wbc is detected, a cihhv-6 should be confirmed. pres is an important differential diagnosis in transplanted patients which developed an encephalitic syndrome. disclosure: nothing to declare background: cytomegalovirus (cmv) infection is a major cause of morbidity and mortality after hematopoietic stem cell transplantation (hsct). it causes end-organ disease, multi-organ dysfunction syndrome, graft failure, increased susceptibility to infections and gvhd. greatest risk of cmv infection in a seropositive host is the reactivation of latent virus. methods: a prospective descriptive study performed at armed forces bone marrow transplant centre, rawalpindi, pakistan from dec 2016 to sep 2018. hundred consecutive patients who underwent hsct were followed with weekly cmv dna quantitative pcr from engraftment till day 100 for cmv reactivation. patients in whom cmv pcr showed more than 2000 copies/ml were treated with antiviral therapy. factors associated with cmv reactivation, outcome of antiviral therapy and effect of cmv on transplant outcome is studied. results: out of 100 cases, 82 were hla matched siblings, 15 were matched family donors and 3 were haploidentical transplants there were 66 males and 34 females. mean age was 11.9 ±8.9 years. fourty-two transplants were done in thalassemia, 35 in aplasia, 09 in leukemias and 14 in other hematological disorders and immune deficiencies. ninety-eight recipients and all the donors were cmv seropositive before hsct. cmv reactivation was seen in 81 patients and 42 of them had cmv viral load more than 2000 copies/ml and 39 patients had cmv viral load less than 2000 copies/ml. nineteen patients had no cmv reactivation. mean time to reactivation since transplant was 36±19 days. valganciclovir was given in 36 patients due to ease of administration and six patients were treated with ganciclovir during their hospital stay. only one patient had resistant disease. mean time to clear viremia was 20±12.8 days. the patients having viral load less than 2000 copies/ml, subsequently cleared cmv without any treatment. antiviral agents; ganciclovir and valganciclovir were equally effective for treating cmv infection with 99% efficacy, however, more adverse effects were seen with ganciclovir. myelosuppression i-iii was seen in 24% patients treated with valganciclovir and in 46% treated with valganciclovir. renal impairment i-ii was seen in 14% of valganciclovir and 20% of ganciclovir treated patients. steroid administration was strongly associated with cmv reactivation (p = 0.003). no statistically significant association was found with the use of atg, gvhd, underlying disease, abo or gender mismatch. os was 81.0 % and 93.1% in with and without cmv reactivation (p=0.1) and dfs was 83.3 % and 96.6 % in with and without cmv reactivation (p=0.06) conclusions: cmv reactivation was seen in 81% of the transplant recipients, this is higher compared to the western world due to high cmv seropositivity is this region. steroids administration in post-transplant period significantly increase the risk of cmv reactivation. preemptive therapy with valganciclovir effectively treats cmv reactivation with acceptable side effects. viral threshold for treatment should be decided considering the regional endemicity. cmv adversely affects the transplant outcome in terms of dfs and os. disclosure: no conflict of interest. acute nephritis requiring nephrectomy caused by adenovirus (hadv) and human polyomavirus bk (bkpyv) following allogeneic hematopoietic stem-cell transplantation in a patient with ph+ all background: adenovirus infection represents an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-hsct), with no established therapy. although different organs may be affected by disseminated hadv infections, kidney involvement has been rarely reported. co-infection of hadv and bkpyv are common complication in patients undergoing allo-hsct, but recent studies demonstrate that bkpyv may facilitate the replication of hadv and lead to elevated viremia with increased virulence and serious clinical consequences. here we report a case of an adult patient who required a monolateral nephrectomy due to hadv pyelonephritis as an early complication of allo-hsct for philadelphia-positive acute lymphoblastic leukemia (ph+ all). methods: in september 2014, an ethiopian gentleman was diagnosed with ph+ all at the age of 24 years. he was treated with polychemotherapy in association with the tyrosin kinase inhibitor imatinib mesylate achieving a complete remission (cr). one year later, due to disease relapse with cns involvement, he was started on vincristine and dexamethasone plus imatinib treatment and in april 2017 he was referred to our bmt center from ethiopia. upon confirmation of the p210 ph+ b-all diagnosis, therapy with the scr/abl dual inhibitor dasatinib associated to intrathecal chemotherapy was started and a salvage treatment with inotuzumab ozogamicin followed by an allogeneic hsct from a hla-identical brother was planned. having achieved a documented molecular cr disease status, in june 2017 the patient underwent allo-hsct following the fludarabine-melphalan reduced-intensity conditioning regimen. graft-versus-host prophylaxis included anti-thymocyte globulin, cyclosporine and mycophenolate mofetil results: on day +24 post-transplantation the patient developed macro-hematuria due to hemorrhagic cystitis and a ct scan unveiled a left pyelonephritis with marked kidney enlargement. kidney microbial investigations were all negative. at the same time, hadv viremia with very high copy number (>25000000 cp/ml) was documented and also elevated bkpyv (>25000000 cp/ml) viruria and viremia (6400 cp/ml). the genotyping of hadv evidenced serotype b11 mainly involved in infections of the urinary tract. treatment with cidofovir was immediately started; nonetheless, due to rapid clinical worsening despite maximal antibiotic therapy, on day +50 a left nephrectomy was performed, which led to a subsequent progressive resolution of the clinical symptoms and negativization of hadv and bkpyv viremia and viruria. pcr real time performed on the kidney tissue unveiled very high concentration of hadv copy number. conclusions: acute pyelonephritis due to disseminated hadv infection may represent a possible cause of severe complication following allo-hsct. monitoring of hadv copy number is helpful to evaluate infection severity and response to treatment. co-infection of hadv and bkpyv in immunocompromised patients should be always considered likely to worsen clinical course and outcome. disclosure: nothing to declare background: infection is a major cause of morbidity and mortality in patients (pts) receiving an allo-hsct. its severity is related primarily to the depth and duration of neutropenia. febrile neutropenia (fn) is defined as a neutrophil count below 500 cells/mm 3 and a fever ≥ 38.3°c at a single measurement or≥ 38°c 2 times at one hour intervals. the objective of our study is to analyze the epidemiological, clinical, biological characteristics of febrile episodes (fe) occurred in 136 pts who benefited an allo-csh over a period of 2 years. methods: from january 2016 to december 2017, 136 allo-hsct were performed in 136 pts including 106 sibling-hla identical, 26 haplo-identical and 04 phenoidentical for essentially acute leukemia (68 pts, 50%), acquired and congenital aplasia (45 pts, 33%). the median age is 22 years (3-59) and sex-ratio (m/f): 1.72. prophylaxis consisted on isolation sterile room with laminar flow, digestive decontamination, fluconazole and aciclovir. nine pts (6.6%) were infected at the time of hospitalization (cellulitis 03, pneumoniae 02, bacterial angina 01, veinitis 01, bronchial pneumonia 01, furuncle cutaneous 01) requiring treatment with antibiotics. conditioning regimen is myeloablative in all pts. anti-thymocyte globulin is used in 61 pts (44.8 %). peripheral blood stem cells (pbsc) are used in 117 pts (86%) with an average level of cd34+ cells: 7,5.10 6 /kg (2. 1-19.8) , bone marrow (bm) in 5 pts with a mean level of nucleated cells: 3.4 x 10 8 /kg (0.8-3.6) and the association of pbsc-bm in 14 pts (haplo-identical). at each fe, are practiced: chest x-ray, procalcitonin test, blood culture, microbiological study of urine and stool (if diarrhea). results: all pts showed aplasia with an average duration of 16 days (6-49), neutrophil engraftment was observed at day 18 (9-31). one hundred and twenty-nine pts (94.8%) presented 205 fe with an average of 1.5 per pt. eleven pts (8%) had 3 fe or more. forty nine (23,9 %) fe are clinically documented (digestive: 19, skin: 14, pulmonary: 11, urinary: 03, oto-rhino-laryngology: 02). the blood cultures are made at 195 fe, 136 fe are microbiologically documented (66 %): gram-positive bacteremia in 77% (mainly coagulase negative staphylococci) and gramnegative bacilli in 23% of cases. procalcitonin test performed during 157 fe: normal (32 cases), probable infection (90 cases), probable sepsis (15 cases), severe sepsis (19 cases) and septic shock (one case). empirical double antibiotic therapy is initiated in 120 pts without waiting for the results of the microbiological study. this association was sufficient in 15 pts (11 %). the transition to a second line was needed in 114 pts (83.8%) and third line in 82 pts (60%). antifungal is added in 36 cases (27%). eight pts benefited from g-csf. the evolution is favorable in 202 fe (98.5%), apyrexia obtained after an average of 3.3 days . three pts died (2%) by severe sepsis on a durable aplasia, 2 of which had a cellulitis before the conditioning. conclusions: fe increase morbidity and mortality in allo-hsct so prophylactic measures are essential. empirical antibiotics treatment has to be instituted very quickly in the absence of documentation. disclosure: nothing to declare p472 abstract withdrawn. atsushi satake 1 , masaaki hotta 1 , ryo saito 1 , akiko konishi 1 , hideaki yoshimura 1 , takahisa nakanishi 1 , shinya fujita 1 , tomoki ito 1 , kazuyoshi ishii 1 , shosaku nomura 1 1 kansai medical university, osaka, japan background: cytomegalovirus (cmv) infection remains a common complication after allogeneic hematopoietic stem cell transplantation (ahsct), which results in increased morbidity and mortality. letermovir is a novel anti cmv drug that inhibits the cmv-terminase complex. the purpose of this retrospective study is to elucidate the efficacy and safety of cmv prophylaxis with letermovir early after ahsct in clinical practice. methods: we retrospectively analyzed the incidence of cmv infection, cmv disease, preemptive therapy, adverse events through week 14 after ahsct, the rates of engraftment and overall survival. all patients underwent ahsct in our institution for hematopoietic malignancies between may 2018 and nov 2018. data collected in this study included patient's characteristics such as age, sex, disease status, donor source and cmv disease risk. cmv infection was evaluated by cmv antigenemia. this study was approved by the research ethics committee of the faculty of medicine, kansai medical university. results: thirteen patients (male 9, female 4) underwent ahsct and received cmv prophylaxis with letermovir. the median age was 54 years (range, 18-65 years). overall, 10 of 13 patients (76.9 %) were considered to be at high risk for cmv, including 5 patients (38.5%) with haploidentical donors, and 1 (7.7 %)with mismatched, unrelated donors. all patients began letermovir from day 0 after ahsct, and achieved engraftment (median 17, 13-22 days). no patient developed cmv disease and required preemptive therapy. one patient died of treatment-related mortality, and 2 patients died of acute gvhd. although one patient discontinued letermovir before day 100 after ahsct because letermovir was suspected to be a cause of persistent nausea, severe adverse events were not observed. conclusions: it is still unknown whether cmv prophylaxis with letermovir improves os and reduces trm; however, our data suggests that cmv infection is considerably inhibited by administration of letermovir early after ahsct. clinical trial registry: not applicable. disclosure: the authors declare noconflicts of interest for this study. background: cytomegalovirus (cmv) is cause of increased morbidity and mortality after transplantation of hematopoietic cells. the pathogenesis of cmv disease or infection is complex with multiple interactions with the immune system, mainly in acute and chronic graft-versus-host disease (gvhd). the aim of this study is to analyze the risk factors for the reactivation of cmv in patients undergoing allogeneic hematopoietic cell transplantation (hct). methods: prospective descriptive study of the risk factors for the reactivation of cmv in the described population. univariate and multivariate analysis of the predisposing factors were performed: donor graft, treatment with corticosteroids, use of antithymoglobin, serologic status, conditioning regimen and the presence of gvhd. results: during the period between august 2014 until january 2017, 71 patients were evaluated. 42.25% (n:30) had reactivation of cmv. average reactivation was 58 days post transplant. both (recipient and the donor) had positive cmv igg in 78.9%. in the univariate analysis, the reactivation of cmv was associated with haploidentical transplantation (p: <0.01), with the use of corticosteroids (p: <0.01) and gvhd (p: <0.01). in the multivariate analysis, the haploidentical transplant maintained its statistical significance in comparison with the related allogeneic transplant (p: 0.0012, or:7.07; ic95%:2.4-20.6) as well as the use of corticosteroids (p: 0.0014, or:9.25; ic95%:2.6-32.4). 100% of patients receiving corticosteroid treatment had grade ii / iii gvhd. the serologicac status, myeloablative conditioning regimen and the use of atg showed no statistically significant association. conclusions: in patients undergoing allogeneic transplantation, were found as risk factor to reactivation, those who received haploidentic transplantation and treatment with corticosteroids. another risk factor that showed greater reactivation was the presence of gvhd. disclosure: nothing to declare methods: a 37 y/o male was referred for allogeneic transplant following 3 cycles of induction therapy for aml with complex karyotype and axsl1 mutation having achieved complete remission following the first cycle of chemotherapy. his first induction cycle was complicated by a perianal myeloid sarcoma which became infected and required surgical drainage and formation of a defunctioning colostomy. results: following 2 allogeneic transplants, the first complicated by secondary graft failure and the second by primary graft failure he presented with two skin lesions, with a third lesion adjacent to his stoma developing shortly after admission. all lesions were erythematous with central necrosis and progressed rapidly in size over 48 hours. biopsy of the skin and para-stomal lesions revealed fungal mycelia, with culture subsequently identifying rhizopus oryzae. initial treatment was with liposomal amphotericin b 5mg/kg/day followed by dose escalation to 10mg/kg/day due to the development of new skin lesions. the patient had been taking posaconazole (tablet) prophylaxis since his first allogeneic transplant and peripheral blood drug levels checked at the time of admission were therapeutic confirming that this was a breakthrough fungal infection. consequently posaconazole was stopped and isavuconazole added to the treatment regimen. surgical assessment was undertaken but surgery was deferred on the basis of high risk due to the extent of the infection and the patient´s profound pancytopenia. the organism was tested for in vitro susceptibilitiy and found to be resistant to posaconazole (mic >16mg/l), with borderline resistance to isavuconazole (mic 16mg/l) and sensitive to amphotericin b (mic 0.25mg/l) (phe mycology reference laboratory, england). isavuconazole was therefore stopped and the patient was managed with liposomal amphotericin b along with daily granulocyte infusions. he underwent a third allogeneic transplant using a different unrelated donor and stable engraftment was achieved. post transplant there was initially an increase in the size of the para-stomal lesion, but no new skin lesions developed. following engraftment he underwent resection of the stomal lesion, with primary closure and re-siting of his stoma. amphotericin b was replaced by isavuconazole prophylaxis on discharge and he continues to make an excellent recovery. conclusions: whilst aspergillus species remain the most common cause of invasive fungal infections in allogeneic transplant patients, other species including the mucorales are seen, and generally associated with poorer outcomes. whilst there are standardised methodologies for susceptibility testing, fungi specific cut offs based on clinical outcomes are only available for a limited number of species/ antifungal agents. in this case, susceptibility testing demonstrated resistance to posaconazole which was consistent with the clinical presentation of invasive infection despite therapeutic levels of posaconazole. it is also worth noting that an estimated 45% of r. oryzae isolates in the uk are resistant to posaconazole. treatment with high dose amphotericin b resulted in improvement in small skin lesions with stabilisation of the larger stomal lesion until count recovery allowed surgical resection. background: total depletion of innate and adaptive immune cell populations occurs after intensive chemotherapy and hematopoietic stem cell transplantation (hsct). both t and b lymphocyte pools are restored slower that myelomonocytic populations. hsct patients are at high risk for bacterial and viral infections at early terms (< 100 days) post-transplant. the reconstitution of the immune system depends on the time required for stem cell recruitment, differentiation, expansion, maturation and release into the bloodstream. restoration terms for myeloid cells after hsct are usually defined as the 1st day with neutrophil count of ≥ 0.5 x 10^9/l with mean recovery terms of 12 to 20 days. high occurrence of cytomegalovirus (cmv) in hsct patients mostly result from reactivation of a latent virus acquired in early childhood. however, delayed immune reconstitution and subsequent infections such as cmv, adenovirus (adv) or herpes 6 (hhv-6) diseases are not unusual and still constitute a major cause of death in peru. methods: peruvian pediatric patients (n=22) diagnosed with aplastic anemia, mds, aml or all underwent a haploidentical hsct performed with the clinimacs device. patients treated were separated in two groups. the group of patients who received viral prophylaxis (ganciclovir) was compared to the group that did not receive any prophylaxis treatment. viral reactivation was confirmed by pcr test twice a week and clinical signs within 15 days after hsct. results: in the group that didn´t received prophylactic treatment, engraftment occurred close to day 10 post haplo-hsct and none of the patients developed gvhd (graft versus host disease). nevertheless, incidences of cmv, hhv-6 and bkv infections before day 15 post haplo-hsct were still high. an overall survival (os) over 65% with an ic 95% was reached at the end of the first year. on the other hand, the group of patients that received prophylaxis with ganciclovir did not developed gvhd and reached the engraftment close to day 10 with a very low viremia incidence after the first month post haplo-hsct. all viral reactivations were caused by cmv and the os was over 91% with an ic 95% at the end of the first year. previous prophylaxis to both the donor and the receptor with ganciclovir (5mg/kg) every 12 hours before and during the conditioning regimen has allowed a better control of viral reactivation. conclusions: the attempts to improve immune function and reduce nonrelapse mortality from infectious complications without increasing gvhd have focused on a partial t cell depleted graft, such as t cell depletion (tcr α/β). this graft retains a large numbers of effector cells, such as tcr γ/δ and natural killer cells. however, delayed immune reconstitution and subsequent infections are a big issue. a novel partial t cell depletion strategy such as depleted naïve t cells (cd45ra+ t cells) could enhance the recovery of immune function after haplo-hsct because donor pathogen memory t cells from the donor are retained. it is necessary to increase the studies and the database to set the scheme of previous prophylaxis to the recipient to contain the viral reactivation and to help a rapid immune reconstitution. disclosure: no conflict of interest is declared information was recovered from the medical records. results: thirty-four patients were included, of them with the following diagnoses: acute leukemia (27), granulocytic chronic leukemia (4), dendritic cell neoplasia (2), aplastic anemia (1). 80% of the patients received a transplant from an identical hla donor and 20% received a haploidentical transplant. mean age's patients was 32 years (19-57). prophylaxis with posaconazole was performed on 44% of the patients with identical hla and 86% on haploidentical group; the rest of the patients received fluconazole. the posaconazole group presented: fever 61%, mucositis gi-ii 94%, gastrointestinal toxicity gi-ii 67% (p= 0.006), hepatic toxicity 11%, kidney toxicity 17%, oral candidiasis 100%. during this period none of the patients presented invasive fungal infection in any group. there were 2 deceases, one on each group and none related to a fungal infection. the overall survival was of the 92% versus 93% on the posaconazole group and the fluconazole group respectively. conclusions: the prophylaxis with posaconazole and fluconazole is effective on the prevention of invasive fungal infection on the first 100 days. the toxicity was similar on both groups. posaconazole can be effective on the prevention of the haploidentical type. is necessary to continue following the patients with infection risk on a long-term period associated with the chronic gvhd. disclosure: none declared lymphoma these results open the question whether allo-sct should still be offered to these patients. methods: we aimed to define the role of allo-sct in refractory or relapsing after two lines de novo or transformed dlbcl patients, and its comparison with zuma-1 car-t cells trial (neelapu et al nejm 2017). we analyse long-term allo-sct results in 40 de novo (n=23) or transformed dlbcl (n=17) out of the 1000 allo-sct performed in our institution between october 1995 to october 2018. results: patients and transplant characteristics are summarized in table 1 . complete response (cr) at 100 days was 67,5% and 83% of them remain in cr at 12 months. with a median follow-up of 46 months, 5-year progression-free survival (pfs) was 54% and 5-year overall survival (os) 48%, with a 3-year transplant-related mortality of 18%. refractoriness at the time of the transplant was associated with a poorer prognosis, with only 2 out of 9 refractory patients being long term survivors (figure 1 ). similar results were reported for zuma-1 trial, with a best response of 55% cr retained in 79% of them at 12 months. with a median follow-up of 15 months, 18-months pfs was 41% and 18-months os 53%. patients characteristics did not differ in our series and zuma-1, except that all the patients in zuma-1 were refractory prior to therapy (table 1) . conclusions: although very few patients with de novo or transformed dlbcl are offering an allo-sct (4% of all allo-sct), this is a curative option in chemosensitive patients and with more mature data and longer follow-up than with car-t therapy; for these reasons, it should still be offer to these poor prognosis patients. moreover, almost all patients have now available donor, better graft-versus-host disease prophylaxis will decrease trm and morbidity, and new therapies will make more patients in sensitive disease before allo-sct. therefore, allo-sct and car-t cells are strategies to be discussed in every young patient with available donor. disclosure: honoraria as advisor or speaker from gilead ( methods: consecutive patients transplanted for hgbl (excluding burkitts lymphoma) between 2007-2017 in the ebmt database were included. data collected included age, sex, pathology subtype (hgbl (including subtypes), tfl, dhl), disease status at sct, conditioning (ma vs beam cam vs flu-mel-cam/atg), engraftment, day 100 outcome, trm, os and pfs and eligibility for emea licensed indication of car-t therapy. results: fifty patients (29m, 21f) with a median age of 47 at diagnosis and 50 at sct were included. the subtypes included hgbl (n=29), tfl (n=11) and dhl (n=10). indications for sct were: primary refractory (n=17), relapse < 12 months after primary treatment (n=12), previous autologous-sct (n=10) and dhl (n=11). the median lines of therapy was 4 (range 1 to 6). conditioning used was cytbi n=21, bu/cy n=1, etop/tbi n=1, flubucy n=1, beamcam n=7, fmc/t, n= 19. all patients engrafted with neutrophil >1.0 10 9 /l at median 15 days and platelets >20 10 9 /l at median 17days. the day 100 mortality was 8% (progressive disease 4%, nrm 4%) with a 5 year os of 56% and mortality due to progressive disease 22% and nrm 16%. disease subtype influenced outcome with an os for primary refractory hgbl, relapsed hgbl, tfl and dhl respectively of 53%, 58%, 57% and 89%. 23 patients were eligible for a licensed car-t product. conclusions: the outcome of these high risk hgbl patients have an acceptable os of 56%, with relapsed disease being the commonest cause of mortality. patients with dhl have a particularly good outcome in this series; recent evidence indicates that some of these patients with a non-immunoglobulin gene associated myc translocation could be managed more conservatively (ash sehn). the outcomes achieved with allogeneic-sct in this series will provide a baseline for outcome assessment with a cart program. disclosure: nothing to declare background: immune checkpoint inhibitors (ici) allow to achieve a durable remission in patients with resistant or refractory (r/r) classical hodgkin lymphoma. however, the information about optimal duration of therapy and the prognosis of the patients after ici cessation is limited (manson, blood 2018). therefore, the optimal role of hematopoietic sct (hsct) in this patient group is not defined. our aim was to determine remission duration in patients who discontinued ici monotherapy after achieving complete remission (cr). methods: this analysis included 20 patients (5 male/15 female) aged 19 to 47 (median 32 years) with r/r classical hodgkin lymphoma who were treated with nivolumab (3 mg/kg every 14 days) and achieved cr. response was assessed by positron-emission tomography/computed tomography (pet/ct) using lyric criteria every 3 month. after nivolumab therapy had been stopped the patients received no other treatment and disease was assessed every 3 months by pet/ct. median follow-up after therapy discontinuation was 20 (10-21) months. results: at the moment of therapy initiation 14 (70%) patients had stage 4 disease, 11 (55%) patients had progressive disease (pd), 4 (20%) patients had stable disease, 3 (15%) patients had partial remission and 2 (10%)complete remission; 12 (60%) patients had b-symptoms and ecog score >1. the median number of previous therapy lines was 5 (3) (4) (5) (6) (7) (8) (9) (10) . before nivolumab initiation high dose chemotherapy with autologous sct was performed in 9 patients (45%) and 8 (40%) received brentuximab vedotin. the median number of nivolumab cycles was 25 (18-30). cr was achieved after median of 6 (6-18) cycles. the median duration of therapy after achievement of cr was 7 (1-15) months. at the time of analysis, all patients were alive, 8 (40%) out of 20 patients relapsed after therapy discontinuation. the median progression-free survival (pfs) for the total group was not achieved. among patients with relapse, the median time before pd was 11 (5-20) months. after relapse all patients were retreated with nivolumab monotherapy or with chemotherapy combination. one patient achieved complete remission; 1 -partial remission; 1 -indeterminate response type 2. other patients are continuing the therapy and their response has not yet been evaluated. conclusions: while complete response was maintained in some patients at median follow up of 20 months after nivolumab therapy cessation, the pfs plateau was not reached. we report that patients with relapse after nivolumab discontinuation sustained sensitivity to nivolumab and achieved a response during retreatment with nivolumab monotherapy or with chemotherapy combination. in patients with unsatisfactory response to nivolumab retreatment, hsct option should be considered. disclosure: nothing to declare high dose chemotherapy with autologous stem cell transplantation in primary central nervous system lymphoma: data from the japan society for hematopoietic cell transplantation (jshct) registry center hospital, tokyo, japan, 4 national cancer institute, bethesda, md, united states, 5 okayama university hospital, okayama, japan, 6 kanazawa medical university, uchinada, japan, 7 kyoto university, kyoto, japan, 8 aomori prefectural central hospital, aomori, japan, 9 yamagata unversity school of medicine, yamagata, japan, 10 tenri hospital, tenri, japan, 11 hiroshima university, hiroshima, japan, 12 japanese data center for hematopoietic cell transplantation, nagoya, japan, 13 nagoya university graduate school of medicine, nagoya, japan, 14 shimane university hospital, izumo, japan background: high-dose chemotherapy (hdt) with autologous stem cell transplantation (asct) has been shown to improve prognosis of patients with central nervous system (cns) lymphoma. whereas the common regimen of hdt for pcnsl in the europe and the us is thiotepa-based regimen, e.g. bcnu-thiotepa, tbc (thiotepa-busulfan-cyclophosphamide), thiotepa-based regimen was only available before discontinuation of thiotepa in 2009 in japan. we report the results of asct for pcnsl from the japan society for hematopoietic cell transplantation (jshct) registry. methods: data from the jshct registry were retrospectively analyzed. 102 patients with pcnsl who received first hdt/asct between 2006 and 2015 were evaluated. distribution differences of clinical characteristics between groups were analyzed with fisher´s exact or mann-whitney u tests. overall survival (os) and progression free survival (pfs) were calculated using kaplan-meier method. two-group analysis of the cumulative incidence of relapse was conducted using the grey test. factors were analyzed in univariable analysis, and all factors with p≤.1 were retained in the multivariable model. all p values were 2 sided, and values were regarded statistically significant if p< .05. results: median age was 54 months (range 20-74) with 15 patients over 64 years of age; 38 males and 64 females. ecog-performance status (ps) at diagnosis was better (ps0-1) in 86 patients and poor (ps2-4) in 16 patients. serum lactate dehydrogenase (ldh) levels at diagnosis were elevated in 17 patients. karnofsky ps and cerebrospinal fluid (csf) protein concentration at diagnosis were not collected in the registry. 71 patients were in complete remission (cr), 21 patients were in partial response (pr), and 7 patients were stable disease (sd) or progressive disease (pd) at the time of hdt/asct. after hdt/asct, additional 20 patients achieved cr. with median follow-up period of 44 months, the 5-year os and pfs were 54.9% and 38.4%, respectively. the was no significant difference in os and pfs between upfront and salvage hdt/asct. since thiotepa, a key agent in hdt/asct for pcnsl, has been unavailable after the discontinuation in japan, the hdt regimens used were not uniform. thiotepa-containing hdt was received by 16 out of 32 patients before 2010, but by 2 out of 70 patients after 2011. thiotepa-containing hdt showed improved pfs (p=.019), lower relapse (p=.042) and a trend toward a survival benefit. in the multivariate analysis, non-complete remission at hdt/asct was an independent predictor for os (hr=2.40, 95%ci:1.25-4.58, p=.008) and thiotepacontaining hdt remained significant for pfs (hr=0.42, 95%ci:0.19-0.95, p=.038). [[p481 image] 1. os(a),pfs(b) in all patients (n=102) and cumulative incidence of relapse in cr patients (c; n=91)] conclusions: our results confirm the activity of thiotepacontaining regimen for hdt/asct in pcnsl patients. currently a pharmaceutical company re-develops thiotepa for new approval of hdt/asct in pediatric solid cancer and adult lymphoma in japan (japiccti-163433). further evaluation with the thiotepa by prospective clinical trials is warranted. disclosure background: t-cell non-hodgkin lymphomas (t-nhl) are rare diseases and they are associated with worse prognosis when compared to their b-cell counterparts. allogeneic stem cell transplantation (allo-sct) may have a curative potential for these patients due to the graft versus lymphoma effect. however, data is limited on the efficacy of allo-sct for these diseases. methods: we identified 53 patients (32% females; median age: 43 y; range,4-67) with t-nhl that underwent allo-sct at university hospital eppendorf between 1992 and 2017. twenty-one patients (underwent allo-sct from a matched sibling donor (msd) and 32 (60%) from a matched unrelated donor (mud). sixteen patients had ptcl (30%), n=8 (15%) anaplastic large-cell lymphoma (alcl), n=8 (15%) angioimmunoblastic large cell lymphoma, n=8 (15%) adult t-cell leukemia/lymphoma, n=5 (9%) hepatosplenic gamma/delta t-cell lymphoma, n=4 (8%) enteropathy associated t-cell lymphoma, n=2 (4%) tcell-prolymphocytic leukemia, and n=1 (2%) each extranodal t/nk-cell lymphoma, cutaneous t-cell lymphoma as underlying diagnosis. the median ann arbour stage at diagnosis was 4 (range, 1-4). ten patients (19%) had bone marrow involvement at diagnosis. all patients were heavily pretreated, 17 (32%) patients relapsed post autologous stem cell transplant (apsct) and one patient post allo-sct. fifteen patients (28%) were transplanted in complete remission (cr) (10 in 1 st cr, 2 in 2 nd cr), n=13 (25%) in partial remission (pr), and n=21 (40%) with advanced disease. most of the patients received myeloablative conditioning (91%). thirty-eight (72%) patients received total body irradiation based regimens and 15 (28%) received chemotherapy based regimens. twenty patients (38%) received anti-t-lymphocyte globulin (atlg neovii), and most patients (87%) received g-csf mobilized peripheral stem cells. results: overall, 49 patients (92%) had neutrophil engraftment (median days: 13; range,9-36) . at day 100, the cumulative incidences of grade ii-iv and grade iii-iv acute gvhd were 42% and 15%, respectively. after a median follow up of 12 months (range, 1-171) the cumulative incidences of chronic gvhd was 12% distributed evenly between limited and extensive. twenty nine patients (55%) achieved cr after allo-sct. median overall survival (os) and disease free (pfs) survival were 44 months and 12 months respectively. the 3 year os and pfs were 50% and 43% respectively. fourteen (26%; 95% ci [0.15-0.40]) deaths were due to non relapse mortality (nrm) and 15 patients (28%; 95% ci [0.17-0.42]) died due to disease progression. patients with a male donor had improved os compared to those with a female donor (3 year os male 56%, female 34%; p=0.038). patient gender, disease subtype, bone marrow involvement, type of allo-sct, donor, patient cmv status, abo incompatibility, disease stage at diagnosis, previous transplant, disease status at transplant, conditioning regimen, atg and stem cell source had no effect on os, pfs, nrm, and post transplant complications. conclusions: acknowledging the retrospective nature, our study shows that allo-sct induces high rates of complete remission, and may have a curative potential even in diseases relapsing post asct. however our findings need to be confirmed in larger prospective studies. disclosure: no funding, no conflict of interest p483 abstract already published. at-home autologous stem cell transplantation in lymphoma patients: clinical impact of non-g-csf administration post-transplant background: severe neutropenia remains the main cause of morbidity and mortality after autologous stem cell transplantation (asct). g-csf administration after asct is a common practice, performed to reduce the duration of neutropenia and its complications. in a previous work in patients with multiple myeloma managed at home after asct, we did not observe a deleterious clinical impact in those patients that did not receive g-csf post-transplant (martinez-cibrian n. et al, bmt 2016) . despite the fact that lymphoma patients receive a more intensive conditioning regimen that multiple myeloma patients, we hypothesized that the use of g-csf in lymphoma patients managed at home during the aplasia phase of asct does not provide a significant clinical benefit. methods: 93 lymphoma patients were managed at-home since day +1 of asct. between february 2010 and july 2016, 68 patients received at-home g-csf 5 μg/kg per day since day +7 until their anc reached 1x10 9 /l (g-csf group) and, since august 2016, 25 patients did not receive g-csf (non-g-csf group). all patients were conditioned with beam and received prophylaxis with a quinolone, fluconazole, aerosolized pentamidine and low-dose acyclovir (hvs+). in all cases we added primary prophylaxis with piperacillin-tazobactam 4.5 g/8h i.v., using a portable intermittent infusion pump (iip), from an absolute neutrophil count (anc) < 0.5x10 9 /l until the first day of fever or until attaining an anc of 1x10 9 /l. first-line therapy at home of neutropenic fever (nf) was refrigerated meropenem 1 g/8h i.v using a portable iip. fever was an indication of immediate visit to the hospital, and those patients presenting with focal infection or signs of severe sepsis were admitted. other indications for readmission were: willingness of the patient or caregiver; uncontrolled nausea, vomiting or diarrhea and mucositis requiring total parenteral nutrition or i.v. morphics. results: the main characteristics of the patients are shown in table 1. there were no differences between groups with respect to gender, diagnosis, stage of disease, comorbidity index (hct-ci), source of stem cells (peripheral blood) and cd34 + cell dose infused. the median (range) age (years) was 44 (19-71) in g-csf group and 52 in non-g-csf group (p=0.03). duration of neutropenia less than 0.5 x10 9 /l was significantly longer in non-g-csf group, with a median of 11 days (range 6-19), compared with 8 (range 6-17) in g-csf group (p < 0.0001). severe neutropenia, less than 0.1x10 9 /l, was also longer in the non-g-csf group (8 days (4-11) vs. 7 (5-13); p=0.014). no differences were observed in the time to platelet engraftment. g-csf post-transplant avoidance did not influence the incidence of neutropenic fever, the first day and duration of fever, the incidence and severity of oral mucositis, bacterial infections documented and number of readmissions. the median duration of the whole procedure at-home was 1 day shorter in the g-csf group (14 vs. 15 days; p=0.12). conclusions: the policy of not administering g-csf post-asct in our home-based program for lymphoma patients, that include intensive bacterial prophylaxis, did not have a deleterious impact on the main results reviewed, which suggests that elimination of its use can be achieved. disclosure the aim of this study was to analyze the spanish experience with patients diagnosed of nhl who received haplosct with pt-cy. methods: sixty patients who received haplosct with pt-cy in 17 spanish centers from 2012 to 2016 were analyzed. patients were followed-up until 2017. gvhd prophylaxis consisted in cyclophosphamide 50 mg/kg/d on days +3 and +4, and mmf and a calcineurin inhibitor from day +5. results: patients' characteristics are summarized on table 1 . median age of patients was 50, 75% male, and diagnosed from t cell lymphoma (37%). most of them didn´t achieve complete response prior to transplant (55%), but only 15% with active disease. up to 63% of patients had received previous transplant, from which 5% was an allogeneic transplantation. source of stem cells was mainly peripheral blood (90%), and reduced intensity conditioning was the preferred (82%) regimen. donors were 43% siblings (26), 38% offspring (23), and 18% parents (11). median neutrophil and platelet engraftment was 18 (16-21) and 28 (20-42) days, respectively. acute gvhd grade ii-iv rate was 55%, with only 6 patients developing grade iii-iv (10%). chronic gvhd rate was 17%, and only in 3 (5%) was extensive. median follow-up was 14 months. the 2-year overall survival and event free survival was 43% and 39%, respectively. the 2-year cumulative incidence of relapse was 14% and 2-year cumulative incidence of nrm was 22%. conclusions: relapsed/refractory nhl are aggressive entities with a fatal course in a short period of time. haplosct with pt-cy permit a new treatment option among these patients, with acceptable outcomes. more studies are needed with a larger cohort of patients and longer follow-up to confirm these results. disclosure: nothing to disclose. higher suv at pre-transplant and day 100 posttransplant pet scan both independently predict inferior survival in patients with diffuse large b cell lymphoma background: autologous stem cell transplant (auto-hct) can cure some patients with relapsed diffuse large b-cell lymphoma (dlbcl) but relapse occurs in about 50% of patients. while our center and others utilize routine surveillance imaging post-transplant, the utility in this setting is unclear. imaging is costly and exposes patients to radiation. novel interventions are now available for patients relapsing after auto-hct making early disease recognition crucial to intervene prior to clinical progression. hence, we studied impact of post-auto-hct surveillance (18)f-fdg-pet ct at day 100 on transplant outcomes. methods: we analyzed a cohort of 131 consecutive auto-hct recipients with relapsed/refractory dlbcl who then underwent pre-transplant pet/ct and surveillance pet ct at day 100 (interquartile range (iqr): 97-103 days) post-hct at the university of minnesota medical center. univariate analysis was performed to analyze pet parameters including deauville score (d), standardized uptake values (suv), total lesion glycolysis (tlg) and total metabolic tumor volume (tmtv) as predictors of relapse and survival after auto-hct. in addition, we assessed outcomes of patients with clinically versus radiographically detected relapsed dlbcl after auto-hct. other pre-hct factors analyzed included age, gender, conditioning regimen, performance status, consolidation radiation therapy, tmtv, suv, tlg. results: five-year cumulative incidence of relapse after auto-hct was 50% (95%ci 39 to 59) and overall survival (os) was 51% (95% ci 41 to 63). twelve (9%) relapsed prior to day 100. d-score for 91 patients with d100 pet/ct were d1 (22%), d2 (55%), d3 (0%), d4 (10%), d5 (13%) with median survival in years for d1, d2, d4 and d5 of 6.0, 6.8, 4.7, and 1.2, respectively. mean suv varied from 1.53 (d1) to 17.9 (d5). suv was predictive of relapse and os. risk of relapse increased with doubling of suv; 2-fold higher suv increased hr by 1.77 (95%ci 1.34-2.33; p= 0.01). mortality increased with doubling of suv in both pre-hct (2-fold increase in suv associated with hr 1.46 [95% ci 1.1 to 1.8]; p=0.01) as well as post-hct pet (hr 1.7 [95% ci 1.3 to 2.3]; p=0) irrespective of the bulk of tumor. in addition, risk of death was 4 times higher in d5 patients relative to d1 (hr 4.10 [95% ci =1.56 to 10.77]; p≤0.01). patients with d5 (n=12) had higher tmtv (137 cm 3 ) compared to d4 (n=9, tmtv 12.5 cm 3 ). the hazard ratio for death following relapse was 2-fold higher (hr 1.8 [95% ci 0.9 to 3.4]; p=0.08) if relapse was detected clinically versus only radiographically over a median follow-up time period of 3.3 years. other pretransplant patient and disease characteristics did not significantly influenced the outcomes. conclusions: in patients with r/r dlbcl undergoing auto-hct, surveillance pet/ct at day 100 identified patients with poor survival~1 year. higher suv in both pre-transplant as well as post-hct pet was predictive of increased mortality. these patients may benefit from novel treatments. [ there are concerns about the risks of nivolumab treatment before and after allo-hsct, due to the risk of heavy gvhd, thus the place of immune checkpoints inhibitors is not yet defined. this report include analysis of our center experience of nivolumab treatment in patients with r/r hl before and after allohsct. methods: we retrospectively evaluated the results of allohsct in 86 patients with r/r chl who had undergone transplant from 2002 to 2018. the analysis included patients received the flube conditioning and ptcy gvhd prophylaxis. in group a patients (n=20) received bridge therapy with nivоlumab and in group b patients (n=34) received bridge therapy with brentuximab vedotin or chemotherapy-based bridges. time from the last nivolumab administration to allohsct was at least 2 months. results: at the time of analysis, median follow-up was 12 (1-20) months for group a, and 15 (1-64) months for group b. there was no difference in two-year os (p=0,39) with significantly better efs (p=0,025) for group a versus group b: 95% and 95% vs 85,3% and 62% respectively. relapse incidence was 0% for group a versus 26,5% in group b (p=0,025). cumulative incidence of non-relapse mortality at 2 years was 5,0% and 13,8% in group a and group b, respectively (p=0,631). there was no difference in grade ii-iv (44% vs 27%, p=0.23) and grade iii-iv (22% vs 13%, p=0.3) agvhd, as well as extensive chronic gvhd (21% vs 28%, p=0,83) in groups a and b, respectively. ten patients with relapse after allohsct were treated with different doses (0,5-3 mg/kg) of nivolumab in cic725 center. at the median follow up of 16 mo (0,6-28) all patients remain alive. the objective response to therapy was assessed in 7 patients noted in all patients (100%), disregard the dose of the nivolumab: cr in 29%, and pr in 71%. the response was lost in four patients, which required nivolumab retreatment. none of the patients developed gvhd after nivolumab administration. in this analysis, there was also no correlation between dose of nivolumab and incidence and severity of adverse events. conclusions: allohsct in combination with immune checkpoints inhibitors is a good option for patients with r/r chl. consideration for immune-mediated toxicities and the potential for increased graft-versus-host disease remain important. early data suggest that nivolumab may be an efficient therapy in patients with r/r chl relapse after allo-hsct. further research needed. disclosure: the authors declare no conflicts of interest. background: transformation to diffuse large b-cell lymphoma (dlbcl) is considered to be one of the most unfavourable events of lymphoma natural history with poorer outcome as compared to de novo dlbcl (alonso-álvarez et al, bjh 2017). in patients suitable for salvage therapy, hematopoietic stem-cell transplantation (sct) could be an option, although its role is not well stablished. we analyse indication and outcome after transplant in transformed dlbcl at a single reference transplant unit. methods: out of 2565 total of transplants performed at our unit between 1995 and 2018 -1564 autologous and 1001 allogeneic-51 were dlbcl transformed from an indolent nhl. of them, 36 received an autologous sct (asct) and 15 an allogeneic sct (allo-sct). results: median age was 60 years old (range 40-69) and 52 (range 35-65) for patients receiving asct and allo-sct, respectively. all asct received beam as a conditioning regimen and most of the patients in the allo-sct group received a fludarabine/melphalan combination (73%). gvhd prophylaxis consisted on tacrolimus/sirolimus combination in 87% and calcioneurin plus methotrexate in 13%. regarding transplant disease status, 28 (78%) of the asct patients were transplanted in complete response (cr). in the allo-sct group, 11 (73%) patients had received three or more treatment lines before transplant and 13 patients (87%) had received a previous asct, being 11 (73%) in cr, 3 in partial response (pr) and 1 in progressive disease. transplant related mortality (trm) was 5.6% in the asct and 27% in the allo-sct group. overall survival (os) and progression-free-survival (pfs) at 25 months were 94% (os), 76% (pfs) for patients receiving asct and 63% (os) and 56% (pfs) for allo-sct. with a median follow up of 57 months for patients receiving an asct, 21 (58%) remain in cr. in the allo-sct group median follow up is 24 months for the whole group and 50 months for alive patients; 9 patients are alive and disease free and 6 have died, 4 due to trm (28%). regarding progression, 12 (33%) have progressed after autologous transplant and 2 after allo-sct. conclusions: indication for hematopoietic sct in transformed dlbcl is stablished in few patients. only 2% of the patients in our transplant unit receive a transplant due to transformed lymphoma, corresponding to a 2.3% of autologous activity and 1.5% of allogeneic activity. according to our results transplant should be considered a curative option. most of our patients were transplanted in cr, so new agents trying to reach best response before transplant should be considered. [[p488 image] 1. eva konirova 1 , antonin vitek 2 , marta krejci 3 , edgar faber 4 , katerina steinerova 5 , david belada 6 , jan novak 7 , juraj duras 8 , petr sedlacek 9 , veronika valkova 2 , andrea janikova 3 , ludek raida 4 , pavel jindra 5 , pavel zak 6 , tomas kozak 10 , marie trnkova 1 , michal karas 5 , marek trneny 1 management. however, differences in patient's characteristics as well as frequency of hsct indication in different lymphoma subtypes have been observed in the last decade. the aim of this study was retrospective analysis of hsct for lymphomas in czech republic. methods: data of adult patients transplanted between years 1993-2016 were retrospectively analyzed using ebmt database. results: between 1993 and 2016, 2816 autologous hsct (asct) were performed in 2651 patients (1511 men, 57%) with different lymphoma subtypes. the median age was 49 years (range 18-75). out of these, 2078 (78%) were patients with non-hodgkin lymphoma (nhl), 569 (21%) with hodgkin lymphoma (hl). the nhl group comprised of diffuse large b-cell lymphoma (dlbcl, 36%), follicular lymphoma (fl, 18%), mantle cell lymphoma (mcl, 16%) and t-nhl (9%). the frequency of asct in lymphomas increased from 1993 to 2000 and has been constant since 2000 (120-130 transplants per year). differences in frequency of asct were observed among lymphoma subtypes -decreasing numbers of dlbcl and fl and increasing numbers of t-nhl and mcl, with asct as part of the induction therapy. between 1996 and 2016 a total of 329 allogeneic hsct (allosct) were performed in 319 patients (200 men, 63%). median age was 46 years (range 19-66). out of these 257 (81%) were patients with nhl, 61 (18%) hl. the most common nhl subtypes were fl (27%), mcl (22%), t-nhl (22%) and dlbcl (16%). in the last 10 years the number of allosct for lymphoma is fluctuating around 20 per year. the median age at asct was significantly higher in the years 2010-2016 vs 1993-2000 [54.5 (18.1-74.8) vs. 40.9 (18.6-72.5), p < 0.0001, fig 1] , while the increase at allosct [46.5 (21.3-65.5) vs 41.6(20.3-64.5)] did not reach statistical significance (p=0.07). with median follow up for allosct, 5 y probability os for patient transplanted in the later period 2010-2016 was in relapsed dlbcl 25.7%, in fl 77.4%, in hl 51.0% and in mcl 45.7%, 5 y os for asct as part of first line therapy in the same period was in mcl 78.1 % and in t-nhl 48.2%. os was significantly better in all patients who underwent asct in the years 2010-2016 vs 1993-2000 (70.% vs. 57 .5%, p < 0.0001) and there was a trend towards better os in patients after allosct (with 44.7% vs 23.5%, p=0.1084) (fig 2) . conclusions: hsct remains important treatment modality for lymphomas in the era of targeted antibody and molecular therapy and we can transplant older patients due to better supportive treatment. acknowledgment: progress q28-9 uk from the czech ministry of education youth and sports disclosure: nothing to declare background: disease chemosensitivity to salvage treatment has been proven to be a major predictive factor for a favorable outcome after autologous stem cell transplantation (asct) for patients with refractory lymphomas. therefore the importance of effective and safe salvageregimens is indisputable. methods: we retrospectively compared the outcomes in terms of safety and efficacy, in 67 (hl:36, nhl:31) patients, with a median age of 34.5(16-75) years, who received as 1 st salvage either dicep [cyclophoshamide (1750 mg/m 2 ), etoposide (350mg/m 2 ), cisplatin (35 mg/ m 2 ), days 1-3, (n=23)] or the widely used regimen eshap (n=44). rituximab was additionally given to all cd-20 positive lymphoma patients. the statistical analysis based on the independent t-test, kaplan meir method and logrank test. results: the reason for salvage treatment was primary induction failure (pif, n=34), early relapse (< 12 months post induction-remission therapy n=14) and late relapsed disease (n=19). more specifically, 19/23 patients (83%) in the dicep-group, and 29/44 patients (65%) in the eshapgroup were assessed with pif or early relapsed disease, however this difference was not statistically significant. both regimens were well tolerated and no major organ toxicities were noticed. eleven patients (48%) from the dicep-group, while only 4(10%) from the eshap-group developed febrile infections. all patients were successfully managed with the appropriate treatment and only one, from the eshap-group, required for short period admission to the intensive care unit. after 1 cycle of dicep and 2 cycles of eshap the disease response was re-assessed by pet/ct scan. the overall response rate (>50% tumor reduction) was significantly superior for the dicep-regimen, reaching 92% (21/23 patients) vs. 64% (27/44 patients) for eshapregimen (p=0,003). eleven patients (48%) from the dicep-group and 14(30%) from the eshap-group achieved complete metabolic remission according to pet/ ct criteria (p=ns). the median hospitalization period was 20(5-25) days for the dicep-group compared to 10(10-19) days for the eshap-group. however, for the eshapgroup, an additional median of 20(6-33) hospitalization days were required, since 12 of the non-responders patients received a 2 nd salvage before asct. the mobilization and stem cell collection was successful for both groups, though significant higher number of cd34+ cells were collected in the dicep-group (17.2x10 3 /kg vs. 5.4x10 3 /kg, p=0,001). all but two patients (due to refractory disease) underwent asct. noticeably, the median period from 1 st salvage treatment to asct was significantly shorter for the dicepgroup (64 vs. 128 days, p=0,013), apparently because 12 non-responders patients from eshap-group treated with a 2 nd salvage. the 3-years overall and progression free survival were similar for dicep-and eshap-groups (95% vs. 88% and 70% vs 80% respectively). two heavily pretreated patients from the eshap-group developed secondary myelodysplastic syndrome post asct conclusions: in our series of patients both regimens proved to be safe. interestingly, despite the fact that more patients in dicep-group had poor risk disease the dicepregiment was significantly more effective, resulting thus in an earlier asct, less exposure to chemotherapeutic agents, that might led in less long-term toxicity. nevertheless, prospective trials with large series of patients are needed to define the role of dicep in the salvage treatment setting. disclosure: no conflict of interest background: although autologous hematopoietic stem cell transplantation (auto-hsct) is one of the best curative strategies for patients with chemosensitive t-cell lymphoma, major limitation remains a tumor contaminated graft-related relapse or residual disease after chemotherapy. several purging methods were introduced in auto-hsct for these limitations, however there are few studies of ex vivo purging of the autograft in lymphomas, especially t-cell lymphoma. therefore, we retrospectively analyzed 59 consecutive t-cell lymphoma patients receiving auto-hsct with/without ex vivo purging. methods: among them, 33 patients underwent autograft manipulation with ex vivo purging by cd34+ cells selection using a clinimacs device. results: with median follow-up duration of 42 months (range, 6-121 months), 3-year overall survival (os; 73.8% vs. 49.0%, p=0.017) and 3-year progression-free survival (pfs; 75.8% vs. 52.4%, p=0.039) in a purged and unpurged group, respectively. transplant-related mortality was observed in both groups (2 patients of a purged group and 1 patient of an unpurged group). neutrophil (10 vs. 9 days, p=0.240) and platelet (30 vs. 24 days, p=0.055) recovery were similar in both group and there was no engraftment failure. on subgroup analysis according to upfront and salvage auto-hsct, while survival outcomes were improved by stem cell purging in the upfront auto-hsct (os with p=0.039 and pfs with p=0.047), there were no different survival outcomes in salvage auto-hsct. the unmanageable late-infectious complications were few in both groups except for predominantly cytomegalovirus reactivation in a purged group (3 vs. 1 patient). conclusions: although cohort was a small number, ex vivo graft-purging method was feasible and safe in t-cell lymphomas. and this purging strategy observed the more favorable survival outcomes in the upfront auto-hsct than salvage setting. therefore, further randomized studies are needed to determine the firm efficacy of cd34+ purification with the large number of patients in auto-hsct for t cell-lymphomas. disclosure: nothing to declare nivolumab-based regimens in relapsed or refractory non hodgkin lymphomas: the role of hematopoietic stem cells transplantation methods: we analyzed data of 18 patients with r/r nhl, among them n11 with diffuse large b-cell lymphoma (dlbcl), n5 with primary mediastinal b-cell lymphoma (pmbcl), n1 with gray zone lymphoma (gzl) and n1 with gamma-delta peripheral t-cell lymphoma (ptcl), who received nivolumab-based regimens. the median age was 37 years (range, 18 -64 years). most of the patients n14 (78%) had a primary chemoresistant disease, the rest patients n4 (22%) had a relapse. the median of lines of prior therapy was 3 lines (range, 2-6 lines). all sixteen patients with dlbcl and pmbcl received 1 -3 cycles of nivolumab in combination with bendamustine, gemcitabine and rituximab (begern). the patient with gzl received 5 cycles of nivolumab in combination with brentuximab vedotin and epoch. and the patient with ptcl received 10 cycles of nivolumab monotherapy. results: at median follow up 8 months (3-16) objective response (or) after nivolumab-based regimens was noted in n10 (56%) patients, complete response (cr) and partial response (pr) in n9 (50%) and n1 (6%) patients, respectively. cr observed in n4 patients with dlbcl, n3 with pmbcl, n1 with gzl, n1 with ptcl. and pr observed in 1 patient with dlbcl. two responding patients with dlbcl underwent auto-hsct. and four responding patients (n1 dlbcl, n1 pmbcl, n1 gzl, n1 ptcl) received allogeneic hematopoietic stem cells transplantation (allo-hsct). the median duration of response for all n10 patients with or was 5 (range: 3-16 +) months. among n4 patients who achieved or without hsct, only n1 remain in cr. two patients who received auto-hsct had a relapse. one patient with dlbcl improved the response after allo-hsct from pr to cr, and all four patients with allo-hsct remain in cr. the probabilities of 1-year os and pfs rates were 49% and 31%, respectively. conclusions: nivolumab-based regimens can lead to an objective response in 56% patients with r/r nhl. however, the durability of response to therapy is not long. nivolumab-based regimens can be used as bridge to allo-hsct disclosure: there are no conflicts of interest to disclose background: patients with aggressive non-hodgkin lymphoma (nhl) who relapse after autologous stem cell transplantation have a dismal outcome and could benefit from radiotherapy, allogeneic stem cell transplantation or experimental treatments. systemic inflammatory parameters at diagnosis have demonstrated to be useful to predict lymphoma evolution. methods: we conducted a retrospective review of patients with aggressive nhl who underwent autologous stem cell transplantation (astc) to evaluate the relationship between ldh, β2-microglobulin, inflammatory parameters (lymphocyte (alc) and monocyte count (amc), ferritin or c-reactive protein) and imaging techniques before and on day +100 post-astc and progression free survival (pfs), as well as the role of residual disease directed radiotherapy (rt). results: one hundred and sixty patients with aggressive nhl received asct as consolidation treatment in our center between 2000 and 2017. the most common diagnosis was diffuse large b-cell lymphoma (dlbcl). one hundred and nine patients received upfront asct for high risk dlbcl (defined as age-adjusted ipi 2-3)(n=28) or for having received two or more lines to obtain first complete remission (n=28), for t-cell lymphoma (n=27) and for mantle cell lymphoma (n=26). the rest was performed in relapsed lymphomas. forty-seven patients (29%) relapsed and pfs was 110 months. pretransplant response was evaluated with ct scan in 66 patients (11 of this with partial remission (ct-pr) and 94 patients were evaluated with fdgpet/ct (27 were pretransplant positive (pet1); of these, 18 patients maintained positivity at day 100 after astc (pet2). pfs in patients with ct-pr was 46 months, in pet1 positive ones 99 months and in pet2 positive ones 21 months. univariate analysis showed pet2 positivity as the most accurate predictor of relapse (hr 3,13, p=0,004) followed by amc at day +100 (hr 1,002, p=0,13), albumin at day +100 (hr 0,93, p=0,03), ldh at day +100 (hr 1,002, p< 0,001) and pretransplant alc/amc ratio (hr 1,068 p=0,008). multivariate analysis only demonstrated an association with pet2 positivity (hr 7,78) p< 001 and ldh in day +100 (hr 1,008) p=0,003 with pfs. five and ten years overall survival were 61% and 37% in pet2 negative patients vs 25 and 5% in pet2 positive ones (p< 0,01). eight out of 19 patients with pet2 positivity did not relapse. salvage radiation therapy was used in 7 patients with positive residual mass and 5 of them did not relapse. two patients relapsed: one patient had residual mass and another had remote affectation from primary site and could be considered as progression before day +100. conclusions: post asct fdgpet/ ct is superior to conventional ct in predicting outcome in aggressive lymphoma after astc. pre and post asct systemic inflammatory parameters didn't help to improve the relapse risk prediction. addition of consolidative rt after astc has demonstrated improvement in pfs in patients with pet positivity. it would be neccesary to develop randomized trials to assess the role of rt in residual disease in advanced aggressive nhl with insufficient response to systemic treatment with pet response evaluation. disclosure: nothing to declare long term outcome of patients with lymphoid malignancy who underwent high dose chemotherapy followed autologous hematopoietic cell transplantation at a single institution over 20 years joanna romejko-jarosinska 1 , ewa paszkiewicz-kozik 1 , lukasz targonski 1 , lidia popławska 1 , jan walewski 1 background: high dose chemotherapy (hdt) and autologous hematopoietic cell transplantation (auto-hct) is a standard of care for relapsed/refractory lymphoma patients (pts) or it is used as a consolidation for myeloma and high risk lymphoma patients in first line treatment. we retrospectively evaluated long-term outcome including late effects and risk factors in patients with lymphoid malignancy who underwent auto-hct. methods: we collected data from 926 consecutive patients with hodgkin lymphoma (hl) (n=326), aggressive b lymphoma (dlbcl) (n=186), myeloma (n=199), indolent lymphoma (n=30), mantle cell lymphoma (n=122) and peripheral t cell lymphoma (n=63) who underwent auto-hct at our institution between 1997 and 2017. at transplant median (range) age was 46 (17-71) years, clinical stage iii/iv was found in 499 of lymphoma pts, complete remission, partial remission and stable/ progressive disease occurred in 462(50%), 397 (43%), 67 (7%) pts, respectively. beam regimen was used in 549 pts (59%), mel200 in 200 pts (23%) and other myeloablative regimens in 177 pts (18%). results 62%) ], respectively. partial remission or stable disease at transplant, clinical stage iii or iv, and age more than 60, were identified as risk factors associated with inferior os and pfs in univariate and multivariate analysis. histopathologic diagnosis was not a risk factor for os and pfs (p=ns). the outcome of patients who underwent auto-hct between 1997-2003 was inferior to the outcome of patients treated in 2004-2010 or 2011-2017. 5year os was 53%, 68%, 73%(p< 0.001) and 5 year pfs was 43%, 54%, 55% (p< 0.01), respectively. we recorded 37 (4%) cases of second primary cancer (18 solid tumors and 19 hematologic cancers). acute cardiotoxicity occurred in 5 patients from 1 to 15 years after transplant, and required heart transplant in 2 patients. 326 patients (35%) died. the main causes of death were progressive disease in 271 pts (89%), second primary malignancy in 22 pts (6.7%) treatment related mortality was 0.8% (8 pts), and mortality within 100 days was 22 (2,4%). [[p494 image] 1. pfs and os in patients underwent hdt and auto-hct 1997 -2003 , 2004 -2010 , 2011 -2017 conclusions: more than 45% of patients who underwent hdt and auto-hct had long term survival without progressive disease. older age, non-complete remission at transplant, advanced stage are associated with poor outcome. patients recently transplanted had a better outcome than patients transplanted before 2004. disclosure: nothing to declare outcomes after haploidentical and matched related hsct in lymphoma do not differ significantly: a single center study nadira durakovic 1,2 , zinaida perić 1,2 , lana desnica 2 , ranka serventi-seiwerth 2 , sandra bašić kinda 2 , ivo radman-livaja 2 , alen ostojić 2 , ante vulić 2 , dražen pulanić 1,2 , pavle rončević 2 , zorana grubić 2 , igor aurer 1,2 , radovan vrhovac 1,2 1 university of zagreb, school of medicine, internal medicine, zagreb, croatia, 2 uhc zagreb, zagreb, croatia background: allogeneic hsct still offers patients with relapsed/refractory lymphoma the best chance of long-term survival. in most such patients timing of hsct is crucial, therefore a related donor is preferred. we analyzed acute and chronic gvhd incidence, relapse and overall survival, but also time to immunosuppression (is) discontinuation and hematopoietic recovery comparing transplantation using haploidentical (haplo) and matched related donors (mrd) in single center in this indication. methods: in the time period between 5/2011 and 5/2018 at uhc zagreb, croatia, 10 mrd and 13 haplo transplantations in lymphoma were done, 15 for hodgkin and 8 for nhl. all patients transplanted from haploidentical donors received ptcy. data were computed using the r package. the probability of gvhd was calculated using the cumulative incidence method and subgroups were compared using the gray test. results: median age was 38 (19-62) years; 36 (19-62) in haplo and 41 (25-56) in mrd group. four patients were in pr and 9 in cr in haplo group, while in mrd group 5 patients were in cr and 5 in pr. in haplo group 12 patients (92%) received bone marrow (bm) and only 1 (8%) peripheral blood stem cells (pbsc). in mrd group all patients received pbsc. all patients in haplo group received nma ("baltimore") conditioning with ptcy while in mrd group 9 patients (90%) received flu-bu2atg, and only one received flutbi as conditioning protocol. in haplo group 85% patients were previously treated with autologus transplantation, 80% in mrd group. there was no significant difference in time to is discontinuation, 149 and 155 days in haplo and mrd group, respectively. patients after haplo recovered slower, recovering anc after 22.6 days (95% ci, 17.7-27.4) and 16.5 (95% ci, 13.9-19) (p=0.04) and recovering platelets after 34.6 days (95% ci, 15.5-53.7) and 10.8 (95% ci, 9.2-12.4) (p< 0.01) in haplo and mrd group. with a median follow up of 469 days, overall survival was 83% (95% ci, 63-100) in haplo and 80% (95% ci, 59-100) in mrd group. trm was 8% in haplo and 20% in mrd group. cumulative incidence of agvhd ii-iv was 31% (95% ci, 9-56) and 32% (95% ci, in haplo and mrd group, respectively (p=0.84). cumulative incidence of cgvhd requiring treatment was 22% (95% ci, and mrd 20% (95% ci, 0-62) in haplo and mrd group, respectively (p=0.46). all cases of cgvhd developed after dli. cumulative incidence of relapse was 32% (95% ci, 9-58) and 38% (95% ci, 7-72) for haplo and mrd group, respectively (p=0.77). conclusions: we found no significant difference in overall survival, relapse incidence, agvhd and cgvhd incidence between these two groups. hematopoietic recovery was slower after haploidentical transplantation, but it did not influence trm as it was higher after mrd. even though limited in number, this data contribute to the growing body of evidence that use of haploidentical donors, particularly in lymphoma setting, is as worthy as using matched related donors and should be at least second choice in donor selection, and in older patients (with older donors) probably the first one. disclosure: nothing to declare. adjuvant involved field radiotherapy post autologous stem cell transplantation for refractory/relapsed lymphomas results in favorable outcome with low toxicity: a single center experience background: involved field radiotherapy (ifrt) to previous bulky or localized residual disease, is a widely used treatment approach to minimize the risk of relapse post autologous stem cell transplantation (asct). however, the proper time for irradiation treatment remains controversial. adjuvant ifrt (adj-ifrt) in pre-asct period could cause undesirable toxicity which might delays or even cancel the asct resulting in increased risk of relapse, or could affect the marrow environmental and marrow niche resulting thus in impaired engraftment. on the other hand, the ajd-iftr in the early post-asct period, upon marrow recovery, offers a potential advantage by delivering irradiation after sufficient disease response, without affecting the engraftment. in this retrospective study we evaluated the safety and efficacy of the ifrt as adjuvant treatment in patients who had previously treated with asct for relapsed or refractory lymphomas. methods: twenty-three patients (hodgkin=14, non-hodgkin=9), aged of 34(16-76) years, underwent asct, for primary refractory (n=15) or relapsed (n=8) disease. patients who had bulky disease at the time of relapse or those with residual mass post salvage treatment, were considered as candidates for adj-ifrt, early (within 2-3 months) after documentation of autologous stem cells engraftment. all patients proceeded to asct with chemosensitive disease after a median of 2 lines of salvage therapy. at the time of asct 20 patients (80%) had residual disease while 4(20%) evaluated to be in complete remission. the preparative regimens were: single-agent melphalan (n=9), busulfan-etoposide-melphalan (n=7), beam (n=5) and bendamustin-etoposide-cytarabine-melphalan (n=3). filgrastim was given till neutrophills recovery, while prophylaxis against bacteria, fungus, viruses and pcp were administered till the completion of adj-ifrt. results: all patients engrafted promptly and successfully. no patient experienced any severe toxicity or active infection before adj-iftr. though our plan was to proceed with adj-ifrt within 3 months post asct, finally it was delivered after a median of 4.5 (2-7) months; the median radiation dose was 30(24-36) gy. ten patients received radiotherapy in the mediastinum, 9 in the abdomen/pelvis/ inguinal area 3 in the neck, and 1 in the left leg. the adj-ifrt was well tolerated. no patient experienced toxicity grade >3 and none required hospitalization. currently, after a median follow-up of 2(2-5) years, 19/23 patients are alive and well; the 5-years overall and progression free survival rates are 75% and 55% respectively. four patients died; 2 due to relapsed disease and 2 heavily pretreated patients due to secondary myelodyspalstic syndrome conclusions: in our study, the adj-ifrt in the early post transplant period demonstrated a safe and well-tolerated profile. taking into consideration the poor risk status of our patients (residual disease post salvage regimen or bulky disease at the time of relapse), the promising overall and progression free survival rates suggested that adj-ifrt post asct is also an effective approach. well designed trials are needed to clarify the role and the appropriate time of radiotherapy in the asct setting disclosure: no conflict of interest adverse prognostic impact of pre-transplant neutrophil/ lymphocyte ratio in lymphoproliferative disorders background: brentuximabvedotin(bv) is a chimeric anti cd30 igg1 antibody, conjugated to synthetic antitubulinmomomethylauristatin. bv is approved for the treatment of classical hodgkin lymphoma (hl) in relapse either after autologous stem cell transplantation (asct) or after two lines of combination chemotherapy in transplant ineligible patients. the aethera trial revealed increased pfs when bv is used as maintenance therapy for 16 cycles in high risk patients after asct. however, this schedule is associated with a high cost and significant toxicity particularly in term of peripheral neuropathy. our primary objective is to assess the efficacy of 4 cycles brentuximab as consolidation therapy after asct for relapsed/refractory (r/r) hl. secondary objectives include side effects, progression free survival (pfs), and overall survival (os). methods: this is a retrospective single center analysis approved by the irb of the american university of beirut medical center. we included in this study consecutive patients with r/r hl who underwent asct between 2014 and 2018, and received bv consolidation post-asct. results: we identified 18 consecutive adult patients with r/r hl treated with bv 1.8mg/kg iv every 3 weeks as consolidation therapy after asct. the indications for bv consolidation was primary refractory disease in 11 patients (61%), early relapse in 6 patients (33%) (after a median time of 10 months; range, 3-11) andextranodalinvolvement in one patient (6%). the median number of lines of therapy pre-asct was 3 (range, 2-5). the median time to bv initiation post-asct was 76 days (range, 35-188). patients received a median of 4 cycles (range, 3-4) of bv post-asct. after a median follow up of 30 months (range, 8-50), five (28%) patients relapsed after asct. the median time to relapse was 7 months (range, 4-21). median pfs and os were not reached. we did not observe any significant toxicities during or after therapy. conclusions: 4 cycles of bv consolidation after asct seem to be safe and effective in preventing relapse, however our findings need to be confirmed with larger prospective studies. chemotherapy or who progress after autohsct is poor. despite introduction of novel agents like brentuximab vedotin (bv) or nivolumab, allohsct appears the most effective treatment option with curative potential. the goal of this study was to evaluate efficacy of allohsct for hl, including patients pre-treated with novel agents. methods: between years 2012-2018, 45 patients (including 23 males) with hl were treated with allohsct in msc institute of oncology in gliwice, poland. median age was 32 (19-57) years. median lines of preceding chemotherapy was 4 (2-8); 34 (76%) patients had been pre-treated with autohsct, 24 (53%) with radiotherapy, 17 (38%) with bv, 7 (16%) -with nivolumab. disease status at allohsct was as follows: cr-19, pr-10, nr-16. patients were treated with hsct from either hla-matched sibling donor (msd, n=21), unrelated donor (urd, n=18) or haploidentical donor (n=6). conditioning was myeloablative in 21 (47%) cases. peripheral blood was used as a source of stem cells. results: all but one patient engrafted with median time of neutrophil recovery of 14 (9-20) days. the incidence of grade 2-4 and grade 3-4 acute gvhd was 16% and 11%, respectively, while the incidence of chronic gvhd was 43%. the probabilities of os and pfs at 2 years were 54% (+/-12%) and 55% (+/-8%), respectively. the incidences of progression and transplant-related mortality were 31% and 14%, respectively. the 2y pfs rates were 42% for msd, 72% for urd and 62% for haploidentical donors. in a univariate analysis pfs was affected by recipient gender (female -75%, male -38%, p=0.009) and disease status at allohsct (cr -78%, pr -47%, nr -38%, p=0.09). in a multivariate model the disease status other than cr was the only factor associated with increased risk of treatment failure (reverse pfs) -hr=3.13, 95%ci 1.45-6.76, p=0.004. neither donor type nor conditioning affected long-term outcome. conclusions: results of allohsct for patients with relapsed/refractory hl are determined by disease status at transplantation. efforts should be done to reduce tumour burden before transplantation, optimally to achieve cr. disclosure: nothing to declare background: brentuximab vedotin (bv), nivolumab and pembrolizumab have been assigned to chemorefractory hodgkin lymphoma treatment. impact of these agents on disease-free-survival after autologous stem cell transplantation (asct) remains under investigation. aim of the study is to compare bv-and nivolumab-treated patients with a control group. methods: clinical characteristics and outcomes of chemo refractory hodgkin lymphoma patients who underwent asct during 2011-2017. results: a total of 56 patients (33 men; 21 women, median age 37 years old, 19-65) were treated with bv: 24 pre-transplant, 30 post-transplant and 7 pre-and posttransplant. pre-transplant bv patients had primary refractory disease or early relapse in the majority (92%). post-transplant treatment occurred in the context of relapsed/refractory disease in 27 patients; 12 (40%) had an allogeneic stem cell transplant. among them, 6 had additional chemotherapy and 2 nivolumab, gaining a complete metabolic response. in the 15 rest of patients, change of treatment due to eventual bv failure occurred. bv was administered as a maintenance treatment in 10 patients. in six of them bv had already been administered pre-transplant as well. out of maintenance treatment patients, 2 relapsed and subsequently received nivolumab. two patients died due to prior chemotherapy complications, whereas 13 are currently on nivolumab treatment. pet-based response was available in 6 patients, 4 having a complete metabolic response (cmr) and 2 a partial metabolic response. stable disease was achieved by ctbased response in the rest patients. no major toxicities were observed. one patient presented with grade 2 asymptomatic hypothyroidism and one with grade 3 anemia attributed to non-inflammatory upper gastrointestinal blood loss. in total, 20 patients received anti-pd1 treatment, all post bv failure. with a median follow-up of 34.3 (1.5-202 .2) months, 5-year overall survival (os) was 65.9% in patients treated only with bv compared to 78.2% in patients treated with additional anti-pd1 treatment (p=0.356, figure) . median os for patients treated only with bv was 113.5 months, whereas median os has not been reached for patients that received anti-pd1 treatment. conclusions: bv pre or post-transplant and anti-pd1 treatment post-transplant after bv failure have outstanding results in chemo refractory lymphoma patients. treatment sequence in allogeneic transplantation eligible patients remains to be further studied. disclosure: nothing to declare background: allogeneic hematopoietic cell transplantation (allo-hct) with reduced-intensity conditioning (ric) has been used in heavily pretreated lymphoma patients with the promise of decreased treatment-related mortality. despite improvements in outcomes of patients with lymphoid neoplasms, several new agents emerge as potential therapies. therefore, we aimed to describe our long-term experience in patients with hodgkin (hl), non-hodgkin lymphomas (nhl) and chronic lymphocytic leukemia (cll) post allo-hct. methods: in this retrospective study, we enrolled consecutive patients who underwent allo-hct for lymphoid neoplasms in our institution (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) . results: in total, 50 patients (male:female=35:25) aged 36 (15-64) years, underwent allo-hct for hl (n=24), nhl (n=21) and cll (n=5). the majority of patients were diagnosed at stage iv (48%); 34% had bone marrow involvement and 66% had undergone autologous hct. most patients were heavily pretreated (median lines=4, range 1-11), 21 of them had received more than 4 treatment lines and at the time of transplantation only 14 had complete response, while 9 had partial response and 27 were refractory. according to disease-risk index (dri), patients were stratified at low (n=11, 23.4%), intermediate (n=12, 25.5%), high (n=20, 42.6%) or very high (n=4, 8.5%) category. among patients with hodgkin lymphoma, brentuximab vedotin was administrated in 7 and 4 of them were effectively bridged to allo-hct. all patients received ric, mainly fludarabine (150mg/ m 2 )-cyclophosphamide (2g/ m 2 ) in cll and nhl and thiotepa (10mg/kg)-fludarabine (120 mg/m 2 )-cyclophosphamide (60mg/kg) in hl from matched sibling (n=27), matched (n=15) or mismatched unrelated (n=8) donors. graft-versus-host disease (gvhd) prophylaxis consisted of cyclosporine or tacrolimus and mycophenolate mofetil or short-term methotrexate and additional low-dose antithymocyte globulin (5mg/kg) in unrelated donors. peripheral blood was the main cell source (48/50) and median number of cd34+ cells infused was 6.37x10 6 /kg (1.33-14.5) . two patients succumbed to advanced underlying disease before engraftment; while the other engrafted successfully. median time until neutrophil and platelet engraftment was 10 and 12 days respectively. eighteen patients (36.7%) developed acute gvhd (grade iii-iv, n=5), steroid sensitive in 10 (62.5%) and 11 relapsed. one-year cumulative incidence (ci) of extensive chronic gvhd was 78.2%, and 13 patients required more than one additional line of immunosuppression (range 1-5). ten patients presented cmv reactivation successfully treated with antiviral medication and 1 patient died from hsv7 encephalitis. with a median follow of 3 years (1-16 years), 10-year os was 40.4%, 10-year non-relapse mortality ci 23.4% and 10year dfs 32%. there was no difference in survival according to original disease (5-year os for nhl=60.2%, hl=46.7%, cll=31%%, p=0.67). multivariate analysis revealed high and very high dri as the single predicting factor of os (hr 9.69, ci 1.55-60.55, p=0.015), when assessing impact of disease, dri, prior treatment lines, gender and bone marrow infiltration at diagnosis. conclusions: our data suggest that ric allo-hct provides encouraging survival rates, potentially offering the chance of cure, with acceptable long-term mortality in selected high-risk patients with lymphoid neoplasms. dri that is mainly associated with disease stage at transplant independently affects survival. therefore, continued efforts are necessary for clinical application of novel agents aiming to lower disease stage pre-transplant. disclosure: nothing to declare results: six pts were identified, with a median age of 30 years at diagnosis: five with hl nodular sclerosis and 1 with lymphocyte depletion. the median number of therapeutic lines prior to allo-hsct was 5 [4-10]; four pts were previously treated with brentuximaband two pts had been submitted to high dose chemotherapy with autologous bone marrow support. at the time of allo-hsct, 4 pts had progressive disease (dp), 1 was in partial response and 1 in complete response (cr). five allo-hsct were performed with a related donor, 4 of wich were haploidentical (2 parents, 1 sibling and 1 descendant) and 1 with an unrelated donor (10/10). prophylaxis for gvhd was performed with tacrolimus and mycophenolate mofetil (with post-transplant cyclophosphamide in haploidentical allo-hsct). on day +100 evaluations, 5 pts had a cr and 1 patient (pt) had dp. the median time to relapse after allo-hsct was of 12 months. at the time of initiation of nivolumab, 5 pts were under steroid therapy for disease control, without other immunosuppressive therapy. the median time between allo-hsct and the beginning of nivolumab was 16 months. the initial dose was 1 mg / kg (associated with corticosteroid therapy), escalated up to 3 mg/kg biweekly, according to patient's tolerance. after the start of nivolumab, 3 patients, with previous gvhd manifestations, presented a worsening of the cutaneous gvhd, which required an escalation of immunosuppressive therapy. as toxicity, 1 pt had a grade 4 pneumonitis, 1 pt had a grade 4 encephalitis/hypophysitis, 1 pt had a grade 4 pancreatitis, 2 pts had headache (grade 1 and 3), 2 pts had a grade 2-3 cutaneous reation. with a median follow-up of 13 months since nivolumab treatment, the overall response rate was 100%: 1 pt obtained cr and 5 pts partial remission. nevertheless, there were 2 deaths after the onset of nivolumab: 1 pt at 4 monts with dp and another one due to acute myocardial infarction at 16 months. at the time of analysis, 3 pts maintained response under nivolumab treatment (median cycles 26) and 1 pt had therapy suspended because of toxicity. conclusions: these results demonstrate the high probability of achieving response with nivolumab treatment in patients with rr-hl relapsing after allo-hsct, but adverse events of grade 4 were frequent in this small group, and the treatment toxicity was significant. disclosure: nothing to declare background: intravascular large b-cell lymphoma (ivlbcl) is a rare form of large b-cell lymphoma with pathological findings of intravascular proliferation and/or sinusoidal involvement of lymphoma cells. according to their geographic distribution, ivlbcl could be dichotomized into asian and western variants. compared with the western variant, where skin involvement was common, the asian variant was reported to involve more frequently the liver, spleen and bone marrow, and hemophagocytic lymphohistiocytosis is more common in asian variant. diagnosis of ivlbl is still difficult because of the lack of overt lymphadenopathy and peripheral blood involvement. thus, timely diagnosis and immediate treatment remain as a challenge to improve outcomes for patients with the asian variant. therefore, we analyzed the clinical features and treatment outcomes of patients with the asian variant of ivlbcl. methods: we analyzed 46 patients who were diagnosed with ivlbcl between 2001 and 2018. all patients were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (r-chop). results: forty-six patients were diagnosed with ivlbcl, and their median age at diagnosis was 62 years (range: 34-82 years). male patients predominated (n=26, 57%), and b symptoms were present in 31 patients (78%). hepatomegaly and/or splenomegaly were observed in 26 patients (57%), whereas lymphadenopathy was less common (n = 22, 48%). bone marrow and liver were the most commonly involved extranodal organs (54%, and 48%, respectively) and were the most common sites of biopsy for diagnosis in this study. all patients received r-chop as a first-line treatment after diagnosis with a median number of six cycles (range one to eight). at the end of treatment, 31 patients achieved a complete response (cr), whereas eight patients showed progression. six patients died after the first or second cycle of r-chop, and the causes of death were treatment-related adverse events including cytopenia, infectious complications, and pulmonary hemorrhage. upfront asct was done for two patients including one patient with cns involvement at diagnosis, and these patients were still alive at the time of analysis without evidence of relapse. on the other hand, the outcome of six patients undergoing salvage asct after relapse was poor; thus, only one patient was alive. likewise, patients with disease progression at the end of treatment with r-chop showed dismal prognoses even after salvage chemotherapy except for one. at a median follow-up of 47.0 months (95% confidence interval, ci 25.0-69.0), the median overall survival was 45.0 months (95% ci 25.8-64.2). the treatment outcome of patients with the asian variant of ivlbcl is still not satisfactory. although upfront autologous stem cell transplantation might be effective for selected patients at high-risk of relapse, its role is still not clear, either. thus, further study should be warranted to develop more effective strategies for diagnosis and treatment. clinical trial registry: not applicable disclosure: nothing to disclare background: peripheral t-cell lymphomas (ptcls) are about 10% of non-hodgkin´s lymphomas usually with an aggressive clinical course and unfavorable prognosis.given their heterogeneity, consensus on the best first-line treatment and the role of autologous/allogeneic (asct/allosct) stem cell transplantation as consolidation is controversial. methods: we evaluated the overall survival (os), progression-free survival (pfs) and toxicities of a cohort of patients with ptcls submitted to asct/allosct intensification at our institution between january 2000 and july 2018. os was calculated from the date of diagnosis until death. pfs was measured from transplant until relapse, progressive disease or last follow-up. os and pfs rates were estimated using the kaplan-meier method and compared with the log-rank test. results: twenty-six patients were identified, 16 female (61%), median age was 46 years (range: 22 to 62). ninetytwo percent of patients presented with advanced-stage disease at diagnosis (ann arbor stage iii or iv), 38% with b symptoms. according to the 2016 who classification, histologic ptcl subtypes included angioimmunoblastic tcell lymphoma (n =12); ptcl not otherwise specified (n =6); anaplastic large cell lymphoma, alk-negative (n =5); anaplastic large cell lymphoma, alk-positive (n =1); nodal peripheral t-cell lymphoma with tfh phenotype (n =2). extranodal nk/t-cell lymphoma, nasal type and primary cutaneous subtypes were excluded. the ageadjusted ipi (aaipi) was low/intermediate low in 15 patients (57%) and intermediate high/high in 11 patients (43%). twenty-seven transplants were performed (19 asct, 8 allosct); 18 were consolidation in 1st response (16 asct and 2 allosct) with 16 in complete remission (cr) and 2 in partial remission (pr). nine transplants were performed as consolidation of 2 nd response (3 asct and 6 allosct) with 4 in cr and 5 pr. in 1 patient allosct was performed after asct, due to early relapse (< 12 months). beam regimen was used in asct as conditioning and flumel in allosct. all patients engrafted, the median time to leukocyte recovery > 1,000/μl was 14 days (range, 11 to 25). four of the 8 pts (50%), submitted to allosct had chronic graft versus host disease which was the most relevant complication of this analysis. considering the whole cohort, the median follow-up was 50.5 months (range, 2 to 217). the estimated 5-year os and pfs were 77% and 39%, respectively. seven patients relapsed (6 early) all after asct, there were no relapses after allosct, however, the results were not statistically significant between the allosct and asct groups; the 5-year os rates were 87% and 62% (p =0,16) and the 5year pfs rates were 85% and 59% (p =0,26) respectively. for the all patients treatment-related mortality (trm) was 3,7%; 7 patients died, 6 with progressive disease (asct) and 1 for hepatic toxicity (allosct) before d+100. conclusions: the results of this retrospective study, taking into account the adverse risk profile of the population, suggest that autologous/allogeneic stem cell transplantation as an effective and safe option for the consolidation of patients with ptcls. these results need to be validated in prospective studies, including a larger number of patients. disclosure background: autologous stem cell transplantation is used as consolidation therapy in relapsed lymphoma patients. however, outcome of lymphoma patients relapsing after autologous stem cell transplantation is poor and allogeneic stem cell transplantation which can be curative is used in the transplant eligible patients in this setting. besides, allogeneic stem cell transplantation can be an option before autologous stem cell transplantation in some high risk patients. in this study, we aimed to compare the survival rates of lymphoma patients older than 60 years of age and patients aged 50-60 who had undergone allogeneic transplantation in our center. methods: we collected the data of lymphoma patients older than 50 years of age who had undergone allogeneic transplantation in our center and analyzed the results by grouping them into 2, namely the ones between 50-60 years of age and the ones over 60 years of age. [[p506 image] 1. figure 1 results: there were 42 patients over the age of 50 who had undergone allogeneic stem cell trasplantation with the diagnosis of lmphoma between 2011 and 2018. 18 of these patients were over 60 years of age. 37 patients had non-hodgkin lymphoma and 5 patients had hodgkin lymphoma. the characteristics of the patients are summarized in table 1 . patients' comorbidity indexes were calculated with augmented hct-ci which includes patients' pretransplant ferritin, albümin and thrombocyte counts as a variable. no difference could be found between groups regarding neutrophil and platelet engraftment times and comorbidity indexes. however, acute graft versus rate and documented bacterial infection rate during the hospitalization period were higher in the 50-60 years age group (p=0,01). 100 day mortality rate and non-relapse mortality rate were not different between groups. more importantly, progression free survival(pfs) and overall survival(os) of patients in the 50-60 years age group and over 60 years of age group were not different (p=0,45) (figure 1) conclusions: in the present study, although the number of patients is low, we showed that lymphoma patients over 60 years of age have similar outcomes and transplant related toxicity as the patients between 50 to 60 years of age. pfs and os were very close in this study. we think that this may be due to low relapse rate in the patients and high mortality rate in relapsing patients. in conclusion, allogeneic stem cell transplantation which has a curative potential may be employed in transplant eligible elderly lymphoma patients disclosure: nothing to declare background: follicular lymphoma (fl) histologic transformation consist on the development of an aggressive lymphoma, usually a diffuse large b cell lymphoma (dlbcl). histological transformation has been considered to have poor prognosis. in pre-rituximab era median os ranged between 1 and 2 years, however, in recent series of patients treated with chemotherapy plus rituximab, the outcome of transformed fl has improved, especially in those that receive autologous stem cell transplantation (asct), who reach 5-year os up to 75% in some series. methods: we have retrospectively studied 19 consecutive patients undergoing asct for transformed fl between 2006 and 2015 in a tertiary center in the basque country, spain. patients were considered to have a transformed fl if they were diagnosed of a dlbcl and they have previous history of fl or histological evidence of a fl in another location. these patients were compared to a retrospective cohort of 38 dlbcl patients with high ipi or stage that received asct in first remission according to our institution strategy. pfs and os were calculated from the time of the asct. in the case of transformed fl, both relapses of the aggressive or indolent lymphoma were considered. survival analysis was performed with kaplan-meyer estimator results: a total of 19 transformed fl and 38 dlbcl patients were studied, with a median follow up of 41.4 and 51.7 months respectively. patient characteristics are described in table 1. 3-year pfs was 68% in transformed fl and 81% in dlbcl, and 3-year os was 81% and 84%, respectively (picture 1). there were no significant differences in pfs or os between this two groups (p = 0.44). in both groups all relapses occurred in the first three years after asct. among the patients with transformed fl 6 relapses were observed. five of them (83%) were aggressive relapses, while only one patient presented relapse as an indolent lymphoma (fl histological grade 3a with an aggressive clinical course). [[p507 image] 1. image 1: transformed fl and dlbcl pfs after asct] conclusions: in our experience, asct in transformed fl offers good results, similar to those in dlbcl. fl presents a natural course akin to that of dlbcl, with relapses occurring early and survival reaching a plateau. this data suggests that some patients with transformed fl can be cured after asct. disclosure: nothing to declare. safety and efficacy of intensive preconditioning regimen containing cladribine in autologous peripheral blood stem cell transplantation of refractory and relapsed young highly invasive lymphoma background: autologous peripheral blood stem cell transplantation (apbsct) is one of the main treatments for patients with non-hodgkin's lymphoma (nhl). effective and safe conditioning regimens can improve the cure rate of nhl. beam is the most common pretreatment scheme, but for refractory and relapsed young highly invasive lymphoma, especially for dual-expression dlbcl, pretreatment needs to be strengthened. studies have shown that the cladribine (clad)+gemcitabine (gem)+busulfan (bu) combination provides synergistic cytotoxicity in lymphoma cell lines.we evaluated the the safety and short-term efficacy of intensive preconditioning regimen containing cladribine (clad+gem+bu) for refractory and relapsed young highly invasive lymphoma undergoing apbsct. methods: ten patients with nhl received apbsct. ca)ctx+ ara-c) therapy followed by g-csf was used for pbsc mobilization. sevenr patients received conditioning regimens of beam(beam group): bcnu 300mg/ m 2 ·d -1 ×1d (-7d), vp16 100mg/m 2 · q12h×4d (-6d--3d), ara-c 200mg/m 2 ·q12h×4 d (-6d--3d), mel 140mg/m 2 ·d -1 ×1d (-2d). three patients received intensive preconditioning regimen containing cladribine (clgb group): clad 5mg/m 2 ·d -1 ×5d (-6d--2d), gem2500mg/m 2 ·d -1 ×2d (-6d, -2d), bu0.8mg/kg q6h×4d (-6d--3d). follow-up date expires on december 1, 2018. results: the age of 3 patients in clgb group was 41, 12 and 50 years, respectively. two patients were diagnosed as diffuse large b-cell lymphoma with double expression and one was diffuse large b-cell lymphoma with two recurrences. the patients of beam group were all high-risk, relapsed and refractory nhl.all patients were successfully engrafted after infusing apbsc. the average lowest leukocyte in clgb group and beam group were (0.023 ±0.023) ×10 9 /l vs (0.237±0.2033)×10 9 /l, respectively. the average lowest leukocyte in clgb group was lower than that in beam group. the average time to anc < 0.5×109/l in clgb group and beam group were 1.33d ±1.53d vs 3.57d±2.07d. the average time to anc≥0.5×10 9 /l in clgb group and beam group were 9.0d±1.0d vs 10.0d±2.6d; the average time to plt≥20×10 9 /l of clgb group was not different to that of beam group (8.0d±1.0d vs 9.6d±2.2d) the average time of neutropenia wasn't significantly different in two groups (8.33d±3.1d vs 8.0d±2.1d). the adverse reactions of gastrointestinal tract and oral mucosa were close in tow groups.vod, hemorrhagic cystitis, pretreatment-related interstitial pneumonia, liver and kidney dysfunction were not happened in tow groups. the rate of infectious fever was close in two groups (2/3 vs 4/7). the median followup period in beam group was 9 (1~19) months. in the beam group, a patient died 20 days after transplantation, because he was diagnosed with recurrent nkt cell lymphoma and intracranial infection caused by severe sinus infection. another case of beam group was diagnosed as double-expressed dlbcl, which relapsed 3 months after transplantation. the remaining patients in ebmt group survived disease-free. the follow-up time of 3 patients in clgb group were 2 months, 3 months and 9 months respectively. all patients survived without disease.however, the follow-up time is short and needs long-term follow-up. conclusions: the treatment of intensive preconditioning regimen containing cladribine (clgb) for refractory and relapsed young highly invasive lymphoma undergoing apbsct is safe. the time of hematopoietic reconstruction is short, and the adverse effects is tolerable for patients with refractory and relapsed young highly invasive lymphoma. the current short-term outcome is good, but the long-term effect need a longer time to follow-up. disclosure: this work was supported by national nature sciences found of china (81300417). there is no disclosure of conflict of interest.the all authors name: xiang-li chen, yu-zhu zang, wen-hui zhang, yin zhang, zhong-wen liu, ping-chong lei, jing yang, yu-qing chen, kai sun. background: small part of children with hodgkin disease (hd) demonstrate initial resistance to the standard and even "salvage" chemotherapy and need innovative drugs for the treatment. methods: a 15-year girl was diagnosed with classical hd (nodular sclerosis)corresponding to stage ii e b (fever >38°c) in april 2016.after two cycles of oepa (vincristine, etoposide, prednisone and doxorubicin) and next two cycles copp (cyclophosphamide, vincristine, prednisone and procarbazine) the patient again had progressive disease. as the patient achieved a partial response (pr) after "salvage"therapy with two cycles of igev (ifosfamide, gemcitabine, vinorelbine, and prednisone), she received auto-sct in february 2017 (patient status before auto-sct was pr). we used ccnu-containing conditioning regimen cem: lomustine (ccnu) 300 mg/m 2 + etoposide 1200 mg/m 2 + melphalan 180 mg/m 2 . at day +90 after auto-sct, the patient again demonstrated progression of the disease: pet/ct-examination showed mediastinal tumor mass enlargement with increased left lung nodule simultaneously to short metabolic activity. patient was under observation. at day +140 the disease had relapsed and progressed -examination by pet/ct demonstrated multifocal progressive disease with multiple pulmonary lesions and increased metabolism in comparison with the previous pet/ct scan. in july-october 2017, the patient had salvage chemotherapy with a combination of brentuximab vedotin (bv) (bv dhap (dexamethasone, cytarabine, cisplatin) + bv (without chemotherapy due to suspected invasive mycosis) + bv dhap), however, only partial pet-positive remission was achieved. because of many times relapsed and progressed disease pembrolizumab therapy was started in october 2017: 2 mg / kg every three weeks four cycles totally. toxic effects and serious complications during and after therapy by pembrolizumab were not observed. in february 2018, after pembrolizumab # 4, the patient showed complete metabolic remission of the disease by control pet-ct. in april 2018, the patient received haplo-sct with post-transplant hd-cyclophosphamide. we used conditioning modes with reduced toxicity (fludarabin 150mg/m 2 + treosulfan 42g/m 2 ), high doses of cy (50 mg / kg) on days +3 and +4. tacrolimus and mycophenolate mofetil started on day +5. mmf was terminated on day 35, tacrolimus -on day 180. patient did not have acute and chronic gvhd. results: at the moment the patient is alive and still in pet-negative cr with duration more than 9 mo. conclusions: pembrolizumab has demonstrated high activity against resistant hd even after post-auto-sct progression with good tolerability for the sick child. disclosure: nothing to declare p510 high dose chemotherapy followed by autolougous peripheral blood stem cell transplantation (asct) in diffuse large b cell lymphoma (dlbcl) median age is 38,9 years (14 to 67) and sex ratio (m/f) 1.14; ann arbor stage iii-iv: 104 pts. before hdt induction chemotherapy (chop, c2h2opa) was instituted and associated with rituximab in 100 pts (66,6%), 49 pts (32,7%) received more than 2 treatment lines and 16 pts (10,7%) received complementary radiotherapy. transplant disease status before hdt was complete remission (cr) in 93 pts, partial remission in 55 pts (rp) and disease progression in 2 pts. the delay from diagnosis to hdt is 11,7 months (4-103). the hdt protocols used are: tutshka : 87 pts, tutshka+vp16 : 42 pts, bam 12 (busulfan +cytarabine+melphalan) :16 pts et beam : 5 pts. all pts received, after thawing, mobilized pbsc obtained by g-csf mobilization (15μg/kg/d, 5 days) alone and froze in liquid nitrogen. the median rate cd34+ cells infused is 3,6 x 10 6 /kg (0. 87-17.36) . the median follow-up at 12/31/2017 is 67 months . results: the median time to graft (pnc > 0.5 x 10 9 /l) was 13 days (9-18). ten early deaths were observed including 8 infection (trm: 6,6%) and 3 in disease progression at 3 months. after 3 months of hdt 137 pts are assessable including 128 pts in cr (93,4%) and 9 pts in pr (6,6%). relapse was observed in 31 pts (22.6%) and it was earlier relapse in a period of 24 months in 15 pts (48%). deaths were among 39/150 pts (26%). persistent cr was achieved in 112/137 pts (81,8%) including 23/30 (76,6%) mlcl and 89/107 (83,1%) others dlbcl. the overall survival (os) and event free survival (efs) at 10 years are respectively 68% and 64%. the os and efs are better in patients who received rituximab in initial therapy : os (79% vs 54%; p< 0,001) et efs (79% vs 46%; p< 0,001). herein, we present one patient with refractory mcl, who were insensitive to chemotherapy and then experienced a dramatic improvement with ibrutinib mono-therapy but later developed ibrutinib resistance,ultimately resulting in the deterioration of disease and death. methods: we give the patient several examinations including ultrasound, bone marrow biopsy, lymph node biopsy, exome sequencing, sanger sequencing, and so on. for the treatment of lymphoma, the patient received chemotherapy, including 1 course of chop(cyclophosphamide 1.3g day 1, doxorubicin 40mg day 1,vinorelbine 40mg day 1, and dexamethasone 15mg from day 1 to 5) and 1 course of r-dhap (rituximab 600mg day0, cytarabine 2g q12 day 1, cisplatin 100mg day 1,dexamethasone 20mg from day 1 to 5)in succession.because of the failure to control disease progression, ibrutinib 560mg qd was used until the patient died. results: the 64-year-old man initially referred to our hospital for complaints of abdominal pain and distention over 2 months. ultrasound showed splenomegaly and multiple enlarged retroperitoneal lymph nodes.excisional biopsy conducted on the right neck lymph node revealed the presence of malignant cells.immunohistochemically, the neoplastic cells were positive for bcl2, bcl6,cd20, cd5, cd79a, cd43, ki-67(50%), sox11, cd21 (fdc) and cyclin d1 and negative for cd10, cd23 and cd3; fluorescence in situ hybridization(fish) showed igh/ ccnd1,t(11;14) 90%.thus a diagnosis of mcl was confirmed. 2 course of therapeutic chemotherapy were applied to the patient but he did not respond well.he suffered recurrent fever, thrombocytopenia, left abdominal pain, splenomegaly and multiple enlarged lymph nodes. then he received ibrutinib mono-therapy, and experienced a dramatic improvement as his body temperature was controlled, his hemogram became normal and his spleen and lymph node tapered.after about 6 months response of ibrutinib, the disease deteriorated rapidly and he died very soon. exome sequencing from the patient peripheral blood at this time detected one missense mutation in exon 5 of tp53 at nucleotide 524g>a, resulting in an argnine to histidine change at amino acid 175 (p.arg175his). but sanger sequencing of the patient bone marrow ffpe sample at the time of original diagnosis did not detect this mutation. conclusions: thus, our study reported a tp53 r175h mutation mcl patient who developed ibrutinib resistance and progressed aggresively, which may open new insight for future effort for alternative therapeutic strategies in ibrutinib-refractory mcl. disclosure: nothing to declare. minimal residual disease, tolerance, chimerism and immune reconstitution peripheral blood samples were obtained for routine analysis at several time points after hsct. all available blood samples between 0.5 and 2 years were used in the analysis. to assess changes in the cd19+b and cd19 +cd27+memory b cell counts over time while accounting for the correlation between the repeated measurements of each patient, we used linear mixed-effects models. wilcoxon rank test, kruskal-wallis test and linear regression were used for univariate analysis. results: at one year after hsct, univariate analysis showed that patients transplanted with a cb graft compared to bm and pbsc had a significantly higher absolute number of b cells (median bm= 501, median cb=1402, median pbsc=502 cells/μl, p=1.4e-5) and memory b cells (median bm= 26, median cb=75, median pbsc=23 cells/μl, . recipients with age under 5 years had significantly higher absolute numbers of b (median=919, median=473 cells/μl, p=2.9e-4) and memory b cells (median=54, median=22 cells/μl, p=2.7e-5) than above 5 years. increase in donor age was associated with a decreasing effect on b cell (r 2 = 0.35, p=5.9e-14) and memory b cell (r 2 = 0.25, p=2.1e-8) reconstitution as determined in regression analysis. following univariate analysis, we analysed these factors in a mixed effects model to assess the relation with differences in b cell or memory b cell numbers 0.5-2 years after hsct. in our analysis we found significant decreasing b cell and memory b cell numbers with increasing donor age corrected for recipient age and source (both p< 0.001). increasing recipient age also showed a significant decrease in b cell and memory b cell numbers (both p< 0.001) but there was no significant influence of donor source ( figure 1) . conclusions: b cell and memory b cell numbers after hsct are influenced by donor and recipient age but not by donor source. older donors and recipients show a decrease in b cells and memory b cells numbers 0.5-2 years after hsct. [[p512 image] 1. figure 1 . b cell development and donor age. green shows cb, red bm, blue pbsc at mean donor age.] 1 copenhagen university hospital rigshospitalet, copenhagen, denmark, 2 leiden university medical center, leiden, netherlands background: the outcome of allogeneic hsct is challenged by a delayed and long-lasting imbalanced t-cell reconstitution increasing the risk of acute gvhd, infections and disease progression. although the role of differentially and functionally distinct t-cell subsets in the development of complications has been addressed, little is known about the factors controlling their recovery. in this study, we investigated the impact of immuneregulating and homeostatic cytokines on the reconstitution of functionally distinct t-cell subsets and associated clinical outcomes. methods: we included 80 children undergoing allogeneic hsct for all (n=46) or aml (n=34) with a median age of 8.3 years (range: 0.8-17.8). donors were either mrd (n=21), mud (n=45) or mmud (n=14). bm (n=70) or pb (n=10) were used as stem cell source. conditioning regimens were based on tbi (n=25) or highdose chemotherapy alone (n=55) and included atg in 58 patients. thirty age-matched healthy children were included as controls. cytokines (il-7, il-15, il-18, scf, il-6, il-2 and tnfα) and active atg in plasma were longitudinally measured from before conditioning until 3 months after hsct (n=80) along with an extended phenotyping of t-cell maturation and differentiation by flow cytometry (n=41). results: the homeostatic cytokines il-7 and il-15 increased from pre-conditioning to peak 1-2 weeks post-hsct and gradually declined thereafter. il-6 levels were shortly elevated, while il-18 and scf remained relatively stable, and il-2 and tnf-α levels were below threshold of detection at all time points. the early rise of il-7 and il-15 was strongly associated with the degree of t-cell depletion by atg, while il-15 also correlated with markers of systemic inflammation. il-7 and il-15 levels were significantly higher in children treated with atg (p< 0.001) and correlated with both longer exposure to atg (p< 0.001) and increased levels of active atg (day +21: il-7: r=0.71, p< 0.0001; il-15: r=0.61, p< 0.0001), indicating that high levels of these cytokines reflected more pronounced t-cell depletion during lymphopenia. higher circulating levels of il-7 and il-15 were associated with a slow recovery of cd3+, cd4+ and cd8+ t-cell counts at day +30 and +60 post-hsct (p< 0.05), while the remaining cytokines did not correlate with immune reconstitution. looking into t-cell subpopulations, increased levels of il-7 and il-15 during the first month post-transplant were associated with lower numbers of naïve t cells and correlated with an increased proportion of cd4+ and cd8+ effector memory cells ( figure) . no differential effect of cytokines on polarization of cd4+ t cells into th1, th2, th17 cells or treg cells was found. in atg-treated patients, il-7 and il-15 levels at day +14 were significantly lower in patients developing acute gvhd grade ii-iv (p=0.0007 and p=0.0007, respectively). in the total cohort, increased il-7 levels were associated with a reactivation of ebv (p=0.040). conclusions: these findings suggest that quantification of il-7 and il-15 can be indicative for the degree of t-cell depletion during the first weeks after hsct and predictive of complications. overall, these results indicate that the lymphopenia-induced elevation of il-7 and il-15 is a major driver of the initial expansion of donor t-cells. background: mathematical kinetic models were adopted to study immune cell reconstitution after allogeneic hematopoietic stem cell transplantation (allo-hsct). the associations between acute graft-versus-host disease (agvhd), relapse and the immune cell reconstitution kinetic models were explored. methods: from june 1, 2011 to may 31, 2015, sixty-five patients with hematological malignancies after allo-hsct were recruited. peripheral blood was collected on +14 day, +28 day, +42 day and in +2 month, +3 month, +6 month, +9 month, +12 month, +18 month, +24 month. lymphocyte subsets were determined by flow cytometry, including in total t lymphocytes (cd3 + ), helper t cells (cd3 + cd4 + ), cytotoxic t cells (cd3 + cd8 + ), cd4/cd8 ratio, nature killer (nk) cells (cd3 -cd56 + ), nkt cells (cd3 + cd56 + ), b lymphocyte (cd19 + ), naive t cells (cd3 + hla-dr + ), static t cells (cd3 + hla-dr -), and regulatory t cells (cd4 + cd25 high foxp3 + ). mathematical kinetic models were calculated for immune cell reconstitution with spss. results: after allo-hsct, a logarithmic curve model was observed for cd3 + t cells. cubic curve models were observed for cd3 + cd4 + t cells, cd4 + cd25 high+ foxp3 + t cell, cd3 + hla-dr -t cells, cd3 + cd56 + nkt cells, cd19 + b cells. cd3 + cd8 + t cells, cd3 + hla-dr + t cells, and cd3 -cd56 + nk cells showed s type curve models. considering t cells were the major mediators for agvhd and graft-versusleukemia effect after allo-hsct. with established immune cell kinetic models, we found that different curve models were observed between patients with and without agvhd after allo-hsct. although the kinetic models were almost the same for leukemia-free and relapsed patients in the first 3 months after allo-hsct, significantly different kinetic curves could be observed thereafter. conclusions: the immune cell reconstitution showed different mathematical curve models after allo-hsct. kinetic reconstitution model of certain immune cell was associated with agvhd and relapse. hence, mathematical kinetic models of immune cell reconstitution may be potential indictor for predicting agvhd and relapse after allo-hsct. disclosure: nothing to declare lineage specific chimerism analysis in pediatric patients following allogeneic hematopoietic cell transplantation (hct background: the outcome of allogeneic hct is dependent on several variables that include patient age, disease and stage, cytoreduction, graft, graft manipulation, and graft versus host disease (gvhd) prophylaxis. one aspect of hct that remains poorly defined and studied is the donor/ host (d/h) chimerism post hct. since 2010, we followed patients with d/h lineage specific chimerism post hct. analyses were performed by short tandem repeat (str) polymorphism analysis at the american red cross blood services (philadelphia, pa). studies were performed on blood total leukocytes, myeloid/neutrophil cells, t-cells, bcells, and nk-cells. methods: in this retrospective study, the charts of 154 consecutive patients who underwent allogeneic hct between january 2010 to june 2015 on the pediatric bone marrow transplant service at mskcc were retrospectively reviewed. lineage specific donor chimerism post hct was studied including d/h chimerism trend, and factors with potential impact on chimerism including: age, disease, graft source, and t-cell depletion (tcd). preliminary analyzes performed on this cohort included wilcoxon rank test and cox proportional hazard analyses. results: 137 patients were selected based on the number of analyses. the median age was 11.3 years. patients had hematologic malignancies (n=95) or non-malignant hematologic diseases (n=27), or immune disorders (n=15). cytoreduction included tbi-(n=42), or chemotherapybased regiments (n=98). patients were recipients of t-cell depleted marrow or peripheral blood grafts (n=101), unmodified marrow or peripheral blood grafts (n=28) or cord blood grafts (n=8). full donor chimerism of myeloid cells, b-cells and nkcells, but not t-cells occurred early post-transplant. there was no difference in the percentage of total donor leukocytes at 3 months vs. 12 months post hsct (n=30), while the median of donor t-cell chimerism was 51% at 3 months and 91% at 12 months post hsct (p< 0.0001, n=42). figure 1 shows the impact of different factors including: (a) the use of tbi-or chemotherapy-based regimens, (b) age (< or > 3 years), and (c) type of graft (t-cell depleted vs unmodified vs cord blood). donor total leukocytes chimerism was significantly lower at 12 months as compared to 3 months for patients < 3 years of age (p=0.012). for most grafts, full donor chimerism of t-cells occurred early, while for t-cell depleted transplants, it took up to one year to complete. cord blood grafts were associated with high t-cell donor chimerism throughout the post-transplant period. there was a significant difference in the % donor t-cells at 3 and 12 months post hct when comparing t-cell depleted and unmodified grafts (p=0.015). conclusions: this preliminary analysis of lineage specific chimerism post-transplant showed that donor tcells may take one year to fully recover post-transplant, mostly following t-cell depleted grafts, without intervention. cord blood grafts were associated with high donor chimerism throughout the post-transplant period. lastly, factors associated with loss of donor chimerism posttransplant were younger age and non-malignant disorders. more in-depth analyses are being performed including the relation of chimerism and hct outcome. disclosure: eileen nicoletti -employee rocket pharmaceuticals, susan prockop -investigator atara biotherapeutics -research funding, susan prockop -mesoblast -research funding, nancy kernan -gentium -support; jazz pharmaceuticals -support, richard o´reilly -atara biotherapeutics -royalty, consultancy and research, jaap jan boelens -bluebird bio -consultancy, avrobio -consultancy; jaap jan boelens -chimerix -consultancy; magenta -consultancy background: the success of hematopoietic stem cell transplantation (hsct) lies with the ability of the engrafted immune system to remove residual leukemia cells via a graft-versus-leukemia effect. despite this, relapse remains the major cause of mortality among patients receiving hsct. one of the immune evasion mechanisms of leukemic cells to escape from donor t cell recognition in haplo-hsct is the genomic loss of the patient specific hla. it has been described in 20-30% of acute myeloid leukemia (aml) and myelodysplastic syndrome (mds) relapses after haplo-hsct. the aim of this study was to analyze hla loss in a large cohort of patients who relapsed after t-cell replete haploidentical transplantation with posttransplant cyclophosphamide. methods: from december 2007 to september 2018, 180 patients with hematological malignancies who received a haplo-hsct were recruited. among them, 31 patients presented a relapse after haplo-hsct. hla typing was performed by real-time pcr using hla-kmr kit (gendx, netherlands).nine patients were excluded from the analysis because the kit employed did not include the recipientspecific hla. thus, a total of 22 relapse cases were analyzed. the analysis of chimerism was carried out using short tandem repeat pcr amplification (ampflstr sgm plus, thermo fisher, usa) with a sensitivity of 1%. results: genomic loss of the patient hla occurred in 6/ 22 patients (27%) ( table 1) . these patients presented different hematological neoplasms. interestingly, 4 patients presented lymphoid neoplasm (1 acute lymphoblastic leukemia (all-t), 1 dentritic cell leukemia (dcl) and 2 hodking´s lymphoma (hl)). hla loss relapses occurred later than classical relapses (370 vs.166 days). regarding the treatment received (table 1) , four patients were studied retrospectively. three of them were treated with donor lymphocyte infusions (dlis) + chemotherapy or other drugs at the time of the relapse. the other patient did not receive any treatment. in the end, all 4 patients died from disease progression. prospectively, we detected hla loss at relapse in other two patients. at the moment of relapse, the first case received brentuximab + haplo-hsct from alternative donor and the other case received daratumumab + haplo-hsct (pending). both patients are alive, the first one in complete remission (cr) and the second one in partial remission (pr). conclusions: the frequency of hla loss at relapse is similar in our cohort to what is described in the literature. hla loss has been identified in patients with lymphoid neoplasms, while this mechanism has not been previously described in such diseases. the analysis of this immune evasion mechanism should be implemented in the routine screening of patients transplanted from haploidentical donors in order to design effective rescue strategies. these treatments should not be based on dlis or second transplantation with the same donor, instead, alternative donors should be used. background: an adequate immune reconstitution (ir) is crucial to reduce transplant toxicity, relapse rate and mortality after allogeneic stem cell transplantation (allohsct). the aim of this, single center retrospective study was to investigate the correlation between the recovery of different lymphocyte subpopulations with the main transplant outcomes, including overall survival (os), disease free survival (dfs) and non-relapse mortality (nrm). methods: we analyzed the ir of 177 adult patients (aml n=62, all n=41, mds n= 24, nhl n=9, hd n=2, cll n=4, cml n=7, mm, n=11, mpn n=10) who underwent (allohsct) between january 2013 and march 2017. median age at transplant was 52 years (range 17. 3-71.3) with male/female ratio of 62%. donors were hlaidentical siblings (n=39, 22%), family haploidentical (n=11, 6%), matched unrelated (109, 62%), mismatched unrelated (n=11, 6%) and cord blood units (n=7, 4%). the stem cell source was the bone marrow (bm) in 30 patients (17%), the cord blood in 7 (4%) and g-csf mobilized peripheral blood in 140 (79%). the conditioning regimen was myeloablative in 99(56%) transplant, reduced intensity in 75 (42%) and immunosuppressive in 3 (2%). gvhd prophylaxis was based on calcineurin inhibitors in combination with methotrexate or mofetil mycophenolate. antilymphocytes immunoglobulins was used in 145 patients (81%) (anti thymocytes globulin, atg sanofi-genzyme in 114 or anti t-lymphocyte globulin, atlg -neovii biotech, in 31). the peripheral blood lymphocyte subsets (cd3+, cd3+cd4+, cd3+cd8+, cd19+ (b cells) and cd16+cd56+ (nk) were analyzed by flow cytometry at 1, 2, 3, 6, 12 and 24 months after hsct. post-transplant engraftment was molecularly determined by vntr analysis. results: as detailed in table 1 the proportion of full donor chimerism analyzed in the peripheral blood t lymphocytes improved progressively after transplantation and the same pattern was observed when the chimeric status was measured in bone marrow mononuclear cells. to favor the achievement of a full donor chimerism, dli were performed in 26 patients starting at the median of 96 days after transplant (range:34-447). with a median follow-up observation of 25 months (range 7-61), the one year os and nrm was 88% and 5%, respectively. at 6 months after allohsct, the achievement of values higher than 75, 175 and 65 /μl for cd4+, cd8+ and nk cells, respectively was significantly associated to a better os (figure 1 ), dfs (p = 0.05), and to a lower nrm (p< 0.001 for cd4+ and cd8+, p= 0.0034 for nk). a better lymphoid reconstitution was observed after the use of either a sibling or a haplo donor than a matched unrelated or cord blood donors. the use of atg was significantly associated with a delayed cd4+ recovery but with a faster nk cells reconstitution. conclusions: at six months after allohsct, recovery of cd4+ and nk cells predicts survival. monitoring of immune recovery may help to guide pre and post-transplant treatment strategies. days infections and disease control. several groups have demonstrated the independent prognostic value of different lymphocyte subpopulations in hsct outcomes. posttransplant cyclophosphamide (pt-cy) effectively prevents gvhd after hla-haploidentical hematopoietic stem cell transplantation (haplo). the use of pt-cy in hla matched related (mrd) or unrelated (mud) donors hsct, although less explored, has also been introduced. the aim of this study was to compare the early immune reconstitution after allogeneic hsct from haploidentical and hla-matched donors using pt-cy. methods: one hundred and sixty-four hsct performed in our center were analyzed: 125 haplo performed between 2011 and 2017 and 39 hsct from hla-identical donors (19 consecutive mrd sct performed with pt-cy between 2016 and 2017 and 20 mud sct with pt-cy between 2014 and 2017). pt-cy was administered at 50mg/kg/d in days +3 and +4 postransplant, followed by mmf 10mg/kg/ d and a calcineurin inhibitor (ciclosporina a or tacrolimus) from day +5 ahead. we retrospectively compared early immune reconstitution at day +30 and day +90 among these three populations. early ir was assessed through the analysis of lymphocyte subpopulations including total t lymphocytes cd3+, cd4 + and cd8+subpopulations, nk cd3-cd56+ cells, cd56 + bright immature subpopulation and total b cd19+ lymphocytes.. lymphocytes subpopulations were determined by multiparametric flow cytometry (fc500 and navios, beckman coulter®). results: all patients, but 1 mud and 1 haplo, received pb as stem cell source. 46 patients received prior transplant in haplo group. patient´s characteristics are shown in table 1. patients who received hsct from mrd showed the fastest ir, with statistically significant differences compared to haplo in almost all lymphocyte populations at day +30 (cd3+, cd4+, cd8+ and nk cells), and also in cd3+, cd8+ and b lymphocytes at day +90. comparison between haplo and mud hsct showed better ir among haplo, demonstrated by higher counts in cd3+,cd4+, cd8+ and nk cell counts at day +30. no differences were seen at day +90. (figure 1 ). percentage of immature cd56 bright nk cells was higher in mud hsct at +30, with no differences between haplo and mrd hsct. conclusions: in our cohort of patients with pt-cy based gvhd prophylaxis regimen, those who received hsct from mrd showed the earliest immune reconstitution compared to haplo and mud at day +30 and +90. haplo showed better ir compared to mud at day +30. nk maturation at day +30 was a little better among haplo and mrd hsct recipients than mud hsct patients. in our experience, using mostly pbsc as graft source, type of donor influenced early ir in pt-cy based hsct, background: cell-free dna (cfdna) isolated from plasma or serum has received increasing interest for diagnostic applications. however, the reported clinical usefulness of cfdna in patients undergoing allogeneic cell transplantation (hsct) is scarce. methods: the chimeric status both in peripheral blood and in cfdna obtained from plasma was investigated in 110 patients undergoing hsct. dna and rna were isolated from plasma within four hours of blood draw. patients were evaluated for chimerism at day +30, +100 and +365 post-transplant. a panel of seven microsatellites was amplified by pcr for chimerism detection and pcr products were analysed by capillary electrophoresis. for further cfdna characterization the micro rna (mirna) 200c was analysed using digital pcr. mutations frequently used for minimal residual disease assessment such as flt3-itd, npm1 and jak2 were also investigated in cfdna. results: the mean cfdna concentration in transplanted patients was 469 ng/ml, while in healthy donors used as control group (n=20) was 119 ng/ml. the mean cfdna concentration difference between both groups reached statistical significance (p= 0.0197). when analysing cfdna from transplanted patients and in the control group we could not detect dna fragments larger than 400 bp and the size range of the analysed fragments was between 80 and 200 bp. in 41 out of 110 patients a mixture of donor and recipient cfdna (mc) was detected. with the exception of three patients relapsing after transplant in which mc was detected both in peripheral blood and plasma in the rest of the patients (n=38) mc was detected only in plasma. the mean percentage of recipient cfdna in the plasma samples was 18% (range: 1-81%). all the patients with acute gvhd (agvhd) (grade: i-iv) (n=15) showed mc in plasma at least in one of the time-point tested. no significant difference was found in the mean recipient cfdna percentage in patients with agvhd grade i-ii when compared with grade iii-iv. meanwhile in the group of patients with chronic gvhd (n=42) mc in plasma was detected in 13 patients. in those patients with clinical improvement of agvhd (n=6) a decrease in the percentage of recipient cfdna was observed during treatment. in patients without improvement or even agvhd worsening (n=5) stable or increasing recipient cfdna percentage was detected. since recipient cfdna can be detected in patients without transplant-related complications we analysed the mirna 200c expression in all patients with recipient cfdna. a significant difference was found in the mirna 200c expression in patients with agvhd (mean mirna 200c: 9.7 mirna 200c copies/10 4 u6 copies) when compared with patients without gvhd (mean mirna 200c: 35.4 mirna copies/10 4 u6 copies). in those patients with extramedullary aml relapse (n=3) frequent mutations (flt3-itd, npm1) were only detected in the cfdna fraction. conclusions: longitudinal analysis of cfdna represents a useful complementary tool in particular for those patients with clinical complications after hsct. disclosure: nothing to declare comparison of the impact of atg/pt-cy-based and tcr αβ-depletion as gvhd prophylaxis regimens on the recovery of memory t-cell compartment background: over recent years haploidentical and hlamismatched donors have been increasingly adopted as a valid donor source. modern graft-versus-host disease (gvhd) prophylaxis regimens such as drug-based (antithymocyte globulin (atg), post-transplant cyclophosphamide (pt-cy)) or graft-manipulated (tcr αβ-depletion) demonstrate effective prevention of gvhd. here we report our data about an influence of different gvhd prophylaxis regimens after allo-hsct with pbsc as a graft source on cd8+ memory t-cells recovery. methods: our study comprised 32 leukemia patients who underwent allo-hsct with pbsc as a graft source in national research center for hematology, moscow, russia. detailed patients characteristics are presented in table 1 . peripheral blood samples were collected on day +30, +60 and +90 after allo-hsct. flow cytometry analysis was performed on bd facs canto ii (becton dickinson, usa) to define t-memory subsets: t-naive and t-stem cell memory (tnv+tscm) -cd8+cd45r0-ccr7 +cd28+; t-central memory (tcm) -cd8+cd45r0 +ccr7+cd28+; t-transitional memory (ttm) -cd8 +cd45r0+ccr7-cd28+; t-effector memory (tem) -cd8+cd45r0+ccr7-cd28-; t-terminal effector (tte) -cd8+cd45r0-ccr7-cd28-. sysmex xe-2100 was used to calculate absolute count of different t-memory cell subsets. mann-whitney u test was used for nonparametric data analysis. a p-value less than 0.05 was considered as significant. results: results of mann-whitney u test (calculated pvalues) to compare absolute number of t-memory cell subsets in terms of different gvhd prophylaxis regimens are presented in figure 1 . during all follow-up period the number of tnv+scm and tcm remains significantly reduced after atg+pt-cy or tcr αβ-depletion compared to atg-based immunosuppressive regimen. on day +30 we observe no difference in the number of tnv+scm and tcm cells after atg+pt-cy or tcr αβ-depletion. terminally differentiated cd8+ cells (ttm, tem, tte) count is significantly lowered in tcr αβ-depletion patients group in comparison to atg+pt-cy. nevertheless recovery of tnv+scm and tcm after pt-cy is faster than after tcr αβ-depletion. conclusions: according to our data the mechanism of pt-cy is seems to be more selective compared to tcr αβ-depletion due to its transient impact just on tnv+scm and tcm without affecting on the effector pool. through this it may lead to delayed reconstitution of adaptive immunity after tcr αβ-depletion compared to using pt-cy. clinical relevance of the quantitative characteristics of immune recovery in the context of different approaches to gvhd prevention remains to be established. background: immune effector cells, belonging to either innate or acquired immunity, play a key role on preventing disease relapse after allogeneic haematopoietic stem cell transplantation (hsct). most of known immune effector are cd3 + cd8 + t-cells and cd3 -cd56 + natural killer lymphocytes, while cd3 + cd4 + cells act as modulatory and regulatory cells. the early post-hsct ratio between these cellular subsets may be an indicator of graft vs-tumor (gvt) effect. methods: we retrospectively revised the immune recovery of 117 allogeneic hsct performed at our institution from 2013 to 2017, analysed on peripheral blood by multiparametric flow cytometry lymphocyte subpopulations panel. diagnosis were acute leukemias (69%), chronic myeloproliferative neoplasms (10%), lymphomas (10%), myelodysplastic syndromes (5%), multiple myeloma (3%) and severe aplastic anemia (3%). we established 2 early time-points of evaluation, 30 and 60 days from the graft infusion, to analyse the differences in disease free survival (dfs) and overall survival (os) between patients according to the cd3 + cd8 + x cd3 -cd56 + / cd3 + cd4 + ratio. results: median ratio at +60 days was of 0,5667. at this time point, patients who showed the ratio higher than the median had both a better dfs (median dfs time not reached vs 12 months; p = 0,018) ( figure 1 ) and os (median os time not reached vs 13 months; p = 0,014). likewise, ratio at +30 showed an advantage on dfs (p = 0,027), and not on os (p = 0,06). other factors possibly affecting both dfs and os were analysed in univariate analysis, such as the use of antithymocyte globulin (atg), conditioning regimen intensity, graft source, hla-matching and disease status at hsct, the latter being the only variable with a significantly detrimental impact on both os and dfs. disease status was confirmed an independent valriable associated with both dfs and os as well as +60 ratio both on dfs (hazard ratio [hr] -2,721; p = 0,015) and os (hr 2,627; p = 0,022). conclusions: our data show that cd3 + cd8 + x cd3 -cd56 + / cd3 + cd4 + ratio assessed at +60 is and independent predictor of transplant outcome, possibly representing a row indicator of anti-leukemic immune surveillance. the integration of this index with other known outcome predictors may help in improving the management of post-transplant phase. [[p524 image] 1. figure 1 background: allogeneic stem cell transplantation (alo-hsct) is a curative treatment but it is associated with lifethreatening complications. most deaths are due to relapse, graft versus host disease (gvhd) and infection. the pattern and quality of the immune reconstitution (ir) after transplantation may affect these outcomes. however, there are limited data on the association of the quality of the ir and either the development of gvhd and survival. methods: eighty-five patients who received a non t-cell depleted alo-hsct in our center from 2011 to 2014 were prospectively studied. most patients received hla-identical grafts. total cd4+ and cd8+ t cells, ccr7+cd4+ and ccr7+cd8+ (which include both naïve and central memory t cells) and naïve ccr7+cd62l+ t lymphocytes were quantified by flow cytometry. data were collected at days +30, +60, +90, +180 and +360 after alo-hsct. the association between ir and the gvhd was studied through an anova. for the multivariate analysis, a logistic regression was performed including those confusing clinical variables that were significant in the univariate analysis (p≤0.10). the study of overall survival (os) versus ir was performed with a cox regression model. results: total cd3+ t lymphocytes reached normal numbers within the first two months. median t cd8+ count was 263 cells/ul after one month, which is within the normal range. conversely, it took nearly one year to get normal counts of cd4+ t cells (542 cells/ul). the only two clinical parameters conditioning a worse recovery of the cd4+ t cells were the previous alosensitization of the donor and the sex, being female donor and male recipient the worst combination for the ir. no parameters influenced the quality of the reconstitution of cd8+ t cells. of note, the age or the hla status did not influence the quality of the ir. when the patients were divided into gvhd and no gvhd, we found no differences in the recovery of either the proportion or absolute count of every t cell subpopulation, including total t cells as well as naïve/central memory t cells, both cd4+ and cd8+. finally, a multivariant analysis confirmed that the absolute counts of cd4+ccr7+ t cells at day +90 as well as the absolute counts of both cd4+ccr7+ t cells and naïve cd4+ccr7+cd62l+ at day +180 were associated with better os. conclusions: in conclusion, neither the development of gvhd nor other relevant parameters seem to play a determinant role in the quality of the ir. to our knowledge this is the first study which demonstrate a clear association between the recovery of naïve cd4+ t cells measured by flow cytometry and the os. disclosure: nothing to declare p526 abstract already published. azacitidine (aza) for prophylaxis or pre-emptive therapy for myeloid neoplasms after allogeneic stem cell transplantation whom 21 were treated prophylactically and 11 preemptively. median age was 55 years [range, 15-69] and all patients had a diagnosis of aml or high-risk mds. prophylactic treatment consisted of aza 32 mg/m 2 for 5 days in cycles of 28 days. in the pre-emptive setting, 5 patients received 75 mg/m 2 for 7 days per cycle and 6 patients 75 mg/m 2 for 5 days per cycle. a median of 6 cycles [range, [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] was administered in the prophylactic group and of 4 cycles [range, 4-22] in the pre-emptive group. ten patients also received at least one dli after the third aza cycle: 8 patients in the prophylactic group and 2 patients in the pre-emptive one. results: during follow-up, 10 patients had significant delays in treatment plan due to transitory mild complications. however, 15% of patients (n=5) presented infectious complications requiring hospitalisation and 40% of patients (n=10 in the prophylaxis group and 3 in the pre-emptive group) presented some form of gvhd. in patients who developed gvhd, 3 had to discontinue treatment (all in the prophylaxis group); also 8 patients discontinued treatment due to disease progression. the overall drop-out rate was 37.5% (n=12). survival was analysed from initiation of treatment with aza and median follow-up was 16 months. one-year efs was 95% in the prophylaxis group, with only one patient relapsing and no deaths. in the pre-emptive group, the 1-year efs was 45% and the median efs was 6 months; 1-year os was 70% and median os was 24 months. conclusions: we conclude that post-transplant aza treatment is a well-tolerated therapy, but the incidence of side effects remains discordant in the literature. results in the prophylaxis group are excellent, but patients with positive minimal residual disease treated pre-emptively had a lower outcome with only stabilisation of the disease. randomised prospective trials are needed to define patients who would benefit the most from this treatment and at what timing, dosage and duration of treatment. disclosure: nothing to declare. abstract already published. results: all subjects experienced hematopoietic engraftment at a median of 17 days (range 14-20) and demonstrated full donor myeloid chimerism. m-mdsc and pmn-mdsc recovery peaked at a median of 22 days posttransplant. the median peak absolute m-mdsc count was 9,828 cell/ml (range 9,180-41,120/ml) representing a range of 1.5% to 4.8% of pbmcs. the pmn-mdsc peak was more robust, with a median absolute peak of 103,730 cells/ml (range 11,330-676,800/ml) representing a median of 25.3% of pbmcs (range 10. 3-44.2%) . of note, the one patient who developed severe, life-threatening gvhd had the lowest absolute and relative pmn-mdsc recovery (11,330 cells/ml and 10.3% of total pbmcs). recovery of m-and pmn-mdscs occurred at a similar tempo and magnitude in two recipients of standard gvhd prophylaxis (tacrolimus/methotrexate). however, while mdscs isolated from ptcy recipients exhibited clear t-cell suppressive capacity, those from the comparison patients did not (see figure) . conclusions: mdscs recover rapidly and robustly after allohct using ptcy as gvhd prophylaxis, and may play a role in mitigating gvhd risk by mediating t-cell suppression. this may be a mechanism by which ptcy results in donor-recipient tolerance. background: high dose chemotherapy followed by autologous stem cell transplantation (asct) offers a cure in the upfront and relapsed setting in both hodgkin (hl) and non-hodgkin lymphoma (nhl). asct also remains standard of care in previously untreated multiple myeloma (mm) patients after induction therapy, if eligible. the availability of new cellular or other immune therapies that can be used after asct underscores the potential importance of monitoring immune reconstitution after asct. methods: immune reconstitution panels (irp) were evaluated retrospectively in all lymphoma and mm patients over a 5-year span (2012-2017) whom underwent asct at our institution. patients were included if they had a pre-asct measured within 30 days of asct and two other irp at any of the following timepoints (1) day 30-45, (2) day 60-90, and (3) at 1-year post-asct. patients in the lymphoma cohort had their irp excluded if they had additional treatment within the first year post-asct (other than maintenance rituximab). mononuclear cells from peripheral blood were analyzed by flow cytometry for assessment of lymphocyte phenotype and numbers. absolute values were compared using the mann-whitney u test. results: the data on 78 patients were available for analysis (50 mm, 24 nhl, 4 hl) . all lymphoma patients were conditioned with beam. all mm patients were conditioned with a standard high dose melphalan regimen. the median pre-asct absolute cd3 counts in the lymphoma cohort were significantly lower than the mm cohort at 1709 cells/μl vs 3309 cells/μl, respectively (p=0.049). however, the mm cohort exhibited a greater percent reduction in cd3 cells on day 30 at 85.3% vs 63.5%, respectively which continued through day 365 at 79.8% vs 70.3%, respectively. this led to nonsignificant changes in absolute cd3 count by day 365 at 667 cells/μl vs 507 cells/μl, respectively (p=0.39) (figure 1). the median absolute cd4 count pre-asct for mm and lymphoma cohorts were 2101 cells/μl and 863 cells/μl, respectively (p=0.007). similarly, a greater percent reduction in cd4 cells led to comparable absolute counts on day 365 at 310 cells/μl vs 298 cells/μl, respectively (p=0.322). the failure of post-asct cd3 reconstitution to pre-asct levels was driven by lack of cd4+ cell recovery, namely cd4+cd45ra+ cells with a median of 21 cells/μl and 20 cells/μl in the mm and lymphoma cohorts, respectively at day 365 (figures 2 and 3). this led to markedly diminished cd4:cd8 ratios through day 365 (figure 4). [[p531 image] 1. conclusions: impaired t-cell reconstitution in both lymphoma and mm continues through 1-year post-asct. as shown, a larger percent reduction in median cd3 and cd4 counts through day 365 was appreciated in mm compared to lymphoma leading to the nonsignificant differences in the post-asct absolute counts despite significantly higher pre-asct counts in the mm cohort. impaired recovery of cd4 t-cells may increase the risk of opportunistic infections, decrease the response to vaccination and lead to ineffective anti-tumor response. further prospective and larger retrospective studies like this should continue in the modern-era as they may help predict responses to further interventions requiring a robust t-cell repertoire for maximal efficacy such as car-t cell and bite therapies. disclosure: nothing to declare p532 peri-transplant detection of measurable residual disease by multicolor flow cytometry is highly predictive for relapse in acute myeloid leukemia patients background: presence of measurable residual disease (mrd) prior to allo-sct has been shown to be predictive for survival in patients in hematological cr of aml. in this study we analyzed the impact of mrd in such patients measured by 8-color multiparameter flow cytometry (mfc) prior to and on day +100 post-transplant. methods: the bone marrow samples immediately prior to allo-sct and on day +100 post-transplant were retrospectively analyzed. mrd evaluation was carried out with antibodies against: (1) results: a number of 55 aml patients (male, n=35) with median age of 57 years (23-77) in hematological cr prior to allo-sct were enrolled in the study. we observed lower survival in patients with mrd by mfc pre-transplant (2y os: 61% (41-81%) vs. 92% (82-100%), p=0.018) due to increased relapse incidence (35% (17-53%) vs. 8% (0-18%), p=0.009). in multivariate analysis, mrd pos prior to allo-sct has strong significant impact on os (hr 6.5 (1.5 -30) , p=0.015). of 55 patients, a sample both before and on day +100 after transplantation was available in 33 patients. of those 33 patients, 15 (46%) were mrd negative prior to transplant and on day +100 (mrd neg/neg ); 14 (42%) patients were mrd positive prior to transplant and negative at day +100 (mrd pos/neg ); and 4 (12%) patients were mrd positive at both timepoints (mrd pos/pos ). dfs and os for these three groups were as follow: 2y dfs: mrd neg/neg : 93% (80-100%), mrd pos/neg : 78% (56-100%); mrd pos/pos : 25% (0-75%, p=0.006); 2y os: mrd neg/neg : 100%; mrd pos/neg : 93% (79-100%); mrd pos/pos : 25% (0-75%, p< 0.001). upon multivariate analysis, the mrd status prior to transplant and on day +100 showed strong significant impact on dfs (hr 3.6 (1.3 -9.9), p=0.01) and os ), p=0.017). we did not observe any significant impact of other factors included in the multivariate analysis (patient's age, patient's sex, and recipient/ donor sex constellation). conclusions: mrd positivity prior to allotransplant and at day +100 by mfc is highly predictive for survival after allo-sct. disclosure: nothing to declaire background: immune reconstitution is a critical factor for risk assessment of life threatening infections and long-term survival in patients undergoing allogeneic cell transplantation (hsct). methods: immune cell subsets (cd19, cd4, cd8, cd56, cd25+cd127-) were quantified by flow cytometry. trec and krec were quantified simultaneously using droplet digital pcr (dpcr). a total of 31 patients were evaluated. mean age at transplant was 56 years (range: 18-74 years) samples were obtained before hsct and at day 30, 100 and 365 after hsct. results: absolute numbers of cd3 and cd19 cells remained below pre-transplant levels until day 100, increasing further and eventually reaching pre-transplant levels one year after hsct. absolute counts of cd4 and cd25+cd127-cells remained below pre-transplant levels beyond one year after hsct. cd56 cells were characterized by fast reconstitution kinetics, reaching pre-transplant levels already at day 30. b cells correlated with krec levels at all time-points tested, whereas t cells correlated with trec levels only one year after transplantation. when we compared cell subsets, trec, krec levels and the reconstitution kinetics thereof between patients with reduced intensity conditioning (n=26) or full conditioning (n=5) no significant differences were observed. patients with pre-transplant trec levels above the mean (200 trec copies/ml blood) showed higher trec levels and a faster t-cell reconstitution after hsct suggesting that tcell reconstitution can be predicted by analysing thymic functionality before transplantation. indeed, in patients with a pre-transplant trec above 200 trec copies/ml blood, the positive predictive value for an efficient t-cell reconstitution was 0.889 (p=0.012). we analysed the recovery kinetics of the cell subsets, trec and krec levels in patients with and without transplant-related complications. patients with either acute graft-versus-host disease or severe infections showed a slower trec reconstitution when compared with patients without complications. conclusions: our data suggest that the analysis of immune cell subsets together with trec and krec quantification can be used to evaluate the immune reconstitution process after hsct. pre-transplant trec levels allow t-cell reconstitution efficiency prediction after hsct. disclosure: nothing to declare background: falling donor / mixed chimerism after allogeneic haematopoetic stem cell transplant (sct) is associated with an increased risk of relapse and the potential for graft rejection. donor lymphocyte infusions (dli) are often administered in patients with mixed chimerism to achieve full donor chimerism but there is little data on long term outcomes for dli given for persistent mixed chimerism. methods: a retrospective analysis of all patients administered dli for mixed chimerism between 2008 to january 2017 was performed. all patients were transplanted at the university hospital of wales within the south wales blood and marrow transplant (swbmt) programme. patients were identified by the swbmt database and additional outcome data gathered by review of patients' medical records. results: 58 patients were treated with 111 donor lymphocyte infusions between 2008 and january 2017. thirty one patients treated for relapse (with or without mixed chimerism) were excluded as was a further patient with a mismatched donor. the rest were 10/10 match. twenty six patients received a total of 54 donor lymphocyte infusions for mixed chimerism alone. the median age was 63 years (range: 27-71) with 65% women. fourteen (54%) of the patients had sibling donor transplants and twelve (46%) from matched unrelated donors. indications for transplant were: for aml or saml (n=16), myelofibrosis (n=4), mds (n=3), hodgkin lymphoma (n=1), non-hodgkin lymphoma (n=1) and all (n=1). escalating doses of donor cd3+ t cells were administered commencing at 5×10 5 /kg to 5×10 6 /kg then increased at half log increments according to chimerism results until full donor chimerism was achieved. the median number of doses administered was 2 (range 1-5). the median interval was 94 days (range 48-408). the median dose was 1×10 6 / kg (range 5×10 5 -6×10 7 ). seventeen patients (65%) achieved full donor chimerism and remained so until most recent follow up (median 25 months, range 12-64). one patient continued to receive dli after the study period and later reverted to full donor. two patients had ongoing mixed chimerism with no evidence of relapse. two patients relapsed; one of whom later achieved remission. there were six cases of gvhd; acute gvhd (grade ii n=2, grade iii n=1) and 2 cases of chronic extensive gvhd. one patient had gvhd features consistent with overlap syndrome. a total of five patients died, four due to infection (one in a patient with gvhd) and one due to cardiac toxicity from previous treatment (confirmed on post-mortem). conclusions: the results of our single centre study help reinforce the evidence for dli in establishing full donor chimerism when mixed chimerism is detected in the absence of relapse. incremental dli dosing is an effective strategy and associated with a low relapse rate. caution should still be given to the risk of gvhd following dli, however the risk appears to be low in this study. larger prospective studies are ongoing to address the optimal dosing strategy for dli post-transplant. disclosure: nothing to declare hypomethylating agents for the treatment of relapsed acute myeloid leukemia after allogeneic blood stem cell transplantation: a single center experience mariarita sciume 1 , giorgia saporiti 1 , elena tagliaferri 1 , nicola fracchiolla 1 , federica grifoni 1 , giorgia levati 1 , luca baldini 1 , francesco onida 1 the post-transplant period with well-balanced profile of good efficacy and moderate toxicity. we retrospectively evaluated the safety and efficacy of hma +/-dli in a reallife cohort of aml patients relapsing after allo-sct. methods: data from all patients with aml who underwent allo-sct at our institution in the last 6 years and subsequently received hma as a salvage treatment for disease recurrence or preemptively for loss of complete donor chimerism were collected. results: eleven patients with a median age of 64 years (range 41-66) were identified; median time between allo-sct and time to hma therapy was 10 months (range 4-42). according to eln genetic risk stratification, 2 patients were classified in the favorable group, 3 in the intermediate-i, 2 in the intermediate-ii and 4 in the adverse one. six patients were treated with aza, whereas the remaining 5 patients with dac. the cycles were repeated every 28 days. ten patients (91%) started hma for morphological aml relapse, while one patient received aza as a sequential treatment after dli administered for loss of complete donor chimerism. median number of cycles was 3 (range 1 -20). treatment strategy included combination with dli in 5 patients (2 in the dac cohort, 3 in the aza cohort), while in one case of flt3-itd + aml sorafenib was also associated to dac and dli. no grade 3/4 toxicities and no acute gvhd occurred. a clinically significant response was observed in four patients (36%), all receiving at least 4 cycles of hma therapy; in particular, a complete remission (cr) was achieved in 3/10 patients treated for morphological relapse, including the one who received the dac/dli/sorafenib combination and one (favorable eln risk) who received aza alone (not eligible for dli due to a concomitant lateonset cutaneous grade 2 gvhd). of interest, the latter patient also displayed a resolution of the cutaneous gvhd. full donor chimerism recovery with no gvhd was observed in the patient who received aza for the progressive donor chimerism loss not responding to dli alone. with a median follow-up of 7 months (range 4-29), the median os from hma treatment in responding patients was 16 months (range 4-29); at the time of data collection responses were maintained in all four patients. seven patients had died, six from aml progression and one for severe intestinal gvhd occurring after failure of dli+aza and a following salvage induction chemotherapy treatment. conclusions: although arising from a limited number of patients, our real-life experience of treatment with hmas +/-dli in aml patients relapsing after allo-sct showed a general very good safety profile and promising antileukemic activity, altogether suggesting a facilitation of the graftversus-leukemia effect (gvl) associated to a possible suppression of the gvh reaction. disclosure: nothing to declare conclusions: in this study, cd4-positive cell count and igg value had recovered about 15 months after sct. in our institute, we have achieved a low incidence of infection by education and medication for patients until recovery of cd4-positive cell count and igg. however, we found a higher incidence of infection after recovery of cd4-positive cell count and igg. at 12-15 months after sct, administration of prophylactic medications such as sulfamethoxazole-trimethoprim were terminated and social comeback such as return to school or work were achieved in most patients. it is possible that the high incidence of community-acquired infection was associated with their comeback. thus, we should consider additional prevention of infection for patients in this period and further evaluation of immunological markers is needed. disclosure: no potential conflicts of interest were disclosed. effect of minimal residual disease before transplantation on the outcome of haplo-identical hematopoietic stem cell transplantation for high-risk acute lymphoblastic leukemia yehui tan 1 , sujun gao 1 , xiaoliang liu 1 , long su 1 , wei han 1 , yu liu 1 , yangzhi zhao 1 background: to analyze the effect of haploid hematopoietic stem cell transplantation (hid-hsct) on high-risk acute lymphoblastic leukemia (all), and to explore the effect of minimal residual disease (mrd) before transplant on the prognosis. methods: a retrospective analysis was made on 39 high risk all patients accepted hid-hsct in our hospital from january 2013 to january 2018. the clinical features, stem cell implantation, complications, survival and recurrence were compared between pre-transplant mrd + and mrdpatients. results: all the 39 patients got successfully implanted. the overall survival (os) was 54.67%, the disease free survival (dfs) was 40.96%, the incidence of acute graft versus host disease (agvhd) was 53.8%, including 23.1% ii~iv degree agvhd and 2.6% iii~iv degree agvhd. there was no significant difference in stem cell implantation, gvhd, cytomegalovirus and hemorrhagic cystitis between mrd + and mrdpatients. dfs and os in mrd + patients were significantly lower than those in mrd -patients, and the cumulative rr rate increased significantly, there was no significant difference in cumulative trm. conclusions: hid-hsct was an effective method to treat high risk all, but mrd + patients had high recurrence rate and poor prognosis. strategy adjustment should be considered to reduce tumor residual and the transplantation strategy should be optimized for these kind of high risk patients, so as to improve survival rate. disclosure: nothing to declare background: lymphocytes are responsible for the cellular and humoral immunity and, consequently, its recovery after allo-hsct might be linked with the survival after the procedure. the aim of this study was to analyze this hypothesis in our series of patients. methods: all the 209 allo-hsct performed in our center from january 2015 through july 2018 were included in the analysis. median age was 52 years (range: 7-69). 122 pts were male (58,4%) and 87 were female (41,6%). baseline diseases were: 69 aml, 49 lpd, 31 mds, 28 all, 16 mpd, 10 mm, and 6 bmf. donor was unrelated in 113 (54,1%), and was family in 96 cases (45,9%) (including 31 haplo-identical). stem cell source was pb in 195 (93, 3%) and bm in 14 pts (6,7%). conditioning regimen was reduced in 111 procedures (53,1%) and intensive in 98 (46,9%) (including just one non-myeloablative). overall mortalities at days +100 and +365 (the latter in patients with follow-up superior to 1 year) were 9,1% and 24,9%, respectively. median follow-up was 25 months (range: 4-47). evolution of absolute lymphocyte counts (alc) and subpopulations at pre-hsct and during the first year after allo-hsct were analyzed. results: as shown in table 1, alc and cd4+ lymphocytes decreased after conditioning therapy, and recovered progressively during the post-hsct period. at day +365, majority of patients had >1000 alc/mcl, clearly improved compared to admission values. cd4+ lymphocytes at day +100 was still very low, but at day +365 around half of the series had 200-500/mcl. we found a strong link between alc, cd4+ lymphocytes, and cd19 + lymphocytes at days +30 and day +100 with overall survival at day +365 of the series (table 2) . conclusions: in our series, immunity recovery was a late event for majority of patients undergoing allo-hsct. in addition, in our experience, the precocity and quality of the alc, cd4+, and cd19+ cells recovery was clearly linked with long-term survival. background: the reconstitution of t and natural killer (nk) cells after hematopoietic stem cell transplantation (hsct) strongly influences the outcome of hsct including viral infection and graft versus-host disease (gvhd). the purpose of this study was to investigate the clinical efficacy of immune reconstitution including t and nk cells after hsct in children. methods: we reviewed the records of 30 patients who undergoing allogeneic hsct in department of pediatrics, pusan national university children's hospital, from january 2013 to july 2017. the counts of t lymphocyte subsets and nk cells was monitored in peripheral blood by flow cytometric technique during 1, 3, 6, and 12 months post-hsct. blood samples for cytomegalovirus (cmv) and epstein-barr virus (ebv) monitoring were tested by real-time pcr assay. results: for total of 30 patients, the mean age was 9.1 years (range, 9 months-19 years), 16 of the patients were boys and 14 was girl. out of a total 30 patients without pre-hsct cmv viremia or cmv infection, 10 (33.3%) recipients experienced cmv infection. the number of cd8 + t cells in 3 and 6 months post-hsct was significantly higher in patients with cmv reactivation compared to patients without (median 1066.11/μl vs. 979.0/μl, p=0.004, and 1047.45/μl vs. 551.44/μl, p=0.002) . in 6 (20%) recipients presented acute gvhd, the number of cd4 + t cells in 1 and 3 months post-hsct was significantly lower in patients with acute gvhd compared to patients without (median 239.13/μl vs. 365.0/μl, p=0.045, and 165.2/μl vs. 344.27/μl, p=0.035) . the number of nk cells in 1 months post-hsct was significantly lower in patients with cmv reactivation and acute gvhd compared to patients without (258.5/μl vs. 501.67/μl, p=0.004, and 162.5/μl vs. 464.88/μl, p=0.027, respectively) . in multivariable analysis, acute gvhd was shown to be the decisive factor influencing total t cells (p=0.028) and cmv reactivation was independently associated with cd8 + t cells (p=0.026). the cd4 + t cells counts were associated with prior hsct history and acute gvhd (p=0.042 and p=0.038), and the cd8 + t cells counts were also significantly associated with donor type (p=0.016). conclusions: overall, our study documents that immune reconstitution of cd4 + , cd8 + t cells and nk cells is strongly associated with cmv reactivation and acute gvhd. additionally, we show that acute gvhd is influenced by lack of sufficient numbers of nk cells as well as cd4 + t cells early after sct. cd8 + t cells, on the other hand, significantly increase after cmv-reactivation and most likely play an important role in reactivation. disclosure: nothing to declare background: curative effect of allogeneic hematopoietic stem cell transplantation (allo-hsct) depends on the alloreactive t-cell immune response toward residual malignant cells -graft-versus-leukemia reaction. however, alloreactive population has not been phenotypically defined. recent studies suggest that alloreactive t cells express both costimulatory and inhibitory receptors simultaneously. exhaustion caused by the inhibitory signaling dampens tcell functionality, which could lead to the disease relapse. here we aimed to investigate the expression of costimulatory and inhibitory receptors on antigen-experienced t cells after transplantation, to isolate subpopulation specific for allo-hsct patients and analyze their t-cell receptor (tcr) repertoire. methods: expression of coinhibitory and costimulatory molecules on pbmcs patients at various time points after allo-hsct was analyzed for expression of: cd3, cd8, cd4, cd45ra, ccr7, cd95, cd27, cd28, klrg1, tigit, pd1, cd137 and ox40 by flow cytometry and compared to healthy donors. cd3+cd8+cd95-cd27 +cd28+pd1+tigit+ fraction and cd3+ cd8+ control fractions were separated on facs aria ii cell sorter. double barcoded cdna libraries of tcr beta-chains for both fractions were prepared and analyzed by sequencing on illumina platform. sequencing results were processed by migec, mixcr and vdjtools software. enriched clones were identified by fisher's exact test (p>10 -10 ). results: we did not find any significant differences between patients after allo-hsct and healthy donors in single marker's expression, but, when considering coexpression of co-stimulatory and inhibitory molecules on t cells we found that cd3+cd8+cd95-cd27+cd28 +pd1+tigit+ subpopulation was significantly increased in allo-hsct patients. moreover it increased with the time since the transplantation (fig. 1 ). this population was isolated by cell sorting and alongside with total cd8+ fraction subjected to tcr beta-chain repertoire sequencing. the population contained clones significantly enriched compared with cd8+ fraction representing potentially alloreactive cells. this hypothesis is further supported by the notion that the level of expression of cd27 and cd28 co-stimulatory molecules is lower in the group of patients who subsequently relapsed, compared with the patients with complete remission, while the expression of inhibitory receptors was high in both groups. conclusions: according to our data patients after allo-hsct have a phenotypically distinct t-cell population characterized by simultaneous expression of costimulatory and inhibitory markers. this population contains specifically enriched clones, which may be specific for alloantigens. further functional assays are needed to confirm the alloreactive potential of this subpopulation. besides low expression of costimulatory molecules combined with high expression of inhibitory receptors on antigen-experienced t-cells of patients after allo-hsct might be associated with a disease relapse. fondazione mbbm, monza, italy, 3 ospedale san gerardo, laboratorio stefano verri, monza, italy background: poor graft function (pgf) is a severe complication after hsct, with a high risk of morbidity and mortality, mainly due to infections. donor cd34+ scb seems to offer high chances of haematological recovery, not jeopardized by gvhd. however, pediatric reports remain scarce. methods: 19 out of 215 patients undergoing transplantation in our pediatric unit from 2012 to 2018 have been retrospectively evaluated for at least 2 line persistent cytopenia (hb< 9.5g/dl, plt< 30000/mmc, n< 1000/ mmc) and/or transfusion-dependency beyond 3 months after hsct in the presence of full donor chimerism. bone marrow cellularity was evaluated through biopsy as further indicator of pgf. (1/8) to donate or medical decision (7/8). bone marrow cellularity was < 5% in 50% of the patients who underwent scb for which the histology was available (8 cases), and 25% in those who have not been treated (2/8). at 30 days after scb 10/11 (91%) patients had hematological response, which was complete in 46% and partial in 45% of the patients. only 1 patient had no response. the infusion was always well tolerated with no adverse events, and no worsening of gvhd. haematological recovery occurred spontaneously at 30 days after bone marrow biopsy in a significantly lower proportion of patients (2/8, 25%, p< 0.05) within the non-scb group. in two cases platelets engraftment was significantly delayed, up to one year after bone marrow biopsy and in one case thrombocytopenia persists and the patient is still receiving thrombopoietin agonists and red blood cells transfusions at 9 months after bone marrow biopsy. conclusions: a stem cell boost matched the goal to yield count recovery in our cohort. viral infections and gvhd may be possible risk factors for pgf.bilinear or trilinear cytopenia with transfusion dependency and bom cellularity < 5% and full donor chimerism are good indications for scb, that can provide a significantly earlier hematological reconstitution, without risks of gvhd. due to the proved early efficacy and safety of cd34+ stem cell infusion, we suggest that this procedure should be taken in consideration in children with severe bone marrow hypoplasia and persistent cytopenia after hsct. disclosure background: as allogeneic hematopoietic stem cell transplantation (hsct) is sometimes performed despite erythrocyte's antigens incompatibility and mismatch, it is essential to carefully track patients' genotypes after it. methods: for the study we used erythrocytes (n=189) and dna (n=20) from patients undergoing abo-or rhesus-mismatch hsct and their donors. we used posttransplant no transfused patients on the periods according transplant protocol by hemagglutination methods in plate and tube using monoclonal antibodies to abo and rhesus antigens (hematolog, russia). we extracted dna with dna kit (bag, germany) and conducted pcr-ssp with kits abo-type, rh-type (bag, germany). chimerism was assessed by the str-pcr analysis with cordis plus multiplex kit for amplification of 19 polymorphic strmarkers and amelogenin loci. the fragment analysis was performed on a 3130 genetic analyzer. informative loci were chosen by comparison of pretransplant patient's and donor's dna. the percentage of donor chimerism was calculated using standard formula. precise rhce and abo genotypes were determined by direct sanger sequencing. we revealed 3 patients with unexpected erythrocyte abo, rhesus phenotypes and genotypes after hsct on +30 (2 patients) and +160 days (all patients). chimerism analyses on str showed in a.e.kh. and g.l.v. patients 99% of donor's dna and less than 1% of recipient's one. b.n.a. patient was relapsed and chimerism analysis revealed 95% of recipient's dna and 5% of donor's one. using serological methods and pcr-ssp we revealed genotypes abo * a1b1; rhd+; rhce * ccee in patient a. e.kh. before hsct, abo * a2o1; rhd+; rhce * ccee in her donor, and abo * a1a2; rhd+; rhce * ccee on +30d after hsct. genotype a1a2 was no recipient's neither donor's origin. direct sequencing did not prove this genotype, but revealed donor's one.on +160d serological methods and pcr-ssp also revealed donor's genotype in this patient. patient b.n.a. had genotypes abo * o01/o01; rhd+; rhce * c w cee before hsct, her donor -abo * b1o1; rhd +; rhce * ccee. on +160d this patient relapsed, but rhesus genotype has been detected as rhd+; rhce * c wcee (lack e gene). direct sequencing revealed gene rhce*ee. abo genotype was recipient's origin -o1o1. in patient g.l.v. using serological and pcr-ssp methods we determined genotypes abo * o01/o01; rhd +; rhce * ccee genotype before hsct, and abo * a1/ o01; rhd+; rhce * ccee genotype in her hsc donor. on +30d patient had unexpected genotype abo * o01/o01 (a lack of a antigen); rhce * ccee (a lack of e antigen). in order to explain unexpected patient's genotypes after hsct we sequenced her rhce and abo genes and found donor's genotype ccee; a1o1 that was in agreement with results of str analysis. to resolve discrepancies between serological, pcr-ssp and sequencing analysis data we sequenced patient's rhce cdna and observed only ce allele. at present time the molecular basis of selective inactivation one of the two rhce alleles is not clear. on +160d patient had donor's genotype. conclusions: what kind of mechanisms led to discrepancies between results obtained by different laboratory methods are still not clear. an interesting case of expression of only one rhce allele in patient g.l.v. allows us to suggest involvement of some epigenetic mechanisms like dna methylation or histone modification in this process. clinical background: in relapsed patients with acute b -lymphoblastic leukemia (all-b) who achieved complete remission (cr) after re-induction therapy, minimal residual disease (mrd; ≥10 -3 all-b cells/ul) is often detected. according to available data, such condition varies from 30% to even 50% of cases, as assessed by pcr or flow cytometry (fc), while the presence of mrd is the most important risk factor for all recurrence. in this abstract, we describe our experience with bridging therapy using blinatumomab infusion after re-induction regimens and before the planned allogeneic stem cell transplantation (allo-sct). the procedure was performed in three young men suffering from relapsed ph (-) all-b at the age of 19, 22 and 34 years. in the first case (19yo), relapse with previous mrd accounting for 2.7% occurred 15 months after cr1 mrd neg . in the next patient (22yo) the second relapse with central nervous system (cns) involvement occurred 5 months after allo-sct performed in cr2 (26 months after cr1, mrd neg ), while in the third patient (34yo), recurrence with cns and testis involvement occurred 12 years after cr1 (mrd neg ). all patients underwent chemotherapy (flam, hypercvad and dnr/vcr/pegasp/dexa regimens respectively) followed by one cycle of blinatumomab (at a dose of 9mcg/d on days 1-7, followed by 28mcg/d on days 8-28 in a continuous infusion) and allo-sct (using eto/cy/tbi/atg/ conditioning regimen for ist and iiird patient and bucy2 for iind patient; using matched unrelated donor (mud, ist and iiird patient) or matched related donor (iind patient)). mrd status was assessed after each cycle of blinatumomab by fc. results: all patients achieved cr mrd pos after reinduction therapy followed by clearance of mrd after blinatumomab course (tab. 1). the second patient, due to positive mrd 6 months after allo-sct received 4 donor lymphocyte infusions additionally. during the administration of blinatumomab, no adverse events (aes) were observed in grade 3 or 4. one patient developed cytokine release syndrome in grade 1. the progression free survival, time to positive mrd and follow up are presented in tab 1. conclusions: the use of blinatumomab as a bridging therapy between re-induction regimens and allo-sct in patients with all-b and mrd pos appears to be safe and leads to the clearance of mrd which may be crucial in os and pfs prolongation after following allo-sct. future studies on larger groups of patients are necessary to confirm this thesis. background: haploidentical hematopoietic stem cell transplantation (hsct) is considered an alternative treatment for hematologic malignancies in patients who do not have an hla-identical sibling donor [1] . since infections and disease relapse resulting from delayed immune reconstitution (ir) are the most common causes of mortality among patients undergoing haploidentical-hsct [2], timely ir is essential in the recovery and survival of these patients. the aim of this study is to describe the evolution of ir after haploidentical-hsct and to estimate survival rates in patients with delayed vs. adequate reconstitution in a single center in colombia, south america. methods: a retrospective cohort study was conducted on 26 consecutive adult haploidentical-hsct recipients at a tertiary referral center. cd4+cells, cd8+cells, cd3+cells, and immunoglobulins levels were monitored before hsct, at first month, and then every three months for the first two years post-transplantation. descriptive statistics were used to analyze patient's clinical characteristics. the kaplan-meier method was used to assess overall survival (os) and relapse-free survival (rfs) rates. results: twenty-six patients were included (50% were male), with a median age of 25.5 years (range 16-59). the most common indication for haploidentical hsct was acute lymphoblastic leukemia (n=21, 80.8%), followed by non-hodgkin lymphoma (n=3, 11 .5%) and myelodysplastic syndrome (n=2, 7.7%). all patients received gvhd prophylaxis therapy with cyclophosphamide, tacrolimus, and mycophenolate mofetil. fifteen patients (57.7%) presented cytomegalovirus reactivation (25/26 at risk), 5 patients (19.2%) epstein-barr virus reactivation, and 4 patients (15.4%) developed adenovirus infection. median time to neutrophil engraftment (neutrophils>0.5×10 9 /l) was 15 days (range12-29) for the 23 patients recipients of peripheral blood progenitor cells (pbpcs) and 21 days (range20-27) for the three remaining bone marrow recipients. platelet engraftment, defined as >20,000 platelets/ mm 3 background: daratumumab is a human monoclonal antibody directed against the glycoprotein cd38 that is overexpressed on the surface of plasma cells in multiple myeloma patients. it is approved as second line therapy either as single agent therapy or in combination with lenalidomide or bortezomib for the treatment of patients with relapsed/refractory multiple myeloma. despite the curative potential of an allo-sct, the high relapse rate remains a clinical problem. data addressing the choice of an optimal salvage therapy regime for these heavily pre-treated patients is missing. methods: from april 2016 till november 2018 a total of 22 patients (male, n=10) with the median age of 64 years (40-72) received daratumumab as a salvage therapy for relapse of multiple myeloma after allo-sct at the university of hamburg. prior to allo-sct all but one patient had received an autograft, 9 patients even ≥2 autografts and 4 patients also a 1. allograft. the median number of salvage lines post-transplant and prior to first daratumumab infusion was 2 (0-4). these salvage regimens included cyclophosphamide, etoposide, bortezomib, lenalidomide, pomalidomide and carfilzomib. daratumumab was started at a median of 19 months (0-43) after relapse/ progress and initiated as single agent therapy in all patients. concomitantly, 14 patients received either an immunomodulatory drug (lenalidomid, n=10; pomalidomid, n=1) or a proteasome inhibitor (bortezomib, n=3) during a later course of daratumumab infusions. combination therapy was initiated when a slow rise of paraprotein and/or free light chains or no response to monotherapy was observed (median at the 11 th infusion). results: the median number of infusions was 14 . twenty adverse reactions were observed in 13 of 22 (59%) patients: dyspnea (n=5), bronchospasm (n=2) shivering (n=3) , cough (n=2), musculoskeletal pain (n=4), acute coronary syndrome (n=1), skin rush (n=1), facial edema (n=1), pressure on eyes (n=1). all adverse reactions appeared during the first infusion and were mostly mild or moderate (ctc 1-2, n=19). tolerance of the following infusions improved and in none of the cases therapy had to be stopped due to adverse events. three patients developed late onset infections (pneumonia, n=2; urinary tract infection, n=1) followed by temporarily therapy interruption. with a median follow-up of 14 months after the first administration 20 of 22 patients remain alive .9%). one patient died due to progress of myeloma and another died due to severe infection/sepsis. 15 of 22 patients responded (68%; pr, n=5; vgpr, n=8; cr, n=2) to the therapy with daratumumab. the responses (decrease of paraprotein and/or free light chains ≥50%) occurred at a median of 7 days (4-372) after the first administration and lasted for 5.0 months (0.5-26.6). conclusions: daratumumab shows an encouraging efficacy and acceptable toxicity profile in patients with relapsed/refractory myeloma after allo-sct. further studies are needed to investigate the role of the combination therapy with immunomodulatory drugs or proteasome inhibitors in this setting. disclosure: nothing to declare p546 clonal plasma cell detection by high sensitive flow cytometry in aphaeresis product is poor prognostic and not increased by use of plerixafor alone background: in an earlier from our center we have demonstrated that residual clonal plasma cells (cpc) decrease both overall survival (os) and disease free survival (dfs) (ash 2018).plerixafor is a selective antagonist of cxc4 chemokine receptor (cxcr4) and able to mobilize human peripheral blood stem cell (pbscs) by acting synergistically with g-csf.the purpose of this study was to evaluate the safety and efficacy of plerixafor in myeloma patients who were proven poor mobilizers and specifically to assess the flow cytometric measurement of residual clonal plasma cells in the apheresis products. methods: patients with a diagnosis of mm who underwent auto hsct at our center between january 2008-november 2018 were retrospectively analyzed.out of 164 patients, 16 patients received plerixafor as mobilization regimen due to poor mobilization with g-csf.pbsc grafts were tested for the presence of clonal pcs (cpc) and the number of normal pcs (npc) by multi-parameter flow cytometry (fcm).the acquisition of the cells was performed using the navios flow cytometer beckmancoulter) .upon the daily checks of the instrument, 3x10 6 cells for each sample were acquired and the collected data was analyzed using the kaluza software (beckmancoulter,usa). results: patient demographics are shown in table 1 .the majority of patients were male and median age was 62 years in the plerixafor group.the median interval from time of diagnosis to mobilization and follow-up from mobilization were 3.3 months and 14.3 months in plerixafor group, respectively. cpc contamination in the pbsc grafts was detectable in 32 and 7 patients with counts ranging between 0-8.7x10 -5 and 0-0.1x10 -5 in g-csf alone and g-csf+plerixafor groups, respectively (p=0.116).there were no significant differences in the proportion of the patients with graft contamination between subtypes of mm in both groups. one hundred (gcsf/plerixafor;93/7) patients had pre-asct pet-ct imaging done with 71 (gcsf/plerixafor;64/7) have active lesion at the time of mobilization. statistically significant association could not be demonstrated between the disease < cr status at mobilization and the number of apc in the apheresis product in both groups (p>0.05).twelve of 16 patients from plerixafor treatment arm proceeded to transplantation within median 9.5 months.the best overall response to induction treatment is shown in table 1 .thirtyfour patients from the g-csf alone arm and 2 patients from the g-csf+plerixafor arm died during the follow-up (p=0.52).disease progression was seen in 48 patients from g-csf alone group and 6 patients from g-csf+plerixafor group of the study(p=0.56).estimated mean os was better among patients w/o apc contamination in plerixafor group, respectively (38.9±5.3mos vs 16.8mos; p=0.52). conclusions: our results on 164 and few plerixafor used patients show that clonal plasma cells are detectable by multiparametric flow more frequently when patients are poor mobilizers and require plerixafor.the clonal pc contamination can be attributed to the myeloma biology as manifested by higher number of lines induction regimens and pet positivity among the plerixafor-required patients. the overall and disease survival was impaired by residual clonal pcs in the graft but not by plerixafor per se. neither was the content of clonal pcs differed from others.thus the cxcr4 shared by hsc and myeloma cells do not cause a myeloma mobilization. clinical trial registry: -disclosure: nothing to declare prognostic factors for overall survival after allogeneic hematopoietic cell transplantation in multiple myeloma patients all factors with significant influence on pts survival were included multivariate analysis (cox regression model) but only re-admission in the first 365 days demonstrated impact on os (hr 4, 082; p=0, 005) . conclusions: we analyzed risk factors for survival in mm pts who received allo-hct. our study identified disease-related risk factors like iss and transplantationrelated factors such as hct-ci and pam, hospital readmission, days of hospitalization and cmv reactivation that were associated with worse long-term survival. in our series, the most frequent death and re-admission cause was infection, so focusing the efforts in reduction of infection could have a beneficial impact on improvement of survival in mm undergoing allo-hct. [[p547 image] 1. figure 1 ] disclosure: there is no disclosure. novel protocol for autologous hsct in multiple myeloma: ambulatory chemomobilization and transplantation of fresh hematopoietic stem cells with backup storage background: autologous hematopoietic stem cell transplantation (ahsct) after melphalan conditioning is still a part of standard treatment of multiple myeloma patients. traditional transplantation of frozen stem cells poses additional risk for the patients connected with dmso and central venous catheter. the transplantation of fresh cells is an option -however, most mobilization protocols are either low-efficient (g-csf), expensive (g-csf + plerixafor) or toxic (standard dose chemomobilization) to directly proceed to transplantation in this fragile group of patients. we describe here the novel combination of ambulatory mobilization with very low doses of ara-c and g-csf connected with direct ahsct with fresh cells. methods: the prospectively collected database of patients after ahsct was searched for patients who underwent ahsct after chemomobilization with ara-c and transplantation with fresh cells (fc) and compared with control group of consecutive patients transplanted with standard protocol (sp) (transplantation with frozen cells) between july 2016 and october 2018. protocol of ambulatory mobilization was: 400mg/m² of arac on days +1 and +2 and g-csf at the dose 5 μg/kg/day from day +5 and escalated to 10 μg/kg/day split into two doses +10 to +13, apheresis started on day +14 (or later) and finished when at least 7.5 x 10e6 cd34+ positive cells were collected. the collected cells were split in three equal parts:1) for use as fresh transplant 2) frozen for possible 2 nd transplant 3) frozen as backup. results: there were 48 transplantations with fresh cells and 49 transplantations with frozen cells compared. both groups had same mobilization protocol -ambulatory low dose ara-c. the median age and number of transplanted cells was similar in both groups (56 vs 57, p=0.99; 5.2 vs 5.3 cd34+/kg, p=0.97 conclusions: we present novel approach that allows direct ahsct after chemo mobilization in all patients who are treated with melphalan. we show that it is not only feasible to do ahsct directly after chemomobilization but also the results may favour this approach when compared with current standard. disclosure: nothing to declare background: high dose chemotherapy followed by autologous hematopoietic cell transplantation (hsct) is considered, since the nineties, the standard of care for patients aged less than 70-75 years old with newly diagnosed multiple myeloma (mm). however, the optimal induction treatment prior to hsct to reduce the tumor burden has changed during the last few years. improved regimens have shown to be able to increase the quality of the pre-hsct response, which might subsequently impact on the post-hsct response, which has been proved to be associated with a longer pfs. we recently changed the induction therapy for pts candidates to hsct. in this analysis, we aimed to check if newer regimens impacted on pretransplant responses, and how auto-hsct changed the pre-hsct status. methods: all the 117 auto-hsct for 99 mm patients performed in our center from january 2014 through august 2018 were included in the analysis. median age was 60 years (range: 38-76). 54 pts were male and 45 were female. durie-salmon stage was distributed as follows: i (9.4%), ii (37.6%) and iii (53%); 13% had creatinine > 2 mg/dl. iss was: 1 (41%), 2 (28.2%), and 3 (30.8%). type of monoclonal component was: igg (51.3%), light chains (23.9%), iga (22.2%) , and non-secretory (2.6%). 51.3% had bence jones proteinuria. conditioning regimen was melphalan 200 mg/m 2 in 106 (90.6%), melphalan 100-140 mg/m 2 in 9 (7.7%), and other in 2 (1.7%). results: pre-transplant therapy was: vcd in 60 (mostly in 2014-6), vtd/vrd/krd in 34 (mostly in 2017-8), and others in 23 cases. status of the disease at transplant was: cr/vgpr in 75, pr in 35, and sd in 7. distribution of pretransplant response based on the type of induction is shown in table 1. peri-transplant mortality was 0%. day +100 mortality was 1.7% (2 pts), due to progressive disease. as shown in table 2, all patients (14/14) who obtained cr pre-hsct, maintained the response at day +100 post-hsct. among the patients in vgpr at hsct, 20 (32.8%) became into cr, and 37 (60.7%) maintained the response. the cr rate at post-hsct increased 264% compared to pre-hsct (37 versus 14 pts). altogether, after hsct 44 pts (37.6%) improved and 65 (55.6%) maintained the pre-hsct response. during the last period of time, 37 pts started on post-hsct maintenance/consolidation, mainly with lenalidomide. conclusions: 1) with the new chemotherapeutic schemes, 76.5% of patients underwent hsct in cr or vgpr; 2) majority of pts (93.2%) consolidated or improved the pre-hsct response; 3) cr increased substantially (2.64 times) after transplant; 4) optimized induction regimens, along with auto-hsct followed by the recently licensed use of maintenance therapy with lenalidomide, might result in a better pfs of patients with mm. background: autologous stem cell transplantation (asct) is commonly used in treatment of patients over 65 years with multiple myeloma (mm), however the safety and efficacy of this procedure is debatable. methods: we conducted a retrospective review of mm patients who underwent asct from 2013 to 2018 at our institution. the purpose of this retrospective study was to compare the 100-day mortality, time to engraftment, and incidence of grade 1-4 toxicities in elderly mm patients with younger patients taking into account comorbidity information. other secondary end points measured were overall survival (os) and progression-free survival (pfs). results: a total of 95 patients were analysed and categorized by age as young patients (37 to 64 y; n=70) or elderly (65 to 71 y; n=25). the compared groups did not differ in terms of gender, ecog, hct-specific comorbidity index (hct-ci), and disease status at asct. melphalan in a dose of 200 mg/m 2 was used as preparative regimen in 65% of younger patients, and in 43% of the elderly (p= 0.18). the remaining patients received 140 mg/m2 of melphalan or lower dose (range, 100-140 mg/m 2 ) due to hct-ci >2 or age, on the physician discretion. in the whole study group there were no transplant related deaths within the first 100 days of asct. stratifying by age, there was no statistically significant difference concerning febrile neutropenia (fn) incidence, which was observed in 14% of younger patients, and 17% of elderly. in contrast, fn was observed more frequently in patients with hct-ci > 2 (38% vs 12%, p=0.05). grade 1-2 infections were more frequent in older patients (35% vs 5%, p= 0.005), but no difference was found in grade 3-4 infections incidence rate such as pneumonia, uroinfections and neutropenic enterocolitis (25% vs 35%, p=0.52), nor grade 1-2 and 3-4 noninfectious toxicities (50% vs 38%, p=0.31, and 4% vs 21%, p=0.06, respectively) . the median time to granulocyte engraftment was 11 days (range, 9-19 days) in elderly and was comparable with younger patients. the time to platelet recovery was also similar. after the median follow-up of 12 months for survivors, os at 18 months was 94% in both groups. pfs at 18 moths was 65% for younger patients, and 55% for elderly (p=0.86).however, the association between pfs and the dose of melphalan used in conditioning was observed. pfs probability at 18 months for patients conditioned with the dose of 200 mg/m 2 , 140 mg/m 2 and 100 mg/m 2 was 71%, 58% and 21%, respectively (p=0.012). conclusions: our data show that asct in transplant eligible mm patients ≥ 65 years of age is safe and provides similar outcomes as seen in younger patients. disclosure: nothing to declare is mobilization with chemotherapy effect response in the multiple myeloma? background: high dose melphalan therapy with autologous stem cell support is a standart approach in symptomatic multiple myeloma patients. response rates increased with the novel anti myeloma agents and the use of chemotherapy for stem cell mobilization should be questioned. the purpose of this study is to determine the effect of cyclophosphamide used during stem cell collection on disease response and transplantation results. methods: we retrospectively collect data from 270 myeloma patients who underwent autologous stem cell transplantation (asct) in ankara university medicine faculty, blood and bone marrow transplantation unit between january 2012 and november 2018. 51 patients who received cyclophosphamide protocol for stem cell mobilization were included in the study. disease response were determined according to international myeloma study group (imwg) criteria before and after the cyclophosphamide. transplant responses and their effects on survival were also indicated. results: after the diagnosis of mm, 51 patients (male/ female: 32/19; median age: 59 years (between 40-74 years)) with median follow-up of 59.1 months (between 7,6 -185,8 months) underwent asct at a mean of 21,7 ±6,2 months.. forty-one patients were evaluated before and after cyclophosphamide (table 1). in 69% of the patients, the disease response was not altered by cyclophosphamide therapy, and 22% of the patients improved their response status. post-transplant response rates of patients who underwent stem cell mobilization with cyclophosphamide are also shown in table-1. the mean survival of the patients was 52,7 ±4,8 months. when patients were grouped according to changes in response status before and after cyclophosphamide; there was no statistical difference between mean calculated survival (improved response, disease progression and stable disease; 59,3±9,0 months, 22,7±14,7 months and 46,4±5,0 months respectively, p=0.425) (figure-1) . the rates of 1-year and 3-year overall survival (os) of the patients with no response to cyclophosphamide treatment were as follows; 74,2%±7,9% and 70,5%±8,3% respectively. thirteen patients who were followed up median 27 months after transplantation died at an average of 3,4±2,5 months; 6 of these deaths were caused by the infection after transplantation. conclusions: in our study, it was observed that the use of cyclophosphamide for cd34+ stem cell mobilization did not change the disease response rates by 69%. the posttransplant survival rates of mm patients who had progressive disease after cyclophosphamide use were lower. however, these results warranted confirmed by randomized controlled trials. clinical trial registry: -disclosure: nothing to declare results of a single center experience: an attempt to augment conditioning regimen in first autologous stem cell transplantation treatment of multiple myeloma (mm) continues to evolve in the era of novel agents. the addition of bortezomib to highdose melphalan (bor-hdm) has been reported by several groups, and it has been outcome and toxicity profile is comparable to high dose melphalan (hdm) alone. the aim of this retrospective study was to evaluate the outcome of the bor-hdm conditioning regimen on overall response for patients with mm undergoing first single asct at our institution. methods: this retrospective single center study reviewed 136 consecutive myeloma patients who had received the first asct either with bor-hdm (n=15, m/f= 11/4) or single agent hdm (n=121, m/f= 76/45) conditioning regimen. in the single agent hdm conditioning regimen, melphalan was administered intravenously at a total dose of 200 mg/m2 on day -3 and -2 and stem cells were infused on day 0. in the bor-hdm group, melphalan 200mg/m 2 was administered on day -2. bortezomib was administered intravenously at a dose of 1 mg/m 2 on day's -6, -3, +1, and +4 as described in a phase 2 study by intergroupe francophone du myeĺome (ifm). results: all consecutive patients who underwent single asct from january 2010 to march 2018 using bor-hdm as conditioning or hdm were evaluated. conditioning regimen was hdm in 121 patients and bor-hdm in 15 patients. median age was significantly lower in bor-hdm conditioned asct compared to hdm group (62 years vs 52 years, p=000). there was no significant difference for mm subtype, iss stage at diagnosis, prior treatment line among hdm vs bor-hdm cohorts (p>0.5). after a median of 4 cycles of induction chemotherapy, patients in the bor-hd exhibited ≥vgpr of 53.8% (n=7) compared to 56.7% (n=51) in the hdm group (p= p>0.5). pre-asct immune response (if (-)) was reported in 22.3% of patients treated with hdm, higher than that seen in the bor-hdm group (6.7%) (p=0.3). nine (69.2%) patients achieved post-asct immune response (if (-)) ≥vgpr compared to 92 (78.6%) in the hdm group. at the time of this analysis, ten patients in the bor-hdm group and 87 in the hdm group had already died, respectively (p>0.5). a total of 5 (45.5%) patients in the bor-hdm group and 50 (44.2%) patients in hdm group had already progressed (p>0.5). estimated mean os and pfs was shorter for group treated with bor-hdm; 48.2±6.9 mos and 40.2±8.5 mos vs. 76.7±3.9 mos and 67.3±3.8 mos, respectively (p>0.5) (figure-1) . we could not demonstrate the impact of pre or post transplant ≥vgpr immune response on survival and disease free survival. there was no engraftment failure observed on either treatment group and no worsening peripheral neuropathy was developed in the bortezomib arm. conclusions: this single center experience on a small patient pool was able to repeat the prospective randomized study results of ifm. further studies are warranted to explore this regimen, especially when induction treatment with novel agents are employed, with special emphasis on the high-risk myeloma patients where response rates are good but sustainability remains an issue. disclosure: nothing to disclosure the efficacy and safety of bortezomib plus busulfan/ melphalan as conditioning regimen in multiple myeloma undergoing autologous stem cell transplantation: phase 1/2 study background: bortezomib have a powerful antimyeloma activity and was regarded as backbone of therapy in the past decade but its safety and efficacy as a part of busulfan/ melphalan conditioning regimen of autologous stem cell transplantation is yet to be shown. methods: a phase 1/2 trial to explore the safety and activity of a bortezomib on days -6, -3, and +1 added to a conditioning regimen with busulfan and melphalan (bumel, 3.2 mg/kg/day and busulfan during day -5 and -3, 140 mg/ m 2 /day of melphalan on the day -2), in multiple myeloma (mm) patients who received autologous stem cell transplantation following bortezomib-based induction chemotherapy. in phase 1, escalating doses (0.7, 1.0, and 1.3 mg/m 2 ) of bortezomib with bumel were administered in each group with three patients. with determined maximum tolerated dose of bortzomib at a 1.3mg/m 2 /day, cohort with 41 patients were analyzed for phase 2. results: in phase 1, no dose limiting toxicity was observed at a 1.3mg/m 2 /day of bortezomib. in phase 2, overall responses at 3 months was shown as 75% of very good partial response (vgpr) or better and 55% of complete response (cr), whereas post-transplant overall best response included 83% of vgpr or better, and 68% of cr, respectively. with median follow-up duration of 31.4 months, median progression-free survival (pfs) was 26.8 months. the probabilities of 2 years-pfs and overall survival (os) were 56.5% and not estimable, respectively. especially, high-risk cytogenetics were associated adverse survival outcome compared to standard-risk cytogenetics, respectively (pfs, 12.2 vs. 35.7 months, p=0.039; os, 26.7 vs. 73.3 months, p=0.086) . with median 11 days and 10 days for neutrophil and platelet engraftments, any graft failure or delayed engraft was not observed. the common grade 3 or severe non-hematological adverse events included neutropenic fever (73.2%) and stomatitis (14.6%). except three cases with transplant-related mortality due to sepsis, other adverse events were manageable. conclusions: these results demonstrate that bortezomib is safe and can be a part of conditioning regimen in combination with bumel, for patients with transplanteligible multiple myeloma. clinical background: allogeneic stem-cell transplantation (allo-sct) is one of treatment option for patients with multiple myeloma (mm) refractory to novel agents. the reports on allo-sct for mm are limited and it is an important issue to argue appropriate conditioning regimens and stem-cell sources, and patient population who will benefit from allo-sct. methods: we retrospectively analyzed 25 consecutive patients who received allo-sct for relapsed and refractory multiple myeloma (rrmm) between oct 2009 and july 2018 at japanese red cross medical center. characteristics of patients, progression-free survival (pfs), and overall survival (os) were analyzed. results: median age at allo-sct was 47 (range 31-61). twelve patients were male and 13 were female. myeloma type were igg:14, iga: 3, igd: 2, and bence-jones: 6. stem-cell sources were peripheral blood from hlamatched related donor (rpbsct): 6, bone mallow from hla-matched unrelated donor (mud): 4, bone marrow from hla-mismatched donor (mmud): 7, and cord blood (cb): 8. twenty-three of 25 patients received flu/mel-base, one patient received bu/mel-based, and one patient received etoposide/cyclophosphamide-based conditioning regimens. twenty-two patients who transplanted after 2012 received 8gy of total body irradiation (tbi). responses before allo-sct were cr: 8, vgpr: 6, pr: 6, sd: 5. five-year pfs was 22% (95%ci: 6-45) and 5-year os was 42% (95%ci: 16-66). ten patients died during observation period and causes of death were primary disease: 8 and treatment-related mortality: 2. patients with vgpr or better before allo-sct showed significantly better pfs (p=0.04) and os (p=0.011) as compared with others. female recipients showed significantly better pfs (p=0.0034) and os (p=0.035) as compared with male recipients. recipients of mmud showed significantly better pfs (p=0.0034). among 15 patients surviving, 10 patients received treatments including maintenance therapy. conclusions: the reason for better pfs and os in female recipients is unknown. it is interesting that recipients of mmud showed better pfs, suggesting graft-versusmyeloma effects. allo-sct can be an effective treatment option if patients and stem-cell sources are appropriately selected. disclosure: authors declare that there are no conflicts of interest. second autologous hematopoietic stem cell tranpslant versus chemoimmunotherapy in relapsed multiple myeloma after first transplantation: single center data background: combination therapy, mostly triple, followed by autologous hematopoietic stem cell transplantation (auto-hct) is widely accepted as the first-line standard therapy for multiple myeloma (mm). despite the availability of agents such as new immunomodulatory drugs (imids), proteasome inhibitors (pis), histone-deacetylase inhibitors and antibodies, it is still possible to achieve longer and deeper responses, however, multiple myeloma is still not cured and relapse is inevitable. the availability of these novel agents has increased questions for determining optimal treatment of patients with relapse after the first auto-hct. methods: we retrospectively analyzed 60 patients who relapsed according to international myeloma working group (imwg) criteria after 1st auto-hct. first group [salvage chemotherapy(ct)] (n=27) was treated with only chemoimmunotherapy because of early relapse or refractory first auto-sct (within 6 months), ineligible to second transplantation because of co-morbidity, unwillingness to transplant. second group (n=33) (salvage transplantion) was treated with second auto-hct as a salvage therapy. consolidation and long term maintenance treatments were used in both groups. results: there was no difference in sex and age between salvage ct and auto-sct groups [female/male: 11 vs 13/ 16 vs 20; ]. the best response after salvage auto-sct was complete remission (cr) in 62,5%, partial remission (pr) in 15,6% patients, while cr in 44%, pr in 14,8% patients treated with salvage ct. progression free survival (pfs) were significantly better in second transplant group (pfs; 71 % on the first year; 46,9 % on the second year after transplant vs 59 % on the first year; 17% on the second year after the salvage therapy in chemotherapy patients)[p: 0,001]. overall survival (os) in salvage auto-sct group was longer than salvage ct (42,3 % 23.7%), although it did not reach a statistical significance (p>0.05). time to achieving the best response after salvage auto-sct and salvage ct was 1 (0-9) month versus 6,5(2-15) months [p:0,02]. grade 3 or 4 nonhematological toxicities were similar (auto-sct 19%, salvage ct 13%) in both groups. conclusions: salvage auto-hct may provides longer progression free survival with similar toxicity profile according to chemoimmunotherapy especially in patients with sensitive to first auto-sct. it is suggested that earlier and better responses, long-term progression free survival can be achieved with salvage auto-sct. we believe that there will be statistical significance in os such as pfs by increasing the number of patients. the authors believe that large scale randomized clinical trials are needed for optimal treatment of relapsing multiple myeloma after first auto-sct. disclosure: nothing to declare background: one of the conditions for successful transplantation of autologous hematopoietic stem cells (auto-hsct) in patients with multiple myeloma (mm) is the timely recovery of hematopoiesis, which is associated with the quantitative and qualitative characteristics of the graft. one of the key indicators is the content of cd34+ cells in the autograft, which depends on many factors. some of them are due to previous treatment, others are directly related to the patient: age, stage of the disease, features of the hematopoietic stem cells (hsc) microenvironment. the aim of the study was to assess the influence of the immune response genes on the autograft cellularity in patients with mm. methods: а retrospective analysis of the genotyping results was performed. evaluation of 21 loci in 15 genes immune response and harvesting of autologous hsc in 78 patients with mm has been made. hematopoietic stem cell mobilization regimen included cyclophosphamide 4 g/m 2 with granulocyte colony-stimulating factor. genotyping of the immune response genes polymorphic regions was carried out by the polymerase chain reaction with allelespecific primers. the number of cd34+ cells was counted on a 6-color facs canto ii flow cytometer. results: according to the results of the autologous transplant harvesting, two groups of patients were identified. first included 65 patients with an autograft cellularity of more than 2×10 6 /kg body weight. the second group consisted of 13 patients examined with the number of cd34 + cells in the autograft ≤2×10 6 /kg of the patient's body weight. comparing the identified haplotypes of the immune response genes with the cellularity of the transplantation material, it was found that the presence of the mutant allele in the homo-and heterozygous haplotypes of the il1β gene (t-511c) increased the chances of harvesting cellular material with a higher content of cd34+ cells in 4 times (χ2=5.04, p=0.02), and the carriage of the wild type allele in the homo-and heterozygous state of the tlr2 (arg753gln) gene is more than in 15 times (χ2=5.06, p=0.02). currently, it has been shown that single nucleotide or amino acid substitutions in genes can lead to changes in the expression pattern of their final products: increased secretion of interleukin 1β (il-1β) or changes in the spatial configuration and functionality of the receptors (tlr2). thus, in the presence of mutations in the il1β gene, the enhanced synthesis of il-1β influences on fibroblasts, immunocompetent, endothelial, epithelial and other cells, by activating hemopoiesis. in turn, the mutational status of the arg753gln locus located within the tir domain of the tlr2 receptor in the cytosol, determines the spatial configuration of the tlr2 acting as a co-stimulatory receptor of cd4+ cells, which ensure the engraftment of the graft. conclusions: identified haplotypical features of the il1β and tlr2 genes in patients with mm may act as predictors of the response effectiveness to mobilization of hscs in their carriers, which may contribute to the mobilization regimen optimization and will contribute to harvesting the optimal cellularity of an autologous graft. clinical trial registry: none. disclosure: authors declare no conflict of interest. differentiating diffuse from focal pattern on computed tomography: added values of a radiomics approach background: focal pattern in multiple myeloma (mm) seems to be related to poorer survival and differentiation from diffuse to focal pattern on computed tomography (ct) has inter-reader variability. therefore the purpose of this study is to assess if a radiomic approach could help radiologists in differentiating diffuse from focal patterns. methods: we retrospectively reviewed imaging data of 70 patients with mm between january 2013 and september 2018 of whom 61(27 men and 34 women; mean age 54.2 ±3.7) with ct, pet-ct or mri available before bone marrow transplant. two general radiologist evaluated in consensus only ct images to define a focal (at least one lytic lesion >5mm in diameter) or a diffuse (lesions < 5 mm, not osteoporosis) pattern. radiomic analysis on ct thinslice images was then applied with regions of interest (rois) done by one researcher not expert in medical imaging or mm blindly to the condition of the patients. the reference standard to differentiate diffuse from focal pattern was done by radiological evaluation of two expert musculosketal radiologists blinded to the clinical data reviewing ct, mri and pet-ct images. n=104 radiomics features were extracted and evaluated with an open source software. mann-whitney u test for unpaired data with 1000 bootstraps samples was used to compare radiomics features of diffuse and focal patterns and then feature reduction was done to avoid over-fitting. receiver operator characteristic (roc) analysis with area under the curve was done to compare radiologists and radiomics evaluation against reference standard. reading time to perform radiomic analysis was also estimated. results: the pathological group included: 22 diffuse and 39 focal patterns. after feature reduction, 9 features were different (p< 0.05) in the diffuse and focal patterns (n=2/9 features were shape-based: majoraxislength and sphericity; n=7/9 were gray level run length matrix (glrlm)). 150mg/kg). a number of eleven patients did not receive any additional immunosuppression except of post-cy. results: after a median follow up of 11.4 months (range 3.3 -34.5) 28 patients were alive. the 2-year probabilities of pfs and os were 42% (21-63%) and 65% (47-83%).the cumulative incidences (cis) of relapse and nrm at 2 years were 28% (11-45%) and 22% (8-36%), respectively. lower serum albumin level at transplantation (≤36 g/dl) was associated with increased relapses (hr 3.8 (1.2-12.7), p=0.028) and nrm (hr 7.9 (2-30), p=0.0026) and resulted in poorer pfs ), p=0.001) and os ), p=0.048). mmud and haploidentical donors were associated with poorer nrm (hr 6.2 (1.9-20.3), p=0.0027), and resulted in decreased pfs ), p=0.001). the high-risk cytogenetic at diagnosis showed no impact on survival. the cis of acute (grade ii-iv) at day +100 and chronic gvhd at 2 years were 26% (10-42%) and 48% (26-70%), respectively. absence of immunosuppressive medication beside post-cy was associated with poorer os ), p=0.01). conclusions: the conditioning with bu, tt and post-cy leads to a favorable pfs and os due to low incidences of relapse and nrm for patients with multiple myeloma relapsing after autografting. disclosure: nothing to declare methods: between january 2011 and may 2017, we included 51 patients with mm who underwent asct and received bortezomib/lenalidomide/dexamethasone (vrd) consolidation and maintenance therapy, mainly lenalidomide(r) 10mg/day for 21 days every 28 days. results: the median age at transplant was 55 years (46-75). forty-six (90%) of patients received r maintenance, 3 patients received vrd maintenance for higher risk features. median duration of r maintenance was 22 months . r dose was changed for toxicity (grade i-ii) in 17 (33%) patients. twenty-nine (57%) patients relapsed: 13 (25%) patients were shifted to different treatment protocols (treatment change). 6 patients (11%) were kept on the same r maintenance (observation group) and 10 (20%) patients had increased lenalidomide dose with dexamethasone (r/ d group). 6 patients (37%) of the last 2 groups required change of treatment later. the median follow up was 38 months . median tnt was 32 months (7-62). at 2 years, the estimated pfs and os were 39% and 97.5% respectively. the median os and pfs2 (from change of therapy) were 54 and 29 months for patients in the observation group, versus 52 and 27 months in the r/d group, and 45 and 33 months with treatment change, respectively. no statistically significant difference was noted. conclusions: our small monocentric study is limited by its retrospective design and small sample size. however, it suggests that increasing lenalidomide dose as well as adding dexamethasone in selected patients can postpone change to different lines of treatment without affecting survival. disclosure: nothing to declare can the drugs used before autologous hematopoietic stem cell transplantation have impact on cmv reactivation that results in decreased os in myeloma patients after asct? more intensive treatment regimens, such as proteasome inhibitors (pi) and/or immunomodulatory (imid) agents. we performed a retrospective, single center study to evaluate the incidence, risk factors, and outcomes of cmv infection in patients with mm who underwent asct with a high-dose melphalan-based regimen. methods: this study involved a retrospective review of all patients with who underwent asct between january 2015 and november 2018 at our stem cell transplantation center. a total of 144 consecutive adult patients with a diagnosis of mm (median age at diagnosis: 58, range: 35-77) underwent asct following induction treatment with novel agents (pis and/or imids). all patients received antiviral prophylaxis with acyclovir 600 mg/day (n=104) or valaganciclovir 1000 mg/day (n=36). results: baseline patient characteristics, according to induction treatment, are summarized in table1. one hundred-five of the 144 patients (97.2%) were cmv iggpositive before asct. overall, 14.6% (n=21) of cmvseropositive patients developed at least one episode of cmv viremia (cmv dna >100 copies/ml) after a median 24 months (range; 3-48 mos) follow-up. persistent cmv viremia (detectable cmv dna load in more than 2 sequential plasma specimens) occurred in 4.9% (7 of 144) of the seropositive asct recipients and all of them were preventive treated with ganciclovir (n=5) or valganciclovir (n=2). the time from stem cell infusion to the development of cmv viremia ranged from 3 days to 48 days. none of the patients with untreated viremia developed identifiable cmv sequelae. no case of primary infection in seronegative patients at transplant was observed. adding to that none of the patients developed cmv disease post asct. if we analyzed the subgroups of patients according to induction therapy (pi-based, imids, pi+imid), the incidence of post-asct cmv reactivation was higher but not statistically significant, in patients who received only pi vs pi+imid (13 (61.9%) vs 8 (38.1%); p=1.00). in univariate analysis, we could not demonstrate the importance of induction therapy with novel agents the occurrence of a post-asct cmv reactivation requiring antiviral treatment. however, statistically significant association found between the disease < vgpr status at asct and cmv reactivation (61.1% vs. 38.9%; p=0.035). after a median follow-up 14.3 months (range; 1-45.9 months), there was no significant impact on pfs, however there was significant decrease in estimated mean os who had cmv reactivation when compared to those without cmv reactivation (34.1±4.5 vs. 41.9±1.3; p=0.002) (figure-1) . conclusions: cmv establishes lifelong latency within host cells and in the setting of impaired cellular immunity; cmv may reactivate from latency, disseminate, and directly cause life-threatening disease. our data suggests that mm patients treated with pi-based induction regimens and immunological response < vgpr at time of asct seem to have higher risk of developing symptomatic cmv reactivation. however, further studies on a large number of patients are warranted to clarify these findings. clinical background: high-dose therapy followed by autologous stem cell transplantation (asct) has been shown to prolong survival in patients with multiple myeloma (mm) in randomized trials. however, these trials only include patients aged < 65 years. data regarding safety and outcomes in this patient population is lacking. methods: the aim of this study was to compare safety profile and outcomes in mm patients younger and older than 65 years-old who underwent asct in our unit from july 2014 to october 2018. patient's demographics, clinical characteristics, transplant related variables and probability of admission to the intensive care unit (icu) were analyzed. patients aged < 65 and ≥65 years-old would be called m1 and m2, respectively, from now on. sorror index was used to estimate risk of mortality in the two cohorts. results: a hundred and eleven patients with mm underwent asct in the study period. median age was 60.9 years-old (range 35-73) and 53.2% were male. thirtythree (28,8%) patients were ≥ 65 years. the probability of having a high risk comorbidity index was similar in both groups (m1 11,7 vsm2 12,1%). the median cells obtained in the apheresis procedure was 5.35 x10 6 (1,70-15.92) in m1 compared to 3.77 x10 6 (1.96-10-48) in m2. there were no differences in median admission lenght between the 2 cohorts (m1:19 days vs m2: 20 days). median days for neutrophil recovery above 1000 was 12 days in both groups with a wider range in m1 (11-23) compared to m2 (11-15) . no differences were found in platelet recovery above 100.000 (m1 20 days vs m2 21 days). median packed red blood cells and platelets transfusions were 1 (0-6) and 3 (0-16), respectively, in m1. in m2 cohort, they were 2 (0-5) and 4 (0-14), respectively. the incidence of grade 3-4 mucositis in m1 and m2 was 50.6% and 44.1%, respectively. there were no statistically significant differences in terms of using morphine for pain control between the two cohorts (m1, 63,4% vsm2, 58,8%). none patient requiered total parenteral nutrition (tpn) in group m2 and only one in group m1. the incidence of icu admission was 1.5 times higher in patients aged ≥65 than in patients < 65 years-old 6,1% vs3,9%), but differences were not statistically significant (p = 0.52). there were no deaths during the transplant procedure in any of the 2 cohorts conclusions: 1) in our series, high-dose therapy followed by autologous hematopoietic cell transplantation in mm patients aged ≥65 was feasible. 2) transplant procedure in older patients was as safe as in patients < 65 years-old. 3) no differences were found in terms of graft, transfusion support, transplant related complications and length of admission. 4) age should not be a limiting factor in considering the modality of asct in this patient population disclosure: nothing to declare the correlation between the kinetics of peripheral blood counts and the response to treatment after high-dose melphalan with stem cell support in multiple myeloma patients background: the long-term survival of mm patients has dramatically increased in the last 20 years, particularly for younger patients. this is attributable in part to the introduction and development of high dose chemotherapy with melphalan with stem cell support (hdm-asct). currently, frontline asct is still considered the standard of care for all eligible patients. many prognostic factors pre and post transplantation have been identified, e.g.: age, comorbidities, cytogenitcs, response to treatment and disease status prior to and post transplantation. to our knowledge there is no data correlating between kinetics of counts response to melphalan and prognosis. our aim was to assess the prognostic significance of the neutrophil and platelets decaying counts after high dose melphalan. methods -we retrospectively analyzed our cohort of 159 multiple myeloma patients who underwent hdm-asct at the hadassah medical center bone marrow transplant department, between the years 2007-2015. the kinetics of neutrophil and platelet decay during the first two weeks after melphalan administration was fitted using linear and exponential mathematical models. methods: we retrospectively analyzed our cohort of 159 multiple myeloma patients who underwent hdm-asct at the hadassah medical center bone marrow transplant department, between the years 2007-2015. the kinetics of neutrophil and platelet decay during the first two weeks after melphalan administration was fitted using linear and exponential mathematical models. results: factors associated with prolonged os in univariate analysis were: iss stage 1(p=0.024), ≤2 lines of treatment prior to asct(p< 0.001), favorable cytogenetics(p=0.004), response to treatment (pr or better, p=0.046) and rapid linear neutrophil decay (p = 0.046). in multivariate analysis, only ≤2 lines of treatment before hdm-asct and rapid linear neutrophils count decay remained statistically significant for os prolongation. no predictive threshold value of the neutrophil decay incline was found. improved pfs was associated with ≤2 lines of treatment prior to asct, and the response status after hdm-asct (p=0.003, p=0.019). additionally, toxicity evaluation showed prolonged neutropenia to be associated with inferior os (hr = 1.139, p=0.03) and rapid exponential decay of neutrophil counts to correlate with higher incidence of mucositis (p = 0.013). fast platelet decay was associated with delayed platelet engraftment (p< 0.01) conclusions: we have shown that rapid linear decay in neutrophil counts predicts better os without a significant benefit in pfs in mm patients undergoing hdm-asct. this discrepancy might reflect the problematic estimation in a retrospective analysis of pfs. rapid decrease in neutrophils and platelet counts was associated with more toxicity: higher mucositis rate and delayed engraftment, respectively. therefore a rapid decay of blood counts after hdm-asct appears to be an in-vivo phamacodynamic marker of higher efficacy and toxicity of melphalan. disclosure: nothing to declare p564 do we need to freeze hematopoietic cells for autotransplants in patients with myeloma conditioned with melphalan? daniel garcia belmonte 1 , beatriz aguado bueno 1 , miguel herrero coderch 1 , rafael de la camara 1 background: multiple myeloma (mm) is the most frequent indication of auto-hsct, representing 51% of all auto-hsct in 2016 (passweg jr. bmt 2018; 53:1139-48) . nearly all are performed with peripheral blood progenitor cells (pbpc), and melphalan 200 mg/m2 is considered the gold standard conditioning regimen. the standard procedure consists in obtaining progenitors, cryopreserved with dimethyl sulfoxide (dmso) and stored and subsequently thawed and re-infused in the patient on day 0. the procedure of cryopreservation is expensive and has some inherent toxicities (dmso) and loss of cells during the procedure. several groups have used non-cryopreserved progenitors showing some benefits compared with cryopreserved transplants, mainly a faster engraftment and a shorter length of hospitalization. objective: to compare noncryopreserved vs cryopreserved auto-hsct in mm methods: we perform an unicentric, retrospective study on 23 consecutive first auto-hsct mm patients transplanted with pbpc between nov-2011 and oct-2018, and conditioned with high dose melphalan (200 mg/m2). the median follow-up was 1620 days (range: 44-2413). 9 patients received non-cryopreserved and 14 cryopreserved auto-hsct. patients characteristics, without differences between non-cryopreserved vs cryopreserved: women/men (15/8); median age was 65 years (range 49-72); in the majority auto-hsct was done as consolidation after first line therapy (78%); year of transplant ≤2014 (61%), ≥2015 (39%). the number of infused cd34 cells were not different: median 3.5 x10 /kg (range 1.7-11.5) in noncryopreserved patients and 3.75 x10 /kg (range 2.1-17) in cryopreserved patients. results: we didn´t observe significant differences in the day of engraftment between non-cryopreserved vs cryopreserved although always was a little bit faster in the noncryopreserved group with a tendency to faster platelet engraftment (>50000/mm3): >20000 platelets/mm3 (median day: 12 vs 12.5, p 0.2), >50000 platelets/mm3 (median: 13 vs 16 days, p 0.09); >500 neutrophils/mm3 (median: 13 vs 13.5 days, p 0.3). the media of days of hospitalization was shorter in non-cryopreserved patients (18 vs 21 days) although not statistically significant (p 0.14). transplantrelated mortality at day +100 was 0% in both groups. overall survival at 5 years was not different: 83.3% in in non-cryopreserved vs 61.5% in cryopreserved patients (kaplan-meier, log-rank p 0.27). the accumulative incidence of relapse at the median follow up (1600 days) was similar: 33.3% in non-cryopreserved vs 35.9% in cryopreserved patients. conclusions: in our short experience, auto-hsct with non-cryopreserved pbpc in myeloma patients conditioned with high dose melphalan obtain similar results to those performed with classical cryopreserved pbpc and might has a faster platelet engraftment and shorter length of hospitalization. if no advantages are associated with cryopreservation, the simplicity of using fresh product is appealing. disclosure: nothing to declare p565 abstract withdrawn. . results: a early death is observed in one pt (group 1) and 5 pts(group 2). the median delay of aplasia is 14 days (10-22) and 12 days (9-17) respectively. in group 1, among the evaluable pts, 30/37 (81%) are in cr, 5 pts in pr and 2 refractory. in group 2, cr: 35/43 (81%), pr: 6 and 2 refractory. a relapse is observed in 32 pts/37 (86,5%) in group 1 and 35 pts/43 (81%) in group 2 with a frequency of 54% and 41% respectively in the first 24 months. at 36 months: 13 pts/38 (34%) in group 1 and 19 pts/49 (38%) in group 2 are dead. at 60 months: 20 pts (52%) and 29 pts (59%). at 120 months: 31 pts (81,5%) and 37 pts (75,5%). the overall survival (os) of the group 1 and group 2 pts were 66% and 62% at 36 months; 47.5% and 38.5% at 60 months; 18% and 24,5% at 120 months respectively (without significant difference). the event free survival (efs) of group 1 and 2 pts were 37% and 36,5% at 36 months, 25% and 13% at 60 months and 8% and 15% at 120 months respectively (without significant difference). conclusions: these 2 protocols with equivalent toxicity allow obtaining of long-term equivalent results on the response rate early transplant, on the rate of relapse and on the overall survival. these results are identical to those of fermand (1999) . disclosure: nothing to declare background: dimethylsulfoxide (dmso) is a major intracellular cryoprotectant, used for cryopreservation of stem cells. it is toxic to both cells and patients at temperatures above 0 o c. reduction of this effect is achieved by either washing of cells after thawing or by reduction of dmso during freezing and storage. the latter requires addition of extracellular cryoprotectants to the freezing media. we assessed the effect of low dmso concentration and different hematocrits of the frozen cells on cell viability and hematologic recovery in patients, transplanted for multiple myeloma. methods: cells were non-programmed frozen and stored at -80 o c in a cryoprotectant solution achieving final concentrations of 5% dmso, 3.6% of hydroxyethyl starch (hes, weight average molecular weight 450 000 da) and 3% of human serum albumin. the cell concentration in the frozen product for the first 77 patients (84 transplantations) varied between 100x10 6 and 250x10 6 cells/ml. in an attempt to reduce the amount of dmso infused, for the rest of the patients (n=172; 192 transplantations) we further decreased the volume of the freezing suspension by removal of the entire plasma. the average age of the transplanted patients was 55 (35 -71). the cells were bedside thawed at 37 o c water bath. the average cell dose was 2,95x10 6 /kg (1,3 -9,2x10 6 /kg). results: viability of the stem cells following thawing assessed by trypan blue exclusion was 95,34% . the hematocrit of the frozen cells had no effect on cell viability (94,80%(low) vs 95,52%(high)). the major complaints, if any, during stem cell infusion were coughing and an increase in nausea and vomiting induced by the prior conditioning. the average time for hematological recovery was 11,66 days (between 9 and 19) for the neutrophils, and 12,17 (between 9 and 20) days for the platelets. there was no significant difference in viability and hematologic recovery (11,86 and 12,60 vs 11,59 and 12,02) between patients receiving cells frozen with low or high hematocrit. conclusions: dimethylsulfoxide, despite its cryoprotective properties, is toxic for stem cells at temperatures above zero c and induces many side effects (cardiac, neurologic, respiratory, etc.) in the patients. to reduce those side effects we use lower dmso concentration, high hematocrit resulting in lower volume of the frozen cell suspension, thus reducing the final quantity of dmso to be infused to the patients. this does not affect the cell viability or the hematologic recovery of patients after transplantation. our easily performed method for unprogrammed freezing of stem cells with final dmso concentration 5% at -80 o c is safe, well tolerated, and provides cryopreservation, which allows high viability and stable cell engraftment, while reducing the undesired side effects of dmso. disclosure: nothing to disclose the conditioning regimen consisted of melphalan for most of the patients. the average age at the time of transplantation was 55 years (35 -71). patients were transplanted with an average cell dose of 2,95x10 6 /kg (1,30 -9,20x10 6 /kg) for the first transplantation and 2,36x10 6 /kg (1,64 -3,19x10 6 /kg) for the second one (every patient received the same cell dose as for the first) with average cell viability 95,34 % (70 -99%), with little difference between first and second transplantation. results: the average time for hematological recovery was 11,60 (between 9 and 19) days for the neutrophils, and 12.11 (between 9 and 20) days for the platelets. we found no correlation between the cell dose and the hematological recovery. there was no difference in the hematopoietic recovery between the first and the second transplantation in the patients, who underwent tandem or two transplantations. conclusions: recovery time is considered by some to be a function of the effective stem cell number. we did not find such correlation, probably because in the analyzed group all the patients, except four of them, received a dose greater than 2 x10 6 /kg cell, which is accepted as a safe dose for autologous stem cell transplantation. disclosure: nothing to disclose p569 plerixafor-mobilized patients have a high risk of noninfectious fever during engraftment after autologous peripheral blood stem cell transplantation background: plerixafor enables rapid and efficient mobilization of hematopoietic stem cells. however, its impact on adverse clinical events after autologous peripheral blood stem cell transplantation (pbsct) is not fully understood. fever is one of the major complications in the preengraftment phase of pbsct. in this research, we focused on non-infectious fever around the time of bone marrow recovery and investigated whether plerixafor as mobilization therapy plays a role in engraftment fever. methods: we reviewed 80 autologous pbscts for treatment of multiple myeloma at the japanese red cross medical center between 2012-2018. non-infectious fever was defined as temperature ≥37°c with onset between two days prior to and two days after engraftment without clinical or microbiological documentation of infection. results: patients were mobilized by cyclophosphamide and filgrastim in 57.5% (n = 46) and filgrastim and plerixafor in 42.5% (n = 34). the median number of transfused cd34+ cells were 2.61×10 6 /kg and 3.45×10 6 / kg, respectively (p=0.002). patients transfused with plerixafor-mobilized grafts had a higher risk of noninfectious fever (85.3% vs 63.0%, p< 0.05). cd34+ cell number or cyclophosphamide pretreatment had no relationship to non-infectious fever. the recovery of lymphocytes was more rapid in plerixafor-mobilized patients (p=0.001). however, the number of lymphocytes was not associated with non-infectious fever. conclusions: combination of filgrastim and plerixafor as mobilization therapy resulted in an increased risk of noninfectious fever during engraftment comparing to mobilization with cyclophosphamide and filgrastim. while the mechanism remains unclear and requires further studies, plerixafor-mobilized grafts may result in an unintended increase in engraftment fever. clinicians should be aware of this possibility if patients are transplanted with those grafts. disclosure: ks received honorarium outside the submitted work from janssen, novartis, celgene, ono pharmaceuticals, takeda, fujimoto pharmaceuticals and srl. ti received honorarium outside the submitted work from janssen, celgene, ono pharmaceuticals and takeda. we assessed the efficacy of a new conditioning regimen consisted of decitabine (dec), busulfan (bu), cyclophosphamide (cy), fludarabine (flud) and cytarabine (ara-c) for allo-hsct in patients with mds and mds/ mpn. fifty patients were enrolled, including 46 with mds and 4 with cmml. patients received dec 20 mg/m 2 /day on days -9 to -5, combining bu/cy/ flu/ ara-c modified preparative regimen. results: at a median follow-up of 522 (15-1313) days, the overall survival (os) was 86%, disease-free survival (dfs) was 78%, and relapse incidence was 11%. the incidence of severe acute (grade iii/iv) graft-versus-host disease (gvhd) was 22%, and that of (predominantly mild) chronic gvhd was 40%. os at 2 years was 74% for mds patients with high risk, 86% for mds patients with very high risk, respectively. the survival was delightful in patients with poor-risk mutations, such as tp53 and asxl1, (86%) and with three or more gene mutations (77%). among the total 12 patients with poor-risk mutations in our research, only one patient (8%) with tp53 relapsed and one (8%) with asxl1and tet2 died. result of continuous observation after transplantation, the percentage of nk cells in the peripheral blood of all patients who had received dec/flu/bu/cy/ara-c conditioning increased at day 28, which may essentially contribute to disease control post-transplantation. conclusions: in summary, the addition of a 5-day schedule of decitabine to a flu/bu/cy/ara-c conditioning regimen has proven feasible, with a low level of toxicity and promising early disease control especially in patients with high risk mds. disclosure: there are no conflicts of interest. the sfgm-tc mds score at day 180 is associated with post-transplant outcomes in patients with myelodysplastic syndrome who underwent cd34+ selected allogeneic stem cell transplant conclusions: in patients with mds undergoing tcd-hct, the sfgm-tc score at day 180 is significantly associated with survival. the lower incidence of acute gvhd in recipients of cd34-selected transplants and the use of myeloablative condition regimens, with lower relapse, may explain the difference with the original finding that the sfgm-tc was predictive at day 100 in unmodified grafts. disclosure the most frequent grade 3, 4 toxicities were thromobocytopenia and neutropenia. infections developed in 12 patients (33.3%), neutropenic fever in 8 (19.4%). five patients (13.9%) either developed or experienced exacerbation of acute graft versus host disease (gvhd), nonechronic gvhd. conclusions: azacitidine use is associated with only modest activity in patients who relapse after allo-hsct. however, in patients who respond to treatment it may allow for a durable disease control. disclosure: the authors declare no competing conflicts of interest background: somatic mutations in mds patients are closely related with clinical phenotypes and prognosis in mds patients. but whether mutations are prognostic for outcomes after allogeneic hematopoietic stem-cell transplantation (allo-hsct) remains to be elaborated. methods: targeted mutational analysis were performed on samples obtained before transplantation from 134 patients underwent hsct. we analyzed the relationship of mutations and clinical outcomes. results: all 134 patients carried more than one somatic mutations, most frequently in kmt2d(67.16%), arid1b (61.94%), ccdc168(52.24%), pclo(47.01%), asxl1/2 (46.27%), srcap(44.78%), u2af1(42.54%), dnah2 (41.79%), ush2a(41.04%) and tet2(34.33%). tp53 mutations were associated with higher ipss-r risk, complex karyotype and monosomal karyotype. dnah2 were more frequent in pediatric patients. in univariable analyses, tp53 mutations were related with decreased disease-free survival (p=0.047); dnah2 mutations were related with increased disease-free survival (dfs) (p=0.038). in multivariable analysis including ipss-r stratification, gvhd, hct-ci and candidate genes, dnah2 mutations were independently associated with better dfs(p=0.027). conclusions: dnah2 mutations is independently associated with better outcomes in mds patients treated with allo-hsct while tp53 may predict unfavorable outcomes. accounting for these somatic mutations may help better selection of candidates for allo-hsct among mds patients. disclosure background: there is a controversy among experts if and how patients with mds and saml should receive cytoreductive therapy before transplant. while aiming to reduce disease burden in order to lower the risk of relapse after transplant cytoreductive therapy is associated with several drawbacks. besides a considerable risk for toxicity and mortality preventing patients to proceed to transplant cytoreductive therapy may also favour the selection of resistant clones which may be difficult to treat at relapse. methods: to address this hypothesis we retrospectively analysed the response and survival following salvage therapy in 73 patients with mds and saml who had relapsed in median 5.6 months (1 to 110 months) after allo-sct according to their pre-transplant strategy (upfront transplantation n=32 44%; induction chemotherapy [ctx] n=26 36%; hypomethylating agents [hma] n=15 20%). results: the majority of these 73 patients received salvage therapy with hma (n=58, 79%; aza n=57, dac n=1) mostly in combination with dli, while the remaining received other salvage treatments (intensive chemotherapy n=1, dli alone n=1, 2 nd transplant n=3, bsc n=5, miscellaneous n=2, missing information n=3). when focussing on those patients treated with hma and dli it became apparent that a significantly higher proportion of patients in the upfront group (58%) achieved cr after salvage therapy in comparison to pre-treated patients (10% cr, p=0.0005; ctx group 5% cr; hma group 18% cr). accordingly, overall survival (os) calculated from the time of relapse was significantly longer in patients in the upfront group than in the group of pre-treated patients (2-year os 59% vs. 19%, p=0.0001). conclusions: overall, these findings imply that pretransplant therapy may favour the iatrogenic selection of resistant clones, which poorly respond to salvage therapy with hma and dli in case of relapse after allo-sct. furthermore, the results support the concept that an upfront transplant strategy is a promising alternative for patients with mds and saml that can be augmented by salvage therapy with hma and dli. disclosure: ts and gk received travel support, lecture fees and research funding from celgene gmbh conclusions: in our country, this procedure has shown to be feasible and we hope to improve it, with better infection control and by acquiring more experience related to the management of these patients. background: extramedullar relapse of mds is a rare complication after allogeneic stem cell transplantation. we present the case of a 69-year-old woman who was admitted into hospital because of insecure walking. paresis of both legs, hypaesthesia of the inner thighs, increased effort at urinating, reduced sphincter tonus, central paresis of the right arm and discreet paresis of the right facial nerve were documented at neurological exam. mri showed a large tumour of the dorsal thorax that immured the adjacent ribs and spine, affected the processus transversus of t9-10 and invaded the spinal canal. the patient had undergone ric allogeneic stem cell transplantation five years ago for mds-eb 2 with complex aberrant karyotype. following an uneventful course and no signs of gvhd, she had been off immunosuppression since 4,5 years. at the time of the admission the patient had slightly lowered wbc (2,9 gpt/l) and plt (111 gpt/l) and clearly increased ldh (793 u/l). methods: histology of a ct-based biopsy of the paravertebral tumour showed an infiltration of the muscles by blastous cells that were cd45-, cd34-, pax5-positive, tdt and cd79a were questionably positive. provisonal diagnosis therefore was lymphoblastic lymphoma, pox tested negative. the bone marrow was hypocellular with increased numbers of mature lymphocytes, but no definite signs of malignancy. cerebrospinal fluid revealed 1574 cells/μl with 90% blasts. immunotype was cd34, cd117, cd13, cd33, hla-dr positive, pox and lymphatic markers were negative. because of this we finally suspected meningeosis leucaemica. we completed the diagnostic workup with genetical and chimaerism tests and compared the result to the patients' mds before allogeneic stem cell transplantation. [[p580 image] 1. mri scan of the large thoracic tumour] results: cerebrospinal fluid (csf) cells consisted of 92% recipient cells, whereas peripheral blood cells were 100% donor. high risk mds at transplant displayed a complex caryotype including trisomy 8 and tetrasomy 8, now 10% of the cells in csf showed trisomy 8 and 70% tetrasomy 8. chimerism and fish of the solid tumour could not be performed, coexpression of myeloid markers within the tumour is pending. conclusions: in conclusion the patient has meningeosis as a result of exclusively extramedullary relapse of myeloid blasts originating from the initial high risk mds with blast excess and complex aberrant caryotype. the evolution of a trisomy 8 clone to tetrasomy 8 clone in relapse is linked to extramedullar manifestations. whether the solid tumour represents myeloid sarcoma with coexpression of lymphoid markers, extramedullary relapse of mds with lymphoid differentiation or, less likely, a separate lymphobastic lymphoma, is not yet clear. disclosure background: adoptive t cell therapy with genetically engineered t cells is a potent innovative immunotherapeutic approach for cancer treatment. unfortunately, the use of t cells redirected against tumor antigens, is severely limited by 1) the difficulty in identifying appropriate cell surface antigens, that could be targeted by car t cells and 2) the paucity of tumor-specific t cell receptors (tcrs) against shared, oncogenic antigens. methods: focusing on wilms´tumor 1 (wt1), a tumorassociated antigen overexpressed by acute myeloid leukemia and several solid tumors, we designed and implemented an innovative protocol for the rapid isolation of wt1-specific t cells and for the generation and characterization of a library of wt1-specific tcrs displaying different human leukocyte antigen (hla) restrictions, to be exploited by tcr gene transfer and tcr gene editing. to this aim, we repetitively stimulated t cells with autologous antigen-presenting cells, including immortalized b cells, pulsed with overlapping peptides spanning the entire wt1 protein. t cell recognition was assessed by flow cytometry in terms of cd107a expression and ifnγ production. recognized peptides were mapped by a deconvoluting grid and t cell clonotypes were longitudinally tracked by tcrαβ sequencing. results: we successfully expanded tumor-specific t cells from 14 consecutive healthy donors, in an average of 4 rounds of in vitro stimulations. the ability of wt1specific t cells to recognize naturally processed epitopes and their on-target specificity was demonstrated upon coculture with antigen-expressing targets including primary leukemic blasts. tracking of the tcrαβ repertoire during culture led to the identification of 20 clonotypes that recognize several tumor-associated peptides and are restricted by more than 5 hla alleles, including hla-a*02:01. tcrs were then expressed via genome editing. briefly, simultaneous editing of endogenous tcr α and β chain genes was achieved using crispr/cas9 technology (efficiency >90%), followed by transduction of t cells with lentiviral vectors encoding wt1-specific tcrs (efficiency >95% of cd8 + t cells). phenotypic characterization of edited t lymphocytes showed a major enrichment of cells harboring t stem cell memory properties. functional validation of the edited t cells is currently ongoing. preliminary results of a 6 hours coculture experiment show that tcr edited t cells kill fresh wt1 + leukemic blasts, harvested from hla-matched patients, with an efficiency up to 70% at an effector to target ratio of 5 to 1, while no killing of controls is observed. conclusions: we set up a protocol enabling consistent and efficient hunting for tumor-specific tcrs with no need for labor intensive t cell cloning. tcr genes can be easily and rapidly used to redirect t cell specificity against cancer cells by tcr gene editing. disclosure: chiara bonini: research funding from intellia therapeutics p583 car t cell therapy targeting relapsed or refractory cd19+ lymphoid disease with third-generation vector rv-sfg.cd19.cd28.4-1bbzeta maria-luisa schubert 1 , anita schmitt 1 , leopold sellner 1,2 , brigitte neuber 1 , angela hückelhoven-krauss 1 , kunz alexander 1 , lei wang 1 , gern ulrike 1 , birgit michels 1 , susanne hofmann 1 , carsten mueller-tidow 1,2 , dreger peter 1,2 , michael schmitt 1, 2 background: t cells genetically engineered to express chimeric antigen receptors (carts) directed against cd19 have demonstrated significant efficacy in patients with iymphoid malignancies including relapsed or refractory (r/r) b-lineage acute lymphoblastic leukemia (all) or r/r b-cell non-hodgkin's lymphoma (nhl). access to cart treatment for patients in europe has been limited so far given that the vast majority of cart trials have been performed in the united states and the p. r. of china. here we present the preliminary results of the first investigator-initiated trial (iit) cart trial in germany. hd-car-1 (eudract no. 2016-00 4808-60; nct03676504) is a phase i/ii trial with in-house cart manufacturing which was initiated in september 2018 at the university hospital heidelberg. methods: adult as well as pediatric patients with r/r all and patients with chronic lymphocytic leukemia (cll) or nhl including diffuse large b-cell lymphoma (dlbcl), follicular lymphoma (fl) or mantle cell lymphoma (mcl) are treated with autologous t lymphocytes transduced with a third-generation car retroviral vector (rv-sfg.cd19.cd28.4-1bbzeta) targeting cd19. the main purpose of hd-car-1 is to evaluate safety and feasibility of escalating third-generation car t cell doses (1-20×10 6 transduced cells/m 2 ) after lymphodepletion with fludarabine and cyclophosphamide. patients are monitored for cytokine release syndrome (crs), car-t-cell related encephalopathy syndrome (cres) and/or other toxicities. in vivo function, survival and anti-tumor efficacy of carts are assessed. results: to date, three patients (cll, dlbcl and mcl, respectively) have been enrolled and subjected to leukapheresis. high numbers of transduced carts were harvested on day 10 of culture (82-123x10 6 carts). transduction efficiency ranged between 33 and 42%. cart products were sterile and free from mycoplasms and endotoxins. no production failure occurred and all patients received the cart product. no signs of crs or cres > grade 2 have been observed. assessments of clinical responses are pending and will be presented at the conference along with updated technical results. conclusions: for hd-car-1, gmp-conform leukapheresis as well as cart manufacturing was effective. administration, patient monitoring and follow-up were performed in-house providing independency from transport or production sites outside the university hospital heidelberg, altogether suggesting that academic cart iits are feasible in germany. clinical background: the prognosis of adult patients (pts) with relapsed/refractory (r/r) precursor b-acute lymphoblastic leukemia (all) is dismal, including with allogeneic hematopoietic stem cell transplantation (allo-hsct). blinatumomab, a bispecific cd19-directed cd3 t-cell engager and inotuzumab ozogamycin (io), a cd22-directed antibody-drug conjugate revolutionized the field, improving their outcomes. anti-cd19 chimeric antigen receptor t (cart) cell therapy has led to further progress and improved outcome (jacoby e; am j hematol, 2018). nowadays, patients with r/r b-all can be offered both therapies, but there are limited data on the safety and efficacy of cart -cell therapy post antibody treatment. we detailed our single center experience in this regard. methods: this report is a part of a single center, phase 1b/2 study on therapy of b-cell malignancies with locally produced cart-cells (nct02772198). the approach uses autologous t cells with car construct that is composed of an anti-cd19 single-chain fv, cd28 co-stimulatory and cd3-zeta intracellular domains. cd19 expression on the blasts was documented prior treatment in all pts by flow cytometry. all pts received 1 x 10 6 /kg cart-cells after lymphodepletion with fludarabin and cyclophosphamide. results: six pts (2 males and 4 females) with r/r b-all were enrolled, including one with ph-positive b-all. the median age was 42 years (25-59). median number of prior therapy was 4 (3) (4) (5) . five pts had prior allo-hsct. four pts were given antibodies as the last therapy prior to cart cells. two pts received blinatumomab resulting in pr in one of them. two additional pts received io (1 after failing blinatumomab) achieving mrdpositive cr. cytokine release syndrome occurred in all pts and was severe in only one patient who required tocilizumab treatment. this patient was also the only patient who experienced neurotoxicity (grade 3), and was treated with dexamethasone. this patient eventually died 18 days post infusion of cart cells due to severe pseudomembranous colitis, toxic megacolon and sepsis. all pts had prolonged neutropenia for a median of 14 days (12-18) after the infusion of cart cells. at day 28 after infusion of cart-cells the cr for the entire cohort was 67%: three pts with mrd-negative and one with mrd-positive response. among the four pts who received antibodies prior the cart-cells, one patient had mrd-positive and two pts had mrd-negative response. the patient with ph positive b-all had progressive disease during the treatment. two pts were referred to second allo-hsct from other donors. one patient with mrd-negative response relapsed after the second transplant and was treated by salvage therapy. the second patient with mrdnegative response demonstrated prolonged remission (20 months) even without second transplantation. with a median follow-up of 11 months (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) the median progression-free and overall survival for the entire cohort were 7.5 and 9 months, respectively. conclusions: autologous anti-cd19 car t-cell therapy after debulking treatment with antibodies, including blinatumomab and/or io, is feasible and results in high response rates in pts with r/r b-all. patients may respond to anti-cd19 car t-cell therapy even after failure to their last salvage therapy with blinatumomab, which demonstrates similar mechanism of action. clinical background: genetically engineered t cells expressing a chimeric antigen receptor (car-t) targeting specific antigens present on acute lymphoblastic leukemia (all) blasts have generated promising results in children and adults with relapsed and refractory disease. the below report provides an insight of lineage switch occuring as a result of intense immunological selection after car-t cell therapy, even with a tumor clone that has no potential for this switch. methods: an eight year old caucasian male with precursor b (pb) cell lymphocytic leukemia was treated with cd 19 directed car-t cell therapy in third remission, and after relapse after previous bone marrow transplantation (bmt) . he was diagnosed with t(12;21) pb cell all at 2 years of age and treated with bfm protocol. he relapsed 8 months after completion of maintenance therapy, and had a 9/10 mmud bone marrow transplant after etoposide, tbi and alemtuzumab conditioning therapy. he had cutaneous acute and chronic gvhd but 8 months post-transplant, he relapsed again with pb cell all, with the same cytogenetic and immunophenotypic disease characteristics.. he was treated with lymphodepleting chemotherapy with fludarabine, cyclophosphamide and alemtuzumab followed by infusion of cd19 directed car t cells. he developed cytokine release syndrome of grade 1 severity manifested as persistent fever, associated with car t cell expansion in the blood. after car-t infusion,there was no detectable b cell all clone in the marrow by pcr and the cytogenetics were negative for t(12:21) translocation. 8 months after the car t cell therapy, he was found to have a mrd positive disease which was monitored closely. results: we document clonal evolution from cd19 negative, mrd positive disease to aml, with the same ig rearrangement (the same clonal disease) but with complete myeloid phenotype mpo, cd33, cd13 positive disease. there was cytogenetic evolution of the underlying clone but the original t(12;21) was retained within the evolved karyotype. sadly, our patient developed fludarabineneurotoxcity during an attempt to induce aml remission, and further curative-intent chemotherapy was not possible. conclusions: there are two case reports of mll rearranged b-all acquiring a clonally related myeloid phenotype associated with cd19-negative escape after cd19 directed car t cell therapy,so far. but, this is the first post car-t cell therapy transformation of all to aml with etv6-runx1 mutation, which is not recognised to have such lineage-switch potential. unlike mll, etv6-runx1 translocation in the pathogenesis of acute myeloid leukemia is not been reported in the literature. the theory behind such transformation is an intense immunological selection of the tumor, driving it to myeloid differentiation with additional clonal cytogenetic events. disclosure: nothing to declare p586 ivac-all-1: interim analysis of a phase i/ii clinical study on personalized peptide vaccination based on patient-individual tumor-specific variants in relapsed pediatric acute lymphoblastic leukemia armin rabsteyn 1,2 , christopher mohr 3 , olaf witt 2,4 , roland meisel 2,5 , cristiane chen-santel 6 , tobias feuchtinger 2,7 , christopher schroeder 8 , jakob matthes 8 , background: acute lymphoblastic leukemia (all) is the most common pediatric malignancy. standard chemotherapy is a successful treatment in 80% of patients, only about 20% develop a relapse, however these patients have a dismal prognosis. prevention of relapse after firstline chemotherapy or stem cell transplantation (sct) is therefore an urgent clinical need. we established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient hla types. we started clinical translation of this approach by starting a phase i/ii clinical trial in 2016 (nct03559413). besides feasibility and toxicity assessments, we aim to assess the capability of the peptide vaccination to induce neoantigen-specific t cell responses in high-risk all patients to target residual tumor cells and prevent leukemic relapses. methods: key inclusion criteria are: pediatric patients with all who suffered from second relapse after standard therapy or first relapse after sct. hematological remission has to be reached prior to vaccination. nonsynonymous mutations are identified by whole exome and transcriptome sequencing of patient leukemic blasts and healthy reference tissue. hla binding peptides harboring the altered amino acids are subsequently predicted in silico by algorithms syfpeithi, netmhc and netmhcpan for the patients' individual hla type. vaccine cocktails consisting of 3-5 individual peptides are produced and formulated under gmp conditions. the vaccination schedule is 16 vaccinations over 33 weeks using gm-csf and imiquimod as adjuvants. response to the vaccination is monitored by detection of t cells recognizing the vaccinated peptides occurring over time in peripheral blood of patients by prestimulation and intracellular cytokine staining. results: until now, 23 patients were recruited, for 17 of those, whole exome sequencing was performed to identify all-specific snvs using a comparative bioinformatics pipeline. we found an average of 118.8 mutations per patient on dna level. based on these data, an average of 178 hla binders derived from neoantigens could be predicted per patient. an average expression of 34.4% of mutations was assessed by rna sequencing. in all cases validated mutations could be identified and cocktail design was feasible. until now, 4 patients received vaccinations. the vaccine was generally well tolerated and no or only mild side effects were observed. immune monitoring was performed for 2 patients until now. in the first patient, we observed a transient cd4+ response against one vaccinated mhc class ii ligand and a sustained cd4+ response against the included wildtype control peptide derived from the antigen survivin. in the second patient, immune monitoring was performed for the first 8 vaccination timepoints, a t cell response was not measurable at this timepoint of vaccination. conclusions: whole exome sequencing of pediatric all patients is feasible and yields small amounts of expressed, tumor-specific mutations. these few mutations are sufficient to predict hla-binding peptides that can be used to formulate individualized peptide vaccine cocktails. we currently conduct a clinical phase i/ii trial in a small cohort of high-risk all patients to assess safety, toxicity and immunogenicity. clinical background: chimeric antigen receptor t cells (cart) are considered as gene therapy medicinal products (gtmp) and genetically modified organisms (gmo). hence, carts manufacturing for clinical application is strictly regulated. appropriate methods assessing car transgene copy number (cn) in a cart product and definition of the frequency of carts in treated cart patient are mandatory. although quantitative real-time pcr-based (qpcr) analysis has been used for this purpose, no standardized procedure to minimize systematic errors and enable comparability has been established yet. here, we report on a single copy genebased (scg) duplex (dp) pcr (scg-dp-pcr) for the determination of the vector copy number (vcn) in cart products as well as patient samples following cart administration. scg-dp-pcr was validated and compared to the broadly used absolute copy number qpcr (acn) approach within the framework of a clinical trial treating patients with good manufacturing practice (gmp)-grade carts (hd-car-1). methods: for conventional acn, primers and probe targeting the car vector rv-sfg.cd19.cd28.4-1bbzeta were designed. standard curves were established via serial dilutions of the sfg.car plasmid. amplification of the standard curve as well as target genomic dna for vcndetermination was performed as singleplex (sp) pcr (sp-car) (method a). on the same qpcr plate, duplex (dp) qpcr reactions were carried out. additionally to the components comprised within method a, the experimental setup contained haploid human genomes as well as primers and probe targeting ribonuclease (rnase) p as human scg. the amplifications for the sfg.car plasmid (dp-car) and the rnasep gene (dp-rnasep) were performed simultaneously (scg-dp-pcr; method b). scg-dp-pcr was performed for standard curves and target samples. target-sample dna was extracted from carts prepared from leukapheresis products of three healthy donors (hd). results: for method validation, efficiency and linearity (correlation coefficient) of the qpcr reactions of method a (sp-car) and method b (dp-car, dp-rnasep) were assessed by linear regression of the pcr signal to the reference standard curve. overall, standard curves were only considered valid if a correlation coefficient (r 2 ) of above 0.98 and efficiencies of 100% ± 10% were achieved. vcns applying method a and b to the same target sample were compared. sp-car pcr reaction displayed efficiency of 103.5 ± 7.1%; 104.2% ± 2.1% and 99.3 ± 1.6% efficiencies were achieved for dp-car and dp-rnase pcr reaction, respectively (table 1) . applying scg-dp-pcr using formula for relative cn assessment (2 -δct (dp-car -dp-rnasep) ) on hd samples resulted in an average of 0.8 ± 0.2 increased cn when compared to method a (table 2) . conclusions: in terms of efficiency and linearity by linear regression qpcr reactions were comparable. validation of scg-dp-pcr was achieved and represents an exact and less error-prone method to fulfil regulatory safety release criteria of cart products. besides of accurately assessing vcn of transduced cells, scg-dp-pcr is also a highly robust method to follow-up carts in treated patients. applying this approach, no standard curve is needed, hence significantly economizing required material as well as time. disclosure: nothing to declare background: t-cells' antileukemic responses in aml-pts need to be improved. dc leu effectively activate t-cells against leukemic blasts, resulting in blast-lysis ex-vivo. factors influencing these activities are not known. methods: we generated dc/dc leu from aml-blastcontaining mononuclear cells (n=74) using standard methods (mcm-mimic/ca-ionophore/picibanil/ifn-α, "mnc-methods") and from blast-containing heparinized whole blood (n=35) using modulatory kits (various combinations of 2-3 clinically approved response-modifiers, "wb-kits", patent 102014014993) and correlated statistically (t-/u-test, pearsons correlation, # means significant) proportions of dc-/t-cell-subtypes/cytokine-profiles with t-cells' antileukemic cytotoxicity (ctx), achieved after mixed lymphocyte culture (mlc) with/without mncmethod-("t*dc mnc "/"t*bla mnc ") or wb-kit-treated cultures ("t*dc wb "/t*bla wb "). ctx was given as proportions of cases with achieved or "improved" blast-lysis compared to control and as frequencies of viable blasts (bla via ) after effector-cell-influence. pooled data and data obtained with single methods in different cohorts are given. results: 1. generation of dc/dc leu : with a) mncmethods: ø18±15% dc and ø11±10% dc leu and b) wb-kits: ø13±8% dc and ø11±13% dc leu without induction of blasts' proliferation. t-cell-proliferation increased (vs uncultured t-cells) after mlc with a) mnc-methods: ø34±38% vs ø7±9% and b) wb-kits: ø63±25% vs ø23±20%. 2. antileukemic reactivity (t-effector-cell-cytotoxicity after mlc): pooling all data: a) mnc-methods ("t*dc mnc " vs "t*bla mnc "): we found a1) blast-cytotoxicity in ø70%(vs50%) of cases with ø59%(vs117%) bla via , a2) blast-cytotoxicity was improved (vs control) in 86% with ø decrease of bla via of 78%; b) wb-kits ("t*dc wb " vs "t*bla wb "): we found b1) blastcytotoxicity in ø80%(vs71%) of cases with ø68%(vs56%) bla via , b2) blast-cytotoxicity was improved (vs control) in 67% of cases with ø decrease of bla via of 43%. in general, these results could be confirmed with single methods: best mnc-methods were picibanil and mcm-mimic, best wbkits were kits containing gm-csf+picibanil or prostaglandins. 3. correlations: pooling all data: cases with "improved" lysis (vs "not improved" lysis) were characterized by a) mnc-methods: increased proportions of mature dc/cells (ø46±29% vs ø35±20%), dc leu /cells (ø10±10% vs ø5±4%) and proliferating t-cells (ø35±40% vs ø19 ±14%), b) wb-methods: dc/cells (r=0.781 # ), dc leu /cells (r=0.815 # ), dc leu /bla (ø40±23% vs ø29±16%), dc leu /dc (ø59±22% vs ø51±25%), cd8 + t-cells (ø58±20% vs ø50 ±24%), ifn-γ (r=0.868 #) , mcp-1 (593±242 vs 547±223 pg/ml). conclusions: blasts are regularly converted to dc leu in the presence of mnc-methods and wb-kits (simulating the in vivo microenvironment). t-cells' coculture with dc/ dc leu after mlc induces and improves antileukemic t-cell activation compared to controls. blast-cytotoxicity correlates with proportions of dc/dc leu -and t-cell subtypes and released cytokines. these data support a role of antigen presentation by leukemic cells (dc leu ) for the stimulation of an immune response in aml in vitro and possibly in vivo. disclosure: nothing to declare evaluation 1 year after the launch of the motion comic immuno-t, explaining patients and their caregivers how immunotherapy strategies work background: one year ago, the first version of immuno-t, a motion comic explaining to patients and caregivers how immunotherapy strategies work, was released. people were informed on and inspired to use the application during (inter)national meetings and events for the general public. meanwhile, the motion comic was further refined and adapted into a second version, based on the evaluations we've collected on the first version. adaptations included a multi-language tool (currently 6 languages), increased user friendliness, and a new supporting musical score. also, a website was launched from which the second version could be downloaded on tablet or smartphone (both android and apple) and a new online evaluation form could be filled in. in 10 months, 40 people have evaluated the motion comic online, and these results are presented here, as well as our future plans within the immuno-t program. methods: through an online questionnaire, 41 participants from belgium (n=38) and the netherlands (n=3) have evaluated the dutch version, and 4 belgian participants evaluated the french version of the motion comic. results: the total group (n=45) consisted of patients (n=15) and their families (n=2), general public (n=10), students (n=7), health care professionals (n=4), researchers (n=5) and kindergarten teachers (n=2). participants' age ranged from 19 to 80 years, with an even distribution amongst the different generations. the majority of the evaluators (97,8%, n=44) thought the motion comic is a good way to explain immunotherapy to patients. 23 individuals (51,1%) felt mainly interested after watching immuno-t, and a total of 32 participants (71,1%) felt hopeful or motivated. focussing on the patient group (n=15), all of the responders think the immuno-t motion comic is a good tool to use in a patient-doctor consultation. 11 patients (73,3%) felt hopeful and/or motivated after watching the whole motion comic, while 3 of them (20%) felt combative and 5 (33,3%) felt gripped and intrigued. as for the new musical score, 35 participants (77,7%) think the music is suitable for the app, while 6 evaluators (13,3%) think the new music is not or not at all fitted to support the motion comic. conclusions: the detailed evaluations allow us to further improve immuno-t, and aid us in the development of other motion comics we plan to release under the cancer-t in motion umbrella. with the current version of immuno-t, a single-center pilot study is being set up, to test the efficacy and usability of immuno-t, based on qualitative research during the experience of the tool, and using validated questionnaires. with this study we want to evaluate the impact of immuno-t on patient empowerment, and the decision making process. the study protocol will be presented at ebmt. disclosure: the development of immuno-t was partly financially supported by celyad, calgene, novartis, roche, amgen, bms, but these companies did not by any means influence the contents and development of the motion comic. a therapeutic strategy to trespass the blood brain barrier for adoptive nk cell therapy in glioblastoma multiforme induced rat: a preclinical study background: glioblastoma multiforme (gbm) is among the most common and aggressive primary brain tumors with very poor prognosis. according to the central brain tumor registry of the united states, central nervous system (cns) tumors in pediatric patients (ages between 0-14 years old) are the second most common malignancies after blood-born malignancies, and the first amongst solid tumors, and known the most common cause of cancer-related deaths. although hematopoietic stem cell transplantation has been exploited to treat many kinds of malignancies, currently its success rate in gbm is limited. therefore, the gbm treatment paradigm needs shifting towards more effective treatments such as immune cell therapy. natural killer (nk) cells have been recognized as potential anti-cancer effector cells, as they can recognize and target tumor cells. since a small percentage of blood cells are differentiated as nk cells, the number of this group of cells is hardly enough to fight tumors, and so their multiplication and activation would be a potential effective cancer treatment. methods: this preclinical study was focused on setting up an optimal protocol for expansion and activation of naïve nk cells and assessing their efficacy towards induced gbm in rat models. ex-vivo expanded and interlukin-2 (il-2)and heat shock protein-70 (hsp-70)-treated nk cells have been exploited. after in vitro study and confirming the efficacy of treated cells through cytotoxicity assays, we induced gbm in 6 male wistar rats (weighted 275-300 gr) using c6 tumor cells injection in rat brain through stereotactic surgery. the tumor formation was proven by mri imaging. following tumor establishment, we analyzed the effect of single injection of il-2-and hsp-70-treated nk cells compared with single injection of non-treated nk cells in two groups of rats. results: systemic intravenous delivery of il-2-and hsp-70-treated nk cells through tail's vein resulted in tumor shrinkage in different time intervals and complete remission in the first group of gbm-induced rat models, whereas in the other group of gbm rats receiving untreated nk cells, the tumor progressed. therapeutic efficacy of the treated nk cells was ascertained compared with non-treated nk cells considering tumor shrinkage observed in mri and survival rates between the two model groups. conclusions: the amelioration of tumor which has been confirmed by mri, proved the migration of activated nk cells through blood brain barrier and homing to cns, and finally targeting gbm tumor cells. our data suggest that nk-cells treated with il2/hsp70 may be a promising immune cell-based therapeutic approach towards treating the fatal gbm. disclosure: nothing to declare p592 abstract withdrawn. long term sorafenib response for extramedullary flt3 + aml relapse after allogeneic stem cell transplantation since june 2017, sorafenib dose has been tapered to 200 mg/day, due to mild skin and gi toxicity. after 3 years of treatment, she maintains cr at medullary and extramedulary levels, with no evidence of a disease that had escaped the mechanisms of action of chemo, hsct and dli. conclusions: in our patient, treatment with sorafenib has provided long-term control of this refractory extramedullary disease, even at adjusted doses. further studies are needed to confirm the efficacy of flt3 inhibitors in the control of relapses after allo-hsct, extramedullary disease and its potential role as maintenance agent. disclosure: nothing to declare background: although chemotherapeutic(ct) agents that used in the treatment of acute lymphoblastic leukemia (all) increase survival, the results are still weak. longterm survival with ct's in relapse all cases is difficult and the prognosis is very weak. inotuzumab ozogamicin is an anti-cd 22 monoclonal antibody and it has the potential to reduce the overall toxicity of intensive regimens for all, as well as to possibly increase the number of patients who may achieve a state of minimal residual disease. methods: 26-year-old male patient was diagnosed with b-cell all in december 2017.after the hoelzer kt protocol was started, maintenance treatment was continued. in the fifth month of treatment,flag ct protocol was started cause of recurrence was seen on 5% blast detection in peripheral blood smear. in august 2018, inotuzumab ozogamicin treatment was started and six cures were completed because the patient was not in remission. in september 2018, he had gone haploidentical bone marrow transplantation from his sibling donor(8/10)with defibrotid prophylaxis for veno-occlusive disease(vod)s. he engrafted succesfully and chimerizm was 99.85% in 30th days of transplantation. he is 60th day of transplantation and in a remission. results: bone marrow transplantation cannot be performed since the complete response cannot be achieved in patients with relapse and resistant b-all.in these patients, new therapies targeting malignant lymphoblasts are needed. inotuzumab ozogamicinis a monoclonal antibody drug conjugate that targets cd22 antigen on malignant lymphoblasts.in many studies, it has been shown that inotuzumab ozogamycin is effective and reliable anti-tumor activity in adults with recurrent and resistant cd22 positive all. however, monoclonal antibody drug conjugates have been shown to be associated with vod's.for this purpose, we used defibrotid to protect our patient from vod. conclusions: treatment with combination ct regimens in b-all is suboptimal and long-term survival is achieved in only 30-40% of patients.targeted molecular therapy and new regimens are needed in relapse and resistant patients.at this point, inotuzumab ozogamycin is an anti-cd-22 monoclonal antibody, as in our case, it provides remission in recurrent and resistant b-all patients and allows patients to complete their treatment with an allogeneic transplant from a fully compatible donor. disclosure: nothing to declare background: mesenchymal stem cells (mscs) are an attractive consideration for therapeutic cures of many difficult diseases on the cellular-level. due to the trophic effects of the cytokines and chemokines that they produce, mscs have shown multiple beneficial properties in the field of oncology. in this study, we will be investigating the effect of mscs derived from human bone marrow (bm), adipose tissue (at), and umbilical cord derived mscs (uc-mscs) on ovarian cancer. to differentiate the mscs, we performed a comparative analysis between the various sources for proliferative capacity, surface antigen expression, differentiation ability, tumor marker and paracrine activity, and their influence on ovarian cancer cell proliferation. methods: measurements of ovarian tumor marker proteins were computed by elisa. proliferative effects, immunomodulatory effects, and apoptosis of the mscs were measured by the culture and counting of colony formations. flow cytometry (fcm) was used to measure the variation of the immunophenotyping and cytokine secretions in co-culture, as well as gene expression. results: cells noticeably proliferated without any modifications to their immunophenotype during the third subculture. the colony-forming unit fibroblast (cfu-f) test showed a proliferation of the mscs along with healthy cells and cancer cell lines with no changes in their phenotype. the supernatant of mscs showed an increase in cellular death of the ovcar3 in ovarian cancer cell lines. a reduction in the level of ca-125 (75-90%; p=0.769) with ovcar3 in co-culture, and a decline of ldh (10-20%; p=0.03) and beta-hcg (10-20%; p=0.04) were observed in co-culture in caov3, skov3 and igrov3 cell lines. a decrease in cd24 of the cancer cell lines in co-culture with the msc supernatant showed a reduction of the cancer tumorigenicity and aggressiveness, while the rate of the cd24 and cd44 asserted their stem state. msc supernatant decreased cell proliferation and mmp-2, mmp-9, and ca-125 mrna expression, while increasing timp 1, 2, and 3. this suggests that mscs have a role in cell death and inhibition of ovarian cancer cell proliferation. an increase of anti-inflammatory il-4 and il-10 cytokines, and a decrease in growth factor gm-csf along with their proinflammatory inf-a, tnf-a, il-9, and il-17a cytokines were also noted. conclusions: the gene and cytokine activity indicate a potential therapeutic anti-inflammatory and antiproliferative role of mscs on ovarian cancer despite their sources. the reduction of ca-125, ldh, and beta-hcg in co-culture, along with the decrease in cd24 and amplified cellular apoptosis demonstrate the beneficial effects of stem cells in ovarian cancer cell lines. disclosure background: hematopoietic stem cell transplant (hsct) is the only cure in sickle cell disease (scd) so far. because of the risk of toxicity, its indication in france is restricted to severe patients with match sibling donor. this study compares the incidence of severe acute toxicity after hsct, between children aged less than 13 years and teenagers aged 13 to 18 years old, treated for scd. methods: all patients suffering from scd, aged less than 18 years at transplant, who received hsct in chu robert debré and necker, between 01/01/2005 and 12/31/2017, were included. severe acute toxicity, defined by onset of severe acute gvhd, organ toxicity or infection, was compared between the two groups of age. results: 73 patients (59 children and 14 teenagers) were included. all patients received a myeloablative conditioning regimen. bone marrow from a sibling donor was the main stem cell source (n=65; 89%). neither death nor rejection was observed with a median follow-up of 29.6 months (range, . the incidence of grade iii-iv acute gvhd was 12.3% and was similar between the two groups; no risk factor was identified in univariate analysis. teenagers had more frequently acute skin toxicity (21.4% vs 0%, p=0.006). in univariate analysis, patients presenting severe organ toxicity were significantly older than others (9.3 vs 7.5 years old, p=0.027). teenagers were more frequently treated for bacterial (64.3% vs 32.2%, p=0.035) or bk virus (42.9% vs 6.8%, p=0.002) infections. in univariate analysis, patients who developed infection were also significantly older at time of transplant (respectively 9.7 vs 7.5 years old, p=0.016). no severe sinusoidal obstruction syndrome was observed. regarding long-term toxicity, 2 patients presented an extensive chronic gvhd, they were both aged less than 13 years old. no cut-off of age could have been defined. conclusions: this study confirms the excellent results of hsct in scd, with a 5-year event-free survival and overall survival of 100%. an older age at transplant seems to be associated with more frequent severe acute toxicity. these results are consistent with previous studies and suggest that hsct should be performed as soon as possible, without any defined "best age". prospective studies are needed, in order to define the place of each therapeutic in scd, with the aim of reducing treatment-related toxicity and developing alternative strategies for patients without match sibling donor. disclosure: nothing to declare p599 new insights into risk factors for transplant-associated thrombotic microangiopathy (ta-tma) in paediatric hsct (n=170) was associated with ta-tma in 1% vs 5.8% vs 7.7% respectively (p=0.067). the presence of comorbidities at d0 (n=86) was significantly associated with an increased risk of ta-tma 10.4% vs3.7% in absence of co-morbidity (n=320); p=0.032. use of csa/tac-based gvhd prophylaxis was associated with less ta-tma with an incidence of 4% vs 13% if these agents were not included (p=0.01). in univariate analysis ta-tma was significantly higher among patients with agvhd grade ii-iv (12/138; 8.6%) vs grade 0-i (6/227;2.6%) (p=0.01). pres was recorded among 10 cases and 50% of them developed ta-tma. two out of the 21 patients with ta-tma had pathological gene mutations in their complement pathway. on multivariate analysis the presence of active comorbidity was a risk factor for ta-tma (or:6.6; 95% ci:1.1-37.6; p=0.032) while the use of csa/tac-based gvhd prophylaxis did not increase the risk for ta-tma (or:0.04; ci:0.004-0.39; p=0.005). in the presence of comorbidities the use of defibrotide as prophylaxis or therapy for vod (n=86) was associated with a drop in the incidence of ta-tma from 12% (9/79) in absence of defibrotide to 0% (0/7). 2-year overall survival was significantly lower among ta-tma cases (58%) in comparison to 81.5% in absence of ta-tma (p=0.001) (figure 1). conclusions: active co-morbidity is a significant risk factor for ta-tma. use of defibrotide prophylaxis in patients with co-morbidities at the time of hsct might offer protection against ta-tma. surprisingly the use of csa/tac based gvhd prophylaxis is not a risk factor for ta-tma probably through limiting the development of high grades agvhd. the association between pres and ta-tma suggests a common pathway of endothelial damage background: gonadal impairment is a severe late effect of myeloablative conditioning regimes with significant impact on quality of life of cancer survivors. the aim of this study was to analyze gonadal function after busulfan (bu) or treosulfan (treo) containing regimens with regard to pubertal stage. methods: this was a retrospective, multicenter study involving patients treated in pediatric ebmt centers between 1992-2012. patients receiving myeloablative doses of bu or treo as part of hsct conditioning were eligible for inclusion. analysis was conducted in two groups according to pubertal status at time of hsct. results: 137 patients (pts) were treated in 25 pediatric ebmt with bu or treo before allogeneic hsct. the median age at transplant was 11.04 years (range 5-18); 89/ 137 (65%) were males (m), 48/137 (35%) were females (f). 89/137 (65%) pts were pre-pubertal at hsct (f= 27;m=62) and 48/137(35%) were post pubertal (f=21;m=27). 118/ 137 (86%) patients received bu (f=38;m=80),77/118 (65%) were pre-pubertal. 19/137 (14%)(f=10;m=9) received treo,12/19 (64%) were pre-pubertal ( figure 1 ). females who received treo in pre-pubertal stage (n=6/6) reached more often spontaneous puberty (100% vs 38%; p=0.016) compared to pre-pubertal bu group (n=8/21) and occurrence of menarche was higher in treo group (p< .01) hormonal replacement therapy was given in 13/27 (48%) females transplanted in pre-pubertal stage and in 15/21 (71%) of those transplanted in post-pubertal stage. 77/89 (86%) males were pubertal at last follow-up and 8 of them (10%) performed sperm analysis (5 oligo-azoospermic,3 unknown). three pregnancies were reported in the population group, all received bu. regarding the evaluation of hormonal levels in pubertal patients at time of hormonal dosage (median 19.54 yrs) (66 bu and 15 treo), males treated with treo had significant lower lh levels (p = 0.045) compared to bu group. females treated with treo had significant lower levels of lh and fsh (p= 0.02 and p= 0.0035 respectively). conclusions: gonadal damage related to treo was significantly lower compared to bu. we observed that: females transplanted during pre-pubertal period had spontaneous puberty more frequently after treo compared to bu and that hypogonadism hypergonadotropic was more frequent after bu than treo. these results must be further confirmed on a larger population. background: viral infections significantly contribute to both morbidity and mortality in patients undergoing hematopoietic stem cell transplantation. traditional antiviral therapy is associated with lack of efficacy, potential toxicity, prolonged hospitalization and increased patient costs. viral specific t cells can be manufactured from donor blood to treat viral infections post-transplant, and are associated with increased clinical efficacy and low toxicity. we postulated that direct costs of vst therapy are lower compared to traditional anti-viral medications methods: vsts were manufactured according to local protocols and fda requirements. total drug cost (as per institutional charges per drug) was calculated for patients who required treatment for viral infections post-hsct. manufacturing costs of vsts are fixed per fda requirements. patients who were treated with investigational antiviral medications (including brincidofovir) were excluded from analysis. patients treated with vsts +/anti-viral medications over a 2 year period were compared to patients treated only using traditional anti-viral medications, including cidofovir, ganciclovir, valganciclovir, foscarnet and rituximab. results: demographics are shown in table 1 . there were no major differences between the two groups treated. the number of anti-viral medications used in the vst group was lower compared to the anti-viral treatment group. median cost of vst treatment was significantly lower compared to those threated with traditional anti-viral therapy ($12,757 vs $16,392, p-value=0.03) . conclusions: treatment with vsts post-hsct for viral infections was lower in cost compared to anti-viral medical therapy. it is likely that overall costs are further reduced with vsts due to reduced inpatient hospital time, less monitoring of labs associated with anti-viral medication side-effects and reduced ancillary costs including nursing and pharmacy. more studies are needed to examine these indirect costs further. background: dock8 deficiency is an autosomal recessive primary immunodeficiency (pid) disease caused by loss-offunction mutations in the dock8 gene (1) . patients with dock8 deficiency present with multiple abnormalities of the immune system, including defective t cell function and impaired production of antigen-specific antibodies. these lead to persistent viral infections of the skin, mucocutaneous candidiasis, recurrent sinopulmonary infections, atopic dermatitis, and other allergic disease, malignancies, and sometimes autoimmunity (2). hematopoietic stem cell transplantation (hsct) is currently the only curative treatment option available (3) . methods: we retrospectively evaluated our patients who underwent allogeneic hematopoietic stem cell transplantation due to dock8 deficiency in ege university pediatric stem cell transplantation unit between 2013 and 2018. results: we identified 16 patients transplanted at a median age of 7.8 years (range: 2-15.5 years). of 16 patients; 4 (%25) received hsct from matched sibling, 11 (%68) from unrelated donors and 1 patient from haploidentical donor. we used busulfan-based myeloablative conditioning regimen to 12 patients (%75), reduced toxicity myeloablative regimen with treosulfan to 2 patients (%12.5) and nonmyeloablative regimen to 2 patients (%12.5). eight of the recipients received bone marrow, 6 of the patients received peripheral blood stem cells, 2 of the recipients received cord blood as stem cell source. fifteen of 16 patients (%93) had achieved engraftment and median follow-up of patients was 29 months (1-64). grade iii-iv acute graft versus host disease (gvhd) occurred in 13% of patients and chronic graft versus host disease was seen 18 % of patients. one patient received cord blood from unrelated donor did not engraft and died from septic shock. four patients died from transplant related toxicity. our patient's survival was %68 ; 11/16 patients alive. conclusions: hsct is the only curative treatment option for dock8 deficiency. in particular, patients with high comorbidity scores have a high risk of toxicity and toxic death. therefore, reduced toxicity conditioning regimens should be used for these patients. references : background: eltrombopag, a low-molecular-weight synthetic nonpeptide thrombopoietin receptor agonist (tpo-r), is a second-generation tpo. it is an oral thrombopoietin mimetic licensed in chronic immune thrombocytopenic purpura that induces platelet maturation and release by binding to c-mpl receptors on megakaryocytes. in a recent study; for patients with refractory saa, eltrombopag induced a response (at 12 weeks) in at least one hematologic lineage in 44% patients and 36% no longer required platelet transfusions. and also 24% patients became rbc transfusion independent and 36% had a neutrophil response. trilineage responses were seen in 24% of patients; although surprising, this might indicate stimulation of c-mpl receptors on remaining stem cells. delayed recovery from thrombocytopenia is common after stem cell transplantation. in a study including 12 adult patients, eltrombopag was used to enhance platelet recovery for post-hsct thrombocytopenia. it is well tolerated and efficacious offering transfusion independence. methods: in our retrospective study, eltrombopag (50mg/day) was started in 19 pediatric patients (age ranging from 4 to 16 years with a median age 12.3 years) for posthematopoietic stem cell transplantation (hsct) thrombocytopenia. all patients fulfilled the following 3 criteria: (1) undergone hematopoietic stem cell transplantation (hsct), (2) had improved total leucocyte counts after leucocyte engraftment, (3) had prolonged thrombocytopenia (< 30.000) needing platelet transfusion. results: four of the patients have received ric while 15 patients ma conditioning regimens before hsct. two haploidentic, 2 autologous, 7 mud, 8 msd transplantations were performed. et (50mg/day) was started in 19 patients who had thrombocytopenia despite neutrophil engraftment on the +30th day of hsct a reduction in platelet transfusions and a platelet count of more than 30,000 were the primary endpoints. before et treatment, bone marrow biopsy was checked in 15/19 patients, 10/15 patients had decreased number of megacocyocytes. none of the patients had active bleeding at the start of eltrombopag but they were all at high risk of bleeding. according to the platelet monitoring, 12 patients had a dose increase starting from the second week. the number of patients in need of platelet transfusions was 7 at the end of the first month; and only 2 at the end of the 2nd month. all patients had a thrombocyte count of more than 30.000 in the third month. in 11 patients, et was discontinued after 2-11 months. no dose limiting toxicities have been observed. conclusions: as a conclusion, et was found highly effective for posthsct thrombocytopenia, with no drug related adverse effects. there was a gradual increase in platelet count, and none of the patients had any complication due to thrombocytopenia. disclosure background: isavuconazole (isa) is a new triazole approved for ifi treatment in the adult population. advantages are activity against both moulds and yeasts spp, excellent bioavailability after oral administration without relevant food or gastric ph effect, a water-soluble prodrug developed to facilitate intravenous administration without nephrotoxic excipients such as β-cyclodextrin, potentially poor drug-drug interactions. isa does not currently appear to require tdm. isa safety and efficacy have not been yet established in children and, in particular, no data are available in the pediatric hsct setting. methods: italian association pediatric hematology oncology (aieop) multicentric analysis of a cohort of allogeneic hsct pediatric patients who received isa as ifi treatment or prophylaxis. due to the lack of recommended dosing in pediatric patients and a clear target isa plasma trough-level range, the therapeutic monitoring (tdm) of isa concentrations was applied by a validated liquid chromatography-tandem mass spectrometry (hlpc-ms/ ms) assay technique. isa trough plasma concentrations (c0) and 3 hours after drug intake (c3h) were measured. results: a total of 16 allo-hsct recipients were included, (m/f 12/4); median age: 13,1 years, range 5-19, median body-weight 48,7 kg (range 17-80). isa was used as ifi treatment in 14 cases and as prophylaxis in 2 patients. donors were haploidentical in 9 patients, matched-sibling in 4, allogenic-unrelated in 4 cases. according to eortc criteria, ifi was proven in 3 patients (1 penicilum, 1 mucor, 1 aspergillus fumigatus), probable in 5 and possible in 7 patients. lungs were the main localization (12 cases), associated with cns involvement in 4 cases and paranasal sinuses in 3; 1 patient had possible hepatic candidiasis. all patients, but one, received isa as rescue treatment for previous therapeutic failure (6 ambisome, 3 voriconazole, 1 combination therapy, 1 posaconazole). seven patients received only iv isa, 3 received only oral isa whereas 6 patients received both iv and oral isa. patients under 20 kg body weight received half isa dose (100 mg tid loading dose on days 1 and 2, 100 mg/die manteinance). the others received adult schedule; only 5 patients received loading dose. isa was administered for a median of 83 days (range: 24-240). in 3 patients isa was administered in combination with caspofungin. tdm was applied to 5 patients including 1 patient with severe vod and 1 with renal failure secondary to ta-tma. the median isa concentrations were 3.94 (1.57-5.85) mg/l and 4.7 (1.29-8.37 ) mg/l for c0 and c3h, respectively. ifi complete remission was achieved in 5 cases, partial remission in 3; treatment failure was experienced by 2 patients. in 3 cases fungal lesions remained stable. ctae grade ii-iii toxicity was observed during treatment in 3 patients, with increased transaminase and/or creatinine levels which resolved after temporary isa withdrawal. no drug-drug interactions were observed in 3 patients receiving csa as gvhd prophylaxis and no modification of csa daily dose was needed. conclusions: isavuconazole use may be considered in the pediatric population, even in the hsct setting, for its safety, efficacy, tolerability, no drug-drug interaction. of course these data deserve further evaluation. disclosure: nothing to declare p608 new treosulfan-based conditioning regimens including epigenetic agents in patients with very high-risk neuroblastoma background: the pts aged 18 mos. or older with disseminated nb involving bone and bone marrow constitute a group of pts with very poor prognosis. although the majority of them are responsive to intensive conventional chemotherapy, most eventually relapse with efs at 5 years of < 10%. at the beginning of the year 2018 we came up with a protocol for this very unfavorable group including epigenetic therapy (5-azacitidine) in the phases of consolidation. methods: seven pts with a median age of 4 (2,6-8) years completed the protocol and received hdct with autologous sct as a consolidation. hdct included two different epigenetic agent containing regimes according to tumor response to the induction therapy assessed by 123 i-mibg and mri (ct-scan). three pts revealing large active residual tumor assessed by 123 i-mibg scan or multiple active bone metastases received a conditioning regimen (regimen a) including 131 i-mibg therapy at a dose 4.0-4.4 mbe/kg on d-14, treosulfan 10000 mg/m 2 /d, on d-6,-5 and -4 (total 30000 mg/m 2 ), melphalan 70 mg/m 2 /d, on d-3,-2 (total 140 mg/m 2 ), 5-azacitidine 75 mg/m 2 /d on d-9 to d-5 (total 375 mg/m 2 ). four pts with cr or vgpr received a «split» conditioning regimen (regimen b) including treosulfan 10000 mg/m 2 /d, on d 11 and -10, and on d-4 and d-3 (total 40000 mg/m 2 ), melphalan 70 mg/m 2 /d, on d-3 and -2 (total 140 mg/m 2 ), and 5-azacitidine 75 mg/m 2 /d, on d -14 to d -12 and on d-9 to -d-5 (total 600 mg/m 2 ). a median number of 5.3 (3.7-7) cd34+/kg was infused on d0. results: the median recovery times to wbc>1.0x10 9 /l and to an unsupported plt>20x10 11 /l were 10 (7-12) and 13 (9-17) days, respectively. all pts experienced grade 4 hematological as well as infectious toxicity assessed by nci-ctc score. there were 7 episodes of severe organ toxicity of grade 3 occurring in 4 pts. in 4 cases we observed a severe mucositis, in 2 cases gi toxicity and 1 episode of the erythema multiforme occurred. one pt revealed a lifethreatening episode of hypotension of grade 4. no transplant-related death occurred. the median number of transfused rbc and plt doses was 3 (1-4) and 6 (3-8), respectively. all pts are alive and well without signs of disease progression in complete hematological recovery with a limited follow-up of 5.3 (4-7) mos. from day 0 of hdct. conclusions: although it is rather early to evaluate the efficacy of the epigenetic agent's inclusion in the induction and/or consolidation phases of a very high-risk nb treatment, we can assume that, first, the hdct combining mibg i 131 and/or high dose of treosulfan with epigenetic agent such a 5-azacitidine was feasible and had an acceptable toxicity. second, the "split" modality of the treosulfan use in conditioning regimen would permit to increase the total dose of the alkylating agent with no inacceptable toxicity. disclosure: nothing to declare pre-and-post magnetic resonance imaging of hips and knees for detecting osteonecrosis in children undergoing hematopoietic cell transplantation: in whom is it necessary? ali y suliman 1 , sue c kaste 2 , ying li 1 , dinesh keerthi 1 , guolian kang 1 , brandon m triplett 1 , ashok srinivasan 1 between april 2016 and august 2017 at the university medical center hamburg-eppendorf, germany, were included. total and active ratg plasma levels were analyzed by elisa and flow cytometry, respectively. primary endpoint of the study was exposure to ratg. secondary endpoints included transplant-related mortality, incidence of acute and chronic gvhd, immune reconstitution, chimerism, rejection and viral infections. patients were monitored at least 100 days post transplantation. statistical analyses were performed using ibm spss statistics 24 software, or graphpad prism 5 software. results: median total grafalon™ and thymoglobuline™ peak plasma levels were 419.0 μg/ml and 60.4 μg/ml, respectively; median active grafalon™ and thymoglobu-line™ peak plasma levels (appl) were 77.9 μg/ml and 8.11 μg/ml, respectively. active thymoglobuline™ plasma levels showed highly variable pharmacokinetics compared to grafalon™. neither grafalon™ nor thymoglobuline™ exposure correlated with lymphocyte count prior to transplantation, cell count in the graft (wbc, mnc, t cells), age, body weight or body surface area (bsa). this is indicative for a saturation effect in both groups. to correlate high or low ratg exposure with clinical outcome parameters, we built two groups within each patient cohort by median appl. the incidence of gvhd was not dependent on high or low ratg exposure. until day 100 post hsct, 49 viral infections or reactivations (ebv, cmv, adv, hhv6, bkv) occurred in the 32 patients. interestingly, adv infections affected only children with high ratg exposure. the median time to leukocyte engraftment was not significantly longer in the high ratg groups compared to the low ratg groups (17 to 16 days for grafalon™, and 14 to 16 days for thymoglobuline™). there was a decreased and/or delayed recovery of cd3 + , cd4 + and cd8 + t cell reconstitution, but not of b cells and nk cells in the high thymoglobuline™ exposure group compared to the low thymoglobuline™ exposure group. overall survival was not statistically significant with 80% in the grafalon™ and 95.5% in the thymoglobuline™ group without influence of ratg exposure. conclusions: high and low exposure to grafalon™ or thymoglobuline™ did not result in significant differences in outcome parameters as incidence of survival, agvhd, cgvhd, rejection, or mixed chimerism in this limited cohort. delayed and decreased immune reconstitution in the high ratg exposure groups did not translate into different clinical outcome parameters. adv infections only occurred in the high ratg exposure group. grafalon™ and thymoglobuline™ showed distinct pharmacological and immunological differences in children and larger cohorts are needed to detect clinically significant differences and adjust dosing regimens individually. disclosure: nothing to declare background: the optimal conditioning regimen for allogeneic hematopoietic cell transplantation (allohct) in children with myeloid malignancies remains undefined, particularly when reduced-intensity conditioning (ric) regimens are utilized. methods: we performed a retrospective review of children undergoing allohct for acute myeloid leukemia (aml) and myelodysplasia-related aml (mdr-aml) over a 10 year period (2008-2018) at our institution, comparing the outcomes of those who received either a busulfan (bu)-or melphalan/thiotepa (mel/thio)-based conditioning regimen. results: a total of 49 patients were analyzed. twentyone received fludarabine/melphalan/thiotepa and 28 received myeloablative busulfan-based conditioning, either in combination with cyclophosphamide (n=19) or fludarabine (n=9). atg was used in 27 patients depending on donor. recipients of mel/thio were selected for ric regimens due to pre-transplant comorbidities (cardiac dysfunction, n=9, 3 requiring peri-transplant milrinone), transplant during chemotherapy-induced aplasia (n=5), underlying diagnosis of treatment-related aml (t-aml) and significant pre-allohct chemotherapy exposure (n=5). proportions of patients with de novo aml (mel/ thio, 57%; bu, 78%) and mdr-aml (19% and 21%) were similar between groups; however, recipients of mel/thio were more likely to have t-aml (24% vs 0%). cytogenetic and molecular risk factors were similar between groups. the majority of patients were transplanted in cr1 (mel/thio, 71% vs bu, 68%) or cr2 (24% vs 38%). more recipients of bu conditioning (71% vs 43%) were mrd-negative at the time of allohct; both groups had comparable proportions of patients with ≥m2 marrow (~10%). donor types and stem cell sources were similar between groups, except unrelated umbilical cord blood which was more common in bu recipients (21% vs 10%). there were no graft failures in mel/thio recipients, compared to 14% (n=4) in those receiving bu-based regimens. engraftment kinetics and immune reconstitution were similar. overall acute and chronic gvhd incidence was higher in recipients of mel/ thio compared to bu (39% vs 21%, and 29% vs 18%, respectively), but rates of grade iii-iv acute or extensive chronic gvhd were comparable. vod requiring treatment was diagnosed in 6 (21%) recipients of bu conditioning and no mel/thio recipients. median duration of follow-up was 30 months (range 2-70) in the mel/thio group, and 18 months (range 1-121) in the bu group. transplantrelated mortality (trm) was similar in both groups (1 patient), occurring before day 100. relapse incidence was comparable (mel/thio, 29% vs bu, 32%); however, relapse occurred at a later time in mel/thio recipients (median d +396 vs d+137). overall survival at 1 and 3 years was superior in mel/thio recipients (95% vs 74%, and 75% vs 50%, respectively). conclusions: in our single institution experience, use of a melphalan/thiotepa-based ric regimen was associated with similar outcomes compared to full-intensity bu-based conditioning, despite higher risk patient and disease characteristics. the majority of recipients of mel/thio conditioning had significant pre-transplant comorbidities, which did not translate into higher trm. while mel/thio recipients had less optimal leukemia control at the time of transplant and high-risk leukemia features (e.g. t-aml), relapse was similar between groups, occurring later in mel/ thio recipients, which may have contributed to better overall survival. disclosure: nothing to disclose methods: pubertal development and biological gonadal parameters were assessed in a retrospective monocentric cohort of pre-pubertal patients who underwent hsct after myeloablative conditioning with total body irradiation (tbi) or busulfan between 1981 and 2017. results: seventy-four patients (28 girls and 46 boys) were included. no spontaneous pubertal development was found in 50% of girls and 10% of boys (p < 0.001) and delayed puberty or no spontaneous pubertal development was found in 57% of girls and 24% of boys (p=0.009). hormone replacement therapy was used in 82% of girls and 24% of boys (p < 0.001). in univariate analysis, tbi conditioning (p=0.05), female sex (p < 0.001), acute gvhd (p=0.05), extensive chronic gvhd (p=0.021), steroid treatment >6 months (p=0.016), and malignant diseases (p=0.016) were associated with no spontaneous pubertal development, whereas tbi conditioning (p=0.003) and extensive chronic gvhd (p=0.005) were associated with delayed puberty. in multivariate analysis, factors independently associated with no spontaneous puberty onset were female sex (p=0.001) and age >10 years (p=0.033). factors independently associated with delayed puberty were extensive chronic gvhd (p=0.041) and age >10 years (p=0.031). tbi was not an independent risk factor for pubertal complications. conclusions: this study confirms the toxicity of myeloablative conditioning on pubertal development and the role of older age and female sex in increased pubertal issues, and suggests a possible role of gvhd in delayed puberty. disclosure: nothing to declare p613 abstract already published. neutrophil elastase activity may serve as a marker for neutrophil extracellular traps formation following stem cell transplantation ronit elhasid 1 , sivan berger-achituv 1 , hila rosenfeld-keidar 1 , szilvia baron 1 1 tel aviv sourasky medical center -tel aviv university, tel aviv, israel background: post-transplant infections rise dramatically in patients with quantitative or qualitative neutrophil defects and constitute a major source of morbidity and mortality following hematopoietic stem cell transplantation (hsct). neutrophils protect the host from microorganisms via multiple processes including phagocytosis and formation of neutrophil extracellular traps (nets). although reactive oxygen species (ros) production seems to be essential for nets formation, the key enzymes of the process are neutrophil elastase (ne) and myeloperoxidase (mpo). methods: ne and mpo activity as well as nets formation were investigated following hsct in 11 patients at week 1 to 6 and 30 after neutrophil engraftment. neutrophils were isolated using easysep direct human neutrophil isolation kit (stemcell technologies inc.) by immunomagnetic negative selection. enzymatic activity of ne and mpo were measured using colorimetric assays. nets formation of phorbol 12-myristate 13-acetate (pma)activated neutrophils was investigated by confocal fluorescence microscopy. all results were compared to those of healthy volunteers. statistical significance was calculated using one way-anova with bonferroni post hoc test. results: 11 patients (median age of 6.8 years [range 2-22 years]) were investigated, 6 following allogeneic hsct (2 acute lymphoblastic leukemia, 1 acute myeloblastic leukemia, 2 epidermolysis bullosa, 1 rhabdomyosarcoma) and 5 following autologous hsct (4 ewing sarcoma, 1 desmoplastic small round cell tumor). all patients experienced fever and neutropenia. at engraftment, average ne activity was significantly decreased compared to the average value of 50 healthy individuals. ne activity improved week by week in patients, reached the lower reference range at 4 weeks following transplantation (fig. 1a) and continued to increase. the enzymatic activity of mpo was comparable to the average value of 50 healthy individuals (fig. 1b) and showed no significant difference between the distinct time points. at neutrophil engraftment, nets formation was absent and comparable to those of non-activated neutrophils (fig. 1c) . although nets formation increased week by week, it did not reach the average of 7 normal controls during the monitored time period. also linear correlation between ne activity and nets formation (r 2 =0.978) was demonstrated. conclusions: impaired ne activity following hsct corresponds to decreased nets formation and could serve as a marker for netosis. strategies to accelerate the recovery of ne function post transplantation might improve nets formation and thereby induce better infection control. a) the average of ne activity (n=11) during 30 weeks following hsct. reference range was measured and calculated from measurements of 50 healthy volunteers using. b) the average of mpo activity (n=11) during 30 weeks following hsct. reference ranges were measured and calculated from measurements of 50 healthy volunteers using the quartile method. c) the average of netosis activity after 100 nm pma activation for 3h (n=11 background: to have a better understanding of incidence, treatment, outcome and risk factors of immune cytopenia in children after allogeneic hsct. methods: between january 2010 and september 2018, 105 pediatric allogeneic hsct have been performed in 99 patients at the ghent university hospital (ghent, belgium). autoimmune hemolytic anemia was defined by a positive direct agglutinin test (dat). dat was performed at moment of engraftment and in case of hemolysis or unexplained anemia. platelets antibodies were evaluated in case of no otherwise explained thrombocytopenia. results: the cumulative incidence of post allo sct autoimmune cytopenia is 9.5% (10/105). in 9 cases there were positive antibodies against red blood cells, and one patient against had antibodies against platelets. of these 10 cases, only 4 (3.8%) were clinically relevant and needed treatment. the median observation period post sct for the whole cohort was 36 months (3 -105) . the clinically significant immune cytopenia started at a median time of day+158 and day +113 in the group without symptoms. the patient who presented the autoimmune thrombopenia developed antibodies against anti-gpiib/iiia, this was resolved after 130 days, the treatment consisted intravenous immunoglobulins (ivig). two of the 3 patients with autoimmune hemolytic anemia had igg mediated antibodies, and 1 had complementmediated dat. these 3 patients were treated with ivig, steroids, rapamune and rituximab. one patient has still dat positive after 36 months, but clinical stable. the other two are also dat positive and have some hemolysis, but the follow up is much shorter (2 months). treosulfan-contained conditioning regimens were more frequently used in patients with significant immune cytopenia. conclusions: immune cytopenia is an infrequent complication after allogeneic hsct. however, its treatment can be challenging, and the hemolysis can persist for years. the association of rapamune and rituximab was adequate to treat this problem in our patients. background: approaches to the management of refractory and relapsed classical hodgkin´s lymphoma (r-r chl) are changing and become more effective. the role of anti-cd30 targeted immunochemotherapy with brentuximab vedotin (bv) has been extensively investigated in adults with r-r chl and is only to be elucidated in children. the study included 14 children and adolescents with r-r chl that were sucessfully treated with bv-based therapy prior to hematopoetic stem cell transplantation (hsct). median age of patients was 16 years (9-19), main histological variant -nodular sclerosis (93%, n=13), advanced stage at diagnosis -86% (n=12). most were heavily pre-treated (median number of previous therapies -4) and progression after autologous hsct was documented in 4 (29%). refractory disease was diagnosed in 8 (57%) and relapsed in 6 (43%). among relapsed patients 4 (67%) were with multiple episodes, 1 (16.5%) -early and 1 (16.5%) -late relapse. treatment regimens consisted of bv in monotherapy 1.8 mg/kg triweekly (n=7) or combination of bv 1.8 mg/kg on day 1 with bendamustine 90-120 mg/ m2 on days 1 and 2 of 3-week cycles (n=5) or combination of bv 1.8 mg/kg on day 1 with dhap (n=2). median number of bv infusions was 3.5 (2-7). all selected patients achieved complete (n=5, 36%) or partial remission (n=9, 64%) prior to hsct. consolidation with autologous hsct was performed in 9 (64%) and with allogeneic hsct -in 5 (36%). primary end points were overall (os) and progression free survival (pfs). response to bv was not assessed in the study as only responders to the bv-based treatment were included. results: with median follow-up of 497 days (105-1368) os and pfs for all patients are 68% and 66%, respectively. pfs after autologous hsct and allogeneic hsct are 70% and 60%, respectively (p=0.7) at present moment 10 (71%) patients are alive and are in remission. three patients died (21%): disease progression (n=1), postransplant idiopathic pneumonia syndrome (n=1) and posttransplant pneumonia (n=1). bv was generally well tolerated with only mild polyneuropathy in 3 patients (21%) as the main reversable documented adverse event. conclusions: in prognostically unfavourable heavily pretreated children and adolescents with r-r chl achievement of response to bv-based therapy prior to hsct is assosiated with promising rates of os and pfs. disclosure provides a treatment by restoring thymidine phosphorylase function and improving disease manifestations. here we report the outcomes of 4 affected siblings who underwent transplantation using an unaffected sibling donor to highlight important experiences in the transplant of such a rare condition. methods: four siblings of consanguineous pakistani descent aged 16, 15, 13 and 5 years underwent myeloablative hsct using fully hla-matched (10/10) peripheral blood stem cells harvested in a single apheresis from an unaffected 9 year old sibling. the oldest sibling, a 16 year old male, first presented in 2016 having emigrated from pakistan with a history of growth failure and abnormal movements. biochemical, nerve conduction and imaging studies confirmed a diagnosis of mngie. testing on three other siblings identified similar biochemical abnormalities, though the 2 youngest children had minimal clinical manifestations of the disease. based on the progressive nature of the disease and the availability of a fully matched donor, a decision was made to pursue transplant for all affected siblings. results: due to the severity of their disease, the 2 oldest siblings were transplanted first using a myeloablative conditioning regime of fludarabine, thiotepa and treosulfan with alemtuzumab. neutrophil engraftment occurred on day + 11 for both, with 100% donor chimerism achieved. there were no significant transplant related complications. the post-transplant course of the 15 year old sibling was complicated by a major stroke-like event characterised by dramatic imaging changes and requiring ventilation, though no cause was identified and the patient's neurologic deficits have since resolved. gastrointestinal symptoms have persisted and both remain tpn dependent, though symptomatically have shown gradual improvement. following the neurologic complications in their older sibling, the 2 younger siblings were conditioned with auc-targeted busulfan and fludarabine plus alemtuzumab. neutrophil engraftment occurred on day + 11, with full donor chimerism achieved. progression to enteral feeding has been much more rapid, with nutrition now fully enteral for both. there were no significant transplant related complications. conclusions: stem cell transplantation represents the only curative option for mngie. due to its rarity and relative infancy as a condition, little is known of the expected course following transplant or the best approach to transplantation itself. despite previous challenges with graft failure in mngie recipients, we were able to gain rapid and sustained donor engraftment using 2 different myeloablative conditioning regimes with minimal transplant-related morbidity and no mortality. in keeping with previous reports, resolution of established gastrointestinal symptoms has been slow, though the 2 siblings transplanted earlier in their disease course have shown more rapid improvement supporting the role of early recognition and access to transplant. it is essential moving forward that specialised transplantation centres collaborate so as to guide clinicians in the management of such a challenging condition. disclosure: there are no conflicts of interest to disclose. g6pc3 congenital neutropenia -biology of inflammatory colitis associated with gcsf use, and disease response to allogeneic transplant, a report of 4 cases background: an autosomal recessive disease, glucose-6phosphatase catalytic subunit 3 (g6pc3) deficiency is a relatively recently identified cause of chronic severe neutropenia. there can be a spectrum to the disease and patients may also present with non-haematological features including prominent chest veins, cardiac, endocrine or urogenital abnormalities. we describe in our patient cohort a response to gcsf but an inflammatory, incapacitating, biopsyproven colitis associated with that g-csf response. we have transplanted 3 children with such colitis, and describe a similar colitis with intestinal failure in a fourth. methods: we investigate the biology of the neutropenic colitis, and demonstrate necrosis of the stimulated neutrophils. in vitro studies demonstrated that unstimulated neutrophils from patients with g6pc3d exhibited significantly increased production of il8, reactive oxygen species (ros) and neutrophil extracellular traps (nets) alongside significantly higher expression of cd11b, cd66b and cd14. in contrast, neutrophils from patients with g6pc3d produced significantly less ros, mmp-9, neutrophil elastase and nets upon stimulation. neutrophils from patients with g6pc3d also exhibited significantly accelerated apoptosis and secondary necrosis which was exaggerated upon stimulation with live escherichia coli bacteria but could only be partially rescued with supplemental exogenous glucose. results: 3 patients have undergone hsct for g6pc3 neutropenic enterocolitis (1 unrelated donor and 2 msd) after fludarabine treosulfan and thiotepa conditioning therapy. alemtuzumab was given as as serotherapy. all 3 patients are alive and well, immune suppression has been discotniuned and there is no gvhd with normal organ function, and resolution of colitis. we describe a 4 th patient with no good donor who has continuing intestinal failure with g-csf use. conclusions: we describe the aetiology of intestinal inflammation and failure with an extensive study of neutrophil biology in this metabolic neutropenia. we describe a novel indication for hsct in this "g-csfresponsive neutropenia". disclosure: nothing to declare p620 does body mass index (bmi) pose a risk to outcome for pediatric non-infantile patients undergoing hematopoietic cell transplantation (hsct)? mona al-saleh 1 , khawar siddiqui 1 , amal al-seraihy 1 , abdullah al-jefri 1 , ali al-ahmari 1 , hawazen al-saedi 1 , awatif al-anazi 1 , mouhab ayas 1 , ibrahim al-ghemlas 1 with no evidence of toxicity. as benefits of stoss therapy in hsct remain unknown, and safety has yet to have been studied extensively in the pediatric population, we hypothesize that stoss therapy is an effective and safe method to reach and attain sufficient levels of vd in pediatric patients undergoing hsct. methods: this is an ongoing prospective, randomized clinical control trial at phoenix children's hospital that commenced december 1 st , 2017. following consent, subjects are randomized to the intervention (stoss) or control arm prior to hsct. stoss therapy consists of a single oral dose of vd (ranging 100,000iu -600,000iu), given based on baseline 25-hydroxyvitamin d [25(oh)d] level and age, followed by standard weekly supplementation. subjects enrolled on the control arm receive standard of care based on endocrine society guidelines of weekly vd supplementation. data collection includes demographics, 25(oh)d levels at baseline, day +30, and day +100, vd toxicity (hyperphosphatemia, hyperkalemia and renal calculi), as well comorbidities were collected. at each time point and for each trial arm, the mean 25(oh)d level and changes from baseline were computed with corresponding 95% confidence intervals (cis) to indicate variability. results: presently, 12 subjects have completed baseline assessment, with day +30 and day +100 follow-up completed for 11 and 9 of these, respectively. at baseline, the mean (95% ci) 25(oh)d was 19.8 ng/dl (10.9, 28.7) among stoss patients and 18.9 ng/dl (10. 3 results: total hrqol scores of transplanted patients were significantly improved compared to those on supportive care and also compared to healthy siblings (p < 0.0001 and 0.0002 respectively), the same was true for physical (p < 0.0001 and 0.0003 respectively) and emotional functioning (p < 0.0001 and 0.0073 respectively). social and school functioning of transplanted children were not different from healthy siblings (p 0.5893 and 0.7603 respectively) while were very significantly improved compared to children with st on supportive care (p < 0.0001 in both cases). conclusions: bmt in a lower-middle income setting may be even more impactful compared to high-income regions. our analysis clearly indicates normalization of hrqol in all major areas of children transplanted for st. a possible resilience effect was noted for physical and emotional scores which were improved compared to healthy sibling controls. we could not however quantify the effect of longer-term issues like fertility impairment after bmt which may eventually adversely impact hrqol, particularly in the indian culture. disclosure: none allogeneic hematopoietic stem cell transplantation in ataxia telangiectasia patients without malignancy background: ataxia telangiectasia (a-t) is a primary immunodeficiency with mutations in atm-gene. besides a slowly progressive neurodegenerative course, a-t leads to increased susceptibility to malignancies which affects 25% of patient (median:12.5 years) with a high mortality mainly due tocomplications of conventional radio-chemotherapy. the incidence of cancer correlates with the extent of immunodeficiency. patients often develop severe progressive granulomatous skin disease with evidence of vaccine-strain rubella-virus in the lesions. prolonged survival, neurologic improvement and malignancy prevention was observed in atm-deficient mice after treatment by syngeneic hsct. nevertheless, pre-emptive hsct is not routinely performed in a-t patients due to concerns about neurodegeneration and toxicity. methods: we present three a-t patients with severe immunodeficiency phenotype, undergoing successful hsct as an individual treatment strategy intending to restore immunodeficiency for long-term malignancyprevention (patient-1) and to treat progressive skin/joint granulomas (patients-2 and -3). results: patient-1 underwent a reduced intensity conditioning (ric) regimen at 4 years of age including fludarabine (150 mg/m 2 ), cyclophosphamide (80 mg/kg), and atg-fresenius (20 mg/kg/d) which was tolerated well. hematopoietic engraftment occurred by day +15. there was an expansion of naïve and memory cd4 + t-cells and cd19 + cells. while initially a mixed donor chimerism in patient's pbmcs (10-20% donor) was observed, patient's tcells (cd3 + ) reached over 90% of donor origin over time. at last follow-up (6 years) he is well, without signs of gvhd and organ toxicity, off immunosuppression with normal levels of atm-protein; his granulomas resolved. patient-2 is a 6 year-old male who was transplanted from his hla-identical sibling, conditioned with fludarabine (150 mg/m²), cyclophosphamide (120 mg/kg), and atg-fresenius (20 mg/kg). hematopoietic engraftment was observed by day +10. t-cell reconstitution started by day +40 with >200μl cd3+ t-cells. his mixed chimerism rapidly turned to donor origin (95% donor cd3+) over time. there was no acute toxicity, however, he developed lumbosacral pain episodes with evidence of urine bk-virus with spontaneous remission. an intermittent metapneumovirus associated pulmonary hypertension was observed with pericardial effusion. treatment included sildenafil and oxygen. at last follow-up (9 months) patient is well without immunosuppression. patient-3 suffered from recurrent chest infections, failure to thrive and progressive and debilitating rubella positive progressive granulomas of the skin. she received allohsct from an hla-identical family donor at 6 years of age. conditioning included busulfan (2.2 mg/kg), fludarabine (150 mg/m²), cyclophosphamide (40 mg/kg), and alemtuzumab (1 x 5mg/m², 3 x 10mg/m²). hsct was complicated by intermittent acute renal failure, cmv reactivation and tma. hematopoietic recovery was observed by day +22. t-cell chimerism increased rapidly over time (> 90% donor). at last follow-up (7 months) patient is well, off immunosuppression and ivig. her skin granuloma resolved with scarring residues. conclusions: pre-emptive allohsct is feasible in a-t when reduced intensity conditioning is used and can correct the immunodeficiency. it might be a treatment option for some a-t patients at high risk of hematological malignancy and severe granulomatous skin disease. to what extent the restored immune system and the increase of atm-protein in these patients could prevent the development of other malignancies needs to be evaluated further. disclosure: nothing to disclose p625 abstract withdrawn. hematopoietic stem cell transplantation in diamond blackfan anemia: brazilian experience background: diamond-blackfan anemia (dba) is a rare inherited red cell aplasia caused by an intrinsic defect of erythropoietic progenitors. the main therapeutic approach is based on repeated red blood cell transfusions and/or corticosteroid therapy. hematopoietic stem cell transplantation (hsct), a potentially curative treatment for dba, is indicated for patients that do not respond to first-line therapy. methods: the aim of our retrospective study is to report the outcomes of 30 brazilian dba patients transplanted between 1990 and 2017 in 9 bmt centers. the median age of the patients was 5 ys (range 1-15) and 60% were male. seventeen patients (57%) were transplanted with matched related donors (mrd) and thirteen (43%) from matched and mismatched unrelated donors (mud/mmud). in the mrd group all patients received bone marrow as hsc source, while in the mud/mmud, eight patients received bone marrow and five received cord blood. all patients with incompatibilities (mismatched) were ucb (5/5). nineteen recipients were conditioned with busulfan plus cyclophosphamide, while the remaining 11 received fludarabine and busulfan, which has been the preferred regimen in brazil in the recent years. after transplant, most (n=24) of the mrd and mud recipients received cyclosporine and short course methotrexate as graft versus host disease (gvhd) prophylaxis. results: twenty-two out of the 30 patients were alive and disease-free at a median follow-up of 24 months (range 1 to 213 months). the 3-year overall survival (os) was 71% (ci 51-91%) (fig 1) . similar results have been demonstrated in studies from europe and from the united states. when analyzed according to donor type, os was 73% (ci 55-100%) and 66% (ci 43-100%) in mrd and mud/mmud respectively (fig 2) . three out of the 5 patients who were transplanted with ucb died. these results are in agreement with those of previously published data showing worse results in unrelated ucb transplants. twenty-nine out of the 30 patients engrafted successfully. in 25 of the evaluated patients, the median time to neutrophil engraftment was 20 days (range 10-27). one patient experienced an early death from hemorrhagic shock on day 12, before neutrophil recovery, and another two patients experienced primary graft failure. post-transplant chimerism was available for 22 patients. sixteen had complete chimera (>90% chimerism), while 6 patients presented with mixed chimerism. acute gvhd was observed in 9 patients (32%), 6 of which classified as grade iv. five patients developed chronic gvhd, considered severe in three of them. eight patients died at a mean of 155 days (range 12-728 days) after hsct and the main causes of death were infections and hemorrhagic disorders. conclusions: hsct is a potentially curative treatment option for dba. in the present study, we report the outcomes of 30 patients with dba transplanted in brazil with a os of 71%, with better results in mrd compared to mud, as expected. despite the small numbers, we observed lower survival after mud/mmud ucb transplantation. since dba is a rare disease, international collaborative studies are essential to better understand the benefits of the hsct in the treatment of these patients. disclosure: nothing to declare p627 treatment of the obliterant bronchiolitis in pediatric allogeneic recipients: two periods compared results: in group 1, the therapies administered for bo included prolonged treatment with steroids in all patients, anti-tnf in 1, azatioprine in 4; while in the group 2, all patients received ima, montelukast and azitromicin, and 4 received i.v. mpd. the median duration of imatinib therapy was 4 years (0.3-7.1 years). after a median follow-up of 4.4 years (range 0.5-12.1 yrs), 9/11 patients of group 1 (82%) died with bo in progress for transplant-related causes. while in the group 2, 2/14 (14%) died in presence of worsening bo. the estimated os at 1 year after hsct was 75% (95% ci; 50-89) in group 1 and 83% (95% ci, 27-97) in group 2 (p=0.103) (figure 1 ), while the os after 4 year decreased at 42% (95% ci; 16-66) in the group 1 while remained stable in the group 2. conclusions: this experience shows a relevant improving in prognosis of children with bo with the use of this protocol including ima, since the significant improving of survival obtained, confirming as reported in adult populations. disclosure results: we presented 20 patients with pres, age ranging from 2 months to 19 years with a average of 9.5 years. there were ten patients with thalassemia major, two patient with acute lymphoblastic leukemia, three patients with sickle cell disease and one patient with myelodysplastic syndrome, one patient with immune deficiency, two patients with acute miyeloid leukemia, one patient with aplastic anemia. ten patients were males, ten were female. all patients were treated with csa or tacrolimus and metilprednisolone for the prophylasix of gvhd. pres occurred at a median of 90 days (range 5-625). clinical findings at onset of leukoencephalopathy were hypertension, headache, seizures, visual disturbance, and altered mental function. eighteen patients alive with normal neurological status. mri showed abnormalities in all patients including patchy bilateral cortical and subcortical lesions, especially in parieto-occipital lobes. conclusions: bmt is associated with several neurological complications that may be underlying diseases, bmt procedure, and severe immunosupression. pres is an uncommon but serious complication after bmt. we report 20 cases of pres who received allogeneic bmt for thalassemia major to emphasize the importance of early recognition and institution of appropriate management of pres during bmt. disclosure: nothing to declare p631 continuous complete molecular remission using three different monoclonal antibodies followed by allogeneic bone marrow transplantation in an infant with chemotherapy-refractory acute lymphoblastic leukemia bernd gruhn 1 , susan wittig 1 , thomas ernst 1 , jana ernst 1 1 university of jena, jena, germany, background: a 10-week-old infant was diagnosed with very immature acute lymphoblastic leukemia (all) with myeloid markers in a foreign university hospital. at the end of induction therapy according to the current lal/shop protocol 10% leukemic cells were detectable in the bone marrow. treatment was changed to fludarabine, cytarabine and granulocyte colony-stimulating factor (flag) in combination with liposomal doxorubicin. after this re-induction still 5% leukemic cells were detected in bone marrow, so the bispecific t-cell engager antibody blinatumomab was given. due to an increasing portion of leukemic cells during the continuous infusion, antibody therapy was stopped and a cycle of clofarabine, cyclophosphamide and etoposide was administered. unfortunately, still 31% leukemic cells were detectable afterwards. because of chemotherapy-refractory leukemia a palliative oral treatment with mercaptopurine was started. however, the parents did not accept the palliative situation and searched for alternative therapeutic options in other university hospitals in europe. after plenty of refusals the infant was admitted to our hospital five months after diagnosis. methods: for molecular characterization genomic dna was isolated from leukemic cells. a mll-mllt3/af9 rearrangement as a consequence of the translocation t(9;11) (p22;q23) was detected and used as a marker for minimal residual disease. for further molecular characterization targeted deep next-generation sequencing was performed for a panel of 54 leukemia-associated genes. interestingly, no mutation was found. to allow precise immunophenotyping of the leukemic cells treatment with mercaptopurine was stopped. results: as in the first immunophenotyping the cd33 antigen was found, we administered the anti-cd33 monoclonal antibody gemtuzumab ozogamicin twice within two weeks. because of the detection of cd38+ leukemic cells after infusion of gemtuzumab ozogamicin, the anti-cd38 antibody daratumumab was given alternating twice within two weeks. unfortunately afterwards, leukemic cells reappeared being negative for cd33 und cd38, but positive for cd22. therefore, we administered the third antibody, the anti-cd22 monoclonal antibody inotuzumab ozogamicin, whereupon our patient developed a tumor lysis syndrome and a severe bone marrow aplasia. shortly after, allogeneic bone marrow transplantation from an unrelated donor using a special conditioning regimen consisting of thymoglobulin, busulfan, fludarabine and clofarabine was conducted. clofarabine was added because an additional antileukemic effect especially in infant all with mll rearrangement was described. after transplantation the patient suffered from a severe hepatic sinusoidal obstruction syndrome with massive ascites, renal and pulmonary dysfunction, but finally the patient recovered completely. the first bone marrow examination 30 days after transplantation revealed a donor chimerism of 100% and a complete molecular remission using the mll-mllt3/af9 rearrangement as marker for minimal residual disease. in all follow-up bone marrow samples we observed a complete donor chimerism and a complete molecular remission. currently, eight months after transplantation the patient is in a very good physical condition with normal development according to the age. background: paediatric chronic graft versus host disease (cgvhd) is a debilitating condition associated with substantial morbidity and mortality. to date, there are no approved therapies for paediatric patients with cgvhd, and current treatments often lack sufficient efficacy or lead to severe/life-threatening toxicities that limit their effectiveness. ibrutinib, a first-in-class, once-daily inhibitor of bruton's tyrosine kinase (btk), is approved in the us for the treatment of adult patients with cgvhd after failure of ≥1 lines of systemic therapy. this phase 1/2 study will evaluate the use of ibrutinib in paediatric patients with moderate or severe cgvhd. methods: this open-label, multicenter, international phase 1/2 study (pcyc-1146) includes patients with moderate or severe cgvhd as defined by the 2014 nih consensus development project criteria. it is divided into two parts: part a will determine the recommended paediatric equivalent dose (rped) of ibrutinib in patients aged ≥1 to < 12 years, and part b aims to evaluate the safety and efficacy of ibrutinib in patients age ≥1 to < 22 years. for part a, patients with cgvhd aged ≥1 to < 12 years who have failed ≥1 lines of systemic therapy will receive once daily oral ibrutinib at a starting dose of 120 mg/m 2 to be escalated up to 240 mg/m 2 after 14 days, if no grade ≥3 toxicities occur, until the rped is determined. for part b, patients aged ≥12 to < 22 years with cgvhd who have failed ≥1 lines of systemic therapy or have newly diagnosed cgvhd will receive once daily ibrutinib (420 mg) until one of the following criteria is met: treatment is no longer required; new systemic treatment for cgvhd is initiated; progression of cgvhd; recurrence of underlying disease; or unacceptable toxicity. patients with newly diagnosed cgvhd will receive ibrutinib in addition to daily corticosteroids (0.5-1 mg/kg prednisone). patients < 12 years of age may be enrolled in part b and treated at the rped after it is determined in part a. key exclusion criteria include uncontrolled active systemic infection or active infection requiring systemic treatment; progressive underlying malignant disease or any post-transplant lymphoproliferative disease; or active hepatitis c/hepatitis b virus. patients must have adequate renal, hepatic, and hematologic function to be enrolled. the primary endpoint of part a is the rped of ibrutinib, as based on pharmacokinetic (pk) data; secondary endpoints include safety and pharmacodynamics (btk occupancy). the primary endpoints of part b are pk and safety of ibrutinib in paediatric patients with cgvhd. secondary endpoints for part b include response rate at 24 weeks as defined by the 2014 nih consensus development project criteria; duration of response; overall survival; and late effects on growth, development, and immune reconstitution. results: this global study is currently enrolling. conclusions: this phase 1/2 study will explore the use of ibrutinib in paediatric patients with cgvhd to potentially meet the high unmet need for proven effective therapies for this population. disclosure enzyme replacement therapy (ert) is the treatment of choice in non-neuropathic hunter syndrome, but as the recombinant enzyme does not cross the blood brain barrier and neuropathic hunter syndrome is left untreated. hematopoietic stem cell transplantation (hsct) is the standard of care in patients with severe mucopolysaccharidosis (mps) type i (mpsih, hurler syndrome) as early transplantation halts cognitive decline in these patients and significantly improves survival. only few case studies have been published on the potential benefit of hsct in mps ii and mostly used busulfan-based conditioning regimens. in one comparative non-randomised multicenter study, hsct might to be superior compared to ert. here, we present our experience in hsct in three children with hunter syndrome using a treosulfan-based conditioning regimen. methods: a retrospective chart review was carried out in patients, who underwent hsct for hunter syndrome. the conditioning chemotherapy regimen included fludarabine, treosulfan, thiotepa and atg. all patients received bone marrow of either related and or matched unrelated donors. gvhd prophylaxis was performed with csa and methotrexat. results: three patients with hunter syndrome were transplanted in our department in 2010. the age was six months, two years and four years, respectively. bone marrow donors were related in one patient and matched unrelated in two patients. the conditioning therapy was generally well tolerated. major complications were fever of unknown origin with need for antibiotic therapy and a mucositis. one patient developed a cmv reactivation. all patients engrafted successfully and recovered well from the hsct. there was no case of acute or chronic gvhd. in 2018 all three patients are alive. donor chimerism is complete in one patient; two patients have a mixed donor chimerism. after application of donor lymphocyte infusions in one patient, donor chimerism is stable at a low level of 16%. the donor chimerism of the other patient still slowly declines to currently 50%. after stem cell transplantation, two patients did not show further progression of the disease and even achieved psycho-motor improvements. interestingly, one of these patients is the one with the low donor chimerism of 16%. one patient suffers from a further progression of the underlying disease with psycho-motoric agitation, aggressive behavior and loss of speech, that occurred within the first year following hsct, but neurocognition stabilized thereafter. conclusions: we found a beneficial effect of hsct on the neuropsychological outcome or at least stabilization of neurocognitive function in our patients with a follow-up of eight years. despite low toxicity of the conditioning regimen, increased donor chimerism may further improve the neurological outcome. disclosure: nothing to declare. tandem sct in pediatric solid tumors, other than brain tumors, has no advantage in terms of efs over single procedure-single center experience , germ-cell tumour (gct), ewing sarcoma (es), nefroblastoma. patients were divided into 2 groups according to the number of procedures: 1st group-single sct procedure, 2nd group-multiple procedures. regimens used for stem cell mobilisation were: topo-cy for nbl and epi-tax for gct, followed by g-csf±plerixafor. conditioning regimens: bu-mel and thiotepa-cy for pts with nbl, thio-tax and ice for pts with gct. patients received antibiotic, antiviral and antifungal prophylaxis, parenteral nutrition and supportive treatment. patients received consolidation treatment, followed up monthly in the first year, then yearly. patients were evaluated for residual disease by imaging tests. parents signed informed consent forms. results: we performed 67 sct procedures to 52 patients: 65.3% nbl, 17.3% es, 11.6% gct and 5.8% nefroblastoma. for this study only patients with nbl and gct were considered. in 1st group were 79% of pts, 21% in 2nd group. patients were diagnosed, staged and treated according to international protocols. sex ratio was 18f/34m. age distribution was 1-4 y 38% (20 pts), 4-10 y 35% (18 pts), > 10 y 27% (14 pts). peripheral stem cell (pbsc) mobilisation was more difficult in patients with multiple courses of chemotherapy±radiotherapy. we found no difference in the period of engraftment following a 2nd or 3rd procedure. hospitalization and supportive measures increased in 2nd and 3rd procedures (26 to 29 days). patients with multiple courses of chemotherapy and multiple hospitalizations had increased infectious risk and during the 2nd or 3rd procedures developed various infectious complications.incidence of severe oral mucositis after the first hsct was 17%, after tandem hsct was 69%. nbl patients : 1st group-6/23 patients alive and efs, 1/23 receives anti g2 treatment; 2nd group-1/7 patients-alive, 2/7 patients-not reached timepoint for mibg scan; 4/7 patients-mibg negative at first, relapsed after 6 mo; 1/7 patient deceased due to pulmonary toxicity. gct patients: 1st group-1/5 patients alive and ef, 1/5 high values in afp levels and receives metronomic therapy, 3/5 patients deceased due to progressive disease, but only had 2 sct. only 1/5 patient had one procedure and died due to progressive disease. conclusions: in our study, tandem hsct in children with solid tumours lead to an increase in survival rates, at least in the first 6 months after sct. most patients (90%) had progressive or relapsed/refractory disease when referred to our department. multiple procedures require a higher number of cd34 cells, very hard to achieve in patients with multiple courses of chemo± radiotherapy. new approaches have to be considered in these diseases, especially in high risk group. disclosure: nothing to declare background: antimicrobial prophylaxis for prolonged neutropenia occurred during the pre-engraftment period is a common practice in allogeneic hsct recipients. data on its effectiveness are few and generally from cases series and not from randomized clinical trials, especially in children. methods: all clinical records of allo-hsct performed from january 1 st 2007 to november 30 th 2018 at hsct-unit of istituto g.gaslini, genoa-italy, were retrospectively reviewed. collected data were underlying diseases, type of donor, antibiotic prophylaxis administration and type, development of fever and pathogen isolated from blood culture, if any, during pre-engraftment neutropenia. antibiotic prophylaxis, usually starting together with the conditioning regimen, was categorized in "standard" (with amoxicillin/clavulanate or ampicillin/sulbactam) or "tailored" (when based on previous bacterial isolations or colonizations). results: 246 allo-hscts were performed in 217 pediatric patients (59% males) with a median age at hsct of 8 years (iqr: 4-13; range: 0-22). hscts were performed from alternative donor (ad) in 55% patients, from relative donor (rd) in 25%, and from haploidentical donor in 20%. table 1 shows the pre-engraftment febrile neutropenia episodes according to type of antibiotic prophylaxis. 224 (91%) hscts received standard prophylaxis, while 20 (8%) the tailored one; only in 2 (1%) did not receive any prophylaxis. fever occurred in 194 (87%) of episodes in patients receiving standard prophylaxis, in 19 (95%) of those treated with tailored prophylaxis and 1 (50%) in the group without prophylaxis; only 13% of patients who received prophylaxis did not develop fever.in 38% of patients, the febrile episodes were diagnosed as bloodstream infections: staphylococcus aureus in 2%; cons in 19%; enterococcus spp in 15%; enterobacteriaceae in 25%; pseudomonas aeruginosa in 11%; other non-fermenting gram negatives in 2% and fungi in10%. conclusions: the occurrence of fever in patients who received antibiotic prophylaxis suggested that it could not be effective in prevention of fever related to neutropenia after allo-hsct. the personalization of prophylaxis could be a possible path to follow these patients. disclosure methods: a total of 15 patients with leukemia or neuroblastoma were included in the study. patients' mothers signed an informed consent for participation in the study. six of study participants were boys and 9 girls, all aged 3 to 6 years. the control group consisted of 18 healthy preschool children (2 groups of 9 children aged 5 to 6 years), 8 boys and 10 girls. results: in most of games the role of a doctor was played by a child. only one child declined to impersonate both a patient and a doctor. younger children mostly agreed to have for a "patient" a toy (proposed by psychologist or one of child's own), child's mother or a medical psychologist. the game lasted for 15 -20 minutes. most patients preferred using real medical consumables and instruments (syringes, adhesive tape, winged infusion sets or, more rarely, pills). most often a syringe or an adhesive tape was chosen. as known from their mothers, among medical manipulations most unpleasant for children are injections and changing implanted catheter dressing. also, most healthy preschool children preferred using real medical instruments over toy ones. group 1 more often used a syringe, a winged infusion set, adhesive tape, gauze or pills. group 2 most often chose syringe or gauze. among medical instruments both groups more often chose a phonendoscope or thermometer.one patient refused to cause pain to a "toy patient". other children sympathized with a "toy patient", stroke injection or dressing location sites or used soothing terms ("wait a little", "it's going to be all right"), wished prompt recovery and hugged their "patients". one child was angry over his "patient" wishing him to "get hurt too". first preschool group children were mostly scolding a toy "patient" for "being guilty of getting sick". second group children were mostly compassionate, encouraged a "toy patient" telling that "all the procedures are needed to get healthy". from children's schoolmasters we know that all first group children received vaccination about a week before a test. children from second group had no injections. overall attitude towards toy "patients" was more mild in the second group. conclusions: 1. during a play children mostly use the medical devices which cause them most discomfort and/or pain. 2. manipulating the items children illustrate their own impression of medical procedures, which are most unpleasant. 3. children may express their negative emotions directed towards medical manipulations via their play actions, these negative reactions may be suppressed in different ways by parents or medical staff. 4. the intensity of child's own traumatic experience and an attitude of nearby adults may influence the child's attitude towards other patients. 5. the mother's wish for a child to tolerate all medical procedures with ease exceeds real capabilities of a small child. disclosure: nothing to declare. allogeneic stem cell transplantation in patients with mucopolysaccharidosis type iiia (sanfilippo): a case series methods: allogeneic sct was performed at the ages of 2, 5 and 4 years, respectively. all three patients received intrathecal enzyme replacement therapy within a clinical trial setting prior to hsct. the conditioning regimen consisted of treosulfan, fludarabine, thiotepa and thymoglobuline. gvhd-prophylaxis was carried out with csa and mtx in two patients and csa and mmf in one patient. stem cell source was bone marrow in two patients and peripheral blood stem cells in one patient. results: the conditioning regimen was well tolerated and all three patients successfully engrafted. two of three patients had an uncomplicated course without occurrence of acute or chronic graft-versus-host-disease (gvhd). at last follow-up 12 and 15 months after hsct, both patients are in good condition and show constant progress of psychomotor development. the third patient experienced severe steroid refractory acute gvhd of intestines (stage 4) and skin (stage 3), which resolved under intensive immunosuppression with cyclosporine, mycophenolate and ruxolitinib. around day 110 after hsct, this patient showed clinical and biochemical signs of transplant-associated microangiopathy (tma) with cerebral seizures and acute renal failure. the cerebral mri showed progressive cerebral atrophy and leukoencephalopathy, also consistent with a progress of the mps iiia. at last follow-up 15 months after hsct, this patient had recovered from tma and was in a stable clinical condition. conclusions: in consideration of the small case number and the short follow-up period in our cohort, allogeneic hsct might be considered as a salvage therapy for patients with mps iiia if other therapeutic options are unavailable for children with this otherwise unfavourable prognosis. however, the early psychoneurological course after transplant seems promising compared to the literature and hsct could become a treatment option for this rare disease. disclosure: nothing to declare methods: for the identification of underlying molecular mechanisms leading to the increased sensibility of rms cells, the activation status of different nf-kb signaling pathways were analyzed using western blot analysis and quantitative real time pcr (qpcr). further, flow cytometry was used to analyze the surface expression of death receptors on either sm treated or untreated rms cells. the overall effect on cell death induction was measured by pi/hoechst staining using a fluorescent microscope. results: treatment with sm led to the suspected degradation of iaps. followed by the activation of both the canonical nf-κb signaling pathway, indicated by the phosphorylation of iκbα and p65, and the non-canonical nf-κb signaling pathway, as indicated by the accumulation of nik and the degradation of p100 to p52. determination of selected target gene transcription revealed an upregulation of the inhibitor iκbα, nik, p100, il-8 and at later time points the death receptors trail-r1 and trail-r2. analysis of gene transcription also led to the finding of neither up-nor downregulation of ciap1 and p65. to evaluate the involvement of trail-r1 and trail-r2 in the sm induced sensitization towards nk cell-mediated killing, surface expression of both death receptors was analyzed. treatment with sm led not only to an induced transcription of trail-r1 and trail-r2, but also to an increased surface presentation of trail-r2. subsequent ligation of trail-r2 by either wt-trail or a specific agonistic antibody (etr-2) resulted in a significant increase in cell death induction. the aforementioned analysis of gene transcription hints towards a bimodal feedback mechanism regulating both, the canonical and non-canonical nf-κb signaling pathway. on the one side, the canonical pathway is negatively regulated by the induced transcription of the inhibitor iκbα. on the other side, the induced transcription of nik, p100 and relb points towards a positive feedback loop of the non-canonical pathway. one mechanism of the increased rms cell sensitivity might be the induced transcription and surface presentation of the death receptor trail-r2. the involvement of trail receptors is further validated by the cytotoxicity data, illustrating a sm mediated sensitization towards a trail induced cell death induction. this mode of cell death fits to the previous research, were trail transcription could be induced in nk cells by sm treatment. the graphical abstract shows the transcriptional upregulation of target genes leading to a putative bimodal nf-kb regulation and increased surface presentation of trail-r2 by treatment with smac mimetics. aim: to investigate the outcome of ucb transplantation in pediatric patients with malignant and non malignant diseases methods: data from 30 patients underwent first allogeneic bone marrow transplantation with ucb from 2/1999 until 6/2013 were retrospectively analyzed. eighteen had malignant disease (md), of whom 15 in complete hematologic response, and 12 non malignant disease (nmd) (scid 5, chronic granulomatous disease 1, severe aplastic anemia 2, s.kostmann 1, osteopetrosis 1, wiskott-aldrich1, amegakaryocytic thrombocytopenia 1). the majority of the patients were male, for md and nmd, as well (m:10/f:8, m:8/f:4, respectively), of median age 6.5 years (range 0.8-11.8 years) and 0.8 years (range 0.4-6.5 years), respectively results: all patients but one, received 1 ucb unit. hla compatibility in antigen/allele level was at least 5/6 and only in 3 patients with md was 4/6. conditioning regimens were myeloablative and tbi 12 gy was given in 4/30. gvhd prophylaxis consisted of cyclosporine and atg was given in all patients pre-transplantation. median value of nucleated cells for md was 3.75χ10 7 /kg (range 2-6.3χ10 7 / kg) and for nmd was 11.05χ10 7 /kg (range 2-27.2χ10 7 /kg). neutrophil and platelet engraftment was achieved in 13/18 and 12/18 patients with md respectively, in a median time of 31 days (range 17-44) και 43 days (range 20-60). in patients with nmd, neutrophil and platelet engraftment was achieved in 7/12 and 6/12 with median day of engraftment 21 days (range 18-28) και 29 days (range respectively. acute gvhd grade ιι-ιv presented in 9/18 patients with md and 4/12 with nmd, although none had cgvhd. the incidence of viral infections was 22 cases in 11 patients with md and 9 cases in 6 patients with nmd. disease relapse occured in 9/18 patients with md. after a median time of 10 years follow up, overall survival (os) and event free survival (efs) for children with md were 22% and 18.5% respectively, while for nmd, os and efs were 33%.treatment related mortality at d+100 was 8% for md and 50% for nmd. among 18 patients with md, 4 are still alive, while the rest died from relapse (n: 8), viral infections (n: 4), septicemia (n: 1) and agvhd(n: 1). among 12 patients with nmd, 4 are alive, while the rest died from viral infections (n: 2), septicemia (n:4) and multiple organ failure (n=2). the median time of hospitalization for patients with md was 76 days (range 32-168), whilst for nmd was 63 days (range 28-114). conclusions: transplantation of unrelated ucb in our unit was combined with high trm in children with nmd and higher probability of relapse for md. disclosure: nothing to declare p643 serum levels of 5-s cysteinyldopa is associated with stem cell transplantation related complications yukayo terashita 1 , mamoru honda 1 , minako sugiyama 1 , yuko cho 1 , akihiro iguchi 1 1 hokkaido university hospital, pediatrics, sapporo, japan background: diffuse hyperpigmentation is common in patients who received chemotherapy or stem cell transplantation (sct). however, there are few reports of the relationship between skin reaction such as pigmentation and the other complications. pigmentation of the skin is thought to be the result of melanin stagnating in the dermic layer due to increased synthesis of melanin and destruction of the basement membrane due to inflammation induced chemoradiotherapy. melanin pigments are classified into two types: brown to black eumelanin and yellow to reddishbrown pheomelanin. 5-s cysteinyldopa (5scd) is precursors of pheomelanin, and its serum level has been used specific biochemical marker for malignant melanoma. here, we examined serially 5scd during the course of sct to determine association with sct related complications, because visual evaluation of skin color is difficult, and there have been no reports about 5scd as sct related biomarker. methods: we prospectively analyzed 41(27 males, 14 females) patients who received sct between may 2011 and march 2015 in hokkaido university hospital. the median age at transplantation of the patients was 7.9 years (range, 0-22). indication for sct were acute myelogenous leukemia in 10 patients, acute lymphoblastic leukemia in 9 patients, and other disease in 22 patients; juvenile myelomonocytic leukemia(2), malignant solid tumor(11), immunodeficiency(6), anaplastic anemia(2), and diamond blackfan anemia (1) . 34 patients received allogeneic sct and 7 received autologous sct. myeloablative conditioning (mac) was used for 30 patients and reduced intensity conditioning (ric) was used for 11 patients. sera were obtained from patients before conditioning therapy, on day 0, +5, +10, +15, +25 and +40. all blood samples were centrifuged at 3,000 rpm for 15 min, and stored at -80˚c until used. we also examined sct related complications such as graft-versus-host disease (gvhd), viral infection, and pre-engraft syndrome (pes). statistical analyses were completed using the mann-whitney u test for unpaired samples, and kruskal-wallis test for three samples. each test was performed with a 5% level of significance. results: the average value of 5scd reached two peaks, day0 (21.6 nmol/l) and day5 (21.7 nmol/l), regardless of stem cell source and intensity of conditioning. in all patients, we found that the level of 5scd on day0 was associated with viral reactivation (p=0.049), 5scd on day5 was associated with pes (p=0.034), and 5scd on day40 was associated with malignant disease (p=0.04). similarly, in patient who received allogeneic sct (n=34), the level of 5scd on day0 was associated with viral reactivation (p=0.048), 5scd on day5 was was associated with pes (p=0.034), 5scd on day 40 was associated with malignant disease (p=0.04). in addition, the level of 5scd on day5 was associated with gvhd of skin (p=0.027), the peak level of 5scd was associated with acute gvhd (p=0.04). conclusions: we found that 5scd can be a biomarker for sct-related complications such as aute gvhd. it is presumed that the production of pheomelanin could be induced by inflammatory procedure in sct. disclosure: nothing to declare p644 hsct in children with bone marrow failure: outcomes from a single singapore centre prasad iyer 1 , michaela seng 1 , vijayakumari k 1 , ah moy tan 1 , mei yoke chan 1 , rajat bhattacharyya 1 1 kk women's and children's hospital, paediatric haematology-oncology, singapore, singapore background: children presenting with pancytopenia often present a challenge to the paediatric haematologist. the underlying diagnosis can be hard to establish as many of the inherited bone marrow failure syndromes (ibmfs) can present with protean manifestations. the large majority of patients with bone marrow failure are often diagnosed with idiopathic severe aplastic anaemia (saa) despite extensive testing. we report our experience of hsct in patients treated with primary and acquired bone marrow failure. methods: we reviewed case notes of all the children who underwent hsct for bone marrow failure in our centre. results: a total of fifteen patients underwent eighteen stem cell transplants in our centre between 2003 and 2016. three patients were diagnosed with fanconi anaemia, one with hoyeraal-hreidarsson syndrome, one with paroxysmal nocturnal haemoglobinuria and the remaining ten children had idiopathic saa. eight children had matched sibling donor transplant, 1 had a matched related donor, 1 had a matched unrelated donor, 3 had umbilical cord blood transplants and the remainder 5 were haploidentical transplants. four of the haploidentical transplants were t-cell depleted and one was t-cell replete. one child with fanconi anaemia had primary graft rejection with cord blood transplant and was successfully rescued with a haploidentical transplant. one child with saa had primary graft rejection twice (t-cell depleted graft) and then was rescued with an alternate haploidentical donor with a t-cell replete graft. of the two patients who died, one had a fatal fungal infection ten months after transplant, and the other died due to a severe influenza pneumonitis three and a half years after bmt. conclusions: haematopoietic stem cell transplant outcomes from our centre are comparable to leading centres in the world. the understanding of underlying conditions that present with bone marrow failure has improved our approach and the way we treat bone marrow failure syndromes. clinical trial registry: not applicable. disclosure: nothing to declare. methods: a retrospective study was performed in children treated with hsct who received pos or flu during early neutropenic period until engraftment from january 2000 to december 2017 at siriraj hospital in thailand. the efficacy, safety and tolerability of pos were compared to flu. results: there were 66 hsct recipients (allo-hsct 62.1%, auto-hsct 37.9%) with mean age of 7.6+4.3 years. most of the patients were thalassemia (34.4%) followed by hematologic malignancy (32.2%) and solid tumor (16.7%). seventeen and 49 cases received pos and flu, respectively. all of patients in pos group were allo-hsct whereas 48.9% in flu group were allo-hsct. in pos group, 2 cases were diagnosed with suspected ifi and 2 cases were probable ifi with total 4 cases (23.5%). in flu group, 10 cases were diagnosed with suspected ifi and 2 cases were probable ifi with total 12 cases (24.5%) which compared 2 groups were not statistically significant (p=0.937). no possible and proven ifi in both groups. in flu group patients received empirical antifungal treatment more than pos group but no statistical significance (20.4% vs.11.76%, p=0.498). both groups had similar rate of elevated liver function test (p=0.567). no early discontinuation of antifungal prophylaxis for intolerance was found in both groups. only 26.7% of patients achieved pos target trough level of 0.7 mg/l after 7 days of treatment with started dose 4 mg/kg three times a day. conclusions: pos and flu are comparably effective, safety and tolerability in ifi prophylaxis in neutropenic children treated with hsct. defining dose recommendation of pos in this setting requires larger studies. disclosure background: severe congenital neutropenia (scn) is typically characterized by anc of <500/μl, maturation arrest of bone marrow myeloid precursors at the promyelocyte-myelocyte stage, and susceptibility to lethal pyogenic bacterial and fungal infections. scn is a rare group of disorders resulting from intrinsic defects in myeloid cell proliferation and maturation caused by mutations in several genes; elane, hax1, gfi1, was, and g6pc3 are among the most common ones. almost 10% of patients are refractory to g-csf, and the only definitive curative approach for such patients is allogeneic hsct. the current absolute indications for hsct is failure to respond to g-csf treatment, or the development of mds/leukemia in patients with scn. here, we present the result of 10 children with scn who received allogeneic hsct . methods: we retrospectively assessed 10 allogeneic hsct in children with severe congenital neutropenia. all patients received busulphan (bu) based myeloablative conditioning regimen. busulphan was used according to weight adjusted dose. in addition, all patients received fludarabine 150 mg/m2 in five days or cyclophosphamide 200 mg/kg in 4 days and atg 30 mg/kg in 3 days. cyclosporin-a and mtx were used for graft versus host disease (gvhd) prophylaxis. donor chimerism was evaluated in either bone marrow or peripheral blood on days +30, +100 and +180. results: the median transplantation age of the patients was 49 months (range 11-167 months). six of them are male. two of the donors were matched siblings and 8 were unrelated two of which were 1 ag 1 ag mismatched. stem cell source was bone marrow in 6 patients, peripheral blood in 2 and cord blood in 2 patients. all patients engrafted. the median time of neutrophil and platelet engratment to was 15 (13-34) days and 16(9-90) days, respectively. graft rejection was experienced in 2 patients, one of them had received unrelated cord blood. all patients are alive, eight of which are with full donor chimerism (between 95-100 %) without any complication (no infection, no gvhd) with a median 40 months (range 24-83 months) follow up. probability of disease free and overall survival were found 80% and 100%, respectively. conclusions: we concluded that hsct is a useful treatment for scn patients, especially those who are unresponsive to gcsf treatment and at high risk for leukemic transformation. however, a larger number of scn patients and longer follow-up are necessary to identify appropriate conditioning regimens and long-term prognosis. disclosure: nothing to declare background: prolonged thrombocytopenia (pt) or secondary failure of platelet recovery (sfpr) are a lifethreatening complications that occurs in 20-40% and 12-20% respectively of the patients following allogeneic hematopoietic stem cell transplantation (allo-hsct). management strategies, including the use of growth factors, cd34+-selected stem cell boost, mesenchymal stem cell (msc) transfusion, and second allo-hsct, are not effective or possible for all patients. eltrombopag, is an oral non-peptide thrombopoietin receptor agonist, that leads to signal transduction and results in promoting the proliferation and differentiation of megakaryocytes. some recently studies show that also can promote haematopoiesis along all three lineages. methods: we described our experience in 4 paediatric patients with poor graft function or secondary failure of platelet recovery after allogeneic stem cell transplantation treated with eltrombopag. results: patients characteristics are detailed in table 1 . all the patients received and allo-hst. the median dose of cd34+ cells infusion was 6.43x10e6/kg (3.95-8.29 ). neutrophils engraftment occurred in +15 day (10-21d) and platelets in +26 day (16-42d). all the patients had an hypoplastic bone marrow with complete chimerism. the median duration from transplantation to spcf diagnosis was 10 months (1.5-24m) . one of the patients received a stem cell boost prior to eltrombopag, without response. the time onset from spgf/sfpr diagnosis to initiating eltrombopag was 16 days (8-32d). eltrombopag was started at a dose of 1mg/kg/d, requiring an increase dose in all cases. the median dose was 50 mg/d (25-100mg). the overall response rate was 50% (2/4). two patients achieved complete response (cr), as defined by platelet ≥ 50 × 109/l. both patients already got neutrophil ≥ 1.0 × 109/l without g-csf. the time from eltrombopag initiation to achieving cr was 21 (10-49d) days. the treatment was given for a median of 81 days (8-203). it was discontinued after 96 and 203 days respectively in the two responder patients. both patients maintain stable blood counts after discontinuing the treatment. the non-responders patients had to stop the treatment because of other reasons not related to eltrombopag. patient 4 had to be rescued with a cd34+ cells boost with a good response. two patients that were in treatment with voriconazole for a fungal infection developed hyperbilirubinemia. there were no grade 3-4 toxicities related to eltrombopag. conclusions: in our experience, according to recently published studies, eltrombopag is a safe and efficacy drug in the treatment of secondary failure of platelet recovery post-hsct. it may be used successfully in children. sometimes higher doses may be considered if no response is achieved. further prospective trials are needed to increase the level of evidence and to identify predictors of response. disclosure: nothing to declare very slow clearance of busulfan in a child with infant leukemia background: busulfan is a drug with a high interindividual variability between dose and exposition. therefore, it is recommended to perform therapeutic drug monitoring (tdm) in the context of myeloablative conditioning, especially in children. methods: we report on a 7-month old boy (7.2 kg, 66 cm) of caucasian decent born to non-consanguine parents with mll-rearranged prob-lymphoblastic leukemia. diagnosis was established one month after birth from peripheral blood and csf tap showed cns involvement. primary chemotherapy was commenced according to the interfant-06 protocol. however, mrd remained positive two months under treatment, leading to an indication for allogeneic stem cell transplantation. in the interfant-06 protocol, we opted for a conditioning regimen comprised of fludarabine (1.2 mg/kg for 5 days), busulfan and thiotepa (2x5 mg/kg). in our institution, busulfan is applied once daily with a target auc of 85-95 h*mg/l in this very high risk situation. according to body weight, busulfan was given with 5.1 mg/ kg as a three-hour infusion on the first day. busulfan concentrations in plasma were measured with gas chromatography-mass spectrometry (gc-ms) and auc was calculated using bayesian curve fitting. results: exact busulfan quantification was not possible after the first dose due to technical reasons. as the levels were estimated to be very high, we decided to reduce the second dose of busulfan by 25%. this resulted in a very high auc of 44 h*mg/l for the second dose, so that busulfan was discontinued after two days, because it was calculated that the patient already received busulfan with a cumulative auc of 90 h*mg/ml. trough levels after the first and second dose were 547 and 572 μg/l, respectively. the patient showed a very slow clearance of 2.1 l/h/sqm, while the volume of distribution was in the usual range (0.86 l/kg). bilirubin and liver transaminases were in the normal range at the time of conditioning, while albumin and quick were decreased on day +18 after transplantation the patient developed clinical und biochemical signs of venoocclusive disease (vod). vod symptoms completely resolved under therapy with defibrotide. leukocyte engraftment was established on day +14. unfortunately, the patients suffered from an early relapse of the leukemia from day +62. attempts to induce a second remission with blinatumomab failed. the patient is currently under palliative chemotherapy. conclusions: busulfan tdm is very important especially in infants receiving myeloablative doses of busulfan to prevent under-or over-exposure. there is evidence that high busulfan trough levels contribute to the development of vod, but anti-leukemic activity of busulfan and cns permeability make it a valuable drug for very high risk patients in childhood leukemia. larger patient cohorts are needed to assess the exposure dependent risks of toxicity versus relapse in infants and toddlers. disclosure: nothing to declare blood (ucb) obtained at delivery from three children who received a diagnosis of cerebral palsy. methods: immunophenotyping of the ucb leukocyte fraction was performed using multicolor flow cytometry. the procedure was performed according to the protocol by shatorje and colleagues (1) . briefly, the ucb samples were labeled with specific antibodies and incubated in the dark for 30 minutes. afterwards, the samples were treated with 5ml of bd facs lysing solution for 10 minutes to preserve the leukocyte fraction only. the cells were washed using pbs (roche) and then centrifuged twice (1500 rpm, 4°c, 4 minutes). the results were analyzed using the facsdiva software (becton dickinson). results: we found an increased white blood cell (wbc) count, lymphocyte count, and cd4:cd8 ratio in all ucb samples. one patient had a low nk cell count and percentage, and another had a low b-cell count and percentage. one sample displayed high t (cd3+) and th cell (cd4+) counts, but with percentages within the limits of the reference values. conclusions: we detected elevated wbc and lymphocyte counts in all ucb samples, despite a lack of intrauterine infection symptoms. many authors have described the pathogenesis of hypoxic-ischemic encephalopathy. briefly, after an acute hypoxia-ischemia insult, activated resting microglia show macrophage-like activity. this leads to a break-down of the blood-brain barrier, infiltration by peripheral leukocytes, and brain exposure, which further exacerbates inflammation. the role of systemic inflammation is being evaluated in the animal model. it is known that systemic inflammation plays a role in traumatic brain injury (tbi) and is an independent risk factor for poor outcome in isolated tbi patients. on the other hand, m2-phenotype microglia inhibit inflammation and protect neurons from secondary damage and death. however, anti-inflammatory mechanisms in neonates are immature and expose them to extremely intensive inflammation. therefore, anti-inflammatory agents, including stem cells, may be beneficial in these patients. disclosure: three of four authors are employees of the polish stem cell bank, warsaw, poland reference: background: under the hypothesis that early natural killer cell infusion (nki) following haploidentical stem cell transplantation (haplo-sct) will reduce relapse in the early post-transplant period, we conducted a pilot study to evaluate the safety and feasibility of nki following haplo-sct in children with recurrent neuroblastoma who failed previous tandem high-dose chemotherapy and autologous sct. methods: we used the high-dose 131 i-metaiodo benzylguanidine and cyclophosphamide/fludarabine/antithymocyte globulin regimen for conditioning and infused 3×10 7 /kg of ex-vivo expanded nk cells derived from a haploidentical parent donor on days 2, 9, and 16 posttransplant. results: seven children received a total of 19 nkis, and nki-related acute toxicities were fever (n = 4) followed by chills (n = 3) and hypertension (n = 3); all toxicities were tolerable. grade ≥ii acute gvhd and chronic gvhd developed in two and five patients, respectively. higher amount of nk cell population were detected in peripheral blood until 60 days post-transplant compared with reference cohort. cytomegalovirus and bk virus reactivation occurred in all patients and epstein-barr virus in six patients. six patients died of relapse/progression (n = 5) or treatment-related mortality (n = 1), and one patient remained alive. conclusions: nki following haplo-sct was relatively safe and feasible in patients with recurrent neuroblastoma. further studies to enhance the graft-versus-tumor effect without increasing gvhd are needed. disclosure: nothing to declare regenerative medicine p652 repeated administration of g-csf using stem cell mobilization protocol could induce improvement of cognitive functions of children with cerebral palsy: phase ii randomized placebo-controlled study background: we performed phase ii randomized placebocontrolled clinical study to reveal the safety and feasibility of repeated granulocyte colony-stimulating factor (g-csf) administration for improvement of cognitive functions of children with cerebral palsy. methods: forty-four children with non-severe type of cerebral palsy were enrolled, and their age were 2-10 years old. g-csf (5μg/kg) was administered for 5 days subcutaneously every 3months during 18 months. we compared their cognitive functions with the magnetic resonance imaging (mri) findings and the following tools between before and 18 months after treatment; zoo location and picture memory as working memory index (wmi) in wechsler preschool and primary scale of intelligence (wpssi), receptive and expressive vocabulary test (revt), and visual motor integration (vmi)/visual perception (vp) test. mobilized stem cell count and cytokine levels were measured before (d+0) and after (d+5) g-csf administration for 5 days every 3 months. results: no significant findings in demography was noticed between g-csf (g-) and placebo (p-) groups. no serious adverse events were observed during the whole study period. the non-severe adverse events such as urticaria (n=1), itching sense (n=3), bone pain (n=1), headache (n=1), fever (n=2), and stomatitis (n=1) were tolerable. the parents felt the clinical improvements of cognition in 10 cases of g-group and 4 cases of p-group (p=0.0367), of language in 8 cases of g-group and 3 cases of p-group (p=0.0632). in zoo location test, we can not find out the significant score (expressed as age equivalent) differences between g-and p-groups. however, in picture memory test, there were significant improvement of age equivalent of 10 months (45.60±19.66→55.60±23.27) during 18 months of study period in g-group compared to 3 months in p-group (p=0.0242). in revt, there were significant improvement of 18 months of age equivalent in expressive tests of g-group (57.87±33.66→76.00±43.38, p=0.0198) compared to 8 months in p-group. no significant findings were noted in receptive test. vmi test showed the increasing tendency of 14 months of age equivalent in g-group (47.75±13.22→61.13±17.78, p=0.0746) compared to p-group. the increment of cd34 + cell counts in peripheral blood were significant in ggroup compared to p-group. the changed levels of interleukin (il)-6, il-10, vascular endothelial growth factor (vegf) as well as g-csf were noted in g-group. we also observed the correlation of cognitive function tests and white matter connectoms of several networks using functionally-defined white matter atlases. conclusions: the repeated administration of g-csf using stem cell mobilization protocol is safe and feasible to improve the language and cognitive functions in children with cerebral palsy. further studies for cellular and paracrine effect of g-csf and/or mobilized peripheral blood stem cells would be needed. background: while high-dose chemotherapy (hdct) with autologous hematopoietic stem cell transplantation (auto-hsct) is an integral part of multimodal therapy for highrisk neuroblastoma (hr nb), there are still subgroups, in which the results are extremely poor. for these patients allografting (allo-hsct) may offer some hope. methods: we summarize the experience of 83 consecutive hr nb patients receiving therapy in our pediatric transplant department in 2008-2017. the median age was 4 years (8 months to 22 years). a total of 78 auto-hscts and 20 allo-hscts were performed. all auto-hsct recipients were characterized by one or several high-risk features: age of more than 18 months at disease (onset n=56), primary disseminated disease (n=59), unfavorable biologic variant (n=29), poor 1 st -line therapy response (n=16) or systemic relapse (n=9). most patients (n=75) received bu-mel hdct (in younger patients oral busulfan was replaced by busilvex), in 3 primary resistant cases a 5d/5d regimen was used. a total of 20 patients with 1st (n=11) or 2nd (n=2) chemosensitive relapse, resistant relapse (n=4) or poor mobilizers with locally advanced resistant tumor (n=3) received allo-hsct from haploidentical donor with fludarabine-based ric. in 12 cases the transplant was modified via immunomagnetic positive or negative selection, 8 patients received post-transplant cyclophosphamide (post-cy)-based gvhd prophylaxis. gvhd prophylaxis also consisted of calceneurin inhibitors and sirolimus. thirteen of 20 allo-hsct recipients received posttransplant immunoadoptive (n=6) or targeted (n=7) therapy. results: the 5-year os and efs in auto-hsct recipients was 48% and 36%, accordingly. all but one patient engrafted with a median time of 17 (11-51) days. bu-mel regimen was characterized by acceptable toxicity with most common toxicities being oral mucositis and infectious complications. the vod/sos incidence was only 3%. four patients dies due to infection (n=2), cns hemorrhage (n=1), and secondary leukemia (n=1). according to multivariate analysis the most important prognostic factors were response to 1 st line therapy and post auto-hsct mibg scan results. the prognosis in initially resistant patients with good response to 2 nd or 3 rd -line therapy was still very poor (all patients relapsed with the median efs of 12 months). all patients receiving a second auto-hsct after relapse died due to disease progression. with a median follow up of 6 (1-80) months 9 allo-hsct recipients are alive, 6 of them with no signs of disease progression. all long-term responders received post-transplant therapy. one patient died due to transplant complications, other deaths were caused by disease progression. there was no obvious difference between outcomes in post-cy based and transplant modification-based transplantations. agvhd more often developed in modified transplant recipients (8 patients vs 2 in post-cy group, 4 of these cases gr iii-iv), 3 patients in post-cy group had grade iii-iv hemorrhagic cystitis. the median time to engraftment was longer for ptcm group compared to transplant modification group (d +23 vs. d+17, accordingly). conclusions: while single hsct with auto-hsct is a golden standard in hr nb patients, the relapse rate is still high and the prognosis in relapsed/refractory patients is dismal. the allografting has some limited effectiveness in these cases and post-transplant therapy has a potential for further improvement. disclosure: nothing to declare p654 abstract already published. veno-occlusive liver disease (vod) is frequent but well treatable with early defibrotide administration in children with neuroblastoma receiving high-dose busulfan and melphalan background: using high-dose intravenous busulfan and melphalan (bumel) prior to autologous stem cell transplantation (sct) in children with high-risk neuroblastoma, seems to decrease toxicity of the myeloablative regimen, except for vod. in this multicenter retrospective study we aimed to assess the outcome of bumel-associated vod with early defibrotide treatment intervention. methods: we retrospectively analyzed 64 children with high-risk neuroblastoma who underwent autologous sct with i.v. bumel regimen in slovakia and prague, czech republic in the period 1/2008 -10/2018. busulfan was administered in q6 hour schedule, with therapeutic drug level monitoring in 84% of patients. all vod patients except one were treated with defibrotide starting at a standard dose of 25 mg/kg/day, given in 4 doses per day. 1 patient was treated with supportive therapy only. ursodeoxycholic acid was used as prophylaxis in all patients. vod was established using the modified seattle clinical criteria (corbacioglu, lancet 2012). results: the incidence of vod was 23% (15/64) in patients treated with intravenous busulfan and melphalan. there was no significant difference in busulfan total dose/kg between patients with (19.2 mg/kg (sd=1,8)) and without (18.0 mg/kg (sd=2,4)) vod manifestation. vod developed at a median of 17 days after sct (range 11 -22 days). anicteric forms of vod were documented, although 73% patients with vod (11/15) presented with increased bilirubin. 73% patients with vod (11/15) developed ascites but only 4 patients (27%) required ascites drainage. no vod patient received renal replacement therapy and only one needed mechanical ventilation. importantly, we successfully treated vod in all patients. relapse or progression of neuroblastoma was the cause of death in 4 vod patients (27%) who died. conclusions: despite targeting busulfan levels to decrease toxicity of the regimen, vod is common (we observed vod incidence exactly in the range of the siopen hr nbl-1 multicenter study (ladenstein, 2017) ). early recognition and early treatment with defibrotide seems to be effective in vod associated with bumel regimen -none of our 15 patients died due to vod. disclosure: nothing to declare results of high-dose chemotherapy (hdct) with autologous hematopoietic stem cell transplantation (auto-hsct) in the treatment of ewing sarcoma family tumors (esft) background: while current dose-intense treatment protocols allow achieving 70-80% survival in localized esft patients, the long-term survival in high-risk cases is still unsatisfactory. although there is a considerable body of data on high-dose consolidation the real effectiveness and optimal indications for this option are still not completely clarified. therefore, a large prospective cohort analysis may still yield useful data. methods: the whole cohort includes 73 consecutive highrisk esft patients with median age of 12 (range 1-23) years receiving hdct with auto-hsct in 2007 to 2018 after obtaining 1 st (n=25) or 2 nd (n=3) cr, pr (n=35), or stable disease (n=10). the high-risk features included lung (n=52), bone (n=25) or bone marrow (n=11) involvement, inadequate local control in primary axial tumors (n=52), large lesions volume or poor treatment response (n=52), and chemosensitive relapse (n=9). most patients had several risk factors. disseminated disease patients were also evaluated according to prognostic score by r.ladenstein et al. highdose busulfan-melphalan followed by autologous stem-cell transplantation (hdt/sct) was used. results: the median observation time was 60 (range 3-130) months. the 5-year overall (os) and event-free (efs) survival were 40% and 37%, accordingly. most important outcome predictors were inadequate local control in chemoresistant cases, a primary tumor volume more than 200 ml, more than one bone metastatic site, bone marrow involvement and additional lung metastases. according to prognostic risk score in disseminated disease esft patients identified three groups with 5-year os rates of 49% for score ≤3 (19 patients), 36% for score 3 to 5 (27 patients), and 8% for score ≥5 (12 patients), (p< 0.01). conclusions: while bu-mel hdct with auto-hsct may still be a feasible option with acceptable toxicity for chemosensitive patients with inadequate local control and some primarily disseminated cases it is ineffective in primary resistant or very high-risk metastatic patients. disclosure: nothing to declare efficacy of tandem high-dose chemotherapy with autologous hematopoietic stem cell transplantation in the treatment of infant embryonal brain tumors day +15 (range, 8-83), after the second auto-hsct was day +17 (range, 9-86). two-year overall survival (os) was 76% and disease free survival (dfs) was 68%. dfs was significantly better among patients with mb (95%) and pnet (75%) in compared to children with etmr (55%), pb (33%) and atrt (0%), (p=0,01). dfs in patients who received tandem auto-hsct was 75% in compare to infants who received only one auto-hsct (44%), (p=0,000). complications grade 4 (according to common toxicity criteria 2014) were observed in 14% of cases. conclusions: employment of tandem hdct with auto-hsct in primary infant embryonal brain tumors may be a feasible option for patients after induction treatment. both conditioning regimens had acceptable toxicity. all patients who had tandem hdct with auto-hsct had better os (75%) in compare with single hdct (44%). patients with mb and pnet had better prognosis with os 95% and 75%, respectively, in compare with other embryonal tumors. disclosure: nothing to declare background: metastatic extra ocular retinoblastoma is carrying a poor prognosis. therapeutic intensification with high-dose, marrow-ablative chemotherapy and autologous hsct has been explored, but its role is not yet clear. this study aimed to evaluate the survival outcome of patients with extraocular retinoblastoma post autologous stem cell transplant, treated at single center methods: this is a retrospective study included all patients with metastatic extraocular retinoblastoma (stages 4a and 4b) that underwent autologous hsct at the children cancer hospital egypt (cche) 57357 from november 2010 to july 2017, the treatment protocol was adopted from cog protocol (aret0321) as all patients received 4 cycles induction chemotherapy followed by consolidation myloablative conditioning, cem (vp16 416.6 mg/m2 x3, melphalan: 40 mg/m2 x4, carboplatin: 500 mg/m2 x3) and stem cell rescue. patients data including initial disease characteristics, transplant data, and survival outcomes were collected and analyzed results: a total of 11 cases were included with median age of 1.7 years, and male to female ratio 2.66. nine patients (81%) were initially presented by extra ocular disease, while 2 patients were presented by intra ocular disease and progressed to metastatic disease. according to cog staging of extra ocular disease, 4 patients had stage 4a, and 7 were stage 4b (5 of them had trilateral disease). after induction therapy, 7(63%) showed complete response and 4 (36%) had ≥ partial response. with average cd34 count of 4x10 6 / kg, the median time to anc and platelet engraftment were 10 days and 19 days respectively, and there was no transplant related mortality. post-transplant radiotherapy was given only to 2 patients. with median duration of follow up of 32 months, the overall and event free survival rates of whole patients were 88.9% and 87.5% respectively conclusions: high dose chemotherapy and stem cell transplantation is a promising potential curative option for patients with metastatic extra ocular only two primary gf (1.4%) occurred, both without dsa. 20 patients developed a primary pgf (15%). 3-years os, 3years pfs and 1-year nrm were analyzed according to the presence of dsa in comparison with negative population. no statistically difference was found. no impact of the presence of dsa on the risk of developing gf and pgf was revealed. major outcomes of transplant was analyzed separately in patients with pgf and good graft function (ggf). 3-years os, 3-years pfs and 1year-nrm in ggf and primary pgf populations were 62% vs 20% (p< 0.0001); 53% vs 20% (p< 0.001), 12% vs 40% (p=0.009), respectively. conclusions: the presence of low level of dsa in the absence of desensitization doesn't correlate with the risk of developing gf and pgf. patients who experienced a pgf had worse outcomes in comparison with patients with ggf. disclosure: nothing to declare. the impact of hla-dpb1 mismatch in t-cell replete unrelated donor allogeneic stem cell transplantation background: high resolution matching of donor-recipient hla improves outcome in allogeneic stem cell transplants. matching for hla-a, -b, -c, -drb1 and -dq is mandatory in our transplant centre, to identify 10/10 or 9/10 matched unrelated donors. high resolution matching for dpb1 has been added over the last 10-15 years. however, the role of dpb1 matching is not yet clearly defined. methods: in this study, we retrospectively analyzed the impact of hla-dpb1 matching on the outcome of t-cell replete allogeneic hematopoietic stem cell transplants with cya/mtx-and without atg as gvhd prophylaxis in patients with hematological malignancies at oslo university hospital between 2005 and 2017. 301 patients with an unrelated donor fully matched (10/10) at hla-a, -b, -c, -drb1 and -dqb1 loci were included. further, 87 patientrecipient pairs were also fully matched on dpb1 (12/12); 118 had permissive and 96 had non-permissive mimatches of one or two dpb1 alleles. the three groups were comparable with respect to diagnosis, gender, age, cytomegalovirus serostatus and conditioning regimen. results: cumulative incidence of relapse at 5 years were significantly higher in the dpb1 matched pairs compared with the permissive and non-permissive mimatched ones, at 40% vs 22% and 13% (p< 0.001) respectively. relpase free survival and overall survival were superior in the nonpermissive and permissive dbp1 mismatched groups vs the fully matched, at 60% and 49% vs 29% (p=0.01) and 59% and 53% vs 40% (p=0.09) respectively. no difference in frequency of acute gvhd grade ii-iv between the three groups were found; dp match 43%, permissive mismatch 40% and non-permissive mismatched 48% (p=0.49). neither was there a difference seen in gvhd grade iii-iv; 12% vs 17% vs 11%, respectively. finally, there were similar outcomes between the three groups regarding chronic gvhd and trm. in corrected multivariate analysis, only dp matching had significant influence on mortality and survival. conclusions: our results show a favorable relapse free and overall survival following a mud allotransplant with a dpb1 permissive or non-permissive mismatched donor compared to a fully dpb1 matched. this is likely due to an increased gvl-effect in dpb1 mismatched groups without the counterbalance of increased acute gvhd and trm. disclosure: nothing to declare p662 a haploidentical may be a better choice than a female genoidentical donor to transplant a patient with high risk acute myelogenous leukemia in first remission norbert gorin 1 , myriam labopin 1 , didier blaise 2 , goda choi 3 , gerard socie 4 , jean henri bourhis 5 , fabio ciceri 6 , emmanuelle polge 7 , arnon nagler 8 , mohamad mohty 1 china, 3 the first affiliated hospital of soochow university, hematology, suzhou, china background: despite the incidence of leukemia increases with age, currently the geriatric population is poorly represented in the standards of care concerning that older adults undergoing hematopoietic cell transplant (hct) may experience higher transplant-related mortality (trm). previous studies have demonstrated that donor age is vital for older patients by affecting trm and survival. accordingly a relevant question is whether outcomes can be improved with a younger hla-haploidentical offspring donor rather than an older hla-matched sibling (msd). in our previous multi-center report under atg+g-csf based protocol for haplo hct, offspring donor is correlated with lower trm and higher leukemia free survival (lfs) as compared with older msd in subgroup analysis for recipients >50years although it did not reach statistical significance. on the contrary, in a recent report from ebmt and cibmtr under ptcy modality for haplo hct, among patients aged 55 to 76 years, despite lower chronic graft-versus-host-disease (gvhd), graft failure, trm, and overall mortality were higher after transplant from offspring compared with an msd although there were differences in transplant platforms between the 2 groups. methods: we extended our multi-center dataset and a matched pair analysis was performed. outcomes of 142 acute leukemia patients (>=50 years) transplanted in cr1/ cr2 who received hct from offspring (n=57) or msd (n=85) between jan, 2013 and june, 2017 present in the multi-center database were analyzed. because the patient population was small, a 1:1 ratio matched pair analysis was implemented with the following matching factors: underlying disease (acute myeloid leukemia, acute lymphoblastic leukemia), disease status (cr1/cr2), age and sex of patients, year of transplant, blood group incompatibilities, and sex of donor. results: we were able to match 41 offspring with 41 msd patients. the two matched groups were comparable in baseline characteristics except for donor age due to the family relationship. all patients achieved myeloid recovery with a median time of 14d and 12d for msd cohort and offspring group (p=0.002). the 100d platelet recovery rate was 95% in both groups. the cumulative incidence of grade ii-iv acute gvhd in msd cohort was significantly lower than in offspring group (12% vs 37%, p=0.009) while the incidence of chronic gvhd in msd cohort was significantly higher than in offspring group (51% vs 27%, p=0.013). the 3-year trm (10% vs 31%, p=0.028) were significantly lower in offspring-hct compared with in msd-hct and relapse incidence was comparable (8% vs 14%, p=0.56). as a result, the 3-year overall survival (57% vs 82%, p=0.033) and lfs (55% vs 82%, p=0.024) ( figure 1 ) were significantly higher in offspring-hct compared with in msd-hct. in a multivariate analysis, msd-hct remained a significant factor for decreased overall survival (hr 2.791(1.141-6.824), p=0.024) by increased trm ), p= 0.032) in comparison with offspring-hct. conclusions: these data favor a young offspring over an older msd in patients >50 years. the current analyses confirm non-hla donor characteristics, rather than hla disparity, predominantly influence survival in older acute leukemia patients. validation of these findings requires a prospective trial wherein the transplant platforms can be closely matched. [[p664 image] 1. figure1. lfs in offspring-hct compared with in msd-hct (55% vs 82%, p=0.024)] disclosure: nothing to declare. impact of sibling donor-recipient sex combinations on rejection after hla-matched bone marrow transplantation for severe thalassemia 1 cure2children foundation, florence, italy, 2 sankalp india foundation, bangalore, india, 3 people tree hospitals, bangalore, india, 4 south east asia institute for thalassemia, jaipur, india, 5 pakistan institute of medical sciences, islamabad, pakistan, 6 central asiri hospital, colombo, sri lanka, 7 nawaloka hospital, colombo, sri lanka, 8 kokilaben dhirubhani ambani hospital, mumbai, india background: severe thalassemia (st), i.e. a thalassemia syndrome with inability to keep spontaneous hemoglobin > 7 g/dl, is a common indication for bone marrow transplantation (bmt) in children in the middle east and south east asia. sex mismatch has been associated with increased risk of solid organ rejection but is not generally considered an important transplant-associated risk factor in the context of fully matched sibling bmt for st. methods: a total of 154 consecutive sibling bone marrow transplants carried out between january 2009 and april 2017 after conditioning with busulfan (14 mg/kg oral, not adjusted to serum levels) and cyclophosphamide (200 mg/kg) (2 patients) in addition to either thiotepa (10 mg/kg) (49 patients), or anti-thymocyte globulin (genzyme 4 mg/ kg or fresenius 16 mg/kg on days -12 to -10) (92 patients) and fludarabine 150 mg/m 2 (11 patients) were analysed. all cases received cyclosporine and methotrexate or mycophenolate mofetil as gvhd/rejection prophylaxis. in the thiotepa group methylprednisolone at 0.5 mg/kg/day was also used during the first 30 days after bmt (lucarelli protocol 6i). bone marrow was the source of hematopoietic stem cells in all cases, in the atg group it was g-csfprimed (5 μg/kg/dose twice daily for 3 to 5 days prior to harvest). all patients were considered low risk based on liver size < 2 cm from costal margin and age less than 15 years (median 4.2 years, range 0.9 to 14.5), all sibling pairs where hla-compatible. results: [[p665 image] 1. sibling donor-recipient sex combinations.] the lowest rejection rate (5%) was observed in the sister to sister (s2s) group of 24 cases, followed by brother to brother (b2b) group of 40 cases with 11%. in the sister to brother (s2b) group of 59 cases, rejection rate was 21%, and 26% in the brother to sister (b2s) group of 31 cases. on univariate analysis the only significant difference at the p 0.05 level by log rank test was b2s vs. s2s groups (rejection proportions of 26% and 5% respectively). interestingly, all 3 patients with rejection and persistent pancytopenia were female recipients of male grafts. conclusions: even though several preparative regimens were employed over an 8-year period, our data suggests that sex mismatch among compatible siblings should be considered as a relevant variable related to bmt decisionmaking. we also recommend to consider autologous back up hematopoietic stem cell collection and storage in sibling sex mismatched transplants, particularly in brother to sister bmts. same-sex fully matched related bmt for severe thalassemia might be the best scenario in which reducedintensity preparation strategies aiming at maximizing fertility preservation might be explored. disclosure: nothing to declare. outcomes of t-cell replete hematopoietic cell transplantation from mismatched related or unrelated donors using high dose post-transplant cyclophosphamide based gvhd prophylaxis background: high dose post-transplant cyclophosphamide (ptcy) based gvhd prophylaxis overcomes immunological barriers in hla mismatched donor transplantation. ptcy has been adopted in many centers as de facto standard for hct from haploidentical donors (haplo hct). it's use in mismatched unrelated donor transplant (mmud hct) is less well established. methods: we analyzed retrospectively outcomes of contemporary cohorts of patients who underwent haplo hct or mmud hct using ptcy + cyclosporine (csa) and mycophenolate mofetil (mmf) at our center. we compared these outcomes with outcomes of cohorts of patients who underwent hct from matched unrelated donors (mud) using atg based gvhd prophylaxis or matched sibling donor (msd) with csa and mmf. patients and donors were considered matched if they background: hla-alloantibodies are a major risk factor for engraftment failure in allogeneic hematopoietic stem cell transplantation (hsct). particularly, complement fixing, donor specific antibodies were shown to be associated with early engraftment failure. prospective antibody-screening, although not currently required for donor search, could permit early identification of high risk patients for positive crossmatch. aim of this study is to set the basis for future applicability of antibody-screening-based definition of acceptable mismatches in donor selection, by creating a large prospective antibody-screening database of patients due to receive an hla-mismatched allogeneic hsct. methods: patients (n=2106) diagnosed with mds/mps, nhl, mm, cll, cml, anaemia (aplastic anemia, hemoglobinopathies, pnh) and hl were prospectively screened for hla-antibodies whenever initial donor search indicated that no completely matched donor would be available. screening was performed with an elisa class i +ii screening assay. all positive screening cases were tested for antigen-specific antibody identification with luminex sab, and acceptable mismatches were defined. the results were subsequently considered in donor search and selection. we now report the frequencies of alloimmunization observed in these patients. results: the highest rate of alloimmunization was observed in patients from the anaemia disease group (overall 40.5%) followed by those from the mds/mpn group (overall 24.8%). the lowest immunization rates were observed in cll (overall 6.9%) and hl (7.0%) patients. alloimmunization rates for hla-class i antigens (p< 0.001) were significantly higher compared to hla-class ii antigens. overall hla-class i immunization rates ranged from 2.3% to 33.3%. hla-class ii immunization rates ranged from 4.7% to 19.8% (table 1) . conclusions: our findings suggest that patients with high transfusion burden like anaemia and mds/mpn patients have the highest risk of hla-alloimmunization with 40.5% and 24.8% anti-hla prevalence rates, respectively. analysis of follow-up data, will enable us to confirm whether prospective definition and consideration of acceptable mismatches in donor selection may lead to similar engraftment failure rates between immunized and non-immunized patients undergoing hlamismatched hsct. background: mothers displaying a persistent fetal microchimerism (fm) proved to be the most suitable donor in t cell-depleted haploidentical stem cell transplantation (hhsct) in children. we presumed that fetal cells leave an imprint in the mothers' immune system which positively affects recognition and elimination of malignant cells in the child by maternal effector cells. distinct killer cell immunoglobulin-like receptors (kir)/hla constellations are not only associated with reduced relapse rates after hsct in children, but also supposedly influence the establishment of an fm. methods: after approval by the local irb and obtaining informed consent, we initiated a protocol to elucidate the factors that influence the establishment, persistence and effect of fm. we established a digital droplet pcr (ddpcr) protocol to determine the fetal microchimerism. for differentiation between maternal and fetal cells, biallelic short insertion/deletion polymorphisms were used. kir and hla-c genotyping was performed by ssp-pcr. parental nk cell alloreactivity against the respective leukemic blasts and kir phenotyping were analyzed by flow cytometry. results: we analyzed 45 parents, whose children were treated for hematological diseases at the university medical center hamburg-eppendorf. a fetal microchimerism was detected in 25% of the mothers. the amount of fetal cells varies between individuals (8x 10 -6 -9x 10 -4 ). we observed a positive correlation between a persisting fm and hla-c1 homo-and heterozygous mothers along with a maternal cen a/b and cen b/b genotype. additionally, fm positive mothers showed a higher surface expression of the hla-c1 respective receptors kir2dl2/s2. the percentage of alloreactive maternal nk cells against fetal cells was higher compared to paternal nk cells; while alloreactivity of fm positive maternal nk cells was similar to nk cells from fm negative mothers. conclusions: persistence of fm was more frequent in mothers carrying at least one hla-c1 allele and a centromeric b/x motif. phenotypically, fm positive mothers had higher expression of kir2dl2/s2 indicating a role of these receptors on the persistence of an fm. in vitro, maternal nk cells showed a higher alloreactivity compared to paternal nk cells. there was no difference in alloreactivity whether the mothers were fm positive or negative, suggesting other mechanisms are responsible for the superior outcome in transplantation from fm positive mothers. disclosure: nothing to declare background: although there have been significant improvements with conventional therapies in beta thalassemia major, hematopoietic stem cell transplantation is only curative therapy. related donors are preferred to diminish transplant risks. in lack of identical related donor, identical unrelated donors are second best choice. in this study, thalassemia major patients transplanted from unrelated donors (mud) were compared with thalassemic patients transplanted from relative donor (mrd) retrospectively. methods: 45 patients who were transplanted between june 2016 and december 2017 in bahçelievler medical park hospital pediatric bone marrow transplantation unit were evaluated retrospectively. all patients were classified according to pesaro risk classification. thirty four of 45 received busulfan, fludarabine, cyclophosphamide, thioteopa for conditioning, 11patients received myeloablative preparation regimen with treosulfan, fludarabine, thiotepa, cyclophosphamide. all patients were given atg, cyclosporine and methoteraxate for gvhd prophylaxis. the patients were compared in terms of acute complications in first 100 days, engraftment, chimerism, acute and chronic gvhd after transplantation. results were evaluated with ibm spss statistics 22 (ibm spss) program. results: a total of 45 patients, 26 (57.8%) male and 19 (42.2%) female, aged between 1 and 18 years (median 5 years) were evaluated. patients were evaluated in two groups as "mud" (n = 15) and "mrd" (n = 30) groups. there was no difference between groups about given stem cells (mud 6,16±1,07x10 6 /kg and mrd 6,25±1,24x10 6 / kg). neither significant difference between different pesaro risk groups in terms of developing acute and chronic gvhd and nor decreased chimerism were detected. neutrophil engraftment time (16,40 days) in mrd group was significantly longer than mud group (13,71 days) (p = 0.006) but no difference between platelet engraftments were observed. gvhd ratio was 33.3% in mud donor group and 13.3% in mrd group and no statistically significant difference was found(p> 0.005). the incidence of engraftment loss in mud group was 13.3% and 36.7% in the mrd group, and there was no statistically significant difference (p>0.05). the rate of decreased chimerism was found to be significantly higher in the mrd group (50%) than in the mud group (6.7%) (p:0.010; p< 0.05). the survival rate was 92.9% in the mud group and 96.7% in the mrd group. the disease-free survival rate was 90.9% in the mud group and 50% in the mrd group. the disease-free survival of mud group was significantly higher than mrd group (p:0.010). conclusions: in our study, transplant related complications and success of transplantation with both mud and mrds were found to be similar. it is promising for mud transplantations to found lower decreased chimerism and similar os and dfss. based on these results, it was concluded that hsct from non-family donors, especially for patients incompatible with chelation therapy and had organ damage, transplantation from unrelated identical donors can be a good choice. although the results of our study seem promising, larger patient groups and prospective clinical trials are required. disclosure: nothing to declare background: use of g-csf stimulation of bone marrow (bm) donors is beneficial in many aspects; it can enhance tnc yield, but also have an immunomodulatory effect on donor t cell function, particularly invariant natural killer t (inkt) cells expansion as well as apcs. we analyzed outcomes of 34 consecutive patients receiving bone marrow from hla-haploidentical donors that were stimulated with g-csf prior to harvest. methods: in the time period between 05/2012 and 05/ 2018, 34 patients received bone marrow from donors stimulated with 10 ug/kg bw of g-csf on days -2, -1 and day of bm collection. four patients (12%) received myeloablative (bucy) conditioning, one (3%) received tec ric conditioning while 29 (85%) received nma ("baltimore") conditioning. all patients received posttransplantation cyclophosphamide (ptcy) on days +3 and +4, tacrolimus and mmf were started on day +5. for 2 patients donors were fathers, 11 mothers, 9 siblings and 12 children. results: median age was 43 years (20-63), there were 14 female and 20 male patients. twelve patients had aml, 10 hodgkin lymphoma, 5 all, 3 mds, 3 nhl and 1 cml. median number of infused tnc in graft was 4.7x10 8 /kg bw (1.8-8.2) and cd34+ cells 1.9 x10 6 /kg bw (1-4.5) . after median follow up of 397 days (range 26-2139), overall survival was 57%, with median survival of 71 months. engraftment was established in 29 (85%) patients, 2 (6%) had primary rejection and 3 patients (9%) died in sepsis prior to engraftment. of 29 patients that engrafted, further 3 (9%) patients had secondary rejection, two of them were transplanted again from a haploidentical donor, both using pbsc as a source of graft. median time to neutrophil recovery (anc>500) was 23 days (12-36), while median time to platelet recovery (plt>20x10 9 /l) was 30 days (12-72) in evaluable patients. cumulative incidence of agvhd ii-iv was 27.7% (95% ci, 13-44);of note is that of 9 patients that developed agvhd only one had grade iii, while remaining 8 patients had grade ii. cumulative incidence of cgvhd requiring treatment was 6.9% (95% ci, [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . cumulative incidence of relapse was 36.1% (95% ci, 20-52) and trm was 25.2% (95% ci, 11-41). conclusions: the use of g-csf mobilized bm graft in the hla-haploidentical setting with ptcy has proven to be useful to us, not only in terms of tnc yield which was more than satisfactory and contributed to adequate hematological recovery, but also in the excellent control of both acute and chronic gvhd, with most patients developing agvhd of grade ii and only one grade iii (actually developed only after dli given for decreasing chimerism). comparative studies are of course warranted to prove benefit, but this data contributes to the growing body of evidence that indeed donor stem cell stimulation with g-csf has potentially powerful immunomodulatory effect. disclosure: nothing to disclose p674 other-relative donors as a reliable bank for allogeneic hsct in countries with culturally accepted cousin-cousin marriages: a two-year report from a pediatric center in iran background: although the optimal donors for patients undergoing allogeneic hematopoietic stem cell transplantation (allo-hsct) are fully-matched siblings, the cousincousin (consanguineous) marriages in some countries have extended the chance to find a matched donor for the hsctrecipient. in this study, an outcome analysis of transplanted patients receiving stem cells from their relatives other than siblings (other-relatives or non-sibling donors) is provided. methods: in this retrospective cross-sectional study, a two-year report of patients who received allo-hsct from their other-relative donors during september 2016 to september 2018 at the department of stem cell transplantation of children's medical center in tehran, iran is presented. the patients were followed up until 1 st december 2018. results: during this time period, 178 patients underwent hsct (both autologous and allogeneic) at this center, of which 159 cases received allo-hsct. out of allo-hsct recipients, the donors of stem cells for 51 cases (32.1%) were their other-relatives. the median (range) age at hsct was 6 (1-11) years and the majority of patients were boys (31/51, 60.8%). according to disease class, the patients were most commonly involved with non-malignant hematologic diseases (18/51 patients, 35.3%) (figure) . the source of hscs for most patients (48 cases, 94.1%) was peripheral blood and for only 3 patients the source was bone marrow. the donors for 50 patients were fully matched and only one patient received the hscs from a one-locus mismatched donor. hsct was successful in 50 patients with most of them achieving full chimerism (44 patients, 86.2%) followed by those developing mixed chimerism (6 patients, 11.8%) and only one patient (2%) experienced graft failure. post-hsct complications included cmv infection in 34 patients (66.7%), other infections in 7 (13.7%), hemorrhagic cystitis in 3 (5.9%) and pres in 2 (3.9%). acute gvhd occurred in 22 patients (43.1%) and chronic gvhd in 3 (5.9%). death occurred in 7 cases and 5 of them were transplant-related, while 1 was due to disease relapse and 1 due to graft failure. the median of overall survival was 469 (40-792) days. conclusions: the likelihood of receiving hscs from an hla-matched other-relative donor in one-thirds of children undergoing allo-hsct, with comparable outcomes to sibling and unrelated donors (as evidenced in this study compared with other studies), introduces family bank as a reliable source for pediatric allo-hsct in countries with culturally accepted cousin-cousin marriages. hence, for transplant physicians, parental consanguinity would be an indication of an extended search for a potential matched donor among the patient's family. [[p674 image] 1. distribution of patients according to disease and disease class] disclosure: nothing to declare. abstract already published. update on the hla frequency distribution of the portuguese bone marrow donor registry eduardo espada 1 , dário ligeiro 2 , hélder trindade 2 , joão forjaz de lacerda 1 frequency distribution varied throughout the country, allowing for analyses of molecular variance and generation of relatively geographically accurate graphical representations of genetic distances between regions and districts. conclusions: with the most recent hla analysis of the portuguese bone marrow donor registry we were able to extrapolate high-resolution haplotype frequencies from the most common low-resolution hla-a/-b/-drb1 haplotypes (corresponding to 50% of the estimated haplotypes at that level), which will lead to an optimization of its use, hopefully limiting the time between donor search and allogeneic hematopoietic stem cell transplant. disclosure: nothing to declare. abstract already published. abstract already published. unmanipulated haploidentical donor transplantation compared to identical sibling donor had better antileukemia effect for refractory/relapsed acute myeloid leukemia in not remission status background: patients diagnosed with saa with no sibling donors and who are refractory to immunosupression are candidates to hematopoietic stem cell transplant using alternative donors. haploidentical donor transplants has been reported using cyclophosphamide (cy) post stem cell infusion as immunephrophylaxis. the present study has the objective of evaluating overall survival and engraftment rates after haploidentical stem cell transplant for saa in a reference center. methods: 15 saa adult patients (≥15 yo) received hsct from haploidentical donors from de january/2010 to august/2018. median age was 19 y (15-28); donor was the father in six, mother in five and a brother in four cases. stem cell source was marrow in 13 cases (87%). conditioning: 10 patients (67%) received cy 29mg/kg, fludarabine 30mg/m² e tbi 200cgy. the remaining received the same drugs but radiotherapy dose varied from 300-400cgy, all them received immunephrophylaxis with post transplant cy 100mg/kg, cyclosporine and mmf. median of infused cells (tcn) was 4,94x10 8 /kg (2,07-12,66). results: eight patients engrafted (53%). among seven graft failures four received a second haploidentical transplant and one received an unrelated donor transplant as salvage regimen. two patients were successfully rescued after the second haplo and the others died from infectious complications. three years overall survival was 55%. death causes included: five infections and two lung hemorrhage. median survival was 93 days (17-1065). no patient had acute graft-versus-host-disease (gvhd) and one patient had mild c-gvhd. conclusions: haploidentical transplant was feasible as therapy for saa refractory to immunessupression with an overall survival of 55% in this cohort. graft failure however is still a problem to be addressed in this setting. disclosure: no disclosure stem cell mobilization, collection and engineering p681 abstract already published. key performance and quality indicators for a successful bone marrow collection marco sampaio 1,2 , ana salselas 2 , fátima amado 2 , filipa bordalo 2 , sérgio lopes 2 , catarina pinho 2 , susana roncon 2 and one from ecc (staphylococcus spp.)presented positive microbiological results. conclusions: bm collection is a challenging strategy because it is a one-time procedure and manually operatordependent technique; simultaneously it is more difficult to control the final cellular content of the bm, which is a risk for donor volume depletion. bm collection is feasible even with donor and recipient weight difference. poorer performance may be found when higher tnc are requested. we respond efficaciously when the request is between 200 and 400*10 8 tnc but we fail to accomplish higher tnc values. we must emphasise that icc tnc demanded was generally lower than ecc. deciding the appropriate tnc for each patient remains a dare and an art. disclosure: nothing to declare. impact of adding plerixafor to mobilization protocol in the immune reconstitution of vδt cells after autologous hematopoietic stem cell transplantation efrat luttwak 1,2 , yael chava cohen 1,2 , odelia amit 1,2 , irit avivi 1,2 , svetlana trestman 1,2 , esti rom 1,2 , rinat eshel 1,2 , ram ron 1,2 1 tel aviv medical center, tel aviv, israel, 2 sackler faculty of medicine, tel aviv university, tel aviv, israel background: multiple myeloma has remained an incurable disease even in the era of novel therapies. front line treatment typically comprises of induction chemotherapy with 4-6 cycles of a bortezomib-based regimen, stem cell mobilization, and harvesting of peripheral blood stem cells (pbsc) by apharesis, followed by high dose melphalan with hct. while brotezomib-based induction regimens have demonstrated no adverse impact on hematopoietic cell harvest number and quality, no study analyzed the impact of timing of the last brotezomib dose prior to collection. in this study we aimed to determine the effect of the timing of the last dose of brotezomib before hematopoietic cell collection and the collection yield. methods: this was a single center historical prospective study, including all sequential newly diagnosed patients with myeloma between 2012 and 2017 that were given a bortezomib-based induction therapy (≤6 cycles) followed by pbsc collection. we excluded patients who either received 1 st line vtd-pace or lenalidomide-containing regimens. peripheral blood cd34+ cells were measured on the day of collection. patients with cd34+ levels of >10 cells/ microliter started collection on the same day, while those with lower levels were given plerixafor. we performed regression analyses to analyze the impact of a variety of precollection factors, including days from last bortezomib therapy on the collection yield. results: we identified 75 patients who fulfilled the inclusion criteria, table. median time from last dose of brotezomib to first leukapheresis was 11 (range, 2-27) days. a statistically significant correlation was found between the days from last dose of brotezomib and both the first collection day-cd34+ cells/kg (r=0.466, p< 0.001), and the total collected cd34+ cells/kg(r=0.341, p=0.03), figure. the optimal cutoff point as indicated by the roc curve was 8.5 days according to collection success with sensitivity of 79% and specificity of 74%, youden´s index 0.52. in multivariate analysis included other factors affecting collection yield (age, gender, status of disease at collection, and prior radiation) -timing of last dose of brotizomib remained significantly associated with the total collected cd34+ cells/kg (p=0.01). increasing age, female gender, and prior radiation were associated with lower collection yield (p=0.006, 0.012, 0.043, respectively). based on this, we developed a model to predict the total collected cd34pos cells = 11.76+ 0.13 (timing in days of last dose of brotezomib) -0.1 (age) -1.39 (if female) -0.01 (≥pr) -1.35 (if prior radation). conclusions: timing of last dose of brotezomib is an important factor for predicting a successful collection. a washout period of 9 days is associated with a better collection yield. these results should be further validated in a prospective study. age (median, range) 63 (37-78) gender -male (%) 44 (59) prior radiation treatment (%) 18 (24) disease status at collection (%) ≥pr -51 ( disclosure: nothing to declare p685 mobilization with plerixafor in "poor mobilizer" related and unrelated donors of hpc-a in case of failed mobilization with g-csf background: in the allogeneic hpc transplantation, both from related and unrelated donors, the most commonly used source is peripheral blood after mobilization with g-csf. it is however known that about 2% of donors are "poor mobilizers"; the rescue strategies are: a third apheretic collection; bone marrow donation. methods: in 2016-2017 in italy a procedure to be adopted in case of failed mobilization of peripheral blood stem cells has been defined and shared between ibmdr, cnt (transplant national center) and cns (blood national center) and the scientific societies simti, sidem and gitmo, using plerixafor, a selective reversible antagonist of the cxcr4 receptor with its binder (the stromal derived factor sdf-1), in combination with standard g-csf dose. moreover since 2017, in accordance with this protocol, the competent authority (aifa) has extended the registration of plerixafor (law no.648/96), also for the mobilization in "poor mobilizer" healthy donors. finally, in 2018 the protocol was extended to "poor mobilizer" family donors, making management equivalent in related and unrelated donors. at the time of the donor´s informed consent for the donation of hpc, the hypothesis of the lack of mobilization or inadequate collection was illustrated and the possible actions proposed as "back-up donation" were anticipated. failed mobilization of cse has been defined as the presence of one of the two criteria: a number of cd34+ circulating on peripheral blood lower than 20/μl on the 5th day of stimulation (5d), or the collection of cd34+ < 1.0x106/kg weight of the recipient at first apheresis. in these cases, a single dose of plerixafor is administered subcutaneously by health professionals under medical supervision, 0.24mg/kg of body weight, 6-11 hours before the start of apheresis. in case of use plerixafor due to failed mobilization of hpc-a or collection of an inappropriate number of cd34+, the notification is made by the collection center to ibmdr (for both family and non-family donors), and to the recipient transplant center : both donor and recipient express their consent; finally once the collection is completed, the collection center informs ibmdr, which in turn notifies cnt/cns/simti/sidem/gitmo. any adverse reactions/events are notified in real time, based on the sop specifications and current regulations. results: since the introduction of the national protocol, 7 donors (4 unrelated donors and 3 related donors) were treated, presenting at least one of the two inclusion criteria (cd34 < 20/μl at 5d or cd34 < 1x10e6/kg after first collection)after use of plerixafor in all donors, the required dose of cd34 was obtained to ensure successful transplantation, with a sufficient increase in the cd34+ cells. no side effects or adverse reactions related to the administration of plerixafor occurred. conclusions: in cases of failed mobilization in the related and unrelated donor, the use of plerixafor according to the methods described in the shared protocol between ibmdr, cns, cnt, simti, sidem, gitmo, proved to be safe and effective. this protocol emphasizes the great value of the sharing of procedures between the register, institutions and scientific societies, ensuring the supervision of the process and the protection of the donor and recipient. disclosure: nothing to declare background: autologous stem cells transplantation (asct) is an effective treatment option for young patients with multiple myeloma (mm). a minority of patients may still experience untoward toxicity due to delayed engraftment. thus, the current policy in many centers is aimed to increase the target dose of collected cd34 + cells up to an "optimal" level of 4x10 6 /kg per procedure. therefore, an ideal mobilization, aimed to collect 4 to 8 cd34 + cells/kg in one apheresis, should achieve a number of circulating cd34 + cells >40/mcl (very good mobilizers). plerixafor may help to maximize the cd34 + collection but its use is limited by high cost. we carried out a retrospective analysis aimed to predict the quality of mobilization and develop an algorithm to optimize both timing of collection and use of plerixafor. methods: we retrospectively collected data from 91 mobilization procedure performed in our center between 2006 and 2016 for mm. all received the same mobilization protocol with cyclophosphamide (range 3-4 gr/sqm) and g-csf 10mcg/kg from +5. cd34 + cell count was started when white blood cells (wbc) count exceeded 1x10 9 /l. patients were excluded from this analysis if 1) showed a cd34+ count >40/mcl (target achieved at first day count) and/or 2) cd34 + count on second day was missing and/or 3) plerixafor was administered on first day according to previous policies. sixty-eight patients were evaluable for the study. univariate and multivariate logistic regression analysis to study ccd34 + kinetics and assess predictors impact on mobilization was carried out. ratio cd34 +/wbc in first day count, gender, disease category and time from mobilization chemotherapy were also included results: among the 68 patients included in the analysis, the threshold of 40 cd34+/mcl cells on the second day was reached by 35 (51,47%) of patients (groupa) whilst the remaining 33 (24,7%) failed the target (groupb). median (range) wbc x10 9 /l and cd34 + /mcl counts in group a and b were 2,01 (1-5,01) and 21,33 (7,56 -39,91), 1,08 (1) (2) (3) (4) (5) (6) 46 ) and 3,31 (0,06-31,14) respectively, with a statistically significant differences among group (mann-whitney p= 0,01 and p=0,02 respectively). only cd34 + /wbc ratio and cd34 + /mcl on first day count had an impact on kinetics and optimal mobilization. logistic regression model highlight cd34+/mcl (or=1,270; 95% ci: 1,060 -1,522) on first count as an independent predictor of second day optimal mobilizer, with auc of 0.9% (0, 99) in roc analysis. two cd34 + thresholds were then calculated: < 7,56/mcl (ppv 0,81; npv 1,0) that identified poor mobilizer, and ≥ 24,06/mcl (ppv 1,0; npv0,59) that exclude probability to fail on second day. for those with a cd34 + count between 7,56-24,06 the cd34+/wbc ratio (or=1,768, 95% ci: 1,307-2,391) was a predictor of optimal mobilization (auc 0,94; 0,885-0,996); cut-off value was 5,63 (sensibility 94,28; specificity 84,84) conclusions: assessment of circulating wbc, cd34+ and their ratio at wbc recovery in a chemo-based mobilization is a valid tool to manage the collection strategy and the on-demand use of plerixafor. we have developed an algorithm aimed to the use of plerixafor to both rescue poor mobilizers and boost cd34 + count in intermediate mobilizers. background: successful autologous stem cell transplantation (asct) requires the infusion of a sufficient number of hematopoietic stem cells (hscs). peripheral blood (pb) is the most commonly used source of hscs, therefore, it is important to optimize methods used to mobilize the hscs. the most clinically used chemotherapeutic agents for effective mobilization are cyclophosphamide and etoposide. recent published studies suggest that etoposide has a better mobilization effect than cyclophosphamide even at lower doses, but it is not clear why this difference occurs. in this study, we tried to determine whether there is a difference in the mechanism of mobilization between cyclophosphamide and etoposide. methods: first, in order to confirm the clinical data for efficacy and toxicity of mobilization, we retrospectively analyzed the data of patients who were diagnosed with lymphoma and performed mobilization using cyclophosphamide or etoposide from january 2011 to december 2018. second, mesenchymal stem cells (msc) were primarily cultured from the healthy controls, then treated cyclophosphamide or etoposide at a concentration of 10% inhibition of cell growth, and cytokine analysis was performed to identify cytokines known to be associated with mobilization. third, mobilization mouse model using cyclophosphamide or etoposide was generated, total blood was collected at the time of hscs collection, and cytokine and network analysis (using ingenuity pathway analysis) was performed. results: the mobilization yields for cyclophosphamide or etoposide were analyzed. etoposide miblized a significantly higher median number of cd34+cells than cyclophosphamide. the rate of successful or adequate mobilization was also significanctly higher for etoposide in univariate and multivariate analysis (table 1 ). in the analysis of toxicity during mobilization, the incidence of neutropenic fever was higher in the cyclophosphamide group (p = 0.007). during mobilization, cyclophosphamide maintained lower wbc counts than etoposide and showed a large increase in wbc counts at the start of collection ( figure 1 ). the cumulative dose of cyclophosphamide or etoposide in patients who underwent autologous stem cell transplantation did not affect leukocyte (anc>1000/microl or platelet (plt >100k/microl) engraftment. in msc treated with etoposide at a concentration of 10% inhibition of cell growth, il-8, which is a cytokine that promotes hematopoietic stem cell mobilization, were shown a statistically significant increase (figure 2 ). in the mouse model of mobilization (figure 3 ), the levels of kc, one of the il-8 homologues in mice, had significantly increased in the etoposide-treated group compared with the levels in the cyclophosphamide-treated group. the levels of other il-8 homologues, mip-2 and lix, also showed increases in the etoposide-treated group compared with those in the cyclophosphamide-treated group; these differences, however, were not statistically significant (figure 4 ). network analysis based on in vivo cytokine results identified that etoposide could promote mobilization in association with matrix metalloproteinase as compared to cyclophosphamide ( figure 5) . conclusions: etoposide has a higher mobilization efficacy when compared to cyclophosphamide, which could due to the different mechanisms of mobilization through the elevation of il8 and the activation of matrix metalloproteinase associated therewith. background: high-dose chemotherapy followed by autologous blood stem cell transplantation (asct) is a standard therapy for wide range of hematologic and solid malignancies. although various methods have been introduced to improve the peripheral blood stem cell (pbsc) mobilization, autologous stem cell collection (ascc) is not successful in every patient. furthermore, even if the ascc is complete, not all of them lead to asct. we evaluated the result of ascc and actual use of pbsc grafts in current practical setting. methods: we retrospectively reviewed the all consecutive ascc procedures performed at the department of oncology in asan medical center, seoul, korea, between january 2000 and october 2018. the targeted number of background: fanconi anemia (fa) is a rare inherited genetic bone marrow (bm) failure syndrome. while abnormal bm cells production occurs very early in life, the usual age of diagnosis is 5-10 years old. gene therapy (gt) might be an alternative to hematopoietic stem cells (hsc) transplantation, but harvest a large number of autologous hsc remains a challenge. we started a mobilization assay, fancomob, to evaluate the safety and the efficacy of fa patients' mobilization with granulocyte-colony stimulating factor (g-csf) and plerixafor. this study is part of the fa's european gt project "eurofancolen". methods: four patients with fanca mutations following the inclusion criteria were selected before pancytopenia. to note, fa4 was diagnosed before clinical manifestation through family screening. they received subcutaneous injection of g-csf (12 μg/kg twice a day) from d-1 and plerixafor (0.24 mg/kg/day) from d-4. the collection protocol targeted 3x10e6/kg of cd34+ cells, based on a predicted future weight in 5 years. cd34+ cells and white blood cells (wbc) blood count were monitored tightly along the mobilization. patients with more than 10 cd34 +/μl or between 5 and 10 cd34/μl with a clustered aspect detected by flow cytometry after plerixafor injection underwent apheresis. cd34+ cells were immunoselected from the collection with clinimacs purification system (miltenyi) and cryopreserved for further gt manipulation. results: the mobilization target was not achieved for the first two included patients (fa1 12 years old and fa2 8 years old). the minimum value of cd34+/μl required wasn't obtained for fa1 and the flow cytometry cd34+ aspect was not clustered for fa2. cd34+ cells were mobilized quickly but transitionally after plerixafor injection for the last two patients, fa3 and fa4, 5 and 2 years old respectively. both patients underwent apheresis procedures. no cd34+ cell rebound was observed after the apheresis was stopped.collection target was not achieved after four days of collection for fa3. it was obtained the first day for fa4 (figure 1 ). back-up for hsc transplantation could not be cryopreserved because of the limited number of cd34+ cells collected in patients fa3 and fa4. no short-term adverse events were observed. following cd34+ immunoselection, cd34+ cell purity and recovery were poor but in the normal range described in the literature for fanconi patients (8-20%)( table i) . one month after the collection hemograms were unchanged. conclusions: our clinical study offer new data showing that mobilization of fa patients with g-csf and plerixafor is safe and more efficient for younger patients, especially before clinical manifestations of bm failure. further efforts are required to establish an effective technic to purify the cd34+ cells after harvesting. basal cd34+ cell and platelets count are a strong predictor for mobilized peripheral blood stem cells on the 4th day of g-csf treatment in donors cryopreserved pbscs. this was associated with considerable efforts for the patients and caused additional treatment costs. on the one hand, having the therapeutic option of an autologous transplantation in the future may represent a clinically relevant advantage. however, a huge number of stem cell products are kept in storage for many years without ever been used for transplantation. our study provides cause for a careful reevaluation of the current clinical practice, which may help to focus more precisely on patients who actually benefit from a cryostored autologous stem cell graft. [[p693 image] 1. fig. 1 : absolute numbers and relative distribution of stem cell grafts] disclosure: the authors confirm that there are no potential conflicts of interest to disclose, except the following: katharina kriegsmann: research funding from bms, celgene, and sanofi. patrick wuchter: membership in advisory boards for sanofi-aventis. reduction of dimethylsulfoxide (dmso) concentration from 10% to 5% in criopreservation of stem cells. influence on the kinetics of engraftment and tolerance to infusion patricia lopez-pereira 1 , beatriz aguado 1 , elena sola 1 , carmen cámara 1 , isabel vicuña 1 , lorena vega 1 , adrian alegre 1 1 hospital universitario de la princesa, madrid, spain background: dmso is the cryoprotectant most used in the cryopreservation of stem cells. it is associated with adverse effects during the infusion of the product, its toxicity being proportional to the volume infused. the most common concentration used has been 10%, although recent publications report that reducing it to 5% leads to lower rate of side effects without impact on the product or the graft. we retrospectively analyzed 207 patients recipients of autologous peripheral blood stem cell transplantation (hct) in our hospital from january 2009 to september 2017. they are divided into two groups according to the concentration of dmso used in the freezing (10% until september 2014 or 5 % since october 2014). the baseline characteristics of the patients, the infused product and the graft are shown in table 1 . the cd34 count was performed by flow cytometry. all freezings were performed with a biological freezer for programmed controlled rate cryopreservation and stored in ultrafreezers at -80ºc. results: both population groups are homogeneous. the t-test was used for statistical analysis. regarding the cd34 + variable, no statistically significant differences were observed (p = 0.636). neither for the variables leukocyte recovery and platelet recovery (p = 0.178, p = 0.991 respectively). the difference in the variable viability is 5.61 units (ci95%: [0.61-10.62]) and is statistically significant (p = 0.028) in favor of dmso 5%. regarding adverse effects, 100% (n = 4) of the serious adverse reactions occurred in the 10% dmso group (hypotension and seizures). the mild and moderate ones were similar in both groups, most were mild nausea, vomiting and flushing. overall, no statistically significant differences were observed due to the low rate of adverse effects found. patients starting with 2014 until october 2018, total of 414 patients attempted collection of autologous pbscs, and 38 poor mobilizers recieved plerixafor during first mobilization cycle. in total, 9 patients required repeated mobilization cycles (2,2%) of which 6 were from the plerixafor group. in total 42 patients recieved pleriksafor; 23 females and 19 males, median age 58 (8-71) with following diagnoses: 28 nhl, 5 mh, 5 multiple myeloma, 1 neuroblastoma, 1 nephroblastoma, 1 sarcoma ewing and 1 seminoma. of 6 repeated mobilizations with plerixafor, 2 patients (33,3%) still failed to collect adequate transplant. in this period we had altogether 3 unsuccessful mobilizations (33,3% in repeated cycles, 0,72% in total). this group of patients consisted of 1 male and 2 female patients, median age 42 (33-58), diagnosis of nhl and failure to collect after 4 leukapheresis procedures each. median number of leukapheresis needed for adequate collection was 2 with preemtive plerixafor use, and 4 in repeated mobilizations. conclusions: our expirience shows that preemtive use of plerixafor in poor mobilizers is efficient and has enhached success of the pbsc collections. due to drug high cost each institution needs to develop its own algorythm in management of poor mobilizers. the factors contributing to plerixafor mobilization failure still need to be elucidated. disclosure: nothing to declare. platelets recovered from mobilized leukapheresis units obtained from hla-haploidentical donors fulfill the criteria of a conventional hemocomponent and can be used for transfusion background: central venous catheter (cvc) related complications may lead to high morbidity and mortality. unlike cvc, peripheral cannulation offers a quick and inexpensive method for safe and non-traumatic vascular access (va) thus its utilization is strongly recommended whenever possible. the ultrasound (us) guidance for acquiring peripheral va is a useful tool for reduction or elimination of the need of using cvc for stem cells collection. we have made an attempt to introduce us method in our apheresis unit having no previous experience with us devices. the aim of the study was to measure the decrease of cvc insertions after introducing us and evaluate the quality of va by comparing average flow rate and confirming that the desired blood volume could be processed. methods: the theoretical education involved a free elearning course in peripheral ultrasound-guided va (pugva, usabcd, aarhus, denmark). subsequently, the personnel have implemented knowledge in practical training on gelatine and silicone phantoms and healthy volunteers. the practical activities also included a fiveday course in an apheresis centre with us-guided cannulation experience. the details concerning va were recorded, including va site, cannula size, average inlet flow rate, number of inlet pressure alarms reported by the apheresis device. the procedure details where traditional approach was applied i.e. palpable cannulation and cvcs have been collected. similarly, the necessary data for procedures where veins were assessed with ultrasound prior to apheresis were recorded. results: before introducing ultrasonography, 58 stem cell collections have been performed in 46 patients. of all these procedures, 18 were accomplished with cvc (31%) and 40 with peripheral va (69%). median cubital vein was the vessel of choice. out of the 40 peripheral va procedures, 14 (35%) were problematic, with 10 or more inlet pressure alarms during every procedure. after the training stage, 34 collection procedures were performed in 25 patients. after introducing us we have observed a significant reduction of the number of cvc insertion required for successful apheresis from 31% to 9% (p=0.01; chi-square test with fisher's exact). thirty one procedures were completed with peripheral va (91%). ultrasound device enabled cannulation not only the superficial veins but also for the deeper veins. cannulation sites included upper arm cephalic vein (39%), median cubital vein (35%), upper arm basilic vein (13%), median antebrachial vein (13%). out of the 34 collections, 13 were considered problematic (42%). no difference in an average flow rate was observed between procedures performed peripherally with and without ultrasound usage (p=0.34; u mann-whitney test). conclusions: despite no previous experience in us guidance, we have successfully managed to introduce the new method in our apheresis unit. within 5 months, we have reduced cvc usage threefold and as the personnel is gaining more experience, we suppose that the cvc usage may be reduced to episodic cases. despite slightly higher number of pressure alarms, all procedures with ultrasound guidance were completed as planned. ultrasound guidance is the most important tool for significant increase in peripheral va usage and may become the only option for patients with difficult va. disclosure: nothing to declare. abstract already published. donor blood management in healthy bone marrow donors: a retrospective single institution analysis background: over the last two decades mobilized peripheral blood stem cells (pbsc) have been established as the main source of stem cells because of improved engraftment and no necessity for hospitalization for the donors. nevertheless, due to the introduction of promising new transplant regimens, especially in the haploidentical transplantation setting bone marrow (bm) donations are regaining importance. although for both donation methods severe side effects are rarely described, bm collection is associated with considerable blood loss and hence symptoms of acute blood loss are commonly observed. therefore autologous blood are collected routinely in some institutions before donation. since the collected bone marrow amount depends on the target dose, the wbc yield in the product influences the required bone marrow volume. therefore we sought to investigate the relationship between collection volume, rbc volume removal, drop in hb and indications for blood transfusion. furthermore, we assessed wbc and cd34+yields in relationship to various donor parameters and to product volume, in order to find prediction tools for collection volumes. methods: 489 allogeneic bone marrow harvests from adult donors were performed at our institution and retrospectively analyzed. complete blood counts, serum iron and ferritin were assessed at work-up and 4 weeks after donation. the bone marrow product quality including wbc, hematocrit (hct) and cd34+ cells were assessed by automatic hemocytometry and single-platform flow cytometry with ishage gating. results: besides local pain most of the side effects were related to blood loss. none of the donors received blood transfusions. the mean reduction of hemoglobin levels was 2.5 g/dl with a minimum hemoglobin level of 8.9g/dl and a persistent anemia according to who criteria after 4 weeks in 7.9% and pathologically low ferritin levels in 29%. no donor presented symptoms with indication for blood transfusion. the median wbc concentration of the bm product was 26.5/nl (5-95% percentile:16.77-38.75/ nl) the cd34+cell concentration 186.5/μl (5-95% percentile: 73.92-346.9/μl). in the linear regression analysis leukocyte counts of the donor before donation correlated significantly with wbc concentration in the product. thus in order to collect with 90% certainty the 300 mio wbc which are a typical per-kg dose for an allogeneic recipient, 16.24 ml of bone marrow must be collected. collection volume did not systematically affect wbc or cd34+ cell concentration. conclusions: achieving high wbc yields in the bone marrow product allowed for collection of relatively modest bm volumes, thus protecting donors from excessive blood loss. acute adverse events were acceptable. optimization of perioperative management in healthy bone marrow donors may be achieved by good collection technique and reevaluation of wbc yields of each institution to calculate required bone marrow amount. the collection of autologous blood is not indicated. furthermore stringent pre-and postoperative hemoglobin management is predicted to limit adverse effects. disclosure: nothing to declare. donor-recipient weight ratio predicts successful stem cell mobilization on day four of gcsf mobilization results: in group 1 median age of donor was 41 years (range 19 to 51 years). in group 2 median age of donor was 31years (range21 to 48 years). table] 1. table 1 ] elaborates other parameters analyzed between the two groups. one patient in group 1 developed grade ii acute gvhd whereas 3 patients in group 2 developed acute gvhd grade ii-iv. at the last follow up no (0/9) patient in group 1 has any symptoms of chronic gvhd whereas (2/5) patients in group 2 have features of chronic gvhd (one extensive, one limited). conclusions: our observation suggests that upfront use of plerixafor in combination with gcsf modifies the graft favorably decreasing the risk of graft failure and graft versus host disease both acute and chronic. it also helps the donor by decreasing the total volume processed, amount of acd exposure and the duration of harvest. disclosure: none. impact of vitamin d levels on peripheral stem cell mobilization in autologous hematopoietic stem cell transplant recipients ferda can 1 , zeynep arzu yegin 1 , zubeyde nur ozkurt 1 , orhun akdogan 1 , lale aydın kaynar 1 and total product cd34 + cell count [6.7(02.8-15) vs 5.9 (2.9-17.2); p=0.047] were significantly higher in patients receiving chemotherapy+g-csf than g-csf only. the study group was divided into two groups based on peripheral cd34 (cut-off level: 20x10 6 /kg) as well as product cd34 levels (cut-off level: 5x10 6 /kg). vitamin d levels were found to be similar among these groups (p>0.05). total product cd34 + cell count was found to be relatively lower in patients with vitamin d levels below 10 μg/l [5.6(3.3-10) vs 6.7(2.8-17.2); p=0,09]. (figure 1 ) conclusions: based on its effect on stem cells in in vitro studies, it may be considered that vitamin d may have a favourable impact on stem cell mobilization. statistically insignificant but relatively lower total product cd34 + cell count in patients who had lower vitamin d levels, which may indicate a role for vitamin d in stem cell mobilization, needs to be confirmed with larger studies. considering the high prevalence of vitamin d deficiency in the general population, the possible role of vitamin d in hematopoietic stem cell mobilization deserves further consideration. disclosure: nothing to declare background: one of the factors, affecting efficiency of autologous hematopoietic stem cell transplantation (autohsct) in hodgkin lymphoma (hl) patients is early recovery of graft, depending on cd 34+ cell count and conditions of cell product cryopreservation and storage. it is well known, that dimethylsulfoxide (dmso), used for cryopreservation, can be cardiotoxic and cause diverse gastrointestinal, pulmonary, kidney, liver side effects and acute hemolysis. lethal for animals dose 30-40 mg/kg leads to life threatening arrhythmias and respiratory arrest. in order to improve dmso toxicity different ways of alternative cryoconservation modes are studied -lower dmso concentration (5% vs 10%), temperature -80˚c instead of ultra-low and washing of cell product. aim of the study is to evaluate the influence of dmso washing on hematopoietic recovery after autohsct. methods: retrospective analysis of hematopoietic recovery of 37 relapse/refractory hl patients after autohsct was performed. mobilization regimen included second line chemotherapy for hl (dhap, begev, igev, ice) with consecutive g-csf administration. cd 34+ cells were assessed, using 6-colour flow cytometer facs canto ii while cell collection, thawing and washing. cells with 10% dmso were stored at -196˚and washed in 14 cases of transplantation with human albumin-dextran (reopolyglukin) and centrifugation. statistical data processing was performed by the χ2 method -pearson criterion; p -the level of significance of differences. results: patient groups had no difference in age, disease stage, gender, time from treatment start to autohsct and cd34+ cell count (p>0,05). time to wbc recovery >1х10 9 /л was 9-29 (median 13,6) days vs 10-34 (median 13,7) days, time to platelet>30х10 9 /л recovery was 11-34 (median 16,9) days vs 11-58 (median 17,9) days in groups without and with cell washing respectively (p=0,507). no difference in blood component consumption was observed (p=0,546). in 18 out of 23 (78%) patients during cell reinfusion without washing nausea, vomiting, arterial hypertension was observed, no reactions were detected after cell washing (p = 0,03). conclusions: washing autologous mononuclear cells from cryopreservant dmso does not lead to low hematopoietic recovery rate after autohsct and can avoid toxicity, thus making autohsct more safe. disclosure: authors declare no conflict of interests. quality assesment of hematopoietic stem cells autografts after cryostorage, harvested using plerixafor background: the introduction of high-dose chemotherapy followed by transplantation of autologous hemopoietic stem cells (hscs) into the treatment program for multiple myeloma (mm) has significantly increased the frequency of achieving complete remissions and overall survival in patients. to obtain a sufficient amount of hscs, hematopoiesis is stimulated with granulocyte-macrophage factors (gm-csf) both in mono mode and after the administration of cytostatics followed by cytapheresis sessions (alone or after the cytostatics followed by cytapheresis sessions) . cryopreservation protocols are used to preserve cells in a viable state, followed by long-term storage of transplants in liquid nitrogen. however, in some patients it is not possible to obtain the necessary amount of hscs. the inclusion of plerixafor in standard mobilization schemes allows you to prepare the sufficient quantity of hscs in most patients with mm. methods: the study included 18 samples of autografts from 18 patients with mm from 2015 to 2017 (median 2.1 ± 0.3). hscs mobilization was performed on the background of unstable blood formation after high doses of cyclophosphamide 6 g/m 2 with the subsequent administration of g-csf at a dose of 12-24 μg / kg (9 samples from 9 patients) and with the addition of plerixaphor at a dose of 0.24 μg / kg (9 samples from 9 patients). immunophenotype viability of hscs in autotransplants after cryopreservation were determined by flow cytometry using the ishage protocol on a flow cytometer (facs cantoii, becton dickinson) by expressing surface markers of antibodies against cd34, cd45, cd90, cd38 and staining with 7aminoactinomycin (7-aad). the colony-forming activity of hscs (cfu-cfu-mix, cfu-gm, cfu-g, cfu-m) was evaluated in methylcellulose (methocult h4435, stemcell technologies, canada) for 1x10 5 transplanted cells for 14 days. results: the viability of hscs in autografts (cd45 + / cd34 + / 7add-) after cryopreservation in both groups was 98 ± 0.7%. in the group of samples using plerixaphor, a higher content of primitive hemopoiesis precursors (primitive cells) (cd34 + cd90 + cd38-) was detected compared with the control group (29.2 ± 2.7% and 13.2 ± 12%, respectively). the cfu count (cfu-cfu-mix, cfu-gm, cfu-g, cfu-m) in the plerixafor group was 100 ± 2.5 per 1x105 explanted cells, in the control group -70 ± 1.8 ( figure 1a-d) . conclusions: the use of plerixafor against the background of standard protocols for the mobilization of hscs allows to obtain high-quality graft with a higher content of primitive cells and proliferative activity. disclosure: no conflict of interest. nothing to declare. comparison of effectiveness of plerixafor plus g-csf in poor and very poor-movilizers: efficacy of the combination of plerixafor and g-csf in poor-movilizer background: healthy donors ocassionally show a poor response to mobilization agents. plerixafor+g-csf can be a salvage strategy in poor mobilizers. some series describe the use of plerixafor to collect greater doses of cd34+ cells in hematopoietic stem cell transplantation (hsct) with tcell depletion. plerixafor use in the mobilization protocol could help collecting higher cd34+ dose in indirect t-cell depletion (cd34+ selection) for ex-vivo manipulated haploidentical transplantation, with less number of apheresis and a rapid engraftment. methods: data of fourteen healthy peripheral-blood donors was retrospectively collected. they received 4 days 10 mcg/kg/day g-csf and 0,24 mg/kg/day plerixafor on 4º day as mobilization treatment. fourteen pediatric patients (median age 13 years, range 8-15) diagnosed with malignant and no malignant hematological diseases received haploidentical hsct with cd34+ selection and cd45ra+ depletion between february 2015 and july 2017 . results: one leukoapheresis procedure was performed in all cases. median processed volume was 11 liters (range 6-14). median of cd34+ cells obtained was 13,73x10 6 /kg (range 7,8-26,47) . after positive selection, >4x10 6 /kg cd34+ cells were infused in all cases (figure 1 ). neutrophil engraftment was achieved after a median of 15 days (range 10-17). few donors presented only plerixafor mild secondary effects. conclusions: our experience showed that a mobilization protocol using g-csf and standard dose of plerixafor (compasive use) is a safe strategy that allows collecting great cd34+ dose in one apheresis procedure. this could be useful for haploidentical transplantation with ex-vivo t depletion, especially if there´s a weight disproportion between donor and patient. background: mesenchymal stem cells (mscs) are selfrenewing multipotent progenitor cells with wide differentiation potential. their ease of isolation and expansion in vitro as well as their unique regenerative therapeutic properties suggest the use of msc as an approach for treating several disorders. extra-embryonic tissues as placenta have been proposed as potential sources of mscs due to the absence of ethical problems neither risks for the patients. furthermore, only protocols using fresh placental tissue have been described so far. a protocol for isolating mscs from delayed-manipulated tissue was designed and tested in order to optimize the use of placental mscs (mscs-p) in an advanced therapies context. methods: full term placentas (n=10) were obtained from healthy mothers in hospital universitario central de asturias (spain). informed consent was obtained from each mother prior to delivery. after dissection of 12 gr decidual tissue it was washing with saline (b. braun, germany) and cut into small pieces. these biopsies were conserved 24 hours in dmem media with 1% antibiotic solution 100x (gibco, usausa) until processing. the day after, tissue was mechanically minced and then enzimatically digested with a combination of 80ui/ml dnase i (sigma aldrich, usa) and 0.25% tripsin-edta solution (w/v) (biochrom, germany) at 37ºc for 1 hour. then, the mixture was filtered with 40μm cell strainer (bd bioscience, usa) and centrifuge at 300xg for 5 minutes. finally, cells were resuspended in 5ml of dmem media suplemented with 10% fbs and antibiotic, seeded in 24-cm flask and incubated in forma stericult co 2 incubator (thermo fisher scientific, usa) at 37ºc, 5%co 2 . culture-expanded mscs cells were phenotipically characterized by flow cytometry (facs aria iiu, bd) with antibodies against cd29, cd44, cd73, cd90, cd105, cd166, cd34, cd45, hla-dr cd14 and cd19 using mesenchymal cell kit (immunostep, spain). afterwards, these cells were differentiated to adipogenic, osteogenic and chondrogenic lineages using stemmacs adipodiff media, stemmacs osteodiff media and nh chondrodiff medium (miltenyi biotec, germany) respectively. after three weeks of differentiation cells were fixed in 4% paraformaldehide (merck, usa) and analyzed. adipogenic, osteogenic and chondrogenic differentiation was visualized after staining with oil red o, alkaline phosphatase and hematoxilin-eosin (sigma-aldrich, usa). results: mscs-p isolated cells were characterized according to the isct criteria for mesenchymal stem cells. they were positive for cd29, cd44, cd73, cd90 and cd105 and negative for cd14, cd34, cd45 and hla-dr, indicative of a typical msc phenotype ( figure 1 ). all the markers showed a high percentage of expression between 84.6 and 99.9%, meaning that msc population obtained with the designed method was very homogenous. similarly, staining for the three studied lineages was positive ( figure 1 ). conclusions: the described protocol allows us to obtain mscs from decidual placental tissue stored and processed 24 hours after the biopsy extraction using a unique enzymatic digestion. this circumstance permits to take advantage of placentas that are discarded after delivery giving us the option to obtain mesenchymal cells that could be used in clinical trials. disclosure: nothing to declare outcomes of umbilical cord transplant in high risk relapsed or refractory acute myeloid leukaemia background: high-risk relapsed/refractory acute myeloid leukaemia (aml) is a fatal disease. allogeneic haematopoietic stem cell transplantation represents the only chance of cure. as the transplant relies on the graft-versusleukaemia (gvl) effect, and if different donors exert different gvl effects, then choosing the right donor assumes great importance. in manchester, a large bmt centre in the north of england, our practice in such aml has been to choose unrelated cord blood (ucbt), without serotherapy in the conditioning therapy, as our preferred donor cell source. methods: we report the results of 16 unrelated ucbt in 15 patients (five boys and ten girls) with high-risk aml, defined as relapsed or refractory disease. thirteen patients (81%) received this as a 1 st transplant, two patients (13%) received this as a 2 nd transplant for relapsed aml post matched unrelated donor transplant, and one (6%) received ucbt twice, once in cr1 and once in cr2. nine patients (56%) had mismatched ucbt, and the rest were fully matched at class-i (hla-a, -b, and-c) and class-ii (hla-drb1). conditioning was given as treosulfan, fludarabine and thiotepa in half of the patients (n = 8), other treosulfanbased regimens were used in two patients (12%), and busulfan-based regimens were used in six patients (38%). no serotherapy was given. results: the median age at transplant was 5 years (range, 5months -15years). neutrophil and platelet engraftment were achieved in 15 and 12 patients at a median of 16 and 35 days, respectively. 13 patients (81%) had engraftment syndrome. all engrafted patients achieved 100% donor chimerism, except one patient who had mixed lymphoid chimerism initially, that was corrected spontaneously to 100% at three months after transplant. acute gvhd grade i-ii developed in six patients (38%), and grade iii-iv developed in three patients (19%). all cases resolved, except two patients where acute gvhd evolved into chronic gvhd (one with grade i skin gvhd which fully resolved, and one with grade iii gvhd gut colitis who was parenteral nutrition dependent till death). two more patients developed chronic grade i skin gvhd and resolved (chronic gvhd developed in 25% in total). three patients (17%) developed veno-occlusive disease (vod), that completely resolved with defibrotide treatment and necessitated ascitic drainage in one of them. viral reactivations occurred in five patients (30%) and were successfully treated. at a median follow-up of 20 months (range, seven months -four years), eight patients (50%) died at a median of 79 (range, 24 to 230 days), with a transplantrelated mortality of 25% and relapse-related mortality of 25%. five patients (31%) relapsed post-ucbt; four died and one had a successful second ucbt (event-free survival was 44%). immune reconstitution in alive patients was achieved at a median of eight months. conclusions: very high-risk patients treated with ucbt with good overall survival and event-free survival, similar to aml treatment rate with low-risk disease. disclosure: nothing to declare in haploidentical transplants is the incidence of acute and chronic gvhd strictly related to the stem cell source? results: the odds ratio was 1.78 with a 95% confidence interval of 0.94 -3.37 (p=0.07). conclusions: the risk of infection of the uc is not related to the microbiological status of the ucb. a possible explanation for this is the presence of antibiotics in the medium used for uc, but not ucb, transport. this means that cryopreservation of ucs from which contaminated cord blood has been obtained is justified. comparison of turkish stem cell coordination center (turkok) with istanbul university bone marrow bank (tris); a single center experience in match unrelated donors azize mergen 1 , selime aydoğdu 2 , başak aksoy 3 , yunus emre savcı 2 , gürcan dikme 2 , funda çipe 2 , ceyhun bozkurt 3 , tunç fışgın 1 older patients are increasingly being transplanted, thanks to improvement in allogeneic hematopoietic stem cell transplantation (allo-hsct) techniques. increasing donor age is associated with greater risk for mortality and graftversus-host disease (gvhd). since sibling donors are of similar age to recipients, we hypothesized that, in older patients, a young matched unrelated donor (mud) would be comparable to an hla-matched sibling donor (msd). methods: we retrospectively compared outcomes of allo-hsct from msd (n=1797) and 10/10 hla mud (n=2212) in patients aged ≥ 60 years with hematological malignancies transplanted between 2006-2015. all patients received reduced-intensity conditioning and graft source was peripheral blood. the primary outcome was overall survival. msds served as the reference category and were compared to muds split into three age groups (≤25 [n=524], 25-40 [n=1072], >40 [n=616] years) using univariable analyses and multivariable cox regression models adjusted for patient, disease, and transplantation features. results: the median age of hsct recipients was 64 years and was similar across groups. median donor age for msd was 60 years and 22, 33, and 45 for the mud age groups ≤25, 25-40, and >40 years. acute leukemia was the leading transplant indication (46%) followed by myelodysplastic syndrome, myeloproliferative neoplasms and indolent non-hodgkin lymphoma. disease risk distribution was similar across donor groups (low [48%], intermediate [42%] , and high [10%] in the complete population; p=0.771). time from diagnosis to hsct was longer with mud compared to msd and increased with an older age of mud. in a univariate analysis, overall survival was 49% (msd), 50% (mud≤25), 45% (mud 25-40), and 43% (mud≥40, p=0.002). corresponding non-relapse mortality (nrm) cumulative incidence was 22%, 25%.,31%, and 32.2% (p< 0.001) (figure) . gvhd-relapse-free (grfs) was 29%, 30%, 26%, and 24% (p=0.012). in a multivariable cox model, young mud (≤25) had a similar risk for mortality compared to msd (hr 1.00, p=0.97), while a monotonic increase in risk was observed with an older donor age (mud 25-40y: hr 1.17,p=0.014; mud≥40y: hr 1.26, p=0.001) (table) . findings were confirmed in a propensity score analysis, matched for key covariates. nrm and grade 2-4 acute gvhd were consistently higher with mud, with the greatest risk associated with older muds. the hazard for grfs was higher with mud aged 25 or higher compared to msd; risk was not higher with younger mud. conclusions: in older patients receiving reduced intensity conditioning, msd remain the optimal choice. however, when not available, young mud provide comparable results. disclosure: nothing to declare background: there is growing evidence that community acquired respiratory virus (carv) increase the risk of pulmonary invasive fungal disease (ifd) in recipients of allogeneic hematopoietic stem cell transplantation (allo-hsct). to date, there is a lack of knowledge regarding the rate of ifd, risk factors (rfs) as well as the most critical period for the development of a later ifd after carv infections in allo-hsct recipients. methods: in this prospective observational study, we retrospectively analyzed the effect of carv on the development of a later ifd in a consecutive cohort of 287 allo-hsct adult recipients who developed 597 carv infectious episodes from december 2013 to december 2018. respiratory virus in upper and/or lower respiratory tract specimens were tested using multiplex pcr panel assays. results: overall, 29 out of 287 allo-hsct recipients (10%) developed ifd within 2 months after a carv episode at median of 21 days (range 0-158 days) from the day of carv detection. all the ifds involved the lungs and in 28 cases (97%) the diagnostic was ia accomplishing criteria of probable (n= 26) or proven (n=2). of note, 26 out of 29 ifd (91%) occurred within the first year after transplantation. the overall rate of ifd after carv episodes was 5% whereas this rate was higher in recipients developing carv during the first year of transplant (7%). ifd was diagnosed in 25 out of 203 with carv lower respiratory tract disease (lrtd) episodes (12%) compared to 4 out of 394 carv upper respiratory tract disease (urtd) (1%) (p= 0.0001). twenty-three out of 133 carv episodes involving the lrtd during the first year after transplant (17%) developed ifd. we did not found differences in ifd rates according to the type of carv identified. multivariate analysis identified 4 rfs for ifd: the use of atg as a part of conditioning [odds ratio (or) 2.7, 95% confidence interval (c.i.) 1.2-3.4, p= 0.01], carv lrtd (or 11.8, 95% c.i. 3.8-36, p= 0.0001), carv infection during the first year of transplant (or 5.9, p731 natural killer cell alloreactive haploidentical stem cell transplantation for multiple myeloma patients catharina elssen 1 , lotte wieten 1 , peter von dem borne 2 , ellen meijer 3 , gerard bos 1 1 maastricht university medical center, maastricht, netherlands, 2 leiden university medical center, leiden, netherlands, 3 amsterdam university medical center, location vumc, cancer center, amsterdam, netherlands background: in the past years many new drugs for multiple myeloma (mm) have been developed and are responsible for a increase in survival. notwithstanding such progress, mm remains incurable. results from allogeneic stem cell transplantation (sct), including haploidentical transplantation, in mm has shown clinical results. however, these responses are only observed in a minority of patients. we hypothesize that this observation might be due to differences in natural killer (nk) cell alloreacitvity, since we have shown in in vivo and in vitro models that mismatched alloreactive nk cells hold the capacity to kill mm cells. the aim of this prospective phase 2 study is to evaluate if kir-ligand mismatched haploindentical bone marrow transplantation (bmt) with post-transplant cyclophosphamide will improve progression free survival (pfs) in poor risk mm patients. methods: poor risk mm patients, aged < 66 years were enrolled if they were responsive to their last line of therapy. poor risk was defined as, high-risk cytogenetics, or relapse within a year after autologous sct, or treated with three or more previous lines of therapy. a prerequisite of enrolment was the possibility of an nk cell mismatch and availability of a mismatched family donor. patients were excluded if donor-specific hla-antibodies were present. patients received a haploidentical bmt with a non-myeloablative conditioning regimen and post-transplant cyclophosphamide. primary endpoint is pfs at 1,5 years. secondary endpoints are engraftment, bone marrow reconstitution, nk cell reconstitution and repertoire, graft versus host disease (gvhd), infections and non-relapse mortality (nrm) at 1,5 years. results: in total 12 poor risk patients were included in the study of which 10 could be evaluated for the primary end point. graft failure and disease progression before transplant rendered the remaining two patients not evaluable. at this interim analysis 7 patients have already reached the 1,5 years of follow up, 5 relapsed within 1,5 years and 2 died due to treatment related infections, without showing progression of disease (20% nrm). average time of progression is 105 days (60-270 days). two of the remaining patients at follow up, still show responsive disease (days 210 en 120). the average time to neutrophil reconstitution is 19 days (14-28 days). all evaluated patients (6/10) show nk cell reconstitution with a mature phenotype in the bone marrow and peripheral blood by day 60. three patients developed acute gvhd (25%) of which 2/12 grade i-ii agvhd and 1/12 patient showed a grade iv agvhd. treatment related mortality was 3/12 (25%), which was in all cases due to infectious disease. conclusions: our interim analysis of mismatched haploidentical bmt in mm showed that the treatment is feasible and forms a possible platform for immunotherapeutic strategies. the majority of patients showed an early disease progression. we predefined that with a pfs of 25% at 1,5 years we would qualify this treatment option successful. with only two patients still in remission this goal will not be achieved and we hypothesize that the late nk cell reconstitution (day 60) is responsible for the lack of response. clinical background: mscs are known to have immune modulatory capacity and may be effective in the treatment of patients with acute gvhd. however clinical studies yielded inconclusive results which was in part due to the great heterogeneity of the msc used. the off-the-shelf msc preparation "msc-ffm", generated by a proprietary pooling process, selection by plastic-adherence, expansion for an aggregate four weeks followed by cryopreservation until use, is available in germany through a national marketing authorization. "msc-ffm" is indicated in steroidrefractory agvhd, dosed at 1-2 x10 6 /kg bw i.v. in four doses one week apart. methods: we report seven consecutive pediatric patients (median age 7.49 y), who received "msc-ffm" from unrelated hla disparate donors between december 2017 and november 2018 in our institution. we gave msc infusions to 6 patients with steroid-refractory grades iii-iv agvhd and one patient who had therapy-refractory background: regulatory t cells (treg) are known for their immunosuppressive function and have proven successful as graft-versus-host disease (gvhd) prophylaxis after allogeneic bone marrow transplantation in a number of preclinical as well as first clinical studies without compromising graft-versus leukemia (gvl) effects. in murine models of acute gvhd lymph node homing capacity via cd62l (l-selectin) proved to be essential for disease prevention. yet, treg recruitment from lymph nodes to peripheral sites of ongoing gvhd also seems necessary to achieve maximum protective as well as therapeutic effects. the chemokine receptor ccr4 directs activated t cells to sites of inflammation, thus high ccr4 expression should facilitate treg homing to affected gvhd target organs. with this project we lay the foundation for future in vivo studies of treg therapy for gvhd by upregulation of ccr4 expression. methods: we performed systematic ex vivo analysis of ccr4 expression on murine naive and memory conventional (tconv) and regulatory t cells isolated from spleen, blood, bone marrow, lymph nodes, liver and lung. cells were stained for characteristic surface and intracellular markers and characterized by multiparametric flowcytometric analysis. ccr4 expression kinetics following stimulation were analysed in tconv and treg isolated from murine splenocytes by facs and polyclonally activated by anti-cd3/cd28-coated beads in the presence of exogenous il-2. expression was monitored by daily flow cytometric analysis. ccr4 overexpression was induced by transduction of expanded treg with ccr4 mrna via electroporation. expression kinetics were monitored by facs, receptor function was tested in transwell migration assays using ccr4 ligands ccl-17 and ccl-22. results: systematic analyses showed higher ccr4 expression on memory treg than on their naive counterpart in all examined organs with bone marrow samples displaying the greatest disparity. memory treg showed higher ccr4 expression than memory tconv in all analysed organs, except for lymph nodes where both memory populations revealed equal expression levels. stimulation of in vitro expanded treg and tconv lead to a strong increase in ccr4 expression with maximum levels on d3 and d2 respectively, whereas restimulation (d7) resulted in no further relevant ccr4 expression on treg. we performed systematic optimization of stimulation and mrna-electroporation conditions to reliably achieve highlevel short-term ccr4 expression. transduction of treg on d11 of in vitro expansion resulted in a strong ccr4 expression, with maximum levels 2h after electroporation and strong ccr4 expression being detectable for at least 8h. transwell migration assays showed enhanced migrational properties of mrna-electroporated treg towards ccr4 ligands. analyses performed 2h and 16h after electroporation showed persistent migration even though measured ccr4 surface expression had already declined significantly. conclusions: we showed that high ccr4 expression can be detected on memory treg in all analysed organs. since in vitro stimulation of murine treg did not reliably induce ccr4 expression, we established a protocol for ccr4 mrna-electroporation. electroporated cells showed stable short-term ccr4 expression and enhanced migrational properties towards ccr4 ligands in vitro. future studies will show whether the induction of short-term ccr4 expression will facilitate in vivo homing of adoptively transferred treg to sites of ongoing gvhd and thus mediate long-term inflammation suppression. disclosure: the authors have no conflict to disclose. survival and immune reconstitution of syngeneic, haploidentical and allogeneic hematopoietic stem cell transplantation in atm-deficient mice ruth pia duecker 1 , patrick c. baer 2 , stefan zielen 1 , ralf schubert 1 from allo-hsct. it´s not necessary to do chemotherapy before transplantation for patients with bone marrow blast cells more than 10% at the time of diagnosis. [[p738 image] 1. figure 1 the hci-ct of patients before transplantation and occurance of grade iii-iv agvhd on overall surviv] background: 1. allogeneic haematopoietic stem cell transplantation (sct) offers the chance of cure for patients with transfusion-dependent thalassemia (tdt). based on the non-neoplastic nature of this condition sct approaches urgently require to prove both efficacious and safe. methods: 2. we report on 13 children, adolescents and young adults (median age: 6 years; range 2-28 years) with tdt receiving sct from an hla-matched donor (mud n=8, msd n=3, mfd n=2) in our center from 2011-2017. all patients received the same treosulfan-based conditioning regimen (treosulfan 3x14g/m2, fludarabine 4x40mg/m2, thiotepa 1x8mg/kg). gvhd prophylaxis was based on atg-fresenius™ (3x10mg/kg, if mud or mfd as donor), csa (with taper from day +270) as well as mtx (day 1,3,6,11) in 9/13 and mmf in 4/13 patients with mtx toxicity, respectively. stem cell source was bone marrow in 8, peripheral blood stem cells in 4 and cord blood in 1 patient. prior to transplantation 6 children received cytoreductive treatment with azathioprine, hydroxycarbamide and intensified erythrocyte transfusion. iron elimination therapy was carried out in 12/13 children with deferasirox. among the 11 patients with available ferris-can™ analysis 4 patients showed substantial liver iron overload (liver iron > 8mg/g) despite intensive chelation prior to sct. results: 3. all patients achieved leukocyte engraftment at median day +23 (range 15-39), however two patients required a cd34-selected pbsc boost on day + 36 and day +50 based on delayed platelet and/or erythrocyte engraftment. nine patients exhibited full donor chimerism in the bm at day +30, the other 4 showed mixed chimerism with < 5% autologous cells. on day +360 peripheral blood chimerism was complete in 12/13 patients with the remaining patient exhibiting stable split chimerism with 100% donor-derived erythrocytes and 60-80% autologous myeloid cells. acute gvhd was observed in three patients (grade 2: n=1, grade 3: n=2). however, all patients responded to immunosuppressive therapy with steroids ± ecp and re-initiation of cni (n=1). one patient suffered background: patients with relapsed or refractory acute myeloid leukaemia (aml) have a poor prognosis. allogeneic hematopoietic stem cell transplantation is the only curative option. however, allogeneic transplantation with active leukemia failed to improve significantly the longterm outcome. to improve the outcome of allo-hsct in such high-risk and refractory patients, sequential schedule of cytoreduction therapy followed by nonmyeloablative conditioning has been developed. methods: to evaluate the outcome of sequential intensified conditioning regimen followed by allogeneic hematopoietic stem cell transplantation (allo-hsct) for refractory acute myeloid leukemia (aml). results: a total of 20 patients with primary or secondary refractory aml transplanted between june 2011 to july 2018 were included. refractoriness was defined as primary induction failure, relapse within 6 months from induction/ consolidation chemotherapy or second relapse. median age is 39 years (1 to 61). the salvage chemotherapy administered was flag-ida. two patients did not receive intensive chemotherapy because of no recovery after induction chemotherapy. seven days after the end of flag-ida, a reduced intensity conditioning consisting of fludarabine,60 mg/m2, thiotepa, 5mg/kg and busulfan 6,4 mg/kg i.v. (n=12) for haploidentical donors or fludarabine plus busulfan (n=8) for hla identical sibling or unrelated donors was administered. graft-versus-host disease (gvhd) prophylaxis consisted of tacrolimus and mycophenolate mofetil. the mycophenolate was withdrawn at day +35 post-transplantation and tacrolimus at day +100. donor lymphocytes (dli) were infused in patients without agvhd at day +120 post-transplantation. seventeen patients achieved complete donor chimerism, 3 patients progressed early and 1 patient died before engraftment. one of the patients which recovery was with persistent leukemia reached donor chimerism after immunosupression discontinuation. ten patients are alive in complete remission. median follow-up of survivors is 28 months (range: 6-43). five patients died of leukemic progression, 3 as result of gvhd and 1 suffered intracranial hemorrhage. five patients received prophylactic dli. the incidence of acute moderate-severe gvhd and moderate-severe chronic gvhd were 70% (n=14) and 50% (n=7), respectively. the non-relapse mortality was 21% (n=4), mainly due to acute gvhd (n=3) . the 2-year cumulative incidence of relapse posttransplantation was 36.8%. the probability of relapse was 57%±13%. the 2-year os and dfs were 46% ± 12% and 38% ± 12% conclusions: the strategy of sequential chemotherapy followed by allohsct ± prophylactic dli has an acceptable toxicity profile and improves both the relapse rate and the survival for refractory aml patients. disclosure: nothing to declare background: the concept of immunological intervention to prevent relapse after hematopoietic stem cell transplantation is associated with the assessment of the chimerism status. distinguishing patient and donor hematopoiesis is usually performed by str-pcr, a powerful method developed for forensic purposes. however, this method shows restrictions with respect to detection limit, preciseness, and the possibility of automated read out. digital pcr could circumvent some of these limitations. methods: recently, validated for the bio-rad droplet digital platforms, the biotype mentype digitalquant assay was released. the assay uses indel polymorphisms on chromosomal dna to distinguish patient and donor hematopoiesis on a fret hydrolysis assay basis ("taqman assays"). thus the assay in principle is applicable on the chamber based 3 d digital pcr system (thermo fisher). due to different reaction chemistry and physical properties of thermal transfer between the digital pcr platforms protocols are reasonably not fully compatible which would lead to lower fluorescence intensities and poor signal resolution on the solid chip based thermo fisher 3d platform. an adjusted pcr protocol was established and optimized using representative markers, followed by determination of tolerable and optimal amount of input dna. specificity, sensitivity and reproducibility testing with artificial mixed samples preceded the extensive verification by comparative measurement of clinical samples (n=59) and ring-trial samples (n=20). source to allogenic bm or pbsc. ucb units are immediately available for transplantation as they are frozen and banked with defined hla typing and it has an advantage for patients who need urgent transplantation. in addition, a higher degree of hla mismatch appears to be acceptable with a comparatively lower risk of acute and chronic gvhd. meanwhile, a higher incidence of engraftment failure, delayed neutrophil and platelet recovery, and posttransplant immune disorders including pre-engraftment immune reactions (pir) are major problems in unrelated ucbt. methods: in our institute, gvhd prophylaxis in ucbt was changed after march 2013. between january 2007 and march 2013, thirty-two patients received tacrolimus plus methylprednisolone (tac/mpsl) and between april 2013 and january 2018, thirty-one patients received tac plus methotrexate (tac/mtx) for gvhd prophylaxis. to investigate better gvhd prophylaxis after ucbt, we compared transplant outcomes after ucbt using gvhd prophylaxis with tac/mpsl (n=32) and tac/mtx (n=31) in single-pediatric transplantation center. results: the cumulative incidence of neutrophil engraftment at day 30 in tac/mpsl group was 70.1 % and 90.3 % in tac/mtx group (p=0.09). median time of neutrophil engraftment was 2 days earlier in tac/mtx group (17 days) than tac/mpsl group (19 days). according to pir, and acute gvhd, tac/mtx group showed superior outcomes; the incidence of pir (p=0.020) and the cumulative incidences of acute gvhd at day 100 (38.7 vs 68.8 %, p =0.045 for grade ii-iv, 9.7 vs 34.4 %, p=0.021 for grade iii-iv) was significantly lower in tac/mtx group than in tac/mpsl. however, the incidences of relapse (p=0.921) and cytomegalovirus viremia (p=0.908), and estimated overall survival (p=0.87) and event-free survival (p=0.88) were comparable between two groups. conclusions: our results indicated that gvhd prophylaxis with tac/mtx had favorable effects; reduced incidence of rip and acute gvhd after ucbt without any negative influences. disclosure: nothing to declare transfer of donor regulatory t-cells after atg reconditioning cures severe refractory gvhd and leads to long term persistence of regulatory t-cells in the recipient cells on a clinimacs® plus device (miltenyi biotec). the cell product contained 83% foxp3 + t-cells. the patient received 3,4x 10 5 /kg t reg on day +177. subsequently intestinal gvhd decreased and finally resolved. three months after the first t reg transfer the patient got a second t reg transfer (3,4x 10 5 /kg) on day +281 due to decreasing t reg levels. thereafter t reg persisted and there was no recurrence of gvhd. the patient is well with low dose sirolimus and prednisone as the only immunosuppressants and is particularly recovering intestinal function. conclusions: this case illustrates an unusually severe acute gvhd after matched sibling sct. transfer of donorderived t reg was able to cure severe and refractory gvhd after t-cell ablation by atg. transferred t reg persisted in the recipient for a long period and did not lead to any adverse events. disclosure: no disclosures to declaim allogeneic hsct for patients with transfusion dependent anemia from matched and mismatched donors julia fekadu 1 , andrea jarisch 1 , jan sörensen 1 , emilia salzmann 1 , eva rettinger 1 , andré willasch 1 , shahrzad bakhtiar 1 , thomas klingebiel 1 , peter bader 1 after first asct. the mean harvest for patients receiving dhap was 10,02x10 6 cd34(+) cells/kg, 3,04 x10 6 cd34 (+) cells/kg for cy, 8.56 x10 6 cd34(+) cells/kg for igev, 6,76 x10 6 cd34(+) cells/kg for ice, 10,52 x10 6 cd34(+) cells/kg for choep. the patient mobilized with vtd-pace achieved 2,2 x10 6 cd34(+) cells/kg after 2 apheresis. 19 of the patients achieved the target number of > 2x10 6 /kg cd34+ cells after 1 apheresis, 15 after two, and 2 after three apheresis. the median time to apheresis was 13 days (8-18) without significant difference between the regimens. the mean wbc count at the time of apheresis was 42,8x10 9 /l after dhap, 34,5 x10 9 /l after cy, 21,2 x10 9 /l after igev, 23,1 x10 9 /l after ice, 45,8 x10 9 /l after choep. there was correlation between wbc and cd34 harvested cells (p=0.005). grate 3-4 thrombocytopenia was found in 8 patient (5 dhap, 1 ice, 1igev, 1 vtd-pace). grate 3-4 anemia was registered in 3 patients (2 dhap and 1 vtd-pace). no correlation was found between the cd 34+ harvest and the age, number of previous lines chemotherapy, the response before mobilization, the type of the lymphoma and the clinical stage. conclusions: our results demonstrate that the chemo-g-csf protocols have comparable effectiveness with accepbackground: cytomegalovirus (cmv) may cause severe complications in recipients of allogeneic haematologic stem cell transplantation (allohsct). letermovir (ltv, 480/ 240mg daily without/with co-administration of cyclosporine) was recently licenced only for cmv prophylaxis in adult allohsct-recipients. paediatric data as well as data on cmv therapy are missing so far. methods: we administered letermovir 240mg orally once daily (with no co-administration of cyclosporine a) to 2 paediatric patients after allohsct. edta-plasma were occasionally obtained at different time points and frozen for determination of letermovir levels using liquid chromatography/mass spectrometry (lc-ms/ms). results: for details on patients, treatment and cmv load see table 1. in short periods of letermovir administration, cmv blood levels became negative in both patients. considering the lacking safety data in paediatric patients, we stopped letermovir treatment in both patients, when liver parameters increased. in patient 1 hepatopathy turned out to represent histologically proven graft versus host disease (gvhd). in patient 2 liver parameters further increased despite withdrawal for another 4 weeks, however, hepatopathy was only mild and self-limiting. both patients additionally received other possibly hepatotoxic substances (mycophenolate mofetil and trimethoprim/ sulfamethoxazole). letermovir plasma levels were 11.900ng/ml (2h), 11.600ng/ml (12h), 1.640-2.190ng/ml (median 1.990ng/ ml, n=3, 24h) and 1.050 ng/ml (36h after administration). conclusions: during short letermovir treatment, we observed fast resolution of cmv viraemia as well as rising liver parameters in both patients. while elevated liver parameters represented gvhd in 1 patient, a causal relationship with letermovir might be considered in the other patient. letermovir peak levels after administration of 240mg were within ranges reported in adults after administration of 480mg while trough levels were higher indicating differences in pharmacokinetics in terms of delayed clearance. inguinal lymphadenomegaly. after failure to respond to seven conventional treatment lines: methotrexate, cop (cyclophosphamide, vincristin and prednisone); gemcitabine; puva; interferon; acitretin and extracorporeal photopheresis), allogeneic hsct from an identical hla male donor was indicated. the non-myeloablative conditioning consisted of fludarabine (200mg / m2), cyclophosphamide (50mg / kg) and total body irradiation(tbi) (400cgy). prophylaxis of graft versus host disease (gvhd) was performed with cyclosporine (3mg / kg) and mycophenolate mofetil (30mg/kg). after conditioning, there was improvement of pruritus and involution of the skin. bone marrow infusion occurred on 2/9/2018 (d0). on d + 83 he presented recurrence of skin lesions of fmf. donor lymphocyte infusion (dli) was performed (1 x 10 7 cd3 + cells / kg / recipient). he presented oral lichen and diarrhea respectively as manifestations of gvhd on d + 104 and d + 112. as infectious intercurrence, hemorrhagic cystitis occurred by bk virus 3 months after the first dli and he received conservative treatment and remained without systemic immunosuppression. nine months after hsct, a second dli (5 x 10 7 cd3 + cells / kg / receptor) was performed and at this time the patient is without clinical manifestations of fmf or gvhd. conclusions: the clinical response of the presented case confirms what has been reported in the literature. ctcls appear to be particularly susceptible to gvl effect, which makes hsct a potential cure for advanced ctcls in eligible patients. the timing to perform hsct in the clinical course of the disease remains a matter to be settled clinical trial registry: not applicable disclosure: no conflict of interest p755 abstract already published. methods: we applied next generation sequencing (pgm, ion torrent/ fischer lifetechnologies) to an unselected cohort of 414 patients (176 female, 238 male, median age 59 (2-80) years) who had been referred for allogeneic stem cell transplantation due to the presence of a high-risk myeloid disorder dnmt3a r882h (3.1%) .98), cebpa (18.1%; ceb-pap198s (16.9%) vus) kras (6.5%; krasr161r (3.1%) vus), and kit (3.9%; kitm541l (3.9%) .74). patients displayed a median of 3 sequence variants (aml, mpn and cmml patients each 3; mds, saa and patients with other, non-malignant hematologic diseases each 4 sequence variants found most frequently in aml were cebpap198s (13.2% of all sequence variants in patients with aml, p< 10 -3 , chi² test) in patients suffering cmml, dnmt3ar882h was particularly frequent (4.8% of all sequence variants in cmml, p=.064). asxl1e1102d (2.1% of all sequence variants in other, non-malignant hematologic diseases, p=.699), idh2r140q (2.1%, p=.677) and krasr161r (4.2%, p=.010) were frequent in other, non-malignant hematologic diseases. in saa, nrasg12d (4% of all sequence variants in saa patients; p=.005) was frequently found, as were dnmt3ak456fs* (8%, p=.007), tet2l1721w (8%, p=.080) and tet2i1762v (8%, p=.001). conclusions: taken together, these data show that vus occur with high abundancy in this high-risk cohort of patients, and that they differ in frequency between various myeloid disorders methods: retrospective analysis of patientswho experienced either hematological relapse or progressed to aml after allo-hsct and were treated with azacitidine for this indication at 7 hematological centers in poland. the primary end-point was overall survival (os), the secondary -response rate. results: 36 patients, 22 males (61.1%), median age 52 (range, 15-66), were enrolled. the primary indication for allo-hsct was aml median time from allo-(95%ci: 4.9-11); with 2-year os of 17.2% (95%ci: 5.1-35.2). for patients stratified according to ebmt aza relapse prognostic score 2-year os was: 9 prophylaxis of agvhd: with atg -atgam 60mg/b.w.-39pts(36,8%), posttransplant cyclophosphomide (ptcy) 50mg/b.w. on d+3;d+4-67pts (63,2%) conclusions: unmanipulated haplo-hsct in 1 st -2 nd cr in children and adolescents with high risk al allows achieving the long-term survival in 64,7%. the use of g-csf stimulated unmanipulated haplo-bm is associated with a satisfactory rate of engraftment. the main cause of death in our study was relapse after allo-hsct. the frequency of acute and chronic gvhd was acceptable, 10-years grfs rate of 35,3% in 1 st -2 nd cr represents good quality of life following unmanipulated haplo-hsct and therefore may be recommended as option for use in children and adolescents with high-risk al. disclosure: nothing to declare background: b7-h3 (cd276) is thought to act as an immune checkpoint and regulates t and nk cell responses. it is highly overexpressed on many solid tumors while on healthy tissue protein expression is limited. this makes b7-h3 an interesting target for cancer immunotherapy. in highrisk neuroblastoma patients, targeting disialoganglioside gd2 with the recently approved monoclonal antibody (mab) ch14.18 after autologous or allogeneic sct, significantly improves survival. however, gd2 expression is heterogeneous and ch14.18 causes severe adverse effects. thus, we evaluated b7-h3 as an alternative or additional target antigen. we investigated different anti-b7-h3 mab constructs and mab-cytokine fusions (immunocytokines) for their ability to elicit antibody-dependent cellular cytotoxicity (adcc) using expanded γ/δ t cells of healthy donors, chex5lf-il2 was confirmed to be the most effective mab construct. interestingly, chek5-il2 showed comparable target cell lysis -however, lysis was only transient while chex5lf-il2 mediated permanent target cell lysis. using patient pbmcs after receiving allogeneic sct, chex5lf-il2 and ch14.18 mediated comparable lysis. calculated specific lysis of lan-1 (after 96 hrs.; in ascending order): targets + effectors w/o mab (22 %) conclusions: b7-h3 is a suitable target antigen in case gd2 expression is low or absent. immunocytokines and fcoptimized mabs targeting b7-h3 might increase the efficacy of immunotherapy in gd2-negative tumors and in combinatory approaches. until now, the low-fucose immunocytokine chex5lf-il2 seems to be the most promising anti-b7 great ormond street hospital, nhs foundation trust petersburg/ raisa gorbacheva memorial institute of children's oncology, hematology and transplantation, rehabilitation medicine, saint petersburg, russian federation, 2 first i. pavlov state medical university of st. petersburg/raisa gorbacheva memorial institute of children's oncology, hematology and transplantation, bone marrow transplantation for pediatric solid tumor petersburg/raisa gorbacheva memorial institute of children's oncology, hematology and transplantation, pediatric hsct outpatients, saint petersburg, russian federation 1 university hospital carl gustav carus james`s hospital diagnosis was aml in 52%, all in 36% and mds in 12% of patients. 90% of patients received pbsc grafts, 10% received unmanipulated bone marrow grafts. os at 2 years was 70% in msd/mud-atg, 76% in haplo-ptcy, 80% in mmud-ptcy and 43% in mmud-atg groups (p=0.0027). in a multivariate cox model non-relapse mortality was 17%, 17%, 4.5% and 42% in msd/mud-atg, haplo-ptcy, mmud-ptcy and mmud-atg groups, respectively. cumulative incidence of acute gvhd grade 3 or 4 was 6%, 9.5%, 3% and 12% after in msd/mud-atg, haplo-ptcy cumulative incidence of chronic gvhd was 26% in the msd/mud-atg group uwe platzbecker 3 , verena wais 4 republic of china background: there are two most noteworthy strategies of haploidentical stem cell transplantations (haplo-hsct), the baltimore post-transplantation cyclophosphamide (ptcy) with or without anti-thymoglobulin (atg), and the beijing g-csf primed bone marrow (bm) plus peripheral blood stem cells (pbsc) (giac). however, the comparison of these two modalities is scarce. in this study, we aim to compare these two approaches for hematological malignancies based on the taiwan blood and marrow transplantation registry (tbmtr) with the comparable cd34 infusion amounts, the neutrophil engraftment time were statistically distinct among these three groups [d+12 (group 1) vs. d+15 (group 2) vs. d+17 (group 3), respectively as to the graft-versus-host disease (gvhd), the patients in group 1 had more grade ii-iv but similar grade iii-iv acute gvhd compared with others (grade ii-iv: 60.0% vs. 37.5% vs. 21.6%, respectively conclusions: haplo-hsct with different strategies is a feasible treatment modality for hematologic malignancies in taiwan. regarding the retrospective nature and limited patient numbers of this study republic of china background: we previously presented a low-resolution hla analysis of cedace, the voluntary portuguese bone marrow donor registry (ebmt 2011, poster p1127) and, more recently, its epidemiological characterization (ebmt 2018, poster b348). currently, cedace is one of the largest bone marrow donor registries in the world, including nearly 4% of the country's population, twice the number of donors present in 2011. the current work is an update on the most common hla haplotypes found in cedace results: of the 396545 donors in the cedace registry, 99.51% were typed in at least 3 loci (hla-a/-b/-drb1), 43.91% in 4 (including hla-cw), and 1.28% in 5 (including hla-dqb1)the 5, 25 and 150 most common haplotypes accounted for, respectively, 8.9%, 23.5% and 50% of the haplotypes found in the entire registry. the five most common haplotypes at the low-resolution at the 3 loci, low-resolution level, out of 394621 donors, 167505 individual genotypes were identified, leading to an hla matching probability at this level of 57.6% hid-hsct) have a stronger anti-leukemia effect compared to identical sibling donor hsct(isd-hsct) in high-risk features .but in refractory/relapsed(r/r) aml patients who not in remission status, it is unclear whether it also augments the gvl effect antithymocyte globulin was used in haploidentical hsct. unmanipulated bone marrow and peripheral blood stem cells for all patients. cyclosporine, short-term methotrexate were employed for gvhd prophylaxis. mycophenolate mofetil included in hid-hsct. performed multivariate analysis for all patients of pretransplantation variables and developed a predictive scoring system for survival according to adverse factors. results: the total survivor median period of follow up was 46 (20-73) months. hid -cohort had higher 5-year actuarial of os multivariate analysis showed isd-hsct,standard conditioning regimen and less than 50% proportional reduction in blast percentage pre-≥2. conclusions: haploidentical donor compared to identical sibling donor had better anti-leukemia effect in allo-hsct for r/r aml in nr status conditioning protocol was melphalan 200mg/m2 for mm and beam for nhl. the quantification and characterization of γδt cells in peripheral blood samples were performed by flow cytometry based on the expression of cd3/cd45/vδ1/vδ2/vγ9/cd27 at 30, 60 and 100 days after ahsct. percentage (%) of γδt cells represented the proportion of these cells among all t cells. results: median age at ahsct was 61 (40-70) years, 63% male. median time from diagnosis to mobilization was 8 (4-52) months, after a median number of therapeutic lines of 1 (1-3); 7pts (13.0%) received radiotherapy. seventeen pts (31.5%) were re-mobilized with plerixafor±g-csf (25% mm vs 60% nhl there was no difference in febrile neutropenia incidence (p=0.766), timeto-engraftment (p=0.093), time-to-neutrophils>500/μl (p=0.174) or erythrocyte transfusions (p=0.076). however, there was more time-to-platelets>20,000/μl (4 vs 8 days; subpopulations did not affected pfs. conclusions: our results showed that pts mobilized with plerixafor need more collection volume (less cd34+cells, higher dmso). plerixafor negatively affected platelet recovery, with similar hematologic and immune recovery for the remaining variables. its use was associated with higher %vδ1+, suggesting that it induces an antineoplastic phenotype. more studies with larger samples and follow-up period are needed to evaluate plerixafor results: total 1156 ascc procedures were carried out in 1072 patients over 18 years, once in 985 patients, twice in 83 patients, three times in 3 patients, and four times in 1 patient. non-hodgkin lymphoma (nhl) comprised 56.7% of all cases (n = 655) 001), total number of chemotherapy cycles before ascc (or 1.06, 95% ci 1.004-1.13, p = 0.035), failure to achieve at least partial response before ascc (or 2.97, 95% ci 1.01-8.73, p = 0.048), total number of days receiving g-csf for mobilization (or 1.10, 95% ci 1.002-1.23, p = 0.045), and salvage use of plerixafor (or 3.78, 95% ci 1.92-7.47, p < 0.001) were found to be independent factors associated with failure of ascc. at the end of the study period, 90.9% of successful collections (n = 962/1058) were used for asct, 4.5% (n=48/1056) were in storage awaiting transplantation hôpital necker-enfants malades melf 140 methods: a total 103 ahsct candidates [median age: 57(18-75) years; male/female: 54/49] were included in this study. twenty-seven patients (26.2%) were diagnosed as non hodgkin's lymphoma, 8 patients (7.8%) hodgkin's lymphoma, 52 patients (50.5%) multiple myeloma, 12 patients (11.6%) acute myeloid leukemia, 3 patients (2.9%) plasmocytoma and 1 patient (1%) testis cancer. premobilization serum 25-hydroxy vitamin d (25-oh d) levels were measured with immunoassay method peripheral cd34 + cell count background: some published data suggest a positive effect of iron chelators on the risk of post-transplant relapse, with an improvement in overall survival after allogeneic hematopoietic stem cell transplantation (hsct) conditioning regimen consisted to intravenous bu 130 mg/m 2 and, flu 40 mg/m 2 d-6 to d-3. gvhd prophylaxis included atg 2.5 mg/kg on d-2 and d-1, ciclosporin and methotrexate. all patients received peripheral blood stem cell transplant from an identical hla-related donor. iron chelation consisted on deferasirox (exjade ® ) 10-20 mg/kg/day, started at day 100, if serum ferritin level ≥ 800 -1000 ug/l and stopped when the level decreased below 600 ug/l or normalized at day 100 after transplant, 35/ 40 patients were evaluable (g1= 20 pts), (g2 =15 pts). patients were abo compatible (66%), had major incompatibility (29%), and 6% had minor incompatibility. median serum ferritin level at day 100, were 1200 ug/l (833-4449) and 1200 ug/l (123-1853) in g1 and g2 respectively with a median follow-up of 34 months, (12-68), disease relapse incidence was higher in patients who did not received iron chelation treatment (g1: 39.8%) versus those who received oral deferasirox (g2:15.6%) (figure 1), but the difference was not statistically significant conclusions: these results deserve more investigation choep (n=5), and 1patient received vtd-pace 10 μg/kg depending on the protocol. the aim was to collect at least 2x10 6 cd34(+) cells/kg body weight. results: forty patients all patients were stage iii and iv at diagnosis. 29 of the patients were mobilized after one line of treatment, six after two lines of treatment and five after 3 and more lines of treatment alexandra martínez-roca 1 , gerardo rodriguez-lobato 1 , gonzalo gutierrez-garcia 1,2 , maria suárez-lledó 1 background: hematopoietic stem cell transplantation (hsct) is an established procedure in lymphoma background: the role of inflammatory cascade in tumor microenvironment has been demonstrated in several studies. as a part of this issue, elevated neutrophil/lymphocyte ratio (nlr) was shown to be associated with an adverse prognosis, particularly in solid tumors. the aim of this study is to determine the impact of pre-transplant nlr on early transplant complications, as well as post-transplant relapse and survival.methods: a total of 119 lymphoma patients [median age: 49(18-71) years; male/female: 64/55] who underwent autologous hematopoietic stem cell transplantation (hsct) were included in this retrospective study.results: the initial diagnosis was hodgkin lymphoma (hl) in 35 (29.4%), b-cell non-hodgkin lymphoma (nhl) in 67 (56.3%) and t-cell nhl in 17 (14.3%) patients. of 116 patients who were evaluated for pretransplant disease status, 52 patients (44.8%) were in complete remission, 45 patients (38.8%) were in partial remission and 19 (16.4%) patients had refractory disease. median pre-transplant nlr was found to be 3.42(0-97) . when the study population was divided into two subgroups as "low-" and "high-nlr", based on median nlr value, number of febrile days were found to be relatively higher in the low-nlr group (p=0.051). a positive correlation was demonstrated between nlr and lactate dehydrogenase levels (r=0.218; p=0.03); and nlr and ferritin levels (r=0.277; p=0.003). at a median follow-up of 24 (1-264) months, overall survival (os) was found to be better in the low-nlr group without statistical significance [25 vs 13(1-91) months; p=0.069]. in univariate analysis, pre-transplant nlr represented a significant impact on os (p=0.021). other prognostic factors were age (p=0.041), platelet engraftment (p=0.018), post-transplant relapse (p=0.009) and pre-transplant ferritin level (p< 0.001). the permanent impact of ferritin on os was confirmed in multivariate analysis (p=0.008).conclusions: in this study, an adverse impact of elevated pre-transplant nlr on os was demonstrated in autologous hsct recipients with lymphoma. as a predictor of prognosis, nlr may be considered as a safe and cost-effective parameter. further studies are required in order to use this predictor in routine clinical practice.background: high cure rates in childhood diseases have been achieved by stem cell transplantation (sct). however, there is little knowledge concerning recovery of the immune system and community-acquired infection after sct. here we studied the long-term reconstitution of the immune system and incidences of community-acquired infection after sct.methods: we reviewed medical records for 44 patients (m/f: 31/13, median age: 5 years (range: 1-22years) who were treated in the department of pediatrics, hokkaido university hospital. we analyzed cd4-positive cell counts, serum immunoglobulin g (igg) levels, and incidences of community-acquired infection until 2 years after sct. indications for sct were all in 8 patients, aml in 4, aa in 6, nb in 5, rms in 2, jmml in 1, nhl in 1, cgd in 5, was in 3, xscid in 2, apds in 2, cd40ld in 2, and other solid tumor in 3 patients. stem cell sources were autologous pb/bm in 6, allogenic bm in 23 (9 related and 14 unrelated) and allogenic cb in 15 patients. in this study, we excluded patients who relapsed after sct.results: the duration of cd4-positive cell counts < 500/ ml after sct was 14.7±11.4 months in all patients. the durations were 12.6±7.1 months in patients with hematologic malignancies, 11.5±7.3 months in patients with hematologic disorders such as aplastic anemia and pid, 28.8±25.9 months in patients with solid tumor, 16.0 ±10.4 months in patients who received autologous sct, 25.1±22.2 months in patients who received related bmt/ pbsct, 13.8±6.7 months in patients who received unrelated bmt, and 10.5±5.8 months in patients who received cbt. the durations of igg < 500mg/dl after sct were 14.9±11.5 months in all patients, 15.8±9.7 months in patients with hematologic malignancies, 11.6±5.1 months in patients with hematologic disorders, 11.0±8.7 months in patients with solid tumor, 19.0±18.6 months in patients who received autologous sct, 12.0±8.3 months in patients who received related bmt/pbsct, 11.5±5.7 months in patients who received unrelated bmt, and 15.9±8.7 months in patients who received cbt. there was a significantly higher incidence of community-acquired infection from 6 months after sct. there were significant differences in the incidence of community-acquired infections between patients with cd4-positive cell counts of < 500/ml and background: allogeneic hematopoietic cell transplantation (allo-hct) has the potential to cure subgroups of patients with multiple myeloma (mm) but its role is controversial due to high transplant-related mortality. while autologous hct is well established as consolidation after induction therapy using novel agents, allo-hct is still considered experimental due to excessive early toxicity.methods: we retrospectively studied 45 mm patients (pts) ; 25 (55,6%) were males with a median age of 52 years (range: 32-60), who underwent allo-hsct in our center between 2007 and 2018.median time between diagnosis and allo-hct was 40 months (range: 20-143).results: the international staging system (iss) was iss i (33,3%), iss ii (28,9%), and iss iii (37,8%). twentyeight (62,2%) pts received cells from unrelated donor and 17 pts from identical siblings. stem cell source were: peripheral blood (n=25) and bone marrow (n=20). grouprisk distribution using hct-ci and pam index can be seen in figure 1. response was evaluated at day +100: cr (53,3%), vgpr (11,1%), pr (22,2%) and sd (2,2%); response was not evaluated in 5 pts. regarding hospitalization, 11 (24,4%) pts and 22 (48,9%) pts needed readmission in the first 100 days and 365 days post-allohct, respectively, and median days of hospitalization was 27 days (range: 4-92). reasons for first re-admission were: infection (68,2%), gvhd (27,3%) and renal insufficiency (4,5%). twenty-seven (60%) died and death causes were: infection (35,6%), progression (15,6%), secondary tumor (4,4%) and the remainder, gvhd and intracranial hemorrhage. in addition, 26 pts (57,8%) suffered cytomegalovirus (cmv) reactivation.median overall survival (os) was 44 months (range: 11-76). univariate analysis showed that re-admission in first 100 days (hr 4, 19; p=0, 001) , re-admission in first 365 days (hr 3, 6; p=0, 001) , cmv reactivation (hr 2,38; p=0,038), iss (iss-i vs iss-ii and iii; hr 0,347; p=0,033), days of hospitalization in the first 100 days and response at +100 day (cr plus vgpr vs the remainder; hr 0,143; p=0,04) were predictor factors for os. other factors like karyotype, response before allo-hct, hla-mismatch or baseline cmv serostatus, among others, do not impact on os.median os in pts with low hct-ci were 56 months (range: 0-156) vs 6 months in intermediate-high hct-ci background: allo-sct is a potentially curative therapy for patients with multiple myeloma as it provides a graftversus-myeloma effect and a myeloma-free graft. due to increased nrm and unclear os benefit the recent guidelines suggested allo-sct to be used in context of clinical trials focusing on the high-risk patients and those who relapsed early after autograft. reduced-intensity conditioning regimens may improve rate of nrm; however, optimal conditioning regimen is still to be determined. here we studied conditioning regimen with 3 alkylating agents consisting of thiotepa (tt), busulfan (bu) and gvhd prophylaxis with post-transplant cyclophosphamide (post-cy) in high risk myeloma patients relapsing after autograft.methods: a total of 40 patients (m, n=23) with median age of 55 years (range 40-67) underwent an allo-sct (mud, n=19; mrd, n=12; mmud, n=6, haploidentical, n=3) during a period from 2014 to 2018 in university of hamburg. the majority of patients had advanced disease (stage iiiab, 73%) and high-risk cytogenetics (56%). the median response durartion after autograft was 1.8 years (range 0.5-8. 3 ). the conditioning included tt (cum. dosage 10mg/kg), bu (median cum. dosage 9.6mg/kg i.v. or 6.4 mg/kg in elderly patients) and post-cy (cum. dosage 100mg/kg, day +3 and +4). eight patients (all of them received allografts from mmud or haploidentical donors) received additionally fludarabine (flu, cum. dosage of background: the overall incidence of active cmv infection in patients with multiple myeloma (mm) receiving background: several scoring systems have been developed to estimate outcomes in mds patients who undergo allohct. however, none of them have been specifically validated in t cell depleted grafts. the aim of this study is to investigate the prognostic ability of a recently published scoring system (sfgm-tc) in a cohort of patients with mds who underwent tcd transplants.methods: 109 patients underwent a first tcd allohct for mds from 2007 to 2018. the sfgm-tc score (caulier et al. curr res transl med. 2018 ) is performed at day 100 and ranges from 0-8, discriminating low (0), intermediate (1) (2) (3) , and high risk (4-8). additional analyses were performed at day 180 and day 365. a landmark analysis was done at each time point for the day 100, 180, and 365 analyses, respectively. background: hematopoietic stem cell transplantation (hsct) is the only curative procedure for the treatment of myelodysplastic syndrome (mds), but among several limiting factors for its accomplishment, such as the patient´s performance status, is a very relevant issue, i.e., the availability of a compatible hla donor and, when available, very often the donor´s age and comorbidities also constitute factors that hinder this medical conduct. considering this scenario, the possibility of a haploidentical transplantation (ht) has emerged as an option. in latin america, ht has been included as a treatment option since 2014. since then, these patients have been included in the latin american registry of transplantation in myelodysplastic syndrome, making it possible to analyze the viability and results of these transplants.methods: from october 2012 to october 2018, seventeen (17) patients were transplanted with a haploidentical donor and included in the latin american registry. none of these patients had an identical hla (8/8 match) related or unrelated donor. data were obtained from the latin american registry of hsct in mds. the statistical analyses were performed using the software spss, version 23.1 and graphpad prism version 5.0, with significance being set at p < 0.05.results: table 1 shows the patients and their characteristics. all donors were haploidentical. there was a predominance of reduced intensity conditioning, which was performed in 13 patients (76.5%), whereas the others received the myeloablative conditioning. cell source was peripheral blood in 10 (58.8%) and bone marrow in 7 (41.2%) of the patients. graft-versus-host disease (gvhd) prophylaxis in post-hsct was carried out with cyclophosphamide 50mg / kg on d+3 and d+5, cyclosporin from d0 and mycophenolate from d+1. complete hematologic recovery was achieved in 16 (94.1%) patients. the incidence of grade ii-iv acute gvhd was 11.7%, whereas chronic gvhd was 5.8%. one death occurred due to graft failure and none of the patients showed autologous recovery. three other patients died. one on d+210 due to a fungal infection, the second on d+90 due to sinusoidal obstruction syndrome and a third on d+61 due to pneumonia caused by pseudomonas. regarding overall complications, there was a predominance of mucositis (47%), overall infections (35.2%) and reactivated cmv in 23.5% of cases. of the total number of living patients, 8 (47%) achieved complete remission and 5 (29.4%) showed disease relapse. the mean follow-up was 39 months (ranging from 5 to 72 months). the lowest probability of disease-free survival at 3 years was 79% (95% ci: 71.48 -88.51).background: relapse is the most important cause of failure after allogeneic hematopoietic stem cell transplantation (hsct) for flt3-itd-positive acute myeloid leukemia (aml). treatment with flt3 tyrosine kinase inhibitors (tki) constitutes a promising clinical approach to induce remission without conventional chemotherapy.methods: a 50 year-old woman diagnosed with aml secondary to myelodisplastic syndrome (mds) with npm1 mutation and internal tandem duplications of the flt3 gene (flt3-itd) in october 2013. after achieving complete remission (cr) with conventional chemotherapy, she received a hla sibling allogenic-hsct in february 2014, with bucy. four months later, aml relapsed only at medullary level (flt3 ratio: 0,67 %), treated with chemotherapy and donor lymphocytes infusions (dli). she achieved 2 nd cr and developed limited chronic graft-versushost disease (cgvhd). nine months later (april 2015), she suffered the first extramedullary relapse only, breast and skin. disappearance of the lesions at all levels was achieved with chemo and radiotherapy. she always had full hematologic donor chimerism.in december 2015, she referred atypical precordial pain irradiated to the back. cardiac mri was performed and several masses were visualized in the pericardial sac, up to 5 cm in diameter. bm remained in cr with full donor chimerism.pericardial fluid showed massive infiltration by leukemic-flt3 positive cells (ratio: 0,7%). she was not considered background: ta-tma is a severe complication that can reduce survival after hsct. risk factors have been variably reported in adults although data on children remains scarce. we aimed to identify a risk profile for development of ta-tma in children undergoing hsct.methods: we retrospectively reviewed clinical charts of 398 children who underwent 406 hscts between 2013-16: at great ormond street hospital (gosh) and the great north children's hospital (gnch). ta-tma was defined according to revised criteria (jodele et al. 2013) . risk factors were categorized into patient derived [age, gender, active co-morbidity at d0 of hsct (uncontrolled viral/ bacterial or fungal infection, pulmonary, cardiovascular instability, steroid therapy >0.3mg/kg beyond d0, bcgiosis or autoimmune disease), transplant related factors (conditioning intensity, stem cell source, hla-matching, use of ciclosporin a (csa) or tacrolimus (tac), cd34+ dose, ex-vivo t cell depletion, use of defibrotide) and post-hsct factors (agvhd, post-hsct viral reactivation, venoocclusive disease (vod) and occurrence of posterior reversible leukoencephalopathy (pres).results: at a median of 7 months post-hsct, ta-tma occurred among 21/406 transplants (5.1%). there was no reported centre variation (5.7% vs 4.6% in gosh vs gnch; p=0.6). gender, underlying disease -primary immune deficiency (pid) (n=273) vs haematological disease (n=133), use of myeloablative (n=153) vs reducedor minimal intensity conditioning (n=229), use of serotherapy or mega doses of cd34 ≥10 x10*6/kg did not influence the development of ta-tma. donor type: msd/ mfd(n=100) vs mud (n=129) vs mmud/haplo-hsct background: we evaluated the outcome of haploidentical hct (hhct) using ex vivo αβ t cell-depleted (tcd) grafts after reduced-intensity conditioning (ric) containing low-dose tbi (ld-tbi) in pediatric patients with acute leukemia (al) in complete remission (cr).methods: between may 2012 and october 2018, 36 patients with acute leukemia (17 all and 19 aml) in cr received haploidentical hematopoietic cell transplantation (hhct) using tcrαβ-depleted graft at asan medical center children's hospital. eighteen patients received hhct between 2012 and 2015 (earlier time period) and the remaining 18 between 2016 and 2018 (recent study period). the conditioning regimens, the dose of αβ+ t cells and gvhd prophylaxis are summarized in table. results: all 36 patients achieved a sustained neutrophil engraftment at a median of 10 days (range, 9-13) . of 36 patients, 11 patients (8 all & 4 aml) relapsed at a median of 6 months (range, 3-16) after transplant. of the 11 patients, 10 patients died of disease. one patient died of disseminated tuberculosis at 11 months after transplant, leading to the trm of 4% at 1 year. as of december 2018, 25 of the 36 patients survive free of disease at a median follow-up of 21 months (range, . at a median followup of 35 months (range, 1-80), efs and os at 2 years for all patients were 59% and 67%, respectively. outcome of hhct in the recent study cohort was significantly better than that in the earlier study period (efs of 85% vs efs of 44%, p=0.05). among the 17 patients with all, the efs of 8 patients, who received hhct in early time period after conditioning with tbi of 600 cgy, was significantly worse than that of 9 patients, who received in recent study period after a higher dose of tbi at 800cgy (13% vs 83%, p< 0.05). the efss of aml were similar between the two study groups (70% for earlier cohort vs 86% for recent study, p>0.05).conclusions: in pediatric patients with acute leukemia in cr, our current haploidentical hct using ex vivo αβ tcd graft after ric containing ld-tbi without gvhd prophylaxis is feasible approach with a low trm. the background: anti-hla antibodies (ahab) have been recently recognized as an important risk factor for graft failure (gf), especially in hla-haploidentical stem cell transplantation (haplo-hsct). although, recently, ebmt consensus guidelines have been published [ciurea, bone marrow transplant 2018] , experience in pediatric t-cell depleted (tcd, another well-known risk factor for gf) haplo-hsct is lacking. in the present study, we report our experience on the use of a desensitization approach based on ebmt guidelines.methods: between june 2017 and august 2018, all patients affected by non-malignant diseases and scheduled for a transplant from an hla-haploidentical donor after negative depletion of αβ t and b cells as previously described [li pira, biol blood marrow transplant. 2016 ], were tested for ahab with luminex® solid-phase immunoassay (one lambda, thermo fisher scientific) 1 month before the hsct. all patients with a mfi higher than 5000, which is considered a cutoff predicting for gf, were treated with a desensitization protocol based on the use of anti-cd20 rituximab (375 mg/m 2 before and immediately after the end of plasma-exchange cycle) and plasma-exchange (pe) ± infusion of irradiated buffy coat (bc) (if after pe ahab mfi was still > 5000 mfi). this latter was obtained by the non-target fraction of the αβ t-cell/b-cell-depletion procedure and consisted of 5 * 10 7 irradiated nucleated cells/kg of the recipient; this was infused 2-4 hours before the infusion of the tcd graft. pe was performed with miltenyi life 18 tm apheresis unit (miltenyi, bergish-gladback) .results: in the study period, 37 patients received αβ tand b-cell depleted haplo-hsct. eighteen (48%) resulted positive for ahab (mfi> 1000); 14 (77%) of them had an mfi > 5000 for either anti-class i or ii ahab. these patients (see table i background: osteonecrosis (on) is a debilitating complication in survivors of allogeneic hematopoietic cell transplantation (hct). limited data is available on its course post-transplantation in children. the purpose of our study was to identify recipients of hct in whom pre-and post-magnetic resonance imaging (mri) is indicated.methods: the retrospective cohort consisted of 436 patients who underwent first allogeneic hct from 1998-2014, and prospectively underwent a total of 1092 pre-and post-transplant mri studies of the hips and knees done annually for 3 years regardless of symptoms. surviving patients were followed for a median time of 8.33 (range 3.93-14.10) years. cases of on were compared to controls matched for age, sex, transplant type, and follow up in a 1:4 ratio for the following variables: ethnicity, underlying disease, on pre-hct, conditioning regimen, graft source, bone mineral density z-scores, body mass index, presence or absence of graft-versus-host disease, steroid use and dosage, and survival status.results: thirty (6.9%) patients had mri findings confirming on post-hct. all patients with on except one were more than 10 years of age. twenty (67%) patients were male. on pre-hct (p < 0.0001) was the only factor associated with presence of on post-hct. epiphyseal on was seen in 9 (30%) patients pre-hct, and 26 (87%) post-hct. eighteen (60%) patients had involvement of more than 30% of articular surface, and were more likely to undergo surgery (p = 0.009).conclusions: the incidence of on in this large pediatric cohort was 7%. the only risk factor for on post-hct was pre-existing on. mri evaluation for on pre-hct is indicated in all patients. mri evaluation for on post-hct is only indicated for patients with on pre-hct and symptomatic patients. this will entail cost savings of usd 500,000 per 100 surviving allogeneic hct patients per year. patients with more than 30% involvement of the articular surface need close follow up.clinical trial registry: none disclosure: none impact of rabbit anti thymocyte globulin exposure on immune reconstitution and outcome after stem cell transplantation in children background: rabbit anti thymocyte globulin (ratg) has been frequently used for many years as gvhd prophylaxis in pediatric stem cell transplantation. precise dosing regimens are crucial but remain challenging due to several pharmacological characteristics in children.methods: ratg levels were measured in pediatric patients undergoing allogeneic stem cell transplantation after obtaining approval by the local irb and informed consent by legal guardians. 32 pediatric patients who received either thymoglobuline™ (n=22) or grafalon™ (atg-f) (n=10) as part of their conditioning regimen background: obesity among children is a growing health problem. malnutrition or being over-weight can be of prognostic impact among children who need hsct.scientific literature shows a lot of controversy in terms of effect of bmi at the time of infusion on the outcome of hsct.methods: we reviewed data of patients who underwent hsct at king faisal specialist hospital & research centre between 2010-2016 to correlate bmi with the outcome and complications of hsct. transplant naïve recipients with age at infusion between 2-14 years who received hsct from matched related donor or autologous hsct, were included in the dataset for analysis. a total of 423 patients' profiles were reviewed of whom 51.1% were boys. median age at transplant was 7.5 years. primary indication of disease was malignancy in 41.6% followed by hemoglobinopathies 26.7%, bone marrow failures 17.3% and immune disorders 12.5%. solid tumors accounted for 29.5% among malignant disorders. myeloablative conditioning was used in 97.2% transplants with 11.8% regimens containing total body irradiation. majority of the patients 81.3% underwent allogeneic transplantation using bm as the source in 80.4% and pbsc in the remaining 19.6% cases. donor was hla-identical sibling in 89%, parents in 10.2% and others in the remaining 0.9% patients. median tnc dose was 2.55 x 10^9 and cd34 was 5.63 x 10^6 per kg of the body weight at the time of infusion. age and gender specific bmi percentiles were obtained and classified according to the definition of centers for disease control and prevention (cdc); 24.8% (105) recipients were categorized as under-weight, 61.2% (259) normal, 5.9% (25) over-weight and remaining 8% (34) as obese.results: based on chimeric studies at day-100, our engraftment rate was 98.3% (400) out of 407 evaluable cases. median time to neutrophil recovery was 14-days from infusion and 26-days for platelets. no statistically significant difference was found for engraftment rate on d-100 as it was 100% (25) among 98.8% (247) in normal, 97% (97) in under-weight and 96.9% (31) in the obese (p-value: 0.467). median time to neutrophil recovery from the infusion date was 15-days in over-weight patients and 14 in the remaining three groups (p-value: 0.841). acute graft vs. host disease (agvhd) of any grade at day-100 was recorded in 19.9% (84). any-grade agvhd was more common in over-weight 24% (6), followed by obese with 23.5% (8), 21% (22) in under-weight and 18.6% (48) in normal bmi-category (p-value: 0.770). chronic gvhd was more frequent in over-weight (14.3%, 3), compared to 9) background: hematopoietic stem cell transplantation (hsct) is the standard treatment for children with severe aplastic anemia (saa) who have hla-identical related donor. there is no standard conditioning regimen for children with saa secondary to non-fanconi anemia (fa) constitutional bone marrow failure syndromes such as telomeropathies. we report the outcome of a consistent reduced intensity conditioning regimen in patients with idiopathic saa or inherited bone marrow failure syndromes other than fa who underwent hla matched related hsct methods: children with saa underwent hsct using the following conditioning regimen: fludarabine 175mg/m2, cyclophosphamide 80 mg/m2, and atg (thymoglobulin) (10 mg/kg) . gvhd prophylaxis included cyclosporin and mycophenolate mofetil. donors were all matched related and bone marrow was the stem cell source. all patients had normal chromosomal fragility testresults: a total of 18 children with saa underwent hsct, 12 females and 6 males. average age was 8.1 (range 0.8-13.8 years). all nine patients who were tested for telomere length had short telomeres. pathogenic or likely pathogenic mutations were reported in 5 patients (2 ercc6l2, 1 ankrd26, 1 tinf2, 1 lztfl1). all donors had normal physical examination, normal cbc, and negative genetic testing if patient mutation is known. all 18 patients engrafted successfully, median time to neutrophil engraftment was 16 (range, 11-29 days) and platelet engraftment 19 (range, 13-45 days). median infused nucleated cell dose was 3.3 (range,0.9-7.3 x10 8 /kg) and cd34 cell dose was 6.7 (range, 1.1-13.1 x10 6 /kg). none of our patients had acute gvhd and one patient had mild classic chronic gvhd of the skin that was controlled with topical therapy for a short period. three patients had secondary graft failure in the first-year post transplant. first patient had pancytopenia with loss of donor chimerism and underwent successful second transplant using fludarabine, atg, and melphalan. the second patient had a nonfunctioning graft despite full donor chimerism suggesting that the related donor might be affected and had silent phenotype. the third patient had homozygous ercc6l2 mutation and developed progressive cytopenia with myelodysplastic features few months post-transplant and subsequently underwent myeloablative matched unrelated transplant using busulfan, fludarabine, thiotepa and atg. however, the patient progressed to have acute myeloid leukemia six months post hsct. fifteen patients (83%) have normal cbc and stable donor chimerism. median follow-up duration of 1140 days (range 330 -2595 days). one patient with lztfl1 mutation developed chronic renal impairment five years post hsct.conclusions: hsct using lower dose cyclophosphamide (80mg/kg) as part of fludarabine based regimen was safe and effective in saa patients with shorter telomeres and described genetic abnormalities. optimal conditioning regimen in ercc6l2-associated bone marrow failure needs to be defined. larger study is needed to confirm our results.clinical trial registry: not applicable disclosure: nothing to declare late effects in patients with hemophagocytic lymphohistiocytosis treated with hematopoietic stem cell transplantation: a review of the literature background:hemophagocytic lymphohistiocytosis (hlh) is an inherited or acquired disorder of immunedysregulation. early diagnosis and immunosuppressive treatment can prevent significant organ-failure. the inherited forms, and some acquired cases can only be cured by hematopoietic stem cell transplantation (hsct). with modern transplant practices, a significant number of patients survive. the purpose of this literature review was to collect data on the frequency and type of late effects in hlh patients surviving after hsct and to examine the association with pre-existing hlh conditions (eg. involvement of the central nervous system (cns) before transplant) and with the pre-transplant conditioning regimens.methods: the medline, embase, web of science and pubmed databases were searched, by two librarians at the karolinska institutet, between may and september 2016 according to the preferred reporting items for systematic review and meta-analysis (prisma) statement. the search terms included "hlh", "fhl", "mas", multiple terms for "hsct" and late-effect conditions. inclusion criteria were publications in english that included children between january 1995 and may 2016. authors of this review screened all the abstracts of studies against the inclusion criteria.results: only nine papers published between 2006 and 2016, with information on late effects in hlh patients who had undergone hsct, were identified. three reports include only small numbers of hlh patients. the remaining 6 papers contain data on 261 long-term survivors with a median follow-up of 5.4 years. five papers address neurological sequelae with a reported incidence from 7-57%. the highest incidence was found after a thorough neurological assessment of 21 hlh patients compared to matched sibling controls. however, the association with cns disease before transplant and age at transplant was not clear. patients with ebvassociated hlh seem to have fewer long-term neurological problems. non-neurological late effects are described in 4 papers only, with endocrinological problems, namely short stature, being the most frequent. one paper specifically analyzed poor growth, thyroid dysfunction and vitamin d deficiency in a cohort of patients with non-malignant disorders including hlh who had undergone hsct after a reduced intensity conditioning regimen and found significant abnormalities in all groups.conclusions: data on late effects in hlh patients is scarce and is mostly based on the retrospective evaluation of small national cohorts. the available information indicates that a significant number of patients suffers from problems which affect their daily life, but lack of information does not allow to analyze the association between pre-transplant conditions and long-term sequelae. therefore, a retrospective comprehensive analysis of patients registered in the ebmt and cibmtr registries is currently performed. it will be crucial to better define the frequency and type of late effects in a large cohort. this knowledge will aid counselling prior to hsct, provide guidance for long-term monitoring of these patients, and potentially identify specific risk factors for late effects in this rare patient population.disclosure: nothing to declare. allogeneic stem cell transplantation in patients with mucopolysaccharidosis type ii (morbus hunter)bernd hartz 1 , nicole muschol 2 , matthias bleeke 1 , johanna schrum 1 , ingo müller 1background: the transplantation of hematopoietic stem cells (hsct) is one of the leading methods of treatment in patients with blood system diseases, primary immunodeficiency syndromes and genetic diseases. at the same time, the quality of life in patients in the long-term after hsct significantly differs from the quality of life of healthy people of the same age. deformations in psychosexual development including problems in the gender identity formation cause social isolation of adolescents, which makes their sexual selfrealization impossible and significantly reduces the quality of their life. the purpose of our study was an assessment of the level of gender identity formation of adolescents and psychosexual development correlation to the normal adolescents of the same age.methods: in a prospective single-center study in 2018, on the base of the department of rehabilitation medicine raisa gorbacheva memorial institute of children´s oncology, hematology and transplantation, we conducted a study of 17 families. the respondents were: 1) parents / guardians of patients accompanying them in the process of examination; 2) adolescents who underwent hsct treatment and undergo planned examinations at the clinic in the posttransplant period (after d + 100), (n = 17, of which 8 girls and 9 boys, age 12 -17 years, from the date of hsct 1 -5 years).the following methods were used to assess gender identity: specially developed questionnaires for teenagers and parents; questionnaire by sandra l. bem (sandra l. bem, 1974) ; projective techniques "the human picture", "the non-existent animal"; max lüscher´s color choices test.results: the traditional type of gender identity, which characterizes high masculinity among male respondents and high female gender indicators in 100% of cases, was not revealed. both among girls and among boys, the androgynous type prevails with a tendency toward femininity.on average, adolescents see themselves as a bit more courageous than their mothers, with rare exceptions, regardless of gender. this confirms the thesis that we received in a previous study that parents tend to see and encourage complacency of adolescents of both sexes, passivity instead of leadership, dedication and independence. all 100% of adolescents who participated in the test demonstrate a shift in the theme of aggression, 77% have some signs of preventing sexual self-determination, abandoning their body, gender, and age. 88% of patients do not communicate with their peers. in 47% of them, negative emotions prevail over positive ones. one third of the test participants demonstrate strong support for rest and minimizing their efforts.conclusions: the characteristics of family upbringing of adolescents who have undergone hsct often contribute significantly to limiting their social experience and lead to specific deformities of individuality, including in the sphere of gender identity. we consider advisable to introduce thematic group counseling of parents within the framework of the "patient's school" in psychological treatment support in the clinic. early diagnosis of the personal aspects of the psychosexual development of adolescents after hsct allows for timely identification of individual problems in this area and identification of general trends in the long term after hsct.disclosure: all authors -nothing to disclose. abnormalities in the morphology of the umbilical cord blood obtained at delivery from children who received a diagnosis of cerebral palsy maciej boruczkowski 1 , izabela zdolińska-malinowska 2 , maciej rojek 2 , dariusz boruczkowski 2background: embryonal brain tumors are the most common malignancies in infants less than 48 months of age. histologically characterized as undifferentiated small round cell tumors, all are similarly aggressive, have a tendency to disseminate throughout central nervous system and very poor prognosis. we tried to assess the effectiveness of tandem highdose chemotherapy (hdct) with autologous hematopoietic stem-cell transplantation (auto-hsct) in this patient group. methods: from 2010 to 2018, 52 infants under 48 months with different primary embryonal brain tumors such as medulloblastoma (n=28), different pnet nos (n=10), pineoblastoma (n=3), atypical teratoid rhabdoid tumor (n=3), etmr (n=8) after surgical resection and induction chemotherapy were planned to receive tandem hdct with auto-hsct. nine patients were conducted only single transplantation because of the development of lifethreatening complications after the first hdct (n=4) or the emergence of early disease progression (n=5). at the moment of hdct 31 patients were in complete remission (cr), 20 patients were in partial remission (pr) and 1 patient had stable disease (sd). the conditioning regimen for tandem auto-hsct were: the first hdct was carboplatin and etoposide, the second was thiotepa and cyclophosphamide, both with intraventricular methotrexate.results: the median follow-up is 24 months (range, 6-85). the median time to engraftment after the first auto-hsct was background: a series of findings suggest that optimizing natural killer (nk) cell reactivity could further improve outcome after allogeneic hematopoietic cell transplantation (allohct). this could be achieved by killer cell immunoglobulin-like receptor (kir) genotype informed donor selection. an enhanced receptor-ligand model which used kir2ds1 and kir3dl1 donor genotype information to augment nk cell activation and minimize inhibition demonstrated improved survival in one large aml study (boudreau et al, jco 2017) . likewise, a second model built on the classification of centromeric and telomeric kir haplotype motifs, also predicted mortality after allohct for aml (cooley et al, blood 2010) . this joint ebmt and cibmtr study aimed at validating the two approaches in an independent cohort of patients with mds or secondary aml.methods: donor samples were retrieved from the collaborative biobank (dresden, germany) and mapped to patient outcome data extracted from the ebmt and cibmtr. genotyping of all kir genes by sequencing exons 3, 4, 5, 7, 8, and 9 was performed by high resolution amplicon-based next generation sequencing. the impact of the classifiers on time-to-event outcomes was tested in cause-specific cox regression models adjusted for patient age, a modified disease risk index, performance status, donor age, hla-match, sex match, cmv match, conditioning intensity, type of t-cell depletion and graft type.results: clinical data from 1704 patients and corresponding donor genotype information were analyzed. the median age at allohct was 59.4 years (range, 18.1 to 79.6 years). the indication for allohct was mds for 72% and saml for 28% of patients. disease risk was low/intermediate and high/very high in 41% and 59%, respectively. donors were 10/10 matched for 79% of patients. myeloablative, reducedintensity and non-myeloablative conditioning regimens were used in 31%, 57%, and 12% of patients, respectively. peripheral blood stem cells were the predominant graft source (93% of patients). atg was administered in 56% and alemtuzumab in 9% of patients. during follow-up after allohct 776 patients died. in univariable and multivariable analyses of the whole cohort, overall survival and the cumulative incidence of relapse of patients with kiradvantageous versus disadvantageous donors were not statistically significantly different. we could not replicate the pattern of outcomes predicted by the kir3dl1/ conclusions: relapse incidence and overall survival after unrelated donor allohct could not be predicted using the kir3dl1/kir2ds1-receptor-ligand model and centromeric/telomeric kir-motif model in this large cohort of patients with mds or secondary aml. this points at the possibility of interactions between nk-cell mediated alloreactivity and disease type or procedural variations of allohct. available information on kir-genes, which have been sequenced but not yet analysed, will be investigated in exploratory analyses.disclosure: the authors have nothing to disclose in the absence of an alternative donor, it is recommended that patients undergo desensitization therapy, especially with high dsa levels (>5,000 mfi). the aim of this study is to analyze the impact of dsa on risk of gf and poor graft function (pgf), and on major outcomes in a consecutive cohort of patients who were systematically screened for dsa before haplo-sct. methods: 141 consecutive patients were candidates for unmanipulated haplo-sct with post-transplant cyclophosphamide (pt-cy) at our center from january 2012 to january 2018 and 135 were analyzed for the presence of hla antibodies.results: 134 patients underwent haplo-sct. hla antibodies were detected in 40 patients,19 of them were dsa, while 21 were non-dsa (ndsa). 10 patients out of 19 with dsa were transplanted using the same donor; 2 underwent a desensitization program before transplant.background: a recent study from ebmt comparing matched sibling (msd) versus haploidentical donors transplantations, showed better outcome with msd in adult patients with intermediate risk aml in first remission (cr1). however, a female donor to a male recipient transplant combination is a poor prognostic factor and this study did not address the question whether in this situation, a haploidentical donor transplant might do better. the present study compared the outcomes of allografted male patients according to whether they received stem cells from a female msd or a haploidentical donor, in the intermediate and high risk cytogenetics groups (mrc classification).methods: the study included 1066 male patients with cytogenetics transplanted between january 2007 and june 2017 and reported to ebmt. 834 received stem cells from a msd female donor and 232 from a haploidentical donor (133 male and 99 female). the follow up was 25 months (12-62). we studied separately intermediate and high risk patients. multivariate analysis was adjusted on factors differing significantly between the 2 groups.results: 1-intermediate risk group: 638 male patients received a female msd and 160 a haploidentical transplant. the distribution of group characteristics was even except that in the haploidentical transplant group, donors were younger (39 y versus 51; p< 0.0001), marrow was more frequently used (45% versus 17%, p< 0.0001) and the interval from diagnosis to transplant was longer (5.4 versus 4.5 months, p< 0.0001). by univariate analysis at two years post transplant, cumulative incidence (ci) of nrm post haplo was higher (26% versus 15%, p=0.002) and ci of extensive chronic gvh lower (14% versus 27%; p=0.002). lfs post msd and post haplo were 64% and 51% (p=0.03), os 69% and 60% (ns), grfs (43% and 43%). by multivariate analyses the only significant poor risk factors were the haplo-identical transplant for nrm (hr: 1.7 (1.1-2-61)) and the patient age for os (hr: 1.15 (1.02-1.28; p=0.02). haploidentical transplantation resulted in less chronic gvhd (hr: 0.43 (0.29-0.64); p < 10 -4 ), but a lower lfs (hr: 1.7 (1.1-2.61); p=0.04). 2-high risk group: 196 male patients received a female msd and 72 a haploidentical transplant. in the haploidentical group, donors were younger (38 y versus 54; p< 0.0001), marrow was more frequently used (42% versus 11%, p< 0.0001) and the interval from diagnosis to transplant was longer (5.1 versus 4.3 months, p= 0.003). by multivariate analysis, haploidentical transplants were associated with a lower relapse incidence (hr: 0,40 (0.21-0.75; p = 0.004),a better lfs (hr: 0,46 (0.28-0.77; p = 0.003),os (hr: 0,43 (0.25-0.75; p = 0.003), and grfs (hr: 0,53 (0.34-0.84; p = 0.006)(see figure) . the only other significant prognostic factor was patient age.conclusions: this study shows that in a male patient with intermediate risk aml, a genoidentical sister donor remains associated with a better lfs. in contrast, in a male patient with high risk aml in cr1, a haploidentical donor may be a better choice than an hla genoidentical sister.disclosure: nothing to declare p663 abstract already published. hematopoietic transplant for older acute leukemia patients: improved survival with offspring donor in comparison with older-aged matched siblingsyu wang 1 , sheng-ye lu 1 , qi-fa liu 2 , de-pei wu 3 , xiao-jun huang 1background: post-transplant relapse remains the major cause of death of treatment failure. therapeutic options for relapse after first allogeneic stem cell transplant (1st hsct) include chemotherapy followed by donor lymphocyte infusion or second allo-hsct (2nd hsct) from the original donor or change to another donor. however, there is unclear outcome for different treatment approach. in this retrospective cohort study, we aim to compare the clinical outcome after different treatment strategy for relapse after first allo-hsct.methods: between 1992 jan and 2018 oct, 1493 consecutive patients receiving 1 st hsct registered to the bmt database in national taiwan university hospital were analyzed. among them, 580 cases had relapsed after first allo-hsct. one hundred and three patients who received no treatment after relapse or with incomplete data were excluded. their transplant data was collected following the ebmt registry data collection forms and manuals. overall survival rate and progression free survival rate were performed by the kaplan-meier method. univariate and multivariate analysis were performed using cox proportional hazard regression model.results: of the 477 patients who experienced relapse after 1 st hsct, total 244 patients (51%) received chemotherapy followed by dli or 2 nd hsct from the same donor (no change group), 36 patient (8%) received chemotherapy followed by 2 nd hsct from different donors (change group), and 197 (41%) had conventional chemotherapy alone. the patients in "change group" were younger (median age 29 vs 37, p = .02), and had more patients achieving complete remission (cr) prior to 2 nd hsct (44% vs 10%, p = < .01) than patients in "no change group". after the 2 nd hsct, the cr was 68% for "no change group" and 91% for "change group". the progression-free survival at 3-year and 5-year were 10.5% and 5.9% (fig 1a, p = .0438%), respectively, for "no change group" and 9.4% and 9.4%, respectively, for "change group". while the overall survival (os) at 3-year and 5-year were 15.8% and 9% (fig 1b, p = .3459%), respectively, for "no change group" and 13.5% and 9%, respectively, for "change group". those who achieved cr prior to 2 nd hsct had a trend of better os than those without cr (17.5% vs 8.7% at 3-year; 8.7% vs 9.1% at 5year, p =.0365)( fig 1c) . there were 1 cases survived for more than 10 years in "change donor group" and 6 cases survived more than 10 years in "no change group". only one had developed relapse after 2 nd hsct but achieved subsequent remission again.conclusions: our study shows that change donor had similar poor outcome comparing to those using the same donor after the 1 st hsct. patients who achieved cr before 2nd hsct had a trend of better os than those without remission and the long-term survivors were only those who achieved cr prior to 2 nd hsct. novel therapy for cr induction would be warrant for this poor prognostic population.disclosure: nothing to declare functional relevance of fetal microchimerism in nk cell cytotoxicity against leukemic blasts in children: a role for hla-c1 and kir2dl2/s2?background: allogeneic stem cell transplantation (allo-sct) remains the most effective curative intent therapy for patients with unfavorable risk acute leukemia. various donor options are available for the patient who lacks an hla-matched sibling donor, such as unrelated donors (urd) and hla-mismatched family (haploidentical) donors. in order to discover the exact role of transplantation type, there are many retrospective analysis, which compared these donor sources, have been reported. recent studies showed some promising results of haploidentical donor transplantation (hidt) using post-transplant cyclophosphamide in comparison with unrelated donor. the goal of this study was to compare the outcome of allo-sct from haploidentical versus matched unrelated (mud 10/10) or mismatched unrelated donor at a single hla-locus (mmud 9/10) for patients with acute leukemia in remission.methods: ninety-six adult (18-65 years) patients with acute leukemia in first or second remission who underwent allogeneic transplantation with a minimum 100 days follow-up at florence nightingale hospital hematopoietic stem cell transplantation center between 2011 and 2017 were included in this study. patient characteristics and medical records of all patients were reviewed retrospectively. thirty-eight patients who received haploidentical donor transplantation were compared with 23 patients receiving a mud 10/10 and 35 receiving a mmud 9/10. patients who completed minimum 100 days post-transplantation follow-up were identified as eligible for survival analysis.results: the characteristics of the patients and transplant donors in this study are summarized in table 1 . median age of patients was 39.4±14 years. proportion of male patients was 39.1%, 74.2% and 57.8% for mud 10/ 10, mmud 9/10 and hidt groups, respectively, which is significantly different (p=0.02). the other baseline factors were similar, including patient age, donor age, recipient cytomegalovirus (cmv) status, donor cmv status, graft versus host disease incidence, median neutrophil and platelet engraftment times and disease status at post-transplant 100 th day. no significant difference was identified in survival analysis among the mud 10/10, mmud 9/10 and hidt groups, even if they were classified according to primary disease (aml vs all) and pre-transplant disease status (cr1 vs cr2). also, donor cmv status (cmv igg positivity or negativity) was not an important factor on survival analysis when compared between these three groups (p=0.406).conclusions: in our study population, clinical outcomes of hidt patients were inferior to mud 10/10 and mmud 9/10 groups. when choosing an alternative donor for patients without an available hla-matched sibling, urgency of transplantation and host/donor features should be considered. we believe that hidt might be a feasible alternative choice in this subset of patients.disclosure: nothing to declare p673 g-csf primed bone marrow in hla-haploidentical transplantation using post-transplantation cyclophosphamide (ptcy) could promote tolerance and further reduce risk of gvhd nadira durakovic 1,2 , zinaida perić 1,2 , lana desnica 2 , ranka serventi-seiwerth 2 , mirta mikulić 2 , brian melamed 3 , alen ostojić 2 , dražen pulanić 1,2 , pavle rončević 2 , zorana grubić 2 , radovan vrhovac 1,2 implementation, development, and coordination of unique quality management systems with evaluation audits, intrahospital and international accreditation and certification processes. quality of health care is a major focus for providers, patients, and accreditors; so, in this study, we aim to compare the quality of bm harvested at ipo-collection centre (icc) with the quality of bm received from external collection centres (ecc) during these 6 last years.methods: this retrospective evaluation included the number of total nucleated cells (tnc) requested by the transplant centre, the tnc collected, and the results of bm microbiological analysis performed; donor age, weight and infectious disease markers (idm); patient demographics and diagnosis. bm collection technique in use at icc was validated according to jacie standards.we consider successful a collection (sc) with tnc between 75 and 125% of the requested value, unsuccessful (uc) if lower than 75% and outstanding (oc) if over 125%.results: a total of 106 bm was collected, 99 for allogenic (75 unrelated) and seven for autologous transplant; 21 unrelated bm were received from ecc (nine from germany, seven from usa and five from portugal). patient main diagnosis were severe aplastic anaemia (n=28), acute myeloblastic leukaemia (n=20), and acute lymphoblastic leukaemia (n=15). donors idm were all negative and nonreactive.mean age (±standard deviation, sd) was 33 (±13.2) and 31 (±9.3) years for icc and ecc donors, respectively. at icc, we were asked to collect an average (±sd) of 221.7*10 8 (±136.0) tnc while ecc were asked for 184.7*10 8 (±105.7). we collected 169.7*10 8 (±82.3) and received 204.5*10 8 (±109.2) tnc. correlation between requested and collected tnc was 0.69 at icc and 0.56 for ecc.we had 41.2% sc and 26.8% oc meaning an accomplishment of 68.0%. we failed to collect required tnc in 32.0%. although 85% of received bm fulfil tnc requirements, bm processing lowered this value to 55% due to erythrocyte removal (seven patients with major abo incompatibility) and plasma reduction (two patients with abo minor incompatibility). these steps reduce final tnc available for transplant. weight difference between donor and patient had no significant impact on final tnc collection performance.sixteen bm from icc (seven staphylococcus spp., five propionibacterium acnesand fourcorynebacterium spp.) background: success of peripheral blood stem cell (pbsc) collections depends on patient biological parameters and stable apheresis device performance. peripheral blood cd34+ cell enumeration is the most reliable predictive factor of apheresis yield however, there are some unexpectedly poor cd34+ cell harvests despite successful mobilization. the aim of the study was assess total collections cd34+ yields and factors influencing main apheresis procedure outcomes including collection efficiency (ce).methods: of 2233 consecutive donors covering the period 1-1-2016 to 30-9-2018 were analyzed for the following parameters: pre cd34 count, cd34 yield per procedure, total cd34 dose collected per patient, cd34 collection targets requested by clinical teams. the efficiency of pbsc procedures was determined by calculating the ce and the correlation coefficient between pre cd34 count and yield per procedure. ce was correlated to preprocedure wbc, platelet count, pre cd34 count and blood volume processed. all pbsc collections were performed by optia spectra across 7 units in uk.results: of the 2233 donors, 1611 were autologous and 622 allogeneic. the autologous donors underwent in total 2543 procedures. the median cd34 target dose for these donors was 4x10 6 /kg.799 (50%) achieved the target dose with 1 procedure and 566 (35%) with 2 procedures. the median pre cd34 count was 30/μl. the median cd34 yield per procedure was 2.54x10 6 /kg and the median total cd34 dose collected per donor was 5.38x10 6 /kg. 92 (5.7%) of autologous donors collected a total cd34 dose < 2x10 6 /kg, of those 27 (1.7%) had a pre cd34 count < 10/μl and 65 (4%) >10/μl.the allogeneic donors underwent in total 878 procedures. the median cd34 target dose for these donors was 5x10 6 / kg. 381 allogeneic donors (61%) achieved the target dose with 1 procedure and 221 (36%) with 2 procedures. the median pre cd34 count was 51/μl. the median cd34 yield per procedure was 4.07x10 6 /kg and the median of total cd34 dose collected per donor was 6.70x10 6 /kg. 17 (2.7) % of allogeneic donors collected a total cd34 dose < 2x10 6 / kg, of those 3 (0.5%) had a pre cd34 count < 10/μl and 14 (2.2 %) >10/μl. the median ce for autologous donors was 55% (range 20-166) and for allogeneic donors was 47% (range12-116). the ce was negatively correlated to wbc (r= -0.29 and -0.37) and platelet count (r=-0.14 and -0.06) for auto and allogeneic donors respectively, but did not correlate to the pre cd34 and blood volume processed. the correlation coefficient between pre cd34 count and cd34 yield per procedure was r 2 =0.67 for the autologous and r 2 =0.34 for the allogeneic collections.conclusions: the majority of autologous and allogeneic donors achieved the target cd34 dose with one procedure. 94.3 % of autologous and 97.3 % allogeneic donors collected a transplantable cd34 dose of > 2x10 6 /kg. 4% of autologous and 2.7% of allogeneic donors did not collect a transplantable dose despite a precd34 count of >10/μl indicating suboptimal procedure performance. the ce was variable and was negatively correlated to the preprocedure wbc and platelet count. the ce and correlation coefficient are lower in allogeneic donors compared to autologous donors.disclosure: nothing to declare the outcome of autologous blood stem cell collection and its actual use in real world: the 21st century experiencekyoungmin lee 1 , jung yong hong 1 , dok hyun yoon 1 , jae-lyun lee 1 , shin kim 1 , kyoung min lee 1 , jung sun park 1 , cheolwon suh 1background: mobilized peripheral blood stem cells (pbscs) have largely replaced bone marrow as the graft source for allogeneic stem cell transplantation. pbscs mobilization with g-csf is highly effective even on the 4th day in order to collect enough number of stem cells. a longitudinal, prospective, observational, single-center, cohort study on healthy donors (hds) was designed to identify predictors of cd34+ cells on the 4th day. methods: as potential predictors of mobilization, age, sex, body weight, height, blood volume as well as white blood cell count, peripheral blood (pb) mononuclear cells, platelet count, hematocrit, and hemoglobin levels were considered. two different evaluations of cd34+ cell counts were determined for each donor: baseline (before granulocyte colony-stimulating factor [g-csf] administration) and in pb after g-csf administration on day 4. a total of 122 consecutive hds with a median age of 47.5 years were enrolled.results: the median value of cd34+ on day 4 was 43 cells/μl (iq 23-68). basal wbc, plt and basal cd34+, are significantly higher for group with cd34+ on the 4th day over the median than below. a multivariate quartile regression analysis, adjusted by gender, age, basal cd34 + and basal plt, shows a, progressively steeper, relationship between baseline cd34+, basal plt and cd34+ on the 4th day. the basal cd34+ cut-off for prevision of cd34+ on the 4th day was < =2 cells/μl and >=3 cells/μl whereas basal platelets count was < =229 x 109/l and >=230 x 109/l.conclusions: g-csf can be highly effective in hds on the 4th day in order to collect enough number of stem cells and we have developed a model for predicting the probability to perform pbsc collection after a short course of g-csf.disclosure: nothing to declare p692 pre-apheresis peripheral blood cd34+ cell counts highly correlates to actual stem cells collected background: prediction of stem cell yield on the basis of pre-apheresis cd34+ cell count and the processed blood volume is essential for the planning and executing of the apheresis process.methods: data analyzed included donor weight, complete blood count and cd34+ count on day of collection, total processed blood volume, cd34 + cell dose collected in the apheresis product and the number of aphereses performed. using the method described by pierelli et al, predicted cd34 + yields were calculated: predicted cd34 + yield x 106/kg = (benchmark ce x volume of blood to be processed x peripheral cd34+ count per μl) / (patient's weight in kg x metric conversion factor).results: in 2017 we established the method described by pierelli to predict the cd34+ cell yield. 323 allogenic aphereses were performed in 2017 with this approach. mean processed volume was 13.71 liters. the mean cd34+ peripheral count before apheresis was 68/ul, the mean collected cd34+count per kg bodyweight recipient was 6.35. pearson´s correlation coefficient (r) between predicted yield using pre-apheresis cd34+count and actually collected cd34+ cells per kg bodyweight recipient was 0.967. the mean difference between predicted and collected cell dose was +4.45%.with knowledge of the predicted stem cell count, we were able to adjust apheresis procedure. in case of marginal predicted yield compared to the requested cell dose, we increased the blood volume to be processed. this proceeding led to a significant reduction of second day donations in 2017 by 38% compared to 2016. in only 3 cases we saw more than -40% lower cd34+ doses collected than initially predicted. all 3 donors showed mild iron deficiency with rbc microcytosis, a factor known to affect apheresis procedure.conclusions: pierellis method of calculating the stem cell yield shows a good correlation between pre-apheresis cd34 + count and actual collected stem cells, making planning and adjusting of the apheresis procedure more feasible and reliable. this proceeding led to significant reduction of second day donations. attention should be paid to iron deficiency anemia, leading to lower than estimated cd34+ dose.[ background: for more than a decade many transplant centers routinely collect and cryopreserve two or more peripheral blood stem cell (pbsc) grafts for a tandem and/ or salvage autologous blood stem cell transplantation (absct) in patients with hemato-oncological diseases. however, subsequent high-dose chemotherapy (hd-cht) and absct is in many cases not performed for a variety of reasons, specifically in patients with aml, all, mpn and burkitt lymphoma. data about the actual utilization rate of the cryostored stem cell products are lacking.methods: we retrospectively analyzed the collection, storage and disposal practices of pbsc products from a large cohort of patients who were treated at the university hospital heidelberg or at the university medical center mannheim during a 12-year period. disease entities included acute myeloid leukemia (aml, n=74), acute lymphoblastic leukemia (all, n=22), mpn (n=18; primary myelofibrosis [pmf], n=9; chronic myeloid leukemia [cml], n=7; secondary fibrosis/essential thrombocythemia [et], n=1; not specified, n=1) and burkitt lymphoma (n=18). patients between 2001 and 2012 were included and followed until 2016.results: an adequate stem cell graft was defined as ≥2.0 x10exp6 cd34+ cells /kg body weight. 97% of the patients were able to collect at least one stem cell graft and the median number of grafts per patient was 1 (range 0-5). we could demonstrate that only 31% of all patients who had collected sufficient pbscs for transplant subsequently underwent an absct. among the disease entities the actual use of the stored pbsc grafts varied considerably from 0% to 56% (figure 1) .conclusions: we could identify striking discrepancies between the collection/storage and actual utilization of background: biosimilars (bio) of granulocyte colony stimulating factors (gcsf) were approved several years ago on the basis of some studies that indicated similar efficacy to the already patented gcsf (neupogen®, neu) both in terms of shortening the neutropenic period after chemotherapy as well as peripheral blood stem cell mobilization in patients with lymphoma and multiple myeloma (mm) treated with autologous stem cell transplantation (auto-hct). however, all these studies are retrospective and there are still concerns about the real efficacy of bio and even more about the real benefit on final costs.methods: we have retrospectively compared the characteristics of the mobilization procedure in both patients with mm and lymphomas, and healthy donors that received either neu or bio (with no chemotherapy) in 4 university hospitals in catalunya from december / 2013 to november 2017. bio replaced neu in june 2016 in all 4 institutions. primary objectives were the mobilization rate (defined as the percentage of patients that achieved ≥ 10 x 10 3 /ml cd34 + cells in peripheral blood on day 4) and the use of plerixafor (plex) in each group as pre-emptive strategy. a multivariable analysis of risk factors influencing the use of plex and mobilization failure (defined as collection of < 2 x 10 6 /kg cd34+ cells) was also performed.results: we treated 216 patients (102 lymphomas and 114 mm) and 55 healthy donors. both groups of patients (138 neu and 78 bio) and donors (33 neu and 22 bio) were comparable regarding pre-mobilization general characteristics. there was a trend for a lower median cd34+ peak on day 4 for bio patients (17 vs 20, p value = 0.1). a total of 53 patients received plex, although 7 of them (13.2%) out of strict theoretical indication, cd34+ cells > 10 x 10 3 /ml (range 10.5-13.5) and were removed for further analysis (n = 46, 31 in the neu group and 15 in the bio group). median number of cd34+ cells on day 4 was significantly lower in the group bio who needed plex (2.4 vs 4.8 for neu+plex, p=002), as well as cd34+ cells finally harvested (2.5 vs 3.3x 10 6 /kg, p=0.03). mobilization failure rate was higher in bio group (0 vs 20%, p=0.01). regarding no plex patients, median number of cd34 +cells on day 4 was also significantly lower for bio patients (33.4 vs 23.7, p=0.03). risk factors for plex use were age, basal disease (lymphoma) and number of prior mobilization therapies. the use of bio was the only risk factor for mobilization failure patients receiving plex [hr 10.3 (95%ci 1.3-77.8), p=0.02]. with respect to healthy donor mobilization, none of them needed plex but 2 cases from the bio group (9%) needed more than one apheresis procedures (2 and 3, respectively).conclusions: we found a lower efficacy of bio in the setting of stem cell mobilization of patients with only gcsf both in terms of a lower cd34+ cells peak on day 4 and a lower number of cd34+ cells in final apheresis product. bio gcsf also seems to be less effective in healthy donors.disclosure: no conflict of interest to be declaredbackground: auto-sct is a common treatment in patients with mm or nhl. the aim of the prospective multicenter goa (graft and outcome in autologous transplantation) study was to investigate the impact of mobilization method used on the cellular composition of collected blood grafts and eventually hematological and immune recovery as well as long-term outcome post-transplant. altogether 282 patients with mm or nhl transplanted between 5/2012 and 12/2016 at four university hospitals were included. the long-term goal of the study is to evaluate characteristics of optimal blood grafts in regard to post-transplant recovery and outcome. methods: altogether 147 patients with mm undergoing first auto-sct were compared with 135 patients with nhl. all nhl patients were mobilized with chemotherapy + g-csf, whereas 39 % of mm patients were mobilized with g-csf only (p < 0.001). mobilization data, graft cellular composition including cd34 + cell subsets and lymphocyte subsets of the blood grafts, post-transplant hematological recovery and outcome were evaluated. the median followup time was 46 months in mm patients and 35 months in nhl patients.results: mm patients mobilized cd34 + cells better (median peak blood cd34 + 65 vs. 40 x 10 6 /l, p < 0.001). the median number of aphereses was 2 in both groups (p = 0.09). altogether 26 % of the nhl patients received plerixafor compared to 12 % in mm patients (p = 0.002). the median number of cd34 + cells collected was higher in mm patients (6.4 vs. 3.9 x 10 6 /kg, p < 0.001).the median amount of cd34 + cells (with 7-aminoactinomycin) in the infused graft was 2.35 x 10 6 /kg in mm group and 2.5 x 10 6 /kg in nhl group (p = 0.02). the grafts contained more nk cells (median 10.1 vs. 6.1 x 10 6 /kg, p = 0.01) and cd19 + cells (median 1.69 vs. 0.00 x 10 6 /kg, p < 0.001) in mm patients. neutropenic fever tended to be more common in nhl patients (88 % vs. 79 %, p = 0.06) but mm patients had significantly more bloodstream infections (31 % vs. 15 %, p = 0.003). the median duration of hospitalization was longer in the nhl patients (22 d vs. 18 d, p < 0.001) and the nhl patients had significantly more often icu admissions (4 % vs. 0 %, p = 0.02).post-transplant neutrophil engraftment was faster in the nhl group (median 9 d vs. 12 d, p < 0.001). the median time to platelet engraftment was 11 days in both groups. platelet count was higher in the mm group from day 15 until 1 year after auto-sct. there were significantly more early deaths (< 100 d from the graft infusion) (4 % vs. 0 %, p = 0.02) and non-relapse deaths (6% vs. 0%, p = 0.003) in the nhl group.conclusions: mm and nhl patients differ in terms of cd34 + cell mobilization, graft cellular composition and post-transplant recovery as well as risk of non-relapse death. thus, the optimal graft may be different in nhl and mm patients.disclosure: the study was supported by vtr fund from north savo hospital district and study grant from sanofi. abstract already published. single-center experience in use of plerixafor for autologous stem cell mobilization: change in practice over 10 years background: plerixafor has been proven to mobilize human periferal blood stem cells (pbscs) alone or acting synergistically with granulocyte-colony stimulating factor (g-csf). it has mainly been used for rescue mobilization after failed regimen of chemotherapy plus g-csf, but lately preemptive use in poor mobilizers has been established as cost-effective. we aim to show ten years of experience and change in practice with plerixafor use in our center.methods: we retrospectively evaluated the outcome of mobilization procedures and leukapheresis collections in our center in the period from january 2009 to october 2018. practice from the first 5 years, when plerixafor was mainly used as rescue agent after failed attempt, was compared to period from 2014 till present when preemptive use in poor mobilizers (defined as cd34+ cell counts <10 x 10 6 /l blood) was established.results: in the period from 2009 to 2013, total of 434 patients underwent collection of autologous pbscs, and 55 patients required repeated mobilization cycles (12,7%). g-csf alone was used in 39 patients and 16 patients (29%) recieved combination g-csf with plerixafor. this cohort consisted of 10 males and 6 females with non-hodgkin (nhl) and hodgkin lymphoma (mh); 13 and 3 respectively. we noted 37 unsuccessful mobilizations (67,3% in repeated mobilization, 8,5% in total) of which one patient was from plerixafor group (6,3%).background: transplantation of hla-haploidentical hematopoietic stem cells (haplo-hsct) is an established procedure for the treatment of several different hematological diseases. one possible strategy to reduce the risk of graft-versus-host disease is represented by the selective depletion of ab t-lymphocytes (coupled with the depletion of cd19+ b-cells in order to reduce the risk of ptld) using the clinimacs device (miltenyi, bergish-gladback). before depletion, leukapheresis units are washed by lowspeed centrifugation, resulting in a platelet (plt) rich supernatant (prs) as a by-product generally discarded. we studied the possibility of recovering plt from prs of the haplo donor for transfusion to the recipient during the aplasia period occurring after hsct.methods: hsc donors were mobilized with g-csf (plus plerixafor in 6 out of 24 donors) as previously described [locatelli et al, blood 2017] . leukapheresis units, obtained with a spectra optia device, were washed twice (producing 2 prs bags) at low speed to remove plt before starting the ab t-cell/b-cell depletion. the two prs were leukodepleted by filtration, centrifuged, resuspended and pooled in a total volume ranging from 95 to 360 ml intersol (is-plt) for overnight incubation at 22°c with agitation (60 cycles/min). the is-plt samples were analyzed for the criteria established by the italian transfusion law. is-plt bags were examined for the following parameters: volume >40ml, plt after leukodepletion >2x10 11 , residual leukocytes < 1x10 6 , ph >6.4 at 22°c at the end of the 5-day storage period. the sterility was tested using bd bactec culture vials. the evaluation of the residual leukocytes was performed with the bd leucocount kit. the absolute plt counts were determined using hemocytometer sysmex xn2000.results: prs bags from 24 donors were processed to produce is-plt units. median resuspension volume in intersol was 220 ml (range 95-360). the absolute mean value of plt counts measured at the end of the storage period was 2.1 x10 11 (range 1.0-6.1 x10 11 ). this value was found below the threshold fixed by italian regulation in 8 cases (33.3%). mean value of residual leukocytes was 1.3x10 5 (range 0-9x10 5 ); the ph value was always > 6.4. sterility was observed in all cases. according to the work of slichter et al. conclusions: we demonstrated that plts recovered from leukapheresis bags can be accepted as a conventional hemocomponent according to the parameters fixed by italian transfusion law and thus can be administered to the haplo-hsct recipients early after transplantation. this strategy carries several advantages. indeed, apart from the obvious advantages in terms of reduced costs, is-plt can be used to desensitize the recipient by absorption of anti-hla class i antibodies, if present in the recipient. moreover, this strategy can avoid the risk of sensitizing the transplanted patients with hla alleles that differ from the donor's ones. finally, the is-plt unit is readily available. a clinical study aimed at testing the use of is-plt units in transplant recipients will be performed to confirm the clinical efficacy of the approach.disclosure: nothing to declare p700 ultrasound guidance as a powerful tool in increasing background: peripheral blood stem cells are generally the preferred graft source for allogeneic stem cell transplantation for malignant disease. in most centers first apheresis is performed on day 5 after 8 to 9 doses of granulocyte colony stimulating factor (gcsf) up to 5ug/kg twice daily. the dose of gcsf and the number of apheresis procedures required contribute to symptom, travel and time burden donors are put through during the process. we hypothesized that taking donor-recipient weight differences into consideration may help reduce this burden methods: a total of 261 healthy donors who donated peripheral blood stem cells on day 4 of gcsf mobilization in the period between january 2015 and august 2018 at the university medical center hamburg-eppendorf were included in this quality control evaluation. the donors were divided into two cohorts. the impact of donorrecipient weight ratio on stem cell harvest was tested in the training cohort (2015-2016) and validated in the second cohort (2017) (2018) . for the training cohort, donors were grouped according to donor-recipient weight ratio < 1.0 vs. 1.0-1.2 vs. > 1.2. for the purpose of this analysis a stem cells dose of 5 x 10 6 cd34+/kg recipient weight was set for successful apheresis.results: in the training cohort including 142 donors, 52 (37%), 36 (25%) and 54 (38%) had a donor-recipient weight ratio of < 1.0, 1.0 -1.2 and > 1.2 respectively. the target stem cells count of 5 x 10 6 cd34+/kg recipient weight was achieved in 36 of 52 (69%), 26 of 36 (72%) and 45 of 54 (83%) donors with donor:recipient weight ratio < 1.0, 1.0 -1.2 and > 1.2 respectively. the cut-off for the validation cohort was therefore set at a weight ratio of 1.2.in der validation cohort including 119 donors, 75 (48%) had a weight ratio > 1.2 while 62 (52%) had a weight ratio ≤ 1.2. overall in this cohort target cell count of 5 x 10 6 cd34 +/kg recipient weight was reached in 92 (77%) cases. this target was reached in 43 of 62 (69%) of donors with weight ratio ≤ 1.2 and in 49 of 57 (86%) donors with weight ratio > 1.2, p = 0.03.conclusions: a donor-recipient weight ratio of > 1.2 is seen in about 40% of peripheral blood stem cell donations for allogeneic stem cell transplantation. in these cases apheresis on day 4 after 7 doses of gcsf is reasonable. donors with lower weight ratios should preferentially donate on day 5 after 8 to 9 doses of gcsf.disclosure: all authors declare no conflicts of interest background: the effect of a second mobilization and collection of peripheral blood stem cells (pbsc) on the cell yield is low, as we previously demonstrated. however, donor safety has been poorly addressed with no changes in the clinical practices.methods: second donations of unrelated and related donors performed between 2001 and 2017 were evaluated (n=24), including pbsc+pbsc (n=18), bone marrow (bm)+pbsc (n=5) and pbsc+bm (n=1). analytical parameters including leukocyte, lymphocyte, hemoglobin and ldh quantification, obtained on the pre-harvest evaluation of first and second donation, were retrospectively analyzed and compared for all donors. the portuguese bone marrow donors registry (cedace) recommends a time between donations no lesser than 6 months. it also states that in very urgent situations like graft failure, donor should be clinical and analytical cleared and its safety ensured.in order to evaluate the impact of time between donations, donor population was divided in 3 groups: < 2 months, 2-6 months, >6 months; to determine the influence of donor age, donors were divided in 2 groups: < 40 and ≥40 years.results: among the total of 24 donors, 13 were volunteer donors of cedace and 11 were familiar. fifteen second donations were performed because of recipient graft failure and 9 due to disease progression or relapse. at the time of second collection, median donor age was 37 years (range 23-57). the median delay between both collections was 262 days (37-1519). time between donations did not seem to substantially impact the analytical donor evaluation: leukocytes, lymphocytes, hemoglobin and ldh results are kept within the reference values. however, donors with less than 2 months between donations showed a slight decrease on leukocyte counts (35%) and hemoglobin values (12%), from the first to the second pre-harvest evaluation. donor age showed no significant influence on the analytical evaluation. nevertheless, when considering only the pbsc+pbsc donations, donors with ≥40 years showed a small decrease on lymphocyte counts (19%) .conclusions: this study demonstrated that the analytical parameters, chosen based on literature, had no significant changes between first and second donation. however, particular attention should be paid when time between donations is lesser than 2 months or donor age is ≥40 years.as we concluded that no significant changes were observed in the group of 2-6 months, it is our opinion that the minimum of 6-12 months established by the registries can be shortened to 2 months ensuring donor safety. an accurate donor risk assessment with a larger population should be accomplished in order to strengthen this recommendation.disclosure: nothing to declare. gaurav kharya 1 , atish bakane 1 , pratibha dhiman 1 , anil khetrapal 1 , vikrant bhar 1 background: t cell replete haploidentical stem cell transplant (hhsct) is complicated mainly by increased risk of graft failure (gf) and graft versus host disease (gvhd). conventionally gcsf has been used to mobilize hematopoietic stem cells (hsc). in tcr hhsct gcsf mobilized graft with megadose of cd34+ cells expose the patient to higher doses of alloreactive t cells increasing the risk of gvhd. plerixafor based mobilization gives an advantage of giving high cd34 cell dose limiting exposure to high alloreactive t cell dose. we share our experience of gcsf + plerixafor based mobilization for tcr hhsct. methods: 9 consecutive patients suffering from scd who underwent hhsct between jan 2018 till date along with the respective donors were enrolled in the study (group 1). all 9 underwent pre-transplant immune suppression (ptis) 2 cycles at 3 weekly intervals using fludarabine+cyclophosphamide+dexamethasone.the graft was mobilized using gcsf@10mcg/kg/day(d1-d5)+plerixafor@0.24mg/kg(d5) 6-8 hours before the pbsch. gvhd prophylaxis included ptcy 50 mg/kg/ day on d3 and 4, sirolimus and mmf starting from d5. group 2 included 5 historical controls where graft was mobilized using gcsf@10mcg/kg/day(d1-d5). various parameters pertaining to mobilization, harvest, engraftment, gf and gvhd were assessed between the two groups.background: poor mobilizers (pm) defined as those with a peripheral blood cd34 + count ≤10cells/μl on day+4 is a significant risk factor for mobilization failure. within these, patients with < 5cells/μl are considered as very poor mobilizers (vpm). use of plerixafor in vpm patient is controversial. the aim of our study is to compare mobilizing and engraftment between pm and vpm who received plerixafor plus g-csf (p+g-csf).methods: in our center, mobilization with g-csf at dose of 10μg/kg/day was used in all pts. apheresis were scheduled on day+5. plerixafor (0.24 mg/kg) was added if the number of cd34 + cells on day +4 was < 10/ul for 2x10 6 cd34 + /kg requested (or < 20/ul for 4x10 6 cd34 + /kg), or if the number of cd34 + cells collected in the first apheresis was < 50% of cd34 + cells requested.between january 2016 and september 2018, 37 out of 157pts (23,6%) received plerixafor for mobilization. we retrospectively studied 30 pts who mobilized with p+g-csfdue to the number of cd34 + cells on day +4 was < 10/ul.results: twelve out of 30 pts were pm, 7 were females, median age 55,5 years (range:32-70). patients' baseline diseases were: 10 non-hodgkin lymphoma (nhl) (83,3%), 1 multiple myeloma (mm) and 1 hodgkin lymphoma. median cd34 + cell count on day +4 was 8/ ul (range:6-10).there was no mobilization failure. eighteen out of 30 pts (60%) were vpm, 9 were females, median age 62,5 years (range:34-69). patients' baseline diseases were: 10 nhl (55,5%), 7 mm and 1 solid tumor. median cd34 + cell count on day+4 was 2,5/ul (range:2-5). two out of 18 pts (16,6%) were considered mobilization failure, in 2 of them did not realized apheresis due to cd34 + cell count on day +5 was 2/ul. no difference was seen between both groups regarding gender, age, patients baseline disease or median cd34 + cells count on day +4.vpm needed more apheresis sessions, 5/16 pts required 2 sessions against 1/12 pts in pm (p=not significant (ns). we obtained enough cells to carry asct in 90% pts, although mean number of cd34 + cells obtained in vpm was lower than in pm (4,89x10 6 /kg vs 6,38x10 6 /kg, respectively) (p=ns).twenty-six pts underwent asct and mean number of cd34 + cells infused were 4,27x10 6 /kg in vpm vs have been the only route used for chpc administration. the appearance of other catheters types made us to reconsider the exclusive use of the cvc for the infusion of chpc. we analyzed the use of a peripheral iv cannula (pivc) as an alternative to cvc for the infusion of chpc in patients with cardiovascular diseases.methods: medical records of 65 patients who received an asct for hematological malignant diseases at the hospital clínic of barcelona from january 2017 to february 2018 were reviewed. of those, eight were infused through a pivc due to cardiac impairment related to previous treatments, ischemic cardiomyopathy or amyloid deposition.hpc were obtained from peripheral blood by apheresis after mobilization with g-csf using acd-a as an anticoagulant. cryopreservation was performed with autologous plasma and dmso 10% by mechanical means and stored in liquid nitrogen. analytical controls were performed including hematocrit, total nucleated cells, total polymorphonuclear neutrophils and platelets using the advia 120 analyzer. the cd34 + / cd45 + population was analyzed by flow cytometry following the ishage single-platform protocol. viability of total nucleated cellularity was carried out by vital staining with acridine orange and the specific viability of the cd34 + population through the technique of 7-aminoactinomycin d. thawing was performed bag to bag by immersion in a water bath at 37ºc and transferred to the bedside of the patient for gravity infusion using an infusion set without filter through pivc of 22 gauche. vital sings monitoring performed before, during and the end of the every infusion bag including: blood pressure, heart rate, oxygen saturation, body temperature and central venous pressure. other aspects assessed during the infusion were pain, cold sensation and signs of extravasation in the area of pivc insertion. after the infusion, the recovery time of the granulocyte series and platelet were evaluated.results: median volume and bags administered was 480 (360-600) ml and 4 (3) (4) (5) . the median of total nucleated cells, total nucleated cells / ml and total cd34+ cells/kg was 462.6 x 10 8 (329.5-657.8), 107.3 x 10 8 (64.66-156.7) and 3.24 (2.46-4.6) respectively. vital signs were within the normal range and allowed to perform the infusion in an average of 20-30 minutes/bag. no patient required stopping the infusion due to pain in the area of peripheral catheter insertion and no extravasations were detected. all patients referred some cold sensation in the insertion vein and its path. median hematopoietic recovery was 14 (11-19) days for neutrophils and 11 (0-19) days for platelets, similar to the recovery experienced from patients who received chpc through cvc.conclusions: based on our data, we conclude that the administration of chpc, through pivc and by gravity is safe for the product and for the patient, being the preferred choice for patients suffering from some type of cardiovascular disease.disclosure: nothing to declare background: by selective depletion of potentially alloreactive cd45ra + cells, t memory cells might be retained in the graft and could mediate pathogen specific immunity. however, cd45ra expression is not restricted to naïve t cells, but also available on b cells, nk cells and cd34 + stem cells to some extent. methods: within this project we aim to analyze cd45ra expression on stem-and nk cells by flow cytometric analysis to estimate the eventual loss of these cells during cd45ra-depletion. furthermore, clinimacs depletion following a one-step approach of direct cd45radepletion and a two-step approach with primary cd34selection followed by cd45ra-depletion of the negative fraction was investigated.results: cd45ra expression on cd34 + stem cells was in median 16.9%. with a median of 99.4% cd45ra expression was measurable on nearly all b cells, which obviates depletion via cd19. a comparably high cd45ra expression of in median 96.0% was detected on nk cells. unfortunately, the amount of nk cells in the cd45ra-depleted product was 0.24%. clinimacs depletion following one-step approach resulted in a stem cell recovery of 52.0%. memory t cell recovery was 24.2% following one-step and 42.0% applying two-step approach. depletion quality measured by log-depletion was 3.9 and 3.8 for cd45ra + t cells and 3.2 and 3.6 for cd19 + b cells for one-and two-step approaches, respectively.conclusions: with regard to stem cell recovery, a previous cd34-selection before cd45ra-depletion is recommendable.background: current clinical practice of routine use of filters for infusion of autologous hematopoietic cell transplantation (ahct) at bone marrow transplant centers across north america and europe is not known. the use of "y" administration tubing without a filter could possibly increase the risk of infusion of macro-aggregates and cellular debris, which may result in increased side effects.methods: we carried out a retrospective chart review of patients (pts) at spectrum health who underwent ahct. group a (gp a) pts received ahct using a "y" administration tubing with 170-micron filter from 3/2013 -3/2017. these patients were compared to a control group (gp b) that received ahct without filter administration tubing from 3/2014-4/2016.this change in clinical practice occurred due to a change in policy at our transplant center as a result of inorganic particles noticed during cryopreservation. we compared the neutrophil and platelet engraftment duration between these groups. we also studied the length of hospital stay and the effect of filter use on any immediate side effects after infusion. due to the retrospective nature of the study it was not feasible to evaluate the difference in duration of infusion between these groups results: the two groups were similar in their age, gender, primary disease distribution and median number of cd34 stem cells infused (table) . there was no difference in median neutrophil (11 vs 11 days) or platelet engraftment (19 vs 19 days) duration for the filter group and the nonfilter group respectively. the median length of hospital stay was also comparable (17 days). there was no statistically significant difference in the immediate side effects (fever, cough, dyspnea, fluid overload, flushing, nausea, vomiting, hypertension, hypotension and anaphylaxis) or confirmed post-transplant infections (viral, bacterial, fungal) experienced by these two groups.conclusions: our results show that the routine use of filter does not prolong hospital stay, and neutrophil/ platelet engraftment duration, thereby, suggesting that viable stem cells are not affected. on the other hand, filter use failed to demonstrate any appreciable decline in the immediate side effects experienced after ahct. gp background: allo-hsct from related haplo-identical donors (haplo-hsct) with post-transplant high-dose cyclophosphamide is increasingly employed in patients who lack a matched related or unrelated donor. the current standard is to use bone marrow grafts (bm) as peripheral blood stem cell grafts (pbsc) have been associated with an increased risk of acute and chronic gvhd. thus, the aim of our study was to compare the main transplant outcomes and especially the incidence of acute and chronic gvhd in recipients of bm and pbsc grafts. methods: thirty-five unselected patients with hematologic malignancy who underwent an haploidentical transplant at our unit between 2011 and 2018 and received bm (n = 17) or pbsc (n = 18) grafts after the same tbf conditioning regimen were analysed in order to assess differences in transplant outcomes.our gvhd prophylaxis consisted in cyclosporine a (csa) from day -1 to +180, a methotrexate "short course" and mycophenolate mofetil (mmf) from day +1 to +28.results: no statistically-significant differences were observed between patients who received bm grafts and those who received pbsc grafts. at transplant fourteen patients were in first complete remission (cr), twelve in advanced cr and 9 had active disease. according to sorror's risk, nine patients were low-risk, nine intermediate-risk and seventeen high-risk. twenty-eight cmv+ patients received the graft from twenty-three cmv+ and five cmv-donors, seven cmv-patients received the graft from five cmv+ and two cmvdonors. mean age at transplant was 51 years (range 23-72), mean donor's age 38 years (range18-39) and mean follow-up 16.9 months (range 1.9-56.7). median cd 34+ cell dose was 5.2x10 6 /kg (range1.4-10.4), 3.6 x10 6 /kg (range 1.4-7.7) in bm recipients and 14.0 x10 6 /kg (range 4.2-10.4) in pbsc recipients. median time to neutrophil recovery (>500/μl) was 22 days (range 16-39) posttransplant, 23 days (range 18-27) for bm recipients and 20 (range 16-39) for pbsc recipients. platelet recovery (>20.000/μl) occurred in all patients except one at a median of 17 days (range 10-151) post-transplant, at a median of 20 days (11-151) post-transplant for bm recipients and at a median of 14 days (range (10-26) for pbsc recipients. seven patients never reached platelets >50.000/μl. three patients developed a poor graft function. acute and chronic gvhd incidence was 28.5% and 22.8% and the risks of acute (hazard ratio [hr], 1.04; p = .955) and chronic (hr, 0.85; p = .816) graft-versus-host disease were similar in the two patient groups. in addition, there were no differences in relapse risks post-transplant (hr, 0.90; p = .898); relapse-free survival was better with pbsc grafts but this difference did not reach any statistical significance. finally, no significant differences were noted in overall mortality by graft type (hr, 0.62; p = .441).conclusions: despite in haplo-hsct the incidence of acute and chronic gvhd is reported to be higher with pbsc than with bm our small patient series does not confirm this assumption that should be clarified by additional studies. instead, our data suggest that pbsc background: since 1988, cord blood (cbu) has become an alternative source of stem cells for transplantation, with approximately 35,000 procedures currently performed. with a 2-year gs of 50%.objective: analyze the outcomes from all the patients transplanted with cbu in our hospital unit.methods: retrospective, longitudinal study. all patients transplanted with cbu in our hospital between 2007 and 2017 were included. we analyzed 40 patients with ages from 7 months to 14 years.results: two of them received doubled cord transplantation the ratio male: female was 1.3: 1. the transplanted pathologies were: bone marrow failure 35%, immunodeficiency 25%, aml 20%, all 17.5%, osteopetrosis 2.5%. ric regimens were used in patients with bone marrow failure and immunodeficiency and myeloablative conditioning regimens were used in patients with malignant hematology diseases. antithymocyte rabbit globulin (atg) based serotherapy was used. one case received cbu from a related donor (sister), the rest received unrelated cbu obtained from centro nacional de la trasfusión sanguínea. the infusion of cd34 + was in a range of 0.11 to of 2.7 x10 6 /kg with and average of 0.43 x 10 6 / kg. compatibility was 4/6 in 70%, 5/6 in 20% and 6/6 in 10%. post-thawing cellularity was not measured. the hla-c was not analyzed. forty two point five percent of the patients had a successful engraftment; the average time of engraftment was 30 days. primary graft failure was detected in 57.5% and secondary graft failure in 10%, for a total success of 32.5%. gvhd was detected in 20% of patients, of which 90% was grade i-ii and 10% grade iii-iv. the overall mortality was 52.5%. causes of death were: infection 45% relapse 30%, hemorrhage 20% and gvhd 5%. the cbsct continues to be an essential alternative in our patients who required transplantation knowing that this stem cell source allows the procedure to be done with less histocompatibility requirements and it is available immediately, which facilitates the process considering the great diversity that exists within our population. however, in our experience, the cbsct has shown a higher mortality risk, which can be improved by analyzing the hla c, choosing in this way the units with better compatibility, and improving cellular dosage since this is key in success.disclosure: nothing to declare the risk of infection of the umbilical cord is not related to the microbiological status of the umbilical cord blood methods: the statistical analysis was carried out on data obtained from samples taken in poland between 01-jan-2017 and 31-dec-2017. the samples were collected in hospitals by external midwifes and sent to the pbkm in accordance with the requirements of the american association of blood banks. after arrival in the laboratory, the blood samples were cultured and the ucs were assessed immediately for visual signs of infection, such as odor, altered color, or visible bloom. the status of both kinds of samples was introduced into the pbkm general database. for the purpose of this analysis, the ucs were considered as microbiologically pure if stored, destroyed after storage, or handed over to the pbkm. samples marked as infected or disqualified for unknown reasons (other than termination of the contract with the customer, viral infection of the mother, and lack of cell growth) were considered as infected. at the time of the statistical analysis, the samples of unknown ucb microbiological culture status were removed from the generated report. the data was summarized as percentages and the odds ratio was calculated. statistical significance was considered at p˂0.05.background: match family donors are the preferable options in allogenic stem cell transplant. however, in the absence of donor relatives match unrelated donors have been an option. in this study, the donor screening, transplant preparation phases of turkish stem cell coordination center (turkok) and the i̇stanbul university bone marrow bank (tris), were compared.methods: the unrelated donor scanning data between march 2015 and november 2018 in pediatric stem cell transplantation unit of altınbas university bahcelievler medical park hospital were evaluated. 93 unrelated transplants were performed in total. 65 % of these transplants (n=61) were included from the donors of turkok registration system and 34,4 % of these transplants (n=32) were included by means of tris from donors outside of turkey. 8 patients (5 tris, 3 turkok) were excluded from the study in consequence of screening update and postpone of transplantation. the statistics were carried out on a total of 85 patients, 67,1 % of whom were in turkok (n=57) and 32,9 % were transplanted via tris (n=28). the day of application to stem cell transplantation unit, reply dates and the transplantation dates were examined for the transplant patients.results: in the current study, the average response time of turkok was found as 0,91±2,96 day (median: 0), the average transplant time after receiving a reply was found as 98,24±63,89 (median: 85) day, the average number of days from date of application to date of transplantation of patients was found as 101,12±63,17 (median: 89). the average response time of tris was 18,78±18,36 (median: 15) day, the average transplant time after receiving a reply from tris was 136,82±63,84 (median: 119) day, average number of days from date of application of tris to date of transplantation of patients 155,61±72,23 (median: 140) day.the average response time of turkok, the average transplant time after receiving a reply from turkok and the average number of days from date of application of turkok to date of transplantation of patients was shorter than tris. the difference between them was found statistically significant (p< 0.05).conclusions: in this study, it was determined that the transplantation processes with turkok were progressing more rapidly. the rapid progress of the process was attributed to the fact that all donor hla tissue groups in the turkok database were studied in high resolution. in international scans carried out through tris, it was thought that the examination of the donor castings coming from bone marrow banks and the time differences between the countries prolong the process. it was thought that hla tests of the registered donors in the tris database and some international bone marrow banks were studied in low resolution but not studied the all hla loci, the centers wanted high-resolution hla, and therefore the involvement of social security institutions and payment procedures were among the factors extending this process.disclosure: nothing to declare background: allogeneic hematopoietic stem cell transplantation (ahsct) is being performed for a group of hematologic diseases with a curative intent. outcomes after ahsct are influenced by the type of donor used. haploidentical transplantation is an emerging option when a fullmatched donor is unavailable. methods: we retrospectively analyzed our transplants performed between january 2015 and november 2018, investigating outcomes and complications among haploidentical stem cell recipients.results: one hundred and nineteen patients underwent ahsct, 9 of them (7.5%) were recipient of a haploidentical stem cell and included in this study. one patient diagnosed with acute lymphoblastic leukemia (all) were performed a haploidentical ahsct for two times due to relapse. among those 9 transplants, 4 of them were diagnosed with acute myeloid leukemia, 2 with all, 1 with chronic lymphocytic leukemia, 1 with myelodysplastic syndrome and 1 with hodgkin lymphoma. the mean age of group was 35.7±15.2 years. three patients (2 aml, 1 all) were in remission at the time of transplantation. patients were given a conditioning regimen based mostly on busulfan, fludarabin and total body irradiation with a myeloablative intent. patients were also given a various combinations of post-transplant cyclophosphamide, calcineurin inhibitors, mycophenolate mofetil and antithymocyte globulin for graft versus host disease (gvhd) prophylaxis; post-transplant cyclophosphamide administered on 6 (67%) of those transplantations. peripheral blood was the source of stem cells in all patients. patients were infused with mean 6.15±0.67x10 6 /kg of cd34+ cells. hematological recovery was achieved with neutrophil engraftment at a mean of 19.2±2.1 days and platelet engraftment at a mean of 17.3±2.7 days. after a median 8month (0.2-40.6 months) follow up, the cumulative rates of grade 3-4 gvhd, relapse and non-relapse mortality were 11%, 28% (n=7) and 71%, respectively. one patient died due to relapse, at the end of the follow up two were still alive with remission. only one patient has died due to chronic gvhd affecting serosa and resulting with a fatal tracheoesophageal fistula. the mean overall survival was 6.9±2.6 months in our study.conclusions: haploidentical transplant is a feasible option in hematologic malignancies with novel gvhd prophylaxis approaches, especially post-transplantation cyclophosphamide. however, these results need to be supported with further investigations with a larger patient group.disclosure: nothing to declare results: a total of 25 patients between 19 and 58 years (median age: 33 years). transplant was done for following disease: acute leukemia (n=9, 36%), aplastic anemia (n=5, 20%), lymphoma (n=4, 16%), myelofibrosis (n=3, 12%), myelodisplastic syndrome (n=2, 8%), chronic myeloid leukemia (n=2, 8%). ten donors were from turkey and fifteen donors were from different countries of europe and america. two of 25 donors were 9/10 and the other 23 was 10/10 hla matched. the conditioning regimen was mostly non myeloablative (n=17, 68%) while eight patients were treated with myeloablative regimen. other than two patients who took tacrolimus and mycophenolate mofetile all of them got cyclophosphamide and methotrexate for graft versus host disease (gvhd) prophylaxis. the median time of neutrophil and platelets engrafman were 18 days (range 11-31) and 13,5 days (range 9-36) respectively. acute gvhd was seen nearly half of the patients (47,8%).overall survival was 40% for all patients and 15 of 25 patiens (60%) died within first month to 18 months (median 3 months). the mortality rate was more higher for the recipients who had donor source from countries other than turkey (30% vs 80% p=0,018). transplant related mortality was the most common reason of mortality (n=7/ 15, 46,7%) and other reasons were gvhd (33,3%), infections and cirrhosis respectively.conclusions: we found the mortality rate more higher in the patients whose donors were from out of our country. however, we need to further multicentric and prospective investigations to confirm our hypothesis, it would be related with impact of ethnicity.disclosure: nothing to disclose late-breaking abstracts p725 targeted twice daily busulfan-based ric-conditioning for allogeneic hematopoietic stem cell transplantation in pediatric patients with chronic granulomatous disease: a 10-year experience with the zurich protocol matthias felber 1 , mathias hauri-hohl 1 , ulrike zeilhofer 1 , federica achini 1 , jana pachlopnik-schmid 2 , janine reichenbach 2 , seraina prader 2 , tayfun güngör 1background: chronic granulomatous disease (cgd) needs sufficient myeloablation to avoid graft failure. for this purpose the ebmt inborn errors working party currently recommends treosulfan or busulfan-based conditioning regimens for cgd-sct. we analyzed the last 10 years of targeted busulfan-based ric-conditioning including engraftment, gvhd rates, chimerism and late term effects in our pediatric sct center in zurich. methods: between 2008 and 2018, n=34 consecutive pediatric cgd patients (median age 6 years, range 1-16 years, n=4 female, n=9 autosomal recessive inheritance) have been transplanted. all patients received therapeutic drug monitoring of twice daily administered iv busulfan (3 or 4 hour infusions; d-5 to -d2) to achieve a targeted cumulative auc of 45-70 mg/l*h. fludarabine (180 mg/ sqm; d-8 to -d3) and serotherapy (thymoglobuline 7.5 mg/ kg total, d-5 to d-3) or alemtuzumab (0.5-0.6 mg/kg total; d-8-to d-6) were used for immunoablation. donors were matched unrelated (10/10 hla; n= 19), mismatched unrelated (9/10 hla; n= 7), mismatched unrelated (hla 8/10; n=1), matched sibling (hla 6/6; n= 5) and haploidentical parental (hla 5/10; n=2). for 2 patients with haploidentical donor post-transplant cyclophosphamide (d-3 and d-4 with 50 mg/kg iv each) and upfront atg-grafalon (40 mg/kg -12 to-d-9) was used. stem cell sources were bm (n=29) and pbsc (n=5). gvhdprophylaxis included iv csa (d-3; continuous infusion) and iv. mmf (d-0, in 2-3 doses).results: follow-up was 6 to 118 months. good overall engraftment was noted, with n=1 secondary graft failure followed by successful retransplantation. in one patient a stem cell boost/dli was necessary due to decreasing myeloid donor chimerism during ebv reactivation, resulting in rapid myeloid donor reconstitution after intervention. low rates of gvhd were documented with n=2 agvhd grade iv and n=2mild/limited cgvhd (nih criteria). with exception of one patient, myeloid donor chimerism at last follow-up was over 82%, mostly over 95%. overall survival was 31/34 (91%). deaths were due to gi-gvhd (n=1), autoimmune hemolytic anemia/sepsis (n=1) and thrombotic microangiopathy (n=1).conclusions: precision dosing of iv busulfan in combination with fludarabine and serotherapy results in excellent outcome of hsct for pediatric cgd-patients with good engraftment, low overall cgvhd rates and stable, mostly excellent donor chimerism. graft failure rate was as low as 3%. low dose alemtuzumab prevented gvhd in the majority of patients. this analysis demonstrates that targeted busulfan-based conditioning is a valid option for pediatric cgd-patients. serum alemtuzumab or atg monitoring could further improve gf and gvhd rates in the future.disclosure: the authors declare no conflict of interest. abstract already published. young hla-matched unrelated donors are comparable to matched sibling donors in elderly patients receiving reduced-intensity conditioning: an analysis on behalf of the ebmt scientific council 95% c.i. 1.7-20.6, p= 0.001) and voriconazol prophylaxis during carv (or 4.2, 95% c.i. 1.1-115.6, p= 0.03). conclusions: we provide evidence that ifd after carv infection. allo-hsct recipients developing a carv lrtd during the first year after transplant may benefit from an adequate antifungal prophylaxis and a close monitoring for the development of a later ifd.disclosure: jose luis piñana has received both, advisory for preclinical/clinical research and financial support to assist to the spanish society of hematology annual meeting 2018 from msd. favorable outcome and engraftment following reducedintensity conditioned allo-hsct in children with primary haemophagocytic lymphohistiocytosis (hlh) and high-risk langerhans cell histiocytosis (lch) laura m. moser, emilia salzmann-manrique, andrea jarisch, jan sörensen, shahrzad bakhtiar, peter bader background: primary haemophagocytic lymphohistiocytosis (hlh) and high-risk langerhans cell histiocytosis (lch) represent two major entities of childhood histiocytoses, which are -although only of rare occurrence -severe in their clinical manifestations. patients present with multisystemic uncontrolled inflammation and multi-organ involvement requiring diverse courses of immunosuppressive and chemotherapy regimens. allogeneic haematopoietic stem cell transplantation (allohsct) is the only available curative option; however, the cumulative treatment toxicity and the underlying inflammatory disease often result in high organ toxicity and inflammatory complications of transplantation, such as graft versus host disease (gvhd) and/or graft failure. especially patients with unrelated donors often deal with high transplant-related mortality (trm) in the setting of conventional intensity conditioning.herein, we present the clinical course of the disease and transplant outcome of 14 children diagnosed with primary hlh (n=12) and high-risk lch (n=3) who underwent allohsct at our centre from 02/2003 to 08/2018.methods: the hlh cohort consisted of 7 cases of familial hlh (fhlh), 4 cases of griscelli syndrome, one xiap-deficient patient and one hlh-patient with inconclusive genetic testing. all hlh patients had developed clinical symptoms prior to transplantation and had been treated according to hlh-protocols 94, 2004 hlh-protocols 94, or 2015 median age at transplantation was 10 months (4 to 128 months). stem cells were derived from hla-matched siblings (msd, n= 4), matched unrelated donors (mud, n = 9) or haploidentical donors (n=1).the majority of patients (9/14) received a ric regimen containing fludarabine, melphalan and thiotepa (n=8) and fludarabine plus cyclophosphamide (n=1). myeloablative treatment (5/14) included a treosulfan-based regimen (n=3) and busulfan-containing treatment (n=2). the entire cohort received serotherapy using either muromonab (n=3), atg-fresenius® (n=9) or alemtuzumab (n=2).results: the overall survival of the entire cohort was 78.6% (11/14) on a median follow-up of 9.9 years ( figure 1 a+b) .all lch patients, being treated with fludarabine, melphalan and thiotepa, survived transplantation and showed complete remission (3/3) . within the hlh cohort the overall survival was 72.7% (8/11). fatalities (n=3) included two patients from the myeloablative group and one ric-treated patient. the cause of death were progressive disease activity during the conditioning phase, leading to multi-organ failure on day +15 despite immunosuppressive treatment (n=1) and complicated cerebral seizures followed by lung haemorrhage, possibly due to aspiration pneumonia with evidence of enterococcus faecium, resulting in septic multi-organ failure on day +4 (n=1). a third hlh patient developed a sudden cerebral edema and ensuing respiratory insufficiency on day +4. whether this was caused by acute neurotoxic damage by fludarabine or a consequence of relapsed hlh could not be conclusively specified. none of our patients suffered from transplant failure or nonengraftment. there was neither severe acute gvhd (iii-iv) nor chronic gvhd observed in this cohort.conclusions: primary hlh and high-risk lch are lifethreatening medical conditions needing rapid allohsct. ric regimens are well-tolerated and sufficient for proper engraftment and disease clearance. disclosure: the authors have no conflicts of interest to disclose. abstract withdrawn. thrombocytopenia following allo-sct concomitant to stem cell boosts. steroid refractoriness was defined as: progression after three days or no response after 5 days of steroid treatment. the median time from sct to the onset of agvhd was 22.5 d (range 9-122 d), and 11 d (range 3-40 d) from the onset of agvhd to the first msc infusion, respectively.the majority of our patients (n=5) suffered from a malignant disease and received a graft from a matched unrelated donor (n=6), while one patient had a haploidentical donor. gvhd prophylaxis was performed in all patients except the patient with the haplo-identical graft. all patients with agvhd were treated with steroids and the patient with thrombocytopenia required regularly transfusions and romiplostin therapy. the median msc dose was 1.7x10 6 cells/kg bw (minimum 1.1 x10 6 ; maximum 3 x10 6 ). three patients received 2 msc doses, two patients 4, one patient 5 and another 7 doses.results: at the time point of agvhd manifestation and msc application, two patients had cmv reactivation, one patient adenovirus infection and one patient ebvreactivation. by day 28, 5/6 agvhd patients responded to msc administration: 3 with complete response and 2 with partial response. at the last follow-up (median: 4.42 months, range 0,82-12.06 months), 4 of 6 patients were alive without acute or chronic gvhd. one patient died soon after msc treatment with no obvious response in the course of a systemic hyperinflammation syndrome. the other patient although complete responder to msc-ffm developed fatal adenovirus sepsis. this based on the profound tcell depletion induced by concomitant application of steroids. the overall survival probability at six month was 66.7%. no acute side effects occurred after msc infusions. the previously mentioned patient suffering from thrombocytopenia did not need any further transfusions after receiving 2 doses mscs combined with stem cell boosts while continuing romiplostin application.conclusions: our data confirm excellent tolerability and high efficacy of the licensed off-the-shelf msc preparation "msc-ffm" in pediatric steroid-refractory agvhd. in our center, current treatment algorithms have escalated "msc-ffm" to the second line, i.e. immediately after steroid refractoriness has been established. besides immunoregulatory properties, this product might facilitate hematopoietic stem cell engraftment.disclosure: novartis (consultancy: included expert testimony, speaker bureau, honoraria), medac (research funding, patents and royalties), riemser (research funding), neovii (research funding), amgen (honoraria) expression of ccr4 modulates migrational properties of in vitro expanded murine regulatory t cells laura m. moser 1,2 , ulrike tischler 1 , christin riegel 1 , julia minderjahn 1 , rüdiger eder 1 , jaqueline dirmeier 1 , isabel zimmermann 1 , evelyn röseler 1 , petra hoffmann 1,3 , matthias edinger 1, 3 background: hematopoietic stem cell transplantation (hsct) as it is carried out successfully at other genetic instability syndromes seems to be an encouraging opportunity for a curative therapy to restore immunity and prevent the development of hematologic malignancies in ataxiatelangiectasia (a-t). however, experience in the conditioning regimen is limited and no transplantation strategy for a-t patients exists, especially in an allogeneic setting. conditioning regimen and donor selection are critical factors in the clinical setting of hsct and incur substantial risks, especially in a-t. the aim of this study was (1) to evaluate whether different approaches of hsct including allogeneic hematopoietic hsct are feasible in regard to graft versus host response (gvhd) and sufficient concerning immune reconstitution (2) and to de-escalate the toxic effects of the conditioning regimen by reducing the dose of cyclophosphamide (cp).methods: t cells from syngeneic, allogeneic and haploidentical donor mice were used to determine gvhd induced t cell proliferation in a mixed lymphocyte reaction (mlr). atm-deficient mice were treated with cp or reduced cp in combination with fludarabine (flu) and transplanted with 5x10 6 cd90.2 depleted bone marrow donor cells from 129/svev gfp-transfected wildtype mice (syngeneic) or from mice of the f1 generation of 129/svev wildtype mice and c57bl/6 mice (haploidentical), or from c57bl/6 mice (allogeneic). tracking of gfp-positive donor derived cells was performed using flow cytometry and atm pcr. oxidative stress and damage were detected by a rt profiler pcr array and 8-hydroxy-2′deoxyguanosine.results: mlr resulted in an increased proliferation of allogeneic donor t cells compared to syngeneic and haploidentical donor cells. response was lower on dendritic cells isolated from atm-deficient mice compared to wildtype controls. in vivo results showed the restoration of t cells in atm-deficient mice accompanied by a prolonged life span and through reduction of thymic tumors. however, allogeneic stem cell transplantation was accompanied with a higher mortality rate, compared to the haploidentical and syngeneic setting. decreased antioxidative capacity and a higher dna-damage were seen in cp treated atmdeficient mice.conclusions: haploidentical hsct seems to be a feasible strategy for a-t. our data provided further evidence for the high sensitivity against ros-inducing agents in a-t and this fact needs to be taken into consideration in the choice of the host-conditioning strategy.disclosure: nothing to declare this research received funding from action for a-t charity (14gou01) background: prognosis of pediatric patients and young adults suffering from refractory or high-risk soft tissue sarcomas remains poor with limited improvement over the last decades despite multimodal treatment strategies. replacing the immune system by an allogeneic hematopoietic stem cell transplantation (hsct) in refractory solid malignancies has been proposed as a potentially curative therapy due to its presumable graft versus tumor effect. based on this concept we additionally performed consecutive donor-derived lymphocyte infusions in allogeneic hsct-patients with refractory or relapsed solid malignancy to further increase anti-tumor efficacy post-transplant.methods: pediatric patients with relapsed and/or refractory cancers or with delayed responses to the respective induction therapies were offered donorderived cellular therapies after immune system replacement by an allogeneic hsct. cellular immunotherapies comprised of donor lymphocyte infusions (dli), natural killer (nk) cell or cytokine-induced killer (cik) cell infusions generated from the original stem cell donors. allogeneic nk cells were generated from unstimulated leukapheresis by a two-step purification procedure using immunomagnetic cd3 t cell depletion, followed by nk cell enrichment (cd56+) with or without in vitro il-2 stimulation and expansion for 9-14 days. for cik cell generation peripheral blood mononuclear cells were isolated and activated by in vitro cytokine stimulation (inf-γ, anti-cd3, il-2 and il-15) an expanded over 10-12 days. expanded cik cells represented a heterogeneous population of polyclonal t cells with in part shared phenotypic and functional properties of nk cells.results: between october 1 st 2003 and january 1 st 2014 a total of 18 patients (eight patients with rhabdomyosarcoma, one patient with synovial sarcoma, two patients with ewing sarcoma, five patients with neuroblastoma, one patient with hepatoblastoma, and one patient with nasopharynx carcinoma) were enrolled. seven of 18 (39%) patients in this study had achieved complete remission (cr) before hsct while another 7 of 18 (39%) patients had obtained at least very good partial or partial response (vgpr or pr). dli was applied in 5 patients, nk cell treatment was offered to another 6 patients, while cik cell therapy was given to 7 patients.1.5-year probabilities of overall survival (os) and progression-free survival (pfs) were 16.2% and 15.9% for all patients with a median follow up of 8.5 months (range, 1.5-115.1 months). patients in cr at the time of immune cell therapy (it) showed estimated 1.5-year os and pfs of 49.6% and 42.7%, respectively. the majority of patients relapsed and ultimately succumbed to their diseases with two of 18 (11%) patients still being alive 9.6 and 9.3 years after it. cumulative incidence of relapse was 79.8% at 1.5 years. t cell engraftment and immune reconstitution (ir) was improved by it, and correlated with treatment response. however, two of 18 heavily pretreated patients (11%) died due to cumulative treatment-related mortality (trm). furthermore, acute graft-versus-host-disease (agvhd) occurred in 9 of 18 patients (50%) with agvhd grade i-ii observed in 6 (33%) and agvhd grade iii seen in three (17%) patients.conclusions: altogether, the results of this study indicate that allogeneic donor-derived cellular therapy at its current state offers curative benefit in selected refractory childhood cancers but warrants further improvement. background: allogeneic stem cell transplantation (allo-sct) is the only curative treatment option for a variety of nonmalignant diseases. the success of allo-sct is strongly associated with rapid and sustained immune reconstitution (ir). we analyzed the ir in patients who received an allo-sct for nonmalignant diseases.ir was assessed on days +30, +60, +90, +180 and +365 after allo-sct analyzing leukocytes, lymphocytes, monocytes, cd3 + t cells, cd3 + cd4 + t helper cells, cd3 + cd8 + cytotoxic t cells, cd3 -cd56 + natural killer (nk) cells and cd19 + b cells.methods: we analyzed ir-data of 116 consecutive patients receiving allo-scts between september 2001 and november 2018. indications of allo-sct were hereditary anemias (thalassemia, sickle cell disease, diamond blackfan anemia; ha, n=35, 30%), inherited bone marrow failure syndrome (fanconi anemia, severe aplastic anemia, others; bmfs, n=34, 29%), hemophagocytic lymphohistiocytosis (hlh, n=12, 10%), immunodeficiency (id, n=28, 24%) and metabolic disorders (n=7, 6%). the median age at allo-sct was 7 years (range, 0.2 -26) and at diagnosis 0.8 years (range, -0.5 -17.8).patients received 1 st allo-sct from msd/mfd (n=53, 46%), mud (n=42, 36%), haploidentical mismatch family donors (n=15, 13%) and mmud (n=6, 5%). conditioning regimens were busulphan-based (n=14, 12%), treosulphan-based (n=45, 39%), flu-mel-thio (n=35, 30%) or others (n=22, 19%). graft sources were bm (n=84, 72%) and pbsc (n=32, 28%).in order to consider the age-dependency of ir we normalized each absolute cell count with its corresponding age-specific expected mean values. (huenecke et al.; eur j haematol; 2008) results: the ir pattern was similar between the ha and bmfs groups. the cd4 + t cells were recovering slightly faster in ha patients compared to the recovery of bmfs patients.monocytes and nk cells proliferate very fast. at day +60 half of the patients already reached their respective monocytes reference value except for id patients, who reached 80% of the reference value at the end of the first year.cd3 + cd8 + cytotoxic t cells recovered significantly faster in patients with hematologic diseases compared to patients with hlh (p< 0.001) and id (p=0.047). half of the patients reached the reference value of cytotoxic t cells in the hematologic diseases group at day +365. by far inferior was the ir for the hlh patients. in this group only 50% of the patients reached the 45 th percentile of the healthy age-matched reference. in the id group 50% of all patients reached the 87 th percentile of the age-matched reference group at day +365.b cells are profoundly decreased at day +30 in all groups. however, the longitudinal expansion of b cells was significantly lower both in the id group and hlh group compared to the hematologic diseases group. at day +365 fifty percent of patients with id, hlh and hematologic diseases reached the 37 th percentile, 52 th percentile and the 100 th percentile, respectively (p< 0.001; p=0.027).conclusions: allo-sct is the only curative option for patients with nonmalignant diseases. ir is dynamic and revealed a complex diversity pattern with regard to the original disease. to investigate factors influencing ir after allo-sct is crucial to improve outcome of these patients.disclosure: nothing to declare. allogeneic hematopoietic stem cell transplantation in patients with myelodysplastic syndrome of relatively high-risk groups: curative effect analysis and optimal timing selection results: among the 135 patients, 133 patients were transplanted successfully. the 3-year overall survival (os) rate and disease-free survival (dfs) rate was 76.8% ±4.2% and 76.7%±4.3% respectively. the 3-year cumulative relapse rate (rr) and the non-relapse mortality (nrm) rate was 13.9%±0.1% and 18.4%±0.1% respectively. the incidence of grade ii-iv acute graft versus host disease (agvhd) was 25.2%±0.1%. for the patients who survived more than 100 days after allo-hct, the 2 years cumulative incidence of chronic graft versus host disease (cgvhd) was 33.4%±0.2%. univariate analysis showed that the hematopoietic cell transplantation comorbidity index(hct-ci) and grade iii-iv agvhd are the high risk factors for os(81.2±4.5% vs 62.8±10%, p=0.027 and 81.9±4.4% vs 51.1±11.8%,p <0.001). multivariate analysis demonstrated that grade iii-iv agvhd and hct-ci are independent risk factors for os(hr=6.206, p <0.001, 95% ci:2.529~15.288 and hr=2.651,p=0.025, 95%ci:1.127~6.232). chemotherapy before transplantation did not improve os or dfs for patients with bone marrow blast cells more than 10% at the time of diagnosis.conclusions: allo-hsct is an effective treatment for mds patients of relatively high-risk groups. the physical condition of the patients and occurrence of agvhd are independent risk factors. for intermediate and high risk ipss-r mds patients, transplantation before the disease progressed into very high risk can achieve better prognosis, high-risk group can still benefit from rebound gvhd after cni tapering which was promptly responsive to treatment steroids, fk506 and ecp. aa one year after sct 12/13 patients were without gvhd and off all immunosuppression while one single patient was still on taper of immunosuppressant after rebound acute gvhd. no chronic gvhd occurred. sctrelated toxicity was common with mucositis in all patients (who grade 4: n=5), elevated liver enzymes (≥grade 3: n=9) and impaired renal function (gfr 40-60ml/min: n=8). five patients developed neurologic symptoms (seizures n=2, pres n=1, pseudotumor cerebri n=2) which all resolved without sequelae. overall survival and transfusion-free survival was 100% with a median observation time of 5 (2-8) years.conclusions: 4. treosulfan-based conditioning followed by sct from hla-matched related or unrelated donors represents a highly efficacious treatment approach for children, adolescents and young adults with tdt and exhibits an acceptable but not negligible safety profile. an individual risk-benefit assessment incorporating hazards such as secondary graft failure, gvhd and long-term toxicity including infertility and 2 nd malignancy has to be executed in the informed consent process for every patient and his/her guardians.disclosure: "nothing to declare" p740 abstract already published. early iron chelation with deferasirox might prevent relapse after busulfan plus fludarabine and atg as a myeloablative conditioning for hla-identical sibling allogeneic hct in aml results: we show an excellent concordance between chimerism assessment on bio-rad and 3 d platforms over the complete range of mixture ratios (r 2 >0,9) and proof the lower detection limit (0,1 %) compared to str-pcr.conclusions: our results promote the transfer of the established mentype assay to a more diverse instrument portfolio. that will allow to implement the analysis of patient and donor hematopoiesis by digital pcr methods in our lab.disclosure background: with the immense progress in therapeutic regimens in pediatric oncology and stem cell transplantation the survivor rates increased up to 80%. at the same time the field of reproductive medicine has achieved substantial advances to offer potential options for fertility preservation. therefore it is of great importance to implement fertility counseling and fertility preserving (fp) procedures for patients facing gonadotoxic therapy. in the report on the expert meeting of the paediatric diseases working party (pdwp) of the ebmt in 2017 counseling related to fp opportunities should be offered to each patient receiving stem cell transplantation (sct), as part of the pre-sct workup by a dedicated and trained team. yet there many medical, ethical, structural and financial issues to consider and overcome. we describe the setting up process to enable fertility counseling for all children with newly diagnosed cancer or those facing stem cell transplantation for malignant and nonmalignant diseases in our department of pediatric oncology and immunology/stem cell transplantation.methods: at our tertiary care center we assembled a multidisciplinary team involved in fertility preservation (pediatric hematology/oncology, pediatric immunology/ stem cell transplantation, reproductive medicine, andrology, psychology and pediatric surgery). we developed an internal grading system for recommendations regarding fertility preservation based on the current recommendations for fertility preservation of leading societies in this field like ebmt, gpoh and dggg. it is important to find a consensus within the team for the counseling to ensure reliable counseling. the third step is to implement structures needed for fertility counseling and performance of invasive procedures including legal aspects (amg). a detailed description of this process is given.results: after setting up structures for the counseling process we counseled 140 oncology and stem cell transplant patients (0-18 years) between january 2017 and may 2018. we analysed data of the patients including age subgroups and disease entities and the results of the counseling process. for those patients undergoing stem cell transplantation the risk of gonadotoxicity is very high, therefore even the very young children underwent fertility preserving procedures in alignment with our recommendations if they suffered from a nonmalignant disease. currently we discourage tissue preserving in malignant systemic disease due to possible contamination with malignant cells. postpubertal female patients were more likely to undergo invasive procedures such as ovarian tissue cryopreservation in the case of oncological diseases, while sperm cryopreservation was recommended in all postpubertal male patients. overall a high percentage of the patients and their family followed our recommendations.conclusions: fertility preservation should be considered as a very important part of the treatment plan for newly diagnosed children and young adults with cancer and those facing stem cell transplantation. unfortunately there is still a great need for setting up structures in institutions taking care of these patients. in addition fertility preservation sadly lacks funding by health insurance in some countries. with the presentation of our experience and data we want to facilitate incorporation of fertility counseling in other pediatric care centers to provide counseling for pediatric patients in need for fertility preservation.disclosure: no conflict of interest regulatory t-cells (t reg ) have been shown to play a role not only in autoimmune diseases and solid organ transplantation but also in gvhd. several mouse models showed a decrease of gvhd incidence after t reg administration. the few clinical trials regarding the application of t reg for the treatment of gvhd are encouraging, however the data is limited. methods: patient: a 14-year-old boy underwent allogeneic sct for chronic myeloid leukemia refractory to imatinib, dasatinib and nilotinib treatment from his 10/10 hla identical brother. freshly derived unmanipulated bone marrow was transplanted after conditioning with of fludarabine (40 mg/m²/d, day -7 to -4), thiotepa (2x 5mg/ kg, day -3) and melphalan (160 mg/m², day -2). cyclosporin a (csa) and mycophenolatmofetil (mmf) were used for gvhd prophylaxis. leukocyte regeneration (>1000/μl) was seen on day +15, granulocyte regeneration (>500/μl) on day +16 and thrombocyte regeneration (>50.000/μl) on day +21. on day +30 after sct he developed acute intestinal gvhd that exacerbated to grade iv°(bloody diarrhea, ileus) and did neither respond to steroids, nor to different immunosuppressive drugs such as cyclosporin, tacrolimus, sirolimus, mycophenolatemofetil and ruxolitinib. extracorporal photopheresis and the administration of immunmodulatory antibodies (adalimumab and tocilizumab) did not succeed either.results: by administration of low-dose interleukin-2 (il-2) in vivo induction of t reg was expected but did not succeed. finally antithymocyte globuline (atg, 20 mg/kg/ d) was administered on day +150 to +152 to eliminate the gvhd-triggering cells. hence, the gvhd declined to grade iii. finally, a decision was made to manufacture t reg from his stem cell donor. from an unstimulated leukapheresis t reg were selected by magnetic depletion of cd8 + t-cells and cd19 + b-cells followed by positive selection of cd25 + background: treatment of patients with transfusion dependent anemia like thalassemia major (tm), sickle cell disease (scd) and diamond-blackfan anemia (dba) has improved over the last decades. for the vast majority of patients, allogeneic hematopoietic stem cell transplantation (hsct) is the only available curative therapy. for a long time, hsct has only been performed from hla-identical sibling donors (msd) or matched family donors (mfd). however, approximately only 25-30 % of affected patients do have a matched sibling donor, therefore hsct from 10/ 10 (mud) and even 9/10 (mmud) matched unrelated donors has gained importance in recent years.methods: 36 patients (age range: 2-27 years) with scd (n=7), dba (n=6) or tm (n=23), receiving hsct from a msd, mfd, mud or mmud between 2010 and 2019 were included in our analysis. 23 patients received transplants from msd/mfd, 13 patients from mud/ mmud. 7 patients were identical for hla-a, b, cw, drb1 and dqb1, 6 patients shared only 9/10 genes. we analyzed extended haplotypes including drb3, drb4, drb5 and dpb1 for all patients with thalassemia. 7 pairs showed non-permissive dpb1 mismatch and 1 pair mismatch for dpb1 and drb4.results: median time for granulocyte recovery was 20 days in patients transplanted from msd/mfd and 22 days in patients transplanted from mud/mmud. platelet recovery was reached after 22 days after hsct from msd/mfd and 24 days after hsct from mud/ mmud. 28/36 (80%) patients showed complete donor chimerism in all controls. 5/36 (13%) patients showed low level mixed chimerism up to 20% during follow up. 1 patient died shortly after hsct, 1 patient showed slowly increasing mixed chimerism and finally developed autologous recovery and one patient rejected the graft.cumulative incidence of grade ii-iv acute graft-versushost disease (agvhd) of mud/mmud was 15,2%, whereas only 2 cases of agvhd grade i occurred in patients transplanted from msd/mfd. as 1 patient rejected the graft from a hla-identical parent, 1 patient transplanted from a hla-identical grandparent developed autologous recovery after 1 year and 1 patient transplanted from a mud lost the graft due to hemophagocytosis, the probability of event-free survival was 89,5 % after hsct from msd/mfd and 83,9 % from mud/mmud.altogether 34/36 patients (94,4 %) are alive and transfusion-independent with complete donor chimerism two years after hsct; resulting in an overall survival probability of 93,1%. in contrast, overall survival probability was 100% in the group of patients transplanted from msd/mfd and 80,1 % in patients transplanted from mud/ mmud after 2 years.there were 2 patients with thalassemia (6,9%) who died from transplantation-related causes. the first patient died 13 days after hsct from a mmud due to candida sepsis with pulseless electrical activity resulting from cardiac iron overload. the second patient died 5 months after hsct from a mud due to graft failure.conclusions: hsct from mud and mmud is a feasible therapy option for patients with transfusion dependent anemia. nevertheless, it should be noted that iron overload can cause severe complications; therefore, measurement of liver and heart iron concentration through mri prior to hsct as well as phlebotomy after transplantation are advisable.disclosure: novartis (consultancy: included expert testimony, speaker bureau, honoraria), medac (research funding, patents and royalties), riemser (research funding), neovii (research funding), amgen (honoraria) background: the therapeutic options for patients with hodgkin´s disease who relapse after first high-dose chemotherapy with autologous stem cell (1 st asct) support are limited. allogeneic stem cell transplantation in this setting is associated with a high level of transplant-dependent mortality rates in excess of 50-65%. new agent, such as brentuximab vedotin, have been approved for the treatment of these patients, however, their efficacy to provide longterm control or cure is still unknown. a second autologous stem cell (2 nd asct) has historically been considered as an option only in a small group of patients so the published experience is scarce. we report our institution´s experience with second autologous transplants in this patient population.methods: we evaluated the outcome of 16 adult patients (7 (44%) female and 9 (56%) male), who received an 2 nd asct between 10/2013 and 08/2018. planned tandem asct were excluded. the median age at 2 nd asct was 32 years (range 21-54), 15 (94%) patients had a karnofsky performance score ≥80%. 12 (75%) patients were in complete remission (cr) and 4 (25%) patients were in partial remission (pr) at day 100 after 1 st asct. seven (44%) relapses within 12 months after 1 st asct. patients received a median of 1 (0-3) treatment lines between 1 st asct and 2 nd asct. only 4 (25%) patients received brentuximab vedotin and none of the patients in our series received checkpoint inhibitors as salvage after 1 st asct. the median interval from 1 st asct to relapse/progression was12,9 months (range 2,9-133,3). the median interval from relapse/progression to 2 nd asct was 9,2 months (range 1,4-46,6). all patients received beam as the conditioning regimen for 1 st asct, and beeam as the conditioning regimen for 2 nd asct.results: the median time to neutrophil recovery (>0.5x10 9 /l) after 2 nd asct were 10 days (range 8-17). best response at day 100 following 2 nd asct included cr in 12 (75%) patients and pr in 3 (19%); 1 (6%) had stable disease. 3 (19%) patients received brentuximab vedotin and none of the patients received checkpoint inhibitors after 2 nd asct. 14 (87,5%) patients are currently alive, with a median follow-up 49,8 months (range 4,2-154,2).2 patient died after 2 nd asct. causes of death were hl progression. the 5-year overall survival was 86%.conclusions: the second asct in patients with a longterm response after the first asct may be the optimal therapeutic option, the effectiveness of which can be enhanced by using new drugs, such as brentuximab vedotin, at all stages of treatment.disclosure: nothing to declare effectiveness of chemo-g-csf protocols for mobilization of peripheral stem cells in patients with non-hodgkin lymphomas and hodgkin disease-single center experienceilina micheva 1 , stela dimitrova 1 , vladimir gerov 1 , trifon chervenkov 2 , liana gercheva 1 , igor reznik 1background: high-dose chemotherapy and autologous stem cell transplantation (asct) play an important role in achieving long-term remission in patients with non-hodgkin lymphoma (nhl) and hodgkin disease (hd). granulocyte colony stimulating factor (g-csf) combined with high-dose chemotherapy is a frequently used mobilization approach; however, the optimal mobilization strategy has not been determined.the objective of the study was to analyze the mobilizing potential of different regimens used for the collection of peripheral stem cells in patients with relapsed or refractory (r/r) nhl and hd. methods: we retrospectively analyzed 40 patients with r/r nhl and hd undergoing stem cell collection after chemo-mobilization in the transplant unit at the university hospital, varna. patients were mobilized after dhap letermovir is promising, even as a therapeutic agent. more paediatric data are urgently needed.disclosure: nothing to declare p752 development of paroxysmal nocturnal hemoglobinuria in a patient after mudallohsct due to jak2v617fpositive myelofibrosis-a case of successful treatment with the second transplantation from another donor agnieszka tomaszewska 1 , barbara nasiłowska-adamska 2 , iwona solarska 2 , kazimierz hałaburda 2background: paroxysmal nocturnal hemoglobinuria (pnh) is a very rare disease associated with pig-a gene mutations in hematopoietic stem cells. there are only single case reports on evolving myeloproliferative diseases to pnh in the literature. there is no data concerning development of pnh de novo after allogeneic hematopoietic stem cell transplantation. in our report we describe a patient with recurrence of jak2v617-positive myelofibrosis 5 years after matched unrelated donor allogeneic hematopoietic stem cell transplantation (mudallohsct) with simultaneous development of clinically significant pnh. a 51 year-old-man with a history of mudal-lohsct in may 2011 due to jak2v617-positive myelofibrosis secondary to essential thrombocythemia was admitted to our department 5 years later with mild anemia (hb-11.0 g/dl) and elevated lactate dehydrogenase (1400 u/l). during last 5 years he remained in complete remission of myelofibrosis with jak2v617 mutation negativisation and 100% donor chimerism. suspecting disease recurrence we performed trephine biopsy confirming myelofibrosis (mf2/mf3) with heterozygous jak2v617 mutation and in flow cytometry analysis of bone marrow we identified cell membrane defect in myeloid line (loss of cd66c). we decided to perform detailed diagnostic tests on pnh -multiparametric flow cytometry of peripheral blood revealed 89% granulocytes and 15% red blood cells with loss of gpi-anchored proteins -pnh clone. these results corresponded with donor chimerism -it was only 20% of donor dna in bone marrow and 33% in blood tests. molecular analysis didn't revealed any mutations in genes: calr, asxl1 and mpl. finally the diagnosis of myelofibrosis recurrence after mudallohsct with presence of pnh clone was established. the therapy with eculizumab was unreachable. so the second allohsct from another matched unrelated donor after fludarabinemelphalan-thymoglobuline-tbi 200 cgy conditioning was performed on 21.10.2016. we didn't observe any complications of this procedure, engraftment was slightly delayed: anc>0.5g/l on the 29 day and plt>50g/l on the 50 day.results: at present, more than 2 years after the second mudallopbsct, the patient remains in a very good condition with 100% of the second donor chimerism and without any features of pnh (clone is undetectable) and myelofibrosis.conclusions: presented case is the first in the literature well documented myelofibrosis recurrence after mudal-lohsct with concurrently development of clinically significant paroxysmal nocturnal hemoglobinuria. the second mudallohsct from another donor was safe and successful treatment strategy in this situation.disclosure: nothing to declare. abstract already published. cutaneous refractory t-cell lymphoma treated with allogeneic hematopoietic stem cell transplantationmarcia silva 1 , ercole orlando 1 , maria claudia moreira 1 , simone lermontov 1 , simone maradei 1 , yung gonzaga 1 , leonardo arcuri 1 , renato araujo 1 , decio lerner 1 1 instituto nacional de cancer, cemo, rio de janeiro, brazilbackground: folliculotropic mycosis fungoides (fmf) is an aggressive clinical course variant of cutaneous t-cell lymphoma (ctcl) -classic mycosis fungoides (mf) 1 , with distinct clinical and pathological characteristics, and it is less responsive to skin-directed therapies. for diseases in advanced stages, chemotherapy, autologous hematopoietic stem cell transplantation (hsct) or immunomodulator drugs may provide remissions with limited duration and the treatment remains substantially palliative 2,3 . these dismal results have induced to explore the therapeutical approach with allogeneic hematopoietic stem cell transplantation (hsct) in such patients. early studies have shown encouraging results also in patients with advanced disease, suggesting a major therapeutical role played by the graft versus lymphoma (gvl) effect 4,5,6 . methods: this is a case report of the use of allogeneic hsct as a potential cure for cutaneous refractory t-cell lymphoma type folliculotropic mycosis fungoides .results: case presentation : a 31-year-old male patient with refractory subtype b fmf t-cell lymphoma 7 , diagnosed in 2007, clinically characterized by exfoliative erythroderma, widespread plaques on the trunk and limbs, solitary tumor on the right shoulder, pruritus and bilateral key: cord-005460-ezrn8cva authors: nan title: physicians – poster session date: 2017-07-28 journal: bone marrow transplant doi: 10.1038/bmt.2017.134 sha: doc_id: 5460 cord_uid: ezrn8cva nan hematopoietic stem cell transplant unit, hematology department, hospital universitario de donostia, donostia/san sebastián and 2 informatics and automatics department, university of salamanca, spain the immature platelet fraction (% ipf) is a relatively new parameter that measures young (reticulated) platelets in peripheral blood (pb). ips rise as bone-marrow (bm) production of platelets increases. several clinical utilities of the %ipf have been already proved, as the treatment response monitoring in aplastic anemia or immune thrombocytopenic purpura. in this study, we aimed to found if ip measurement might be useful during the grafting phase of hsct. this study includes 141 patients who underwent allo-hsct in our center during the last 2.5 years. 79 were male (56%) and 62 female (44%). median age was 52 years (range: 7-69). baseline diseases were: acute leukemias (78), lymphoproliferative disorders (22), myelodysplastic syndromes (15), chronic myeloproliferative diseases (12), multiple myeloma (8) and bone marrow failures (6) . donor was unrelated in 79 cases, and related in 62 (including 23 haplo-identical). conditioning regimen was: busulphan-based (93), melphalan-based (17), tbi-based (17) and others (14) . progenitors source was pb in 128, and bm in 13. platelet count, %ipf and absolute ip count (aipc) from day +1 to the day of stable graft were analyzed. 52.4% patients reached plat ⩾ 20 000/mcl at day +14, 82.1% at day +21 and 86.9% at day +28. median first day of plat ⩾ 20 000/mcl was day +14 (range: . median %ipf was 2.6% (range: 0-15.4), 2.5% (range: 0-28.4) and 3.65% (range: 0-15.3) at days +9, +10 and +11, respectively. median aipc was 292/mcl (range: 0-2835), 336 (range: 0-2840) and 504 (range: 0-3660) at days +9, +10 and +11, respectively. among the time points analyzed, aipc at day +11 showed the best positive correlation with platelets counts at day +14 (r = 0.72). interestingly, patients with lower aipc at day +11 showed a delayed platelet graft (see table 1 ). contrarily, patients with higher aipc at day +11 had an earlier platelet graft. absolute immature platelet count before the graft seems to predict the precocity of the platelet graft for the majority of patients undergoing allo-hsct. this finding might help physicians for the patient management (anticipation of hospital discharge and so on). disclosure of conflict of interest: none. [p001] p002 analysis of genetic polymorphism for cardiovascular diseases (cvd) in placental and maternal blood in hypertension and hypercholesterolemia c khalil 1 , a azar 1 and a ibrahim 1,2 1 reviva stem cell research and application center, lebanese university, middle east institute of health hospital and 2 faculty of medical sciences, lebanese university, lebanon cardiovascular diseases are the world's leading cause of death representing 30% of the total global mortality. the genetic polymorphism of the 12 cvd genes, especially the ace: angiotensin converting enzyme gene risky alleles (ins/del) which are associated with a high and inappropriate level of ace can be considered as a genetic model in the development of hypertension and its complications in cvd. we evaluated the mutation impact of the 12 cvd genes in the lebanese population, based on 40 samples derived from placental blood (pb) and 40 samples derived from peripheral blood of postpartum mothers. adult females (age ⩽ 35 years) were divided (n = 20 per group) into group1 (normotensive, normocholesterolemia: nn), and group 2 (hypertension, hypercholesterolemia: hh). buffy coat were extracted from the 40 pb. all tests on pb and maternal blood were done by using the test strip assay to identify the most relevant genetic variations to estimate the risk for cvd. the presence of a double mutation (ins+/del+) related to the ace gene in the hh group was 75%. the presence of a single mutation (ins − /del+) was only associated to the hh by 25%. (ins − /del − ) was absent in 100% of the pb and nn. despite the presence of double mutation ins/del for cvd in maternal blood, pb was free of this mutation. therefore, beyond genetic mutations, other factors can play a major role in the occurrence of cvd. disclosure of conflict of interest: none. s124 b e mt automated red blood cell depletion in abo incompatible grafts in the pediatric setting c del fante, l scudeller, s recupero, g viarengo, f compagno, m zecca and c perotti fondazione irccs policlinico san matteo red blood cell (rbc) depletion by apheresis is employed to reduce the rbc content from abo major or bidirectional mismatch bone marrow (bm) grafts mainly to avoid severe haemolysis 1. rbc depletion results in a significant volume reduction (due to both rbc and plasma depletion) and buffy coat concentration 2.3. in pediatric setting, both rbc depletion and volume reduction before transplantation or cryopreservation can avoid fluid overload and renal impairment, especially in low/very low body weight recipients. the aim of this study was to evaluate the quality of the graft and immediate post infusion complications in rbc depleted bm in major and minor abo mismatch recipients using an automated device. patients and methods: bm aspirates for transplantation in pediatric setting were processed at our centre using the spectra optia (terumo bct) automated device. the initial collection preference was set at level 30 and then was adjusted in order to maintain a haematocrit of 5% (colorgram) in the collection bag. flow speed was set at 120ml/min for 10 cycles. mean recipients' body weight was 31 kg (range:11-72). pre and post procedure bm bag volume, hct%, mononuclear cells (mncs) count, (including b and t lymphocytes), cd34+ cell and cell viability were calculated. moreover, post procedure rbc volume and procedure time were registered. on the patient's side, post infusion complications (renal impairment, fluid overload, fever and haemolitic reactions) and time to engraftment were evaluated. results: a total of 20 rbc depletion procedures were consecutively performed on 20 bm grafts (13 major and 7 minor abo incompatibility, 16 mud and 4 related donors). data about pre and post procedure graft composition are reported in table 1. mean time to engraftment for pmn was 22.6 days (range:17-34) and for plt was 33.5 (range: 21-43). pre and post-procedure cell viability were always 497%. mean procedure time was 80.6 minutes (range:59-115). no bacterial or fungal contamination was detected. no infusion complications were recorded. one graft failure was observed. conclusions the spectra optia automated system is efficient in rbc depletion of abo mismatched grafts, permitting an effective volume reduction and an excellent mncs and cd34+ cell recovery in pediatric setting. automated rbc depletion may be proposed in low/very low body weight recipients both in abo major and minor incompatibility setting to minimize graft infusion side effects. building up a stem cell transplantation program in an emergent country, in the public setting, with limited economic resources, is not an easy work to do. international cooperation may be essential for the development of the program, in training, technological support and implementation of international guidelines. after 20 years, we show an experience of international cooperation between a highly developed center in france (institut paoli calmettes, marseille) and the stem cell transplantation department of hospital maciel, a public assistance service in montevideo, uruguay. fourteen persons between doctors and nurses have been trained in france in stem cell collection and processing, patient's clinical handling, nursing, outpatients care and quality management. french missions of experts have been also received in hospital maciel every year since 2002 for in situ human resources training. in last 5 years we developed a program for optimizing transplant results and reducing transplant related mortality (trm), based on several measures: improvement of patients selection, applying the sorror comorbidy index; adjustment of conditioning regimen doses, in order to reduce toxicity; development of a program to improve interaction with the intensive care unit; protocolization of the standard proceedings treatments; and initiating a program of quality and safety at the national institute of quality of uruguay inacal. 456 adult patients have been treated with autologous (asct) (347) or allogeneic (allosct) (109) sct, with hematological malignancies. different modalities of allosct have been included progressively, becoming the only center accredited by the national regulation authorities (fnr) to perform unrelated donor sct and the haploidentical donor sct. this increased the proportion of allogeneic transplants from the historical 20% until 33% in last 2 years. regarding patients health coverage, 45% comes from the private assistance system and 55% from the public health system. the major indications are lymphoid malignancies and acute leukemia, for asct and allosct, respectively, showing the same trend than cibmtr. three-year overall survival (os) for acute myeloid leukemia after allosct is 61%. considering asct for diffuse large b cell lymphoma, 3 years os after autologous sct is 82% and 52% for chemo sensitive and resistant disease, respectively. threeyears os after asct for hodgkin disease is 87 and 67% for sensitive and resistant disease, respectively. asct in multiple myeloma shows an os of 69 and 50% at 3 and 5 years, respectively. in trm, results during the last 5 years (after the described strategy) are shown in figure 1 . the development of the-program of continuous improvement in quality-and the impact of results was locally recognized by two annual prices from inacal in 2013 (bronze) and 2014 (silver) in the category ‛commitment to public service.' a successful mirna-223 and the level of proangiogenic cytokines: angiopoietin-1 (angpt1), matrix metalloproteinase-9 (mmp-9) and vascular endothelial growth factor (vegf) in patients with lymphoproliferative malignancies prior to autologous hematopoietic stem cell transplantation (hsct) and in early posttransplant period. twenty-four patients were enrolled to the study (11 f,13 m). the median (me) age was 57 years. the investigated group consisted of 19 multiple myeloma and 5 lymphoma patients. the plasma samples were collected on 4 time points: before chemotherapy-‛bc', on the day of hsct -‛0', 7 days after hsct-‛+7' and 14 days after hsct-‛+14. ' the cytokines were evaluated using elisa method, while mirna levels were estimated by qpcr method. the wilcoxon matched-pairs test was used to compare groups of dependent continuous variables: mirna's relative quantification (rq) levels or cytokines expression at two different time points. spearman rank correlation coefficient (r) was used to compare independent variables. we observed continuous decline of cytokines and mirnas level after conditioning treatment. the deepest decrease of expression was marked on ‛+7' day ( table 1) . we noticed a positive correlation between mirna-16, mirnacells in pbsc product. among the autologous transplanted patients between march 2011 and october 2016, we have selected 170 according to diagnosis, conditioning regimen and number of infused bags of cryopreserved pbsc. this group included 77 females and 93 males with median age of 56 (range: . most of them, 105 (61.76%), had multiple myeloma (mm), 41 (24.12%) had non-hodgkin's lymphoma (nhl) and 24 (14.12%) had hodgkin's disease (hd). after harvesting, cd34+ cell and leukocyte number in pbsc product were enumerated on flow cytometer and blood cell counter, respectively. pbsc were cryopreserved with 10% dymethil sulfoxide (dmso) and cell viability was measured with trypan blue exclusion test before and after adding dmso, and as well after thawing in water bath on 37°c. as a conditioning regimen for the mm patients, melphalan was used and for the nhl and hd patients we used beam regimen. all received one bag of cryopreserved pbsc and pegfilgrastim 6 mg on the first or the second post-transplant day. time to hematopoietic recovery was measured; for neutrophils 40.5 × 10 9 /l, leukocytes 41 × 10 9 /l and platelets 420 × 10 9 /l with at least 2 days without platelet transfusion. the median number of total leucocytes infused was 91.88 × 10 9 /l (range: 29.27-284.87 × 10 9 /l) of which cd34+ cells were 2-24.09 × 10 6 /kg of patient's body mass (median 4.75 × 10 6 /kg). pre-freezing cell viability before and after adding dmso was with a median of 100% (74.1-100) and 81, 75% (26.7-100), respectively, and post-thaw viability 57.37% (16.7-100) . the average time to engraftment was 9.8 days (6-26) for neutrophils, 10 days (6-27) for leucocytes and 10.8 days (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) for platelets. our results confirmed the known correlation between the number of infused cd34+ cells and engraftment of neutrophils (po 0.0001), leukocytes (p o0.0001) and platelets (p = 0.0005). we found inverse correlation between the infused leukocytes and cell viability with dmso (p = 0.0035) and after thawing (p = 0.0019). no correlation was found between prefreezing and post-thaw viability with hematopoietic recovery, and also between the cd34+ number and these viabilities. no differences were found considering patients' age, gender, diagnosis, conditioning regimen or day of applying pegfilgrastim. we can indirectly infer good survival of cd34+ cells and higher sensitivity of other nucleated cells to preparation of pbsc product. trypan blue exclusion assay, due to its inability to distinguish type of stained cells, is not relevant for cd34+ cells survival determination. disclosure of conflict of interest: none. chronic granulomatous disease (cgd) is a kind of primary immunodeficiency disorder of phagocytic cells which resulting in failure to kill a defined spectrum of bacteria and fungi and in concomitant chronic granulomatous inflammation. allogeneic hematopoietic stem cell transplantation is the only treatment proved to be potentially curative in cgd. unrelated umbilical cord blood (ucb) is increasingly used as an alternative to bone marrow. methods: unrelated ucbt was performed 14 consecutive cgd children at our center between 2015 and 2016. median age was 18.5 months (range: 5-143 months), median body weight was 10.3 kg (range: 8-34 kg). all patients received myeloablative conditioning regimen consisting of busulfan, fludarabine, cytarabine, cyclophosphamide and g-csf. all patients received tacrolimus as prophylaxis for graft-versus-host disease (gvhd). median nucleated cells were 9.2 × 10 7 /kg (range: 4.5-15.9 × 10 7 /kg), and median cd34+ cells were 3.0 × 10 5 /kg (range: 0.9-7.0 × 10 5 /kg). median follow-up time was 9.5 months (range: 6-23 months) results: 10 of 14 patients engrafted. median time to neutrophil engraftment was 30 days, and median time to platelet engraftment was 33.5 days. 13/14 patients were alive, and 10/14 had full donor engraftment. overall survival rate was 92.8%. disease-free survival was 71.4%. 2 of 14 patients had grades iii-iv acute gvhd. no patients developed chronic gvhd. only one patient died from multi-organ failure related to adenovirus infection. conclusion: unrelated ucbt should be considered as potential curative methods in children with cgd. myeloablative conditioning regimen has improved the engraftments of the ucb. disclosure of conflict of interest: none. reduced muscular mass and excess visceral fat in patients undergoing hsct are associated with higher mortality, longer hospitalization, longer use of immunosuppressive drugs, graftversus-host disease (gvhd) and comorbidities leading to shorter survival time. a recent study of patients undergoing allogeneic hsct showed that occurrence of enlarged areas of visceral and peripheral fat is inversely associated with the disease-free interval after the transplant. reduced muscle mass has also been associated with higher prevalence of chronic gvhd and low rates of success following allogeneic hsct. objectives: to investigate whether amount muscle mass and muscle strength (ms) as well as the amount of visceral fat (vf) of patients undergoing hsct would influence the duration of the engraftment time (en). we evaluated 14 hsct patients (⩾18 years) at hospital israelita albert einstein, são paulo, brazil, on their first day of hospitalization, before hsct. the thickness of the right femoral quadriceps muscle (rfq), measured at 6 cm from the top edge of the patella was measured using ultrasound (us) in b-mode. the dominant upper limb strength of the patients was evaluated by the hand grip test. the vf was measured in the abdominal region, by the thickness of the fat layer between the linea alba and the anterior wall of the aorta. most patients were women (57%) with a mean age of 50 years (±16 years) and 50% of our patients were elderly (⩾60 years). the haploidentical (57%) was the predominant hsct, autologous (36%) and allogeneic (7%). most patients were overweight, with body mass index (bmi) of 27 kg/m 2 (±4 kg/m 2 ). the average time en was 16 days (±6 days). rfq was 1.5 cm (±0.3 cm), ms was 31 kgf (±7.0 kgf) and the vf was 5.3 cm (±1.4 cm). patients with lower rfq had a longer engraftment time that was statistically significant as the negative correlation between rfq and en was rs = 0.8, p o0.05), independent of the age and the hsct type as analyzed by linear regression. no significant correlation between vf or ms with en was found. in this cohort of patients we found that longer engraftment times were significantly correlated to reduced muscle mass but no positive or negative correlation was found with superior limb muscular force or with the amount of visceral fat. disclosure of conflict of interest: none. 1 hematopoietic stem cell transplant unit, hematology department and 2 pharmacy department. university hospital of donostia. donostia/san sebastiań introduction: lymphocytes are the cells responsible for the cellular and humoral immunity and, consequently, critical for hematological patients. the aim of this study was to analyze the eventual conexion between lymphocyte recovery and survival (srv) after allogeneic hematopoietic stem cell transplantation (allo-hsct). patients and methods: we retrospectively analyzed data from 223 consecutive patients who underwent allo-transplants in our unit. in total, 126 patients were male (56.5%) and 97 female (43.5%). median age was 53 years old (range: . baseline disease was: acute leukemia (56.9%), lymphoma (11.2%), myelodysplastic syndrome (10.3%), chronic myelogenous leukemia (8.9%), multiple myeloma (4%), aplastic anemia (3.58%), chronic lymphocytic leukemia (3.13%) and others (1.79%). 55.1% of allo-hscts were from an unrelated donor, and 44.9% from a family donor (25% of them haplo-identical). the sc source was pbsc in 89.6%, and bm in 10,4%. a variety of conditioning regimens were employed, including: busulphan-based (69.5%), melphalan based (10.4%), tbi-based (9.86%) and others (9.86%). evolution of absolute lymphocyte counts (alc) and subpopulations during the first year after allo-hsct were analyzed. results: as shown in table 1 , alc decreased abruptly during conditioning therapy and recovered up to baseline at days +30 and +100; at day +365 median alc had clearly improved compared with admission values. median cd4+ cells were lower than 500/mcl in two thirds of pts at day +100 and in only one third at day +365. as shown in table 2 , we found a significant link between alc at day +30 and srv, as well as between cd4+ cells at day +100 and srv. in our series, immunity recovery was a late event for the majority of patients undergoing allo-hsct. in addition, in our experience, the precocity and quality of the alc and cd4+ recovery was clearly linked with long-term survival. disclosure of conflict of interest: none. although there is experimental evidence suggesting the presence of a common mesoderm cell as origin of both hematopoietic (hsc) and mesenchymal progenitor cells (msc) in an animal model, it is still controversial if durable engraftment of native donor-derived mscs without ex vivo treatment can occur in the recipient of allogeneic hsct. to assess the presence of donor-derived msc following hsct. between july 2015 and july 2016, a total of 33 recipients of hsct were analyzed for hsc and msc chimerism. eighteen patients received bm grafts (54%), 11 patients had peripheral blood as stem cell rescue (33%) and finally 3 patients had a cord blood transplantation (9%). patients received myeloablative (91%) or reduced intensity conditioning (9%) for malignant (91%) or nonmalignant disease (9%). bm aspirate cells were plated and expanded in α-mem with 10% human platelet lysate at 10 000 cells/cm 2 . after 5-7 days, nonadherent cells were removed, while the adherent cells were expanded until they reached confluence. after 2 weeks we quantified msc precursors as colony forming unit fibroblast (cfu-f). finally the amplified sequences were resolved by capillary electrophoresis (3500 ruo genetic analyzer, applied biosystems) and analyzed by comparing genotypes of bmt recipell detachment, nuclear dna was extracted (dneasy blood and tissue kit-applied biosystems) and specific polymorphic tandemly repeated regions (strs) were amplified by means of the polymerase chain reaction(pcr) following the specific manufacturers' instructions. (ampfℓstr identifile kit, applied biosystems following hsct (hsc and msc) to those of donors. we cultured 54 whole bm aspirates from patients following hsct with a median time of 244 day (range: 41-1606). cfu-f/1 × 10 6 growth was observed in a majority of bm the prevalence of human pegivirus in recipients of allogeneic hematopoietic stem cell olga koroleva 1 , e parovichnikova 1 , l kuzmina 1 , m drokov 1 , v vasilyeva 1 , z konova 1 , ekaterina mikhalcova 1 , d dubnyak 1 , n popova 1 , tamara romanova 2 , d tikhomirov 2 , t tupoleva 2 and v savchenko 1 1 bone marrow transplant department, national research center for hematology and 2 virology department, national research center for hematology human pegivirus (hpgv; previously named as gb virus c/hepatitis g virus) was discovered more than 20 years ago. it is an rna virus referred within the genus pegivirus of the family flaviviridae. hpgv rna is found in liver, spleen, bone marrow and peripheral blood mononuclear cell, including t-and b-lymphocytes, nk-cells and monocytes. despite of the fact that it is a molecular structure, mechanism of replication and transmission routes are very well understood but the clinical significance of hpgv is still not determined. recipients of allogeneic hematopoietic stem cell have a high risk infection of hpgv. it is known, that hpgv is a nonpathogenic virus, however, it may play a role in immunocompromised individuals. to investigate the frequency of occurrence of hpgv and its clinical significance in recipients of allogeneic hematopoietic stem cell. blood samples were obtained from 101 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-hsct): all n = 21, aml n = 53, mpn n = 7, cll n = 1, mm n = 1, lpd n = 4, aa n = 6, mds n = 8. a median of age was 33 years (19-64 years) . forty five patients were males and 56 patients were females. conditioning regimen was ric in 75 cases, mac in 26. bone marrow as a graft source was used in 68, pbsc-33. all patients received multiple transfusions of blood components at the previous stages of treatment. hpgv rna had been assayed by polymerase chain reaction real time (rt-pcr) on plasma samples before started pre-transplantation conditioning. despite the diagnosis incidence of hpgv was high 53.5% (rna-hpgv was positively in 54 patients). patients with piercings and tattoos had incidence of hpgv in 64% that was not statistically significant (p-0.5). hpgv is known as nonhepatotropic virus. in our study there was also no statistical reliability of specific changes in liver function test such as elevating the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and total bilirubin due to the rna-hpgv. liver enlargement was also not statistically significant according to ultrasound scan results in patients infected with hpgv. we also analyzed the co-infection with hepatitis b and c virus. results are presented in table 1 . coinfection was not statistically significant. however, only one patient with hepatitis c was coinfected hpgv. leukocytes recovery median was 22 days (14-55). thrombocytes recovery median was 23 (11-82). the presence of rna-hpgv did not affect the recovery of peripheral blood cells in patients after allo-hsct. according to our study the frequency of hpgv infection in recipients of allogeneic bone marrow was quite high (53.5%), and it did not depend on the presence of any other hepatotropic viruses. clinical significance of hpgv infection in recipients of allogeneic hematopoietic stem cell has not been revealed, it is possible due to the short follow-up. it needs further clinical research. disclosure of conflict of interest: none. quantification of cd31+ recent thymic emigrants and t cell receptor excision circles (trecs) in umbilical cord blood transplanted patients v devlia 1,2 , j gridlestone 3 , m raymond 3 , s tulpule 1 , d tewari 1,2 , r hough 4 , c navarrete 3 , a madrigal 1,2 , b shaw 1,2 , r danby 1 and a saudemont 1,2 1 anthony nolan research institute, london, uk; 2 ucl cancer institute, london, uk; 3 nhsbt colindale, london, uk and 4 ucl, london, uk reconstitution of t lymphocytes is a limiting factor in the regeneration of an effective immune system in adult patients following hematopoietic stem cell transplantation. cd31 (pecam-1) is a transmembrane glycoprotein expressed on naive t-cells that have recently emigrated from the thymus into the periphery. in peripheral blood, cd31 + t lymphocytes also contain high numbers of t-cell receptor excision circles (trecs); excision loops of dna excised during t-cell receptor gene rearrangement during t cell maturation within the thymus (1) (2) (3) . however, quantification and correlation of cd31 and trec has not been formally investigated in patients following umbilical cord blood (cb) transplantation. quantification of cd31 and trecs post cb transplant will provide an insight into the immune reconstitution of t cells from the thymus. we therefore sought to measure cd31 and trecs in patients after cb transplant and assess whether these markers provided evidence of thymic recovery. we followed 67 adult patients (median age 52.9 years) who underwent cb transplant in the uk. patient samples were collected 28, 60, 100, 180, 365 and 730 days post transplant. using flow cytometry, we determined absolute counts of cd4+cd31 +cd45ra+ and cd8+cd31+cd45ra+, and quantified the copy numbers of trec genes in peripheral blood mononuclear cells (pbmcs) via real time pcr. results: at the six time points, the number of samples collected were the following: 44, 39, 37, 22, 24 and 14. in all of the samples, the overall median number of cd4+cd31+cd45ra+ was 29 cells/μl (range: 0-827 cells/μl). the median level of cd4+cd31+cd45ra+ cells increases from 16 to 50 cells/μl from day 28 to day 365. absolute counts of cd4+cd31+cd45ra+ at all of the six time points is 10-fold lower compared to healthy controls (median: 279 cells/μl, range: 105-523 cells/μl). the overall median number of cd8+cd31+cd45ra+ cells is 30 cells/μl (range: 0-2222 cells/μl). there is an increase in the median number of cd8+cd31+cd45ra+ cells between days 28 and 720 posttransplant from 2 to 92 cells/μl. however, the absolute median counts of cd8+cd31+cd45ra+ cells in patients are twofold lower, 2 years post transplant, compared to healthy controls (median: 252 cells/μl, range: 133-503 cells/μl). in the majority of the patient samples throughout all time points the trec gene copy numbers were undetected (n = 132). in a few patient samples (n = 9) trec gene copy numbers were quantified but with this limited sample size no correlations can be made between the absolute counts and trec gene copy numbers. our data suggests that cord blood transplant patients within the uk have reduced levels of cd4+cd31 introduction: common variable immunodeficiency (cvid) is a highly heterogeneous group of primary immunodeficiency characterized by defective antibody production, recurrent infections, lymphoproliferation and autoimmunity. autosomal recessive mutations in lrba, encoding lps-responsive beigelike anchor protein were first described as a cause of cvid-like disease in 2012. although hsct is accepted as a standard treatment modality for long-term resolution of severe primary immunodeficiencies, its role is less established in patients with lrba deficiency. patients and methods: whole exome sequencing of patient's genomic dna obtained prior to the hsct revealed a homozygous deletion in lrba (c.5527delt:p. c1843fs). immunological analyses including serum immunoglobulin levels, flow cytometry analyses of lymphocyte subsets, cytotoxicity/proliferation assays, vaccine responses were studied at several time points throughout the disease course, prior to and after hsct. a 14-year-old boy, born to consanguineous healthy parents of turkish origin became symptomatic at the age of 6 months. he hospitalized several times due to recurrent pulmonary infections. he developed pancytopenia, lymphadenopathy, hepatosplenomegaly and autoimmunity (autoimmune hemolytic anemia and thyroiditis) with low serum immunoglobulin levels at the age of 4. as a result, he received several courses of steroid and prophylactic immunoglobulin and wide-spectrum antibiotics. over time he manifested growth failure and diagnosed with ibd-like colitis. due to the cumulating severe cvid-related complications, a hsct was performed at the age of 14 years with the bone marrow stem cells from his hla identical brother after a conditioning regimen including fludarabine, busulfan and atg. severe intractable colitis with hypoalbuminemia continued till the engraftment despite vigorous fluid-electrolyte replacement therapy and accompanied with severe episodes of acute gastrointestinal bleeding. after the achievement of full donor chimerism, diarrhea episodes resolved. he received three doses of abatasept because of persistent cytopenia thinking about unresolved immune dysregulation. he is in complete remission at 1-year post-hsct with no signs of graft versus host disease. allogeneic hsct should be considered in patients with lrba deficiency prior to the development of disease-related severe cumulative manifestations. disclosure of conflict of interest: none. inflammatory bowel disease (ibd) is a chronic disorder of the gastrointestinal tract. very early onset ibd (veo-ibd) represents those severe children with disease onset occurring before 6-years-old. interleukin-10 receptors (il-10ra, il-10rb) mutation are considered to be one of the very important genes for veo-ibd. currently variant treatment, such as steroid medication, immunosuppressive agents and biological agents could not get complete remission. allogeneic hematopoietic stem cell transplantation (allo-hsct) was reported to induce remission in those with veo-ibd. we performed unrelated umbilical cord blood transplantation (ucbt) in five consecutive children with veo-ibd due to il-10 receptor mutation between 2015 and 2016. median age of five children was 15 months (range: 6-46 months), and median body weight was 7 kg (range: 3.2-9.5 kg). all patients received reduced intensity conditioning (ric) regimen consisting of busulfan, fludarabine and cytarabine. prophylaxis for graft-versus-host disease (gvhd) was tacrolimus. most patients (80%) received a 1 or 2 hla alleles-mismatched cord unit. median nucleated cells of the cord blood were 14.3 × 10 7 /kg (range: 11.2-51.5 × 10 7 /kg), and median cd34+ cells were 4.5 × 10 5 /kg (range: 3.6-14.9 × 10 5 /kg). median follow-up time was 10 months (range: 6-24 months). all patients engrafted, median time of neutrophil engraftment was 22 days, and median time of platelet engraftment was 27 days. four of five patients were alive with continuous donor engraftment, and achieved complete clinical remissions. colonoscopy at 6 months after transplantation in two children revealed the mucosa healing. two children had grade iii acute graft-versushost disease (gvhd). one child developed severe chronic gvhd of both lungs and died of ards at 6 months after transplantation. it is the first clinical trial that unrelated ucbt was performed in veo-ibd children in china. our data should unrelated ucbt with ric should be considered as a potentially curative therapeutic option in children with veo-ibd. disclosure of conflict of interest: none. patients with refractory primary induction failure and resistant relapse are poor candidates for hematopoetic stem cell transplantation (hsct) . additional attempts at remission induction with various combinations of chemotherapy will unlikely improve the outcome and will contribute to excess toxicity. a major goal of sct has been to develop strategies to reduce the risk of gvhd while maintaining or enhancing gvl. tcrαβ+/cd19+lymphocytes depletion is a technology of graft manipulation with a potential to increase gvl effect and improve gvhd control and immune reconstitution in this group of patients. a total of 31 pts with refractory aml (primary induction failure (n = 10), refractory relapse (n = 21)), 17 female/14 male, median age 9.7 years (1. [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] , underwent allogeneic sct between may 2012 and august 2016, median fu 1.5 years (0.3-3.8). 26 pts were transplanted from haploidentical donors and 5 from mud. all pts had active disease (ad) at the moment of sct and received treosulfanbased high-intensity conditioning regimen. three regimens of gvhd prophylaxis were used. regimen 1 (n = 7): atgam 50 mg/kg with (n = 5) or without (n = 2) post-transplant tacro/ mtx; regimen 2 (n = 9): thymoglobulin 5 mg/kg, rituximab 200 mg/m 2 and post-transplant bortezomib on day+2,+5 (n = 9); regimen 3 (n = 15): tocilizumab 8 mg/kg on day-1 and post-transplant bortezomib (n = 15), 4 pts receive additional abatacept 10 mg/kg on day+2, +7, +14, +28. tcrαβ+/cd19 +-depletion of sct with clinimacs technology was implemented in all cases. the median dose of infused cd34+ cells was 8 × 10 6 /kg (range: 4.2-17), tcra/b-14 × 10 3 /kg (range: 2-52). all engrafted pts received additional post-transplant courses of low-dose chemotherapy, including hypomethylating agents and dli. primary engraftment was achieved in 27 of 31 pts(three pts had disease progression, one died at the moment of engraftment), the median time to neutrophil and platelet recovery was 12 days (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) . early mortality within 100 days was 3.2% (one pt with aml had acute lung injury after engraftment on day +14), 1.5-years ptrm-6.7% (95%ci: 1.7-25) . there were no allergic or infusion-related adverse events associated with tocilizumab or abatacept. ci of gvhd grades ii-iv and iii-iv was 25.8% (95% ci:14-47), and 9.7% (95%ci:3.3-28), respectively. ci of cgvhd was 23% (95% ci:12-42). ci of acute gvhd was lower in a group with prophylaxis regimen without serotherapy: 20% (95% ci:2-28) vs 31.3% (95% ci:7-54) in atg group. no correlation between graft composition, donor type with the incidence of agvhd and cgvhd was noted at 1.5 years ppfs (event = death or relapse or progression) was 37% (95% ci:19-54), 1.5-years pos -48% (95% ci:30-67). median time of fu for survivors is 1.5 years (range: 0. [3] [4] . we confirm that the depletion of tcrαβ +/cd19+lymphocytes from the graft ensures high engraftment rate and low transplant-related mortality in pediatric pts with refractory aml. we suggest that tocilizumab and abatacept can be safety administered to children with acute leukemia in the context of treosulfan-based conditioning regimens. long-term follow-up will demonstrate if the gvhd prophylaxis without serotherapy and combined administration of tocilizumab, abatacept and bortezomib post-tcrαβ+/cd19 +depleted grafting will improve gvl effects without extensive gvhd-related morbidity and mortality in pts with refractory aml. disclosure of conflict of interest: none. the jacie experience at the university hospital of amiens l marie-noelle 1 , w brigitte 2 , f isabelle 3 , g bérengère 3 , h muriel 4 , h anne 3 , v elsa 5 , m jean-pierre 3 and c amandine 3 1 lacassagne; 2 oncopôle, chu amiens picardie; 3 hématologie clinique et thérapie cellulaire-chu amiens picardie; 4 oncopôle-chu amiens picardie and 5 the jacie (joint accreditation committee of isct and ebmt) accreditation aims to improve the management of patients benefiting from autologous or allogenic hematopoietic stem cell (hsc) transplants. usually, candidates' centers for jacie accreditation have already existing clinical activity when they have willingness to comply with jacie standards. here, we present our new experience in the implementation of jacie quality process, at the same time as allograft clinical activity. implementing process autograft clinical activity existed at amiens hospital, since 1991, but in lack of center cellular therapy laboratory and hsc collection that were outsourced. the collection activity for autologous transplant was set up in 2009, the cellular therapy laboratory in november 2011 and then the allogenic transplant was started in july 2012. as early as march 2011, we set up a steering committee with hematological clinicians, managers of each sector, a transplant coordinator nurse, the head of the processing laboratory and a part-time quality engineer recruited part-time. each actor had to become familiar with standards to obtain information from accredited centers in order to evaluate objectives and their prioritization. steering committee decided on deadlines and established a roadmap including the following: the list of jacie required standard operating procedures and their writing; assignment of the tasks for each actors in order to evaluate, writing and approval each document; organization of documents diffusion; information to all staff on the approach; creation of feedback committee for adverse events management; establishment of morbidity and mortality review; formalization of initial and continuing training for medical and paramedical staff; and organization of cross audits with external teams. at the same time, we assessed requirements for starting activity: training for medical and paramedical staff; training for the transplant coordinator nurse; circuits for taking care of donors; the organization of the in-patient department; the organization of follow-up of post-transplant patients; and authorizations of the national regulation agencies for processing facility on manipulations and cellular qualifications and for regulatory collection and transplant. allogenic transplant clinical activity started in july 2012. accreditations jacie visit occurred on 8 and 9 june 2015 and our center has been officially accredited since 16 march 2016. neither quality approach, nor clinical activities were easy to implement. medical and paramedical staff had to get acquainted with a new organization and restrictions. despite difficulties, implementing jacie quality process, concomitantly with allograft activity allowed to create a true team dynamics with a common reflection on the means to be implemented. moreover, quality approach has assured us best ensure to care graft patients. the result is true satisfaction, which be credited to all. disclosure of conflict of interest: none. previously published p024 three-dimensional co-culture of peripheral blood monocytes supports and expands functional hematopoietic stem/progenitor cell without immobilization y xu 1 , x li 1 , b wang 1 , w shan 1 , h chen 1 , s liu 1 , r tie 1 , y long 1 , s cai 1 , h xu 1 , x yu 1 and h huang 1 1 bone marrow transplantation center, the first affiliated hospital, school of medicine, zhejiang university very low numbers of circulating hematopoietic stem/progenitor cells (chspcs) are found in normal human peripheral blood (pb) without mobilization. here, we developed a three dimension co-culture system to seize and expansion chspcs from pb monocytes without mobilization. flow cytometry analysis was carried out to identify chspc phenotypes. multipotential properties of chspcs were determined using colonyforming unit assay in methylcellulose and reconstitution ability in the compromised animals. the critical regulation mechanism underlying chspcs was identified with transcriptome analysis based on next-generation sequencing technology at total or single cell levels. loose cobble stone colonies (lcs), round or vessel-like compact colonies (rccs or vccs) were presented in three dimension co-culture system after about 2 weeks. the colonies lasted for at least six passages with no obvious apoptosis sign, and expanded more than~10 000fold during the period. we studied the niche-mediated regulation mechanism of chspc fate at molecular level compared to the conventional method of two dimension culture. furthermore, chspcs were capable of forming all types of hematopoietic colonies, including cfu-gemm, and especially held short term engraftment capacity for compromised nogs by radiotherapy. transcriptome analysis by deepsage identified 167 genes significantly associated with regulating the function of chspcs. figure 1 : the cellular morphology in three dimension culture system for peripheral blood monocytes without mobilization during the culture for 2-3 weeks. figure 4 : short transplantable potential analysis of chspcs. figure 5 : (a) static of differentially expressed genes between three-and two-dimensional culture systems for peripheral blood cells. (b) go functional analysis classifies those genes by biological process, cellular component and molecular function. (c) the significant differences between the molecular phenotypes of three-and two-dimension chspcs indicating that chspcs from three dimension culture hold stem properties. our system may provide a more ideal and balanced approach which not only seizes circulating chspcs, promotes selfrenewal and expansion of chspcs, but also holds phenotypic and functional attributes of chspcs. 6. to wash or not to wash? comparison of neutrophil and platelet engraftment after infusion of cryopreserved autologous stem cells before and after the implementation of bedside thawing am halldorsdottir 1 , s atladottir 2 , m thorsteinsdottir, na arnason 1 , g runarsson 2 , t jonsson 1 , oe sigurjonsson 1,3 and s reykdal 2 1 the blood bank, landspítali, the national university hospital of iceland; 2 department of hematology, landspítali, the national university hospital of iceland and 3 school of science and engineering, reykjavik university cryopreserved autologous peripheral blood stem cell (pbsc) grafts are widely used after high-dose chemotherapy in the treatment of patients with myeloma or lymphoma. prior to infusion, cryopreserved grafts can be thawed at the bedside, or thawed and washed at the cell therapy laboratory. at our institution the practice of routine washing of stem cell grafts in the laboratory was discontinued in april 2012 and bedside thawing implemented instead. this was done to minimize the time thawed cells are exposed to toxic dmso. this study was performed at a single center, at landspítali-the national university hospital of iceland, which is the only transplant center in iceland. autologous pbsc transplants have been performed in iceland since 2004. the study compares outcome for two groups of patients, who received either; (a) thawed and washed autologous pbsc cell grafts from january 2008 to [p024] april 2012, or (b) autologous pbsc grafts thawed at the bedside from april 2012 to november 2016. the following outcomes were compared; days to neutrophil engraftment (absolute neutrophil count (anc) 40.5 per μl), and platelet engraftment (20 and 50 × 10e9/l). data on mean cd34+ cell content/kg of the infused grafts, measured prior to cryopreservation, were also compared. all patients have received premedication with solucortef, clemastine and ondansetron prior to infusion of the graft. from january 2008 to april 2012 a total of 84 patients received thawed and washed autologous pbsc grafts, and between april 2012 and november 2016 83 patients received autologous pbsc grafts thawed at the bedside. majority of the patients were diagnosed with either multiple myeloma or related disorders (n = 86) or lymphoma (n = 67) whereas the remaining patients (n = 14) had miscellaneous diagnoses. days to engraftment and the dose of cd34+ cells infused are compared in table 1 . there was no significant difference in the mean cd34 content of infused autologous stem cells in the two groups (6.9 vs 6.5 × 10e6 cd34+cells/kg, p = 0.41). there was also no difference in the mean number of days to engraftment of neutrophils (12.8 vs 13.3 days, p = 0.14), platelets at 20 days (19.2 vs 18.1 days, p = 0.64) or platelets at 50 days (33.1 vs 28.1 days, p = 0.62) after transplant. one hundred day mortality was comparable in the two groups or 2.4%. additional data on transfusion requirements, infections and use of granulocyte-colony stimulating factor will be presented. [p025] there was no difference in neutrophil or platelet engraftment after changing the autologous stem cell graft thawing procedure from post-thaw washing in the laboratory to bedside thawing. bedside thawing of stem cells is a safe procedure that results in acceptable cellular engraftment. disclosure of conflict of interest: none. the procedure of autologous hematopoietic stem cell (hsc) transplantation requires cryopreservation of hscs. addition of dmso (dimethyl sulfoxide) is necessary to secure the viability of such cells, but this cryoprotectant causes adverse reaction during infusion into patient. the concentrations of dmso in cryopreservation mixture vary strongly between different transplant centers. usually, the hscs are stored in mixtures containing 10% dmso, however, many centers successfully use lower concentrations. the main aim of the study was to evaluate the clinical impact of different dmso concentrations in cryopreservation mixture (5%, 7.5%, 10%) on reconstitution of hematopoiesis after autologous hsc transplantation. the project was approved by the local bioethics committee. written informed consent obtained from all of patients. the study is registered to clinicaltrials.gov (identifier: nct02452099). between january 2014 and july 2016, 150 consecutive patients with hematological malignancies or solid tumors, referred for autologous hsc transplantation, were recruited in the study. the patients were randomly assigned to one of three study arms (50 patients each). hscs obtained by leukapheresis were cryopreserved in three concentrations of dmso: 5%, 7.5%, 10%, respectively. study groups did not differ significantly with regard to the diagnosis (mostly mm, nhl or hl), age or conditioning regimen (chemo-or radiotherapybased). all patients received granulocyte-colony stimulating factor (g-csf, filgrastim) starting from day +4 after transplantation to support neutrophil recovery. in case of 7 patients, the transplantation was cancelled due to progression or other medical reasons. four patients died shortly after transplantation, due to refractory infections. data for 139 patients were subjected to statistical analysis. the viability of nucleated cells on the day of transplantation was similar in all groups (median 96%, range: 85-99% for 10% dmso group; 97%, range: 85-99% for 7.5% dmso; 97%, range: 90-99% for 5% dmso; p = 0.52). the dose of transplanted cd34+ cells was comparable in all group: (median 4.70 × 10 6 /kg of recipient body weight for 10% dmso, 4.35 × 10 6 /kg for 7.5% dmso and 3.97 × 10 6 for 5% dmso, p = 0.44). the median time to leukocyte recovery, defined as the first day with wbc count exceeding 1.0 × 10 9 /l was 10 days in all groups (ranges: 9-12 for 10% dmso; 7-11 for 7.5% dmso; and 9-12 for 5% dmso; p = 0.36). similar results were obtained in case of neutrophil recovery-the median day, when the anc exceeded 0.5 × 10 9 /l, was 10 in all arms (ranges: 9-12; 8-11 and 9-12, respectively; p = 0.20). the day when the platelets level were greater than or equal to 20 × 10 9 /l (sustained without transfusion within 7 days) was similar in all groups: medians were 15 days in 10%, 7.5% and 5% dmso (ranges: 8-20; 0-19; 0-24; p = 0.61). no serious adverse effects were observed during hscs infusion and during 24 h after transplantation. reduction of dmso concentration from in cryoprotective mixture 10% to 7.5% and 5% has no negative impact on cell viability during cryopreservation and engraftment after auto-hsc transplantation. disclosure of conflict of interest: none. a real-world cost-effectiveness analysis demonstrates that introducing plerixafor to improve mobilization in multiple myeloma patients who behave as poor mobilizers is cost-effective considering the whole mobilization and transplant procedure r touzani 1,2 , a-m stoppa 3 plerixafor, a cxcr4-antagonist, is efficient to improve cd34 + cell mobilization and collection in candidates for autologous transplantation who behave as poor-mobilizers. the cost of the drug is however of concern. published medico-economics studies were mostly conducted in the us, and few including detailed and comprehensive micro-costing of the collection and transplantation process; conclusions may thus not apply to european countries where cost structures are different. to compare costs and effectiveness of plerixafor-free and plerixafor-replete management strategies for multiple myeloma patients who behaved as poor-mobilizers after adequate administration of a standard rhg-csf mobilization regimen. sixty patients diagnosed with multiple myeloma were consecutively identified during years 2009-2011, immediately before and after ema granted marketing authorization for plerixafor. poor-mobilizers were defined as having circulating cd34+ cell counts below 20/μl. plerixafor was introduced or not as a result of the attending physician's decision, reflecting progressive changes in medical practices over this transitional period. the historical and study groups were matched over four criteria: disease stage at diagnosis, age, gender and number of chemotherapy treatments received before mobilization. two cost-effectiveness analyses (cea) were conducted; the primary cea looked at the criterion ‛collecting at least 2 × 10 6 cd34+ cells'; a secondary cea looked at the criterion ‛successful autologous transplant administered'. detailed micro-costing evaluations (2015 figures) did not or did include transplantation costs for the first and second cea, respectively. the two groups were similar in terms of age, sex distribution, disease characteristics or previous treatments. 27/30 and 26/30 patients proceeded to high-dose melphalan and autologous transplantation in the study and historical groups, respectively. there was a trend to a higher number of collected cd34 + cells in the control group; however, the proportion of patients who met the minimal target number of 2 × 10 6 collected cd34 + cells/kg was identical (28/30). length of hospitalization, times to neutrophil and platelet recoveries, numbers of prbc and platelet transfusions were identical in the two groups. mobilization and collection costs per patients were more important in the plerixafor group that in the historical group (8.757 vs 5.460 €, p o0.0001), and proportionally higher in patients who received plerixafor as part of a remobilization treatment rather than pre-emptively (10.401 vs 8.162€, respectively). the main cea concluded to a 3.237€ increase in costs for the same number of patients achieving a minimal target number of 2 × 10 6 collected cd34 + cells/kg. the second cea found a decrease in the cost of transplant, with 12.724€ in the study group vs 13.634€ in the historical group (ns). in total, the 2.035€ increase for the complete procedure cost (22.866€ per successfully autografted patient in the study group vs 20.831€ in the historical group) was not statistically different. cost-effectiveness arguments should not been used against the administration of plerixafor in multiple myeloma patients in the european context. future prospective researches looking at patients reported outcome criteria and labour organization in apheresis facilities are needed. disclosure of conflict of interest: this work was supported by a grant from sanofi s.a.; cc: research support, honorarium & hospitality from sanofi s.a. administration of plerixafor for peripheral blood cd34+ stem cell content of o30 × 10 6 /l for autologous stem cell mobilization leads to decreased apheresis days and increased total yield m kamdar 1 , s abebe 1 , gr gonzalez fontal, l gates 1 , a hammes 2 , d abbott 2 , j gutman 1 , b haverkos 1 , d sherbenou 1 and c smith 1 1 division of hematology and transplantation and 2 department of biostatistics and informatics, university of colorado, denver, colorado, usa autologous stem cell transplantation (asct) is an effective treatment for lymphoma and plasma cell neoplasm (pcn) (multiple myeloma and amyloidosis). granulocyte-colony stimulating factor (g-csf) is the most commonly used upfront mobilizing agent with plerixafor-based higher cost approaches reserved for poor/unsuccessful mobilizers. several mobilization algorithms utilizing g-csf and plerixafor have been published however the most efficient and cost effective strategy is yet to be determined. most transplant centers administer plerixafor for peripheral blood (pb) cd34+ stem cell content of o10 × 10 6 /l on day (d) 4 of g-csf mobilization. at the university of colorado (uch) we changed our programmatic approach in 2015 and administered plerixafor for pb cd34+ count of o30 × 10 6 /l on d4 of g-csf mobilization. in this study we evaluate the impact of this novel mobilization algorithm on apheresis days and total stem cell yield. patients (pts) with lymphoma and pcn who underwent asct at uch until 3/2015 received plerixafor if pb cd34+ cells on d4 of g-csf mobilization was o10 × 10 6 /l. based on our institutional review of poor/unsuccessful mobilizers and using logistic regression analysis this algorithm was revised in 4/2015. in the new algorithm all pts received plerixafor if pb cd34+ cells on d4 of gcsf mobilization was o30 × 10 6 /l. demographics were compared between pts with lymphoma and pcn before (group 1: 9/2013-3/2015) and after (group 2: 4/2015-4/2016) the new algorithm was implemented. the primary goal of this analysis was to assess the total days of apheresis and total stem cell yield between the two groups. we also sought to analyze days to wbc engraftment and platelet engraftment. a total of 131 pts were included in this analysis. group 1 consisted of 77 pts (26 pts had lymphoma and 51 pts had pcn). group 2 consisted of 54 pts (20 pts had lymphoma and 34 pts had pcn). we found that there was a significant increase in total yield (p = 0.0017) in group 2 as compared to group 1. on further disease subtype assessment we noted that pts with pcn in group 2 had a significant increase in total yield (p = 0.0014). in lymphoma pts on univariate analysis group 2 showed a significant decrease in apheresis days (0.468 days, p = 0.044, 95% ci: (−0.91, − 0.026)). on multivariate analysis there was still a marginally significant decrease in group 2 (0.47 days, p = 0.052, 95% ci: (−0.916, − 0.024)) compared to group 1. in pcn pts on univariate analysis group 2 showed a significant decrease in apheresis days (0.387 days, p = 0.025, 95% ci: (−0.718, − 0.056)). on multivariate analysis group 2 continued to show a significant decrease in apheresis days (0.426 days, p = 0.02, 95% ci: (−0.772, − 0.081) compared to group 1. we found no significant difference between the two groups in days to neutrophil engraftment and platelet engraftment. our analysis showed that a mobilization algorithm of administering plerixafor for a pb cd34+ stem cell count of o30 × 10 6 /l on d4 of g-csf mobilization led to a decrease of roughly 0.46 days in the lymphoma cohort and a significant decrease of 0.43 days in the plasma cell neoplasm cohort. we also noted a significantly increased total yield of stem cell collection in group 2. overall our programmatic approach led to decreased chair-time for apheresis and better resource utilization. pharmacoeconomic impact of this approach will be updated at the meeting. disclosure of conflict of interest: mk: speakers bureau, seattle genetics; remaining authors declare no conflict of interest. administration of stem cell boosts (scbs) from the original donor offers a therapeutic option. we report on 50 pediatric patients with pgf who received a total of 61 boosts with cd34 + selected peripheral blood stem cells (pbsc) after transplantation from matched unrelated (n = 25) or mismatched related (n = 25) donors. median time between hsct and infusion of the 61 scbs was 94 days (13-519). boosts contained a median number of 3.15 × 10 6 cd34+ progenitor cells/kg body weight (range: 0.71-27.9 × 10 6 ) with a median number of 2417/kg (range: 100-23 630) residual cd3+ t cells. within 8 weeks after application, a significant increase in median neutrophil counts (600 vs 1516/mm 3 , po 0.05) and a decrease in erythrocytes and thrombocytes transfusion requirement (median frequencies 1 and 7 vs 0, p o0.0001 and o 0.001), were observed, and 78.8% of the patients resolved one or two of their initial cytopenias whereas 36.5% had a complete hematological response. additionally median lymphocyte counts for cd3+, cd3+cd4+, cd19+ and cd56+ increased 8.3 fold, 14.2 fold, 22.3 fold and 1.6 fold, respectively. the rate of de novo acute gvhd grade i-iii was only 6% and resolved completely after treatment. no gvhd iv or chronic gvhd occurred. patients who showed a response to scb displayed a trend toward better overall survival (os) (p = 0.07). administration of cd34+ selected scbs from alternative donors is a safe and effective procedure. we hypothesize that the cd34+ progenitor boosts may have an enhancing effect on maturation of committed lymphoid precursors already present in the host or generate another wave of thymic seeding with accelerated t-cell differentiation process in the absence of any immune suppression. further studies are warranted to better define the impact on immune reconstitution and survival. disclosure of conflict of interest: none. plerixafor plus granulocyte-colony stimulating factor (g-csf) has been shown to mobilize more cd34+ cells than g-csf alone for autologous hematopoietic stem cell transplantation (hsct). however, there are few studies that analyze the impact of this strategy in engraftment. the aim of our study is to compare mobilization and engraftment between patients who received a combination of plerixafor plus g-csf and patients (pts) who mobilized with g-csf alone. a retrospective casecontrol analysis was performed in 24 pts with myeloma who mobilized with plerixafor plus g-csf (group p/g-csf) and was compared with 24matched for sex and age controls who mobilized with g-csf alone (group g-csf). all pts underwent hsct between 2009 and 2015. mobilization with g-csf at dose of 10 μg/kg/day was used in all pts. the aphaeresis was scheduled on day +5. plerixafor (0.24 mg/kg) was added if the number of cd34+cells on day +4 was o10/μl for 2 × 10 6 cd34+/kg requested (or o20/μl for 4 × 10 6 cd34+/kg), or if the number of cd34+cells collected in the first apheresis was o50% of cd34+ requested. conditioning and supportive care were similar in both groups. in p/g-csf group, 13 were male and 11 female. median age was 60.92 years (range: 49-71). in group g-csf, 15 were men and 9 female. median age was 60.67 years (range: 50-73 years). there were no differences between both groups. disease status at time of mobilization was different between groups (p = 0.023). in p/g-csf group: 11 (45.83%) pts were in complete remission (cr), 4 (16.66%) very good partial responses (vgpr), 7 (29.16%) partial response (pr) and 2 (0.08%) had no response to treatment. in g-csf group: 7 (29.16%) pts had reached cr, 13 (51.16%) vgpr and the remainder in pr. sixteen (66.67%) pts in p/g-csf group had received ⩾ 2 lines of treatment vs 9 (37.5%) pts in g-csf group (p = 0.046). no difference was seen on mean day-dose of g-csf (14 μg/kg/24 h in p/g-csf group vs 12 μg/kg/24 h) (p = 0.067). there was no difference on cd34+/kg requested (19/24 pts in p/g-csf were requested 2 × 10 6 /kg vs 18/24 in g-csf group) (p = 0.73). p/g-csf group needed more apheresis sessions, 17 (70.83%) pts required ⩾ 2 sessions against 4 (16.67%) pts in group g-csf (p o0.001). we obtained enough cd34+ cells to carry out hsct in all patients, although mean number of cd34 + cells obtained in p/g-csf group was lower than in g-csf group (2.92 × 10 6 /kg vs 4.98 × 10 6 /kg, respectively) (p o0.001). also, mean number of cd34+ infused in p/g-csf group was lower (2.92 × 10 6 /kg vs 3.55 × 10 6 /kg) (po 0.001). however, engraftment results were similar in both groups, as represented in table 1 . patients who required mobilization with plerixafor plus g-csf got an engraftment as good as patients who do not require the combination despite of worse baseline parameters. given that the number of cd34+ infused in the p/g-csf group has been lower than g-csf group, these results might suggest that the different composition of graft cell with plerixafor plus g-csf mobilization, described in some studies, could impact on engraftment outcomes. high-dose chemotherapy following autologous hematopoietic stem-cell transplantation (autohsct) is an effective method of treatment both recurrent and primary refractory lymphoma patients. however, some patients have mobilization failure (‛poor mobilizers') with inadequate collection of peripheral blood stem cell (pbsc). aim: to evaluate the efficacy and factors influencing pbsc mobilization and collection for the autohsct in patients with lymphomas. thirty patients were included in this study: 17-with hodgkin lymphoma, 7-with non-hodgkin lymphoma, 6-with multiple myeloma; 17 women and 13 men of them. the median age of patients was 36 years (24-64 years). the mobilization of pbsc with only colony-stimulating factors (csf) was carried out for 17 patients, chemotherapy (cyclophosphamide, etoposide) in combination with csf-for 13 patients. only one patient had plerixafor mobilization. the concentration of cd34+ in peripheral blood (pb) was studied on the day of the intended cytapheresis. cytapheresis was commenced when cd34+ concentration had been greater than 0.01 × 10 6 cells/ml. twenty-four patients (80%) from 30 had collection of pbsc. the collection was not performed in six patients (20%) because the concentration of cd34+ in pb on the day of the intended cytapheresis was lower than 0.01 × 10 6 cells/ml. there was no possibility to use plerixafor in these cases for economic reasons. the median concentration of cd34+ in pb on the first day of the intended cytapheresis in the group of patients that had cytapheresis was 0.013 × 10 6 cells/ml whereas in the group of failed-0.005 × 10 6 cells/ml (p o0.05). fifty-nine tests of cd34+ in pb were done. distribution and test results by days from the first day of the intended cytapheresis are presented in table 1 . the total number of the cytapheresis was 36. the majority of patients had 1 procedure of pbsc collection (n = 22), 13 patients had 2 procedures and only 1 had 3. the last patient had had two previous failed cytapheresis procedures and the adding of plerixafor helped him to collect necessary number of cells. the median of cd34+ cells on patient's kilo was 2.85 × 10 6 cells/kg. sex, age, mobilizing regimen, previous radiation therapy, the count of lines of chemotherapy before autohsct were not significantly associated with poor pbsc mobilization and collection. only tumor response before autohsct (complete/ partial response or stabilization) was significantly associated with cd34+ cell count in the product of cytapheresis. patients with complete or partial response had significantly better cd34+ count. [p034] disclosure of conflict of interest: none. factors associated with failure in mobilization of peripheral blood hematopoietic progenitor cells in autologous transplantation je dulon-tarqui, bl acosta-maldonado, l rivera-fong, sa sánchez-guerrero, jf zazueta-pozos, ja padilla-ortega, wj ladines-castro and lm valero-saldaña high dose therapy followed by autologous stem cell transplantation (asct) obtained from peripheral blood is currently the standard model for treatment consolidation in various hematologic malignancies. a global incidence of 5-40% of failure to mobilization is reported, and some factors associated with poor mobilizers in hodgkin's lymphoma (hl), non-hodgkin's lymphoma (nhl) and multiple myeloma (mm) when the yield in peripheral blood stem cells (pbsc) collection is unsatisfactory, the effects for the recipient can be serious. the donor's age, gender, body surface area (bsa), processed blood volume and the method of g-csf dose calculation may affect the cd34+ yield. as g-csf has a low distribution volume in the peripheral blood (pb), it might be appropriate to calculate the doses by using the bsa instead of per kg body weight. 175 consecutive allogeneic pbsc donations performed in 170 healthy donors at the karolinska university hospital in stockholm were included. a complete medical history, physical examination, electrocardiogram, chest x-ray and laboratory testing were done before pbsc donation. relevant data for analysis were collected from the institutional quality database for a retrospective review. the total blood volume was calculated using the formula by nadler et al. the bsa was calculated using the formula by du bois and du bois. the concentration of cd34+ cells in the pb and the processed volume of blood were significantly correlated to cd34+ cells yield (po 0.00005 and po0.001, respectively, see table 1 ). the g-csf dose per m 2 was significantly correlated to the concentration of cd34+ cells in the pb (p = 0.0003) and in the product (p = 0.01, see table 1 ). smaller bsa (p o0.001) and less processed volume (p o0.001) were found among female donors, who were given lesser g-csf dose per m 2 (p o0.001) and showed lower yield compared to men (po0.05). however, multivariate analysis of the yield showed that only the concentration of cd34+ cells in the pb and the processed volume remained independent significant (see table 1 ). [p036] in this study, we found the concentration of cd34+ cells in the pb and the processed volume of blood to be independent predictors of yield. we recommend to get a high concentration of cd34+ cells in the pb, and to process adjusted volumes of blood when needed. an evaluation if the calculation of g-csf dose per m 2 is more appropriate than per kg body weight should be done in future studies. autologous stem cell transplantation (asct) has been widely used in the treatment of hematological malignancies over the last two decades. despite its broad use, some characteristics that might influence engraftment have not been exhaustively investigated, particularly graft purity with respect to contamination by platelets (plts) and white blood cells (wbc). here we report collection characteristics and engraftment kinetics of a single center consecutive series of 510 asct. we retrospectively collected clinical records of 481 patients who underwent leucapheresis procedures (la; followed or not by asct) and data on 510 asct at our institution over 16 years (2000-2016) ( table 1 ). the impact on engraftment kinetics of conditioning chemotherapies, amount of infused cd34+ cells and wbc/plts graft contamination were analyzed. absolute neutrophil count (anc) engraftment was defined as the duration of neutropenia (from day 0 to the first of 3 consecutive days of anc4500/μl post asct). regarding cd34+ cell collection, no impact of mobilizing regimens and wbc count during la was observed. on the other hand, we observed a difference in the number of total cd34+ cells collected among different diagnoses: the median overall collection was 7.2 (0.65-64.06) × 10 6 /kg cd34+ cells for nhl patients, 5.66 (0.71-23.31) × 10 6 /kg for mm patients, 6 .15 (0.51-23.24) × 10 6 /kg for hl patients and 3.56 (0.64-20.3) × 10 6 /kg for aml patients) (p = 0.001). considering cd34 + cells/kg harvested on the first day of la, 59.2% of nhl and hl, 57.5% of mm patients and 34% of aml patients harvested ⩾ 5 × 10 6 /kg cd34+ cells. of note, among aml patients, 40.6% collected o 2.5 × 10 6 /kg. the differences were statistically significant (p = 0.003). moreover, an inverse correlation between collected cd34+ cells and age was shown (p = 0.001). anc recovery after asct was not influenced by conditioning regimen whereas diagnosis impacted on the duration of neutropenia (aml patients displayed a longer aplasia, po0.01). we observed that the median days with anco 500/μl were 10, 11 and 12 in patients who received 45.3 × 10 6 /kg, 3.5-5.3 × 10 6 /kg and o3.5 × 10 6 /kg cd34+ cells, respectively (po0.0001). furthermore, the same finding was observed considering the duration of thrombocytopenia (median number of days with plts o50 000/μl: 15, 18 and 20 in patients who received 45.3 × 10 6 /kg, 3.5-5.3 × 10 6 /kg and o 3.5 × 10 6 cd34+ cells, p o0.0001). looking at the apheresis product, we analyzed the impact of harvest contaminating wbc and plts on engraftment kinetics. notably, when the asct collection contained 4100 × 10 3 /μl wbc, anc engraftment (days with anc o 500/μl) lasted longer (median days 11) compared to patients who received a graft with lower wbc count (po 0.0001). a faster anc engraftment was also observed in patients receiving harvests with plts levels 4600 × 10 3 /μl compared to those who infused a collection bag with plts o600 × 10 3 /μl (p = 0.005). herein, we confirmed that the disease and the amount of infused cd34+ cells significantly influence time of anc and plts engraftment; furthermore, we observed for the first time that quality and purity of the graft have a substantial impact on engraftment kinetics. a combination of chemotherapy with growth factor is a commonly used strategy for hematopoietic stem cell (hsc) mobilization. the collection of timely and adequate numbers of hscs is a prerequisite for proceeding to transplantation. a variety of mobilization strategies are currently used. the knowledge of efficacy, safety and predictability of different hsc mobilization strategies might help blood and marrow transplantation (bmt) programs to effectively schedule patients for mobilization. given the many variables associated with the mobilization of hsc, collecting an adequate stem cell dose in a timely and effective manner is an art and science. factors that might affect the process includes type of disease and mobilization protocol, financial clearance, availability of chemotherapy beds, scheduling various diagnostic procedures and transplant urgency. to evaluate the effectiveness and related coordination efforts of ‛just-in-time' strategy of hsc mobilization and collection, we performed a retrospective study comparing all patients in whom peripheral hsc mobilization was attempted at khcc from january 2005 through november 2016. data collected included the disease type, mobilization protocol, days to and number of collections, cd34+ cell dose, calendar of the mobilization and collection. the records of a total of 1042 mobilizations were reviewed. 364 were of healthy allogeneic donors, and the remaining 678 were of patients undergoing autologous transplantation. table 1 depicts the overall summary of number of days and collection procedures per each protocol. detailed mobilization kinetics per disease type and mobilization protocol were also captured and evaluated. [p037] s140 [p038] detailed analysis of mobilization kinetics comparing different mobilization strategies aids in prediction of number of days of mobilization and anticipated number of collections. this helps in proactively scheduling patients based on collection predictability. a seamless communication through a shared calendar between key parties, primarily bmt physicians and nurse coordinators, bmt and flow cytometry laboratories and chemotherapy unit can be achieved. autologous stem cell transplantation is still a standard of care in the treatment of multiple myeloma. lenalidomide-based regimens are commonly used in both transplant-ineligible as well as -eligible patients. prolonged lenalidomide-exposure is known to affect mobilization of cd34 + cells, although the basic mechanisms are poorly understood. limited prospectively collected data is available on the effect of lenalidomide in the capacity to mobilize cd34 + cells for transplantation as well as graft cellular composition and post-transplant hematological recovery compared to the lenalidomide-naive patients. this prospective study included 60 newly diagnosed myeloma patients who received mobilization with low-dose cyclophosphamide + g-csf, were successfully apheresed and transplanted before the end of 2014. twenty-six patients had received a median of three cycles of lenalidomide-based induction (43 %), whereas 34 patients were lenalidomide-naive and served as the control group. both baseline characteristics and collection targets were similar between the groups. cd34 + mobilization and apheresis yields were analyzed and compared between the groups. blood graft cellular composition was analyzed from the thawed cryopreserved samples with a flow cytometry. graft function was evaluated by collecting engraftment data as well as by total blood counts at day +15 and at 1, 3, 6 and 12 months after post-transplant. the patients in the lenalidomide group had both lower median peak b-cd34 + counts and about 40% lower cd34 + yields of the first apheresis but without statistical significance ( table 1 ). the median number apheresis was significantly higher in the lenalidomide arm (2.0 vs 1.5, p = 0.039). the number of cd34 +cd133+cd38-, cd3+cd4+, cd3+cd8+ cells and nk cells in the cryopreserved grafts were comparable between the arms. time to neutrophil engraftment was 12 days in the both groups. the median time to platelet engraftment was 12 d in the lenalidomide group and 13 d in the control group. hematological recovery was comparable between the groups within 12 months post-transplant. lenalidomide-based induction therapy seems to have an impact in the number of apheresis needed, but not in the total yield of cd34 + cells in the graft. neither the graft cellular composition nor posttransplant recovery in myeloma patients was affected by the limited duration of lenalidomide used before mobilization and collection of blood grafts. between september 2015 and november 2016. siemens hematek 3000 system was used for luc count. luc numbers and percentage was measured before leukapheresis. we used pearson test for the correlation and roc curve for cut off value. patients' characteristics were shown in table1. there was not a correlation between luc number and mobilized cd34 positive stem cell number. but luc percentage was positively correlated with mobilized stem cell number (p:0.01). a count of 5 × 10 6 /kg collected stem cells are optimal for autologous stem cell transplantation. we found 2% luc percentage as a cut-off value for prediction of collecting optimal number of stem cells with 61% sensitivity and 60% specificity. as expected luc percentage was negatively correlated with white blood cell count. there was no correlation between mobilized cd34 positive stem cell number and age. both luc percentage and mobilized cd34 positive stem cell number did not differ with underlying disease. we found only one study in the literature that evaluated luc percentage as a tool for the prediction of successful stem cell collection. they found that baseline luc numbers negatively correlated with stem cell mobilization in healthy donors (1) . but we measure luc on apheresis day and found a positive correlation between luc percentage and stem cell mobilization. and we found a cut-off value for optimal stem cell mobilization with acceptable sensitivity and specificity. in our study we demonstrate that luc percentage measurement on apheresis day may be a very simple and cheap tool for the prediction of optimal stem cell mobilization. the spectra optia (so) apheresis system performs a wide range of therapeutic procedures, including peripheral blood stem cell (pbsc) collection in mobilized donors and patients (pts). the device was studied to evaluate the cellular composition of pbscs harvested in pts with multiple myeloma (mm), non hodgkin's lymphoma (nhl) and hodgkin's lymphoma (hl) planed for autologous peripheral stem cell transplantation (apbsct), and to optimize the collection of pbscs using the cd34+ precount and collection efficiency (ce2) of apheresis device which is calculated as follows: ce2 = total cd34 + cells collected × 10 6 /kg; cd34+ precount/ μl × blood processed (liters). the blood volume processed is calculated as follows: desired cd34+ × 10 6 /kg × recipient weight (kg): ce2 × cd34+ precount/μl in our study enrolled pts undergoing pbsc mobilization and planed for apbsct. we evaluated so system's mononuclear cell (mnc) collection performance, with respect to cd34+ cells and mnc collection efficiency, platelet reduction pre to post apheresis, and product purity in view of using prediction algorithms to optimize the procedure and predict the cd34+ yield, blood volume processed and platelets loss. we also evaluated neutrophil and platelet recovery in pts who underwent apbsct. results: between 30/3/2015 and 30/11/2016, 45 pts underwent pbsc harvesting by so device. median age was 46 years (20-71). there were 19 females and 26 males. diagnosis was mm in 21 pts, hl in 17 pts and nhl in 7 pts. the number of ahereses procedures was 59. mobilization consisted in g-csf alone in36 pts, chemotherapy and g-csf in 8 pts, and g-csf + cxcr4 inhibitor in one patient. median count of cd34 + cells pre-collection was 58/μl (16.5-372) . median total blood volume processed was 12.4l (6.3-19.9 ). median count of cd34 + cells collected was 4.1 × 10 6 /kg (1-23.6). median mnc collection efficacy was 48% .median cd34+ cell collection efficacy was 45.5% (15-95%). median platelet reduction pre to post apheresis was 30% (0-50%). median product hematocrit and granulocytes product was 5% (3-9) and 52% (5-93), respectively. twenty-six of the 45 pts underwent myeloablative high dose chemotherapy followed by apbsct which was performed for mm in 18 pts, hl in 6 pts, and nhl in 2 pts. the median count of cd34+ cells infused was 2.5 × 10 6 / kg (1.15-10.6). all the pts received g-csf post-apsct until neutrophil recovery. the median day for neutrophil recovery was 10 (8) (9) (10) (11) (12) (13) (14) . median duration of severe neutropenia (anc o0.5 × 10 9 /l) was 7 days (4-10). the median day for platelet recovery was 10 (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) . median duration of severe thrombocytopenia (platelets o20 × 10 9 /l) was 5.5 days (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) . conclusion: the study results confirm that the so apheresis system's mnc collection protocol is safe and effective. the neutrophils and platelets recovery in pts auto-transplanted was not inferior compared to historical controls. in addition, this system help to use prediction algorithms for whole blood processing to achieve a desirable and optimal yield based on cd34+ precounts and ce2 of the apheresis device. disclosure of conflict of interest: none. peripheral blood stem cell apheresis in small children is difficult! aa hedayati-asl 1 , m emam-jome, p dinarooni, v fallah, a mehrvar and r zangooei 1 in low-weight children with cancer and healthy donor children, peripheral blood progenitor cells (pbpcs) have largely replaced bone marrow as source of autologous and allogeneic stem cells in part because of their relatively easy collection. however, there is a concern regarding medical, psychosocial and technical difficulties in small children. we retrospectively analyzed peripheral blood stem cell apheresis in 38 collections. 30 patients were with cancer (17 patients = neuroblastoma, 4 patients = retinoblastoma, 5 patients = germ cell tumor, 1 patient = hepatoblastoma, 3 patient = wilm's tumor) and 8 healthy children donors. the study was conducted between 2012 and 2016. peripheral stem cell apheresis was performed in the mahak cancer children's hospital in a nice room for children where the patients stayed with their families. patients s142 were not routinely sedated. pbpc were collected by a cobe spectra cell separator (cobe, denver, co, usa). harvesting was performed after 5 days mobilization. mean body weight was 11.6 kg (range: 8-15 kg) for a median age of 3 years (range: 10 months-5 years). mean duration of harvesting was 205 min (range: 164-270 min). mean volume of stem cell collection was 135 ml (range: 110-240 ml). the mean number of total nucleated cells collected was 5.4 × 10 8 /kg (range: 3.2-9.9 × 10 8 /kg recipients). no side effects occurred. children didn't require an additional haematopoietic progenitor mobilization or additional apheresis in other day. pbsc collection was without transfusion in healthy donor children. pbsc collection may be difficult in small children owing to the large volume apheresis compared to the child's weight. various problems, such as metabolic or haemodynamic disorders may be were seen. peripheral stem cell harvest can be performed in low-weight children under safe and effective conditions even when systematic priming by blood is avoided. processing with increase of blood volume may to increase in the yield by recruiting progenitor cells. disclosure of conflict of interest: none. peripheral blood stem cell collection in low body weight children: a single centre experience g del principe*, g leone, s lazzaro, a meschini, k feri, p marchitelli, d carasso, f locatelli and m montanari department of pediatric hematology/oncology and transfusion medicine, irccs bambino gesù children's hospital, rome, italy pbsc became preferred source for autologous transplantation because of easier collection and faster engraftment. however apheresis for low body weight children ( o12.5 kg) is affected by some issues: venous access, extracorporeal volume, metabolic and hemodynamic complications, citrate toxicity, so is crucial to standardize harvesting procedure both maximizing stem cells collection and reducing adverse events. a dual lumen central venous catheter was used to obtain a minimal blood flow of 10-15 ml /min and pbsc collection was performed with spectra optia mnc v6.1 apheresis system, starting with cd34+ cell ⩾ 20 μl in peripheral blood. the priming of extracorporeal circuit was made with compatible, irradiated, leucodepleted packed red cell to avoid hypovolemic state. citrate dextrose solution a(acd-a), with a ratio of 1:24 to whole blood, and a bolus of heparin 50 ui/kg were used as anticoagulants. all patients, treated without sedation, were monitored by ecg, pulse oximetry and non invasive blood pressure; electrolytes panel (na, k, ca) and act (activated coagulation time) were assessed at the beginning, 30 minutes after and then every hour during apheresis. hypocalcemia was managed by 350 mg calcium gluconate slow infusion. we report our experience of pbsc collection in low body weight children ( o12.5 kg) treated in our apheresis department between january 2015 and november 2016. a total of 37 pbsc collections were performed in 17 children (8 m/9 f, median age 21 months, median weight 10.5 kg) affected by medulloblastoma (n = 4), germ cell tumor (n = 2), neuroblastoma (n = 8), retinoblastoma (n = 2), brain cancer (n = 1). total blood volume processed ranged from 2.0 to 4.55 tbv (median 3.05) and median count of cd34+ collected was 5.5 × 10 6 /kg(range: 1.5-54). all procedures were performed with a median duration time of 199 minutes (range: 114-293 min) and no serious adverse events occurred. in our experience pbsc collection is safe and feasible also in low body weight children using a tailored apheresis procedure. disclosure of conflict of interest: none. plerixafor on demand in the first or in the second attempt of cd34 mobilization j romejko-jarosinska, e paszkiewicz-kozik, l targonski, m szymanski, z pojda and j walewski 1 1 high-dose chemotherapy and autologous hematopoietic cell transplantation (auto-hct) is a recommended strategy for patients with relapsed, refractory or high risk lymphoma. mobilization failure of cd34+ cells after granulocyte colonystimulating factor (g-scf) with or without chemotherapy is a factor limiting patient access to this potentially curative procedure. the use of plerixafor with g-csf may improve cd34+ cell harvest in poor mobilizing patients. we evaluated the clinical effectiveness of plerixafor and g-csf ± chemotherapy administered on demand in the first and second attempt of mobilization in lymphoma or myeloma patients who were eligible for auto-hct. we evaluated data on 59 consecutive patients with hodgkin lymphoma (17), dlbcl (17), mantle cell lymphoma (10) , myeloma (8) and other lymphoma subtypes (7) who were mobilized with plerixafor between january 2011 and october 2016. median (range) age of patients was 47 (27-69). patients received a median of 2 (1-4) chemotherapy lines. radiotherapy was applied in 13 patients. all patients received g-csf (10 μg/kg/day) ± chemotherapy and plerixafor (240 μg/kg/day) on demand in the absence of increase in the number of cd34+ cells in peripheral blood above 10/μl on the day of the scheduled apheresis (within 20 days following the chemotherapy and after at least 4 days of g-csf). plerixafor was given to 36 patients in the first attempt of mobilization and to 23 patients during the second mobilization. the mobilization was considered effective if the harvest cell dose was 2 × 10 6 /kg cd 34 or more. after plerixafor administration circulating cd34+ cells increased to 410/μl in 21 patients (58%) and in 11 patients (43%) in the first and in the second mobilization, respectively (p = 0.26). the cd34+ cell collection was performed in 52/59 patients (88%): in 32/36 (89%) patients in the first and in 20/23 patients (87%) during the second mobilization cycle. the median number of apheresis was 3 (range: 1-6), for both mobilizing cycles. the median (range) cd34 cell dose collected in the first and second cycle was 3.03 (range: 0.77-16.87) × 10 6 /kg and 1.89 (range: 18-11.53) × 10 6 /kg, respectively (p = 0.007). the harvest was successful in 27/32 patients (84%) in the first and in 10/20 patients (50%) in the second cycle (p = 0.007). three patients (9%) who failed the collection with plerixafor in the first attempt, succeeded in the second cycle. additional second mobilization with plerixafor was successful in five patients (25%) who failed the first mobilization. in total, 30/32 (93%) and 15/20 (75%) of patients given plerixafor in the first or in the second mobilizing cycle harvested at least a minimum cd34 cell dose for auto-hct (p = 0.05). these results show that plerixafor administered on demand is an effective rescue strategy for poor mobilizing patients. each mobilization cycle with plerixafor resulted in the increase of circulating cd34 cell count. successful harvest is more frequent if plerixafor is administered in the first than in the second mobilization attempt. the evaluation of the prognostic factors for mobilizing failure with plerixafor is necessary to identify the poor mobilizers precisely. disclosure of conflict of interest: jr-j, ep-k, lt and jw: sanofi (travel grants); ms and zp: none; jw: lecture, honoraria cryopreserved stem cell grafts are still widely used both in the autologous or allogeneic settings. cryopreserved grafts can be thawed at the bedside or thawed and washed at the cell therapy laboratory. we recently reported that post-thaw washing did not impair hematopoietic engraftment, in a cohort of 2930 autologous transplanted patients receiving either unwashed or washed grafts (calmels b et al, bone marrow transplant. 2014). post-thaw washing can be implemented using various methods such as manual centrifugation, automated centrifuge-based (sepax 2, biosafe) or spinningmembrane devices such as lovo (fresenius kabi). we here report a step by step implementation of the lovo biomedical device (bmd) for washing thawed stem cell grafts. having defined a washing program, we aim to compare this protocol to our routine process, using the sepax 2 bmd. we took advantage of 11 apheresis products intended for destruction and cryopreserved in 2 identical bags; after dry-thawing (plasmatherm, barkey), bags were connected to the sepax 2 or to the lovo bmd, diluted volume to volume with +4-8°c 6% hydroxyethylstarch 130/0.4 (voluven, fresenius kabi) and processed using the smartwash program (sepax 2) or a 2cycles standard wash protocol on lovo (a cycle referring to one pass through the spinning membrane). the lovo settings were customized for this application: reduction retentate pump rate 75 ml/min, desired inlet pcv 10%, and automated volume to volume dilution. after processing, cd34 and cd45 absolute counts and viability were evaluated by single platform flow cytometry (stem-kit, beckman coulter) and dmso was quantified by capillary zone electrophoresis (p/ace, beckman coulter). post-wash data show comparable cd34+ cell recovery, viability and effective dmso depletion. we conclude that lovo enables high efficiency dmso depletion while preserving optimal cd34 viability and recovery. comparison with sepax 2, a widely used automated centrifugebased device, reveals comparable efficiency. moreover, the length of the procedure when using the lovo does not significantly delay the process as compared to bedside thawing. we are currently evaluating lovo for the processing of multiple bags and higher cell contents, due to its ability to concentrate large volumes of cells suspension. post-thaw washing using automated cell processing systems have thus to be preferred over bedside thawing, since they provide multiple benefits including a short processing time, efficient dmso and cell debris removal, precise determination of infused cd34+ cell dose, and improved cellular stability. [p047] disclosure of conflict of interest: none. using bone marrow (bm) as the graft source results in lower graft-versus-host disease incidence, which is particularity important in haploidentical (haplo) stem cell transplantations (sct). nonetheless achieving adequate cd34+ cell count might be complicated in cases of donor-recipient weight differences. priming with g-csf may partly solve this problem. also there are reports of immunomodulatory effect of bm priming. in the retrospective study we have evaluate the effect of priming on stem cell yield and the outcomes of sct. patients and methods: 50 patients with primed bm graft were matched in the ratio 1:1 to non-primed grafts. the criteria for matching were type of the donor, age of the recipient, underlying disease and disease status at the time of sct. priming was performed with three injections of filgrastim 5-7 mcg/kg daily for 3 days prior to bm harvesting. median recipient age was 18 years (range: 1-58). 60% of patients received the graft from haplo donor, 40% from matched related donor (mrd). 39% had acute lymphoblastic leukemia, 38% had acute myeloid leukemia, 10% had aplastic anemia, 13% had other malignancies. 68% were classified as salvage patients. 27% received myeloablative conditioning, 73% received reduced intensity. post-transplantation cyclophosphamide (ptcy) was used as graft-versus-host disease prophylaxis in 83% of patients. results: the yield of cd34+ × 10 6 cells /kg of recipient weight was only non-significantly higher in the priming group: 6.0 ± 3.4 vs 5.0 ± 2.5, p = 0.12. the yield of cd34+ cells per kg of donor weight was also not different: 4.3 ± 4.0 vs 4.3 ± 5.8, p = 0.55. there was no difference in the incidence of primary graft failure (14% vs 20%, p = 0.42). median time to neutrophil (21 vs 23 days, p = 0.02) and platelet (19 vs 23 days, p = 0.05) engraftment was shorter in nonpriming group. there was no differences between priming and non-priming groups in the incidence of acute grade ii-iv gvhd (14% vs 4%, p = 0.11), moderate and severe chronic gvhd (12% vs 11%, p = 0.88), 1-year non-relapse mortality (12% vs 18%, p = 0.46), relapse incidence (36% vs 38%, p = 0.91), overall survival (64% vs 54%, p = 0.52), event-free survival (52% vs 44%, p = 0.62) and gvhd-relapsefree survival (38% vs 36%, p = 0.62). conclusions: priming of the bone marrow with reported schedule did not result in higher cd34+ cell yield and was not associated with any differences in the outcomes of sct. nonetheless, these results should be interpreted with caution, because our study included large proportion of pediatric patients, patients with active disease and ptcy as gvhd prophylaxis, and they may not translate to the other groups of patients. disclosure of conflict of interest: none. priming with granulocyte-colony stimulating factor preserves the contents and abundant ifn-γ production capacity of γδ t cells z bian 1 , q fu 1 , m huo 1 , xj huang 1 and j liu 1 1 peking university people's hospital the increasing evidences indicate that removal of αβ t-cell and b-cell from grafts was efficient and reproducible in allogeneic hematopoietic stem cell transplantation (allohsct). γδ t cell is one of the functional subpopulations preserved by this graft manipulation and supposed to play a role in improving the transplant outcomes. thus, comprehensive understanding the subsets and functional capacities of γδ t cells in graft becomes important. although there is increased attention paid on this special t-lymphocyte subpopulation, the contents and cytokine production capacities of peripheral γδ t cells before and after granulocyte-colony stimulating factor (g-csf) mobilization for allohsct have not been reported. peripheral blood (pb) before g-csf treatment, g-csf-primed pb and bone marrow (bm) grafts were obtained from 19 healthy donors. the proportions of total γδ t cells and various γδ t-cell subsets were detected by flow cytometry. furthermore, effects of g-csf on the contents and cytokines production by γδ t-cell subsets were also determined. the percentages of most γδ t-cell subsets including cd27+, cd27-, vδ1+, vδ1+cd27+, vδ1+cd27-, vδ2+, vδ2+cd27+, vδ2 +cd27-, and non-vδ1/δ2 were preserved in the g-csf-primed pb grafts compared with those before g-csf mobilization. interestingly, we found that peripheral γδ t cells and various subsets all predominantly expressed ifn-γ in response to stimulation. this abundant ifn-γ production capacity of peripheral γδ t cells were maintained after g-csf treatment. in contrast, production of il-17 by γδ t cell and its subsets were decreased in the same context. priming with g-csf preserved the contents and abundant ifn-γ production capacity of γδ t cells. our data suggests a reasonable role of γδ t cells in preventing from allohsct associated complications and may help establish an effective γδ t cell-based immunotherapeutic approach to improve the overall survival of allohsct. disclosure of conflict of interest: none. processing of hematopoietic stem cells grafts: towards automation of cryopreservation/thawing steps a-l chateau 1 , j gaude 1 , c malenfant 1 , a autret 2 , c lemarie 1 , c chabannon 1 and b calmels 1 1 centre de thérapie cellulaire-institut paoli-calmettes and 2 unité de biostatistiques-institut paoli-calmettes autologous hematopoietic stem cells (hsc) support is still widely used to allow for high-dose chemotherapy in the context of myeloma and lymphoma treatment. in the autologous setting, mobilized aphereses are systematically cryopreserved. currently, cryopreservation and subsequent thawing rely on manual and largely operator-dependent processes such as manual addition of dmso for cryopreservation or thawing in standard water baths. these operations are thus hampered by significant intra-and inter-facility variability and have to be replaced whenever possible with automated and harmonized processes. the aim of our study was to evaluate a recent, versatile device: smartmax (biosafe, eysins, switzerland), based on the peltier-seebeck effect, for its ability to automatically add the dmso-containing solution to the cell product and to thaw hsc bags. we thus compared three different cryopreservation/thawing protocols ( figure 1 ). we first evaluated the use of the smartmax at the thawing step by comparing 110 cryopreserved apheresis products thawed using our routine device: the plasmatherm (barkey), an automated dry-thawing device that contains water (protocol a), with 51 products thawed with the smartmax (protocol b); after thawing, all products were washed using the smartwash program of the sepax2 (biosafe). we then evaluated the smartmax for its ability to automatically add the dmso solution: 9 autologous grafts were processed with the smartmax, both for cryopreservation and thawing (protocol c); we compared these 9 ‛fully automated processes' to 51 apheresis processed with protocol b. absolute cd34+ and cd45+ cell counts and viability were measured before cryopreservation and after washing using single platform flow cytometry. for all three protocols, the quality of the collected product was comparable in terms of median cd45+ cell and neutrophil contents. when comparing protocols a and b, viable cd34+ cell recovery after thawing and washing was slightly lower in the smartmax group (77%) as compared to the plasmatherm group (82%, p = 0.009). when comparing protocols b and c, viable cd34+ recovery was comparable (p = 0.8) when the cryopreservation solution was automatically added by the smartmax (81%), as compared to the manual technique (77%). these preliminary data need to be validated on larger numbers of procedures, however suggest that smartmax use can safely be substituted both to the manual addition of the cryoprotectant and to the traditional thawing step in water baths; potential advantages include complete water removal from sensitive clean rooms and gmp environments. full automation of previously manual and operatordependent technical processes will ultimately allow for improved standardization and reproducibility across cell processing facilities. [p050] disclosure of conflict of interest: none. reduced efficacy of mobilisation using gdp compared to ive a hunter, w merrison, am martin, k hodgson, f miall, r moore and r lewin university hospitals of leicester, nhs trust the use of ive ± rituximab for relapsed/refractory disease in lymphoma is well established. stem cell mobilisation using g-csf post ive administration has been the standard of care in our unit for 20 years. recent interest in cisplatnin-based treatments has seen a change in practice with the use of gdp ± rituximab increasingly common. we have assessed the success of stem cell mobilisation post gdp and compared it to ive using g-csf. patients were eligible for augmentation with plerixafor if their peripheral blood cd34 levels were between 5-10 × 10 6 cells/l at the time of collection. from sept 15 to oct 16 21 patients with progressive or relapsed lymphoma underwent stem cell collection. patients characteristics: 12 dlbcl, 4 follicular and 5 t-cell nhl. had a median age 65 (37-74 years). 13 received gdp, 8 ive. overall 23% patients failed to mobilise a sufficient cd34 cell dose to proceed to hdt. all the patients who received ive mobilised successfully but 5/13 (38%) patients receiving gdp failed to mobilise. of the patients who did mobilise the average cd 34 collection was higher in the patients who received ive 7.1 (4.5-25.4) and the number of apheresis procedures was lower, median 1 (1-2) compared to 3.4 (2.6-9.1) and 2 (13), respectively, in the gdp group. patients in the gdp group who failed to mobilise were not eligible for plerixafor because cd34 levels were below 5 × 10 6 /l. taking age into account the median age in the ive group was higher 65(48-74) than the gdp group 62 (37-74) and the lines of previous therapy were not different. patients who had successful stem cell collections went on to receive hdt with leam and all patients engrafted. in this small collection of patients we have experienced a higher failure of mobilisation post a cisplatnin-based protocol compared both to our historical controls pre plerixafor usage (data not shown) but also to current patients. further investigation is needed to ascertain the impact of cisplatnin on stem cell mobilisation and its impact of treatment strategies. disclosure of conflict of interest: none. single centre experience of zarziotm biosimilar granulocyte-colony stimulating factor (gcsf) for the mobilisation of healthy donors demonstrates good leukapheresis yields and safety profile at 24 month median follow-up jg taylor 1,2 , t seddon 2 , k alizadeh 2 , c agrawal 2 , l kempster 2 , jg gribben 1,2 and sg agrawal 2,3 1 centre for haemato-oncology, barts cancer institute, 2 dept. haemato-oncology, st bartholomew's hospital, london, uk and 3 experimental pathology, blizard institute, queen mary university of london, uk biosimilars have led to significant improvements in the affordability of growth factors such as granulocyte-colony stimulating factor (gcsf). data has shown similar performance and efficiency to parent drugs but concern has been raised about their use in healthy donors due to lack of data examining adverse effects in this setting. we conducted a retrospective analysis investigating mobilisation and adverse effects in 51 healthy sibling donors of adults undergoing an allogeneic haematopoietic stem cell transplant at st bartholomew's hospital from 2011 to 2014. harvest data were gathered from hospital records. adverse effects data were gathered from hospital records and telephone follow up. 58% of donors were male with a median age at harvest of 46 (13-65). all donors were mobilised using zarziotm biosimilar gcsf at a dose of 10 μg/kg/day. median number of apheresis required was 1 (1) (2) (3) . median cd34+ cell count was 5.6 × 10 6 /kg bodyweight (1.3-13.9) with 1664 × 10 6 cd34+/μl (168-3779) in peripheral blood. the target cd34+ count (45 × 10 6 /kg) was achieved in 59% of donors and an adequate yield (2-5 × 10 6 /kg) in 33%. in four donors (8%), the harvest was deemed to have been unsuccessful as the cd34+ count was o2 × 10 6 /kg. the patients with donor harvest yields o2 × 10 6 /kg proceeded to transplant; all four patients engrafted and one patient had mixed chimerism at day 28 but was fully donor by day 75. median cd3+ cell count was 2.7 × 10 6 /kg bodyweight (0.8-6.4) . median days to neutrophil engraftment (40.5 × 10 9 /l) was 16 . median days to platelet engraftment (420 × 10 9 /l) was 5 (0-23) with one patient never engrafting. forty (80%) of 51 donors were contacted at a median of 24 months (1-54) post mobilisation to establish incidence of adverse effects. three donors were uncontactable as they had moved overseas. eight donors were not contacted to avoid distress as their sibling had died since transplant. among contacted donors 42.5% reported side effects including bone and lower back pain controlled with analgesia, constipation and low mood. other side effects included chest pain which was considered to be musculoskeletal in origin on day 3 of gcsf administration associated with taking an increased dose due to patient error (n = 1) and abdominal contractions like labour while receiving gcsf (n = 1). three (7.5%) reported side effects lasting beyond one month post mobilisation: lower back pain lasting 2 months (n = 1), fatigue of 3 months duration (n = 1), and cough of 8 months duration (n = 1). our data demonstrates good mobilisation using 10 μg/kg/day zarziotm biosimilar gcsf without significant adverse effects at 2 years median follow up. this supports its ongoing use for the mobilisation of healthy donors. disclosure of conflict of interest: sga has received honoraria from sandoz and grant support from sandoz and amgen. stem cell mobilization in poor mobilizers with multiple myeloma (mm) or non-hodgkin lymphoma (nhl) before and after introduction of plerixafor: single center comparative analysis using a cost-efficient single fixed-dose schedule r wäsch 1 , c greil 1 , c kiote-schmidt 1 , s hildenbeutel 1 , k kühbach 1 , r bosse 1 , j duyster 1 and m engelhardt 1 1 department of hematology, oncology and stem cell transplantation, university medical center, freiburg, germany collection of hematopoietic stem cells (hsc) from the peripheral blood (pb) is routinely conducted prior to highdose chemotherapy and autologous transplantation. despite safety and efficiency of current apheresis procedures including mobilizing chemotherapy and granulocyte colony-stimulating factor (g-csf), there is a significant rate of mobilization failures due to different patient-dependent factors necessitating additional agents like plerixafor. while plerixafor is approved for patients with mm or nhl based on prospective studies using steady state mobilization with g-csf − /+ plerixafor, prospective studies using chemo-mobilization are lacking. here we compared the outcome of poor mobilizer from the pre-plerixafor era with poor mobilizers who received additional plerixafor in a real world analysis. we analyzed 50 consecutive patients with mm or nhl who were mobilized at our academic center between 2011 and 2016 and received plerixafor, because they were expected to be poor mobilizers, due to 1. low counts of cd34+ cells in pb samples prior to apheresis, 2. after a first apheresis day with insufficient yield or 3. as a rescue strategy after insufficient harvest with previous mobilizing chemotherapy (greil c,…engelhardt m, wäsch r. leukemia & lymphoma 2017, in press). we examined cd34+ cell counts in pb and in apheresis products to identify those patients who were able to collect a sufficient cd34+ cell count for transplantation after application of plerixafor. we compared these data with 100 consecutive poor mobilizers from the pre-plerixafor era, who were mobilized between 2000 and 2011 without plerixafor. the median pb cd34+/μl count at first apheresis was significantly higher after the first dose of plerixafor when compared to the pre-plerixafor group with 19.9 vs 9.8 (p o0.001). accordingly, the median collected cd34+ cells/d (×10 6 /kg bw) and total cd34+ cells (×10 6 /kg bw) were significantly increased with 1.67 vs 0.88 (p o0.001) and 4.13 vs 2.66 (p o0.001), respectively. the rate of 42 × 10 6 cd34+ cells/kg bw in first apheresis (%) increased from 11% in the pre-plerixafor era group to 38% after the first dose of plerixafor in the plerixafor group. consistently, the successful transplantation rate increased from 58% in the preplerixafor group to 90% in the plerixafor group. successful stem cell mobilization could be achieved with only a single fixed-dose of plerixafor in 62% of poor mobilizers as previously reported by our group. the addition of plerixafor to chemomobilization in poor mobilizers with mm or nhl significantly increased pb cd34+/μl counts, apheresis yields and transplantation rates when compared to poor mobilizers from the pre-plerixafor era. these favorable apheresis results can be obtained using our cost-efficient, single fixed-dose plerixafor schedule in the majority of the patients leading to a 90% transplantation rate in poor mobilizer. disclosure of conflict of interest: rw received research funding, advisory and speaker's honoraria from sanofi-aventis. high-dose chemotherapy followed by autologous peripheral blood stem cells transplantation (pbsct) is the standard of treatment for patients with hematological malignancies. recombinant granulocyte colony-stimulating factors (g-csfs) are widely used alone or in combination with chemotherapy, in order to mobilize patient's stem cells (cd34+) for autologous and allogeneic peripheral blood stem cells transplantation. aim: the aim of our study was to compare effectiveness and safety of different biosimilar products of filgrastim used in autologous pbsc mobilization in patients with hematological malignancies. our retrospective analysis included 282 patients (118 women and 164 men) with median age 54 years ( range: ,who underwent the procedure of autologous pbsct in years 2012-2014 in the haematology, blood neoplasms, and bone marrow transplantation clinic of medical university in wrocław. there were three different biosimilar products of filgrastim used: tevagrastim (teva) in 95 patients, nivestim (hospira) in 92 patients and zarzio (sandoz) in 95 patients. 90 (32%) patients were diagnosed with plasma cell neoplasms, 145 (51%) with hodgkin's and non-hodgkin's lymphomas, 20 (7%) patients had acute myeloid leukemia and 27 (10%) had other hematological malignancies. statistical analysis was conducted using statistica 12 (statsoft polska) statistical software. for quantitative variables arithmetic means and standard deviations were calculated for the estimated parameters in the studied groups. distribution of variables was tested using w-shapiro-wilk test. p0.05). there were also small variations in the mean number of leukapheresis necessary to obtain the minimum cd34+ cell count: 1.32 in zarzio group, 1.37 in nivestim group and 1.66 in tevagrastim group. however, there were no difference between biosimilar g-csfs. the highest rate of successful mobilizations (defined as 42 × 10 6 /kg cd34+ cells collected) was observed in 88.2% patients received zarzio, in 86.2% received nivestim and in 84.9% patients received tevagrastim. the safety profile was comparable between the biosimilar g-csf and included bone pain in 30 (10%) patients and headache in 25 (9%) patients. the results are shown in table 1 . all three used biosimilar g-csfs demonstrated similar efficacy and safety in stem cell mobilization in patients with hematological malignancies. therefore, it seems that all the analyzed products can be used interchangeably. presented observations should be verified with wider prospective research. [p055] disclosure of conflict of interest: none. use of g-csf stimulation of bmt donors might prove to be beneficial in many respects, improving tnc yield but also through immunomodulatory effect on donor t cell function and apcs1. we analyzed outcomes of 14 consecutive patients receiving bone marrow transplants from hla-haploidentical related donors that received g-csf stimulation prior to harvest. fourteen patients received hla-haploidentical bmt with pt-cy between 5/2012 and 10/2016. five donors were siblings, 4 children, 4 mothers and 1 father. donors received g-csf at the dose of 10 mcg/kg bw sc. on days − 2, − 1 and 0 before bm collection. twelve patients received nonmyeloablative conditioning according to baltimore protocol2, while two patients received myeloablative conditioning (bucy). along with post-transplantation cyclophosphamide, all patients received tacrolimus and mmf form day +5, as described earlier 2. median age was 41 years (range: 19-63), 7 female and 7 male patients. eight patients had aml, 1 cml, 4 mh and one all. ten of them were in remission, while 2 mh patients were in pr, and 2 aml patients had residual disease as evident by immunophenotyping. median number of infused tnc was 5.17 × 10 8 /kg bw (range: 1.84-8.21); cd34+ cells 1.88 × 10 6 /kg bw (range: 1-4.47 ) and cd3+ cells 1.35 × 10 7 /kg bw (range: 0.37-6.04). median follow up was 362 days (range: 26-1654). eleven patients engrafted (79%), one patient had primary rejection, one had overt disease relapse at day +35 and one patient died in aplasia due to sepsis. median day to neutrophil recovery (anc 40.5 × 10 9 /l) was 21 (range: 15-29), median days to platelet recovery (plt 420 × 10 9 /l) was 31 (range: 12-45). in all patients mmf was discontinued at d +35. two patients developed acute gvhd in our cohort (18%), one after receiving dli for falling chimerism at day +169. one patient (9%) developed chronic gvhd, after having received dli due to disease progression. at the time of analysis 10 patients are evaluable; 4 patients had disease relapse/progression (40%), 6 patients are alive and in remission. one patient died due to sepsis in aplasia (accounting for 7% non-relapse mortality). one patient that rejected the graft was transplanted again from the same donor, using myeloablative conditioning and peripheral stem cells as graft source and engrafted. overall survival median is 2.7 years, with significantly shorter survival if patient was not in complete remission at time of transplant (p o0.01). even though the experience with g-csf mobilized bm graft in the hlahaploidentical setting with pt-cy is relatively small, in our series it has been beneficial in terms of tnc yield. also, the incidence of acute and chronic gvhd in our patients has been low, particularly agvhd with one case developing only after dli. whether the observation is the result of limited number of patients, or it reflects the immunomodulatory effect of g-csf on bm graft as previously suggested1 remains to be seen as further studies are warranted. autologous transplantation of haematopoietic stem cells (ahsct) is usually perceived as a fully standardized and safe procedure; however, a minority of patients experience a delayed engraftment and seldom even an engraftment failure, possibly related to a poor quality of the graft. therefore the current policy in many centers is aimed to increase the target dose of collected cd34+ cells up to an ‛optimal' level of 4 × 10 6 /kg. plerixafor was introduced in the clinical practice to maximize the mobilization of hsc, in order to collect an optimal number of cd34+ cells in a limited number of collections also in poor and slow mobilisers. we carried out a retrospective analysis of our case series aimed to individuate mobilization predictors optimize the ‛on demand' use of plerixafor. we analyzed 162 patients who underwent mobilization with cyclophophamide (4 g/sqm) and filgrastim 10 mcg/kg from +5 in our unit from 2009 and 2016. diagnosis were multiple myeloma (mm) 74 (45.7%), non-hodgkin lymphoma (nhl) 46 (28.4%), hodgkin lymphoma (hdg) 14 (8.6%) and 28 (17.3%) autoimmune disease (ms 14.8%; ssc 2.5%). median age (range) was 53 years ; male/female ratio 82/80. circulating cd34+ cell count was started at white blood cells (wbc) recovery, which was defined as the first day when their count exceeded 1 × 10 9 /l. the primary goal was to identify at wbc recovery one or more factors predicting a suboptimal mobilization, which was defined as the failure to exceed 40 cd34+/mcl circulating cells in the day after the wbc recovery. patients were excluded from this analysis if 1) showed a cd34+ count 440/mcl at wbc recovery (very good mobilizers) and/or 2) had received plerixafor and/or 3) did not proceed to another cd34+ count the day after wbc recovery. binary logistic regression was used to obtain the factors that increased the odds for an optimal mobilization. overall 80 out 162 (49.4%) patients were shown as very good mobilisers as their cd34+ count exceeded 40/mcl at wbc recovery. on the remaining 82, 7 were excluded for the lack of a second assessment and 2 for the lack of data. among the remaining 73 patients, the threshold of 40 cd34+/mcl cells on the second day was reached by 55 (75.3%) of patients (group a) while the remaining 18 (24.7%) failed the goal (group b). median (range) wbc × 10 9 /l and cd34+/mcl counts in group a and b at wbc recovery were 2.01 (1-6.43) and14.65 (0.70-39.91) and 2.84 (1-20) and 7.39 (0-33), respectively, with a statistically significant differences among group (mann-whitney u test with p = 0.01 and p = 0.02, respectively). wbc (or = 2.193; 95% ci: 1.197-4.019) and cd34+/mcl (or = 0.858; 95% ci: 0.77-0.955) in first day count, but not gender, disease category and time from mobilization chemotherapy to first cd34+ count, were predictors of optimal mobilization. combining these two predictors we found that wbc/cd34+ ratio has a sensitivity of 82.4% with an auc 83.7 in roc analysis. assessment of circulating wbc, cd34+ and their ratio at wbc recovery in a chemo-based mobilization strategy can predict sub-optimal mobilization of hsc and support the decision of adding plerixafor. these data will be prospectively validated in a broader set of patients. disclosure of conflict of interest: none. human platelet lysate (hpl) is rich in growth factors (gf) and nutritive elements and represents a powerful xeno-free alternative to fetal bovine serum (fbs) notably for mesenchymal stem cell (hmsc) proliferation. however, there is a large variability in hpl preparations (various sources, use of different and non-standardized production protocols, with variable and limited number of donors), resulting in discrepancies in product quality, low management of product safety and poor batch-to-batch standardization. we describe here the development and the characterization of a standardized hpl prepared from outdated transfusional grade screened normal human donor platelet concentrates (pcs), manufactured on an industrial scale (batch size of 250 donors) and following a highly qualified process (clean room, trained operators, validated aseptic filtration). pcs were frozen at − 80°c and thawed at +4°c to lyse platelets. cell debris were removed by centrifugation and the supernatant (hpl) was recovered. clinical grade 10l batches of aseptic filtered hpl were characterized. first, we showed that hpl prepared from a limited number of donors displayed a variability in terms of gf contents. on the contrary, we observed a robust standardization between industrial batches of hpl (250 donors) in terms of gf contents (bfgf, egf, vegf, pdgf-ab, tgf-beta1 and igf-1), biochemical analyses (total proteins, albumin, fibrinogen, vitamins and iron) and efficacy on bone marrow (bm)-hmsc proliferation. secondly, we compared expansion and functional characteristics of bm-hmscs grown in clinical grade hpl vs msc-screened fbs batches. we showed a reproducible increase in cell growth kinetics using hpl, a maintenance of bm-hmsc clonogenic potential and membrane marker expression (with however a strong overexpression of cd90). we observed a similar adipogenic and osteogenic differentiation potential and finally that immunosuppressive properties of bm-hmscs (inhibition of t-cell proliferation) cultivated in parallel in both conditions also remained identical. finally, we demonstrated the stability over time of hpl stored at − 80°c and − 20°c. in conclusion, we demonstrated the feasibility to use a standardized, characterized, efficient and clinical grade hpl for research and cell therapy applications. disclosure of conflict of interest: sv, se, lc, pb, tb, al, fg and bd are employees of macopharma. previously published p061 alpha/beta t cell depleted donor lymphocyte infusion m karakukcu, e ünal, l kaynar, s özcan, g tezcan karasu and mb acar the main objective of this project is to improve a safe and efficient new donor lymphocyte infusion (dli) with depletion of αβ+ t cells which cause graft versus host disease (gvhd), and enrichment of anti-leukemic γδ+ t cells, nk cells and dendritic cells to build an effective and permanent anti-tumor effects for patients relapsed hematological cancers after allogeneic hematopoietic stem cell (hsc) transplantation who have blasts and mixed chimerism. this study is conducted with collaboration of erciyes university pediatric and adult hsct units, and bahcesehir university, medical park hospital pediatric hsct unit. the tcr αβ+ t cell depleted dli product that is used in the study was collected and separated at erciyes university apheresis unit. the cell contents obtained for tcr αβ+ t cell depleted dli used for patients were cd3 cells were reduced to 1.4-23.5 × 10 6 cells/kg, γδ+ t cells were reduced to 2.58-23.48 × 10 6 cells/kg, αβ+ t cells were reduced by 99.99%, and were obtained at 5-4100 cells/kg. a total of 10 patients (4 female, 6 male) were included in the study, consisting of an adult and 9 children. nine patients had hematological malignancies. five patients were referred for all, three for aml, one for mds and one for griscelli syndrome. efficiency: the clinical response to the αβ+ t cell depleted dli treatment was achieved in 7/10 patients (70%). in these patients, although the increase of chimerism was limited in 2 patients, no recurrence was occurred. one of the two patients who previously responded to the treatment but experience of decreasing chimerism had relapsed after 2 months, and 9 months later. one of these two patients died after relapse. the other was managed by the second transplant. the most important objective of this study was to show that αβ + t cell depleted dli treatment is reliable. none of the patient showed severe gvhd except one patient with mild grade ii gvhd. despite the presence of severe gvhd after hsct in two patients, reactivation for gvhd was not observed after treatment with αβ+ t cell depleted dli. none of the patients had a bone marrow aplasia. as a result, αβ + t cell depleted dli treatment seems to be highly safe, and effective in selected patients. disclosure of conflict of interest: none. hematopoetic stem cell transplantation (hsct) is associated with several potentially lethal complications; for example, relapse of the malignant disease, graft rejection, infectious complications and graft versus host disease (gvhd). higher levels of cd3+ cells in the graft have clearly been associated with increased risk of gvhd, but also superior gvl effect and less infectious complications. to tackle post-transplant complications such as graft failure and relapse, donor lymphocyte infusion (dli) have successfully been used for decades but with an associated risk of gvhd. to decrease the risk of gvhd but still use facilitating cells in the cell product we performed αβ depletion of grafts for use as stem cell booster after allogeneic hsct to treat infections or poor immune reconstitution. in this study, 11 patients were infused post-hsct with αβ t-cell depleted grafts. the indication for infusion of αβ t-cell depleted graft in all patients was poor immune reconstitution with associated infectious complications. for all 11 patients, the original hsct donor was used for the αβ t-cell depleted boost. to characterize the αβ-depleted stem cell grafts, samples were stained for various cellular subsets and analyzed by flow cytometry. we could show a median log depletion of αβ cells of 4.1 and a median yield of γδ t-cells (%) of 61. 4 . the median cd34+ cell dose (×10 6 /kg) was 5.5. all 11 patients were alive 3 months after infusion. after 1 year only one patient succumbed. despite that the majority of patients suffered from agvhd grade 2 or 3 before infusion of αβ t-cell depleted graft none showed increased symptoms afterwards. in more than 70 % of the patients there was a increase in granulocytes, thrombocytes and white blood cells 3 months after infusion. in conclusion, we describe the use of αβ t-cell depleted grafts as stem cell booster in 11 patients suffering infectious complications due to graft failure after hsct with encouraging results. disclosure of conflict of interest: none. delayed engraftment or graft failure still remains a concern in bone marrow transplantation (bmt). graft composition may predict engraftment after infusion. this study aims to determine which quality control parameters used for the characterization of bone marrow grafts are the most predictive in order to minimize the risk of engraftment delay or graft failure. we conducted a multicenter retrospective study in pediatric patients who underwent first allogenic bmt at two centers in barcelona (catalonia, spain) between 2011 and 2015. quantitative variables considered for the study were: total nucleated cells (tnc), mononucleated cells (mnc), cd34 + cells, cd3+ cells and granulocyte-monocyte (gm) colonies enumeration. qualitative variables considered for the study were viability assessed by flow cytometry and clonogenic efficiency of the cd34+ cells (clonegm) which is the ratio between gm colonies and cd34+ cells. 85 patients were included (median age (range) were 7 years old (0) (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) ). the median tnc(range) was 4.26e8/kg (0.51-29.18e8/kg) and 1.1e8/kg (0.48-5.88e8/kg) for mononuclear cells (mnc). on the other hand, the median (range) cd34+ cell dose was 5.03e6/kg (0.85-24.91e6/kg) and t-cell dose (cd3+) was 0.41e8/kg (0.10-44.86e8/kg). the median (range) colonyforming unit granulocyte macrophage (gm/kg) dose was 4.35e5/kg (0.33-48.62e5/kg). the median (range) of cd34+ cell viability, was 95% (63-99%) and the median(range) of the clonogenic potential of cd34+ cells (clonegm) was 10.35% (1.22-76.17%). the median (range) of engraftment was 22 days for neutrophils and 20(11-38) days for platelets. 1 patient was considered as primary graft failure. in the univariate analysis, cd34+ (p = .049) and mnc (p = .045) cell dose predicted a faster neutrophil engraftment and female donor a slower neutrophil and platelet engraftment (p = .012 and p = .040). cell viability also correlated to a better platelet engraftment (p = .030). in the multivariate analysis we observed a trend for a faster neutrophil recovery according cd34+ cells infused. again, female donor was associated with slower engraftment. in order to establish a safety threshold, we did a quartile analysis of cd34 dose and found 3.18e6/kg (quartile 25) discriminates a faster neutrophil engraftment [median 25 days vs 21 days for those with higher cd34+ cells (p = .026)]. in conclusion, we found an association between mnc and cd34+ cell dose and time to engraft, and established a safety threshold of 3.18e6 cd34+/kg. also, bm grafts from female donors were associated with slower engraftment. no other qualitative parameters were predictive of engraftment. disclosure of conflict of interest: none. plasma cell myeloma (pcm) is currently treated with chemotherapy and autologous stem cell transplantation (asct) but relapse rates remain high. adoptive transfer of mature haploidentical natural killer (nk) cells is a promising approach to provide pcm patients with highly immunocompetent effector cells with anti-myeloma function early post transplantation. here we report on the current clinical phase i/ii trial of multiple preemptive infusions of good manufacturing practice (gmp) expanded nk cells to pcm patients (clinicaltrials.gov nct01040026). ten pcm patients were recruited (seven males, three females, median age: 59y). all patients received four cycles of vtd chemotherapy (reaching cr: 4 × , vgpr: 5 × and pr: 1 × ) before high dose therapy with melphalan 200 mg/m 2 and asct. after successful stem cell mobilization and cryopreservation of patients' stem cells after the third vtd cycle, nk cells from haploidentical family donors were purified from unstimulated leukapheresis by t cell depletion and nk cell selection using clinimacs. highly pure nk cells (mean: 4.8 × 10 8 cells) were obtained with a minimal t cell contamination corresponding to a 6.1 log t cell depletion. nk cells were expanded ex vivo for 19 days in gmp-medium containing autologous irradiated feeder and interleukin-2 and -15. nk cell numbers increased 54-fold (range: 38-76-fold). in three nk cell products t cell contents were 11 × 10 5 cells/kg body weight (bw: 10 × above limit of clinical trial) and were successfully reduced by 2°cd3-depletion to 0.3 × 10 5 cells/kg bw. nk cell products were cryopreserved in escalating doses (1.3 × 10 6 , 1.3 × 10 7 and rest as multiple doses of maximal 1.0 × 10 8 cells/ kg bw). the pcm patients received 65-460 × 10 8 expanded nk cells (median: 3.8 × 10 8 cells/kg bw, range: 0.9-5.7 × 10 8 cells/ kg bw) as 3-8 infusions (median. 6 dlis). the nk-dlis were administered between day 2 and 21 after asct and were well tolerated without any acute adverse events. no signs of acute or chronic graft-versus-host disease were observed in any of the patients after a total of 57 nk-dlis. engraftment occurred between days 13-24 (median: 16 days). infused donor nk cells were monitored by short-tandem repeats pcr. donor nk cells were detected in peripheral blood one and 20 h post infusion (% donor nk of enriched blood nk cells: mean: 30%, range: 9-90%, and mean: 17%, range: 0-33%, respectively) indicating significant nk cell survival in recipients in the absence of il-2 support in vivo. clinical responses at last follow-up compared to a retrospective cohort of matched control patients will be presented. these results demonstrate the feasibility of large-scale gmp expansion and safety and immunotherapy with third-party leukemia-specific t cells (leuk-sts) represents an attractive approach for acute leukemia (al) patients lacking a fully matched donor or relapsing after allogeneic hematopoietic cell transplantation (hct). its application however, is limited by the demand for high numbers of antigen presenting cells (apcs), capable to produce clinically relevant numbers of leuk-sts. low volume, non-transplantable cord blood units (cbus) could theoretically serve as an easily accessible source to generate high numbers of dendritic cells (dcs) and subsequently leuk-sts, providing also the advantage of reduced alloreactivity, even in cases of partial matching. our goal was to generate clinically relevant doses of leuk-sts targeting al-related antigens, the wilms tumor protein (wt1) and the preferentially expressed antigen in melanoma (prame), through the exploitation of non-transplantable cbus. to generate dcs, immunomagnetically enriched cd34+ cells from cbus ⩽ 70ml were cultured in g-rex devices in the presence of scf, gm-csf and il-4. dcs matured by toll-like receptor ligand 3 and 7/8 were immunophenotypically characterized by flow cytometry (fcm). secreted cytokines were measured with elisa. matured dcs were activated with a peptide-mix of wt1 and prame and used as apcs to repeatedly stimulate naive t-cells (derived from the cd34fraction of the same cbu). the phenotype and the specificity of generated leuk-sts were determined by fcm and ifn-γ/ elispot, respectively. starting from mean 4.2 × 10 5 ± 1.1 × 10 5 cd34+ cells, from 4 non-usable cbus, we generated 3.3 × 10 9 (range:1.9-5.7 × 10 9 ) myeloid dcs (cd33+/cd11c+:76.8 ± 5.5%) in 35 days (fold change~11.000). the produced cells highly expressed maturation markers (cd11+/cd40+:79 ± 12%; cd11c+/hla-dr+:78 ± 10%) and secreted high levels of th1cytokines (ιl-12:224 ± 185 pg/ml; il-6:1.9 ± 0.1 × 10 5 pg/ml, tnf-α:5268 ± 1316 pg/ml) and low levels of the th2-cytokine, il-10. the average number of cd34-cell-derived leuk-sts after 4 week-culture was 7.5 ± 3.4 × 10 7 (~2 logs above clinical doses). the produced cells were enriched in cd3+ polyclonal cells (80 ± 7%), comprising of cd4+ (28 ± 10%) and predominantly cd8+ cells (52 ± 17%), expressing effector memory (cd45ra − /cd62l − :52.8 ± 5%) and effector memory ra markers (temra: cd45ra+/cd62l − :46 ± 4%), while containing insignificant numbers of cd4+/cd25+cells (1 ± 0.5%). specificity was seen after the second stimulation at the earliest and was increasing after each stimulation [mean spot forming cells (sfc)/2 × 10 5 cells at second, third, fourth stimulation: 106 ± 33; 422 ± 111; 1335 ± 314; respectively]. in particular, produced cells were highly specific for both targeted antigens (prame:1019 ± 275, wt1:316 ± 55), while they expressed low the programmed cell death protein-1 (cd3+/pd-1+:9 ± 4%), implicating absence of cell exhaustion after repeated stimulations. we report a paradigm of ‛circular economy' in science, by the exploitation of non-usable cbus, towards scalable generation of cb-cd34+-cell-derived dcs and cb-cd34-cellderived leuk-sts from the same cbu and establishment of leuk-sts banks. whether similarly produced leuk-sts could significantly advance the treatment of al or leukemic relapse after hct, will be ultimately determined in vivo. disclosure of conflict of interest: none. comparison of two different methods to generate antifungal-specific t-cells under pre-clinical-scale conditions r geyeregger 1 , s tischer 2, 3 invasive infections with aspergillus fumigatus constitute a major cause of morbidity and mortality in immunocompromised patients after haematopoietic stem cell transplantation. although adoptive immunotherapies against viral pathogens are already in phase i/ii trials, clinical-grade methods for the generation of aspergillus-specific t-cells (asp-t-cells) from healthy transplant donors or even related or unrelated thirdparty donors are still under development. in this study, two different strategies interferon-gamma (ifn-g) cytokine capture system (ccs) vs short-term in vitro expansion (ste) were performed from the same healthy volunteers in order to evaluate the most suitable approaches for the in-time generation of clinical applicable asp-t-cells. pbmcs from leukapheresis of healthy donors (n = 6) were first prepared in hannover for the ifn-g-ccs and then sent to vienna to prepare the ste. all donors belong to the allocell registry (www.allocell.com) of hannover medical school and the frequency of asp-t-cells was pretested by high-throughput ifn-g elispot assay. for the ifn-g-ccs, 1 × 10 7 cells were stimulated for 16 h with gmp-conform aspergillus lysate followed by magnetic selection of ifn-g-producing t cells. cells were characterized for phenotype and function by multicolour flow cytometry. for the ste, 20 × 10 7 cells were cultured in g-rex devices and stimulated for 12 days with either the aspergillus lysate alone or with pooled overlapping pepmixes (catb, crf1, f22, gel1, pmp20, shmt and sot) and il-15. to further characterize the final cell products, multicolour flow cytometry, ifn-g elispot and ifn-g/granzyme b flurospot analyses were performed. ifn-g-ccs: frequency of ifn-g positive asp-t-cells pre-magnetic enrichment ranged between 0.07 and 0.16%. recently we defined t-cell donors as eligible if ⩾ 0.03% specific ifn-g+ t cells are detectable. the purity of asp-t-cells among cd3+ cells, obtained from three donors after magnetic selection was in mean 64% ± 3 (range: 58-69%). the absolute number of selected ifn-g+ cd3+ t-cells was 706 ± 194. this could be approximately multiplied by a factor of 100, if 41 × 10 9 pbmcs are used for the generation of clinically applicable t cells using the ccs and the prodigy device. ste: after 12 days, asp-t-cells (n = 3) showed highly specific activity against the lysate (in mean 1339 ± 79 spot forming colonies (sfc)/105 cells) and pooled pepmixes (in mean 892 ± 276 sfc/105 cells). in both methods (lysate vs pooled pepmixes), predominantly cd4+ t-cells were expanded (84% ± 2.3 vs 82% ± 5.3 of cd3+) compared to cd8+ t-cells (12.6% ± 2,9 vs 14.7% ± 5.3). interestingly, whereas after ste, cd4+ t-cells include mainly central memory t-cells (mean 40%; cd62l+cd45ra − ), cd8+ t-cells include mainly effector memory t-cells (27%; cd62l − cd45ra − ). generated t cells were highly functional and cytotoxic as determined by the secretion of effector molecules granzyme b and ifn-g. based on the purity of up to 69% after the ifn-g-ccs and the high number of sfc received after ste with lysate and pepmixes, both methods seem to be suitable for clinicalscale productions. for patients who are in need for high asp-tcell numbers the application of first in-time ccs-purified asp-t-cells followed by the administration of ste cells might be a promising way to boost antigen-specific t-cell response. disclosure of conflict of interest: none. complete computerization of cell therapy product files (‛zero paper') in the qap 10 software o christéle 1 , r catherine 2 , r aline 3 , k mathias 4 , m lavinia 3 , d vincent 5 , m jean-pierre 6 and l marie-noelle 7, 8 1 hematologie clinique et thérapie cellulaire-chu amiens picardie, 2 simedia-ver, 3 hématologie clinique et thérapie cellulaire-chu amiens picardie, 4 simédia-ver, 5 direction système informatique-chu amiens picardie, 6 hématologie clinique et thérapie cellulaire, 7 lacassagne and 8 the computerized management of cell therapy products (ctp) is an obligation for processing laboratories to meet regulatory requirements. the software used is often independent of institutional systems in view of the specificity of cellular therapies and do not always allow the implementation of the ‛zero paper' policies that are being put in place. we report here our experience with the qap10 software (quality assurance partner) developed by the company simédia (www. qap10.com) in open source (mit license) allowing the management of fully computerized ctp files. the qap10 software has been developed to ensure the traceability of ctp for both preparation and quality control by combining the product preparation environment (personnel, premises, reagents, consumables, equipment). initially, with the help of the company simédia, we parameterized the software in accordance with our procedures for the preparation and quality control of ctp. we built a file that we printed out for archiving on paper. it soon seemed necessary to reverse this mode of operation to add to the software the documents papers to obtain a file completely computerized and to avoid paper archiving. the close collaboration between the cell therapy laboratory staff, the software referent within the information system department of the amiens hospital and the company simédia enabled: set up a document backup server sufficiently proportionate in memory. have simedia carry out the necessary developments so that all documents can be integrated into the software, set up a coherent working circuit, organize the registration of documents, put in place a rigorous verification of the mandatory elements of the file. the reflection on the computer file made it possible to evolve the software to widen its use to all documents of management of the laboratory: maintenance of equipment, control of premises, housekeeping, staff training, quality control of automatons, reagents and consumables, process, reception, distribution. rigorous formalization was mandatory to ensure that the record was organized in a uniform manner. an intermediate paper record is still necessary for a period of about 1 month: from the programming of the graft to the final validity of the injected product. this folder consists only of transient elements that cannot be integrated into the qap 10 software immediately. the transition from the paper file to a computer file took place in several stages, calling into question our functioning. the difficulties of this implementation are of several natures: the heterogeneity of the documents components a cell therapy product file, the impossibility of benefiting from an interface between all computer software used on the hospital, the psychological barrier prompting us to keep a paper copy, work habits, the guarantee of computer backup quality as well as its verification. but the complete computerization of the ctp file has the following advantages: easy and secure accessibility of information, resolution problems archiving paper files, a single backup media folder. disclosure of conflict of interest: none. conditioned media from allogenic mesenchymal stem cell culture (msc-cm) enhances wound healing in an allogenic 3d skin model moyasasr al-shaibani, x wang 1 , p lovat, a tulah and a dickinson 1 newcastle university migration of the epidermal layer towards the wound centre is an important step in the healing process. full thickness in vitro skin models can be used to investigate epidermal migration towards an injury site. since wound healing therapies often require allogenic transplantation of primary keratinocytes, an allogenic 3d skin model was developed to investigate epidermal migration. the effect of mesenchymal stem cell conditioned media (msc-cm) was assessed for wound healing using this in vitro human 3d skin model. human mscs were derived from human hip joints, and characterised using standard protocols. at 80% confluence, msc secretions were collected in serum free medium and referred to as msc-cm which were then analysed for protein content using elisa. fully humanised allogenic 3d skin models were developed (n = 3) and a 3 mm punch was induced into each model followed by daily treatment with msc-cm to investigate the migration of the epidermal layer towards the punch centre over the dermal layer at different time points (1 week, 2 weeks, and 4 weeks). intact and wounded models were characterised structurally by haematoxylin/eosin (h&e) staining and immunofluorescence (if) was used to validate the dermal and epidermal biomarkers such as collagen 3 (col3), cytokeratin 14 (k14), keratin 10 (k10), loricrin and involucrin. mscs were characterised as stipulated by the international society for cell therapy, that is, fibroblast like cells with the ability to differentiate into tri-lineages (adipocyte, chondroblast and osteoblast). phenotypically, over 95% of the cells were able to express phenotypic markers for variant stem cells such as cd73, cd90 and cd105. over 95% of the cells were negative for the expression of cd14, cd19, cd34, cd45 and hla-dr (p = 0.025). msc-cm contained different concentrations of a variety of growth factors such as keratinocyte growth factor (kgf), hepatocyte growth factor (hgf), platelet-derived growth factor (pdgf), stromal-derived factor-1 (sdf-1) and macrophage stimulating protein-1 (msp-1). h&e staining showed that the models had distinct dermal and epidermal layers similar to that of real skin. additionally, if showed that the models expressed dermal and epidermal biomarkers, for example, col3, k14, k10, loricrin and involucrin. after treatment with msc-cm, the epidermal multilayers of the punched models started to migrate towards the punch centre and covered the whole punched area after 4 weeks of treatment with recovered expression of the epidermal biomarkers, for example, k14, k10, loricrin and involucrin. a fully humanised allogenic 3d skin model is a useful tool to mimic the in vivo environment and evaluate the wound healing process. it could also be used as a screening method to test candidate wound healing drugs. allogenic keratinocytes could be used as a cellular sheet to cover the wound area with the ability to migrate towards the wound centre and promote wound healing. a possible explanation for promoting epidermal migration at the injury site is that msc-cm contains cytokines which accelerate cell migration such as kfg, sdf-1 and msp-1, in addition to other cytokines which promote both migration and proliferation of epidermal cells, for example, hgf and pdgf. disclosure of conflict of interest: none. before each freezing and after each thawing, a quality control is performed including a minima: (i) cd34+ quantification; (ii) estimation of the percentage of hsc cd34+ viability, via 7aminoactinomycin-d (7-aad) staining and (iii) evaluation of hsc functional ability to form colony ‛cfu-gm' (colony forming unit-granulocyte macrophage). apoptosis, or programmed cell death, involves complex pathways in part the path fas-fas ligand (fasl), mitochondrial components and caspase enzymes. the involvement of apoptosis dependent on caspases activation pathway in hsc cd34+ after thawing remains unknown. here, we assess the extent of apoptosis caspase-dependent before and after cryoconservation of hsc cd34+, using a fluorescent labeled inhibitor of caspases ‛flica. ' we tested the induction of apoptosis caspasedependent, before and after hsc cd34+ cryoconservation from patients with different hematological malignances: multiple myeloma (n = 21), lymphoma (n = 19). caspases pathway activation status was evaluated by flow cytometry, using a fluorescent labelled inhibitor of caspases ‛flica' staining test, in hsc cd34+, lymphocytes cd3+, monocytes cd14+ and natural killer cells cd56+. in order to assess cell viability, cells were stained in parallel with 7-aad. we determined positive cells %, that is, showing caspase activation in viable cells (flica+ cells), before and after cryoconservation. caspase pathway activation level was then correlated with hsc functional ability to form colony ‛cfu-gm,' and day's number of clinical aplasia. in our cohort, we showed a significant caspases pathway activation, with 18.9% cd34+ flica+ cells after thawing, compared with the 2.4% described in fresh cd34+ cells (p o0.0001). moreover, caspases pathway was significantly activated in thawing cd3+, cd56+ and cd14 + cells: flica+ cells % in thawing cells were, respectively, 16.8%, 31.1% and 6.2% vs 3%, 9.7% and o1% in fresh cells. we also report a significant increase of apoptosis caspasedependent in lymphoma patients (22.6% of cd34+ flica+) in comparison to myeloma patients studied (18.6% of cd34+ flica+) (po 0.0001). in contrast, no correlation has been established between observed caspases pathway activation and hsc cd34+ capacity to form cfu-gm, or still day's number of clinical aplasia. our results show substantial cell death, induced by the increase in caspases pathway activation, secondary to the thawing process, and across all study cell types. this advance of apoptosis caspase-dependent may affect the immune response quality during recipient aplasia, without detecting a clinical impact. moreover, caspases pathway activation through cd3+ and cd56+ subpopulations could modify the therapeutic result of donor lymphocytes infusion dli, though yet untested. thawing process in autologous graft induces apoptosis caspase-dependent in all apheresis product cells, particularly in hsc cd34+, without clinical impact in graft fate. disclosure of conflict of interest: none. donor-derived nk cell infusion combined with hla halpoidentcial blood stem cell transplantation to decrease leukemia relapse for high risk acute myeloid leukemia patients b wu, y huang, j xu, y he, jxm zhang*, z wu* hematology department, zhujiang hospital of southern medical university, guangzhou, china 510280 *shenzheng hank biologoical engineering co.ltd. hla halpoidentcial blood stem cell transplantation have solved the donor deficiency for patient who need to treat by transplantation. the high relapse of leukemia especially for high risk patient post transplantation affect the outcome of haploidentical stem cell transplantation. natural killer (nk) cells are part of the innate immune system and play a scavenger role to detect targets marked by ‛missing self' induced by viral infection or malignant transformation. infusion nk cells into receipt prior to stem cell transplantation could decrease the gvhd in mouse bone marrow transplantation model. in an effort to decrease the leukemia relapse and gvhd after halpoidentical stem cell transplantation for high risk acute myeloid leukemia patients, we evaluated the addition of donor-derived nk killer cells before halpoidentical stem cell transplantation in high risk acute myeloid leukemia patient. here we report interim results for five patients enrolled last year. five high risk acute patients received halpoidentcial stem cell transplantation combined with donor-derived nk cells infusion. all patients received an fbca conditioning regimen, which consisted offludarabine (25 mg/m 2 /day, intravenous) on days − 9 to − 5, busulfan (3.2 mg/kg/day, intravenous) on days − 8 to − 5, cyclophosphamide(60 mg/kg/day, intravenous) on days − 3 to − 2 and rabbit antilymphocyte globulin (atg 2.5 mg/kg/day, intravenous) on days − 5 to − 1. donor-derived nk cells were infused into patient prior to stem cell transplantation. gvhd prophylaxis was a combination of cyclosporine a (csa) and short term methotrexate. five high risk patients (2 patients with aml m5 cr2, 1 patient with aml m5 nr, 1 patient with aml m0 cr2 and 1 patient with aml m2 cr2) enrolled from jan 2015 to nov. 2015; the donors are parents and sibling. hla were mismatched between donor and patients. median cd34+ dose infused was 5.06/kg (range: 2.3-106/kg) and the nk cell dose infused was 1 × 10 8 /kg (0.8-1.2 × 10 8 /kg). all five patients got hematology recovery and achieved hematology cr. only one patient occurred grade ii agvhd post transplantation and controlled by methylprednisolone. at a median time of 12 months (range: 9-16 months) post peripheral blood stem cell transplantation, the incidence of acute gvhd grade ii is 20% (1/5) . no chronic gvhd observed. four patients are still cr and survival with event free survival with median 1 year follow up. one patient with aml m5 who had not achieved remission before transplant relapsed after 6 months and got cr with second nk infusion and still survival. nk infusion prior to transplantation was found to be safe and feasible. there was no increase acute gvhd or chronic gvhd risk. there was a trend towards increased 1-year survival for high risk leukemia patient. the potential benefit on overall survival remains to be further evaluated with additional patient enrollment and longer follow up. however, given the favorable safety profile of nk cells, future strategies to enhance efficacy such as repeat dosing or modification of nk cells are worth potential exploration. disclosure of conflict of interest: none. donor lymphocyte infusion (dli) is a therapeutic option in the treatment or prevention of relapse after allogeneic stem cell transplantation (allohsct).of note, the risk of graft-versus-host disease (gvhd) associated with the graft-versus-tumor (gvt) effect may be influenced by the level of hla disparity between donor and recipient. data on use of dli after unmanipulated haploidentical hsct (haplohsct) with post-transplant cyclophosphamide (pt-cy) are still currently limited. we report 7 patients (pts) receiving dli between 2014 and 2016 for prevention or treatment of relapse after haplohsct. seven pts were given 11 haplodli doses, as treatment for relapsed disease (n = 4) or as preventive therapy of relapse for high risk disease (n = 3). four pts had acute myeloid leukemia (aml), 1 had acute lymphoblastic leukemia and 2 lymphomas -1 hodgkin (hl) and 1 non-hodgkin dlbcl. 1 pt had intermediate risk disease features, 1 adverse risk and 5 pts had refractory disease at time of haplohsct. 4 pts had a previous hsct (2 allogeneic and 2 autologous). 6 of the 7 pts received a ric regimen and the source of stem cells was peripheral blood s153 (n = 5) and bone marrow (n = 2). gvhd prophylaxis was cyclosporine and mycophenolate mofetil (mmf), atg and pt-cy. median follow-up after haplohsct was 27 (range: months. median time to neutrophil and platelet (450g/l) recovery were 16 and 24 days, respectively. after haplohsct, 3 pts developed acute gvhd (agvhd) of grade i (n = 1) or ii (n = 2), at a median of 26 days after haplohsct. the median time from haplohsct to first dli was 204 days (range: 71-624). all pts had full donor chimerism at time of dli. before dli 3 pts relapsed at a median time of 149 days (range:86-177), of whom 2 pts had aml and received salvage chemotherapy and 1 pt with hl being treated by dli alone. of the 3 relapsed pts,1 showed progressive disease after first dli dose and 2 achieved a sustained cr (with duration of cr of 6 and 9 months at last follow-up). the remaining 4 pts were given dli in cr, in 1 case (of aml) associated with azacytidine. 5 pts received 1 dli dose and 2 pts were given 3 dli injections with escalating doses. the first dose of dli was 1 × 10 6 cd3/kg in 4 pts, 5 × 10 5 in 1 pt and 1 × 10 5 in 2 pts. the 2 pts who received 3 dli doses (lymphomas) were given: (1) 5 × 10 5 -1 × 10 6 -5 × 10 6 ; (2) 1 × 10 5 for 2 doses followed by 1 dose of 5 × 10 5 . four pts developed chronic gvhd (cgvhd, 57%) in a median time of 23 days (range:11-42) after dli (3 of them had presented previously agvhd grade i-ii). cgvhd was limited in 1 case, moderate in 1 pt and severe in 2 pts. 3 of these pts presented features of an overlap syndrome (acute/chronic gvhd) with signs of agvhd de grade i,ii and iii in 1 pt each. involved organs were skin/mucosal (n = 4), liver (n = 3), gastrointestinal tract (n = 2), lung (n = 1) and joints (n = 1). all patients experiencing gvhd after dli were treated by systemic corticotherapy, extracorporeal photopheresis and cyclosporine or weekly low dose methotrexate. median follow-up after first dli was 10 months (range: . none of the 4 pts receiving prophylactic dli relapsed during the follow-up period. 2 pts died,1 of relapse and 1 of severe cgvhd. 5 pts were in cr at last follow-up,3 with no signs of gvhd and 2 with limited cgvhd. despite the limited cohort, dli after haplohsct appears to be a therapeutic option in high risk pts allowing enhancement of gvt in the setting of haplohsct with post-cy infusion. disclosure of conflict of interest: none. previously published p075 early and sequential ctla4ig primed donor lymphocyte infusions (dli) following post-transplantation cyclophosphamide (ptcy)-based haploidentical pbsc transplantation for advanced hematological malignancies promote proliferation of mature natural killer (nk) cells with cytotoxic potential and markedly reduces relapse-risk without increase in gvhd sr jaiswal, s zaman, p bhakuni, s bansal, s deb, s bhargava and s chakrbarti 1 we have earlier shown that cd56 enriched cell infusion following ptcy resulted in rapid proliferation of mature nk cells with attenuation of gvhd and early use of prophylactic g-csf mobilized dli resulted in improved disease-free survival. ctla4ig has been shown to be effective in attenuating t cell activation and induce transplantation tolerance in preclinical models. it has recently been employed to induce transplantation tolerance and reduce early alloreactivity in patients with nonmalignant disorders undergoing ptcy-based haploidentical hsct. nk cells on the other hand are resistant to ctla4ig and in fact might demonstrate better anti-tumour effect in presence of ctla4ig as cd86 is a putative activation receptor. to explore this phenomenon, we employed sequential ctla4ig primed dli following ptcy-based haploidentical hsct in patients with relapsed/refractory hematological malignancies.15 patients (12-60 years; aml-6, all-5, nhl-4) received abatacept (ctla4ig) as a part of gvhd prophylaxis at 10 mg/kg on day − 1 followed by pbsc and sequentially on days +6, +20 and +35 followed 12 h later by dli of 5 × 10 6 cd3 cells/kg containing 0.03-1 × 10 6 /kg cd56+ cells. ptcy was administered on days +3 and +4 with cyclosporine from day +5 to day +60 and subsequent rapid tapering. the immune reconstitution of the study group (ctla4ig-dli) was compared with the cohort of patients with both malignant and nonmalignant diseases who received abatacept but not dli (n = 12; ctla4ig group) and those receiving cd56 enriched donor cell infusion on day +7 (n = 10; nki group). results: there were no acute infusion related toxicities. all patients engrafted at a median of 15 days (12-20 days). the incidences of acute and chronic gvhd (all mild) were 20% and 25%, respectively. three patients reactivated cmv and there was only one non-relapse mortality (6.9%). only 4 patients relapsed (27.8%) with a disease-free survival of 72.6% at 1 year. these cells had greater expression of cd107a compared to normal healthy donors. the recovery of cd56+, cd56+16+ and cd56 +16 − cells were similar in the ctla4ig-dli and nki groups at days 28, 60 and 90 post-transplant and this was significantly higher than the ctla4ig group ( figure 1 ). in contrast to ctla4ig group, nk cells recovered at day +28 with predominantly cd56dim cd16+ phenotype with significant population of cells expressing kir+nkg2a phenotype in both ctla4ig and nki groups with higher expression of cd107a. interestingly, the 4 patients who relapsed had attenuated recovery of cd56+16+ cells at 28 and 60 days(21/μl and 15 cells/μl) without cd107a expression, in contrast to the rapid and sustained recovery of this population of nk cells in those not experiencing relapse (cd56+16+ cells 181/μl and 103/μl). however, the recovery of tregs was prompt and sustained in the comparator groups, which remained low in the ctla4ig-dli group until day +90. there were no differences in the recovery of other t cell subsets between the three groups. the study demonstrates the unique ability of ctla4ig to augment nk cell proliferation, maturation and cytotoxicity and reduce relapse with attenuation of t cell activation and gvhd in the context of the early use of ctla4ig primed dli following ptcy-based haploidentical hsct without ex vivo selection or expansion. we hope this novel strategy might offer less expensive and yet a viable alternative in the field of nk cell therapy. [p075] disclosure of conflict of interest: none. enhanced cytotoxicity of γδ-cytokine induced killer cells against hematologic malignancies n bloom, s eldror, s caspi, s teihuman,h vernitsky, e jacoby, b bielorai and a toren cik cells are ex vivo expanded by scheduled addition of anti-cd3 mabs and a cytokine cocktail that contains ifn-γ, il-2 or il-15. cells represent an in vitro generated heterogeneous population consisting of different effector cells-cd3poscd56pos, cd3negcd56pos and cd3poscd56neg-t cells that mainly (495%) express α/β t-cell receptor (tcr) s154 and to a lesser extent (o 5%), tcr γδ phenotype. these nklike t cells product show a dual functional activity, retaining their original t cell specificity and nk cytotoxic capacity via marked up regulation of the nk cell receptor, nkg2d. pre and clinical studies showed that the optimal cytotoxic effect of cik cells against different malignancies (target cells) is achieved at 40:1 e:t ratio, which means high numbers of αβ t-cells that might increase the risk of gvhd. here we produced ciks from αβ tcr depleted cellular products (defined as γδcik) and tested their phenotype expression and in vitro cytotoxic activity against hematological malignancies. fresh apheresis products were processed using the clinimacs depletion reagent, according to manufacturer instructions. target product was cultured with rpmi1640 supplemented with 10% fcs and ex vivo expanded by scheduled addition of cytokine cocktail that contains ifn-γ (1000 iu/ml), anti-cd3 mabs (50 ng/ml) and 500 iu/ml il-2. the cells were cultured for 14 days. cytotoxic activity of the γδcik was evaluated against various target hematological malignant cell lines (k562, reh, jurkat, and u937). after 14 days, the αβ depleted cik cultures resulted in 97.5% γδ t-cells (41 folds expansion) compared to 1.0% of γδ t-cells immediately after depletion, and compared to only 1.6% in non-selected cik cells. the percentage of αβ t cells in γδcik cell cultures started from 0.002% (immediately after depletion) to 0.5% compared to 95.1% αβ t cells were found in non-selected cik cells cultures. γδcik cells produced robust cytotoxic activity at a 10:1 e:t ratio against reh cells (22.6 ± 5.3%), jurkat cells (51 ± 7.9%); u937 (62.5 ± 8.5%) and k562 (43.4 ± 2.0%), compared to nonmanipulated cik cell activity against the same targets (5 ± 1.0%; 8.3 ± 1.4%; 12.4 ± 6.1%; 7.3 ± 2.9%, respectively). we found higher degranulation capacity of γδcik cells compared to non-selected cik cells against reh (45.3 ± 16.1% vs 17.6 ± 3.8%), jurkat (42.3 ± 18.4% vs 6.8 ± 3.5%), u937 (37.6 ± 15.5% vs 17.1 ± 3.1%) and k562 (29.2 ± 16. centre de thérapie cellulaire, institut paoli-calmettes; 2 unité de transplantation et de thérapie cellulaire, institut paoli-calmettes; 3 centre d'immunologie de marseille-luminy and 4 laboratoire d'immunomonitoring, institut paoli-calmettes during the past 15 years, the major improvements in the field of allogeneic hematopoietic stem cell transplantation (hsct) (reduced intensity conditioning regimen, high level hla typing, alternative donors, gvhd prophylaxis…) significantly extended the feasibility of this procedure. in contrast, disease recurrence after hsct remains a main issue. thus, many post-hsct prophylactic interventions are under investigation. unmanipulated donor lymphocyte infusion (dli) remains one of the most frequently used post-hsct treatment, but its potential benefit in increasing gvl effect may be counterbalanced by the induction of gvhd. in this setting, the use of adoptive transfer of ex vivo enriched and activated nk cell infusions from the same donor (dli-nk) may induce gvl effect without causing gvhd. we therefore report on a single-center phase 1 clinical trial (nct01853358) evaluating the safety of ex vivo activated allogeneic nk cells infused between days 60 and 90 after hsct. the aim was to determine the maximum tolerated dose (mtd) of ex vivo highly purified and activated dli-nk after matched related donor hsct. the schedule plan a first phase of 3+3 dose escalation method using 3 dose levels (1.10e6/kg, 5.10e6/kg and 45.10e6/kg). grade 3-4 secondary adverse events according to nctci classification and severe gvhd occurring within 30 days after dli-nk were considered as dose-limiting toxicities (dlt). a second step allowed enrolling patients at the mtd. over a period of 3.5 years, 14 patients with various hematological malignancies (aml, all, hl, nhl, mds) were infused with activated nk cells at a median time of 91 days (range: 61-106) post-hsct. apheresis products were collected from the hsc donor, cd3-depleted and cd56-selected by immunomagnetic separation using clinimacs. selected nk cells were cultured for 7 days in medium supplemented with 10% fetal calf serum in the presence of 1000 u/ml of il-2 in air-permeable cell culture bags. after immunomagnetic separation, cd56enriched products had a median cd56+ cell purity of 94% (range: 77-100) and viability of 96% (93-99). after il-2 activation, the median cd56+ cell dose was 4.8 × 10e6/kg (1.2-21.4) , with a viability of 81% (71-94) and a residual cd3 + cell content of 0.4 × 10 4 /kg (0-1.5 × 10 4 /kg). all release criteria to be met were fulfilled for the 14 preparations infused : viability 490%, negative microbiological testing, cd56 + cell count ⩾ 1 × 10 6 /kg, and cd3 + cell content o5 × 10 4 /kg. standardized quality controls were employed at all steps of the manufacturing process, adding a level of consistency to the product testing before release. activated-nk cells were well tolerated in all 14 patients, with no occurrence of dlt. thus, mtd was not reached. two patients presented with a moderate chronic gvhd, both of them during cyclosporine a dose reduction. relapse occurred in 2 patients with aml. one patient died from idiopathic pneumoniae, without evidence of relapse, gvhd or infectious disease. with a median follow up of 30 months (1-41), 2 year os was 83% ( figure 1 ). therefore, infusion of highly purified, activated-nk cells of donor origin as a substitute to standard dli does not induce gvhd nor other side effects after hsct: the demonstration that modulation of nk cell activity can achieve disease control after hsct deserves to be investigated in larger trials. [p077] disclosure of conflict of interest: none. feasibility, safety, rapid production and efficacy of institution-produced cd19 car staff were trained on site for collection, processing and cryopreservation by regional experts. a total of 101 units were collected and processed as part of the initial validation of the project. ucb units were processed on either axp or sepax systems, and all cryopreserved in bioarchive (an automated, robotic cryopreservation system that can archive up to 3623 units). the characteristics of which as well as the post processing data are depicted in table 1 . [p079] we shared a successful story of establishing the first public cord blood bank in jordan. the first 101 units collected showed excellent sterility, viability, collection volume and total nucleated cells. a very good recovery of both nucleated and cd34+ cells were obtained using axp and sepax cell separation systems. the process of validation of equipments and methodology is complete. we anticipate moving to permeant facility of the cord blood bank in the new expansion in early 2017. we look forward for steady progress in ucb recruitments, hla typing, cryopreservation and adherence to netcord-fact standards as well as participation in international registries. functionally active ifn-gamma secreting cmv pp65 specific t cell therapy as an alternative for clinically urgent cmv related diseases n kim, y-s nam 1 , k-i im 1 , j-y lim, y-w jeon 1 , y song and s-g cho 1 the catholic university of korea, seoul cytomegalovirus (cmv) related diseases are a serious cause of morbidity and mortality following hematopoietic stem cell transplantation (hsct). it has been reported over the last two decades that cmv-specific cytotoxic lymphocytes (cmv-ctls) can provide long-term cmv-specific immunity without major side effects as an alternative to antiviral drugs. however, its application has been limited by prolonged manufacturing process of cell therapy. in this study, we apply the ifn-γ cytokine capture system (ccs) using the fully automated clinimacs prodigy device to rapidly produce cmv-ctls that may be applicable in clinically urgent cmv-related diseases. five validation runs were performed using apheresis samples from randomly selected cmv-seropositive healthy blood donors. then, clinimacs prodigy automatically performed successive processes including antigen stimulation, anti-ifn-γ labelling, magnetic enrichment, and elution which took~13 h. the original apheresis samples consisted of 0.3% ifn-γ secreting cd3+ t cells in response to cmv pp65 antigen (cd3+ifn-γ+ cells) which were mainly cd45ra+cd62l+ naive t cells. following ifn-γ enrichment, the target fraction contained 51.3% cd3+ifn-γ+ cells with reduction in naive t cells and the selection of cd45ra − cd62l − and cd45ra +cd62l − memory t cells. furthermore, extended culture of these isolated cells revealed functional activity including efficient proliferation, sustained antigen-specific ifn-γ secretion and cytotoxicity effect against pp65 pulsed target cells. therefore, we suggest ifn-γ ccs by clinimacs prodigy as a simple and robust approach to produce cmv-ctls, which may be highly feasible and applicable in clinically urgent cmvrelated diseases. disclosure of conflict of interest: none. in vitro generation of tumor antigen-specific t cells from patient and healthy donor stem cells s bonte 1 , s snauwaert 1 , g goetgeluk 2 , b vandekerckhove 2 and t kerre 1 1 hematology, ghent university hospital, ghent, belgium and 2 department of clinical chemistry, microbiology and immunology, ghent university, ghent, belgium acute myeloid leukemia remains a therapeutical challenge, as many patients relapse after chemotherapy. allogeneic stem cell transplantation is in most of these patients the only option for cure, but carries a high risk of morbidity and mortality and a suitable donor may be lacking. recently, advances are being made in the field of t cell immunotherapy. the classical protocol, in which peripheral blood lymphocytes (pbl) are transduced with a tumor antigen-specific t cell receptor (tcr), can generate t cells with low and possibly hazardous specificities (due to mispairing of the endogenous and introduced tcr α and β chains). therefore, we have developed a novel protocol in which we generate tumor antigen-specific t cells from cd34+ hematopoietic stem cells. we have already succeeded in generating large numbers of tumor-specific, naive and resting t cells that only carry the introduced tcr, starting from postnatal thymus and cord blood cd34+ cells. now we are optimizing this protocol for clinically more relevant samples, such as mobilized peripheral blood from healthy stem cell donors and from patients in remission after chemotherapy and/ or other treatments, and leukapheresis samples from patients at diagnosis. in our protocol, cd34+ cells were isolated from hla-a2+ fresh patient and healthy donor samples and cultured on op9-dl1 in the presence of scf, flt3l and il-7, until t cell commitment. subsequently, the cells were transduced with a tumor antigen-specific tcr and again co-cultured until cd4+ cd8+ double positive cells were abundantly present. at that point, agonist peptide was added, which induces maturation. finally, cells were polyclonally expanded on feeder cells. for hla-a2 negative samples, cd4+ cd8+ double positive cells were co-cultured with a cell line (t2 pulsed with the agonist peptide or a cell line with endogenous expression of the agonist peptide) which can present the agonist peptide to the maturing t cells. using the above protocol, we were able to generate tumor antigen-specific t cells from 3 out of 3 healthy donor samples, 1/1 sample from a patient in remission and 2/4 samples from patients at diagnosis, who were all hla-a2+. for most samples, multiple rounds of agonist peptide stimulation were necessary to obtain further maturation. in contrast, generation of mature t cells from cd4+ cd8+ double positive cells in postnatal thymus or cord blood co-cultures, requires only 1 round of agonist peptide stimulation. for the hla-a2 negative samples, we were able to generate an adequate cd4+ cd8+ double positive population from 1/1 healthy donor sample, 3/3 samples from patients in remission and 0/1 sample from a patient at diagnosis. agonist selection using a cell line seems inefficient as cd27 is not upregulated and cells did not mature to cd4+ or cd8+ single positive mature t cells. we are currently co-culturing more samples using our protocol. furthermore, we are investigating the effect of freezing and thawing on the in vitro t cell generation process (cell numbers and efficiency). finally, we are also working on optimizing the protocol for generation of tumor antigen-specific t cells from hla-a2 negative patient and healthy donor samples. disclosure of conflict of interest: none. increase of polyspecific immune responses against leukemia-associated-antigens (laa) and reduction of regulatory cytotoxic t-cell (ctl) responses against malignant cells play a major role in maintaining remission and prolonging overall survival in patients with hematologic malignancies after allogeneic stem cell transplantation (allo-sct) and/or donor lymphocyte infusions (dli). graft versus leukemia (gvl) effects after allogeneic stem cell transplantation and/or dli are considered to be t cell-mediated. many groups described specific t-cell responses against several leukemia associated antigens (laa) in different hematological malignancies. however, t cell responses after allo-sct and dli are not well characterized. in this study, we analyzed laa-specific t cell responses after allo-sct and dli. to this end, we assessed the frequency and diversity of laa-specific cd8+ t cells using elispot analysis and tetramer assays in 12 patients (5 patients (pts) with acute myeloid leukemia, 2 pts with chronic myeloid leukemia, 3 pts with multiple myeloma and 2 pts with chronic lymphatic leukemia) before and after dli. epitopes derived from prame, npm1mut, rhamm, wt-1 and other laa were tested. moreover, the frequency of regulatory t (treg) cells was measured and the course of cytokine profiles before and after dli was analyzed. these immunological findings were correlated to the clinical course in the respective patients. in elispot and tetramer assays, an increase in frequency and diversity of laaspecific t cells was observed in all patients. importantly, there was a significant increase from a median of 1 to 4 laa-derived t cell epitopes (p = 0.03) in clinical responders (r) when compared to non-responders (nr). these positive results in r vs nr where s157 confirmed by tetramer-based flow cytometry assays, where an increase in frequency from 0.5 to 2.3% in the r group of laaspecific t cell/all cd8+ t cells was observed. interestingly, the frequency of tregs in clinical responders decreased significantly from a median 72.9 % to 54.6 % (p = 0.008) while the frequency of tregs kept stable over time in non-responding patients. t cell subset analysis did not reveal significant differences before vs after dli administration. in cytokine assays using elisa for the detection of more than 10 cytokines before and after dli we found a shift towards proinflammatory and t cell stimulating cytokines. taken immunologic surveillance of leukemia is employed for the prevention and treatment of relapse post allohsct. to augment this effect donor lymphocytes are infused (dli) in patients at risk. this procedure is associated with a high risk of agvhd and we believe that this route of administration may not make the direct contact between infused cells and blasts the optimal one. to address these issues, we started delivering donor lymphocytes directly to the bone marrow cavity (ib-dli) in patients post allohsct at relapse. three with aml and one with cll, all relapsed post allohsct: 3 allosib: 50-year-old female aml patient (relapsed 2 years post hsct), 22-year-old aml male 7q31 del (relapsed in 3 years, traumatic brain injury), 25-year-old male aml flt3 itd+ received mud hsct (relapsed 9 months) and 64-year-old cll male, tp53 del, ebv reactivation (progressed 7 years). two patients (26% and 12% blasts in the marrow) received ib-dli up-front and two others due to higher proportions of leukemic cells received either flag (aml case) or anti-cd20 moab (cll case) followed by ib-dli. tcr clonotyping revealed in all 4 patients the presence of the prevailing oligoclonal response on the polyclonal background (characteristic for each individual) which was identified in the marrow and in the blood. however, in two out of 4 patients a distinct oligoclonal peak was seen at first in the marrow and then in the blood. microarray analysis of the transcriptome in the marrows of patients who received three ib-dli courses revealed in all patients preferential use of genes associated with lymphocyte or lymphocyte activation pathways. the patients who responded favorably (cr or pr) clustered with the transcriptomes of normal individuals, but those who failed to respond clustered separately. ib-dli was safe and not associated with gvhd. selective accumulation of cd8+cd279+ as well as the presence of a distinct oligoclonal peak in the marrow suggest that tcrbeta clonotypes may be private to leukemia cells recognition. the response may result in cr or pr and the patients were in a good physical shape during the treatment, which makes it possible to deliver the salvage chemotherapy if required. broad spectrum antibiotics were started. after the orthopedic consultation, the fourth finger was amputated and amputation from the left ankle was recommended. a stem cell transplantation option was offered to patients and their relatives as one of the therapeutic approaches. upon acceptance by patient, 10 μgr/kg of colony stimulating agent was started to patient. when the stem cell was 20/μl, the stem cells were collected. the obtained stem cell product was injected intra-lesionally (picture b). granulation tissue began to develop from the second week in the foot floor of the patient. after from 8th week, the necrotic tissue was disappeared and the granulation tissue was appeared. at 24 weeks, 50% of the lesion healed. at 48th week, there was normal tissue instead of necrotic tissue on plantar surface at left leg (picture c). this case report suggests that diabetic foot/ulcer can be healed with intralesional application of stem cells in patients with diabetes mellitus. [p085] disclosure of conflict of interest: none. and third (n = 5) cell infusions were cryopreserved. cells were infused following conventional chemotherapy (ia, mec, hdac) in 15 cases (44%), chemotherapy plus hypomethylating agents in 8 cases (24%) and hypomethylating agents alone in 11 cases (8 azacytidine, 3 decytabine; 32%). the procedure was well tolerated, with mild and transient ‛haploimmunostorm syndrome' (fever 84%, rash 28%, diarrhea 14%). only the two patients with cmml received corticosteroid. one patient suffered early infusional reaction that was resolved with support treatment. none of the patients showed acute or chronic gvhd or persistent donor engraftment in chimerism tests. four patients had bacterial infections, but no other significant invasive fungal or viral infections were observed. all aml/raeb patients treated achieved complete remission with microhct treatment (13; 87%). only one patient, with cmml, died during microhct induction (7%). four patients relapsed at 7, 9, 10 and 15 months after the infusion; two of them achieved a second sustained complete remission with another micro-hct from a different donor (one of them had developed anti-hla antibodies). as described in figure 1 , median overall survival is 16 months and overall survival at 2 years is 40%. microhct is a well tolerated procedure in elderly aml/mds patients who are not candidates to allogeneic hct. infectious complications are insignificant and the remission rates are very encouraging in very high risk cases, with no evidence of gvhd. patients can undergo a second microhct from a different donor. in addition to the experience by ai et al, we have also shown that microhct can be safely administered following a hypomethylant agent course instead of conventional chemotherapy. a large, international, randomized clinical trial will address the safety and efficacy of microhct for elderly aml/ mds patients (nct02171117). [p086] disclosure of conflict of interest: none. wilms tumor protein 1 (wt1) is expressed in a variety of solid tumors and is found in more than 80% of patients with acute myeloid leukemia which makes it an attractive target for immunotherapy. previously it was shown that t cells recognizing wt1 are suitable for adoptive t-cell therapy by increasing the graft versus leukemia effect. however, the efficiency of this therapeutic strategy is still limited due to the low precursor frequency and specificity of wt1-specific t cells in the peripheral blood of healthy donors. the ubiquitous antioxidant inducible enzyme heme oxygenase-1 (ho-1) and its products have immunomodulatory effects, which render it as a potential target for the modification of t-cell responses. recently, we found that inhibition of ho-1 enzyme activity via tin-mesoporphyrin (snmp) results in activation and proliferation of antiviral t cells from healthy donors. in this study we aimed (1) to identify the mechanism of ho-1 modification in the generation of wt1-specific t cells and (2) to develop strategies for the sufficient generation of wt1specific t cells from healthy donors to augment effective t-cell immunity in leukemia patients and to broaden the applicability of adoptive t-cell therapy to the majority of patients. the frequency of wt1-specific t cells in peripheral blood of healthy donors (n = 50) was examined before and after snmp treatment via ifn-γ elispot using the wt1-overlapping peptide pool (ppwt1). enrichment efficiency of wt1-specific t cells after ho-1 inhibition was verified in response to ppwt1 and the hla-a*02:01-restricted wt1 peptides 37 (vldfappga, wt137) and 126 (rmfpnapyl, wt1126) by ifn-g secretion assay and expression analysis of the t-cell activation marker cd137. phenotypic and functional characterization of wt1specific t cells were further assessed by multicolor flow cytometry, luminex assays and elisa with respect to t-cell subsets, cytotoxicity, proliferative capacity and secretion of effector molecules. in 24% of donors we found specific t cells against ppwt1 by ifn-γ elispot (10 spots/250.000 pbmcs). the frequency of wt1-specific t cells in these donors could be increased fivefold after inhibition of the enzymatic activity of ho-1 via snmp. to assess the possibility that ho-1 modulation might be clinically applicable in conformity with good manufacturing practice, enrichment of snmp-treated wt1-s159 specific t cells was evaluated based on ifn-g secretion and cd137 expression. compared to snmp-untreated cells there was a 3.74-fold higher response of ho-1 modified wt1-specific t cells pre-enrichment and an up to 16-fold higher enrichment efficacy, while snmp treatment did not affected the t-cell functionality. in conclusion, modification of the enzymatic activity of ho-1 resulted in a more effective generation of functionally active wt1-specific t cells suitable for adoptive t-cell therapy. this makes ho-1 a promising therapeutic target to boost antigen-specific t-cell responses for treatment we recently developed and characterized a standardized and clinical grade human platelet lysate (hpl) that constitutes an advantageous substitute for fetal bovine serum (fbs) for human mesenchymal stem cell (hmsc) expansion required in cell therapy procedures, avoiding xenogenic risks (virological and immunological) and ethical issue. because of the progressive use of pathogen reduced (pr) labile blood components, we evaluated the impact of the novel procedure theraflex uv-platelets for pathogen reduction on hpl quality (growth factors content) and efficacy (as a medium supplement for hmsc expansion). this technology is based on short-wave ultraviolet light (uv-c) and has the main advantage not to need the addition of any photosensitizing additive compounds (that might secondary interfere with hmscs). we applied theraflex uv-platelets procedure on fresh platelet concentrates (pcs) suspended in platelet additive solution and prepared hpl from these treated pcs. we compared the quality of pr-hpl with the corresponding non-pr ones, in terms of growth factor contents. then, we evaluated the efficacy of pr-hpl, in comparison with hpl and msc-screened fbs. we performed large scale culture of hmscs during 3 passages and evaluated the proliferation of cells and the maintenance of their properties: profile of membrane marker expression, clonogenic potential, immunosuppressive properties (inhibition of t-cell proliferation) and potential to differentiate in adipocytes and osteoblasts. we showed no impact on the content in 5 growth factors tested (egf, bfgf, pdgf-ab, vegf and igf) and a significant decrease in tgf-b1 (−21%, n = 16, p o0.01). a large scale culture of hmscs during 3 passages showed that hpl or pr-hpl at 8% triggered comparable hmsc proliferation than fbs at 10% plus bfgf (n = 3). moreover, after proliferation of hmscs in hpl or pr-hpl containing medium, their profile of membrane marker expression, their clonogenic potential and immunosuppressive properties (inhibition of t-cell proliferation) were maintained, in comparison with hmscs cultured in fbs conditions. the potential to differentiate in adipogenic lineage of hmscs cultured in parallel in the 3 conditions, evaluated using oil red o and nile red stainings and the measurement of triglyceride accumulation, remained quantitatively identical. we also showed that the potential to differentiate in osteoblasts (quantified using alizarin red s and von kossa stainings and alp activity measurement) of hmscs grown in hpl or pr-hpl was not impaired, in comparison with fbs. in conclusion, we demonstrated the feasibility to use uv-c treatment to subsequently obtain pathogen reduced hpl, while preserving its optimal quality and efficacy for hmsc expansion for cell therapy applications. although it is still not used widely in clinical practice. in this paper, we demonstrated a case of ada-scid who received hsct as an adolescent from matched unrelated donor (mudd) after termination of her peg-ada treatment due to severe intractable thrombocytopenia induced by peg-ada. patient showed good engraftment and incremental clinical improvement. her post transplantation course was complicated with multiple complications including: grade i gut gvhd as well as hemorrhagic cystitis (btk related) and ebv infection, additionally, she developed several cns complaints like headache, vomiting and dizziness which were found to be due to increased intracranial pressure with multiple enhancing cerebral lesions found on brain imaging. further investigations for the brain lesions confirmed the diagnosis of malignant diffuse large b cell lymphoma (dlbcl) involving the brain. the lymphoma was highly suggested to be originated from donor cells giving the timing relationship between transplant and establishment of the diagnosis. this lymphoma was successfully treated with full recovery and good final immune reconstitution but with lack of b cell engraftment and need for monthly ivig. we conclude that, peg-ada can rarely induce thrombocytopenia in an autoimmune manner by forming antibodies against platelets and good recovery of thrombocytopenia can be achieved after discontinuation of peg-ada. hsct can be considered as modality of treatment even in older patients with scid due to ada deficiency keeping in mind high possibility of complications including, autoimmunity and malignancy. disclosure of conflict of interest: none. (1) (2) (3) (4) . based on the pre and post-apheresis cd34+ cell counts, the collection efficiency of the apheresis amicus device was median 89.5% (54-170) and of the comtec median 82% (32-95). in mm the apheretic collections were started on median day 5 (4-6), while in lymphoma patients, due to chemotherapy, the day of apheresis start was 12 (9-18). after cryopreservation and thawing, viability (7-aad, bd) was median 87.5% (43-100). with these cell products, up to now we engrafted 9 patients following high-dose chemotherapy (5 mm autografted after mel200, 2 hl and 2 nhl autografted after beam). engraftment was prompt and stable in all with anc 0.5 and 1.0 × 10 9 /l on median day 11 (10-12) and 12.5 (11-15), respectively, and with platelet count 20 and 50 × 10 9 /l on median day 14 (11-17) and 17.5 (13-44), respectively. these results are similar to those obtained by most experienced centers in europe and us, and confirm the fact that autologous transplantation may be implemented also in developing countries when appropriate technology and application of standard procedures are employed. with this experience our center is also developing allogeneic transplantation, and the initial results in thalassemia will be reported in a separate abstract. disclosure of conflict of interest: none. extracorporeal photopheresis (ecp) is a safe and effective immunoregulatory therapy for steroid-refractory graft-versushost disease (gvhd) but its mechanism of action is poorly understood. ecp is a non-immunosupressive therapy whose modulating mechanism is thought to result in an increase in t-regs in the patient and in inversion of the cd4/cd8 ratio at the end of treatment. in this study, we evaluated the effect of ecp on t cell response in a cohort of steroid-refractory gvhd patients. from november 2009 to november 2016, 40 patients (28 con acute gvhd and 12 with chronic gvhd) treated with ecp in our unit, were retrospectively evaluated. patient characteristics are shown in table 1 . we performed an ‛off-line' system ecp using a cell separator (spectra optia, teruno bct) for the cmn apheresis; after 8-methoxypsoralen was added, the product was photoinactivated in the ultraviolet a irradiator (uvamatic-g1, macopharma). ecp procedures were performed for two consecutive days, initially weekly (agvhd), or every two weeks (cgvhd) and afterwards monthly according to clinical response. anthracycline-induced cardiotoxicity (aic) is irreversible, which has limited the use of this anthracycline in cancer chemotherapy. to explore the therapeutic effect and its possible mechanism of bone marrow derived mesenchymal stem cells (bmscs) on cardiac damage induced by anthracyclines in a rat model. study selects sd rats aged 2-3 weeks to isolate and culture bmscs, and flow cytometry was used for phenotypic identification of bmscs. 48 female sd rats were first randomly divided into 6 groups: the sham control, bmscs control, 4.0 mg/kg daunorubicin (dnr), dnr with bmscs, dnr with dexrazoxane (dzr), dnr with bmscs and dzr. left ventricular (lv) function before, during and after chemotherapy were assessed by echocardiography. at the end of 4 weeks, animals were euthanized and organs were collected in 10% buffered formalin for histopathology using hematoxylin and eosin staining and immunohistochemical analysis was used to identify the cellular subpopulations that infiltrate the cardiac tissues. after the construction of microrna-21 (mir-21)modified bmscs with lentiviral vector, 50 sd rats were randomly assigned into 5 groups: the normal control, the empty vector control, dnr, dnr with bmscs, dnr with mir-21-modified bmscs. the density of new blood vessels of rats in each group was detected by immunohistochemical method. mir-21, bcl-2, bax and vegf mrna expressions were detected by qrt-pcr. bcl-2, bax and vegf, cx43, troponin t and bnp protein expressions were detected by western blotting. all procedures performed in studies involving animals were in accordance with the ethical standards of the institutional. an animal model of drug-induced cardiomyopathy was built in the dnr treated rats.lv ejection fraction (lvef) and lv fractional shortening (lvfs) were significantly decreased compared to that of the sham control (p o0.001), and the signs of the myocyte injury (myocytolysis, vacuolization and disruption) in paralleled with the inflammatory infiltrates, marked by cd3 and hla-dr, were observed in the dnr group, while bmscs alone or synergistic with dzr facilitate the anthracycline-induced lv dysfunction returning to the baseline values and the recovery of myocarditis (p o0.001). in the mir-21-modified bmscs transplant group, mir-21 expression, cell migration and proliferation ability were higher than that in the bmscs and empty vector groups (p o0.05). the cardiac regenerative capacity of bmscs following significant myocardial injury were further enhanced by mir-21 compared to that of the dnr group and the control groups (all po 0.05), revealed by the significantly higher density of new blood vessels and upregulation of vegf expressions, during which the pro-apoptotic protein bax were down-regulated and the anti-apoptotic protein bcl-2 function were upregulated in the mir-21 overexpression group compared to that with the bmscs, dnr group and the control groups (all po0.05). western blotting demonstrated that the expression of c × 43 were significantly decreased, while expressions of troponin t and bnp were significantly increased in the mir-21 overexpression group in contrast to that with the dnr group (all p o0.05). these results showed that bmscs could reverse cardiac damage induced by anthracycline, and the cardioprotective efficacy was further enhanced by mirna-21-mediated regulation of apoptosis and angiogenesis. disclosure of conflict of interest: none. effective adoptive t cell therapy against cancer is dependent on long-lived tumor-specific stem cell-like t cells with the ability to self-renew and differentiate into potent effector cells. however, current protocols for ex vivo generation of tumorspecific cd8+ t cells result in terminally differentiated effector t cells. it was found that minor histocompatability antigen (miha)-specific cd8+ t cells with an early memory-like phenotype and long-lived memory transcription profile could be expanded from naive precursors using akt-inhibitor viii1. importantly, these akt-inhibited tumor-specific cd8+ t cells showed a superior expansion capacity and anti-tumor effect multiple myeloma bearing mice. for the clinical exploitation of ex vivo generated akt-inhibited tumor-specific cd8+ t cells, we tested the effect of potential clinical grade akt-inhibitors azd5363, gdc0068, gsk2110183, gsk2141795, mk2206 and triciribine in polyclonal stimulations, allogeneic mixed lymphocyte reactions (mlr), and antigen-specific t cell assays. polyclonal stimulation with anti-cd3/cd28 beads on cd8 naive t cells was used for a first screening of the akt-inhibitors. for all inhibitors, a dose dependent effect on the naiveassociated receptors ccr7, cd62l and cxcr4 was observed. this had limited effect on viability, activation and proliferation except for triciribine, which was therefore excluded for further assays. moreover, in the mlr, treatment of naive cd8+ t cells with remaining akt-inhibitors resulted in a dose dependent effect associated with higher ccr7, cd62l, cxcr4 and cd28 expression. furthermore, the akt-inhibited cd8+ t cell products showed a 10-25 fold increased expansion capacity upon restimulation in vitro. when expanding miha-specific cd8+ t cells from the naive repertoire in the presence of one of the akt-inhibitors, the miha-specific cd8+ t cells showed a more early memory phenotype compared to controls. this was displayed in higher levels of the naive-associated receptor cd62l ( figure 1 ). in addition, these miha-specific cd8+ t cells were shown to be functional, as antigen-specific restimultation resulted in degranulation (cd107a) and ifn-γ production. based on this ifn-γ production, the akt-inhibited antigenspecific cd8+ t cells can be selected using the cytokine capture assay (miltenyi, for enriched infusion in patients suffering from hematological malignancies. using aktinhibition in the generation of tumor-reactive t cells results in a more early memory tumor-specific cd8+ t cell product. this adoptive immunotherapy product retains superior proliferation capacity upon infusion, and its potential selfrenewal capacity could result in a long-term anti-tumor effect in patients suffering from a hematological malignancy. chimeric antigen receptors (cars) are composed of an extracellular domain-derived from a tumour-reactive monoclonal antibody, linked to one or more signalling endodomains. in early clinical trials, cd19 car-t cells have demonstrated impressive anti-tumour activity against different b-cell malignancies, including chronic lymphocytic leukaemia, acute lymphoblastic leukaemia (all) and non-hodgkin lymphoma. conventional alpha-beta car-t cells are however hla-restricted and could cause graft-versus-host disease (gvhd) when used across major mismatches, as expected in the highly anticipated setting of off-the-shelf car-t cells from third-party donors. besides being non-hla restricted, gammadelta t cells possess intrinsic anti-tumour reactivity, making them attractive effectors for next-generation car-t cell therapies. so far, however, attempts at exploiting gammadelta t cells in patients have been largely disappointing, possibly because of sub-optimal ex vivo culture conditions. the aim of our study was to optimise the generation of gammadelta car-t cells and to test their anti-tumour potency both in vitro and in vivo. starting from peripheral blood mononuclear cells of healthy donors, we stimulated gamma-delta t cells with zoledronate and il-2/il-15, and transduced them with retroviral vectors encoding for cd19 cars carrying either cd28.z or 4-1bb.z signalling endodomains. we assessed antitumour activity in vitro by measuring killing, secondary expansion and cytokine production after co-culturing gamma-delta car-t cells with different cd19+ all cell lines, and in vivo in nsg previously engrafted with a b-all semi-cell line. although allogeneic hematopoietic stem cell transplantation (allosct) is a curative option to treat hematologic malignancies, disease recurrence remains a concern in the setting of high risk diseases. thus, post allosct therapeutic strategies are needed to treat and/or prevent disease progression. in this setting, donor lymphocytes infusion (dli) is an option as post allosct immunotherapy aiming to enhance graft versus leukemia (gvl) effect. although dli may induce persistent remission, graft versus host disease (gvhd) is a potential complication following dli. because of the suspected higher incidence of gvhd in the presence of hla mismatches, few series focused on dli following haploidentical stem cell transplantation (haplosct) so far. we therefore report our experience of dli following haplosct using post-transplantation cyclophosphamide (pt-cy) platform. we included in this single center study all consecutive adult patients with hematological malignancies who received dli after haplosct with pt-cy as part of gvhd prophylaxis from 2013 to 2016 (n = 21). conditioning regimens were non-myeloablative (low dose tbi-based) or with reduced toxicity (various dose of busulfan according to disease and patient characteristics). ciclosporine a and mycophenolate mofetil were given as additional gvhd prophylaxis in all cases. dli were given at escalating doses, expressed as cd3+cells/kg, without gvhd prophylaxis, and ranged from 1 × 10 5 our study suggests that dli following haplosct with pt-cy is feasible. gvhd is frequent but with a relatively low incidence of severe forms. no response rate was achieved in the context of hematological relapse, underlining that preemptive or prophylactic strategy might be preferred. indeed, the overall good outcome in patients receiving prophylactic dli is promising taking into account the poor prognostic of the diseases indicated for alternative donor transplantation. further prospective studies are needed in specific disease settings to assess the benefit for using such post allohsct immune-intervention. [p103] disclosure of conflict of interest: none. dual specific cytokine-induced killer cell therapy as a treatment option for life-threatening ptld-a case report of the frankfurt experience l-m pfeffermann 1 infection with epstein-barr virus (ebv) is a frequent complication after allogeneic hematopoietic stem cell transplantation (hsct) and besides relapse remains a significant cause of morbidity. prolonged immunosuppression or delayed t-cell recovery may favor ebv reactivation after transplantation, which under these circumstances can lead to life-threatening lymphoproliferative disease (ptld). consensus is lacking on the optimal treatment of ptld. adoptive immunotherapies with both anti-tumor capacity and restored virus-specific cellular immunity may represent optimal treatment options especially when considered in the context of ptld. in this case report we applied in vitro activated t-cells namely cytokineinduced killer (cik) cells with dual specific cytotoxic capacities transferring both anti-cancer potential and donor t-cell memory against ebv infection for the treatment of ebvassociated ptld which progressed to highly proliferative large b cell lymphoma during delayed t-cell recovery after allogeneic hsct. the reported patient had received an allogeneic hsct for secondary myelodysplastic syndrome following acute myeloid leukemia, and due to delayed t-cell recovery had developed ebv-related ptld two months after transplantation. treatment with rituximab, conventional ebvspecific t-cells and wildtype cik cells failed, therefore the patient was offered ebv-specific cik cells on a compassionate use basis. ebv-specific cik cells were generated from peripheral blood mononuclear donor cells. cells were activated and expanded in the presence of ifn-γ, il-2, anti-cd3 antibody and il-15. on day 0 and 2 of culture an ebv peptide pool was added for additional priming. follow-up analysis included in vitro and in vivo monitoring of ebv-specific cik cells. with above mentioned protocol we were able to generate cik cells containing 1 × 10 4 cd8 + ebv-specific t-cells/kg body weight of the patient. infusion of ebv-specific cik cells resulted in rapid clearance of plasma ebv dna level and sustained disappearance of large (vol. 27cm 3 ) ptld-malignant lymphoma. during one-year follow-up analysis we were able to detect ebv-specific cik cells (cd4 + and cd8 + ) in vivo by flow cytometry using specific mhcdextramers. facs-monitoring of the patient´s blood revealed besides cd8 bright t-cells also an increasing cd8 dim t-cell population with a remarkable percentage of t emra cells within this compartment (up to 95%) indicating virus-specific t-cells. no cytokine release syndrome appeared after ebv-specific cik cell treatment, but cytokine secretion patterns, analyzing serum of the patient, reflected cytotoxic and anti-virus capacity provided by this treatment. cytotoxic potential, as well as t h 1 cell differentiation and function offered by ebvspecific cik cell treatment were further confirmed by in vitro analysis. ebv-specific cik cells revealed an 1. s168 hematologic disease. the global survival ratio in the follow-up was 56.8% (with 66.6%, 60.6% and 42.8% survivals in 3, 6 and 12 months, respectively). the variables significantly associated with greater survival were: type of gvhd (cgvhd), number of affected organs (an organ had to be moderately or severely affected to be included in this category) and steroid dependence as the main reason to initiate ecp (see figure 1 ). there was a trend towards significance for the degree of gvhd and cutaneous involvement to be factors associated to enhanced survival ratios. extracorporeal photopheresis is a safe treatment option for patients with gvhd, generating a response and decreasing immunosuppression in an important percentage of them. the presence of cgvhd rather than agvhd, a lower severity degree of the condition, having a lower number of affected organs, skin as main affected organ and steroid dependence as the reason to start the ecp treatment were all factors associated with greater survival in our sample. disclosure of conflict of interest: none. tx) . we report the case of a patient refractory to chemotherapy treated with ibrutinib as debulking therapy before allo-tx. in june 2014, a 62 years old woman was diagnosed with mcl. the staging performed by whole body ct scan, colonoscopy, egds and bone marrow (bm) biopsy was conclusive for stage iva with bulky lymph node over and below the diaphragm, bm, enteric and peripheral blood (pb) localization. the planned treatment included 3 cycles of r-chop, 1 cycle of high dose (hd) cy, 2 cycles of hd arac and autologous sct. after completion of hd cy, the restaging showed progressive disease, with a thyroid involvement and histologic switch in a blastoid variant. disease continued to progress even after 2 cycles hd arac, so we tried to control the disease with r-bendamustine (90 mg/mq on days 1-2 of 21-d cycle), but after the first cycle the neck circumference increased. we shift to lenalidomide (25 mg on days 1-21 of 28-d cycle) without any response after two cycles. we excluded patient from autologous sct programme because of chemo-refractoriness and we searched matched unrelated donor because no hla identical sibling was available. we started a therapy with ibrutinib (560 mg/die on days 1-21 of 21d cycle). after the first cycle we observed a rapid response with decrease of neck size and the disappearance of superficial lymphnode; we performed 6 cycles of ibrutinib, and we reached a good partial remission with lymphnode of max 4 cm, and a bm and pb involvement of 20%. meantime an unrelated donor with 7/8 hla matching was identified, so in december 2015 we performed allo-tx with reduced-intensity conditioning (thiotepa 5 mg/kg-fludarabine 90 mg/m 2 -melphalan 100 mg/m 2 ) and cyclosporine and short term methotrexate as gvhd prophylaxis. engraftment was at day +17. in the first 100 days after allo tx she experienced a clostridium enteritis, transient cmv reactivation and acute gastrointestinal gvhd on day +60 with rapid response to steroid therapy. main complication happened on day 100, when sudden fever and stupor, progressive to coma, occurred; subsequently pneumococcal encephalitis was diagnosed, with positive csf microbiological exam and two signal alteration in the right cerebral hemisphere at mri. the patient was treated with ampicillin and ceftriaxone with a favourable outcome. the mcl revaluations performed at 2, 5 and 9 months showed complete remission with disappearance of all pathological lymph node and pb involvement. currently the patient is at 1 year post asct, she is enrolled in a rehabilitation program and mcl is in complete remission. our experience seems to indicate that ibrutinib is safe and can be used as bridge to allo-tx therapy in refractory mcl. we will investigate side effects of this platform of therapy, and, given the early occurrence of pneumococcal infection, we will consider to perform capsulated bacteria vaccination before allo-tx. disclosure of conflict of interest: none. [p107] pt 2 was diagnosed with c-all in 2014 and received a mud-hsct (tbi 8gy, cyclosphosphamide) in 1/15 due to persistend mrd. following early rel 6/15, 2 cycles of blina led to mrd+ cr, for which a 2nd hsct from a haploidentical family donor (busulfan, thiotepa, fludarabine) was performed in 10/15. molcr lasted 3 months and rel3 was treated with 3 cycles of weekly io followed by one dli (5x10-4 cd3+ cells/kg), resulting in mrd+ cr and complete donor chimerism. five weeks after the last io cycle, the pt was admitted with ascites, hyperbilirubinemia and reduced general condition. vod was suspected, but diagnostic paracentesis revealed malignant ascites demonstrating fatal progressive disease. conclusion. our data suggest that the sequential use of io and dli is feasible even for heavily pretreated patients with r/r all after hsct and can induce molecular remissions. we observed an unusual case of late onset, severe vod responding to defibrotide and one all relapse manifesting itself with ascites in our patients. we therefore suggest close monitoring of liver function tests in the setting of this therapy and extensive diagnostic work-up for any developing liver abnormalities or ascites. disclosure of conflict of interest: ng: advisory board (pfizer, amgen); research support (amgen); gb: honoraria (amgen). [p108] p108 while allogeneic hematopoietic stem cell transplantation from matched related and unrelated donors has become a standard of care treatment for patients with hematological malignancies, transplantations from mismatched or haploidentical family donors remain challenging. currently t-replete and t-deplete transplantation strategies are applied aiming to improve the outcome after haploidentical transplantation. despite high rates of relapse many centers regard post-transplant cyclophosphamide, a t-replete strategy, as a standard of care approach. we have developed a t-depleted transplant approach where donor lymphocytes selectively depleted of alloreactive t-cells (atir101) using th9402, arhodamide-like dye, are infused after cd34-selected haploidentical hsct, to overcome the challenges of infectious complications, gvhd and relapse. in phase i (cr-air-001) we have demonstrated safe infusion of these lymphocytes at doses up to 2 × 10 6 viable t-cells/kg. recently, we reported a promising 1-year grfs was 57% from a phase ii trial (ash 2016),that is awaiting final results soon. here, we introduce a randomized, multicenter phase 3 study (cr-air-009), where 200 patients with acute myeloid leukemia (aml), acute lymphoblastic leukemia (all) or myelodysplastic syndrome (mds) are planned to undergo a haploidentical hsct with either a t-cell depleted graft and adjunctive treatment with atir101, or with a t-cell repleted graft and use of posttransplant cyclophosphamide. inclusion and exclusion criteria are listed in table 1 . all patients will undergo myeloablative conditioning consisting of either tbi (12 gy) or melphalan/ busulfan, in combination with thiotepa and fludarabine. patients in the atir101 study group will receive atg (2.5 mg/ kg once daily for 4 days) during conditioning and atir101 infusion at a dose of 2 × 10 6 viable t-cells/kg is given between 28 and 32 days after the hsct. patients in the ptcy group will receive cyclophosphamide (50/mg/kg) on day 3 and 4 (or 5) with subsequent use of immune suppression up to 6 months post-hsct. the primary endpoint of the study is gvhd-free, relapse-free survival (grfs). grfs is defined as time from randomization until grade iii/iv acute graft-versus-host disease (gvhd), chronic gvhd requiring systemic immunosuppressive treatment, disease relapse, or death, whichever occurs first. this endpoint captures both safety and efficacy. additional secondary endpoints are overall survival (os), progression-free survival (pfs), relapse-related mortality (rrm) and transplantrelated mortality (trm). patients are planned to be randomized in study centers in europe and north america. a number of 40-50 sites are planned to participate in the study. enrolment is expected to continue until mid-2018 with initial results being available first half 2019. results of this study will determine which transplant regimen provides most clinical benefit in haploidentical donor transplantation, with the promise of an effective regimen without the use of post-transplant immune suppression. disclosure of conflict of interest: jr is an employee of kiadis pharma. multipotent mesenchymal stromal cells (mscs) are used for prevention and treatment of graft versus host disease after allogeneic hematopoietic stem cells transplantation due to their immunomodulatory properties. mscs fate in vivo after infusion is unknown. the aim of this study was to analyze the changes in mscs and allogeneic lymphocytes properties when co-cultured in vitro to simulate their interactions in vivo. the bone marrow from 13 donors (7 male and 6 female aged 22-62 years, median 27 years) was used. mscs were cocultured with allogeneic lymphocytes in a ratio of about 1:10 for 4 days and their basic properties were analyzed over time. lymphocytes were activated by adding to the culture medium 5 mg/ml of pha (pha-lymphocytes). some mscs were treated for 4 h with 500 u/ml ifnγ (γmscs). determination of gene expression levels was performed by reverse transcription polymerase chain reaction in real time (modification of the taq-man) and of antigen expression on mscs and lymphocytes by flow cytometry. significant reduction in the proportion of viable cells was observed in mscs co-cultured with pha-lymphocytes. in γmsc co-cultured with pha-lymphocytes no reduction in the proportion of living cells was revealed. this indicates the sensitization of mscs by ifnγ to factors secreted by pha-lymphocytes. in mscs co-cultured with pha-lymphocytes and lymphocytes mean fluorescent signal intensity level (mfi) of cd90 gradually decreased. ifnγ treatment and co-cultivation with lymphocytes led to significant increase of hla-dr mfi on mscs. co-cultivation with lymphocytes increase the hla-dr mfi on mscs much stronger than ifnγ treatment. relative expression level (rel) of ido1 gene increased dramatically in both mscs and γmscs when co-cultured with lymphocytes. at a day 1 in γmscs rel of ido1 increased 500 fold, and then gradually declined. in mscs cocultured with lymphocytes il-6 rel increased almost 20-fold and then decreased 2-fold at the fourth day. the csf1 rel in γmscs showed twofold increases, upon incubation mscs and γmscs with lymphocytes csf1 rel increased fourfold and sevenfold, respectively. co-culture of msc and γmscs with lymphocytes led to decrease in the proportion of cd25, cd38, cd69, hla-dr, and pd-1 positive cells (both cd4 and cd8) after one day, compared with pha-lymphocytes without mscs. proportion of cd25+, hla-dr+ and pd-1+ cells also decreased after 4 days of co-culturing with msc or γmscs (compared with pha-lymphocytes without mscs), but anyway number of activated cells increased 1.2-3 folds compared with first day. number of cd69+ lymphocytes after 4 days of co-culturing with mscs or γmscs did not vary significantly from control and decreased in comparison with first day. main inhibition of activated lymphocytes by co-culturing with mscs occurs during the first day of their interaction, and then the inhibition became less effective, moreover in mscs decreased the rel of the main immunomodulating factors, and most of them were eliminated. mscs treatment with ifnγ resulted in improved survival and resistance of these cells to lymphocytes action. the results indicate that the effect of mscs injected intravenously to patients is limited to several days. disclosure of conflict of interest: none. autologous adipose-derived mesenchymal stem cells (admscs) embedded in platelet-rich fibrin (prf) promote healing in different types of wounds. by avoiding the needlerelated complications, prf-embedded autologous admscs graft provides a new effective stem cell-based therapeutic strategy for wound healing. adult male (age ⩽ 75yo) were equally divided (n = 5 per group) into group 1 (prf only), and group 2 (prf+admscs). regular dressing (without any agent) was used for both groups with a frequency of changing every 3 days. rpf with or without admscs was patched on the wound (maximum surface area 77 cm 2 ). all patients were followed up until complete healing. a complete healing was noted in both groups; however, the healing in group 1was very slow (after 10 weeks), compared to a quicker one in group 2 (after 6 weeks). control of the moisture was very well noted in group 2, less in group 1. group1 showed a lot of exudates on the wound; less exudate in group2 were noted. infections were absent. both groups had a colonized wound. signs of inflammation were very well noted in group 1; no signs of inflammation in group2. admscs embedded in prf offered rapid wound healing responses then prf alone. keywords: mesenchymal stem cells, platelet-rich fibrin, engineered tissue wound healing disclosure of conflict of interest: none. stem cell source p112 additionally cryopreserved g-csf primed pbsc can substitute the second transplantation for the patients with acute leukemia who lately relapsed after hematopoietic stem cell transplantation y lee 1 , j moon 2 , ih lee 3 and s sohn 2 1 department of hematology, kyungpook national university hospital; 2 department of hematology,kyungpook national university hospital and 3 kyungpook national university hospital although allogeneic hematopoietic cell transplantation (allo-hct) is a potentially curative therapy for acute leukemia, survival outcomes of the patients relapsed after transplantation remains poor with high early mortality and a small percentage of second remission. this study evaluated the efficacy of dli using g-csf primed pbsc additionally cryopreserved for the patients who relapsed after allo-hct. we retrospectively reviewed the medical records of the 255 patients who received allo-hct for acute myelogenous leukemia (aml), myelodysplastic syndrome (mds), and acute lymphoblastic leukemia (all) between 1998 and 2013 in kyungpook national university hospital. among the 93 patients who had relapsed after allo-hct, the 39 patients received dli using the additionally harvested cells. at the time of harvest for the first hct, collecting targeted pbscs (greater than 6 × 10 6 /kg cd34+ cells) allowed us to cryopreserve surplus pbscs, including cd3+ cells with dimethylsulfoxide in a nitrogen tank. then, we analyzed the efficacy of dli for the patients who were classified into early relapse or late relapse group by the median time of relapse after transplant. the median age at transplant was 38.5 years (range 15-68 years) and male was 111 patients (44.4%). primary diseases for allo-hct were aml/mds (n = 175, 70%) and all (n = 75, 30%). one hundred forty three patients (57.2%) were in cr1 (complete remission), 25 (10%) in further cr, and 87 (33.2%) in relapsed and refractory status. one hundred seventy one patients (68.4%) received myeloablative conditioning regimen. the median dose of cd34+ cell was 5.21 × 10 6 /kg (range: 1~20.6 × 10 6 /kg). almost 95% of patients achieved the neutrophil engraftment with a median time of 13 days (range: 9-24days). the 1-year overall survival (os), relapse free survival (rfs), non-relapse mortality (nrm) and graft-versus-host disease (gvhd)-free/relapse-free survival (grfs) since hct was 55.3 ± 3.1%, 66.0 ± 3.2%, 28.2 ± 0.3%, and 32.9 ± 3.1%, respectively. there was no significant difference according to s172 the infused cd34+ cell dose (lower o6 × 10 6 /kg vs higher ⩾ 6 × 10 6 /kg). the incidence of chronic gvhd was more frequent in higher cd34+ group (32.9% vs 48.2%, p = 0.042). median time from hct to relapse was 139 days (range: 21~1801 days). after relapse, 46 patients (49.4%) were treated with salvage chemotherapy, 9 patients (9.6%) with second allo-hct, and 38 patients (40.8%) with dli. the median number of cd3+t cell was 2.95 × 10 7 /kg (range: 0.1~5.43 × 10 7 /kg). fourteen patients (23.6%) achieved dli induced cr, 20 patients progressed, and 6 patients were not evaluable for response. dli induced acute gvhd was observed in 24 patients (63.1%) and chronic gvhd developed in 4 patients (10.5%). in late relapse group, the 1-year os since post-transplant relapse was significantly higher in dli group than non-dli group (figure 1 , 53.4 ± 7.4% and 26.7 ± 7.4%, p = 0.039) but, early relapse group had no difference. the patients treated with dli showed significantly survival benefit in late relapse group (median 286 days vs 103 days, p = 0.002). the incidence of dli-induced gvhd does not differ between two groups. dli for the patients who lately relapsed after allo-hct can be a feasible and an effective option in terms of response, donor convenience and it's cost. in the late relapse group, g-csf primed dli may replace second transplantation. disclosure of conflict of interest: none. cord blood transplantation-19 years of experience c alves 1 , f amado 2 , f bordalo 2 , s ferreira 2 , s lopes 2 , c pinho 2 , t rodrigues 2 , l antunes 3 and s roncon 2 1 serviço de imuno-hemoterapia; 2 serviço de terapia celular and 3 registo oncológico do norte, instituto português de oncologia do porto francisco gentil, epe allogeneic haematopoietic stem cell transplantation (hsct) is a well-established treatment for patients with malignant and non-malignant haematological disorders. cord blood transplantation (cbt) has extended the availability of hsct to patients that would not otherwise be eligible for this curative approach because of the lack of human leucocyte antigen (hla) identical donor. the aim of this retrospective study was to analyse and characterize 19 years of cbt activity in our institution (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) . we examined patient electronic files and created a database in excel to register cord blood unit (cbu) parameters and patient characteristics. after thawing, cbu was washed/diluted with validated procedures; the cellular content was evaluated by immunophenotyping and followed ishage recommendations; sterility was assessed by bacterial/fungal cultures, viability by trypan blue exclusion assay and functionality by clonogenic capacity. the transfusion of blood products after transplant was quantified and the hematological recovery (hr) established using cibmtr criteria. correlation between continuous variables was assessed with spearman coefficient. overall survival (os) was determined according to cellular content and hla-disparity by the kaplan-meier estimator. survival between groups was compared using the log-rank test. a total of 59 cbu were administered to 57 patients (table 1) : 30/57 female, the main diagnosis was acute leukaemia (35/57). a sibling cbu was used for 3 patients; the unrelated were imported from europe (66%), usa (32%) and oceania (2%). hla-matching was 6/6, 5/6, 4/6 and 3/6 for 8, 21, 15 and 6 patients, respectively; 47% were abo-identical. after thawing, 95% were washed and presented no microbial growth. the majority of patients were submitted to a bussulfan-based myeloablative conditioning regimen; graft versus host disease prophylaxis was performed with a calmodulin inhibitor+mycophenolate mofetil. complete and mixed chimerism was verified in 42% and 7% of patients; 5% had graft failure; the rest were unknown results (46%). at the moment, we reported 27 patients alive (22 in complete remission, 5 with evidence of disease relapse) and 30 dead at a median of 6 (0.4-198) months after cbt; the most frequent cause (43%) was recurrence of the initial clinical condition. the correlation between nucleated cells (nc) and cd34+ cells per kg/hr (p = 0.277; p = 0.123) and number of cd34+ cells per kg/os (p = 0.123) was not statistically significant. however, the engraftment and os was associated with hla-mismatch (p = 0.025; p = 0.012) and os was related to nc per kg (p = 0.012). our clinical results suggest that despite increased hla disparity, ucb offers promising results. ucbt is feasible in patients when the unit contains a high number of cells. there are several strategies for the future, related to cbu expansion and homing techniques, nurturing procedures, selection of optimal cbu unit and enhancement of immune recovery, in order to improve the application of cbu. s173 received sirolimus and mmf. median time to neutrophil and platelet engraftment was 10 days (9-13) and 18 days (17-124), respectively. one patient died from parainfluenza pneumonia (d111), one patient from ptld (d 203), one patient from late pulmonary vod (d379), and one patients from relapse (d89). with a median follow up for survivors of 17 months, one year survival is 64%. three patients had grade 2-3 gvhd and none of the survivors have chronic gvhd. though unrelated donor chimerism was dominant early after transplant and contributed to early count recovery, definitive engraftment was dominated by ucb chimerism in all but one patient. conclusion: among older adult patients with hematological malignancies,~20% lack haplo-identical relatives. for these patients, double or single ucb transplant is challenging because of delayed engraftment. cd34 selected partially matched grafts from unrelated donors hasten hematopoietic recovery and are over time outcompeted by ucb grafts which provide robust hematopoiesis with low risk of chronic gvhd. the combination of mismatched unrelated hematopoietic progenitors and ucb grafts provides an attractive alternative for older patients lacking hla-idental donors or haploidentical relatives. in planned trials, mismatched donors may be selected based on kir type to further enhance gvl effects. disclosure of conflict of interest: partially supported by miltenyi biotec. haploidentical stem cell transplantation (haplo-sct) is an attractive option for patients who do not have an hlamatched donor. historically it has been associated with high rates of graft rejection, relapse and low incidence of graft versus host disease (gvhd). to decrease these issues we have considered the use of primed bone marrow as stem cell source, early withdrawal of immunosuppressive therapy and the use of donor lymphocytes infusions (dli) in haplo-sct with high-dose post-transplantation cyclophosphamide (ptcy) as main gvhd prophylaxis. to analyse our incidence of acute and chronic gvhd and overall survival (os) in patients with haplo-sct with short course of inmunosupressive therapy. we retrospectively analyzed a cohort of 28 patients who underwent haplo-sct with primed bone marrow as stem cell source, between years 2012 and 2016 in our centre. gvhd prophylaxis consisted in ptcy (50 mgr/kg on days +3 and +4) and tacrolimus plus mycophenolate (mmf) from day +5 as recommended by baltimore group. mmf was stopped on day +28. tacrolimus was tapered off from day +50 with withdrawal on day +120 in patients without gvhd or with active disease. dli were considered if mixed chimerism, relapse or disease progression appeared. the characteristics of the patients are shown in table 1 . results: there was no primary graft failure. eight of 27 patients (29.6%) developed agvhd (grade ii-iv) and it was severe (grade iii-iv) in 2 patients (7.4%). cutaneous agvhd location was the most common presentation (9 patients (33%)) and it was associated with intestinal gvhd in 2 patients. twenty two patients were evaluable for cgvhd. thirteen patients (59%) developed chronic gvhd that was mild, moderate and severe in: 8 (36.3%), 3 (13.6%) and 2(9%) patients, respectively. the median time of onset cgvhd was 6 months (range: 3-33) and it was related with previous withdrawal of the immunosuppression in 5 (22.7%) patients, tapered off immunosuppression in 6 (27.2%) patients and dli in 2 (9%) patients. systemic treatment was required in 8/13 patients but only 2 patients were treated with high doses of steroids (41 mg/kg/day). the median days of is therapy in patients who developed gvhd was 172 days (range: 81-244). dli were used in 7 (25%) patients because of: relapse/disease progression in 5(17.8%) and secondary graft failure in 2(7.1%). two patients achieved complete remission and 2 patients developed cghvd. the median number of dli per patients were 2 (1-3) with a median cd3+cell of 3 × 10 6 /kg (range: 1 × 10 5 -1 × 10 7 /kg). with a median follow-up of 12 months (range: 1-56), the estimated os at 1 and 2 years after haplo-sct were 74% and 52%, respectively. at the moment of this study 17 patients (60.7%) were alive, 14 patients in complete remission, 2 in partial response and 1 in progression. eleven (39.3%) patients died due to: disease (4), infections (4), pleuropericarditis (1), hepatic veno-occlusive disease (1) and refractory gvhd. five patients (17.8%) are without is therapy and without gvhd symptoms. in our experience, early withdrawal of immunosuppression following haplo-sct with primed bone marrow and posttransplantation cyclophosphamide facilitates the development of chronic gvhd and can decrease the relapses in patients with high-risk hematological malignancies. it is necessary more follow up and more studies to confirm this preliminary results. [p115] disclosure of conflict of interest: none. s174 myeloid malignancy (n = 20 except 1 patient with saa) received fludarabine (flu)/busulfan ± tbi 4gy, while lymphoid malignancies (n = 9) received flu/tbi 8gy or cy/ tbi 12gy. all patients received g-csf-mobilized t-cell replete pbsc from a haplo donor. gvhd prophylaxis was ptcy 50 mg/ kg on day +3/+4, tacrolimus (d+5 to +180), and mycophenolate (d+5 to 35). the median duration of follow up of surviving patients is 21 months. median age was 45 (17-66) years, 17 patients (59%) were male, 14 (48%) were african american, and 14 patients (48%) had comorbidity index (hct-ci) ⩾ 3. all patients had hematological malignancy (except 1 patient with saa) including 12 patients (43%) not in cr. disease risk index was high/very high in 12 (42%) and intermediate in 14 (48%). on the day of transplant, 14 patients (48%) did not receive steroid premedication ( = no-steroid group), while 15 patients (52%) received 125 mg of methylprednisolone 30 minutes prior to pbsc product infusion ( = yes-steroid group). all the following outcomes are described in the ‛no-steroid' vs ‛yessteroid' group, respectively. cumulative rate (ci) of anc engraftment (⩾500/μl) on day +28 was 79% (95% ci 60-100%) and 93% (95% ci 81-100) (p = 0.07). ci of platelet engraftment (⩾20 000/μl) on day +56 was 71% (95% ci 51-99%) and 90% (95% ci 74-100%) (p = 0.2). primary engraftment failure was observed in 3 patients; 1 in yes-steroid and 2 in no-steroid. no primary engraftment failure was observed with myeloablative tbi (8-12 gy) (n = 9). ci of agvhd gii-iv (day+100) was 21% (95% ci: 8-58%) and 28% (95% ci: 12-64%) (p = 0.77). ci cgvhd (1 year) was 42% (95% ci 20-90%) and 49% (95% ci 27-87%) (p = 0.48). ci of relapse (1 year) was 22% (95% ci 7-75%) and 7% (95% ci 1-47%) (p = 0.71). ci of non-relapse mortality (nrm) (1 year) was 51% (95% ci 29-90%) and 38% (95% ci 19-77%) (p = 0.48). post-infusion noninfectious fever (d0 to +3) was observed in 12/14 (86%) and 12/15 patients (80%). median tmax was 103f and 102f (p = 0.38). only 1 patient in the no-steroid group developed life-threatening cytokine release syndrome and survived. no difference of viral reactivation was noted between groups. cmv reactivation occurred in 7 (50%) and 13 patients (87%), bk reactivation in 20% (in both groups), hhv6 in 36% and 40%, ebv in 14% and 7%. the 18-month overall survival was 46% (95% ci 17-74%) and 60% (95% ci 35-85%) (p = 0.53). the 18-month disease-free survival was 41% (95% ci 15-68%) and 53% (28-79%) (p = 0.46). t-cell replete haplo pbsc transplant is effective therapy for patients with high-risk hematological malignancies. high-dose steroid premedication with pbsc infusion neither influences transplant outcome nor prevents post-infusion febrile reaction. disclosure of conflict of interest: as discloses grant support (american porphyria foundation), consultation (medpace inc), research support (astellas and fate therapeutics), honoraria (alxion, and spectrum), and royalty for licensing of intellectual property (incysus biomedical). intrabone transplant of unwashed cb in hematological malignancies: engraftment and safety f giglio 1 , s marktel 1 , r greco 1 , m morelli 1 , mt lupo-stanghellini 1 , e xue 1 , l lazzari 1 , m marcatti 1 , m zambelli 2 , c parisi 2 , r milani 2 , s piemontese 1 , a assanelli 1 , c corti 1 , m bernardi 1 , f ciceri 1 and j peccatori 1 1 unit of hematology and bone marrow transplantation, irccs san raffaele scientific institute, milano, italy and 2 immunohematology and transfusion medicine unit, irccs san raffaele scientific institute, milano, italy cord blood transplant (cbt) in adult patients (pts) is limited by the risk of graft failure or delayed engraftment due to low cell counts. to improve the capacity and speed to engraft, intrabone (ib) cbt technique has been investigated, showing high rate of engraftment and low acute gvhd, also when compared with double cb transplant. cb units washing procedure has been suggested to remove dmso toxic potential effect. we report our experience in 15 adult pts with hematological malignancies receiving ib unwashed cb in an attempt to reduce the loss of progenitor cells and the risks associated with cell-washing procedure. between 2009 and 2016 we performed 15 allogeneic hematopoietic stem cell transplant (hsct) using unwashed cbu as a source and infusing them ib. all pts were adult and suffering from hematological malignancies. this population was characterized by very high-risk and advanced phase disease. all pts received a cb hsct because of unavailability of sibling or matched unrelated donors. eleven pts received a treosulfanbased myeloablative conditioning regimen and a sirolimusbased ghvd prophylaxis; four pts received busulfan-based myeloablative conditioning and a cyclosporine-based ghvd prophylaxis. cb units were thawed and diluted with albumindextran solution immediately before the transplant. this ‛nowash' dilution was implemented to reduce product manipulation that may results in cell loss. furthermore, graft manipulation risks potential contamination, requires increased technologist time, and delays time to infusion. the ib infusion was performed under local anesthesia and with short conscious sedation, at bedsite in the bmt ward. the infusion was preformed monolaterally or bilaterally according to the volume to be infused. starting from a 10% dmso concentration in the cb units before the dilution, the graft products contained a median of 3.6% dmso (range: 2.3-6.9) at ib-hsct. the median cd34+ cells infused were 0.09 × 10 6 /kg b.w. (range: 0.03-0.82). the median mono-nucleated cells were 15.66 × 10 6 /kg b.w. (range: 4.7-36.8). the median cd3+ t-cells were 2.6 × 10 6 /kg b.w. (range: 0-5.54). the median infused volume was 75 ml (range: 60-215). no procedure-related adverse events were observed, nor related to the ib technique, neither to the sedation. of the 15 transplanted pts, 9 were evaluable for engraftment (1 patient rejected the graft and 5 patients died before day +30 because of severe infections). all 9 achieved anc 40.5 × 10 9 /l after a median of 24 days (range: 16-45) and 8 achieved plt 420 × 10 9 /l at a median of 45 days (range: 25-141). three patients developed grade iii-iv acute gvhd grade. according to extreme heterogeneity of the population no correlations with relapse incidence and diseasefree survival could be evaluated. ib infusion of unwashed cb is feasible, safe, easy to perform. no adverse events related to the procedure were documented. no dmso toxicity was documented. engraftment was obtained in all evaluable pts. our data confirm that direct ib cbt overcomes the problem of graft failure even when low numbers of cb cells were transplanted, thus leading to the possibility of using of this technique in a large number of adult pts, for whom this approach represents the sole possibility of long-term survival. the ‛no wash' cb dilution can also help the implementation of ib transplant thanks to the easier graft manipulation. [p117] disclosure of conflict of interest: none. lower incidence of cgvhd after cord blood transplantation for hematological malignancies in comparison with hematopoietic stem cell transplantation from other donors: 20 years' experience in a single institute m yoshino, m obiki, m osakie, s ikeno, t sato, m nasashima, y kagaya, n kawashima, t morishita, y ozawa and k miyamura department of hematology, japanese red cross nagoya first hospital the outcome of cord blood transplantation (cbt) for hematologic malignancy was investigated. however the incidence of gvhd is not accurately known. the goal of this study was to compare the outcome of cbt with allogenic hematopoietic stem cell transplantation (allo-hsct) from other sources, mainly unrelated bone marrow (urbm). patients' characteristics: 755 patients who underwent allo-hsct, between 1990 and 2015 in our hospital were retrospectively analyzed. donor sources were cord blood cell (n = 112), urbm (n = 372), hla matched sibling bone marrow (sibling bm) (n = 166), and hla matched sibling peripheral blood stem cell (sibling pbsc) (n = 105). in cbt, the median age was 38.5 (17-68), and the diagnosis included aml (69), all (15), mds (13), cml (4) and other (11). the disease risk was good in 58 and poor in 54. disease risk was slightly higher in comparison with other sources. prophylaxis of acute gvhd was tacrolimus, short-term methotrexate (88), cyclosporine, short-term methotrexate (22) and others (2) . the 5-year overall survival (os) rate after cbt (1990 cbt ( -2004 10 .0%, engraftment failure 6.7%, acute gvhd 3.3%), relapse 10.0% and other 6.7%. in cbt cases, engraftment failure after allo-sct was observed in 24 cases (21.4%) which is higher than that among urbmt (8.6%), 32 out of 372. 10 cbt cases underwent the second allo-hsct and 9 patients achieved engraftment and 7 patients were alive at 100 days after allo-hsct. 6 of them survived at 2 years after allo-hsct. our results suggest that the outcome of cbt has improved in recent years. moreover, cbt has an advantage in the least cumulative incidence of acute/chronic gvhd. cbt may well create the best outcome in the future. disclosure of conflict of interest: none. chronic active epstein-barr virus (ebv) infection is a major type of ebv-associated t/nk-cell lymphoproliferative disorders (lpd) in childhood. however, young adults rarely develop chronic active ebv infection (caebv), and shows more aggressive features than that of childhood. umbilical cord blood transplant (ucbt) is a possible treatment option for caebv patients who have no hla-matched donor, but there is little information available about the efficacy and safety of ucbt for adult patient with caebv. we analyzed six adult patients with caebv who underwent a single-unit ucbt between 2010 and 2016 at our institute (including a case reported in 2014 [1] ). the diagnosis of caebv was made according to the criteria proposed in 2005 [2] ; persistent infectious mononucleosis (im)-like symptom and detection of increased ebv genomes in peripheral blood mononuclear cells (pbmc). ebv-dna load was measured using real-time quantitative pcr. median patient age at diagnosis was 27 (23-39) years. target cells of ebv-infection were cd4+t cells (n = 4) or nk cells (n = 2). median ebv-dna load was 2.6 × 10 4 copies per microgram of dna in pbmc (range: 1.6 × 10 3 -1.3 × 10 5 ) at the diagnosis. all patients were given prednisolone and cyclosporine, and then etoposide (n = 2) or combination chemotherapy (n = 4) before transplant. ebv load has slightly decreased to a median of 1.3 × 10 4 copies (range: 4.0 × 10 2 -7.1 × 10 4 ), but disease status was active at ucbt in all. median time from the diagnosis to ucbt was 101 days (range: 62-440). one patient received total body irradiation (tbi) 12gy + cyclophosphamide (cy) 120 mg/kg + cytosine arabinoside 8 g/m 2 , and the other five patients received fludarabine (flu) + melphalan (lpam) 80-140 mg/m 2 or cy 120 mg/kg with tbi 4gy before ucbt. umbilical cord blood (ucb) was 4/6 hla-matched to the recipients. median number of infused ucb cd34+ cells was 0.87 × 10 5 /kg (range: 0.76-1.93 × 10 5 ). gvhd prophylaxis was consisted of tacrolimus + methotrexate or mycophenolate mofetil. neutrophil engraftment and complete donor chimerism were achieved in four patients, but two of them developed secondary graft failure (gf) early after engraftment. the other two patients developed primary gf. second ucbt was successfully performed in the 4 patients with gf a median of 31.5 days (range: 28-58) after the first ucbt. ebv genomes in pbmc became undetectable immediately after ucbt. at a median follow-up of 873 days (range: 54-2446), ebv-dna was undetectable or very low, and im-like symptoms were resolved in all cases. however, at 7-9 months after ucbt, two patients developed ebv+ b-cell lpd derived from donor cells, that was successfully treated with rituximab therapy. this study suggested that ucbt could eradicate ebv-infected cd4+ t cell-or nk cell-clones. ucbt can be a treatment option for adults with caebv. rituximab monotherapy was effective for post-transplant lpd from donor b cells. however, a high incidence of gf was observed in patients receiving reduced-s176 intensity conditioning of flu/lpam or cy /low-dose tbi. further studies are needed to find more optimal regimens for stable engraftment of ucb in adult patients with caebv. there is an increased incidence of ab0 incompatibility-50-60%, in allogeneic hematopoietic stem cell transplantation (allohsct) in patients who are russian citizens as a result of the variability of genetic polymorphism in the multi-ethnic population and a significant number of unrelated donors from international bone marrow registries. ab0 incompatibility in different types of allohsct may be an additional aggravating factor for the development of immunological complications and decrease effectiveness of treatment, but the data is still controversial [1] . from may 1999 to december 2015 in raisa gorbacheva memorial institute for children oncology, hematology and transplantation 1131 patients with leukemia, malignancies and hereditary diseases were included to the study, who were performed 1428 hsct: allogeneic unrelated -814 (57%); allogeneic related-344 (24.1%); haploidentical -267 (18.7%); umbilical cord blood in 3 patients (0.2%). age was 0-76, median-25 years. patients were predominantly with acute myeloid leukemia-37% (n = 602), acute lymphoblastic leukemia-30% (n = 501) and chronic myeloid leukemia -6% (n = 94). results: in 54.6% of cases (n = 780) ав0 incompatibility was determined: major-37.8% (n = 295); minor-45.4% (n = 354); combined-16.8% (n = 131). ав0 incompatibility in allohsct did not influence overall survival (p = 0.56) and frequency of acute graft versus host disease (gvhd) (p = 0.2). also there was no difference in overall survival depending on combination of condition regimen and ab0 incompatibility: reduced intensity (ric) or myeloablative (mac) (p = 0.7). an increased frequency of acute gvhd was observed in ric and ав0 incompatibility (30.8%) compared to mac (15.3%, p = 0.002). ab0 incompatibility was not a major factor (log worth 0.87) which influenced the fact and speed of donor's transplant engraftment in comparison to level of hlacompatibility (15.1), hematopoietic stem cell source (7.05) and type of hsct. but the presence of major ab0 incompatibility increase the period of erythroid recovery (p = 0.01) as reflected in the higher amount of blood transfusions. complications caused by ab0 incompatibility were identified in 2.4% of all cases (n = 34) including acute and delayed hemolysis, partial red cell aplasia and immune thrombocytopenia. conclusion. the presence of ав0 incompatibility is not a limiting factor to perform allohsct, however, it demands high quality prophylaxis and accurate transfusion therapy depending on ab0 incompatibility type to prevent immune complications. keywords: allogeneic hsct, ab0-incompatibility. poor graft function or graft failure have become common indications for infusion of immune-selected cd34 + cells (‛boost') or second unprocessed allo-hsct, creating the need for remobilization of the same related or unrelated. we retrospectively compared the results of two consecutive cycles of rh-g-csf treatment and peripheral blood progenitor cell collections in 20 related donors cared for at our institution between 2008 and 2016. mobilization consisted of the administration of rh-g-csf at a dose of 10 μg/kg per day injected in the evening, and apheresis was started in the morning of the fifth day after the fourth dose of rh-g-csf. collection was performed with a spectra or spectra optia cell separator (terumo bct). eleven out of 20 were haplo-mismatched donors and 9 were hla matched donors. four donors were re-collected because of recipient graft failure and 16 because of poor graft function; in the latter situation, immunomagnetic selection of cd34+ cells was performed on the collected cell product prior to infusion into the recipient, using the clinimacs medical device, as previously published. median donor age was 43 years (range: 18-66) at time of first donation, median weight 70kg (45-112) and bmi 24 (18-34). median delay between mobilizations 1 and 2 was 192 days (28-1530). interestingly, the median delay between collections was 119 days (43-701) in the haplomismatched setting and 231 (28-1530) in the matched setting. median number of circulating cd34+ cells/μl after the first 4 injections of rhg-csf was 61 vs 37 at the first and second mobilization cycles (po0.001, table 1 ). seven out of 20 donors (35%) requested more than one apheresis session to obtain the target number of collected cd34+ cells during the first cycle, as compared to 12 out of 20 (55%) for the second cycle: this is largely due to the higher target of cd34 + cells for the second collection, expecting that the median cd34 recovery after immunomagnetic selection is 60% in our experience. our study shows that a second cycle of mobilized peripheral blood progenitor cell collection from related donors is associated with a significant reduction in response to hematopoietic growth factors and mobilization capacity. this information allows planning the number of aphereses at the second cycle-and subsequently the number of immunoselection procedures to be carried out-taking into account the higher cd34+ cell dose target needed for subsequent immunomagnetic selection. cmv reactivation remains one of the main complications after allogeneic stem cell transplantation (hsct), requiring antiviral therapy, causing myelosuppression, prolonged hospitalization, higher treatment costs and mortality. cmv seronegative donors are recommended for cmv seronegative recipients. however data about donor selection for cmv positive (cmv-pos) recipients is not conclusive. some studies showed that selecting cmv-pos donors for cmv-pos patients might be beneficial. cmv-seropositivity is very high in lithuania among healthy blood and bone marrow donors (65%) and even higher among hsct recipients (up to 90%), so donor selection for cmv-pos recipients is an object of interest. retrospective analysis of cmv reactivations in cmv-pos allogeneic hsct recipients (transplanted during 2005-2014 year in vilnius university hospital) who survived at least 6 months post hsct was performed. data about cmv reactivation frequency, time post hsct, duration, maximal cmv dna copy number at each reactivation collected. cmv reactivation was considered when cmv dna copies detected 4500/ml in patient's blood. statistical analysis conducted using sas 9.2; student's tests for statistical significance; kaplan-meier methods for overall survival. among 316 allogeneic hsct recipients 286 (90.5%) were cmv-pos. 286 cmv-pos allo-hsct recipients were further analysed. 150 of them received graft from cmv-pos (pos/pos group) and 136-from cmv-seronegative donors (pos/neg group). more patients in pos/neg group experienced cmv reactivation in first 6 months post hsct in comparison to pos/pos group (83.1% vs 65.3%, p o0.05). pos/neg group patients had more cmv reactivations (2.5 vs 2 times in 6 months post transplant period, po 0.05), reactivations were diagnosed earlier post transplant (50.7 vs 123 days post hsct, p o0.0001), had longer duration (60.9 vs 40.3 days, po0.0001) and larger maximal cmv dna copy number (285107,9 vs 24339.4 copy/ml, p o0.0001) in comparison to pos/pos group patients. pos/pos group patients showed tendency for better survival than pos/neg group patients, however did not reach statistical significance. in a univariate analysis only hla mis-match and donor cmv seronegativity were factors statistically significantly associated with cmv reactivation. donor cmv serostatus is significant factor selecting donor for allo-hsct recipients. according to our findings, selecting cmv-pos donor for cmv-pos recipient may reduce cmv reactivation frequency and duration. disclosure of conflict of interest: none. selection of the best hsc donor when a matched donor is not available is still a matter of debate, and reports in pediatric population are scarce. this is a retrospective study conducted by brazilian society of hsct (sbtmo), including 11 centers, aimed to compare matched unrelated (matched-urd), mismatched unrelated (mm-urd) and unrelated cord blood (ucb) hsct. all or aml/mds patients o 18 y/o who have received first unrelated hsct between 2010-2014 were included. hla 4-digit typing was available for urd; for ucb, hla class-i 2-digit typing. overall survival (os), and cumulative incidence (ci) of agvhd, cgvhd, nrm and relapse were analyzed. on an unplanned analysis, we fitted a lognormal bayesian survival model with random effects, imputing the probabilities of ucb matching at 8 loci. a total of 212 patients were included (93 matched-urd, 47 mm-urd and 72 ucb). median age was 8.7 y/o. most patients had all (61%). proportion of early disease in matched-urd was higher (37%, against 19 and 21%). matched-urd were 10/10 4-digit hla matched (except one, for whom dq was not available), while most of mm-urd (89%) were 9/10 hla matched. ucb were 6loci (58%) or 8-loci typed (42%). based on previous report 1 on extending 6-loci hla typing to 8-loci ucb, we estimated that 23 ucb were 0-1 mismatched at 8 loci, 26 had 2 mismatches and 23 had 3 or more mismatches. conditioning regimens were mainly myeloablative, tbi-(59%) or bu-(33%) based. grafts were in vivo t-cell depleted in 67% of the patients, not balanced between groups (p = 0.01). with median follow-up of 3.3 years, 3y os was 56%, 52% and 39% for matched-urd, mm-urd and ucb, respectively (p = 0.02, log rank test). for matched-urd, mm-urd and ucb, ci of grades iii-iv agvhd at 6 months were 18%, 13% and 17% (p = ns); moderate/severe 3y-cgvhd, 8%, 20% and 4% (po 0.05); 3y-relapse, 26%, 21% and 14% (p = ns); and 1y-nrm, 26%, 24% and 49% (p = 0.06). we found out that primary graft failure occurred in 14 (19%) of ucb, compared to 9% in mm-urd and 3% in matched-urd. when ucb matching probabilities at 8 loci were imputed and analyzed in a bayesian model (controlled for age, gender, disease status and diagnosis, and t-cell depletion), survival was inferior in ucb with 3+ mismatches (9.8-times lower median survival, 95 ci 2.6-39.4), but not with up to 2 mismatches (1.3-time higher median survival, 95 ci 0.4-3.8). of note, in vivo t-cell depletion marginally impaired survival (2.4-times decrease, 95 ci 1.0-6.0). discussion: in our population, overall survival achieved with mm-urd was not different to 10/10 matched-urd, despite higher incidence of moderate/ severe cgvhd. on the other hand, survival with ucb was significantly lower. recent report 2 have shown excellent os with ucb compared to 8/8 hla-matched urd. ucb cohort inferior results may have been due to hla disparity degree, since survival in ucb with up to 2 mismatches (out of 8) was not worse. one limitation of our study is that tnc and cd34 from ucb units were not available, impairing primary graft failure analysis. we have also found that in vivo t-cell depletion might have a detrimental effect on survival and should be studied further in prospective trials. in conclusion, mm-urd, especially hla 9/10, is a suitable option when a fully hla 10/10 matched-urd is not available. ucb matched at least 6/8 may also be a good option. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) is a proven treatment for patients with high risk or relapsed hematological malignancy. the probability of having a hla matched family donor is about 30%. in populations with high consanguinity rates, the probability of having non-sibling matched family donor(mfd) is much higher. to explore the impact of msd vs non-sibling mfd on outcome of hsct recipients, we undertook a single center retrospective analysis of pediatric patients transplanted with the diagnosis of hematological malignancy at our center in the last five years. a retrospective cohort from 2011 to current included 113 pediatric patients with hematological malignancies transplanted from family donors, of which 98 were from msd and 15 from non-sibling mfd. hla matched family donors were identified by high resolution allelic typing and were matched 10 of 10 hla loci. diseases were all (n = 58), aml (n = 37), mds (n = 7), jmml (n = 5), kml (n = 2), nhl (n = 1) and hodgkin's disease (n = 3). conditioning regimens were tbi or busulphan-based myeloablative in all patients. the median age of the patients was 9.4 years (range: 5 month-18.9 years). although peripheral stem cell seemed to be used more commonly in non-sibling mfd recipients (64% vs 42%), the difference was not statistically significant. the median follow up time for alive patients 24 months (1-67 months) . two year overall survival and leukemia free survival did not differ between patients with transplantations from msd or nonsibling mfd ( 67% ± 5.0 vs 60 ± 5.1, p = 0.31) similarly, leukemia free survival was not different between msd and non-sibling mfd transplants (58.2% ± 13.1 vs 58.1% ± 13.2, respectively). the incidences of grade ii-iv acute gvhd in msd and nonsibling mfd transplants were % 18 and %13, respectively. the incidences of relapses were 28% in msd transplants and 20% in mfd transplants and the difference was not significant (p = 0.49). these data show that the results of hsct from nonsibling mfd is comparable to hsct of msd in children with hematological malignancy. our data emphasize the need for extended high resolution family typing for patients in regions where there is high rate of consanguinity. disclosure of conflict of interest: none. [p125] p127 donor-recipient rh incompatibility is a risk factor for mortality after pediatric matched related allogeneic hematopoietic stem cell transplantation k ghanem 1 , n hariss 1 , z merabi, n kreidieh 2 , n tarek, r saab 1 , s muwakkit 1 , h el-solh 1 and m abboud 1 1 american university of beirut, department of pediatrics and adolescent medicine and 2 american university of beirut, optimal donor selection is critical to achieve the best outcome after allogeneic hematopoietic stem cell transplantation (allo-hsct). there is no consensus regarding the effect of donor-recipient rh incompatibility on survival after matchedrelated donor (mrd) allo-hsct in children and adolescents. this abstract aims to study this effect in a single-institution cohort over a period of 11 years. this is a retrospective chart review for all patients aged o21 years who underwent allo-hsct at the american university of beirut medical center between august 2005 and june 2016. a total of 70 patients with a median age of 11 years (range: 0.6-21 years) underwent allo-hsct from mrd for the following diseases: leukemia (n = 36), bone marrow failure (n = 15), thalassemia (n = 8), scid (n = 5), metabolic diseases (n = 4) and lymphoma (n = 2). the stem cell source was bone marrow for 56 patients (80%) and mobilized peripheral blood stem cells for 14 patients (20%). the grafts contained a median of 5.3 × 10 6 /kg total cd34 cells. tbi was used in 13 patients (18%). all but 4 patients achieved sustained neutrophil and platelet engraftment. after a median follow-up of 47 months (range: 4-135 months), the 2-year overall survival rate was 70% (95% ci: 57-79%). by multivariate analysis using cox proportional hazard regression model looking at the following factors for overall mortality: diagnosis, recipient's age, donor's age, the use of tbi, stem cell source, cd34 count, donor-recipient abo incompatibility, donorrecipient rh incompatibility, and donor-recipient sex-mismatch, the only statistically significant risk factor for mortality was donor-recipient rh incompatibility (hr: 3.26, p: 0.04). this risk was not statistically significant when looking at transplantrelated mortality (hr: 3.9, p: 017) and relapse-related mortality for malignant diseases (hr: 3, p: 0.12). there was no association between the incidence of acute or chronic gvhd and rh incompatibility. donor-recipient rh incompatibility was associated with an increased risk of mortality in children and adolescents undergoing mrd allo-hsct. further studies with larger number of patients are needed to confirm this finding. disclosure of conflict of interest: none. effect of iron or vitamin b12 deficiencies on in vitro colony forming capacity of peripheral blood-derived hematopoietic stem cells in children ny özbek, mm zabun, y köksal and m özgüner iron deficiency (id), id anemia (ida)and vitamin b12 deficiency (vit-b12d) are common disorders in developing countries. in urgent situations, children with these disorders could be donors before treatment. in this study, we investigated capacity of peripheral blood-derived hematopoietic stem cells to develop colony-forming units (cfu) in children with id and vit-b12, in vitro. patients and methods: we included 102 children (age 6 months-18 years) in the study in 5 groups: children with id (n = 15); children with ida (n = 20); children with vit-b12d (n = 12); children with both id and vit-b12d (i/ vit-b12d; n = 18); and control children (n = 37) who has normal peripheral blood findings, and normal ferritin and vit-b12 levels. from each child complete blood counts (cbc), and levels of ferritin, vit-b12, and cero-reactive protein (crp) have been obtained. who criteria, adjusted for age and sex, have been used for definition of anemia, id, ida and vit-b12d. four ml peripheral blood drawn into tubes with edta has been used for cfu analysis. mononuclear cell suspension (1.5 × 10 6 cell/ml), obtained from peripheral blood by ficoll-hypaque density gradient separation method, has been cultured in dishes containing semi-solid agar culture medium (methocult, h4434 classic, stem cell technologies, canada) in appropriate conditions. after 2 weeks, number of cfu colonies [burst forming erythroid (bfu-e); colony forming unitgranulocyte macrophage (cfu-gm); colony forming unitgranulocyte-erythrocyte-monocyte-megakaryocyte (cfu-gemm)] have been investigated by an inverted microscope. results: statistical analysis showed no difference between groups for age, sex, crp levels, and cfu-e, cfu-gm and cfu-gemm numbers. however, expected differences between groups were present concerning mean values of hemoglobin, ferritin and vit-b12 levels, mean corpuscular volume (mcv), and red cell distribution width (rdw) ( table 1) . discussion: this study shows in vitro proliferation capacity of peripheral stem cells has not been influenced by id, ida, vit-b12d, or i/vit-b12d. our results may indicate normal grafting ability of peripheral stem cells obtained from donors with iron, vit-b12 or i/vit-b12 deficiencies for hematopoietic stem cell transplantation. however, in vivo analysis should also be performed in order to reach a definite conclusion. [p128] disclosure of conflict of interest: none. efficiency of day 4 compared to day 6 stem cell mobilization in allogeneic donors h al-gaithi 1 , s al-mamar 2 , m al-huneini 2 , d dennison 2 , s al-kindi 2 , k al-farsi 2 and m al-khabori 2 1 hematology residency training program, oman medical specialty board; 2 hematology department, sultan qaboos university hospital granulocyte colony stimulating factor (g-csf) given for 4-6 days is commonly used for mobilization of allogeneic stem cell donors. the optimal days of g-csf administration is still debatable. the primary objective of this study is to compare the yield of stem cell mobilization, assessed using cd34+ cell count, between day 4 and day 6. secondary objectives include the assessment of the impact of donor's age, weight, mean corpuscular volume and blood group on the difference in the cd34+ cell count. in this retrospective study we included all allogeneic stem cell donors mobilized with g-csf for 6 days from january 2003 till october 2015 in the bone marrow transplantation unit at sultan qaboos university hospital. of 106 donor records reviewed, 84 were with available data and selected for the study. descriptive and analytical statistics were performed using stata 13.1. we included 84 donors with median age and weight of 19 years and 60 kg, respectively. the median day 4 wbc and cd34+ cell count were 37.4 × 10 9 /l and 54 × 10 6 /l respectively; while the median day 6 wbc and cd34+ cell count were 44.4 × 10 9 /l and 86 × 10 6 /l, respectively, (figure) with a statistically significant difference from day 4 (p o0.001). in the multivariable model, there were no significant impact of donor's age (p = 0.215), weight (p = 0.108), height (p = 0.428) and mean corpuscular volume (p = 0.263) on the difference in cd34+ cell yield. however, donor's blood group ab predicated a significantly higher difference (p = 0.036). six days of g-csf mobilization achieves higher cd34+ cell count than 4 days in allogeneic stem cell donors especially in donors with blood group ab. however, cd34+ cell count on day 4 is high enough to allow for successful mobilization. appropriately designed prospective trial is needed to confirm these results. disclosure of conflict of interest: none. there are known differences between individuals on an unrelated hsc donor register who decide to proceed with verification typing (vt) vs those who choose not to. in the anthony nolan registry, white british donors are more than twice as likely as other ethnic groups to continue with testing at vt (or 2.44; po 0.001 (unpublished data)). the purpose of this study was to explore differences in key characteristics between white british donors and british donors from other ethnic groups with a view to developing interventions to reduce vt stage attrition. study recruitment occurred april 2013-may 2016. all donors not proceeding at vt were invited to participate, and a stratified random sample of those proceeding at vt were recruited to meet pre-determined targets for each ethnic group. data were collected via structured interview (telephone or online). 4 broad categories of participant characteristics were assessed: demographic, culturally related, psychosocial, and donation-related. measures were previously validated scales with established psychometric properties either created for, or used in other donation-related settings. for analyses donors were divided into two groups based on ethnicity: white british (wb), and non-white british (nwb). results: 170 wb donors and 187 nwb donors completed interviews donors proceeding at vt were more likely than their counterparts to participate in the study (66% vs 25%, p o0.001). mean donor age was 33.8 with no difference between ethnic groups and 43% of donors in both groups were female. nwb were statistically more likely to have completed higher education, and have a stronger religious affiliation. in contrast they were less likely to be blood or organ donors. nwb also described greater mistrust of the medical system and of hsc allocation. nwb donors were more likely to have joined the register at a recruitment event (p=0.012) or a place of worship (p=0.012), while wb donors were more likely to have joined online (p=0.013). wb donors reported significantly higher scores regarding feeling well informed about donation both at the point of joining, and at the point of vt and were more likely to remember joining the register and the two donation methods. this study highlights important differences in demographics, culturally related variables and donor interaction with the register between white british donors and donors from other ethnic backgrounds. given the higher rate of vt attrition in nwb donors, these findings could be used to tailor interactions/information given to donors on the register to ensure their priorities are addressed. disclosure of conflict of interest: none. data on mismatched family donor transplants for myelofibrosis are scarce due to the risk of poor engraftment, gvhd and exclusion from trials. outcomes from such transplants performed between 2001 and 2015 reported to the ebmt are presented. sixty-nine patients, median age 58 (27-71) years; 44 (64%) male, 50 (74%) had primary, 18 (27%) had secondary myelofibrosis (6 from et, 5 from prv and 7 others) and unknown 1(2%). jak2 v617f was mutated in 15/25. karnofsky performance status was 470% in 98 %; median time from diagnosis to allograft was 41.4 (range: 0.72-213) months. the donors were predominantly male 47 (68%), median age 42 (22-75) years, hla mismatched at 1 locus in 12 (17%) and 2 or more loci in 48 (70%). donor-recipient serology was cmv − / − in 8 (12%) ± in 4 (6%), − /+ in 15 (22%) and +/+ in 34 (49 %) missing 8 (12%) . bone marrow was used in 34 (49%) and peripheral blood in 35 (51%). the median total nucleated cell count (tnc) was 7.5 × 10 8 /kg (range: 2.3-21 × 10 8 /kg) (n = 17). the median cd34+ cell dose was 6.9 × 10 6 /kg [p130] s182 (range: 1.9-18.18 × 10 6 /kg)(n = 19). patients. conditioning was myeloablative in 48 (70%) and ric in 21 (30%). predominant conditioning regimes were fludarabine, busulphan, atg (fbatg) and thiotepa, busulphan, fludarabine (tbf n = 33). tbi was administered in 8 (12%) and t cell depletion in vivo in 22 (32%) and ex vivo in 5 (7%) patients. gvhd prophylaxis varied with post transplant cyclophosphamide administered in 33/67 (48%) and atg in 19/67 patients (28%).neutrophil engraftment occurred in 53 (82%) patients at a median of 20 days (range: 11-83). primary graft failure ensued in 8 (12%) and secondary graft failure in 4 (6%) patients at a median of 12 (range: 4.5-35) months. eleven patients had a second allograft at a median interval of 4 (1-20) months. responses to the first allograft censoring for a second allograft, data available in 45 patients, showed that complete remission was achieved in 35 patients (78 %), 6 (13 %) were never in cr and 4 (9 %) were not evaluable. relapse occurred in 8 (12%) of patients at a median interval of 3 (2.8-21.8) months. the cumulative incidence (ci) of grade ii-iv acute gvhd (agvhd)was 12% (95% ci 4-21%) and for grade iii-iv agvhd at was 5% (95% ci 3-11%). data for chronic gvhd (cgvhd) was valid in 49 patients of whom 47% developed cgvhd. the ci of cgvhd at 2 years was 58% (95% ci 43-72%):ci of limited cgvhd was 45% (95% ci 31-59%) whereas the ci of extensive cgvhd was 10% (95% ci 2-19%). median follow-up was 24 (95% ci 13-35) months. the 2 and 5 year os was 53% (95% ci 40-66%) and was 40% (95% ci 23-57%). the 2 and 5 year rfs was 43% (95% ci 30-56%) and 31% (95% ci 15-47%). the 2-year ci of relapse was 20% (95% ci 10-30%). the 2 year nrm was 37% (95% ci 25-49%), which increased to 49% (95% ci 32-65%) at 5 years. thirty patients died due to infection (16, 53%), gvhd (7, 23%), organ damage or failure (3, 10%), relapse/disease progression (1, 3%) and secondary malignancy or ptld (1, 3%) unknown 2. there was no significant effect (univariate analysis) of recipient or donor gender, degree of hla mismatch, cmv matching, primary or secondary mf, chronic vs advanced disease at transplant, conditioning intensity or regimen, gvhd prophylaxis with atg or post transplant cyclophosphamide or stem cell source on overall survival. the data are encouraging for patients with myelofibrosis, with engraftment, pfs and os being attained with limited severe chronic gvhd from family mismatched donors. disclosure of conflict of interest: none for all other authors, fc consulting with molmed. feasibility of salvage second allogeneic stem cell transplantation for disease relapse or graft failure: a single centre experience g battipaglia 1,2 , d salvatore 3,1 , r dulery 3 , f giannotti 3 , f malard 2 , e brissot 2 , s sestili 2 , f isnard 2 , s lapusan 2 , a-c mamez 2 despite high rates of toxicity and mortality, a second salvage allogeneic stem cell transplantation (second allohsct) might be an option to consider in patients experiencing disease relapse or graft failure after first allohsct. we retrospectively analyzed outcomes after second allohsct in a cohort of 30 patients (18 males and 12 females) transplanted either for disease relapse (group 1, n = 19) or graft failure (group 2, n = 11) between 2007 and 2015 in a single centre in france. median age at second allohsct was 38 (range: 17-64) years. diagnoses were acute myeloid leukemia (group 1: n = 12; group 2: n = 3), acute lymphoblastic leukemia (group 1: n = 2; group 2: n = 4), myelodysplastic syndrome (group 1: n = 2; group 2: n = 1), myeloproliferative neoplasm (group 1: n = 3; group 2: n = 1), bone marrow failure (group 1: n = 0; group 2: n = 2). median time from first allohsct to second allohsct was 38 (range: 2.5-230) months in group 1 and 1.5 (range: 1-34) months in group 2. graft source for the second allohsct were: haploidentical bone marrow (group 1: n = 3; group 2: n = 1), haploidentical pbscs (group 1: n = 5; group 2: n = 1), cord blood (group 1: n = 8; group 2: n = 8), matched unrelated pbsc (group 1: n = 3; group 2: n = 1). at time of second allohsct, 11 patients were in cr and 8 presented active disease in group 1. conditioning regimen was myeloablative in 5 patients (group 1: n = 3; group 2: n = 2), reduced intensity (ric) in 20 cases (group 1: n = 11; group 2: n = 9). a sequential schema consisting of a combination of thiotepa, etoposide and cyclophosphamide followed by a fludarabine and busulfanbased ric was used in 5 out of 8 patients with active disease in group 1. sixteen patients received atg as part of the conditioning regimen for second allohsct (group 1: n = 10; group 2: n = 6). all but one patient engrafted, at a median time of 18 (range: 6-51) days. cumulative incidence of acute and chronic gvhd were 27 ± 17% and 42 ± 19%, respectively, 2-107) months, non-relapse-mortality (nrm) and relapse incidence (ri) were 24 ± 15% and 27 ± 15%, respectively, while disease-free (dfs) and overall survival (os) were 49 ± 19% and 48 ± 15%, respectively, for the entire cohort. in all, 15 patients died of infections (n = 8), hematological disease (n = 4), gvhd (n = 1), hemorrhage (n = 1) and for unknown causes (n = 1). main outcomes of patients in group 1 were: ri 16 ± 15%, nrm 28 ± 18%, agvhd 26 ± 15%, cgvhd 56 ± 20%, dfs 56 ± 20%, os 55 ± 20%, respectively. main outcomes of patients in group 2 were: ri 45 ± 22%, nrm 18 ± 24%, dfs 36 ± 29%, os 36 ± 29%, agvhd 27 ± 23%, cgvhd 18 ± 23%. historically, a second allohsct was hampered by significant morbidity and mortality. however, the advent of reduced-toxicity conditioning regimens and improved supportive care allowed to significantly improve the results of patients receiving a second allohsct as suggested from the above results. therefore, a second allohsct could be considered as an option to rescue a certain number of patients experiencing disease relapse or graft failure, for which prognosis is very poor. decision is to be discussed on a case-by-case basis. disclosure of conflict of interest: none. haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide for patients with high-risk hematologic malignancies am carella department of oncology and hematology, irccs casa sollievo della sofferenza, san giovanni rotondo allogenic hematopoietic stem cell transplantation (sct) has been increasingly used for treatment of adult with high risk hematologic malignancies. for patients lacking an hlamatched related or unrelated donor, unmanipulated haploidentical (haplo)-sct is a potential alternative. haploidentical transplantation performed with post-transplantation cyclophosphamide (ptcy)-based graft-versus-host disease (gvhd) prophylaxis has been associated with favorable outcomes for patients with acute leukemia and lymphomas we analyzed outcomes of 45 patients with hematologic malignancies who received t-cell-replete haematopoietic stem cells and posttransplantation cyclophosphamide after myeloablative or nonmyeloablative hla-haploidentical donor transplantation. the median age was 37 years (14-68); twelve patients were in first remission (cr1), 4 in second remission (cr2) and 29 had an active disease. ). the diagnosis was acute leukemia (n = 32), myelodisplastic syndrome (n = 3), hodgkin disease (n = 7) non hodgkin lymphoma (n = 2) and multiple myeloma (n = 1). median follow-up was 260 days. stem cell source was bone marrow (bm) for 42 patients, and peripheral blood (pb) for 3. myeloablative conditioning (mac) was used in 37 patients and reduced intensity regimen (ric) in 8 patients. thirty one patients were first grafts, the others underwent previous autologous sct (n = 11) or mud (n = 3). gvhd prophylaxis s183 consisted in pt-cy on days +3 and +4, cyclosporine (from day +5), and mycophenolate (from day +5). the median day for neutrophil engraftment was day +20 (14-29). no graft failure was observed. chimerism was evaluable in 39 patient; on day + 30 all patients had 100% donor chimerism on marrow cells median follow-up was 260 days. the cumulative incidence of acute gvhd grade ii-iv was 22%, grade iii-iv 9% and chronic gvhd 15%. one-and 2-years os was 56.53% and 53.39 %, respectively. with a median follow-up for the surviving patients of 752 days (130-2207), the cumulative incidence of transplant-related mortality (trm) is 22%, and the relapserelated death is 26%. thus, we demonstrate excellent rates of engraftment, gvhd, and trm in adult patients treated with haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide. this approach is a widely available, safe, and feasible option for adult patients with high risk hematologic malignancies, including those with a prior history of myeloablative bmt and/or those with co-morbidities or organ dysfunction, also for patients with active disease at the time of transplant. disclosure of conflict of interest: none. it has recently been shown that t-replete allogeneichematopoietic stem-cell transplantation (allo-hsct) from a haploidentical donor (haplo-id) could be a valid option when a matched donor is not available. unfortunately, the worldwide donor registries comprise mainly donors of caucasian origin and patients of non-caucasian origin have a much lower chance of finding a matched unrelated donor (mud). the lengthy period of international search when required and the financial burden of this process are considered as additional significant limitations. at the american university of beirut medical center (aubmc) in lebanon, we started the mud program in 2011 and haplo-id hsct program in 2014. we report here our experience in this two groups of patients. patients and methods: we have transplanted 21 patients from a haplo-id donor since 2014 and compare their outcome with the 6 patients transplanted from a mud since 2011. the patients and transplant characteristics are listed in the table 1 . the 2 groups were comparable except for conditioning. patients in haplo-id group received two days of posttransplant high-dose cyclophosphamide (pt-hdcy) followed by cyclosporine a (csa) and mycophenolate-mofetil while patients in the mud group received pre-transplant antithymocyte-globulins and csa starting on day-3. all patients engrafted in the mud group, while one patient did not engraft in the haplo-id group, the patient had refractory all transplanted with progressive disease, and died on day +47. the median of anc 4500/mm 3 was 14 days (12-20) vs 17 days (12-29) in the haplo-id and mud groups, respectively. fourteen patients from the haplo-id group developed grade 2 acute graft-versus-host disease (agvhd) vs one after mud-hsct. two patients haplo-id group developed limited cgvhd and none after mud grafts. six patients relapsed in the haplo-id group vs three patients in the mud group. two and three patients died from non-relapse mortality in the haplo-id and mud group, respectively. at the last follow-up, 13 patients are still alive in the haplo group vs 2 patients in mud group and all of them are in cr. we conclude that t-replete haplo-id hsct followed by pt-hd cy is associated with promising results or at least comparable to patients transplanted from mud. haplo-id hsct seemed to be safe and feasible in patients with high risk hematological malignancies. finally, because of the obvious advantage in rapidly finding a donor (21 haplo transplants in three years vs 6 mud transplants in 5 years), development of haplo-id hsct is warranted to satisfy the regional needs. [p134] disclosure of conflict of interest: none. haploidentical hematopoietic stem cell transplantation (haplo-hsct) using t-cell-replete (tcr) grafts and posttransplantation cyclophosphamide (ptcy) provides a curative approach for patients with high-risk mds/aml lacking a conventional hla-matched donor. in children and adults haplo-hsct using ptcy as gvhd prophylaxis seems to be safe with low treatment related morbidity and mortality (trm). however, few data are available for elderly patients with advanced disease. we retrospectively analyzed the outcome of 49 patients with mds (n = 5)/aml (n = 44) age 50-74 years (median age 60 years; 24 patients 50-59 years, 25 patients ⩾ 60 years; 21 male), who underwent tcr haplo-hsct with high-dose ptcy at our institution between january 2009 and november 2016. disease was active in 41 patients while 8 had achieved cr. 12 patients failed previous allo-hsct. pretransplantation risk factors were scored using the hematopoietic cell transplantation-specific comorbidity index (hct-ci) which was ⩾ 3 in 19 patients (median hct-ci = 2, range: 0-8). a sequential therapeutic concept using either flamsa (n = 28) or clofarabine (n = 18) as cytoreduction was used prior to reduced intensity conditioning (ric) in all but 3 patients. ric consisted of fludarabine/cyclophosphamide combined with either melphalan (n = 32), busulfan (n = 1) or 4 gy tbi (n = 12). post-grafting immunosuppression consisted of cyclophosphamide, tacrolimus and mmf in all patients. 57% received a bone marrow graft. one graft rejection occurred. neutrophil and platelet engraftment was achieved in 95% and 77% of evaluable patients, respectively at a median of 19 (13-89) and 33 (11-103) days. acute gvhd grade i-iii occurred in 29% of the patients whereas no grade iv agvhd was observed. chronic gvhd presented in 33%. it was most frequently assessed as mild to moderate (13 pts). only 3 patients developed severe cgvhd; no gvhd related death was observed. cmv reactivated in 22 of 36 patients at risk, one patient developed cmv disease (pneumonia). no ebv reactivation or ptld occurred. one-year trm was 24%. 12/49 (24%) patients relapsed, three within the first 100 days after haplo-hsct. at a median follow up of 27 months (range: 4-74 months) estimated one-and two-year overall survival (os) was 55/46 %, respectively. when stratified by age, estimated one-and two-year os was 65/42% in patients o60 years and 47/47 % in patients ⩾ 60 years (p = 0.771/p = 0.794). one-and two-year progression-free survival (pfs) was 50/45%, respectively. stratified by age estimated one-and two-year pfs was 53/41% in patients o 60 years and 47/47% in the elderly (p = 0.836/p = 0.887). unmanipulated haploidentical allografting using ptcy-based gvhd prophylaxis in high-risk mds and aml patients aged over 50 years is safe and well tolerated resulting in acceptable trm. a remarkable survival outcome can be achieved in elderly high-risk aml/mds patients with significant comorbidities. disclosure of conflict of interest: none. key performance indicators to assess the quality of a collection facility: experience of a single center s roncon*, c pinho 1 , f bordalo 1 , s lopes 1 , s ferreira 1 and f amado 1 1 allogeneic hematopoietic stem cell transplantation (allo-hsct) has evolved into an effective immunotherapy for the treatment of a variety of disorders. when patients do not have a familiar matching donor, transplant centers (tc) search for an unrelated and volunteer donor. this one must be previously evaluated by the collection center (cc) to donate peripheral blood stem cells (pbsc) or bone marrow (bm); lymphocytes can also be asked after allo-hsct. this work aims to evaluate our performance as cc, ensuring donor safety, quality of cell therapy products (ctp) and the accomplishment of tc requirements. we retrospectively analyzed all the requests of ctp collections sent by the portuguese registry from 2012 to 2016. countries of destination, number and type of ctp were determined. we established eight key performance indicators (kpi) classified into four categories: response time; product quality; satisfaction of patients and donors; and on-site donor motivation. the intended target was defined by the mean result obtained in the first half of 2012 (excluding kpi-7). written comments from donor center (dc) and tc were received by email or written in the local notebook. the donor's answers were obtained through a survey given on the collection day. a total of 349 requests were assessed: 231 pbsc, 61 bm, 16 lymphocytes and 41 cancellations; 84% were sent to europe (98/259 to portugal), 14% to america and 2% to oceania; 30/41 were withdrawn by tc (14 patients died, 14 presented progressive disease and 2 had a better hlamatched donor) and 11/41 by dc (7 donors not cleared and 4 refused). the results obtained with kpi-1, -2, -4 and -7 exceeded the intended target (table 1) . after the first kpi-1 results, we verified a positive evolution. we took an average of 3 days of delay in sending donor clearance. however, there is no holdup in the ctp delivery, as demonstrated by kpi-2. regarding kpi-3 it is important to notice that 60% of ctp with a cell number less than requested were bm and lymphocytes; when pbsc was considered separately, the result increased (87% vs 75%). analyzing kpi-4, 85% (n = 11/13) of the contaminated ctp were bm. concerning kpi-5, -6 acknowledgments and -8 commitment, we recognize that our initial targets were too ambitious (100%). the kpi-6 shows a low number of complaints (n = 4): one due to a misreading of the request and three to communication failures; all were properly examined and rectified. a good general status was guaranteed in almost all the donors (kpi-7). the decrease of kpi-8 is due to the fact that one donor refused to proceed after three postponements of the collection date by tc. table 1 -key performance indicators of the quality of our activity as a cc. the overall good level of our results reflects an extremely professional performance as a cc. we consider that these kpi should be continuously monitored with the purpose of earlier detect any deviation of the stated goals and assess the progress against settled strategies. we further suggest the establishment of universal indicators in order to standardize [p136] practices, share expertise and improve the quality of services and products provided to patients and donors. two year later, the patient had a genoidentical allogenic stem cell transplant (from the bone marrow stem cell of his sister, who was 35 years old, hla compatible). he had a reduced intensity conditioning, associating busulfan, fludarbine and antilymphocyte serum. 6 months from the asct, he was in complete remission with 100% donor chimerism. 4 years after the asct, the patient presented a progressive thrombocytopenia without any other peripheral causes. the bone marrow aspiration initially showed a refactory cytopenia with multilineage dysplasia. the patient was followed up during 12 months, and then a second bone marrow aspiration has shown a refractory anemia with excess blasts2 raeb2. a cytogenetic study has every time demonstrated a female karyotype (44,xx) on 20 mitoses out is 20, and chimerism was 100% donor. the diagnosis of the myelodsplastic syndrome of the donor cells was approved. the patient was treated by azacitidine (75 mg/m 2 , from j1 to j7, j1 = j28). after 6 cycles, the patient was in complete hematologic response (normalization of the platelet count) and a partial bone marrow response (normalization of the blasts rate but persistence of the signs of dysplasia). he received 6 more cycles, and presented hematologic relapse (reemerging of thrombopenia). a phenoidentical allogenic stem cell transplantation was suggested. conclusion the occurrence of mds on the donor cells is rare. these anomalies are secondary to intrinsic factors (of donor) or extrinsic factors )of the transplant recipient). the treatment is not definitely determined. disclosure of conflict of interest: none. nk-cell alloreactivity based on kir/ligand mismatch in the donor vs recipient direction provides better graft-versustumor effect in patients with active hematological malignancies undergoing allogeneic t-replete haploidentical transplantation followed by post-transplant cyclophosphamide a wanquet 1,2 , s bramanti 1,3 , s harbi 1 , s fürst 1 , f legrand 1 , c faucher 1 , a granata 1 , p-j weiller 1,2 , c picard 4 , b calmels 5 , c lemarie 5 , c chabannon 2,5,6 , l castagna 1,3 , d blaise 1,2,6 and r devillier 1, haplo-sct have been developed in the past years with very interesting results in high risk patients. gvhd prophylaxis using post-transplant cyclophosphamide (pt-cy) recently allowed extending the use of unmanipulated haplo-sct. it was shown that nk alloreactivity, triggered by donor-recipient inhibitory kir gene-gene mismatches, could lead to better outcomes and survival in the setting of in t-cell-depleted haplo-sct. however, few data is available on the impact of kir-ligand mismatch on the outcome after t-replete haplo-sct with pt-cy. we thus assessed the impact of nk alloreactivity on the outcome of patients who received haplo-sct followed by pt-cy. we retrospectively collected the data from patients from two centers who were treated for various high risk hematological diseases and underwent a haplo-sct with pt-cy from december 2009 to december 2014. we assessed the kir-binding epitope in hla-c and hla-b molecules for all patients, and we predicted nk cell alloreactivity in the donor vs recipient direction via the immune polymorphism database kir ligand calculator, based on the kir-ligand mismatch between donors and patients. because disease status at the time of haplo-sct is one of the most important predictor of outcome, we separately analyzed two cohorts of patients: those transplanted in complete remission (cr group) and those transplanted with active disease (no cr group). using a multivariate cox model (adjusted by disease type, age and conditioning), we therefore evaluated the impact of nk alloreactivity on outcome in both cr and no cr groups. we analyzed 144 patients with a median age of 54y (20-74). they were mostly transplanted for lymphoma (n = 72, 50%) or aml/ mds (n = 47, 33%). patients mostly received a tbi-based nonmyeloablative conditioning regimen (n = 94, 65%) and pbsc as graft source (n = 91, 63%). eighty one and 63 patients were transplanted in cr and in no cr, respectively. nk alloreactivity was found in 30/81 cr patients (37%) and 22/63 no cr patients (35%). with a median follow up of 30 months (12-77), cr patients had a significantly better outcome than those in the no cr group (2-year pfs 65% vs 32%, respectively, p o0.001). in no cr patients, multivariate analysis showed that nk alloreactivity was significantly associated with reduced the risk of relapse (hr = 0.25, p = 0.019, figure 1a ) with no increase of both acute (hr = 1.30, p = 0.648) and chronic gvhd (hr = 2.61, p = 0.232), and nrm (hr = 0.60, p = 0.277). this led to significantly better pfs (hr = 0.41, p = 0.014, figure 1b ) and a trend for better os (hr = 0.52, p = 0.069). in contrast, in cr patients, we found no difference in outcome according to nk alloreactivity for all end points (acute gvhd: hr = 1.78, p = 0.204; chronic gvhd: hr = 2.11, p = 0.321, nrm: hr = 1.69, p = 0.313, relapse: hr = 0.85, p = 0.762, figure 1c ; pfs: hr = 1.19, p = 0.637, figure 1d ; os: hr = 0.83, p = 0.672). our results suggest that nk alloreactivity provides better disease control with no increase of gvhd, especially in patients transplanted with active disease. thus, donor selection should rely on the prediction of nk alloreactivity. this may contribute to improve outcome of these patients with high risk of relapse after transplantation, underlining the need of a specific strategy of donor search, and the promising perspective of early post-transplant nk-cell-based immunotherapy. haploidentical bone marrow transplantation (haplo-bmt) with post-transplant cyclophosphamide (pt-cy) is being increasingly used, in the last five years, for patients lacking a suitable hla-matched donor. genoa study (eudract number: 2012-000703-32) provides for a modified gvhd prophylaxis platform compared to the original baltimora protocol. aim of the study: in this study we assessed outcomes in 282 consecutive patients transplanted from a haploidentical donor for haematological malignancies. all patients received a uniform gvhd prophylaxis: cyclosporine (csa) starting on day 0, mycophenolate (mmf) starting on day +1, and post transplant cyclophosphamide (pt-cy) 50 mg/kg, on days +3 and +5. all patients received a myeloablative conditioning consisting of thiotepa, fludarabine, busulfan (three doses n = 116 or two doses n = 111), or tbi, fludarabine (n = 55). the median age was 48 years (17-74); at transplant 145 (51%) patients were in remission of disease (cr1 and cr2), and 137 had an active disease (49%); all patients were first grafts. the diagnosis were acute myeloid leukemia (n = 111), myelodisplastic syndrome (n = 31), acute lymphoblastic leukemia (n = 56), myelofibrosis and myeloproliferative diseases (n = 43), non hodgkin lymphoma (n = 19), chronic lymphocytic leukemia (n = 9) and multiple myeloma (n = 13). the median follow up was 562 days (range: 6-2241 days). the median infused mononucleated cells was 3.4 × 10e8/kg (range: 1.1-7.7). seven patients died before engraftment, and 21 (7%) had autologous recovery: 15 (5%) after conditioning with 2 doses of busulfan. full-donor chimerism on day +30 was reached in 254 (90%) patients. the median day for neutrophil engraftment was day +18 (range: 13-60 days). the cumulative incidence of grade ii-iv and iii-iv acute gvhd (agvhd) was 17% (n = 49) and 5% (n = 15), respectively. two years cumulative incidence of moderate-severe chronic gvhd (cgvhd) was 13% (n = 39).sixty one (21%) patients experienced haemorragic cystitis. at 3 years the cumulative incidence of non relapse mortality (nrm), relapse and relapse related death was 17% (n = 47), 32% (n = 91) and 25% (n = 69), respectively. causes of death were infections (n = 34), hemorrhage (n = 7), gvhd (n = 5), secondary neoplasia (n = 1) and relapse (n = 69). at 4 years of follow up overall survival and disease free survival was 55.7% and 47%, respectively. at the same time overall survival rate was 73% for patients in remission and 29% for patients with active disease at transplant(p o0.001). in conclusion, a modified pt-cy as gvhd prophylaxis and ma conditioning regimen followed by haploidentical bmt results in a low risk of agvhd and cgvhd and encouraging rates of trm and dfs. disclosure of conflict of interest: none. the italian bone marrow donor registry (ibmdr), in collaboration with admo (associazione donatori di midollo osseo) since 2009 has implemented, as part of the donor enrollment strategy, public enrollment events (pe). our donor center (dc) has taken part to those events since the first years. one or more clinician (or trained biologist) has been present to pe to inform the potential donors, evaluate the candidates and supervise the collection of biological fluids. all the local permission where obtained. aim: aim of this study was to compare the compliance of the donor enrolled in pe with donors enrolled at our dc institutional site. we prospectively evaluated all the donors recalled for further evaluation and/or for requalification in the years 2014 and 2015 at our dc itmi07. we defined 3 possible results for the call: ‛success' (the donor was eligible and accepted to be evaluated, or only temporarily ineligible) ‛not eligible' (the donor was definitively ineligible) and ‛consent denied'. results: a total of 286 donors were called back in the years 2014 and 2015 (16 not found). eightyfour recalled donors had been enrolled after 2009. among them 53 (63.1%) had been enrolled at the dc and 31 during pe (36.9%). the two populations were not different for age at the call, age at enrollment and gender (table 1) . [p140] when evaluating the probability of obtaining a "success", no significant difference was found between the two populations: 86.8% vs. 83.9% (chi square p=0.23). no significant difference was also found for the "not eligible" and the "consent denied" categories. of note, when we turned to the whole 286 donor population we had called back (median age 34, range 21-55), the probability of "success" and "consent denied" were not related to donor age, and time from enrollment to recall, whereas donor ineligibility was (spearman test p=0.02 and 0.002). public events with the presence of an adequate trained medical team represent a valid option for the enrollment of new unrelated donors. disclosure of conflict of interest: none. the search for hematopoietic stem cell unrelated donors in patients with malignant hemopathies with not-sibling matched family donor: the experience of a center a pérez 1 , r goterris 1 , m gómez 1 , s blanco 1 , a segado 1 , c arbona 1 , jch boluda 1 , m poch 1 and c solano 1 1 hematology department, hospital clínico universitario, valencia unfortunately, as few as 30-35% of patients will have an hlaidentical matched sibling donor available for hematopoietic stem cell (hst) donation. the search for an unrelated donor (urd) (adult or cord blood) is often the best option for those patients lacking a suitable matched donor. below we describe the experience with the search for an unrelated donor in our center. between september 1995 and march 2016 the search for urd was activated for 263 patients. the median age of the patients was 46 years (range: 0.4-69), 10% were under 18 years and 60% were males. acute myeloid leukemia (n = 67), acute lymphoblastic leukemia (n = 43), non-hodgkin's lymphoma (n = 60), chronic/prolymphocytic lymphocytic leukemia (n = 23), hodgkin's lymphoma ((n = 13), multiple myeloma (n = 14), chronic myeloid leukemia (n = 15), philadelphianegative myeloproliferative neoplasms (n = 9), myelodysplastic syndrome (n = 8), aplastic anemia/paroxysmal nocturnal hemoglobinuria (n = 6), others (n = 5). the disease status in hematological malignancies was: first cr (n = 77), 4 second cr (n = 49), pr (n = 46) and refractoriness (n = 82). the donor type requested at the activation of the search was an adult (n = 110), umbilical cord blood (n = 7) and two options (n = 146). results: a compatible donor was found in 197 patients (76% of the series) after a median of 44 days (range: 1-847) from the activation of the search. the degree of adult donor compatibility (not available in 7 cases) was: complete hla identity (8/8: n = 49, 10/10: n = 37); an hla difference (7/8: n = 12, 9/10: n = 31); lower degree of compatibility (n = 13). the degree of umbilical cord blood compatibility: identity ⩾ 4/6 (n = 48). a total of 151 patients (57%) were transplanted, 103 from adult donor and 48 from umbilical cord blood. the median time between the activation of the search and the hst transplantation was 4 months (range: 0.7-29), being 3.2 months for acute leukemia and 5.1 months for other pathologies, and between the location of the donor and the hst transplantation 70 days (range: 5-412), being 45 days for umbilical cord blood and 76 days for an adult donor. there were 108 cancellations of the urd search (41% of the total) for the following reasons: clinical status of the patient (n = 63), performing a haploidentical transplant (n = 20), transplant center does not consider (n = 9), norms of the registry (n = 8) and loss of indication of transplantation (n = 8). the median time from the beginning of the search to its cancellation was 4.5 months (range: 0.3-53). at the time of analysis, the median follow-up of the 263 patients is 17 months. the survival of the series in the 5 years is 37% and 43% for patients transplanted from urd. 76% of the searches activated in our center allowed the localization of a urd with an adequate degree of hla compatibility. however, only 57% of the patients for whom the search was activated were finally transplanted. the most frequent cause of cancellation of the procedure was the clinical deterioration of the patient. disclosure of conflict of interest: none. the leukemic transformation of otherwise healthy donor stem cells provides a useful in vivo model to study the mechanisms involved in leukemogenesis. we report two cases of donor cell-derived haematological malignancy in which wholeexome sequencing (wes) was performed in bone marrow (bm) samples from recipient at different times after allogeneic hematopoietic stem cell transplantation (allo-hsct) in order to study the dynamics of emergence of mutations that precede the development of donor cell leukemia (dcl) and donor cell myelodysplastic syndrome (dc-mds). case 1: a 43-year-old female diagnosed with lymphoblastic leukemia-b t(1;19), who developed acute myeloid leukemia (aml) with normal karyotype, npm1+of donor origin 16 months after unrelated cord blood transplantation (ucbt). case 2: a 65-year-old male diagnosed with mantle cell lymphoma, who developed mds 45,xx,-7,del(12)(p12) of donor origin, 57 months after allogeneic bm transplantation from his hla-identical brother. the donor also developed mds several months later. wes (sureselect-xt human-exon 50mb) was performed by next generation sequencing (hiseq) on donor stem cells (scs) infused as well as on bm samples from recipient after allo-hsct. the exome of donor scs and 5 bm samples, from case 1, were aligned to the human reference genome (grch 37/hg19) and donor scs and 9 bm samples were aligned to grch 38/ hg38 in the second case. in both cases non-synonymous variants in the coding regions or synonymous variants in splice regions of genes related to leukemia were selected. in addition, bm samples were matched to their scs and to prior bm samples to identify the acquired variants. variants meeting such criteria were evaluated with 3 functional predictor software's (sift, polyphen2 and mutation taster). wes analysis revealed progressive emergence of multiple somatic mutations probably related to the development of leukemia in bone marrow samples post allo-hsct ( figure 1 ). both scs showed alterations that may be involved in leukemogenesis. (case 1: sh2b3 and case 2: kmt2c, kmt2a, arhgap26 and monosomy 7). somatic mutations, acquired over time, fall into genes that play well-established roles in signalling pathways (ras-mapk, pre-mrna splicing factor, apoptosis, dna doublestrand break repair, dna replication and so on). mutations in leukemic subclones that disappear after chemotherapy were indentified, as well as the acquisition of new mutations in resistant subclones. we propose a possible model of leukemogenesis in these cases ( figure 2 ). the present study reveals a process of sequential clonal expansions, promoted by the acquisition of additional somatic mutations in donor hematopoietic cells. detection of heritable or acquired gene mutations in donor associated with predisposition to haematological malignancies could have clinical implications for the patients undergoing to allo-hsct. although the cause of donor cell-derived haematological malignancy onset seems to be multifactorial, the infusion of a scu with pre-leukemic potential in a context of residual toxicity in recipient as a result of pre-transplant chemotherapy, a post-transplant environment characterized by a decreased immune surveillance may well have played role in these cases. the study of a greater number of dcl cases by next generation sequencing could help to understand this process and to detect new mutations involved in the emergence of aml. disclosure of conflict of interest: none. the impact of donor and recipient sex in allogeneic stem cell transplantation-single center experience (cic 859) y petrov 1 , p ganeva 1 , g arnaudov 1 , s lozenov 1 , y davidkova 1 , v stoeva 1 , i tonev 1 , m guenova 1 and g mihaylov 1 1 national hospital for active treatment of hematological diseases allogeneic hematopoietic stem cell transplantation (hsct) has been one of the most effective therapeutic modalities for patients with hematological malignancies and bone marrow failure syndromes. optimal donor selection is one of the key factors to enhance the success rate of this procedure. we [p142] s189 retrospectively investigated whether and how donor-recipient sex affects transplantation outcomes of 73 patients transplanted between 2010 and 2015 in our center. the median age of the patients was 37 years (range: 23-51). thirty-nine of the patients (53%) received a pbsc from a hla-identical sibling, and 34 patients (46.5%) received pbsc from matched unrelated donor. forty-six percent were male recipients with male donors (m-m), 11.9% were female recipients with male donors (m-f), 23.8% male recipients with female donors (f-m), and 17.8% female recipients with female donors (f-f). we performed a crosstab analysis and χ 2 tests to observe whether the donor sex affects our study population. patients with male donor had superior overall survival and progression-free survival compared to those with female donor (66.7% vs 29.0% p = 0.001 for os, and 52.3% vs 34.2% p = 0.003 for pfs; cramer`s v = 0.372). we further investigated how the disparity of the donor in the four groups (m-m, m-f, f-m and f-f) affects the os, pfs and nrm. the f-m group had a worse overall and progression-free survival comparing the other groups (11% 4-year os and 17% pfs; p o0.0001).this group had 27% relative increase in the non-relapse mortality compared with m-m group (p = 0.009). for m-m group there was a 2% relative increase in the subdistribution hazard of nrm compared with m-f group (p = 0.02). the f-f group and m-f group had similar subdistribution hazard of nrm (39% vs 40% p = 0.009). the incidence of acute gvhd and chronic gvhd for the groups was: 34% and 41% (m-m), 37% and 32% (m-f), 41% and 40% (f-m), 32% and 7% for the (f-f) group. the appearance of either acute or chronic gvhd did not show statistical significance regarding the os and pfs in the groups (p = 0.07). we examined the effect of donor-recipient sex incompatibility on the outcome of hsct in out center. our results showed inferior os and pfs for f-m group and a higher incidence of nrm compared with other groups. these effects might be associated with allogeneic immune responses against h-y antigens. key words: stem cell transplantation, donor sex, recipient sex, overall and progression-free survival [p143] disclosure of conflict of interest: none. from 2011 to 2014, 66% of the 3834 patients affected by hematological malignancy searching for an unrelated donor through the italian registry successfully identified a suitable donor. this proportion increases up to 71% when searching for a cord blood unit was considered, corresponding to total transplant efficiency of 62%. from april 2006, the rome transplant network adopted a unique policy for the identification of a potential alternative donor, following a hierarchical selection that considered as first choice a volunteer unrelated donor, secondly a cord blood unit and last a haploidentical related donor. before starting the unrelated donor search, a preliminary query through the bone marrow donor worldwide database was performed for all the patients referred to the rome transplant network. based on the low resolution hla typing (a, b and drb1) it was possible to arbitrary assign a good or poor score that might predict the identification of a full matched (8/8 a, b, c and drb1) donor. therefore, aims of the present study were to assess the utility of the preliminary query and the impact of the use of high resolution hla typing since the starting of donor search on the timing for the unrelated donor identification. moreover, the final aim was of comparing donor identification and transplant efficiency between the national registry, that considers only the unrelated donor and the rome transplant network, whose policy includes also haploidentical donor as third choice in the donor search process. at rome transplant network 79% out of 417 adult patients met criteria of a good preliminary query corresponding to a matched unrelated donor identification in 50% of cases vs only 12.5% for patients with poor preliminary query. our policy led to 78% and 74%, respectively, of alternative donor identification and transplant efficiency, significantly higher than the corresponding data of 71% (p = 0.007) and 62% (p o0.0001) reported by the national registry. moreover, the median duration of search process for mud identification has been significantly reduced by the use of hr hla typing patient at the start of the formal search activation from 88 (range: 1-1016) to 66 (range: 8-905) days at ibmdr (po 0.001) and from 61 to 41 days (20-321) at rtn (po 0.001). in conclusion, the preliminary query represents a useful tool to address the search towards the best donor choice and to perform transplant in adequate time. moreover, the timing of donor identification has been significantly reduced with the use of high resolution typing at the start of donor search. a search and selection donor policy should be basically established and should include the haploidentical donor to improve the transplant efficiency. disclosure of conflict of interest: none. the long term prognosis of elderly acute myeloid leukemia (aml) patients remains poor. advances in the uses of alternative donors and reduced intensity conditioning regimens have extended the use of allogeneic hematopoietic stem cell transplantation (hsct) to a wider number of patients. however, few studies have reported data on the efficacy of hsct from alternative donors in elderly aml patients. we retrospectively analyzed the transplantation outcome in 93 consecutive elderly aml patients aged 460 years who received hsct (2005 hsct ( -2015 at the catholic blood and marrow transplantation center. donor types were autologous (n = 18) or hla matched related (mrd, n = 28), unrelated (mud, n = 22), or haploidentical (n = 25). for graft-versus-host disease (gvhd) prophylaxis, methotrexate and cyclosporine (mrd) or tacrolimus (mud/haploidentical donor) were used. mud and haploidentical donors were given antithymocyte globulin. the median age was 63 years, with 23 patients (25%) 465 years. intermediate-or adverse cytogenetic risk was observed in 91% of patients. with a median follow-up of 44.7 months, overall survival (os) and disease-free survival (dfs) at 3 years after transplantation were 37% and 38% for autologous, 40% and 35% for mrd, 67% and 62% for mud, and 67% and 67% for haploidentical hsct, respectively. the 3-year relapse was significantly higher for autologous hsct compared to allogeneic hsct (40% vs 14%, p = 0.012), while it was similar among allogeneic donors: mrd, 13%; mud, 14%; haploidentical, 15% (p = 0.925). the 3-year non-relapse mortality (nrm) for mud (24%) or haploidentical donor (18%) hsct was comparable to that of autologous hsct (22%), while it was relatively higher for mrd hsct (52%, p = 0.056). of the 75 patients receiving allogeneic hsct, the 1-year cumulative incidence of moderate to severe chronic gvhd was significantly increased for mrd (64%) compared to alternative donor hsct (35%, p = 0.001). in multivariate analysis, patient age (hr 0.8, 95% ci 0.8-1.0, p = 0.005) and donor type (hr 3.5 95% ci 1.0-13.0, p = 0.056 for mud; hr 6.2, 95% ci 1.7-22.6, p = 0.006 for mrd compared to haploidentical donor) were significantly associated with the cumulative incidence of moderate to severe chronic gvhd, while female-to-male hsct showed a borderline significance (hr 2.1, 95% ci 0.9-4.7, p = 0.075). incidence of acute gvhd was similar according to donor type. in the multivariate analysis for nrm, patient age (hr 1.4, 95% ci 1.1-1.6, p = 0.001), mrd (hr 4.5, 95% ci 1.4-14.4, p = 0.011), and hematopoietic cell transplantation-comorbidity index high risk (hr 6.4, 95% ci 2.3-17.5, p = 0.001) were significantly associated. in conclusion, our results showed significantly higher relapse rate for elderly aml patients receiving autologous hsct compared to allogeneic hsct, responsible for the lower survival rate in autologous hsct. we observed that nrm rate for mud and haploidentical donors for elderly aml patients were lower than expected and similar to autologous hsct. relatively higher incidence of nrm for mrd hsct seemed responsible for the low long term dfs. these results suggest a need for strengthening of gvhd prophylaxis in mrd hsct for elderly aml patients. our results suggest a potential role of alternative donor hsct to improve long term survival rates in elderly patients with aml. disclosure of conflict of interest: none. for patients with saa, transplantation from an unrelated donor (ud) is usually considered after failure of at least one course of immunosuppression. this strategy is based on a relatively high risk of complications for ud transplant recipients, such as graft rejection, graft-versus-host disease (gvhd) and infections. however, the outcome of unrelated donor transplants has significantly improved in recent years, due to better donor selection, conditioning regimen optimization and better supportive care. the authors describe results from 51 patients with saa who receive unrelated allogeneic transplants in a single reference institution from 1997 to 2014. data was retrieved from the center databasis and there were 30 females and 21 males. median age was 15 years old . median total number of cells infused was 3.4 × 10 8 /kg.61% of the patients have received more than 50 transfusions previously. conditioning regimen were: cy 120 + tbi 1320 ± atg in 16 (31%) patients, bu 12 mg/kg+ cy 120+ atg in 18 (35%), and fludarabine + cy+atg in 8 (16%), fludarabine, cy+tbi 200 in 9 (18%) patients. stem cell source was marrow in 84%, cord blood in 13% and peripheral blood in 3% of patients. transplants were full matched in 32 (62%) patients, had one mismatch (out of 12) in 12 (24%) and 2 mismatches in 7 (14%) patients. engraftment was complete as evaluated by donor chimerism at day 30 and 100 post transplant in 36 patients (71%), partial in 4 (8%) and graft failure was observed in 9 (18%) patients. acute gvhd grade ii-iv was seen in 9 patients ( 18%) and nih moderate to severe chronic gvhd was seen in 8 (16%) patients. median overall survival was 328 days (4-4287) and estimated 5 years overall survival was 55%. risk factors for survival identified were: hla mismatch and stem cell sources other than marrow. unrelated transplants are a feasible salvage therapy for patients with saa refractory to immunosuppression, being hla compatibility and marrow stem cell source factors with a positive impact on survival. disclosure of conflict of interest: none. use of haploidentical stem cell transplantation continues to increase, the 2015 european society for blood and marrow transplant activity survey report jr passweg 1 , h baldomero 1 , p bader 2 c bonini 3 , rf duarte 4 , c dufour 5, , a gennery 6 , n kröger 7 , j kuball 8 , f lanza 9 , s montoto 10 , a nagler 11 , ja snowden 12 , j styczynski 13 and m mohty 14 for the european society for blood and marrow transplantation (ebmt) 1 hematopoietic stem cell transplantation (hsct) is an established procedure for many acquired and congenital disorders of the hematopoietic system, including disorders of the immune system, and as enzyme replacement in metabolic disorders. the annual activity survey of the ebmt describes the status of hsct in europe and affiliated countries and has become an instrument used to observe trends and to monitor changes in technology use. teams were invited to report their transplant activity for 2015 by indication, stem cell source and donor type using a single paged survey. a record number of 42 '171 hsct in 37 '626 patients (16 '030 allogeneic (43%), 21 '596 autologous (57%)) were reported by 655 centers in 48 countries in 2015. trends include continued growth in transplant activity during the period 2005 and 2015, with the highest percentage increase seen in middle income countries (allo 209%, auto 215%), and the lowest in very high income countries (allo 64%, auto 28%), for both allogeneic and autologous hsct. in contrast the absolute growth is highest in the very high income countries (growth allo rates 114 transplants per 10 × 10 6 inhabitants, auto rates 85 for very high income countries; allo rates 35, auto rates 38 for middle income). main indications for hsct were myeloid malignancies 9 '413 (25%; 96 % allogeneic); lymphoid malignancies 24 '304 (67%; 20% allogeneic); solid tumors; 1 '516 (4%; 3% allogeneic); and non-malignant disorders; 2 '208 (6%; 90% allogeneic). remarkable is a decreasing use of allogeneic hsct in cll from 504 patients in 2011 to 255 in 2015 and is most likely due to the development of potentially very effective cll drugs. use of haploidentical donors for allogeneic hsct continues to increase 2 '012 in 2015; a 291% increase since 2005. the highest growth is seen in myeloid malignancies 1 '008, with lymphoid malignancies 636, nonmalignant disorders 316 and 52 others. in aml, haploidentical hsct increases similarly for patients with both advanced disease and those in cr1. both marrow and peripheral blood is used as stem cell source for haploidentical hsct with higher numbers reported for the latter. this year's activity survey shows continued increase in the use of haploidentical hsct across europe within the main indication groups and cell source. it reflects in a timely manner current trends in stem cell transplantation and is an essential tool for health care planning and health policy makers. human bone marrow mesenchymal stromal cells derived exosomes (hbmmdes) are small membrane vesicles secreted from mesenchymal stromal cells that may serve as a vehicle for protein, mrna and microrna (mirna) transfer to distant cells; affecting gene expression, proliferation, and differentiation of the recipient cells. therefore, mdes may possess some of the immunoregulatory properties of their parental cells. in the present study we aim to explore the immunomodulatory function of mdes and understand the molecular mechanisms enabling it. for this purpose, we co-cultured hbmmdes with activated human lymphocytes. using ultracentrifugation, hbmmdes were isolated from expanded human bone marrow derived mesenchymal stromal cells (hbmmscs). using em and zeta sizer, particles were shown to be in the range of 80-120 nm. pha activated human peripheral blood lymphocytes (pbls), r-848/il2 activated b cells and anti cd3/cd28 activated t cells were co cultured with purified mdes. cell proliferation was tested using thymidine incorporation assay. we found that exosomes derived from 1 × 10 3 to 1 × 10 6 mscs exhibited a dose-dependent inhibition of lymphocyte proliferation. exosomes derived from 1 × 10 6 mesenchymal stromal cells co cultured with pha activated pbls, activated b cells and activated t cells showed proliferation inhibition of 53%( p ⩽ 0.001), 34.37% (p ⩽ 0.05) and 47.41 % (p ⩽ 0.01), respectively. in order to understand the molecular mechanism behind the immunomodulatory effect of mdes, we have profiled mde's mir content using illumina hiseq 2500 platform and we are currently profiling co cultured activated lymphocytes mrna content using next-generation sequencing system, illumina. preliminary results demonstrate some higher abundance of specific mscs derived mirs in the mdes. hbmmscs have been shown to serve as immune modulators in patients with acute and chronic graft versus host (gvhd). in the future, mdes may provide an alternative therapy for gvhd. compared with bmmscs, mdes are more stable, have no risk of aneuploidity or ectopic proliferation and have less probability of immune rejection. additional studies are needed to explore the applicability of mdes to serve as modulators of the immune response. disclosure of conflict of interest: none. graft-versus-host disease (gvhd) is the major complication after allogenic haematopoietic stem-cell transplantation s192 (hsct). extra virgin olive oil (evoo) is a source of phenolic compounds such as glycoside oleuropein, hydroxytyrosol and tyrosol. olive oil polyphenols have shown antioxidant, immunomodulatory, antiproliferative, anti-apoptotic and antiinflammatory properties that might be useful in the prophylaxis and treatment of gvhd. polyphenolic extract (pe) of evoo was obtained by the method described by vazquez roncero et al. with some modifications. briefly, fifty grams of evoo (oleoestepa, seville, spain) was extracted with methanol/water (80:20, vol/vol, 125 ml ). the mixture of evoo, methanol and water was decanted and the methanolic extract was concentrated and lyophilized. then, the effect of pe in cell viability and activation of t lymphocytes from healthy donor's buffy coats either resting or activated with anticd3 plus anticd28 was analyzed by flow cytometry after staining with 7aad, anexin-v and cd25. proliferation assays were performed with pkh and the quantification of il-2, il-4, il-6, il-10, tnf-α and ifn-γ cytokines in cell culture supernants with bd cytometric bead array (cba). signaling pathways were analyzed by western blot. finally, in a mouse model of acute gvhd (c57bl/6 in balb/c), mice were randomized into two experimental diet groups: standard diet (2014s harlan laboratories) and standard diet (2014s harlan laboratories) supplemented with 600 ppm of pe obtained of evoo. the severity of gvhd was assessed by a scoring system described by cooke et al. that incorporates five clinical parameters: weight loss, posture (hunching), activity, fur texture, and skin integrity. pe did not affect t cell viability. by contrast, pe decreased t-cell activation and proliferation of t-lymphocytes stimulated with anticd3 plus anticd28. in addition, there was a decreased production of th1 (ifnγ, il-2 and tnf) and th2 cytokines (il-4, il-6 and il-10) in the presence of pe. regarding the signaling pathways analyzed, pe inhibited phosphorylation of akt and nuclear translocation of nfkb in activated t cells. in the mouse model of acute gvhd, animals which received the pe supplemented diet had an increased survival as compared to mice receiving a standard diet. also, gvhd incidence was significantly lower among mice receiving the pe supplemented diet as assessed by both the presence of gvhd signs as well as pathological examination. polyphenols obtained from evoo are an important immunomodulatory agent capable to reduce the proliferation and activation of activated t cells and the production of proinflammatory cytokines. in a mouse model of acute gvhd, pe supplemented diet reduced the incidence and severity of the disease and increased the survival of mice. disclosure of conflict of interest: none. graft-versus-host disease (gvhd) is a leading cause of postallogeneic haematopoietic stem cell transplantation (hsct) morbidity and mortality (1) . extracorporeal photopheresis (ecp) is an alternative therapeutic strategy that appears to act in an immunomodulatory fashion, potentially involving regulatory t lymphocytes and dendritic cells in patients who are refractory to steroids. dendritic cells (dcs) are the most important antigen-presenting cells, playing a pivotal role in t-cell function and in the link between innate and adaptive immunity. moreover, dcs are also critical mediators of immune tolerance and energy. they can be divided into two major subsets, plasmacytoid dcs (pdcs) and myeloid dcs (mdcs) which have distinct functions. pdcs play a pivotal role in peripheral tolerance through generation of regulatory t (treg). on the other side mdcs promote, as well as pdcs, th2 and th0/tr1 responses (1-4). our study was performed to understand the mechanism of action involved in immunomodulatory effect of ecp. as the modulation of dcs and tregs number and function (7, 8) may be a central mechanism of ecp in maintaining self-tolerance, down-regulating immune responses, and limiting inflammation (9). eight patients affected by gvhd were included in this pilot study. in ecp apheresed mononuclear cells are exposed to 8methoxypsoralen and uva radiation. after this photoactivation, which induces dna damage and apoptosis, the cells exposed are re-infused into the patient inducing an immunomodulatory effect. all patients or their legal guardians gave their consent for this study. a sample of peripheral blood (pb) (basal condition), a sample of apheresis pre-uva photoactivation (pre-pa) and a sample of photoactivated apheresis (pa) were collected at the first day of ecp and every week for the first month of treatment. circulating dcs, mdcs (cd14/16-cd85+cd33+), pdcs (cd14/16-cd85+cd123+) and tregs (cd4 +cd25+foxp3+) were directly enumerated and phenotypically characterized. the assays were performed at day+1,+8, +15,+21,+30 data are expressed as mean ± s.d. of absolute number of cells/μl. at day +1 there were no differences in the absolute number of both mdcs and pdcs between pre-pa and pa. consequently there were no differences between pb and pa. from day +8 till +30 we observed an increase of these two cellular populations at every date of treatment. comparing the basal pb of day +1 vs day +30 we observed an increment of 40% and 120%, respectively for mdcs and pdcs (mdc from 11247 cell/μl to 15742 cell/μl; pdc from 6983 cell/μl to 15263 cell/μl). comparing the basal pb of day +1 vs day +30 we observed an increment of 115% of tregs (from 4257 cell/μl to 9142 cell/μl) while we observed a median increment of 34% calculated between pre-pa and pa of each day of treatment from day 1 to day +30. no firm conclusions can be drawn from a clinical point of view, however a biological effect has certainly highlighted. in particular no substantial differences in basal pb mdc or pdc emerged during the first month of treatment while a significant increase of mdc and pdc can be observed since day +15 following uva photoactivation. regarding tregs we observed an increment of 115% of tregs between pb from day +1 to day+30 and a median increment of 34% calculated between pre-pa and pa of each day of treatment. disclosure of conflict of interest: none. [p151] p152 impact of th17 cells on xenogeneic graft-versus-host disease l delens, s servais 1 , g ehx 1 , l vrancken 1 , g fransolet 1 , c gregoire 1 , m hannon 1 , s dubois 1 , c daulne 1 , f baron 1 and y beguin 1 1 giga i3 : hematology, university of liege acute graft-versus-host disease (gvhd) is a severe complication of allogeneic hematopoietic stem cell transplantation. its pathophysiology is complex and not yet fully understood. in particular, the impact of th17 cells on murine acute gvhd has yielded conflicting results, while demonstration of increased levels of th17 cells at the site of acute gvhd provided only indirect evidence of their involvement in humans. here, we assessed the potential implication of th17 cells in a humanized mouse model of xenogeneic gvhd (x-gvhd). methods: x-gvhd was induced by infusing human peripheral blood mononuclear cells (pbmcs) into nod-scid il-2rγnull (nsg) mice given 2.5 gy total body irradiation 1 day prior transplantation. th17 cells were generated by culturing naive cd4+ t cells with anti-cd3/anti-cd28 coated beads under th17-skewing cytokines (tgf-β1, il1-β, il-6, il-21, il-23, neutralizing anti-il-2 and anti-ifnγ antibodies) in hypernatremic conditions (nacl 40 mm). results: after 8 days of culture, a median of 21.75% of il-17a+ cells was obtained. we confirmed the expression of il-17a, rorc and il-23r by these cells by rt-qpcr. we next assessed the co-injection of human pbmcs (1.106) with in vitro differentiated cells under th17 skewing conditions (1 × 10 6 ) (co-injection group, n = 20), in comparison with the injection of pbmcs alone (2 × 10 6 cells, pbmcs group, n = 17). we observed higher x-gvhd score (p60%) of cells expressing both il-17a+ and ifnγ+ cells (th17/ th1-like phenotype) among cd4+ il-17a+ cells while coinjected mice had higher blood concentration of il-17a (p = 0.026) than pbmc mice. these results demonstrate that addition of th17 cells worsened x-gvhd confirming their role in acute gvhd pathogenesis. disclosure of conflict of interest: none. although survival from allogeneic stem cell transplantation (hsct) has significantly improved, acute graft-versus-host disease (gvhd) remains a major cause of death. intestinal dysbiosis has been associated with acute gastrointestinal gvhd and poor outcome after hsct. we reported a correlation between microbiota (gm) composition and short chain fatty acid (scfa) production and gvhd in transplanted children. 1 to assess how the metabolic pathways of gm change during transplantation and identify modulators of immune response, we perform first longitudinal metagenomic analysis in children undergoing hsct. 8 patients (pts) (6male; mean age: 10y) with hematologic malignancies (7 all, 1 aml), who received busulphan-based myeloablative conditioning and t-cell replete bone marrow graft were enrolled. pts were prospectively enrolled in a protocol with at least 3 specimens fecal samples collected: one before and two after hsct, in order to build a proper trajectory. gvhd prophylaxis was cyclosporine for 3 pts receiving a matched related donor and cyclosporine, short-term mtx and atg for 5 pts receiving a matched unrelated donor. non-gvhd and gvhd patients had similar exposures to antibiotics during the stool collection. of these pts, 50% developed gvhd within the first 100 days. we applied shotgun metagenome sequencing to total fecal dna from samples collected. functionalities were assigned by reads mapping at different levels of the kegg database. 2 relative abundance was calculated and statistical analysis was performed. according to our findings, core functional profiles were overall conserved through the time-points in all patients ( figure 1a ), in contrast to the phylogenetic profiles behavior, this finding confirming the overall redundancy of gut microbiome core functionalities. analyzing the single metabolic pathways in subjects who developed gvhd, we found in the pre-hsct period a higher relative abundance of nucleobasis (purine and pyrimidine) metabolism (p o0.05) and branched-chain amino acids biosynthesis (p o0.05). functions related to the production of branched-chain amino acids are involved in the biosynthesis of the cell wall of gram-negative bacteria, microorganisms including subgroups with well know opportunistic pro-inflammatory. in addition, post-hsct samples of gvhd patients showed a lower abundance of genes involved in polysaccharides metabolism, as glycan biosynthesis and glycosaminoglycan degradation (p o0.05) ( figure 1b ). glycosaminoglycan degradation activity gets bacteria able to survive during extreme situations, as fasting using mucus polysaccharides as energy source, contributing to maintain a mutualistic composition of gm and scfa production by the saccharolytic functions of the endogenous mucus polysaccharides. this study detects functional peculiarities in the gm of non-gvhd pts. the gut metagenome configuration of non-gvhd patients is structured to derive scfa after hsct. the production of these metabolites promotes peripheral regulatory t-cell generation 3 , potentially explaining the protective role of gm from gvhd. although intestinal epithelial cells (iecs) are crucial regulators of barrier function and immune homeostasis, they also facilitate inflammation in exaggerate responses to proinflammatory mediators by pretransplant conditioning regimen, which plays a critical role in amplifying graft-versus-host disease (gvhd). thus inhibition of the converting to pathogenic iecs by conditioning may represent a novel approach to inhibit gvhd. aryl hydrocarbon receptor (ahr) is the ligandactivated transcription factor which has the ability to mediate the biochemical, metabolic, and toxic effects of environmental chemicals. recently, it has been demonstrated that ahr is an important regulator of cell development, differentiation, and function of both innate and adaptive immune cells. the ability of ahr is induced by respond to endogenous ligands generated from the host cell, diet, and microbiota. here, we investigated the regulatory role of ahr in iecs under inflammatory responses and its therapeutic activity for modulation of gvhd. ahr and cyp1a1 expression in mouse iecs were determined by real-time pcr. mouse iecs were pretreated with endogenous ahr ligands l-kynurenine (l-kyn, 300 mm) or pbs for 6 h and then stimulated with lps or il-1b for 24 h. cytokine levels were measured using the mouse flex-set cytokine bead array or real-time pcr. b6d2f1 (h-2b/d) recipients were administrated l-kyn daily by i.p. injection for 3 days. then the recipients were lethally irradiated and transplanted with 5 × 10 6 tcd-bm plus 2 × 10 6 t cells from b6 (h-2b) donor. mice were monitored every other day for survival and clinical score. colons were collected and stained with hematoxylin and eosin (h&e) for histopathological scoring. we found that ahr was constitutively expressed in the mouse iecs. cyp1a1 (an ahr target gene) was significantly increased by treatment of l-kyn under un-stimulatory condition. we further observed that l-kyn completely abrogated il-1β-mediated il-6 or lps-mediated tnf-a expression in iecs. administration of bdf1 recipient mice with l-kyn before transplantation significantly reduced the lethality and severity of gvhd. histopathology clearly revealed that treatment of l-kyn inhibited intestinal gvhd. our results demonstrate that 1) ahr is constitutively expressed in iecs, 2) treatment of endogenous ligand l-kyn induce ahr activation in the steady status, 3) ahr activation blocks conversation of the epithelial cells into pathogenic cell type, and 4) pre-administration of ahr ligand reduces gvhd. our study suggests that activation of ahr pathway in iecs before allogeneic hematopoietic stem cell transplantation (hsct) is a possible strategy to reduce intestinal gvhd. disclosure of conflict of interest: none. [p156] s196 relating with acute graft-versus-host disease (gvhd) after allogeneic hematopoietic stem cell transplantation (allo-hsct), protecting endothelial cells (ecs) from damage may be a potent prophylaxis and therapeutic strategy of acute gvhd (agvhd). conventional agvhd therapies may cause many adverse side effects because of their multiple targets. therefore, we explored the therapeutic efficacy of simvastatin, a lipid-lowering drug, which has been demonstrated endothelial protection. our previous clinical observation has found patients with agvhd had lower angiopoietin-1 (ang-1) level at day 7 but higher ang-2 level at day 21 than those without agvhd. in this study, we explored changes in ang-1 and ang-2 expression in an agvhd mouse model and determined whether simvastatin prevents gvhd through regulating ang-1 and ang-2 expression. we preincubated ea.hy926 ecs with simvastatin (1mmol/l) 12 h before stimulated with tnf-a, then ang-1 and ang-2 concentration in the cell supernatant was measured by elisa. ang-1 and ang-2 mrna and protein level of treated and untreated cells were examined simultaneously. in vitro simvastatin increased ang-1 production and release but conversely inhibited ang-2 release from ea.hy926 ecs. donor mice spleen cells were injected along with bone marrow cells into recipient mice after lethal irradiation to induce agvhd. simvastatin was administered orally once daily to mice (10 mg/kg) for 7 days after allo-hsct and started −1 day after allo-hsct. then mice survival time was monitored and organ damage was evaluated. the plasma level of ang-1 and ang-2 was measured by elisa, expressions of ang-1 and ang-2 in aortic endothelium were assessed by immunohistochemistry. simvastatin improved the survival and attenuated the histopathological gvhd grades of agvhd mice. plasma levels of ang-1 were significantly decreased, while plasma levels of ang-2 obviously increased in agvhd mice after transplantation. simvastatin reduced plasma levels of ang-2, elevated the plasma levels of ang-1 as well as the aortic endothelial levels of ang-1 and ang-2. in summary, simvastatin represents a novel approach to combat gvhd by increasing ang-1 production while suppressing ang-2 release to stabilize endothelial cells. there is a growing evidence of safety and efficacy of posttransplantation cyclophosphamide (ptcy) in stem cell transplantations (sct) from different donors and graft sources. still the optimal combination of immunosuppressive agents with ptcy should be elucidated for different types of scts. we report the 2-year update of the prospective nct02294552 single-center trial that evaluated risk-adapted graft-versushost disease (gvhd) prophylaxis with ptcy in related, unrelated and haploidentical scts. 200 adult patients (median age 32 y.o., range: 18-62) with hematologic malignancies, including aml (47.5%), all (26.5%), cml (10.5%), mds (4%), and lymphomas (11.5%), were enrolled in the study. 23% of patients were classified as salvage. 26% received the graft from matched related (mrd), 65% from matched/mismatched unrelated (mud/mmud), and 9% from haploidedntical (haplo) donor. 43% received bone marrow graft (bm) and 57%peripheral blood stem cell (pbsc) graft. 18.5% had myeloablative conditioning and 81.5%-reduced-intensity conditioning. gvhd prophylaxis for matched bm grafts consisted of single-agent ptcy 50 mg/kg days+3,+4, for matched pbsc graft-ptcy+ tacrolimus+ mycophenolate mofetil (mmf) 30 mg/kg days 5-35, and for any mismatched graft-ptcy+ tacrolimus+ mmf 45 mg/kg days 5-35. median follow-up was 20 months (range: 4-40). grade ii-iv (10% vs 18% vs 11%, p = 0.37) and grade iii-iv acute gvhd (4% vs 6% vs 0%, [p157] p = 0.59) were not different in mrd, mud/mmud and haplo groups, respectively. moderate and severe chronic gvhd was infrequent in all groups with slightly lower incidence after mud/mmud graft: and 22% vs 9% vs 21%, p = 0.046. nonrelapse mortality (nrm) was not different after mrd, mud/ mmud and haplo sct (8% vs 14% vs 24%, respectively, p = 0.19), while relapse incidence was higher after mrd and haplo grafts: (45% vs 22% vs 52%, p = 0.0017). 2-year overall survival (os), event-free-survival (efs), and gvhd-relapse free survival (gfrs) were 73% vs 71% vs 44% (p = 0.0015); 48% vs 65% vs 33% (p = 0.0008); 29% vs 56% vs 22% (p = 0.0002) for mrd, mud/mmud and haplo groups, respectively. in the multivariate analysis only disease risk index (hr 2.2 95%ci 1.6-3.0, p = 0.0001), severe sepsis (hr 3.8 95%ci 1.8-8.0, p = 0.0004) and chronic gvhd (hr 0.53 95%ci 0.30-0.93, p = 0.02) were predictive for efs, while type of donor was not a significant factor (hr 1.0 95%ci 0.6-1.7, p = 0.99) (figure 1 ). the incidences of complications were: hemorrhagic cystitis-23%, sepsis-24%, severe sepsis-8%, invasive mycosis-8%, cmv reactivation-45%, veno-occlusive disease-2.5%, transplant-associated microangiopathy-3.5%, grade 3-4 liver toxicity-14%, grade 3-4 kidney toxicity-1%. more than one third of patients experienced poor graft function during 100 days after sct, and in 83% of them cmv, hhv and bk virus reactivations were identified as the cause. the reported risk adapted strategy alleviates the risk of gvhd and nrm after mmud and haplo grafts. the observed differences in the relapse incidence, os and efs were predominantly due to unbalanced disease risks in the groups. the relapse of underlying malignancy with this prophylaxis still significantly influences the outcome. substantial number of patients experience poor graft function, which doesn't translate into nrm. disclosure of conflict of interest: none. a high migratory capacity of donor t-cells in response to the lymph node homing receptor ccr7 increases the incidence and severity of graft-versus-host disease vg garcía de soria 1 , ip sainz 2 , e jiménez 1 , a arriero 1 , c fernández-arandojo 1 , c cuesta 2 , b colom 2 , a marcos 2 , a rosendo 2 and cecilia muñoz calleja 2 1 department of hematology hospital universitario de la princesa and 2 department of inmunology. hospital universitario de la princesa graft-versus-host disease (gvhd) pathogenesis involves migration of the donor t-cells into the secondary lymphoid organs (slo) in the recipient, which is steered by two homing molecules: cd62l and ccr7. therefore we investigated whether the migratory capacity of donor t-cells is associated with gvhd. this single center prospective study included 85 donor-recipient pairs. in vitro chemotaxis assays of the lymphocytes of the apheresis product were performed in parallel with the analysis of cd62l and ccr7 by flow cytometry. the potential of activation of ccr7+ t-cells was assessed through ex vivo activation assays with peripheral blood monuclear cells (pbmc) from healthy donors using anti-cd3 and anti-cd28mabs. the migratory index to the ccr7 ligands, ccl19 and ccl21, was higher in t-cells from donors whose recipients will develop gvhd. these data indicated that the migratory capacity of the donor t-cells is clearly related to the development of gvhd. this prompted us to study the relationship between gvhd and the expression of two of the most relevant molecules in the trafficking of lymphocytes towards slo, cd62l and ccr7,as a subrogate index of the migratory potential of t-cells. consequently, we quantified the numbers of cd62l+ and ccr7+ t-cells in the graft. the initial transversal analysis of our data revealed that the percentage of cd62l+ lymphocytes in the apheresis product was very low compared to healthy lymphocytes. the analysis also confirmed that cd62l undergoes plasma membrane shedding after g-csf mobilization thus making it a non-valid biomarker. the analysis of ccr7 molecule revealed that the acute gvhd group received higher percentage of cd4+ccr7+ t-cells, whereas chronic gvhd patients were transplanted with higher percentage of cd8+ccr7+ t-cells compared to the non gvhd group. these results were confirmed when patients were subdivided into degrees of severity. a multivariate analysis was performed to investigate the real value of ccr7 to predict the development and severity of gvhd, and confirmed that ccr7 expression is a risk factor for the development of gvhd. thus, the percentage of ccr7+cd4+ t-cells increases the probability of developing acute gvhd (or = 1.08, c.i (95%) = 1.01-1.16, p = 0.019) and suffering a higher degree (or = 1.08, c.i (95%) = 1.01-1.15, p = 0.014). similarly, the or of the percentage of ccr7+cd8+ t-cells was 1.17 (c.i (95%) = 1.01-1.36, p = 0.0031) and 1.21 (c.i (95%) = 1.05-1.39, p = 0.006) for the development of chronic gvhd and its degrees, respectively. finally, to study the potential of activation of ccr7+ t-cells, we carried out ex vivo activation assays with pbmc from healthy donors using anti-cd3 and anti-cd28mabs and the expression of cd40l on cd4+ t-cells and of cd69 on cd8+ t-cells as markers of activation, demonstrating that ccr7+ t-cells exhibited higher potential of activation than ccr7-t-cells. to our knowledge this is the first analysis of the influence of the migratory capacity of the donor t-cells on clinical outcome following allogeneic hsct. our data show that ccr7 could be considered a subrogate biomarker of the migratory capacity of the donor lymphocytes for predicting the risk of suffering gvhd. based on the previous findings, we propose that the selective depletion of ccr7 expressing cells could be an effective preventive therapy for gvhd. disclosure of conflict of interest: none. previously published p160 a single center research for outcome in patients receiving imatinib for steroid-refractory chronic gvhd after allogeneic stem cell transplantation l ni, y luo, y tan, y hu, y zhao, j shi and h huang despite of major progress in allogeneic stem cell transplantation over the last decades, steroid-refractory chronic graftversus-host disease (sr-cgvhd) remains a leading cause of late morbidity and mortality. pre-clinical evidence confirms cgvhd has antibodies activating the platelet-derived growth factor receptor (pdgf-r) pathway. since this pathway can be inhibited by imatinib, we performed a study including 16 patients with sr-cgvhd given imatinib at a dose of 300 mg per day. all patients with a median age of 25 years (range: 16-53) underwent allogeneic hematopoietic stem cell transplantation in our single center between 2008 and 2015, and chronic gvhd occurred at a median time of 10 months (range: 3-29) after transplantation. patients had active cgvhd with measurable involvement of skin, lung or other districts and had previously failed in first-line immunosuppressive therapy. the major organs involved were lung (n = 11), skin (n = 10) and mouth (n = 1), including 5 cases involving both lung and skin, 8 cases involving 3 or more organs. according to the 2014 national institutes of health (nih) criteria and nih global severity, 13 patients were evaluated as severe cgvhd, and the other three were moderate. meanwhile, the 2014 nih working group had updated its recommendations for overall responses, consisting of complete remission (cr), partial remission (pr), and lack of response (unchanged, mixed response, progression). cr was defined as resolution of all manifestations in each organ or site, and pr was defined as improvement in at least 1 organ or site without progression in any other organ. after 3 months treatment, 14 patients receiving sufficient dose of imatinib revealed overall response rate (orr) at 78.6%, and orr remained unchanged at 6 months assessment, but with cr rate increased to 28.6%. two patients couldn't meet the response of cr or pr were considered as a lack of response, including one evaluated as unchanged and one mixed response because of pr in lung accompanied by progression in eyes. with a median follow-up of 9 months, 14 patients were alive, with a 1 year estimated overall survival was 87.1%. 2 patients eventually died of pneumonia. except 1 patient discontinued imatinib because of grade 2 toxicity as gastrointestinal discomfort at the first month, no one had imatinib-related grade 3 to 4 toxicity. this study suggests that imatinib is a promising and better tolerated treatment for patients with sr-cgvhd. disclosure of conflict of interest: none. acute graft-versus-host-disease (agvhd) is a major complication after allogenic hematopoietic transplantation (allo-sct). in recent years, a number of tissue-specific proteins have been described as biomarkers that could contribute to anticipate and/or diagnose this complication earlier and more accurately. reg3α (regenerating-islet-derived-3-alpha) has been directly related to gastrointestinal (gi) agvhd. our objective was to analyze plasma levels of reg3α at days +15 and +30 in patients who underwent unmanipulated haploidentical transplantation with reduced conditioning regimen (haplo-ric), and to correlate the results with the development of agvhd. we retrospectively analyzed 63 consecutive patients (2009-2016) who underwent haplo-ric with post-transplant cyclophosfamide (days +3, +4), mmf and csa as gvhd prophylaxis. seven cases were excluded due to early death (before day +30) and 4 cases due lack plasma sample. characteristics of the 52 patients included in the analysis are described in table 1 . reg3α detection was performed by elisa (mbl international corp, woburn, ma) according to manufacturer's instructions on 200 μl of plasma obtained at day +15 and +30. the association of the incidence of agvhd with known clinical variables and plasma reg3a levels were performed by cox regression and mann-whitney u-test, respectively. the determination of the best cut-off of reg3α levels to stratify patients with gi agvhd was performed with roc curves. the stadistical program used was r v2.15.0. the cumulative incidence of grade ii-iv and grade iii-iv agvhd was 52% and 17%, respectively. characteristics of agvhd are shown in table 2 . no association was found between agvhd and usual clinical variables (stem cells source, age, sex, conditioning regimen, donor/recipient sex and number of infused cd34 + cells), and with plasma reg3α levels at day +15.plasma reg3α levels at day +30 were higher in patients who devolved gi agvhd compared to patients who did not showed gi agvhd (median [p161] s199 and range: 2483 (2022-5904) vs 1110 (0-7797) pg/ml, p = 0.14, figure 1 ).the best cut-off selected on day +30 was 1989 pg/ml (s85%, e71%). patients with levels higher than 1989 pg/ml at day +30 had a significantly higher incidence of gi agvhd grade ii-iv (hr 6.9, p = 0.008, figure 2 ). plasma levels of reg3α at day +30 after haplo-ric correlated with the occurrence of gi agvhd grade ii-iv. therefore, plasma levels of reg3α could be use for the prediction and/or diagnosis of gi agvhd. disclosure of conflict of interest: none. anti-fibrotic treatment with pirfenidone in patients with gvhd-associated bronchiolitis obliterans syndrome ke hostettler, s gerull, g nair 1 , j passweg 1 , m tamm 2 and j halter 1 1 hematology, university hospital basel, switzerland and 2 pneumology, university hospital basel, switzerland prognosis of lung gvhd remains poor due to progressive decrease of lung function and repeated infections. pirfenidone exhibits anti-fibrotic effects and has been shown to reduce disease progression in patients with idiopathic pulmonary fibrosis. five patients with established bos (nih criteria 2014) and stable or deteriorating lung function under standard immunosuppressive treatment without active infection were treated with pirfenidone (2403 mg/d) in addition to their current therapy. clinical assessments and pulmonary function tests were performed every three months. five patients (4m, 1f), median age 60y (range: 29-65y) that were diagnosed with bos at a median time of 13.5 months post-transplant started pirfenidone at a median time of 51months (8-102) after diagnosis of bos. two patients are currently still under treatment after 611 and 638 days. two patients had to stop treatment due to financial reasons after 189 and 206 days of therapy. one patient never reached more than 20% of the planned dose due to gastro-intestinal symptoms and was excluded from further analysis. at the start of treatment median fev1 was 0.94l (0.72-1.34); 34.5% predicted (range: 21-44%) and median fvc 2.59 l (1.62-3.24); 46-84 % predicted. median fev1 trajectory was − 0.65 % predicted/ month during median 6months before start of pirfenidone (median − 19ml/month) and +0.33% predicted/month (+9.8ml/month) during treatment with pirfenidon. the treatment was well tolerated except in one patient with gastrointestinal complaints, no phototoxic reactions or serious drugrelated adverse events occurred. in our small number of patients pirfenidone was rather well tolerated and generally safe. the observed, albeit small trend in change of fev1 trajectory justifies further studies of anti-fibrotic therapy as a new therapeutic option in bos after allogeneic hsct. disclosure of conflict of interest: none. anti-thymocyte globulin has been widely used for the prevention of severe graft versus host disease in patients undergoing hsct from unrelated donor. however, the optimal dose remains to be defined. in samsung medical center (seoul, korea), institutional strategy for the atg use has been changed since april 2013, and we hypothesized that the incidence of chronic gvhd may differ by atg strategy. before april 2013, atg 4.5 mg/kg was routinely used in allogeneic hsct from unrelated donor, whereas, the dose of atg was escalated to 7.5 mg/kg since april 2013. in this study, a total of 170 patients who underwent allogeneic hsct from matched or unmatched unrelated donor between jan 2010 and dec 2015 were retrospectively analyzed. peripheral blood was used as the source of stem cells in all patients. after a median follow up of 30.9 months, the cumulative incidence of moderate to severe chronic gvhd was 30.2% (95% confidential interval [ci], 18.3 to 43.0) in the low-atg group and 23.7% (95% ci, 14.5 to 34.1) in the non-atg group (p = 0.655). the rate of 2year overall survival (os) was not significantly different between the groups (49.3% in low-atg group vs 48.1% in high-atg group, p = 0.841), as was the rate of disease free survival (dfs) (40.8% in non-atg group vs 42.3% in atg group, p = 0.867) and cumulative incidence of relapse (cir) (23.9% in non-atg group vs 24.5% in atg group, p = 0.776). in allogeneic hsct from unrelated donor, larger atg dose (7.5 mg/kg) did not reduce the incidence of chronic gvhd when compared to lower atg dose (4.5 mg/kg). disclosure of conflict of interest: none. allogeneic hsct provides a curative chance for patients with hematological fatal disease. however, substantial risks remain for morbidity and mortality caused by disease relapse and graft-versus-host disease. in samsung medical center (seoul, korea), institutional strategy for the atg use has been changed since april 2013, and we hypothesized that the incidence of chronic gvhd may differ by atg strategy. before april 2013, atg was not routinely used in matched sibling donor (msd) transplantation, whereas, atg 5 mg/kg has incorporated into hsct process in transplantation from msd thereafter. in this study, a total of 182 patients who underwent allogeneic hsct from msd between jan 2010 and dec 2015 were retrospectively analyzed. peripheral blood was used as the source of stem cells in all patients. after a median follow up of 40.5 months, the cumulative incidence of moderate to severe chronic gvhd was 22.0% (95% confidential interval [ci], 13.5 to 31.8) in the atg group and 55.2% (95% ci, 42.9 to 65.8) in the non-atg group (p = 0.0018). the rate of 2-year overall survival (os) was not significantly different between the groups (62.5% in non-atg group vs 58.7% in atg group, p = 0.624), as was the rate of disease free survival (dfs) (61.1% in non-atg group vs 53.3% in atg group, p = 0.377) and cumulative incidence of relapse (cir) (23.4% in non-atg group vs 28.3% in atg group, p = 0.463). in allogeneic hsct from msd, atg use was significantly associated with less occurrence of chronic gvhd, but not linked to increasing risk of relapse, with showing similar os and dfs between atg and non-atg group. disclosure of conflict of interest: none. long-term follow-up from the prospective randomized phase iii multicenter trial comparing a standard gvhd prophylaxis with cyclosporine a and methotrexate with or without additional pretransplant atlg (grafalon, previously atg-fresenius s) (given 20 mg/kg/day, days − 3 to − 1) in unrelated donor hematopoietic cell transplantation after myeloablative conditioning resulted in a significant reduction of acute and chronic gvhd without compromising relapse rate and survival [1, 2, 3] . here we report on a subsequent prospective non interventional observational study evaluating the outcome of patients receiving atlg in unrelated donor transplantation in day to day clinical practice without the selective measures of a clinical trial (german clinical trials register drks00004581). thirteen transplant centers included 165 patients with haematological malignancies (median age 54 years, iqr 45-61 years, range: 18-77 years) in early (n = 75, 45%), intermediate (n = 29; 18%) or advanced (n = 61; 37%) disease status receiving marrow (n = 6) or pbsc (n = 159) from 10/10 matched (128; 78%) or mismatched (37; 22%) unrelated donors (n = 4 related) after myeloablative (n = 100, 61%) or ric (n = 65, 39%) conditioning. gvhd prophylaxis consisted of calcineurin inhibitors, mainly csa (n = 154, 93%) with mtx or mmf and atlg. different dosing regimens were allowed according to current practise of centers. median total atlg dose was 46 mg/kg (iqr 32-60 mg/kg, range: 15-91 mg/kg). median follow-up was 12 months (range: 8-14 months). as compared to patients in our randomized phase iii multicenter trial [1, 2, 3] , patients in this study were older; advanced disease status, 10/10 match, pbsc transplantation were more frequent, and given median atlg dose was lower. acute and chronic gvhd, nrm, relapse risk, dfs and os at one year were similar to the results obtained in our randomized trial: incidence of°ii-iv agvhd: 27%, iii-iv agvhd: 13%; moderate/severe cgvhd: 24%; nrm: 17%; risk of relapse: 23%; relapse mortality: 10%; os: 73%. the experience in day to day clinical practice confirms the results shown in our randomized trial, namely the gvhd protective effect of atlg without compromising nrm or relapse rates. baseline calprotectin as a predictor for acute gastrointestinal graft versus host disease (gvhd)-a prospective study n schmidlin 1 , a holbro 1,2 , jp halter 1 , d heim 1 , l infanti 1,2 , a plattner 2 , r plattner 2 , c rothen 3 , a buser 1,2 , c bucher 1 and jr passweg 1 1 division of hematology, university hospital basel, switzerland; 2 blood transfusion center, swiss red cross, basel, switzerland and 3 rothen medical laboratories, basel, switzerland graft versus host disease (gvhd) is a major complication after allogeneic stem cell transplantation. so far there is no good validated predictor for the incidence and severity of gvhd. fecal calprotectin (cpt) is a protein in leukocytes with antibacterial properties. it has been shown to be elevated in acute gastrointestinal gvhd. additionally, cpt may be predictive for treatment response. the aim of the current prospective study was to investigate the role of baseline cpt in predicting incidence and severity of intestinal gvhd. in this prospective study conducted at the university hospital basel, switzerland, we included all adult patients undergoing hsct. the institutional review board approved the study. data were collected prospectively. cpt was measured twice before conditioning and at transplantation. fecal samples for cpt were obtained before conditioning and on the day of transplantation and assessed twice by standard elisa. between march 2012 and april 2016 a total of 194 patients (55% males, 154 patients with both baseline and transplant cpt values) were included. patient, disease and transplant characteristics are described in table 1 . median age at transplant was 55 years (range: 21-73 years). most patients had myeloid neoplasia and 46% received myeloablative conditioning. gvhd prophylaxis consisted mainly of cyclosporine containing regimens (98%). cpt levels ranged from 19 to 1500 μg/g both at baseline (median: 100 μg/g) and at transplantation (median: 101 μg/g), with a good consistency between the two measurements performed (internal quality control). on the other hand, cpt did not correlate with c-reactive protein. the two measurements were taken in median 7 days apart, depending on the conditioning regimen. eighty-five patients had an increase of at least 50 μg/g between baseline and transplantation. overall 61 (31.4%) patients developed acute intestinal gvhd (grade 1: 19; grade 2: 18; grade 3: 16, and grade 4: 8 patients, respectively). cpt both at baseline and at transplantation was not predictive for the incidence of gvhd, acute intestinal gvhd, and for acute intestinal gvhd grade 3-4 ( figure) . additionally, we did not find a significant association between cpt levels and the above mentioned endpoints for patients showing an increase of cpt of at least 50 μg/g between baseline and transplantation. in the current prospective study, we didn't find any correlation between baseline cpt values and the incidence and severity of gvhd and intestinal gvhd. further studies identifying early markers and predictors of gvhd are urgently needed. [p166] disclosure of conflict of interest: none. calcinuerin inhibitor (ci) free graft-versus-host disease (gvhd) prophylaxis: its effects on resource utilization, renal function, and the cost of care m muilenburg 1 , k cole, m abidi 1,2 , s williams and as al-homsi 1,2 1 spectrum health blood and marrow transplantation and 2 michigan state university, college of human medicine effective gvhd prevention following allogeneic hematopoietic stem cell transplantation (ahsct) is vital to reducing transplant morbidity and mortality and improving overall outcomes. several strategies are currently utilized for gvhd prophylaxis including mtx, mmf, cis, post-transplant cyclophosphamide (cy), and proteasome inhibitors. recently, we described the results of a phase i-ii trial of cyclophosphamide (cy) and bortezomib (bor) where patients (pts) received cy (50 mg/kg) on days (d) +3 & +4 and bor on d 0 & +3. the incidences of grade ii-iv and grade iii-iv acute gvhd were 35% and 11%. the incidence of chronic gvhd was 28%. in addition to gvhd, there are other factors that affect patients' quality of life and cost of care and that should be considered. it is well documented that cis have an unfavorable toxicity profile. this includes nephrotoxicity and electrolyte disturbances. furthermore, the cis need serial level monitoring. thus, we endeavored to compare the effects of cybor combination against ci-based regimens by focusing on electrolyte requirements, specifically mg, and renal function. we also sought to better understand financial considerations surrounding the need for ci drug level monitoring. sixteen pts were randomly selected from the cybor group and 16 patients from an internal control group of patients who received mmf and cyclosporine or tacrolimus following reduced-intensity ahsct. the groups were well matched in regards to age, sex, disease status, pam score, and baseline renal function. on each pt, mg results from d 0 to +90 were compiled. based on institutional protocol, a mg replacement value was assigned as well as the corresponding drug and infusion charges. next, the number of immunosuppressant (is) trough levels from d 0 to +90 was tallied and the internal lab charges calculated. to compare renal function, gfr was calculated at baseline, d 0, and d +30. χ 2 tests and wilcoxon rank square tests were used to analyze the data. for the cybor group, median mg value was 1.9 mg/dl (iqr 0.3) vs 1.7 (0.3) in the control group (po0.0001). cybor pts required a median of 7 grams (16) vs 49 grams (55) in the control group (p = 0.001). the cost of mg replacement and infusion was significant (p = 0.001) ( table 1) . for is checks, drug levels were checked a mean of 0.625 times per patient in the cybor group compared to 18.75 times in the control group (po0.0001), which also translates to significant savings ( table 2) . considering these costs, the cybor group saved~$6000. for gfr, 3 cybor pts and 1 control pt had reduced gfr at baseline. on d +30, cybor pts had better renal function in comparison to the control group (p = 0.018) ( figure 1 ). in summary, cybor significantly reduced the use of resources post-transplant and thereby the associated cost related to mg replacement and need for drug level monitoring. furthermore, cybor preserved renal function at d +30. these findings could also impact patient's quality of life. although our cost analysis was restricted to certain aspects of care and did not take into account other factors, it highlights specific important benefits of ci-free gvhd prophylaxis and supplicates further study. a formal prospective comparison of cost and qol is warranted. . related donor n = 11 and unrelated donor n = 5 (compatibility 9/10 n = 1), with conditioning regimen: myeloablative n = 5 and non-myeloablative n = 11. median interval between transplantation and diagnosis of cgvhd of 11 months(5.1-40.2); and between cgvhd diagnosis and sativex 23 months (9.1-105.3), with a median of 4 prior treatment lines (2-9). at the time of beginning, the cgvhd was extensive in all patients, severe cgvhd n = 5 and moderate cghvd n = 11. all patients except one had cutaneous involvement (n = 13 with sclerodermal features). in addition, other organs were affected: digestive n = 2, pulmonary n = 7, hepatic n = 4, ocular n = 8, oral n = 9, genital n = 2 and muscular n = 7. drug was started because of pulmonary affectation in 3 patients and due to sclerodermal/muscular involvement in 13 patients. concomitant therapies during treatment were: topical cutaneous treatment n = 11, topical ocular treatment n = 10, pulmonary n = 7, sirolimus n = 8, tacrolimus n = 3, oral corticosteroids n = 9, extracorporeal photopheresis s n = 4, ruxolitinib n = 2, imatinib n = 1, mesenchymal stem cells n = 1. the mean dose were three puff/day (2) (3) (4) (5) , with good tolerance, only two discontinuations of treatment because of adverse effects. median time of treatment 156 days (45 to 561). at the time of the analysis 11 patients were still under treatment. responses mainly occurred within the first 60 days, s203 with a median time of duration of 106 days (20 in 450). responses after two months of treatment were: 6 partial organ response, 4 mixed responses, 4 unchanged and 2 organ progressions; at 120th day (14/16) only two patients maintained their responses (one pr and one mixed response). it must be pointed out that one patient who reached pr with sativex in monotherapy maintain response after 18 months of treatment. in addition, cramps were resolved in 5 patients. sativex appears to be an effective treatment option in patients with chronic gvhd, particularly in those having cramps, sclerodermal features and pulmonary affectation. as seen in multiple sclerosis context, the main issue with its use is the loss of response in the long-term follow up. the median dose is inferior to the one described in ms, leaving the question if higher doses can deepen the response. these results should be confirmed in prospective trials. disclosure of conflict of interest: none. several risk factors associated with acute and chronic graftversus-host-disease (gvhd) have been identified in multiple studies. most commonly associated factors are human leukocyte antigen (hla), mismatch between recipient and donor, as well as several other characteristics such as age, conditioning regimen and prior acute gvhd. objective: the aim of this study was to evaluate the characteristics of acute and chronic gvhd in patients who underwent an allogenic hematopoetic stem cell transplantation (hsct), identify differences in the profile risk factors for acute and chronic gvhd and their impact in post-transplant morbidity and mortality. this retrospective study included 90 mexican adult patients who received an allogenic hsct between january 2010 and march 2016, at instituto nacional de cancerologia. we analyzed 90 patients with a median age of 30 years (15-64), from which, 60% were male patients. among the participants with hematologic malignancies, 39 were previously diagnosed with acute lymphoblastic leukemia, 20 acute myeloid leukemia, 11 chronic myeloid leukemia, 8 lymphoblastic lymphoma and 3 with myelodysplastic syndrome. because bone marrow transplants are not performed at this institution, all transplants were from peripheral blood stem cell harvest. acute gvhd prophylaxis consisted in a triple immunosuppressive drug regimen for all patients. 86.7% of the patients had high risk disease prior to hsct. myeloablative conditioning represented 82% of the applied regimens, which consisted of iv busulfan in 63.3% of the cases. 44.9% of patients, were transplanted within 12 months from diagnosis. the cumulative incidence of acute gvhd at 100 days was 21.1% (19 patients). patients with acute gvhd had 42% grade a, 15% grade b and 42% grade c, according to the ibmtr grading system. 12 patients had skin involvement, with grade 1-2 acute gvhd in 83% of the cases, 4 patients developed liver involvement and 6 patients had gastrointestinal tract disease. 19% of the patients developed chronic gvhd, from which, 57% were classified as severe, 10.5% as moderate and 21.6% as mild. 36% of the patients who developed chronic gvhd had a single organ involvement, while 26.3% had 3 or more organs/sites. prior acute gvhd was associated with de development of chronic gvhd. the multivariate analysis identified hla unrelated donor as the only risk factor associated with the development of acute gvhd (hr, 5.1; 95% ci, 3.3-7.9, p = 0.043). the overall survival at 5 years was of 69% poor patients who developed acute gvhd and of 34% for those who didn´t (p = 0.065). our analysis showed that the incidence of acute and chronic gvhd at our center is lower than the reported at other centers, but we were not able to identify risk factors usually associated with the development of gvhd, perhaps due to the small population that we evaluated. graft versus host disease (gvhd) remains one of the main obstacles to broader application of allogeneic stem cell transplantation (sct). despite the routine use of prophylactic therapies, chronic gvhd (cgvhd) occurs in 10 to 80% of patients undergoing allogeneic sct. ciclosporin a (csa) remains the backbone for gvhd prophylaxis in both myeloablative (mac) and reduced intensity conditioning (ric) sct. however, in a significant proportion of patients, csa causes important side effects and needs to be discontinued. in this study we have evaluated the impact of substituting csa for mycophenolate mofetil (mmf) as immunosuppression (is), on the incidence of cgvhd. we have compared the outcome of 87 consecutive patients that underwent allogeneic sct from march 2011 to november 2015 at the bmt unit of the hammersmith hospital and received csa as part of the gvhd prophylaxis. of them, 54 patients (62%) remained on csa prophylaxis for the duration of the planned post sct immunosuppression period and 33 patients (38%) required a switch to mmf before day +100. the reason for changing the is was nephrotoxicity in the majority of cases (n = 25, 70%), neurological toxicity (n = 2, 6%), disease relapse (n = 1, 3%), intolerance (n = 2, 6%) or not determined (n = 3, 9%) . we excluded from the analysis those patients whose is was changed due to the presence of acute gvhd. both groups had similar patient and transplant characteristics (see table 1 ). [p171] however, distribution according to diagnosis showed a predominance of aml (43%) in patients that remained on csa and mds (2%) for those that switched to mmf. the mean survival rate of the entire cohort was 902.897 days (±87) . the mean survival of each group was: csa 996.86 days (±109.076) and mmf 602.474 (±94.779). this difference in survival reached statistical significance (p:0.04). we graded cgvhd using the nih scoring system as mild, moderate and severe. out the 54 patients that continued with csa, 55.6 % (n = 30) had no cgvhd; 16.7 % (n = 9) had mild cgvhd; 20.4 % (n = 11) had moderate and 7.4 % (n = 4) had severe cgvhd. in patients that switched to mmf 45.5 % (n = 15) did not develop any cgvhd; 9.1 % (n = 3) developed mild; 24.4 % (n = 8) moderate cgvhd and 21.25 % (n = 7) developed severe cgvhd. (p: 0.093). the cumulative incidence of any cgvhd at 2 years post sct was 58% for the csa/mmf group and 42% for the csa only group (p = 0.04). csa is one of the standards of care for gvhd prophylaxis in both ric and mac sct. in our cohort of patients, those who remained on csa had a better overall survival and a reduced incidence of chronic gvhd compared with those patients that stopped csa and replaced it by mmf. csa toxicity should be prevented to avoid gvhd-related complications. disclosure of conflict of interest: none. although the outcome of allogeneic stem cell transplantation (sct) from an unrelated donor (ud) has considerably improved over the recent years, graft versus host disease (gvhd) still represents a severe and potentially lethal complication. in vivo t-cell depletion with anti-thymocyte globulin (atg) has been shown to significantly decrease the risk of both acute and chronic gvhd without compromising survival, however the optimal dose has not been defined yet. aim of present retrospective study was to evaluate the impact of two different doses of rabbit atg (thymoglobulin) on gvhd incidence, infectious complications and outcome of 156 patients undergoing sct from ud. between february 2004 and september 2015, 40 patients received thymoglobulin 5 mg/kg (atg-5 group) and 116 received thymoglobulin 7 mg/kg (atg-7 group) in addition to cyclosporin and short course mtx as gvhd prophylaxis. the two groups were comparable regarding sex, age, diagnosis and disease phase at transplant, comorbidity index, stem cell source and antimicrobial prophylaxis. conditioning treatment was myeloablative in 90% of atg-5 group patients and in 78% of atg-7 group patients. donor and recipient pairs were 10/10 hla matched in 75% of the cases of the atg-5 group and in 39% of the cases of the atg-7 group (p 0.001). netrophil engraftment occurred in 150 (96%) patients at a median of 17 days post transplant (range: 11-41 days); six patients (2 in the atg-5 group and 4 in the atg-7 group) died before engraftment. overall, 48 patients (31%) developed grade ii-iv acute gvhd, without significant differences between the two groups (atg-5 32% and atg-7 30%, p 0.939). similarly, chronic gvhd was not significantly different between the two groups: moderate to severe chronic gvhd occurred in 30% of the patients in the atg-5 group and in 27% of the patients in the atg-7 group (p 0.846). univariate logistic regression analysis didn't show any significant differences between the two groups respect the incidence of bacteremia, invasive fungal infections acute and chronic gvhd. with a median follow-up of 66.6 months, 84 patients (54%) are alive, 79 in complete remission and 5 after disease relapse. transplant related mortality was superimposable in the two groups (atg-5 17% vs atg-7 20%). kaplan-meier estimates of overall survival and event free survival were 54% and 52%, respectively, without statistically significant differences between the two groups and between hla matched and hla mismatched sct. the results of our study suggest that different doses of atg tailored on hla compatibility might be effective for preventing gvhd with any detrimental effect on overall survival and incidence of infectious complications. a prospective randomized study is mandatory to confirm our preliminary results. disclosure of conflict of interest: none. c-reactive protein levels at acute gvhd diagnosis predict steroid-resistance, treatment related mortality and overall survival after allogeneic hematopoietic stem cell transplantation l minculescu 1 , ls friis 1 , bt kornblit 1 , sl petersen 1 , i schjødt 1 , ns andersen 1 and h sengeløv 1 department of hematology, rigshospitalet, copenhagen, denmark acute graft versus host disease (agvhd) remains an excessive cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (hsct). primary treatment consists of high-dose corticosteroids, but a small group of patients are steroid-resistant and their prognosis is especially poor. a predictor of patients at risk of steroid-failure would aid the decision of additional immunosuppressive treatment at an early stage. there is experimental evidence that co-existing inflammation aggravates agvhd. since c-reactive protein (crp) is a systemic inflammatory marker, we aimed to investigate whether crp levels at agvhd diagnosis could predict the risk of failing first-line therapy and developing steroid-resistance. we retrospectively studied 461 patients transplanted between 2010 and 2015, table 1. acute gvhd was diagnosed in 204 patients, 149 of whom had grade ii-iv. crp, total white blood cell-, lymphocyte-and neutrophil counts were available for all patients at the time of agvhd diagnosis. according to local protocol, patients with failed response to high-dose steroid (2 mg/kg) were treated with the tumor necrosis factor (tnf) alpha inhibitor infliximab and categorized as steroid-resistant. of 149 grade ii-iv agvhd patients 28 (19%) developed steroid resistant disease. crp levels at diagnosis among these where between o1 and 263 mg/l. crp levels where significantly higher in patients who developed steroid resistance compared to patients responding to high-dose corticosteroids, p = 0.001, hr 1.011 (95% ci 1.005, 1.018). this translated into significantly increased transplant-related mortality (trm) and decreased overall survival in patients with high crp levels, figure 1 . total white blood cell-, lymphocyte-and neutrophil counts were not associated with steroid resistance in agvhd patients. cxcr3 is chemokine receptor expressed on activated t lymphocytes, in particular on th1 cells, nk cells, dendritic cells, and subsets of epithelial and endothelial cells. cxcr3 ligands attract th1 cells into inflamed tissues and concomitantly block the migration of th2 cells. furthermore, inhibitory functional autoantibodies against cxcr3 occur in humans which play an important role in cxcr3-dependent immune regulation. in addition, cxcr3 regulates endothelial cell homeostasis. there are two variants of cxcr3: cxcr3-a and cxcr3-b. overexpression of cxcr3-a on endothelial cells is associated with an increase in cell survival, whereas overexpression of cxcr3-b dramatically reduced dna synthesis and up-regulated apoptotic endothelial death. here we have studied if a dysfunctional cxcr3 axis might be involved in gvhd pathogenesis and could link endothelial and t cell pathology in acute gvhd. we assessed concentrations of the cxcr ligands cxcl9, cxcl10 and cxcl11 as well as anti-cxcr3 autoantibodies in 98 patients with high grade (3) (4) acute intestinal gvhd for whom serum was available at gvhd onset. furthermore, anti-cxcr3 autoantibodies and cxcl9 levels were measured in sera stored before conditioning therapy. all variables were tested for influence on post-gvhd survival using cause-specific cox regression analysis. at gvhd onset, we observed a strong inter-correlation of cxcr3 ligands, but no correlation with anti-cxcr3 auto-antibodies. compared with pre-conditioning probes, cxcl9 levels strongly increased (median 303 to 721 pg/ml, p o0.001), whereas antithese results suggest crp levels at diagnosis as a valid predictor of developing steroid resistant disease in agvhd grade ii-iv and survival in allogeneic hematopoietic transplant recipients. [p173] s206 cxcr3 decreased (median 4.4 to 2.6 u/ml, po 0.001). anti-cxcr3 levels before conditioning and at gvhd onset correlated (coeff. 0.497, p o0.001), whereas cxcl9 levels did not. in multivariable analyses, low anti-cxcr3 and high cxcl9 measured at disease onset were strongest predictors of survival after acute gvhd. notably, high levels of the proinflammatory chemokine cxcl9 were particularly prognostic of an adverse outcome of gvhd in the presence of a high endothelial risk as assessed by the previously published easix score, while high anti-cxcr3 levels were most protective in patients with low easix score (that is, low endothelial risk). a score based on cxcl9, anti-cxcr3, and easix allowed an effective prediction of acute gvhd outcome ranging from mortality 490% (high cxcl9 + high easix) to mortality o20% (low cxcl9, low easix, high anti-cxcr3. our data suggest a strong role for the cxcr3 axis in the pathology of acute high grade gvhd. the opposing effects of cxcl9 and anti-cxcr3 indicate a functional, attenuating role for these auto-antibodies. the overall prognostic impact of the immunemodulating cxcr3 axis appears to depend on the underlying integrity of the patients' endothelial homeostasis. despite some progress in acute lymphoblastic leukemia (all) treatment including modern chemotherapy modalities, monoclonal antibodies and newer tyrosine kinase inhibitors (tki) for ph positive cases, the final success is still difficult to reach. allogeneic hematopoietic stem cells transplantation (allohsct) has remained an essential approach in attempts to cure all. tki routinely used for all ph(+) pre-and post-transplant treatment are also described as an alternative and adjunctive approach for chronic gvhd especially with fibrotic features due to their antifibrotic activity targeting the platelet-derived growth factor receptor (pdgfr) pathways. in this study we have tried to estimate the potential influence of pretransplant tki treatment on gvhd occurrence comparing all ph(+) and all ph(− ) cases treated with allohsct. a cohort of 119 all patients consisted of 93 all ph( − ) and 26 all ph(+) cases treated with allohsct was retrospectively analyzed. all patients were transplanted from sibling or unrelated donor (no haploidentical procedures were included). all ph(+) patients achieved pretransplant treatment with imatinib and chemotherapy, and ph( − ) patients with chemotherapy alone. the median age in ph( − ) and ph(+) group was 28 vs 35 (p = 0.04), the percentage of hla mismatched transplantations -4,.3 vs 19.2 (p = 0.00004), the percentage of acute gvhd cases-48.4 vs 76.9 (p = 0.01) and extensive chronic gvhd cases-80.5 vs 50.0, respectively. there were no significant difference between groups in patients sex (f/m-41/52 vs 14/12 respectively), ric/mac conditioning, unrelated/sibling donor, donors age, bm/pbpc transplantation, number of cd34 cells and chronic gvhd incidence. all patients received cyclosporine-and methotrexate-based gvhd prophylaxis. gvhd occurrence was analyzed in subgroups as previously described: all ph( − ) and all ph(+). as mentioned above the incidence of acute gvhd was higher in ph(+) group (higher number of hla mismatched transplantations in this group) but the incidence of extensive chronic gvhd was higher in ph (-) group. cox proportional hazard model analysis revealed death risk caused by gvhd higher in ph negative group (hazard ratio = 2.3; ci 95% = 1.02-5.18; p = 0.04). the analysis of competing events was performed to estimate the probability of death caused by gvhd vs other complications (transplant related mortality, infections and relapse). the impact of conditioning was not significant on gvhd related deaths vs other complications (p = 0.234 vs 0.009, respectivelyfigure 1 ). the same results were achieved with donor cmv status (p = 0.09 vs 0.04figure 2 ). we have not found any significant difference either in gvhd or other complications related deaths taking into account patient s sex/age, donor sex/age, patients cmv status, number of cd34 cells transplanted. on the other hand, the influence of agvhd and chgvhd on deaths related to other complications was not significant (p = 0.242 vs 0.147). cumulative probability of overall survival was higher in ph(+) group but the difference was not significant. the impact of pretransplant treatment with imatinib on gvhd occurrence has not been estimated so far. we are aware of our results to be preliminary and variety of data is still to be evaluated. however our results, if confirmed, may suggest the influence of imatinib on decreasing the extensiveness of chronic gvhd. disclosure of conflict of interest: none. seta-tsukinowa, otsu, 520-2192, japan; 3 department of pathology and laboratory medicine, emory university hospital, atlanta, ga, usa; 4 department of biostatistics and bioinformatics, rollins school of public health, emory university, atlanta, ga, usa; 5 pathology and pediatrics, emory university school of medicine, atlanta, ga, usa, aflac cancer center and blood disorders service, children's healthcare of atlanta, atlanta, ga, usa and 6 bloodworks northwest research institute, seattle, wa, usa more than 90% of allogeneic hematopoietic stem cell transplant (allo-hsct) patients receive red blood cell (rbc) and platelet (plt) transfusions in the peritransplant period. preclinical models indicate that rbc and plt transfusions trigger inflammation, raising the question of whether such transfusions are associated with development of severe acute graft-versus-host disease (grade iii/iv agvhd) and mortality in allo-hsct recipients. we conducted a retrospective analysis of rbc and plt transfusions, agvhd incidence, and mortality among 322 consecutive adult patients receiving non-t celldepleted allogeneic bone marrow (11%) or g-csf-mobilized blood stem cell grafts (89%). common underlying diseases were acute myeloblastic leukemia (41%), myelodysplastic syndrome (14%), and acute lymphoblastic leukemia (12%) . underlying disease risk was ranked as low (41%), intermediate (26%) or high (32%). allografts were obtained from 10/10 hlamatched sibling donors (35%), unrelated donors (43%), or from donors mismatched at 1-2 hla alleles (22%). graft sources were bone marrow (11%) or mobilized pbsc (89%). the cumulative incidences of grade iii-iv agvhd and mortality prior to day 150 without developing grade iii/iv agvhd were estimated using the cumulative incidence function and a cox proportional hazards regression model. covariates included in multivariable analysis was limited to baseline covariates associated with grade iii/iv agvhd at the p median number of rbc or plt transfusions ( figure 1 ). univariate analysis showed a lower hematocrit on admission (median of 5 rbc units transfused (p = 0.001) were significantly associated with the risk of developing grade iii/iv agvhd, while a longer time to neutrophil engraftment was inversely associated with grade iii/iv agvhd ( ⩾ median of 15 days, hr 0.58, p = 0.03). multivariate cox regression analysis showed only larger numbers of rbc units transfused and hla mismatch independently associated with severe agvhd (p = 0.02 and p = 0.008, respectively), while underlying disease risk and larger numbers of transfused rbc units were independently associated with overall survival in a multivariate analysis that excluded agvhd grade. overall mortality rate was lowest for the group with fewer rbc and plt transfusions (43%), and greatest for the group with more rbc and plt transfusions (67%). groups that received more rbc units had higher rates of mortality due to gvhd, while patients who received more plt transfusions and fewer rbc transfusions had greater mortality from relapse ( figure 2 ). these data support the hypothesis that peritransplant rbc transfusions are associated with the risk of developing severe agvhd and worse overall survival following allo-hsct. prospective studies are warranted to whether rbc transfusions promote t-cell activation and inflammation in allo-hsct recipients, leading to increased severe agvhd. disclosure of conflict of interest: none. early high umbilical cord blood cd3 chimerism associated with acute gvhd at time of onset in haplo-cord transplantation h choe 1 , j hsu 1 , s mayer 1 , u gergis 1 , a phillips 1 , t shore 1 and k van besien 1 1 department of hematology/oncology, weill cornell medicine, new york, ny 10065, usa introduction: haplo-cord transplantation is a combined haploidentical and cord blood transplant that allows for more rapid engraftment by the haplo with eventual loss of the haplo graft upon engraftment of the cord. haplo-cord transplants are associated with an approximate 25-43% incidence of agvhd. reported, using chimerism assessments at approximately day 56 after transplant for aml and mds, that lower umbilical cord blood (ucb) chimerism in the cd3 or cd33 lineages were associated with increased rates of relapse. we did not find a statistically significant association between day 56 chimerism and risk for acute gvhd.(2) here we report our analysis of chimerisms at the onset of agvhd. patients and methods we retrospectively reviewed all patients who underwent haplocord sct for all hematologic malignancies between july 2012 and march 2016. ucb for haplo-cord transplants were selected based on hla-typing and cell count. grafts were matched for at least 4 of 8 hla loci by the standard criteria and contained a minimum cell count of 1 ×10 7 nucleated cells per kilogram (kg) of the recipient's body weight before freezing. the haploidentical donor was a relative in the large majority of cases. we identified 90 patients evaluable for agvhd (onset before day 100) without preceding relapse or early death. of the total 90 patients, 31 patients were diagnosed with agvhd of any stage and grade. fractionated chimerisms including cd3 and cd33 components were routinely sent to evaluate for engraftment of the recipient vs haplo vs ucb. chimerism data was collected for both agvhd and no agvhd patients. the two-sided student's t-test was used to compare the agvhd cohort to the no agvhd cohort. chimerisms collected on patients with agvhd were within median ± 4 days of onset of agvhd. the median time to onset of agvhd was 47 days (range: 15-99 days). the median post-transplant chimerism recorded for comparison with the no agvhd patients was 57 days. the agvhd cohort had significantly lower cd3 recipient (p = 0.005) and higher cd3 ucb engraftment (p = 0.006). all other fractions, including the cd33 chimerisms, were not significantly different between the two cohorts. the agvhd vs no agvhd cohorts were further compared by degree of hla mismatch (4-6 out of 8 vs 7-8 out of 8). the frequency of agvhd was similar in the 4-6 out of 8 (23/62, 39%) and the 7-8 out of 8 (8/28, 28%) groups. within these subgroups, cd3 ucb chimerism was higher for those with agvhd (p = 0.03 and p = 0.03, respectively). conclusion the onset of agvhd in haplo-cord transplantation is associated with a significantly higher cd3 ucb chimerism and lower cd3 recipient chimerism. higher ucb chimerism may indicate that full ucb chimerism poses a higher risk of agvhd development. vice-versa persistent recipient chimerism may protect from acute gvhd. il-6 is a pleiotropic cytokine with both pro-and antiinflammatory properties (scheller 2014). the proinflammatory properties are mediated through trans-signaling that depends on the soluble il-6 receptor. il-6 trans-signaling is involved in several autoimmune diseases and in regulation of tissue regeneration of the gi-tract. specific snps in the il-6 receptor have been associated with increased baseline crp levels, severity of autoimmune diseases and response to interleukin-6 inhibition in rheumatoid arthritis. so fare little is known about the role of trans-signaling in graft-versus-host-disease (gvhd). in this study we investigated how 4 specific snps in the il-6 receptor influence pretransplant levels of crp, il-6 sil-6r and the risk of grade ii-iv acute gvhd in allogeneic stem cell transplantation (asct) in patients with family donor. dna was available for 103 patients (65 male, 40 female median age 48, range: 15-70) and 101 donors, that underwent asct with a matched related donor (97 sibling) at haukeland university hospital in the period 2006-2016. the majority received conditioning with either bycy (79) or flubu (17) and only 2 patients were transplanted with tbi-based conditioning. four different snps in the il-6r gene (rs2228145, rs4845617, rs4845618, rs4845374) were chosen on the basis of (i) documented or suspected roles in autoimmune disorders; and (ii) allele frequency between 0.10-0.49 and r 2 o0.5 between the different snps. genotyping was done using kaspar assays with viia7 instrument (life technologies). the overall genotype call rate was 98%. no departures (p-values o0.01) from hardy-weinberg equilibrium were observed. pretransplant serum levels of il-6, sil-6r and were analyzed with bio-plex kits (bio-rad, hercules, usa). both serum and dna analyses were performed in duplicates. patients being homozygous for the rs4845618 minor allele had significantly higher pretransplant serum sil6r levels but lower crp levels compared with patients homozygous for the major allele. the overall incidence of agvhd requiring high-dose steroid treatment (grade ii gastrointestinal, grade iii-iv liver and skin) in the cohort was 48%. when analyzing the conventional clinical and laboratory parameters only transplantation with a non-sibling donor was associated with increased risk of agvhd (p-value o0.01 hr 3,20 confidence interval 1.42-7.17). the presence of the rs4845617 in donor or recipient was associate with a significant increase in the rate of aghvd (p-value 0.027 hr 1.93 confidence interval 1.08-3.47). the snp rs4845617 (p-value 0.04 hr 1.86, confidence interval 1.04-3.35) was also significant in an adjusted model including both donor type and rs4845617. none of the evaluated snps were associated with an increase in early or late trm and did not influence os either. this study suggests that snps in the il-6r influence pretransplant biochemical characteristics and clinical outcomes after asct. future studies investigating the effect of il-6 inhibition as gvhd prophylaxis or treatment should include analyses of il-6 receptor snps to investigate their possible influence on treatment outcomes. graft-versus-host disease (gvhd) continues to be the major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant (allo-hsct). the prophylaxis scheme varies according to the center and the country. in ours institution we use triple-prophylaxis based on cyclosporin a (cya), metrotexate (mtx), and mycophenolate mofetil (mmf). this scheme has been used for more than one decade in asian centers where it has proven adequate effective and safe to prevent gvhd. we evaluated 90 patients undergoing allogeneic hematopoietic stem cell transplantation treated at the national cancer institute from january 2010 to december 2015. the triple-prophylaxis scheme consists in cya (adjusted serum levels, mtx (5 mg/m 2 days +1, +6, +11, +18) and we evaluated different doses of mmf, one of them includes 500 mg bid × 35 days and the other has high doses (15 mg/kg bid × 180 days), as gvhd prophylaxis. the response characteristic was analyzed using the pearson test, fisher's exact test on categorical variables and student's t-test, mann-whitney u on continuous tests. kaplan-meier method was used to estimate the probabilities of os, sle with the differences compared by the log-rank test. we analyzed 90 patients with median of age of 30 years (range: 15-64), 60% male gender, all were transplant with peripheral blood progenitor cells as a source. 52.2% were acute lymphoid leukemia and 25.5% acute myeloid leukemia, 12.2% chronic myeloid leukemia, 3.3% myelodysplastic syndromes, 3.3% aplastic anemias, 2.2% non-hodgkin's lymphomas and 1.1% hodgkin's lymphomas. myeloablative conditioning was used in 82% (bucy, cfm-gat) and 18% reduce intense conditioning (flubu, flucy, flucy-gat), 94.4% related hla compatibility. mmf 500 mg twice daily (bid) for 35 days (group 1) and of mmf 15 mg/kg bid for 180 days (group 2), in the group 2 the 85.3% developed febrile neutropenia vs 64.3% in group 1(p = 0.03). the frequency of gvhd was 19.6% group 1 vs 23.5% group 2 (p = 0.6), chronic gvhd was 23.5% vs 19.6% respectively (p = 0.7). at the moment of analysis 58.9% vs 26.5% were free of disease (p = 0.01). there no difference at 5-year overall survival was 37% (group 1) vs 49% (group 2) (p = 0.85), neither free-survival disease (p = 0.85). the mmf regimen shows noninferiority scheme for gvhd. the low doses and for shorter administration did not show differences in the incidence and severity of acute or chronic gvhd, os, dfs compared to the mmf regimen at 180 days with high doses. the high doses shows higher incidence of febrile neutropenia, but there were no differences in documented infections. disclosure of conflict of interest: none. a protein-losing enteropathy can develop due to conditioning regimen related gut toxicity and can cause albumin decline during peritransplant period in allogeneic stem cell transplantation (allohct). damaged intestinal mucosal barrier results in alloactivation of donor t cells and this situation is considered a primary event in the pathogenesis of acute graft-versus-host disease (agvhd). peritransplant albumin decline, as a result of conditioning regimen related protein-losing enteropathy, may predict agvhd (1) . in this retrospective study we tested this hypothesis. we evaluated 249 patients who received allohct between 2011 and 2016. albumin decline from the day of conditioning initiation until its nadir in the first 2 weeks of post-transplant period was calculated as delta albumin. acute gvhd was proven by biopsy in all patients. chi square and mann-whitney test were used for statistical analysis. patients' characteristics were shown in table-1. acute gvhd was developed in 78 patients and severe agvhd was developed in 15 patients. delta albumin was not different between agvhd patients and no agvhd patients. delta albumin was not related with severe agvhd. delta albumin was not different between patients who received myeloablative and reduced intensity conditioning regimens. when we used a cutoff value of 0.9 gr/dl for delta albumin, we could not find a relation between delta albumin and development of both agvhd and severe agvhd. we repeated the analysis for acute myeloid leukemia (aml) and myelodysplastic syndrome (mds) patients who receive myeloablative conditioning regimen and we found the same results, there was no difference between agvhd patients and no agvhd patients in terms of delta albumin. there was a number of studies that used albumin as a predictive and prognostic marker in the setting of agvhd. but albumin may decrease in patients due to many reasons like malnutrition, proteinuria, enteropathy, liver disease or being negative acute phase reactant. because of albumin value can show variability between patients, albumin decline may be a more objective criterion. rashidi et al. showed that 0.9 gr/dl decline in albumin may be a predictor of severe agvhd in 88 patients who was diagnosed with aml and mds and received myeloablative conditioning regimen. we repeat this analysis in our mds and aml (n = 98) patients but we couldn't find this relation. when we evaluated all our 249 patients, again there was no relation between delta albumin and development of both agvhd and severe agvhd. in conclusion, our study did not support rashidi et al.'s findings. because serum albumin level shows variability due to many reasons, it is hard to use albumin as a predictor of agvhd. sclerodermatous chronic graft-versus-host disease (scl-cgvhd) in its severe manifestation affects the patient quality of life and, due to complex pathomechanism, does not respond to standard immunosuppressive therapy-calcineurin inhibitors (cni) with corticosteroid. methotrexate (mtx) and rituximab appeared to be effective in some patients but the novel strategies, including extracorporeal photopheresis (ecp), imatinib, m-tor inhibitors (for example, sirolimus) and ruxolitinib seem to become the real breakthrough. we retrospectively analysed data of 33 patients with scl-cgvhd, who underwent allogeneic hematopoietic cell transplantation (hct) between 2009-2015 in 5 transplant centres. the study group consisted of 32 patients with haematological malignancies and one with aplastic anaemia, 14 female and 19 male, with the median age 36 (18-64). donors' median age was 40, with predominance of matched sibling donors (21 donors) and even distribution of the donors' gender. in 22 patients (67%) acute gvhd (agvhd) was diagnosed with skin involvement observed in 19 ones. acute gvhd directly progressed to cgvhd in 13 cases. in 11 patients (33%) cgvhd developed ‛de novo' and in 2 cases cgvhd was induced by dli. median time from hct to cgvhd diagnosis was 8 months and to scl-cgvhd diagnosis-32 months. seven patients (21%) were scored as moderate cgvhd and 26 patients (79%) as severe cgvhd according to nih-2014 cgvhd activity classification. in 14 patients sclerotic features had superficial form and in 19 ones deep sclerosis was observed. chronic gvhd manifestation in other organs includes: mouth (94%), joints and fascia (77%), liver (74%), eyes (64%), gi tract (33%) and lungs (21%). 26 patients were treated with ecp and/or sirolimus and /or imatinib with 80% response rate (complete-cr, partial-pr or minimal-mr). median duration of ecp therapy, sirolimus and imatinib treatment was 20 months (2-40), 3 months (1-30), and 5 months (1-18), respectively. sirolimus was added more likely (9 patients) as the first in case of suboptimal response to ecp after median 15 weeks and in 4 patients was subsequently replaced by imatinib with no favourable outcome in 3 cases. in 3 patients imatinib was initially used in combination with ecp therapy, leading to pr or mr. mtx without novel therapies was effective in 4 patients with limited skin involvement, 3 patients responded to mtx plus imatinib and 1 patient to mtx plus sirolimus. two patients, after failure of other therapies, have been receiving ruxolitinib with improvement. only 3 patient (15%) were nonresponsive to ecp (progressive or stable disease), 4 patients (36%) to sirolimus and 4 patients (33%) to imatinib. toxicity incidence was equally observed in case of sirolimus and imatinib and lead to the therapy discontinuation in altogether 4 patients. infectious complications were observed in 20 patients (60%). ecp confirms to be the most effective therapeutic strategy in severe forms of scl-cgvhd with favourable safety profile. imatinib and sirolimus, targeting different fibrotic pathways, both play important role in nonresponsive patients, improving the outcome in ecp and non-ecp group. in case of limited access to ecp, mtx remains to be beneficial in combination therapy of moderate scl-cgvhd and an alternative to cni. disclosure of conflict of interest: none. post-transplant morbidity and mortality are majorly determined by gvl effect counter-balanced by gvhd. treatment with systemic steroids represents the first-line therapy for gvhd, but is associated with increased incidence of infection and relapse. ecp can reduce the extent of gvhd while preserving anti-virus/-tumor activity. to elucidate this clinical phenomenon on an immunological level, we correlated clinical data with immunological findings in 20 patients under ecp treatment. nine patients with acute gvhd (agvhd) of the gut ii-iv suffering from severe diarrhea were treated by ecp in addition to triple-drug immunosuppressive therapy. furthermore, 11 patients with chronic gvhd (cgvhd) of the skin or lung despite triple-drug received ecp treatment. patients were evaluated according to their individual response and clinical condition. phenotypical analysis of different cellular subsets of patients and healthy donors was performed by multicolor flow cytometry. functional properties of virus-specific cd8+ t and nk cells were evaluated by inf-γ-elispot and 51cr-release assay. about 20 patients were treated by ecp in this study. however, two agvhd and two cgvhd patients had to be withdrawn from ecp treatment after a few ecp cycles due to pancytopenia or poor clinical condition. for patients with agvhd 8 up to 25 ecp cycles were needed for response. all patients achieving a complete response (cr) were still alive 1 year after initiating ecp therapy. overall response, that is, cr or partial response (pr) according to nih criteria, was obtained in 5 of 7 patients with agvhd (71.4%) including cr in 3 of 7 (42.8%). out of 9 cgvhd patients 7 (77.8%) reached pr, and 2 (22.2%) remained stable under ecp treatment. after 1 year, overall survival (os) was 60% for agvhd patients responding to ecp, while only 25% for non-responders. os for cgvhd patients was 91%. during intensive ecp treatment for patients with agvhd of the gut, the average stool volume and frequency decreased and consistency changed from loose to formed stool. steroids could be tapered down to a mean of 22% of the initial dosage. cgvhd patients were stabilized under ecp treatment and steroid dosage could be reduced to a mean of 38%. clinically responding patients showed increased numbers of regulatory cells including mdscs, foxp3+cd8+ and foxp3+cd25+cd4+ tregs, as well as cd4 − cd8 − cd3+ t, vδ2+ t cells and regulatory b lymphocytes. furthermore, loss of cd62l expression on effector cells like cd4+ te, cd8+ te, nk and nkt cells was observed under ecp treatment. interestingly, ecp treatment did not dramatically influence the frequency of cd4+cd8+cd3+ t, γδ t cells and nkt cells, which possess anti-virus/-tumor function. elispot and 51cr-release assays revealed stable anti-viral activity of cd8+ t cells as well as functional cytotoxicity of nk cells. moreover, cd8+ t, cd8+ tem, cd62l+cd4+ temra, cd56 +cd3 − nk and cd56brightcd16 − nk cells could serve as reliable biomarkers for prediction of response to ecp. conclusion: ecp treatment might stabilize or even improve clinical situation of patients suffering from gvhd. in clinically responding patients an immunomodulation was observed in terms of increasing numbers of regulatory cells with loss of migratory capacity of effector cells while anti-virus/-leukemia t-cell function was preserved. disclosure of conflict of interest: none. extracorporeal photopheresis affects dendritic cells by reducing total numbers and blunting cytokine production in patients with graft versus host disease tj altmann 1,2 , m bickerton 1 , am flinn 1 , u cytlak 1 , p milne 1 , s pagan 1 , m collin 1,2 , v bigley 1,2 and ar gennery 1,2 1 institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom and 2 newcastle upon tyne hospitals nhs foundation trust, newcastle upon tyne, uk graft versus host disease (gvhd) and concomitant immunosuppression is a leading cause of morbidity and mortality post hematopoietic stem cell transplantation (hsct). the pathophysiology of gvhd is complex, involving presentation of histo-incompatible antigen by activated recipient dendritic cells (dcs), activation and proliferation of donor t cells and resultant tissue damage. extracorporeal photopheresis (ecp) is a second-line treatment for steroid refractory or dependent gvhd that facilitates the reduction of immunosuppression. ecp's mechanism of action is unclear and is likely to be multifaceted. apoptosis of lymphocytes, induction of a th2 favoured environment and increased numbers of regulatory lymphocytes have been implicated 1 . although ecp has been shown to modify the function of in vitro monocyte-derived dcs 2 , its effect on primary (non monocyte-derived) dcs has not been studied. our aim was to determine whether ecp had immediate or long-term affects on primary dc numbers or function. we enumerated monocyte and dc subsets (cdc1, cdc2 myeloid dcs and plasmacytoid dcs) in whole blood before, during and after ecp cycles, and developed a novel dc function assay, suitable for use on clinical samples. four adults with immunosuppression withdrawal gvhd and four children with acute gvhd, received ecp during the study. all received ciclosporin gvhd prophylaxis and corticosteroid treatment at onset of gvhd. children received ⩾ 1 dose of infliximab prior to starting ecp. adults received two ecp treatments (one cycle) every 2 weeks and children received two ecp treatments (one cycle) weekly. whole blood was taken before and after each cycle of ecp. trucount flow cytometry analysis of whole blood was used to enumerate mononuclear leukocytes. to assess function, peripheral blood mononuclear cells were isolated by density centrifugation and stimulated with toll-like receptor agonists. cell-specific cytokine production was then analyzed by flow cytometry. samples were compared to healthy controls and pre-ecp samples. median time to first ecp treatment from gvhd diagnosis was 33.5 days. no gvhd flares were experienced during study period. (1) adult had a cycle of treatment delayed due to intercurrent pneumonia. numbers of cdc2, pdcs and classical monocytes were significantly reduced after each ecp treatment in the adult group. dc numbers followed the same trend after ecp in the paediatric group but were not significantly different before and after ecp. this is perhaps due to initial lower dc count compared to adults in the children before the first ecp cycle. functional analysis showed a reduction in cytokine production in dcs and monocytes in both groups over the course of ecp treatment. our data support a cell-intrinsic effect of ecp on monocytes and dcs, with numerical and functional consequences. this may contribute to the beneficial effect of ecp both through reduction of inflammatory effector function and through modulation of interactions with other immune cells. correlation with immunosuppression withdrawal and clinical events during treatment may provide further insight into the role of monocytes and dcs in gvhd and ecp, which may aid in the development of novel targeted therapies for gvhd. extracorporeal photopheresis as early second-line treatment for patients with steroid-dependent or refractory acute graft-versus-host disease: a single-centre experience i sakellari 1 , i batsis 1 , e gavriilaki, a-k panteliadou, a lazaridou, k leontopoulos, d mallouri, a bouinta, v constantinou, e yannaki, c smias and a anagnostopoulos 1 1 department of hematology bmt unit, g. papanicolaou hospital, thessaloniki, greece acute graft-versus-host disease (agvhd) remains a severe complication of allogeneic haematopoietic cell transplantation (allohct). corticosteroids as the backbone of initial therapy for agvhd result in varied complete responses (25-69%). traditional secondary treatments lead to profound immunosuppression without improved survival. on the basis of our experience in chronic gvhd, we aimed to prospectively assess the role of extracorporeal photopheresis (ecp) as early secondline treatment in steroid-dependent and refractory agvhd. we enrolled consecutive patients with steroid-dependent or refractory grade (gr) ii-iv agvhd post allohct from january 2013 to august 2016. all patients with unrelated or haploidentical donors received thymoglobulin (atg) 5 mg/kg as prophylaxis. post-transplant gvhd prophylaxis included cyclosporine-methotrexate in myeloablative and cyclosporine-mycophenolate mofetil in reduced toxicity or intensity regimens. ecp was commenced after assessment of response to 5 days of steroid treatment according to our protocol: two sessions per week for 1 month, one session per 2 weeks for 3 months, evaluation of response and one session per month for 6 months. we studied 20 patients, aged 35 (18-65), post allohct with myeloablative (14), reduced toxicity (4) and intensity (4) conditioning, from sibling (3), matched (8) or one locus mismatched (8) volunteer unrelated and haploidentical (1) donors. disease risk index was high (10), intermediate (9) and low (1). acute gvhd was observed at day +17 (8-50) in 15 patients, late onset at + 130 (110-160) in 4 patients and induced at +38 post donor lymphocyte infusion in a relapsed aml patient. skin, intestine and liver involvement was evident in 6 patients, skin and intestine in 10 and skin only in 4 patients. nine patients (2 with grii, 7 with griii agvhd) were steroid-dependent and 11 (8 with griii, 3 with griv) steroidrefractory. atg was administered simultaneously with ecp initiation in six refractory patients that further developed ebv reactivation (p = 0.032) treated pre-emptively with rituximab. ecp was commenced at day +51 for 15 (4-20) sessions. the majority of patients (16/20) presented partial (6), very good (9) or complete (1) response to ecp. with 8.3 (1.7-51) months of follow-up, immunosuppression was reduced in 10/20 and ceased in 1 patient. clinically significant bacterial infections were found in 17 patients, fungal in 2, cmv and ebv reactivation in 14 and 13, respectively, and other viral in 5 patients. cumulative incidence (ci) of chronic gvhd was 77.4 at 1 year. one-year ci of agvhd-related mortality was 21%. one-year overall survival (os) was 53% and significantly increased in steroid-dependent vs refractory patients (80% vs 36%, p = 0.039). reduction of immunosuppression (p = 0.008) and response to ecp (p = 0.034) were associated with improved os, irrespectively of other factors. our data indicate that ecp should be considered early in the course of steroiddependent or refractory agvhd, before irreversible end organ damage has been established. optimal timing of intervention, frequency, duration and tapering schedule of ecp remain important unanswered questions. [p186] disclosure of conflict of interest: none. extracorporeal photopheresis for treatment of chronic graft versus host disease m lanska, a zavrelova, j radocha and p zak faculty of medicine, 4th department of internal medicine-hematology, university hospital hradec kralove, czech republic allogeneic stem cell transplantation represents a curative approach to many hematologic disorders. graft versus host disease (gvhd) is a1 complication with significant morbidity, mortality and decreased quality of life. extracorporeal photopheresis (ecp) represents possible treatment approach. mononuclear cells (mnc) collected by apheresis are photosensibilized with 8-methoxypsoralenem ex vivo, irradiated with uva and transfused back to the patient. aim of the study: evaluation of patients treated with ecp for gvhd at our center. thirteen patients (8 females and 5 males, median age 44 years) were treated with ecp. about 12 patients (pts) had matched sibling donor and 1 patient had unrelated donor. about 10 pts had sclerodermic form of gvhd, 2 had concomitant pulmonary gvhd, 2 had pulmonary gvhd alone. one pts had mild, seven moderate and five severe gvhd according to nih. about 11 patients were treated with steroids. mnc separation was prepared on cobe spectra and spectra optia (terumo bct, usa). 8-methoxypsoralen was added, irradiation was done on macogenic g2 (macopharma, mouvaux, france). about 696 procedures in 13 pts were performed (median 41 procedures, 12-120 procedures). the schema was as follows: ecp on 2 consecutive days every 2-3 weeks first 3 months with subsequent increase in interval. median follow up was 35 months. in sclerosing form two pts reached cr, six pts pr, one is stable and one patients progressed. in pulmonary gvhd one reached cr, two partial improvement, one is stable. seven pts are still alive, six died (two due to relapse, one secondary malignancy and three infections). it was possible to withdraw steroids in 10 pts. adverse events were clinically negligible. ecp is an effective treatment for chronic gvhd. especially sclerodermic form responds to ecp very well. it is safe and well tolerated procedure with minimal toxicity. supported by prvouk p-37. disclosure of conflict of interest: none. allogeneic haematopoietic stem cell transplantation (hsct) is a potentially curative treatment option for children with a variety of haematological, oncological and immunological diseases. graft versus host disease (gvhd) represents a major cause of post-transplantation mortality and morbidity affecting multiple organs including skin, gut, liver and lungs. gvhd is considered a succession of inflammation and donor t-cell activation initiated by translocation of gastro-intestinal microorganisms through impaired mucosal barriers after chemotherapeutic conditioning and/or infection. diagnosis of gvhd is based on clinical symptoms and histological findings, necessitating invasive and potentially harmful procedures including endoscopy and biopsy. as yet, no non-invasive markers are available for diagnosis or treatment monitoring in children with gvhd. faecal calprotectin (fc) reflects intestinal mucosal inflammation of any origin. in the setting of allogeneic hsct in adults, fc has shown to be a marker for acute (steroid-resistant) gvhd. we aimed to evaluate the feasibility of prospective fc measurement as a non-invasive marker for diagnosis and treatment in children with gvhd. a prospective, observational, single centre study was started in july 2015. by december 2016, 21 paediatric allogeneic hsct patients (age 0-17 years) were included after informed consent. faecal samples were collected from 2 weeks before to 6 months after hsct. fc levels were measured by elia, according to manufacturer's instructions. clinical symptoms were prospectively evaluated and managed according to local guidelines. if gvhd was suspected on clinical grounds, histological confirmation was obtained. first-line therapy for gvhd consisted of corticosteroids. in case of steroid-resistant disease, more advanced immune modulation was applied. a total of five patients developed histologically confirmed gvhd: acute gvhd of skin and gut (n = 2, one patient with steroidresistant disease), acute gvhd of skin only (n = 1); chronic gvhd of lung only (n = 1) and acute gvhd of skin followed by chronic oromucosal gvhd (n = 1). without exception and regardless of gut involvement, gvhd occurrence was accompanied by rises in fc levels to values 4100 μg/g (range: 108-1600 μg/g). fc levels correlated with clinical and histological grading. moreover, adequate response to therapy was consistently reflected by return of fc levels to values o100 μg/g. sensitivity of fc levels to diagnose gvhd was poor due to increased fc levels in patients with posttransplant complications other than gvhd such as viral reactivation and pulmonary or gastro-intestinal infections. fc levels reflect gvhd occurrence and correlate with clinical and histological grading in paediatric allogeneic hsct patients. fc levels increase in case of gvhd regardless of gut involvement, supporting a central role for (subclinical) intestinal inflammation in gvhd initiation. although, in this interim analysis, fc lacks sensitivity to diagnose gvhd, fc may serve as a noninvasive marker for monitoring therapy response and, thereby, reduce the need for repeated invasive procedures including endoscopy and biopsy. acute graft-versus-host disease (agvhd) is a major complication of allogeneic hematopoietic cell transplantation (hct), and glucocorticoids are typically used as first-line treatment. the aim of our study was to evaluate the effect of first-line ecp +/ − steroid therapy in order to reduce the incidence of infections and toxicity. from december 2010 to january 2016, 48 of 180 pts (27%), were diagnosed with agvhd grade ⩾ 2 following allosct. 40 pts were treated with ecp +/ − steroid as first-line therapy. about 8 (20%) pts were treated with ecp only and 32 (80%) with ecp + steroid 1-2 mg/kg/day. we compare this cohort with an historical group of patients, transplanted between 2001 and 2011, who were treated with steroid only for grade 2-4 agvhd (n = 23 out of 130). the two cohorts were well balanced in terms of median age (p = 0.4), disease type (p = 0.9), disease status (p = 0.09), graft source (p = 0.2), conditioning regimen (p = 0.1) and hct-ci (p = 0.3). there were more female patients (p = 0.03) and more haploidentical transplant (haplo-sct) (p = 0.0005) in the cohort treated with ecp+/ − steroid. ecp was performed using the offline technique, and was started as soon as possible with a treatment schedule consisting of four rounds of two procedures per week, three rounds of two procedures every other week and finally two procedures every month. steroid was tapered as soon as possible after starting ecp. the clinical response was evaluated at day +28. median follow-up for alive patients was 28 months for ecp group and 97 months for control group. there was no difference in terms of median time of agvhd onset (38 vs 39 days) and number of pts with grade 2 or 3-4 agvhd ( figure 1 ). ecp was started after a median of 4 (0-30) days from agvhd diagnosis. every patient underwent a median of 19 (2-83) ecp procedures, during a median time of 6 months. on day 28 after starting agvhd treatment with ecp+/ − steroid, 24 pts (70%) achieved cr or pr, 10 pts did not respond and 2 experienced agvhd relapse to front-line therapy. one year cumulative incidence (ci) of agvhd relapsed/refractory was 28.5% for ecp+/ − steroid. these percentages were not different from the cohort receiving steroid alone. ci of moderate-severe cgvhd was lower in the ecp group, probably due to the higher frequency of haplo-sct with pt-cy in the ecp group. about 100 days after agvhd onset, ci of infection (49% vs 74%), especially cmv reactivation (34% vs 67%), was lower in the ecp group, but was not statistical significant. ecp allowed a faster taper of steroid: 17 (3-98) vs 75 days (23-338) (p o0.0001). overall survival, progression-free survival, non-relapse mortality and ci of relapse rates did not differ in the two groups. in multivariate analysis, visceral involvement by agvhd was associated with an increased risk of failure to front-line therapy (hr: 5.5; range: 0.7-41; p = 0.09). this observational study suggests that the overall response rate of ecp +/ − steroids is similar to steroid alone for front-line treatment of grade 2-4 agvhd, but is potentially associated with lower incidence of infection, and in particular of cmv reactivation. a prospective phase 2 clinical trial is warranted to address whether augmentation with ecp may be beneficial for agvhd frontline treatment. [p189] a 4-year-old girl with neuroblastoma received autologous stem cell transplant (asct), followed by antibiotic prophylaxis and filgrastim. her transplant preparative regimen consisted of busulfan and melphalan. engraftment of neutrophil took place on day 11 after asct. twentieth day after asct, she experienced nausea and diarrhea. there was neither skin rash nor elevation in liver enzymes. the diarrhea continued to worsen day by day and reached to a daily volume of 1500 ml/ m 2 . infectious studies for stool and blood including testing for influenza a and b, parainfluenza, adenovirus, epstein-barr virus, amebiasis, cryptosporidium parvum, cytomegalovirus, clostridium difficile, salmonella, campylobacter, yersinia and shigella were all negative. colonoscopy and endoscopy were performed by an experienced pediatric gastroenterologist and findings were suspicious for severe graft versus host disease (gvhd). colonoscopy and rectoscopy revealed severe inflammatory changes, friability and patchy dark exudates on the mucosa of rectum. endoscopy revealed erosions, ulcers in the esophagus and a pale mucosal surface with reticulated submucosal vessels accompanied with erosion and erythema in the antrum of stomach. grade 3 gvhd was confirmed by pathologic analysis that revealed diffuse crypt dropout and mucosal erosion on rectal mucosal biopsy. mucosal erosions, apoptosis of epithelial cells and small lymphocytic infiltration of the lamina propria were found on duodenal biopsy. after these results, we started methylprednisolone intravenously at a dosage of 2 mg/kg/day. on the fourth day of treatment we increased the dosage to 5 mg/kg/day and added cyclosporine to treatment. because of unresponsiveness to treatment we decided to administer thirdparty mesenchymal stem cells (msc) (1 × 10 6 cd73+/cd105+ cells per kg). these were given intravenously at day +49 asct as single infusion. the second dose was given at day +56. within 5 days after first application of mscs, the frequency of diarrhea decreased to one-third. at day +16 after second dose of mscs, the patient's stool became nearly normal. we tapered the steroids first and stopped cyclosporine at +92th days after asct. discussion and conclusion: to our knowledge, this is the second case report of spontaneous severe autologous gvhd in a child with a solid tumor malignancy. regarding the pathogenesis of autologous graft-versus-host disease, there may have multiple causes for the loss of tolerance to self because of disrupted immune system. alteration of t regulatory cells by previous chemotherapy may be key point. endogenous cells that survive conditioning and assist in post-transplant maintenance of self-tolerance may be affected. microchimerism due to maternal cells transmitted during fetal development and persisting throughout adult life has also been postulated as a cause. however it is not very clear for factors that may contribute to the pathogenesis of this rare disease. autologous gvhd has the potential to cause critical illness in the hematopoietic stem cell transplantation patient population. in patients with multiple myeloma some experts report pathologically verified gastrointestinal gvhd as high as 6%. responses to steroids are variable. however, a significant proportion improve dramatically after early therapeutic intervention. so clinicians and pathologists should be aware in suspecting and recognizing gvhd in patients with diarrhea to guide therapy as soon as possible. disclosure of conflict of interest: none. haplosct patients did not receive additional gvhd prophylaxis aside from the ex vivo t-cell depletion (tcd) with clinimacs system. of the bud bmts 36 out of 39 patients engrafted, 92% of which had gvhd. the 8% who did not have any gvhd, relapsed. eighty one percent had agvhd, of which majority (51%) were grade 2 ( table 1) three patients did not have agvhd (10/10 mud and two cord blood grafts), but developed cgvhd. one of the patients who had grade 4 agvhd died and one still has intermittent cgvhd 12 years post bmt. there were 55% who had cgvhd. currently, all of our haplosct receive a cd3/cd45ra tcd grafts (n = 14). the depletion techniques for the 14 others were either cd3tcd, cd3/ cd45ra /tcrab tcd+cd34 +/cd45ra tcd; tcrab tcd. all 28 patients who received haplosct engrafted and 57% had agvhd (56% grade 1) ( table 1 ). we noted that some of the patients presented with nonclassical agvhd signs (upper gut gvhd, oral gvhd, blood). there were no patients who presented with grade 4 agvhd. cgvhd in the cohort was 35%. of note, 35% of patients did not have any gvhd and did not receive any form of immunosuppression post bmt. only eight patients received further immunosuppression for agvhd, median duration 131 (range: 91-344) days. at the time of this report, there are four patients with agvhd still receiving immunosuppression all o100 days and all on tapering doses. haplosct using ex vivo tcd techniques has a lower risk of gvhd with comparable, if not superior outcome to bud. the degree and duration of immunosuppression is also much less. this may translate to earlier immune reconstitution and less viral reactivation. [p191] disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (allo-hsct) offers a potential cure for several hematological diseases, but it is burdened by severe life-threatening complications, being gvhd the major cause of morbility and mortality. recently, more have been understood of the physio-pathologic relationships between endothelium and graft-versus-host disease (gvhd), showing that vascular endothelium is an early phase target of gvhd. in recent years, the direct count of circulating endothelial cells (cec) has emerged as a valuable biomarker of endothelial damage in a variety of disorders. however, due to their rareness and complex phenotype, different published techniques have showed variable degrees of uncertainty, reporting a wide range of cec values in healthy subjects. by means of the commercially available rare cell isolation platform cellsearch system, for cec identification and count, we correlated cec count changes to gvhd onset and response to treatment in allo-hsct patients. cec were analysed in 90 allo-hsct patients (37 aml, 15 all, 11 hd, 4 nhl, 4 cll, 5 mds, 4 cms, 8 mm, 2 saa) at the following time points: t1 (pre-conditioning), t2 (pre-transplant), t3 (engraftment), t4 (day+28 or onset of gvhd), t5 (1 week after steroid treatment). the median cec/ml at t1 was 24 (range: 2-786), in comparison to a value of 2 (range: 1-14) in healthy controls (p 0%: or 4.2, 95% ci 1.6-10.8; p = 0.002). we confirm that cec count represent a valid biomarker to monitor endothelial damage in patients undergoing allo-hsct and can be a valuable tool in supporting the diagnostic definition of gvhd and in monitoring responsiveness to treatment. moreover, the use of the cellsearch system can be crucial in order to move routinely cec monitoring into clinical practice of allo-hsct. reference clinicaltrials.gov nct02064972. disclosure of conflict of interest: this research was conducted with the support of the investigator-initiated study program of janssen diagnostics, llc to ca. kb is employee of janssen diagnostics. results of hla mismatched unrelated donor (mmud) hematopoietic cell transplants (hct) are worse than results of fully matched hct due to higher risk of gvhd, infection and graft failure. atg during conditioning reduces incidence of gvhd but can increase risk of infection and relapse. high doses posttransplant cyclophosphamide (2 × 50 mg/kg) prevent gvhd in haploidentical hct. we initiated this approach instead of atg in hct from one alelle or antigen mismatched unrelated (7/8)mmud-hct in 2014. here we present outcome of 21 patients (cy-group) transplanted between 2014 and 2016, comparing it with outcome of 54 patients transplanted between 2010 and 2016 from 7/8 mmud with atg-f (fresenius) 40 mg/kg given during conditioning. 21 patients in cy-group (12 males, 9 females) were transplanted from mmud mismatched for hla ( a-8, b-3, c-5, dr-5). about 14 patients had aml, 2 mds, 2 cll, 1 cml, 1 all and 1 mps. med. age of patients was 43 years (25-60). about 16 patients received myeloablative (flu-175 mg/m + iv bu 12.8 mg/kg) and 5 nonmyeloablative(flu-175 mg/m+mel 100-140 mg/kg +-tt 5 mg/kg) conditioning. about 18 patients received pbpc and 3 bm as a graft. graft versus host prophylaxis consisted of cyclophosphamide (50 mg/kg aibw) on d+3 and +5, cyclosporine a from d 0 and mmf from d+1. all patients received antibacterial, antifungal, hsv and pcp prophylaxis. historical control (atg) group consisted of 54 patients (32 males, 22 females), med. age 54 y (19-65) who had mmud-hct for aml-21, mds-9, nhl-5, mf-5, all-4, cll-4, cml-2, h.d-1, saa-1, mps-1 and mm-1. there were 13 mismatches for a, 11 for b, 20 for c and 10 for dr. myeloblative conditioning was used in 32 and nonmyeloablative in 22 patients. all patients received atg-f 20 mg/kg × 2 given d-2 and-1. cyclosporin was initiated d-2 and mmf d-1. all patients received anti-infectious prophylaxis as described previously. three of 21 patients from cy group died so far. two of them due to relapse and one due to toxicity and infection during aplasia. five patients relapsed . two achieved cr after dli and one is alive in relapse expecting second hct. about 18 patients are alive, 17 of them in cr. eight patients experienced agvhd (gr.i-3, gr.ii-5,gr.iii-0, gr. iv-0) and eight developed clinically mild cgvhd). about 22 patients from atg group are alive 11-80 m (med. 32 m) posthct. about 32 patients died 1-74 m postransplant (med. 16 m) due to vod, gvhd, infections and relapse. 100-day mortality is 5% (1/21) in cy group and 19% (10/54) in atg group. one year mortality is 14% (2/14) in cy and 30% (13/44) in atg group. patients from cy group have 78% probability of os at 24 months posthct vs 47% from atg group. cyclophosphamide 2 × 50 mg/kg instead of atg fresenius(40 mg/kg) for gvhd prophylaxis reduced 100-day and 1-year mortality, and improved probability of 24 m os significantly in our cohort of patients. this approach seems to be safe and effective in 7/8 mmud-hct. disclosure of conflict of interest: none. high transplanted cd34+ cells are not associated with beneficial effect on graft-versus-host disease-free, relapse-free survival (grfs) after allogeneic hematopoietic cell transplantation y lee 1 and ih lee 2 1 department of hematology, kyungpook national university hospital and 2 inhee lee the success of allogeneic hematopoietic cell transplantation (allo-hct) is comprehensively assessed by individual comorbidity, relapse, graft-versus-host disease (gvhd) and death. besides, inconsistent results have been reported regarding the dose of cd34+ cells. in the current study we have addressed the issue of the potential effect of stem cell dose on the of gvhd-free/relapse-free (grfs) associated with cd34+ cells doses. we retrospectively reviewed the medical records of the 255 patients who received allo-hct for acute myelogenous leukemia (aml), myelodysplastic syndrome (mds) and acute lymphoblastic leukemia (all) between 1998 and 2013 in kyungpook national university hospital. the grfs included grade 3-4 acute gvhd, systemic therapy-requiring chronic gvhd, relapse or death. the patients were reclassified into two groups according to the targeted cd34+ cell doses (6 × 10 6 per kg) by knuh protocol. a lower cd34+ group (n = 165, 64.7%), patients who underwent allo-hct with cd34+ cell dose o6 × 10 6 per kg; and a higher cd34+ group (n = 90, 35.3%) patients who underwent allo-hct with cd34+ cell ⩾ 6 × 10 per kg. the median age at transplant was 38.5 years (range: 15-68 years) and male was 111 patients (44.4%). primary diseases for allo-hct were aml/mds (n = 175, 70%) and all (n = 75, 30%). one hundred forty-three patients (57.2%) were in cr1 (complete remission), 25 (10%) in further cr and 87 (33.2%) in relapsed and refractory status. one hundred seventy-one patients (68.4%) received myeloablative conditioning regimen. gvhd prophylaxis consisted of methotrexate and cyclosporine a or mtx and tacrolimus. the median dose of cd34+ cell was 3.94 × 10 6 per kg (range: 0.46-6 × 10 6 per kg) in lower cd34+ group and 7.54 × 10 6 per kg (range: 6.01-20.6 × 10 6 per kg) in higher cd34+ group. there was no significant difference in neutrophil, platelet engraftment between two groups. the incidence of chronic gvhd was more frequent in higher cd34 + group (32.9% vs 48.2%, p = 0.042). the median follow-up duration was 18.1 months, with a range of 0.2-209.7 months. the 1-year overall survival (os), relapse free survival (rfs), nonrelapse mortality (nrm) and graft-versus-host disease (gvhd)free/relapse-free survival (grfs) since hct was 55.3 ± 3.1%, 66.0 ± 3.2%, 28.2 ± 0.3% and 32.9 ± 3.1%, respectively. there was no significant difference according to the infused cd34+ cell dose (figure1). the relapse rate was not proportionally affected by the cell dose (22.4% vs 26.7%, p = 0.712). and there was no significant correlation between the number of cd3+ and cd34+ cells infused (spearman correlation coefficient: p = 0.307). in a univariate analysis, patients transplanted with the higher cd34+ cell doses and higher cd3+ cell doses had no increased grfs (p = 0.623 and p = 0.158). an independent factor associated with worse grfs was risk status at transplant (hr = 1.782, 95% ci:1.267-2.509, p = 0.001). these results suggest that careful assessing the cd3+ and cd34+ graft content and tailoring the cell dose infused may help in reducing cgvhd risk without negative impact on grfs. a large and prospective study in a homogenous population will be needed to confirm the effect of stem cell dose. disclosure of conflict of interest: none. imatinib associated with extracorporeal photopheresis can fully reverse severe sclerotic-type lesions in patients with chronic graft-versus-host disease: the lille university hospital experience l magro 1 , j gauthier, b catteau 1 , l mannone 2 , a lionet 1 , v coiteux and i yakoub-agha 1 1 lille university hospital and 2 nice university hospital severe sclerotic-type chronic graft-versus-host disease (cgvhd) is difficult to reverse and can dramatically alter the quality of life of patients after allogeneic hematopoietic cell transplantation (allo-hct). imatinib or extracorporeal photopheresis (ecp) used separately yield sustained responses in s218 only about 30% of patients with steroid-refractory cgvhd. given their respective modest efficacy we hypothesized that the combination of imatinib with ecp could lead to higher response rates. we are reporting here on seven patients with severe steroid-refractory sclerotic-type cgvhd treated at our institution using this combination. we retrospectively analysed all patients treated at our institution (n = 7) with the combination of imatinib with ecp for severe steroidrefractory scgvhd. imatinib was started at 200 mg/day and increased to 400 mg/day if well tolerated. the cellex closed system was used for ecp. ecp was initiated twice weekly during 4 weeks. after this « induction » period, ecp sessions were scheduled less frequently according to the response to treatment. additional immunosuppressants were tapered gradually in responding patients. initial grading and response evaluation was determined according to the nih 2014 criteria. steroid-refractoriness was defined as progression of gvhd on high-dose steroids (⩾1 mg/kg) or progression during corticosteroid tapering. patient characteristics are displayed in table 1 . patients received an allo-hct between may 2004 and april 2011. median age at allo-hct was 47 (range: 23-57). a variety of myeloablative (n = 2) and non-myeloablative conditioning regimens were used (n = 5). antithymocyte globulin was used before allo-hct in one patient. gvhd prophylaxis consisted of ciclosporine and methotrexate in six patients. one patient received tacrolimus and methotrexate. five patients had prior history of acute gvhd. nih global severity grade was severe in all patients (n = 7) due to severe sclerotic features. the median number of previous therapies was 3 (range: 2-5). all patients were steroid-refractory. after a median follow-up of 54 months (range: 13-76 months) the overall response rate was 100%. the complete response rate was 57%. median time on ecp associated with imatinib was 36 months (range: 4-60 months). median time to best response was 7 months (range: 3-22 months). corticosteroids could be discontinued in all patients after a median time of 8 months (range: 6-44 months). patients #1, #5 and #6 received maintenance therapy with ecp upon discontinuation of imatinib. in four patients, both ecp and imatinib led to complete response and could be discontinued after 38, 74, 4 and 53 months for patients #3, #4, #5 and #7, respectively. patient #4 and #6 passed away after due to a myocardial infarction and the development of a solid tumour, respectively. patient #4 was off therapy while patient #6 remained on maintenance with ecp. both remained in complete response. patient #2 remained in response during 25 months before progression of cgvhd while on imatinib and ecp. none of our patients experienced adverse events related to either imatinib or ecp. despite the limited number of patients in this report, we observed that the combination of imatinib and ecp can lead to complete and sustained reversal of severe steroid-refractory sclerotic-type cgvhd. these encouraging results should be confirmed in a larger cohort. disclosure of conflict of interest: lm: therakos (honorarium). allogeneic hematopoietic cell transplantation (allo-hsct) is an established treatment modality that is potentially curative for many patients (pts) with acute myeloid leukemia (aml). aml itself is the most common indication for pts undergoing hsct nowadays. for pts with high-risk disease, allo-hsct is, perhaps, the most effective curative treatment and is considered the standard post-remission therapy in first complete remission (first cr). this is a retrospective study to analyze those variables which were associated with patients' overall survival (os) after allo-hsct. the study population consisted of 31 pts who were diagnosed of aml from january 2010 to july 2016 at the hospital universitario central asturias, and submitted to allo-hsct in first cr. risk status based on validated cytogenetics and molecular abnormalities following recommendations of european leukemianet was performed. sixteen (51.6%) were male. median age was 42 years old (range: 1-64). clinical characteristics at transplantation are represented in table 1 . median follow-up was 27 months (5-75). considering the donor type, os at 1 year was higher in pts receiving sd (91.8%) compared to 66% in those who received urd (p = 0.012). regarding graft source, os at 1 year was 88.9% who received pbsc compared to 48% in pts receiving bmsc (p = 0.012). gender also showed significant association with os, which was higher among men, os at 1 year was 100%, compared to 47.4% for women) (p = 0.002). the presence of minimal residual disease (mrd) detected using multiparametric flow cytometry was performed prospectively after induction and consolidation, and before transplantation. thirteen pts had negative mrd before transplantation. median os was greater in pts with negative mrd before transplantation compared to the group with positive mrd (67 vs 27 months, respectively) (p = 0.24). this difference did not reach statistical significance probably because the low number of the sample. thirteen pts developed agvhd. only 4 (28.6%) pts receiving sd developed agvhd compared to 8 (50%) pts among those who had an urd; however this association was not statistically significant (p = 0.23). also, we observed higher incidence of agvhd in bmsc group (6 pts; 60%) whereas only 7 (36.8%) in pbsc group developed agvhd. this tender did not reach significant association (p = 0.2). one year os was 59.8% in pts who developed agvhd and 87.1% who did not (p = 0.05). all factors that had a significant influence on pts survival were included in a multivariate analysis (cox regression model): graft source, donor type, pts gender and agvhd development. developing agvhd kept an independent association with mortality (or 6.12, 95% ci 1.39-27.29, po0.001) and male gender also persisted as an independent protective factor (or 0.12, 95% ci 0.02-0.06, p = 0.003). in our series, agvhd has shown a significant and independent association with os over other parameters such as graft source, type of donor or mrd before transplantation. identifying reliable predictors for agvhd development, controlling well known risk factors for this disease, as well as improving management of immunosupressors should still be the key to potentiate longer os in our patients. larger studies are needed to confirm our results. acute and chronic graft-versus-host diseases (gvhd) are associated with increased morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-hsct). older patients undergoing allo-hsct may experience a high degree of transplant-related complications and this concern has historically limited the use of allo-hsct for some older patients. in many studies, age has been shown to be a negative prognostic factor for survival and associated with higher transplant-related mortality (trm). however, in others, age was not shown to be a significant factor if appropriate adjustments for other comorbidities are incorporated in the analyses. there are very few studies that evaluated the relationship between patient's age, the presence of gvhd and long-term transplantation outcome. the aim of this study is to evaluate the impact of age in patients who develop acute and/or chronic gvhd after allo-hsct for hematological malignancies on the trm incidence. we included in the study 595 patients with hematological malignancies who received allo-hsct and were followed in our center between january 2008 and january 2016. for the purpose of this study, only patients who developed grade ii-iv acute gvhd and/or limited or extensive chronic gvhd where considered for analysis (n = 306). patients were split into three homogeneous groups according to age at transplantation taking into consideration the underlying disease, type of conditioning and disease response at transplantation. group 1 (younger) included patients aged o40 years (n = 103), group 2 (intermediate) included patients aged between 40 and 55 years (n = 102) and group 3 (older) included patients older than 55 years (n = 101). gvhd evolution over time was followed as well as the cumulative incidence of trm was calculated in case of acute or chronic gvhd in each group. thirty seven percent of grade ii gvhd occurred in the younger group (n = 29), 32% (n = 25) in the intermediate group and 31% (n = 24) in the older group; majority (69%) resolved in the younger group as well as in 24 and 46% in the latter two groups, respectively, while trm rates at 1 year were 10%, 30% and 30%, respectively, sdhr = 4.8, p = 0.01. among patients who had acute gvhd grade ii, 51, 36 and 59% in the three respective groups developed chronic gvhd later. grade iii-iv gvhd occurred in 21% (n = 20) in the younger group, 38% (n = 38) in the intermediate group and 38% (n = 35) in the older group; with a respective resolution in 20%, 26% and 17% of patients and were associated with comparable trm rates at 1 year of 39%, 40% and 47%, respectively, p = 0.5. among patients who had acute gvhd grade iii-iv, 40, 37 and 38% in the three respective groups developed chronic gvhd later. de novo chronic gvhd was observed with a higher rate in the intermediate and in the older group (table) while patients with extensive chronic gvhd older than 55 years had significantly higher trm at 2 years (47%) compared to 32% in those younger than 55 years, sdhr = 1.9, p = 0.04. patients who develop acute gvhd grade iii-iv could incur over 40% of trm at 1 year independently of age. resolution of acute gvhd grade ii was significantly better in younger patients while older patients with grade ii acute gvhd or with extensive gvhd had higher mortality compared to younger ones. in addition to an adapted prophylaxis, a better preemptive gvhd strategy should be warranted in older patients. [p197] disclosure of conflict of interest: none. in vivo effects of nilotinib on lymphocyte subpopulation and function following allogeneic stem cell transplantation e marinelli busilacchi 1 , a costantini 2 , j olivieri 1 , n viola 2 , s coluzzi 3 , e pirro 4 , g mancini 5 chronic graft versus host disease (cgvhd) is a major complication of allogeneic stem cell transplantation and is characterized by frequent multiorgan involvement resembling autoimmune diseases; its pathogenesis is still incompletely defined and a standard treatment is lacking. donor-derived cd4+ and cd8+ t lymphocytes have been considered the main effector cells mediating cgvhd pathogenesis; however, recent studies suggest that b cells might also play an important role. in vitro data indicate that tyrosine kinase inhibitors (tkis) such as imatinib and nilotinib affects both innate and adaptive immune response by interacting with different cell populations (t cells, b cells, dendritic cells, mast cells and macrophages). we sought to evaluate the impact of different doses of nilotinib on the distribution and function of lymphocyte subpopulations. we analyzed 44 samples obtained from 15 patient with steroiddependent/refractory cgvhd enrolled in a phase 1-2 study with nilotinib in steroid-refractory cgvhd (nct01810718): triplets of patient were treated with escalating doses starting from 200 mg/die (5), 300 mg/die (5), up to 400 mg/die (5) . blood and plasma were collected at baseline and at day 90 and 180 of therapy. trough plasma nilotinib concentrations had been previously determined by hplc (abstract c039, haematologica: evaluation of nilotinib safety in patients with steroid-refractory chronic graft-versus-host disease: a phase i-ii gitmo study). peripheral blood mononuclear cells were isolated by density gradient centrifugation using ficoll biocoll. six color flow cytometry analysis (facs canto ii) was performed using conjugated antibodies (anti-cd3, cd4, cd25, cd16, cd56, cd19). inflammatory cytokine analysis was performed on plasma samples according to the instruction of bioplex pro human cytokine 17plex assay (bio-rad). statistical analysis was performed by 2-tailed student's t-test; differences were considered statistically significant for po0.05. flow cytometry analysis showed that nilotinib did not exert any significant impact neither on the proportion of t lymphocytes subpopulation (cd3+cd4+ t helper, cd3+cd4+cd25+ t regulatory, cd3 +cd4 − t cytotoxic), nor on b lymphocytes and nk cells. on the contrary, a statistically significant and dose-independent decrease of pro-inflammatory and th-17 cytokine production was observed ( figure 1 ): reduction of il2 (po0.02), il10 (po0.05) and ifnγ (po0.02) were already significant after 90 days; decreases of il17 (po0.05) and tnfα (po0.02) become significant after 180 days. interestingly, after 180 days of therapy, among the 21 patients enrolled (according to the itt criteria) ten patients showed cgvhd improvement and the other five remained stable. this study shows that therapeutic doses of nilotinib can reduce plasma levels of inflammatory cytokines without affecting the proportions of lymphocyte subpopulations. these findings correlate with clinical response and suggest that besides the previously demonstrated anti-fibrotic effects, nilotinib has also potent anti-inflammatory and immune regulatory properties, supporting its role in patients with cgvhd. disclosure of conflict of interest: none. [p198] previously published p200 infectious gastro-enteritis after allogeneic hematopoietic transplantation after reduced intensity conditioning (allo-ric): incidence and possible role in gastro-intestinal acute gvhd i garcía-cadenas 1 , r martino 1 , a esquirol 1 , a bosch 1 , n rabella 2 , s saavedra 1 , c muñoz 2 , j briones 1 , s brunet 1 and j sierra 1 1 hematology and microbiology departments and 2 hospital de la santa creu i sant pau, autonomous university of barcelona, spain enterotoxigenic c. difficile-associated associated disease or infection (cdi) is a common cause of diarrhea after hematopoietic stem cell transplantation (sct). recent studies have suggested the relationship of cdi with gastro-intestinal (gi) graft-versus-host disease (gvhd). the possible role of other types of infectious gastro-enterocolitis (g-ec) in gvhd development has not been studied. as a prior investigation to a national prospective observational study on this issue, we conducted a single-center retrospective analysis including all adult patients who received an allo-ric sct between january 2010 and march 2016. the aim was describing the cause(s) (if known), timing and outcomes of recipients with possible g-ec (defined as new onset acute diarrhea grade ⩾ 2) in the first year after sct. of the 123 patients studied (median age: 54 years, 62% male, 49% aml or mds as underlying disease), 97 (79%) had a total of 148 episodes of acute diarrhea, with 35 (28%) developing more than one event. these acute diarrheas occurred at a median of 39 days (range: 1-363) after sct. overall, a g-ec causing pathogen was identified in 33 of 148 stool specimens (22%) and included: cdi (7), c. jejuni (7), rotavirus (7), adenovirus (2), norovirus (2), b. hominis (3), s. stercoralis, g. lamblia, a. caviae, salmonella enterica and cryptococcus (one in each case). most postransplant diarrheas (68/148; 46%) occurred during the 4 weeks after infusion and were attributable to mucosal damage caused by the ric (negative microbial screening and no evidence of gvhd).the rate of infectious g-ec among the diarrheas occurring after day +30 was 41% (33/80). the overall incidences of enteric infection were 12.7% (95% ci: 6.5-18.9) and 17.6% (95% ci:10.4-24.8) at +6 and +12 months after sct, respectively. all the infected patients had mild to moderate disease, and no deaths were attributable to this complication. there were no differences in 2 year-os and nrm between the infected and uninfected patients (81% vs 73%, p = 0.6 and 16% vs 19%, p = 0.7, respectively). in univariate analysis age o50 years, prior sct, donor type, atg administration and prior grade 2-4 agvhd were associated with development of infectious gastro-enteritis. in multivariate analysis, unrelated donor and grade 2-4 agvhd were the only factors significantly associated with gastrointestinal infection (hr 2.7; 95% ci: 1.1-6.5, p = 0.02 and hr 3.6, 95% ci 1.5-8.5, p = 0.004; respectively). acute gvhd occurred in 46% of patients (n = 56), with a median onset of 54 days (range: 4-231). the cumulative incidences of 2-4 acute gvhd at 100 days and 6 months post-sct were 21% (95% ci: 15.3-32%) and 32.6% (95% ci: 23.4-42%), respectively, and there was a trend toward a higher risk of 2-4 gvhd in the group of patients with an enteric pathogen (48.2% vs 27% at 1 year, p = 0.06). more importantly, an enteric infection occurred just before the onset or aggravation of gvhd in 12/33 infected patients in our study (36%) at a median interval of 8 days after the infection (range: 0-24). in summary, our results confirm that enteric infections are a common complication after allo-ric, representing at least 20% of the episodes of acute diarrhea during the first year post-sct. a possible interplay between infectious g-ec and gvhd was observed in this study. disclosure of conflict of interest: none. long-term efficacy of extracorporeal photopheresis in chronic graft versus host disease m nygaard 1 , t karlsmark 1 , n smedegaard andersen 2 , i schjødt 2 , s lykke petersen 2 , l smidstrup friis 2 , b kornblit 2 and h sengeløv 2 1 department of dermatology, bispebjerg hospital, copenhagen, denmark and 2 department of hematology, rigshospitalet, copenhagen, denmark chronic graft versus host disease (cgvhd) activity is known to fluctuate over time, so we evaluated cgvhd continuously throughout the extracorporeal photopheresis (ecp) treatment course and after stopping ecp. patients with at least 1 year follow-up, who were treated with ecp at department of dermatology, bispebjerg hospital between 2009 and 2015 were evaluated. a single investigator retrospectively evaluated response to ecp monthly for 6 months, every 3 months until 2 years and every 6 months until 3 years. prednisolone doses were recorded every 3 months. responses were defined as complete remission (cr) if no symptoms of cgvhd were present, partial remission (pr) as improvement in cgvhd or stationary cgvhd with more than 50% reduction in prednisolone, no change (nc) as no difference in symptom burden and o50% reduction in prednisolone. progressive disease (pd) was defined as worsening of symptoms with unchanged or intensified immunosuppressive medication. ecp was performed with therakos uvar xts or cellex. there were 45 evaluable patients with moderate (n = 20) or severe (n = 25) steroid-refractory, dependent or -intolerant cgvhd. the median age was 58 years (range: 19-71) and there were 22 females and 23 males. conditioning regimen was myeloablative (n = 10) and non-myeloablative (n = 35). seventeen had related donors and 28 had unrelated. stem cell source was peripheral blood (n = 36), bone marrow (n = 7) or umbilical cord blood (n = 2). number of organs affected by cgvhd was one (n = 8), two (n = 16), three (n = 11), four (n = 9) or five (n = 1) and involved organs were skin (n = 36), eyes (n = 26), mouth (n = 24), lungs (n = 8), genitals (n = 6), liver (n = 6), musculoskeletal system (n = 5) or gastrointestinal tract (n = 2). time from diagnosis of cgvhd to first ecp was median 444 days (range: 11-2760) and time from referral to ecp and the first ecp procedure was median 52 days (range: 11-178). at the time of the first ecp procedure patients were also treated with prednisolone (n = 43), sirolimus (n = 21), calcineurininhibitor (n = 18), mycophenolate mofetil (n = 5), imatinib (n = 4), methotrexate (n = 1) or rituximab (n = 1). one patient received no immunosuppression. total number of ecp cycles was median 20 (range: 1-61). responses over time are shown in figure 1 . overall response to ecp was seen in 25 (56%) of the patients. most responses were seen after more than 3 months ecp treatment. in univariate analysis of possible baseline predictors of response, no significant associations were found. prednisolone dose was significantly reduced at every 3 months after start of ecp (p o0.01). additional cgvhd treatment was administered to 14 (31%) patients during ecp treatment (sirolimus n = 6, calcineurininhibitor n = 6, uva1 n = 4, methotrexate n = 3, rituximab n = 3, mycophenolate mofetil n = 2). about 6 (13%) patients had more than 1 additional treatment. prednisolone dose was increased at least once in 20 (44%) patients during ecp treatment. overall survival at 5 years was 80%. follow up was median 694 days (range: 62-2416). more than half the patients with cgvhd (56%) improve overall after treatment with ecp, but flares in cgvhd activity still occur. prednisolone dose is significantly reduced at all time points after starting ecp, but short term increased doses or additional immunosuppression was necessary in more than one-third of the patients. larger prospective studies with long-term end points are warranted. disclosure of conflict of interest: marietta nygaard has received a travel grant and speaker's fee from therakos/ malinckrodt. chronic graft versus host disease (cgvhd) remains a major cause of morbidity and mortality after hematopoietic stem cells transplantation despite the improvement of the immunosuppressive prophylaxis. skin, buccal, lacrymal and hepatic disorders are the most frequent. sclerotic gvhd remains a severe form and often refractory to standard treatment lines such as corticosteroids and calcineurin inhibitors. the antifibrotic activity of imatinib by the inhibition of pdgf-r and tgfb-β pathways has been used in the treatment of refractory gvhc with sclerotic features and systemic scleroderma. here, we report the results of imatinib treatment in 28 patients (pts) with refractory cgvhd. over a period of 13 years (january 2000-december 2012), 1308 pts received allogeneic stem cells transplantation from related donors, 28 of whom were treated with imatinib for refractory cgvhd: 24 pts for malignant diseases (14 cml, 9 aml, 1 nhl) and 4 pts for aplastic anemia. the median age is 31 years (6-55), the sex ratio m/f: 2.1. conditioning regimen used with chemotherapy alone: myeloablative (14 pts with gvhd prophylaxis combining ciclosporin and methotrexate), reduced intensity (14 pts with prophylaxis combining ciclosporin-mycophenolate mofetil). all pts received peripheral blood stem cell transplant with an average of cd34 cell count: 7.1 × 10 6 /kg (1.23-21.8). the median duration of the cgvhd is 8 months (3-27). the firstline treatment consisted of the combination of steroidsciclosporin with or without mycophelonate mofetil. imatinib was administered to these pts after median treatment duration of 48 months (9-108) for moderate (2 pts) and severe (26 pts) cgvhd according to the nih classification. treatment with imatinib, at doses ranging from 100 to 400 mg/d, was introduced in the second line for all pts. the evaluation is conducted in october 2016 after a median follow-up of 128 months (76-189). tolerance was good except in a one pt with severe thrombocytopenia that led to a transient cessation of treatment. after 6 months, analysis of pts who received imatinib according to couriel criteria and nih criteria: complete remission (cr): 1 pt (18%), partial remission (pr): 20 pts (71%), stable disease (sd): 5 pts, failure: 2 pts (7%). a long-term evaluation performed after a mean duration of treatment 62 months (6-91) finds similar results with a cr: 4 pts (14%), pr: 18 pts (64%), sd: 2 pts (7%) and failure: 4 pts (14%). corticosteroids were tapered or discontinued in 12 pts (cr or pr). at october 2016, 26 pts (93%) were alive and 2 pts (7%) died of severe infections. treatment with imatinib seems to be a good therapeutic option in the treatment of cgvhd in its moderate or severe form refractory to a minimum of two immunosuppressive agents according to the nih criteria as shown by our results in terms of response and survival with good tolerance. disclosure of conflict of interest: none. atg significantly reduces the risk of cgvhd both in unrelated and in hla identical sibling. the finke's study 1 randomised pts undergoing an allogeneic unrelated stem cell transplant (sct) after a myeloablative regimen to receive or not 60 mg/kg atg-grafalon reporting a significant reduction of cgvhd without increase of relapse and no os and dfs effect. however a successive study 2 didn't confirm those results (significant reduction of acute and chronic gvhd but poorer survival mainly due to higher relapse probability in the atg arm). the conflicting data reported on urd sct have several explanations, one is about the dose and the timing of atg. the timing of atg infusion has been demonstrated to be crucial for cb transplant 3 : an earlier administration is still active in preventing gvhd while ensuring engraftment and low hampering of immune reconstitution. here we report a large (193 sct) retrospective monocentric analysis on low atg doses (and 15-25 mg/kg for bm according to the degree of hla matching and 30 mg/kg for all pbsc sct) given early (from day − 6 to − 2). pts in the study were aml (n = 112, 58%), all (n = 57, 30%), hr mds (n = 21, 11%), cr1 (n = 111, 66%) cr2 or 4(n = 31, 18%), active disease (n = 27, 16%) for al; median age was 46 (range: 18-66). myeloablative conditioning were bu-cy120 (n = 72, 37%), bu-flu (n = 61, 32%), edx-tbi (n = 20, 10%), other (n = 40, 21%); pbsc was used in 41% (n = 80); sct were performed between 2005 and 2015 at the bologna transplant center. sct were performed from hla 10/10 identical urd (n = 63, 33%), or from 9/10 (n = 93, 48%), 8/10 (n = 30, 16%) and o8/10 (n = 7, 4%). median follow up was 55 months. overall, grade 2-4 agvhd was 26%, grade 3-4 agvhd 9%; cumulative incidence (ci) of cgvhd of any severity was 25%, for moderatesevere cgvhd 18%. ci of relapse and nrm was 28% and 21%, respectively. the 3-year overall and disease-free survival were 60% (95% ci: 52-67%) and 60% (95% ci: 51-68%). the gvhd (agvhd grade 3-4 and moderate-severe cgvhd) and relapse free survival (grfs) of the entire population ( figure 1 ) was 45% at 3 years (95% ci: 37-52%). restricting the analysis to patients in cr1-2, we found that cgvhd (any severity), gfrs and os at 3 years were 23%, 50% and 64%, respectively. comparing transplants with 10/10 urd to mismatched ones (9/10 or less) we found a trend for increased mod/sev cgvhd in pts undergoing transplant with mismatched urd (shr 2.32, 95% ci: 0.95-5.65, p = 0.06); agvhd grade 3-4 and cgvhd overall were not significantly increased; relapse incidence according to hla mismatches resulted 33% and 26% in 10/10 and ⩽ 9/10, respectively; grfs was 52% in 10/10 and 41% in ⩽ 9/10. the data reported show that low and early administration of atg is able to effectively prevent acute and chronic gvhd without increasing relapse thus ensuring really convincing grfs, even for o10/10 matched urd transplants. graft versus host disease (gvhd) is one of serious complications in patients after allogeneic hematopoietic stem cell transplantation. the application of mesenchymal stem cells (msc) represents a promising method for the treatment of severe steroid refractory gvhd. we present the data from an interim analysis of clinical trial, within which we applied msc in 28 patients with acute or chronic gvhd after allogeneic transplantation. the diagnoses included aml (12 pts), mds (5 pts), all (2 pts), cll/nhl (5 pts), mpn (4 pts). the patients underwent sibling hla-compatible (7), haploidentical (4), unrelated hla-compatible (13) or hla-mismatched (4) transplants. the median interval between the transplantation and msc was 6 months (1-95). the indications for msc infusion were steroid resistant acute gvhd (11 pts), steroid-dependent gvhd (agvhd 3 pts, cgvhd 11 pts) or chronic gvhd with the need for long-term immunosuppression and corticosteroid intolerance (3 pts). msc were applied as a single infusion at a median dose of 3.45 (0.9-5.0) × 10 6 /kg. response to treatment was assessed on day 14, 30, 60 and 100. the severity of gvhd prior to msc was graded as clinical stage 3 (2-3) in acute and stage 2 (1-3) in chronic gvhd, respectively. the median dose of corticosteroids was 0.92 (0.3-1.2) mg/kg/day in agvhd and s224 0.25 (0.1-0.5) mg/kg/day in cgvhd patients. on day +14 the partial response (pr) was achieved in 85% of patients with agvhd, the stabilisation of gvhd (sd) was found in 85% of patients with cgvhd. the dose of corticosteroids was reduced in most patients with agvhd (to 62% of the starting dose; 30-97%), while the early reduction was possible only in 36% of cgvhd patients. on day+100 only 19 patients were evaluable. the agvhd patients (7 pts) achieved a significant clinical response: 4 pr, cr 3 and dose of corticosteroids was reduced in all of them (to 17%; 10-60%). the minor responses were achieved in cgvhd patient (12 pts.) with 3 pr and 9 sd. however the dose of corticosteroid was reduced in 83% of these cases (to 56% of the initial dose; 21-71%). a total of 10 patients died because of infectious complications. most of them (8/10) were agvhd patients who expired early up to day +60. there were observed no side effects of msc application neither during the infusion nor later during the follow-up of 16 (2-45) months. the analysis of lymphocyte reconstitution revealed the changes of kinetics of some subsets as compared to the day +0 benchmark. the b-lymphocyte count tended to decrease in 82% of patients from chronic gvhd subgroup (vs 60% in agvhd). conversely nk cells declined in most agvhd patients (80% vs 36% in cgvhd). also the pro-inflammatory th17 cell was affected especially in agvhd (decrease in 63% pts vs 50% pts in cgvhd). the counts of myeloid/plasmocytoid dendritic cell increased in 80%/80% agvhd and 50%/91% ccvhd patients. the screening testing of cytokines (raybiotech, 42 cytokines, 6 pts, day +0 to +60) revealed changes of some analytes after msc infusion, including a decrease of proinflammatory cytokines such as ifn-γ, tnf-α, il-6. our experience with the treatment of gvhd using msc confirmed the safety of this immunotherapy. the favourable clinical effect with reduction of severity of gvhd and steroids dose was observed, especially in patients with acute form of gvhd. methotrexate day +1 omission is not associated with higher incidence of acute graft-versus-host-disease mm rivera franco, e leon rodriguez 1 and a campos castro 1 1 allogeneic hematopoietic stem cell transplantation (allo-hsct) remains a high-risk procedure due to its related morbimortality, limiting the broader application of this important treatment modality. despite extensive research over the years, acute graft-versus-host-disease (agvhd) affects the majority of patients undergoing allo-hsct, and up to 50% will develop clinically significant grades (ii). over the years, several methods for gvhd prophylaxis have been implemented, including immunosuppresive agents. methotrexate (mtx) is one of the earliest drugs used for gvhd prophylaxis. frequently, a short course of intravenous methotrexate (given on days +1, +3, +6 and +11 after hsct) is combined with a 6-month tapered course of cyclosporine. there is no consensus on which drugs and schedules for prevention of gvhd are best and clinical practice varies by institution. further, it is not clear whether omission of the day +1 dose of mtx has a negative effect on outcome in terms of morbidity. to describe the frequency of acute and chronic gvhd, mucositis and engraftment in patients receiving methotrexate (plus csa) as prophylaxis, omitting day +1. ninety-five consecutive patients who underwent allo-hsct from 1999 to september 2016, and received mtx as immunosuppressive prophylaxis were included. all patients received three doses of mtx, always excluding day +1. mtx was administered iv, either 10 mg/m 2 day +3, +6, +11 or 15 mg/m 2 day +3, and 10 mg/m 2 during days +6 and +11. we included 95 patients (55% male). the most frequent underlying diseases were aplastic anemia (21%) and acute lymphocytic leukemia (21%). ninety-nine percent of patients had a matched related donor. forty patients (42%) had gender disparity with their donor, and 13% presented abo incompatibility (major in 75%). most of the patients received myeloablative conditioning regimens (n = 73, 77%). the median of cd34+ infused cells were 2 × 10 6 (range: 0.8-6.8). the median neutrophil and platelet engraftment was 20 (11-43) and 15 (range: 5-46) days, respectively. from all the cohort, only 15 patients (16%) developed acute gvhd (53% grades i-ii) ( figure 1 ). thirty patients (32%) developed chronic gvhd, which was limited in 73%. most of the patients, 83% (n = 79), presented acute toxicity after the conditioning regimen, from which 76% (n = 60) corresponded to superior mucositis (50% grade i-ii and 50% grade iii-iv. the 10-year overall survival was 59% and the 10-year relapse free survival was 68%. our results showed a low incidence of acute gvhd, mostly grades i-ii, and similar survivals compared to previously reported studies, proposing that the administration of day +1 mtx as gvhd prophylaxis is not mandatory, however, prospective studies might be necessary to test our results. [p205] disclosure of conflict of interest: none. outcome of refractory graft versus host disease (gvhd) treated with extracorporeal photopheresis (ecp) as second line: a single-center experience j cornago navascues, b aguado bueno, av arriero garcía, edc jimenez barral, i vicuña andres and a alegre amor hematology department, university hospital la princesa, madrid, spain gvhd is a common and, sometimes, life-threatening entity related to hematopoietic stem cell transplantation (hsct). steroids remain the first-line therapy but they are not always enough to control it, or their side effects are simply unacceptable. both acute and chronic gvhd are responsible of impairment occurred in different organs that can lead to increase morbidity and mortality in our patients. different options are available as second line, but it is a well known fact that ecp, due to its inmunomodulatory mechanism, yields satisfactory response rates and presents excellent safety profile. from may 2012 to october 2016, 30 patients with steroid-dependent or refractory gvhd have been treated in our centre with ecp. we have performed 305 ecp procedures with the therakos cellex device, an integrated 'on line' system. the transplant was from a sibling donor in 13 cases and 17 from an unrelated donor. the median of cd3+ infused was 247.35 × 10 6 cd3/kg. eight patients (16.7%) presented agvhd, 17 (56.7%) cgvhd and finally, 5 (16.7%) had an overlap gvhd syndrome. most of patients (87.5%) with agvhd had a severe intestinal involvement as the main manifestation of the disease. however, all patients with cgvhd had a multiorgan involvement with a median of four organs affected, being skin, mouth, eyes and lungs the most common implicated. ten patients in our series have died, 7 for gvhd complications or infections and 3 due to relapse of aml. as firstline treatment they all received steroids and cyclosporine or mycophenolate mofetil. median ecp per patient has been 18 (2-31). ecp procedures were performed for 2 consecutive days, in initial phase weekly (in those with agvhd), or every 2 weeks (cgvhd) and then monthly according to clinical response, evaluated by clinical assessment and reduction in immunosuppression. about 75% of patients with agvhd had a significant clinical response to ecp so that steroid doses could be tapered and even in 37.5% of them withdrawal was possible. in the cgvhd group overall response rate (orr) to ecp was 94.1%. in 35% of these patients steroids could be suspended after a median of 8.5 ecp procedures. all patients who responded to ecp in cgvhd are still alive. independently of gvhd type, 81.4% of patients responded to ecp and 37% of them even could stop steroid therapy. those who had no response are dead. in cgvhd, 82.35% of patients remain alive, in contrast with agvhd or overlap syndrome patients whom survival is around 40%. about adverse events, 60% of patients did not present any complication associated with ecp. complications were mostly related to central venous catheter, with 12 cases of bacteremia and 2 thrombosis, easily recovered. in our experience, ecp is effective as second line treatment in gvhd, obtaining the best results in the chronic gvhd group. in fact, cgvhd patients with a good clinical response to ecp, specially when steroid doses can be tapered, have the better outcomes and longer survival. the tolerance to the procedure is excellent without severe adverse events. more experience is required to determine the best scheme of ecp and its role as prophylactic treatment. mesenchymal stromal cells (mscs) possess immunomodulatory properties and may play important roles in graft-versushost disease (gvhd) and engraftment. this study examined co-transplantation of mscs and hscs (hematopoietic stem cells). we investigated co-administration of ex vivo expanded mscs along with hla-identical sibling-matched hscs in β thalassemia major patients. we recruited 70 patients from january 2010 to january 2015 in our study. all participants received cyclophosphamide-based or fludarabine-based conditioning regimens and short-course methotrexate and cyclosporine as gvhd prophylaxis. mscs were administered intravenously (1.0-2.0 × 10 6 /kg) into patients (n = 41) 4 h before infusion of hscs. the outcomes were then compared to those of 29 patients transplanted with hscs alone. the median follow-up in the msc and non-msc group was 2.98 and 2.62 years, respectively. median time to wbc engraftment 40.5 × 10 9 /l was17.7 days (range: 15-20 days) in both groups (p-value = 0.83) and median time to platelet engraftment 420 × 10 9 /l was 27.2 days (range: 22-31 days) in the msc group, while it was 36.6 days (range: 22-50 days) in the non-msc group (p-value = 0.26). fifty-six percent of patients had acute gvhd in the msc group compared to the non-msc group where 65.5% developed acute gvhd (p-value = 0.42). meanwhile, chronic gvhd was 21% in the msc group and 37% in the non-msc group (p-value = 0.14). although the incidence of acute and chronic gvhd was lower in co-transplantation of hscs and mscs, no statistically significant difference was noted between the two groups. three-year overall survival rate was 70% and 61% in the msc and non-msc group, respectively (p-value = 0.78). three-year thalassemia-free survival rate was 54% in the msc group and 61% in the non-mscs group, showing no statistically significant difference (p-value = 0.35). the 3-year rejection incidence in the msc and non-msc group was 19% and 3 %, respectively (p-value = 0.07). there was no statistically significant difference between the two groups in terms of 3-year transplant-related mortality (pvalue = 0.79). this study indicates that co-transplantation of hla-identical sibling hscs with mscs does not inflict harm on bone marrow transplantation procedure and seems to be safe and secure. on the other hand, differences between the two groups in acute and chronic gvhd, engraftment, overall survival, thalassemia-free survival and rejection incidence did not reach statistical significance. therefore, despite the immunomodulatory activity of mscs and their role in gvhd amelioration and engraftment improvement resulted from in vitro studies, their efficacy in the clinical setting has not been conclusively proven which indicates further multicenter randomized clinical trials are required. keywords: β-thalassemia major, co-transplantation of mesenchymal and hematopoietic stem cells, engraftment, graft-versus-host disease. hematology-oncology and stem cell transplantation research center, tehran university of medical sciences, shariati hospital, tehran, iran. disclosure of conflict of interest: none. a number of studies were published with contradictory results comparing tacrolimus (tac) and cyclosporine a (csa) for graftversus-host disease (gvhd) prophylaxis, but there are only few that accounted for pharmacokinetic (pk) parameters. in this retrospective study we have identified pk parameters that affected gvhd incidence and incorporated them in the s226 multivariate comparison of tac-and csa-based prophylaxis. the retrospective study included 95 consecutive patients with csa and 239 consecutive patients with tac prophylaxis. 36% were grafted from matched related donor (mrd) and 64% from unrelated donor (ud). about 30% received busulfanbased myeloablative conditioning (mac) and 70% reducedintensity conditioning (ric). second agent for gvhd prophylaxis was short-course methotrexate (mtx) 10-15 mg/m 2 on days +1, 3, 6, 11 in 66% of patients and mycophenolate mofetil 30 mg/kg days − 1 to +30 in 34%. unrelated graft recipients also received antithymocyte globulin (atgam, pfizer, ny, usa) 60 mg/kg. the pk parameters analyzed were mean and median concentrations, pk variability parameters and number of concentrations below the targeted limit (nlow) within 21, 30 and 50 days after hsct. for tac the highest predictive value for acute gvhd was observed for median concentration during first 21 days (auc = 0.575), and for absolute skewness (auc = 0.567) of concentration data. for csa parameters with highest predictive value were median concentration (auc = 0.547) and variability coefficient (auc = 0.736) during first 30 days. nlow was also a significant parameter for tac current gvhd prevention regimens are partially effective, delay immune reconstitution, impair graft versus tumor effect and are cumbersome to use. therefore, there is a pressing need to develop innovative approaches for the prevention of gvhd. we completed a phase i-ii study employing a calcineurin and mtor inhibitor-free regimen based on a combination of post-transplant cyclophosphamide and bortezomib (cybor) in patients receiving fludarabine and busulfanbased reduced-intensity conditioning followed by peripheral blood, matched related or unrelated transplant. patients receiving grafts from unrelated donors also received ratg. we reported that the regimen was feasible and safe and yielded promising outcomes. (1,2) herein, we compare the results to those of a quasi-contemporaneous matched group of patients receiving a calcineurin-based gvhd prophylaxis. the experimental and control groups were well-matched in terms of age, sex, donor type, disease status, renal function and pam score. the cybor group (n = 28) was treated during a timeframe spanning from 2012 to 2016 and the control group (n = 15) from 2013 to 2016. gvhd prophylaxis for the control group was mmf and csa (n = 2) or tacrolimus (n = 13). both groups received supportive care according to standard institutional protocols. median follow-up for the cybor group was 28.7 months as opposed to 11.2 for the control group. median times to neutrophil engraftment for the cybor and control groups were 16 days (13-23) and 12 (10-21), respectively (p = 0.001). two patients from the cybor arm died before achieving platelet engraftment. for the remaining s227 patients, median time to platelet engraftment was 27 days (15-38). for the control group, five patients never dropped their platelet count below 20 × 109/l. for the remaining patients, median time to platelet engraftment was 17 days (10-29) (p = 0.002). there was no primary or secondary graft failure in either of the two groups. the incidences of acute grade ii-iv and grade iii-iv for the cybor group were 35.7 and 10.7%. for the control group, the incidences were 60 (p = 0.12) and 20% (p = 0.4). the incidence of chronic gvhd for the cybor and control groups were 28% and 14.3%, respectively (p = 0.62). treatment-related mortality was 14.3% and 20% for the cybor and control groups, respectively (p = 0.13). the incidences of cmv, ebv and bk reactivation for the cybor group were 57.1%, 32.1% and 17.9%, respectively. for the control group, the incidences were 46.7% (p = 0.49), 26.7% (p = 0.68), 0% (p = 0.09). the 2-year progression free survival and overall survival were 49.0% and 49.9% for cybor group and 18.8% and 31.3% for the control group ( figure 1 ). the 2year gvhd and disease-free survival (grfs) were 45.6% and 20%, respectively. despite the limitations of our study that include its size and its design and the delayed neutrophil and platelet engraftment associated with the cybor regimen in comparison to calcineurin-based prophylaxis, our data confirm the promising outcomes previously reported with the cybor combination and reaffirm the need for a large randomized study comparing cybor to a standard calcineurin-based regimen. clostridium difficile infection (cdi) causing enterocolitis may represent a serious clinical problem in patients undergoing allogeneic hematopoietic cell transplantation (allo hct). the reported prevalence varies substantially among heterogeneous patient cohorts. although cdi has been proposed as a risk factor for the development of gastrointestinal (gi) acute graft-versus-host-disease (agvhd), limited knowledge on the prevalence of cdi, occurrence of gi agvhd in cdi patients, relapse incidence and mortality of cdi patients in large patient cohorts is available. the aim of this analysis was to study the implications of cdi in a homogenous cohort of patients with either aml or mds undergoing allo hct. at our center all patients undergo stool test once a week for clostridium difficile antigen while in aplasia until discharge, irrespective of clinical symptoms for enterocolitis. patients with positive stool antigen tests (that is, toxin test) in the absence of clinical symptoms were referred to as cd+, in contrast to patients without a positive test and without clinical symptoms which were referred to as cd − . we retrospectively analyzed the data of a total of n = 727 patients with either aml or mds undergoing allo hct in our institution between 2004 and 2015. overall survival (os) was measured from allo hct to the date of death or last follow-up. after hsct, relapse and nonrelapse mortality were considered as competing events. eventprobabilities were calculated according to kaplan-meier for os and using competing event statistics for the cumulative incidence of relapse (cir), non-relapse mortality (nrm) and agvhd. 95% confidence intervals (ci) were provided for major endpoints. statistical analyses were performed using the r environment for statistical computing version 3.1.3 (r core team 2015, vienna, austria, www.r-project.org). from a total of n = 727 patients with either aml or mds who underwent allo hct, we identified n = 528 (73%) who were cd − , n = 103 (14%) who were cd+, and n = 96 (13%) who had cdi. interestingly, n = 33 (34%) of patients with cdi were diagnosed having gi agvhd as compared to n = 13 (13%) of patients who were cd+ and compared to n = 95 (18%) of patients who were cd − , p = 0.001. the three groups harbored no differences when comparing incidences of liver and skin agvhd or chronic gvhd, respectively. when dissecting gi agvhd according to ctcae criteria, only n = 8 (24 %) of cdi patients vs n = 7 (54 %) of cd+ patients, and n = 53 (56 %) of cd − patients had grade 3-4 gi agvhd, p = 0.007. with regard to os and trm, no statistical differences were observed between the three groups. the cir was 13% for patients with cdi, 15% for cd+ patients and 9% for cd − patients, p = 0.02, respectively. this analysis represents the largest published analysis of clostridium difficile in patients with aml or mds who underwent allo hct. the prevalence of cdi in this patient cohort was 13%. patients with cdi developed significantly more often gi agvhd as compared to patients who were either cd+ or cd − , respectively. however, this did not translate into differences in os or trm. disclosure of conflict of interest: friedrich stölzel has received research funding from astellas. the majority of studies on cytokines in allogeneic hsct were performed with classical gvhd prophylaxis, consisting of nonspecific immunosuppressive agents. with this type of prophylaxis almost in all studies published, higher levels of proinflammatory cytokines are associated with development of acute gvhd, while lower levels indicate the success of immunosuppressive agents in abrogation of alloreactive response. currently, there is no data, whether the dynamics of cytokines after ptcy is similar to the situation of classical gvhd prophylaxis. out of 192 adult patients transplanted at first state medical university with ptcy between 2014 and 2015 we have identified 20 cases with acute gvhd and plasma samples available. these patients were matched in the ratio 1:2 to patients who did not develop acute gvhd. the study group was comprised of 60 adult patients with hematological malignancies who underwent hsct. all patients received ptcy-based gvhd prophylaxis. five plasma biomarkers were studied by elisa: il-17a, il-6, il-8, tnf-α and ifn-γ. blood samples were obtained from patients on days − 7, 0, +7, +14. the fifth time point varied between day +21 and +28 to represent the sample after engraftment, but before onset of acute gvhd. about 10 (50%) out of 20 gvhd patients had a grade i, 7 (35%) grade ii, 5 (25%) grade iii agvhd, 6 (30%) patients developed multiorgan agvhd. about 13 patients (21.6%) had chronic gvhd. there was no difference between gvhd + and gvhd − groups in any of the clinical parameters. the median of engraftment for all patients was 21 (9-43: range). the median agvhd was 30 days (23-92: range). neither of the cytokine levels was significantly different in patients with agvhd grades i − iv and without gvhd. however, for patients with agvhd grade ii − iv we found that low levels of il-8 on day +7 (126.83 ± 43.794 vs 276.89 ± 310.51 pg/ml, p = 0.04) and ifn-γ on day +21-28 (34.70 ± 23.71 vs 60.96 ± 41.37 pg/ml, p = 0.03) were associated with increased risk of gvhd. the roc analysis was performed to determine the cut off values for il-8-133.56 pg/ml (auc = 0.714) and ifn-γ -35.94 pg/ml (auc = 0.720). the incidence of agvhd grade ii-iv was significantly higher in patients with levels of cytokines lower than cut off (40% vs 5.7%, p = 0.008 and 43.7%, p = 0.012 for il-8 and ifn-γ, respectively). the same pattern was observed for patients with agvhd grade iii-iv. low levels of il-8 (96.12 ± 39.79 vs 303.52 ± 346.19 pg/ml, p = 0.008) and ifn-γ (21.69 ± 14.78 vs 58.80 ± 39.92 pg/ml, p = 0.012) on day +28 were especially predictive. the cut off values for il-8 was 147.09 pg/ml (auc = 0.869) and for ifn-γ-25.71 pg/ml (auc = 0.858). the incidence of agvhd grade ii-iv was also significantly higher in patients with levels of cytokines lower than cut off (p = 0.004 and p = 0.0006 for il-8 and ifn-γ, respectively). for chronic gvhd only higher level of il-17 at day +28 (209.17 ± 329.59 vs 106.06 ± 210.65 pg/ml, p = 0.037 for patient with and without gvhd, respectively) was significantly predictive. in this pilot trial we have demonstrated that dynamics of cytokines after gvhd prophylaxis with ptcy may be different from conventional one, and well-known predictive biomarkers might not work after ptcy. further large prospective trials are warranted to elucidate reliable biomarkers for gvhd after this type of prophylaxis. disclosure of conflict of interest: none. graft versus host disease (gvhd) remains one of the main obstacles to broader application of allogeneic transplantation. gvhd prevention and treatment techniques are poorly standardized. the 1st-line treatment of newly diagnosed chronic (c) gvhd is corticosteroid. there is no standard 2ndline treatment for cgvhd. approximately 50-60% of patients (pts) with cgvhd require secondary treatment within 2 y after initial systemic treatment. recently the jak1/2 inhibitor ruxolitinib emerged as an efficacious treatment for corticosteroid-refractory (sr) acute and c-gvhd with a 24% of sr-cgvhd patients reporting a long lasting immunosuppression-free complete response. the current study seeks to analyse the efficacy and safety of ruxolitinib in highly pre-treated sr-cgvhd pts in our centre. ruxolitinib treatment was given off label after provision of an informed signed consent and in the absence of alternative therapeutic options including clinical trials. we analysed data prospectively collected at our long-term follow-up clinic between 2015 and 2016. a written consent was given by pts allowing the use of medical records for research in accordance with the declaration of helsinki. overall 5 pts (median age 57 y-range: 39-67 years; mean karnofsky score 70%) with sr-cgvhd were treated s229 with ruxolitinib. median time from transplant was 46 months (range: 9-68). ruxolitinib was initiated at a starting dose of 5 mg twice daily-median time on ruxolitinib 4 months (range: 2-15)-4/5 pts increased the dose up to 10 mg twice daily. four pts had a classic and 1 an overlap sr-cgvhd. all of them had skin sclerodermatous involvement and 4/5 joint and fascia involvement with significant decrease of range of motion and limitation of adl. all pts were previously treated with several lines of immunosuppression (3-11) including high-dose prednisone in 1st line (5/5), rapamycin (5/4), tk-inhibitor imatinib (4/5), extracorporeal photopheresis (4/5). all pts were pre-screened for risk of infection and regularly checked on a fortnightly basis. all pts were under active prophylaxis according to recommendation for gvhd pts and ruxolitinib therapy. after a cumulative follow-up of 867 days we reported only one serious adverse event represented by a cmv pneumonia requiring hospitalization with complete recovery. early time point evaluation (5/5 pts evaluable) at +1 month underlined how all pts were reporting subjective improvement at the patient global ratings according to nih 2014. data were confirmed at the health care provider global ratings. month 3 evaluation (3/5) confirmed meaningful responses (partial responses 3/3) according to nih 2014, with both patient and health care provider global ratings improvement and concomitant enhancement in lee skin symptoms score and sf-36 health-related qol. at last followup no evidence of myelosuppression, infections, pml, nonmelanoma skin cancer was registered. considering the concomitant treatment (with reference to azoles and rapamycin or cyclosporine) no cases of toxicity due to drug-druginteraction was reported. ruxolitinib is well tolerated in highly pre-treated sr-cgvhd. its safety profile seems to be reassuring. the efficacy data observed also at this early time point is preliminary but promising in this subset of pts with a long history (⩾3 lines) of treatment for cgvhd. confirmatory study in a larger number of patients is underway on a multicentre basis. disclosure of conflict of interest: none. severe acute enteral graft-versus-host-disease (gvhd) is a lifethreatening complication of allogeneic bone marrow transplantation. in case of resistance to corticosteroids as the firstline treatment severe enteral gvhd harbors a high morbidity and mortality. retrospective analyses indicate efficacy of the jak1/2-inhibitor ruxolitinib in the treatment of acute or chronic gvhd in adults, but experience in paediatric patients is limited. here, we report a small cohort of paediatric patients with stage 4 steroid-refractory gvhd of the gut who received ruxolitinib as salvage therapy within a multimodal immunosuppressive regimen. we retrospectively analysed four patients aged 8-16 years with severe, steroid-resistant acute gvhd of the gut who were treated with ruxolitinib in our institution. all patients were transplanted for non-malignant haematologic disorders, graft source was 2 × mmud, 1 × mud, 1 × msd. the conditioning regimen consisted of treosulfan, fludarabine and thiotepa. serotherapy with thymoglobuline was administered in all patients transplanted from unrelated donors. all patients received mtx and cyclosporine as gvhdprophylaxis. gvhd was staged according to the glucksberg-scale. ruxolitinib was added to the immunosuppressive regimen when acute stage 4 gvhd was reached and became resistant to treatment with methylprednisolone (2 mg/kg/day) as well as infliximab and mycophenolate (mmf) as second-line immunosuppressants. acute stage 4 enteral gvhd developed at a median of 38 days after transplant (30-58 days) and ruxolitinib was started at a median of 53 days post-transplant (48-85 days). the starting dose varied between 10 mg/day and 40 mg/day, that is, 0.25-0.5 mg/kg/day, taking into account the expectedly low bioavailability of the oral drug during severe diarrhea. upon improvement of gvhd symptoms and/ or increasing side effects the dose was gradually tapered and ruxolitinib was discontinued after a median of 39 treatmentdays (19-83 days) . after addition of ruxolitinib to the immunosuppressive regimen, the symptoms of acute gut gvhd gradually improved in all four patients with decreasing abdominal pain and stool volumes. immunosuppression with steroids and mmf could slowly be tapered. all patients are alive after a median follow-up of 392 days (95-571 days) from diagnosis of acute stage 4 gut gvhd. the most prominent side effect attributable to ruxolitinib was thrombocytopenia with a nadir in platelet counts after 30 days of ruxolitinib treatment in 3/4 patients. platelets recovered within 2 weeks after ruxolitinib was discontinued. neutropenia was observed in one patient with anc dropping o0.5/nl after 30 days of ruxolitinib treatment. mild to moderate elevation of liver transaminases was observed in all four patients during ruxolitinib treatment. one patient developed imminent acute renal failure, another patient showed symptoms of hemolytic uraemic syndrome. however, due to the multimodal treatment of these critically ill patients, these complications could not clearly be attributed to ruxolitinib. ruxolitinib is potentially beneficial in severe acute enteral gvhd in children refractory to corticosteroids as well as second-line immunosuppressants. however, randomized trials are warranted to verify safety and efficacy of ruxolitinib in this patient cohort. disclosure of conflict of interest: none. steroid refractory acute gvhd is a major cause of mortality after allogeneic stem cell transplantation. until date, no agent or treatment strategy has demonstrated superior efficacy in this patient group. the dose and duration of steroid treatment is associated with several short and long-term side effects, therefore concepts facilitating rapid steroid taper may be beneficial. both ruxolitinib and ecp have been reported to be effective in treatment of steroid refractory (sr) agvhd. we analyzed data from consecutive adult patients who received ruxolitinib for sr agvhd between march 2015 and august 2016 in our institution overall, 19 patients (male n = 12; female n = 7) with a median age of 58 years (range: 18-74) were included. donors for allogeneic sct were msd (n = 3), mud (n = 12) and mmud (n = 3). median time to gvhd onset after stem cell transplantation was 29 days (range: 7-154 days). about 14 patients had agvhd grade iii or iv (all with gi involvement), while 5 patients had skin grade 3 involvement. sr agvhd was diagnosed if agvhd manifestations were progressive after 3 days or persistent and without improvement after 7 days or no partial remission after 14 days of treatment with 2 mg/kg bw of systemic steroids. patients received additional ecp (n = 11), if response to ruxolitinib was lacking or slow (n = 9) or instead of ruxolitinib due to cytopenias (n = 2). ruxolitinib was first-line treatment for sragvhd in 11 patients (58%). median initial dose of ruxolitinib was 10 mg (range: 5-15 mg) twice daily. steroids were tapered and stopped, even if agvhd was still active. primary end point was non-relapse mortality at 6 months. secondary end point was response on day 28 after initiation of ruxolitinib. response occurred relatively slowly, resulting in a day 28 overall response rate of 58% (cr = 6, pr = 5). however, a total nine patients (47%) attained a complete response (cr), five with ruxolitinib alone and four others in combination with ecp. about 12 patients (63%) required dose reduction or interruption of ruxolitinib mainly due to cytopenias. after a median follow-up of 210 days, 8 patients are alive. causes of death were relapse of malignant disease (n = 1), gvhd (n = 2), infections (n = 7) and other (n = 1). median survival from diagnosis of sr agvhd was 61 days for non-responders and 252 days for responders ( figure 1 , p = 0.0096). in univariate analysis, non-response was associated with higher risk of nonrelapse mortality (rr; 5.6, 95% ci: 1.51-20.6, p = 0.01). ruxolitinib and ecp are two effective promising treatment options, which may be complementary in patients with sr agvhd. cytopenia is the most frequent side effect of ruxolitinib while infections remain the major cause of death. [p215] disclosure of conflict of interest: ayuk-therakos: honoraria; kröger: novartis: honoraria, research funding. steroid-refractory acute graft-versus-host disease (sr-agvhd) is associated with a dismal outcome. janus kinase (jak) 1/2 signaling has been shown to be instrumental in multiple steps leading to inflammation and tissue damage in gvhd. jak1/2 inhibitor ruxolitinib was studied in the treatment of sr-gvhd by zeiser et al. (leukemia 2015) , and the overall response rate was reported to be 81.5%. we have now studied ruxolitinib in the treatment of six adult patients with steroid-refractory, grade iii-iv, intestinal agvhd. all the patients were male. the median age of the patients was 50 (range: 19-59) years. three of the patients were transplanted for aml, one for all, mds and mm each. all the patients had been given a myeloablative conditioning treatment (cytbi 2, treosulfan+fludarabine 4). two patients had a sibling donor and four a matched unrelated donor. the graft was from peripheral blood in all the patients. gvhd prophylaxis consisted of cyclosporine and a short course of methotrexate, and in addition antithymocyte globulin in the unrelated donor setting and methylprednisolone in one sibling recipient. agvhd of the intestine manifested on days + 17, +25, +39, +60, +63 and +136 with diarrhea. in two patients it was preceded by agvhd of the skin by 7 and 49 days, respectively. gi-biopsy showed acute gvhd of grade iii and of grade iv in three patients each. treatment of intestinal gvhd was started with methylprednisolone 10 mg/ kg/day, tapering the dose to 5 and 2 mg/kg after 12 doses each. gastroduodenoscopy and colonoscopy were performed at the onset of symptoms indicating intestinal gvhd. biopsy confirmed the diagnosis in all cases. because the diarrhea continued in spite of methylprednisolone treatment, ruxolitinib was started 7, 9, 10, 10, 20 and 90 days from the first day of diarrhea. the dose of ruxolitinib was 10 mg × 2 per day orally. four patients showed a clear response to ruxolinitib, normalization of bowel function, after 3, 4, 16 and 27 days from the start of ruxolitinib treatment. the healing of the intestinal lesions was verified by biopsy. two of these patients had received extracorporeal photopheresis simultaneously. two patients did not benefit from ruxolinitib treatment. one of them had continuous infectious complications and therefore ruxolitinib was only started after 90 days from the start of diarrhea. the other patient died of fulminant diarrhea after 3 weeks of ruxolitinib treatment. cmv reactivation was detected in three of the responders, and two of them had also polyoma virus cystitis. one patient developed a pulmonary aspergilloma, which is under control with drugs. corticosteroid-resistant gastrointestinal acute gvhd was treated in six patients, out of whom four showed a good response. disclosure of conflict of interest: none. although methotrexate (mtx) is commonly used in the prophylaxis of graft-versus-host disease (gvhd) after allogeneic hematopoietic stem cell transplantation (allo-hsct), some small studies have also reported its use in the treatment of chronic gvhd. the aim of this study was to evaluate the efficacy and safety profile of low-dose mtx for treatment of sclerodermatous chronic gvhd (sgvhd) after the failure of first and second line treatments. we retrospectively evaluated 23 adult patients who received low-dose mtx as salvage treatment of sgvhd during the period elapsed between june 2006 and june 2016 in a tertiary referral university hospital in spain. there were 17 (73%) males and 6 (27%) females. the median age was 54 years (range: 28-69). all had received an allo-hsct for hematologic malignancies. the median time from allo-hsct to sgvhd was 666 days (range: 334-2679). thirteen patients (56%) had presented previous acute skin gvdh. superficial skin lesions mimicking lichen planus (lichenoid gvhd) were diagnosed in 19 (82%) patients, while lesions resembling lichen sclerosus, morphea or fasciitis (sgvhd) where seen in all 23 (100%) patients. the total body surface area was affected by more than 50% in 15 patients (65%). besides the skin, other organs/tissues involved were the eyes (65%), mouth (52%), nails (34%), lungs (17%), liver (8%) and gastrointestinal tract (4%). treatment lines prior to mtx administration were: prednisone (pdn) in 23 patients (100%), phototherapy (pht) in 4 (17 %), cyclosporine (cya) in 2 (8%), mycophenolate mofetil (mfm) in 2 (8%), pht + pdn + cya in 3 (13%), pdn + mfm + cya in 3 (13%), extracorporeal photopheresis + pdn in 1 (4%). the median time from sgvhd onset to mtx treatment was 308 days (range: 19-937 days). mtx was administered subcutaneously in 21 patients (91%) and orally in 2 patients (9%). median dose of mtx was 13.74 mg/week (range: 7.73-18.48 mg/week) and median length of treatment was 61 weeks (range: 2-148 weeks). in two patients (8%) early withdrawal of mtx occurred (one due to early death secondary to septic shock and other due to rapid disease progression). mtx-related toxicity occurred in three patients (13%): megaloblastic anemia, asymptomatic increase of liver enzymes and mucositis, respectively. response to mtx was evaluated in the 21 patients (91%) s231 who did not suffer early mtx discontinuation. seventeen patients (73%) presented a partial response; of them, two are still under mtx treatment for 26 and 59 weeks, respectively. fourteen patients (60%) received pdn concomitantly to mtx (median dose 20 mg/day, range: 5-60); 1 year after mtx treatment, only four patients were receiving pdn (median dose 5 mg/day, range 5-15). seven patients have finished mtx treatment without reappearance of symptoms, receiving only topical treatment with emollients, tacrolimus or corticoids for short periods. in four patients (26%) sgvhd progressed despite mtx administration. our data suggest that mtx is a safe, inexpensive and effective alternative for refractory sgvhd. its potential used in earlier phases of sgvhd deserves further investigation. disclosure of conflict of interest: none. severe chronic gvhd has a major influence on late morbidity and mortality after hematopoietic stem cells transplantation (hsct). ecp is a good approach to treat refractory-gvhd: leucocytes are obtained from peripheral blood by apheresis, incubated with 8-mop, irradiated and then infused to the patient where they undergo apoptosis and induce tolerance. it is a promising alternative that reduces doses of immunosuppressive therapy and their side effects in the treatment of gvhd. this study shows its efficacy in persistent refractory cgvhd. the procedure was applied to three patients (pts) aged 24, 28 and 32 years (two aml and one cml), sex ratio (1m/2f) who underwent allogeneic-hsct with myeloablative conditioning regimen based on chemotherapy alone from a peripheral blood stem cells with cd34 levels: 1.24, 7.6 and 8.23 × 10 6 /kg respectively. prophylaxis of gvhd combined ciclosporin and methotrexate in short cycle. severe extensive cgvhd (according to nih criteria) was observed in the three cases after an average delay of 5.3 months (3-7) with involvement of 1-6 organs (mouth, eyes, skin, liver, joints and lungs). all pts are refractory to three lines of immunosuppressive agents (ciclosporin-corticosteroids, mmf and imatinib), with an average of 3 thrusts/pt (2-4) over an average period of 65 months (09-103). ecp was performed under the open system or dissociated system (macopharma) for two sessions per week for 4 weeks, one session per week for 8 weeks, one session every 2 weeks for 12 weeks and one session per month for 3 months. after a median period of 6 months (3-10), an average of 22 sessions/pt (15-26) was performed. in terms of tolerance, a red blood cell transfusion was required in one pt, spontaneously resolved lymphopenia was observed in another pt, and a poor venous approach led to the pause of a central catheterization in one pt. the 3month and 6-month evaluation according to the couriel response criteria shows a partial response observed as of the first month with net improvement especially on skin sclerotic features and joints retractions initially refractory to all therapeutic lines. this allowed gradual reduction doses of corticosteroids. pce is recommended in the curative treatment for refractory chronic gvhd from the second line. this encouraging study on a small series shows its efficacy in persistent and late refractory forms. it is nevertheless necessary to evaluate it on a larger number of pts. disclosure of conflict of interest: none. successful treatment of steroid-refractory acute gastrointestinal graft-versus-host-disease by fecal microbiota transplantation p neumeister 3 steroid-refractory acute gastrointestinal (gi) graft-versus-host disease (agvhd) is a severe complication of allogeneic hematopoietic stem cell transplantation (allo-hsct) associated with a high mortality rate. loss of intestinal bacterial diversity is thought to be associated with severity of gi-agvhd and an impaired intestinal microbiota with reduced diversity is an independent predictive factor for mortality. fecal microbiota transplantation (fmt) is the application of a fecal suspension derived from a healthy donor into a patient's gi tract. it has been successfully applied in recurrent clostridium difficile associated diarrhea including patients who underwent allo-hsct. we report the complete resolution of lower gi-agvhd following colonoscopic fmts in three patients that had been refractory to 4-6 lines of immunosuppressive therapies. microbiota analysis by 16s rdna before fmts revealed a severely depleted microbiota in all patients. donors (different persons for each patient) were healthy adult subjects. repetitive (1-6) colonoscopic fmts were necessary to permanently establish the donor's microbiome. all patients responded clinically by reduction/normalisation of stoll volumes, stopping total parenteral nutrition and tapering of steroids. a possible causative relationship of fmt in the reversal of severe intestinal dysbiosis and subsequent resolution of gi-agvhd can therefore be hypothesized. the establishment of donors' microbiota and increase in bacterial richness was associated with disease control. no immediate procedure-related infections or other side effects were observed. besides restoration of an initially severely reduced microbial richness by fmts, response of gi-agvhd was sustained despite reduction and discontinuation of concomitant immunosuppressive treatments. restoration of dysbiosis by fmt might represent a promising novel therapeutic approach for refractory lower gi-agvhd. disclosure of conflict of interest: none. tear film proteomics reveals important differences between patients with chronic ocular gvhd and healthy controls k plattner 1 , n gerber-hollbach 1 , s moes 2 , p jenoe 2 , d goldblum and j halter 1 ophthalmology, university hospital basel, ch-4031 basel and 2 proteomics core facility of the biozentrum, university of basel, ch-4056, basel chronic gvhd frequently involves the eyes, leads to important decrease of quality of life and may threaten visual capacity. sicca syndrome is one of the hallmark of ocular cgvhd. analysis of tear protein composition may help to identify biomarkers for early diagnosis and prognosis of ocular cgvhd. tear fluid of 42 patients with ocular cgvhd were compared with 10 healthy individuals in this single center study. results of the first 10 patients are reported here. tryptic digests from schirmer strips were analyzed on an orbitrap mass analyzer. clinical examinations included slit lamp examination, fluorescein staining, schirmer test, break-up-time (but) and a quality of life questionnaire (osdi). outcome measures were differences and consistency of proteins in human tear fluid s232 between patients with ocular gvhd and healthy controls. statistical analysis was performed by one sample wilcoxon-tests, p-values o 0.01 was considered significant. ten patients (eight males, two females) with a median age of 47 years (range: 24-69) were analyzed. all underwent pbsct, eight from an unrelated donor. cgvhd overall score was moderate in three and severe in seven. eye organ score was 2 in six and 3 in four patients. all patients had more than one organ manifestation of cgvhd. eight were under systemic immunosuppressive therapy at the time of analysis, two had topical treatments only. in total 306 different proteins were detected in tears analyzed. compared to controls, 172 were differentially expressed in ocular cgvhd. expression was highly significantly different in 75 proteins. compared to controls, expression of 41 proteins was at least 10-fold increased, representing 11 different categories. among them, more than 75% of all proteins belong to one of three categories: cytoskeletal proteins, nucleic acid binding or structural proteins. albumin, cluster or keratin (keratin type i-iii) and cluster of pyruvate kinase were most highly overexpressed. expression of 14 proteins was decreased to o1 to 50%, belonging to 12 different protein classes. half of them belong to defense/immunity proteins, enzyme modulators, hydrolases, nucleic acid binding and carrier proteins. expression of lactotransferrin, proline-rich protein and prolactin-inducible protein was most profoundly decreased. compared to healthy controls, a high number of protein is found to be differentially expressed in tears in ocular cgvhd. among them high expressions are observed for proteins that may indicate disturbed integrity of ocular surface and leakage of conjunctival capillaries. most profoundly decreased proteins include proteins with important functions in host defense and immunomodulation. more detailed pathway analysis is necessary to identify biomarkers for ocular cgvhd. disclosure of conflict of interest: none. steroid-dependent chronic gvhd after allogeneic peripheral blood stem cell transplantation is a great problem. nonresponders to corticosteroid therapy are at high risk of mortality. we hypothesized that such patients could benefit from treatment strategy using in patients with primary severe autoimmune diseases like multiple sclerosis and crohn's disease. patient z., 4 y.o. was diagnosed in july 2011 with myelomonocytic leukemia (jmml). initially he was treated with low-dose of cytarabine and epigenetic agents. in september 2013, jmml progression was observed with leukocytosis, thrombocytopenia, splenomegaly. bone marrow aspirate showed 19.2% monocytes and 19.8% blast cells. splenectomy was performed in november 2013 due to refractoriness to blood components transfusions. in december 2014 unmanipulated haploidentical peripheral blood stem cell transplantation from mother with 3.2 × 10 6 /kg cd34+ and 3.5 × 10 8 /kg cd3+ was performed. the conditioning regimen was myeloablative including melphalan 100 mg/m 2 day − 5 and treosulfan 14 000 mg/m 2 days − 4, − 3, − 2. no organ toxicity 4grade 2 was observed. gvhd prophylaxis consisted of hatg 10 mg/kg on days − 5, − 3, − 1, +1, i.v. tacrolimus from d − 1 and mmf 25 mg/kg from d 9. engrafted was fast and prompt (100% donor) with wbc41.0 × 10 9 /l on d +8, plts420 × 10 9 /l on d +10. acute gvhd of stage ii was observed in early posttransplant period and treated with steroids and tnf-α inhibitor (infliximab). patient also received five procedures of ecp. all attempts of immunosupression tapering failed and the patient was staying on high dose of tacrolimus, mmf and courses of steroids till october 2015. in october 2015, gvhd stage ii flare with blood eosinophilia occurred after another attempt of steroids withdrawal. clinical examination showed that the patient was in complete remission with full donor chimerism. mild response of gvhd to steroids was observed. in april and may 2016 patient received two doses of rituximab 375 mg/m 2 with no significant response. in order to restore naive immune system first course of chemotherapy with cyclophosphamide 2000 mg/m 2 was performed in the end of may 2016. no toxicity 4grade 2 was observed. the patient recovered wbc41.0 × 10 9 /l on d +12, plts420 × 10 9 /l on d +11. in the phase of hematological recovery he was mobilized with g-csf and two leukaphereses of pbscs were performed. in june 2016 our patient was transplanted with previously collected 0.5 × 10 6 cd 34+/kg following nonmyeloablative regimen including cyclophosphamide 1500 mg/m 2 on day − 3 and fludarabine 10 mg/kg, on days − 3, − 2, − 1. second dose of cyclophosphamide was not administered because of severe hyponatriemia with seizures due to the cpm administration. no other significant toxicity was observed. the patient did not require either blood product or i.v. antibiotics. doses of tacrolimus and mmf were picked on 2 months late and no more steroids were given. the patient is well in cr with no signs of gvhd for 7 months. we speculate that pbsc collection from patients under massive immunosupression underwent allogeneic transplant is difficult but feasible. the nonmyeloablative regimens in such group of patients could be well tolerated and ensure the restoration of naive recipient immune system. this option could be discussed as an attractive alternative for treating resistant gvhd in steroid resistant patients. disclosure of conflict of interest: none. severe acute gi-gvhd is a serious early complication of allotransplants, and still remains a clinical diagnosis.(1) endoscopic biopsies provide the best supportive evidence, but are invasive and morbid in patients who are already medically compromised. 18 f-fdg pet/ct may be able to stratify patients who require endoscopy and biopsy. to evaluate the performance of 18 fdg pet/ct in differentiating moderate to severe gi-gvhd from no or mild disease in pediatric patients with suspected gi-gvhd. retrospective chart review of all paediatric allo-transplant patients referred for 18 f-fdg pet/ct with suspected gi-gvhd from 2009 to 2015. clinical follow-up, endoscopy and biopsy findings were correlated with 18 f-fdg pet/ct. regional suv parameters were extracted by placing rois around stomach, duodenum, distal ilium, caecum, ascending, transverse, descending colon, recto-sigmoid colon and rectum. regional, and average large and small bowel suv data were statistically compared between patients with no or mild git-gvhd vs moderate to severe disease. the clinical and biopsy-supported diagnosis of acute gi-gvhd was taken as the true positive diagnosis for acute gi-gvhd. roc curves were generated for whole bowel suvmax values. about 50 scans in 34 patients, median age of 9 years (6 mths to 18 y), were performed at a median of 71 days post bmt. there were 15 stage 1, 13 stage 2-4 and 22 with no acute gi-gvhd. transverse colon suvmax was significantly higher in the stage 2-4 gi-gvhd compared to no or stage 1 disease (mann-whitney-u-test p o0.05). there was a non-significant trend for average large bowel suvmax to be higher in the stage 2-4 group than the no or stage 1 disease group (mean suvmax 4.16 compared to 2.94, p = 0.07). a cut off whole bowel suvmax 2.74 had a sensitivity of 79% and specificity of 61% for detecting moderate to severe gi-gvhd. 18 f-fdg pet/ct is a feasible and potentially useful non-invasive tool in the diagnosis and monitoring of therapeutic efficacy in acute gi-gvhd (2) . large bowel suvmax may be higher in patients with stage 2-4 gi-gvhd, and transverse colon suvmax could have the ability to differentiate children with no or stage 1 gi-gvhd from those with stage 2-4 disease. a negative 18 fdg-pet/ct could serve as a criteria to avoid invasive endoscopic procedures and observe for the persistence of gastrointestinal symptoms before subjecting these patients to an imageguided biopsy. in patients too unwell for endoscopy, suvmax 44 (roc curve specificity 75%) and a high suvmax in the transverse colon could serve as supportive evidence for moderate to severe acute gvhd, in the absence of biopsy findings. a major advantage of a pre-endoscopic 18f-fdg pet/ ct is to guide the procedurals to sample areas with the best diagnostic yield. prospective controlled studies are needed. oral mucosal progenitor cells (omlp-pcs) possess immunomodulatory and antibacterial properties, suggestive of their in vivo function in healthy tissue and their potential contribution to scarless wound healing in the buccal mucosa (2, 3). our aim was to establish whether the function of oral stromal progenitors is impaired in chronic graft versus host disease (cgvhd) and restored with response to treatment. a patient with grade 3 oral cgvhd was treated with systemic thalidomide for 9 weeks (200 mg/day). punch biopsies of buccal mucosa were taken before and after treatment. oral progenitor cells were isolated and expanded in vitro. numbers of progenitors was assessed using colony forming unitfibroblast (cfu-f) assays. stem cell markers (cd90, cd105, cd73, cd29, cd34, cd45, hla i and ii) were evaluated by flow cytometry. wound healing and antibacterial potential were assessed using a collagen gel lattice assay and bacterial cocultures as previously described (2, 4) . secreted levels of relevant cyto-and chemokines associated with wound healing were assessed by elisa. significant clinical improvement with reduced inflammation in the oral mucosa and healing of ulcers was seen after 3 weeks of thalidomide treatment, with continued improvement after 9 weeks. cell surface expression of cd90 and cd105 on omlp-pcs was elevated postthalidomide; markers correlated with stemness and angiogenesis in mesenchymal stromal cells. this correlated with a restoration of wound healing potential and antibacterial function after thalidomide treatment ( figure 1 ). figure 1 : antibacterial testing demonstrated a loss of antibacterial function against (a) gram positive and (b) gram negative micro-organisms in the cgvhd omlp-pcs that could be completely or partially restored to levels comparable with healthy controls after thalidomide treatment. *p ⩽ 0.05, **p ⩽ 0.01, ***p ⩽ 0.001. we demonstrate, for the first time a correlation between clinical improvement of oral cgvhd with thalidomide treatment and restoration of endogenous progenitor cell function. this study highlights the importance of a dysfunctional oral mucosal stroma in the pathogenesis of cgvhd. further studies should focus on the role of the stroma in promoting cgvhd and the precise mechanisms by which thalidomide is able to restore its functions. chronic graft-versus-host disease (cgvhd) is a major cause of late morbidity and treatment-related mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (hsct). cgvhd is driven by a th2 biased t-cell mediated alloreactive immune response that leads to chronic inflammation and fibrosis in various organs. thymic stromal lymphopoietin (tslp) is an epithelial cell-derived cytokine that mainly affects myeloid cells. upon stimulation with tslp, dendritic cells are polarized towards a dc2 phenotype driving th2 biased immune response. we hypothesized that tslp is involved in the pathogenesis of cgvhd and that elevated levels of tslp post-transplant may lead to an increased risk of cgvhd. in the present study, we measured plasma tslp levels during hsct to study associations with clinical outcomes including cgvhd. about 38 adult patients undergoing myeloablative hsct at rigshospitalet, denmark, from 2011 to 2013 were included. diagnoses included aml (n = 15), all (n = 11), myelodysplastic syndrome (n = 6), other malignancies (n = 4) and anemias (n = 2). donors were either hla matching siblings (n = 11) or mud (n = 27). grafts were either bmsc (n = 24) or pbsc (n = 14). conditioning included tbi (n = 31) or high-dose chemotherapy alone (n = 7). plasma tslp was measured by elisa (abcam) before transplantation, at the day of transplantation and at day +7, +14, +21 and +90 post-hsct. monocytes were counted daily, and t, b and nk cells were measured at day +30 and +90 using flow cytometry. about 35 (92%) patients engrafted; acute gvhd grade 2-4 was seen in 20 (53%) patients, and 16 (42%) patients developed severe cgvhd. oas was 57.9 %, trm 26.3% and relapse rate 15.8%. median plasma tslp levels increased from before conditioning (101 pg/ml) to reach a peak at day +21 (313 pg/ ml, p = 0.03), followed by a gradual decline. the plasma levels of tslp at day +21 were positively correlated with same-day monocyte counts (ρ = 0.58, p = 0.006). approximately half of the patients (n = 14) experienced an overall rise in tslp from baseline (100 pg/ml) to day +90 (328 pg/ml). this increase in tslp was not significantly associated with any transplantrelated baseline characteristics. however, patients, who had an increase in tslp levels from baseline to day +90, had a significantly higher risk of extensive cgvhd compared to those in whom tslp levels at day +90 were similar or below baseline levels (cumulative incidence of cgvhd: 77% (increased tslp at day +90) vs 38% (normal/low tslp at day +90), p = 0.01). development of cgvhd was also associated with the nucleated cell dose infused (p = 0.04) and transplant using pbsc (p = 0.08). tslp plasma levels were not associated with acute gvhd, oas, trm, relapse rate or numbers of t cell, b cell or nk cells posttransplant. we have found that increased levels of tslp from baseline to day +90 were associated with an increased risk of extensive cgvhd. this association may be due to the ability of tslp to polarize the immune system toward a th2 response. importantly, the increase in plasma tslp levels was not associated with any transplant-related characteristics suggesting that tslp may be an independent predictor of cgvhd. these findings indicate that anti-tslp treatment may be a new approach to fight severe cgvhd. disclosure of conflict of interest: none. thymopoiesis following hct: a retrospective review comparing interventions for agvhd in a paediatric cohort c roberts 1 , am flinn 1 , ma slatter 1,2 , r skinner 2 , h robson 2 , j lawrence 2 , j guest 2 and ar gennery 1,2 1 institute of cellular medicine, newcastle university and 2 great north children's hospital, newcastle-upon-tyne, uk acute graft-versus-host disease (agvhd) is a life threatening complication of allogeneic haematopoietic cell transplantation (hct), treated with topical and/or systemic corticosteroids. in steroid-refractory agvhd extracorporeal photopheresis (ecp) can be effective. ecp exposes apheresed mononuclear cells to 8-methoxypsoralen and ultra-violet radiation. systemic corticosteroids and agvhd are damaging to thymic tissue. delayed immune reconstitution, especially of the t lymphocyte compartment, is associated with increased morbidity and mortality. 1 therefore, management strategies must be effective in treating agvhd but endeavour to minimise resulting thymic damage. we compare the effect of topical steroid therapy, corticosteroids and ecp on thymic reconstitution following hct in paediatric patients. statistical analysis was performed using the kruskal-wallis test. about 155 paediatric allogeneic hcts were performed between june 2010 and april 2016, at the great north children's hospital, newcastle for malignant and non-malignant disease. we reviewed computerised records to categorise patients into four groups: no agvhd, mild agvhd treated with topical steroid, agvhd treated with systemic steroid, agvhd treated with ecp. laboratory data were reviewed to provide values of naive (cd4+ and cd4 − )cd45ra+cd27+ t-lymphocytes at 3, 6, 9 and 12 months post-hct. values for thymic output for the ecp group were additionally recorded at 3, 6, 9 and 12 months during ecp. excluded were patients with no available data, those with o12 months follow-up, those with chronic gvhd, recipient of 41 hct or received dli post-hct. about 104 patients were included, 42 (40.4%) had no agvhd, 49 (47.1%) had agvhd treated topically or systemically, 13 (12.5%) had agvhd and received ecp. for analysis, the group treated with steroids were divided into those treated with topical therapy and those given systemic steroids. the median values of all groups at each time point (3, 6, 9 and 12 months) are shown ( figure 1 ). there was a significant difference between the rate of thymopoiesis (measured by the addition of cd4+ and cd4 − cd25+cd27+ cells) between all groups (no agvhd, agvhd treated with topical or systemic steroids, and agvhd treated with ecp) at 3, 6, 9 and 12 months post-transplant (p = 0.002, p o0.001, p o0.001, p = 0.001 respectively). further analysis excluded those treated with ecp (so including the no gvhd (n = 42), topical treatment (n = 23) and systemic steroid treatment group (n = 26)). at each time point p = 0.001, p = 0.019, p = 0.021and p = 0.019, respectively, demonstrating a statistically significant difference in time to thymopoiesis between those that had developed agvhd and those that had not. acute graft-versus-host disease (agvhd) is a significant cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. which despite first line treatment is well-established (esteroids), second line is not well defined. evaluate the results with different second line treatment used and the risk factors associated with of sr-agvhd. we review the clinical records of 281 consecutive patients undergoing allogenic hsct from 2005 to 2015 in our hospital. about 53% presented agvhd. sr-agvhd was defined as progression after 3 days, no clinical change in 5 days or incomplete response after 14 days of treatment. about 34 patients (25%) met s235 criteria for sr-agvhd. there were no significant differences between both groups (sr-agvhd vs no sr-agvhd) respect to age (recipient/donor), unrelated donor, prophylaxis of gvhd, cd3 lymphocyte and cd34 cell. the median time between transplantation and agvhd diagnosis was 25 days (7-123). patients who did not respond on fifth day of steroid treatment have an 80% rate mortality vs 33% on no sr-agvhd group (p = 0.03). sr-agvhd: 34 patients presented sr-agvhd and this was related to: hla mismatch (35% sr-agvhd vs 15% no sr-agvhd, p = 0.008), male recipient/female donor (38% sr-agvhd vs 17% no sr-agvhd, p = 0.02) and advanced underlying disease (56% sr-agvhd vs 22% no sr-agvhd, p = 0.001). second line: basiliximab (82.4%); extracorporeal photopheresis (ep) (2.9%), timoglobulin (8.8%) and others therapies (5.9%). two patients (6%) obtained complete response (cr) and 10 patients (29%) partial response (pr). global response (cr, pr) after second line (mainly basiliximab) showed better overall survival (p = 0.009). third line: basiliximab (8.3%); ep (41.7%), mesenchymal cells (msc) (8.3%), ruxolitinib (16,7%) and others (24.9%). ruxolitinib improve gvhd cutaneous and hepatic but not intestinal. the best results were achieved with ep (2 cr, 1 pr) and basiliximab/msc (1 pr, respectively). only patients who achieved cr survived. fourth line: msc (50%)/ ruxolitinib (50%) does not improve the prognosis. no serious adverse effects were observed with msc therapy, basiliximab and ep. about 14% of patients showed cmv reactivation with basiliximab. about 27 patients died (80%), 21 patients with early mortality ( o6 months) due to refractory agvhd (40%) or secondary infections (60%). overall survival at 6 months and 2 year was 28 ± 8% and 0%, respectively. in multivariate analysis the main factor for trm was the steroid-refractory vs steroidsensitive (hr 2.00, 95% ci 0.91-4.39; p = 0.083) and was unfavorable the association of hepatic and intestinal agvhd (hr 2.24, 95% ci 0.90-5.57; p = 0.082) no sr-agvhd: 115 patients. trm-100 was 18% (n = 7), mainly due to infection (71%). trm-1 year was 37% (n = 15), mainly by gvhd (40%) and infections (40%). median follow-up of 26 months, os-6 months and 2 year were 84 ± 3%/75 ± 4%, respectively. trm was associated with not obtained cr/pr after second line (p = 0.001), no cr after third line (p = 0.018) and relapse of gvhd despite achieving cr initially (p = 0.004). in our series only the patients that obtained cr/pr after second-line or cr after third-line improved os. the best results in sr-agvhd were obtained with basiliximab and extracorporeal photopheresis. trm was associated with relapse of gvhd and advanced disease to the transplant. randomized clinical trials are needed to assess different treatment modalities for sr-agvhd. [p226] disclosure of conflict of interest: none. extracorporeal photopheresis (ecp) is a therapy for steroidrefractory chronic graft versus host disease (cgvhd). therapeutic response to ecp has been linked with a progressive increase in circulating granulocytic myeloid-derived suppressor cells (g-mdsc) in acute gvhd, but not in cgvhd 1 . low density neutrophils (ldn) phenotypically resembling g-mdsc (putative g-mdsc) show marked flux in cgvhd patients receiving ecp, and a reduction in their frequency is associated with a sustained therapeutic response to ecp 2 . recent data has identified lectin-type oxidized ldl receptor-1 (lox-1) as a specific marker of ldn with t-cell (tc) suppressive activity 3 . using this marker we have conducted a cross-sectional study to assess whether putative g-mdscs in this patient cohort have suppressive activity. about 15 patients with steroid refractory or steroid-dependent cgvhd (mean treatment duration of 9 months) receiving ecp and 8 healthy controls were recruited. patients had gvhd affecting skin (15/15), liver (3/15) and gut (2/15). pbmc were isolated and immunophenotyped by flow cytometry for markers of g-mdscs (cd14 − ve , cd16, cd66b, hla-dr − ve , cd33 int ) and lox-1 expression. suppressive function was determined by measuring the inhibition of proliferation of anti-cd3/cd28-activated purified cd3 tc from healthy donors by 4-day co-culture with g-mdscs from patients. statistical analysis was conducted using graphpad 6. ecp patients had substantially greater frequencies of circulating putative g-mdsc than healthy controls (p o0.0001; median: 13% and iqr 2%-32% vs 0.2% and iqr 0.1%-0.6%, respectively). while there were substantially greater frequencies of circulating lox-1 + cells in pbmc from ecp patients than healthy controls (p o0.0001; median: 1.5% and iqr 0.39%-35% vs 0.053% and iqr 0.029%-0.062%, respectively), these were mainly the minority population within the putative g-mdsc fraction with no significant difference between ecp patients and healthy controls in the proportion of lox-1 + cells (29% ± 16% vs 21% ± 9%, respectively). ecp had no significant effect on circulating putative g-mdsc frequency measured before and the day after treatment (median: 8.4% and iqr 4%-44% vs 16% and iqr 6%-25%; n = 11, respectively) nor on lox-1 frequency (median: 1% and iqr 0.29%-12% vs 2.8% and iqr 0.88%-7.3%; n = 9, respectively). at a tc:g-mdsc ratio of 1:1, isolated g-mdscs from ecp patients suppressed cd3 tc proliferation (mean ± sd: 52% ± 23 %; n = 14). however, the potency of suppression was highly variable (min-max: 18-82%). the pattern of lox-1 expression suggests that only a subset of putative g-mdscs in ecp patients are suppressive and may explain why suppressive function in this cell fraction is so highly variable. however, the relatively high frequency of lox-1 cells in this patient cohort might contribute to immunosuppression resulting in increased susceptibility to opportunistic infections. according to the revised eortc/msg criterion, 29 patients were diagnosed with cns-ifd. among those patients without cns-ifd (3826 patients), cns-ifd were matched in a 1:3 ratio for analyzing the risk factors of cns-ifd. and among 594 (15.4%) patients who occurred pulmonary ifd without cns involvement, 87 patients were selected as control group for analyzing the risk factors associated with involvement of cns in pulmonary ifd. we selected the control group using a 1:3 ratio matched-pair method with the variates of (1) age; (2) sex; (3) underlying disease. we retrospectively reviewed 29 patients complicated with cns-ifd after hsct in our single center during a 10 years period. most patients received haploidentical stem cell transplantation. the median onset time of cns-ifd was 173 (24-972) after hsct, and most (82.7%) of them have prior pulmonary ifd. the most frequent pathogen was aspergillus, while no crypoccosis and candidas were found. the most common clinical presentation was space-occupying symptoms and signs. brain abscess were the most common imaging finding. prior pulmonary ifd (po 0.001, hr 62.746(95% ci, 14.28-275.27)) was the only risk factors associated with occurrence of cns-ifd. while poor response at 6 weeks (p = 0.045, hr 2.574 (95% confidence interval: 1.021-6.487)) was the only risk factor predicting the involvement of cns in pulmonary ifd. the response (complete and partial response) at 12 weeks and last follow-up was 27.6% and 20.6%, respectively. the overall survival was 24.2% at the last follow-up with a median 289 (27-3341) days after transplantation. in conclusion, patients with pulmonary ifd had higher risk of cns-ifd, especially in those with poor response after 6 weeks of treatment. and the prognosis of cns-ifd was very poor after hsct. disclosure of conflict of interest: none. adv may cause severe infections in hsct recipients, especially from unrelated donors or cord blood particularly in pediatrics. disseminated infections usually occur after digestive reactivations. at 3 mo.post-hsct, the incidence of adv digestive infection and viremia in pediatric hsct is about 30% and 15%, respectively. therapeutic strategies to control adv infections are limited to the use of infusion of cidofovir (cdv) or ex vivo anti-adv selected cytotoxic lymphocytes (ctl). however cdv is not labeled for adv treatment, presents a renal toxicity and has shown limited efficacy. specific-ctl remain difficult to produce. brincidofovir (cmx001, bcv, chimerix, usa) is an orally-available lipid conjugate of the nucleotide analog cdv that has demonstrated broad clinical antiviral activity against double-stranded dna viruses (that is, herpes-, adeno-, orthopox-and polyomaviruses. the drug has an increased bioavailability compared to cdv and has shown encouraging results. we report here the results obtained with this compound in 20 patients treated in six centers from january 2015. there were 20 pts (8m/12f), median age at hsct: 65 mo. (15-202). hsct indication was all in nine, pid in six, aml in two, fa in two and ibmf in one. donor was 9/10 or 10/10 mud in four and six pts, respectively; haplo-identical familial donor in 4; 5/6 or 6/6 unrelated cb in two and three pts, respectively; msd in one. stem cell source was bm for 11 pts, cb in 5 and pbsc in 4. two pts underwent a second hsct. cond' regimen were mac in 16 pts. all pts received either ex vivo or in vivo t-cell depletion. three pts presented with adv-disseminated disease, seven pts with blood + other site (throat, urine or stools) adv infection, three with adv-related gut disease, three with blood infection and three with gut infection. the remaining patient received bcv for jc viremia with fever. median time for virus infection diagnosis was d20 post-hsct (range: d-126 to d+300). about 13 pts experienced 24 other viral infection episodes after hsct (cmv: 8; ebv: 5; bk: 5; hsv: 3; hhv6: 2; influenzae: (1). about 12 pts received 1-6 injections of cdv prior to bcv treatment. one pt received specific-adv ctl before bcv without efficacy. the reason to switch from cdv to bcv was uncontrolled adv infection (n = 11) or cdvinduced renal failure (n = 1). two additional pts experienced renal impairment after cdv. about 14 pts received 1-4 lines of immunosuppressive therapies (including ecp) in addition to calcineurin inhibitor at time of bcv therapy due to grade iii and iv acute gvhd in seven and seven pts, respectively. median adv load at time of bcv initiation was 4.5log copies/ ml (range: 3-9) in blood and 5log copies/ml (3.3-7.5) in stools. median duration for bcv therapy was 3 weeks (range: 1-18). about seven pts with blood adv infection or disseminated disease experienced adv disappearance as well as four pts with gut disease or infection. three of them experienced adv infection relapse and received thereafter cdv, bcv or advspecific ctl. five pts presented with grade 2-4 diarrhea during bcv treatment. about 13 were alive at end point where seven died from septis (n = 2), multi-organ failure (n = 2), gvhd (n = 2) and adv disseminated infection (n = 1). adenovirus infections occur often in immunocompromized pts receiving concomitant nephrotoxic drugs that may avoid cdv use.bcv appears as efficient therapy against adenovirus infection in such pediatric pts since here 13 out of 20 pts where alive after adv infection and bcv treatment. in this study we retrospectively analyzed cmv reactivation determined by pcr and response to pre-emptive therapy in patients receiving an haplo (n = 68, 24%) comparing them with a control group of non haplohsct (110 mrd and 106 mud) (n = 216, 76%). median age was 52 years (range: 16-71), 56 for haplohsct and 52 for control group. conditioning regimen was myeloabaltive (mac) in 33.5% and reduced intensity (ric) in 66.5%. haplohsct characteristics: haplo conditioning was fludarabine (30 mg/m 2 or 50 mg/m 2 × 4 days in ric or ma regimen) and busulfan (3.2 mg/kg × 2 in ric or 3 days in ma) (19.1% mac, and 80.9% ric). cyclophosphamide-post was used for gvhd prophylaxis in 94%. median of days to reach more than 500 × 10 9 granulocytes and more than 20 × 10 9 platelets were 17 (13-31) and 22 (0-103), respectively. incidence of acute gvhd was 70% (grade i-ii 64.2%, and iii-iv 5.7%), with two steroid-refractory cases. cmv reactivation: 66.2% of haplohsct patients presented cmv reactivation, vs 29.8% in control group (p = 0.000). median number of cmv reactivation episodes was 1 in both groups. median time to cmv pcr detection was 35 days (4-70) and 40 (0-186) in haplohsct and control group respectively (p = 0.042). average maximum cmv iu by pcr was 17.499 in haplo vs 8.206 in the control group (p = 0.035). first antiviral pre-emptive therapy (valganciclovir in 65.7%) was effective in 82% in haplohsct vs 65% in control group (p = 0.064). main reason for antiviral treatment switch was failure in cmv iu reduction, and foscarnet was the most used therapy in refractory cases. twenty patients developed cmv disease (5 in haplo and 15 in control group) (gi disease 90% and pulmonary disease 10% in both groups). in a multivariate cox-regression model, receiving an haplohsct, serological cmv status (positive patient/negative donor), mac regimen and development of acute gvhd grade i/ii or grade ii/iv were variables associated with a higher risk of cmv reactivation. based on these results, haplohsct is associated with a higher cmv reactivation compared to non-haplohsct, despite a lower incidence of all other risk factors as agvhd or mac in the haplo group. although it is not statistically significant, response to pre-emptive therapy is higher in haplohcst and no differences in cmv disease were observed. disclosure of conflict of interest: none. although a number of patients with hiv infection and hematological disease have successfully undergone allogeneic hsct together with combination anti-retroviral therapy (cart), short and long-term outcomes remain not well known. we report the spanish experience treating hiv-infected adult patients with high-risk hematological malignancies with allogeneic hsct. we retrospectively reviewed 17 hiv-positive patients who received allogeneic hsct in three institutions in spain within geth (grupo español de trasplante hematopoyético y terapia cellular). seventeen patients have been transplanted between 1999 and 2015. median age was 44 (30-57), 82% male. diagnosis and transplant characteristics are summarized in table 1 . cumulative incidence of neutrophil and platelet engraftment were 88% at 30 days (median 15 days), and 76% at 60 days (median 13 days), respectively. with a median follow-up of 42 months (22-87), os and efs were 35%. trm was 17% at 12 months and 32% at 36 months. grade ii-iv agvhd rate was 41%, and moderate/severe cgvhd rate was 41%. all patients received cart. two patients showed severe toxicity related to interaction of immunosuppressive s238 drugs and protease inhibitors. about 76% of patients showed infectious complications. viral infections were the most frequent cause: cmv (9), bk (2), adv (1), hhv-1 (2), hcv (1), hhv-8 (1), parainfluenza (1). two patients had invasive aspergillosis and one patient presented disseminated tuberculosis. causes of death were: relapse (4), infection (3), gvhd (2) and toxicity (1). all surviving patients showed undetectable hiv load after hsct. allogeneic hsct is an effective therapy for high-risk haematological malignancies in patients with hiv infection, and long-term hiv suppression with cart is feasible. however, interactions between immunosuppressive agents and anti-retroviral drugs, high rates of significant gvhd, and frequent infectious complications account for a complex procedure in this population. selected hiv-infected patients with hematologic malignancies should be considered for allo-hsct when indicated, in experienced centers. disclosure of conflict of interest: none. clostridium difficile infection (cdi) is one of the most common causes of nosocomial infectious diarrhea in europe and usa, which results in high morbidity and mortality among hospitalized patients. allogenic hematopoietic stem cell transplant (hsct) recipients remain at high risk for cdi. incidence rate ranges from 2 to 27%. numerous risk factors including acute graft-versus-host disease (agvhd), hla matching status, conditioning-intensity, use of total body irradiation (tbi) may play an important role in the course of cdi in these patients. the aim of this study was to evaluate the prevalence of cdi in children, and to assess the influence of such factors as gender, age, diagnosis, hla matching status, conditioning-intensity, use tbi-containing regimen, source of graft (bone marrow/bm/ vs peripheral blood/pb/)or agvhd on course, duration of treatment and outcome in children undergoing hsct. between 2014 and 2015 a total of 342 hscts were performed in five polish pediatric transplant centers, including 267 allogeneic and 75 autologous. all patients were followed up to 6 months post hsct. we analyzed retrospectively 29 episodes of cdi infection in the group of 342 children. twenty-one of 29 children were diagnosed with hematological malignancies: acute lymphoblastic leukemia (all), acute myeloblastic leukemia (aml), myelodysplastic syndrome (mds), two were diagnosed with severe aplastic anemia (saa), one with chronic granulomatous disease (cgd) and 5 of them-with solid tumors. the median age was 9.3 years (range: 2.5-19.0 years). majority of patients underwent myeloablative conditioning protocol (24/29). in allogeneic setting 21/24 patients underwent mud-hsct, 2/24 pts msd-hsct and one patient was given a haploidentical pbsct. in this series, in 7 out of 24 cases bm was a transplant source, and pb in 17 out of 24. cdi was defined as having diarrhea that tested positive for c. difficile via pcr, cytotoxin assay, or dual enzyme immunoassays. kruskal-wallis test, wilcoxon test and χ 2 -test were used to estimate the influence of risk factors on severity of disease, duration of treatment and outcome. we observed 29 episodes of cdi (8.5 %) in hsct recipients: in 24 allotransplant recipients (8.9% of all transplants) and in 5 autotransplant recipients (6.7% of all auto-hsct). nine patients responded to therapy with metronidazole, seven patients responded to vancomycin alone, and in two patients rifaximine was administered. six children required adding second drug: vancomycin or metronidazole, five patients were not given any medications. there was no significant correlation between such factors as diagnosis, gender, age, conditioning regimen, hla matching, agvhd and severity of disease, and duration of treatment. recurrence rate was difficult to assess due to lack of data. we observed three deaths. one of them was connected with cdi. there was one 17-year-old boy with saa (mud-pbsct, hla 10/10) with no agvhd. the other two deaths were due to progression of s239 disease. cdi occurred in nearly 9% of pediatric patients undergoing hsct, surprisingly often in autologous hsct too (6.7%). almost all patients experienced mild cdi with adequate response to antibiotic therapy. cdi is a rare cause of death among transplant recipients. disclosure of conflict of interest: none. antifungal prophylaxis in high-risk paediatric patients with haematological malignancies: a monocentric experience k perruccio, i capolsini 1 , a carotti 2 , n albi 3 , l pitzurra 4 , a velardi and m caniglia 1 pediatric haematology oncology section; 2 haematology and clinical immunology section; 3 transfusion medicine and 4 the choice of antifungal prophylaxis in high risk paediatric haematological patients (according to the latest ecil6/seifem guidelines) remains an open question. a recent retrospective survey from associazione italiana ematologia oncologia pediatrica (aieop) showed that, in these patient categories, the only variable which significantly impacted on invasive fungal infection (ifi) occurrence was the presence or not of antifungal prophylaxis at the ifi onset (unpublished data). from january 2012, in our pediatric hematology oncology unit, 31 allogeneic hematopoietic stem cell transplantations (hsct) were performed (median age: 10 years; range: 6 months-23 years), mainly for acute leukaemia (median follow-up: 24 months; range: 4-50). patients received liposomal amphotericine b (n = 20), micafungin (n = 10), or fluconazole (n = 1) as primary antifungal prophylaxis until neutrophil recovery (41 × 109/l). seven patients developed acute gvhd (22%) which evolved in gvhd in 5 (16%). as outpatients, they continued with posaconazole (n = 17), voriconazole (n = 4), or micafungin (n = 10) until cd4+t-cell recovery (4200/cmm) or gvhd immune suppressive prophylaxis/treatment withdrawn. during the last year, according to ecil6/seifem guidelines1, we administered primary antifungal prophylaxis also to 10/12 high risk (hr) acute leukaemia patients. two patients with aml were treated with posaconazole, four patients with hr-all received micafungin, four relapsed all patients received micafungin (n = 2), or liposomal amphotericine b (n = 1), or posaconazole (n = 1). one aml patient was then transplanted; all relapsed patients are waiting for transplant. no differences were observed in terms of breakthrough proven/probable (pp)-ifi incidence, according to antifungal prophylaxis in the various patient groups. in particular, in the early phase, we observed a pp-ifi incidence of 10% in both treatment arms (micafungin vs liposomal amphotericine b, p = ns). in the late phase, we observed 1 case of pp-ifi who were receiving posaconazole as prophylaxis. overall survival (os) was 96%, with 4% mortality rate. in hr leukaemia patient group, we observed pp-ifi in the only two patients who were not receiving any antifungal prophylaxis at the ifi onset. antifungal prophylaxis is strongly recommended in paediatric patients with haematological malignancies who are at high risk of ifi. the choice of antifungal drug depends on the treatment phase, drug interactions (particularly for azoles), patient compliance and clinical conditions which interfere with intestinal absorption. in our experience, as no differences were observed in term of efficacy, micafungin resulted the best choice in terms of tolerability, toxicity, compliance and cost saving. antifungal prophylaxis with micafungin and bridging to inhaled liposomal amphotericin b after engraftment in patients undergoing allogeneic hematopoietic stem cell transplantation d rivera* ,1 , c de ramon 1 , a avendaño 1 , j carrillo 1 , d caballero 1 , l lopez 1 , i ormazabal 1 , a navarro 1 , a martin 1 micafungin is an effective antifungal for prophylaxis, active against candida spp. including those resistant to other antifungals (c. glabrata and c. krusei) and also active against aspergillus spp. guidelines focused on antifungal prophylaxis, recommend its use during preengraftment and early postengraftment period in allogeneic hematopoietic stem cell transplant (allo-hsct) recipients. moreover, its profile of low drug interactions and side effects, makes it a suitable alternative for patients who need concomitant treatments, present hepatic insufficiency and in those who do not tolerate oral drug administration. the addition of inhaled liposomal amphotericin b (lamb) after engraftment, provides an alternative way to effectively prevent mold infections, that are acquired mainly by inhalation. inhaled lamb has good tolerance with absence of drug interactions and low toxicity. the aim of this study was to describe the experience in the hsct unit of the university hospital of salamanca with micafungin and lamb as primary prophylaxis in patients undergoing allo-hsct with reduced intensity conditioning (ric) and graft-versus-host disease (gvhd) prophylaxis with tacrolimus and sirolimus. thus evaluating efficacy and tolerability in our population. retrospective observational study from january 2013 to august 2016, including all adult patients undergoing allo-hsct with ric and gvhd prophylaxis with tacrolimus and sirolimus, in whom an azole derivative is not indicated, due to drug interactions. therefore received prophylaxis with micafungin during the preengraftment period and bridging to lamb after engraftment at discharge, and continuing it during the first 100 days post-transplant. data from 106 patients from our hsct unit. ten (9.4%) patients who had invasive fungal infection before undergoing allo-hsct, and 16 (14.8%) patients who received prophylaxis with drugs other than micafungin-lamb were excluded. underlying disease was grouped by leukemia in 44 (41.5%) patients, lymphoma in 23 (21.7%), myelodysplastic syndromes in 18 (17%), multiple myeloma in 11 (10.4%) and other diseases in 10 (9.3%) patients. eighty patients underwent peripheral blood allo-hsct, of whom were related donor in 40 (50%) patients and unrelated donor in 40 (50%). prophylaxis with micafungin in 80 (75.5%) patients, dose of 50 mg per day, with a mean of 19 days (±6 days) with postengraftment bridging with lamb 24 mg weekly, continuing it during the 100 days posttransplant. days of neutropenia during preengraftment, o14 days in 24 (31.2%) patients, 14-28 days in 49 (63.7%), more than 28 days in 4 (5.2%). during follow-up there were three cases (3.8%), two catheter related candida infection, and one esophageal candidiasis. there were no reported aspergillosis cases (possible, probable or proven), according to the european organization for research and treatment of cancer (eortc) criteria. finally, prophylaxis with micafungin and inhaled lamb, was considered an effective and safe strategy in 77 (96.3%) patients, with no side effects reported. according to our experience with micafungin and the addition of inhaled liposomal amphotericin b, the results indicate that, this is an appropriate alternative for antifungal prophylaxis, in patients undergoing allo-hsct, because of their efficacy, few side effects and drug interactions. disclosure of conflict of interest: none. recipients of allogeneic hematopoietic cell transplantation (allohct) are at high risk of developing invasive fungal infections (ifi). in the early phase (o100 days) after allohct, the use of antimold prophylaxis has been generalized, although there is no consensus on the best therapeutic strategy. the use of nebulized liposomal amphotericin b and fluconazole has been shown to be effective, safe and associated with low economic costs in lung transplantation 1 . however, the use of this prophylactic strategy in the early phase of allohct setting has not been evaluated. we included all consecutive patients who received their first allohct in our center from january 2013 to august 2016 and who underwent antifungal prophylaxis according to the prospective ambineb protocol (nebulized liposomal amphotericin b 24 mg administered three times per week as loading dose and once per week and fluconazole 200 mg per day until day +90). patients with a previous ifi were excluded. patients with graft-versus-host disease (gvhd) receiving high dose corticosteroids were allowed to be changed to voriconazole or posaconazole at physician's discretion. the primary objective of the study was the incidence of ifi at day +180. the secondary objectives were to assess adherence and toxicity of the ambineb protocol. only cases with proven or probable ifi according to eortc-msg criteria were considered. a multidisciplinary team of experts in hematology, infectious diseases, microbiology and radiology prospectively evaluated and categorized each case. we included 102 patients with a median age of 50 years (range: 18-70) and a median follow-up for survivor of 14 months (range: 2-47). patients received allohct mainly for acute leukemia (55%), non-hodgkin lymphoma (16%) and myelodysplastic syndrome (12%) . patients received allohct from hla unrelated (59%) or related donors (28%) mostly using a reduced intensity conditioning (57%). graft-versushost disease (gvhd) prophylaxis was performed with calcineurin inhibitors mainly in combination with sirolimus (57%) or methotrexate (17%). after the comprehensive review, only one case of proven or probable ifi at day +180 was diagnosed. prophylaxis with ambineb was completed in 75 patients (74%) while 27 (26%) stopped the treatment. the most frequent causes of discontinuation were possible ifi (6%), gvhd (5%), admission in intensive care unit (5%) and toxicity (6%) (figure) . ninety-four patients (92%) did not have adverse advents associated with ambineb. eight patients presented organ toxicity which was at least partially attributed to ambineb, including gastrointestinal symptoms (n = 4), liver function test abnormalities related to fluconazole (n = 3) and cough (n = 1). of the 27 patients who discontinued ambineb, 22 (81%) were switched to other antifungal drugs including (echinocandins (12, 54%) posaconazole (4, 18%), voriconazole (2, 9%) or others (4, 18%)). overall survival and non-relapse mortality of all patients at median follow-up were 64% (95% ci 58-69) and 25% (95% ci 20-30), respectively. the combination of nebulized ambisome and fluconazole is effective in preventing ifi in the early phase of allo-hct and is associated with high adherence and low toxicity. neutropenia-related infections is a common complication of apsct in patients (pts) with mm and dlbcl. our study aims are to: (1) assess antibacterial susceptibility patterns of isolated organisms, to guide antibiotic prophylaxis (2) identify the epidemiology of bacteremia with susceptibility patterns to direct empiric therapy of febrile neutropenia (3) assess the interval between the occurrence of neutropenia and the isolation of resistant bacteremia to identify the best timing to start prophylaxis (4) identify contributing factors for the development of bacteremia and mortality. our retrospective study included 191 adult pts who underwent apsct for mm and dlbcl between 2005 and 2015. we recorded the following: age, gender, basic illness, comorbidities, number of cd34 + cells infused, a central venous catheter, duration of neutropenia, diarrhea and mucositis, mechanical ventilation, positive bacterial cultures with susceptibility profiles and history of broad-spectrum antibiotic intake for more than 72 h for the past 3 months on hospital setting, and mortality. statistical analysis was carried out using spss (version 19). about 102 isolates were obtained: urine (46.5%), blood (28.5%), sputum (9%), wound (7%), venous catheter (7%) and stool (2%). gram-negative (gn) species were predominant (73.5%) with e. coli (32.5%), klebsiella (k) (11%) and pseudomonas (pseudo) (11%). isolates sensitive to third generation cephalosporins (3gc) represented 83% of the enterobacteriaceae (entero) including 78% in e. coli and 73% in k. all entero isolated were susceptible to carbapenems (carba), pipercillin/ tazobactam (pip/taz), amikacin and ciprofloxacin (cipro). all pseudo (n = 11) and acinetobacter (n = 4) isolates were susceptible to carba, pip/taz, amikacin, cipro, colistin and tigecycline. as for gram-positive (gp) bacteria (26.5%), coagulase negative staphylococci (cns) were predominant (14%) . oxacillin susceptibility reached 29% and two isolates methicillin resistant s. aureus were identified. all gp were susceptible to glycopeptides. a total of 29 bacterial isolates were identified (29 episodes of bacteremia) from 24 pts. gn were predominant (72.5%) with e. coli being most common (24%). all gn were susceptible to 3gc, carba, pip/taz, amikacin and cipro. as for gp (27.5%), cns predominated (24%) including 28% oxacillin-susceptible causing seven episodes of bacteremia with six central line-associated. no glycopeptide resistance was identified. none of the clinical features and pts' characteristics reached statistical significance as risk factor for bacteremia. however, the need for mechanical ventilation and mortality were higher in bacteremic vs non-bacteremic pts (16.7% vs 3%, p = 0.004, and 12.5% vs 3%, p = 0.036, respectively). all bacteremic episodes occurred after developing neutropenia (median = 2.5 days, range: 1-9) except for one case of clabsi caused by e. coli occurring 1 day before neutropenia. pip/taz was prescribed in 21% of bacteremia episodes followed by quinolones (11%) and carbapenems (4%). no previous use of third and fourth generation cephalosporins was observed. we recommend quinolone prophylaxis in apsct pts. for empiric therapy, antibiotics recommended by international guidelines including, cefepime and pip/taz still fit. thus, we could spare the use of carba and other last-resort antibiotics to other conditions. we also recommend continuous surveillance of resistance. disclosure of conflict of interest: none. fever is an almost universal complication in patients undergoing autologous stem cell transplantation (asct), however, microbiological documentation is only achieved in 20-30% of such febrile episodes (fe). 1 this low diagnostic efficiency makes epidemiological assessment in transplant units difficult, and may lead to a suboptimal empirical treatment. we have studied the utility of strict blood culture (bc) extraction as a mechanism to improve microbiological documentation of fe in these patients. we conducted a retrospective study over 66 consecutive asct performed in our centre between june 2015 and may 2016 (1 year). about 33 patients were male and 27 female, with age between 27 and 72 years (mean 57.8). diagnosis was 4 hodgkin lymphoma, 27 non hodgkin lymphoma and 35 multiple myeloma. ascts were performed in reverse isolation conditions, in rooms equipped with hepa and pall filters. prophylaxis against herpes virus and p. jirovecii with acyclovir and pentamidine was used. no prophylaxis against gram negative bacteria or filamentous fungi was performed. bc were extracted at the beginning of every fe and every 48-72 h if fever persisted (or more frequently, following clinical criteria). blood samples from intravascular devices and peripheral blood were collected in two bactec bottles each (for aerobic and anaerobic microorganisms). complementary diagnostic techniques and empiric antibiotic therapy were performed following our institution's guidelines. fe were classified in microbiologically documented infections (mdi), clinically documented infection (cdi) and fever of unknown origin (fuo) following his criteria. 2 85 fe were studied (average 1.28 fe per patient, 6.04 days of fever duration per fe). about 28% of fe were classified as mdi, 26% as cdi and 46% as fuo. mdis were cause by gram positive bacteria (56%), gram negative bacteria (26%) and polymicrobial infections (17%). no viral or fungal infections were observed. an average of 7.2 bc per fe and 7.6 per patient were extracted. the proportion of fe classified as mdi was related to the number of blood cultures extracted during the episode. only 8% of fe with three or less bc extracted were classified as mdi, 21% if 4-6 bc were extracted, and 55% if 7-9 bc were extracted (p o0.05). no significant difference in proportion of mdi classified fe between the extraction of 10 or more bc and 7-9. all patients were discarded in good clinical conditions. according to our experience, a strict 7-9 blood culture extraction is related to a high rate of microbiological documentation of febrile episodes. moreover, we have not observed the rise in gram negative bacteria reported by other studies and gram positive cocci persist as the main infection cause in our centre. candida which has been traditionally related to duration of neutropenia, emerges as a pathogen beyond the aplastic period in allogeneic haematopoietic cell transplantation (allohct). in the setting of alternative transplants and aggressive immunosuppressive therapy, these infections are a challenging problem. there is scarcity of data regarding the significance of breakthrough candidaemia in allohct. to that end, we aimed to determine the incidence, clinical and microbiological characteristics and outcome of candidaemia in allohct recipients. we studied consecutive allohct recipients from january 2014 to june 2016. blood cultures were obtained from peripheral vein or central venous catheters (cvcs) routinely and on febrile patients. well-known risk factors for candidaemia were studied: neutropenia, type of transplant, moderate to severe graft-versus-host disease (gvhd) and coexisting infections. among 108 allohct recipients, we identified seven patients with candidaemia: five post matched unrelated (four myeloablative and one reduced intensity conditioning) and two post haploidentical transplant. in median time of 3.5(1.3-8) months, 20 episodes of candidaemia were noted, despite antifungal prophylaxis with echinocandins or azoles. infections with non-albicans candida spp. occurred more frequently (19/20) and c. parapsilosis was the predominant microorganism (11/19). other species were isolated: c. famata (5), c. krusei (2) and c. haemulonii (1). all candida spp. isolates were phenotypically susceptible to antifungal agents already administered to patients. there was no resistance to echinocandins indicated by minimum inhibitory concentrations (mics). all patients had severe acute or late-onset gvhd with intestinal involvement and cvcs prior to candidaemia. although cvcs were removed in 7/7 and patients were treated with echinocandins, new cvcs were re-contaminated in 4/7 with the same or other species. all patients presented well known risk factors for candidaemia (use of broad spectrum antibiotics due to severe bacterial infections, total parenteral nutrition due malnutrition, long-term high-dose corticosteroids and other immunosuppression), but no neutropenia. one patient survived, whereas five patients succumbed to gvhd and multi-organ failure and one patient to sepsis due to bacteremia. candidaemia was observed in non-neutropenic patients with agvhd and cvcs on antifungal prophylaxis, despite difficulties in diagnosis due to poor sensitivity of blood cultures. the epidemiology of candidaemia has changed in the last decade and its risk is more diverse and complex. the irreversible intestinal gvhd lesions might be the main source of candida in patients receiving antifungal prophylaxis. our data show that candidemia remains an important issue in profoundly immunosuppressed patients contributing to excessive morbidity. our aim was to compare the rate of neutropenic sepsis, defined as fever of 438 o c and a neutrophil count of o0.5 × 10 9 /l, before and after the introduction of ciprofloxacin prophylaxis. one hundred and eight adult patients, of which 60 had acute myeloid leukaemia, 17 had acute lymphoblastic leukaemia, 12 had lymphoma and 19 had other haematological malignancies, were identified through our admission database. of these 108 patients, 48 received oral ciprofloxacin during their neutropenic phase. the median duration of neutropenia was 13 days in both the no-prophylaxis and ciprofloxacin groups. there was a significant reduction in the rate of neutropenic sepsis from 88.3% (53/60) in the no-prophylaxis group to 68.8% (33/48) in the ciprofloxacin group (p = 0.012). prolonged infection, suggested by the use of broad-spectrum antibiotic treatment for more than 10 days, was more common in the group which did not receive prior ciprofloxacin prophylaxis (45.0% vs 20.8%, p = 0.009). rate of intensive care admission (15.0% vs 0.0%, p = 0.005) was also reduced by the use of ciprofloxacin. however, there was no significant difference in the length of stay (mean of 28 vs 25 days, p = 0.187), or in the 30-day infection-related readmission rate (17.9% vs 13.3%, p = 0.536) between the two groups. in terms of the cause of neutropenic sepsis, escherichia coli, klebsiella pneumoniae and pseudomonas aeruginosa were the most common bacteria isolated from cultures in the no-prophylaxis group. eighty percent of these organisms showed sensitivity to ciprofloxacin. in the ciprofloxacin group, staphylococcus epidermidis was the most frequently found bacteria. with regards to treatment related adverse effects, none of the patients who received ciprofloxacin prophylaxis developed clostridium difficile diarrhoea. in conclusion, ciprofloxacin is still an effective antibacterial prophylaxis during neutropenia following allogeneic stem cell transplantation. clinicians should have a high suspicion of a gram-positive infection in patients who develop neutropenic sepsis on ciprofloxacin prophylaxis. disclosure of conflict of interest: none. s243 hematopoietic stem cell transplantation (haplohsct) to cure leukemia, malignancy and some inherited diseases, different additional reasons interfere microbiota metabolism and integrity. among them are radiation and chemotherapy, mucositis, infection and graft versus host disease (gvhd). the curative mechanism of fmt is based on the ability of donor intestinal microbiota to substitute and to provide all necessary functions of altered patient's microbiota. three patients (3, 10 and 28 years old) after haplohsct, who observed pseudomembranous colitis (toxin b-positive) as gvhd of intestine outcome, were enrolled to the study and performed fmt. relatives (mother, father and brother) were used as microbiota donors. donor and patient examination have included routine clinical and biochemistry laboratory data, microbiota cultural methods, pcr of most common intestinal microorganisms. additional for patient-level of fecal calprotectin by elisa was tested, identification of drug resistant bacteria and histology of intestine were made. patient's preparation for fmt included-probiotic (inulin) administration 72 h prior procedure, discontinuation of all antibiotics 24 h prior procedure and antiemetics (5ht3agonist), prokinetics and proton pump inhibitor. delivery of donor's microbiota was performed in two consecutive steps under total intravenous anesthesia: with esophagogastroduodenoscopy-to the duodenum; with colonoscopy-to the caecum. all patients observed complete clinical response in 14-28 days after fmt (table 1 ). in 10 days we have revealed significant quantitative and qualitative changes in microbiota composition, which was matched to donor's microbiota. in 35 days after fmt we identify microbiota changes in oropharynx and urogenital tract similar to donor microbiota. this leads to substitution of multidrug resistant klebsiella pneumoniae strains by drug sensitive microorganisms and helps to treat severe infection complications after haplohsct. platelet aphereses were carried out in 83 donors (50 males and 33 females with median age 38.5 years) using haemonetics instrument with simultaneous leucoreduction. quantitative detection of cmv, ebv and hhv-6 dna was performed by multiplex real-time quantitative pcr kit (interlabservice, russia) in donors' whole pb, plasma and platelet aphereses at the time of platelet collections. viral load in hsct recipients was monitored weekly after hsct and 7 days before hsct by the same pcr kit. lower limit of detection (llod) of the applied kit for all viruses was 500 copies/ml. in specimens of platelet donors we additionally performed ultra-sensitive pcr with llod 100 copies/ml. only one patient (2.4%) was cmv-positive by pcr prior to hsct. cmv reactivation after hsct ⩾ 500 copies/ml was noted in whole pb of 9 patients (22.0%) with median time of 41 days (range: 0-71). donor source in cmv-reactivated patients was as follows (3 mud, 1 msd, 3 haplo). cmv viremia ⩾ 1000 copies per ml was detected in seven patients (17.1%). cmv disease was found in five cases (12.2%). none of patients were positive by pcr for ebv or hhv-6 prior to hsct. ebv reactivation ⩾ 500 copies/ml was found in six cases (14.6%), ⩾ 1000 copies/ml in 5 (12.2%) with median time of 27 days (range: 20-56). no signs of ptld or other ebv-dependent clinical symptoms were observed. hhv-6 level after hsct ⩾ 500 copies/ml was detected in 17 patients (41.5%), ⩾ 1000 copies/ml in 11 ones (26.8%) with median time of reactivation 19 days (range: 13-36). hhv-6 disease was observed in one patient. none of platelet donors were cmv-positive in plasma, whole blood or platelet aphereses products. ebv ⩾ 500 copies/ml was detected in whole pb specimens of five platelet donors (6.0%). application of ultra-sensitive pcr revealed low level of ebv-viremia in additional 11 pb cases with median ebv level 260 copies/ml (range: 115-410). none of platelet donors have any clinical signs of ebv disease. there is no any ebv-positive case among platelet concentrate specimens. in two cases low levels of hhv-6 was found in a whole pb (110 and 180 copies/ ml). none of hhv-6-positive case was observed among plasma and platelet concentrate specimens. despite high incidence of cmv, ebv and hhv-6 reactivation after hsct in pediatric patients we could not show that source of viral reactivation was contamination of platelet apheresis products by donorderived herpes viruses. disclosure of conflict of interest: none. conventional respiratory virus (crv) infections are known to be major causes of morbi-mortality after stem cell transplantation (sct) due to the increased risk of progression to lower respiratory tract infection (lrti) in this setting. risk of developing severe lrti is mostly related to factors specific to the patient and the underlying disease, although the intrinsic virulence of crvs may also determine their outcomes. we conducted a single-center retrospective study including all adult sct recipients who had crv disease (defined as patients with symptomatic respiratory disease and crv identification) during a 7-year period (2009-2016) with the main objective of evaluating epidemiological changes over time and their association with infection outcomes. during the study period 137 episodes of crv disease were diagnosed in 104 patients (median age: 48 years, 58% male, 49% aml or mds as baseline disease). 83 patients (80%) received an allogeneic-sct (allo-sct) (30% had a prior sct) and 21 (20%) an autologous sct. crv disease was diagnosed at a median of 343 days after sct (range: 1-4361), with 33 cases (24%) occurring before day +100. during the infectious episode 31 of 83 allo-sct recipients (37%) had active gvhd and 39 (47%) were under s244 prednisone (pdn)415 mg/kg. most of the patients (84%) had symptoms compatible with an upper respiratory tract infection (urti), with 15 of them (11%) progressing to a lrti, while 7 (5%) had a lrti only. hospital admission was required in 46 episodes (33%) with a median duration of hospitalization of 11 days (range: 1-30), 17% required supplemental oxygen, 6% were transferred to the intensive care unit and 4% required mechanical ventilation. the most commonly identified pathogens over time are shown in figure 1 . twenty-four cases (17%) had concomitant bacterial or fungal infections. influenza a virus was the most frequent crv detected (49 episodes, 36%) followed by human respiratory syncytial virus (37 episodes (27%) and human parainfluenza virus type 3 (18 episodes, 13%). during the 2009 flu pandemic, only 2 of the 13 crv infections diagnosed in sct recipients (15%) were associated to influenza a virus h1n1. antiviral treatment was started in 62 episodes (45%), antibiotics in 56% and combined therapy in 25% during a median of 7 days (range: 4-21). the rv resolved in 129 cases (94%) at a median of 12 days (2-72) from onset, with crv being considered the leading cause of death in only 3 patients (3% of all cases and 3/22 (14%) in those with a lrti). predictors of severe crv infection (including icu admission, need for supplemental oxygen, need for mechanical ventilation requirement or death) in multivariate analysis were lymphocyte count o 200 cells/μl (hr: 4.2, 95% ci: 4-26, p = 0.01) and co-infection with other pathogens (hr: 4.8, ci95%: 1.5-16, p = 0.00). no specific crv nor period post-sct of the infection influenced the risk of severe infection. our results confirm that crv infections are a frequent cause of morbidity after sct with a high need for hospital-based care. temporal changes in the principal circulating crvs has been identified during the 7-year study period, with influenza a virus being the most common. profound lymphocytopenia and presence of co-pathogens are associated with infection severity. [p246] disclosure of conflict of interest: none. the 127 consecutive hsct performed from 2012 to 2016 are being analysed retrospectively. out of them 60 (47%) performed in hepa filter room and 67 (53%) in non-hepa filter room, criterion was purely financial to make this decision. 96/127 (76%) were allogeneic and 31/127 (24%) were autologous hscts. blood cultures both bacterial and fungal were taken at onset of fever and with every change of antibiotics till patient became afebrile. chest x-ray and if required hrct chest was done for all patients who had respiratory complaints. we did not use antibacterial prophylaxis; however, antifungal prophylaxis was administered along with conditioning; and at the onset of fever systemic antibiotics were started. antifungal agents were added if fever persisted for 3 days pre empatively. extremely well trained nurses were looking after both the groups. all treatment protocols, antibiotic/antifungal policies were same in both the groups. median time for neutrophil engraftment was 12 days in hepa filter room and 13 days in non-hepa filter room. total 4/127 (3 %) patients did not engraft till 30 days. out of them 02/60 (3 %) were in hepa filter room and 2 /67 (2.9 %) in non hepa filter room. blood cultures were positive in total 20/127 (16%) patients, 17 were positive for bacterial and 3 for fungal organisms. in hepa filter hsct 11/60 (18%) were positive and in non-hepa filter hsct were 09/76 (13%) positive. total 34/127 (27%) patients developed pneumonia, out of them 14/60 (23%) were in hepa filter and 20/67 (30%) hsct were in non-hepa filter room. statistically not significant. no central venous access cather issues or infections were documented in any groups gr 2-4 agvhd : hepa rooms 11/60 (18.3%),non hepa rooms 14/67 (20.8%) :was not statistically significant the 30-day mortality was 25/127 (20%), 07/60 (12%) patients were from hepa filter rooms and 18/67 (27%) were from non-hepa filter rooms. cost : average cost of allogeneic hsct in hepa room : usd 22 000. average cost of allogeneic hsct in non hepa room: usd 13 700. average cost of auto hsct in hepa room: usd 12 000. average cost of auto hsct in non hepa room : usd 8500. incidence of blood culture positivity & incidence of pneumonia was not different. these are two very important issues in outcome of hsct. agvhd incidence did not depend on the room type. these are significant findings from this study. results were slightly better in hepa filter rooms compare to non-hepa filter rooms, which was statistically insignificant. our study had few confounding factors hence we could not be concluded that hepa-filtered rooms are not necessary. nevertheless, our experience suggests that availability of dedicated hepa units with special air-handling equipment should not be considered a critical and essential precondition for providing allogeneic hsct to patients even in developing world with financial constraints. these would otherwise succumb to potentially curable hematological illnesses with background of financial constraints and wait list of hepa filter rooms. early hsct in a clean patient in non hepa rooms is extremely cost effective with comparable outcomes. nursing care, experience of the team, experience in hsct program & well established protocols are more important in outcome of hscts. disclosure of conflict of interest: none. we present five cases of cytomegalovirus (cmv) pneumonitis occurring in patients after recent allogeneic stem cell transplantation (allohsct). these cases were complicated by an organising pneumonia (during the recovery period) with a predominantly central peribronchial pattern. all patients presented with evidence of active cmv pneumonitis which was treated successfully with anti-viral therapy but was followed by persistent severe dyspnoea, cough and hypoxia. high resolution computed tomography (hrct) imaging showed widespread central peribronchial consolidation with traction bronchiectasis. in most cases there was a marked clinical and physiological improvement after treatment with systemic corticosteroids. however, in all patients the lung function remained abnormal and in some cases imaging revealed a fibrosing lung disease. these cases represent a previously undescribed central peribronchial pattern of organising pneumonia complicating cmv pneumonitis that can result in chronic lung damage. disclosure of conflict of interest: none. cytomegalovirus reactivation in pediatric acute leukemia after stem cell transplantation has an effect on relapse and survival in aml but not in b-precursor all j-s kühl 1 , l sparkuhl 1 and s voigt 1 department of pediatric oncology/hematology/sct, charité universitätsmedizin berlin, berlin, germany several studies have indicated better survival after stem cell transplantation (sct) for acute leukemias, especially acute myeloid leukemia (aml), in case of cytomegalovirus (cmv) reactivation. here, we investigated if cmv reactivation had an impact on survival after sct for aml or acute lymphoid leukemia (all) in children. 177 pediatric allogeneic stem cell transplant recipients from our institution who received myeloablative conditioning were included. transplant indications included aml, t-all and b-precursor all. cmv reactivation was correlated with relapse, mortality as well as acute graft-versus-host disease (gvhd) and was analyzed by fisher's exact test or χ 2 -test (if n4100). from the 177 patients included, 42 were transplanted for aml (24%), 22 for t-all (12%), and 113 for b-precursor all (64%). mortality and relapse rates (27-37% and 18-26%, respectively), cmv reactivation rates (21-36%) as well as numbers of negative cmv serology status (19-32%) of donor and recipient were comparable between different acute leukemias. when patients were analyzed altogether, cmv reactivation had no effect on relapse rates or mortality. however, a tendency towards fewer relapses after cmv reactivation was observed in aml patients (no relapse (0%) with cmv reactivation vs 11 relapse cases (33%) without cmv reactivation; p = 0.083). in those 128 leukemia patients capable of reactivating cmv (that is, donor or recipient cmv seropositive prior to sct), cmv reactivation had a protective effect on relapse rates in aml (no relapse (0%) with cmv reactivation vs 11 relapse cases (44%) without cmv reactivation; p = 0.017). a similar tendency could be seen in t-all whereas no effect in patients with b-precursor all was documented. numbers of acute gvhd cases grade 4i between aml and t-all with or without cmv reactivation were similar. different effects of cmv on relapse rates and mortality in aml vs b-precursor all were noticed in 79 patients who were either not capable of cmv reactivation or who did reactivate cmv post sct. in 17 aml patients, there were no relapses (0%) and 2 deaths (12%) in contrast to 17 relapse cases (27%) and 24 deaths (39%) in 62 children with b-precursor all (p = 0.017 and p = 0.044, resp.). latently cmv infected aml patients without documented cmv reactivation after sct have a significant worse prognosis compared with all other aml patients. this is also likely to be the case in patients with t-all, however, patient numbers in our cohort were too few. the protective effect of cmv reactivation in aml and possibly t-all does not appear to be gvh-related since the rate of relevant acute gvhd cases was comparable. cmv reactivation after sct for b-precursor all lacks significance. disclosure of conflict of interest: none. infections are among the most frequent and relevant complications of hematopoietic stem cell transplantation (hsct). little is known about the role of dental foci for the prevalence of infections in hsct. dental status was prospectively evaluated in all patients at our center before undergoing hsct. a total of 132 different patients before undergoing 163 hsct (83 allogeneic and 80 autologous), with a median age of 52 years (range: 4-70 years) were evaluated. for evaluation a panoramic x-ray evaluation was performed. dental findings included the status of third molars and root fillings as well as caries, periodontitis, destructed teeth and apical bone loss. as non-dental parameters we used age, sex, type and status of central venous line, mucositis, and type of transplantation. these were correlated with neutropenic fever, bacteremia and pneumonia in a bivariate manner before a multivariate analysis was performed. no correlation of initial dental status to neutropenic fever, bacteremia or pneumonia was found. however, bacteremia and suspected infection of central venous lines was a significant predictor of neutropenic fever. in conclusion, dental surgery should only be performed prior to hsct if urgently required and limited to those individuals with overt infection. disclosure of conflict of interest: none. [p250] early experience with clinimacs prodigy ccs method in generation of virus-specific t-cells for pediatric patients with severe viral infections after hematopoietic stem cell transplantation k kallay 1 viral reactivation especially in children is a frequent complication of allogeneic hematopoietic stem cell transplantation. most of these episodes can effectively be controlled by an antiviral or antibody therapy; in refractory cases a novel virusspecific t-cell therapy could be a promising management option. in our pediatric cohort of 43 allogeneic transplantation during 1 year 9 patients fulfilled criteria for virus-specific t-cell therapy (5 boys, 4 girls, median age of 11 (1.5-16) years). six patients were transplanted because of hematological malignancies and 3 for inborn errors. donor distribution was the following: 7 matched unrelated, 1 sibling and 1 haploidentical donor. in 5 cases bone marrow, 3 cases peripheral blood and 1 case cord blood was used as a stem cell source. the underlying viral illness was cmv in 3, ebv in 2 and adenovirus in 1 case, while more than one virus was detected in 3 cases (cmv+adenovirus 2 cases, cmv+ebv 1 cases). viral diseases necessitating a t-cell therapy were cmv pneumonitis and colitis, adenovirus enteritis and cystitis and ptld. patients initially received cidofovir for adenovirus, rituximab for ebv and a combination of gancyclovir and foscarnet for cmv infections. the indication for t-cell therapy was progressive viral disease in 8 of the 9 cases and uncontrollable viral load in 1 case. the procedure was performed on a median of 63 (49-113) day post transplant. donors were 1st degree relatives in 5 cases, 2nd degree relatives in 3 cases and an unrelated person in 1 case, the best hla match was haploidentical. the median age of the donors was 47 (33-60) years. cells were produced by the clinimacs prodigy cytokine capture system (ccs) method after mononuclear leukapheresis. the system produced a median of 9.9 (6.7-25) times 103/kg cd4+ and a median of 32.6 (16-125.1) times 103/kg cd8+ interferon producing cells while the non-interferon producing cells were far below gvhd limit with a median of 3.6 (1.5-12.4) times 103/kg cd4+ and a median of 3.85 (1.4-4.5) times 103/kg cd8 + cells. the t-cell products were administered uneventfully in all but one case. we observed a manageable cytokine storm in one patient. glucocorticoid treatment was ongoing due to acute gvhd in 5 children; however we could manage to keep the steroid dose below 1 mg/kg in all cases. eight patients became completely asymptomatic, while 7 also cleared the virus. we experienced decreasing viral load in all cases, the first negative viral results were achieved on a median day of 13 (13-55). six patients are alive without viral illness or sequale, and complete viral dna clearance in peripheral blood with a median follow up of 329 (144-580) days. one patient with cmv pneumonitis improved during the first week but deteriorated on the second week and died of respiratory insufficiency despite of mechanical ventilation. in 2 cases the viral illness improved or cleared, but the patients died of invasive aspergillosis. no cases of gvhd, rejection, organ toxicity or recurrent infection were noticed. virus-specific t-cell therapy produced by the clinimacs prodigy ccs is a feasible, fast, safe and effective way to control resistant viral diseases after pediatric hematopoietic stem cell transplantation. this treatment can be implemented within a week in most cases. in order to define the appropriate place of this approach for patients with viral reactivations more data should be collected. disclosure of conflict of interest: none. central venous catheter (cvc) is essential for the treatment of recipients of stem-cell transplant. it is usually placed for the administration of conditioning regimen, stem cell infusion, intravenous antibiotics, immunoglobulins, electrolyte and nutritional support and blood concentrates. this patient group is at high risk for catheter-related bloodstream infections that can result in substantial morbidity and mortality. the neutropenia secondary to the conditioning regimen determines the risk of catheter-related infections, which may serve as an entry into the blood circulation, leading to bacteremia, fungemia, and consequently to septic shock and death. the risks of infection and the spectrum of infectious syndromes differ according to the type of transplant, conditioning regimen, type of implant of stem cells and therapies used after the procedure. gram-positive bacteria, particularly coagulase-negative staphylococcus spp, remain the leading cause of catheter-related bloodstream infection, although an increase in gram-negative bacteria as the causative agent has been noted. aim of the study: to evaluate the impact of the early cvc removal on the frequency of febrile episodes and infections in our group of patients. during a 15 years period we have treated 351 patients with hematologic neoplasm with high-dose chemotherapy and stem cells transplantation. patients were treated in sterile room conditioned with hepa filtration. in every patient was introduced double-lumen cvc (321 subclavia, 7 jugular, and 23 femoral). 44% were febrile (10% fuo), catheter-related infection was present in 9%, while positive culture from cvc was present in 28%. the most frequent isolated bacteria from cvc were gram positive-staphylococcus coagulasa negative. the catheter was removed on the day of discharge. trm is 2.4%. from 01 january 2016 to 01 november 2016 we have transplanted additional 40 patients. to aim to decrease infection related mortality we perform strategy to remove cvc on day +2 after stem cell transplantation. the febrile episodes decreased on 25% (10/40), there were no early post-transplant mortality due to infection. early removal of the cvc and adequate handling from the nursing staff is essential for outcome of this patient population in regard of infective complications efficient prevention, early diagnosis, and effective treatment of catheter related infection are essential to providing the best care to these patients and can minimized morbidity and mortality. disclosure of conflict of interest: none. fever in patients with agranulocytosis during autologous hematopoietic stem cell transplantation (autohsct) can be associated with non-infectious causes due to g-csf, vancomycin, engraftment syndrome. in this case biochemical markers, such as presepsin (psp), procalcitonin (pct) and c-reactive protein (c-rp), can help in differential diagnosis of fever of infectious and non-infectious genesis. psp, pct and c-rp were assessed on the day of admission to the hospital (da), on d+1, on d+3, on d+7 and on the day of discharge (dd). if patients developed neutropenic fever (nf), the markers were assessed at the beginning of the fever, 6 h after, then on the second, third, fourth days after. if patients developed nf immediate empirical antibiotic therapy (at) was implemented with meropenem. in cases of ineffective 1st line ab, 2nd line at was added or totally changed. there were 100 patients included in the study: 41 patients with hodgkin lymphoma, 27 with non hodgkin's lymphoma, 32 with multiple myeloma, out of 100 patients there were 51 women and 49 men. the median age was 41 years (18-66 years). the conditioning regimens were cbv, beeac or hd melphalan. 69 patients developed infectious complications (ic): 2 of them had sepsis and others-nf. the median of nf development was 5.5 days. depending on the efficiency of at therapy patients were divided into two groups: group 1: patients that have had effective at (they 've had fast clinical response and they haven't needed to change medicine (n = 45)); group 2: patients that have had ineffective 1st line at, they haven't had response to 1st line at and they've needed to change another at (n = 24)). there were significant differences in psp levels on the third day after ab had been admitted: 365.3 pg/ml in group 1 and 750.6 pg/ml in group 2 (p = 0.0019). similar differences between the analyzed groups were observed on the fourth day: 350.5 and 569.7 pg/ml, respectively (p = 0.024). pct and c-rp didn't show any significant changes between group 1 and 2 on each day of the study (table 1) . disclosure of conflict of interest: none. enterovirus related immune reconstitution inflammatory syndrome (iris) following haploidentical stem cell transplantation in an mhc class ii deficient child r shah 1 , s waugh 2 , k foong ng 1 , z nademi 1 , t flood 1 , m abinun 1 , s hambleton 1 , a gennery 1 , m slatter 1 and a cant 1 1 paediatric immunology and bmt, great north children's hospital, newcastle upon tyne, uk and 2 department of virology, great north children's hospital, newcastle upon tyne, uk immune reconstitution inflammatory syndrome (iris) has been described after hsct in association with fungal, viral and bcg infections. we describe a case of post-hsct iris associated with enterovirus infection. case: a girl with mhc ii deficiency (rfxp2c.362 mutation) underwent treosulfan/fludarabine/ thiotepa/atg conditioned tcrαβcd3+ depleted haploidentical hsct at 1.8 years of age. pre-transplant work up did not reveal any viral or fungal infections except norovirus in stool. cyclosporine (csa) was given as gvhd prophylaxis. neutrophil and platelet engraftment occurred on d+15 and d+9, respectively. on d+6, her stool was tested positive for enterovirus (taqman pcr), however; she was asymptomatic. the child started having fevers and irritability from d+21 which persisted despite the use of antimicrobials. no evidence of fungal or bacterial infection was found. enterovirus pcr in blood was found positive on d+31 (cycle threshold value, ct 32.4) and further typing showed it to be echovirus 13. at this time, symptoms progressed with diarrhoea, developmental regression and signs of radiculopathy. mri (brain and spine) was normal and csf showed pleocytosis (815 wbc/mcl-100% lymphocytes, protein 4.23 g/l) with positive enterovirus pcr (ct 17). subsequently, immunoglobulin prophylaxis was increased to 0.5 g/kg bi-weekly, and with supportive measures, the patient slowly recovered. blood enterovirus pcr remained positive. with no evidence of gvhd, csa was tapered off by day+105 and child was discharged on d+112 on a bi-weekly ivig replacement. she presented 5 days later with signs of raised intracranial pressure. mri showed hydrocephalus, and vp shunt was placed and broad spectrum antibiotics administered. csf showed wbc o1/mcl, protein 0.23 g/l and enterovirus positive. methylprednisolone 2 mg/kg/day was started suspecting iris. in subsequent csf testing 3 days later, enterovirus was negative. enterovirus pcr remained positive in blood during this period. patient's clinical deterioration correlated with a rise in cd4/cd8 counts and c reactive protein with clearance of enterovirus from csf, blood and stool ( figure 1 ). subsequently, the child showed gradual but marked improvement and discharged home. discussion: the clinical features of index case fits into criteria for iris 1 . markedly raised crp suggests high il-6 levels without any bacterial or fungal pathogens being isolated. in addition, iris occurs at the site of prior active infection (brain in index case) and viral clearance and clinical recovery demonstrated with the continuation of steroids. the incidence of enterovirus infection in hsct recipients is around 10% 2 . iris, in this case, had a temporal correlation with discontinuation of csa, and it has been shown that discontinuation of immunosuppression is associated with higher risk of iris. a high index of suspicion for iris is necessary during immune recovery post-hsct especially when immunosuppression is being tapered in a patient with pre-existing infection. aggressive antiviral treatment (when available) and judicious immunosuppression are the keys to managing iris complications. posttransplantation lymphoproliferative disease (ptld) is a significant cause of morbidity and mortality in allogeneic stem cell transplant patients. identifying high risk patients, routine pcr screening, early diagnosis and therapy are crucial for successful management. patients and methods primary objectives of this study were to describe epidemiology of ebv associated ptld and to assess risk factors in our paediatric cohort. additionally, role of immunoglobulin (ig) levels as a possible diagnostic/prognostic marker was analyzed. between 1 january 1 2011 and 30 june 2016, 140 allogeneic transplantations were performed in 118 pediatric patients (82 boys and 36 girls) at our center. median age was 7.68 years (0.03-18). underlying diseases were hematological malignancies (68%), nonmalignant hematological conditions (13%), immunodeficiencies (11%) and others (8%). stem cell source was bone marrow (62%), peripheral blood (19%) and cord blood (19%). donors were unrelated (75%), sibling (18%), haploidentical (4%) or other matched family donors (3%). routine ebv pcr screening and ig level detection were performed weekly. rituximab prophylaxis was given only in nine cases. results ebv dnaemia was found in 16/118 patients (13.6%), while ptld was diagnosed in 11/118 patients (9.3%). all ptld cases were related to ebv infection, median of highest viral load was 11 790 copies/ml (688-6 670 000). diagnosis was confirmed by biopsy in 6/11 cases, further five fulfilled criteria of probable ptld (positive pcr with appropriate clinical symptoms). ptlds occurred at a median of day +48 (19-85) after transplantation. all patients received rituximab treatment along with a reduction of immunosuppressive therapy. four patients died of ptld (mortality 36%), all confirmed by autopsy. a higher incidence of male gender (10/11; 90.9% vs 67.3%), bone marrow graft (10/11; 90.9% vs 59.8%), hematological malignancy (10/11; 90.9% vs 67.3%) and second transplantation (5/11; 45.5% vs 14%) could be detected among ptld patients when compared to the non-ptld group. elevated igg, iga or igm levels were observed in 13/118 patients. nine out of 13 had positive ebv pcr testing, eight of them developed ptld. five of the ptld patients had monoclonal or biclonal immunoglobulin elevation, two of them died. in 3 cases, elevated ig level preceded the positive ebv pcr results by at least 1 week. conclusion: at our centre incidence and mortality of ptld was similar to published data. we observed a tendency that a higher representation of male gender, hematological malignancy, bone marrow graft and second transplantation could be confirmed in the ptld group however due to small number of patients, a correlation and statistical significance could not be calculated. elevation of immunoglobulin levels do not seem to be specific for ptld but in selected cases it could predict ebv disease earlier than pcr testing. disclosure of conflict of interest: none. autologous peripheral hematopoietic stem-cell transplantation is a procedure of a stem cell rescue with patients' own previously collected hematopoietic stem cells, after myelotoxic therapy. the purpose of stem cell reinfusion is to ensure adequate recovery of hematopoiesis, shorten the period of profound neutropenia and to reduce the risk of infections. the transplantation itself carries a moderate risk for infection but some patients have higher risk due to the nature of underlying disease, earlier treatment and in case of severe mucositis. for these reasons, all treated patients are in isolated clean rooms and receive ciprofloxacin, fluconazole and acyclovir prophylaxis. in the 3.5-year period, 177 autologous transplantations were performed. the patients were 20-72 years old, with median of 55.18 years. of all transplanted patients, 106 or 59.88% had multiple myeloma, 66 or 37.3% had lymphoma and 5 or 2.82% had acute myeloid leukemia. all of the patients received pegfilgrastim 6 mg on the first or the second posttransplant day. febrile neutropenia (ne o0.5 × 10 9 /l) was reported if patient's temperature was above 38.3°c in one measurement or above 38°c in two consecutive measurements. these patients were treated empirically with piperacillin/tazobactam 4.5 g four times a day with the addition of vancomycin in the case of severe mucositis or pulmonary infiltrates. in all cases blood and urine cultures were performed, as well as testing for seasonal flu. time to neutrophil recovery (ne40.5 × 10 9 /l) was 7-20 days, with a median of 10 days, and average of 10.25 days. febrile neutropenia was reported in 76 patients (42.94%) and in 39 (51.31%) patient's samples pathogen was isolated. gramm negative bacteria caused sepsis in 54.29% of patients. we had to change empirical therapy according to antibiogram in 37.2% patients. in 1 month follow-up period, there were two (1.12%) infection related deaths. our data on incidence of infections is consistent with literature data but large number of papers show satisfactory results of safety of patients discharged from hospital immediately after the autologous stem cell transplantation and who were treated at home during the phase of profound neutropenia. there is still an ongoing debate whether it is possible to conduct this procedure in such manner in our health system. disclosure of conflict of interest: none. fluconazole was equal to mold-active drugs in preventing early invasive fungal disease after allogeneic stem cell transplantation regardless of transplantation type y sun, j hu, h huang, j chen, j-y li and x-j huang there are still controversies that whether mold-active drugs is better than fluconazole in preventing invasive fungal disease (ifd) after allogeneic stem cell transplantation (hsct). we hypothesis that the optimal prophylaxis might be different in patients with different risk profile, such as in different time period after hsct or received alternative donor transplantation. in the prospective china assessment of antifungal therapy in haematological disease (caesar) study database, 661 out of 1401 patients received primary antifungal prophylaxis were analyzed. the ifd incidence of different time period after transplantation (early, late and very late) and survival were compared among different drug groups. in patients with fluconazole, itraconazole, voriconazole or micafungin prophylaxis, the overall incidence of ifd after transplantation were 7.2%, 12.6%,1.4% and 5.2%, respectively (p = 0.0379). however, there is no difference in early ifd (o40 days post hsct) among 4 groups of patients. the risk factors associated with occurrence of ifd were neutropenia duration 414 days (po0.01, or 3.73 (1.66-8.36)), adult (p = 0.02, or 3.37 (1.23-9.18)) and alternative donor (unrelated donor or haploidentical donor) transplantation (p = 0.01, or 5.88 (1.48-23.32)). in the sub-group analysis with only alternative donor (unrelated donor and haploidentical donor), it also demonstrated that fluconazole is equal to other mold-active drugs in preventing early ifd. patients received fluconazole prophylaxis has even better overall survival. the overall survival in patients with fluconazole, itraconazole, voriconazole or micafungin prophylaxis were 88.3%, 83.5%,78.9% and 72.4%, respectively (p = 0.0047). our current [p259] study suggests that fluconazole is equal to mold-active drugs to prevent early ifd in hsct patients, even in high-risk patients received transplantation from alternative donors. however, further prospective randomized study was warranted to confirm this conclusion. disclosure of conflict of interest: none. (9.5%) received autologous hsct and 38 (90.5%) allogeneic hsct. sixty-five out of 107 patients (60.7%) were affected by different haematological diseases: 36 by lymphoma, 11 by multiple myeloma, 7 by chronic lymphocytic leukaemia and 11 by others diseases including mastocytosis, amyloidosis and essential thrombocythemia. hbv reactivation prophylaxis prescribed was entecavir for 4 hbsag+ inactive carrier patients and prolonged lamivudine (lmv) course for 103 (96%) patients. in 6 patients (5.6%) lmv prophylaxis was withdrawn 12-18 months after the end of immunosuppressive therapy. eight out of 107 patients (7.4%) experienced hbv reactivation: 4 of them during lmv treatment and then they were switched to entecavir or tenofovir therapy, 4 patients reactivate hbv after lmv interruption (3.7%). in these patients reactivation was observed after an average time of 4 months (range: 3-6) after discontinuation of lmv prophylaxis. median duration of prophylaxis was 49 months (range: 40-60) after the end of immuno-suppression. three out of 4 patients (75%) underwent allogeneic hsct and 3 patients (75%) received rituximab. one out of 4 (25%) seroreverted in hbsag positive and hbsab negative status, with hbv-dna42000 ui/ml (table 1) . two patients out of 4 (50%) experienced hbv-dna detection below 20 ui/ml. disclosure of conflict of interest: none. [p260] [p260] the severity is measured on grades (grade 1: microscopic hematuria to grado 4: clots cause urinary tract obstruction). the treatment is based on support measures: hyperhydration, continuous bladder irrigation, instillation of topical agents and in severe cases must be performed a cystoscopy for clot evacuation. in the case of the presence of poliomavirus virus (bk virus) the use of cidofovir had been demonstrated in vitro studies to have activity against bk virus. we performed 42 allogenic transplants of which 10 are haploidentical from 2010 to october 2016. we realized a retrospective case study to analyses the experienced in the management of hc. results: of a total of 42 allogenic transplants realized, developed a hc: 1 patient received an identical hla transplant and the 4 patients remaining haploidentical allotrasplant. all cases were male, with an age range of 18-42 years. the status of the disease was: 3 were in complete remission and 1 had visible disease. 3 of the 4 patients received cyclophosphamide as immunosuppressive therapy and all patients received cyclosporine and mofetil micofenolate also. the onset of the symptomatology was between day 9 and day 82 post transplant and the range of duration was from 14 to 45 days. the four patients precised continuous bladder irrigation but because of the poor response they received instillation of hialuronic acid (4 doses). two patients required the use of cidofovir (3 doses). one of the four patients required urinary tract catheterization because of hydronephrosis and renal impairment. in our review we confirmed that this entity is more frequent in the haploidentical transplant and bkv is the most prevalent cause in the late hc. -the three patients received 3 doses of cidofovir (1mg/kg) without probenecid and had a good response. -three patients present acute renal failure associated to hc. the four patients needed bladder instillations with saline but they had poor response and received at least 4 doses of hyaluronic acid. disclosure of conflict of interest: none. hsv infection in allo-hsct setting is mostly reactivation of latent virus. hsv disease commonly presents as mucocutaneous lesions of the oral cavity. however some patients develop serious fatal visceral dissemination. prophylactic use of acyclovir has markedly reduced the incidence of hsv disease during the period of neutropenia after allo-hsct. in this study, our aim is to demonstrate the incidence, clinical outcome and risk factors for hsv disease in adult allo-hsct. between 2015 and 2016, 89 patients who underwent allo-hsct in our center were included to the study. all hsct candidates and donors were tested for hsv-1/2 immunoglobulin g (igg) antibodies prior to transplantation. all patients received acyclovir prophylaxis (related transplants 400mg tid, unrelated transplants 800mg tid) during conditioning and after allo-hsct up to 3 months. chlorhexidine oral solution as well as bioadherent oral protective gels was used for oral hygiene. all patients were followed for symptoms of reactivation. hsv1/2 igg seropositivity was detected in 66 recipients (74%) and 48 donors (54%). the distribution of hsv status was as follows: recipient and donor seropositive in 34 (38%), recipient and donor seronegative in 9 (10%), recipient seropositive and donor seronegative in 32 (36%), recipient seronegative and donor seropositive in 14 (16%) transplants. the median age of the patients was 41 (range: 18-67), 48 patients were male (54%) and 79 (89%) had malign disease. the stem cell source was peripheral blood in 73 (82%) patients and 48 (54%) received grafts from related donors. sixty four patients (72%) received myeloablative conditioning regimen. the most common graft-vs-host disease (gvhd) prophylaxis administered was cyclosporine (csa) and methotrexate (mtx) in 60 patients (67%). acute graft vs host disease was detected in 29 patients (33%).four patients from seropositive 66 patients (6%) had hsv reactivation, the patient characteristics are given in the table. all patients had hsv reactivation within 1 month of allo-hsct except one patient had symptoms at sixth month posttransplant when he suffered from oral gvhd. all patients s252 and donors were seropositive prior to allo-hsct and responded well to antiviral treatment. the incidence of hsv reactivation in allo-hsct was detected as 6% which is lower to previous studies. successful primary prophylaxis and oral hygiene might reduce the incidence. all patients were responded to antiviral treatment and no visceral dissemination was detected. disclosure of conflict of interest: none. patients who have received hematopoietic stem cell transplantation (hsct) may suffer, to some extent, losses in humoral and cell immunity against antigens to which they had been previously exposed naturally (infection caused by wild microorganisms) or artificially (through vaccination). the conditioning regimen for hsct replaces the patient's immune system and involves the loss of previous immunity. this study analyzed patients included in the vaccination program for hsct recipients in the salamanca health care complex during the period 2010-2016. we assessed the serological status prior to hsct for the following immunopreventable diseases (hepatitis b, hepatitis a, varicella), and the study after hsct also included measles, rubella and parotitis, prior to their inclusion in the hsct vaccination program. the study included 302 patients, 53.8% of which (n = 168) were men. 83.3% of the patients (n = 260) were allogeneic hsct recipients with an average age of 47 ± 16 years, and 16.7% (52) were autologous hsct recipients with an average age of 43 ± 16 years. prior to hsct, 40% of the patients showed immunity against hepatitis b (hbv antibodies 410 ui/l), 82.6% against hepatitis a (positive for hav igg) and 85% against varicella (positive for varicella igg). no statistically significant differences were observed regarding this variable hepatitis b anti-hbs 410 ui/l, hepatitis a, igg positive, varicella igg positive, measles igg positive, rubella igg positive, parotitis igg positive. table 1 compares the serological status before and after transplantation. in the pre-transplant serological study we observed that less than half of the patients are protected against hepatitis b, while over 80% of them are protected against hepatitis a and varicella. regarding the diseases in which we know the serological status before and after transplantation (hepatitis a, hepatitis b and varicella), we observed that most patients maintain immunity. in the case of rubella, measles and parotitis we only have access to the serological status after transplantation, and we observed that parotitis is the disease with the lowest seroprotection. therefore, vaccination would be indicated, just as in the case of hepatitis b. the clinical results support the need to adapt the vaccination schedule to the immunological status of the patients after hsct individually. disclosure of conflict of interest: none. impact of cumulative steroid dose on infectious diseases after allogeneic hematopoietic cell transplantation m watanabe 1 , j kanda, t kitano, t kondou, k yamashita and a takaori-kondo 1 after allogenic hematopoietic cell transplantation (hct), highdose steroids are used to treat transplantation-related complications such as graft-versus-host disease (gvhd). however, the use of high-dose steroids is associated with an elevated risk of infectious diseases. information on the association between cumulative steroid dose and infectious diseases after hct is scarce. a total of 238 patients who underwent their first hct in kyoto university hospital from 2005 to 2015 and survived at least 30 days after transplantation were included in this study. we analyzed the association between cumulative steroid dose used within 30 days after transplantation and the occurrence of infectious diseases, including invasive fungal infection (possible/probable/proven cases), cytomegalovirus (cmv) antigenemia, and bacteremia through 180 days after transplantation. sixty-three patients received transplantation from a related donor, 114 received unrelated bone marrow grafts, and 61 received unrelated cord blood units. their median age was 51 (range: 17-66) years and median day of neutrophil engraftment after transplantation was 21. patients were categorized into 3 groups according to their cumulative steroid dose within 30 days: no steroid administration (n = 183), low-dose cumulative steroid administration under 500 mg of prednisolone in total (n = 27), and high-dose cumulative steroid administration over 500 mg of prednisolone in total (n = 28). reasons for steroid administration were treatment for gvhd in 35 patients, engraftment syndrome in 11, and other reasons including lung complications in 9. the rate of invasive fungal infection was 6% (12 possible cases with pneumonia and 1 proven case of candida blood stream infection) and we found no apparent association between fungal infection and steroid use regardless of dose. cmv antigenemia was diagnosed in 41%, 67% and 60% of patients in the 3 groups respectively, and both low-dose and high-dose steroid groups were significantly associated with a high risk of cmv antigenemia (low-dose group, adjusted hazard ratio (ahr), 2.37, p = 0.004; high-dose group, ahr 1.84, p = 0.01). bacteremia was diagnosed in 9.8%, 11% and 21% of patients in the 3 groups, respectively. high-dose steroid use was a risk factor for bacteremia (ahr 1.70, p = 0.027). seven patients died from infection (fungal, 2; viral, 2; bacterial, 3). two of three bacterial infection-related deaths occurred in the highdose steroid group, although the number of events was too small to analyze. our data confirmed that steroid administration is itself a risk factor for cmv antigenemia and close observation to detect cmv antigenemia is mandatory for patients using steroids regardless of its cumulative dose. high-dose cumulative steroid use is a risk factor for bacteremia. contrary to our expectations, steroid administration showed no apparent association with invasive fungal infection in our study, perhaps because of its generally low incidence in our hospital. disclosure of conflict of interest: none. impact of different t-cell depletion techniques on the incidence of infectious complications after allogenic hematopoietic stem cell transplantation k aikaterini 1 , s federico 1 , d-l vu 2 , e boely 2 , c dantin 1 , a pradier 1 , y tirefort 1 , a-c mamez 1 , o tsopra 1 , c stephan 1 , y beauverd 1 , e roosnek 1 , s masouridi-levrat 1 , c van delden 2 , y chalandon 1 1 department of oncology, hematology unit, university hospital of geneva and 2 department of medicine specializations, infectious diseases, university hospital of geneva t-cell depletion (tcd), obtained by either in vivo antithymocyte globulin (atg) administration or ex vivo depletion, is a well-established strategy for graft-versus-host-disease (gvhd) prevention after allogeneic hematopoietic stem cell transplantation (hsct)1-4. however, the prolonged lymphopenia associated with tcd can result in increased incidence of disease relapse1 and infections. although many studies investigated the impact of tcd on disease relapse 1-4, little is known about the impact of tcd strategies on the incidence of infectious complications after allogeneic hsct. we retrospectively evaluated the incidence of infectious complications in 236 consecutive patients who underwent allogeneic hsct at our center from september 2010 to december 2015. 100 patients received tcd grafts obtained by in vivo atg administration as part of the conditioning regimen (atg group). 17 patients received partially tcd grafts obtained through incubation with alemtuzumab in vitro washed before infusion followed on day +1 by an add-back of donor t cd3+ cells5 (ptcd group). 60 patients received grafts tcd by both methods combined. 59 patients did not receive any form of tcd (no-tcd group). cumulative incidence estimates of infectious complications were calculated and compared using the gray test. given the increased risk of infection associated with gvhd and its treatments, gvhd or death from other causes were defined as competitive events in the analysis. we didn't observe any significant difference in the 1-year cumulative incidence of bacterial infections in patients receiving tcd by atg (45% (95% ci 35-54.5%)) ptcd (58.8% (95% ci 31.2-78.5%)) or both (55% (95% ci 41.4-66.7%)) compared with patients receiving no tcd (52.5% (95% ci 38.9-64.5%)). similarly, the 1-year cumulative incidence of viral infections or reactivations was comparable in patients receiving no-tcd grafts (80.3% (95% ci 66.8-88.8%)) compared with patients receiving tcd grafts (atg: 82.2% (95% ci 72.9-88.6%); ptcd: 76.5% (95% ci 45.7-91.2%); atg+ptcd: 81.7% (95% ci 68.9-89.6%)). finally, no significant impact of tcd was observed on 1-year cumulative incidence of fungal (no-tcd: 6.8% (95% ci 2.2-15.2%); atg: 18.1% (95% ci 11.2-26.3%); ptcd: 11.8% (95% ci 1.8-31.9%); atg+ptcd: 18.3% (95% ci 9.7-29.1%)) and parasitic (no-tcd: 1.7% (95% ci 0.1-8%); atg: 1% (95% ci 0.1-4.9%); ptcd: 5.9% (95% ci 0.3-24.2%); atg +ptcd: 1.7% (0.1-8.3%)) infections. image/graph: 1-year cumulative incidence estimates of infectious complications depending on the tcd strategy employed. the results of our retrospective analysis indicate that the cumulative incidence of bacterial, viral, fungal and parasitic infectious diseases are similar in patients receiving tcd grafts compared to those receiving no-tcd graft, suggesting a favorable toxicity profile of different tcd strategy in respect of infections. these results should be confirmed by similar analysis in large scale, prospective clinical trials assessing the potential benefits of tcd on transplantation outcomes. 57 patients with aml were considered eligible for hsct, 7 died before transplantation. 13 patients (26%) underwent transplantation from hla-identical sibling, 13 (26%) from haploidentical family donor and 19 (38%) from matched unrelated donor, while 5 patients (10%) received unrelated cord blood cells. twenty (35%) out of 57 eligible patients have had an ifi episode before transplant: 6 were proven, 4 probable and 10 possible; (9 (47%) pneumonia, 4 (19%) gastroenteritis, 3 (15%) sinusitis, 2 (10%) candida sepsis, 1(5%) meningitis and 1(5%) cutaneous abscess were registered). five (25%) out of 20 patients with a previous ifi and 2 (5%) out of 37 without previous ifi did not receive hsct (or 5.83 95% ci 1.02-33.96, fisher test p: 0.04). the majority (55%) of patients with a previous ifi waited hsct more than 6 months from the date of eligibility in comparison with those without a previous ifi (55% vs 30%; or 0.37, 95% ci 0.12-1.13, p-value 0.08 fisher test) overall a post transplant ifi episode was diagnosed in 13 (26%) of transplanted patient; 4 (26%) had a relapse of a past ifi vs 9 (25%) of the patients without a previous ifi who had a new episode. (or 1.53, 95% ci 0.39-5.90, p-value 0.4 yates test).a higher number of patients with ifi (10 out of 15, 66%) respect to those without a previous ifi (10 out of 35, 30%) died in a median time of 160 days(range: 22-480 ) after hsct. furthermore, those who had a previous ifi had a lower median survival (317 days (range: 22-1095)) compared to patients without a previous ifi (530 days period (range: 20-1490)) (student's t-test p: 0.014)). a previous ifi episode in the pre transplant period slows and limits the accessibility to hsct, and is significantly associated with an increased mortality. disclosure of conflict of interest: none. s254 delayed immune reconstitution has been described for haploidentical hematopoietic stem cell transplantation (hsct) compared to conventional hsct, nevertheless the incidence of invasive aspergillosis infections (iai) in haploidentical sct and the efficacy of primary prophylaxis are not well defined. our objective is to describe the incidence, risk factors and mortality of iai in our patients, using as prophylaxis micafungin during the conditioning and neutropenia period, switched to posaconazole or voriconazole when oral intake is feasible. we retrospectively analyzed 40 consecutive patients from 2014 to 2016 who received haploidentical grafts: unmanipulated for 20 adults, tcrab depleted in 6 children and cd45ra depleted in 14 children. the stem cell source was peripheral blood in all cases. adults (22-70 yo) were treated for aml/mds (n = 8), all (n = 2) and lymphoma (n = 10). children (6 mo-15 yo) were treated for aml (n = 6), all (n = 9), aplastic anemia (n = 2) and immunodeficiencies (n = 3). conditioning regimen was bu-flu-cy (n = 18, adults), thio-bu-flu (n = 2, adults), flu-mel-thio for all pediatric patients; atg was used in 6 children and tli in 14 children. median follow up was 12 months (1-30) for adults and 7 months (1-28) for children. we used eortc criteria for iai and analyzed probable or definite as cases. there were 6 events of iai, with a bimodal presentation: 3 events (7.5%) during neutropenia period and 3 (7.5%) after 6 months of hsct ( figure 1 ). five of them were probable and one definite (aspergillus niger). site of infection was mainly pulmonar; cns was suspected in two adult patients and skin was proven in one adult patient. all 3 patients at the late period had chronic gvhd at diagnosis. one patient had primary graft failure. severe cmv disease (hepatitis and colitis) was present in one adult. mortality related to iai was high (5/6), patients died at a median of 35 days. figure 1 . iai patients characteristics the global incidence of iai in haploidentical hsct is similar to conventional hcst. primary prophylaxis with micafungin switching to oral triazole is successful (92.5%) during the early period. late cases (7.5%) had clearly known risk factors (chronic gvhd, steroids and cmv), and primary prophylaxis had been modified due to toxicity or interactions. iai mortality in our patients is very high (84%) despite effort in prophylaxis, diagnosis and treatment. visceral intractable abdominal pain prior to skin lesions from herpes zoster can be misdiagnosed as gvhd post stem cell transplantation which may lead to initial increase in immunosuppression and hence high mortality if we don't suspect. case report and literature review through pubmed results: 13year-old male with relapsed all post mud pbsct (10/10) transplant in following cy tbi atg conditioning presented at day +117 with intractable diffuse abdominal pain with constipation. no history of nausea, vomiting or skin rash. on physical examination his abdomen was soft, diffuse tenderness but no rigidity, muscle guarding and rebound tenderness. laboratory tests including liver function test, amylase, lipase were normal. usg abdomen and mri abdomen showed no abnormalities, except for presence of fecolith. during the stay his pain worsened needing morphine infusion, pca and later ketamine. he had previous history of acute gut gvhd controlled on budesonide and cyclosporine which was later being weaned once his symptoms were controlled. in view of previous history of gvhd, gi consultation was sought and he underwent ugi endoscopy and biopsy which was non-significant. on day 7 of his admission he developed a pustular skin lesion on thigh and scrapping from that showed vzv and his blood pcr was also positive, he was started on intravenous acyclovir. his lesions improved and crusted and his abdominal pain subsided after 72 h of acyclovir and was discharged on oral acyclovir after 14 days of intravenous therapy. review of literature illustrated in table 1 . severe abdominal pain in patients who received an allogeneic stem cell transplant has a broad differential. here we describe a case of vzv presenting with intractable abdominal pain needing opioids. because of the poor prognosis and life-threatening nature of disseminated vzv disease, it should be considered and included in the patient's workup. intravesical cidofovir (5 mg/kg, diluted in 90 ml sterile water) was once weekly applied until symptom control for 60 min. via a transurethral catheter, i.v. cidofovir was initiated if no symptom control was achieved after 3 local applications. in patients with hc 3 or 4 a lavage catheter was added. bkv cystitis (dysuria (n = 8) or dysuria combined with hematuria (n = 10)) developed in 18 out of 152 transplants (12%). median age was 54 years, 89% were female and 50% received a mismatch transplantation after 1 mac or 17 ric conditioning regimens. in 67% of bkv cystitis cases also cmv reactivation within the first 180 days could be detected. 67% had acute gvhd ii°-iv°at the onset of bkv cystitis and 83% received steroid medication. the median time to symptom occurrence was day +40 after hsct (iqr 25-75: 31-136). 5 patients (3 with dysuria and one either hc 1°and 2°) didn´t require therapy due to self limiting symptoms. 3 (23%) of 13 treated patients showed only dysuria, 3 (23%) hc 1°, 5 (38%) hc 2°, 1 (8%) hc 3°and 1 (8%) hc 4°. the first patient was treated with i.v. cidofovir twice and symptoms relieved. all the following 12 patients were exposed to intravesical cidofovir as 1st line therapy. 8 patients (67%) achieved a complete remission with a median of 2 intravesical procedures (range: 1-3). 1 patient showed symptom improvement and all 9 patients didn´t require further therapy. 3 patients had to be switched to i.v. application due to bladder spasms during intravesical application (n = 1) or to insufficient symptom control (n = 2). 2 out of these 3 responded to i.v. treatment, whereas 1 patient receiving 2nd transplant didn´t respond at all. in patients with spontaneous symptom relieve the median bkv concentration at the time of symptom onset was 2 log lower compared to those requiring antiviral therapy. local therapy reduced bkv viruria by 2 log. pain during cidofovir instillation in 50% of patients was the only significant side effect of local therapy compared to creatinine increases by 450% in 66.6% of i.v. treated patients. intravesical treatment of symptomatic bkv cystitis with cidofovir (5 mg/kg) is safe and effective with an 75% symptom improvement rate and no systemic side effects. in patients without sufficient symptom or bleeding control i.v. cidofovir is still an option, which however induces significant renal toxicity. we therefore recommend intravesical cidofovir as 1st line therapy in case of dysuria or hematuria induced by bkv after hsct. disclosure of conflict of interest: none. haemorrhagic cystitis is a recognised complication of stem cell transplant (sct), with a reported incidence of 5-25% of cases (1) . the majority of cases are associated with bk polyomavirus (bkv), and less often adenovirus and cytomegalovirus. there are a lack of high quality studies on the optimal prevention and management of haemorrhagic cystitis. treatment options are restricted by conditioning toxicity, immunosuppression and other co-morbidities such as renal impairment. cidofovir has an inhibitory effect on bkv replication and has been used extensively in the treatment of haemorrhagic cystitis. however, severe nephrotoxicity limits routine intravenous use in sct patients. alternative options include using low dose intravenous cidofovir or intravesical administration. we conducted a retrospective case review of post sct patients presenting with bk virus associated haemorrhagic cystitis in our institution between january 2010 and november 2016. we identified 5 patients in total (4 male,1 female). the indications for stem cell transplant were as follows: severe aplastic anaemia 1 high risk aml 1 relapsed aml 1 relapsed all 2 onset of symptoms (haematuria and painful micturition) ranged from day − 4 to day +27, and the time to resolution of symptoms varied from 16 days to 68 days. four of the patients were treated with intravesical cidofovir only, with the number of doses required varying from 3 to 7. one patient received combination treatment with both intravenous (7 doses), and intravesical cidofovir (5 doses). all patients had a good clinical response with complete resolution of symptoms and no major complications. however, the level of bk virus in the urine did not always correlate with clinical response. some of the patients did not tolerate urethral catheterisation and required a general anaesthetic for the placement of the urethral catheter; 1 patient required a supra-pubic catheter. currently 3 out of 5 patients are alive and well; 2 patients died from causes not related to bk virus associated haemorrhagic cystitis. our experience shows that intravesical administration of cidofovir is a safe and effective option for the treatment of bk virus associated haemorrhagic cystitis. an allogeneic stem cell graft from a cytomegalovirus (cmv) seronegative donor puts recipients at high risk of cmv reactivation which can lead to cmv disease and mortality. based on the immunogenicity of cmv phosphoprotein 65 (cmvpp65) we initiated a clinical phase i trial with a novel vaccine designed by our group: a cmvpp65-derived peptide in water-in-oil emulsion (montanide) plus administration of granulocyte-macrophage colony stimulating factor. ten patients received four vaccines s.c. at a biweekly interval after allogeneic stem cell transplantation. we monitored the patients for their clinical outcome and cmvpp65 antigenemia. multi-color flow cytometry test were performed to assess cmvspecific cd8+ and gamma-delta t cells. novel neutralizing anti-cmv antibody assays were established and correlated to clinical parameters. findings: in general, patients tolerated the peptide vaccination well, no drug-related adverse events others than rash or induration at the site of injection were detected. seven of nine patients with cmvpp65 antigenemia cleared the cmv after four vaccinations and were hitherto free from antigenemia. two patients with cmv reactivation showed persisting cmv antigenemia. one of these two refractory patients received additional four injections and remained hitherto free from cmv antigen. another patient obtained a prophylactic vaccination and did not develop antigenemia. an up to six-fold increase in frequency of both cmv-specific cd8+ t cells or vdelta2-gamma-delta t cells was detected in five patients. moreover, titers of neutralizing antibodies increased in four patients up to 10-fold over the time of vaccination. humoral and cellular immune responses correlated with clearance of the cmv load. cmvpp65 peptide vaccination was safe and well tolerated in patients after allogeneic stem cell transplantation at high risk for cmv reactivation. the vaccine showed encouraging immunological and clinical results. a prophylaxis study using the vaccine in solid-organ transplant patients is ongoing. disclosure of conflict of interest: none. sporopachydermia cereana is a rare yeast found in necrotic cactus tissue, predominantly in the americas. infection in humans has only been reported in 4 neutropenic patients with fatal course, either directly from the pathogen or other complications of immunosuppression. treatment is complicated by difficulties in pathogen-identification with conventional diagnostic techniques and by resistance to echinocandins. here we present a patient with acute myeloid leukemia (aml) and s. cereana infection. this is the first patient who was successfully treated with antifungal therapy and who survived s. cereana infection. case presentation we present the case of a 50-year-old female patient who was diagnosed with normal karyotype aml with dnmt3a and idh2 mutations in december 2015. she achieved complete remission after two cycles induction chemotherapy. during the 2 nd induction cycle the patient developed persistent fever in neutropenia despite broad-spectrum antibiotics and the replacement of prophylactic fluconazole to caspofungin. blood cultures showed growth of s. cereana, shown to be sensitive to azoles (mic fluconazole o1 mg/l, mic voriconazole o0.12 mg/l) as well as amphotericin b (mic o0.25 mg/l), but resistant to caspofungin (mic44 mg/l). following the susceptibility profile the treatment was changed first to liposomal amphotericin b, and with the availability of mic results to voriconazole. metastatic fungal infection (that is, endocarditis, endophthalmitis, hepatosplenic candidiasis) was excluded. after regeneration of peripheral blood values the treatment was switched to oral voriconazole. a ct scan of the chest and abdomen prior to allo-hsct after 6 weeks of treatment with voriconazole revealed new multiple necrotic mesenteric lymph nodes. an ultrasound-guided biopsy of a node revealed no growth on fungal cultures, a grocott stain revealed no hyphae or spores. a panfungal pcr of an its (internal transcribed spacer) fragment revealed fungal dna, which could be confirmed as s. cereana. at this time the level of voriconazole in serum was found to be sub-therapeutic (0.4 mg/l), and the dosage was increased accordingly. subsequent ct scans 4 and 6 weeks later revealed a regression of the affected abdominal lymph nodes. in the further course non-myeloablative conditioning with fludarabine and busulfan prior to allo-hsct using pbsc from her hla-matched brother was performed. under prophylaxis with cyclosporine, methotrexate and antithymocyte globulins (atg) graft-versus-host disease (gvhd) remained absent. the allo-hsct was performed under voriconazole treatment with no further complications and the patient engrafted at day 20. the treatment was changed to fluconazole 400 mg daily before discharge. due to the complete radiological regression of the infection in follow-up scans and excellent general condition of the patient 5 months after hsct, fluconazole was discontinued. the patient remains in morphological complete remission 6 months after hsct and has a 100% donor chimerism. the first published case of survival of infection with s. cereana exemplifies the continual progress made in treating infections in the severely immunocompromised patient. diagnosis via its sequence-analysis seems reliable but a high index of suspicion is required for neutropenic patients who do not respond well to standard antimycotic therapy. the increased availability of the technology may lead to more frequent diagnoses in the future. disclosure of conflict of interest: none. neutropenic enterocolitis (ne) is a clinical syndrome characterized by fever and abdominal pain in patients who received chemotherapy for hematological malignancies and who treated with stem cell transplantation (sct). the aim of this study was to determine the incidence, risk factors and outcome of ne after autologous sct (auto-sct). we retrospectively evaluated 226 patients with non-hodgkin lymphoma (nhl), hodgkin lymphoma (hl) and multiple myeloma (mm) who underwent auto-sct between january 2013 and december 2016 in our center. patients with lymphoma were conditioned with carmustine, etoposide, cytarabine, melphalan (beam) or thiotepa, etoposide, cytarabine, cyclophosphamide, melphalan (tecam). patients with multiple myeloma were treated with melphalan as conditioning. diagnosis of ne was established in case of neutropenic fever, abdominal pain or diarrhea, and bowel wall thickening 44 mm on abdominal ultrasonography. febrile neutropenia was seen in 199 (88%) patients of all. the median time from transplantation to neutropenia was 4.5 days (range: 0-9 days). ne occurred in 22 (9.7%) in all neutropenic patients. the median time to ne after auto-sct was 7 days (range: 2-9 days). the median neutrophil engraftment time was 12.5 days (range: 9-18 days). abdominal pain was seen in all patients with ne. twenty one patients (95%) had diarrhea. ileus was seen in 1 (4.5%) patient and septic shock was developed in 3 (13.6%) patients. five (22.7%) of 22 patients had bloodstream infection. klebsiella pneumoniae in 1, pseudomonas aeruginosa in 1, escherichia coli in 1, staphylococcus aureus in 1 and coagulasenegative staphylococcus in 1 patient were documented in patient's blood stream. early diagnosis was made by abdominal ultrasonography in all patients at a day of median 7 days (range: 2-9). twenty (91%) patients were resolved completely with good supportive care and proper antibiotherapy. two (9%) patients died of septic shock and ileus. ne is a rare but serious complication in patients underwent high dose chemotherapy followed by auto-sct. gramnegative bacteria are the main causative pathogens. abdominal ultrasonography is the simple, cheap, fast diagnostic and noninvasive procedure that allows the early diagnosis and effective treatment. disclosure of conflict of interest: none. [p274] neutrophil transfusions in the treatment of neutropenic patients submitted to allogeneic hsct: possible role on graft failure s giammarco, p chiusolo 1 , l laurenti 1 , f sorà 2 , n piccirillo 2 , l teofili 2 and s sica 2 1 hematology department, università cattolica del sacro cuore and 2 hematology departement, università cattolica del sacro cuore granulocyte transfusions (gtx) from g-csf-stimulated donors have been shown to increase the absolute neutrophil count (anc) before expected haematopoietic recovery in neutropenic patients after chemotherapy or haemopoietic sct. thus gt offers a therapeutic option along with antimicrobial agents and growth factors to improve clinical outcome of neutropenic patients with severe infections. the primary limitations of gt include low component cell dose and leukocyte incompatibility. the transfusion of g-csf-mobilized, hla-matched granulocyte components resulted in sustained anc increments, but the efficacy of this procedure has not been established by convincing randomized control trials. aim: we focused our attention on gt in the setting of allogeneic hsct, in particular on the feasibility and safety of this procedure on the rate of engraftment. between 2006 and 2016 our centre performed 211 allogeneic hsct. we analyze data from 59 transplanted patients receiving gt at some point during their disease. indication for gt was severe sepsis mainly due to mdr gram-bacteria. patients received a median of 4 gt (1-34), in different phase: 24 patients during induction therapy, 18 during hsct, 9 at diagnosis and during hsct and 8 after hsct. patients' characteristics are summarized in table 1 . median cd34+ cells dose was 6.4 × 10 6 /kg (range: 1.2-24). donor source was in 49 patients g-csf mobilized peripheral blood, 6 bone marrow and 4 cord blood. median neutrophil recovery (4500/mmc) was 16 days and platelet recovery (420 000/ mmc) was 14 days. sepsis were documented in 36 pts and 45 pts developed fuo. relapse was documented in 21 pts (35%). twenty-two pts are still alive and in complete remission (37%), death occurred in 37 pts: 19 due to trm and the remaining 18 for disease relapse. graft failure occurred in 16 of the 211 pts submitted to hsct. among the 11 patients (18%) who experienced graft failure, six (54%) received gt before hsct, because of sepsis during the induction therapy, and the remaining 5 after hsct, during aplasia period. in the remaining group (152 pts) not receiving gt, only 5 (3%) graft failure were observed. thus a statistically difference (p = 0.0005 fisher's exact test) increase in the rate of graft failure was detected in patients receiving gt. the role of gt in the treatment of infections in neutropenic patients remain still unclear for several reasons including the lack of clinical trials convincingly and consistently demonstrating efficacy, by availability of gt donors and by center's experience. gt has been successfully used in our center in patients with severe sepsis from mdr gram-bacteria during severe neutropenia but an increase number of graft failure has been registered in patients subsequently receiving hsct. alloimmunization to hla antigens in patients receiving gt might lead to an excess of graft failure requiring hla antibodies detection and attempt to reduce titer prior to hcst and maximizing stem cell dose. disclosure of conflict of interest: none. viridans streptococci are microorganisms frequently isolated from blood cultures of patients undergoing myeloablative allogeneic hematopoietic cell transplantation (allohct). poor dentition status has been associated with an increased risk of streptococcal bacteremia in the immediate post-allohct neutropenic period. the objective of this study was to evaluate the impact of oral health status on bacteremia risk in a cohort of patients undergoing therapy for acute myeloid leukemia (aml). a retrospective study was conducted in patients with aml treated at dana-farber/brigham and women's cancer center (df/bwcc) from 2007 to 2011. there was no formal dental assessment prior to aml induction therapy. all patients underwent protocol directed pre-allohct dental evaluation that included a standardized examination, comprehensive dental radiographs, and detailed treatment planning guidelines. poor oral health status was defined as presence of acute or chronic odontogenic infection, and it was assumed that oral health status at the time of induction therapy was the same as the pre-allohct evaluation findings. oral health status at the time of allohct was determined by the completion of required dental treatment. positive blood cultures were recorded from aml induction to day +60 post allohct. organisms that caused bacteremia were classified as 'of possible oral source' by a blinded microbiologist. two-sided fisher's exact test was used to compare the oral health status of the entire cohort to patients with blood cultures of potential oral source. from january 2007 to january 2011, 181 patients with aml underwent myeloablative allohct at df/bwcc and were s260 followed through today +60, and of these, 92 patients met the inclusion criteria and were included in the cohort. the median age was 48 years (range: 24-66) and there was similar distribution of genders. the most common aml induction regimen was daunorubicin and cytarabine (63/92; 68%) and of those that received consolidation therapy (49/92; 53%), almost all patients were treated with cytarabine. nearly all patients (90/92; 98%) received cyclophosphamide and total body irradiation for allohct conditioning and the majority of patients (83/92, 90%) received tacrolimus/methotrexate (n = 51) or tacrolimus/sirolimus (n = 32) for gvhd prophylaxis. over half of patients (51/92, 54%) experienced mucositis during their course of therapy for aml. pre-allohct dental evaluations were completed in 91/92 (99%) of patients. of the 13/91 (14%) patients identified as having poor oral health status, 13/13 (100%) completed all required dental treatment prior to allohct. bacteremias occurred in 63/92 (68%) patients, and 12/63 (19%) had positive blood cultures of potential oral source. of the 12 patients with positive blood cultures of potential oral source, 1/12 (8%) patient developed bacteremia during induction and 11/12 (92%) patients developed bacteremia during allohct. of the 13/91 (14%) patients identified as having poor oral health status, one patient (1/13; 8%) had a positive blood culture with a bacteria of potential oral source during induction/consolidation (p = 0.68). oral health status was not associated with risk of bacteremia of potential oral source at either aml induction/consolidation or allohct. risk of such bacteremia in the setting of myeloablative allohct may be related more to overall gastrointestinal translocation. disclosure of conflict of interest: none. is one of the main alternatives to trimethoprimsulfamethoxazole (tmp-smx) for prophylaxis of pneumocystis pneumonia (pcp)(maertens et al. jac 2016). ato is less effective than tmp-smx to prevent pcp1 but the reasons of this lower efficacy are not well understood. ato acts on pneumocystis, plasmodia and toxoplasma species by inhibiting mitochondrial pyrimidine biosynthesis. ato is highly lipophilic and its absorption in volunteers is improved by a fatty meal. there is a wide inter-individual variability in bioavailability and many drug interferences. the aim of this study was to assess the plasma concentrations of ato in patients under pcp prophylaxis with ato oral suspension and explore the factors which might impact its bioavailability. all adult patients receiving ato for pcp prophylaxis in the hematology and clinical immunology wards between may and september 2016 were included in the study. the prescribed dose was 750 mg of oral suspension twice a day. blood samples were collected around 12 h after the evening dose (cmin) and 1-5 h after the morning dose (cmax). plasma was immediately separated after each sample and frozen at − 20°c until proceeding to the assay. ato plasma levels were measured by uv-high-performance liquid chromatography. clinical and biological data, exact timing and modalities of intake (during a meal or not), and concomitant medications were collected. cmin and cmax results are presented as median (iqr 25-75%) and compared by mann-whitney u-test or signed rank test when appropriate. patients: a total of 85 measurements were performed in 33 patients (allogeneic hsct patients: 19; hematology non-transplanted patients: 6; hiv-infected patients: 7). the mean age (range) was 53 years (33-75), the m/f ratio was 21/12. only two patients were neutropenic. the median cmin was 11.3 μg/ml (6.2-27.8) and the median cmax was 13.4 μg/ml (6.0-28.3). thirteen of the 33 (39%) patients had a cmin. disclosure of conflict of interest: none. presepsin as a marker of infectious complications during high-dose chemotherapy following autologous hematopoietic stem cell transplantation in lymphoma patients y dubinina, v sarzhevskiy and v melnichenko national pirogov medical surgical center lymphoma patients, who undergo high-dose chemotherapy following autologous hematopoietic stem cell transplantation (autohsct), are at high risk of developing infectious complications (ic). mortality from ic during the transplantation, according to various data ranges from 12 to 42%. thus the development of models of early prognosis of ic during autohsct has become more urgent. it's reasonable to include the dynamics of biochemical markers of inflammation in these models. presepsin (psp), procalcitonin (pct) and c-reactive protein (c-rp) were assessed on the day of admission to the hospital (da), on d+1, d+3, d+7 and on the day of discharge (dd). if patients developed neutropenic fever (nf), the markers were assessed at the beginning of the fever, 6 h after, then on the second, third, fourth days after. there were 100 patients included in the study: 41 patients with hodgkin lymphoma, 27with non-hodgkin's lymphoma, 32with multiple myeloma, out of 100 patients there were 51 women and 49. the median age was 41 years (18-66). the conditioning regimens were cbv, beeac or hd melphalan. depending on the presence of ic, the patients were divided into 2 groups: group 1patients without infectious complications (n = 31), group 2patients with the development of infectious complications (n = 69). the median of the nf development was 5.5 days. 53 patients from group 2 had no microorganism growth in blood stream, either in repeated studies. gram+ flora was detected in 12 patients, 1 patient had gram-, 2 patients had mixed flora and 1 patient had pneumocystis jirovecii infection with respiratory insufficiency grade 3. significant differences in psp level between groups 1 and 2 were determined on d+3, on d+7 and the dd after autohsct. considering the median day of the nf appearance (5.5 days), it's supposed both the prognostic value (differences on d+3, that is, 2 days before the clinical manifestation of infection) and the diagnostic value of psp (differences on d+7 and on the dd) ( table 1 , graph 1). [p278] disclosure of conflict of interest: none. hc is often a serious complication and occurs in 70% of allo-hsct recipients. early bleeding is usually the result of chemotherapy toxicity however late occurring hc is multifactorial. bk virus infection has been shown to be related with hc. most studies demonstrate bk virus at the time of bleeding therefore not allowing the risk imposed by asymptomatic infection to be estimated. in this study, our aim is to show the effect of risk factors as well as pre-transplant bk viral load in asymptomatic recipients on development of hc in allo-hsct. between 2014 and 2016, we prospectively evaluated 59 allo-hsct. in order to detect the bk viral load, we performed quantitative bk virus pcr (altona diagnostics, germany) from blood samples at days 0, 30, 60 and 90 after allo-hsct. informed consents were obtained from all participants. bk virus pcr was considered positive if any number of copies were detected above the analytical sensitivity of the tests. the patients were monitored for signs and symptoms of hs. the risk factors for the development of hs were evaluated by univariate and multivariate analysis. p o0.05 was considered statistically significant. the median age of the group was 41 (range: 22-71), 18 of the patients (31%) were aged 450. male to female ratio was 1.36 (34/25). fifty two patients (88%) had diagnosis of malign hematological disease. stem cell source was peripheral blood in 51 (86%), bone marrow in 8 (14%) allo-hsct. patients received stem cells from 26 related donors (44%) vs 33 (56%) unrelated or haplo donors. myeloablative conditioning was administered in 47 patients (80%). forty-four of the conditioning regimens (75%) included cyclophosphamide. hc was diagnosed in 22 patients (37%) at a mean of 100 days (range: 0-367), early hc was detected in 4 of 22 patients (18%). the frequency of bk viremia and number of viral copies are given in detail in table. the frequency of bk viremia increases during transplantation in relation to clinical hc (66%, 66%, 87%, 100%; p = 0.007). acute graft vs host disease (agvhd) was diagnosed in 37 patients (63%) at a median time of posttransplant day 67: grade i-ii gastrointestinal/skin/liver in 31 (84%), grade iii-iv gastrointestinal/ skin/liver in 6 patients (16%). the most common gvhd prophylaxis preferred was cyclosporine and methotrexate in 50 patients (85%). in univariate and multivariate analysis (age450, sex, diagnosis, stem cell source, donor type, conditioning regimen, agvhd, cy administration, bk virus pcr at days 0, 30, 60) bk virus titer positivity at day 0, 30, 60 (p = 0.008, p o0.001, p o0.001), myeloablative conditioning (p = 0.018), the presence of agvhd after day 30 (p = 0.018) and conditioning regimen that includes cyclophosphamide (p = 0.024) are found to be related with increased risk of hs. patients with hc and clots were treated with continuous bladder irrigation as well as 8 of patients with bk viremia received cidofovir and six of them responded to treatment (75%). our study showed that, bk titer positivity, myeloablative conditioning, presence of agvhd, cyclophosphamide containing conditioning are associated with hc. detection of bk viremia in later transplant period is more sensitive for clinically proven hc. prophylactic treatment might be considered in patients with asymptomatic bk viremia in pretransplant period. [p279] disclosure of conflict of interest: none. this project has been granted by ankara university scientific research committee numbered as 15b0230007. high-dose chemotherapy with peripheral blood progenitor cell (pbpc) collection followed by a myeloablative conditioning and autologous stem cell transplantation (asct) is considered the standard of care of relapsed/refractory non hodgkin/hodgkin lymphoma (nhl/hl). a widely adopted conditioning regimen is the combination of carmustine etoposide cytarabine and melphalan (beam), whose feasibility and efficacy has been largely demonstrated. high dose fotemustine plus etoposide, cytarabine and melphalan (feam) has in some cases replaced beam conditioning. neutropenic enterocolitis (nec) is a life threatening complication of patients (pts) treated with chemotherapy (cht) with mortality rate up to 50%. it's a clinical syndrome in neutropenic patients (pts) characterized by abdominal pain (ap), fever (f) and diarrhoea (d). ultrasound (us) was used to evaluate bowel-wall thickening (bwt), and 44 mm is considered diagnostic of nec. early diagnosis is crucial to start conservative medical management (cmm), which appears the optimal strategy for most cases. objective: 1. to evaluate if nec incidence and outcome differs in beam vs feam and 2. to evaluate prospectively if bed-side-us (bus) can detect early signs of nec and guide a prompt treatment (cmm or surgical) in order to reduce mortality. in the last 5 years all pts with nhl/hl admitted in our bmt unit wards at university of pisa (italy), undergoing asct were prospectively enrolled. abdominal us was performed, baseline before treatment, and as only one symptom (or a combination) appeared within 12 h from onset: f and/or d and/or ap in cht-related neutropenic pts. 95 pts were conditioned with beam and 52 pts with feam. nec was diagnosed in n = 19/52 feam and in n = 25/95 beam patients. incidence was 36% and 25% respectively, without a statistically significant difference (p = 0.234). two pts died/19 in feam arm (10.5%) and 2pts/24 in beam arm (8.3%), without a statistically significant difference (p = 0.778). at time of diagnosis (dx) symptoms were: f+ap+d 45%, f+d 4%, f+ap 3%, ap+d 35%,d 3%,ap 10%. f alone was never present at diagnosis of nec. at dx, f was absent in 18/44 nec episodes (40%). all pts were treated promptly as bus allowed diagnosis with cmm except one 1 pts who underwent surgery, guided by us features, during neutropenia. the likelihood of nec dx in a discriminant st model (bayes theorem) for pts with bwt and ap = 98.8%, ap+d = 99.9%, ap+d+f = 100%, ap+f = 99.9%, d+f = 5%. bus allowed to detect early signs of nec and to start prompt treatment in this life threatening complication, of nhl/hl pts undergoing asct. this is a prospective study thus the true incidence of nec in nhl/hl undergoing asct should not be underestimated. there is not a statistically significant difference in incidence and outcome of nec in pts conditioned with beam in respect to feam. with bus pts do not live the isolation room. fever is not a condition sine qua non for nec diagnosis. early diagnosis allows most of pts to be treated with cmm. images of bus and ct were superimposable with lower costs, and less radiation exposure. a low mortality rate in pts with a 25-36% chance of developing this life threatening complication suggests that a prompt bus in neutropenic patients as just one symptom presents allows to make early diagnosis of this life threatening complication and guide prompt treatment (conservative or surgical), reducing mortality. disclosure of conflict of interest: none. quantiferon-cmv in the evaluation of cmv-specific immunity after autologous and allogeneic hsct j moreno 1 , ltesta 1 , l zanetti 1 , l serra 1 , b pereira 2 , m souza 1 , a carolina souza 2 , mp souza 1 , vr colturato 1 and cm machado 1,2 1 hsct program, amaral carvalho foundation and 2 virology laboratory, institute of tropical medicine, university of são paulo cytomegalovirus (cmv) is a major cause of morbidity and mortality after allogeneic hsct. the same is not observed in autologous hsct recipients who do not need to receive immunosuppression after transplantation. in the present study, we compared the reconstitution of cmv-specific immunity in autologous and allogeneic hsct recipients. patients were invited to participate in the study and signed the informed consent. cmv surveillance with the antigenemia (ag) test (cmv brite, biotest, germany) was done weekly in the first 3 months of transplant in allogeneic hsct recipients. preemptive ganciclovir therapy was initiated whenever a positive antigenemia was detected. the presence or absence of cmvimmunity was determined by a commercial interferon (inf) gamma release assay (quantiferon cmv, qiagen) before hsct and monthly thereafter up to d+90. from january to october 2016, 106 hsct recipients (29 auto and 77 allo) were included in the study. ag was positive in 54 (70%) of the allohsct recipients at a median of 39 (range: 14-146) days. ag recurrences occurred at a median of 81.5 (38-249) days, in 15 of the 54 pts (27.8%) who had at least one episode of positive ag. 103 hsct recipients were included in the analysis of qtf-cmv. in the pre-hsct sample, qtf-cmv was reactive in 60 of the 85 allohsct (70.6%) and in 25 of the 28 autohsct (89.3%). significantly less allo hsct recipients recovered cmvimmunity at day +30 (31.8%) and day+60 (59.3%) in comparison with autohsct (95% and 100%, respectively, p o0.01). up to day +90, all autohsct have recovered cmvimmunity, in comparison to 68% of the allohsct recipients (p = 0.096, figure 1 ). the qtf-cmv test performed at d+30, d +60 and d+90 did not predict the risk of cmv reactivation in the following month. similarly, the test did not anticipate the risk of ag recurrences: 80% of the hsct recipients with undetermined or non-reactive qtf-cmv test at d+60 had ag recurrence after this period, in comparison with 70% of the patients with a reactive result (p = 0.56). in the present study, the qtf-cmv test alone could not predict the risk of cmv reactivation or recurrences. [p281] disclosure of conflict of interest: qiagen. recovery of vδ2+ γδ t cells is critical to epstein-barr virus reactivation after haploidentical hematopoietic stem cell transplantation j liu, z bian, q fu, l xu, x zhang, y wang and x-j huang peking university people's hospital, peking university institute of hematology, beijing, china epstein-barr virus (ebv) reactivation and its related disease are life-threatening complications in patients undergone haploidentical hematopoietic stem cell transplantation (haplohsct). our previous studies found that impaired cd4 − cd8 − t-cell recovery correlated to the increased occurrence of ebv infection after haplohsct. γδt cells make up 50-90% of cd4 − cd8 − t cells in the peripheral blood of healthy donors. expansion of vδ1+ γδt t cells after hsct has been reported and this subset could respond against autologous ebv-lcl in vitro. selective activation and expansion of vγ9vδ2-t cell could inhibit ebv-lpd development in humanized mice. however, the association of γδ t-cell recovery with ebv reactivation after allohsct remains unknown. this is a prospective cohort study including 110 consecutive patients who were diagnosed as hematological malignancy and underwent haplohsct. recovery of t lymphocyte and a panel of subsets, including cd3+, cd4+, cd8+, cd4-cd8-, tcrαβ+, tcrγδ+, vδ1+, and vδ2+ t cells, were determined by flowcytometry at 30, 60, 90, 180 days after haplohsct. all recipients and donors were tested negative for ebv dna in the peripheral blood before transplantation. recipients were monitored weekly for ebv dna load until day 100 after transplantation. recipients with peripheral blood plasma ebv dna load 41000 copies/ml at least on two consecutive occasions were diagnosed as ebv reactivation (ebv +). ebv − cohort generally represents patients whose ebv dna loado1000 copies/ml in peripheral blood. within 100 days after haplohsct, 17 of 110 (15.5%) recipients were diagnosed as ebv reactivation. compared to recipients with negative ebv dna load, the counts of cd3+, cd8+, and tcrαβ+ t cells were not statistically different in the ebv+ cohort from 30 to 180 days after haplohsct. in contrast, recoveries of cd4+ and cd4-cd8-t cells in ebv+ patients were significantly hampered at 30 days after transplantation (p = 0.021 and p = 0.046, respectively). although the tcrγδ+ t-cell counts were also decreased at 30 and 60 days in the ebv+ cohort, the comparisons did not reach the statistical significance (p = 0.072 and p = 0.082, respectively). notably, recoveries of vδ2+ γδ t cells at 30, 60 and 90 days were continuously delayed in recipients with ebv reactivation (p = 0.029, p = 0.001 and p = 0.046, respectively). whereas the counts of vδ1+ γδ t cells were similar between the two groups from 30 to 180 days in this context. in this prospective and large cohort study, we showed that the occurrence of epstein-barr virus (ebv) reactivation was associated with the hampered recovery of vδ2+ rather than vδ1+ γδ t cells after haplohsct. our findings will help explore γδt subset-dependent therapeutic strategies to control the serious complications due to ebv infection post transplantation and improve the overall survival of haplohsct recipients. disclosure of conflict of interest: none. in particular, bloodstream infection (bsi)is a frequent complication in the pre-engraftment phase with an impact on the morbidity and mortality of these patients. objectives: to analyze the incidence of bsi in patients undergoing hsct in our center, and to identify predisposing factors for the development of bsi in pre-engraftment phase patients after hsct. fifty-one consecutive patients undergoing hsct were analyzed retrospectively in our center during the period of 1 july 2015 and 30 june 2016. the characteristics of the sample are shown in table 1 . we have reported all the bsi between day 0 and day 30 after stem cell infusion. 70.5% (36 patients) received antibacterial prophylaxis with ciprofloxacin, five patients with broad spectrum antibiotics and five did not received any drug. the average days of fever have been 3.30 days (0-12 days). a total of 184 blood cultures has been collected (3.6 per patient). there have been 15 bsi (21.5% of the patients) with 8 (53.4 %) of cases caused by gram-negative organism (4 escherichia coli, klebsiella pneumoniae, acinetobacter baumanii, proteus vulgaris and delftia acidovorans) and 7 (46.6%) by gram-positives (1 enterococcus faecium, 1 enterococcus faecalis, 3 staphylococcus epidermidis, streptococcus mitis and streptococcus viridians group). one patient presented 3 different episodes of bsi, two patients 2 independent episodes and the rest eight, only one microorganism isolated. we have identified two bsi by extended-spectrum betalactamases (esbl-producing organism) and one isolation of carbapenem-resistant gram-negative bacteria. the rate of quinolone-resistant is 80% in all the sample. in univariate analysis, several factors like presence of comorbidities, presence of severe mucosits, type of catheter and antibacterial prophylaxis modality don't increased the risk to develop bsi (p40.05). the place where the procedure is performed does not influence the development of bsi. although the presence of previous infections is not a risk factor, hospitalization for infection in the 90 days before hsct does influence the development of bsi with statistical significance (po0.05). the crude mortality rate of the sample has been very low (2%), with only one death related to bloodstream infection. bsi are a common relative complication in the patient undergoing hsct but with an extremely low mortality in our sample. hospitalization for infection in the 90 days before hsct does influence the development of bsi. it is important to note that outpatient model and conventional rooms don't increased the incidence of bsi. although the use of quinolones in prophylaxis does not result in an increase in infections caused by multiresistant micro-organisms (esbl and carbapenemias) with acceptable resistance rates (80%), it also does not reduce the incidence of bsi in our sample. according to our analysis, his routine employment still throws light and shadows. [p283] disclosure of conflict of interest: none. septic episodes with multiple bacterial strains during antithymocyte globulin (atg) therapy for conditioning for allogeneic stem cell transplantation under rifaximin gut decontamination d markel 1 , c schultze-florey 1 , t brockmeyer 1 , v panagiota 1 , c lück 1 , a schwarzer 1 , m beck 1 , e dammann 1 , a ganser 1 , g beutel and m eder 1 recent evidence demonstrates the importance of the enteric microbiome for the development of gastrointestinal graftversus-host disease (gvhd) and mortality after allogeneic stem cell transplantation (sct) (1, 2) . accordingly, the usage of the non-absorbed rifamycin derivate rifaximin for gut decontamination has been reported to preserve the intestinal microbiota composition with a positive effect on overall survival in a single centre retrospective analysis (3). we here report severe septicaemia requiring therapy at the intensive care unit (icu) during atg application for conditioning in three patients with rifaximin used as single agent for gut decontamination within 6 months. after changing our gut decontamination from a chinolon-metronidazole regimen to rifaximin, three cases of severe septicaemia by gram-negative and gram-positive bacteria during atg treatment occurred within 6 months. patient #1 was a 52-year-old woman with tmds/aml after breast cancer conditioned according to the flamsa-bu protocol. the second (#2) and third (#3) patient were 55and 37-year-old males with a complex karyotype secondary aml after omf and relapsed inv(16) aml with meningeosis leucaemica, respectively. patients #2 and #3 were treated with flamsa-bu and flamsa-tbi, respectively. all patients received rabbit atg (atg fresenius/grafalon) at a dose of 3 × 10 mg/kg body weight and rifaximin (2 × 200 mg) for gut decontamination. patient #1 developed severe escherichia coli and pseudomonas aeruginosa septicaemia on day − 3 of the conditioning regimen and had to be transferred to the icu with septic cardiomyopathy for therapy with vasopressants and levosimendan. in patient #2 escherichia coli, klebsiella oxytoca, staphylococcus hemolyticus and staphylococcus epidermidis were simultaneously detected in blood cultures at day − 2. the patient was transferred to the icu and treated with vasopressants for septic shock. patient #3 developed septic shock due to klebsiella pneumoniae and enterobacter cloacae on day − 4 under atg therapy. mechanical ventilation and vasopressor therapy were required. fortunately, all three patients survived and completely recovered without any sepsis related disabilities under escalated anti-infective and intensive care therapy. all were discharged from the hospital in the outpatient clinics. interestingly, all isolated gram-negative pathogens were found to be sensible for a chinolon based gut decontamination. the reasons for these septic complications under atg therapy are not exactly understood but raise a note of caution on the use of rifaximin as single agent gut decontaminant during atg application in conditioning for allogeneic sct. infections with mycobacterium genavense were described for the first time in 1990. since then, several cases have been reported, but almost exclusively in patients with aids. most patients who underwent hsct have insufficient cellular immunity. here we report a mycobacterium genavense infection in a patient mimicking a lymphoma-relapse after hsct. a 58year-old female patient was diagnosed in july 2013 with stage ivb alk-negative anaplastic t-cell-lymphoma with cervical, retro-/supraclavicular, mediastinal, axillary and retroperitoneal lymphadenopathy as well as pulmonary manifestation. two chemotherapy treatment lines and autologous stem cell transplantation resulted in a partial remission. to improve remission prior to hsct the patient received 2 courses of brentuximab-vedotin. after conditioning therapy with fludarabine, busulfan, cyclophosphamide and atg, hsct from a hla compatible unrelated donor was performed in april 2014. a pet-ct-scan in november confirmed complete remission. after hsct the patient remained lymphocytopenic with cell count of cd4+ cellso100/μl. after acute stage iii gastrointestinal graft-versus-host disease (gvhd) low dose immunosuppressive therapy was maintained due to mild chronic gvhd of the liver and the upper gastrointestinal tract. beginning in june 2015 the patient experienced increasing fatigue, general weakness, loss of appetite, nausea, night sweating and fever. abdominal ultrasound, urine and blood culture as well as ct scans revealed no focus of infection. different lines of empirical antibiotic therapy resulted only in short term improvement. several blood culture tests remained sterile. a fdg-pet-ct scan showed a paraaortal and parailiacal lymphadenopathy with a high fdg uptake (suv between 19.7 and 20.1), highly suggestive of lymphoma relapse. endoscopic evaluations revealed two polypoid lesions in the bulbus duodeni. histology of duodenal biopsies revealed a massive accumulation of weakly pas-positive bacilli. pcr analysis confirmed an infection with mycobacterium genavense. despite several attempts mycobacteria were not recoverable on solid media even by long term culture. treatment was started with rifampicin, ethambutol, ciprofloxacin and clarithromycin. lymph node manifestation responded to therapy with decreasing fdg-uptake (suv 8.2) in a control fdg-pet-ct scan 3 months later. after 9 months treatment was terminated due to therapy refractory nausea. lymphocytopenia was persisting with cd4+ cellso100/μl. six weeks after stopping the antibiotic therapy, symptoms as fever and weakness reappeared. duodenal biopsy could not confirm persistent mycobacterial infection. fdg-positive intraabdominal lymph nodes (suv 11.2) and spleen (suv 8.2) were detected in a control fdg-pet-ct-scan. five lymphnodes were surgically removed. immunohistology detected histiocytic cell proliferation with no sign of lymphoma relapse. pcr confirmed the presence of mycobacteria-dna. consequently, antibiotic treatment was resumed. mycobacterium genavense can present with all the symptoms of a lymphoma relapse and should be considered in immune compromised patients. reliable diagnosis can only be obtained from lymph node biopsies and/or endoscopic evaluation. treatment has to be accompanied by restoring cellular immunity and should only be stopped after pcr-negative biopsies. disclosure of conflict of interest: none. stratification of patients with multiple myeloma and lymphoma undergoing autologous hematopoietic stem cell transplantation in term of antifungal prophylaxis r moghnieh, s khaldieh, l awad, d abdallah, n droubi, a youssef, a mougharbel, t jisr and a ibrahiim makassed university hospital, beirut, lebanon autologous hematopoietic stem cell transplantation (ahsct) is at intermediate risk for invasive fungal infections (ifi). the recommendations of international scientific societies are not homologous regarding prophylaxis against ifi in patients (pts) undergoing ahsct. the primary end point was to assess risk factors for the need of empiric/preemptive antifungal therapy in ahsct recipients, and to extrapolate to the subgroup of pts that requires antifungal prophylaxis in our population of ahsct pts. the secondary endpoint was to determine the fungal species distribution infecting or colonizing the pts. our study included adult pts (418 yo) who underwent ahsct for lymphoma and multiple myeloma (mm) between 2005 and 2015. all febrile neutropenic pts are being managed according to the 2010 infectious diseases society of america (idsa) guidelines regarding the use of antimicrobial agents in neutropenic pts with cancer. eligible pts were divided into two groups: those who received empirical antifungal therapy and those who did not need it. we recorded demographic and baseline clinical characteristics including: age, gender, comorbidities, stage, disease status at ahsct, high-dose therapy regimen, the presence of mucositis and its grade, the number of cd34 + cells transfused, the presence of central line or portacath, the need for mechanical ventilation, the presence of diarrhea, the duration of neutropenia, and the presence of bloodstream infections. pts who had lung infiltrates suggestive of ifi were analyzed separately. the causative fungal pathogens and colonizers were analyzed. univariate and multivariate analysis of potential risk factors to assess further significance was performed using spss. 190 patients were included.106 pts (56%) had lymphoma and 84 pts (44%) had mm. the need of empiric antifungal therapy was statistically more significant in lymphoma than mm pts (po0.01).the presence of mucositis grade ⩾ 3 showed a statistical significance for the need of antifungal therapy (p = 0.02). in the lymphoma group, remission status (pr vs cr) was not a significant factor for the need of empiric antifungal therapy (p = 0.49).the presence of mucositis grade ⩾ 3 was at the limit of significance ( p = 0.05). in the mm group, remission status (pr vs cr) did not affect the need of empiric antifungal therapy (p = 1). however, mucositis grade ⩾ 3 was found to be a significant risk factor for the need of empiric antifungal therapy (p = 0.02). following factors: the number of cd34 + cells transfused, the presence of central line and portacath, the need for mechanical ventilation, the presence of diarrhea, the duration of neutropenia, and bloodstream infections did not show any significance for the need of antifungal prophylaxis in both groups. all recovered fungal isolates (n = 14) were not from deep seated tissues biopsies or blood, and were identified as candida albicans in 7 with lymphoma, and in 7 with mm. they reflected the candida ecology in this pts series rather than deep seated fungal infections. we suggest to give antifungal prophylaxis to all lymphoma pts because of the higher need of empirical antifungal therapy, and give antifungal prophylaxis to mm pts having a predisposition for severe mucositis. fluconazole is the antifungal of choice for prophylaxis since all the fungal isolates were candida albicans. keywords: autologous hematopoietic stem cell transplantation, antifungal prophylaxis. disclosure of conflict of interest: none. a 48-year-old previously fit woman from a rural area of eastern europe was admitted to the hospital for severe aplastic anemia. steroids, csa, antinfective prophylaxis and supportive therapy were administered without response; therefore rabbit atg was then administered, with minor response; the year later, she underwent allogeneic-hsct (mud 9/10, ric: tbi, cyclophosphamide and fludarabine; gvhd prophylaxis: atg, csa, mtx). several days after transplantation she developed left migraine with ipsilateral back-eye pain. brain mri and ct showed a diffuse opacification of paranasal sinuses, mainly in the sphenoid sinus. the symptoms gradually improved with a specific treatment. the patient achieved a quick and complete haematological recovery and she was discharged. at follow-up visits she complained a flare of the migraine, with a left-sided headache that did not improve with nsaids. the headache gradually intensified until vision in the left eye became blurred with conjunctival injection. after consultation with ophthalmologist, for suspected toxoplasma retinitis, administration of intravitreal steroids and clindamicine was begun with partial benefit. however 15 days after (d +90) she was admitted in hospital because of worsening headache, irradiated in the occipital area, and weakness in the right hemibody. tests on csf were negative for neurotropic pathogens. an mri showed a complete occlusion of the intracranial tract of left internal carotid artery, with likely infectious material localized in the left lateral cerebral fissure. a chest tc showed a nodule with initial excavation in the right superior pulmonary lobe. for suspected tuberculosis she started antitubercular therapy. despite a second lumbar puncture confirmed pleocytosis compatible with acute purulent meningitis, microbiological research for bacteria, fungi and bk were negative. so antitubercular and antitoxoplasma therapy were stopped and the patient underwent surgical biopsy within the sphenoid sinus. pathological examination of the biopsy specimens showed acute and chronic inflammation of the respiratory mucosa, periodic acid. schiff and grocott staining ( figure 1 ) highlighted several septate fungal hyphae. cultural analysis revealed colonies of scedosporium apiospermum so the patient started targeted voriconazole intravenous therapy. nevertheless, 10 days later, she developed aphasia and right hemiparesis. a brain angio-mri confirmed the appearance of new lesions compatible with infectious localizations associated to an increased defect of left internal carotid artery vascularisation and complete left choroid detachment. after 2 weeks of voriconazole a significant clinical improvement have been observed and she was discharged, continuing oral antifungal therapy with voriconazole. at the last follow-up she achieved a complete resolution of neurologic symptoms, with permanent left eye blindness. 8 months later (d +370) she was asymptomatic, with normal haematological and neurological conditions and was able to stop the antifungal therapy. this case-report confirms that the risk of invasive fungal infection (ifi) is relevant in patients receiving hsct for aa, probably due to the prolonged neutropenia and association of other risk factors such as the immunosuppressive therapy and the iron overload. in this very poor prognosis infection, the early diagnosis of cns ifi remains challenging, but the administration of voriconazole was extremely effective. disclosure of conflict of interest: none. in this study, we aim to present the seroprevalence of ebv and incidence of posttranplant lymphoproliferative disease as well as to evaluate the relation with gvhd. between 2006 and 2015, the ebv serology of 364 patients that underwent allogeneic hematopoietic stem cell transplantation and their donors were evaluated in the study. ebv ig g (vca-igg, ebna ig g, ea-igg) and igm (vca-igm) antibodies were detected by chemolluminesance method (abbott, abd). all patients were followed for reactivation. ebv igg seropositivity was detected in 338 patients (93%) and 238 donors (77.7%). there was no statistically difference in related vs unrelated transplants in seropositivity. the median age of the patients was 37 (range: , 217 patients were male (60%) and 295 (81%) had malign disease. the stem cell source was peripheral blood in 299 (82%) patients and 258 (71%) received grafts from related donors. myeloablative conditioning regimen was received by 273 of patients (75%) (table) . all patients received acyclovir prophylaxis (related transplants 400mg tid, unrelated transplants 800 mg tid) during and after allo-hsct up to 3 months. twenty six-yearold pretransplant ebv seropositive aplastic anemia patient had ebv ig m positivity after 3 months of allo-hsct and developed lymphoproliferative disease. he was in complete remission after 4 courses of rituximab and methylprednisolone. three patients were ebv igm seropositive in 4th, 9th and 24th months of allo-hsct and received symptomatic treatment. acute gvhd was detected in 223 patients (61%) whereas 285 patients (78%) had chronic gvhd. acute gvhd and chronic gvhd incidences were similar in comparison of donor ebv seropositive vs seronegative status (78% vs 22%, p = 0.72; 80% vs 20%, p = 0.199). ebv seropositivity was detected in 92.8% of patients. the donor ebv serology was not related with acute or chronic gvhd. [p290] disclosure of conflict of interest: none. the umc utrecht pediatric experience with brincidofovir after allo hsct ca lindemans, m bierings and jj boelens pediatric blood and marrow program, dept. of pediatrics, university medical center utrecht, the netherlands viral reactivation with dna viruses form a considerable complication of allogeneic hematopoietic stem cell transplantation (hsct). there are little effective antiviral therapies and most have considerable toxicity. especially for adenovirus, there is no satisfactory therapeutic option. recently a new oral antiviral agent, the cidofovir prodrug brincidofovir became available to european patients only on the basis of urgent medical need and after a case by case approval by the health authorities. the aim was to describe our single center experience with brincidofovir in the pediatric allogeneic hsct setting. in the umc utrecht, pediatric patients receive t-replete bone marrow or unrelated cord blood (ucb) as the donor source after mostly myeloablative conditioning regimens (+ serotherapy in unrelated-hct). as gvhd prophylaxis patients receive cyclosporine a (csa) and mtx for bone marrow, csa and prednisone for ucb. patients are by standard weekly monitored for the presence of adenovirus, ebv, cmv en hhv6 viremia by rt pcrs in the plasma. extensive immune reconstitution measurements are performed every 2 weeks. since 2015, patients that developed viral reactivation with adenovirus, or a combination of other dna viruses (cmv, bk or hhv6) were offered brincidofovir if the viremia was progressive or in the context of poor immune reconstitution. brincidofovir was given in suspension (10 mg/ml) at the dose of 2 mg/kg biw, or 100 mg biw for larger children. de drug was discontinued when the viral load was below detection level. in total, six pediatric patients (age range: 0-18) received brincidofovir (2 patients tablets, 4 the suspension). four received it for adenovirus reactivation, a 5th patient for cmv and bk and a 6th patient for cmv en hhv6. the median day post-hsct of the first administration was 29 days post hsct (range: − 4 to 101), the median day post detection of viral reactivation 14 days . the median duration of administration was 36 days (10-98) with two patients being discontinued because of death. in no patient the drug was discontinued due to toxicity issues. the patients that died had multi-organ failure due to a combination of severe agvhd and multiple infectious issues. the patients were discontinued when the viral load was low and when they had cd4 counts of at least 50/μl. none of the four alive patients reactivated after the drug was discontinued. urgent medical need administration of brincidofovir is feasible. in our limited series we found the drug was well tolerated. disclosure of conflict of interest: i am a medical consultant for brincidofovir (chimerix). reactivation of herpes simplex virus 1 (hsv-1) or varicellazoster virus (vzv) occurs frequently after allogeneic stem cell transplantation (asct). here, we report three unusual cases, two with reactivation of hsv-1 and one with vzv. patients and methods: patient (pt) 1 (50-year-old, male) was allografted for high risk acute lymphoblastic leukemia in first complete remission after conditioning with total body irradiation (12 gy) and etoposide (60 mg/kg). graft-versus-host disease (gvhd) prophylaxis was performed using cyclosporine a, short course methotrexate and anti t-lymphocyte globulin (atg). pts 2 (44year-old, female) and 3 (67-year-old, male) were allografted for acute myeloid leukemia in second and first complete remission, respectively. conditioning regimens used were flamsa-ric in pt 2 and fludarabine/busulfan in pt 3. in both cases, gvhd prophylaxis consisted of cyclosporine a, mycophenolate mofetil, and atg. pts 1 and 2 had already experienced hsv-1-positive oral mucositis following induction chemotherapy and had successfully been treated with acyclovir. both developed hsv-1-positive oral mucositis again after asct. in both cases, initial therapy with acyclovir i.v. at a dose of up to 10 mg/kg t.i.d. was ineffective. to explore the mechanism leading to clinical acyclovir resistance, the thymidine kinase genes of both viral strains were sequenced. pt 3 presented with severe abdominal pain and nausea 11 months after asct. in this case, acyclovir prophylaxis post asct had been stopped 2 months before due to side effects. moreover, low dose prednisolone therapy was necessary for chronic gvhd. the hsv-1-strain from pt 1 showed a single base pair deletion in the region from nucleotide position 430 to 436 of the thymidine kinase gene (which consists of a guanosine repeat). in pt 2 a single base pair insertion in the same region was found. both genetic alterations lead to a loss of enzyme activity and acyclovir resistance. in both pts treatment was changed to foscarnet which led to rapid improvement. in the case of pt 3, multiple mucosal erosions were found on endoscopy of the esophagus. in these vzv dna was detected by polymerase chain reaction (pcr). only 4 days later, a vesicular skin eruption developed, which did not follow a dermatomal distribution. again, in the vesicular fluid vzv dna was detected by pcr. in this patient, acyclovir (10 mg/kg i.v., t.i.d.) resulted in rapid improvement. reactivation of hsv-1 and vzv after asct is a frequent finding. usually, hsv-1 strains respond well to acyclovir. in some cases, resistance can develop, especially in patients that had been treated with acyclovir before. acyclovir resistance of hsv-1 caused by mutations in the thymidine kinase gene can be overcome by treatment with foscarnet which directly inhibits the viral dna polymerase. disseminated vzv reactivations after asct have been described. clinical presentation can be misleading, for example, beginning with severe abdominal pain that precedes the vesicular eruption by several days. disclosure of conflict of interest: none. toxoplasmosis is a rare but severe complication after hematopoietic stem cell transplantation (hsct) (1) . it can involve the central nervous system alone or can manifest as a disseminated disease. in the paediatric population the mortality rate is high and sequelae are often severe. new diagnostic tools, such as the pcr assay, may allow for rapid diagnosis and preemptive therapy (2, 3) . we retrospectively analysed all children who underwent allogeneic hsct in our centre between january 2011 and december 2015. patients lost to follow up before day +100 were excluded. patients and donors were tested before transplant in order to assess their immunological status against t. gondii. a total of 187 allo-hsct were analysed. before transplant, 28.8% of recipients (r) were toxo-igg positive and 71.2% were toxo-igg negative. among donors (d), serology was available only for 152/187: 23% were toxo-igg positive, 77% were toxo-igg negative. we found a high number of not tested donors (18.7%, 35/187) which included, in most cases, mud from foreign registries. the group at higher risk for toxoplasmosis, d − /r+, included 21.7% pairs, whereas d − /r − were 55.2%, d+/r-were 15.1% and d +/r+ were 7.9%. in our series the cumulative incidence of toxoplasmosis disease was 2.1%, with 4 cases out of 187 transplants. two of them (case 1 and 3) had cerebral toxoplasmosis, one (case 2) had disseminated toxoplasmosis and case 4 had toxoplasmic chorioretinitis. mortality rate was 50%: two patients died because of multiorgan failure and disseminated toxoplasmosis respectively. in no case localized cerebral toxoplasmosis was the main cause of death. no complications were seen in surviving patients. all patients who developed toxoplasmosis were toxo-igg positive before hsct and three of them were transplanted from a toxoplasma igg negative donor (fourth donor not tested). in the two fatal cases the interferon-gamma releasing assay (igra) never became positive, confirming the absence of specific cellular immunity. toxoplasmosis disease can affect hsct outcome in paediatric recipients and pre-hsct seropositivity is the most important risk factor for toxoplasma disease in the post transplant period. in our cohort seroprevalence was higher than expected, probably due to the high number of patients coming from eastern europe. in order to reduce the burden of toxoplasmosis disease in our population we decided to implement a real-time pcr screening protocol for d − /r+ pairs, to provide rapid diagnosis and early therapy. all positive recipients with a seronegative donor will undergo real-time pcr screening starting on the day of stem cells infusion, and regularly until cd4+ t cell recovery. in the future we will analyse the impact of this strategy in this particular subset of immunocompromised patients. treatment with brincidofovir for adenovirus disease in pediatric hematopoietic transplants introduction adenovirus may cause serious morbidity and mortality after allogeneic hematopoietic transplants in children. severe lymphopenia is the main risk factor associated with progression to disseminated and often fatal disease. treatment with unlicensed cidofovir is based on monitoring of plasma viral load by pcr. however, cidofovir is only moderately effective at controlling adenovirus and it is associated with significant renal toxicity. brincidofovir is a lipid conjugate of cidofovir. it has a good oral bioavailability and achieves higher intracellular levels of active drug than cidofovir with a better safety profile. it is a potent inhibitor of viral dna synthesis so it could be indicated in immunocompromised patients with adenovirus disease. patients and methods we present three children of 3, 5 and 9 years old diagnosed of acute lymphoblastic leukemia (all) in 2nd complete remission (the first two patients) and severe aplastic anemia the last one. there were 2 girls and 1 boy. they underwent a peripheral blood hematopoietic stem cell transplantation using αβ/cd19 depletion with a haploidentical donor in the two patients with all and cd45ra depletion with a matched unrelated donor in the other patient. patients that underwent haploidentical transplants developed early acute graft versus host disease grade iii with gut and skin involvement so immunosuppressive treatment with corticoids was started. they developed severe lymphopenia ( o300/ mm 3 ). in the first month after transplant an adenovirus disease was diagnosed in the three patients from the weekly monitoring of plasma viral load by pcr. adenovirus was also tested in stools, urine and respiratory sample. in all patients adenovirus was also detected in urine sample. in one of them adenovirus was detected in nasal exudate too and in the other the virus was isolated in stools and in a skin biopsy. results: all of them were initially treated with cidofovir with poor results. foscarnet and gancyclovir was also used without improvement. finally they started a treatment by compassionate use with oral brincidofovir twice a week. with the first dose of brincidofovir plasma viral load started to go down until its complete disappearance. brincidofovir tolerance was good with only mild and limited diarrhea in two cases in the day they were taking brincidofovir. two of the three patients were alive without signs of adenovirus disease. in the other patient blood adenovirus load by pcr decreased below 1000/ml, but remain high in urine. she died of respiratory failure due to pulmonary graft versus host disease. conclusion brincidofovir may be a promising therapeutic option for the treatment of severe adenovirus disease in immunocompromised patients with a good toxicity profile. disclosure of conflict of interest: none. table 1 . all patients were transplanted with pbsc for haematological malignancy, and s269 received reduced intensity conditioning (ric) regimens with in vivo t-cell depletion. the proportion of patients with baseline and post-vaccination hi titres ⩾ 1:40 were 28.6 and 25% for a(h1n1)pdm09, 14.3% at both time points for a (h3n2), and 32.1 and 25% for b/phuket. pre and postvaccination geometric mean titres gmt) were higher by mn than hi for a(h1n1)pdm09 and a(h3n2), but lower for b/ phuket (p = 0.05). no post-vaccination seroconversions were detected by hi, while a single seroconversion to a(h1n1) pdm09 was detected by mn in a patient vaccinated at 0-3 months. the mn assay did not detect any additional low-titre seroresponses (negative to detectable titre) below hi threshold. none of patient age, lymphocyte count, days from transplant to vaccination, donor type, and gvhd or ist at vaccination correlated with baseline or post-vaccination titres by either assay. response to iiv was virtually absent throughout the first year post-hsct, with a single seroconversion to a(h1n1)pdm09 detected by mn but not hi, although the sample size was small and half of patients were vaccinated at 0-3 months. there is a clear need for a novel, immunogenic seasonal iiv and/or novel vaccination regimens in this population. vaccination of recipients' relatives and close contacts, and hsct healthcare workers should be strongly encouraged. pre-and post-transplant iron overload (io) has been associated with considerable long-term morbidity and mortality in pts undergoing transplantation. classically, management of io in the post-allo-hsct setting has been based in the performance of therapeutic phlebotomies (tp), which are inconvenient for the patient and are often not feasible due to ongoing anemia. we recently published the first prospective study of deferasirox in adult allo-hsct pts with io (vallejo, et al. haematologica 2014). in this retrospective analysis, we analyzed the real-life management of io in the post-allotransplant setting. this study includes the last 113 pts with a minimum follow-up of 6 weeks, who underwent allo-hsct in our center (october 2014-october 2016). 63 pts were male (55.8%) and 44 female (44.2%). median age was 53 years (range: 7-69). baseline diseases were: aml (44.2%), lymphoproliferative disorders (16.8%), mds (12.4%), all (8.8%), chronic myeloproliferative diseases (7.1%), mm (5.3%), and bm failures (5.3%). donor was unrelated in 61 cases (54%; 14 of them hla mismatched), and related in 52 (46%; 21 of them haplo-identical). conditioning regimen was: busulphan-based (68.1%), melphalan-based (13.3%), tbi-based (7.1%), and others (11.5%). progenitors source was pb in 102 (90.3%), and bm in 12 (9.7%). pre-hsct: pts had been transfused with a median of 23 prbc (range: 0-147), and their median serum ferritin (sf) was 1359 ng/ml (range: 22-5116). day +180 post-hsct: 15 pts had died, and 24 pts had not reached that day yet, so 74 pts were evaluable. they had been transfused with a median of 31 prbc (range: 0-157), and their median serum ferritin (sf) was 1127 ng/ml (range: 56-7993). 55% pts had sf superior to 1000 ng/ml. liver mri (by sir method) to assess liver iron concentration (lic) was performed in 44 pts at day +180. seven pts (15.9%) had no io (lic 0-2 mg/g), 12 pts (27.3%) had moderate io (lic 2.1-4.4 mg/g), and 25 pts (56.8%) had severe io (lic superior to 4.5 mg/g). median lic was 4.66 mg/g (range: 0.6-11.34). among the 29 cases with history of more than 20 prbc transfused and sf higher than 1000 ng/ml at day +180, 28 (96.6%) were proved to have liver io by mri; the other pt had io in spleen. 30 pts started some kind of therapy to treat the io: 6 pts with severe io initiated a tp program and 24 pts (6 out of 12 with moderate io, and 18 out of 25 with severe io) initiated chelation therapy with deferasirox. the drug was started at low dose (2.5-5 mg/kg/ day), and was increased if tolerated up to a maximum of 20 mg/kg/day. of note, the majority of pts were also taken a number of medications (immunosuppressants, statins, antimicrobials, etc). 3 of those 24 pts (12.5%) did not tolerate the drug, and were changed to tp. for more details, see the table. (1) the combination of the history of prbc transfusions and serum ferritin levels was, in the majority of cases, enough to assess the io in the post-allo-hsct setting. (2) liver mri (by sir method) helped to assess io in doubtful cases. (3) deferasirox, initiated at low doses and increased if tolerated, was safe and its use helped to avoid the need of therapeutic phlebotomies for the majority of patients. this study reproduces, in a real-life setting, our previous findings in a prospective clinical assay. [p297] disclosure of conflict of interest: none. a case-control study of risk factors of primary graft failure with a focus on associated early-onset severe infections v alcazer 1 , a conrad 2 , f-e nicolini 1 , s ducastelle-lepretre 1 , f barraco 1 , x thomas 1 graft failure (gf) is a rare but devastating event after allogeneic haematopoietic stem cell transplantation (ahsct), exposing the recipient to disease relapse, drawbacks of marrow aplasia, infections and death. the aim of this study was to analyse the risk factors associated with graft failure after ahsct, with a specific focus on early-onset severe infections (esi). we conducted a retrospective, observational, single-centre, matched case-control (1:2) study among adult s270 ahsct recipients transplanted at the haematology department of our institution between 2008 and 2015, with a subsequent follow-up of 12 months. engraftment was assessed at day+42 post-ahsct. gf cases were classified as primary gf (pgf), defined as failure to achieve donor-derived absolute neutrophil count (anc) ⩾ 0.5 × 109/l or lasting more than 3 consecutive days without evidence of disease relapse and early-secondary gf (esgf), referring to the loss by day 42 post-ahsct of a previously functioning graft associated without evidence of disease relapse. each case was matched with two controls according to underlying haematological disease, hla matching, stem cell source, intensity of conditioning and temporal proximity of ahsct. demographics, haematological and graft characteristics as well as esi report were retrieved. esi were classified in invasive fungal infections, viral infections (cmv, ebv, hhv-6, other viruses), toxoplasmosis and severe sepsis of bacterial origin. during the study period, 598 ahsct were performed at our center. seventeen (3.1%) gf cases were identified, of which 15 pgf and 2 esgf, and were matched with 34 controls. in the descriptive analysis, gf and control populations did not significantly differ when considering demographics, haematological characteristics and hematopoietic stem cell source. regarding pretransplantation status and graft characteristics, only disease status (progressive disease) and cell dose (both cd34+ and cd3+ cells number/ kg) were associated with graft failure. the proportion of patients with ⩾ 1 esi before day 42 was significantly higher in cases than in controls (11/17 vs 11/34, p = 0.038), with an overall number of esi events of 19 and 12 among cases and controls, respectively. five cases had ⩾ 2 concurrent esi. the median time from ahsct to the first esi event for gf cases was 17 days (interquartile range (iqr), 11-24) vs 15 (iqr, 8-34) days for controls (p = 0.779). in the gf setting, the most prevalent infections were herpesviridae infections (n = 7 including hhv-6 n = 4, ebv n = 2, cmv n = 1), probable ifi (n = 4), severe sepsis of documented bacterial origin (n = 3), toxoplasmosis (n = 2) among whom one patient developed haemophagocytic syndrome. when further analysing subsets of esi using logistic regression, only toxoplasmosis was a significant risk factor for gf (p = 0.018). death related to an infection was proven for 8 gf patients vs 5 control patients (p = 0.012). the overall survival probability at 12 months was significantly lower in the gf setting than in control patients (hr = 2.59 (95% ci 1.25 − 5.36), p = 0.01). the survival rates at 12 months were 35.3% and 57.7% for gf and control patients, respectively. at our center, graft failure is statistically associated with early-onset severe infections, and already known graft characteristics such as cell dose and disease status. however, our study would need more power to increase its significance. disclosure of conflict of interest: none. allogeneic stem cell transplantation (asct) is a curative option for hematological disorders, especially malignancies. in immunosuppressed women after asct, the progression from cervical dysplasia to invasive carcinoma is accelerated, and cervical cancer is likely a more aggressive disease. therefore, follow-up protocols after asct should include regular gynecologic evaluation with papanicolaou (pap) smears. we retrospectively evaluated 32 pap smears in 20 women who underwent asct and searched the risk factors for abnormal cervical cytology. the median age at transplantation was 44.5 years (range: 22-65 years). the most frequent indication for asct was leukemia (70%), and 85% of the patients received a transplant from a sibling hla-matched donor. stem cell source was peripheral blood in all patients. myeloablative conditioning regimen was used in 50% of patients. cyclophosphamide, busulfan and fludarabin were used in 20 (100%), 18 (90%) and 10 (50%) patients, respectively. acute graft versus host disease (gvhd) occurred in 7 patients (35%) and chronic gvhd in 4 patients (20%). secondary cancer (1 breast cancer) was reported in only one patient at 40 months after asct. the follow-up time was 23 months (range: 3-104 months). after asct, benign and abnormal pap smears were found in 12 (60%) and 8 (40%) women, respectively. the median time between asct and development of abnormal cytology was 2 months (range: 1-11 months). four (20%) women had at least one smear with atypical squamous cells of unknown significance (asc-us), one (5%) had a low-grade squamous intraepithelial lesion (lsil), one (5%) had atypical squamous cells/high-grade lesion (asc-h) and one (5%) had asc-us and asc-h. one (5%) patient had malign smear. two patients with asc-h showed high-grade atypia mimicking cancer but had a negative follow-up. patient who had malign smear died because of aorta dissection. cervical biopsy showed cervical intraepithelial neoplasia (cin) i in 3 (15%) women who had asc-us or asc-h. one patient was hpv-positive. we did not find any relationship between cervical cytological abnormality and clinical factors. after asct, patients are high risk for abnormal cervical cytology and secondary gynecological cancer. regular surveillance of patients is the most important factor for decreasing the risk of developing cervical and other secondary cancers. gynecologic examinations and cervical cytological testing after asct allows early diagnosis and effective management of cervical abnormalities. disclosure of conflict of interest: none. kidney dysfunction is a frequent complication of allogeneic stem cell transplantation (sct) and contributes to the morbidity and mortality of the procedure. incidence of severe acute kidney injury (aki) in patients undergoing nonmyeloablative allogeneic sct for malignant diseases ranges from 14 to 47%. lymphoma patients are often heavily pretreated through both chemotherapy and autologous sct and may be at increased risk of developing kidney injury. we performed a retrospective analysis of 108 consecutive patients with lymphoma undergoing nonmyeloablative allogeneic sct between 2004 and 2016 (table 1) . acute kidney injury (aki) within 100 days of allogeneic sct was diagnosed and staged according to rifle-criteria, and severe aki was defined as rifle stage i-e (4doubling of creatinine or 450% decrease of egfr). chronic kidney disease was defined as an estimated glomerular filtration rate (egfr) o60 ml/min/1.73 m 2 1 year after allogeneic sct. we performed multivariate logistic regression to evaluate potential risk factors for severe aki. severe aki developed in 75 patients (69.4%). reduced overall survival was observed in these patients, although not statistically significant. no significant associations were seen with age at transplantation, baseline kidney function or prior autologous sct. severe aki was associated with acute graft versus host disease (gvhd) (or 2.8, p = 0.026) and the use of an unrelated donor (or 2.8, p = 0.025). chronic kidney disease was observed in 20 (18.5%) of patients alive after 1 year. we report a substantially higher incidence of severe aki after nonmyeloablative allogeneic sct for lymphoma than has been reported for other malignancies. acute gvhd and unrelated donor stem cell s271 source were associated with severe aki, while prior autologous sct, age and baseline kidney function were not. [p301] disclosure of conflict of interest: none. patients with acute myeloid leukemia who are treated with conventional chemotherapy still have a substantial risk of relapse. we, therefore, retrospectively analyzed data to investigate the effects and some risk factors of allogeneic hematopoietic stem cell transplantation in relapsed and refractory acute myeloid leukemia patients, and to provide some suggestion for the clinical treatment. a total of 84 refractory and relapsed acute myeloid leukemia patients receiving allogeneic hematopoietic stem cell transplantation in our center between february 2005 and december 2014 were retrospectively analyzed, including 23 patients in no-remission (nr) and 61 patients in second complete remission (cr2) at the time of transplant. the median age was 35 years (range: 9-55). conditioning was myeloablative using cyclophosphamide, busulfan and total-body irradiation (bu/cy, n = 44; tbi/cy, n = 3), and others were underwent nonmyeloablative stem cell transplantation. 81 patients had successful engraftment. acute-gvhd and chronic-gvhd appeared in 47and 37 patients. the 3year overall survival (os), relapse rate and disease-free survival (dfs) of the cases was 50 ± 6.0%, 44.8 ± 5.8% and 37.1 ± 0.3%, respectively. the 3-year dfs were higher for patients in cr patients (52.2 ± 6.7%) than in nr patients (21.3 ± 10.1%), and the relapse rate in nr group and cr group were 60.4 ± 1.6% and 29.7 ± 0.4% respectively. there was no significant difference in treatment-related mortality compared cr group with nr group. sex, age, related-donor graft were not independent factors affecting os, dfs and relapse rate. it is concluded that allo-hsct is an effective salvage therapy for patients with refractory and relapsed aml. non-remission before transplant and severe agvhd are high risk factors of poor prognosis for allo-hsct. patients in cr group who accept reinduction chemotherapy before transplantation have better prognosis than those in nr. the overall outcome seems related to the disease status. hsct during refractory and relapsed can achieve long-term survival in selected patients with individual therapy. disclosure of conflict of interest: none. the incidence of most hematologic malignancies increases with age. aging is related with a greater prevalence of impaired functional status and comorbidities. although cure of malignant and non-malignant hematological diseases is potentially possible with allo-hsct, it could lead to significant transplant-related mortality. decision making about referral to allo-hsct in older adults is a challenging task. in this study we aim to present our geriatric allo-hscts. from 2007 to 2016, 33 [p303] patients (age 3 60) underwent allo-hsct in our center included to this retrospective study. pre-transplant status as well as posttransplant toxicities, complications and outcomes were determined. the age distribution of the group: 27 patients was aged 3 60 and o65, 5 patients was aged 465 and o70, 1 patient was 71 years old. the median age of donors was 49 (range: 21-73). the pre-transplant patients' characteristics are given in the table. remission was achieved in twenty-three (70%) patients. twenty-six patients (79%) had neutrophil engraftment (40.5 × 10 9 /l) at a median day of 19 (range: 10-41) and platelet engraftment (20 × 10 9 /l) at a median day of 20 (range: 14-54). post-transplant complications are detailed in the table. acute graft vs host disease (gvhd) was occurred in 10 patients (31%) and chronic gvhd in 12 patients (36%). eight patients (24%) were diagnosed with a relapse and 1 year relapse-free survival was 15%. the 1-year and 2-year os were detected as 30% and 12%. the most common reason for mortality was sepsis. the 1-year os was higher in patients who had reduced intensity conditioning regimen and remission status pre-transplant however they were not statistically significant (30% vs 21%, p = 0.6; 31% vs 25%, p = 0.9) (figure) . since increasing number of older patients being diagnosed with hematologic malignancies, this trend of increasing number of allo-hsct will continue. tolerability and effectiveness are lesser, toxicity is higher in older adults. although study population is relatively small, reduced-intensity conditioning and pre-transplant remission status may be related to better survival. comprehensive geriatric assessment may be considered prior to allo-hsct for global evaluation. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (asct) is a procedure with high morbidity and mortality (10-20%) requiring a complex hospital infrastructure. improved support measures and development of homecare units has allowed that asct at-home programs may be possible. our center has launched a pioneering program in our country in patients with asct to perform at home the following of aplasia, control of immunosuppressive therapy (ist) and intravenous support from the d+1 of asct until the engraftment and independent ambulatory patient. to evaluate the patient safety, we compared the group of patients at-home (asct-op) with a cohort of asct 'in patient' with similar characteristics (asct-ip). 26 asct patients between january 2014 and october 2016 at the hospital clinic of barcelona. 13 patients performed asct-op and 13 had an asct-ip. all patients received conditioning (myeloablative-mac-or reduce intensity-ric-) in the hospital with fludarabine 40 mg/m 2 (d1-4) and busulphan 3.2 mg/kg (2-4 doses), prophylaxis of gvhd was performed with tacrolimus/mycophenolate (mmf) in asct-op group and cyclosporine(csa) and methotrexate (mtx) or mmf in asct-ip group. in all patients, the infectious prophylaxis was conventional (levofloxacin, fluconazole and acyclovir). moreover, the asct-op group received prophylaxis with ceftriaxone 1 g intravenous (iv) once daily and liposomal amphotericin b inhaled 25 mg twice a week during neutropenia. the asct-op group from d+1 received a nurse visit once daily and physician visits twice a week in the hospital. baseline characteristics were analyzed those related to toxicity and patient outcomes. the median age (range) was 56 years (23-69), male/female 16/10; (62% male). the source of the progenitors was peripheral blood in all cases and analysis of the results detailed in the table: disclosure of conflict of interest: none. an increase in rdw-sd after allogeneic hematopoietic transplantation is associated with a poor prognosis s leotta, a cupri, a di marco, a spadaro, l scalise, g sapienza, mg camuglia, g avola, g moschetti 1 and g milone 1 istituto oncologico del mediterraneo red cell distribution width (rdw), is an erythrocyte index influenced by stress erythropoiesis, inflammation and antioxidants. rdw predict mortality in sepsis, chronic kidney diseases and in cardiovascular disease. no data are available on rdw after hematopoietic transplantation. in a retrospective study we collected data on changes of rdw-sd in a group of 81 patients who received allogeneic hematopoietic transplantation. fortyeight patients were affected by acute leukemia, 13 by lymphoma, 9 by mm, and 11 by other diagnosis. rdw was studied at baseline and monthly for the first 3 months. a subset of 34 patients were studied prospectively for clinical and laboratory signs of microangiopathy. at baseline before the transplant a rdw-sd higher than normal upper limit was observed in 43% of allogeneic candidates. a high co-morbidity score (htc-ci score 2-5) at the pre-transplant screening was a factor associated to high rdw-sd (χ 2 p = 0.01). a value of rdw-sd higher than normal range, at baseline, was not associated to any other factors, such as age, diagnosis, phase of the disease, previous transplantation, c-reactive protein, bilirubin, creatinine and arterial hypertension. early after allogeneic transplant we noticed at day +30 a significant reduction of rdw-sd but subsequently (at day +60) the proportion of patients showing an abnormal rdw-sd increased to 63%. an abnormal rdw-sd at s274 day +60 was registered in 70% of allogeneic transplant patients who presented an acute gvhd while in only 30% of patients who did not presented during the first 3 months an acute gvhd (χ 2 p = 0.02). in allogeneic transplantation group, patient who, at day +60, had a rdw-sd higher than normal value had a inferior outcome in respect to patients having a rdw-sd within normal ranges (os was 70% vs 30%; logrank: p = 0.04;), (ci of trm: 45% vs 18%).these two groups were not significantly different for pretransplant features in the subset of patients studied prospectively, abnormal rdw-sd was associated to presence of schystocytes in pb (chi test: 0.004) and patients having ⩾ 2% schystocytes had a median rdw-sd of 71 (iqr 31) vs a median rdw-sd of 46 (iqr 12.2) in patients who did not show schystocytes in pb (mann-whitney u-test p = 0.004). rdw-sd was significantly correlated also to serum triglycerides (r = +0.4, p = 0.0004) and to red blood cell mean corpuscular volume (r = +0.32, p = 0.02). abnormal rdw-sd is frequent after allogeneic transplantation. abnormal rdw-sd is associated to acute gvhd and its value obtained at day +60 marks a group of patients with poor prognosis because of high trm. this simple parameter warrant further studies to determine its clinical usefulness in monitoring of patients suffering acute-gvhd and in diagnosis and monitoring transplant associated microangiopathy. [p305] disclosure of conflict of interest: none. sickle cell disease (scd) poses a lot of psychological burden for the patient and the caregiver. it also poses a significant financial burden over the family. ohaeri et al. developed a 16 point questionnaire to asses sickle cell disease burden called as sickle cell disease burden index (scdbi) and its impact on caregiver's quality of life (qol). we used this questionnaire to assess the impact of hematopoietic stem cell transplant (hsct) on caregiver's qol. 16 point questionnaire was sent to 15 set of parents whose child underwent hsct between january 2016 and june 2016. scdbi contained 16 questions in various domains (3:family finances, 3:family interactions, 5:routine family activity and 5:parental coping ability). answers were graded on a score of 0-3 (0:never occurred and 3:occurred regularly or had a severe impact on the family). the results were interpreted in two headings a. family finances and interactions (0: no impact; 1-3: insignificant impact; 4-6: moderate impact; 7-9: severe impact) and b. routine family activity and parental coping ability (0: no impact; 1-5: insignificant impact; 6-10: moderate impact; 11-15: severe impact). all these domains were assessed before and after hsct. ten parents replied with duly filled questionnaire. mean age at hsct was 8.1 years (range: 1-14), m/f:7/3. all were symptomatic for 46 months before hsct with 90% having more than 2 hospital admissions. majority of parents were from middle class with median family income of 30 000 usd per annum (range 16 000-200 000 usd). median score for family finances and interactions (a) before hsct was 6 (range: [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] which decreased to 0 (range: 0-3) after hsct. median score for routine family activities and parental coping ability (b) before hsct was 13 (range: which decreased to 0 (range: 0-6) after hsct. our results suggest that before hsct there was a moderate impact on family finances and interactions which reduced to no impact after hsct. similarly there was severe impact on family activities and parental coping ability before hsct which changed to no impact after hsct. our study suggests that hsct not only improves the qol of the child but also of the caregivers. chronic graft versus host disease (cgvhd) is a late complication of allogenic hematopoietic stem cell transplantation (hsct) that affects many tissues and organs and manifests with polymorphic clinical features similar to autoimmune diseases. poorly understood pathophysiological mechanisms are implicated in inflammation and tissue fibrosis which is a hallmark of cgvhd. the affection of lachrymal glands is frequent and contributes to ocular manifestations presenting as dry eye syndrome. autologous serum eye drops (aesds) are used topically to facilitate tissue healing and ease the symptoms in a variety of ocular diagnosis. it is unclear if the serum of a patient with cgvhd is suitable for remedy preparation and if the transplanted patient himself can meet the criteria for autologous donation. aim is to show the safety, feasibility and efficacy of autologous serum preparations in ocular lesions after allogenic hsct. donors should meet criteria for autologous blood donation (infectious disease status, complete blood count hgb4110 g/l, hct433%, adequate venous access). aesds are prepared from 150 ml of autologous blood left to clot, irradiated and centrifuged to separate serum which is diluted with saline in 1:5 ratio or 1:1 if requested. product is dispensed into 1.5 ml ampules, stored at − 20°c and a 3-month supply is released to the patient after receiving negative results of sterility testing. in period from 2005 to 2016. in the aesds program 12 patients (4 female, 8 male) with ocular symptoms were included. all met required predonation criteria. of 29 collections performed, one failed due to venous access problem and one product had to be discarded due to hemolysis. cgvdh global nih score of the patients at start of the program was: 4 severe, 6 moderate, 1 mild and 1 not scored. all patients presented with moderate to severe dry eye symptoms. in 6 (50%) patients aesds alleviated dry eye symptoms. in 3 (25%) out of 12 patients referred to aesd program, more than 3 autologous blood collections were performed (range: 4-10) and aesds were used regularly through period of 10-36 months, which points to the beneficial effect of the long-term use of the serum. three patients dropped out because aesds showed no advantage compared to commercial lubricant eye drops preparations. one patient dropped out because of a venous access problem, 4 patients had disease s275 progression and needed other therapies: 3 cases of amniotic membrane application of which 2 continued with aesds to facilitate the healing effect. one patient was recently included and the effect of aesd is still evaluated. autologous donations in cgvhd patients are feasible, safe and autologous serum preparations can help relieve symptoms of dry eyes. it needs to be further elucidated specifically in which patients and at what point of the disease course the effect of the aesds is the most beneficial to make optimal use of these preparations. disclosure of conflict of interest: none. idiopathic pneumonia syndrome (ips) is a non-infectious pulmonary complication with diffuse lung injury that develops in 5-10% of patients who undergo hematopoietic cell transplantation (hct) and the mortality rate remains high at 80%1. the major aim of this study was to identify prognostic biomarkers for ips and establish positive and negative predictive values (ppv and npv) of ips. in a case-control study, we compared 41 patients with ips with available samples (transplanted between 1988 and 2014 at fhcrc) with 162 hct control recipients who did not require bronchoscopic examination and who did not grow any bacterial or fungal blood cultures. for each subject, plasma samples at day 7 post hct and onset of ips or matched time points for controls were analyzed. the 'onset sample' for controls was the sample closest to day 24 (median day of onset for patients with ips). we measured six proteins by elisa: suppressor of tumorigenicity 2 (st2), tumor necrosis factor receptor 1 (tnfr1), interleukin-6 (il-6), lymphocyte vessel endothelial receptor (lyve)-1, endothelial protein c receptor (epcr), and herpes virus entry mediator (hvem). multivariable logistic regression models were used to evaluate the association of each protein with ips vs controls. cytokine cutoff values that maximized discrimination between ips and controls were identified using receiver operating characteristic (roc) analysis. ppv and npv of ips were calculated using the identified cytokine cutoffs across a range of hypothetical ips prevalence values (0-15%) day 200 weighted kaplan-meier survival curves were estimated for high/low cytokine subgroups. similarly, a weighted log-rank test was used to evaluate p-values. a multivariable logistic regression model including six cytokines showed that st2 and il-6 were significantly important markers to identify ips at the onset (table 1) . st2 value at day 7 post hct was significantly associated with occurrence of ips and il-6 had a marginal association. predictive values for ips by a plausible percentage of the actual hct population (up to 15%) are shown in figure 1 . of the six proteins, st2 showed the highest ppv both at onset and day 7 post hct followed by tnfr1, and il-6. npv were high in all the markers. to analyze whether st2 and il-6 at day 7 after hct can predict survival following ips, we dichotomized the patients into cytokine high and low groups (cutoff level: st2, 19 ng/ml; il-6, 35 pg/ml) and compared survival after downweighting the observations to represent a plausible percentage of the actual population (ips prevalence, 5%). day 200 survival rate were significantly lower in st2 high value group than in st2 low value group (80% vs 88%, p = 0.015). similarly, il-6 high value was associated with high mortality (day 200 survival rate, 78% vs 88%, p = 0.003). st2, il-6, and tnfr1 were good prognostic markers for occurrence ips. especially, st2 and il-6 at day 7 after hct can be a predictor for both ips occurrence and survival following ips. these results require validation in an independent prospective hct population. body composition parameters are sensitive nutritional indicators that influence response to treatment and mortality in cancer patients. research is not conclusive on the changes in muscle attenuation and adipose tissue areas in the stem cell transplantation (sct) phases. objective is to assess the changes in adipose tissues, skeletal muscle index (smi) and waist circumference (wc) among stem cell recipients in the peri-transplantation phase. study design: institutional review board approved this retrospective study with 61 adult patients (age416 years) having b and t lymphoma who underwent sct. each patient was imaged by pet/ct scan pre-sct and 3 months post transplantation. a cross sectional image was analyzed at the level of the l3 to calculate total adipose tissue (tat), visceral adipose tissue (vat), intra-muscular fat (imf), smi and wc. data was analyzed by gender since body composition parameters differed significantly between the two categories in the literature. the study sample consisted of 61 patients (mean age: 38.2 ± 13.7 years, 35 (57%) males, 51(83.6%) autologous sct, median overall survival in months: 39.8 in males and 40.5 in females). death was observed in 6 (17.1%) males and 1(3.8%) female. patient characteristics were similar for males and females except for weights (kg) and body mass index (kg/m 2 ): 86.7 and 28.6 vs 63.5 and 24.1 in males and females respectively. changes from pre-sct to 3 months post sct revealed that tat, vat, smi and wc decreased with mean differences of 42 ± 61.2 cm 2 , 18.28 ± 37.6, 3.3 ± 7.5 cm 2 /m 2 and 4.58 ± 5.4 cm, respectively in males (po0.01). in females, tat and wc significantly decreased with mean differences of 18.4 ± 37 cm 2 and 3.1 ± 4.3 cm, respectively (po0.01). in females, vat and smi decreased clinically but did not reach clinical significance. in multivariate analysis, no significant associations were shown with mortality and progression rates. this study fills a research gap by providing data on the evolution of body composition parameters in the peri-transplantation phase. tat, vat, smi and wc decrease 3 months post transplantation. future studies should evaluate the associations of these parameters with major outcomes on larger sample sizes. [p310] disclosure of conflict of interest: none. . 5 patients received tbicontaining preparative regimen. all these patients were exposed to calcineurin inhibitors for prevention and treatment of gvhd. 16 patients suffered from cgvhd-grade moderate or severe. all patients required systemic corticosteroids, because of gvhd (16pts) or during basic treatment of lymphoma (2 pts). all patients had deficient states of vitamin d initially and required replacement. all of them, except for 2 patients, had balanced adrenal insufficiencies and 2 patients had balanced hypothyroidism. all women had premature ovarian failure (2 received hrt). according to measurements of bone mineral density (bmd), low bone mass was detected in 15 patients; osteopenia (11pts), osteoporosis (4pts). bone loss of femoral neck (8-osteopenia, 3-osteoporosis) occurred more often than lumbar vertebral ( 6-osteopenia, 2-osteoporosis) or radius (3osteopenia, 1-osteoporosis). presence of avascular necrosis of bone (avn), confirmed by mri, was detected in 12 patients and the most common site of involvement was the femoral head(all patients), knee(3 pts) and shoulder(1 pts). one of the first symptoms of avn was pain and functional limitation. all patients required intensive analgesic treatment, usually nsaids and 4 patientsfentanyl. fractures occurred in 12 patients. the femoral neck (7 pts) and thoracic or lumbar vertebral (3 pts) were two most common fracture sites. all patients were qualified for surgery; 6 patients required hip replacement, 6patients still awaited to perform surgery or were disqualified because of severe, skin cgvhd. bone complications may occur in about 17% of allo-hct survivors (including 30% patients with gvhd, and up to 60% patients with severe or moderate cgvhd) within first 4 years after allotransplantation. bone loss, particularly at the femoral head, is the most common complication. avascular necrosis usually requires surgical intervention because of fractures. exposure to higher doses of corticosteroids (during treatment of gvhd) increases risk of bone complications. early diagnosis by mri and dxa may help to detect bone complications. (41%) and (e) 3 (21%). a total of 41 (30%) pts failed to meet these criteria but remained alive on day 5 and 39 (28%) died before day 5. the overall survival (figure 1 ) for the 58 pts was 28% at 3 years with an overall mortality in icu of 33% (19/58) compared to 71% (29/41) for those who did not meet our criteria. the overall survival for pts that met our criteria at fifth day and were discharged to the haematology ward (n = 39), was 49% at 3 years. in this study, 50% of patients survived their icu admission. patients could be stratified according to the reason for admission and given an individualized 5-day trial: those who met our criteria for successful icu trial (42%) had a low icu mortality (33%) and those who were subsequently discharged home had a overall survival of 49% at 3 years. this study raises the possibility of offering a short-term icu stay to oncohematologic patients and perhaps allows for the ceiling of intensive care for those who fail these criteria. [p312] disclosure of conflict of interest: none. in contrast, only 2.98% of patients without cgvhd showed a thromboembolic complication in the later time course, with one patient showing an additional thrombotic risk factor. in multivariate analysis cgvhd was an independent risk factor for thromboembolic complications after hsct. 1.8% of patients with thrombosis before hsct showed one afterwards. thrombosis before hsct was not found as risk factor for thromboembolic complication after hsct. our retrospective analysis showed an increased risk for thromboembolic complications after allogeneic hsct, with substantial higher risk in patients with chronic gvhd (8.9%). in ongoing studies we currently investigate a vascular screening procedure with additional biomarkers according to inflammation and endothelial damage in patients with cgvhd prospectively. we hope to identify patients at risk for thromboembolism and prevent future complications on an individualized basis. disclosure of conflict of interest: none. (1). pts with cns involvement received intrathecal therapy with cytarabine and in one case additional cns irradiation was applied. 10/11 pts died after a median time of 9 mts (range: 1-25 mts) due to resistant systemic relapse, infectious complications or extensive graft-versus-host disease following allohsct. 1 patient remains alive and disease-free at 88+ mts following secondary allohsct. conclusions: our data indicate that em disease following allohsct affects a significant proportion of pts with aml. sites of em relapses vary widely among the pts with skin and cns being frequently involved. an aggressive approach combined of local and systemic therapy including secondary allohsct may produce favorable response in a small proportion of pts, however, overall prognosis for pts with isolated em relapses still remains poor. due to the lack of effective treatment strategies, there is a need for novel approaches to manage isolated em relapses after allohsct. disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct)-associated thrombotic microangiopathy (tma) is a multifactorial complication, and has variable incidence in study populations due to different diagnostic criteria. aim: our aim was to identify pediatric patients with hsct associated tma using 4 different diagnostic tma criteria published in literature and to compare the various groups for tma parameters and outcomes. we enrolled 33 pediatric patients who underwent allogeneic hsct using treosulfan based or reduced intensity conditioning therapy. 4 different tma diagnostic criteria, the bmt ctn toxicity committee consensus definition (1), the overall thrombotic microangiopathy grouping (2), the diagnostic criteria created by city of hope (3) and the criteria proposed by jodele et al. (4) were used to startify the patients. we determined and registered the following tma activity markers: presence or development of increased ldh and decreased haptoglobin levels, new onset anemia, thrombocytopenia, fragmentocytes, coombs test, kidney function, proteinuria, hypertension and terminal complement complex (sc5b-9). complement pathway activities, components and sc5b-9 were measured during early hsct period. two/33 (1), 7/33 (2), 3/33 (3) and 10/33 (4) subjects met the different tma diagnostic criteria according to the four different systems on day 12 and 34 (1) and on median 44 (2), 43 (3), 61 (4) post-hsct days. all of the 6/10 patients who were defined with the first three criteria, met the forth definition. due to normal haptoglobin levels and kidney function, 4/10 patients fulfilled only the forth criteria. tma coexisted with acute graft-versus-host disease in 7/10 cases (7/10 vs 4/23; p o0.01). patients who met any of the different tma diagnostic criteria had higher sc5b-9 level on day 28 (411 vs 201 ng/ml; p = 0.003) compared to those without. all of the 10/33 subjects defined with tma had elevated sc5b-9 (4250 ng/ml) level during the early hsct period. two patient died before day 100 after hsct, out of which one patient met all of the four tma diagnostic criteria. after a median 2.29 (1.2-3.1) year follow-up time, overall survival was 24/33. 8/10 patients with tma survived, compared to 16/23 patients without tma. relapse related mortality was the most common cause of death (n = 7/9, po0.05), while tma was not a significant cause of mortality after reduced toxicity conditioning therapy. hsct-associated tma has a variable and complex pathophysiology. using the different diagnostic criteria may influence the incidence and the time of diagnosis of this transplant-related complication. monitoring all of the published tma activity parameters, including complement terminal pathway activation marker, may help to guide physicians to recognise tma after hsct. disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct) is associated with a risk of non-relapse mortality (nrm). it's important to assess the risk of complications and mortality before the hsct. some indexes quantify the impact of patients' comorbidities on hsct outcome. the most frequency used is the hct comorbidity index (hct-ci) and the european group for blood and marrow transplantation score (ebmts). this study tried to determine which of the two indexes best predicts the outcome in a series of patients submitted to hsct in a single center. between 2011 and 2015, 259 hsct were performed in our center. a total of 215 hsct have been analyzed (we excluded patients o18 years (yr), 2 nd hsct, haploidentical donors and hsct for specific diseases with very low number ( o3%) of hsct performed: aplastic anemia, cll, prolymphocytic leukemia, mycosis fungoides, sezary syndrome, dendritic cell neoplasia, plasma cell leukemia and poems syndrome). the hct-ci and ebmts were calculated retrospectively (yr 2011-2013) and prospectively (yr 2014-2015). overall survival (os), relapse incidence (ri) and nrm were analyzed in the overall series and separately according to the type of hsct: autologous hsct (auto-hsct) or allogeneic hsct (allo-hsct). male: 89 (59%) patients. median age: 54 yr (range: 18-71). diseases: aml 54 (25%), all 19 (9%), mm 67 (31%), nhl 41 (19%), hl 12 (5%), mds 17 (8%), cmpd 5 (3%). disease status: 1 st complete remission (cr) or 1 st chronic phase 102 (48%), ⩾ second cr 30 (14%), first partial remission (pr) 55 (26%), ⩾ second pr 12 (5%), no response 11 (5%) and without previous treatment 5 (2%). auto-hsct in 120 patients (56%) and allo-hsct in 95 (44%) patients. related and unrelated donor were 52 (55%) and 43 (45%), respectively. the conditioning regimen was standard in 57 (60%) cases and reduced intensity in 38 (40%). hct-ci and ebmts grouped 0-2, 3 and ⩾ 4 were 57 (48%), 29 (24%), 34 (28%) and 38 (32%), 51 (42%), 31 (26%) in auto-hcct and 61 (64%), 18 (19%), 16 (17%) and 34 (36%), 32 (34%), 29 (30%) in allo-hsct, respectively. median follow-up was 3.15 yr (0.66; 5.73) for the overall series, 3.18 yr (0.66; 5.73) for auto-hsct and 2.60 yr (0.97; 5.70) for allo-hsct. significant differences in os and nrm were found according to the ebmts in patients submitted to auto-hsct. one-yr-os and 3-yr-os were 91% (95% ci: 85%; 97%) and 68% (95% ci: 57%; 79%), respectively, in patients with ebmts 0-3, vs 73% (95% ci: 57%; 87%) and 56% (95% ci: 37%; 75%), respectively, in patients with ebmts ⩾ 4 (p = 0.033). one-yr-nrm and 3-yr-nrm were 2% (95% ci: 0%; 7%) in patients with ebmts 0-3, vs 13% (95% ci: 4%; 28%) in patients with ebmts ⩾ 4 (p = 0.015). no significant differences were observed for ri according to ebmts in patients submitted to auto-hsct. no significant differences in os, ri and nrm were observed according to ebmts in patients submitted to allo-hsct. no significant differences regarding os, ri or nrm were found when the hct-ci was assessed. in our series, only the ebmts was predictive of os and nrm in patients submitted to auto-hsct. failure to find statistically significant differences for the hct-ci and for ebmts in allo-hsct recipients could be due to an insufficient number of patients or to a partial retrospective collection of data. infertility is common after hct predominantly as a result of the chemoradiotherapy used in conditioning. nonetheless, some patients do retain or recover fertility. newer reduced intensity regimens may be less gonadatoxic. in addition, patients are increasingly encouraged to store gametes, or embryos before transplant. we sent questionnaires to 602 ebmt centers requesting retrospective details of number of pregnancies and pregnancy outcome for all patients treated between 1995-2015. 27 centers responded from 13 countries detailing 234 patients who became pregnant/partners conceived. the most frequent underlying diagnoses were acquired bone marrow failure (n = 44, 25f) aml (n = 42, 15f), hd (n = 26, 15f), cml (n = 25, 7f), all (n = 19, 4f) and b nhl (n = 18, 10f). other diagnoses included mds, mps, solid tumours, autoimmune disease, cll, t-nhl, haemoglobinopathy. of 110 females (f), 35 (32%) involved assisted reproductive techniques (art). 30f had tbi ( seven o4 gy) of which 16 (50%) had art. 25f had reduced intensity conditioning of whom 6 (24%) had art. 70f were specified as having standard conditioning of whom 24 (34%) had art. 73f had allogeneic (26 art, 36%) and 37f had autologous transplants (9 art, 24%). of 124 men (m) whose partners conceived, 61 (49%) had art. 54m received tbi of which 36 (67%) had art. where specified, 19 had reduced intensity hct (3 art, 16%) and 94 had standard conditioning (48 art, 51%) .93 had had allogeneic hct (43 art) and 31 autologous (12 art). 19 men had reduced intensity transplants. 53 men received tbi (two o4 gy) of whom 36 (68%) had art compared to 69 men without tbi, 16 (23%)of whom had art. data on return of menstruation was available for 84. 64 indicated yes and 12 (19%) had art. 20 indicated amenorrhoea of whom 14 (70%) had art. 224 specifying number of children had 324 live births (lb) and 87 (39%) patients had more than one child after hct. 146 lb occurred in female patients (41 art, 28%) and 178 lb were in partners of male patients (88 art, 49%). the median gestational age for 61 female patients was 39 weeks (range: 22-42) and the median birth weight was 3 kg (range: 0.3-4.19). there were 3/80 congenital anomalies. the median follow up of the offspring was 5 y (range: 0-15). developmental problems were indicated for 1/71 (fine motor skills) and learning difficulties in 1/70 (adhd). in partners of male patients the median gestational age for 62 offspring was 39 weeks (range: 26-43). the median birth weight for 56 offspring was 3 kg (range: 0.87-4.16). congenital malformations occurred in 4/89. one infant died of pulmonary infection. in women, several methods of assisted conception were used including hormone stimulation, ivf, cryopreserved embryos, donor embryos and cryopreserved ovarian tissue. the most frequent method was use of donor embryos (22/35) in which a minimum of 30 attempts led to 21 lb. the median number of attempts was 1 (range: [1] [2] [3] [4] [5] . art were frequently used in this group of posttransplant patients particularly in male patients vs female, tbi vs non-tbi, amenorrhoeic vs menstruating women, standard conditioning vs ric. in patients who conceive after hct, successful pregnancy leading to healthy offspring is the likely outcome. disclosure of conflict of interest: none. reduction of trm after sct was observed over the transplant periods and supportive care with danaparoid was found to be significantly effective to reduce trm. therefore, prophylactic administration of danaparoid is considered to be a reasonable option to improve the transplant outcomes for children. [p320] disclosure of conflict of interest: none. the attainment of transfusion independence after transplant is sometimes hampered by a combination of factors, ranging from infections to the need of combined therapy for clinical complications, as well as control of gvhd. iron overload is frequently observed in hematological patients before and after hematopoietic stem cell transplantation (hsct). whereas several reports have focused on iron overload before transplant, up to now, this is the only report that show full recovery of hematopoiesis and correlate this to deferasirox chelation performed on this particular subset of patients. we report on 19 patients, transplanted for hematological diseases (17 acute leukemia, 1 aplastic anemia, 1 multiple myeloma) heavily transfused before transplant that, considering the iron overload, were treated with deferasirox after hsct. before starting deferasirox, the patients were fully engrafted and in complete remission, although transfusion dependent, and with incomplete hematological reconstitution after allogeneic hsct. patients were selected according to the following inclusion criteria: (1) transfused pre-transplant with more than 20 rbc units; (2) incomplete hematological recovery; (3) transfusion-dependence; (4) serum ferritin 41000 ng/ml; (5) normal creatinine value. the workup for other aetiologies resulted negative. all patients received an initial dose of deferasirox 10 mg/kg/day, later adjusted according to side effects. all patients experienced an increase in hemoglobin levels, with a reduction in the frequency of rbc transfusions, followed by transfusion independence (median time: 24 days from the first dose of deferasirox). in addition, it was promptly (median time: 27 days) associated with hematological improvement, with sustained values and no further platelet support or growth factors administration. no relevant modifications with immunosuppressive or myelosuppressive drugs were made during deferasirox treatment. deferasirox was well tolerated. basing on our results, we think that deferasirox determined stimulatory, and/or depressive effects on hematopoiesis after allo-hsct. this clinical experience raises the possibility of a potential additive benefit on hematopoiesis after transplant following iron chelation therapy with oral deferasirox. further long term studies, in larger cohorts of patients are needed to confirm these data and to design an efficient strategy to reduce iron loading after transplant. disclosure of conflict of interest: none. supported in part by ail pesaro onlus. (4) hypertension (n = 2). all five patients had normal adamts13 levels and negative testing for shiga toxin. complement mutation genetic studies were obtained for four patients including 10 genes (n = 2) and 12 genes (n = 2) and were all negative. testing for complement pathway including c5b-9 were obtained for 2 patients and were normal. all five patients were treated with eculizumab with induction treatment at 900 mg weekly × 4 doses, followed by one dose of 1200 mg on the fifth week, and 1200 mg every 2 weeks thereafter. patients had a recovery of hemoglobin and platelets and a rise in haptoglobin and a normalization of ldh within 4-6 weeks from the start of eculizumab. eculizumab was discontinued for 3 of the 5 patients without recurrence of their tma; they are now 18-24 months since the discontinuation of eculizumab. in summary, there is a subacute syndrome of thrombotic s282 microangiopathy that can occur late post transplant. this syndrome appears to be complement mediated as shown by its response to a terminal complement inhibitor. it also appears to be transient without recurrence following treatment discontinuation. disclosure of conflict of interest: none. transplant associated microangiopathy (tam) is a very severe complication occurring after allogeneic bone marrow transplantation (bmt), burdened by a high case-fatality rate. it is characterized by abnormal complement activation, triggered by various agents (calcineurin inhibitors, acute gvhd, infections) with subsequent endothelial damage. in the literature, 6 cases of mutations in recipient complement genes are described, but none in donor dna. here we describe for the first time 3 patients affected by tam, carrying mutations in donor complement genes. in our lab, we studied 6 patients affected by tam; they were screened for cfh autoantibodies, adamts13 function and variants and macro-rearrangements in cfh (and related), cfi, cfb, cd46, c3, dgke, thbd genes and at-risk haplotype (cfh-h3 and mcpggaac) by means of next-generation sequencing (ngs) and multiplex ligationdependent probe amplification analysis (salsa mlpa p236 armd mix-1; mrc holland). ngs was used to sequence dna by haloplex kit (agilent) on a miseq (illumina) platform with 450-fold coverage of every target base. the bioinformatic analysis was performed using sophia genetics and the pathogenicity was assessed by means of in silico predictions (polyphen2, sift, mutationtaster, aligngvgd). all of the predicted pathogenetic variants were confirmed using sanger sequencing. the same genetic screening was extended also to donor dna in all 6 cases. the screening for known causes of tam revealed mutations in recipient complement genes in one case; no mutations were found neither in recipient nor in donor dna in two cases; instead, donor genetic alterations were found in 3 patients whose characteristics are summarized in table s283 donor hematopoietic cells. in the three cases presented, tam was relatively delayed with respect to hsct, in particular in two cases (6 months) and this timing is compatible with the concept of reticulo-endothelial 're-population' by donor cells of monocytic lineage, responsible for the production of regulatory proteins of the alternative pathway of the complement. we also underline the response to anti-c5 inhibition in the 2 patients who were treated with eculizumab; this fact further supports the hypothesis that the disease was related to complement dysregulation. we therefore suggest that both the recipient and the donor should be screened for complement gene mutations, so that more cases could be identified and the pathogenesis of tam could be further clarified. among these we observed one autologous engraftment, one death due to septic shock before engraftment and two primary gf. we used a desensitization treatment based on 4 plasma exchange procedures, intravenous immunoglobulin (1 g/kg) and rituximab (375 mg/sm) in 2 patients. one of these patients (aml, haploidentical donor) had dsa against hla-b50 (mfi 900). she experienced primary gf with increasing titles of dsa (maximum mfi 10 500); so, on day 38, a second transplant from the same donor was performed after a desensitization treatment. a progressive decrease in dsa was documented (up to mfi ⩽ 200). on day 12 patient achieved neutrophil count over 500/μl and on day 23 platelet count over 20 000/μl. the second patient (mds, haploidentical donor), instead, received a desensitization procedure before the first transplant. she had dsa against hla-a24 (mfi 3700), and after desensitization dsa levels decreased and reached 0. on day 20 patient achieved neutrophil count over 500/μl and on day 38 platelet count over 20 000/μl. dsa were detected in 1/9 of usct candidates (11%) and 4/17 of haplosct candidates (24%) and they were associated with failure to obtain allogeneic engrafment in 3 cases. desensitization treatment achieved dsa clearance and engraftment in the 2 patients in which it was performed, underlining the potential benefit of this procedure in the setting of hsct with dsa that has to be validated by prospective and controlled studies. disclosure of conflict of interest: none. early complications and late effects and quality of life at myeloma multiplex patients z trajkovska-anchevska, a pivkova, s genadieva-stavrich, l chadievski, z stojanoski, l chevreska and b georgievski university hematology clinic, skopje, macedonia the subject of this research is the quality of life at patients with myeloma multiplex at diagnosis and during therapy within 6-12 months. the research aims to analyze patients to be able to continue activities which will contribute for improving their quality of life as a priority task placed before the patient, his family, health institutions and social environment. this research was conducted at the university clinic for hematology skopje in the period from june 2009 to march 2012. it covers patients infected with multiple myeloma, diagnosed and treated during this period. a total of 80 patients analyzed, using the eortc qlq c30 ver. 3.0 standardized questionnaires for hr quality of life that analyzed the physical, cognitive, emotional, personal and social functions related to the patients. it also analyzed and general health and quality of life. analysis of physical functioning at diagnosis is 27.5 during treatment 59.5, significantly improved. personal functioning at patients at the diagnosis is 17.9, during therapy − 36.4. analyzing emotional functioning in patients at diagnosis is 39.9, during the therapy over 73.3 significantly improved. in examining the cognitive functioning is also a significant difference at diagnosis 55.2, during treatment 72.5. social functioning of the patients was 26.2 at the diagnosis; during the treatment grow to 50.8. significant improvement was notices in these patients' symptoms like fatigue, nausea and vomiting, pain, dyspnea, insomnia, loss of appetite, constipation and diarrhea. the analysis of the financial difficulties of patients at diagnosis is 76.2 and 72.5 during treatment, meaning no significant difference in the time given. the analysis of the overall health and quality of life at patient has a value of 23.9, and during therapy 58.8. quality of life at patients with myeloma multiplex that makes the research group was significantly improved as a result of on time diagnosis and treatment with modern medicaments and the role of social worker with the application of certain social skills, continuous counseling, guidance and education for their reintegration in the community. installing the quality of life as a separate category and investigating the factors that affect its expression in the daily functioning of the patients within the changed framework of action, as like this example for malignant disease. the needs of clearly defined interactions patient illness and treatment, quality of life and specifying the segments where it can effectively act and improve in order to achieve positive progression towards improving the qualitative features of this category is a clear and primary objective that must be inserted into the current approaches to monitoring patients with malignant hematological diseases. acute graft-versus-host disease (agvhd) is a common and severe complication after allogeneic stem cell transplantation. since the current first-line treatment is based on treatment with systemic glucocorticoids (gc), steroid-induced hyperglycaemia develops frequently in patients with (agvhd) potentially impacting on their outcome. we performed a retrospective analysis on 104 patients who received systemic gc for agvhd and thoroughly investigated the consequences of aberrant glucose metabolism. in particular, we focused on glucose parameters early after initiation of gc. with a median of 50 (range: 4-513) blood glucose measurements during gc treatment, increasing mean, median and maximum glucose levels as well as the need for insulin treatment were associated with decreased overall survival (os) in simple and multiple survival analysis. early hyperglycaemia, as defined by mean blood glucose levels 4125 mg/dl during the first 3 days of gc therapy, was also found to be highly associated with adverse outcome: in multivariate analysis, the hazard ratio (hr) for death was 2.5 (95% ci 1.32-4.87, p = 0.005) in patients with early hyperglycaemia. while the risk of death due to relapse was not increased, the hr for death due to non-relapse mortality was 3.26 (95% ci 1.53-6.92, p = 0.0021) in a competing risk analysis. a score based on early hyperglycaemia and non-response to gc within 7 days allowed the identification of three risk groups: patients with both risk factors had an inferior os at 5 years of 4.1% as compared to 75.4% in patients with none. patients with one risk factor had a 5-year os rate of 32.0% (p = 0.0002 for trend). in this retrospective study, we identified early hyperglycaemia after gc initiation as a prominent factor predicting increased nonrelapse mortality in agvhd patients. in addition, a score based on early hyperglycaemia and lack of response to gc was highly predictive for overall survival in these patients. disclosure of conflict of interest: none. early toxicity because of infectious complications not relapse is the main cause of death after allogeneic transplantation in aplasia for patients with refractory or relapsed acute myeloid leukemia high-dose cytarabin was given in 13/25 pts with induction failure. the search for a stem cell donor was started immediately after results of high-risk cytogenetic, no achievement of bone marrow aplasia on day 14 of induction therapy, or immediately after diagnosis of relapse. four patients had a related 10/10 donor, for 17 patients a 10/10 matched unrelated donor was identified and 10 patients received a transplant from a 9/10 unrelated donor. the interval between diagnosis of primary disease or relapse and tx was 3 (1-7) months (mo) for both groups . in 24 patients melphalan (100-140 mg/m 2 ) was used to induce an aplasia before starting conditioning therapy. the interval between melphalan and conditioning therapy was 13 (9-21) days. three pts started the conditioning therapy while in aplasia after previous chemotherapy. the conditioning therapy was of reduced intensity in all pts. and consisted of treosulfan (30 g/m 2 )/fludarabin(flu) in 19 pts, tbi(8gy)/flu in 7 pts and busulfan(8 m/(kg)/flu in 5pts, respectively.atg was given to all pts with an unrelated donor. most pts (21/31) had a severe neutropenia with a median of 0.3/nl ( 0.1-5.2) before starting melphalan because of refractory leukemia. after a median follow-up of 21 (4-68) mo 11 pts (35%) were alive without relapse. 6 (19%) pts died because of a relapse after a median of 6 (3-25) mo. the nonrelapse mortality was 45% (14/31 pts). most of these pts (10/14, 71%) died because of infectious complications early after transplantation (med 1; 0-19mo). in 4 pts graft versus host disease was the main cause of mortality. in this retrospective 'real-life' analysis, we showed that an early allogeneic transplantation is feasible for patients with primary refractory or relapsed aml. a reduced intensity conditioning after induction of aplasia with melphalan offers a chance of long-term relapse-free survival for about 30% of patients with an otherwise dismal prognosis. nrm is high, especially because of infectious complications early after transplantation, probably related to the long period of severe neutropenia. therefore, the focus has to be set on early recognition and intervention of infectious complications. disclosure of conflict of interest: none. recent evidence supports the effector role of complement activation in several types of thrombotic microangiopathythe atypical hemolytic uremic syndrome (ahus) as well as the transplantation-associated thrombotic microangiopathy (ta-tma). the blockade of the terminal complement complex formation by anti-c5 monoclonal antibody eculizumab provides an effective treatment option in severe and devastating cases of ta-tma. the experience with the use of eculizumab in this indication is slowly accumulating in the hsct community, however the published data originate from small case series or uncontrolled trials and sharing of emerging real-life observations may be valued. on case reports of two pediatric patients treated with eculizumab for ta-tma with very detailed followup of multiple complement parameters, including terminal complex sc5b-9 and eculizumab drug levels we would like to demonstrate: (1) achieving therapeutic levels of eculizumab (499 μg/ml) may be unsuccessful even with initially intensified dosing interval. furthermore, we documented rapid eculizumab clearance from circulation which allowed only for short periods ( o48 h) of efficient drug levels during the weekly dosing. (2) we did not observe tightly correlated sc5b-9 and eculizumab levels within the dosing intervals; however the long-term sc5b-9 formation suppression was achieved concomitantly with improved eculizumab levels and slowed drug clearance. (3) classical complement pathway activity assay (ch50) may not reliably substitute for therapeutic efficiency monitoring in case of hypocomplementaemia due to protein losses (profound diarrhea, proteinuria, gi bleeding, catabolism). this holds true also for the alternative pathway activity which remained low during treatment in both patients. (4) mycotic infections may represent serious therapy related risks in eculizumab treatment after hsct (both patients achieved control of complement activation after multiple doses of eculizumab, however suffered fatal infections subsequently). besides, we observed a significant increase in c3a concentrations correlated with clinical onset of infection which invites for further investigation of this complement cascade product as early indicator of mycotic infection. in conclusion, we would like to highlight the great added value of timely available complement assay results, including sc5b-9 and especially eculizumab drug level values-to be used together with detailed clinical parameters for directing effectively these highly personalized (and also costly) treatments. [p329] disclosure of conflict of interest: none. table 1 . no difference in terms of drug-related adverse events was observed in the three patient cohorts with no reported serious adverse events. similar results were obtained performing two separate sub-analysis only for lymphoma or myeloma patients. despite the limitations due to the non-randomized nature of the study, from our data on a large cohort of patients s286 with a long-term follow-up biosimilar filgrastim (zarzio®) could be considered substantially equivalent in terms of efficacy and safety to lenograstim (myelostim®) and peg-filgrastim (neulasta®), when used for hematological recovery and febrile neutropenia prophylaxis after asct in adult patients with hematologic malignancies. disclosure of conflict of interest: none. we studied all 107 adults who underwent allo-hsct during a 23-month period (01 january 2015 to 22 november 2016) in our center. a total of 6 pts (5.6%) received epag for pfg with thrombocytopenia. three pts were male, and three female. median age was 59 years (24-67). the baseline diagnoses were: alm (2), mds-raeb (1), idiopathic myelofibrosis (1), aa (1), and cll (1). three transplants were from family donor (all of them haplo-identical), and 3 from unrelated donor (the three of them hla 9/10). sc source was pb in 5 cases, and bm in 1. epag was started at 50 mg/day and escalated each 2 weeks to 75, 125 and 150 mg if platelet count was o50 × 10 9 /l. we analysed the platelets, anc, and hgb at epag initiation and 90 days after being with the maximum dose. median time between allo-hsct and eltrombopag initiation was 120 days (17-155). median maximum dose used of epag was 150 mg/day (125-150). median platelets, anc and hgb before starting treatment were 13 × 10 9 /l (5-28), 1 × 10 9 /l (0.07-11.2) and 8.6 g/dl (7.6-12.1), respectively. five patients (83%) were severely thrombocytopenic (platelet count ⩽ 20 × 10 9 /l), 4 (67%) were anemic (hbg o 10 g/dl), and 3 (50%) were neutropenic (anc o1.0 × 10 9 /l). median platelets, anc and hgb at day +90 of maximum dose were: 37 × 10 9 /l (8-108), 2.4 × 10 9 /l (0.93-9.62) and 11.8 g/dl (7.9-14.5), respectively. the 5 thrombocytopenic pts (100%) responded to epag, with increases of 120 00, 25 000, 28 000, 39 000 and 96 000 × 10 9 /l in the platelet count. three anemic pts (75%) responded and achieved increases of hgb of 1.1, 4.7 and 6.8 g/dl. finally, the 2 neutropenic pts (66.6%) responded and achieved increases of anc of 4080 and 9550 × 10 9 /l. at the moment of the analysis close, pts are at a median of +11.5 months post-hsct (8) (9) (10) (11) (12) (13) (14) (15) (16) , and all but one (who died from a septic shock) are alive and outpatient. this survival is striking for subjects who develop a complication with such a high expected mortality as pfg. pgf is a life-threatening complication, relatively frequent after alternative donor hsct, whose treatment has been very disappointed. we report our experience in pts who developed pgf during the last 2 years. epag induced responses in platelets in all pts of the studied group. bilineal and trilineal responses were also seen. in our opinion, prospective studies are warranted in order to confirm epag as a new efficient treatment of post-hsct poor graft function. disclosure of conflict of interest: none. s287 ing 19 patients who developed es with an equal number of patients who did not between january 2013 and november 2016. we analyzed variables such as cd34+ cells per kg infused, use of granulocyte colony-stimulating factor (csf-g) and engraftment day. analytical data, including baseline and maximum determination of serum glutamic oxaloacetic transaminase (got) and glutamic pyruvic transaminase (gpt), c-reactive protein (crp) and procalcitonin (pct), as well as clinical data fever, weight gain, digestive and respiratory symptoms, pulmonary infiltrates were analyzed. sixty-eight patients were women. median age was 59 years old (range: 39-73). patients were conditioned with beam (52%), melphalan 200 mg/m 2 (42%) and bcnu-tt (5%). nineteen patients developed es in our series, which correspond to eight percent of all asct. case and control groups were matched according to age, sex, diagnosis and conditioning regimen. the most prevalent baseline disease in the group with es was myeloma (42.1%), followed by mantle cell lymphoma (26.3%). all patients who developed es had fever, 79% skin rash, 37% respiratory symptoms, 16% pulmonary infiltrate an 9% digestive symptoms. a summary of the comparison of data analyzed in subgroups is shown in table 1 . we found significant difference in the percentage of weight gain (p = 0.007), increase of tgo (p = 0.0001), increase of tgp (p = 0.001) and increase the number of cd34+ cells per kg infused (p = 0.043), we found an inverse correlation between the number of cd34+ cells per kg infused and incidence of es. however, in terms of post-transplant csf therapy (p = 0.075) and crp and pct valor (p = 0.85 and p = 0.25, respectively) we did not find significantly difference to develop es. in our series, weight gain and tgo and tgp rise were risk factors for es development. therefore, we should be aware of es in patients who develop fever, elevated liver enzymes and weight gain during graft phase. we did not find a significant difference in crp and pct suggested in other studies. further studies are required to better characterize risk factors of es development. busulphan-based (155), melphalan-based (122), beam (58), tbi-based (22), and others (43). weight at hospital discharge was significantly lower than at admission (5.6% in allo-hsct, and 5.4% in auto-hsct). weight at day +100 was also significantly decreased compared with the admission (8.6% in allo-hsct, and 6.7% in auto-hsct). weight at day +100 was lower than the ideal for their sex and height in the allo-hsct setting. contrarily, among the patients undergoing auto-hsct, the weight at day +100 remained higher than the ideal for their sex and height in a high proportion of cases. regarding serum albumin, it was significantly decreased at discharge (9% in allo-hsct, and 17.5% in auto-hsct), but recovered values similar to admission at day +100. in the auto-hsct setting, prealbumin levels were significantly reduced at discharge (39%), and in lower proportion at day +100 (8%), compared with admission values. in the allo-hsct patients, prealbumin levels were significantly reduced at discharge (5%), but had been recovered at day +100, compared with admission values. disclosure of conflict of interest: none. recently, blood and marrow transplant clinical trials network has proposed a composite endpoint: gvhd-free, relapse-free survival (grfs) for hsct outcomes. this endpoint includes as event: iii-iv acute gvhd (agvhd), relapse, death or chronic gvhd (cgvhd) requiring systemic treatment. in the last embt annual meeting a redefinition of this endpoint was proposed changing cgvhd event from those patients with cgvhd requiring systemic treatment (the original one) to those with just severe cgvhd (the redefined one). we retrospectively analysed 603 patients consecutively transplanted (1995-2014) excluding non-malignant diseases, second allo-sct and those o16 years old age. we had generated two composite endpoints: in both iii-iv agvhd, relapse or death were considerated events but we defined grfs1 as the one with cgvhd event including those who required systemic treatment (as the original one) and in grfs2 just those with severe cgvhd (the ebmt redefined one). the median age was 49 years (16-69) and 59% (362) were males. other characteristics of patients are resumed in table 1 . with a median follow up for patients alive of 39 months , the median estimated survival in months and the % at +1 year and +2 years was: 114 months, 71% and 62% overall survival (os); 24 months, 58% and 50% event free survival (efs); 6 months, 35% and 26% grfs1; 11 months, 46% and 38% grfs2. 138 (23%) and 210 (35%) hadn't any event in grfs1 and in grfs2, respectively. in grfs1, event's incidence was: 90 (15%) for iii-iv agvhd, 170 (28%) for cgvhd, 151 (25%) for relapse and 54 (9%) for death; in grfs2 was 90 (15%), 65 (11%), 173 (28%) and 65 (11%), respectively. considering those patients with cgvhd as event in grfs1, 105 of them hadn't the event as cgvhd at the same time in grfs2 (since they had cgvhd requiring systemic treatment but not severe cgvhd). for these patients, the alternative event in grfs2 was: 72 without any event, 22 relapsed and 11 died. in the multivariate, the factors associated with better outcomes were: in grfs1 early ebmt stage (p o0.001 with early as reference; intermediate p factor with more impact in both, but it is interesting to point it out that haploidentical donor had an advantage in grfs1. these results are being validated in a large series and the definitive results will be available at the moment of the meeting congress. [p334] disclosure of conflict of interest: none. steroid refractory acute graft-versus-host disease (gvhd) remains a major complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). affected patients have a very poor prognosis. gvhd has been associated with transplant-associated thrombotic microangiopathy (ta-tam). endothelial damage mediated by radiation, viral reactivation, drug exposure or alloreactivity results in exposure of subendothelial collagen, activation of coagulation and small vessel occlusion to a degree that results in organ failure. complement is thought to be a major mediator of endothelial damage. although a consensus exists about the exceedingly high morbidity and mortality of ta-tam and diagnostic criteria have been converging to a consensus, no biomarkers to diagnose tam and predict outcome have been established. we hypothesize that a ta-tma, related to dysregulation of the alternative complement pathway correlates with organ damage. a retrospective analysis of 660 consecutive patients with hematological malignancies receiving an allo-hsct at the university hospital basel in the period from 2003 to 2013 was performed. data on the occurrence, risk factors and outcome of patients with ta-tma and the correlation with acute gvhd was collected. available biopsies of organs suspected to be affected by tam and/or gvhd will be performed. routine bone marrow biopsies for histological, immunohistochemical signs of ta-tam and complement activation will be analyzed. serum samples will be used to characterize markers of complement activation using plasma levels of c5b-9 and c5b-9 deposition in tissues biopsies. 660 patients (aml n = 260; all n = 152; mds/mpn n = 93; lymphoid neoplasm n = 85; plasma cell disorder n = 53; bone marrow failure n = 17) underwent myeloablative (n = 432) and non-myeloablative (n = 228) allo-hsct at a median age of 47 years (range: 19-71 years). forty-eight (7.3%) patients matched the established diagnostic criteria for tam (increased ldh, platelet count o50 g/l or o 50% of normal baseline, schistocytes 42 per high power field, creatinine increase). the median time to onset of tam was 36 days post-transplant (range: 22-67 days). subjects with ta-tam had significantly higher 3-year nonrelapse mortality compared to those without (47.8% vs 18.2%, p o0.001). grades 2-4 agvhd and cytomegalovirus viremia were independent risk factors for ta-tam, and serum ldh level 4500 u/l as well as arterial hypertension were early signs of ta-tma occurrence. patients with clinically relevant agvhd (⩾ grade 2) had more ta-tam than patients without agvhd (45% vs 24%; po 0.001). tam correlated with agvhd severity; the higher the agvhd grade, the more the patients who suffered from tam. allo-hsct recipients with grades 2-4 agvhd or cytomegalovirus viremia should be closely monitored for the presence of ta-tma. at the meeting first results of histological, immunohistochemical and complement activation analyses will be presented. disclosure of conflict of interest: none. hemorrhagic cystitis (hc) after stem cell transplantation (sct) can cause significant morbidity and prolonged hospitalization. early bleeding occurs almost exclusively when using cyclophosphamide (cy) (5-25% of cases), while late onset hc are classically attributed to bkv infection, and occurs up to 58% of patients (pts) receiving myeloablative haplo-sct who had positive bk viruria (1, 2). we retrospectively studied hc cases among pts submitted to haplo-hsct in our department. thirty-eight pts receiving an haplo-sct with post-transplant cy (pt-cy) were included (table 1) . prophylaxis for cy included hyperhydratation (3 l/m 2 of 0.9% saline) and mesna administration (200 mg for each 1000 mg of cy/daily divided into three doses). hematuria was graded as follows: grade i, microscopic; grade ii, macroscopic; grade iii, with clots; and grade iv, leading to urinary retention or requiring surgical intervention (1). pts with hc and clots were treated with continuous bladder irrigation. twenty-three pts (60.5%) developed hc at a median of 9.5 days post-sct (range: 1-57). clinical severity was grade i in 6 cases (26.1%), grade ii in 13 cases (56.5%), grade iii in 2 cases (8.7%) and grade iv in 2 cases (8.7%). at the onset of hc diagnosis, bk viruria was investigated in 13/23 pts. five pts (38.5%) had bkv negative (bkv − ) hc and 8 pts (61.5%) bkv positive (bkv+) hc. bkv-hc occurred after a median of 5 days (range: 5-52) while bkv+ hc after 14.5 days (range: 5-57), respectively (p = 0.06). among bkv+ pts, 4 received iv cidofovir 5 mg/kg once a week for 2 weeks and then once every 2 weeks. median number of administrations was 3 (range: 2-4). oral probenecid was given at the dose of 2 g 3 h before and 1 g 2 and 8 h after cidofovir administration. two pts obtained a complete response (cr) after 70 and 110 days, respectively, one patient reached a partial response after 31 days and one pt failed to obtain a response. no pts developed renal toxicity during treatment. one pt received ganciclovir for concurrent cmv viremia and bkv+ hc resolved in 55 days. three patients did not receive any treatment for mild or asymptomatic cystitis. all of them achieved remission after a median of 10 days from the onset (range: 5-57.) among bkv-hc, 3 pts obtained spontaneous resolution after a median of 4 days (range: 1-52), while two pts died early after sct. finally, among pts for whom bk viruria was not available, a remission was reached in 6 of them after a median of 28.5 days (range: 12-43), while 4 pts died early after sct. in our cohort of pts, hc occurrence was of 60.5% and bkv was responsible for the 61.5% of cases. contrary to its high incidence, hc showed a relative benign course, with an overall remission rate of 87.5%, regardless of treatment. finally, we found a trend for a longer interval between sct and hc onset in pts with bkv+ hc, as compared to cy-related hc (p = 0.06). sos is a rare and serious complication of hematopoietic stem cell transplantation (hsct). it is diagnosed using the modified baltimore criteria of hyperbilirubinemia, weight gain or ascites 45% over baseline, hepatomegaly or right upper quadrant pain of liver origin. only defibrotide has been approved for the treatment of veno-oclusive disease. hdmp has been described as effective sos therapy in a few case series (1, 2). we describe our experience of treating adult sos using hdmp. objective is to retrospectively analyze the treatment efficacy and overall survival of patients diagnosed with sos after hsct and treated with hdmp. we used vilnius university hospital data base to identify patients diagnosed with sos under baltimore criteria and treated with hdmp over 2007-2016 period. patient demographics, transplant and clinical data, response, survival (kaplan-meier survival analysis) and hdmp infusion related complications were analyzed. we identified 11 patients (9 males) of whom 10 had had allogeneic hsct (6 reduced intensity conditioning) and one had received a double autologous hsct. sos was diagnosed on the median day +22 (+7 to +81 days). the median bilirubin value was 61.7 μmol/l (11.1-137 μmol/l). all patients had liver enlargement of median 210 mm (160-235 mm) on ultrasound. two patients had normal bilirubin values but displayed the remaining signs and symptoms of sos at diagnosis. patients received intravenous methylprednisolone 500 mg/m 2 every 12 h for 3 days. none received defibrotide. seven (64%) patients responded on median day +12 (+3 to +20 days) after the start of hdmp. four responded by decrease in serum bilirubin by 50% and resolution of symptoms without the need of further treatment. the remaining three responders received maintenance treatment after one course of hdmp with reduced doses of methylprednisolone until resolution of symptoms. four patients failed to respond and died of multiorgan failure on median day +12 (+5 to +41). the median observation time was 6 months (0-44 months). the median overall survival for the sos group was 8 months (range: 0-18) and it was 27 months among the responders. no adverse reactions related to hdmp infusion were observed. hdmp therapy in adult sos results in clinically relevant response rate. further prospective trials are required to assess hdmp efficacy in comparison to defibrotide or as add on therapy. prevalence of hypertension (ht) in general pediatric population is~4%, while in children treated with hematopoietic stem cell transplantation (hsct) it is up to 30%. we assessed factors contributing to the development of ht in children treated with hsct and usefulness of ambulatory blood pressure monitoring (abpm) in this population of patients. the study included 30 children (21 boys, 9 girls; mean age 10.9 years) treated with hsct for neoplasms (n = 22; 73%) or non-neoplastic disorders (n = 8; 27%). control group included 19 children (8 boys, 11 girls; mean age 12 years). abpm measurements (spacelab device) were performed before hsct and after a mean of 7 months after hsct (in 16 of the 30 children). blood samples were collected from 10 children treated with hsct and all controls. total rna extraction was performed and microarray analysis was conducted using genechip human gene 1.0 st arrays (affymetrix). in patients after hsct no antihypertensive treatment was used. mean systolic blood pressures (sbp) before and after hsct did not differ significantly from the control group. mean diastolic blood pressures (dbp) before and after hsct were 68.7 ± 12.2 mm hg and 65.4 ± 9.42 mm hg, respectively, and mean dbp percentiles were 77.1 ± 7.9 and 78.3 ± 6.7, respectively; the differences between the study group and the control group were significantly higher before hsct. mean 24-hour arterial pressure (map) percentiles were 83.3 ± 11.1 and 79.2 ± 8.7, respectively; the differences between the study group and the control group were significantly higher before hsct. before hsct and after the procedure, the european society of hypertension criteria for high normal blood pressure (bp) and ht were fulfilled in 16%/12% patients and 20%/0% patients, respectively. nocturnal bp decrease o 10% was found in 46%/ 53% patients and 420% nocturnal bp decrease in 3%/7% patients, respectively. in the control group o 10% nocturnal bp decrease was found in 10% of children and 420% nocturnal bp decrease in 5% of children. when the groups of patients before and after hsct were compared, highly significant differences were found in gene expression levels for mthfr ( in children referred for hsct a trend towards higher bp values was seen. in children assessed 6 months after hsct more abnormalities in nocturnal bp measurements were seen, which may be a predictor of ht. in children treated with hsct significant differences in the expression of ht-related genes were found. abpm was useful in bp monitoring in children treated with hsct. hypothyroidism may complicate of allogeneic hematopoietic stem cell transplantation (allo-hsct); risk factors are analysed. we studied 229 patients with aml who underwent an allo-hsct between 2003 and 2013 with different conditioning regimens (myeloablative, reduced-intensity, chemotherapybased, total body irradiation-based). thyroid stimulating hormone (tsh) and free thyroxin levels (ft4) were available in 104 patients before and after allo-hsct. median age at transplantation (n = 104) was 47 years (iqr 40-59), 37 (35.6%) were female and overall mortality was 34.6% (n = 36) ( table 1) ursodeoxycholic acid (udca) has been shown to have a protective effect in the liver complications after allogeneic stem cell transplantation (allo-sct), but it also has other immunomodulatory effects; it has been described also a potential benefice as graft-versus-host disease (gvhd) protection. we retrospectively analysed 618 patients consecutively transplanted between 1995-2014 excluding second allo-sct and those o16 years old. we analysed the differences between those with and without prophylactic udca using spps v20. results: the median age was 49 years (16-69) and 59% (362) were males. other patient characteristics are resumed in table 1 objective of study was to evaluate the impact of disease status on the outcomes of allogeneic hematopoietic stem cell transplantation (allo-hsct) in the treatment of patients with refractory and relapsed acute lymphoblastic leukemia(all). 52 patients with refractory and relapsed all, including 19 cases in advanced stage (nonremission, nr) and 33 cases in more than or equal to second complete remission(⩾ cr2), received allo-hsct after myeloablative conditioning regimen in our department. results: 51 patients engrafted successfully. the transplantation-related mortality (trm) rate of nr and ⩾ cr2 was 10.5% vs 12.1% (p = 0.815). the incidence of agvhd was 52.6% vs 57.6% (p = 0.730), including 42.1% vs 33.3% (p = 0.527) with mild (grade i-ii) and 10.5% vs 24.3% (p = 0.399) with severe (grade iii-iv) agvhd. the incidence of cgvhd was similar also(41.6% vs 57.9%, p = 0.660). with a median follow-up of 12(1.8-44.5) months, the cumulative relapse rate of nr and ⩾ cr2 was 47% vs 34.3%(p = 0.425), respectively. the estimated 2 year overall survival (os) and 2 year leukemia-free survival (lfs) rate were 42.6% vs 45.7% (p = 0.487) and 46.3% vs 46.2% (p = 0.571), respectively. multivariate analysis results showed that cgvhd was independent favorable risk factor for os and lfs of r/r all. for relapsed patients, os was significantly better with first cr duration 46 months and time to transplant ⩽2 months. alio-hsct is an effective salvage treatment option for patients with refractory and relapsed all. our retrospective analysis showed that r/r all with different status prior transplant had similar outcome post transplantation. disclosure of conflict of interest: none. the deleterious effect of intensive care unit (icu) admission during hematopoietic stem cell transplantation (sct) on patient survival is well established. however, it is unknown whether admission into the icu during the chemotherapy for the underlying disease has any impact on survival after sct. we reviewed patients who had received a first sct between the years 2000 and 2016 in our institution, and we compared the overall survival (os), relapse incidence (ri) and non-relapse mortality (nrm) between patients who required icu admission during the chemotherapy prior to the sct (icu group) with matched patients (1:2) who did not need it (no-icu group). sixty-six patients were included, 22 of them in the icu group and 44 in the no-icu group. as shown in table 1 , the main clinic-biologic variables and the sct procedure were comparable between the patient groups. the causes of icu admission for the icu group patients were: 11 (50%) respiratory failure, 4 (18%) septic shock, 4 (18%) neurological disturbance, 2 (9%) post-surgery and 1 (5%) tumor lysis syndrome. seventeen patients (77%) needed mechanical ventilation. the median time between icu admission and the sct procedure was 144 days (range: 106-1097), and the median days of icu stay were 12.5 . with a median follow-up after sct of 5.47 years (0.50-16.22) for the icu group and 4.52 (0.73-15.85) for the no-icu group, the 5 year os (ic 95%) probabilities were 49% (28-70%) and 45% (29-61%) in the icu and no-icu patients (p = 0.353), the 5-yr probabilities of relapse were 34% (14-56%) and 42% (25-58%)(p = 0.755) and the 5-yr probabilities of nrm were 32% (14-52%) and 24% (12-38%)(p = 0.333), respectively. there were no differences in either os, ri or nrm between icu and no-icu in the allogeneic or autologous subgroups considered separately. at the moment of the study, s295 12 (54%) of icu and 22 (50%) of no-icu group had died. the causes of death in the icu group were: relapse in 5 (42%), infection in 4 (33%), gvhd in 1 (8%) and gvhd plus infection in 2 (17%). the causes of death in the no-icu group were: relapse in 8 (36.4%), infection in 4 (18.2%), gvhd in 3 (13.6%), gvhd plus infection in 5 (22.7%) and veno-occlusive disease and secondary malignancy, one each (4.55%). in this series, admission to the icu before sct did not have an impact on outcomes after sct. these results suggest that these patients benefit from this treatment as much as the other patients, without expecting worse outcomes as a result of a previous icu admission. supported in part by grants rd12/0036/0029 (rticc, fejer), pi14/01971, instituto carlos iii, and sgr225 (gre), spain. disclosure of conflict of interest: none. autologous stem cell transplant (asct) is a well established treatment for several haematologic and non haematologic malignancies, either as front-line or rescue therapy. however it is associated with toxicity and complications which might lead to treatment-related mortality (trm). although decrease in trm has been reported, data about the precise reduction and detailed analysis of causes of mortality throughout years are scanty. the aim of this study was to evaluate early trm and its causes in patients who underwent an asct in a single center along the last three decades. data of all consecutive adults (415 years old) asct recipients were prospectively collected at a single center from december 1988 to august 2016 and then retrospectively analysed. trm was defined as mortality happened into the 100 days post asct or during conditioning treatment due to any cause except relapse or progression of main diagnosis. demographic characteristics, diagnosis, conditioning regimen and cause of death were analysed. data were compared for two periods: from december 1988 to december 2000 and from january 2001 to august 2016. a total of 849 patients were included, median age was 45 years (16-71) and 50.3% were male. diagnoses were: lymphoma (n = 391), multiple myeloma (mm) (n = 216), acute myeloid leukaemia (aml) (n = 93), amyloidosis (al) (n = 15), acute lymphoblastic leukaemia (all) (n = 39), solid tumours (including breast cancer and germ-cell tumours) (n = 89), chronic myeloid leukemia (cml) (n = 3), thrombotic thrombocytopenic purpura (ttp) (n = 2) and autoimmune disease (n = 1). the most frequent indication for asct was lymphoma (46.1%) and mm (25.5%). twenty patients died within 100 d from asct (trm). demographic characteristics and causes of death for this patients are shown in table1. the cumulative incidence of trm at day +100 was 2.8% (95% ci 1.9-4.1). comparing both periods, trm cumulative incidence was 7.9% (95% ci 4.9-11.8) in first period (1988-2000) vs 0.8% (95% ci 0.3-1.8) in last period (2001-2016) po 0.001. (figure 1 ). according to main diagnosis trm cumulative incidence was higher in patients diagnosed with solid tumour 6.7% (95% ci 2.7-13.2) and al 6.7% (95% ci 0.4-26.9) followed by acute leukaemia (aml/all) 4.5% (95% ci 1.9-9.1), mm 2.8% (95% ci 1.1-5.6) and lymphoma 1.3% (95% ci 0.5-2.8) p o0.03 (figure 2 ). sepsis (65%) was the main cause of death in both periods of time, and the only one cause of death in the last period. the second cause was sinusoidal obstruction syndrome (sos/vod) (20%), which only appeared in the first period. this study shows a low incidence of trm in asct recipients, with a significant decrease in the last period (2001-2016), despite the higher risk in some groups of patients such as those with amyloidosis and solid tumours. in our experience, infection is the main cause of early death in asct recipients and sos/vod has disappeared in last years as a cause of early transplant related mortality. disclosure of conflict of interest: none. incidence and risk factors for hepatic sinusoidal obstruction syndrome after allogeneic hematopoietic stem cell transplantation: a retrospective multicenter study of turkish hematology research and education group (threg) hepatic sinusoidal obstruction syndrome (hsos) is a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). the mean incidence of hsos was found to be 13.7% (0-40%) in the literature. we examined the incidence and risk factors for hsos after allo-sct. eight centers from turkey were enrolled in the study. we retrospectively evaluated the medical records of patients who were treated with allo-sct between january 2012 and december 2015. a baltimore criterion was used for assessment of hsos. two hundred eighty three (96%) of 295 patients who were treated with prophylaxis with defibrotide alone or one or more of the n-acetylcysteine, diuretics and heparin used defibrotide (10-25 mg/kg/day). the study included 889 patients (514 males/348 females) with median age of 37 (15-71) years. the demographic and clinical characteristics of patients were summarized in table 1 . the incidence of hsos was 9.3% (83). prophylaxis for hsos was used in 40 (48.1%) of patients, who developed hsos. defibrotide as prophylaxis was received by 32 of 40 (80%) of patients. hsos developed in a median of 13 (0-34) days after stem cell infusion. seventy-five (90.3%) of patients who developed hsos had infection at the time of diagnosis. forty-five of them had ascites, 63 had hepatomegaly and, 74 had weight gain. seventy-two (86.7%) of patients with hsos were treated with defibrotide after diagnosis. the median time of starting defibrotide in these patients was 14 (2-29) days. thirty-six (43.3%) of patients with hsos recovered completely and forty-seven (56.7%) of them died as a result of multi organ failure. the incidence of hsosrelated mortality in allo-hsct cohort was found to be 5.3%. in univariate analysis, statistically significant associations were not found between hsos incidence and age/sex of recipient, type of conditioning regimen, stem cell source and type of gvhd prophylaxis. on the other hand primary diagnosis of myelofibrosis, donor type, engraftment status and prophylaxis for hsos were significantly associated with hsos development. hsos prophylaxis was significantly decreased hsosassociated mortality (p = 0.006). hsos still remains a serious life-threatening complication of allo-sct. although the incidence is low, hsos is associated with increased 100-day nonrelapse mortality. hsos prophylaxis especially with defibrotide, seems to reduce hsos associated mortality in high risk patients. [p346] disclosure of conflict of interest: none. hemorrhagic cystitis (hc) is a serious complication occurring after allogenic hematopoietic stem cell transplantation (hsct) more frequent on haploidentical (haplo) hsct, with an incidence of 10-70% 1 associated mainly with the effect of cytotoxic agents such as cyclophosphamyde (cy). the conditioning regimen, bkpyv infection and graft versus host disease have an implication in the incidence. other authors related the reactivation of cmv and a previous transplantation as risk factors to hc development2. with this study we aim to describe the hc incidence and risk factors in all haplo-hsct performed in the canary islands. we analyzed all consecutive haplo-hsct from family donors performed at our hospital between 2013 and 2016. the conditioning regimen used for the transplant was the hopkins haplo protocol with high dose cy (50 mg/kg on days 3 and 4) posttransplantation (ptcy). we used as hc prophylaxis intense hydratation on the cy administration day and the following 24 h (using bladder wash only in 1 patient with cardiac dysfunction) and perfused mesna at 100% of cy dose beginning 15 min before the cy administration on 16 pts and at 20% of the last dose at 0, 4 and 8 h on all pts. we used spss v.23 to determine the cumulative incidence (ci) of hc. we performed 20 haplo-hsct, of which 10 were males (1 was transplanted 3 times) and 8 were women. the mean age was 40 (range: 16-64). the pts presented the following diagnosis: aml (10), all (1), eh (5), nhl (3), am (1). 45% of pts received the haplo-hsct in remission, 50% with refractory disease and 5% of pts did not receive previous treatment. 6 pts developed hc (36.5% ci at day +80) (figure 1a ) with a median time from haplo-hsct to onset of 23 days (range: 3-42), 1 (17%) was grade i, 4 (66%) grade ii and 1 (17%) grade iv. the grade i case did not received the mesna infusion like most of the other pts. no pts died due to hc and all cases resolved without sequelae. 12 pts received cy pre-and post-transplant and only 8 pts received ptcy. the ci at day +80 for the pts with ptcy was 33.3% and for cy preand post-transplant 38.3% (figure 1b) . we found no statistically significant difference on the ci of hc between these two groups. the development of hc was related to cy in 1 patient, who suffered from this complication on the second and third haplo-hsct. for the rest of the pts (after day +30) the hc was related to bkpyv infection, as a consequence of the immunosuppression state of the patient, we also observed all these pts had positive serum viral load for cmv. the incidence of hc associated to post-hsct high cy dose in our series is 15% lower than other ones. most of them on grade 1 or 2 and without mortality associated. the risk of hc is high, particularly in the setting of highly pre-treated patients (especially those undergoing a 2nd transplant). the development of hc after day +30 is evidently associated to bkpyv as a contributing factor for continuous inflammation and cmv reactivation (as an immunosuppression marker). in our study, hc did not have an impact on mortality of high-risk patients after haplo-hsct. the hc remains frequent with a high morbidity in particular when it is severe, often causing prolonged hospitalization and resource use. we need further studies to recognize the at-risk population early. [p347] ta-tam is not a rare post-transplant complication and it is potentially fatal. in survivors, it was often associated with longterm morbidity and chronic organ damage, mostly to the kidney with poor renal prognosis. our retrospective study showed ta-tam associated risk factors included t reg haplo hsct as the incidence was highest in this group, tbi-based conditioning or tbi based conditioning plus cyclophosphamide. although acute gvhd and infection were associated with ta-tam in retrospective studies, no association emerged between acute gvhd or infection preceding diagnosis in our series of patients. in order to prevent ta-tam we need to understand its underlying biological mechanism so we are investigating its pathogenesis by means of cytokine assays, histology and murine models. disclosure of conflict of interest: none. mortality in children requiring invasive mechanical ventilation (imv) after allogeneic hematopoietic stem cell transplantation (hsct) is known to be high. little is known about the longterm outcome of those who survive imv. we therefore reviewed the medical records of 55 children who survived s298 imv after they had received a hsct between 2000 and 2012 in the two pediatric hsct centers in the netherlands. retrospective multi-center cohort study in two university hospitals that perform all pediatric hscts in the netherlands. long-term survival of hsct recipients who had received imv was assessed. health status was reviewed more in detail for those who were still alive 2 years after discharge from the pediatric intensive care unit (picu). in the absence of standardized use of quality of life questionnaires, health status was expressed as the number of affected domains (cardio-respiratory, motor and miscellaneous, regardless of the degree of dysfunction) and level of education. health status was categorized as follows: no health problems when all four domains were normal; mild health problem when there was an abnormal score in one of the four domains; moderate health problems when there was an abnormal score in two domains; severe health problem when there were abnormal scores in three or all four domains. between january 2000 and december 2012, 641 patients underwent a hsct in the two study centers together. a total of 89 hsct recipients received imv within 1 year after hsct (14% of all hsct recipients). median time between hsct and picu admission was 59 days (iqr 17-102 days). the most common indication to start imv was respiratory failure (73%). median duration of imv was 10 days (iqr 5-18 days). 34 patients (38%) died during their picu admission. of the 55 patients who were discharged alive from picu, 27 patients were still alive 2 years after picu discharge (49% of those who survived picu admission). health status of these long-term survivors was assessed in december 2014 by hospital database review, using the most recent hospital contact. follow-up time varied from 2 to 11 years (median 6.5 years) after picu discharge. two patients (8%) had no health impairment, eight patients (33%) had mild health problems, five patients (21%) had moderate health problems, and nine patients (38%) had severe health related problems. very little is known about long-term mortality and morbidity of hsct recipients who survived imv. survival of picu treatment in pediatric hsct recipients is limited. however, long-term outcome of those who survive picu treatment seems promising: a considerable proportion of them still is alive 2 years later without obvious sequelae. this is the first study which assessed long-term outcome of imv after hsct. further studies in this population are urgently required to counsel parents and to optimize quality of life outcomes in these children. disclosure of conflict of interest: none. long-term surveillance data support lack of increase in mortality or cancer risk in brincidofovir clinical trial participants m morrison, k fitzgerald 1 , t brundage, a harrison and wg nichols 1 chimerix brincidofovir (bcv) is an orally bioavailable lipid conjugate of cidofovir (cdv), with broad-spectrum activity against doublestranded dna viruses, including cytomegalovirus (cmv), adenoviruses (adv), polyomaviruses (bk and jc viruses), and orthopoxviruses. bcv is being evaluated for prevention of cmv and other dna viruses in high-risk hematopoietic cell transplant (hct) recipients, and for the treatment of serious adv infection. bcv is also being developed for the treatment of smallpox under the us fda's animal efficacy rule. because bcv, cdv, and other marketed nucleoside analogs are reported to be carcinogenic in rodents, a registry was established to evaluate the long-term safety of bcv in subjects who have participated in bcv clinical studies. to date, the registry includes prior participants from study 301 (a placebo (pbo)-controlled study of bcv for cmv prevention) and study 304 (a single-arm study of bcv for adv treatment). subjects are encouraged to consent for long-term follow-up in the registry following participation in bcv clinical studies. registry participants are followed at 6-month intervals for a minimum of 3 years from the time of completion of the parent study. development of malignancies (new or relapsed), lifethreatening or fatal adverse events (aes) assessed as potentially related to bcv, and subjects' vital status are collected. a total of 649 subjects were enrolled in the parent studies (302 bcv and 148 pbo from study 301, 199 bcv from study 304). of these, 291 are enrolled in the registry as of 24 october 2016 (155 bcv and 80 pbo from study 301, 56 bcv from study 304). bcv recipients in the registry are 60% male, 84% white, with a median age of 47 ( o1-76) years, similar to the bcv recipients in the parent studies. the median duration of follow-up is 12 (0-30) months, with 80% of subjects continuing in follow-up at the time of analysis. all-cause mortality from the time of first dose in the parent study through current registry follow-up is 25% for bcv vs 22% for pbo (p = 0.559) in study 301, and 51% for bcv in study 304. all-cause mortality in the registry since completion of the parent study is 21% bcv vs 24% pbo for subjects from study 301 (p = 0.618) and 14% bcv for subjects from study 304 (figure 1) . the incidence of a new malignancy in registry subjects from study 301 is 17% bcv vs 23% pbo (p = 0.295), and the incidence of relapsed primary malignancy is 12% bcv vs 21% pbo (p = 0.055). in registry subjects from study 304, 7% developed a new malignancy and 4% had a relapse of the primary malignancy. no bcv-related life-threatening or fatal aes have been reported to date in the registry. registry data collected to date support no increase in late mortality or increased risk for carcinogenicity in patients treated with bcv. long-term surveillance for cancer risk and other drivers of mortality is important for novel compounds undergoing development in hct and other immunocompromised patient populations, with high background risk for these outcomes. [p351] disclosure of conflict of interest: all authors of this abstract are employees and stockholders of chimerix. hematopoietic stem cell transplantation (hsct) is a medical procedure that allows the cure of many paediatric diseases. it has been described an increased risk of new malignancies in this population and it represents an important cause of late mortality. we analyzed the late evolution of 371 patients submitted at pediatric age to hematopoietic transplantation (hsct) (allogeneic or autologous) in santa creu i sant pau hospital between 1984 and 2013. a total of 434 hsct was analyzed. it has been calculated the cumulative incidence of secondary malignancies at 30 years of follow-up. it has been done univariate and multivariate analysis of risk factors through χ 2 -test and binary logistic regression method (odds s299 ratio, or). it has been studied the relative risk (rr) for new malignancies through comparison of observed cases in our cohort with the expected cases in the general population. we observed 19 cases of secondary malignancies with a cumulative incidence of 6% at 15 years, 12% at 20 years and 36% at 30 years of follow-up. the risk was higher of expected in general population for each tumor type and in the different range of age, being the rr for malignancies in our cohort of 51.4 at 30 years of follow-up. solid tumors were the most prevalent malignancies (16 out of 19 cases). the median time of latency from hsct to diagnosis of malignancy was 16 years (1-31 years) . the thyroid tumors were the later ones and hematologic malignancies the earliest to be developed. chronic graft versus host disease was a statistically significant risk factor in univariate (or = 16; p = 0.006) and multivariate analysis (or = 15.4; p = 0.000). total body irradiation of conditioning was a significant risk factor only in multivariate analysis (or = 4.3; p = 0.03). previous radiotherapy was a significant risk factor only in univariate analysis (or = 3.1; p = 0.04). mortality was 42% (8 out of 19) between patients with a new malignancy and it was the cause of death for all the cases. we observed an incidence of secondary malignancies after hsct of 5.1% that is significantly higher compared to the expected in the general population (p = 0.000). the factors that have been related to an increased risk were chronic gvhd, tbi and previous radiotherapy. microalbuminuria defined, as urinary albumin: creatinine ratio (acr) of 30-300 mg albumin/g creatinine is a marker of endothelial dysfunction and inflammation. in general populations albuminuria predicts development of chronic kidney disease (ckd) and cardiovascular disease (1). in the general population microalbuminuria is associated with the metabolic syndrome (2) . in patients with hypertension, diabetes and the critically ill, it is a marker of adverse events and poor outcomes. following hematopoietic cell transplant (hct), microalbuminuria at day 100 was associated with a four-fold increased risk of chronic kidney disease (ckd) at 1 year in a single centre study; macroalbuminuria at day 100 was associated with a 6.8-fold increased risk of non-relapse mortality (3) . international guidelines for adult and children survivors of hct recommend that proteinuria is assessed at least annually (4, 5) . there is a paucity of data on the prevalence and implications of micro and macroalbuminuria in long-term survivors (410 years) of adult hct, however. this was a single-centre retrospective study conducted in patients attending a dedicated clinic for long-term (minimum 10 years) survivors of hct. we investigated prevalence of albuminuria and its association with renal disease, cardiac disease and the metabolic syndrome. of 55 patients, 8 were treated for acute leukemia, 2 for aplastic anaemia and 46 for cml. the median follow up time was 24 years (range: 13-37 years) and the median age at follow up was 55 years (range: 24-81 years). for 36/55 urinalysis was normal (group a) and 19 (34%) had microalbuminuria (group b). none had macroalbuminuria. group b were significantly more likely to have ckd grade 2-4 (egfr o 60) compared to those in group a (p = 0.001). group b patients were significantly more likely to have diabetes or impaired glucose tolerance 7/19 (37%) vs 2/35 (6%) in group a (p = 0.005). group b patients were also significantly more likely to have dyslipidaemia (p = 0.019 ) with 14/19 (70%) affected vs 23/35 (37%) in group a. cardiac disease and hypertension were more frequent in group b, 4/19 (21%) and 7/19 (37%), respectively vs group a 3/35 (9%) and 8/35 (22%) but these data were not statistically significant. the more features of the metabolic syndrome present, however, (elevated hba1c, /glucose, dyslipidaemia, hypertension) the more likely a patient was to have microalbuminuria (p = 0.007). our data demonstrates that microalbuminuria is a frequent finding in long term survivors of hct. patients with microalbuminuria are more likely to have ckd grade 2 or below. they are also more likely to have diabetes and dyslipidaemia. as this was a retrospective study we are not in a position to comment on whether microalbuminuria is predictive of the development of renal disease, metabolic syndrome or cardiovascular disease in this group of patients. this warrants further study as intervention, for example with ace inhibitors, may have the potential to reduce morbidity. the purpose of the study is the improvement of transplantation techniques and supportive care lead to an increasing number of long-term survivors after allogeneic hematopoietic stem cell transplantation (ahsct). recipients of ahsct have a higher prevalence of cardiovascular risk factors. ambulatory blood pressure measurement (abpm) is the 'gold standard' to diagnose arterial hypertension (ht). the prevalence and treatment control of ht by abpm is unknown in ahsct patients (pts). this prospective single center study at university hospital basel included all pts ⩾ 1 year after ahsct in complete hematological remission during annual follow-up consultation. office blood pressure (obp) was measured on both arms after 5 minutes rest. abpm by noninvasive continuous bp monitoring (pulse transit time method) was performed on the same day. ht was defined as obp ⩾ 140/90 mm hg, mean systolic bp ⩾ 130mm hg on abpm s300 (bp 24 ) and/or current use of antihypertensive drugs. 175 pts (39% female) were included with median age of 53 years (range: 19-75) and 9 years (range: 1-33) after transplantation. 108 (62%) pts received total body irradiation-based conditioning, 70 (40%) pts had chronic graft-versus-host disease, and 39 (22%) required immunosuppression. mean bmi (kg/m 2 ) (± sd) was 25 ±5, with 22 (13%) pts 430. twenty-seven (15.4%) pts were current smokers. fourty-three (25%) pts had chronic kidney disease (egfr o60 ml/min/1.73 m 2 ) and 17 (10%) diabetes. 82 (47%) pts were on antihypertensive drugs consisting of ace/at-ii-inhibitors in 55 (31%), calciumchannel blockers in 18 (10%), beta-blockers in 32 (18%) and diuretics in 24 (14%) pts. thirty-nine (22%) pts were on ⩾ 2 drugs. among our cohort 47 (27%) pts were normotensive without antihypertensive treatment (mean age 46 ± 13 years, 62% female and mean bp 24 (systolic/ diastolic bp) 113 ± 8/76 ± 8 mm hg). 128 (73%) pts were hypertensive and/or on antihypertensive treatment. untreated ht was diagnosed in 46 (26%) pts (mean age 52 ± 13 years, 41% female and mean bp 24 of 147 ± 22/91 ± 12 mm hg), including 14 (8%) with white-coat hypertension and 9 (5%) masked hypertension (normal obp, high abpm). in the group of pts with current antihypertensive medication 32/82 (39%) were controlled (mean age 55 ± 13 years, 25% female, and mean bp 24 119 ± 9/77 ± 7 mm hg) whereas 50/82 (61%) were hypertensive on abpm (mean age 55 ± 11 years, 24% female, mean bp 24 146 ± 13/90 ± 10 mm hg). thirty-four (68%) pts with uncontrolled ht were already hypertensive at obp. although long-term survivors after ahsct are known to be at elevated cardiovascular risk, diagnosis of arterial hypertension was missed in every fifth patient. the proportion of controlled hypertension is poor with only 39%. disclosure of conflict of interest: none. myasthenia gravis (mg) is a rare complication of allogeneic stem cell transplantation (sct) and is often associated with graft-versus-host disease (gvhd). we report a 49-year-old man who presented with oculobulbar and neck weakness 30 months after an unrelated donor, allogeneic sct for chronic myeloid leukaemia (cml). he was diagnosed in 2009 with chronic phase cml. this responded poorly to tyrosine kinase inhibitors (tkis) and he was found to carry the t315i mutation with additional monosomy 7. he underwent a fully hla matched unrelated donor sct with y 90 -anti cd66 targeted radiotherapy, fludarabine, melphalan and alemtuzumab conditioning. he had grade 1 cutaneous gvhd on ciclosporin withdrawal but no other significant gvhd. he has an immune mediated neutropenia since 4 months post sct and has reduced immune reconstitution as demonstrated by a sub-normal absolute cd4 level. he remains on pneumocystis prophylaxis and has not experienced increased infection. chemotherapeutic agents have a cytotoxic effect on the oral mucosa and is a major problem following cancer treatment. cooling the oral mucosa in conjunction with chemotherapy infusion, using ice chips, is known to reduce the severity of oral mucositis (1, 2) . although effective, ice chips are perceived as uncomfortable. the aim of the present study was to determine the optimal cooling temperature to prevent adverse effect of chemotherapeutic agents using tissue engineered oral mucosal models (teom). teom were incubated at 35°c, 30°c, 25°c or 20°c for 30 min followed by exposure to 162 μg/ml of 5-fu for 2 h (control models were incubated at 35°c). teom were then washed and further incubated for 48 h at 37°c co 2 . cell viability and inflammatory cytokine production (il-6 and tnf-α) were measured using (prestoblue) and (elisa), respectively this study demonstrates an increased capacity to restore cell viability with decreasing temperature (figure 1a ). teom treated with 5-fu further showed an increased secretion of the pro-inflammatory cytokines tnf-α and il-6 at all temperatures compared to un-treated controls. for il-6, secretion increased markedly when cells were incubated with 162 μg/ml 5-fu at 35°c and 30°c compared to cells incubated with medium alone at 35°c (figure 1b) . for tnf-α, secretion was significantly higher (p o0.05) in cells treated with 162 μg/ml 5fu at 35°c compared to untreated mucosal models and mucosal models treated with162 μg/ml 5fu but incubated at 20°c (figure 1c ). teom models incubated at 20°c has an increased capacity to restore cell viability following exposure to 5-fu. incubation at 20°c further reduces the release of pro-inflammatory cytokine compared to those incubated at 35°c. (2) and one received fludarabine and cyclophosphamide. all patients received campath-1h as part of the conditioning regimen. stem cell source: peripheral blood stem cells 73 patients and 3 bm. comorbidity was assessed using the haematopoietic cell transplantation co-morbidity index (hct-ci), with 16 patients (21%) having no co-morbidities, 35 (46%) a co-morbidity index of 1-3 and 25 (33%) had a score ⩾ 4. follow up of survivors ranged from 1 to 148 months (median: 32 months). at the specified end point 29 patients had relapsed (38%) with an actuarial 3-year relapse rate of 55%. there were 34 deaths (44%). relapse (23) was the main cause of death with transplant related mortality of 5% (4) at day 100, 8% (6) at 6 months and 13% (10) at 1 year. the actuarial os at 3 years was 48%, with a 3-year dfs of 39%. of the surviving relapsed patients all received chemotherapy and donor lymphocyte infusions resulting in effective recovery of remission, showing the utility of this approach. in terms of co-morbidity, actuarial survival rates were 60% in those with an hct-ci index of 0, 39% with an index of 1-3 and 50% with an index ⩾ 4. the results of this retrospective study indicate that allosct using reduced intensity conditioning regimens can be an effective treatment strategy for older patients with high risk myeloid malignancies including those with significant co-morbidities. relapse remains the main cause of treatment failure and strategies to reduce relapse risk are required. patients that relapse post allosct may respond to further treatment such as azacytidine or intensive chemotherapy and donor lymphocyte infusions. (3) whether patient-related variables were associated with disagreement. this is a secondary analysis of a cross-sectional multicenter study where patients and clinicians completed an identical qol questionnaire (fact-bmt) at day 90. clinical and demographic variables as well as anxiety and depression (hads) were collected. agreement was analyzed with the intraclass coefficient correlation (icc). rates of under-and over-estimation were calculated. logistic regression models identified predictors of disagreement. we analyzed 96 pairs of questionnaires, filled in by 96 patients and 11 clinicians. patients' median age was 54 years, 50 (52%) were men, and 50 (52%) received an allogeneic hct. clinicians' median age was 42 years, 7 were men and had worked on the transplant field for a median of 12 years (range: 3-23). agreement on qol was moderate (icc = 436). exploratory analyses revealed that agreement for emotional (icc = 092) and social (icc = 270) wellbeing was poor, whereas it was moderate for physical (icc = 457), functional (icc = 451) and bmt concerns (icc = 445). patients' wellbeing was overestimated in 41-59% of the categories of wellbeing parameters, and underestimated in 10-24%. patient-related variables explained 12-17% of the variance on disagreement across scales. specifically, anxiety contributed to disagreement in all subscales, except in social wellbeing, where non-significant univariate associations were observed (p40.05). type of transplant (allogeneic vs autologous), performance status, and graft-versus-host disease were not associated with disagreement (p40.05). patients and clinicians agreement on qol is suboptimal, particularly on emotional and social wellbeing. patients' wellbeing cannot be estimated from other sources than themselves. these results highlight the unmet needs of hct recipients with respect to qol-related issues; an outcome that must be addressed by hct programs since their wellbeing is as important as survival endpoints. disclosure of conflict of interest: none. . we wanted to test the function and the safety of picc device as alternative to standard cvc in patients submitted to autologous stem cell transplantation (abmt). the primary end point of the study was to individuate the cause leading to the failure of picc (its removal or the needing of another cvc during the abmt procedure). secondary end points were the correct function of the device and its praticity. twenty patients submitted to abmt for multiple myeloma (18) or lymphoma (2) experienced a double lumen picc device (17) or a single lumen (3) if the patient already carried a permanent single lumen cvc such as hickman or port-a-cath. we excluded from this experience patients with high risk of life-threatening situation or high risk of intensive care already before abmt. picc devices were placed from a specialistic nurse team by ultrasound identification of a deep venous vessel in upper arms. melphalan 200 or ceam were the standard conditioning regimens employed in myeloma and lymphoma abmt respectively. we considered a failure all the causes leading to the removal of picc or requiring another cvc before the end of the transplant procedure. at last we collected nurses and clinicians opinions about the picc functionality. no complication has been recorded in positioning phase. 19/20 patients maintained the picc device for all the time of transplant procedure. only one patient needed to remove the device for infection. the opinion of nurses and clinicians about the picc device was a significatively slower speed of infusion and resistance to the flow; in fact, 11/20 patients needed an infusional pump. the idraulic resistance of the catheter was particular evident against cellular fluids (stem cells suspension, transfusions of blood and platelets). for this reason picc seems to be less indicated in patients requiring many endovenous infusions (nurses' opinion). the rate of infection of picc devices seems to be lower compared to cvc, but the number of cases tested in this experience is too limited for definitive conclusions about it. for other aspects picc is similar to other cvcs. picc seems to be a valid alternative to standard cvc in patients who do not require intensive care, and in particular in patients with low intensity abmt who do not present a high number of endovenous infusions. maybe picc is less burdened of infections respect to normal cvc. this fact, summed to the lower risk during the positioning of the device, leads to consider the use of this device in abmt setting for standard risk patients. disclosure of conflict of interest: none. there are only few algorithms for the selection of hlamismatched unrelated donors, when no fully matched donor is available. indirect recognition of hla-mismatches can be predicted using the model of 'predicted indirectly recognizable hla epitopes' (pirche). the pirche model is a recently developed computer-based strategy, which classifies hladerived epitopes that are potentially presented by patientdonor shared hla-molecules. we performed a multicenter retrospective study evaluating the impact of pirche on outcome after allogeneic stem cell transplantation from hla 9/10 matched unrelated donors. the study cohort included 1997 adult patients who had undergone allogeneic stem cell transplantation for aml or mds. pirche scores were computed for 424 recipients of hla 9/10 matched unrelated donor transplants (9/10mud) using a web-based tool. primary endpoint was overall survival at 2 years. patients with a 9/10 mud were divided into 2 groups according to the sum of pirche i+ii values (pirche score). eighty-five (85) patients had a pirche score of 0 (no pirche detected), 339 a pirche score 40. km estimate of 2 year os was higher for 9/10 mud with pirche score = 0 compared to pirche score 40: 57% (95% ci: 51-63%) vs 47% (95% ci 41-53%), p = 0.04. os was similar for 9/10 mud with pirche score = 0 and 10/10 mud (57% vs 55%). cox regression analysis revealed poorer os for pirche scores 40 (rr 1.5, 95% ci: 1.0-2.1, p = 0.03). cumulative incidence of nrm at 2 years was lower for 9/10 mud with pirche score = 0 compared to pirche score40 (20% vs 32%, p = 0.05). multivariate cox regression analysis revealed poorer nrm for pirche score40 (rr 1.7, 95% ci: 1.0-2.9, p = 0.03). cumulative incidence of agvhd grade 2-4 at 6 months was not significantly different for 9/10 mud with pirche score 0 compared to pirche score40 (23% vs 30%, p = 0.2). cumulative incidence of cgvhd at 2 years was lower for 9/10 mud with pirche score 0 compared to pirche score40 (31% vs 49%, p = 0.04. our findings require confirmation, ideally in a large prospective cohort study. if validated, the pirche model would allow selection of permissible hla-mismatches that may be associated with an improved transplant outcome in terms of reduced nrm and better os. [p364] disclosure of conflict of interest: none. this study was supported by a research grant from pirche-ag to the university medical center, hamburg-eppendorf. pretransplant liver dysfunction has been recognized as a risk factor for complications and mortality after allogeneic hematopoietic cell transplantation (allo-hct). however, there is no consensus on the optimal way to evaluate liver function in hct candidates. transient elastography (te) is a noninvasive method for diagnosing liver damage and cirrhosis. while elastography is widely used in the setting of viral hepatitis, its possible role in allo-hct recipients has not been deeply evaluated. patients receiving allo-hct in our center from may 2014 are scheduled to receive pretransplant evaluation by a hepatologist under a prospective protocol. the evaluation includes a hepatologist consultation, liver function and infectious serology tests and te. all patients receive ursodiol from hct admission to day +30. this study constitutes the first evaluation of the ongoing protocol for patients receiving their first allo-hct from may 2014 to august 2016. sixty patients received a first allo-hct during the study period. sixteen patients did not undergo hepatologist evaluation due to timing issues (n = 6), unstable medical condition (n = 4) or other reasons (n = 6). finally, 44 patients received pretransplant evaluation by a hepatologist under the current protocol and constitute the study population. median age at transplantation was 51 years (range: 21-69). most patients received a transplant for acute leukemia (n = 23, 52%) or non-hodgkin's lymphoma (n = 10, 23%) mainly from hla matched unrelated donors (n = 21, 48%). thirty-two patients received reduced-toxicity regimens (73%). graft-versus-host disease (gvhd) prophylaxis consisted of tacrolimus in combination with another agent. median follow-up for survivors of 14 months (range: 3-29). median elastography was 5.6 kpa (range: 2.9-13.7). considering the hct-ci categories on hepatic dysfunction, 38, 6 and 0 patients scored 0, 1 and 3 points, respectively. there were two cases of veno-oclusive disease (vod). overall survival and non-relapse mortality of all patients at median follow-up were 76% (95% ci 69-83) and 22% (95% ci 14-30), respectively. in the univariate analysis, median elastography was not associated with a higher risk of nrm (p-value = 0.13), os (p-value = 0.11) or hepatic chronic gvhd (p-value = 0.32). the two patients with vod had normal pre-hct transaminase levels and te. this first analysis of an ongoing protocol with universal pre-hct evaluation of hepatic function indicates that increased values of transient elastography are not associated with higher nrm or lower os after the procedure. further studies including a larger number of patients are needed in order to clarify the possible role of elastography in the hct setting. disclosure of conflict of interest: none. allogeneic hematopoetic stem cell transplantation (hsct) remains associated with a high morbidity and mortality in spite of advances in hsct management. specifically, pulmonary complications account for a substantial proportion of deaths within the first 100 days after hsct. therefore, identification of lung dysfunction and additional comorbidities are crucial for preventive strategies in hsct. given the inconsistent association of pretransplant lung function s305 parameters on mortality after hsct and the significant changes in hsct care over the last decades, the aim of our study was to assess the effect of pulmonary function and comorbid conditions on mortality in patients undergoing hsct for hematological disorders. we retrieved relevant clinical data of all consecutive patients at the hematology division of the basel university hospital with a transplant for hematological disorders between 2008 and 2015. we examined the lung function at baseline and 3, 6 and 12 months after hsct-including the 1 s forced expiratory volume (fev1% of predicted), fev1/vcmax and diffusing capacity for carbon monoxide (dlco, adjusted for hemoglobin concentration). in addition, we assessed pretransplant conditions such as age, sex, karnofsky performance status (kps), donor type and various risk scores in hsct (hematopoietic cell transplantation comorbidity index (hct-ci), european society for blood and marrow transplantation (ebmt), revised pretransplant assessment of mortality score (pam)). using uni-and multivariate cox proportional-hazards regression analysis, we evaluated patient-and transplant-related risk factors for all-cause mortality by including the following categorical candidate variables: fev1 (⩾80% vs 50-79% vs o50% of predicted), kps ( o90% vs ⩾ 90%), age ( o54 vs ⩾ 54 years), conditioning intensity and donor type (matched-related vs mismatchedrelated vs matched-unrelated vs mismatched-unrelated). within the study period, 429 patients with predominantly acute leukemia (64%) or lymphoproliferative disorders (28%) underwent myeloablative (n = 330) and non-myeloablative hepatic veno-occlusive disease/sinusoidal obstruction syndrome (vod/sos) is a potentially life-threatening complication of conditioning for hematopoietic stem cell transplantations (hsct). recombinant thrombomodulin (rtm) is a new drug for treating disseminated intravascular coagulation (dic) and is an endothelial anticoagulant cofactor that promotes the thrombin-mediated formation of activated protein c (apc). rtm has been used to treat vod/sos, but its ability to prevent vod/sos has not been established. we evaluated the cases of 19 pediatric hematology and oncology patients (8 (43%) acute myeloid leukemia, 3 (16%) acute lymphoblastic leukemia, and 4 (21%) neuroblastoma patients, and 1 (5%) patient each with myelodysplastic syndrome, rhabdomyosarcoma, hemophagocytic syndrome (hlh), and wiskott-aldrich syndrome) who underwent hsct at our institution between 2007 and 2014 and had ≧ 1 risk factors for vod/sos. these risk factors included previous treatment with gemtuzumab ozogamicin (go), receiving 42 hsct, undergoing conditioning with busulfan (bu), and being diagnosed with hlh. the patients who received hsct after 2012 (n = 8; rtm group) were treated with rtm as a prophylaxis against vod/sos (380 u/kg per day for 7 days; from days 7 to 13) together with ursodeoxycholic acid (urso) and low-molecular-weight heparin (lmwh), and the others (n = 11; control group) were only treated with urso and lmwh. the incidence of vod/sos was evaluated, and various coagulation parameters and markers of endothelial injury (plasminogen activator inhibitor type (pai-1) and apc) were measured in both groups. the patients' median age was 2 (range: 0-18) years, and 11 (58%) were male. clinical characteristics, including vod/sos risk factors, were wellmatched in both groups. the risk factors possessed by the patients included receiving 42 hsct (9/19, 47%), previous go treatment (6/19, 32%), conditioning with bu (3/19, 16%), and a diagnosis of hlh (1/19, 5%). although vod/sos occurred by post-hsct day +35 in 3 (27%) patients in the control group, vod/sos was not seen in the rtm group. two of the former 3 patients (2: previous treatment with go, 1: a diagnosis of hlh) suffered severe vod/sos, and 1 (a diagnosis of hlh) died of the condition. no grade 3/4 adverse events involving bleeding or severe organ damage were reported in the rtm group. interestingly, the mean peak value of pai-1 and apc (markers of endothelial injury) were significantly lower in the rtm group (table 1) . [p367] disclosure of conflict of interest: none. protective effect of early human cytomegalovirus reactivation on relapse of myeloproliferative disorders after allogeneic hematopoietic stem cell transplantation z peric 1,2 , j wilson 2 , n durakovic 1,2 , l desnica 2 , a ostojic 2 , vv rezo 2 , v marekovic 1,2 , r serventi-seiwerth 2 and r vrhovac 1,2 1 school of medicine, university of zagreb, zagreb, croatia and 2 university hospital centre, zagreb, zagreb, croatia there have been conflicting results regarding the association between early cytomegalovirus (cmv) reactivation and decreased incidence of relapse after allogeneic hematopoietic stem cell transplantation (allo-hsct). this prompted us to retrospectively evaluate the potential impact of cmv reactivation on transplantation outcomes in a study population of 161 consecutive adult patients who underwent allo-hsct in our institution and were treated and followed in a homogenous manner. patients were monitored for cmv reactivation once weekly for the first 100 days after allo-hsct. monitoring was done with a real time qpcr with lower limit of detection of 150 genome copies per ml of blood. when cmv viremia was detected, all patients were treated with intravenous ganciclovir or oral valganciclovir untill two consecutive negative qpcr assays. univariate and multivariable proportional hazards models using the fine and gray approach were considered to evaluate the variables for relapse, treating death as competing event. between 2011 and 2014, 97 male and 64 female patients underwent allo-hsct at a median age of 46 years (range: 18-64). among them, most patients were treated for myeloid malignancies (74 aml, 11 mds and 24 mpn with 11 cml, 11 mf and 2 cmml), while the rest had lymphoproliferative disorders (24 all, 10 nhl, 6 mm, 5 mh and 6 cll) and one patient had aplastic anemia. the donors were unrelated in 79 cases, related in 77 patients and haploidentical in 5 patients. most of the patients (70%) received peripheral blood stem cells after a reduced-intensity conditioning regimen (56%). with a median follow-up of 23 months, early cmv reactivation occured in 62% patients at a median of 27 days after transplantation and did not affect relapse incidence in patients with lymphoproliferative disorders. on the contrary, the cumulative incidence (ci) of hematologic relapse in patients with myeloproliferative disorders (aml and mpn) at 20 months after allo-hsct was 36% (95% ci, 21-52%) in patients without, opposed to 18% (95% ci, 10-29%) in patients with cmv reactivation (p = 0.04). however, cmv reactivation did not significantly affect (p = 0.21) overall survival between patients with (64%; 95% ci 53-77%) and without cmv reactivation (48%, 95% ci 34-68%). a striking and previously unreported correlation between cmv reactivation and relapse was found in patients with mpn; the ci of relapse was 50% (95% ci, 12-80%) in patients without, opposed to only 6% (95% ci, 25-100%) in patients with cmv reactivation (p = 0.01). a substantial and independent reduction of the relapse risk in myeloproliferative disorders (aml+mpn) associated with early cmv reactivation was confirmed by multivariate analysis using time-dependent covariate functions for high-risk disease, use of atg, chronic graft-versus-host disease (hazard ratio 3.33; 95% ci, 1.09-10.09, p = 0.03), and cmv reactivation (hazard ratio 2.37; 95% ci, 1.05-5.37, p = 0.04). in summary, this report supports an independent role of cmv reactivation on relapse in patients with myeloproliferative disorders. to our knowledge, we are the first to show a significant reduction of relapse incidence in patients with mpn, even though our findings are based on a relatively small number of patients. however, this putative virus-versus-myeloproliferation effect definitely warrants further research. [p368] disclosure of conflict of interest: none. final result of fact-bmt is score ranged 0-148 point (the higher the score, the better qol). for qualitative assessment of donor-recipient relationship, the adult sibling relationship questionnaire (asqr) in polish version was used. the asrq-s consists of 47 items which are spread over eight scales designed to investigate three factors: warmth, conflict and rivalry. the questionnaires were given to both subgroups, donors and recipients of msd-hsct and the results were compared to each other. the overall result of the fact-bmt questionnaire was 109.0 ± 7.5 points, which means that the examined group generally described their qol as 'quite good'. the best results were found in functional well-being (25.6 ± 0.9), while the worst in emotional well-being (20.7 ± 0.5) dimension. statistically, the qol score was not influenced by age at hsct (p = 0.256), current age (p = 0.378) or gender (p = 0.117) of the respondents. the recipients scored highest on warm factor (62.6 ± 7.8), while donor respondents scored slightly higher rivarly (60.3 ± 6.0) than warm (45.7 ± 5.4). the second dimension scored by recipients was rivarly (40.7 ± 6.8). conflict scores were lowest, although donor respondents scored higher on these than recipient respondents (38.6 ± 5.5 in donors vs 32.9 ± 3.6 in recipients). statistical analysis revealed that the being a donor or recipient of msd-hsct determines the level of rivarly in the sibling relationship (p = 0.007) with no impact on warm and conflict dimension. health-related qol in transplanted patients is quite good. sibling donor-recipient relationship is unbalanced with recipient respondents being more likely to assess a warm relationship, while rivalry was more likely to be present among donor. further multicenter studies based on larger cohort of patients are necessary to assess sibling relationship after transplantation life experience. disclosure of conflict of interest: the authors have nothing to disclose. this work was supported by grant from poznan university of medical sciences (502-14-01104119-10398). rate of re-admission in patients undergoing allogeneic transplants from identical siblings, unrelated donors or haploidentical donors f sora 1 , s sica, l laurenti, p chiusolo, s giammarco, i innocenti, e metafuni, a corbingi and a bacigalupo 1 department of hematology, catholic university of rome hla identical siblings (sib), unrelated (ud) and family hla haploidentical donors (haplo) are currently being used for patients undergoing an allogeneic transplant (hsct) for hematologic disorders. gvhd prophylaxis is usually different, and is commonly based on a calcineurin inhibitor (cni) and methotrexate (mtx) with or without atg for sibs and uds, wheres post-transplant cyclophosphamide (pt-cy)+a cni and mycophenolate (mmf) is used for haplos. we will refer as sib, ud, haplo platform, the combination of a given donor and a given gvhd prophylaxis. the outcome of these three different platforms is usually measured in terms of gvhd, non relapse mortality (nrm) and survival. days of admission and readmissions are important in terms of morbidity, but also of costs, and are usually not reported. aim of the study: assess the duration of the first admission and the incidence of a new re-admissions, in the first 100 days after the transplant. we retrospectively analyzed 151 patients from 2012 to2016. sixtyone received peripheral blood stem cell graft from an ud, and gvhd prophylaxis with cya+mtx+atg; 54 received a peripheral stem cell graft from a sib and gvhd prophylaxis with cya +mtx; 36 patients received bone marrow hsct from haplorelated donor and pt-cy+cya+mtmf. patients characteristics are shown in table 1 . relapses were excluded from the readmission analysis. the median time from the transplantation to discharge was 25 days for ud, 27 for haplo and 21 days for sib: there was no significant difference between haplo vs ud (p = 0.6), whereas the admission of both haplo and ud was longer than sibs (po 0.01). first readmission. fiftyone patient out of 151 required of a new admission for complications after tranplant (28 out of 61 after mud (46%), 13 out of 54 (24%) using a sibling donor and 10 out of 36 using an haploidentical donor (28%)). there were significantly more re-admissions in the ud vs sib group (0.01) and a trend for more ud readmissions vs haplo (p = 0.08); siblings had the lowest number of readmissions. time to neutrophil engraftment was comparable in haplo vs ud patients (p = 0.1) and in sib vs ud (p = 0.1); the time was longer in haplo vs sibs (p o0.01). the reason to re-admitted the patients in the hospital after tranplantation was fever in 14 out of 28 (50%) new admissions in ud setting,11 out of 13 (85%) in sib and 7 out of 10 (70%) in haplo; acute gvhd was the cause for re-admission in 5 out of 28 (18%) ud, 1 out of 13 (8%) sib and none in haplo. the other causes for re-admission in the hospital were hemorragic cistitis, thoracic or abdominal pain. second re-admission. of hospitalization is registered in 10 out of 61 patients in ud (7 for aghvd and 3 fever), 2 out of 54 (4%) in sib (2 episodes of fever) and 1 out of 36 (3%) patients in haplo (1 for fever and 1 progressive disease). also for second episodes, ud grafts had significantly more admissions compared to haplo and sibs. third re-admission was recorded only in ud patients (5 out of 61-8%). this study shows a comparable duration of admission for transplant for haplo and ud patients, both significantly longer than sib grafts. the number of re-admissions is comparable in haplo vs sibs and there is a trend for lower number of re-admission as compared to uds. we interpret this outcomes with caution given the relatively small sample size and heterogeneous disease population included. future studies need to confirme our results. disclosure of conflict of interest: none. prolonged thrombocytopenia (pt) is frequent event after allogeneic haematopoietic stem cell transplantation (hsct), especically in haploidentical transplantation, which could be up to 15% according to our previous report. pt has significant negative impact on long-term outcomes, mainly due to increased non-relapse mortality. however, there are no efficious treatment. in this study, we report the preliminary results of recombinant human thrombopoietin (rhtpo) in treating this kind of patients. from 2016.7 to 2016.10, 16 patients were enrolled under the following inclusion criterion: (1) diagnosed with dpe or sfpr after allogeneic stem cell transplantion; (2) no sign of minimal residual disease or recurrence of hematological malignancy; (3) not using other tpo receptor agonist or il-11 within 1 month of enrollment. pt include delayled platelet engraftment (dpe) and secondary failure of platelet recovery (sfpr). the former was defined as failure to achieve platelet counts ⩾ 20 000/μl for 7 consecutive without transfusion until 35 days after transplantation, while the latter was defined as a decline in platelet counts below 20 000/μl for 7 consecutive days, or requiring transfusion support after achieving sustained counts without transfusions for 7 consecutive days after hsct. the prescription of rhtpo was 15 000 iu once daily for 28 days, or if patients achieve platelet ⩾ 50 000/μl for 3 consecutive days with a duration o28 days. response was defined as success of achieve platelet counts ⩾ 20 000/μl for 7 consecutive days. the response time was defined as the first day achieve response from the start of prescription. the primary end point was response rate, and the secondary end point was reponse time. a total of 16 patients were enrolled, including 7 males and 9 females. the median age was 30 (18-50) years. all patients received haploidentical transplantation. among these patients, 10 patients were dpe and 6 were sfpr. all patients received a 28-day prescription. the overall response rate was 50% (8 out of 16) in the overall population, while 60% (6 out of 10) in dpe and 33.3% (2 out of 6) in sfpr, respectively. among the 10 patients with response, the median response time was 21 (10-28) days from the first dose of rhtpo. after 4 weeks of the last dose of rhtpo, none of the responsed patient lose response. since the followup time is too short, the impact of relapse, gvhd were not reported. this single-arm preliminary result suggest that rhtpo could be a efficious method to manage pt after stem cell transplantation. however, these result need further confirmation. disclosure of conflict of interest: none. reproductive health in long-term female survivors after allogeneic hematopoietic stem cell transplantation z peric 1,2 , a samardzic 2 , n durakovic 1,2 , d tina 1,2 , l desnica 2 , r serventi-seiwerth 2 and r vrhovac 1,2 1 school of medicine, university of zagreb, zagreb, croatia and 2 university hospital centre zagreb, zagreb, croatia most female recipients of allogeneic hematopoietic stem cell transplantation (allo-hsct) suffer from premature menopause, infertility and endocrine imbalance owing to gonadal damage from myeloablative conditioning. in order to evaluate ovarian recovery and long-term endocrine complications in our institution, we performed a retrospective study of female patients who received a myeloablative allo-hsct during their reproductive age. we identified 50 female patients who underwent myeloablative allo-hsct in our institution between 1983 and 2009 and were still alive with available follow-up at the time of this study. among them, 37 patients accepted to participate and responded to a query designed for this s308 purpose. the median age of our patients at transplantation was 32 years (range: 12-47 years). they were interviewed at a median of 20 years (range: 7-33 years) post allo-hsct. the majority of patients were transplanted for a myeloid malignancy (14 acute myeloid leukemia, 7 chronic myeloid leukemia, 3 myelodysplastic syndromes and 1 chronic myelofibrosis), while 7 patients had aplastic anemia and 5 had acute lymphoblastic leukemia. all patients received bone marrow transplant from a hla-matched related donor after a myeloablative conditioning. conditioning regimen consisted of cyclophosphamide with or without total body irradiation (tbi) or in combination with busulfan. only 6 patients (16%) resumed a normal menstrual cycle after allo-hsct, without the need for hormonal replacement therapy (hrt). all these patients were transplanted for aplastic anemia and none of them received tbi in the conditioning regimen. eight patients (22%) remained amenorrheic indefinitely and never started hrt, even though most of these women were transplanted under the age of 40 years. 25% of these patients were diagnosed with osteoporosis later in life. the remaining 23 patients (62%) started hrt at a median of 11 months after allo-hsct (range: 3-27 months). however, only seven patients on hrt (30%) resumed regular menstrual cycle. a median duration of hrt therapy was 6 years (range: 3-20 years). none of the women receiving long-term hrt had severe cardiovascular complications or breast cancer. finally, five women gave birth to eight healthy children in our study population. three unassisted pregnancies were observed in two female patients after spontaneous recovery of ovarian function (both patients with aplastic anemia). the remaining two patients restored ovarian function with the use of hrt and gave birth after an assisted pregnancy (one woman gave birth to triplets after an in vitro fertilization (ivf), while other became pregnant with a donated oocyte). in spite of the fact that almost all women who undergo allo-hsct develop an ovarian failure, spontaneous recovery is sometimes possible, particularly following conditioning regimen without tbi. in patients without spontaneous recovery, hrt should be initiated promptly to prevent the early and late unwanted effects related to estrogen deficiency. moreover, recovery of normal ovarian function and even a viable pregnancy is a realistic possibility in patients placed on hrt, particularly with the use of potential therapeutic interventions as ivf or oocyte cryopreservation. it is therefore crucial to provide adapted pre-transplant counselling and recommendations for regular post-transplant follow-up in female patients who undergo allo-hsct. disclosure of conflict of interest: none. transplant-associated thrombotic microangiopathy (ta-tma) is a multifactorial disorder caused by systemic vascular endothelial injury leading to end-organ damage often involving the kidney. ta-tma occurs in up to 30% of patients undergoing hsct, and may be associated with poor outcome. although pathogenesis has not been fully clarified, activation of the complement system has been suggested to play a central role, and eculizumab, a monoclonal antibody (mab) that mediates terminal complement blockade, has shown therapeutic benefit in cases unresponsive to immunosuppression modulation. we report the case of a pediatric allogeneic hsct recipient with severe ta-tma, who did not tolerate treatment with eculizumab, now successfully treated with oms721, a novel human mab targeted to the mannan-binding lectin-associated serine protease-2 (masp-2), a molecule central to the activation of the lectin pathway of complement. a 14-year-old girl received an allogeneic hsct from a hla-compatible unrelated donor for the treatment of diamond-blackfan anemia. at month +5 of the posttransplant course, she developed progressive deterioration of renal function, microhematuria and serositis, that prompted the cyclosporine discontinuation. from month +7, the patient experienced progressive trilinear cytopenia, elevated ldh, schistocytes, undetectable haptoglobin, hypertension, increased serum creatinine, nephrotic range proteinuria, and serositis, and a diagnosis of ta-tma was established. laboratory investigations documented no abnormalities in the patient but identified a stop-codon heterozygous 43 variant in cfhr5 c.485_489dupaa (p.glu163lysfs*10) in the donor's dna. the patient was initially treated with eculizumab, but she developed acute pulmonary edema soon after eculizumab administration as the consequence of a possible reaction to the drug which had to be discontinued. the patient was subsequently treated with plasma exchange, with only limited benefit. upon ta-tma relapse at month +11, eculizumab was re-administered at lower doses, but she developed a new episode of acute pulmonary edema, preventing further eculizumab continuation. renal function progressively deteriorated and she was started on hemodialysis, reaching a 3 times weekly regimen. the patient received oms721, kindly provided on a compassionate use basis by omeros corporation, seattle, usa, starting with an iv dosing schedule. she did not experience any adverse events, and was able to tolerate the treatment well. at 2 months from oms721 initiation, she has shown improvement in ldh and haptoglobin levels, and, more importantly, her creatinine levels have normalized, allowing for complete discontinuation of hemodialysis and partial outpatient management. anti-masp-2 mab oms721 is a promising new option for the treatment of ta-tma occurring after hsct, and seems to have a safe profile also in the pediatric/adolescent setting. disclosure of conflict of interest: none. severe cytokine release syndrome after t-cell replete haploidentical transplantation with post-transplant cyclophosphamide is associated with increased death rate d taurino 1 , j mariotti 1 , b sarina 1 , l morabito 1 , s bramanti 1 , c carlo-stella 2 , a santoro 2 and l castagna 1 1 bone marrow unit, humanitas cancer center, istituto clinico humanitas, rozzano, italy and 2 hematology department, humanitas cancer center, istituto clinico humanitas, rozzano, italy haploidentical stem cell transplant (haplo-sct) represents a potential curative strategy for several hematological malignancies. haplo-sct may represent an alternative option when a hla matched-identical sibling (hlaid) or a matched unrelated donor (mud) is not available. the syndrome of systemic inflammation, characterized by fevers, vascular leak, hypotension, and respiratory and renal insufficiency, in the context of elevated inflammatory markers and cytokine levels was previously described as cytokine-release syndrome (crs)1. recent publications have elicited the occurrence of crs after haploidentical transplant, especially after peripheral blood stem cell graft, and its high-related mortality 2-4. here we report the experience of our institution with crs after haplo-sct. between march 2014 and october 2016, we treated 29 patients with haplo-sct with a graft source represented by peripheral blood stem cells. we monitored the occurrence of crs symptoms and utilize a previously described grading system 1, 4 starting from day 0, up to day 14 after transplant. severe crs is defined as grade 3 or higher because it requires aggressive interventions and is characterized by oxygen requirement ⩾ 40%, 43 l nasal cannula, hypotension requiring high dose or multiple vasopressors, grade 3 renal toxicity or grade 4 transaminitis. other characteristics comprise newonset altered mental status without other explanation and new cardiomyopathy without wall motion abnormality. results: 27 out of 29 patients experienced fever between day 0 and day 14 post transplant with most episodes (24 patients) occurring between day 0 and day 4. on day 7 after transplant, 3 patients had grade 3, 6 grade 2 and 19 grade 0 crs, respectively. by day 14 post haplo-sct, 5 patients had crs grade 43, 5 grade 2 and 1 grade 1. overall, the incidence of crs any grade was 43% (95% ci 21-55%). 1 year after transplant 8 patients died because of non-relapse related side effects. with a median follow-up for alive subjects of 10 months, 1-year overall survival (os) was 64% (95% ci: 42-80%). 1-year os was 73% for patients with a crs 3 on day 7 (p = 0.007). conclusions: crs represent an important complication after haplo-sct. crs score 43 on day 7 after hst apparently correlates with long-term survival. better strategies need to be implemented for an early detection of severe crs in order to develop effective treatments, such as tocilzumab, for this important side effect. further studies are ongoing at our institution in order to correlate post-haplo crs with graft composition, laboratory parameters and immunereconstitution. hematopoietic cell transplantation (hct) is associated with significant morbidity that impairs survivor's sexual functioning. however, few studies have specifically addressed it. thus, we examined (1) sexual functioning during the first year post hct, (2) differences between allogeneic and autologous hct, and (3) whether demographic, clinical and psychological variables were associated with sexual functioning. this is a prospective multicenter study assessing patients before hct, at day 90, 180 and 360. sexual functioning was assessed with the changes in sexual functioning questionnaire, which yields a total score, along with scores for the dimensions of frequency, pleasure, orgasm, desire and arousal. anxiety and depression (hads) were also collected. we included 159 consecutive hct recipients: 91 (53%) were men, with a median age of 51 years (range: 18-71), 93 (58%) received an allogeneic hct and 66 (42%) an autologous hct. sexual functioning was significantly affected: 86% of the sample reported impairment at pre-hct, 91% at day 90, 87% at day 180 and 86% at day 360. mixed model analysis indicated that sexual functioning was not associated with time from hct (p = 0.802) or hct type (p = 0.538). however, there was an interaction between these two variables (p = 0.022), particularly at day 90, since sexual functioning had improved among autologous survivors and worsened among allogeneic survivors leading to nonsignificant differences between hct type (p = 0.082). frequency of sexual functioning improved during the study period (po 0.001), and no differences were observed between hct type (p = 0.111). again, there was a borderline interaction between post-hct time and hct type (p = 0.059), since autologous survivors reached higher frequencies than allogeneic survivors, with significant differences at day 90 (p = 0.003). pleasure significantly improved during the study period (p = 0.035), without observing differences between hct groups (p = 0.121). again, however, autologous survivors reported significant improvements in pleasure at day 90 (p o0.001) and a trend at day 180 (p = .093) when compared with allogeneic survivors. orgasm did not improve during the study period (p = 0.837), and no differences were obtained between hct groups (p = 0.413). allogeneic survivors had higher orgasm scores at pre-hct (p = 0.020), which worsened during the study period, particularly at day 90 (p = 0.028). in contrast, autologous survivors reported improvements in orgasm by day 90. non significant results were obtained in the sphere of sexual desire and arousal (p40.1). bivariate analyses indicated that women, older age and depression were associated with impaired sexual functioning at all assessed time-points (p o0.05). chronic graft-versus-host disease (gvhd) was associated with worse sexual functioning at day 180 (p = 0.045) and 360 (p = 0.020). no differences were obtained when considering diagnosis, having received previous hct, intensity of the conditioning regimen and whether patients lived with a partner (p40.05). stepwise multivariate regression analyses indicated that gender (p = 0.001) and extensive chronic gvhd (p = 0.012) predicted for worse sexual functioning at day 360. sexual functioning should be routinely assessed and considered for eventual targeted intervention in both hct populations, particularly during the first year post transplant. additional clinical efforts should focus on patients more vulnerable to impaired sexual functioning. disclosure of conflict of interest: none. significant improvement of qol by using atg as part of the conditioning regimen followed by hla-identical peripheral stem cell transplantation in acute leukemia patients. results from a prospective, randomized phase iii study (atg family study) b francesca 1 , s carlos 2 , w christine 3 , s mariarosaria 1 , p massimo 4 , s carmine 5 , m giuseppe 6 , b wolfgang 7 , cm angelo 8 , p francesca 9 , m nicola 10 cgvhd is a major complication after allogeneic sct. we previously demonstrated that the addition of anti-tlymphocyte globulin (atlg neovii, formerly atg-fresenius) to a myeloablative preparing regimen followed by peripheralblood sct from an hla-identical sibling for pts with acute leukemia resulted in a significant reduction of cgvhd, without increasing the risk of relapse or infection. 1 the study protocol included quality of life (qol) questionnaires (eortc qlq-30 and hdc29) before and after sct (day+ 100, 6, 12 and 24 mos). the qlq-c30 includes a global qol scale, five functional scales (physical, role, emotional, cognitive and social function) and nine symptom scales (fatigue, nausea-vomiting, pain, dyspnea, insomnia, appetite loss, constipation, diarrhea and financial problems). the qlq-hdc29 includes six multi-item scales and eight single items that describe impairment through highdose treatment. mixed models for repeated measures (mmrm) and linear mixed models (lmm) were used to analyze the time courses and the slopes of the outcomes depending on treatment arm (atg vs non atg), age, country, sex, and cgvhd. (clinicaltrials.gov: nct00678275). pts with a qol form returned decreased by visit (70% pre-sct, 45% at 100 days and 29% at 24 mos after sct). forty-nine percent in the atg and 60% in non atg arm provided any qol forms after sct. return of any post-sct qol forms by country was 68% for germany, 62% for italy and 25% for spain. pts with cgvhd were more likely to return qol questionnaires (66% vs 45% w/out cgvhd) while neither age nor sex were closely associated with qol form return. the majority of subscales of the qlq-30 indicated an average improvement of qol and reduction of symptoms over time, notably in the atg group. in an mmrm model controlling for country, age, sex and cgvhd, pts treated with atg showed significantly more pronounced improvement of global health status/qol over time compared to non-atlg (p = 0.02), with a treatment group difference of 2.8 ± 3.9 points (marginal mean ± sem) at day 100 and increasing to 10.5 ± 5.3 points at month 24 favoring atg. significant superiority of atg (po0.05) was also observed for four of the five functional scales as well as for several symptom scales scores including appetite loss, insomnia, nausea-vomiting and dyspnea. for the qlq-hdc29, significant treatment effects favoring atg were observed for gi side effects and impact on family. lmm analyses of qol by country indicate that patients from italy generally gave more favorable ratings for all functional scales and lower scores for most symptom scales than those from germany while the time courses and slopes were similar for most scales. these results underline the importance of the habits and cultural environment which are distinctive of each country. males and females showed similar qol ratings at pre-and post-sct. patients up to 34 years tended to provide more favorable functional ratings and less severe symptom scores than older patients and also showed more pronounced improvements of qol. pts receiving atg in a randomized study have significantly less cgvhd and improved grfs, resulting in an improved qol regarding global health status and most functional scales. notably, we also observed a significant difference in qol assessment between pts from germany and italy. oral mucositis (om) is a well-known side effect of high-dose chemotherapy and radiotherapy in hematological patients, which influences the health-related quality of life (hrqol) of the affected patients. the purpose of this study is to demonstrate the impact of om on hrqol in stem cell transplanted patients in routine care. prospective, noninterventional single-center observational study was performed at a german university hospital. inpatient allogenic and autologous stem cell transplant patients ⩾ 18 years with high-dose chemotherapy. om was assessed with the who oral toxicity scale, pain using the numeric rating scale (nrs) and the performance status with the ecog score. hrqol was captured with the eortc qlq-c30 and the qlq-oh15 questionnaires (3 days before hematopoietic stem cell transplantation (hsct); 7 days after hsct; 14 days after hsct). statistical significance was assumed p o0.05. a total of 20 patients (11 autologous and 9 allogenic) was included from august to december 2016. a total of 11 (55%) patients developed om. of these 11 patients, 3 suffered from grade 1, 3 from grade 2, 4 from grade 3 and 1 from grade 4 om. three days before hsct, the mean qol of all 20 patients was 45%, the mean qlq-c30 summary score 60.3% and the mean oral health related quality of life 82.3%. most of the patients suffered from om around day 7 after hsct. after 7 days, quality of life (qol) was higher in patients with no om (32.3%) than in patients with om (30.0%). the qlq-c30 summary score was significantly (p = 0.004) lower in patients affected by om (43.1%) than in patients who did not develop an om (65.8%). om affected patients had significantly more limitations in emotional (no om 84.4%; om 56.7%; p = 0.038) and cognitive functioning (no om 93.8%; om 51.7%; p = 0.002) and in fatigue (no om 58.3%; om 80%; p = 0.045), pain (no om 14.6%; om 55%; p = 0.005) and insomnia (no om 12.5%; om 60%; p = 0.001), they had a significantly higher rate of problems. oral health-related quality of life was significantly (p = 0.003) lower in patients who were affected by om (57.9%) compared to patients who did not develop an om (83.9%) and patients with an om had significantly more problems with a sore mouth (no om 8.3%; om 46.7%; p = 0.028), sticky saliva (no om 29.2%; om 63.3%; p = 0.045) and sensitive mouth (no om 8.3%; om 56.7%; p = 0.003). after 14 days, qol was higher in patients with no om (47.9%) compared to patients with om (46.7%). patients with no development of om had a higher but not significant physical functioning, cognitive functioning and social functioning. patients affected by om had higher levels of fatigue and pain and more often suffered from a sore mouth. oral health-related quality of life was higher in patients without om (75%) compared to patients with om (68.3%). comparing all assessed days patients with om had higher scores on the nrs increasing with a higher grade of om (mean nrs score grade 1; 2-2.3, grade 3; 4-4.5), the ecog index was higher in om affected patients during episodes with om (mean ecog score-2.3) compared to episodes without om (mean ecog score-1.9). om has a major impact on the hrqol, health-related symptoms and functionality. in the future, there has to be a higher awareness from clinicians and patients of the prevention, assessment und causes of om. more research has to be initiated to ease the symptomatology and to improve patients' quality of life. disclosure of conflict of interest: none. according to ebmt data, chronic gvhd (cgvhd) occurs in 40-70% of all patients after allogenic hematopoietic stem cell transplantation (allo-hsct). pulmonary cgvhd is the most severe form. but it is very unpredictable to use due to the fact that many factors can affect it (breath-dependent; need experience not only from physician but from patients also and so on). here we show that routine software-based image analysis algorithm can provide data that highly correlated with pft results and have excellent sensitivity and specificity in pulmonary cgvhd diagnosis. we blindly analyzed 120 ct scans (made without additional expiration) in 24 allo-hsct patients at different time points. all scans were performed on ct scanner aquilion 64, toshiba, japan. according to hounsfield units (hu) definition, − 1000 hu ('air') have approximate density at 0 g/ml; 0 hu ('water') have approximate density at 1 g/ml. the analysis of ct scans (heart, vessels and bronchi were excluded from analysis) was based on automated software conversion (image-analysis algorithm providing by multivox software, msu, moscow, russia) of each ct-image pixel from hu to density units (g/ml). pft were performed using standard procedures at same as ct scans time points (spirolab iii, italy). all patients with hematological malignancies (acute leukemia-17, aplastic anemia-1, chronic myeloid leukemia-1, t-cell lymphoma-1, chronic myeloproliferative disorder-1, myelodysplastic syndrome-3) were transplanted in national research center for hematology between 2012 and 2015. median of age was 41.5 years (range: 19-60 years). eight patients were males, 16-females. seventeen received reduced-intensity and 7-myeloablative conditioning regimen. graft from match unrelated donor (mud) were used in 17 cases, 'mismatch' mud-2, match related donor (mrd)-9, 'mismatch' mrd-1. median follow-up is 44.8 months. we analyzed lung tissue experimental density in patients before and after allo-hsct at different time points. median of lung tissue experimental density were 0.178 (interquartile range (iqr), 0.148-0.186), 0.17 (iqr, 0.153-0.185) and 0.147 (iqr, 0.131-0.172) for patients before allo-hsct, after allo-hsct with cgvhd (except pulmonary cgvhd) and with pulmonary cgvhd, respectively. mann-whitney u test was used to reveal significant differences between these groups (see figure 1 ). also, we found strong correlation between pft and experimental density (spearman's correlation coefficient r = 0.537) (see figure 2 ). forty-five ct scans of patients with pulmonary cgvhd and 59 ct scans of patients without pulmonary cgvhd at the time of ct scan as control subjects were included in roc analysis to assess the clinical values of our model. we generated an roc curve and found that the area under the curve (auc) was 0.77 (95% ci, 0.67-0. 86) (p o0.0001) (figure 3 ). standard ct scan is presented as easy to perform, breath-independent, standardized and wide spread method for every patient after allo-hsct. it can be performed many times during all their post-hsct life. ct scan with a simple software analysis allows to select a group with high probability of pulmonary cgvhd and who can be suspected of cgvhd development by this method with sensitivity-60% and specificity-81.36%. disclosure of conflict of interest: none. the choice of effectiveness criteria affects conclusions of economic evaluation of newer allogeneic bone marrow transplantation modalities :example based on a randomised multicenter trial comparing two reduced intensity conditioning regimen (flu-bu-atg) vs (flu-tbi) for matched related allo-sct s le corroller*, anne-gaelle 1 , c siani 2,1 , r tabrizi a re-evaluation of the per-diem hospitalization cost was performed in 2016 and included the utilization of hospital technical facilities and a more precise estimation of overheads costs. we performed three separated cost-effectiveness analysis, using, respectively, pfs, os and qaly as end point. when using pfs as effectiveness, relapse costs were not included. weighting coefficients for the cost per qaly analysis came from the literature. at 5 years, os and pfs were 41% and 29%, respectively, and did not statistically differ between groups. the mean total cost per patient was not statistically different between groups (111725€ for fba vs 98316€ for ftbi, ns). using pfs as end point, the icer of fba compared to ftbi is 35 034€ per year of pfs gained. using os, the icer became non-statistically significant, signifying that when handling uncertainty, no difference in term of cost-effectiveness was observed between fba and ftbi with os as end point. using s312 qaly, the icer was statistically ns again, showing no advantage in terms of cost per qaly of one conditioning regimen over the other. this result was obtained both considering three weighted health states (dfs, progression and death) and four weighted health states (dfs without gvhd, dfs with gvhd, progression and death) for the qaly calculation. using os and qaly, the two conditioning regimens were not different in terms of cost-effectiveness, while fba may be considered as more cost-effective using pfs as effectiveness criterion. using intermediary end points allows economic evaluation to be available earlier in the life cycle of an innovation. however, it implies strong hypotheses about the predictive value of the pfs over the os. longer period evaluation and qaly may reverse preliminary results. this situation is likely to exist in the hematology setting where alternatives between chances of cure and toxicities of treatment are often observed. research about allogeneic sct modalities is archetypical of such situations and decisions makers should be aware of the necessity of further economic re-evaluation along the development and diffusion process of innovative treatments. disclosure of conflict of interest: none. the impact of corticosteroids prophylaxis for the engraftment syndrome incidence during autologous stem cell transplantation in multiple myeloma and amyloidosis the es is a complication of asct characterized by an inflammatory response during peripheral blood recovery. the standard treatment is based on corticosteroid therapy. the incidence of es after asct increases in chemotherapy lowtreated patients such as those with multiple myeloma (mm) and amyloidosis (al).moreover, the es is associated with the use of g-csf after infusion of stem cells. therefore, our bmt team does not use g-csf since 2009 in this population reducing the incidence and severity of es. therefore, it makes sense to use low-dose prednisone to prevent this complication. in this study, we compared two consecutive cohorts of patients with mm/al that performed an asct while evaluating the corticosteroids prophylaxis (cp) in the es incidence and its effect on other clinical variables. we included 120 patients with mm (n = 96; 80%) and al (n = 24; 20%) that performed an asct between january 2011 and november 2016 in a single institution. the median age (range) was 56.7 (33.8-71.7) years. during the procedure, all patients received melphalan as conditioning chemotherapy and none received g-csf. fortyseven patients (39%) received intravenous methylprednisolone or oral prednisone 0.5 mg/kg/day from day +7 until reaching a neutrophil count ⩾ 500 per mm 3 for 3 consecutive days (cs group), and 73 (61%) patients did not receive corticosteroids (noncs group). the characteristics of patients in both groups (age, gender, status performance and previous treatment were similar (p40.05)). the cs group, received higher doses of cd34 + than the noncs group (3.68 × 10 6 /kg vs 2.92 × 10 6 /kg, respectively, p = 0.006). the median (range) days of neutropenia ( o 500 per mm 3 ) was 9 (4-25) days. es was diagnosed in 43 (36%) patients. fifty-seven (48%) patients had fever, showing infectious focus or microbiological isolation in 24 (20%) cases, whereas the incidence of grade iii-iv oral mucositis and relevant gastrointestinal toxicity was 8% and 2.2%, respectively. the complete analysis between groups (cs versus noncs) for the whole series and in the mm/al subgroups is detailed in table 1 . the administration of corticosteroids as prophylaxis seems to reduce the incidence of es in the overall series or in the analysis for the subgroups (mm and al) without increasing infection. [p381] disclosure of conflict of interest: none. chronic gvhd is a condition that might occur after allo-hsct and has been proved to impair long-term survival and quality of life of patients. graft failure is also a major potential complication for patients undergoing transplant for an aplastic anemia/bone marrow failure (bmf). partial in vivo t-cell depletion, employing anti-thymocyte globulin (atg) during conditioning, has been proved to successfully prevent the mentioned potentially life-threatening complications in highrisk patients. however, the possibility of developing epstein-barr virus (ebv)-induced post-transplant lymphoproliferative disorders (ptlp) has been a limiting factor to use atg. this study includes the last 100 pts with a minimum follow-up of 100 days, who underwent allo-hsct in our center (november 2014-august 2016). a total of 56 pts were male and 44 female. median age was 53 years (range: 7-69). baseline diseases were: acute leukemias (54), lymphoproliferative disorders (17), myelodysplastic syndromes (12), chronic myeloproliferative diseases (7), multiple myeloma (5) and bone marrow failures (5) . donor was unrelated in 57 cases, and related in 43 (including 18 haplo-identical). conditioning regimen was: busulphan-based (70), melphalan-based (13), tbi-based (8) and others (9). progenitors source was pb in 89 and bm in 11. patient/donor ebv pre-transplant serology was: +/+ in 94 cases, +/ − in 5 and − /+ in 1. rabbit atg (thymoglobuline) was employed in 58 cases: 54 at 4.5-6 mg/kg (urd transplants) (low dose), and 4 cases at 7.5 mg/kg (all of them pts with bmf) (intermediate dose). family donor (including haplo-identical) transplants of those pts with diagnosis different from bmf (42 cases) did not receive atg. systematic monitoring of ebv using quantitative pcr was employed. ebv reactivation was considered when dnaemia was superior to 1000 copies per ml. a total of 5 pts presented ebv reactivation: 0/42 (0%) in cases without atg, 4/54 (7.4%) in cases with low-dose atg and 1/4 (25%) in cases with intermediate-dose atg. median time of reactivation was the day +34 (range: +32 to +151). there was one single case of ebv-induced ptld which belonged to the intermediate-dose atg group. all cases (including the one with ptlp) were successfully treated with rituximab at 375 mg/m 2 /week. median number of doses employed were 3 (range: [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] . mortality due to ebv was 0% in our series. limited donor availability in the form of either matchedrelated or unrelated donors drew attention to haplo-hct. donors of haplo-hct shares an exact haplotype with the recipient but is mismatched for hla genes on the unshared haplotype. most studies have shown promising results in terms of graft success and survival. in this study our aim is to present the early and late outcome of our haplo-hct patients. between 2012 and 2016, we retrospectively evaluated 16 haplo-hct in terms of post-transplant outcome, survival and complications who diagnosed and followed in our center. the median age of patients was 37 (range: 19-61), 10 (63%) of them were male recipients. the patient characteristics were given in table 1 . thirteen patients (81%) had pre-transplant active disease. neutrophil and platelet engraftment was achieved in 7 patients (44%) at a median day of 21 (range: 16-40) and 34 (range: 13-116). eight of 16 patients (50%) died within 1 month after transplant because of sepsis without achieving engraftment. haplo-hct is the second transplant in four of 16 patients (25%): 1 patient relapsed after full-matched related transplant, 1 patient relapsed after 9/10 matched unrelated transplant, 1 patient had engraftment failure after full-matched unrelated transplant, 1 patient underwent haplo-hct in another center, followed in remission for 2 years and relapsed. acute graft vs host disase (agvhd) was diagnosed in 6 patients (37%), whereas chronic gvhd in 4 patients (25%). four patients were relapsed (25%) during follow-up with median rfs of 6 months. three patient had bk virus-positive hemorrhagic cystitis (18%). the distribution of infections is shown in figure, viral infections were detected later than fungal and bacterial infections. previous history of invasive pulmoner aspergillosis was detected in 5 of the patients (31%) (2 of them were re-transplanted) and received secondary prophylaxis. overall survival (os) of 6 months and 1 year were 25% and 18%, respectively. the choice between alternative graft sources depends on the urgency of the transplant on each institutional preference. higher complication and infection rates in addition to decreased survival compared with previous studies since our patient population consisted of refractory patients with comorbidities. preferable patient profiles undergoing haplo-hct may have better outcomes. disclosure of conflict of interest: none. the third month risk factor score: detection of disease at day +100 of allogeneic stem cell transplantation is the most important risk factor of worse prognosis m celis 1 , c fernández 1 , l yáñez 1,2 , a bermúdez 1,2 , a insunza 1 , m colorado 1 , m lópez-duarte 1 , i romón 1 , s garcía-ávila 1 , a cabero 1 , a casado 1 , m sánchez-escamilla 1 , c richard 1 and e conde 1,2 1 hematology department, hospital universitario marqués de valdecilla and 2 university of cantabria before allogeneic stem cell transplant (sct), several index can provide prognostic information (ebmt risk score and hcti score). however, there is scarce data for the impact of the procedure during the first 100 days of transplant, in which opportunistic infections and the acute graft versus host disease (gvhd) can induce harmful effects. our purpose is to create a risk factor score, measured at day +100 post sct, to give information about the prognosis of the patient. we retrospectively analyzed seven clinical (disease, fungal and cmv infection, acute gvhd, treatment with corticosteroids, karnosfsky status and length of hospitalization) and eight analytical (related to immune status, liver and lung function, nutritional status, iron overload and platelet count) risk factors in 131 patients who underwent sct in our center between 2011 and 2015 and were alive at day +100. data were collected as categorical variables and compared by χ 2 -test. significant variables (p o0.05) were evaluated in a multivariate logistic regression model. those who maintained statistical significance were then assigned a point value calculated with their β-coefficient. summation of the points resulted in a weighted risk score. median age was 51 years (range: 4-73) and 80 were males (61.1%). the most frequent disease was aml, 49 patients (37.4%). the conditioning regimen was myeloablative in 97 patients (74%) and bone marrow was the principal stem cell source (71%). donor was mrd in 39 (29.8%), mud in 51 (38.9%) and mmd in 41 (31.3%). the median followup was 26 months (range: 3-66). the univariant model identified five prognostic variables: detection of disease by molecular, cytogenetic or flow cytometry asses in leukemias, myelodisplastic syndrome and multiple myeloma or image (ct scan ± pet) in lymphoma, dose of corticosteroids ⩾ 0.5 mg/kg/ day, ferritin 42500 ng/ml, albumin o3.0 g/dl and platelet o100 000 per mm 3 . table 1 shows variables evaluated. in the multivariate model, the detection of disease (hr 3.80, 95% ci 1.90-7.61, p 2500 ng/ml (hr 2.39, 95% ci 1.30-4.42, p = 0.005), and platelet o100 000 per mm 3 (hr 2.06, 95% ci 1.11-3.89, p = 0.022) were associated with higher risk of death and according with their-coefficient 4, 2 and 2 points were, respectively, assigned. the third month risk score (tmrs) was calculated in all patients and they were stratified into three groups: low risk of death (a, 0-2 points), intermediate risk (b, 4 points) and high risk (c, ⩾ 6 points). at 2 years post sct, the estimated overall survival according with the tmrs was 78.9% ± 4.3 in group a, 31.0% ± 8.6 in group b and 36.4% ± 14.5 in group c, po 0.001. although the harmful effect of the first 3 months of transplant can impact in the survival, the detection of disease at day +100 is the most determinant risk factor of death. this fact gives us the need of transplant in the best response and, in those who cannot, to plan promptly rescue strategies. the next objective is to confirm our risk score in a validation group. disclosure of conflict of interest: none. recombinant human soluble thrombomoduline alpha (rhtm) is a novel anticoagulant agent and approved for disseminated intravascular coagulation in japan. the aim of the study is to evaluate the therapeutic potential of rhtm for sinusoidal obstructive syndrome/hepatic veno-occlusive disease (sos/ vod). we retrospectively studied 878 times of allogeneic hematopoietic cell transplantation in toranomon hospital from june 2008 to june 2015. we extracted the patients who used rhtm for dic and satisfied the diagnostic criteria of sos/ vod around the same time, because the use of rhtm for sos/ vod alone is off-label. data on the patients who used rhtm for 43 days within 100 days after transplantation were analyzed. the patients who were already treated with rhtm before the emergence of the first symptom or sign of sos/vod, and who started rhtm over 30 days after the emergence of the first symptom or sign of sos/vod, were excluded from the [p383] analysis. to diagnose classical sos/vod (⩽21 days after transplantation), we used two classical criteria of the modified seattle and the baltimore. for late-onset sos/vod (4day 22 of transplantation), we used the criteria of ebmt. we defined as severe sos/vod, if the patients had renal (cr ⩾ 2 times of baseline), respiratory (spo2 ⩽ 90% or the need for positive pressure) or central nervous system failure until 2 weeks after the diagnosis of sos/vod. complete response (cr) was defined as the resolution of all the symptoms and the signs in sos/vod diagnostic criteria. a total of 39 patients were extracted. the median age was 60 years (range: 27-72) and 27 patients (69%) was male. donor cell sources were ucb (n = 34) and ubm (n = 5). most of the prophylaxis regimen was the combination of ursodeoxycholic acid and dalteparin in 36 patients (92%). classical sos/vod was diagnosed in 3 (8%) and 8 patients (21%) by the criteria of the modified seattle and the baltimore at the median day of 14 (range: 11-14) and 16 (range: 11-20), respectively. twenty-eight patients (72%) were diagnosed as late-onset sos/vod at the median day of 44 (range: 22-89). severe sos/vod developed in 33 patients (85%) (renal, n = 32; respiratory, n = 7; central nervous system, n = 15). the elevation of transaminase was observed in 18 patients (46%). the median interval from the emergence of the first symptom or signs of sos/vod to rhtm administration was 7 days (range: 0-23). the median duration of rhtm use was 11 days (range: 3-63). rhtm was used alone in 20 patients (51%), in combination with dalteparin in 7 (18%), with atiii in 5 (13%), with dalteparin and atiii in 3 (8%), with atiii and pge1 in 2 (5%), and with pge1 in 2 (5%). corticosteroid was used concomitantly in 32 patients (82%). finally, 13 patients achieved cr of sos/vod. the cumulative incidence of cr of sos/vod was 33.3 % at 1 year after the administration of rhtm (95% confidence interval, 18.5-48.9%). the median interval from the administration of rhtm to cr of sos/vod was 51 days (range: 6-141). at 1 year after transplantation, overall survival was 25.6% (95% confidence interval, 13.3-69.9%). from the administration of rhtm to 2 weeks after the cessation of rhtm, 23 hemorrhagic adverse events were observed. seven out of 23 events were at grade 3-5, and 5 out of 7 events were fatal (intra-abdominal in 2, gastrointestinal in 1, lung in 1 and brain in 1). we concluded that rhtm had a therapeutic potential for sos/vod. disclosure of conflict of interest: none. thrombopoietin receptor agonists for delayed and prolonged clinically-relevant severe thrombocytopenia after allogeneic hematopoietic stem cell transplantation v bosch vilaseca 1 , i garcía cadenas 1 , e roldán 2 , s novelli 1 , r martino 1 , p barba 2 , a esquirol, l díaz polo 1 , g orti 2 , d valcárcel 2 and j sierra 1 1 hematology department, hospital de sant pau, barcelona, spain and 2 hematology department, hospital de la vall d'hebron, barcelona, spain persistent thrombocytopenia is a common complication after allogeneic stem cell transplantation (allosct), which dramatically increases the patients' dependence on hospital-based healthcare. thrombopoietin receptor agonists (tpoa) increase platelet counts in other clinical settings; however, the experience regarding their use after allosct is limited. we retrospectively evaluated tpoa efficacy in 15 consecutive adult allosct recipients who received tpoa as a compassionate use for severe thrombocytopenia post-engraftment. five patients (33%) had primary and prolonged failure of platelet recovery, while 10 had secondary thrombocytopenia: in seven of these cases, gvhd and/or a viral infection were the 'attributed' cause, while three were classified as post-allosct itp. all 15 patients were dependent on platelet transfusions (median: 2 times per week, range 1-5), with severe bleeding episodes in nine cases (60%) before tpoa onset. tpoa was started at a median of 160 days after allosct (range: 65-1041). romiplostim was used in 13 (87%) cases. the median starting dose was 2 μg/kg once a week (range: 1-5 μg/kg), while the final dose identified as most beneficial was 4 μg/kg (range 1-10 μg/kg). eltrombopag was used in 2 cases (13%), with an initial dose of 50 mg daily; while the final doses were 100 and 150 mg daily. overall, 9/15 patients responded to tpoa therapy (defined as a stable platelet recovery to ⩾ 30 000/μl without transfusion support). the 180-day cumulative incidence of successful platelet recovery to ⩾ 30 000/μl and ⩾ 50 000/μl was 70% (95% ci, 67-73%) and 56% (95% ci, 42-69%), respectively, which were reached at a median of 21 and 48 days from start of therapy. five of the 9 patients (56%) with severe bleeding at onset responded to tpoa (4 of them without further hemorrhages) at a median of 93 days (range: 1-299). at a median follow-up of 511 days from start of therapy, three patients who responded continue tpoa treatment, while four other responders were able to discontinue it without recurrence of thrombocytopenia. among these 4 patients, s317 the median total duration of treatment was 201 days (range: 113-300). one patient lost his response within 5 months after tpoa onset when he developed thrombotic microangiopathy associated with progressive gvhd. the remaining responder experienced disease relapse on day +88 after allosct. among the 6 non-responders, 1 had leukemia relapse during tpoa treatment, 1 switched from romiplostim to eltrombopag without success and the remaining cases had active severe infections at tpoa onset (2 hemorrhagic cystitis and 1 cmv colitis) or non-controllable intestinal bleeding due to progressive gvhd. tpoa were well tolerated, with only 2 patients showing adverse events (grade 3 liver toxicity and grade 3 fatigue), which did not lead to any change in therapy. six patients (40%) underwent follow-up bone marrow biopsies that did not display any increase in marrow fibrosis, including the 1 patient who had myelofibrosis prior to allosct. although six patients in the study had active gvhd when tpoa was started, no patients showed worsening of gvhd. our results support the safety and efficacy of tpoa for the treatment of persistent thrombocytopenia in allosct recipients. further studies should compare the efficacy of romiplostim and eltrombopag and identify surrogate clinical and laboratory variables that are predictive of response to one (or both) of these tpoa. disclosure of conflict of interest: none. . clinical response in both groups was defined as improvement of organ function (no neurological residues; normalization of kidney function) and independence of red blood cell and platelet transfusions. results: the median time of ta-tma onset was 8.2 months (0.5-32.8) after hsct. thirty-five of 37 patients (95%) were under treatment with calcineurin-inhibitors or sirolimus at the time the ta-tma occurred. in all cases, the immunosuppressive drug was stopped promptly. in 28 patients, classical treatment was the primary therapy with a response rate of 52% (including four patients who switched to ec), whereas the response rate to ec treatment was significantly higher with 92% (p = 0.017). all patients receiving ec showed sufficient blockade of the terminal complement pathway after the second ec application (ch50 o 10%). despite the increased response rate for ec therapy, there was no difference seen between these two groups according to overall survival in weeks: classical treatment 9 (95% ci 0-19.3) vs ec treatment 14.9 (95% ci 6.9-22.7) p = 0.84. the main cause of death differed significantly between this two treatment approaches with a therapy-related mortality due to infection with 70% in the ec group during tma therapy and none seen in the classical treatment group (p = 0.001). progressive gvhd was identified as an adverse prognostic factor in both groups (p = 0.048 and p = 0.005). conclusion: in our analysis, we show that ec shows a significant higher response rate in severe ta-tma patients compared to the classical treatment approach. however, in both groups the outcome remained very poor. since most patients presented with advanced, severe ta-tma, especially in the ec group, we hypothesize that earlier diagnosis and treatment of ta-tma and more effective prevention and treatment of infections will improve the outcome of patients with this complication. however, randomized studies are essential for comparison of these two treatment strategies to identify patient groups that benefit from a treatment with ec. disclosure of conflict of interest: none. tocilizumab as an effective treatment in cytokine release syndrome as an early peri-transplant complications in patients subjected to allogeneic stem cell transplantationproinfammatory/autoimmune patient/donor hla haplotype life-threatening early allogeneic hsct complication risk factor hypothesis m-g patrycja 1,2 , p-j beata 1,2 , s marcin 1 , k ksenia 1 , s-k agnieszka 1 and sb aleksander 1,2 1 bone marrow transplantation unit, department of haematology, krakow university hospital and 2 jagiellonian university collegium medicum cytokine release syndrome (crs) is classical complication of car t cells therapy, but also can be connected with early peritransplant complications in patients subjected to allogeneic stem cell transplantation. it can be connected with atg infusion, but also with inflammatory response during periengraftmetnt period (pre-engraftment syndrome and engraftment syndrome) and septic infections. severity of these complication can differ depending on patient's performance status and therapeutic options from just observation and vigilance to mechanical ventilation need. we would like to present small patient series (n = 5) subjected to msd (n = 1) and mud (n = 4) with early transplant-related complications treated with combination of steroids (dexamethason) and tocilizumab. in two of them, tocilizumab was used after second dose of atg. both patients present hypotonia with decreased urine output, prompt increase of creatinine level and presence of acute inflammatory parameters crp, beta2microglonulin and procalcitonin level, fluid retention and decreased oxygen saturation. in another one patient, these symptoms were connected with pbsc infusion from unrelated donor. in later two patients, we observe almost the same clinical presentation in preengraftment phase. in every of patients infection was ruled out-blood cultures were negative. all these patients were treated with tocilizumab in a single dose of 8 mg/kg. in all patients, we observed prompt response-normalization of clinical state, renal function, oxygen saturation and decrease of inflammatory factors-crp, procalcitonin and beta2microglobuline. discussion: crs is a rare complication connected with early phase of allogeneic stem cell transplantation. there were no results of treatment with steroids, reduction of a dose of cyclosporine a according to decreased renal function, but all patient completely/fully recovered after single dose tocilizumab treatment. all our patients were subjected to reduced intensity protocols, what might be a risk factor to develop crs because non complete depletion of the patient origin monocytes/macrophages active population. we also analyzed other factor connected with crs in early peritransplant period finding possible connection with s318 proinflammatory hla phenotype. it was obvious in the patient one our patients with peri-engraftment phase crs-he was diagnosed previously with rheumatoid arthritis b27 pos, dr4. in three of five, we have found sle predisposition in hla phenotype (drb1*1501/dqb1*0602 or drb1*0301/ dqb1*0201), in later one-ra associated hla antigen drb1 0101.these patients were analyzed correlating with historical cohort of additional five patients with mortal and another three with very severe early peri-transplant complications and in all we have found the same 'sle or ra hla phenotype'. because small number of analyzed patients and documented high frequency of these haplotype in population, this is still an opened question is proinflammatory/autoimmune hla phenotype connected pathogenically with predisposition to develop severe transplant complications and are we able to treat all these patients with combination of steroids with tocilizumab. further analysis is needed. disclosure of conflict of interest: none. transplant-associated thrombotic microangiopathy (ta-tma) is a severe complication post haematopoietic cell transplantation (hct) leading to high mortality rates. however, outstanding questions regarding its diagnosis, pathophysiology and treatment remain in the literature. recent studies suggest evidence of complement activation, implicating that complement inhibition may be an effective alternative treatment strategy in refractory patients. therefore, we hypothesized that increased complement activation can be detected in ta-tma patients using a functional assay, the modified ham test. we enrolled consecutive patients with ta-tma according to the international working group criteria from january 2015 to june 2016. as controls, we studied patients with graft-versushost-disease (gvhd). complement activation was detected using the modified ham test, a cell proliferation assay based on the susceptibility of a pnh-like cell line to complement activated serum. normal human serum was used as a negative control and lipopolysaccharides(lps)-incubated normal serum as a positive control. all samples were tested in triplicates and twice. we studied 10 ta-tma patients transplanted from unrelated 8/8 matched (4) or 7/8 mis-matched (3) donors, identical (2) and haploidentical (1) siblings. all patients presented severe acute and/or chronic gvhd. ta-tma presented at median +109 (9-930) day post-transplant. in the control group, we studied two patients with steroidsensitive grii and two with steroid-refractory griv acute gvhd. we were able to detect significantly increased complement activation in the serum of ta-tma compared to gvhd patients (p = 0.039). based on previous studies and present controls, percentage of non-viable cells higher than 20% was considered a positive modified ham test, indicating increased complement activation in four ta-tma patients. regarding treatment outcomes, two patients with a negative modified ham test responded to cyclosporine cessation and steroid administration. plasma infusion with/without plasma exchange was initiated in seven patients. however, only three of them responded to second-line treatment. the modified ham test result was significantly increased in refractory patients (p = 0.048). the terminal complement inhibitor eculizumab was administered in one refractory patient with a positive modified ham test and renal failure at presentation. despite delayed initiation (28 days post ta-tma diagnosis), response was observed after three doses of eculizumab including evidence of reduced hemolysis, schistocytosis and transfusion needs. however, the patient succumbed to complications of end-stage renal disease (54 days post ta-tma diagnosis). among 10 ta-tma patients, 8 succumbed at a median +215 (73-430) day to transplant-associated complications, related to gvhd and infections from multi-resistant pathogens. ta-tma is associated with increased morbidity, mortality and severe complications, including gvhd. unlike gvhd, increased complement activation was observed in a significant portion of ta-tma patients. complement inhibition seems an encouraging therapeutic option in these patients. given the lack of robust functional assays for complement activation, the modified ham test may be useful for early recognition of patients that would benefit from complement inhibition. . this proposal includes, along with the 'classical sos' (cases diagnosed before day +21), the new type 'late onset sos' (cases diagnosed afterwards). new ebmt criteria for severy grading classify cases of sos into four grades (mild, moderate, severe, and very severe). the aim of this retrospective study is to analyze the cases of severe/very severe, both classical and late onset sos, occurred in our unit during the most recent period of time. we studied the last 100 pts, with a minimum follow-up of 100 days, who underwent allo-hsct in our center (november 2014-august 2016). 56 pts were male and 44 female. median age was 53 years (range: 7-69). baseline diseases were: acute leukemias (54), lymphoproliferative disorders (17), myelodysplastic syndromes (12), chronic myeloproliferative diseases (7), multiple myeloma (5), and bone marrow failures (5) . donor was unrelated in 57 cases, and related in 43 (including 18 haplo-identical). conditioning regimen was: busulphan-based (70), melphalan-based (13), tbi-based (8) , and others (9). all patient received prophylactic [p390] s320 ursodeoxycholic acid. progenitors source was pb in 89, and bm in 11. five patients developed severe/very severe sos (5% incidence); 3 were classical (at days +8, +11 and +20), and 2 were late onset (at days +34 and +44) (see table 1 ). four cases had received conditioning with a busulphan (iv)-based regimen (doses from 6.4 to 12.8 mg/kg), and one case with tbi plus cyclofosfamide at high doses. all cases presented with right upper quadrant pain, jaundice, ascites, weight gain, hiperbilirrubinemia, and renal function impairment. all but one had increased transaminases. the five cases were treated with defibrotide, in spite of which all of them died. considering that overall day +100 mortality was 9%, severe/ very severe sos was the most important cause of death of the series. [p391] although milder forms of sos might resolves within weeks, the most severe forms are still associated with a very high mortality rate. prophylaxis with defibrotide (the drug currently licensed for treatment) for high-risk patients has not been sufficiently studied yet. therefore, a high index of suspicion, early detection and early therapy are the only ways to try to reduce mortality due to sos in the hsct setting. disclosure of conflict of interest: this research has been performed entirely with public financial support. the royal marsden hospital, sutton, uk; 2 anthony nolan research institute, london, uk and 3 university college london, london, uk secondary poor graft function (spgf) complicates up to 15% allogeneic hcts, and is associated with increased mortality and poor quality of life due to recurrent infections and the need for ongoing blood product support. potential interventions include a second allograft using further conditioning, however many patients with spgf have a reduced performance status and are at an increased risk of complications from this procedure. unconditioned haematopoeitic progenitor cell (hpc) top-ups are associated with a high risk of gvhd if unmanipulated cellular products are used. cd34+ selection offers an attractive alternative, but incurs a loss of up to 20% hpcs and is an expensive procedure, unavailable to many centers internationally. alemtuzumab, a monoclonal anti-cd52 antibody, is routinely used in allogeneic transplant conditioning in the uk to prevent gvhd. we report the results of a retrospective study examining the efficacy of alemtuzumab conditioned hpc top-ups for spgf. data pertaining to patients who had undergone a second infusion of hpcs from their original donor were identified from our hospital-specific promise database. those who met the criteria of spgf defined as ⩾ 2 of hemoglobin 20 × 10 9 /l without support. 10 patients (4 pediatric, 6 adult) who underwent initial allogeneic transplants for malignancy (8) or bone marrow failure (2) received an alemtuzumab conditioned hpc top-up for spgf at our center 2005-2016. the diagnosis of spgf was made at a median 2.6 months post allograft (range 2-60) with trilineage cytopenias in 7 patients and bilineage cytopenias in 3 patients. all patients had received transplants from 10/10 (7 patients) or 9/10 (3 patients) matched unrelated donors. the median interval between initial transplant and top-up was 187 days (range 87-1853), and a median cd34 dose of 2.7 × 10 6 /kg recipient weight (range 0.3-7.63) was infused. 90% patients achieved haematological improvement (hi) at a median 20 days post-top-up (range 13-179), with the only failure to achieve hi seen in the patient who had received the lowest cd34 dose (0.3 × 10 6 /kg). one patient developed grade i agvhd post top-up but no grade ii-iv agvhd was observed. 1 year os was 80% and 2 year os 60% following hpc top-up. 3 deaths occurred due to infection at 1, 5 and 23 months post top-up, and one due to relapse of a prior non-haematological malignancy. 9 patients had an aplastic or hypocellular bm trephine pre-top up, which was repeated at 100 days post topup in 6 patients, of whom 5 had a normocellular bm trephine, while 1 remained hypocellular. alemtuzumab conditioned hpc top-up appears an effective intervention for spgf with results comparable to those of cd34 selected top-ups, and therefore represents a feasible alternative. larger studies are needed to exclude complications including viral reactivation and to investigate immune reconstitution following this procedure. disclosure of conflict of interest: none. high dose chemotherapy (hdt) followed by autologous stem cell transplantation (asct) has shown to improve outcome in patients with relapsed/refractory diffuse large b cell lymphoma (dlbcl). in the rituximab era, the benefit of asct has been debatable as prior study (coral study) has shown that patients who received r-chop as induction chemotherapy & responded to salvage chemotherapy had a poorer outcome following asct compared with those who received chop alone. in addition, it remains unclear whether addition of rituximab to standard high dose beam regimen provides any additional benefit. we retrospectively analyzed 63 dlbcl patients receiving high dose beam (n = 27) or rituximab +beam (r-beam) (n = 36) followed by asct for relapsed/ refractory dlbcl since 2002. all patients who received chop (n = 15) ± rituximab (n = 48) as first line therapy and who received ⩽ 2 lines of salvage chemotherapy before asct were analyzed. rituximab was given at the dose of 375 mg/m 2 on day +1 and +8 of asct. twenty-two (81%) patients in beam group and all the patients (100%) in r-beam group received rituximab-based salvage chemotherapy prior to asct. the 10year overall survival (os) was 71% and event-free survival (os) was 67% for the whole cohort. r-chop induced patients did not fare any worst after asct than chop induced patients (10 year os 73 vs 68 %; p = 0.91). there was a trend towards better survival in patients with pre-transplant disease free interval (dfi) 412 months compared to those with dfi 500/μl) time was 10 days and 11 days, respectively. median platelet recovery (420 000/μl) time was 17 days and 11 days, respectively (p = 0.11). ten year os (67% r-beam vs 77% s321 beam, p = 0.38) and efs (65% r-beam vs 73% beam, p = 0.28) were also comparable between both groups. hdt with beam and asct remains beneficial for patients with relapsed/ refractory dlbcl. it should be offered to all patients who respond to salvage chemotherapy with the expectation that they fare no worse than patients who do not receive rituximab in the induction chemotherapy. addition of rituximab following the standard beam for hdt and asct does not compromise haematopoietic recovery, but does not result in improved outcome in our study. prior use of rituximab during first-line or salvage therapy in most of the patients of r-beam group might have negated the beneficial effect of r-beam over beam. (1) . in this study, we aimed to develop a cns targeted chemotherapy regimen, which has lower toxicity and higher complete remission rates, in combination therapy. eight patients with secondary cns lymphoma (scnsl) and two with primary cns lymphoma (pcnsl), followed between the years 2010 and 2015, were included in the study, retrospectively. the patients were histologically diagnosed with biopsy and underwent autologous stem cell transplantation (apkht). all patients were treated with r-idaram/ rt (radiotherapy)/subsequently autologous stem cell transplantation (apsct) with r-beam protocol. the r-idaram regime consists of the following substances: rituximab 375 mg/m 2 , 50 cc/h infusion, day 1; cytosine arabinoside 1.0 gr/m 2 i.v., 1 h infusion, days 2 and 3; dexamethasone 100 mg, 12 h infusion, days 2, 3 and 4; idarubicin 10 mg/m 2 i.v.,15 min infusion, days 2 and 3; methotrexate 3 gr/m 2 (2 gr/m 2 at 445 years old-patients), 6 h infusion, day 4; and cytosine arabinoside 70 mg plus methotrexate 12 mg, intrathecally, days 2 and 8. the patients included seven males and three females. the median age was 44 years (range: 23-67). six scnsl patients were diagnosed in the application and two of them were diagnosed during r-chop chemotherapy (ct) protocol. five patients (57%) were stage ivb, and the others (43%) were stage iiib at diagnosis. after two or three chemotherapy cycles, patients were mobilized with growth factor support and median 5.510 6 cells per kg (range: 4-8) stem cells were collected. then, at a dose of 3600-4140 cgy cranial rt was administered for 14 days. after the third cycle of r/idaram, the state of remission was evaluated by cranial mri and lumbar puncture (lp). all patients achieved complete remission. neutrophil engraftment occurred at a median of 12 days (range: 10-18) and platelet engraftment occurred at a median 16 days (range: 11-21). after apkht, three patients relapsed and died at the fourth, ninth, and thirteenth months. grade i-ii manageable neurological toxicity occurred in two patients. the median follow-up time was 24 (range: 2-74) months. the five-year overallsurvival (os) was 63%. serious signs of infection were not observed in patients during transplantation. in pcnsl and scnsl, a standard treatment regimen has not yet been found. apsct with r-beam following modified r/idaram/rt is a curative and applicable therapeutic regimen with low toxicity, which can provide high rates of long-term survival and disease-free survival. despite the advent of novel therapies, autologous hematopoietic stem cell transplantation (ahsct) following melphalan (m)-based conditioning remains the standard of care for patients with multiple myeloma who are eligible. still, the majority of patients experience disease progression and ultimately succumb to their disease. we hypothesize that integrating novel agents in the conditioning is feasible and safe and may increase complete remission rates and overall survival. we completed a phase i, dose escalation study of carfilzomib (c) added to a backbone of bendamustine (b) and melphalan. all patients received a fixed dose (20 mg/m 2 ) of c on days (d) − 29, − 28, − 22, − 21, − 15 and − 14. in addition, patients were conditioned as described in table1. due to dose-limiting toxicity in cohort 2, the study was amended after the first 6 patients. subsequently, the dose of m was reduced to 140 mg/m 2 and the d +6 dose of c was omitted, per oversight of a data safety monitoring board. fifteen patients were enrolled, 9 males and 6 females. median age was 56 years (39-68). performance status was ⩾ 80% (kps) in all patients. per the international staging system (iss), 3 patients had stage i disease, 5 had stage ii, 6 had stage iii, and 1 had unknown staging. three patients had high-risk cytogenetics: 2 with t(4;14) and 1 with deletion 17p. four patients had undergone a prior ahsct. disease status at enrollment was stable disease (sd) (n = 3), partial response (pr) (n = 8), or very good partial response (vgpr) (n = 4). median cd34+ cell dose infused was 3.11 × 10 6 /kg (2.23 − 6.92 × 106). median follow-up was 18.2 months (1.4-28.7). all fifteen patients are evaluable s322 for engraftment. median time to neutrophil engraftment was 12 d (11-15). one patient died before achieving platelet engraftment. for the remaining patients, median time to platelet engraftment was 16 d (12-20) . non-hematologic toxicities included grade 3 acute mucositis (n = 1), lower gi complications (n = 7), electrolyte disturbances (n = 7), transaminase elevation (n = 1) renal insufficiency (n = 1), atrial fibrillation (n = 1), hypoxia (n = 1), prolongation of the qtc interval (n = 1), and grade 4 acute sepsis (n = 2), including 1 death (cohort 2) on d +44. eight patients went on to receive maintenance therapy: 3 with bortezomib, 3 with lenalidomide, and 2 with lenalidomide, dexamethasone, and c. posttransplant disease status was assessed per protocol by spep, spif, serum free light chains, and light chain ratio. twelve patients were evaluable on d +100. two patients had sd, 7 had vgpr, and 3 had complete response (cr). eight patients were evaluable on d +365. two patients had progressive disease, 1 had pr, 3 had vgpr, and 2 had cr. the combination of cbm prior to ahsct appears feasible, with manageable toxicities, at the doses described in cohort 3b. a prolonged follow-up and a phase ii study are warranted to determine response rates and long-term outcomes. disclosure of conflict of interest: none. beam (carmustine, etoposide, cytarabine, melphalan) is the most frequently used high-dose chemotherapy regimen for patients with lymphoma referred for autologous hematopoietic cell transplantation (autohct). in recent years a novel conditioning protocol containing bendamustine instead of carmustine (beeam) has been proposed in order to potentially increase the efficacy. so far, however data on its safety are limited. the aim of this study was to retrospectively compare the safety profile of beam and beeam based on single center experience. 174 consecutive patients with lymphoma treated with beam and 63 patients treated with beeam between year 2011 and 2016 were included in the analysis. the median age was 47 (19-69) years and 46 (22-73) years, respectively (p = ns). clinical characteristics of both groups were comparable. patients with hodgkin's lymphoma constituted 49% in the beam group and 40% in the beeam. among those with non-hodgkin lymphoma the diagnosis of dlbcl predominated. beam treatment consisted of carmustine 300 mg/m 2 on day − 6, etoposide 400 mg/m 2 /d on days − 5 to − 2, cytarabine 400 mg/m 2 /d on days − 5 to − 2, and melphalan 140 mg/m 2 on day − 1. in the beeam regimen carmustine was substituted by bendamustine administered on days − 7, − 6 at the total dose of 400 mg/m 2 i.v. peripheral blood was used as a source of stem cells. cd34+ cell dose was 5.1 (1.5-42.6) × 10 6 /kg in the beam group and 4.1 (2 − 16.8) × 10 6 /kg in the beeam group (p = ns). time to engraftment and the rates of adverse events up to day +100 after autohct were the study endpoints. all patients engrafted in both study groups. median time to neutrophil 40.5 × 10 9 recovery was 11 (7-37) days after beam and 10 (7-12) days after beeam (p = 0.13). median time to achieve platelet count 450 × 10 9 was 13 (7-44) days and 14 (7-33) days, respectively (p = 0.29). two patients died without progression before day +100 in the beam group, both due to bacterial infections. no early deaths were reported in the beeam group. the rates of grade 3 or 4 adverse events were comparable (see: table 1 ). administration of bendamustine instead of carmustin as part of conditioning does not affect engraftment as well as toxicity profile of the regimen. therefore beeam may be safely used in patients with lymphoma undergoing autohct. its efficacy requires evaluation in prospective studies focused on homogenous patient populations. [p398] disclosure of conflict of interest: none. the baltimore group reported a low dose tbi-based nonmyeloablative conditioning regimen followed by t cell replete bone marrow, with post-transplantation cyclophosphamide (pt-cy) to control gvhd and graft rejection. based on the fact that in our facility conventional low dose tbi was not available, we wanted to explore whether tmi/tli could be a potential substitute the aims of our study was to explore if tmi/tli can be considered an effective substitute of tbi in terms of os, pfs and nrm. retrospective analysis was applied in 159 cases of haploidentical hsct from april 2009 to october 2016. all patients underwent baltimore conditioning associating fludarabine (30 mg/m 2 /day) day − 6 to − 2, cy (14.5 mg/kg/day) on days − 6 and − 5, and tbi 2 gy in 135 patients and tmi/tli 2 gy in 24 patient at day − 1. unmanipulated bone marrow graft was infused at day 0. postgrafting immunosuppression consisted of cy (50 mg/kg/day) on day +3 and +4, and mycophenolate mofetil for 30 days, and tacrolimus or cyclosporine. no differences between the two groups was observed in term of age, gender diagnosis, disease status and donor type. 93% of patients engrafted in both arm (23/24 and 125/135). in tbi cohort vs tmi/tli cohort, the median time to anc 4500/μl and platelet recovery 420 000/μl was not different (22 and 27 days vs 20 and 27.5 days, p = 0.14 and 0.32, respectively). in all tmi/tli evaluable patients, full chimerism was observed at days +30. after a median followup of 17 months in tmi/tli cohort and 34 months in tbi arm, 1-year nrm was 25.9% and 13.9% (p = 0.16), respectively. the 1 years os and pfs were not statistically different in the two groups 70% vs 57.1%, p = 0.12 and 62.5% vs 48.1%, p 0.09, respectively). the 1-year relapse incidence was 26% in tmi/tli group and 23.6% in tbi group, p = 0.61. no difference in incidence of both agvhd and cgvhd was observed between the two groups. this retrospective analysis suggests that tmi/ tli could be considered an effective substitute of low dose tbi, with a sufficient degree of immunesuppression of recipient, allowing engraftment and full chimerism. the gvhd both acute and chronic as well as the 1-y nrm were not different. disclosure of conflict of interest: none. comparison of the beeam conditioning regimen and the beam conditioning regimen in the autologous transplantation for hl and nhl s lozenov 1 , p ganeva 1 , y petrov 1 , g arnaudov 1 and g mihaylov 1 1 the beam has established itself as a standard of care conditioning regimen in the autologous lymphoma hsct setting for most transplant centres in europe. yet however various other regimens are being compared with it in order to achieved better safety profile, better os and dfs, in order to improve results with chemoresistant and unfavourable patients. one such regimen is beeam (bendamustine, etoposide, cytarabine, melphalan).we aimed to compare the efficacy of the beam and beeam conditioning regimens and to compare their myelotoxicity profile. we evaluated retrospectively 114 adult patients (mean age 41.1197 with sd 11.12404), receiving auto-hsct at the national specialized hospital for active treatment of hematological diseases in sofia, bulgaria for relapsed/refractory hl or nhl (of them mh -57, dlbcl -26, pmbcl -15, fl -3, lbl -3, ptcl-nos -3, aitl -2, alcl -2, mcl -2, mzl -1) for the period from 1.01.2013 to 1.07.2016 with a follow-up of patients up to 1.11.2016. ninety-two of the patients received the beam (as previously described -bcnu 300 mg/m 2 i.v. day − 6, etoposide 200 mg/m 2 i.v. days − 5 to − 2, cytarabine 400 mg/m 2 i.v. days − 5 to − 2, and melphalan 140 mg/m 2 i.v. day − 1) regimen and 22 received beeam regimen (bendamustine on days − 7 and − 6 (160 mg/m 2 ); cytarabine, 400 mg/m 2 intravenously daily, from day − 5 to day − 2; etoposide, 200 mg/m 2 intravenously daily, from day − 5 to day − 2; and melphalan, 140 mg/m 2 intravenously on day − 1). the overall survival at the second and third years of follow-up (os-2, os-3) and dfs at the third year, the cr rates and the average time periods to hematological recovery, were compared. the os at 2 and 3 years, respectively, was 86.1% and 86.1%, for beeam and 78.8 % and 71% for beam, the dfs at 3 years was 76.4% for beeam and 73.2% for beam, provided that the differences did not have statistical significance (p 0.851 for os and p 0.890 for the dfs). the cr rate was 63.63% in the beeam group versus 50% in the beam group. from the patients who received autologous hsct in stable disease or progression pre-transplant status (chemoresistnat patients), 22.72% of the patients receiving beeam achieved cr at the first post-transplant evaluation versus 10.86% respectively for the beam group. the mean time to hematological recovery for neutrophils was 11.1765 ± 4.91471 days (beeam) versus 10.2469 ± 3.56216 days (beam) and 12.6471 ± 6.04091 days (beeam) versus 11.1235 ± 2.52677 days (beam) for platelets. beeam appears to be a non-inferior alternative conditioning regimen to the standard beam, it shows a trend towards higher myelotoxicity, but also a trend towards better response rates in chemoresistant patients. [p400] disclosure of conflict of interest: none. autologous hematopoietic stem cell transplantation (asct) is widely used as a consolidation therapy in aggressive non-hodgkin's lymphoma (nhl) and recurrent or refractory classic hodgkin's lymphoma (hl). in mexico, the use of carmustine (bcnu) in the conditioning regimen of these patients is limited due to the lack of access to the drug and its high costs. this study aims to compare results in terms of toxicity, disease-free and overall survival between a group of patients treated with the standard regimen beam and another group treated with a scheme in which carmustine was replaced by cisplatin (peam regimen). a comparison of two groups with lymphoma was performed and the clinical aspects of cisplatin 100 mg/m 2 d . the characteristics were well balanced between the two groups. the mean time for neutrophil grafting (4500 per mm 3 ) was significantly slower with beam than with peam (12 vs 11 days, p = 0.001), hospitalization time was longer with beam compared to peam (25 vs 22, p = 0.015). on the other hand, proportion of patients who require red blood cell s324 transfusion was significantly higher in beam group (58%) versus peam group (20%) (po 0.001), but total amount of platelet transfusion did not differ between groups. about the toxicity, beam patients had significantly more frequent incidence and severity of nausea/vomiting (95% vs 53.8%) and diarrhea (61.5% vs 90%) compared to peam (p o 0.01). no significantly differences were observed in incidence of mucositis (p = 0.65). at the moment of the analyses, 75% of patient of the peam group were in complete response versus 59% of the patients treated with beam, but it did not represent a significant difference. disease-free survival and 5-year overall survival in the peam vs beam scheme were similar with 64% vs 59% (p = 0.69) and 84% vs 76% (p = 0.35) respectively but with less toxicity using the peam scheme. peam regiment is not inferior scheme compared with beam, because it shows similar outcomes in disease-free survival and overall survival. additionally, peam is a well-tolerated regime and beam scheme was associated with greater gastrointestinal toxicity such as nausea, vomiting and diarrhea, also greater hematology toxicity such as more requirement of red blood cell transfusion. [p401] disclosure of conflict of interest: none. cumulative busulfan exposure is associated with relapse following busulfan and cyclophosphamide myeloablative allogeneic stem cell transplantation for acute myeloid leukaemia e wong, d kliman 1 , m chau 2 , j szer 2 , c nath 1 , p shaw 1 , d ritchie 2 , d gottlieb 1 and a bajel 2 1 westmead hospital, new south wales, australia and 2 royal melbourne hospital, victoria, australia the optimal busulfan exposure to reduce disease relapse in adult patients with acute myeloid leukaemia (aml) undergoing busulfan/cyclophosphamide myeloablative allogeneic stem cell transplant (allosct) is poorly defined. we retrospectively analysed busulphan pharmacokinetics (pk) and outcomes of patients who underwent busulfan/cyclophosphamide conditioned allosct for aml from 2010 to 2016. busulfan was administered intravenously over 5 days (1.6 mg/ kg/d for 2 days followed by 3.2 mg/kg for 3 days). peripheral blood was obtained for busulfan pk after the first dose. subsequent doses of busulfan were decreased if daily busulfan exposure (area under the curve; auc) was anticipated to exceed 5000 μm per min/day. cyclophosphamide was dosed at 120 mg/kg. the primary outcome was the cumulative incidence of relapse (cir) accounting for non-relapse mortality (nrm) as a competing risk. independent variables analysed included age, sex, cytogenetic risk group, disease risk index (dri), donor type, stem cell source, t-cell depletion, and cumulative busulfan auc (cumauc) calculated as previously described. 1 (figure 1) . t-cell depletion was also associated with increased cir (hr 3.3; p = 0.0049). patient age, sex, cytogenetic risk, dri and graft type were not significantly associated with cir. on multivariate analysis, cumauc 415 000 μm per min remained significantly and independently associated with lower cir (hr 0.38; p = 0.036). cumauc was not associated with nrm, rfs, os, or the incidence of acute or chronic gvhd. figure 1 . cumulative incidence of relapse in patients stratified by total busulfan exposure. [p402] cumulative busulfan exposure 415 000 μm per min is independently associated with reduced relapse following busulfan/cyclophosphamide allosct for adults with aml. these findings support further evaluation of the optimal busulfan exposure to reduce aml relapse in a prospective clinical trial, whereby patients could be randomised to target cumauc 415 000 μmol per min versus standard practice. hematopoietic stem cell transplant with busulfan and cyclophosphamide (bucy) based conditioning has a relatively high incidence of liver toxicity and sinusoidal obstruction syndrome (sos). busulfan and cyclophosphamide metabolites share the same glutathione conjugation in the liver metabolism. a small number of studies addressed different sequence of both drugs bucy vs cybu during conditioning. differences in liver toxicity, sos, transplant related mortality (trm), relapse incidence (ri) and overall survival (os) were reported favoring cybu conditioning. we decided to address the above issues at the umc ljubljana, slovenia. this was a retrospective study following patients with myeloid malignancies (aml, mds, mpn) with bucy (n = 30) and cybu (n = 14) conditioning through a three year period in a single institution. primary endpoint was detecting difference in liver toxicity by measuring levels of liver enzymes. secondary endpoints were incidence of sos, difference in trm, ri and os. patients characteristics between groups at the time of the transplant did not differ significantly. we observed significantly higher liver toxicity through elevated bilirubin and alt in the bucy 73.3% than cybu 64.3% patient group (picture 1). the highest probability of liver toxicity was around d0 in the bucy group and in the second week after the transplant in the cybu group. the incidence of sos, trm and ri were comparable between the groups. there was no difference in os between the patient groups during the 40-month follow-up. bucy conditioning for hematopoietic stem cell transplant causes higher incidence of liver toxicity compared to cybu conditioning. there is no difference in sos frequency, trm, ri and os between bucy and cybu conditioning. prospective controlled comparison would be needed for further study of the subject. disclosure of conflict of interest: none. early monocyte recovery is associated with better overall survival after busulfan containing myeloablative conditioning allogeneic hematopoietic cell transplantation in patients with acute myeloid leukemia a lojko-dankowska 1 the outcomes of allogeneic hematopoietic cell transplantation (allohct) in acute myeloid leukemia (aml) depend on different patient-, disease-and transplant related factors, including the dose and combination of agents used for conditioning. the aim of the study was to analyze the outcomes of allohct in patients with intermediate or high risk aml according to disease risk index (dri) who received myeloablative conditioning consisted of intravenous busulfan (9.6-12.8 mg/kg) combined with cyclophosphamide (120 mg/ kg) or fludarabine (150 mg/m 2 ) between 2010 and 2016 in our institution. the published data indicate that the combination of busulfan (bu) and fludarabine (flu) seems to have more favorable toxicity profile than combination of bu and cyclophosphamide (cy), so bucy regimen has been substituted with buflu as the myeloablative conditioning for aml patients in our institution practice since 2014. we evaluated the influence of type of regimen on transplant outcomes along with the impact of other potential prognostic factors, including age of patient, dri, donor type, hla and gender mismatches, stem cells source, and lymphocyte and monocyte recovery. the study group consisted of 71 aml patients, median age 37 years (range: 19-59), classified as intermediate (n = 57) or high (n = 14) risk according to the dri, who were conditioned with bucy (n = 38) or buflu (n = 33) followed by allohct from hla identical sibling (n = 23) or 9-10/10 matched unrelated donor (n = 48). the stem cell were collected from peripheral blood (n = 44) or bone marrow (n = 27). gvhd prophylaxis consisted of calcineurin inhibitor combined with mtx plus atg in allohct from unrelated donors. engraftment was observed in all patients. the median time to neutrophil count (0.5 g/l) and platelet count (20 g/l) recovery was shorter after buflu in comparison with bucy (18 days vs 22 days; p = 0.045 and 13 days vs 20 days; p o0.001), however peripheral blood stem cells were used more often after buflu regimen than after bucy (88% vs 40%, p340/mm 3 on+21 day after transplant (2-year os 77% vs 55%, p = 0.034) and intermediate vs high dri (2-year os 78% vs 53%, p = 0.068). in multivariate analysis higher amc after allohct remained the only independent favorable prognostic factor for os (rr 0.35 (95% ci 0.13-0.97), p = 0.04). our results suggest that early monocyte recovery after myeloablative bu containing conditioning allohct is significant favorable predictor of outcome. in our experience both bucy and buflu myeloablative regimens result in similar long-term survival after allohct in aml patients. [p403] disclosure of conflict of interest: none. the use of t-cell depletion as part of the conditioning protocol has the potential to improve the tolerability of allogeneic stem cell transplantation (hsct) through the reduction in graft versus host disease (gvhd). despite the wide spread adoption of this practice in many parts of the uk and europe, definitive recommendations regarding the most appropriate dose remain elusive. previous experience by our group with 100 mg of alemtuzumab combined with fludarabine and busulfan based conditioning demonstrated good long-term outcomes with low rates of gvhd. however, due to concerns of high relapse risk especially in patients with high-risk myelodsypastic syndrome and acute myeloid leukaemia, we instituted a policy change in 2013 to reduce the dose of alemtuzumab in the conditioning protocol from a total of 100-60 mg. we conducted a retrospective analysis of all consecutive patients undergoing reduced intensity unrelated allogenic stem cell transplantation with fludarabine (150 mg/m 2 ), busulfan (6.4 mg/kg iv or 12.8 mg/kg iv) and alemtuzumab (fb2c or fb4c, respectively) conditioning for neoplastic myeloid disorders between 2010 and 2016. patients were subsequently analysed in two cohorts; those receiving 60 mg of alemtuzumab (n = 84) and those receiving 100 mg of alemtuzumab (n = 95). apart from a decreased proportion of females in the 60 mg alemtuzumab group, the cohort was balanced across the different dose levels ( table 1 ). the longterm overall survival (os) of the entire cohort was good with a 5 year os of 51%. no significant differences in overall outcomes across the two groups were observed with a 5 year os of 51% in the 60 mg group vs 49% in the 100 mg group (p = 0.39). cumulative incidence of relapse (cir) and nonrelapse mortality (nrm) was 42% and 21% and 39% and 24% in the 60 mg and 100 mg groups, respectively. interestingly, age had a significant effect on nrm in the 100 mg (7% age o50, 28% age 50-65 and 41% age 465 p = 0.04), but not in the 60 mg group (11% age o50, 19% age 50-65 and 24% age 465 p = 0.8). the effect on relapse rate was not significant in either group (p = 0.6 and p = 0.11, respectively). this retrospective analysis did not demonstrate an overall improvement in transplant outcomes with dose de-escalation of alemtuzumab from 100 to 60 mg. in particular, we did not see the anticipated improvement in relapse rate in this cohort. notably older patients seem to tolerate the 60 mg dose better due to the lower nrm. prospective trials with accompanying translational work are required to determine the optimal dosing and schedule for this group of patients. disclosure of conflict of interest: none. bendamustine was given at 200 mg/m 2 /d for the first 4 pts then 100 mg/m 2 /d for the 4 subsequent pts and finally at 120 mg/m 2 /d for the remaining pts (22 pts). among the beam group, 68% had non-hodgkin's lymphoma (nhl) and 32% hodgkin's lymphoma (hl) compared to 87% and 13%, respectively, in the beeam group (p = 0.014). hhv-6 detection was performed by pcr for symptomatic pts (fever, rash or prolonged cytopenia). patients were housed in single bedrooms with air filtration and received the same supportive care. median age was 50 (18-66) and 56 (20-67) in the beam and beeam groups respectively and median of previous chemotherapy regimens was 2 (range: 1-5). fifty two out of 90 patients were male (37/60 in the beam group and 15/30 in the beeam group). pts were in cr (46.7% vs 56.7%) or pr (53.3% vs 43.3%) at time of transplant. there was no difference in terms of hematologic recovery (median = 11 days (range: 7-22)), blood and platelets transfusion, mucositis toxicity. there was no statistical difference in the incidence of acute renal failure when comparing the two groups. however, there was a very striking difference when considering the highest dose of bendamustine when compared as well to the two others doses of bendamustine (po 0.00001) as to the beam group (p = 0.005). additionally, we also observed a high incidence of symptomatic hhv-6 infections (53.3% vs 8.3%, p o0.00001), digestive toxicity (36.6% vs 15%, p = 0.03) and a longer hospitalization duration (25 days (range: 18-59) vs 21 days (range: 18-32), p = 0.001) for patients in the beeam group overall. with a median follow up of 18.3 and 9.7 months for beam and beeam respectively, overall survival (93% vs 86%), transplant related mortality (0% vs 3%) and event free survival (83% vs 78%) were comparable. overall, beeam regimen was associated with longer duration of hospitalization, higher rate of digestive toxicity and increased risk of symptomatic hhv-6 infection as compared to the beam regimen. in addition, higher doses of bendamustine (200 mg/m 2 /d for two consecutive days) were associated with unacceptable high rate of acute renal toxicity. with a still short follow-up, the absence of benefit on disease control together with higher short term toxicity does not allow to recommend the use of beam instead of classical beam. should it be used, we suggest that pts should be carefully monitored for renal toxicity and for hhv-6 infection in case of symptoms. disclosure of conflict of interest: none. high-dose treosulfan and melphalan for consolidation therapy in high-risk ewing sarcoma me abate, a paioli, a longhi, m cesari, e palmerini and s ferrari musculoskeletal department, rizzoli orthopaedic institutes, bologna, italy common toxicities observed after high dose chemotherapy with busulfan and melphalan for high risk ewing sarcoma (es) are generally well managed by current supportive care but some patients can develop severe complications. treosulfan is an alkylating agent that has recently been used as a substitute of busulfan to prevent potential serious complications related to busulfan. medical records of 7 es patients undergoing autologous peripheral blood stem cell (apbsc) transplantation after intravenous treosulfan (treo) and melphalan (mel) from 2011/6/1 to 2016/2/29 were analyzed with regard to toxicity and outcome. patients were included into the study if they were eligible for the protocols activated in our institution for es and presented reasons that did predict potential complications related to busulfan, such as previous radiotherapy on axial skeleton/pelvis or coexistence of high risk of epilepsy. as consolidation treatment patients received intravenous treo 12 g/m 2 over 3 days and mel 140 mg/sqm with support of apbsc transplant and use of granulocyte colony stimulating factor. in those patients with lung metastases total lung irradiation was performed at least 2 months after treomel. frequency of toxicity for treomel was recorded with at least 6 months of follow-up and was evaluated according to nci ctg common toxicity criteria. the median age at diagnosis of patients receiving treomel was 16 years (range 13-25 years), 3 males and 4 females. 5 patients had localized disease at diagnosis with poor radiological or histological response to standard chemotherapy; one patient had lung metastases at diagnosis and one patient had relapsed disease with lung metastases. before receiving treomel the primitive tumour underwent radiation therapy in 5 cases (3 pelvis, 1 cervical vertebra, 1 sacrum), surgical resection in one case(tibia) and surgical resection plus radiation therapy in one case (fibula). 2 patients showed eeg abnormalities at high risk of developing epilepsy. the median number of infused cd34+ cells was 5.9 × 10 6 /kg (range 3.4-15.9). febrile neutropenia occurred in 5/7 patients and lasted one day in 3 patients and 2 days in 2 patients. median time to granulocyte engraftment was 10 days (range 10-12 days); median time to platelet engraftment 420 000 was 10 days (range 9-16 days). only one patient needed 2 red blood cells transfusions; 5 patients needed 1 platelet transfusion and 2 patients needed 2 platelet transfusions. none developed grade 3-4 stomatitis or grade 3-4 [p406] hematuria or grade 3-4 liver toxicity. surprisingly, a patient became pregnant after 1 year and 10 months from transplantation. with a median follow-up of 24 months (range 8-65 months) 5 patients are alive in complete remission, one patient is alive with relapsed disease and one patient died for disease progression. these results, related to a limited cohort of patients, confirm the lower toxicity observed for treosulfan with respect to busulfan. although more data are needed to clarify the role of treosulfan in es, the impact of potential severe complications observed with busulfan, including infertility, should suggest its replacement with treosulfan in selected cases. disclosure of conflict of interest: none. immunoadsorption procedures prior to haploidentical allogeneic pbsct could prevent graft failure in patients with hematological malignancies displaying anti-donorspecific hla antibodies donor-specific anti-hla antibodies (dsa) have been shown to be associated with a high risk of rejection in solid organ transplantation and with graft failure (gf) in allogeneic hematopoietic stem cell transplantation. a combination of anti-cd20 (rituximab), plasma exchange (pe), and ivig in 12 patients with additional buffy coat infusion in 5 among them prevented graft loss in all patients that became c1q negative before sct. we addressed the question whether immunoadsorption in combination with rituximab can also be applied in patients with dsa to prevent graft failure in haploidentical pbsct. four patients with acute myelocytic leukemia or myeloma in second complete remission were enrolled. the presence of dsa was determined by luminex at pre bmt checking. immunoadsorption was performed with polyclonal sheep anti-human igg adsorbers (miltenyi biotec gmbh, germany) on life 18 apheresis system. in addition all patients received rituximab 375 mg/kg bw in a single dose. patients were conditioned with a reduced intensity regimen comprising tbi 2 gy, cyclophosphamide 29 mg/kg, and fludarabin 150 mg/m 2 . all patients received cyclophosphamide post bmt (ptcy) 100 mg/kg. non-t-cell depleted pbsct were transfused in a sequential manner in 2 doses each. the data of the patients and treatments is summarized in table 1 . two patients had a normal hematopoietic reconstitution and are alive at +21 and +15 months post-transplantation, one with hepatic gvhd; chimerism was 100% in peripheral blood on last follow up. one patient died following a graft failure. by a combination of rituximab and repeated immunoadsorption prior to allogeneic pbsct the titer of dsa could be lowered sufficiently to enable engraftment. ia turned out to be a safe procedure without relevant clinical side effects. hematopoietic reconstitution was in the normal range in 2 of 3 evaluable patients. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative option for patients with beta thalassemia major. however, the availability of hla-matched related donor remains the main obstacle for allogenic hsct. although, a few studies have been reported, experience with hla matched unrelated donors is limited. we present the result of 35 children with beta thalassemia major who received allogeneic hsct from hlamatched unrelated donors with using a novel conditioning regimen. we retrospectively assessed 35 unrelated hsct in children with beta thalassemia major. all patients received busulphan (bu) based myeloablative conditioning regimen. busulphan was used according to weight adjusted doses. in addition, all patients received fludarabine 150 mg/m 2 in 5 days, cylophosphamide 120 mg/kg in 3 days, thiotepa 10 mg/kg in one day and atg 30 mg/kg in 3 days. cyclosporin-a and mtx were used for graft versus host disease (gvhd) prophylaxis. donor chimerism was evaluated in the peripheral blood on days +30, +100 and +180. the median age of the patients was 6.9 years (range 14 month-15 year). two of the patients were grouped in class i and rest of them were class ii. the median serum ferritin level was 1.255 ng/ml (range, 585-5832). all of s329 the donors were matched 10/10 with high-resolution hla typing in gvhd direction but three of them 9/10 with graft failure direction. twenty-three of them received bm (median tnc: 6.2x108/kg) and 12 pbsc (median mnc:7.3x108/kg) with median cd34+ cell number 7.20x106/kg. the median neutrophil and platelet engraftment days were 13 and 14 days in pbsc and 17 and 20 days in bm group, respectively. grade i-iv acute gvhd was observed in 7 patients (26%) and only one experienced limited chronic gvhd with only skin involvement. mild to moderate vod was seen in 13 patients (37%) and treated with defibrotide successfully. all patients except one are alive with full donor chimerism (between 95-100 %) with a median 12 months (range 3-49 months) follow-up. one patient died because of cmv pneumonia. these data show that the results of hsct from unrelated donors in selected low risk thalassemia patients may be comparable to hsct of matched sibling donors. however, it needs further studies with long term follow up and larger study population. disclosure of conflict of interest: none. table 1 . data about cytogenetic risk of group 1 patients were available only in 9 individuals. differences between groups were analyzed by t-student and chi square tests. survival was analyzed by kaplan-meier method and differences in survival between groups were evaluated by log rank test. no differences were found between groups regarding gender, sc source, disease status at sct, type of donor and number of cd34+ cells infused. patients in group 2 were significantly older (median age for groups 1 and 2: 32 vs 38, p = 0.021). gvhd prophylaxis protocols included atg in a higher frequency in group 2. no differences between groups 1 and 2 were observed in neutrophils recovery (median days to anc4500/μl: 9 vs 8 respectively, p = 0.78) and platelets recovery (median days to platelets 420 000/μl: 8 vs 5 respectively, p = 0.51). patients in group 1 required more red cell transfusions (median packed rbc: 3 vs 1, p = 0.048). no differences were observed regarding platelets transfusion requirements or length of hospitalization. post-sct os was significantly better in group 2 (3 years-os group 1: 23%; group 2: 61%; p = 0.029) (figure 1). there were no significantly differences between groups regarding frequency of mucositis, diffuse alveolar haemorrage, sepsis, acute and chronic gvhd. vod was more frequent in group 1 (4/21 vs 0/20, p = 0.03). trm mortality was higher in group 1 (6/21 vs 3/20), being this difference no statistically significant (p = 0.29). as it was reported by others, the use of fludarabine-based conditioning regimen was associated with a significantly better post-sct os and a reduced frequency of vod in aml patients. reduction in trm and differences in the frequency of described complications are not statistically significant probably due to the small size of this sample. since march 2016, given the limited availability of melphalan, we administer the beac regimen (carmustine, etoposide, cytarabine, and cyclophosphamide), instead of the gold standard conditioning regimen beam, followed by autologous haematopoietic cell transplantation (ahct) in relapsed or refractory hodgkin (hl) and non-hodgkin (nhl) lymphoma patients. the primary goal of this analysis was to assess the immediate related toxicity of this alternative regimen. we used beac (carmustine 300 mg/m 2 , etoposide 800 mg/m 2 , cytarabine 800 mg/m 2 , and cyclophosphamide 140 mg/kg) in 22 consecutive lymphoma patients (13 hl, 9 nhl) who underwent ahct for relapsed or refractory disease. the median age of the patients was 42.5 years (17-64). they all received peripheral stem cell grafts with a median cd34+ cell dose of 5.13 × 10 6 /kg cells (2.07-15.78). disease status post salvage treatment (at ahct) was complete remission (cr) in 6, partial remission (pr) in 11 and progressive disease in 5. the disease was chemosensitive to salvage therapy in 17/21 patients. median follow up was 112 days (31-224). toxicity was assessed according to the who toxicity scale grading. all patients engrafted successfully. median time for engraftment was day +10 (d+10) for neutrophils (4500/mm 3 ) and d+11 for platelets (420 000/mm 3 , without transfusion within the previous 3 days). patients were hospitalized for a median of 19 days (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) . no treatment-related mortality occurred. two patients died due to disease progression (both nhl patients, on d+143 and d+63). toxicity assessment until d+30 is presented in table 1 : moreover, no hemorrhagic cystitis or macroscopic hematuria, and no cardiac events were encountered. febrile neutropenia was recorded in 4 and bacteremia in 9 patients (7 gram+, 2 gram-, 2/9 related to central venous catheter), with fever ≤ grade 2 in all cases. during d+30-100 two patients presented fever of unknown origin, and 3 patients had upper or lower respiratory infections, with no other adverse events being recorded. in terms of disease best response within 3 months post ahct (18/22 patients evaluated), 11 patients achieved or sustained cr, 5 pr (1 of these patients eventually died due to disease progression), 1 relapsed and 1 succumbed due to disease progression (no response). according to our preliminary results, the early toxicity profile of beac is very low, the regimen is easily tolerable for the patients, and without any treatment-related mortality. its use as an alternative conditioning regimen in ahct for lymphoma patients seems feasible. further investigation including more patients and comparative analysis to other conditioning regimens are warranted for reliable conclusions on the toxicity and efficacy of beac. disclosure of conflict of interest: none. veino-occlusive disease (vod) is a potentially fatal adverse event caused by intravenous (iv) busulfan used in bone marrow transplantation (bmt) conditioning. the objective of this study was to identify determinants of vod in children treated by iv busulfan. this was a retrospective analysis of data collected in 293 children from two bmt centers over 10 years. vod was diagnosed according to modified seattle criteria. individual pharmacokinetic data, including busulfan area under the concentration-time curve (auc) and maximal concentration (cmax) were estimated in all children by using a validated bayesian approach. 1 we examined the relationships between the occurrence of vod and available data in a learning (n = 243 patients) and validation set (n = 50 patients) obtained by random splitting. logistic regression was used as a continuous statistical model. in addition, we used classification and regression tree (cart) analysis, a machine learning and binary partitioning technique, to identify determinants of vod and their optimal cut-off values. the predictive performance of variables within both models was assessed by these results are compared with historical data from our service using beam as conditioning followed by auto-sct in lymphoma patients. nine patients were enrolled to receive neam: mitoxantrone 6 mg/m 2 day -6 to -4, etoposide 100 mg/m 2 every 12 hours and cytarabine 100 mg/m 2 every 12 hours day -6 to -3, and melphalan 140 mg/m 2 day -2, followed by auto-sct. the median age was 51 years (19-63); five non-hodgkin lymphomas (nhl) and four hodgkin lymphomas (lh). six patients were in partial remission (pr), two in complete remission (cr), and one with progressive disease at time of auto-sct. neam patients were compared with a historical control group of patients receiving beam regimen (n = 167). differences between groups were analyzed by t-student and χ 2 -tests. median cd 34+ cells infused in neam and beam groups was 3.48 × 10⁶/kg (3.09-8.72) and 4.14 × 10⁶/ kg (1.02 -25.6), respectively (p = 0.42). the median time to neutrophil recovery (4500/μl) was 16 days (11-22) and 10 days (3-30) (p = 0.000017) and median time for platelets recovery (420 000/μl) was and 15 days (9-30) and 8 days (1-49) (p = 0.018) respectively, for neam and beam patients. median duration of hospitalization was 34 days (24-55) with neam and 27 days (15-59) with beam (p = 0.002). among neam patients, 88% had one or more febrile episodes during neutropenia. no case of grade iii or iv mucositis was described. there was no transplant-related mortality (trm: 0%) associated with the use of neam regimen. at the present, all neam patients are alive, two of them in relapse (22%). due the difficulties in obtaining carmustine in our region, neam can be considered as a feasible alternative to beam. however, despite the sample was small enough to draw conclusions, we find that neam presents prolonged aplasia of significant value, we are currently exploring conditioning regimens followed by auto-sct in hodgkin's and non-hodgkin's lymphomas based on bendamustine, etoposide, cytarabine and melphalan. disclosure of conflict of interest: none. at present, decision-making about conditioning regimens for allogeneic hsct is based on patient's and donor's features, and disease characteristics. during the last years, terms as 'myeloablative/non-myeloablative/reduced-intensity' have been frequently employed in a confusing and unequal way among the different centers. knowing the expected intensity and myeloablative effect from each regimen is very useful and constitutes the aim of this analysis. we have analysed the severe neutropenia (anc o500 per mcl), and thrombocytopenia durations (platelets o20 000 per mcl), the need for platelet concentrates transfusion and the duration of the inpatient period of the 223 allo-hsct carried out during the last four years in our centre. these data are reported according to the conditioning regimen used and to the type of transplant performed. then, they are compared among them in order to stablish intensity ranks. results: population characteristics are described in table 1 : conventional intensive regimens (bu-cy2, cy-bu2, tbi-cy) reported more days of severe neutropenia and greater need of platelet concentrates transfusion. the regimens with less days of severe neutropenia, less need of platelet concentrates transfusion and fewer days of admission were flu-bu3 and flu-bu2. allotransplants carried out with stem cells from bm presented more days of severe neutropenia and longer hospital stay. similar platelet transfusion need was reported. haplo-identical allotransplants reported more days of severe thrombocytopenia, but were not asociated to longer neutropenia or longer hospital stay than the others. these data are described in detail in table 2 . flu-bu: the number (4, 3 or 2) expresses the doses of busulphan administered at 3.2 mg/kg/day. pc: platelet concretates data is expressed in medians. within the analysed conditioning regimens, an intensity rank is stablished regarding the myelosupression induced (in descending order): conventional intensive regimens (cy-bu2, bu-cy2, tbi-cy), flu-mel, flu-bu4, flu-bu3 and flu-bu2. all of them induced severe neutropenia and thrombocytopenia, and for that reason they must be considered myeloablative regimens. disclosure of conflict of interest: none. myeloablative allogeneic stem cell transplant for aml and mds: the impact of advanced age in the outcome m sánchez-escamilla* 1,2 , s garcía-ávila 1 , l yáñez san segundo 1,2 , ma bermudez rodriguez 3,2 , mm colorado araujo 1,2 , a casado diez 1 , m celis alvarez 1 , a cabero martinez 1 , c fernandez martinez 1 , a insunza garminde 1,2 , c richard espiga 1,2 and e conde garcía 1,2 1 allogeneic hematopoietic stem cell transplantation (allo-hsct) is the only curative option in high risk myeloid hematological malignancies. myeloablative conditioning (mac) regimen has been proven to be effective in the control of high risk diseases in advanced age patients. objective: the aim of this study was to analyze the efficacy of myeloablative allo-hsct in two cohorts of patients considering their age at transplant. we also analyzed the incidence of acute and chronic graft versus host disease (gvhd) and procedure related outcomes who underwent to myeloablative allo-hsct were retrospectively analyzed. the median age was 49 years (iqr 36-57). both groups were divided regarding their age at allo-hsct [group 1, age ⩾ 55 years (n = 41) and group 2, age o55 years (n = 93)]. patient´s characteristics are shown in picture 1. data were collected as either continuous data and compared by two-tailed unpaired t-test or mann-whitney test, or as categorical variables and compared by chi-square. the procedure related outcomes were analyzed with the kaplan-meier test. the incidence of acute gvhd grade ii-iv was similar in both groups (43.9% in group 1 and 42.2% in group 2, p = 0.761 ). the mean day to acute gvhd (grade ii-iv) development was 38 days in group 1 and 40 days in group 2. the most involved organs in both groups were skin (group 1: 94.4% and group 2: 72.1% [p = 0.258]) and gut (group 1: 55.6% and group 2: 62.8% [p = 0.598]). at day +100 post-transplant 123 patients were alive and evaluable for chronic gvhd. the incidence of cgvhd development was similar between group 1 and 2 (61.1% versus 68.4%, respectively, p = 0.140). however, severe grade s333 of cgvhd was high in group 1 patients (25.0% versus 18.4%). with a median follow up of 43 months (iqr, 9-70) the probability of os was significantly low (p = 0.004) in group 1 (46.3% +-7.8) compared with group 2 (67.0% ± 4.9). pfs was also significantly low (p = 0.003) in group 1 (41.5% +-7.7) compared with group 2 (66.3% ± 4.9). trm at 43 months was higher in group 1 compared with group 2 (34.1% versus 17.2%). mortality due to relapses was also higher in group 1 (17.0% versus 12.9%). most of the patients died during the first 24 month. comparing both groups at this time (24 months post-transplant), trm was higher in group 1 compared with group 2 (26.8% versus 14.0%). deaths due to relapse were also higher in this group (17.1% versus 10.7%). in our series, myeloablative conditioning regimen provides good survival rates and disease control in high risk hematopoietic diseases, however in patients aged ⩾ 55 years confers high toxicity. it may be necessary to evaluate other strategies in this group of patients. disclosure of conflict of interest: none. allogenic hematopoietic cell transplantation (hct) is reserved for a group of high risk multiple myeloma (mm) patients having relapsed after high-dose melphalan and autologous transplantation. in general, reduced-intensity conditioning (ric) regimen are applied in this patient group with the aim of reducing transplant related mortality (trm). however, relapse of disease remains a major challenge after allogeneic hct. to address this issue, we added radioimmunotherapy (rit) to a conventional ric regimen. we have used a 188 rhenium anti cd66 antibody in combination with a ric conditioning regimen. this ß-emitter leads to a so called 'cross-fire' effect allowing for bone marrow doses of 20 gy and in parallel may target cd66 on myeloma cells. we hypothesized that this strategy may decrease the incidence of relapse. so far, we have treated nine patients with high risk relapsed multiple myeloma. all patients had received one (n = 7), two (n = 1) or three (n = 1) high-dose regimens and autologous hct. conditioning therapy was flu/mel (n = 5), flu/ bu (n = 2) or flu/treo (n = 1). flu/cy and 2 gy tbi were applied before haploidentical transplantation. patients received g-csf mobilized pbsc from unrelated (n = 8) or haploidentical (n = 1) s334 donors. either tacrolimus/ methotrexate/ bortezomib or cyclosporine a/ methotrexate were used for gvhd prophylaxis. early extramedullary toxicity was limited. neutrophil and platelet engraftment was timely and complete in time in seven of nine cases. all patients achieved full donor chimerism around day fifteen after hct. severe acute graft-versus-host-disease (gvhd grade iii-iv) occurred in two patients and was lethal in both cases. two patients have experienced extramedullary relapse, one of them in the central nervous system and the other in the soft tissue. in two patients, a transplantation-associated thrombotic microangiopathy (ta-tma) was diagnosed. four patients are alive and in complete remission. we conclude that the combination of a ric regimen with a 188 rhenium anti-cd66 radioimmunotherapy is save and feasible. the incidence of gvhd, ta-tma and extramedullary relapse will be monitored closely and will be presented in a larger patient cohort. disclosure of conflict of interest: none. the sirolimus/tacrolimus (sir/tac) combination has been associated with a better outcome after allogeneic hematopoietic stem cell transplantation (allo-hsct) when compared with conventional prophylaxis for graft vs host disease (gvhd) as cyclosporine/methotrexate in the true reduced-intensity conditioning (ric) setting but not in the myeloablative setting. in moderate-intensity regimens as thiotepa/busulphan/fludarabine (tbf), the sir/tac combination has not been evaluated. from january 2009 to december 2015, all consecutive ric-allo-hsct recipients who received sir/tac combination to prevent gvhd in three spanish institutions were included in the study. the reduced-toxicity regimens used in this study where: (a) intravenous busulphan (6.4 mg/kg) and fludarabine 150 mg/ m 2 (bf), or (b) thiotepa days -4 and -3 and 10 mg/kg if 455 yrs old or 5 mg/kg if o55 yrs old) on days -6 and -5 added to the bf regimen (tbf). the gvhd prophylaxis with sir/tac was given as detailed elsewhere (cutler c blood 2014) and was consistent within the 3 center. the outcomes of the procedure according to the conditioning regimen were analyzed. overall, 125 patients were included: 66 tbf and 59 patients in the bf group, with a median follow-up of 22 months (range 3-83) and no difference in the median age (56 vs 58 years old). there were more males (71% vs 47%, p = 0.007) and more female donors to male recipients (35% vs 13%, p = 0.006) and more patients with lymphoid diseases and previous asct in the tbf group (27% vs 12%, p = 0.03), whereas there were more unrelated donors in the bf group (56% vs 76%, p = 0.02). other baseline characteristics were balanced between the 2 groups (table 1) . sir/tac prophylaxis had to be discontinued in 48% and 65% patients in the tbf and bf groups, respectively. toxicity was the main reason for discontinuation in the tbf group. the most frequent toxicities were renal injury (tbf 30% and bf 10%) and neurologic impairments (tbf 6%, bf 5%). in the bf group, the main reason of discontinuation was relapse or a mixed chimera. patients who received tbf presented higher incidence of extensive chronic gvhd (65% vs 39%, p = 0.02), higher nrm at 100 days (21% vs 4%) and at 2 years (42% vs 13%, p = 0.001). there were no differences in os (2 years) between both groups (49 ± 6.9% vs 80 ± 5.5%, p = 0.001) (figure) . there were no differences regarding to acute gvhd 2-4 (39% vs 36%, p = 0.31), acute gvhd 3-4 (23% vs 13%, p = 0.08), or relapse (up to 2 years, 22% vs 14%, p = 0.3) between the 2 groups, either. the combination of sir/tac as gvhd prophylaxis was associated with higher incidence of chronic gvhd and nrm in patients receiving conditioning regimen with tbf compared to those receiving bf. there were no differences in os between both groups. [p418] cr complete remission, pr partial remission, nr non remission, hct-ci hematopoyetic cell transplant comorbility index. disclosure of conflict of interest: none. reduced-intensity conditioning regimen with fractionated total body irradiation of 6 gy and cyclophosphamide 60 mg/kg for allogeneic hematopoietic stem cell transplant is well tolerated and offers a potential disease control as treatment of acute leukemia and lymphoproliferative disorders m adler, t girinsky 1 , s koscielny, g ferini, s wittnebel, s mayeur, c chahine, m vanghele, s pilorge, c castilla-llorente and j-h bourhis 1 the use of reduced-intensity conditioning regimens (ric) before allo-hsct is widely extended since it preserves the graft-versus-leukemia effect but reduces treatment related mortality. however, there exist different ric regimens with diverse outcomes and the choice of the ric regimen relies on the type of disease treated, experience of the center and previous therapies. this is a retrospective study of patients treated in our institution within 01/2000 and 12/2015. the ric regimen consisted of fractionated total body irradiation (ftbi) of 6 gy administered in 3 consecutive days (2 gy/day) and cyclophosphamide 60 mg/kg given in 2 days (30 mg/kg/day). post-transplant immunosuppression consisted of csa started the day before allo-hsct and short mtx on days 1, 3 and 6 after transplantation. for patients receiving transplant from unrelated donors, anti-thymocyte globulin at a dose of 5 mg/ kg (2.5 mg/kg/day for 2 days at day -2 and -1) was used as part of the immunosuppressant therapy. 78 patients (median age: 54 years: range: 36-64 years) were included. the median hct-ci was 0.5 (range: 0-4). primary disease was multiple myeloma (mm) in 45 (58%), al/mds in 14 (18%), cll in 10 (13%), nhl in 9 cases (12%) . 51 patients (65%) received transplant from matched related donors, 22 (28%) from matched unrelated donors and 5 (6%) from mismatched unrelated donors. female to male mismatch incidence was 23% (n = 18). most of the patients (n = 77) received a peripheral blood graft. 1 patient received a second allogeneic transplant. mm patients were transplanted in a "tandem" autologous-allogeneic hsct program in 42 cases. the median number of chemotherapy lines prior to transplant was 3.5 in cll, 2.8 in mm and 2.5 in nhl. 62 patients (91%) engrafted by day 28 post transplant. neutrophil engraftment occured at a median of 19 days (range: 14-35 days) and platelet engraftment at a median of 18.5 days (range: 9-103 days). full donor chimerism was observed in 62 out of 67 patients (92%) having survived by day 180. primary graft rejection was observed in 4 patients. treatment related toxicities consisted of grade 3/4 mucositis in 53 patients (68%), grade 3 (range: 2-4) cardiac toxicity in 6 patients (8%), grade 3 (range: 3-4) hemorrhagic complications in 4 patients (6%) including 3 cases of hemorrhagic cystitis and secondary malignancies in 4 patients, this within a median follow-up of 6.6 years. infectious complications during aplasia included fever of unknown origin (n = 52), bacteremia (n = 17) with 3 cases of bacteremia with severe sepsis and 8 cases of infections defined by bacterial foci. incidence of agvhd was 33% with 3 cases of grade 3/4 refractory agvhd. cgvhd occurred in 30 pts (39%). the non-relapse mortality (nrm) at 100 days was 5% including 2 cases of septic shock, 1 case of acute cardiac toxicity and 1 case of agvhd. the nrm at 1 year was 10%. 1-year survival rates were 60% in al, 80% in cll and 88% in nhl with extended survival benefit. in al patients, the relapse incidence was 36% comprising 2 patients who progressed during conditioning. the 1-year survival rate in mm patients was 77%. in mm patients who were in complete response prior to transplant, median overall survival was 4.6 years. the used ric regimen resulted in durable donor engraftment with an acceptable toxicity profile permitting efficient disease control in the described cohort. disclosure of conflict of interest: none. graft manipulation using selective depletion of αβ-t cells provides a source for haploidentical hematopoietic stem cell transplantation (haplo-hsct) enriched in effector cells. initial reports demonstrated safety and rapid immune reconstitution using this method, in malignant and non-malignant disorders using several proposed conditioning regimens. no specific considerations were given to hematologic malignancies. we reviewed a total of twenty seven pediatric patients who underwent haplo-hsct using αβ-t cell depletion between 2013-2016 in a single tertiary referral center. we report the results of 22 procedures performed in eighteen patients transplanted for malignancies. twenty two haplo-hsct were performed in eighteen patients. the indication for hsct was acute leukemia in sixteen (all = 11, aml = 5) and neuroblastoma in two. median age at hsct was 5.6 years. six patients had failed a prior hsct, and the remainder had no matched donor. the initial reduced-toxicity conditioning regimen consisted of melphalan, fludarabine, thiotepa and atg, and resulted in a high rate of graft rejections (7 of 9). thus, a totalbody irradiation (tbi)-based regimen was implemented, with prompt engraftment in all the patients. we observed rapid neutrophil and platelet engraftment kinetics (median time to engraft, 10 days and 11.5 days, respectively). significant treatment related complications were all due to graft failure in patients receiving reduced-toxicity conditioning, with two infection-related mortalities in the presence of prolonged neutropenia. none of the patients developed hepatic sinusoidal-obstruction syndrome, and no grade 3-4 acute graft-versus-host disease (gvhd) or chronic gvhd were observed with either regimen. importantly, the majority of patients with acute leukemia were free of immunosuppression in the first 100 days post hsct. the 2-year actuarial event-free and overall survival of the entire cohort were 34% and 55% respectively, with results for tbi-based conditioned patients being 58% and 88%. overall, we demonstrated that a tbibased conditioning for haplo-hsct using αβ-t cell depletion for malignant diseases resulted in acceptable outcomes in these high-risk patients without increased toxicity. disclosure of conflict of interest: none. high-dose chemotherapy conditioning regimens followed by autologous stem cell transplantation (auto-sct) generally provide good results in relapsed and refractory lymphomas. we evaluated the efficacy and safety of tecam regimen as conditioning with autologous stem cell support in patients with relapsed/refractory lymphomas. thirty-two (16 patients were refractory, 15 patients were relapse and one frontline treated) patients (21 m, 11 f) with lymphoma at various stages (stage ii, 19%; stage iii, 22%; stage iv, 59%) who underwent asct were included in this retrospective study. the median age at transplantation was 52.5 years (range, 28-69 years). the diagnosis were as follows: 9 diffuse large b-cell non-hodgkin lyphoma (nhl), 9 hodgkin lymphoma (hl), 5 mantle cell lymphoma, 3 follicular lymphoma, 3 marginal zone lymphoma and 3 t-cell nhl. all patients received tecam as conditioning regimen that consist of thiotepa (40 mg/m 2 × 4 days), etoposide (200 mg/m 2 × 4 days), cyclophosphamide (60 mg/ kg × 1 day), ara-c (200 mg/m 2 × 4 days) and melphalan (60 mg/m 2 × 2 days). median cd34(+) cells were 6.7 × 10 6 /kg (range; 1.9-19.3 × 106/kg) which were infused at day 0, followed by recombinant human granulocyte colonystimulating factor (rhug-csf) at a dose of 5 μg/kg/day. the median time between mobilization and auto-sct was 2 months (range; 1-13 months). the median time to recovery of absolute neutrophil and platelet counts independent of transfusion were 11 (range; 9-19) and 14 (range; 10-41) days, respectively. the median stay in hospital was 28 days (range, 11-108 days). bacterial, sitomegalovirus and invasive fungal infection were detected in 11 (34%), 4 (13%) and 2 (6%) patients, respectively in first 100 days of auto-sct. three s336 patients (9.3%) died from transplant-related complications. the overall response rate was 75% (22 cr, 68.8%; 2 pr, 6.2%) after auto-sct. relapse developed in 7 patients during median follow-up period of 6.5 months (range; 1-21 months) after auto-sct. the 1-year estimated dfs ( figure 1 ) and os were 70% and 45%, respectively. no statistical significance was observed for os and pfs in terms of gender, patient age ( o60 and ⩾ 60 years) and nhl and hl lymphoma group (p ⩾ 0.05). the tecam regimen for auto-sct in lymphoma seems to provide encouraging results in terms of response and its good tolerance with acceptable toxicity. [p421] disclosure of conflict of interest: none. allogeneic hematopoietic cell transplantation (allosct) is the only curative treatment for myelofibrosis. however, its widespread use is limited by early non-relapse mortality (nrm). the optimal modalities of the conditioning regimen are a major unmet clinical need. in an attempt to reduce early nrm, we used a tbf conditioning regimen (thiotepa, busulfan (bu), fludarabine (flu) and antithymocyte globulin (atg)). our aim was to reduce nrm and improve engraftment by using such tbf conditioning. thirty consecutive patients with a median age of 56 years (range, 32-69) who underwent allosct for primary (n = 18) or secondary (n = 12) myelofibrosis were included. according to the refined dynamic international prognostic scoring system (dipss-plus), patients were stratified as intermediate-1 (n = 3), intermediate-2 (n = 6), and high (n = 16) risk. five patients had blast transformation. ruxolitinib was given to 14 patients (47%) prior to allosct. graft source was pbscs in 26 patients (87%) and bm in 4 patients (13%). donors were matched related (mrd, n = 6), unrelated (n = 19) and haploidentical (n = 5). conditioning regimen was tbf in 18 patients (60%). in our historical cohort 8 patients (27%) received fb (flu, bu, atg). in addition, 4 patients received a 'tec-ric' sequential conditioning (thiotepa, etoposide, cyclophosphamide, and after 3 days rest, flu, bu and atg) for blast transformation (n = 2) or refractory proliferative myelofibrosis (n = 2). gvhd prophylaxis consisted of cyclosporine (csa) and mycophenolate mofetil in 26 patients (87%), csa and short course methotrexate in 3 patients (10%) with abo mismatch and csa alone in 1 patient (3%) with mrd. high dose posttransplant cy (pt-cy) was added in haplo cases. no significant difference was observed between tbf, fb and tec ric patients in terms of age, gender, karnofsky score, comorbidity index, number of previous treatment line, history of ruxolitinib administration and source of stem cells. median follow-up was 20 months (range, 3-75). two tbf patients died of septic shock before engraftment at day +12 and +19 after allosct, respectively. one fb patient died of graft failure at day +108 post allosct. median time to neutrophils and platelets (420 g/ l) recovery was 15 days (range, 9-28) and 20 days (range, 1-55) with tbf, 17 days (range, 14-53) and 18 days (range, 7-50) with fb, and 19 days (range, 15-24) and 14 days (range, 14-58) with tec ric. grade ii-iv acute gvhd occurred in 27.8% of tbf patients, 37.5% of fb patients, and 25% of tec ric patients (p = 0.90). moderate chronic gvhd developed in 1/13 evaluable tbf, 2/7 fb and 0/4 tec ric patients. no severe forms of chronic gvhd were observed. at last follow-up, 1 patient relapsed, 12 died and 18 are still alive. main causes of death were disease progression (n = 1), infection (n = 9) and gvhd (n = 2). nrm at 2 years was 33.3% in tbf patients, 50% in fb patients, and 25% in tec ric patients. the 2-year os were 66.7% in tbf patients, 37.5% in fb patients, 75% in tec ric patients, respectively. cd34+-selected stem cell boost without further conditioning allowed to 4 patients for poor graft function, with significant hematological improvement in 3 patients. tbf conditioning regimen seems to be efficient in allosct for patients with myelofibrosis and compares favorably with previously published fb regimens. these preliminary results give a rationale to support a prospective evaluation of this platform. disclosure of conflict of interest: none. we proposed here to compare the outcome of patients receiving either thymoglobuline (atg), a rabbit anti-human thymocyte immunoglobulin or campath, a recombinant dna-derived humanized monoclonal antibody directed against cd52. campath and atg are both commonly used as in vivo tcd before hsct, respectively in united kingdom and france but very few comparing data are available. all consecutive patients with acute myeloblastic leukemia (aml), myelodysplastic syndrome (mds) or myeloproliferative neoplasia (mpn) who received a reduced intensity hsct from an unrelated donor transplanted between 2006 and 2015 were included in this study. a propensity score was used to identify and control potential confounding to relate the treatment group to the outcomes. in the matched sample, cox regression model was used to describe the association between treatment and outcomes. 322 patients have been included. all patients received fludarabine and busulfan with either atg (n = 153) or campath (n = 169). patients treated by atg received cyclosporine plus mycophenolate mofetil or methotrexate and patients treated by campath received cyclosporine alone as gvhd prophylaxis. comparing patient and transplant characteristics, atg patients were older (62 vs 60 years), had less often aml (52 vs 69%), had higher disease risk (adverse dri: 14 vs 9%; poor cytogenetics: 25 vs 11%; high cibmtr score: 41 vs 28%), were less often in complete remission at time of transplant (62 vs 76%) and were transplanted less often from a mismatched hla donor (16 vs 26%). cumulative incidence of sustained engraftment was in 98% and 99% campath and atg patients. time to neutrophil engraftment was longer in atg patients (19 vs 13 days). acute gvhd ii to iv rate were higher after atg (44% vs 19%) as well as chronic extensive gvhd (26% vs 13%). relapse rate was higher after campath (44% vs 27%). disease-free survival (dfs) was higher after atg (53 vs 37%) and the gvhd-free relapse free survival (grfs) was similar (35% vs 32%). according to the prognostic factors for outcome, a propensity score was developed selecting 234 patients from the original cohort. the estimation of tcd effect was than studied. relapse risk was higher in patients treated by campath while there is a non-significant advantage for atg in dfs (table 1) . [p423] tcd with atg or campath gives similar os, dfs and grfs. severe acute or chronic gvhd is lowered by campath but the higher relapse risk counterbalances the potential benefit of campath finally given similar os. nevertheless, lower risk disease patient might benefit from campath while higher risk patients might benefit from atg. disclosure of conflict of interest: none. high-dose chemotherapy (hdt) with autologous stem cell transplantation is the standard of care for relapsed/refractory (rr) or high grade non-hodgkin-lymphoma (nhl) and hodgkin-lymphoma (hl)2. the standard hdt in autologous stem cell transplantation (asct) for lymphoma is carmustinebased hdt using a combination of carmustine, etoposide, cytarabine and melphalan (beam); this standard conditioning programme is used by most groups worldwide1. we have designed novels hdt regimens in which carmustine was substituited by an equal dose of fotemustine (feam)3 or thiotepa (team) and we compared these two hdt regimens in terms of engraftment times, toxicity, tolerability and frequency of relapse after asct. from february 2011 to september 2016 we consider a total of 67 relapsed/refractory patients affected by hl and nhl respectively 18 hl and 49 nhl with different grade of initial disease (grade i-iv) and different response to prior treatments. the all other drugs were administered according to a standard beam regimen1. after a day of rest, autologous peripheral blood progenitor cells were infused on day 0, followed by s.c. g-csf (5 mg/kg) from day 1 of asct until 2 consecutive days when the ancs were 4500 × 109/l3. the primary objectives of the study were to assess the feasibility and safety of the feam and team regimens in terms of acute toxicity, grade of mucositis, hemopoietic engraftment and relapse after asct. acute toxicity include chemotherapy-induced nausea and vomiting, diarrhea, hepatotoxicity, nephrotoxicity and infection complication. in all 67 patients cd34+ cells were collected from peripheral blood and the median number of infused cells per patient was 5.79 × 10e6/kg. the median time of engraftment was 9 days for neutrophil recovery (n4500 × 10 9 /l) and 11 days for plt recovery (420 000 × 10 9 /l). acute toxicity occurred in 14 total patients (20.8%), mucositis grade 3-4 occurred in 34 patients (50% of cases). frequency of relapse in all 67 cases was 43.2%. feam conditioning regimen was used in 41 cases showing a median time of neytrophil recovery of 10 days and a median time of plt recovery of 11 days. acute toxicity occurred in 4 of these cases (9.7%), mucositis grade 3-4 occurred in 18 patients (43.9% of cases). frequency of relapse in feam group of patients was 41.4%. team conditioning regimen was used in 26 cases showing a median time of neytrophil recovery of 9 days and a median time of plt recovery of 11 days. acute toxicity occurred in 10 of these cases (38.4%), mucositis grade 3-4 occurred in 16 patients (61.5% of cases). frequency of relapse in team group of patients was 50%. relapse/progression of lymphoma and conditioning regimen toxicities remain limitations to treatment success. the two novels hdt regimens feam and team are safe and feasible and show similar engraftment times, tolerability and frequency of relapse. maybe the team regimen shows toxicity slightly higher than feam regimen but longer follow-up is needed to evaluate fully its efficacy and long-term safety. disclosure of conflict of interest: none. treosulfan is a prodrug of a bifunctional alkylating cytotoxic agent. there are few reports regarding toxicological side effects of treosulfan-based conditioning prior to hsct. here we report on incidence of early potential treosulfan-related toxicity in 118 patients treated with treosulfan-based conditioning before hsct. treosulfan was given at a dose of 14 g/m 2 /d for 3 days in combination with fludarabin 30 mg/m 2 /d for 5 days prior to hsct. most patients (n = 93) had a haematological malignancy, while 25 patients had a non-malignant disorder as hsct indication. an hla-a, -b and -dr matched unrelated donor (mud) was used in 80 cases, 33 patients had a hla-identical sibling donor and 5 received an hla-a, -b or -dr allele mismatched unrelated donor. as graft versus host-disease (gvhd) prophylaxis, most patients (n = 93) received cyclosporine and methotrexate. patients medical records were scrutinized retrospectively to collect laboratory tests (aspartate aminotransferase (ast), alanine aminotransferase (alt), creatinine) before hsct and then weekly until 5 weeks after hsct. levels of ast and alt were significantly increased 1 week after hsct compared to before hsct. however, only a few patients had transaminase levels over 2 or 3 times the upper normal level (unl) levels decreased sharply after the first week. most of the cases with high levels of ast/alt at one week had normal or close to normal levels before hsct. creatinine levels increased after week 2 but no patient had levels ⩾ 2 × unl. clinical features of all oral mucositis (om) were recorded using the world health organization (who) scoring system. most patients (68%) had no or very limited (grade i) om, 22% had grade ii and 10% had grade iii or iv of om. according to our toxicological results this is low-toxic protocol. however, all patients became neutropenic, 61% already at the time of graft infusion, indicating that the protocol has a myelo-toxic effect comparable to conventional mac protocols. all patients engrafted, except three patients who died very early. median time to neutrophil and platelet engraftment was 18 (range 10-31) and 15 days (9-55), retrospectively, which is significantly later when compared to engraftment data for other ric protocols used at our centre (data not shown). median duration of neutropenia (o0.5 × 10 9 /l) was 17 days , comparable to what is expected after conventional mac conditioning. secondary graft failure (gf) occurred in 8 (6.8%) patients, all having a nonmalignant disorder and 6/8 having a urd. non-relapse mortality (nrm) was 7.5% (95% ci 3.7-13.3%) at 100 days and 11.9% (6.8-18.5%) at one year after hsct. causes of death within one year after hsct were: relapse 7, epstein-barr virus associated posttransplant lymphoproliferative disease (ptld) 4, other infections 4, organ failure 2, gvhd 2, hemophagocytic lymphohistiocytosis (hlh) 1. other infections occurring within 100 days after hsct were cytomegalovirus (cmv) reactivation 46 (39%), invasive fungal infection 6 (5.1%) and blood stream infection 47 (40%). veno-occlusive disease of the liver or sinusoidal obstruction syndrome (vod/sos) occurred in one patient and haemorrhagic cystitis in two patients. this study shows that early regimen-related toxicity after hsct was low despite similar marrow toxicities compared to mac regimens. disclosure of conflict of interest: none. allogeneic stem cell transplantation from haploidentical donors (haplosct) is an increasingly adopted option for patients (pts) with high-risk hematological malignancies. in our institution, we previously described a platform for unmanipulated peripheral blood stem cell (pbsc) haplosct using a calcineurin-free gvhd prophylaxis with sirolimus, micophenolate and anti-human t-lymphocyte immunoglobulin (atg) after conditioning with treosulfan and fludarabine (trramm; peccatori et al., leukemia 2015) . as an attempt to decrease relapse rate, especially in advanced-phase diseases, we designed a new phase ii prospective clinical trial intensifying conditioning regimen with the addition of 4gy total-body irradiation (tbi) (trramm4gy; eudract#2011-001534-42). we report results on 75 pts. 75 pts affected by aml (n = 49), other myeloid (n = 8) and lymphoid (n = 18) malignancies were prospectively enrolled from may 2010 to june 2015. median pts age was 45 y (range 17-67). revised disease risk index (r-dri) was low or intermediate in 31 pts, high in 34 pts and very-high in 10 pts. twenty-five pts had previously received an allogeneic stem-cell transplantation with a median time from 1st to 2nd sct of 17 months . median hct-comorbidity index by sorror et al. was 1 (0) (1) (2) (3) (4) (5) . pts received a myeloablative conditioning regimen consisting of treosulfan (14 g/m 2 /d from -6 to -4), fludarabine (30 mg/m 2 /d from − 6 to − 2) and tbi 4gy (fractionated in 2 doses, from − 1 to 0). source of stem cells were unmanipulated g-csf-mobilized pbsc from haploidentical donors. gvhd prophylaxis consisted of atg-fresenius (grafalon, neovii) 10 mg/kg/d from − 4 to − 2, rituximab 200 mg/m 2 on − 1, mycophenolate mofetil 10 mg/ kg from − 1 to +30 and sirolimus (target concentration 8-15 ng/ ml) from − 7. median infused cd34+ and cd3+ cell doses were 6.9 × 10⁶/kg and 2.15 × 10⁸/kg, respectively. median follow-up for survivors was 48 months (5-74). neutrophil engraftment occurred in 95% of pts with a median of 17 d (9-47), platelet engraftment was reached in 76% of pts with a median of 17 d (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) . the 100-d cumulative incidence (ci) of grade ⩾ 2 acute gvhd (agvhd) was 31 ± 10% and of grade ⩾ 3 agvhd 27 ± 10%; the 2 years ci of chronic gvhd was 34 ± 11%. the ci of transplant-related mortality (trm) at 1 y and 4 y were 31 ± 10% and 34 ± 11%, respectively. the ci of relapse at 1 y and 4 y were 27 ± 10% and 34 ± 11%, respectively, with a median time to relapse of 90 d . interestingly, we did not observe any extramedullary relapse; loss of mismatched hla-haplotype occurred in 33% of relapses. among 36 pts who were in active disease at time of haplosct and who were evaluable, 94% achieved complete remission (cr) and full donor chimerism at day+30. the 1y and 4y probabilities of disease-free survival (dfs) were 42 ± 11% and 33 ± 11%, respectively. at 4 y, 27% of pts are alive, disease-free and immunosuppression-free minimal residual disease, tolerance, chimerism and immune reconstitution the number of human leukocyte antigen (hla)-mismatched hematopoietic cell transplantation (hct), including cord blood transplantation, has been increasing. hla-flow method can discriminate mismatched hla antigens between donor and recipient by using flow cytometry, and can evaluate minimal residual disease (mrd) or chimerism after hla-mismatched hct. by developing more simple methodology, hla-flow might be more widely applicable. we have developed modified 6-colorbased hla-flow method. the aim of this study is to evaluate the utility of the 6-color-based hla-flow for monitoring of mrd and chimerism after hla-mismatched hct in children. from june 2013 to november 2016, serial monitoring of mrd or chimerism by the 6-color-based hla-flow was performed in twelve patients undergoing hla-mismatched hct (46 tests). nucleated cells obtained from bone marrow were stained by immunofluorescent antibodies against hla antigens mismatched between donors and recipients. these cells were also stained by immunofluorescent antibodies against surface antigens such as cd3, cd19, cd33, cd34 and cd16/cd56 for determining lineage of the cells. these surface antigens were also used as a marker of leukemic blasts in the mrd study. we used 6-color-based flow cytometry (facs-navious) and the data were analyzed with flow jo. erythroblasts and dead cells were excluded from the analysis. in each study, at least 105 cells were analyzed. for mrd analysis, we concurrently tested real-time quantitative polymerase chain reaction (pcr) of peripheral wt1 mrna or leukemia-specific fusion genes. pcr of polymorphic short tandem repeats or fluorescent in situ hybridization of x/y chromosomes was concurrently tested for chimerism study. age of patients ranged from 0 to 16 years. donor sources included bone marrow (n = 9) and cord blood (n = 3). for mrd monitoring of acute leukemia (n = 9), the 6-color-based hla-flow could detect mrd in three patients. five patients have not experienced relapse. no discordance with other mrd markers was observed in these patients. hla-flow could not separate donor-derived cells from recipient-derived ones in one patient receiving bone marrow transplantation. as for chimerism testing (n = 3), the 6-colorbased hla-flow could successfully evaluate quantitative lineagespecific chimerism in all patients. there is no discrepancy between hla-flow and other methods. we could complete evaluation of the 6-color-based hla-flow within two days in all tests. the 6-color-based hla-flow is a simple, quick and useful method for the quantitative evaluation of mrd and lineagespecific chimerism after hla-mismatched hct in children, irrespective of donor sources. it is thought that our method is applicable in all institutions owing 6-color-based flow cytometry. acknowledgement: we thank drs. nobukazu watanabe, eri watanabe and natsuko sato (university of tokyo) for their technical advices. we also thank drs. tomoko okunushi (chiba university), hidefumi hiramatsu and katsutsugu umeda (kyoto university) for their care of patients involved in this study. disclosure of conflict of interest: none. survival (pfs), cumulative incidence of relapse (cir) and acute/ chronic gvhd incidence in aml patients (pts) submitted to allo-hsct at our institution between january 2003 and december 2014. this retrospective study evaluated 122 aml pts submitted to allo-hsct from 56 matched sibling donors (msd) and 66 matched unrelated donors (mud) who provided bone marrow (bm) or peripheral blood as stem cell grafts.ir was evaluated at 100, 180 and 365 days post-transplant in all 122 pts. cmv-dna copies were determined in peripheral blood by quantitative pcr twice weekly in the first 100 days post-transplant and subsequently once weekly. cmvi/r was analyzed as a timedependent covariate.effect of cmvi/r on os and pfs was estimated by cox proportional hazard model. cir and gvhd incidence were analized with a competing risk approach, considering death from any cause as a competing event. effect of cmvi/r on cir and gvhd were evaluated by fine & gray model. median age at allo-hsct was 50.7 years (19.1-68.9 ). in our population 68% of donors were seropositive for a previous cmv infection and pts transplanted from these donors showed a significantly lower cumulative incidence of cmvi/r than pts transplanted with seronegative ones (shr = 0.56, 95%,ci:0. our study demonstrates that cmvi/r influences the success of allo-hsct by determining a better ir characterized by a higher cd8+ cell number that might exert an immune protective control on disease outcome by improving os,pfs and cir with no effect on gvhd. another factor of utmost importance to achieve this same goal might be constituted by the significantly increased nk cell number six months after allo-hsct. to assess the dynamics of molecular response to treatment in aml adult patients with concomitant flt3 and npm1 mutations. this retrospective single center studystudy was approved by the institutional review board of american university of beirut medical center. twelve consecutive newly diagnosed (n = 11) or relapsed (n = 1) aml patients received idarubicin/cytarabine induction and one or two consolidation (s) ( table 1) . seven patients received allogeneic stem cell transplant (allo-sct) and 3 had haploidentical-sct (hap-lo_sct); all followed by post-transplant sorafenib maintenance. median follow-up was 11.5 (6-27) months. all transplanted patients remain alive and disease free.flt3 mutation was tested on dna using a qualitative method with a sensitivity of 0.01%. npm-1 mutation was tested on cdna using a qualitative or a quantitative rt-pcr with a sensitivity of 0.01% and 0.008 ncn respectively. patients were tested at diagnosis, after induction, after each consolidation, before and s341 at days 30, 60 and 100 after allo-sct for kinetics of npm1 and flt3 molecular response. after induction, flt3 became negative in all tested patients (n = 10). after first consolidation, flt-3 was not tested in 3 patients who had a negative result after induction, was negative in 8 patients including the 2 patients who were not tested after induction, whereas a molecular relapse was noted in one patient who developed a hematological relapse and rapidly died. another patient died after the third consolidation, in complete remission, due to septic shock. no molecular positivity for flt-3 was noted later on, whether after second consolidation or post-transplant. conversely, npm-1 mutation became negative in 2 out of 12 tested patients after induction, in 1 additional patient after first consolidation and in 7 additional patients after sct, mostly after starting sorafenib. npm-1 mrd value remained elevated in 3 out of 4 patients with quantitative assessment at diagnosis and post induction (figure1). flt3 become negative early after induction while npm1 negativity lags behind. persistent npm-1 mrd does not seem to predict post-transplant outcome and may indeed become negative after sorafenib. these results need confirmation in larger studies. disclosure of conflict of interest: none. in allogeneic stem cell transplantation (allo-sct), an early detection of the transplant outcomes such as overall survival (os), event-free survival (efs), cumulative incidence of relapse (cir) and non-relapse mortality (nrm) is fundamental regarding the use in time of additional therapy after sct. therefore, we investigated the association between early immune reconstitution (ir) on day +30 after allo-sct and outcomes in children suffering from acute leukemia or myelodysplastic syndrome (mds). this study collected data from 188 allo-sct from january 2005 until december 2014 in our institution. the median survival follow-up was 38 months. indications of allo-sct were all (n = 113, 60%), aml (n = 44, 23%) and mds (n = 31, 17%). the median age was 10 years (range, 0.6 -18). patients were transplanted in cr (n = 131, 70%) and pr/nr (n = 57, 30%). patients included in the study received 1st sct (n = 170, 90%), 2nd sct (n = 15, 8%) or 42 sct (n = 3, 2%). grafts were from sibling (msd; n = 42, 22%), matched unrelated (mud; n = 95, 51%), haploidentical (n = 45, 24%) or mismatched unrelated (mmud; n = 6, 3%). conditioning regimens were tbi-based (n = 87, 46%) or chemo-based (n = 101, 54%). stem cells were from bone marrow (n = 118, 63%) or peripheral blood (n = 70, 37%). we analyzed the absolute count of lymphocytes (alc), monocytes, cd3+ t cells, cd3 +cd4+ t-helper cells, cd3+cd8+ cytotoxic t-cells, cd3-cd56+ natural killer (nk) cells and cd19+ b cells assessed on day 30 ± 5 after sct. we used the percentiles of the lymphocyte subsets of the same cohort to categorize the samples throughout the study. patients with alc over the 50th percentile of alc (alc o 327 cells/μl) had a 1.97-fold increased hazard ratio (hr) to develop relapse (p = 0.017). nk cell counts on day 30 after sct were strong associated with os, efs, cir and nrm. patients with nk cell count over the 75th percentile (nk 4 268.8 cells/μl) had increased hr for os (hr = 1.8, p = 0.01) and efs (hr = 2.01, p = 0.017) compared to patients with nk count under the 75th percentile. patients with nk cells over the 25th percentile (nk o52.5 cells/μl) had a hr = 3.3 (p = 0.009) for relapse and hr = 0.364 (p = 0.016) for nrm compared to patients with nk cell count under the 25th percentile. monocyte cell count on day 30 correlated with os, efs and cir. patients with cd14+ cells count under the 15th percentile of cd14+ (cd14+ o242 cells/μl) has an increased hazard ratio for os, efs and relapse compared to patients with cd14+ cell counts over the 15th percentile. no association between absolute cell count of cd3+, cd4+, cd8+ and cd19+ on day +30 after allo-sct and any outcomes either os, efs, cir or nrm was found. the study confirms the strong association between early ir and outcomes after allo-sct in children. our study suggests that especially nk cell and monocyte cell count on day +30 may have significant prognostic implications. our findings suggest that the cells count of alc, nk cells and monocytes on day +30 after allo-sct could be useful to predict outcomes after allo-sct and should be taken into account in considering alternative treatment. disclosure of conflict of interest: none. early immune reconstitution (eir) has proven to be a significant determinant for the outcome of allogeneic hematopoietic stem cell transplantation. in the setting of unmanipulated haploidentical transplantation (haplo-hsct), some groups have identified the absolute leukocyte count on day +30 (alc30) as an independent prognostic factor in terms of overall survival (os), disease free survival (dfs) and infectious mortality (im). the aim of this study was to evaluate the impact of eir on os, dfs and im among patients who underwent haplo-hsct with postransplant cyclophosphamide (ptcy) at our institution. from july 2011 to april 2016, 83 haplo-sct were performed at our institution. threedied before day 30 after haplo-sct, and 13 patients had missing data. conditioning regimen consisted of fludarabine, cyclophosphamide and busulfan. twenty-nine patients received a reduced intensity conditioning regimen (1-2 days of busulfan) while 37 a myeloablative regimen (3-4 days of busulfan). gvhd prophylaxis comprised ptcy, cyclosporine and mycophenolate mofetil. patients were assessed for eir by means of alc30, cd3+ t lymphocyte count on +30 (cd3), nk lymphocyte count on +30 (nk) and nk cd56 bright percentage on +30(cd56 br ). we analyzed 66 pts, with a median follow-up of 21 months (9-36). the median age of the pts was 43 (range 30-57), 76%men. diagnosis were: aml(32%), hl (23%)non-hl (17%), all (8%),mds(8%), cml(6%), others(6%). 55% were in complete remission at the time of transplant, 21% in partial remission and 24% had overt disease. in terms of infectious complications, cmv reactivation was documented in 76% of the pts, 1% developed a proven invasive fungal infection and 26% suffered from bk+hemorrhagic cystitis. median os and dfs were 21 (9-36) and 17 months (7-31), respectively. im rate was 21% at the end of follow up. median follow-up was 21 months (9-36). roc curves were used to determine the optimal cut-off values for each of the studied parameters: 300 cells/μl for alc30, 120 cells/μl for cd3, 41 cells/μl for nk and 83% for cd56 br were chosen. pts with alc30 ⩾ 300/μl had better os (p = 0.005) and dfs (p = 0.05), than those with alc30 o 300/μl. median os and dfs were 25 months vs not reached (nr) and 22 months vs nr, respectively. pts with cd56 br ⩾ 83% had better os (p = 0.04) than those with lower values. median os was 25 months vs nr; however no difference was seen in terms of dfs. we didn´t observe statistically significant differences in os or dfs, among pts with different levels of cd3 and nk on +30. cumulative incidence of im was significantly lower in pts with an alc30 ⩾ 300 (p = 0.04), pts with cd3 ⩾ 120/μl (p = 0.006) and pts with nk ⩾ 41 (p = 0.04); patients with cd56 br ⩾ 83% showed tendency to have lower cumulative incidence of infectious mortality (p = 0.24, non-significant). cumulative incidence of relapse was not affected by alc30, cd3, nk or cd56 br . our study supports the independent prognostic value of early immune reconstitution after unmanipulated haploidentical transplantation with ptcy, especially in terms of lower infectious mortality. os and dfs were better among patients with alc30 ⩾ 300 cells/μl. pts with cd56 br ⩾ 83% also showed better os. no correlation was found between cd3 or nk on +30 with os or dfs. cumulative incidence of infectious mortality was affected by alc30, cd3 and nk on +30; while cd56 br seems to have less impact. [p431] disclosure of conflict of interest: none. an early absolute lymphocyte count (alc) recovery after autologous stem cell transplantation (asct) for hematologic malignancies has been related with an improved transplant outcome due to a faster autologous immune restoration. in this retrospective study we analyze post-transplant survival of non hodgkin lymphoma (nhl) patients and its relation with alc at day +15 post-asct. we analyzed 53 consecutive adult nhl patients who underwent asct at the hematology and sct department of hospital maciel (montevideo, uruguay). only individuals with at least 6 months post-transplant follow up were included. all patients received beam (bcnu, etoposide, cytarabine and melphalan) conditioning regimen followed by peripheral blood stem cells previously collected by apheresis. median cd34+ cell dose was 4.13 × 10e6/kg (1.62-12.58). median alc at day +15 was 500/μl. patients were divided into two groups: alc at day +15 inferior than 500/ul (group 1) and alc at day +15 superior or equal than 500/ul (group 2). differences between groups were analyzed using t-student and chi-square tests, with statistical significance determined at p o0.05. disease free survival (dfs) and overall survival (os) were analyzed by kaplan meier method. differences in survival between groups were determined by log-rank test. no differences were observed between groups regarding gender, histology, disease status at transplant and cell dose. patients in group 1 were older and more heavily pretreated. neutrophils and platelets engraftment were significantly faster in group 2 (table 1) . after a median follow up of 36 months, disease-free survival (dfs) and overall survival (os) were superior in group 2. median dfs was 47 months and not reached (p = 0.019) and os was 51 months and not reached (p = 0.016) in groups 1 and 2 respectively (figure 1 ). an early alc recovery after asct was associated with a superior dfs and os in nhl patients. individuals with alc major or equal than 500/ul had also a shorter time to neutrophils and platelets recovery and a shorter hospital stay. in this study, cd34+ cell dose does not seems to be a determinant factor for lymphocyte recovery. the load of previous treatment may influence lymphocyte recovery after asct. these results support the association between early post-asct lymphocyte recovery and improved survival in nlh patients. [p432] disclosure of conflict of interest: none. t cell depletion (tcd) reduces the risk of graft versus host disease (gvhd) but also the graft versus leukaemia (gvl) effect, thus increasing the risk of relapse. donor lymphocyte infusions (dli) can be given to boost donor chimerism, with the intention of enhancing the gvl effect. 1 it is not currently known whether giving dli based on bone marrow chimerism (bmc) influences survival, or whether certain groups of patients benefit more from dli than other groups. in addition, it is not known whether the overall aim of achieving 100% bmc associates with improved survival. we investigated whether day 100 (d100) bmc was predictive of survival, and whether giving dli based on this result was associated with improved overall survival. data were retrospectively collected from case notes and laboratory reports for patients who underwent allogeneic stem cell transplant (allosct) for aml or mds at the northern centre for bone marrow transplantation between 2010 and 2015. patients who died prior to d100 were excluded from the analysis. of the 147 patients analysed (aml 117, mds 30), 68% were male and 38% female. the median age was 59 years (range 22-74). conditioning was with flu/bu/ alemtuzumab (63), flu/mel/alemtuzumab (45), cy/tbi alemtuzumab (7), flamsa tbi/bu atg/alemtuzumab (27), other (5) . 103 (70%) received a graft from an unrelated donor, 42 (29%) a matched sibling donor and 2 (1%) another source. 143 (97%) received mobilised pbscs, 3 (2%) bone marrow and 1 (1%) cord blood. statistics were performed using graphpad prism. p values were calculated using the chi square test and taking po0.05 to determine significance. bmc was divided into 3 groups 100%, 90-99% and o 90%. 100% bmc at d100 was associated with a significant increase in 2 year overall survival (os) (74.6% vs 57.3% and 33.4% for 90-99% and o90%, respectively, p o0.0012). patients with a d100 bmc o80% had a 2 year os of o10% (with relapse the cause of death in 90%). in patients whose d100 bmc was o 90%, there was a significant improvement in 2 year os seen in those who received dli (61.7% survival at 2 years vs 0% with no dli, po0.0026) (figure 1 : os by d100 bm chimerism (with and without dli). attainment of 100% bmc at a subsequent time point also significantly improved survival in those with a d100 bmc of 90-99% (79.4% 2 year survival vs 33.6% who never attained 100%, p o0.001) and o90% (100% 2 year survival vs 14.7%, p o0.006). we found d100 bmc to be predictive of os in this population. in addition, dli was associated with an improvement in os, especially in patients whose bmc at d100 was o90%. there was also a statistically significant improvement in os seen in patients who subsequently attained a 100% bmc, where it was o100% at d100. the objectives of this analysis were to examine the optimal alc recovery cutoff utilizing receiver operator characteristics (roc) analysis and to examine infused allograft characteristics associated with early alc recovery. after due irb approval, patients (pts) with aml and all who underwent hct at our institution between 2010-2015 were identified. pts with t-cell depletion or maintenance post hct were excluded. data were collected retrospectively from the patient's records. cellular contents of infused products (cd34, cd3, tnc, mnc, alc and amc) in addition to alc post hct were analyzed and optimal cutoff, if present, was established using roc analysis for the end point of relapse. time to end point analysis was computed using the kaplan-meier with log ranks. for competing events, cumulative incidence was computed using grey's model. univariable and multivariable analyses were performed using cox proportional hazard regression. a total of 72 pts met the inclusion criteria and were analyzed. optimal alc cutoff by roc analysis was established to be on day +14 (d14) with alc 40.3 × 10 9 /l and was subsequently defined as early lymphocyte recovery (erl). pts with alc ⩽ 0.3 × 10 9 /l were deemed to have delayed lymphocyte recovery (dlr). patients were subsequently stratified accordingly and patient, disease and transplant related factors were well balanced between the groups. median follow up of the entire cohort was 17 (2-64.8) months. graft characteristics: roc analysis established optimal cellular cutoff, if present to predict elr. pts in the elr group were more likely to receive cd 34 × 10 6 /kg o6 (0.018), cd3 4 24 × 10 7 /kg (0.017) and alc 41.3 × 10 8 /kg (p = 0.015). we did not find a significant threshold for other allograft variables i.e. (tnc, mnc or amc). post hct outcomes: at 2 years, corresponding cumulative incidence of relapse (cir) and non-relapse mortality (nrm) was16.9% vs 46.9% (p = 0.025) and 23.2% vs 14.2% (p = 0.51), for elr and dlr cohorts, respectively. there was a trend towards improved progression free survival (pfs) and overall survival (os) in favor of elr vs dlr at 61.9% vs 40.1% (p = 0.09) and 70.1% vs 53.9% (p = 0.12), respectively. median time to neutrophil and platelet engraftment was 17 and 24 days, respectively for both groups. incidence of acute graft vs host disease (agvhd) was similar (p = 0.4); however, chronic gvhd (cgvhd) was more prevalent in the elr group at 70% vs 27%, respectively (p = 0.0006). on s344 multivariable analysis for relapse, elr retained its prognostic significance with hr 0.27 (0.05-0.94; p = 0.038). cgvhd and first complete remission (cr1) at the time of hct were also protective factors from relapse in multivariable analysis. we observed that elr is an independent predictor for relapse in patients receiving allogeneic hct for acute leukemia with a trend towards improved os. this is possibly related to higher incidence of cgvhd. elr was influenced by infused allograft characteristics particularly cd34 count. given the sample size and retrospective nature of the analysis, these important observations should be examined prospectively. disclosure of conflict of interest: none. allosct is the only curative option for the treatment of hematological disorders with depression of hematopoiesis and primary immunodeficiencies.non-myeloablative conditioning (mac) regimens lead to long persistence of mixed chimaerism (mc) in the majority of patients. purpose: to estimate the relationship between type of hematopoietic chimaerism and appearance of gvhd in patients with non-malignant diseases after allosct eleven patients (8 boys and 3 girls) with median age of 9 years (range 3-17) were included in the current study. among them there were 7 patients with severe aplastic anemia (saa), 2 with fancony anemia (fa), 1 with thalassemia, 1 with nijmengen syndrome, treated in our center from 2008 to 2016. donors' sources were as follows: siblings in 7 cases, mud (10/10) in 4 ones. in 5 cases bone marrow aspirate were used, in 6 mobilized peripheral blood hematopoietic stem cells. conditioning regimens included fludarabin, cyclophosphamide and horse atg for saa patients, in fa and nijmengen syndrome patients this scheme was augmented by low-dose busulfan. in thalassemia patient we used mac with busulfan, fludarabin and horse atg. in majority of case gvhd prophylaxis consisted of tacrolimus and methotrexate combination. when allosct was performed form mud patients were additionally administered with mycophenolate mofetil. median of follow-up period was 32 mo (range 7-93). quantitative evaluation of chimerism was done by multiplex amplification of str loci with subsequent fragment analysis using «cordis plus» kit («gordiz llc», russia). we analyzed whole bone marrow and peripheral blood together with cd3+ and cd19+ lymphocyte subpopulations. presence of ⩾ 99% donors' hematopoietic cells was considered as complete donor chimerism (cc), less than 99% was considered as mc. all patients engrafted in time and all of them are alive at the time of current analysis. there were no severe life-threatening complications, infections or graft rejections. only 2 patients achieved cc at day +28. at day +100 only these 2 patients stayed in cc. at this time point mc was mainly revealed in cd3 + lymphocytes. in 1 year after hsct proportion of cc patients enlarged to 82% (2 patients did not achieved this time point). there is no any correlation between time of engraftment and chimerism value at day +28, either between the dose of transplanted cd34+ cells and chimerism level ((p40.05 in both cases). severe gvhd was noted only in 2 female patients with cc at day +28. in the first case it was acute gvhd grade iii after hsct from mud, in the second case extensive form of chronic gvhd in 1 year after hsct from sibling was observed. there are no other cases of grade iii-iv acute gvhd in the observed cohort of patients. localized form of chronic gvhd [p434] was revealed in 4 (36%) patients. in other patients there were no signs of chronic gvhd. despite limited number of observations we assumed that fast achievement of cc corresponds to severe gvhd. and vice versa, long persistence of mc prevented emergence of gvhd. however our findings need to be confirmed in a larger group of patients and preferably in a multicenter setting. disclosure of conflict of interest: none. interest of quantitative assessment of hematopoietic chimerism by real-time quantitative polymerase chain reaction after hematopoietic cell transplantation for hematological malignancies: a retrospective analysis on 347 adult patients from rennes university hospital g laure 1 , c mathilde 2 , b marc 1 , n stanilsas 1 , d charles 1 , l christine 2 , a mehdi 2 , lb laetitia, s gilbert 3 , l thierry 1 and r virginie 2 1 department of hematology, chu pontchaillou, rennes; 2 immunogenetics and histocompatibility laboratory, etablissement français du sang, rennes and 3 etablissement français du sang-bretagne chimerism (percentage of recipient versus donor-derived blood cells) is used to document engraftment after hematopoietic stem cells transplantation (hsct). detection of persistant host cells, 1-2 as well as an increase in recipient cells chimerism has been associated with impaired dfs and os. 3 quantitative real time pcr (qrt-pcr) 4 is a highly sensitive, reproducible method, which can detect very low levels of recipient cells. the aim of this study was to evaluate the prognostic impact of early chimerism 30 days (d30) and 100 days (d100)) after hsct and the meaning of detection of an increase of chimerism, even at low levels, during follow-up. 347 adult patients who underwent hsct in rennes between 2002 and 2013 were included in this retrospective study. all chimerism analyses were done with qrt-pcr using whole blood sample. complete chimerism (cc) was defined by less than 0.1% recipient cells detected. with a median follow-up was 653 days, 101 patients relapsed with a median time of 116.5 days after hsct. both d30 and d100 mixed chimerism (mc) (40.1 % recipient cells detected) were associated with an increased relapse risk (p = 0.0003 and p o0.00001 respectively) compared to patients with cc in univariate analysis. however, when looking at subgroups analysis, d30 and d100 mc vs cc was significantly associated with increased relapse risk in this cohort for myeloid diseases (p = 0.0049 and p o0.0001) but not for lymphoid diseases (p = 0.506 and p = 0.059). no difference in os was observed (p = 0.32 and p = 0.34). more important, detection of an increased of mc (imc) was associated with an increased relapse risk in univariate and multivariate analysis (or = 9.69 [5.42; 17 .34], or = 10.05 [5.35; 18 .90]), (po0.0001), as well as impaired os (p = 0.0043) and dfs (p o0.0001). among the 103 patients with aml and at least 2 chimerism analysis available, only 3 relapsed without imc detected but the patients' last available chimerism analysis was 75, 84 and 138 days before relapse respectively. median levels of recipient cells detected was 1.2 %. altogether, these results indicate that serial analyses of chimerism with qrt-pcr are a useful tool for post-transplant monitoring and might help identify patients at highest risk to relapse after transplant, especially in myeloid disease. 3 monitoring frequency is critical in order to obtain the highest clinical impact, and the timing of monitoring as well as the safety and type of pre-emptive interventions still need to be explored. considering the kinetics of the disease, frequent analysis in myeloid pathology might improve the detection of impending relapse. although an approximately 1-log higher sensitivity of quantitative pcr (qpcr) compared to short tandem repeat (str) has been documented in different studies, the latter remains the standard procedure for hc assessment to date. we hypothesized that qpcr could be superior to str for monitoring the molecular kinetics of donor cell engraftment, response to donor lymphocyte infusions (dli) and development of relapse post-hct. we analyzed 30 patients (pts) who underwent mainly 10/10 hla-matched unrelated hct mostly for acute myeloid or lymphatic leukemia at the university hospital essen between 2006 and 2013. transplant conditioning was mostly myeloablative and gvhd prophylaxis was by cyclosporin a and methotrexate without anti-thymocyte globulin (atg). median follow-up of pts was 1504 days (d) (317-2891). cytomegalovirus (cmv) reactivation in the first 100d posttransplant was measured by pp65 (n = 22) or pcr (n = 8). a total of 459 retrospective genomic dna samples from peripheral blood (pb; n = 364) or bone marrow (n = 95) collected between d21 and d2302 post-transplant were available for hc analysis in parallel by str (mentype chimera, biotype) and qpcr (alleleseq, abbott). threshold for hc positivity in qpcr was set at 0.1% following published protocols. concordance in hc analysis between qpcr and str was found in 365/459 (79.5%) samples, with all 94 discordant cases positive in qpcr but negative in str. engraftment could be assessed in 110 samples drawn at d21-d213 from 18 pts without relapse in the first 6 months post-hct. these samples showed concordant negative or positive qpcr and str results reflective of full donor engraftment or persistent mixed chimerism (pmc) in 5 and 3 pts, respectively. in 2 pts, qpcr but not str documented delayed conversion to full donor chimerism until d200. in the remaining 8 pts, positive results in qpcr but not str during early engraftment were observed exclusively in bm, in particular those drawn before d35 post-hct. qpcr but not str was also able to document the kinetics of conversion to full donor chimerism which took 126d and 196d in 2 pts receiving dli for treatment of pmc and relapse, respectively. 8 informative relapses could be assessed in 33 samples drawn at least 192d before onset. 6/6 bm and 23/26 pb were positive in qpcr, compared to 2/6 bm and 2/23 pb in str, with a sensitivity of 8/8 (100%) and 3/8 (37.5%) relapses, respectively. consistent with previous reports on a protective effect of early cmv reactivation on relapse in gvhd prophylaxis regimens without atg, relapse occurred in 1/10 (10%) pts who experienced cmv reactivation in the first 100d post-transplant, compared to 10/20 (50%) pts who did not. no apparent associations were observed between early cmv reactivation and engraftment kinetics post-hct. hc assessment by qpcr is highly concordant with str, but markedly superior for molecular monitoring of engraftment kinetics and relapse. positive qpcr results in bm should be interpreted with caution during early engraftment, while both bm and pb were highly informative for relapse in our series. these results advocate the feasibility and clinical utility of qpcr for post-hct hc monitoring in routine use. disclosure of conflict of interest: the commercial assays for qpcr chimerism analysis were provided by abbott molecular free of charge. tyrosine kinase inhibitor (tki) has become the standard of care in patients (pts) with chronic myeloid leukemia (cml) and an unavoidable tool in the combined therapy for pts with philadelphia chromosome positive (ph+) acute lymphoblastic leukemia (all). nevertheless, allogeneic stem cell transplantation (hsct) remains the standard therapy of all ph+ and of cml pts failing 1st line therapy with tki, with failure or insufficient response or intolerance or mutations resistant to 2nd generation tki, or in the advanced phase at diagnosis. in the past decade the feasibility and safety of post-hsct imatinib administration as prophylactic or therapeutic strategy was confirmed. second and 3rd generation tki administration after hsct is under investigation. here we are reporting our experience in post-hsct treatment with the 3rd generation tki ponatinib in 5 pts treated between 2011 and 2016 at our institution. pts data and information were collected from institutional database and chapters revision. a written consent was given by pts allowing the use of medical records for research in accordance with the declaration of helsinki. pts and diseases features are reported in table 1 . pre-transplant treatment for the all ph+ patient consisted of chemotherapy combined with dasatinib, followed by a 1st mud hsct and dasatinib in maintenance. the patient relapsed 1 year after hsct with documentation of mutation v299l. ponatinib was introduced as salvage treatment to bridge 2nd haplo hsct. pre-transplant treatment for the cml patients consisted of tki therapy with combination of chemotherapy in case of uncontrolled progression of disease. two pts received a 1st mud hsct but relapsed respectively 5 months and 4 years later. ponatinib was introduced as salvage treatment to bridge 2nd haplo hsct. four pts received ponatinib 45 mg daily before the last hsct: one patient achieved sustained major molecular response, 4 pts obtained transient response. all pts were presenting 2nd generation tki resistant mutations (table 1) . ponatinib was started at a median of 157 days after hsct (range, 117-583) as salvage treatment in overt relapse (3 cases), prophylaxis (1 case) or preemptive therapy (1 case). acute gvhd was diagnosed in 4 pts before ponatinib administration, 2 of them also experienced chronic gvhd. no new cases of gvhd were observed after initiation of ponatinib. immunosuppressive treatment and azoles treatment were discontinued before ponatinib in all but one patient who was under combined treatment for chronic gvhd: therapeutic drug monitoring was closely performed without evidence of drug-drug interaction. pts were regularly evaluated for toxicities and monitored for cardiovascular events. no serious adverse events were reported in our experience: we administered ponatinib at a median maximum dosage of 30 mg daily (range, 15-45 mg), for a median of 24 weeks (range, 4-132 weeks). at last evaluation one patient maintained the status of molecularly undetectable leukemia (follow-up post hsct: 34 months) and one major molecular response (follow-up post hsct 29 months). three patients who received therapeutic ponatinib in overt relapse did not respond and died for progressive disease. ponatinib is safe and well tolerated as bridge to hsct and to maintain disease control after transplant. prophylaxis targeted therapy and preemptive therapy with ponatinib may lead to the reduction of disease relapse for high-risk ph+ leukemia. disclosure of conflict of interest: none. at nuh singapore we have adopted the cd45 ra depletion to ameliorate graft versus host disease. materials and methods: we have transplanted 14 leukemia patients with cd3 depleted hsct followed by cd45ra depleted donor lymphocyte infusion. no additional gvhd prophylaxis or gcsf was used. results: 100% patients achieved primary engraftment. median time for neutrophil engraftment (4500/μl without gcsf) was 14 days (range 7-17 days), platelet engraftment (450 000) was 13 days (range 10 to 29 days). immune reconstitution was rapid with median cd4 and cd8 cell counts 4200/μl at day 30. by day 200 median cd4 count was 390/μl (range 312-817/μl). no patient developed grade iv acute gvhd. there was a significantly reduced incidence of invasive viral infections as compared to conventional transplants. importantly, all patients achieved complete remission (cr) on day +21 and remained in cr for longer time as compared to conventional transplants. conclusion: our preliminary data suggests that rapid immune-reconstitution of nk cells and t cells with this strategy correlates with reduced infection related mortality without loss of graft versus leukemia effects. disclosure of conflict of interest: none. the use of post-transplantation high-dose cyclophosphamide (pt-cy) has overcome the need for extensive depletion of t lymphocytes from haploidentical donor grafts, which traditionally resulted in severe and prolonged immunosuppression. it is therefore relevant to investigate the degree and the tempo of immune reconstitution after t cell replete haploidentical stem cell transplantation (haplo-sct) by use of pt-cy. we prospectively monitored cellular immunity in 15 consecutive adult patients (male/female: 9/6), who underwent haplo-sct with pt-cy for myeloid (n = 12) or lymphoid (n = 3) malignancies. the median age at transplant was 56 (range, 27-68) years. the conditioning regimen was myeloablative in 10, reduced-intensity in 4, and non-myeloablative in 1 case. the source of the graft was peripheral blood (n = 7) or bone marrow (n = 8). in addition to pt-cy, graft-versus-host disease (gvhd) prophylaxis included tacrolimus and mycophenolate mofetil. absolute counts of cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ cells were measured by flow cytometry at 1, 3, 6, 9, 12, 18 and 24 months post transplant. the median doses of infused cd34+ and cd3+ cells were 4.13 (range, 1.16-9.61) × 10 6 /kg and 4.45 (range, 2.2-38) × 10 7 /kg, respectively. neutrophil engraftment (4500/ul) was achieved at a median of 18 (range, 16-35) days, whereas platelet engraftment (420 000/ul) was observed at a median of 23 (range, 13-54) days. seven patients developed acute gvhd (grade i/ii: 6, grade iii: 1). chronic gvhd occurred in 3 patients, and was extensive in the 2 of them. cytomegalovirus infection was detected in 9/15 cases at a median interval of 42 (range, 30-89) days post transplant. two patients were administered rituximab for epstein-barr virus reactivation at 10 months, whereas one patient developed bk virus-associated hemorrhagic cystitis at 2.5 months following haplo-sct. there was 1 death due to gvhd and infection at 7 months post transplant. at a median follow-up of 12 (range, 3-33) months, 14/15 patients remain alive and disease-free. the absolute counts of t and b cells were extremely low early post transplant, while nk cells recovered from the first month (mean count, 254/μl). the number of cd8+ t cells started to increase beyond the first month, and exceeded lower normal limit at 3 months (mean count, 296/μl). cd4+ t cells remained in general low ( o100/μl) for the first 3 months, increased moderately by 6 months (mean count, 186/μl), and approached lower normal values at 9 months (mean count, 325/μl) [ figure 1 ]. of note, cd4+cd45ra+ naïve t cells remained significantly impaired (absolute count range, 4-52/μl) in all 7 patients in which they were assessed beyond the 1st year from transplant. cd19+ b cells were suppressed for the entire first trimester (mean count at 3 months, 68/μl), but increased rapidly between 6 and 9 months (mean counts, 165/μl and 366/μl, respectively). in haplo-sct with pt-cy, reconstitution of cellular immunity can be achieved at adequate levels by 6-9 months following transplant. the observed deficit in the recovery of naïve t-helper cells may be related to a possible effect of pt-cy on thymopoiesis and warrants further investigation. [p440] disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct) is a curative therapy for patients with sickle cell disease (scd). hemoglobin a in scd ameliorates the manifestations of the disease and this could be achieved with stable mixed chimerism after a reduced intensity hsct. this study aims to estimate the proportion of patients who develop mixed chimerism after hsct for scd and to characterize its progression in patients who develop it. this is a retrospective cohort study conducted at sultan qaboos university hospital (squh) bone marrow transplant unit in oman. we included all patients with scd who received hsct over the course of 10 years between may 2007 to may 2016. patients who received second hsct were excluded. short tandem repeat polymerase chain reaction was used for chimerism assessment. mixed chimerism was defined as 5 − 95% chimerism at 6 months from hsct. the data was analyzed by r program 3.1.2. χ 2 or student t-test were used to assess the impact of acute graft versus host disease (agvhd) prophylaxis, age at transplantation, gender, red blood cell antigen alloimmunization, preparative regimen, and ferritin on the development of mixed chimerism. we included 56 eligible patients. the median follow-up time after hsct was 26 months (interquartile range: 17.3 − 50.3 months). the mean age at transplant was 19.9 years (standard deviation: 8.44). fifty-nine percent of patients were male. most patients had s/s genotype (77%), followed by s/beta-thalassemia mutation (20%). the indications for bmt were: stroke in 7%, acute chest syndrome (acs) in 9%, recurrent vaso-occlusive crisis (voc) in 38%, stroke and acs in 7%, acs and voc in 31%, orbital compression syndrome in 2%, stroke and moyamoya disease in 4%, and moyamoya disease in 2%. the two most frequently used preparative regimens were busulfan/fludarabine/atg in 49% and thiotepa/treosulfan/fludarabine in 42%. twenty-five percent of patients developed mixed chimerism at six months after hsct. on follow up of patients with mixed chimerism, 10% rejected the graft, 20% developed complete chimerism, and 70% continued to be in mixed chimerism. preparative regimen and the development of agvhd were statistically significant predictors of mixed chimerism at 6 months (p values: 0.00079 and 0.01817 respectively). age at transplant, gender, red blood cell antigen alloimmunization, and ferritin were not statistically significant predictors of the mixed chimerism (p40.05). the study confirmed that mixed chimerism can commonly be achieved in patients with scd after hsct and in majority, it remains stable on long-term follow-up. reduced intensity preparative regimen and lack of agvhd predicts the development of mixed chimerism. larger prospective studies are needed to confirm these results. disclosure of conflict of interest: none. there was a majority of male patients (69%). the median hbf level was 15% (0-77), median monocyte count was 5.109/l (range: 0-74.8), median platelet count was 64.109/l (4-377), median marrow blasts was 5% (0-37) above 96 patients who were explored by marrow karyotype, 27% of them had a monosomy 7. mutations in ptpn11 were detected in 33 patients. fifty patients (43%) were treated with the bu/cy/mel regimen, whereas 22 patients (19%) received the bu/flu/mel regimen. at 6 years, the overall survival (os) was 62% (95% ci: 53-72). nineteen and 9 patients developed vod after bu/cy/ mel and bu/flu/mel conditioning regimen, respectively. the cumulative incidence of agvhd 3-4 was 26% (95% ci: 19-35). the 6-year cumulative incidence of relapse and non-relapse mortality was 30% (95% ci: 22-39) and 18% (95% ci: 11-25), respectively. the median delay of relapse was 90 days (range 15-1330). among relapsing patients, 16 were transplanted twice and one underwent 3 hsct. in multivariate analysis, female donor to male recipient sex-mismatch, cmv status, total body irradiation and ras-double mutation/other additional mutation predicted poorer outcomes. the bu/flu/mel conditioning regimen was associated with a decreased risk of relapse. however, there was no statistical difference for os between the two main preparative regimens, bu/cy/mel vs bu/ flu/mel. our results show that allogeneic hsct may cure approximately 60% of patients with jmml and are similar to the best results published by other groups. relapse represents the main cause of treatment failure and a second hsct should be proposed. despite a decreased risk of relapse with the bu/ flu/mel regimen, there was no statistical difference in terms of os between the two main conditioning regimens, bu/flu/mel vs bu/cy/mel. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (ahsct) is the only curative treatment modality for the majority of pediatric patients with myelodysplastic syndrome (mds) and myeloproliferative neoplasms (mpn). the purpose of this study is to evaluate overall (os) and failure (relapse or death from any cause) free survivals (ffs), non-relapse mortality (nrm) and relapse incidence in children who underwent ahsct for mds or mpn in a single center from turkey. we retrospectively analyzed 45 ahsct carried out in 43 patients (median age: 9.2 years; range: 0.4-18; 24 males). thirty four had primary mds and 9 had secondary mds. according to the modified who mds and mpn classification, 18 had refractory cytopenia (rc), 12, refractory anemia with excess blast (raeb), 1, refractory anemia with excess blast in transformation (raeb-t) and 12, juvenile myelomonocytic leukemia (jmml). amongst patients with secondary mds, 4 had been treated for acute myeloid leukemia, 2 had been treated for non-hodgkin's lymphoma and 1 had been treated for acute lymphoblastic leukemia, retinoblastoma and osteosarcoma, each, previously. donors were related in 18 transplantation (5 haploidentical transplantation) and the stem cell resources were bone marrow (n = 27), peripheral blood (n = 14), cord blood (n = 2) and bone marrow +peripheral blood (n = 2). three-year ffs and os for patients with mds were 55% and 57.0%, respectively; and for patients with jmml, 50% and 64%, respectively. crude incidence of nrm and relapse for entire group were 33% and 22%, respectively. ahsct offers durable ffs and os for a significant group of pediatric patients with mds and mpn. less toxic conditioning regimens could result in better results in some patients. disclosure of conflict of interest: none. allogeneic stem cell transplantation in children with autism z antonella, g alessandra, ma beatriz and r vanderson bone marrow transplantation unit, hospital sirio-libanes, são paulo, brazil autism spectrum disorders (asd) are severe heterogeneous neurodevelopmental abnormalities characterized by dysfunctions in social interactions and communication skills, restricted interests, repetitive, and stereotypic verbal and non-verbal behaviors. the etiology of asd remains unknown, but recent studies suggest a possible association with altered immune responses and asd. inflammation in the brain and central nervous system has been reported with microglia activation and increased cytokine production in postmortem brain specimens of individuals with asd. other studies have established a correlation between asd and family history of autoimmune diseases, associations with mhc complex haplotypes, and abnormal levels of various inflammatory cytokines and immunological markers in the blood. the paracrine, secretome, and immunomodulatory effects of stem cells would appear to be the likely mechanisms of application for asd therapeutics. we describe two cases of patients with asd who underwent hsct for acute lymphoblastic leukemia (all) and whose symptoms were markedly decreased like an improvement of social interaction, communication, and behaviors. the first patient is an 11-year-old girl with asd who was diagnosis with ph-positive all in october 2011 (at the end of treatment, bcr-abl remained positive). she underwent a matched sibling hsct in march 2015. the conditioning regimen was total body irradiation (tbi) and cyclophosphamide. during the 20-month follow-up period, we observed improvement in social interaction, communication, and behaviors (according to the childhood autism rating scale-cars). the second case is a 7-year-old boy with asd, asperger syndrome, who was diagnosis with all in september 2012. he presented with bone marrow and testicular relapse in may 2015 and underwent a matched unrelated hsct in november 2015. the conditioning regimen used was etoposide, atg and tbi. during the 12-month follow-up period, we observed improvement in social interaction, communication, and behaviors (according to cars). there is no treatment for asd thus every effort to minimize the symptoms are valuable. in both cases, social interaction was significantly increased, and the aggressive behaviors decreased. clinical cases have reported responses in autistic children receiving cord blood cd34+ cells. several incurable neurological disorders have shown benefits with cellular therapy. thus, autism should be explored as an indication. clinical studies are an immediate need to fully explore its potential in autism. disclosure of conflict of interest: none. conditioning is the initial phase of hematopoietic stem cell transplantation, based on high dose chemotherapy, combined or not with total body irradiation, aiming to eradicate the disease and prepare the environment of the bone marrow for the new cells. conditioning regimens can be characterized as myeloablative or non-myeloablative. during the period of conditioning and immunological reconstitution, signs and symptoms of the gastrointestinal tract are frequent, negatively influencing oral food intake, and may require the use of complementary nutritional therapies, aiming at an adequate caloric intake with the objective of avoiding decreasing in the nutritional status. the study aims to describe the association between the regiment intensity and the nutritional aspects during hospitalization of children and adolescents undergoing allogeneic hematopoietic stem cell transplantation (hsct) at a tertiary hospital. a retrospective study with medical records of patients undergoing allogeneic hsct, aged between 0 and 19 years of age (incomplete) between january 2009 and december 2014. data were collected (regimen intensity, clinical signs of mucositis and nutritional therapies used) during the hospitalization and analyzed by the relative risk (rr). sixty-three patients were evaluated, being 56% male, with a median age of 10 years. nineteen types of conditioning protocols were used. of these, 64% were high intensive regimen and 36% were low intensive regimen. the four most applied (59% of cases) were bucy (busulfan + cyclophosphamide) with and without atg (thymoglobulin), as well as cytb (cyclophosphamide+total body irradiation), also with and without atg. mucositis were observed in 83% of patients, being 50% grade 3 and grade 4. the association was positive when analyzed the regimen intensity (myeloablative) with mucositis (rr = 1.51 (1.10-2.08)) as well with the use of parenteral nutrition (rr = 2.49 (1.17-5.13)). patients showed high prevalence of mucositis during hospitalization decreasing food intake, being necessary to use the parenteral nutrition. myeloablative regimen needed more nutritional therapy intervention when compared to non-mieloablative regiment. results demonstrate that an appropriate nutritional screening tool considering these aspects could help to intervene earlier maintaining an adequate nutritional status. autoimmune cytopenia (aic) is a potentially serious complication of hematopoietic stem cell transplantation (sct). autoimmune hemolytic anemia (aiha) is the most common aic, followed by immune thrombocytopenic purpura and autoimmune neutropenia. aic after sct is considered difficult to treat and associated with high morbidity and mortality. the aim of this cohort study is to evaluate incidence, outcome, potential risk factors and current treatment strategies and to explore the immune dysregulation predisposing to aic. the ebmt-promise database was accessed to identify all pediatric scts between 2000 and 2016 complicated by aic at our center. potential risk factors (i.e., age, gender, diagnosis, donor type, stem cell source, conditioning regimen) for aic after sct were assessed using univariate and multivariate cox regression analysis. in addition, we summarized treatment decisions of all aic patients. a nested matched case-control study was performed to search for possible biomarkers for aic. of 531 consecutive scts, 27 were complicated by the development of aic (cumulative incidence 5.2%) at a median of 5 months post-sct (figure) . aiha was the most common aic (48%), followed by combinations of two or more aics (evans syndrome, 33%). non-malignant disease, young age, alemtuzumab serotherapy pre-sct, non-tbi based conditioning regimen and cmv reactivation were associated with aic in univariate analyses. using multivariate cox regression analysis, non-malignant disease (hr 3.6, p = 0.028), alemtuzumab use (hr 2.4, p = 0.035) and cmv reactivation (hr 3.7, p = 0.013) were independently associated with aic (figure) . for patients with cmv reactivation, diagnosis of aic was made at a median of 4 months (iqr [1] [2] [3] [4] [5] [6] [7] [8] ) after detection of maximum viral load. in our nested case-control analysis, serum levels of individual anti-and proinflammatory, and regulatory cytokines did not differ significantly between patients and controls. however, the cytokine profile of aic patients appeared to skew towards a more pronounced th2 response, compared to controls. firstline treatment, usually with prednisone and/or ivig, or a waitand-see approach led to resolution of aic in 9 (33%) cases. second and subsequent-line therapies, often in combination with continuation of other treatments, consisted of rituximab (n = 16), bortezomib (n = 7) or sirolimus (n = 3) and eventually led to resolution of aic in 44%, 57% and 100% of cases, respectively. overall survival of aic patients was 78%. in this retrospective cohort study, we identified cmv reactivation post-sct, alemtuzumab use and non-malignant disease as independently associated clinical risk factors for the development of aic. treatment with first-line therapy was mostly insufficient. for patients with severe aic, rituximab, bortezomib or sirolimus can be regarded as promising step-up therapies. figure (bkv) may cause polyomavirus-associated nephropathy or polyoma virus-associated hemorrhagic cystitis in bone marrow-transplant patients.we present 19 patients with bk polyoma virus (bkv) ascociated hemorrhagic cystitis and 2 patients with bk polyoma virus associated hemorrhagic cystitis and nephritis. between 2013 and 2016, 124 patients received an allogeneic bmt at acıbadem adana hospital pediatric bone marrow transplantation unit. 21 patients occurred bkv associated hemorrhagic cystitis and nephritis. bkv was detected in the urine analysis and blood by pcr (polymerase chain reaction) in all patients. we presented 21 patients with bkv infection, age ranging from 3 to 20 with a average of 13.1 years. they underwent allogeneic bmt due to thalassemia major (13 patients), aplastic anemia (4 patients) and acute lymphoblastic leukemia (4 patients). the patients were treated with hydration, continuous bladder irrigation, ciprofloxacin, and weekly intravesical hyaluronic acid instillation for four weeks, and cidofovir. fourteen patients showed complete resolution of hematuria. one patient with refractory above these therapy also received hyperbaric oxygen and recombinant factor viia (rfviia, novoseven; novo nordisk,bagsvaerd, denmark). hemodialysis was performed in two patients who developed renal failure due to nephritis. bkv is ubiquitously present in the general population. 1 reactivation of infection occurs under conditions of immunosuppression, particularly hsct or renal transplantation, and causes late-onset hc. bkv the management of bkv cystitis and nephritis sometimes may be very difficult and refractory all treatments, we presented our experience of bkv infection and management in transplanted patients in our center. patients with high-risk hematologic malignancies (hrhm) are among those in the highest risk group for developing invasive fungal disease (ifd), especially mold infections. allogenic hematopoietic stem cell transplantation (alsct), acute myeloid leukemia (aml), refractory and relapsed acute lymphoblastic leukemia (all), myelodysplastic syndromes and chronic extensive graft-versus-host disease are considered hrhm. ifd are a major cause of morbidity and mortality in these patients, however, the optimal strategy for antifungal p448 s352 prophylaxis in this population is not well defined yet. we performed a retrospective, observational study to investigate documented ifd during antifungal prophylaxis in children with hrhm who were admitted in our unit between 2010 and 2016. demographic and clinical data were collected from patient's electronic medical records. all patients were treated with prophylactic voriconazole (vcz) according to our local practice. oral administration was preferred when available. vcz therapeutic drug monitoring (tdm) was not available in our center until june 2016. breakthrough ifd was defined as occurrence of a proven or probable ifd according to eortc/ msg criteria while on vcz prophylaxis (⩾7 days of treatment) or within 15 days after discontinuation of prophylaxis. during the study period, 75 hrhm patients were treated with prophylactic vcz in our unit. 4 patients out of 75 developed a breakthrough ifd. patient's demographic characteristics, main diagnosis and treatment are collected in table 1 . initial and maintenance vcz doses are adjusted by weight in all patients except in patient-4 (adjusted according to vcz plasma level). adherence and tolerance to treatment was excellent in all patients. disclosure of conflict of interest: none. (3) stable mixed chimerism (smc) when fluctuations of ac were o5%; and (4) mixed progressive chimerism (pmc) when ac were ⩾ 15%. 2-3 101 hscts performed in 85 patients (pts) were included: 72 children with a median age of 2.01 yrs (iqr 0.62-7.35 yrs) at diagnosis and 6.2 yrs (iqr 2-11 yrs) at hsct received one hsct (84.7%),10 pts two hsct (11.8%), and 3 pts three hscts. primary diagnosis were bone marrow failures in 37 pts (43.5%), primary immunodeficiencies in 25 (29.4%), inborn errors of metabolism in 15 (17.5%) and haemoglobinopathies in 8 (9.41%). the donor was match related in 23 (23%) procedures, match unrelated in 63 (62%), and haploidentical in 15 (15%); stem cell source was bone marrow in 55 (54%), peripheral blood in (26%) and cord blood in 20 (20%). conditioning regimen (cr) included busulfan in 19 hscts (18.8%), treosulfan in 33 (32.7%), while 48 hscts (47.5%) were conditioned with reduced intensity crs (including low dose of tbi in 9); 1 pt did not received cr. gvhd prophylaxis was based on csa/mtx (or mmf) in association with atg (69) or alentuzumab (16) ; recipients of tcrαβ/cd19 depleted haploidentical graft did not received post transplant immunosuppression. engraftment was observed in 87 hscts (79 after 1 st , 7 after 2 nd and 1 after 3 rd hsct) after a median of 18 day (iqr 14-23 days). acute gvhd occurred in 45 hscts at risk (52%), and it was severe (gr. iii-iv) in 20 (23%), chronic gvhd in 31 (31%). at last follow-up (median 4.35 yrs), 75 (88%) pts were alive, while 10 pts are dead for infections (n = 5), vod (n = 1), c-gvhd (n = 3) and vascular event (n = 1). figure 1 reported the evolution of chimerism over time. in our experience in children with non malignant disease, cc increased from 36% to 67% at subsequent analyses. 60% of pts with mc at 1 st evaluation became cc, 16% remained smc, 5% evolved in pmc, and 19% rejected. only 2 pts with cc at first time point rejected the graft. this study highlight the extreme variability of chimerism in the early post transplant course of children with non malignant disease and confirmed the relevance of performing serial analysis to monitor and, if necessary, improve graft function. naive t-cells identified by cd45ra expression are believed to cause graft-versus-host-disease (gvhd), while cd45ra-t-cells are memory cells that provide anti-infection and anti-tumoral effects. depleting cd45ra+ naïve cells and retaining memory t-cells in the graft is a novel approach to haploidentical hsct for children. 18 children with high risk leukemia (6 aml, 12 all) received cd45ra-depleted haploidentical hsct following non-myeloablative conditioning. cell-selection performed on g-csf-mobilized peripheral blood. two cellular products obtained using clinimacs device, infused to each patient: a cd34 selection and a cd45ra depletion from the cd34negative fraction. product infused contained a median of 6.04 (range 4.04-9.93) x106/kg cd34+ cells and a median of 6.5 (range 1.3-490) × 10 3 /kg of cd3+ cells in the cd34-selected s353 graft. the second product was the cd45ra depletion, cd45ra +/kg was a median of 6.15 × 10 3 /kg (range 0-498 × 10 3 /kg) and a median 4.70 (range 2.21-6.37) depletion log of cd45ra + cells. median dose of cd45ro+ cells (memory t-cells) infused was 8 (range 3.8-102) × 10 7 /kg. seventeen patients achieved neutrophil engraftment at median of 10 days (range 8-12) post-transplant. one patient could not achieve engraftment, died at day +8 due to sepsis. two patients presented secondary graft failure (day +18 and +20), both received a second hsct. three patients developed agvhd 4grade ii with gastrointestinal tract involvement, all steroids responsive. three patients presented clinical features of cgvhd. patients have an extensive skin involvement, with hepatic findings in one and pulmonary affection in other, at day +315, +130 and +330 post. ten of 18 patients (55.5%) remain alive in remission with median follow-up 156 (range 8-597) days post-transplant. eight patients died, 3 due progression at day +128, +117, +162 (2 presented positive minimal residual disease at hsct), 4 due to infectious complications (days +8, +44, +50, +55) and 1 due to cardiogenic shock at day +253. four patients relapsed, 3 of them died afterward with progressive disease. t-cells led immune recovery, achieved values higher than 500, 600, 1500 and 2400 cells/mcl at day 30, 60, 90 and 210 respectively. most of t cells were cd8+cd45ra-(median of 288, 370 and 2334 × 10 6 /mm 3 respectively on day +30, +60 and +90) and cd4+cd45ra-t cells (median of 129, 161 and 767 × 10 6 /mm 3 respectively on day +30, +60 and +90), while cd8+45ra+ and cd4+45ra+ cells remained low recapitulating the cd45ra depleted graft composition. six patients presented cytomegalovirus reactivation, one progressed to cmv disease. five patients with hhv-6 encephalitis. probable aspergillosis in 1 patient (aml-m7 with secondary graft failure) at day +16 after second hcst. two cases of toxoplasmosis (1 cns, 1 pulmonary). cd45ra-depleted haplo-hct showed acceptable tolerability with rapid and sustained engraftment as well as a full donor chimerism, minimal risk of acute gvhd and accelerated inmunologic reconstitution. to note the high incidence of hhv-6 encephalitis seen. disclosure of conflict of interest: none. collection of peripheral blood stem cells in teenager sibling donors: a single center experience c carvalho 1 , f amado, f bordalo, s ferreira, s lopes, c pinho and s roncon 1 serviço de terapia celular, instituto português de oncologia do porto francisco gentil, epe human leukocyte antigen (hla) compatibility is important in allogeneic haematopoietic stem cell transplantation in order to reduce post-transplant complications; however, siblings only present a 25% chance of hla-match with the patient. the well-known advantages and the low risk of complications associated to peripheral blood stem cells (pbsc) collected by apheresis made this procedure the first option in teenagers. the aim of this retrospective study was to analyse and characterize the paediatric sibling pbsc donor population assuring safety during the collection procedure, providing a high-quality product and accomplishing patient needs. we consulted the clinical files of donors under 18 years old since 1995-2015; a database in excel ® was created to register population characteristics, collection parameters and graft requirements. the informed consent was obtained from parents before procedure. the leukapheresis were performed with a cobe spectra system; since 2009, we use a spectra optia apheresis system. the donor/patient weight ratio (proposed by styczynski et al.) was determined for each pair. the collection was programed based on clinical and analytical donor's features as well as transplant requirements. the analytical assays were done by a certified laboratory. we performed 29 pbsc apheresis in 23 healthy donors, 10 females and 13 males ( table 1) . all of them started on the 5 th day after mobilization with granulocyte colony-stimulating factor (g-csf) administered subcutaneously, bidaily. the weight ratio was o1 in eight situations. most of donations were performed by peripheral vein; a central venous catheter (cvc) was placed into a femoral vein in six adolescents. a median of 4 (3) (4) (5) blood volemias were processed during 174(115-318) minutes; the anticoagulation used was citrate+heparin (ratio 25:1). in general, one-collection day was enough to obtain the number of cd34+ cells required; six donors had to perform a 2 nd collection. in 19 cases, we cryopreserved the exceeding cells after graft infusion. the procedure was well tolerated, with only 2 adverse reactions registered (one hematoma in the puncture local; one paraesthesia due to hypocalcaemia induced by citrate). no blood products were used after the procedure or needed for the priming of the extracorporeal circuit. so far, no serious long-term adverse events were observed. table 1 . median (range) of donors and leukapheresis products data. our long lasting experience shows that pbsc collection in the teenage population is safe and feasible, allowing us to obtain a high-quality product for the patients. there were no adverse events associated with the g-csf mobilization or cvc placement which is different from the experience of other groups. we recognize that leukapheresis by peripheral vein is a lengthy procedure but no complaint was reported to the collection team. [p451] disclosure of conflict of interest: none. correspondence between clinical and hystological grading of gastro-intestinal grading acute graft versus host disease in children m faraci 1 , a rizzo 2 , p gandullia 3 , s arrigo 4,3 , a barabino 3 , e lanino 1 , s giardino 1 and c coccia 5 1 hematopoetic stem cell transplant unit, institute g gaslini, genoa, italy; 2 pediatric department, institute g gaslini, genoa, italy; 3 gastroenterology and digestive endoscopy unit, institute g gaslini, genoa, italy; 4 gastroenterology and digestive endoscopy unit, institute g gaslini, genova and 5 department of pathology, institute g gaslini, genoa, italy diagnosis of gastro-intestinal acute graft versus host disease (gi-agvhd) is frequently confirmed by apoptosis findings on mucosal biopsies. 1 aims of this single center retrospective study is to evaluate the correlation between clinical and histological grading of gi-agvhd in children undergoing allogeneic haematopoietic stem cell transplantation (allo-hsct), and to describe histological findings obtained by gi endoscopies in order to evaluate usefulness in the diagnosis of gi-agvhd. 348 allo-hscts were performed in our department between january 2000 and december 2015. gi biopsies were performed in 26 pts (7.4%) because of suspected gi-agvhd. 14 pts were transplanted for malignant (53.8%) and 12 for not malignant diseases. the median age at hsct was 9.5 years (0.5-16.9).14 pts (54%) received myeloablative and 12 (46%) reduced intensity conditioning regimen. 21 pts (80.7%) received an unrelated donor (ud), 4 pts a related donor (rd) (15.3%), and 1 an haploidentical donor (3.8%). at onset of diarrhea, microbiological examinations of stool were performed and pcr research for cmv, adenovirus, hhv6, ebv were evaluated in blood and in mucosal biopsies. mucosal biopsies were obtained with esophago-gastro-duodenoscopy in 4 pts (15.3%),esophago-gastro-duodeno-colonscopy in 3 (11.5%), pancolonscopy in 11 (42.3%), flexible sigmoidoscopy in 3 (11.5%), and rectal suction biopsy in 5 pts (19.2%). all mucosal biopsies, except in case of rectal suction, were obtained under sedation. the interval between mucosal biopsies and onset of gi acute symptoms was 23 days (from − 66 to 103 days). biopsies were taken from different sites in the gi tract, were stained using hematoxylin-eosin and evaluated using histological grading of agvhd. 1 in these 26 pts the maximum grade of agvhd was: grade 2 in one (4%), grade 3 in 14 (54%), and grade 4 in 11 pts (42%). at time of histological evaluation, diarrhea was the most common gi symptom (84.6%); 2 children had also cutaneous agvhd and 5 hepatic agvhd. pcr-cmv was positive in 2 mucosal biopsies obtained with pancolonscopy, pcr-adenovirus in other 2 obtained with upper and pancolonscopy, pcr-hhv6 in 2 rectal biopsies, and pcr-ebv in one with upper and pancoloscopy. the comparison between clinical and histology grading of gi-agvhd is shown in table 1 . mucosal biopsies were positive in 1/4 pts evaluated with esophago-gastroduodenoscopy (25%) (grade 1 agvhd), in 3/3 pts undergone esophago-gastro-duodeno-colonscopy (grade 1 in 2 and grade 3 in 1), in 8/11 (73%) who received a pancolonscopy (grade 1 in 5, grade 2 in 1, grade 4 in 2), and 7/8 (87%) of rectal biopsy obtained by sigmoidoscopy or rectal suction biopsy (grade 1 in 3, grade 2 in 1, grade 3 in 1, and grade 4 in 2). one patient developed duodenal intraparietal hematoma after upper endoscopy. in our experience, we did not demonstrated a overall correlation between clinical and histological grading of agvhd showing that hystological examinations underestimated the grade mild or moderate of agvhd. we confirmed 2,3 that rectal biopsies represent to be more effective and safe diagnostic method for the confirm of diagnosis of gi-agi. during the past few decades, hematopoietic cell transplantation (hct) as a treatment modality for primary immunodeficiencies (pid) has undergone remarkable advancement mainly due to better availability of alternate donors resulting in increase in not just matched unrelated donor (mud) but also increased haploidentical (haplo) and cord blood transplants (cbt). additionally, refinement of the conditioning regimens and better graft versus host prophylaxis have presumably led to better survival outcomes. however, a literature gap is identified in evaluation of these outcomes in general with respect to donor and conditioning regimens. we conducted a systematic review by performing a comprehensive search of the pubmed and ovid library from its inception to august 2016. mesh terms included 'primary immunodeficiency (immunodeficiencies)', 'stem cell transplant', 'bone marrow transplant' and 'hematopoietic cell transplant'. all pid studies which used hct as a treatment modality were included. experimental cellular therapies were excluded. both cellular immunodeficiencies (e.g. scid, was, a-t), and innate immunity disorders (e.g. ifngr, cgd) were included in the search. reviews, case reports, meta-analysis and non-english language articles were excluded from our electronic search. publication bias was excluded by performing a methodological search of unpublished conference abstracts from the annual meetings of cis, aspho, asbmt, ebmt, and siop from 2000 to 2016. the data were analyzed considering the outcomes -overall survival and gvhd. 21 studies fulfilled the strict selection criteria for the electronic search comprising of 1010 pid patients. in majority of the hcts, a myeloablative conditioning regimen (mac) was utilized (47% of the evaluable) but a shift towards more reduced intensity conditioning (ric) was observed in the later years. 120 cbts were identified. 27% of patients developed some degree of acute gvhd, whereas chronic gvhd was identified in 15% of the patients. total number of haplos was 317. overall survival was found to be 71% post-hct. a meta-analysis could not be performed due to the heterogeneity of both the predictor variable data (combined stem cell sources were also used for hct) and due to the extremely small number of the patients when categorized in subgroups (e.g. for omenn syndrome, rag deficiencies). this is the largest study of hcts in pid, and we observe that alternate donor hcts have increased significantly over the past decade for the treatment of pid. while the incidence of chronic gvhd was low, acute gvhd still remains a problem in about a third of the pid patients transplanted. disclosure of conflict of interest: none. hepatic veno-occlusive disease (vod) is a common and serious complication of hemotopoietic stem cell transplantation (hsct) in children. we aimed to assess prospectively the use of prophylactic defibrotide in pediatric patients undergoing hsct. in this study, 113 patients who underwent hsct were given defibrotide prophylaxis as 25 mg/kg per day in four divided intravenous infusions over 2 h, starting on the same day as the pretransplantation conditioning regimen. the mean duration of use of defibrotide is 20 days as a prophylaxis. in this study, 113 patients were recruited, 66 male patients and 47 female patients, with the average of 9.1 years, range 1-20; 8% infants, 55% children and 37% adolescent. there were 50 patients with thalassemia major, 41 patients with leukemia, 11 patients with aplastic anemia, one patient with diamond blackfan anemia, two patients with congenitale dyserythropoetic anemia, one patient with osteopetrosis, four patients with famial hemophagocytic lymphohistiocytosis, two patienrs with severe immune deficiency and one patient with kostman syndrome. all transplants were allogeneic. no serious side effects were seen. in eight patients developed clinical vod (seattle criteria). in these patients, defibrotide dose was increased to a treatment dose of 40-60 mg/kg per day. one infant patient with kostman syndrome died due to hepatic and pulmonary veno-occlusive disease. after 36 months of follow up, 7 patients who developed vod are being well and no patient have transplant related complications. hepatic veno-occlusive disease, which is caused by hepatocyte and sinusoidal vessel endothelium damage, can ocur early after hsct, and in its severe form, may lead to liver faillure, hepatorenal syndrome, portal hypertension, and eventually death from multiorgan faillure. in this prospective study, we demonstrated that the use of defibrotide is safe and effective in preventing and treating vod in pediatric patients at high risk. immune reconstitution (ir) is critical for clinical outcome after allogeneic hematopoietic stem cell transplantation (hsct). host and proceeding-related factors affect the ir dynamics and survival. isolated ir parameters are commonly correlated and proposed to predict clinical outcomes after hsct, but these approaches only confer prognostic value at single time points or for single markers. we aim to demonstrate an appropriate methodology to assess the capability of combined serial measurements of lymphocyte subsets to reflect the impact of infections on ir after paediatric hsct. retrospective data of patients receiving a first hsct for any indication with any cell source in the paediatric hsct program from 2006 to 2015 were included. to characterize the kinetics of immune reconstitution, cd3+, cd4+, cd8 + t-cells, b-cells, nk-cells and their naive and memory subsets were measured and analysed at various time points at 2 years post-hsct to stablish a joint model for the evolution of cell subpopulations. slope per month (cellular increase or decrease) of each lymphocyte subsets were calculated and compared with clinical outcomes and cumulative risk of infections. a total of 88 children (range from 0-15 y.o. median 5 y.o.) were included, with cb (n = 19) pb (n = 22) and bm (n = 47) as cell sources. the cumulative incidences after early period were 45% for viral infections (ebv 27%, cmv 22%, bk 11%, adv 4%) and 30% for bacterial infections. data on ir were available for 77%, of the diseasefree survivors. in a exploratory multivariate analysis we detected mainly differences in cd8+, cd8+cd45ro+ memory and nk cells at 1 year after hsct, with dependent tendency according on the cell source and hla compatibility. analysis of the slope tendency patterns were stablished for the analysis of the impact of infections in the ir. delay in cd8+ and cd8+ra+ appearance (mean slope/m = − 7.1% and − 1.8% respectively) remarks the ir profile for bacterial infections, and delayed in nk, cd8 and cd8ro+ (−8.4%, − 5.8%, − 26% respectively) for overall viral infections. additional correlations allow differences in ebv (cd8+ra+ high mean slope/m = 15.9%), cmv (delayed in cd8ro+ slope/m = 10%), and bk infection (cd8+ra + plus cd4ro+ and nk high mean slope/m = 24.9%, 36% 22%). understanding the dynamics of reconstitution by integrating information from the monitoring of lymphocyte subpopulations allows the establishment of kinetic profiles that may help to evaluate the risk of infections and adjust infection prophylaxis in the follow-up of transplanted patients. mortality rate in hsct patients admitted to intensive care unit (icu) is still as high as 20% to 70%. this rate increases when respiratory complications progress to acute respiratory failure (arf) requiring mechanical ventilation (mv). 1 the aim of this study was to determine the feasibility and effectiveness of early continuous positive airway pressure (cpap) delivered in a pediatric hematology-oncology ward to prevent occurrence of arf requiring mv. we retrospectively analysed children treated with cpap in our pediatric hematology-oncology ward between october 2011 and october 2016. thirty-two patients received cpap delivered with helmet during the study period. data were available for 26 patients, 15 males and 11 females, median age 12 years [range 2-20]. eighteen patients underwent allogenic hsct: 1 from sibling donor, 11 from matched unrelated donor, 4 from haploidentical family donor, 2 from cord blood unit. seven patients had a malignant disease: 5 all, 1 aml,1 ewing sarcoma. infectious pneumonia was the main cause of arf in 16/26 patients (61.5%): 9 viral pneumonitis (4 rhinovirus, 3 parainfluenzae virus, 1 respiratory syncitial virus and 1 cmv). five patients had proven/ probable invasive fungal infection according to eortc criteria (3 aspergillosis and 2 mucormycoses). other causative agents were pneumocystis jiroveci (1), bacillus of calmette and guerein (1), toxoplasma gondii (1) and st. mitis (1) . non infective causes of arf were acute transfusion related lung injury (2), hemorragic alveolitis (2), cryptogenic organizing pneumonia (3), tumor lysis syndrome (1), and alveolar oedema due to renal failure (1). according to chest imaging, 13/26 patients (50%) presented with pulmonary consolidations, while 31% had both interstitial infiltrates and pulmonary consolidations. at baseline median neutrophil count was 2.05 × 10 3 /μl (range 0-21.0 × 10 3 /μl), mean heart rate 128 bpm, pulsiossimetry saturation in room air 86%. h-cpap was applied in 19/26 patient with a curative aim, in 7/26 patients as palliative support to reduce respiratory distress. median positive pressure delivered was 10 cmh 2 o (7-12 cmh 2 o), median fio 2 was 40% (30-100%). h-cpap was applied for a median of 11 days . no patient failed h-cpap because of agitation or adverse events (skin breakdown, conjunctivitis, gastric distension or epistaxis). ten patients were transferred to icu (34.6%), 8/10 because of hsct complications. median icu stay was 8.7 days (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) . only 3 patients required mechanical ventilation, in 2 cases associated to ecmo. nether psao 2 in room air (p 0.98 ci 95%) nor cpap level (p 0.76 ci 95%) correlated with the need of icu admission. patients requiring higher fio 2 during cpap demonstrated a not statistically significant trend to higher icu admission rate (p = 0.149).there was a higher rate of mv in patients with higher cpap fio 2 level (p = 0.008). mv prolonged icu stay (p 0.0049). cumulative mortality was 34.6% (9/26); only 1 patient died in icu (10%), because of post hsct parainfluenza virus pneumonitis requiring ecmo. helmet cpap delivery in pediatric hsct ward is feasible and safe, both for curative and palliative aim. if applied early, cpap could reduce icu admission rate for mv and icu mortality. veno occlusive disease (vod) and graft versus host disease (gvhd) are both dreadful complications of hematopoietic stem cell transplantation (hsct). although they have different clinical signs, it is suggested that they share similar pathophysiological pathway. defibrotide is used in the treatment of vod for a long time but it is very less known about its effect on gvhd. in this study, we analyzed a 'high risk' patient population in pediatric hsct to show the effect of defibrotide on acute gvhd. between june 2014-august 2016 totally 75 'high risk' pediatric allogenic hsct procedures were enrolled in this study. 'high risk' definition involves busulphan/ melphalan usage in conditioning regimen, second myeloablative hsct, pre-existing liver disease, allogenic hsct for leukemia with second relapse and diagnosis of hemophagocytic lymphohistiocytosis (hlh) or osteopetrosis. defibrotide prophylaxis group (n = 22) received 25 mg/kg/day per day and continued for at least 14 days after transplantation. the control group (n = 53) received only continuous infusion of low-dose heparin until 30 days after transplantation. for the comparison between groups, the fisher's exact test was used. all analyses were performed using spss 20 and p-value of 0.05 was considered statistically significant. we analyzed totally 75 hsct procedures with different diagnosis; 17 beta thalassemia major, 14 leukemia, 9 hlh, 18 primary immunodeficiencies, 3 osteopetrosis, 4 fanconi aplastic anemia (faa), 2 myelodysplastic syndrome, 2 neuroblastoma, 1 congenital amegakaryocytic thrombocytopenia, 1 krabbe disease, 1 aplastic anemia, 1 hypereosinophilic syndrome and 1 sickle cell disease. all the procedures meet the 'high risk' definition; most of them (n = 50) have busulphan for conditioning, also there are 9 hlh and 3 osteopetrosis patients, 2 neuroblastoma patients had the second myeloablative regimen, faa and aplastic anemia patients had pre-existing liver disease, and 2 of the leukemias had beyond second relapse. the mean age was 6.7 years old (0.25-19.6), 27 hsct performed from match sibling donor (msd) (36%), 3 hsct from match family donor (mfd) (4%), 43 hsct from match unrelated donor (mud) (57%) and 2 hsct from haploidentical mother (3%). we especially focused on gvhd and vod. totally 13 vod cases (17%) in these 75 hsct procedures were detected. only two of them detected in the prophlaxis group (9%) and 11 cases in the control group (20%). there were 32 cmv reactivation cases detected in 75 hsct procedures (42%). in the prophlaxis group there were 11 cases (50%) and in the control group 21 cases (39%). we detected 36 acute grade i-iv gvhd cases in 75 hsct procedures (48%). only 4 of them were in the prophlaxis group (18%) and 32 cases were in control group (60%). the prophlaxis group's agvhd ratio was significantly lower than the control group (p = 0.001). defibrotide for vod prophylaxis is confirmed by several studies, but its benefits for agvhd is not clear. in this study, we show the significant effect of defibrotide on agvhd. we speculate that the protective effect of defibrotide on both vod and agvhd could be explained by the similar pathophysiology of these complications. we need larger studies on the pathophysiological pathways, then we could invent more effective interventions. disclosure of conflict of interest: none. conventional extracorporeal photopheresis (ecp) has proven efficient for the treatment of graft-versus-host-disease (gvhd) but is limited to patients with sufficient body weight. a mini buffy coat ecp (mini-ecp) 'off line' technique that allows treatment of small children has been developed, using various methods for mononuclear cell (mnc) separation from whole blood. we present treatment of low body weight child with mini-ecp 'off line' technique using sepax system for mononuclear cell (mnc) selection and macogenic irradiator. a toddler with juvenile myelomonocytic leukemia (jmml) developed acute gvhd, after a matched unrelated stem cell transplantation (sct) performed at the age of 12 months. acute gvhd of the skin occurred three months after sct and responded to high dose steroids, but recurred six months after sct (biopsy of the skin confirmed acute gvhd) together with gvhd of the liver. because of the resistance to steroids and cyclosporine, mini-ecp was introduced as therapy. the patient weighed 8 kilograms. blood was collected from tunneled central venous catheter, and collected volume was replaced with saline infusion. the cord blood collection bag (macopharma, france), which contains 21 ml citrate phosphate dextrose (cpd) anticoagulant solution was used for whole blood collection. whole blood was processed using sepax system separator (biosafe, switzerland), and final volume of buffy coat was set on 25 ml. extracted buffy coat was transferred into the uv-a illumination eva bag (macopharma, france) and diluted with saline solution up to 200 ml. 8methoxypsoralen (gerot, austria) was injected directly into the uv-a illumination bag, and cells were irradiated by the uv-a illumination device macogenic 2 (macopharma, france). irradiated cells and autologous residual blood were reinfused back to the patient. during the whole procedure patient's vital signs were monitored. ecp procedures were performed 3 times per week for 4 weeks, followed by 2 times per week at 2 weeks intervals for 2 months. in 3 month period 28 mini-ecp procedures were performed. median of collected whole blood was 92 ml (range 52-100). median of total nucleated cell (tnc), and mononuclear cell recovery after sepax separation were 88.3% (range 66.7-104), and 90.8% (range 63-102), respectively. median of hematocrit in final irradiated product was 4% (range 3.8-6%). patient was reinfused with median of 1.0 (range 0.66-1.8) tnc × 10 8 /kg bw, and median of 4.95 (range 3.62-13.72) lymphocyte × 10 8 /kg bw. after one month of ecp together with steroids and cyclosporine, gvhd of the skin improved, and the steroids were gradually weaned, with no recurrence. gvhd of the liver showed no improvement, and other therapies had to be introduced, but without steroids. for the first time in croatia, mini-ecp was performed in a child with gvhd, in whom conventional ecp could not be used because of insufficient body weight. mnc separation using automated closed system sepax separator has proven efficient and safe. mini-ecp treatment was continued for three months, without technical difficulties. positive effect was experienced concerning the skin gvhd, but not the liver gvhd. after the first experience in our country, in future we plan to use this technique for low-weight patients or patients with contraindications for apheresis, which are in need of second-or third-line therapy for gvhd. disclosure of conflict of interest: none. gonadal failure represents one of the late effects of haematopoietic cell transplantation (hsct) with a negative impact on quality of life in young patients (pts) undergoing hsct1,2. the aim of this retrospective multicentre ebmt study was to assess gonadal function in untreated pts undergoing allogeneic hsct between 5 to 20 years (yrs) of age, after a preparative regimen with busulphan (bu) or treosulfan (treo). eighty-seven pts (32 females, 55 males) were reported from 17 out of 123 contacted ebmt centers: 26/87 (30%) received allogeneic hsct during pre-pubertal and 61/87 (70%) in pubertal phase. of the 87 pts, 76 (87.4%) received bu in myeloablative dose [25 pre-pubertal, (median age of 6.7 yrs) and 51 pubertal, (median age of 13.4 yrs)] and 11 pts (12.6%) received treo (1 in pre-pubertal and 10 in pubertal period). underlying diseases were primary immunodeficiency (34.5%), chronic myeloid leukemia (33.3%), myelodisplastic syndrome (24.1%), familial haemophagocytic lymphohistiocytosis (6.9%) and shwachman-diamond syndrome (1.1%). 17/26 of prepubertal pts (71%) developed spontaneous puberty (69.5% in the bu group and 100% in treo group). 21/28 (75%) females undergoing hsct during puberty completed their pubertal development (71.4% in bu group and 100% in treo group). none of females (4/4) with bu during pre-pubertal phase developed spontaneous menarche (sm), while 33.3 %(7/21) of females who received bu in pubertal period had sm. all females (n = 5) treated with treo during pubertal phase had sm (100%). for both conditioning regimens, the 42.8% (12/28) s358 of females treated during the puberty experienced sm. among the remaining 14 females (for 2 pts the information is missing) who did not developed sm, 13 received hrt 2.5 yrs after hsct and 5 of them had ovarian recovery after a median of 2.3 yrs from hsct (1.43-6.72). the median age at last follow up was 15.8 and 13.2 yrs in bu and treo pre-pubertal group, and 22.2 and 19.9 yrs in bu and treo pubertal group respectively. in the pubertal group, 18 females (69.5%) are still receiving hormonal replacement therapy (hrt) (16 in the bu group and 2 in treo group). 2 pts (7.4%) had spontaneous pregnancy. no problems in newborns are reported. sperm analysis was performed in 18.2% of pubertal pts (6/33) of males, and 66% (n = 4/6 treated with bu) were azoospermic (data regarding 2 pts were missing). the sperm analysis was repeated in half of the males. until now no paternity was reported. in this experience, the pubertal development in pts who received treo (n = 6) was normal, and in the bu group the majority of females (70%) had normal puberty. the rate of sm is higher (100%) in females after treo than bu (28%). the hrt is ongoing at last follow-up in 76% of females treated with bu and in 40% of those who received treo. our data suggests that treo may have a better outcome than bu in young girls receiving allogeneic hsct and larger studies are warranted. male patients require longer follow-up. prevention in patients transplanted from partially matched donors. we report the single centre experience in haploidentical sct. in years 1999-2016 in the department of pediatric bmt, oncology and hematology at wroclaw medical university, 72 children underwent sct from partially matched, haploidentical parental donors. graft manipulation in 38 patients consisted of cd34sel, in 22 patients the cd3 immunomagnetic depletion (tcd-cd3) was performed, and in 12 -tcr alpha-beta depletion (tcd-ab). we analysed the impact of type of manipulation procedure, conditioning regimen, demographic factors, and kir genotype on survival and probability of neutrophil recovery. the probability of engraftment and neutrophil recovery was 86% vs 77% in cd34sel group (p = ns). probability of 5 year overall survival in the tcd group was similar to the cd34sel group (45% vs 34%, p = ns). in the tcd patients, neither use of busulfan vs treosulfan, nor kir genotype, nor donor sex had noticeable impact on sct result and survival. patients transplanted after tcd due to non-malignant disease had higher survival probability, than those with malignancies (69% vs 28%, p = 0.04). the trm in tcd patients was reduced in comparison to cd34sel (24 vs 58%, p = 0.003). the trm after tcd resulted mostly from severe viral infections in tcd-cd3 patients. in 4/34 tcd patients spontaneous acute, skin (stage 2) gvhd was diagnosed and successfully treated. two patients received unmanipulated donor lymphocyte infusions (dli) and developed severe acute steroid-resistant (grade 4) gvhd, in one of them with fatal outcome. tcd methods are superior to cd34sel due to significant reduction in treatment related mortality. haploidentical sct after tcd can result in durable engraftment, but warrants intensive post-transplant monitoring for infectious complications and cautious approach to dli therapy. disclosure of conflict of interest: none. median of 15 days for neutrophils in both groups, 17 for platelets (23 in ptcy,12 in αβ+cd3+/cd19+depleted, p 0.005). donor chimerism was complete in 22 patients (84.6%). in αβ +cd3+/cd19+depleted group, 4 patient rejected (33.3%:1 primary and 3 secondary reject, 22, 28, 55 and 195 days after haplo, respectively) and were rescued with a second transplant. seven patients (50%) developed acute (a-) gvhd in ptcy group (grade 1-2 in 4; grade 3-4 in 3), compared to one (8.3%: grade 4) in αβ+cd19+depleted group (p 0.02). among patients at risk, 3 out of 9 in ptcy group developed chronic (c-) gvhd (33.3%:1 score-3, 1 overlap, 1 score-1), compared to 0/9 patients in αβ+cd3+/cd19+depleted group (p 0.08). the cumulative risk of cmv-reactivation was 72% and 58% in ptcy and αβ+cd3+/cd19+depleted groups, respectively (p 0.63). t-cell reconstitution was significantly different in the two groups,with a median absolute number of cd16+56 +cd3-and γδ+cd3+ higher in αβ+cd3+/cd19+depleted group on day +120 (p 0.03) and a median number of cd3 +cd8+ higher in ptcy group on day+180 (p 0.04). length of hospitalization was shorter in the αβ+cd3+/cd19+depleted group, with a median time from haplo to discharge of 23 days compared to 31 days in the ptcy group (p 0.003). some children have not donor and an urgent need to proceed to transplantation because of disease status. we reviewed the role of haploidentical transplantation in children and report our single center experience. ten children were transplanted from haploidentical family members donors (median age:12.5 years). we performed alfa beta t cell depleted transplantation in three patients and unmanipulated bone marrow transplantation with posttransplant cyclophosphamide in seven patients. the diagnosis were eight high risk leukemias (three all and five aml) and two severe aplastic anemia. patients were myeloablative conditioned with cyclophosphamide, fludarabine and total body irradiation in aplastic anemia received alfa beta t cell depleted grafts with a median cd34 cell dose of 2.9 × 10 6 /kg (range:2.6-3.2) and busulphan, cyclophosphamide in high risk leukemias received unmanipulated bone marrow grafts with posttransplant cyclophosphamide in 3rd and 5th day of posttransplant with a median cd34 cell dose of 7.4 × 10 6 /kg (range:2.38-16.1). median follow up of our patients 10 months. six of 10 patients are alive and in disease free follow up. four patients were relapsed and dead median 5.5 months of transplantation. the rate of relapse was 50 % for leukemia patients in remission and 50% for patients with active disease. myeloablative conditioning regimen followed by haploidentical bone marrow transplantation with posttransplant cyclophosphamide may be an option in high risk leukemia patients need urgent transplantation because of desease status who have not donor. table 1 . all patients received hd-cy 50 mg/kg on d+3 and d +4. cyclosporine a 3 mg/kg/d i.v., then 6 mg/kg/d p.o. adjusting for blood levels 200-400 ng/ml and mycophenolate mofetil 15 mg/kg 2 times daily po were started on d+5. mmf was discontinuated on d+35, csa-after d+100. all pts received anti-microbial prophylaxis for bacteria, fungal, herpes infection and pneumocystis according to institutional practices. analysis for donor chimerism and mrd were performed at d+30, +60, +100, +180. pts, donors and stem-cell harvest characteristics are described in table 1 . 4 pts had high risk hematological malignancies, and 1 relapsed after auto-sct neuroblastoma (hr-nb). 1 pt was transplanted in 1st cr (aml m7) and others in 2nd cr. 3 pts had full engraftment (neutrophil engraftment at 17,18 and 22 days). 1 pt (hr-nb) was concerned as a primary failure for achieving neutrophil and platelet engraftment by d+30, despite of complete donor chimerism in bone marrow. he was transplanted additionally with the same donor at d+49 after 1st transplant. 1 pt died before engraftment at d+20 (fulminant ps. aeruginosa-sepsis). 2 pts remain alive in cr (2ndcr-aml and 1st cr-m7 aml) with follow-up of 375 and 164 days (05/12/2016) without cgvhd with complete donor chimerism. 2 pts relapsed after d+100 ( were transplanted in 2nd cr-flt3 aml and 2nd cr-nb) and died. 1 pt died because of infectious complication at d+20 (transplanted in 3d cr-all). 4/5 pts had grade 2 acute gvhd. hla-haploidentical hsct with post-transplant t-cell in vivo repletion grafts by using hd-cy is feasible and effective in children with hr-haematological malignancies. [p463] who were match unrelated donor. thalassemic reconstitution occurred in three patients. acute graft-versus-host disease (gvhd) of grade ii-iv occurred in 17 % and chronic gvhd in 12%. acute and chronic gvhd were seen more frequently in patients with class 2-3 compared to class 1. mortality rate was also higher in these groups. seven patients died. one patient died on post-transplant day 26 due to intracranial bleeding. the other 6 patients with chronic gvhd died between 182 and 257 days, on average 219 days post-transplant. these data suggest that allogeneic bmt remains an important treatment option for children with beta-thalassaemia major, particularly when compliance with iron chelation is poor. the society to support children suffering from cancer, also known as mahak, was set up in 1991 as a non-governmental and non-profit organization. in the past two decades, the organization has attracted a vast public support and fulfilled a great part of its mission which is to support children with cancer, reduce the child mortality rate and create an appropriate environment that empowers families who have children with cancer. pediatric stem cell transplant also is used to treat many types of conditions affecting children and adolescent, including cancer and certain hematologic, immune reconstitution inflammatory syndrome (iris) is a clinical condition emerging after immune recovery of an immunocompromised status, mostly after the initiation antiretroviral therapy (art) in human immunodeficiency virus (hiv) infected patients, but also in several other settings, such as the recovery from the severe combined immunodeficiency (scid) status after hematopoietic stem cell transplantation (hsct). herein, we report a patient transplanted for scid who developed iris for two times, namely shortly after transplantation and after donor lymphocyte infusion (dli) ( table 1 ) in our patient, t cells passing from the donor probably contributed to the immediate post-transplant increase in the size of granulomas. this inflammatory response waned after the institution of immunosuppressive and methylprednisolone therapy. however, immunosuppressives were stopped due to lowered chimerism at follow-up, and the inflammatory response re-appeared after administering stem cell support containing a large amount of t cell from the donor for dli purpose. although the mechanism by which dli results in clinical responses is unclear, it is presumed to be a t cellmediated process. several studies have been performed to strengthen our understanding of the immunopathogenesis of iris. while some of those studies put forth t cell-associated causes, others implicated cytokines and non-t cells. the reaction that developed in our patient is suggestive of t cellassociated causes. immune reconstitution inflammatory syndrome remains a poorly understood entity. the dli procedure in our case provides a unique clue supporting a t cell mediated process. pediatric transplant teams need to be s364 aware of the previous iris phenomenon of bcg-adenitis while making the decision of dlis. [p469] disclosure of conflict of interest: none. pediatric patients treated with a hematopoietic stem cell transplantation (hsct) often suffer from late side effects caused by the treatment. the aim of this study is to investigate the late effects of a hsct on dental development. in addition, patients and parents awareness on this topic was investigated. 42 young adults treated and followed at the ghent university hospital who were under the age of 12 y at the time of hsct were examined clinically and radiographically (planmeca promax 2d). transplants (11 autologous/31 allogeneic) were done for malignant disease in 34 pts. eight patients received a hsct for a non-malignant disease. twelve patients underwent a conditioning regimen with total body irradiation (tbi), 21 patients with busulfan and 9 patients with other chemotherapeutic agents. 16 patients were o3 y, 9 patients were 3-6 years and 17 patients were 4 6 years at hsct. every patient was evaluated on dental agenesis, microdontia and rootcrown ratio. patients and their parents were asked about their knowledge and interest for dental screening at the follow-up clinic using a questionnaire. overall, the prevalence of agenesis and microdontia of one or more dental elements is respectively 51.3% and 46.2% in our study population. 76.3% of patients have a strongly aberrant root-crown ratio of at least one element. patients treated o3 years of age show significantly more microdontia (76.9%; po0.001) as well as agenesis (92.3%; p o0.001) compared to patients treated at an older age. the first premolar of the mandible is the most vulnerable element for agenesis as well as for microdontia. more microdontia is found in patients treated with a busulfan conditioning regimen compared to the other conditioning regimens (68.4% versus 25%). patients older than 6 years, treated with busulfan have statististically more microdontia compared to patients 46 y treated with tbi conditioning regimen (p = 0.044). there was no difference of the conditioning regimens on agenesis nor on root-crown ratio. almost all patients/parents find it important to receive information about the dental late effects of a hsct and are interested in dental screening at the follow-up clinic. treatment with hsct has an explicit negative impact on dental development. the degree of this effect depends on age at hsct and used conditioningregimen. dental follow-up of these patients is essential and should be incorporated in the follow-up program. disclosure of conflict of interest: none. importance of body composition in the outcome of hematopoietic stem cell transplantation in elderly patients l koch 1 , n hamerschlak 1 , r garcia 1 , c prado 1 , c silva 1 and a pereira 1 hospital israelita albert einstein the loss of muscular mass is a well recognized cause of the decline in muscle strength and functionality that accompany the aging process. in 1989, irwin rosenberg proposed the term 'sarcopenia' to describe the decline in muscle mass associated with aging. changes in body composition after hematopoietic stem cell transplantation (hsct) have been the subject of previous studies. immunosuppressive therapy and corticosteroids are known to alter skeletal muscle metabolism. infections and graft-versus-host disease (gvhd) that can occur after hsct may also affect body weight and composition. therefore, both the treatment and complications after hsct exert large negative effects on lean muscle mass, especially in elderly patients. patients with hematologic malignancies are usually well nourished before undergoing hsct. objective: the aim of this study is to determine in an elderly population whether parameters of body composition could be correlated to outcomes after hsct. we performed a retrospective longitudinal study through review of medical records of 48 patients ⩾ 60 years old undergoing hsct at hospital israelita albert s365 einstein, from 2013 to 2015, that were subject to tomography scans (cts) in a period ranging from 60 days before and 15 days after hsct. table 1 . there were no differences between groups with respect to age, gender, diagnosis, stage of disease, and source of stem cells. in ly patients, the quantity of peripheral cd34+ cell dose (×10 6 /kg) infused was different between groups (group ly-ct the incidence of nf was significantly higher in group mm-g (19 (59.4%) vs 19 (37.3%); p = 0.049). no differences were observed in the incidence and severity of mucositis, first day and duration of fever, documented bacterial infections or readmission rate between mm patients groups. this study suggests that in at home asct, the use of piperacillintazobactam prophylaxis significantly reduces the incidence of neutropenic fever and hospital readmission in patients with ly, and also that no administration of g-csf in mm patients reduces significantly the incidence of neutropenic fever. disclosure of conflict of interest: none. [p472] allogeneic stem cell transplantation (sct) has been recognized as a curative treatment for patients with wiskott-aldrich syndrome (was). in sct for was, myeloaberative conditioning (mac) has been indicated to avoid a mixed chimera. however, risk factors for a mixed chimera in patients with was who have received sct have not been evaluated. here, we analyzed the outcomes of sct and risk factors for a mixed chimera in 108 patients with was who underwent sct in japan since 1985. we reviewed medical records of 108 consecutive was patients who received sct since january 1985 who were registered with the japan society for hematopoietic cell transplantation. the age of the patients at transplantation ranged from 3 months to 23 years, and the mean age was 3.81 years. the origin of the stem cells was related bone marrow (bm) or peripheral blood stem cells (pbsc), unrelated bm or pbsc, and unrelated cord blood (cb) for 36, 41 and 27 patients, respectively. a preparative conditioning regimen consisting of mac was provided to 76 patients, and reduced-intensity conditioning was provided to 30 patients. fifty-one patients received prophylaxis against graft-versus-host disease (gvhd) with cyclosporine in combination with methotrexate (mtx) or a steroid, and 51 patients received tacrolimus (tac) with mtx or a steroid. chimerism analysis had been performed in 91 patients. neutrophil engraftment was achieved in 91 patients (82.7%). the engraftment rate was significantly higher in patients who received tac for gvhd prophylaxis, (p = 0.0001) overall survival rate was significantly higher in patients with complete chimerism than in patients with mixed chimerism (88.2 ± 6.1% and 66.7 ± 9.9%, respectively, p = 0.003), though there was no significant difference in stem cell sources. using multivariate analysis, the rate of complete chimerism in patients who received mac including cyclophosphamide (cy) at more than 200 mg/kg was significantly higher (p = 0.02) than the other conditioning. not only patients with mixed chimerism but also patients with complete chimerism were complicated with auto-immune diseases. in this study, achievement of complete chimerism after sct was important for survival in patients with was. we found that patients who underwent mac including cy at more than 200 mg/kg had a higher rate of complete chimerism. we also found a higher neutrophil engraftment rate in patients who received tac for gvhd prophylaxis. thus, mac including cy at more than 200 mg/kg and tac for gvhd prophylaxis are optimal conditions of sct for patients with was. disclosure of conflict of interest: none. adenosine deaminase (ada) deficiency is an inherited autosomal recessive immunodeficiency which represents about 10-15% of scid. since 1992 we diagnosed 29 patients affected by ada-scid: 10 underwent hematopoietic stem cell transplantation (hsct), 10 were treated with replacement therapy with peg-ada and 4 received gene therapy; 5 patients died before or after treatment. maternal t lymphocyte engraftment is frequently detected in scid patients, but this is never been found in ada deficient patients. a 3-months-old italian girl, from non-consanguineous parents, presented to our hospital with a history of frequent bronchiolitis associated with dermatitis, mycosis, hypogammaglobulinaemia, marked lymphopenia (t cells cd3, 171/mmc; cd3/cd4, 158/mmc; cd3/ cd8, 8/mmc, b cells 15.2/mmc, and nk cells, 110/mmc) and in vitro absence of proliferative response to pha. level of immunoglobulins was almost normal (igg 439 mg/dl, iga 87 mg/dl, igm 57 mg/dl). high levels of toxic metabolites were found: axp, 1.573 micromol/ml rbc; daxp, 0.629 micromol/ml rbc; %daxp, 28.5. ada activity in rbc lysates was abnormally high for scid-ada (0.54 u/g hb). molecular analysis confirmed diagnosis: the sequencing of exon 10 revealed two mutations: a missense mutation previously reported called p.ser291leu (c. 872c4t) and a new missense mutation defined p. leu298pro (c.893t4c). t-cells str analysis of patient showed 54.1% maternal t lymphocytes engraftment never reported before in ada-scid patients. the girl was transferred to the isolated bmt unit and the respiratory symptoms progressively improvement. replacement therapy with peg-ada was started immediately at a dose of 30 u/kg/twice per week. ig therapy was started at a dose of 200 mg/kg every two weeks. after three months of treatment patient showed an increase in t cells count (cd3, 411/mmc), and a decrease of toxic metabolites: axp, 1.652 micromol/ml rbc; daxp, 0.011 micromol/ml rbc; %daxp, 0.7 maternal t-cell engraftment persists, despite a good response to the peg-ada therapy. the last examination before hsct reveals maternal t-cell engraftment of 9.2%. patient underwent hsct from mud hlaidentical donor after a myelo-ablative reduced intensity conditioning regimen protocol d ebmt/esid guidelines. the number of infused cd34+ cells was 14.29 × 10 6 /kg and 69.47 × 10 6 cd3/kg. she is actually at day+108 post hsct, is doing well and shows 100% engraftment of donor cells. disclosure of conflict of interest: none. graft versus host disease (gvhd) is a frequent complication in patients undergoing haematopoietic stem cell transplantation. while the exact pathophysiology of gvhd is not known, the gut microbiome has been implicated in its development since it was shown that total gut decontamination (tgd) decreases the incidence of gvhd. with this study we aim to get insight into the diversity of the gut microbiota before, during and after total gut decontamination in comparison with selective gut decontamination (sgd). secondly, we want to identify changes in microbiota composition that relate to the occurrence of graft-versus-host disease. for this prospective cohort study we recruited 22 children (o 18y) that were eligible for a stem cell transplantation at the leiden university medical center between january and december 2015. of these, 64% (n = 14) received tgd (consisting of piperacillin/ tazobactam and oral amphotericin b), whereas the other 36% (n = 8) received selective gut decontamination with polymyxin /neomycin and oral amphotericin b. in total, 129 fecal samples were collected, weekly during admission for the stem cell transplantation and monthly thereafter up to 6 months after transplantation. also samples were collected from family stem cell donors as healthy controls. samples were processed within 24 hours and stored in the -80 freezer, after which 16s v4 amplicon sequencing (illumina hiseq, rapid mode, 250 bp read length) was applied. data analysis (taxonomy and shannon diversity) was primarily done using qiime (ref). compared to microbiota diversity in stem cell donors (mean shannon index (si) 3.43), we observed an overall lower mean si during tgd (1.90) and slightly higher mean si during sgd (2.43). microbiota diversity months after sgd (2.45) was similar to diversity during sgd (2.43), while diversity months after tgd (2.63) was higher than during tgd (1.90). further analysis of repopulation dynamics demonstrated no differences in repopulation duration after both decontamination strategies. however, we did observe differences in the type of bacteria that repopulated, with bacteroidales being more prominent in sgd and lactobacillales more prominent in tgd patients. actinomycetales (genus rothia) was exclusively present in tgd patients during decontamination. also, the clostridiales (blautia, lachnospiraceae and peptostreptococcaceae) were bacteria that appeared after the decontamination period. four patients (18%) in this cohort developed gvhd grade 1 or more. in these patients we did observe individual compositional changes of the gut microbiota at the time of ghvd diagnosis, e.g very low diversity or dominance of enterobacteriales. considerable microbiota diversity is observed in patients that received tgd. different repopulation dynamics were observed between tgd and sgd. no common pattern was found in the gvhd cases. disclosure of conflict of interest: none. minimal residual disease (mrd) pre-and post-hct for children with aml is highly predictive of event-free survival: a pediatric blood and marrow transplant consortium study d jacobsohn 1 johns hopkins all children's hospital, 16 children's hospital los angeles, usc keck school of medicine multicenter data regarding the significance of mrd in children with aml pre-and early post-hct are lacking. we hypothesized that pre-and post-hct mrd assessments using wt1 pcr combined with multi-dimensional flow cytometry (mdf) would be predictive of disease relapse and event-free survival (efs) at 2-yrs post-hct. subjects were o21 yrs with aml in morphologic cr undergoing ma allogeneic hct. stem cell sources included bm, pbsc, or cb. bm and pb samples were collected at 3 time points: baseline ( o3 weeks prior to preparative regimen); day+42 (±14 days); and day+100 (±20 days). bm samples were analyzed for both wt1 expression and mdf mrd (single reference lab using a 'difference from normal' approach without access to diagnostic phenotype); pb samples were analyzed for wt1 only. mdf detection limit was 0.02%; however, we required that 2 independent analysts certify that the abnormal population was aml. in addition, sorted mrd+ cells were tested for chimerism. wt1 positivity was defined as ⩾ 1300 copies for bm and ⩾ 200 copies for pb. results were not available to the treating clinician. 150 subjects were enrolled at 34 centers in us and canada. 20 enrolled subjects did not undergo hct and 6 were excluded for progression prior to hct or other ineligibility. in 124 eligible subjects, 2-yr efs and os were 52% and 61%, respectively. the 2-yr ci of relapse and trm were 36% and 13%, respectively. mdf identified 7 subjects pre-hct having 0.2-14% residual disease. the 2-yr relapse rate in subjects with +mrd by mdf pre-hct was 100% vs 32% (23-40%) in those who were negative. 2-yr dfs and os were 0% and 29% (4-65%) for positive mdf pre-hct, and 54% (45-63%) and 62% (53-71%) for negative mdf. pre-hct mdf sensitivity for 2-yr dfs was 10%; specificity was 100%. mdf mrd at days 42 and 100 were similarly predictive of outcome. sorted mrd+ cells from 19 post-hct samples were all noted to be of recipient origin. pb wt1 had no correlation with dfs or relapse; bm wt1 at day+100 correlated with 2-yr os (79% (68-88%) low/neg vs 57% (39-75%) high). other wt1 cutoffs studied demonstrated no correlation with outcomes. figure 1 : relapse probability by flow cytometry mrd at 3 time points. mdf mrd pre-hct and at days +42 and +100 was significantly associated with lower efs and os in children with aml undergoing hct. mdf is specific but not sensitive, as many negative mdf patients relapsed. our goal was to define a reproducible assay that did not depend on having the initial aml profile. this would facilitate multi-institutional studies aimed at decreasing relapse. given that constraint, we were able to detect clear mdf mrd in a small percentage of patients that was highly predictive and can be used in trials. wt1 level was not predictive in this multi-institutional trial. the sensitivity of flow was significantly affected by not having the initial flow available. future attempts to improve sensitivity should include initial flow and/or test higher channel flow or molecular pcr techniques. in addition, we confirmed that mrd + cells obtained by cell sorting post-hct were of recipient origin. future testing of 'suspicious' sorted cells by fish, molecular, or comparative genomic hybridization could possibly increase mfd sensitivity. novel cellular or targeted therapies should be tested in clinical trials to improve outcomes in patients with mfd mrd noted either pre-or post-hct. [p480] disclosure of conflict of interest: none. novel mutations were identified with ngs and low intensity of conditional regimen succeeded in children with fanconi anemia who received allo-hsct s hu 1 , h hou, j lu, p xiao, x bian, h liu, y hu, j ling, l li, l kong, z zhai and y yao 1 children's hospital of soochow university to explore the possiblity of applying next-generation sequencing (ngs) to diagnose the disease of fanconi anemia (fa) and evaluate the efficiency and safety of low intensity conditional regimen on children with fa receiving allogenic hematopoietic stem cells transplantation (allo-hsct). five patients initially suspected as severe aplastic anemia were diagnosed as fa by the method of next-generation sequencing (ngs)-based genetic diagnosis panel. one patient received hla-identical sibling donor hematopoietic stem cell transplantation (mrd), three patients underwent unrelated donor matched (ud) hsct, and one patient received unrelated cord blood transplantation (ucb). the conditional regimen consisted of either 300 cgy tbi or 3.2-3.6 mg/kg of busulfan with 20-40 mg/kg of cyclophosphamide. meanwhile, atg at 10 mg/kg and fludarabin at 140-180 mg/m 2 were included as well. cyclosporin or tacrolimus as well as mycophenolate mofetil (mmf) were used for the prophylaxis of graft versus host disease (gvhd). engraftment of neutrophil and platelet and complications followed transplantation such as infection, gvhd, and hemorrhagic cystitis (hc) were observed. of 5 cases diagnosed as fa by ngs, only 1 case showed the abnormality of chromosome fragility test which has been regarded as golden criteria in the diagnosis of fa. meanwhile, we found 5 novel mutations in 3 cases of fa which enriched chinese national database with data of rare diseases by ngs. the counts of mononuclear cells (mnc) were (3.87-18.57) × 10 8 /kg for non-ucb and 9.83 × 10 7 /kg for ucb. the counts of cd34+ were (4.01-9.57) × 10 6 /kg for non-ucb and 2.56 × 10 5 /kg for ucb. all 5 cases succeeded in allo-hsct with the low intensity of conditional regimen. the median time for neutrophils engraftment was 11 days (range 9~15 days), median time to platelets (plt) engraftment was 14 days (range 8~28days). one case occurred with grade i of agvhd, 2 cases with hemorrhagic cystitis. after transplantation, all patients were monitored the copies of ebv-dna and cmv-dna of whole blood, and five case with ebv positive and 3 cases with cmv positive. no patient suffered of ebv or cmv disease. the hepatic veno-occlusive disease (vod) and hc were observed in 5 fa patients after transplantation. ngs showed much more specific and facilitated for the diagnosis of fa. low intensity of conditional regimen is efficient and safe which should be recommended for the treatment of fa patients. disclosure of conflict of interest: none. outcome of alternate donor stem cell transplantation in children: an indian experience sp yadav 1,2 , n rastogi 1,2 , d thakkar 1,2 , s kohli 1,2 , s nivargi 1,2 , r misra 1 and s katewa 2 1 in india due to lack of donor registries and cord blood banks very few alternate donor stem cell transplants (sct) are performed. haploidentical sct has become feasible with availability of post-transplant cyclophosphamide (ptcy) technique. here we present our experience of setting up alternate donor program for sct for children in india and report the outcomes of the same. we collected data retrospectively of all children who underwent alternate donor sct during jan 2013 to dec 2016 in two centres. a total of 47 sct were performed for 43 children; median age 6 years (1-18 years) and 37 were boys and 6 girls. of these, 41 underwent haploidentical (35 ptcy and 6 tcr alpha-beta/cd19 depleted), 4 matched unrelated donors (mud) and 2 unrelated cord blood (ucb) sct. the diagnosis was: primary immunodeficiency-10, thalassemia major-14, sickle cell disease-3, inherited bone marrow failure-4, acquired aplastic anemia-3, acute lymphoblastic leukemia-3, acute myeloid leukemia-4, neuroblastoma-4, ewings sarcoma-1 & leukodystrophy-1. the conditioning was with fludarabine, cyclophosphamide and total body irradiation backbone in 36 children (thiotepa added in 24), fludarabine and treosulfan in 5, fludarabine and busulfan in 2, busulfan and cyclophosphamide in 4. serotherapy was part of conditioning, rabbit anti-thymoglobulin 4.5 mg/kg in 38 and campath 1 mg/kg in 9. graft vs host disease (gvhd) prophylaxis was ptcy along with tacrolimus and mycophenolate mofetil in 37 patients (35 haploidentical, 1 mud & 1 ucb) and ex-vivo tcr alpha-beta depletion in 6 and cyclosporine and methotrexate in 4. all were transplanted after a signed informed consent. a median of 8 million of cd34cells/kg was infused (range 5-24 million/kg).graft source was peripheral blood in 39 and bone marrow in 6 and ucb in 2. five children died before engraftment. the remaining 42 had neutrophil engraftment by median of 14 days (range 8-35) and platelet engraftment by median of 14 days (range 9-48). chimerism at day+100 was available in 34 cases; 31 of them had full donor hematopoiesis. one had mixed chimerism and 3 fully recipient. four children underwent a second haploidentical sct after rejection of which 2 are alive and disease free. the median follow-up of remaining patients is 9 months (range 1-40); the cumulative incidence of graft versus host disease (gvhd) acute and chronic extensive was 26% and 20% respectively. grade-iii-iv acute gvhd was seen in 3 patients. a total of 15 patients have died (sepsis-4, stroke-1, gvhd-3, vod-3, encephalitis-3 and progressive disease-1). among encephalitis deaths, one child had undergone ucb with ptcy and another tcr alpha-beta depleted second sct.; both had bk virus in the csf.there were 11/41 deaths in haploidentical (ptcy-10/35 & tcr alpha-beta-1/6), 3/4 in mud and 1/2 in ucb sct. overall survival is 68% and disease free survival is 66% at last follow up. alternate donor sct is an acceptable curative option for children lacking a matched sibling donor. haploidentical donor sct is more feasible in the setting of lack of donor registries having indian ethnicity donor. disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct) from an unrelated donor (ud) is largely used for pediatric patients with all in second complete remission (cr) lacking an hlaidentical sibling. in this study, we retrospectively analyzed outcome of patients (pts) given ud-hsct in centers affiliated to the associazione italiana di ematologia ed oncologia pediatrica (aieop) network between 2000 and 2013. three hundred fifty-six pts with all in second cr experiencing either bone marrow (bm), isolated extramedullary or combined relapse were included in the study; 139 were males and 217 females, median age at hsct being 4.8 years (range 0.5-19). bm, peripheral blood (pb) and cord blood (cb) were the stem cell source in 64%, 16% and 20% pts, respectively. all children received a myeloablative conditioning regimen, either tbi-(293 pts) or chemotherapy-based (63 pts). as gvhd prophylaxis, the combination of cyclosporine a, short-term mtx and atg was employed in most pts. according to the berlin-frankfurt-munster (bfm) classification of first leukemia recurrence, 59% and 42% of pts were assigned to the s1+s2 and s3 +s4 groups, respectively. level of pre-hsct minimal residual disease (mrd), measured within 30 days before hsct through flow cytometry (fcm) in the laboratory of padova, is available in 37 children; more data will be presented during the s372 meeting. with a medium follow-up of 6.5 years (range 0.5-15), the overall survival (os) was 52%, while the event-free survival (efs) was 50%. the cumulative incidence of transplant-related mortality (trm) and leukemia recurrence were 24% and 25%, respectively. the efs probability for children transplanted in the time period [2000] [2001] [2002] [2003] [2004] [2005] [2006] [2007] [2008] [2009] and 2010-2013 was 45%, 56% and 52%, respectively (p = ns). patients who received a tbi-based conditioning regimen had a significantly better outcome in comparison to children who received chemotherapy-based treatment, efs being 53% and 36%, respectively (p = 0.01). efs of pts belonging to s1+s2 and s3 +s4 groups was 66% and 35% respectively (p = 0.0001). the difference in efs is largely explained by an increased incidence of leukemia recurrence in s3+s4 (34%) compared to s1+s2 pts (23%) (p = 0.0002). efs of pts who experienced grade ii acute gvhd was 68%, while that of pts with either absent/grade i acute gvhd or grade iii-iv acute gvhd was 52% and 11%, respectively (p = 0.0001). pts with limited chronic gvhd had a better efs as compared to those with either extensive or absent chronic gvhd (77%, 35% and 61%, respectively; p = 0.039). the choice of stem cell source (bm, pbsc, cb) did not influence the probability of efs, which was 56%, 46%, 40% respectively (p = ns). importantly, among pts with evaluable mrd before hsct (n 37), the group with detectable levels 0.001% (n 9), respectively 54% and 17% (p = 0.038). conclusions. outcome of children with 2nd cr all who underwent transplant from an ud is significantly influenced by the presence of tbi in the conditioning regimen, limited severity of acute and chronic gvhd and bfm classification at time of 1st relapse. notably, mrd level before transplant, namely with a cut-off of 0.001%, influences efs. disclosure of conflict of interest: none. the median mononuclear cell dose was 5.5 × 10 8 /kg. the median time to reach absolute neutrophil count 40.5 × 10 9 /l was 13 days, and the median time to platelet count 420 × 10 9 was 16 days. grade 2 and grade 3 mucositis was seen in 61% of our patients. transplant-related mortality at 100 days not occurred. only three patients relapsed 15, 18 and 30 months after transplant (mean 21.5 m.). with a median follow-up of 39 months (4-48 months) after transplant the event free survival were 84%. only one patient had death, two years after transplantation. no significant different between cbv group vs ceam group in engraftment day. high-dose therapy with stem cell rescue can lead to durable remissions in children and adolescents with advanced hd. future investigations should focus on strategies designed to decrease relapse after auto-transplantation, particularly in patients at high risk for relapse. our analysis suggests that these regimens (ceam, cbv) are feasible in pediatric patients with acceptable engraftment and toxicity. pbsc collection may be difficult in small children owing to the large volume apheresis compared to the child's weight. various problems, such as metabolic or haemodynamic disorders may be were seen. peripheral stem cell harvest can be performed in lowweight children under safe and effective conditions even when systematic priming by blood is avoided. processing with increase of blood volume may to increase in the yield by recruiting progenitor cells. disclosure of conflict of interest: none. outcomes of children with hemophagocytic lymphohistiocytosis given allogeneic hematopoietic stem cell transplantation in italy allogeneic hematopoietic stem cell transplantation (hsct) is the only curative treatment for patients with familial hemophagocytic lymphohistiocytiosis (hlh) or relapsed/ refractory hlh. we analyzed outcomes of a cohort of 109 patients (65 m, 44 f) with hlh given hsct between 2000 and 2014. median age at hsct was 2 years (range 0.4-20). genetic testing was performed for 94/109 patients (86%). mutation of prf1 was found in 31 patients (32%), of unc13d in 32 (33%), of stxbp2 in 2 (2%), of rab27a in 6 (6%), of sh2d1a in 5 (5%), of birc4 in 2 (2%) and of lyst in 1 patient (1%). no known gene abnormality was found in 15 patients who had recurrent/ refractory hlh. central nervous system (cns) involvement at diagnosis was recorded for 79 patients (72%) and was present in 30 of them (38%). the primary endpoint was event-free survival (efs), defined as the probability of being alive and in continuous complete remission (cr) at last follow-up. in order to determine efs, death from any cause, relapse or graft failure were considered events. ninety-five patients received one transplant, while 14 received more than one hsct, because of rejection in 8 patients or disease relapse in 6 (preceded by rejection in 1 case): 2 hsct were performed in 12 cases, while 3 and 4 hsct were performed in 1 case each. donor for first transplant was an hla-matched sibling for 25 patients (23%), an unrelated donor for 73 patients (67%) and a partially matched family donor for 11 patients (10%). conditioning regimen was busulfan-based for 61 patients (56%), treosulfan-based for 21 patients (20%) and fludarabine-based for 26 patients (24%). the 5-year probability of overall and efs were 71% and 60% respectively (fig. 1) . twenty-six (24%) patients died due to transplant-related causes, while 14 (13%) and 10 (9%) patients experienced graft rejection and/or relapse, respectively (see also fig. 1 ). twelve out of 14 children (86%) given a 2nd hsct after graft failure/relapse are alive and disease-free. active disease at hsct was not statistically associated with adverse outcomes, while patients had a trend for a worse outcome if the interval between diagnosis and hsct was 46 months. patients transplanted from partiallymatched family donors (pmfd) had a significantly worse efs (9%) than recipients of a matched family donor transplant (73%) or a matched unrelated donor allograft (63%, po 0.001). the main reason for the dismal efs of pmfd recipients was graft rejection, which, however, was largely rescued by a 2nd hsct. patients given peripheral blood stem cell transplantation had a lower efs probability (39%) as compared to bone marrow (60%) or cord blood recipients (76%, p = 0.0185). children given hsct o o/u46 months from diagnosis had a better efs as compared to those transplanted 46 months from diagnosis (69% vs 50%, p = 0.069). in multivariate analysis, only the use of a pmfd predicted a worse efs probability (relative risk:12.26, p = 0.0008). these data suggest that in patients with hlh allogeneic hsct is able to cure 2/3 of patients. haploidentical hsct in patients with hlh is currently associated with unsatisfactory rate of engraftment, new approaches being needed to ameliorate this outcome. active disease does not preclude the chance of benefiting from transplantation, which should be ideally performed within 6 months from diagnosis. [p485] defibrotide shows efficacy in the prevention of sinusoidal obstruction syndrome (sos) after allogeneic hematopoietic stem cell transplantation: a retrospective study on 237 patients. disclosure of conflict of interest: none. standard gvhd prophylaxis regimens impair the graft-versustumor (gvt) effect, delay immune reconstitution and are associated with high rate of infections. high-dose posttransplantation cyclophosphamide (ptcy) targets alloreactive donor t cells proliferating early after bmt, promotes regulatory t cell and permits rapid immune reconstitution. in this pilot trial we evaluate the safety and effects of ptcy in unmanipulated haploidentical and matched unrelated transplantation (mud) in pediatric patients with all. fifteen pediatric patients with high risk all underwent unmanipulated allogeneic bone marrow (bm) (n = 11) or peripheral blood stem cell (pbsc) (n = 4) transplantation followed by ptcy between april 2014 and march 2016 with a median follow-up of 24 months (7-27). eight patients were transplanted from haploidentical donors and 7 from mud. the median age was 9.5 years (range 1.9-17) and were in complete remission (cr) at the moment of bmt. in 2 patients this was a second bmt. all pts. received myeloablative conditioning regimen (treosulfan-based n = 14, tbi based n = 1) and ptcy on day +3, +4, posttransplant prophylaxis consisted of tacrolimus from day +5 (n = 5), tacrolimus/mmf (n = 8), atg (rabbit, thymoglobuline) at 5 mg/kg without other posttransplant prophylaxis(n = 2, both from mud). primary engraftment was achieved in 100% of pts., the median time to neutrophil recovery was 20 days and to platelet recovery was 22 (7-69) days. all pts. had full donor chimerism on day +30. causes of death included viral infections (n = 1); gvhd and viral infection (n = 1). cumulative incidence (ci) of acute gvhd grade ⩾ ii was 33% (95% ci: 16-68), grade iii-iv-13.3% (95% ci: 3.7-48) and chronic gvhd-21.2% (95% ci: 7.7-58.4). two-year event-free survival (efs) and overall survival (os) were 86.7% (95% ci: 69.5-100) and were equal. median time of follow-up for survivors is 2 years (range 0.7-2.3). we demonstrate that unmanipulated hsct and posttransplantation cyclophosphamide allows for high rate of engraftment with acceptable transplant-related mortality in pediatric patients with all. all major outcomes were equivalent between transplantation from unrelated and haploidentical donor. gvhd prophylaxis including ptcy was effective. event-free survival was high despite chemotherapybased conditioning in most patients. disclosure of conflict of interest: none. serotherapy with atg is frequently used in allogeneic hsct to prevent gvhd and rejection. however, the choice of the two most frequently used rabbit atg brands depends on country, disease protocol, national recommendations and/or physician's preference. atg-genzyme (atg-g, thymoglobulin) is prepared by immunizing rabbits with human thymocytes, whereas rabbits are immunized with a jurkat cell line for production of atg-fresenius (atg-f, recently named as antihuman t-lymphocyte immunoglobulin atlg, grafalon, noveii biotech). the recommended dose of both brands differs a factor 4-5. we have previously reported the pharmacokinetics/ pharmacodynamics (pkpd) of atg-g in a large cohort of pediatric hsct recipients and concluded that the clearance of the active component of atg, which is the portion of atg binding to lymphocytes, had a major impact on immune recovery post-hsct, while total atg did not. 1 both atg brands have frequently been compared according to disease outcome, without detailed analysis of composition and clearance of the active components. in the present study, we compared clearance of the active component and immune recovery after atg-g and atg-f, respectively. the serum concentrations of total and active atg were measured longitudinally after hsct in 56 children (40 atg-g, 16 atg-f), transplanted with bm or pbsc of unrelated donors for all or aml between january 2010 and june 2016 in leiden (n = 36) or copenhagen (n = 20). atg-g treated patients received a total dose of 6-10 mg/kg and atg-f was given at a total dose of 30-60 mg/kg in both cohorts administration was divided over 3-4 days. serum samples (pre-conditioning, day of hsct, +1; +2; +3; +4 and +6 weeks and +2 and +3 months after hsct) were analyzed by elisa for total atg and by quantitative flow cytometry on hut78 cells for active (lymphocyte binding) atg. lymphocyte (sub-)populations were analyzed at +1, +2 and +3 months post-hsct by flow cytometry. as reference group for immune recovery, 22 children transplanted for all or aml with an hla-identical donor and not receiving serotherapy were included. the median serum concentration of total atg at the day of hsct was 6 times higher for atg-f (atg-g 67 μg/ml, atg-f 403 μg/ ml; figure a) as the result of the higher dose of atg-f given. the active atg concentration was twice as high for atg-f (atg-g 11.1 au/ml, atg-f 23.4 au/ml figure b ). three weeks later at the expected time of engraftment, the total atg concentration was decreased with the same factor for both atg brands (atg-g from 67 to 25 μg/ml, factor 2.7; atg-f from 403 to 139 μg/ml, factor 2.9). however, the active atg concentration showed a much faster decline for atg-f (atg-g from 11.1 to 1.03 iu/ml, factor 10.8; atg-f from 23.4 to 0.23 iu/ml, factor 100). correspondingly, the number of cd3 t-cells at 1 month post-hsct was higher after atg-f than after atg-g (atg-g, atg-f and no-serotherapy 41, 169 and 386 cells/μl, respectively. figure c) . this is the first study to compare the pkpd of total and active atg-genzyme and atg-fresenius. active atg-f showed a much faster clearance than atg-g, which was associated with a significantly faster cd3 t-cell recovery at 1 month post hsct. thus, atg-f is not only quantitatively but also qualitatively very different from atg-g, which will clearly impact hsct outcomes. reduced toxicity myeloablative conditioning regimen in pediatric hematologic malignancies not associated with improved outcomes s chaudhury 1,2 , i helenowski 2 , r duerst 1,2 , wt tse 1,2 , m kletzel 1,2 , j schneiderman 1,2 and d jacobsohn 3 1 ann and robert h. lurie children's hospital of chicago; 2 northwestern university feinberg school of medicine, chicago and 3 children's national health system, washington dc allogeneic (allo) hematopoietic cell transplantation (hct) is the only curative potential therapy in refractory and relapsed pediatric leukemias. poor outcomes in allo hct are associated with treatment-related mortality (trm), mostly due to regimen-related toxicities (rrt) and graft-versus-host disease (gvhd) after myeloablative conditionings (mac), but high relapse rate with reduced-intensity or nonmyeloablative regimens. 1 to improve trm, without compromising conditioning intensity, we prospectively explored the feasibility and efficacy of a mac but reduced-toxicity conditioning (rtc) regimen, consisting of fludarabine 30 mg/m 2 /d (given first) × 5d, daily busulfan dosed to target an auc of 4000 microm*min/d × 4, ratg 1.5 mg/kg/d × 3 and 400cgy of total body irradiation in 30 patients (table 1) with hematologic malignancies. gvhd prophylaxis was cyclosporine and mmf. all patients tolerated the rtc well, with no graft failures. rrt included moderate mucositis (67%), infections (bacterial 33%, viral reactivation 63%, fungal 20%) and 3 cases of venoocclusive disease (vod). cumulative incidence d100 ⩾ gr 3 acute gvhd was 32% (95% confidence interval [ci], 16-50), extensive chronic gvhd was 3.5% (95% ci, 0.2-16). mortality at 100 days was 10.7% (95% ci 3-25), 2 due to infections with agvhd and 1 vod. with a median follow-up of 1.5 y (range, 0.6-5), the cumulative incidences of relapse at 1 years was 29% (95% ci, 14-47). mortality due to severe agvhd was 90%. overall survival (os) and progression-free survivals (pfs) for 1year was 63% (95% ci, 42-78), and 56 % (95% ci, 36-72) respectively. on univariate analysis there was no association of outcomes with donor type, graft source, disease or busulfan exposure except significantly higher cgvhd in unrelated donors, agvhd severity with peripheral blood. in summary, the use of the myeloablative rtc resulted in comparable trm, with high relapse rate was high, including in those developing chronic gvhd. this suggested a less robust graft-versusleukemia effect resulting in poor pfs and os. nonetheless, this regimen may be used as a lower-trm platform to combine with other strategies, intensive disease monitoring pre and post hct, addition of post hct maintenance therapy in combination with marrow as the stem cell source to decrease relapse or gvhd. specific immune response to vaccinations decline after hematopoetic stem cell transplantion (hsct). re-vaccination of all hsct recipients is recommended in all guidelines but bcg vaccination is not recommended due to safety concerns after hsct. mycobacterium tuberculosis can cause severe disease in children including meningitis and milliary tuberculosis (tb). the bacille-carmette-guerin (bcg) is a liveattenuated vaccine with documented efficacy against milliary disease and meningitis. routine vaccination of all infants residing in countries with high tb incidence is recommended by world health organization. however, there is no data in literature regarding its safety in post hsct setting. here, we report 34 children who underwent matched related allogeneic hsct at ankara university pediatric bone marrow transplant (bmt) unit and received bcg 24-months post-transplant. all patients were free of graft versus host disease (gvhd) and immunosuppressive therapy (ist) and had negative ppd skin test prior to vaccination. none of the recipients developed local or disseminated tuberculosis as a complication of bcg with a median follow up of 10 years. we conclude that the bcg vaccine is safe in the post hsct period when administered at least 24 months out of transplant to a selected group of patients who are free of gvhd and ist. disclosure of conflict of interest: none. single centre experience of harvesting bone marrow from donors o3 years of age r raj, r uppuluri 1 , d subburaj 1 , d jayaraman 1 , k mullanfiroze 1 , v swaminathan and l vaidhyanathan 1 1 department of paediatric blood and marrow transplantation, apollo speciality hospital harvesting bone marrow for allogeneic marrow transplantation from donors o15 kg presents special challenges. we present data on 67 sibling donors from our institution between 2006 and 2016. the mean age was 23 months with a range between 8 months to 48 months. children less than one year accounted for 25% of our donors with the youngest being 8 months of age and the smallest donor weighed 5.5 kg. all aspirations were performed from iliac crests and all donors were given general anaesthesia by a paediatric anaesthetist. irradiated blood was transfused in 97 % of the donors during the procedure. the volume of marrow obtained ranged from 5 to a maximum of 20 ml/kg donor weight. the product contained an average cd34 count of 5.5 × 10 6 /kg recipient weight with a range from 1.6 to 12 × 10 6 /kg. only on one occasion was a second harvest needed, where the donor weighed 12 kg and recipient 42 kg with major blood group incompatibility requiring red cell reduction. the yield of cd34 cells per ml of bone marrow was on average 22% higher than children above 3 years of age. all recipients showed brisk engraftment in 2 weeks. none of these donors experienced major difficulties following the aspiration procedure. thus, very young children may safely donate marrow for allogeneic transplantation and the yield of stem cells obtained is substantial. this data is particularly relevant in transplantation for haemoglobinopathies like thalassaemia major and sickle cell anaemia, where families are being counselled about a target of 15 kg for the donor in order to plan transplantation. disclosure of conflict of interest: none. sinusoidal obstruction syndrome-veno-occlusive disease in pediatric patients given either autologous or allogeneic hematopoietic stem cell transplantation (hsct). a retrospective study of the aieop-sct (italian haematology-oncology association-stem cell transplantation) group m faraci 1 , r luksch, e calore 2 , f saglio 3 , a prete 4 , mc menconi 5 , v trevisan 6 , g de simone 7 , v tintori 8 , s cesaro 9 , s santarone 10 , mg orofino 11 , e lanino 1 , m zecca 12 and a bertaina 13 sinusoidal obstruction syndrome (sos), known as venoocclusive disease (vod), is a potentially life threatening complication that can develop after hsct. although sos progressively resolves within few weeks in most patients, the severe forms result associated with multi-organ dysfunction and high mortality rate (480%). aim of this survey is to evaluate incidence and management of sos in a large cohort of children receiving either allogeneic or autologous hsct. we retrospectively reviewed pediatric hscts performed in 12 (46%) out of 26 aieop affiliated centers, between january 2000 and april 2016. new ebmt criteria have been used for the diagnosis of sos (serum total bilirubin ⩾ 2 mg/dl and 2 of the following criteria: painful hepatomegaly, weight gain 45%, and ascites) and for the classification of severity grading. 1,2 among a total number of 6208 hsct procedures (2980 autologous and 3228 allogeneic), we identified 94 (1.5%) patients with sos. this complication occurred in 37 and 55 cases after autologous and allogeneic hsct, respectively. fiftytwo pts (55%) received iv busulphan (bu) at myeloablative dose, 28 (30%) oral bu, while 14 (15%) were treated with different conditioning regimen. the median time of sos occurrence was 16 days after hsct. details about prophylaxis and therapy are reported in figure 1 . out of the 92 children, 54 (59%) fulfilled all sos-ebmt criteria. bilirubin ⩾ 2 mg/dl, gain of weight 45%, ascites, and painful hepatomegaly did not occurred in 16, 3, 4 and 2 patients, respectively. thrombocytopenia was present in 85 pts (92%), thickening of gallbladder in 63 (68%) and abnormalities of coagulation parameters in 78 (84%). according to sos ebmt severity grading, levels of transaminases were mild in 18 pts (19%), moderate in 21 (22%), severe in 13 (14.1%), and very severe in 42 (45.6%). notably, creatinine was mild in 67 pts (71%), while 5 (5.4%), 10 (10.8%), and 12 (13%) children showed moderate, severe and very severe grade of renal failure. thirty-three pts (36%) had respiratory failure, and 28 (85%) of them experienced right pleural effusion. six out of the 25 patients who developed acute kidney injury, required dialysis. severe encephalopathy occurred in 8 pts (8.6%) and 22 (24%) out of the 92 pts evaluated, were admitted in intensive care unit. as therapy of sos, 69 pts received defibrotideâ (df); the dosage was 25 mg/ kg/day in 63% of them. the median duration of df treatment was 15.5 days (range 4-104). thirty-three (35%) pts received methylprednisolone (median dose of 2 mg/kg). fifteen pts (16.3%) died due to mof (2 in moderate, 6 in severe, and 7 in very severe group) at a median time of 6 days from sos diagnosis (range 3-75 gg). our multicenter survey showed that, at least in our experience, there is a significant variability in the management approaches to sos/vod in children, while, diagnostic evaluations are more homogeneous. interestingly, in our cohort, the increase of bilirubin may be an absent criteria, while thrombocytopenia and abnormalities of coagulation parameters are more frequent. as expected, mof occurred mostly in patients experiencing severe sos. df represents first strategy to treat sos in the majority of patients, even if steroids and ursodeoxycholic acid are still used. the hyper-ige syndromes are characterized by marked elevations in plasma ige levels and eosinophilia with impairment in t cells which clinically results in combined immune deficiency. dock8 deficiency, the autosomal recessive form, brings about allergic/atopic manifestations and unusual susceptibility to infections with herpesvirus family members (herpes simplex virus, human papilloma virus) and molluscum contagiosum. symptoms in patients with dock8 deficiency typically emerge during childhood, and the majority results in death because of infections and malignancy by the third decade. hematopoietic stem cell transplantation (hsct) is now considered a standard of care for dock8 deficiency when an appropriate donor is available. in this study, we present the 5 unrelated hsct results of 4 children with dock8 mutation. the demographic and clinical data of the 4 patients with 5 transplantations studied are shown in table 1 . hsct was administered between august 2013 and august 2015 at bahçeşehir university medical park antalya hospital and the clinical data of the hscts are presented in table 2 . all patients were transplanted from unrelated donors with bone marrow, except one with cord blood. the cord blood transplantation´s regimen was non-myeloablative which resulted with rejection. despite existence of serious morbid problems before transplantation, all the patients engrafted successfully. majority of the complications mentioned in the table 2 were improved and they are in the follow-up in an outpatient basis. discussion dock8 deficiency has high mortality, and hsct should be considered as early as possible before development of significant organ damage. despite myeloablative conditioning and high morbidity before the transplantation, survival was very good in our patients. myeloablative and nonmyeloablative transplants have been performed from related and unrelated donors and have reported successful results even without the preparative regimen. in our center, all transplants performed from unrelated donors by myeloablative regimen have been successful but have resulted in transplant rejection with cord blood transplantation after nonmyeloablative regimen. in all of our patients, stable full chimerism has been detected, however mixed chimerism have also been shown to be useful in several reports. whether hsct also cures the autoimmune complications and reduces the risk of cancers is as yet undetermined. however, a myeloablative conditioning regimen followed by allogeneic hematopoietic stem cell transplantation from unrelated donors in dock8 deficiency results in improvement of the clinical phenotype with a low incidence of regimen-related toxicity. disclosure of conflict of interest: none. successful bone marrow transplantation after myeloablative conditioning in a child with ipex syndrome b kuşkonmaz 1 , d ayvaz 2 , mh abur 3 , fv okur 1 , g karagüzel 4 , f orhan 5 , i̇ tezcan 2 and du çetinkaya 1 immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome is a rare disorder. although most patients present in infancy with a clinical triad of intractable diarrhea, insulin-dependent diabetes, and eczematous dermatitis, some patients present with severe food allergies and other autoimmune manifestations. the disease is caused by mutations in the forkhead box p3(foxp3) gene, a transcription factor that is essential for the development and function of regulatory t (treg) cells. this cells plays an essential role in controlling immune responses and preventing autoimmunity. patients usually die in the first years of life without treatment. the only effective cure is hematopoietic stem cell transplantation (hsct). here we report a patient with ipex syndrome who underwent hsct after myeloablative conditioning. 9 months of age boy with the history of diarrhea, insulin-dependent diabetes, eczematous dermatitis, pneumonia, coombs positive hemolytic anemia, referred to our hospital for investigation of immunodeficiency. on admission physical examination showed eczematous skin rash, submandibular lymphadenopathy, hepatosplenomegaly. before hsct the patients treated with immunosuppressive agents including methylprednisolone, mycophenolate mofetil and monthly intravenous immunoglobulin. complete blood count revealed anemia (hb: 7.7 g/dl), and eosinophilia (1900/mm 3 ). serum immunoglobulins were: ig g: 1550 mg/dl (463-1006), igm: 172 mg/dl (46-159), iga: 60.9 mg/dl (17-69), ige :1538 iu/ml. lymphocyte subset analysis showed cd3 64%, cd4 22%, cd8 40%, cd16+56 13%, cd19 19%. foxp3 gene analysis showed c.748_750delaag mutation. at the age of 1 year, patient underwent hsct from his hla matched sibling. myeloablative conditioning regimen including busulfan (20.4 mg/kg) and fludarabine (160 mg/m 2 ) was given to the patient. cyclosporine a and methotrexate (day +1, day +3, day +6) were used as graft versus host disease prophylaxis. bone marrow was used as the stem cell source and the number of cd34+ cells was 4.5 × 10 6 /kg. neutrophil and platelet engraftment were achieved on day +13 and +35 [p493] s378 respectively. acute and chronic gvhd were not observed, but patient developed veno-occlusive disease treated with defibrotide, sepsis treated with broad spectrum antibiotics. chimerism analysis showed %98 donor profile at the third month of hsct. after hsct, autoimmune hemolytic anemia, eczematous dermatitis, food allergies, diarrhea and type 1 diabetes resolved completely within two months after hsct. now the patient is in good clinical condition without any symptoms 5 months after hsct. early hsct provides better outcome in patients with ipex, before the organ damage due to autoimmunity and/or adverse effects of immunosuppressive therapy. myeloablative conditioning is associated with substantial transplantation-related mortality whereas nonmyeloablative conditioning carries an increased risk of rejection because of dysregulated effector t-cell function. in this patients, myeloablative conditioning was preferred because of the risk of rejection. although the required levels of donor chimerism and conditioning intensity are unknown, engraftment of donor treg cells seems to be sufficient to control the disease. the patient is well without any symptoms of ipex after hsct with full donor chimerism. disclosure of conflict of interest: none. interferon gamma receptor 1 deficiency (ifnr1) is a rare autosomal recessive immune deficiency disorder associated with very poor outcome secondary to severe and disseminated mycobacterial infections. hematopoietic stem cell transplantation (hsct) has been proposed as a curative option. however, hsct for these patients is particularly difficult owing to a high rate of graft rejection. the use of a non t-cell depleted transplant from an hla-identical sibling and fully myeloablative conditioning regimen has been shown to have improved outcomes. we report the first successful hsct with a t-depleted haplo-identical donor, performed in a girl with severe ifnr1 deficiency. we reviewed the medical chart of a 2-year-old hispanic girl with ifnr1 deficiency who was diagnosed at birth, since her brother had previously been diagnosed with the same complete ifnr1 deficiency. they were found to have a novel mutation variant detected at c.201-1g4t. as expected with this disorder, she developed disseminated infection with mycobacterium abscessus infection at 2 months of age and was subsequently found to have mycobacterium abscessus osteomyelitis. she was treated with multiple antibiotics including: amikacin, linezolid, meropenem and clarithromycin while tigecycline was added a few weeks prior to admission for hsct. she was continued on this therapy until day + 30 following which antimicrobials were gradually weaned off. she was enrolled on the bp-004 trial, a multicenter, prospective phase i-ii trial (enrolling both malignant and non-malignant diseases) evaluating αβtcr +/cd19+ depleted haplo-transplantation followed by administration of bpx-501 t cells containing the ic9 suicide gene, (clinicaltrials.gov nct02065869). her conditioning regimen included busulfan (4 mg/kg/day for 4 days) and cyclophosphamide (50 mg/kg/day for 4 days). fludaragbne, tli (900 cgy) . gvh prophylaxis with atg/rituximab. the patient received a graft with: tnc-9.98 × 10 8 cells/kg, cd34+ cells-16 × 10 6 cells/kg, and αβtcr+ t cell content of 4.78 × 10 4 cells/kg. as per protocol, since the αβ tcr+ t cells in the product was below threshold of 1 × 10 5 cells/kg, she did not receive any post-transplant immune suppression. bone marrow recovery occurred at day +10 with anc 4500/mm 3 and platelet recovery at day +18. full engraftment with 100% donor chimerism based on cytogenetic analysis was observed at day +28 after transplantation and has remained stable. she is currently 14 months post-transplant, and has done well without major complications and or signs of mycobacterial infection. there is limited data in patients receiving hsct for ifnr1 deficiency with very poor outcomes either relating to graft failure, transplant complications and progressive mycobacterial infection. to our knowledge, this is the first patient with ifnr1 deficiency transplanted successfully with a haploidentical donor and alive without any active mycobacterial infection. this report suggests that using a highly immunopotent graft depleted of only αβtcr+ t cells while retaining other immune effectors might offer a potential strategy to engraft these high risk patients using haplo-identical donors thereby allowing access to virtually all patients in need. disclosure of conflict of interest: none. tandem autologous stem cell transplantations for high risk pediatric embryonal central nervous system tumors: a single center experience k rosenfeld 1 , r dvir 1 , s constantini, j roth 2 , s edelman 1 , a tal 1 , d levin 1 , m manisterski 1 , s achituv 1 and r elhasid 1,3 1 department of pediatric hematology-oncology, tel aviv medical center; 2 department of pediatric neurosurgery, tel aviv medical center and 3 sackler faculty of medicine, tel aviv university pediatric embryonal central nervous system tumors are highly malignant tumors, which tend to disseminate through the cerebrospinal fluid to the brain and spinal cord and include: medulloblastoma, pinealoblastoma and primitive neuroectodermal tumors (pnets). the recommended treatment for these tumors is a complete surgical excision, craniospinal radiation and chemotherapy. the use of high dose chemotherapy with tandem autologous hematopoietic stem cell transplantation (hsct) has been advocated for high risk patients, and infants who could not be irradiated. between july 2010 and november 2016, 16 pediatric patients (11 males, 5 females) suffering from high risk medulloblastoma, pnet or pinealoblastoma underwent tandem autologous hsct. they were treated according to two protocols: group a consisted of ten patients with median age of 8.2 years (range 3.9-15.5 years) received the st jude sjmb03 protocol, while group b consisted of six patients with median age of 2.1 years (range 1.4-3.5 years) who received the children's oncology group -acns0334 protocol. all patients engrafted with median time for neutrophil engraftment of 11 days (range 7-14 days) and for platelets engraftment (420 000) of 13 days (range 13-24 days). median follow-up was 3.5 years (range 1 week-6 years). neurological toxicity: two group a patients had convulsions episodes, one occurred during infusion of cryopreserved stem cells, and the other was a result of progressive disease during the last course of hsct. gastrointestinal toxicity: seven patients required total parenteral nutrition due to mucositis. diarrhea occurred in seven patients, two of them were diagnosed with rota virus and two with clostridium difficile. infectious complications: all patients suffered from at least one episode of neutropenic fever which was treated with broad spectrum antibiotics. there were 7 documented bacteremia in 6 patients. (1 klebsiella pneumonia, 1 proteus mirabilis, 3 staphylococcus aureus, 1 streptococcus viridans and 1 staphylococcus epidermidis). metabolic complications: four patients in group a developed reversible syndrome of inappropriate anti-diuretic hormone secretion (siadh) during chemotherapy, and all group a patients developed hypomagnezemia. four patients died, one due to progressive disease, one due to early relapse 3 months post treatment, one due to late relapse 5 years post treatment and one due to sepsis 4 months post treatment. another patient relapsed 1.5 years s379 post treatment, underwent surgery and radiotherapy and is now 3 years post therapy. late effects: four group a patients developed endocrinological sequelae at a median of 20 months (range 17-21 months) and require hormone replacement therapy. tandem autologous hsct is a feasible treatment for pediatric high risk embryonal tumors, with good engraftment and acceptable toxicities using sjmb03 and acns0334 protocols, with overall survival of 75%. long follow-up is needed in order to diagnose and treat late effects. disclosure of conflict of interest: none. the diagnostic role of liver stiffness measurement in predicting hepatic veno-occlusive disease (vod) in pediatric hematopoietic stem cell transplantation (hsct) k kleinschmidt 1 , f ravaioli 2 , r rondelli 1 , g marasco 2 , r masetti 1 , a prete 1 , a colecchia 2 , d festi 2 and a pession 1 1 pediatric oncology and hematology unit, department of pediatrics, university of bologna, sant 'orsola-malpighi hospital and 2 department of medical and surgical sciences, university of bologna vod is a potentially life-threatening complication associated with hsct in which immediate therapeutic action is crucial for patients' outcome. liver stiffness measurement (lsm) using fibroscan represents a non-invasive method to detect the grade of liver fibrosis and portal hypertension as in case of vod. to evaluate the predictive potential of lsm in pediatric patients (pts) at risk for developing vod, a prospective, ongoing, single-center study has been performed at the university hospital of bologna. lsm was performed by using the fibroscan device, which consists of a 3.5 mhz ultrasound transducer probe that transmits low-frequency vibrations (50 hz) to the liver tissue. the propagation velocity is proportional to the stiffness (elasticity) of tissue. lsm will obtain pathological high values (47.5 kpa) when the tissue is altered like in liver fibrosis, or post-sinusoidal portal hypertension. from november 2014 -september 2016, 25 pediatric pts (18 male, 7 female), aged 3-20 years (mean 11.7), affected by hemato-oncologic disease, eligible to allogeneic (22) or autologous (3) sct conditioned with busulfan-based chemotherapy, were enrolled. pts were scheduled for 4 study examinations with lsm: at t0 (baseline) before chemotherapy, t1 (day 7-10 after sct), t2 (day 17-20) and t3 (day 27-30). the diagnosis of vod was defined according to modified seattle/baltimore criteria. twenty-five pts were enrolled in the protocol, 22 of which were evaluable for the study (pts characteristics table 1 ). 4 out of 22 pts (18%) developed vod. the cumulative incidence (se) of vod in our setting was 19 % (8.6). baseline lsm values on t0 of all pts were normal (7.5 kpa at t2 (p = 0.002) and t3 (p = 0.004). from our observations, an anticipating pattern of pathological lsm in presence of clinical and laboratory parameters within normal ranges in patients who develop vod can be derived. preliminary data indicate a high predictive potential of lsm in the diagnosis of vod, however the number of cases is not sufficiently representative to draw definitive conclusions. to optimize the predictive potential of the method, more frequent (daily) measurement in the critical time frame are currently investigated. [p497] all= acute lymphoblastic leukemia, aml= acute myeloid leukemia, bu= busulfan, treo=treosulfan, fluda= fludarabine. disclosure of conflict of interest: none. [p497] the exact role of extra-corporeal photopheresis in children with gvhd: an unanswered question ss anak, h bilgen 1,2,3,4,5,6,7 , y yaman, et saribeyoglu, k ozdilli, v hazar, m elli, am kokrek, h hizli and k payalan ecp continues to be a controversial treatment, probably due to the mechanism of action not being identified, the varying photopheresis procedures and treatment schedules, and the difficulty of conducting trials on relatively rare diseases with involvement of clinically heterogeneous organs. ecp was performed in our pediatric transplant center to 8 patients mean age of 12 years ( 4-18) diagnosed to have all ( 3 pts), thalassemia ( 2 pts), aplastic anemia (1), blacfan diamond (1), refractory hodgkin disease (1) following our internal protocol for 152 ecp sessions. five of the patients had mud, 3 had hla id sibling transplants. chronic gvhd was diagnosed in 2 of the patients 6 had acute gvhd. skin was involved in all the patients, liver in 6 of the patients, lung in 3, gut in 6 and mucous membranes in 7 patients. the ecp treatment consisted essentially of three steps: (1) collection of mncs from the patient, (2) processing of mnc buffy coat, and (3) return of mncs to the patient. collection was performed using a cell separator (haemonetics mcs plus), processing two blood volumes. our protocol provides for a maximum final mnc volume to be collected at 150 ml, with a hematocrit (hct) value below 5%. the maximum procedure time was set at 180 min. the mncs collected were adjusted to a constant volume of 300 ml by the addition of saline and 3 ml of 8-mop in aqueous solution, to always obtain a final concentration of the drug of 200 ng/ml. the diluted buffy coat was transferred into a special uv-a-permeable bag (pit-kit medtech solutions), and uv-a radiation at 2 j/cm 2 was performed (uva-pit irradiator). the photoactivated mncs were returned to the patient within 30 minutes using a blood transfusion set. during ecp procedure, patients' vital signs were monitored. anticoagulation consisted in acidcitrate-dextrose formula a set at a variable ratio (1:14-1:20) according to the patient's characteristics (clinical conditions, body weight, coagulation values) and platelet (plt) count. prophylaxis of hypocalcemia consisted of the administration of calcium gluconate (5 ml diluted in 5-10 ml saline) every 30 to 45 minutes. all procedure related side effects were recorded. during the reinfusion and postreinfusion phases, the patients were monitored for fever, chills, headache, rash, erythema, urticaria, itching and edema. no serious complication was detected. all the patients had also steroids, 4 had concurrent mesenchimal stem cells. ecp was applied on 2 consecutive days every 2-4 weeks which is continued for approximately 6 months followed by a maintenance schedule tapered to an every 2-to 4-weeks. the mean session cycle was 19 ( 6-51) between february 2015 to november 2016 . the most commonly involved organ was the skin which demonstrated a response rate of 75%, followed by liver (66%), lung (25%), gut (18%) and mucous membranes (68%) the concurrent immunosuppression could be reduced during ecp therapy, and no increase in opportunistic infections was detected. 1/8 patient died after a relapse, 7/8 are alive with chronic mild gvhd. however, despite our good response rates, our understanding of ecp remains limited. patients who suffer from acute and chronic gvhd have limited treatment options. ecp remains an important therapeutic option. future basic, translational, and clinical research studies will provide a better understanding of its mechanism of action and optimize its therapeutic potential. disclosure of conflict of interest: none. tolerability and responses to ex vivo il2 activated nk cells from haploidentical parental donors in paediatric patients with refractory leukaemia/lymphoma pl tan 1 prognosis for patients with refractory leukaemia/lymphoma ineligible for transplants and those who relapse posttransplant is poor. in adult settings, adoptive transfers of ex vivo il2 activated natural killer ('ank') cells from nk alloreactive donors, especially for nk sensitive cancers, has been successful in bridging patients to curative transplants.(1) this approach has not been reported in paediatric patients. we report our experience in 8 consecutive patients, of median age 9 (range, 1-15) years, with refractory leukaemia/ lymphoma (aml, 2; all, 2; mixed phenotype acute leukaemia, 2; lymphoma, 2) who received 9 treatments with 'ank' from haploidentical parental donors on institutional protocol, between aug 2012 and 2016. parents/legal guardians/patients provided informed consents as per institutional guidelines for donors and patients procedures. donor lymphocytes harvested at steady state were cd3 depleted followed by overnight culture in il2 before being infused into patients lymphodepleted with fludarabine and cyclophosphamide. additional rituximab were given to 3 patients and another received tbi 2 gy. subcutaneous il2 injections at doses 1-3 mu/m 2 /dose started on d-1 and were planned for 6 doses, as tolerated. nk alloreactive donors (kir-ligand mismatch) and kir b/x genotype were available to all except 2 patients. two patients were treated for post-transplant relapse; 1 of whom also received 'ank' pre-transplant; other 6 patients had failed best conventional therapy including cd19/cd3 bispecific t cell engager (blinatumomab) in 1. lymphodepletion was well tolerated. a median tnc and cd56+ dose of 9.8 (range, 2.9 to 38) × 10 7 /kg and 1.8 (range, 1.2-18) × 107/kg, respectively were administered. cytokine release syndrome (crs) was observed in 8 of 9 treatments (6 grade 1, 1 grade 3, 1 grade 4). the patient with dock8 deficiency, disseminated ebv+ cerebral lymphoma had grade 4 crs and robust tumour lysis syndrome but succumbed to neurotoxicity. of the 9 treatments, there were 7 responses, including the 2 given posttransplant. excluding the 2 treatments given post-transplant and 2 non-responders, median peak donor chimerism was 93% (range, 7-100%) occurring at a median of 12 (range, 7-22) days. five patients (4 responders, 1 non-responder) proceeded to transplants at a median of 43 (range, 35-96) days after 'ank.' responders had longer survival time compared to nonresponders (median 314 vs 88 days). two responders (25%) achieved sustained minimal residual disease (mrd) remission after transplants and are alive 138 and 380 days from 'ank.' five eventually died of their primary leukaemia/lymphoma; 1 from crs. our preliminary experience in a small cohort of 8 paediatric patients with refractory leukaemia/ lymphoma showed that adoptive transfers of ex vivo il2 activated nk cells from haploidentical parental donors were tolerable; with responses seen in 75% of patients; and 25% achieving prolonged mrd remissions after transplants. patients with cerebral diseases might be at increased risks of neurotoxicity with this approach; and care must be taken in patient selection and the design of the lymphodepletion therapy. alternative donor choices are limited in multi-racial, multiethnic societies with small families such as singapore. unrelated cord blood transplant provides a feasible alternative to patients lacking adult stem cell donors in children with primary immunodeficiency diseases. method: we describe our experience using unrelated cord blood transplant (ucbt) for 9 children with pid from august 2005 to november 2014. during this period we performed hsct for 12 children with pid: 9 with unrelated cord blood (75%); 2 with msd and i mud. out of 9 cases of ucbt there were 5 severe combined immunodeficency (scid), 2 chronic granulomatous disease (gcd), 1 hyperigm syndrome and 1 wiskott aldrich syndrome (was). the median age of transplant was 13.7 months (range 1.3 to 83.3 months). all presented with multiple infections ranging from disseminated bcg infection to parainfluenza /rsv /rotavirus infection to pseudomonas sepsis, staphylococcal endocarditis to pulmonary aspergillosis for scid. hyper igm presented with pnemocystitis carini pneumonia while cgd conditions presented with perianal abscess and fungal pneumonia. the child with was had life threatening git bleeding and a hemorrrhage trachaebronchial cast removed after a failed initial extubation for gastroscopy. conditioning regimes consisted of reduced intensive (fludarabine based) conditioning regime for scid and myeloablative regime for the rest. the median tnc dose was 12.7 × 10(7)/kg (range 4.2 to22.5) and median cd34+ cells dose was 3.68 × 10(5)/kg (range 1 to 8.9). results: all engrafted well except for one graft failure in cgd. he refused 2nd transplant and died 1.5 years post transplant from fungal pneumonia. median engraftment time for neutrophil was 21 days (range 13 to 33) and platelet was 30 days (range 18 to 65 days). grade 1 skin aghvd occurred in one patient while another patient died of agvhd of liver and lungs. chronic gvhd was found skin and liver in one patient. trm was 11 % (due to agvhd). median follow up was 1255 days (ranged 327 to 4109). overall 5 years survival was 78%. post-transplant complication with life threatening puemonitis was not uncommon. one patient developed biopsy -proven idiopathic interstitial pneumonitis and required ecmo for one month. he received immunosupressive drugs including methylprednisolone, infliximab, oral imatinib (tk inhibitor), azithromycin and nebulised becotide. he was weaned off oxygen after 2-3 months. conclusion: our limited experience showed unrelated cord blood is good source of stem cell for transplant in pid in a multiracial population. one case of graft failure was likely due too low cell dose cd34 +cells dose 1 × 10(5)/kg. the expertise in icu has enabled us to support several patients who presented with infective pneumonia pre-transplant and post -transplant. with better technology like alpha/beta depletion haploidentical transplant may be a better option to achieve engraftment earlier so as to avoid stormy post-transplant infections seen in unrelated cord blood setting. disclosure of conflict of interest: none. in spite of these recommendations, literature from developing countries suggest that pbscs are used more and more frequently without compromising the transplant results, as they seem to be preferred graft source for donors in many countries incl. poland. therefore we analyzed the efficacy of mud-hsct in children with saa transplanted in our centre. clinical data of 44 saa and pnh children and adolescents (27 boys and 17 girls), who underwent mud-hsct between october 2000 and july 2016 were retrospectively analyzed. the median age was 11.2 years (range 0.7-20 years) according to the graft source, the patients were divided into pbsct group (37 patients) and bm group (7 patients). four patients required second mud transplant due to graft rejection. overall survival for all patients was 66 %. estimated 5-year overall survival (os) was not statistically different between pbsct group and bm group [(68% vs 54% ) p = 0.42]. there was no significant difference in os between group who had ist before transplant and the group, who had an upfront transplant as a first line of therapy [65% vs 62%, p = 0.79].the time to neutrophil and platelet engraftment was statistically longer in bm group than in pbsc group [(anc 16 vs 15 days, plt 29 vs 16 days, respectively) p = 0.017]. the incidence of grade iii-iv acute graft-versus-host disease (gvhd) in pbsct group was similar to that in bm group [37% (14/37) vs 29% (2/7)]. the incidence of chronic gvhd in pbsct group was similar to that in bmt group [8% (3/37 ) vs 14% (1/7)]. other transplantrelated complications like heart failure, central nervous bleeding, incidence of infections were comparable within the two regimens. there were 15 deaths in the whole group. the main reason of death were infectious complications or multiorgan failure (mof) in severely pretransfused patients in this historical cohort of patients. unrelated donor pbsct in children and adolescents with saa seems to be not inferior to unrelated donor bmt. the incidence of chronic gvhd was surprisingly low in saa recipients of mud pbsc. increased morbidity and mortality due to infections was due to individual poor clinical situation of patients before transplant (i.e. fungal infections, contamination with resistant bacteria, prolonged neutropenia). disclosure of conflict of interest: none. dyskeratosis congenita (dc) is characterized by the clinical triad of reticular skin pigmentation, nail dystrophy, and oral leukoplakia. the majority of patients with dc develop bone marrow failure (bmf), which is the main cause of death in dc patients. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative treatment for bmf associated with dc. transplant-related morbidity/mortality is common, especially after myeloablative conditioning regimens. hsct has been introduced into the management of dc, which has had remarkable clinical results. we report our experience in 4 children with dc who underwent allogeneic transplantation at a single medical center. patients received a fludarabine-based reduced intensity conditioning (ric), and the graft source was unrelated peripheral blood stem cells. median age at the time of hsct was 5.5 years (range, 4-13 years). the numbers of infused mononuclear cells and cd34+ cells were 15.62 ± 3.04 × 10 8 /kg and 5.80 ± 3.37 × 10 6 /kg, respectively. the median time of neutrophil and platelet recovery were 13.5 days (range, 12-17 days) and 21.5 days (range, 19-26 days). two patients experienced grade ii-iii acute graftversus-host disease (gvhd), and chronic gvhd was only observed in one patient. all four patients remained alive and transfusion independent at the median follow-up of 18.5 months (range, 9-31 months). correction of previously existing physical defects was observed in two patients. unrelated peripheral blood hsct can be a curative option for dc. ric based on the type of disease is important to s382 achieve successful hsct. a larger sample size and extended follow-up of this rare patient population are needed to determine whether the changes in therapy will improve longterm survival. disclosure of conflict of interest: none. autosomal recessive hyper-ige syndrome due to dock8 mutation is a combined primary immunodeficiency, characterized by severe eczema, recurrent infections, and susceptibility to autoimmunity, malignancy, and multiple allergies, in addition to unusual high serum ige level. dock8 patients tend to have a progressive severe clinical course with mostly fatal outcome during second to third decade of life without hematopoietic stem cell transplantation (hsct). in our center we have a large number of dock8 patients. during a period of 11 years (2006-2016), we transplanted 14 patients with documented dock-8 mutation confirmed by molecular genetics. one patient did not receive any conditioning because of poor clinical condition and he died from severe cutaneous and gut gvhd and another patient received cbt with bu/flu with zero engraftment. the rest of the patients received hsct from hla full matched donor with chemoablation with bu/cy for all with 100% lymphoid and myeloid engraftment (str). among those patients who received chemoablation, gvhd developed in 6 patients mostly grade i and ii. in addition 3 patients died: one died of severe gvhd and the other two died of sepsis. for dock8 patients we highly recommend early hsct if fully matched donor is available to prevent the high mortality associated with the disease. alloreactivity triggered by interactions between killer cell immunoglobulin-like receptors (kir) and natural killer (nk) cells plays a role in graft-versus-tumor (gvt) effects after hematopoietic stem cell transplantations (sct). in particular, kir-ligand mismatching between the donor and recipient might promote nk cell alloreactivity after unrelated cord blood transplantations (ucbt) in adult patients with acute myeloid leukemia (aml). recently, it has been suggested that allogeneic nk cells could be the effector cells that mediate gvt effects after mismatched allogeneic transplants for refractory childhood solid tumors. however, there are few reports about the efficacy of kir-ligand mismatched sct in pediatric cases. here, we report the excellent outcomes of kirligand mismatched cbt (kir-cbt) in pediatric patients with refractory malignant disease. we evaluated the cases of 9 pediatric hematology and oncology patients [4 (44%) aml, 1 (12%) myelodysplastic syndrome [mds] , and 4 (44%) neuroblastoma [nbl] patients] who underwent kir-cbt between 2010 and 2016 at our institution. among the 4 aml cases, one involved refractory disease (induction failure), and the other three involved relapsed aml (one patient relapsed after the 1st sct because of 5q-). all 4 nbl patients underwent kir-cbt followed by auto-peripheral blood stem cell transplantation (pbsct) because of stage 4 disease. the mds patient underwent kir-cbt because of refractory anemia with excess blasts. kir mismatching was defined as incompatibility between the donor kir and recipient kir ligand, and only inhibitory kir that interacted with human leukocyte antigen (hla)-bw4, -c1, or -c2 group ligands were considered. the median age of the patients was 4 (range 2-18) years. all 4 of the aml patients were in complete remission (cr) at the time of the hsct (cr1 = one case, cr2 = 3 cases). the mds patient was in a non-cr state, and all of the nbl patients were in their 1st cr at the time of the hsct. the aml patients received total body irradiation (tbi)-based conditioning (12 gy tbi and 120 mg/kg cyclophosphamide [cy]), and the mds patient received busulfan (bu)-based conditioning (19.2 mg/kg bu and 120 mg/kg cy). the nbl patients received reducedintensity conditioning regimens (125 mg/m 2 fludarabine, 140 mg/m 2 l-pam, and 2 gy tbi). the cb exhibited hla 2-4 locus mismatches (dna typing), including at least one inhibitory kir gene mismatch. the prophylaxis for graftversus-host disease (gvhd) consisted of tacrolimus and shortterm methotrexate. anti-thymocyte globulin (atg) was not used as a gvhd prophylaxis in any case. after the median follow-up period of 25 months (range: 4 -54 months), all 9 patients were alive, and none of them had relapsed after the kir-cbt. although grade ii-iv gvhd was observed in 6 patients (67%), it was controlled with prednisolone. chronic gvhd was not seen in any case. the present findings suggested that nk cell alloreactivity plays a role in preventing childhood myeloid leukemia and nbl relapse after kir-cbt. although our results are limited, this report provides novel data to support further investigations into the use of kir-cbt for the treatment of pediatric refractory malignant disease. disclosure of conflict of interest: none. impact of fcm-based minimal residual disease on transplant outcomes in patients with aml in hematological complete remission t oka, j kanda 1 , k ohmori 2 , m hishizawa 3 , t kitano 4 , t kondo 5 , k yamashita 6 it is reported that the presence of minimal residual disease (mrd) before hematopoietic stem cell transplantation (hsct) is associated with poor overall survival in patients with acute myelogenous leukemia (aml) in hematological complete remission (cr). we retrospectively analyzed the association between flowcytometry (fcm)-based detection of mrd and transplant outcomes. we included 56 adult patients with aml in hematological cr, who underwent their first allogeneic hsct between april 2005 and may 2015 at kyoto university hospital. mrd of bone marrow before hsct was measured using fcm. to search for target antigens to detect mrd, threecolor fcm analyses were performed using a differential panel for every disease and patient, which allowed us to detect ⩾ 0.1% of mrd. of the 56 patients (median age: 48.5, range: 18-66), 41 patients were included in the mrd-negative group (mrd o0.1%), whereas 15 were included in the mrd-positive group (mrd ⩾ 0.1%). in the latter group, 6 patients were included in the mrd-low group (mrd o0.6%), and 9 were included in the mrd-high group (mrd ⩾ 0.6%). there was no significant difference in the patient background between the mrd-negative and mrd-positive groups. the 3-year overall survival rates for the mrd-negative, mrd-low, and mrd-high groups were 82%, 63%, and 30%, respectively (p = 0.007, figure 1 ). in a multiple regression analysis, the mrd-high group was significantly associated with higher overall mortality than the mrd-negative group (mrd-low vs mrd-negative, hazard ration [hr] 1.62, p = 0.554; mrd-high vs mrd-negative, hr 8.47, po 0.001). the 3-year relapse rates for the mrdnegative, mrd-low, and mrd-high groups were 15%, 0, and 67%, respectively (p o0.001). there were no significant differences in non-relapse mortality among the three groups. the analysis of fcm-based detection of mrd revealed that an mrd positivity of ⩾ 0.6% was significantly associated with high risk of relapse and death even in patients with aml with hematological cr. the stronger consolidation or conditioning therapy before hsct based on mrd could improve transplant outcomes in these patients. [p506] disclosure of conflict of interest: none. post-transplant cyclophosphamide (ptcy) and megadose t cell depleted (tcd) haplohsct for tolerance induction f aversa 1 , e bachar-lustig 2 , n or-geva 3 , y zlotnikov klionsky 2 , l prezioso 1 , s bonomini 1 , a monti 1 , i manfra 1 , c schifano 1 , s pratissoli 4 , f lohr 5 , r lamanna 6 , v sgobba 6 , n giuliani 7 and y reisner 2 1 hematology and bmt unit, university hospital of parma, italy; 2 department of immunology, weizmann institute of science, rehovot, israel; 3 neurology department, stanford school of medicine, stanford, california; 4 radiotherapy unit, university hospital of modena, italy; 5 radiotherapy unit, university hospital of modena, italy; 6 genetic unit, university hospital of parma, italy and 7 hematology and bmt unit, university hospital of parma, italy the use of ptcy is associated with reduced risk for gvhd in t cell replete nma haplo-hsct; however, this intervention is still not sufficiently safe to justify treatment of non-malignant diseases or as a platform for organ transplantation. experimental data: in a total of 66 mice, we showed that combining the power of megadose tcd hsct with high dose ptcy (fig.1a) , enables marked and durable chimerism following nma conditioning, while each modality alone was ineffective (figure 1a) . chimerism included all myeloid and lymphoid lineages, and lda analysis of alloreactive t cells revealed specific immune tolerance towards donor stimulators (fig.1b) , also associated with acceptance of donor but not 3 rd party skin. clinical trial: a similar protocol was developed for clinical use. the first patient, a 54 yr old male with high-risk multiple myeloma in cr after autohsct, received megadose (15.4 x10 6 cd34+ cells/kg) cd3/cd19 depleted (1.17 x10 5 cd3+t cells/kg) haploidentical pbpcs after atg, fludarabine and 2 gy single frcation tbi. ptcy was given to control both hvg and gvh reactions (fig. 1c) . hematopoietic engraftment was achieved at day +15 with over 97% donor type chimerism during the first 6 months in the myeloid and b cell lineages. t cells during this period were predominantly of host type (10-23% donor type), gradually increasing to 63-72% at 9-12 months post transplant (fig. 1d) . the patient overcame cmv and subsequently ebv reactivation without any treatment (fig. 1e-1g) . dextramer facs analysis revealed that cmv and ebv specific cd8 t cells were exclusively of host origin (fig. 1f-1h) . at +18 months, cr and normal free light chain ratio were confirmed. the second patient, a 50 year-old male with high risk heavily pretreated multiple myeloma (tandem auto-hsct, 3 yr maintenance with lenalidomide, salvage therapy with vd) received a similar hsct (10.8 x10 6 cd34+ cells/kg, 1.2x10 5 cd3 +t cells/kg). despite transient engraftment (50% donor cell on day +17), graft failure with autologous recovery (0.04% donortype chimerism) was documented on day +30. this may be due to the extended treatment (3 yrs) with lenalidomide, but rejection cannot be excluded. after 5 months, this patient tolerated a second haplo-hsct (different donor) after myeloablative conditioning (atg, treosulfan, thiotepa and fludarabine) and alfa/beta tcr/cd19-depleted pbpcs. at 8 month follow up, he shows no sign of gvhd, good immunological reconstitution, excellent quality of life, and remains in complete remission. collectively, our murine proof of concept data supported by clinical experience in the first high risk mm patient. the marked level of host t cells persisting over the first year after hsct can provide anti-viral immune protection until thymus-derived donor t cells are generated. avoiding additional post transplant immune suppression ensures a robust anti-viral immunity and a graft vs tumor effect. the rejection experienced by the 2 nd patient, although corrected by a 2 nd myeloablative tcd hsct, indicates that the conditioning must be fine-tuned to optimize engraftment in every patient. we are therefore testing, increasing tbi from 2 gy to 3 gy. further studies will determine the efficacy of this approach in elderly mm patients, in non-malignant hematopoietic diseases, or as a prelude for organ transplantation and cell therapy. over the last decade the addition of alemtuzumab to fludarabine-based reduced intensity conditioning regimen is common practice in the unrelated donor allograft setting. in recent years, however, its use has extended to reduced intensity hla-identical sibling donor allografts with the aim of providing an additional prophylaxis against gvhd. it is difficult to assess though whether this practice has any negative influence in the relapse rate or whether it has any net benefit or disadvantage in terms of overall survival. in this retrospective study we have analysed a historical cohort of 65 patients [41 males, 24 females, mean age 59.05 (40-73)] who s385 received a ric fully matched unrelated donor (49 patients) or sibling donor (16) hsct as consolidation treatment for hr aml in 2 transplant centres in uk and greece. the conditioning regimen included fludarabine in all cases, together with melphalan and alemtuzumab(29 patients), busulphan and campath (21 patients), busulphan and thiotepa (1 patient), melphalan (3 patients), busulphan with and without atg (9 patients) total body irradiation (200 cgy, 2 patients). in total, 50 patients received alemtuzumab (35 mud 50 mg alemtuzumab and 15 sibling donor hsct recipients 30 mg alemtuzumab) and 9 patients (7 mud and 2 sibling donor hsct recipient) received atg with 5 patients receiving t replete allografts. gvhd prophylaxis was ciclosporin for patients receiving alemtuzumab based or atg based regimen and ciclosporin with low dose methotrexate for t-replete allografts. the median follow up was 32.3 months (range 3-154 months).all but four patients were transplanted in cr1 overall, patients receiving conditioning without alemtuzumab suffered more frequent (po0.0001) and more severe (po0.0001) acute gvhd. this group, however, had a significantly (po 0.05) lower relapse rate. the overall survival remained unaffected. the subgroup of patients receiving allografts from mud had a clear benefit in terms of a lower incidence (p o0.0001) and severity (p o0.0001) of acute gvhd: none of the patientsreceiving alemtuzumab experienced grade iv agvhd, but up to 5/14 patients not receiving alemtuzumab suffered severe grade iv gvhd. however, the use of campath was associated with a significantly higher rate of relapse or progression of the aml (po 0.02), so that none of the 6 mud recipients not having campath relapsed, while 9/26 patients having alemtuzumab relapsed. although none of these factors had a net impact on survival, there was a nonsignificant (p = 0.07) trend towards a higher survival in patients who received alemtuzumab. in the sibling donor allograft setting, alemtuzumab had no significant impact on the incidence of acute gvhd, relapse or survival. finally, in diseases where cytogenetic or molecular markers of high risk were available, our results showed a better overall survival (po0.06) in ric alemtuzumab conditioning undergoing fully matched unrelated donor hsct, probably as a result of the protection against graft versus host disease while maintaining graft versus leukaemia effect. overall, alemtuzumab is a highly protective agent against agvhd in mud hsct recipients while it maintains the graft versus leukaemia effect.however it did not show any clear benefit of its use in the identical sibling donor setting. larger prospective studies are required in order to determine the need for this agent in this particular setting. disclosure of conflict of interest: none. blastic plasmacytoid dendritic cell neoplasm (bpdcn) is a rare disease which constitutes o1% of all hematologic neoplasms annually. majority of bpdcn present with diverse skin involvement prior to leukemic dissemination, whereas a minority (~10%) have systemic involvement at diagnosis. there are no established therapies for bpdcn and most pts receive acute leukemia, myeloid or lymphoblastic, induction regimens; but responses are short-lived and prognosis is poor upon relapse. allogeneic hematopoietic cell transplantation (allo-hct) is offered to bpdcn cases based on small retrospective or registry case series. we retrospectively analyzed outcomes of bpdcn pts who received an allo-hct at 5 transplant centers in the usa. a total of 20 pts were eligible for analysis ( table 1 ). the primary endpoint was overall survival (os). twenty patients (m = 18, 90%), median age of 51 (14-71) yrs, received an allo-hct from a matched related (n = 9, 45%), matched unrelated (n = 7, 35%), mismatched-unrelated (n = 2, 10%), umbilical cord (n = 1, 5%) or haploidentical (n = 1, 5%) donor using myeloablative (mac) (n = 13, 65%) or reducedintensity (ric) (n = 7, 35%) conditioning. fifteen pts received hyper-cvad as pre-allograft therapy (front-line = 14, salvage = 1). the majority (n = 17, 85%) were allografted in cr1. median f/u for survivors was 26.3 (3.9-128.8) months. median time-to-neutrophil and platelet engraftments were 16 (12-26) days and 15 (7-45) days, respectively. five pts never dropped s386 platelet counts below 20 000/μl. three pts (mac = 2, ric = 1) relapsed at 6, 7, and 99 months, respectively. all 3 relapsed with marrow involvement (1 had also skin involved). mean os was 81.9 (53.1-110.8) months. one-year and 3-year os were 85% (95% ci = 64-95%) and 70% (95% ci = 48-85%), respectively. there was no difference in 3-year os when comparing mac versus. ric (hr = 1.68 (95% ci = 0.34, 8.4), p = 0.53). median time to onset of acute gvhd was 35 (10-117) days; grade ii-iv acute gvhd occured in 6 cases. chronic gvhd was seen in 5 cases (mild = 2, mod/severe = 3). allo-hct is an effective therapy for bpdcn resulting in durable remissions. encouraging outcomes observed in this analysis may be explained by offering allo-hct early in the disease course and in the setting of complete remission. larger studies are needed to better understand risk factors for relapse to develop post-transplant strategies to improve outcomes. disclosure of conflict of interest: none. a risk-factor analysis for overall survival in patients with acute leukemia that relapse following t-replete haploidentical transplantation: on behalf of the acute leukemia working party of the european society for blood and marrow transplantation s piemontese 1,2 , m labopin 2,3 , f ciceri 1,2 , c schmid 2,4 , a ruggeri 2,3 , w arcese 5 , z gulbas 6 , y koc 7 , j tischer 8 , b bruno 9 , w depei 10 , d blaise 11 , d beelen 12 , g ehninger 13 , a boumendil 2,3 , m houhou 2,3 , m mohty 2,3 and a nagler 2, 14 relapse of acute leukemia is the leading cause of transplantation failure with devastating results. relapse post t-replete haploidentical transplantations (haplo-sct) is not well characterized. the objective of this study was to identify riskfactors for overall survival in patients with al that relapsed after a haplo-sct. from 2007 to 2014, 1660 haplo-sct were performed in 186 ebmt centers as first allogeneic transplantations for adults with acute leukemia. out of 657 patients for whom we were able to receive updated data, 208 relapsed and were included in this analysis. median follow-up among survivors was 25 months after haplo-sct (2-97) and 7.2 months (1-71) after relapse. median time from haplo-sct to relapse was 5 months (11 d-36m). diagnosis was acute myeloid leukemia (aml) in 72% and acute lymphoblastic leukemia (all) in 28% of the patients, respectively .fifty-two (25%) patients were transplanted in first complete remission (cr1), 42 (20%) in cr2 or cr3, while 114 (55%) were transplanted in active disease. ric regimen was used in 116 (57%) patients and 85 (41%) received bone marrow as stem cell source. post-transplant cyclophosphamide (pt-cy) was used for graft-versus-host disease (gvhd) prophylaxis in 121 patients (58%). fifty-two (25%) of the patients who relapsed post haplo-sct experienced previously acute gvhd and 42 (21%) chronic gvhd post transplantation. treatment of relapse varied and included: none in 42 (21%), ist withdrawal only in 15 (8%), chemotherapy (ct) only in 59 (30%), tyrosine-kinase inhibitor (tki) only in'(2%), tki and ct in 6 (3%), dli only in 9 (4%), subsequent transplant in 12 (6%), ct and dli in 37 (19%), ct and subsequent transplant in 8 (4%), tki ct and subsequent transplant in 1 (0.5%), dli and subsequent transplant in 5 (2.5%) patients. donors for second allogeneic transplant were unrelated (n = 1), haploidentical (n = 23) and cord blood (n = 2). second transplant was performed in cr for 8 patients and in relapse for 18 patients. only 2 patients who received a second haplo were alive at 47 and 69 months post second transplant. the majority of patients who received dli were in relapse at time of dli (81%), and 26% achieved cr after dli. os 1y after dli was 27%, 7 patients being alive at a median time of 18 mo (4-55) post dli. overall, the one-year overall survival (os) following relapse was 17% (95% ci: 11.6-22.3). in univariate analysis disease status at haplo-sct (cr vs active disease), cytogenetics (good/intermediate vs poor) and median time from haplo-sct to relapse (4 or o 5.03 months) were associated to a higher os at one year after relapse: 27% (p = 0.003), 27% (p = 0.03) and 25% (p o10 -4 ), respectively. in multivariate analysis complete remission at haplo-sct (p = 0.004; hr 0.64; ci:0.47-0.87) and time from haplo-sct to relapse higher than 5.03 months (p = 0.001; hr 0.58; ci: 0.41-0.81) were risk factors for a higher os after relapse. in the 58 patients transplanted in cr and relapsing more than 5 month after haplo, 1 and 2 y os were respectively 33% and 20%. these findings suggest that similar to other transplantation setting os for acute leukemia that relapse post haplo-sct is dismal. disease status at transplant and time from transplant to relapse are the two important prognostic factors that can predict somewhat better survival. indication for second transplant should be carefully evaluated. integrations with novel therapies are in unmet need to prevent and treat relapse post haplo-sct. disclosure of conflict of interest: none. patients (pts) with aml who relapse after autologous stem cell transplantation (asct) have a dismal outcome but some can be rescued with an allogeneic transplantation (allohsct). yet, available evidence presently stems from analyses of limited patient numbers. we decided to analyze the ebmt registry to evaluate the outcome and determine the prognostic factors in a large series of such pts. the ebmt registry was screened for adult pts with de novo aml (non-apl) who received an allograft in cr2 or first relapse (2000-2015) after being autografted in cr1. pts receiving ex vivo t cell depletion (tcd) were included only if they received a haploidentical allohsct. inclusion criteria were met by 537 pts (48% female, median age 45 [range 18-78] years). median time from asct to relapse was 10 (range 0.6-176, iqr 5.8-19.1) months. at allohsct, pts were in 1 st relapse (25%) or cr2 (75%). donors were matched sibling (18%), unrelated (57%), haploidentical (13%), or cord blood (12%), respectively. conditioning was myeloablative in 46% and reduced intensity in 54% of the pts, respectively. the median follow up was 52 months (range o1-167 months). at 3 years post allograft (figure), leukemia free survival ( 4 -18] of the pts. all factors significantly associated with ⩾ 1 endpoint in univariate analysis were entered in a multivariate cox regression model (table 1) . ri was lower in pts transplanted in cr2 rather than in relapse (31.1% vs 44.7%; hr 1.76, p = 0.004) and in pts who relapsed later (410 months, median value) as opposed to those who relapsed early post asct (25.5% vs 43.2%; hr (per month) 0.97, p o10 -3 ). ri was lower in pts transplanted with an unrelated donor (ud) in comparison to those transplanted from a matched sibling donor (29.1% vs 50.5%; msd, hr: 0.49, p o10 -3 ). patient age, poor cytogenetics, transplantation in relapse, previous tbi for asct, myeloablative conditioning (mac) vs reduced intensity (ric) and ud, haplo or cbt vs msd all significantly increased nrm. lfs was significantly better in pts with good risk (47.3%) than in pts with intermediate risk or poor risk cytogenetics (29%; hr 0.62, p = 0.007) or in pts who relapsed late (per month: hr 0.99, p = 0.003) post asct. lfs was worse in pts who previously had received tbi (20% vs 45%; hr = 1.82; po10 -3 ). the same prognostic factors were significant for os. haploidentical (hr 2.25, p = 10 -3 ) and cord blood (hr 2.09, p = 0.003) transplants resulted in lower os than those from msd. finally, date of transplant significantly influenced os which was higher in pts transplanted after january 2008 vs those allografted before; 48.2% vs 31.7%, hr (per year) 0.96, p = 0.02). about one third of adult patients with aml who relapse post asct can be rescued with an allogeneic transplantation, especially if the duration of persisting cr post asct is long and no tbi was received in the past. transplantation from an msd while in cr2 rather than at relapse offers the best outcome. disclosure of conflict of interest: none. high incidences of graft-versus-host disease (gvhd) and relapse have seriously impeded the widespread application of haploidentical hematopoietic stem cell transplantation (haplo-hsct) for high-risk acute leukemia lacking conventional hla-matched donors. one hundred and ten high-risk acute leukemia patients underwent haplo-hsct with idarubicin (ida) intensified conditioning regimen (ida intensified bucy2 for acute myelocytic leukemia (aml) and ida intensified tbi-cy for acute lymphoblastic leukemia (all)). for donor-recipient hla 3/6 or 4/6 transplant, we separately administered a total of 9 mg/kg or 6 mg/kg antithymocyte globulin (atg) and basiliximab for gvhd prophylaxis. all enrolled patients were observed longitudinally until death or lost to follow-up. the 100-day cumulative incidences of ⅱ-ⅳ and ⅲ-ⅳ agvhd for all patients were 30.3%, 14.7%, respectively. the 2-year cumulative incidence of extensive cgvhd was 12.2%. the relapse rate was 18.2%. the 3-year probability of overall survival (os) reached 63.1%. the patients in non-complete remission (nr) showed significantly higher relapse and worse survival than complete remission (cr) minimal residual disease (mrd) (-) and cr mrd (+) patients. however, the relapse, 3y-os and disease-free survival (dfs) of cr mrd (-) did not differ from cr mrd (+) patients, indicating our intensified transplant technique could overcome the poor prognosis of mrd. for whatever aml or all patients, the relapse rates, agvhd, cgvhd and the estimated 3-year os and dfs between two atg group were equivalent, except that all patients in atg 9 mg/kg experienced higher relapse (33.0% vs 19.2%, p = 0.226). although the incidence of cytomegalovirus (cmv) reactivation in atg 9 mg/kg and 6 mg/kg was 77.4%, 72.9%, the average episodes of cmv reactivation were remarkably [p512] higher in 9 mg/kg. our ida intensified haplo-hsct technique could improve the outcome of high-risk acute leukemia and could be recommended as a good alternate for patients lacking hla-matched sibling donors. diagnosed secondary aml were randomized 1:1 to cpx-351 or standard 7+3 therapy. cpx-351 induction was 100 units/m 2 on days 1, 3, 5 (first induction) and days 1, 3 (reinduction); 7+3 first induction was cytarabine 100 mg/m 2 /day × 7 days and daunorubicin 60 mg/m 2 on days 1, 2, 3, and reinduction was cytarabine 100 mg/m 2 /day × 5 days and daunorubicin 60 mg/ m 2 on days 1, 2. a dynamic allocation procedure stratified patients by age group (60-69 or 70-75 years) for each study arm. patients with complete response (cr) or cr with incomplete platelet or neutrophil recovery were considered for allogeneic hct, based on institutional criteria. overall survival (os) landmarked at the time of hct was assessed. a total of 309 patients were enrolled on the induction trial. . patient and aml characteristics in the hct age subgroups were generally similar between arms. in both age subgroups of patients receiving hct, median os was longer in the cpx-351 arm than in the 7+3 arm (table 1 ). in the 60-69 group, serious adverse events (saes) prior to hct in the cpx-351 and 7+3 arms occurred in 28% and 22% of patients, respectively; in the 70-75 group, in 13% and 67%, respectively. the most common sae was febrile neutropenia (cpx-351, 11.5%; 7+3, 5.3%), occurring in all age groups. relapse after allogeneic haematopoietic stem cell transplant (allo-hsct) for acute myeloid leukaemia (aml) and myelodysplastic syndrome (mds) remains the main cause of treatment failure. it is associated with dismal prognosis and short survival. proposed salvage strategies are tapering of immunosuppressive therapy, re-induction with chemotherapy and consolidation with donor lymphocyte infusion (dli) or second allo-hsct, although, results remain disappointing. azacitidine (aza) and dli has proved to be an effective and well-tolerated outpatient approach in this setting, and results in at least temporary disease control in the majority of patients, thus, representing a valuable alternative to current treatments. between january 2010 and november 2016, 16 patients with relapsed aml or mds after allo-hsct were treated with subcutaneous aza 100 mg/m 2 days 1-5 every 28 days and escalating doses of dli if feasible at manchester royal infirmary, uk. aza was continued until cr or disease progression. patients characteristics: median age 60 (range 45-69) years, 56% males, diagnoses were aml (n = 12) and mds (n = 4). five (31%) patients had either monosomal or complex karyotype. fifty percent of patients were in cr1 before transplant, 12.5% in cr2, 12.5% had a partial response and 25% did not receive any chemotherapy before the transplant. fifteen out of 16 received fludarabine-base reduced intensity conditioning regimen and all but one had a t-cell depleted graft. at relapse 88% had mixed donor chimerism. median time to relapse was 9.5 (range 2-21) months after allo-hsct. with a median follow up of 6.5 (range 1-25) months a median of 5 (range 1-16) courses of aza were administered and median of 2 (range 1-6) dli were infused. doses of dli were administered starting at 0.1 x107/kg and escalating by log5. aza and dli infusions were well tolerated; only two patients withdrew due to intolerance. seven patients were admitted at least once due to infections (86%) or progressive disease. only two patients developed mild gvhd grade 1. complete remission was achieved in 12.5% patients and stable disease in 56%. patients in cr had full donor chimerism. median overall survival for patients in cr was 25 months compared to 9 months for those who did not respond (p = 0.031). patients with more than 20% blasts on bone marrow at time of relapse after allo-hsct had a worse outcome than those with less than 20% blasts (11 months and 25 months respectively, p = 0.09). no differences were seen when compared time to relapse ( o 6 months vs ⩾ 6 months) s390 and outcome, or disease and overall response, although numbers in this series are small. image/graph: overall survival following azacitidine and dli, patients in complete remission, stable disease and disease progression. azacitidine and dli can provide long term remissions in patients with relapsed aml/mds post allo-hsct with low toxicity. lower disease burden at relapse carries better outcomes. low rates of gvhd are seen following azacitidine and dli most likely showing the immunomodulatory effect of azacitidine described by other groups. acute myeloid leukemia (aml) is a frequent complication in patients affected by telomere maintenance disorders ('telomeropathies') such as dyskeratosis congenita (dkc). treatment of aml in dkc patients by chemotherapy and hematopoietic stem cell transplantation is characterized by frequent remission failure, high organ toxicity and poor outcome. a 27-yearold patient with aml was admitted to our hospital in december 2014. he had been treated with 6 cycles beacopp for hodgkin´s lymphoma (hl) in 2009. on admission, the patient presented clinical signs of premature aging with hair greying and lack of fully recovered hair growth after chemotherapy (cx) for hl. flow-fish analysis revealed tl below the 1% percentile within leucocytes in line with the suspected diagnosis of telomeropathy. retrospective tl analysis by confocal q-fish from bm at hl diagnosis confirmed short tl before the start of any chemotherapy. he received standard aml induction cx (3+7), but follow-up revealed persistence of aml. salvage cx with flag-ida was applied resulting in partial remission with only weak regeneration of normal hematopoiesis. the patient received an allogeneic stem cell transplantation (asct) after conditioning with 140 mg/m 2 melphalan and fludarabin from his hlamatched brother whose tl was found to be normal. after asct, he developed sinusoidal obstructive syndrome and progressive liver failure treated with defibrotide and he was admitted to icu for sepsis. leucocyte count showed sufficient engraftment on day 14; however, liver function recovered only partially. during critical care treatment, the patient showed cardiomyopathy, renal failure and extensive wound healing problems without epithelial proliferation indicative of severe replicative exhaustion. finally, he died due to sepsis with acute liver failure on day 91 after asct. aml arising from dkc is a rare event with substantial impact on patients´prognosis. therapy remains challenging due to poor bm function and high risk of organ toxicity, especially liver failure and lung fibrosis. dose reduction of alkylating agents and avoidance of total body irradiation are necessary in conditioning prior to asct in patients with dkc and aml, however no clear data or recommendations exist for the management of these patients. tl screening can help to identify patients with suspected dkc related bm failure or aml and to identify family donors without telomeropathy. physicians should be aware of possible dkc related aml, especially in familial cases of aml or bone marrow failure, impaired or prolonged recovery following cytoreductive treatment or coincidence of solid (e.g. oral cavity carcinomas) and hematological malignancies. disclosure of conflict of interest: none. chronic graft-versus-host disease and donor lymphocyte infusions in patients with non-de novo acute myeloid leukemia or advanced myelodysplastic syndromes after allogeneic stem cell transplantation pg hemmati 1 , k pfeifer 1 , lg vuong 1 , cf jehn 1 , p le coutre 1 , b dörken 1 and r arnold 1 1 medizinische klinik mit schwerpunkt hämatologie, onkologie und tumorimmunologie, charité-universitätsmedizin berlin, campus virchow-klinikum, berlin, deutschland aml with myelodysplasia-related changes and therapy-related aml (taml), collectively termed secondary aml (saml) in daily clinical routine, represent distinct subgroups in the 2016 revised who classification of myeloid neoplasm and leukemias. as compared to de novo-aml, saml is associated with a poor survival when using conventional chemotherapy approaches. this is mainly due to unfavorable cytogenetics, older age and/or the presence of comorbidities as well as poor response to induction therapy. furthermore, cumulative organ toxicity resulting from treatment of the antecedent solid malignancy in patients with therapy-related disease has to be taken into account. allogeneic stem cell transplantation (allosct) represents the only option to achieve long-term disease control and definitive cure. we retrospectively analyzed 204 patients with saml or advanced mds (eb-2 according to who) transplanted at our center between 1995 and 2015. at the time of allosct, 98 patients (48%) were in complete hematologic remission (chr), whereas 106 patients (52%) had active disease. cytogenetic risk was categorized according to the swog/ecog classification and was favorable (n = 3; 2%), intermediate (n = 94; 46%), unfavorable (n = 84; 41%), or unknown/undetermined (n = 23; 11%). standard myeloablative conditioning (mac) using 12 gy total body irradiation (tbi) and cyclophosphamide was used in 41 patients (20%), whereas fludarabin/busulfan/atg-based reduced intensity conditioning (ric) was applied in 163 patients (80%). grafts were from related (n = 51; 25%) or unrelated (matched: n = 112; 55% or mismatched: n = 41; 20%) donors. the median follow-up of the surviving patients was 46 (5-24) months. a graft failure occurred in 5/204 patients (3%). at last day of follow-up 72/204 patients (35%) were alive and in chr. relapse occurred in 77/204 patients (38%) after a median interval of 4.6 (range: 0.1-135) months. cause of death were either relapse or nrm (gvhd and/or infections) in 69/204 patients (34%) or 56/204 patients (28%). at 1, 3, 5, and 10 years after allosct overall survival (os) or disease-free survival (dfs) of the entire cohort was 56%, 46%, 38%, and 29% or 50%, 38%, 36%, and 27 %, respectively. at the same time points, the cumulative incidence of relapse (ci-r) or non-relapse mortality (ci-nrm) was 30%, 37%, 37%, and 40% or 20%, 25%, 27%, and 33%, respectively. extensive uni-und multivariate analyses revealed a number of factors associated with inferior outcome, e.g. poor-risk cytogenetics, the presence of taml, advanced age, reduced physical performance, and comorbidities, whereas donor type (unrelated versus unrelated), and remission status had no significant impact on overall outcome. furthermore, the development of gvhd, especially the presence of cgvhd, and the use of donor-lymphocyte infusions (dli), either in a prophylactic or pre-emptive setting, were identified as independent predictors for a reduced relapse incidence, which in turn, led to an improved os and dfs. our results indicate that allosct represents an important treatment option for patients with saml. however, a relapse rate of 30% at 12 months prompts the development of novel approaches to prevent early disease recurrence. strategies to augment the graft-versus-leukemia (gvl) effect of allosct may help to improve the results. disclosure of conflict of interest: none. myeloid sarcoma (ms) is a rare hematologic myeloid neoplasm that can involve any site of the body. it can occur as an exclusively extramedullary form or it can be associated with an acute myeloid leukemia (aml), a chronic myeloproliferative neoplasm (mpn) or a myelodysplastic syndrome (mds) at onset or at relapse. the rarity of ms does not enable prospective clinical trials and therefore a specific multicenter register can be useful for the clinical and biological studies of this rare disease. we report the clinical characteristics and outcome of 48 histologically confirmed ms, diagnosed and treated in 9 italian hematological centers in the last 10 years. the patient's median age was 46 years. there were 9/48 de novo extramedullary ms, 24/48 de novo aml-related ms and 15/48 were secondary aml-related ms. the most common extramedullary anatomic sites of disease were: skin, lymph nodes and soft tissues. forty-three patients (90%) underwent a program of intensive chemotherapy including flai, hdac-ida, hypercvad and mec schemes, with a cr rate of 44% (19/43). twenty-two (46%) patients underwent allogeneic sct, 13 from a mud, 8 from an hla-identical sibling donor and 1 from an haploidentical donor. the median os of the whole population (48 pts) was 16.7 months. the os probability at 1, 2 and 5 years was 64%, 39% and 33%, respectively. the os was better in patients that underwent an intensive therapeutic program (median os: 18 months vs 5 months). among the intensively treated patients, in univariate analysis, the os was better in young patients (p = 0.008), in patients that underwent allo-sct (p = 0.009) and in patients that achieved a cr during treatment (p = 0.001), and was worse in pts with secondary aml-related ms (p = 0.007). age, response to intensive chemotherapy and allo-sct were the only three variables that significantly influenced dfs (p = 0.02, p = 0.01 and p = 0.04, respectively). in multivariable analysis, allo-sct and response to intensive chemotherapy remained significant in predicting a better os (p = 0.04 and p = 0.001, respectively), and response to intensive chemotherapy was the only significant variable in predicting dfs (p = 0.01). after allo-sct we observe a survival advantage in patients who achieved a pre-transplant cr (p = 0.008) and in those who developed a chronic gvhd (p = 0.05). patients with ms, both with de novo and secondary forms, still have a very unfavorable outcome and require an intensive therapeutic program, that includes allo-sct, whenever possible. the outcome after allo-sct is positively influenced by the development of chronic gvhd suggesting a graft versus ms effect. disclosure of conflict of interest: none. relapse of acute lymphoblastic leukemia (all) after allogeneic stem cell transplantation (sct) is associated with poor prognosis. blinatumomab may enhance the efficacy of donor lymphocyte infusions (dli) in this specific situation but data on the concurrent use of dli and blinatumomab are sparse. the patient presented here was diagnosed with standard risk pre-b-all (presence of t(3;9); bcr-abl and cd20 negative) at the age of 23. during treatment according to the german multicenter all-study group (gmall) protocol he presented with molecular relapse and 8 months after initial diagnosis he received a tbi-based myeloablative sct from an unrelated hla-identical (10/10) donor. post sct he was negative for minimal residual disease (mrd) with 100% donor engraftment. given the high relapse risk he received 3 prophylactic dli without occurrence of graft-versus-host disease (gvhd). one year after 1st sct he presented with an extramedullary (testes) and molecular relapse. after remission induction resulting in negative mrd he received a 2nd sct from an alternative, hlaidentical (10/10) donor after reduced intensity conditioning. this again resulted in negative mrd with 100% donor chimerism without any gvhd. six months after 2nd sct he presented with bone marrow relapse. we decided on the concurrent use of blinatumomab and dli. the first cycle of blinatumomab was initiated at standard dose including dose escalation without relevant toxicities. on day 40 of the 2nd cycle, i. e. in the infusion-free interval before the 3rd cycle the patient received the first dli at 1x107 cd3/kg. no toxicities or gvhd occurred. the 3rd cycle of blinatumomab was initiated and a second dli at 2.5x107 cd3/kg was applied on day 3 of the 3rd cycle. on day 32 of the 3rd cycle, i. e. day 29 after 2nd dli the patient presented with signs of overlap gvhd (mouth, skin) and topical steroids were started. upon progression of clinical gvhd systemic steroids were initiated with immediate response. steroids were rapidly tapered and a 4th cycle of blinatumomab was started. gvhd did not recur. current staging after the 4th cycle blinatumomab, i.e. on day +413 after 2nd sct and 7 months after initiation of blinatumomab treatment revealed complete remission with negative mrd, 100% donor chimerism and no signs of extramedullary relapse. counts of cd4-cells at that time point were 147/μl. no relevant infections or relevant blinatumomab-associated toxicities were present during the entire course after the 2nd sct. in this case concurrent treatment of blinatumomab and dli resulted in the longest disease-free interval for our patient compared to preceding chemotherapy or dli alone. together with the small number of reported cases (ueda et al.) this supports the concept of concurrent blinatumomab and dli as an effective post sct treatment. the objective of the study is to evaluate the clinical efficacy and safety of decitabine (dac) in combination with haag regimen [homoharringtonine (hht), cytarabine (ara-c), doxorubicin (acla) and recombinant human granulocyte colony stimulating factor (g-csf)] for advanced patients with acute myeloid leukemia (aml). thirty-six patients with advanced aml receiving dac combined with haag chemotherapy in our center from december 2012 to august 2015 were enrolled in this study. eighteen of them were refractory or relapsed aml, and another 18 patients were those who didn't achieve complete remission (cr) after a course of induction chemotherapy. the therapeutic responses, side effects and longtime survival were retrospectively analyzed. after a course of treatment, the rate of cr and partial response (pr) was 58.3% (21/36) and 22.2% (8/36) respectively, while the overall response rate (orr) was 80.6% (29/36) in the cohort. for the patients with refractory or relapse aml, cr was 61.0% (11/18), pr was 22.2% (4/18), and orr was 83.3% (15/18). while for the other not getting cr after a course of induction chemotherapy, cr was 55.6% (10/18), pr was 22.2% (4/18), and orr was 77.8% (14/18). grade 4 hematological toxicities were observed in all patients, and 72.2% cases experienced infection. and all non hematological side effects were mild and well-tolerated. with a median follow-up of 7.5 (0.5~33.3) months, the 1-year overall survival (os) rate was 43.3%, 24.2% for the refractory or relapsed aml patients, and 61.6% for those not achieving cr after a course of induction chemotherapy. the difference was significantly (p = 0.01). conclusion dac combined with haag regimen is safe and effective salvage treatment for advanced stage aml patients. disclosure of conflict of interest: none. aml patients harboring flt3-itd mutation are associated with decreased survival compared to patients without flt3-itd mutation. nevertheless, whether flt-itd mutation also has negative impact on the post-transplant survival is less clear. for flt3-itd mutated aml, a decreased leukemia-free survival (lfs) after allogeneic hsct was observed in ebmt analysis but not cibmtr. in this study, unlike studies of ebmt or cibmtr which only pre-specified populations of patients were analyzed (cr1 in ebmt, cr1+cr2 in cibmtr), we examined the prognostic impact of flt3-itd mutation on post-transplant outcome of "all" the adult aml patients reported to taiwan bone marrow transplant registry (tbmtr). tbmtr is a research collaboration affiliated to the taiwan society of blood and bone marrow transplantation. it comprises all the 14 transplantation centers in taiwan that contribute detailed data on hsct. adults aged ⩾ 18 years with a diagnosis of aml and with known flt-itd mutation status in the registry were included. patient characteristics and transplant outcome following allogeneic hsct for flt3-itd mutated and nonmutated aml were compared. kaplan-meier estimates were used to calculate the probability of lfs and overall survival (os). multivariable analyses for lfs and os were performed using cox proportional hazards model. 365 patients who met the eligibility criteria were enrolled for analysis. the median follow-up of survivors was 21 months. of the 365 patients, 94 (25.8%) were positive and 271 (74.2%) were negative for flt3-itd mutation. flt3-mutated patients had significantly more transplantation at cr1 (57.4%), shorter time interval between diagnosis and hsct (5.6 months), and higher wbc count at diagnosis (51.7 × 10 9 /l) comparing to patients without flt3 mutation (43.5% at cr1, 6.5 months from diagnosis to hsct, and 11.8 × 10 9 /l wbc count at diagnosis). significant more flt3 mutated patients had intermediate-risk (80.9%) and normal (64.9%) karyotype at diagnosis. the age, donor type, stem cell source, conditioning regimen, and atg use were not significant different between flt3-mutated and non-mutated patients. of the whole population, flt3 mutation status did not negatively impact the transplant outcome (2 years os for flt3 mutated and non-mutated patients: 45.2% vs 50%, log rank p = 0.624; 2 years lfs for flt3 mutated and non-mutated patients: 40.2% vs 32.4%, log rank p = 0.192). when different pre-transplant conditions (cr1, subsequent cr, and no cr) were analyzed separately, flt3-itd mutation status is still not a significant prognostic factor of os and lfs for patients in cr1 (equally good) and no cr (equally bad). however, for patients in subsequent cr, flt3-itd mutation is the only significant factor predicting poor os and lfs in multi-variable analysis (median os and lfs for flt3 mutated and nonmutated patients: 378 vs 1252 days, log rank p = 0.005; 204 vs 1049 days, log rank po 0.001 respectively). the incidence of non-relapse mortality, grade 3/4 acute gvhd and extensive chronic gvhd is comparable between flt3-mutated and nonmutated patients. flt3-itd mutation is a significant and strong predictor of poor survival for aml patients in subsequent cr at hsct. for flt3-itd non-mutated aml, a sizable portion of patients can have disease free survival after allogeneic hsct at subsequent cr. however, allogeneic hsct at cr1 should be strongly recommended for flt3-itd mutated aml. [p523] disclosure of conflict of interest: none. allogeneic stem cell transplantation (asct) is a curative strategy in acute myeloblastic leukemia (aml) and myelodysplastic syndrome (mds). however, relapse keeps being the main cause of treatment failure. extramedullary relapse (er) is a rare event and its management is not well standardized. we retrospectively analyzed patients who received asct from 2006 to 2016 and developed er in our centre. we performed a descriptive study to analyze characteristic of these patients, post-relapse treatment and survival. statistic analysis was performed using spss v.22. we found a total of 18 patients with er, one of them with 2 er after 2 consecutive asct, so we analyzed 19 cases of er. patient and transplant characteristics are summarized in table 1 . at day +100, 95% of patients were in complete response (cr). er occurred after a median of 13 (2-98) months post-asct. eleven patients (58%) presented with a bone marrow relapse concomitant with the er. er affected central nervous system (cns) in 7 patients (36.8%), bone in 4 patients (21%), skin or soft tissue in 3 patients (15.8%), mama in 2 patients (10.5%), ocular globe in 2 patients (10.5%) and teste in 2 patients (10.5%). two of them presented with multiple sites affected. between the 7 patients who developed cns relapse, 2 of them had received intrathecal prophylaxis. regarding post-er management, immune modulation was conducted in 16 patients (immunosupression tapering in 9, donor lymphocyte infusions in 4 and both strategies in 3). all patients except one received systemic treatment (salvage chemotherapy in 11, azacitidine in 5, low dose arac in 1 and atra in 1 patient with a promyelocytic leukemia). together with systemic treatment, 12 received radiotherapy and intrathecal therapy was used in all 7 patients with cns involvement. response: 12 out 18 patients treated, 12 (63.2%) achieved cr and 6 (31.6%) progressed. two responding patients received a 2 nd asct. after a median follow-up of 67 months (15-123), 8 patients are alive and disease free, with an estimated overall survival of 45% at 5 years. patients receiving salvage chemotherapy followed or not by a 2 nd asct experienced a significantly better os than those receiving other therapies (median os 92 vs 10 months; p = 0.005). patients with bone marrow involvement at relapse show a worse prognosis (median os 39 vs 54 months; p = 0.23) although not statistically significant due to small number of patients (image 1). ten patients died due to disease progression. er must be considered in patients receiving an asct in case of organ symptoms. patients can be rescued with salvage chemotherapy followed or not by a 2 nd asct achieving good results in terms of long term os. it seems that involvement of bone marrow at relapse confers a worse prognosis, what should be confirmed in a larger series of patients. [p524] disclosure of conflict of interest: none. flag-ida regimen as bridge therapy to allotransplant in refractory/relapsed aml patients: a single-center experience c pasciolla, m delia, d pastore, p carluccio, a ricco, a russo rossi, a mestice, f albano and g specchia university of bari, italy although treatment outcome in acute myeloid leukemia (aml) adult patient has improved over the past decade, relapse still occurs in up to 50-70% of cases. furthermore, 15-30% of patients fail to achieve complete remission (cr) because of treatment-resistance. the management of primary refractory and/or relapsed disease remainschallenging for clinicians. in our study, we reviewed the outcome of 116 refractory and/or relapsed aml patients who underwent salvage therapy with the flag-ida regimen between 2005 and 2015 at our institution. the study aim was to determine the efficacy of the flag-ida regimen in order to clarify which variables (who ps, ldh, bone marrow, peripheral blood blasts and platelets counts, white blood cells (wbc), pmn, molecular-cytogentic risk, duration of response and relapsed or refractory disease), present before starting flag-ida treatment, might have an impact both on cr and on os. we analyzed 116 consecutive adult patients (56 males, 60 females; median age 48 years, range 17-72) with newly diagnosed acute myeloid leukemia refractory to standard induction regimens or relapsed after cr, who received the flag-ida protocol as salvage therapy between january 2005 and december 2015. sixty-eight of the 116 patients (58%) were in first relapse, forty-seven patients (42%) were refractory to conventional chemotherapy. median wbc count before salvage therapy was 10.1 x109/l (range 0.56-88). median bone marrow and peripheral blasts counts were 52 and 20%, respectively; median platelets count was 91x10e3/μl. according to the fab classification, 14 patients had m0, 5 m1, 53 m2, 16 m4, 22 m5, 4 m6, 2 had biphenotype acute leukemia. according to molecular-cytogenetic risk stratification 51 (44%), 44 (38%) and 21 (18%) patients belonged to poor, intermediate and good risk group, respectively. sixty-nine of 116 patients (59%) achieved complete remission (cr); forty-seven 41%) patients were refractory to the salvage therapy. in multivariable analysis, variables with positive impact on response rate were lower wbc counts (o10e3/μl, p = 0.0047), higher platlets counts (450x10e3/μl, p = 0.046), molecular-cytogenetic risk (p = 0.032), duration of response in relapsed aml (p = 0.006) and relapsed rather than primary refractory disease (p = 0.042), respectively. median os was 17 months (m). cox regression analysis confirmed that both higher platlets counts, p = 0.002 (17 (450x10e3/μl) vs 11 m (o50x10e37ul), log rank, p = 0.05) and relapsed disease, p = 0.041 (23 (relapsed) vs 17 m (refractory), gehan-breslow, p = 0.021) correlated with better survival. of note, molecular-cytogenetic risk evaluated before starting treatment was associated with cr, while no correlation was found with os. our data seem to confirm the value of flag-ida in relapsed amland may suggest its best usage as bridge-therapy in patients awaiting allotransplantation. disclosure of conflict of interest: none. s395 leukemia relapse is the major cause of death in patients received allogeneic hematopoietic stem cell transplantation (allo-hsct). the precise etiological mechanisms of leukemia relapse remain unclear. both leukemia cells themselves and hematogenesis micro-environment are involved in the relapse event. in our previous study, we reported a case of donor derived relapse of acute myeloid leukemia (aml) after allo-hsct. the patient and his donor-sister both harbored a germline mutation(c.584-589dup) in cebpa gene. donor hematopoietic cells transformed to aml by developing two somatic cebpa mutations (247dupc and 914-916dup) in the patient's microenvironment. hence we suspect that 584-589dup mutation of cebpa gene may altered hematopoiesis microenvironment and increased the survival of aml cells. to conform our hypothesis, we transfected mesenchyme stem cells (mscs) with cebpa 584-589dup or wide type and took vector as control. aml cell line hl60 cells were co-cultured with transfected mscs and then treated with 40ng/ml doxorubicin. apoptosis and cell cycle were detected at day 3. mscs protected hl60 cells from toxicity of doxorubicin. this protection was enhanced by overexpression of cebpa 584-589dup . apoptosis rates of hl60 cells in group of msc-vector and msc-cebpa 584-589dup were 61.7 ± 5.8% vs 30.9 ± 5.6% (p＜0.05). a larger part of hl60 cells remains quiescent with s396 higher rate of g0/g1 phase in msc-cebpa 584-589dup group, which may reduce the sensibility of hl60 cells to doxorubicin. to explore mechanisms involved in the alteration of microenvironment, we performed rna sequence with each group of mscs. we found that col1a1, col1a2 and col3a1 were upregulated in msc-cebpa 584-589dup group compared with msc-cebpa wt group (col1a1:cebpa wt vs cebpa 584-589dup was 1713.65 vs 2317.88, p = 4.70e-19; col1a2:cebpa wt vs cebpa 584-589dup was 2260.02 vs 2755.81, p = 2.76e-10; col3a1: cebpa wt vs cebpa 584-589dup was 746.20 vs 964.82, p = 1.06e-06). furthermore, we found that ddit3 and herpud1 genes, which were important factors in cellular unfolded protein response(upr) and to topologically incorrect protein, failed to augment in cebpa 584-589dup group (ddit3 : vector vs cebpa wt the cure rate of childhood acute lymphoblastic leukemia (all) has improved considerably and approaches 80% today. however, the outcomes of patients who suffer from leukemic relapse remain unsatisfactory. despite the high cure rate of children and adolescents with all a subgroup of patients benefit from allogeneic hsct. allo hsct remains the standard treatment for intermediate/high risk aml patients. 59 patients, all = 42 and aml = 17 age 1 to 20 years with median age 11 years, m/f = 33/26(m/f all = 15/18, aml = 8/9) underwent sct in our hospital (from 2012 to 2016). fifty-eight patients transplanted allo hsct and 1pt aml auto hsct. conditioning regimens consisted of busulfan (iv) +cyclophosphamide for allo and cyclophosphamide + vp16 +cytarabine for auto hsct. peripheral blood (pb) was the source of progenitor cells in 47 patients, bone marrow (bm) in 11 patients and cord blood in one patient. in allo hsct, 50 patient transplanted 6/6 matched and 8 patients 5/6 matched. gvhd prophylaxis regimen was cyclosporine + mtx. all patients engrafted. in allogeneic pbsct all patients' median time to absolute neutrophil count (anc) 40.5 × 109/l was 12 days, and the median time to platelet count 420 × 109 was 15 days vs 17 and 21 days in allo bm all patients. in allogeneic pbsct aml patients median time to anc 40.5 × 10 9 /l was 12 days, and the median time to platelet count 420 × 109 was 14 days. (all patients with aml transplanted with pb). at present 47 pts are alive (36 all, 11 aml) and 12 pts died due to ards, vod, hemorrhagic stroke, sepsis and relapse. trm was 9% at 100 days. median time of death after transplantation was 195 days in all and 153 in aml. in allo pbsct all patients hospitalization period were 36 days vs 45 in allo bm all patients. acute gvhd appeared in 78% pts. chronic gvhd appeared in 55% pts. with a median follow-up of 35 months (3-51 months) after transplant the event-free survival were 73% and four years overall survival 75% in all patients. a median follow-up of 32.5months (4-48months) after transplant the event-free survival were 68% and three years overall survival 63% in aml patients. hematopoietic stem cell transplantation can lead to durable remissions in children and adolescents with leukemia and increase in survival of children. pbsct in childhood all was consistent with significant faster anc and platelet recovery in allogeneic pbsct, hospitalization was shorter. longer follow-up is required to evaluate fully efficacy and long-term results. disclosure of conflict of interest: none. group hla-c1/c2 subtypes were defined as previously practiced. median age of patients was 41, 47% of them were male. allo-hscts were performed from 60% unrelated donors vs 40% related donors. remission status was detected in 67% of patients whereas 12% had active disease pre-transplant. stem cell sources were as follows: 88% peripheral blood, 7% bone marrow, 3% cord blood, 2% bone marrow plus peripheral blood. the most frequent fab subtype was aml-m4. patients were grouped by hla-c status: 23% c1/c1 homozygote, 57% c1/c2 heterozygote and 20% c2/c2 homozygote. the frequency of hla c donor/recipient mismatch allo-hscts was 19%. relapse was detected in 26% of patients. the relapse risk was significantly lower in c1/c1 homozygote patients compared to c2/c2 homozygotes (13% vs 36%, p = 0.02). lfs was similar between c1/c1 homozygote group and c2/c2 homozygote group (p = 0.324) (figure 1 ). in multivariate analysis (age, sex, remission status, related/ unrelated transplant, aml subtype), lfs was increased by pre-transplant remission status (p1 year). there was no difference detected between 2-years os in c1/c1 homozygote group and other groups (57% vs 50%, p = 0.51) (figure 2) . when similar analysis were repeated with donor hla type results were not significant. our results confirm two earlier published reports on aml and all. even in the absence of kir genotyping, hla group c1 has a protective effect. if hla matched donor is not possible a donor-recipient hla-c mismatch favoring c1 to c2 may be preferable. disclosure of conflict of interest: none. immunomodulatory kits do not induce aml-blasts' proliferation ex vivo: ipo-38 is an appropriate and reliable marker to detect and quantify proliferating blasts c plett, dc amberger, a rabe, d deen, z stankova, a hirn, y vokac, j-o werner, j schmohl, d krämer, a rank, c schmid and h schmetzer aml-blasts can be converted to dcleu by immunomodulatory 'kits' (ex vivo). t-cells' energy can be overcome after stimulation with dc/dcleu and results in antileukemic reactivity. a potential induction of blast-proliferation (e.g. by immunomodulatory kit-application in vivo) in aml-pts has to be excluded. 8 kits containing combinations of gm-csf with 1-2 additional factors (pge-1/2, picibanil, ifnα, tnfα, calciumionophore) were studied with respect to the generation of dc/dcleu from blasts, mediation of antileukemic reactivity (after dc/dcleu-stimulation) and with respect to their potential to induce blast-proliferation in a whole blood (wb) culture-system. we studied 3 different markers (ipo-38, ki-67, cd71) and quantified blast proliferation before/after culture. we correlated findings with (ex vivo) antileukemic functionality, with disease-entities and the course of the disease. dc-generation: we could generate dc/dcleu regularly from wb culture from 36 aml-pts. detection of blast proliferation: ø65.6 % (range46-82%) of uncultured blasts expressed ipo-38, 33.1% (16-50%) cd71, 25.4% (8-43%) ki-67. induction of blast proliferation: pooling all results we found lowest amounts of proliferating blasts after culture with kit i (gm-csf+picibanil, 10% ± 13.32), kit k (gm-csf+pge2, 9.14% ± 12.01), kit m (gm-csf+pge1, 7.67% ± 11.79). amounts of proliferating blasts were lower compared to uncultured cells. highest expression of proliferating blasts was found with ipo-38 followed by cd71 and ki-67.we found few individual aml-samples with increased blast-proliferation after ex vivo kit-culture. antileukemic activity: t-cells stimulated with dcleu (generated with kits) improved antileukemic activity. correlations between blast-proliferation and antileukemic activity will be presented. clinical correlations: pts with bad (vs good) cytogenetic risk were characterized by higher proportions of proliferating blasts in uncultured blasts; in some pts with iron-deficiency anemia (ida) proportions of cd71+unculured blasts were lower than of ipo-38/ki-67+ blasts. ipo-38 is a stable marker to be used to quantify proliferating blasts in aml-pts. cd71 is also a good marker, although not suitable for some pts with ida, ki-67 is no reliable marker for every given pt. subtypes of pts correlated with proportions of proliferating blasts. in general kit treatment of blasts did only exceptionally induce blast proliferation ex vivo. in general lowest risk for blast proliferation was seen after culture with kit i, k and m. t-cellstimulation with dc/dcleu generated after kit-treatment resulted regularly in antileukemic reactivity. we conclude that an in vivo treatment of aml-pts with kits i, k or m might be safe (no induction of blast proliferation). disclosure of conflict of interest: none. the occurrence of additional cytogenetic abnormalities (acas) is common in philadelphia chromosome-positive acute lymphoblastic leukemia (ph+ all), but is of unknown significance in the tyrosine kinase inhibitor era. recent study [aldoss et al., 2015] has revealed the acas appear to have a significant deleterious effect on outcomes post-hsct in adult ph+ all patients only. we retrospectively analyzed data from adult and pediatric patients with ph+ all who had undergone allogeneic hematopoietic stem cell transplantation (allo-hsct) at our university between 2008 and 2015. among 65 patients with ph+ all, 53 patients had available data on conventional cytogenetics before allo-hsct. all patients and transplant characteristics are listed in table i . thirty-three of 53 patients (51%) had isolated t(9;22). acas were revealed in 20/53 (31%) pts, including 13/53 (20%) pts with ⩾ 3 cytogenetic abnormalities (with complex karyotype, ck). the median follow-up was 645 (26-2461) days. overall survival (os) and event free survival (efs) were 48% (95% ci 7-33) and 30% (95% ci 17-44) at 4 years, respectively. in univariate analysis, prognostic factors associated with increased os and efs were donor type (match related/match unrelated vs haploidentical; p = 0.02 for both), the disease status at transplant (cr1 vs beyond cr1; p = 0.01, only for efs), acas (aca-vs aca+; p = 0.04, only for os) and, especially, the complex karyotype (ck-vs ck+; p = 0.01, only for os) (figure 1 ). multivariate analysis showed that the independent prognostic factors for os and efs remained the complex karyotype (hr-2.79, 95% ci, 1.23-6.34; p = 0.01) and disease status at transplant (hr-2.15, 95% ci, 1.13-4.09; p = 0.01), respectively. the study demonstrates the acas and disease status at allo-hsct to be independent prognostic factors not only for adult, but for pediatric ph+ all patients too. up to 20% of newly diagnosed acute myeloid leukemia (aml) patients (pts) present initially with hyperleukocytosis consequently placing them at increased risk for morbidity and mortality during induction. 1,2 whereas early publications 3 have indicated that hyperleukocytosis is an adverse prognostic factor associated with poor long term outcome, it is currently unknown whether hyperleukocytosis still retains prognostic value for aml patients undergoing allogeneic stem cell transplantation. furthermore, it is unknown whether hyperleukocytosis retains prognostic value when modern molecular markers such as flt3 and npm1 are accounted for. we hypothesized that hyperleukocytosis at initial diagnosis is still an independent adverse prognostic factor influencing long term outcome in aml pts undergoing allogeneic stem cell transplantation. we performed a retrospective analysis using the multicenter registry of the acute leukemia working party (alwp) of the european society for blood and marrow transplantation (ebmt). pts included in the analysis were over 18 years of age, with de-novo non-m3 aml, a presenting white blood cell count of over 100k, with an hla matched related or unrelated donor, transplanted between 2005 and 2014. clinical outcome indices of hyperleukocyotosis pts namely, non-relapse mortality (nrm), graft versus host disease (gvhd), relapse incidence (ri), leukemia free survival (lfs), overall survival (os) and gvhd-free/relapse-free survival (grfs) were compared to a cohort of pts without presenting leukocytosis. multivariate analyses were used to assess whether hyperleukocytosis was independently associated with ri, nrm, os, lfs, and grfs. age, gender, number of chemotherapy inductions, cytogenetics, donor type, fms-like tyrosine kinase-3 (flt3) status, nucleophosmin (npm1) status, and conditioning intensity were covariates for regression modeling. a cohort of 357 pts with hyperleukocytosis (159 patients with wbc over 50k and less than 100k, and 198 patients with wbc over 100k) was compared to 918 pts without hyperleukocytosis. pts with hyperleukocytosis were younger, had an increased rate of favorable risk cytogenetics, were more likely to be flt3 and npm1 mutated, and had an increased rate of myeloablative conditioning. on univariate analysis pts with hyperleukocytosis had an increased rate of ri (30% vs 22.7%, p = 0.013), and decreased incidence of grfs (36.6% vs 45.3%, p = 0.022). in multivariate regression analysis, hyperleukocytosis was significantly associated with increased ri (hazard ratio [hr] of 1.55, 95% confidence interval [ci], 1.145-2.124; p = 0.004), s399 poorer lfs (hr of 1.38, 95% ci, 1.071-1.785; p = 0.013), decreased grfs (hr of 1.38, 95% ci, 1.117-1.71; p = 0.002), and poorer os (figure 1) (hr of 1.4, 95% ci, 1.073-1.846; p = 0.013). image/graph hyperleukocytosis at initial presentation retains a significant prognostic role for aml patients undergoing allogeneic stem cell transplantation even in the current era of advanced molecular prognostication. outcome after hematopoietic stem cell transplantation for philadelphia-positive aml: relatively favorable outcome in patients allografted in first complete response; a survey from the acute leukemia working party of the european society for blood and marrow transplantation (ebmt) v lazarevic 1 , m labopin 2 , w deipei 3 , i yakoub-agha 4 , a huynh 5 , p ljungman 6 , n schaap 7 , d blaise 8 , jj cornelissen 9 , n maillard 10 , p pioltelli 11 , t gedde-dahl 12 , s lenhoff 1 , m houhou 2 , j esteve 13 , m mohty 2 and a nagler 2,14 aml with t(9;22) and bcr-abl rearrangement (ph-pos aml) is a very rare aml subtype, recognized as a new provisional entity in the recent who 2016 classification. the role of stem cell transplantation (sct) in the era of abl tyrosine-kinase inhibitors (tkis) is mostly unknown. we analyzed long-term outcome in patients ⩾ 18 years after allogeneic or autologous sct performed between 2000-2013 in ebmt centers responding to a designated survey. patients with blast crisis cml and philadelphia-positive all were excluded. primary end-point was os. secondary end-points were nrm, acute gvhd, chronic gvhd, lfs, ri, and the effect of tki on outcome. 65 patients (median age, 48 years, range: 19-67; 31 males and 34 females) with ph-pos aml undergoing sct (allogeneic, 57; autologous, 8) were identified. median wbc count at diagnosis was 57x10 9 /l (1.2-366) and 25% had splenomegaly (data missing on 10 patients). translocation t(9;22) was the sole abnormality in 36 patients (55.5%). the majority of the patients received one or two courses of chemotherapy before transplant and 79% attained cr after one course. the majority (70%) received a tki (mostly imatinib, 34/43) before transplant, with a median period of exposition of 86 days (iqr 60-173), while 21 (34%) received tki after the transplant either as maintenance (n = 11) or treatment for relapse (n = 9). at time of transplant, 56 patients were in complete response (cr1-53, including all autosct; cr2-3) and the remaining 9 patients were allografted in advanced phase. among 40 patients with available information, 14 achieved a mrd negative status at transplant (ratio bcr-abl/abl o10 4 ).regarding allosct, conditioning regimen was myeloablative (mac) in 29/30 (96%) patients o50 years, while in patients 450 years 15 received a reduced intensity regimen (ric) and 9 mac. cell source was peripheral blood stem cells in 39 and bone marrow in 18 allogeneic transplants. the donor was a hla matched sibling (msd) in 32 cases and unrelated (ud) in 25, amongst whom 18 were 10/10 and 7 were 9/10 hla matched, respectively. in the 8 patients undergoing autologous sct the majority received busulfanbased conditioning (n = 6) and peripheral stem cells (n = 7). median follow-up was 6. 36.4%, 46.5%, 59.4%, and 34.9%, respectively. by the univariate analysis, age ( o50 vs ⩾ 50) was associated with ri (5-yr: 22.7 vs 50%), lfs (5-yr: 61.9 vs 31.8%, and grfs (5-yr: 52.4 vs 18.2%), whereas mrd-negative status before allosct was associated with an improved grfs (38.9 vs 16.7%). in the 8 patients autografted, ri, nrm, lfs and os at 5-year was 50% (12.5-79.4), 0%, 50% (15.4-84.6), and 62.5% (29-96), respectively. outcome of patients with ph-pos aml who received allosct in cr in recent years was relatively favorable, especially among younger patients, probably reflecting the beneficial effect of tki. disclosure of conflict of interest: none. outcome of allografting for aml-cr2 is equivalent across the bsbmt and ebmt and is associated with encouraging os and dfs across all age groups j byrne, j perry 1 , k kirkland 1 , r pearce 1 and g jackson 2 1 bsbmt and 2 newcastle university and freeman hospital, newcastle relapsed aml has a very poor prognosis with a high mortality, even if a second cr2 is achieved. the only curative treatment is with an allogeneic hsct but allografts for aml in cr2 are considered to have a worse outcome compared to those performed in cr1, especially in older patients for whom this therapy may not be considered. the bsbmt undertook a bench-marking study analysing the outcomes for all patients allografted for aml-cr2 from 2006 to 2011. the uk outcomes from 534 paediatric and adult patients were compared to 3070 non-uk patients transplanted for the same indication reported to the ebmt during the same period. allogeneic transplants for aml-cr2 represent an important part of any allograft program and numbers referred for allograft were stable between 2006-2011 in both programs. the median age of patients was 45.8 yrs and 43.3 years in the bsbmt and ebmt cohorts respectively, with 14% and 15% of patients aged o 18 years and 19% and 16% aged 4 60 years in the 2 groups. the length of first remission was missing in many of the ebmt registrations so time from diagnosis to transplant was used as a surrogate for this and was similar in both cohorts (18 m and 19 m respectively). similarly the presence of comorbidities was poorly reported in both databases but was similar. the bsbmt cohort included fewer patients undergoing ric conditioning protocols (55% vs 66%), fewer sibling transplants (25% vs 38%) and more pbsc allografts (79% vs 20%). transplant related morbidity and mortality were similar across the two cohorts (bsbmt v ebmt) with rates of severe acute gvhd (grade iii and iv) 6% v 10%, limited chronic gvhd 35% v 28%, relapse at 1 year 22% v 20% and death in cr at 1 year 20% v 18%. chronic gvhd (both limited and extensive) appeared more common in the bsbmt cohort (50% v 36%) although reporting of cgvhd was more comprehensive and complete within the bsbmt registry and may be under-reported in the ebmt registry. outcomes were excellent in both cohorts with outstanding rates of leukaemia free survival in this high risk cohort at day 100 (bsbmt v ebmt) 86% v 83%, at 1 year 63% v 67%, at 3 years 49% v 53% and at 5 years 41% v 43%. although os and lfs was significantly shorter in patients aged 4 60 years, at 37% and 30% the results in this high risk age group are acceptable and warrant its continued use. multivariate analysis of the combined cohorts showed that age at transplant ( o 18 yrs/18-60/460 yrs), time from diagnosis to transplant and the presence of agvhd were important factors affecting dfs. risk factors for relapse included the type of conditioning used, presence of agvhd and time from diagnosis to transplant, whereas those for trm included age, agvhd, source of stem cells and time to transplant. there was no significant difference in outcomes between the bsbmt and ebmt for this indication. outcomes for patients allografted for aml-cr2 are excellent and appear superior to those reported for patients not undergoing an allograft in both the bsbmt and ebmt cohorts. the os and dfs observed are comparable to those reported for allografts in aml-cr1 and, although this study has not considered outcomes for patients who did not achieve a 2 nd cr, it nevertheless supports the practice of risk stratification of aml patients such that only high risk patients are offered an allograft in cr1 with the remainder being offered an allograft as salvage after relapse. disclosure of conflict of interest: none. to evaluate the efficacy and safety of intensive chemotherapy followed by allogeneic hematopoietic stem cell transplantation (allo-hsct) for atl. a total of 21 evaluable atl patients treated at our center from 1995 to 2015 are retrospectively analyzed. the htlv-i proviral dna load in peripheral blood mononuclear cells using pcr assay was developed, in comparison with htlv-1 tax protein expression measured by western-blot, to confirm the diagnoses and monitor the disease control. 13 patients were male and 8 patients were female. median age was 50.8 (range 28-66) years. all obtained patient samples were subjected to flow cytometric examination and karyotype analysis. 19 patients received chemotherapy as the induction therapy while 2 quit at the time of diagnosis, 5 with da-edoch regimen while 14 with other regimens such as chop, vcap and amp. da-edoch regimen is a variation of dose-adjusted epoch regimen with the replacement of prednisone (60 mg/m 2 per day) by dexamethasone (15 mg/m 2 per day). before the conventional regimens bucy followed by prophylaxis donor lymphocyte infusion, both received a course of salvage chemotherapy including fludarabine and cytarabine for 5 days registered on http://clinicaltrials.gov (nct02328950). the gvhd prophylaxis consisted of atg, csa and mtx. the patient characteristics, therapeutic effect and survival data were collected. all patients came from the coastal area in the south-east of china. subtype classification of these 21 atll were 18 acute, 2 lymphoma and 1 chronic type. the main manifestations were characterized with cutaneous and respiratory involvement, hepatosplenomegaly, lymphadenopathy and the laboratory abnormalities as leukocytosis with atl cells, hypercalcemia and elevated serum ldh. typical morphological characterisitic of "flower cells" were observed in 85.7% cases and most of the atll cells are cd4+cd8-. chromosomal abnormalities were detected in 4 cases. all 14 patients who didn't receive da-edoch regimen died of disease progress, while among 5 patients with da-edoch regimen, 2 achieved cr, 2 pr and 1 died. with a median follow-up of 18.6 (10-24.1) months, 3 patients respond to da-edoch are still alive. 2 patients in cr achieved successful engraftment with complete donor chimerism in one month post haplo-identical transplant. both were received prophylaxis donor lymphocyte infusion and the immunosuppressive agents were abruptly discontinued for induction of a graft-verus-atl (gvl) effect. they keep remain alive and disease free longer than 2 years so far without severe graftversus-host (gvhd), and the htlv-1 proviral dna became undetectable after allo-hsct. conclusion: it shows great promise of da-edoch regimen followed by allo-hsct to the long-term cure of atl with apparent clearance of the virus. haplo-identical transplantation can be an alternative option for the atl patients without increasing non-relapse mortality. [p537] disclosure of conflict of interest: none. in the absence of an hla-matched related donor or a good matched unrelated donor in time, haploidentical stem cell transplantation (haplo-sct) is an option for patients requiring an allogeneic hematopoietic stem cell transplant. substantial progress has been made in the last two decades with a dramatic improvement in patient outcomes, with some groups reporting preliminary beneficial effects similar to the ones in hla matched unrelated donor and cord blood transplant. several strategies have been adopted through the years for the procedure. the two strategies used in haplo-sct are ex vivo t-cell depletion and t-cell replete transplantation. the latter can be done with a combination of immunosupressive drugs ( beijing approach) or with post-transplantation highdose cyclophosphamide (post-cy). due to of its lower cost, feasibility and practicality, post-cy has become the most often used platform for haplo-sct in the majority of allogeneic transplantation units worldwide. we analyzed our experience in haplo-sct, since the first one in march 2104 to the last one that has just been done in october 2016. we collected all complications reported, also mortality related to treatment and to the disease. we analyzed the overall survival (os). 9 transplants were treated, with different sct indications, the most common being acute myeloblastic leukemia (n = 5, 55%), the rest of indications are exposed in figure 1. all our patients, independent of the conditioning receive post-cy as t cell depletion measure and stem cells were collected from peripheral blood. age at the time of transplant was 40.6 years, 77% were males, 23% females, the rest of patient characteristics are listed in table 1 . in our series the treatment related mortality (trm) was low with only 1 patient (11%) that died before the day +100. as complications, we reported 33% of hemorrhagic cystitis, 22% of sinusoidal obstruction syndrome, 22% reports systemic inflammatory response syndrome, 55% of citomegalovirus (cmv) reactivation. neutrophils graft is 18.2 days (r = 14-23) and the platelets graft is 24.2 days (r = 13-34). in our series we haven't reported any case of graft failure, one of the transplantes the patient had antihla antibodies, this was treated with a plasmapheresis previous the stem cell infusion, and was infused with a high number of cd34+ cells (8 × 10 6 /kg), no graft problems, and has had no complications since then with the graft. the os for the whole group is 22 months, with a median not reach at 36 months, with 2 patient's dead at time of analysis. two patients had a relapse after the haplo-sct (22%), both of them received lymphocyte donor infusion, sadly, neither of them responded. the haplo-sct procedure is been adopted by many centers for high risk hematology malignancies, mainly because the fast availability of donors, and because of the preliminary results that have been reported place it as good as the unrelated donor or cord unit transplant. our center is getting experience in these types of transplants, and our results reflect similar outcomes as larger studies. with longer follow ups we will be able to keep the trend of good results both in procedure safety and disease efficacy. os, toxicity and trm are expected for these high risk malignancies. in overall, the haplo-sct seems a reasonable technique that is reflecting in our short series, the results being reported in studies worldwide at bigger scale. disclosure of conflict of interest: none. complex karyotype, the presence of flt-3 itd and losses of genetic material in chromosome 7, are all considered high risk markers in aml. patients bearing these abnormalities could undergo a myeloablative allogeneic transplantation in cr1 whenever this is possible, although this could significatly reduce the chances for cure in older patients due to increased transplant related mortality. ric allografts could be performed in older patients in order to overcome the deleterious effects of these individual abnormalities but its effect still remains controversial in the high risk group. between 2005 and 2015, a total 65 patients [41 males, 24 females, mean age 59.05 (40-73)] received a ric allograft (49 from fully matched unrelated donors, and 16 from an identical sibling) for high risk aml in 2 transplant centres. high risk disease was classified according to their response to treatment, the presence of complex karyotype, the presence of individual cytogenetic/molecular abnormalities or a combination of these. in particular, 24 patients presented one single karyotype abnormality and 17 presented three or more. ten patients presented alterations of chromosome 7 and 7 patients presented flt-3 itr. the conditioning regimes included fludarabine in all cases, together with melphalan and campath (29 patients), busulphan and campath (21 patients), busulphan and thiotepa (1 patient), melphalan (3 patients), busulphan with and without atg (9 patients) total body irradiation (200 cgy, 2 patients). graft versus host disease prophylaxis was ciclosporin for patients receiving alemtuzumab or atg but for patients who had a t-replete allograft ciclosporin and low dose methotrexate was the preferred prophylaxis. all but four patients were transplanted in cr1 patients were followed-up for a mean 32.3months (range 3-150). thirty-one (47%) patients remain alive. the causes of death (34 cases) include relapse or progression of the original disease (13), transplant-related causes (17) and unknown in 4 cases. the influence of the genetic abnormalities on survival was analysed, showing there were no significant differences between patients with normal karyotype, single chromosomal abnormalities and two or more abnormalities. likewise, the kaplan-meier survival analysis of patients bearing flt-3 itd was not significantly different to the rest of the cohort (p = 0.4; 3/7 died, with only one case being related to relapse). patients bearing chromosome 7 abnormalities (with or without other chromosomal aberrations) had a comparable, not significantly (p = 0.6) different survival to the rest of the cohort: 6/10 patients bearing this abnormality died although, most interestingly, none of these deaths were related to relapse. our results indicate ric allografts can provide an adequate consolidating effect in hr aml with complex karyotype, alteration of chromosome 7 or flt-3 itr, yielding clinical results that are comparable to those obtained in patients with aml allografted for other indications. this is particularly important as these alterations are more frequent in patients whose age prevents them from having myeloablative grafts. disclosure of conflict of interest: none. early detection of inapparent replicative human cytomegalovirus (hcmv) infection together with its preemptive antiviral treatment has led to a marked reduction of life-threatening hcmv disease after allogeneic hematopoietic stem cell transplantation (allosct). we first reported in a retrospectively performed study that hcmv reactivation is associated with a reduced risk for relapse in patients with aml after transplant. now, we evaluate the impact of early hcmv replication on the risk for leukemic relapse in patients with aml after t cell depleted transplantation in a prospectively performed observational study (registration trial drks00004300). between january 2012 and march 2015 we enrolled 251 patients with aml in this trial who were consecutively transplanted at the university hospital of essen. 239 patients received a myeloablative regimen (tbi based conditioning n = 83, chemotherapy based conditioning n = 156) and 12 patients a ric (n = 12 chemotherapy based regimen). patients were transplanted in 1.cr (n = 123), 2.cr (n = 51) or more progressive disease stages (n = 77) from hla-identical sibling donors (n = 60) or hlaidentical unrelated donors (urd) (n = 126) or mismatched unrelated donors (n = 65). patients who received a second transplant were excluded from the study. the median age of patients was 54 years (range 18-72) and that of the donors 35 years (range 14-69). gvhd prophylaxis was performed with mtx and csa, or csa and mmf with (n = 182) or without atg (n = 69) (30-60 mg total dose). the incidence of acute gvhd grade 2-4 was statistically not different in both groups (46% versus 40%). hcmv-reactivation (hcmv-r) detected by pcr occurred between 23 and 87 days (median 45 days) after allo sct. only patients surviving day 60 after transplant were included in the study for estimation of relapse incidence (cir) or overall survival (os). hcmv status of recipients (r) or donors (d) were in 29% r-/d-, 12% r-/ d+; 28% d+/r-and 31% r+/d+. patients with a documented hcmv-r had a cir at 4-year after transplant of only 30% as compared to 47% in patients without a hcmv-r (p = 0.016). estimates for 4-year os were in favor for patients with hcmv-r (61% for patients with hcmv-r versus only 48% for patients without hcmv-r), but this did not achieve statistical significance. non-relapse mortality was greater in patients with hcmv reactivation (23% versus 12%, ns) a substantial and independent reduction of relapse risk associated with early hcmv replication was confirmed by multivariate analysis including competitive factors as unfavorable cytogenetics according to eln, advanced disease stages of aml, hla-identical donor versus mismatched donor, sibling versus unrelated donor, presence of acute gvhd grades ii-iv, chronic gvhd, and hcmv-r [(hazard ratio: 0.61, 95% ci: 0.38-0.98, p o0.042) for occurrence of hcmv-r]. the final result of this first prospective performed study confirms an independent advantageous effect of early hcmv replication on the leukemic relapse risk in patients with aml after transplant. disclosure of conflict of interest: none. . primary engraftment of wbc and platelets was achieved in 64 pts, one patient (pt) died at day +3 after hsct, and one had a secondary graft failure. a median time to wbc engraftment was 13 days (9-27), to platelets -14 days (9-32). cumulative incidence (ci) of acute gvhd (agvhd) grade ⩾ ii was 25% (95% ci:16-38): from haplo-23%(12-45), from mud 26% (15-45),p = 0.7. ci of agvhd4iii 8%(95% ci:3-18): haplo vs mud -7% (95% ci:2-25) vs 9%(95% ci:3-25), respectively,p = 0.7. ci of chronic gvhd (cgvhd)-16% (95% ci:9-28). ci of agvhd was significantly lower in a group with regimen 2 (2014-2016) of gvhd prophylaxis: 10% (95% ci:4-25) vs 32%(95% ci:18-57), po 0.0001. regimen 2 was also effective in prevention of cgvhd: ci at 2 year after hsct was 10% vs 24%, p = 0.14. сi of trm was 12%(95% ci:6-24): haplo-7%(2-25), mud -14% (6-32). early mortality (before +100-day) was relatively low (n = 3): 1 pt died from bacterial sepsis; two pts died due to adv infection. thirteen pts died after 100 days: 9 pts relapsed and died due to complications of second hsct; bacterial sepsis in 1 pt and viral infection (adv and cmv) in 3 pts (2 with extensive chronic gvhd). ci of relapse was 24% (95% ci:15-38) at 2 year: from haplo-12% (95% ci:4-36), from mud-32% (95% ci:20-52),p = 0.086. event-free survival (efs) at 2 years was 64% (95% ci: 52-77): haplo -81%(95% ci: 66-97), mud -54% (95% ci:37-70), p = 0.06. os was 70% (95% ci: 58-82) at 2 years: haplo-77% (95% ci: 57-77), mud -65% (95% ci: 48-81), p = 0.3. relapse-gvhd-free survival at 2 years was different among recipients of haplo and mud hsct: 51%(95% ci: 28-75) vs 32% (95% ci: , p = 0.3. we confirm that the depletion of tcrαβ+/cd19+ depletion from the graft ensures high engraftment rate and acceptable transplant-related mortality in pediatric aml pts. there is a trend towards better efs for haploidentical transplantation. gvhd prophylaxis including ratg/rituximab/bortezomib improves gvhd control s404 in recipients of tcrαβ+/cd19+depleted grafts in comparison to hatg/tacro/mtx apparently without loss of anti-leukemic activity. disclosure of conflict of interest: none. results of t-cell depleted haploidentical stem cell transplantation in adults with acute leukemia improve with time: a study from the acute leukemia working party of the european society of blood and marrow transplantation (ebmt) s simona 1,2 , m labopin 3 , a ruggeri 1,3,4 , a velardi 5 , f ciceri 6 , j maertens 7 , l kanz 8 , f aversa 9 , d bron 10 , d bunjes 11 , m mohty 1,3 and a nagler 3, 12 t cell-depleted (tcd) transplants from haploidentical donors are increasingly used in the absence of a hla full-matched donor for patients (pts) with high risk acute leukemia (al). progress has been made in optimization of conditioning regimens and post-transplant cellular therapy to potentiate the graft-versus-leukemia effect with no graft-versus-host disease (gvhd). however, relapse incidence (ri) and non relapse mortality (nrm) remain the main obstacles for pts outcomes. we report 308 adults with de novo al, given a tcd haplo from 2005 to 2015. to analyze the effect of transplant period on tcd haplo outcomes, pts were analyzed in two separate groups: 2005-2011 (n = 191) and 2012-2015 (n = 117). tcd haplo were performed in 66 ebmt centers. median follow-up was 11 months with no difference according to transplant periods. median age was different between groups, being 41 and 46 years respectively (p = 0.044). the majority of pts had acute myeloid leukemia (aml) (75% vs 69%, p = 0.261) and disease status at haplo was first complete remission (cr1) in 55% and 64% of pts respectively (p = 0.115). pts transplanted before 2012 had more frequently a karnofsky performance status o90% (19% vs 10%, p = 0.041). conditioning was myeloablative in 76% and 77% of tcd haplo before and after 2012 (p = 0.935), mainly based on fludarabine(flu)-tbi, flu-melphalan-thiotepa or cyclophosphamide-tbi. as for ric it was flu-melphalan-thiotepa, flu-tbi or cyclophosphamide-tbi. the cumulative incidence (ci) of neutrophil engraftment, grade ii-iv acute gvhd and chronic gvhd were not different according to transplant period, being 93% and 90%, p = 0.302; 20% and 22%, p = 0.667; 19% and 11%, p = 0.119, respectively. in the whole population 2-year ri and nrm were 20% and 48%, with no difference before and after 2012 (21% vs 19%, p = 0.722; 54% vs 38%, p = 0.109, respectively). ri was 20% before 2012 versus 15% after 2012. the main cause of nrm was infection, with no difference over time (46% vs 50%, p = 0.316). in multivariate analysis, disease status was the only factor associated with ri (hr 2.05, 95% ci 1.09-3.84, p = 0.025). tcd haplo after 2012 (hr 0.57, 95% ci 0.38-0.86, p = 0.008), younger age (hr 0.82, 95% ci 0.63-0.98, p = 0.023) and ric (hr 0.53, 95% ci 0.32-0.88, p = 0.014) were independently associated with lower nrm. 2-year os was 36% with a marked improvement for tcd haplo performed after 2012 (47% vs 29%, p = 0.024), while lfs and grfs were 31% and 24%, respectively. according to disease status, 2-year lfs, os and grfs were higher for pts transplanted in cr1 (38% vs 22%, p o0.001; 41% vs 29%, p = 0.006; 30% vs 17%, respectively p = 0.004). in multivariate analysis, tcd to further establish the role of haplosct in high-risk aml, we performed a retrospective matched-pair comparison of hlamatched sct vs haplosct/ptcy in two german centers. highrisk aml was defined by any of the following criteria: refractory or relapsed aml, secondary aml, or genetic aberrations classified as intermediate-high or adverse accordingly to the eln classification. all consecutive adults, who fulfilled ⩾ 1 of these criteria before either hla-matched or haplosct/ptcy were included (n = 200). recipients of haplosct were pair-matched with patients receiving a matched donor sct. matching variables were (1) stage at sct, (2) genetic subgroups accordingly to eln, and (3) age (±5y). 39 patients (pts) undergoing haplosct/ptcy could be successfully pairmatched (p = 1.0 for stage and genetic subgroup, 0.9 for age) with 39 recipients of matched sct (12 family, 27 unrelated sct). within the entire cohort, median patient age was 57y (24-70). at start of conditioning, 20% of patients were in cr, 18% had refractory, 52% had relapsed, and 10% had untreated disease. genetics were favorable (16%), intermediate i (51%), intermediate ii (12%) and unfavorable (21%). hla-matched sct was uniformly performed following flamsa-ric. 34 recipients of haplosct/ptcy (87%) received cytoreductive chemotherapy with flamsa (n = 16) or clofarabine (n = 18) before ric was started. median follow-up among survivors was 33 months. overall cr rate at d+30 was 95%, 4 patients suffered from refractory disease or early death, (n = 2 each). overall-survival (os) for the entire cohort was 74%/53.9% at 1y/3y from sct. the corresponding 1y/3y leukemia-free survival (lfs) was 63.5%/46.6%. median time to engraftment was 18.0 and 17.5 days after matched and haplosct, respectively (p = 0.8). with respect to outcome, no difference was observed between the two groups: os at 1y/3y was 78.5%/54.5% after matched sct, and 61.5%/55.2% after haplosct/ptcy (p = 0,71, figure 1 ). lfs at 1y/3y was 76.2%/ 42.6% within the hla-matched group and 56.4%/50.8% within the haploidentical group (p = 0,99). recipients of haplosct showed a higher incidence of agvhd ⩾ ii°(46% vs 18%, p = 0.014), as well as a trend towards increased 1y-nrm (18% vs 5%, p = 0.08), whereas 1y-relapse rates were comparable (23% after haplosct/ptcy vs 26% after matched sct, p = 0.5). relapse was the most frequent cause of death in both cohorts, main causes of nrm were gvhd and infections (no difference between the two groups). allogeneic sct following sequential conditioning can achieve excellent results in high-risk aml. in our study, results after haploidentical transplantation were comparable to those obtained after hla-matched sct. hence, haplosct/ptcy following sequential conditioning can be considered as a reasonable option for patients with high-riaml. [p544] disclosure of conflict of interest: none. a significant proportion of patients with acute myeloid leukemia ( aml) will either be refractory to initial chemotherapy or will suffer refractory relapse. the role of allogeneic transplantation (hct) in active disease is contentious. there is a growing body of literature that sequential chemotherapy, pioneered by the german flamsa regimen, followed by ric hct is a safe and efficacious modality in these patients, and there have been numerous modifications of this regimen, especially as amsacrine is not widely available. fludarabine, cytarabine and etoposide (vp16) (flav) have been reported as an an effective salvage regimen. here we report on single center outcomes of a variation of the flamsa regimen, substituting amsacrine for etoposide with mainly myeloablative conditioning. patients were consented for a clinical protocol if they had aml that was refractory to 2 cycles of chemotherapy, or 1 cycle and considered to be at risk of complications of a second cycle, and if they had a matched related donor. patients with myelodysplasia received flav if they had high or very high risk cytogenetics. cytoreductive chemotherapy consisted of fludarabine 30 mg/m 2 / day × 4 days, cytarabine 2g/m 2 /day × 4 days, etoposide 100 mg/m 2 /day × 3days, commenced simultaneously. after 3 days of rest, conditioning chemotherapy consisted of fludarabine 30 mg/m 2 × 2 days and and iv busulfan 0.8 mg/ m 2 q 6 hours; the number of busulfan doses varied between 8-12, depending on patient comorbidity. ten patients (76%) received myeloablative doses of busulfan. patient received 2 doses of atg at 2.5 mg/m 2 /day on day -3 and -2. patients received gcsf mobilized peripheral blood hematopoietic cells. post-transplant gvhd prophylaxis was csa and mmf. csa was tapered from day+60 and stopped at day +90 in the absence of gvhd. mmf was discontinued between day +30 and day +40. donor lymphocyte infusions were collected for planned prophylactic dli. thirteen patients received a flav-sct between march 2014 and october 2016. the median age was 39(15-57); male:female ratio was(7: 6). 10 patients (77%) had aml and 3 (23%) pts had mds. all patients had detectable disease prior to flav. the median time for plt engraftment was 19 days (9-50). the median time for anc engraftment was15 days (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) . cytogenetic cr rate on a day 28-30 bone marrow was 46%, and morphological cr was 60%.3 patients (23 %) developed veno-occlusive disease. acute gvhd grade ii-iv occured in 4 pts ( 30%). 3 (23%) patients developed chronic gvhd. death was due to disease relapse in 5 (38%) and nrm in 1(7.7%) patients, resulting from h1n1 pneumonia. 5 patients (38%) received dli for post transplant relapse, and one of these is in molecular remission. at a median follow up of 4.8 months post transplant (1.6-29m), 1 year dfs was 23%. the 1 year and 2 year os was 51% (±14%) (figure 1 ). our experience, consistent with published data, demonstrates that for patients with active aml refractory to chemotherapy, transplantation is an effective modality of disease control and may be the only curative therapy in a significant proportion. etoposide appears to be a suitable alternative to amsacrine. our patients tolerated busulfan at myeloablative doses, and this may be required for adequate disease control. our report is limited by small numbers and relatively short follow-up, but is encouraging enough for us to continue offering flav hct for these high-risk patients. [p545] disclosure of conflict of interest: none. sequential high-dose chemotherapy reinduction followed by haploidentical transplantation in acute leukemias l brunello 1,2 , cm dellacasa 1 , l giaccone 1,2 , e audisio 3 , d ferrero 3,2 , s d'ardia 3 , b allione 3 , s aydin 3 outcomes after t-cell replete haploidentical stem cell transplantation (haplo hsct) have been encouraging and haplo hsct has become an alternative option for patients without a hla-identical related or unrelated donor. 1,2 as previously published, the sequential use of intensive chemotherapy and allogeneic transplantation represents a possible approach to the treatment of high-risk acute myeloid leukemia (aml). 3, 4 between 2010 and 2015, 19 acute leukemia (al) patients received sequential therapy (s.t.) consisting of high-dose chemotherapy reinduction and haplo hsct during the chemotherapy-induced neutropenia. median age at transplant was 50 years (range: 21-62); median number of previous therapy lines was 2 (range: 1-3) and 5/19 (26%) patients had received a previous allogeneic hsct. twelve and 5 out of 19 patients had de-novo aml and secondary aml, respectively; furthermore two patients presented blastic crises of chronic myeloid leukemia. all patients had active disease at the time of s.t. and median marrow blast count before reinduction was 25% (range: 6-88%). hematopoietic cell transplantation comorbidity index was ≥ 3 in 12/19 patients (63%). all patients received high-dose cytarabine (≳1 g/sqm) containing regimens as reinduction therapy. the conditioning regimen was started after a median of 9 days from the end of reinduction (range: 4-15). sixty-eight percent of patients (13/19) were conditioned with a myeloablative regimen (thiotepa tot. 10 mg/kg, busulfan tot. 9.6 mg/kg, fludarabine tot. 150 mg/ sqm), while 6/19 (32%) patients received a non-myeloablative conditioning. bone marrow was used as stem cell source in 18/19 (94%) patients. graft-versus-host disease (gvhd) prophylaxis consisted of post-transplant cyclophosphamide with calcineurine inhibitors and mycophenolic acid. all patients engrafted but one, who was rescued with a second haplo-hsct with peripheral blood stem cells from the same donor. median day of neutrophil recovery was day +18 (range: 14-24). median follow-up of survivors was 4.2 years (range: 1.6-5.4); 1-year overall and event-free survivals were 37% and 32%, while 1-year relapse incidence and non relapse mortality were 42% and 26%, respectively. overall cumulative incidences of acute and chronic gvhd were 39% and 38% at day +100 and +400. among patients who developed gvhd, 2 grade iii-iv acute gvhd and 2 moderate-severe chronic gvhd were observed. at 4.2 years post haplo-hsct, 20% of patients are alive and disease free. in our cohort of heavily pre-treated and high-risk patients, s.t. with a myeloablative conditioning was safely used to reduce leukemic burden pre-transplant and enhance graft-versus-leukemia effects. only the prompt availability of a haploidentical donor allowed to implement this treatment modality. though small the patient cohort, our findings suggest that transplant-related toxicity was acceptable and early relapse was the major treatment-failure. however, long-term survival and disease-free rates of 20% in these very poor prognosis patients are highly encouraging. in elderly patients with acute lymphoblastic leukemia (all) and kinase activating lesions allogeneic stem cell transplantation (allo-sct) is considered to be the only curative option. 1 however, high risk of relapse and non-relapse mortality (nrm) often withholds elderly patients with existing comorbidities from definitive therapy. 2 even though, age alone as the most important eligibility criterion for allo-sct has become less important. 3 a 61-year old patient with a medical history of adipositas, hypothyreosis, arterial hypertension, hypercholesterolemia and coronary artery disease with stent implantation was diagnosed with common-b-cell-all based on immunophenotype. cytogenetic analysis showed two coexisting clones with an abnormal karyotype of 47,xx; t(1;9)(q22;q34), +17 [3] and 46,xx; t(1;9)(q22;q34),der(6)(6;17)(p23;q21) [2] and no evidence of bcr-abl1 positive disease using fluorescence in situ hybridization (fish) technique. rt-pcr and sequencing of the fusion transcript was performed to validate the rcsd1-abl1 t(1;9)(q22;q34) fusion between exon 3 of rcsd1 and exon 4 of abl1. western analysis of phosphorylated abl1 and its downstream target crkl was performed to investigate the in vivo activity of dasatinib. clinical monitoring of minimal residual disease (mrd) levels has been performed via rt-pcr of the rcsd1-abl1 fusion transcript followed by nested pcr of the amplicon to detect early relapse or mrd. as positive control the plasmid pcr2.1-topo_rcsd1-abl1(626bp) was synthesized encoding rcsd1-abl1 amplicon with the fusion site. our patient was enrolled on gmall elderly (01/2003) and treated accordingly. thereby, no sustained remission could be achieved. flag-ida re-induction and study treatment with an oral pi3k/mtor inhibitor remained futile. cytogenetics and verification of the rcsd1-abl1 fusion gene prompted salvage treatment with the tyrosine kinase inhibitor (tki) dasatinib as single agent. the in vivo activity of dasatinib was highlighted by a decrease of rcsd1-abl1 amplicon and inhibition of phosphorylated abl1 and its downstream target crkl was shown. clofarabine and cyclophosphamide complemented treatment and mrd negative remission was achieved due to administration of the bi-specific t-cell engager blinatumomab. consolidation with allo-sct was performed. ongoing remission has been achieved for more than 30 months now. we demonstrate that monotherapy with tki like dasatinib is effective in refractory rcsd1-abl1 positive all. to the best of our knowledge, this is the first elderly patient with rcsd1-abl1 positive all with a sustained and ongoing complete remission. thereby, we suggest allo-sct after successful tki even for elderly patients with existing comorbidities and uncommon cytogenetics. relapse is the most important cause of failure of allogeneic hematopoietic stem cell transplantation (hsct) for flt3-itdpositive acute myeloid leukemia (aml). in those cases, treatment with flt3 tyrosine kinase inhibitors (tki) constitutes a promising clinical approach to induce remission without conventional chemotherapy. forty-eight-year-old woman was diagnosed of aml secondary to myelodisplastic syndrome with npm1 mutation and internal tandem duplications of the flt3 gene (flt3-itd). after achieving complete remission (cr), she received a sibling allogenic-hsct. four months later, aml relapsed at the medullary level, without central nervous system (cns) involvement, treated with conventional chemotherapy and donor lymphocytes infusions (dli). she achieved second cr and developed chronic graft-versus-host disease (cgvhd). nine months later, she suffered the first extramedullary relapse, at the mammary, cutaneous and probably pericardial levels. there was not medullary involvement. disappearance of the lesions at all levels was achieved with conventional chemotherapy and radiotherapy, and full donor chimerism. eight months later, she referred atypical precordial pain irradiated to the back. cardiac mri was performed in which several masses were visualized in a pericardial sac up to 5 cm in diameter (dec-15). bm was maintained in cr. in study of pericardial fluid, infiltration by leukemic flt3 positive cells was observed. the patient was not considered candidate for further systemic chemotherapy nor radiotherapy treatment. then, treatment with the flt3 sorafenib inhibitor was started, by compassionate use, at dosage of 200 mg/12 h, which maintains after one year. after first month with sorafenib, pericardial lesions decreased considerably, ranging from 5 cm in diameter to 1.7 cm (jan-16). in the subsequent ct controls, progressive decrease of the lesions has been observed and no new lesions have appeared in other locations. in the last ct (oct-16) pericardial thickening is almost non-existent, without new lesions. after one year of treatment, she maintains cr at medullary and extramedullary levels images. in our patient, treatment with sorafenib has achieved sustained control of extramedullary disease, which had escaped the mechanisms of action of conventional chemotherapy, allotransplant and dli. further studies are needed to corroborate the efficacy of flt3 inhibitors in the control of aml extramedullary disease and in the treatment of relapses after allo-hsct. all pts with tbi-based regimen received rabbit atg. tcrαβ+/cd19+ depletion of hsct with clinimacs technology was implemented in all cases according to manufacturer's recommendations. the median dose of cd34+ cells in transplant was 10 × 10 6 /kg (range: 3.9-18.8), tcrα/β-23 × 10 3 /kg (range: 0.2-300). primary engraftment was achieved in 68 of 71 pts. (2 pts died before engraftment, one received second hsct), the median time to neutrophil and platelet recovery was 13 and 14 days, respectively. early (100 day) mortality was 7%(95% ci: 3-17), 2-year ptrm-17% (95% ci: 10-30). the three early deaths were due to bacterial sepsis (n = 2) and viral infections(n = 1), seven late: viral infection in 4 pts (adv = 2, adv+cmv = 1, cmv = 1), bacterial sepsis in 2 pts and rhinocerebral mucormycosis in 1 pt, all late deaths were associated with cgvhd and prolonged corticosteroid therapy. ci of acute gvhd grades ii-iv and iii-iv was 22.5% (95% ci: 9.6-53), and 7% (95% ci: 1.3-37), respectively. ci of cgvhd was 17.6% (95% ci: 6.4-48). regimen 2 was more effective in prevention of agvhd ii-iv: ci at 2 year after hsct was 14% vs 35,7% in regimen 1, p = 0.033 and in cgvhd 7% vs 35.7%, p = 0.006. ci of relapse at 2 years was 32% (95%ci:15.7-64.5). two-year pefs(event = death or relapse) was 49.9% (95% ci: 37-62), 2-year pos-54% (95% ci:41-67). in patients, who received tbi-based conditioning pefs was 59% (95% ci: 38-81), as compared with treosulfan-based 47% (95% ci: 32-61), p = 0.65. median time of follow-up for survivors was 2.2 years (range: 0.3-4.4). we confirm that the depletion of tcr-alpha/ beta and cd19 lymphocytes from the graft ensures high engraftment rate and acceptable transplant-related mortality in pediatric all patients. viral infections and leukemia relapse await further improvement of control. all major outcomes were equivalent between transplantation from unrelated and haplo donor. disclosure of conflict of interest: none. the prognosis of patients (pts) with relapsed/refractory acute lymphoblastic leukemia (all), especially after allogeneic hematopoietic stem cell transplantation (allo-hsct), is very poor. therapeutic options for these pts are limited. blinatumomab is a bispecific t-cell engager (bite) antibody construct with dual specificity for cd19 and cd3. bite therapy may help to overcome the resistance to chemotherapy (ct) with minimal toxicity, and may be a bridge to allo-hsct. we analyzed data of 34 pts from 4 hematologic centers in russian federation with relapsed cd19 positive all, who received bite. the median age was 22 (range: 1-62) years, 9 (26%) pts o18 yrs, 25 (74%) pts ≥ 18 yrs. the diagnosis was all b-i (egil) subtype in 11 pts, b-ii-in 16, b-iii-in 5 pts, and 1 patient had mixed phenotype leukemia (m5 (fab) and b-all). three (9%) pts had philadelphia positive (ph+) all. in 11 pts it was the first relapse of all, in 13-second, in 10-third. thirty pts had isolated bone marrow relapse, 4 pts-combined relapse (bone marrow and extramedullary sites). the bone marrow blast infiltration was o 50% in 15 pts, 450% in 19 pts. disease relapse was revealed after ct in 19 pts (7 (37%) pts received allo-hsct after the therapy of relapse), after allo-hsct (3-from related, 9-from unrelated, 3-from haploidentical donors)-in 15 pts (4 pts received second allo-hsct after the therapy). in 8 pts with posttransplant relapse donor lymphocyte infusion (dli) was used in combination with bite. every patient received from 1 to 7 cycles (median 2) of bite. complete remission (cr) was achieved in 18 (53%) pts (in 14 (41%) pts it was minimal residual negative remission): in 8 (42%) pts with all relapse after ct, in 10 (67%) pts-after allo-hsct. pts with less than 50% blasts in bone-marrow at baseline experienced substantially higher response rates compared with patients with 50% blasts or higher (67% (10/15) vs 52% (10/19)). response rates were similar although the number of relapse-45% (5/11) in first relapse, 61% (8/13) in second relapse and 70% (7/10) in third relapse. pts with posttransplant all relapse who received bite in combination with dli had higher response rate than pts, who received bite as monotherapy: 87.5% (7/8) vs 43% (3/7), respectively. one-year os was 50% (95% ci 23-77%). one-year dfs was 48% (95% ci 21-75%). grade ≥ 3 hematological toxicity (neutropenia, thrombocytopenia) was observed in 13 (38%) pts, grade ≥ 3 liver toxicityin 4 (12%) pts. five patients (15%) developed toxic neuropathy during the therapy. cytokine release syndrome occurred in 3 (9%) pts. one patient after allo-hsct (but not after dli) developed grade i agvhd. there were no fatal treatment related toxicity. tree (17%) pts who responded to bite had relapse. eighteen (53%) pts died: 14 pts-of disease relapse/ progression. the treatment of relapsed/refractory all with bite is effective and has acceptable toxicity. we demonstrated high efficacy in therapy of posttransplant all relapses, especially when bite was combined with dli, perhaps, due to additional immunological effect of the transplant. disclosure of conflict of interest: none. the mechanism of sorafenib anti-leukaemic effect seen in aml patients relapsing post allohsct involves the augmentation of alloreactivity which includes infiltration of the affected marrow by cd8+ cells having pd-1 receptor which presence characterize lymphocytes with antitumour potential multikinase inhibitor (mki) sorafenib is clinically active in acute myeloid leukaemia (aml) patients, especially in those with flt3 itd who receive allohsct as a part of their primary treatment. to throw some light on the mechanism of this antileukemic post-transplant sorafenib effect we studied the fate of two patients (flt3 itd-positive, npm1-positive) who relapsed 56 (53-year-old male) and 256 (50-year-old female) days post-transplant and their salvage treatment included sorafenib. the multikinase inhibitor (400 mg twice daily) was given either together with flag or da 2+5. the response was prompt. the patients became, after completion of the chemotherapy, leukaemia free. both patients continued the sorafenib (200 mg twice daily) treatment together with the aml standard maintenance chemotherapy (female case) or without any chemotherapy (male patient, substantial comorbidity and liver toxicity). (1) . the patients responded well to the therapy and were free of leukaemia for 16 and 32+ months after initiation of the mki treatment (flt3 itd negative, 100% chimerism documented in the blood and in the marrow). (2) . in both patients, 3 and 25 months on sorafenib, skin lesions appeared either in the context of cgvhd, which progressed to a life-threatening level in a male patient or as a photodermatitis-like cheek eruption. histopathology revealed the presence of severe cd3+ cells infiltration in affected tissues in both patients. (3) . cd8 positive lymphocytes colonized the marrow of both patients. these marrows infiltrating cells co-expressed cd279 (pd-1 receptor) in proportions which were higher than those seen in the blood (14.72% ± 1.45% vs. 3.63% ± 1.21%, p = 0.002). a similar observation was made for cd8+cd69+ cells (37.26% ± 3.50% vs. 1.58 ± 0.43%, po 0.002). 5. transcriptome analysis of the marrow cells, which addressed the genes involved in lymphocyte activation, revealed the presence of proinflammatory profile which included a higher expression of tlr9 and il-12. (1) sorafenib given with or without moderate chemotherapy was effective in two patients in maintaining the anti-leukaemic effect of salvage chemotherapy. (2) this was associated with the presence of alloreactivity (affected tissues infiltration with cd3+ cells) clinically seen as a severe fatal cgvhd aggravated by sorafenib treatment associated unwanted effects in one cases and with rather mild skin lesions appearing later during the treatment. the outcome of elder patients with acute myeloid leukemia or high risk myelodysplastic syndrome treated with allogeneic hematopoietic stem cell transplantation ch tsai 1,2,3 1 division of hematology, department of internal medicine, national taiwan university hospital; 2 tai-cheng stem cell therapy center, national taiwan university and 3 genome and systems biology degree program, national taiwan university allogeneic hematopoietic stem cell transplantation (hsct) is a curative-intent treatment for patients with high-risk hematologic diseases, including acute myeloid leukemia (aml) and myelodysplastic syndrome (mds). both the incidences of aml and mds increase with age and patients elder than 60 years of age were traditionally excluded from hsct because of high possibilities of therapy related morbidity/mortality. recently, with the introduction of reduced intensity conditioning regimens and the improvement of hsct care, more and more elder patients could undergo hsct for consolidation or salvage purposes. however, literature regarding the treatment outcome of elder patients receiving hsct is scarce. patients diagnosed as aml or high risk mds aged equal or more than 60 years were recruited consecutively at national taiwan university hospital. the high risk mds was defined to include myelodysplastic syndrome with excess of blasts-1 and 2 according to the 2016 world health organization (who) criteria. the cytogenetic risk stratification was based on original medical research council classification. from 2008 to 2016, a total of 51 patients were enrolled consecutively. the median age was 63.1 (range: 60-73) years and the gender distribution was even. among them, 11 (21.6%) patients had high risk mds, 27 (53%) had de novo aml, 10 (19.6%) had secondary aml, and three (5.9%) had therapy related aml. at diagnosis, four (7.8%) patients had extramedullary disease. nine (17.6%) had unfavorable-risk cytogenetics, 12 (23.5%) had either unfavorable-risk cytogenetics or intermediate-risk cytogenetics but with flt3-itd mutations. regarding the pre-hsct disease status, nine patients had the first complete remission (cr), 11 had the second cr, and 31 patients were treatment naive or had refractory disease. the graft-versushost-disease(gvhd)-free/relapse-free survival (grfs) in which events include grade 3-4 acute gvhd, chronic gvhd with moderate severity according to cibmtr criteria, relapse, or death of any cause. with median follow-up of 38 months (range: 0.8-85.2), the median overall survival (os) for all patients was 13.9 months, the relapse-free survival (rfs) was 11.3 months, and the grfs was 9.0 months. in univariate analysis for os and rfs, high-risk mds was a favorable prognostic factor but secondary or therapy related disease (p = 0.013 for os and 0.007 for rfs, respectively), unfavorablerisk cytogenetics or intermediate-risk cytogenetics but with s410 flt3-itd mutations (p = 0.005 and 0.002, respectively), pre-hsct refractory disease (p = 0.018 and 0.037, respectively), and grade 3-4 acute gvhd (p = 0.001 and 0.002, respectively) were unfavorable prognostic factors. however, for grfs, only unfavorable-risk cytogenetics or intermediate-risk cytogenetics but with flt3-itd (p = 0.020) and pre-hsct refractory disease (p = 0.018) were unfavorable prognostic factors. in multivariate cox proportional hazards regression analysis for os and rfs, grade 3-4 acute gvhd was a significant unfavorable risk factor; for gfrs, pre-hsct refractory disease status was a significant unfavorable risk factor. our results showed that the choice of hsct should not solely based on the age factor and pre-hsct disease status. incorporating cytogenetics and genetic mutation status could risk-stratify elder patients with hsct. further prospective trials are warranted to validate these findings. disclosure of conflict of interest: none. children affected with acute lymphoblastic t cell leukaemia (t-all) and relapse after allogeneic stem cell transplantation (sct) have limited treatment options and a poor prognosis. immune checkpoint inhibitors targeting the programmed death (pd-1) receptor pathway may enhance the graftversus-leukaemia (gvl) effect by blockade of inhibitory signals to t cells mediated by its ligand pd-l1. we report a 9-year old girl with refractory t-all after allogeneic sct, who was treated off-label with the pd-1 inhibitor pembrolizumab. the girl was diagnosed with t-all (8.2 g/l wbc, 82% bone marrow infiltration, cns negative, t (6;11)) and underwent hlahaploidentical bone marrow transplantation from her mother with post-transplant cyclophosphamide since she failed to achieve molecular remission despite an intensified chemotherapeutic regimen. on day 100 post sct, she had a 100% donor chimerism and decreasing minimal residual disease (mrd) marker (minimal 1 × 10 − 6 ). 140 days post sct she had a molecular relapse with an mrd of 5 × 10 − 3 and a subsequent morphological relapse as well as mixed donor chimerism. further treatment regimens included chemotherapy, intrathecal therapy and four donor lymphocyte infusions (dlis). initially, she displayed a good morphological response to dlis but the leukaemic burden eventually remained stable with an mrd of 2 × 10 − 2 . considering 54.2% pd-1 expression on cd3+ t cells in the patient's bone marrow and the encouraging data in other hematologic malignancies an off-label therapy with the pd-1 inhibitor pembrolizumab1-4 was initiated. the patient and her parents gave informed consent and she received a single dose of pembrolizumab at 3.3 mg/kg 343 days after sct. one week after administration of pembrolizumab, the patient developed acute gvhd grade iv of the skin, mucosa, liver, lung, central nervous system and eyes. she had a severe generalized inflammatory reaction with high inflammatory markers, increased hepatic transaminases and lymphocytic infiltration of the liver, cerebrospinal fluid and bronchoalveolar lavage fluid. magnetic resonance imaging (mri) of the brain revealed periventricular white matter lesions and hyperintensities of basal ganglia and bilateral temporal lobe consistent with autoimmune encephalitis. treatment with high-dose corticosteroids, cyclosporine and the anti-interleukin 6 receptor antibody tocilizumab slightly improved her clinical condition. her mrd value significantly decreased to 4 × 10 − 4 two weeks after administration and she achieved a 100% donor chimerism in bone marrow. despite this promising response her medical condition deteriorated and the severe inflammatory reaction caused fatal multi-organ failure. this is to our knowledge the first report on a remarkable and fast response to pd-1 inhibition in a patient with pediatric t-all refractory to multiple lines of therapy including allogeneic sct. this case illustrates the potential risk of checkpoint inhibitors to trigger severe gvhd that is not responsive to steroids. induction of inflammatory gvl responses without causing severe gvhd by therapeutic checkpoint inhibition needs to be addressed in future clinical trials. in recent years, there is a remarkable trend in the use of haploidentical-related hematopoietic cell transplantations (haplo-hct) in patients who do not have a hla matched related or unrelated donor. here, we report our single-center experience, in patients who underwent haplo-hct for acute leukemia. between 2011 and 2016 seventeen consecutive adult patients, seven males and ten females, median age 42 years (range: 18-61 years) with high-risk acute leukemia underwent unmanipulated, bm or pbsc transplantation from an haploidentical family donor. eleven patients transplanted for acute myeloid leukemia (5 in cr1, 1 in cr2, 1 in minimal active disease after cr1, 1 second trasplant in cr2, 1 transformed mds in cr1, 2 aml secondary to myelofibrosis in cr1), 5 for acute lymphoblastic leukemia (2 in cr1, 3 in active disease) and 1 mastcell leukemia (secondary to aml) in active disease. sixteen patients received myeloablative conditioning, and 1 reduced intensity, respectively. in five patients stem cells source was bm, in 12 were g-csf mobilized pbsc. the median infused cd34+ cell dose was 4.47 × 10 6 (range: 1.0 × 10 6 -8.2 × 10 6 ). conditioning regimens were: bu-flu-mac (n = 9), tbf-mac (n = 7), tbf-ric (n = 1) the regimens for gvhd prophylaxis were: ptcy as sole gvhd prophylaxis (n = 1), mtx-csa-atg (n = 9), methylpred-atg-tacrolimus (n = 6), atg-csa-mtx-mmf (n = 1). sustained trilineage engraftment occurred in 15 patients (88%), two patients died of transplantation-related complications before day 21 after transplantation without myeloid recovery. for patients receiving bm or pbsc grafts, the median time to 4500 neutrophils recovery was 16 days (range: 10-36), and 420,000 platelets recovery was 16 days (range: 13-37). 7/15 patients (46.6%) and 2/15 (13.3%) had ii-iv and iii-iv grade of acute gvhd, respectively.the incidence of grade ii-iv cgvhd was 27%. after a median follow-up of 11 months, 4/17 patients (23.5%), 4 out 5 patients transplanted in cr1, are alive and disease free at 50, 28, 19, 16 months (inclusing the patient transplanted for aml after imf). the 2-year probability of overall and progression-free survival was 40% (95% ci, 4.0-58.0%) and 28.6% (95% ci, 2.0-20.0%), respectively. causes of death were: sepsis (n = 1 ), fatal agvhd (n = 1), pneumonia (n = 1), toxicity (n = 2), progression (n = 4) and relapse (n = 4). in our experience unmanipulated bm or pbsc transplantation from haploidentical family donor is feasible approach with high engraftment rates and acceptable trm (23%) and rate of grade iii-iv agvhd, associated with durable remission in a proportion of patients with high-risk acute leukemia, specially in patients with aml transplanted in first remission. it is generally recognized that allogeneic hematopoietic stem cell transplantation (allo-hsct) should not be administrated to patients with severe aplastic anemia (saa) or very severe aplastic anemia (vsaa), when they got active infection. however, without neutrophil, severe infection is usually difficult to control and even fatal. under these circumstances, rapid recovery of neutrophil by allo-hsct might be an alternative to control infection. from january 2002 to december 2015, there were 175 young patients received allo-hsct for saa or vsaa at shanghai children's medical center in china. among them, 22 patients (11 males and 11 females) with a median age of 7.0 years (range: 3.0-14.0 years) received allo-hsct with refractory active infections. refractory active infection was defined as persistent neutropenic fever with nonresponse to standard doses of broad-spectrum antibacterial agents and antifungal agents for more than three weeks, with or without definite focus of infection. prior to allo-hsct, four patients had persistent fever of unknown origin, 11 patients with singlesite infection, and 7 patients with multiple-site infections. sites of infection included lung, sinus, cellular tissue, peritoneum, liver, spleen and skin. the conditioning regimen consisted of fludarabine, cyclophosphamide and rabbit-antithymocyte globulin with or without total body irradiation (tbi) (2-3 gy). twelve patients were transplanted from mismatched unrelated donors, 3 from matched sibling donors, and 7 from haploidentical donors. sixteen patients received g-csf mobilized peripheral blood stem cells, three patients g-csf mobilized peripheral blood stem cells plus g-csf primed bone marrow stem cells, two patients bone marrow stem cells, and 1 patient umbilical cord blood stem cells. a median of 11.4 × 10 8 /kg mononuclear cells with 4.6 × 10 6 /kg cd34+ cells were transfused, except the patient who underwent ucbt with a total of 1.3 × 10 8 /kg mononuclear cells and 1.5 × 10 6 /kg cd34+ cells transfused. eighteen patients achieved recovery of neutrophil and finally control of infections, including one patient who suffered primary graft failure and had autologous marrow recovery. three patients died of infection and one patients died of acute renal failure before recovery of neutrophil. one patient died of pneumonia 8 months after allo-hsct. one patient become thrombocytopenia after allo-hsct. the other 16 patients are all disease-free. there were five patients developing grade i-ii acute gvhd, and 4 patient grade iii-iv acute gvhd. all were cured at last. three patients had localized chronic gvhd and one patient had extensive chronic gvhd. with a median of 2 years follow-up, the overall survival rate and disease-free survival rate are 77.3% ± 8.9% and 71.3% ± 10%, respectively. allo-hsct could be a feasible way to control infection for children with saa or vsaa in the present of refractory active infections. disclosure of conflict of interest: none. paroxysmal nocturnal hemoglobinuria (pnh) may present hemolysis isolated (classical pnh) or associated with aplastic anemia (aa; aa/pnh syndrome). while classical pnh patients require anti-complement treatment (eculizumab), the treatment of aa/pnh patients should target their underlying aa by immunosuppression (ist), or even bone marrow transplantation (bmt). however, in a few patients clinically meaningful aa and hemolysis may be concomitant, eventually justifying both ist and eculizumab. to date there is no standard treatment for s413 this rare condition. amongst a large cohort of 145 pnh patients (between 2007 and 2016) at our reference centers, st. louis hospital (paris) and federico ii university (naples), we retrospectively assessed characteristics and outcomes of patients diagnosed with aa/pnh who received intensive ist during or immediately before (3-6 months) eculizumab treatment. nine patients were identified. eight patients fulfilled the criteria of severe aa, and one had an immunemediated isolated agranulocytosis. since no patient had a hla-matched related donor for bmt, all patients received intensive ist according to institutional guidelines. six out of 9 patients were already on eculizumab treatment at the moment of starting intensive ist (concomitant treatment) whereas 3 patients received ist in the 3-6 months (median time of 3 months) before the introduction of anti-complement therapy (sequential treatment). for all patients already on treatment, eculizumab was not discontinued to minimize the risk of rebound intravascular hemolysis and thrombotic complications. eculizumab was administered at the standard dose of 900 mg fortnightly in all but one patient, who needed an increased dose (1200 mg) because of pharmacokinetic breakthrough. six patients (5 aa and 1 agranulocytosis), including the three undergoing a sequential treatment, received standard ist with horse-antithymocyte globulin (h-atg, 40 mg/kg for four consecutive days) combined with cyclosporine a (csa). the remaining three aa patients received alemtuzumab (3-10-30-30 mg subcutaneously in four consecutive days) and csa within the prospective trial nct00895739; one of these patients a few months later also received a second ist course with rabbit-atg (3.5 mg/kg for five consecutive days) and csa. all the patients completed the scheduled treatment without any side effect, including infusion-related reactions. lymphocyte depletion (o 100/μl) was observed in all patients irrespective of sustained therapeutic complement blockade. all the patients were available for response assessment at 6 months. among the six patients receiving a concomitant treatment we observed one partial response (pr) and two complete responses (cr), whereas the three remain patients were non-responders (nr). of them one was rescued with an unrelated bmt, while two remained on eculizumab treatment. one of the cr relapsed at 3 years showing clonal evolution and finally died. all the other patients are alive, keeping their hematological response. patients receiving a sequential therapy were one in pr and two in cr 6 months after introduction of eculizumab. in conclusion, for patients diagnosed with severe aa/pnh syndrome intensive ist and eculizumab treatment, can be safely delivered either concurrently or sequentially, with an overall response rate of nearly 70%. this is the first systematic description of the management of severe aa in hemolytic pnh patients receiving eculizumab treatment. disclosure of conflict of interest: none. (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) in aa, 12 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) in rcc, and 8 (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) years in rcmd. sixty-five patients underwent bmt from hla-matched (related 41, unrelated 24) and 23 from hla-mismatched (related 6, unrelated 17) donors. conditioning regimens were used as follows; cyclophosphamide (cy)+antithymocyte globulin (atg) ± total body irradiation (tbi) (n = 29), fludarabine (flu)+cy ± atg ± tbi (n = 42), and flu +melphalan (mel) ± atg ± tbi (n = 17). all patients got engraftment after bmt. however, late graft failure was found in 6 patients with rcc, and 7 with rcmd, but none with aa. out of 13 patients who developed late graft failure, 8 patients used flu+cy ± atg ± tbi, 3 used cy ± atg ± tbi, and 2 used flu +mel ± atg ± tbi for conditioning regimens. five-year cumulative incidence (ci) of graft failure was higher in rcmd (36 ± 6.0%) than in aa (0%) and rcc (20 ± 1.8%), significantly (po0.01). five-year ci of graft failure tended to be higher in flu regimen (23 ± 2.2%) than in cy+atg ± tbi regimen (10 ± 0.65%), but not significant (p = 0.20). five-year ci of graft failure did not differ between with (21 ± 2.1%) or without tbi (29 ± 6.3%) (p = 0.57). multivariate analysis revealed that the morphological classification was a significant risk factor for graft failure (po0.01). five-year failure free survival rate (63 ± 11%) in rcmd was significantly lower than in aa (96 ± 3.8%) and rcc (74 ± 7.9%) (p = 0.02). graft failure, second malignancy, and death were considered as failure events. one patient with aa died of infection, four with rcc died of infection (n = 2), bleeding (n = 1) and myocarditis (n = 1), and one with rcmd died of infection. five-year overall survival rates were not different among 3 groups (aa, 96 ± 3.9%; rcc, 89 ± 5.2%; rcmd, 95 ± 4.7%) (p = 0.53). high incidence of graft failure in rcmd may be due to higher bm cellularity than in aa and rcc. the optimal conditioning regimen of bmt should be established for children with abmf based on the bm cellularity and morphological classification. disclosure of conflict of interest: none. recent studies have suggested inferior outcome of patients treated with rabbit atg (thymoglobulin, sanofi) as compared to horse atg (atgam,pfizer or lymphoglobulin, genzyme), and a higher early mortality with rabbit atg has been suggested to explain this difference. aim: to assess early mortality, response rates at 3,6 and 12 months and long term outcome, in a large cohort of aa pts, treated in europe or asia with rabbit atg and cyclosporin, as first line treatment. eligible for this study were pts with aa, treated with thymoglobulin between 2001 and 2012 in europe (n = 519) or asia (n = 457). median year of treatment, was 2008: characteristics were comparable : median age 20 and 21 years, interval diagnosis treatment (23 and 25 days) and severity of the disease (46% and 48% with vsaa). early mortality was analyzed for all 976 patients.long term outcome was also analyzed for 800 pts for who response data (no, pr, cr) were available. mortality o90 days was 5.5% and 2.1%, respectively, in the time period 2001-2008 and 2009-2012 (p = 0.007). in these 2 time periods, early mortality for patients aged 0-60, was reduced from 3.5% to 1.4% and for patients over 60, from 22% to 9%. overall response was recorded in 800 patients. at 6 months the cumulative incidence of response was comparable in the 2 time periods: 62% vs 66%, and at 1 year, 73% vs 75% (p = 0.8). response rates at 6 months were age dependent: 68%, 66%, 62%, 40% respectively in patients aged 0-20, 21-40, 41-60, 460 (p = 0.0006). when non responders at 3 months were reevaluated at 1 year, 59% had responded, 26% were non responders, 5% had died, and 10% had received other treatment. responses at 6 months, were 60%, 66%, 74%, in pts with very severe, severe and non severe aa (p = 0.0001). the actuarial 10 year survival for the entire population was 71%, and 70%, when pts were censored as surviving at transplant. actuarial 10 year survival in univariate analysis was as follows: 89% vs 61% for day 90 responders vs non responders (p o0.01), 68% vs 80% for males versus females (p = 0.07); 82%, 72%, 66%, 27% in pts aged 0-20, 21-40, 41-60, 460 years (p o0.001); 67%, 78%, 76% in pts with neutrophils o0.2 × 10 9 /l, 02-05 × 10 9 /l and 40.5 × 10 9 /l (p o0.001); 77%, 75%, 68% for pts with an interval diagnosis-treatment of o30 days, 31-60 days or 4 60 days (p = 0.002). finally pts treated 42008 had a 5 year survival superior to pts treated before 2008 (84% vs 77%, p = 0.01). survival at 5 years, in the recent period (2009-2012), was 83% for pts aged 1-60 and 60% for pts over 60 years. in multivariate cox analysis the following variables remained independent predictors of survival: patient age, year of treatment, severity of the disease, interval diagnosis treatment, and gender. thymoglobulin +csa is effective and safe in patients with aa. the outcome is mainly age dependent. the inrerval between diagnosis and treatment remains a strong predictor: the earlier the better. for pts o60 years old current early mortality. 4. for pts 460 years of age, current early mortality is higher (9%), response rate (40%) and 5 year survival (70%) are lower. 5. the actuarial 10 year survival for the enire population was 71%. survival at 5 years has improved from 77% (o o/u42008) to 84% (2009-2012), especially for pts over 60 years (37% vs 70%, p = 0.006). [p560] s415 disclosure of conflict of interest: we thank centers for providing up date follow up of their patients . this study was supported by a grant of sanofi-genzyme. both of the patients have extremely low anc o0.05 × 10 9 /l, reto0.5%, plt o20 × 10 9 /l. both was given antibiotic treatment with carbapenem, vancomycin/linezolid, voriconazole or amphotericin b liposome and got no response. no pathogenic bacteria or fungus was found from either of the patients. both of them had no full sibling or matched unrelated donor and had their father as their haploidentical donor. bone marrow combined with peripheral blood stem cell (pbsc) was adopted. conditioning: fludarabine days − 5 through − 2, (40 mg/m 2 × 4), intravenous busulfan (1.1 mg/kg q6h) on days − 4 to − 2. gvhd prophylaxis: high-dose cyclophosphamide 40 mg/kg on days +3 and +4, mmf and tacrolimus since days +5. rabbit anti-thymocyte globulin (thymoglobulin) 2.5 mg/kg on days − 4 to − 2. stable neutrophil engraftment (anc 40.5 × 10 9 /l) occurred on day +13 and day+19 respectively. platelet achieved 20 × 10 9 /l on day +11 and day +55, respectively. both transplant course was complicated by febrile neutropenia without detected etiology, while both children have no fever since the first day anc 40.5 × 10 9 /l.the facial swelling was resolved in both patients except for palatal fistula and fistula of maxillary sinus as the sequela of severe nasosinusitis. no acute or chronic gvhd. case1 had hemorrhagic cystitis on day +30 which last for about 30 days, and suspected thrombotic microangiopathy (tma) with hypertension, thrombocytopenia, elevated ldh and creatine on day +51 which was resolved soon after discontinue of tacrolimus. case2 had delayed engraftment of platelets and herpes simplex virus 6 encephalitis on days +40 which was cured by ganciclovir and high dose intravenous immunoglobulin. now they are 9 and 7months post-hsct respectively and are doing well with 100% chimerism and no gvhd. alternative donor hsct may be considered as the first line salvage therapy for patients of vsaa with extremely low anc and active infection. fast reconstruction of neutrophil helped to control the infection. hallo-identical hsct make sure nearly every patients can find a donor. ptcy is proved to be efficient and safety in gvhd prophylaxis and facilitating engraftment in these two challenging cases. disclosure of conflict of interest: none. long-term outcome of patients with severe aplastic anemia receiving allogeneic hematopoietic cell transplantation using nonmyeloablative conditioning with fludarabine and low dose total-body irradiation l cheryl xiu qi 1 , l yeh ching 2 , p michelle li mei 1 , t lip kun 1 , h william ying khee 2 , g yeow tee 2 , t patrick huat chye 2 and k liang piu 1 1 department of haematology-oncology, national university cancer institute, singapore and 2 department of haematology, singapore general hospital, singapore allogenic haematopoeitic stem cell transplant (ahct) offers the best prospect of cure in patients with severe aplastic anaemia (saa). the use of myeloablative hct is however limited by the toxicity of preparative regimens, the lack of matched sibling donors, transplant related mortality and graft rejection. the introduction of non-myeloablative (nm) conditioning offers the possibility of extending this potentially curative treatment to patients in whom ahct was previously contradindicated. in 2006, we reported the outcome of 8 patients with saa who have received ahct using nonmyeloablative conditioning comprising of 3 days of fludarabine at 25mg/m 2 and total body irradiation at 2 gy (flu + tbi 2gy). here, we report a longer follow-up, with 6 additional patients who had recevied ahct with this regimen. fourteen patients with a median age of 37 years old (range: 17-48 years old) received filgastrim-mobilised peripheral blood stem cell transplant from either hla identical sibilings (n = 12) or matched unrelated donor (n = 2) after receiving nm conditioning consisting of flu + tbi 2gy. the first two patients received cyclosporine (cya) and mycophenolate mofetil (mmf) for the post-transplant immunosuppessive therapy. the remainining 12 patients received cya, mmf and a short course of methotrexate (mtx) for additional graft-versus-host dsease (gvhd) prophylaxis. results all patients achieved prompt engraftment. the median time for engraftment of neutrophils (40.5 × 10 9 /l) and platelets (420 × 10 9 ) were 16 days (range: 13-20 days) and 13 days (range: 8-25 days), respectively. chimerism analysis on day 28 and subsequently showed 495% donor cells in all patients except 1, who developed secondary graft failure at 3 months and required salvage hct. none of the patients experienced grade 3 and above regimenrelated toxicity. five patients developed grade ii-iv acute gvhd and 2 patients developed limited chronic gvhd. with a median follow-up of 8.8 years (range: 0.54-14.52 years), the estimated overall survival and event free survival were 86% and 79% respectively. the two patients who did not receive mtx developed acute gvhd of the liver and succumbed to infective complications. the remaining 12 patients who had received triple immunosuppressive therapy were well, with limited chronic gvhd seen only in 2. our results suggest that the nm conditioning regimen comprising of flu + tbi 2gy provides sufficient immunosuppression to allow prompt and stable engraftment with minimal regimen-related toxicity. it is an attractive option for patients with saa who require ahct but are at increased risk of regimen-related complications from more intensive cyclophosphamide-based regimens. disclosure of conflict of interest: none. paroxysmal nocturnal hemoglobinuria (pnh) is a rare acquired clonal disorder of hematopoietic cells characterized by the triad of hemolytic anemia, cytopenias and high risk of venous thrombosis. due to the rarity of the disease, most reported data derive from multicenter studies. we describe the natural history of the disease in a 30-year (yrs) long single center series of pnh patients (pts). we performed a retrospective analysis of 42 consecutive pts followed at our center from 1985 to 2016. since 1985, the diagnosis was made by ham test; starting from 2000, flow cytometry (fc) analysis was used to diagnose new pts and to confirm pnh in pts previously diagnosed by the ham test. at diagnosis, 26 pts had classic pnh, 9 aplastic pnh and 7 intermediate form. the cumulative incidences of thrombosis, cytopenia and clonal neoplasm were 39%, 18% and 10%, respectively. except for 1 pt with aplasia, no severe infections were diagnosed, nor renal failures or pulmonary hypertention. the 30 yrs overall survival (os) was 84%. a nonsignificant better os was associated to the absence of thrombotic events (96% vs 80%) and to a diagnosis made during the last decade (100% vs 90% vs 75%).up to 2005 the treatment options were supportive care or allogenic bone marrow transplantation. since 2005, eculizumab was used in transfusion-dependent patients and/or in case of a thrombotic history. overall, 14 pts were transfusion-independent for the entire period of the illness, 28 were transfusion-dependent and/or had thrombotic events(8pts). six of the latter pts never received eculizumab but only transfusion support (3 pts) or allogeneic bone marrow transplant (3 pts), while 22 pts received eculizumab (the first 4 pts were included in the phase iii triumph and shepherd trials).considering the increased risk of meningococcus infection for pts on eculizumab, vaccination with conjugated anti-meningococcus serotypes acwy was employed and, since 2016, conjugated antimeningococcus serotype b was added. overall, 18 pts treated with eculizumab became transfusion-independent and four remained transfusion-dependent. no thrombotic event was observed after eculizumab, even if 8 pts had recurrent thromboembolisms prior to receiving the drug. no severe infection was documented. one patient developed extravascular hemolysis and receive a successfully selective splenic artery embolization. the 10 yrs os in the eculizumab group was 92%.no pnh-associated death occurred. our study confirms that thrombosis is a major complication in pnh pts not receiving eculizumab, influencing os. the better os in the last decade is probably due to the use of eculizumab and to lack of thrombotic events. in particular, for 22 pts on eculizumab the 10 year os was 92%, even though half of the pts had thromboembolism and diagnosis made prior to the last decade. although kidney failure and lung hypertension have been reported, we did not observe these complications in our long follow-up case series. we can assume that the availability of a dedicated emergency room at our center allows to perform, promptly, hyper-hydration or transfusion support in case of hemoglobinuria crisis, reducing the risk or organ damage. no infections have been observed after eculizumab, probably due to the vaccination program schedule recommended in the literature, plus the addition of conjugated anti-meningococcus serotype b. however, shared guidelines are needed. disclosure of conflict of interest: none. mortality following hsct in saa pts over the age of 40 is reported to be in the order of 50%, without taking in to account long term sequelae such as chronic gvhd, known to be more frequent in older patients. this has prompted international guidelines to recommend first line immunosuppressive therapy above 40 years of age. the question is whether this is still true in 2017. the aim of the study is to assess whether trm in saa patients grafted 2010-2015 is reduced,as compared to the era 2001-2009. we used the wpsaa ebmt registry, and identified 748 pts aged 40 years or more, with acquired saa, grafted between 2001 and 2015. we divided pts in 2 transplant eras:2001-2009 (n = 327) and 2010-2015 (n = 407). in the more recent period (2010-2015) pts were older (53 vs 49 year, p o0.01), were more often grafted from alternative donors (alt) (64% vs 43%, po0.01), with a greater use of bm (54% vs 41%, p o0.01), and with a longer interval dx-tx (317 vs 258 days, p0.01), and more often received a fludarabine containing regimen (55% vs 42%, p o0.01). the os 5 year of pts grafted in 2001-2009 was 57%, compared with 55% for pts grafted 2010-2015 (p = 0.7). in multivariate analysis, including the interval diagnosis transplant, patient's age, donor type, stem cell source and conditioning regimen, the lack of improved survival in 2010-2015 was confirmed (p = 0.3). a very strong age effect was shown both in univariate and multivariate analysis: survival of pts aged 40-50 years, 51-60 years and 461 years, was respectively 64%, 54%, 41% (p o0.0001) and this was confirmed in multivariate analysis. the conditioning regimen, also proved to be a significant predictor, with improved survival for alt transplants receiving flu containing regimens (56% vs 46%, po0.001). in general pts receiving either cy200 or a flu containing regimen, did significantly better than pts receiving other preparative regimens (58% vs 50%, p = 0.02). the use of a sibling donor (sib) did not prove to predict survival in multivariate analysis. pts receiving campath in the conditioning,did significantly better than pts not receiving campath (65% vs 54% po0.01); similarly survival of patients with atg was superior 59% vs 41% compared to patients not receiving atg (p o0.01). when pts receiving either campath or atg (n = 564) were compared to patients not receiving either (n = 161), the difference in survival was 61% vs 41% (p o0.0001), and this was significant also in multivariate analysis. combined primary and secondary graft failure was reduced from 16% to 12% in the two time periods (p = 0.02), acute gvhd grade ii-iv was reduced from 15% to 11% (p = 0.1) and chronic gvhd was also reduced from 32% to 26% (p = 0.04) infections remain the leading cause of death in both transplant eras (18% and 22% respectively), followed by gvhd (5% and 4%) and graft failure (5% and 2%), whereas ptld have been reduced from 3% to 0.5%. hsct in pts with acquired saa aged 40 and over, continues to carry a significant risk of trm also in 2010-2015, ranging from 36% in younger pts (40-50) to 59% in older pts (460 years). survival is predicted in multivariate analysis, by two crucial predictors: patients' age and the use of either campath or atg,the latter giving a 20% survival advantage over no campath/atg. alt and sib donors produce similar survival. this study gives further support to current guidelines, suggesting first line therapy with atg+csa, in pts over the age of 40. [p565] disclosure of conflict of interest: none. autoimmune diseases p566 allogeneic haematopoetic stem cell transplantation as curative therapy for early-onset, refractory crohn's disease e groene 1 , p bufler 1 , k krohn 1 , s immler 1 , g marckmann 2 , t feuchtinger 1 , s koletzko 1 and m albert 1 1 dr. von hauner university children's hospital and 2 institute of ethics, history and theory of medicine, lmu results of a recent randomized trial suggest that autologous hsct is an option in adult patients with severe, therapyrefractory crohn's disease (cd) with an associated mortality risk of 4%. however, relapse of the disease is frequent (1). in contrast allogeneic hsct has resulted in long-term cure of cd in affected patients transplanted because of haematological malignancy (2). we report a 17 year old girl who was diagnosed with severe cd at age seven (paris classification l3, l4a, b1). neither next generation sequencing nor immunological work up identified a monogenetic cause of cd. progressive chronic inflammation manifesting ubiquitously in the gastrointestinal tract resulted in severe complications, such as perianal fistulas with rectal stenosis, intestinal abscesses, dysphagia, severe weight loss, failure to thrive, delayed puberty and the need for ileostomy and long-term exclusive enteral nutrition via tube feeding. despite multiple lines of therapy, including repeated nutritional therapy, steroids, immunosuppressants (methotrexate, azathioprine) and biologicals (infliximab, adalimumab, certolizumab) a lasting remission could not be achieved resulting in poor quality of life. after careful risk/benefit assessment including ethical counselling allogeneic hsct was offered. she underwent allogeneic hsct from a matched (10/10) unrelated bone marrow donor (4.3 × 10 8 /kg total nuclear cells). conditioning was performed according to a protocol successfully applied in adolescents with chronic granulomatous disease (3) with alemtuzumab (3 × 0.2 mg/kg/d), targeted busulfan (tauc 53770 ng × h/ml) and fludarabine (6 × 30 mg/m 2 ). cyclosporine a and mycophenolate mofetil were used as gvhd prophylaxis. neutrophil and platelet engraftment were observed on days +20 and +24, respectively. the post hsct course was complicated by grade i acute skin gvhd treated with topical steroids and toxic megacolon secondary to scarring stenosis on both ends of the unused colon on day +130 requiring surgery and a colostomy. at 12 months post hsct the patient is well, off immunosuppressive medication, without gvhd and exhibiting 495% donor chimerism. the cd is in complete clinical and histological remission as proven by endoscopy and biopsies. stoma reversal with restitution of intestinal continuity is planned for the next 12 months. refractory cd can lead to life-threatening complications and severely reduced quality of life. although long-term outcome in our patient will need to be carefully assessed, allogeneic hsct may offer a curative therapy in children and young adults with severe cd, even in the absence of an identified monogenetic cause. current ebmt recommendations include consideration of ahsct in exceptional circumstances for patients with severe refractory cd. the only randomised trial of ahsct in cd (astic) confirmed substantial short-term benefits but failed to meet its primary 1 year endpoint. to further clarify the longterm safety and efficacy of ahsct in cd we performed a retrospective analysis of patients undergoing ahsct for cd outside the astic trial using the ebmt registry. patients were identified from the ebmt registry. all adult patients undergoing ahsct for a primary diagnosis of cd from 1997 to 2015 were eligible for inclusion. patients who were enrolled in the astic trial were excluded. from a total of 99 patients (across 27 centres) on the ebmt registry, data were obtained from 76 patients transplanted in 14 centres in 7 countries. median patient age was 30 yrs (range: 20-51) and 63% were female. median age at first diagnosis of cd was 18yrs (range: 2-48). patients were heavily pre-treated, having failed or been intolerant to a median of 6 previous lines of therapy (range: 3-10). 55% had received experimental therapy prior to auto-hsct. 80% of patients had undergone at least 1 operation. the median time from first diagnosis of cd to auto-hsct was 12.3 years (range: 1.3-25.8). all patients received peripheral blood stem cells following conditioning with cyclophosphamide 200 mg/kg and 84% received anti-thymocyte globulin (atg). the median cd34+ dose infused was 5.5 (range: 2.4-40.6) × 10 6 /kg. twelve percent of patients underwent cd34+ selection. neutrophil and platelet engraftment occurred at a median of day 10 (range: 6-18) and day 9 (range: 1-44), respectively. sixety-one percent received post transplant g-csf. median length of follow-up following auto-hsct was 42 months (range: 6-174). at 100 days post auto-hsct, 64% of patients were in clinical remission (cr), defined as no abdominal pain and normal stool frequency. a further 27% experienced significant improvement, defined as improvement in abdominal pain and stool frequency. for 5% there was no appreciable change in disease and in 4% the disease worsened compared to baseline. at 1 year post auto-hsct, 39% were in cr, 19% were improved, 20% were unchanged and 22% had worsened. at last follow-up, 37% were in cr, 23% were improved, 25% were unchanged and 15% had worsened. overall 74% restarted medical therapy post auto-hsct and 38% required further surgery. overall 26% developed an infection requiring treatment post auto-hsct (11% bacterial, 12% viral). ebv and cmv reactivation occurred in 7% and 4% respectively and herpes zoster occurred in 4%. a secondary autoimmune disease developed in 13%, most commonly thyroid disease (63%). malignancy developed in 5%, of which skin cancer accounted for 75% of cases. one patient died at 56 days post auto-hsct due to cmv infection, sepsis and multiorgan failure. this large retrospective series further supports the safety and efficacy of ahsct in a population with severe and treatment-refractory crohn's disease, 60% of patients experienced complete remission or significant improvement in cd symptoms with long-term follow-up. trm observed was similar to ahsct for other indications. in summary, ahsct appears to be an extremely promising therapy for severe refractory cd. further follow up of astic patients and future randomised trials are warranted. disclosure of conflict of interest: none. memory stem t cells (tscm) are long living self-renewing memory t cells with long-term persistence capacity, which play a relevant role in immunological memory and protection against infectious diseases and cancer1,2,3,4,5,6. the aim of this work is to investigate the potential role of tscm as a reservoir of arthritogenic t cells in rheumatoid arthritis (ra). we analysed the dynamics of circulating tscm (here identified as cd45ra+ cd62l+ cd95+ t cells) and other memory t-cell subpopulations by multiparametric 11-color flow cytometry in 27 patients with active ra and in 14 of them also during treatment with anti-tnfα biological agents (etanercept). to analyse cytokine productions, functional assays were performed stimulating peripheral blood mononuclear cells (pbmcs) with pma/ionomycin and brefeldin a. after the stimulation, cells were stained for surface markers, fixed and stained for intracellular cytokines. we traced circulating antigen specific cd4+ t cells for the vimentine-derived citrullinated peptide (vimcit) 65savracitssvpgvr77 7,8 in hla-drb1 × 04:01 ra patients before and during the anti-tnfα treatment using custom mhc class ii tetramers. viral antigen specific cd8+ t cells were traced using mhc class i dextramers. age-matched healthy donors (hds) were used as control for all the experiments. we found a significant expansion of cd4+ tscm in patients with active ra both in terms of frequency and absolute counts. notably, cd4+ tscm significantly contracted upon anti-tnfα treatment, suggesting a role of tnfα in tscm accumulation. in contrast to cd4 +t cells, cd8 compartment did not show significative alterations compared to (hds). furthermore, cd4+tscm in ra patients displayed an enrichment in the th17 phenotype, largely implied in autoimmune disorders, while the other t cell subpopulation were not enriched in the th17 phenotype. at the antigen specific level, we were able to trace in hla-drb1 × 04:01 patients antigen specific cd4+ t cells, comprising tscm, specific for the vimentin-derived citrullinated peptide. of notice, citrullinated vimentin specific cd4+ t cells, including tscm, contracted during anti-tnfα administration, while viral-specific cd4+ t cells (ebvbhrf-1) and antiviral cd8 specificities (cmvpp65, flump, ebvbmlf-1) were not affected by etanercept administration, thus suggesting a possible role of cd4+ tscm as reservoir of arthritogenic autoreactive t cells. overall, our results suggest that tscm, by representing a long-term reservoir of undesired specificities, might play a non redundant role in sustaining ra and possibly other t cell mediated disorders, thus representing novel biomarkers as well as therapeutic targets. ongoing experiments will characterize the tcr repertoire on sorted tscm and cd4+ memory subsets in order to identify a possible oligoclonality in tscm repertoire. in conclusion, the analysis of tscm dynamics in autoimmune disorders could have relevant clinical implications as new biomarkers and for devising innovative therapeutic strategies. ebv and cmv reactivation following autologous haematopoietic stem cell transplantation (hsct) for autoimmune neurological diseases resolves spontaneously and rarely requires treatment c mapplebeck 1 , b sharrack 1 , h kaur 1 , y ezaydi 1 , h jessop 1 , l pickersgill 1 , l scott 1 , m raza 1 and ja snowden 1 1 departments of haematology, neurology and virology, sheffield teaching hospitals nhs foundation trust, sheffield, uk autologous haematopoietic stem cell transplantation (hsct) for severe autoimmune diseases involves immunosuppressive conditioning regimens and current guidelines recommend monitoring for viral reactivation of cytomegalovirus (cmv) and epstein barr virus (ebv) (snowden et al 2012) . however, the incidence, degree and management of viral reactivation are not established. we performed a retrospective observational service evaluation study of all patients receiving cyclophosphamide 200 mg/kg + rabbit anti-thymocyte globulin 6 mg/kg (atg, thymoglobulin) followed by autologous hsct for various autoimmune neurological diseases between 2011 and 2016 at our centre. data collected included the baseline serological status of the patient prior to transplant and serial blood pcr quantitation (copies/ml). if ebv and cmv reactivation occurred details of further management was collected and descriptive statistics were used to summarise outcomes. twenty-three patients received autologous hsct between january 2011 and october 2016; 21 patients with multiple sclerosis (ms), 1 with chronic inflammatory demyelinating polyneuropathy (cidp) and 1 with stiff person syndrome. twenty-two patients had positive ebv igg serology prior to transplant and 1 patient had an equivocal result. seventeen patients had evidence of ebv reactivation and a further patient had ebv dna detected post-transplant but with less than 250 copies/ml. the average time to peak ebv pcr was 26.5 (range: 12-44) days post-transplant and a range: in ebv pcr peak level from 623 to 577 000 copies/ml. the 4 patients who had ebv pcr results of over 100 000 copies/ml had ct scans of chest, abdomen and pelvis performed which did not demonstrate significant lymphadenopathy or hepatosplenomegaly. in all patients monitored for a detectable ebv reactivation, the ebv pcr spontaneously began to fall within 2 months (average 36 days, range: 18-60 days) post-transplant and no specific treatment was required. one patient had late ebv reactivation of 3480 copies/ml at 6 months post-hsct associated with chronic tonsillitis and tonsillectomy specimens showed follicular hyperplasia without evidence of post-transplant lymphoproliferative disorder (ptld) and ebv pcr levels normalised without other treatment. 8 (35%) patients had positive cmv igg serology prior to transplant and one patient had an equivocal result. only 1 of 23 patients had a significant reactivation of cmv with 51 300 copies/ml at 21 days post-transplant, successfully treated with intravenous immunoglobulins and valganciclovir. two other patients had low level cmv reactivation with 94 and 476 copies/ml, respectively which resolved spontaneously without treatment. ebv reactivation in patients with neurological autoimmune disease undergoing autologous hsct is common and usually resolves spontaneously without treatment. asymptomatic cmv reactivation occurs in approximately 13% of patients in this setting and may require treatment. autologous hematopoietic stem cell transplantation (hsct) has been utilised for the treatment of severe multiple sclerosis (ms). it results in significant improvement of neurological function, although patients can experience exacerbations of ms-related symptoms during the procedure. we reviewed 17 patients with ms who underwent stem cell mobilisation and collection from march to november 2016. the median age was 40 years (24-55). nine patients (53%) were male. the interval from diagnosis to hsct was 114.3 months (range: 11.6-128.3). 9 patients (53%) had relapsing-remitting (rrms), six patients (35%) secondaryprogressive (spms) and two patients (12%) primary-progressive (ppms) multiple sclerosis. only 2 patients (12%) had not received any prior treatment, whereas 10 patients (59%) received two prior treatments, three patients (17%) received three treatments and two patients (12%) received four treatments. the median expanded disability status scale (edss) score was 6 (range: 2-6). peripheral blood stem cells were mobilised with cyclophosphamide (cy) 2 g/m 2 on day +1 and daily gcsf (5 μg/kg subcutaneously) from day +3 until the completion of the harvest. hsct was performed at a median of 33 days after mobilisation (range: 25-59). the conditioning regimen consisted of cy (50 mg/kg/day from day − 5 to − 2) and atg (2 mg/kg/day from day − 4 to − 2). exacerbation of ms symptoms was defined as the appearance of new or worsening of old symptoms for at least 24 h duration in a previously stable (4 weeks) patient. of the total cohort, 13 patients (76%) underwent mobilisation with cy+gcsf uneventfully. only two patients (12%) had an exacerbation of ms requiring hospital admission after collection (one with fatigue and increase of spasticity, other with worsening weakness). no patient required hospital admission during the mobilisation procedure. the median cd34+ cell dose was 8.39 × 10 6 /kg (range: 2.2-24). the median number of apheresis was 1(1-2). a total of 14 patients have undergone hsct at the time of this analysis. during transplant a total of 11 patients (78%) experienced an exacerbation of ms. of these, 54% (n = 6) before day 0 and 46% (n = 5) between day +4 and +7. symptoms of exacerbation were: muscle spasms in 4 patients (36%), weakness and reduced power of limbs in 4 patients (36%), increase instability and tremor in two patients (18%) and one patient (10%) with worsening of neuropathic pain. only three patients (28%) received treatment with methylprednisolone for ms exacerbation and symptoms had fully resolved by discharge in all patients. other transplant complications included neutropenic fever in all, invasive fungal infection in 1, fluid overload in 9 (64%) and atg related complications in 11 (78%) such as fever (n = 10) and pericarditis/serositis (n = 1). the median time to neutrophil engraftment was 10 days (10-14) and the median duration of hospital admission was 20 days (15-25). exacerbation of ms symptoms is common during hsct and can also occur during mobilisation. in our hands, after cy and gcsf mobilisation only two patients (11%) developed an exacerbation of ms symptoms compared with 11 patients (78%) after ct and atg conditioned hsct. it is possible that the addition of atg to cy triggers an immunological response involved in this transient deterioration of the ms symptoms. further studies are required to confirm this hypothesis. disclosure of conflict of interest: none. inflammatory immune response syndrome (iris) is a noninfectious worsening of neurological condition during immune recovery and has been documented to occur in hiv and in multiple sclerosis following alemtuzumab. the manifestation of iris includes headache, nausea, weakness, neurologic deficits, and mri enhancing lesion. we report three cases of iris after autologous non-myeloablative hematopoietic stem cell transplantation (hsct) in patients for which the transplant indication was an inflammatory neurologic disease: neuromyelitis optica (nmo), chronic relapsing inflammatory optic neuritis (crion), and multiple sclerosis (ms). mobilization was with cyclophosphamide 2 gr/m 2 and gcsf. conditioned regimen was 200 mg/kg cyclophosphamide (50 mg/kg/d) and 6.0 mg/kg ratg (thymoglobulin). the conditioning regimen for nmo and crion also included 1000 mg rituximab. case 1. a 22 years old african-american female with systemic lupus erythematosus (sle) and nmo was discharged day 10 and readmitted on day 14 for fever, headache, progressive altered mental status with dysarthria and legs. brain mri had numerous t2/flair hyperintense and enhancing lesions in the subcortical and periventricular white matter. a lumbar puncture was negative for infection including jcv. complete recovery occurred after treatment included high dose of steroids and plasmapheresis. case2. a 48 years old female with crion experienced blindness, weakness and slurring of speech three months post hsct. mri showed a large enhancing brain stem lesion. lumbar puncture was jcv negative. complete recovery occurred after solumedrol and rituximab. mri 6 months later demonstrated complete resolution of the enhancement with return of vision to baseline. case3. a ten year-old boy diagnosed with paediatric ms developed hemichorea seven days after hsc reinfusion. brain mri revealed a gadolinium-enhancing lesion in the contralateral basal ganglia. lumbar puncture was negative for infection including jc virus. symptoms resolved spontaneously after seventeen days. the appearance of new neurologic symptoms and mri enhancing lesions early after autologous hsct is unexpected and may be related to lymphocytes in the graft, immune recovery post engraftment, and or persistent auto-antibodies. it is mandatory to perform a lumbar puncture to exclude the possibility of infections including progressive multifocal leukoencelopathy (pml) due to jcv. the timing of presentation, the negativity of jc viral load, and the complete recovery with or without immune suppression suggest the hypothesis of iris, as an epiphenomenon of the immune reconstitution following autologous hsct for neurologic diseases disclosure of conflict of interest: none. hematopoietic stem cell transplantation is the effective method of therapy for cns autoimmune disorders in children. long-term outcomes and late effects estimation required. the aim of the study is to estimate long-term outcomes and late effects at children underwent auto-hsct for multiple sclerosis (ms) and allo-hsct for neuromyelitis optica (nmo). twelve pts. with ms and 3 pts. with nmo were included to the analysis. ms pts. gender: female -75% (n = 9), male -25% (n = 3 allogeneic haematopoietic stem cell transplantation (hsct) remains the sole curative option for patients with myelofibrosis (mf). although a spectrum of conditioning regimens has been used, the optimal preparative treatment before hsct remains to be defined. we did a phase ii randomized study at 21 transplant centers in italy with the aim of comparing the reduced-intensity conditioning (ric) fludarabine-busulfan (fb) (conventional arm), that had been already tested in the prospective ebmt study (1) with the ric fludarabine-thiotepa (ft) (experimental arm), that has been widely used in italy in the last two decades (2) . eligible to this study were patients with primary mf or a mf subsequent to a previous essential thrombocytemia or polycyhemia vera, an age ≥ 18 ≤ 70 years, a karnofsky performance status 4 60, a comorbidity index o o/u4 5 and with at least one of the following unfavorable prognostic factors: anemia (hb o 10 g/dl), leukocitosis (25 × 10 9 /l), circulating blasts 41% or constitutional symptoms. patients were randomized to receive intravenous busulfan 0.8 mg/kg for 10 doses or thiotepa 6 mg/kg for two doses associated to fludarabine 30 mg/m 2 for impact of pre-transplant ruxolitinib in myelofibrosis patients on outcome after allogeneic stem cell transplantation syed abd kadir, sharifah shahnaz, author 1,2 , zabelina, tatjana, co-author 1 , christopeit, maximilian, co-author 1 , wulf, gerald, co-author 3 , wagner, eva, co-author 4 , bornhaeuser, martin, co-author 5 , schroeder, thomas, co-author 6 , crysandt, martina, co-author 7 , mayer, karin tina, co-author 8 , stelljes, matthias, co-author 9 , badbaran, anita, co-author 1 , wolschke, christine, co-author 1 , ayuk ayuketang, francis, co-author 1 , triviai, ioanna, co-author 1 , wolf, dominik, co-author 8 ruxolitinib (rux) is the first approved drug for treatment of myelofibrosis. because spleen size and constitutional symptoms may influence outcome after allogeneic stem cell transplantation (asct), rux is recommended before stem cell transplantation in order to reduce therapy-related morbidity and mortality and improve outcome (ebmt/eln recommendation, leukemia 2015) the aim of this retrospective study was to evaluate the impact of pretreatment with rux in comparison to transplantation of rux-naïve mf patients with regard to outcome after asct. we included 149 myelofibrosis patients (pts) with a median age of 59 years (range: 28-74) who received asct between 2000 and 2015 from related (n = 23), matched (n = 86) or mismatched (n = 50) unrelated donor. all patients received busulfan-based reduced intensity conditioning. while 113 pts (66%) did not receive rux, 46 pts (34%) received rux at any time point prior to asct. the median daily dose of rux was 30 mg (range: 10-40 mg) and the median duration of treatment was 28 days (range: 12-159 days). in 11 pts rux was stopped before stem cell transplantation because of no response or loss of response, while in 35 pts rux was given until start of conditioning. gvhd prophylaxis consisted of cni plus short course mtx or mmf and anti-lymphocyte globulin. according to dynamic ipss (dipss) (n = 170) the patients were either low (n = 2), intermediate-1 (n = 36), intermediat-2 (n = 72), or high risk (n = 36). as the median follow up was shorter for patients treated with rux (15 vs 73 months, po0.001). primary graft failure was seen in 2 pts in the rux and three in the non-rux group. the median leukocyte engraftment was 13 days (range: 9-32) in the ruxolitinib and 14 days (range: 7-34) in the non rux group (p = 0.7). the incidence of acute gvhd grade i to iv was significantly lower in the rux group (49% vs 64%, p = 0.05), while agvhd grade ii-iv (33% vs 44%, p = 0.14) and grade iii/iv (23% vs 25%, p = 0.48), did not differ significantly. the ci of nrm at 1 year was 18% (95% ci: 6-30%) for the rux group and 22% (95% ci: 14-30%) for the non-rux group (p = 0.58), and the ci of relapse at 2 years was 8% (95% ci: 0-16%) vs 20% (95%ci: 12-28%, p = 0.25). the 2 years rfs and os was 66% (95%ci: 50-82%) and 69% (95%ci: 51-87) for the rux group and 59% (95% ci: 49-69%) and 70% (95% ci:62-78%) for the non-rux group (p = 0.29 and p = 0.45, respectively). within the rux group (n = 53), 24 pts responded to rux (more than 25% spleen size reduction), while 29 pts did not respond or lost response prior to stem cell transplantation. here, no significant difference could be seen between the responding and non-responding group for nrm (19% vs 17%, p = 0.69), relapse (4% vs 13%, p = 0.62), rfs (61% vs 72%, p = 0.81) and os (63% vs 75%, p = 0.89). in a multivariate analysis including rux treatment as variable there was a non-significant trend in favor for in the tyrosine kinase inhibitor (tki) era, allogeneic haemopoietic stem cell transplantation (allo-hsct) has become the later-line therapy but still remains the only known curative treatment for chronic myeloid leukemia (cml). since the introduction of tki in our centre in 2004, the trend of allo-hsct among our cml cohort has changed over time. the purpose of this study is to examine hsct outcomes of our cml cohort who was either tki naïve or has received tki therapy prior hsct. between may 1999 and december 2015, 98 cml patients in our center received allo-hsct with 39% were tki naïve. the time of diagnosis to transplant was significant shorter among the tki naive group as compared to those received tki prior hsct (17.29 ± 7.29 months versus 42.33 ± 31.92 months, respectively). there were no gender different (60% males) but the median age at hsct was younger among tki naïve group, 29.50 years (range: 14-44 years) versus 33.50 years (range: 16-59 years) respectively. malays remained majority ethnic group but the percentage was reduced among patients received tki prior hsct (60.5% versus 46.7% respectively). the disease phase at hsct was significant different whereby majority of tki naïve group was in first chronic phase (cp1) (60.5%) as compare to patients with prior tki exposure (35.0%). all the patients in the tki naïve group received hla-matched related siblings donor (mrd) with 81.6% marrow stem cell source whereas only 88.8% of patients who have prior tki exposure received mrd with 93.3% were from peripheral blood stem cell (pbsc). all patients in the tki naïve group but only 73.3% among patients who have prior tki exposure received full myeloablative conditioning regimen. there was slower neutrophil and platelet engraftment (19.97 ± 4.50 days versus 15.02 ± 3.55 days and 20.03 ± 6.72 days versus 13.93 ± 4.70 days respectively) among tki naive group. at 30 june 2016, the 1-year overall survival (os) of cml at all disease status was 50% in tki naive group versus 32% for patients who have prior tki exposure and transplanted in more advance disease stage. in general, patients in cp1 have the best os. there was higher incident of grade 2 to 4 acute graft-versus-host-disease (gvhd) among the tki naïve group (48.6% versus 16.7%, respectively) likely due to intensity of conditioning regimen with no significant different in chronic gvhd incident. similarly, there was higher relapse rate among tki naive patients (44.7% versus 16.7%, respectively) as upfront post transplant tki was not routinely given to this group of patients in the past. further multivariate analysis to ascertain predictors of transplant outcome among this cohort of patients included disease status, donor-recipient gender combination, ethnic difference will be presented. in conclusion, despite emergent of effective and potent next generation tki, hsct still has it role as curative modality for patients who failed tki. as showed in our data, the transplant outcome is excellent for patients who remain in cp1 at the time of hsct and it is important to identify patients earlier, before disease progression, especially young patients, in order to optimize transplantation outcomes. disclosure of conflict of interest: none. the purpose of this analysis was to provide 10-year follow-up of the gcllsg cll3x trial which aimed at evaluating reducedintensity conditioning (ric) hsct in patients with poor-risk cll. the cll3x trial included 100 patients (median age 53 (27-65) years), of whom 90 patients were allografted with blood stem cells from related (40%) or unrelated donors (60%) using fludarabine-alkylator-based ric regimens. 24% had refractory cll at hsct, and 35% had a tp53 deletion and/or mutation. the 6-year follow-up of the trial including the observation that genetic risk factors such as tp53 lesions and sf3b1 and notch1 mutations had no prognostic impact has been previously reported. survival and relapse information was requested for all patients who underwent hsct within the cll3x trial in 9 german centres (the canadian centre was unavailable for follow-up) and were alive at the 6-year followup. results: follow-up information was received for 37/44 patients (84%) alive at the 6-year follow-up. of these, 5 patients had died (3 cll, 1 chronic gvhd, 1 secondary cancer), and 3 had experienced disease recurrence. with a median follow-up of survivors of 9.7 (0.6-15.2) years, 10-year nrm, relapse incidence (rel), event-free survival (efs), and overall survival (os) of all 90 patients allografted was 25%, 55%, 31% and 51%, respectively, without significant effects of tp53 lesions on outcome. absence of minimal residual disease (mrd) at the 12-month landmark post hsct was highly predictive for a reduced relapse risk, in particular if mrd eradication occurred only after immunosuppression withdrawal, suggesting of effective graft-versus-leukemia activity (gvl; 10-year rel 12%). in the 32 patients who were alive and event-free 6 years post allohct, nrm, rel, efs, and os 4 years after this landmark (or 10 years after transplant) was 3.4%, 18%, 79%, and 94% with a median follow-up of 4.3 years (1.2-9.2) after the 6-year landmark. notably, no relapse event occurred beyond 10 years post hsct. of those who remained event-free beyond 10 years, all 8 patients who were available for mrd assessment at their most recent follow-up were mrdnegative. altogether 39 of the 90 allografted patients had cll recurrence after transplant; 34 between 2003 and 2010, and 5 from 2011 onwards. whilst the median survival of those patients who relapsed during the earlier period was 19 months, all 5 patients with late relapse are currently alive 4-62 (median 28) months after the event. conclusions: long-term observation of patients allografted in the cll3x trial confirms that ric hsct can provide gvl-mediated sustained disease control in a sizable proportion of patients with poor-risk cll independent of the tp53 status. patients who are in mrdnegative remission one year after hsct have an 87% probability of remaining disease-free at least for 10 years. however, late relapses do occur but may benefit from strategies involving innovative pathway inhibitors. hallek: consultancy and speakers bureau for pharmacyclics, llc and an abbvie company; speakers bureau for janssen; m. kneba: consultancy, honoraria, travel grants and research funding from gilead and roche; consultancy, honoraria and travel grants from abbvie and janssen-cilag; research funding from amgen; travel grants from glaxo-smithkline; p.dreger: consultancy for roche and janssen; consultancy and speakers bureau for novartis and gilead. no evidence for an increased gvhd risk associated with post-transplant idelalisib given for relapse of chronic lymphocytic leukemia or lymphoma: first results of a survey by the ebmt chronic malignancy and lymphoma working parties p dreger 1,2 , a boumendil, l koster 2 , c scheid 3 idelalisib is a kinase inhibitor (ki) approved for the treatment of cll and follicular lymphoma (fl). idelalisib has a specific adverse effect profile including immune-mediated inflammatory conditions such as colitis and pneumonitis, raising concern about the safety of this ki if administered for treatment of malignancy recurrence after allogeneic hematopoietic cell transplantation (allohct). the purpose of this ongoing study is to provide information on the safety and efficacy of idelalisib in this setting. we included in this study adult patients who had been registered with the ebmt for an allohct for cll or lymphoma and who received idelalisib for treating disease relapse or persistence at any time after transplant as indicated by participating investigators upon request by the ebmt study office in leiden. baseline patient, disease, and transplant data were collected from med-a forms. centers were requested to provide additional treatment and follow-up information. as of november 29, 2016, a total of 19 patients have been registered, of whom a full dataset as required for this analysis was available for 14 patients (cll 9, fl 2, diffuse large b-cell lymphoma (dlbcl) 1, peripheral t-cell lymphoma 1, unspecified 1) who had undergone allohct between july 2009 and april 2015. all patients except one were male. median age at transplantation was 52 (36-63) years and the median interval from diagnosis to allohct was 3.5 (0.8-12.2) years. prior to allohct, 3 patients (1 cll and 2 lymphoma) had received an autohct and two other patients had been exposed to ki (idelalsib 1, ibrutinib 1). disease status at allohct was sensitive in 71% of the patients. conditioning was reduced-intensity in 71% of the transplants and included in vivo t cell depletion in the majority of cases (71%). donors were identical siblings in 43% with pbsc being the stem cell source in all cases. the interval between hct and idelalisib commencement was 18 (2-68) months in the cll group but only 3 (1-57) months in the lymphoma group. prior to idelalisib, grade ii-iv acute gvhd and chronic gvhd had been observed in 7% and 36% of the patients, but was still active at the time of idelalisib commencement in only two cases (14%) . four patients with cll had already failed ibrutinib given for post-hct relapse prior to idelalisib. the median time on idelalisib until documented withdrawal or event (progression, retreatment, death) was 237 (9-569) days. after start of idelalisib, one patient developed grade 2 acute gvhd and subsequently chronic gvhd, however, in this patient idelalisib was started as early as 30 days after transplant. efficacy of idelalisib in this high-risk patient sample was limited with only one pr in the cll group (stable disease 4, progressive disease 1, not available 3; lymphoma not available), translating into a median event-free survival after start of idelalisib of 240 days. five patients with cll underwent a subsequent treatment with an alternate ki (ibrutinib 3, venetoclax 2). altogether, there were five deaths, all due to diease progression (cll 2, lymphoma 3). median overall survival was 360 days for the whole sample and not reached for cll. this preliminary data does not support concerns about the safety of idelalisib in the post allohct setting. updated results of this ongoing study will be presented at the meeting. tested patients (67%) achieved a ccyr and at least a mmolr, respectively. the response to transplant by day 30 assessment correlated significantly with the disease status before transplant. a higher percentage of patients who experienced cytogenetic response before transplant experienced molecular response post-transplant (77%) compared with those who did not (61%; p = 0.027). for the entire group, the 1-year cumulative incidence (ci) of acute gvhd grade ii-iv and grade iii-iv were 41% and 15%, respectively; 5-year ci of extensive chronic gvhd was 31%. there was no significant difference in the ci of severe acute or chronic gvhd between donor types. the ci of nrm at 100 days and 1 year was 14% and 30%, respectively. the ci of cytogenetic and molecular relapse at 5 years was 22% and 31%, respectively. overall the 5-year os, pfs and gvhd-free, relapse-free survival (grfs) were 49%, 34%, and 22%, respectively. in multivariable analysis for grfs, transplant in cp2 and the use of haploidentical donor significantly associated with better grfs. the 5-year grfs of patients in cp2, ap and bp before transplant was 24%, 16% and 14%, respectively (p = 0.013). ( figure 1a ) patients receiving a haploidentical donor had a better 5-year grfs when compared with hla matched transplants (53% vs 21%, p = 0.019). ( figure 1b ) for pfs, transplantation in cp2, using a haploidentical donor and mac regimen associated with better pfs while age, cytogenetic and molecular response before transplant did not predict survival. ahsct is curative for a proportion of patients with advanced cml. pfs and grfs are favorably influenced by percentage of bm blasts and donor type, with haploidentical donor having at least as good outcomes as hla matched donors, while molecular and cytogenetic response before transplant do not appear to correlate with survival posttransplant disclosure of conflict of interest: none. allogeneic stem cell transplantation (sct) has been considered as the treatment of choice for younger patients (pts) with high-risk chronic lymphocytic leukemia (cll). role of allogeneic sct in era of novel drugs is widely discussed. here we present our results after sequential use of chemotherapy and reduced-intensity conditioning (ric) in cohort of 25 high-risk cll pts. high-risk cll was defined by one of the following: disease refractory to purine analogs, short response or early relapse (within 24 months) after purine analog combination treatment, and/or progressive disease with unfavorable genetic abnormalities (del [17p]/tp53 mutation). we analyzed 25 pts with high-risk cll undergoing chemotherapy and ric sct in our centre from august 2007 to june 2016. the median of pretransplant lines were 3 (range: 2-4), novel drugs (idelalisib, ibrutinib) were used in 20% of pts (5/25). fludarabine (30 mg/m 2 ) and cytarabine (2 g/m 2 ) for 4 days (fc) were used for cytoreduction in all pts. after 3 days of rest, ric consisting of 4 gy tbi, anti-thymocyte globulin 10-20 mg/ kg/day for 3 days, and cyclophosphamide 40-60 mg/kg/day for 2 days followed. median age of pts was 53 years (range: five-year overall (os) and relapse-free survival (rfs) was 59% and 57%. ci for cgvhd in pts surviving more than 3 months post-hct was 35% after 5 years and 49% after 10 years. in a multivariate cox-regression model the occurrence of cgvhd independently improved os (p = 0.001, hr 0.27; 95% ci 0.12-0.59%) as well as rfs (po 0.001, hr 0.19; 95% ci 0.08-0.46). high risk dipss plus score demonstrated significant inferior survival compared to intermediate-2 (os p = 0.006; rfs p = 0.009), int-1 (os p = 0.037; rfs p = 0.042) and low risk (os p = 0.021; rfs p = 0.014) which could be confirmed in multivariate analysis for os (p = 0.001, hr 3.13; 95% ci 1.56-6.3) and rfs (p o0.001, hr 4.84; 95% ci 2.05-11.43). rfs additionally was improved by splenomegaly (n = 60) vs. normal spleen size (n = 11) at time of hct (p = 0.01, hr 0.29; 95% ci 0.1-0.75). ruxolitinib (n = 20) or none (n = 45) pre-treatment compared to other drug therapy (n = 19) resulted in improved os (p = 0.013) and rfs (p = 0.031) and was an independent factor for rfs in multivariate analysis (p = 0.046, hr 0.39; 95% ci 0.16-0.99). non-relapse mortality (nmr) was significantly determined by high-risk dipss plus score (p = 0.001) or dipss high and int-2 (p = 0.009). relapse incidence was significantly lower in pts with splenomegaly compared to asplenic or normalspleensized pts prior to hct (p = 0.027). our data point out that pre-therapy and dipss or dipss plus score are relevant pre-transplant outcome factors while chronic gvhd is the most important independent hct-related factor. furthermore, splenomegaly at hct reduces risk of relapse and therefore improves rfs. [p582] disclosure of conflict of interest: none. thalassaemia major affects 10 000 new babies in india each year and haematopoietic stem cell transplantation offers the only chance of cure. we present data on 179 children with thalassaemia major aged between 9 months and 17 years using a uniform conditioning regimen consisting of thiotepa 8 mg/kg, treosulphan 42 gm/m 2 and fludarabine 160 mg/m 2 . equine antithymocyte globulin at a dose of 45 mg/kg was added to children who were undergoing transplantation from an unrelated donor source. there were eight deaths before engraftment due to sepsis or bleeding and two related to graft versus host disease. all patients showed complete chimerism on day 28. however, in 21 children there was an acute drop in donor chimerism between day 60 and 120 post transplantation. immunosuppression was abruptly stopped when donor chimerism dropped below 95% in all children. seven children responded well and re-established complete chimerism with this measure. seven children progressed to develop complete graft loss. donor lymphocyte infusion (dli) in the form of small aliquots of peripheral whole blood from the donor was administered in seven children. dli was used in a graded fashion every 2 weeks starting from 1 × 10 4 /kg of cd3, followed by 1 × 10 5 /kg and 5 × 10 5 /kg. all of them continued to maintain their graft with these interventions. drop in chimerism was seen particularly in children less than 3 years at the time of transplantation comprising 14 out of 21 children. older children with lucarelli class iii were also prone to rejection in our earlier series and this complication has now been eliminated with pre-transplant immunosuppression and hypertransfusion. children above the age of 7 years were more prone to graft versus host disease and required on average 18 months of immunosuppression. treosulphan based protocol has been equally well tolerated by all age groups, all lucarelli classes of children with thalassaemia major and different donor sources. the transplant related mortality and graft rejection rates have been low at 5.5% and 3.9%, respectively. however, children less than 3 years need to be monitored carefully during the first 4 months of transplantation as early withdrawal of immunosuppression can prevent graft rejection resulting in excellent outcomes. disclosure of conflict of interest: none. 16 institute of cellular medicine, newcastle university, newcastle-upon-tyne, uk hemophagocytic lymphohistiocytosis (hlh; hemophagocytic syndrome) is a rare syndrome of potentially fatal, uncontrolled hyperinflammation. allogeneic stem cell transplantation (allosct) is indicated in familial, recurrent or progressive hlh. additional recommendations include central nervous system involvement and unknown triggering factor. while data for allosct outcome are available for the pediatric setting, information for adults is very limited. the aim of this study was to retrospectively analyze the information from the ebmt databases about adult hlh patients who underwent allogeneic stem cell transplantation. we obtained data of 67 adult (≥18 years of age) patients transplanted due to hlh. additionally, an hlh-oriented questionnaire was sent to the clinical centers, with 23 responses received so far. median age at transplantation was 28 (range: 18-65). there was a slight male predominance 43/67 (64%). the majority of patients were reported with secondary hlh 33/67 (49%), the familial disease was reported in 29/67 (43%) patients. in two patients triggering factor was attributed to malignancy. the majority of patients received stem cells obtained from the peripheral blood (52/67; 78%) while for the remaining ones it was bone marrow. reduced intensity conditioning was used since 2004 in 23/63 (37%) of patients. thirteen (19%) patients received tbi. donor choice was: 33 matched unrelated (50%), 7 mismatched unrelated (11%), 26 identical sibling (39%). engraftment was observed in 55/61 (77%). the cumulative incidence of acute graft versus host disease (gvhd) at 100 days was 26% (95% ci 15-37%). the cumulative incidence of chronic gvhd at 1 year after allosct was 13% (95% ci 2-23%) and increased to 31% at 3 years (95% ci 16-47%). the 3-year probability of overall survival is shown in fig.1 . the median survival time was 8 months. the 3-year os was 41% (95% ci 28-55%). for patients who survived until 3 months, this proportion was more favorable with an os of 62% (95% ci 45-78%) at 3 years after transplantation. among 19 patients with observation times longer than 15 months, only one patient died (in the 48th month after allosct due to relapse which occurred in the 12th month. after 12 months no more relapses of hlh were recorded-the cumulative incidence reached 19%. the non-relapse mortality reached 42% after 15 months. the familial disease was associated with a better prognosis than secondary hlh (p = 0.04). unlike the pediatric population, where reduced intensity conditioning (ric) was associated with higher survival, in adult patients there was no difference between the conditioning types. data form the 23 questionnaires confirm clinical picture typical for hlh at the diagnosis: fever in 21/22 (95%), splenomegaly in 19/20 (95%), hemophagocytosis in 18/20 (90%) and hyperferritinemia with median concentration of 4215 ng/ml (range: 63-260 160). image fig.1 overall survival after allogeneic stem cell transplantation for adult hlh patients until 36 months (95% confidence intervals are shown in grey). the number of patients at risk is indicated below the time axis at the corresponding time points. to our knowledge, this is the largest group of adult patients with hlh who underwent allogeneic stem cell transplantation. relatively low relapse incidence shows that allosct can effectively cure hlh. patients who survive the first period after this procedure can expect a long disease-free survival. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative option for children suffering from various life-threatening inherited non-malignant diseases with best results using hla-identical family donor. hsct from unrelated or mismatched family donors is associated with increased risk of agvhd and graft rejection.use of post-transplantation cyclophosphamide (ptcy) with or without additional immunosuppression has been shown to be effective prophylaxis against gvhd in patients with hematological malignancies. there are limited reports of hsct using pt cy for patients with non-malignant disorders. we retrospectively analyzed results of 18 hsct in patients with life-threatening non-malignant diseases using ptcy-based gvhd prophylaxis. patients characteristics are presented in table 1 . thirteen patients (72.2%) were transplanted upfront, 5 patients (27,8%) were rescued after primary or secondary graft failure after first hsct. donors were hla-matched (n = 8) or mismatched (8-9/10) (n = 4) unrelated, haploidentical (n = 5) or hla-identical family (n = 1). bone marrow was used as graft source in 13 (72.2%) patients and peripheral blood stem cell in 5 (27.8%). median cd34+/kg recipient weight-7.25 × 10 6 (3.0-13.5), cd3+/kg-9.785 × 10 7 (0.78-90.6). the conditioning regimen was myeloablative in 12 patients (conventional-3, reduced toxicity-9), reduced intensity-6. the gvhd prophylaxis consisted of a combination of ptcy at dose of 50 mg/kg on days +3 and +4 with calcineurin inhibitors (tacrolimus-6 pts, cyclosporine a-10 pts) or sirolimus (1 pt) and mmf (15 pts) starting on day +5. all but one patients received also serotherapy with rabbit (12 pts) or horse atg (5 pst) and rituximab (4 pts). with a median follow-up of 8 months (range: 1-33), the kaplan-meier estimates of os − 81.5%. one patient with thalassemia died before engraftment on day+11 from severe vod. 15/17 pts (88%) achieved engraftment. the median time for neutrophil and platelet engraftment was 23 (16-28) and 19 days (12-32), respectively. primary graft failure was observed in 2 patients (1 was successfully retransplanted from another haploidenticle donor, 1 was not eligible for a second transplantation, but alive). at last follow up, 10 (67%) patients had full donor chimerism, 4 (27%) had stable mixed chimerism without signs of disease progression. one patien with wiscott-aldrich syndrome had secondary graft failure with progressive loss of donor chimerism and were successfully rescued with second haploidentical transplant from the same donor. of 15 engrafted patients, agvhd ii-iv was seen in 4 (26.7%) patients. one patient developed grade ii (gut stage ii) agvhd, which resolved with systemic steroids. severe (griii-iv) agvhd was observed in 2 pts after second hsct, both had calcineurin and mtor-inhibitors induced toxicity leading to discontinuation of this drugs, but responded on combined (steroids and ruxolitinib) therapy. one patient with was developed grade iii gvhd (gut stage 4) after severe cmv-colitis and died on day from multiple organ failure (suspected tma). one patient developed extensive chronic gvhd of kidney (minimal change [p587] disease) after tapering of immunosupression. one patient with hurler syndrome had seizures, died on day+29 from multiple organ dysfunction syndrome. conclusion: ptcy is a promising option for agvhd prophylaxis in patient with non-malignant disease, lacking an hla-matched family donor. disclosure of conflict of interest: none. an exploratory, open-label study to evaluate the safety and feasibility of atir201, a t-lymphocyte enriched leukocyte preparation depleted ex vivo of host alloreactive t-cells (using photodynamic treatment), as adjuvant treatment to a t-cell depleted haploidentical hematopoietic stem cell transplantation in patients with beta-thalassemia major c selim 2 , w rob 3 , l sarah 4 , f josu de la 5 previous studies demonstrated that donor lymphocytes, selectively depleted of alloreactive t-cells (atir), could be given safely in adult patients receiving a haploidentical hsct. in 42 patients a single dose of atir, at doses up to 2 × 10 6 viable t-cells/kg, was given and no grade iii/iv acute gvhd has been reported. this confirms the efficacy of the elimination method of allo-reactive t-cells and attributes to its beneficial safety profile. in an ongoing phase 2 study, cr-air-007 (nct01794299), infusion of atir101 at 28 days post-hsct results in a reduction of transplant-related mortality (trm) and improvement of overall survival and event-free survival. adjunctive treatment with donor lymphocytes in patients receiving a t-cell depleted, haploidentical hsct for nonmalignant diseases such as beta thalassemia major, could provide early immunological support and better immune reconstitution in the absence of gvhd. in an open-label, multicenter phase 2 study (cr-bd-001; eudract 2016-002959-17), 10 patients age ≥ 2 years and ≤ 25 years with beta thalassemia major will undergo a haploidentical hsct with adjunctive administration of atir201. patients will receive a t-cell depleted graft (cd34-selected, or cd3/cd19 depleted, or tcr-αβ depleted, depending on the experience of the study center) from a related, haploidentical donor, patient conditioning will be myeloablative following standard practices at the study center. atir201 infusion at a dose of 2 × 10 6 viable t-cells/kg is given between 28 and 32 days after the hsct. to assess safety, patients will be evaluated for the occurrence of dose limiting toxicity (dlt), defined as acute gvhd grade iii/iv within 180 days post hsct. efficacy will be primarily evaluated by transfusion-free survival (tfs), occurrence of severe infections, and time to t-cell reconstitution, taking into account hematologic and sustained engraftment. all patients will be closely monitored for cmv, ebv and adenovirus titers, with initiation of pre-emptive treatment upon rising blood titers. regulatory authorities in the united kingdom and germany have approved this clinical study protocol. enrolment of the study is expected to continue during 2017, with first report of safety of atir201 to be expected first half 2018. disclosure of conflict of interest: j. rovers is employee of kiadis pharma, sponsor of the study. sickle cell disease (scd) can be cured with haematopoietic cell transplantation (hct), yet progress in the practice and research of hct for scd has not come without risks and uncertainty. the information and decisions that families and physicians encounter in this field are complex and hanging. in this hermeneutic study, we analyze the case of one family who advocated for hcts for two of their four children knowing the potential risks. these experiences have had a profound impact on both the family and the medical team. this study was conducted through the research method of hermeneutic phenomenology. hermeneutic inquiry is described as the practice and theory of interpretation and understanding in human contexts and aims to make sense of the particulars of these contexts and arrive at deeper understandings. data collection: in-depth interviews were conducted with the mother of the family, the hct nurse coordinator, and the hct physician. the interviews were audiotaped and transcribed verbatim. the transcribed interviews were later reviewed by the physician, who then wrote an additional reflection. this work culminated in approximately 70 pages of single spaced data in textual form. in hermeneutics, interpretation takes place through a careful reading and re-reading of the data, looking for statements and instances that resonate with the researcher. initial individual interpretations of researchers are then raised to another level of interpretive analysis in the research team's communal attention to the data. particular criteria guide the analysis: agreement, coherence, comprehensiveness, potential, and penetration. the following excerpts and interpretations are provided as examples of the analysis, with names changed for confidentiality. "being heard" arose repeatedly in this family's experience, including at the time of their request for a transplant without meeting the traditional criteria for hct. they persisted in their belief that their children would benefit from hct. "they gave us options to see if there was a chance for a transplant...how life would look…. and then we figured…a transplant for him was better at the time…worth the risks…. and you wouldn't even know. he plays basketball now, he plays sports, he's active and he can exercise and run. i never had any regrets because i felt it was better and the most important thing is his organs were really intact; none of the organs were destroyed…so i think it's the right decision we made" (mother). "this family has changed my career, and my life as a result. they challenged my practice and way of thinking. they did so in a considerate way, out of a duty to advocate for their children. we worked through the tension of different viewpoints with respect and all of us grew in the process. at least i can say our team did. i certainly did... i am humbled by their trust and respect…i am grateful to them" (physician). patients and providers are deeply impacted by their interactions. dr. robert buckman stated that it was the individual case that changed his practice always. he claimed he could not walk into a new patient's room without his practice being forever changed. in presenting this hermaneutic analysis, we aim to remind ourselves of the opportunity for growth that can result from reflection on this sacred patientprovider relationship. disclosure of conflict of interest: none. defibrotide (df) prophylaxis and adjustment of busulfan schedule to prevent veno-occlusive disease and thrombotic microangiopathy in an infant with a membrane cofactor protein (mcp) gene mutation and metachromatic leukodystrophy undergoing hematopoietic stem cell gene therapy (hsc-gt) v calbi 1,2 , f fumagalli 1,3,4 , r penati 3 , g consiglieri 3 , m migliavacca 3,1 , d redaelli 1 , s acquati 1 , v attanasio 1 , r chiesa 5 , f ferrua 1,3 , f barzaghi 1,3 , m cicalese 3,1 , a assanelli 3,6 , p silvani 7 , s tedeschi, r arora 8 , a soman 8 , f ciotti 3 , m sarzana 3 , g antonioli 3,1 , c baldoli 9 , s martino 10 , gl ardissino 11 , mg natali sora 4 , l naldini 1,12 , f ciceri 12,13 , a aiuti 1,12,3 and me bernardo hepatic veno-occlusive disease (vod) and thrombotic microangiopathy (tma) are life-threatening complications that can occur after hsc transplantation. expert consensus guidelines support use of df for treatment and prophylaxis of vod due to its ability to restore thrombo-fibrinolytic balance and protect endothelial cells. presymptomatic monozygous twins affected by late infantile metachromatic leukodystrophy (mld) underwent investigational hsc-gt after conditioning with busulfan. no comorbidities were evident at baseline. the dose of transduced cd34+ cells was similar in both patients (18.2 × 10 6 cd34+/kg for patient 1 and 14.1 × 10 6 cd34+/kg for patient 2). patient 1 (p1): at 8 months of age, received conditioning with iv busulfan 80 mg/m 2 /dose for 4 doses (target auc 85 mg × h/l). on day (d) +18 after gt, he developed severe vod and was treated with diuretics, fresh frozen plasma, paracentesis and df. on d+39 schistocytes in peripheral blood, marked proteinuria, complement factor consumption, and increases ldh and bilirubin were observed. the patient's condition worsened, with reduced urine output and generalized oedema with pleural effusion. stool, urine and blood cultures were negative and adamts13 activity was 35%; anti-complement factor h (cfh) antibodies (ab) were positive (1371 ui/ml). these findings led to the diagnosis of atypical hemolytic uremic syndrome (ahus; a form of tma) and eculizumab (300 mg/weekly dose) was started on d +40. patient subsequently developed pulmonary oedema and needed non-invasive ventilation. molecular analysis revealed a heterozygous deletion of cfhr3-r1 and ala353val mutation in the mcp gene, a defect previously shown to be associated with ahus. due to the presence of ab anti-cfh and antiplatelet, 4 weekly doses of rituximab (375 mg/m 2 ) were administered. after treatment, p1 progressively improved although he showed prolonged severe anaemia and thrombocytopenia and bone marrow (bm) hypoplasia, secondary bleeding which required reinfusion of unmanipulated autologous bm cells on d +66. nine months after hsct-gt p1 has shown good hematopoietic recovery, stable engraftment of the transduced hscs, no signs of renal damage or complement activation, albeit with neurodevelopmental delay. patient 2 (p2): given his twin history and genetics, this 9 month old infant was considered at increased risk of vod, so prophylaxis with df (25 mg/kg/d) was administered from d-4 to d+30 and the busulfan conditioning was modified by adjusting the auc to a lower target (1 mg/kg/dose for 14 doses; target auc 67.2 mg × h/l). the child had a good clinical recovery and didn't develop signs of vod or tma after hsc-gt. on d+12 and +14, respectively, anti-cfh and anti-platelet ab were positive. considering the history of the twin, 4 weekly doses of rituximab were administered. p2 is currently 8 months after gt with persistent engraftment of transduced hscs and no signs of tma. data from this case-control report of monozygous twins diagnosed with mld, and subsequently shown to also harbor mutations in complement regulator gene, suggest that df prophylaxis and busulfan adjustment may have helped prevent systemic microangiopathic damage in the second twin. patients with rare disease may have mutations in genes in addition to those that cause their disease. patients at risk of post-transplant tma following hsc-gt for genetic diseases may require tailored df prophylaxis and treatment. disclosure of conflict of interest: a. aiuti is the principal investigator of the tiget-mld clinical trial of gene therapy. the mld gene therapy was licensed to glaxosmithkline (gsk) in 2014 and gsk became the financial sponsor of the trial. all authors declare no other competing interests. hematopoietic stem cell transplantation (hsct) using an optimized conditioning regimen is essential for the longterm survival of patients with inherited bone marrow failure syndromes (ibmfs). we report hsct in 25 children with fanconi anemia (fa, n = 12), diamond-blackfan anemia (dba, n = 7), dyskeratosis congenita (dc, n = 5) and shwachman-diamond syndrome (sds, n = 1) from a single hsct center. the graft source was peripheral blood stem cells (n = 20) or cord blood stem cells (n = 5). fa, dc and sds patients received reduced-intensity conditioning, while dba patients had myeloablative conditioning. the median numbers of infused mononuclear cells and cd34+ cells were 14.4 × 10 8 /kg and 4.5 × 10 6 /kg, respectively. the median time for neutrophil and platelet recovery was 12 and 17 days, respectively. there was one primary graft failure. after median follow up 3 years the overall survival was 96%. the incidence of grade ii-iii acute and chronic graft versus host disease (gvhd) was 32% and 16% respectively. in a multivariate analysis, the type of conditioning regimen was the only factor identified as significantly associated with grade ii-iii acute gvhd (p = 0.01). we conclude that hsct can be a curative option for patients with ibmfs. disease specific conditioning regimen was important to disease the transplant-related mortality. [p591] disclosure of conflict of interest: none. homozygous sickle cell disease (scd) patients suffering from end-stage renal disease (esrd) show a variable outcome after kidney transplantation as underlying disease can cause poor allograft survival and disease-specific problems. we present a case of a 27-year old patient with severe scd and esrd who underwent haploidentical bone marrow transplantation (bmt) with consecutive living kidney transplantion (lkt). the patient suffered from multiple complications of scd including stroke with secondary hemorrhage, symptomatic epilepsy, esrd and uncontrolled hypertension. the patient had been on hydroxyurea without success and required regular blood transfusion due to severe renal anemia. the rationale for bmt was uncontrollable iron overload. a reduced intensity conditioning regimen was used with (fludarabine, cyclophosphamide and 2gy of tbi, dose-adjusted to esrd). graft-versus-host disease (gvhd) prophylaxis consisted of post-transplant high-dose cyclophosphamide, cyclosporine a (cya) and mycophenolate mofetil (mmf). the donor was her 56-year old mother with hbs trait, the stem cell source was bone marrow, the cell dose 4.74 × 10 8 nucleated cells/kg. during conditioning daily hemodialysis was performed to keep drug levels stable. neutrophil engraftment occurred on day +26, chimerism at day +19 was 98%. hbs increased from 1.3% pre-hsct to 40.0% 6 months after hsct. hemoglobin values increased from 70 g/l pre-hsct to 110 g/l post-hsct and reticulocytes from 16 g/l to 124 g/l. erythropoietin levels increased from 2.3 iu/l pre-hsct to 178 iu/l 6 months after hsct. during the follow-up, the patient did not show any sign of acute gvhd or vaso-occlusive crisis, hemolysis or sickling. relevant complications were disease-related (therapy resistant hypertension and epileptic seizure due to former brain damage). on day +151 a lkt from the same donor was performed. the initial immunosuppressive treatment with mmf was continued, cya was switched to tacrolimus and steroids were added for 3 months. the post-transplant period was uneventful. currently, 12 months after haploidentical bmt and 6 months after lkt there are no signs of gvhd, the blood chimerism is 100%, the kidney allograft function is very good (gfr 73 ml/min/1.73 m 2 ) and immunosuppression is withdrawn. iron overload is being corrected by regular phlebotomies. the patient no longer requires antihypertensive medication and there is evidence of vascular remodeling. this is the first report of a successful haploidentical bmt followed by kidney transplantation from the same donor in a patient with scd. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) can cure non-malignant diseases, such as primary immune deficiency (pid), severe aplastic anemia (saa) and osteopetrosis (op). in the absence of a well-matched donor, transplantation from a haplo-identical donor maybe considered. post-transplant cyclophosphamide (ptcy) is a new strategy derived from the treatment of malignant diseases in adults that has been little studied in high-risk pediatric nonmalignant diseases. fifteen children (2.2 years, range: 0.19-10.88) underwent hsct in the pediatric immunology and hematology unit of necker hospital, paris, between december 2014 and september 2016. these children were suffering from op (n = 3), saa (n = 2), hemophagocytic lymphohistiocytosis (hlh) (n = 3), immunodysregulation polyendocrinopathy enteropathy x-linked (ipex) syndrome (n = 2), combined immune deficiency (n = 4) and leukocyte adhesion molecule deficiency (n = 1). three patients received a low-intensity conditioning regimen (cr) (based on fludarabine, cyclophosphamide, and total body irradiation) whereas the other 12 received myeloablative cr (based on busulfan auc targeted and fludarabine). fourteen patients received serotherapy before hsct. post-transplant cyclophosphamide (50 mg/kg/ day) was given on d3 and d4 and graft versus host disease (gvhd) prophylaxis with cyclosporine and mycophenolate mofetil was initiated on d5. the transplanted stem cells were obtained from bone marrow in all cases. engraftment with full donor chimerism was observed in 13 patients. the median cd34+ cell dose was 14.8 × 10 6 cells/kg body weight (range: 7.4-35.9 × 10 6 ). neutrophils recovered after a median of 18 days (range: 14-32), and overall survival (os) was 80% after a median follow-up of 1 year (range: 0.18-1.98). three patients died due to graft failure (n = 2) or infectious complications related to gvhd (n = 1). grade ii acute gvhd occurred in 8 of the 13 patients displaying engraftment (62%), and chronic gvhd and/or autoimmune complications were observed in four patients (31%). viral complications were frequent, occurring in 10 patients (77%) with cmv infection (n = 8) /disease (n = 1), adenovirus disease (n = 1) and bk virus cystitis (n = 4). haploidentical transplant with ptcy is feasible in high-risk patients with non-malignant diseases. chronic gvhd and autoimmunity were more frequent than for more conventional approaches in such patients. infection rates were high. disclosure of conflict of interest: none. sickle cell disease (scd) remains associated with high risks of morbidity and early death. even best of supportive care fails to improve quality of life. hematopoietic stem cell transplant (hsct) can be considered for selected group of patients. in long run it is not just economical but also substantially improves quality of life (qol). we report our experience with hsct for scd from india. seventy three consecutive patients suffering from scd who underwent hsct between january 2006 and november 2016 were included in the study. fifty two underwent matched sibling donor (msd), 2 matched family donor (mfd), 3 matched unrelated (91/0 or 10/10), 2 cord blood transplant cbt (1 matched sibling cord blood and 1 matched unrelated) and 15 patient underwent haploidentical transplant. different conditioning regimens were used and so was the graft versus host disease prophylaxis depending on institutional protocols as depicted in table 1. a total of 73 patients underwent sct. the median age was 9 years (10 months-29 years). m/f ratio was 45/28. majority of patients were either from african union or oman. all patients suffered from one or other severe symptoms making them eligible for sct. graft source was bone marrow (bm) in 30 with median cd34 count of 5.3x106/kg (0.92-10.7), peripheral blood (pb) in 36 with median cd34 count of 8.5x106/kg (3.9-20.18), cord blood in 2 with median cd34 count of 1.27x105/kg (0.44-2.1) and combined bm & pb in 5 with median cd34 count of 6.37x106/kg (1.5-23.3). of the 73 patients, 61 are alive and disease free with lansky/karnofsky scores of 100. there were 8 deaths (4 msd/mfd/mud; 3 haploidentical and 1 matched unrelated cbt). four patients rejected the graft (2 haploidentical and 2 msd/mfd/mud). at the last follow up, the probabilities of survival, scd-free survival, and transplantrelated mortality were 89%, 83.5%, and 11%, respectively. outcome of hsct in scd has improved significantly. with better conditioning regimens, improved supportive care, the outcome of alternative donor transplant and adult scd has improved and matches sibling donor transplant. hsct should be strongly considered as a curative modality for selected patients suffering from scd. disclosure of conflict of interest: none. s434 staff jointly defined more than 50 local standard operating procedures. patients with low-risk characteristics (age ≤ 7 years, liver size ≤ 2 cm below costal margin) and a hla matched sibling donor were considered eligible in this initial phase of activity. a downstaging protocol with hydroxyurea and deferoxamine or deferasirox was adopted. conditioning regimen included iv busulfan and cyclophosphamide. gvhd and rejection prophylaxis included atg from day − 12 to − 10 and csa, mtx and methylprednisolone. gcs-f primed bone marrow was chosen as stem cell source. the first allogeneic hsct of the whole iraq was performed in a child with thalassemia at hiwa hospital in october 2016. up to now, 3 patients (2 females, 1 male) underwent hsct; median age at transplantation was 2 years; median infused tnc 18.5 × 10 8 /kg, cd34+ 10.1 × 10 6 /kg. all of them engrafted. no major complication were observed. one of them developed grade ii agvhd (skin only) which resolved after increasing the dose of steroids. a huge number of patients with low-risk thalassemia are now in the waiting list and some of them have already started downstaging having planned hsct in a short time. a matched sibling transplant program in children with thalassemia is feasible and safe in kurdistan. such a program can provide many advantages: far less psychosocial and financial burden for the families and significant saving for the government. the estimated costs of performing locally hsct are much less than in the countries where patients were previously referred. the continuation of cooperation is of paramount relevance for further implementing the activity and extending the transplant accessibility to patients with other hemato-oncological disorders of childhood. disclosure of conflict of interest: none. long term follow-up after reduced-intensity conditioning and stem cell transplantation for thalassemia major r rihani, a natsheh, sm abu, e khattab, r najjar, f sheab, s sharma, n hussein, a tbakhi and m sarhan bone marrow and stem transplantation program-king hussein cancer center, amman, jordan hematopoietic stem cell transplantation (hsct) is the only curative treatment for thalassemia major (tm). reducedintensity conditioning (ric) before hsct for high risk tm patients results in fewer complications, when compared with myeloablative regimens. one hundred and three tm patients received hscts from an hla-identical related donor at king hussein cancer center, between january 2002 and november 2016. of those, 62 were high risk tm (60%) who received ric hscts. in this report, we describe follow-up beyond 2 years (median, 42; 24.6-126 months) post ric hscts. forty-four class ii-iii patients (58%) were identified (25% with hepatitis c); with a median age of 14 (13.4-28) years. females accounted for 59% (n = 26). conditioning regimen consisted of oral busulfan 8 mg/kg, fludarabine 175mg/m 2 ,tli 500cgy and atg followed by pbsct. gvhd prophylaxis consisted of mmf and csa. median infused stem cell dose was 5.4 × 10 6 /kg. all patients attained neutrophil and platelet engraftment (median, 15.3 and 21.3 days, respectively). persistent mixed donor or full donor chimerism were observed in 95.5% (n = 42) and 4.5% (n = 2), respectively. immune-suppressive therapy for gvhd treatment was required in 16 (36.4%) patients (agvhd, n = 8; cgvhd, n = 8). moreover, veno-occlusive disease occurred in 4 patients (9%) that resolved completely. secondary graft failure was noted in 11 (25%) patients. the 5-year overall survival was 100%, while the 5-year probability of thalassemia-free survival was 72.2%. other factors evaluated include: growth parameters, endocrine and other organ functions, in addition to functional status. this report confirms the safety and efficacy of ric regimens in hscts for high risk tm patients. those regimens are associated with excellent engraftment and sustained mixed donor chimerism; and lead to excellent thalassemia-free and overall survival rates. [p596] disclosure of conflict of interest: none. in-time hsct for pts. with hurler syndrome (hs) can significantly improve the results. long-term follow-up and late effects estimation required to prepare a special observation and rehabilitation programs. aim. to analyze our experience with hsct for hs in the field of special observation and rehabilitation programs. forty hsct during the 2004-2016 were performed for 38 pts. with hs. median age at the diagnosis was 15 months (3-38 months), at hsct-24 months (9-48 months). bm used in 62.5% (n = 25), pbsc-32.5% (n = 13), cb-5.0% (n = 2). mac conditioning was used for 29 hsct, ric-for 11. ric regimen: flu+mel+atg, mac: bu/treo +flu+thio/mel (bu was used in early 2000) and atg +rituximab (in case of mud hsct). all pts. with ric received mud hsct, pts. with mac mud-18 pts., mrd-5 pts. pts. received csa/tacro-based gvhd prophylaxis. mmf/mtx was additionally added in all cases. in 3 ric hsct immunomagnetic сd3/сd19+ depletion of pbsc (by clinimacs) was used. a special observation protocol including somatic and neurocognitive estimation was developed. all pts. engrafted with full donor chimerism on d+30. median of engraftment day-20 (11-29 days). thirty three pts. survived. reasons of death-mac: infections-3 pts., ric: trali-1 pt., agvhd-1 pt. trm improved, over the years, with improving of supportive care and donor selection as well as pre-transplant screening. no early severe toxicity revealed. pulmonary infection episodes was registered in 30% of pts. in our study. gvhd: grade iideveloped 14 pts., grade iii-iv-2 pts. (after ric), local cgvhd-3 pts. (ric). no extensive cgvhd. 5 pts. rejected (mac and ric rejection rate was same). at median follow up of 60 months (8-160 months), the estimated 5 years pos was 81%. best response correlated with early hsct (and better status before hsct) and higher level of aidu after. late effects estimation showed that 47.4% (n = 18) of patients experienced late effects: cardio-vascular-16 pts., skeletal-15 pts., endocrine-3 pts. all pts. with cardio-vascular effects received mac. skeletal effects affected patients of older age, pts. transplanted in younger age do not have such effects. median period of late effects arising after hsct was 15 month (3-27 months). only 3 pts. experienced serious pulmonary late effects (infections), all episodes was before 2010. no pts. in our study have progressive retinal degeneration. 80% of pts. improved in the neurosensory component and all pts. improved in neurocognitive status and development after hsct. best response correlated with neurocognitive rehabilitation based on unique computer model used by our group in russian national rehabilitation center "russkoe pole." in-time hsct is an effective and safe way to stop neurodegenerative process for pts. with hs. both mac and ric regimens can be used with the same effectiveness. mac regimens associated with bigger number of cardiovascular late effects. long-time follow-up showed that these patients require the special observational protocol including estimation of cardio-vascular, skeletal, endocrine and neurocognitive risks. better neurocognitive response correlated with intensive rehabilitation using computer model. russian joint study showed effective cooperation for treatment pts. with hs in the national setting. disclosure of conflict of interest: none. little is known about pathogenesis of solid tumors after hsct but, intensive cytotoxic conditioning therapy with defective dna repair of persisting stem cells/stromal cells, viral infection, and immunosuppression may play a role. 3/6 patients with solid tumors had a melphalan-based conditioning. melphalan was linked to sarcoma and lung cancer in animal model. there are few data linking parotid mec to infection by cmv and hhv6 which can remain dormant in the salivary glands. both affected patients had hhv6 during the transplant period. p8 and p11 had a family history of solid tumor pointing to a possible genetic factor. whilst secondary malignancy post-hsct for patients with malignant disorders is well recognised, non-ptld malignancy post-hsct for pid has not previously been reported. a larger study is needed to evaluate incidence and risks. allogeneic hsct is a treatment of choice for the bone marrow failure in patients with sds. hsct from unrelated or mismatched family donors is associated with higher morbidity and mortality compared with matched sibling. combined pgd and hla antigen testing is a possible option to preselect a compatible donor for an affected sibling requiring hsct. we describe a case report demonstrating first successful hsct for 6 years girl with sds by using preimplantation genetic diagnosis and hla matching. diagnosis of sds was suspected at 5 m.o., based on clinical features, family history, laboratory studies. at 6 m.o., bone marrow (bm) aspiration revealed hypocellular marrow with signs of dysplasia and expansion of blasts (15.6% blasts). the sanger sequencing of sbds gene showed c.183-184ta4ct and c.258+2t4c mutations. the patient had recurrent infections, including bilateral pneumonia caused by phaeohyphomyces, bloodstream infection, cmvdisease. due to the lack of matched related or unrelated donors, hsct with ric (flu, mel, atg) from haploidentical father was performed at 16 months of age. after the 1st allo-hsct, engraftment was achieved on d+13, initial str study showed full donor chimerism. post-transplant period was complicated with severe cmv-infection and signs of secondary hlh. at d+135, graft rejection was registered. the girl became dependent on regular rbc and platelet transfusions, bm examination revealed hypocellularity with moderate signs of myelodysplasia without elevated blast count. due to lack of available hla-compatible donors, an option of in vitro fertilization (ivf) with preimplantation selection of a normal hla-matched embryo was considered. after controlled ovarian hyperstimulation 2 embryos were hla-compatible and healthy (first, wild-type; second, heterozygous for sbds gene mutation c.183-184ta4ct). hence, the only unaffected hla-identical embryo was transferred resulting into full-term pregnancy. at the age of 5.5 years after 1st hsct, the 2nd s437 transplant was performed with a combination of cb and bm as a source of hematopoietic stem cells. the donors' age was 2 years a reduced toxicity conditioning regimen (rtc) based on flu150 mg/m 2 , treo42 g/m 2 , thiotepa 10 mg/kg with serotherapy (thymoglobuline 7.5 mg/kg) was used. because of neurotoxicity, arterial hypertension, since d+1 csa was changed to sirolimus +mmf for gvhd prophylaxis. the total number of infused nc was 2.5 × 10 8 /kg; cd34+, 3.85 × 10 6 /kg; cd3+, 2.4 × 10 7 /kg. engraftment was achieved on d+28. any signs of gvhd, severe infectious or toxic complications were not observed. eight months later, the patient is alive, has full donor chimerism in bm and is not transfusion-dependent. in the absence of hla-identical donor, ivf with preliminary pgd and hla-typing could be a chance for matched donor to cure patients with non-malignant genetic diseases. in case of low cord blood cellularity, a combination of cb and bm from the same sibling could be used. our experience showed a successful engraftment of sds patient and stable donor chimerism after second hsct of cb and bm from pgd-selected sibling with rtc. disclosure of conflict of interest: none. the safety and efficacy of familial haploidentical (fhi) stem cell transplantation utilizing cd34 enrichment and cd3 addback in patients with high risk sickle cell disease (scd) ( figure 1a ). probability of 1yr efs is 87.4% (ci95: 58-97%) ( figure 1b ). immune cell reconstitution has been robust and similar to rtc and msd allosct in scd (table 1 ). there have been 3 deaths, vod, steroid refractory agvhd and cgvhd. mac followed by fhi utilizing cd34 enrichment and t-cell addback in patients with high-risk scd is safe, tolerable and results in long-term donor chimerism and absence of scd symptoms or complications. a larger cohort and follow-up will be required to confirm these preliminary findings. disclosure of conflict of interest: none. supported by r01fd004090-01a1. [p601] s438 lymphoma p602 a clinical prognostic index for assessing patients aged 460 being considered for high-dose therapy and autologous stem-cell transplant in relapsed or refractory high-grade non-hodgkin lymphoma d edwards 1 , k kirkland 1 , r pearce, s robinson 1 and g cook 1 bsbmt patients with relapsed high-grade nhl or disease refractory to first-line therapy can still be cured with high-dose therapy and autologous stem cell transplant if they respond to salvage chemotherapy. this aggressive algorithm is accepted in younger patients but is less well established in the elderly. age 460 has a negative predictive score in the international prognostic index (ipi) and there are concerns that the outcomes of hdt in these patients are significantly worse. deciding which older patients will benefit from hdt is challenging and there are no established predictive tools to guide physicians. we present a clinical prognostic index derived from information readily available at the time a patient is being assessed for asct the bsbmt audited the outcomes for uk patients aged 460 transplanted between 2004-2009 (n = 371) and benchmarked against the european bone marrow transplant (ebmt) database for the same period (n = 2695). the primary outcome was progression-free survival (pfs) but data was also analysed for overall survival (os), relapse rate (rr) and non-relapse mortality (nrm). we included all patients with a diagnosis of high grade nhl and the following demographic features were also analysed: age at diagnosis; age at transplant; m/f; year of transplant; cr/not cr at transplant; no. of prior therapies; no. of cells infused; clinical staging; karnofsky status at transplant; histology; ipi at diagnosis; mobilising regime and conditioning regime. candidate prognostic indices were factors achieving significance in univariate and multivariate analyses of the main outcomes by regression analysis. the best prognostic index was selected based on the bsbmt dataset and then applied to the rest of the ebmt dataset (the validation dataset). there were no significant differences in patient characteristics between the uk and non-uk groups nor in outcomes of pfs, os, rr or nrm. (figure 1 ). in both univariate and multivariate analysis the following features were associated with a significantly worse outcome for pfs, os, rr and nrm : age466, karnofsky score. disclosure of conflict of interest: none. underwent an allo-sct at our center after a treosulfan-based conditioning regimen. eleven pts received a mrd, 19 pts a mud, and 11 pts a haplo unmanipulated pbsc allo-sct. at allo-sct 17 pts were in cr, 10 pts were in pr, and 14 pts had sd/pd. hct-ci was evaluable for 34 pts, 18 had a score ≥ 3. the backbone conditioning regimen consisted of treosulfan 14g/m 2 from day − 6 to − 4, and fludarabine 30 mg/m 2 from day − 6 to − 2; twenty-five pts were treated with this reduced toxicity conditioning (rtc) regimen. intensification with other alkylating agent (melphalan, thiotepa, or cyclophosphamide) or radiotherapy (4gy total dose) was applied on the remaining 16 pts (myeloablative conditioning, mac). gvhd prophylaxis was based on cyclosporine a and methotrexate (17 pts) or rapamycin and mycophenolate mofetil (24 pts), plus anti-thymocyte globulin or post-transplant cyclophosphamide accordingly to donor type. median numbers of infused cd34 +/kg and cd3+/kg were 6.66 × 10 6 (range: 2.72-12.24) and 2.34 × 10 8 (range: 0.03-6.89), respectively. median follow-up was 61 months (range: 18-139). thirty-nine pts were evaluable for engraftment; median time to neutrophil ≥ 0.5 × 10 9 /l was 16 days (range: 10-30), and 16 days (range: 10-59) to platelet ≥ 20 × 10 9 /l. treosulfan conditioning provided a cr in 3 and 6 pts respectively in pr and sd/pd at transplant. no graft failure was observed. one and 5 years overall survival (os) was 58.5% and 52.6%, respectively. progression free survival (pfs) and gvhd-free/relapse-free survival (grfs) were respectively 51.2% and 41.5% at 1 year, 44.3% and 23% at 5 years. one and 5 years relapse/progression incidence (ri) was 29.3% and 36.1%, respectively. transplant related mortality (trm) was 14.6% at 100 days, 19.5% at 1 year and for the entire follow-up. the 100-day cumulative incidence (ci) of agvhd grade ≥ 2 was 4.9%; ci of moderate to severe cgvhd was 24.8% at 2 years. the outcome of pts in cr at 5 years was significantly better compared to that of pts with active disease in terms of both os (82.4% vs 33.3%, p o0.005), pfs (68.6% vs 25%, p o0.005), grfs (36.3% vs 12.5%, p o0.005), and ri (19.6% vs 50%, p o0.05). no statistical differences in os, pfs, and ri were found when pts were stratified according to donor type and [p602] the use of rtc or mac regimen. at last follow-up, 22 patients are alive and disease free; 3 of them obtained a durable cr using chemotherapy and/or dli for disease progression after allo-sct. treosulfan-based conditioning regimen is effective and well-tolerated in patients with advanced b-nhl undetgoing allo-sct. disclosure of conflict of interest: none. systemic anaplastic large cell lymphoma (salcl) is a very infrequent well-defined histological entity that comprises around 11% of all t-cell non-hodgkin lymphoma. in the absence of prospective clinical trials, autologous stem cell transplantation (autosct) is considered the standard of care as consolidation therapy after first line therapy for those patients not expressing the alk protein (alk neg salcl) and for patients with relapsed disease. the objective of this retrospective analysis was to analyse the long-term outcome of patients diagnosed with salcl and being treated with autosct during the course of the disease, making special emphasis on the potential impact of the administration of brentuximab vedotin (bv). eligible for this study were patients 18 years or above with salcl who underwent autosct between 2010 to 2014 and were reported to the ebmt. baseline patient, disease, and transplant data were collected from ebmt med-a standard forms. centers with potentially eligible patients were contacted to provide additional treatment and follow-up information including a written histopathology report for central review. seventy-nine patients (48 males) with a median age at diagnosis of 43 years (range: 14-70) and at transplantation of 45 years were included in the final analysis. thirty-nine patients were alk negative, 38 alk positive and in 2 patients expression of alk protein was unknown. at diagnosis, 60 patients (76%) presented with advanced stage and 48 (61%), with b symptoms. sixty-three patients (80%) received 1-2 lines of therapy before autosct. ten patients were treated with bv at some point before autosct; two patients as second line therapy, three as third line, one as fourth line and four as fifth line therapy. the median number of bv doses was 5 (range: [3] [4] [5] [6] [7] [8] . the median time between diagnosis and transplantation was 12 months (range: . most patients had chemosensitive disease at autosct [65 patients (82%)] and in all but 2 patients peripheral blood was used as the source of stem cells. conditioning regimen consisted on beam / beam-like protocols in 72 patients (91%). all patients engrafted. with a median follow up for surviving patients of 34 months (range: 2-71), 57 patients are alive (72%), 20 patients died (25%) and 2 patients (3%) are lost for follow up. disease relapse after transplantation was the most frequent cause of death after the procedure. cumulative incidence of non-relapse mortality for the whole series was 3% (95% ci, 0.5-9) at 100 days, 1 year and 3 years. cumulative incidence of relapse was 27% (95% ci 17-27) and 32% (95%ci 21-44) at 1 and 3 years, respectively. 1 and 3years progression free survival (pfs) was 70% (95% ci 60-82) and 65% (95% ci 54-78), respectively and 1 and 3-years overall survival (os) was 82% (95% ci 73-91) and 71% (95% ci 61-83), respectively. there were no significant differences in any of the outcomes between bv treated and non-treated patients. autosct results in a promising pfs and os in patients with salcl. the potential impact of the administration of bv as salvage strategy before the procedure needs to be further elucidated. disclosure of conflict of interest: none. coeliac disease (cd) is a t-cell immune-mediated enteropathy to dietary gluten, characterized by small bowel villous atrophy resulting in malabsortion. the enteropathy is reversible with a gluten-free diet (gfd), however symptoms and signs which persist 41 year are defined as refractory coeliac disease (rcd). rcd is divided into type i and ii, depending on absence/ presence respectively of clonal intra-epithelial t-lymphocytes (iels) with an aberrant phenotype (cytcd3 pos, membranous cd3, cd4 and cd8 neg). rcdii patients have a 5 year survival of 1.0, plts 420) was successful at a median of 11.5 (range: 10-15) days and no transplant-related mortality occurred. all patients achieved a clinical complete remission, with normalization of nutritional indices at 100 days, but persistently abnormal iels and clonal t-cells on duodenal biopsy. with a median follow-up of 42.5 (range: 22-56) months, 5 patients remain in clinical remission, 1 patient relapsed with rcd and no patient progressed to eatl. chemotherapy and asct is a safe and effective strategy for the treatment of rcd offering the possibility of sustained clinical responses. clonal tcr in duodenal biopsy/blood and iel flow cytometry form part of the patient evaluation prior to the chemotherapy/asct program. most patients with hodgkin lymphoma (hl) are cured with conventional chemotherapy. however, approximately 20% of patients relapse after primary treatment. for those, high-dose chemotherapy (hdc) followed by autologous stem cell transplantation (asct) is the standard of care. fifty seven adult patients with relapsed or refractory hl submitted to asct between 2000 and 2015 were reviewed. variables examined were sex, age, ann arbor stage (i-ii vs iii-iv), b symptoms, bulky disease, extranodal involvement, nodal areas involved (≥3vs12vs ≤ 12 months) and response to the treatment prior to asct. log-rank test was used to compare differences in survival for each factor. patients median age was 31 (17-64) years at diagnosis. ann arbor stage iii-iv in 35 (61%) patients, b symptoms in 25 (44%), extranodal involvement in 20 (35%) and bulky disease in 13 (23%). all patients were treated according to the abvd protocol in first line. indications for asct were relapsed disease (n = 30, 52.6%) and lack of complete response (cr) or progressive disease with 1st line treatment (n = 26, 45.6%). there were a median of 2 (2-5) treatment-lines before asct (protocols eshap, ice, beacoop, gvd and others). the disease was chemosensitive in 86% cases: cr in 24 and partial response (pr) in 25 patients prior to asct. refractory disease (rd) in 14% (n = 9). in 84.2% patients, the hematopoietic cells mobilization was performed under stimulation with granulocyte-colony stimulating factor in hematologic recovery after the cycle of 2nd line chemotherapy, and most of which required 1 (1-4) apheresis. conditioning regimens were beam (93%) and gmb (7%). the median time to hematologic recovery was 11 days (8-14) for neutrophils4500/ul and 13 days (9-25) for platelets420,000/ ul. three months after asct, thirty-nine (68.4%) patients had cr, one (1.8%) patient maintained pr and 6 (10.5%) patients had disease progression. status unknown in 7 patients and four (7%) patients died. relapse rate 32% (n = 15/47). with a median follow-up time after asct of 52 (0-169) months, median disease-free survival (dfs) was 26 (0-169) months and overall survival (os) was 52 (0-169) months. there were 19 deaths (33.3%), four (7%) related to early infectious complications of asct, two (2.6%) due to late infectious complications, eleven (21.1%) due to disease progression and 1 (1.8%) in context of secondary acute myeloid leukemia. response to the treatment prior to asct was the only factor with survival influence. the dfs and os differed significantly in chemosensitive disease compared with rd (dfs mean: 51 vs 21 months,p = 0.025, os mean: 64 vs 22 months, p = 0.007). the response to salvage treatment prior to asct is the main prognostic factor for survival after asct. prognosis remains poor in patients with rd or early and disseminated relapses. for these patients, the therapeutic approach should include intensive treatment with tandem hdc and stem cell transplantation, allogeneic transplant or early consolidation with brentuximab-vedotin after asct. hodgkin's lymphoma (hl), although considered a curable neoplasm in adults, could be associated with a very poor prognosis when refractory to primary induction therapy or when it relapses within 12 months from an autologous stem cells transplant (auto-sct). the optimal treatment of patients with heavily pretreated/refractory hl is controversial. brentuximab vedotin (bv) is an active single agent in this context; unfortunately, there are no well established therapies when patients fail to respond or progress after bv. encouraging results were recently described with checkpoint inhibitors. similarly, data pertaining to efficacy of bendamustine (benda) shows encouraging activity in various refractory lymphomas. we included in this study adult patients with hl who relapsed post auto-sct and were refractory to or progressed after salvage bv and were treated with benda as salvage therapy with an intention to proceed with an allo-sct. this study was [p606] conducted in two major centers in lebanon, the american university of beirut medical center (aubmc) and makassed university hospital. we identified 12 eligible cases. the primary study endpoint was objective response rate (orr). the secondary endpoint evaluated successful rate of bridging into an allo-sct. the median follow-up times from auto-sct and from benda salvage were 35 (14-59) and 10 (4-35) months, respectively. the median age of patients was 27 years (17-54). all patients had bv as salvage therapy post auto-sct, and all of them progressed after a median of 4 (3-6) cycles. clinical characteristics are outlined in table 1 . patients received a median of 6 cycles (2-8) of benda. the treatment was well tolerated, with rather infrequent adverse events and transient and manageable toxicities. the orr was 75%, in 9 of 12 patients, with 43% obtaining a complete response. eventually, 6 of 9 proceeded to allo-sct using a matched related donor, and the remaining 3 patients are planned for allo-sct. only one patient died from disease progression after 24 months post allo-sct. two of 3 patients who progressed following benda received salvage therapy with nivolumab and are being planned for haplo-identical transplant while the third one is being planned for therapy with nivolumab. from the initiation of benda, the median duration of response for the 9 patients was 10 months (4-29); all these patients had maintained a continuous response at the last follow-up examination. conclusion: notwithstanding the limitations associated with our analysis, namely a small sample size and its retrospective nature, these results suggest a role for bendamustine in post bv failures. these findings also provide the basis to evaluate the concept of benda as a bridge to allo-sct in a large prospective study. [p607] disclosure of conflict of interest: none. brentuximab vedotin for relapsed or refractory hodgkin lymphoma, single center experience king faisal specialist hospital and research center, riyadh, kingdom of saudi arabia ms rauf 1 , i maghfoor 1 , a badran 1 , mn zahir 1 and s akhtar 1 1 hodgkin lymphoma (hl) patients with relapsed or progressive disease after high dose chemotherapy (hdc) and auto-sct have limited curative options. fda granted approval of brentuximab vedotin (bv) for the treatment of hl and anaplastic large cell lymphoma (alcl) patients who fail auto-sct or have had at least 2 prior multiagent chemotherapy regimens and are not candidates for auto-sct. we are reporting single center experience of bv usage in this "approved" setting. medical records were reviewed to collect required data. kaplan-meier (km) method was used to calculate overall survival (os) and progression free survival (pfs) from date of first dose of bv. from 2013-2015, 25 patients received bv. 24/25 had hl (22 classic hl-nodular sclerosis, 2 hl-mixed cellularity) and 1 alcl. 19/25 (76%) pts were primary refractory or had early relapse after initial treatment. 15/25 (60%) pts received bv were refractory to the last treatment. all the baseline characteristics of patients are mentioned in table 1 . median bv cycles administered were 6 (2-16). overall response rate (orr) was 40% (10 patients): cr in 6 (24%), pr in 4 (16%) (5/10 were primary refractory or early relapsed). median pfs for whole group was 5 months (95% ci, 3.6-6.3). km estimated 1-year os was 74% and 2 year was 68%, median os has not been reached yet. for 10 patients who responded, pfs at 12 months was 68% (95% ci, 38%-98%), median pfs not reached. for 15/25 patients with progressive disease (pd) or non responders after bv, median pfs was only 3 months (95% ci, 1.1-4.8). there was no difference in os between patients with responders and non responders. median os has not yet been reached in either group as mentioned in survival curves. at the median follow up of 19 months (range: 4-55 months) 18 patients are alive, 10 patients are alive without disease, 4 patients received consolidation bone morrow transplant (2 auto-sct and 2 allo-sct). 2 patients completed 16 courses and achieved cr. rest of 4 patients who are alive without disease; they had pd on bv but achieved cr with other treatments. 8 patients are alive with disease; 1 patient is on bv and 7 are on another treatment. 7 patients have died, 2 because of pneumonia while being on bv and 5 due to pd. 3/15 patients who received bv, achieved cr after failing all previous treatments and are in cr. peripheral sensory neuropathy developed in 3 patients; one required dose reduction. 1 patient stopped treatment due to pulmonary toxicity. we are reporting largest single center data from middle east which confirms that bv as a single agent is effective and safe. overall response rate is lower as compare to pivotal trial but cr rate is comparable to other reported case series. this analysis also concludes that bv can be used as bridge to transplant in patients who don't respond salvage chemotherapy. disclosure of conflict of interest: none. was used to diagnose hiv infections. cox proportional hazards models were used to evaluate risk factors of overall mortality. fifty-six patients with nhl (1.4%) and 23 patients with mm (0.8%) were positive for hiv antibody. in patients with nhl, overall survival was significantly lower in the hiv-infected patients than in the hiv-negative patients [5year overall survival: hiv-infected patients, 44% (95% confidence interval, 29%-58%) vs. hiv-negative patients, 65% (95% confidence interval, 63%-67%), p o0.001)]. in a multivariate analysis, hiv infection was significantly associated with an increased risk of mortality (hazard ratio 2.39, p o0.001), and this effect was consistent regardless of transplant year. on the other hand, overall survival in patients with mm was similar between the 2 groups [61% (95% confidence interval, 31%-82%) vs. 63% (95% confidence interval, 63%-67%), p = 0.988]. previous studies in europe and the united states showed comparable survival rates between hiv-infected and hiv-negative patients with nhl. however, our study showed that hiv infection was associated with a higher risk of mortality in patients with nhl in japan. suppression of t cell-mediated immunity or hiv related diseases might affect transplant outcomes in japanese patients. [p609] disclosure of conflict of interest: none. while beam and beac regimens (bcnu, etoposide, cytosar in both regimens and melphalan or cytoxan, respectively) are commonly used as conditioning high-dose therapy (hdt) in patients with non-hodgkin lymphoma (nhl), there have been few reports comparing these regimens. a retrospective analysis found the superiority of beam over beac in terms of overall survival (os) and event-free survival (efs). toxicities were similar, except that beam was associated with more frequent lower gastrointestinal (gi) mucositis. other studies reported that these regimens had similar efficacy and outcome. recently, a concern regarding cardiotoxicity of beac has risen. the current study aimed to compare efficacy and toxicity of beac and beam as consolidation hdt in young patients with mantle cell lymphoma (mcl) undergoing autologous stem cell transplantation (asct). this is a retrospective analysis of outcomes in mcl patients who received hdt with beam or beac followed by asct at 3 bone marrow transplant centers in israel. os, disease-(dfs) and progressionfree survival (pfs) and regimen toxicity were compared. seventy seven mcl patients who were diagnosed between 1995-1/2016 and received consolidation with beac or beam were included in the analysis. forty nine patients were treated with beam and 28 patients-with beac. no significant differences between the groups were revealed in terms of age, sex, the mantle cell lymphoma international prognostic index (mipi) risk score, induction protocol and% of patients transplanted in first complete response (cr1) (mean age 57 yrs in beam vs 59 yrs in beac group; 69% of patients in beam group had mipi risk score 2-3 vs 62% in beac group; 68% of patients in beam group were transplanted in cr1 vs 71% in beac group). the amount of infused cd34 cells was significantly higher in the beam group (median cd34 cells/ kg: 8.2 in beam vs 4.6 in beac groups; p = .001); the number of days to platelet engraftment was significantly greater in the beac group (median 12 days in beam vs 14 days in beac group; p = .02). there were no differences in the number of blood transfusions or hospitalization days between the groups. the rate of grade 3-4 upper mucositis was significantly higher in the beam group (41% in beam vs 18% in beac group; p = .046); no other differences in toxicity (grade 3-4 lower mucositis, pulmonary congestion, infections) were observed between the regimens. non-relapse mortality by day 30 posttransplant was 0% in both groups. a median follow-up was 29 (range: 1-119) months. the 3-yr dfs in beam and beac groups was 58% and 64%, respectively (p = .65). there was no difference in the 3-yr os between the groups (70% in beam and 84% in beac group; p = .51). there was a trend to improved dfs and os in patients transplanted in cr1 receiving beam (p = .09, figure) . in multivariate analysis, low-to-intermediate mipi and transplant in cr1 were found to significantly increase pfs (p = .04 and.01, respectively), while the hdt regimen did not affect pfs. beac and beam hdt regimens followed by asct had similar efficacy in mcl patients. there was a trend to improved dfs and os in patients transplanted in cr1 and treated with beam vs beac. the toxicity profile was similar in both groups, except a significantly higher rate of grade 3-4 upper gi mucositis. [p610] disclosure of conflict of interest: none. early or refractory relapsed ( o1 year) diffuse large b-cell lymphoma has a very poor prognosis especially for those not responding to salvage chemotherapy. allogeneic stem cell transplantation is potentially curative. even though this is less likely in those not responding or having frank progression pretransplantation. methods: at our institution we identified all patients with aggressive b-cell lymphoma (diffuse-large b-cell lymphoma and blastoid mantle cell lymphoma) who were refractory or progressive to salvage chemotherapy with r-dhap and who had peripheral blood stem cells (42 × 10 6 cd34+/kg body weight) collected after the 1st or 2nd cycle. after high-dose melphalane and autologous stem cell transplantation 13 patients had a partial and 6 a complete remission. 1 patient died due to neutropenic infection, 2 patients died due to progressive disease leading to a transplant related mortality of 3.5%. median progression-free survival after autologous transplantation was 4.6 months. 24 proceeded to allogeneic stem cell transplantation. 8 patients had a matched related sibling, 9 had a matched unrelated donor and 7 had a mismatched unrelated donor. transplant related mortality was 42% in this heavily pretreated population. 2-year overall survival of all patients intended for treatment is 21%. one of these patients with relapsed mediastinal lymphoma after allogeneic transplantation was cured by salvage radiotherapy and is in long-term remission (42 years). conclusions: salvage high-dose melphalane and autologous peripheral blood stem cell transplantation for diffuse large b-cell lymphoma as a bridge to allogeneic transplantation is potentially curative for a minor fraction of these patients. however, the remission rate of 79% (46% pr, 21% cr) and progression-free survival of 4.6 months after high-dose melphalane and autologous stem cell transplantation provides a window of opportunity to use new drugs and cellular therapies in these poor prognosis patients. high dose chemotherapy and autologous stem cell transplantation is the treatment of choice for patients with relapsed refractory hodgkin lymphoma. several factors including number of chemotherapy lines received before conditioning, time of relapse and remission status before transplantation can predict survival and pfs in patients undergoing autologous stem cell transplantation. in 2012, we reported on a 63 patients who underwent high dose chemotherapy followed by autologous stem cell transplantation from 2003 to 2008. all patients with relapsed or refractory hodgkin lymphoma in the period of 2009-2013, who underwent high dose chemotherapy followed by autologous transplantation were retrospectively analyzed. the main outcomes of the study were complete remission (cr) at day 100, overall survival (os) and relapse-free survival (rfs). the impact of the following variables on os and rfs: (a) disease status at the time of transplant, (b) number of chemotherapy lines prior to conditioning and (c) time of relapse 12 months and (d) age. a total of 78 patients were identified. the median age was 31 year. there were 50.6% females and 49.4% males. complete remission (cr) was achieved in 48.7% of patients and 49.9% with chemotherapy sensitive disease at the time of transplantation. prior to conditioning regimen, 43.2% received two chemotherapy lines, and 56.8% received more than two lines. 41% relapsed in less than 12 months and 57% relapsed more than 12 months after completion of therapy cr at day 100 was 69.2%. the median os for the whole group was 62.0 months; the median rfs was 10,6 months. the number of chemotherapy lines significantly impacted os and efs. cr status before conditioning, favorably influenced os and efs with a trend toward better os in favor of those who underwent abmt while in complete remission. the time of relapse and the age did not affect survival outcomes. [p614] the outcome of patients with relapsed or refractory hodgkin lymphoma is favorable and the number of chemotherapy lines received before conditioning is the only factor that had a statistically significant impact on os and efs. since the identification of human immunodeficiency virus (hiv), a clear association between hiv and specific malignancies has been recognized. high-grade b cell lymphomas are the most common malignancy complicating hiv infection and one of three aids defining malignancies. diffuse large b cell lymphoma (dlbcl) accounts for 80% of cases. before 1996, lymphomas were the cause of 16% of all deaths attributable to aids. after the introduction of highly active antiretroviral therapy (haart) overall incidence of adm declined, however longer survival and exposure to environmental risk factors have increased the incidence of non adm (adm) such as hodgkin's lymphoma (hl). since haart has improved overall survival substantially, the aim of chemotherapy should be complete remission rather than palliation with careful consideration of drug interactions and side of haart. between 2011 and 2016 a total of 510 patients were detected hiv positive. twenty-one of these patients were diagnosed with a malignancy and 8 patients referred to our department with a hematologic malignancy were evaluated retrospectively. diagnosis, stage, treatment, survival data were recorded. haart during chemotherapy, nadir cd4 count and cd4 count at diagnosis of malignancy was evaluated. four patients were diagnosed with high grade b cell lymphoma, 2 patients with primary central nervous system lymphoma (pcnsl), 1 patient with hl and 1 patient with multiple myeloma (mm). all patients were male and median age at diagnosis was 40.5 (24-63). hiv seropositivity was identified during evaluation of malignancy in both pcnsl patients. median duration of hiv seropositivity before diagnosis of malignancy was 18 months for the remaining patients. patient characteristics, treatment modification and cd4 counts are summarized in tables 1 and 2 . lymphoma was fatal in 5 and the cause of death was identified as lymphoma progression in all patients including one patient diagnosed with hodgkin's lymphoma. a patient presented with multiple plazmositomas was diagnosed with multiple myeloma is currently receiving induction treatment together with haart. hiv related lymphoma patients frequently present with extra nodal disease, incidence of central nervous system involvement is also higher and prognostic score tends to be in the intermediate or high-risk groups. prognosis is also worse than hiv negative population. degree of immunosuppression is implicated and the duration of immunosuppression is directly correlated with the risk of developing lymphoma rather than hiv itself. haart allowed the use of aggressive chemotherapy since it improved immune system and decreased infectious complications. multiple myeloma is a rare neoplasm observed in hiv infection and the treatment is based on data obtained from hiv negative patients. treatment of such patients as well as lymphomas should take into consideration the toxic effects of haart combined with chemotherapy. since hiv positive [p615] patients are excluded from most studies, there are no guidelines to direct treatment and avoid toxicities. drug interactions should be monitored closely and modifications should be made accordingly. interruption of haart may not be mandatory since studies have shown safety of continuation of haart during chemotherapy. for newly diagnosed hiv and malignancy, careful clinical and laboratory evaluation should be made before postponing haart until after chemotherapy. disclosure of conflict of interest: none. the outcome of hdct and asct in refractory hodgkin lymphoma (r-hl) is not as encouraging as in relapsed hl. ten years ago we analyzed and reported outcomes of asct in our r-hl patients, however the follow-up was short. now we a reporting long term outcomes in r-hl after asct in one of the largest numbers reported to date. between 1996 and 2014, patients with hl who underwent hdc and asct for r-hl in adult medical oncology (age 4 14 years) were identified. r-hl is defined as partial response (pr), no response (nr), stable disease (sd), progressive disease (pd), relapsing within 3 months (relapse o3 m) of finishing the planned (chemotherapy + radiation therapy (xrt)) treatment or refractory to salvage chemotherapy. kaplan-meier (km) method was used to estimate progression free survival (pfs) and overall survival (os) from the day 0 of asct while progression is defined as progression of disease, relapse and death from any cause. all percentages are rounded to nearest. 307 patients underwent hdc and asct during 1996-2014 and 177 of them met the criteria of r-hl. male 97 (55%), female 80 (45%), median age at diagnosis was 22.2 years (8-61 years) and at asct was 24 years (14-62 years). initial therapy was abvd in 153 (86.4%), mopp/copp alternating with abv or abvd in 11 (6%) and others in 13 (7%). 49 (28%) had xrt after initial chemotherapy. response to initial chemo + xrt was pr in 80 (45%), pd in 51 (29%), cr in 38 (21%) (28/38 relapsed within 3 months and others have refractory relapse) and no response in 4 (2%) and others in 4 (2%). prior to salvage chemotherapy, 119 (67%) had stage iii-iv, 90 (51%) extra-nodal involvement, 43(24%) bulky disease and 31 (18%) had b symptoms, spleen involvement in 43(24%), performance status 0, 1 in 155 (88%). eshap was used as first line salvage in 153 (86%) or 3 rd line 13 (7%). post salvage / prior to hdc and asct disease status was pr in 112 (63%), cr in 53 (30%) and nr/sd in 12 (7%). 111 (63%) patients had a fdg-pet scan prior to asct, 45 (25%) were in cr. beam was used as conditioning regimen. median follow-up for all alive patients is 81 months (15-224) from asct. response rate post asct: cr in 105 (59.3%), pr in 16 (9%), nr/sd in 1 (0.6%) and pd in 45 (25.4%) patients, others /unknown in 10 (5.6%). 61 (35%) patients had xrt post auto-sct. type of first post hdc auto-sct event was no event in 81 (46%), persistent disease in 17 (10%), pd in 45 (25%), relapsed disease in 23 (13%), treatment related mortality in 6 (3%) and died of other cause 5 (3%). at last follow-up in november 2016, 94 patients (53%) are alive with no disease, 6 (3%) alive with disease, 64 (36%) died of disease and 13 (7%) died of treatment related mortality or other causes. for entire group, km estimated median os is 129 months, 1,2,3,5 and 7 year survival is 83%:73%:64%:59%:55% respectively. median pfs is 48.5 month, 1,2,3,5 and 7 year pfs is 57.6%:53.6%:51%:50%:47% respectively. we are reporting a very high risk group of patients with a very long follow-up. in patients with r-hl, eshap + beam combination resulted in high response rate (68.3%). these remissions are durable. a 5 year os survival of greater than 50% in our population is higher than most reports with similar numbers. although our cohort has a 7 year os survival of 55%, 45% patients have either relapsed or died underscoring need for improvements in the management refractory hl. [p616] disclosure of conflict of interest: none. here we update the previously reported results of our reduced-intensity conditioning (ric) allo-hsct experimental program, initiated in 2002. as of november 2016, in our centre 34 patients underwent ric allo-hsct. donors were hla-identical sibling in 16, fullymatched unrelated in 7, 1 or 2-mismatch-unrelated in 9 and haploidentical relative in 2. median age was 53 years (range: 19-66). all patients (22 m and 12 f) had stage iib/iv refractory mf (n = 22) or refractory ss (n = 12). median number of previous treatment lines was 6 (range: 2-12). source of stem cells was peripheral blood in 31 patients and bone marrow in 3. median time from diagnosis to hsct was 46 months (range: 13-264). conditioning included flu/ctx/tbi200, pentostatin +tbi200 and flu/mel in case of hla-identical or unrelated donor, whereas the tt/flu/ctx/tbi200 regimen was used in the haplo setting. gvhd prophylaxis included csa/mmf in all patients, with the addition of atg in cases with unrelated donor and post-transplant ctx (50 mg/kg giorni +3 e +4) in cases with haploidentical donor. full donor chimerism was obtained in 28/33 of the evaluable patients, in a median time of 2 months (range: 1-12). grade ii-iv acute gvhd occurred in 16 patients (57%), while grade iii-iv was observed in 8 patients (28%). chronic gvhd occurred in 10 patients (36%), being extensive in 4 (14%), all transplanted from hla-identical sibling (no atg). following transplantation, a complete remission (cr) was achieved in 22 out of the 33 evaluable patients (67%), of whom 2 experienced relapse at +25 and +35 months, respectively. transplant-related death occurred in 6 patients (17%), of whom 4 were in cr. out of the 11 patients who did not achieve cr, 9 died from progressive disease (median follow-up of 12 months, range: 3-31), 1 from a secondary malignancy and 1 is still alive with disease 41 months after transplant. of note, all pts who died in progression had chemoresistant disease at time of transplant. at the last follow-up, 18 patients were alive and 16 (89%) maintained cr after a median time of 66 months (range: 4-189). in the whole population, the 5-year overall survival was 52% (95% ci 34-70) and the 5-year disease-free survival (dfs) was 44% (95% ci 27-62). however, when mf and ss were analysed separately, 5-yrs dfs were 32% (95% ci 12-51) and 69% (95% ci 38-99), respectively (figure) . apart from diagnosis, outcome appeared to be primarily associated with the disease status at transplantation, with a 5-yr dfs of 100% in the group of patients (n = 8) who were in cr before starting the conditioning. after a median follow-up longer than 5 years, we confirm the efficacy of ric allo-hsct as a powerful therapeutic strategy in inducing and maintaining remission in selected patients with chemosensitive advanced-stage ctcl, with results particularly encouraging in ss. [p617] disclosure of conflict of interest: none. outcomes of allogeneic hematopoietic stem cell transplantation for hodgkin lymphomas: a retrospective multicenter experience of the rete ematologica pugliese (rep) f gaudio 1 , p mazza 2 , am carella 3 , d pastore 1 , g pisapia 2 , a mele 4 , p galieni 5 , n cascavilla 3 , g specchia 1 and v pavone 4 1 hematology, university of bari, bari, italy; 2 hematology, ospedale "san giuseppe moscati", taranto, italy; 3 hematology, ospedale "casa sollievo della sofferenza", san giovanni rotondo, fg, italy and 4 hematology, ospedale "cardinale panico", tricase, le, italy; 5 hematology, ospedale "c. g. mazzoni", ascoli piceno, italy hodgkin's lymphoma (hl) is a potentially curable disease, and modern therapy is expected to successfully cure more than 80% of the patients. second-line salvage high-dose chemotherapy and autologous stem cell transplantation (sct) have an established role in the management of refractory and relapsed hl, leading to long-lasting responses in approximately 50% of relapsed patients and a minority of refractory patients. patients progressing after intensive treatments, such as autologous sct, have a very poor outcome. allogeneic sct represents the only strategy with a curative potential for these patients; this study reports a retrospective multicenter experience of the rete ematologica pugliese (rep) over the past 16 years aiming to define the impact of patient, disease, and transplant-related characteristics on outcomes. 67 patients with histologically confirmed diagnosis of hl who received allogeneic sct from 2000 to 2016 were retrospectively studied. the median age was 34 years (range: 16-57 years) and 36 (54%) were male. the majority of patients (84%) had had a prior autologous sct. at the time of allogeneic sct, 28 (42%) patients had a chemosensitive disease and 39 (58%) were chemorefractory. most (93%) patients received reduced-intensity conditioning, 54% received matched sibling donor and 46% matched-unrelated donor grafts. the disease status at day 100 post-transplant was reported in 62 out of 67 evaluable patients. of the 26 patients with chemosensitive disease, 18 (70%) achieved a cr, 7 (27%) had a pr or stable disease and 1 (3%) had progressive disease. of the 36 patients with chemorefractory disease 7 achieved a cr (20%), 26 had a pr or stable disease (72%) and 3 (8%) had progressive disease. although the overall survival has improved significantly in mantle cell lymphoma (mcl) according to advanced treatment options, relapsed or refractory disease remains a challenge. recently, lots of targeted agents actively have been tried clinical studies and adapted to clinical practice in indolent lymphoma. however, the role of frontline autologous hematopoietic stem cell transplantation (auto-hsct) has not been fully understood in patients with mcl, compared with a few impressive published data about auto-hsct as salvage treatment option for patients with relapsed mcl. so, we retrospectively evaluated consecutive patients diagnosed mcl, and compared the clinical outcomes of high-dose chemotherapy followed by auto-hsct and conventional chemotherapy alone. between january 2003 and december 2014, consecutive patients with newly diagnosed with mcl at catholic blood and marrow transplantation center in south korea were included in this study. all of the patients received high-dose cytarabine-containing regimen or chop with/without rituximab regimen for induction therapy regardless of transplant eligibility. the treatment approach in our institution for patients was based on the physician discretion for transplant eligibility or ineligibility that depend on patient age, comorbidities, and disease status. seventy patients were included in the analysis. initial chemotherapy regimens were consisted of chop (n = 12, 17%), r-chop (n = 44, 63%), r-hypercvad (n = 10,14%), and hypercavd (n = 4, 6%). demographics and disease characteristics of both groups are shown in table1. patients received auto-hsct were superior s449 overall survival (os; p = 0.015) and progression-free survival (pfs; po0.001). the subgroup analysis according to high-risk of mcl international prognostic index (mipi) or bone marrow involvement was performed. between the two treatment arms among the high-risk mcl group, the clinical parameters were not different. the high-risk mcl patients with frontline auto-hsct showed superior os (p = 0.0216) and pfs (po0.001) compared with conventional chemotherapy alone. although mcl is classified within indolent lymphoma, frontline auto-hsct can be considered for patients diagnosed with mcl in the group of high-risk mipi or bm involvement with the favorable survival outcomes. disclosure of conflict of interest: none. nasal type extranodal nk/t-cell lymphoma (enktl) is a very rare and agressive malignancy characterized by a poor outcome. current standard therapy is not yet established. the role of high dose therapy followed by haematopoietic stem cell transplantation (hsct) is still controversial. we evaluated the outcomes of all the enktl patients undergoing hsct in a multicenter analysis on patients registered by the société francophone de greffe de moelle et de thérapie cellulaire (sfgm-tc) and compared them with a population of french patients who received chemotherapy alone. sixty four enktl (48 males and 16 females) received hsct, including 19 allogeneic (allosct) and 45 autologous transplantations (autosct). median age at the time of hsct was 43 years (range: 17 to 70 years). overall, 57% of the patients presented with disseminated disease (64% and 55% in the allosct and autosct, respectively), 61% were in complete response (cr) at the time of hsct (74% and 61% in allosct and autosct groups, respectively) and 82% had received l-asparaginase regimen prior to hsct (73% and 84% in allosct and autosct groups, respectively). five (26%) and 20 (44%) patients of the allosct and autosct groups underwent upfront hsct therapy, respectively. four patients received tandem autologous/ allogeneic transplants. in allosct, stem cell source was a matched related donor in 13 patients, an unrelated donor in 3 patients and an umbilical cord blood in 3 patients. reduced intensity conditioning regimens (based on fludarabine-busulfan combination) and beam regimen were used in 42% and 84% of patients from the allosct and autosct groups, respectively. median overall survival for the whole cohort was 17.1 months (range: 1 to 131 months). the 3-year non-relapse mortality was 16.1% and 22.7% in the allosct and autosct groups, respectively (p = 0.58). the 3-year overall survival (os) and progression free survival (pfs) were 47.5% and 40.8% in the autosct and 43.4% and 47.4% in the allosct group, s450 respectively ( figure 1a) . the absence of cr prior to hsct was associated with a poor prognosis (p = 0.008). as compared to allosct, autosct resulted in a better outcome in patients who didn't achieve cr before transplant (p = 0.002) and tended to have better outcome in high pink risk score (figure 1 b-c) . finally, at 3 years pfs and os of patients who have been treated by chemotherapy alone (ct) (n = 55) or followed by allosct (n = 12) or autosct (n = 17) in cr1 were 55%, 81% and 50%, 54% and 44%,50%, respectively ( figure 1d ). in this french cohort, more patients received autologous hsct in upfront therapy than allogeneic hsct. in cr1, there is no evidence suggesting that transplantation is associated to a better outcome than chemotherapy alone. however, a precise matching based on the pink score will be evaluated to ensure that patients who were intensified were not of worst prognosis. in refractory patients there is also no clear advantage to perform allosct when compared to autosct. however, in relapsing disease after ct or autosct allosct, allowed to obtained durable control of the disease. disclosure of conflict of interest: none. high relapse rate is one of concerns for allo-sct in pts with relapsed/refractory aggressive lymphoma. an optimal conditioning regimen designed for aggressive lymphoma may reduce relapse, especially during early post-transplantation period. however, it is not established yet. results of a german phase 2 study of allo-sct with conditioning regimen of fludarabine, busulfan (12 mg/kg po or 9.6 mg/kg iv), and cyclophosphamide with or without post-transplantation rituximab for relapsed/refractory aggressive lymphoma suggested the role of myeloablative busulfan-containing regimen in reducing relapse rate in pts with aggressive lymphoma. based on these results, we conducted a single institution prospective study to explore feasibility of the bmf regimen consisted of full-dose busulfan, melphalan, and fludarabine in pts with relapsed/refractory aggressive lymphoma (umin000013940). patients with aggressive lymphoma who achieved at least sd with salvage chemotherapy after experiencing either pd during first-line therapy, early relapse ( o12 mo) after firstline therapy, late relapse (≥12 mo) but refractory to salvage therapy, relapse after auto-sct,; age 20-65; ecog ps of 0-2; and without severe organ dysfunction were eligible. donor source could be 6/6 matched related or unrelated donor pb/bm or cb with ≤ 2 antigen mismatch; the bfm regimen was consisted of busulfan 12.8 mg/kg iv, fludarabine 180 mg/m 2 , and melphalan 80 mg/m 2 (yamamoto h. bbmt 2016). gvhd prophylaxis was csa + mtx (related pb), tac + mtx (unrelated bm), and tac + mmf (cb). primary end point of the study was survival with engraftment at day 60, and secondary end points were engraftment rate at day 100; nrm and relapse rate at day 100 and 1 y; progression free survival (pfs), overall survival (os), and gvhd at 1 y. protocol was approved by irb and written ic was obtained from all pts. twelve pts (male 10, female 2) with a median age of 55 y (33-63) were enrolled. ps was 0-1 in 11 pts. diagnosis were dlbcl (n = 4), transformed fl (n = 3), enktcl (n = 3), ptcl (n = 1), and aitl (n = 1). median number of previous line of therapy was 3.5 (2-5) and 5 pts had failed previous auto-sct. diseases status at transplantation was cr (n = 6), pr (n = 4), and sd (n = 2). donor source was cb (n = 6), unrelated bm (n = 5), and related pb (n = 1). survival with engraftment at day 60, primary endpoint of the study, was achieved in 100%. neutrophil engraftment was achieved at a median of day 18 (13-32). full donor chimerism at day 30 was achieved in all of the 11 pts evaluated. two pts developed vod which was manageable. with a median follow-up of 20 mo, 3 pts had progression of lymphoma at 2, 5, 6 mo. five pts died and cause of death were progression of lymphoma in 3, interstitial pneumonitis in 1 (at 5 mo), systemic adenovirus infection in 1 (at 5 mo), and agvhd in 1 (at 4 mo). os and pfs at 1y were 54% and 46%, respectively. relapse and nrm rates were 8% and 0% (day 100), and 27% and 27% (1y), respectively. agvhd of grade ii-iv was observed in 7/12 pts and 2 pts developed limited cgvhd. this prospective study shows that allo-sct using myeloablative conditioning regimen with full-dose busulfan, melphalan, and fludarabine for relapsed/refractory aggressive lymphoma is feasible and deserves further evaluation. disclosure of conflict of interest: none. for patients with advanced ctcl, the allogeneic hsct seems to be curative with graft versus lymphoma effect playing a major therapeutical role. in this retrospective study, 16 patients with a median age of 52 years (range: 24-67) affected by ctcl underwent allogeneic hsct after a median of 4 (range: 1-8) lines of chemotherapy, including autologous transplant for 2 of them. the median time from diagnosis to hsct was 46 months (range: 9-309). the diagnoses were: sezary syndrome (ss, n = 8). mycosis fungoides (mf, n = 2), primary cutaneous cd30+ lymphoma (n = 4), panniculitis-like t-cell lymphoma (n = 1), nk t cell lymphoma (n = 1). at time of hsct, 2 patients (12.5%) were in complete remission (cr), 9 (56.3%) in partial remission (pr) and 5 (31.2%) had active disease. the patients were transplanted from an hla-identical (n = 7), mismatched (n = 1) or haploidentical (n = 1) sibling, from matched unrelated donor (n = 5) or from a single cord blood unit (n = 2). different pre-transplant regimens were used as myeloablative (mac) in 6 (th-bu-flu, n = 4; bu-cy, n = 2) or as reduced intensity (ric) in 10 (th-flu-cy, n = 7; th-bu-flu, n = 3). al patients engrafted for neutrophils at a median of 17 days (range: 12-46) and 14 patients engrafted for platelets at a median of 14 days (range: 12-77). acute gvhd was of grade 0-i in 9 patients and ii-iv in 7 (40.7%). skin was the most common organ involved. five of 11 evaluable patients experienced chronic gvhd which was mild in 3 and severe in 2. at a median of 3 months (range: 1-11), 7 patients died (4 mac and 3 ric) because of gvhd (n = 4), vod (n = 1), pneumonia (n = 1) or multiorgan failure (n = 1). all 12 patients surviving at 3 months from transplant were in cr. only patients prepared with a ric (n = 4) relapsed respectively at 3, 9, 10 and 11 months from hsct. these patients received dli associated or not to chemotherapy. three achieved cr, which remained stable in 2, while one patient died in cr from post dli acute gvhd. one patient (nk-t cell) not achieving cr is still alive with active disease. for all 16 patients the median survival was 10 months (range1-130). with a median follow up of 76 months (range: 4-130), 9 patients (2 mac, 7 ric) are alive, 8 in cr and 1 with active disease. at 10 years, the os was 54 ± 13%; at 5 years dfs was 34 ± 12%. according with the median time (46 months) from diagnosis to transplant, the 10-year os was 88 ± 12% for patients transplanted early and 25 ± 15% for the others (p o0.04), while dfs was respectively s451 58 ± 19% and 13 ± 12% (p o0.05). despite the small number of patients, our results confirm the high susceptibility of ctcl to the graft versus lymphoma effect and point out the time to transplant as a crucial prognostic factors for the outcome. finally, the long-term follow up of our series strongly supports hsct for the cure of ctcl. disclosure of conflict of interest: none. recently, a new prognostic score, the nccn-international prognostic index (ipi) has been developed 1 to stratify patients affected by diffuse large b cell lymphoma (dlbcl), and in high-intermediate and high risk groups the survival was equal or less than 50%. the aim of this analysis was to evaluate the outcome of a cohort of dlbcl patients undergoing high dose chemotherapy (hdc) as consolidation following first line chemo-immunotherapy, after their re-classification according to the nccn-ipi. we performed a retrospective study on 221 patients diagnosed with dlbcl, with a high/intermediate or high-risk disease according to the ipi (2-5), who received upfront hdc with asct, in 2 institutions. the patients were then re-stratified according to the nccn-ipi and arbitrarily classified in 2 groups: low risk (nccn-ipi ≤ 3) and high risk (nccn-ipi ≥ 4). the pre-transplantation disease status was assessed by positron emission tomography (pet) or computed tomography (ct). the primary endpoints were non-relapse mortality, progression-free survival (pfs), overall survival (os) and relapse risk. the estimated 3-year pfs for all patients was 80.1% (95% confidence interval [ci] 74.2-86.0) and the 3-year os was 91.0% (95% ci 86.7-95.3). of these patients, 93 had a low risk ipi score (ipi = 2) and 128 were considered high risk (ipi ≥ 3). subsequently, the whole cohort was re-stratified according to the nccn-ipi: 133 patients were allocated to the high-risk (nccn-ipi ≥ 4) group, and 88 to the low-risk group (nccn-ipi ≤ 3). the analyses were then carried out for both groups. the 3-year pfs was 92.0% (95% ci 85.7-98.3) in the low-risk group and 71.2% (95% ci, 62.2-80.2) in the highrisk group (po 0.01), whereas the 3-year os was 98.8% (95% ci 96.4-100) in the low-risk group and 85.0% (95% ci 77.9-92.1) in the high-risk group (p = 0.02). the significant difference in os and pfs between the two groups was mainly due to the cumulative incidence of relapse at 3 years (graph 1): 8.0% (95% ci 3.2-15.7) in the low-risk group and 27.1% (95% ci 18.7-36.2) in the high-risk group (po 0.01). non-relapse mortality was comparable in both cohorts: 1% (95% ci 0.2-3.3) for all patients. figure 1 : cumulative incidence of relapse following hdc and according to nccn-ipi. patients affected by high-risk dlbcl still have an unsatisfactory prognosis after treatment with conventional therapy regimens, even in the rituximab era. the 5-year os and pfs in patients with nccn-ipi score ≥ 4 range: from 33% to 64% and from 30% to 51% respectively 1 . although this is a retrospective analysis subject to all related biases, our results suggest that upfront intensive therapy with autologous stem cell transplantation may significantly improve the outcome of these patients compared to conventional chemotherapy. the role of hdc in the treatment of dlbcl is controversial. however, new entities or new risk stratifications, as the one reported here, could allow to identify high risk subpopulations that could benefit from this approach. enteropathy-associated t-cell lymphoma (eatl) is an exceedingly rare and often rapidly fatal subtype of peripheral t-cell lymphoma, arising from intraepithelial lymphocytes. eatl type i is associated with celiac disease; type ii occurs in patients without inflammatory pre-conditions (according to who 2016 classification now called monomorphic epitheliotropic intestinal t-cell lymphoma (meitl)). surgical debulking and anthracyclinebased chemotherapy (ctx) followed by high-dose chemotherapy (hdctx) and autologous cell rescue (asct) are pursued when possible in this often malnourished and frail patient cohort. yet, even with intensive consolidation relapse occurs in 40-70% of patients. the value of allogeneic hematopoietic cell transplantation (hct) is not clarified as of today due to limited reports. here, we report on a patient with meitl who was rescued with an allo-hct for his 2nd relapse following prior asct. moreover, we summarize the available literature on the use and outcomes of allo-hct for eatl and meitl. a 48y old man with spontaneous intestinal perforation was diagnosed with meitl following emergency partial resection of the small intestine. histology revealed infiltration by monotonous medium-sized lymphocytes with abnormal immunophenotype (cd3+, cd56+, cd8+, cd5-, cd30-, tia-1+) consistent with type ii eatl. post-surgical 18f-fdg pet-ct scan showed abnormal uptake in gastric antrum and pyloric region but no other manifestations. ctx with cho(e)p (6 × ) followed by beam hdctx and asct was performed and achieved a complete remission (cr1). however, 9m post asct disease relapsed and was treated with 2 × dhap, and 4 × dhaox. cr2 was achieved after the 3rd cycle of salvage therapy. due to anthracyclineinduced cardiopathy allo-hct could not be performed at that time. 5m after completion of salvage therapy, disease relapsed again, and was progressive under pralatrexat treatment (1 cycle, 6 infusions). by then cardiac function had recovered and therapy was switched to dose-reduced mini-beam (2 × ). in cr3 reduced intensity conditioning (ric; fludarabine, busulfan, atg) and allogeneic hct from a matched sibling donor was performed. ciclosporin a (csa) and mycophenolate mofetil (mmf) were given as gvhd prophylaxis. prompt engraftment in blood (day+14) and full donor chimerism in the marrow (d+100) were achieved. immunosuppression was tapered and discontinued on d+55 (mmf) and d+193 (csa), respectively. 18f-fdg pet-ct scan at 3m post-hct showed cr, but at 6m relapse was suspected (under work-up). only few cases of patients with eatl/meitl treated with allo-hct are reported in the literature (n = 9, table 1 ), and the value of this highly aggressive therapy is not clear at this point. of note, the patients listed in table 1 were given allo-hct instead of asct. long-term complete remission (cr) could be achieved in 4/9 patients, while 5 patients suffered from early relapse and died of the disease (n = 4 before d+100 post allo-hct). asct following surgery and ctx appears to cure 33-60% of patients in available series. no treatment concept is available for relapse following asct, and no published data are available for allo-hct for relapse post asct. the disease is exceedingly rare and is afflicted with very poor outcomes. therefore, patients given this aggressive treatment should be reported, even when treatment outcomes are not positive. disclosure of conflict of interest: none. strong graft versus lymphoma effects with low toxicicty of haploidentical hematopoietic stem cell transplantation comparing with hla-identical in t cell lymphomas: a retrospective multicenter study s bramanti, r devillier, s fuerst, b reda, a granata, s harbi, c faucher, i legrand, a santoro, d blaise and l castagna istituto clinico humanitas rozzano consolidation treatment of relapsed/ refractory t-nhl with allogeneic stem cell transplant (sct) is considered a curative options but few patients manage to undergo this procedure, due to the highly refractory nature of the disease. the primary aim of this work is to evaluate the gvl effects among t-nhl with both hla identical and haploidentical donors. we have retrospectively analized the long term outcome of 43 consecutive patients affected by t-nhl, received hlaidentical or t-cell replete haplo-sct with pt-cy, in 2 european centers, between february 2010 and october 2015. the patients received nonmyeloablative (nmac) or reduced intensity (ric) conditioning regimen. gvhd prophylaxis consisted of 50 mg/kg of pt-cy (day +3 and +4) in haplo setting and atg plus cyclosporine a in the hla identical setting. patients characteristics were reported in the table 1 . no differences were founded in the two groups . most of the patients were transplanted in complete remissions but only 8 as consolidation of first line. no graft failure occurred. the cumulative incidence of acute gvhd grade 3-4 was 5% in the haplo setting vs 11% in the hla id .extensive chronic gvhd was seen in 7% of haplo, and in 28% in the hla id . 9 patients had cmv reactivation, 3 hemorrhagic cystitis, and 1 ebv reactivation. after a median follow-up of 3 years os was 83% and 71% and pfs was 77% and 64% in the haplo vs hla id group see figure 1 . nrm was 5% in haplo setting and 11% in hla identical one. the 3 years cir is 16% and 23% in haplo and hla id setting respectively. this study confirm a strong anti-lymphoma effect of allo hsct without prohibitive toxicities. haplo-hsct with pt-cy shows low rate of cgvhd in a contest of poor prognosis t-nhl patients. [p625] disclosure of conflict of interest: none. ibrutinib is the first-in class bruton tyrosine kinase inhibitor that has been approved for the treatment of relapsed mantle cell lymphoma. however, despite the high response rate of 68% including 21% of complete response, the median duration of response is relatively short with an overall survival of 58% at 18 months (wang ml, et al, n engl j. med 2013). we report a single experience of 3 patients with relapsed mcl who underwent allogeneic stem cell transplant (allosct) after ibrutinib monotherapy salvage. all 3 patients had previous autologous stem-cell transplantation (asct) before and were given ibrutinib at a dose of 560 mg daily after the second or subsequent relapse. all patients had to be at least in pr according to cheson 2014 criteria before allosct. patients had an unrelated 10/10 (2) or 9/10 (1) allo-sct from peripheral hematopoietic stem cells after a reduced conditioning regimen with busilvex, fludarabine and antithymocyte globulin in association with zevalin according to our recent published phase 2 study protocol (bouabdallah k, et al. ann oncol 2015). graft versus host disease (gvhd) prophylaxis consisted on ciclosporine and methotrexate. patients (2m/1f) were aged from 62 to 65 years and received between 2 and 3 previous chemotherapy regimens including asct in their last treatment strategy before introduction of ibrutinib. all patients had extensive disease with gastric involvement in 2 patients and pulmonary localization in 1 patient. median time between diagnosis and ibrutinib introduction was 5 years (3-7) and the median time between asct and allosct was 4 years (4) (5) . median duration of ibrutinib treatment was 5 months (5-6) and it was stopped one week before proceeding to allo-sct. it was not planned to restart btk inhibitor after transplant. patients were assessed for response after at least 3 months of treatment with ibrutinib. at time of evaluation, all patients were in complete (2) or very good partial response (1) before allosct. the patient in partial response had 92% tumor reduction with persistent gastric ulcer where histology examination shows cd5+ but negative cycline d1 lymphoid cells. all patients engrafted (median duration of pnn o500g/l = 11 days (10-15) and median duration of platelets o20g/l = 2 days (0-14)) with fulldonor chimerism at 1 month. one patient had a grade ii cutaneous chronic gvhd (cgvhd) with favorable outcome and developed 8 months later a bronchopulmonary obstruction syndrome related to cgvhd. with a median follow-up of 9 months (9-23) after allo-sct, all 3 patients are alive in cr. one patient, in complete metabolic response before transplant had a gastric relapse 3 months later but achieved again a cr 3 months after reintroduction of ibrutinib. after the first case reported by furtado m et al (leuk lymphoma 2016), we report here 3 additional cases with longer follow-up after allogeneic transplantation. the excellent tumor control after treatment with ibrutinib together with a very good outcome after allosct should drive to consider this approach in young patients with mcl relapse after asct. disclosure of conflict of interest: none. autologous hematopoietic stem cell transplantation (autosct) is considered the standard approach for high risk or relapsed/ refractory non-hodgkin and hodgkin lymphoma. although a large variety of conditioning regimens are available, including the widely used beam (carmustine, etoposide, cytarabine, melphalan), there is no consensus regarding a standard approach. in the context of carmustine shortage, we have chosen to replace it by thiotepa. however, clinical data about thiotepa-based autosct conditioning are still sparse, except some retrospective data for primary central nervous system lymphoma. thus, we designed a multicenter prospective study (nct02504190) to assess the efficacy and toxicity of a team (thiotepa, etoposide, cytarabine, melphalan) conditioning regimen. team regimen consisted in total dose thiotepa of 8 mg/kg on day-6; etoposide 100 mg/m 2 /12 h and cytarabine 200 mg/m 2 /12 h (day-5 to -2); melphalan 200 mg/m 2 on day-1. patients underwent autosct with team conditioning, and were included in this analysis if they have fullfilled the following criteria: age older than 18 years, biopsy-proven hodgkin or non-hodgkin lymphoma, hiv seronegative, and first autosct. thirty-three male and nine female with a median age of 59 years (range: 19-72) were analyzed thus far. karnofsky score was 20g/l was 13 days (range: 8-48). of note, 5 patients received thrombopoietic agents after engraftment because of persisting thrombocytopenia. the most significant regimen-related toxicities were mucositis in 100% of patients (median grade = 3, range: 2-4) and diarrhea in 98% of patients (median grade = 1, range: 0-3). other non-hematologic grade 3 adverse events occurred in 13 patients (31%) and no grade 4 adverse events were observed. central line-associated bloodstream infection occurred in 11 patients (26%). surprisingly, 16/36 evaluable patients (44%) developed human herpesvirus 6 reactivation. only 2 patients required intensive care unit transfer. the median duration of hospital stay was 29 days (range: 20-62). after a median follow-up of 8 months (range: 2-20), the non-relapse mortality (nrm) was 0%. only one patient relapsed of refractory aitl 2 months after autosct and died 1 month after. the estimated 1-year overall survival and progression-free survival were 98% and 98%, respectively. a team conditioning regimen seems to be a safe and valid platform in autosct for patients with high-risk or relapsed/ refractory lymphoma. although mucositis and diarrhea were frequent, there were no grade 4 adverse events and no deaths related to the treatment. updated results with updated followup will be presented. disclosure of conflict of interest: none. the cell of origin has no prognostic impact on high-dose chemotherapy with r-beam and autologous stem cell transplant for diffuse large b cell lymphoma s lozano cerrada, r saliba, s srour, s ahmed, c hosing, r champlin and y nieto university of texas, md anderson cancer center, department of stem cell transplantation and cellular therapy diffuse large b-cell lymphoma (dlbcl) is a biologically heterogeneous disease that can be classified according to its cell-of-origin (coo). the germinal center b-cell (gcb) subtype has better outcome with frontline r-chop than the activated b cell (abc) subtype. however, the prognosis of these two types of dlbcl after high-dose chemotherapy and autologous stem cell transplant (asct) is less clear. the purpose of our study was to evaluate progression-free survival (pfs), event-free survival (efs) and overall survival (os) in a cohort of dlbcl patients treated with r-beam (rituximab, carmustine, etoposide, cytarabine, melphalan) and asct according to coo. we have the dicep regimen effectively reverses the poor outcome for lymphoma patients with suboptimal response or failure post 1st salvage treatment p kaloyannidis 1 the outcome of patients (pts) with refractory hodgkin's (hl) and non-hodgkin lymphomas (nhl) post 1st salvage treatment (salv1) is considered poor. the published data, have shown extremely low survival rates (15-20%) even after 2nd salvage treatment (salv2) followed by autologous stem cell transplantation (asct), due to the low response rates post salv2 and the high relapse rates post asct, confirming that the management of these pts remains a major challenge. we herein evaluated the dicep regimen [dose intesified cyclophoshamide (1750 gr/m 2 ), etoposide (350 mg/m 2 ) and cisplatin (35 mg/m 2 ), days 1-3] as a salv2 treatment, in terms of safety and efficacy regarding disease response and stem cell mobilization/collection. moreover, we evaluated pts' long term outcome post asct. we retrospectively analyzed the data of 27 (11 hl, 16 nhl) pts, with a median age of 32 (16-61) yrs. twenty-one had suboptimal response (75% reduction): 4 and minor response (≤50% reduction): 2). three pts had stable disease while 3 experienced progression. overall 23/27 pts underwent asct after a median of 44 days (range: 22-70) post dicep. no pt was considered ineligible for the asct due to unacceptable toxicity post dicep; 4 did not undergo asct because of progressive (n = 3) or stable (n = 1) disease. the 5-yr overall survival (os) was 70% for the whole cohort of pts (71% for hl and 66% for nhl, p = ns) while the 4-yr progression free survival (pfs) from dicep administration (± asct) was 62% (60% for hl and 64% for nhl p = ns). in particular, for the 23 autografted pts, the 5-yr os was 70% (80% for hl, 70% nhl p = ns) and the 4-yr pfs was similar, 70% (65% for hl, 72% for nhl, p = ns) our data demonstrate that dicep is an effective salvage regimen with acceptable toxicity and no negative impact on the cd34+ collection. the promising response rates post dicep in combination with the very encouraging pfs rates achieved post asct, in this unfavorable and heavily pretreated group of patients, strongly support the rationale for using dicep as 1st line salvage regimen in selected pts in order to proceed to a successful asct. for the treatment of aggressive lymphoma, high dose chemotherapy followed by autologous stem cell transplant (asct) is an important component. however, the role of upfront asct in patients with diffuse large b cell lymphoma (dlbcl) is still controversial. furthermore, there is currently no consensus on a single best conditioning regimen for asct in patients with dlbcl. we retrospectively analyzed the records of 184 patients with dlbcl who underwent upfront asct in state of complete remission (cr) or partial remission (pr) from 17 institutions in korea. we evaluated the outcomes and prognostic factors of upfront asct in patients with dlbcl. we compared the outcomes of most widely used two conditioning regimens for asct; carmustine based regimens and busulfan containing regimens. total 141 patients (81.0%) achieved cr after asct and overall response rate (orr) was 86.7%. with median follow up of 34 months, 65 patients (35.3%) had progression or relapse. the 3-year overall survival (os) rates and progression free survival (pfs) rates were 75% and 65%, respectively. infection events were found in 99 patients (54.5%) and treatment related mortality was 3.8%. these outcomes were comparable with the results of previous other studies. cox multivariate analysis for os showed that eastern cooperative oncology group performance status (ecog ps) ≥ 2 (p = 0.003) and rituximab based induction therapy (p = 0.062) were significant prognostic factors. in addition, the following factors were significantly associated with pfs in multivariate analysis; female (p = 0.031), ps ≥ 2 (p = 0.018) elevated β2-microglobulin (p = 0.008), failure to achieve cr with induction chemotherapy (p = 0.004), carmustine based conditioning regimen (p = 0.012) and melphalan based conditioning regimen (p = 0.031). there were no significant differences in os and pfs according to stage, b symptom, bulky disease, high lactate dehydrogenase, bone marrow involvement, high or high-intermediate international prognostic index (ipi), absolute lymphocyte count and absolute monocyte count. therefore, it is considered that upfront asct can overcome the poor prognosis in patients with advanced stage or high risk ipi. in the analysis with conditioning regimen, neutrophil and platelet engraftment were slower in the carmustine group compared to the busulfan group. there were no significant differences in os between busulfan group and carmustine groups with 3-year os rates of 74.4% and 77.9%, respectively (p = 0.797). pfs at 3years was 66.0% in busulfan group versus 59.9% in carmustine group (p = 0.241). however, carmustine based conditioning regimen was poor prognostic factors for pfs in multivariate [p631] s457 analysis (p = 0.004). in subgroup analysis, busulfan group had significantly higher pfs compared to the carmustine group especially in female patients (67.9 months vs. 7 months, p = 0.002), with b symptom (67.9 months vs. 7.4 months, p = 0.033) and abnormal serum ldh level (70.2 months vs. 25 .9 months, p = 0.045). the outcomes of upfront asct in patients with dlbcl after induction therapy were acceptable. it is considerable in selected high risk patients who achieve cr with induction treatment, and have good performance status at diagnosis. in cases of conditioning regimen, busulfan based regimen resulted in improved outcomes compared with carmustine based regimen especially in patients with disseminated disease or female patients. disclosure of conflict of interest: none. no heavy chain was present in 25%). the predominant light chain was kappa (60%). 80 patients had bence-jones positive myeloma. 109 received bortezomib as induction therapy before transplant. we analyzed overall survival (os) and progression-free survival (pfs) in 5 groups of patients. we separated the groups according to improvement in grade of response from preasct to postasct. the post-asct grade of response was measured 3 months after asct. the os and pfs were estimated by the kaplan-meier method. pfs was measured from diagnosis to disease relapse and os was measured from diagnosis to death by any cause. results by subgroups of patients are detailed in table 1 . median os and pfs of the whole group was 8 years and 54 months, respectively. if we analyze groups only by their grade of response before asct we find the following results: rc (5years os rate 78.9%, median pfs 95 months); pr/vgpr (median os 7.31 years and pfs 40 months); sd/progression (median os 6.46 years and pfs 21 months). according grade of response after asct, instead: rc (5 years os rate 78%, median pfs: 66 months); pr/vgpr (median os 7,31 years, and pfs 40 months); sd/progression (median os 1.05 years and pfs 11 months). in our experience, the grade of response before asct is a capital predicting factor for patients os and pfs. patient in cr before asct that preserve it after transplant, have a median pfs of 95 months, the 5 years os rate being 80.4%. patients in situation of progression after asct have a very dismal prognosis (median os 1.05 years, pfs: 11 months), however, patients who change from sd/progression to pr after asct have a median pfs of 29 months and a os of 6.4 years. comparing these results we observe that this second group is particularly benefited by transplant. autologous peripheral blood hematopoietic stem cell transplantation in elderly patients with multiple myeloma as a standard therapeutic procedure. is it feasible? a single-center experience l cadievski, s genadieva stavric, z stojanoski, a pivkova veljanovska, d miloska, b kocoski, l cevreska and b georgievski university clinic of hematology, department for hematopoietic stem cell transplantation, university ss. cyril and methodius, skoje, republic of macedonia autologous peripheral blood stem cell transplantation (pbsct) represents a standard therapeutic approach in the treatment protocol of myeloma patients. it is known that multiple myeloma is a hematological disease that is a characteristic for the older population. autologous pbsct ideally should be performed in every myeloma patient, but with the elderly myeloma patients the procedure might be risky if know the possible comorbidities, or the possibility of the body to fully compensate the side effects of the conditioning regimen, the procedure or its possible complications. we present our experience in using high dose conditioning with melphalan 200 mg/m 2 followed by autologous pbsct for elderly myeloma patients, using the age limit od 65 years. our retrospective analysis of our data during 16 years of experience, shows that we have performed autologous pbsct on 12 patients with myeloma at the age of 65 or older. 11 males (91.6%), and 1 female (8.4%). 6 patients (50%), were diagnosed with igg type myeloma, 5 patients (41%) with iga myeloma, and 1 patient (9%) with light chain myeloma. median age of the patients was 66.9 years (65-73). all patients were initially treated with cy-thal-dex regimen. in 5 (41%) patients complete response (cr) was achieved, in 5 (41%) very good partial response (vgpr), and in 2 (18%), partial response (pr). in all patients the mobilisation of hematopoietic stem cells was performed with g-csf, and a median of 2 apheresis procedures were performed, and the average number of collected cells was 3.11 × 10 8 /kg tt mononuclear cells (range: 6.0-2.1). days to confirmed engraftment in our group of patients was 11.5 (range: 9-15). the number of blood transfusions was on average 2.8 (range: 0-6), and the number of transfusion of thrombocytes 38.1 units (range: 10-74). in the majority of patients, mainly after the year 2012 (that represents 10 patients of the whole group), we used noncryopreserved hematopoietic stem cells, kept under the temperature of 40c, for median of 2 days, thus avoiding the toxicity of dmso. additionally, we used central venous catheter inserted in the femoral vein for apheresis and application of the stem cells afterwards. the day after, the catheter was removed, thus avoiding catheter associated infections. all patients received standard infectious prophylaxis with fluconasole 200 mg/daily, ciprofloxacin 500 mg/ two times daily, acyclovir 200mg/ three times daily, cefixime 400 mg/once daily, and ursodeoxycholic acid for vod prevention. no serious infectious complications were reported. our transplant related mortality was 0%. in the group with noncryopreserved stem cells no graft failure was reported. in two patients we even performed tandem autologous pbsct with no major complications. of the group of 12 patients, the majority, 8 patients (66%), had hta as comorbidity, 1 (8%) with cardiomyopathy, and 1 (8%) with inserted prosthetic aortic valves. three patients (24%) have died because of relapse of the disease. our oldest patents were 72 and 73 years old, and are still alive 1 year posttransplant, in cr. we can conclude the performing autologous pbsct in elderly myeloma patients can be safe and effective therapeutic option, but with careful selection of the patients, balancing the risk profile of the patient and the benefit, or the risk of the procedure. affective supportive care, monitoring and reducing the risk of complications is an imperative to a good result. disclosure of conflict of interest: none. autologous stem cell transplantation program for patients with multiple myeloma in an outpatient setting k lisenko 1 , s sauer 1 , g egerer 1 , j schmier 1 , m witzens-harig 1 , a schmitt 1 , ad ho 1 , h goldschmidt 1,2 , j hillengass 1 and p wuchter 1,3 1 department of medicine v, heidelberg university hospital, heidelberg, germany; 2 national center for tumor diseases heidelberg (nct), heidelberg university, heidelberg, germany and 3 institute of transfusion medicine and immunology, mannheim, german red cross blood service baden-württemberg-hessen, medical faculty mannheim, heidelberg university, germany the first and second authors contributed equally. high-dose chemotherapy with melphalan and autologous blood stem cell transplantation (absct) for treatment of symptomatic multiple myeloma (mm) is performed in the usa and canada mostly on an outpatient basis, whereas in germany and western europe an inpatient setting is the standard. we report on a german single-centre program to offer the procedure on an outpatient basis to selected patients. major inclusion and exclusion criteria for eligibility were defined as follows: patients had to be able to reach the hospital within 45 minutes, had reliable support from their family at home, had an ecog performance score of 0-1 and were willing and able to comply with the demands of the program. patients with severe co-morbidities were not included. all patients were treated on our outpatients' clinic and examined on daily visits by a team of physicians. feedback from patients was obtained by means of a questionnaire. from september 2012 to september 2016, 26 patients with mm stage iiia were enrolled. all engrafted within the expected time range: (median time to leukocyte 41,000 /μl and neutrophil recovery 4500 μ/l: 14 days; median time to platelet recovery 420/nl: 10 days, 450 /nl: 14 days). twenty patients (77%) had an episode of neutropenic fever but only in 5 patients (19%) blood cultures were found to be positive. there occurred no cases of infection with multiresistent bacteria. although rather liberal criteria for hospital admission were applied, 18 of 26 patients (69%) could be treated entirely on an outpatient basis. eight patients (31%) were temporarily admitted for inpatient treatment with a median duration of 4.5 days (range: 2-18 days), mainly because of neutropenic fever. no severe adverse events occurred. feedback from patients revealed a high level of satisfaction with the outpatient setting. high-dose chemotherapy and absct on an outpatient basis is safe and feasible if conducted in a comprehensive surveillance program. the feedback from patients was very positive, thus encouraging further continuation and expansion of the program. disclosure of conflict of interest: none. high dose of melphalan (bor-mel). we retrospectively analyzed 69 patients with mm who underwent asct between january 2014 and march 2016. in these patients, conditioning regimen consisted of a high dose of melphalan (140-200 mg/ m 2 ) intravenously on day -2 and two doses of intravenous bortezomib at 1.3 mg/m 2 administered on days − 1 and +2. this cohort was compared with patients underwent asct between 2003 and 2013, conditioned with high dose of melphalan alone. response rate was evaluated according to imwg criteria. all patients were evaluated after induction therapy and 3 months after asct. all patients were followed until death or reference date (november, 2016). results: patients' demographics and baseline disease-related characteristics are shown in table 1 . [p636] no difference was found in terms of neutrophil and platelet engrafment, hospitalization days (p=0.723) and use of mechanical invasive ventilation (p=0.415). bor-mel regimen did not enhance severity of preexisting peripheral neuropathy (pn) in any patients, and only one presented de novo grade 2 pn. non relapsed mortality was 1.4% and 1% in the bor-mel and mel cohorts, respectively (p=0.673). complete response rate after transplant was significantly better in the bor-mel cohort than in the mel cohort (65.2% vs. 40.7%; p=0.001) ( figure 1b) . when the analysis was restricted to patients who received bortezomib-based therapy, this difference was also statistically significant (64.1% vs. 44.4%; p=0.024) ( figure 1d ). median of follow-up was 12 months in the bor-mel vs. 42 months in the mel cohort. no difference was found in terms of overall survival (os) and progression free survival (pfs) between both groups. for all patients, a post-transplant deeper response was associated with better os and pfs (p=0.008 and p o0.001, respectively). our results are in line with previous studies demonstrating that bortezomib combined with melphalan is a well tolerated conditioning regimen and may enhance the response rate after transplant, even in patients receiving bortezomib in the induction therapy. these results should be confirmed in a randomized trial. for newly diagnosed patients (pts) with multiple myeloma (mm), the triple-agent induction treatment based on bortezomib plus dexamethasone in combination with cyclophosphamide (vcd) or lenalidomide (vrd) represent extremely reliable regimens, which in combination with early autologous stem cell transplantation (asct) result in high response rates and prolonged long-term outcomes. however, though both regimens are widely used, there are extremely limited studies that compare the vrd vs. vcd in terms of safety and efficacy. in the present study we compared the outcomes of 19 newly diagnosed mm pts who received induction treatment vrd (n = 10) or vcd (n = 9) and proceeded early to asct. the vrd and vcd pts groups were similar regarding age at diagnosis (52 vs.54 ys, p = ns), interval between diagnosis-asct (5,8 vs. 5,7 months, p = ns) and maintenance treatment post asct (8 vs. 6 pts, p = ns). per revised international scoring system (riss), the vrd-group had slightly more advanced disease (stage i: 1, stage ii: 7 and stage iii: 2), compared to vcd-group (stage i: 3, stage ii: 5 and stage iii: 1), however this difference was not statistical significant. the conditioning regimen consisted of single agent melphalan: 200mg/m 2 . the t-test, kaplan-meir and cox regression were utilized for the statistical analysis. following a median of 4 cycles of treatment (range: 4-6 for vrd vs. 2-6 for vcd, p = ns), in the vrd-group 4 pts achieved complete remission (cr), 5 pts very good partial remission (vgpr ≥ 75% reduction of m-band) and 1 pt partial remission (pr: 50-75% reduction of m-band) while in the vcd-group cr:3, vgpr:2 and pr:3 pts (p = ns). the toxicities in terms of peripheral neuropathy, myelosuppression, liver and renal function were well tolerated and no patient discontinued treatment due to severe side effects. the 5-yr overall survival (os) was 100% for the vrdgroup vs. 75% for the vcd-group; nevertheless, the difference was not significant due to the size sample of the pt groups. the stage at diagnosis, the disease status pre-asct and the maintenance post-asct did not influence the os. interestingly, the 4-yr progression free survival (pfs) was significantly superior for patients who had been induced with the vrd regimen (75% vs. 36% p = 0.05) and for patients who achieved cr or vgpr before asct (pfs: 50%) while no pts with pr pre-asct was progression-free 2 yrs post asct (p = 0.001). in multivariate analysis, only the cr or vgpr status before asct favorably affected the long term pfs. our results are in line with the limited published data from other studies with larger series of patients. in our study, very low disease burden before asct proven to be an independent factor for prolonged pfs. taking into consideration that vrd resulted in more cr or vgpr status, it is reasonable to conclude that vrd is a highly effective regimen and could be first treatment choice for newly diagnosed mm patients who are fit for early asct post induction. lenalidomide cohort and 64 in the maintenance bortezomib cohort. baseline characteristics and outcome data were obtained via chart review. the primary outcome was pfs. the secondary outcomes were overall survival (os) and treatment-related toxicities. the median follow-up time was 33 months. median time to death (4.28 years vs 5.77, p = 0.47) and median time to progression (1.71 years vs 1.74, p = 0.77) were not significantly different in the maintenance lenalidomide cohort compared to the maintenance bortezomib cohort. in the multivariate analysis, pfs was worse in patients at international staging system (iss) stage 3 at diagnosis compared to those at iss stages 1 and 2 (hr, 1.86; 95% ci, 1.11 to 3.12; p = 0.02) and worse in patients with less than very good partial response (vgpr) to last prior therapy compared to those with a response to prior therapy of at least vgpr (hr, 2.05; 95% ci, 1.14 to 3.69; p = 0.02) [see figure 1 ]. pfs was improved in patients with more than two years of maintenance therapy compared to those with less than two s464 years of maintenance therapy (hr, 0.40; 95% ci, 0.22-0.70; po0.01), but this result does not account for patients who ended maintenance therapy due to disease progression. os was worse in patients at iss stage 3 at diagnosis compared to those at iss stages 1 and 2 (hr, 3.87; 95% ci, 1.44 to 10.39; p = 0.01). peripheral neuropathy was more common in the bortezomib cohort (39% vs 9%, p o0.01), while cytopenias were more common in the lenalidomide cohort (30% vs 3%, po0.01). figure 1 kaplan-meier curve for pfs for the maintenance lenalidomide group versus the maintenance bortezomib group by log-rank test (p = 0.16). lenalidomide and bortezomib maintenance after transplantation have equal efficacy in prolonging progression-free and overall survival in patients with multiple myeloma. iss stage significantly affects time to progression and overall survival, and response to last prior therapy affects time to progression. length of maintenance therapy may be a significant predictor and warrants further analysis. these findings suggest that both lenalidomide and bortezomib are acceptable maintenance therapy options for post-transplantation multiple myeloma patients. autologous stem cells transplantation (auto-hct) is an accepted method in multiple myeloma (mm) patients, but usually it is not curative. the issue of allogeneic hematopoietic stem cells transplantation (allo-hct) is challenging yet for myeloma. we investigated allo-hct in mm and compared with auto-hct. in this retrospective study, we recruited 272 patients from january 2011 to january 2015 (218 (80.15%) patients in autologous group and 54 (19.85%) in allogeneic group). we performed allogeneic hct with peripheral blood stem cells source in our center for patients who are relatively young (less than 55 years old) with good performance, have match sibling donor and accepted allogeneic hct. the conditioning regimens in autologous group was melphalan 200 mg/m 2 only and in allogeneic groups was fludarabine 30 mg/m 2 plus melphalan 140 mg/m 2 in 5 consequent days. gvhd prophylaxis consisted of methotrexate and cyclosporine. the outcomes then compared between two groups using log-rank and gray tests and cox proportional hazard regression. the median follow-up in the autologous and allogeneic group was 17.02 months. three years disease-free survival of auto-hct was 38.61% (ci: 27.37%, 49.72%) and 68.88% (ci: 50.74%, 81.47%) for allo-hct patients (p value = 0.0363). three years overall survival of auto-hct was 77.26% (ci: 66.08%, 85.16%) and 82.15% (ci: 64.92%, 91.44%) for allo-hct patients (p value = 0.6363) showing no significant statistical difference between two groups. mortality rate was 11.01% for auto-hct and for allo-hct was 12.96%. the most common cause of death between two groups was relapse of primary disease. three year relapse incidence was 20.83% (ci: 9.04%, 35.30%) for allo-hct and 54.33% (ci: 42.02%, 65.09%) for auto-hct (gray's test p value = 0.018). the three year trm incidence was 10.36% (ci: 2.92%, 23.33%) and 7.01% (ci: 3.14%, 12.98%) in allogeneic and autologous patients respectively (gray's test p value = 0.42). despite there was no statistically significant difference between two groups in terms of os but dfs and relapse incidence was meaningfully better in allogeneic group. so, perhaps the reason of non-significant os improvement in allogeneic group is higher early death due to higher trm. we suggest that this study needs longer follow up to see whether allo-hct resulted in os improvement. disclosure of conflict of interest: none. myeloablative allogeneic hematopoietic stem cell transplantation from unrelated donors for patients with relapsed or refractory multiple myeloma n tsukada 1 , s shingaki, m ikeda, t ishida and k suzuki 1 division of hematology, japanese red cross medical center allogeneic hematopoietic stem cell transplantation (allo-sct) for patients with multiple myeloma (mm) is increasing in number despite in the era of novel agents, especially as a second line treatment and beyond. it has been reported that allo-sct for patients with mm resulted in high incidence of treatment related mortality (trm). high incidence of disease relapse is also a major problem especially after reducedintensity stem cell transplantation (rist). it is an important issue to reduce the incidence of trm while preventing disease relapse. the use of stem cells from unrelated donors is required for those without hla-matched sibling donors. the purpose of this study is to evaluate the feasibility of an intensified conditioning regimen incorporating both 140 mg/ m 2 of melphalan and 8 gy of total body irradiation (tbi), followed by allo-sct from unrelated donors for patients with relapsed or refractory mm. we retrospectively analyzed eight consecutive patients who received allo-sct from unrelated donors with the conditioning regimen including 8gy of tbi, fludarabine 25 mg/m 2 for five days, and melphalan 140 mg/m 2 between april 2013 and july 2015 at the japanese red cross medical center. six patients received unrelated bone marrow transplantation (bmt) and two patients received cord blood transplantation (cbt). graft-versus-host disease (gvhd) prophylaxis was consisted of tacrolimus and short term methotrexate. the median age at allo-sct, the time from diagnosis of myeloma to allo-sct, and the numbers of prior treatment lines were 48.5 years (range: 31-60 years), 38.5 months (range: 8-64 months), and 3.5 lines (range: 1-7 lines), respectively. five patients are female. no episode of either grade ≥ iii toxicity or non-relapsed mortality was documented during the median follow-up period of over two years. cumulative incidence of grade ≥ ii acute and severe chronic graftversus-host disease were 37.5% (95% confidence interval [ci] 7.2%-69.4%) at 100 days and 25.0% (95% ci 2.9%-58.1%) at 180 days, respectively. probabilities of progression-free survival and overall survival were 22.5% (95% ci 0.0%-58.7%) and 58.3% (95% ci 22.0%-94.7%), at 3 years, respectively. the results suggest that allo-sct conditioned with this intensified regimen may be tolerable for patients with relapsed or refractory mm. disclosure of conflict of interest: none. the role of allogeneic stem cell transplantation (allosct) in the era of novel myeloma drugs remains controversial. it is the only curative treatment option but non-relapse mortality makes the decision making difficult as opposed to achievements with autologous sct and new mm drugs by which the median survival is nowadays nearing 10 years. aim of this study was retrospectively evaluate the outcome of allosct for mm performed at our institute, including evaluation of factors affecting survival. all 66 consecutive patients allotransplanted for mm between 1986 and 2014 were included. the data were collected from our transplant registry. frequencies and medians were produced as appropriate. kaplan-meier method was used to calculate os and pfs and log rank test for comparisons. univariate analysis for factors affecting survival was performed with cox proportional hazard model. median age of all 66 patients was 55 (36-66) years. half of the patients had igg myeloma, 23% had iss score 3 (score available for 30 patients), and 33% had high-risk cytogenetics (data available for 39 patients). response to treatment at sct was at least vgpr in 80% of patients, and transplant timing was early (within 15 months from dg) in 58% of patients. sibling donors were used in 58% and muds in 42% of transplants, and conditioning was ma for 50% and ric for another 50% of patients. acgvhd grade 0-2 occurred in 78% and grade 3-4 in 22% of patients; 32% of patients had extensive chrgvhd. posttransplant cr rate was 83%. 45% of pattients have relapsed after allosct, and 42% are alive with the median follow-up of 5,6 years. non-relapse mortality has been 30% (35% until 2005, 27% since then). the median survival of patients up to age of 60 years is 4.9 years vs 3.0 years for patients 460 years (n = 8) with survival plateau after 6 years at 40% level. transplant period, cytogenetics, donor type, conditioning intensity or occurrence of chrgvhd had no statistical impact on survival. significant differences in os were observed between disease status at scr 4 vgpr vs o15 months from dg vs later), grade of acgvhd 0-2 vs 3-4, and best resonse post-transplant cr vs not less than cr. the respective differences for pfs were in sct timing, grade of chrgvhd, and best post-transplant response. in univariate cox regression analysis the only significant factors for os were severity of acgvhd and cr vs other responses after sct, and for pfs allosct timing, severity of chrgvhd, and best response to sct. with allosct ca. 40% of mm patients can be cured but at the cost of high non-relapse mortality. the occurrence of grade 3-4 acgvhd and less than cr response to sct predict poor survival. considering the increasing survival expectations with modern standard therapy for mm, allosct may be recommended for younger patients with high-risk features, and allosct should be done early in the disease course. disclosure of conflict of interest: none. angiogenesis plays an important role in the pathophysiology of hematological malignancies including plasma cell myeloma (pcm). microrna-21 (mir-21) is overexpressed and displays oncogenic activity in cancers. however, little is known about the role of mir-21 in pcm. the aim of the present study is to examine the expression level of peripheral mir-21 in pcm patients and to determine its role in angiogenesis. vegf serum levels and mir-21 in pbmcs was measured in 93 patients with pcm directly before melphalan 200 mg/m 2 followed by autologous hematopoietic stem cell transplantation (auto-hsct) and 2 months after hsct; and 35 healthy controls. the study population was divided into two groups after therapy: responders (stringent complete response, complete response, very good partial response, partial response) and nonresponders (stable disease, progressive disease). gene expression of mir-21 was quantified by sybr green real-time fluorescent quantitative pcr. further tube formation of huvecs and vegf secretion was measured in mir-21 mimic or inhibitor transfected human plasma cell myeloma cell lines h929 and rpmi-8226. the expression level of mir-21 was significantly increased (2.7 ± 0.55 versus 0.78 ± 0.22; p o0.01) in pbmcs of pcm patients compared with healthy controls. further, serum vegf levels were increased in pcm patients (477 ± 145 pg/ml versus 178 ± 78 pg/ml in normal controls; p o0.01). after auto-hsct, the expression level of mir-21 was significantly different in responders compared to nonresponders. responders had a lower expression of mir-21 compared to non-responders. further, serum vegf levels decreased in responders to auto-hsct compared to nonresponders. vegf expression was increased in the supernatant from mir-21 mimic transfected human pcm cell lines h929 and rpmi-8226 compared with the negative control, while s467 vegf was decreased in the mir-21 inhibitor transfected cell lines. the angiogenic ability of huvecs was increased under pretreatment with the supernatant from h929 and rpmi-8226 cells transfected with mir-21 mimic compared with negative controls and decreased when pretreated with mir-21 inhibitor transfected cells (fig. 1) . this study demonstrated that mir-21 was upregulated in pcm patients. responders to auto-hsct had a decrease of mir-21 expression and vegf levels. further, mir-21 regulated angiogenesis. therefore inactivation of mir-21 or activation of its target gene may be a potential therapeutic approach in pcm. fig. 1 : in vitro matrigel tube formation assay. (i), normal control (ii, iii), mir-21 mimic transfected h929 cells (iv), mir-21 mimic transfected rpmi-8226 cells (v), mir-21 inhibitor transfected h929 cells. original magnification × 100. disclosure of conflict of interest: none. [p647] pilot study of busulfan/thiotepa as conditioning regimen followed by allografting and post transplantation cyclophosphamide in advanced relapsed myeloma patients c wolschke, e klyuchnikov, d janson, m heinzelmann, m christopeit, f ayuk and n kröger university medical center hamburg-eppendorf despite the significant improvement in outcomes has been observed for myeloma patients, the disease still remains incurable. due to limitations, such as trm and gvhd, the role of allogeneic stem cell transplantation as salvage therapy in this setting remains unclear. in present pilot study we provide data on the use of post cyclophosphamide (ptcy) as gvhd prophylaxis after a busulfan/thiotepa based conditioning regimen in patients who relapsed after autologous stem cell transplantation. between 11/2014 and 08/16 17 myeloma patients (male n = 10, female n = 7) with a median age of 55 years (range: 45-66) (pts), who relapsed after autologous stem cell transplantation received allogeneic stem transplantation with with ptcy as gvhd prophylaxis after busulfan (9.6 mg/kg for age 460y and 6.4 mg/kg for age 460years) and thiotepa (10 mg/ m 2 )and for haploidentical and mmud additional fludarabin (90 mg/m 2 ). all pts. were relapsed after one or two autologous stem cell transplantations. donors were haploidentical (n = 1), mmud (n = 4), mud (n = 6) and hla-identical sibling (n = 6). stem cell source was pbsc (n = 15) or bm (n = 2). all patients received cyclophosphamide 50 mg/kg of body weight on day +3 and +4, which was in 10 pts (n = 5 mrd, n = 5 mud) the only gvhd prophylaxis, while patients with mmud and haploidentical donor received also cyclosporine a from day +5 and mmf (until day 35) and 2 patients (mrd and mud) received additional cyclosporine. we observed no primary or secondary graft failure. the median time for neutrophil and platelet engraftment was 19 (range: 15-24) and 51 days (range: 22-279), respectively. major toxicities grade 3 and 4 were: renal (n = 1) and mucositis (n = 1). major infectious complications were: cmv: n = 10 cmv-reactivations (n = 10), sepsis (n = 5), pneumonia (n = 3) rsv-(n = 2) and hsv (n = 1). acute gvhd grade ii to iv and ii/iv was noted in 29% and 24%, respectively and mainly seen in patients with cyclophosphamide as single gvhd prophylaxis. remission rate were n = 8 complete remission, n = 7 vgpr, n = 1 partial remission, n = 1 n.a. after a median follow up of 12 months 3 pts progressed and 7 patients (n = 1 relapse, n = 6 trm) died. the 1 year pfs was 18% (n = 3). busulfan/thiotepa is an active conditioning regimen for advanced relapsed myeloma patients. post cyclophosphamide might increase anti-myeloma activity, but as single gvhd prophylaxis it causes significant agvhd in mrd and mud and additional immunosuppressive agents such as cyclosporine should be added. disclosure of conflict of interest: none. magnetic resonance imaging (mri) for multiple myeloma (mm) is a sensitive, non-invasive and non-toxic method for detecting myeloma lesions. the goal of the study was to assess whether quantitative mri metrics can detect treatment response and replacement of neoplastic cells by fat marrow. the study was hipaa-compliant and irb-approved. we retrospectively identified all patients who achieved a complete response (cr) after induction therapy between 2000 and 2014. inclusion criteria for the study was total spine mri imaging at diagnosis and after achieving cr. cr was determined using the imwg criteria. spinal vertebrae t12 through l5 were outlined with imagej software. fractures and lesions were excluded. images were analyzed using histogram-based (entropy, skewness, kurtosis) and texture-based statistics. a two-sided t-test was used to compare quantitative mri metrics from before therapy and after achieving cr. cox regression was used to explore the association between progression free survival (pfs) and change in each quantitative mri metric based on a median split. pfs was defined as the time from the second mri to death or progression of disease. nineteen patients met the above criteria. median age was 61.5 years (range: 37.5-72.2). majority of patients (68%) were male. majority of patients had iss stage 1 disease (57.9%) and standard-risk cytogenetics (89.5%). an induction regimen containing an imid and/or a proteasome inhibitor was commonly used (73.7%). all patients received an autologous stem cell transplant (asct) consisting of high dose melphalan followed by autologous stem cell rescue. three patients received a planned second asct. seven patients (36.8%) were in cr before asct. nine patients (47.4%) were treated with imid maintenance after planned initial therapy. median time to repeat mri imaging after cr was 10 months (range: 4.4-19.8). mean change in measurements of kurtosis, skewness, entropy and 6 texture analyses are shown in table 1 . no significant change was detected between preand post-cr mri. furthermore, no significant association was seen between the change in any quantitative metric and pfs. [p649] despite promising results by other groups, we could not find a significant association between quantitative t1 image analysis and cr or pfs. there was heterogeneity in the time of repeat mri imaging which may have limited our ability to study interval change. although no definitive conclusions can be made from this small sample, correlation between pfs and kurtosis or texture d may be promising and should be investigated in a larger group prospectively. multiple myeloma (mm) treatment (tx) has evolved in recent years. solid data on the impact of new tx on patient (pt) outcomes outside clinical trials, however, is lacking. this study aimed at investigating tx practices, pt journeys, and outcomes in the real-world in countries with different access to new tx. the study was conducted between 04/2015 and 06/2016 in bulgaria, croatia, czech republic, poland, romania, and slovakia. it consisted of a cross-sectional (x) and a retrospective (r) phase. for the x-phase, investigators included all symptomatic mm pts seen during a 3-week counting phase to provide a snapshot of where in the pathway pts were at a given moment. for r-phase, investigators collected data on current and past tx, including symptoms, dosages, administration schedule, tx durations, tx interruption, reasons for change/discontinuation, and tx response. pts were selected in reverse chronological order with a quota of a maximum of 3 pts who completed first-line (1l) tx within the past 3 months (mo), 4 pts in second-line (2l) and 7 pts in third or higher lines (3+l). pts included in the x-phase could also be included in the r-phase, if they met the respective inclusion criteria. in total, 39 physicians included 522 pts in the x-and 35 physicians included 277 pts in the r-phase. in the x-phase, 52% of pts were o 65, 36% were 65-75, and 12% were 475 years; the median time since diagnosis was 27 mo. 57% of pts were currently undergoing tx, 41% were previously treated and 2% had never been treated. of currently-treated pts, 37% received 1l, 30% 2l, 19% 3l and 14% 4+l. in the r-phase, 47% of pts were o65 years. of pts receiving 1l, 59% continued to 2l, 33% to 3l, 15% to 4l and 8% to 5l. of the 38% of pts eligible for stem cell transplantation (sct), 55% ( = 21% of all pts) received sct at 1l; these proportions were similar across countries. the most frequently-used regimens in 1l and 2l were bortezomib-based (57% and 53%, respectively), in 3l and 4+l lenalidomide-based (47% and 35%, respectively). median duration of 1l was 6 mo, followed by a median disease-free interval (dfi) of 4 mo. median dfi was longer in pts with sct than in those without (6.5 mo vs 1.5 mo). time to progression (ttp) decreased with later tx lines, from median 9 mo at 1l to 4 mo at 3l. depth of response, as assessed by the treating physician, decreased with each additional line of tx: 50% of pts achieved at least very good partial response (≥ vgpr) in 1l, while only 25% achieved ≥ vgpr at 3+l. ttp was longer in pts with better response levels: in 1l, median ttp for pts with ≥ vgpr was 22 mo versus 6 mo for pts with 6 mo for pts with ovgpr. the most common ( ≥20%, all grades) adverse events (aes) and co-morbidities in 1l were anemia (42%), thrombocytopenia (29%), neutropenia (25%), neuropathy (25%), and fatigue (22%). these aes disrupted treatment in 57% in 1l, 41% in 2l and 55% in 3+l. the study found that of sct eligible pts, only slightly more than half were transplanted. poorer outcomes and increasing ae incidence with each tx line highlight the challenges of mm tx. information on real-world pt management may be valuable for physicians to plan their tx strategies and can provide input for health economic evaluations of existing and new tx. disclosure of conflict of interest: daniel coriu declares to have received consulting fees or other remuneration (payment) from novartis, amgen, pfizer, takeda, janssen. ivan spicka declares to have received research grants from celgene, consulting fees or other remuneration (payment) from bms, takeda, celgene, janssen-cilag, and amgen, and to be a member of the speakers bureau of celgene, janssen-cilag, amgen, and bms. zdenka stefanikova declares to have received consulting fees or other remuneration (payment) from amgen, celgene, and takeda and be a member of the speakers bureau of amgen, celgene, and takeda. daniela niepel, krisztian szabolcs toka, and paul schoen are amgen employees and hold amgen stock. dominik dytfeld, and georgi mihaylov have nothing to declare. safety and efficacy of autologous stem cell transplantation in elderly patients with multiple myeloma t maia 1 , c marini 1 , p medeiros 1 , e aguiar 1 , j cancela pires 1 , r bergantim 1 , f trigo 1 and je guimarães 1,2 1 hematology department, centro hospitalar de são joão and 2 faculty of medicine, university of porto autologous stem cell transplantation (asct) is considered standard treatment for multiple myeloma (mm) patients under the age of 65 years, but its safety and efficacy still uncertain for patients over this age. retrospective analysis from one single centre concerning mm patients under, equal or over 65 years who underwent asct between january/2010 and july/2016. it was also compared to 65-70 years old mm patients diagnosed in this period of time who were not transplanted. we analysed a total of 160 patients, 135 of which underwent asct. onehundred-and-six of the transplanted patients were aged 65 years or less (median 56, iqr 10 years), 29 patients were aged more than 65 years (median 67, iqr 2 years) and 25 patients were non transplanted (median 68, iqr 4). the conditioning regimen for younger patients who underwent asct consisted mainly of melphalan 200mg/m 2 (mel200) while half of the elder patients received melphalan 140mg/m 2 (mel140). regarding transplant-related myelotoxicity there were no statistical differences between patients aged 65 years or less and over 65 years old, however the first group needed less days of g-csf (p = 0.04). non-hematopoietic toxicity measured by infections and mucositis was not influenced by age. patients 465 years conditioned with mel200 had more days of aplasia (p = 0.05), greater need of g-csf (p = 0.01) and transfusional support (p = 0.04) than patients ≤ 65 years. there were no differences on non-hematopoietic toxicity. in the elderly group, patients conditioned with mel200 presented more aplasia days (po0.01), higher grade of mucositis (p = 0.03) and more days of iv antibiotics (p = 0.02) than those transplanted with reduced dose of melphalan. comorbidities had no effect on transplant-related toxicity, either by age or by dose of melphalan. days of hospitalization and post-transplant complications did not differ according to age group. transplant related mortality was 2% at day 100 posttransplant. survival after transplant in patients 65 years old or under vs older patients (median follow-up time, 30 months), was not influenced by age (os, 83mo vs 59mo, p = 0.17; pfs, 38mo vs 37mo, p = 0.59). regarding the non-transplanted elderly group, these are patients with more renal disease (p = 0.02) and poorer performance status (p = 0.04) than the transplanted cohort. there is also higher cytogenetic risk (p = 0.01). induction regimens were similar in transplanted group and non-transplanted group 465 years old, and response to first line therapy (before asct of transplanted group) revealed no differences. infections were the most common complication in both groups. transplanted patients needed less days of hospitalization (p = 0.04). comparing the long term outcome of these two groups, survival curves of the elderly patients transplanted were clearly superior to the nontransplanted (os, 62mo vs 21mo, p o0.01; pfs, 45mo vs 20mo, p o0.01) although one has to consider that the non-transplant group has worse features than the elderly transplant group. transplantation in the elderly still debatable but this study shows that it might bring benefit. globally, transplant related toxicity is not influenced by age. regarding dose of melphalan, higher dose in elderly patients has higher toxicity, without apparent benefit in survival. therefore, age should not restrict the access to asct, but instead selection must be based on individual clinical and functional status. disclosure of conflict of interest: none. second autologous stem cell transplantation for relapse after allografting in multiple myeloma using cd 34+ selected donor cells without immunosuppression p novak 1 number of patients receiving a second allogeneic stem cell transplantation (sct) in europe is increasing despite high treatment related mortality (trm). in multiple myeloma only very few reports of second allogeneic sct exist with limited number of patients and substantial mortality. while in most hematological malignancies, the donor cell chimerism is dropping down if patients are relapsing, in myeloma donor cell chimerism remains complete despite relapse. to reduce trm we thought that full donor cell chimerism may allow us to perform a second high dose busulfan based chemotherapy followed by "autologous transplantation" after stem cell mobilization and collection. however, because two consecutive patients failed to collect sufficient cd34+ cells for an autologous transplantation even with plerixafor, we used donor t cell depleted cd34+ selected cells and transplanted those patients in an "autologous" fashion without any immunosuppression. to enhance graft-versus-myeloma effect, we added donor lymphocyte infusion (dli) at day 100. we report here on 11 myeloma patients with a median age of 58 years (range: 48-68) who relapsed after allogeneic sct and underwent a second "autologous" sct with cd34+ selected donor cells. all patients had received one (n = 8) or two (n = 3) autologous sct before 1. allografting. 6 patients received an upfront auto-allo protocol and 5 patients received 1. allogeneic sct as a salvage therapy. 73% of patients received a reduced intensity melphalan based conditioning regimen for 1. allogeneic sct and the median pfs was 39 months (range: 22-56). before 2. allograft patients had received overall a median of 5 (range: 3-7) treatment lines. at the time of 2. allogeneic sct all patients had a full or nearly full donor cell chimerism and remission status was very good partial remission (n = 1), partial remission (n = 5), stable disease (n = 4), progressive disease (n = 1). 82% of patients received a myeloablative busulfan based conditioning regimen and all received cd 34+ selected stem cells with a median number of 5.3 × 10 6 /kg cd34+ cells (range: 1.4-7.5) and 5 × 10 3 /kg cd3+ cells (range: 1.6-6). engraftment was noted in 100% at a median of 10 days (range: 9-14). no further graft-versus-host disease (gvhd) prophylaxis was performed and no acute gvhd (agvhd) was observed. according to treatment plan, 9 patients received escalating dli around day +100, starting with a median dose of 2 × 10 6 /kg (range: 0.5-5) in combination with lenalidomide maintenance in 6 patients. 4 patients experienced agvhd ii-iv after dli. two patients had a severe gvhd (grade iii) which resolved completely after steroid therapy. no nonrelapse mortality after sct and dli was observed. after a median follow up of 43 months (range: 6-49) the median pfs was 16 months (range: 8-24) which translates into a pfs for all patients of 61% at 1 year and 13% at 2 years. median os was 31 months (range: 13-48) and an os of 69% at 2 years and 27% at 3 years was observed. for patients with advanced multiple myeloma relapsing after allografting, a second "autologous" sct with cd34+ selected donor cells without immunosuppression followed by dli is an encouraging treatment option with low toxicity. disclosure of conflict of interest: none. second autologous stem cell transplantation as treatment option for relapsed patients with multiple myeloma: a single center experience (cic 859) p ganeva, y petrov 1 , m mincheff 2 , i tonev 3 , m guenova, l gartcheva 4 , a michova 5 , g arnaudov 6 and g mihaylov 7 1 ya. petrov; 2 m. mincheff; 3 i. tonev; 4 l. garcheva; 5 a. michova; 6 g. arnaudov and 7 g. mihaylov the use of modern therapies such as bortezomib, lenalidomide, thalidomide coupled with upfront high-dose therapy and autologous stem cell transplantation (asct) has resulted in improved survival in patients with newly diagnosed multiple myeloma (mm). the role of second asct as salvage therapy for relapse is unclear because of the availability of new agents to treat progression in multiple myeloma (mm). as the treatment options for management of patients with relapsed mm has become increasingly complex, physicians must consider both disease-and patient-related factors when choosing the appropriate therapeutic approach, with the goal of improving efficacy while minimizing toxicity. we retrospectively reviewed all mm patients who received a second asct as salvage therapy at our center from 2009 to december 2015. for this period we performed 211 transplants for mm patients. twenty five (11.8%) patients received second asct (18 patients were relapsed) and for 7 patients asct was performed as tandem transplant. we analyzed only second asct for relapse. the median time to relapse after first transplant was 18.8 months (range: 8-50 months). all patients received reinduction therapy before the second asct. conditioning was performed with melphalan with two different doses (200 mg/m 2 and 140 mg/m 2 ). the median age at second transplant was 51.8 years (range: 40-67 years), and female/man ratio was 4/14. median interval between first and second asct was 28.3 months (range: 12-60 months). we have no observed early deaths. until now 8 (45%) patients are dead because of progression disease. response rate was assessed three months after asct, nine (50%) patients achieved vgpr, three (16.6%) patients achieved at least a partial response, three (16.6%) had sd and three (16.6%) progressed despite salvage asct.median overall survival (os) was 35.6 months ( relapse ≥ 24months = 47.7; ≤ 24 months = 20). second asct is a feasible and safe option for salvage therapy in mm, especially in bulgaria where the possibility of using novel agents such as carfilzomib, lenalidomide, daratumomab for relapsed patients is limited to clinical trials, because of no reimbursement. the best results were observed in patients whose time to progression was more than 24 months after first asct. advances in treatment of multiple myeloma (mm) has improved overall survival in these patients (pts). a steady increase in the risk of secondary malignancies has been reported over the last decades in mm survivors. estimated incidence of secondary acute myelogenous leukemia or myelodysplastic syndrome (t-mds/aml) after treatment with alkylating agents is 1%-1.5% per year 2-10 years after primary chemotherapy. no specific risk factor has been recognized, but genetic instability, natural history of the disease as well as induction therapy and autologous stem cell transplantation (hct) have been implicated. recently, novel anti-myeloma treatments have been linked with an increase in secondary malignancies, but no solid relationship has been established yet. in a retrospective study, we analyzed the incidence of secondary malignancies (t-aml/mds and solid tumors) in patients suffering from mm who had undergone autologous hct using high-dose melphalan conditioning regimen in our bmt unit. study population consisted of 192 consecutive pts with median age of 55 years (29-70), 56.5% of them being male, who were transplanted during a period of 28 years . type of myeloma was igg/a/d in 56%, 18.8% and 0.5% respectively, while 17.2% was light chain and 7% nonsecretory. the majority of pts presented with k light chain myeloma (62.8%). there was almost equal distribution between iss stages i and ii (45%/38.5%) and only 16.6% were diagnosed with advanced stage myeloma. most pts received two lines of chemotherapy (60%) and all of them more than one. treatment regimens before autologous hct included vad (63pts), bortezomib-based (133pts), dcep (8pts) and rd (29pts) and 34 pts received radiotherapy. chemotherapy administration for mobilization was used in 18 pts (9.3%). conditioning regimen before autologous hct consisted of high-dose melphalan (200 mg/m 2 ) and in case of renal insufficiency 140 mg/m 2 . incidence of a secondary malignancy was 5.7% after a median follow up period of 46 months. t-aml /mds was diagnosed in 9 (4.68%) pts and 2 (1.02%) were diagnosed with breast and lung cancer respectively. pts diagnosed with secondary malignancy were previously exposed in induction therapy to melphalan (6), vad (3), bortezomib (3), high-dose cyclophosphamide as mobilization treatment (4) and radiotherapy (4) . cytogenetic analysis was available in 6 patients diagnosed with t-mds/aml and the majority (4/6) presented complex karyotype. abnormalities mainly observed were deletions and insertions in chromosomes 5,7,17. pts with secondary malignancies had an overall survival of 68 months (26-178), however, after malignancy diagnosis, survival was very poor, four months only . secondary malignancies in pts with multiple myeloma after autologous hct occur with a substantial frequency and have a dismal prognosis. the role of novel treatment agents has to be elucidated. further studies are needed to identify new risk factors and develop better surveillance strategies. [p654] disclosure of conflict of interest: none. survival analysis after allogeneic hematopoietic stem cell transplantation in patients diagnosed with multiple myeloma: a single center experience p patricia hernandez* 1 , r maria calbacho 2 , a laura posada 3 , g fabio augusto ruiz 2 , r anabelle chinea 2 1 hospital universitario nuestra señora de candelaria, santa cruz de tenerife (spain); 2 hospital universitario ramón y cajal, madrid (spain) and 3 hospital universitario cruces, barakaldo (spain) allogeneic hematopoietic stem cell transplantation (allohsct) may provide long term remission cures for patients diagnosed with high-risk multiple myeloma. however, its use is limited since it has a high rate of treatment-related mortality (trm), and because its efficacy compared to autologous hsct is not fully established. we studied 16 patients that underwent allohsct between 2002-2015. population characteristics are in table 1 . all patients were treated at least with one prior therapy lines (1) (2) (3) (4) (5) , all including autohsct (43.75% underwent 2 prior autohsct). 83% had 2 or 3 prior therapy lines. 11 of them received bortezomib as part of treatment regimens. donor characteristics: 2 non-related; 14 hla-identical. gvhd prophylaxis: methotrexate plus a calcineurine-inhibitor: 12 cyclosporine and 4 tacrolimus. median follow-up 15.5 months (1.3-174.5), average was 35.9 months. seven patients died (43.75%); 2 because of progression (12.5%), and 5 (31.25%) due to trm, including infections and immediate complications of transplantation, such as toxicities, icu admission and agvhd: infections: 7 cmv reactivations, 3 invasive fungal and 7 bacterial infections. disease status: 6 patients were in cr prior to allohsct. 3 of them maintained it after. remaining patients died before disease was evaluated. seven patients were in pr prior to transplant, and 4 reached cr after allohsct. one had progressive disease and reached cr after the procedure. two had stable disease and progressed after allo; one of them is in cr after additional therapy lines, and the other one died 4 months after due to it. donor characteristics: hla-identical sibling donors: 87.5% (1 hla-mismatch, passed away 2.7 months after allo due to trm). one of the nonrelated donors, had an hla-mismatch, and died 4 months after allohsct due to trm, the other one is alive after 21 months. gvhd: 10 (62.5%) developed agvhd and 3 of them maintained it chronically. two suffer from cgvhd, plus 3 that initiated it as agvhd. 9 were refractory to steroids. longterm survivors: 3 patients had overcome three years after allohsct. they were among 39 and 50 years old at the time transplant was performed. none of them received bortezomib as part of therapy protocols for the disease. all had 2 therapy lines prior allograft. 2 were submitted to 2 prior autologous hsct. relapse: 4 patients relapsed after allohsct (25%, median time to relapse 6.2 months), being alive 50% at the end of the study. allogeneic hsct is associated with a high incidence of nrm and a low incidence of relapse. rates of acute and chronic gvhd are high. in our cohort, besides that more than 50% are alive until now, they suffer from extensive chronic gvhd and are in need of treatment. long-term survival may be related with patient factors such as young age, but also low tumor burden, or less prior therapy lines; in our group there are no differences in this aspect. studies including high-risk abnormal cytogenetics should help to define which patients are best candidates to allohsct. high-dose melphalan followed by autologous haematopoietic cell transplantation (ahct) remains the standard of care for eligible multiple myeloma (mm) patients. the majority of patients in clinical practice and trials receive a melphalan dose of 200 mg/m 2 (mel200), but a reduced dose of 140 mg/m 2 (mel140) is often used in patients perceived to be unable to tolerate mel200. it remains to be determined whether this considerable dose difference results in different clinical outcomes. we therefore analysed 1978 patients with mm who underwent a first single mel140 or mel200-conditioned ahct between january 2008 and december 2012. all patients were included in the calm study, an analysis of a prospectively defined cohort of patients with data reported retrospectively to the ebmt, covering ahcts for mm and lymphoma. patients in the mel140 group were older than patients in the mel200 group at the time of ahct (median 64 years [range: 28-73] vs 59 years ; p o.001). compared to the mel200 group (n = 1733, 87.6%), fewer patients in the mel140 group (n = 245, 12.4%) were overweight or obese (49.5% vs 63.9%; p = .003). compared to the mel200 group, more patients in the mel140 group had received proteasome inhibitor-containing induction therapy (71.7% vs 57.5%; p = .001), had a karnofsky score of ≤ 80 (38.2% vs 28.1%; p = .002), and were transplanted in less than pr (13.0% vs 7.8%; p = .025). overall survival (os) from the time of ahct was not significantly different between the mel140 and mel200 group (6significantly different between mel140 and mel200 (12 days in both groups for neutrophil recovery; 16 vs 15 days for platelet recovery). however, late neutrophil recovery was noted in a small proportion of patients in the mel200 group. neutrophil recovery 421 days post ahct was not observed in any engrafting patient in the mel140 group, but occurred in 37 (2.2%) engrafting patients in the mel200 group (p = .011). a cox proportional hazards model that included melphalan dose, age, and remission status at ahct showed that melphalan dose had no effect on os, pfs and relapse risk. the findings suggest that mel140 is not inferior to mel200 in younger and older mm patients and may reduce the risk of delayed haematological recovery in some patients. further analyses in relevant subgroups such as patients with high-risk features or renal impairment are required. disclosure of conflict of interest: none. high-dose therapy (hdt) followed by autologous stem cell transplantation (asct) remains the standard of care for patients younger than 65 years of age with multiple myeloma (mm). different agents are being used to control the disease before asct, including the older thalidomide based combination or the newer bortezomib and lenalidomide based combination. the relation between the initial induction regimen and outcomes after asct is not completely clear. to evaluate the effect of different induction regimens on asct outcome, we retropsectively evaluated the outcomes of a low cost older regimen of thalidomide based combination in doublets or triplets with newer novel agents like bortizomib or lenalidomide based combination in a low resources country in transplant-elegible patients with multiple myeloma who underwent autologous stem cell transplantation at king hussein cancer center bmt program we retrospectively reviewed the files of patients diagnosed with mm from january 2008 till december 2015, who received induction treatment followed by hdt and asct and followed up in a single institution. we compared the effects of different induction regimens, disease stage, and remission status before transplantation on over-all survival (os), event free survival (efs) and progression free survival (pfs) using kaplan meier curves. a total of 94 patients were included, 54 (57.4%) of them received thalidomide based induction (group 1) and 40 (42.6%) received bortezomib and lenalidomide based induction (group 2). patients also offered no consolidation nor maintenance therapy. 35 (37.2%) patients were stage i, 34 (36.2%) stage ii and 15 (16%) were stage iii. stage was not documented for 10 (10,6%) of cases. 58 (61.7%) were in complete remission (cr) and 36 (38.3%) were in partial remission (pr). the estimated 5-year os for the whole group was 57.7%. there was no statistically significant difference between both groups in regards to initial iss stage of disease (p = 0.332) or cr status before asct. 32 patients (59.3%) in group 1 achieved complete remission ( cr ) or very good partial response (vgpr), while 25 (62.5%) patient in group 2 achieved cr or vgpr. there was no statistically significant difference between group 1 and group 2 in 5-years os ( 5-year os was 60% vs 57%, p = 0.5007), efs (39.6% vs 52.6%, p = 0.8029) or pfs (45.2% vs 57.8, p = 0.8033). the use of an old, low-cost, thalidomide-based regimen in a low-resources country achieved a favorable transplantation outcomes in patients with multiple myeloma who received hdt and asct. double autologous stem cell transplantation (asct) is a useful treatment for multiple myeloma (mm) patients. we can make the second asct (2asct) without reinduction treatment (tandem regimen) or after a reinduction treatment after first asct (1asct) relapse (salvage regimen). we have conducted a retrospective study over 61 mm patients undergoing a double asct performed in our centre from 1996 to 2016. we have compared the different conditioning regimens used, and if there are any difference between tandem or salvage asct. we do not use maintenance treatment systematically. characteristics of patients and conditioning regimens in table 1 . the overall survivals (os) of our patients are 139 months (m) from treatment start till last control. the most important prognostic factors are the duration of the progression free survival (pfs) after 1asct (hr: 0.96 (0.94-0.99); p = 0.006), and the use of bumel like conditioning regimen at the 1asct or at the 2asct vs another conditioning regimens (hr: 3.43 (1.4-8.39); p = 0.007). today there are 27 patients alive (43%), but only 10 (37%) are free of mm now. the 25 patients who were treated with tandem have a little better os than salvage patients (166m vs 103 m; p = 0.55. not significative). patients at tandem group who received different conditioning regimen at the 1asct and at the 2asct live more time than patients treated only with melphalan 200 (mel200) at both asct. at salvage group the duration of pfs after 1asct is better than the pfs after 2asct (28 m vs 13 m). the use of the same conditioning regimen at the both asct has worse results than if we use different treatment. patients who were treated with s474 bumel at the 1asct or 2asct have better os than patients treated with cbv or mel200. patients who not responded to reinduction treatment before 2asct have worst pfs after 2asct (rc:29 m, response; 19m and not response; only 10 m). attention is drawn to the fact that patients who received bumel at 1asct have large os, but they are very few (8) patients. only one patient has died during the 2asct, and was a patient of salvage group treated with bumel. double autologous transplantation continues to be a useful treatment despite the new mm treatments, and allows to prolonged the os. tandem asct probably is a useful treatment in high risk mm patients. salvage treatment is most useful in patients with a large pfs after 1asct, and good response to reinduction treatment. although mel200 continue to be the standard conditioning regimen for asct in mm patients, we have observed that patients treated with different conditioning regimen at 1asct and 2asct have better prognostic, and bumel has the best results in our serie. disclosure of conflict of interest: none. allogeneic haematopoietic stem cell transplantation (allo-hsct) is an effective treatment for myelodysplastic syndrome (mds) and acute myeloid leukemia (aml). the prognosis of elderly patients with mds and aml after chemotherapy is poor. allo-hsct is feasible in these patients; however the management of elderly patients with mds and aml for allo-hsct is difficult. we performed a retrospective survey of allo-hsct for elderly patients with mds and aml in our institution. we retrospectively analyzed the records of elderly patients ≥ 60 years with mds and aml who underwent allo-hsct in our hospital between january 2011 and december 2015. in this study, we assessed the ipss-r (revised international prognosis scoring system) cytogenetic score and the ipss-r score against the outcome of elderly mds and aml patients who treated with allo-hsct. fifty-one elderly patients with mds and aml were treated with allo-hsct in our institution, 47 patients with mds (29 with mds overt aml) and 4 with de novo aml. ages ranged from 60 to 71 years (median 64), 18 patients were female and 33 were male. there was a history of malignant disease in 14 patients. according to the ipss-r cytogenetic scores of mds patients, 10 patients fell in the good risk group, 7 were in the intermediate risk group, 7 were in the poor risk group, and 23 were in the very poor risk group. regarding the ipss-r score, 1 patient fell in the low risk group, 5 in the intermediate risk group, 6 in the high risk group, and 35 in the very high risk group. sixteen patients were in 1st complete remission (cr), 1 patient was in 2nd cr, 9 patients were in partial remission, and 25 patients were not in remission (nr) upon administration of allo-hsct. all patients received a reduced intensity conditioning regimen. 45 patients [p658] were treated with fludarabine (flu), melphalan and low dose tbi-containing regimens; 5 patients were treated with flu, intravenous busulfan and low dose tbi; and one patient was treated with flu, cyclophosphamide and low dose tli. graftversus-host disease (gvhd) prophylaxis consisted of tacrolimus plus methotrexate in 46 patients, and tacrolimus, methotrexate and mycophenolate mofetil in 5 patients. thirty-four patients received anti-thymocyte globulin (atg). the donor source was sibling bone marrow (bm) in 1 patient, sibling peripheral blood stem cell in 7, unrelated bm in 36 and unrelated cord blood in 7. relapse-free survival (rfs) and overall survival (os) were 40.7% (95% confidence interval (ci): 27.2-53.8%) and 49.7% (95% ci: 35.1-62.7%) at 1 year, 31.4% (95% ci: 18.2-45.5%) and 33.6% (95% ci: 19.2-48.5%) at 3 years, respectively (figure 1.) . in this study, 4 patients died before engraftment. non-relapse mortality (nrm) was 19.6% at day 100. twenty-five patients developed chronic gvhd (3 patients limited and 22 extensive). the causes of death were disease progression (10 patients), treatment-related mortality (13 patients), infection (4 patients) and other causes (3 patients). we suggest that many elderly allo-hsct patients with mds and aml were in the very poor risk group when the ipss-r cytogenetics score was assigned, in the very high risk group when the ipss-r score was assigned and nr upon administration of allo-hsct. rfs and os were 31.4% and 33.6% at 3 years, respectively. there is a need for novel treatment strategies to manage elderly mds and aml patients for allo-hsct. [p659] disclosure of conflict of interest: none. counting bone marrow blasts as a percentage of nonerythroid cells provides superior risk stratification for mds patients with erythroid predominance a sun 1 , y yu 1 , t zhang 1 , q wang 1 , d liu 1 and s chen 1 1 the first affiliated hospital of soochow university, jiangsu institute of hematology, suzhou, china patients with erythroid predominance (≥50% erythroblasts, mds-erythroid) compose a significant proportion of patients with mds. the erythroid/myeloid subtype was divided from the aml category into mds-erythroid by the 2016 who classification of myeloid neoplasms. at that time, there was no consensus on a more appropriate way of enumerating bone marrow (bm) blasts from tncs or necs in mds-erythroid patients. to clarify these questions, 1283 mds patients were retrospectively analyzed in our center. mds-erythoid was observed in 27.0% of patients (346/1283), and these patients had similar clinico-pathological features and overall survival, with 937 cases of mds with o50% encs. by calculating the percentage of bm blasts from necs, 73 of 200 patients (36.5%) with mds-erythroid who were diagnosed within who subtypes without excess blasts (eb) were moved into higherrisk categories and showed shorter os than those who remained in the initial categories (p = 0.041). recalculating the international prognostic scoring system-revised (ipss-r) by enumerating blasts from necs, 40 of 168 (23.8%) mdserythroid patients with relatively lower risk were re-classified as higher-risk and had significantly poorer survival than those who remained in the lower-risk category (p = 0.030). this was especially true for the intermediate risk group that was stratified by ipss-r (unchanged patients vs. shifted patients, p = 0.007). however, the impact of enumerating bm blasts from necs on classification and prognostication was not evident in all mds patients. in conclusion, our results suggested that enumerating the percentage of bm blasts from necs significantly improved the prognostic assessment of mds-erythroid, especially for patients within the intermediate risk group stratified by ipss-r. disclosure of conflict of interest: none. myelodisplastic syndrome (mds) is a group of clonal and heterogeneous diseases, characterized by ineffective hematopoesis. the incidence of mds is about 5% of all blood disorders in children, approximately 40% of them develops acute leukemia. allogeneic hematopoietic stem cell transplantation (allo-hsct) is effective curative treatment of mds in children, but depends on disease status, type of clonal chromosomal abnormalities presented at the time of allo-hsct and graft quality. the aim of this study: to analyze the influence of graft quality on the outcome of childhood mds after allo-hsct. allo-hsct were performed in 58 patients (pts) p662 hypomethylating agents vs. allogeneic sct in elderly patients with advanced myelodysplastic syndromes: a single center study j cermak, a vitek, m markova-šťastná, j soukupova-maaloufova and p cetkovsky institute of hematology and blood transfusion, prague, czech republic a group of 26 patients older than 50 years of age with myelodysplastic syndrome (mds) raeb ii or with acute myeloid leukemia with multilineage dysplasia with less than 30% of bone marrow blasts (mds raeb-t according to the fab classification) was treated with hypomethylating agents (hma) and the results were compared to those obtained in an age and diagnosis matched group of 16 patients who underwent allogeneic stem cell transplantation (sct). in the hma group, 22 patients received azacytidine (vidaza) in the dose of 75 mg/ m 2 × 7 every 28 days and 4 patients were treated with decitabine (dacogen) in the dose of 20 mg/m 2 × 5 every 28 days. median number of cycles administered was 10.8 (range: 3-31). in the transplanted group, 8 patients were transplanted upfront and 10 patients were pretretated with combination chemotherapy, 8 patients received myeloablative conditioning and 8 patients were transplanted after reduced conditioning regimen. a hematologic response to hma (cr,pr, hematologic improvement) was observed in 15 patients (61.5%), cr was achieved in 8 patients (31.8%). in sct group, engraftment was achieved in 14 out of 16 patients, 8 patients died after sct ( 5 on complications related to sct, 3 patients relapsed). no difference in 1 year survival was observed between both the groups (65.6% for hma vs. 62.5% for sct), however, median overall survival (os) was 19.0 months in hma treated group compared to 47.6 months in sct group (p = 0.03). in a recent analysis performed at 48 months after starting the treatment, 2 patients treated with hma (7.7%) and 6 transplanted patients (37.5%) were alive, 16 patients in hma group and 3 patients in sct group relapsed. estimated 5 years survival was 31.3% in sct group and only 3.8% in hma group (p = 0.001). no significant differences in results and adverse effects of treatment were observed between patients aged 51-60 years and those older than 60 years in both hma and sct groups. our results confirm previous observations showing that despite a promising effect of hma resulting in hematologic response in more than 50% of elderly patients with advanced mds, allogeneic sct still represents the only potentially curative treatment connected with long-term survival in a significant number of patients even in this mds patients subgroup. disclosure of conflict of interest: none. immunophenotypic assessment of erythroid dysplasia by a simplified cocktail in myelodysplastic syndromes in taiwan c-c li 1 , p-f weng 1 , c-t lin 1 , j-l tang hypomethylating agent (hma) is commonly used as a bridge therapy to prevent leukemic transformation prior to selection of a donor for allogeneic stem cell transplantation (sct) in patients with myelodysplastic syndrome (mds), and showed low toxicity. although its roles are known, the underlying genetics and clonal dynamics upon hma treatment has not been systematically examined using serial samples, especially in allogeneic stem cell transplantation (sct) setting. in this study, we performed targeted serial sequencing on 66 bone marrow samples from 22 mds patients treated with hma for bridging of allogeneic sct. to perform targeted deep sequencing, bm mononuclear cells before hma treatment and, and fractionated t-cell samples (cd3+ fraction) as controls were taken before hma treatment. analysis of genetic mutations were performed using targeted resequencing by illumina hiseq 2000 (sureselect custom probe set targeting [p664] entire exon regions of a myeloid panel consisting of 84 genes). all 22 patients received hma (decitabine: 15, azacitidine: 7), and the median number of cycles was four (range: 2-12). the overall response rate for hma pre-treatment was 55%: there were four cases of complete remission (cr) (18%), six cases of marrow cr (27%), and two cases of hematologic improvement (9%). targeted sequencing revealed 37 mutations in 16 patients (16/22, 73%) with median of 2 mutations per patient (range: 2-5). mutated genes were then grouped into 8 biological pathways, defined in the cancer genome atlas (tcga) aml study. the most frequent biological pathway at diagnosis was dna methylation (32%), followed by activated signaling (27%), chromatin modifiers (18%), tumor suppressors (18%), spliceosome (14%), cohesin complex (9%), npm1 (4%), and myeloid transcription factors (tfs) (4%). when assessing the difference in pattern of variant allele frequency (vaf), we found the significant reduction of vafs in four (25%) patients after hma. with a median follow-up of 63.4 months, 5-year overall survival (os) were 69.6% (95% ci, 49.0-90.2). there was no significant difference in os according to the presence of mutations in each biological mutational pathway (all, p40.05). to identify prognostic value of mutational dynamics, we subclassified the change of variant allele frequencies (vafs) after median fourth cycles of hma [no mutated or reduction of vafs (11 patients) vs. stable or increased (11 patients)]. however, there was no significant association between the dynamic of mutation and os (p = 0.374). these data show that hma using as bridge therapy for allogeneic sct in mds patients is insufficient to achieve the sufficient molecular responses and, mutational pathway and dynamics may not prognostic in this clinical setting. to clearly demonstrate the role of hma pre-treatment in mds, systematic assessment on a larger cohort is necessary. disclosure of conflict of interest: none. any role of high-dose chemotherapy in mediastinal nonseminoma germ cell tumors? p pedrazzoli, s secondino, a necchi 1 , f lanza 2 and g rosti 1 istituto nazionale tumori, milano and 2 ospedale santa maria delle croci, ravenna among germ cell tumors, primary mediastinal nonseminoma germ-cell tumors (pmnsgcts) have the poorest outcome with 5-year overall survival ranging from 40 to 45%. indeed, the presence of mediastinal location defines per se a "poor prognosis" category according to the igcccg classification. this clinically and biologically distinct disease entity is associated with lower complete response rates to chemotherapy (ct), high rates of relapse and disappointing results from salvage ct. current standard first line treatment for patients with mediastinal primary location is still four cycles of peb, as for all igcccg poor-prognosis patients. we have reviewed available data present in the literature, including recommendations and expert opinions, on the use of high-dose chemotherapy (hdc) with autologous stem cell support in pmnsgcts. the use of hdc as both early intensification (that is, first-line setting) and at disease recurrence (salvage setting) have been reported in small cohorts of patients. according to the largest retrospective comparison, it has been suggested that hdc, given up-front, may produce a 15% to 20% absolute improvement in survival compared with standard dose ct. studies of the ebmt suggest that responsive disease after induction therapy may have a better outcome. mediastinal primary had salvage rates by hdct of less than 15% based on an international multicenter analysis and an ebmt study. the use of hdct in pmnsgcts warrants further investigation, preferably with the use of modern hdct strategies (that is, multiple carboplatin and etoposide courses). while hdc cannot be routinely recommended in pmnsgcts, selected patients with chemosensitive disease may benefit from early intensification. a retrospective analysis evaluating the large ebmt database is ongoing; results will be presented at the meeting. disclosure of conflict of interest: none. high dose therapy and autologous stem cell transplantation in gynaecological malignancies: a monocentric retrospective study m nderlita 1 , i vergote 1 and d dierickx 2 1 university hospitals leuven, department of gynaecology; university hospitals leuven and 2 department of hematology high-dose chemotherapy (hdt) followed by autologous stem cell transplantation (asct) has been established as a treatment option in many relapsed hematologic malignancies. however, in spite of many small trials, there still is no proven role for this treatment in solid tumors including most gynaecological epithelial carcinomas. however, in some recurrent non-epithelial ovarian cancers, such as sex cord stromal tumors, germ cell tumors, neuroendocrine gynaecological tumors and gestational trophoblastic disease, some studies suggest a possible role for hdt followed by asct. we performed a monocentric retrospective descriptive analysis of all patients diagnosed with gynaecological malignancies and treated with hdct followed by asct in our center. clinical, laboratory, pathological and imaging data were collected and analysed, together with information on treatment and outcome. eleven patients were included in this analysis, with a median age of 29 years (range: 14-56) at time of diagnosis. eight patients suffered from ovarian neoplasia. at time of diagnoses 6 patients showed metastatic disease. first line therapy consisted of surgery (n = 4), chemotherapy (n = 2) or a combination of both (n = 5). median time to progression after first line therapy was 39.8 months (range: 0-192) with a median time between primary diagnosis and start hdt of 54.7 months (range: 4-306). three patients underwent single ast, whereas the other 8 patients had a tandem ast, with a median time of 2 months between first and second hdt (range: 1-4). treatment related toxicity was manageable, although there was 1 treatment-related death. at last follow up 5 patients (45%) were still alive with a median follow up of 3.9 years (range: 0.25-15.1) after last asct for all patients. of the 6 deceased patients 5 died with progressive disease. although the number of patients is very small, this retrospective study shows that hdt and asct is feasible in heavily pretreated patients with relapsed/refractory gynecological malignancies, although further studies are mandatory for optimal selection of patients, histological subtype and timing of hdt during the disease course. disclosure of conflict of interest: none. the human endogenous retroviruses (hervs) are remnants of ancient exogenous retroviral infections of the humans: they represent about 8% of the human genome 1 . the basic genes of hervs are group-specific antigen (gag), polymerase (pol) and envelope (env); there are also two regulatory regions, long terminal repeat (ltr), located at 5' and 3' ends. several reports have shown that hervs may play a role in the development of autoimmune diseases, such as multiple sclerosis 2 . additionally the existence of a strong relationship between hervs expression and cancer, based on the mrna expression profile of hervs in normal and cancer tissues has been suggested 2 . the increased level of expression level of herv-h in colorectal cancer (crc), a major cause of cancer death worldwide has been already shown. the aim of the study was to analyse the expressions of env genes of herv-r, herv-h, herv-k and herv-p in the peripheral blood mononuclear cells (pbmcs), in the tumor and in the adjacent normal tissues of 20 colorectal cancer patients. a group of control composed by pbmcs from 46 healthy subjects was also included. rna was isolated from the biological samples and a reverse transcription assay was conducted. quantitative real time pcr was performed to evaluate the expression of the hervs env gene. all the env genes were related to the expression of an housekeeping gene, gapdh. the quantification was carried out using comparative ct method and the difference between the levels of env gene expression in pbmcs, cancer and adjacent normal tissue was given by fold difference. fold difference values were relative to a calibrator: first the pbmcs of patients and then pbmcs of control healthy group. δct values were analysed using the paired sample t-test, followed by a bonferroni correction. higher levels of expression of herv-h, herv-k and in particular herv-p were found in tumor tissues, as compared to pbmcs and to adjacent normal tissues of patients, with an increase of 24-, 10-and 78folds, respectively. the δct distribution of herv-h, herv-k and herv-p in cancer tissues were statistically significant (po0.05) ( table 1 ). the expression of herv-h, herv-k and herv-p env gene resulted increased in the colorectal tumor tissues also when compared with the pbmcs of the healthy controls (5-, 15-and 26-folds, respectively). the δct distribution of herv-h, herv-k and herv-p in tumor tissues were statistically significant ( ρ < 0.05). no difference of expression was observed between pbmcs of healthy controls, pbmcs and normal adjacent tissues of patients (figure 1 ). hervs env gene expression cannot be used as a diagnostic biomarker, but it is conceivable that hervs are directly involved in the pathogenic process of cell transformation and, if the protein expression will be demonstrated, the protein of hervs env gene could be the target for new immunotherapy strategies against colorectal cancer. [p668] disclosure of conflict of interest: none. a biosimiliar g-csf filgrastim is as effective as a reference drug however itis not as cost effectiveas it supposed to be and by the way no impact on the health care system m kurt yüksel, g pekcan, u sahin, s bozdag, s toprak, p topcuoglu, o arslan, g gurman and m beksac ankara university school of medicine biosimiliars are up to 1000 times the size of small molecule generic drugs and far more structurally complex. additionally biosimiliars are manufactured in living cell lines using processes that cannot be exactly replicated from one manufacturer to the next. a biosimiliar cannot be identical to its reference biologic drug. with 67billion dollars in global sales of biologic medicines anticipated to go off patent by 2020.this lead to fast production of biosimiliar drugs. besides, it is expected that biosimiliar drugs will be more cost effective than the reference drugs and will have a meaningful impact on health care systems around the world. aim: to compare biosimiliarfilgrastim (leucostim) with two reference g-csf filgrastim (neupogen) and lenograstim(granocyte) in the context of safety, efficacy and cost effectivity. records of patients with multiplmyeloma(mm) whom underwent autologous stem cell transplantation(asct) and received g-csf sc5mikrogram/kg/day from +day 5 until engraftment were [p668] retrospectively evaluated 60 mm patients were treated with high dose melphalan and asct at the ankara university school of medicine bone marrow transplantation unit between 2013 and 2016. the median age was 59 (38-75 years) with 55% male. patients were divided into three groups (n = 20) whom received reference filgrastim (neupogen), lenograstim (granocyte) and biosimiliarfilgrastim (leucostim): groups a, b and c respectively. the total cost of each g-csf in dollars was calculated by one package of g-csf multiplied by total used days . chi-square, mann-whitney u and kruskal-wallis tests were used for analyses of variance. the percentage of patients who received melphalan 200 mg/m 2 were%80, 85, 80in groups a, b, c respectively (p = 0.9).there was no statistically significant difference between the engraftment day of neutrophil 500 and 1000; platelet 20 000 and 50 000 in the groups. (p = 0.07, p = 0.55, p = 0.33, p = 0.81 respectively) themedian numbers of g-csf administered days were 7(5-18), 8 (5-12), 7 (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) in groups a, b, c, respectively .eventhough there was no statistical difference between the numbers of days( p = 0.23), the total cost in dollars was statistically difference between a vs b and c vs b (both p o0.0001) and there was no statistical difference between a vs c (p = 0.89), total cost in dollars as follows: 155$ (112-288$), 416$(260-624$) and 166$(81-250$) for the group a, b and c respectively. our results demonstrate that biosimiliar gcsf leucostim is highly similar to existing licenced biologic products in turkey with no clinically meaningful difference interms of safety and efficacy. on the other hand it as a biosimiliardoes not have a meaningful impact on the cost savings to the health care system as expected when compared with reference filgrastim. disclosure of conflict of interest: none. in this study, we investigated the roles of prx ii, one of 3 critical peroxidases besides catalase and gpxs, in cml primary cells at diagnosis and remission while patients were treated with sti (signal transduction inhibitor) and tested the same roles in imatinib(im) sensitive ph+ cell lines and resistant cell lines as well. newly diagnosed cml cells, im resistant k562 cells and parental k562 cells were treated stis and analyzed western blot assay to detect bcr-abl, phosphorylated bcr-abl and prx2 protein expression level. we added n-acetylcysteine (0-5mm, 6hr) to k562 cells to show antioxidant effect of imatinib and analyzed dcf-da detection for intracellular ros level and western blot for prx2 protein level. mtt assay was performed to detect cell death by nac time-dependent treatment of 5mm nac(0, 24, 40, 48hr). imatinib resistant k562 cells were established by treatment of gradual increment of imatinib. we also repeatedly investigated the effects of im therapy using prxii overexpressed k562 cells by transfection. at diagnosis of cml, ros level was elevated and prx ii was either absent or significantly suppressed. as ph chromosomes were decreased with stis, suppressed or absent prxs levels were restored to the level of normal individuals. these findings were also inversely correlated with the level of ph chromosomes in the cases of disease progression and re-remission with further treatment. when sti were treated in ph positive cell line, we found deceased cell survival and ros level by mtt assay and dcf-da methods respectively, but elevated prx ii by western blot. by the treatment of nac into ph+ cell lines, the level of dcf-da was decreased and mtt level was down, but prx ii level was elevated. interestingly, the level of bcr-abl oncogene were decreased in prx ii tranfected cells. meanwhile, we observed that prx ii restoration was mild or weak in imatinib resistant k-562, which we established in our lab. the importance of the roles of ros and its prx ii, antioxydant enzymes in cml is further established by our work. our finding may contribute to find a new pathway on which tkis are working besides the mechanisms of atp binding competitively, blocking the binding of abl-bcr kinase to the substrate resulting apoptosis of ph+ cells. furthermore, our finding may be useful to overcome the stis resistant cml in the clinics in the future. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation for the treatment of mucopolysaccharidosis f shunqiao, s xiaodong and l junhui department of hematology, capital institute of pediatrics, china mucopolysaccharidosis (mps) is a lysosomal storage disorder caused by deficient activity of the iduronate-sulfatase.this leads to accumulation of glycosaminoglycans(gags) in the lysosomes of various cells,which causes progressive multisystem involvement with ensuing death.the aim of this study was to exploit the effect of treatment with allogenic hematopoietic stem cell transplantation and administration of high doses of cyclophosphamide early after haplobmt in these cases. we retrospectively reviewed data from 3 mps patients (2 cases mps ii, and 1 case mps i). the two mps ii patients were 44-month-old and 35-month-old boy and the mps i patient is a 84-month-old girl at the time of transplantation. the reduced-intensity of bu+flu conditioning regimen in allo-hsct for these patients was as follows: busulfan 4 mg/kg at 5-2 days before transplantion,fludarabine 40 mg/m 2 at 6-3 days before transplantion.graft-versus-host disease(gvhd) prophylaxis:rabbit antithymocyte globulin 2.5 mg/kg daily at 5-3 days before transplantation,shortcourse methotrexate,posttransplantation high-dose cyclophosphamide on days +3 and +4 was followed by mycophenolate mofetil and cyclosporine.the donors all were their hlahaploidentical father. these three patients' neutrophil engraftment occurred on +14d, +12d and +15d after transplantation respectively, platelet engraftment occurred on day +14d, +10d and +15d after transplantation respectively.complete donor type engraftment was confirmed by short tandem repeat-polymerase chain reaction(str-pcr) on day 14 after transplantation. no regimen-related toxicity occurred,gvhd and graft failure were not observed. 1 month after transplantation, the activity of the iduronate-sulfatase was increased to normal. the motion of metacarpophalangeal joints ameliorated, regression of hepatosplenomegaly, the neurocognitive function improved. allogeneic hematopoietic stem cell transplantation is an effective measure to treat patient with mps at least mps ii and mps i. the reduced-intensity conditioning regimen was helpful to decrease the regimen-related toxicity. posttransplant cyclophosphamide approach successfully used and reduced the incidence of gvhd. this study aimed to evaluate the feasibility of alternative donor hematopoietic stem cell transplantation (hsct) using busulfan, fludarabine, and thymoglobulin conditioning for patients with chronic granulomatous disease (cgd) who lack an hla-matched familial donor. medical records of 11 consecutive patients who received alternative donor hsct between may 2010 and may 2016 were reviewed, and the transplant-related outcome measures were analyzed retrospectively. the donor source was unrelated peripheral blood (pb) in 4, unrelated cord blood (cb) in 4, and haploidentical father in 3 patients. only 2 transplants (8/8 allele-matched unrelated pb) were hla-matched according to current standards relevant to the donor type. the conditioning regimen was uniform; fludarabine 40 mg/m 2 on days -8 to -4, busulfan 3.2 mg/kg/d (or 120 mg/m 2 /d) on days -6 to -3, and thymoglobulin 2.5 mg/kg/d on days -3 to -1 (or on days -8 to -6 in cb recipients). all but one patient were male and their median age at transplantation was 6.5 y (range: 1.1-26.3). one patient who received a cord blood graft suffered from primary engraftment failure, while the other 10 patients were successfully engrafted with their chimerism levels ranging from 66% to 100% (median 100%) at 1 month post-transplant. the median days to neutrophil and platelet engraftment were 12.5 (range: 11-22) and 27 (range: 11-47), respectively. among the 10 patients engrafted, one patient experienced secondary graft failure which was rescued by a second transplantation. the remaining one patient who failed to engraft was also rescued with a haploidentical graft from his mother. eight patients (73%) developed cmv antigenemia, and one of those patients developed cmv hepatitis. three patients developed grade 3 acute gvhd which were manageable. one patient who developed grade 4 hepatic gvhd eventually died. two patients developed extensive chronic gvhd, but became free of immunosuppressants after a complete resolution in one and with remaining stable mild joint contractures in the other. including 2 patients who were rescued by additional transplantation, 10 patients are alive with their latest chimerism levels ranging from 86.8% to 100% (median 100%). the estimated 5-y overall survival rate was 85.7% with a median follow-up of 49 months (range: 6-72). even though the majority of our cohort underwent a mismatched transplantation, the survival rate was excellent. while conditioning with busulfan, fludarabine, and thymoglobulin seems feasible for alternative donor hsct in patients with cgd, special attention needs to be payed on cmv infection and severe gvhd which might offset the high survival rate. disclosure of conflict of interest: none. diarrhea is a common infectious complication in patients who had hematopoetic stem cell transplantation 2 so, we aimed to detect entamoeba histolytica ratio before engraftment, amoung 375 patients who had diarrhea after periferic hematopoetic stem cell transplantation (phsct) in our clinic. allogenic phsct patients had a median age of 29 (range: 15-63) and autolog phsct patients had a median age of 54 (range: 18-74). diarrhea is described as an abnormal increase in the frequency (3 or more times per day), volume or liquidity of stools. we based upon this description in this study. we made stool examination in the first day of diarrhea. as stool examination, we used direct microscopic evaluation and adhesin antigen test specific for e.hystolytica with enzyme linked immunosorbent assay (elisa), e. histolytica ii, techlab, blacksburg, usa). we accepted e.hystolytica positivity as detecting cyst or/and trophosoit in stool and antigen test positivity at the same time. in our study, 185 of 375 patients had diarrhea in the first 28 days of phsct. diarrhea was found in 139 of 242 in autologous phsct patients (%57), 21 of 63 patients in allogenic phsct with non-myeloablative conditioning regiment (%33) and 25 of 70 patients in allo phsct with myeloablative conditioning regiment (%36). diarrhea occured at +8th day of transplantation and the median duration of diarrhea was 3 days. e. histolytica positivity was found 46 of 185 patients (25%) who underwent phsct within first 28 days of transplantation. infection is an important mortality and morbidity factor for patients who had hematopoetic stem cell transplantation, when especially before engrafment (between 0-30 days). 1 autologous phsct patients were elderly, with poor self-care and low socioeconomic status individuals. e. hystolytica is a frequent pathogen in posttransplant diarrhea at endemic regions. prophylactic metronidasole treatment should be used routinely for autologous phsct as in allogenic phsct. 3 patients and companions sholud be tested for e.hystolytica before autologus/allogenic phsct in endemic regions. prophylactic treatment for amebiasis and scanning patient/companions could be a part of solution for post phsct diarrhea. despite the emergence of disease modifying therapies (dmts) for multiple sclerosis (ms) a cohort of patients with aggressive disease have ongoing progression/relapse, associated with progressive disability. autologous haematopoietic stem cell transplantation (ahsct) has been used worldwide for aggressive ms with inflammatory changes on mri. we update on a uk single centre experience of ahsct in ms. a retrospective audit of ahsct performed for ms from 2012 to 2016 at 1 uk centre (king's college hospital) was undertaken. patients were selected for transplantation based on persistent clinical relapses (relapsing-remitting ms) or secondary progressive neurological disability with mri lesion activity despite use of at least 1 dmt. primary progressive patients were also eligible if new/active mri lesions were demonstrable. followup included clinical evaluation, edss assessment and mri scanning. we report our preliminary findings. as of november 2016, 30 patients (16 female, 14 male, 18 rrms, 10 spms, 2 ppms) had received ahsct. mean age at transplant was 40.6 years (range: 22-57). the mean baseline edss was 5.3 (range: 2.5-8.0). 29 patients underwent cyclophosphamide/atg conditioning, while 1 received beam/atg. whilst conditioning and stem cell infusion were well tolerated there was a high rate of infections, with 23/30 patients developing a culture confirmed infection. reactivation of ebv and cmv were observed in a number of patients (21 and 8, respectively) while a number of delayed herpes zoster infections were also seen (4 cases of shingles and 2 of disseminated varicella infection in patients who had previously experienced it in childhood). median follow-up was for 361 days (63-1479). of patients with a formal 6 month assessment (n = 16), 4 had a stable edss, 6 had an improved score (median improvement 0.5, range: 0.5-2.5) and 6 had a deterioration in their score (median 0.5, range: 0.5-1.0). at 12 months (n = 11), 1 had a stable edss, 4 had an improved score (median 0.75, range: 0.5-1) and 6 had a deterioration in score (median 0.5, range: 0.5-1.5). at 24 months, two patients assessed both had improvements in edss scores (median 1, range: 0.5-1.5). for patients who underwent mri at 6 month follow-up (n = 14), 10 had a stable lesion load, 2 demonstrated improvement in lesions and 1 had a new lesion (the remaining mri was difficult to read due to a high baseline lesion load in this patient). 4 patients had mri's at 12 months; 3 were stable and 1 demonstrated a reduction in lesion load. to date, no patients have developed secondary malignancies or autoimmune diseases. of patients with followup data, 4/18 rrms patients experienced suspected clinical relapses following hsct-only one had a new lesion on mri (with no gadolinium enhancement). 3 of the 4 received steroids to treat these relapses (it is unclear if the remaining patient received treatment). 1 patient tried a new disease modifying therapy (1 dose of rituximab) following hsct. ahsct in this cohort was feasible with universal mobilisation and harvest. whilst conditioning and stem cell infusion were well tolerated, there was a high rate of infectious complications in the neutropenic phase. however, the transplant related mortality was 0% despite significant levels of disability amongst this patient cohort. ahsct remains a treatment option to be further investigated in this difficult cohort of patients. disclosure of conflict of interest: none. peripheral blood (pb) stem cells (scs) mobilized with g-csf are the first-choice source for allogeneic transplantation. we carried out a prospective study on healthy donors (hds), to identify donor characteristics that could influence the effectiveness of mobilization with special focus on the value of the basal cd34+ cell count. sibling hds were analyzed in a prospective study. we tested somatic variables (sex, age, weight, height, volemia) and, basal blood counts (white blood cell, peripheral blood mononuclear cell, platelets, hematocrit, hemoglobin, cd34+ cell). hds received g-csf subcutaneously at a dose of 10 μg/kg day. two different determinations of cd34+ cells were done in each donor: baseline (before g-csf administration) and in pb on the morning of the fifth day (after g-csf administration). 128 consecutive hds (65 males) with a median age of 43 years were enrolled. the mean value of cd34+on day 5 was 90.8 cells/μl, while the median value was 75.5 cells/μl. we performed two multivariate analyses either by using median regression (to predict the median value of cd34+on day 5) according to the values of cd34+ at baseline, the first adjusted by gender, age and blood volume and the second by gender, age and bmi. results of both models indicate that from basal cd34+ values o = 1 to values ranging between 3 and 4 cells/μl, predicted median values of cd34+ on day 5 significantly increase, from 54.6 to 92.8 cells/μl for model adjusted by blood volume, and from 49.9 to 92 cells/μl for model adjusted by bmi. baseline, pb cd34+ cell count correlated with the effectiveness of allogeneic pbscs mobilization and could be useful to plan the collection. disclosure of conflict of interest: none. comparison of efficacy between chemotherapy plus granulocyte colony stimulating factor (g-csf) and chemotherapy plus g-csf and granulocyte-macrophage colony stimulating factor (gm-csf) for mobilization of peripheral blood stem cells (pbsc) and hematological recovery post-transplantation in patients with multiple myeloma (mm). a retrospective study of autologous peripheral blood stem cell (apbsc) mobilization data of 56 mm patients who treated with chemotherapy plus g-csf or chemotherapy plus g-csf and gm-csf from may 2008 to july 2016. the mobilization efficacy and hematopoietic recovery were analyzed. a total of 65 stem cell mobilizations were performed in 56 mm patients. in the univariate analysis, successful collection rate of single harvest in female and in patients at iss stage iii, r-iss ii/iii and chemotherapy plus g-csf was lower(po0.05). however, age(≦60 yrs vs 460 yrs), subtype, d-s staging (i+ii vs iii), cycles of chemotherapy before mobilization (≦6 cycles vs 46 cycles), disease phase before mobilization (pr vs cr) and interval diagnosismobilization (≦18 months vs 418 months) were not correlated with the cd34+ cell collection and successful mobilization rate(p＞0.05). in the multivariate model, rate of successful mobilization in patients who received chemotherapy plus g-csf+gm-csf mobilization regimen was high (or = 12.009, 95%ci 1.961-73.537). the effect of mobilization regimen remained significantly (p = 0.007). all patients successfully underwent hematopoietic reconstruction without transplantation-related mortality. chemotherapy plus g-csf +gm-csf mobilization regimen can significantly increase the effect of apbsc mobilization and ensure the reconstruction of hematopoietic function after transplantation. this mobilization regimen is a safe and effective method of mobilizing apbsc. disclosure of conflict of interest: none. clinical efficacy of bk virus specific t-cells in treatment of severe refractory hemorrhagic cystitis after hla haploidentical transplantation om pello 1 , a bradshaw 2 , a innes 2 , s-a finn 2 , s uddin, e bray 2 , e olavarria, jf apperley and j pavlu 1 centre for haematology, imperial college at hammersmith hospital, london, uk and 2 department of clinical haematology, hammersmith hospital, imperial college healthcare nhs trust, london, united kingdom hemorrhagic cystitis caused by bk virus (bkv) is a significant complication of allogeneic hematopoietic cell transplantation (hct). it is particularly common in the setting of hla haploidentical transplantation and can be challenging to manage. here we present a post haploidentical hct patient who developed severe bkv haemorrhagic cystitis resistant to standard therapy and who responded to adoptive transfer of donor t cells enriched with anti-bkv specific cells. a 40 year old man underwent hct for acute myeloid leukaemia with inversion of chromosome 3 and monosomy of chromosome 7 while in first complete remission. as he had no related or unrelated hla identical donor, cells from his hla haploidentical sister were used. on day +32 of this procedure he developed haemorrhagic cystitis. supportive treatment was initiated and cystoscopy showed diffuse bleeding from his urinary bladder with blood clots. urine pcr for bkv showed 5.2 billion copies/ml. despite bladder irrigation, local therapy to s483 bladder mucosa and intravenous hydration, he failed to improve, so treatment with weekly intravenous cidofovir was initiated on day +38. his symptoms improved, but on day +72 he again deteriorated on weekly infusions of cidofovir. his immunosuppression was slowly tapered off without any graft versus host disease (gvhd) but without any significant effect on his hemorrhagic cystitis. he underwent bladder diathermy, was treated with intravesicular hyaluronate and with intravenous cidofovir, but continued to have frank haematuria with blood clots and significant lower abdominal pain. although there was no evidence of obstruction his renal function deteriorated on cidofovir therapy. hence we elected to trial adoptive anti bkv therapy. a leukoapheretic lymphocyte collection was used to prepare an anti-bkv t cell enriched product using the clinimacs prodigy and the cytokine capture system from miltenyi biotec. the eluted product contained 50% and 5% of cd4+ and cd8+ lymphocytes expressing ifng+ respectively and the cd4+/cd8+ dose adoptively transferred on day +86 of transplantation was 0.34 × 10 4 /kg. in vivo expansion of anti-bkv t cells in the patient was analysed weekly for the first month using the research grade peptivators bkv lt and bkv vp1 and the rapid cytokine inspector (cd4/cd8 t cell) kit. bk viral load was monitored by pcr in urine samples twice weekly. ifng+ anti-bkv reactive t cells were undetectable in the patient for the first two weeks after adoptively transfer of donor t cells. twenty days after the adoptive transfer an increase in the cd4+ ifng+ population was observed, in response to the bkv vp1 peptivator. this observation correlated in time with a substantial decrease of the urine bkv viremia from 3.3 million copies/ml to 1360 copies/ml and a complete resolution of patient's symptoms. no gvhd, recurrence of urinary symptoms or any other problems have been observed to date (day +260 of transplantation, +174 days after the adoptive transfer). we are not aware of any other reports of successful adoptive anti bkv cellular therapy. our experience suggests safety and efficiency of the use of anti-bkv t cell enriched products using the clinimacs prodigy and the ifng capture system in hla haploidentical hct where bkv cystitis constitutes a significant complication. this opens the possibility of further clinical trials. disclosure of conflict of interest: none. haploidentical donor (hd) has been used as an alternative stem cell source when patients do not have a hla-matched related or unrelated donor. to overcome the hla barrier, haploidentical stem cell transplantation (haplosct) using post-transplantation cyclophosphamide (ptcy) has been conducted. here, we compared the clinical outcomes of haplosct using ptcy with those of unrelated donor transplantation. eighty-two patients (28 from hd and 54 from unrelated donor [ud]) who underwent allogeneic hematopoietic stem cell transplantation (hsct) in seoul national university children's hospital from january 2013 to june 2016, were analyzed. there were no significant differences between hd and ud patients with respect to median age of patients, sex distribution, and diagnosis [42. 6%], p = 0.081). the conditioning regimen of haplosct included targeted busulfan, fludarabine and cyclophosphamide using ptcy, tacrolimus and mycophenolate mofetil for graftversus-host disease (gvhd) prophylaxis. all patients showed engraftment except for a patient who underwent unrelated hsct. neutrophil engraftment of ud was faster than hd (median 11 days versus 15.5 days, respectively, po0.001). however, there was no significant difference of platelet engraftment. incidences of complications, such as hepatic venoocclusive disease, cmv infection, and hepatic dysfunction, between both groups, were comparable, except hemorrhagic cystitis (hd: 32.1%, ud: 7.4%, p = 0.004). moreover, cumulative incidence of acute gvhd (hd: 32.3%, ud: 44.7%, p = 0.260), severe chronic gvhd (hd: 8.4%, ud: 26.7%, p = 0.059), relapse (hd: 28.6%, ud: 25.1%, 7p = 0.323) and non-relapse mortality (hd: 0%, ud: 9.7%, p = 0.151) were not significantly different. the overall and event-free survival of hd and ud were 85.4% vs 86.2% (p = 0.703) and 75.0% vs 75.9% (p = 0.509), respectively. the clinical outcomes of haplosct using ptcy were comparable with those of ud, and a trend of lower cumulative incidence of severe chronic gvhd and non-relapse mortality was encouraging. it could be a promising alternative therapeutic option in pediatric hsct. disclosure of conflict of interest: none. , median number of apheresis procedures was 2,15 (1-6), median amount od dmso infused 20 ml (7-60). time to engraftment was median 11 days (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) . statistical comparison between cryopreserved pbsc grafts and bm showed benefit for pbsc in the terms (po0.01) of faster engraftment, less infective complications, less transfusion support and less hospital stay. in 253 patients (86%) dmso related events were not registered during graft administration. in 41 patients (14%) mild to moderate dmso related events were registered, as nausea in 34 patients (83.3%), vomitus in 20 patients (50%), tachycardia in 8 (20.8%), hematuria in 6 patients (16%) and 2 patients (4.16%) with bradycardia, hypotension, fever and high temperature during graft infusion. cryopreservation of stem cells is a feasible procedure at our institution. there are some issues that have to be improved. the process is standardized with achieved engraftment in all transplanted patients. disclosure of conflict of interest: none. effectivity of a fludarabine based conditioning regimen in allogenic hematopoietic stem cell tranplantation for patients with severe aplastic anemia and over twenty years old p mustafa 2,3 , k melya pelin 1 , s handan haydaroglu 2 and g ilknur 1 1 gaziantep university, faculty of medicine, department of internal medicine; 2 hematology and 3 bone marrow tranplantation unit, gaziantep, turkey severe aplastic anemia (saa) is an anemia with bone marrow hypocellularity and caused by hematopoietic stem cell failure (1) . allogenic periferic hematopoietic stem cell transplantation (aphsct) is a curative treatment choice (2) . although cyclophosphamide (cyc) and anti thymocyte globulin(atg) is accepted as standart conditioning regimen, especially for patients with high rejection risk, using fludarabin (fu) based regimens show increased successful engraftment ratio with minimal toxic side effects (3) . to the study, 20 saa patiens who were transplanted from hla matched sibling donors between the years 2010-2015 were included. the patients comprised of 13 male (%65) and 7 female (%35). median age was 22 (range: 20-42). the median time from diagnosis to transplantation was 3 (range: 2-108) months. conditioning regimen consisted of cyc (1200 mg/m 2 ), fu (120 mg/m 2 ), atg (fresenius rabbit, 15 mg/kg). the median dose of stem cells was 7 × 10 6 stem cell/kg (range: 5-12). methotrexate (10 mg/ m 2 given four days) and cyclosporine (cyca) (3-5 mg/kg given 18 months) were applied for graft versus host disease (gvhd) prophylaxis. all 20 patients ecog performance status were good (0-1). prior to transplantation only one of the patients received atg-csa, the others received only supportive treatment. after aphsct, neutrophil engraftment was occured at a median of 16 days (range: 11-20) and thrombocyte engraftment was occured at a median of 14 days (range: 11-20). posttransplant graft failure was observed in only one patient at tenth month and this patient had aphsct again from the same donor with the same conditioning regimen. acute gvhd didnot occur in any patient. the 5 (%25) of patients had common chronic skin/oral mucosa gvhd. these 5 patients received methylprednisolone (mp) and/or mycophenolate mofetil (mmf) in addition to the cyclosporine treatment. extracorporal photopheresis was applied to the two patients with chronic gvhd. all chronic gvhd patients had complete response to the immunsupresive treatment with a median follow up time 46 months (range: 1-64). one patient died from sepsis. at 5 year overall-survival rate was 90%. fu based conditioning regimen in aphsct with young saa patients has favorable results. fu based regimen might be a gold-standard treatment in the future. cgvhd; tgfb-induced factor homeobox 1, interleukin 2 receptor gamma, tetra trico peptide repeat domain 37, carbonic anhydrase i, serpin peptidase inhibitor clade a and myod family inhibitor. we established a 3-gene model (myod family inhibitor, tgfb-induced factor homeobox 1, tetra trico peptide repeat domain 37) to diagnose cgvhd. our 3-gene model showed 81.00% sensitivity, 90.40% specificity, 80.8% precision, 81.03% accuracy and 86.90% roc area in diagnosing cgvhd. tgfb-induced factor homeobox 1 increased in expression after rituximab treatment in responders. myod family inhibitor was found to be able to predict rituximab responses in steroid-refractory cgvhd patients. we could demonstrate that gene expression studies were useful in the diagnosis of cgvhd after allo-hsct. we developed a 3-gene model to diagnose cgvhd. hematopoietic stem cell transplantation (sct) is physically and psychosocially demanding. however, exercise interventions may have positive impact on sentiment and psychological well-being in patients undergoing sct. we report on a prospective, randomized study comparing the influence of a multimedia sensor-based practice with classical physiotherapeutic treatment (pt) on psychological aspects and quality of life (qol). patients undergoing sct were randomized into the control group (n = 23) receiving pt or the experimental group exercising on the nintendo-wii (n = 19). patients of both groups performed the exercises under the supervision of a physiotherapist and completed the functional assessment of cancer therapy -bone marrow transplantation (fact-bmt), hospital anxiety and depression scale (hads-d) and distress thermometer at the date of hospital admission (t1) and on day 14 (t2), 28 (t3) and 100 (t4) after sct. questionnaires were completed by the participants independently and without supervision. groups were compared using the mann-whitney u-test. a p value o0.05 was considered statistically significant. the median age of patients was 59 years in the control group and 57 years in the experimental group. results of fact-bmt generally showed a decline of the qol domains measured on t2 and t3 and a raise at t4 in both groups. physical well-being (pwb) showed the strongest fluctuation of all domains. it declined significantly between t1-t2 in both groups (pt p = 0.015, wii p = 0.019), followed by a significant increase between t2-t4 (both groups p = 0.001). however, only in wii-group results of pwb at t4 ranked significantly above t1 (p = 0.028). highest scores were proved for social and emotional well-being (swb/ewb) in both groups. in wii-group ewb increased significantly between t1-t4 (p = 0.015) and ranked above pt-group at all times. functional well-being (fwb) scored lowest in both groups at all times. the score of bone marrow transplant scale (bmts), the second lowest score in both groups, was always higher in wii-group. the level of distress was comparable between both groups. however, at t2 distress increased above the cut-off level of 5 in both groups (wii-group p = 0.006, pt-group p = 0.276). this was accompanied by an increase of anxiety (p = 0.705) and depression (p = 0.006) in the pt-group, while both parameters decreased in the wii-group (p = 0.087 and p = 0.220), respectively. anxiety in intervention group 5,8/4,4/5,0/,4 at t1/t2/t3/t4 stayed below standard group 5,9/6,4/5,9/6,4 at all times. depression averaged out at 4,9/6,5/5,4/5,7 in physiotherapy group and 5,5/4,3/5,9/3,8 in wii-group. to the best of our knowledge, this is the first study to show that exergaming using the nintendo-wii is feasible in the immediate phase after hsct. exergaming may be regarded as beneficial since our data indicate less psychological distress and higher qol in sct recipients exercising with nintendo-wii. therefore, it may be used in addition to pt. disclosure of conflict of interest: none. acute graft versus host disease (agvhd) is the most frequent and serious complication following haematopoietic stem cell transplantation (hsct), with a high mortality rate. a clearer understanding of the molecular pathogenesis may allow for improved therapeutic options or guide personalised prophylactic protocols. circulating micrornas are expressed in body fluids and have recently been associated with the etiology of agvhd, but global expression profiling in a hsct setting is lacking. this study profiled expression of n = 799 mature micrornas in patient serum, using the nanostring platform, to identify micrornas that were dysregulated at agvhd diagnosis. selected micrornas (n = 10) were replicated in independent cohorts of serum samples taken at agvhd diagnosis (n = 42) and prior to disease onset (day 14 post-hsct, n = 47) to assess their prognostic potential. sera from patients without agvhd were used as controls. dysregulated micrornas were investigated in silico for predicted networks and mrna targets. profiling identified 61 micrornas that were differentially expressed at agvhd diagnosis. mir-146a (p = 0.03), mir-30b-5p (p = 0.007), mir-374-5p (p = 0.02), mir-181a (p = 0.03), mir-20a (p = 0.03) and mir-15a (p = 0.03) were significantly verified in an independent cohort (n = 42). mir-146a (p = 0.01), mir-20a (p = 0.03), mir-18 (p = 0.03), mir-19a (p = 003), mir-19b (0.02) and mir-451 (p = 0.01) were differentially expressed 14 days post-hsct in patients who later developed agvhd (n = 47). high mir-19b expression was associated with improved overall survival (os) (p = 0.008), while high mir-20a and mir-30b-5p were associated with lower rates of non-relapse mortality (p = 0.05 and p = 0.008) and improved os (p = 0.016 and p = 0.021). pathway analysis associated the candidate micrornas with haematological and inflammatory disease. circulating biofluid micrornas are dysregulated at agvhd onset and have the capacity to act as prognostic and diagnostic biomarkers. their differential expression in serum suggests a role for circulatory micrornas in agvhd pathology, which warrants further investigation. disclosure of conflict of interest: none. factors associated with medication adherence amongst allogeneic hematopoietic stem cell transplantation recipients j lehrer 1 , e brissot 2,3 , a ruggeri 2,4 , r dulery 2 , a vekhoff 2 , g battipaglia 2 , f giannotti 2 , c fernandez 1,5,6 , m mohty 2,3 and m antignac 1 1 ap-hp, hôpital saint-antoine, service de pharmacie, paris, f-75012; 2 service d'hématologie et de thérapie cellulaire, hôpital saint antoine, assistance publique-hôpitaux de paris; 3 sorbonne patients with median ages of 23 years (range: 11-54 years) between december 2006 and march 2016, which including 7 case of primary hlh (homozygous missense mutation in unc13d: n = 3; homozygous missense mutation in prf1: n = 1; heterozygous missense mutation in prf1 in the combination with hemizygous missense mutation in sh2d1a: n = 1; mutation in rab27a: n =1; mutation in itk: n = 1). 5 cases of unknown causes hlh, 10 cases of lympgoma -hlh (nk/t-cell lymphoma: n = 6, primary γδt cell lymphoma in skin: n = 1; subcutaneous panniculitis-like t cell lymphoma: n = 2; primary t cell lymphoma in skin: n = 1) and 39 cases of ebv associated hlh. 41 patients achieved cr+pr before hsct, and 20 patients nr. 47 patients were transplanted from hla-haploidentical family donors, 13 from hla-identical sibling donors, and 1 from a matched unrelated donor. conditioning regimen include tbi and non-tbi. the median overall survival rate was 65.6% with a median survival time of 38 months (range: 5-119 months). os of primary hlh is 85.7%, os of unknown causes hlh is 60%, os of lymphoma-associated hlh is 60%, os of ebv-hlh is 64.1%. os of cr+pr is 80.5%, os of nr is 35.0% 6 patients without engraftment died because of 2 graft failure and 4 toxicity of conditioning regimen. 15 patients with engraftment died. of those, 1 patient died of hsct-associated tma, 3 patient died of grade iv agvdh, 5 patients died of relapsed hlh or organ failure as results from unsuccessful treatment of the progressively elevated ebv-dna load. 2 patient died of tumor relapse, and 4 patient died of infection. acute gvdh occurred in 42 patients with grade i-ii agvdh in25 patients and grade iii-iv agvdh in 17 patients; chronic gvdh occurred in 19 patients. 46 patients achieved completed chimerism, 9 patients appeared with mixed chimerism,and2 patient presented with graft failure. of 34 ebv-hlh with engraftment, reactivated ebv infection was found in 33 (97%) with the whole blood ebv-dna load at 103-107 copy numbers per ml. ptld occurred in 3 patients confirmed by pathology. after reduced immunosuppressors, negative result of ebv infection was obtained while patients developed gvdh. for 39 ebv-hlh, patients who carry with ebv loading ebvdna ≤ 105 copies/ml before transplantation, overall survival rate was significantly higher than that of ebvdna4105 copies/ml (po0.05); who achieved cr+pr os was significantly higher than that of nr (po0.05); who range: from diagnosis to transplantion ≤ 6 months os was significantly higher than that of 46 months (po0.05). allogeneic hematopoietic stem cell is an effective method for primary hlh and lymphoma-hlh, ebv-hlh,even haploid transplantation. the remission status before transplantation is decisive for the prognosis. disclosure of conflict of interest: none. hepatic veno-occlusive disease after allogeneic hematopoietic stem cell transplantation in a single centre: revised diagnosis and incidence according to new ebmt classification s santarone, a natale, p olioso, g papalinetti, t bonfini, p accorsi, s angelini, g iannetti, m di ianni and p di bartolomeo dipartimento di ematologia, medicina trasfusionale e biotecnologie, bmt center, ospedale civile, pescara, italy sinusoidal obstruction syndrome, also known as venoocclusive disease (sos/vod), is a potentially life threatening complication that can develop early after hematopoietic cell transplantation (hct). in this study we retrospectively investigated the incidence, risk factors and outcomes of sos/vod in 978 transplants, performed in 896 patients between march 1982 and may 2016, on the basis of the new diagnostic criteria and classification of the ebmt. the patient's median age was 31 years (1 to 71). of them, 536 were males and 442 females. 896 patients received one transplant and 82 two transplants. a diagnosis of hematological malignant and nonmalignant disease was present in 784 and 194 cases, respectively. the disease risk at hct was standard in 397 cases, intermediate in 237 and high in 344. an hla identical sibling donor was used in in 691 cases, an unrelated donor in 166 and a haploidentical family donor in 121. conditioning was myeloablative in 813 transplants and at reduced intensity in 165. source of hematopoietic stem cells was bone marrow in 680 transplants and peripheral blood in 298. we did not limit the diagnosis of sos/vod to the classical 21 days after hct, but all suspicious cases appearing in the first 100 days were evaluated. sos/vod was diagnosed in 56 cases, of which 47 in the first 21 days after transplant and 9 between day 22 and 50 (median day 9). their main clinical characteristics are shown in table 1 . the severity of sos/vod was mild in 5 patients (9%), moderate in 6 (11%), severe in 8 (14%) and very severe in 37 (66%). the cumulative incidence (ci) of sos/vod was 5.7+0.005%. among the most relevant variables studied in univariate analysis (recipient age and gender, ferritin level at hct, type of hematological disease, disease risk at hct, type of donor, number of transplants, time of transplant, drugs used in the conditioning regimen, intensity of the conditioning regimen, source of stem cells), there was no factor with an adverse impact on sos/vod incidence. of 56 patients with diagnosis of sos/vod, 41 (73%) died. sos/vod was the main cause of death in 9 patients and a relevant contributing cause of death in 10. of relevance, 6 of 8 patients (75%) with severe sos/vod and 34 of 37 patients (92%) with very severe sos/vod died, whereas only one patient with moderate sos/vod died and no patient with mild sos/vod died. among 56 patients with sos/vod, 19 received defibrotide therapy and 37 the best supportive available therapy. defibrotide was given for a median of 20 consecutive days (range: 5 to 87), starting at day 18 post-hct (range: 3 to 49) with a median total bilirubin level of 3,16 mg/dl (range: 1.4-20.7). the 1-year overall survival (os) of patients treated with defibrotide was better as compared to that of patients who received the supportive therapy (47% versus 27%) although the difference doesn't reach the significance (p = 0.25). the occurrence of sos/vod does influence significantly the 1-yr os considering that it was 72 +1.5% for patients without sos/vod and 33+6% for patients with sos/vod (p = 0.0001). in conclusion, the new ebmt diagnostic and severity criteria for sos/vod has been very useful in identifying patients with severe and very severe forms of this complication. if validated in prospective studies, these criteria will allow an earlier selection of patients requiring immediate therapeutic intervention. [p692] disclosure of conflict of interest: none. the prognosis of patients with newly diagnosed ewing's sarcoma family of tumors (esft) has improved significantly over the last few decades. nonetheless, the long-term survival is still below 35% patients with high risk features.the role of s490 high dose chemotherapy and autologous stem cell transplantation (hdct and asct) for high risk and relapsed esft was analyzed. a retrospective medical chart review was done on patients with efst who underwent hdct and asct between september 1998 and january 2015 at seoul national university children's hospital. indications for hdct and asct included metastasis at diagnosis, bulky primary tumor (4100 ml), axial/ central primary site, and relapsed disease. single hdct and asct was performed in the earlier period, and the regimen was changed from mec (melphalan, etoposide, carboplatin), to topothiocarbo (topotecan, thiotepa, carboplatin), and to bumel (busulfan, melphalan). tandem hdct and asct was performed in the recent period, 1st hdct with bumel and 2nd hdct with modified mec (melphalan, etoposide, carboplatin). twenty-one patients who were diagnosed with esft at a median age of 8.7 years old underwent conventional chemotherapy, radiation therapy and/or surgery and received hdct and asct in complete response (n = 14) or partial response (n = 7). the overall survival of the patients was 70.0% at median 3.6 years and the event free survival (efs) of the patients was 55.0% at median 2.9 years from the last asct. the efs of the patients who underwent single hdct and asct with mec (n = 11), topothiocarbo (n = 1), and bumel (n = 4) was 54.5%, 0.0% and 75% respectively. the efs of the patients who underwent tandem hdct and asct (n = 5) was 50.0%. seven patients relapsed at median 6.6 months from the last asct. despite further treatment, 5 patients died of disease and 2 patients are currently alive without disease. one patient developed acute myeloid leukemia at 17.8 months from the last asct and is currently alive without disease after additional chemotherapy, hla-haploidentical stem cell transplantation and donor lymphocyte infusions. one patient died of transplantation-related mortality due to septic shock and lung infection. hdct and asct may be a promising treatment option for patients with high risk or relapsed esft. further refinements may be needed to identify the optimal regimen and number of hdct and asct. disclosure of conflict of interest: none. post transplant cyclophosphamide (pt-cy) has expanded the use of unmanipulated haploidentical grafts which have a high hla disparity between host and donor. one of the consequences of hla disparity is the development of engraftment syndrome (es). this is an immunological reaction characterized by non-infectious fever and skin erythema that develops after neutrophil engraftment. es resembles an infectious process but treatment involves the use of high dose steroids. our hypothesis is that pts undergoing haploidentical transplants (haplo) with pt-cy should have a high rate of es given the high hla disparity between donor and recipient. objectives: to determine the incidence, symptoms, morbidity and mortality of es in patients undergoing haplo with pt-cy at our institution. retrospective analysis of 22 patients with highrisk hematological diseases undergoing haplo with pt-cy at clinica santa maria between november 2012 and august 2016. es was diagnosed using the spitzer criteria (1). es was diagnosed if pts met 3 major criteria or 2 major plus 1 minor criterion. symptoms could occur prior to or after neutrophil engraftment (neutrophils over 500 cells / ul). all patients signed informed consent and the study was reviewed by our institutional review board. 22 patients received haploidentical grafts ( table 1) . all patients had neutrophil engraftment at a median of 18 days. 9/22 patients (41%) had symptoms that met criteria for es ( table 2 ). 2/9 were transferred to icu due to hypoxemia and 1 patient died after diagnosis of es. 5/9 pts were treated steroids. all patients received broad spectrum antibiotics during the febrile period and neutropenia. blood cultures, ebv and cmv pcr were negative in all es pts. there were no significant differences in hospital stay or one-year overall survival (os) between patients who developed and pts who did not develop es (median 37 vs. 35 days respectively, p = 0.68; one-year os 56% vs. 57%, p = 0.86, respectively). es is a frequent complication in patients undergoing hsct haplo with pt-cy. the incidence of es in our study was higher when compared to historical full match related donors series and lower when compared to cord blood transplant studies (2) there was no increased morbidity and mortality associated with es diagnosis. prompt institution of steroids is recommended in es patients after ruling out an underlying infectious process to avoid further complications. haploidentical allogenic hematopoietic stem cell transplants (haplo-hsct) is an alternative transplant procedure for patients with hematologic malignancies that are in need of transplant and do not have a compatible donor. due to the broad hla disparity, the haplo-hsct can be performed with t-cell depletion and megadose of cd34+. alternatively haplo-hsct can be performed with non t-cell depleted transplants (t-replete) either in combination with anti-thymoglobuline serum (atg) or post-transplant cyclophosphamide (pt-cy) as gvhd prophylaxis strategy. center effect is a known risk factor for outcomes of haplo-hsct in both t-cell depleted (tcd) and t-replete settings. however, many centers tend to specialize in one gvhd prophylaxis strategy making it difficult to differentiate the treatment effect from the center effect. the objective was to investigate the role of center effects in gvhd prevention strategy, on leukemia-free survival (lfs) and overall survival (os) in a population of adult patients with acute leukemia receiving haplo-hsct. a retrospective multicenter study was conducted on patients reported to the ebmt registry. inclusion criteria were: age 418 years, lymphoblastic or myeloid acute leukemia (all or aml) in first or second complete remission (cr1 or cr2), receiving a haplo-hsct between 2005 and 2014. in this population (n = 606), in order to assess the interaction between center and gvhd prevention treatment, we then included in the study selected patients from the centers that had performed more than 20% of both tcd and t-replete haplo-hsct during the study period. center effects on the outcomes consisted of 1) center effect on the baseline risk of event and 2) interaction between center and strategy of gvhd prevention. these center effects were estimated using cox mixed-effects models and tested using permutation tests. all models were adjusted on age, cmv statuses, disease (all or aml), secondary leukemia, previous autologous transplant, disease status (cr1 or cr2), peripheral blood vs. bone marrow transplant, conditioning regimen. after selection, 226 patients were available across 29 centers in europe. one hundred and one (45%) patients received tcd, 125 t-replete haplo-hsct (62 (27%) using atg and 63 (28%) using pt-cy). overall, 175 (77%) patients had aml. there were 86 (69%) peripheral blood transplants in the tcd group and 92 (91%) in t-replete. median follow-up was 2.7 years. in adjusted analyses, without accounting for center effect, t-replete tended to be associated with better lfs (hazard ratio (hr): 0.70 (95%ci 0.45-1.07), p = 0.10) and os (hr = 0.67 (95%ci 0.43-1.04), p = 0.076). when center effects were included, there was significant heterogeneity across centers on the baseline risk of both outcomes (lfs: p = 0.036 and os: p = 0.048). when accounting for interaction between center by strategy for gvhd prevention, the effect of t-replete vs. tcd on the outcomes did vary across centers (p = 0.065 and p = 0.031 for interactions in lfs and os, respectively) ( figure 1 ). we found an interaction between center and strategy for gvhd prevention on outcomes of patients who received a haplo-hsct. the difference between the 2 strategies (tcd or t-replete) varied across centers, in size and direction. this could be in part related to the increase in expertise with each technique in some centers and with the different management of complications, such as infections-related and relapse. disclosure of conflict of interest: none. adherence included recognition of spuriously high levels (typically from contaminated lines) and delayed dose adjustment due to late reporting of levels by the laboratory. the most common cause of unjustifiable non-adherence was failure to increase the dose in response to a low level. inadequate or excessive dose adjustments may be due to lack of experience or unfamiliarity with the sop. two interventions were launched with the aim of improving adherence to the sop for therapeutic tacrolimus dosing. firstly, to provide a rapid and user-friendly calculation method, we developed a mobile phone application (tacrocalc, a dose calculator based upon the sop algorithm) for android and ios devices using python and swift, respectively. secondly, to reduce the number of spuriously high levels, all nurses responsible for specimen collection participated in an educational module delivered by medical and senior nursing staff. key messages included the need to: use only the dedicated colour-coded tacrolimus lumen to infuse iv tacrolimus; avoid sampling from this lumen; sample peripherally when other lumens are known to be contaminated (reasons for this are being explored); suspend infusion of iv tacrolimus 15 minutes before taking a level; send only immediately pre-dose levels for oral tacrolimus. initial re-audit of 16 episodes post intervention (data collection is ongoing) demonstrated a 40% increase in sop adherence (p = 0.03; fisher's exact test), with no cases of unjustifiable non-adherence and a significant reduction in spuriously high levels. in conclusion, the use of tacrocalc by doctors and the implementation of targeted teaching for nurses dramatically improved adherence to the tacrolimus sop. this should ultimately improve therapeutic dosing whilst avoiding toxicity, which may result in better transplant outcomes. tacrocalc is now being adapted to include an option for paediatric dosing, with the potential to dose related medications such as cyclosporine. disclosure of conflict of interest: none. king's college hospital, 2 imperial healthcare, charing cross hospital and 3 imperial healthcare, hammersmith hospital managed with calcium and vitamin d alone in 28/43 cases (65%) and together with bisphosphonates in 9/43 (21%). osteoporosis was managed with bisphosphonates ± calcium/ vit d in 19/36 and with calcium/vit d alone in 10/36. 8 /34 indicated that they would give bisphosphonates in the absence of osteoporosis, if a patient with osteopenia was receiving long term steroids. dissemination and implementation of existing guidance on investigation and managing low bmd post hct appeared to be poor amongst respondents to our survey. routine dxa scanning was underused; the trigger for dxa in the context of steroids is inappropriately high at many centres at 1 mg/kg daily for 3 months; in established osteoporosis, bisphosphonates were used less frequently than would be anticipated. these findings may reflect the limited data on which current recommendations have been made, or the large number of non-transplant guidelines for investigating and managing low bmd which confound management of this post-hct patient group. hematopoietic stem cell transplantation (hsct) still remains as the most efficient therapy for adult patients with acute myeloid leukemia. for older patients and those lacking a hlacompatible donor, autologous hematopoietic stem cell transplantation (auto-hsct) is a valid therapeutic option. 1 authors aimed for determining the effect of auto-hsct for acute myeloid leukemia patients and analyze group of patients who underwent auto-hsct. the study has been set as a retrospective single center study. clinical information included age, gender, aml type and cytogenetic risk. pretransplantation treatment, mobilization and conditioning were analyzed and thus subsequently authors used kaplan and meier method to calculate the actuarial overall survival rate. table 1 describes patients' characteristics. majority of patients received similar induction therapy based on combination of cytarabine and anthracycline. timespan from the diagnosis to auto-hsct varied from 74 days to 1791 days, median was 175 days. seventy (88,6%) patients received a preparative regimen consisting of busulfan at 1mg/kg orally, four times daily for 4 days for a total dose of 16 mg/kg administered on day -6 through day -3 and melphalan 100-150 mg/m 2 intravenously for over 4 hours on day -2. patients achieved an absolute neutrophile count (anc) of ≥ 0.5 × 109/l in between 10 to 40 days; median was 14 days. patients achieved not transfused platelet count ≥ 20 × 109/l in between 10 to 209 days; median was 19 days. median of patients' discharge from hospital was 19 days (range: from 13 to 44 days) since auto-hsct. hundred day mortality after autologous transplant was at 6.32% (5/79). on the date of our evaluation (april 30, 2016), 48 patients were alive and in continued cr. the relapse rate was 39.5% (32 patients) and 7 patients (8.6%) were lost from follow-up. the 5-year overall survival (os) was 60.8%, so the target median of overall survival has not been reached. [p701] the development of dyslipidaemia is commonly observed after haematopoietic stem cell transplantation (hsct). few data are available concerning lipid profiles over a long followup period or with regard to the different transplantation types (autologous vs. allogeneic) or the effect of multiple transplantations on the development of dyslipidaemia. a retrospective, single center cohort study including 1239 adult patients (416 years) who underwent hsct at the university hospital basel s496 1973-2013 and who survived ≥ 100 days was performed. patients with at least a baseline lipid measurement were included (n = 1096) and grouped according to the type of their first hsct (autologous or allogeneic). for the examination of the effect of subsequent hscts, patients with consecutive transplantations of the same type were included and other patients were censored when a different transplantation type was performed. serial lipid profiles (total-, ldl-and hdl-cholesterol and triglycerides) before and after transplantation were examined. of the 1096 patients, 407 underwent a first, and 89 of these at least one subsequent autologous hsct. 689 underwent a first, and 85 of these at least one subsequent allogeneic hsct. median age of patients at autologous hsct was 52y (iqr 39-61) and 43y (32-53) at allogeneic hsct. 62% and 58% were males, median bmi pre-transplant was 25 (22-28) and 24 (22-27). the majority of patients underwent s497 intensive conditioning before hsct. median follow-up time was 3.0 years in the autologous and 4.8 years in the allogeneic group, with a maximum follow up time of 26.1 and 34.3 years, respectively. table 1 shows the number and percentage of patients with dyslipidaemia (1st autologous and allogeneic transplants). the distribution of exact total cholesterol values along with comparisons with baseline measurements according to group are presented in the figure 1 . *% based on number of measurements available total, ldl-and hdl-cholesterol and tg increased within 3 months of transplantation, regardless whether autologous or allogenic transplantation or a first or a subsequent transplantation was performed. the percentage of patients with dyslipidaemia accordingly rose significantly within 3 months of transplantation and persisted throughout follow-up. although patients undergoing an autologous hsct presented with higher baseline values of total cholesterol, a significantly greater increase post-transplant was observed after allogeneic hsct. first and subsequent transplantations seem to behave similarly with respect to changes in lipid profiles. disclosure of conflict of interest: none. nuremberg, erlangen, germany; 4 department of cancer immunology, institute for cancer research, oslo university hospital, radiumhospital, oslo, norway; 5 kg jebsen center for cancer immunotherapy, institute of clinical medicine, university of oslo, oslo, norway; 6 department of haematology and oncology, university hospital of the goethe university, frankfurt, germany and 7 childrens hospital, goethe university, frankfurt, germany natural killer (nk) cells are lymphocytes of the innate immunity with a potent anti-tumor capacity. in tumor patients, such as multiple myeloma (mm) patients, an elevated number of nk cells correlates with a higher overall survival (os) rate. our study adressed nk cells characteristics and anti-tumor ability in mm patients. especially cytotoxicity of patientderived, cytokine-stimulated nk cells against mm cells has been analyzed at various time points (at diagnosis, before/ after chemotherapy and/or auto-sct). nk cells from patients were analyzed by facs after pbmcs isolation via ficoll separation at different time points: tp1, before the start of high dose chemotherapy (hdc)/auto-sct; tp2, after early leukocyte recovery (leukocytes 41000/μl) and tp3: at least 2 weeks after tp2. for testing nk cell cytotoxicity against mm cells, nk cells were purified via negative selection and expanded in vitro for 1-2 weeks in low doses il-2 and il-15. nk cells were divided into the cd56 ++ cd16 − or cd16 + and cd56 + cd16 ++ subsets. while the major nk cell subset at tp1 was the cd56 + cd16 ++ nk cell subpopulation (71.86%), after leukocyte recovery at tp2 cd56 ++ cd16 − /+ nk cells were the main subsets (cd16 − : 22.85%; cd16 + : 36.51%). we further evaluated the nk cell function upon tumor interaction at the defined time points. cd56 ++ cd16 − nk cells were the main subset to produce ifn-γ upon interaction with k562 cells at all different time points. the percentage of ifn-γ-positive cd56 + + cd16 − nk cells was slightly decreased at tp2 compared to tp1 but significantly increased from tp2 to tp3 (p-value: 0.0008). similarly, mip-1β-and cd107a-positive cd56 ++ cd16 − cells remained constant between tp1 and tp2, whereas their percentages increased from tp2 to tp3 [p-values: 0.0056 (mip 1β) and 0.0232 (cd107a)]. moreover, in a small group of mm patients, we isolated nk cells and expanded them for 1-2 weeks prior to the functional assays. as expected, the expansion rate was reduced after chemotherapy compared to nk cells from healthy controls, but the patients nk cells increased their ability to kill mm cells due to the ex vivo cytokine expansion. conclusion: our data demonstrate that nk cells have an altered phenotype and function after hdc/auto-sct. remarkably, these nk cells were able to secrete cytokines and still displayed cytotoxic capacity against different types of tumor cells. however, as the proliferative capacity of nk cells seemed to be reduced following chemotherapy, innovative nk cell therapeutic approaches further improve the patients nk cell activity by an ex vivo cytokine stimulation procedure. finally, we suggest that an additive cell therapy with cytokinestimulated autologeous nk cells might improve the outcome of mm patients. lymphoid and myeloid acute leukemia are the most frequent type of cancer and the most frequent cause of cancer related death in children. relapse and refractory disease are the main clinical problems that current therapies are still unable to solve. one of the main nk cell activating receptors is nk cell group 2d (nkg2d). nkg2d receptor recognizes human mica/ulbp1-6 ligands. these nkg2d ligands are expressed in leukemia cells and constitute suitable targets for immunotherapy. the expression of nkg2d ligands was analyzed in peripheral blood mononuclear cells from 61 pediatric patients suffering from acute leukemia (21 acute myeloid leukemia, 25 b cell acute lymphoid leukemia and 15 t cell acute lymphoid leukemia), as well as in 7 leukemia cell lines (k562, rs4-11, jurkat, nalm-6, molt-3, reh and cem), by flow cytometry using specific monoclonal antibodies directed against mica, micab, ulbp-1, ulbp-2, ulbp-3 and ulbp-4, and by quantitative pcr using taqman probes. peripheral blood mononuclear cells from healthy donors were labeled with cd45ra microbeads and depleted using automacs device. the hl20i4r-mndanticd19bbz lentiviral vector was derived from the clinical vector cl20i4r-ef1a-hgcopt27 but contained the extracellular domain of nkg2d, the hinge region of cd8a and the signaling domains of 4-1bb and cd3-z. the cassette was driven by mnd promoter. viral supernatant was produced by transient transfection of hek293t cells with the vector genome plasmid and lentiviral packaging helper plasmids pcagg-hivgpco, pcagg-vsvg and pcag4-rtr2. cytogenetic studies and array comparative genomic hybridization were performed to analyze the genetic stability of lentiviral-transduced memory t cells. the in vitro cytotoxicity of cd45ra − t cells against leukemia cells, healthy pbmc and mesenchymal stem cells (msc) was evaluated by performing conventional 4-hour europium-tda release assays or by flow cytometry using cfse and 7aad labeling of target cells. nkg2dl were heterogeneously expressed in leukemia primary cells and cell lines. for b cell all primary samples, we found expression of mica/b, mica and ulbp1 decreased in refractory disease compared to remission (p = 0.01, p = 0.03 and p = 0.02, respectively). lentiviral transduction of nkg2d-4-1bb-cd3z markedly increased nkg2d surface expression in cd45ra − memory t cells, which became consistently more cytotoxic than untransduced cells against leukemia cells. additionally, no chromosomal aberrations nor cytotoxic activity against healthy pbmc or mesenchymal stem cells was observed in nkg2d car expressing t cells. our results demonstrate nkg2d-car redirected cd45ramemory t cells target nkg2dl expressing leukemia cells in vitro and could be a promising and safe immunotherapeutic approach for acute leukemia patients. peripheral blood stem cell mobilization and collection from elderly patients (≥65 years) with multiple myeloma: a single center experience g cengiz seval 1 , sk toprak 2 , s civriz bozdag 2 , m kurt yuksel 2 , p topcuoglu 2 , o arslan 2 , m ozcan 2 , t demirer 2 , g gurman 2 , h akan 2 , m beksac 2 and o ilhan 2 1 clinic of hematology, yildirim beyazit university yenimahalle education and research hospital and 2 department of hematology, ankara university school of medicine high-dose melphalan followed by autologous hematopoietic cell transplantation (auto hsct) has become the standard procedure for patients with symptomatic multiple myeloma (mm). the ability to mobilize stem cells from healthy donors shows little deterioration with age, the influence of patients' age on auto hsct is uncertain and studies in patients' ≥ 65 years are scarce. severe studies specific to mm have failed to show an independent effect of patient age on cd34+ mobilization. we retrospectively compared myeloma patients below the age of 65 with patients above 65 years of age, analyzing cd34 mobilization into peripheral blood and the number of leukapheresis needed to collect at least one single stem cell graft. material and methods: from february 1999 through april 2016, data from 501 myeloma patients below the age of 65 were compared to 52 myeloma patients above 65 years of age. all these data were obtained from the ankara university faculty of medicine center for therapeutic apheresis and written informed consent was signed according to our institution regulations. most of the patients received only gcsf at a dose of 5 μg/kg bw twice-daily s.c. until stem cell procurement. patients underwent further pbsc collections until we obtained the target dose 420 cd34+ cells/μl blood. a maximum of 3 collections were performed in the first mobilization; if the cell dose was not achieved, we submitted patients to a second mobilization. fifty two of 553 patients were above 65 years of age (median age 66, range: 65-73) and 501 patients were below the age of 65 (median age 54, range: 29-64). baseline characteristics of the older and younger patient cohorts are summarized in table 1 . mobilization regimens for the younger patient population were cyclophosphamide based (n: 122), g-csf only (n: 372) and +plerixafor (n: 7). mobilization in the older population was with cyclophosphamide based (n: 10), g-csf only (n: 41) and +plerixafor (n = 1). the chemotherapy regimens were not statistically different between both age groups. there were no significant statistical differences in time from diagnosis to mobilization, number of prior therapies or disease status between both patient groups. the number of cd34+ circulating cells before scheduled leukapheresis was mean 69.28 cells/μl (median 49 cells/ μl, range: 2-397; sem ± 46.875) in all patients (including patients who failed mobilization). our data support the observation that after a standard mobilization regimen with anti-myeloma chemotherapy and once-daily growth factor support, patients above 65 years of age show an impaired cd34 mobilization into peripheral blood compared to a younger population. this can be overcome by an increased number of leukaphereses. still the number of progenitor cells in the actual graft is inferior compared to the younger population. [p712] disclosure of conflict of interest: none. donor and/or recipient citomegalovirus (cmv) seropositivity has been associated with a poor overall survival (os) in patients who have received an allogeneic hematopoietic stem cell transplantation (allohsct). in comparison with seronegative donors, hsct from seropositive donors has been associated with decreased disease-free survival (dfs) and increased non-relapse-related mortality (nrm). we analyzed the prognostic impact of cmv serology status (donor/ recipient) in patients diagnosed with acute leukemia (al) [p713] s503 who had received an allohsct in our institution. retrospective unicentric study of patients diagnosed with al between 2001 and 2015 who received allohsct.the following outcomes were studies: os, dfs, and cumulated incidences of relapse (ri), nrm, acute graft-versus host disease (agvhd) and chronic gvhd (cgvhd). the series included 163 patients (86 males, 77 females), median age of 44 years . al type: 42 (26%) all, 121 (74%) aml. type of transplant: 88 (54%) related donor, 42 (26%) unrelated donor and 33 (20%) unrelated umbilical cord blood. the majority, 111 (68%), received myeloablative conditioning. stem cells source: peripheral blood 124 (76%), cord blood 33 (20%) and bone marrow 6 (4%). cmv serology status: positive receptor 124 (76%), negative receptor 39 cases (24%); positive donor 100 (61%), negative donor 63 (39%). serology status combinations (d/r): +/+ 84 (52%), +/ − 40 (24%), − / − 23 (14%), − /+ 16 (10%). 56 patients developed agvhd and 14 (9%) cgvhd. the impact of donor/recipient cmv serology status on os, dfs, ri, nrm and incidence of agvhd and cgvhd for the overall series is reported in table 1 . no statistically significant differences were detected in any of the analyzed variables. in this study, donor/recipient cmv serology showed no influence on the analyzed variables os, dfs, al relapse, nrm, acute and chronic gvhd. however, the sample size limits the validity of the results. disclosure of conflict of interest: none. supported in part with the grants pi10/01417 from fondo de investigaciones sanitarias and rd12/0036/0029 from rticc, instituto carlos iii and 2014sgr225(gre), generalitat de catalunya, spain. petersburg, russia during the last two decades ahsct has been used as a treatment option for ms with promising outcomes. qol is an important outcome of ms treatment. its assessment gives the patient's perspective on the overall effect of treatment. we aimed to study qol in ms patients before and after ahsct and search the value of the data obtained for decision-making. a total of 135 patients with different types of ms were enrolled in the study: mean age-34 (range-17-54) years old; male/ female-53/82; mean edss-3.5 (range: 1.5-8.5). all patients were treated by ahsct. reduced-intensity beam-like conditioning was used (bcnu 300 mg/m 2 , etoposide 100 mg/m 2 , ara-c 100 mg/m 2 and melphalan 100 mg/m 2 ). mean follow-up was 24 months (range: 12-53 months). qol was assessed using generic questionnaire sf-36. for comparisons t-test for independent samples or mann-whitney test was used. qol parameters in ms patients at 12 months after ahsct improved in comparison to base-line: physical functioning-66.3 vs 52.6, role-physical functioning-62. 8 6 . further qol improvement was registered at long-term follow-up: integral qol index exhibited 0.50 at long-term follow-up as compared to 0.32 at base-line. qol improvement was more dramatic in relapsing-remitting ms than in progressive ms. we found a significant increase of all eight sf-36 scales in a year posttransplant as compared with base-line in relapsing-remitting ms patients (po 0.05). in progressive ms patients statistically significant improvement was registered for six out of eight sf-36 scales (except bodily pain and role-emotional functioning) (p o0.05). improved qol parameters were preserved over the entire study period in all the patients who did not have disease progression or relapse. in conclusion, qol monitoring in ms patients after ahsct provides clinicians with the unique information regarding the changes in physical, psychological and social well-being of patients who have been treated with this new treatment modality. it allows to evaluate risks/ benefits of ms patients undergoing ahsct and might influence decision-making. further studies are needed to examine the trajectory of qol changes in this patient population to better define treatment outcomes after ahsct. disclosure of conflict of interest: none. pediatric patients with leukocyte adhesion deficiency type-i (lad-i), a rare autosomal recessive primary immunodeficiency disorder, experience severe and recurrent lifethreatening bacterial infections. allogeneic haematopoietic stem cell transplantation (hsct) offers the possibility of curative therapy although the conditioning regimen used for hsct in lad-i is still a controversial issue. this study provides evaluation of outcome of the lad-i pediatric patients who underwent reduced-intensity conditioning (ric) hsct. twenty four patients (14 female) with severe lad-i who received 26 hscts between februay 2007 and september 2016 at our center were enrolled. the median age at hsct was 30 months (range:4 months-14 years). patients received bone marrow (n = 9), peripheral blood progenitor cells (n = 14) or umbilical cord blood grafts (n = 3) from hla-matched related donors (n = 18), mismatched related or unrelated donors (n = 4), unrelated fully matched donors (n = 1) and haploidentical relative donors (n = 1). ric regimen was provided with fludarabine, melphalan and anti-thymocyte immunoglobulin. cyclosporine a and prednisolon were used as graft-versus-host disease (gvhd) prophylaxis. engraftment occurred in 23/26, of which one patient experienced graft rejection.the median times to neutrophil and platelet engraftments were 12 days (range: 10-23days) and 15 days (range: 10-32days), respectively. with a median follow-up of 43 months (range: 2-95months), overall survival (os) was 70.8%.the main causes of death were gvhd and infection. acute gvhd occurred in ten patients (4 grade i-ii, 6 grade iii-iv) and 3 patients also developed chronic gvhd. there were no significant differences in acute gvhd occurence and also os regarding to the stem cell sources. at this time,10 patients with full chimerism and 6 patients with mixed chimerism are alive and disease free. conclusion: hsct offers long term benefit in lad-1 and should be considered as an early therapeutic option if a suitable hla-matched stem cell donation is available. as pretransplant infections in primary immunodeficient patients especially those affected by lad-1 lead to rise in mortality rate, ric regimen is found to be safe and mixed donor chimersim appears sufficient to prevent significant symptoms. disclosure of conflict of interest: none. tregs based immunotherapy may be beneficial in several immune mediated diseases including graft versus host disease (gvhd). the possibility of cryopreserving tregs might lead to the administration of multiple doses, thus potentially increasing their efficacy in chronic diseases. however, there are few and controversial data on the functionality of tregs after cryopreservation. here, we evaluated the phenotype and the inhibitory capacity of thawed tregs. tregs were purified from leukapheresis of normal donors (n = 3) by double immunomagnetic depletion (cd8 and cd19) followed by cd25 enrichment using the clinimacs system (miltenyi biotec) under gmp condition. the cells were cryopreserved in saline solution containing 10% human serum albumin (hsa) and 10% dmso with a controlled-rate freezing. cell viability was assessed by 7-aad staining. number/phenotype and function were evaluated on fresh and thawed tregs. cryopreserved autologous t effector (teff) cells were used in mlr assays. before cryopreservation the tregs enriched product mean viability was 95 ± 4% and the mean percentage of cd45+cd4 +cd25+cd127low and cd45+cd4+cd25+cd127lowfoxp3+ cells was 74 ± 13% and 66 ± 10%, respectively. we then analysed the tregs enriched product after thawing. mean viability of thawed tregs, by 7-aad staining, was 85 ± 7%. the viable tregs were almost totally cd4+cd25+ (97 ± 2%). the mean percentage of cd4+cd25+cd127low and cd4+cd25 +cd127lowfoxp3+ thawed cells was 73 ± 14% and 71 ± 20% respectively. the contaminant cells present in the treg enriched product were mostly cd4+cd25+cd127+ (around 18%). we further characterized the phenotype of the cd4 +cd25+cd127low population. this population was almost totally foxp3+ (93 ± 6%) and expressed selected markers at various degree (cd62l (50 ± 2%), cd15s (6 ± 2%), cd45ra+ (19 ± 3%), hla-dr+ (15 ± 10%), ccr7+ (74 ± 5%), cd49d (52 ± 14%), cd26+ (1 ± 0.4%), cd196+cd161+ (4 ± 1%). notably, viable thawed tregs were able to induce inhibition of autologous teff cells in a 1:2 tregs:teff ratio as freshly isolated tregs: 44 ± 16% (thawed) vs 55 ± 24% (fresh) of inhibiton (p4 0.1). in conclusion, here we demonstrated that thawed tregs from healthy donors mantain a stable phenotype. in addition, in our hands tregs show good suppressive ability after thawing despite lower expression of cd62l and cd15s relapsed and refractory malignant b cell diseases: evidence for therapeutic efficacy via subcutaneous administration of anti-cd20 × anti-cd3 antibody lymphomun r buhmann, p ruf, j hess, h lindhofer, u jacob 1 and m dreyling 1 the trifunctional antibody anti-cd20 × anti-cd3 lymphomun represents a chimeric immunoglobulin scaffold (mouse igg2a/ rat igg2b) with promising treatment outcome in patients suffering from malignant b cell diseases. by changing the lymphomun administration route from intravenous (i.v.) to subcutaneous (s.c.) the proinflammatory cytokine-mediated side effects were considerably slighter and generally welltolerated. most importantly, s.c. lymphomun showed outstanding responses in b cell depletion even in the absence of elevated cytokine levels (e.g. il-6) that are required for cytotoxic t cell activation. in summary, the clinical tolerability of s.c. lymphomun may result in a considerable improvement of the subjective well-being and in enhanced mobility due to decreased pain symptomatology. intestinal microbiota disruption is associated with acute gastrointestinal (gi) gvhd and inferior outcome in patients after allogeneic stem cell transplantation (asct). the wide use of systemic broad spectrum antibiotics adds a further risk factor contributing to major microbiota shifts. here, in a retrospective analysis of 200 patients undergoing asct at the regensburg university medical center we assessed the relative expression of paneth cell antimicrobial peptides (amps) in 292 human intestinal biopsies in relation to acute gi gvhd and systemic antibiotic treatment. the relative expression of paneth cell amps was significantly higher in biopsies of the upper gi tract than in the lower gi tract for reg3α (p ≤ 0.001), human defensin (hd) 5 (p ≤ 0.001) and hd6 (p ≤ 0.001). regarding the distribution of paneth cell amps in the gi tract we observed significantly higher expressions of all three paneth cell amps in the duodenum, jejunum and ileum compared to the stomach, colon and rectum (po0.001, figure 1 ). in the presence of acute gi gvhd, paneth cell amps reacted contrarily in the upper and lower gi tract: we observed a decrease of hd5, hd6 and reg3α in the upper gi tract (p ≤ 0.01), similarly paneth cell count dropped in case of severe gi gvhd stage 2-4 (po0.001). however in the lower gi tract severe acute gi gvhd was associated with an increase of paneth cell amps (p ≤ 0.03). initiation of additional systemic antibiotic treatment prior to day 10 after asct correlated with a significantly higher expression of hd5 (p = 0.002) and reg3α (p = 0.01) in intestinal biopsies compared to patients without or with initiation of systemic antibiosis after day 10. however, no significant differences were found in terms of hd6 expression in intestinal biopsies and start of systemic antibiotic therapy. the expressions of hd5, hd6 and reg3α in intestinal biopsies seem to respond to major microbiota disruptions caused by acute gi gvhd or systemic antibiotic treatment. while observations in the upper gi tract seem to reflect paneth cell damage, the relative increase in the lower gi tract may indicate inflammatory induction of amps in colonic epithelial cells in the course of gvhd. [p718] disclosure of conflict of interest: none. patients (55%) were in complete remission at the time of pcy haplo-sct. hematopoietic cell transplantation-comorbidity index was ≥ 3 in 20 patients (74%). thirteen patients (48%) received non-myeloablative conditioning regimen (as baltimore schema, luznik et al. bbmt 2008) prior to haplosct while remaining patients received busulfan-based regimen. all patients were given pcy and both csa and mmf as gvhd prophylaxis. day+100 cumulative incidence of grade 2 to 4 and 3 to 4 acute gvhd was 15% and 7%. 2-year cumulative incidence of chronic gvhd was 12%. the cumulative incidence of non-relapse mortality and relapse at 2 years were 38% and 27%, respectively. with a median follow up of 25 months (range: 4-63), 2-year progression-free and overall survivals were 36% and 39%, respectively. disease status at the time of haplosct was a major determinant for outcome. indeed, 2year nrm and os were 58% and 25% in patients transplanted with active disease, respectively, while corresponding values in patients transplanted in cr were 21% (p = 0.036) and 49% (p = 0.041), respectively ( figure 1a and 1b) . we can conclude that in selected patients who could be candidate for second transplantation, haplosct is feasible and may represent a curative option. the overall incidence of relapse of 27% is promising in this situation for which no alternative for cure is available, with relatively good survival in patients transplanted in cr. however, the very high nrm (58%) in refractory patients should make us consider second transplant with caution in this setting. for these patients, specific developments are needed to avoid procedure-related toxicity. [p722] disclosure of conflict of interest: none. secondary solid tumors following hematopoietic cell transplantation for thalassemia major a natale, s santarone, a meloni, a pepe, m di ianni, s angelini, p di bartolomeo 1 1 dipartimento di ematologia, medicina trasfusionale e biotecnologie-ospedale civile, pescara, italy secondary solid tumors (sst) have been described after hct, in particular for patients affected by hematologic malignancies. there is limited information about the incidence of sst following hct for thalassemia major (tm). the aim of this study was to determine the incidence of sst in 134 patients with tm who received hct in our center between 1983 and 2013. 117 patients survived more than 3 years after hct and were enrolled in the study. of them, 57 were males and 60 females. their median age at time of hct was 10 years (1-29). as conditioning regimen, they received busulfan (14 mg/kg) and cyclophosphamide (200 mg/kg). the gvhd prophylaxis included cyclosporine and methotrexate. all patients received bone marrow cells from an hla identical donor. at time of this report, 112 patients were cured, whereas 5 patients rejected their graft and are now under regular transfusion treatment. overall, the median follow-up after hct was 24 years (3-34). seven patients developed a malignancy 3.2 to 28 years (median 16.4 years) after hct including 2 carcinomas of the tongue, 1 oral squamous cell carcinoma, 1 colorectal cancer, 1 thyroid carcinoma, 1 carcinoma of the uterine cervix, and 1 parotid carcinoma. the 30-yr cumulative incidence (ci) of developing sst was 10+0.17%. all patients underwent surgical resection of the tumor and in addition 4 of them received chemotherapy and/or radiotherapy. of relevance, the 3 patients with cancer of the oral cavity were affected by severe chronic gvhd with buccal cavity involvement. 2 patients (1 with parotid and 1 with tongue carcinoma) died of tumor progression and 5 are living. we compared these results with 2 case control populations. first of all, we investigated the occurrence of solid tumors in the 117 individuals (64 males, median age 10 years at time of marrow donation), who served as stem cell donors for hct. one donor developed breast cancer 29 years after marrow donation at age of 38. the 30-yr ci of developing solid tumor for donors was 4.5+0.21% with a statistically significant difference (p = 0.03) as compared to that of transplanted patients. the second case control population consisted of 117 patients affected by tm treated with transfusions and iron chelation. the matching technique applied was based on the variables age and sex. one control per case (transplanted patient) was randomly selected from the miot (myocardial iron overload in thalassemia) registry and matched by sex and age with the transplanted patient population. 2 patients developed an hepatocellular carcinoma (hcc) at age of 39 and 44 years, respectively. one patient died and one is living. using the event rate measure, we observed an event rate of 0.102 at 30 years for the transplant group and 0.041 for the nontransplant group (p = 0.106). this study shows that the magnitude of increased risk of sst is twofold to threefold for patients treated with hct as compared with an age-and sex matched nontransplant tm patients or with stem cell donors. notably, among the transplanted patients we didn't observe any case of hcc, which is one of the most frequent solid tumor in nontransplant tm patients, whereas we observed 4 cases of head/neck cancers. in our series, cgvhd seems to be a strong risk factor in the development of new solid tumors. patients with cgvhd, especially those with involvement of the oral cavity, must receive a very long careful monitoring and surveillance in order to prevent the development of secondary cancers. disclosure of conflict of interest: none. sequential treatment with bortezomib plus thalidomide plus dexamethasone followed by autologous hematopoietic stem cell transplantation (hsct); consolidation and maintenance therapy in patients with multiple myeloma a bachiri 1 , ma bekadja 2 , s talhi 2 , s abderrahmani 1 , h ouldjeriouat 3 and r bouhass 2 1 department of hematology, hmru oran, oran, algeria; 2 department of hematology and cell therapy, ehu1st november, oran, algeria and 3 department of hematology and cell therapy, oran, algeria the management of multiple myeloma (mm) has been significantly improved in recent years in young patients, where ahsct and advent of new molecules was introduced as first line treatment. the sequential treatment (induction followed by autologous hematopoietic stem cell transplantation; consolidation and maintenance therapy) has increased rates response (cr and vgpr) as well as the overall survival. our purpose was to assess the efficacy and adverse effects of sequential treatment with vtd chemotherapy and autologous hsct followed by consolidation and maintenance therapy. in a prospective multicenter study, we evaluated this mm management strategy at oran, in two hematology centers. patients aged under 65 years with de novo mm, were treated with induction included: bortezomib (1.3 mg/m 2 , d1-d4-d8-d11), thalidomide (100 mg/ m 2 d1-d21) and dexamethasone (40 mg, d1-d4; d8-d11). a total of 3 to 4 cycles where delivered every 21 days. autologous stem cell was mobilized using g-csf alone (15 μg/kg/day for 5 days). leukapheresis to harvest stem cells were performed on day -2 and -1. the conditioning regimen consisted of melphalan 200 mg/m 2 . a consolidation phase was initiated two months later with the same protocol (vtd), followed by a maintenance treatment with thalidomide 50 mg/day given orally for 12 months. this study was done over a 6-years period (january 2010-december 2015). fifty patients were included. they include 19 women and 31 men (sex ratio = 1.63). the median age at diagnosis was 53 years (32-64). according to durie salmon staging, 80% of patients were in stage iii, while 38% were in stages iii according to iss staging. the monoclonal component was igg in 56% of patients. postinduction overall response rate in the 50 eligible patients was 100%, including 52% vgpr and 38% cr/ and 10% pr rates. the median of cd34 + rate was 3.88x10 6 /kg (1.41 to 11). all patients had engraftment on the median of day 10 (range; 7 to 14) and platelet transfusion independence on the median of day 13 (range; 9 to 19). there was no graft failure. one patient died following the procedure (trm). posttransplantation on day 100, cr and at least vgpr remained significantly higher (98%). in the 49 evaluable patients, the estimated os at 79 months was 82%, the estimated dfs at 43 months was 66% and the pfs at 78 months was 66.5%. at the 30/09/2016, 43 (86%) patients are alive and 39 (80%) without disease activity after a median follow-up of 33 months (range; 3-79). the main hematological toxicities post transpland (grade 3/4) were thrombocytopenia (49%), neutropenia (50%), and anemia (8%). the most frequently observed nonhematological toxicities (all grades) included peripheral neuropathy (66%). our experience suggests that the sequential protocol used in first line produce a better outcome with fewer adverse events and is an interesting therapeutic option in term of efficacy and tolerance. disclosure of conflict of interest: none. micrornas are small, non-coding single-stranded rnas and regulate approximately 50% of all genes by repressing translation. they are present in bodily fluids, where they are protected from rnase-mediated degradation by encapsulation into extracellular vesicles (evs) and demonstrate a novel capacity to regulate the cellular differentiation of blood cells and immune function. candidate micrornas mir-377, mir--199, mir-93* and mir-423 have previously been associated with acute graft versus host disease (agvhd) in posthematopoietic stem cell transplant (hsct) patient plasma. however, validation in independent cohorts is necessary, and their presence within extracellular vesicles (evs) has not been explored. microrna expression was evaluated in a prognostic cohort (n = 81) of day 14 (d14) post-hsct patient serum samples by taqman qrt-pcr. further assessment in an independent cohort of serum samples taken at the time of agvhd diagnosis was also performed. expression was also assessed in serum evs at sequential time points (pre-hsct, d0, d7 and d14) and an independent verification cohort of d14 serum samples by ev isolation, rna extraction and taqman qrt-pcr analysis. this study replicated elevated serum expression of mir-423 (po 0.001), mir-199 (p = 0.04), mir-93* (p o0.001) and mir-377 (p = 0.03) in agvhd, in a prognostic cohort of d14 post-hsct patient samples (n = 81). expression was also associated with disease severity. further analysis at agvhd diagnosis in an independent cohort (n = 65) confirmed high expression of mir-423 (p = 0.02), mir-199 (p = 0.007) and mir-93* (p = 0.004) at disease onset. investigation of microrna expression patterns during early hsct at sequential time points (pre-hsct to d28) identified elevated micrornas at d7 post-hsct in all transplant patients. in a novel investigation of microrna expression in serum evs (n = 15), mir-423 (p = 0.09), mir-199 (p = 0.008) and mir-93* (p = 0.001) levels were lower at d14 in patients who later developed agvhd, and this was replicated for mir-423 (p = 0.02) and mir-199 (p = 0.04) (n = 47). comparing serum to circulating evs, at d14 patients remaining agvhd-free had significantly higher expression of mir-423 (p = 0.03), mir-199 (p o0.001) and mir-93* (p = 0.001) in the ev fraction. results validate the capacity for circulating serum mir-423, mir-199 and mir-93* to act as diagnostic and prognostic biomarkers for agvhd. novel findings of differential expression between whole serum and the ev compartment prior to disease onset suggest a role for ev micrornas in the biology of agvhd, which warrants further investigation. disclosure of conflict of interest: none. prior data indicate similar outcomes after transplants from hla-haplotype-matched relatives, hla-idntical siblings and hla-matched unrelated donors. we used our dataset to answer a clinically important question: who is the best donor for a person with acute leukemia. we analyzed data from persons with acute leukaemia in 1 st complete remission treated in a prospective, multi-centre study. patients were randomly divided into training (n = 611) and validation (n = 588) sets. 1199 consecutive subjects received a transplant from an hla-haplotype-matched relative (n = 685) or an hlaidentical sibling (n = 514). 3-year leukaemia-free survivals (lfss) were 75% (95% confidence interval [ci], 72, 78%) and 74% (70, 78%; p = 0.95). the multivariate model identified 3 major risk factors for transplant-related-mortality (trm): older donor/recipient age (donor440years/recipient430 years; hazard ratio [hr] = 1.88; [1.05, 3.35]; p = 0.03), female-to-male transplants (hr = 2.11; [1.50, 2.98; p = 0.01) and donor-recipient abo major-mismatch transplants (hr = 1.55 [1.08, 2.23; p = 0.02). a risk score was developed based on these three features. trms were 8% (5, 10%), 15% (12, 18%) and 31% (19, 43%) for subjects with scores of 0-1, 2 and 3 (p o0.001). 3 year lfs were 78% (75, 81%), 74% (70, 78%), and 58% (45, 71%; p = 0.003). the risk score was validated in an independent cohort. in recipients 450years, lfss were 69% and 86% (p = 0.08) after transplants from identical-sibling or children. our data confirm donor source or degree of hla-disparity is not significantly correlated with transplant outcomes. selection of the best donor needs to consider donor-recipient age, sex-matching and abo-incompatibility amongst persons with acute leukemia receiving transplants from family members. [p726] disclosure of conflict of interest: none. synergistic effect of kir ligands missing and cytomegalovirus reactivation in improving outcomes of haematopoietic stem cell transplantation for treatment of myeloid malignancies d cardozo, a marangon, r da silva, fj aranha, j visentainer, s bonon, s costa, e miranda, c souza and f guimarães. the lack of one or more hla class i alleles, whose protein products are the ligands for kir receptors, has been exploited as a prognostic factor for the outcome of patients with haematological malignancies treated by haematopoietic stem cell transplantation (hsct). although it has been accepted that kir-hla interactions may influence the outcome of the hlamismatched hsct, there is no consensus regarding the settings of hla-matched transplantation. there are studies that have reported either benefits, or no effects, under the influence of inhibitory kir-hla interactions. additionally, certain activating kirs and/or reactivation of cytomegalovirus (cmv) infection have been reported to affect the outcome of hla-matched transplantation. the goal of this study was to evaluate the influence of kir-hla genotypes on the outcome of patients undergoing treatment for haematological malignancies by non-t-depleted lymphocyte haematopoietic stem cell transplantation (hsct) from hla-matched sibling donors. the prospective study was conducted at the center of hematology, university of campinas, and 50 patients and their donors were followed up from 2008 to 2014. kir and hla class i genes were genotyped and patients grouped based on the presence of kir ligands combined with kir genotype of their respective donors. patients with all kir ligands present (n = 13) had a significantly higher (p = 0.04) incidence of acute graft-versus-host-disease (gvhd) than patients with one or more kir ligands missing (n = 37). the overall survival following transplantation of patients with myeloid malignancies (n = 27) was significantly higher (p = 0.035) in the group with one or more kir ligands missing (n = 18) than in the group with all ligands present (n = 9). presence of kir2ds2 was associated with a worsening of hsct outcome while reactivation of cytomegalovirus (cmv) infection improved the outcome of patients with one or more kir ligands missing. our results indicate that kir-hla interactions affect the outcome of the hla-matched transplantation, particularly in patients with myeloid malignancies. disclosure of conflict of interest: none. p = 0.015), lower disease-free survival (p = 0.015 and p = 0.015) and lower overall survival (p = 0.01 and p = 0.014). one-year cir of the above two groups were 5.6 ± 5.4% vs. 57.1 ± 18.7% in mrd negative and positive patients, respectively (p = 0.001). in addition, those who had consistent positive mrd prior to hlamatched sibling hsct showed even worse outcomes compared to patients without pre-mrd. unmanipulated haploidentical hsct might have the stronger graft-versus-leukemia effect compared to hla-matched sibling hsct. it suggested that those who received unmanipulated haploidentical hsct with pre-mrd might not need more intensive relapse intervention after transplantation. disclosure of conflict of interest: none. the retrospective study of allogeneic hematopoietic cell transplantation for 36 patients with mixed-phenotype acute leukemia in toranomon hospital, japan in the real clinical setting, however, there are substantial number of patients who can not achieve cr after chemotherapy. we conducted a retrospective study including such patients to elucidate the outcome of allogeneic hct in toranomon hospital, japan. we studied the patients with mpal diagnosed from july 2008 to september 2015. mpal was diagnosed according to who classification in 2008. from june 2013, we examined cytoplasmic myelo-peroxydase (cmpo) routinely for flowcytometric analysis in all the patients, to distinguish mpal from acute lymphoblastic leukemia (all). we included the patients who were diagnosed as mpal in toranomon hospital, regardless of their diagnosis or clinical course in the previous hospitals. a total of 36 mpal patients underwent their first allogeneic hct with related bone marrow or peripheral blood stem cells (r-bm/pb) (n = 9), unrelated bone marrow (u-bm) (n = 6), and unrelated umbilical cord blood (u-cb)(n = 21). the median patient age was 41 years (range:17-69). the immunophenotype of leukemia cells included 23 cases of b and myeloid (b/my) (64%) and 13 cases of t and myeloid (t/my) cell lineage(36%).eleven patients(31%) harbored philadelphia chromosome. the remission induction chemotherapy was performed with all-type regimens in 31 patients, and acute myeloid leukemia (aml)type regimens in 5 of 36 patients, 18 patients(50%) were not in cr at the time of transplantation. myeloablative conditioning (mac) regimens were used in 30 pantients(83%). the 2-year overall survival (os) rate was 43.1% (95% confidence interval (ci), 25.7-59.4%). to identify the factors that influenced os, we performed univariate analysis and compared the following pre-transplantation factors: age at the time of transplantation ( o41 vs.4 = 41 years), committed immunophenotype (b/my vs.t/my), karyotype (philadelphia chromosome (ph vs.non-ph), disease status at the time of transplantation (cr vs.non-cr), donor cell source (r-bm/pb vs.u-bm vs.u-cb, cb vs.non-cb), and conditioning regimen (mac vs.reduced intensity conditioning). cr at the time of transplantation was extracted as a significant predictive factor for the better os(2-year os; cr vs. non-cr, 63.0% (95% ci, 32.1-82.8%) vs.22.2% (95% ci, 6.9-42.9%), p = 0.009). the cumulative incidence of relapse rate (rr) at 2 years after transplantation was 53.3% (95% ci, 31.8-70.8%). to identify the factors that influenced relapse rate, we performed univariate analysis and compared pretransplantation factors same as above. harboring philadelphia chromosome was extracted as a significant predictive factor for lower relapse rate (2-year rr; ph vs.non-ph, 27.3%(95% ci, 0.1-77.3%) vs. 66.5%(95% ci, 41.4-82.8%), p = 0.003). the older patients(p = 0.07) and the patients in cr (p = 0.05) also showed a trend towards lower relapse rate. allogeneic hct provided 63.0% of 2-year os for mpal patients in cr at the time of transplantation. on the other hand, for patients not in cr, 2year os was approximately 20%. the use of tyrosine kinase inhibitors along with chemotherapy before transplantation might prevent relapse after transplantation in mpal patients with ph chromosome. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (allo-hsct) is a standard of treatment for many patients with hematological malignancies. however, the disease relapse and graft failure after first allo-hsct (1 st allo-hsct) lead to poor outcomes almost in all cases. second allo-hsct (2 nd allo-hsct) is one of primary options that can decrease the mortality in this group of patients. here we report our experience of 15 patients who underwent 2 nd allo-hsct. the aim of the study was to estimate a clinical efficiency and practicability of 2 nd allo-hsct. we included 15 patients (9 males/6 females) with acute myeloid leukemia (aml, n = 10), acute lymphoblastic leukemia (all, n = 3) and myeloproliferative disease (mpd, n = 2) who underwent 2 nd allo-hsct for relapse (66,7%) or graft failure (33,3%) from the same (n = 8) or another donor (n = 7) between november 2012 and october 2016. median age was 31 years (range: 18-42 years). three (20%) patients had a matched related donor (mrd), nine (60%) patients had a matched unrelated donor (mud) and three (20%) patients had a mismatched unrelated (mmud) at the second transplant. to evaluate time gap affecting outcomes all patients were divided into two groups: who underwent 2 nd allo-hsct in more/less than 6 months after 1 st allo-hsct. in "less than 6 months" group three patients were re-transplanted for relapse and one-for graft failure, in other group there were seven patients who received 2 nd allo-hsct for disease relapse and four-for graft failure. fisher's exact test were performed to exclude probability of imbalance between groups (p40.05). median of overall survival (os) and disease-free survival (dfs) after 2 nd allo-hsct was 13.5 months and 10.59 months respectively. (see figure 1a ,1c) two patients (13.3%) developed graft failure and three relapsed (20%). acute graft-versus host disease (agvhd) incidence was extremely low as 13.3% (n = 2) even despite use of mud/mmud in 80% of cases. mortality rate were 53.3% in a group of 2 nd allo-hsct. it should be noted that only 3 (20%) patients died because of disease progression. five patients (33.3%) died in complete remission due to severe infections or previous toxicity (e.g. heart failure). the effect of donor change on dfs was not significant (p = 0,88). our statistical analysis reveal significantly differences in os in patient with long-term interval (46 months) between 1 st and 2 nd allo-hsct. median of os in patients who underwent 2 nd allo-hsct in more/less than 6 months after 1 st allo-hsct was 20,59 vs 7,02 months respectively. (see figure 1b ,1d) for hazard ratio (hr) estimation mantel-haneszel approach were used hr for group who were transplanted in less than 6 months from 1 st allo-hsct was 8.36, (95% ci, 1.054 s511 to 66.38, p = 0.04). as for dfs difference was not significant (p = 0.07). according to our analysis, performing 2 nd allo-hsct in a period less than 6 months after 1 st allo-hsct seemed not very reasonable due to extremely high mortality even in young patients (hr-8.36, p = 0.04). as for "more than 6 months" group it can be considered even despite hla-disparity between donor-recipient pair due to extremely low agvhd rate (13.3%). donor change was not associated with better outcome (p = 0.88) disclosure of conflict of interest: none. hemopoietic stem cell transplantation (hsct) is an effective treatment for many hematologic disorders, and globally over 70 000 procedures/year are performed in more than 70 countries. however, not all the countries have enough resources and expertise to establish an hsct program, and patients are often forced to emigrate for transplantation, with heavy social and economic consequences. in the year 2015 the iuc (an italian ngo) identified the hiwa cancer hospital (hch) in sulaymaniya (iraqi kurdistan) as a possible site for the establishment of a new hsct transplant center. a hsct expert from italy (mi) following a visit to the hch, reported a positive conclusion on the feasibility of an hsct project. this was mainly due to the fact that many of the required technologies were already available at hch, including a 6-bed positivepressure, hepa-filtered-air clinical unit, 2 last-generation cell separators and a well equipped hla laboratory. following this preliminary survey, a capacity building project was rapidly made and submitted to the italian agency for development cooperation, that approved and funded it in march 2016 with the specific aim to cure thalassemia patients either of kurdistan and of the refugees population from syria and other parts of iraq. in april 2016, the joint italian and kurdish team started the project. a first autologous transplant was done in june 2016 followed by 8 more autografts (overall, 5 myeloma and 4 lymphoma patients). in october, following appropriate downstaging, a first low-risk thalassemia patient was allografted from her hla-id sibling, followed by 2 more patients. all the patients engrafted promptly, with one death occurring on day +23 with acute cardiac failure and a major toxicity recorded in a single patient (nhl, severe enterocolitis with perforation) that was successfully treated. the full process for the start-up included the following activities developed during 8-month time: (1) s512 of transplants, the hch group also submitted to ebmt an application for full membership, that was promptly approved. in all this project, the italian counterpart provided over 30 highly-experienced volunteer specialists (physicians, nurses, technicians and one physicist), each with a specific mission plan. despite the many difficulties and obstacles encountered, the clinical results obtained so far appear encouraging, though there is still need to furtherly support the hch in order to make it totally independent. following this intervention, the hch is the only one center performing both auto and allo hsct not only in the iraqi kurdistan region, but also in all the iraqi nation. we conclude that international cooperation may be fruitful also in the field of high-technology medicine, and may contribute to improve the capabilities of centers even in critical geographic areas, representing a valuable instrument also to implement nation-to-nation scientific exchanges. disclosure of conflict of interest: none. the use of plerixafor with g-csf in conditioning regimen for hematopoietic stem cell transplantations with tcr alpha/beta and cd19 depletion of graft in wiscott-aldrich syndrome patients: a single-center experience b dmitry 1 , l alexandra 2 , s larisa 1 , g elena 1 , s irina 1 , t pavel 1 , k rimma 1 , n galina 3 , m michael 1 and m alexei 1 grade 2 acute gvhd (agvhd) was 13% (2 pts). no pt experienced a grade 42 agvhd. three patients presented a limited form of chronic gvhd (21%). incidence of oral mucositis and gastrointestinal/liver toxicity has been extremely low in this population of patients, even in those with active disease and heavily treated at the time of transplant. eight out of fifteen pts (53%) are alive with a median follow-up of 31 months (range: 12 -48 m). seven (47%) are in cytogenetic/molecular remission. six out the eight patients who were transplanted in cr1 or cr2 are alive (75%), while two out the seven patients who were transplanted in advanced phase are alive (29%). in this preliminary clinical experience, we find that unmanipulated haploidentical transplants with post-transplant cyclophosphamide are a valid alternative and have outcome comparable to unrelated and match sibling transplants, in pts with hematologic malignancies. advanced disease is the only adverse factor for diseasefree survival. we therefore consider this therapeutic option when a match sibling or a 10/10 ag mud donor is not immediately available. disclosure of conflict of interest: none. autism spectrum disorder (asd) is a group of neurodevelopmental disorders characterized by impaired social communication and interactions with restricted and repetitive behaviors. although asd is suspected to have either heritable or sporadic genetic basis, its fundamental etiology and pathogenesis are poorly understood. recently researchers have suggested that stem cells have therapeutic potential for asd. wharton's jelly-derived msc (wj-msc) from third-party donors (tpd) have high proliferation and differentiation potential. this cell population has also non-immunogenic and immunomodulatory properties, thus seem to be a promising treatment stem cell source. the polish stem cell bank (pbkm) has provided wj-msc for clinical application in a medical therapeutic experiment for children with asd. twenty-three patients (pts) with asd aged from 3 to 18. 10/12 (median age: 7 years and 7 months), after bioethical committee approval, received intravenous injections of wj-msc, obtained from tpd. the cells were previously collected from healthy newborns, then processed, screened for bacterial contamination as well as endotoxin content, and frozen in liquid nitrogen vapour. wj-msc immunophenotype was confirmed using flow cytometry assay. the pts received from 1 to 5 injections in intervals from 4 to 12 weeks. the average cell dose per infusion was 1.01 × 10^6/kg of body weight (bw). each pt was examined by the same neurologist at the day of infusion. comorbidities present in some patients: unspecified speech disturbances, flaccid paralysis, flaccid tetraplegia, unspecified encephalopathy, epilepsy, sensorineural hearing loss. one patient was diagnosed with 2 comorbidities: conductive hearing loss and intellectual disability. almost 80% of pts, after their treatment with wj-msc, revealed positive changes in neurological examination. an improvement in speech was observed in 10 pts and improvement of cognitive functions ensued in 7 pts. what is more, 26% of children showed progress in self-reliance, social interactions and improved their ability to concentrate. there was a reduction of aggressive behavior in 4 pts and 2 pts have experienced better quality of sleep. there was only one adverse event after wj-msc infusions -psychomotor agitation occurred in 24 hours after the administration. five follow-ups have not yet been completed. the administration of thirdparty donor wj-msc seems to be safe and efficient procedure with promising preliminary results in patients with asd. hematopoietic stem cell transplantation between red cell incompatible donor-recipient pairs red blood cell depletion from bone marrow and peripheral blood buffy coat: a comparison of two new and three established technologies human bone marrow processing using a new continuous-flow cell separation device disclosure of conflict of interest: none. references 1. zama d, et al. gut microbiota and hematopoietic stem cell transplantation: where do we stand? bmt the kyoto encyclopedia of genes and genomes-kegg metabolites produced by commensal bacteria promote peripheral regulatory t-cell generation disclosure of conflict of interest: none antifungal prophylaxis in hematopoietic stem cell transplant recipients: the unfinished tale of imperfect success guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective differences in aspergillus-specific immune recovery between t-cell-replete and t-cell-depleted hematopoietic transplants toxoplasmosis following allogeneic hematopoietic stem cell transplantation diagnosis of toxoplasmosis after allogeneic stem cell transplantation: results of dna detection and serological techniques implementation of molecular surveillance after a cluster of fatal toxoplasmosis at 2 neighboring transplant centers management of high blood pressure genes for blood pressure a prospective studyon the predictive value of plasma bk virus-dna load for hemorrhagic cystitis in pediatric patients after stem cell translantation cidofovir for bk virusassociated hemorrhagic cystitis:a retrospective study hemorrhagic cystitis after bone marrow transplantation bcsh/bsbmt guideline: diagnosis and management of veno-occlusive disease (sinusoidal obstruction syndrome) following haematopoietic stem cell transplantation drug safety evaluation of defibrotide defibrotide for prophylaxis of hepatic veno-occlusive disease in paediatric haemopoietic stem-cell transplantation: an open-label, phase 3, randomised controlled trial safety and effects of prophylactic defibrotide for sinusoidal obstruction syndrome in hematopoietic stem cell transplantation disclosure of conflict of interest: none university children's hospital basel, division of paediatric oncology/haematology late complications subcommittee of translated related complications and quality of life wp; 5 clinic of paediatric haemato-oncology, department of women's and children's health, university of padova, italy; 6 department of surgery, division of transplantation division of blood and marrow transplantation, the children's hospital at westmead ovarian function after bone marrow transplantation during childhood pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning haploidentical hematopoietic transplantation:current status and future perspectives t-cell replete haploidentical donor transplantation using post-transplant cy: an emerging standard-of-care option for patients who lack an hla-identical sibling donor hematopoietic stem cell transplantation in thalassemia major and sickle cell disease: indications and management recommendations from an international expert panel allogeneic stem cell transplantation for thalassemia major killer-cell immunoglobulin-like receptors reactivity and outcome of stem cell transplant kir b haplotype donors confer a reduced risk for relapse after haploidentical transplantation in children with all kir/hla interactions negatively affect rituximab-but not ga101 (obinutuzumab)-induced antibody-dependent cellular cytotoxicity reduction of minimal residual disease in pediatric b-lineage acute lymphoblastic leukemia by an fcoptimized cd19 antibody diagnoses: hodgkin's lymphoma(hl)-38 pts (refractory 22; relapsed 16); non-hodgkin's lymphoma(nhl disease status before asct: 1st (after refractority prior radiotherapy to the mediastinum -19/48 (39.6%); heavily pretreated patients with advanced disease (x 3 lines previous treatment) 18/48 (37.5%). grafts: pbsc -43/48 pts with median of cd34+cells-3 ccnu dose: 47/48 pts 300mg/m2; 1pt 400 mg/m2. engraftment: anc>500 cells/mkl: median=d+11(8÷24), 47/48pts. plt>50 000 cells/ mkl: median=d+ 28(13÷122), 43/48pts. full engrafted 43/ 48pts /8 pt required a short-term mechanical ventilation (2 of them died because of lung infection ad d+68 and d+82) aeruginosa associated sepsis on a background of graft failure); 2pts (4.2%)-d+68 and d+82 (pulmonary toxicities +infection; both had prior mediastinal radiotherapy). relapse/ progression after asct-8/48 pts (16.6%), 6 of them died. 1 pt achieved secondary mds (diagnosed 4.5 mo after asct). for this group of pts with relapsed/refractory lymphomas (n=48) 11-year os=0 for nhl(n=10) efs=0.88 (se ± 0.2) lomustine-containing conditioning regimen cem (lomustine, etoposide, melphalan) is effective and feasible in autologous stem cell transplant efficacy and toxicity of a ccnu-containing high-dose chemotherapy regimen followed by autologous hematopoietic cell transplantation in relapsed or refractory hodgkin's disease champlin re reduced-toxicity conditioning therapy with allogeneic stem cell transplantation for acute leukemia idarubicin-intensified bucy2 conditioning regimen improved survival in high-risk acute myeloid, but not lymphocytic leukemia patients undergoing allogeneic hematopoietic stem cell transplantation: a retrospective comparative study comparison of outcomes of idarubicin intensified tbi-cy and traditional tbi-cy conditioning regimen for high-risk acute lymphoblastic leukemia undergoing allogeneic hematopoietic stem cell transplantation: a single center experience inhibition of cd25 (il-2r alpha) expression and t-cell proliferation by polyclonal anti-thymocyte globulins csf-primed bone marrow transplantation for patients with high-risk hematologic malignancies in an exploratory analysis, os after hct appeared to be longer in the cpx-351 arm in both age groups. these results suggest that cpx-351 may provide an effective bridge to successful transplant for a high-risk subgroup of aml patients. support: celator pharmaceuticals, inc., a subsidiary of jazz pharmaceuticals plc consulting ambit biosciences, amphivena therapeutics, ariad, astellas pharma sunesis, tolero; institutional research funding abbvie chiarella and louie: employment celator/jazz; stock jazz pharmaceuticals plc. hoering disclosure of conflict of interest: none. inkt-/nk-/cik-cell (subsets) are important for immunesurveillance. antibody 6b11 targets the vα24-jα18-invariant-t-cell-receptor (tcr) in the cdr3-region, which is semiinvariantly rearranged in inkt-cells. we characterized: i.) inkt-/nk-/cik-subsets in pb-samples from healthy donors (n = 9 subsets under stimulation with dendritic-cells of leukemic origin (dc leu ), generated from aml-blasts in mononuclear cells(mnc) and whole-blood (wb, containing soluble/cellular components of pts' pb) with 'cocktails' (dc-generating-methods/kits). 1.1) compared to healthy mnc (significantly) lower proportions of inkt-cells comparable correlations were seen in adultall-and cll-pts. 2.1) we quantified inkt-/nk-/cik-subsets before/after mixed-lymphocytecultures (mlc) of t-cell-enriched immune-reactive cells stimulated with mnc/wb (with or without pretreatment 'cocktails' inducing blasts' conversion to dc leu ) from aml-pts. our findings show, that 1)inkt-/nk-/cik-cells increase after mlc independent of the stimulator-cells-suspension (under the influence of il-2); 2) pretreatment of mnc/wb-blasts with 'cocktails' increases inkt-counts and induces a shift in the composition of inkt-/nk-/cik-subsets after mlc, that might correlate with an improved antileukemic potential; 3) individual samples showed varying, however higher inkt-, cik-cell-counts after pretreatment with different (especially prostaglandin-containing) 'cocktails'; 4) dc-/inkt-/nk-/cikcells-values after mlc were comparable in physiological hypoxia vs normoxia; 5) in cases with antileukemic blast-lytic activity after mlc t-/inkt-/nk-/cik-cells were significantly increased-pointing to an involvement of these cells in antileukemic reactions. in summary: (1) healthy mnc present with significantly higher inkt-/nk-/cik-cells compared to aml/all/cll-leukemic mnc. (2) subtypes of inkt-cells differ in healthy vs leukemic samples, resembling a shift in the composition of inkt-cells. (3) amounts of inkt-/ nk-/cik-cells in aml/all/cll-mnc-samples correlate with prognosis. (4) 'cocktail'-treated aml-blasts (resulting in dc leu ) lead to a shift in t-,inkt-/nk-/cik-cell-counts/compositions, what correlates with improved antileukemic activity against aml-blastspointing to a cross-talk of these cells. proportions of inkt-/ nk-/cik-cells management of philadelphia chromosome-positive acute lymphoblastic leukemia (ph+ all) outcome of allogeneic stem cell transplantation for aml and myelodysplastic syndrome in elderly patients (⩾60 years) comorbidity-age index: a clinical measure of biologic age before allogeneic hematopoietic cell transplantation high rate of hematological responses to sorafenib in flt3-itd acute myeloid leukemia relapsed after allogeneic hematopoietic stem cell transplantation phase i trial of maintenance sorafenib after allogeneic hematopoietic stem cell transplantation for fms-like tyrosine kinase 3 internal tandem duplication acute myeloid leukemia haematopoietic cell transplantation with and without sorafenib maintenance for patients with flt3-itd acute myeloid leukaemia in first complete remission quantitative monitoring of minimal residual disease (mrd) after sct was performed by four-colour flow cytometry and/or real time pcr. the median time of neutrophil engraftment (above 0.5 × 10e9/l) was 16 days, 96% of pts (24/25) engrafted, one patient died in aplazia. non-relapse mortality (nrm) after 1 year and 2 years was 8% (2/25) and 13% (3/25). causes of death were refractory gvhd (n = 1), infection (n = 1) and multiorgan failure (n = 1). incidence of acute gvhd was evaluated in 23 pts: 52% (12/23) of pts had gvhd (grade i+ii in 7 pts, grade iii in 5 pts). incidence of chronic gvhd was evaluated in 22 pts, 41% (9/22) of pts had gvhd with median follow-up from sct 37 months (range: 4-90), 64% of all pts (16/25) were alive (14 in remission of cll with mrd negativity, 2 with relapse), 9 pts died (3 from nrm, 6 from cll relapse/progression), 8 relapses (32%; 8/25) occurred. sequential use of chemotherapy and ric regimen with allogeneic sct is safety and effective treatment of high-risk cll with reponse rate 86% and low nrm. progression-free survival and overall survival at 3 years from sct were 56% and 64% 1 department of hematology, hemostasis, oncology and stem cell transplantation hannover deutsche klinik für diagnostik helios klinik wiesbaden, germany; 19 imperial college london at hammersmith hospital du cane road centre for haematology london disclosure of conflict of interest: none. references 1. sibon d, brice p. optimal treatment for relapsing patients with at our institution, pr-hl is defined as partial response (pr), no response (nr), stable disease (sd), progressive disease (pd), relapsing within 3 months of finishing the planned treatment. progression free (pfs) and overall survival (os) from the day 0 of auto-sct was estimated by kaplan-meier (km) method. from 1996 to 2014, 234 patients with aethera trial criteria were identified. male 121 (52%), female 113 (48%), median age at diagnosis:22 yrs (12-61), at auto-sct: 24.3 yrs (13.8-63)(90% o40 yrs). initial chemo: abvd in 194 (83%). 70 (30%) had radiation therapy (xrt) after initial chemo. response to initial chemo + xrt was refractory disease:152 (65%), relapse between 3-12 months:49 (21%) and relapse after 12 months:33 (14%) aethera had 28% stable disease before sct vs we have only 6%. aethera 24 months pfs (45% control arm, 65% brentuximab arm, investigator assessment) and our 56.4% is not much different. despite having similar selection criteria, our median pfs is higher than both aethera trial placebo and experimental arm. clinically, rate of progression in both studies are very high and comparable at 24 months. given the very high cost of this drug and while waiting for survival fifty-nine (82%) and 13 (18%) patients had relapsed and primary refractory chemosensitive dlbcl, respectively. secondary ipi was 0-1 in 23 (44%) patients, 2 in 13 (25%) patients and 3-4 in 16 (31%) patients. fifty-one (71%) and 21 (29% patients had gcb and abc tumors, respectively. abc patients received more prior lines of chemotherapy than gcb patients (76% vs 48% received 4 2 lines of chemotherapy, p = 0.03). the rest of characteristics were equally distributed between both groups (table 1) disclosure of conflict of interest: none disclosure of conflict of interest: none. p631 upfront autologous stem cell transplantation in patients with diffuse large b cell lymphoma: focused on risk factors for survival and conditioning regimens ds kim 1 association between complete response and outcomes in transplant-eligible myeloma patients in the era of novel agents e jantunen 1 and v varmavuo 12 1 department of medicine disclosure of conflict of interest: none. and 6 hematology department lenalidomide after stem-cell transplantation for multiple myeloma bortezomib induction and maintenance treatment in patients with newly diagnosed multiple myeloma: results of the randomized phase iii hovon-65/gmmg-hd4 trial disclosure of conflict of interest: none we performed a retrospective study to investigate survival outcomes and toxicities of l maintenance therapy compared with b maintenance in mm patients post-ahct. this study included 156 patients who received ahct for mm between 2008 and 2015 after induction with l-or b-based therapy. all patients received ahct within 12 months of mm diagnosis and received melphalan 200 mg/m 2 conditioning. patients who received tandem transplantations (autologous or allogeneic) were excluded. only patients initiating maintenance therapy within 6 months post-ahct were included. maintenance therapy was defined as monotherapy with either l or b. the primary outcome was pfs. secondary outcomes were overall survival (os) and treatment-related toxicities. 92 patients received l maintenance and 64 b maintenance post-ahct. at baseline there were no differences in iss stage, ds stage or cytogenetic risk between maintenance cohorts. at time of analysis, 49% (n = 45) receiving l maintenance and 52% (n = 33) on b maintenance experienced disease progression. median time to progression (1.71 vs 1.74 yrs, p = 0.77) was not significantly different between cohorts. by multivariable analysis, choice of maintenance (l vs b) was not significant for pfs or os. variables significant for improved pfs were iss stage i disease response improved while on maintenance in 38% (n = 24) with l and 34% (n = 31) with b. median os was not statistically different between maintenance cohorts (4.28 vs 5.77 yrs, p = 0.47). iss stage i/ii vs iii while cytopenias were more common in the l cohort (30% vs 3%, p o 0.01). the median follow-up time for survivors was 33 months. these findings suggest that both lenalidomide and bortezomib are equivocal maintenance therapy options for post-transplantation mm patients. choice of maintenance therapy post-ahct for mm did not demonstrate a difference in survival outcomes. based on these data, maintenance choice should be guided by patient specific anticipated tolerance rather than drug type alone. iss stage and post-ahct disease response continue to be significant predictors for outcomes. toxicities recorded on maintenance were as anticipated. length of maintenance therapy may be a significant predictor and warrants further analysis. the analysis was underpowered to disclosure of conflict of interest: none. p650 real-world multiple myeloma management practice patterns and outcomes in six central and eastern european countries d coriu 1 , d dytfeld 2 , d niepel 3 , i spicka 4 second autologous stem cell transplantation as salvage therapy for multiple myeloma: impact on progression free survival and overall survival second autologous stem cell transplantation: an effective therapy for relapsed multiple myeloma second auto asct for treatment of relapsed multiple myeloma the role of second autografts in the management of myeloma at first relapse moving beyond autologous transplantation in multiple myeloma ebmt data office bortezomib-based versus nonbortezomib-based induction treatment before autologous stem-cell transplantation in patients with previously untreated multiple myeloma: a meta-analysis of phase iii randomized, controlled trials first-line therapy with thalidomide and dexamethasone in preparation for autologous stem cell transplantation for multiple myeloma mechanism of action of bortezomib and the new proteasome inhibitors on myeloma cells and the bone microenvironment: impact on myeloma-induced alterations of bone remodeling boys; 19 girls) with following mds types: refractory cytopenia of childhood-11 (19%), refractory anemia with excess blasts -14 pts (24%), refractory anemia with excess blasts in transformation-22 pts (38%), juvenile myelomonocytic leukemia in 11 pts (19%). the median of age was 7 years (1-19 years) mac consisted busulfan (bu) 16 mg/kg + cyclophosphamide 120 mg/kg. ric included fludarabine (flu) 150 mg/m 2 + melphalan (mel) 140 mg/m 2 , flu 150-180 mg/m 2 + bu 8mg/ kg. the bone marrow (bm) was used in 38 pts (66%), peripheral blood stem cells (pbsc) in 13 pts (22%), combination of bm and pbsc in 7 pts (12%). 5-years overall survival (os) was 45% os was in pbsc group -28%; bm group-50%, combination of bm and pbsc-25% there were two cases of mds, eb-2, although erythroid aberrancy can not be found, fc did disclose significant aberrancy on myelomonocytic lineages. on the other hand, all the normal control bm samples revealed no any erythoid phenotypic abnormality. our study suggests this simplified cocktail of 2-tube, 4-color, fc is very sensitive and useful in the assessment of erythroid phenotypic abnormalities in mds we analyzed 62 consecutive patients (44% were female, median age of 48 (range: 18-70) allografted for mds (median ebmt risk score of 3, median disease risk index of intermediate risk) over a 19-year period (1998-2016) with mac conditioning for 66% and ric for 34% pfs 40 ± 14%, grfs 26 ± 12%, ri 37 ± 13% and trm similarly, there was not difference between tdep and non tdep patients for 3-years pfs (48 ± 18% and 28 ± 20%, p value 0.321), 3-years gfrs (32 ± 17 vs 19 ± 17, p value 0.111) (graph), 3-years ri (36 ± 18% and 37 ± 20%, p value 0.622) and 3-years trm (26 ± 16% and 23 ± 18%, p value 0.933). finally, tdep had no significant impact on 3-years grade 2-4 agvhd when compared to the non tdep (26 ± 18% and 31 ± 16%, p value 0.656). it had not either on 3-years cgvhd (26 ± 18% and 28 ± 34%, p value 0.637). our study shows that tdep is feasible on patients undergoing hsct for mds disclosure of conflict of interest: none. p665 mutational pathway and dynamics may not be prognostic in patients with myelodysplastic syndrome receiving hypomethylating agent pre-treatment for allogeneic stem cell transplantation republic of korea; 3 department of computer science; 4 the donnelly center for cellular and biomolecular research amebiazis after bone marrow transplantation use of a five-agent gvhd prevention regimen in recipients of unrelated donor marrow impact of age on outcomes after bone marrow transplantation for acquired aplastic anemia using hla-matched sibling donors treatment of acquired severe aplastic anemia: bone marrow transplantation compared with immunosuppressive therapy-the european group for blood and marrow transplantation experience disclosure of conflict of interest: none. leukemia, myelodisplastic syndrome, juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia s bondarenko hla-mismatched unrelated (n = 5, 10%), and haploidentical (n = 8, 15%) donors. response was achieved in 30% (n = 16) of pts after 1-5 (median 3) courses of hma therapy: complete remission (cr) in 2 (4%), partial remission (pr) in 14(26%) of pts. stabilization (s) was documented in 30 (56%) pts, in 8 (14%) pts there was disease progression (p) after beginning of hma therapy mismanaging the gift of life: noncompliance in the context of adult stem cell transplantation l'adhésion thérapeutique et at. des lieux en allogreffe de cellules souches hématopoïétiques (csh) dans des services de pédiatrie et d'adulte. rapport de la sfgm-tc predictive validity of a medication adherence measure in an outpatient setting data is limited to small case series, transplant registries and a single prospective multicenter observational study. here we report our institutional experience with auto-hct in patients with hrl. twenty patients with hrl [non-hodgkin = 14 (70%), hodgkin = 6 (30%)] and treatable hiv infection underwent hdt consisting of carmustine, etoposide, cytarabine and melphalan (beam) followed by peripheral blood auto-hct from 04/2006 to 07/2015. in 2 cases rituximab was administered as part of the preparative regimen. patient-, disease-, and transplant-related characteristics are summarized in table 1. median age was 48 years (range: 35-61). the median follow-up for surviving patients was 42 months (range: 6-110) abbreviation: n: number of patients; m: male gender; auto-hct: autologous hematopoietic cell transplant; nos: not otherwise specified; dlbc: diffuse large b-cell lymphoma ara-c), melphalan; cr1: first complete remission; cr2 :second complete remission disclosure of conflict of interest: none. p696 incidence of secondary primary malignancies (spm) in patients with multiple myeloma m curly 22 , g laurent 23 and k nicolaus 24 1 city of hope igm 21 (0.6%), lines of induction regimens prior to hsct one in 2003 pts (53%), two in 724 pts ( 19.3%), 4 2 in 348 pts (9.3%), and missing in 682 pts (18%). induction regimens included imids and proteasome inhibitor (pi)s with alkylating agents in 1266 pts (33.7%), imids and pis with no alkylating agents in 1328 (35.5%), and alkylating agents with no imids or pis in 478 (12.7%) and missing data in 685 (18%). radiotherapy was used pre hsct in 614 pts (16.3%), no radiation in 2461 pts (66%) and missing data in 682 (18.2%). plerixafor (p) was administered mostly for poor hsc mobilization as defined by the centers number of hsc collected o3 × 10 6 in 239 pts (6.4%), 3-5 in 397 pts ( 10.6%), 45 × 10 6 in 1394 pts (37%), and data missing in 1727 (46%). the number of cd 34+ hsc infused o3 × 10 6 in 760 pts (20%), 3-5 × 10 6 in 1055 pts (28%0, 45 × 10 6 in 799 pts (21%), and missing in 1143 (30%). a total of 141 pts developed spm with cumulative incidence of 5.4% (95%ci 4.4,6.3) at 72 mo. data are missing in 414 pts (11%) use of radiotherapy, type of induction, hsc cell dose did not influence the cumulative conflict of interest: f. sahebi, none declared, s. iacobelli, none declared, l. koster none declared l. gardaret none declared, n. kroger received research fund from sanofi, curly morris, none declared p697 interaction between center effect and strategy for gvhd prophylaxis on outcome of t-cell depleted and t-cell replete haploidentical transplant inserm u1153 ecstra team expanding transplant options to patients over 50 years-improved outcome after reduced intensity conditioning mismatched-unrelated donor transplantation for patients with acute myeloid leukemia: a report from the acute leukemia working party of the ebmt nkg2d ligands in tumor immunity comprehensive analysis of nkg2d ligand expression and release in leukemia: implications for nkg2d-mediated nk cell responses nkg2d cars as therapy for cancer russian federation high incidence of mixed chimerism with impaired graft function remains a significant issue in patients with wiskott-aldrich syndrome (was) after hsct. simultaneous use of plerixafor with g-scf is efficient in inducing stem cell release and opening of bone marrow niches. the use of plerixafor/g-csf in conditioning demonstrates better levels of donor chimerism in patients with acute myeloid leukemia. we report our experience of plerixafor/g-csf usage in patients with was as an addition to myeloablateive conditioning to improve stem cell engraftment p = 0,85. events were considered: death in 2 patients, graft rejection in 5 patients, mixed myeloid chimerism (less than 20% donor) in 2 patients. median time of event was 3,2 months after hsct (1.23-8.6) all patients are alive, median fu is 3 months, range: 0.43-6.3. 2 patients had acute gvhd: 1-grade 2 (gut), 1-grade 1 (skin), in both cases resolved after a short course of steroids. all patients had more than 95% donor chimerism monthly till the time of last fu. the comparison of peripheral blood chimerism (% of donor cells) in was patients transplanted with and without plerixafor/g-csf in conditioning is shown (figure 1). the additional use of plerixafor with g-csf references 1. moratto et al disclosure of conflict of interest: none. is undesirable. fifteen pts (3 males, 12 females, median age 48, range: 17 -65 years) with high risk hematologic malignancies ( acute myeloid leukemia n.10, 66%; acute lymphoblastic leukemia n 2, 13% pretransplant conditioning regimen consisted of thiotepa 10 mg/kg in two days, busulfan 9.6 mg/kg in three days, and fludarabine. source of stem cells was g-csf stimulated bone marrow in all. dose of marrow nucleated cells and cd34+ were 5.4 (range: 3.4-6.7) × 10 8 /kg and 3.5 (range: 2.1-5.8) × 10 6 /kg respectively. post-transplant cyclophosphamide at 50 mg/kg/ day was given on days 3 and 5 after transplantation, together with cyclosporine (starting at day − 1 until day 180 posttransplant) and mycofenolate (from day +1 to day +20) modeling autism spectrum disorders with human neurons autism spectrum disorders neurobiology and genetics of autism: a developmental perspective. the development of autism: perspectives from theory and research wharton's jelly-derived mesenchymal stem cells treatment in children with cerebral palsy: our second preliminary results of the clinical application in poland a mucha 1 , k kosterna 1 , m chroscinska-krawczyk 2 , m kotarska 2 , k mitosek-szewczyk 2 , m murzyn 3 the polish stem cell bank cases application potential of bone marrow mesenchymal stem cell (bmscs) based tissueengineering for spinal cord defect repair in rat fetuses with spina bifida aperta sensory neuron differentiation potential of in utero mesenchymal stem cell transplantation in rat fetuses with spina bifida aperta: sensory neuron differentiation of in utero mscs analysis of post allo-hct relapse in acute leukaemia patients, a comparative on second allo-hct and donor lymphocyte infusions g orti 1 , j sanz 2 , i garcia-cadenas 3 , i sanchez-ortega 4 , mj jimenez 5 , p barba 1 , c ferra 6 , r parody 4 , j sierra 3 , ma sanz 2 , s querol 7 and d valcarcel 1 1 hospital universitari vall d´hebron; 2 hospital universitario la fe; 3 hospital de sant pau i la santa creu; 4 hospital duran i reynals ico, 5 hospital germans trias i pujol ico; 6 hospital germans trias i pujol and 7 banc de sang i teixits acute leukaemia relapse after allogeneic hematopoietic cell transplantation (allo-hct) associates poor prognosis. in this scenario, lowering the tumour burden prior to a second allo-hct (2 nd allo-hct) or donor lymphocyte infusions (dli) is essential to improve survival. thus, patients that respond to chemotherapy and subsequently receive a dli or 2 nd allo-hct appear to associate better outcomes compared to patients receiving only chemotherapy, but data regarding this particular group of patients is lacking. we retrospectively analysed a cohort of post allo-hct relapsed acute leukaemia patients, who, after tumour reduction, were treated with either a 2 nd allo-hct or dli. data was collected from 5 centers, 42 patients were consecutively included from 1995 to 2016. patients were treated to reduce the tumour burden and received the 2 nd allo-hct or dli on morphological remission or postchemotherapy aplasia. 26 patients (62%) were diagnosed with aml and 16 (38%) with all. 23 patients (55%) underwent 2 nd allo-hct and 19 (45%) received dli. median patient age was 38 (4-66) years. the median follow-up was 674 (9-5823) days. since data regarding time from first allo-hct to relapse was unavailable, we calculated the time from allo-hct to 2 nd allo-hct or dli (time to 2 nd allo-hct or dli). median time to 2 nd allo-hct/dli was 336 (9-8823) days, and was 674 days and 336 days for 2nd allo-hct and dli respectively (p = 0.004). regarding the dli group, the median dli dose was 1.1x10 7 / cd3+ (0.01-10x10 7 ) cells and the mean number of infused dli was 1.4/patient. one-year os was 51% (se ± 8%). in os univariate analysis, longer time to 2 nd allo-hct/dli associated better survival rates (p = 0.003). the 1-year dfs was 39% (se ± 8%). a longer time to 2 nd allo-hct/dli (p = 0.006) and 2 nd allo-hct compared to dli (p = 0.047) (figure 3 ) associated better dfs. the 1-year nrm was 24% (se ± 8%). univariate analysis identified pb as stem cell source as linked to better nrm (p = 0.086). the 1-year relapse incidence (ri) was 35% (se ± 9%). ri univariate analysis related longer time to 2 nd allo-hct/dli (p = 0.013) to lower ri. on os multivariate analysis, longer time to 2 nd allo-hct/dli was associated to better survival (p = 0.042). this association was also observed on dfs multivariate analysis (p = 0.017). table 1 summarizes 2 nd allo-hct and dli univariate analysis. grade ii-iv acute gvhd was diagnosed in 8 (35%) and 5 (26%) patients post 2 nd allo-hct and dli, respectively. chronic gvhd was diagnosed in 8 (4 extensive) and 3 patients after a 2 nd allo-hct and dli, respectively. in this study, longer time to 2 nd allo-hct/dli associated better dfs. 2 nd allo-hct (compared to dli) associated better dfs on univariate analysis, but this association was not observed on multivariate analysis. of note, the 2 nd allo-hct group included more patients with longer time to 2 nd allo-hct/dli. this might be explained by 2 nd allo-hct patients relapsing later or by the fact that the preparation of a 2 nd allo-hct might require longer time than dli. results of this analysis warrant further study with larger number of patients.advancing age is associated with worse prognosis in acute myeloid leukemia (aml). intensive induction chemotherapy in patients aged ⩾ 60 years results in lower aml remission rates with increased induction mortality vs younger patients. cpx-351 is a liposomal formulation of cytarabine and daunorubicin encapsulated at a 5:1 molar ratio. a phase iii, randomized, open-label study of cpx-351 vs 7+3 (cytarabine and daunorubicin) in newly diagnosed older patients with high-risk secondary aml showed superior survival in the cpx-351 arm (hazard ratio 0.69; p = 0.005). in that trial, eligible patients went on to allogeneic hematopoietic cell transplantation (hct). an exploratory analysis of those patients by age strata is reported here. patients aged 60 to 75 years with newly p534 number, composition and/or antileukemic activity of (dc-stimulated) invariant nkt-, nk-and cik-cells is predictive for outcome of patients with aml, all and cll cl boeck 1# , dc amberger 1# , f doraneh-gard 1 , w sutanto 1 , t guenther 1 , j schmohl 2 , f schuster 3 , h salih 2 , f babor 3 , a borkhardt 3 myelofibrosis (mf) is a hematolgic malignancy which is characterised by extramedullary hematopoiesis due to bone marrow fibrosis resulting in spleno-and/or hepatomegaly. allogeneic stem cell transplantation (allo-hsct) is the only curative treatment for mf but is associated with therapy related morbidity and mortality. retrospective studies suggested an increase of liver toxcicity in mf patients in comparison to other diseases following allo-hsct. the aim of this prospective study was to evaluate the impact of liver stiffness measured by transient elastography (fibroscan) on liver toxicity after allo-hsct. between 2013 and 2015 we included 39 patients (male 64%, female 36%) who underwent allo-hsct due to primary mf(72%), postpv/et-mf (23%) or mf in transformation (5%). the median age of the patients was 62 y@@@ears (range: 35-74). conditioning regimen was mainly busulfan based reduced intensity. all patients received atg. gvhd prophylaxis was csa/mmf in all patients. stem cell source was peripheral blood in 95% and bone marrow in 5% of the patients. donor sources were as follows: mrd (18%), mud (77%) and haploidentical relative (5%). fibroscan was performed prior to conditioning. elevated liver enzymes, bilirubin above the normal value or the onset of veno-occlusive disesae (vod) from the time of conditioning start and within the first 100 post-transplant days were considered as indicators for liver toxicity. the median stiffness of the liver measured by fibroscan on the day before conditioning treatment start was 7.6 kpa (range: 4.4-39.7). six patients (15%) had prior liver diseases such as cirrhosis (n = 1), viral hepatitis (n = 3), steatosis (n = 1), or vod (n = 1). the median onset of liver toxicity was day 0 (range: − 2 until +92). the median bilirubin level of all 39 patients was 4 mg/dl (range: 0-17). the median ap level was 153 u/l (range: 80-833), the median ggt level was 343 u/l (range: 88-1647), the median alt level was 108 u/l (range: and the median ast level was 67 u/l (range: 20-12292). the pearson-test revealed a positive correlation between liver stiffness and the elevation of the ap (r = 0.55, p = 0.001) and ggt levels (r = 0.54, p = 0.008). the comparison of the median maximum enzyme and bilirubin levels is shown in table 1 . in two patients who developed severe vod requiering defibrotide, the liver stiffness level was 6.9 kpa and 13.8 kpa, respectively. the patient with the highest stiffness level (39.7 kpa) developed acute gvhd of the liver, which completely resolved after steroid treatment. only one of those five patients who had stiffness levels 413 kpa died due to liver toxcity and concurrent septic shock, he suffered from viral hepatitis prior to transplantation. liver stiffness measured by transient elastography (fibroscan) positively correlates with the elevation of the cholestatic enzymes ap and ggt in myelofibrosis patients after allo-hsct and may predict liver toxicity. disclosure of conflict of interest: none.[p574]in the era of tyrosine kinase inhibitors (tki) as superior first line treatment in the therapy of cml, the concept of allogeneic hsct has been pushed to the role of salvage therapy. to date, data on allogeneic hsct after tki-therapy are scarcely available. in this study, we report single center data on the outcome of 52 cml patients, for the most part pretreated with tki, who underwent allogeneic hsct between 1999 and 2015 with a follow-up of 12 months to 17 years. upon obtaining written informed consent 48 patients diagnosed with bcr-abl-positive cml and 4 patients with bcr-abl-negative atypical cml were included in this analysis. the majority of patients underwent myeloablative conditioning regimen. the median age at time of hsct was 47 years with a range: from 19 to 67 years. twenty-one patients were transplanted from a matched related donor, and 31 received stem cell grafts from an unrelated hla-compatible donor. 36/48 patients received tki-therapy before transplantation, 23 patients received more than 1 tki prior to hsct. 10/48 patients were treated with interferon prior to hsct. twenty-two patients were transplanted due to acceleration or blast crisis. twenty-six patients received an allogeneic hsct in chronic phase (cp, n = 16) or complete hematologic (chr, n = 7) or cytogenetic remission (ccyr, n = 3). kinase domain mutations could be identified in seven patients including t315i-mutation in four patients. seven patients showed "major route" cytogenetic aberrations. next to advanced disease status, tki intolerance (n = 4) and tki resistance (n = 11) were the main indications for hsct after 2001. after a median follow up of 5 years and 3 months, those 26 patients transplanted in cp, chr or ccyr showed an overall survival (os) of 79%. 3/27 patients died in remission and two patients died after cml relapse. after 2004 none of the 15 patients transplanted in cp, chr or ccyr died or relapsed so far, with a median follow-up of 1672 days. all of these patients received tki therapy prior to transplant. twenty-two patients transplanted in advanced stage cml (bc and ap) had after a median follow up of 5 years an os of 44%. the difference between survival curves is significant (log rank test p = 0.041; hr 0.3407, 95% ci of ratio 0.1211-0.9585). prior to transplantation 19 of these patients received a tki-therapy. in this group, four patients died due to cml relapse, one died after development of donor cell leukemia and five patients died in remission. one patient with atypical cml was transplanted in bc and died of progressive disease shortly after transplantation. the other three patients with atypical cml were transplanted in cp-phase. with a median follow-up of 648 days these patients are in ongoing remission. even in times of tkitherapy allogeneic hsct remains a successful and safe therapy option for cml patients with tki intolerance or resistance. patients transplanted in cp or complete remission had an excellent long-term outcome. allogeneic hsct should be considered in tki resistance or intolerance before the development of blast crisis. despite tki therapy, overall survival deteriorates in patients with advanced disease. however, this treatment modality can improve survival rates substantially compared to other available therapies. tkimaintenance therapy could be a possible strategy to prevent cml relapse, although randomized data on tki-maintenance therapy after allogeneic hsct are still lacking.[p583]disclosure of conflict of interest: none. use of first or second generation tki for cml after allogeneic hematopoietic stem cell transplantation: a study by the cmwp of the ebmt y chalandon, s iacobelli 1 , j hoek 2 , l koster 2 , l volin 3 , j finke 4 , jj cornelissen 5 , i yakoub-agha 6 patients (pts) relapsing with cml after allogeneic hematopoietic stem cell transplantation (allohsct) may be treated with tki and/or dli. as nowadays the majority of cml pts would have received at least imatinib prior to transplantation, we were interested in analizing (a) the type of tki used after allohsct, (b) the indication for tki treatment, (c) the outcome of this treatment and d) the temporal relationship with dli if given. 435 pts received tki after first allogeneic hsct for cml. transplants had been performed in cp1, n = 194, ap, n = 60 or for more advanced disease (bc/4 cp1, n = 177) from hla identical siblings (n = 231) or ud (n = 204) between 2000 and 2013. tki given prior to transplant was imatinib (n = 268), dasatinib (n = 162), nilotinib (n = 88), bosutinib (n = 4) and ponatinib (n = 7). median age at transplant was 44 (18.5-68) years, 274 pts (63%) were male. tki post allohsct were given between 2000 and 2015. first tki given was either imatinib (n = 223), dasatinib (n = 131), nilotinib (n = 67), bosutinib (n = 2) or ponatinib (12). the indications for tki therapy were the same as for transplantation (n = 25), for relapse/progression/ persistent disease (n = 246), for prophylaxis/pre-emptive (n = 147), planned (n = 5), others (n = 8) and missing (n = 4). median follow-up from start of tki was 55 (1-171) months. the median time interval from transplant to tki was 6 (0.2-165) months. it was longer for tki given for relapse/progression with 15 (1-89) months and shorter for tki given for prophylaxis/pre-emptive with 1.6 (0. haematopoietic cell transplant (hct) is the only curative approach for scd. due to concerns regarding the toxicities associated with myeloablative conditioning regimens in adults, a non-myeloablative protocol was developed by hsieh et al. (national institutes of health, nih protocol). the use of this novel regimen was able to achieve a curative degree of mixed donor chimerisms with minimal transplant-related complications. the alberta children's hospital (ach) has adopted this conditioning regimen in children due to the efficacy and low rates of toxicities published by the nih group. with generally lower rates of gvhd in younger recipients, our group had no reason to believe rates of toxicities would be greater in a younger population with fewer comorbidities secondary to scd than those described in the nih cohort. to our knowledge, there is no published literature describing the utilization of the nih protocol in a paediatric population. we describe our experience in children with scd who underwent matched sibling donor (msd) peripheral blood hct using nih protocol. this retrospective cohort describes outcomes of msd hct in children with scd who underwent hct with the nih conditioning regimen between 2013-2017. a total of 13 potential subjects were identified. eight subjects have consented to the analysis to date. msds with either normal haemoglobin or sickle cell trait were considered appropriate for donation. the transplant procedure: the conditioning regimen consisted of alemtuzumab 0.2 mg/kg/dose administered subcutaneously daily for five days (days − 7 to day − 3). patients received a tbi dose of 300 cgy on day -2, with testicular shielding for male recipients. gvhd prophylaxis consisted of a sirolimus load of 3 mg/ m 2 /dose (po) on day − 1, followed by 1 mg/m 2 /dose once a day starting on day 0. unmanipulated peripheral blood stem cells were infused on day 0. sirolimus was used for gvhd prophylaxis post-hct and continued until at least one year. weaning of sirolimus was initiated no earlier than 1 year post-hct and if donor t-cell chimerisms were greater than 50%. institutional supportive care protocols for scd hct were followed. patients were eligible for early discharge post-hct even prior to neutrophil engraftment. eight patients (5 f, 3 m) have been registered on this retrospective study. follow-up ranges from 1 to 52 months post-hct. there were no failed stem cell mobilizations. all patients had donor neutrophil engraftment at a median of 21 days. all patients are currently alive. there have been no cases of graft failure to date and no sickling crises post-hct. one patient has dropping myeloid chimerisms but still 4 20% donor. no cases of veno-occlusive disease, idiopathic pneumonia syndrome, cerebral hemorrhage, pres, or posttransplant lymphoproliferative disease were observed. three cases of cytomegalovirus (cmv) reactivation required pre-emptive therapy. only one patient did not initiate sirolimus weaning at 1 year post-hct due to donor t-cell chimerisms of 42%; this patient is 52 months post-hct and is likely to start weaning sirolimus soon. there have been no cases of acute or chronic graft-versus-host disease. nonmyeloablative conditioning regimen is safe and effective as curative therapy for scd. long-term follow-up of these children to assess organ function post-hct is underway. disclosure of conflict of interest: none.the number of new hiv/aids cases has been declining in developed countries, whereas it is still increasing in japan, with the cumulative number reaching 26,607 as of june 28, 2016. hiv infection is associated with an increased risk of hematological malignancies such as non-hodgkin lymphoma (nhl). autologous hematopoietic cell transplantation (auto-hct) is a treatment option for hiv-infected patients with nhl and multiple myeloma (mm). however, the prognosis after auto-hct in hiv-infected japanese patients remains unclear. the aim of this study is to evaluate the effect of hiv infection on transplant outcomes after auto-hct in japan. using the national database of the japan society for hematopoietic cell transplantation, we retrospectively evaluated patients with nhl (n = 3862) and mm (n = 2670) who underwent their first auto-hct between 2001 and 2014. presence of hiv antibodyperipheral t-cell lymphomas (ptcl) comprise a heterogeneous group of diseases among which ptcl-not otherwise specified (ptcl-nos) represents the most common histology. patients with ptcl are typically offered high-dose chemotherapy followed by autologous hematopoietic cell transplantation (auto-hct) as front-line consolidation. allogeneic hct (allo-hct) is generally offered in the relapsed setting; however, in selected cases it is also offered as front-line consolidation. no randomized controlled trial (rct) have been performed to date comparing offering an allo-hct versus other treatment modalities either in the front-line or in the relapsed setting. thus, we performed this systematic review/meta-analysis to assess the totality of evidence pertaining to the role of allo-hct in ptcl. search of the literature was undertaken via pubmed and web of science from inception until september 6, 2016. no search limits were applied but studies presented only in abstract form were excluded. data were collected on treatment benefits (complete remission (cr), progression-free survival (pfs), overall survival (os)) and harms (non-relapse mortality (nrm), grade ii-iv acute graft-versus-host disease (gvhd), and chronic gvhd). the search identified 1271 references; however, only 17 studies (6 in front-line (n = 132 pts), 11 in relapsed/refractory setting (n = 330 pts)) were eligible based on our inclusion criteria and had extractable data. three studies included both frontline and relapsed/ refractory cases but data for certain outcomes were reported separately. the median follow-up time for studies evaluating allo-hct in the front-line or relapsed/refractory setting ranged from 30-45 months and 12-85 months, respectively. in the front-line setting, allo-hct resulted in cr rates of 64% ((95%ci = 50-77%), 2 studies, n = 49 pts), pfs rate of 64% ((95% ci = 49-78%), 5 studies, n = 100 pts), and os rate of 72% ((95% ci = 62-81%), 5 studies, n = 95 pts). nrm rate was 6% ((95% ci = 0-15%), 3 studies, n = 68 pts). acute (grade ii-iv) and chronic gvhd rates were 39% (95% ci = 24-56%), 2 studies, n = 38 pts) and 33% (95% ci = 16-53%), 3 studies, n = 64 pts), respectively. in the relapsed/refractory setting, allo-hct resulted in cr rates of 68% ((95% ci = 70-97%), 5 studies, n = 75 pts), pfs rate of 39% ((95% ci = 31-47%), 6 studies, n = 203 pts), and os rate of 52% ((95% ci = 43-60%), 6 studies, n = 307 pts). nrm rate was 21% ((95% ci = 13-31%), 7 studies, n = 194 pts). acute (grade ii-iv) and chronic gvhd rates were 34% (95% ci = 23-46%), 7 studies, n = 197 pts) and 37% (95% ci = 30-44%), 8 studies, n = 199 pts), respectively. notwithstanding the need to perform a rct to compare the efficacy of allo-hct versus auto-hct as front-line consolidation in ptcl, the results of this systematic review/meta-analysis show very encouraging os rates of 72% following allo-hct. moreover, allo-hct also offers an encouraging os rate of 52% in patients with ptcl in the relapsed/refractory setting. the higher nrm rate in the relapsed/refractory setting probably reflects the adverse effect of a higher number of prior prescribed therapies. one of the limitations of our analysis is the inability to analyze outcomes for individual histologic subtypes due to the aggregate nature of the published data. disclosure of conflict of interest: none. high-risk patients with relapse or refractory hodgkin lymphoma do significantly better after hdc auto-sct compared to control arm of aethera trial. mature results from a cohort of 234 patients s akhtar, s rauf, tam elhassan and i maghfoor king faisal specialist hospital and research center, riyadh, kingdom of saudi arabia brentuximab vedotin use in hodgkin lymphoma (hl) patients who had hdc auto-sct has been reported to improve progression free survival (pfs) but not the overall survival (os) in a phase 3 trial (lancet 2015;385:1853-62). in this trial, after hdc auto-sct, 329 high risk hl patients were randomized to receive placebo (control gp) vs brentuximab (experimental gp) as consolidation therapy. we are reporting our experience of patients with similar selection criteria as control gp. hl patients z14 yrs who received hdc auto-sct with similar selection criteria as defined in aethera trial were identified that is, patients had at least one of the following risk advanced lymphomas still represent a therapeutic challenge and allo-hsct is among treatment options. between march 2007 and august 2016, seventy-three patients (pts) affected by r/r lymphomas (34 nhl and 39 hl) underwent an allo-hsct after a treosulfan-based conditioning regimen and sirolimus as calcineurin-inhibitor-free prophylaxis of gvhd. six pts received a mrd, 18 pts a mud, and 49 pts a haplo unmanipulated pbsc. at allo-sct 30 pts were in cr, 13 pts were in pr, and 30 pts had sd/pd; sixty patients underwent autologous sct before allo-hsct. hct-ci was evaluable for 64 pts, 33 had a score ≥3. thirty-three pts received treosulfan and fludarabine reduced toxicity conditioning regimen (rtc) and intensification with other alkylating agent or with 4 gy total body irradiation was added on the remaining 40 pts (myeloablative conditioning, mac). all pts received a backbone gvhd prophylaxis with sirolimus and mycophenolate mofetil; atg or pt-cy or both were added in 41, 25, and 3 pts respectively. median numbers of infused cd34+/kg and cd3 +/kg were 6.01 × 10 6 (range: 2.72-9.06) and 2.39 × 10 8 (range: 0.3-6.89), respectively. median follow-up was 44 months (range: 3-111); median time to neutrophil ≥ 0.5 × 10 9 /l was 17 days, and 20 days to platelet ≥ 20 × 10 9 /l. sixteen out of 43 patients with pre-transplant active disease obtained a cr after treosulfan conditioning; nine of them (6 hl and 3 b-nhl) achieved durable cr without post transplant treatment. oneand 3-years os was 62% and 48%, pfs was 47% and 37% at 1 and 3 years respectively; cumulative incidence of relapse/ progression was 32% and 42% at 1 and 3 years. grfs was 31% and 19% at 1 and 3 years, respectively. transplant related mortality (trm) was 15% at 100 days, 21% at 1 year and for the entire follow-up. the 100-day cumulative incidence (ci) of agvhd grade ≥ 2 was 19% and ci of agvhd grade ≥ 3 was 10%; ci of moderate to severe cgvhd was 23% at 2 years and for the entire follow-up. no differences in ci of agvhd or cgvhd were found if pts were stratified according to donor type, but ci of moderate-severe cgvhd was significantly higher in pts after mac regimens (p o0.0005). as expected, the outcome of pts in cr was significantly better compared with active disease, in terms of os (p = 0.0061), pfs (p = 0.00022), ri (p = 0.0028). in multivariate analysis, intensity of conditioning regimen (rtc vs mac), gvhd prophylaxis (use of atg, pt-cy or none), donor sex and age at allo-sct did not impact the transplant outcomes; both os and pfs were reduced by active disease at allo-hsct (hr = 4.37, ci 95% 1.76-10.86, p = 0.01 and hr = 4.37, ci 95% 1.97-9.7, p = 0.00, respectively) and by nhl histology (hr = 3.88, ci 95% 1.65-9.19, p = 0.02 and hr = 2.43, ci 95% 1.09-5.42, p = 0.03, respectively); grfs and ri were impacted only by active disease (hr = 2.25, ci95% 1.19-4.25 and hr = 4.89, ci 95% 1.87-12.62, p = 0.01, respectively). allo-hsct after treosulfan conditioning and sirolimus gvhd prophylaxis is feasible even in heavily pretreated pts affected by lymphomas. complete remission status before transplant remains crucial for better outcomes and in the era of new targeted treatments should be pursued. disclosure of conflict of interest: none.university of eastern finland, kuopio, finland and 12 department of medicine, kymenlaakso central hospital, kotka, finland autologous stem cell transplantation continues to have an important role in the treatment of patients with multiple myeloma (mm). in mm patients the most commonly used mobilization method is granulocyte-colony stimulating factor (g-csf) ± cyclophosphamide (cy). generally, up to 10-20% patients mobilize poorly with these methods and plerixafor may be used to enhance mobilization. the most important parameter of graft quality has usually been the number of cd34+ cells, but there are also significant numbers of other cell subsets in the grafts and they may also be of special interest in regard to post-transplant recovery and outcome. for example, a higher number of lymphocytes and nk cells in the grafts has been associated with improved lymphocyte as well as nk cell recovery, respectively. the mobilization methods used seem to affect the graft composition. however, there is currently no prospective data on the effects of plerixafor on the graft composition, post-transplant hematological and immune recovery or outcome in patients with mm. altogether eighty-seven patients with mm were included into this prospective study. seventy-seven patients were mobilized with g-csf ± cy (control group) and ten patients received also plerixafor due to poor mobilization (plerixafor group). in the control group 57/77 (74%) and in the plerixafor group 3/10 (30%) of patients were mobilized with g-csf+cy (p = 0.009). there were no statistically significant differences between the groups according to age, gender, paraprotein type, initial iss, induction therapy used or disease status at the time of mobilization. by imwg risk stratification, there were more high risk patients in the plerixafor group (5/10 vs. 13/77, p = 0.066). cryopreserved graft samples were analyzed with flow cytometry for t and b cells (cd3/cd8/cd45/cd19) as well as for nk cells (cd3/cd16+cd56). also, cd34+ cell subclasses were analyzed (cd34/cd38/cd133). complete blood counts were evaluated at +15 days, 1, 3, 6 and 12 months posttransplant. to evaluate immune reconstitution, flow cytometry of lymphocyte subsets (t, b, nk) was performed in a subset of patients at 1, 3 and 6 months after the graft infusion using the same antibody panel as for graft analysis. there were no significant differences between the groups in the number of cd34+ cells in the grafts. also, the median number of aphereses was two in the both groups (p = 0.086). the proportion of the more primitive cd34+ cells (cd34 + cd133 + cd38 -) was significantly higher in the plerixafor group (p = 0.001). in addition, the number of various lymphocyte subsets analysed was significantly higher in plerixafor group table 1 ). there were no statistically significant differences in the course of hematological recovery. the recovery of blood cd3+cd4+ t cells was significantly faster in the plerixafor group at one at three moths post-transplant. there was no significant difference in the progression-free survival (pfs) (log rank, p = 0.408) with the median follow-up time of 703 days in the plerixafor group and 882 days in the control group (0.099). in the present study plerixafor added to g-csf ± cy seemed to significantly alter the cellular composition of autologous blood grafts in poorly mobilizing mm patients. hematological recovery was comparable but the cd3+cd4+ t lymphocyte recovery was faster in the plerixafor group. the pfs was comparable between the groups. disclosure of conflict of interest: dr. valtola has received honoraria from sanofi and jansen-cilag. dr. silvennoinen has received a research grant from celgene and janssen, honoraria from genzyme and sanofi and participated in advisory board organized by amgen, janssen and takeda. dr. siitonen has received honoraria from amgen and celgene. dr. jantunen has received honoraria from genzyme, amgen and sanofi and has participated in eu leadership meeting organized by genzyme as well as medical advisory board meeting organized by genzyme and amgen. dr. varmavuo has received consultancy fees from abbvie, roche, celgene, amgen and sanofi. the other authors declare no conflicts of interest. bortezomib after high-dose melphalan as conditioning regimen before autologous stem cell transplantation in patients with multiple myeloma: a comparison with the historical conditioning regimen with melphalan alone ga ferini; ja arbelbide; al basquiera; e nucifora; n schutz; v otero; d fantl hospital italiano d buenos aires, buenos aires, argentinahigh dose of melphalan followed by autologous stem cell transplantation (asct) is the standard of care for younger patients with multiple myeloma (mm). to enhance the efficacy of the conditioning regimen, the intergroupe francophone du myelome added bortezomib to melphalan showing improved response rates, without significant toxicity. bortezomib has shown synergistic effects with melphalan, mainly if the bortezomib is administered 24 hours after the melphalan. since 2014, we have changed our conditioning regimen for patients with mm undergoing asct by adding bortezomib toallogeneic stem cell transplantation (allosct) is a potencially curative option for patients with multiple myeloma (mm). despite the improvement of reduced-intensity-conditioning (ric), transplant-related mortality (trm) remains high. there is no consensus on which graft versus host disease (gvhd) prophylaxis regimen is superior. some studies have suggested that tacrolimus-based prophylaxis is more effective than cyclosporine (csa) in terms of lower incidence of severe acute gvhd (agvhd), with no impact on overall survival (os). herein, efficacy and toxicity between two gvhd prophylaxis regimens is analyzed. we retrospectively analyzed 14 patients (pts) with relapsed mm who received allosct ric in the period from 2003 to 2015 in a single centre (table 1) . population: age, 51 years (40-73); median follow-up: 19 months (1-175). conditioning regimen: allo-ric (fludarabine + busulfan or melphalan regimens) and 100% was bortezomib-based in the tacrolimus group. donor: matched related (11 pts), unrelated (1), mismatch unrelated (1) and haploidentical (1) donor. gvhd prophylaxis: all patients received a short course of methotrexate + csa (9 pts, 64%) or tacrolimus (5 pts, 36%). complete response at transplant was 33% at csa group and 60% at tacrolimus group. all pts underwent toxicity related to chemotherapy (mainly mucositis and neutropenic fever) with organ impairment (renal or liver) in 100% tacrolimus arm as well as 4 pts in csa group. the incidence of agvhd was 80% and 77.8% in tacrolimus and cyclosporine groups, respectively (p = 0.99). grade iii-iv agvhd were reported in 2 pts (40%,tacrolimus) and 4 pts (44%, cyclosporine), with severe gastrointestinal and liver involvement. glucocorticoid resistance was observed in 75% in both groups. patients with refractory agvhd received other immunosuppressive therapies: more than 3 second-lines agents (3) (4) (5) (6) were necessary in fifty percent of pts in both groups to control gvhd. two patients had to interrupt tacrolimus due to neurological toxicity and suspected thrombotic thrombocytopenic purpura. no patients had to discontinue treatment in the csa arm because of toxicity. the 12-months os was 78.6% (80% in tacrolimus vs 66.7% in csa (p = 0.78)) and the 24-months was 68.8%. a total of 4 pts died because of gvhd. during follow-up, only 2 patients relapsed (10 and 126 months after allosct, respectively) in csa group. no relapse were seen in tacro group. in our experience, no significant differences were observed between both calcineurin inhibitor in terms of os, toxicity and gvhd incidence. an explanation could be our small number of patients. allosct is an effective therapy for selected patients but it is associated with high rates of gvhd and trm. a long-termsafety and effective prophylactic regimen is necessary as main objective.[p638]disclosure of conflict of interest: none. several parameters, including early lymphocyte, neutrophil, platelet recovery, and infused dose of cd34+ cells, have been associated with clinical outcome of patients with haematological malignancies. however, their prognostic significance remains uncertain. the aim of current study was to evaluate prognostic significance of clinical and laboratory parameters that might influence survival after autologous stem cell transplantation (asct) in hodgkin lymphoma (hl) and multiple myeloma (mm). this retrospective study included a total of 90 with hl and 114 mm patients (median age 32 years, 55 years, respectively) who underwent asct between november 2005 and june 2016. hl patients were conditioned with beam (84.4%) and cbv (15.6%) regimen, while mm patients received conditioning with high dose of melphalan. high ips (international prognostic score) at diagnosis had 68.9% hl patients and high iss (international scoring system) had 27.2% of mm patients, of which 8.8% had renal impairment. the average of transplanted cd34+ cells in hl patients was 7.15 × 10 6 /kg (range: 2-25.0 × 10 6 /kg), and 6.6 × 10 6 /kg (range: 2-15.51 × 10 6 / kg) in mm patients. after asct, favourable treatment response (partial/complete remission) achieved 83.3% hl patients, of whom 22.7% had infused o5 × 10 6 /kg cd34+ cells. median time to recovery of absolute lymphocyte count 500 × 10 6 /l or greater (alc500) was 16 days (range: 9-31 days), recovery of absolute neutrophil count ≥ 500 × 10 6 /l (anc500) was 12 (range: 6-26 days), and platelet recovery ≥ 20 × 10 6 /l (plt20) was 12 days (range: 5-44 days). after asct, 93.6% mm patients achieved favourable treatment response, of whom 26.5% had infused cd34+ cell dose48.7 × 10 6 /kg. median time to alc500 was 15 days (range: 9-23 days), anc500 was 13 (range: 9-24 days), and plt20 was 11 days (range: 5-26 days). median follow up of patients with hl was 67 months, while after asct, median event free survival (efs) was 20 months, and overall survival (os) was 38 months. treatment response after asct strongly influenced both efs and os after asct (po0.0001). in patients who achieved favourable treatment response, os and efs after asct were influenced by infused cd34+ cell dose (o5 × 10 6 /kg vs. ≥ 5 × 10 6 /kg), prolonged recovery of alc500 by day+20, plt by day +13, and achieving of anc500 by day +11(po0.05). multivariate analysis among significant variables showed that infused cd34+ cell dose was the most important parameter that influenced os and efs (po0.05). median follow up of mm patients was 50 months, while after asct, median efs was 26 months and os was 34 months. regarding patients who achieved favourable treatment response, os and efs after asct were influenced by the presence of renal impairment, infused cd34+ cell dose (≤8.7 × 10 6 /kg vs. 48.7 × 10 6 /kg) and plt20 recovery by day +13 (po0.05). among these significant parameters, multivariate analysis pointed out infused cd34+ cell dose as the most important parameter that influenced both os and efs (po0.05). these data suggest that number of infused cd34+ cells is an independent factor that may contribute to outcome of patients with hl and mm. disclosure of conflict of interest: none. high-dose therapy with autologous stem cell transplantation (asct) has become the treatment of choice for symptomatic eligible patients with multiple myeloma (mm). we studied an induction regimen of cyclophosphamide, bortezomib and dexamethasone (cybord) and showed rapid and deep responses after 4 cycles in patients with newly diagnosed mm and we subsequently done asct with melphalan (mel) conditioning. cost is the major limiting factor in developing world.all the drugs used are generic brands manufactured in india. a total of 25 mm patients (median age: 54.5 years, 76% male and 24% female) were transplanted between 2012 and 2016. in all, patients had igg kappa-12 (48%), igg lambda-03 (12%), iga lambda-05 (20%), iga kappa-02 (08%), kappa light chain 02 (08%), lambda light chain 01 (04%) patients. prior to autograft, all cases had received cybord with generic medicines. median time diagnosis to asct was 7.5 months (5 to 21 months). stem cell mobilization was done with g-csf alone in 17 (68%), g-csf plus plerixafor in 07 (28%) and chemo mobilization in 01 (04%) patients. all patients received asct support after conditioning with 200 mg/m 2 generic melphalan alone (dose adjustment was done according to renal status). all patients received thalidomide maintenance from march 2012. bortezomib used was manufactured by dr. reddy's lab, hyderabad and melphalan used was manufactured by emcure pharmaceuticals, pune, india. 68 patients from 1999 to 2011 received cyclophosphamide, vincristine, adriamycin and dexamethasone (cvad) protocol of originator medicines followed by originator melphalan conditioning and asct (cvad-mel-asct). at the time of autograft, 21 (84%) of patients were in complete remission, 02 (08%) in partial remission, 02 (08%) very good pr. median day of engraftment was 10 for neutrophils and 14 for platelet. transplant related mortality was 16% (4/25) out of which 2 died of infection and 2 deaths of cardiac events. the pfs and os rates were 80% and 84% at median follow up of 18.6 months. patients who were treated with cvad-mel-asct had efs of 74% at 2yrs and 52% at 5yrs. cost of bortezomib showed significant difference, generic was 4000usd where as for originator drug was 20000usd for 4cycles of chemotherapy. cost of melphalan also showed difference with 450usd for generic and 2000usd for originator drug. generic cybord showed excellent response rate and allows excellent stem cell collection and transplantation which can further consolidate response and improve outcome. cybord induction and melphalan conditioning with generic medicines can be considered a standard regimen for transplant-eligible patients with newly diagnosed mm in resource constraint situation. generic cybord-mel-asct is more cost effective than originator cvad-mel-asct. generic medicines produced in india are of good quality and cost effective. this study needs long term follow up to assess survival parameters at a median. disclosure of conflict of interest: none. and an extra copy of one or more odd-numbered chromosomes and as intermediate risk(ir) if they had t(4;14) or del(13) (q).overall survival (os) and relapse-free survival (rfs) were calculated from the time of allo hsct and auto hsct on day 0, from diagnosis to death or disease progression. the median age at presentation was 53.86 (range: 20-80) years, and 72 (63.7%) were men. at a median follow-up time of 18 months, 73% were alive.45 of the 113 patients with available fish samples underwent auto hsct. 24 patients (53.3%) achieved cr and 21 patients (46.7%) relapsed. of the 13 patients who had received allo hsct, five patients (38.5%) achieved cr and five patients (38.5%) remained alive. in patients who received auto hsct, the risk of relapse was 56% less than those never transplanted (p = 0.02), but the difference was not significant in patients who received allo hsct. the relapse-free survival in hr patients was 6 months (po 0.001), in ir was 11 months (p o0.001) and in sr was 37.67 months (p o0.001). in transplant patients, rfs in hr patients was 5.73 times more than sr group (po0.001) and in ir group was 3.35 times more than sr (p o0.001). the survival time in transplant patients was significantly better than non-transplanted patients (p o0.001). the median overall survival (os) in hr patients was 25.45 months, in standard risk group 30 months and in sr patients was 31 months. cytogenetic abnormalities detected by fish are of significant value in classification, risk stratification and management of patients with mm. we can use cytogenetic data to provide prognostic information and also to guide management and clinical practice. these data indicate that autologous stem cell transplantation could potentially be of benefit to myeloma patients. disclosure of conflict of interest: none. chronic graft-vs-host disease (cgvhd) is the most troublesome complication developing after allogeneic hematopoietic stem cell transplantation (allo-hsct). diagnosis of cgvhd has largely been based on clinical features only. we previously reported gene expression profiles in patients with cgvhd after allo-hsct. we extended our study to develop a molecular diagnostic method of cgvhd. we selected six most commonly expressed genes from the former dna expression study. and, a home-made 6-gene pcr array were used to evaluate gene expression profiles in the peripheral blood mononuclear cells of 39 patients given allo-hsct (20 cgvhd patients, 19 non-cgvhd patients) and 19 normal controls. the gene expressions of the allo-transplanted patients were compared to those of the stem cell donors. sybr green qpcr and multiplexqpcr were performed to confirm the usefulness of the selected genes in the diagnosis of cgvhd. infogainattributeeal and ranker were used to develop a gene model to diagnose cgvhd. k-nearest neighbor model and weka classifiers lazy ibk module were applied to evaluate the performance of the gene model. in another 21 steroid-refractory cgvhd patients (14 responders, 7 non-responders), the gene expression changes were analysed using our 6-gene pcr array before and 57 days after rituximab treatment. we identified six genes most accurately delineating cgvhd patients from those without treatments after allogeneic hematopoietic stem cell transplantation (hsct) are long and constraining for patients. medical adherence in hsct patients is of major concern in daily practice but it has been not yet described. 1, 2 the aims of our study were to evaluate treatment adherence and to identify factors associated with adherence behaviors. an observational single-center study was based on self-reported questionnaires completed by patients in a hematology day hospital between november 2015 and july 2016. the patientreported adherence was evaluated using the eight-item morisky medication adherence scale (mmas-8). 3, 4 individual item scores were summed: patients with a score of 8/8 were considered as good adherents to medication whereas a poor adherence referred to a score under 8. among the latter, medium adherence ranged to a score of 6-8, while a score of o6 was considered low adherence. socio-demographic and medical characteristics were collected by health records. a univariate model was used to evaluate if some of patients' characteristics were associated with adherence. statistical analysis was performed using r software (version 0.98.1103 -2009-2014 rstudio, inc). fifty-six patients were included in the current study. median age at transplantation was 55 years (range: 16-72 years). diagnosis were aml (n = 28), all (n = 10), myelofibrosis (n = 8) and other hematological diseases (n = 10). 24 patients received a hsct from a related donor (13 haploidentical). myeloablative conditioning was used in 18 patients and reduced intensity regimen in 38 patients. a total of 64.3% (36/56) of the patients were poor adherent according to mmas-8. among these patients, 6/36 were low adherent and 30/36 were medium adherent. the results of univariate analysis showed that a poor adherence was associated with a longer time since hsct and discharge at home. however elderly patients, patients treated with cyclosporine and patients with daily hydration at home were associated with a better adherence (po 0.05). our study presents the first data on adherence among patients undergoing hsct. risk factors associated with a poor adherence have been identified in order to determine patients' profiles that will benefit more from interventions to improve adherence. particular attention has to be paid to younger patients. efforts to establish a regular follow-up of these patients are needed in order to sustain patients in the treatment adherence to prevent the occurrence of severe complications. we studied the effect of basic fibroblast growth factor (fgfb) and dexamethason on expansion and immune modulation of mscs in patients with lymphomas. mscs were generated from bone marrow aspirates obtained from the patients with hodgkin's lymphoma (hl; n = 8) and non-hodgkin's lymphoma (nhl; n = 4). the adherent fraction of marrow aspirate was cultivated with/without the basic fibroblast growth factor (fgf-b, 10 ng/ml) or dexamethason (10 − 5 м or 10 − 8 м) to reach 80-90% confluence. then mscs were passaged with accutase and used for experiments after 1-2 passages. the number of msc precursors (cfu-f) in bone marrow of lymphoma patients was found to be significantly decreased both in patients with nhl (17 ± 5, p o0.01) or hl (26 ± 5, p o0.05). the time until 80-90% confluence was significantly increased and took on average 26 ± 2 days (vs 15.4 ± 0.6 in donors). finally, the immunosuppressive ability of patient msc was significantly lowered and was only registered at the high concentrations of mscs (1:1 and 1:2). the expansion of patient mscs was significantly promoted with fgfb resulting in a significant decrease of primary cell cultivation (from 25.4 ± 1.52 to 18.6 ± 1.21 days; p = 0.041) and a statistically significant twofold increase in the number of cells received at the first passaging. in addition, in cultures with fgfb there was a decrease in the relative amount of resting mscs and a threefold increase of cycled cells in cd73+ mscs. dexamethasone has also provided a moderate stimulating effect on the msc growth. in fact, the use of 10 − 5 м of dexamethasone resulted in the increase of the cell yield by 1.6 times and of 10 −8 м-by 1.9 times. however, fgfb and dexamethasone differed in their effect on the msc ability to inhibit the proliferative response of t lymphocytes upon stimulation with mitogens or alloantigens. indeed, fgfb failed to correct the impaired immunosuppressive activity of patient mscs, and median percentage of suppression still remained lowered-17% vs 16% without fgfb. in contrast to fgfb, dexamethason could increase the immunosuppressive activity of patient mscs by 1.5 times (in dose of 10 − 5 м) and by 2.1 times (for 10 − 8 м). our data indicate that fgfb and dexamethasone used during the generation of mscs exert a stimulating effect on the msc expansion. in contrast to fgfb, dexamethasone, in the broad range: of doses, was able to enhance the suppressive properties of mscs that are initially reduced in patients with lymphoma. these findings suggest the existence of at least two mechanisms of impairments in immunoregulatory function of mscs in lymphomas-dependent and independent of the msc proliferation. disclosure of conflict of interest: none. free nonabsorbable antibacterial digestive decontamination is associated with a low incidence of gastrointestinal acute gvhd and better gvhd-free/ relapse-free survival (grfs) in the atg-based conditioning regimens nabil yafour 1 commonly antibacterial prophylaxis based of oral no absorbable antibiotic such as (neomycin colistin, gentamicin, vancomycin) used before and after engraftment, other fluoroquinolone such as levofloxacine were recently used to prevent invasive infection. however the exact interaction with gastrointestinal acute graft versus host disease (gi-agvhd) remains unclear. the objective of this study was to evaluate a novel composite endpoint of gvhd-free/relapse-free survival (grfs), in which events include grades 3-4 gi agvhd, chronic gvhd requiring systemic therapy, relapse, or death in atg based-conditioning regimens, with free no absorbable antibacterial digestive decontamination prophylaxis. a total of 39 evaluable consecutive patients with hematological disease were included in period of february 2013 to mai 2016. patients with malignancies disease (n = 33) received myeloablative conditioning regimens plus atg (5 mg/kg); including once daily busulfan (130 mg/m 2 ,-6d to -3d, iv ) + fludarabine (40 mg/m 2 /d, -6d to -3d, iv) (aml = 26, all = 3, cml = 1) or melphalan (140 mg/m 2 , d-1, iv) (all = 3). six patients received cy/atg for saa. gvh prophylaxis consisted to; ciclosporine a (csa) + mtx. csa was maintain levels between 150-400 ng/ml and tapered at the discretion of the treating physician. all patients were received peripheral blood stem cells (pbsc) graft from a matched related donor. since december 2015 levofloxacine and voriconazole was administered as antibacterial and antifungal prophylaxis. diagnostic, clinical grading and treatment of gi-agvhd and gi-cgvhd were performed according to established criteria and nih recommendations. probability of grfs was estimate by kaplan-meier method. median age was 35 years (range: 6-60). median dose of cd34+ and cd3+ cell doses were 4.9 × 10 6 (range: 2.5-7) and 1.84 × 10 8 (range: 0,036-5,28). the median time to neutrophil and platelet recovery were13 days (range: 6-33) and 14 days (range: 10-43) respectively. at time of transplant 12/39 (31%) had an intestinal colonization with extended-spectrum betalactamase (esbl) producing bacteria. only 5/39 (13%) developed infectious diarrhea during the period of transplant. incidence of grade iii/iv gi-agvhd and gi-cgvhd requiring systemic therapy were 5% and 5% respectively. for patients with malignancies diseases (n = 33), 23 (70%) were alive at a median follow up of 12 months (range: 5-43). incidence of relapse, disease free survival rates were 30%, 67% respectively. the grfs rate as defined previously was 48% at 30 months. these results confirm that free no absorbable antibacterial digestive decontamination and atg-based conditioning regimens were associated with very low incidence of gi-gvhd and better grfs in patients with malignancies diseases. diverse bacterial populations of the gastrointestinal tract remain important factors to promote immune tolerance after allogeneic sct. disclosure of conflict of interest: none. g-csf primed hla haploidentical transplantation from maternal or collateral donor using atg plus reduced dose of posttransplantation cyclophosphamide: results of a phase ii prospective trial y wang 1 , x-j huang 1 1 peking university people's hospital, peking university institute of hematologythe transplantation milieu using granulocyte colony-stimulating factor (g-csf), and anti-thymocyte globulin (atg) for hlahaplotype-mismatched transplants from related donors has resulted in favourable outcomes with low transplant-related mortality (trm), without increased relapse rate. however, in this transplant modality, the poorer outcome owing to high incidence of graft-versus host disease (gvhd) related to maternal donor or collateral donor remains a concern. meanwhile the use of post-transplant cyclophosphamide (pt/cy) in recent years appears to be protective against severe acute and chronic gvhd. we performed a prospective pilot study of hla haploidentical stem cell transplantation (sct) from maternal or collateral donors with intensified conditioning including g-csf and atg, followed by two lower doses of pt/cy (14.5 mg/kg × 2 doses). outcomes were compared with those of 160 controls from matched-pair analysis who undergone haploidentical sct from other donors than mother or collateral relatives at the same time period. a total of 40 patients with myelodysplastic syndrome (mds) or leukaemia undergoing haploidenticla sct from maternal or collateral donors were enrolled in the study. incidence of grade ii-iv and grade iii-iv acute gvhd at day 100 were comparable between the study group and the control group (17.5% vs. 33.3%, p = 0.07; 5.0% vs. 12.5%, p = 0.24). incidence of cmv and ebv reactivation at day 100 were also comparable between the study group and the control group (75.0% vs. 85.0%, p = 0.16; 15.0% vs. 29.2%, p = 0.09). after a median follow-up of 303 days and 341 days, the incidence of trm and relapse at 1 year were comparable between the study group and the control group (5.0% vs. 13.3%, p = 0.16; 10.0% vs. 6.7%, p = 0.54); the probability of overall survival and lfs at 1 year were comparable between the study group and the control group (84.2% vs. 79.8%; p = 0.24; 83.0% vs. 77.7%, p = 0.48). in conclusion, conditioning with atg and low-dose pt/ cy might be a feasible option for patients undergoing hla haploidentical, t-cell replete sct from maternal or collateral donors. trial registration: the study is registered at www. clinicaltrial.gov as nct02412423. disclosure of conflict of interest: none. hematopoetic stem cell transplantation (hct) is a lifesaving treatment option for eligible patients with hematological malignancies. hct is inherently associated with a risk of nonrelapse mortality that varies greatly depending on transplant and patient characteristics. the assessment of the risk of complications and mortality before the procedure is extremely important. the hct comorbidity index (hct-ci) introduced by sorror m. is one of the tools proved to predict hct outcomes and was shown to be significant in various disease and hct settings. the objective is to evaluate hct-ci index of hct recipients, determine impact of different variables on ci score, particularly those, showing pulmonary and cardiac function. data of hct-ci of autologous (auto) and allogeneic (allo) hct recipients, transplanted during period january 2015-october 2016 were analyzed. impact of pulmonary and cardiac function values on ci score was evaluated: dlco (diffusing capacity of the lung for carbon monoxide), fev1 (forced expiratory volume) and ef (cardiac ejection fraction) are parameters, reflecting pulmonary and cardiac function, which values are included into hct-ci score. the statistical data analysis was conducted using spss program. the differences were considered statistically significant at p ≤ 0.05. records of 100 allo and 220 auto hct recipients, transplanted during 01.2015 -10.2016 in vilnius university hospital were revised. median age of allo hct and auto hsc recipients was 50 (19-75) and 58 (19-74) years respectively. main indication for allo hct was acute myeloid leukemia 48 (48%) patients and for auto hct -multiple myeloma 133 (60.5%) patients. hct-ci was completely calculated (no values missing) in 64 allo and 102 auto hct recipients. only patients with available complete hct-ci data were further analyzed. hct-ci in hct recipients was as shown in table 1 . hct-ci score o3 was calculated in 37 (57.8%) and ≥ 3 in 27 (42.2%) allo hct recipients. hct-ci score o3 was calculated in 45 (44.1%) and ≥ 3 in 57 (55.9%) auto hct recipients. hct-ci score did not differ statistically significant between male and female recipients in both hct categories as well as in different age groups of patients (below and above 40 years in allo and below and above 60 years in auto hct). dlco was found to be below normal values (o80%) in 43 (67.19%) allo hct and in 67 (65.7%) auto hct recipients. fev1 was less affected and found to be lower 80% in 6 (9.3%) allo hct and in 17 (16.7%) auto hct recipients. ef below 50% detected in 1 (1,6%) allo hct and in 6 (5.9%) auto hct recipients. low dlco was found to cause the greatest impact on hct-ci score and was statistically significantly associated with higher hct-ci (po0.001). the most common hct-ci in both hct groups was score 3. dlco was found to be below normal ranges in relatively large patient group and had the greatest impact on hct-ci score. further studies on reasons of pulmonary function impairment and it's impact on hct outcomes are warranted.[p690]disclosure of conflict of interest: none. haemophagocytic lymphohistiocytosis (hlh), a life-threatening hyper-inflammation syndrome, is classified into primary and secondary forms. primary hlh is caused by gene mutations resulting in impaired cytotoxicity of natural killer (nk) cells and cytotoxic t lymphocytes (ctls). secondary hlh arises in the setting of autoimmunity, infection, malignancy, or less commonly, may be idiopathic. treatment of hlh has two major goals: halting the triggering event and controlling the overactive immune system. however, patients with primary or recurrent secondary hlh should subsequently undergo allogeneic hct for long lasting disease remission. we retrospectively evaluated 61 hematopoietic stem cell transplantation (hsct) might be a valid treatment option for adults suffering from aggressive t-cell malignancies providing long term disease control. since a suitable hla-matched donor cannot be identified for all patients (pts) in need for transplantation, alternative donors graft sources such as related hla-haploidentical donors are considered. through introduction of t-cell-replete (tcr) hlahaploidentical transplantation (haplo-hsct) using post transplantation cyclophosphamide (ptcy) successful treatment with low non-relapse mortality rate (nrm) has been observed in lymphoma patients (luznik et al., bmt, 2008) . however, less data are available on the outcome of this haplo-approach in the treatment of t-cell malignancies, in particular when disease is refractory. we retrospectively evaluated the outcome of haplo-hsct using tcr grafts and ptcy in 8 pts with peripheral t-cell lymphoma treated between 2010 and 2015 at our institution (t-nhl = 6, t-all = 2; male n = 5; median age: 37 years). disease was refractory/active at time of transplantation in 7 pts, while one had achieved second cr. all patients received at least 2 prior treatment lines and one patient failed previous allogeneic transplantation. while fludarabine and cyclophosphamide served as backbone for conditioning, 3 pts received a tbi-based and 5 a drug-based conditioning regimen which was myeloablative in 50%. if disease was active at time of haplo-hsct, a sequential therapeutic concept was performed involving intensive chemotherapy (clofarabine n = 6) shortly preceding conditioning (zoellner ak et al., bmt, 2015) . post-grafting immunosuppression consisted of cyclophosphamide, tacrolimus and mycophenolate mofetil in all patients. graft source was bone marrow in 3 pts. no primary graft rejection occurred; 7/8 pts engrafted, one died early in aplasia. neutrophil/platelet engraftment was achieved at a median of 20 (range: 14-36) and 42 (range: 17-117) days, respectively. acute gvhd grade ii-iii was observed in 4 pts, whereas no patient developed grade iv agvhd. mild chronic gvhd occurred in one patient. 50% of the pts developed grade ii-iii treatment-related toxicities most commonly diarrhea (33%) and mucositis (25%); grade iv toxicity (mucositis) was observed in one patient only. no vod occurred. cmv reactivated in 4/5 pts at risk, whereas no ptld was seen. proven invasive aspergillosis was diagnosed in one patient. at day +30 seven pts achieved cr. 3 pts relapsed and 3 died (relapse n = 1, infection n = 2). 1-year nrm was 25%. at a median follow up of 46 months (range: 15-76) the estimated 1-year and 3-year overall survival (os) and progression-free survival (pfs) were 63%/63% and 50%/33%, respectively. three pts received haploidentical dlt pre-emptively (n = 2) and therapeutic (n = 1), leading to sustained cr in two, while no severe gvhd occurred. sequential therapy in the setting of tcr haplo-hsct using ptcy as gvhd prophylaxis is feasible, well tolerated and shows low rates of gvhd and acceptable nrm in patients with relapsed/refractory t-cell lymphoma/ leukemia providing an effective anti-lymphoma/leukemic activity. thus, we suggest that intensified tcr hapo-hsct using ptcy should be considered as an alternative for patients suffering from aggressive t-cell malignancies, lacking hlamatched donors. disclosure of conflict of interest: none. tacrolimus is a calcineurin inhibitor increasingly used as immunosuppression following allogeneic stem cell transplantation; maintenance of therapeutic serum levels is essential to reduce the risk of graft rejection and graft versus host disease. however, tacrolimus can be associated with serious side effects and potential drug interactions. regular monitoring of serum levels and appropriate dose adjustment is essential to ensure therapeutic levels and to avoid toxicity. in our adult bmt unit, an established standard operating procedure (sop) provides a prescriptive dosing algorithm for: (i) initiation of tacrolimus therapy; (ii) conversion between iv and oral routes; (iii) dose adjustment based upon tacrolimus serum level and interacting medications. we performed an audit assessing adherence to the sop dosing algorithm. 97 inpatient tacrolimus dosing episodes from five consecutive haploidentical transplants were retrospectively analysed. 17 episodes were excluded due to insufficient records. for the remaining 80 episodes, tacrolimus serum levels and corresponding doses were identified. the response of the medical team to each serum level was compared with the sop dosing recommendation. to account for sensible rounding of doses, a margin of error of ± 10% was permitted. adherence to sop dosing was 54%. non-adherence to the sop (46%) was subcategorised as justifiable (21%) or unjustifiable (25%). justifiable non-autologous hsct is currently being explored for its efficacy and safety in the treatment of multiple sclerosis (ms). as more experience is gained in treating this cohort, treatment related mortality has steadily improved although the procedure still carries a degree of risk. ebv reactivation is well described in allogenic stem cell transplants although less so in autologous transplantation. we investigated the frequency of ebv reactivation in patients with ms undergoing autologous hsct at a single uk site. 30 patients underwent autologous hsct for treatment of ms at king's college hospital between feb 2012 and aug 2016. all were mobilised with cyclophosphamide 4g/ m 2 and g-csf. 29 were conditioned with cyclophosphamide and atg, and one with beam/atg. previous exposure to ebv (ebv igg) was assessed prior to transplant and local posttransplant ebv monitoring was performed on whole blood samples by means of quantitative pcr in 26 patients. data was collected retrospectively. all 26 (100%) patients were positive for ebv igg pre-transplant. overall, 194 samples were tested for monitoring post-transplant. 20 (76.9%) patients demonstrated positive pcr post-transplant on local testing with one further patient being negative on local tests but later becoming positive on testing in their parent hospital (full results unavailable). of these 20, the median time to positive testing post-transplant was 24 days (7-91). maximal ebv dna titre was reached at a median time of 40 days post-transplant (7-101) with a mean maximum titre of 5.17 log (3.3-8.2). 2 patients experienced symptomatic reactivation with an associated large paraproteinemia. one of these developed hyper-viscosity requiring plasma exchange and developed neurological symptoms mimicking an ms relapse (max ebv titre of 8.2 log). this patient received rituximab and ebv level is declining, the other was observed carefully but developed right leg weakness which is slowly improving. the patient with raised ebv at their parent hospital also received rituximab (unclear if this reactivation was symptomatic). we have developed a protocol to pre-emptively treat ebv reactivation with rituximab once a 6 log titre is reached and one patient has so far been treated according to this. of the 18 patients with locally confirmed reactivation who did not receive rituximab, 9 (50%) self-resolved at a median time of 98 days (44-182), 5 (33.3%) have ongoing re-activation (4 with improving, 1 with stable titres) and 4 (22.2%) have not had any local bloods performed ≥ 6 months. 1 patient with selflimiting reactivation later had a further positive titre (370 days post-transplant and 182 days post initial resolution). ebv reactivation appears to be common in patients with ms in the first 3 months post autologous hsct. unlike in other patient groups such as aplastic anaemia patients receiving allogeneic transplants it can cause significant neurological symptoms which may be confused with ms relapse. the mechanism of this reactivation is probably related to atg administration but may be exacerbated by prior immune suppression in this heavily pre-treated group, the majority of whom have received highly active disease modifying therapies in the past. these results demonstrate the importance of monitoring for ebv reactivation following autologous hsct and the consideration of pre-emptive therapy. disclosure of conflict of interest: none. reduced bone mineral density (bmd) is a well recognised complication of hct. guidelines recommend scanning by dual energy x-ray absorptiometry (dxa) one year after transplant in all hct patients 1 or else specific groups of high risk patients. 2, 3 it is recognised that both dose and duration of steroids are risk factors for low bmd and it is recommended that prednisolone doses greater than or equal to 5mg/day for more than 3 months should prompt a dxa scan. for patients with osteopenia it is recommended that calcium/vitamin d supplements are given together with lifestyle advice including diet, smoking cessation and weight bearing exercise. 1 in this survey we have investigated the current practise in investigating and managing bone health in the context of hct. a survey was sent to all 453 centres including 45 countries registered with ebmt as of november 2016. 63 centres replied from 14 countries. response numbers to each question were variable and are indicated by the denominators. 5/36 used a national guideline to guide their practise, and 3/34 used an international guideline. no single guideline was quoted more than once. 25 low testosterone has been demonstrated to be an independent determinant of endothelial (dys)function in men. graftversus-host disease (gvhd) is a major contributor to nonrelapse mortality (nrm) after allogeneic stem cell transplantation (allosct). vulnerability of the recipients' endothelial cell system is a novel concept to explain why a proportion of patients with acute gvhd fail to respond to escalating immunosuppressive therapy and ultimately succumb to gvhd and related complications. this retrospective study investigated the prognostic impact of pre-transplant testosterone levels on nrm after allosct in male patients. between 2002 and 2014, a total of 277 male patients undergoing allosct at heidelberg university (median age 55 years) provided informed consent to participate in this observational study (training cohort). a total of 71 patients (26%) received transplants from related donors (rd). diagnoses were aml (48%), mds (29%), lymphoid malignancies (33%) and multiple myeloma (12%) . a total of 176 patients (78%) received statin treatment post allosct as per institutional standard policy. for validation, an independent patient cohort of 205 men allografted for aml and mds (median age 57 years, 18% rd, no statin treatment) at essen university was analysed. pretransplant serum samples were collected between 0 and 2 months before allosct and cryopreserved at − 80°c. testosterone and suppressor of tumorigenicity-2 (st2) levels were measured by radioimmunoassay and elisa, respectively. median pre-transplant testosterone level in the training and validation cohort was 13.6 nmol/l (range: 0.3-41.7 nmol/l) and 16.0 nmol/l (0.8-38.1 nmol/l), respectively. in the training cohort, lower pre-transplant testosterone as continuous variable was associated with shorter os (p = 0.009). lower testosterone levels showed a trend towards higher nrm (p = 0.09) and a significant association with nrm after onset of acute gvhd (p = 0.02). multivariate analysis confirmed lower pre-transplant testosterone levels as a significant predictor of an increased nrm risk after gvhd onset (p = 0.03). in the subgroup of patients not receiving statins post-transplant, lower testosterone levels were associated with increased incidence of transplant-associated microangiopathy (p = 0.01), and, in addition, with higher pre-transplant st2 levels indicating endothelial vulnerability. in the validation cohort, similar results with regard to overall survival (os, p = 0.02), nrm (p = 0.04), nrm after acute gvhd onset (p = 0.03) in univariate analysis, and to nrm after gvhd onset (p = 0.02) in multivariable analysis could be observed. the association of pre-transplant testosterone levels (in quartiles) and incidence of nrm after gvhd onset in the training and validation cohort is depicted in figure 1a and 1b, respectively. our study suggests that low pre-transplant testosterone is associated with serological and clinical evidence for endothelial damage and is an independent risk factor for a fatal outcome of gvhd. prospective studies in the allosct setting investigating testosterone and testosterone supplementation in deficient patients are highly warranted. disclosure of conflict of interest: none. nk cells anti-tumor ability in multiple myeloma patients s tognarelli 1,2 , b jacobs 3,4,5 , i von metzler 6 , h serve 6 , p bader, t klingebiel 7 , a mackensen 3 and e ullrich 1,2 1 department of pediatric stem cell transplantation and immunology, childrens hospital, goethe university, frankfurt, germany; 2 cellular immunology, loewe centre for cell and gene therapy, goethe university, frankfurt, germany; 3 department of hematology and oncology, university hospital erlangen-busulfan is one of essential drugs for hematopoietic stem cell transplantation (hsct). because of its narrow therapeutic range: targeted busulfan using therapeutic drug monitoring (tdm) has been used. generally, the initial dose of busulfan is determined by patients' body surface area as 120 mg/m 2 except for infants (80 mg/m 2 ). however, pharmacokinetic evidence of these initial doses is scarce. therefore, we investigated the full pharmacokinetics of busulfan in infant and child, and attempted to validate that these initial doses are acceptable. one hundred ninety-five pediatric patients undergoing hsct using four-day targeted busulfan were enrolled. of them, 6 patients received hsct when their age was ≤ 1 year old (infant group [ig]), and 19 patients received when 1-2 years old (toddler group [tg]). the remaining 170 patients were defined as a child group (cg). busulfan was administered intravenously once daily for 4 consecutive days. tg and cg received 120 mg/m 2 as the first dose, and ig received 80 mg/m 2 . using daily tdm, we adjusted the next dose of busulfan. target daily and total area under the curve (auc) were 18 750 μg*h/l/day and 74 000-76 000 μg × h/l, respectively. median first-day busulfan auc of ig, tg, and cg were 18 416, 22 529 and 20 410 μg × h/l, respectively, which was significantly different (p = 0.031). however, there was no significant difference in median total busulfan auc (ig; 74 180, tg; 73 406, and cg; 74 482 μg × h/l, respectively, p = 0.089). the coefficient of variance (cv) of four-day busulfan aucs in ig and cg was similar (median cv: 22.1% and 24.7%, respectively), whereas cv of tg was 40.4%. in sub-analysis of tg and cg who received equally 120 mg/m 2 as the first dose, there was an inverse correlation between age and first-day busulfan auc (r = − 0.148, p = 0.042), as well as between age and cv of four-day busulfan aucs (r = − 0.210, p = 0.004). initial busulfan dose as 80 mg/m 2 for infant could be acceptable in aspect of first-day auc and cv of four-day busulfan aucs. however, higher first-day auc and cv were shown in tg. although target total busulfan auc could be achieved safely by tdm, we suggest that reduction of initial dose less than 120 mg/m 2 is also necessary to patients with 1-2 years old to lower the relatively higher first-day auc. taken together, tdm is highly recommended to reduce busulfan toxicity, especially in younger children. disclosure of conflict of interest: none. post-induction treatment strategy of acute myeloid leukemia (aml) is currently driven by european leukemia net (eln) risk assessment at diagnosis. if it is well established that patients belonging to favourable-risk group can be treated with chemotherapy and/or autologous stem cell transplantation (sct) and that those belonging to the unfavourable-risk group should be addressed to allogeneic (allo) sct, for patients included in the intermediate-risk groups the best post-induction treatment has not been established yet. we report here a 6years (2010-2015) allo-sct single center experience in 78 aml patients. median age was 53 years (range: 20 -68), 17%, 23%, 9% and 51% were grouped in the eln favourable, intermediate-i, intermediate-ii and unfavourable risk category, respectively and 47% of the patients were allotransplanted in advanced disease-phase (2nd complete remission). half of the patients received a sibling hla compatible donor, 76% of the cases received peripheral blood stem cells and half of the patients received a myeloablative conditioning regimen. graft versus host disease prophylaxis was conventionally based on cyclosporine and shor-course methotrexate, with the addition of antilymphocyte immunoglobulin in case of matched unrelated donor. the clinical and transplant characteristics of the patients according to the eln-risk group were well balanced. with a median follow up of 20 months (range: 8-58 months), the projected 2 years overall survival (os) and disease free survival (dfs) is 45% (95% ci: 32-57%) and 43% (95% ci: 30-54%). the median os and dfs in favourable/intermediate-i vs intermediate-ii/unfavourable is 21.8 and 14.8 months ( figure 1a ; p = 0,67) vs 18 and 14,8 months ( figure 1b ; p = 0.66). the relapse rate (rr) and the non relapse mortality (nrm) at two years are 38% (95% ci: 26-50%), and 15% (95% ci: 8-26%), respectively. non differences were observed comparing the 2 years rr and the 2 years nrm of patients in the favourable/intermediate-i vs intermediate-ii/unfavourable eln risk group (36% vs 40%; p = 0.66 and 16% vs 18%; p = 0.95). interestingly, the percentage of patients allotransplanted in advanced phase of the disease was higher in those included in low/intermediate-i with respect to intermediate-ii/unfavourable eln-risk group (73% vs 43%; p = 0.001). our data suggest that allo-sct can cure approximately 40-50% of aml patients, with no difference within the eln risk groups. disease recurrence remains the major problem and this is highly correlated to the percentage of patients in advanced phase of the disease at transplant, particularly in eln favourable/intermediate-i patients. we are currently collecting the data on minimal residual disease (mrd) status of these patients during chemotherapy and before transplant using moelcular biology on target genes and/or multiparametric flow cytometry on leukemia associated immunophenotype, in order to assess if the prognosis of these patients may be refine by the prospective application of mrd data.[p707]disclosure of conflict of interest: none. outcome of allogeneic stem cell transplantation for patients with high-risk acute leukemia according to donor type and graft-versus-host disease prophylaxis s lindner, t berg 1 , j riemann 1 , s ajib 1 , z jedlickova 1 , s gueller 1 , f lang 1 , a sackmann 1,2 , n goekbuget 1,2 , h martin 1 , a bacigalupo 3 , h serve 1,2 and g bug 1 1 department of medicine ii, hematology and oncology, university hospital frankfurt, goethe university, germany; s500 2 german cancer consortium (dktk), german cancer research center, heidelberg, germany and 3 università cattolica del sacro cuore, fondazione policlinico universitario gemelli, roma, italyin high-risk acute leukemia (hr-al), allogeneic hematopoietic stem cell transplantation (hsct) is the only potentially curative treatment. increasingly, hsct is being performed utilizing alternative donors. we retrospectively analyzed the outcome of 148 consecutive patients (pts) with hr-al (aml/all, n = 118/30) undergoing first allogeneic hsct in our transplant unit between 1/2011 and 6/2015 according to donor type and graft-versus-host disease (gvhd) prophylaxis: in the matched related donor group (mrd, n = 26), hsct was performed with standard immunosuppression (is), that is, calcineurin inhibitor (cni) plus methotrexate or mycophenolate mofetil (mmf). for 10/10 hla-allele matched unrelated donors (10/10 mud, n = 84) or 9/10 hla-allele mud (9/10 mud, n = 24) we used is and anti-thymocyte globulin (atg fresenius/neovii). hsct with a haploidentical family donor or an 8/10 hla-allele mismatched unrelated donor was performed using is with cni plus mmf and post-transplant cyclophosphamide (pt-cy, n = 14). a myeloablative (n = 75) or reduced-intensity (n = 73) conditioning regimen was applied in complete remission (cr, n = 106) or active disease (n = 42). pts had a median age of 52 years (range: 19-72) and hematopoietic cell transplantation comorbidity index of 3 (range: 0-11). patient and treatment characteristics were well balanced between the groups except for a higher percentage of pts transplanted in cr in the pt-cy group (93% vs. 54-76%, p = 0.03). peripheral blood stem cells were preferred for mrd, 10/10 mud and 9/10 mud (81%, 95% and 96%, respectively) and bone marrow for 93% of pt-cy based hsct. all pts engrafted. with a median follow-up of 28 months (range: 1-60), probability of overall survival (os) at 3 years was 54 ± 10% for the mrd, 65 ± 6% for the 10/10 mud, 41 ± 11% for the 9/10 mud and 93 ± 7% for the pt-cy group, without significant differences (p = 0.05). however, the probability of achieving the combined endpoint gvhd-and relapse-free survival (grfs) at 3 years varied significantly between the groups (mrd 8 ± 5%, 10/10 mud 43 ± 6%, 9/10 mud 29 ± 10% and pt-cy 57 ± 13%, po0.01), reflecting the high cumulative incidence (ci) of chronic moderate and severe gvhd at 1 year in the mrd (58 ± 11%) as opposed to the other groups (10/10 mud 12 ± 4%, 9/10 mud 12 ± 8% and pt-cy 33 ± 14%, p o0.01). of note, donor type had no impact on ci of transplant-related mortality (trm) at 3 years (12 ± 3%), acute gvhd g3-4 at day +100 (10 ± 3%) or leukemic relapse at 3 years (34 ± 4%). overall, aml pts 460 years of age had a significantly inferior relapse-free survival compared to younger pts (50 ± 9% vs. 71 ± 6%, respectively, p o0.01) without a higher ci of trm (p = 0.37). median time to aml relapse was 6 months. our results suggest that pt-cy-based alternative donor hsct is safe in hr-al pts and provides a solid basis for a randomized clinical trial comparing hsct from haploidentical family donors and 9/10 mud, currently in preparation. while os did not vary between groups, grfs was dismal after mrd transplants without atg, due to high rates of severe chronic gvhd, consistent with published data. as leukemic relapse remains the major cause for treatment failure especially in elderly pts, maintenance strategies using novel drugs or cellular therapies are warranted. disclosure of conflict of interest: none. relapse following hematopoietic stem cell transplant (hsct) is the leading indication for a second transplant in patients with malignant disease. hsct has been shown to be superior to chemotherapy alone or palliative measures in these patients. for non-malignant disease a second transplant may be considered for graft failure after first transplant. data regarding the outcome of a second hsct for non-malignant disease is scarce. we retrospectively analyzed 29 patients who underwent a second hsct, for survival and toxicity data. twentynine patients (age 0-19 years) who received a second hsct at our institution during 1998-2015 were included in the analysis. thirteen patients had an underlying malignancy and 16 patients were transplanted for non-malignant indications, including inborn errors of metabolism, non-malignant hematologic diseases and immune deficiency. median follow up was 14 months (range: 1-180). there were 10 deaths (77%) in the malignant group, 7 (53%) were due to disease relapse and 3 (23%) were transplant related. fifty percent of deaths occurred within the first year following the second hsct. in the non-malignant group there were 5 deaths (31%), of which 2 (12%) were attributed to the underlying disease and 3 (18%) were transplant related. all deaths but one occurred within the first year post hsct. treatment related mortality following second hsct is higher compared to first transplant. the higher survival rate in the non-malignant group suggests that transplant following graft failure should be considered ins501 patients with otherwise incurable underlying disease. though the outcome for patients with relapse of malignant disease following hsct is poor, a second transplant may benefit a subset of these patients. attempts to achieve complete remission prior to transplant should be made to improve outcome. due to the small number of patients in our cohort, further multi-center trials are needed. disclosure of conflict of interest: none. disseminated bcg infection (bcg-osis) is a rare but most serious complication in vaccinized especially immunocompromised children. severe combined immunodeficiency disorder (scid) is probably the commonest primary immunodeficiency associated with bcg-osis, though there is no such definitive data as most of the cases described in literature are in the form of reports. hematopoietic stem cell transplantation (hsct) is a life-saving treatment for patients with scid, especially if therapy is instituted early, prior to onset of infections.as bcg vaccine is routinely given to all iranian children at birth, the likelihood of having an active infection at the time of transplant would be significantly high. the main objective of this study was to evaluate the outcomes of hsct in scid patients with disseminated bcg infection . sixteen scid patients underwent hsct in our center since 2007 to 2016, of which nine patients (7 male, 2 female) were enrolled in this analysis. all the 9 patients had received bcg vaccination according to the national vaccination protocol, and had undergone anti-tuberculosis (tb) treatment prior to transplant due to disseminated bcg infection. the mean age at hsct was 9.3 months (range: 6-13 months). patients received bone marrow (n = 1), peripheral blood progenitor cells (n = 6) or umbilical cord blood grafts (n = 2) from hla-matched related donors (n = 7) and mismatched unrelated donors (n = 2). three patients received unconditioned matched sibling donor transplants and ric regimen was provided with fludarabine, melphalan and rabbit anti-thymocyte immunoglobulin (thymoglobulin) in others . cyclosporine a and prednisolon were used as graft-versus-host disease (gvhd) prophylaxis. they also continued to receive anti-tb treatment. all patients but one engrafted. the median times to neutrophil and platelet engraftments were 12 days (range: 11-39), and 18 days (range: 17-90), respectively. engraftment with full chimerism (495%) occurred in 5 patients and the other 3 patients had mixed chimerism. with a median follow-up of 24 months (range: 3 -48 months), overall survival was 66.7%. the main cause of death was disseminated bcg infection.three out of 8 patients who achieved engraftment, developed acute gvhd (grade i-ii), while one patient developed extensive chronic gvhd. although anti-tb treatment continued, tuberculous dactylitis occurred in 3 patients post-hsct that were successfully treated. on last post-hsct follow-up, 4 patients with full chimerism and 2 with mixed chimerism are alive and disease free. scid is called as a pediatric emergency as it invariably leads to fatality in infancy without early aggressive therapy and hsct. in hsct recipients, the impaired cellular immunity renders these patients more susceptible to infection. as previous reports suggest, our study demonstrates that with appropriate anti-tb cover, immunological reconstitution with complete recovery from bcg infection can be achieved by early hsct. disclosure of conflict of interest: none. paraproteinemia occurrence after allogeneic hematopoietic stem cell transplant as a possible marker for chronic gvhd onset f monaco 1 , s tamiazzo 1 , f dallavalle 1 , l calcagno 2 , m pini 1 and m ladetto 1 1 hematology and 2 transfusion medicine, azienda ospedaliera ss. antonio e biagio e cesare arrigo, alessandria, italy transient monoclonal gammopathy is commonly reported after solid organ or stem cells transplant (sct) for hematologic malignancies. however the clinical significance of a paraproteinemia appearance is not fully understood, because the attempts to correlate its effect on survival rates, graft versus host disease (gvhd) occurrence and viral reactivations have led to controversial results. starting from these reports we decided to evaluate among our allogeneic transplanted patients the incidence of m-component and its possible relationship with chronic gvhd. one-hundred and one patients undergoing allosct at the hematology unit of alessandria (italy) between 2006 and 2015 were evaluated. 55% of patients were male and 45% were females. pretransplantation diagnosis included: 62 acute myeloid leukaemia/ high-risk myelodisplastic sindromes (62%), 14 acute lymphoblastic leukaemia (14%), 13 lymphoproliferative disorders (13%) and 12 other less common malignancies (12%) . patients with multiple myeloma were excluded from the study. all patients had, at least, two pre-transplantation serum electrophoresis with no evidence of pre-existing monoclonal component. serum electrophoresis was scheduled to be performed at 90, 180 and 360 days and 2 years after transplantation. forty-nine patients were submitted to allo-sct from a sibling donor and 52 from a matched related donor (mud); in vivo t-cell depletion with anti-thymocyte globulin was used in 63 patients. thirty-four patients relapsed after allosct, 52 (52%) developed chronic gvhd and 56 patients (56%) are currently alive at the last follow-up. posttransplantation follow up ranged from 81 to 2695 days with a median of 496 days. paraproteins were detected in 52 out of 101 patients (52%), being monoclonal in 28 patients, and bi or tri-clonal in the remaining cases; the immunoglobulin subclass most commonly observed was igg. ten-year overall survival of the whole population was 50%; splitting the population in two cohorts (with or without paraproteinemia) we did not detect any statistical differences in overall survival, gvhd development and relapse incidence at +90 and +180 days posttransplant; viceversa, after 360 days, a statistically significant difference was observed in chronic gvhd occurrence in patients with or without paraproteinemia (85% vs 42%, respectively, po 0.001). ten-year overall survival curves were significantly better in patients with paraproteinemia as compared with the paraprotein-free group (59% vs 45%, p = 0.04), and an even more evident significance was seen in ten-year relapse free survival curves (66% for patients with paraprotein vs 48% for patients without paraprotein, p = 0.009). monoclonal gammopathy, also in our experience, is frequent following allo-sct. we observed a strong correlation between the occurrence of paraproteinemia, chronic gvhd and a significantly better overall and relapse-free survival. recently many evidences showed that b cells are involved in the pathogenesis of chronic gvhd (cgvhd) and anti-b-cell therapy has been suggested for the treatment of cgvhd. we speculate that the presence of a monoclonal gammopathy after allogeneic transplant is expression of the activation of the b-cell compartment. a prospective study with a larger population should be considered, in order to confirm our results and assay post-transplantation monoclonal gammopathy as an early marker for gvhd development. disclosure of conflict of interest: none.inherited bone marrow failure (ibmf) syndromes are rare pediatric disorders that characteristically associate physical abnormalities, progressive bone marrow failure and predisposition to cancer. the most common of these disorders is fanconi anemia (fa). stem cell transplantation (sct) using related or unrelated donors are the only curative therapeutically approach when severe marrow failure is established. the aim of the study was to analyze the results of sct for patients with ibmfs in a single center. we performed a retrospective study in pediatric patients with ibmf admitted in pediatric hematology and bone marrow transplant department, fundeni clinical institute between january 2000 and september 2016. diagnosis and severityof ibmfs were established based on hematological results, bone marrow biopsy and clinical findings. genetic testing for ibmfs is not currently available in our country. indication for sct was established when patients developed moderate/severe aplastic anemia and became transfusion dependent.in case of dba, sct indication was established for steroid resistant disease. the donors were selected from family members or unrelated donors, 10/10 matched.the conditioning regimens used were reduced intensity (fludarabine 120-150 mg/m 2 , cy 20 mg/kg, f-atg 40 mg/kg) for af, dc and myeloablative (busulfan i.v., fludarabine 150 mg/m 2 , thiotepa 20 mg/kg, f-atg 30 mg/kg) for dba. gvhd prophylaxis consisted of standard methotrexate and csa/tacrolimus. all parents signed informed consent forms. in our center, between 2000 and 2016, 20 patients with ibmf were diagnosed: 10 (50%) patients with fa, 6 (30%) patients with diamond blackfan anemia (dba), 2 (10%) patients with diskeratosis congenita (dc), and 2(10%) patients with not classifiable ibmfs. the patient data is available in table 1 . seven out of 20 patients (35%) performed sct procedures: sibling 3 patients (2 patients with af, 1 patient with dc), mud 4patients (3 patients with af, 1 patient with dba). all patients (100%) engrafted for pmn (median = 17, range: 12-29 days) and platelet (median 21, range: 13-46 days).2/7 (42%) presented reactivation of cmv and received valganciclovir, 1/7 developed cmv disease (encephalitis and pneumonia), 2/7 (28%) developed bkv cystitis and required extensive hydration and levofloxacin. 4/7 (57%) developed grade i-ii skin acute gvhd day +100, which responded to topical treatment and low dose of corticosteroids. 1/7 (14%) developed grade iii intestinal acute gvhd, which responded to high-dose corticosteroids.1 /7 (14%) developed grade iv intestinal chronic gvhd (day +160), without response to high-dose corticosteroids, mmf and later died on day +221, due to infectious complications (severe pulmonary and cerebral aspergillosis). 6/7 patients (85%) are alive, with 100% donor chimerism 4/6(66%) or stable mixed chimerism 2/6 (33%). median follow-up for sct patients was 515 days (26 days-5y 6mo). conclusions in our study we observed a low incidence of severe complications associated with low mortality rate (14%) . sct is a procedure that associates multiple risk situations, but it remains the only curative cytomegalovirus (cmv) infections remain a significant cause of morbidity and mortality in patients whose immune systems are compromised, including hematopoietic stem cell transplant (hsct) recipients. although the adoptive transfer of third party cmv-specific t cells has proven both safe and clinically beneficial in treating even drug-refractory infections/disease, broader implementation and commercialization of this strategy has been hampered by (i) the postulated need for extensive cell banks generated from donors representing diverse hla profiles, and (ii) lack of large scale t cell manufacturing processes. to address these limitations we have developed a proprietary decision tool (cytomatch™) to identify a small panel of healthy donors who should provide almost universal hla coverage; and optimized a simple, scalable manufacturing process to generate large numbers of cmv-specific t cells. to assess the robustness of our strategy we generated a bank of cmv-specific t cells (viralym-c™) from 8 carefully selected healthy donors. the lines were polyclonal, comprising both cd4 + (21.3 ± 6.7%) and cd8 + (74.8 ± 6.9%) t cells, expressed central cd45ro+/cd62l+ (58.5 ± 4.2%) and effector memory markers cd45ro+/cd62l-(35.3 ± 12.2%), and were specific for the immunodominant cmv antigens ie1 and pp65 (ie1: 419 ± 100; pp65 1070 ± 31 sfc/ 2 × 10 5 , n = 8). a fixed-dose (2 × 10 7 cells/m 2 ) phase i clinical trial was subsequently initiated to test the safety and efficacy of these "ready to administer" t cells in pediatric and adult hsct recipients with drug-refractory cmv infections. using our bank of just 8 lines, we have identified a suitable line for 21 of 22 patients screened. of these, 7 patients have been treated with viralym-c cells; 6 received a single infusion and 1 patient required 2 infusions for sustained benefit. there were no immediate infusion-related toxicities; and despite the hla disparity between the viralym-c™ lines and the patients infused, there were no cases of de novo or recurrent graft versus host disease (gvhd). based on viral load (measured by quantitative pcr) and/or symptom resolution, viralym-c cells controlled infections in all patients with 5 complete (cr) and 2 partial responses (pr) achieved within 4 weeks of infusion. one patient with cmv retinitis had complete resolution of symptoms following viralym-c™ infusion. our results demonstrate the feasibility, preliminary safety and efficacy of "ready to administer" viralym-c™ cells that have been generated from a small panel of healthy, eligible cmv seropositive donors identified by our decision support tool. these data suggest that cost-effective, broadly applicable t cell anti-viral therapy may be feasible for patients following hsct and potentially other conditions. disclosure of conflict of interest: drs. juan vera, ann leen and brett giroir hold equity and drs. ifigeneia tzannou, sunitha kakarla are employed by viracyte. haploidentical stem cell transplantation (hsct) protocols utilizing ex vivo t-cell depleted grafts have been proven efficient in preventing graft versus host disease (gvhd), but cause a delay in early t-cell recovery that increases the risk of graft rejection, leukemia relapse and viral infections. conventional donor lymphocyte infusion (dli) after hsct transplantation is conditioned because of the high prevalence of gvhd even with low dose of t cells. here we present preliminary data of escalating cd45ro+ memory t cells as dli in three patients that received a selective graft depleted of naïve (cd45ra+) t-cells. three children that were transplanted following nonmyeloablative conditioning regimen with a graft consisting of cd34+ and cd45ra-cells, with mixed chimerism, lymphopenic and viral/opportunistic infections and minimal residual disease positive before hsct received dose scalating cryopreserved haploidentical cd45ra-memory t cell starting with a initial dose of 1 × 10 5 /kg, until a maximal dose of 1 × 10 8 /kg with a 21 days interval. we infused 10 products with a naïve (45ra+) t-cell dose less than 1 × 10 4 /kg with 499.9% purity of cd3 + cd45ro+ memory t-cells in all cases. all infusions were well tolerated without any side effect during infusions neither gvhd. following the dli, a progressive increase in t cell counts was observed. our preliminary data suggest that dose escalating of haploidentical memory t cells (45ro+) as dli provides a safety platform, even with high dose of t cells (1 × 10 8 /kg), for adoptive immunotherapy in haploidentical 45ra+ depleted grafts with no gvhd complications, and allows an increase in t cell reconstitution. however, efficacy of this strategy requires longer studies. relapse after allogeneic hematopoietic stem cell transplantation (allohsct) remains a major therapeutic challenge: outcome is very poor, without curative option in most cases. second allohsct may be considered in few selected patients because of anticipated limitations: (1) donor availability; (2) high toxicity due to previous treatments; (3) low efficacy considering the very advanced disease situation. we hypothesized that the use of post transplantation cyclophosphamide (pcy) haplo-sct after relapse following allohsct may deal in part with these limitations. in particular, the presence of full haplotype hla mismatch could provide a decisive antileukemic effect relative to alloreactivity. in absence of large series in this setting, we report here the outcome after haplosct for patients who relapse after a first allohsct. we retrospectively studied adult patients, who received a second pcy haplo-sct for hematological malignancies. patients were treated between 2009 and 2016. the objective was to assess both the feasibility and the efficacy of haplosct in this setting. twenty seven patients were included: median time between first allohsct and relapse was 11 months (range: 1-82). median age at second transplantation was 49 years old (range: 21-61). most of patients had acute myeloid leukemia (n = 12, 44%) or hodgkin lymphoma (n = 6 patients, 22%). fifteen the impact of minimal residual disease and its kinetics prior to different types of allogeneic hematopoietic stem cell transplantation on clinical outcomes in patients with acute myeloid leukemia z xiao-su 1,2 , l yan-rong 1 , yan-hong 1 , p xu-ying 1 , qian-jiang 1 , hao-jiang 1 , lan-ping, xu 1 , xiao-hui zhang 1,2 , yu-wang 1,2 , h xiao-jun 1,2 and c ying-jun 1 this study investigated the impact of minimal residual disease (mrd) and its kinetics prior to different types of allogeneic hematopoietic stem cell transplantation (hsct) on clinical outcomes in patients with acute myeloid leukemia (aml) in complete remission (n = 132). 107 patients who received unmanipulated haploidentical hsct and 25 patients who received hla-matched sibling hsct were enrolled. mrd measured using 8-color flow cytometry (fcm) at fixed time points before transplantation was retrospectively analyzed. the patients were divided into four groups based on mrd kinetics before transplantation: consistent negative, positive to negative, negative to positive and consistent positive. during the follow-up, total twenty (15.2%) patients underwent relapse. through unique variate analysis, none of mrd status at various time points before unmaipulated haploidentical transplantation was associated with clinical outcomes, as well as the dynamic change of mrd before hsct (p40.05), although the patients with consistent positive mrd before hsct seemed to have a relatively higher incidence of relapse (p = 0.151). one-year cumulative incidence of relapse (cir) were 11.2 ± 4.7% vs. 31.1 ± 13.8% in mrd consistent negative and consistent positive groups, respectively (p = 0.202). however, patients with positive mrd after the second chemotherapy or pre-mrd before hla-matched sibling hsct showed a significant poor outcomes including higher cir (p = 0.015 and both neuroblastoma (nrb) and rhabdomyosarcoma (rms) in childhood are the aggressive malignant disease with higher mortality. this paper aims to study the efficacy of autologous peripheral blood stem cell transplantation (apbsct) in the treatment of high risk advanced nrb and rms. 34 patients with high-risk stage iv nrb and 9 patients with advanced childhood rms were treated by apbsct in our hospital from october of 2010 to may of 2016. in the subgroup of nrb patients, 16 patients got complete remission (cr) and 1 patient got cru while 17 patients had tumor residual disease after intensive induction therapy before asct. the median age was 5.55 (1-9) years old. primary sites of the tumors included submaxilla (n = 2), cervical (n = 5), adrenal gland (n = 16)and retroperitoneal (n = 11). the conditioning regimen consisted of busulfan and melphalan (busulfan 1 mg/kg × 4d, melphalan 140mg/m 2 x1d) or cem regimen (carboplatin 600 mg/m 2 × 3d, etoposide 500 mg/m 2 × 3d, cyclophosphamide 1800 mg/ m 2 × 2d); the pathology of 9 stage iii childhood rms patients was embryonal rhabdomyosarcoma. there were 8 cases in cr and 1 case in partial remission (pr). the median age was 6.56 (3-13) years old. primary sites of the tumors included bladder (n = 1), left forearm (n = 3), retroperitoneal (n = 1), pelvic (n = 3) s513 and talus (n = 1). the conditioning regimen consisted of melphalan, cyclophosphamide and dactinomycin (melphalan 60 mg/m 2 × 3d, cyclophosphamide 1800 mg/m 2 × 2d, dactinomycin 0.0 13 mg/kg × 3d).there were 13 double apbsct cases (nrb n = 12, rms n = 1). all the relapse patients were treated with chemotherapy and radiation therapy. all the patients successfully underwent mobilization, collection and reinfusion. the time of hematopoietic reconstitution was (11. 0 ± 3) days, no severe toxicity was observed, no transplant-related death was found. with a median follow-up of 25.35(2-60) months, one of the patients was lost to follow-up. in the subgroup of nrb patients (n = 33): the 2-year event-free survival and total survival rate of all patients were 66.2% and 79.4%, respectively. the survival time of no recurrence was significantly different between the double transplantation group and single transplantation group (p o0.05). in the subgroup of rms patients (n = 9), 1 patient died, 6 patients live without pd(1 patients had double apbsct), 2 patients suffered recurrence but still alive. apbsct achieved good outcome in patients with high risk advanced nrb and rms. transplantation-related toxicities were tolerable. double apbsct significantly improved the depth of remission. disclosure of conflict of interest: none. transplantation outcomes of a once-daily intravenous busulfan and fludarabine conditioning for allogeneic hematopoietic stem cell transplantation in pediatric aml and high risk mds: single center experience in korea y-t lim, e-j yang and k-m park department of pediatrics, pusan national university children's hospital, yangsan, koreathere have recently been some reports suggesting that oncedaily intravenous busulfan as a conditioning regimen for hematopoietic stem cell transplantation (hsct) possibly reduces the toxicities without influencing the clinical outcome as compared with the traditional 4 times daily dosage schedule. but until recently there has been little research and limited data available on the safety and efficacy of oncedaily intravenous busulfan and fludarabine in pediatric allogeneic hsct. we report the outcomes for allogeneic hsc recipients, evaluating engraftment status, regimen related toxicities (rrt), and event free survivals (efs) after use of oncedaily intravenous busulfan and fludarabine conditioning for allogeneic hsct in children with aml and high risk mds in a single pediatric center of korea. from january 2005 to december 2015, 22 aml and 2 high risk mds children who received once daily iv busulfan/fludarabine based conditioning regimen for allogeneic hsct were reviewed, bu/flu ± atg consist of intravenous fludarabine (40 mg/m 2 ) and busulfan (110~130 mg/m 2 , once daily iv) on days -6 to -3, and antithymocyte globulin (atg) (3 mg/kg) on days -3 to -1. all patients received tacrolimus and mini-dose methotrexate (5 mg/m(2)) for graft versus host disease (gvhd) prophylaxis. 17 boys and 9 girls were enrolled with median age of 10.1 years (range: 0.6-17.9 years). the median period from diagnosis to transplantation was 7 months (range: 5-49 months). more than half of the patients had a matched sibling donor (n = 16, 62%), 27% patients (n = 7) had a matched unrelated donor, 8% patients (n = 2) had a mismatched unrelated donor, and the remaining 1 patient had a mismatched family donor. as a stem cell source, peripheral blood stem cells (pbsc) were 22 cases (85%), bone marrow and cord blood were 2 cases in each. the median follow-up for patients was 40 months. the median number of infused total nucleated cells and cd 34+ cells except cord blood transplantation were 9.2 × 10(8)/kg and 7.8 × 10(6)/kg. all patients including who received cord blood were successfully engrafted. the median time to absolute neutrophil count (anc) recovery (anc 4 500 × 10(6)/l) and platelet recovery (platelet4 20,000 × 10(6)/l) were 13 days, 18 days in each. the incidence of acute gvhd was 19.2%, while severe grade iii/iv gvhd was observed in only 2 patient (7.7%). there were only two cases (8.7%) of extensive chronic gvhd in this study. transplant-related toxicities were acceptable, there was no case with cns toxicity, eleven patients (42.3%) developed grade ii,iii mucositis and grade i-iii hepatic toxicity in twenty four (92.3%), but transient. there was 3 clinically diagnosed veno-occlusive disease (vod), but most recovered by fluid restriction and diuretics. nine patients (36%) showed positive cytomegalovirus (cmv) antigen/pcr but only one patient developed cmv colitis. eight patients died: 7 due to relapse/disease progression, 1 due to extensive chronic gvhd. the 5-year efs and overall survival were 62.2% and 66.1% respectively. at 3 year, the cumulative incidence of relapse was 19.2%. overall, once-daily intravenous busulfan and fludarabine was less toxic and effective as conditioning regimen in aml and high risk mds patients undergoing allogeneic transplantation in children. disclosure of conflict of interest: none. haploidentical stem cell transplantation from unmanipulated graft has becoming a practiced option for high risk hematological malignancies who lack a matched related or unrelated donor. lack of a matched sibling or unrelated donor (mud) can be a significant barrier to allogeneic transplantation in patients who stand to benefit from this procedure. hlahaploidentical donors are readily available for nearly all such patients. haplo transplantation has inherent advantages over mud transplantation including the lower cost of graft acquisition, greater availability of donors for ethnic minorities, and immediate access to the donor in patients in whom delay cerebral palsy (cp) is a heterogeneous group of conditions that result in permanent motor disability. it may occur due to perinatal hypoxic insults, developmental brain abnormalities, genetic diseases, traumatic or infectious causes. in general the condition is non-progressive, but improvement over time is rarely seen. various treatment methods have been used for the management of this disorder. however, there has been no absolute cure for cp. the ultimate goal of stem cells therapy is to use the regenerative capacity of the stem cells causing a formation of new tissues to replace the damaged tissue. the polish stem cell bank (pbkm) has provided wharton's jellyderived msc (wj-msc) for medical therapeutic experiment application in children with cp. wj-msc from third party donors were administered to 27 patients (pts) with cp aged from 1.6/12 to 16.9/12 (median age: 6 years and 1 month). twenty two pts have received infusions intravenously (i.v.), 1 pt intrathecally (i.t.), and 4 pts via both routes (first i.v., next i.t.). the cells were previously collected from healthy newborns, processed, screened for bacterial contamination as well as endotoxin content, and frozen in liquid nitrogen vapour. msc immunophenotype was confirmed using flow cytometry assay. the pts have received from 1 to 6 infusions in intervals from 4 weeks to 6 months. median i.t. dose was 15 × 10 6 cells per infusion, while median i.v. dose was 1 × 10 6 cells/kg of body weight per infusion. each patient has been examined by the same neurologist at the day of each infusion and the result of examination has been described in a follow-up. twelve patients were diagnosed with epilepsy as comorbidity. eighteen pts (67%) showed positive changes in neurological examination after their treatment with wj-msc. almost half of the children experienced improvement of cognitive functions (12 out of 27 pts). muscle tension was reduced in 6 pts. improvement in the ability to concentrate, better contact with others and improved social interactions were observed in 19% of pts. correction of motility was noticed in 5 pts, 2 pts have experienced better quality of sleep. in 3 cases there has been a reduction in the number of epileptic seizures (1 pt even discontinued some of his medicines). there were no s515 noticeable changes in neurological examination of 2 patients. seven follow-up forms have been not received yet. the experiment data provide evidence that third-party donor wj-msc are suitable and efficient stem cells for treatment in patients with cp. however further and more extensive examination, with a greater number of patients is needed, which will be beneficial for far-reaching results. spina bifida (sb) is a congenital malformation resulting from failure of fusion in the spinal neural tube during embryogenesis. despite surgical repair of the defect, most patients who survive with spina bifida have multiple system damage due to neuron deficiency in the spinal cord. it has been confirmed that the mesenchymal stem cells (mscs) have the ability to survive, migrate and differentiate into cells of a neural lineage. wharton's jelly-derived mscs (wj-mscs) from third-party donors have high proliferation and differentiation potential along with non-immunogenic features, thus seem to be a promising stem cell source. the polish stem cell bank (pbkm) has provided wj-msc for clinical application in a medical therapeutic experiment for children with sb. eleven patients (pts) were qualified for administration of wj-mscs. three pts have been waiting so far for their therapy after bioethical committee approval. seven pts were in the middle of stem cell therapy (after 1 or 2 injections), 1 pt had finished one cycle of stem cell therapy (5 injections -ijs) and resumed therapy by administering a first dose of wj-mscs. the cells were previously collected from healthy newborns, processed, screened for bacterial contamination as well as endotoxin content, and frozen in liquid nitrogen vapors. six pts have received infusions intravenously (median dose: 1.01 × 10 6 /kg body weight per infusion), and 1pt was given 1 injection of 40 × 10 6 cells intrathecally. each patient has been examined by the same neurologist at the day of each infusion and the result of examination has been written in a follow-up. there were 6 pts, who received at least 2 doses of wj-msc, and all of them showed positive changes in neurological examination. the important improvement, declared by pts, was in areas: pronunciation and/or self-reliance (3pts), movement of arms and/or legs (4pts), quality of life (3pts), core stabilization (1pt). only one adverse event occurred after third injection of wj-msc: 1 pt had nausea and a fever. in case of other pts it was too early to provide reliable feedback. the transplantation of wj-mscs could stimulate the mscs to differentiate towards sensory neurons. this could be one of the reasons of observed improvement of many vital functions in patients, after mscs treatment. this approach might have value in the experimental treatment of sensory neuron deficiency in spina bifida.